Ecology and classification of North American Freshwater Invertebrates

Andrea de Leon Oliver

The littoral community of lotic and lentic environments of the Middle Paraná River is complex and dynamic, providing shelter to a high biological diversity and abundant populations. A numerically important group and with active participation in the community structure is that of macrocrustaceans, and, specifically, the order Decapoda, superorder Eucarida Calman 1904, subphylum Crustacea Brünnich 1772. The taxonomic unit Decapoda records more than 8,500 species, most of them restricted to marine areas (Brusca and Brusca 1990); however, some of them have successfully conquered freshwater and brackish environments. Among the latter, mangrove swamps and lenitic environments of large river floodplains provide the highest diversity and density of individuals due to their environmental heterogeneity (Bliss 1989).

Ecology and Classiﬁcation of North American Freshwater Invertebrates Second Edition This Page Intentionally Left Blank Ecology and Classiﬁcation of North American Freshwater Invertebrates Second Edition Edited by JAMES H. THORP Bayard and Virginia Clarkson Professor of Biology Department of Biology Clarkson University Potsdam, New York and ALAN P. COVICH Department of Fishery and Wildlife Biology Colorado State University Fort Collins, Colorado San Diego San Francisco New York Boston London Sydney Tokyo Front cover images: From Jurine, L. (1820). Histoire des Monocles, qui se trouvent aux environs de Geneve. J. J. Paschoud, Paris, France. This book is printed on acid-free paper. Copyright © 2001, 1991 by ACADEMIC PRESS All Rights Reserved. No part of this publication may be reproduced o transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. Requests for permission to make copies of any part of the work should be mailed to: permissions Department, Harcourt Inc., 6277 Sea Harbor Drive, Orlando, Florida 32887-6777 Academic Press A Harcourt Science and Technology Company 525 B Street, Suite 1900, San Diego, California 92101-4495, USA http://www.academicpress.com Academic Press Harcourt Place, 32 Jamestown Road, London NW1 7BY, UK http://www.academicpress.com Library of Congress Catalog Card Number: 00-109117 International Standard Book Number: 0-12-690647-5 PRINTED IN THE UNITED STATES OF AMERICA 00 01 02 03 04 05 06 EB 9 8 7 6 5 4 3 2 1 “For aminals and callapiters and other wonders of nature seen through our children’s eyes.” Dr. James Thorp “To my family and to Professor G. E. Hutchinson for their inspiration and patience.” Dr. Alan Covich This Page Intentionally Left Blank Contents Contributors xiii Preface xv 2 AN OVERVIEW OF FRESHWATER HABITATS James H. Thorp and Alan P. Covich I. II. III. IV. 1 INTRODUCTION TO FRESHWATER INVERTEBRATES James H. Thorp and Alan P. Covich I. Introduction 1 II. Approches to Taxonomic Classiﬁcation 1 III. Synopses of the North American Freshwater Invertebrates 4 IV. Taxonomic Key to Major Taxa of Freshwater Invertebrates 16 Literature Cited 18 Introduction 19 Lotic Environments 19 Underground Aquatic Habitats 30 Lentic Ecosystems 33 Literature Cited 39 3 PROTOZOA William D. Taylor and Robert W. Sanders I. Introduction 43 II. Anatomy and Physiology 45 vii viii Contents III. Ecology and Evolution 58 IV. Collecting, Rearing, and Preparation for Identiﬁcation 69 V. Identiﬁcation of Protozoa 70 Literature Cited 89 4 PORIFERA Thomas M. Frost, Henry M. Reiswig, and Anthony Ricciardi I. II. III. IV. Introduction 97 Structure and Physiology 97 Ecology and Evolution 105 Collecting, Rearing, and Preparing Sponges for Identiﬁcation 115 V. Classiﬁcation 116 Literature Cited 130 VI. Identiﬁcation of North American Genera of Freshwater Turbellaria 163 NEMERTEA VII. General Characteristics, External and Internal Anatomical Features 173 VIII. Ecology 174 IX. Current and Future Research Problems 176 X. Collection, Culturing, and Preservation 176 XI. Taxonomic Key to Species of Freshwater Nemertea in North America 176 Literature Cited 176 7 GASTROTRICHA David Strayer and William D. Hummon I. II. III. IV. Introduction 181 Anatomy and Physiology 182 Ecology and Evolution 183 Collecting, Rearing, and Preparation for Identiﬁcation 188 V. Taxonomic Key 189 Literature Cited 192 5 CNIDARIA Lawrence B. Slobodkin and Patricia E. Bossert I. Introduction 135 II. General Biology of Cnidaria 136 III. Descriptive Ecology of Freshwater Cnidaria 140 IV. Collection and Maintenance of Freshwater Cnidaria 150 V. Classiﬁcation of Freshwater Cnidaria 151 Literature Cited 153 6 FLATWORMS: TURBELLARIA AND NEMERTEA 8 PHYLUM ROTIFERA Robert Lee Wallace and Terry W. Snell I. II. III. IV. Introduction 195 Anatomy and Physiology 197 Ecology and Evolution 207 Collecting, Rearing, and Preparation for Identiﬁcation 230 V. Classiﬁcation and Systematics 233 Literature Cited 248 9 NEMATODA AND NEMATOMORPHA Jurek Kolasa George O. Poinar TURBELLARIA I. Introduction: Status in the Animal Kingdom 156 II. Anatomy and Physiology 156 III. Ecology 158 IV. Current and Future Research Problems 162 V. Collecting, Rearing, and Identiﬁcation Techniques 163 NEMATODA I. Introduction 255 II. Morphology and Physiology 256 III. Development and Life History 260 IV. Ecology 261 V. Collecting and Rearing Techniques 265 VI. Identiﬁcation 269 Contents NEMATOMORPHA VII. Introduction 280 VIII. Morphology and Physiology 282 IX. Development and Life History 288 X. Sampling 290 XI. Identiﬁcation 291 Literature Cited 292 BRANCHIOBDELLIDAE VI. Introduction to Branchiobdellidae 456 VII. Anatomy and Physiology of Branchiobdellidans 456 VIII. Ecology and Evolution 458 IX. Identiﬁcation of Branchiobdellidans 459 Literature Cited 461 10 13 MOLLUSCA: GASTROPODA ANNELIDA: EUHIRUDINEA AND ACANTHOBDELLIDAE Kenneth M. Brown I. II. III. IV. Introduction 297 Anatomy and Physiology 298 Ecology and Evolution 302 Collecting and Culturing Freshwater Gastropods 315 V. Identiﬁcation of Freshwater Gastropods of North America 315 Literature Cited 325 Ronald W. Davies and Fredric R. Govedich I. Introduction to Euhirudinea and Acanthobdellidae 465 II. Taxonomic Status and Characteristics 466 III. Euhirudinea and Acanthobdellidae 468 IV. Ecology 474 V. Collection and Rearing 486 VI. Identiﬁcation 486 VII. Taxonomic Keys 489 Literature Cited 497 11 MOLLUSCA: BIVALVIA Robert F. McMahon and Arthur E. Bogan I. II. III. IV. Introduction 331 Anatomy and Physiology 333 Ecology and Evolution 354 Collecting, Preparation for Identiﬁcation, and Rearing 393 V. Identiﬁcation of the Freshwater Bivalves of North America 397 Literature Cited 416 12 14 BRYOZOANS Timothy S. Wood I. II. III. IV. V. VI. Introduction 505 Anatomy and Physiology 505 Ecology and Evolution 511 Entoprocta 515 Study Methods 516 Taxonomic Key 517 Literature Cited 523 ANNELIDA: OLIGOCHAETA, INCLUDING BRANCHIOBDELLIDAE 15 Ralph O. Brinkhurst and Stuart R. Gelder Diane R. Nelson OLIGOCHAETA I. Introduction to Oligochaeta 431 II. Oligochaete Anatomy and Physiology 432 III. Ecology and Evolution of Oligochaeta 438 IV. Collecting, Rearing, and Preparation of Oligochaetes for Identiﬁcation 444 V. Taxonomic Keys for Oligochaeta 446 TARDIGRADA I. II. III. IV. V. VI. Introduction 527 Anatomy 528 Physiology (Latent States) 535 Reproduction and Development 536 Ecology 539 Techniques for Collection, Extraction, and Microscopy 543 ix x Contents VII. Identiﬁcation 544 Literature Cited 546 16 WATER MITES (HYDRACHNIDA) AND OTHER ARACHNIDS II. Anatomy and Physiology of Crustacea 780 III. Ecology and Evolution of Selected Crustacea 787 IV. Collecting, Rearing, and Preparation for Identiﬁcation 798 V. Classiﬁcation of Peracarida and Brachiura 799 Literature Cited 800 Ian M. Smith, Bruce P. Smith, and David R. Cook I. Introduction to Arachnids 551 II. Water Mites (Hydrachnida) 552 III. Other Arachnids in Freshwater Habitats 651 Literature Cited 653 17 DIVERSITY AND CLASSIFICATION OF INSECTS AND COLLEMBOLA William L. Hilsenhoff I. II. III. IV. V. Introduction 661 Aquatic Orders of Insects 664 Partially Aquatic Orders of Insects 681 Semiaquatic Collembola — Springtails 699 Identiﬁcation of the Freshwater Insects and Collembola 700 Literature Cited 721 20 OSTRACODA L. Denis Delorme I. II. III. IV. V. VI. VII. VIII. IX. X. XI. XII. XIII. XIV. 18 Introduction 811 Anatomy and Physiology 812 Life History 817 Distribution 820 Chemical Habitat 821 Physical Habitat 823 Physiological and Morphological Adaptations 826 Behavioral Ecology 827 Foraging Relationships 827 Population Regulation 828 Collecting and Rearing Techniques 829 Uses of Freshwater Ostracoda 831 Current and Future Research Problems 832 Classiﬁcation of Freshwater Ostracodes 832 Literature Cited 842 AQUATIC INSECT ECOLOGY Anne E. Hershey and Gary A. Lamberti I. II. III. IV. V. VI. Introduction 733 Aquatic Insect Communities 733 Aquatic Insect Life Histories 747 Insect-Mediated Processes 751 Secondary Production 761 Effects of Land Use on Aquatic Insect Communities 763 VII. Role of Disturbance 765 VIII. Aquatic Insects in Biomonitoring Studies 767 IX. Summary 768 Literature Cited 768 19 INTRODUCTION TO THE SUBPHYLUM CRUSTACEA Alan P. Covich and James H. Thorp I. Introduction 777 21 CLADOCERA AND OTHER BRANCHIOPODA Stanley I. Dodson and David G. Frey I. Introduction 850 CLADOCERA II. Anatomy and Physiology 851 III. Distribution 860 IV. Behavior 862 V. What Cladocerans Eat and What Eats Cladocerans 866 VI. Population Regulation 867 VII. Functional Role in the Ecosystem 868 VIII. Paleolimnology and Molecular Phylogeny 868 IX. Toxicology 871 X. Current and Future Research 873 XI. Collecting and Rearing 873 XII. Taxonomic Keys for Cladoceran Genera 875 Contents OTHER BRANCHIOPODS XIII. Anatomy and Physiology of the Other Branchiopods 891 XIV. Ecology of the Other Branchiopods 893 XV. Current and Future Research Problems 899 XVI. Collecting and Rearing Techniques 899 XVII. Taxonomic Keys for Non-Cladoceran Genera of Branchiopods 899 Literature Cited 904 22 COPEPODA Craig E. Williamson and Janet W. Reid I. II. III. IV. V. Introduction 915 Anatomy and Physiology 916 Ecology and Evolution 920 Current and Future Research Problems 933 Collecting and Rearing Techniques 934 VI. Identiﬁcation Techniques 935 Literature Cited 942 23 DECAPODA H. H. Hobbs III I. II. III. IV. V. Introduction 955 Anatomy and Physiology 955 Ecology and Evolution 964 Current and Future Research Problems 986 Collecting and Rearing Techniques 989 VI. Identiﬁcation 991 Literature Cited 993 Glossary 1003 Subject Index 1021 Taxonomic Index 1037 xi This Page Intentionally Left Blank Contributors Numbers in parentheses indicate the pages on which the authors’ contributions begin. Arthur E. Bogan (331) North Carolina State Museum of Natural Sciences, Research Laboratory, Raleigh, North Carolina 27607. Patricia E. Bossert (135) Science Department, Northport High School, Northport, New York 11768. Ralph O. Brinkhurst (431) Hermitage, Tennessee 37076. Kenneth M. Brown (297) Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana 70803. David R. Cook (551) 7836 North Invergordon Place, Paradise Valley, Arizona 85253. Alan P. Covich (1, 19, 777) Department of Fishery and Wildlife Biology, Colorado State University, Fort Collins, Colorado 80523. Ronald W. Davies (465) Faculty of Science, Monash University, Clayton, Victoria 3168, Australia. L. Denis Delorme (811) National Water Research Institute, Canada Centre for Inland Waters, Burlington, Ontario, L7R 4A6. Stanley I. Dodson (849) Department of Zoology, University of Wisconsin, Madison, Wisconsin 53706. David G. Frey† (849). Thomas M. Frost† (97). Stuart R. Gelder (431) Department of Biology, University of Maine at Presque Isle, Presque Isle, Maine 04769. Fredric R. Govedich (465) Department of Biological Sciences, Monash University, Clayton, Victoria 3168, Australia. † Deceased. xiii xiv Contributors Anne E. Hershey (733) Department of Biology, University of North Carolina–Greensboro, Greensboro, North Carolina 27402. William L. Hilsenhoff (661) Department of Entomology, University of Wisconsin, Madison, Wisconsin 53706. H. H. Hobbs III (955) Department of Biology, Wittenberg University, Springﬁeld, Ohio 45501. William D. Hummon (181) Department of Biological Sciences, Ohio University, Athens, Ohio 45701. Jurek Kolasa (155) Department of Biology, McMaster University, Hamilton, Ontario L8S 4K1 Canada. Gary A. Lamberti (733) Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana 46556. Robert F. McMahon (331) Department of Biology, The University of Texas at Arlington, Arlington, Texas 76019. Diane R. Nelson (527) Department of Biological Sciences, East Tennessee State University, Johnson City, Tennessee 37614. George O. Poinar, Jr. (255) Department of Entomology, Oregon State University, Corvallis, Oregon 97331. Janet W. Reid (915) Department of Invertebrate Zoology, National Museum of Natural History, Smithsonian Institution, Washington, DC 20560. Henry M. Reiswig (97) Redpath Museum and Department of Biology, McGill University, Montreal, Quebec H3A 2K6. Anthony Ricciardi (97) Department of Biology, Dalhousie University, Halifax, Nova Scotia B3H 4J1. Robert W. Sanders (43) Department of Biology, Temple University, Philadelphia, Pennsylvania 191ZZ. Lawrence B. Slobodkin (135) Department of Ecology and Evolution, State University of New York, Stony Brook, New York 11794. Bruce P. Smith (551) Biology Department, Ithaca College, Ithaca, New York 14850. Ian M. Smith (551) Biodiversity Section, ECORC, Central Experimental Farm Agriculture and Agri-Food Canada, Ottawa, Ontario, Canada K1A 0C6. Terry W. Snell (195) School of Biology, Georgia Institute of Technology, Atlanta, Georgia 30332. David Strayer (181) Institute of Ecosystem Studies, Millbrook, New York 12545. William D. Taylor (43) Department of Biology, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada. James H. Thorp (1, 19, 777) Department of Biology, Clarkson University, Potsdam, New York 13699. Robert Lee Wallace (195) Department of Biology, Ripon College, Ripon, Wisconsin 54971. Craig E. Williamson (915) Department of Earth and Environmental Sciences, Lehigh University, Bethlehem, Pennsylvania 18015. Timothy S. Wood (505) Department of Biological Sciences, Wright State University, Dayton, Ohio 45435. Preface It is a pleasure to write the preface to the second edition of this book because it indicates that our authors produced a ﬁrst edition that met the needs of our audience. To justify having those same professionals ﬁnd the second edition useful, as well as attracting a new generation of readers, it was incumbent upon us to develop a text that improves and updates the ﬁrst edition. We hope that you will ﬁnd our authors have done so! The reader will discover that almost every chapter has undergone major changes, with updated literature references, new coverage of invertebrate biology and ecology, and extensive revisions to taxonomy and classiﬁcation. In a few cases, authors have made important improvements in ﬁgures and have introduced more detailed taxonomic keys. We have expanded the number of authors (for Chapters 4, 11, 13, 16, and 22) and added a new chapter (Chapter 18 by Drs. Anne Hershey and Gary Lamberti). As in the ﬁrst edition, we limited our coverage of aquatic insects to save space and avoid excessive duplication with current textbooks on aquatic insect ecology and classiﬁcation. However, we opted to add a chapter on ecology to provide an expanded coverage of aquatic insects at the introductory level to accompany a more taxonomically centered chapter on insects (Chapter 17 by Dr. William Hilsenhoff). This edition is strongly focused on freshwater taxa found in surface waters of the United States and Canada. This encompasses at least 10,000 – 15,000 named species while avoiding the broad diversity of tropical aquatic invertebrates in North America. Groundwater and estuarine species are typically ignored or barely mentioned. We have also severely limited coverage of parasitic species and taxa that are only occasionally associated with aquatic habitats. As in the ﬁrst edition, we have asked the authors to set the taxonomic keys in their chapters at a level xv xvi Preface where the average reader could reasonably be assured of reaching an accurate organismal identiﬁcation without being an expert on that group. This approach is counter to a tendency in some texts to write dichotomous keys to the species level for the nonexpert. In many highly speciﬁc keys, nonspecialists use the keys to obtain both a species name and, sometimes, a false sense of security that accuracy has been achieved. To supplement information at the generic or higher level in our text, the chapters provide information on where the reader can ﬁnd more detailed, regional taxonomic keys. As in our ﬁrst book, many people contributed to this edition besides the authors. At Academic Press, we appreciate, in particular, efforts of our editor, Dr. Charles R. Crumly, and his able colleague and assistant, Ms. Donna James. At Clarkson University, we especially thank Linda Delosh and Bobbi Baillargeon for help in managing aspects of the book’s production and chapter distribution. Each chapter was reviewed by one to three outside experts; their help is acknowledged in some chapters and through individual letters from editors and authors. We also express our appreciation again to the following reviewers of the second edition, while sincerely apologizing in advance for any we have overlooked: Drs. Thomas A. Langen, Denis H. Lynn, Michael L. Pace, Henry M. Reiswig, Anthony Ricciardi, David Strayer, Mitchell J. Weiss, John W. Fleeger, W. T. Edmondson, Jeffrey D. Jack, Elizabeth J. Walsh, Andreas Schmidt-Rhaesa, Dreux J. Watermolen, Eileen H. Jokinen, Charles Lydeard, Caryn C. Vaughn, Mark J. Wetzel, Dean W. Blinn, Byron T. Backus, Judith E. Winston, Clark W. Beasley, William R. Miller, John C. Conroy, Thomas Simmons, Sharon K. Jasper, Manuel L. Pescador, Arthur C. Benke, Timothy B. Mihuc, David C. Culver, B. Brandon Curry, Alison J. Smith, John E. Havel, William R. DeMott, Anna M. Hill, and David M. Lodge. We continue to appreciate suggestions from readers on how individual chapters or the book as a whole could be improved. Future comments and suggestions can be directed to authors of individual chapters or to the editors. One of the sad tasks not faced in the ﬁrst edition is announcing the demise of past authors. We greatly regret the passing of Professors David G. Frey (a coauthor of Chapter 21: Cladocera and Other Branchiopoda) and, at a relatively young age, Thomas M. Frost (senior author of Chapter 4: Porifera). Dr. Stanley I. Dodson handled all revisions to Chapter 21, and Tom Frost had ﬁnished coauthoring Chapter 4 before he died in a tragic accident in the summer of 2000. They will be sorely missed by the two of us and by their many other colleagues and friends. As premature as it may sound, we have already begun thinking about our third, and probably last edition of this book with the current editors. While we expect another ten years will have elapsed before the third edition is completed, we realize it will require new authors in a number of areas as more senior people retire, shift research focal areas, or move into academic administration. If you are a reader who has the expertise, interest, and drive to write such a chapter, please feel free to contact one of us . . . but wait a few years so we have time to relax and handle those other tasks that have piled up on our desks while we completed work on the second edition! James H. Thorp and Alan P. Covich 1 INTRODUCTION TO FRESHWATER INVERTEBRATES James H. Thorp Alan P. Covich Department of Biology Clarkson University Potsdam, New York 13699 Department of Fishery and Wildlife Biology Colorado State University Fort Collins, Colorado 80523 I. Introduction II. Approaches to Taxonomic Classiﬁcation III. Synopses of the North American Freshwater Invertebrates A. Protozoa B. Porifera C. Cnidaria D. Flatworms: Turbellaria and Nemertea E. Gastrotricha F. Rotifera G. Nematoda and Nematomorpha H. Mollusca I. Annelida J. Bryozoa K. Tardigrada L. Arthropoda IV. Taxonomic Key to Major Taxa of Freshwater Invertebrates Literature Cited I. INTRODUCTION II. APPROACHES TO TAXONOMIC CLASSIFICATION Next to their more vibrant, and often larger, marine relatives, freshwater invertebrates may initially seem drab and uninspiring. Yet once students of freshwater biology penetrate beyond superﬁcial appearances and thoroughly examine the diverse structural, physiological, behavioral, and general ecological adaptations of freshwater invertebrates, few fail to be impressed by the enticing nature of these fascinating animals. This chapter serves as a very brief introduction to freshwater invertebrates of Canada and the United States. Following a short discussion of procedures for naming and classifying organisms, we brieﬂy describe the taxonomic groups covered in Chapters 3 – 23. We also include a taxonomic key to help you begin the process of classifying freshwater invertebrates. This dichotomous key will lead you to the appropriate chapter for more detailed biological and taxonomic information. Additional information on freshwater invertebrates can be found in books by Hutchinson (1993) and Merritt and Cummins (1996) as well as in various texts on limnology and stream ecology (e.g., Wetzel, 2001). No phylum is totally immune to an occasional reshufﬂing of species, renaming of constituent taxa, and recalculation of total species richness (number of species). This dynamic feature of the discipline is, in part, a necessary response to the acquisition of new knowledge, but it also results from the ubiquitous and almost inherent disagreements that usually typify groups of two or more scientists! Taxonomists are frequently labeled as “splitters” or “lumpers” according to whether, respectively, they tend to acknowledge more or fewer species within an assemblage. Classiﬁcations they produce based on diverse scientiﬁc approaches, such as cladistics and numerical ( phenetics) taxonomy, can produce dramatically different views of relationships among taxa. Moreover, molecular systematics has sometimes shaken the traditional taxonomic schemes based on classical phenotypic characters. Although arguments at the species level are especially rampant, debates occasionally break out regarding the categorization of higher tiers, including classes and phyla. While reading through this book, you will often Ecology and Classiﬁcation of North American Freshwater Invertebrates. 2nd Edition Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved. 1 2 J. H. Thorp and A. P. Covich encounter references to current taxonomic debates. The ﬁnal classiﬁcation of taxa has been left to the authors of individual chapters. As editors, we have allowed the authors a great amount of leeway in this area, requesting only that they inform the reader about the principal areas of dispute. Species are named today using a system of binominal nomenclature ( names composed of two parts) based on the 18th-century proposal of the brilliant Swedish naturalist Carl von Linné (or Carolus Linnaeus, the latinized form he preferred to use). This is the “genus and species” designation familiar to most biology students (e.g., Homo sapiens for the human species). You will occasionally see a species designated with three names (a trinomen), which is permissible under guidelines of an international code described below. For example, Bosmina (Sinobosmina) freyi is the genus, subgenus, and species name of a water ﬂea, or cladoceran, common in rivers (it is one of a species complex formerly called Bosmina longirostris). In contrast, Pheidole xerophila tucsonica is the genus, species, and subspecies name of a harvester ant living in the desert near Tucson, Arizona. Scientiﬁc names are either in Latin or in a form that has been latinized (e.g., B.(S.) freyi was formed by latinizing the last name of Dr. David G. Frey, to honor this now-deceased coauthor of Chapter 21). The ﬁnal arbiter for naming taxa according to this system is the International Commission on Zoological Nomenclature, a judicial body elected periodically by the International Congress of Zoology to evaluate taxonomic names proposed by scientists. They use a set of agreed-upon rules, termed the International Code of Zoological Nomenclature (ICZN). The commission bases its decisions on rules covering the priority of names and designation of “type” taxa. As the name implies, “priority” speciﬁes that the ﬁrst name assigned in a publication is generally the ofﬁcial name of that taxon. The commission has, however, plenary powers to set aside the “rightful” name of a species if doing so would further the cause of science. For example, alteration of a familiar, long-standing name could cause major confusion in the scientiﬁc literature. Occasionally, two distinct taxa are inadvertently assigned the same binominal name by scientists working in different institutions. In that case, the species described last loses its name as a junior homonym. To lessen the state of confusion and diminish possibilities for future arguments, one individual of each species is designated as the prime example of that taxon. Such “type” specimens are deposited in one of many internationally recognized museums and made available for study by responsible scientists. Type specimens can be replaced only if the original is inadvertently lost or destroyed. Taxonomic arguments among scientists fall into two broad categories: “What is a species?” and “How do you properly recognize evolutionary relationships among species?” In the ﬁrst case, phylogenetic (including are they interfertile), evolutionary, and recognition deﬁnitions are employed. In the second instance, scientists use phenetics, cladistics, and phylogenetic taxonomy to establish relationships, as described below. Systematists, which include many authors of our text, are involved in naming species (alpha taxonomy) and establishing phylogenetic relationships among groups (beta and gamma taxonomy). Most of these scientists can be divided in modern times into those employing numerical taxonomy ( numerical phenetics) and those using the more popular cladistics ( phylogenetic systematics), although there is unacknowledged blending of the two approaches in some cases (especially when gene sequencing data are being used). Each approach analyzes attributes of a species, or “characters,” (e.g., morphological features or genetic sequences), to determine relationships among groups. In a very simpliﬁed deﬁnition, numerical taxonomists sum a large number of randomly selected characters possessed by multiple species and determine phylogenetic relationships based on the degree of similarity among species (i.e., shared characters), without placing relative evolutionary values on different characters. In contrast, cladists emphasize the recency of common descent (shared “derived” characters), a genealogical technique that places relative evolutionary values on different characters. Their approach depends signiﬁcantly on identiﬁcation of primitive ( ancestral) vs derived (i.e., more recently evolved) characters. Information on systematic zoology is published in various journals, such as the Zoological Record, Zeitschrift fv̈r Wissenschaftliche Zoologie (Abteilung B), and Bulletin Signaletique (CNRS), as well as in numerous society publications and journals dealing with a broad range of subjects including classiﬁcation. Readers interested in knowing where best to search for papers on a given taxon should consult the Literature Cited section in the appropriate chapter of this book, or see more specialized texts, such as Merritt and Cummins (1996) on genera of freshwater insects. The International Code of Zoological Nomenclature passes judgment on names of genera and species but does not strictly control higher classiﬁcation. For that reason, some disagreements exist about the numbers and names of higher classiﬁcation tiers, especially at the ordinal level and above (e.g., the phyla Cnidaria vs Coelenterata, Bryozoa vs Ectoprocta, and disagreements on whether Crustacea is a phylum, subphylum, or class). The system for adding sufﬁxes to taxonomic names as a means of designating classiﬁcation level has 1. Indroduction to Freshwater Invertebrates TABLE I Principal Zoological Ranks and Sufﬁxes Where Designated Kingdom Phylum Subphylum Superclass Class Subclass Cohort Superorder Order Superfamily (-oidea)a Family (-idae) Subfamily (-inae) Tribe (-ini)a Subtribe (-ina)b Genus Subgenus Species Subspecies a An ending recommended but not mandatory according to the International Code of Zoological Nomenclature. b An ending customary but not cited in the code. been confusing in the past but is beginning to change for the better. A nonexclusive list of the principal zoological ranks and sufﬁxes is shown in Table I. TABLE II 3 The importance of correct identiﬁcation and consistent approaches to classiﬁcation soon becomes apparent to all people interested in learning about general biotic relationships. Vast amounts of information are catalogued in research libraries and museums that use the Linnean system of binominal classiﬁcation. Once a specimen is correctly identiﬁed, a large number of pertinent references in specialized journals can be accessed, not only in systematics but also in ecology, evolution, animal behavior, and medical sciences. Furthermore, the global revolution known as the Internet has opened a tremendous resource for taxonomists and other biologists. By using computer-based information retrieval systems present in most libraries or your personal access to the World Wide Web, you can rapidly track down biological and taxonomic research on an incredible number of species. The ease with which student and advanced scientists can now obtain information on many groups was almost unimaginable 10 years ago. To help you get started, we have included in Table II some of the favorite sites recommended by our authors. Keep in mind, however, that these are only a small minority of available sites and that the life span of a particular Web address is unpredictable and relatively ﬂeeting compared to many of the sources used by investigators in the past (unless the Web site is directly maintained by a museum). Brief Selection of Web Sites for Freshwater Invertebrates Taxonomic group and site General sitesa Aquatic Sciences and Fisheries Abstracts Cambridge Biological Abstracts Current Contents Science Citation Index Web of Science Zoological Record Invertebrate Classiﬁcation and Ecology in Generalb benthos.org / nysfola.org / links.html ucmp.berkeley.edu / help / taxaform.html york.biosis.org / zrdocs / zoolinfo / grp_dipt.htm Protozoa uga.edu / protozoa / home.htm 130.158.208.53 / WWW / Servers / Protistologists.html megasun.bch.umontreal.ca / protists / protists.html Porifera mailbase.ac.uk / lists / porifera / ﬁles / ucmp.berkeley.edu / porifera / porifera.html Rotifera member.aol.com / bdelloid1 / deloid.htm nazca.mmtlc.utep.edu / ~rotifer / main.html Nematoda and Nematomorpha ianr.unl.edu / son / Site maintained by Corporate Corporate Corporate Corporate Corporate Corporate North American Benthological Society Individuals Paleontology Museum, UC Berkeley BIOSIS (not-for-proﬁt organization) and the Zoological Society of London Society of Protozoologists Corporate / University University of Montreal Individuals Paleontology Museum, UC Berkeley Individuals Individuals Society of Nematologists (Continues) 4 J. H. Thorp and A. P. Covich TABLE II (Continued) Taxonomic group and site Mollusca: Gastropoda ﬂmnh.uﬂ.edu / natsci / malacology / malacology.htm#Top ummz.lsa.umich.edu / mollusks / index.html Mollusca: Bivalvia courses.smsu.edu / mcb095f / gallery / inhs.uiuc.edu / cbd / collections / mollusk.html Annelida inhs.uiuc.edu / ~mjwetzel / mjw.inhsCAR.html keil.ukans.edu / ~worms / annelid.html Ectoprocta (Bryozoa) civgeo.rmit.edu.au / bryozoa / default.html Arthropoda: Insecta and Collembola afn.org / ~iori / oinlinks.html entweb.clemson.edu / database / trichopt / coleopsoc.org / famu.org / mayﬂy / mc.edu / ~stark / stoneﬂy.html ouc.bc.ca / eesc / iwalker / intpanis / Arthropoda: Crustacea: Crustacea: General and Peracarida odu.edu / ~jrh100f / amphome / york.biosis.org / zrdocs / zoolinfo / arthrop.htm#crustaceans nmnh.si.edu / gopher-menus / Isopods.html Arthropoda: Crustacea: Ostracoda uh.edu / ~rmaddock / IRGO / irgohome.html Arthropoda: Crustacea: Branchiopoda (Cladocera) cladocera.uoguelph.ca / Arthropoda: Crustacea: Copepoda nmnh.si.edu / iz / copepod / Arthropoda: Crustacea: Decapoda bioag.byu.edu / mlbean / crayﬁsh / crayhome.htm nmnh.si.edu / gopher-menus / Crayﬁsh.html Site maintained by Florida Museum of Natural History Museum of Zoology, University Michigan Individuals Illinois Natural History Survey Illinois Natural History Survey Individuals / University of Kansas Individuals / Royal Melbourne Institute of Technology Individuals International Trichopteran Committee Coleopterists Society Florida Agriculture & Mechanical University Individuals Individuals Individuals BIOSIS National Museum of Natural History International Research Group on Ostracoda, University Houston Individuals National Museum of Natural History Monte Bean Life Science Museum, BYU National Museum of Natural History Note. Most of these sites have links to many other sites. a We do not guarantee the accuracy or quality of any of these sites. All of these sites were open in the spring of 2000, but we have no idea how long they will remain accessible. Sites maintained by museums are likely to be long-lasting. Sites designated as being maintained by “individuals” are usually maintained by someone at a university, but the commitment of that university to the long-term survival of the site is usually unknown. b These require that your institution subscribe to the service (i.e., someone pays!). c Many of these Internet Web sites begin with http: / /www. Delete the www if those letters do not work. III. SYNOPSES OF THE NORTH AMERICAN FRESHWATER INVERTEBRATES All major phyla of invertebrates, other than Echinodermata, have some freshwater representatives, but only a few are more diverse in freshwaters than in the world’s oceans. Some salient features of these freshwater taxa, as discussed in Chapters 3 – 23, are summarized in the remainder of this section. A. Protozoa Protozoa are unicellular (or acellular) eukaryotes existing either as individuals or as members of a loose-knit colony (Fig. 1). The term protozoa is a taxon of convenience, having no signiﬁcance in classiﬁcation schemes within the kingdom Protista (or Protoctista) other than as a traditional functional grouping of all heterotrophic and motile species. Modern taxonomic treatments divide the kingdom Protista into 27 phyla (more or less), 2 of which contain signiﬁcant numbers of free-living, phagotrophic species. These two phyla, Ciliophora (ciliates) and Sarcomastigophora (the majority of amoeboid protozoa), are the subject of Chapter 3, by William D. Taylor and Robert W. Sanders. Protozoa play important, though often overlooked, roles in freshwater ecosystems. For example, ﬂagellate protozoans are the predominant predators of picoplankton (primarily bacteria and small cyanobacteria) 1. Indroduction to Freshwater Invertebrates 5 of canals permeating the entire sponge body. Water is propelled through this canal system both passively, as a result of its basic hydrodynamic structure, and actively by choanocytes (collar cells). These cells also extract bacteria and algae from the water column by suspension (ﬁlter) feeding. Many species also contain symbiotic algae (green algae, except for one sponge that shelters a yellow-green algal symbiont), which provide supplemental nutrition. The annual life cycle of a freshwater sponge is characterized by shifts between periods of active growth and dormancy in the gemmule stage. Availability of suitable hard substrate and adequate food seem to be the critical factors regulating the spread and growth of sponges, as described by Thomas M. Frost, Henry M. Reiswig, and Anthony Ricciardi in Chapter 4. Few animals prey on sponges, and most of those graze only a small portion of the sponge’s tissues, leaving the sponge alive to grow and reproduce. FIGURE 1 Solitary and colonial protozoa: (A) The zooﬂagellate Khawkinea; (B) Amoeba; (C) the colonial ciliate Vorticella; and (D) the solitary ciliate Stentor. See Chapter 3. [These and subsequent ﬁgures in this chapter were redrawn and modiﬁed from illustration in Chapters 3 – 22.] and thus are essential components of the “microbial loop” within lakes and rivers. Many genera consume bacteria within the surﬁcial layer of sediments and in the water column, and larger taxa capture and eat algae or other protozoa. They, in turn, are preyed upon by some species of oligochaetes, chironomids, and rotifers. B. Porifera About 27 species of sponges colonize freshwater habitats in Canada and the United States, with a general east-to-west and north-to-south decline in species richness. Although less diverse than protozoa, freshwater members of the phylum Porifera — the sponges — nonetheless are commonly found on hard substrates in many lakes and streams (Fig. 2). Except for the marine mesozoa and placozoa, sponges have the simplest structure of all multicellular phyla, showing little variation in external form and having a cellular-level (or incipient, tissue-level) organization. The most prominent feature of their anatomy is the progressively ﬁner series FIGURE 2 Freshwater sponges (phylum Porifera): (A) Drawing of a living sponge on a stick; (B) representative sponge spicules (all 25 – 150 m in length). See Chapter 4. 6 J. H. Thorp and A. P. Covich FIGURE 3 Freshwater coelenterates (phylum Cnidaria): (A) A polypoid Hydra; (B) a medusoid Craspedacusta. See Chapter 5. C. Cnidaria Cnidarians, or coelenterates, have been relatively unsuccessful in adapting to freshwater, being represented only by one class (Hydrozoa) with four or ﬁve purely freshwater genera: the common hydras (brown Hydra and green Chlorohydra, or H. viridissima) and the rare medusoid Craspedacusta and polypoid Calposoma and Polypodium (Fig. 3). Cordylophora, a mostly estuarine taxon, occasionally penetrates relatively freshwaters, where it lives in a colonial, sessile form. Despite their low diversity, cnidarians — or at least hydras — are routinely present on almost any reasonably hard substrate in permanent ponds and lakes and in streams ranging from headwater streams to large rivers. The cnidarian body consists of a generally thin, acellular layer of mesoglea sandwiched between two thicker cellular layers (ecto- and endoderm), all surrounding a central body cavity (the coelenteron). Coelenterates dispatch their mostly invertebrate prey (both zooplankton and some benthos) with ectodermal batteries of stinging or sticky nematocysts. They are, in turn, fed upon by a few ﬂatworms and sometimes crayﬁsh. Green hydra are named for the presence of endosymbiotic Chlorella, algae that provide a signiﬁcant source of nutrition for cnidarians. The biology of freshwater Cnidaria is described in Chapter 5, by Lawrence B. Slobodkin and Patricia E. Bossert. more than 200 species of turbellarians (phylum Platyhelminthes) and three species of ribbon worms (phylum Nemertea, or Rhynchocoela) found in North America (Fig. 4). He discusses the acoelomate Turbellaria, the simplest bilaterally symmetrical eumetazoan with three embryonic cell layers, and the more complex, coelomate Nemertea, which has an anus and a closed circulatory system. These latter ﬂatworms possess an D. Flatworms: Turbellaria and Nemertea In Chapter 6 on unsegmented ﬂatworms, Jurek Kolasa describes the biology and classiﬁcation of the FIGURE 4 Turbellarian ﬂatworms (phylum Platyhelminthes) and a ribbon worm (phylum Nemertea): (A) Macrostomum; (B) Dugesia; and (C) the numertean Prosotoma. See Chapter 6. 1. Indroduction to Freshwater Invertebrates eversible, muscular proboscis resting in a rhynchocoel. Despite their common name, ﬂatworms are usually cylindrical in cross section! Flatworms are well represented in the benthos of lakes and streams. Ribbon worms of the single North American genus Prostoma are rarely reported but can be common in their typical shallow, littoral zone habitats; all are carnivores that capture their prey with a sticky proboscis. The much more diverse turbellarians are distributed more widely than ribbon worms; but they are not much better known, in part because they are very difﬁcult to identify with the collection and preservation techniques normally employed in ﬁeld studies. The great majority of these ﬂattened or cylindrical worms are free-living denizens of lakes and streams from shallow to deep waters. Most are small (1 mm long), especially the more diverse microturbellaria (~150 species in North America). Better known, however, are the larger triclads (most 10 mm long), which are often called planaria. Turbellarians consume prey in accordance with their size, variously eating bacteria, algae, protozoa, and small or large invertebrates. E. Gastrotricha The pseudocoelomate gastrotrichs (Fig. 5) are common residents of epigean waters throughout North America, with densities ranging from 100,000 to 1,000,000 animals per square meter. Although fewer than 100 freshwater species from North America have been reported, the actual diversity of the phylum 7 Gastrotricha is probably much greater in freshwater (and many marine species are also present). Possibly because gastrotrichs are so small (50 – 800 m), our ecological knowledge of this group has not advanced very far. They colonize both aerobic and anoxic surface sediments, thrive as members of the aufwuchs assemblage on aquatic plants, and occasionally venture into the plankton. Oddly enough, given their size, they are very rare in groundwater (other than the hyporheic zone of streams). Gastrotrichs thrive on a diet of bacteria, algae, protozoa, and detritus. Not much is known about their enemies, but these certainly include amoeba, cnidarians, and predaceous midges. Their ecology and classiﬁcation are examined in Chapter 7 by David L. Strayer and William D. Hummon. F. Rotifera Perhaps no other phylum is as clearly associated with freshwater as is Rotifera (Fig. 6). Nearly 2000 species of rotifers, or “wheel animals,” inhabit freshwaters throughout the world, whereas only about 50 species are exclusively marine. These unsegmented, pseudocoelomates are distinguished by two principal anatomical features: an apical, ciliated region known as the corona and a muscular pharynx, termed the mastax, with its complex set of hard jaws. The ciliated corona is employed for both locomotion and foodgathering. Rotifers range in size from minute creatures barely 100 μm long to giants of 2 mm or more! As Robert L. Wallace and Terry W. Snell point out in Chapter 8, rotifers are one of the three principal animal taxa in the plankton (along with protozoa and microcrustaceans). Because they are more efﬁcient than cladocera when feeding on minute algae, rotifers can exert a greater grazing pressure on the small picoplankton. Other rotifers are important predators on bacteria, protozoa, and small metazoa in the plankton. Rotifers play a critical role in the microbial (nutrient) loop within freshwater lakes and rivers. Rotifers are the numerical dominants of most large river zooplankton communities. Many species are also benthic or nearly so. Larval ﬁsh, some protozoa, insect larvae, microcrustaceans, and other rotifers are numbered among their predators. G. Nematoda and Nematomorpha FIGURE 5 Representative freshwater Gastrotricha: (A) Chaetonotus; (B) Stylochaeta. See Chapter 7. Nematodes, which are best known to the average person for damaging crops, are also abundant in freshwater lakes and streams where they spend all or a large part of their life cycle (Fig. 7A). Chapter 9 by George O. Poinar covers 66 genera (encompassing 300 – 500 species), representing over 95% of the 8 J. H. Thorp and A. P. Covich FIGURE 6 “Wheel animals” of the phylum Rotifera: (A) A Solitary Keratella; (B) a colony of Sinantherina. See Chapter 8. known species of roundworms. Nematodes are unsegmented worms with a body cavity (pseudocoel) and complete alimentary tract; they lack jointed appendages and specialized circulatory and respiratory systems. Roundworms are signiﬁcant components of the micro- and macrobenthos of many aquatic ecosystems. For example, in Mirror Lake, New Hampshire, they constitute 60% of all individuals in the benthic metazoa, although only about 1% of the total biomass (Strayer, 1985, as cited in Chapter 9). Many nematodes are parasitic in plants and animals, but these are only brieﬂy mentioned in Chapter 9 by George O. Poinar. Free-living roundworms, the focus of that chapter, obtain nourishment from bacteria, algae, and protozoa. While ﬂatworms and crayﬁsh feed occasionally on roundworms, their greatest threat comes from other, predaceous nematodes. Another phylum of worms that, in a very rough way, resembles nematodes is Nematomorpha (Fig. 7B). These hairworms, or horsehair worms, are free-living as adults but parasitic as larvae in various invertebrates. Like nematodes, they are unsegmented pseudocoelomates, but their intestine is nonfunctional, and they are generally much larger than roundworms. The evolutionary position of nematomorphs remains controversial, but they probably have a distant relationship with nematodes. All but one genus occurs in freshwater, and seven genera from North American lakes and streams are reported. H. Mollusca Two classes within the phylum Mollusca contain large numbers of freshwater species: the classes Gastropoda (snails) and Bivalvia (mussels and clams). These ubiquitous and abundant molluscs are discussed separately in Chapters 10 (snails) and 11 (mussels and clams). 1. Gastropoda FIGURE 7 Illustrated is a common roundworm (A; phylum Nematoda) and a less frequently observed, adult horsehair worm (B; phylum Nematomorpha). See Chapter 9. Snails are one of the more diverse freshwater groups (having about 659 species in North America) and are certainly among the most easily recognized by the public. These soft-bodied, unsegmented coelomates have a body organized into a muscular foot, distinct head region, and visceral mass: a ﬂeshy mantle covers the viscera and secretes a spiraled, univalve, calcareous shell (Fig. 8). They are commonly found in littoral regions of lentic and lotic habitats, where they collect bottom detritus, graze on the algal ﬁlm covering rocks, wood snags, and plants or even ﬂoat upside down at the water surface, consuming trapped and neustonic algae. Recent ecological work on freshwater gastropods, 1. Indroduction to Freshwater Invertebrates 9 species are listed on state and federal lists of “threatened and endangered species” as a result of human disruption of their aquatic habitats, loss of ﬁsh hosts for larval stage, and invasion of Asian clams (Corbicula ﬂuminea), zebra mussels (Dreissena polymorpha), and quagga mussels (D. bugensis). These exotic species have become the numerical and/or biomass dominants in many North American waters, especially east of the great plains, and they are exerting substantial ecological and economic effects. I. Annelida FIGURE 8 Three types of shell morphologies in freshwater snails and limpets (phylum Mollusca, class Gastropoda): (A) The most common, spiral shell, as shown by Pomacea: (B) two views of the planospiral snail Planorbella (Helisoma); and (C) the conical freshwater limpet Ferrissia. See Chapter 10. as described by Kenneth M. Brown in Chapter 10, has focused on their important roles as grazers on periphyton and as prey of benthic-feeding ﬁsh and invertebrates, especially crayﬁsh but also others like leeches and predaceous water bugs (Belostomatidae). Freshwater limpets are included in Gastropoda, although they are not related closely to their marine relatives. The phylum Annelida consists primarily of three relatively diverse freshwater groups (oligochaete worms, branchiobdellidans, and leeches) and a few minor freshwater taxa. Annelids are legless, segmented coelomates with serially arranged organs; they occur in both lentic and lotic environments, where they tend to be either detritivores (oligochaetes) or predators (leeches and branchiobdellidans). The taxonomy of Annelida is in great dispute — an observation reﬂected by the diverse views of authors in Chapters 12 and 13. A strong area of controversy involves the rank of higher groups, beginning at whether Clitellata (which encompasses all annelids except polychaetes) is a superclass, class, or subclass; this then affects ranks of lower groups like the oligochaetes and leeches. Unless the reader is a student of annelid biology and evolution, 2. Bivalvia The North American fauna of freshwater bivalves is the richest in the world, with over 250 species of mussels and clams (Fig. 9; see also Chapter 11, by Robert F. McMahon and Arthur E. Bogan). Bivalves, or pelecypods, are major deposit and suspension feeders within permanent lakes, streams, and large rivers, where they are often the largest invertebrates in body mass. Except for the smaller pea clams (Sphaeriidae), native mussels (Unionacea) are uniquely capable of growing large enough to escape predation by almost all aquatic vertebrates and invertebrates. Bivalves are much less motile than gastropods, and their body is enclosed in two hinged, calcareous shell valves. A radula, which is employed by snails for grazing, is absent in clams and mussels. Unlike the vast majority of North American freshwater invertebrates, bivalves have been of direct economic importance to humans; they were formerly used for food (by Native Americans) and button production and are now harvested for freshwater pearls or for the core elements needed to grow marine-cultured pearls. Many native FIGURE 9 One native and two invadere species of freshwater bivalves (phylum Mollusca,class Bivalvia): (A) The native mussel Quadrula; (B) the ubiquitous Asiatic clam, Corbicula; and (C) a newcomer to North America, the rapidly spreading zebra mussel, Dreissena. See Chaper 11. 10 J. H. Thorp and A. P. Covich one can set aside these arguments for the time being and concentrate on learning other details of annelid biology, ecology, and classiﬁcation. 1. Oligochaeta and Branchiobdellida In Chapter 12, Ralph O. Brinkhurst and Stuart R. Gelder discuss the free-living oligochaete worms (~150 species) and the related branchiobdellidans, a lesswell-studied group of 107 species in North America (Fig. 10). Oligochaetes are among the more widely distributed freshwater taxa and one of the very few invertebrates to colonize moist terrestrial environments. In addition to the characteristics of the phylum as a whole, oligochaetes lack suckers and generally have four bundles of setae on every segment but the ﬁrst. Oligochaetes are best known for their ingestion of sediments in the littoral and profundal zones (a process prominent in nutrient recycling), but some species feed on benthic algae and other epiphytic organisms. Their great abundance and ease of consumption and assimilation make them favorite prey items for many invertebrate and vertebrate predators. Branchiobdellidans are ectocommensals of mostly astacoidean crayﬁsh, where they consume epibionts living on crayﬁsh, spare food lost by the host in the feeding process, tissue from host wounds, and other branchiobdellidans. They have a posterior sucker for attaching to their hosts. 2. Leeches and Acanthobdellids Another ecologically important group of annelids is represented by 73 North American species of leeches, FIGURE 11 Schematic view of the leech Placobdella. See Chapter 13. as described in Chapter 13, by Ronald W. Davies and Fredric R. Govedich. A single species of leech-like Acanthobdellidae occurs in North America; it is a temporary ectoparasite of salmonid ﬁsh. Unlike most oligochaetes, leeches have suckers (anterior and posterior) and lack setae (Fig. 11). Another major distinction between these two annelids is their feeding niches. Most leeches are predators of invertebrates, such as chironomid midges, oligochaetes, amphipods, and molluscs; but nonscientists best know this group for the minority that are ectoparasites, feeding on the blood of vertebrates (sanguivory), including incautious humans. Predators of leeches include ﬁsh, birds, snakes, salamanders, and a large variety of freshwater invertebrates. J. Bryozoa FIGURE 10 Two freshwater annelids with distinctive lifestyles: (A) The free-living oligochaete Branchiura, showing its posterior gill ﬁlaments; (B) Ceratodrilus, a branchiobdellidan which is an ectocommensal of crayﬁsh. See Chapter 12. Bryozoans, or “moss animals,” are generally sessile, colonial invertebrates with ciliated tentacles (called “lophophore”) for capturing suspended solitary algae, protozoa, and organic particles (Fig. 12). All but one North American species are in the phylum Ectoprocta, or Bryozoa, and are composed of animals whose mouth, but not anus, is located within the whorl of feeding tentacles. Although the diversity of freshwater Bryozoa is not high (24 species in North America), they are actually quite common occupants of hard, relatively stationary, and biologically inactive substrates. Most live in relatively warm water (18 – 28°C). They occur in lakes but thrive better in ﬂowing water, where 1. Indroduction to Freshwater Invertebrates 11 FIGURE 12 Freshwater bryozoans: (A) Plumatella colony (phylum Bryozoa or Ectoprocta;) (B) close-up of two individuals in an ecotoproct colony; and (C) the ubiquitous entoproct Urnatella. See Chapter 14. a constant source of suspended food encounters their tentacles. Our ecological knowledge of Bryozoa is relatively sparse despite the long-standing familiarity of scientists with this group. The only nonectoproct bryozoan is the cosmopolitan, suspension-feeding Urnatella gracilis. It is classiﬁed in the phylum Entoprocta, a taxon with very dubious relation to the ectoprocts. Both the mouth and anus of entoprocts are placed within the whorl of feeding tentacles; other important structural differences from the ectoprocts also exist. The biology, ecology, and classiﬁcation of bryozoans are described in Chapter 14, by Timothy S. Wood. while 6 genera with 13 – 14 species are typically freshwater. The actual number of water bear species in North America is probably considerably higher than this low value. Tardigrades are segmented micrometazoa with four ﬂeshy legs terminating in claws (Fig. 13). They have piercing/sucking mouthparts and obtain nutrition from plants, animals, and detritus. An important part of the water bear life cycle is the latent stage (cryptobiosis) entered into when environmental conditions become inhospitable. The biology and systematics of these little-known creatures are described by Diane R. Nelson in Chapter 15. K. Tardigrada Tardigrades, or “water bears,” are easy to overlook because of their diminutive nature (adults average 250 – 500 m), but they are widely dispersed in freshwater and terrestrial environments, and a few of the ~800 species worldwide occur in the ocean. Although the taxonomy of Tardigrada needs to be revised, the diversity of freshwater species in North America is low compared to most other phyla with freshwater members: 3 genera with 5 species are strictly freshwater, FIGURE 13 Lateral view of a heterotardigrade (phylum Tardigrada). See Chapter 15. 12 J. H. Thorp and A. P. Covich L. Arthropoda The most successful terrestrial phylum and one of the most prominent freshwater taxa is Arthropoda. Its three subphyla with freshwater members — Chelicerata (water mites and aquatic spiders), Uniramia (aquatic insects), and Crustacea (crayﬁsh, fairy shrimp, copepods, etc.) — are all diverse and important components of lakes and streams. (Some authors prefer to group the insects, myriapods, and crustaceans as classes within a single subphylum, Mandibulata.) Arthropods occupy every heterotrophic niche in benthic and pelagic habitats of most permanent and temporary aquatic systems and are well represented in groundwater habitats. As adults, these metameric coelomates are characterized by a chitinous exoskeleton and stiff, jointed appendages modiﬁed as legs, mouth parts, and antennae (except in water mites). 1. Water Mites The class Arachnida (subphylum Chelicerata) is represented in freshwater by a single genus of semiaquatic, true spiders (Dolomedes) and a colossal number of water mites (subclass Acari; Fig. 14). In Chapter 16, Ian M. Smith, David R. Cook, and Bruce P. Smith, discuss the biology and classiﬁcation of several distantly related groups of mites, the most diverse of which is Hydrachnida. As they point out, a single square meter of substrate from a littoral weed bed in a eutrophic lake may contain 2000 mites representing up to 75 species and a comparable area of rocky stream bed may yield 5000 individuals in 50 or more species! Over 1500 species are estimated to be present in Canada and the United States. Because of their prodigious numbers and wide distribution, mites probably play a major, albeit often ignored, role in aquatic ecosystems as predators (e.g., on ostracodes, early instars of insects, various zooplankton, and insect eggs), ectoparasites (mostly on insects), and prey of ﬁsh, other mites, and a few other invertebrates. For example, FIGURE 14 Generalized larval (A) and adult (B) water mites (phylum Arthropoda, Acari). See Chapter 16. mites interact intimately with all life stages of aquatic insects, parasitizing 20 – 50% of adults in natural populations. Mites differ from members of other arthropod subphyla in lacking antennae; adults generally have four pairs of jointed walking legs and segmentation is greatly reduced or not immediately apparent. 2. Aquatic Insects and Collembola Perhaps the most diverse and best studied groups of freshwater invertebrates are the 10 insect orders containing the roughly 6000 species of aquatic insects (Fig. 15). William L. Hilsenhoff introduces the classiﬁcation and general characteristics of aquatic insects in Chapter 17, and Anne E. Hershey and Gary A. Lamberti tackle the daunting task of summarizing some basic ecological aspects of aquatic insects in Chapter 18, leaving the more detailed discussions to books devoted to all or speciﬁc groups of aquatic insects (e.g., McCafferty, 1981; Ward, 1992; Merritt and Cummins, 1996). Like the water mites, insects occupy every freshwater habitat and heterotrophic niche and are present with tremendous densities and diversities. The majority spend most of their lives as larvae, only brieﬂy departing the freshwater milieu to mate. Aquatic insects feature mandibles and one pair of antennae; adults have three pairs of legs and generally possess functional wings. Chapter 17 also covers the semiaquatic springtails, or Collembola. These wingless arthropods were once classiﬁed as insects but are now generally regarded as members of the separate class Entognatha within the superclass Hexapoda, which also includes insects. They are usually associated with the water surface (neuston) or riparian habitats. Although a number of species are sometimes collected in or on water, only about 15 species are regarded as semiaquatic or riparian. 3. Crustaceans Nearly 4000 species of crustaceans inhabit freshwaters around the world, occupying a great diversity of habitats and feeding niches. Within pelagic and littoral zones, water ﬂeas and copepods are the principal macrozooplankton, and benthic littoral areas shelter vast numbers of seed shrimps, scuds, and other crustaceans. An omnivorous feeding habit is typical of crustaceans, although there are many strict herbivores, carnivores, and detritivores. Members of the subphylum Crustacea are characterized by a head with paired mandibular jaws, a pair of maxillae, and two pairs of antennae. Their appendages are often biramous. a. Peracarida and Branchiura Several groups of Crustacea, most in the class Malacostraca, superorder Peracarida, have a relatively small number of freshwater representatives. Two of these, Amphipoda and 1. Indroduction to Freshwater Invertebrates 13 FIGURE 15 Five arthropods in the superclass Hexapoda: (A) Baetis, a mayﬂy nymph (animals drawn in a-d are all in the class Insecta); (B) the caddisﬂy larva Polycentropus; (C) and adult elmid beetle Stenelmis; (D) the midge larva Chironomus; and (E) a semi-aquatic springtail (class Entognatha, order Collembola). See Chapter 17. Isopoda, are important taxa within marine systems. Chapter 19 (by Alan P. Covich and James H. Thorp) includes both an introduction to the subphylum Crustacea and coverage of the ecology and systematics of three malacostracan orders representing less than 200 species: Amphipoda (scuds), Isopoda (aquatic sow bugs), and Mysidacea (opossum shrimp) (Fig. 16). Of these three taxa, the scuds are the most widely distributed and diverse within North American freshwaters, for example. The peracaridans are omnivorous and opportunistic, consuming items ranging from algae and organic matter to small, living invertebrates and ﬂesh of dead bivalve molluscs. They occur in ecosystems ranging in size from large rivers to small ponds and are also well represented among subterranean fauna. The subclass Branchiura, or “ﬁshlike,” attach themselves to the gills of ﬁsh; only one genus occurs in North American freshwaters (Argulus). b. Ostracoda Approximately 420 species of mussel and seed shrimps (class Ostracoda) found on this continent are easily distinguished from other crustaceans by the typical, bivalved carapace enveloping their bodies (Fig. 17). This protective covering is composed of chitin and calcite. Ostracodes occur within the benthos of nearly every conceivable aquatic system from temporary ponds to large rivers to groundwater habitats, but they rarely enter the plankton even though many species are good swimmers. Almost all are free living and most are herbivores on attached algae or detritivores. They are, in turn, consumed by ﬁsh, waterfowl, and various benthic and planktonic invertebrate predators, as discussed by L. Denis Delorme in Chapter 20. c. Cladocera and Other Branchiopoda The class Branchiopoda includes the common Daphnia and other cladocera frequently studied in the laboratory component of introductory biology courses, along with lesser known representatives of the class such as the elusive fairy shrimp of ephemeral pools and saline lakes (Fig. 18). Branchiopods are a heterogeneous group linked by similar mouth parts and leaf-like thoracic legs (phyllopods). Virtually all species within the eight extant orders of Branchiopoda are limited to freshwaters (mostly lentic systems). As pointed out by Stanley I. Dodson in Chapter 21, branchiopods occupy key positions in aquatic communities. As consumers, they are algivorous herbivores, detritivores (often assimilating bacteria on benthic or suspended organic matter), and occasionally predators of small invertebrates. They are important prey items in the diets of many ﬁsh, waterfowl, and certain other vertebrate and invertebrate predators. Many species are planktonic, while others live in shallow-water benthic habitats. Some, like the fairy 14 J. H. Thorp and A. P. Covich FIGURE 16 Representative freshwater crustaceans: (A) Gammarus, a scud (order Amphipoda); (B) Caecidotea, an aquatic sow bug (order Isopoda); and (C) Mysis, an opossum shrimp (order Mysidacea). See Chapter 19. shrimp Artemia, are adapted to temporary ponds and to hypersaline ponds and lakes. Scientists estimate that the United States and Canada contain ~400 species of cladocera and 78 species of noncladoceran branchiopods. d. Copepoda Copepods are very common in aquatic and semiaquatic habitats, spanning the gamut FIGURE 17 Female ostracode Candoma (crustacean class Ostracoda) with left value of carepace removed. See Chapter 20. from ephemeral pools, groundwaters, and wetlands to the benthos and plankton of large lakes and rivers. They are also major constituents of marine zooplankton and can be abundant in moist terrestrial environments. Most are free-living, but two of the seven orders contain species parasitic on freshwater ﬁsh. Slightly over 300 species from North American fresh waters are described, 10 of which are considered to be introduced. They typically represent more than 50% of the zooplankton biomass in rivers and lakes. As was the case with cladocera, copepods have an integral role in aquatic food webs both as primary and secondary consumers (most are omnivorous) and as a major source of food for many young and adult ﬁsh, other copepods, and other invertebrates, such as larval phantom midges (Chaoborus). Copepods can be distinguished from other small (0.5 — 2.0 mm) crustaceans by their possession of: (1) a cylindrical, segmented body; (2) two setose, caudal rami on the posterior end of the abdomen (which are used in swimming); and (3) a single, simple anterior eye (Fig. 19). The biology and classiﬁcation of this important crustacean are 1. Indroduction to Freshwater Invertebrates 15 FIGURE 18 Tow branchiopod crustaceans: (A) The widely distributed “water ﬂea” Daphnia; (B) the more localized fairly shrimp Eubranchipus, showing an adult male (C) Triops, a notostracan tadpole shrimp. See Chapter 21. discussed by Craig E. Williamson and Janet W. Reid in Chapter 22. e. Decapoda Decapoda is the most diverse order of the class Malacostraca in marine and freshwater FIGURE 19 ecosystems. It encompasses 342 species of crayﬁsh and 17 species of shrimp in epigean and subterranean freshwaters north of Mexico (Fig. 20). All are characterized by terminal claws on the ﬁrst three of ﬁve pairs of thoracic appendages and a branchial chamber Two copepod crustaceans: (A) A generalized calanoid species; (B) a representative cyclopoid copepod. See Chapter 22. 16 J. H. Thorp and A. P. Covich enclosed within the carapace (see Chapter 22 by Horton H. Hobbs). Decapods are the largest motile invertebrates in North American freshwaters. Their size, activity, and omnivorous diet make them key players in the benthos of many ecosystems. For example, they can inﬂuence species distribution of snails within a community by selectively preying on certain size classes or shell morphologies, and they can be voracious herbivores that devastate vascular plants in ponds. Decapods, unlike practically all other North American invertebrates, are increasingly consumed by humans, especially in the southern United States. M. Taxonomic Key to Major Taxa of Freshwater Invertebrates FIGURE 20 Crayﬁsh (A) are extremely common decapod crustaceans, while palaemonid shrimp (B) have a more restricted distribution (although occasionally abundant). See Chapter 23. The following dichotomous, taxonomic key is designed speciﬁcally to lead the reader to appropriate chapters within this book (i.e., it should not be employed in other contexts). It can be used for the larval and adult stages of aquatic insects, but it generally applies to the adults of other taxa. 1a. Unicellular (or acellular) organisms present as individuals or colonies; heterotrophic and / or autotrophic; protozoans (Fig. 1) ...............................................................................................................................................................kingdom Protista (Chapter 3) 1b. Multicellular, heterotrophic organisms (sometimes with symbiotic autotrophs)................................................................................. 2 2a(1b). Multicellular animals without discrete organs and with a cellular-level (or incipient tissue-level) construction; sponges (Fig. 2) ................................................................................................................................................................phylum Porifera (Chapter 4) 2b. Multicellular animals with organ or organ-system construction and two or three embryonic cell layers ...................................... .......................................................................................................................................................................................................... 3 3a(2b). Two embryonic cell layers; adults with a central body cavity opening to the exterior and surrounded by cellular endoderm, and acellular mesoglea, and a cellular ectoderm; polyps (e.g., hydra) and medusae (e.g., Craspedacusta) (Fig. 3) ................................ ...............................................................................................................................................................phylum Cnidaria (Chapter 5) 3b. Three embryonic cell layers; adults with cellular ectoderm, mesoderm, and endoderm ..................................................................... 4 4a(3b). Flattened or cylindrical, bilateral, acoelomate worms with only one opening to the digestive tract; ﬂatworms (some occasionally called planaria) (Fig. 4) ....................................................................................phylum Platyhelminthes: class Turbellaria (Chapter 6) 4b. Pseudocoelomate or coelomate eumetazoa with a complete digestive tract........................................................................................ 5 5a(4b). Flattened, unsegmented worms with an eversible proboscis in a rhynchocoel and a closed circulatory system; ribbon worms (Fig. 5) ............................................................................................................................................................. phylum Nemertea (Chapter 6) 5b. Not with above characteristics ......................................................................................................................................................... 6 6a(5b). Pseudocoelomates; legs, lophophorate tentacles, and segmentation absent; gastrotrichs (Fig. 5), rotifers (Fig. 6), roundworms (Nematoda; Fig. 7a), or hairworms (Nematomorpha; Fig. 7b). ......................................................................................................... 7 6b. Coelomates; legs, lophophorate tentacles, and/or metameres present; snails and mussels (Mollusca; Figs. 8 and 9), segmented worms (Annelida; Figs. 10 and 11), bryozoans (Fig. 12), water bears (Tardigrada; Fig. 13), and arthropods (mites, insects, and crustaceans; Figs. 14 – 20) ................................................................................................................................................................................... 10 7a(6a). Small (50 – 800 m), spindle- or tenpin-shaped, ventrally ﬂattened pseudocoelomates; more or less distinct head bearing sensory cilia; cuticle usually ornamented with spines or scales of various shapes; posterior of body normally formed into a furca with distal adhesive tubes; gastrotrichs (Fig. 5) ................................................................................................. phylum Gastrotricha (Chapter 7) 7b. Not with above characteristics .......................................................................................................................................................... 8 1. Indroduction to Freshwater Invertebrates 17 8a(7b). Nonvermiform pseudocoelomates with apical (and usually ciliated) corona and muscular pharynx (mastax) with complex set of jaws; wheel animals, or rotifers (Fig. 6) .................................................................................................. phylum Rotifera (Chapter 8) 8b. Not with above characteristics; nonsegmented worms ..................................................................................................................... 9 9a(8b). Alimentary tract present; cylindrical body usually tapering at both ends; noncellular cuticle without cilia, often with striations, punctuations, minute bristles, etc.; most less than 1 cm long (except family Mermithidae); roundworms (Fig. 7a)........................ ........................................................................................................................................................... phylum Nematoda (Chapter 9) 9b. Degenerate intestine; anterior and posterior tips normally are obtusely rounded or blunt; cuticle opaque and epicuticle usually crisscrossed by minute grooves; pseudocoel frequently ﬁlled with mesenchymal cells with large clear vacuoles, giving tissue a foamy appearance; length several centimeters to 1 m, width 0.25 – 3 mm; only adults with free-living stage; hairworms or horsehair worms (Fig. 7b) ..................................................................................................................................... phylum Nematomorpha (Chapter 9) 10a(6b). Soft-bodied coelomates, usually with a hard calcareous shell; most with ciliated gills, a ventral muscular foot, and a ﬂeshy mantle covering internal organs; snails and mussels (Figs. 8 and 9) ............................................................................ phylum Mollusca ........................................................................................................................................................................................................ 11 10b. Body not enclosed in a single, spiraled shell or in a hinged, bivalved shell, or if a bivalved shell is present, then animal has jointed legs.................................................................................................................................................................................................. 12 11a(10a). Body enclosed in a single shell, which usually has obvious spiral coils (limpets are the exception); body basically partitioned into head, foot, and visceral mass; radula present; snails and limpets (Fig. 8) ............................................................................................................................... phylum Mollusca: class Gastropoda (Chapter 10) 11b. Body enclosed in two hinged shells; head and radula absent; mussels and clams (Fig. 9) ..................................................................................................................................... phylum Mollusca: class Bivalvia (Chapter 11) 12a(10b). Legs absent in all life stages ............................................................................................................................................................ 13 12b. Adults and most larval stages with legs; if larvae without legs or prolegs (some insects), then cephalic region with paired mandibles .........................................phylum Arthropoda .............................................................................................................................. 16 13a(12a). Lophophorate tentacles absent; segmented worms . ........................................................................................................................ 14 13b. Ciliated, lophophorate feeding tentacles present.............................................................................................................................. 15 14a(13a). No suckers and four bundles of chaetae on each segment except the ﬁrst (Oligochaeta and Aphanoneura; Fig. 10A) “or” posterior, disk-shaped sucker on segment 11 and head composed of peristomium and three segments, ectoparasites on crayﬁsh (Branchiobdellida; (Fig. 10B) ....................................................................................................................... phylum Annelida (in part) (Chapter 12) 14b. Body with anterior and posterior suckers and no chaetae; leeches (Euhirudinea; Fig. 11) “or” posterior sucker on four segments, ectoparasites on salmonid ﬁsh (Acanthobdella peledina)........................................................... phylum Annelida (in part) (Chapter 13) 15a(13b). Anus opens outside a generally U-shaped lophophore; bryozoans (Fig. 12A) ....................................................... phylum Ectoprocta (Bryozoa) (Chapter 14) 15b. Both mouth and anus open within lophophore; calyx with single whorl of 8 – 16 ciliated tentacles (Fig. 12b) ................................. ........................................................................................................................................................ phylum Entoprocta (Chapter 14) 16a(12b). Minute adults with four pairs of clawed, nonjointed legs; water bears (Fig. 13) ........................................................................... ......................................................................................................................................................... phylum Tardigarda (Chapter 15) 16b. Adults and most larvae with jointed legs; arthropods ..................................................................................................................... 17 17a(16b). Antennae absent; body divided into cephalothorax (prosoma) and abdomen (opisthosoma); adults generally with four pairs of legs; water mites or the pseudo-aquatic water spiders (Fig. 14) ..........................subphylum Chelicerata, class Arachnida ...................... ........................................................................................................................................................................................ (Chapter 16) 17b. Antennae present; insects, springtails, and crustaceans . .................................................................................................................. 18 18a(17b). Mandibles and one pair of antennae; adults (most of which are terrestrial) generally with functional wings, larvae with wing pads or without any indication of wings; larvae and adults normally with three pairs of legs (some larvae without legs); wingless adults in class Entognatha (order Collembola, the springtails) and winged adults are in class Insecta (aquatic insects) (Fig. 15) ..........................................................................................................................................subphylum Uniramia (Chapters 17 and 18) 18b. Two pairs of antennae and mandibles (at some life stage); adults with variable numbers of legs, never with wings ............................ ....................subphylum Crustacea ................................................................................................................................................. 19 19a(18b). Body with 19 segments (head: 5; thorax: 8; abdomen: 6); abdomen with pleopods; carapace covers head and all or part of thorax ..............................class Malacostraca ........................................................................................................................................... 20 19b. Body enveloped in a bivalved carapace (ostracodes), “or” thoracic legs ﬂattened and leaf-like (water ﬂeas and other branchiopods), “or” 11 postcephalic segments and a single cephalic eye (copepods) .............................................................................................. 21 18 J. H. Thorp and A. P. Covich 20a(19a). Malacostracans with ﬁrst three of ﬁve pairs of thoracic appendages modiﬁed as maxillipeds (including usually large chelae on ﬁrst pair in crayﬁsh) and carapace encloses branchial chamber; crayﬁsh and atyid and palaemonid shrimp (Fig. 20) .............................................................................................................................................................. order Decapoda (Chapter 23) 20b. Malacostracan crustaceans with other characteristics, including scuds (order Amphipoda), aquatic sow bugs (order Isopoda), and opossum shrimp (order Mysidacea) (Fig. 16) ................................................................................................................. (Chapter 19) 21a(19b). Body enveloped in laterally compressed, bivalved carapace; mussel and seed shrimp (Fig. 17) ...................................................... .............................................................................................................................................................. class Ostracoda (Chapter 20) 21b. Body not enclosed within a bivalved carapace ................................................................................................................................ 22 22a(21b). Thoracic legs ﬂattened and leaf-like (phyllopod), mandibles are simple unsegmented rods with inner corrugated grinding surfaces; last body segment with a pair of spines or claws; water ﬂeas (cladocera) and other branchiopods (Fig. 8) .......................................... ......................................................................................................................................................... class Branchiopoda (Chapter 21) 22b. Cylindrical, segmented body, two setose caudal rami on posterior end of abdomen, and a single, simple anterior eye; copepods (Fig. 19) ................................................................................................................................................ class Copepoda (Chapter 22) LITERATURE CITED Hutchinson, G. E. 1993. A treatise on limnology, Vol. IV, The zoobenthos. Wiley, New York. McCafferty, W. P. 1981. Aquatic entomology. Science Books International, Boston, MA. Merritt, R. W., Cummins K. W., Eds. 1996. An introduction to the aquatic insects of North America. 3rd ed. Kendall/Hunt, Dubuque, IA. Ward, J. V. 1992. Aquatic insect ecology. 1. Biology and habitat. Wiley, New York. Wetzel, R. G. 2001. Limnology, 3rd ed. Academic Press, San Diego, CA. 2 AN OVERVIEW OF FRESHWATER HABITATS James H. Thorp Alan P. Covich Department of Biology Clarkson University Potsdam, New York 13699 Department of Fishery and Wildlife Biology Colorado State University Fort Collins, Colorado 80523 I. Introduction II. Lotic Environments A. The Physical – Chemical Milieu B. Ecosystem Changes Along the Stream Course III. Underground Aquatic Habitats A. Hyporheic and Phreatic Zones B. Aquatic Habitats within Caves and Other Karst Topography IV. Lentic Ecosystems A. Geomorphology and Abiotic Zonation of Lakes B. Biotic Zonation of Lakes C. Wetlands, Ephemeral Ponds, and Swamps D. Hypersaline Lakes Literature Cited I. INTRODUCTION II. LOTIC ENVIRONMENTS The contribution of inland waters to the total biospheric water content is insigniﬁcant in terms of percentage ( 1% according to Wetzel, 2001) but absolutely crucial from the perspective of terrestrial and freshwater life. Although inland lakes contain 100 times as much water as surface rivers, most lake water is held within massive basins, such as the Laurentian Great Lakes of North America, Lake Baikal of Siberia, and Lake Tanganyika of East Africa. Because most freshwater invertebrates are clustered within shallow, well-lighted zones of lakes, the relative importance of small ponds, creeks, and rivers as habitats for aquatic invertebrates is much greater than their volume percentages would otherwise indicate. Composition, species richness, and total density of invertebrates vary considerably among inland water habitats, as discussed in this chapter. Distributions of invertebrates are inﬂuenced by interactions among physical, chemical, and biological characteristics. The general importance of these abiotic and biotic factors is examined in this chapter. Detailed information on the ecology of individual taxa in inland water ecosystems can be gleaned from Chapters 3 – 23. Flowing waters, or lotic environments, were a principal pathway for evolutionary movement of animals from the sea to lakes and land. Even today, many taxa of freshwater invertebrates are restricted to headwater streams and rivers by unique environmental characteristics of these ecosystems. Compared to nonﬂowing waters, or lentic ecosystems, streams are generally more turbulent than lakes and, therefore, stratiﬁcation of the water mass with a thermocline is rare. High turbulence generally maintains high oxygen concentrations, reduces within-stream temperature differences, and more evenly distributes plankton and suspended or dissolved nutrients. Temperatures in streams ﬂuctuate over a smaller range than is typical of shallow littoral zones of lentic ecosystems, where most lakedwelling animals reside (Hynes, 1970). Except in the northernmost rivers, ice is less commonly encountered and is generally not as thick as in lakes. Flowing water habitats frequently possess more habitat heterogeneity, and the food web in forested drainage basins is more dependent on allochthonous production (externally produced plant matter), even though instream production can be important (Thorp and Delong, 1994). Lotic Ecology and Classiﬁcation of North American Freshwater Invertebrates. 2nd Edition Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved. 19 20 J. H. Thorp and A. P. Covich ecosystems are also more permanent on both evolutionary and ecological time frames than most lentic habitats. Both heterogeneity and permanence are thought to increase diversity within these ecosystems. In this section, we review features of epigean, or surface waters. Characteristics of aquatic environments where water ﬂows underground (hyporheic, phreatic, and cave environments) are discussed in Section III. A. The Physical – Chemical Milieu 1. Basin Morphometry and Characteristics of Flowing Water Although stream invertebrates are inﬂuenced directly by the physical features of the local habitat and only indirectly by geological forces shaping the morphometry of the entire ecosystem, ecologists and invertebrate zoologists must consider all those factors when attempting to understand what controls community composition. Some elements of stream morphometry that inﬂuence aquatic communities are stream order, channel patterns (e.g., meandering and braiding), erosion, deposition, and formation and characteristics of pools and rifﬂes. Classifying streams by their order, or pattern of tributary connections, is a simple technique that aids analysis of longitudinal (i.e., upstream – downstream) changes in stream characteristics within a single catchment; however, it is less useful, and indeed often misleading for comparing streams in different catchments. For example, communities within streams in the eastern coastal plain bear no close resemblance to communities within similar-order streams in the southwestern deserts (when ﬂooded) (cf. Figs. 1C and 1D respectively). According to this scheme, as reﬁned by Strahler (1952) and illustrated in Figure 2, a continuously running, headwater creek with no permanent tributaries is classiﬁed as a ﬁrst-order stream (permanency is deﬁned here as not being ephemeral on a seasonal or other regular basis). Two ﬁrst-order streams combine to form a second-order segment; two second-order creeks join as a third-order stream, etc. Stream order increases only when two streams of equivalent rank come together; hence, a third-order stream that ﬂows into a fourth-order system has no effect on the subsequent numbering scale of the larger stream. The Mississippi River, the largest river in North America, attains a stream order of 11 – 13 in its lower reaches (depending on which maps are used as the basis for analysis). Unfortunately, this scheme is not as useful for many streams in arid portions of the continent where a dry stream bed (Fig. 1D) may be replaced by a raging torrent in a matter of minutes or a few hours. Viewed from the air, streams clearly do not ﬂow in a simple, straight direction for signiﬁcant distances (Fig. 3A), nor do stream beds maintain constant depths for long stretches. Instead, the channel traces a meandering pattern of gentle and sharp bends through the basin with alternating bars, pools, and rifﬂes (Fig. 4) as the water travels downstream along a roughly helical path. The distance between meanders is a function of several factors, especially stream width. The river channel also has a tendency to undergo braiding (i.e., the division of the channel into a network of branches) in response to the presence of erodible banks, sediment transport, and large, rapid, and frequent variations in stream discharge (Gordon et al., 1992). Water ﬂows in a somewhat helical manner along the surface toward an undercutting bank where the deepest pool is usually located and then passes along the bottom toward a “point” or “alternate” sediment bar on the opposite bank from the pool. Sediment is continually being eroded from some areas (e.g., undercut banks and the head of islands) and deposited in other regions (such as on bars and the foot of islands). Successive deepwater pools are separated by shallow rifﬂe regions near the midpoint of two pools. This sequence of bars, pools, and rifﬂes is characteristic of both straight and meandering segments of most rivers, especially those with poorly sorted loads. (See Calow and Petts, 1992; Gordon et al., 1992; and references cited therein for additional details on ﬂuvial geomorphology). Scientists have appreciated the inﬂuence of alternating pools, rifﬂes, and (to a much lesser extent) sediment bars for some time. In comparison to pools, rifﬂes are characterized by greater habitat heterogeneity, sediment size, stream velocity, and sometimes oxygen content. In general, well-ﬂushed, stony rifﬂes support more species and individuals than silty reaches and pools (Hynes, 1970). This richness is a consequence, in part, of the different adaptations required to live in these physically distinct areas, especially with regard to dissolved oxygen. Our initial separation of aquatic ecosystems into ﬂowing- and standing-water environments recognizes the fundamental importance of this character to the biotic composition of inland water environments. Scientists measure water movement in streams in several ways, including stream discharge, turbulence, and velocity. Mean and peak values of stream discharge inﬂuence various ecosystem characteristics such as substrate particle size, bed movement, nutrient spiraling, and rate of animal drift. Stream discharge is calculated by the equation Q wdv where “Q” is the discharge in cubic meters per second (cms) or cubic feet per second (cfs), “w” the width, “d” 2. An Overview of Freshwater Habitats A B FIGURE 1 Lower-order streams: (A) Rocky-bed mountain stream in Glacier National Park, Montana; (B) rocky-bed stream from the Smoky Mountains of North Carolina; (C) sandy, coastal plain stream from South Carolina; (D) intermittent, dry-bed stream from Arizona (photographs by J. H. Thorp). 21 22 J. H. Thorp and A. P. Covich C D FIGURE 1 (Continued ) the depth, and “v” the velocity. As stream ﬂow increases, shear stress is magniﬁed. When the stress reaches a critical point, the substrate is picked up, rolled, or generally moved downstream. Various aspects of stream discharge, including mean annual discharge and yearly and historical extremes, are described annually for most rivers in the United States in the Water Data Reports published by the U.S. Geological Survey. This information is derived from multiple gaging stations located near the mouth of each river and often extending into the headwaters; waterquality data are also available for a few gaging stations in some of these rivers. These reports are available in the government documents section of most major libraries and are accessible through the USGS Web site. For more details on hydrologic processes, consult 2. An Overview of Freshwater Habitats FIGURE 2 Bifurcation of streams illustrating the system for classiifying lotic ecosystems according to stream order. The maximum order of the hypothetical stream shown here is sixth. articles or books on this subject, such as Allan (1995), Calow and Petts (1992, 1994), Gordon et al. (1992), Newbury (1984), and Statzner et al. (1988). In addition to being affected by the mean and range of stream discharge, freshwater invertebrates are strongly inﬂuenced by water velocity. An invertebrate’s ability to maintain its position on a rock and the amount of food passing its way (especially for suspension feeders) are both affected by current velocity. The mean current velocity of a stream at any given time usually occurs at a depth of about 0.6 of the maximum depth, as measured downward from the water surface (or 0.4 of the maximum depth as measured upward from the stream bottom). The current velocity diminishes progressively toward the bottom from that point and drops dramatically near the substrate in a 1- to 3mm-thick area known as the boundary layer. For this reason, velocities experienced by two invertebrates living within boundary layers of separate streams — one with high and the other with low average current velocity — may not differ appreciably. Many stream invertebrates are partially protected from dislodgment by having a low proﬁle that keeps their bodies mostly within the boundary layer. Other possible adaptations for life in fast ﬂow include streamlining, reduction of projecting structures, development of suckers, friction pads, hooks or grapples, and secretions of adhesive products. Under normal ﬂow conditions, the average current velocity varies from the headwaters to the mouth of a 23 river. Although many people believe that headwater streams ﬂow faster than large-order rivers, this popular idea has been repeatedly disproved (e.g., Leopold, 1953; Ledger, 1981). This erroneous view of streams may have arisen because the waters of a turbulent headwater stream (Figs. 1A and 1B) can “appear” to casual observers to be moving faster than the waters of a higher order, laminar ﬂow river (Fig. 3A), unless the large river is shallow and thus more turbulent (Fig. 3B). It is important to note, however, that ﬂow within microhabitats in any river order can substantially exceed the average current velocity of the stream reach. Fluctuating current velocity rather than constancy is typical of all lotic systems. Except in the largest rivers, zero or near-zero ﬂow rates have been recorded at least once in most lotic environments since the U.S. Geological Survey began recording stream discharges in the 19th century. The smaller the stream and the drier the surrounding area (e.g., desert streams), the more likely that a lotic system will be intermittent on at least a seasonal basis. At the other extreme, all streams have peak discharges on seasonal and multiyear bases. Flood events, or spates, may be very predictable, such as those produced by seasonal snow melt in mountainous streams, or they may occur dramatically over unpredictable time periods (Resh et al., 1988). Current velocities during ﬂoods and normal periods are related to the width, depth, and roughness of the stream bed, but rarely exceed 3 m/s even during ﬂoods (falling water seldom surpasses 6 m/s) (Hynes, 1970). Above 2 m/s, most rivers begin to enlarge their beds by erosion, unless constrained by rock banks or human construction. Invertebrates are often adapted morphologically or in life-history traits to moderate, seasonally predictable spates, but large unpredictable ﬂoods may eliminate all or most of the fauna of a stream by causing bed movement and severely scouring the bottom. When these scouring events occur, the sources for recolonization may be assemblages found in the adjacent hyporheic zone, in surface waters located upstream or downstream, or in separate, nearby stream catchments (Gibert et al., 1994; Jones and Mulholland, 2000). 2. The Importance of Substrate Type The nature and provision of substrates are of prime importance to survival because the overwhelming majority of lotic invertebrates are benthic (see references in Hynes, 1970; Allan 1995). Substrates provide sites for resting, food acquisition, reproduction, and development (e.g., places for pupal case attachment), as well as refuges from predators and inhospitable physical conditions. Hard substrates are generally valuable only for their surface qualities (except for species that bore into wood), whereas relatively soft substrates 24 J. H. Thorp and A. P. Covich A B FIGURE 3 (A) Ohio River above Louisville, Kentucky; here the river is meandering with a sandy-gravel bottom and a more laminar ﬂow; (B) Rio Grande near Santa Fe, New Mexico; here this large river is more turbulent than the Ohio River (photographs by J. H. Thorp). (ranging from sand to silt) provide a three-dimensional environment. The desirability of a given substrate type can be evaluated for a speciﬁc taxon using a combination of at least the following interrelated characteristics (not necessarily in order of importance): 1. Whether it is mineral (ranging from large boulders to ﬁne clay particles), living (algae, mosses, vascular plant stems and roots, and, rarely, animal hosts), or formerly living (e.g., abscised leaves and snags from tree limbs and trunks). 2. The silt and organic content of, or on, the substrate, including whether it is bare or covered by signiﬁcant amounts of algae, mosses, or vascular plants. 3. Its particle size, surface texture, and porosity. 4. Its stability, as judged by its retention in the 2. An Overview of Freshwater Habitats FIGURE 4 Schematic drawing of a stream showing straight reaches and meanders. In both sections of a river, a sequence of deep (pools) and shallow (rifﬂe) areas is manifested. Point bars and alternate bars develop as bed material is deposited in areas opposite from the pools. The thalweg, a line connecting the deepest part of the channel, migrates back and forth across the river. system (e.g., leaves are a short-lived substrate, whereas boulders are retained almost indeﬁnitely) and its tendency to shift position (ranging from almost constantly shifting sand to boulders that rarely turn). 5. The physical-chemical milieu in which the substrate resides, such as the water depth at which it is located, the ambient oxygen content, and the range of current velocities surrounding it. Even if a particular site meets all the physical and chemical criteria for a desirable substrate, it may not be appropriate for an individual invertebrate if a competitor has preempted the space or if it is located in an area that would make a resident especially susceptible to predators or abiotic disturbances. For these reasons, the distribution of a species across a spectrum of substrate types may not reﬂect so much the selectivity of the invertebrate species as the effects of substrate limitation. While a particular taxon may not beneﬁt greatly from substrate heterogeneity, the species diversity of the entire community tends to rise with an increase in habitat complexity. In a like manner, an individual may beneﬁt from maximum substrate, but the community as a whole may ﬂourish better with some disturbances. This latter concept, embodied in part by the “intermediate disturbance hypothesis” (Connell, 1978; Ward and Stanford, 1983; Thorp and Cothran, 1984), suggests that intermediate levels of biotic or abiotic disturbances (e.g., frequency of substrate shifting) can promote maximum species diversity under certain circumstances. For a more detailed discussion of the general effects of substrate characteristics, the reader should initially consult Allan (1995). 3. Thermal and Chemical Constraints on Distribution a. The Thermal Environment Because temperatures vary on a seasonal basis and a gradient exists from headwaters to mouth, it is not surprising that the distri- 25 bution and abundance of freshwater invertebrates are correlated with the annual mean and range of temperatures (e.g., Hynes, 1970; Ward, 1986). A general stream temperature pattern on an annual basis is a progressive increase from headwaters to mouth, especially in streams originating in the mountains and fed primarily by terrestrial runoff. [This general pattern may not hold for low-gradient, north-ﬂowing streams.] Streams originating from coldwater springs, however, with their relatively constant temperatures in the headwaters, can be warmer in winter near their source than somewhat farther downstream; in the summer, the opposite pattern exists (Minckley, 1963). In many temperate zone rivers, annual ﬂuctuation in temperature increases downstream, while diel temperature changes peaks in mid-order reaches (Statzner and Higler, 1985). In contrast, diel and annual temperature amplitudes are very low in middle and lower reaches of tropical streams, although high-elevation, tropical streams have signiﬁcant seasonal temperature gradients (Covich, 1988). Predictions of thermal patterns must be modiﬁed to account for effects of tributaries (often colder) and dams on downstream reaches. Likely differences in thermal regimes between natural and regulated rivers are described by the “serial discontinuity concept” (Stanford et al., 1988), which predicts that the thermal character of the stream below a dam will be “reset” toward that typical of reaches above the dam. This pattern is evident if you calculate the number of degree-days (sum of the average daily temperatures over a period) in stream reaches above and below the dam. The timing of insect emergence, an invertebrate’s tolerance of speciﬁc habitats, and annual productivity of individual species and communities are all inﬂuenced directly or indirectly by the number of degree-days in those habitats (Sweeney and Vannote, 1978). Species richness and the nature of invertebrate fauna vary along a continuum from headwaters to the mouth. Crustaceans (benthic and planktonic), molluscs, and ﬁsh increase in abundance and diversity with stream order, whereas insects generally decrease downstream. Because of previously noted patterns for temperature means and ﬂuctuations with stream order, it is not surprising that aquatic ecologists saw a link between thermal regimes and species diversity. Temperature may inﬂuence organisms directly or have an indirect effect through changes in oxygen saturation levels (the oxygen-carrying capacity of water decreases at higher temperatures). While it is true that a great many lotic invertebrates are more stenothermal (i.e., surviving only in a narrow temperature range) than their lentic counterparts (Hynes, 1970), it has not been demonstrated that the relationship between invertebrate diversity and temperature is more than coincidental. The problem in distinguishing 26 J. H. Thorp and A. P. Covich causative factors is that many biotic and abiotic parameters other than temperature also change along river gradients. For example, habitat complexity, which has often been demonstrated to signiﬁcantly inﬂuence diversity, varies downstream, often reaching a maximum value in mid-order sections of rivers. b. The Chemical Environment Certain natural chemical features of streams and lakes signiﬁcantly affect invertebrate abundance and diversity by inﬂuencing habitat quality. Of these, the most crucial are probably dissolved oxygen and salinity (water hardness). Hydrogen ion concentration, or pH, is occasionally important in natural systems. Anthropogenic pollution has had a severe impact on the integrity of aquatic ecosystems by affecting these chemical parameters as well as by introducing organic and inorganic toxicants. As a general rule, dissolved oxygen decreases downstream. This pattern occurs because the upper reaches of a river are usually more turbulent, the surface to volume ratio is more favorable for diffusion, and temperatures are lower. In the last case, oxygen solubility is related in an inverse, nonlinear manner to temperature; this gas is markedly more abundant in cold waters. Pure water in equilibrium with air at standard pressure holds 12.770, 10.084, 8.263, and 6.949 mg O2/L at 5, 15, 25, and 35°C, respectively (see Mortimer, 1981, in Wetzel, 1983, p. 158). Because oxygen is produced and respired by plants, it may be locally more abundant or even supersaturated during the day in regions with heavy plant growth. However, oxygen potentially decreases at night to lethal levels in weedchoked streams because of high oxygen demands for decomposition and plant respiration. The probability of lethal oxygen concentrations occurring for the invertebrate species typical of a stream reach rises as currents diminish; hence, the problem is more serious in lakes and seasonally sluggish streams than in fast-ﬂowing lotic habitats. Invertebrates differ both in their respiratory needs and in their anatomical and behavioral adaptations for obtaining oxygen. Respiratory rates of lotic species are generally dependent on external oxygen concentration (tension) at medium to low levels, while oxygen consumption rates of animals in lentic or typically sluggish lotic environments tend to be independent of ambient tensions, at least at medium-to-high concentrations (Hynes, 1970). Currents provide a steady renewal of oxygen to the respiratory surfaces of invertebrates. Where velocities are too low, the animal may need to ventilate its gills or obtain atmospheric oxygen. For example, crayﬁsh normally move water over their respiratory surfaces with “gill balers” but some species climb partially or completely out of water if oxygen tensions drop too low (returning when gills need moistening). Because invertebrates have different oxygen requirements, it is tempting to assume that interspeciﬁc differences in distribution are partially caused by oxygen limitations (e.g., contrasting assemblages of caddisﬂies in mountain streams and ﬂoodplain rivers). Certainly many stream animals, such as larvae of the blackﬂy Simulium, cannot obtain adequate oxygen in still water (Hynes, 1970), but this explanation for restriction from lentic habitats is less effective in resolving species differences in distribution along the entire stream reach. Even if one demonstrated in the laboratory that the oxygen tensions in high-order rivers were inadequate to support a caddisﬂy from a mountain stream, this is not evidence that oxygen was the principal factor leading to the present pattern of distribution. Other factors, such as interspeciﬁc competition, resource type (e.g., seston particle size), substrate form, or predators, may have been initially responsible, with ﬁne tuning of respiratory adaptations coming later. The ionic content of inland waters is usually extremely dilute in comparison to seawater, with four cations (Ca2, Mg2, Na, and K) and four anions 2 2 (HCO 3 , CO3 , SO4 , and Cl ) representing the total ionic salinity in most freshwater systems, for all practical purposes (Wetzel, 2001). Soft waters are generally found in catchments with acidic igneous rocks, whereas hard waters (relatively high salinities for freshwater) occur in drainage regions with alkaline earths, usually derived from calcareous rocks. The global mean salinity of river water is 120 mg/L; North American waters are slightly harder, averaging 142 mg/L [from Livingstone (1963) and Benoit (1969) in Wetzel (1983, p. 180)]. Most soft waters in North America have salinities 50 mg/L. Salinities are slightly elevated during periods of low ﬂow, and they generally increase downstream because of bedrock erosion (Hynes, 1970) and human inﬂuences on watersheds. Except for a few hypersaline environments, such as mineral springs and alkaline and brine lakes, inland waters are hypotonic to the internal ﬂuids of freshwater species; therefore, all freshwater animals need some ability either to osmoregulate (almost all species) or, at the minimum, to control the relative abundances of certain ions (necessary even for freshwater Cnidaria; see Chapter 5). Because animals with either partially calcareous exoskeletons (e.g., crayﬁsh, ostracodes, and cladocera) or hard calcareous shells (snails and mussels) have an added need for calcium ions, one might expect them to be particularly excluded from soft-water habitats (see Chapter 18). Indeed, molluscs are rare in softwater lakes and less abundant in soft-water streams, but this limitation is not as severe as in lentic ecosystems. Molluscs can live in some soft-water streams, because 2. An Overview of Freshwater Habitats they can tolerate extremely dilute tissue ﬂuids (the lowest of any metazoa; see Chapter 11). The minimum concentration of Ca2 tolerated by mussels (2 – 2.5 mg Ca/L) is just adequate to prevent the rate of shell dissolution from exceeding the rate of shell deposition. Despite their heightened need for calcium, crayﬁsh can be abundant in many soft-water streams of the Southeast where these ions are difﬁcult to obtain. The problem of ionic loss at the critical period when the old exoskeleton is molted is partially overcome by the crayﬁsh habit of consuming its shed exuvia as a way of extracting additional calcium ions. This behavior and the efﬁcient osmoregulatory system of the crayﬁsh (with gastrolith formation) allow this decapod to colonize a greater chemical range of habitats (see Chapter 23). In the last decade, the catastrophic effects of acid precipitation and mine drainage on freshwater ecosystems have drawn great attention from scientists and politicians alike, but relatively unpolluted rainwater and runoff from thick forest litter are also slightly acidic (though not at lethal levels). Bogs and the streams draining them are among the most naturally acidic ecosystems; sphagnum bogs usually have a pH below 4.5 and occasionally below 3.0 (Wetzel, 2001). Streams draining limestone catchments are well buffered by carbonates and therefore have moderate to high pH values as well as plentiful supplies of calcium. In contrast, acidic waters are normally low in calcium, causing potential osmotic problems for their residents. Despite probable ionic and pH difﬁculties which may have eliminated certain taxa, naturally acidic ecosystems often support a surprisingly diverse invertebrate assemblage. B. Ecosystem Changes along the Stream Course 1. Patterns along the Continuum Physical and biological characteristics clearly change from headwaters to the river mouths. The river widens, deepens, and becomes more turbid downstream, often removing part of the bottom from the photic zone. Stream shading from riparian trees lessens, with a corresponding increase in percentage of the surface receiving direct solar radiation. Concurrently, the relative importance values for autochthonous (internally produced) and allochthonous carbon to energy budgets alter along with the ratio of production to respiration. As a result of these and other habitat changes, the invertebrate fauna undergoes dramatic shifts in species composition, relative abundances, and functional feeding groups. This holistic view of the river was not synthesized until formation of the river continuum concept (RCC) by a group of aquatic ecologists working in temperate 27 deciduous streams of North America (Vannote et al., 1980; Sedell et al., 1989). This important theory has proved to be one of the most hotly debated in recent decades, and that fact alone contributes to the pivotal role it has played in aquatic ecology. Individual hypotheses in the RCC have received both support and criticism (as examples of the latter, see Winterbourn, 1982; Statzner and Higler, 1985; Junk et al., 1989; Thorp and Deong, 1994). It is not our purpose to defend or assail this holistic, ecosystem concept; instead, we wish to acquaint you with some of its predictions as they could inﬂuence your understanding of the habitat and ecology of lotic invertebrates. Because the RCC was originally developed from studies of relatively undisturbed, North American streams with forested headwaters (cf. Fig. 1B), the following comments may not be as applicable to stream invertebrate communities in other biomes or regions of the world. The river continuum concept relies on three theoretical pillars of support. The ﬁrst principle is that stream communities originate in response to a continuous gradient of physical variables present from headwaters to the mouth; this view contrasts with a previous portrayal of natural streams as a series of disjunct communities proceeding downstream in a stepwise fashion. The second canon is that biotic communities within the “wetted channel” of the river cannot be divorced from either the adjacent riparian zone or the surrounding biotic and geomorphic catchment area that funnels water, nutrients, and other material into the aquatic ecosystem. The ﬁnal tenet is that the nature of a downstream assemblage is inextricably linked with processes occurring upstream (this has been challenged for ﬂoodplain rivers by Junk et al., 1989). Of the many physical, chemical, and biological predictions made by the RCC, the one most relevant to understanding invertebrate ecology concerns longitudinal changes in the relative abundance of functional feeding groups and their food resources. The proposed relationships between stream size and progressive alterations in community composition are illustrated in Figure 6. As heterotrophs, invertebrates consume living and/or dead organic matter, processing it in ways characteristic of several “functional feeding groups.” These groups include “shredders,” which break up coarse particulate organic matter (CPOM) such as abscised leaves; “collectors,” which gather or ﬁlter CPOM, ﬁne particulate organic matter (FPOM), and / or live, drifting organisms; “grazers” or “scrapers,” which remove algae or other aufwuchs growing on rocks or other substrates; and “predators,” which may include carnivores and some herbivores. The traditional means of assigning a functional feeding label to a life stage of a particular species has 28 J. H. Thorp and A. P. Covich been either to observe the organism’s feeding behavior (preferably in the ﬁeld) or to analyze its gut contents on a seasonal basis (e.g., Benke and Wallace, 1997). This task is time consuming and may be impractical for some taxa. References to the food resources and feeding behavior of species or larger taxonomic categories are reported in many research papers (for examples, see chapters in this book and the tables and references on aquatic insects in Merritt and Cummins, 1996). The danger in relying on general sources, however, is the well-established observation that functional feeding classiﬁcations can vary signiﬁcantly with life-history stage, season, and other ecological features; it is also difﬁcult to assign omnivores to any single or consistent functional feeding group (see summary of some limitations of this method in Mihuc, 1997). An approach that overcomes some of these problems is to construct food webs using data on stable isotope and/or fatty acid content of producers (dissolved organic matter through living terrestrial and aquatic plants) and aquatic consumers; these data reﬂect the cumulative feeding patterns of freshwater invertebrates over periods of up to 3 months (e.g., Thorp et al., 1998). The RCC predicts that the invertebrate assemblages of headwater streams, with their heavy canopy of riparian trees, will be dominated by shredders and collectors. As the stream widens and more light reaches the water surface, autochthonous production from benthic algae and rooted macrophytes increases, allowing the development of a signiﬁcant grazer fauna; shredders should diminish dramatically in these mid-order reaches of the river. Farther downstream, the river deepens so that much of the bottom is below the photic zone (supposedly limiting autotrophic production in the river), and the channel is hypothesized to be sufﬁciently wide to preclude any signiﬁcant input of allochthonous production from the riparian zone. In this high-order section of the river, collector – ﬁlterers are expected to dominate the ecosystem. These conclusions on primary sources of organic matter have been strongly challenged for large rivers by both proponents of the ﬂood pulse concept (Junk et al., 1989), who emphasize the importance of allochthonous production generated on river ﬂoodplains, and proponents of the riverine productivity model (Thorp and Delong, 1994), who stress the greater importance of instream (autochthonous) primary production to secondary (animal) production, especially at higher trophic levels. 2. Nature of the River’s Source One weakness in the original formulation of the RCC was its reliance on data collected from a narrow range of stream sizes (mostly stream orders 1 – 3). Even today, our knowledge of assemblages living at opposite ends of the continuum — springs and intermittent streams versus large rivers — is woefully inadequate (Meyer, 1990). Research on intermittent, or ephemeral, streams (Fig. 1D) is more common in arid regions of North America (e.g., Matthews, 1988; Forrester et al., 1999), but has expanded into headwater streams in more humid regions of eastern North America (e.g., Delucchi and Peckarsky, 1989). Almost all streams are subject to seasonal and / or aperiodic disturbances from unusually high current velocities and stream discharges, but only intermittent streams ﬂuctuate regularly from dry to wetted basin. When a stream stops ﬂowing, a resident invertebrate may have several options for its survival or that of its future progeny: it may move downstream, seek shelter in deep pools, enter the hyporheic zone (i.e., the wetted substrate below the surface waters, as described in Section III.A), emerge as an adult (some aquatic insects), develop a resistant stage, or lay eggs and then die. Although the composition of the invertebrate fauna in permanent and intermittent streams may differ, life-history patterns of invertebrates in ephemeral streams are not particularly unique (Delucchi and Peckarsky, 1989), perhaps because options for migrating downstream or entering the hyporheic zone are readily available for many species. The ecology of that portion of the ecosystem, however, may be signiﬁcantly different from that of a community functioning within permanent streams (e.g., Forester et al., 1999). There is a tendency to think of streams as originating exclusively from terrestrial runoff, but many headwater systems are derived from springs. Near their source, spring-fed streams often vary in physical, chemical, and biological features from other types of ﬁrst-order streams (e.g., Covich, 1988). Temperatures within spring-fed streams are relatively more constant, and oxygen concentrations are occasionally lower, sometimes to near-lethal levels for metazoa. In other cases, hot springs may provide habitats that range into the upper lethal limits for eukaryotes (see Chapters 19 and 20) and even prokaryotes. Metazoan life near the upwelling region is rarely possible; but, as ambient temperatures approach 40°C or lower in side channels and pools and in areas farther downstream, conditions become suitable for some invertebrates (e.g., Barnby and Resh, 1988). These include a very restricted number of species of nematodes, oligochaetes, mites, dipterans (such as brine ﬂies), and ostracodes and a few other crustaceans. Geothermal springs are often extremely vigorous environments not only because temperatures may approach the boiling point but also because their waters are frequently laden with high concentrations of sulfur. The ionic content of springs can differ from that of from nearby streams supplied by runoff from land. Springs in limestone regions are well buffered and are 2. An Overview of Freshwater Habitats frequently highly charged with calcium bicarbonate (Hynes, 1970). Saline springs exist where the ionic content is too high for many invertebrates. The biota of springs often vary from other ﬁrstorder streams as a result of their connection to underground rivers and their aquatic constancy (Gibert et al., 1994; Jones and Mulholland, 2000). A net placed near the spring source can capture unusual subsurface species that have been washed out of their subterranean environments. In this way, blind troglobitic shrimp have been retrieved from surface waters in the springs of the San Marcos River of Texas (Longley, 1986; Strenth et al., 1988). Because springs are relatively uniform environments over long ecological periods, it is not unusual to ﬁnd relict species in these environments that have survived the retreat of the glaciers only in these limited habitats (Hynes, 1970). For discussion on endemic groundwater fauna in unglaciated eastern North America, see Strayer et al. (1995). 3. Large Rivers At the opposite extreme of the continuum, the invertebrate biota and ecological processes in large rivers (e.g., Figs. 3A, 3B, and 5) have rarely been studied in the past (although this is beginning to change) despite their importance to humans. Hynes (1989) pointed out that only 4% of all ecological publications of ﬂowing waters have involved large rivers. Consequently, certain misconceptions about the nature of large rivers have arisen, such as descriptions of the “typical” large river as a slow, meandering, soft-bottomed stream. In con- 29 trast, observations by one of us (JHT) in the Mississippi, Ohio, and Tennessee Rivers suggest that the bottom is more likely to be sand, gravel, or cobble except in the shallow, slow-moving water near banks and in the mouths of tributaries where silt often accumulates. Furthermore, evidence for differences in meandering patterns along a river continuum has not yet been generated, and in comparison to upstream reaches, the current velocity is generally higher in large rivers. Because governments have been removing fallen trees and regulating both ﬂow and channel depth in navigable rivers since the mid-1800s, the present nature of these ecosystems may be considerably different from the original state (Minshall, 1988). For example, removal of snags has greatly reduced the abundance of shallow-water hard substrates, thereby inﬂuencing composition of both the insect assemblage and the ﬁsh that normally fed upon these insects (cf. Benke et al. 1994). Many aquatic ecologists lacking experience in large rivers (other than reservoirs) often think of them as “lakelike,” perhaps expecting similar physical and chemical conditions. Unlike lentic environments, however, the helical ﬂow of riverine currents prevents formation of thermoclines and major zones of oxygen depletion within the water column; this has considerable inﬂuence on processes of nutrient cycling (or spiraling in rivers). The greater depth and turbidity of sedimentladen rivers reduce the amount of benthos receiving light so that only a narrow photic zone exists. The resulting restriction on phytoplankton net production, along with microbial breakdown of allochthonous FIGURE 5 Photograph of a large river (the Ohio River upstream of Louisville, Kentucky), showing the laminar ﬂow nature of its waters (photograph by J. H. Thorp). 30 J. H. Thorp and A. P. Covich detritus, is thought to be a primary factor producing a heterotrophic metabolic state in most large rivers (Howarth et al., 1996). The potential importance of physical factors in an advective environment (Pace et al., 1992) and their inﬂuence on the role of biotic interactions (Thorp and Casper, unpublished data) are currently being investigated. The RCC appears generally correct in predicting the important role for suspension feeders in large rivers, but it underestimates the role of some other groups, such as grazers. As predicted by the RCC, suspension feeders predominate in both the benthos (e.g., mussels and midges) and pelagic zones (various crustacean and rotifer zooplankton) of large rivers, especially since the invasion of zebra and quagga mussels into eastern rivers (Thorp et al., 1998). However, the predicted absence of grazers, as would be anticipated in the Ohio River from Figure 6, is certainly incorrect. Abundant populations of snails in the Ohio consume various types of benthic algae, other aufwuchs, and detritus (depending on the depth distribution of snails; K. Greenwood and J. H. Thorp, unpublished data). Invertebrates in general tend to be clustered in nearshore (especially on rocks, as in Fig. 5), shallow-water regions of rivers (Thorp and Delong, 1994), where they have access to both benthic algae and allochthonous material entering from the riparian zone. Because the North American Benthological Society focuses on nonpelagic ecology and members of the American Society of Limnology and Oceanography are primarily oriented to lentic and oceanic environments, studies of plankton ecology in rivers are relatively rare in North America, though more common in Europe. However, the increasing importance of zebra mussels in rivers has contributed to studies of these potamoplankton (from the Greek potamos, or river) and benthic – pelagic coupling processes (e.g., Pace et al., 1998; Jack and Thorp, 2000; Thorp and Casper, unpublished data). One of the more interesting trends in large-river research, that seems to be developing includes freshwater zooplankton. This trend revolves around the importance of lateral slack-water areas, or storage zones (Reckendorfer et al., 1999, Thorp and Casper, unpublished data), to community and ecosystem processes in the river as a whole. III. UNDERGROUND AQUATIC HABITATS A. Hyporheic and Phreatic Zones FIGURE 6 Predicted relationship between stream size and structural and functional attributes of a freshwater ecosystem as embodied in the river continuum concept. P/R refers to the ratio of production to respiration (from Fig. 1 in Vannote et al., 1980). Until recently, aquatic ecologists considered the boundaries of rivers to extend only from the air-water interface down to the silty, sandy, or rocky “bottom” of the river. We now realize that this deﬁnition is overly limiting and view streams as four-dimensional ecosystems with longitudinal (upstream-downstream), vertical (deeper into the sediments), lateral (ﬂoodplain), and temporal components (cf. Ward, 1989). Early this century, scientists recognized the potential importance of interstitial spaces within sediments of lakes and streams as refuges for small animals. This interstitial habitat is termed the “hyporheic zone” within lotic systems and the “psammon zone” in lentic environments. The depth of the hyporheic zone varies greatly according to bed topography, substrate porosity, and water velocity (Boulton et al., 1998), being greater, for example, in rivers having a gravel bed rather than a mud bottom (the habitable interstitial space is inﬂuenced by, but is not directly related to, sediment porosity). The overlying surface, or epigean, waters strongly inﬂuence the nutrient content, oxygen tension, and other physical and chemical characteristics of this zone and, consequently, the nature of its fauna, 2. An Overview of Freshwater Habitats or “hyporheos.” Likewise, upwelling from the hyporheic zone can provide nutrients to stream organisms. While many organisms dwell much of their lives in the hyporheic, it is not clear whether this zone serves as the refuge from stream spates that it was once thought to be (Gayraud et al., 2000). Almost all freshwater invertebrate phyla have representatives in hyporheos, with the probable exceptions of Cnidaria and Porifera. They include several orders of insects (e.g., midges, elmid beetles, and stoneﬂies), mites, crustaceans (including cladocera, ostracodes, and amphipods), oligochaetes, turbellarians, rotifers, snails, tardigrades, and protozoa. Hyporheic organisms are usually abundant in streams unless the substrate is bedrock, clay, or other material containing pore sizes under 50 m (Williams, 1984), but their densities reﬂect differences in organic content and oxygen, among other factors. Below a few centimeters, the density of hyporheos is generally inversely related to substrate depth, with very few individuals occurring much below 1 m into the sediment. Because the hyporheic food web is based on detrital inputs from surface waters (Boulton et al., 1998), animal density is strongly related to the amount of organic carbon present in the interstitial spaces (Brunke and Gonser, 1999). Organisms in the hyporheic, and presumably the phreatic, are more resistant to low oxygen conditions (hypoxia) than are epigean species, but they cannot permanently tolerate suboxic conditions (Malard and Hervant, 1999). The distribution and local diversity of hyporheic invertebrates are also inﬂuenced by historical patterns of glaciation (Strayer and Reid, 1999). The hyporheic zone also extends laterally into the stream bank, where often the distance it penetrates horizontally from the river proper, is similar to that it penetrates vertically into the river bed. Data from the Flathead River, a gravelly stream of the northern Rocky Mountains, have shown, however, that the hyporheos permeates groundwater as far as 2 km from the surface water of the river (Stanford and Ward, 1988). It has been known for some time that a unique fauna can occupy the phreatic zone — a saturated underground area where the water moves slowly laterally in the absence of direct inﬂuence from surface water rivers. The study by Stanford and Ward, however, demonstrated that the groundwater community within up to 2 km from the Flathead River was hyporheic rather than phreatic because there were physical (ﬂow), nutrient, and biotic connections between the two areas. The biota of the adjacent phreatic zone was dominated by subterranean crustaceans, while the hyporheic zone supported stoneﬂies and other typically riverine taxa. 31 B. Aquatic Habitats within Caves and Other Karst Topography Regions of the world containing large amounts of soluble limestone and adequate underground water often form complex subterranean cavities; such karst topography is widespread in North America (Fig. 7). Karsts develop when rocks are fractured and are especially susceptible to solution (e.g., crystalline, high calcite limestone). Over time, small cavities can enlarge to produce huge underground caverns. The public generally becomes aware of solution processes only when an above-ground entrance exists or a sinkhole develops (also called sinks or, more properly, dolines). Caves can be deﬁned as karsts with an entrance, passageways, rooms, and a terminal blockage to all but water and material it carries. Cave environments provide habitats (Fig. 8) for many types of invertebrates which differ in several signiﬁcant ways from surface, or epigean, aquatic habitats. They have very constant temperatures, that are equal to the average yearly surface temperature of the local geographic region, light is dim or absent, and abiotic disturbances are probably less frequent and severe (although ﬂoods occur in caves). Food is quite scarce in caves, as it usually arrives only through groundwater or periodic movement of animals that seek refuge within an open cave after having fed outside (e.g., bats which deposit organic guano in the cave). The principal forms of organic matter supporting the food web are particulate (POM) and dissolved organic matter (DOM) along with heterotrophic bacteria growing in water or on suspended and benthic detritus and some chemoautotrophic bacteria. Consequently, food webs are relatively simple, consisting mostly of detritivores and their predators. The density and species diversity of a single cave community are usually quite low. In contrast to the simplicity of a cave community, the species composition of one cave can be markedly FIGURE 7 Karst topography in the 48 contiguous states of the United States (from Ritter, 1986). 32 J. H. Thorp and A. P. Covich FIGURE 8 (A) A cave habitat in southern Indiana; (B) The blind, troglobitic crayﬁsh Orconectes testii (both photographs courtesy of H. H Hobbs, III). different from that of an adjacent but unconnected neighboring cave. Endemism is especially prominent in caves because of inherent geographic isolation. The species radiation of amphipods in North America has been much greater in karsts than in surface waters (see Chapter 19). A total of 927 species of invertebrates that live exclusively in caves or associated subterranean habitats in the contiguous 48 states in the United States, have been described so far, with more than 50% of the aquatic species and subspecies derived from only 18 counties representing less than 1% of the land area (Culver et al., 2000)! Just over 50% of these subterranean species are from only a single county, and 95% of the cave species are listed by the Nature Conser- 2. An Overview of Freshwater Habitats vancy as vulnerable or imperiled. It should be evident, therefore, that habitat protection at those sites is absolutely critical to species survival. Obligate cave species, or troglobites (as opposed to the facultative troglophiles), usually have low metabolic rates and a much longer lifespan than their surface relatives. They are also frequently blind and lack body pigmentation. Reproductive yield is spread over longer periods with fewer but more yolky eggs formed during each reproductive bout. For example, a female crayﬁsh of the troglobitic species Orconectes inermis in Indiana caves may carry only a few dozen large eggs, while nearby epigean, congeneric individuals can bear hundreds of small eggs (J. H. Thorp, personal observation, and H. H. Hobbs, III, personal communication; see also ecologically similar species in Fig. 8). Information on karst communities can be gleaned from journals such as the National Speleological Society Bulletin. See also Chapters 19 and 23 in this book for details about cave amphipod and decapod crustaceans, respectively. For other information on the ecology of groundwater communities, see specialty texts such as Gibert et al. (1994) and Jones and Mulholland (2000). IV. LENTIC ECOSYSTEMS Unlike the situation for lotic habitats, the chemistry, physics, geology, and biology of lentic (or lacustrine) environments are well covered in several standard limnology textbooks (e.g., Cole, 1994; Lampert and Sommer, 1997; Wetzel, 2001), most of which devote very little space to stream studies. Lake limnology is also discussed in more depth in various specialty texts (e.g., Taub, 1984; Carpenter, 1988) and in four volumes on limnology by Hutchinson (1957, 1967, 1975, 1993). For this reason, our discussion of lake habitats is briefer than that accorded to stream ecosystems. A. Geomorphology and Abiotic Zonation of Lakes Although rivers and streams are important biologically, more than 99% of the inland surface waters are in fresh and saline lakes. These standing water, or lentic, ecosystems occur on every continent but are concentrated in the formerly glaciated regions of the Northern Hemisphere. Except for ~20 lakes with depths 400 m, most natural and human constructed lentic systems have average depths of 20 m (Wetzel, 2001). Most are geologically young, dating from the last glacial period in the case of natural lakes or from the last century for human-made lakes. According to Hutchinson (1957), natural lakes are formed by over 33 70 distinct processes. Most result from catastrophic phenomena (landslides and glacial, tectonic, and volcanic activities), but some form less violently from the action of rivers (e.g., oxbow lakes), waves, and rock solution. Before the advent of extensive, commercial fur trapping in North America, ponds built by beavers were a pervasive feature of the headwaters of many North American streams (Naiman et al., 1986). Lentic ecosystems can be divided into several abiotic zones, primarily based on distance from shore, light penetration, and temperature change (Fig. 9). The photic zone extends downward to the depth of 1% light penetration; all primary producers and most heterotrophic animals live within this zone. Areas below these depths are variously called the aphotic or profundal zone (the latter often in reference to benthos). The shallow, nearshore region of the photic zone where rooted macrophytes exist is termed the littoral zone. The entire mass of open water located away from both the shore and the littoral zone is known as the limnetic or pelagic zone. On a seasonal basis, most lentic ecosystems in North America become stratiﬁed with a layer of lighter water, the epilimnion, ﬂoating over the denser hypolimnion (Fig. 9). At least during the summer, the epilimnion is considerably warmer than the hypolimnion. These two zones are separated by a layer of rapid temperature change called the metalimnion. The boundary between the epilimnion and the hypolimnion, where temperature changes occur most rapidly with depth, is deﬁned as the thermocline (it is typically within the metalimnion). Because there is minimal exchange of water between upper and lower zones during stratiﬁcation, the hypolimnion is frequently lower in oxygen, higher in nutrients, and different in pH and other chemical concentrations. When lake stratiﬁcation breaks down for a short period during one or more seasons, much or all of the water mass recirculates in a process referred to as lake turnover. If a lake mixes completely twice a year, it is referred to as dimictic; if it turns over only once a year it is called monomictic. In other lentic systems, complete mixing either rarely takes place (in oligomictic lakes) or occurs more than twice per year (polymictic lakes). In addition to thermal zones, some lakes are chemically stratiﬁed (commonly with a heavier layer of saline water in the hypolimnion). Some of these lakes never mix completely and are termed meromictic; they only circulate in the upper zones. As a consequence, they are typically devoid of oxygen in deeper zones, where nutrients can accumulate over time. The benthic zone can be further divided into somewhat arbitrary categories based on other ecosystem characteristics, such as substrate type, wave action, and 34 J. H. Thorp and A. P. Covich FIGURE 9 Biotic and abiotic zones within a lake along with a list of some representative freshwater invertebrates found within these zones. vegetation type. The importance of substrate type for lentic environments is similar to that described earlier for lotic ecosystems. ﬂeas (cladocera; Chapter 20), alternate periods of resting or foraging on the bottom with intervals of swimming and foraging in the water column. 1. Neuston B. Biotic Zonation of Lakes Lake invertebrate assemblages can be subdivided primarily into “zooplankton”, within the water column, and “benthos,” which live in, on, or just above the bottom (commonly in association with macrophytes). A third, smaller group encompasses the “neuston,” which live at the air – water interface. These ﬁrst two categories remain quite distinct except in three general cases. A few species, such as the phantom midge Chaoborus, migrate upward at dusk into the plankton and return to the benthos near dawn. Meroplankton, in contrast to the much more diverse holoplankton, spend only a portion of their life cycle in the water column. For example, chironomid midges are benthic for most of the larval period (the short-lived adults are aerial), but swim within the water column during the earliest stages of their larval existence. Dreissenid mussels, which invaded North America in the late 1980s, have a veliger stage that is planktonic for up to 3 weeks (Garton and Haag, 1993; see also Chapter 11). Although not true members of the plankton, many nearshore species, such as littoral zone water The term neuston refers to the assemblage of organisms associated with the surface ﬁlm of lakes, oceans, and slow-moving portions of streams. It generally includes species that live just underneath the water surface (hyponeuston), individuals that are above but immersed in the water (epineuston), and taxa that travel over the surface on hydrophobic structures (superneuston or, more properly, a form of epineuston). This name is similar to, or a subset of the older name, pleuston (sometimes neuston is used in reference to the microscopic components of the more encompassing pleuston). The density of neustonic organisms decreases with increasing turbulence. The neustonic food web is primarily supported by a thin bacterial ﬁlm on the upper surface of the water, a concentration of phytoplankton near the surface, and allochthonous inputs from trapped terrestrial and aquatic organisms. Protozoa are common in this assemblage (which also includes algae and ﬂoating macrophytes), whereas other typically planktonic taxa are rare (one exception is the cladoceran Scapholeberis). Moving over the water surface are springtails (Collem- 2. An Overview of Freshwater Habitats bola), some arachnids (mites and water spiders), and various families of heteropteran bugs (e.g., water striders, Gerridae) (refer to Chapter 16 for information on arachnids and to Chapters 17 and 18 for details about insects and springtails). Larger neustonic species are especially vulnerable to predation from both aquatic and terrestrial predators, and all species must be adapted to the higher ultraviolet radiation present near the water surface (see Williamson et al., 1999, for recent perspectives on UV and lake plankton). 2. Zooplankton of Pelagic and Littoral Habitats Zooplankton include all animals in the water column that ﬂoat, drift, or swim weakly (i.e., they are at the mercy of currents). Fish, as powerful swimmers, are the principal members of freshwater “nekton” in littoral and pelagic environments. Some predatory zooplankton are active swimmers and comprise the “nektoplankton”; examples are the large cladoceran Leptodora kindtii (Chapter 21) and mysid “shrimp” Mysis relicta (Chapter 19). Zooplankton thrive within two distinct habitats: open-water epilimnion and the nearshore littoral zone. The littoral habitat is the more heterogeneous of the two in terms of spatial and temporal complexity. The seasonal presence of macrophytic plants (algae and vascular plants) and proximity of planktonic and benthic habitats provide important aspects of physical complexity to the littoral zone. Additionally, temperatures ﬂuctuate more rapidly in shallow water, and wave turbulence is greater. Oxygen is rarely limiting nearshore (except, sometimes in eutrophic lakes), but the effects of ice formation can be more severe. The majority of lentic research has dealt with the pelagic zone. Biological interactions in the two planktonic habitats are different in response to those spatial and temporal differences and to variance in bases of the two food webs. Pelagic plankton rely on nutrients regenerated by lake turnover, offshore transport, and internal recycling; photosynthesis is exclusively by phytoplankton. Littoral plankton derive nutrients from the same three sources, but they have ﬁrst access to allochthonous carbon and nutrients. They can also gain energy and recycled nutrients from the benthos on a continual basis (not possible for pelagic zooplankton except during lake turnover). Phytoplankton, periphyton, and macrophytes provide photosynthetic products to support the invertebrate assemblage of the productive littoral zone. Predator – prey relationships and zooplankton behaviors vary between pelagic and littoral zones. Openwater copepods, cladocera, mysids, and a few rotifers migrate vertically on a diel basis. Fewer littoral zoo- 35 plankton migrate, and among those that do, a typical pattern is for offshore movement at dusk and inshore migration at dawn. Ecologists believe that an important cause for both horizontal and vertical migration is predator avoidance. The types of predators in the two habitats are considerably different. Aquatic insects in general are of minor importance in the pelagic zone, but insect predators, such as dragonﬂies and predaceous beetles, are extremely abundant nearshore. Although predaceous adult and larval ﬁsh are usually more numerous in shallow, vegetated regions, the greater habitat complexity of the littoral habitat reduces foraging efﬁciency relative to open-water regions. Microcrustacean predators are present in both habitats. 3. Littoral and Profundal Benthos Benthic invertebrates are often divided into three arbitrary size classes based on the mesh dimensions of the sieve used to process the samples. Macroinvertebrates are retained by coarse sieves ( 200 m mesh) and are usually sorted with No. 60 or 35 U.S. Geological Sieves (250 and 500 m mesh, respectively). Meiofauna, which are plentiful but rarely identiﬁed, need to be collected with ﬁne sieves ranging from 40 to 200 m mesh (usually 100 m); microbenthos pass even these minute openings. The effort required to process benthic samples, especially from vegetated and / or silty habitats, increases tremendously with diminishing mesh size; consequently, species smaller than macrofauna are often ignored. While these size categories in themselves have no natural ecological or taxonomic signiﬁcance, the frequent exclusion of the smaller forms from ecological analyses may have severely inﬂuenced our perception of how benthic assemblages function. For example, Strayer (1985) estimated that the micro- and meiobenthic animals contributed 68% of the species, 98% of the individuals, 25% of the biomass, and 35% of the production of the zoobenthos in Mirror Lake, New Hampshire. Studies in Mirror Lake revealed that over half of the benthic micro- and meiofauna live in the top centimeter of sediments (Strayer, 1985, 1986). Most freshwater benthos reach their maximum densities and diversity in shallow water and decline perceptibly with increasing depth in the profundal zone. Few macroinvertebrates tolerate conditions in the profundal zone beneath the seasonal thermocline, but micro- and meiofauna can be abundant in deep water. This pattern probably reﬂects gradients of oxygen availability, habitat heterogeneity, and food resources — all of which are greater in the littoral zone. Studies of vegetated and nonvegetated regions of the littoral zone demonstrate the great value of macrophytes in reducing predation rates on benthic macrofauna (e.g., Hershey, 1985). This refuge is especially crucial because benthic 36 J. H. Thorp and A. P. Covich animals are generally poor swimmers and have difﬁculty escaping highly motile predators. A variety of distinctive habitats are available for benthic invertebrates. Nematodes, gastrotrichs, ﬂatworms, and other meiobenthos frequent infaunal habitats where they move between sediment particles (Chapters 6 – 9). Those living on or among sand grains are called psammon or interstitial organisms. Macroinvertebrates, such as freshwater mussels, oligochaetes, and some crayﬁsh, regularly burrow in the substrate. Many motile and sedentary species live on the surface of the mud, rocks, or submerged plant debris. Smaller animals and algae that live on ﬁrmer substrates are sometimes called aufwuchs. In addition to the attached aufwuchs, macrophytes support some larger, motile invertebrates which either feed on the aufwuchs (e.g., grazing snails; Chapter 10) or use the plant stems and leaves as perching sites for resting or foraging (e.g., sprawling dragonﬂy nymphs; Chapters 17 and 18). C. Wetlands, Ephemeral Ponds, and Swamps For many years the public has ignored or labeled as undesirable the vast acreage of wetlands, ephemeral ponds, and swamps in North America, considering them breeding grounds for mosquitoes and snakes. Many have been drained, ﬁlled, and bulldozed for housing and commercial development without regard to their intrinsic value to wildlife and the environment. Our knowledge of these fascinating aquatic ecosystems is relatively limited in comparison to information available about lakes and streams. Interestingly enough, one factor that has begun reversing this trend has been the implementation of strict environmental laws to protect the broad category of wetlands. Such laws have also led to an inﬂux of research funds to study these ecosystems which have spawned a huge variety of journal articles (e.g., Zimmer et al., 2000), books (e.g., Batzer et al., 1999; Mitsch and Gosselink, 2000), and even a new journal (Wetlands). In its broader deﬁnition, the term freshwater wetlands refers to nontidal ecosystems whose soils are saturated with water on a permanent or seasonal basis. Emergent aquatic vegetation is prominent and may alternate with annual terrestrial plants in these marshy, or palustrine, habitats. The extensive biomass of trees, herbaceous vegetation, grasses, and other plants in many wetlands is responsible for their recently recognized abilities to help ﬁlter pollutants from the environment. Wetlands vary from shallow, seasonally ephemeral ponds (such as Carolina bays and other pocosins, Sharitz and Gibbons, 1982) to relatively permanent vegetation-choked marshes to semi-lotic alluvial swamps (Fig. 10; see also Mitsch and Gosselink, 2000). Because wetlands are generally more ephemeral than other lentic ecosystems, their biotic communities are strongly inﬂuenced by water cycles. Wetlands invertebrates are affected by ecosystem permanency (e.g., years since last exposure), predictability of drying, and season of drying (if at all). The nature of the community also reﬂects volume and depth of open water and sometimes current velocity (for alluvial swamps). See Batzer et al. (1999) for habitat speciﬁc information on invertebrate ecology from most types of freshwater wetlands in North America. Invertebrates of seasonal wetlands, such as the Carolina bays of the southeastern United States, have evolved various characteristics that have directly or indirectly adapted them for ecosystems that alternate between aquatic and terrestrial states. Wiggins et al. (1980) divided animals of temporary pools into four groups based on their adaptations for tolerating or avoiding drought and their period of recruitment. “Group 1 are year-round residents incapable of active dispersal, which avoid desiccation either as resistant stages or by burrowing into pool sediments. Group 2 are spring recruits which must oviposit on water but subsequently aestivate and overwinter in the dry basin in various stages. Group 3 are summer recruits ovipositing in the dry basin and overwintering as eggs or larvae. Group 4 are nonwintering migrants leaving the pool before the dry phase, which is spent in permanent water; they return in spring to breed.” When a temporary pool reﬁlls after a drought, the ﬁrst colonizers are usually detritivores which exploit the abundant biomass of recently dead and decaying terrestrial vegetation; these species provide the animal tissue that supports the later arrival of predaceous invertebrates (Wiggins et al., 1980). Many species in ephemeral environments are ecological generalists, because they live in both temporary and permanent aquatic ecosystems. Alluvial swamps are at an opposite continuum from temporary wetland pools. Contrasting with the typical picture of a swamp as a stagnant marsh, alluvial (or riverine) swamps contain many areas of slowly to rapidly moving waters; portions of these wetlands somewhat resemble a highly braided stream. While it is true that southeastern alluvial swamps, such as those bordering the Savannah River between South Carolina and Georgia, are infested with alligators and poisonous snakes, they are otherwise extremely beautiful ecosystems with very few aerial insect pests once you have moved inward from the surrounding brush and terrestrial forest. Alluvial swamps are extremely heterogeneous, organically rich environments; oxygen does not appear to be limiting, at least in the shaded, ﬂowing water regions. 2. An Overview of Freshwater Habitats A B FIGURE 10 (A) Photograph of a low-ﬂow portion of a bald cypress — water tupelo, alluvial swamp of the Savannah River. Floating platforms in center are suspending wood snags as part of a colonization study by Thorp et al. (1995). (B) Small wetland area in northern New York created by a beaver dam. Shown are cattails (foreground), open water covered by duckweed (middle), and various emergent vegetation and woody plants (photographs by J. H. Thorp). 37 38 J. H. Thorp and A. P. Covich The many soft- and hard-sediment habitats of swamps are home to a great diversity and density of aquatic invertebrates. In a study of an alluvial swamp in South Carolina, Thorp et al. (1985) showed that invertebrates rapidly colonized wood snags, reaching a rough steady state in numbers within the ﬁrst week of an 8-week study. Peak densities were equivalent to nearly 18,000 animals/m2! “Filter-feeding taxa were numerically dominant early but soon were subordinate to gatherer and scraper functional feeding groups. Current velocity, seston particle size [as a food resource], dispersal capacity and competition for space may be important factors affecting community structure and colonization patterns in these aquatic ecosystems” (p. 56). D. Hypersaline Lakes Natural saline lakes are common worldwide but generally have neither the average size nor abundance of freshwater lakes (Eugster and Hardie, 1978; Hammer, 1984). They are frequently encountered in the prairies and the Great Basin of the western United States and in the provinces of Alberta, Saskatchewan, and British Columbia of western Canada (Hammer, 1984). The Great Salt Lake of Utah is the largest saline lake in North America. Saline lakes usually occur in more arid regions where closed basins promote high concentrations of salts (Fig. 11). These lentic ecosystems include saltern lakes, which are high in sodium chloride; lakes with large concentrations of sulfates and borates; and soda lakes characterized by abundant sodium carbonates and bicarbonates. Salinities in the Great Salt Lake have been recorded as high as 200 g/L (versus an average of 35 g/L in the world’s oceans). In addition to high salinities, hypersaline lakes differ in pH, metal concentrations, and alkalinity from more typical inland lakes. The abiotic characteristics of saline lakes make them extremely rigorous environments. Cyanobacteria colonize saline lakes as do some sedges, but the submerged macrophyte ﬂora is relatively sparse in comparison to that in freshwater lakes. Because invertebrate density and diversity are enhanced by abundant littoral plants, the depauperate ﬂora of saline lakes and the lack of certain other microhabitats undoubtedly depress the invertebrate fauna of briny pools. Few invertebrates of inland waters can regulate osmotic and ionic concentrations of their tissues in a hypertonic environment. As a result, hypersaline environments contain very few species, although their densities may be high. For example, the only permanent metazoa in Mono Lake, California (Fig. 11), are the ubiquitous brine shrimp, Artemia salina, and the brine ﬂy genus Ephydra (see also Herbst, 1999). High densities of brine shrimp in Mono Lake are crucial to survival of FIGURE 11 Saline Mono Lake in arid central California. The light “rocks” in the foreground are actually calcium carbonate mounds known as “tufa”; many of these arise within the lake proper. Visible on the island in the background is a small volcanic cone (photograph by J.H. Thorp). 2. An Overview of Freshwater Habitats migratory eared grebes, Podiceps nigricollis, which molt ﬂight feathers while at Mono Lake, depending on these grazing crustaceans as their sole food source (Cooper et al., 1984). This simple but important food chain is threatened by the possible demise of the brine shrimp as a result of rising salinities brought on by removal of freshwater from the Mono Lake catchment to provide water for the human population in Southern California. Other saline lakes support more species, including the rotifer Brachionus plicatilis, the calanoid copepod Diaptomus nevadensis, and several families of ﬂies, beetles, and true bugs. As saline lakes become less salty, the diversity and density of their ﬂora and fauna are enhanced. For example, the meromictic Waldsea Lake of Saskatchewan, which is one of the better studied saline ( 3 ppt total dissolved solids) inland lakes in Canada, has a much higher species diversity than the much saltier Mono Lake. It contains two macrophyte species, some ﬁlamentous algae, a sparse phytoplankton population, and a dense but rather uniform population of zooplankton, dominated by the calanoid copepod Diaptomus connexus, the rotifers Hexarthra fennica and Brachionus plicatilis, and the water ﬂea Daphnia similis (Hammer, 1984). The littoral zone supports high numbers of relatively few species, including several species of true bugs, nine genera of beetles, midges (mostly Cricotopus), a few caddisﬂies, the damselﬂy Enallagma clausum, one snail species (Lymnaea stagnalis), and several genera of crustaceans (see references in Hammer, 1984). Although Waldsea Lake is certainly diverse compared to the hypersaline Mono Lake, its community is depauperate in comparison to most permanent, freshwater lentic environments. The inland waters of North America are sufﬁciently diverse and abundant to prevent the valid formation of any simple, all-encompassing theory on how biotic communities of these aquatic ecosystems are regulated. This does not imply, however, that we should abandon the task of assimilating and evaluating data from a diversity of ecosystems in order to understand better the effects of a few identiﬁable factors, such as the inﬂuence of habitat characteristics. A comparative approach, combined with intensive descriptive and experimental studies, will in the long run enhance our ability to develop a holistic view of stream and lake ecosystems. 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Sanders Department of Biology University of Waterloo Waterloo, Ontario N2L 3G1, Canada Department of Biology Temple University Philadelphia, Pennsylvania 191ZZ I. Introduction II. Anatomy and Physiology A. Protozoa as Cells and Organisms B. Protozoan Organelles C. Anatomy of Flagellates D. Anatomy of Amoeboid Protozoa E. Anatomy of Heliozoa F. Anatomy of Ciliates G. Environmental Physiology III. Ecology and Evolution A. Diversity and Distribution B. Reproduction and Life History C. Ecological Interactions D. Evolutionary Relationships IV. Collecting, Rearing, and Preparation for Identiﬁcation I. INTRODUCTION Protozoa are ubiquitous; they are present in an active state in all aquatic or moist environments, and cysts are present everywhere in the biosphere, ready to give rise to active populations. They play an important role in many natural communities, although they are often overlooked. In the laboratory, protozoa often play the role of model organisms; cell biologists, physiologists, geneticists, and even developmental biologists frequently turn to protozoa to address questions that would be more difﬁcult to answer with metazoa, but in the hope that the answers are relevant to metazoa. The result is a surprising amount of information about a relatively few species. Accordingly, detailed monographs are available on several taxa, including Blepharisma (Giese, 1973), Paramecium (Wichterman, 1986), Tetrahymena (Elliott, 1973) and Amoeba (Jeon, 1973). Each monograph contains thousands of references. Ecologists also have used protozoan populations and communities as Ecology and Classiﬁcation of North American Freshwater Invertebrates. 2nd Edition Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved. V. Identiﬁcation of Protozoa A. Notes about the Key B. Taxonomic Key to Functional Groups of Protozoa C. Taxonomic Key to Major Groups and Common Genera of Flagellated Protozoa D. Taxonomic Key to the Major Groups and Common Genera of Protozoa with Pseudopodia E. Taxonomic Key to Major Groups and Common Genera of Ciliates Literature Cited model systems to investigate processes, such as competition and predation, or to test models, such as island biogeography, that would be much more difﬁcult to study using larger organisms. Again, the result has been an extensive body of literature that is largely restricted to a few species and/or to unusual or artiﬁcial habitats. In the last 20 years, there has been an explosion of information about the ecology of free-living protozoa, especially the planktonic species that were virtually ignored in the earlier literature. This increase in interest followed the development of direct-count methodologies for aquatic bacteria, and an appreciation of the importance of bacteria and their predators in aquatic systems. The ﬁrst focus was on the heterotrophic ﬂagellates and ciliates that consumed bacterial production and passed it to larger consumers, the “microbial loop.” As aquatic ecologists came to realize that protozoa are not only present, but play a major quantitative role in material and energy ﬂow, this interest expanded to other feeding guilds and freshwater environments. 43 44 W. D. Taylor and R. W. Sanders The goal of this chapter is to provide an introduction to the ecology and classiﬁcation of protozoa for ecologists who wish to include them in studies of aquatic environments. The chapter focuses on the biology of the animal-like protozoa, largely ignoring the related pigmented organisms which are covered in phycological sources. The chapter is oriented toward the organisms, not the freshwater habitats themselves. Readers wishing to learn about the fauna of particular habitats might consult the bibliography on freshwater protozoa prepared by Finlay and Ochsenbein-Gattlen (1982) or some more recent works on particular habitats: lake plankton (Beaver and Crisman, 1989; Laybourn-Parry, 1992; Riemann and Christoffersen, 1993; Carrias et al., 1996 Foissner et al. 1999), streams (Bott and Kaplan, 1990; Carlough and Meyers, 1991; Sleigh et al., 1993), and wetlands (Pratt and Cairns, 1985; Decamp and Warren, 1999). What are protozoa? Protozoa are unicellular or colonial eukaryotes, including all of the heterotrophic and motile ones. It is a taxon of convenience. Although the protozoa were considered to be a single phylum of eukaryotic animals in early classiﬁcations, modern treatments distribute the protozoa among many phyla. This reﬂects the immense diversity of this assemblage, and the fact that differences among these unicellular organisms are at least as profound as those which separate plant and animal phyla (see Section III.D). Margulis and Schwartz (1988), in their ﬁve-kingdom classiﬁcation of living organisms, place the unicellular eukaryotes and some of their multicellular descendants, 27 phyla in all, in the kingdom Protoctista. Other modern classiﬁcations partition the protists into more or fewer phyla. The classiﬁcation of unicellular eukaryotes is in a state of ﬂux, with much work to be done. In any event, it is among these groups that the organisms we know as protozoa are scattered. For the purpose of this chapter, we deﬁne the term protozoa as those unicellular or colonial eukaryotes that are heterotrophic, and will restrict ourselves to a discussion of free-living, phagotrophic forms in freshwater. Among the protozoan phyla we will not discuss here are the marine Foraminifera and Radiolaria, the soil-dwelling Labyrinthulamycota, Plasmodiophoromycota and Acrasida, the saprophytic Hyphochytridiomycota, Oomycota and Chytridiomycota, and the parasitic Apicomplexa, Microspora, and Myxospora. The classiﬁcation we use, and citations to ﬁgures illustrating the major taxa, are outlined in Table I. It is designed to be as current as possible, but still retain the older names. Therefore, it emphasizes classes, subclasses, or orders, largely according to which retains the most familiar name for the group in question. Our major TABLE I Major Taxomomic Categories of Protozoa as Used in This Chapter Functional Group Phylum Class subclass Order Phytoﬂagellates (largely autotrophic, but some mixotrophic and heterotrophic) Cryptophyta Dinoﬂagellata Euglenida Chrysophyta Chlorophyta Zooﬂagellates (heterotrophic) Zoomastigina Choanoﬂagellida Kinetoplastida bicoecids diplomonads cercomonads Ameboid Protozoa Karyoblastea Rhizopoda Heterolobosea Schizopyrenida Lobosea Gymnamoebia Euamoebida Leptomyxida Acanthopodida Testacealobosia Arcellinida Himatismenida Filosea Aconchulinida Testaceaﬁlosida Granuloreticulosida Athalamea Monothalamea Foraminiferea (marine only) Actinopoda Heliozoa Actinophryida Desmothoracida Ciliophryida Centrohelida Rotosphaerida Ciliated Protozoa Ciliophora Karyorelictea Heterotrichea Spirotrichea Hypotrichia Choreotrichia Stichotrichia Oligotrichia Odontostomatida Armophorida Litostomatea Figure reference 13J 13A – C 13D, F – I 13K – N; 14A 13E 14B – H 14J, L – P 14I 14M 14K 16V 15A – C 15D – M,O,P 15N 16B – Q 16A 16R, 17A 16S – W; 17B – K 17P – R 17L – O 18B,C 18A 18E 18D,F,H 18G 23J 22A,B,D – F 20X,Y 21S,T,U,X 21A – I,N – Q 21V,W 21,J,M,L 21K,R 22G,I,K,L,N,P – W; 23D – I (Continues) 3. Protozoa 45 TABLE I (Continued) Functional Group Phylum Class subclass Order Phyllopharyngea Phyllopharyngia Suctoria Nassophore Nassulida Microthoracida Colpodea Prostomatea Prostomatida Prorodontidae Plagiopylea Oligohymenophorea Peniculia Scuticociliatia Hymenostomatia Peritrichia Figure reference 24T 19A – P 20Z 20V,W 22C; 23K; 24N,P – S 22H,J,O 23A – C 24M 24A,B,D,E,G,I,J 23L – X 24 C,F,H,O 20 A – U points of reference were Page (1998), Lynn and Small (1997), and various chapters in Lee et al. (1985) and Margulis et al. (1989). Despite the fact that the term protozoa is perhaps not a proper taxonomic group, it remains a useful functional one. For ecologists conducting studies involving aquatic animals, including those of microscopic proportions, it is natural to include the animal-like protozoa in those studies. After all, it is functional similarity that generally concerns the practising zoologist or ecologist. II. ANATOMY AND PHYSIOLOGY A. Protozoa as Cells and Organisms Large protozoa that have many nuclei, or large, amitotic, polyploid nuclei, may be best thought of as acellular rather than unicellular creatures. Nonetheless, some species have been studied extensively by cell biologists as model cells, and ecologists interested in protozoa can glean much useful information from their labors. This section will rely heavily on information from those few taxa. We will ﬁrst describe some of the organelles which are important and widely distributed among protozoa, then discuss major groups of protozoa with respect to their more unique organelles. Lastly, we will revert to an overall view in discussing environmental physiology of protozoa. In all sections, we will emphasize those organelles that relate to FIGURE 1 The formation and fate of food vacuoles, autophagic vacuoles, and pinocytotic vacuoles in Tetrahymena pyriformis. (From Elliott and Clemmons, 1966.) feeding, locomotion, ecology, and morphology at the light-microscope level. B. Protozoan Organelles 1. Food Vacuoles Phagocytosis in protozoa leads to the formation of food vacuoles (Fig. 1). Digestion is, as one would expect, primarily intracellular in free-living protozoa. The formation and behavior of food vacuoles is best known in the ciliates Tetrahymena (see Rasmussen, 1976; Nilsson, 1976, 1987) and Paramecium (Allen, 1984). A more general review is provided by Nisbet (1984). The formation of food vacuoles is stimulated by the presence of particulate food. In protein broth medium, Tetrahymena shows very slow rates of food vacuole formation, ingestion, and growth unless particles are present (Ricketts, 1972), even though it can grow without forming food vacuoles in complex media via carrier-mediated uptake of dissolved compounds across the plasma membrane. During starvation, vacuoles resembling food vacuoles may be created to digest cellular components. Formation of such “cytolosomes” is also known from Amoeba (Chapman-Andresen, 1973). 46 W. D. Taylor and R. W. Sanders Many protozoa have a ﬁxed site of food-vacuole formation; a cytostome with accompanying structures to aid in ingestion. In others, for example, Rhizopoda and Heliozoa, the site of ingestion is ﬂexible, perhaps only limited to a characteristic region of the cell. The Suctoria have many sites for ingestion. The contents of a single vacuole may vary from a single large food item in macrophagous species to thousands of small items, such as bacteria, in microphagous forms. Similarly, individual protozoa may have one-to-many food vacuoles. The vacuoles of microphagous forms may be perfectly spherical, while larger vacuoles containing single prey may be quite irregular, conforming to the contours of the prey. Macrophagous protozoa may distend themselves to surround large food items, leave them protruding from their cytoplasm, or even share a prey item among several individuals. Large food items may be digested by cytoplasmic extensions in dinoﬂagellates and possibly other protozoa (Fig. 2). Digestion may be initiated by the toxicysts of some predatory ciliates. In others, it begins when granules (acidosomes) fuse with the developing food vacuole. The pH of the new vacuole drops precipitously, then increases minutes later. Hydrolytic enzymes, such as FIGURE 2 Phagocytosis of large food items by protoplasmic extensions in the dinoﬂagellate Ceratium hirudinella (A – C), and the consumption of a large diatom by a Paraphysomonas-like chrysophyte (D). (A – C from Hofeneder, 1930: D after Suttle et al. 1986.) acid phosphatase, are introduced as the food vacuole fuses with lysosomes. The size of the food vacuole may diminish, reﬂecting the absorption of water and nutrients, and vacuoles may coalesce. Some protozoa, such as ciliates, have a ﬁxed site of egestion or cytoproct. When the contents of the food vacuole are released, the membrane surrounding the vacuole is retained and recycled (Allen, 1984). It is unlikely that a cellular equivalent of a digestive track exists in protozoa. Nevertheless, it has been possible to estimate the feeding rate of protozoa in the ﬁeld from the rate at which food vacuoles are eliminated (Goulder, 1972). 2. Contractile Vacuoles These organelles are widely distributed in protists and are almost universal in freshwater protozoa and sponges. They are absent in dinoﬂagellates and are generally lacking in those protists with rigid cell walls and/or from marine or endosymbiotic habitats. A large body of evidence suggests that contractile vacuoles eliminate water and, thereby, counteract the tendency of naked protists to swell in freshwater (Patterson, 1980). The activity of the contractile vacuole is correlated with changes in the osmotic balance between the cell and its medium, and a loss of contractile function results in swelling of the cell. Of course, the occurrence of contractile vacuoles in naked, freshwater protists and their absence in most rigid-walled, marine or endosymbiotic ones also support this theory. Associated with the contractile vacuole is a specialized cytoplasm, the spongiome, which collects the solute to be expelled by contractile vacuole. The spongiome may contain vesicles or canals visible with the light microscope, and therefore be of diagnostic value (Fig. 3). Patterson (1980) recognized four types of contractile vacuole complexes in protozoa, with six different patterns of behavior. Permanent contractile vacuole pores are present in ciliates and are readily visible in silver-stained specimens. The contractile vacuole may have a ﬁxed location in some other groups, for example, in the gullet or reservoir of cryptomonad or euglenoid ﬂagellates, but the ﬁxed pores, if they exist, are not visible. Several important functional aspects of the contractile vacuole complex remain unknown; for example, the composition of the ﬂuid expelled, and, therefore, the role of the contractile vacuole complex in salt balance and excretion. It is also debatable whether expulsion of contractile vacuole contents is active or passive, and how the activity of the contractile vacuole is controlled. Dinoﬂagellates possess a functionally homologous organelle, the pusule. This organelle consists of a single 3. Protozoa 47 FIGURE 3 Top views of three morphologies of contractile vacuoles found in protozoa, showing their cycles of activity from left to right as seen with a light microscope. (A) A vacuole which forms by the coalescing of smaller vesicles, as is common in Rhizopoda. Vacuole (B) has ampullae in ﬁxed locations, while vacuole (C) also has visible canals. (After Patterson, 1980.) chamber or tubule associated with a ﬂagellar opening, and it is lined with vesicles (Dodge and Gruet, 1987). 3. Cilia and Flagella Flagella, cilia, and derived structures are widely distributed in eukaryotes, including protozoa. Their ultrastructural similarity, including the basic 9 pairs 2 pattern of microtubules in the shaft, or 9 triplets in the base, indicates homology despite the great diversity in form and function. Many animal sense organs contain cilia, which emphasizes that they are probably intrinsically sensory as well as motile. Cilia or ﬂagella which are thought to be sensory occur in many protozoa, especially the “brosse” kinetids of prostomateans, and the ﬂagellum of phototactic euglenids. It is well established that the motile force in cilia and ﬂagella is the sliding of microtubules in the shaft against each other. Therefore, the active movement is the bending of shaft and not the movement of shaft relative to the base. The bending of shaft takes on the appearance of a beat or stroke in short cilia or a wave in long ﬂagella. Flagella usually occur in pairs and are relatively long (often longer than the cell that carries them, and reaching lengths of up to 50 m). A major functional and morphological dichotomy among ﬂagella is between those with and without mastigonemes (Fig. 4). Mastigonemes are thin, hairlike projections perpendicular to the shaft of the ﬂagellum. They are not visible with light microscopy, but are functionally important. A distally directed wave of bending in a smooth ﬂagellum will push the cell in the opposite direction to which the ﬂagellum is pointed, as in animal sperm. The same wave form in a ﬂagellum with mastigonemes will pull the cell in the direction in which the ﬂagellum is pointed. To understand the motility of protozoa, especially those using cilia and ﬂagella, it is necessary to appreciate that the apparent viscosity of water increases as the size of organisms moving through it decreases (Holwill, 1974; Fenchel, 1987). To ﬂagellates, water is a liquid so viscous that they sink only very slowly and they not glide from inertia. The difference between cilia and ﬂagella is one of form and function. Cilia are generally short and densely packed. They are organized in parallel rows (kineties) to produce ﬁelds of cilia whose activities are coordinated (Fig. 5A). The movement of adjacent somatic cilia is not quite synchronous; rather, each is slightly out of phase with its neighbors, or metachronous. A ﬁeld of cilia, therefore, moves in waves that may or may not correspond in direction to the actual power stroke of the cilia. Compound ciliary organelles or polykinetids are restricted ﬁelds of cilia acting as a single unit in locomotion, as in the cirri of hypotrichs (Fig. 20Y), or feeding, as in the oral apparatus of hymenostomes (Fig. 5B). They may be relatively long compared to other cilia and fused so that their compound nature is not obvious under light microscopy, except as indicated by their diameter. Ciliary movement is faster than ﬂagellar movement, even accounting for ciliates generally being larger than ﬂagellates (Sleigh and Blake, 1977). Indeed, within both these groups there is relatively little increase in absolute swimming speed with body size; 48 W. D. Taylor and R. W. Sanders FIGURE 5 The distribution of cilia on Tetrahymena (A). The compound ciliary organelles associated with the mouth (B) are used to collect bacteria into the forming food vacuole. Somatic cilia beat metachronously to propel the cell (C). (D) The path of a single cilium viewed from the side, showing the power stroke and the recovery stroke in different planes. FIGURE 4 Different uses of ﬂagella in two sessile bacterivores, and two swimming forms. Arrows indicate movement of water relative to the cells. Note that distally directed waves of beating produce opposite forces in smooth ﬂagella and ﬂagella with mastigonemes. (A) The choanoﬂagellate Monosiga, modiﬁed from Fenchel (1982a); (B) Actinomonas, which is probably a heliozoan, also modiﬁed from Fenchel; (C) a dinoﬂagellate; (D) a chrysophyte. ﬂagellates average about 0.2 mm/s, while ciliates average about 1 mm/s. 4. Extrusomes Extrusive organelles or extrusomes are membranebound organelles associated with the pellicle of protists and containing material which can be ejected or extruded from the cell. Most are visible at the lightmicroscope level, at least in the extruded state. They are widespread in occurrence, and diverse in structure and function; they are probably not homologous. The function of many is in doubt (Haackbell et al., 1990). However, following Hausman (1978) we will group them according to their functional and structural similarity for discussion, omitting those restricted to marine groups. Spindle trichocysts of nassophorean ciliates, especially those of Paramecium, are well known. They are abundantly distributed over the cortex (e.g., 8000 per cell in Paramecium) and each may discharge a pointed projectile on a proteinaceous shaft in response to mechanical or physical stimulation. These are probably effective deterrents to some potential predators (Harumoto, 1994) although specialists, such as the predatory ciliate Didinium, are undaunted (Fig. 6). Expulsion of the trichocyst is extremely rapid and is driven by a change in the paracrystalline structure of protein in the shaft of the trichocyst. The increase in length of the shaft following expulsion is approximately eightfold. Extrusomes are found in several ﬂagellate groups; most dinoﬂagellates have spindle trichocysts similar to those of ciliates. Until recently, these groups were not considered to be even remotely related. However, recent studies of ribosomal RNA suggest they are less distant than one might suppose based on gross morphology (Gunderson et al., 1987). Ejectisomes are found in most 3. Protozoa FIGURE 6 (A) Capture of Paramecium by Didinium. Note the use of toxicysts by Didinium, and the spindle trichocysts released by Paramecium in response. (B) The oral apparatus and zone of contact, as revealed by electron microscopy. Note that there are two varieties of extrusomes involved in Didinium’s attack, and that the deciliation of Paramecium is part of the result. (C) The ultrastructure of spindle trichocysts of Paramecium in the resting (left) and discharged (right) state. (D)Ultrastructure of Didinium pexicysts (left) and toxicysts (right). Both are shown in resting and discharged states. c; Capsule. ci; cilia. olm; outer limiting membrane p; discharged pexicysts. pr; protuberance of Paramecium cytoplasm. t; discharged toxicyst. tc; terminal cap. tr; discharged trichocyst. (B and D are from Wessenberg and Antipa, 1970; C is from Hausman, 1978.) 49 50 W. D. Taylor and R. W. Sanders FIGURE 7 Examples of extrusomes in other protozoa. (A) Kinetocysts from the axopodia of the heliozoan Hetrophrys (from Lee et al., 1985). (B) Discobolocysts from the cell surface of the chrysophyte Ochromonas, and the steps from formation (1) to discharge (5). (C) Ejectisomes from the gullet of Cryptomonas. a; undischarged. b-d; discharging, showing uncoiling in two directions and rolling of the uncoiled ejectisome into a tube. e; discharged ejectisome. (D) Haptocysts from the tentacles of a suctorian. Arrows indicate the location of the extrusomes to the right. Extrusomes in A – C are from Hausman (1978). Haptocysts in D are drawn from Rudzinska (1965). 3. Protozoa cryptomonads and in a few other phytoﬂagellates (Fig 7). They are typically dimorphic, although of similar morphology; larger ejectisomes are associated with the gullet, while smaller ones are external to the gullet and near the anterior of the cell. Because some cryptophytes are heterotrophic, it is tempting to speculate that the ejectisomes are used in food capture. However, the gullet is not the site of phagotrophy, so an offensive role of these extrusomes is doubtful. The mechanism of expulsion of ejectisomes is very different from that of spindle trichocysts; the nonexpelled ejectisome is a tight coil of ribbonlike material which uncoils and rolls into a tube on expulsion. A similarly coiled secondary structure forms a pointed tip. Discobolocysts are also extrusomes of ﬂagellates, in this case primarily of chrysophytes. They are almost spherical, rather than spindle-shaped, but their expulsion is reminiscent of spindle trichocysts; the shaft of the discobolocyst elongates, apparently through a conformational change in the material in the shaft (Fig. 7). Although their function is unknown, it is possible that they discourage phagotrophs. Extrusomes are used for capturing food by predatory ciliates in the classes Prostomatea and Litostomatea, which have toxicysts, and Phyllopharyngea, which have haptocysts. Toxicysts are extruded by eversion, and in doing so release material which appears to be toxic to the prey. Several types of toxicysts are known, and up to three kinds may be found within a species. The use of toxicysts by Didinium in capturing Paramecium is illustrated in Figure 6. Although the toxicysts of Didinium are concentrated on the proboscis, they may be elsewhere in other ciliates. Loxophyllum (Fig. 23I) has them in clusters along its lateral margin, while Actinobolina (Fig. 22G) has toxicysts on tentacles that are widely distributed over the body. Haptocysts are found in the tips of the feeding tentacles of Suctoria (Fig. 19). Prey (mostly other ciliates) which touch the feeding tentacles are caught and held by the extrusion of the haptocysts (Fig. 7) which penetrate the prey, inject substances (probably including hydrolytic enzymes) and aid in the fusion of membranes of the predator and prey. The cytoplasm of the prey is then drawn into the suctorian through the feeding tentacles. Some Heliozoa possess extrusomes of various types, which may give the elongate pseudopodia (axoopodia) a bumpy appearance (Fig. 7). Kinetocysts resemble haptocysts or small trichocysts. Extrusomes in Heliozoa also include mucocysts (see below) and electron-dense bodies that also expel amorphous material onto the cell surface. All probably facilitate prey adhesion and capture. Mucocysts are widespread in ciliates and ﬂagellates. They secrete an amorphous material onto the cell 51 surface that, at least in many species, is involved in cyst formation. Tetrahymena can use mucocysts to create a temporary capsule in response to introduction of a dye; this is probably a defensive reaction, although it is not known which other stimuli cause this response. Wolfe (1988) suggested that mucocysts may be the protozoan’s ﬁrst line of defense. Mucocysts may also be involved in nutrition; the mucous secreted may be reingested after attracting dissolved compounds or ﬂocculating small food items (see Section III.C). Euglenoid ﬂagellates have larger muciferous bodies in addition to mucocysts, although some authors do not differentiate between the two. These provide a continuous coat of mucilage on the surface of the cell and, in some species, participate in cyst formation, adhesion to the substratum, or stalk formation. C. Anatomy of Flagellates The term ﬂagellates is applied to protists with oneto-many ﬂagella. In free-living taxa, as opposed to parasitic species, the number of ﬂagella is limited; Paramastix has two rows of 8 – 12 ﬂagella, but most others have 1 – 4 (usually 2). There are several groups of heterotrophic ﬂagellates in freshwater: choanoﬂagellates, kinetoplastids, diplomonads, cercomonads, and bicoecids. These are raised to phyla by some authors, while bicoecids are occasionally classiﬁed with chrysophytes. Some Rhizopoda, the schizopyrenids or amoeboﬂagellates, also have ﬂagella. Other groups of ﬂagellates contain mostly or entirely autotrophic forms with chloroplasts. However, many of the pigmented, autotrophic taxa are capable of phagotrophy, a condition referred to as mixotrophy (Sanders, 1991). Among these autotrophic and mixotrophic groups there are nonpigmented, wholly heterotrophic species. The groups with many mixotrophic or heterotrophic taxa include cryptophytes, chrysophytes, dinoﬂagellates, and euglenoid ﬂagellates. These groups are usually considered as phyla. The functional signiﬁcance of mixotrophy varies widely. Different species use the ability to phagocytose other cells to supplement photosynthesis in low light, to gain access to nutrients, or to survive long periods in the dark (Jones, 1997). In keeping with our desire to cover the animal-like protists, we will survey heterotrophic and mixotrophic ﬂagellates, ignoring primarily autotrophic groups. Pigmentation and chloroplast morphology are important taxonomic characters for some groups, but here we will emphasize features found in heterotrophs. Choanoﬂagellates, or collared ﬂagellates, are distinctive for the collar that surrounds the single ﬂagellum (Figs. 4, 14C–H). They bear a strong resemblance 52 W. D. Taylor and R. W. Sanders to choanocytes of Porifera. The collar may be difﬁcult to distinguish with light microscopy; but when examined with electron microscopy, it is evidently composed of microvilli. The microvilli intercept bacteria drawn past the cell by the ﬂagellum. Because most choanoﬂagellates are attached or colonial, the distally directed waves of the smooth ﬂagellum serve to push water past the protozoan. Bacteria caught on microvilli are transported to the cell and ingested by pseudopodia at the base of the collar (Leadbeater and Morton, 1974). Many choanoﬂagellates attach to the substrate, and many have an external, loose-ﬁtting covering or lorica (although, again, it may be difﬁcult to see with the light microscope). In one marine family (Acanthoecidae), the lorica is basketlike. Bicoecids (Fig. 14I) resemble choanoﬂagellates, although they lack a collar. Like choanoﬂagellates, they are enclosed in a lorica and have a ﬂagellum that is used to create a feeding current. A second ﬂagellum lies along the cell and continues posteriorly to become an attachment to the base of the lorica. Kinetoplastids (Fig. 14J,N – P) are known mostly as parasites, especially Trypanosoma and its relatives, but many members of the suborder Bodina live in freshwater (Vickerman, 1976). They have a unique single mitochondrion which runs the length of the cell and one or more DNA-rich organelles, kinetoplasts, associated with this mitochondrion near the ﬂagella. The best known genus is Bodo, which, like other bodonids, has two ﬂagella (Fig. 14J). One trails, often in contact with the substrate, while the other extends ahead. The trailing ﬂagellum may attach temporarily to the substrate. In the related, but sessile and colonial, genus Cephalothamnium (Fig. 14L), the attachment is permanent. Diplomonads and cercomonads are relatively unimportant groups to the freshwater ecologist; diplomonads are largely parasitic, with a few freeliving forms that may be found in organically enriched water (e.g., Hexamita, Fig. 14M). There is one freshwater cercomonad, Cercomonas (Fig. 14K), and a common soil ﬂagellate, Heteromita. Cercomonas, like the diplomonadida, tends to be found in organically enriched environments. The cryptomonads include many common heterotrophs and autotrophs; mixotrophy has been reported, but is not common. The two ﬂagella are unequal in length and different in appearance (Fig. 7). The longer one has two rows of mastigonemes, while the shorter has only one. These ﬂagella arise from a subapical invagination commonly referred to as a “gullet,” although it does not appear to be the site of ingestion in heterotrophic forms. The contractile vacuole empties into this vestibule. Ejectisomes (see above) may not be visible to the light microscopist unless they are discharged, which they sometimes are after ﬁxation. The pellicle is covered with plates, although these also are not generally visible. The dinoﬂagellates (Fig. 13A – C) are a very large and unique group; they are probably more important in marine than in freshwater environments. Certainly, our knowledge of this group is mostly based on marine studies. Their unique arrangement of ﬂagella — one spirals around the cell in a groove (girdle) and the second is distally directed in another groove (sulcus) — makes them distinctive. Again, heterotrophy and mixotrophy are common (Fig. 2). Because dinoﬂagellates sometimes ingest large prey, their phagotrophic habits have been more often recognized. A covering of plates may or may not be present (hence “armored” and “naked” dinoﬂagellates). Ingestion in some species is assisted by an appendage, the peduncle, which emerges from the sulcus and allows them to subdue prey many times their size (Gaines and Elbrächter, 1987; Jacobson and Anderson, 1986). The trichocysts also assist in prey capture in some forms. Very large prey may be digested by protoplasmic extensions, and this tendency intergrades with ectoparasitism, which is common in this group. Marine and brackish species are noted for the production of toxins. Chrysophytes also contain both colorless heterotrophs and pigmented mixotrophs. Even among the mixotrophs, the degree to which different taxa rely on phagotrophy varies greatly. Chrysophytes are generally small, and their prey are bacteria. They have two unequal ﬂagella: one long and directed anteriorly with rows of mastigonemes; and the other short, smooth, and directed laterally (Fig. 4D, Fig. 13K – M). They are naked or covered with ﬁne siliceous scales which are not always visible with light microscopy; many are amoeboid. Their carbohydrate storage product, chrysolaminarin, occurs in liquid globules and may be useful in recognizing members of this group. Euglenids are generally large ﬂagellates with two ﬂagella, although in many taxa only one ﬂagellum emerges from the gullet (Fig. 13D). Several heterotrophic species creep over the substrate with the second ﬂagellum trailing and hidden beneath the cell (Fig. 15F – H) as in some bodonids and Cercomonas. Indeed, molecular genetic studies indicate that the euglenids may be related to the kinetoplastids, and both may be rather remote from other ﬂagellates (Gunderson et al., 1987). Heterotrophic species may have an ingestatory apparatus consisting of two rods, allowing them to swallow relatively large items. Again, the gullet is not used in ingestion. 3. Protozoa FIGURE 8 Types of pseudopodia. (A) The cylindrical pseudopodia of Amoebidae. (B) The cylindrical, granular, eruptive pseudopodia of Pelomyxidae. (C) The ﬂattened, hyaline, eruptive pseudopodia of some Entamoebidae. (D) The axopodia of Heliozoa. (E) The ﬁliform pseudopodia of some Mayorellidae. (F) The broad, ﬂattened pharopodia of some Mayorellidae. (G) The ﬁlopodia of athalamids. (Modiﬁed from Bovee and Jahn, 1973.) 53 around the cell. Locomotion may be achieved by extending many pseudopodia simultaneously, as in Amoeba (Fig. 15F), or by moving as a single mass on a broad front (e.g., Figs. 8F, 15E,P), or as a cylinder (limax amoebas, Figs. 8A, 15I,K,L). Not only do pseudopodia have characteristic shapes, but the tail end or a uroid may be distinctive (Fig. 15J,L), and the cell surface may be distinctly sculptured, as in Thecamoeba (Fig. 15D). The dynamics of movement also varies; some amoebas move continuously and smoothly, while the cytoplasm of others seems to burst forward intermittently; this motion is called eruptive. Members of the subclass Testacealobosia are distinguished by their external coverings, called tests (Fig. 16A – Q). These are generally vase-shaped, with a single opening through which pseudopodia emerge. The test may be proteinaceous, but is often covered with secreted plates or inorganic particles. Many are terrestrial, but benthic forms are common and a few are planktonic. Planktonic species have oil droplets or gas-ﬁlled vacuoles to compensate for the weight of the test (Meisterfeld, 1991). Others use the gas vacuoles, which can appear in a matter of minutes, to leave the substrate and rise to the surface ﬁlm where pseudopodia are extended at the air-water interface (Ogden, 1991). Schizopyrenida are small amoebas, many of which are capable of transforming into ﬂagellates (Fig. 9C). D. Anatomy of Amoeboid Protozoa These are the protozoa we generally refer to as amoebas, in reference to the well-known Amoeba proteus. Locomotion is mostly by pseudopodia. Flagella are restricted to temporary swimming stages in the order Schizopyrenida. Rhizopoda and Karyoblastea mostly have blunt pseudopodia called lobopodia, where as the Filosea and Granuloreticulosida have pseudopodia ending in ﬁne points (Fig. 8). Amoebas range in size from only a few micrometers to 2 mm in diameter. Although many lack a ﬁxed external morphology, the characteristic morphologies shown by the various taxa are surprisingly distinctive even if difﬁcult to quantify (Fig. 15). By using also the number, size, and structure of organelles and characteristics of tests (where present), identiﬁcation is not as difﬁcult for living specimens as might be imagined. The morphology of amoebas is plastic. Many adopt a stellate morphology (Fig. 9) if suspended in water. Few are truly planktonic, but rather they live on surfaces or in sediments. Most show an inner granular cytoplasm and an outer hyaline cytoplasm, or hyaloplasm, with a characteristic thickness and distribution FIGURE 9 Some amoebas and their “ﬂoating forms” on the right. (A) Ameoba proteus, scale 100 m. (B) Polychaos timidum, scale 20 m. (C) Naegleria gruberi, scale 10 m. (After Page, 1976.) 54 W. D. Taylor and R. W. Sanders The ﬂagellated form may have two or four ﬂagella. As amoebas, they are small, uninucleate, and usually simple in form. The amoeboid form may be a soildweller, transforming into a ﬂagellate on being suspended in water. The best known amoeboﬂagellate of this kind is Naegleria. Naegleria gruberi was considered by Page (1976) as possibly the most common amoeba in freshwater. At one time, Naegleria was best known as a model system for microtubule assembly, since it could be induced to assemble its ﬂagellum in the laboratory. The similar Naegleria fowleri was discovered as a cause of fatal encephalitis in humans in 1958, and the genus is now better known in infamy as an unwanted resident of air conditioners and swimming pools. Pathogenic strains are able to survive at 37°C, which, fortunately, most free-living amoebas cannot. Increased abundance of pathogenic N. fowleri is a dangerous consequence of thermal pollution (Tyndal et al., 1989). The interesting history of this discovery, as well as other aspects of this group, can be found in Carter (1968) and Singh (1975). Filosea and Granuloreticulosida are amoebas with ﬁne, pointed pseudopodia. Filosea (Figs. 16S – U,W, 19A – K) have single or branched ﬁlopodia with ﬁne points (Fig. 8G), while Granuloreticulosida have a network of branching and anastomosing reticulopodia (Fig. 17L – R). Most members of these two classes have tests. The freshwater granuloreticulosids are among the least studied protozoa, although their marine relatives are very well known. Naked amoebas feed by phagocytosis, the smaller being bacterivores and the larger being predators of algae, other protozoa, and small metazoa. Particulate food is surrounded by ectoplasm, sometimes engulfed by a pseudopod, and enveloped in a food vacuole. In contrast to most other phagotrophic protozoa, there is no ﬁxed site of ingestion or cytostome. Testacealobosia are largely algivores, and many feed on ﬁlamentous algae, drawing in ﬁlaments through the opening in their test. Pinocytosis or “cell drinking” can be induced in Amoeba by dissolved organic substances. Channels appear in the ectoplasm through which substances concentrated on the cell surface are imbibed. Although Amoeba does not have mucocysts, a mucous coat on the cell surface appears to be involved (ChapmanAndresen, 1973). The signiﬁcance of this form of nutrition in the ﬁeld is unknown, but one must conclude that Amoeba uses this ability somewhere other than on microscope slides in undergraduate laboratories! E. Anatomy of Heliozoa The Heliozoa are primarily freshwater counterparts of the better known marine Radiolaria (classes Polycystinea and Phaeodarea) and Acantharia. All these groups have unique pseudopodia, called axopodia (Fig. 8D). Axopodia are strengthened by a microtubular array called an axoneme or stereoplasm. [The term axoneme is also used to describe the microtubular core of cilia and ﬂagella, but this does not imply homology.] The origin and ultrastructure of axonemes is diverse; indeed, the Heliozoa may well be polyphyletic. Most Heliozoa lack the skeleton that is so characteristic of Radiolaria and Acantharia, although some are covered in siliceous or organic scales (Fig. 18H,F), and some have a perforated shell or capsule (order Desmothoracida, Fig. 18A). As diverse as the evolutionary origin of the stereoplasm may be, the axopodia supported by the microtubles are similar in function. Their surface is sticky, possibly because of extrusomes on the axopodia or near their bases (Fig. 7). The outside cytoplasmic layer of the axopods appears to be in constant motion, streaming out and returning on opposite sides of each axopod. Food items adhere and are transported with the ﬂow of cytoplasm until they reach the surface of the cell body and are engulfed. In this way, they are functionally analogous to the microvilli of choanoﬂagellates. Food items may range from picoplankton to mesozooplankton in different Heliozoa. Large prey items, such as rotifers and copepods in the case of Actinosphaerium, may be entangled in several axopodia and engulfed by pseudopods. Several individuals may participate in the capture of one prey. Although Heliozoa are frequently planktonic, they are found primarily on or near the benthos. Some Heliozoa traverse the bottom with a unique tumbling motion resulting from controlled changes in the length of the axopods. Many sessile forms with stalks are known. In sessile forms, cell division is likely to be unequal, producing a dispersal stage that may be ﬂagellated or amoeboid. F. Anatomy of Ciliates Unlike many of the other protozoan groups, the phylum Ciliophora is certainly monophyletic; despite their diversity, they share many features. After discussing these common characteristics, we will consider the important differences among subphyla and classes, emphasizing organelles involved in locomotion and feeding. The presence of cilia is an obvious and distinctive feature. The cilia may be reduced in number, especially in sessile forms, or organized into larger compound ciliary organelles, such as cirri. The only large group that does not always possess cilia is the Suctoria, sessile predators whose dispersal stages are, however, ciliated. 3. Protozoa This distinctive group is easily recognized by its feeding tentacles. The novice should take care not to confuse small, ciliated animals with ciliates; the size range of ciliates overlaps that of several metazoan groups, such as turbellarians, rotifers, and gastrotrichs. The cortex of Ciliophora is a wonderful example of the complex cell ultrastructure to be found among protozoa. We refer the reader to Lynn (1981) and Lynn and Corliss (1990). This ultrastructure is important in the deﬁnition of higher taxa, while the arrangement of both somatic cilia and the compound ciliary organelles, including those associated with the cytostome, are important at all taxonomic levels. The other distinctive characteristic of ciliates is their unique nuclear organization or nuclear dualism. One or more large amitotic, highly polyploid macronuclei are functional in RNA synthesis, while one or more smaller, diploid micronuclei function as gametic nuclei during conjugation (see Section III.B). The ciliates are divisible into ten classes (Small and Lynn, 1985). Class Karyorelictea is thought primitive for the group, with numerous macronuclei that are not highly polyploid. They are largely benthic. The freshwater genus Loxodes (Fig. 23J) is perhaps better known in its environmental physiology and ﬁeld ecology than any other heterotrophic protozoan (reviewed in Goulder, 1980; Finlay 1990). Compound ciliary organelles associated with the cytostome are prominent in the classes Heterotrichea and Spirotrichea. Large heterotrichs, such as Stentor and Spirostomum (Fig. 22F,A) are familiar as teaching material. Spirotrichs are abundant in many freshwater habitats, from plankton (choreotrichs and oligotrichs, Fig. 21S – W) to anaerobic benthos (odontostomes and armophorids, Fig. 21J – N,R). Most consume algae and other nanoplankton-sized food particles. Stichotrichs and hypotrichs (Fig. 21A – H,N – Q, 20X – Z) are mostly dorsoventrally ﬂattened crawlers, whose somatic cilia are all compound cirri. Like spirotrichs and heterotrichs, Nassophorea tend to be algivores except for one small and poorly known group, the microthoracids. The Nassophorea are named for their basket-like “nasse” or cyrtos supporting the cytopharynx (Fig. 12). Classes Prostomatea and Litostomatea are largely predators, often of other ciliates(Fig. 22G-W, 23A-I). Prostomes generally have apical cytostomes, while many litostomes have subapical, sometimes slitlike cytostomes. The mouth is encircled by a crown of cilia from whose bases (kinetosomes) arise the “rhabdos,” a cylinder of microtubules surrounding, and supporting the cytopharynx. Toxicysts are found in most species and are used to subdue active prey. Toxicysts may be found around the cytostome, on a proboscis, on tentacles, or elsewhere on the body. A number of 55 short, specialized kineties (rows of kinetosomes) are often found near the anterior. This “brosse” (brush) probably assists in prey recognition. Class Phyllopharyngea contains the distinctive Suctoria (Fig. 19), sessile or free-ﬂoating predators of other ciliates. Suctoria are unusual in that most have several feeding tentacles rather than a single mouth. Prey ciliates stick to these tentacles because of the ﬁring of haptocysts (see extrusomes, above), and their cytoplasm is withdrawn through the tentacles. Suctoria reproduce by unequal binary ﬁssion (budding), which yields a ciliated dispersal stage or “swarmer.” The other subclass, the Phyllopharyngia, contains surface-associated algivores (e.g., Chilodonella, Fig. 24T), plus a diverse array of marine epizooic forms. Colpodea (Fig. 24K,L,N,P – S) are not as frequent in freshwater environments, most being terrestrial bacterivores. They are more likely to be encountered in small, temporary waters, and can be readily obtained by incubating terrestrial vegetation or soil in water. Plagiopylea (Fig. 24M) were formerly placed in the Colpodea, and they do resemble colpodids in form. Unlike colpodids, they are strict anaerobes. The Oligohymenophorea are mostly microphagous. These are named for the compound ciliary organelles that are found in a buccal cavity surrounding the cytostome. The most common pattern (subclasses Hymenostomatia, Scuticociliatia, Peniculia; Figs. 24C,H, 23L – X, 24A – E,G,I,J) comprises three polykinetids on the left side of the buccal cavity and an undulating membrane on the right. The net result is three brushes, the polykinetids, working against a curved wall, the undulating membrane, to deliver small particles to the cytostome. Most are effective bacterivores, although some are histophagous (consuming tissue of injured or recently killed animals), parasitic, or algivorous. The large subclass Peritrichia (Fig. 20A – U) contains sessile bacterivores in which the buccal cavity is deepened as an infundibulum, and the polykinetids wind down it to the cytostome after encircling a prominent peristome. Somatic ciliature is absent in most species. Many are attached to the substrate by a contractile stalk, as in the common Vorticella. Many are colonial, and a few secondarily free-swimming. G. Environmental Physiology 1. Factors Affecting the Distribution of Protozoa Protozoa are found over an extremely broad range of environmental conditions, but individual species, especially those adapted to unusual circumstances, may have relatively narrow limits to their distribution. Several microbial communities with characteristic 56 W. D. Taylor and R. W. Sanders fauna have been described. Biological, physical, and chemical factors must interact to govern the distribution of these assemblages in a manner that deﬁes simple description. Nonetheless, several physical – chemical factors are important to the growth and/or distribution of protozoa. Among these temperature and oxygen are probably the most important and best known. Light is obviously important to autotrophic forms, and the negative inﬂuence of UV light on protozoa is probably an important factor that has received too little attention (Sommaruga et al., 1996). An extensive and still rapidly expanding literature concerns the importance of environmental contaminants. The distribution of protozoa with respect to organic pollution and its associated environmental conditions has been used as part of a system for classifying the state of aquatic habitats according to their fauna (Sládecek, 1973). Foissner (1988) and Foissner and Berger (1996) have clariﬁed the taxonomy of ciliates used as indicators and provided a summary of the “saprobity values” of ciliates. Similarly, Bick (1972) provided a guide to ciliates useful as biological indicators in freshwater, along with their ecology and distribution with respect to several parameters. A list of species occurrences versus environmental parameters was assembled by Noland (1925). His conclusion that “the wide range of tolerance that most ciliates show toward the physico-chemical factors studied, together with the evident correlation of several of these factors with the food habits of the species, suggests that the nature and amount of available food has more to do with distribution of the freshwater ciliates than any other one factor” still appears to be warranted. The physical nature of the substrate, including the biological substrate of periphyton, is also important to the community of surface-oriented protozoans that develops on it (Picken, 1937; Ricci, 1989). Indeed, the relative importance of biotic components of the environment to the distribution of species versus the structural and chemical – physical components, is difﬁcult to ascertain from survey data. 2. Temperature As in all organisms, metabolic processes and growth in protozoa are dependent on temperature. Because of the difﬁculty of measuring growth rates in situ for protozoa, several workers have investigated the relationship of temperature and growth rate in the laboratory to facilitate the extrapolation of laboratory data on growth rate to ﬁeld situations (Finlay, 1977; Baldock et al., 1980). Generally, Q10 values for protozoa are about 2.0, as they are for many biological processes, but important deviations occur (Baldock and Berger, 1984; Müller and Geller, 1993). Active protozoa are found in freshwater environments at temperatures between 0 and 50°C. For example, protozoa contribute to the plankton community of almost permanently ice-covered lakes in Antarctica (Laybourn-Parry et al., 1995). Some protozoa can be cultured in the laboratory at or near 0°C, but it is doubtful whether all protozoa can function at such low temperatures, or whether those that can perform any better than one would expect from extrapolating the metabolic rate versus temperature curve for species collected from more moderate temperatures to 0°C. Lee and Fenchel (1972) found that Euplotes collected from Antarctic, temperate, and subtropical waters had similar growth – temperature relationships; but the Antarctic strains did not multiply above about 12°C, while the subtropical strain multiplied even above 35°C, but could not reproduce below 6°C. A similar situation is to be expected with respect to high temperatures; most species continue to increase their rate of cell division as temperature increases to 25°C or more and tolerate at least 30°C. But there are numerous records of species from hot environments, up to 50°C (Noland and Goldjics, 1967). Tolerance to extremes of temperature and some other variables have been induced by gradual acclimation (see Fauré-Fremiet, 1967). 3. Oxygen Protozoa are sufﬁciently small that they do not need organelles to increase the surface area for respiration. Aerobic forms appear to function at extremely low oxygen tensions and, thereby, avoid competition and predation from larger animals. It is in these circumstances, especially when they are generated by high densities of bacteria in response to organic enrichment, that protozoa are most abundant. Aerobic protozoa that are typically found under such conditions are called microaerophilic. The succession of species following organic enrichments and the resulting changes in oxygen tension were a focus for some of the earliest research in protozoan ecology (Noland and Goldjics, 1967). A great deal of research has been done on the autecology of Loxodes, a microaerophilic ciliate living in the benthos and plankton of hypereutrophic waters where it consumes nanoplanktonic algae (Fenchel and Finlay, 1991). One of the most surprising outcomes of this research is the discovery that Loxodes can use nitrate rather than oxygen as an electron acceptor. It undergoes diel vertical migrations between the anoxic hypolimnion and oxygenated epilimnion, but cannot survive in either if constrained. It aggregates at low O2 concentrations guided, in part, by unique organelles (Müller’s vesicles) now known to be responsible for geotaxis. The direction of the geotactic response is determined by the oxygen tension, which is probably 3. Protozoa detected by cytochromes, and by light, which is detected by pigment granules (pigmentocysts) in the pellicle. Although other ciliates have been intensively studied in the laboratory, the work on Loxodes is unusual in the degree to which laboratory studies have been guided by ﬁeld observations. This suggests that autecological work on other species could be very rewarding. Some microaerophilic ciliates contain endosymbiotic green algae (see Section III). Despite their photosynthetic symbionts, they aggregate at depths where O2 and light are low (Finlay et al., 1987). At very low O2 tensions, they move into dim light suggesting a role for the symbionts not only as producers of carbon for their hosts but as a means for maintaining a low intracellular level of oxygen. Many free-living protozoa are tolerant of anaerobic conditions (Fenchel and Finlay, 1991). Among the ciliates, this diverse assemblage of species have been referred to as “sapropelobionts” (e.g., Jankowski, 1964a,b), although some of the species embraced by this term are microaerophilic rather than anaerobic. Fenchel (1969) suggested that the term “sulﬁde ciliates” is more appropriate for the truly anaerobic ciliates of reducing sediments. These are intolerant of oxygen and lack mitochondria and cytochrome oxidase. Instead of mitochondria, some anaerobic protozoa have hydrogenosomes, organelles that resemble mitochondria, but reduce pyruvate and generate hydrogen. Epi- and endozooic bacteria are commonly associated with anaerobic protozoa, and methanogenic endosymbionts, in particular, may be closely associated with the hydrogenosomes (Fenchel and Finlay, 1991). Ecological studies of freshwater species have appeared in recent years (Wagener et al., 1990, Massana and Pedrós-Alió, 1994; Guhl et al., 1996). Other protozoa which can probably exist under anaerobic conditions include the giant amoeba Pelomyxa palustris, which has been said to consume at least some oxygen (Bovee and Jahn, 1973) although it has no mitochondria (Daniels, 1973). Oxygen consumption in some anaerobes may be a protective mechanism (Fenchel and Finlay, 1991). The diplomonads, which have two free-living genera (Hexamita and Trepomonas) also lack mitichondria. Flagellated protozoa in the anaerobic hypolimnia of eutrophic lakes may reach abundances as great or greater than those in the surface waters (Bennett et al., 1990). Many true anaerobes among the protozoa belong to taxa that include gut-dwelling endosymbionts. 4. Environmental Contaminants The relative abundance and diversity of protozoa has also been proposed as a useful indicator of organic 57 and toxic pollution (Cairns et al., 1972), and the response of protozoa to contaminants has been reviewed in that light (Cairns, 1974). Others have advocated the use of protozoa in single-species bioassays (Nilsson, 1989). Relatively quick respiration bioassays may reveal both decreases and transient increases in respiration (Slabbert and Morgan, 1982). Because tests with protozoa are relatively rapid, they are particularly useful for studies of quantitative structure – activity relationships (QSAR) where the toxicity of many compounds must be compared. The response of Tetrahymena to a wide variety of compounds is similar to that of a cyprinid, the fathead minnow (Bearden and Schultz, 1997). Protozoan predators and prey have been used to explore complex effects of contaminants on communities (Doucet and Maly, 1990, Fernandez-Leborans and Novillo-Vajos, 1993). Among environmental contaminants, the response of protozoa to oil and other hydrocarbons is relatively well known. They are generally resistant to hydrocarbons (Rogerson and Berger, 1981); the ciliate Colpidium colpoda accumulated hydrocarbons in intracellular inclusions, up to about 20% of the cell volume. The number of mucocysts increased, implicating these organelles as having a protective function (see Section II.B.4). Scott et al. (1984) found that freshwater ponds treated with oil alone or oil and oil-dispersant mixtures had numbers and biomass of protozoa similar to those in control ponds, although some taxa were reduced while others increased. The toxicity of various hydrocarbons to Tetrahymena was similar when differences in solubility and partitioning between the water and ciliates were considered, indicating a similar mode of action once they were in the cell (Rogerson et al., 1983). Biomagniﬁcation of chlorinated hydrocarbons may be substantial. For the pesticide Mirex, the concentration in Tetrahymena was 193 times that present in the medium, and 82% of the available contaminant was in the cells (Cooley et al., 1972). For Arochlor™ 1254, the biomagniﬁcation was 60 -fold. A wide variety of insecticides have been shown to cause dose-dependent inhibition of growth and biomagniﬁcation in Tetrahymena (Dhanaraj et al., 1989; Lal et al., 1987). Some were toxic at higher concentrations. The toxicity of heavy metals to protozoa also has been explored in some detail (Ruthven and Cairns, 1973; Doucet and Maly, 1990; Fernandez-Leborans and Novillo-Vajos, 1993). Intraspeciﬁc variation in heavy metal tolerance appears to be related to environmental variation in contamination (Nyberg and Bishop, 1983). To some degree, tolerance to metals is speciﬁc, but is also generally greater in outbreeding species (Nyberg, 1974; see Section III.B for discussion of 58 W. D. Taylor and R. W. Sanders breeding systems). Free-ion concentration, rather than the total amount of metal, appears to determine toxicity (Stoecker et al., 1986). 5. Energetics of Protozoa Because protozoa are fast-growing and easily cultivated in the laboratory, their energetics have been studied extensively. Ingestion, growth, respiration, egestion, and other parameters have been measured or estimated, and quotients cataloged. We deal with some of these parameters elsewhere; for example, maximum growth rates are discussed in Section II.B, some ﬁeld estimates of growth rate are mentioned in Section III.B and egestion in Section III.C.10). Laybourn-Parry (1984) summarized much of the existing information on protozoan energetics, and Calow (1977) integrated it into his wider review of animal energetics. Fenchel’s (1974) classic paper drew attention to the relationship between body size and rate of population increase for organisms, and the more rapid growth rate of the smaller as compared to the larger ones. His analysis also suggested a cost associated with multicellularity; both net growth efﬁciency (growth/assimilation) and growth are generally larger for protozoa than for metazoa of similar size. Calow’s summary of conversion efﬁciencies conﬁrms that protozoa are generally efﬁcient converters of energy, with many species showing growth efﬁciencies of 50% or more. However, efﬁciencies for some taxa are much lower (e.g., Scott, 1985, and references therein) and it is difﬁcult to generalize. Given the high growth rates of protozoa, we expect that ﬁeld populations of protozoa will be found to have high production-to-biomass (P/B) ratios relative to metazoa, on the order of one per day. However, few ﬁeld estimates of growth rate are available. Field estimates of growth rates include Schönborn (1977) and Taylor (1983b) for benthic populations, and Taylor and Johannsson (1991) and Carrick et al. (1992) for planktonic populations. It appears that planktonic ciliates undergo periods of consumer and resource control, and these may be different for different species (Havens and Beaver, 1997; Wang and Heath, 1997). III. ECOLOGY AND EVOLUTION A. Diversity and Distribution The concept of species within the protozoa has always been problematic. The deﬁnition of species as interbreeding or potentially interbreeding populations is irrelevant to the many asexual forms. On the other hand, studies of the genetics of what were once believed to be species, such as Paramecium aurelia, Tetrahymena pyriformis, and Euplotes patella, have revealed groups of interfertile mating types (Corliss and Daggett, 1983). These groups of mating types are called syngens by protozoologists, but are essentially sibling species. New species names have been created to recognize them in some cases (e.g., P. monaurelia, P. biaurelia, and so on). Careful morphological analysis has uncovered differences among some of these species, but in practice, they are not morphologically separable. They must be identiﬁed by mating reactions with known lines, or by using electrophoretic differences in some cytoplasmic enzymes. At the same time, the phenotypic plasticity of protozoa has also led to the description of invalid species names (Gates, 1978). Finlay et al. (1996) provided a carefully reasoned analysis of this problem, and concluded that “the biological species concept is neither appropriate nor practicable” for ciliated protozoa. They concluded that we must instead be pragmatic and consider morphological species. The same would be more true of phyla where sex is less common or absent. It remains that some protozoan groups have as many species as the more familiar animal taxa. For example, ciliates comprise some 7000 species (Corliss, 1979), with about 3000 known freeliving species (Finlay et al., 1996), of the tens of thousands of protozoan species. At this point it should be admitted that the taxonomy of free-living protozoa, at least as practiced by ecologists, is in a primitive state. A textbook on protozoa will advise the student to collect protozoa at the margin of a pond or in some other small and protected waters rich in organic material. Collections from these environments are likely to yield rich assortment of large protozoa, readily identiﬁable at least to genus under the dissecting or compound phase microscope. The working freshwater ecologist will more likely be interested in lake plankton, littoral or profundal benthos, or stream aufwuchs. Samples from these situations will more likely yield minute forms too small to be seen under a dissecting microscope, and too few to be examined under a compound microscope without concentration. They may not be ﬁgured in the textbooks. Many will be undescribed species, or species descriptions will be available only in specialist journals, not to be found in most libraries, or so outdated as to be of dubious value. Largely because of these problems, most ecologists who include protozoa in their studies of aquatic habitats do not identify protozoa, even if they do count them and measure them for biomass estimates. Techniques of sample preservation, concentration, and enumeration (see Section IV), if they exist for a particular biotope, may not allow for identiﬁcation. Molecular techniques such as species-speciﬁc oligonucleotide probes have been developed and may prove to be 3. Protozoa useful tools for identiﬁcation of particular protozoan groups in natural samples (Lim, 1996). The distribution of most free-living protozoa is probably broad. Certainly, some morphological species are worldwide in distribution and are essentially ubiquitous. Experiments using enriched and incubated ﬁeld samples led Fenchel et al. (1997) to conclude that although local diversity of protozoa may be high, global diversity may not be; morphological species are usually present in appropriate habitats, even if not detected by initial sampling. Protozoa quickly colonize new environments, as was shown by Maguire’s (1977) study of new volcanic islands. On the other hand, sibling species of the Paramecium aurelia complex have limited distributions on a world wide scale (Sonneborn, 1975) even though the species complex is ubiquitous. B. Reproduction and Life History Most protozoa reproduce by equal binary ﬁssion, and therefore their life histories are simple. Multiple ﬁssion is more common among endosymbionts but does also occur in free-living forms. Among free-living protozoa, multiple ﬁssion appears to be related to intense temporal heterogeneity in the environment. For example, it is found in some terrestrial forms which are active only for brief periods when their environment is moist, or in histophages whose food resources are very patchy and ephemeral (Fig. 10). Therefore, some analogy can be drawn between the signiﬁcance of multiple ﬁssion in certain free-living protozoa and some endosymbionts using the common theme of dispersal (Taylor, 1981). A phenomenon perhaps related to multiple ﬁssion, in that it is related to dispersal, is unequal ﬁssion or budding. Binary ﬁssion is only approximately equal, and differences in the partitioning of cytoplasm occur and are adjusted in the next cell cycle. But a consistent asymmetry of daughter cells is widely distributed among sessile forms; the larger daughter cell remains attached, while the smaller is motile. This pattern undoubtedly has evolved independently a number of times. It is perhaps a reﬂection of the probability of survival of the two daughter cells not being equal, and resources being partitioned according to the odds. The smaller daughter cell is usually a propagule, capable of establishing itself in a new location. In some cases, (e.g., Zoothamnium, Fig. 20H,I), it may function as a gamete. Generation times are highly variable among protozoa, but the maximum rates of population increase attainable when food is not a constraint generally increase with temperature and decrease with body size (see Section II.5). Multiple regressions of growth rate on cell size and temperature are sometimes used to 59 estimate production despite shortcomings (Müller and Geller, 1993). However, even for protozoa of the same size and growing at the same temperature, maximum rates of cell division vary widely among taxa. Some of this variation is correlated with genome size and may reﬂect a causative relationship. Among most eukaryotic cells with diploid, mitotic nuclei, there is a positive relationship between DNA content and cell size, and a negative relationship between DNA content and division rate. It appears that, among such cells, a large cell must have a proportionately large DNA content and nucleus, and a slow division rate. This relationship holds among most plant and animal cells and among most protozoa. However, it has also been established in plants that polyploidy allows a large DNA content and large cell size, but without a concomitant decrease in division rate. Division rate is apparently related to the size of the genome, not the number of copies. The diverse nuclear arrangements of protozoa can be viewed as various solutions to the genome size/cell FIGURE 10 Life cycle of the histophagous ciliate Ophryoglena. Change from the theront (hunting morphology) (A) to the trophont (feeding) morphology (B,C) occurs when food is found. The prototomont stage (D) seeks a suitable place to settle, and leads to the cyst or tomont stage (E,H). The tomont usually produces 4–8 new theronts, or may produce a much larger number of microtheronts (F,G). The function of microtheronts is unknown. Theronts may encyst if they do not ﬁnd food, and re-emerge as small secondary theronts. (Modiﬁed from Canella and Rocchi-Canella, 1976.) 60 W. D. Taylor and R. W. Sanders size/ﬁssion rate constraint (Cavalier-Smith, 1980). Multinucleate cells, nuclear dualism, amitotic nuclei, and colony formation may represent ways to get larger without slowing growth. For example, in ciliates, cell size is correlated with macronuclear DNA content, but cells divide as frequently as one would expect from the size of their micronucleus (Shuter et al., 1983). In this context, both multinuclearity and multicellularity can be seen as means of avoiding the constraints imposed by the basic eukaryotic plan. Encystment is a feature of many protozoan life cycles, especially those that live in environments that dry out (Corliss and Esser, 1974). Many protozoa also produce cysts at times when environmental conditions are unsuitable for other reasons, such as lack of food. Encystment under these conditions can be considered as a means of coping with temporal heterogeneity. Still other protozoa incorporate encystment into the life cycle following feeding (the “digestive cyst”) or during cell division (the “reproductive cyst”). Digestive cysts are common in ciliates that rapidly consume large amounts of food, possibly after a long period of searching, e.g., histophages and predators. Some species enter a cyst after feeding and emerge after cell division, combining these functions. The encysted stage may reduce energy expenditure and/or the risk of predation when the acquisition of food is either not possible or unnecessary. Nonencysting protozoa may persist days or weeks in the absence of food; this survival time is not closely related to cell size, is density-dependent, and probably reﬂects differences in energy costs associated with locomotory behavior (Jackson and Berger, 1985). Survival time was found to be negatively correlated with intrinsic rate of population increase (see Section III.C.2) among eleven species, but there were exceptions to this trend. Crawford and Stoecker (1996) examined survival and respiration during starvation in a marine mixotrophic ciliate Strombidum capitatum. There was no decrease in dark respiration with starvation, and the starving population comprised both larger cells with low speciﬁc respiration rates and small cells with high speciﬁc respiration rates. Survival time was longest for the large cells. Sex is widespread, but far from universal among protozoa. For example, it is common among the Ciliophora, Chlorophyta, Chrysophyta, and Granureticulosida, limited among Dinoﬂagellata and Heliozoa, and absent in most if not all Zoomastigina and Rhizopoda. Grell (1973) and Margulis and Sagan (1986) discussed the occurrence and variety of sexual phenomena among the protists. Grell organized sexual phenomena into three basic types: (1) gametogamy, in which gametes fuse as free-swimming cells; (2) autogamy, where gametes or gametic nuclei of the same individual fuse; and (3) gamontogamy, where mates (gamonts) unite to exchange gametes or gametic nuclei. Gametogamy is found in ﬂagellates where, typically, isogametes are produced by multiple ﬁssion. However, anisogamy also occurs. Autogamy is present in Heliozoa and some Ciliophora. Gamontogamy is the usual form of sex in Ciliophora. As in most small metazoa, sexuality interrupts asexual reproduction, and is associated with environmental change. It should be emphasized that sexuality is not necessarily associated with reproduction in protozoa and is best understood as a separate phenomenon (Margulis and Sagan, 1986). Indeed, sex usually marks the end of the existence of an individual protist; it becomes the gamete or gametes, and its genome ceases to exist. The restricted occurrence of sex among protozoa, and the separation of sex from reproduction in many taxa, make protozoa a promising group for further study concerning the evolution and signiﬁcance of sex. The relationship between sex and asexual reproduction has been carefully examined in some Ciliophora, especially Paramecium (see Wichterman, 1986, for a review of this topic). Exchange of haploid nuclei between two conjugants (gamontogamy) leads to two individuals with new and different genotypes. Each is the founding member of a new clone, expanding by asexual binary ﬁssion. Individuals of a new clone will undergo a period of “sexual immaturity” when sex does not occur. The duration of this immature period varies among species; species with short immature periods are considered to be “inbreeders,” as mating with close relatives is favored, whereas species with long immature periods are considered to be “outbreeders.” The immature period is followed by a period of sexual maturity when conjugation will occur under the following circumstances: (1) a partner of a complementary mating type is available; (2) both are at the right stage of the cell cycle; and (3) both are slightly starved. If conjugation does not occur before the end of the period of maturity, a period of sexual senescence follows. The rate of binary ﬁssion declines, cells are less likely to survive conjugation if a mate is found, and autogamy (self-fertilization) may occur. Although autogamy may prolong vigor, the clones ultimately die out. An analogy can be drawn between the life of a clone of ciliates and the development of a metazoan from a zygote — a large population of cells develops asexually from the single cell resulting from sexual fusion, but ultimately the size and life span of that population are limited by senescence which must be circumvented by another sexual episode. As some ciliates do not undergo conjugation and others vary greatly in the frequency necessary to prevent senescence (a strain of Tetrahymena without a 3. Protozoa micronucleus and, therefore, not capable of sex was isolated in 1923 and is still going strong), one is tempted to speculate on the function of both sex and senescence. Is sex required to postpone senescence by replacing irreparably damaged DNA by recombination? Or, is senescence a mechanism to eliminate micronuclear or gametic genes that have not been expressed for many generations? Or, is the need for sex a function of the unique amitosis of the ciliate macronucleus? The ﬁnite life span of animal cells in tissue culture versus the immortality of cultures of cancer cells begs similar questions. That autogamy in ciliates is a stop-gap measure to postpone senescence contrasts with sex in the heliozoan Actinophrys sol, where fusion of the products of meiosis within the same cell is the normal pattern. Bell (1988) reviewed this fascinating subject and concluded that senescence of both sexual and asexual protists can be viewed as a common phenomenon, explicable in terms of the genetic deterioration of ﬁnite populations. Although sex is a necessary event in the life cycle of some ciliates, it is a dangerous undertaking; genetic load is high in many ciliates, as is the mortality following conjugation. As noted previously, some species avoid close matings by having a long immaturity period. As if conjugation was not dangerous enough, prokaryotic endosymbionts render some ciliates “matekillers”; their partners in conjugation pass on their genes but are lethally affected (see Section III.C.4). C. Ecological Interactions 1. Intraspeciﬁc Interactions among Protozoa Protozoa have been used extensively in the study of competition and predator – prey interactions. Their obvious advantages for this type of study include small size, short generation time, and, for some species, ease of maintenance in the laboratory. These advantages do not carry over to ﬁeld experiments, and little work has been done in situ. Intraspeciﬁc competition in protozoa is evident in the dynamics of batch cultures, where a few individuals are introduced into a large, but ﬁnite environment. If inoculated into batch cultures as starved individuals, some ciliates will respond to this situation by growing rapidly and increasing in size past the biovolume at which the cell might be expected to divide. This has been interpreted as a strategy to sequester the maximum amount of food should the supply become exhausted. However, if food is still available, these cells enter into a phase of exponential growth where they divide at a similar size in each cycle. When the food supply is depleted, some individuals will continue to 61 divide at the expense of cell size. Again, this can be interpreted as an adaptation for an r-selected or colonizing species to produce as many propagules as possible (Taylor, 1981; Fenchel, 1987). Curds and Cockburn (1971) found that, in order to model the population dynamics of Tetrahymena in batch cultures, it was necessary to include interference among individuals in their equations. This was not necessary for continuous cultures. The exhaustion of food in some species induces cannibalism, sometimes with abrupt changes in morphology for the cannibals (see Giese, 1973, for a thorough discussion of this phenomonen in the ciliate Blepharisma). Another form of interference is the “killer” phenomenon. Some protozoa harbor prokaryotic endosymbionts that render them lethal to conspeciﬁcs (mate-killers were mentioned above). 2. Interspeciﬁc Competition among Protozoa Interspeciﬁc competition in laboratory cultures of ciliates has been studied by Gause and numerous authors since. Gause (1934) provided clear evidence that environmental factors could inﬂuence the outcome of competition. The Lotka – Volterra equations predict that the winner of exploitative competition for resources in stable environments should be the species with the greater K or carrying capacity, that is, the more efﬁcient user of the resource. However, K is usually measured as numbers, not biomass, so smaller species will tend to have a higher K. In his studies of competitive interactions of bacterivorous ciliates, Luckinbill (1979) conﬁrmed that K did not predict the outcome of competition. But, in accordance with part of the r/K selection hypothesis, the intrinsic rate of increase, r, was negatively related to competitive ability. [The r/K selection hypothesis holds that r, which measures ﬁtness in density-independent or stochastic environments, is negatively correlated among species with K, which measures ﬁtness in stable environments.] Thus the theorem seems to hold for ciliates, except that K, being highly correlated to body size, does not measure competitive ability in stable environments. As noted above, r is also correlated with body size. To factor out this correlation, Taylor (1978a,b) expressed intrinsic rate of increase as r/re, where re is the expected r for a ciliate based on a regression of r on body volume. Therefore, r/re is a measure of how fast-growing or r-selected a ciliate is for its body size. This measure was found to be negatively correlated with frequency of occurrence in the ﬁeld, and positively correlated with macronuclear ploidy (Taylor and Shuter, 1981). This research suggests that protozoan communities contain fast-growing but ephemeral 62 W. D. Taylor and R. W. Sanders opportunists, as well as more conservative, specialist species that have more modest growth rates, but stable populations. A similar picture emerged from a comparison of the population dynamics of a relatively fast and a relatively slow-growing species in the laboratory (Luckinbill and Fenton, 1978). Field studies of exploitative competition in protozoa are few, as are ﬁeld measurements of growth rates for comparison to laboratory estimates under conditions of excess food. Accordingly, we know little about the relative importance of density-dependence or independence. Work on planktonic protists suggests that ﬁeld populations may experience periods of rapid growth and periods of little or no growth (Taylor and Johannsson, 1991; Carrick et al., 1992). Maguire (1963a,b) found strong evidence for the exclusion of colonizing species in the genus Colpoda by Paramecium and associated species. Hairston and Kellerman (1965) and Hairston (1967) found that one member of the Paramecium aurelia species complex dominated sympatric sibling species in competition experiments and predominated in ﬁeld collections. The ﬁeld population they studied appeared to be foodlimited. In contrast, Taylor (1983b) found that sessile ciliates on an artiﬁcial substrate in a stream had high growth and mortality rates, implying little competition. The extensive studies on colonization by Cairns and co-workers (reviewed in Cairns and Yongue, 1977) provide indirect evidence for species interactions, including competition. There is also competition between protozoa and metazoa consuming similar resources. Potential metazoan competitors include some rotifers and crustaceans, although it is difﬁcult in some cases to distinguish between the effects of competition and predation (Wickham and Gilbert, 1993). Likewise, the presence of predatory protozoa may affect the outcome of competition between other protozoan species (Lawler, 1993). 3. Predatory Interactions among Protozoa Many protozoa are predators of other protozoa. For example, Chardez (1985) listed a diverse assortment of protozoa consuming testaceans. Protozoa have been used extensively in laboratory studies of predator – prey interactions, including such topics as selectivity, stability of predator – prey interactions, and the behavioral and energetic determinants of functional and numerical response. Much of this research has been done with Didinium nasutum. Hewett (1980a,b) and Berger (1979) showed that Didinium changes its body size, feeding behavior, and feeding and growth rate when fed different prey and may encounter some difﬁculty handling large prey after adjusting to small ones. The extrusomes which enable Didinium and other predatory ciliates, dinoﬂagellates, and Heliozoa to handle their prey were mentioned in Section II. Although extrusomes are important weapons of many other ciliates, especially in the Litostomatea and Suctoria, other predators are simply swallowers, including the giant cannibal forms of Blepharisma and Tetrahymena, large ciliates such as Bursaria, and some other predators such as Peranema among the Euglenida and the large amoebas. Interest in the “microbial loop” (the route by which picoplankton production reaches larger consumers) has focused attention on the contribution of protozoa, as consumers of picoplankton, to production at higher trophic levels. A microbial loop leading to ﬁsh via crustacean zooplankton is likely to be inefﬁcient because of the number of steps involved, and even more inefﬁcient if predatory protozoa process some of that production. Nevertheless, protozoa are ingested by many metazoa and often appear to be a high-quality food (Sanders and Wickham, 1993). Relatively little is known about the quantitative signiﬁcance of predatory protozoa in planktonic food webs. In some instances ciliates appear to be the major predators of heterotrophic nanoﬂagellates (Weisse et al., 1990), but the relative predation impact on ﬂagellates by ciliates versus metazoa appears to vary seasonally (Weisse, 1991; Sanders et al., 1994). Known predators of other protozoa, such as Dileptus and Actinosphaerium, are commonly observed in the plankton. The prey of many abundant litostomes is not known, but they are likely to feed on other protozoa. Use of ﬂuorescently labeled prey has identiﬁed small ciliates, including Strobilidium, Halteria, and tintinnids, as predators of heterotrophic nanoﬂagellates in lakes (Cleven, 1996). In some estuarine systems, dinoﬂagellates may be important predators of ciliates (Bockstahler and Coats, 1993) but this is not documented in freshwaters. Presence of predatory ciliates, amoebas, and turbellaria elicits morphological changes in the ciliate Euplotes that reduce predation (Kusch, 1993). Predation within the protozoan microplankton is still an unknown loss in the transfer of energy to higher trophic levels and therefore bears on the debate concerning the importance of the microbial loop. 4. Symbionts of Protozoa Symbionts of protozoa include both ecto- and endosymbiotic prokaryotes and eukaryotes, including a wide variety of parasites (Curds, 1977; Jeon, 1983; Lee and Corliss, 1985). The most obvious eukaryote symbionts of freshwater protozoa are algae of the genus Chlorella, often called zoochlorellae. These are most widely studied in the ciliate Paramecium bursaria, but are also known from a wide variety of other ciliates as well as ﬂagellates, amoebas, and many metazoa. In 3. Protozoa 63 some cases, the symbiosis appears to beneﬁt the protozoan because of the photosynthates released by the algae; P. bursaria, for example, probably receives maltose from its symbionts and can grow better at low food concentrations when they are present. Although some alga-bearing protozoa display positive phototaxis and aggregate at the water surface (Berninger et al., 1986), others aggregate deeper, in dim light. Some of these microaerophilic forms are negatively phototactic, unless the medium is anaerobic (Finlay et al., 1987), and zoochlorellae may provide low internal oxygen tensions in anaerobic external environments. Therefore, there may be two functional types of protozoan – algal symbioses: one in which the primary beneﬁt for the host is to receive photosynthate, characterized by positive phototaxis, and one in which the primary beneﬁt for the host is oxygen production, characterized by oxygen-dependent phototaxis. Some choreotrichs (Strombidum spp.) harbor isolated chloroplasts as symbionts (Rogerson et al., 1989; Stoecker et al., 1989). The chloroplasts may be from more than one species of alga. In some cases, whole cells may be retained as symbionts. The host ciliate becomes functionally mixotrophic, able to photosynthetically ﬁx signiﬁcant amounts of carbon. The haptorid ciliate Perispira ovum ingests the ﬂagellate Euglena, incorporating its chloroplasts, mitochondria and paramylon bodies (Johnson et al., 1995). It probably uses oxygen produced by the chloroplasts to live in low-oxygen environments. Prokaryotic endosymbionts are widely distributed in protozoa. For example, several species of anaerobic protozoa have endosymbiotic methanogenic bacteria (Section II.H.3). Some endosymbionts are infectious parasites which may kill their hosts, while some are necessary for the survival of their hosts. In some cases, the function of the symbionts is unknown. Perhaps the most interesting endosymbionts from an ecological point of view are the intensively studied “killer” particles of ciliates. Some transform their hosts into “killers” that cause the death of conspeciﬁc “sensitives” simply by their proximity. Others transform their hosts into “mate-killers,” whose partners in conjugation are lethally affected. The signiﬁcance of these endosymbionts to ﬁeld populations of Paramecium has been investigated by Landis (1981). He concluded that the infection must be maintained by natural selection, because killers rarely have a chance to mate and transmit their infection to new hosts. Mating and cell division are the only known means of transmission. to be rather nonspeciﬁc, involving species that can also be found attached to, or crawling on, nonliving substrates as part of the aufwuchs community. Other associations (e.g., peritrichs of the order Mobilida that are epizooic on ﬁsh) are perhaps better left to the parasitologists. However, some cases deserve brief mention here because they are common and likely to be noticed. One common association is between planktonic, colonial cyanobacteria and peritrich ciliates (Pratt and Rosen, 1983; Kerr, 1983). Peritrichs are frequently so abundant as to rival the biomass of their host colony, and they likely affect its nutrient environment and motility. Smaller peritrichs, choanoﬂagellates, and bicoecids are common epizooics on diatoms and ﬁlamentous cyanobacteria (Fig. 11A). Peritrichs and Suctoria colonize the exoskeleton of planktonic crustacea. Kankaala and Eloranta (1987) 5. Epizooic and Epiphytic Protozoa FIGURE 11 (A) Choanoﬂagellates epiphytic on ﬁlamentous cyanobacteria from Georgian Bay, Lake Huron. Scale bar 10 m. (B) Tokophrya quadripartita (Suctoria) epizooic on Limnocalanus from Lake Michigan. (From Evans et al., 1979.) Many freshwater protozoa have developed epizooic associations with animals. Some associations are likely 64 W. D. Taylor and R. W. Sanders FIGURE 12 The cyrtos of Nassula, and how it is used to ingest ﬁlamentous cyanobacteria. (From Tucker, 1968.) found that the clearance rates of Vorticella epizooic on Daphnia can rival those of their host, suggesting that competition for food with Vorticella could be signiﬁcant to the Daphnia. Epizooic suctoria (Tokophrya spp., Fig. 12B) on mysid and calanoid Crustacea in the Great Lakes may be heavy enough to impair their hosts (Evans et al., 1981) and may also compete with them for food. Henebry and Ridgeway (1980) suggested that epizooic protozoa on Crustacea may be useful pollution indicators. Benthic invertebrates may also be infested with peritrichs (see Baldock, 1986, for a brief summary). The biomass of these may be a signiﬁcant portion of the protozoan biomass in streams. 6. Protozoa as Bacterivores Protozoa are most widely known as bacterivores. Bacterivorous protozoa are abundant in activatedsludge sewage treatment plants, and, indeed, are neces- sary for their proper functioning (Curds and Cockburn, 1970). At the opposite extreme, there is considerable evidence that in plankton communities, especially oligotrophic ones, heterotrophic ﬂagellates are the dominant bacterivores and that much of the carbon ﬂow passes through them (e.g., Sanders et al., 1989; Nagata, 1988; Carrias et al., 1996). Although ciliates may be abundant bacterivores in enriched environments (hence, the old name “infusorians” or animals from organic infusions), they are thought to be quantitatively unimportant in many planktonic ones. However, in some planktonic environments, especially those where cladoceran crustacea can seasonally reduce ﬂagellate abundances signiﬁcantly, ciliates may become the dominant bacterivores (Simek and Straskrabová, 1992). Heterotrophic ﬂagellates, and to a lesser degree ciliates, also may be important bacterivores in the water column of rivers (Carlough and Meyers, 1991), although bacterivory by protists in this environment has barely been addressed. 3. Protozoa The relative importance of ﬂagellates and ciliates as bacterivores in different habitats and during different seasons is, therefore, an open question. The question should probably be phrased in terms of the size distribution of grazers of picoplankton, and the answer probably involves species interactions and food web structure as well as trophic status. The small Heliozoa are an overlooked group of largely planktonic bacterivores, and small amoebas are bacterivorous also. Mixotrophic ﬂagellates may play a signiﬁcant role as bacterivores in some freshwater plankton (Bird and Kalff, 1987; Sanders and Porter, 1988; Berninger et al., 1992). The importance of mixotrophs as bacterivores varies considerably both temporally and spatially, and the factors determining their abundance and feeding rates in natural systems are not entirely clear. However, light, temperature, dissolved nutrient concentrations, and food levels are likely to affect both the abundance and the relative importance of phagotrophy versus photosynthesis in these organisms (Bird and Kalff, 1987; Sanders et al., 1989, 1990; Jones and Rees, 1994). Many protozoa glean bacteria from surfaces or attached to particles (Albright et al., 1987; Caron, 1987), but the quantitative role of bacterivorous protozoa in benthic habitats is relatively unknown. Pioneering studies in this area include Baldock and Sleigh (1988) and Bott and Kaplan (1989). In experiments with stained lake sediment, Starink et al. (1994) found over 90% of protozoa ingested bacteria at rates up to 73 bacteria protist1 hour1. Small protozoa or “heterotrophic nanoplankton” (ﬂagellates, amoebas, and some small ciliates) capture and consume bacteria individually, with rates of ingestion of tens to hundreds per hour (e.g., Fenchel, 1982b). Most ciliates, in contrast, use their complex oral ciliature to collect hundreds into each food vacuole, producing feeding rates up to thousands of bacteria per hour (e.g., Taylor, 1986). Although the former are likely to be selective in their feeding, the larger ciliates are probably very limited in their ability to discriminate. The use of ﬂuorescent microspheres to measure bacterivory (McManus and Fuhrman, 1986) resulted in renewed interest in the ability of protozoa to discriminate against inorganic particles in favor of bacteria (Nygaard et al., 1988) and in the inﬂuence of chemical coatings and surface charge (Sanders, 1988). Both ciliate and ﬂagellate bacterivores discriminate among bacteria on the basis of size (Sanders, 1988; Simek and Chrzanowski, 1992; Simek et al., 1994). Several studies have documented that ciliates differ in their ability to use different bacteria as food. Some may be unable to consume ﬁlamentous bacteria, while other 65 bacteria, especially pigmented ones, produce toxins that render them unsuitable to support the growth of some ciliates (Dive, 1973; Taylor and Berger, 1976). A poorly understood and usually ignored facet of bacterivory in protozoa is the role of extracellular products. The presence of some ciliates leads to the ﬂocculation of bacteria (Hardin, 1943; Curds, 1963; Nilsson, 1976). Mucous-like extracellular materials are involved in uptake of dissolved material and possibly small particles by pinocytosis in Amoeba. Small amoebas secrete various hydrolases into their medium (Schuster, 1979) as do ﬂagellates. Porter (1988) suggested that ﬂocculation and external digestion may be important in bacterivorous ﬂagellates. The involvement of mucocysts or other extrusomes is likely, but not conﬁrmed. As one might expect, feeding rate (functional response) and growth rate (numerical response) of bacterivores increase with bacterial density, and the nature of these response curves has been studied because of their relevance to interspeciﬁc competition and the control of bacterial numbers. In addition to the expected hyperbolic functions, thresholds or sigmoid responses have also been observed, and attributed to changes from feeding to searching behavior at low particle concentrations (e.g., Taylor, 1978a; Sanders, 1988). Accordingly, low concentrations of bacteria (104 – 107 per milliliter, usually 105 –106) can persist even in stationary-phase cultures of bacterivorous protozoa. The behavioral and physiological mechanisms responsible for this phenomenon are still in doubt (Sambanis and Frederickson, 1988); however, it is probably relevant to the densities of picoplankton observed in ﬁeld situations. 7. Algivorous Protozoa Larger protozoa are less likely to be bacterivores, and most consume algae or other protozoa. Suspension-feeding ciliates each ingest a distinct size-spectrum of particles, related to the form and function of the oral apparatus (Fenchel, 1980), and this spectrum generally shifts in larger ciliates toward larger particles, i.e., algae and other protozoa. However, even small prostomes can be important predators of ﬂagellates (Sommaruga and Psenner, 1993). Larger ﬂagellates, amoebas, and Heliozoa also consume other algae and protozoa. Protozoa feeding on relatively large items are more likely to be selective, as the rate at which particles are consumed is much reduced. For example, Rapport et al. (1972) documented selective feeding by the heterotrich ciliate Stentor consuming other protozoa at up to about ﬁve prey per minute. Stentors consistently biased their feeding toward Tetrahymena relative to FIGURE 13 (A) Peridinium; (B) Gymnodinium; (C) Gyrodinium; (D) Khawkinea halli; (E) Polytomella citri; (F) Entosiphon sulcatum; (G) Petalomonas abcissa; (H) Peranema trichophorum; (I) Urceolus cyclostomas; (J) Chilomonas paramecium; (K) Paraphysomonas vestita; (L) Spumella ( Monas) vivapara, two cell shapes; (M) Ochromonas variabilisa; (N) Dinobryon sertularia(mixotrophic). (After: Bourrelly, 1968, L; Calaway and Lackey, 1962, E, F, J, N; Eddy, 1930, A; Jahn and McKibben, 1937, D; Leedale, 1985, H; Pascher, 1913, M; Lemmermann, 1914, K; Shawhan and Jahn, 1947, G; Smith, 1950, I.) Scale 5 m for E; 10 m for A, B, C, G, I, J, K, L, M; 20 m for D, F, H, N. 66 3. Protozoa three autotrophic ﬂagellates when mixed versus single species feeding suspensions were compared. The behavioral response of algivorous choreotrich ciliates appears to involve both mechanical and chemical stimuli, and suggests that although ciliates may ingest inert particles, they may discriminate among them as well as among food organisms (Buskey and Stoecker, 1989). Selective feeding has been noted for Amoeba (Mast and Hahnert, 1935) and Thecamoeba (Page, 1977). Ciliates may have relatively large ecological impacts on phytoplankton populations; in Lake Constance ciliates consumed about 14% of the daily primary production during a phytoplankton spring bloom, which was equivalent to the amount removed by metazoan zooplankton (Weisse et al., 1990). Although most phagotrophic ﬂagellates are bacterivores, larger species may be effective algivores. Dinoﬂagellates, for example, may consume diatoms and other large phytoplankton (see Section II.B.1). Zoomastigina are probably under-rated as consumers of algae (Canter, 1973; Goldman and Caron, 1985; Suttle et al., 1986). The colorless chrysophyte Paraphysomonas (Fig. 2D) was observed to feed selectively on diatoms, phagocytosing cells that were nearly their own volume (Grover, 1989). The phagotrophic ﬂagellate Peranema (Fig. 13H) feeds on related autotrophic euglenids. The consumption of ﬁlamentous algae (including cyanobacteria) would appear to be mechanically difﬁcult for protozoa, but many have specialized to this role. Large amoebas, such as Pelomyxa include ﬁlamentous algae in their omnivorous diets, and some Testacealobosia, such as Arcella and Difﬂugia, are readily cultured on ﬁlamentous algae which they engulf through the aperture of their tests. The nassophorine ciliates contain many forms which feed on ﬁlamentous algae, including Nassula, Pseudomicrothorax, and Frontonia. Nassula spp. (Fig. 11) appear to limit their diet to Oscillatoria. Detailed ultrastructural studies have revealed the role of the cyrtos in feeding, especially how it is used to break the algal ﬁlament so that ingestion can begin (Tucker, 1968; Peck, 1985). Abundant epibenthic diatoms, especially noncolonial and unattached pennate diatoms such as Navicula, usually attract abundant herbivorous ciliates and amoebas. Many dorsoventrally ﬂattened and thigmotactic ciliates in the Phyllopharyngia and Nassophorea are diatom feeders. Their functional response has been described by Balczon and Pratt (1996). 8. Histophagous Ciliates Several genera and species of ciliates feed on metazoan tissue. Some, like Coleps hirtus and Prorodon spp., are omnivores and only opportunistic histophages. 67 Strict histophages, feeding only on fresh tissue, include all hymenostomes of the genus Ophryoglena (Fig. 10) and some Tetrahymena spp. (e.g., Corliss, 1973; Lynn, 1975). Deltopylum is also a histophage; but in our experience, it is relatively rare. Philaster appears to be marine only. These forms feature complex life cycles with reproductive cysts and multiple ﬁssion, which are probably adaptations to an extremely patchy food resource (Section III.A). Like other carrion feeders, they increase the efﬁciency of food webs by bypassing the detrital pathway. Ophryoglena, and the related ﬁsh parasite Ichthyophthirius, share the unique organelle of Lieberkühn (Canella and Rocchi-Canella, 1976), or watchglass organelle, whose function is unknown but probably related to their feeding mode. Related hymenostomes are insect parasites. Lambornella can be a parasite of the cuticle of mosquito larvae (Washburn et al., 1988), and, it and some Tetrahymena spp. may have signiﬁcant impacts on aquatic Diptera (Golini and Corliss, 1981; Egerter et al., 1986). Espejoia feeds on the egg masses of aquatic insects. 9. Metazoan Predators of Protozoa Protozoa, as quantitatively important components of plankton, aufwuchs, or sediment communities, probably contribute to the nutrition of many animal-feeding guilds, such as ﬁlter-feeders, scrapers, and depositfeeders. However, because most protozoa are fragile and without structures that resist digestion, they are seldom recorded among the gut contents of animals despite their importance (McCormick and Cairns, 1991). Among benthic animals, minute predators, such as Naididae (Oligochaeta), Orthocladinae (Diptera, Chironomidae), Turbellaria, and Copepoda consume protozoa selectively (Taylor, 1983b; Archbold and Berger, 1985; Kusuoka and Watanbe, 1989). More is known about the feeding of planktonic animals on protozoa (Porter et al., 1985; Sanders and Wickham, 1993; Jack and Gilbert, 1997; Stoecker and Capuzzo, 1990), reﬂecting the recent interest in the microbial loop (Section III.C.3) and how picoplankton production reaches higher trophic levels (e.g., Pomeroy, 1974; Sherr et al., 1987). Most planktonic rotifers consume heterotrophic ﬂagellates, and many prey on ciliates (Arndt, 1993; Gilbert and Jack, 1993). Copepoda especially are known to be consumers of protozoa, and recent studies suggest that copepods may actively select protozoa from among similar-sized plankton (Burns and Gilbert, 1993; Wickham, 1995a,b). Cladocera can ingest ciliates as well as both photosynthetic and nonphotosynthetic ﬂagellates (Sanders and Porter, 1988; Wickham and Gilbert, 1993). There is evidence, however, that ciliates are not always suitable as food 68 W. D. Taylor and R. W. Sanders for Cladocera (Debiase et al., 1990). Fish would seem to be unlikely predators of protozoa, but protozoa are of important food for some larval ﬁshes (Kornienko, 1971; Kentouri and Divanach, 1986). Many planktonic protozoa are capable of rapid or saltatory movements that reduce their probability of being captured. Speeds attained during these movements exceed 100 body lengths per second, and they reduce the frequency of capture compared to species that lack such movements (Gilbert, 1994). Morphological adaptations that reduce probablity of ingestion can be induced by the presence of predators in only a few hours in some ciliates (Wicklow, 1997). The ciliate Lambornella reacts to the presence of predatory mosquito larvae by shifting morphologically and behaviorally to become a mosquito parasite (Washburn et al., 1988). energetics of protozoa (Section II) suggests that protozoa are more rather than less efﬁcient in converting the food they ingest into cytoplasm. Recent studies on nutrient recycling by ﬂagellates suggest that mineralization of nitrogen and phosphorus by protozoa may be particularly low when it is most needed, that is, when their food organisms are themselves N- or P-limited (Goldman et al., 1985; Caron et al., 1988). In situ measurements of nutrient release relative to ingestion are few, but release of P for planktonic ciliates appears to be close to 50%, or so we expect based on conversion efﬁciencies (Taylor and Lean, 1991). Protozoa probably make an important contribution to the regeneration of nutrients in many environments, but no more so than one would expect from their contribution to consumption and production. 10. Protozoa and Nutrient Cycling D. Evolutionary Relationships Regeneration of the inorganic constituents of living organisms occurs through a process involving ingestion, digestion and egestion by consumers, catabolism and autolysis of all organisms, and the enzymemediated breakdown of nonliving organic matter by decomposers. Autotrophs are net consumers of these inorganic constituents, whereas phagotrophs regenerate them. Decomposers, the heterotrophic bacteria and fungi, regenerate carbon; but they may or may not be net producers of other inorganic constituents, depending on the composition of the organic matter they decompose. If it is poor in other constituents, such as organic nitrogen and phosphorus (as when it is mostly carbohydrate), then decomposers will compete with autotrophs for these other constituents. In the ﬁnal analysis, the incorporation and regeneration of inorganic constituents will be in balance unless organic matter is stored or exported. Heterotrophic protozoa ﬁt into this picture as typical consumers, eating other organisms and releasing both unassimilated organic material and end products of catabolism, and as major consumers of the decomposers. The latter role is manifested in the ability of bacterivorous protozoa to accelerate the breakdown of organic material by bacteria (Stout, 1980). This property of protozoa is mostly due to their keeping the bacteria in an actively growing state at moderate abundances, rather than directly due to their contribution to the remineralization process. Protozoa also contribute directly to mineralization, and some early studies suggested protozoa were particularly active in releasing inorganic nutrients. However, the basis of this view has been challenged (Taylor, 1986). Indeed, the relatively independent literature on The relationship of protozoa to plants and animals, as well as the evolutionary relationships among the protozoa, are controversial areas of current research. New tools for the study of phylogeny coming from molecular biology are contradicting morphologybased classiﬁcations, but the number of organisms examined is still small. Here, we will brieﬂy introduce some of the major theories and controversies. One change in view which has occurred over recent years is that the major dichotomy among living things is not between plants and animals, but between prokaryotes and eukaryotes. The evolution of eukaryotes from the prokaryotes is perhaps the major event in organic evolution on earth, and may have occurred by essentially Lamarckian means: the inheritance of acquired characteristics (Taylor, 1987). That is, the eukaryotic cell represents the symbiotic amalgamation of several distantly related prokaryotes (Margulis, 1981). Although this concept is controversial, the controversy is largely one of degree in that the symbiotic origin of some organelles, such as double-membraned mitochondria and chloroplasts, is well supported by molecular techniques and is now widely accepted. Gupta et al. (1994) argued on the basis of a 70-kDa heatshock protein that the eukaryotic nucleus (also double-membraned) may also have been derived by engulﬁng a bacterium. Within the unicellular eukaryotes, or Protista, one of the most primitive forms is the giant amoeba Pelomyxa (Karyoblastea); it lacks mitochondria and has amitotic nuclear ﬁssion. Many consider the ﬂagellum to be as old as the eukaryote cell, but disagree whether colorless ﬂagellates are primitive or derived from pigmented forms. Analyses of ribosomal RNA 3. Protozoa strongly support the former argument for most Zoomastigina. Some protists without mitochondria, including the colorless (and parasitic) ﬂagellate Giardia, are clearly very close to the prokaryotes relative to other protozoa. This strongly suggests that their features are primitive rather than derived (Sogin et al., 1989, 1996). The same analysis reveals that the chlorophyte Chlamydomonas is more closely aligned with higher plants than other protozoa, including other autotrophic phyla (Sogin et al., 1989). Although our view of the relationship among unicellular eukaryotes is in a state of ﬂux, the molecular techniques currently being applied reveal that these groups have had long and independent evolutionary histories. For example, the genetic distances within ciliates or ﬂagellates are greater than the distances between chlorophytes and higher plants, or between plants and animals (Sogin and Elwood, 1986; Gunderson et al., 1987). There are two major schools of thought with respect to the relationship between protozoa and metazoa (Hanson, 1977). One considers that the protozoan ancestor of metazoa is a colonial ﬂagellate, probably a choanoﬂagellate, and that Porifera, Placozoa, Cnidaria, some gastrotrichs, and other animals with monociliated epidermal cells are among the most primitive animals. Others believe that primitive animals were derived by the compartmentalization of a ciliated, multinucleate protozoan. The early development of animals best supports the former hypothesis, as do the data collected thus far on ribosomal RNA; the protozoa least distant from animals are ﬂagellates, not ciliates, which seem to have had a very long independent history. Perhaps the most important conclusions an ecologist can draw from the dynamic ﬁeld of protist systematics is that although the protozoa are all unicellular or colonial and comprise one to many phyla in different classiﬁcation schemes, they are more distant among themselves than multicellular animals from the evolution point of view, and at least as morphologically and physiologically diverse. IV. COLLECTING, REARING, AND PREPARATION FOR IDENTIFICATION The small size, delicacy, and lack of hard parts in most protozoa makes the quantitative collection and enumeration of protozoa particularly difﬁcult. The methods for sample collection and examination vary with the group of interest and the habitat, and there is room for improvement in almost all the procedures mentioned below. General techniques for the beginner 69 are provided in Lee et al. (1985) and Finlay et al. (1988). For some groups — especially amoebas and heterotrophic flagellates, when identification is required or when the sample contains sediment or some other material — examination of live organisms in fresh collections under a dissecting and/or compound microscope is the only method available. With care, this can be done in a quantitative manner (e.g., Bott and Kaplan, 1989). The “most probable number” method of enumeration, based on inoculating culture medium with small aliquots of sample and scoring the fraction of such inocula producing cultures, is common in the study of soil protozoa and has been applied to freshwater. However, the resultant estimates sometimes have been very low relative to direct counts (Bott and Kaplan, 1989). Given the difficulty in culturing many freshwater protozoa, this method should be reserved for estimating species known to be able to grow on the medium used from small inocula. Extraction procedures have been devised for benthic protozoa, but are not widely used (Hairston and Kellerman, 1964; Fenchel, 1967). Fixed samples are not useful in benthic studies, as the task of enumerating fixed but motionless protozoa in sediment is virtually impossible. Studies on epibenthic or aufwuchs protozoa have generally resorted to artiﬁcial substrates. Those that allow for microscopic examination are particularly useful: glass slides (Schönborn, 1977), petri dishes (Spoon and Burbank, 1967; Taylor, 1983b), and strips of nylon mesh (Taylor, 1983a). Polyurethane foam has been extensively used as a substrate in studying protozoan community interactions and response to pollution (Cairns et al., 1979). Planktonic samples present fewer problems. Large protozoa can be settled from samples and counted by inverted microscopy as for phytoplankton. Useful ﬁxatives are Lugol’s Iodine (1% v/v, or higher concentration in saline or hard water, e.g., Taylor and Heynen, 1987) or mercuric chloride (5% saturated mercuric chloride plus a drop of 0.04% bromothymol blue, Pace and Orcutt, 1981). These procedures do not lend themselves to the identiﬁcation of ciliates, nor to the detection of chromatophores in small ﬂagellates. Filtration methods may aid with both of these problems. The quantitative protargol or QPS method of Montagnes and Lynn (1987a,b) and Skibbe (1994) produces permanent, quantitative, stained preparations for identiﬁcation of ciliates and ﬂagellates, although it requires that samples be ﬁxed in a concentrated Bouin’s ﬁxative. The use of epiﬂuorescence microscopy on ﬁltered samples (Caron, 1983) can facilitate the separation of 70 W. D. Taylor and R. W. Sanders heterotrophic and phototrophic protists and is generally the method of choice for enumerating very small planktonic protozoa, even though it does not lend itself to identiﬁcation. Culture of most protozoa is best begun by maintaining samples collected in the ﬁeld. Samples kept in the laboratory will usually undergo succession, with different species waxing and waning in abundance. Screening out metazoan predators, such as copepods, may encourage protozoa to increase in number. Supplementing the organic or nutrient content of the sample may help. Cereal grains (rice or wheat, boiled for quicker effect or plain), broth from boiled vegetable matter (hay, lettuce), or nutrient broth are commonly used. Remember that adding too much is easy, and results in foul conditions that may encourage species different from those important in the original sample; or one may simply kill all the aerobic protozoa. Generally, additions of organic matter to cultures should not be more than about 0.1% w/v for whole vegetable matter, and no more than a tenth as much (100 mg/L) for more labile material. The ﬁrst step in isolating species of interest is to prepare some protozoa-free medium by ﬁltering or boiling water from the site where the protozoa were collected, and inoculating individuals of the desired species back in. Mineral water is frequently used for culturing heterotrophic forms. Again, rice or wheat grains or organic infusions are useful in promoting the moderate growth of bacteria for bactivorous species. Algae or other protozoa may be required as food for others. White worms (enchytreids) are convenient food for histophagous forms. Ultimately, you may wish to establish monoxenic (the organism of interest and its prey only) or axenic cultures, possibly cloned from a single individual. Artiﬁcial freshwater media are described in Finlay et al. (1988) and Thompson et al. (1988). Although involving marine protozoa, the recent efforts by Gifford (1985) might prove a useful template for attempts to culture difﬁcult freshwater forms. A manual of elementary techniques is found in Finlay et al. (1988). General and speciﬁc techniques for studying the ecology of freeliving protozoa are also compiled in Kemp et al. (1993). V. IDENTIFICATION OF PROTOZOA A. Notes about the Key It is often difﬁcult and time consuming to identify protozoa to the level of species. In many cases, unambiguous identiﬁcation requires specialized staining techniques or the use of electron microscopy. The taxonomic grouping used in this key is an amalgamation of publications by specialists on the different groups (e.g., Page, 1998; Lynn and Small, 1997; Lee et al., 1985; Margulis et al., 1989). While the taxonomy is based on ultrastructural features and morphometric attributes that in many cases are not readily discerned with light microscopy, this key is designed to make possible the identiﬁcation of many protozoa to a family or genus level using light microscopy alone. Although observation of living organisms is important for identiﬁcation, the key should still be useful for many ﬁxed samples. Most of the specialized terms used in the key are deﬁned in the general glossary. If one becomes more interested in identifying protozoa to a species level, specialized techniques and keys in the publications cited earlier can be used. The genera listed in the key are representative of the families and are not complete. Likewise, the illustrations used as examples here are of one or more species considered typical of a genus, but do not necessarily represent all of the species in the genus. Advances in the systematics of the protozoa are continuous. Due to the heterogeneity of the group, it is often difﬁcult to remain abreast of the changes. A new edition of Lee et al. (1985), An Illustrated Guide to the Protozoa, is expected to be published by the Society of Protozoologists at any time, and will likely have changes in systematic organization. Classiﬁcation below the class level in the Zoomastigina and the ordinal level in Heliozoa are not conclusively agreed upon, or are under revision. Thus, the use of familial names was avoided for these groups in the key. Phyla of ﬂagellates marked with an asterisk (*) contain many or mostly photosynthetic members; genera listed have one or more species that lack chloroplasts and are thus heterotrophic, or contain chloroplasts, but also exhibit heterotrophic nutrition. B. Taxonomic Key to Functional Groups of Protozoa 1a. Locomotion via pseudopodia as in Figure 8. Rarely, ﬂagella may be present as well (e.g., Fig. 9 C) .................................................... Amoeboid Protozoa (Section V.D) 1b. Simple cilia or compound ciliary organelles characteristic (but see Fig. 19A – P) and present in at least one part of life cycle; subpellicular infraciliature present even when cilia are not; two types of nuclei (macronucleus and micronucleus) with rare exceptions (Fig. 23J). Ciliated Protozoa, Phylum Ciliophora (see Section V.E) 3. Protozoa 1c. 71 Commonly 1 to 4 ﬂagella, 16 to 24 in one free-living genus (Paramastix); binary ﬁssion................................................... Flagellated Protozoa (see Section V.C) C. Taxonomic Key to Major Groups and Common Genera of Flagellated Protozoa 1a. With median groove (annulus and sulcus); two ﬂagella, one extending transversely around the cell............................................... Phylum Dinoﬂagellata* (Peridinium, Gymnodinium, Gyrodinium; Fig. 13A – C) 1b. Without a median groove .................................................................................................................................................................. 2 2a (1b). Often large ﬂagellates with pharyngeal rods (sometimes difﬁcult to see) or containing the reserve material paramylon; one or usually two ﬂagella arise from within an anterior invagination (reservoir); contractile vacuole associated with reservoir; elongate; body shape often plastic when living. Phylum Euglenida* (Khawkinea, Entosiphon, Petalomonas, Peranema, Urceolus; Fig. 13D,F – I) 2b. Cells not as above ..............................................................................................................................................................................3 3a (2b). Flagellates with two or four equal ﬂagella. Phylum Chlorophyta* (Polytoma, Polytomella; Fig. 13E) 3b. Cells with two unequal ﬂagella...........................................................................................................................................................4 4a (3b). Small cells with a deep, subapical gullet; two nearly equal length ﬂagella arise from gullet................................... Phylum Cryptophyta* (Chilomonas; Fig. 7C, Fig. 13J) 4b. Cells without deep gullet ....................................................................................................................................................................5 5a (4b). Typically with two unequal ﬂagella; long ﬂagellum usually directed forward during swimming, with short ﬂagellum directed backwards if emergent; compact Golgi body frequently visible anterior to nucleus of larger cells; chrysolaminarin vescicle(s) often ﬁll posterior of cell; contractile vacuole usually present in extreme anterior end of cell; many species capable of simultaneous photosynthesis and phagocytosis. Phylum Chrysophyta* (Dinobryon, Monas, Ochromonas, Paraphysomonas, Uroglena; Fig 7B, 13K – N, 14A) 5b. Cell not as above. Phylum Zoomastigina ...........................................................................................................................................6 6a (5b). Single anterior ﬂagellum encircled laterally by a tentacular, funnel-shaped collar; solitary or colonial; with or without theca. Class Choanoﬂagellida (Codosiga, Desmarella, Sphaeroeca, Diploeca, Monosiga, Salpingoeca; Fig. 4A, 14B – H) 6b. Without collar, or with indistinct collar..............................................................................................................................................7 7a (6b). Like Choanoﬂagellida above, but with second trailing ﬂagella attached to the base of lorica; protoplasmic collar rudimentory. Bicoecid ﬂagellates, afﬁnities uncertain (Bicoeca; Fig. 14I) 7b. Without protoplasmic collar...............................................................................................................................................................8 8a (7b). Flagellates with two unequal length ﬂagella, one trailing; ingestion by pseudopodia. (Cercomonas; Fig. 14K) 8b. Not as above ......................................................................................................................................................................................9 9a (8b). With one or usually two ﬂagella arising from a ﬂagellar pocket (depression); characteristically elongate or bean shaped. Unique organelle, the kinetoplast, usually associated with the ﬂagella. Class Kinetoplastida (Bodo, Cephalothamnium, Pleuromonas, Rhynchomonas; Fig. 14J,L,N – P) 9b Cells with one or two nucleus-ﬂagella complexes (karyomastigonts) each with 1 – 4 ﬂagella; no Golgi apparatus; when two karyomastigonts, mirrored symmetry of nuclei and ﬂagella. Diplomonads (Hexamita; Fig. 14M) D. Taxonomic Key to the Major Groups and Common Genera of Protozoa with Pseudopodia 1a. Large, cylindrical or ovoid multinucleate amoeba; bacterial symbionts; usually containing mineral particles; nonmotile ﬂagella-like extensions; oxygen poor habitats Phylum Karyoblastea. (one genus, Pelomyxa; Fig. 8B, Fig. 16V) 1b. Pseudopodia hyaline, usually blunt, eruptive (Fig. 8A,C,F) but ﬁliform process may occur. May be ﬂagellated stages........................2 1c. Hyaline, ﬁliform pseudopodia (ﬁlopodia) sometimes branching, not anastomosing (Fig. 8E); no known ﬂagellate stages or spores. Class Filosea.....................................................................................................................................................................................21 1d. Threadlike and delicate pseudopodia with ﬁnely granular appearance, often branching and anastomosing to form complex reticulum (reticulopodia, Fig. 8G). Test often present. Phylum Granuloreticulosea ...................................................................................27 1f. Long slender axopods (Fig. 8D) radiating from cell; with or without skeletal elements. Phylum Heliozoa .......................................32 FIGURE 14 (A) Uroglena americana(mixotrophic); (B) Desmarella moniliformis; (C,D) Sphaeroeca volvox, individual cell and colony; (E) Codosiga botrys; (F) Diploeca placita; (G) Salpingoeca fusiformis; (H) Monosiga ovata; (I) Bicoeca lacustris; (J) Bodo caudatus; (K) Cercomonas sp.; (L) Cephalothamnion cyclopum; (M) Hexamita inﬂata; (N,O) Pleuromonas jaculans, attached and amoeboﬂagellate forms; (P) Rhynchomonas nasuta. (After: Bourrelly, 1968, L; Calaway and Lackey, 1962, N, O, P; Lackey, 1959, B, F; Lee, 1985, K; Pascher, 1913, A; Pascher, 1914, C, D, E, G, H, I, J, M.) Scale 2.5 m for P; 5 m for F, G, H, I, K, L; 10 m for A, B, C, J, M, N, O; and 20 m for E. 72 3. Protozoa 73 2a (1b). Small, usually cylindrical amoebae, many with a ﬂagellated stage (Fig. 9C). Phylum Rhizopoda, Class Heterolobosea, Order Schizopyrenida ...................................................................................................................................................................................3 2b No ﬂagellated stages. Class Lobosea ..................................................................................................................................................4 3a (2a). Amoeboid form cylindrical, monopodial, eruptive; temporary ﬂagellate stages common; nucleolus divides to form polar masses in mitosis...................................Vahlkampﬁidae (Valkampﬁa, Naegleria; Fig. 15A – B) 3b Nucleolus disintegrates during mitosis, nuclear membrane does not; single known freshwater species; commonly ﬂattened; no ﬂagellate stage known...................................Gruberellidae (Stachyamoeba; Fig. 15C) 4a (1b). Lacking external test. Subclass Gymnamoebia ...................................................................................................................................5 4b. Incompletely enclosed in a test or other ﬂexible cuticle of microscales. Subclass Testacealobosia .....................................................13 5a (4a). Cylindrical or ﬂattened; ﬂattened forms with regular outline; no trailing uroidal ﬁlaments, with rare exceptions; not strikingly eruptive. Order Euamoebida ..............................................................................................................................................................6 FIGURE 15 (A) Vahlkampﬁa avaria; (B) Naegleria; (C) Stachyamoeba lipophora; (D) Thecamoeba sphaeronucleolus; (E) Vanella miroides; (F) Amoeba proteus; (G) Mayorella bigomma; (H) Vexillifera telemathalassa; (I) Hartmanella vermiformis; (J) Chaos illinoisense; (K) Saccamoeba lucens; (L) Trichamoeba cloaca; (M) Echinamoeba exudans; (N) Acanthamoeba; (O) Filamoeba nolandi (P) Hylodiscus rubicundus. (After: Bovee, 1985, A, B, C, D, H, I, J, M, N, O; Kudo, 1966, F; Page, 1988, E, G, K, L, P.) Scale 10 m for A, B, C, E, I, M; 15 m for H, N, O, P; 30 m for D, G, K, L; 50 m for F; and 100 m for J. 74 W. D. Taylor and R. W. Sanders 5b. Usually ﬂattened; frequent changes in shape typical; sometimes eruptive; subpseudopodia usually present, often furcate ..............................................................................................................................................................................................11 6a (5a). Without subpseudopodia....................................................................................................................................................................7 6b. With subpseudopodia.........................................................................................................................................................................9 7a. (6a). Cell body ﬂattened .............................................................................................................................................................................8 7b. Cell body subcylindrical ...................................................................................................................................................................10 8a (7a). Cell usually oblong; cresent-shaped hyaline margin at anterior end; pellicle-like layer, with dorsum often wrinkled and/or ridged; usually uninucleate; no cytoplasmic crystals. Thecamoebidae (Thecamoeba; Fig. 15D) 8b. Body usually fan-shaped, oval, or spoon-shaped, with hyaline margin occupying up to half of length. Vannellidae (Vannella, Fig. 15E) 9a (6b). Subpseudopodia hyaline, blunt, digitiform, usually from anterior hyaline margin; uninucleate; nucleolar material in central body. Paramoebidae (Mayorella, Fig. 15G) 9b. Few slender, conical, or linear subpseudopodia, from anterior hyaline margin or cell surface; uninucleate. Vexilliferidae (Vexillifera, Fig. 15H) 10a (7b). Most species polypodial; length usually more than 75 m; uni- or multinucleate; numerous cytoplasmic crystals. Amoebidae (Amoeba, Chaos, Trichamoeba; Fig. 15F,J,L) 10b. Cell monopodial, pseudopods rare; uninucleate with central nucleolus; cytoplasmic crystals in some; cysts common. Hartmannellidae (Hartmannella, Saccamoeba; Fig. 15I,K) 11a (5b). Cell regularly discoid, ﬂattened ovoid, or fan-shaped; usually broader than wide; postcentral granular mass, usually surrounded, sometimes completely, by hyaline border with short subpseudopodia. Hyalodiscidae (Hyalodiscus; Fig. 15P) 11b. Not as above ....................................................................................................................................................................................12 12a (11b). Cell ﬂattened, broad and irregular in outline, though sometimes elongate during locomotion; slender tapering subpseudopodia, sometimes furcate, produced from broad, hyaline lobopodium; often with small lipid globules; uninucleate. Order Acanthopodida, Acanthamoebidae (Acanthamoeba; Fig. 15N) 12b. Several to many ﬁne, sometimes furcate subpseudopodia, ﬁner than in Acanthamoebidae. Echinamoebidae (Echinamoeba, Filamoeba; Fig. 15M,O) 13a (4b). Test more or less rigid with distinct aperture. Order Arcellinida.......................................................................................................11 13b. Discoid or sometimes globose amoeba incompletely closed in a ﬂexible tectum, no well deﬁned aperature. Order Himatismenida, Cochliopodiidae (Cochliopodium, Fig. 16A) 14a (13a). Pseudopods conical, clear, sometimes anastomosing. Suborder Phryganellina, Phryganellidae (Phryganella; Fig. 16B – C) 14b. Pseudopods digitate and ﬁnely granular ...........................................................................................................................................15 15a (14b). Test membranous or chitinoid, pliable or rigid; no plates or scales, but may have attached debris. Suborder Arcellina...........................................................................................................................................................................................16 15b. Test chitinoid or not, rigid, with embedded and/or attached plates, scales, siliceous granules. Suborder Difﬂugina .........................................................................................................................................................................................18 16a (15a). Test ﬂexible to semi-rigid; aperture ventral with variable shape. Microcoryciidae (Penardochlamys; Fig. 16H – I) 16b. Test rigid, chitinoid ..........................................................................................................................................................................17 17a (16b). Test often areolar, smooth; aperture ventral, round. Arcellidae (Arcella, Pyxidicula; Fig. 16D,F – G) 17b. Test oval to ﬂask-shaped, nonareolar, clear; aperture terminal. Hyalospheniidae (Hyalosphenia; Fig. 16L – M) 18a (15b). Aperture round, broadly oval or wavy. Difﬂugiidae (Difﬂugia, Lesquereusia; Fig. 16J – K,N) 18b. Aperture slit-like or narrowly oval ...................................................................................................................................................19 19a (18b). Aperture terminal .............................................................................................................................................................................20 19b. Aperture antero-ventral, invaginated, slit-like with overhanging lip. Plagiopyxidae (Plagiopyxis; Fig. 16E) 20a (19a). Test particles rectangular. Paraquadrulidae (Quadrulella; Fig. 16O – P) 20b. Test particles not rectangular. Nebelidae (Nebela; Fig. 16Q) 21a (1d). Without distinct test, sometimes with scales. Order Aconchulinida ..................................................................................................22 21b. With test. Order Testaceaﬁlosida......................................................................................................................................................23 3. Protozoa 75 FIGURE 16 (A) Cochliopodium bilimbosum; (B, C) Phryganella nidulus; side and oral views; (D) Pyxidicula operculata; (E) Plagiopyxis callida; (F, G) Arcella vulgaris, side and dorsal views; (H, I) Penardochlamys arcelloides, side and oral views; (J, K) Difﬂugia corona, side and oral views; (L, M) Hyalosphenia cuneata; (N) Lesquereusia spiralis; (O, P) Quadrulella symmetrica; (Q) Nebela collaris; (R) Penardia granulosa; (S) Chlamydophrys minor; (T, U) Lecythium hyalinum, dorsal and side; (V) Pelomyxa palustrus; (W) Pseudo-difﬂugia gracilis; (After: Bovee, 1985, A, B, C, H, I, J, K, N, O, P, R, T, U; Deﬂandre, 1959, D, E, F, G, L, M, Q, S, W; Kudo, 1966, V.) Scale 10 m for C, R, S; 25 m for A, H; 30 m for L, T, W; 45 m for N, O; 60 m for Q; 90 m for B, E, F, J; and 500 m for V. 22a (21a). Filopodia nonanastomosing and more or less radiate. Vampyrellidae (Vampyrella; Fig. 17A) 22b. Filopodia distally branching and anastomosing. Reticulosidae (Penardia; Fig. 16R) 23a (21b). Test without scales; may have spines, attached debris.......................................................................................................................24 23b. Test with secreted, siliceous scales arranged in deﬁnitive patterns.....................................................................................................25 24a(23a). Test round, thin; may have spines or spicules. Chlamydophryidae (Chlamydophrys, Lecythium; Fig. 16S – U) 24b. Test not round. Pseudodifﬂugiidae (Pseudodifﬂugia; Fig. 16W) 76 W. D. Taylor and R. W. Sanders FIGURE 17 (A) Vampyrella lateritia; (B) Paraeuglypha reticulata; (C) Euglypha tuberculata; (D, E) Trinema enchelys, oral and side views; (F) Sphenoderia lenta; (G, H) Cyphoderia ampulla; (I, J) Campascus triqueter; (K) Paulinella chromataphora; (L) Diplophyrys archeri; (M) Lieberkuehnia wagnerella; (N) Microcometes paludosa; (O) Microgromia haeckeliana; (P) Biomyxa vagans; (Q) Chlamydomyxa montana; (R) Reticulomyxa ﬁlosa. (After: Bovee, 1985, B, D, E, F, G, H, I, J, K, L, M, N, O, P, Q, R; Deﬂandre, 1959, A, C.) Scale 10 m for L, N, O; 15 m for K; 25 m for A, B, C; 40 m for D, F, G; 50 m for I, M, Q; 80 m for P; and 10,000 m for R. 25a (23b). Scales round to elliptical, thin, overlapping, adjacent or scattered. Euglyphidae (Paraeuglypha, Euglypha, Trinema, Sphenoderia; Fig. 17B – F) 25b. Scales not as above ...........................................................................................................................................................................26 26a (25b). Scales thick, cylindrical; test usually with aperture at end of a neck bent to one side. Cyphoderiidae (Cyphoderia, Campascus; Fig. 17G – I) 26b. Scales long, with long axes perpendicular to pseudostome; with two symbiotic cyanobacterial ﬁlaments. Paulinellidae (Paulinella; Fig. 17K) 3. Protozoa 77 27a (1e). Phylum Granuloreticulosea. With a test. Class Monothalamea ........................................................................................................28 27b. Without a test. Class Athalamea...................................................................................................................................................... 30 28a (27a). Test with one pseudostome (opening)...............................................................................................................................................29 28b. With more than one pseudostomes. Amphitremidae (Diplophyrys, Microcometes; Fig. 17L,N) 29a (28a). Test ﬂattened on one side. Microgromiidae (Microgromia; Fig. 17O) 29b. Test not ﬂattened on one side. Lieberkuehnidae (Lieberkuehnia; Fig. 17M) 30a (27b). Naked amoebae with or without anastomosing pseudopodia; one or a few nuclei Order Athalamida..............................................31 30b. Naked, multinucleate and highly reticulate plasmodia. Order Promycetozoida, Family Reticulomyxidae (Reticulomyxa; Fig. 17R) 31a (30a). Body mass changeable in form. Biomyxidae (Biomyxa; Fig. 17P) 31b. Body mass more or less round and central. Chlamydomyxidae (Chlamydomyxa; Fig. 17Q) 32a (1f). Class Heliozoa; Axopodia without visible axonemes........................................................................................................................33 32b. Axonemes usually visible with light microscopy ...............................................................................................................................34 33a (32a). Cell enclosed in latticed organic capsule (skeleton); generally stalked; no centroplast; cell body spherical in adults. Order Desmothoracida (Clathrulina; Fig. 18A) FIGURE 18 (A) Clathrulina elegans; (B) Actinophrys sol; (C) Actinosphaerium eichhorni; (D) Heterophrys myriopoda; (E) Ciliophrys infusionum; (F) Acanthocystis turfacea; (G) Lithocolla globosa; (H) Raphidiophrys elegans. (After: Deﬂandre, 1959, H; Kudo, 1966, B, C, D, E; Rainer, 1968, A, F, G.) Scale 15 m for E; 30 m for B, D, G; 50 m for A; 75 m for F, H; and 160 m for C. 78 W. D. Taylor and R. W. Sanders 33b. Lack centroplast and apparently lack axonemes, the lack of microtubules (axonemes) in axopods has not been conﬁrmed and this order may be abandoned. Order Rotosphaerida (Lithocolla; Fig. 18G) 34a (32b). Stalked or not; long thin granule-studded axopods that usually arise from centroplast; axonemes frequently discernible with light microscopy; usually with skeleton of siliceous or organic plates and/or spicules; highly contractile axopods or stalk. Order Centrohelida. (Heterophrys, Acanthocystis, Raphidiophrys; Figs. 7, 18D, F,H) 34b. No skeleton ......................................................................................................................................................................................35 35a (34b). No centroplast or axoplast; axopods granual-studded and thicker at bases; large central nucleus surrounded by lacunar ectoplasm or several nuclei at periphery of central area with vesicular ectoplasm. Order Actinophryida (Actinophrys, Actinosphaerium; Fig. 18B – C) 35b. Microtubule organizing center of dense plaques from the nuclear membrane or from centroplast; may be confused with Actiniphryida without knowledge of ﬁne structure (origin and pattern of axopod microtubules). Order Ciliophyrida (Ciliophrys; Fig. 18E) E. Taxonomic Key to Major Groups and Common Genera of Ciliates 1a. Suctorial tentacles present; no true cytostome or cytopharynx; adults (trophonts) usually sessile, many species ectosymbiotic, some planktonic species; cilia absent except in free-swimming dispersal stages; with or without lorica. Class Phyllopharygea, Subclass Suctoria ..............................................................................................................................................................................................9 (Note: Patterns of division and release of the larvae form the basis for dividing the subclass into orders. Other characters are useful in identiﬁcation, but knowledge of the full life cycle is often required before suctorians can be conﬁdently assigned to a genus.) 1b. Cilia present in unencysted stages; no suctorial tentacles ....................................................................................................................2 2a (1b). Conspicuous buccal ciliature at apical pole; buccal ciliature winds clockwise toward the center when viewed from oral end; somatic cilature reduced or absent; mobile or sessile; solitary, gregarious or colonial; some species loricate; oral region can contract and withdraw in most species. Class Oligohymenophora, Subclass Peritrichia ........................................................................................17 2b. Not as above ......................................................................................................................................................................................3 3a (2b). Oral area usually bordered by a well-developed adoral zone of membranelles (AZM) consisting of more than three membranelles (polykineties); with or without ventral cirri ......................................................................................................................................26 3b. Oral area not as above; no ventral cirri ..............................................................................................................................................4 4a (3b). Cytostome at or near surface; buccal cavity, if present, without cilia or paroral membranes ..............................................................5 4b. Cytostome at end of a buccal cavity with cilia or paroral membrane(s) associated .............................................................................6 5a (4a). Cytostome at or near anterior end, or continuing down side as a slit ...............................................................................................52 5b. Cytostome lateral or ventral ...............................................................................................................................................................7 6a (4b). Paroral membrane(s) within or leading into buccal cavity ................................................................................................................65 (Sometimes difﬁcult to see; follow both alternatives in key when in doubt) 6b. No paroral membrane; simple cilia in buccal cavity .........................................................................................................................82 7a (5b). Cytostome a barely visible lateral slit on the convex side of a tapering front end, or a lateral opening at the base of an anterior proboscis..........................................................................................................................................................................................63 (see also Spathidiidae, 55) 7b. Circular mouth located mid-ventrally; no proboscis; large cyrtos .......................................................................................................8 8a (7b). Body nearlly ellipsoid, rounded in cross section, sometimes ﬂattened ventrally; medium to large (some 100 m); densely ciliated all over; oral depression present. Class Nassophorea, Order Nassulida, Nassulidae (Nassula; Fig. 20Z) 8b. Body usually ﬂattened; ventrum ciliated, dorsum bare or with a few cilia; anterior preoral arcs of right ventral ciliary rows continuous with more posterior parts. Class Phyllopharyngea, Order Cyrtophorida, Chilodonellidae (Chilodonella; Fig. 24T) 9a (1a). Budding and cytokinesis on surface of trophont. Order Exogenida ..................................................................................................10 9b. Budding begins in a pouch ...............................................................................................................................................................12 10a (9a). Trophont basally attached to bottom of lorica near stalk. (Thecacineta; Fig. 19A) 10b. Not as above ....................................................................................................................................................................................11 11a (10b). With vase-like lorica, sometimes with stalk-like base; tentacles extend through one or more slits. Urnulidae (Metacineta, Paracineta; Fig. 19B – D) 3. Protozoa 79 FIGURE 19 (A) Thecacineta cothurniodes; (B, C) Metacineta mystacina, top and side views; (D) Paracineta crenata; (E) Podophyra ﬁxa, showing trophont, encysted form, and swarmer; (F) Acineta limnetis; (G) Sphaerophyra magna; (H) Trichophyra epistylidis; (I) Dendrocometes paradoxus; (J) Heliophyta reideri; (K) Tokophyra quadripartita; (L) Multifasciculatum elegans; (M) Squalorophyra macrostyla; (N) Discophyra elongata; (O) Stylocometes digitalis; (P) Dendrosoma radians. (After: Corliss, 1979, P; Goodrich and Jahn, 1943, F, K, L, M; Kent, 1880 – 1882, G, I; Matthes, 1972, J, O; Noland, 1959, A, B, C, D, N; Small and Lynn, 1985, E, H.) Scale 15 m for E, H, J, O; 30 m for A, D, F, G; 50 m for I, L, M, N; 75 m for B, K; and 200 m for P. 11b. Trophont small, pyriform to spherical; usually stalked; tentacles apical or evenly distributed; often attached to other ciliates. Podophryidae (Podophyra, Sphaerophyra; Fig. 19E,G) 12a (9b). Budding and cytokinesis completed in brood pouch. Order Endogenida . . . ....................................................................................13 12b. Cytokinesis begun in pouch, completed exogenously. Order Evaginogenida ....................................................................................15 13a (12a). Trophont without stalk or stalk-like process; attached to substrate by broad part of body. Dendrosomatidae (Dendrosoma, Trichophyra; Fig. 19H,P) 80 W. D. Taylor and R. W. Sanders FIGURE 20 (A) Hastatella radians; (B) Astylozoon faurei; (C) Urceolaria mitra; (D) Trichodina pediculis; (E) Scyphidia physarum; (F) Cothurnia imberbis; (G) Vaginicola ingenita; (H, I) Zoothamnium arbuscula, individual and colony; (J) Ophrydium eichhorni; (K) Vorticella campanula; (L) Pyxicola afﬁnis; (M) Platycola longicollis; (N) Thuricola folliculata; (O) Epistylis plicatilis; (P) Rhabdostyla pyriformis; (Q, R) Carchesium polypinum, individual and colony; (S) Opercularia nutans; (T, U) Campanella umbellaria, individual and colony; (V) Pseudomicrothorax agilis; (W) Microthorax pusillus; (X) Aspidisca costata; (Y) Euplotes patella; (Z) Nassula ornata. (After: Corliss, 1959, V, Y; Kahl, 1930 – 1935, A, B, C, D, E, H, L, N, Q, R, T, U, W; Kent, 1880 – 1882, I, J, K, O, S, X; Noland, 1959, F, G, M, P.) Scale 15 m for V, W; 20 m for A, B, G, P; 25 m for D, H, F, X; 30 m for C, Z; 40 m for E, L, M, N, S; 50 m for O, Y; 75 m for K, Q, U; and 200 m for J. 3. Protozoa 13b. 81 Not as above ....................................................................................................................................................................................14 14a (13b). With lorica, usually stalked; tentacles in a few fascicles or distributed over body. Acinetidae (Acineta; Fig. 19F) 14b. Lacks lorica; tentacles single, fascicles, or evenly distributed. Tokophryidae (Tokophyra, Multifasciculatum; Fig. 12 and 19K,L) 15a (12b). Conical tentacles; body often hemispherical . Dendrocometidae (Dendrocometes, Stylocometes; Fig. 19I,O) 15b. Not as above, capitate tentacles .......................................................................................................................................................16 16a (15b). Body discoidal, ﬂattened against substrate, with peripheral attachment ring. Heliophryidae (Heliophrya, Fig. 19J) 16b. Thin tentacles; body spherical to ovoid; some species loricate and/or stalked. Discophryidae (Discophrya, Squalorophyra; Fig. 19M,N) 17a (2a). Trophont mobile; symbiotic on other organisms; with complex, aboral holdfast. Order Mobilida...................................................18 17b. Trophont only rarely motile, attached with stalk; often attached to other organisms; many gregarious or colonial species. Order Sessilida............................................................................................................................................................................................19 18a (17a). Denticles of holdfast with hooks and spines; ectosymbionts on Hydra, ﬁshes, Amphibia. Trichodinidae (Trichodina; Fig. 20D) 18b. Denticles of holdfast simple, toothed; elongated macronucleus, ectosymbionts on turbellarians. Urceolariidae (Urceolaria; Fig. 20C) 19a (17b). Free-swimming; aboral end drawn to point with one or two rigid bristles or cilia; swim with oral end forward. Astylozoidae (Astylozoon, Hastatella; Fig. 20A,B) 19b. Aborally attached to substratum directly or by stalk ........................................................................................................................20 20a (19b). Colonial, with zooids in gelatinous matrix. Ophrydiidae (Ophrydium; Fig. 20J) 20b. Not in gelatinous matrix ..................................................................................................................................................................21 21a (20b). Stalk absent, tapering body may resemble short stalk. Scyphidiidae (Scyphidia; Fig. 20E) 21b. Stalked, or, if stalkless, in lorica .......................................................................................................................................................22 22a (21b). With contractile stalk; solitary or colonial........................................................................................................................................23 22b. If stalked, stalk not contractile; solitary or colonial ..........................................................................................................................24 23a (22a). Each zooid independently contractile; if solitary, entire stalk contractile. Vorticellidae (Carchesium, Vorticella; Fig. 20K,Q – R) 23b. Entire colony contracts in unison due to shared, continuous myonemes. Zoothamniidae (Zoothamnium; Fig. 20H – I) 24a (22b). With vase-shaped lorica, stalkless or short stalked; slender body attached to lorica aborally. Vaginicolidae (Cothurnia, Platycola, Pyxicola, Thuricola, Vaginicola; Fig. 20F,G,L – N) 24b. Not as above ....................................................................................................................................................................................25 25a (24b). Stalked; peristomal area raised on short neck with furrow separating it from margin of zooid. Operculariidae (Opercularia; Fig. 20S) 25b. Stalked; solitary or colonial; peristomal area not on neck, no furrow. Epistylididae (Campanella, Epistylis, Rhabdostyla; Fig. 20O, P, T, U) 26a (3a). Locomotor cirri present on ventral surface; generally dorso-ventrally ﬂattened. Class Spirotrichea, subclass Hypotrichia................27 26b. Not as above ....................................................................................................................................................................................36 27a (26a). Adoral zone of membranelles well developed, prominent .................................................................................................................28 27b. AZM reduced; no marginal or caudal cirri, ventral cirri conspicuous. Class Spirotrichea, Subclass Hypotrichia, Order Euplotida, Aspidiscidae (Aspidisca; Fig. 20X) 28a (27a). Right marginal row of cirri absent, one or more left marginal cirri; large caudal cirri; paroral membrane only as a ﬁeld on right of oral area. Class Spirotrichea, Subclass Hypotrichia, Order Euplotida (Euplotes; Fig. 20Y) 28b. Left oral cilia as a “collar” and “lapel”, right oral cilia variable with one or more paroral membranes. Class Spirotrichea, Subclass Stichotrichia .....................................................................................................................................................................................29 29a (28b). Ventral cirri not in distinct ﬁle from anterior to posterior (exception: Gastrostyla) Oxytrichidae (Gastrostyla, Oxytricha, Stylonychia; Fig. 21A,C,E) 29b. Ventral cirri in distinct linear or zig-zag ﬁle(s) ..................................................................................................................................30 30a (29b). Frontoventral cirri run length of ventrum as zig-zag ﬁles. Urostylidae (Urostyla, Uroleptus; Fig. 21B,D) 82 W. D. Taylor and R. W. Sanders 30b. Ventral cirri in one or more linear ﬁles of varied length....................................................................................................................31 31a (30b). Body in lorica; without marginal cirri; anterior end long, narrow, contractile, Chaetospiridae (Chaetospira; Fig. 21N,O) 31b. Not as above ....................................................................................................................................................................................32 32a (31b). Files of cirri curved or spiralled along body......................................................................................................................................33 32b. Files of cirri straight or oblique, ventral only....................................................................................................................................35 33a (32a). Files of cirri end on dorsum; oral region extends over one-fourth of body length. Strongylidiidae (Strongylidium; Fig. 21P) 33b. Not as above ....................................................................................................................................................................................34 FIGURE 21 (A) Gastrostyla steini; (B) Uroleptus piscis; (C) Oxytricha fallax; (D) Urostyla grandis; (E) Stylonychia mytilus; (F) Gonostomum afﬁne; (G) Amphisiella oblonga; (H) Stichotricha aculeata; (I) Hypotrichidium conicum; (J) Discomorphella pectinata; (K) Metopus es; (L) Myelostoma ﬂagellatum; (M) Saprodinium dentatum; (N, O) Chaetospira mülleri, contracted and extended forms; (P) Strongylidium crassum; (Q) Psilotricha acuminata; (R) Caenomorpha medusula; (S) Tintinnidium ﬂuviatile; (T) Tintinnopsis cylindricum; (U) Strombidinopsis setigera; (V) Strombidium viride; (W) Halteria grandinella; (X) Strombilidium gyrans. (After: Jankowski, 1964a, b, J, M; Kahl, 1930 – 1935, F, G, H, I, K, L, N, O, P, Q, R, V, W, X; Kent, 1880 – 1882, A, B, C, D, E; Noland, 1959, S, T, U.) Scale 15 m for L; 25 m for H, W, X; 30 m for F, I, J, P,Q, R, T; 40 m for G, K, M, N, O, S, U, V; 60 m for B, E; 80 m for A, C; and 140 m for D. 3. Protozoa 83 34a (33b). Ventral ﬁles of cirri curve to left rear. Psilotrichidae (Psilotricha; Fig. 21Q) 34b. Ventral ﬁles of cirri spiral obliquely around body. Spiroﬁlidae (Hypotrichidium, Stichotricha; Fig. 21H,I) 35a (32b). Files of cirri parallel to long axis of oral region along its right border. Gonostomatidae (Gonostomum; Fig. 21F) 35b. Files of cirri not parallel to oral region, one or more of the ventral cirral ﬁles extends from anterior to well past mid-ventrum. Amphisiellidae (Amphisiella; Fig. 21G) 36a (26b). Body surface ciliated ........................................................................................................................................................................46 36b. Somatic ciliature sparse or absent.....................................................................................................................................................37 37a (36b). Body ﬂattened, rigid, often with spines; body ciliature present as short, generally obvious rows; oral ciliature relatively inconspicuous; mainly anaerobic. Class Spirotrichea, Order Odontostomatida ................................................................................................38 37b. Not as above ....................................................................................................................................................................................40 38a (37a). Body box-shaped, generally with short posterior spines; short rows of body cilia at front and rear, those in front parallel to and forward of oral opening. Epalxellidae (Saprodinium; Fig. 21M) 38b. Body round to discoidal ...................................................................................................................................................................39 39a (38b). Distinct anterior spine; band of cilia on ridge overhanging buccal cavity. Discomorphellidae (Discomorphella; Fig. 21J) 39b. Body ciliature very sparse; lacks spines; pair of cirri at posterior end. Myelostomatidae (Myelostoma; Fig. 21L) 40a (37b). Membranelles numerous in complete or almost complete circle at oral end; body generally conical or bell-shaped; mostly planktonic. Class Spirotrichea, Subclasses Choreotrichia and Oligotrichia................................................................................................41 40b. Body rounded or conical, spirally twisted to left; twisting of body less prominent than Metopidae; often with long caudal spine; oral region spiralled; dense preoral cilia along anterior border of oral cavity; body cilia absent except for caudal tuft and anterior cirruslike tufts; anaerobic. Class Spirotrichea, Order Armophorida (Metopus, Caenomorpha; Fig. 21M,R) 41a (40a). Oral ciliature forms a closed circle. Subclass Choreotrichia..............................................................................................................42 41b. Oral cilature forms an open anterior collar with an anterioventral “lapel”. Subclass Oligotrichia ...................................................45 42a (41a). Loricate, attached to inner wall of lorica ......................................................................................................................................... 43 42b. Not loricate ......................................................................................................................................................................................44 43a (42a). Delicate, gelatinous or mucoid tubular lorica, with attached debris, often translucent . Tintinnididae (Tintinnidium; Fig. 21S) 43b. Lorica rigid, agglomerate of mineral grains and diatom fragments. Codonellidae (Tintinnopsis; Fig. 21T) 44a (42b). Rows of somatic ciliature equally distributed around body, extending length of body; body cilia may be long. Strombidinopsidae (Strombidinopsis; Fig. 21U) 44b. One or more usually short rows of body cilia; body ciliature short. Suborder Strobilidiina, Strobilidiidae (Strobilidium, Fig. 21X) 45a (41b). Somatic ciliature reduced to a few bristle-like cirri arranged circumferentially around the equator effecting a jumping motion. Halteriidae (Halteria; Fig. 21W) 45b. Somatic ciliature greatly reduced or absent, no bristles; sometimes with prominent girdle close to middle of cell. Strombidiidae (Strombidium; Fig. 21V) 46a (36a). Uniform somatic ciliation; often with conspicuous tuft of caudal cilia; buccal membranelles large, but not always obvious; anterior end of body twisted left; oral region spiralled with dense preoral ciliation; in richly organic environments with low oxygen. Class Spirotrichea, Order Armophorida, Metopidae (Metopus; Fig. 21K) 46b. Not as above ....................................................................................................................................................................................47 47a (46b). Somatic ciliature well developed, uniform, usually inserts on oral region; body size large; many contractile species; rarely loricate; some species pigmented. Class Heterotrichea, Order Heterotrichida ................................................................................................48 47b. Somatic ciliature well developed; body large (500 –1,000 m), broad; buccal cavity prominent, funnel-like; macronucleus elongate; carnivorous. Class Colpodea, Order Bursariomorphida, Bursariidae (Bursaria; Fig. 22C) 48a (47a). Body trumpet-shaped; highly contractile; oral ciliature spirals clockwise around ﬂared anterior end; often pigmented and/or with algal endosymbionts. Stentoridae (Stentor; Fig. 22F) 48b. Body not trumpet-shaped .................................................................................................................................................................49 49a (48b). Body laterally compressed, pyriform to elipsoid; often pigmented pink, red, or purple; long, narrow peristome on left margin; noncontractile. Blepharismidae (Blepharisma; Fig. 22B) FIGURE 22 (A) Spirostomum minus; (B) Blepharisma lateritium; (C) Bursaria truncatella; (D) Climacostomum virens; (E) Condylostoma tardum; (F) Stentor polymorphus, half extended; (G) Actinobolina radians; (H) Coleps hirtus; (I) Bryophyllum lieberkühni; (J) Metacystis recurva; (K) Lacrymaria olor; (L) Askensia volvox; (M) Urotricha farcta; (N) Mesodinium pulex; (O) Vasicola ciliata; (P) Trachelophyllum apiculatum; (Q) Enchelyodon elegans; (R) Homalozoon vermiculare; (S) Enchelys simplex; (T) Chaena teres; (U) Spathidium spathula; (V, W) Didinium nasutum, live and silver stained. (After: Dragesco, 1966a, K, S, V, W; Dragesco, 1966b, P, R; Kahl, 1930 – 1935, A, B, D, E, F, G, H, I, J, L, M, N, O, Q, T, U; Kent, 1880 – 1882, C.) Scale 10 m for M, N; 20 m for H, J, L, P, S; 30 m for G, O, U; 40 m for B, K, T; 60 m for E, Q, R; 80 m for D, V, W; 100 m for A, F, I; and 200 m for C. 84 3. Protozoa 49b. 85 Body shape not as above ..................................................................................................................................................................50 50a (49b). Body large, very long; oral cavity shallow; peristomal area long, narrow, oral ciliature sometimes inconspicuous; contractile. Spirostomidae (Spirostomum; Fig. 22A) 50b. Buccal ciliature prominent; oral cavity deeper than above ................................................................................................................51 51a (50b). Buccal cavity occupying much of anterior part of body; paroral membrane inconspicuous; somatic ciliation dense; with or without endosymbiotic algae. Climacostomidae (Climacostomum; Fig. 22D) 51b. Paroral membrane prominent; contractile; somatic ciliation dense. Condylostomatidae (Condylostoma; Fig. 22E) 52a (5a). Body ovoid; with tentacles extending from the cell in all directions when at rest, but retracted and hardly visible when swimming. Class Litostomatea, Order Pharyngophorida, Actinobolinidae (Actinobolina; Fig. 22G) 52b. Tentacles absent ...............................................................................................................................................................................53 53a (52b). Translucent CaCO3 plates (armor) in cortex; typically barrel-shaped; body frequently spiny, often with prominent anterior and caudal thorns; long caudal cilium common; brosse present, but inconspicuous. Class Prostomatea, Order Prorodontida, Colepidae (Coleps; Fig. 22H) 53b. No such armor, not as above ............................................................................................................................................................54 54a (53b). Sessile in pseudochitinous lorica; one or more caudal cilia; paratenes obvious. Class Prostomatea, Order Prostomatida, Metacystidae (Metacystis, Vasicola; Fig. 22J,O) 54b. Not as above ....................................................................................................................................................................................55 55a (54b). Mouth at anterior end, rounded or only slightly elongated ..............................................................................................................56 55b. Apex fan-shaped to varying degree; mouth a long slit, beginning at anterior end; slit may extend down side of cell. Litostomatea, Order Haptorida, Spathidiidae (Byrophyllum, Spathidium; Fig. 22I,U) 56a (55a). Mouth at anterior pole, surrounded by an unciliated area and often raised as a blunt cone; circumferential ciliary girdle of closely apposed cilia, otherwise naked or with short cilia. Class Litostomatea, Order Haptorida ................................................................57 56b. Uniform ciliation around mouth, and typically over the entire cell ...................................................................................................58 57a (56a). Girdle ciliature of one type, one or two girdles. Didiniidae (Didinium; Figs. 6, 22V,W) 57b. Body ciliature of two types, one cirrus-like, as a single girdle. Mesodiniidae (Askenasia, Mesodinium; Figs. 22L,N) 58a (56b). Rear one-third to one-ﬁfth of body unciliated, except for one or more long caudal cilia; other somatic ciliation evenly distributed. Class Prostomatea, Order Prorodontida, Urotrichidae (Urotricha; Fig. 22M) ...................................................................................... 58b. Not as above ....................................................................................................................................................................................59 59a (58b). Body uniformly ciliated at rear; mouth with oral dome or bulb; cytopharynx not permanently inverted, but inverts during ingestion. Class Litostomatea, Order Haptorida ..............................................................................................................................................60 59b. Not as above; lack oral dome ...........................................................................................................................................................62 60a (59a). Anterior tapering to a ciliated neck; neck set off by groove and a circle of longer cilia; contractile . Lacrymariidae (Lacrymaria; Fig. 22K) 60b. Not as above ....................................................................................................................................................................................61 61a (60b). Body long, generally 4 times width; ovoid or ﬂask-shaped; oral region simple dome, sometimes pointed. Trachelophyllidae (Chaenea, Trachelophyllum; Fig. 22P,T) 61b. Oral region ﬂattened at apex; cytostome circular to ovoid; body usually shorter than four times width. Enchelyidae (Homalozoon, Enchelys, Enchelyodon; Fig. 22Q,R,S) 62a (59b). Short “dorsal brush” (brosse) extends backward from apical mouth on one side (may not be obvious without silver staining); body ellipsoid. Class Prostomatea, Order Prorodontida, Prorodontidae (Prorodon, Pseudoprorodon; Fig. 23A,B) 62b. Dorsal brush lacking, may have reduced brosse; similar to, and easily confused with Prorodon. Class Prostomatea, Order Prorodontida, Holophryidae (Holophyra; Fig. 23C) 63a (7a). Body long, ﬂattened; mouth on convex side of tapering anterior end; ciliated only on right side of body. Class Karyorelictea, Order Loxodida, Loxodidae (Loxodes; Fig. 23J) 63b. Not as above ....................................................................................................................................................................................64 64a (63b). Cytopharynx permanent, round, located at base of proboscis or tapering front end; ciliature along proboscis; toxicysts on ventrum of proboscis. Class Litostomatea, Order Pharyngophorida, Tracheliidae (Dileptus, Paradileptus, Trachelius; Fig. 23D,E,H) FIGURE 23 (A) Prorodon teres; (B) Pseudoprorodon ellipticus; (C) Holophyra simplex; (D) Trachelius ovum; (E) Paradileptus robustus; (F) Amphileptus claparedi; (G) Litonotus fasciola; (H) Dileptus anser; (I) Loxophyllum helus; (J) Loxodes magnus; (K) Cyrtolophosis mucicola; (L, M, N) Philasterides armata, live, silver stained, and oral detail of silver-stained specimen; (O) Loxocephalus plagius; (P) Urozona bütschlii; (Q) Balanonema biceps; (R) Pleuronema coronatum; (S) Histiobalantium natans; (T) Cohnilembus pusillus; (U) Uronema griseolum; (V) Cinetochilum margaritaceum; (W) Cyclidium glaucoma; (X) Calyptotricha pleuronemodies. (After: Corliss, 1959, R; Dragesco, 1966a, I; Grolière, 1980, M, N; Kahl, 1930 – 1935, A, B, C, F, G, J, K, O, P, Q, S, V, W, X; Kudo, 1966, D, E, H; Noland, 1959, L, T, U.) Scale 10 m for K, Q; 15 m for P, V; 20 m for T, U, W, X; 25 m for G, H, L, M; 30 m for C, I, S; 40 m for B, R; 50 m for F; 60 m for A, O; and 75 m for D, E, J. 86 3. Protozoa 87 64b. Mouth slit-like, located along convex side of tapering anterior end, evident only when feeding; body laterally compressed; both sides ciliated, cilia may be only bristles on 1 side. Class Litostomatea, Order Pleurostomatida, Amphileptidae (Amphileptus, Litonotus, Loxophyllum; Fig. 23F,G,I) 65a (6a). With linear oral furrow or groove leading from anterior end to mouth, bordered on right by paroral membrane ............................66 65b. Without linear preoral groove bordered by membranes, but generally with membranes inside oral cavity .......................................71 66a (65a). With an apparent double paroral membrane on the right side of the furrow (actually the paroral membrane plus a row of somatic cilia). Class Oligohymenophora, Order Scuticociliatida, Cohnilembidae (Cohnilembus; Fig. 23T) (Many genera of the Order Scuticociliatida, e.g., lines 66 – 70 and lines 75 – 76, are difﬁcult to identify without special preparation techniques such as Protargol or other silver staining) 66b. Paroral membrane along furrow not “double”.................................................................................................................................67 67a (66b). Preoral furrow long, shallow, ciliated with three ciliary ﬁelds; generally a single caudal cilium; body long, ovoid. Class Oligohymenophora, Order Scuticociliatida, Philasteridae (Philasterides; Fig. 23L – N) 67b. Bottom of preoral furrow not ciliated, paroral membrane curves around rear of mouth ..................................................................68 68a (67b). Dwells in lorica that is open at both ends; resembles Pleuronema or Cylidium. Class Oligohymenophora, Order Scuticociliatida, Calyptotrichidae (Calyptotricha; Fig. 23X) 68b. Not in a lorica as above ...................................................................................................................................................................69 69a (68b). Small (15 – 60 m long); with few somatic cilia; distinct caudal cilium. Oligohymenophora, Order Scuticociliatida, Cyclidiidae (Cyclidium; Fig. 23W) 69b. Larger than above ............................................................................................................................................................................70 70a (69b). Paroral membrane a prominent velum, extends from anterior to well past equator of cell; one to many stiff, long caudal cilia; a single contractile vacuole. Class Oligohymenophora, Order Scuticociliatida, Pleuronematidae (Pleuronema; Fig. 23R) 70b. Somatic ciliation uniform; long stiff cilia distributed over body surface; paroral membrane less prominent than above. Class Oligohymenophora, Order Scuticociliatida, Histiobalantiidae (Histiobalantium; Fig. 23S) 71a (65b). Oral cavity huge, covers most of ventrum; dorsal side convex; cilia of paroral membrane long. Class Nassophorea, Order Peniculida, Lembadionidae (Lembadion; Fig. 24G) 71b. Mouth smaller than above, not over half of body length ..................................................................................................................72 72a (71b). With one or more long caudal cilia...................................................................................................................................................73 72b. Without caudal cilia .........................................................................................................................................................................77 73a (72a). With one or two girdles of cilia ........................................................................................................................................................74 73b. Cilia not in girdles, usually in longitudinal rows ..............................................................................................................................75 74a (73a). Oral cavity equatorial; distinct constriction in the ciliated girdle; ends bare except for caudal cilium. Class Oligohymenophora, Order Scuticociliatida, Urozonidae (Urozona; Fig. 23P) 74b. Oral cavity deep, equatorial; two distinct ciliary girdles; ciliary tuft on posterior end. Class Nassophorea, Order Peniculida, Urocentridae (Urocentrum; Fig. 24D) 75a (73b). Mouth at, or anterior to, middle of cell ............................................................................................................................................76 75b. Oral area large, toward posterior half of cell (⬇ mid-ventral); body usually ﬂattened; cilia denser ventrally. Class Oligohymenophora, Order Scuticociliatida, Cinetochilidae (Cinetochilum; Fig. 23V) 76a (75a). Small, 50 m; anterior pole ﬂat, unciliated. Class Oligohymenophora, Order Scuticociliatida, Uronematidae (Uronema; Fig. 23U) 76b. Somatic ciliation even; body long-ovoid; oral area small, closer to anterior end. Class Oligohymenophora, Order Scuticociliatida, Loxocephalidae (Balanonema, Loxocephalus; Fig. 23O,Q) 77a (72b). Body small, ovoid to ellipsoid; in mucilaginous case from which it can emerge freely; cytostome near anterior end. Class Colpodea, Order Cyrtolophosidida, Cyrtolophosidae (Cyrtolophosis; Fig. 23K) 77b. Not associated with mucilaginous envelope .....................................................................................................................................78 78a (77b). Body distinctly heart- or cone-shaped; oral region relatively large, covers most of ventral surface. Class Nassophorea, Order Peniculida, Stokesiidae (Stokesia; Fig. 24B) 78b. Body shaped otherwise .....................................................................................................................................................................79 79a (78b). Body ovoid to ellipsoid; mouth in anterior half of body, pointed in front, with pre- and/or post-oral sutures, sometimes hard to see; 88 W. D. Taylor and R. W. Sanders FIGURE 24 (A) Frontonia leucas; (B) Stokesia vernalis; (C) Glaucoma scintillans; (D) Urocentrum turbo; (E) Disematostoma bütschlii; (F) Turaniella vitrea; (G) Lemabadion magnum; (H) Colpidium colpoda; (I) Paramecium caudatum; (J) Clathrostoma viminale; (K, L) Maryana socialis, individual and colony; (M) Plagiopyla nasuta; (N) Bresslaua vorax; (O) Tetrahymena pyriformis; (P, Q) Tillina magna, live and line drawing of silver-stained specimen; (R, S) Colpoda steinii, live and silver stained; (T) Chilodonella uncinata. (T) (After: Corliss, 1959, O, R; Dragesco, 1966b, B; Kahl, 1930 – 1935, A, C, D, E, F, G, H, I, J, K, L, M, P; Kudo, 1966, N; Lynn, 1976, S; Lynn, 1977, Q; Noland, 1959, T.) Scale 15 m for G. O. R; 25 m for C, H; 30 m for D, F; 40 m for B, E, J, M; 60 m for I, N; and 75 m for A, K, Q. 3. Protozoa 89 nematodesmata prominent to side and rear of mouth; contractile vacuoles with long collecting canals. Class Nassophorea, Order Peniculida, Frontoniidae (Disematostoma, Frontonia; Fig. 24A) 79b. Mouth typically rounded or blunt in front; body ciliature with preoral suture, no postoral suture; one paroral membrane and three membranelles, which are typically inconspicuous. Order Hymenostomatida, Suborder Tetrahymenina ...........................................80 (Genera of this suborder, lines 80 and 81, are difﬁcult to identify without special preparation techniques, i.e., silver staining) 80a (79b). Body pyriform to cylindrical; bases of membranelles of uniform width; mouth roughly triangular. Tetrahymenidae (Tetrahymena; Fig. 1, 24O) 80b. Not as above ....................................................................................................................................................................................81 81a (80b). Right ventral cilia curve left and twist forward parallel to suture. Turaniellidae (Colpidium, Turaniella; Fig. 24F,H) 81b. Right ventral cilia curve left, but do not twist forward toward mouth. Glaucomidae (Glaucoma; Fig. 24C) 82a (6b). In a gelatinous tube-like lorica, which may have dichotomous branches; with a terminal cone-like protuberance bearing long cilia and surrounded by a circular adoral groove leading to the buccal cavity. Class Colpodea, Order Colpodida, Marynidae (Maryna; Fig. 24K,L) 82b. No gelatinous case; mouth area not as described above ...................................................................................................................83 83a (82b). Body small (100 m), laterally ﬂattened; ciliation relatively sparse, on pellicular ridges................................................................84 83b. Not as above ....................................................................................................................................................................................85 84a (83a). Cytostome in rear half of body; small cyrtos hidden by ventral pellicular fold. Class Nassophoria, Order Microthoracida, Microthoracidae (Microthorax; Fig. 20W) 84b. Cytostome opens laterally in anterior one-third of body, long tubular cyrtos; body nearly oval. Class Nassophoria, Order Propeniculida, Leptophrygidae (Pseudomicrothorax; Fig. 20V) 85a (83b). Body ciliation uniform; ventral cytostome in an oral vestibule; nematodesmata form a ring or “basket” around mouth; similar to Frontoniidae. Class Nassophorea, Order Peniculida, Clathrostomatidae (Clathrostoma; Fig. 24J) 85b. Mouth area sunken in, with oral groove leading to it, or with ciliated pharyngeal tube, or both......................................................86 86a (85b). Medium to large (150 m in some species); body foot-shaped or ellipsoidal, with pointed or rounded caudal end; uniform body ciliation; long, broad, ciliated oral groove leads into buccal cavity; two contractile vacuoles; one species with algal symbiont. Class Nassophorea, Order Peniculida, Parameciidae (Paramecium; Fig. 6 and 24I) 86b. Not as above; with single contractile vacuole ...................................................................................................................................87 87a (86b). Oral groove transverse, in anterior part of body; cytostome at base of deep tubular pocket; densely ciliated; common in anaerobic habitats. Class Plagiopylea (Plagiopyla; Fig. 24M) 87b. Body reniform, indented in middle on one side; oral groove passes around groove toward mouth; division in a cyst. Class Colpodea, Order Colpodida, Colpodidae (Bresslaua, Tillina, Colpoda; Fig. 24N,P – S.) LITERATURE CITED Albright, L. J., Sherr, E. B., Sherr, B. F., Fallon, R. D. 1987. Grazing of ciliated protozoa on free and particle-associated bacteria. Marine Ecology Progress Series 38:125 – 129. Allen, R. D. 1984. Paramecium phagosome membrane: from oral region to cytoproct and back again. Journal of Protozoology 31:1 – 6. 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External Morphology B. Internal Anatomy and Physiology III. Ecology and Evolution A. Diversity and Distribution B. Reproduction and Life History C. Ecological Interactions D. Evolutionary Relationships I. INTRODUCTION Sponges in fresh water? Many people react this way when they are told that there might be sponges in a lake or stream. Most people have seen sponges from the ocean but are unaware that they also occur in freshwater. Yet sponges are common and sometimes abundant inhabitants of a wide variety of freshwater habitats. In some situations they comprise a major component of the benthic fauna and may play important roles in ecosystem processes in freshwater. Sponges are the simplest of the multicellular phyla. They lack organs and tissues are their highest level of organization. Specialized cells accomplish many basic biological functions in sponges. Despite their simplicity, however, sponges display a variety of 1 The preparation of this chapter was supported by several grants from the National Science Foundation. We thank Janet Blair, Joan Elias, Susan Knight, and Yolanda Lukaziewski for their assistance in its preparation. † It is with profound regret that we learned in the late summer of 2000 of the death of Tom Frost who gave his life saving another person from drowning. He will be missed by his many friends and colleagues. Ecology and Classiﬁcation of North American Freshwater Invertebrates. 2nd Edition Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved. Anthony Ricciardi Department of Biology Dalhousie University Halifax, Nova Scotia B3H 4J1 IV. Collecting, Rearing, and Preparing Sponges for Identiﬁcation V. Classiﬁcation A. Freshwater Sponge Species of Canada and the United States B. Key to the Freshwater Sponges in Canada and the United States Literature Cited elegant adaptations to freshwater habitats including a strong capacity for osmoregulation, complex life cycles, a capability to feed selectively on a broad range of particulate resources, and, in many species, an intimate association with symbiotic algae. This chapter will introduce you to the structure, function, and diversity of freshwater sponges. It will emphasize their taxonomy and their basic ecology. For those whose interests are whetted by this introduction, Bergquist (1978) and Simpson (1984) provide detailed treatments of the biology of the phylum Porifera in general. II. STRUCTURE AND PHYSIOLOGY The simplicity of a sponge’s external features contrasts sharply with the complexity of its internal structure and function. Viewed macroscopically, freshwater sponges exhibit a limited range of non-distinct body forms, but a microscopic examination reveals a variety of internal features. This contrast derives from the basic organization of sponges in which tissues rather than organs represent the highest level of 97 98 Thomas M. Frost, Henry M. Reiswig, and Anthony Ricciardi FIGURE 1 Gemmulated colonies of Spongilla lacustris and Duosclera mackayi exhibiting an encrusted growth form. The distinct zone between the two species illustrates the typical nonmerging front that occurs when two different freshwater species grow into contact with each other on a substratum. morphological complexity. In fact, specialized cells operating independently or in association with related cells accomplish such primary functions as food gathering, digestion, and reproduction. An appreciation of the utility of sponge structure, therefore, is possible only by including microscopic examinations. Likewise, it is difﬁcult to discuss the physiological functions of sponges independently of their microscopic structure and these topics are presented jointly, in a general overview. There has been substantial debate as to whether sponges should be considered as colonies or individuals (Hartman and Reiswig, 1973; Simpson, 1973). This reﬂects the continuing difﬁculties that ecologists, evolutionary biologists, and zoologists in general encounter in deciding on what an individual is (Santelices, 1999). From an ecological perspective, it is most important to consider that sponges are much more like colonies, such as corals or bryozoans, in terms of their ecological function than they are like individual arthropods or vertebrates. Overall, however, concepts of colony or individual are not really appropriate for sponges, because of their level of biological organization. A. External Morphology Freshwater sponges display a variety of morphologies that range from encrusting (Fig. 1), to rounded (Fig. 2) and ﬁnger-like (Fig. 3) growth forms. Because form can vary substantially within a species (Penney and Racek, 1968), external morphology is of very limited use in sponge taxonomy. Variation in growth form is inﬂuenced by environmental conditions such as water movement (Jewell, 1935; Palumbi, 1984) and the availability of light (Frost and Williamson, 1980). Differences can be particularly dramatic between lentic and lotic habitats (Neidhoefer, 1940). The surface structure of sponges varies from ﬂat (Fig. 1) to strongly convoluted (Fig. 2). Although there are some species-associated differences in surface characteristics, this feature, too, is strongly inﬂuenced by habitat conditions. Finer-scale characteristics of external sponge anatomy are related to internal features. 4. Porifera FIGURE 2 A colony of Ephydatia muelleri exhibiting a rounded growth form encrusted on a branch from a fallen tree (from Neidhoefer, 1940). FIGURE 3 A colony of the common freshwater sponge Spongilla lacustris exhibiting a characteristic, ﬁngerlike growth form. 99 100 Thomas M. Frost, Henry M. Reiswig, and Anthony Ricciardi Examples, discussed below, include the incurrent and excurrent portions of the feeding canals and extensions of the skeletal system. B. Internal Anatomy and Physiology The basic organization of a sponge consists of a surface epithelium, composed of pinacocytes, surrounding an organic matrix, termed the mesohyl, which contains a broad variety of specialized cells. Cells within the mesohyl interact with the epidermal cells to accomplish basic functions including the processing of water for food uptake and gas exchange, digestion, structural support, and reproduction. Some sponge cells are highly plastic in their behavior and can shift their function with changing environmental conditions. De Vos et al. (1991) provided a detailed atlas of the overall general morphology of sponges illustrating most cell types with scanning and transmission electron micrographs. All sponge cells, including those in the epidermis and in the mesohyl, appear to be involved in osmoregulation (Weissenfels, 1975). This capacity has been critical in allowing the invasion of freshwater by sponges from their original marine habitats. FIGURE 4 1. Water Processing System A sponge can be considered, essentially, as a series of progressively ﬁner ﬁlters and a mechanism that circulates water through them. The sponge ﬁlters are capable of removing particles ranging from large algae to bacterial cells less than 1 m in diameter and, possibly, to colloidal organic matter (Frost, 1987). In addition, the movement of water through the canal system, with its extensive surface area, fosters the transport of gases and excreted materials. The main features of the sponge water-processing system (Figs. 4 and 5) have been identiﬁed through the efforts of numerous investigators (reviewed in Frost, 1976b, 1987) with several papers by Weissenfels and his co-workers (Weissenfels, 1975, 1976, 1980, 198l, 1982,1992; Langenbruch and Weissenfels, 1987) providing particularly important details on structure. Although there are some subtle differences in structures among species, the summary below provides a general overview of the structures associated with sponge water processing. Water ﬁrst enters a sponge through a large number of 50-m diameter openings termed ostia (single: ostium) that are spread across its surface epithelium. Passing through the surface, inﬂowing water next enters a large subdermal cavity. From there, water ﬂows A schematic diagram indicating the primary components of the sponge feeding canal system. The diagram is not drawn to scale, but Figure 5 provides an indication of the size and arrangement of the feeding system in an actual sponge. Water ﬂow patterns are indicated by arrows. 4. Porifera 101 FIGURE 5 A scanning electron micrograph of a cross section of the freshwater sponge Ephydatia ﬂuviatilis indicating the form, size, and arrangement of the feeding canal system. Illustrated are a sudbdermal cavity (SD), an incurrent canal (IC) with lateral branches into the sponge (i), and excurrent canal (EC) and its lateral branches (e), an atrium (At), and an osculum (Os). Arrows indicate the ﬂow path of water and numerous choanocyte chambers are visible throughout the sponge. Magniﬁcation, approximately 100 (from Weissenfels, 1982). into incurrent canals which branch repeatedly into canals of progressively narrower diameters, until they reach a choanocyte chamber (Fig. 4). Choanocyte chambers are the keystones of the sponge feeding system. Each chamber consists of numerous choanocytes that are characterized by the presence of a ﬂagellum and a collar of microvilli. Beating by the ﬂagella within these chambers is primarily responsible for setting up the ﬂow of water through the sponge. Water ﬂow is also facilitated by a sponge’s basic hydrodynamic structure. Vogel (1974) has demonstrated that water will actually ﬂow passively, albeit at a reduced rate, through the feeding canals of killed sponges because of this structure. Choanocytes also serve as the ﬁnal ﬁlters in the feeding system. The microvilli that make up the collars of the choanocytes are spaced to form openings less than 0.1 m in diameter. Once water has passed the choanocyte chambers, it enters the excurrent portion of the water processing system. Exiting the choanocyte chambers, the canals join together in an anastomosing fashion forming larger and larger diameter vessels, ﬁnally entering a broad chamber termed an atrium. From this chamber water exits the sponge body through a specialized structure termed an osculum. Such oscula channel water from large portions of a sponge and each sponge is likely to have several oscula. Water is forced from the oscula with sufﬁcient force so as to enable materials 102 Thomas M. Frost, Henry M. Reiswig, and Anthony Ricciardi that have passed through the sponge to be transported far enough away from the sponge that these materials are unlikely to be recaptured by the ﬂow of water entering that sponge. Although the description above may suggest that the structure of the sponge canals was ﬁxed, the system is actually dynamic. The positions of both the incurrent and excurrent canals, as well as their components, can shift as the sponge grows or as portions of the feeding canals are occluded by ingested materials (Mergner, 1966). Similarly, the mesohyl of freshwater sponges exhibits rhythmic condensation that assists the choanocyte chamber in pumping water (Weissenfels, 1990). 2. Digestion A second key feature of sponge feeding involves the capacity of the cells within the canal network to engulf particles through phagocytosis. Cells ranging from the porocytes that form the ostia on the sponge surface to the pinacocytes lining the incurrent canals and the choanocytes exhibit this capacity for phagocytosis. Digestion and the transport of nutrients within the sponge body also involve phagocytic activities in which materials are exchanged between cells. Information on activities by cells in sponge digestion has been gained by several detailed studies employing light and electron microscopy (van Weel, 1949; Schmidt, 1970; Willenz, 1980). Phagocytosis can occur when a particle makes contact with a surface within the canal system. Food particles large enough to occlude a sponge’s ostia are taken up directly at the epithelium. Smaller particles are taken up after contact with the cells lining the canal walls or with the collars of the choanocytes. Following initial uptake, food particles are transferred to digestive cells, termed archaeocytes, which move freely within the mesohyl. As digestion proceeds, archaeocytes bearing ingested materials move through the sponge interior and transfer nutritional materials to other cells, such as those involved with reproduction or skeleton formation. After the digestion of ingested materials is completed, archaeocytes move to the excurrent portion of the canal network. Nondigested material is released into the water ﬂowing out of the sponge by a reverse phagocytic action. Egested particles are then carried by excurrent ﬂow out through oscula at the sponge surface. Detailed analyses of cell activities in sponge digestion have revealed a surprising degree of complexity. The time necessary for a particle to traverse the digestive system completely, from initial uptake to release into an excurrent canal, can vary markedly with the nature of the food particle and with environmental conditions. Ordinarily, digestible food particles may be held within archaeocytes for more than 12 h prior to release into the sponge excurrent. Under certain circumstances, sponges can sense indigestible particles and release them after much shorter time periods (Frost, 1980a). The capacity to speed the processing time for ingested materials is most evident when a sponge is faced with a dense suspension of particles. At such times, food particles may be taken up, cycled, and released almost immediately (Schmidt, 1970). The sophistication of the sponge feeding system is particularly evident in digestive activities because the rapid cycling of ingested particles can be highly selective. While overabundant particles are being quickly cycled through the sponge, other food particles, present in lower concentrations in the feeding suspension, are held within the sponge for periods that are sufﬁciently long to allow normal digestion (Frost, 1980a, 1981). Sponges, therefore, are selective feeders, not in their initial uptake of particles as selective feeding is typically deﬁned, but in their ultimate use of these resources (Frost, 1980b). A sponge’s capacity to vary particle transit time is linked to the large number of cells that are operating within it. Digestive cells are sufﬁciently numerous so that, under normal feeding conditions, each cell handles only one ingested particle at a time (Willenz, 1980). Each individual archeocyte then, can be considered as a separately functioning digestive unit with the capacity to tune its actions to the characteristics of the food particle that it is processing. 3. Skeleton The gross structure of freshwater sponges derives from an interplay between two fundamentally different components — a mineral skeleton made up of siliceous structures termed spicules and an organic skeleton made up of collagen. Collagen binds spicules together into rigid structures yielding the sponge’s basic framework. All freshwater sponges exhibit skeletal systems comprised of siliceous spicules and collagen. Marine sponges exhibit a broader variety of supporting structures (Berquist, 1978). a. Siliceous Spicules Some major groups of sponges are unique among multicellular organisms in their use of silica as a primary component of their skeleton. Freshwater sponge spicules are composed of opalescent silica that has been laid down along an axial, organic ﬁlament by specialized cells termed sclerocytes. Considerable diversity exists in spicule morphology, and, because much of this variability is species-speciﬁc, their structure plays a critical role in sponge taxonomy. Three general classes of spicules are recognized: (1) megascleres, which make up the main framework of 4. Porifera 103 FIGURE 6 A scanning electron micrograph of megascleres from Spongilla lacustris collected in a lake with a high concentration of dissolved silica. A spined microsclere is also present. The bar is 10 m in length. a sponge; (2) microscleres, which appear to add structural reinforcement for sponge tissues; and (3) gemmoscleres, which form part of the resistant coat of gemmules (see below). Megascleres are needle-shaped structures (Fig. 6 and Fig. 7) that range in length from 150 to 450 m. Microscleres are similar in form to megascleres but are usually less than one-ﬁfth their FIGURE 7 length (Fig. 6). Gemmoscleres are similar in size to microscleres and both exhibit a variety of forms ranging from needlelike to dumbbell or star-shaped. Any of these three spicule forms may be smooth or spined depending on the species. An elaborate terminology has been developed to describe the varied shapes of spicules. These terms have been summarized in Boury-Esnault A scanning electron micrograph of a megasclere of Spongilla lacustris collected in a lake with low concentration of dissolved silica. The bar is 10 m in length. 104 Thomas M. Frost, Henry M. Reiswig, and Anthony Ricciardi and Rützler (1997), but their use has been kept to a minimum in this chapter. The ﬁne-scale structure of the spicules, particularly the gemmoscleres, plays a critical role in the taxonomy of sponges, and its importance cannot be overemphasized. The classiﬁcation of freshwater sponges is based fundamentally on the structure of gemmule spicules (Penney and Racek, 1868). b. Collagen The predominant form of collagen in sponges is spongin, which serves primarily to bind the spicules of the inorganic skeleton together. A second form of collagen provides smaller-scale structure within the sponge mesohyl. Finally, collagen, often combined with gemmoscleres, forms the resistant coat of gemmules. 4. Reproduction Both sexual and asexual processes can play major roles in sponge reproduction. Sexual reproduction involves the activities of numerous, isolated reproductive cells functioning throughout the body of an active sponge. Asexual reproduction often involves major changes in all of the cells within a sponge. a. Sexual Reproduction Although detailed observations have been made for only a few freshwater species, it appears that the mode of sexual reproduction is usually gonochoristic, with each separate sponge being entirely male or female. In at least some cases, however, the gender of a particular sponge is not ﬁxed but appears to depend on environmental conditions. In at least one species, S. lacustris, sponges have been shown to switch sex from one year to the next (Gilbert and Simpson, 1976a). Bisbee (1992) did report one hermaphrodite specimen in a population of S. lacustris in South Carolina where most sponges exhibited only male or female elements. Sexual reproduction is accomplished by specialized cells that develop within the mesohyl during limited portions of the year. Reproductive cells are derived from other, more common sponge cells. Spermatogenesis occurs within distinct spermatic cysts which appear to be formed from choanocytes. Egg cells (oocytes) seem to develop either from archeocytes or choanocytes. Oocyte growth is dependent upon a variety of nurse cells that are phagocytized as the egg develops (Gilbert, 1974). Because these events do not occur for extensive periods, successful sexual reproduction requires synchronous timing of egg and sperm development across sponge populations in a habitat. This has been documented for some freshwater species (Simpson and Gilbert, 1973). This requirement leads to spectacularly synchronized events of extensive sperm production in some marine species (Reiswig, 1970). Fertilization occurs when sperm that have been released into the open water by other sponges are brought into the canal systems of female sponges by their feeding currents. Sperm are then taken up by the sponge, probably by choanocytes, and conveyed to an egg cell where fertilization takes place. As is the general case within the Porifera, freshwater sponges are viviparous. Larvae undergo extensive development prior to their release from the mother sponge. Material for growth is provided by nurse cells. In the ﬁnal stages of development, the sponge larva, termed a parenchymula, contains archeocytes, pinacocytes, sclerocytes, and choanocyte chambers. In addition, its surface is covered by ﬂagellated epithelial cells which allow the larva to swim upon release. Once development is completed, larvae are released through the excurrent portion of the feeding canal system. The larvae swim until they settle onto a substratum, where they undergo metamorphosis and quickly develop the structures typical of adult sponges. b. Asexual Reproduction Asexual reproduction in sponges may be simple or complex. On one extreme, clonal development of separate, independently functioning sponges can take place through fragmentation. The plastic nature of sponge cells makes it possible for even very small pieces to develop into active, fully functional sponges. Propagation in this fashion occurs commonly in some habitats (Frost et al., 1982). On the other extreme, most freshwater sponges typically form gemmules in a complex process of dedifferentiation in which the structures of the active sponge regress into masses of cells surrounded by resistant coats (Figs. 1 and 8). This process, which often occurs prior to the onset of rigorous environmental conditions, permits sponges to weather periods of stress and to disperse into other habitats. The coat of a mature gemmule usually consists of three collagen layers. In most species, specialized spicules, gemmoscleres, which are formed only during gemmule formation, are also embedded within the gemmule coat. The coat of each gemmule is continuous with a much thinner layer at a single specialized structure, termed a micropyle or foraminal aperture, through which cells emerge during germination. Within the gemmule are a large number of specialized cells, thesocytes, which contain numerous yolk inclusions. Thesocytes can store energy-rich materials which are used for growth following germination. The aggregation of cells to form gemmules takes place within the mesohyl. Gemmules are often present in large numbers within the vestigial skeletons of previously active sponges. Completely formed gemmules exhibit low metabolic rates until germination is induced, and are 4. Porifera 105 FIGURE 8 Numerous gemmules (the small whitish spheres) of Spongilla lacustris in a formerly active ﬁngerlike growth of the sponge. extremely resistant to environmental stress. Gemmules of a common freshwater species Ephydatia muelleri survived exposure to 80°C for more than 9 weeks (Barbeau et al., 1989) although cold tolerance has been shown to vary substantially among species (Ungemach et al., 1997). Gemmules can also survive anoxic conditions for several months (Reiswig and Miller, 1998). Upon germination, the thesocytes increase their metabolic rate, exit from the gemmule coat through the micropyle, and differentiate into the varied cells typical of an active sponge. At least one freshwater sponge species is also capable of another form of asexual reproduction, budding. Buds exhibit many of the structures of active sponges, but in a smaller form that is capable of dispersal within water (Saller, 1990). They do not, however, exhibit the same resistance to adverse environmental conditions as gemmules. 5. Biochemistry Sponges exhibit a variety of unusual chemical constituents. Some, particularly those identiﬁed as secondary metabolites, have been linked with an ability to deter predation (Pennings et al., 1994). Particularly unusual among chemical features of sponges has been a major portion of ﬂuorine in the body of a marine species (Gregson et al., 1979). Most unusual chemicals have been documented in marine species, but some are also likely to occur in freshwater species. It has been suggested that they may provide some defenses against predation and overgrowth by epizooic organisms for freshwater sponges (Frost, 1976a). III. ECOLOGY AND EVOLUTION A. Diversity and Distribution 1. Diversity The diversity of sponges in freshwater is low in comparison with that in the marine habitats from which they have evolved. Although the entire phylum Porifera consists of more than 5000 species (Bergquist, 1978), there are probably fewer than 300 freshwater species worldwide. In the most recent taxonomic revision of the Spongillidae, the largest family of freshwater sponges, Penney and Racek (1968) list a total of 95 species in 18 genera . While it is certain that there are additional, undescribed species, particularly in tropical regions that have not been thoroughly investigated (e.g., the Amazon Basin; Volkmer-Ribeiro, 1981), it is clear that freshwater forms represent a small group within the sponge phylum. In turn, North American freshwater sponges represent only a restricted subset of the world’s freshwater species. Overall, within both the Spongillidae 106 Thomas M. Frost, Henry M. Reiswig, and Anthony Ricciardi and Metaniidae, we have generated a list of 27 clearly distinct species in 11 genera that appear to be valid reports for the portion of North America north of Mexico and the Caribbean. 2. Distribution From the broadest perspective, the distribution of sponge species is inﬂuenced by biogeographic factors. On an intermediate level, the general environmental conditions that exist within a lake or stream will control the presence of a particular sponge species there. Finally, conditions such as light, substrata, and wave action will determine sponge distribution within a particular water body. The freshwater sponge species in North America exhibit a variety of distribution patterns (Penney, 1960; Penney and Racek, 1968). Several species have been reported throughout the United States and Canada, while others have been observed in only one or a few habitats. In some cases, a broad distribution in North America represents only a subset of a more extensive range. Ephydatia ﬂuviatilis and Eunapius fragilis are distributed worldwide, while Spongilla lacustris, Ephydatia muelleri, and Trochospongilla horrida occur throughout temperate regions of the Northern Hemisphere. Some species exhibit more restricted crosscontinental distributions. Anheteromeyenia ryderi occurs primarily in eastern North America but has also been reported once in Central America and from several locations in Western Europe (Økland and Økland, 1989). Other species are broadly distributed throughout North America, but have not been observed on other continents (e.g., Duosclera mackayi and Trochospongilla pennsylvanica). Of the remaining North American species, most appear to be restricted to limited regions within Canada and the United States; and, in the most extreme cases, several species have been reported from single locations. It is important to note that this chapter provides limited coverage of North America. It considers only sponges in Canada and the United States. Limited information on Mexican sponges is reported in Rioja (1953). Smith (1994) presented the ﬁrst report of a freshwater sponge from the West Indies. Abundant sponges have been observed in a wetland in Costa Rica (T. Frost, personal observation), and it is certain that there are sponges throughout Mexico and Central America. Within Canada and the United States, there have been numerous reports on the occurrence of sponges in provinces and states or regions within them. Some recent examples include reports for Alberta (Clifford, 1991), Arizona (Sowka, 1999), Connecticut (De Santo and Fell, 1996), eastern Canada (Ricciardi and Reiswig, 1993), western Montana (Barton and Addis, 1997), and southern Lake Michigan (Lauer and Spacie, 1996). Penney (1960) presented an extensive review of earlier reports on freshwater sponges. Despite these reports, information available on the occurrence of sponges does not match their overall distribution. The apparently limited ranges of some species may result more from a lack of observations rather than actual restrictions in sponge distributions. Accepting this limitation, however, there appear to be some general trends in the distribution of sponges across North America. For example, relatively few species of sponges have been reported in western regions, and there appears to be a general east-to-west decrease in sponge diversity. A similar trend occurs between northern and southern regions, with more species reported from the northern United States and southern Canada than from the more southern regions of the continent. In some situations, the limited distribution of a particular species may involve adaptations to climatic conditions. In many other cases, biogeographic factors, particularly limitations on dispersal, are likely to control regional distribution patterns. There are some notable exceptions to limitations on dispersal, however (e.g., Jones and Rützler, 1975, Økland and Økland, 1989). Within regional distribution patterns, the factors that regulate the occurrence of a species in a particular lake or stream are less well understood. While Spongilla alba appears to be restricted to brackish water habitats (Poirrier, 1976), other species are inﬂuenced by a variety of environmental factors. Jewell (1935, 1939) provided the most detailed quantitative information available on this topic. In comparing species across lakes in northern Wisconsin, she found considerable variation in each species’ habitat requirements. Some species were restricted to a narrow subset of environmental conditions, while others occurred throughout a broad range of habitats. Chemical factors were correlated with the distributions of some species; but for other species, more recent analyses indicate that there is wide tolerance of chemical conditions (Colby et al., 1999). In addition, some species were favored by ﬂowing water, while others were more typically associated with standing water. In general, it is difﬁcult to pinpoint any speciﬁc factors that control the distribution of sponges. Many species are sufﬁciently broad in their distribution so that many lakes can be expected to contain at least some small specimens. This is particularly true in some regions, e.g., northern Wisconsin, where sponges occur in nearly every lake. In some cases, particularly in smaller lakes, large populations of several sponge species may be common. Aside from cases of geographic isolation, sponges would be 4. Porifera excluded only from habitats with frequent physical disturbances, with high pollution levels, or with high loadings of silt or particulates that can clog their feeding systems (Harrison, 1974). Not all pollutants affect sponges, however. They appear to be tolerant of high levels of some contaminants, particularly heavy metals (Richelle et al., 1995). Within a lake or stream that is suitable for growth, sponge distribution is controlled by ﬁner scale environmental features. For most sponges, a hard substratum is essential for growth, and the absence of a surface on which to grow will limit the distribution of sponges even where other conditions are highly favorable. Only a few species, notably Spongilla lacustris and, to a lesser extent, Ephydatia muelleri, can grow in soft sediments. In rivers and streams, sponges are excluded from regions with high ﬂow due to physical disruption. In lakes, sponges can be limited in shallow waters by wave action or ice scour (Bader, 1984). In deeper waters, sponges can be limited by low oxygen or by colder temperatures. Some species with symbiotic algae may be limited by light availability, particularly where water is darkly stained by dissolved organic materials, but species with less dependence on symbionts may thrive in such habitats. B. Reproduction and Life History The annual life-history pattern for most freshwater sponges involves periods of active growth alternating with dormancy and includes both asexual and sexual reproductive processes. Transitions to, and from, dormant stages usually involve the asexual processes of gemmule formation and hatching. Asexual reproduction also occurs during active growth periods, both in the formation of gemmules and in the generation of separately functioning sponges through fragmentation. Sexual reproduction occurs only for a limited time during periods of active growth. Key features of the freshwater sponge life cycle are summarized in Figure 9 and discussed in detail below. Overall, reproduction in sponges involves responses to two distinct problems, propagation within a single habitat and dispersal among habitats. Within-habitat processes are discussed separately below prior to a consideration of dispersal. 107 either situation, sponge feeding ceases and respiratory activities are greatly reduced. Most studies of dormancy in sponges have focused on transitions involving gemmules. Despite the common role of gemmules in dormancy, their presence does not in itself necessarily signify a transition to a dormant life-cycle period. In many cases, particularly where sponges are large, a few gemmules are produced during most of the active season. Most freshwater sponges undergo a period of dormancy at some time during the year, typically during periods of environmental stress. In temperate habitats, dormant periods occur most commonly during winter. This is the case for the majority of sponges in North America. However, at lower latitudes this pattern can be reversed with dormancy occurring during hot, summer periods and active growth taking place during cooler seasons (Poirrier, 1974; Ilan et al., 1996). Dormant periods have also been observed in response to drying in ephemeral habitats. While dormancy is a common feature of freshwater sponge life cycles, it does not always occur; some sponges maintain active growth even under winter ice cover (Frost, personal observation). The timing of dormancy varies both among species within particular habitats and among habitats for individual species. In Lake Pontchartrain, Louisiana, Spongilla alba exhibits a typical pattern of gemmule formation during winter months (Poirrier, 1976), while a co-occurring population of Ephydatia ﬂuviatilis exhibits a reciprocal pattern of summer dormancy (Harsha et al., 1983). At the same time, populations 1. Dormancy In most cases, dormant periods for freshwater sponges are characterized by the complete transformation of all active tissue in a sponge into gemmules (Simpson and Fell, 1974; Simpson, 1984). More rarely, tissue regression yields a growth form that functions as a gemmule without its specialized structures. In FIGURE 9 General features of the annual life history of freshwater sponges (see text for detailed explanations). 108 Thomas M. Frost, Henry M. Reiswig, and Anthony Ricciardi of E. ﬂuviatilis in more northern habitats form dormant stages in winter. S. lacustris populations in New Hampshire gemmulated during winter and were active during summer (Simpson and Gilbert, 1973), while population in South Carolina were inactive during summer periods (Bisbee, 1992). Sexual reproduction occurred at different times during the seasonal cycle in these areas as well. In Little Rock Lake, Wisconsin, S. lacustris undergoes a winter dormancy period while co-occurring populations of Corvomeyenia everetti and Ephydatia muelleri maintain active growth during the entire winter. Populations of E. muelleri in nearby Wisconsin lakes undergo typical winter dormancy. The transition from an active to a dormant state for sponges usually appears to be cued by environmental factors, such as changing temperatures or declining water levels. In some species this transition may also involve a complex response to previous growth conditions (Gilbert, 1975). Within a region, the timing of gemmulation is nearly synchronous across habitats for all populations of a species that exhibit dormancy (Frost, personal observation). In contrast, different species within a region can vary substantially in the seasonal timing of dormancy and in the rate at which the transition from active tissue to gemmules proceeds. In Mud Pond, New Hampshire, Trochospongilla pennsylvanica begins forming gemmules in mid-August when water temperatures are at their maximum while S. lacustris does not form gemmules until October when water temperatures reach 10°C (Simpson and Gilbert, 1973). Sponge species also vary in the factors that induce their release from dormancy (Simpson and Fell, 1974). Temperature appears to be the primary environmental cue that induces gemmule hatching in temperate habitats. Gemmules of some sponges hatch while water temperatures are at near-winter levels suggesting that another cue such as photoperiod or the availability of light is operating. In some species, gemmules exhibit a form of diapause in which a certain period at cold temperatures is necessary before hatching can occur (Simpson and Fell, 1974). Such a delay prevents hatching during short, warming periods prior to the onset of winter. Hatching at such times could lead to major population reductions if time or energy was insufﬁcient for a second formation of gemmules. Not all species form diapausing gemmules and, in these cases, hatching will take place during any period of increasing water temperature. The occurrence of diapause appears linked to the timing of gemmule formation. Species that form gemmules later in the year are less likely to exhibit diapause. While there are obvious costs to hatching at the wrong time, there may also be advantages to being able to hatch quickly without diapause under some circum- stances. Studies of dormancy in freshwater sponges have generally focused on cold-water populations, and less is known about the factors that control the transitions to, and from, dormant stages when they occur in response to hot or dry conditions. In general, while it is clear that the formation of dormant stages plays a crucial role in the overall life history of most sponges, substantial variability is apparent in: (1) the morphological forms involved in dormant stages; (2) the timing of transitions to, and from, dormant periods; and (3) the occurrence of diapause. This variability suggests that the overall value of dormancy and the successful strategies by which it can be employed differ markedly among habitats and that sponge responses to such differences involve a diversity of adaptations. 2. Growth, Reproduction, and Dispersal within Habitats Nondormant periods in the freshwater sponge life cycle appear to be characterized by continuous growth. Although quantitative studies have been limited, sponge growth in some habitats can be extremely proliﬁc. In a pond in New Hampshire, 10 mg dry mass of gemmule tissue of S. lacustris that hatched in spring produced an average of 18 g of active sponge tissue just prior to gemmule formation the following fall (Frost et al., 1982). Ephydatia ﬂuviatilis exhibited comparable proliﬁc growth rates in a European stream (Pronzato and Manconi, 1995). Growth is largely indeterminate for individual sponges, which may reach surprisingly large sizes. In some optimal habitats, for example, a single specimen of S. lacustris, consisting of intertwined ﬁngers (as in Fig. 1), can occupy more than a cubic meter of space (Frost, personal observation). Sexual reproduction for most freshwater sponge populations occurs synchronously throughout a habitat in a 1 – 3 month period following hatching from dormant conditions (Simpson and Gilbert, 1973). Synchrony across a population is particularly important, because otherwise the sperm released by male sponges would have a low probability of successfully fertilizing an egg (Gilbert, 1974). The motile larvae produced by sexual reproduction in freshwater sponges can play an important role in the establishment of new sponges. In particular, the colonization of new substrata will often depend on such motile forms. While settling on an appropriate substratum is clearly critical for the successful growth of a new sponge, little information is available on substrate choice by settling sponge larvae. Studies of a variety of marine organisms suggest, however, that substrate selection by freshwater sponge larvae may be possible. Fragmentation of intact specimens into separately functioning units may also play an important role in 4. Porifera freshwater sponge life cycles. In some cases, such fragmentation occurs during periods of tissue regression and may not lead to growth on new substrata. In other habitats, however, particularly those with large amounts of aquatic vegetation to which sponges can attach, fragmentation and growth may occur repeatedly during an active season leading to dispersal throughout a habitat (Frost et al., 1982). Although gemmules are frequently associated with dormant periods, they may also be present in freshwater species during periods of growth. Many sponge species routinely contain a few gemmules within otherwise active tissue, particularly in areas of thick growth. In an extreme case, S. lacustris develops specialized, summer gemmules which have much thicker coats than the gemmules formed for overwinter periods (Gilbert and Simpson, 1976b). These investigators suggested that summer gemmules may serve in a “bet-hedging” strategy against some environmental catastrophe which could occur prior to more extensive gemmule formation. It seems likely that summer gemmules may also serve in dispersal among lakes or streams. 3. Dispersal Among Habitats As is the case for most freshwater organisms, an ability to disperse among different habitats is critical for the continued success of sponges in freshwater environments. Most lakes and streams are ephemeral from an evolutionary perspective. Nearly all lakes in North America are less than 20,000 years old (Frey, 1963), a brief period compared to the 100 million year history of freshwater sponges (Racek and Harrison, 1975). While there has been little documentation of dispersal by freshwater sponges among habitats, it seems likely that gemmules are the primary agent for such movement. The two other life-cycle stages that might function in dispersal among habitats, larvae and sponge fragments, possess neither the structural nor the physiological mechanisms necessary for movement out of water or across signiﬁcant distances. It is conceivable that these forms, particularly the larvae, could function in short-range dispersal among connected water bodies, but their fragile structure would preclude any out-of-water transport. Gemmules, on the other hand, with their resistant coats and ability to withstand harsh environmental conditions appear to be well suited to dispersal among habitats. Ricciardi and Reiswig (1993) have reported rafting of S. lacustris gemmules during ﬂooding in the Ottawa River. C. Ecological Interactions As is the case with all organisms, the size of a freshwater sponge population, and its subsequent 109 impact on an ecosystem, results from an interplay of growth and loss processes. Growth is regulated by the availability of nutrients and suitable habitat. Losses are inﬂuenced by physical environmental conditions and, potentially, by interactions with other organisms. Although quantitative studies are few, it appears that the dynamics of freshwater sponge populations are mediated largely by the availability of suitable microhabitats within a lake or stream. Where substrates for sponge growth are short lived or where physical disturbances are frequent, growth and loss rates can be extremely rapid and may operate primarily in a density independent fashion. Under such circumstances, sponge populations would be regulated by an interplay between the physical environment and their own growth and life history processes. Concomitantly, interactions with other organisms would exert only minimal inﬂuences on sponge population processes. In contrast, where substrates are permanent, sponge populations appear more likely to be regulated by intra- or interspeciﬁc biotic interactions (Jackson and Buss, 1975). In many sponge habitats where growth on permanent substrates is rare, such biotic interactions could be expected to exert only a secondary role in their population dynamics. 1. Availability of Substrates The development of freshwater sponge populations usually depends on the availability of hard substrates. Most species must be attached to a substrate in order to grow. In addition, all species seem to depend on the presence of some solid surface for the successful settling and growth of their free-swimming larvae. A variety of surfaces are suitable. Examples range from boulders or exposed bedrock to the branches of fallen trees to the leaves and stems of aquatic macrophytes. Human built structures in water, such as bridge foundations, dams, spillways, and ﬂoats, can also serve as suitable surfaces for sponge growth. Potential substrates vary markedly in their permanence relative to the longevity of sponges, and these differences, in turn, have important consequences for sponge population dynamics. Among North American species, only S. lacustris has been routinely observed growing out of soft sediments. A quantitative investigation of its population dynamics (Frost et al., 1982), however, illustrated a crucial role for substrates even where sponges did not depend upon such substrates for their growth. In Mud Pond, New Hampshire, S. lacustris grows either directly from soft bottom sediments or attaches to any of several species of aquatic macrophytes which exhibit large summer populations, but which die back completely before winter. Due to the near absence of any 110 Thomas M. Frost, Henry M. Reiswig, and Anthony Ricciardi permanent substrates in Mud Pond, gemmulated sponges there overwinter almost exclusively in sediments on the pond bottom. Successful hatching in spring depends upon the presence of enough gemmulated tissue to grow out of these sediments, and many sponges are not large enough to make this transition. Most sponges are unsuccessful in hatching, and spring populations are sparse. Hatching from sediments, therefore, is the limiting step in the Mud Pond population’s dynamics. Despite remarkable growth during summer (a 1,000-fold increase in biomass), the population of S. lacustris in Mud Pond was maintained at nearly constant levels throughout ﬁve years of observations. Essentially, the population is maintained in a density-independent fashion by a combination of the lack of permanent substrates and the difﬁculty of gemmule hatching from soft sediments. Sponge population dynamics appear to be substantially different in habitats where more permanent substrates are available. Populations of Ephydatia ﬂuviatilis growing on hard substrates in a river on Sardinia, Italy exhibited growth rates that were still high, but were substantially lower than those reported for S. lacustris (Pronzato and Manconi, 1995). Populations were persistent on rocks over several years making transitions from active to gemmulated tissue. They were substantially disrupted, however, by a major ﬂood but did recolonize habitats fairly quickly (Pronzato and Manconi, 1995). In general, there appear to be periods of substantial population growth with substantial degeneration during other parts of the year, a process that can be categorized as density-independent (Pronzato and Manconi, 1995). Finally, in Mary Lake, Wisconsin, S. lacustris grows attached to trees that have fallen into the lake and sponges repeatedly make the transition from active tissue to gemmules and back to active tissue within the same skeleton over the course of several years. In contrast with the large ﬂuctuations in sponge density observed in Mud Pond, it seems that the populations of S. lacustris in Mary Lake exhibit higher densities throughout the year and lower growth rates during the course of the summer than those seen in Mud Pond (Frost, personal observation). Under such circumstances, population dynamics can be characterized primarily as density-dependent. Overall, the availability of substrates exerts a major control over sponge population dynamics. For many species, the lack of substrates limits growth completely. But, even where species are not wholly dependent on substrates, their nature and availability can exert a major inﬂuence over sponge population dynamics. The longevity of an individual sponge, including repeated transitions between active and gemmulated states, would appear to be directly related to the permanence of its substrate. In addition, it seems likely that the importance of both intra- and interspeciﬁc biotic interactions in sponge population dynamics will be inﬂuenced by the time that a particular substrate has been available for colonization. Only on long-lived substrates would sponges be likely to reach sufﬁcient densities to interact with each other. 2. Nutritional Ecology When suitable habitats are available, sponges are capable of substantial growth. In some cases, these high growth rates appear to be linked primarily to a sponge’s efﬁcient particle-gathering mechanisms. In other situations, however, sponges have an alternative means of obtaining resources. Many freshwater sponges contain large numbers of intracellular algae. They form symbiotic associations that combine autotrophy and heterotropy. In addition, because sponges require substantial amounts of silica for their spicules, population growth and maintenance have the potential to be inﬂuenced by silica availability. a. Sponge Feeding Although there have been several detailed studies of the mechanisms by which sponges feed and of the resources they consume (Frost, 1987), much less is known about the inﬂuence of food availability on sponge growth under natural conditions. The rates at which sponges ﬁlter water can be extremely high. During summer, specimens of S. lacustris typically ﬁltered more than 6 ml/h per miligram dry mass of tissue (Frost, 1980b). At this rate, a ﬁnger-sized sponge ﬁlters more than 125 L per day . Because a sponge’s feeding effectively removes all particles ranging in size from bacteria to large algae from the water it ﬁlters, these rates suggest that sponges may exhaust available food under some circumstances. Food limitation would be particularly likely in still water or where particulate resources are sparse. Pile et al. (1997) demonstrated that such depletion occurred in Lake Baikal, Russia, where sponges consumed a variety of picoplankton (cells 2 m in diameter) in substantial quantities. b. Algal Symbionts In many cases, freshwater sponges are bright green due to the presence of large quantities of chlorophyll contained within extensive populations of algal symbionts (Gilbert and Allen, 1973a, b). This situation is so common that sponges are frequently mistaken for plants in freshwater habitats. The algal symbiosis in freshwater sponges is representative of a large number of algal – invertebrate associations that are common in marine and freshwater habitats (Reisser, 1984). In these mutualistic 4. Porifera associations, the nutritional processes of algae and invertebrates are closely coupled. In freshwater sponges, and in many other invertebrates, algae are maintained endosymbiotically within the cells of their host. Algal – invertebrate symbioses combine autotrophic processes with normal animal heterotrophy. In the resulting, mixotrophic nutrition, symbiotic algae provide photosynthetically ﬁxed carbon to the invertebrate host which, in turn, supplies nutrients such as nitrogen or phosphorus or carbon dioxide to the algae. This combination has proved particularly successful in nutrient poor conditions, such as coral reefs (Muscatine and Porter, 1977) but occurs over a broad range of habitats. Algal symbionts play a major role in the growth of some freshwater sponges. Contributions from symbiotic algae accounted for 50 – 80% of the growth of S. lacustris in Mud Pond, New Hampshire (Frost and Williamson, 1980). The contribution of algae was determined by maintaining sponges in situ under darkened conditions, which led to the loss of their algae. Comparisons were then made between the growth of such aposymbiotic sponges and that of normal green sponges maintained under lighted but otherwise identical conditions. In similar experiments in Little Rock Lake, Wisconsin, S. lacustris and two other sponge species were incapable of any growth and died after a few weeks under darkened conditions (Frost, personal observation). Sand-Jensen and Pedersen (1994) also demonstrated a substantial contribution by symbiotic algae to the growth of S. lacustris in a Danish stream. Not all sponges depend on algal symbionts, however. Some species can live in darkness, where their nutrition is based solely on heterotrophic processes. The factors that control the proportional contribution of autotrophic and heterotrophic processes to overall sponge nutrition are less clear. That sponges failed to grow under darkened conditions in only some habitats indicates that the relationship between a sponge and its algal symbionts can range from facultative to obligate depending on environmental conditions. Even for species that sometimes depend to a large extent upon their algae, the amount of algal chlorophyll within a sponge (a rough measure of the potential contribution of its algal symbionts) varies markedly, both among habitats for a species and among species within a single habitat. In a survey of northern Wisconsin lakes, the average concentration of algal chlorophyll in S. lacustris varied among lakes by more than a factor of three (Frost, personal observation). Similarly, in Little Rock Lake, Wisconsin, three sponges exhibited consistent species-speciﬁc differences in their chlorophyll content that were coupled with signiﬁcantly different rates of both photosynthesis and feeding (Frost and Elias, 1990). These differences 111 occurred even though the three sponge species shared identical environmental conditions. The symbiotic relationship between sponges and algae differs fundamentally among sponge species. Some species depend, to a large extent, on algae for growth, while others exhibit little dependence. These differences among species may involve specializations to particular environmental conditions, in that sponges that contain large amounts of algal chlorophyll may be capable of growth only where light is readily available. Comparing habitats, it seems likely that sponges would vary the density of symbionts that they contain in response to the relative availability of light and particulate resources. However, this relationship is not simple. Detailed examinations of several sponge species indicate that increasing particulate resources may favor higher rather than lower concentrations of algae (Frost and Elias, 1990). Until recently, it had been thought that all symbiotic associations of algae and invertebrates in freshwater involved the presence of one group of green alga termed zoochlorellae (Reisser, 1984). Invertebrates hosting this alga include protozoans, hydra, ﬂatworms, clams, and sponges. It has now been determined, however, that at least one freshwater sponge species, Corvomeyenia everetti, and likely its congener C. carolinensis, contain a yellow-green alga as their symbiont (Frost et al., 1997). Volkmer-Ribeiro (1986) has suggested that the Corvomeyenia species have an evolutionary history that is long distinct from sponges with green-algal symbionts. These distinct histories combined with numerous overall similarities between sponges with green and yellow-green symbionts indicate that two, closely convergent symbiotic associations have evolved separately in the freshwater sponges. Morphological afﬁnities between marine algae and the yellow-green symbiont suggest that the symbiotic association in Corvomeyenia may have developed in an ancestral marine form and been maintained during a subsequent invasion of freshwater. It is important to note that differences between zoochlorellae and yellowgreen algae cannot explain the observed differences in algal contributions to overall sponge nutrition described above. Substantial differences in the contribution of algae have been observed in comparisons of sponge species that contain only green algal symbionts. The variety of ecological interactions between sponges and algae and the complexity of their evolutionary history indicate that algal symbioses have been important in the diversity and distribution of freshwater sponges. c. Potential Alternative Nutritional Modes Additional means of gathering food resources have been documented recently for some marine sponges. Vacelet 112 Thomas M. Frost, Henry M. Reiswig, and Anthony Ricciardi and Boury-Esnault (1995) have described a deep-sea sponge that lacks a typical sponge water-processing system and feeds on small animals with a mechanism that can be considered as carnivorous. However, some carnivorous sponges near deep-sea hydrothermal vents exhibit a symbiotic relationship with methanotrophic bacteria (Vacelet et al., 1995). Neither nutritional mode has been documented in freshwater species, but their existence in marine habitat raises interesting possibilities. d. Inﬂuence of Silica on Skeleton Structure The availability of silica within a habitat may exert both direct and indirect effects on sponge growth. As discussed previously, some sponges are restricted to high or low silica habitats. However, some species are distributed across habitats exhibiting a broad range of silica conditions, where they exhibit major responses to its availability. For example, in populations of S. lacustris in different lakes in northern Wisconsin, the total amount of biogenic silica and both the number and morphology of megascleres vary directly with silica concentrations in the lake (Frost et al., in preparation). Total biogenic silica and spicule width (Fig. 6) decrease as less silica is available. In contrast, however, the number of megascleres increases as their width decreases (Fig. 7). With this combination of changes, the total surface area of megascleres remains constant as silica decreases — a situation that increases the structural support gained per unit of available silica. This suggests a direct and complex response by sponges to different availabilities of silica. Responses to low silica conditions are related to changes in the stiffness and growth form of sponges. Specimens of S. lacustris from high silica habitats are much stronger and more resistant to breakage than those growing where silica is low. Sponges in low silica habitats, therefore, may be limited to growing closely attached to substrates or in waters with little wave action. Also, because spicules may play a role in the resistance of sponges to predation (see below), populations in low silica habitats may be more vulnerable to consumption. 3. Biotic Interactions a. Competition for Substrates The availability of substrate plays a crucial role in the population dynamics of freshwater sponges. As such, competition for surfaces would seem to have a potentially important effect on sponge distribution and abundance. A sponge has the potential to interact with a substrate any time that its growth leads to contact with another sponge or any other organisms. There are reports of signiﬁcant interactions for a substrate within, and between, sponge species, as well as between sponges and other organisms. Such interactions do not appear to occur commonly, however, and the role of competition for a substrate in the regulation of freshwater sponge populations remains largely unknown. In part, this is due to the minimal attention that interactions for substrates have received in freshwater in general. In addition, because freshwater sponge populations are often limited by density-independent processes, the abundance of sponges within a habitat is usually sparse with little potential for interactions on substrates. When specimens of same sponge species grow into contact with each other on a substrate, two distinctly different results have been observed (Van de Vyver, 1970). In some cases, the separate sponges will form a single, functionally integrated unit. In other instances, a distinct structural barrier is erected in areas of contact and the two sponges continue to function as completely separate units. The genetic relationship between sponges appears to control whether they will merge or develop a barrier. Separate sponges grown from fragments or gemmules taken from the same original sponge will always merge with each other. In contrast, experimental manipulations have indicated the existence of distinct strains within a sponge species which will not merge with each other (Van de Vyver and Willenz, 1975). Studies of marine sponges originally suggested that only sponges that are genetically identical would merge with each other (Niegel and Avise, 1983). However, subsequent analyses have indicated that, while the probability of merging appears to be a function of the genetic similarity of sponges, specimens need not be genetically identical in order to join into a single unit (Stoddart et al., 1985). Such fusion interactions in sponges operate at a cellular level (Van de Vyver, 1979) and have attracted the attention of researchers interested in the basic process of cell – cell recognition. These studies have direct implications for the understanding of immune-response systems in more complex invertebrates and in vertebrates (Smith and Hildemann, 1984). Interactions for substrates among different sponge species have been thoroughly documented in marine systems while they have received little attention in freshwater. Marine studies have demonstrated a complex hierarchy in which certain sponge species can completely overgrow and kill other species (Jackson and Buss, 1975). These interactions have a major inﬂuence on overall community ecology on substrates (Jackson, 1977). Observations of interspecies interactions in freshwater sponges have been limited to instances where sponges have been shown to develop a nonmerging front similar to that described above for intra-species interactions (Fig. 1). In habitats where 4. Porifera substrates are extremely long-lived, it seems likely that some sponge species may be able to completely overgrow other sponges, but the signiﬁcance of such interactions in freshwater habitats remains to be demonstrated. Interactions for substrates between sponges and other organisms had likewise received little attention in freshwater. Recently, however, sponges have been shown to overgrow and kill the zebra mussel, Dreissena polymorpha, a species whose recent invasion of North America via the Laurentian Great Lakes has been extensive and has raised substantial concerns (Ricciardi et al., 1995). Unfortunately, sponge overgrowth does not appear to be a solution to zebra-mussel related problems. More generally, for attached organisms overgrowth by sponges must be a factor in other freshwater habitats. The spreading of a sponge across any surface must involve the overgrowth of microscopic algae and bacteria. Sponges often encrust on aquatic macrophytes, but do not seem to harm the plants. In a few cases, freshwater sponges have been observed to overgrow and kill colonies of bryozoans (Frost, personal observation). The overall importance of sponge overgrowth for other organisms remains to be assessed. b. Predation and Infaunal Organisms Conspicuous predation upon sponges, in which major portions are consumed by a larger organism, is rare. A variety of smaller organisms consume portions of sponges, but they appear to act as grazers or parasites, leaving the sponge that they consume largely intact. Such species that feed on sponges represent a subset of the diverse infaunal community that typically occurs in freshwater sponges. Other infaunal organisms appear only to make use of the structure provided by a sponge in a commensal relationship. Resistance to predation by larger organisms appears to be characteristic of the Porifera in general (Randall and Hartman 1968; Resh, 1976). Mechanical and chemical defenses combine to make sponges resistant to predation. Spicules provide mechanical deterrence to predators; these sharp spines are thought to act as an irritant to its mouth and digestive system. The effectiveness of spicules in deterring predation was evident in experimental tests in which snails would not consume S. lacustris from habitats where it contained robust spicules, but would consume specimens from habitats in which low silica availability limited the size of spicules (T. Frost and A. Covich, personal observation). Chemical deterrence to predation has been attributed to a variety of toxic and pharmacologically active compounds that are well documented in marine sponges (Cecil et al. 1976) and are also likely to occur in freshwater species. 113 Despite the general effectiveness of sponge defense mechanisms, a few organisms prey effectively upon sponges. In some cases sponge predators have developed a nearly complete dependence on sponges. The few large predators on freshwater sponges include ﬁshes, crayﬁshes, ring-necked ducks and, possibly, snails. Extensive predation by ﬁsh on sponges has been documented in Africa (Dominey, 1987) and South America (Volkmer-Ribiero and Grosser, 1981), but is not common in North America. Crayﬁsh were implicated as causing major reductions in a sponge population in a Massachusetts stream (Williamson, 1979), but widespread consumption of sponges by crayﬁsh has not been reported. Juvenile ring-necked ducks have been reported to sometimes depend on sponges as a food source (McAuley and Longcore, 1988). Snails will consume and can subsist on freshwater sponges under laboratory conditions (T. Frost and A. Covich, personal observation); but here too, their signiﬁcance as predators under natural conditions remains unknown. Numerous small invertebrates reside within, or attached to, freshwater sponges. Examples include protozoans, oligochaetes, nematodes, rotifers, bivalves, water mites, and aquatic insects (Berzins, 1950; Resh, 1976; Volkmer-Ribiero and De Rosa-Barbosa, 1979). A variety of relationships exist between these infaunal organisms and their sponge hosts. Some infaunal species feed upon sponges while others use the sponge body as a habitat. In some cases the relationships are obligate, at least for certain life cycle stages, and such species may show a striking degree of specialization to the sponge host. Other organisms are only occasionally associated with sponges. Several groups of aquatic insects typify organisms with obligate, specialized feeding on freshwater sponges. Numerous species within three insect orders, the Diptera, Neuroptera, and Trichoptera, depend on freshwater sponges during major portions of their life cycle (Resh, 1976). One neuropteran family, the Sisyridae, is so commonly associated with sponges that its members are referred to as Spongilla Flies. Not all relationships between insects and sponges are obligate. Some insect genera that contain obligately, spongefeeding species also contains species that are only occasionally or never associated with sponges (Resh et al., 1976). The variety of interactions evident between sponges and related insect species indicates that the evolutionary history of insect – sponge relationships is diverse and complex. Obligate relationships with freshwater sponges have also been documented for water mites (Proctor and Pritchard, 1990) that apparently depend on the sponge primarily for structure. The water mites live and lay their eggs within the feeding canal system of 114 Thomas M. Frost, Henry M. Reiswig, and Anthony Ricciardi the sponge. These and other organisms that live within sponges (e.g., clams, insects and protozoans) may be taking advantage of the lack of predation on sponges. Spicules and chemical defenses may provide a general refuge against predators on infauna as well as sponges. The community ecology of the infaunal organisms themselves is also complex. The presence and abundance of a particular infaunal species results from an interplay of its own life cycle, the life cycle of the host sponge and potentially, the abundance of other infaunal organisms. Data from northern Wisconsin sponge populations indicate that most other infaunal species are rare or absent during periods when water mites are abundant within sponges (J. Elias and T. Frost, personal observation). Again, this is an area that has received little attention. tative of sponges in a variety of other lakes and streams. Sponge populations in some northern Wisconsin lakes, for example, appear to be substantially larger than those in Mud Pond (T. Frost, personal observation). At the same time, sponges in many ecosystems represent only a minor component when compared with other organisms. Even when sponges account for substantial portions of an ecosystem’s primary and secondary production, they may not interact directly with higher trophic levels. The lack of predation on sponges can lead to an accumulation of material in sponge biomass that is not passed directly up the food chain. Interaction of sponge with other ecosystem components may be indirect, operating primarily to limit the availability of materials to other organisms within the food web. 4. Functional Role in Ecosystems 5. Paleolimnology In most lakes and streams, populations of freshwater sponges are sufﬁciently sparse that they are unlikely to play a major role in ecosystem function. However, there are marked exceptions to this general pattern. In some habitats, sponges are the dominant component of the benthic community and exert a substantial inﬂuence on total nutrient cycling and primary production. Quantitative analyses of freshwater sponge populations are rare, but a detailed study of one population illustrates the potentially major impact of sponges on ecosystems (Frost, 1978; Frost et al., 1982). At the peak of its population size in early October, S. lacustris in Mud Pond, New Hampshire reached an average biomass of greater than 3.5 g dry mass per square meter. At this density, the total sponge population in the pond would ﬁlter more than ten million liters of pond water per day, a rate that would cycle a volume equivalent to that of the entire pond every 7 days. Since the sponges are capable of removing most phytoplankton and bacteria from the water that they ﬁlter, their potential impact on Mud Pond is substantial. Because of their algal symbionts, sponges can also account for a substantial portion of the primary production in Mud Pond. During late summer and fall, the chlorophyll within the sponges is approximately equivalent to that in the entire phytoplankton community (Frost, 1978; Frost and Williamson, 1980). Sponges also play a major role in ﬂowing-water ecosystems. Mann et al. (1972), in a detailed study of the River Thames in England, found that sponges accounted for nearly 40% of the total production by benthic animals. Clearly, sponges can be a major component of the biota of some ecosystems. While detailed quantitative studies are rare, observations indicate that the populations in Mud Pond and the River Thames are represen- The siliceous nature of sponge spicules makes them resistant to decomposition under most circumstances. As such they are usually well preserved in lake sediments and have the potential to serve as a useful tool in indicating historic lake conditions. In situations where speciﬁc habitat requirements can be deﬁned for a species (Jewell, 1935), the presence of its spicules in the sediments of a lake from which it is currently absent can indicate changes over time in the environmental conditions of that lake. Likewise, even when a sponge species is present throughout an extended period within a lake, quantitative changes in spicule morphology over time can indicate shifts in the availability of silica within that lake. For example, lakes throughout northern Wisconsin appear to have exhibited a more than 50% reduction in silica availability over the last 12,000 years (Kratz et al., 1991). Paleolimnological investigations employing sponge spicules are a potentially fruitful area for study (Harrison, 1986). Studies in 28 Connecticut water bodies revealed that most lakes had maintained the same sponge populations over the past century, but that a few lakes had exhibited changes in sponge populations perhaps indicating a decline in water quality (Paduano and Fell, 1997). Spicule presence and distribution has been used to infer the history of selected Florida soils (Schwandes and Collins, 1994). Further studies have the potential to reveal other important features of lake history. D. Evolutionary Relationships All freshwater sponges belong to the class Demospongiae, the most diverse and morphologically complex of the four major groups in the sponge phylum. Within the demosponges, the evolutionary history of the freshwater sponges is long and polyphyletic. Fossil 4. Porifera freshwater sponges are reported at least 100 million years before (Racek and Harrison, 1975); and, although there is still debate in this area, there appear to have been at least three, and possibly four, separate, successful invasions of freshwater from marine habitats. The separate invasions are presently classiﬁed into distinct families among the freshwater sponges (VolkmerRibeiro and De Rosa-Barbosa, 1979, Volkmer-Ribeiro, 1986). The phylogenetic diversity of freshwater sponges is not well represented in North America where species are conﬁned primarily to the most cosmopolitan family, the Spongillidae (Penney and Racek, 1968). Volkmer-Ribeiro (1986) proposed that species in the genus Corvomeyenia should be grouped, along with several South American species, in the family Metaniidae with an evolutionary history in freshwater that is distinct from the spongillids. Only two North American species, Corvomeyenia everetti and C. carolinensis, occur in this proposed family which has a worldwide, primarily tropical distribution (Volkmer-Ribeiro, 1986). Not all sponge biologists have recognized the Metaniidae as a separate family (e.g., Ricciardi and Reiswig, 1993), however. The two other freshwater sponge families are absent from North America. Species in the Lumbomerskiidae appear to be conﬁned to a few large and ancient lakes in Europe and Asia (e.g., Lakes Baikal and Ochrid), and the family is characterized by a large degree of endemism. Species in the Potamolepidae are widely distributed throughout Africa and South America (Volkmer-Ribeiro and De Rosa-Barbosa, 1979). IV. COLLECTING, REARING, AND PREPARING SPONGES FOR IDENTIFICATION Collecting sponges is usually straightforward. In many cases, sponges grow in shallow water and can be obtained simply by hand or with a long-handled rake. Where sponges are rare, it may be easiest to collect them while snorkeling. This may be the case particularly when collecting in areas with numerous hard substrates where small stones must be overturned and the undersides of fallen trees and large boulders must be examined. Where sponges grow in deeper waters, scuba techniques afford the most efﬁcient means of collection. Dredges or nets dragged across substrates in deeper waters are likely to miss most sponges and, at the same time, are likely to disrupt substantial bottom areas. In very deep waters, however, these may be the only practical means of collecting. When searching a lake or stream for sponges, it is best to examine as broad a variety of substrates as 115 possible. Aquatic macrophytes, rocks, and fallen logs are common sites for sponge growth. In bog habitats, the roots of vegetation growing at the edges of bog mats or on the undersides of the mats themselves are likely sites for sponges. Where sponges are rare, it may be necessary to examine a large number of different substrates before ﬁnding any specimens. In other habitats, however, sponges will be conspicuous. Growing or maintaining sponges under controlled environmental conditions is much more difﬁcult than collecting them. Some investigators have grown small sponges from gemmules in the laboratory for cytological investigations (Rasmont, 1962; Fell, 1974). Such specimens can also be particularly useful in examining smaller scale structural details of sponges. Poirrier et al. (1981) have developed an effective, continuous-ﬂow system for sponges using bacteria as a food source. Laboratory investigations of this sort can provide important insights into sponge ecology (e.g., Harsha et al., 1983), particularly when controlled conditions are essential. Sponges appear to be highly sensitive to environmental conditions, however, and the responses of sponges obtained in laboratory studies must be compared carefully with their behavior under ﬁeld situations. As such, to evaluate sponge behavior under natural conditions, it is often best to work with freshly collected sponges and to conduct experiments in situ whenever possible. The identiﬁcation of freshwater sponges depends primarily on characteristics of spicules and on features of intact gemmules. Successful species identiﬁcations are dependent on obtaining all of the spicules (megascleres, gemmoscleres, and, if present in a species, microscleres) that can occur in a species. Thus, it is absolutely essential to obtain samples of sponges that include all spicule types. Gemmoscleres are particularly important, and this can present a problem because gemmules (Figs. 1 and 8) may only occur during certain times of the year. Some gemmoscleres may remain in active sponge tissue after gemmules have hatched, so there is some chance of identifying sponges without gemmules visible in them. Gemmules should be sought carefully in any survey, however. As the primary step in species identiﬁcation, samples of all types of spicules that occur in a species should be prepared for microscopic examination. To obtain spicule preparations, organic portions of a sponge are digested with acid and the remaining spicules are mounted on a glass microscope slide. Sponge tissue can be digested with nitric acid in a centrifuge tube that is immersed in boiling water for 1 h. The spicules can then be concentrated by gentle centrifugation, after which the acid is poured off and the spicules are then washed with ethanol or methanol. 116 Thomas M. Frost, Henry M. Reiswig, and Anthony Ricciardi Washing and centrifugation should be repeated at least two times after which the spicules are mounted on a microscope slide using a cover slip and a permanent medium (e.g., Permount). Reiswig and Browman (1987) described a similar, but more precise technique for a quantitative method of processing sponge spicules. Intact gemmules can be removed from sponge tissue, dried, and mounted directly on microscope slides. Specimens of entire sponges are necessary for museum collections. Whole sponges can be dried or preserved in alcohol. Simpson (1963) describes methods that can be used for preparing freshwater sponges for cytological observations, such as are necessary for examining reproductive cycles. V. CLASSIFICATION The classiﬁcation scheme reported here is derived primarily from Penney and Racek (1968), who have completed the most recent taxonomic revision of the family Spongillidae, including the genus Corvomeyenia. Their monograph provides more detailed descriptions of most of the species reported here and should be consulted for rigorous taxonomic investigations. Information for several species in this chapter is based on reports other than those Penney and Racek (1968), most of which have been published since 1968. In the cases where substantial information about a species has been obtained from a source other than, or in addition to, Penney and Racek, these sources have been cited along with the species description in the list of species below. In addition, there are a number of cases where species reported for Canada and the United States by Penney and Racek (1968) have been judged subsequently as invalid (e.g., Poirrier, 1974, 1977; Harrison and Harrison, 1977; Harrison, et al., 1977; Harrison, 1979), and these species have not been included here. Ricciardi and Reiswig (1993) provide another valuable source of information on 15 sponge species that occur in eastern Canada, and this report should be consulted for more detailed information on these species. In general, the freshwater sponge species in Canada and the United States can be distinguished by characteristics of their spicules. Taxa are separated primarily by the presence, shape, and spination of microscleres and / or gemmoscleres, although the presence or absence of spines on megascleres can be a useful characteristic in some cases in distinguishing species within a genus. Spicules are usually either needle or rodlike (Fig. 6) or dumbell-shaped (Fig. 10). The latter are termed birotulate, and their rounded ends are called rotules. In the descriptions here, a spicule should be assumed to be needle or rodlike, unless it is described as otherwise. Also, the descriptions reported here are based on examinations with light microscopes. More detailed examinations with scanning electron microscopes can reveal ﬁne structures, primarily spines, that are not apparent in light microscope observations. Such ﬁner scale observations should be interpreted with caution when employing the descriptions and key that are provided here. Gemmoscleres are critical in the classiﬁcation of freshwater sponges, and it is important to include gemmules when preparing sponge specimens for identiﬁcation. Obtaining gemmoscleres may be a problem, particularly in young sponges derived from larvae, where gemmules and their spicules may not be present during certain times of the year. Examine a collected specimen closely to insure that gemmules are present (Figs. 1 and 8). It is possible, particularly in larger specimens, that some gemmoscleres may have been left in a sponge body from a previous year’s gemmules. However, it is also possible that a few stray gemmoscleres or FIGURE 10 Megasclere (M, scale bar 50 m) and gemmoscleres (g, scale bar 25 m;) of Anheteromeyenia argyrosperma (from Ricciardi and Reiswig, 1993). 4. Porifera even entire gemmules from another sponge species may have been incorporated into a sponge specimen. The growth of the tissue of one sponge species over the gemmules of another species has lead to some erroneous species identiﬁcations (Ricciardi and Reiswig, 1992). Thus, identiﬁcations based on only a few gemmoscleres should be made with caution. In some species with microscleres, it may be difﬁcult to distinguish between microscleres and gemmoscleres on the basis of structure alone. By separating gemmules from intact sponges and making separate spicule preparations of them, it is possible to obtain samples in which gemmoscleres predominate. Alternatively, by carefully sampling tissue from the surface of a sponge in areas of new growth it may be possible to obtain material in which gemmoscleres are rare and in which microscleres and megascleres predominate. Because of the important role that microscleres play in separating species, identiﬁcations based on only a few microscleres should also be made with caution. It is important to consider that ecomorphic variation can be substantial for some sponge species, particularly Anheteromeyenia ryderi, Ephydatia ﬂuviatilis, and Spongilla lacustris. Cautions about particularly troublesome situations are raised in the species descriptions below. Ricciardi and Reiswig (1993) provide more detailed information on the eastern Canadian species. In using the key presented here, there are only two cases in which characteristics other than the structure of spicules are required to distinguish among taxa. In contrasting the genera Ephydatia and Radiospongilla, it is necessary to obtain a cross section of intact gemmules in order to view the arrangement of gemmoscleres within the gemmule coat. Similarly, within the genus Heteromeyenia, it is necessary to employ the size, shape, and structures of the foraminal aperture of entire gemmules to distinguish among species. Particularly important for Heteromeyenia are the forms of structures on the end of the foraminal tubule termed cirrous projections. Ricciardi and Reiswig (1993) reported an extensive survey of sponges in eastern Canada in which they reported 15 species. The report is extremely useful and should be consulted for detailed information on the species that they report. It even includes a key for distinguishing, to a certain extent, among sponges for which gemmoscleres are not available — a critical requirement of the key here and for using Penney and Racek (1968). A. Freshwater Sponge Species of Canada and the United States Summarized below are the spicular characteristics and distribution patterns of freshwater sponges reported 117 from the United States and Canada. Dimensions presented for spicules are the ranges that have been reported for them; average values to be expected are very close to the mean of the numbers listed below. In some cases, mean dimension values are reported as xx(mean)-xx, where xx represents ranges. It is important to note that these mean values are from Ricciardi and Reiswig (1993). They may be speciﬁc to sponges from eastern Canada and may not be representative of sponges growing in other regions. Values for ranges are the widest values that have been published in the papers reported here. Some sponge spicule features may vary from habitat to habitat. For a few species information is also provided on features of gemmules, such as the distribution of gemmoscleres within them, their opening structures (foraminal apertures), and their overall distribution within a sponge body. Geographic distribution patterns emphasize the occurrence of sponges in the United States and Canada, but also describe the overall distributions reported for each species. Anheteromeyenia argyrosperma (Potts) Spicules: megascleres slender, 240-(284)-304 m in length, and sparsely covered with small, sharply pointed spines; microscleres absent; gemmoscleres birotulates of two distinct length groups, 65-(81)-89 or 110-(130)-160 m, with both size classes similar in form exhibiting spines on their entire shaft and conspicuous recurved, clawlike hooks on their ends. Distribution: reported from the eastern half of North America from Florida to Canada but conﬁned to this region. Anheteromeyenia ryderi (Potts) Spicules: megascleres are extremely variable from habitat to habitat with lengths ranging from 141-(220)279 m; variable shape and coverage with broadly conical spines; microscleres absent; gemmoscleres birotulates of two distinct length groups with distinct differences in their shapes, 28-(34)-41 or 45-(64)-75 m in length; shorter forms have shafts with only one or a few spines and ﬂattened rotules with a large number of small teeth; larger forms are robust with numerous recurved spines on their shaft and with strongly recurved hooks on their ends. Distribution: reported from the eastern half of North American, from Louisiana to Canada, but conﬁned to this region. Corvomeyenia carolinensis (Harrison) Spicules: megascleres slender, straight to slightly curved and entirely smooth, ranging 194 – 280 m in length; microscleres birotulate with straight to strongly 118 Thomas M. Frost, Henry M. Reiswig, and Anthony Ricciardi FIGURE 11 Megasclere (M, scale bar 50 m) and gemmoscleres (g, scale bar 25 m;) including a rotule (r) of Anheteromeyenia ryderi (from Ricciardi and Reiswig, 1993). curved ( 80%), smooth shafts, 15 – 25 m in length terminating in rotules 4 – 7 m in diameter with 4 – 6 recurved hooks; gemmoscleres birotulates with straight to slightly curved, smooth shafts 60 to 158 m in length and terminating in rotules of 13 – 22 m diameter with 5 – 8 recurved hooks (Harrison, 1971). Distribution: reported from one pond in South Carolina and one lake in Connecticut. gemmoscleres birotulate with straight to slightly curved, smooth shafts, 33-(59)-78 m in length and terminating in rotules 10-(20)-26 m in diameter bearing 5 – 7 recurved hooks. See information on Corvospongilla novaeterrae below and in Ricciardi and Reiswig (1993). Distribution: reported only from the eastern half of the northern United States and Canada. Corvomeyenia everetti (Mills) Spicules: megascleres slender, slightly curved and entirely smooth, 143-(218)-285 m in length (in rare cases, a variable number of megascleres may be sparsely spined); microscleres birotulates, 14-(18)26 m in length, terminating in rotules 3-(5)-7 m in diameter with 3 – 6 small, distinctly recurved spines; Corvospongilla becki (Poirrier) Spicules: megascleres stout, 130 – 218 m in length, usually curved, covered with spines, the spines being larger near the ends of a spicule; microscleres birotulate, 25 – 44 m in length, rotules 9 – 17 m in diameter, usually with four recurved hooks; gemmoscleres of two distinct length classes, the smaller class is 28 – 56 m in length, slightly to strongly curved and spined, except in the inner curved region, the larger class 71 – 139 m in length, straight to slightly curved and completely spined (somewhat similar in form to the gemmoscleres of S. lacustris — Fig. 29) (Poirrier, 1978). See information on Corvospongilla novaeterrae below. Distribution: reported from one lake in Louisiana. FIGURE 12 Microscleres (m) and gemmoscleres (G) of Corvomeyenia carolinensis (after Harrison, 1971). Corvospongilla novaeterrae (Potts) Spicules: megascleres stout, smooth, 112-(154)-170 m in length and relatively scarce; microscleres are abundant birotulates, 13-(21)-32 m in length with smooth shafts, rotules dome shaped with 3 – 6 spines; gemmoscleres highly variable, bearing numerous large, recurved spines which tend to be concentrated near the ends of the shaft, sometimes making spicules appear birotulate with lengths 21-(39)-63 m and widths, excluding spines, 3-(6)-9 m. This species is remarkably similar to Corvomeyenia everetti in its basic growth form, except that C. novaeterrae gemmoscleres are not thin, elongate birotulates and C. novaeterrae gemmules 4. Porifera 119 FIGURE 13 Megasclere (M, scale bar 50 m), microscleres (m, scale bar 25 m) including a rotule (r) and gemmosclere (g, scale bar 25 m) of Corvomeyenia everetti (from Ricciardi and Reiswig, 1993). have a very poorly developed outer layer. See detailed information on C. novaeterrae in Ricciardi and Reiswig (1993). Distribution: reported from a few lakes in the maritime provinces of Canada and one lake in Connecticut (De Santo and Fell, 1996). Dosilia palmeri (Potts) Spicules: megascleres slender, 370 – 450 m in length, slightly curved to nearly straight, covered with sparse spines in their central portion; microscleres stellate with 8 – 12 usually smooth rays arising from a central nodule, length is extremely variable; gemmoscleres birotulates, 55 – 85 m in length, occurring in two subtly distinct size classes, with strong spines on their central shaft and with equally sized rotules, 23 – 25 m in diameter, bearing numerous blunt recurved teeth (Penney, 1960). Distribution: reported from Florida in North America north of Mexico and from other locations in Central America. Dosilia radiospiculata (Mills) Spicules: megascleres slender, 290 – 400 m in length, entirely smooth or covered with minute spines; microscleres stellate with 6 – 8 microspined rays projecting from their center, length is extremely variable; gemmoscleres birotulates of two distinctly different size classes with longer forms exhibiting nonspined shafts 120 – 230 m in length and shorter forms exhibiting strongly spined shafts 45 – 82 m in length. [There is some question as to whether the two species of Dosilia are distinct or simply ecomorphic variants of a single species.] Distribution: reported from the Canadian border south to Mexico, but only from this region. Megasclere (M, scale bar 50 m), microscleres (m, scale bar 10 m) and gemmoscleres (g, scale bar 10 m) of Corvospongilla novaeterrae (from Ricciardi and Reiswig, 1993). FIGURE 14 120 Thomas M. Frost, Henry M. Reiswig, and Anthony Ricciardi FIGURE 15 Microscleres of Dosilia radiospiculata (after Penney and Racek, 1968). Duosclera mackayi (Carter) Spicules: two distinct classes of megascleres, the ﬁrst is relatively scarce, straight or slightly curved, 177(200)-302 m in length, 7-(12)-18 m in width (excluding spines), covered with coarse spines and the second is somewhat shorter, 79-(156)-267 m in length and generally broader, 2-(8)-20 m in width (excluding spines) and densely covered with spines that are long, pointed, strongly recurved near the tips of the spicule and perpendicular near its center; microscleres absent; gemmoscleres are the same as the second class of megascleres, which are always present in a specimen, even in the absence of gemmules. When intact gemmules are present, gemmoscleres are arrayed tangentially to the surface of the gemmule except near the foraminal aperture. The overall orientation of gemmules can help distinguish Duosclera mackayi from a similar species, E. fragilis. In D. Mackayi the foraminal apertures are always oriented inward or towards a substrate while those in E. fragilis are always directed outward or upward from a pavement layers. This species has recently been classiﬁed as a new genus (Reiswig and Ricciardi, 1993). Previously reported as Eunapius igloviformis in Penney and Racek (1968) and as Eunapius mackayi in Ricciardi and Reiswig (1993). Distribution: throughout the United States and Canada, but conﬁned to these regions. Ephydatia ﬂuviatilis (Linneaus) Spicules: megascleres usually slightly curved, 210(343)-439 m in length, and usually entirely smooth, although in some cases, some sparsely spined megascleres co-occur with smooth forms; microscleres absent; gemmoscleres birotulates of one class, 20-(23)-30 m in length with a slender, smooth shaft, which sometimes has 1 – 4 large spines, with ﬂat irregularly shaped rotules of equal, 13-(18)-24 m diameters and usually more than 20 teeth that are not deeply incised. E. ﬂuviatilis can sometimes be confused with E. muelleri. These species can be most surely distinguished by comparing gemmosclere length to rotule diameter. Megascleres (M, scale bars 50 and 25 m for the magniﬁed section), gemmoscleres, which are actually a second size category of megasclere (g, scale bars 25 m) and gemmules of Duosclera mackayi, the arrowhead indicates the foraminal aperture on the underside of a gemmule cluster (from Ricciardi and Reiswig, 1993). FIGURE 16 4. Porifera 121 Megasclere (M, scale bar 50 m) and gemmoscleres (g, scale bar 25 m) and a rotule (r, scale bar 10 m) of Ephydatia ﬂuviatilis (from Ricciardi and Reiswig, 1993). FIGURE 17 Gemmosclere length is always greater than rotule diameter in E. ﬂuviatilis while gemmosclere length is less than or equal to rotule diameter in E. muelleri (Ricciardi and Reiswig, 1993). Distribution: appears to be truly cosmopolitan with more frequent occurrence in temperate than in tropical zones. Ephydatia millsii (Potts) Spicules: megascleres slightly curved to nearly straight, 180 – 270 m in length, with numerous small spines except at the tips; microscleres absent; gemmoscleres birotulates of one size class, 36 – 48 m in length, with smooth shafts that are clearly broader near the rotules and with distinctly ﬂat, disk – shaped rotules of equal, 22 – 28 m diameters and only very small incisions at their margins. Distribution: reported only from Florida. Ephydatia muelleri (Lieberkühn) Spicules: megascleres straight to slightly curved, 171-(245)-350 m in length, usually with small spines, typically lacking at the tips, but smooth in rare cases; FIGURE 18 Racek, 1968). Gemmoscleres of Ephydatia millsii (after Penney and microscleres absent; gemmoscleres birotulate of one class, 8-(17)-28 m in length, with thick smooth shafts and with ﬂat irregularly shaped rotules of equal, 8-(15)-27 m diameters and usually have fewer than 12 teeth that are deeply incised into long rays. E. muelleri can sometimes be confused with E. ﬂuviatilis. These species can be most surely distinguished by comparing gemmosclere length to rotule diameter. Gemmosclere length is always greater than rotule diameter in E. ﬂuviatilis while gemmosclere length is less than or equal to rotule diameter in E. muelleri (Ricciardi and Reiswig, 1993). Distribution: widely distributed throughout the Northern Hemisphere with a preference for temperate regions. Eunapius fragilis (Leidy) Spicules: megascleres entirely smooth, 165-(189)271 m in length; microscleres absent; gemmoscleres straight to slightly curved, covered with conspicuous spines often more dense near the tips, 32-(57)-140 m. Mature gemmules enclosed in a common brown coat forming a pavement layer cemented to the substrate (Gp in Fig. 20) or in individual clusters of 2 – 4 gemmules. The overall orientation of gemmules can help distinguish E. fragilis from a similar species, Duosclera mackayi. In E. fragilis, the foraminal apertures are always directed outward from a cluster or upward from a pavement layer while those in D. mackayi are always oriented inward or toward a substrate (Ricciardi and Reiswig, 1993). Distribution: truly cosmopolitan. Heteromeyenia baileyi (Bowerbank) Spicules: megascleres slender, 216-(247)-320 m in length, smooth or with sparse microspines except near the tips; microscleres 53-(67)-85 m in length, delicate, 122 Thomas M. Frost, Henry M. Reiswig, and Anthony Ricciardi Spined and smooth megascleres (M, scale bar 50 m) and gemmoscleres (g, scale bar 10 m), and a rotule (r, scale bar 10 m) of Ephydatia mülleri (from Ricciardi and Reiswig, 1993). FIGURE 19 slightly curved to almost straight with spines that occur throughout their length, but which increase in length towards the central region where they are often knobbed and distinctly perpendicular to the main axis; gemmoscleres birotulate of two intergrading length classes: (1) short birotules with ﬂat, serrated rotules (13-(18)-22 m in diameter), 38-(51)-60 m in length, and (2) long birotules 49-(70)-86 m in length with rotules (18-(22)-28 m in diameter) composed of long recurved hooks giving an umbrellalike appearance (hooks often have knobbed tips). Foraminal apertures of gemmules do not have terminal cirrous projections like those for H. tubisperma (Fig. 24), H. latitenta (Fig. 22), and H. tentasperma (Fig. 23). The long Megasclere (M, scale bar 50 m), gemmoscleres (g, scale bars 25 m), and a gemmule pavement of Eunapius fragilis, the arrowhead indicates a foraminal aperture on the dorsal surface of the gemmule pavement (from Ricciardi and Reiswig, 1993). FIGURE 20 4. Porifera 123 FIGURE 21 Megasclere (M, scale bar 50 m), microsclere (m, scale bar 25 m) and gemmoscleres (g, scale bar 25 m) of Heteromeyenia baileyi (from Ricciardi and Reiswig, 1993). middle spines on the microscleres of H. baileyi are also useful in distinguishing it from other species in the genus. Their maximum length is on average greater than the width of the microsclere. Distribution: widely distributed throughout the eastern United States and Canada with a few reports from Europe. Heteromeyenia latitenta (Potts) Spicules: megascleres straight, 265 – 285 m in length, smooth to sparsely microspined; microscleres slender and entirely spined with those in the central region only slightly larger than those on the ends, 85 – 100 m; gemmoscleres birotulates of two length groups, 50 – 55 or 60 – 78 m with shafts bearing numerous stout and pointed spines, both rotules of equal, 16 – 18 m diameters, margins forming numerous conspicuous, recurved teeth. Foraminal apertures of gemmules with one or two, very long, cirrous projections starting from a disk that is initially ﬂat but rounded in its terminal regions. FIGURE 22 Foraminal aperture of Heteromeyenia latitenta (after Neidhoefer, 1940). Distribution: reported only from northeastern United States. Heteromeyenia tentasperma (Potts) Spicules: megascleres very slender, 260 – 280 m in length, with sparse microspines; microscleres slender with sparse microspines, 75 – 80 m; gemmoscleres birotulates of two length groups, 50 – 55 or 65 – 72 m with stout shafts bearing a small number of acute spines and with both rotules of equal, 15-18 m diameters and consisting of an arrangement of lateral spines. Foraminal apertures of gemmules distinctly tubular and relatively short with 3 – 6 long and irregular cirrous projections. Distribution: reported only from northeastern to midwestern United States. Heteromeyenia tubisperma (Potts) Spicules: megascleres slender, 190-(290)-337 m in length, smooth to sparsely microspined; microscleres, 73-(100)-118 m in length, slender and entirely spined with spines near the tips small and recurved and those near the central portion distinctly larger, straight, and with knobs; gemmoscleres birotulates of two roughly deﬁned size classes, with both classes 33-(44)-70 m in length with stout shafts bearing a small number of acute spines and with both rotules of equal, 12-(19)-25 m FIGURE 23 Foraminal aperture of Heteromeyenia tentasperma (after Neidhoefer, 1940). 124 Thomas M. Frost, Henry M. Reiswig, and Anthony Ricciardi FIGURE 24 Megasclere (M, scale bar 50 m), microsclere (m, scale bar 25 m), and gemmoscleres (g, scale bar 25 m) and a foraminal aperture (GF) of Heteromeyenia tubisperma (from Ricciardi and Reiswig, 1993). diameters, consisting of an arrangement of lateral spines. Foraminal apertures of gemmules distinctly tubular, slender and very long (0.5 – 0.9 times the diameter of the gemmule) with 5 or 6 cylindrical cirrous projections. It is important to note that developing gemmules may have stunted foraminal development such that they resemble H. latitenta and H. tentasperma. Specimens should be thoroughly inspected for fully developed gemmules. H. tentasperma can be distinguished from H. baileyi which has shorter microscleres with longer spines. Distribution: reported only from the eastern half of North America. Radiospongilla cerebellata (Bowerbank) Spicules: megascleres straight to slightly curved, 240 – 330 m in length, entirely without spines; microscleres absent although immature gemmoscleres may be abundant in some portions of the dermal membrane; gemmoscleres usually distinctly curved and rarely straight, 72 – 110 m in length, covered with abundant spines that are pronouncedly recurved toward the terminal ends (in intact gemmules, gemmoscleres are arrayed in two distinct layers with those in the inner layer arranged in a radial fashion and those in the outer layer arranged tangentially to the inner layer) (Poirrier, 1972). FIGURE 25 Megasclere (M, scale bar 50 m), gemmoscleres (g, scale bar 25 m), and a gemmule (G) showing a crater like depression around the foraminal aperture of Radiospongilla crateriformis (from Ricciardi and Reiswig, 1993). 4. Porifera 125 Distribution: reported only from Texas in the United States, but widely distributed in tropical and subtropical Asia and Africa. Radiospongilla crateriformis (Potts) Spicules: megascleres slender and slightly curved, 240-(278)-300 m in length, sparsely covered by very small spines, except at their tips; microscleres absent; gemmoscleres slender and covered with small conical spines, except at the tips where slightly recurved spines are sufﬁciently dense to form pseudorotules, 60-(71)80 m in length. Gemmoscleres in intact gemmules are arranged radially around the gemmule coat except in the immediate vicinity of the foraminal aperture where they form a craterlike depression leaning away from the aperture. Distribution: reported primarily from the eastern half of the United States but as far west as Wisconsin and Texas; eastern Canada (Ricciardi and Reiswig, 1993), also reported in China, Japan, Southeast Asia, and Australia. Spongilla alba (Carter) Spicules: megascleres entirely smooth, 144 – 420 m in length; microscleres slender and slightly curved with erect spines that are longer in the central region, 49 – 124 m; gemmoscleres slightly to moderately curved with stout, sharp, recurved spines that are more dense at the ends, forming distinct heads, 48 – 130 m (Poirrier, 1976). Distribution: occurs in warmer regions worldwide with a strong preference for brackish water, re- FIGURE 26 Microsclere (m) and gemmosclere (G) of Spongilla alba (after Penney and Racek, 1968). ported from the southeast coastal regions of the United States. Spongilla aspinosa (Potts) Spicules: megascleres slender and entirely smooth, 155-(274)-338 m in length; microscleres range from rare to abundant in number, smooth or very sparsely microspined, 21-(50)-78 m in length; gemmoscleres smooth, resembling small megascleres, 129-(274)306 m in length. Distribution: reported from the United States from Florida to Michigan, and from eastern Canada. Spongilla cenota (Penney and Racek) Spicules: megascleres stout and completely smooth, 310 – 410 m in length; microscleres numerous and FIGURE 27 Megasclere (M, scale bar 50 m), microscleres showing a range of forms (m, scale bar 25 m) and gemmosclere (g, scale bar 25 m) of Spongilla aspinosa (from Ricciardi and Reiswig, 1993). 126 Thomas M. Frost, Henry M. Reiswig, and Anthony Ricciardi Spongilla lacustris (Linneaus) Spicules: megascleres entirely smooth, 158-(254)362 m in length; microscleres densely covered with small spines, 30-(61)-130 m in length; gemmoscleres absent from winter gemmules and slightly curved, usually covered with strong, curved spines that tend to be concentrated near the tips, 18-(32)-130 m long in thick-coated summer gemmules (Poirrier et al., 1987). Distribution: throughout the Northern Hemisphere including frequent reports from throughout the United States and Canada. FIGURE 28 Microsclere (m) and gemmosclere (G) of Spongilla cenota (after Penney and Racek, 1968). slender, covered with small spines at their tip and with a group of clearly larger spines in their center, 68 – 123 m; gemmoscleres extremely stout, entirely covered with stout, sharp, recurved spines 65 – 86 m (Poirrier, 1976). Distribution: reported only from Florida, and the Yucatan in Mexico. Stratospongilla penneyi (Harrison) Spicules: megascleres slightly curved, 215 – 296 m in length, smooth to very delicately microspined, microscleres slender, 38 – 75 m in length, covered with very small spines, gemmoscleres curved to distinctly bent, 48 – 123 m in length, smooth to delicately microspined with sharply pointed apices. Distribution: reported from only one location, a canal in southern Florida (Harrison, 1979). Trochospongilla horrida (Weltner) Spicules: megascleres straight to slightly curved, 155-(187)-250 m in length, covered with stout, FIGURE 29 Megasclere (M, scale bar 50 m), microscleres showing a range of aberrant forms in the right plate (m, scale bar 25 m) and gemmosclere (g, scale bar 25 m) of Spongilla lacustris (from Ricciardi and Reiswig, 1993). 4. Porifera 127 Megascleres (M, scale bars 50 and 25 m for magniﬁed portion which shows the truncated spines typical of the genus) and gemmoscleres (g, scale bar 10 m) with rotule (r) of Trochospongilla horrida (from Ricciardi and Reiswig, 1993). FIGURE 30 blunt, truncated spines; microscleres absent; gemmoscleres small birotulates, 8 – 10 m in length, with stout smooth shafts and with rotules of nearly equal size, with the smaller between 9 and 13 m in diameter and the larger between 13 and 16 m with circular margins (overall length of gemmoscleres is never greater than the diameter of the smaller rotule). Compare with information for T. pennsylvanica to confirm an identification. Distribution: widely dispersed throughout the Northern Hemisphere although with few reports in eastern Canada. to this chapter. In such cases, the two species can be distinguished by the length of the gemmosclere shaft relative to the rotule diameter. The shaft length is longer than, or in rare cases, equal to, the diameter for the smaller rotule’s diameter in T. pennsylvanica and shorter than the rotule’s diameter for T. horrida (Ricciardi and Reiswig, 1993). Distribution: reported throughout, but apparently restricted to, the North American continent. Trochospongilla leidii (Bowerbank) Spicules: megascleres straight to slightly curved and entirely smooth, 150 – 170 m in length; microscleres absent; gemmoscleres small birotulates, 11 m in length, terminating in rotules with circular margins and equal, 12 – 14 m diameters. Distribution: reported from limited regions of the eastern United States, with one report of a population from the Panama Canal (Jones and Rützler, 1975). Please note that the successful use of this key depends on obtaining a fully representative sample of all Trochospongilla pennsylvanica (Potts) Spicules: megascleres slender and slightly curved, 100-(253)-432 m in length, entirely covered with blunt, truncated spines; microscleres absent; gemmoscleres small birotulates with slender shafts, 11(17)-41 m in length, terminating in rotules with circular margins and usually with two distinctly different diameters, 3-(9)-23 m and 13-(24)-41 m. In some cases both rotules of the gemmoscleres of T. pennsylvanica appear to have the same diameters leading them to be identiﬁed incorrectly as T. horrida using the key B. Key to the Freshwater Sponges in Canada and the United States FIGURE 31 Megasclere (M) and gemmosclere (G) of Trochospongilla leidii (after Penney and Racek, 1968). 128 Thomas M. Frost, Henry M. Reiswig, and Anthony Ricciardi Megasclere (M, scale bars 50 m) and gemmoscleres (g, scale bar 10 m) with rotules (r) or aberrant forms (ga) of Trochospongilla pennsylvanica (from Ricciardi and Reiswig, 1993). FIGURE 32 of the types of spicules that can occur within a sponge species (megascleres, gemmoscleres, and, if they occur in a species, microscleres). If a species ordinarily exhibits gemmoscleres or microscleres and they are not contained within a specimen, it will not be possible to identify even its genus. The one exception to this rule occurs for Spongilla lacustris which is unique among the species in this key in its lack of gemmoscleres in its winter gemmules. If gemmules are clearly present in a specimen but gemmoscleres are absent, it is most likely S. lacustris. [Caution is necessary here in that some foreign bodies in sponges may resemble gemmules superficially, for example, the eggs of water mites. Gemmules can be distinguished by their highly resistant coats.] It is important to note that S. lacustris. may also have gem- moscleres; they occur when summer gemmules are or have been present. Also note that this key is intended only for use with the species listed above from Canada and the United States. The couplets in this key have been designed only for this particular subset of the world’s freshwater sponges. Those examining specimens from other regions will need to consult Penney and Racek (1968) and more recent taxonomic work (e.g., Volkmer-Ribiero, 1986, and the references cited in the species list). The key is arranged systematically so that genera are separated prior to species within a genus. In most cases, therefore, a valid identiﬁcation to genus may be possible even if a species has not previously been reported in Canada or the United States. Even to separate genera, however, it is necessary to have included all the possible spicule types from a specimen. 1a. Microscleres present. ..........................................................................................................................................................................2 1b. Microscleres absent. .........................................................................................................................................................................16 2a(1a). Microscleres star-shaped (Fig. 15) or birotulate (Figs. 10 and 11). ...............................................................................................................................................................3 2b. Microscleres rod-shaped to needlelike in structure. (Fig. 21 and Fig. 24)...........................................................................................................................................................................8 3a(2a). Microscleres star-shaped with several rays extending from central region of the spicules (Fig. 15)......................................................................................................................................................................Genus Dosilia 4 3b. Microscleres birotulate (Figs. 12 and 13) ...........................................................................................................................................5 4a(3a). Gemmoscleres birotulate in two distinctly different size categories (45 – 82 m in length and 120 – 230 m in length). ..........................................................................................................................................................................Dosilia radiospiculata 4b. Gemmoscleres birotulate in two size categories that are nearly equal in length (55 – 85 mm). .....................................................................................................................................................................................Dosilia palmeri 5a(3b). Gemmoscleres birotulate (Figs. 12 and 13). .......................................................................................................................................................................Genus Corvomeyenia 6 4. Porifera 5b 129 Gemmoscleres rod- or needle-shaped (Fig. 14). .......................................................................................................................................................................Genus Corvospongilla 7 6a(5a). Microscleres straight to slightly curved birotules with a predominance of straight forms (Fig. 12). ..........................................................................................................................................................................Corvomeyenia everetti 6b. Microscleres a mixture of straight to strongly curved birotules with a predominance of curved forms (Fig. 11). ..................................................................................................................................................................Corvomeyenia carolinensis. 7a(5b). Megascleres covered with spines ............................................................................................................................................................................Corvospongilla becki 7b. Megascleres smooth ..................................................................................................................................................................Corvospongilla novaeterrae 8a(2b). Gemmoscleres birotulate (as in Fig. 21). .......................................................................................................................................................................Genus Heteromeyenia 9 8b. Gemmoscleres needlelike (as in Fig. 17) or absent. ...........................................................................................................................12 9a(8a). Foraminal aperture of gemmules without terminal cirrous projections (contrast with Figs. 22, 23, and 24). ..........................................................................................................................................................................Heteromeyenia baileyi 9b. Foraminal aperture of gemmules with distinct, terminal cirrous projections (Figs. 22, 23, and 24). .................................................10 10a(9b). Foraminal aperture of gemmules with one to two very long cirrous projections starting from a ﬂat disk, ﬂat and ribbonlike at the base and cylindrical at the end. (Fig. 22) .........................................................................................................Heteromeyenia latitenta 10b. Foraminal aperture of gemmules with 3 – 6 cirrous projections that are short when compared with those of H. latitenta. ...............11 11a(10b). Foraminal aperture of gemmules distinctly tubular and very long ranging from 0.5 to 0.9 times the diameter of the gemmule (Fig. 23) .....................................................................................................................................................Heteromeyenia tubisperma 11b. Foraminal aperture of gemmules distinctly tubular, but short, less than 0.4 times the diameter of the gemmule (Fig. 24). .................................................................................................................................................................Heteromeyenia tentasperma 12a(8b). Gemmoscleres smooth or covered with very ﬁne spines....................................................................................................................13 12b. Gemmoscleres absent or covered with robust, conspicuous spines (Figs. 28 and 29). .............................................................................................................................Genus Spongilla, except for S. aspinosa ..............14 13a(12a). Microscleres smooth or very sparsely microspined (Fig. 27). ...................................................................................Spongilla aspinosa 13b. Microscleres covered with very small spines. ...................................................................................................Stratospongilla penneyi 14a(12a). Gemmoscleres clublike with spines conspicuously more dense on the ends than in the center (Fig. 26). .........................Spongilla alba 14b. Gemmoscleres absent or with spines distributed evenly across the length of the spicule (Figs. 28 and 29). .......................................15 15a(14b). Microscleres with conspicuously denser and longer spines in the central region (Fig. 28). ..........................................Spongilla cenota 15b. Microscleres with spines distributed evenly across the length of the spicule (Fig. 29). ..............................................Spongilla lacustris 16a(1b). Megascleres of a single size category with distinctly different forms of gemmoscleres ......................................................................17 16b. Megascleres in two distinctly different size categories (Fig. 16) with the smaller category serving also as gemmoscleres although both sizes of megascleres are always present even when gemmules are absent. .......................................................Duosclera mackayi 17a(16a). Gemmoscleres birotulate (Fig. 30) ....................................................................................................................................................18 17b. Gemmoscleres rod shaped or needlelike (Fig. 20) .............................................................................................................................25 18a(17a). Margins of rotules completely smooth (Figs. 30 and 31). ....................................................................................................................................................................Genus Trochospongilla 19 18b. Margins of rotules distinctly spined or serrated (Figs. 17 and 19) ....................................................................................................21 19a(18a). Megascleres conspicuously spined (Fig. 30) ......................................................................................................................................20 19b. Megascleres smooth or with very ﬁne spines (Fig. 31) ........................................................................................Trochospongilla leidii 130 Thomas M. Frost, Henry M. Reiswig, and Anthony Ricciardi 20a(19a). Gemmoscleres with rotules of two distinctly different diameters (Fig. 32) ............................................Trochospongilla pennsylvanica 20b. Gemmoscleres with rotules of nearly equivalent diameters. (Fig. 30) ..............................................................Trochospongilla horrida 21a(18b). Gemmoscleres occurring in two distinct size classes (Fig. 10) ..................................................................................................................................................................Genus Anheteromeyenia 22 21b. Gemmoscleres occurring in only one size class (Fig. 17) .............................................................................................................................................................................Genus Ephydatia 23 22a(21a). Gemmoscleres of two classes that are similar in shape, but clearly different in length (Fig. 10) ...........................................................................................................................................................Anheteromeyenia argyrosperma 22b. Gemmoscleres of two classes that are distinct in both shape and size (Fig. 11). .......................................................................................................................................................................Anheteromeyenia ryderi 23a(21b). Rotules of gemmoscleres with margins that are nearly smooth bearing numerous, very small incisions (Fig. 18). ..................................................................................................................................................................................Ephydatia millsii 23b. Rotules of gemmoscleres with clearly indented margins (Fig. 17). ....................................................................................................24 24a(23b). Gemmoscleres less than 20 m in length with rotules bearing fewer that 12 teeth deeply incised into long rays, gemmosclere length less than or equal to rotule diameter (Fig. 19). ...............................................................................................................................................................................Ephydatia muelleri 24b. Gemmoscleres greater than 25 m in length with rotules bearing greater than 20 teeth that are not deeply incised (Fig. 17). .............................................................................................................................................................................Ephydatia ﬂuviatilis 25a(17b). Most gemmoscleres in intact gemmules arrayed in a distinctly radial fashion within the gemmule coat (Fig. 25). .....................................................................................................................................................................Genus Radiospongilla 26 25b. Gemmoscleres in intact gemmules arrayed only tangentially to the surface of the gemmule. ..................................................................................................................................................................................Eunapius fragilis 26a(25a). Megascleres sparsely covered with small spines (Fig. 25). ................................................................................................................................................................Radiospongilla crateriformis 26b. Megascleres entirely smooth. ....................................................................................................................................................................Radiospongilla cerebellata LITERATURE CITED Bader, R. B. 1984. Factors affecting the distribution of a freshwater sponge. Freshwater Invertebrate Biology 3:86 – 95. Barbeau, M. A., Reiswig, H. M., Rath, L. C. 1989. Hatching of freshwater sponge gemmules after low temperature exposure: Ephydatia mülleri (Porifera: Spongillidae). Journal of Thermal Biology 14:225 – 231. Barton, S. H., Addis, J. S. 1997. Freshwater sponges (Porifera: Spongillidae) of western Montana. Great Basin Naturalist 57:93 – 103. Bergquist, P. R. 1978. Sponges, University of California Press, Berkeley, CA. Berzins, B. 1950. Observations on rotifers on sponges. Transactions of the American Microscopical Society 69:189 – 193. Bisbee, J. W. 1992. 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This Page Intentionally Left Blank 5 CNIDARIA Lawrence B. Slobodkin Patricia E. Bossert Department of Ecology and Evolution State University of New York Stony Brook, New York 11794 Science Department Northport High School Northport, New York 11768 I. Introduction II. General Biology of Cnidaria A. Body Plan B. Nematocysts C. Feeding D. Reproduction and Metamorphoses E. Ecological Interactions III. Descriptive Ecology of Freshwater Cnidaria A. Hydra B. Craspedacusta C. Calposoma D. Cordylophora E. Polypodium Hydriforme IV. Collection and Maintenance of Freshwater Cnidaria A. Collecting Techniques B. Maintenance Procedures V. Classiﬁcation of Freshwater Cnidaria A. Species Groups of Hydras B. Taxonomic Key to Genera of Freshwater Cnidaria Literature Cited and Further Readings I. INTRODUCTION Medusae, anemones, corals, and other polyps compose the ancient and remarkably successful phylum, Cnidaria. They occur as fossils in the lithographic stone of the Mid-Cambrian Burgess shale, and have not changed very dramatically since then. Fossilized cnidarian embryos are reported from the lower Cambrian (Kouchinsky et al., 1999). The phylum name, Cnidaria, is derived from the Greek term for “nail”, based on their possession of nematocysts, which look like rods attached to a round capsule. The other name for the phylum is Coelenterata, a term alluding to their saclike internal space, the coelenteron. A general overview of the phylum and survey of older literature is provided by Hyman (1940) and the papers in various conference proceedings (e.g., Mackie, 1976). The freshwater representatives of Cnidaria are small animals belonging to the class Hydrozoa, with relatively few species and somewhat monotonous anatomy. They consist of the following taxa: 1. The common and familiar hydras, a group of secondarily simple, solitary polyps; Ecology and Classiﬁcation of North American Freshwater Invertebrates. 2nd Edition Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved. 2. The sporadically common Craspedacusta and Limnocodium, jellyﬁsh with minute, polypoid larvae (Boulenger and Flower, 1928; Fuhrman, 1984). 3. Calposoma, a tiny, colonial polyp, which is so small and inconspicuous that it is probably more common than it appears to be (Rahat and Campbell, 1974; Fuhrman, 1984). 4. Polypodium, which spends part of its life cycle as a parasite in the eggs of sturgeon and part as an ambulatory, predaceous polyp (Lipin, 1926; Raikova, 1973, 1980); this species has been described primarily from eastern European rivers, but should also be found in North American sturgeon. 5. Various estuarine coelenterates, which may occasionally occur in relatively fresh water; colonial, sessile animals of the genus Cordylophora will serve as examples of these (Hubschman and Kishler, 1972; Roos, 1979). Hydra and Cordylophora belong to the order Hydroida of the class Hydrozoa. Craspedacusta, Limnocnida, and Calposoma are classiﬁed as members of the order Limnomedusae. The parasitic Polypodium 135 136 Lawrence B. Slobodkin and Patricia E. Bossert has been assigned to the order Trachylina of the same class (cf. Hyman, 1940). The fact that the other three orders of the class Hydrozoa (Actinulida, Siphonophora, and Hydrocorallina) and the other three classes of coelenterates (Scyphozoa, Cubozoa, and Anthozoa) are not represented outside of the sea is curious. There is not even any serious speculation as to why freshwater invasion by coelenterates has been so severely limited. There is no special osmoregulatory organ in the phylum, but this is not an explanation since its absence has not stopped the successful invaders. Because of their small size, soft bodies, and often sessile habits, freshwater Cnidaria are either not collected or not well preserved in most routine collecting procedures. They are, however, widely distributed and can be found in most ponds and streams when a speciﬁc search is made. When they are abundant, they can be major predators of small invertebrates and even of tiny ﬁsh. In turn, Hydra is fed upon by ﬂatworms, and crayﬁsh eat Craspedacusta. Probably, other animals prey on freshwater Cnidaria, but this has not been carefully studied. II. GENERAL BIOLOGY OF CNIDARIA A. Body Plan All cnidarians share a simple body plan of a central cavity surrounded by two cellular layers (Fig. 1). The endoderm lines the interior cavity, the coelenteron. Between the endoderm and the ectoderm is an intermediate, noncellular mesoglea. Nematocysts aid in feeding and in repelling predators, and are present in all freshwater and marine cnidarians. FIGURE 1 Cross section through digestive cavity (coelenteron) of generalized cnidarian. The feeding aperture or mouth leads into the coelenteron, which functions as a gut. By convention, the end of the animal with the mouth is termed “oral”, the opposite end “aboral.” The oral aperture serves at different times as mouth and anus. When the mouth is closed, the pressure of ﬂuid in the coelenteron can stiffen the body, even in the complete absence of any hard tissue. The coelenteron, therefore, functions also as a hydrostatic skeleton. The mesoglea varies enormously in thickness among different members of the group. At its most meager, as in Hydra, it is not more than 200 m thick, containing only wandering cells, nonliving ﬁbrous components, and ﬁbers from neuromuscular cells. It is more fully developed in medusae, such as Craspedacusta, and may contain a great deal of collagenous or gelatinous material. The ectodermal body wall has neural and contractile properties and is also the location for ripe nematocysts. The presence of deﬁnite cell layers with differentiated functions distinguishes these animals as true metazoa. The absence of mesoderm implies that they do not have organs like higher metazoa; therefore, such terms as “tentacles” and “gut” are used in a functional sense. The basic body plan can be manifested as either a polyp or a medusa (Fig. 2). Polyps are typically elongated along the oral – aboral axis. Medusae are approximately bell-shaped and usually have their greatest body dimension perpendicular to the oral – aboral axis. The coelenteron of a polyp is usually deeper than its body is wide, while a medusa is usually wider than its coelenteron is deep. Also, medusae generally have relatively thicker mesoglea. In some species, different generations, and, in some cases, the same individual organism at different stages of its development, can adopt the form of either a polyp or a medusa. Around the feeding aperture of polyps or the edge of the bell of medusae is typically a ring of tentacles. Tentacles are extensions of the two cellular layers into more or less elongated projections. The coelenteron FIGURE 2 (B) medusa. Basic body plan of Cnidaria showing (A) polyp and 5. Cnidaria may or may not extend into the tentacles. Tentacular ectoderm is especially rich in nematocysts, which may be arranged in rosette or ring-shaped batteries. Tentacles are used in food capture, defense, and in some cases, locomotion. B. Nematocysts The phylum is characterized by the presence of cnidoblasts, ectodermal cells that produce the cell products called cnida or nematocysts. Because there are many kinds of elaborately spined nematocysts, these structures are valuable characters for the classiﬁcation of coelenterates, particularly in such morphologically monotonous groups as Hydra. Nematocysts consist of a capsule containing a threadlike tube (Fig. 3A). Near the base of the tube is a projection reminiscent of a trigger. Remnants of the living cnidoblast are absent from mature nematocysts. Many nematocysts come to lie on the tentacles, often after having been moved through the mesoglea from some other region. After ﬁring, a nematocyst consists of a long thread with a capsule at its base (Fig. 3B). Some nematocysts FIGURE 3 137 (the stenoteles or penetrants) are open at the thread tip, giving the appearance of a hypodermic syringe with a long needle. Stenoteles may have a complex of thornlike structures around the base of the thread. Stenoteles eject a neurotoxin, which partially paralyzes the prey. Desmonemes (or volvonts), another form of nematocyst, seem to lack poison, but rather eject sticky threads that wind about the spines and hairs on the body of the prey, interfering with movement. The capsules of some volvonts remain ﬁxed to the tentacles after ﬁring so that their exploded threads fasten prey to the tentacles as if by many tiny ropes or grappling hooks. At least 17 morphologically distinct forms of nematocysts are present in the phylum (Fig. 4). The penetrants and volvonts of Hydra come in several forms, classiﬁed in terms of capsule size, spination of the threads, and shape and distribution of the basal spines. A single animal may have ﬁve or more types of nematocysts. Nematocysts of basically the same type may differ among species in how the thread is coiled inside the capsule prior to eversion. Some may appear like a coiled spring, with gyres at right angles to the longest dimension of the capsule, while others are coiled parallel to Discharged nematocyst from Hydra. 138 Lawrence B. Slobodkin and Patricia E. Bossert making them of interest as examples of complex cell differentiation. Also, the mechanism by which the thin thread of the nematocyst everts from its coiled state within the capsule (like an enormously elongated, inverted ﬁnger of a glove suddenly turning itself inside out) is a difﬁcult problem in ﬂuid pressures. The poison that is secreted by some nematocysts through the open tip of the thread is of medical interest. Furthermore, the enormous diversity of shapes, sizes, and spination of nematocysts within single organisms and among the different coelenterates, poses a problem in cell differentiation and genetics. Readers interested in more details on nematocysts should consult the large review volume by Hessinger and Lenhoff (1988), which is too lengthy to summarize here. C. Feeding FIGURE 4 Some of the types of nematocysts present in Cnidaria (redrawn from Hyman, 1940); (A) rhopaloneme (only in Siphonophora); (B) spirocyst; (C) same as B, unraveling (not discharged); (D) desmoneme [(H) and (J) same as D but not discharged]; (E) atrichous hydrorhiza [(L) same as E, but discharged]; (F) holotrichous isorhiza [(K) same as F but discharged]; (G) stenotele inside its cnidoblast [(M) same as G but discharged]; (N) microbasic amastigophore; (O) homotrichous microbasic eurytele; (P) heterotrichous microbasic eurytele; (Q) macrobasic mastigophore; (R) teleotrichous macrobasic eurytele; (S) heterotrichous anisorhiza; and (T) microbasic mastigophore. 1, Capsule; 2, tube; 3, butt; 4, cnidoblast; 5, its nucleus; 6, lid; and 7, stylet. the long dimension of the capsule. Nematocyst structure was initially considered a central taxonomic character; but recent evidence indicates that details of shape and coiling, and also the relative abundance of nematocysts of different types, are somewhat variable even within clones of Hydra (Campbell, 1987). The microanatomy and function of nematocysts is a rich research area. The enormous interest about nematocysts and their production arises in part from the following observations. On the level of electron microscopy, they are extremely elaborate structures After a prey has been stung and encumbered by the nematocysts, the tentacles move the victim to the mouth, which opens to admit it into the coelenteron. Often the prey is still alive and active, but this ceases as soon as the gastric cells lining the coelenteron secrete digestive juices. Since there is no anus, food cannot be passed along the gut while digestion continues (as in higher metazoa). Instead, feeding stops until the digestion process is completed and the indigestible remnants have been regurgitated. In hydra, ingested food decomposes within an hour into a slurry of particles. The role of food vacuoles is reminiscent of the feeding process in protozoa and sponges (Barnes, 1966). The free borders of the digestive cells ingest particles by pinocytosis. Individual food particles are enclosed in vacuoles that are moved through endodermal cells. Eventually, their indigestible residues are ejected by the endodermal cells and returned to the coelenteron to leave ultimately through the mouth. D. Reproduction and Metamorphoses In the coelenterates, many kinds of reproduction exist (Hyman, 1940). Like all metazoa, they can reproduce sexually. The fertilized eggs may produce larvae differing anatomically and ecologically from the adult sexually reproducing stage. In marine coelenterates, there may be an elaborate succession of larval stages, some of which may form colonies or reproduce vegetatively by budding or fragmentation. In any particular species, one or more of these stages may be missing. There are medusae that produce eggs that go through various larval stages to produce new medusae (Fig. 5A). Some polyps produce gonads (Fig. 5A), and others bud off medusae that either swim away to become sexual (Fig. 5C) or remain attached to 5. Cnidaria 139 FIGURE 5 (A) Sexual medusa produces larva; larva forms new medusa; (B) sexual polyp generates new polyp; and (C) polyp produces medusa which becomes sexual and forms new polyp [redrawn from Barnes, 1966.] their parents and become sexual without ever feeding independently. Polyps or medusae may produce new individuals that may or may not resemble their “parent.” A new individual may separate from its parent; but in many coelenterates, the asexually produced individuals stay attached and form a colony. The development of colonies is absent in hydra, but occurs among all other freshwater Cnidaria. In the anatomically simplest coelenterate colonies, such as the colonial microhydra, “larvae” of the freshwater Craspedacusta, all attached individuals are essentially similar in both form and function (Payne, 1924). Each has its own mouth and coelenteron and each is capable of budding new polyps. Microhydra larvae can produce medusae by budding. Colonies of many coelenterates are much more elaborate. In a common modiﬁcation of this process, certain members of the colony become “gonozooids” that neither feed nor have tentacles; instead, they consist of a stalk rising from the common stolon that buds off medusae. These medusae, in turn, either form gonads while still attached to their colony or leave the colony and then produce gonads. The best known freshwater coelenterates, the hydra, have lost the medusa stage entirely. In hydra, polyps may produce new polyps by asexual budding or may temporarily switch to sexual reproduction using 140 Lawrence B. Slobodkin and Patricia E. Bossert gonads. Fertilized eggs derived directly from polyps may then hatch to produce new polyps. There are several marine examples, but only one freshwater example of medusae budding new medusae. This is in a species presumed endemic to Lake Tanganyika, in the great African rift (Thiel, 1973). E. Ecological Interactions Although some species may gain part of their nourishment from intracellular algal symbionts, all cnidarians are carnivores. Their prey consists primarily of small coelomate animals, organisms that evolved long after the appearance of coelenterates. They generally do not feed on protozoa, nematodes, or sponges, nor will these animals trigger the nematocysts. This raises a curious question, for which there is no evident answer: “What did the ﬁrst coelenterates eat?” Hydra are extremely effective predators. Prey includes crustaceans, worms, and ﬁsh and insect larvae. They have been seriously considered as a biological control organism for mosquitoes. Hydra and Craspedacusta can both sting ﬁshes very badly. Fish that are too big to swallow often cannot survive being stung. For this reason, hydra are sometimes serious pests of ﬁsh hatcheries. Nematocysts are sufﬁciently unpleasant that relatively few predators attack coelenterates. Turtles, ﬁsh, crabs, worms, echinoderms, and ﬂatworms are among the predators on marine coelenterates. Predation on the freshwater coelenterates is not well studied. Crayﬁsh eat Craspedacusta (Dodson and Cooper, 1983). Flatworms are reported to eat Hydra (Hyman, 1940; Kanaev, 1969), but we have seen small ﬂatworms withdraw from contact with hydras. Some marine coelomates are not affected by nematocysts and are commensal with coelenterates. On coral reefs, clown ﬁsh live among tentacles of large anemones and other ﬁsh occur only in close association with the Portuguese man-of-war. In freshwater, Anchistropus, a chydorid cladoceran, has been observed clinging to, and apparently feeding on, the body wall of hydras (Grifﬁng, 1965; Slobodkin, personal observation). The amoeba Hydramoeba hydroxena feeds on hydra (Stiven, 1976), and the hypotrichous ciliate Kerona lives on the surface of hydras (Hyman, 1940). Polypodium hydriforme is parasitic on a ﬁsh for part of its life (Raikova, 1980). III. DESCRIPTIVE ECOLOGY OF FRESHWATER CNIDARIA Their anatomical simplicity and the ease with which hydra can be cultured in the laboratory make freshwater Cnidaria very important as experimental material in cellular and developmental biology, as well as in neurobiology, biochemistry (Ciernichiari et al., 1969), and genetics (Sabine et al., 1987; Schummer et al., 1992). Hydra also lends itself to studies in cell growth, morphogenesis, microanatomy, and symbiosis (Ciernichiari et al., 1969; Dunn, 1986, 1987, 1988; Bossert and Dunn, 1996). In the present chapter, we will focus on cnidarian ecology and natural history, which have been less well studied. A. Hydra 1. General Biology of Hydra Hydra are small polyps from 1 to 20 mm in body length. The body is crowned by up to 10 or 12 tentacles. Usually the tentacles are of approximately the same length as the body, but may be somewhat shorter, particularly in the green hydra, and can exceed 20 cm in length in hungry brown hydra in quiet water. There are no medusae. Reproduction is by budding or by gametes produced directly from ectoderm. The fertilized eggs may enter a resting stage. When development proceeds, the egg immediately develops into a polyp (see Fig. 5). Hydra are found attached to almost any reasonably hard surface. Slight bacterial ﬁlms may make surfaces more attractive, but heavy growth of microalgae may be avoided. Water lily stems, charophytes, dead leaves, sticks, and stones are favored substrates. They may appear as single animals or as dense furlike aggregations. There are reports of ﬁshing nets becoming completely covered with brown hydra, resulting in rashes on the hands and arms of the ﬁshermen (Batha, 1974). If the wait for food is longer than approximately 12 h, the hydra begin to change their locations on the substrate (Ritte, 1969; Lenhoff and Lenhoff, 1986). They have two different methods of movement. Smallscale movements on a substrate may occur by attaching the stretched tentacles to the substrate or, in shallow water, to the surface ﬁlm, releasing the pedal attachment and contracting the tentacles and reattaching. Sufﬁciently crowded or hungry hydras ﬂoat off their substrate (Lomnicki and Slobodkin, 1966). They may appear in the plankton or be found ﬂoating upside down with the pedal disk in the water surface ﬁlm and their tentacles trailing. Batha (1974) extensively documented the existence of planktonic hydra in Lake Michigan through use of divers and of suitable attachment surfaces suspended in midwater. In addition, the reports of ﬁshnets covered by hydra and observations by Grifﬁng (1965) of 5. Cnidaria sudden relocations of hydras within a single pond suggest that hydras are probably much more important components of lake plankton than has been generally realized. Floating animals will sink and settle either from wave and current action or from having just fed. This behavior keeps hydra in areas of abundant food. It is also evolutionarily important as a dispersal mechanism. From the perspective of a naturalist, it has the effect of making it relatively easy to collect hydra at the outﬂow of lakes and ponds. They are not tolerant of heavy metals, but they can thrive in even highly eutrophic water. They can survive at temperatures from near freezing to 25°C. Several Hydra species may coexist in a pond; often a small green hydra and, at least, one large brown species will co-occur. Also, strains of Hydra may replace each other seasonally. Bossert (1988) showed that green hydra collected several months apart from the same small pond had very different size and growth characteristics when maintained under very similar conditions in the laboratory. Despite many years of collection and observation, we have never found a species of green hydra that was consistently larger than any strain of asymbiotic brown hydra, nor have we found any contradictory account in the scientiﬁc literature. If it occurs, it certainly seems rare. Some large brown hydra can be caused to become green in the laboratory (Rahat and Sugiyama, 1993), but it is not clear that this is signiﬁcant in nature. Hydra occur in freshwater from the Amazon to Alaska and from Siberia to Africa at depths from shallow water to 60 m or more. This cosmopolitan distribution may be a result of the portability of the thecate eggs; or perhaps, as has been suggested by Campbell (1987), it might be due to the four species groups (see Section V) having differentiated before the primeval continental masses separated in the Mesozoic era. A recent study of the hydras of Madagascar (Campbell, 1999), which has been an island for more than 100 million years, shows that they differ very little from mainland hydra, indicating that either the rate of evolution of the genus Hydra is very slow or that they are surprisingly well dispersed. One species group (oligactis) is missing from Madagascar. Since there is no obvious reason why hydra of this group should be less mobile than the others, their absence strengthens the interpretation of slow evolution. This implies that hydras are evolutionarily very old. 2. Reproduction and Mortality Asexual budding is the primary reproductive mechanism of hydra during periods of population increase. Under optimal conditions of food supply, temperature, 141 FIGURE 6 Hydra. (A) Male gonad; (B) female gonad; and (C) egg [redrawn from Campbell, 1987.] and water quality, each adult hydra polyp can produce two buds per day and each bud can mature and begin reproducing in a week or less. Generally, the smaller strains of hydra bud more rapidly than the larger ones, at equal feeding rates. Buds are from 12 to 20% of the size of the mother, varying with the strain. While Hydra usually reproduce by producing freeliving buds, under deleterious environmental conditions they may develop testes or single egg ovaries and engage in sexual reproduction. External fertilization by free-swimming sperm occurs while the egg is attached to the body wall of the mother. The embryo may enter a resting period of days or even months before proceeding with direct development into a new polyp. During sexual reproduction, gonads develop along the stalk in place of buds (Fig. 6). Mature male gonads are a mound of tissue with a distinct apical nipple from which sperm extrude. Mature female gonads consist of a single large egg cell resting on a cushion of smaller cells. The fertilized eggs are surrounded by a theca, which may be smooth or ornamented or may consist of polyhedral plates. The features of the theca are of taxonomic importance. Sexuality in hydra seems to occur only under deleterious environmental conditions, although the precise cues are unknown. Chemical changes due to temperature ﬂuctuations, and, perhaps, sudden nutritional variation can all induce sexuality at certain times (Lenhoff and Loomis, 1961; Kanaev, 1969). Individual hydra have been reported as unisexual and some bisexual individuals have been noted (Hyman, 1940; Kanaev, 1969). If conditions improve, sexual hydra can return 142 Lawrence B. Slobodkin and Patricia E. Bossert to asexual reproduction. Buds and gonads may occur concurrently in the same individual. Most studies of hydra sexuality are made on animals that have been maintained in the laboratory. Batha (1974) has suggested that sexual reproduction is very rare in nature, based on the complete absence of gonads among thousands of brown hydra collected in Lake Michigan. Although we have found eggs in green hydra collected from a small pond and other ﬁeld reports of gonads do exist (Kanaev, 1969), it seems clear that in hydra the numerically most important reproductive process is budding. In most plants and animals, population size and genetic recombination are associated through the process of sexual reproduction, but sexual reproduction in hydra is not signiﬁcant to population dynamics. It seems important for maintaining genetic heterogeneity and permitting escape from temporarily unsuitable conditions (cf. the chapter on “Escape in Time and Space” in Slobodkin, 1980). In a single pond or stream, most of the animals are likely to belong to a rather small number of vegetative reproductive lines, being genetically identical except for occasional mutations. Even those that have emerged from eggs are likely to be the result of relatively close inbreeding, since sexuality is usually found in crowded local populations whose members are descended from a very small number of clonal lines. There is no direct evidence on sperm survival in nature, but it seems unlikely that sperm can travel great distances. Hydra that are very small, either because of youth or starvation, will not produce buds. If nutrition is limited, animals may be kept indeﬁnitely in a condition of neither growing nor budding. Both body size and budding rate are proportional to feeding rate up to a point of food saturation. The capacity of hydra to reduce its body size is of ecological interest. Animals with hard skeletons are committed to a particular body size in the sense that they cannot shrink below a given point during periods of food shortage. The inability to reduce size may contribute to death by starvation. By contrast, hydras shrink in size when starved, but can be restored to full reproductive size by increasing their food supply. At a given temperature, hydras will come to a steady-state body size and budding rate if food supply is sufﬁcient and constant. The time for complete size adjustment to either temperature change or feeding level change on the part of an individual brown hydra is approximately 3 weeks. Reduction of temperature decreases growth rate, but increases body size. These size differences seem to be due to changes in cell number rather than cell size (Fig. 7) (Hecker and Slobodkin, 1976). Because FIGURE 7 Relative sizes of hydra raised at different temperatures. Scale shown is the same size at each temperature [redrawn from Hecker and Slobodkin, 1976.] larger animals have greater food requirements, the smaller green hydra can produce buds when fed one or two Artemia salina nauplii per day while the larger brown hydra require 5 – 10 per day before they will bud. 3. Feeding The tentacles extend above and lateral to the body. They are generally motionless except in the presence of potential food or during locomotion. In healthy animals, the tentacles are cylindrical or tapered, but never clubbed. The length of the tentacles varies somewhat with species, but considerably more with environmental circumstances. In quiet water, animals with bodies no longer than 2 cm have been observed to constrict their tentacles into thin threads extending at least 20 cm (L. B. Slobodkin, personal observation). This is unusual however, and tentacles equal to three body lengths or less are more typical. Tentacles are quickly retracted if the animal is disturbed or if organisms brush against them. Whole prey or extracts of their body ﬂuids will initiate active waving movements of the tentacles (Lenhoff, 1983). If a prey organism brushes against the tentacles, nematocysts will discharge, poisoning the prey and attaching it to the tentacle. Other tentacles and their nematocysts then join the attack. In a matter of 1 – 4 min, the prey will have been pressed against the mouth surface by the tentacles and will have entered the coelenteron. Meanwhile, the tentacles for subsequent swallowing may have caught other prey. The number of prey swallowed in one feeding encounter varies with the size of the hydra, the size of the 5. Cnidaria prey, and the previous feeding condition of the hydra. One large Daphnia magna may ﬁll the coelenteron completely, stretching its walls to give the appearance of a Daphnia stuffed into a thin expandable sack of hydra tissue. During the digestion process, the body may become rounded as the swallowed prey are reduced to a slurry. After approximately 1 h, material is suddenly discharged from the coelenteron through the mouth, the hydra momentarily appearing like a punctured balloon. The columnar shape is then restored, tentacles regain their virulence, and the animal waits for its next meal. There is evidence that several days of starvation will increase the appetite of a brown hydra. Brown hydra can survive more than 40 days without food, and green hydra can live without food 4 months or longer. In general, hydra eat small, open-water plankters, but are less effective at capturing animals that normally inhabit underwater surfaces. The common cladoceran genera Simocephalus, Scapholebris, and Chydoris and at least some ostracods are immune to the activities of hydra (Schwartz et al., 1983). Hydra can eat very small ﬁsh and insects, but sufﬁciently large animals with hard skeletons and strong swimming force can escape after being stung. The long bristles on small midge larvae have been found to impede predation by hydra (Hershey and Dodson, 1987). Apparently, hydra primarily feed on the kinds of prey that they are least likely to encounter. This suggests that hydras are sufﬁciently important as natural predators that only those crustaceans that have evolved immunity to hydra can coexist closely with them. 4. Immortality and Regeneration Trembley, in the eighteenth century, had already demonstrated the capacity of hydra to regenerate perfectly, even after severe mutilations. The regenerative powers of hydra make them favorite classroom objects. They can be decapitated, bisected, have their tentacles amputated, or even be turned inside out and in a matter of a few days regenerate missing parts or regain their proper organization. Within a clone, rings of hydra stalk can be threaded on hairlike quoits and may fuse to form a single tube (Burnett, 1973). Even without operations, different accidental conditions in the ﬁeld or laboratory will produce hydra with various mutilations: more or fewer tentacles, missing heads and so on. All of these hydras will reorganize themselves neatly if water chemistry is not deleterious and if they have been reasonably well-nourished before the mutilations occurred. Since hydras can regenerate so well, are they potentially immortal? Immortality in any organism is impossible to demonstrate during a research program of ﬁnite duration, and therefore the question cannot be 143 unequivocally settled. However, what we generally mean by immortality in organisms is the absence of any signs whatsoever of senescence or permanent scars of the past. Martinez (1997) maintained laboratory cohorts of hydra for more than 3 years and demonstrated the absence of any symptoms of senescence. Single polyps have been maintained in our laboratory without budding for at least a year, their lives terminating only from human error. There is no clear evidence for senescence of any kind in hydra. If hydra are starved they will become smaller; but do not die until they are too small to feed. Newborn hydra are approximately at this minimum size (Slobodkin et al., 1991). It seems likely that hydra polyps are potentially immortal. All deaths in hydra can be assigned to such things as alterations in water quality or food supply, temperature shocks, excessively severe starvation, or predation. 5. Symbiosis While species of Hydra may be difﬁcult to distinguish, there is a very clear distinction between the brilliant green color of some species of Hydra and the yellow, brown, and gray colors of those that do not have symbiotic algae. The green hydra are accepted as a taxonomically distinct group and have been assigned their own generic status as Chlorohydra, which we use here interchangeably with “green hydra.” No certainty exists as to the monophyletic character of this genus. The green color arises from Chlorella-like cells, unicellular algae each occupying a vacuole in the endodermal cells of their hosts. Each endodermal cell contains 10 – 35 algae-laden vacuoles (Fig. 8). The precise number varies with species, but is nearly constant within a hydra strain and a set of environmental circumstances (Bossert, 1986). Algae are also found in the central cells of the tentacles, but in somewhat smaller numbers. The endoderm of the buds contains algae like those of the mother. Ample evidence exists showing that green hydra gain nourishment from their symbionts. Radioactivetracer experiments have demonstrated that maltose, a secondary photosynthetic compound, leaks from the algal symbionts to their hosts (Muscatine, 1965; Ciernichiari et al., 1969; Lenhoff, 1974). Also, there is microscopic evidence that algal cells can be attacked by host lysozymes (Dunn, 1986, 1987, 1988). In competition experiments between brown hydra and green hydra populations, light provides a signiﬁcant advantage to the green hydra (Slobodkin, 1964). The algae can be removed from some strains of green hydra by use of a variety of techniques, including photosynthetic poisons, prolonged darkness or extremely strong light, and dilute glycerin solutions 144 Lawrence B. Slobodkin and Patricia E. Bossert FIGURE 8 An algae-laden endodermal cell from Hydra (viridis) (1000 ). (Pardy, 1983). Such hydras are referred to as aposymbiotic and are susceptible to reinvasion by algae. It is possible to develop green patches in brown hydra by injecting their coelenteron with algae or by feeding green hydra to brown ones (L. B. Slobodkin, personal observation), but this coloration fades with time. If the association with algae is advantageous, why are big hydras not green? Symbiotic relationships involve a delicate interaction between the two partners, particularly when one partner lives within the cells of the other. A primary requirement for stable endosymbiosis is a mechanism providing balanced rates of mitosis of the host and symbiont cells. If there were no control of the algal rate of increase, they would be expected to kill their host cells by ﬁlling them with algae; and if the hydra cells excessively limited the algae, they would be eliminated and the hydra would be brown. While the number of algae per host cell stays constant in all green hydra, maintaining this constancy seems more difﬁcult in the larger green hydra strains. Bossert and Dunn (1996) have shown that algal cells are increasing faster than host cells in green hydra strains of all sizes, but the disparity between algal and animal mitotic rates is greatest in the largest strains. Dunn (1986, 1988) suggested that algal cells are being actively expelled or digested by all green hydra, but especially by larger strains. Size in green and brown hydra is apparently regulated by the relative amounts of several hormones, some of which activate the formation of buds while others inhibit budding (Sabine et al., 1987). Bossert (1988) found that one of the hormones implicated in producing smaller hydra size also inhibits algal mitosis within green hydra cells. Apparently, the same hormonal mechanism that produces small size aids in maintaining the balance between algal cell and hydra cell increases, while a hormonal balance that permits larger size makes control of algae more difﬁcult and, in the largest strains, impossible. More investigation of this problem is needed. 5. Cnidaria Green hydras are never found in nature as aposymbionts and seem to have evolved a dependence on algae. However, these algae have probably not evolved a dependence on hydra. In fact, how the algae beneﬁt from the association is not at all clear. Certainly, the Chlorella in a green hydra are immune to being eaten by ﬁlter feeders, have an assured source of mineral nutrients, CO2, and nitrogen, and are moved into light as the hydra moves. However, the actual rate of increase of algal cells inside hydra is probably lower than those of algal cells outside, and it is not obvious that the number of algal cells contained in the entire green hydra population is a signiﬁcant fraction of the natural algal population in the lake or pond. 6. Toxicology With the development of modern techniques, there has been an impressive increase in the number and signiﬁcance of research programs focused on hydra. For example, like any small, easily cultured animal, hydra has been attractive for use in ecotoxicology. In these tests, the survival or death rate of organisms is typically related to concentrations of particular toxins. Toxicity tests using specially prepared hydra can provide evidence as to whether or not a particular toxin is likely to cause cancer. The procedure involves mass culture of Hydra attenuata, and disassociation of the hydra into individual cells that are aggregated into “artiﬁcial embryos.” The small, mixed-cell hydra patties organize themselves into complete and perfect animals in less than a week. It has been found that toxins which do not produce cancers are approximately equally toxic to intact hydra and to the reconstituting patties while known carcinogens are much more toxic to the regenerating cell mass than to the intact hydra (Johnson et al., 1982). 7. Molecular Biology The regeneration studies, and the toxicological studies as well as mathematical analyses of hydra population dynamics and evolution (Slobodkin et al., 1991), are all possible because of the extreme anatomical and behavioral simplicity of hydra. Loewenhoek ﬁrst described hydra in 1704. They became an object of intense scientiﬁc concern with the work of Trembley (Lenhoff and Lenhoff, 1986). Trembley collected and described green and brown hydra, feeding them on small invertebrates and dissecting them with a razor. He established their remarkable capacity to recover from damage that would kill most animals. Among the ingenious dissections were removal and splitting of the head, and producing multiheaded animals. He also sliced hydra into rings. In 145 those cases, a new head developed if the slice had been nearer the original head, while a foot developed if the slice had been closer to the original foot. He even threaded whole hydra onto bristles and watched as they turned themselves inside out, leaving the bristle behind. There existed in the 18th century a belief that animals at birth were endowed with a kind of uniqueness, an animal soul. Transforming a single hydra into 10 others identical with itself by slicing it into pieces effectively eliminated the idea of a unique animal soul (Burnett, 1973). It was also generally assumed in those days that green organisms that beneﬁted from light were plants. Trembley demonstrated that green hydra were animals that in certain respects acted as if they were plants. Studies of hydra continued to contribute to our understanding of basic biological questions through the next two hundred years. Hydra research has gained in importance in the past two decades since it has been found that the molecular and genetic apparatus of hydra is also amenable to analysis. The current volume of work is very large; hence, only a brief indication of what is being done will be included here. The biochemical and genetic properties of Cnidaria are of signiﬁcance in studies of the evolution of metazoa. Some early workers felt that hydra, arguably the simplest metazoa, must be very close to the ancestor of all metazoa. It is now clear that neither hydra itself nor other cnidarians, such as Scyphozoa, Cubozoa, and Hydrozoa (to which hydra belongs), are on the main branch that led from Cnidaria to other metazoa. This conclusion is based on the fact that they are the only metazoa in which mitochondrial DNA is arranged in a linear, rather than circular pattern. However, another major group of Cnidaria, the anthozoans, have circular molecules of mitochondrial DNA like all other metazoa and would, therefore, seem closer to the main line of metazoan evolution than hydra (Bridge et al., 1992; Warrior and Gall, 1985). Despite this ﬁnding, the extreme simplicity of hydra anatomy suggests that it may provide a useful molecular model for higher metazoa. Signal transduction pathways mediated by cAMP or inositol triphosphate are present in hydra as they are in virtually all higher organisms. The control of basic body form in hydra involves an interaction of homeotic genes, some of which are largely identical with body form genes in mice and fruit ﬂies (Bernﬁeld, 1993; Aerne, 1995; Finnerty and Martindale, 1999; Galliot et al., 1999). The implications of this are that the basic developmental processes of the ﬁrst metazoa (or perhaps advanced premetazoan ancestors of the ﬁrst metazoa) evolved a developmental control mechanism which has remained 146 Lawrence B. Slobodkin and Patricia E. Bossert FIGURE 9 (A) Craspedacusta sowberii, also named Limnocodium victoria (see text); (B) larval stage of A, also named Microhydra ryderi (see text); and (C) stages in development of Craspedacusta [redrawn from Payne, 1924.] essentially intact throughout all subsequent metazoan evolution! B. Craspedacusta The ﬁrst scientiﬁc accounts of freshwater medusae were based on specimens found in the giant water lily tank of Regents Park in London in 1880 (Fig. 9A). Two authors described these separately. Lankester named them Craspedacusta sowberii after the discoverer, Sowber, and Allman named the same organisms Limnocodium victoria after the lily. In the same year and in the same tanks, a tiny colonial hydroid was found. This animal had no tentacles; each polyp terminated in a bulbous “capitulum” studded with nematocysts. In the center of the capitulum was the mouth. These little polyp colonies (Fig. 9B) were initially, and correctly, assumed to be the larval stage of the medusae. However, very similar polyps were discovered in a water tank in Philadelphia and were described and named as a separate species. It was not until 1928 that it became once again obvious that the polyp named Microhydra ryderi was the larva of Craspedacusta (Boulenger and Flower, 1928). Since their initial description, Craspedacusta medusae have been found in many locations around the world, apparently transported with ornamental aquatic plants and with the water hyacinth, a recently spread pest species. They have also been found in locations free of imported plants; for example, Lake Gatun in the Panama Canal zone (Slobodkin, personal observation). Since ﬁrst documented in the United States in 1908, these unpredictable and sporadic organisms have been reported in 35 states. The jellyﬁsh have been 5. Cnidaria found in lakes, farm ponds, quiet coves of rivers, and old water-ﬁlled quarries. When they occur they are locally abundant but sufﬁciently rare to call to public attention (Anonymous, 1999). 1. Life Cycle The most conspicuous stage of the life cycle is the small medusa (0.5 – 1.5 cm). Except in the Yang-tse river system of China, the occurrence of noticeable populations of the medusae of Craspedacusta is sporadic and often surprising to local naturalists. As the water warms up in a pond or in the slow current of a stream backwater, a swarm of medusae appears, often where it has never been seen before or at least has not been apparent for many years. These medusae feed on zooplankton. As they grow, the number of tentacles increases from 8 – 12 up to as many as 100. After several weeks of growth, gonads develop in pouches of the radial canals. Fertilized eggs produce a small crawling planula, which then differentiates into a microhydra (Payne, 1924). The planula consists of two cell layers forming a double-walled, sausage-shaped sac. The differentiation of the planula into a microhydra is rather simple. It stands on end and both a mouth and a capitulum develop on the unattached end, thereby forming a polyp. The new polyps may continue to bud off new microhydra or medusae. Buds may remain attached after they have developed a capitulum, producing colonies of up to 12 polyps attached to a common stolon. Starvation and severely abnormal temperatures cause the polyps to shrink to a cellular ball, surrounded by a chitinlike membrane. This can persist through the severe conditions and then redifferentiate as a polyp. The buds of medusae appear initially as rounded swellings of the polyp wall (Fig. 9C). Over a period of several weeks, they enlarge and develop a central manubrium. An endoderm-lined circular canal with four radial canals leads to the base of the manubrium. Eight tentacles emerge from the circular canal. The medusa bud is initially covered by a layer of tissue that eventually perforates centrally, remaining as the vellum of the adult. The mouth opens into the manubrium. The medusa is by now at least as large as the polyp, to which it is still attached at the aboral end. Eventually, it begins locomotory pulsation and separates from the polyp, completing the life cycle (Payne, 1924). Medusae usually reproduce sexually; fertilized eggs develop into planulae, which transform into microhydra. 2. Ecology The microhydra polyps feed in essentially the same fashion as hydra. Their small size limits their prey, but this is partially compensated for by their colonial 147 growth pattern, which permits several polyps to make a simultaneous attack. Free-living medusae feed on various crustacean zooplankters. They can even kill, but apparently not swallow, the large (0.5 cm), predaceous cladoceran Leptodora (Dodson and Cooper, 1983). As a medusa feeds, it grows larger, adding additional tentacles at both the vellum margin and its inner edge. Several hundred tentacles are found on mature animals. Medusae appear sporadically in shallow ponds, natural lakes, and artiﬁcial reservoirs throughout the north and south temperate zones. Often entire medusa populations are unisexual. The polyps are so inconspicuous that they probably have a much broader distribution than the literature reports. Many locations containing polyps are reported not to have medusae. This bewildering picture has been clariﬁed by Acker (1976) and Kramp (1950) . Acker suggested that medusa production by the polyps requires temperatures greater than approximately 20°C during a period of increasing temperature and adequate, but not enormous, food levels. Other environmental alterations may be signiﬁcant but have not been tested. These conditions are reminiscent of those that produce sexuality in hydra. Acker and Kramp maintain that the original natural habitat of Craspedacusta sowerbii is the Yangtse-Kiang region of China. In the upper river valley, two Craspedacusta species, C. sowerbii and C. sinensis, coexist, whereas only C. sowerbii reaches the downstream areas. From this habitat, C. sowerbii traveled with water hyacinths and other plants to its present worldwide distribution. Shallow pools exist along the lower Yang-tse valley which are subject to large temperature changes and to sudden ﬂooding from the main river during high water. Plankton populations in these ponds ﬂuctuate strongly. In these ponds, medusae are a recurring phenomenon throughout the year. The occurrence of medusae in the spring is so regular that they are given a common name that translates to “peach blossom ﬁsh.” The generic name Limnocodium is usually applied to freshwater medusae of the Old World and Craspedacusta to those of the New; but it is not clear if this is more than a geographic distinction nor has there been enough investigation to determine how many species of freshwater medusae actually exist. Due to their sporadic occurrence, the changes in size and tentacle number with developmental stage, the simplicity of larval anatomy, and the difﬁculties of preserving specimens, morphologic studies of freshwater medusae are difﬁcult. As in hydra, many species have been described, but perhaps some of these are based on nutritional or developmental history or preservation artifacts. The strongest evidence for multiple species is that sympatric 148 Lawrence B. Slobodkin and Patricia E. Bossert populations of two species of medusae are known from the Yang-tse and that a medusa from Lake Tanganyika has an extraordinarily different life cycle. In this latter case, a mature medusa buds new medusae from its manubrium in addition to reproducing sexually (Bouillon, 1957). C. Calposoma This is a colonial polyp not much bigger than a paramecium and similar in general appearance to the microhydra except that it is considerably smaller and has tentacles rather than a capitulum. As far as is known, there is only one species, Calposoma dactyloptera (Fuhrman, 1984). Some of its properties have been described by Rahat and Campbell (1974). These organisms are so small that their tentacles consist of a single cell, a tentaculocyte, which contains a row of miniscule nematocysts. The full natural history of these animals and the details of their life cycle are not known. Their general anatomy is reminiscent of a miniaturized hydra with stiff tentacles, but this does not necessarily indicate taxonomic or evolutionary proximity. D. Cordylophora The genus Cordylophora is an athecate member of the primarily brackish water and marine hydrozoan family Clavidae. It grows as a branching colony up to 5 cm high. The feeding polyps have a conical hypostome on which ﬁliform tentacles are irregularly arranged. Colonies also include gonophores, which produce gonads. There is a chitinous periderm (Fig. 10). During periods of stress, the animals regress to “metanonts”, masses of resting tissue in the hydrorhizae (Naumov and Stepan’iants, 1980). The metanonts appear when the plant stalks on which they grow begin decomposing in the fall (Roos, 1979). Cordylophora was ﬁrst found in the Caspian and Black seas. It has been suggested that it evolved during the time that these two bodies of water were connected. It now occurs in rivers of Europe and America, apparently having spread during the last century attached to ship bottoms. The recent expansion of range may be attributed to the relatively greater speed of seagoing vessels during the last hundred years which would shorten the time of salt water immersion between brackish water ports (Roos, 1979). Industrial and navigational developments have extended the region of brackish water in some estuaries, and the increasing pollution of rivers may have duplicated estuarine conditions. This may be expected to encourage the geographic spread and the upriver FIGURE 10 Cordylophora [Redrawn from Roos, 1979]. movement of not only Cordylophora but also other estuarine Cnidaria (Hubschman and Kishter, 1972). The possibility of foreign Cnidaria appearing in habitats where they have never been seen is also enhanced by rapid vessels and the use of water ballast discharged in ports of arrival. Cordylophora is one example of an expanding range. We expect that there are many others, which we have not attempted to survey. E. Polypodium Hydriforme The remaining freshwater coelenterate that we will consider is the strange and poorly understood Polypodium hydriforme, originally described by (Lipin, 1911, 1926) and later examined in detail by Raikova (1973, 1980). This tiny Hydrozoan was ﬁrst discovered in Eastern Europe as a parasite inside the eggs of the European sterlet (Acipenser ruthenus), a small member of the sturgeon family. It is particularly interesting as it is perhaps the only endoparasitic coelenterate. 5. Cnidaria 149 FIGURE 11 (A) Polypodium stolon with apex of knobs directed toward center of egg; (B) emerging polyp; and (C) mature polyp [redrawn from Lipin, 1911.] Obviously there are modiﬁcations associated with its parasitic habit that make classiﬁcation difﬁcult, but it is reported to have only one type of nematocyst. These match the type found in the narcomedusae, an extremely toxic group of hydrozoans. The development of the larval Polypodium (Fig. 11) is closely coordinated with that of the sturgeon egg. A binucleate, single cell stage is known from immature sterlet oocytes. The two nuclei are unequal in size and chromosome number. The smaller, reportedly haploid nucleus is surrounded by the large polyploid nucleus, which develops into a trophic envelope around the embryo formed by the division of the small nucleus and its surrounding cytoplasm. By the time the host oocyte has started to accumulate yolk, the Polypodium is a two-layered planula, approximately 1-mm long. It has a ﬂagellated external layer, which eventually will become the endoderm and an internal layer of ultimate ectoderm, all surrounded by a capsule that serves as a digestive organ for consuming yolk (Raikova, 1980). After a month, the planula has developed into a stolon with internally directed buds and tentacles. At this stage, the Polypodium is a colony consisting of a straight stolon from which project as many as a dozen knobs, arranged linearly with their apices toward the center of the egg (Fig. 11A). Each knob develops two indentations. From each indentation, 12 tentacles, four of which are short and stubby, project into the stolon itself (Fig. 11B). As the ﬁsh eggs ripen and are released from the ﬁsh, the tentacles evert through a slit in the stolon. Simultaneously, the knobs invert, developing a coelenteron lined by what had been the surface exposed to the egg yolk. The stolon breaks up and the knobs now appear as somewhat bifurcated polyps with 12 tentacles on each head and a coelenteron full of ﬁsh egg yolk (Fig. 11C). The free-living polyps subdivide by longitudinal ﬁssion. They crawl on the bottom using tentacles as walking legs, aided by nematocysts (isotrichous isorhiza) that hold the substrate. They feed on turbellaria and oligochaetes. Gonads form on the polyps: both single sex and hermaphroditic individuals are known. The genital anatomy is considerably more complex than that of hydra. These gonads and their accessory structures arise from endoderm. The presumed ovule is diploid and is released into the gastric cavity. The presumed male gonads become ﬁlled with binucleate cells. One nucleus remains haploid, while the other becomes polyploid. These may fall out of the coelenteron, but there are observations of polyps crawling onto young sterlets and placing these gonads on the ﬁsh. The transition from ﬁsh surface to immature oocyte has not been observed. Polypodium parasitizes at least ﬁve species of Acipenser in all of the major rivers of the former Soviet Union. Eighty percent of sterlet (Acipenser ruthenus) and 20% of the sturgeon (A. guldenstadti) are infested, 150 Lawrence B. Slobodkin and Patricia E. Bossert with sporadic infections in other species of the genus. A careful search of North American sturgeon will probably reveal the presence of this cnidarian. IV. COLLECTION AND MAINTENANCE OF FRESHWATER CNIDARIA A. Collecting Techniques Hydras are so ubiquitous that their presence should be expected in any reasonably unpolluted body of water. Unfortunately, since they are usually sedentary, they are not easily found in plankton tows. Also, lacking hard body parts, they are often badly damaged by preservatives. A careful examination of suitable substrates is required. If rocks, leaves, submerged vegetation (e.g., Myriophyllum, Elodea) are collected and placed overnight in a glass or enamel pan, and the pan is carefully examined under a low-power dissection microscope, hydras are usually found. Abundance will depend on the seasonal distribution of zooplankton and may vary among lakes. Since hydras ﬂoat when hungry and attach again after they have been fed, the best place to took for them is often the downstream end of a lake or the pools in the stream immediately below the lake. If there is a dam, this should be examined with particular care. In a typical body of water, there may be at least three species of Hydra: a green one, a large brown one, and an intermediate-sized brown one. Collecting of the medusae of Craspedacusta must be done with buckets rather than nets. The greater the sample volume the better (to prevent anoxia and excess temperature change). A compromise must be found between excessive agitation of the water, which will damage the animals, and insufﬁcient stirring, which will permit them to settle or to become anoxic. The microhydra larvae can be found on fragmentary organic debris, much like hydra, but we have not collected them. B. Maintenance Procedures Craspedacusta and Cordylophora can be kept in natural water and fed on ﬁeld-collected zooplankton. Even the free-living stage of Polypodium can be kept in the laboratory and fed on oligochaetes and turbellaria (Raikova, 1973). Dodson and Cooper (1983) have fed a range of foods to Craspedacusta, including large rotifers and copepods. Generally, it is extremely difﬁcult to maintain jellyﬁsh in aquaria for any length of time. Their tissues are so fragile that they are battered to pieces by most aquarium aerating or stirring systems. The more or less sedentary polyps are therefore easier to study. However, the microhydra larvae of Craspedacusta have not been extensively studied, perhaps due to their small size. Hydras are laboratory animals par excellence. General directions for most of the things one might want to do with hydra are discussed in detail in the papers collected by Lenhoff (1983). They can be maintained in artiﬁcial pond water or bottled spring water and fed on either live natural foods, such as cladocerans (e.g., Daphnia), copepods, or larvae of midges and mosquitoes. They will not eat protozoa. Although the brine shrimp, Artemia, do not normally occur in freshwater, many pond animals eat them avidly in the laboratory. Many investigators use Artemia nauplii as food for hydra, Craspedacusta, planarians, and other small freshwater invertebrates. The Artemia eggs are collected at the edges of salt ponds and lakes. They are sold in vacuum-packed cans for use by aquarists and may be stored for long periods at cool temperatures. The procedure for feeding hydra with Artemia consists of following the package directions for hatching the brine shrimp (a process requiring about 1 day), draining and rinsing the hatched nauplii free of the salt water, and adding them to the hydra. The nauplii must be alive and vigorous or the hydra will not eat them. Dead Artemia can be fed to ﬂatworms! Artemia are best drained in a net that can be made by inserting a taut sheet of bolting silk in an embroidery hoop. The Artemia and their salt solution are poured into the net, which should be wet on both sides to speed drainage. The shimmering mass of Artemia and eggs are then rinsed into a new container of hydra culture water. If this is permitted to stand for 15 min, the active nauplii will swim toward a light, leaving the unhatched eggs behind. These active swimmers can be taken with a medicine dropper and fed to the hydra. This keeps salt and hydra separate and also permits elimination of most of the unhatched Artemia eggs. These small prey may be eaten in great numbers. Large brown hydra can eat as many as 100 Artemia at a single meal, and even the smallest green hydra can consume 1 or 2. No more than 10 min after the ﬁrst prey is swallowed, the swallowing process stops even if some prey remain on the tentacles. We have seen that organisms may brush against the tentacles with impunity for some time after a hydra has been satiated. This suggests either that nematocysts are no longer discharging or that the nematocyst supply has been temporarily exhausted. The primary danger in this feeding process is that water contaminated with either dead Artemia or with 5. Cnidaria the regurgitation products of previous feedings is highly detrimental. The live food must therefore be added to the hydra; and after approximately 1 h, the hydra must be placed in clean water, either by moving them or by discarding the tainted water. Since hydra usually stick to the substrate, the old medium can be simply poured off and a new medium added. The glass surface will eventually become coated with bacteria. This can be prevented somewhat by scraping the glass with a rubber spatula before discarding the old medium, but this is not usually effective for more than about 1 week, at which time it is best to provide new, clean containers. Hydras are remarkably sensitive to heavy metals and to detergents. Even a very short length of copper tubing in a water supply will kill hydra. Therefore, in cleaning the dishes, physical dirt is often less dangerous than the detergents. Dishes must be very thoroughly rinsed in metal-free water. To obtain nontoxic water, one may either use water from the collecting site, pretest the water with hydra to make sure that the water is innocuous, or use glass distilled or deionized water. When rinsing dishes containing hydra, appropriate salts must be added to the distilled water. Various recipes for culture media are available. We use two stock solutions adapted by K. Dunn (personal communication) from solutions developed by Loomis, Lenhoff, and others. Solution A contains 81 g NaHCO3 in 1 L of distilled water. Solution B contains 7.46 g KCl, 20.33 g MgCl2 6H2O, and 147.02 g CaCl2 2H2O in 1 L of distilled water. These stock solutions are added at the rate of 1 mL/L to distilled water to make artiﬁcial pond water. Hydras die at temperatures above approximately 30°C, but most seem healthy at temperatures as low as 5°C. The growth and reproductive rates are proportional to temperature, as are the food demands. Stocks are therefore best maintained at low temperatures except when rapid growth is desired. V. CLASSIFICATION OF FRESHWATER CNIDARIA The ﬁve groups of freshwater Cnidaria are clearly distinct from each other. Except for the hydras, they are relatively rare and sporadic in their distribution. In North America there is no evidence, at present, for more than one species of Craspedacusta, Polypodium, or Calposoma. There may be several species of Cordylophora, but we are considering Cordylophora as one example of a brackish water cnidarian rather than a completely freshwater coelenterate. Our taxonomic key, therefore, consists of ﬁve very coarse divisions, one of which, that for the hydras, is then subdivided in somewhat greater detail. 151 A. Species Groups of Hydras There are two closely related genera of hydras: the brown genus known as Hydra and the green hydras assigned to the genus Chlorohydra. By convention, the term hydra refers to members of both genera, unless otherwise speciﬁed. Hydras tend to look superﬁcially similar, except for the distinction between the asymbiotic and green species. On more careful examination, differences become apparent, not only among apparently unrelated species but even within a clone. Asymbiotic animals from the same clone change color depending on the color of their food. All hydra will also change body size as a function of the amount of food and of temperature (Hecker and Slobodkin, 1976). Despite the within-clone plasticity, unrelated hydra maintained under identical conditions, side by side in the laboratory, retain consistent differences. Batha (1974) noticed that ﬁeld-collected hydra differ in appearance from their own clonal descendants maintained for long periods in the laboratory. Even animals collected from the same pond at different seasons and kept in the laboratory sometimes show consistent differences in laboratory cultures, despite apparent similarities of the ﬁeld-collected specimens. The effect of all this is to make taxonomy extremely difﬁcult. Some investigators have tended to describe new species on the basis of rather unstable, variant appearances, while others have despaired of making any precise identiﬁcations. Campbell (1987) has provided a compromise position, in which he has classiﬁed the hydra into clearly distinct “species groups,” each composed of an uncertain number of more closely related species whose precise identity may have to await new techniques of examination. Color, presence or absence of a body stalk, nematocyst shape, and the order of appearance of tentacles on the new buds are the characters used. The structure of the theca around the fertilized eggs, the appearance of gonads, proportions of different kinds of nematocysts, microscopic details of symbiotic algae, and certain physiological characteristics are all likely to be important in subdividing the various species groups. Unfortunately, these subdivisions are difﬁcult for all but the most serious students and professionals. Since the species group is adequate for most purposes, it seems advisable to quote extensively from Campbell’s descriptions. The four species groups are given as follows. 1. The easily recognized group Hydra viridissima (also known as Chlorohydra). In addition to the brilliant green color, Campbell lists the following characteristics: “small to moderate body size and tentacles, stalk not distinct from the rest of the column; hermaphroditic; 152 Lawrence B. Slobodkin and Patricia E. Bossert FIGURE 13 (A) Stenotele and (B) tentacle formation in Hydra oligactis group. [Redrawn from Campbell, 1987.] FIGURE 12 (A) Holotrichous isorhiza and (B) nonspiny embryotheca. [Redrawn from Campbell, 1987.] embryotheca spherical, made of polygonal plates; nematocysts very small; tentacles arising simultaneously on buds.” 2. The stalked Hydra or Oligactis group: “large size; pronounced translucent stalk in large individuals; long tentacles; dioecious; spherical embryo with simple theca; slender stenotele (capsules) and large, blunt, cylindrical holotrichous isorhiza (capsules); distinct golden color in culture due to yellow crystals in the ectoderm; two lateral tentacles arising before the others in the bud” (see Fig. 12). 3. The vulgaris group or common hydra “11 moderate size tentacles of moderate length; dioecious or monoecious, sometimes switching between the two conditions; spherical embryotheca ornamented with spines; broad stenotele (capsules); slender holotrichous isorhiza; often slipper-shaped, with the anterior end broadly pointed; tentacles arising on the buds simultaneously or nearly so” (see Fig. 13). 4. The “gracile Hydra” or braueri group: “small to medium size; tentacles moderately short; hermaphroditic; embryotheca and egg ﬂattened, with the embryotheca adherent to the substrate; embryotheca smooth or papillate; tentacles arising simultaneously on the buds; holotrichous isorhiza (capsule) broader than half its length; and stenotele (capsule) plump; body often palecolored during laboratory cultivation.” No single character, not even the nematocysts, can by itself distinguish one group from another. Every group probably contains a large number of species, each of which has been described under a large number of names. Perhaps biochemical procedures may eventually sort them out, but for the moment and for most purposes, strains collected from the ﬁeld should be classiﬁed to a species group and then supplied with enough ancillary description so that other workers can at least know if they have the same or a similar organism. Campbell (1987) provided a key to the four groups of Hydra, which we have incorporated in a key to the other freshwater Cnidaria. B. Taxonomic Key to Genera of Freshwater Cnidaria The polyp and medusa stages of a single species of coelenterate can differ greatly, both morphologically and ecologically. The key has been arranged to give the same results whether one starts with polyps or medusae. Also, the key refers to “species groups” of Hydra as names in parentheses. 1a. Parasitic in ﬁsh eggs ..........................................................................................................................................................Polypodium 1b. Nonparasitic ......................................................................................................................................................................................2 2a(1b). Medusae ........................................................................................................................................................................Craspedacusta 2b. Polyps ................................................................................................................................................................................................3 3a(2b). Polyp without basal attachment, with oral surface downward ..........................................................................................Polypodium 3b. Polyps with basal disk ........................................................................................................................................................................4 4a(3b). Solitary polyps with single circle of multicellular tentacles; no medusa, gonads are body stalk...........................................................7 5. Cnidaria 153 4b. Colonial polyps ..................................................................................................................................................................................5 5a(4b). Filiform tentacles, irregularly arranged on conical hypostome; branching colony with gonophores ...............................Cordylophora 5b. Colonial; atentacular or tentacles unicellular......................................................................................................................................6 6a(5b). Atentacular, oral capitulum; polyps from common stolon; may bud medusa from polyp; “microhydra” ................................................................................................................................................................Craspedacusta 6b. Minute with unicellular tentacles ........................................................................................................................................Calposoma 7a(4a). Bright green...................................................................................................................................................................Hydra (viridis) 7b. Not green ...........................................................................................................................................................................................8 8a(7b). Holotrichous isorhiza nematocysts at least half as broad as long; embryotheca without spines...........................................................................................................................................................................Hydra (braueri) 8b. Holotrichous isorhiza nematocysts slender or if plump, then embryotheca spined..............................................................................9 9a(8b). Buds acquire two lateral tentacles before others appear; otherwise stenotele nematocysts at least 1.5 times as long as broad .........................................................................................................................................................................Hydra (oligactis) 9b. Buds acquire tentacles in some other order; otherwise stenotele nematocysts less than 1.5 times as long as broad ...................................................................................................................................................................................Hydra (vulgaris) LITERATURE CITED AND FURTHER READINGS Acker, T. S. 1976. Craspedacusta sowerbii: an analysis of an introduced species, in: Mackie, G. Ed., Coelenterate biology and behavior, Plenum, New York, pp. 219 – 226. Aerne, B. 1995. Cnidarian hox genes. Developmental Biology 169:547 – 56. Angradi, T. R. 1998. Observations of freshwater jellyﬁsh, Craspedacusta sowerbii Lankester (Trachylina: Petasidae), in a West Virginia reservoir. Brimleyana 25:34 – 42. Anonymous. 1999. Fresh water jellyﬁsh in Rhode Island, URI Watershed Watch Home Page. Barnes, R. D. 1966. Invertebrate Zoology, Saunders, Philadelphia, PA. Batha, J. V. 1974. The distribution and ecology of the genus Hydra in the Milwaukee area of Lake Michigan. Ph. D. thesis, Zoology Department, University of Wisconsin, Milwaukee. Bernﬁeld, M. 1993. Molecular basis of morphogenesis. [Symposium with the Society for Developmental Biology] Wiley-Liss, New York. Bossert, P. 1986. Regulation of intracellular algae by various strains of the symbiotic Hydra viridissima. J. Cell Sci. 85:187 – 195. Bossert, P. 1988. The effect of Hydra strain size on growth of endosymbiotic alga. Ph.D. thesis, Department of Biology, State University of New York, Stony Brook, NY. Bossert, P., Dunn, K. 1996. Regulation of intracellular algae by various strains of the symbiotic Hydra viridissima. Journal of Cell Science 85:187 – 195. Bouillon, J. 1957. Etude monographique du genre Limnocnida (Limnomedusae). Annales de la Societe Royale Zoologique de Belgique 87:254 – 500. Boulenger, C. L., Flower, W. U. 1928. The Regent’s Park medusa, Craspedacusta sowerbii and its identity with C. (Microhydra) ryderi. Proceedings of the Zoological Society of London 66:1005 – 1015. Bridge, D., Cunningham, C., Schierwater, B., DeSalle, R., Buss, L. 1992. Class level relationships in the phylum Cnidaria: Evidence from mitochondrial genome structure. Proceedings National Academy of Science USA 89:8750 – 8753. Burnett, A. L. 1973. Biology of Hydra, Academic Press, New York. Campbell, R. D. 1987. A new species of Hydra (Cnidaria: Hydrozoa) from North America with comments on species clusters within the genus. Zoological Journal of the Linnean Society 91:243 – 263. Campbell, R. D. 1999. The Hydra of Madagascar (Cnidaria: Hydrozoa). Annales de Limnologie-International Journal of Limnology 35(2):95 – 104. Ciernichiari, E., Muscatine, L., Smith, D. C. 1969. Maltose excretion by the symbiotic algae of Hydra viridis. Proceedings of the Royal Society of London Series B 173:557 – 576. Colasanti, M. 1995. NO in hydra feeding response. Nature 374(6522):505. Devries, D. R. 1992. The freshwater jellyﬁsh Craspedacusta sowerbyi: a summary of its life history, ecology, and distribution. Journal of Freshwater Ecology 7(1):7 – 16. Dodson, S. I., Cooper, S. D. 1983. Trophic relationships of the freshwater jellyﬁsh Craspedacusta sowerbii Lankester 1880. Limnology and Oceanography 28:345 – 351. Dunn, K. W. 1986. Adaptations to endosymbiosis in the green Hydra, Hydra viridissima. Ph. D. thesis, Department of Biology, State University of New York, Stony Brook. Dunn, K. W. 1987. Growth of endosymbiotic algae in the green hydra, Hydra viridissima. Journal of Cell Science 88:571 – 578. Dunn, K. W. 1988. The effect of host feeding on the contribution of endosymbiotic algae to the growth of green hydra. Biology Bulletin 175:193 – 201. Elliott, J. K., Elliott, J. M., Leggett, W. C. 1997. Predation by hydra on larval ﬁsh: ﬁeld and laboratory experiments with bluegill (Lepomis macrochirus). Limnology and Oceanography 42(6): 1416 – 1423. Finnerty, J. R., Martindale, M. Q. 1999. Early evolution of Hox and ParaHox genes: evidence from the Cnidaria. Developmental Biology 210(1):41. Fuhrman 1984. Craspedacusta sowerbii Lank. et un noveau coelentere d’eau douce, Calpasoma dacryloprera. n.g., n.sp. Revue Suisse de Zoologie 46:363 – 368. Galliot, B., de Vargas, C., Miller, D. 1999. Evolution of homeobox 154 Lawrence B. Slobodkin and Patricia E. Bossert genes: Q(50) Paired-like genes founded the Paired class. Development Genes and Evolution 209(3):186 – 197. Grifﬁng, T. C. 1965. Dynamics and energetics of populations of brown hydra. Ph.D. thesis, Zoology Department, University of Michigan, Ann Arbor, MI. Grosvenor, W., Rhoads, D. E., Kassimon, G. 1996. Chemoreceptive control of feeding processes in hydra. Chemical Senses 21(3):313 – 321. Hecker, B., Slobodkin, L. B. 1976. Responses of Hydra oligactis to temperature and feeding rate, in: Mackie, G. O. Ed., Coelenterate ecology and behavior, Plenum, New York, pp. 175 – 186. Hershey, A., Dodson, S. 1987. Predator avoidance by Cricotopus: cyclomorphosis and the importance of being big and hairy. Ecology 68:913 – 920. Hessinger, D. Lenhoff, H. M. Eds. 1988. The biology of nematocysts, Academic Press, London. Hubschman, J. H., Kishler, W. J. 1972. Craspedacusta sowerbii Lankester 1880 and Cordylophora lacustris Allman 1871 in Western Lake Eric (Coelenterata). Ohio Journal of Science 72:318 – 332. Hyman, L. H. 1940. The invertebrates: Protozoa through Ctenophora, McGraw-Hill, New York. Johnson, E. M., Gorman, R. M., Gabel, B., George, M. E. 1982. The Hydra attenuata system for detection of teratogenic hazards. Teratogenesis, Carcinogenesis, and Mutagenesis 2:263 – 276. Kanaev, I. I. 1969. Hydra: essays on the biology of freshwater polyps. Lenhoff, H. M. Ed. [Translated by Burrows, E. T. and Lenhoff, H. M.] Published by H. T. Burrows and H. M. Lenhoff, University of Miami, Coral Gables, FL. [Originally published by the Soviet Academy of Sciences, Moscow, 1952.] 453 pp. Kouchinsky, A., Bengtson, S., Gershwin, L. A. 1999. Cnidarian-like embryos associated with the ﬁrst shelly fossils in Siberia. Geology 27(7):609 – 612. Kramp, P. L. 1950. Freshwater medusae in China. Proceedings of the Zoological Society of London 120: 165 – 184. Lenhoff, H. M. 1974. On the mechanism of action and evolution of receptors associated with feeding and digestion, in: Muscatine, L. Lenhoff, H. M. Eds., Coelenterate biology: reviews and new perspectives, Academic Press, New York, pp. 211 – 214. Lenhoff, H. M. 1983. Hydra: research methods, Plenum Press, New York, 463 pp. Lenhoff, H. M., Loomis, W. F. 1961. The biology of Hydra and of some other coelenterates, University of Miami Press, Coral Gables, FL. Lenhoff, S. G., Lenhoff, H. M. 1986. Hydra and the birth of experimental biology, 1744: Abraham Trembley’s Memoires concerning the polyps, Boxwood Press, Paciﬁc Grove, CA. Link, J., Keen, R. 1995. Prey of deep-water hydra in Lake Superior. Journal of Great Lakes Research 21(3):319 – 323. Lipin, A. 1911. Morphologie und biologie von Polypodium. Zoologische Jahrbuecher, Abteilung fuer Anatomie und Ontogenie der Tiere 31. Lipin, A. 1926. Polypodium. Zoologische Jahrbuecher, Abteilung fuer Anatomic und Ontogenie der Tiere. Lomnicki, A., Slobodkin, L. B. 1966. Floating in Hydra littoralis. Ecology 47:881 – 889. Mackie, G. O. 1976. Coelenterate ecology and behavior, Plenum, New York. Martinez, D. 1997. On senescence in asexual metazoans. Ph.D. thesis, Department of Ecology and Evolution, State University of New York at Stony Brook, Stony Brook, NY. 181 pp. Muscatine, L. 1965. Symbiosis of Hydra and algae. III Extracellular products of the algae. Comparative Biochemistry and Physiology 16:177 – 192. Naumov, D. V., Stepan’iants, S. D. 1980. Teoreticheskoe i prakticheskoe znachenie kishechnopolostnykh: sbornik. Leningrad, Zool. in-t. Pardy, R. 1983. Preparing aposymbiotic Hydra and introducing symbiotic algae into aposymbiotic Hydra, in: Lenhoff, H. M. Ed., Hydra: Research methods, Plenum, New York, pp. 393 – 401. Payne, F. 1924. A study of the freshwater medusae, Craspedacusta ryderi. Journal of Morphology 38:397 – 430. Rahat, M., Campbell, R. 1974. Nematocyst migration in the polyp and 1 celled tentacles of the minute freshwater coelenterate Calposoma dactyoptgera. Transactions of the American Microscopical Society 93:379 – 385. Rahat, M., Sugiyama, T. 1993. The endodermal cells of some brown Hydra are autonomous in their ability to host or not to host symbiotic algae: an analysis of Chimera. Endocytobiosis and Cell Research 9(2 – 3):223 – 231. Raikova, E. 1973. Life cycle and systematic position of Polypodium hydriforme Ussov (Coelenterata, a cnidarian parasite of the eggs of Acipenseridae. Publications of the Seto Marine Biological Laboratory 20:165 – 174. Raikova, E. 1980. Morphology, ultrastructure and development of the parasitic larva and its surrounding trophamnion of Polypodium hydriforme Coelenterata. Cell Research 206:487 – 500. Ritte, U. Z. 1969. Floating and sexuality in laboratory populations of Hydra littoralis. Ph. D. thesis Zoology Department. University of Michigan, Ann Arbor, MI, 350 pp. Roos, P. 1979. Two stage life cycle of a Cordylophora population in the Netherlands. Hydrobiologia 62:231 – 239. Sabine, A., Hoffmeister, H., Chica, H., Schaller, H. C. 1987. Head activator and head inhibitor are signals for nerve cell and differentiation in hydra. Developmental Biology 122:72 – 77. Schummer, M., Scheurlen, I., Schaller, C., Galliot, B. 1992. Hom Hox Homeobox genes are present in hydra (Chlorohydra viridissima) and are differentially expressed during regeneration. Embo Journal 11(5):1815 – 1823. Schwartz, S. S., Hann, B. C., Herbert, D. N. 1983. The feeding ecology of Hydra and possible implications in the structuring of pond plankton communities. Biological Bulletin (Woods Hole, Mass) 164:136 – 142. Slobodkin, L. 1964. Experimental populations of Hydrida. British Ecological Society Jubilee Symposium. Journal of Animal Ecology 33 (Supplement):131 – 148. Slobodkin, L. 1980. Growth and regulation of animal populations, Dover, New York. Slobodkin, L. Bossert, P., Mattessi, C., Gatto, M. 1991. A review of some physiological and evolutionary aspects of body size and bud size of Hydra. Hydrobiologia 216:377 – 382. Spadinger, R., Maier, G. 1999. Prey selection and diel feeding of the freshwater jellyﬁsh, Craspedacusta sowerbyi. Freshwater Biology 41(3):567 – 573. Stiven, A. 1976. The quantitative analysis of the hydra-hydramoeba host – parasite system, in: Mackie, G.O. Ed., Coelenterate ecology and behavior, Plenum, New York, pp. 389 – 400. Thiel, H. 1973. Limnocnida indica in Africa. Publications of Seto Marine Biological Laboratory 20:73 – 79. Thorp, J. H., Barthalmus, G. T. 1975. Effects of crowding on growth rate and symbiosis in green hydra. Ecology 56:206 – 212. Walsh, E. J. 1995. Habitat-speciﬁc predation susceptibilities of a littoral rotifer to two invertebrate predators. Hydrobiologia 313:205 – 211. Warrior, R., Gall, J. 1985. The mitochondrial DNA of Hydra attenuata consists of two linear molecules. Archives des Sciences (Geneva) 38:439 – 445. 6 FLATWORMS: TURBELLARIA AND NEMERTEA Jurek Kolasa Department of Biology McMaster University Hamilton, Ontario L8S 4K1 Canada TURBELLARIA I. Introduction: Status in the Animal Kingdom II. Anatomy and Physiology A. General External and Internal Anatomical Features B. Environmental Physiology III. Ecology A. Life History B. Distribution C. Behavioral Ecology D. Foraging Relationships, Predators, and Parasites E. Population Regulation and Density F. Functional Role in the Ecosystem IV. Current and Future Research Problems A. Dispersal B. Rhabdoids C. Endosymbiosis D. Community Ecology V. Collecting, Rearing, and Identiﬁcation Techniques VI. Identiﬁcation of North American Genera of Freshwater Turbellaria A. Taxonomic Key to Orders and Suborders of Turbellaria B. Taxonomic Key to Catenulida Turbellaria and Nemertea are common and often very numerous inhabitants of freshwaters. Even though more than 200 species of Turbellaria and three species of Nemertea live in North America, their ecology and systematics have been less studied than that of many other common aquatic invertebrates. An obvious reason for this limited attention is Ecology and Classiﬁcation of North American Freshwater Invertebrates. 2nd Edition Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved. C. Taxonomic Key to Macrostomida D. Taxonomic Key to Lecithoepitheliata E. Taxonomic Key to Prolecithophora, Proseriata, and Tricladida F. Taxonomic Key to Typhloplanoida G. Taxonomic Key to Kalyptorhynchia and Similar Forms H. Taxonomic Key to Dalyellioida NEMERTEA VII. General Characteristics, External and Internal Anatomical Features VIII. Ecology A. Life History and General Ecology B. Physiological Adaptations C. Behavioral Ecology D. Functional Role in the Ecosystem IX. Current and Future Research Problems X. Collection, Culturing, and Preservation XI. Taxonomic Key to Species of Freshwater Nemertea in North America Literature Cited Appendix 1. List of North American Species of Microturbellaria the difficulty posed by preservation. Most turbellarians become unrecognizable after a routine preservation of field samples in alcohol or formalin. Study of live specimens is the best method and many interesting discoveries lie ahead in the areas of reproductive biology, dispersal, endosymbiosis, and community structure. 155 156 Jurek Kolasa TURBELLARIA I. INTRODUCTION: STATUS IN THE ANIMAL KINGDOM The turbellarian ﬂatworms are the lowest acoelomate Bilateria with only a single opening to the digestive tract; that is, they lack a deﬁnitive anus. Cestoidea (tapeworms), Trematoda (ﬂukes), and Turbellaria constitute the phylum Platyhelminthes. Because of tradition and practical considerations, turbellarians are divided into microturbellarians and macroturbellarians. This division does not reﬂect phylogenetic relationships, but rather superﬁcial morphological similarities. Also, depending on the methodology chosen, the taxa discussed may have a rank different from the one used here (see Ehlers, 1986). Several orders and suborders of Turbellaria are known from freshwaters (Table I, Fig. 1). One order, Acoela, has been recently excluded from Turbellaria on the basis of DNA sequence analysis (Ruiz-Trillo et al., 1999) but we retained it in this chapter for practical reasons. Of the approximately 400 species of freshwater microturbellaria, about 350 have been recorded in Europe (Lanfranchi and Papi, 1978), and approximately 150 occur in North America (Appendix 6.1), many of which are the same as those found in Europe. Many species and genera of microturbellarians remain TABLE I to be discovered in North America. Records from other regions of the world are sparse. Interestingly, Africa, South America, and Papua New Guinea trail closely behind North America in the number of species. Differences in species richness are clearly due in great part to research effort; and for this reason, the number of species in North America must be highly underestimated. Triclads (often called planarians) are more thoroughly studied. The number of recorded species in North America is approximately 40, but the actual number of taxa is likely to be substantially higher. II. ANATOMY AND PHYSIOLOGY A. General External and Internal Anatomical Features Most freshwater microturbellaria are less than 1 mm in length, although some can reach several millimeters. Triclads are distinctly larger, with most species exceeding 10 mm and the largest being several centimeters long. The turbellarian body is elongated, relatively soft, and usually tapered at the ends. Sometimes lateral ﬂaps near the cerebral region or a short tail like section are present. With the exception of triclads, ﬂatworms are generally not ﬂat — despite their common name. Most Selected Characteristics of Commonly Recognized Turbellarian Orders and Suborders Occurring in Freshwaters Taxona order suborder superfamily Approx. number of species Average body size (mm) Comments and special features Microturbellarians Catenulida 60 0.5 – 1 Acoela Macrostomida Prolecithophora Lecithoepitheliata 3 50 5 10 0.5 – 1 1.0 – 3 5.0 – 10 3.0 – 10 Proseriata Rhabdocoela Dalyellioida Temnocephalida 4 2.0 – 5 Thin chains of zooids; common in various habitats Most species marine Common in various habitats Many marine species Many marine species; some are terrestrial or semiaquatic Many marine species Typhloplanoida Typhioplanida Kalyptorhynchia Macroturbellarians Tricladida a 100 0.8 – 1 1.0 – 14 Common in various habitats Commensals on crust aceans, snails, turtles, one parasitie 150 0.5 – 6 15 1.0 – 2 Common in various habitats including terrestrial and semiaquatic Rare; most species marine 100 5 – 20 Taxonomical rank is indicated by indentation. Greatest diversity associated with karst habitats 6. Flatworms: Turbellaria and Nemertea 157 FIGURE 1 Schematic representation of higher turbellarian taxa found in freshwaters with an emphasis on the most useful diagnostic features. (A) Acoela (one unidentiﬁed species); (B) Catenulida (e.g., Catenula, Stenostomum, I and II represent two successive zooids); (C) Macrostomida (Macrostomum, Microstomum); (D) Lecithoepitheliata (e.g., Prorhynchus, Geocentrophora); (E) Dalyellioida (Gieysztoria, Microdalyellia); (F) Typhloplanoida (e.g., Mesostoma, Olisthanella, Castrada); (G) Kalyptophynchia (e.g., Gyratrix; intestine omitted for clarity); and (H) Proseriata and Tricladida (e.g., Bothrioplana, Dendrocoelopsis). Co, Copulatory organ; cp, ciliated pits; i, intestine, m, mouth; n, protonephridial duct; o, ovary; ph, pharynx; pr, proboscis; s, stylet, st, statocyst; t, testes; v, vacuolized tissue; and y, yolk glands. microturbellaria are circular in cross section, with some differentiation of shape of the dorsal and ventral surfaces. Moreover, species that reproduce asexually may be composed of several zooids (Figs. 3B, 4A,B, 12A), giving a chainlike appearance to an animal. Turbellarians may be colorless, white, red, bluish, green, black, brown, or yellowish depending on epidermal and parenchymal pigments, gut content, and symbiotic algae. Some species, such as Dugesia tigrina and Hydrolimax grisea, develop characteristic patchy patterns of pigments. Anatomically, the most prominent turbellarian features are a ciliated epidermis, rhabdoids, an intestine without anus, ventral mouth, and a complex reproductive system (Figs. 1, 9, 12D). The ciliated epidermis covers the entire body surface. Rhabdoids are rodlike, light-refracting structures produced in the epidermis (dermal rhabdoids) or in the parenchyma (adenal rhabdoids). It is often difﬁcult to determine the type of rhabdoids in live specimens. Adenal rhabdoids are most abundant at or near the front extremity. They often aggregate in juxtaposed groups that are known in the taxonomic literature as rod tracts. Wrona (1986) reviewed hypotheses on the role of rhabdoids in triclads and has provided experimental evidence that rhabdoids contribute to mucus production. Rhabdoids are also poisonous and used in prey immobilization or in deterring predators. Other components of the body walls include a basal membrane and muscle ﬁbers. The ﬁbers are longitudinal, circular, or in larger forms, diagonal. Some ﬁbers go through the parenchyma. The parenchyma is a loose tissue ﬁlling the body cavity and containing various cell types, including secretory, excretory, reproductive, and formative cells and their insoluble excretions, that fulﬁll important physiological functions (Hyman, 1951). In large turbellarians, the parenchyma is usually more compact than in smaller forms, where it can be quite loose and vacuolated. Turbellarians possess numerous glands, of which the frontal, pharyngeal, shell-producing, and rhabdoidproducing glands are most common. A vast array of sensory organs enables turbellarians to react to environmental stimuli. These organs include sensory hairs, eyes, statocysts, and chemoreceptors of various kinds. Some species possess aggregations of sensory cells, or ciliated pits, in lateral depressions situated in front of the brain ganglion. Exact functions of the ciliated pits and structures associated with them are unknown, but at least some of these structures (i.e., the refractive bodies of Stenostomum) have been implicated in phototaxis (Marcus, 1951). Some evidence exists that these refractive bodies function as lenses (Tyler and Burt, 1988). Thigmotaxis is common and plays a role in a range of behaviors such as choosing substrate, hunting prey, and avoiding predators. Among North American freshwater Turbellaria, a statocyst is present only in Catenula, Rhynchoscolex, Otomesostoma, and an unidentiﬁed representative of Acoela (Strayer, 1985; Kolasa et al., 1987). Turbellarians have two types of simple eyes: inverted and direct cup eyes (Bedini et al., 1973). The number of eyes is variable, but one pair of eyes is most frequently present. Locomotion in the Turbellaria is based on the movement of cilia. Some triclads, however, are capable 158 Jurek Kolasa of fast muscular contractions allowing them to crawl in bursts of leechlike behavior. Smaller forms can both swim and glide on submersed objects, while heavier forms are restricted to gliding only. Adhesive organs in the form of pseudosuckers or papillae are often present. B. Environmental Physiology Gaseous exchange in turbellarians occurs through their body walls. Such exchange depends on the surface / volume ratio and is not very efﬁcient. Consequently, ﬂatworms may have a limited ability to adapt to low-oxygen conditions. This conclusion appears to be true for most ﬂatworms and this limitation is often exploited in extraction of specimens from samples (e.g., Young, 1970; Schwank, 1981). However, the ability to cope with reduced oxygen levels does not seem to differ substantially from that of other aquatic invertebrates. Oxygen adaptability differs more among species adapted to diverse habitats than among major taxa (cf. Heitkamp, 1979b). For example, species of Macrostomum normally found in stream headwaters are more sensitive to low oxygen levels, and perhaps high temperature, than are representatives of the same genus from physically more ﬂuctuating habitats (J. Kolasa, personal observation). Some species, particularly in the genus Phaenocora, have developed adaptations to cope with anaerobic conditions (Young and Eaton, 1975). They showed that the presence of algae in Phaenocora typhlops enhances its ability to survive low-oxygen conditions. This same species, as well as a related P. unipunctata (Öersted) produces hemoglobin, allowing them to store oxygen during burrowing in the anoxic mud. Many marine species, however, live in low-oxygen sediments, but have neither hemoglobin nor symbiotic algae. Heitkamp (1979a, b) studied respiration rates of Opistomum pallidum, two species of Mesostoma, and a species hosting symbiotic zoochlorellae — Dalyellia viridis. He showed the respiration to be related to the body size and shape (surface in a very ﬂat Mesostoma ehrenbergi, or weight in cylindrical Mesostoma lingua and other species). Interestingly, Dalyellia viridis that are symbiotic with zoochlorellae had the highest respiration rate of all studied species; possibly their symbiotic algae contribute signiﬁcantly to the observed rate. Heitkamp (1979b) found the respiration rate of microturbellarians to vary depending on the temperature and diurnal rhythm. A considerable effort has been devoted to studying nutrition and digestion in Turbellaria, particularly by Jennings (1977). In this context, the lipid storage capacity of ﬂatworms and its importance to population dynamics and competition is discussed for Dugesia polychroa by Boddington and Mettrick (1977). Speciﬁcally, they show that populations are regulated by intraspeciﬁc competition for food, which affects individual size, cocoon production, and cocoon viability. Triclads clearly avoid light. Microturbellarians appear to be either indifferent or positively phototactic (e.g., Typhloplana viridata which contains symbiotic algae), although if a sample is suddenly illuminated, many will attempt to hide in sediments. Desiccation of eggs can be tolerated by some species and it may be an obligatory factor in releasing them from diapause and stimulating embryonic development. Similarly, some triclads such as Phagocata may undergo encystment as entire or fragmented animals. Temperature may also have an important effect on reproduction. Dumont and Schorreels (1990) showed that clutch size, total offspring, and daily offspring all depended on temperature rather than food in Mesostoma lingua. Although the majority of Turbellaria appear to have some habitat preferences, within a limited range of physical conditions many species have adaptations conferring considerable ﬂexibility. Some members of the Catenulida are found in habitats as different as fast-ﬂowing streams and temporary pools (e.g., Stenostomum leucops, Catenula lemnae). Mesostoma species have been reported from both warm rice ﬁelds in the Sacramento Valley in California as well as from snowmelt pools at elevations above 2400 m (Collins and Washino, 1979). Even though microturbellarians are delicate and fragile organisms, they appear to have a signiﬁcant potential for dispersal. Results of an experiment conducted by Jan Ciborowski (University of Windsor) were surprising. Ciborowski studied colonization of artiﬁcal ponds (ﬂexible swimming pools) situated on the roof of a university building for several weeks. About 50% of these ponds were successfully colonized by six different species of microturbellaria (unpublished data). With only one exception, these species are not known to produce resistant disseminules such as eggs or cocoons. III. ECOLOGY A. Life History In most species, miniature replicas of the adult hatch directly from eggs; these juveniles differ from adults chieﬂy by the absence of reproductive systems. Some freshwater turbellarians, however, may be ovoviviparous (Mesostoma) or may have a larval stage distinctly different from the adult (Rhynchoscolex). Numerous modiﬁcations of the life cycle have evolved among turbellarians. Many seasonally occurring species 6. Flatworms: Turbellaria and Nemertea are univoltine, particularly those associated with temporary habitats or at the extremes of their geographical ranges. Other species are multivoltine, with the number of generations depending on habitat availability (see Heitkamp, 1982, for an excellent account). A similar diversity of life cycles is observed in triclads, but the ecological reasons for this diversity remain hidden. Reproductive patterns may sometimes coincide with major taxonomic subdivisions. Calow and Read (1986) studied European representatives of the three families that are also represented in North America: Dugesiidae, Planariidae, and Dendrocoelidae. These investigators have found that species breeding over several seasons (Dugesiidae, Planariidae) invest less in reproduction than members of Dendroceolidae, which reproduce once and die. As a rule, turbellarians are hermaphroditic. Various modiﬁcations of both egg formation and development are found and may include self-fertilization, as in Mesostoma ehrenbergi (Fiore and Ioalé, 1973; Göltenboth and Heitkamp, 1977), protandry or progyny in many other species, or loss of either the male or female gonad as in Catenulida (Rieger, 1986). All of these modiﬁcations have implications for habitat adaptability, clonal diversity, rate of population growth, and ability to colonize. Asexual reproduction by means of paratomy, that is transverse division of the body, is common in several genera of microturbellaria, particularly in Catenulida and Macrostomida. In macroturbellarians, architomy (transversal ﬁssion of the caudal region) occurs frequently in Dugesiidae and in some Planariidae (Calow and Read, 1986; Rieger, 1986). In some species of Stenostomum, and in some populations of the common triclad Dugesia tigrina, sexual organs have never been observed. B. Distribution Freshwater turbellarians are largely free-living animals, although a few European freshwater species such as Varsoviella kozminski (Gieysztor and Wiszniewski) and Phaenocora beauchampi (Sekera) are ectoparasitic on crustaceans. Several other freshwater forms, including triclads, occurring in Europe and Australia are commensal on crustaceans and turtles (e.g., Jennings, 1985). The great majority of freshwater turbellarians are free-living and live in various aquatic systems such as ponds, lakes, streams, hyporheic water, ditches, and temporary puddles. However, some may be found in aquatic habitats normally excluded from the domain of freshwater ecology, such as water ﬁlms among fallen leaves in a mesic forest or in capillary soil water of a grassy meadow (Sayre and Powers, 1966). 159 In North America, microturbellarians are known as far north as Igloolik, a small island at 68°N latitude off Melville Peninsula. Two species, Mesostoma and an unidentiﬁed mesostomid, were collected by P.D.N. Hebert and associates (unpublished). In Alaska and in the area of Hudson Bay in northern Canada, as many as six different species are known (Holmquist, 1967; Schwartz and Hebert, 1986). Further south, in temperate climatic zones, the species diversity increases and can easily reach 20 to perhaps 60 species in a single lake or pond. For example, there are 23 species of ﬂatworms in the oligotrophic Mirror Lakes, New Hampshire (Strayer, 1985) and as many as 57 species occur in the polytrophic Lake Zbechy in Poland (Kolasa, 1979). Streams also have rich ﬂatworm fauna. In a short section of Wappinger Creek, an eastern tributary of the Hudson River in New York, 15 species were found (Kolasa et al., 1987). A survey of several streams, including springs and underground water in the same area revealed 32 species of microturbellarians, some of which are still undescribed. Because of a dearth of systematic studies, few microturbellarians are known from the West Coast. Case and Washino (1979), Collins and Washino (1979), and other related papers report Mesotoma lingua from California, together with some other common species in rice ﬁelds. Many species, particularly among the Catenulida and the Macrostomida, are widely distributed worldwide, while species of the “higher” Turbellaria, such as the Typhloplanida and Tricladida, are geographically more restricted. About 3% of all species known from Europe are designated as cosmopolitan in distribution (Lanfranchi and Papi, 1978), but studies in the tropics and Southern Hemisphere (Mead and Kolasa, 1984) suggest that this ﬁgure is too low. Caves and underground waters hold many unique and endemic species, particularly of triclads (e.g., Kenk, 1987). Some troglobiotic triclads and microturbellarians have recent or remote marine origins (e.g., Kawakatsu and Mitchell, 1984a; Kolasa, 1977b). An extensive study of European underground triclads (Gourbault, 1972) revealed a suit of distinct distributional, reproductive, and physiological characteristics. These often include specialized underground microhabitat selection, greater frequency of sexual reproduction, and slower metabolism as compared to epigean (or surface-dwelling) triclads. In contrast, a number of interstitial and underground water microturbellarians, for example Bothrioplana semperi, Limnoruanis romanae, and Stenostomum pegephilum, occur on more than one continent. Distributions of most genera appear to be worldwide among microturbellarians, but not triclads, many genera of which are known exclusively from either North 160 Jurek Kolasa America, Eurasia, South America, or Australia (Ball, 1974). The majority of triclads from North America are described from the eastern and southern United States and Canada (Kenk, 1972). Four triclad species inhabit Alaskan waters and 10 live in Tennessee (Chandler and Darlington, 1986). Several species of triclads with recent marine origins occur in freshwater caves of Mexico (Kawakatsu and Mitchell, 1984b; Benazzi and Giannini, 1971). Boreo-alpine distribution of many cold-water species has been tied to glaciations in Europe and similar patterns also seem to occur in North America (Hampton, 1988). At lower latitudes, similarities between South American and North American microturbellian composition (cf. Marcus, 1945) suggest considerable interchanges of species. The ecological distribution of both microturbellarians and triclads has been studied more intensively in Europe, but most of the results are directly relevant to North American fauna. The distribution of microturbellarians in lakes has been investigated by Strayer (1985), Chodorowski (1959), Kolasa (1977a, 1979), Rixen (1968), Schwank (1976), and Young (1970, 1973a). These studies show that microturbellarians differentiate among substrate types and depth zones. The ﬂatworm assemblages of sand and gravel bottoms are distinctly different from assemblages in habitats characterized by bottoms of silt, coarse organic matter, or vegetation. Plant successional zones also differ in their species assemblages. Although the littoral zone has the greatest density and diversity of ﬂatworms, there is apparently no limit to how deep some species may be found as long as oxygen is available. For example, Otomesostoma auditivum lives at a depth of 15 – 45 m in Echo Lake in the Sierra Nevada range, but it occurs at a depth of 145 m in some European alpine lakes (Hyman, 1955). Ecological differentiation of microturbellarians in running waters is more pronounced than in lakes. European studies showed distinct assemblages of species associated with springs, headwaters, and lower stretches of streams and rivers (epirhithron, metarhithron, hyporhithron, and potamon) (Schwank, 1981; Kolasa, 1983). These regularities in zonation of ﬂatworm assemblages are consistent with patterns observed among other lotic taxa. These patterns, however, still need to be related to newer ecological approaches to running waters, i.e., to the river continuum concept (Vannote et al., 1980). Among those classical zones, epirhithron shows the greatest species richness. No comparable studies have been published on North American microturbellarians. Zonation of triclads in streams was studied in a greater detail, including experimentation (Reynoldson, 1982). Competition, as well as thermal requirements were implicated as important factors separating several species of ﬂatworms along a water course and in standing waters (Claussen and Walters, 1982; Pattee, 1980; Reynoldson, 1982). Both microturbellarians and triclads live in habitats where the physical conditions require additional adaptations in comparison to an average pond or stream environment. Wet mosses and leaves on the water edge, water containers at the base of bromeliad leaves, the high Arctic, ice melt ponds at elevations over 4000 m in Central Asia, and hot springs are habitats for one or more species of ﬂatworms. Representatives of freshwater families are also commonly found in humid terrestrial habitats. C. Behavioral Ecology Laboratory studies reveal that turbellarians have a limited ability to learn simple tasks such as choosing white over dark branches of a maze (McConnell, 1967). Observations of prey handling (personal observation), however, indicate that their learning ability may be much greater for tasks that might be advantageous under natural conditions, such as prey or predator recognition. Possible behavior modiﬁcation or behavior determination associated with symbiosis is a potentially rewarding research area. Typhloplana viridata, a common spring pond species, is unique in being positively phototactic. This behavior may be associated with the presence of symbiotic algae. It might be of general interest to know control mechanisms of this behavior. D. Foraging Relationships, Predators, and Parasites Microturbellarians eat bacteria, algae, protozoa, and small invertebrates, while triclads feed predominantly on larger invertebrates. Scavenging is common in both morphological groups. Various Mesostoma species have been studied in detail and illustrate the diversity of feeding habits and techniques within one genus. Leaf-shaped M. ehrenbergi usually suspend themselves in the water column on a mucous ﬁlament attached to the surface ﬁlm (Göltenboth and Heitkamp, 1977). In that position, the worm waits for a cladoceran or an insect larva to come into contact. Using its front end, M. ehrenbergi traps the prey with sticky mucus, wraps around it, and proceeds to feed. According to Dumont and Carels (1987), M. lingua uses neurotoxins to immobilize its prey in addition to mucus. Mesostoma can also glide on the surface of underwater objects or swim directly in the water column. Both activities enhance its chances of encountering 6. Flatworms: Turbellaria and Nemertea 161 prey. Unlike M. ehrenbergi, many other species are spindle-shaped (e.g., M. arctica). These species hunt more often on the bottom or among submersed objects. Although several Mesostoma studied spend more than 50% of their time resting on various objects, they can catch prey as soon as it comes close enough to create vibrations in the water (J. Kolasa, personal observation). At that point, Mesostoma can erect the front portion of its body and actively move it around in search for contact with the prey. Several species of Mesostoma, such as M. vernale (Hyman) and M. californicum (Hyman), as well as species of Bothromesostoma have a ﬂat ventral surface with which they cling to the surface ﬁlm. These species are more agile and much faster than other species of Mesostoma and they prefer hunting at the water surface. Immobilizing the prey and sucking its body ﬂuids is a common feeding behavior in microturbellarians equipped with a strong pharynx rosulatus, or in triclads with a pharynx plicatus. Many other turbellarians, however, swallow the whole victim or bite chunks of prey. For example, species of Stenostomum and Macrostomum that feed on algae, protozoa, rotifers, and other meiofauna usually swallow the prey whole. Other, such as Stenostomum predatorium and various Phaenocora species may attack and partly consume larger turbellarians (Kepner and Carter, 1931), oligochaetes, or other soft invertebrates. Finally, a few microturbellarians (e.g., Gyratrix hermaphroditus and Prorhynchus stagnalis) use their copulatory stylets to stab prey. Chemical detection of prey undoubtedly plays an important role in feeding. Injured invertebrates attract species of both triclads and microturbellarians under laboratory conditions. The presence of injured or recently killed organisms allows scavenging by many individuals that played no role in immobilizing the prey. Turbellarians themselves are subject to predation and parasitism. Ciliates and ﬂagellates are frequent parasites of Catenulida and Typhloplanida, and nematodes have been found in Lecithoepitheliata (Prorhynchus; J. Kolasa, unpublished). Fish occasionally eat triclads (Davies and Reynoldson, 1971). Microturbellarians (Phaenocora typhlops) may occasionally be eaten by invertebrates, such as a chironomid Anatopynia (Young, 1973b) or other Turbellaria (e.g., Stenostomum predatorium). (Peter, 1995). The few studies conducted in Britain indicate that intraspeciﬁc competition for food may be very important for Phaenocora typhlops (Young, 1975), while interspeciﬁc competition may play a role in regulating several planarians (Reynoldson, 1982). In European Polycelis tenuis and, perhaps, in American Cura foremanii (Girard), this regulation is expressed by a negative relationship between the worm body size and density. In fact, long-term changes in the population size of Polycelis suggest that limit cycles or cyclical ﬂuctuations of the density due to intraspeciﬁc competition are involved (Reynoldson, 1982, and references therein). Densities of microturbellaria vary according to the habitat, season, and species. No major generalizations have been made, but some patterns are beginning to emerge, particularly along seasonal and successional axes. In small, second- or third-order streams, the greatest densities of microturbellaria are found in the lower reaches, i.e., in the metarhithron and hyporhithron, while the highest richness is found in the epirhithron zone (Kolasa, 1983). For example, a mean density of 1280 individuals / m2 (of all species) was recorded in Wappinger Creek, New York, in an artiﬁcal substrate after a week of colonization. Schwank (1981), using volume instead of surface to quantify turbellarian abundance in submontane streams in Germany, found densities of about 80 microturbellarians / L. If one assumes an average sample depth into the substrate of 5 cm, this translates into over 4000 individuals / m2. His data suggest a corresponding density of triclads of 32 individuals / L and 1600 individuals / m2. In oligotrophic Mirror Lake, turbellarian densities varied from 40,000 individuals / m2 in shallow littoral regions to almost zero in profundal zones; the lakewide mean was 27,000 individuals / m2 (Strayer, 1985). Other studies reported densities of 800 individuals / m2 of Lake Michigan (Nalepa and Quigley, 1983), 3500 individuals / m2 (annual mean) in shallow littoral areas of Zbechy Lake in Poland (Kolasa, 1979), 3100 individuals / m2 in Lake Paajarvi in Finland (Holopainen and Paasivirta, 1977), to 9500 individuals / m2 in ponds in Germany (Heitkamp, 1982). These values have to be interpreted cautiously, however, in the light of seasonal succession and abundance changes, which ordinarily produce the greatest richness and abundance in early summer (Bauchhenss, 1971; Strayer, 1985; Chodorowski, 1960). E. Population Regulation and Density F. Functional Role in the Ecosystem It is not clear whether there is any single mode of population regulation that applies to ﬂatworms. Asexual reproduction of Dugesia tahitiensis was found to be density-dependent under laboratory conditions The functional role of species is typically discussed in terms of their trophic interactions. All triclads are predatory. Several invertebrates and vertebrates may consume triclads and are a signiﬁcant source of their 162 Jurek Kolasa mortality. However, triclads constitute a relatively minor component of the diet of their predators (Davies and Reynoldson, 1971). Triclads themselves consume isopods, midges, oligochaetes, caddisﬂies, mayﬂies, ostracodes, and cladocerans. The ability to exploit various resources differs among species and may result in strong competition with a simultaneous expansion of invading species (e.g., Gee and Young, 1993). Heitkamp (1982) reports intense predation on Mesostoma and Rhynchomesostoma by Dalyellia in temporary pools. He has not determined the frequency of such predation nor established whether this predation results in population regulation. High densities of both triclads and microturbellarians suggest that their role in biotic interactions of benthic communities may be greater than their contribution to the diet of other organisms. Densities of Mesostoma species (lingua) in excess of 1000 individuals / m2 were observed in more than 30% of the rice ﬁelds studied by Collins and Washino (1979) in California. In some cases, microturbellaria may regulate population dynamics of zooplankton in ponds, as demonstrated by Maly et al. (1980). They found that the feeding rates of Mesostoma ehrenbergi were two zooplankton per day (estimated from Fig. 1). However, Schwartz and Hebert (1982) reported that this species can consume about ten cladocerans per day. Blaustein (1990) and Blaustein and Dumont (1990) found that Mesostoma lingua might have major numerical impact on mosquito larvae, cladocerans, and ostracodes in rice ﬁelds, but little effect on copepods. More important, perhaps, is the functional role of microturbellaria as consumers of protozoa, rotifers, and algae, especially by the usually abundant Stenostomum leucops and similar forms. Unfortunately, no quantitative data on this subject are available. Although there are no explict studies of the energy ﬂow through lake meiobenthos, the detailed study of the energy budget of Mirror Lake (Strayer and Likens, 1986) indicated this ﬂow to be equivalent to that to zooplankton. Furthermore, Strayer (1985) showed that Turbellaria, and Rhynchoscolex in particular, were a substantial component of meiobenthos in Mirror Lake. This statement might be somewhat misleading because meiobenthos itself is a minor component of the whole budget. IV. CURRENT AND FUTURE RESEARCH PROBLEMS A. Dispersal Understanding the geographical and ecological distribution of Turbellaria is important because their distribution has evolutionary and ecological implications. Yet this understanding will suffer seriously unless the dispersal capabilities of ﬂatworms are assessed in terms of distance and rates. Hebert and Payne (1985) found low levels of gene ﬂow in Arctic Mesostoma. Their calculated dispersal rates are inconsistent with both the unpublished data from Ciborowski’s experiment mentioned earlier and with common observations that even newly created water bodies have a rich complement of species. A series of questions may be posed. Is dispersal different at various latitudes? Do dispersal strategies and efﬁciency differ along major taxonomic lines? Are habitat generalists better dispersers than specialists? Are species associated with a particular habitat type (e.g., temporary ponds) better dispersers as opposed to cave dwellers? If yes, what in their life cycle makes them so? These questions point out the limits in factual knowledge and ecological interpretation of the many aspects of turbellarian dispersal. B. Rhabdoids Wrona’s (1986) study demonstrated a lack of support for hypotheses that rhabdoids play a major role in contacts with prey or predators in triclads. In many microturbellarians however, rhabdoids, and adenal products in particular are channeled toward the anterior of the body. The front end usually plays a crucial role in exploration of the environment and in initiation of attack against prey. This special position is highly suggestive of more active applications of rhabdoids than the mere production of mucus. Therefore, the role of rhabdoids warrants further research. C. Endosymbiosis Green algae occur in Phaenocora, Dalyellia, Typhloplana, Castrada, and some other microturbellarians. The nature of the association between the algae and turbellarians is not fully understood. However, it could be used as a convenient general model for laboratory manipulation. D. Community Ecology Comparative research on the density and richness of turbellarian assemblages in various water types might suggest which factors have relatively greater inﬂuence in structuring ﬂatworm communities. Quantiﬁcation and standardization of sampling procedures may be a major hurdle for comparative ecologists. Different density estimates obtained in quantiﬁcation of lake Turbellaria may be due to minor differences in techniques (e.g., Strayer, 1985). 6. Flatworms: Turbellaria and Nemertea V. COLLECTING, REARING, AND IDENTIFICATION TECHNIQUES A variety of techniques can be used to obtain qualitative and quantitative samples of Turbellaria. These techniques may involve: scooping with a plankton net over aquatic vegetation or sediments; collecting bottom sediments by means of bottom samplers, such as Ekman, Ponar, or multiple corer (Schwank, 1981; Strayer, 1985); ﬁltering water taken from wells, springs, hyporheic interstitial, or ponds or puddles using a plankton net (Kolasa et al., 1987); or band baiting (e.g., Mesostoma, triclads) into traps using pieces of liver or injured aquatic insects (Case and Washino, 1979; Kenk, 1972). In streams, a Surber sampler or similar devices can be applied. Samples should be cooled, topped with water, and transported to the laboratory as quickly as possible. In the laboratory, samples can be transferred to jars or beakers and left for a couple of hours for the water to stagnate. When the water clears, animals swim toward the surface or glide on the glass where they can be picked up with a pipette for further examination. Alternatively, turbellarians can be directly separated from the sediments under a stereomicroscope, but this procedure is more time consuming. Turbellaria can also be removed from mineral substrates having a low organic matter content by gently heating the sample on a hot plate so that the surface of the sediment reaches 30 – 32°C (Kolasa, 1983). Other methods of extracting ﬂatworms from sediments were developed for marine sands (Martens, 1984; Noldt and Wehrenberg, 1984). These methods are based on various combinations of anaesthetizing worms (with MgCl2 or ethanol), stirring water to separate mineral particles, and sieving to catch worms. Some of these methods may be adaptable to freshwater fauna. Many triclads and microturbellarians can be maintained in the laboratory in small glass containers as long as the water is regularly changed and appropriate food provided. Synthetic pond water is best for common species. Temperatures between 17 and 25°C may be adequate for most species, except for cold-water forms. Food requirements vary with the species and variety of easily available aquatic invertebrates must be tested to ensure success. These may include cladocerans, mosquito larvae (for Mesostoma, Dugesia), oligochaetes (for Phaenocora), protozoa, or other small turbellarians (Stenostomum) (e.g., Kolasa, 1987; Yu and Legner 1976). Often, pieces of larger invertebrates, such as Asellus, Gammarus, and Tubifex, can be successfully used. McConnell (1967) provided many useful hints on rearing triclads. Most turbellarians can be identiﬁed by squash mounts of live animals. Identiﬁcation is difﬁcult for a 163 novice. Squash mounts are prepared by placing a live individual in a drop of water under a microscope coverslip. Next, excess water is removed with a ﬁne pipette or a strip of ﬁlter paper until the specimen is immobilized. Pressure on the specimen can be varied by removing or adding water. Such mounts, with a little bit of practice, reveal the arrangement and appearance of internal organs — information usually necessary for species determination. Whenever further study is desirable, animals can be ﬁxed and preserved as in the following steps. 1. Anaesthetizing with 7% ethanol, 0.1% chloretone, 1% hydroxylamine hydrochloride, or slowing their locomotion by placing them in a small volume of water on ice (this step is optional); 2. Killing with hot ﬁxatives, such as Stieve’s, Gilson’s, Bouin’s, room temperature 70% ethanol, or (optional) killing cooled worms with glutaraldehyde (see electron microscopic techniques for details); 3. Rinsing mercury residues of the above ﬁxatives with 50% ethanol solution of iodine (except Bouin’s); 4. Storing specimens in 70% ethanol. Animals so prepared can be sectioned and analyzed with standard histological methods, including staining with Mallory’s stain, Delaﬁeld’s hematoxylin, and other common stains. VI. IDENTIFICATION OF NORTH AMERICAN GENERA OF FRESHWATER TURBELLARIA The key provided uses a combination of both phylogenetic and superﬁcial morphological and anatomical characters. Although the phylogenetically important characters permit greater conﬁdence, they are often very difﬁcult to use by an inexperienced researcher. It is strongly recommended that identiﬁcation be conﬁrmed by comparing the specimen at hand with exact taxonomic descriptions of the species available in other publications. Monographs by Luther (1955, 1960, 1963, for Dalyelliidae, Macrostomidae, Typhloplanidae), Nuttycombe (1956), Nuttycombe and Waters (1938, for Catenula, Stenostomum), Ferguson (1939, for Macrostomum), and Gilbert (1938, for Phaenocora) are particularly useful. A. Taxonomic Key to Orders and Suborders of Turbellaria As some characters may be difficult to determine, it may be advisable to match the general body plan of 164 Jurek Kolasa the individual being identified with one of the pictures in Figure 1A – H. For additional schematics one should consult a guide to world Turbellaria (Cannon, 1986). 1a. Simple female gonad; egg entolecithal (Acoela, Macrostomida, Catenulida; freshwater members of the latter two often reproduce asexually and ovary is absent) (Figs. 1A – C, 12A, B)..........................................................................................................................2 1b. Heterocellular female gonad, with yolk-producing part separate from the oocyte-producing part (all other Turbellaria) (Figs. 1D – H, 2) ...........................................................................................................................................................................................4 2a(1a). Mouth opens directly or through a pharynx simplex into the body; no protonephridia; no distinct intestine; gonads without clear walls (Fig. 1A); rare in freshwater..............................................................................................................................................Acoela [Only one species (unidentiﬁed) collected in North America (from Mirror Lake in New Hampshire; Strayer, 1985).] 2b. Mouth opens to a pharynx simplex; protonephridia present; epithelial, ciliated intestine present.......................................................3 3a(2b). Protonephridia with a single, central excretory duct, often two or more zooids; male gonopore, if present, situated on the dorsal side and in the front of the body; ovary without oviducts and supplementary organs; statocyst present in some species (Figs. 1B, 12A, B) ...................................................................................................................................................Catenulida (see Section VI.B) 3b. Protonephridia with a pair of main excretory ducts; male gonopore on the ventral side of the posterior part of body; female gonopore usually separate and in front of the male one, more than one zooid in the family Microstomidae only; no statocyst (Figs. 1C, 5A, B) ...............................................................................................................................................Macrostomida (see Section VI.C) 4a(1b). Single germovitellarium where ova are surrounded by yolk cell epithelium; pharynx variabilis; copulatory organ armed with a sclerotized stylet near the pharynx (Figs. 1D, 2A)..............................................................................Lecithoepitheliata (see Section VI.D) 4b. Ova not surrounded by yolk epithelium; ovary separate or combined with vitellarium (Figs. 1E – H, 2B, C)......................................5 5a(4b). Pharynx plicatus or variabilis; testicles and ovaries dispersed; germ cells dispersed or aggregated (Fig. 1H)..................Proseriata, Prolecithophora, and Tricladida (see Section VI.E) 5b. Pharynx bulbosus (doliiformis or rosulatus, Fig. 1E – G) ....................................................................................................................6 6a(5b). Pharynx doliiformis, directed forward (Fig. 1E) ...................................................................................Dalyellioida (see Section VI.H) 6b. Pharynx rosulatus directed ventrally or, exceptionally as in Phaenocora, ventrally and forward ........................................................7 7a(6b). Without a proboscis (Fig. 1F) ..........................................................................................................Typhloplanoida (see Section VI.F) 7b. Proboscis present (Fig. 1G) ............................................................................................................Kalyptorhynchia (see Section VI.G) FIGURE 2 Examples of different types of heterocellular female gonads. (A) Prorhynchus, Geocentrophora; (B) Bothrioplana; and (C) Typhloplana, Dalyellia. 6. Flatworms: Turbellaria and Nemertea 165 B. Taxonomic Key to Catenulida 1a. Brain simple, compact, oval ...............................................................................................................................................................2 1b. Brain composed of paired frontal and posterior lobes; a group of sensory cells in front of the brain, often arranged pseudometamerically, i.e., in parallel rows .........................................................................................................................family Stenostomidae 5 2a(1a). Brain situated usually at the base of, but not further than in the middle of prostomium (frontal body section without the intestine); with ciliated ventral or lateroventral furrow separating prostomium from the rest of body ...........................family Catenulidae 3 2b. Brain situated in the ﬁrst 1/3 of prostomium ..............................................................................family Chordariidae 3a(2a). A ring of strongly ciliated longitudinal grooves developed on the prostomium (Fig. 3A) ........................................................Suomina 3b. No such grooves present ....................................................................................................................................................................4 4a(3b). Clearly open intestine extends, approximately, to the middle of the postpharyngeal section of body (Figs. 3B,C, 12A)..........Catenula 4b. Open intestine extends much further, leaving no more than 1/5 to 1/4 of the postpharyngeal section closed; usually the intestinal lumen of individual zooids is connected ...........................................................................................................................Dasyhormus 5a(1b). Adult individuals composed of two or more zooids; gonads often absent...........................................................................................6 5b. Adult individuals (with gonads) without signs of asexual divisions; with long prostomium, ciliated pits absent (Figs. 3D,F, 12B)........ .....................................................................................................................................................................................Rhynchoscolex 6a(5). A section of the intestine near the pharynx with a strong ring of muscles (Fig. 4E) ..................................................Myostenostomum 6b. No such structure, intestine uniformly ciliated (Fig. 4A – D)............................................................................................Stenostomum FIGURE 3 Some representatives of Catenulida. (A) Suomina turgida; (B) Catenula lemnae; (C) Catenula leptocephala; and (D) and (E) Rhynchoscolex simplex. Length of species varies between 0.35 and 5.00 mm. B, Brain; f, furrow; i, intestine; m, mouth; n, protonephridial duct; o, ovary; ph, pharynx; and st, statocyst. Chordarium 166 Jurek Kolasa FIGURE 4 Some representatives of Catenulida. (A) Stenostomum leucops; (B) S. beauchampi; (C) S. glandulosum; (D) S. brevipharyngium; and (E) Myostenostomum tauricum. B, Brain; cp, ciliated sensory pits; g, muscular ring of intestine or gizzard; i, intestine; lo, light-refracting organs; m, mouth; n, protonephridial duct; pg, pharyngeal glands; ph, pharynx; and tc, tactile cilia. C. Taxonomic Key to Macrostomida 1a. Ciliated pits present; intestine extends in front of mouth; numerous zooids separated by division planes (Fig. 5B,C) family Microstomidae .............................................................................................................................................................................Microstomum 1b. No ciliated pits; intestine entirely behind the pharynx; no signs of asexual division (Fig. 5A, D, E) ..................family Macrostomidae Macrostomum D. Taxonomic Key to Lecithoepitheliata 1a. Stylet of the copulatory organ straight; no eyes ................................................................................................................Prorhynchus 1b. Stylet of the copulatory organ curved; eyes present except in some cave forms ...........................................................Geocentrophora E. Taxonomic Key to Prolecithophora, Proseriata, and Tricladida 1a. Mouth situated in front and directed forward ...................................................................................................................Hydrolimax 1b. Mouth situated elsewhere and directed backward or ventrally ...........................................................................................................2 2a(1b). Mouth situated near the middle and directed ventrally; statocysts present....................................................................Otomesostoma 2b. Mouth directed backward; no statocyst .............................................................................................................................................3 6. Flatworms: Turbellaria and Nemertea 167 FIGURE 5 Representatives of Macrostomida and copulatory organs of various families. (A) Macrostomum tuba; (B) Microstomum lineare; (C) copulatory organ of M. lineare; (D) stylet of Macrostomum gilberti; (E) stylet of an unidentiﬁed Macrostomum from New Haven, CT; (F) sclerotized copulatory apparatus of Microdalyellia rossi (Dalyelliidae); (G) sclerotized copulatory apparatus of M. tennesseensis; and (H) copulatory organ of Opistomum pallidum containing a spiny reversible cirrus (Typhloplanidae). Ci, Cirrus; co, copulatory organ; cp, ciliated pits; e, eye; i, intestine; m, mouth; o, ovary; ov, egg; ph, pharynx; ps, prostatic secretions; s, sperm; t, testes; and vs, seminal vesicle. 3a(2b). Intestine split into two branches behind the pharynx (Figs. 6 – 7) ........................................................................................Tricladida1 3b. Intestine not composed of three branches (Fig. 8A) 4a(3a). Internal pharyngeal muscles in two distinct layers (Planariidae) (Fig. 6) .............................................................................................5 4b. Internal pharyngeal muscles in one layer of mixed longitudinal and circular ﬁbers (Dendrocoelidae) (Fig. 6)...................................12 5a(4a). Oviducts, separate or united, open into end part of the bursa stalk ....................................................................................................6 5b. Oviducts united, the common oviduct opens in the genital atrium (Fig. 6) .........................................................................................7 6a(5a). Testes extend to the level of pharynx ............................................................................................................................................Cura 6b. Testes extend to the posterior end of body...............................................................................................................................Dugesia 7a(5b). Numerous eyes arranged in a band around the anterior end of body (Fig. 7G) .......................................................................Polycelis 7b. Eyes absent or one pair only; if numerous, then not along the head margin .......................................................................................8 8a(7b). Adenodactyl present (Fig. 6)....................................................................................................................................................Planaria 8b. Adenodactyl absent ............................................................................................................................................................................9 9a(8b). Anterior end with an adhesive organ ................................................................................................................................................10 9b. Anterior end without an adhesive organ...........................................................................................................................................11 10a(9a). Body elongated, ﬂat, with a well-developed postpharyngeal section .................................................................................Sphalloplana 1 Substantial parts of the key to Tricladida follow Kenk (1972). Separation of Hymanella from Phagocata is based on Ball et al. (1981). 168 Jurek Kolasa FIGURE 6 Schematic representation of the major anatomic features used in taxonomy of Tricladida. Ad, Adenodactyl; b, bursa; bd, bursal duct; ca, common genital atrium; im, internal muscle layer of the pharynx (two possible types shown); m, mouth; ma, male atrium; odc, common oviduct; ph, pharynx; pb, penis bulb; pp, penis papilla; sd, sperm duct; v, vagina; and vs, seminal vesicle. 10b. Body turtle-shaped, with reduced postpharyngeal section .........................................................................................................Kenkia 11a(9b). Prepharyngeal, fused testes in male phase; large atrium lined by papillose epithelium in female phase; head round and broad; the anterior ramus of the intestine does not reach beyond eyes .................................................................................................Hymanella 11b. Other set of characters .........................................................................................................................................................Phagocata 12a(4b). Anterior end of the body with a deeply invaginated adhesive organ .................................................................................Macrocotyla 12b. Adhesive organ absent, or an adhesive disk present..........................................................................................................................13 FIGURE 7 Some common representatives of Tricladida. (A) Hymanella retenuova; (B) Phagocata velata; (C) Dugesia tigrina; (D) D. dorotocephala; (E) D. polychroa; (F) Procotyla ﬂuviatilis; and (G) Polycelis coronata (A and B modiﬁed from Ball et al., 1981). 6. Flatworms: Turbellaria and Nemertea 169 FIGURE 8 Representatives of (A) Proseriata (Bothrioplana semperi); (B) Typhloplanoida (Mesosomta craci); and (C – F) Kalyptohynchia; (C) Gyratrix hermaphroditus and (D) its stylet; (E) Opisthocystis goettei and (F) its copulatory organs. Ar, Adenal rhabdoids; b, brain; co, copulatory organ; cp, ciliated pits; dr, dermal rhabdoids; e, eye; ev, excretory vesicle; i, intestine; n, protonephridial duct; o, ovary; pg, pharyngeal glands; ph, pharynx; pr, proboscis; s, stylet; t, testes; vg, granular vesicle; vs, seminal vesicle; and y, yolk glands. 13a(12b). Penis papilla present .........................................................................................................................................................................14 13b. Penis papilla absent, bulb large........................................................................................................................................Rectocephala 14a(13a). Penis bulb rounded, containing a seminal vesicle........................................................................................................Dendrocoelopsis 14b. Penis bulb elongated, contains a prostatic vesicle ..................................................................................................................Procotyla F. Taxonomic Key to Typhloplanoida The basic plan of the freshwater typhloplanoid reproductive system is given in Fig. 9. 1a. Yolk glands paired, situated on both sides of the intestine..................................................................................................................2 1b. Yolk glands developed as single strand of cells above the intestine (Fig. 10E, F)...............................................................Limnoruanis 2a(1a). Pharynx developed as a typical, round or slightly elongated pharynx rosulatus and oriented ventrally or somewhat forward ...........3 2b. Pharynx elongated or cylindrical, oriented backward; copulatory organ with a spiny cirrus (Fig. 5H) ..............................Opistomum 3a(2a). Testes under yolk glands.....................................................................................................................................................................4 3b. Testes above yolk glands ..................................................................................................................................................................11 4a(3a). Protonephridial ducts open separately on the body surface (Protoplanellinae) (as in Fig. 10C) ..........................................................5 4b. Protonephridial ducts combined with mouth or gonopore (as in Fig. 10A) ........................................................................................7 5a(4a). Front extremity with a central depression and set slightly apart by two lateral, ciliated pits; pharynx in the ﬁrst third of body (Fig. 10C) Prorhynchella 5b. Front extremity not set apart, rounded; pharynx in the second to third portion of body ....................................................................6 6a(5b). Reproductive complex with a bursa copulatrix; ductus ejaculatorius usually surrounded by gelatinous matrix (Fig. 11E, F; cf., Fig. 9) ...............................................................................................................................................................................Krumbachia 6b. Bursa copulatrix absent ...................................................................................................................................................Amphibolella 7a(4b). Protonephridial ducts joined and combined with mouth; no retractable proboscis (Typhloplaninae) .................................................8 170 Jurek Kolasa FIGURE 9 A schematic representation of the main reproductive organs and structures of freshwater Typhloplanoida. In some species, additional structures may be present or the relative arrangement among the organs may be slightly different. 7b. Protonephridial ducts open into the gonopore; retractable front extremity (Fig. 10D) ..........................................Rhynchomesostoma 8a(7a). With a bursa copulatrix and an atrium copulatorium (Fig. 11D)............................................................................................Castrada 8b. One of the above organs missing ........................................................................................................................................................9 9a(8b). With eyes (Figs. 10A, 12C) ..........................................................................................................................................Strongylostoma 9b. No eyes ............................................................................................................................................................................................10 10a(9b). Atrium copulatorium absent; bursa copulatrix present..................................................................................................Typhloplanella 10b. Both bursa copulatrix and atrium copulatorium absent; always with green algae; pharynx in the ﬁrst half of body; copulatory organ pear-shaped.......................................................................................................................................................................Typhloplana 11a(3b). Pharynx developed as a typical, round pharynx rosulatus ................................................................................................................13 11b. Pharynx barrel-shaped, directed clearly forward, and situated near the front extremity (Fig. 11B)...................................................12 12a(11b). Ductus copulatorius absent; inferior genital atrium inconspicuous (Fig. 11B) ....................................................................Phaenocora 12b. With ductus copulatorius; exceptionally large inferior atrium; occurs in sulfuric springs ........................................Pseudophaenocora 13a(11a). An elongated supplementary organ, the ascus, present in the proximity of the gonopore; or with a well-deﬁned gland complex in its place (Ascophorinae) (cf. Fig. 9) .......................................................................................................................................................14 13b. Ascus absent.....................................................................................................................................................................................16 14a(13a). Pharynx in the middle of the ﬁrst half of body ....................................................................................................................Protoascus 14b. Pharynx approximately in the middle of body or slightly behind .....................................................................................................15 15a(14b). Gonopore near the middle of body ......................................................................................................................................Ascophora 6. Flatworms: Turbellaria and Nemertea FIGURE 10 Some representatives of Typhloplanoida. (A) Strongylostoma simplex; (B) copulatory organ with testes of S. simplex; (C) Prorhynchella minuta; (D) a juvenile of Rhynchomesostoma rostratum; (E) Limnoruanis romanae; and (F) sclerotized part of the copulatory organ, partly reverted (upper picture), and the copulatory organ with the gonopore and glands (lower picture) in L. romanae (C modiﬁed from Rusebush, 1939). Ar, Adenal rhabdoids; e, eye; g, glands; i, intestine; n, protonephridial duct; o, ovary; pg, pharyngeal glands; ph, pharynx; s, stylet,; sph, sphincter; t, testes; vg, granular vesicle; vs, seminal vesicle; and y, yolk glands. FIGURE 11 Representatives of the Dalyellioida (Castrella) and Typhloplanida (others); (A) Castrella pinguis; (B) Phaenocora sp. (from Churchill, Manitoba); (C) Mesostoma vernale (the habitus of this species is also characteristic of various Bothromestoma); (D) Castrada virginiana; (E) Krumbachia hiemalis; and (F) copulatory complex of Krumbachia hiemalis. ar, Adenal rhabdoids; bc, copulatory bursa; bs, blind sac of the atrium; co, copulatory organ; de, ductus ejaculatorius; gp, gonopore; i, intestine; m, matrix; n, protonephridial duct; o, ovary; ov, egg capsule; ph, pharynx; r, seminal receptacle; s, stylet; t, testes; vs, seminal vesicle; and y, yolk glands. 171 172 Jurek Kolasa FIGURE 12 Microphotographs of live Turbellaria and Nemertea. (A) Several partly framed individuals of Catenula sp. showing chains of zooids; (B) frontal portion of body of Rhynchoscolex sp.; (C) Strongylostoma sp., a representative of Typhloplanoida; (D) squash preparation of the portions of the reproductive system of Mesostoma craci, with the copulatory organ in the middle; and (E) a frontal portion of body of a nemertean Prostoma sp. B, Brain; BC, bursa copulatrix; CD, common duct; ED, ejaculatory duct; prostatic granulations are visible in its proximity; I, intestine; M, mouth; N, protonephridium; O, ovary; PH, pharynx; R, rhynchocoel; S, sensory pit; ST, statocyst; T, testicle; Y, yolk glands; I, ﬁrst zooid; and II, second zooid. 6. Flatworms: Turbellaria and Nemertea 173 15b. Gonopore in the third posterior part of body ...............................................................................................................Dochmiotrema 16a(13b). Protonephridial ducts open separately on the body surface; eyes often present..................................................................Olisthanella 16b. Protonephridial ducts open into mouth ............................................................................................................................................17 17a(16b). With a ventral pit and a canal connecting the bursa copulatrix to the ovovitellin duct; often ﬂat on the ventral side and strongly convex on the dorsal ................................................................................................................................................Bothromesostoma 17b. Without the above characters (Figs. 11C, 8B, 12D) ............................................................................................................Mesostoma G. Taxonomic Key to Kalyptorhynchia and Similar Forms 1a. Excretory vesicle absent; protonephridial ducts open directly at the surface of body; single ovary (Fig. 8C).......................................2 1b. Protonephridial ducts open into an excretory vesicle in the rear end of body; paired ovary (Fig. 8E, F).........................Opisthocystis2 2a(1a). Copulatory organ with an almost straight stylet (Fig. 8C, D) .................................................................................................Gyratrix 2b. Copulatory organ with a curved stylet.............................................................................................................Microkalyptorhynchus3 H. Taxonomic Key to Dalyellioida 1a. With a paired ovary; copulatory organ with a straight, funnel-like tube..............................................................................Pilgramilla 1b. With a single ovary; copulatory organ with a more of less complex sclerotized structure (e.g., Figs. 5F, G, 11A) ...............................2 2a(1b). Sclerotized structure directly attached to the rest of the copulatory organ ..........................................................................................3 2b. Sclerotized structure in a separate pocket adjacent to the bulb of the copulatory organ (Fig. 11A).........................................Castrella 3a(2a). Sclerotized structure typically with two handles and two lateral branches carrying spines, auxiliary parts may be present (Fig. 5F, G) ...........................................................................................................................................................................................................4 3b. Sclerotized structure different; usually in the form of a spiny ring ......................................................................................Gieysztoria 4a(3a). One to several eggs in the parenchyme; normally, older individuals green due to symbiotic zoochlorellae..............................Dalyellia 4b. Never more than one egg; no zoochlorellae ...................................................................................................................Microdalyellia NEMERTEA VII. GENERAL CHARACTERISTICS, EXTERNAL AND INTERNAL ANATOMICAL FEATURES Nemerteans are coelomate (Turbeville and Ruppert, 1985) and unsegmented worms possessing an eversible muscular proboscis resting in the rhynchocoel, a walled, longitudinal dorsal cavity connected with the mouth (Fig. 13 and 14, see also Figure 12E in Section VI.F.4 Unlike the Turbellaria, Nemertea have an anus as well as a closed-blood circulatory system. A monolayered, ciliated epidermis and several layers of muscles enclose the body. The excretory system is protonephridial. Sensory structures include eyes, statocysts 2 An unidentiﬁed kalyptorhynchid occurring in a tributary of the Hudson River (Kolasa et al., 1987) also meets this criterion, although it may belong to a family other than that containing Opisthocystis. Klattia is a synonym of Opisthocystis (Karling T.G. 1956. Ark. Zool. 9:187 – 279). 3 Taxonomic placement within Kalyptorhynchia is provisional. Name (absent in freshwater nemerteans), and cerebral, frontal, and lateral neural organs. The reproductive system is much simpler than in Turbellaria. Freshwater nemerteans are hermaphroditic and often protandric. Spermatozoa and ova are produced in numerous gonads situated in diverticulae of the intestine. As a rule, fertilization is external and self-fertilization can also occur. Development is direct. Most information provided in this section on nemerteans is based on an excellent review of freshwater and terrestrial nemerteans by Moore and Gibson (1985). That review will be an invaluable source of data and references for anyone working on the ecology and biology of freshwater nemerteans. discussed by W.E. Hazen (1953. Morphology, taxonomy, and distribution of Michigan rhabdocoeles. Ph.D. Thesis, Univ. of Michigan, Ann Arbor. 122 pp.). 4 Until 1985, Nemertea were considered to be acoelomate. With the new ﬁnding (Turbeville and Ruppert, 1985), they can no longer be associated with other ﬂatworms. Our presentation here is more for convenience than systematic reasons. 174 Jurek Kolasa FIGURE 14 Sections through the anterior extremity of three North American nemerteans. (A) Prostoma eilhardi; (B) P. graecense; (C) P. canadiensis (after Gibson and Moore, 1976, 1978). bw, Body wall musculature; cg, cephalic gland lobular region; dc, dorsal cerebral commissure; ep, epidermis; fo, frontal organ; id, improvised ducts from cephalic gland lobules; oe, oesophagus; pr, proboscis; rc, rhynchocoel; rd, rhynchodaeum; rl, rhychodaeal longitudinal muscle ﬁbers; st, stomach; and vc, ventral cerebral commisure. VIII. ECOLOGY A. Life History and General Ecology FIGURE 13 A semi-schematic view of Prostoma sp. b, Brain; e, eye; fo, frontal organ; g, gonad; i, intestine; ov, egg; s, stylet. Twelve species of freshwater nemerteans are classiﬁed into two, possibly polyphyletic families and six genera (Moore and Gibson, 1985). In the family Heteronemertea, three monotypic genera are recognized: Planolineus (Beauchamp, 1928), Siolineus (du BoisReymond Marcus, 1948), and Apartronemertes (Wilfert and Gibson, 1974). In the family Hoplonemertea, the genus Prostoma (Duges, 1828) contains most of the species. Two other genera, Campbellonemertes (Moore and Gibson, 1972) and Potamonemertes (Moore and Gibson, 1973) are known from islands in the southern Paciﬁc, Campbell Island and South Island, New Zealand, respectively. Most nemerteans are marine, although a small minority are terrestrial or freshwater. The freshwater forms are benthic, predatory worms 10 – 40 mm in length and of pink coloration. Prostoma feed readily on oligochaete worms and, occasionally, on crustaceans, nematodes, turbellarians, midge larvae, and other small invertebrates (McDermott and Roe, 1985). Feeding activity is most intense at night and the sticky proboscis captures prey. Little is known about habitat selection by freshwater nemerteans. North American Prostoma species have been found in both streams and lakes, including Lake Huron. In lakes, Prostoma species appear to be associated with ﬁlamentous algae. Even though some nemerteans have strong regenerative capabilities, reproduction is exclusively sexual, with both male and female organs present at the same time. Since only limited quantitative studies have been conducted on the biology of freshwater nemerteans in 6. Flatworms: Turbellaria and Nemertea the ﬁeld, mechanisms of population regulation remain unknown. Clearly, Prostoma can reproduce rapidly with up to 210 eggs per reproductive episode throughout the year as long as the temperature is above 10°C (Young and Gibson, 1975). A study of one population in England suggests that Prostoma population density increases during the cold months of the year and declines during the summer, with smaller individuals recorded during the winter (Gibson and Young, 1976). It is not clear, however, if this reproduction pattern is general or due to cattle trampling of the shore vegetation where the study was conducted. Distribution of nemerteans in North America is not well known. (Strayer, 1985) reported Prostoma rubrum from Mirror Lakes, New Hampshire. However, P. rubrum is no longer a valid species (Gibson and Moore, 1976), and this observation has to be treated as Prostoma sp. Prostoma canadiensis is known from one site in Lake Huron (Gibson and Moore, 1978), where is occurs to a depth of 20 m. Prostoma species are probably much more common in freshwaters than the infrequent records from eastern North American might indicate. A Prostoma species (P. rubra) was also reported in vegetation of slow coastal streams, marshes, and estuarine pools in St. Vincent, Caribbean (Harrison and Rankin, 1976). So far, no Prostoma have been found in temporary water bodies. Three established species of nemerteans are known from North America (Fig. 14); a fourth, P. asensoriatum, is a questionable species. Distribution of these species in North America is summarized in Figure 15, but they are probably present in most standing and running waters. With the exception of the dubious P. asensoriatum, all three remaining species have been 175 recorded in other parts of the world, including South America, Europe, Africa, and Australia. Zoologists do not know whether the worldwide pattern of distribution is natural or, instead, a result of recent humanmediated dispersal. Analysis of genetic diversity of various populations might shed some light on this question. B. Physiological Adaptations Osmoregulation is an ecologically important function in nemerteans as in all other freshwater invertebrates with permeable body walls. It is controlled by the cerebral organs and involves several organs and enzymatic systems associated with blood vessels (Moore and Gibson, 1985). Well-developed nephridia may play a role in osmoregulation as well as in the removal of nitrogenous wastes (but the latter has not yet been demonstrated). Gases, particularly oxygen, are exchanged across the surface of the body. The role of blood vessels in oxygen transport is unclear. Nemerteans, like turbellarians, release copious mucus whose various functions probably include defense, locomotion, physiological barrier, and encystment. C. Behavioral Ecology There are no studies devoted exclusively to the behavior of freshwater nemerteans. Nevertheless, some observations on feeding behavior, escape reactions, and locomotion are available. Adult nemerteans can only crawl, while small juveniles also swim. Forward locomotion of Prostoma may involve ciliary movement alone, ciliary movement in combination with muscular waves, sinusoidal curves when the animal pushes through the vegetation, or peristalsis when the animal moves backward. The proboscis provides the fastest movement forward. This long organ is rapidly everted and attached to substrate, and the body is then pulled toward the point of attachment. Typically, prey are captured by a sticky proboscis and then swallowed whole by the widely distended mouth (McDermott and Roe, 1985). The mechanisms of prey detection are poorly understood. In terrestrial nemerteans, these mechanisms are unusual in that they involve ambushing strategy and reliance on mechanical stimuli. D. Functional Role in the Ecosystem FIGURE 15 Known distribution of Prostoma species in North America (after Gibson, 1985; modiﬁed and updated). C, P. canadiensis; E, P. eilhardi; A, P. asensoriatum; G, P. graecense; and U, unidentiﬁed Prostoma. Necessarily, the functional importance of nemerteans in the ecosystem can only be indirectly inferred from their trophic position and relative densities. I have found densities of Prostoma species in Wappinger Creek, a tributary of the Hudson River, to vary 176 Jurek Kolasa between 50 and 590 individuals / m2, which constitutes a small fraction of all predatory invertebrates identiﬁed at the study site. Similarly, low densities of P. canadiensis (up to 140 individuals / m2) were observed on gravelly and muddy substrates in Lake Huron. IX. CURRENT AND FUTURE RESEARCH PROBLEMS In view of the limited knowledge of nemertean ecology, almost any area of research will provide valuable information. New information on phenology, population dynamics, and habitat selectivity would permit a better evaluation of the role of nemerteans in freshwater communities. Speciﬁc studies in the biology of nemerteans appear particularly promising in the areas of reproductive biology and evolutionary ecology. The ecological role of femaleness later in the life cycle, the production of resting eggs, and the environmental versus genetic cues of life stages all may offer exciting and general models for evolutionary ecology. X. COLLECTION, CULTURING, AND PRESERVATION Collection of nemerteans is similar to collection of any soft-bodied, benthic invertebrates such as turbellari- ans or oligochaetes living in the substrate or on aquatic plants. Sieving and vigorous washing may damage specimens. Placing samples in beakers or jars and topping with water allows nemerteans to crawl toward the edges of water where they can be collected with a pipette. Freshwater nemerteans are relatively easy to culture. They may be maintained and will reproduce in aquaria or even in petri dishes in clean, oxygenated water and on a diet of live aquatic invertebrates. Chopped tubiﬁcid oligochaetes provide a good diet. Worms can be preserved in 80% ethanol. Before preservation, however, worms should be narcotized using 7% ethanol or chloretone until their movement ceases. According to Moore and Gibson (1985), gin can be used in the ﬁeld if pure ethanol is unavailable, but only as a last resort! XI. TAXONOMIC KEY TO SPECIES OF FRESHWATER NEMERTEA IN NORTH AMERICA It should be noted that Gibson and Moore (1976, 1978) provided keys to freshwater nemerteans of the world. Those keys are more suitable if you encounter a species that has not yet been recorded in North America. The present key is an adaptation of the keys by Gibson and Moore (1976, 1978) pertinent to established North American Prostoma. 1a. Cephalic glands open via frontal organs and by improvised ducts (Fig. 14C); esophagus distinct but unciliated; rhynchodaeum with isolated longitudinal muscle strands; proboscis with twelve nerves. P. canadiensis (Gibson and Moore, 1978) 1b. Cephalic glands open only via frontal organ, no improvised ducts .....................................................................................................2 2a(1b). With a distinctive ciliated esophagus; cephalic glands reach back to the brain (Fig. 14B); rhynchodaeum with a well-developed layer of longitudinal muscle ..............................................................................................................................P. graecense (Bohmig, 1892) 2b. Different combination of characters; indistinct and unciliated esophagus (Fig. 14A); rhynchodaeum without speciﬁcally associated layer of longitudinal muscle ﬁbers; proboscis with 9 – 10 nerves..........................................................P. eilhardi (Montgomery, 1894) ACKNOWLEDGMENTS I am very grateful to L.R.G. Cannon, J. Moore, R. Gibson, A.P. Mead, R. Kent, S. Schwartz, D. Strayer, and S. Tyler for helpful comments on the manuscript, additional references, and permission to reproduce ﬁgures, glossary entries, and fragments of keys. LITERATURE CITED Ball, I. R. 1974. A contribution to the phylogeny and biogeography of the freshwater triclads (Platyhelminthes: Turbellaria), in: Riser, N.W., Morse, M.P. Eds., Biology of the Turbellaria. McGraw-Hill, New York, pp. 339 – 401. Bauchhenss, J. 1971. Die Kleinturbellarien Frankens. Ein Beitrag zur Systematik und Ökologie der Turbellaria excl. Tricladida in Süddeutschland. 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Regulation of aquatic diptera by planaria. Entomophaga 21:3 – 12. 6. Flatworms: Turbellaria and Nemertea APPENDIX 6.1 List of North American Species of Microturbellaria The arrangement of orders and families follows with some modiﬁcations from that of Lanfranchi and Papi (1978). Catenulida Family Catenulidae Catenula confusa Catenula lemnae Catenula leptocephala Catenula sekerai Catenula virginia Suomina turgida Family Chordariidae Chordarium europaeum Family Stenostomidae Myostenostomum tauricum Rhynchoscolex platypus Rhynchoscolex simplex Rhynchoscolex sp. 1 Rhynchoscolex sp. 2 Stenostomum anatirostrum Stenostomum anops Stenostomum arevaloi Stenostomum beauchampi Stenostomum brevipharyngium Stenostomum ciliatum Stenostomum cryptops Stenostomum glandulosum Stenostomum grande Stenostomum kepneri Stenostomum leucops Stenostomum mandibulatum Stenostomum membranosum Stenostomum occultum Stenostomum pegephilum Stenostomum predatorium Stenostomum pseudoacetabulum Stenostomum simplex Stenostomum temporaneum Stenostomum tuberculosum Stenostomum unicolor Stenostomum uronephrium Stenostomum ventronephrium Stenostomum virginianum (probably synonymous with S. unicolor) Macrostomida Family Macrostomidae Macrostomum collistylum Macrostomum curvistylum Macrostomum gilberti Macrostomum glochostylum Macrostomum lewisi Macrostomum norfolkense Macrostomum ontarioense Macrostomum orthostylum Macrostomum phillipsi Macrostomum reynoldsi Macrostomum riedeli Macrostomum ruebushi Macrostomum sensitivum Macrostomum sillimani Macrostomum sp. 1, indeterm. Macrostomum sp. 2, indeterm. Macrostomum sp. 3, indeterm. Macrostomum stirewalti Macrostomum tenneseense Macrostomum tuba Macrostomum lineare Macrostomum virginianum Family Microstomidae Microstomum lineare Lecithoepitheliata Family Prorhynchidae Geocentrophora cavernicola Geocentrophora sphyrocephala Prorhynchus stagnalis Proseriata Family Plagiostomidae Hydrolimax grisea Prolecithophora Family Bothrioplanidae Bothrioplana semperi Family Otomesostomidae Otomesostoma auditivum Dalyellioida Family Provorticidae Genus indet. species indet Pilgramilla virginiensis Family Dalyelliidae Castrella pinguis Castrella grafﬁ Dalyellia viridis Microdalyella abursalis Microdalyella circobursalis Microdalyella fairchildi Microdalyella gilesi Microdalyella rheesi Microdalyella rochesteriana Microdalyella rossi Microdalyella ruebushi Microdalyella sillimani Microdalyella tennesseensis Microdalyella virginiana Typhloplanida Family Typhloplanidae Amphibolella spinulosa 179 180 Jurek Kolasa Ascophora elegantissima Bothromesostoma personatum Castrada hofmanni Castrada lutheri Castrada virginiana Castrada sp. indet Krumbachia cf. hiemalis Krumbachia minuta Krumbachia virginiana Limnoruanis romanae Mesostoma arctica Mesostoma californicum Mesostoma columbianum Mesostoma craci Mesostoma curvipenis Mesostoma ehrenbergii Mesostoma macroprostatum Mesostoma platygastricum Mesostoma vernale Mesostoma virginianum Olisthanella truncula Opistomum pallidum Phaenocora agassizi Phaenocora falciodenticulata Phaenocora highlandense Phaenocora kepneri Phaenocora lutheri Phaenocora virginiana Prorhynchella minuta Protoascus wisconsinensis Pseudophaenocora sulﬂophila Rhynchomesostoma rostratum Strongylostoma cf., elongatum Strongylostoma radiatum Typhloplanella halleziana Kalyptorhynchia Family Polycystidae Gyratrix hermaphroditus Opistocystis goettei Family undetermined Microrhynchus virginianus 7 GASTROTRICHA David Strayer William D. Hummon Institute of Ecosystem Studies Millbrook, New York 12545 Department of Biological Sciences Ohio University Athens, Ohio 45701 I. Introduction II. Anatomy and Physiology A. External Morphology B. Organ System Function III. Ecology and Evolution A. Diversity and Distribution B. Reproduction and Life History C. Ecological Interactions D. Evolutionary Relationships IV. Collecting, Rearing, and Preparation for Identiﬁcation V. Taxonomic Key Literature Cited I. INTRODUCTION The gastrotrichs are among the most abundant and poorly known of the freshwater invertebrates. Gastrotrichs are nearly ubiquitous in the benthos and periphyton of freshwater habitats, with densities typically in the range of 100,000 – 1,000,000 individuals/m2. Nonetheless, the remarkable life cycle of freshwater gastrotrichs was worked out only recently, and we know almost nothing about how the distribution and abundance of these animals are controlled in nature. The impact of freshwater gastrotrichs on their food resources and on freshwater ecosystems has not yet been investigated. Twelve genera and fewer than 100 species of freshwater gastrotrichs are now known from North America. Because the North American gastrotrich fauna has received so little study, these numbers understate the real diversity of the fauna. Formerly placed in the Aschelminthes or Nemathelminthes, gastrotrichs usually are now considered to constitute a phylum of their own. The evolutionary placement of gastrotrichs is unclear; some authors (e.g., Lorenzen, 1985; Wallace et al., 1996) believe that they are most closely related to nematodes, kinorhynchs, loriciferans, nematomorphs, and priaulids, while others (e.g., Conway Morris, 1993) think that gastrotrichs are more closely related to rotifers and acanthocephalans. Important general references on Ecology and Classiﬁcation of North American Freshwater Invertebrates. 2nd Edition Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved. gastrotrichs include Remane (1935 – 1936), Hyman (1951), Voigt (1958), d’Hondt (1971a), Hummon (1982), Ruppert (1988), Schwank (1990), and Kisielewski (1991, 1998). The phylum contains two orders: the Macrodasyida, which consists almost entirely of marine species, and the Chaetonotida, which contains marine, freshwater, and semiterrestrial species. Unless noted otherwise, the information included in this chapter refers to freshwater members of the Chaetonotida. Macrodasyids usually are distinguished from chaetonotids by the presence of pharyngeal pores and more numerous adhesive tubules (Fig. 1). Macrodasyids are common in marine and estuarine sands, but are barely represented in inland freshwaters. Two species of freshwater gastrotrichs have been placed in the Macrodasyida. Ruttner-Kolisko (1955) described an aberrant gastrotrich, Marinellina ﬂagellata (Fig. 1A), from the hyporheic zone of an Austrian river. Unfortunately, she was able to ﬁnd only two specimens, both of them apparently immature. Because Marinellina has a pair of anterior lateral structures that Ruttner-Kolisko interpreted as adhesive tubules, she placed this species in the Macrodasyida. Remane (1961) rejected the assignment of Marinellina to the macrodasyids and placed it instead in the chaetonotid family Dichaeturidae. Kisielewski (1987) reafﬁrmed Ruttner-Kolisko’s 181 182 David Strayer and William D. Hummon A. External Morphology Gastrotrichs are colorless animals, spindle- or tenpin-shaped, and ventrally ﬂattened (Fig. 2). Freshwater gastrotrichs are 50 – 800 m long. Conspicuous external features include a more or less distinct head, which bears sensory cilia, and a cuticle which, in most species, is ornamented with spines or scales of various shapes. In the most common freshwater family, the Chaetonotidae (as well as in the rare Dichaeturidae and Proichthydiidae), the posterior end of the body is formed into a furca, which contains distal adhesive tubes that allow the animal to attach itself tenaciously to surfaces. In other families, these structures are absent, but the posterior end of the body may bear long spines or sensory bristles. The ventral side of the animal bears longitudinal rows or patches of cilia, which provide the forward-gliding locomotion of the animal. B. Organ System Function FIGURE 1 Freshwater macrodasyid gastrotrichs: (A) Marinellina ﬂagellata, and (B) Redudasys fornerise. AT, adhesive tube; and PP, pharyngeal pore. From Ruttner-Kolisko (1955) and Kisielewski (1987). original placement of the species. An animal similar to Marinellina was just discovered in the hyporheic zone of another Austrian stream (Schmid-Araya and Schmid, 1995); but until it is studied critically, the systematic placement of these enigmatic Austrian gastrotrichs will remain unclear. Kisielewski (1987) discovered an undoubted macrodasyid, Redudasys fornerise (Fig. 1B), from the psammon of a Brazilian reservoir. Additional freshwater macrodasyids probably will be found when appropriate habitats (psammon, hyporheic zone) are explored. The distribution, biology, and evolutionary relationships of any such species will be of great interest. II. Anatomy and Physiology The following brief account of gastrotrich anatomy is summarized chieﬂy from Remane (1935 – 1936), Hyman (1951), Hummon (1982), and Boaden (1985), which should be consulted for greater detail. The digestive system begins with a subterminal mouth, which may be surrounded by a ring of short bristles. Between the mouth and the pharynx lies a cuticular buccal capsule, which is often somewhat protrusible. The muscular pharynx is similar to the nematode pharynx, with a triradiate, Y-shaped lumen. Often, there are anterior and posterior swellings, but these lack the valves characteristic of the pharyngeal bulbs of nematodes. Posterior to the pharynx is an undifferentiated gut, which empties into the anus. The paired reproductive organs lie lateral to the gut in the posterior half of the body. In young animals, large, developing, parthenogenetic eggs are present. As there are no oviducts, the egg is released through a rupture in the ventral body wall. Older animals become hermaphrodites (see below) and bear both sperm sacs and developing sexual (i.e., meiotic) eggs lateral to the gut. An unpaired organ of unknown function, the X-organ, lies posterior to the gonads near the anus. The brain is bilobed, straddling the pharynx. Sensory organs include long cilia and bristles, which presumably are tactile. Balsamo (1980) demonstrated that Lepidodermella squamata is photosensitive, although it does not appear to have distinct multicellular photoreceptors. Gray and Johnson (1970) found evidence of a tactile chemical sense in a marine macrodasyid; it seems probable that freshwater chaetonotids possess similar chemosensory abilities. The excretory system consists of a pair of protonephridia (Brandenburg, 1962) in the anterior mid- 7. Gastrotricha 183 FIGURE 2 Schematic illustration of a typical chaetonotid gastrotrich showing (A) dorsal view, (B) ventral view, (C) posterior end of a hermaphrodite, showing sexual organs, and (D) egg. AT, adhesive tubes; AN, anus; BR, brain; E, egg; F, furca; G, gut; M, mouth; PH, pharynx; PN, protonephridium; SC, sensory cilia; SS, sperm sac; and X, X-organ. Modiﬁed from Remane (1935 – 1936), Voigt (1958), and Kisielewska (1981). body, which empty through pores on the ventral body surface. There is no circulatory or respiratory system, per se. III. ECOLOGY AND EVOLUTION A. Diversity and Distribution Gastrotrichs are widely distributed in freshwaters in surface sediments and among vegetation. Some species of the Neogosseidae and Dasydytidae are good swimmers and are occasionally reported from the plankton of shallow, weedy lakes (e.g., Hutchinson, 1967; Green, 1986; Kisielewski, 1991). However, none of the gastrotrichs has become as truly planktonic as the daphnid cladocerans or ploimate rotifers. Gastrotrichs have been found in a wide range of freshwater and semiterrestrial habitats. They are abundant in most lakes, ponds, and wetlands (Tables I and II). Kisielewski (1981, 1986a) showed that gastrotrich density and species richness are positively correlated with the productivity of the habitat, and several workers have found that gastrotrich density is highest in highly organic sediments (e.g., Strayer, 1985). Apparently, most gastrotrichs live very near the sediment 184 TABLE I David Strayer and William D. Hummon Number of Species of Gastrotrichs Found in Some Freshwater Habitats. Habitat Total Chaetonotus European lakesa 18 12 Mirror Lake, NH Ponds, Polandb 20 – 32 21 8 – 20 10 Peat bogs, Polandc Bog pools, Polandd Phragmites mats, Romania Ponds, Brazile Rivers, Brazile 27 24 28 38 22 Other Chaetonotidae Dasydytidae Neogosseidae Source 5 1 0 12 7 0 3 0 1 16 12 16 9 8 7 2 4 5 0 0 0 Preobrajenskaja, 1926; Balsamo, 1981, 1990, Bertolani and Balsamo, 1989 Strayer, 1985 Nesteruk, 1986; Kisielewski, 1986a Kisielewski, 1981 Kisielewska, 1982 Rudescu, 1968 14 5 12 12 10 4 2 1 Kisielewski, 1991 Kisieleswki, 1991 a Mean of four lakes. Mean of seven ponds. c Mean of four bogs. d Mean of two pools. e Sum of several collecting sites. b surface in lakes (Fig. 3). Gastrotrichs also are abundant in unpolluted streams, where they inhabit sand bars, sometimes in great numbers (Hummon et al., 1978; Hummon, 1987). Apparently, they are scarce in groundwaters other than the hyporheic zone (RenaudMornant, 1986). Their rarity in underground waters is surprising, because many marine gastrotrichs are interstitial in habit (d’Hondt, 1971a; Renaud-Mornant, 1986), and because the small size and bacterial diet of gastrotrichs would seem to preadapt them to the groundwater habitat. Gastrotrichs are among the few animals commonly found in anaerobic environments (Moore, 1939; Cole, 1955; Strayer, 1985), remaining abundant even during TABLE II extended periods (i.e., months) of anoxia. The physiological basis of the anaerobiosis of freshwater gastrotrichs has not yet been studied. It seems likely that some freshwater gastrotrichs possess a sulﬁde detoxiﬁcation mechanism similar to that demonstrated for marine gastrotrichs (Powell et al., 1979, 1980) and freshwater nematodes (Nuss, 1984; Nuss and Trimkowski, 1984) to deal with the elevated concentrations of H 2S that often accompany extended anoxia. Little is known of the factors that control the distribution of individual species of gastrotrichs in freshwater. Arguing largely from analogy with marine work (d’Hondt, 1971a), we might expect factors of primary importance to include the granulometry, stability, Density and Biomass of Gastrotrichs in Some Freshwater Habitats. Site Density (No. / m2) Biomass (mg DM / m2) Source Lake Bikcze, Poland Lake Brzeziczno, Poland Lake Piaseczno, Poland Mirror Lake, NH Lake Suviana, Italy Lake Erie, OH Three small ponds, Poland Mississippi River, MN 1,160,000a 920,000 a 910,000 c 130,000 d 57,000 e 50,000 f 1,600,000 – 2,600,000 130,000 – 230,000g 23a,b 30a,b 23b,c 1d — — 25 – 78b — Nesteruk, 1996 Nesteruk, 1996 Nesteruk, 1996 Strayer, 1985 Madoni, 1989 Evans, 1982 Nesteruk, 1996 Hummon, 1987, and unpublished a Littoral zone. Converted from wet mass by multiplying by 0.15. c Mean of three stations. d Lakewide mean. e Mean of two deepwater stations. f Beaches. g Sand bars. b 7. Gastrotricha FIGURE 3 Vertical distribution of gastrotrichs within the sediments of Lake Brzeziczno, Poland, (dashed line) and Mirror Lake, New Hampshire, (solid line) as a function of depth from the sediment surface. From data of Strayer (1985) and Nesteruk (1991). packing, and organic content of the sediment, the amount of dissolved oxygen, and the density and composition of communities of microbes and predators. Also, culture work suggests that the inorganic chemistry of the water and the presence of anthropogenic contaminants can exert a strong inﬂuence on gastrotrich populations (Hummon, 1974; Faucon and Hummon, 1976; Hummon and Hummon, 1979). Many genera of freshwater gastrotrichs are known to have intercontinental or cosmopolitan distributions. Exceptions include several genera so far known only from Brazil (the macrodasyid Redudasys, the chaetonotids Arenotus and Undula, and the dasydytid Ornamentula); Dichaetura and Marinellina, rare species known only from Europe, and the proichthydiids Proichthydium and Proichthyioides, which have been found only at their type localities in Argentina and Japan, respectively. The geographic distribution of individual species is not well known, because of the primitive state of the species-level taxonomy of freshwater gastrotrichs and the paucity of ﬁeld studies throughout most of the world. Even in North America, only Arkansas (Davis, 1937), Illinois (Robbins, 1965, 1973; Horlick, 1975), northern Indiana (Pfaltzgraff, 1967), Michigan (Brunson, 1947, 1948, 1950), and eastern Ohio (Craft, 1993) have received even cursory 185 surveys of freshwater gastrotrichs. Thus, the 76 species that Schwank (1990) reported from North America probably are a small fraction of North America’s freshwater gastrotrich fauna. Some species have been reported to occur over broad ranges, including in some cases more than one continent (e.g., Kisielewski, 1991). Until further studies are made, such reported intercontinental distributions should be regarded with caution (cf. Frey, 1982; Todaro et al., 1996), especially because “conspeciﬁcs” collected from different continents often exhibit marked morphological differences from one another (e.g., Robbins, 1973, Emberton, 1981). Chaetonotus typically dominates gastrotrich faunas everywhere in freshwater, both in terms of numbers of species and numbers of individuals (Table I). Other genera of the Chaetonotidae are common in all kinds of freshwaters, but are usually less abundant than Chaetonotus. The Dasydytidae and Neogosseidae are less widespread than the Chaetonotidae, usually living in weedy, productive waters, where they may, however, become numerically abundant (e.g., Blinn and Green, 1986; Kisielewski, 1981, 1986a; Nesteruk, 1986). Dasydytids and neogosseids also are more abundant and speciose in the tropics than in temperate waters (Table I; Kisielewski 1991). The very rarely seen Dichaeturidae and Proichthydiidae have been collected from cisterns, underground waters, tree holes, and among moss (Remane, 1935 – 1936, Ruttner-Kolisko, 1955; Sudzuki, 1971). B. Reproduction and Life History Until recently, populations of freshwater gastrotrichs were thought to consist entirely of parthenogenetic females (e.g., Hyman; 1951; Pennak, 1978). However, more detailed recent studies have dispelled this notion, and have revealed a remarkable life cycle among freshwater gastrotrichs (Fig. 4) (Weiss and Levy, 1979; Hummon, 1984a – c, 1986; Levy, 1984). Newly hatched gastrotrichs are relatively large (approximately two-thirds the length of adults — Brunson, 1949) and already contain developing parthenogenetic eggs. These eggs develop rapidly under favorable conditions. At 20°C, the ﬁrst egg may be laid within a day after the mother hatches. Typically, a total of four parthenogenetic eggs is laid over a 4-day period. Apparently, parthenogenesis is apomictic, so that offspring are genetically identical to their mother. There are two kinds of parthenogenetic eggs. The more common kind, tachyblastic eggs, develop immediately and hatch quickly (within a day of being laid, at 20°C). Occasionally, the ﬁnal parthenogenetic egg laid by a female is not a tachyblastic egg, but a resting, or opsiblastic, egg. Opsiblastic eggs are thick-shelled, a 186 David Strayer and William D. Hummon FIGURE 4 Schematic diagram of the proposed generalized life cycle for freshwater chaetonotid gastrotrichs, based predominately on study of Lepidodermella squamata. Dashed lines show hypothetical events which have not yet been demonstrated. From Levy (1984), after ideas presented by Levy and Weiss (1980), with permission of the authors. OPSI, opsiblastic egg; and TACHY, tachyblastic egg. little larger than tachyblastic eggs, and are very resistant to freezing and drying (Brunson, 1949). The factors that induce the production of opsiblastic eggs are not well known, although such eggs are often produced by animals in crowded cultures. Opsiblastic eggs are almost always the ﬁnal egg produced by an animal, even if the total number of eggs is fewer than four. Following the production of parthenogenetic eggs, animals develop into hermaphrodites (Fig. 2C) (Hummon and Hummon, 1983; 1988). During this time, sperm and meiotic sexual eggs are produced, and the X-body grows. These changes occur slowly, over the period of a week after the last parthenogenetic egg is laid. No one has yet observed sperm transfer or fertilization, although Levy and Weiss (Levy and Weiss, 1980; Levy, 1984) reported ﬁnding a third kind of egg (the “plaque-bearing egg”) in cultures of Lepidodermella squamata. They suggested that plaque-bearing eggs may be the product of sexual reproduction. Because the sperms are few in number (32 – 64 per animal) and nonmotile, fertilization probably is internal. Animals reared in isolation do not appear to produce fertilized sexual eggs, so cross-fertilization probably is the rule. The absence of ducts associated with the male or female reproductive system makes it difﬁcult to suggest a mechanism of sperm transfer (Hummon 1986, described one bizarre possibility). Probably the enigmatic X-organ is involved. Much remains to be learned about the postparthenogenetic sexual phase and its importance in nature. The life cycle just described is unique among invertebrates and offers considerable ecological ﬂexibility to gastrotrich populations. The initial parthenogenetic phase allows for explosive population growth under favorable conditions: workers have commonly reported growth rates (r) of 0.1 – 0.5 per day in laboratory cultures (e.g., Hummon, 1974; Faucon and Hummon, 1976; Hummon and Hummon, 1979; Hummon, 1986). Production of parthenogenetic resting eggs (opsiblastic eggs) buffers the population against unfavorable conditions and presumably allows for dispersal among habitats. Finally, the subsequent sexual phase introduces genetic recombination. Sexual reproduction is most likely to occur in populations in which rates of mortality are low enough to allow some gastrotrichs to reach the age required for sexual development. C. Ecological Interactions Gastrotrichs feed on bacteria, algae, protozoans, detritus, and small inorganic particles. Bacteria probably are of primary importance. Bennett (1979) demonstrated that Lepidodermella squamata readily digested bacteria and found that this gastrotrich would not 7. Gastrotricha survive in laboratory cultures in the absence of bacteria. He reported that L. squamata could digest the green alga Chlorella as well, but suggested that algae were of secondary importance in gastrotrich diets. Gray and Johnson (1970) showed that the marine macrodasyid Turbanella hyalina could choose among various strains of natural bacteria, apparently on the basis of a tactile chemical sense. Thus, the quality as well as quantity of bacterial populations was important to the gastrotrichs. Freshwater gastrotrichs are most likely capable of similar ﬁne discrimination among bacteria and other prey. Reported predators of gastrotrichs include heliozoan and sarcodine amoebae, cnidarians, and tanypodine midges (Brunson, 1949; Bovee and Cordell, 1971; Moore, 1979), but many other benthic predators presumably feed on gastrotrichs. Nothing is known about the importance of predation in regulating populations of freshwater gastrotrichs or about the quantitative importance of gastrotrichs as a food item for various predators. We have no direct information on what regulates gastrotrich populations in nature. Gastrotrichs are capable of enormous population growth (10 – 50% per day) in laboratory cultures. If potential growth rates are anywhere near this high in nature, as seems likely in some circumstances, then there must be an equally high counterbalancing mortality, perhaps from predation. There are no detailed studies of the population dynamics of freshwater gastrotrichs in nature. The few quantitative studies of the seasonal dynamics of freshwater gastrotrich populations (Fig. 5; see also Nesteruk, 1986) have shown that population densities are usually (but not always) lowest during the winter. We do not know what drives these sea- FIGURE 5 Seasonal trends in gastrotrich abundance. Left: density (number / cm3) of two species of dasydytids in the surface sediments of a boggy pool (“Complex B”) in Poland: Setopus dubius (䊉) and Dasydytes ornatus (䉱). From data of Kisielewska (1982). Right: density (number / cm2) of all gastrotrichs (䊉), an unidentiﬁed species (probably of Heterolepidoderma) (䊏), and Lepidodermella triloba (䉱) on the gyttja sediments of Mirror Lake, New Hampshire (original). Plotted points are means s.e. (n 16). 187 sonal dynamics, but seasonal changes in water temperature, food supply, and predation pressures are obvious possibilities. Gastrotrichs, along with nematodes and rotifers, are among the most abundant animals in the freshwater benthos, often having densities on the order of 10 – 100 individuals/cm2 ( 100,000 – 1,000,000 individuals/m2) (Table II). However, because there have been no direct measurements of the roles of gastrotrichs in freshwater ecosystems, their importance can only be guessed at. There is only a single, tentative estimate of gastrotrich metabolism in freshwater: Strayer (1985) estimated secondary production and respiration of gastrotrichs in Mirror Lake, New Hampshire, each to be 50 – 100 mg dry mass per square meter per year, which is less than 1% of total production or respiration of the zoobenthic community. This estimate suggests that gastrotrich metabolism, and processes such as nutrient regeneration that are correlated with metabolism, are of minor importance in freshwater ecosystems. It would be imprudent, however, to dismiss gastrotrichs as quantitatively unimportant to ecosystem functioning without actually measuring gastrotrich activities under deﬁned conditions. The extraordinarily high rates of population turnover and potentially highly selective feeding behavior of gastrotrichs suggest that they may exert a considerable inﬂuence on the composition of natural bacterial communities. D. Evolutionary Relationships The phylogenetic relationships among the various aschelminth groups are not yet clear. Several points of morphological similarity between nematodes and gastrotrichs (summarized by Lorenzen, 1985, and Wallace et al., 1996) suggest that these two groups are allied, but other analyses (Conway Morris, 1993; Garey et al., 1996) have placed gastrotrichs closer to rotifers and even platyhelminthes than to nematodes. Within the Gastrotricha, only the broad outlines of phylogeny have been sketched out. Three major groups of gastrotrichs are widely recognized: the order Macrodasyida and the suborders Multitubulata and Paucitubulata of the order Chaetonotida. The Multitubulata (which includes only the marine Neodasys) exhibit many primitive characteristics and are in some ways intermediate between the Macrodasyida and the Paucitubulata (which contains almost all of the freshwater gastrotrichs). Therefore, Boaden (1985) suggested that modern Macrodasyida and Paucitubulata are descended from a Neodasys-like ancestor. Nevertheless, on the basis of digestive tract ciliature and other characters, we believe that the ancestral 188 David Strayer and William D. Hummon gastrotrich was more likely similar to the dactylopodolid macrodasyids. Evolutionary relationships among the families, genera, and species of freshwater gastrotrichs are still largely unknown because of a paucity of basic morphological, biochemical, and zoogeographical information about most species. An enormous number of species have not even been described, let alone studied. Even in North America, perhaps 75 – 90% of the species of freshwater gastrotrichs are undescribed. The correct systematic placement of some genera (e.g., Dichaetura, Marinellina) will require much additional study. Furthermore, there is growing concern (e.g., Remane, 1935 – 1936; Ruppert, 1977; Kisielewski, 1981, 1987, 1991, 1997; Schwank, 1990) that some of the traditionally deﬁned genera of the Chaetonotidae may be inadequate. Better morphological information and the discovery of species having characters intermediate between genera is leading to a blurring of distinctions between genera such as Chaetonotus, Heterolepidoderma, and Lepidodermella. Further, it is becoming increasingly clear that some of the characters traditionally used to deﬁne gastrotrich genera (e.g., adhesive tubes, cuticle ornamentation) are subject to convergent evolution (e.g., Kisielewski, 1991; Balsamo and Fregni, 1995), and so might not reﬂect common lines of descent. Finally, it seems likely that Ichthydium, which is deﬁned by an absence of cuticular ornamentation, is polyphyletic. In response to these concerns, Schwank (1990) and Kisielewski (1991) erected new genera of chaetontotids and redeﬁned existing genera, but the classiﬁcation of this group may need to be redone once more complete information becomes available. IV. COLLECTING, REARING, AND PREPARATION FOR IDENTIFICATION Gastrotrichs may be collected by taking samples of sediments or vegetation. For quantitative work on sediment-dwelling species, small-diameter (2 – 5 cm) cores are preferable. For plant-dwelling forms, quantitative samples probably could be obtained by modifying sampling methods developed for macroinvertebrates (e.g., Downing, 1984; Jackson, 1997) to use very ﬁne mesh and small sample volumes or subsampling. Because living animals are preferable to preserved animals for many purposes, it often is desirable to extract the animals from the sample prior to preservation. If the sample must be preserved immediately, workers recommend narcotizing the animals with 1% MgCl2 for 10 min, then ﬁxing them in 10% formalin with rose bengal (e.g., Hummon, 1981, 1987). It is difﬁcult to extract or count the gastrotrichs from a sample. Some workers have handpicked or counted animals under a dissecting microscope (e.g., Hummon, 1981, 1987; Evans, 1982; Strayer, 1985), but this procedure is tedious. Density gradient centrifugation (Nichols, 1979; Schwinghamer, 1981) ought to be useful in extracting gastrotrichs from sediment, but has not yet been tested on freshwater gastrotrichs. Nesteruk (1987) found that a modiﬁed Baermann funnel was useful for extracting chaetonotids, but not dasydytids, from pond sediments. Sieves should be avoided or used cautiously, because gastrotrichs are too small to be retained quantitatively on even very ﬁne mesh sieves. For example, Hummon (1981) found that a 37-m mesh sieve retained only 31% of the gastrotrichs in a series of samples taken from the upper Mississippi River. For quantitative work, it is important to check the efﬁciency of whatever extraction or counting method is used, because gastrotrichs are so small and can be easily overlooked (cf. Strayer, 1985, pp. 295 – 296). Living gastrotrichs are preferable to dead gastrotrichs for taxonomic work. Living gastrotrichs often are too active for critical observations to be made, so they must be slowed down. This can be accomplished by gently squeezing the animal (either with a rotocompressor — Spoon, 1978 — or by removing some of the water from beneath a cover slip with a tissue), by placing it in a viscous medium such as methylcellulose, or by narcotizing it. Cocaine was the traditional narcotic of choice (Brunson, 1959), but it is now difﬁcult to obtain for laboratory use. d’Hondt (1967) recommended using MS 222, and we have had very good success with narcotizing gastrotrichs by bleeding 1.8% neosynephrine (available at pharmacies) under the cover slip. Animals may be killed with formalin or fumes of osmium tetroxide (e.g., Brunson, 1950) following narcotization. Osmium tetroxide is a superior ﬁxative, but is dangerous and should be used with extreme care. It is sometimes necessary to examine individual scales, which can be isolated from an animal by bleeding 2% acetic acid under the cover slip. For serious taxonomic work, videomicroscopy of living animals may provide better permanent documentation of species characters than killed specimens or slides. Procedures for culturing gastrotrichs were described by Packard (1936), Brunson (1949), Townes (1968), Hummon (1974), and Bennett (1979). Gastrotrichs have been cultured on 0.1% malted milk, raw egg yolk, wheat grain infusion, baked lettuce infusion, and baker’s yeast. Brunson (1949) recommended that animals be acclimated gradually to culture media when collected from the wild. Hummon (1974) described a procedure for starting individual cultures of known-age 7. Gastrotricha animals from eggs that may be especially useful for bioassay work. V. TAXONOMIC KEY It is relatively easy to identify most North American freshwater gastrotrichs to genus and very difﬁcult to identify them to species. Species identiﬁcation requires a keen eye, careful observation, a cooperative gastrotrich, and some luck, because most of the freshwater gastrotrichs of North America undoubtedly are undescribed. The following works are helpful in species identiﬁcation: Brunson (1950, 1959), who keyed and illustrated species then known from North American 189 freshwaters; Robbins (1965, 1973), who gave additional information and drawings of North American species; d’Hondt (1971b), who gave a key to the species of Lepidodermella and deﬁned three subgenera of Ichthydium; Kisielewski (1981), who made a critical evaluation of the morphological characters that must be measured to describe (or identify) a species; Kisielewski (1986b), who gave a recent treatment of Aspidiophorus; Schwank (1990), who provided illustrated keys (in German) for all known freshwater gastrotrichs worldwide; and Kisielewski (1991), who described many Brazilian species and addressed several important issues in gastrotrich systematics. The following key includes genera of freshwater gastrotrichs known from or likely to be found in North America. 1a. Animal with at least three pairs of adhesive tubules (one anterior and two posterior) and a pair of pharyngeal pores, body strapshaped (Fig. 1A,B); an almost entirely marine group not yet reported from North American freshwaters.............order Macrodasyida 1b. Animal lacking adhesive tubules (Fig. 7A– H ) or with one pair (very rarely two pairs) of adhesive tubules posteriorly (Fig. 6A– L), and no pharyngeal pores, body strap-shaped or tenpin-shaped; common and widespread in freshwater ...............order Chaetonotida ...........................................................................................................................................................................................................2 2a (1). Posterior end of body usually with furca and adhesive tubules; body usually strap-shaped or tenpin-shaped (Fig. 6A-L) ...................3 2b. Posterior end of body without furca or adhesive tubules, although sometimes bearing spines or pegs; body usually tenpin or bottleshaped (Fig. 7A– H)..........................................................................................................................................................................12 3a (2). Furca doubly branched; scales and spines sparse or absent (Fig. 6A); a rare genus not yet reported from North America .................... Dichaeturidae, Dichaetura 3b. Furca singly branched; scales and spines present or absent (Fig. 6B– L); common and widespread .....................................................4 4a (3). Branches of furca heavy, sickle-shaped, curved, and tapered, not distinctly divided into a cone-shaped basal part and a distal duct; head with long cilia that are not arranged in bundles; head plates absent (Fig. 6B); a rare family not yet reported from North America ................................................................................................................................................................................Proichthydiidae 4b. Branches of furca usually with a cone-shaped base and a distal adhesive duct; body often with numerous spines or scales; head with cilia that are arranged in bundles; cephalic plates present (Fig. 6C – L); common and widespread ................................Chaetonotidae ...........................................................................................................................................................................................................5 5a (4). Branches of furca ringed and often very long; body often large and without a distinct neck (Fig. 6C) ..............................Polymerurus 5b. Branches of furca not ringed (Fig. 6D – L)...........................................................................................................................................6 6a (5). Dorsal surface of body without spines or scales (except for dorsal sensory bristles or a few scales at the base of the furca (Fig. 6D) ... ...........................................................................................................................................................................................Ichthydium 6b. Dorsal surface of body with numerous scales or spines (Fig. 6E – L) ...................................................................................................7 7a (6). Spines or spined scales present and often numerous (Fig. 6E – G, J) ....................................................................................................8 7b. Spines absent (occasionally a few thin spines are present at the base of the furca) (Fig. 6H,I ,K,L) .....................................................9 8a. (7). Ventral scales different from dorsal scales; spines of various types (Fig. 6E – G); common and widespread.......................Chaetonotus 8b. Ventral scales similar to dorsal scales; posterior part of body with several long spines that reach beyond the end of the furca (Fig. 6J); not yet reported from North America .............................................................................................................Lepidochaetus 9a (7). Furca without adhesive tubes; body markedly tenpin-shaped, with groups of long cilia on the head and posterior part of the body (Fig. 6H); a semiplanktonic genus known only from Brazil ......................................................................................................Undula 9b. Furca with adhesive tubes; body strap-shaped or tenpin-shaped; without groups of long cilia on head and posterior body (Fig. 6I,K,L) .....................................................................................................................................................................................10 10a (9). Dorsal surface of body covered with stalked scales (Fig. 6I) ..........................................................................................Aspidiophorus 190 David Strayer and William D. Hummon FIGURE 6 Genera of freshwater Dichaeturidae, Proichthydiidae, and Chaetonotidae. (A) Dichaetura; (B) Proichthydium; (C) Polymerurus, showing detail of ringed branches of furca; (D) Ichthydium, (E – G) Chaetonotus, showing examples of spination; (H) Undula; (I) Aspidiophorus, with detail of coat of scales and a single scale; (J) Lepidochaetus, in dorsal (left) and ventral (right) views, (K) Heterolepidoderma, with detail of scales; and (L) Lepidodermella, with detail of scales. From Remane (1935 – 1936), Brunson (1950), Hyman (1951), Voigt (1958), Robbins (1965), and Kisielewski (1991). 10b. Scales on dorsal surface of body unstalked (Fig. 6K,L) .....................................................................................................................11 11a (10). Scales elongate, with longitudinal keels (Fig. 6K) ...................................................................................................Heterolepidoderma 11b. Scales not keeled (Fig. 6L)............................................................................................................................................Lepidodermella 12a (2). Head with club-shaped tentacles (Fig. 7A,B) .............................................Neogosseidae..................................................................13 12b. Head without club-shaped tentacles (Fig. 7C – H) .......................................Dasydytidae..................................................................14 13a (12). Posterior end of body with two groups of long spines (Fig. 7A); elements of mouth-ring jointed.........................................Neogossea 13b. Posterior end of body with single medial group of spines (Fig. 7B); elements of mouth-ring unjointed .............................Kijanebalola 7. Gastrotricha 191 FIGURE 7 Genera of freshwater Neogosseidae and Dasydytidae. (A) Neogossea; (B) Kijanebalola; (C) Stylochaeta; (D) Ornamentula; (E) Anacanthoderma; (F) Setopus; (G) Haltidytes, and (H) Dasydytes. From Brunson (1950), Krivanek and Krivanek (1958), Balsamo (1983), Schwank (1990), and Kisielewski (1991). 14a (12). Posterior end of body with pair of peglike protuberances (Fig. 7C) ....................................................................................Stylochaeta 14b. Posterior end of body without peglike protuberances (Fig. 7D – H) ..................................................................................................15 15a (14). Body enclosed in a “lorica” of large, thick, ornamented scales (Fig. 7D); known only from Brazil..................................Ornamentula 15b. Scales absent or small and inconspicuous (Fig. 7E – H) .....................................................................................................................16 16a (15). Head much narrower than body and scarcely wider than neck; lateral spines absent or identical to dorsal spines; pharynx with two bulbs (Fig. 7E); not yet reported from North America ...............................................................................................Anacanthoderma 16b. Head distinctly wider than neck; body with long lateral spines; pharynx with one bulb or no bulb (Fig. 7F – H) .............................17 17a (16). Some lateral spines movable and sharply bent basally; posterior end of body rounded, without rear spines (Fig. 7G)..........Haltidytes 17b. All spines ﬁxed, straight or bent distally; posterior end of body usually with spines (Fig. 7F,H).......................................................18 18a (17). Lateral spines with 1 – 3 lateral denticles and often terminally bifurcated; spines of uniform thickness from their base to the last lateral denticle; pharynx usually with a distinct posterior bulb (Fig. 7H) .................................................................................Dasydytes 18b. Lateral spines tapered, with at most one weak lateral denticle and never terminally bifurcated; pharynx without posterior bulb (Fig. 7F) ...................................................................................................................................................................................Setopus 192 David Strayer and William D. Hummon VI. LITERATURE CITED Balsamo, M. 1980. Spectral sensitivity in a freshwater gastrotrich (Lepidodermella squammatum Dujardin). Experientia 36: 830 – 831. Balsamo, M. 1981. Gastrotrichi della Toscana: il lago di Sibolla. Bolletino del Museo Civico di Storia Naturale Verona 7: 547 – 571. Balsamo, M. 1983. 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Kisielewska, G. 1981. Hermaphroditism of freshwater gastrotrichs in natural conditions. Bulletin de l’Académie Polonaise des Sciences, Série des Sciences Biologiques 29:167 – 172. Kisielewska, G. 1982. Gastrotricha of two complexes of peat hags near Siedlce. Fragmenta Faunistica 27:39 – 57. Kisielewski, J. 1981. Gastrotricha from raised and transitional peat bogs in Poland. Monograﬁe Fauny Polski 11:1 – 143. Kisielewski, J. 1986a. Freshwater Gastrotricha of Poland. VII. Gastrotricha of extremely eutrophicated water bodies. Fragmenta Faunistica 30:267 – 295. Kisielewski, J. 1986b. Taxonomic notes on freshwater gastrotrichs of the genus Aspidiophorus Voigt (Gastrotricha: Chaetonotidae), with descriptions of four new species. Fragmenta Faunistica 30:139 – 156. Kisielewski, J. 1987. Two new interesting genera of Gastrotricha (Macrodasyida and Chaetonotida) from the Brazilian freshwater psammon. Hydrobiologia 153:23 – 30. Kisielewski, J. 1991. Inland-water Gastrotricha from Brazil. 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Freshwater invertebrates of the United States. 2nd ed., Wiley-Interscience, New York. Pfaltzgraff, G. H. 1967. A preliminary study of the Gastrotricha of northern Indiana. Proceedings of the Indiana Academy of Sciences 76:400 – 404. Powell, E. N., Crenshaw, M. A., Rieger, R. M. 1979. Adaptations to sulﬁde in the meiofauna of the sulﬁde system. I. 35Sulﬁde accumulation and the presence of a sulﬁde detoxiﬁcation system. Journal of Experimental Marine Biology and Ecology 37:57 – 76. Powell, E. N., Crenshaw, M. A., Rieger, R. M. 1980. Adaptations to sulﬁde in the sulﬁde-system meiofauna. Endproducts of sulﬁde detoxiﬁcation in three turbellarians and a gastrotrich. Marine Ecology Progress Series 2:169 – 177. Preobrajenskaja, E. N. 1926. Zur Verbreitung der Gastrotrichen in den Gewässern der Umgebung zu Kossino (in Russian, with a German summary). Arbeiten der biologischen Station zu Kossino (bei Moskau) 4:3 – 14. Remane, A. 1935 – 1936. Gastrotricha und Kinorhyncha. 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The benthic micrometazoans of Mirror Lake, New Hampshire. Archiv für Hydrobiologie Supplementband 72: 287 – 426. Sudzuki, M. 1971. Die das Kapillarwasser des Lückensystems bewohnenden Gastrotrichen Japans I. Zoological Magazine 80: 256 – 257. Todaro, M. A., Fleeger, J. W., Hu, Y. P., Hrincevich, A.W., Foltz, D. W. 1996. Are meiofaunal species cosmopolitan? Morphological and molecular analysis of Xenotrichula intermedia (Gastrotricha: Chaetonotida). Marine Biology 125:735 – 742. Townes, M. M. 1968. The collection, identiﬁcation, and cultivation of gastrotrichs. Turtox News 46:99 – 101. Voigt, M. 1958. Gastrotricha. Die Tierwelt Mitteleuropas, Band I, Lieferung 4a, pp. 1 – 45. Wallace, R. L., Ricci, C., Melone, G., 1996. A cladistic analysis of pseudocoelomate (aschelminth) morphology. Invertebrate Biology 115:104 – 112. Weiss, M. J., Levy, D. P. 1979. Sperm in “parthenogenetic” freshwater gastrotrichs. Science 205:302 – 303. 8 PHYLUM ROTIFERA Robert Lee Wallace Terry W. Snell Department of Biology Ripon College Ripon, Wisconsin 54971 School of Biology Georgia Institute of Technology Atlanta, Georgia 30332 I. Introduction II. Anatomy and Physiology A. External Morphology B. Organ-System Function C. Environmental Physiology III. Ecology and Evolution A. Diversity and Distribution B. Reproduction and Life History C. Ecological Interactions D. Evolutionary Relationships IV. Collecting, Rearing, and Preparation for Identiﬁcation I. INTRODUCTION The phylum Rotifera or Rotatoria comprises of approximately 2000 species of unsegmented, bilaterally symmetrical, pseudocoelomates, possessing two distinctive features (Fig. 1). First, at the apical end (head) is a ciliated region called the corona, which is used in locomotion and food gathering. In adults of some forms, ciliation is lacking and the corona is a funnel or bowlshaped structure at the bottom of which is the mouth. Second, a muscular pharynx, the mastax, possessing a complex set of hard jaws, called trophi, is present in all rotifers. When viewing the anterior end of most rotifers one is struck with the idea of a rotating wheel. This is due to the metachronal beat of cilia on the corona, a structure usually composed of two concentric rings: trochus and cingulum (Fig. 2). This same image provided early microscopists with the name for the phylum: the etymon is Latin, rota, “wheel” and Latin, ferre, “to bear” equals “wheel bearers.” Although rotifers are often confused with ciliated protozoans and gastrotrichs by beginning students, those organisms do not possess trophi and their ciliation is not distributed in the same Ecology and Classiﬁcation of North American Freshwater Invertebrates. 2nd Edition Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved. A. Collections B. Culture C. Preparation for Identiﬁcation V. Classiﬁcation and Systematics A. Classiﬁcation B. Systematics C. Taxonomic Keys D. Taxonomic Key to Families of Freshwater Rotifera Literature Cited way as in rotifers. Rotifers are small organisms, generally ranging from 100 – 1,000 m long, although a few elongate species may surpass 2,000 m or more. Very few rotifers are parasitic (May, 1989); nearly all are free-living herbivores or predators. Collectively this phylum is widely distributed, being found in all freshwater habitats at densities generally ranging up to about 1,000 individuals/L. However, rotifers occasionally become abundant if sufﬁcient food is available, and can attain population densities of 5,000 individuals/L. In some rather unusual water bodies, exceedingly large populations can develop; sewage ponds may contain about 12,000 individuals/L (Seaman et al., 1986), and at certain times in soda water bodies in Chad, much more than 100,000 individuals/L may occur (Iltis and Riou-Duvat, 1971)! Although most inhabit freshwaters, some genera also have members that occur in brackish and marine waters. For example, about 20 of the 32 species comprising the genus Synchaeta are described as marine (Nogrady, 1982). However, only about 50 species of rotifers are exclusively marine. In general, rotifers are not as diverse or as abundant in marine environments as microcrustaceans, but they occur in many nearshore 195 196 Robert Lee Wallace and Terry W. Snell FIGURE 1 Lateral view of a generalized rotifer. (Modiﬁed from Koste and Shiel, 1987, with permission.) marine communities (Egloff, 1988) and occasionally comprise the dominant portion of the biomass (Schnese, 1973; Johansson, 1983). One unusual group of rotifers, the bdelloids (Fig. 3), may be found inhabiting the ﬁlm of water covering mosses, lichens, and liverworts. Additionally, they are often abundant in soils (Pourriot, 1979); estimates of their densities range from about 32,000 to more than 2 million individuals/m2, depending on soil moisture levels. Because of their feeding habits, and the fact that they are sometimes more numerous than nematodes, rotifers play an important role in nutrient cycling in soils (Pourriot, 1979). Most rotifers are free-moving, either swimming as members of the plankton or crawling over plants or within the sediments; however, some sessile species live permanently attached to freshwater plants (Wallace, 1980). The vast majority of rotifers are solitary, but about 25 species form colonies of various sizes (Wallace, 1987). All freshwater rotifers are either exclusively parthenogenetic or produce males for a limited time each year. Therefore, unless collections are made FIGURE 2 Female and male Brachionus plicalitis. (Modiﬁed from Pourriot, 1986, with permission.) 8. Phylum Rotifera 197 ologia. Some of the papers discussed in this chapter were presented at those meetings. II. ANATOMY AND PHYSIOLOGY A. External Morphology FIGURE 3 Typical bdelloid rotifer (Philodina). (Modiﬁed from several sources.) frequently, male rotifers may never be seen. Three very different classes of rotifers are commonly recognized (Seisonidea, Bdelloidea, Monogononta). Additional accounts of this phylum may be found in most texts of general and invertebrate zoology, and in some specialized books about freshwaters (Edmondson, 1959, pp. 420 – 494; Hutchinson, 1967, pp. 506 – 551; Pennak, 1989, pp. 169 – 225). For detailed reviews of the biology of rotifers consult the works of de Beauchamp (1965), Hyman (1951, pp. 59 – 151), Koste (1978), Ruttner-Kolisko (1974), and Nogrady et al. (1993). In the 1800s there were some beautifully illustrated works that still offer an excellent view of these animals (e.g., Hudson and Gosse, 1886). There is no single scientiﬁc journal or set of journals in which researchers publish their work on rotifers; the ﬁeld is simply too diverse. However, every three years, since 1976, a small group of workers (approximately 50 – 100) have gathered to hold the International Rotifer Symposium. To date, nine such meetings have been held and most of the proceedings have been published as a special volume of the journal Hydrobi- Rotifers are saccate-to-cylindrical in shape, sometimes appearing wormlike (e.g., many bdelloids, Fig. 3). Typically, the body is comprised of four regions: head (with corona); neck; body; and foot (Fig. 1). Although these regions may be marked by folds in the body wall, which function like joints, rotifers are not metameric (segmented). Unfortunately, the generalizations noted here do not represent all rotifers well. In some forms, the neck and foot may be quite prominent, while in others, they are absent (Fig. 4). In the class Monogononta, male rotifers (Fig. 2) and juveniles (larval females) of sessile rotifers are usually much smaller than adult females (Fig. 5). In addition, male rotifers are often structurally simpler (e.g., the gut commonly functions only as a food reserve) and the larvae of sessile forms generally have a different morphology from that of the adult (Wallace, 1980). When either males or larvae are found in plankton tows, their strikingly different morphologies may lead to improper identiﬁcation. The foot is an appendage that extends from the body ventrally (Fig. 2). It usually possesses two toes, but the number may vary from 0 to 4. The foot also may possess pedal glands whose ducts exit near the toes. These glands secrete a sticky cement for temporarily attaching the rotifer to substrates. In larvae of sessile rotifers, the cement forms a bond with the substrate that is not easily detached; if dislodged, sessile forms do not reattach (Wallace, 1980). Rotifers possess a syncytial integument or body wall containing a ﬁlament layer of varying thickness called the intracytoplasmic lamina (Clément, 1985). This feature apparently is shared with the parasitic phylum, Acanthocephala, indicating a phylogenetic relationship (see Section III.D.). The integument of a halophile rotifer, Brachionus plicatilis, was examined biochemically by Bender and Kleinow (1988) and their work indicates that it contains two ﬁlamentous, keratinlike proteins (Mr 39,000 and 47,000) cross-linked by disulﬁde bonds. Species in which major portions of the integument are thickened are termed “loricate;” those in which the integument is thin and very ﬂexible are termed “illoricate.” In general, thickness of the body wall is of little taxonomic signiﬁcance since extremes may be found within a single family or genus (e.g., Cephalodella). Further, even in loricate forms portions of the integument (lorica) have a less welldeveloped intracytoplasmic lamina, making the lorica 198 Robert Lee Wallace and Terry W. Snell FIGURE 4 Representative rotifers. Monogononts: (A) Asplanchna; (B) Euchlanis; (C) Keratella (lateral view); (D) Polyarthra; and (E) Synchaeta. Bdelloids: (F) Philodina; and (G) Rotaria. (From Koste, 1976, with permission.) ﬂexible in that region. Flexibility is found in the region of the corona, often in the foot, and at articulations between movable spines and the body. In some rotifers, the integument has various projections (e.g., bumps and ﬁxed or movable spines) that serve various functions, chief among these being protection from some predators (see Sections III.A.1 and III.C.4). B. Organ-System Function Internally, rotifers possess a perivisceral cavity (pseudocoelom) in which are suspended muscles, nerves, and digestive, reproductive, and protonephridial organs (Figs. 1, 2, and 3). Respiratory and circulatory systems are absent. Rotifers possess two remarkable features. First, all postembryonic tissues are syncytial. Second, all individuals of a species have a consistent number of nuclei in each organ throughout life. [N.B.: There are approximately 900 nuclei per female (Hyman, 1951).] This feature, called “eutely,” is also seen in a few other invertebrates (e.g., nematodes, Chapter 9). Most nuclei may be seen using a standard light microscope, but special optics such as differential interference contrast (Nomarski) enhance their visibility. 1. Corona There is considerable variation in the shape of the anterior ends of rotifers, and at least seven different 8. Phylum Rotifera 199 FIGURE 5 Adults and larvae of some sessile species (family Flosculariidae). (A) Adult Octotrocha speciosa (from Koste, 1989, with permission); (B) larval Octrotrocha speciosa (dark ﬁeld illumination, length 1200 m); (C) colony of adult Lacinularia ﬂosculosa (bright ﬁeld illumination, individual length 1200 m); and (D) larval Lacinularia ﬂosculosa (bright ﬁeld illumination, larvae somewhat compressed by the cover glass, length 450 m). types of coronae have been described based on the placement of the mouth and the distribution of cilia (Koste and Sheil, 1987). The corona form of many rotifers is comprised of two ciliated rings called the trochus and cingulum (Figs. 2 and 5). These structures are responsible for the production of water currents that are used in locomotion and feeding. However, not all rotifers possess this coronal ciliation. Adults of the family Collothecidae exhibit the most extreme varia- tion from the typical plan; in most collothecids, cilia are nearly or completely lacking from the corona and long setae surround the rim of a funnel-shaped structure known as the infundibulum (Latin, a funnel) (Fig. 6). These setae prevent escape of prey when the edges of the infundibulum fold over the victim, capturing it in a fashion similar to the Venus ﬂytrap. However, some adult atrochidae possess no setae on their corona (e.g. Cupelopagis). In other groups, ciliation 200 Robert Lee Wallace and Terry W. Snell FIGURE 6 Three representatives of the order Collothecacea. (A) Collotheca trilobata, a sessile form; (B) Collotheca mutabilis, a planktonic form; (C) Collotheca sp. with two developing embryos (width of corona 100 m); and (D) Collotheca cornuta, dark ﬁeld illumination (length of setae 200 m). (A, modiﬁed from Wallace et al., 1989, with permission; and B, modiﬁed after several sources.) 8. Phylum Rotifera may be limited to a ventral ﬁeld or several lobes, as is seen in some creeping forms and some bdelloids. Other structures that may be present on the corona include cirri, sensory antennae, and palps. 2. Trophi and Gut Once food is captured by the corona, it enters a ventral mouth by passing through a short, ciliated tube into a muscular pharynx, termed the “mastax.” The mastax possesses a chitinous lining on the inside, developed as a set of translucent jaws called trophi. The trophi may work the food in various ways (e.g., grinding) before it is passed to the esophagus and swallowed (Figs. 1 and 2). In the family Collothecidae, a portion of the mastax is enlarged into a food-storage organ known as the proventriculus (Fig. 6). The mastax leads posteriorly to an esophagus, stomach, and in most species, intestine and anus, but the gut ends in a blind stomach in some genera (i.e., Asplanchna, Asplanchnopus). The posterior portion of the intestine (cloaca) receives eggs from the oviduct and ﬂuid from either a bladder or directly from paired protonephridia (Fig. 1). Often the gut is pigmented, depending on the nature of recently ingested material. Different species found in the same sample may possess guts that vary in color due to differences in diet. Rotifer trophi are composed of several hard parts and associated musculature, which articulate in a speciﬁc spatial arrangement. In their basic form, trophi consist of three functional units: an incus (Latin, anvil) and paired mallei (Latin, hammer) (Fig. 7). The incus is composed of three pieces: a fulcrum and a pair of rami (Latin, branch) that move like forceps and articulate with the fulcrum at their bases. Each malleus consists of two parts: manubrium and uncus. The manubrium (Latin, handle) resembles a club-shaped structure extended at one end into a cauda (Latin, tail) and ﬂared at the other end (head). The manubrium articulates with a toothed structure called the uncus (Latin, hook). The plane of movement of the pieces comprising the malleus is at right angles to that of the rami. In some species, the trophi may be modiﬁed by reduction of the basic parts, addition of accessory structures, or by asymmetrical development of one or more of the pieces. The trophi of rotifers have been recognized as important taxonomic features: classes, orders, and families, and even species may be determined based on the details of the trophi alone (e.g., Salt et al., 1978). Nine different types of trophi are recognized based on the size and shape of the seven pieces and the presence of any accessory parts (Figs. 7 – 10), transitional and aberrant types also are known. In “Malleate” trophi (Fig. 8A – C, E, F, P), all parts of the incus and mallei are well developed and functional, but the rami are charac- 201 teristically massive and may possess teeth along the inner margin. Further, the unci have 4 – 7 large teeth. This form works by grasping food and grinding it before pumping the crushed material into the esophagus. Malleate trophi are present in such common rotifers as Keratella and Kellicottia. “Malleoramate” trophi (Fig. 8I) are found only in the order Flosculariacea and resemble the malleate form except that, in the malleoramate form, the rami are strongly toothed and the unci possess many thin teeth. Similar to the malleoramate form, “ramate” trophi (Fig. 9) have large, semicircular shaped rami and unci with many teeth. Ramate trophi are generally considered to be limited to the Bdelloidae (Melone et al., 1998a). Only members of the order Collothecacea (family Collothecidae) possess “uncinate” trophi (Fig. 8H). These trophi are characterized by unci possessing few teeth, usually with one large and a few small ones. “Virgate” trophi (Fig. 8D, K – O, Q, S) are modiﬁed for piercing and pumping and generally can be recognized by the long fulcrum and manubria and the presence of a powerful hypopharyngeal muscle. Some trophi of this form are asymmetrical. Virgate trophi are found in the common genera Notommata, Polyarthra, and Synchaeta. “Forcipate” trophi (Fig. 8J) as the name implies, have an action like forceps, whereby the trophi are projected from the mouth to grasp prey, which are then brought into the mouth and swallowed. Forcipate trophi are limited to the family Dicranophoridae. “Incudate” trophi (Fig 8G, R) function by grasping prey with a forcepslike action, but this form has a different morphology than the forcipate type; the rami are quite large and the mallei very small. The mastax actually initiates prey capture by creating a suction, drawing prey into the mouth, which is then stuffed into the stomach with the aid of the trophi. Asplanchna, which has no anus, uses its trophi to extract undigestable materials from its FIGURE 7 Generalized rotifer trophi. (Modiﬁed from Wallace et al., 1989, with permission.) 202 Robert Lee Wallace and Terry W. Snell FIGURE 8 Rotifer trophi types. (A) malleate trophi of Epiphanes, ventral; (B) malleate trophi (Epiphanes), ventral elevated; (C) malleate trophi (Epiphanes), lateral; (D) virgate trophi of Notommata, dorsal; (E) malleate trophi of Proales; (F) showing hypopharynx (lateral); (G) incudate trophi of Asplanchna; (H) uncinate trophi of Collotheca; (I) malleoramate of Ptygura; (J) forcipate trophi of Dicranophorus; (K) asymmetrical virgate trophi of Trichocera rattus; (L) virgate trophi of Eothinia; (M) virgate trophi of Itura, oral plates enlarged; (N) virgate trophi of Synchaeta with the powerful hypopharynx muscle; (O) virgate trophi of Ascomorpha; (P) malleate trophi of Proales gigantea, an intermediate form between malleate and virgate types; (Q) virgate trophi of Cephalodella, dorsal; (R) incudate trophi of Asplanchna; and (S) virgate trophi of Cephalodella, lateral. Bars 20 m. (From Koste and Shiel, 1987, with permission.) stomach. Incudate trophi are limited to the family Asplanchnidae. “Cardate” trophi (Fig. 10) are found only in the family Lindiidae and function by producing a pumping action, without the hypopharyngeal muscle. The “fulcrate” type of trophi have been described as an aberrant form and are incompletely understood (Edmondson, 1959, p. 432). This form is found only in the class Seisonidea, a very small group of marine rotifers. 3. Organ Systems Research on the ultrastructure of rotifers has proceeded rapidly during the past decade and promises to continue to do so. For a comprehensive overview on the topic of ultrastructural research on rotifers, consult the works of Amsellem and Ricci (1982), Clément (1977, 1980, 1987), Clément et al. (1983), Clément and Wurdak (1991), Wurdak (1987), Wurdak and Gilbert (1976), and Wurdak et al. (1977, 1983). FIGURE 9 Ramate trophi of bdelloid rotifers. (Modiﬁed from several sources.) a. Muscular System The muscular system consists of small groups of longitudinal and circular muscles inserted at various points on the integument or between the integument and viscera (Fig. 2). In loricate species, 8. Phylum Rotifera 203 FIGURE 10 Cardate trophi of the family Lindiidae. (From Harring and Myers, 1922, with permission.) the integument provides a ﬁrm structure against which the muscles work. Muscle contraction can increase the pressure of the pseudocoel, which then acts as a hydrostatic skeleton. In some species, muscles retract the corona, which increases pressure within the pseudocoel, thus expanding ﬂaccid portions of the integument, known as body-wall outgrowths (Asplanchna, Fig. 11). In others, such as Brachionus (Fig. 12), retracting the corona makes spines, that articulate with the body in the posterolateral region, swing forward. When in this extended position these spines interfer with predators like Asplanchna which would otherwise consume the Brachionus (Gilbert, 1966, 1967). Muscles also are present in the viscera, particularly in the mastax and stomach. Striated, longitudinal muscles are responsible for retracting the corona and foot and for moving certain articulating spines that are not positioned by hydrostatic pressure. Some species possess powerful muscles that control movement of certain locomotory appendages (e.g., Hexarthra, Fig. 13). Contractions of these muscles cause a swift downward sweep of the FIGURE 12 Spined and unspined Brachionus calyciﬂorus. (From Koste, 1976, with permission.) appendages which results in a rapid displacement or jump of the rotifer (see Section II.C.1). b. Nervous System The nervous system is simple, consisting of only a cerebral ganglion or brain (Fig. 3) located dorsally on the mastax, a few other ganglia present in the mastax and foot, and three types of sensory organs, namely mechano-, chemo-, and photoreceptors. Mechanoreceptive bristles are situated on the corona, while several antennae are located elsewhere on the body surface, usually laterally and caudally (Fig. 2). Chemoreceptive pores are also present on the corona. Many species possess one or more photoreceptive eyespots, sometimes accompanied by a pigmented spot (Fig. 3). When present, eyespots are located in the anterior end, usually near the brain (Clément, 1980; Clément et al., 1983). While most rotifers retain the eyespots throughout life, larvae of sessile rotifers often lose them during metamorphosis. Paired, ventral nerve cords proceed from the brain along the length of the body into the foot. Several other ganglia are usually found in the nerve cords at the exit points for lateral nerves. One interesting structure found in the apical region of many bdelloid and monogonont rotifers is the FIGURE 11 Body form variability in the genus Asplanchna. (b.w.o. body-wall outgrowths). (Modiﬁed from Gilbert, 1980a, with permission.) FIGURE 13 Hexarthra showing positioning of muscles that initiate jumps. (Modiﬁed from several sources.) 204 Robert Lee Wallace and Terry W. Snell retrocerebral organ. This structure consists of paired subcerebral glands and an unpaired retrocerebral sac, both with ducts that lead to the surface of the corona (Fig. 8.1). While the function of the retrocerebral organ is unknown, Edmondson (1959, p. 422) has noted that there are fewer protonephridia in rotifers that possess well-developed retrocerebral organs. However, Clément (1977) suggests that it may function as an exocrine gland, perhaps lubricating the anterior part of the body. Although information on neurobiochemistry is very limited, research has shown that acetylcholine functions as a neurotransmitter (i.e., a cholinergic system) in twelve species of rotifers from six families (Nogrady and Alai, 1983, Nogrady and Keshmirian, 1986a, b). In addition, norepinephrine neuroreceptor sites (i.e., an adrenergic system) have been reported in Brachionus plicatilis, and a widespread catecholaminergic neuronal systems have been observed in species of Asplanchna and Brachionus (see Keshmirian and Nogrady, 1988). c. Protonephridium A paired protonephridial system comprised of tubules and ﬂame cells functions in excretion and osmoregulation in all rotifers (Figs. 1 and 2). Usually there are only a small number (6) of ﬂame cells, but large rotifers may possess many more. For example, Asplanchna sieboldi may have up to 100 ﬂame cells (Ruttner-Kolisko, 1974, p. 11). Normally, the tubules drain into a urinary bladder which leads to a cloaca, but the bladder is absent in some species and a contractile cloaca assumes its function. d. Reproductive System Besides their separation by anatomical details of the trophi, the three classes of rotifers are differentiated based on the anatomy of their reproductive systems (Wallace, 1998). The gonads are paired in both the Seisonidea and the Bdelloidea, but in the latter class, males are completely unknown and reproduction is always asexual. Members of the third class (Monogononta) have only one gonad. Although males are present in this group, they have not been described for a large number of species. However, it is generally assumed that most (all) monogononts are capable of producing males given the proper conditions, or at least that the ancestral forms were capable of male production. When males are produced by monogonont populations, they are usually limited to a few days or a week, so that during the year most reproduction is parthenogenetic (see Section III.B.1). The reproductive organs of female rotifers comprise three units, which may be seen with a light microscope: ovary, vitellarium, and follicular layer (Amsellem and Ricci, 1982). The ovary of a rotifer is a small, syncytial mass that is closely associated with the yolk-producing vitellarium. At birth the adult complement of ovocytes already has been formed in the ovary. The vitellarium is also syncytial with a constant number of nuclei, a characteristic useful in the taxonomy of some species. The follicular layer surrounds both the ovary and the vitellarium; in some species, this layer forms the oviduct, which connects with the posterior portion of the gut forming a joint exit, the cloaca (Fig. 1). In general, monogonont males are much smaller (about 100 m long) than females. In nearly all forms, the gut of males is reduced to a rudiment or is absent entirely (Fig. 2). In those forms with a rudimentary gut (e.g., Asplanchna), it serves as an energy source for the fast swimming, nonfeeding male. The single testis is large and saccate, usually containing 50 freely ﬂoating mature sperm. A ciliated vas deferens leads from the testis to the penis, usually with one or rarely two pairs of accessory (prostate) glands that discharge into it (see Section III.B.2). Commonly, rotifers are oviparous, that is, they release their eggs outside the body where the embryos develop. Many planktonic rotifers carry their eggs attached to the body of the mother by a thin thread (Brachionus), while others ﬁx them to a substrate (Euchlanis) or release them into the plankton (Notholca). A few species retain the embryo in the body until the offspring hatches: ovoviviparous (e.g., Asplanchna and Cupelopagis). C. Environmental Physiology 1. Locomotion All rotifers swim during at least a portion of their life cycle and some swim continuously, never attaching even temporarily to surfaces. Swimming inﬂuences the acquistion of food and mates, increases the number of encounters with predators, and promotes the dispersal of the larvae of sessile forms. Some rotifers are unusual in that they can swim, but routinely remain in mucus tubes attached to a substrate (e. g., certain Cephalodella species), while others are free-ﬂoating mucus sheaths (Ascomorpha). Most rotifers swim in a helical pattern, so that the actual distance traveled is greater than the linear displacement (see Starkweather, 1987). However, for practical reasons, most researchers do not attempt to calculate absolute distance when considering the distance traveled. Although the theoretical power requirements of swimming (i.e., the theoretical energy required to overcome water resistance) is 1% of the total metabolism, the actual energetic cost appears to be much greater. 8. Phylum Rotifera In Brachionus plicatilis, Epp and Lewis (1984) calculated this cost to be approximately 38% of the total metabolism (See also Epp and Lewis, 1979). Swimming speeds of male and female Brachionus plicatilis have been measured by Luciani et al. (1983), Epp and Lewis (1984), and Snell and Garman (1986). Swimming speeds also have been measured for Asplanchna brightwelli (Coulon et al., 1983), two species of Keratella (Gilbert and Kirk, 1988), and for Anuraeopsis ﬁssa, Asplanchna girodi, Brachionus calyciﬂorus, Euchlanis dilatata, K. americana, Lecane quadridentata, Brachionus patulus, and Trichocerca pusilla (Rico-Martinez and Snell, 1998). These studies have shown that swimming speed is temperature – dependent and varies among strains. Values recorded for B. plicatilis at 25°C range from 0.6 to 0.9 mm/sec for females and from 1.3 to 1.5 mm/sec for males, with young and old females swimming about 30% slower than mature females. Asplanchna brightwelli and Keratella females normally swim at 0.9 and 0.5 mm/sec, respectively. Euchlanis dilatata is one of the fastest species, with males and females both swimming at 0.98 mm/sec at 25°C. Little is known about the swimming speeds of the larvae of sessile rotifers, but the swimming speeds of the larvae of Ptygura beauchampi are know to vary with age. New born (0 – 2 h) through mid-aged (ca., 3 h) larvae swam about 2 – 2.5 mm/sec, faster than the rates reported for Brachionus males. However, older larvae (4.5 h) swam at approximately 1.0 mm/sec. Concomitant with these changes in swimming speed was an increase in turning frequency (Wallace, 1980). Correlations of rotifer ultrastructure with turning frequency, locomotion in general, and other behaviors have been a ﬁeld of systematic investigation by Clément and his co-workers (Clément, 1987; Clément et al., 1983; Chauoy, 1995). Their efforts have demonstrated that rotifers are excellent models for comparative neurobehavioral studies, but much more work remains to be done before a complete synthesis of structure, function, and behavior will be possible. Spines and other appendages inﬂuence both swimming speed and sinking rates in rotifers. For example, Stemberger (1988) has found that unspined Keratella testudo generally swim faster and sink more slowly than spined forms. On the other hand, Polyarthra normally swims at a much slower velocity (0.24 mm/sec) than any species previously discussed, but is capable of very short bursts (about 0.065s long) of rapid movements called jumps. During a jump, Polyarthra may attain velocities greater than 50 mm/sec, with a mean velocity of 35 mm/sec, or 100 times its normal swimming speed (Gilbert, 1985a, 1987; Starkweather, 1987). Jumps are produced by movements of 12 appendages, called paddles, which articulate with the 205 body near the head. When Polyarthra detect a local disturbance in the water a few body lengths away, powerful striated muscles rapidly ﬂex some of the paddles upward and then during the jump they return to their original positions. During this process the rotifer is displaced an average of 1.25 mm (ca. 12 body lengths). Jumping helps this rotifer escape invertebrate predators, such as Asplanchna (Gilbert, 1980b; Gilbert and Williamson, 1978) and ﬁrst instar Chaoborus (Moore and Gilbert, 1987) as well as the ﬁltering currents of microcrustaceans, such as Daphnia (Gilbert, 1985b, 1987). They are also effective against naive workers attempting to remove Polyarthra from plankton samples using micropipets! The genera Filinia and Hexarthra also can jump rapidly, but movement in these forms has not been well studied (see Section III.C.4). In contrast, Keratella cannot jump to avoid the ﬁltering currents of daphnids and, as a result, are severely damaged or killed when swept into the branchial chambers of cladocerans (Burns and Gilbert, 1986a,b; Gilbert and Stemberger, 1985b). However, Keratella is not without some escape abilities; it is capable of increasing its swimming speed by a factor of about 3.5 when it encounters inhalant currents of Daphnia or the predatory rotifer Asplanchna (Gilbert and Kirk, 1988). Another source of phenotypic variation in body size is polyploidy. Substantial intrapopulational variation in body size was reported for Euchlanis dilatata (Walsh and Zhang, 1992). Two distinct body sizes were observed in Devils Lake, Oregon over 12 months of intensive sampling. The smaller morphotype was about 225 m in length and the larger averaged about 275 m. The larger morphotype had a chromosome number of 21 and the smaller morphotype had 14. The haploid chromosome number found in males of this species is n 7. This is the ﬁrst case of polyploidy reported for rotifers, but new techniques for staining and counting rotifer chromosomes may make more systematic explorations possible. 2. Physiological Ecology Physiological tolerances of organisms prescribe environments where survival and reproduction are possible. Thus, an environmental tolerance curve for a species summarizes the range of environments where reproduction occurs and culminates at the upper and lower lethal limits for the species (e.g., temperature range). Within the tolerance curve, the environmental optimum for a species is that environment where survival and reproduction are maximal. Therefore, a set of tolerance curves (including temperature, pH, etc.) indicate the breadth of adaptation of a species and its niche width. Environmental tolerances and niche widths have been characterized for a few rotifer 206 Robert Lee Wallace and Terry W. Snell species. For example, Epp and Lewis (1980) described the response of Brachionus plicatilis to temperature. That study determined respiration rate over temperature ranging from 15 to 32°C and recorded Q10s of 1.9 – 2.4 for broad temperature intervals. Respiration levels off between 20 and 28°C, indicating that B. plicatilis can maintain a constant metabolic rate over this temperature range. At higher and lower temperatures, respiration rate increased, presumably because of thermal stress which was beyond the homeostatic capability of this rotifer. Snell (1986) showed that amictic and mictic females have similar temperature tolerance curves. Amictic B. plicatilis females reproduced at 20 and 40°C, whereas mictic females did not. In general, amictic females reproduced over a broader environmental range of temperatures salinity, and food level than did mictic females. The effect of pH on the distribution and abundance of rotifers is a topic that has received a good deal of attention. However, since hydrogen-ion concentration is related to other important chemical parameters in freshwaters, studies of rotifer occurrence as a function of pH alone are of limited value. Nevertheless, some extensive early work by Myers in the 1930s demonstrated that rotifer species can be classiﬁed into a few broad groups based on pH alone: alkaline species, acid species; and those with a broad range (see also Edmondson, 1944). More recently, Berzins and Pejler (1987) concluded that species found in oligotrophic waters had pH optima at or below neutrality (pH of 7.0) and those species common to eutrophic waters had optima at or above neutrality. Further, they noted that acid-water species were often nonplanktonic or semiplanktonic. Much less is known of the speciﬁc metabolic responses of rotifers to pH. The inﬂuence of pH on B. plicatilis was described by Epp and Winston (1978). They found that swimming activity and respiration rate were not signiﬁcantly different at pH values of 6.5 – 8.5. Snell et al. (1987) examined the pH from 4.0 to 9.9 and found swimming activity depressed below pH 5.6 and above pH 8.7. Alkaline waters depressed swimming activity more than acidic conditions. Increasing acidiﬁcation of lakes as a result of acid rain has been widespread in North America. Acidiﬁcation leads to predicable changes in the rotifer zooplankton, such as the increasing dominance of Keratella tauracephala (Brett, 1989). This observation has been repeated in the experimental acidiﬁcation of lakes (Gonzalez and Frost, 1994). In general, it appears that the importance of rotifers within lakes increases with acidity (Frost et al., 1998). Osmoregulation has been investigated by Epp and Winston (1977) for B. plicatilis, a species that is common in highly alkaline waters and salt lakes. This species was an osmoconformer capable of tolerating osmolarities exceeding 957 mOsm/L (33.5 ppt). Ito (1960) suggested that the upper osmotic limit for B. plicatilis may be as high as 2860 mOsm/L (97 ppt). Transfer of this rotifer directly from 41 to 957 mOsm/L caused considerable mortality, but acclimation to high osmolarities was achieved by gradually increasing ionic concentrations. Brachionus plicatilis does not tolerate low osmolarities well, and this probably accounts for its restriction to alkaline and brackish waters. Although most rotifers require oxygen concentrations signiﬁcantly above 1.0 mg/L, some can tolerate anaerobic or near-anaerobic conditions for short periods. Other species routinely live in oxygen-poor regions, such as the hypolimnion of eutrophic lakes or in sewage ponds. The physical and chemical factors described above, along with food (Bogdan and Gilbert, 1987) and predation (Williamson, 1983), deﬁne the niche boundaries for rotifer species. Niche boundaries for ﬁeld populations have been described by Edmondson (1944), Makarewicz and Likens (1975, 1979), Miracle (1974), and Miracle et al. (1987). These investigations have generally revealed that rotifer species extensively partition the environment, thus avoiding negative interactions. 3. Environmental Toxicology Because rotifers ﬁll an important ecological role and are relatively easy to culture, interest is growing in their use for aquatic toxicity testing. The response of rotifers to a variety of toxicants has been characterized in both natural and laboratory populations. For example, effects of various insecticides, herbicides, and wastewater on natural rotifer populations have been investigated (Snell and Janssen, 1995). In general, rotifers seem to serve as good indicators of environmental water quality (Sladecek, 1983), and the use of rotifer population dynamics as sensitive indicators of toxicity has been promoted (Halbach, 1984; Halbach et al., 1981, 1983). A multispecies approach to toxicity assessment with a laboratory microcosm consisting primarily of rotifers has been attempted by Jenkins and Buikema (1985). Many short-term, acute toxicity tests with a variety of substances have been conducted using rotifers, e.g., insecticides, heavy metals, free ammonia, sodium dodecyl sulfate, crude oil and petrochemicals (Snell and Janssen, 1998). Unfortunately, the results of these studies are not directly comparable, as their methodologies varied considerably. However, testing protocols are comparable from several studies of the halophile B. plicatilis and a number of median lethal concentrations have been reported. (LC50 is the concentration of a compound which kills 50% of a cohort within a speciﬁed 8. Phylum Rotifera exposure time.) Capuzzo (1979a, b) found B. plicatilis to be very sensitive to free chlorine and chloramine. After a 30-min exposure at 25°C, LC50 values of 0.09 and 0.01 mg/L were recorded for chlorine and chloramine, respectively. Brachionus is more sensitive to mercury (LC50 of 0.045 mg/L; Gvozdov, 1986) than to chromium (LC50 500 mg/L; Persoone et al., 1989). Free ammonia is also toxic, with LC50 values of 20.4 and 17.7 mg/L at salinities of 15 and 30 ppt, respectively (Snell and Janssen, 1995). Pesticides, in contrast, are not very toxic to B. plicatilis. The 24-h LC50 values for ﬁve organophosphate pesticides and one organochlorine pesticide ranged from 0.9 to 150 mg/L for three different strains of B. plicatilis (Serrano et al., 1986). These same investigators observed that pesticide resistance of this rotifer is about 1000 times greater than that reported for other aquatic organisms. Since pesticides are generally targeted to arthropods, the low sensitivity of rotifers to these compounds is not surprising. Several median lethal concentrations for a variety of compounds have been reported for freshwater rotifers, mainly in the genus Brachionus. Dad and Kant Pandya (1982) recorded 24-h LC50 values for B. calyciﬂorus exposed to two insecticides, and Couillard et al. (1989) found the following relative metal toxicities: Hg Cu Cd Zn Fe Mn. LC50 values of 600 and 0.16 mg/L for phenol and pentachlorophenol, respectively, were reported by Halbach et al. (1983) for B. rubens. A standardized rotifer toxicity test for marine and freshwater that employs test animals derived from hatching B. plicatilis or B. calyciﬂorus resting eggs has been described (ASTM, 1991). This test is less expensive because stock cultures are not necessary to obtain test animals, and it is simple, rapid, and sensitive, so the use of rotifers in aquatic toxicology is expected to expand. 4. Anhydrobiosis The ability of rotifers to tolerate desiccation and then be revived sometime later has been known since the early 1700s, when Leeuwenhoek observed rehydration of rotifers found in dry sediments of rain gutters. Anhydrobiosis, which is also termed cryptobiosis and osmobiosis, is limited to bdelloids. Unlike monogononts, bdelloids cannot produce cysts. The term cryptobiosis refers to the slow or hidden metabolism which is characteristic of these animals, while anhydrobiosis and osmobiosis emphasize the processes whereby loss of water is accomplished through evaporation and external osmotic pressure, respectively. While in the desiccated state, these rotifers resemble a wrinkled barrel (or tun, as in the tardigrades, see Chapter 15), with the head and foot retracted into the animal’s trunk (Fig. 14). The signiﬁcance of this phenomenon is clear, 207 FIGURE 14 Body of a desiccated bdelloid rotifer (Habrotrocha rosa) with the corona withdrawn into the trunk. (Bar 50 m.) as many bdelloids inhabit environments that dry completely at irregular intervals. A few strictly aquatic bdelloids cannot withstand desiccation (Ricci, 1998b). Anhydrobiosis involves more than simple drying; unless loss of metabolic water proceeds slowly, the rotifer usually dies. During anhydrobiosis, the ﬁne structure of cells is retained, but in a greatly modiﬁed state (Ricci, 1987; Schramm and Becker, 1987). Changes that occur internally include a 50% reduction in the volume of the pseudocoel, a condensation of cells and organs, and a decrease in cytoplasmic volume, so that the entire animal is only about 25 – 30% of its original size. Nuclei, mitochondria, endoplasmic reticula, and other organelles form a compact mass within cells of the anhydrobiotic animal (Schramm and Becker, 1987). Desiccated bdelloids have been reported to be viable even after more than two decades in the anhydrobiotic state. Recovery from anhydrobiosis may require as little as 10 min, or it may take several hours, according to prevailing environmental conditions. Survival is negatively affected by starvation before desiccation and by moist environments and high temperatures during the desiccation period (Ricci 1987; Ricci, et al., 1987, Schramm and Becker, 1987). III. ECOLOGY AND EVOLUTION A. Diversity and Distribution 1. Phenotypic Variation Phenotypic variation is an important adaptive mechanism in rotifers, but has posed difﬁcult problems for systematists. This variation arises by several mechanisms including cyclomorphosis, dietary- and predatorinduced polymorphisms, polymorphisms in hatchlings 208 Robert Lee Wallace and Terry W. Snell from resting eggs, and dwarﬁsm. Cyclomorphosis is the seasonal phenotypic change in body size, spine length, pigmentation, or ornamentation found in successive generations of zooplankton. These changes are phenotypic alterations in a single population that are related to physical, chemical, or biologic features of the environment. Each different morphological form is called a morphotype. Speciﬁcally excluded from cyclomorphotic change are seasonal succession of sibling species and clonal replacements of genotypes, both of which are genetic changes in populations. A striking phenotypic change in morphology that is associated with a dietary polymorphism was described for three Asplanchna species (brightwelli, intermedia, sieboldi) by Gilbert (1980a). Diets that include the plant product a-tocopherol (vitamin E) induce saccate females, the smallest morphotype, to produce cruciform daughters. Cruciforms have lateral outgrowths of the body wall (Fig. 11) that protect them from cannibalism by conspeciﬁcs by making them larger and, thus, more difﬁcult to ingest if captured. In the presence of a-tocopherol and certain prey types, cruciforms can produce a third morphotype called campanulates (more prevalent in A. sieboldi and A. intermedia). Campanulates are very large females ( 2000 m), which heavily cannibalize saccate females. Female polymorphism is much less pronounced in A. brightwelli where there is a 50 – 60% increase in body size, but no campanulates are produced and body wall outgrowths are slight. Dietary polymorphism in Asplanchna (gigantism) may have evolved originally as a generalized growth response to larger prey typical of eutrophic waters (Gilbert, 1980a; Gilbert and Stemberger, 1985c). The tocopherol response probably is adaptive, because it signals the availability of nutritious rotifer and microcrustacean prey. Another source of phenotypic variation is predator-induced polymorphisms. Spined and unspined forms had been recognized in several rotifer species for many years, but the cause(s) and signiﬁcance(s) of these variations remained an enigma (Fig. 12). However, Gilbert (1966, 1967) was the ﬁrst to show that spine production could be induced in the offspring of female B. calyciﬂorus if adults were exposed to culture medium which had previously held the predatory species Asplanchna. Gilbert (1967) also demonstrated that such spines were strong deterrents to predation by Asplanchna (see Section III.C.4). However, Stemberger (1990) has shown that food concentration can dramatically modify the development of spines in B. calyciﬂorus. Two additional sources of phenotypic variation are polymorphisms called “aptera generations” in the hatchlings of resting eggs and dwarﬁsm, both of which have been reported in rotifers of some tropical crater lakes (Green, 1977). Aptera morphotypes were initially thought to be different species of Polyarthra, but later were shown to be forms lacking the paddles that are characteristic of this genus. Only the generation hatching from resting eggs lacks paddles; their parthenogenetic offspring develop into typical morphotypes. Similar polymorphisms between resting egg hatchlings and parthenogenetic generations were described for Keratella quadrata and are suspected for Notholca acuminata (Amrén, 1964). Dwarﬁsm in Brachionus caudatus in Cameroon crater lakes was described by Green (1977) and is characterized by reduced body size and spination as compared to normal morphotypes. Green speculated that high temperature combined with reduced food supply may cause this condition. 2. Distribution and Population Movements Water bodies are not uniform habitats with respect to concentrations of food and predators and abiotic factors such as dissolved oxygen concentration, light intensity, temperature, and water movements. Therefore, it is not surprising to ﬁnd that rotifers are not evenly distributed in lakes and ponds; often there is considerable variability with respect to their horizontal and vertical distribution. For example, Hoffman (1982) showed that two Filinia species had very different vertical distribution patterns over the course of a year in a small lake in Germany (Fig. 15). Striking horizontal variabilty in rotifer distribution has been reported by a variety of researchers: for example, by Green (1985) for a tropical lake. Thus, we ﬁnd that some rotifers are strictly littoral, being found in open waters only as occasional migrants, whereas others are pelagic; however, the depth at which they are found is a function of season. Consequently, experienced researchers can determine to some degree where and when a water sample has been taken, merely by the composition of the rotifer species it contains. Although these major distribution patterns are the result of differential population growth and water currents and other large-scale water movements, within-lake distribution patterns can be inﬂuenced to a lesser degree by locomotory behaviors. One commonly recognized behavior of marine and freshwater zooplankton is a daily (diel) vertical migration in the water column in which the animals usually come to the surface only during the night. During the day, zooplankton avoid visual predators (ﬁsh) that occupy near-surface waters, but when they return to the surface at night they can exploit the rich algal resources present there. In rotifers, diel migrations are never as dramatic as those typical of microcrustaceans; the population maximum usually changes only about 1 – 2 m 8. Phylum Rotifera 209 FIGURE 15 Temporal and spatial distribution of two species of Filinia in Lake Plußee, Germany. Solid line indicates limits of the population at one individual per liter; dotted line indicated boundaries of higher population levels (numbers of individuals per liter). (Modiﬁed from data in Hofmann, 1982, with permission.) over a daily cycle (Fig. 16). However, ovigerous (eggbearing) and nonovigerous females may have different migration patterns, and this can cause serious errors in the calculations of birth rates. Errors of nearly an order of magnitude depending on the sampling protocol may occur, if the population is sampled at only one depth or at different times during the day (Magnien and Gilbert, 1983). Horizontal movement of rotifers has been investigated by Preissler (1980) who showed that some pelagic rotifers apparently avoid the inshore region. Preissler demonstrated this phenomenon, known as “avoidance of the shore,” by using a circular plexiglass arena in which he simultaneously monitored the swimming direction of several zooplankton species. When he artiﬁcally altered the shadow produced by the natural elevation of the shoreline by adding a black collar around the arena, both Asplanchna priodonta and Syn- chaeta pectinata swam away from the shadow. The littoral rotifer Euchlanis dilatata apparently showed no preference based on light intensity. Research on the abundance and distribution of rotifers in lotic systems (ﬂowing waters) has lagged far behind that of lentic ones, but we do know that both rotifer diversity and abundance are usually much lower in rivers than in lakes. Further, most of what is present seems to be derived from upstream lakes or secondary channels with minimal reproduction within the channel (Saunders and Lewis, 1989). In some instances, the number of species is very high, but usually only a few of these are numerically signiﬁcant (10). The density of rotifers encountered in lotic (ﬂowing) systems is usually much smaller than that commonly present in lentic (lakes) ones (ca. 5 – 400 individuals/L), but in some instances it may be quite high (ca. 3,000 individuals/L). 210 Robert Lee Wallace and Terry W. Snell FIGURE 16 Depth distribution of Keratella crassa in a small, shallow lake during a 48 h period in the summer of 1980. (A) Total population; (B) nonovigerous population; (C) ovigerous population; (D) egg ratios calculated for each sampling point; (E) egg ratio and mean density of the entire population throughout the water column. Kite diagrams express the population as relative percentages for each sampling point, while the line indicates the mean depth of the entire population. (From Magnien and Gilbert, 1983, with permission.) 3. Biogeography Early studies of rotifer distributions were dominated by the belief that all rotifers are cosmopolitan, an idea supported by the fact that the resting eggs of monogononts and the anhydrobiotic stages of bdelloids are transported by birds and other mechanisms. Such passive dispersal, it was argued, is very effective at ensuring that most species become globally distributed. However, data has accumulated suggesting that this conclusion was premature. Rotifers may not be as easily dispersed as previously thought (Jenkins and Underwood, 1998). Green (1972) showed a latitudinal zonation in certain planktonic rotifers, and Pejler (1977b) provided data demonstrating that some species have restricted distributions. In general, endemism seems to be an important theme in several genera, including Keratella, Notholca, and Synchaeta (Dumont, 1983). Exhaustive inventories of rotifer fauna in a few lakes has suggested that 150 – 160 rotifer species reside in a typical temperate lake as compared to more than 210 species for a tropical lake (Dumont and Segers, 1996). These numbers are likely to go higher as sessile and contractile species become more well known. These authors argue that the theory of cosmopolitanism must be abandoned for rotifers and cladocerans. In comparison to the substantial research effort developed elsewhere, there has been relatively little work on rotifer biogeography in the United States and most of that work was done prior to the 1950s or is limited to speciﬁc geographic areas (e.g., the Great Lakes). Nevertheless, in North America there are several examples of species with restricted distributions in the genera Anuraeopsis, Brachionus, Lecane, and Lepadella. Central America and the southern United States have close afﬁnities to tropical South American fauna, while further north, afﬁnities with Europe are apparent and endemism is strong in Keratella, Notholca, and Synchaeta (Chengalath and Koste, 1987). Dumont (1983) suggested that continental drift and Pleistocene glaciations best explain the current biogeography of rotifers. Most biogeographical analyses of rotifers have been limited to collating information on the location of populations from descriptions found in the literature. This problem has additional difﬁculty because many species undergo periodic polymorphism (cyclomorphosis) (see Section III.A.1), with each population having a slightly different morphology. In the past, this phenomenon confused some workers who considered each morphotype to be a new species. Furthermore, the question of what constitutes a rotifer species becomes problematic as males have never been described for many taxa. Future efforts to clarify these matters probably will be greatly aided by modern techniques in gel electrophoresis (e.g., King, 1977; King and Zhao, 1987; Snell and Winkler, 1984) and by using mating behavior to deﬁne species boundaries (Snell and Hawkinson, 1983; Snell et al., 1988; Snell, 1989). In the future, biogeographic research may play an important role in monitoring environmental conditions by examining the structure of rotifer communities in a variety of lakes subject to environmental pertubations: 8. Phylum Rotifera e.g., heavy metal input (MacIsaac et al., 1987) and acid precipitation (Frost et al., 1998). 4. Colonial Rotifers Most rotifers are solitary and interact only as potential prey or mates, but about 25 species (in eight genera) of the class Monogononta form permanent colonies (Wallace, 1987). All colonial forms are members of two families (Flosculariidae and Conochilidae) of the order Flosculariacea (Fig. 17). None of these taxa are predators and all reproduce in a fashion typical of monogononts. There appears to be a relationship between coloniality and sessile existence. About 70% of colonial forms (18 species) are sessile, but even more 211 striking is the fact that all seven genera of the family Flosculariidae have colonial species. This has led some workers to suggest that sessile species are somehow preadapted for the evolution of coloniality (see Wallace, 1987 for a review). The form of colony production has signiﬁcant implications. Because colonial rotifers do not reproduce by budding or the formation of specialized zooids, colony members are not intimately connected, as are the colonial bryozoans (Chapter 14); therefore, energy resources cannot be shared among colony members. Some colonial rotifers produce tubes from either hardened secretions (e.g., Limnias), pellets (e.g., Floscularia conifera), or gelatinous secretions (e.g., Lacinularia and Conochilus). These tubes are FIGURE 17 Colonial rotifers. (A) A portion of a small, planktonic colony of Conochilus unicornis: animals live within a gelatinous mass which they secrete, also present are many small euglenoids; (B) a small, sessile colony of Floscularia: adults reside within tubes they construct of tiny pellets of compacted detritus; and (C) a large sessile colony of Sinantherina. 212 Robert Lee Wallace and Terry W. Snell important in the overall structure of the colony as they provide a substrate or matrix for the addition of new members to the colony (see Section III.A.5). The number of individuals in a colony varies greatly among genera. Floscularia ringens usually builds colonies with fewer than ﬁve individuals, as do some members of the family Conochilidae. In this family, small colonies, composed of only an adult and oneto-several young, are produced by some species, while others may have more than 250 individuals within a colony. Although most colonial species construct small colonies of fewer than 5 – 24 individuals, some routinely produce large colonies of 50 – 200 individuals (e.g., Floscularia conifera, Sinantherina socialis, Conochilus hippocrepis), and a few produce colonies of truly gargantuan size (1000; Lacinularia). Colonies form by one of three different methods, but these methods produce colonies that may have a different degree of genetic relatedness among colony mates (Table I). In allorecruitive (Greek, allo, “other”) colony formation, free-swimming young (larvae) produce colonies by settling on tubes of sessile adults. Thus, the colonies of allorecruitive species grow in size (numbers of individuals per colony) and in the number of colonies within the habitat (colony density) by intercolonial recruitment of young. Because the larvae joining these colonies may come from females belonging to another colony, genotypic relatedness within the colony is probably low. These colonies are transitory, beginning when larvae attach to a previously settled adult and ending when larval recruitment to an old colony ceases. Late recruits to an allorecruitive colony may suffer reduced fecundity, because death of the founding individual might lead to dislodgment from the substrate and death of all members of the colony. A few allorecruitive species produce interspeciﬁc colonies of two or more species. Some species of the genus Floscularia reproduce this way. TABLE I In autorecruitive (Greek, auto, “self”) colony formation, the young remain with their mother in the colony. Thus, the colonies of autorecruitive species grow in size (numbers of individuals per colony) by intracolonial recruitment of young back into the parental colony and in the number of colonies within the habitat (colony density) by colony ﬁssion. Because the young joining these colonies come from the parent colony, genotypic relatedness within the colony is probably high. Here autorecruitive colonies are long-lived and develop continuously throughout the season, increasing in size as new individuals are added and decreasing only if the colony divides. Late recruits to these colonies probably do not suffer diminished fecundity because the colony continues though individuals die. Autorecruitive species rarely produce interspeciﬁc colonies. Species of the genera Conochilus and Conochiloides reproduce this way. In geminative (Latin, gemin, “double”) colony formation, all the young born within a span of a few hours leave the parent colony as a free-swimming larval colony. Members of this young colony subsequently explore and attach to a new substrate in concert, but they may join an older colony, thus genotypic relatedness within the colony is probably moderate to high depending on the particular history of the colony. Thus, because the size of the larval colonies depends mainly on the fecundity of the parental colony, the number of individuals in a colony does not increase over its life time. Geminative species rarely produce interspeciﬁc colonies, but it is possible. Species of the genera Lacinularia and Sinantherina reproduce this way. The consequences of high genetic relatedness have not been explored, but may include increased vulnerability to parasites, uniformity of behaviors, and decreased genetic diversity of resting eggs. Colony Formation in Rotifers Colony formation category Increase in colony size (individuals/colony) Allorecruitive (intercolonial) Autorecruitive (intracolonial) Young are recruited to established colonies or remain solitary Young stay within the parental colony Geminative Rare-to-absent Mode of increase of colony density in the habitat Predicted genotypic relatedness within colonies Frequency of interspeciﬁc colony formation Young leave the parental colony and may establish a new colony Fission: adult colonies fragment producing smaller colonies Cohorts of larvae collectively establish new colonies Low Common Floscularia (Fig. 17B & 43A) High Absent Conochilus (Fig. 17A) Moderate-to-high Rare-to-absent Lacinularia (Fig. 5C) Sinantherina (Figs. 17C) Examples 8. Phylum Rotifera Two hypotheses have been offered to explain the adaptive signiﬁcance of coloniality in rotifers. One hypothesis suggests that colonial animals possess an energetic advantage over solitary individuals of the same species; for example, colonial F. conifera apparently live longer and mature faster than the solitary individuals (Edmondson, 1945). Juxtaposition of ﬁltering currents produced by two individuals may permit increased ﬁltering rate and/or enhanced ﬁltering efﬁciency. Although experiments have not supported the idea that coloniality affects ﬁltration rates, Wallace (1987) has provided some information that supports the view that coloniality increases ﬁltering efﬁciency. A second hypothesis argues that colonial existence can protect individuals from certain predators. Gilbert (1980b) and Diéguez and Balseiro (1998) showed that large Conochilus colonies are less vulnerable to attack by certain invertebrate predators, because they are too large to be engulfed whole and because individual rotifers can retract into the refuge of the gelatinous matrix of the colony. 5. Sessile Rotifers Sessile species are found in two families of rotifers: Flosculariidae (7 genera) and Collothecidae (5 genera). Although they are often overlooked because their habitats generally are not examined thoroughly, these forms are actually quite common in lakes and rivers in North America, occasionally reaching very high densities on plant surfaces (6 individuals/mm2) especially in bogs and small eutrophic ponds (Wallace, 1980; Wallace and Edmondson, 1986). FIGURE 18 213 The juvenile motile stages of sessile rotifers (Fig. 5) are not considered to be larvae by some researchers, but others have used this term and there is a conceptual parallel to other sessile invertebrates that undergo an extensive metamorphosis after settlement (Wallace, 1980). Not surprisingly, the behavior of larvae changes dramatically once they come into contact with a potential attachment site. These new behaviors have been described using terms such as selection, choice, and preference. However, it should be recognized that the use of such words does not imply cognition by the rotifer; they are merely convenient terms to describe this phenomenon. Several workers have demonstrated that larvae can select a particular substrate from among those available for settlement (Wallace, 1980). For example, larvae of F. conifera settle with a greater frequency on the tubes of conspeciﬁcs than on aquatic plants, although there is substantially more plant surface available. This propensity leads to the formation of intraspeciﬁc colonies, each with 50 or more individuals. During the growing season, about 75% of the entire population may be colonial (Wallace, 1977). Some species apparently attach to a surface based on the chemistry of the water in the vicinity of the substrate. Wallace and Edmondson (1986) have shown that greater than 90% of Collotheca gracilipes larvae prefer the undersurface of Elodea canadensis leaves to the upper surface (Fig. 18). Choice of the undersurface is apparently in response to the way Elodea can alter the concentration of calcium ions in the water immediately around the leaf. In water having a pH in the Collotheca gracilipes colonizing the apical meristern of the aquatic macrophyte Elodea canadensis. Many (25) individuals may be seen attached to the under surfaces of the leaves. (Length of the lowest leaf is ⬇ 1 cm.) (From Wallace and Edmondson, 1986, with permission.) 214 Robert Lee Wallace and Terry W. Snell neutral-to-alkaline range, Elodea acts as a polar plant, removing Ca2 from beneath the leaf and releasing it above. This choice provides a superior habitat in comparison to the upper surface. In short-term, laboratory experiments, young C. gracilipes that attached to undersurfaces of Elodea leaves grew signiﬁcantly taller and produced more eggs per female than those attached to the upper surfaces of the same leaves. Several other species of sessile rotifers exhibit strong preferences for particular substrates, but the signiﬁcance of these associations has not been fully elucidated (Wallace, 1980). Larval substrate selection behaviors have been described for three species: Ptygura beauchampi, Sinantherina socialis, and C. gracilipes. In general, larvae appear to react to potential surfaces in similar ways. Very young larvae avoid settling for periods up to several hours after hatching. Once this refractory period is past, all surfaces are explored, but some (i.e., the preferred substrates) receive much more attention and elicit different behaviors, including some reminiscent of male mating behavior (see Section III.B.2). A larva will traverse these surfaces with both its corona and foot in contact with the surface, occasionally stopping in this slightly bent position. The larva may continue exploration of the surface for several minutes, but eventually it attaches to the surface using cement from glands in the foot and then undergoes metamorphosis. Immediately after attachment and metamorphosis have occurred, the young of most sessile rotifers begin to secrete a protective tube. Often this secretion is in the form of a clear, gelatinous material (e.g., Collotheca and Stephanoceros), but in others the tube becomes somewhat opaque by the adherence of debris and colonizing microorganisms (e.g., several Ptygura species and Lacinularia). One species of Limnias forms a cement tube that looks like a series of rings, placed one on top of another. Perhaps the most fascinating example of tube construction is found in the genus Floscularia. Some species in this genus possess a small ciliated cup on the ventral side of the head to which some tiny mineral particles and other small debris collected by the corona are shunted. The ciliated cup appears to be in constant motion, mixing gelatinous secretions with the particles to form small pellets, either in the shape of bullets or balls. Once a pellet is fully formed, the rotifer places it on top of the tube in an action resembling the movements of a bricklayer. In this way the tube is constantly elongated as the animal grows. Because pellets are manufactured from particles collected from the water, they may have a greenish tint, but they are usually brown. When a heavy rainstorm temporarily suspends soil in the water, the pellets that are produced by Floscularia usually turn out to be very dark. Thus, the event of the storm is marked as a dark ring in the tubes of all the animals alive at that time. Edmondson (1945) noted this and used suspensions of powdered carmine and carbon black to mark the tubes of F. conifera, so that he could study the dynamics of population growth and various aspects of rotifer life history in a ﬁeld population (See Fig. 17B). B. Reproduction and Life History 1. Reproduction The type of reproduction varies among the three classes of rotifers (Wallace, 1998). Species in the class Seisonidea reproduce exclusively through bisexual means, with gametogenesis occurring via classical meiosis and the production of two polar bodies. At the other extreme, members of the class Bdelloidea reproduce entirely by asexual parthenogenesis, a process which includes two equational divisions producing two polar bodies. No males have been observed in bdelloids. Species in the class Monogononta exhibit cyclical parthenogenesis where asexual reproduction predominates, but sexual reproduction also occurs occasionally. a. Cyclical Parthenogenesis Cyclical parthenogenesis in monogononts (Fig. 19) takes place in the absence of males (amictic phase), but periodically males are FIGURE 19 Generalized life cycle for monogonont rotifers. The life cycle of bdelloids consists of only the parthenogenetic portion. 8. Phylum Rotifera produced and sexual reproduction takes place (mictic phase). Amictic females are diploid and produce diploid eggs (called amictic eggs) that develop mitotically via a single equational division into females (Wallace, 1998). The term “summer egg” has been applied to the eggs produced by amictic females, but this term is misleading because amictic eggs can be produced at any time of the year, depending on the species in question. Most of the monogonont rotifer life cycle is spent in the amictic phase, but in certain conditions, sexual reproduction occurs concurrently within the population, presumably initiated by speciﬁc environmental stimuli. Upon receiving the mictic stimulus, amictic females begin producing both mictic and amictic daughters. The proportion of mictic daughters and the duration of their production, depends on the strength of the mictic stimulus. Mictic female production is only the initial step in mictic reproduction and is followed by male production, fertilization, and ﬁnally formation of a diapausing embryo called a resting egg or cyst (also winter or fertilized egg) (Fig. 19). Mictic females produce haploid eggs via meiosis that, if unfertilized, develop into haploid males. Males are typically smaller, faster swimming, and shorter lived than females. The males of some species are structually reduced, but extremes exist from fully developed males (Rhinoglena) to a condition in which certain organs are degenerate (Conochilus). Among sessile species the male is freeswimming while the female is permanently attached to a substrate. Fertilized mictic eggs are diploid and develop into resting eggs possessing thick, often sculptured, walls (Fig. 20). These dormant stages are very resistant to harsh environmental conditions and may be dispersed over wide areas by the wind, water, or migrating animals. After a period of dormancy, which varies among species, resting eggs respond to species-speciﬁc environmental cues and hatch, releasing diploid, amictic females that enter into the asexual phase of the life cycle (Fig. 19). The stimuli that induce hatching may include changes in light, temperature, salinity, and oxygen concentration (Wallace, 1998). With a few notable exceptions, the stimulus for initiating sexual reproduction is still poorly understood. Dietary -tocopherol (vitamin E) controls the shift from amictic to mictic reproduction in most Asplanchna species (Gilbert, 1980a), and photoperiod plays a similar regulatory role in Notommata (Pourriot and Clément, 1981). In the genus Brachionus, population density has been most frequently cited as a stimulus for mictic female production (Wallace, 1998; cf, Pourriot et al., 1986). In addition to environmental factors, genetic factors also play a major role in determin- 215 ing the sensitivity of particular strains to mictic stimuli (e.g., Snell and Hoff, 1985; Lubzens et al., 1985). An unusual variation of the standard monogonont life cycle has been observed in three genera: Asplanchna, Conochilus, and Sinantherina. In some populations amphoteric females (individuals that produce both female and male offspring) have been recorded (e.g., Ruttner-Kolisko, 1977b; King and Snell, 1977). Amphoteric rotifers have the ability to produce both diploid (female) and haploid (male) eggs, with some producing both females and resting eggs, or males and resting eggs (Fig. 17). The presence of amphoteric reproduction in other rotifers has not been fully investigated, and its signiﬁcance in the life history of those genera that possess it remains to be determined. There have been reports of the production of pseudosexual eggs; that is, the production of resting eggs via parthenogenesis in the absence of males. However, little in known of this phenomenon. Two different types of parthenogenetic amictic eggs have been described in Synchaeta pectinata (Gilbert, 1995). The traditional type is thin shelled (1.4 m) that develops without diapause. The second type has a thick-shelled (9 m) amictic egg that enters an obligatory diapause after 1 – 3 cleavage divisions. The mean duration of diapause is about 14 days, and it is not greatly extended by low temperature. These eggs are induced immediately after starvation and seem to be produced at no additional energetic cost compared to traditional amictic eggs. The adaptive signiﬁcance of diapausing amicitic eggs seems to be to increase the ability of populations to survive short-term food limitation. Synchaeta pectinata also produce resting eggs following sexual reproduction and these have the capacity for dormancy extending up to several years. b. Resting Eggs The density of resting eggs in sediments has not been routinely examined and the few published studies have used different methods for estimating density. Because of their capacity for extended dormancy, resting eggs theoretically could accumulate to high densities in sediments. Nipkow (1961) found resting egg densities for seven species to range from 100 to 600 eggs/cm2 in lake sediments, but Synchaeta oblonga densities ranged from 3,000 to 4,000 eggs/cm2. Snell et al., (1983) found an average of 200 resting eggs/cm2 for Brachionus plicatilis in the surface sediments of a subtropical brackish pond. These workers showed that the highest densities were on the sediment surface, and numbers declined exponentially to a depth of 7 cm. May (1987) examined resting egg densities in sediments from Loch Leven, Scotland, by recording the number of animals hatching from sediments incubated at temperatures of 5, 10, and 15°C. This technique is 216 Robert Lee Wallace and Terry W. Snell FIGURE 20 Resting eggs of several monogonont rotifers. (A) Asplanchna girodi; (B) Hexarthra fennica; (C) Brachionus calyciﬂorus; (D) Conochiloides natans; (E) Sinantherina socialis; and (F) Kellicottia bostoniensis. (Bars 10 m; the wrinkled backgrounds in panels B and F are the supporting membranes.) (Original scanning electron photomicrographs by Hendrik Segers.) not directly comparable to the others just noted, and probably yields lower estimates of resting egg density (see also May, 1986). Marcus et al. (1994) observed B. plicatilis resting eggs at densities up to 1532 individuals/cm2 in anoxic and sulﬁdic sediments of Pettaquamscutt estuary in Rhode Island. Rotifer resting eggs were abundant as deep as 12 cm into these sediments and were the most abundant among any of the zooplankton taxa present. B. plicatilis resting eggs older than 40 years could be hatched and used to initiate viable populations. The number of resting eggs in sediments is a function of previous resting egg production, mortality in the sediments, and hatching rates. Certain abiotic features 8. Phylum Rotifera 217 such as sedimentation rates and the uneven deposition of sediments in the benthos (sediment focusing) may affect the ﬁnal density at particular sites in a lake or estuary. 2. Reproductive Behavior Detailed descriptions of mating behaviors are available for Asplanchna brightwelli (Aloia and Moretti, 1973), three species of Brachionus (e.g., Gilbert, 1963b; Snell and Hawkinson, 1983), and Anuraeopsis ﬁssa, Asplanchna girodi, Euchlanis dilatata, Keratella americana, Lecane quadridentata, Brachionus patulus, and Trichocerca pusilla (Rico-Martinez and Snell, 1998). Males and females show pronounced sexual dimorphism, males being smaller and faster swimmers (Fig. 2). Lacking a functional foot, males swim constantly without attaching. Because males and females swim randomly, the probability of male – female encounters in planktonic species can be modeled mathematically (Snell and Garman, 1986). In the colonial, sessile rotifer Sinantherina socialis, males may copulate with several females of one colony in succession. Females take no active role in locating a mate or reacting to the male once an encounter occurs. Males, in contrast, display a distinct mating behavior upon encountering conspeciﬁc females. Mating behavior begins only when the corona of the male squarely contacts the female. However, not all head-on encounters result in mating; indeed, the probability of copulation in laboratory cultures generally varies from 10 to 75% in Brachionus plicatilis, depending on the strain (Snell and Hawkinson, 1983). This requirement for head-on contact by the male is due to the presence of chemoreceptors in his coronal region, which apparently respond only to a speciesspeciﬁc glycoprotein on the surface of the female (Snell et al., 1995). Mating begins with the male swimming circles around the female, skimming over the surface of her lorica (Fig. 21). During this phase, the male maintains contact with the female with both his corona and penis (this requires the male to remain in a slightly bent position). After several seconds of circling, the male attaches his penis to the female, usually in the region of her corona, and loses coronal contact. After about 1.2 min of copulation in B. plicatilis (Snell and Hoff, 1987), sperm transfer is completed and copulation is terminated when the male and female break apart and swim away. Newborn B. plicatilis males only have about 30 sperm and transfer two or three at each insemination (Snell and Childress, 1987). 3. Aging and Senescence Rotifers have long been used as models of aging and senescence. Jennings and Lynch (1928) conducted FIGURE 21 Male mating behavior in brachionid rotifers. Arrows indicate general swimming movements by male and female rotifers. (Male not drawn to scale.) the ﬁrst major study of aging in rotifers using the monogonont Proales sordida. They found the mean lifespan to be 8 days at 20°C, but a maternal effect was noted. Offspring from older mothers had shorter lifespans and more variability in their developmental rates and fecundities. Maternal effects were a theme expanded upon by Lansing who, in a series of papers during the 1940s and 1950s, showed that parental age also inﬂuenced longevity in the bdelloid Philodina citrina (see King, 1969 for a review). Lansing established “orthoclones” by isolating the ﬁrst offspring of parental females, then the ﬁrst offspring of F1 females, and so forth, yielding a clone derived exclusively of the ﬁrst-born females. Orthoclones derived from the offspring of older females also were developed. This protocol is a powerful tool for investigating effects on aging because the only difference among orthoclones is the age of their mothers. In P. citrina, mean lifespans of 5-, 11-, and 16-day orthoclones were 23.8, 18.1, and 16.6 days, respectively. Lansing’s general conclusion was that young orthoclones have increasingly longer lifespans, while older orthoclones have progressively shorter lifespans, resulting eventually in clonal extinction. Rate of senescence, Lansing hypothesized, was controlled by a maternal effect that was transmissible and cumulative. However, the effect was reversible since the ﬁrst-born females from an old orthoclone outlived their parents. Based on 218 Robert Lee Wallace and Terry W. Snell these and other studies, Lansing proposed that calcium is the maternal factor responsible for what is now known as the “Lansing Effect.” Rate of calcium accumulation and its importance in rotifer senescence became the reigning hypothesis of the day. Since Lansing’s work, researchers have reported similar results in some species, but not in others. King (1983) re-examined Lansing’s data using life table analysis and concluded that Lansing probably did not observe an aging effect. King showed that shortlived lines derived from old parents reproduced earlier and at higher rates in succeeding generations. Longlived lines derived from young parents delayed initial reproduction to later age classes. King argued that the Lansing effect actually resulted from shifts in fecundity patterns that concentrated reproduction in a few age classes in short-lived lines. Snell and King (1977) demonstrated that concentrated reproduction in rotifers could shorten lifespan. Until more is known about the physiological basis for the “Lansing Effect”, King suggested that it be viewed with skepticism. The pattern of reproduction also modiﬁes longevity (Snell and King, 1977). Female Asplanchna brightwelli with high rates of reproduction concentrated early in life have shorter mean lifespans (2.5 days at 25°C) than females with slower reproduction distributed over a greater portion of the life- span (mean lifespan 4.5 days). The length of prereproductive and postreproductive periods were similar for short- and long-lived individuals. However, length of the reproductive period was about twice as long in long-lived individuals. These data suggest that reproduction inﬂuences individual survival negatively, but deleterious effects are minimized when the reproductive period is spread over a greater portion of the lifespan. Further, Rougier and Pourriot (1977) found that maternal age is negatively correlated with mictic female production. When Brachionus calyciﬂorus females began reproducing at age 2 days at 15°C, 80 – 90% of their daughters were mictic. By age 8 days, only 25% of daughters were mictic, and reproduction by 12 day old females yielded no mictic daughters. This decline in mictic female production with age was linear in 2-, 6-, and 10-day orthoclones. Male lifespan has been examined far less thoroughly than that of females, but available data indicates that males live only about half as long as females (Fig. 22) (King and Miracle, 1980; Snell, 1977). Other experiments on aging in rotifers have indicated that vitamin E (a-tocopherol) can extend mean lifespan about 10% at 23°C in the bdelloid Philodina sp., but maximum lifespan is unaffected (Enesco and Verdone-Smith, 1980). Vitamin E, at a concentration of 20 g/mL, also was found to extend the lifespan FIGURE 22 Female and male survival of three clones of Brachionus plicatilis at 25°C. Clone LA is from LaJolla, CA; clone MC is from McKay Bay near Tampa, FL; clone SP is from Castellon, Spain. Note that the Y axis (survivorship %) is a log scale. (From King and Miracle, 1980, with permission.) in A. brightwelli (Sawada and Enesco, 1984). Of several other antioxidants tested, however, only thiazolidine-4-carboxylic acid (TCA) extended the mean life span signiﬁcantly, presumably by quenching free radical reactions. Other factors that affect mean life span in A. brightwelli are photoperiod (light:dark cycle, L:D), dietary restriction (Sawada and Enesco, 1984), and ultraviolet (UV) radiation (Enesco, 1993). Rotifers exposed to a 6:18 or 0:24 L:D photoperiod showed an 18% and a 22% increased life span, respectively, over a control photoperiod of 12:12 Dietary restriction has lifeextending effects in a wide variety of animals. In A. brightwelli, two different modes of dietary restriction yielded longer life spans. Reduction of food intake increased life span by 14.2%, whereas intermittent feeding produced an 11.8% longer life span. Ultraviolet irradiation in the 200 – 4800-J/m2 range shortened life span signiﬁcantly, and life span declined logarithmically as UV dose increased. 4. Population Dynamics Studies of rotifer population dynamics attempt to explain the causes of changes in population size. By separating and quantiﬁng the relative contributions of reproduction, mortality, and dispersal, ecologists are able to identify the factors regulating population size and the determinants of average abundance. Because some species are easily cultured and experimentally manipulated, rotifers have proven to be useful models for investigating the dynamics of animal populations. Techniques also exist for investigating the dynamics of natural rotifer populations, further broadening their usefulness in ecological studies. a. Life Tables The most rigorous approach to studying population dynamics is lifetable analysis. Life 8. Phylum Rotifera tables are age-speciﬁc records of all reproduction and mortality occurring in a population. From these observations, vital population statistics can be calculated such as intrinsic rate of increase (rm), net reproductive rate (Ro), life span, generation time, reproductive value, and stable age distribution. The general protocol for gathering lifetable data on rotifers is to collect a cohort of newborn females, isolate them in small volumes of culture medium (ca. 1 mL), and make daily observations of their survival and reproduction. Maternal females usually are transferred daily to fresh medium with a speciﬁc food level, and offspring are counted and removed. A table is then constructed of age, number surviving in each age class, and number of offspring produced in each age class. At least 10 replicate females are grouped into each population and probability of surviving to age x (lx) and age-speciﬁc fecundity (mx) are calculated. The detailed observations required for life-table analysis are usually only possible in laboratory populations and a number of genera have been examined in that way, including several species of Brachionus (Halbach, 1970; Pourriot and Rougier, 1975; King and Miracle, 1980; Walz, 1987), Asplanchna (Gilbert, 1977; Snell and King, 1977), and bdelloids (Ricci, 1983). In certain cases, it is possible to perform life table analyses on ﬁeld populations (i.e., Floscularia conifera, Edmondson, 1945). Although these and other studies have examined survival and reproduction of parthenogenetic females, survivorship curves for male rotifers also are available for A. brightwelli (Snell, 1977) and B. plicatilis (King and Miracle, 1980). The major environmental factors affecting age-speciﬁc survival and reproduction are temperature, food quantity and quality, genetic strain, and reproductive type (either amictic, or fertilized or unfertilized mictic females). Increased temperature shortens survivorship. In B. calyciﬂorus, for example, 50% of a cohort survive to age 16 days at 15°C, 11 days at 20°C, and 5 days at 25°C (Fig. 23). Age-speciﬁc fecundity also is compressed at higher temperatures. At 15°C, fecundity occurs at a low rate over 20 days. In contrast, at 25°C reproduction occurs at a high rate and is compressed into 10 days. Females produce a total of 13 offspring at 15°C, 16.6 at 20°C, and 12.9 at 25°C. The intrinsic rates of increase (rm) calculated from these lx and mx values are 0.34, 0.48, and 0.82 offspring per female per day at the respective temperatures (Halbach, 1970). The effect of food quantity on lx and mx schedules is illustrated in Figure 24. Brachionus calyciﬂorus was fed the green alga Chlorella at densities of 0.05 – 5 106 cells/mL at 20°C. The best survival occurred at algal concentrations 0.5 106 and 1.0 106 cells/mL, where mean life span was 9 days (Halbach and 219 Halbach-Keup, 1974). Life span decreased at lower food levels, reaching 2.5 days at 0.05 106 cells/mL. Lower life spans also were recorded at food concentrations above 1.0 106 cells/mL, probably the result of accumulation of algal metabolic products that are toxic to rotifers. Fecundity also peaked at 1.0 106 cells/mL, with a mean of 17 offspring per female. In contrast, at 0.05 106 cells/mL, lifetime fecundity was only 0.5 offspring per female whereas 3 offspring per female were produced at 5.0 106 cells/mL. Intraspeciﬁc differences in survival among strains is illustrated in Figure 22. King and Miracle (1980) examined three strains of B. plicatilis and found that their mean life spans at 25°C ranged from 6 to 13.5 days. Because these strains were acclimated to, and tested in, a common environment, the observed differences must be genetic. Also shown in the same ﬁgure are male survivorship curves; interstrain differences are again apparent with male life spans. The three different female types (amictic, and unfertilized and fertilized mictic) differ markedly in their agespeciﬁc survival and fecundity. In Brachionus urceolaris, for example, amictic females live at 20°C an average of 9 days and produce 20 female offspring; unfertilized mictic females live 9.5 days and produce 25 male offspring; and fertilized mictic females live 10 days and FIGURE 23 Effect of temperature on age-speciﬁc survival and fecundity of Brachionus calyciﬂorus. The left Y axis is the percent of the cohort surviving (open circles) and the right Y axis is the offspring per female per hour (closed circles). (From Halbach, 1970, with permission.) 220 Robert Lee Wallace and Terry W. Snell FIGURE 25 The fecundity schedule and egg developmental times of three types of Brachionus urceolaris females at 20°C with excess food. Bottom panel: amictic females producing diploid female eggs; middle panel: unfertilized mictic females producing haploid male eggs; top panel: fertilized females producing diploid resting eggs (solid circles). Arrows in the top panel represent the time of resting egg attachment to fertilized mictic females(hrs). Each bar in the middle and lower panels represents one egg and the length of the bar indicates the developmental time for that egg(days). (Modiﬁed from Ruttner-Kolisko, 1974, with permission.) FIGURE 24 Effect of food level on age-speciﬁc survival and fecundity of Brachionus calyciﬂorus. The left Y axis is the percent of the cohort surviving (closed circles) and the right Y axis is the offspring per female per hour (open circles). The food is Chlorella pyrenoidosa provided at algal doses (AD) in the ( 0.05 – 5) 106 per ml/range. (From Halbach and Halbach-Keup, 1974, with permission.) produce four resting eggs (Fig. 25). The greatly reduced fecundity of fertilized females is typical of other rotifer species and is likely due to higher energy requirements for resting egg formation (Gilbert, 1980a). Also reported in Figure 25 are development times for each egg (time from egg extrustion to hatching) and the birth intervals between eggs. Birth intervals are shortest in the middle of the reproductive period. Individuals within a single rotifer population differing in their growth forms (e.g., spined and unspined) can possess very different intrinsic rates of increase under identical culture conditions. Stemberger (1988) has shown that the rm value for spined and unspined forms of Keratella testudo can differ signiﬁcantly, but the difference depends on food concentration (Fig. 26). At low food levels, there is no signiﬁcant difference in rm for the two forms of this rotifer. At higher food concentrations, however, the unspined form had much greater values of rm. Presumably, a similar phenomena will be found in other species in which there are spined and unspined forms (see Sections III.A.1, and III.C.4). When life-history patterns of zooplankton are compared, rotifers have higher rm values than either cladocerans or copepods (Allan, 1976). The rm values of these groups range from 0.2 to 1.6, 0.2 to 0.6, and 0.1 to 0.4 offspring per female per day, respectively. Rotifers achieve their high population growth rates because of short development times, which more than compensate for their small clutch sizes. Rotifers also FIGURE 26 Intrinsic rates of population growth of spined (open Figures) and unspined (closed Figures) Keratella testudo. Dashed line, r 0. (Modiﬁed from Stemberger, 1988, with permission.) 8. Phylum Rotifera show the greatest response to increased temperatures. The high population-growth rates of rotifers may be an adaptive response to predation, against which most rotifer species have few defenses (Stemberger and Gilbert, 1987a). As a result of these characteristics, rotifers are regarded as being able to quickly exploit new conditions. b. Dynamics of Field Populations Annual cycles of natural rotifer populations have been characterized for several species. One particularly thorough multiyear study was conducted by Herzig (1987) in Neusiedlersee, a shallow, well-mixed Austrian lake. Figure 27 shows 12 years of data for the abundance of 13 rotifer species. Some species, like Keratella quadrata, have relatively stable population sizes and occupy the lake nearly continuously. In contrast, abundances of other species, such as Rhinoglena fertoensis, Filinia longiseta, and Hexarthra fennica, ﬂuctuate much more. Fluctuation of rotifer abundance over two or three orders of magnitude during a seasonal cycle is typical of many natural populations. Techniques have been developed to characterize the dynamics of natural zooplankton populations (Edmondson, 1993). The instantaneous rate of increase of a population (r) is the difference between instantaneous birth (b) and death (d) rates rbd (1) 221 The value of r for a given population may be estimated from the equation r ln Nt ln N0 T (2) This procedure requires two successive estimates of population size (No and Nt) separated by time interval T. The estimate of r is based on several assumptions about the population, including that it is growing exponentially with a stable age distribution. One can also estimate the birth rate in this population by counting the number of eggs carried per female. The ﬁnite hatching rate is B E D (3) where E is the number of eggs per female and D the developmental rate at a speciﬁc temperature. The value E is determined directly from samples of the population and values of D by observing hatching of eggs from a sample brought back to the laboratory. For example, Herzig (1983) reports values of D for several species. When E and D have been calculated, the instantaneous birth rate, b, can be estimated from the following equation by Palohiemo (1974) b ln (E 1) D FIGURE 27 Phenologies of several important rotifer species in Neusiedlersee, Austria. The X axis is the season (winter, spring, summer, and autumn) and the Y-axis the rotifer abundance per cubic meter. (From Herzig, 1987, with permission.) (4) 222 Robert Lee Wallace and Terry W. Snell With these data, it is possible to estimate the death rate, d, by subtraction dbr (5) The egg ratio technique makes it possible to predict growth of natural rotifer populations where reproduction by amictic females is parthenogenetic. A few simple measurements allow ecologists to estimate important population parameters that summarize processes of birth and death occurring in the population. This technique has proven useful for investigating the population dynamics and secondary production of zooplankton (e.g. Edmondson and Litt, 1987). However, before applying the egg ratio method, the works of Edmondson (1993) and Palohiemo (1974) should be consulted. The dynamics of natural rotifer populations are affected by a number of environmental factors including temperature, food quantity and quality, exploitative and interference competition, predation, and parasitism. Temperature is a major factor affecting fertility, mortality, and developmental rates. In general, higher temperatures do not increase the number of offspring produced per female, but instead, shorten birth intervals by decreasing developmental time (Edmondson, 1993; Pourriot, 1965). Life span also is reduced, but the net effect of higher temperatures is an increased population growth rate. In laboratory studies, higher temperatures and elevated food levels combine synergistically to increase signiﬁcantly mean rate of population increase (r) of Brachionus calyciﬂorus (Fig. 28). Other abiotic environmental factors, such as oxygen concentration, light intensity, and pH, also inﬂuence rotifer population dynamics (see Hofmann, 1977 for a review). Food availability is a major biotic factor regulating rotifer population growth. Species have markedly different food requirements for reproduction. Threshold food concentration, the food level where population growth is zero, was determined for eight species of planktonic FIGURE 28 Synergistic effects of temperature and food density on mean rate of population increase (rm) of Brachionus calyciﬂorus. (From Starkweather, 1987, with permission.) FIGURE 29 Threshold food levels where population growth rate (rm) 0. Food concentration is algal dry mass/mL; rm is offspring per female per day. The solid line indicates food concentrations required to maintain a population growth of zero. Values above the dashed line (positive rm) indicates increasing population densities; values below the solid line (negative rm) indicate decreasing population densities. Species code: Kc Keratella cochlearis, Ke Keratella earlinae; Kcr, Keratella crassa; So, Synchaeta oblonga; Sp, Synchaeta pectinata; Bc, Brachionus calyciﬂorus; Pr, Polyarthra remata; and Ap, Asplanchna priodonta. (From Stemberger and Gilbert, 1985b, with permission.) rotifers by Stemberger and Gilbert (1985). Small species like Keratella cochlearis had lower threshold food concentrations than larger species like Asplanchna priodonta or Synchaeta pectinata (Fig. 29). The logarithm of the threshold concentration was positively related to the logarithm of rotifer body mass, so the smallest species had the lowest food thresholds. The food concentration required to support 50% of the maximum population growth rate (rmax/2) varied 35-fold among the eight rotifer species and also was positively related to body mass. Small rotifer species, therefore, appear best adapted to food-poor environments because those species possess low food thresholds. Larger species, in contrast, are better adapted to food-rich environments, where they have higher reproductive potentials. c. Genetic Variation Our knowledge of rotifer genetics is in its infancy. An early worker, Hertel (1942) observed strong inbreeding depression in ﬁve strains of Epiphanes senta after one generation of inbreeding by self-fertilization. Net fecundity (R0) was reduced 70 – 80% in the F1 generation and continued to decline slowly in F2 and F3 generations. Birky (1967) found similar results in Asplanchna brightwelli, with a 25 – 45% reduction in percentage of resting eggs hatching after one generation of selﬁng. Inbreeding increases homozygosity, thus potentially revealing deleterious recessive alleles previously masked by dominance. Inbreeding depression suggests that substantial amounts of genetic variability exist in natural rotifer populations. Electrophoretic analysis of allozymes may be used to characterize genetic variation in natural populations. 8. Phylum Rotifera Although application of these techniques to rotifers has been very limited, allozymes have been used to distinguish strains. Snell and Winkler (1984) analyzed ﬁve enzymes representing seven loci in 17 strains of Brachionus plicatilis from all over the world. This work showed no relationship between geographic proximity and genetic similarity. In the same species, Serra and Miracle (1985) found low allozyme variation, with only one or two electrophoretic patterns present in each population. These researchers suggested that this low variability resulted from intense inbreeding and interclonal competition, both of which lead to stabilization of just a few highly adapted genotypes. Although genetic variance within populations was low, Serra and Miracle found substantial variance between populations. In contrast, King and Zhao (1987) demonstrated that several electromorphs were present in each collection of B. plicatilis from Soda Lake in Nevada, rather than a few dominant types as reported by Serra and Miracle (1985). King and Zhao also observed no seasonal succession of genotypes and concluded that B. plicatilis in Soda Lake follow a pattern of incomplete genetic discontinuity. In this pattern, several genotypes persist throughout the year, but their frequencies increase or decrease in response to environmental changes (King, 1972). Allozyme variation was analyzed in ﬁve populations of the bdelloid Macrotrachela quadricornifera using six enzymes (Pagani, et al., 1991). Relatively high polymorphism was found, with only 27% of alleles shared by all strains. A variety of perspectives on the genetic structure of rotifer populations have been offered. Ruttner-Kolisko (1963) argued that most rotifer populations are highly homogeneous due to founder effects and strong selection eliminating all but a few genotypes. As a result, bisexual reproduction in rotifers is characterized by extreme inbreeding. Such a genetic structure produces fragmented gene pools where geographically separate populations also are isolated genetically. Birky (1967) disagreed with this view, stating that the substantial heterozygosity revealed by inbreeding experiments suggests that rotifer populations are not genetically homogeneous. The great morphological similarity among geographically separated populations (Pejler, 1977a; Dumont, 1983) further supports the idea that rotifer gene pools are better integrated than Ruttner-Kolisko proposed. Seasonal changes in rotifer population structure have been characterized by King (1972) who proposed three models; one based on physiological adaptation; and two based on genetic changes. Working with Asplanchna girodi in a small pond in Florida, King (1977, 1980) concluded that the complete genetic discontinuity model best described his observations. Asplanchna girodi actually had a succession of genetically distinct populations, each hatching from resting eggs, complet- 223 ing their life cycle, and returning to dormancy as the next population was emerging. Little overlap occurred between populations, thus making them completely discontinuous. Competition (Snell, 1979) and the presence of cyanobacteria (Snell, 1980) were major ecological factors promoting succession among these rotifer populations. The population structure differs from the incomplete genetic discontinuity structure that King and Zhao (1987) reported for B. plicatilis in Soda Lake. Experimental data from interpopulation crosses also illuminate the genetic structure of rotifer populations. Some strains of rotifers can mate successfully with strains from widely separated habitats, while others such as Asplanchna brightwelli (Birky, 1967; King, 1977) are reproductively isolated. Behavioral reproductive isolating mechanisms are well developed in rotifers and can be used to deﬁne species boundaries (Snell, 1989). Gilbert (1963b) investigated this phenomenon in Brachionus calyciﬂorus and found that mating behavior was strictly speciesspeciﬁc; B. calyciﬂorus males failed to mate with females of B. angularis, B. quadridentatus, Synchaeta or Euchlanis. Although Snell and Hawkinson (1983) reported no behavioral reproductive isolation among temporally separated populations of B. plicatilis from the same bay, reduced probabilities of copulation among spatially and geographically isolated populations revealed incipient behavioral isolation. Reproductive isolating mechanisms are not always effective barriers to gene ﬂow. Pejler (1956) argued that the morphological intermediates of Polyarthra vulgaris and P. dolichoptera he found in several Swedish tarns were the result of interspeciﬁc hybridization. RuttnerKolisko (1969) provided some direct evidence of interspeciﬁc hybridization when she successfully hybridized B. urceolaris and B. quadridentatus. Resting eggs were formed from this cross (in both directions), but they could be hatched only when female B. urceolaris were crossed with male B. quadridentatus. Asymmetry in hybridization is typical of closely related species and suggests that reproductive isolation between B. urceolaris and B. quadridentatus has yet to be completed. C. Ecological Interactions 1. Foraging Behavior Although the diets of some rotifers are highly specialized (Bogdan and Gilbert, 1987), many species consume a wide variety of both plant and animal prey and may be described as generalist suspension feeders (Walz, 1997). For example, Asplanchna species generally are considered predatory, yet their main food often can be large algae. In contrast, the “herbivorous” Brachionus and Ptygura will also eat small ciliates. 224 Robert Lee Wallace and Terry W. Snell Thus, distinctions between predator and herbivore in rotifers often are not clear; it is more constructive to consider the ways in which rotifers encounter and process food items rather than what they eat. Research on food selection by rotifers began by correlating the density of natural populations of rotifers to the food available (Edmondson, 1965) and by determining food selectivities using laboratory cultures and/or natural populations (Bogdan and Gilbert, 1982, 1987; Salt, 1987). In the latter studies, natural foods (i.e., algae, bacteria, yeast) or artiﬁcial materials (e.g., latex microspheres) are used to observe rotifer feeding behavior and to calculate feeding rates. Natural foods have been labeled using radioisotopes, but loss of label from the food can result in signiﬁcant errors. However, even the simple procedure of adding a few drops of food suspension to a Petri dish with rotifers may lead to interesting observations concerning how rotifers process food. For example, using powdered carmine, Wallace (1987) demonstrated that adult Sinantherina socialis do not act independently of one another when in a colony. Instead, the coronae of the animals in a large section of the colony all face in the same direction for several minutes. Animals exhibiting this behavior are called an array. Arrays determine where the dominant water currents ﬂow to the colony, how the water currents ﬂow around the colony, and where the currents leave the colony. Rotifers feed in ways that are directly related to their general life history. Although most planktonic rotifers, such as Asplanchna, Brachionus, Polyarthra, and Rhinoglena, swim at similar rates through comparable areas of the water column, the ways in which they encounter and capture food can be quite different. Brachionus uses its swimming currents to sweep small food particles into the buccal region for processing; this is often termed ﬁlter or suspension feeding. Asplanchna, on the other hand, does not use its swimming currents to gather food. Once this rotifer contacts a potential food item with its corona, it may or may not attempt to ingest the item based on such factors as hunger level and the size and type of prey (Gilbert and Stemberger, 1985b; Salt et al., 1978). Some benthic rotifers creep along algal ﬁlaments and feed by piercing the ﬁlament and sucking the cytoplasm from the cells; examples are Notommata copeus (Clément et al., 1983) and Trichocerca rattus (Clément, 1987). Sessile rotifers capture food in one of two ways, depending on the family. Members of the family Flosculariidae (e.g., Floscularia, Ptygura, Sinantherina) create feeding currents in a manner similar to the planktonic suspension feeders. In contrast, all collothecid rotifers are ambush raptors (e.g., Collotheca, Stephanoceros). Once a prey enters the infundibular region of the corona of these rotifers, long setae (Collotheca) or arms (Stephanoceros) fold over the prey, capturing it much like the action of a Venus ﬂytrap. All members of the family Atrochidae lack setae (e.g., Cupelopagis). In these forms, an enlarged, sometimes sheetlike corona folds over prey to capture them. In both cases, prey are pushed through the rotifer’s mouth and into the proventriculus where they are stored until the mastax transfers them into the stomach. When prey are particularly abundant, several live prey may be seen in the proventriculus. Once food is captured, the process of ingestion begins, but not all captured food items are consumed. By observing individual Brachionus calyciﬂorus in various densities of suspended food particles, Gilbert and Starkweather (1977, 1978) showed that this rotifer regulated its ingestion of food particles by three different mechanisms. First, rotifers used pseudotrochal cirri to screen certain large particles away from the mouth. Second, particles collected by the corona may be rejected later by cilia within the buccal ﬁeld. Finally, particles that gain entrance to the oral cavity may be returned to the buccal ﬁeld by action of oral cilia for subsequent rejection. The mastax also may actively reject particles. Microphagus rotifers, such as Brachionus and Ptygura, use the mastax to push food (usually larger particles) out of the oral cavity. Members of the genera Asplanchna and Asplanchnopus also use their trophi to remove materials from their stomachs, but the materials removed by these raptors are empty carapaces of hard-bodied prey such as cladocerans. Occasionally, predatory rotifers such as Cupelopagis, may become so engorged that attempting to ingest another prey item results in the loss, from the proventriculus, of one previously captured. 2. Functional Role in the Ecosystem Freshwater zooplankton are dominated by three taxonomic groups (protozoans, rotifers, and microcrustaceans), yet it is the microcrustaceans that usually receive the most attention from researchers. Such a disparity is probably to be expected for two reasons, one signiﬁcant and one trivial. First, microcrustaceans commonly account for a greater proportion of the total zooplankton biomass than either protozoans or rotifers. Second, microcrustaceans are easier organisms to manipulate and observe in laboratory situations. However, although rotifers usually have a smaller standing biomass than microcrustaceans, their high turnover rate makes them very important to the trophic dynamics of freshwater planktonic communities (e.g., Bogdan and Gilbert, 1982, Neil, 1984). Feeding rates of zooplankton generally are referred to as ﬁltration or clearance rates and are measured as 8. Phylum Rotifera microliters of water cleared of a certain food type per animal per unit time (i.e., L/animal/h). Usually rotifer clearance rates are lower than those of cladocerans and copepods, although the rates depend heavily on food type, temperature, and animal size (Bogdan and Gilbert, 1982). For most rotifers, clearance rates are commonly between 1 and 10 L/animal/h, whether determined in the laboratory or in the ﬁeld. However, a few species can achieve levels exceeding 50 L/animal/h (e.g., Bogdan and Gilbert, 1987). Using estimates of clearance rates, Starkweather (1987) noted that even moderately sized rotifers with body volumes of about 10 3 L process enormous amounts of water with respect to their size: 103 times their own body volume each hour! Ingestion rates (biomass consumed per animal per unit time) also are very high for rotifers. An adult rotifer may consume food resources equal to 10 times its own dry weight per day. Therefore, by having assimilation efﬁciences (i.e., assimilation divided by ingestion) of between 20 and 80%, rotifers convert a good deal of their food to animal biomass, which may be passed on to the next trophic level (Starkweather, 1980, 1987). Although microcrustaceans generally have higher clearance rates than rotifers (about 10 – 150 L/animal/h for cladocerans and 100 – 800 L/animal/h for copepods) rotifers can exert greater grazing pressure on phytoplankton than some small cladocerans. Bogdan and Gilbert (1982) report that in a small eutrophic lake, Keratella cochlearis accounted for about 80% of the community grazing pressure on small algae during the year. These two workers also showed that K. cochlearis had ﬁltering rates about 5 – 13 times higher than that for the cladoceran Bosmina longirostris (Gilbert and Bogdan, 1984). Therefore, under certain conditions, rotifers may be important competitors to small, ﬁlter-feeding microcrustaceans and are important in nutrient recycling in aquatic systems (Makarewicz and Likens, 1979). Furthermore, rotifers can alter the species composition of algae in certain systems. Schlüter et al. (1987) showed that intense feeding by Brachionus rubens can cause a shift in the dominant algal species from Scenedesmus to a spined algae, Micractinium. Apparently, this shift is based on the inability of B. rubens to consume algae with protective spines. Although generally not ingested, cyanobacteria are increasingly recognized as playing an important role in determining zooplankton species composition. Large cladocera are more sensitive to the cyanobacteria Anabena afﬁnis than rotifers (Gilbert, 1990). The mechanism for this differential sensitivity to cyanobacteria toxicity is based on different tendencies to ingest cyanobacterial ﬁlaments and different physiological tolerances of the toxin (Kirk and Gilbert, 1992). 225 Having the ability to reproduce rapidly, rotifers may account for 50% or more of the zooplankton production, depending of the prevailing conditions (Herzig, 1987). This production, in turn, can be an important food source for other rotifers, Asplanchna (Gilbert and Stemberger, 1985b), cyclopoid and calanoid copepods (Williamson, 1983), malacostracans (Mysis, Threlkeld et al., 1980), insect larvae (Chaoborus, Moore, 1988; Moore and Gilbert, 1987), and ﬁsh (Hewitt and George, 1987). Abundance and species composition of rotifers often reﬂect the trophic status of lakes. For example, Hillbricht-Ilkowska (1983), Walz et al. (1987), and others have reported changes in the maximal totalpopulation density of several orders of magnitude when lakes were subject to intense eutrophication. Individual species sometimes undergo dramatic population changes during those periods. Walz et al. (1987) showed that the density of Asplanchna in Lake Constance increased its maximum population levels 280fold over a period of 28 years. Population declines also have been seen. Edmondson and Litt (1982) indicated that Keratella cochlearis was abundant during the years when Lake Washington had elevated concentrations of dissolved phosphorus, low water transparency, and high algal densities. However, as these waterquality parameters improved, the population of this rotifer declined dramatically. Overall, there was at least a 20-fold increase, followed by a decline during a period of 15 years. Studies of the interactions of rotifers with other organisms will probably continue to receive attention in the future, especially predator – prey interactions (Gilbert, 1966 1967; Salt, 1987; Williamson, 1983), interference competition between other herbivorous zooplankton and rotifers (Burns and Gilbert, 1986a, 1986b; Gilbert and Stemberger, 1985a), the toxic effects of cyanobacteria (Snell, 1980; Starkweather and Kellar, 1987), mate recognition (Snell et al., 1995), and life-history strategies (Walz, 1995). One underutilized tool for the study of energetics and trophic interactions is the chemostat, which, unlike batch cultures, maintains constant experimental conditions (Boraas and Bennet, 1988; Walz, 1993). 3. Competition with Other Zooplankton Rotifers, cladocerans, and copepods often compete for limited food resources and, in general, rotifers are relatively poor exploitative competitors because their clearance rates are usually many times lower than those of daphnids (see Section III.C.2). Rotifers also have a more limited size range of particles they can ingest compared to cladocerans and are less resistant to starvation (Kirk, 1997). Most rotifers eat algal cells in the 226 Robert Lee Wallace and Terry W. Snell 4 – 17 m range and are much less efﬁcient at ingesting smaller or larger cells (Gilbert, 1985b). In contrast, most cladocerans eat algal and bacterial cells in the 1 – 17 m range, and some can ingest much larger cells (Hall et al., 1976; Bogdan and Gilbert, 1982). Thus, cladocerans generally have broader food niches than rotifers in terms of food type and size, and through direct competition may suppress rotifer population growth. This outcome may be reversed, however, when a sufﬁcient quantity of suspended sediments is present in a lake (Kirk, 1991). Exploitative competition between rotifers and daphnids is readily demonstrated when these zooplankton are grown in single and mixed cultures. Brachionus calyciﬂorus and Daphnia pulex both grow well on the alga Nannochloris oculata in single species cultures. When both species are present, however, Daphnia removes an increasingly larger proportion of algal cells until the rotifers gradually starve to extinction after 2 – 3 weeks (Fig. 30). Daphnia is unaffected by the presence of rotifers. Population growth of certain rotifers also is inhibited by Daphnia through interference competition (Burns and Gilbert, 1986a, b; Gilbert and Stemberger, 1985b; Gilbert, 1989a). For example, in the presence of any one of four Daphnia species, Keratella cochlearis suffers mechanical damage (i.e., killed, wounded, or loss of eggs) when swept into the branchial chamber of the daphnids. No species differences were detected among the Daphnia used, but the rate at which rotifers were killed was directly proportional to daphnid body length. Keratella also was found in the guts of some Daphnia, documenting a new pathway for trophic interactions. Daphnia have a major impact on Keratella populations when the cladocerans are larger than 2 mm and present in densities of 1 – 5 individuals/L. Similar effects have been reported for short-term bottle experiments involving the cladoceran Scapholeberis kingi and the rotifer Synchaeta oblongata (Gilbert, 1989b). Of course, interference and exploitative competition may occur simultaneously (Gilbert 1985b, 1988). Predation is another important regulatory factor in rotifer population dynamics, as rotifers are prey for several aquatic predators including protozoans, other rotifers, insect larvae, cladocerans, copepods, and planktivorous ﬁsh. From many studies we know that predation affects rotifer population dynamics both directly (by contributing to mortality) and indirectly as a selective force shaping rotifer morphology, physiology, and behavior (see the next section). However, one area that deserves more study is the predatory interactions of protozoans and rotifers (e.g., protozoans as predators — Finlay et al., 1987 and Nogrady et al., 1993; rotifers as predators — Arndt, 1993; and Gilbert and Jack, 1993). 4. Predator – Prey Interactions FIGURE 30 Competition between Brachionus calyciﬂorus and Daphnia pulex. Brachionus and Daphnia were grown in single species (closed symbols) and mixed-species (open symbols) batch cultures at 20°C, daily renewed with 5 106 Nannochloris cells/mL. Population size (Y axis) is the number of individuals in the 80 mL culture. Error bars ( 1 se) are visible when they exceed the size of the symbols. (From Gilbert, 1985, with permission.) Rotifers possess several types of defensive mechanisms that have apparently evolved in response to predation. These mechanisms include morphological features, escape movements, and other ways of avoiding potential predators (see Stemberger and Gilbert, 1987a; Williamson, 1983, 1987). Most rotifers are transparent and quite small, some comparable in size to protozoans (ca. 60 – 250 m long). While these features beneﬁt limnoplanktonic rotifers by reducing their visibilty to ﬁsh, a small body size renders rotifers more vulnerable to invertebrate predators, which feed by touch rather than by sight. Many rotifers produce a thickened integument (lorica) and/or spines and other projections, or carry their eggs, all of which have been shown to reduce the ability of predatory zooplankton to prey upon them (e.g., Gilbert, 1966, 1967, 1980b; Stemberger and Gilbert, 1984b). Spines are produced in some rotifers (e.g., Brachionus calyciﬂorus, Figs. 12 and 31; Keratella cochlearis; Keratella testudo, Fig. 26) in response to a build-up of soluble substances released by invertebrate predators such as Asplanchna and copepods (Gilbert, 1967; Stemberger and Gilbert, 1984b; Stemberger, 8. Phylum Rotifera 1984). We now know that exposure to a water-soluble factor secreted by the predatory rotifer Asplanchna or the copepods Epischura, Mesocyclops, and Tropocyclops induces de novo spine formation or spine lengthening in several planktonic rotifers (Stemberger and Gilbert, 1987a, 1987b) (see Section III.A.1). Rotifers for which such polymorphisms have been observed include Brachionus calyciforus, B. bidentata, B. urceolaris, Filinia longiseta passa, Keratella cochlearis, K. slacki, and K. testudo. The adaptive signiﬁcance of spined morphotypes is a signiﬁcant reduction in capture and ingestion by invertebrate predators by making the rotifer more difﬁcult to manipulate and swallow. The presence of spines on B. calyciﬂorus is a good example of the phenomenon (Fig. 12). The way this works is as follows. When disturbed by a potential predator, B. calyciﬂorus will retract its corona and by doing so increases the hydrostatic pressure within its pseudocoelom. The elevated pressure causes the posterolateral spines to swing forward and outward (Fig. 31). Thus, the apparent body volume is larger. For Asplanchna, this increase in the size of its prey is sufﬁcient to prevent ingestion after capture. After a period of time during which Asplanchna attempts to swallow B. calyciﬂorus (usually 60 sec), the predator will release (reject) the prey which then swims away unharmed. Thus, the invertebrate predator releases a biochemical cue (an allelochemic) that initiates a developmental change in subsequent generations of the rotifer (spine production) reducing the effect of predation. Some forms of K. cochlearis also possess posterior spines, which make them four times more likely to be rejected after capture by A. girodi than unspined forms (Stemberger and Gilbert, 1984b). Most rejected prey swim away unharmed. However, occasionally spined forms are swallowed by Asplanchna or a similar genus (Asplanchnopus) and the spines become lodged in the pharynx or esophagus of the predator. FIGURE 31 Extension of the postero-lateral spines in Brachionus calyciﬂorus. (Modiﬁed from Koste, 1976, with permission.) 227 FIGURE 32 Filinia (lateral view). This rotifer possesses long spines which are used in making rapid jumps to escape predators. (Modiﬁed from several sources.) Presumably, both predator and prey die when that happens. Spine production has additional consequences beyond predatory avoidance. Stemberger (1988) has demonstrated that, in the absence of predators, reproductive effort (survivorship and fecundity) and hydrodynamic characteristics (sinking rates and swimming speeds) vary in spined and unspined Keratella testudo. These relationships were further complicated by food concentration (see Section III.B.4.a, and Fig. 26). Transparent mucus sheaths produced by some planktonic rotifers (e.g., Conochilus and Lacinularia species) appear to function in a similar way to spines. The sheaths make the rotifers effectively larger and, therefore, less vulnerable to invertebrate predators (e.g., Asplanchna and predatory copepods) without making them more visible to ﬁsh (Gilbert, 1980b; Stemberger and Gilbert, 1987a, 1987b; Wallace, 1987). Three rotifer genera can often escape predators by making rapid jumps using a variety of appendages: long spines (Filinia, Fig. 32), setous armlike appendages (Hexarthra, Fig. 13), and paddles (Polyarthra, Fig. 33) (see Section II.C.1). Many rotifers assume a passive posture, displaying what has become known as the “dead-man response,” rather than ﬂeeing when predators attack (e.g., Asplanchna, Brachionus, Keratella, Sinantherina, and Synchaeta). This simple behavior is merely a retraction of the corona into the body and passive sinking. Contraction of the corona stops the animal from swimming, thus eliminating the vibrations it produces in the water that may be detected by the predator. In addition, this behavior may make the rotifer more difﬁcult to grasp in its turgid state. In Sinantherina spinosa, this passive posture exposes a group of 228 Robert Lee Wallace and Terry W. Snell FIGURE 33 Polyarthra. This rotifer possesses long paddlelike appendages which are used in making rapid jumps to escape predators. (Modiﬁed from several sources.) cases, it appears that parasites can cause the demise of an entire population within a few days (Edmondson, 1965). The sporozoan parasite, Microsporidium (Plistophora aerospora) frequently infects planktonic rotifers possessing thin loricas (Ruttner-Kolisko, 1977a). For example, members of the genera Asplanchna, Brachionus, Conochilus, Epiphanes, Polyarthra, and Synchaeta have been reported to be parasitized. Apparently, water temperature is an important mediating factor in the spread of the parasite as infection rate drops off at water temperatures below 20°C. In infected rotifers, the pseudocoel of the animal becomes nearly ﬁlled with sausage-shaped cysts of Microsporidium (Fig. 34). Several workers have described endoparasitic fungi which attack soil rotifers of the genera Adineta and Philodina. These reports describe three avenues of parasitic attack: adhesion, ingestion, and injection. Tzean and Barron (1983) have described one fungus that forms peglike, adhesive appendages on both small conidia (spores, ca. 30 m long) and long vegetative hyphae. small spines on its ventral body surface, which may function in defense against planktivorous ﬁsh (see Wallace, 1987). To date, only one species of rotifers, Sinantherina socialis, has been shown as unpalatable to small zooplanktivorous ﬁshes (Felix et al., 1995). While neither the nature nor the location of the unpalatability factor(s) is known, these authors speculate that this colonial species possesses an unidentiﬁed chemical held in the glandlike “warts” which are located at the anterior end of the animals. Defenses against predators like spines, mucus sheaths, thickened loricas, and escape movements are energetically demanding. Some species, like Synchaeta pectinata, are not well defended against predators, but have evolved very high, maximal population growth rates that offset mortality from predation. Avoiding potential predators in space and/or in time is another simple yet effective defense mechanism against predators. Some rotifers occupy the habitat at a different time of year than an important predator, migrate vertically or horizontally in the habitat, or live in zones with low oxygen concentration, thereby missing predatory pressures altogether. 5. Parasitism on Rotifers The importance of parasites in controlling population density in rotifers has not been examined thoroughly, although a few studies have correlated parasitic infection with a decrease in population density of planktonic species (Ruttner-Kolisko, 1977a). In certain FIGURE 34 Photomicrograph of Brachionus infected Microsporidium within its body cavity (pseudocoel). with 8. Phylum Rotifera FIGURE 35 Fungal parasites of rotifers. (A) Four rotifers trapped by adhesive pegs on the vegetative hyphae of Cephaliophora; (B) unicellular, assimilative hyphae of Triaculus; and (C) zoosporangium of Haptoglossa with two evacuation tubes and ten encysted zoospores. (Bars ⬇ 100 m.) Once the adhesive pegs attach to a rotifer, they germinate and rapidly colonize the pseudocoel (Fig. 35A). At least three different genera of fungi produce spores (Fig. 35B) that initiate parasitic attack when ingested (e.g., Barron, 1980a; Barron and Tzean, 1981). A third avenue of infection occurs via hypodermic injection of a vegetative cell into the rotifer (Barron, 1980b; Robb and Barron, 1982). Once inside the host, these fungal cells grow into assimilative hyphae, producing more infective cells either inside or outside the rotifer (Fig. 35C). 6. Aquaculture In freshwater communitites, rotifers serve as food for several invertebrate predators which, in turn, are consumed by many economically important ﬁsh. They also are used directly as food by many planktivorous adult ﬁsh (O’Brien, 1987). Perhaps the most important ecological link between rotifers and ﬁsh is the consumption of rotifers by larval ﬁsh. At an early age, 229 most ﬁsh larvae feed on microzooplankton, rotifers being one of the most important components of the diet. In general, rotifers are highly nutritious and their biochemical composition can be further improved by specialized diets (Watanabe et al., 1983). Many ﬁsh are well adapted to locate, pursue, capture, and ingest rotifers, and rotifers are easy prey because they swim slowly and frequently lack predator defenses (Stemberger and Gilbert, 1987a). Aquaculturists have exploited this important relationship between planktivorous ﬁsh and their rotifer prey in intensive aquaculture systems. This ﬁeld has developed into a major technical discipline in several countries, most notably Israel, Japan, and China. Most of this work has the practical goal of determining the correct biotic and abiotic factors necessary to maintain mass cultures of rotifers (usually Brachionus plicatilis) as food organisms for larval shrimp and ﬁsh (Lubzens, 1987; Lubzens et al., 1989). Most systems for mass culturing of rotifers are simple batch cultures capable of producing kilogram quantities of rotifer biomass each day (Hagiwara et al., 1997). A substantial amount of effort has been devoted to identifying optimal culture conditions for the most commonly grown species (Brachionus rubens and B. calyciﬂorus in freshwater and B. plicatilis in saltwater). For mass cultures of B. rubens raised at 20°C, the optimal pH is 6.0 – 8.0, with upper and lower lethal limits of 9.5 and 4.5, respectively (Schlüter, 1980). Oxygen levels as low as 1.15 mg/L sustained good growth, but 0.72 mg oxygen/L was strongly limiting (Schlüter and Groeneweg, 1981). Schlüter (1980) also tested eight algal species and found that Scenedesmus costato-granulatus, Kirchneriella contorta, and Chlorella fusca produced the best rotifer growth. Algal concentrations of 70 mg/L dry weight of Scenedesmus were optimal. Brachionus rubens reproduction in mass cultures is inhibited by ammonia levels of 3 – 5 mg NH3-N/L (Schlüter and Groeneweg, 1985). Because about 50% of the nitrogenous wastes excreted by Brachionus is ammonia, waste product removal is a serious consideration in mass culture design (Hirata and Nagata, 1982). Although rotifers have been important in marine ﬁsh aquaculture for many years (Lubzens, 1987), freshwater rotifers are just beginning to be used. Nauplii of brine shrimp (Artemia) have been used extensively as larval feed, but these marine organisms do not live more than about 6 h in freshwater, and their decomposition contributes to the organic load in culture tanks. A freshwater organism is preferred for freshwater aquaculture, and rotifers ﬁll this role well. Rotifers also are being used as food for young ﬁsh larvae too small to begin feeding on Artemia. For these reasons the use of rotifers in freshwater aquaculture is expected to grow. 230 Robert Lee Wallace and Terry W. Snell D. Evolutionary Relationships The phylogenetic position of the eight tradition “pseudocoelomate” phyla represents a problem that continues to generate debate among workers. Within this larger question several opinions have been expressed concerning the evolution of rotifers, including some that propose a polyphyletic origin of the phylum. One theory suggests that rotifers evolved from an ancestor possessing a ﬂuid-ﬁlled body cavity (Lorenzen, 1985), perhaps a regressed coelom, while another proposes that rotifers were derived from an ancestral acoel turbellarian (phylum Platyhelminthes, Chapter 6) (Hyman, 1951). We believe that the evidence favors the FIGURE 36 Phylogenetic relationships within phylum Rotifera at the class level. Illustrated here are the three different hypotheses regarding the relationship of acanthocephalans and rotifers. (A) The traditional hypothesis based on an analysis of morphological data concludes that acanthocephalans are a sister group to rotifers (Melone et al., 1998). (B) A recent hypothesis based on an analysis of molecular data (nuclear 18S rRNA and mitochondrial 16 S rRNA) concludes that acanthocephalans are a highly modiﬁed sister group to bdelloid rotifers (Garey et al., 1998). (C) Results of recent research of molecular data (nuclear gene: 82 kd, heat-shock protein) concludes at acanthocephalans are highly modiﬁed rotifers related to the sister group of bdelloids plus monogononts (Mark Welch, 2000). Numbers refer to changes in character state as gains or losses. Character states are numbers as follows: (1) intracytoplasmic lamina; (2) females with uterine bell, ciliated epidermal cells absent, cephalic gland absent; (3) ciliated corona present, mastax with trophi present, retrocerebral apparatus present and paired, dorsal antenna present; (4) Eurotatoria: urinary collection bladder present, manubrium present in trophi, paired retrocerebral apparatus fused into a single unit; (5) male genital ducts U-shaped, penis absent; (6) male haploidy, single gonads; (7) adult capable of a type of diapause, loss of male, stomach syncytial, fulcrum absent in trophi; and (8) specialized parasitic features. view that rotifers and platyhelminthes are related; these phyla share several common features including protonephridia, a ciliated integument which possesses mucus glands, and a cerebral eyespot with a pigment cup (Wallace et al., 1996; Garey et al., 1998). The most critical issue regarding the origin of the Rotifera is the question of its relationship to phylum Acanthocephala. Presence of the intracytoplasmic lamina within a syncytial epidermis as well as other morphological features in both phyla suggests that they are closely related even though acanthocephalans are 5 – 1500 times larger in size than the largest rotifer (Wallace et al., 1996). Lorenzen (1985) argues that structures identical to the rostrum and lemnisci of acanthocephalans are found in bdelloids, thus making the two monophyletic. However, Ricci’s (1998a) recent critique argues against homology of these structures and a recent cladistic analysis of some 60 morphological characters rejected Lorenzen’s thesis that acanthocephalans and bdelloid rotifers are related (Melone et al., 1998b). Nevertheless, molecular analyses of 18S rRNA genes from a few well-known species place the acanthocephalans ﬁrmly within the Rotifera. One view places the acanthocephalans as a highly modiﬁed sister group to the bdelloids (Garey et al., 1996, 1998), while another places them as highly modiﬁed rotifers related to the sister group of bdelloids and monogononts (Mark Welch, unpublished observations). If additional genetic analysis conﬁrms these studies, the deﬁnition of what constitutes the phylum Rotifera will need to be completely re-interpreted. A composite overview of these hypotheses is illustrated in Figure 36. IV. COLLECTING, REARING, AND PREPARATION FOR IDENTIFICATION A. Collections Collecting rotifers does not require complex or expensive equipment. One can almost always collect several species of planktonic rotifers by towing a ﬁnemesh (25 – 50 m) net through any body of water. [N.B.: Nets with greater mesh sizes tend to miss smallbodied forms.] Productive lakes and ponds usually provide especially good sampling sites. More elaborate equipment such as closing nets and Clarke – Bumpus samplers work well, but are not necessary unless required by a speciﬁc experimental protocol. Water collected by discrete sampling devices (e.g., Van Dorn sampler, Kemmerer bottle, plankton trap, pumps) is then ﬁltered through a net of appropriate size. In weedy areas, a dip net or ﬂexible collecting tube (Pennak, 1962) are very useful. Another simple method to 8. Phylum Rotifera collect rotifers is to submerge a 3 – 4 L (1 gallon) glass jar in a weedy region and to arrange loosely a few aquatic plants in it. If using this technique, ﬁll the jar to about 2 – 5 cm from the top and place it near a subdued light source, such as a north window. Rotifers which swim to the surface on the lighted side may be removed using a transfer pipette. Certain aquatic plants, such as Elodea, Myriophyllum, and ﬁlamentous algae are good substrata to examine for the presence of sessile rotifers, but Utricularia usually provides the richest diversity (Wallace, 1980). Plants with highly dissected leaves may be examined in small dishes using a dissecting microscope. Broadleaved plants must be cut into strips and examined on edge. The upper few centimeters of moist sand taken just above the water line along a lake or a marine shoreline or the hyporheic interstitial zone of a stream bed usually provides several species of rotifers. Unfortunately, very few studies have been conducted on rotifers from this habitat termed “psammon,” in part because of the difﬁculty in separating the organisms from the sand. Recent studies have revealed a remarkable diversity (35 – 85% of the fauna) and abundance (up to 105 individuals/L) of rotifers in this habitat. Under certain conditions rotifers may be found at depths of up to 60 cm into the interstitial. For a review of these topics consult Schmid-Araya (1995). Sediments collected from the bottom using a dredge, coring apparatus, or suction device usually provide several bdelloid and monogonont species. The upper few centimeters of sediments from a core collected in the winter or early spring normally contain resting eggs, which can be induced to hatch within a few days when several milliliters of sediment are incubated at ambient spring or summer temperatures (May, 1986, 1987). Do not overlook laboratory aquaria or holding tanks as potential sources of material. We have found some unusual species in aquaria that had remained almost unattended for months. One might try adding a small amount of sediments from several sites as a way of adding variety to the rotifer community within the aquarium. Sessile rotifers may be present if the aquarium contains aquatic plants recently collected from the ﬁeld. However, if you attempt to keep sessile forms, be sure ﬁrst to remove all snails from the aquarium. Populations of rotifers may be maintained or even increased by adding a small amount of food once or twice a week. B. Culture King and Snell (1977), Ricci (1984), and Stemberger (1981) have summarized the general procedures 231 for culturing rotifers. Some species of rotifers may be cultured in the laboratory quite easily, obtaining densities of 105 individuals/L in a few weeks. Others seem impossible to keep even for short periods. Several species are commonly raised under a variety of conditions (Table II). Most of these need regular care a few times per week (changing the medium, feeding, etc.). However, some species require little care. For example, Habrotrocha rosa is easily cultured in dilute broths of decaying powdered baby food and may be nearly ignored for weeks at a time without extinction of the culture (see Bateman, 1987). Techniques used to culture protozoans (e.g., making extracts and infusions of various grains, manure, soil, etc.) have been adopted for the culture of some rotifer species with great success. Most rotifer cultures can be maintained xenically (i.e., containing several contaminating organisms) without much problem. In fact, sometimes rotifers are found contaminating cultures of protozoans, microcrustaceans, etc., that have been provided by commerical biological supply companies. Rotifers have been maintained under axenic (Dougherty, 1963) or monoxenic culture (Gilbert, 1970) conditions. Sophisticated culture techniques using single- and two-stage chemostats have been described by several workers (Starkweather, 1987; Walz, 1993). C. Preparation for Identiﬁcation Three preservatives are commonly used to preserve rotifers: formalin and Lugol’s iodine at concentrations of 5% or less, and ethanol at about 30 – 50%. Lugol’s has two advantages over formalin. It is less toxic and it stains the specimens slightly; the staining makes the animals more visible during sorting procedures. Unfortunately, Lugol’s iodine makes the lorica skrink and deforms the specimens. Other stains such as rose bengal are commonly used to stain specimens preserved in either formalin or ethanol. Unless special precautions are taken before ﬁxation, illoricate rotifers (especially bdelloids) will contract into a completely unidentiﬁable lump, making identiﬁcation difﬁcult if not impossible. Such specimens have lost all value for taxonomic purposes, although the trophi still may be useful (see later discussion). Histological ﬁxatives (e.g., Bouins) or sugar-formalin solutions (used to prevent osmotic shock in microcrustaceans) are not particularly helpful in preventing contraction in rotifers. Whenever possible, live specimens should be identiﬁed ﬁrst, then ﬁxed to determine the effect of a particular ﬁxative on body shape. Fortunately, several anesthetics have been found to work well on rotifers (Edmondson, 1959; May, 1985; Nogrady and Keshmirian, 1986a, b; Pennak, 1989 p. 232 Robert Lee Wallace and Terry W. Snell TABLE II Examples of Rotifers That Have Been Cultured Species Bdelloids Habrotrocha rosa Philodina acuticornis several bdelloids Monogononts Asplanchna spp. Brachionus angularis B. calyciﬂorus B. patulus B. plicatilis B. rubens Cephalodella forﬁcula Encentrum linnhei Euchlanis dilatata Keratella cochlearis Keratella testudo Lecane inermis Notommata copeus Polyarthra major Polyarthra vulgaris Synchaeta cecelia Culture conditions Reference Batch cultures of tap water and powdered baby food, dog food, etc. Monoxenic culture Batch cultures Bateman (1987), Schramm and Becker (1987), Wallace personal observations Meadow and Barrow (1971) Ricci (1984), Pourriot (1958) Batch cultures Aloia and Moretti (1973), Gilbert (1967), Stemberger (1981), Stemberger and Gilbert (1984a) Walz (1983a) Gilbert (1963a) Gilbert (1970) Boraas and Bennet (1988) Rao and Sarma (1986) Hirayama and Ogawa (1972) Lubzens (1987) Droop and Scott (1978) Pilarska (1972) Schlüter and Groeneweg (1981) Rothhaupt (1985) Dodson (1984) Scott (1983), Scott (1988) King (1967) Walz (1983a,b) Stemberger and Gilbert (1987b) Dougherty (1963) Pourriot (1977) Stemberger (1981) Buikema et al. (1977) Egloff (1988) Continuous culture Batch culture Monoxenic cultures Chemostat culture Batch culture Batch culture Mass culture Chemostat culture Batch culture Mass culture Chemostat culture Batch culture Monoxenic and Chemostat cultures Batch culture Continuous and batch cultures Batch culture Batch culture Batch culture Batch culture Batch culture Batch culture (For additional references see Hydrobiologia volumes 255/256 and 313/314; Pourriot (1965: 122) provides a list of species that have been cultured prior to about 1959.) 195), but no single anesthetic has been shown to be universally effective. Care should be taken when working with anesthetic agents; some are controlled substances and/or are toxic. An alternative approach is to use the simple hot-water ﬁxiation technique described by Edmondson (1959, p. 433) which generally gives excellent results with a number of species. It is generally a good idea to place live samples in jars over ice for the return trip to the laboratory, although we have found that a few species suffer when cooled (e.g., Sinantherina socialis). Techniques for preservation and mounting of rotifers for examination using light microscopy (Edmondson, 1959, p. 433 Pennak, 1989, p. 196; De Smet, 1998) and transmission and scanning electron microscopy (Amsellem and Clément, 1980; Melone, 1998; Kleinow, 1998) have been described. However, the striking beauty of the living rotifer is utterly lost during almost any ﬁxation and mounting procedure. Observations of live rotifers are not always easily accomplished. The goal is to retard their movements without crushing or distorting the specimen with the cover glass. Both objectives may be accomplished with a compression microscope slide (Taylor, 1993). If a compressor is unavailable, then tiny pieces of broken cover glass (not recommended) or clay supports (recommended) work well as corner supports. If the clay supports are too high, a slight pressure from a pencil on each corner will reduce the height of the cover glass to the desired level. With some practice, one can trap a planktonic rotifer sufﬁciently to prevent swimming without undue constriction. Sessile rotifers are handled more easily. Plant material with attached rotifers can be trimmed with iridectomy scissors to a size suitable for placement on a microscope slide. The animal will remain in place without the need of compression as long as the plant material is large enough to act as an anchor. Methylcellulose or other viscous agents and ﬁbrous material, such as glass wool or shredded ﬁlter paper, also may be used to impede swimming species. Unfortunately, methylcellulose interferes with ciliary function, and ﬁbers reduce observation to a game of hide-and-goseek. Any lighting conditions may be used, as long as you are careful not to overheat the specimens. Strobe lighting provides a marvelous view of ciliary movements, and darkﬁeld illumination is often spectacular! 8. Phylum Rotifera The ﬁnal identiﬁcation of many rotifer species requires examination of the trophi which, in certain species, may be done by compressing an intact animal. However, Myers (1937) described a method to extract trophi from surrounding soft tissues using a small volume of bleach (sodium hypochlorite) in a depression slide (see also Edmondson, 1959, pp. 432 – 433, Pennak, 1989, pp. 195 – 196; Stemberger, 1979). A regular microscope slide may be used, if the cover glass is supported so the arrangements of the pieces of the trophi are not disturbed by compression. Because the trophi are liberated rather quickly when the bleach comes into contact with the rotifer, it is necessary to ﬁnd the animal rapidly; otherwise it becomes necessary to scan the entire slide for the small trophi. Be aware that bubbles may form and obscure your view when bleach comes into contact with some biological materials. Methods for preparing trophi for scanning electron microscope (SEM) work have been described by several workers including a recent methodology by Kleinow (1998) that gives stereoscopic pictures. In all work with trophi, it is important to remember that these structures are very small (50 m long) and that they are three dimensional objects with a particular spatial arrangement among all seven pieces comprising the trophi. V. CLASSIFICATION AND SYSTEMATICS A. Classiﬁcation Classiﬁcation schemes differ slightly in the ways that they treat the three basic groups of rotifers. In this key, three classes (Seisonidea, Bdelloidea, Monogononta) are recognized. The ﬁrst two are comprised of rotifers with two gonads and are sometimes considered orders within the class Digononta, leaving class Monogononta separate (e.g., Pennak, 1989 p. 196). Another method of classiﬁcation emphasizes the unique anatomy and obligatory sexual reproduction of the Seisonidea, separating it from Bdelloidea and Monogononta, which are then placed in the class Eurotatoria (Koste, 1978, p. 48; Melone et al., 1998b). European workers tend to rank rotifers as a class within the phylum Aschelminthes, while North American authors treat rotifers as a phylum (in which case the group known as aschelminths does not receive taxon status). B. Systematics 1. Class Seisonidea This monogeneric taxon (Seison) comprises only two recognized species of large (2 – 3 mm) dioecious, 233 marine rotifers that are epizoic on the gills of a leptostracan crustacean, Nebalia. The corona is very reduced and is not used in food gathering or locomotion. Both species have paired gonads and a functional gut in both sexes. The trophi are fulcrate. Females have ovaries without vitellaria. Sexes are of similar size and morphology. These unusual marine rotifers are not included in the following key. For a detailed review of this class consult the work of Ricci et al., (1993). 2. Class Bdelloidea Comprising 18 genera and just over 360 species (Ricci 1987), bdelloids possess a rather uniform morphology and are exclusively females, reproducing by parthenogenesis. Bdelloids are characterized by having paired ovaries with vitellaria, more than two pedal glands, and ramate trophi. Most bdelloids are microphagous with a corona of either two trochal disks or a modiﬁed ciliated ﬁeld. Bdelloids often have a vermiform body with a pseudosegmentation consisting of annuli, which permit shortening and lengthening of the body by telescoping. One order (Bdelloida) comprising four families (Adinetidae, Habrotrochidae, Philodinavidae, Philodinidae) is recognized. Bdelloids generally are not caught in plankton tows, although they may be found in waters with dense vegetation. Bdelloids often occur in sediments or among plant debris or crawling on the surfaces of aquatic plants. Some forms inhabit the capillary water ﬁlms formed in soils (Pourriot 1979) or covering mosses. Many species are capable of becoming desiccated and then rehydrated (see Section II.C.4). Unfortunately, there has been no systematic review of North American bdelloids. The works of Bartos (1951), Burger (1948), Donner (1965), Koste and Shiel (1986), and Pourriot (1979) should be consulted for more detail on bdelloids. 3. Class Monogononta Class Monogononta comprises the largest group of rotifers with more than 1600 species in about 95 genera of benthic, free swimming, and sessile forms. All are assumed to be dioecious with one gonad. In many species, males have never been observed. Females possess one ovary with a vitellarium. Males usually are structurally reduced with a vestigal gut that functions only in energy storage. Males generally are ephemeral, usually being present in the plankton for only a few days or weeks each year. Monogononts are microphagous or raptorial, but a few are parasitic. The corona is modiﬁed as broad-to-narrow disks or earlike lobes or is vase-shaped with reduced ciliation and long setae used in prey capture. In this key, three orders are recognized (Collothecacea, Flosculariacea, Ploimida), collectively comprising 24 families. Other workers 234 Robert Lee Wallace and Terry W. Snell TABLE III List of the Rotifer Families Recognized in the Key Presented Here Order Bdelloidea Adinetidae (4’) Habrotrochidae (2) Philodinavidae (4) Philodinidae (3) Order Collothecacea Collothecidae (5) Order Flosculariacea Conochilidae (8) Filiniidae (11) Flosculariidae (9) Hexarthridae (7) Testudinellidae (11’) Trochosphaeridae (10) Order Ploimida Asplanchnidae (15) Birgeidae (14’) Brachionidae (23’) Colurellidae (19) Dicranophoridae (12) [De Smet, 1997] Epiphanidae (22) Euchlanidae (21) Gastropidae (27’) Lecanidae (21’) [Segers, 1995a] Lindiidae (14) Microcodonidae (26) Mytilinidae (20) Notommatidae (26’) [Nogrady and Pourriot, 1995] includes Ituridae [Pourriot, 1997] and Scaridiidae [Segers, 1995b] Proalidae (22’) [De Smet, 1996] Synchaetidae (27) Trichocercidae (24) Trichotriidae (23) References in square brackets indicate recent publications done as a part of an ongoing series of guides that are updating rotifer taxonomy. include the ﬁrst two orders as suborders within order Gnesiotrocha (de Beauchamp, 1965; Koste, 1978; Koste and Shiel, 1987). Table III lists the families recognized in the subsequent key and their relevant couplet number. C. Taxonomic Keys There are a variety of keys to rotifers including those by Edmondson (1959) and Pennak (1989), covering the North American fauna, that may be followed to the level of genus. However, the specialized keys of Koste and Shiel (1987) for Australian waters, Pontin (1978) for the British Isles, Stemberger (1979) for the Great lakes, Ruttner-Kolisko (1974) for planktonic rotifers in general, and Koste’s (1978) revision of Voigt for the rotifers of central Europe are also important. This second group of works deals nearly exclusively with monogonont rotifers. While these keys may not be comprehensive enough to cover all of the variations in size and morphology that are sometimes found within a species, they will probably prove to be more than adequate for most work where detailed descriptions are needed. A comprehensive work of providing a worldwide guide to the rotifers is currently underway under the auspices of H.J.F. Dumont as coordinating editor. Volume 1 in the series (Nogrady et al., 1993) provides a general key to the level of family. To date four subsequent volumes have been published by SPB Academic Publishers. These volumes collectively cover the following taxa: Volume 2, Lecanidae; Volume 3, Notommatidae and Scaridiidae; Volume 4, Proalidae; and Volume 5. Dicranophoridae. A very simple key is available from Ward’s Natural Science. One of the fundamental differences among the higher taxonomic levels in phylum Rotifera is the structure of the trophi, with characteristic types of jaws being recognized in each family (see Section II.B.2). [Transitional forms are also known (Koste, 1978; Koste and Shiel, 1987).] Although the following key is designed with the nonspecialist in mind, commensurate with the central importance of the trophi, we have used both the structure of the trophi and other obvious characters of anatomy and morphology as priniciple points of separation. In keeping with the nature of this text, the key is taken only to the level of family following, for the monogononts, the recent taxonomy of Koste (1978), Koste and Shiel (1987), and Nogrady et al. (1993). D. Taxonomic Key to Families of Freshwater Rotifera 1a. Rotifers with paired ovaries (Digononta) and ramate trophi (Fig. 9) .......................................................................order Bdelloidea 2 1b. Rotifers with a single ovary and trophi other than ramate .................................................................................................................. ...........................................................................................................................................................................class Monogononta 5 [Although this step obviously is very important, special care is generally not necessary to resolve this couplet. The ramate trophi of bdelloids are usually identifable in whole animals without resorting to their isolation (see Melone et al., 1998b and Section IV.C). If the type of trophi and number of ovaries cannot be determined, there are other clues that may be useful for live organisms. Upon contacting a substrate, many bdelloids will crawl on the surface in a manner reminiscent of a leech, hence the etymon of the name (Greek, bdella, leech). Further, the corona of some bdelloids has the appearance of two separate wheels, while only a few sessile monogononts of the family Flosculariidae give this impression.] 8. Phylum Rotifera FIGURE 37 Representatives of Family Habrotrochidae (Habrotrocha). (From Koste, 1976, with permission.) 235 FIGURE 38 Representatives of the Family Philodinidae. (From Koste, 1976, with permission.) 2a(1a) Stomach without lumen, as a syncitial mass of food vacuoles that gives the gut a frothy appearance ................................................... .........................................................................................................................................................................family Habrotrochidae [Three genera (e.g., Habrotrocha) reported in North America; mainly on moss or benthic, includes about 120 species (e.g., Fig. 37.] 2b Stomach with lumen...........................................................................................................................................................................3 3a2b. Corona with the appearance of two separate wheels (trochus) extended on pedicels..............................................family Philodinidae [About 10 genera and 150 species, of which Philodina and Rotaria are common; also Dissotrocha and Macrotrachella (Fig. 38).] 3b Corona not as above ..........................................................................................................................................................................4 4a(3b). Corona ciliated lobes or regions near mouth; rostrum ciliated............................................................................family Philodinavidae [One rare species reported from North America (Abrochta, Fig. 39).] 4b. Corona as ﬂat ciliated ﬁelds on ventral side (no cingulum) .......................................................................................family Adinetidae [Two genera (Adineta, Bradyscela) of about 15 species (Fig. 39).] 5a(1b). Trophi uncinate (Fig. 8H); corona lacking typical ciliated regions, but is open as an infundibulum with or without elongate setae around the margin ..............................................................order Collothecacea comprising 2 families: Atrochidae and Collothecidae [Collothecidae (Collotheca and Stephanocerous) possess elongate setae (Figs. 6 and 40); fewer than 50 species; mostly sessile, ca. ﬁve species are free swimming, many produce clear gelatinous tubes; none are colonial, but may colonize substrata forming dense groupings (cf. Flosculariidae). Atrochidae (Acyclus, Atrochus, Cupelopagis) lack setae; each genus monospeciﬁc (Fig. 41).] 5b. Trophi other than uncinate; corona with typical ciliated lobes or ﬁelds, or ciliated bands ..................................................................6 6a(5b). Rotifers possessing malleoramate trophi (Fig.8I), order Flosculariacea...............................................................................................7 [Malleoramate trophi possess unci with numerous ﬁne teeth nearly completely overlying the rami (unlike the malleate type; see the description in couplet 16); teeth close to the fulcrum are usually larger than those more distant. In live animals, the nearly constant movement of the trophi in a grinding or poundinglike action is characteristic.] 236 Robert Lee Wallace and Terry W. Snell FIGURE 39 Representatives of two bdelloid families. (A) Family Philodinavidae, Abrochta, ventral view of corona (adapted from several sources); and (B) family Adinetidae, Adineta, dorsal view of adult (from Koste, 1976, with permission). FIGURE 40 Representative of Family Collothecidae (Collotheca sp. within its gelatinous tube which is embedded with detritus and diatoms). 6b Rotifers with trophi other than malleoramate .........................................................................................................order Ploimida 12 7a(6a). Conical body with six hollow, armlike, setose appendages that are outgrowths of the integument; appendages inserted with powerful muscles ............................................................................................................................................................family Hexarthridae [Monogeneric family, genus Hexarthra (Pedalia) with about eight species (Fig. 13), some of which inhabit salt or brackish waters. Although illoricate, Hexarthra spp. preserve well in formalin without serious contraction.] 7b. Body lacking hollow, armlike, setose appendages ...............................................................................................................................8 8a(7b). Rotifers with a corona of horseshoe or U-shaped ciliated bands; possessing a ventral gap in the coronal ciliation; free-swimming; solitary or small to very large colonies (150 individuals) within a gelatinous matrix..........................................family Conochilidae FIGURE 41 Representatives of family Atrochidae (Cupelopagis vorax). (Adapted from several sources.) 8. Phylum Rotifera 237 FIGURE 42 Coronae of the family Flosculariidae. (Modiﬁed from several sources.) [Two genera recognized, based on the position of antennae (Conochilus and Conochiloides), but Koste (1978) and Ruttner-Kolisko (1974) do not consider antennal position to be a signiﬁcant feature and subsume Conochiloides within Conochilus (Fig. 17).] 8a. Rotifers with corona other than a horseshoe or U-shaped ciliated band; solitary or colonial; with or without a gelatinous matrix ................................................................................................................................................................................................9 9a(8b). Rotifers typically with elongate bodies and large, circular to lobate, earlike corona (Fig. 42); solitary or colonial; with or without a tube or gelatinous matrix; mostly sessile, but some free swimming .....................................................................family Flosculariidae [Seven genera of mainly sessile species (Figs. 5, 17C, and 43), a few are free-swimming; includes the genera Floscularia, Lacinularia, Ptygura, and Sinantherina.] 9b. Rotifers lacking a large, circular to lobate, earlike corona ...............................................................................................................10 10a(9b). Spherical rotifers with a corona as a circular band around the equator or toward one end............................family Trochosphaeridae [Genus Trochosphaera (Fig. 44) with two species in eutrophic waters, rare, but when present may be very abundant. A second rare genus (Horaëlla) apparently not reported in the United States.] 10b. Rotifers not spherical in shape .........................................................................................................................................................11 11a(10b). Rotifers with two movable anterior spines (bristles) below the corona, of varying lengths often much longer than the body and one (rarely two) rigid caudal spines; foot absent ..............................................................................................................family Filiniidae [Genus Filinia (Fig. 32) with two North American species and apparently many hybrids. The long spines point posteriorly when the animal is swimming, but the anterior ones point forward when the corona is withdrawn.] 11b. Rotifers without long spines as described above ...............................................................................................family Testudinellidae [Two dissimilar genera. (1) Genus Testudinella, lorica greatly ﬂattened dorsoventrally, with dorsal and ventral plates fused along the lateral margin; foot annulated and retractile ending in a ciliated cup, which is difﬁcult to see; approximately 12 littoral species up to 250 m long (Fig. 45). (2) Genus Pompholyx, body having 4 nearly equal lobes in cross section; with a pair of frontal eyespots, (Fig. 45) common in lakes and ponds.] 12a(6b). Rotifers with forcipate trophi (Fig. 8J) ...........................................................................................................family Dicranophoridae [About 12 genera of creeping, littoral rotifers (Fig. 46) with symmetrical or asymmetrical forcipate trophi, a feature easily determined by gentle compression of the specimen; no planktonic or semiplanktonic species are present in this family.] 12b. Rotifers with trophi other than forcipate .........................................................................................................................................13 13a(12b). Mostly littoral rotifers possessing cardate trophi (Fig. 10) having a sucking action or trophi highly modiﬁed and stomach with zoochlorellae ...................................................................................................................................................................................14 13b. Rotifers with trophi other than cardate ...........................................................................................................................................15 14a(13a). Manubria of the trophi with hooks that may be determined in lateral view of the animal (Fig. 10); body spindle-shaped.................... family Lindiidae [Monogenetic family, Genus Lindia (Fig. 47); mostly littoral.] 14b. Highly modiﬁed trophi, possessing pseudunci; stomach with zoochlorellae, gastric glands absent (Fig. 48) ................family Birgeidae [Monospeciﬁc family (Birgea enantia, a rare littoral species).] FIGURE 43 Representatives of family Flosculariidae. (A) Floscularia conifera, portion of a sessile colony, with two adults inside a tube constructed of pellets made of detritus (tube length may reach 2000 m); (B) Ptygura barbata attached to a ﬁlamentous alga (total animal length ⬇ 300 m); (C) Limnias melicerta, sessile in its clear cement tube (individuals ⬇ 900 m); and (D) Ptygura mucicola, sessile within the colonial cyanobacterium, Gloeotrichia (body length ⬇ 100 m). 238 8. Phylum Rotifera 239 FIGURE 44 Representative of family Trochosphaeridae. (A) Photomicrograph of Trochosphaera (specimen slightly distorted); and (B) line drawing interpretation. (Diameter of the individuals ⬇ 800 m; original photomicrograph by Tom Nogrady.) 15a(13b). Saccate rotifers, with incudate or modiﬁed incudate trophi, (Fig. 8G,R) some lacking intestine and anus...........family Asplanchnidae [Three genera. Two lacking an intestine: Asplanchna, foot absent, with six species (Figs. 11 and 49) and Asplanchnopus, foot present, with three species. One rare, benthic genus with intestine and foot, Harringia, (Fig. 50) with two species.] 15b. Rotifers not possessing incudate trophi ............................................................................................................................................16 16a(15b). Rotifers possessing malleate trophi...................................................................................................................................................17 [In malleate trophi (Fig. 8A,B,C,E,F,P), each uncus has only 4 – 7 teeth (unlike the condition found in malleoramate trophi; see description couplet 6). The fulcrum may be short (malleate) or long (submalleate).] 16b. Rotifers possessing virgate trophi ....................................................................................................................................................24 [Virgate trophi are often asymmetrical (Fig. 8D,K,L,M,N,O,Q,S), with a long fulcrum and manubria and generally small rami.] FIGURE 45 Representatives of family Testudinellidae. (Ventral views of Testudinella and Pompholyx.) (Modiﬁed from several sources.) FIGURE 46 Representative of family Dicranophoridae (Dicranophorus). (From Koste, 1976, with permission.) 240 Robert Lee Wallace and Terry W. Snell FIGURE 47 Representative of family Lindiidae (Lindia). (From Harring and Myers, 1922, with permission.) FIGURE 48 Representative of family Birgeidae (Birgea). (From Harring and Myers, 1922, with permission.) 17a(16a). Loricate rotifers: body wall thickened and ﬁrm ................................................................................................................................18 17b. Illoricate rotifers: body wall not thickened or ﬁrm ...........................................................................................................................22 [Interpreting whether the body wall is thickened (loricate) or not (illoricate) can be difficult and it does take some experience to judge this characteristic. To get an indication as to how firm the lorica is, follow this procedure. When working with fresh material gently, apply pressure from the point of a pencil or probe onto the cover glass while observing the specimen. If the body puffs out under pressure and returns to its original shape when the pressure is released, the specimen is illoricate. Loricate forms will exhibit much less flexibility of the body wall. In preserved materials, illoricate forms tend to shrivel up, while the body wall of loricate rotifers will retain its shape even if the inner organs separate from the body wall, collapsing into a central mass of tissue.] 18a(17a). Lorica possessing furrows, grooves, or sulci; or with a dorsal, semicircular head shield covering the corona; or with a very strongly developed lorica and a dorsal transverse ridge..................................................................................................................................19 18b. Lorica lacking furrows, grooves, sulci or dorsal head shield; without a strongly developed lorica and dorsal transverse ridge ........23 FIGURE 49 Representative of family Asplanchnidae (Asplanchna). [From Koste, 1976, with permission.] FIGURE 50 Representative of family Asplanchnidae (Harringia). [From Koste, 1976, with permission.] 8. Phylum Rotifera FIGURE 51 Representative of family Colurellidae. [Modiﬁed from several sources.] 241 FIGURE 52 Representative of family Mytilinidae (Mytilina). [Modiﬁed from several sources.] 19a(18a). Lorica with medial, ventral furrow (but no lateral furrow or grooves) extending the full length of the animal or with a ventral notch in which the foot lies, or with a dorsal, semicircular head shield covering the corona ............................................family Colurellidae [Four genera found in the littoral zone including Colurella (possessing a medial ventral furrow, Fig. 51A), a very common genus of some 15 species, which browse on epiphytic microorganisms; Lepadella (possessing a ventral notch in which the foot lies, Fig. 51B), very common with many species; Squatinella (possessing a dorsal semicircular head shield covering the corona, Fig. 51C). [N.B.: Diplois daviesiae (family Euchlanidae), a rare monospeciﬁc genus present in Sphagnum bogs will key to family Colurellidae, if the lateral furrows of this species are missed.] 19b. Lorica lacking medial, ventral furrow or notch, and lacking a head shield .......................................................................................20 20a(19b). Lorica with dorsal, medial sulcus (double keel) or with a strongly developed lorica usually possessing a dorsal transverse ridge ........ family Mytilinidae [Two littoral genera: Mytilina (Fig. 52) possessing a laterally ﬂattened lorica with a dorsal longitidutinal sulcus, spines on all four anterior corners, about 10 species common, and Lophocharis with a strongly developed lorica, usually having a dorsal transverse ridge.] 20b. Lorica lacking a dorsal, medial sulcus and lacking a dorsal transverse ridge ....................................................................................21 ▼ FIGURE 53 Representative of family Euchlanidae (Euchlanis). [Modiﬁed from several sources.] 242 Robert Lee Wallace and Terry W. Snell ▼ FIGURE 54 Representatives of the family Lecanidae. (A & B) Lecane spp.; (C) Lecane lunaris (dorsal); and (D) lateral view. (A and B from several sources; C and D from Dartnall and Hollowday, 1985, with permission.) 21a(20b) Foot projecting from between dorsal and ventral plates at the posterior end of the lorica; dorsal and ventral plates separated by a deep furrow or groove ...........................................................................................................................................family Euchlanidae [Four genera common in the littoral rotifers, including Euchlanis (Fig. 53).] 21b. Foot projecting through a hole in the ventral plate at the posterior end of the lorica; dorsal and ventral plates connected by a weak furrow or groove ......................................................................................................................................................family Lecanidae ▼ FIGURE 55 Representative of family Epiphanidae (Epiphanes). (From Dartnall and Hollowday, 1985, with permission.) 8. Phylum Rotifera 243 FIGURE 56 Representatives of family Proalidae. (A) Proales (from Chengalath, 1985, with permission); and (B) photomicrograph of adult of Proales werneckii (dark body) with numerous eggs within a gall of Vaucheria sp. [Gall ⬇ 600 m long.] [A large number of littoral species in two genera (Fig. 54): Lecane (with two toes which may be partially fused at the base) and Monostyla (one toe or toe divided distally); Koste (1978) has subsummed Monostyla within Lecane.] 22a(17b). Mouth set in a funnel-shaped buccal ﬁeld ..............................................................................................................family Epiphanidae [Six genera including Epiphanes (Fig. 55), Cyrtonia, Mikrocodides, and Rhinoglena all littoral species or in small, shallow lakes. Epiphanes, sometimes incorrectly described as a ‘typical rotifer’ in textbooks, is not common, but may be found in ponds receiving animal wastes.] 22b. Mouth set in an oblique, ciliated ﬁeld on ventral side; body wormlike to fusiform ....................................................family Proalidae [Four genera, including the large genus Proales; generally found in littoral zones and sandy beaches (Fig. 56A). Proales daphnicola is planktonic and may be attached to cladocerans; Proales werneckii is parasitic in galls that it induces in ﬁlaments of aquatic Vaucheria spp (Fig. 56B).] 23a(18b). Lorica extending beyond body to head, foot, and toes ..........................................................................................family Trichotriidae [Three genera, including Macrochaetus (Fig. 57A), with numerous bilaterally placed spines (about seven littoral species) and Trichotria (Fig. 57B), with a pair of heavy spines on the dorsal side of the foot (about 10 littoral species).] 23b. Lorica not extending beyond body .......................................................................................................................Family Brachionidae [A large family comprising 6 genera of common rotifers. Brachionus, a very common genus with about 25 species of littoral and planktonic rotifers (Fig. 58). B. plicatilis is frequently found in salt and brackish waters. Kellicottia, with two common planktonic species, possessing long anterior spines of unequal length (Fig. 59). Keratella, with more than 15 species, having the dorsal 244 Robert Lee Wallace and Terry W. Snell ▼ FIGURE 57 Representatives of family Trichotriidae (Macrochaetus and Trichotria). [Modiﬁed from several sources.] plate decorated with a characteristic facet pattern (Fig. 60). K. cochlearis is very common. Notholca a genus common in cool waters (Fig. 61).] 24a(16b). Body twisted as a partial helix (asymmetrical) and/or trophi asymmetrical; or small, saccate animals, predatory within the colonial alga, Volvox .......................................................................................................................................................family Trichocercidae [Three genera: (1) genus Trichocera (body twisted as a partical helix; unequal toes; asymmetrical trophi) with some 90 species, may be important in the plankton (Fig. 62); (2) genus Elosa (body showing in cross section three lobes, two lateral and one dorsal, plus ventrally an inconspicous lobe; asymmetrical trophi) with two species common in the psammon and with Sphagnum moss; (3) Ascomorphella volvocicola, predatory within Volvox (Fig. 63).] 24b. Body and trophi not as described above ...........................................................................................................................................25 25a(24b). Free-swimming rotifers.....................................................................................................................................................................27 25b. Crawling or creeping rotifers in the littoral (ocassional species in the plankton) ..............................................................................26 FIGURE 58 Representatives of brachionid rotifers (family Brachionidae; genus Brachionus. [From Koste, 1976, with permission.] FIGURE 59 Kellicottia (dorsal view) a common genus of the family Brachionidae. (From several sources.) 8. Phylum Rotifera 245 Variation in the genus Keratella, a common genus of the family Brachionidae. (Individuals ⬇ 100 – 200 m in length.) (A) K. cochlearis (from Koste, 1976, with permission); (B) K. quadrata; and (C) K. testudo (from Stemberger, 1988, with permission). FIGURE 60 26a(25b). Purple plates positioned anterior to the mastax; corona wide, ﬂat, and somewhat circular; foot jointed and long, about 50% of the total length of the animal, with a single toe ....................................................................................................family Microcodonidae [Monospeciﬁc family of one uncommon species, Microcodon clavus, mostly littoral, but may be found in the plankton.] 26b. Lacking purple plates as described earlier, corona ventral and not circular ........................................................family Notommatidae [A varied family with many species in 13 genera including Cephalodella (Fig. 64), Notommata (Fig. 65), and Pleurotrocha. Also included at this point in the key are the families Ituridae and Scaridiidae.] 27a(25a). Corona with four prominant setae (sensory bristles) and auricles (earlike structures); or possessing 12 movable, ﬂattened, sword-topaddle- or feather-shaped appendages (paddles); or with a sculptured lorica having ridges, grooves, or areolations ............................ family Synchaetidae [Four genera: with sensory bristles and auricles, Synchaeta (Fig. 66), comprising some 12 freshwater species plus others of marine and brackish waters, may be important in the plankton; with paddles Polyarthra (Fig. 33), fewer than 10 species separated based on paddle morphology, nuclei of vitellarium, and lateral antennae; Pseudoploesoma (Fig. 67A) and Ploesoma (Fig. 67B).] FIGURE 61 Notholca, a common genus of the family Brachionidae, usually found in cool waters. (A) Variation within the genus (modiﬁed from several sources); and (B) N. squamula. (Modiﬁed from May, 1980, with permission). 246 Robert Lee Wallace and Terry W. Snell FIGURE 62 Trichocera (lateral view) an important genus of the family Trichocercidae. Lateral view. [From Wallace et al., 1989, with permission.] FIGURE 63 Predatory activity of Ascomorphella volvocicola (family Trichocercidae) on its prey, the colonial alga Volvox. Seen here are at least three rotifers feeding on the cells of a colony. Gaps in the cells of the alga represent the damage done by the rotifer; an egg is seen just to the right of center. (Photomicrograph by courtesy of Russ Shiel.) 8. Phylum Rotifera FIGURE 64 Representatives of family Notommatidae (Cephalodella). (Cephalodella sp. from Dartnall and Hollowday, 1985; others from Koste, 1976, with permission.) 27b. 247 FIGURE 65 Representative of family Notommatidae (Notommata, dorsal view). (From Koste, 1976, with permission.) Lacking prominant setae, auricles, and paddles, and lorica not sculptured .........................................................family Gastropodidae [Two genera: Ascomorpha (Fig. 8.68A) (incorporating Chromogaster) comprising 6 species, possessing a ﬁngerlike projection (palp) from the corona; Gastropus (Fig. 8.68B) with three species having a laterally compressed body, may be important in the plankton.] FIGURE 66 Representatives of family Synchaetidae (Synchaeta). [From Koste, 1976, with permission.] FIGURE 67 Representatives of family Synchaetidae. (A) Pseudoploesoma (side view) and (B) Ploesoma (dorsal view). Bars ⬇ 75 m. (Modiﬁed from several sources.) 248 Robert Lee Wallace and Terry W. Snell Representatives of family Gastropodidae. (A) Ascomorpha sp.; (B) Gastropus, (bar 100 m). (A from Koste, 1976, with permission; and B modiﬁed from several sources.) FIGURE 68 ACKNOWLEDGMENTS We wish to thank our mentors J. J. Gilbert and C. E. King for all the help and encouragement they have given us over the years. We also acknowledge Dr. W. Koste, a good friend for advice and inspiration; a worker who knows more about rotifers than we ever thought possible. Drs. W. T. Edmondson, J. J. Gilbert, T. Nogrady, P. L. Starkweather, R. S. Stemberger, L. J. Walsh, and G. H. Wittler reviewed the manuscript for this chapter, a task very much appreciated by the authors. However, we remain responsible for any errors which may still be present. We thank H. L. “Chico” Taylor for his insight in preserving and mounting rotifers for taxonomic work. LITERATURE CITED Allan, J. D. 1976. Life history patterns in zooplankton. American Naturalist 110:165 – 180. Aloia, R. C., Moretti, R. L. 1973. Sterile culture techniques for some species of the rotifer Asplanchna. Transactions of the American Microscopical Society 92:364 – 371. Amsellem, J., Clément, P. 1980. 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Resting egg ultrastructure and formation of the shell in Asplanchna sieboldi and Brachionus calyciﬂorus. Archiv für Hydrobiologie Beiheft 8:298 – 302. 9 NEMATODA AND NEMATOMORPHA George O. Poinar, Jr. Department of Entomology Oregon State University Corvallis, Oregon 97331 NEMATODA I. Introduction II. Morphology and Physiology A. Cuticle B. Hypodermis C. Muscular System D. Digestive System E. Excretory System F. Respiration G. Nervous System H. Sense Organs I. Reproductive System III. Development and Life History IV. Ecology A. Fossil Record B. Habitats of Freshwater Nematodes C. Enemies V. Collecting and Rearing Techniques A. Sampling Methods B. Extraction NEMATODA I. INTRODUCTION The phylum Nematoda comprises a wide range of roundworms that can be grouped either in nutritional (microbotrophic, predaceous, and parasitic in plants, invertebrates, or vertebrates) or ecological (soil, marine, freshwater, ectoparasitic, endoparasitic, or freeliving) categories (Poinar, 1983). Relatively little attention has been devoted to this important group of invertebrates by freshwater biologists because of difﬁculties associated with sampling, extraction, and identiﬁcation. Nematode representatives of essentially all of the nutritional categories mentioned above occur in freshwater during one or more life stages. However Ecology and Classiﬁcation of North American Freshwater Invertebrates. 2nd Edition Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved. C. Fixing and Mounting D. Culturing VI. Identifcation A. Taxonomic Key to Families and Selected Genera of Freshwater Nematoda B. Taxonomic Key to Extant Genera of Freshwater Mermithidae NEMATOMORPHA VII. Introduction VIII. Morphology and Physiology IX. Development and Life History X. Sampling XI. Identiﬁcation A. Taxonomic Key to Extant Genera of Nematomorpha Literature Cited Appendix 9.1 Systematic Arrangement of the Nematode Genera nematodes are treated here only if all or a large part of their life cycle occurs in freshwater habitats (these habitats are discussed elsewhere). Nematode parasites of vertebrates that live in or frequent freshwater usually occur in the freshwater habitat only as eggs or within intermediate hosts and, therefore, are not discussed in this chapter. Mermithid parasites of insects are included since the eggs, infective stages, postparasitic juveniles, and adults occur in freshwater habitats. Only nematode genera containing obligate aquatic species (those which live nowhere else) that have been reported from North America are included in the keys in the present work. The systematic arrangement of these genera is listed in Appendix 9.1. As more studies are undertaken, it is likely that genera described from other continents will also be represented in North America. In addition, the reader should note that 255 256 George O. Poinar, Jr. undescribed or unreported genera may be encountered in routine sampling. The related group, Nematomorpha, or hairworms, are frequently encountered by freshwater biologists. Superﬁcially, they resemble mermithid nematodes, and share similar life history strategies with certain species of the latter group. II. MORPHOLOGY AND PHYSIOLOGY Nematodes are nonsegmented, wormlike invertebrates lacking jointed appendages but possessing a body cavity and a complete alimentary tract (Fig. 1). While they lack specialized respiratory and circulatory systems, nematodes possess a well-developed nervous system, an excretory system, and a set of longitudinal muscles. With the exception of the family Mermithidae, most freshwater nematodes are under 1 cm in length. Although the basic body shape and anatomic plan of all nematodes are similar, there are some characteristics of most freshwater nematodes that are lacking in many terrestrial forms. One of these is the presence of three unicellular hypodermal glands commonly located in the upper part of the tail, immediately behind the anus (Figs. 1 and 2D). These caudal glands produce a secretion that is carried by a canal to the tip of the tail. At this point, the canal is attached to a valve (spinneret), which passes through a pore in the tail terminus (Figs. 1 and 6d). The secretion produces an adhesive deposit by which the nematodes can attach themselves to the surfaces of submerged rocks, plants, invertebrates, and debris. Mucus may also be produced by pharyngeal glands of aquatic nematodes. The deposits from the caudal and pharyngeal glands can be used to form slimy traces over the substrate. Microorganisms that become entrapped in these deposits serve as food when the nematodes ingest the mucus together with the attached microorganisms (Riemann and Schrage, 1978). A. Cuticle The exterior body covering on all nematodes is a noncellular, ﬂexible, multilayered structure called FIGURE 1 Adult female of Plectus (Plectidae) [center insert shows tail of male]. M, mouth; S, stoma; P, pharynx; N, nerve ring; E, excretory pore; B, valvated basal bulb of pharynx; I, intestine; R, rectum; A, anus; C, caudal glands; T, spinneret; V, vulva; O, ovary; F, egg; D, spicule; G, gubernaculum; L, genital papillae. [Drawing by A. Maggenti, Univeristy of California, Davis; labeling by G. Poinar.] FIGURE 2 (a) Gradually tapering pharynx of a freshwater Dorylaimus sp. Note absence of distinct valve in basal portion of pharynx. Arrow shows the nerve ring. (b) Fossil tracts thought to be made by freshwater nematodes. From the 50 million-year-old (Middle Eocene) Green River sediments of Lake Uinta in the Soldier summit area near Provo, Utah. (c) A juvenile freshwater nematode capable of swimming by rapid vibratory body movements. (d) Tail of a freshwater nematode showing three caudal glands (CG), the caudal gland tube (T) and spinneret (arrow). 257 258 George O. Poinar, Jr. the cuticle. It is secreted by a layer of underlying hypodermal cells and covers the entire external surface of the nematode, with portions entering various external openings such as the stoma, rectum, vagina, amphids (lateral sense organs), and sometimes the excretory pore. The cuticle is composed of an outer cortex, a middle matrix, and an inner ﬁber or basal zone. All these regions are constructed of sublayers, which vary in thickness according to the species and stage. The outermost lipoidal layer of the cortex is important because it serves as a semipermeable membrane, allowing water and solutes to pass in and out of the body cavity. The cuticle of freshwater nematodes may bear longitudinal striations, punctations, bristles, setae, or somatic alae (Figs. 6b and 7a). When present, the bursa is attached to the male tail and is composed of ﬂattened ﬂaps of cuticle which guide and hold the male in place during mating (Fig. 6c). All these cuticular structures are useful characters for identifying nematode groups or species. The cuticle is normally shed four times during development. At each molt (ecdysis), the old cuticle splits and is discarded. In a few forms, the old cuticle is retained, producing a double cuticle or sheath around the nematode (Fig. 8b). B. Hypodermis The hypodermis is a layer of tissue that is located beneath the cuticle and is responsible for the formation of the cuticle. It is generally a thin layer of tissue that expands into the coelom to form longitudinal cords between the muscle ﬁelds. Most nematodes possess lateral, dorsal, and ventral cords that contain the nuclei and other cytoplasmic inclusions of the hypodermis. The hypodermal cords originate as distinct cells but later lose their identity and form a syncytium. C. Muscular System Nematode movement is controlled by longitudinal muscles because these organisms do not possess circular muscles. These somatic muscles are composed of many adjacent cells which are innervated by nerves lodged in the ventral and dorsal hypodermal cords. Most nematode movement superﬁcially resembles that of many other elongate, stiff-bodied, appendageless animals, whether they be vertebrates (snakes) or invertebrates (biting midge larvae). In locomotion, the sideto-side bending of the body is produced by alternating contractions and relaxations of opposite muscle sectors that are controlled by the central nervous system. When placed on ﬂat surfaces in the laboratory, nematodes normally crawl on their sides. Some aquatic nematodes can swim by rapid vibratory or thrashing side-to-side movements of the body. D. Digestive System All freshwater nematodes, except the Mermithidae, contain a continuous alimentary tract composed of a stoma, pharynx, and intestine (Fig. 1). The stoma (mouth) is the most anterior part of the digestive tract and is usually lined with cuticle. The stomal walls are formed by a fusion of various smaller segments or rings (rhabdions), which may be separated as is found in the Cephalobidae, or fused, as in the Rhabditidae (Fig. 8a). The structure of the stoma generally reﬂects the food selection of the bearer. For example, predaceous nematodes tend to have a wide stoma armed with teeth (Fig. 7b), a stylet, or a spear (Fig. 9b), whereas microbotrophic forms generally have a small tubular stoma (Fig. 8a), and omnivores a funnel-shaped mouth cavity (Fig. 10a). Plant parasites have protrusible stylets which can be inserted into plant tissues (Fig. 8b), and the infective stages of invertebrate parasites often have a small stylet used in penetrating the cuticle or intestinal wall of the host. The pharynx (or esophagus) lies between the stoma and intestine and passes food from the mouth into the intestine. It is usually composed of radial muscles, which upon dilation allow the pharynx to function as a pump or vacuum. The pharynx also contains glands that produce secretions used in digestion, escape from the egg shell, molting, and penetration of host tissue. There are normally three portions to the pharynx: (1) the corpus (sometimes enlarged to form a distinct metacorpus or median bulb); (2) the isthmus (usually surrounded by the nerve ring); and (3) the basal bulb (often containing valves to keep food particles from reversing their direction of ﬂow) (Fig. 8a). When nematodes feed, material is sucked up into the mouth by reduced pressure resulting from contraction of the radial muscles of the pharynx. Food is passed along the lumen by waves of alternating contractions and relaxations of the pharyugeal muscles. The pharyngeal lumen opens behind the food, thus creating suction pressure, and closes in front of the food, forcing it through. The intestine is a single-cell-thick, cylindrical tube that runs from the pharynx to the anal opening (cloacal opening in males). It is composed of an anterior ventricular region, a midintestine or intestine proper, and a posterior rectal region (not always obvious). The inner surfaces of the cells facing the lumen are lined with microvilli which seem to be responsible for the major uptake of nutrients. A pharyngeal-intestinal valve is located in the anterior portion, behind the basal bulb 9. Nematoda and Nematomorpha of the pharynx. In some freshwater forms, this valve is quite distinct and elongated (10c). An intestinal – rectal valve is located at the junction of the intestine and rectum. This unicellular sphincter muscle controls the amount of water passing out of the body. Many rhabditid-type nematodes have three rectal glands, which empty into the rectum. These glands are not to be confused with the caudal glands, which produce secretions from the tail terminus (Fig. 2d). The Mermithidae are unusual in having the intestine detached from the remainder of the alimentary tract and modiﬁed into a trophosome or food storage organ. During development, the trophosome separates from the pharynx and rectum, thus remaining as a separate, isolated organ. E. Excretory System Although variable in structure, the excretory system of nematodes falls into two basic types. The ﬁrst is frequently found in freshwater forms and consists of a ventral excretory gland (renette cell) connected by an excretory duct to the excretory pore on the surface of the cuticle. The ventral pore usually opens in the pharyngeal or anterior intestinal region of the body. The second type is a tubular system consisting of a series of longitudinal excretory canals; pooled contents of these canals pass into a joining canal which connects with the excretory duct and pore. Most freshwater nematodes excrete nitrogen in the form of ammonia or urea. The excretory system also has an osmoregulatory function of removing water that diffuses into the pseudocoelom and so regulates the amount of turgor pressure in the body cavity; some scientists believe that this is the primary function of the “excretory” system. F. Respiration Most nematodes are aerobic organisms, at least during their developmental period. Many nematodes can survive short periods of anaerobic conditions but only a few forms can survive anoxia indeﬁnitely. Strayer (1985) listed representatives of three nematode genera that he considered as benthic species found under anoxic conditions in Mirror Lake. A specialized respiratory system is lacking in nematodes. Instead, they obtain and lose gases by simple diffusion. Body cavity cells, when present, appear to play no role in oxygen transport. G. Nervous System The nervous system of nematodes centers around a central anterior mass of ganglia or “brain,” which 259 connects with nerve cords extending anteriorly and posteriorly through the body. The central ganglionic mass is closely associated with the circumpharyngeal commissure or nerve ring (Fig. 2a). Nerves extending anteriorly from the nerve ring innervate the amphids and cephalic sense organs, while those running posteriorly control the male tail papillae. H. Sense Organs The size, shape, position, and spatial arrangement of sense organs are all characteristic of nematode species or groups and are therefore frequently used as taxonomic characters. The anterior sensory papillae are found on the nematode head. The basic number is 16, but usually some have become vestigial. There is normally an inner circle of six labial papillae around the mouth, a second ring of six labial papillae slightly behind the ﬁrst set, and a third circle of four cephalic papillae still further back on the head. In mermithids, the cephalic papillae may also include adjacent enlargements of the hypodermal tissue. The amphids are paired, lateral sense organs that open to the exterior on the nematode cuticle. They may be located on the same circle as the labial or cephalic papillae or further back in the neck region. The term amphid generally refers to the exterior configuration of the amphidial opening, although the amphid also consists of an amphidial pouch, amphidial gland, and amphidial nerves. The structure of the amphidial openings and their location are very important taxonomic characters. They can vary from small pores to large circles (Fig. 7c), pockets (Fig. 10b), or spirals (Fig. 7d). Sexual dimorphism may be evident with either the male or female possessing a considerably larger amphid usually of the same general shape. Amphids may be multifunctional and evidence suggests that they serve as photoreceptors, olfactors, or sensory glands. Some are sensitive to pH and ions. Other innervated papillae on nematodes are deirids (paired papillae located laterally near the nerve ring), postdeirids (similar structures located at the midbody), and genital papillae (found on the ventral surface around the cloacal opening of males). When elongate, the genital papillae are usually attached to thin membranous outgrowths of cuticle (bursa) (Fig. 6c). The presence or absence of a bursa and its structure are important taxonomic characters. A few freshwater nematodes possess what are thought to be light receptor organs. These receptors normally consist of pigmented areas in the neck region, which may or may not be associated with a cuticular-structured lens. If a lensatic body is present, 260 George O. Poinar, Jr. then the organ is sometimes referred to as an ocellus or pseudocellus. I. Reproductive System The majority of nematodes, including the freshwater forms, reproduce by amphimixis (sperm and eggs come from separate individuals). However, some forms have developed uniparental reproduction (i.e., autotoky), and this condition also occurs in aquatic genera. In the latter, autotoky usually expresses itself as parthenogenesis, where progeny arise from unfertilized eggs. Hermaphroditism seems quite rare among freshwater nematodes (Poinar and Hansen, 1983), apparently occurring in Chronogaster troglodytes (Poinar and Sarbu, 1994) and some others. Asexual reproduction does not occur in the Nematoda. In amphimictic reproduction, the male nematode always places sperm inside the female; external fertilization has never been demonstrated. Some aquatic species exhibit “traumatic insemination” when the male penetrates the female cuticle with his spicules and releases sperm into her body cavity. Female nematodes contain one or two gonads, which open to the exterior on the ventral side of the body at the vulva (Figs. 1 and 6a). The vulva is connected to a muscular tube, the vagina, which in turn leads into the uterus followed by the oviduct and ovary. A spermatheca (sperm-collecting area) is usually present between the uterus and oviduct. With doubleovary species (didelphic), the gonads are usually opposite and join at a common vagina (Fig. 6a). Singleovary forms (monodelphic) have retained the anterior ovary, which is often reﬂexed into the posterior part of the body (Fig. 8d). The male may have a single testis (monorchic) or two testes (diorchic), which lead to a common seminal vesicle and vas deferens before entering the cloacal chamber, a common opening for the reproductive and digestive systems. Males of freshwater nematodes all possess one or two sclerotized structures termed spicules (Figs. 1 and 6c). These are inserted into the vagina of the female at insemination (except in traumatic insemination when they penetrate the cuticle of the female). Some males also have another sclerotized structure, the gubernaculum. The spicules are contained within the walls of the cloacal chamber while the gubernaculum is attached to the ﬂoor of the cloacal chamber. The latter structure supports the spicules during their movement in and out of the cloacal opening (Fig. 6c). On the ventral surface of the male tail is usually some type of genital papillae, which may or may not be supported by a bursa (Fig. 6c). Through the release and detection of sex attractants, male and female nematodes are able to locate each other. Once together, the nematodes intertwine and the posterior region of the male contacts the female vulva. Sometimes an adhesive compound is produced to seal the union. Spicules are then inserted and sperm is transferred from the vas deferens of the male into the vagina and uterus of the female. The pair may remain joined for only several minutes or may be united for days. It is common to ﬁnd “balls” of mermithids in stream bottoms all coiled together in continuous copulation. Individual sperm as well as secondary spermatocytes may be transferred during mating. In many nematodes, sperm maturation is completed in the female. Nematode sperm is variable in size and shape, but is always nonﬂagellated and usually amoeboid. Parthenogenetic forms can reproduce by mitotic or meiotic parthenogenesis. In the former, the diploid somatic number of chromosomes is retained, whereas in the latter, two maturation divisions occur which are similar to oogenesis in amphimictic species. A diploid chromosome number is established by fusion of the nonextruded polar nucleus with the egg pronucleus or by doubling of the chromosome before the ﬁrst cleavage division occurs. III. DEVELOPMENT AND LIFE HISTORY The eggs of nematodes differ from all other stages in containing chitin in their shells. During embryonic development, nematodes show predeterminate cleavage, which means that cells destined to form speciﬁc tissues appear early in the embryo. Freshwater forms like the mermithids may deposit eggs with fully formed juveniles ready to hatch upon receiving the right stimuli. Other species deposit eggs in the singlecell stage, with embryonic development occurring in the environment. Postembryonic development in nematodes (development after hatching) is similar to the gradual type of metamorphosis in insects. Aside from an increase in size, proportional changes of various organs, and the development of the gonads and second sexual characteristics, there are few differences between juvenile and adult nematodes (Fig. 11c). For this reason, the immature stages of nematodes should be called juveniles and not larvae, because the latter usually implies complete metamorphosis where the immature stages differ radically from the adults. [Despite these differences, however, the term larva is widely used today in referring to the immature, postembryonic stages of nematodes.] Hatching is inﬂuenced by temperature and external stimuli. The eggs of the mermithid Pheromermis pachysoma hatch when ingested by aquatic insects. 9. Nematoda and Nematomorpha Eggs of other freshwater nematodes hatch when the water reaches a certain temperature. Eggs that undergo anabiosis as a result of desiccation will not hatch until the area is ﬂooded. This probably enhances survival for nematodes in temporary water sources, although this point still requires elucidation. In most cases, it is the ﬁrst-stage juvenile that emerges from the egg; however, in mermithids, juveniles molt once in the egg and then emerge as second-stage juveniles. If equipped with a spear or tooth, the young nematode will use these to break out of the shell, otherwise it simply employs pressure and pharyngeal secretions to soften the shell wall. All nematodes undergo four molts and have six stages during their development (i.e., egg, ﬁrst-stage juvenile, second-stage juvenile, third-stage juvenile, fourth-stage juvenile, and adult). During each molt, the cuticle is shed and replaced by another secreted by the hypodermis. Sometimes two molts may occur almost simultaneously, as in postparasitic juvenile Mermithidae (Fig. 9a), and occasionally the shed skin is retained around the body of the next stage (Fig. 8b). Free-living nematodes generally complete their development rapidly in comparison to other metazoans. This can be 3 – 5 days for rhabditids (under optimum conditions) and generally from 1 to 6 weeks for most other freshwater forms. Therefore, nematode populations can turn over very quickly (see Schiemer, 1983, 1987 for energetic studies on nematodes). Aside from premolt and quiescent stages, most nematodes feed continuously throughout the growth period. Most freshwater nematodes take food in through the mouth and absorb nutrients into the intestinal cells. During their parasitic development in the hemolymph of invertebrates, mermithids absorb nutrients directly through their body walls. Nematode growth entails both enlargement and multiplication of cells. Growth is frequently nonproportional from stage to stage. For example, the intestine often will grow faster than the pharynx, so the proportion of the two organs differs in each stage. This information can be used to determine nematode stages, along with gonad development. The types of food vary among the freshwater nematode groups. Microbotrophic forms ingest bacteria, algae, single-celled fungi, and protozoa. Predaceous species attack small metazoans such as nematodes, annelids, early insect stages, and molluscs. Many are omnivorous and will consume microbial life as well as metazoans. Plant parasites feed on the cytoplasm of higher plants, algae and ﬁlamentous fungi. Parasites of invertebrates absorb nutrients from the hemolymph of various taxa, especially insects in the case of the Mermithidae and snails in the case of Daubaylia. Vertebrate parasites may occur in the guts or body cavities 261 of ﬁsh, aquatic birds, or amphibians, where they feed on host ﬂuids and tissues. Only the egg stages are freeliving and these are not likely to be encountered or recognized during routine collecting activities. The dependence of nematodes on moisture has brought a tremendous selection pressure to withstand periods of desiccation. Some nematodes form resistant stages, while in others, all life stages can tolerate drought conditions for various periods. Resistant stages include the egg and the second- through fourthstage juveniles. Some nematodes have been maintained for years (up to 25 years) in anabiosis and then revived in water. Very little is known about the resistant stages, dispersal, and survival of freshwater nematodes. Jacobs (1984) mentions the “ability for passive dispersal” of resistant forms, but does not discuss how this dispersal occurs. It is very likely that the eggs and, possibly, some juvenile stages of forms adapted to semitemporary water sources can enter a resistant, quiescent stage capable of surviving desiccation and possibly temperature extremes; these have been demonstrated in some soil and plant nematodes. Passive dispersal may occur when these resistant stages are blown by heavy winds or washed to new areas by ﬂash ﬂoods. The possibility of freshwater nematodes being carried in mud attached to the body parts of various water-frequenting animals, such as wading and swimming birds, is also feasible. The presence of freshwater nematodes in temporary ponds has not been investigated. IV. ECOLOGY A. Fossil Record The oldest known possible fossil record of aquatic nematodes consists of tracks dating from the Upper Precambrian of Australia and Europe (Hantzschel, 1975). Tracks similar to these in the Green River sediments of Lake Uinta (Middle Eocene) (Fig. 2b) were considered to be made by nematodes (Moussa, 1969). These more recent tracks occur in the Soldier summit area near Provo, Utah, and were made during the second lacustrine phase of the Green River sediments. Lake Uinta was a large freshwater lake that occupied the Uinta and Piceance Creek Basins in Utah and Colorado. These tracks could have marked the passage of fairly large freshwater nematodes moving near the shore. The early Precambrian tracks could well be nematodes since it is highly probable that the phylum dates back to that period when representatives of the now predominantly aquatic order Araeolaimida probably existed (Poinar, 1983) and biting midges 262 George O. Poinar, Jr. (Ceratopogonidae: Diptera) were not present, which are a group of insects whose legless larvae move in a fashion similar to nematodes. The aquatic mermithids represent the earliest and most frequenty encoutered undisputed nematode fossils. The earliest of these occurs in 120 – 135 million-year-old amber from Lebanon (Fig. 12a). It was originally described as occurring in a biting midge (Ceratopogonidae: Diptera) (Poinar et al., 1994), but according to Art Borkent (personal communication) the host is actually a member of the Chironomidae. Since the generic placement of this specimen was based in part on its host family, it is now prudent to transfer the species libani from Heleidomermis Rubstov into a new genus Cretacimermis Poinar. Thus, this nematode, which is the oldest deﬁnite fossil nematode, should now be called Cretacimermis libani Poinar, Acra and Acra (1994). Another aquatic mermithid fossil species is Heydenius dominicanus from Dominican amber (Poinar, 1984). Two specimens of this species were found in close association with an adult craneﬂy (Tipulidae: Diptera) and an adult mosquito (Culicidae: Diptera) (Fig. 12b). Based on the record of present-day mermithid infections of these two host groups, and the discovery of a female mosquito with a mermithid still coiled in its abdomen in Dominican amber, it can be assumed that H. dominicanus was a parasite of the adult mosquito. Such rare fossils show the antiquity of some parasitic associations. B. Habitats of Freshwater Nematodes Nematodes constitute an important and signiﬁcant portion of the zoobenthic community of freshwater habitats. This community has been separated by benthic ecologists into three size categories (Strayer, 1985). Animals retained on sieves with a mesh ranging from 200 to 2000 m compose the macrofauna, those that are retained on sieves with a mesh ranging from 40 to 200 m construct the meiofauna, and those that pass through a 40-m mesh sieve make up the microfauna. Freshwater nematodes covered in the present chapter could be considered as belonging to all groups; the large free-living stages of the Mermithidae would be considered macrofauna, the majority of the adults and juveniles of the other groups would fall under meiofauna, and the young juveniles and eggs of many forms would fall into the microfaunal category. Thus, for nematodes in general, these size categories are little used, although they are helpful if only certain stages are being studied. Nematodes constituted 60% of all the benthic metazoans in Mirror Lake, New Hampshire, and are probably the most abundant benthic animals in freshwater (Strayer, 1985). In Mirror Lake, nematode abundance showed a distinct depth distribution, with a strong minimum at 7.5 m. More than 90% of the nematode biomass at 7.5 m was composed of species of the large predatory nematodes belonging to the genus Mononchus. Strayer (1985) also reported that 63% of the nematodes in Mirror Lake lived in the top 2 cm of sediment, while only 18% penetrated to a depth greater than 4 cm. Species of the genera Monhystera and Ethmolaimus, which constituted 55% of the nematodes in Mirror Lake, were found in the benthic zone. Although the nematodes outranked all other animals in abundance (constituting 59% of all metazoan individuals in the benthos), they still represented only 1% of the zoobenthic biomass. Nematodes normally contribute between 1 and 15% of the zoobenthic biomass in lakes, and other studies show that they constitute from 40 to 80% of all meiobenthic animals (Holopainen and Paasivirta, 1977; Oden, 1979; Nalepa and Quigley, 1983). Nematodes that occur in freshwater have traditionally been called free-living nematodes. The term freeliving is an ecological ranking, which means that these nematodes have no symbiotic association with multicellular plants or animals. Such nematodes obtain nourishment from bacteria, algae, protozoa, and other unicellular organisms. Nematodes with this type of nutrition are best referred to as microbotrophic, although they have been called saprophytic, saprophagous, bacteriophagous, microbivorous, and microphagous. Such nematodes act as secondary consumers, feeding on bacteria and fungi. They may serve to maintain these microbial populations, although many of the free-living forms will die if bacterial populations reach high numbers, which is why most of the normal aquatic nematodes are absent from areas high in sewage. The term free-living, however, is also used to refer to a particular stage in the life cycle of a nematode — often a stage in the life cycle of a plant-parasitic or animal-parasitic nematode — when it has either ended or not yet begun its parasitic relationship. Examples in the freshwater habitat are the mermithids. The egg, infective second-stage juvenile, postparasitic juvenile, and adult mermithids are free-living and nonfeeding. Nourishment is obtained from the hemocoel of insects only during a portion of their juvenile development. Thus, free-living in the present work refers to those nematodes that have some developmental stages occurring in freshwater, irrespective of their nutritional requirements or their status at other developmental stages. The habitats of freshwater nematodes vary as do the categories of freshwater resources. Two important requirements for freshwater nematodes to complete their development are continuation of the water source and availability of food and oxygen. Many freshwater nematodes feed on microorganisms that ﬂourish in the gyttja, an organic deposit composed of excretory 9. Nematoda and Nematomorpha material from benthic animals. It is most obvious in lakes and ponds, where it is often combined with decomposing plant debris. This sediment increases in density with increasing depth. Core samples are used for quantitative measurements of zoobenthos in the gyttja (Strayer, 1985). Annual productivity of freshwater nematodes in an Austrian alpine lake was studied by Bretschko (1973). In the deeper benthos, below 20 m, there were 2 – 4 annual generations with the total yield of nematode biomass estimated at 66 kg per year. It is interesting that the production was higher in shallow water and during the winter when the lake was covered with ice. For example, Tobrilus grandipapellatus was collected at populations of 235,000 individuals / m2 in the winter, but only 60,000 individuals / m2 in the summer. This could be related to the availability of oxygen and density of micribial populations. Nematodes of all environments, including freshwater, are among the most numerous animals feeding on both primary decomposers such as bacteria and fungi, as well as primary producers such as algae and higher plants (Nicholas, 1984). The signiﬁcance of nematodes in the general economy of the ecosystem is not known, and attempts to estimate their contributions to energy ﬂow have been made only with terrestrial and marine forms. Such estimates have used the equation: CPREU where C is the consumption, P the production, R the respiration, E the ejecta (feces), and U is excretion. Although freshwater nematodes have been little studied, estuarine forms have been examined in detail (Warwick and Price, 1979). Some 40 species of TABLE I 263 nematodes occur on a mud ﬂat in the Lynher Estuary in southern Britain. The population varies from 8 – 9 106 individuals / m2 in winter to nearly 23 106 individuals / m2 in late spring (15% of the macrofauna). Warwick and Price (1979) calculated a total annual oxygen (O2) consumption of 28 liter O2 /m2 per year, which is equivalent to some 11 g of metabolized carbon (C). The annual production was calculated at about 6.6 g C / m2 per year. Previous calculations with the soil nematode Caenorhabditis briggsae showed that the conversion of bacteria into nematode tissue varied from 13 to 20% (Nicholas, 1984). The question of nematodes living under anoxic conditions has been addressed by several workers. In deep lakes, the hypolimnion may become anaerobic for certain periods, resulting in a reduction of nematode fauna, but not eradication. In Lake Tiberias, in Israel, Por and Moary (1968) found large numbers of Eudorylaimus andrassyi at a depth of 43 m during the winter oxygen-free period. In the Neusiedlersee in Austria, Tobrilus gracilus occurs in muddy anaerobic zones beyond the limits of emergent vegetation (Schiemer, 1978). In his study of the nematodes in Mirror Lake, Strayer (1985) found most species in the littoral zone, but species of Ethmolaimus, Monhystera, and unidentiﬁed Tylenchidae were highly abundant in the anaerobic sediments at a depth of 10.5 m. Extreme habitats for freshwater nematodes include high-temperature hot springs. Table I lists some freshwater nematodes collected at unusually high temperatures. The listing of 61.3°C for Aphelenchoides sp. is not only a record for nematodes, but for all metazoan life forms! Survival of Freshwater Nematodes in Hot Water Springs at High Temperatures Nematode species Temperature (0C) Location Reference Aphelenchoides sp. Aphelenchoides sp. Aphelenchus sp. Doryalimus atratus Linstow Dorylaimus atratus Linstow [D. thermae Cobb] Dorylaimus atratus Linstow [D. thermae Cobb] Euchromadora striata (Eberth) Monhystera ocellata Bütschli Monhystera gerlachii Meyl Plectus sp. Rhabdolaimus brachyuris Meyl Theristus pertenuis Bresslau and Schuurmans Stekhoven Tylocephalus sp. 61.3 35.0 57.6 47.0 40.0 New Zealand New Zealand Chile Italy Wyoming, USA Rahm (1937) Winterbourn and Brown (1967) Rahm (1937) Issel (1906) Hoeppli (1926) 53.0 Wyoming, USA 52.0 52.0 52.0 57.6 52.0 52.0 Italy Italy Italy Chile Italy Italy Cobb, in Hoeppli (1926) Meyl (1954) Meyl (1954) Meyl (1954) Rahm (1937) Meyl (1954) Meyl (1954) 45.3 New Zealand Winterbourn and Brown (1967) 264 George O. Poinar, Jr. Other specialized habitats encompass brackish and estuarine waters, inland saline lakes, cave streams, and associations with speciﬁc aquatic plants. “Artiﬁcial water sources” include canals, waste water, tap water, wells, ﬁlter beds of sewage treatment plants, and temporary water sources. Nematodes found in these habitats often do not have any relation to true freshwater forms or a freshwater habitat, and the majority are not treated in this work. A unique cave habitat has resulted in what appears to be the ﬁrst aquatic nematode specialized for survival in hydrogen sulﬁde-rich thermo-mineral waters. These conditions are found in the Movile cave, located in a limestone plateau in Southern Dobrogea, Romania (Poinar and Sarbu, 1994). The nematode, Chronogaster troglodytes, lives in ﬂoating microbial mats comprised primarily of mycelia of fungi belonging to the class Oomycetes and various bacterial populations. Selection for this habitat could have undergone millions of years since a Miocene age for the origin of Movile cave has been suggested. Also, while members of the genus Chronogaster are mostly aquatic, they, like many freshwater nematodes, are quite broad in their selection of habitat. Other species of Chronogaster have been FIGURE 3 collected from sandy or sandy loam soils, clay soils, pasture soils and even forest soils. In fact one species, C. africana is mentioned as occurring in multiple aquatic and terrestrial habitats (Heynes and Coomans, 1980). Such plasticity was probably an important feature in the specialization of C. trogoldytes in its unique underground cave habitat. A list of nematodes recovered from caves and subterranean waters is presented by Poinar and Sarbu (1994). Previous workers have attempted to separate the freshwater-dependent nematodes from the facultative forms. Micoletzky (1922) distinguished between completely aquatic, principally aquatic, amphibious, and principally terrestrial species; while more recently, Jacobs (1984) presented an ecological classiﬁcation with stenohygrophilic nematodes representing the strictly aquatic forms and enhygrophilic nematodes representing the semi-aquatic forms. The strictly aquatic or stenohygrophilic taxa covered here occur in many microhabitats (Fig. 3). As examples, periphytic forms live among plant roots, bryophilic taxa associate with moss and liverworts, endobenthic species thrive within the sediment and bottom debris, epibenthic groups live on the surface of the bottom, haptobenthic forms exist on Scene of the bottom of a typical fast-ﬂowing stream showing several nematode microhabitats. (A) Stream bed containing endobenthic forms; (B) bed surface containing epibenthic groups; (C) rock surface containing haptobenthic groups; (D) Nostoc algal pads containing mermithids that parasitize Cricotopus midges (Chironomidae) living inside the algae. 9. Nematoda and Nematomorpha the surface of submerged rocks, debris, and aquatic plants, and planktonic taxa live continuously in the water column. The latter group is poorly known and mainly found in turbid waters where their occurrence may represent more of a dispersal than a habitat mode. Many genera of freshwater stenobygrophilic nematodes are widely dispersed, occurring on more than one continent. Some species are eurytopic, occurring in diverse habitats, while others are stenotopic and restricted to a few specialized habitats. Freshwater nematodes have been considered as indicators of water pollution (Zullini, 1976). Heavy metals and other pollutants settle and are taken up by organic matter in the sediments. Nematodes ingest this material and those that are sensitive to the pollutants may die. Representatives of the Chromodorida are apparently more sensitive to pollution that those of the Rhabditida (Zullini, 1976). In extremely polluted waters, only the Rhabditoidea may be quite abundant. A two-year study sampling nematodes along two streams in Indiana indicated that an analysis of the benthic nematode community structure could be useful in evaluating disturbance to aquatic habitats (Ferris and Ferris, 1972). C. Enemies Perhaps the greatest enemies of freshwater nematodes are other predaceous nematodes. Representatives of the Mononchida, Dorylaimida, and Enoplida are known to attack and devour a range of small invertebrates in their surroundings. The crayﬁsh Pacifastacus leniusculus was observed to feed occasionally on nematodes (Flint, 1976). The freshwater, rhabdocoel turbellarian, Microstomum feeds on nematodes (Stirewalt, 1937), as will the freshwater nemertean worm, Prostoma (Coe, 1937). The role of disease organisms in regulating populations of freshwater nematodes is not known, but the author has frequently collected specimens of Tobrilus infested with what appeared to be microsporidian spores (Fig. 9c). Other records of probable protozoan diseases in freshwater nematodes have been reported in Chromadora, Dorylaimus, Ironus, Monhystera, Plectus, Theristus, Trilobus, Tripyla, Prodesmodora, Achromadora, Paraphanolaimus, and Desmolaimus (Poinar and Hess, 1988). V. COLLECTING AND REARING TECHNIQUES 265 are present in the ecosystem. The latter method is used to evaluate the components of a population, the proportion of each in the environment, and the dynamics of the population over time. Qualitative methods for sampling freshwater nematodes depend on the water ﬂow and the zone to be sampled. In sampling nematodes from beds of fast-ﬂowing streams, a net, an instrument to turn over rocks and debris on the stream bed, and a set of sieves, are necessary (Fig. 4a). The net is ﬁrst anchored with the opening facing upstream. A pick or geologist’s hammer is used to disturb the stream bed slightly upstream from the net (Fig. 4b). The stream washes nematodes and other debris from the disturbed area into the net. The contents of the net are then placed in enamel collecting pans with water and set aside for extraction. Nets can be pulled over aquatic plants to obtain nematodes living on their surfaces. The type of nematode being collected will determine the mesh size of the net selected. It is recommended that, in order to obtain a fair sample of all nematodes, a mesh size no larger than 40 m be used. A number of devices have been devised to collect freshwater nematodes (Filipjev and Schuurmans Stekhoven, 1959). These include dredges, wormnets, bottom catchers, mudsuckers, swab or sledge trawls, and plankton nets. Their use depends on the type of material desired. For sluggish or stationary water sources, samples of the bottom (mud, sand) can be placed in a water-ﬁlled container (Fig. 4c) which is then shaken, the suspension allowed to settle for 3 – 4 sec, and the supernatant then poured into a second container for extraction. Rocks and other submerged material can be placed in buckets and the nematodes washed off with a water spray or by vigorous shaking or brushing. Quantitative sampling methods involve the use of augers or probes to remove core samples of measurable sizes from the beds (Strayer, 1985). Other quantitative methods can be devised on the basis of need and the desired biotype to be investigated. Techniques applied to marine nematodes may also be suitable for freshwater forms. For further information, see Hulings and Gray (1971), Holme and McIntyre (1971), and Downing and Rigler (1984). It should be remembered that any estimate of the nematode population depends on the type of sample taken. Thus, a biased sample will produce a biased estimate of the nematode population. Also, it should be noted that nematodes are sensitive animals and all sampling and extraction techniques should be as gentle as possible. A. Sampling Methods There are two basic types of sampling: qualitative and quantitative. The former method is used in preliminary studies to determine which kinds of nematodes B. Extraction The process of extraction involves removing nematodes from samples collected in various freshwater 266 George O. Poinar, Jr. FIGURE 4 (a) Equipment needed to sample freshwater nematodes. A, pail; B, set of gradated sieves; C, net; D, pick; E, squeeze bottle; F, shovel, G, geologist’s hammer. (Hip boots are not shown.) (b) With the geologist’s hammer, the stream bed is disturbed upstream from the positioned net. (c) With a shovel, samples of the stream bed are removed together with roots of aquatic plants. (d) Large nematodes (mermithids and dorylaimids) and debris are transferred from the net into an enamel examination pan. habitats (Figs. 4d, 5a–d). The basic principles of nematode extraction are based on the fact that most nematodes have a speciﬁc gravity between 1.10 and 1.14, thus they will slowly sink in freshwater. They are denser than ﬁne clay particles, but lighter than sand. The extraction methods used for soil and plant-parasitic nematodes are wholly adequate for freshwater forms. Although the majority of freshwater nematodes are too small to view with the naked eye, the free-living stages of many aquatic mermithids are large enough to be handpicked from the samples (Fig. 5c). They can be carefully lifted with a ﬁne needle containing an Lshaped bend at the tip or with a pair of ﬁne forceps (Fig. 5d). Other mermithids that are small enough to 9. Nematoda and Nematomorpha FIGURE 5 (a) Nematodes and debris collected from the stream bed are passed through a series of three sieves with openings ranging from 0.4 – 0.04 mm across. (b) Nematodes are washed off the surface of the ﬁnal screen with a spray of water from the plastic squeeze bottle. (c) Free-living stages of mermithids are large enough to be seen with the naked eye and can be picked up with forceps. (d) Larger nematodes can be transferred directly to a small vial for later killing and ﬁxing. 267 268 George O. Poinar, Jr. escape visual spotting can be collected with the extraction processes. One of the oldest and simplest methods of extracting nematodes from a wide range of samples is the Cobb sieving and gravity method, or a modiﬁcation of the latter. This consists of making a water suspension of the nematodes and pouring it through a series of screens of different mesh sizes to collect the nematodes. The suspension is made by placing the samples in a pail or other suitably large container (Fig. 4c), adding 2 – 3 L of water (preferably from the original source), allowing the mixture to set for 10 – 20 sec to allow the heavier particles to sink, and then pouring the suspension containing the nematodes through a series of screens (Fig. 5a). If extraneous large debris (wood particles, rocks) is present, the samples should ﬁrst be poured through a 20 – 40 mesh sieve (openings are 0.840 – 0.350 mm). The sizes of sieves selected will, of course, inﬂuence the size range of nematodes extracted. If a sample of all available nematodes is desired, then a ﬁnal ﬁne screen of 400 – 500 mesh (openings are 0.035 – 0.026 mm) can be used. The problem with the ﬁne screens is that they become easily clogged with debris. Such screens should be tapped rapidly and gently on the under surface with the ﬁngers to assist passage of the water. The nematodes, together with debris, will be trapped on the surface of the 400- or 500-mesh sieve. Both can be removed by directing a small jet of water on the back of the sieve and washing the contents into a separate container (Fig. 5b). Certain biases are inherent in sieving extraction methods (Hummon, 1981). Since they may be unsuitable for quantitative studies (Viglierchio and Schmidt, 1983), the more tedious method of sorting through unsieved samples under a dissecting microscope may be desirable (Strayer, 1985). The ﬁnal step consists of separating the nematodes from the ﬁne debris. For this, the Baermann funnel method, or a modiﬁcation thereof, can be used. All that is needed is a funnel with a piece of rubber tubing attached to the stem. The tube should be closed with a screw clamp. The nematode – debris mixture is placed on a ﬁne cloth or facial tissue, which is supported by a piece of screen held in the top, wide portion of the funnel. Water is slowly added to the funnel until the nematode – debris mixture is just covered. The clamp should be opened to eliminate the air trapped in the funnel stem and to allow a continuous column of water to ﬂow into the funnel. The nematodes crawl through the debris and facial tissue, drop through the screen, and settle at the base of the funnel stem, where they can be drawn off by releasing the clamp. The nematodes should be drawn off every 6 h; the apparatus can be operated in this fashion for several days. Then the nematodes can be counted or handpicked under a dissecting microscope. A more recently devised method of isolating nematodes from samples involves the principle of increasing the speciﬁc gravity of the solution to make the nematodes ﬂoat to the surface. Substances such as sugar, salt, or Ludox can be added to the nematode – water mixture in the centrifugal-ﬂotation and sugar-ﬂotation techniques (Ayoub, 1977). If sugar is used, then 673 g added to 1 L of water will result in a speciﬁc gravity of 1.18, which is greater than that of nematodes (causing them to ﬂoat on the surface). Detailed instructions for these and other methods of extracting nematodes from soil and plant samples are presented by Ayoub (1977) and Nichols (1979). Quantitative methods of extraction for monitoring nematode populations over a period of time often involve the use of elutriators, which use an upcurrent of water to separate nematodes from the medium. Such methods are rather elaborate and are discussed by Southey (1978). C. Fixing and Mounting After the nematodes have been extracted, they should be held in water (preferably water from their original collecting source) prior to ﬁxation. The nematodes should be killed before being placed in ﬁxative; otherwise they become distorted and difﬁcult to examine. They are most easily killed with heat, which also tends to relax and extend them. Too much heat will destroy the internal tissues, but good results can be obtained by pouring hot water (60 – 70°C) over the nematodes. They should then be transferred to the ﬁxative as soon as possible. The best ﬁxative for nematodes is TAF (7 mL 40% formalin, 2 mL triethanolamine, and 91 mL distilled water). However, 3 – 5% formalin or 70% ethanol can also be used if TAF is not available. Some distortion may occur with ethanol, however. The nematodes should be ﬁxed for at least 2 – 3 days before transfer to glycerin for mounting on microscope slides. Nematodes are most easily conveyed to glycerin by the evaporation method, which requires little handling. Fixed specimens are transferred to a dish containing a solution of 70 mL ethanol (95%), 5 mL glycerol, and 25 mL water. The dish is partly covered for the ﬁrst 3 days to allow the alcohol and water to evaporate at a slow rate; then the cover is removed for the next 14 days. Finally, the containers (now with mostly glycerin) are placed in a desiccator or an oven (35°C) for another 2 weeks to drive out the remaining water. The nematodes will then be in a relatively pure solution of glycerin and can be mounted directly on 9. Nematoda and Nematomorpha microscope slides. Such slides are termed permanent since they can be kept for years for continuous study. Eventually, the nematode tissues tend to become transparent. Temporary slides are made by mounting the nematodes directly after ﬁxation in the ﬁxing solution (formalin or alcohol). They may last several months if the ringing seal is tight. The following steps can be taken to make permanent slides with specimens transferred to glycerin (modiﬁed from Poinar, 1983): 1. With a small pointed instrument (dental pulp canal ﬁle, small insect pin mounted on a wooden splint, needle), transfer the nematode(s) to a small drop of glycerin placed in the center of a microscope slide. 2. Push the nematode to the bottom of the drop. Then add at three equidistant points around the nematode, small supports (coverslip pieces, wire) with a width equal to or slightly wider than that of the nematode. Push these also to the bottom of the drop. 3. Place a cover slip over the drop and lower it slowly at a 30° angle so that air bubbles will not become entrapped in the glycerin as the slide touches the mounting medium. 4. Add more glycerin, if needed, by placing a small drop at the edge of the coverslip and allowing it to move under the glass and spread throughout. If the coverslip is ﬂoating on glycerin, then remove the excess by blotting it with a moistened (water) piece of tissue. 5. Carefully seal the edges of the coverslip with a ringing compound, such as nail polish or Turtox slide ringing cement (General Biological Supply House, Chicago, II). 6. Label the slide with information regarding the date, locality, and collector. D. Culturing Some free-living, microbotrophic freshwater nematodes can be cultured in the laboratory if they are presented with a growing colony of their preferred food (i.e., bacterial or algal species) and maintained under environmental conditions (temperature, oxygen) comparable to those present at the collecting site. Nuttycombe (1937) successfully cultured several species of freshwater nematodes using a wheat grain infusion method. Between 200 and 300 grains of wheat seeds were added to 250 mL of spring water in a ﬂask. This mixture and a separate ﬂask of pure spring water were heated to a boil and allowed to cool. The spring water was poured into 200 mL Petri dishes with 3 – 4 grains 269 of the boiled wheat. The dishes with the wheat seeds were allowed to stand for 2 – 3 days before the nematodes were added. Microorganisms brought in with the nematodes grew on the wheat seeds, and the microbotrophic nematodes fed on the microbes. Postparasitic juveniles of freshwater mermithids can be held in the laboratory until they mature for identiﬁcation. Care should be taken so that both the temperature and the amount of dissolved oxygen parallel those of the collection site. Periodic transfers to new containers with freshwater may be necessary to prevent the buildup of fungi which will destroy the nematodes. Petersen (1972) devised a method of culturing a mosquito mermithid parasite (Romanomermis culicivorax) through its entire life cycle. The mosquito chosen as a laboratory host was Culex pipiens quinquefasciatus (Say), because it could be reared continuously in crowded conditions, was easily maintained in colony, and was highly susceptible to the nematode. Postparasitic juvenile mermithids were placed in wet sand within plastic trays (36 25 100 cm). The postparasites molted to the adult stage, mated, and oviposited in the sand. The eggs, which embryonated in the moist sand, could be maintained in their resting state for several months. When the trays were ﬂooded with water, the infective stages emerged from the sand and were capable of infecting newly hatched mosquito larvae. The infected insect larvae were kept in a separate container and fed. By the time the mosquitoes were ready to pupate, the nematodes had completed their development and started to emerge. The emerging postparasitic juveniles were then transferred to wet sand to continue the cycle. Adequate numbers of mermithids were available for both scientiﬁc investigations and biologic control studies with this rearing method. VI. IDENTIFICATION Once the specimens are mounted, they can be examined and identiﬁed with a compound microscope. Because the identiﬁcation of nematodes involves the use of many ﬁne details, a microscope equipped with oil immersion should be employed (1000 ). The use of dioscopic illumination after Nomarski or differential interference contrast better reveals minute features of the cuticle (such as the amphid structure). If possible, living or just-killed nematodes should also be examined under the microscope. Their movements can be reduced by removing water from under the coverslip and compressing the nematode between the microscope slide and coverslip. Certain characters are much clearer on living specimens and there is a certain thrill in watching these creatures move under a microscope 270 George O. Poinar, Jr. that is absent with preserved material. However, measurements can best be made on ﬁxed nematodes. In preparing the following taxonomic key, the following works dealing with freshwater nematodes were consulted: Cobb (1914); Chitwood and Allen (1959); Ferris et al. (1973); Tarjan et al. (1977); Pennak (1978, 1989); Esser et al. (1985); Esser and Buckingham (1987); Jacobs (1984); Tsalolikhin (1983); Gerber and Smart (1987). The ﬁrst ﬁve references also have keys which can be consulted by the interested reader for cross-reference. For descriptions of various genera and a listing of species, Goodey (1963) is still very helpful, although now out of date. Some 66 genera of freshwater nematodes are included in the present key. Between 300 and 500 species of nematodes are probably included within these 66 genera. Only those genera that contain species that are obligately aquatic (live nowhere else) and have been reported from North America are included. Over 95% of the aquatic nematodes encountered should be included in the following key. A. Taxonomic Key to Families and Selected Genera of Freshwater Nematoda The following key pertains to the families and selected genera of freshwater nematodes and freeliving stages of parasitic nematodes found in freshwater habitats of North America. Following this is a separate key to the genera of aquatic Mermithidae. 1a. Elongated and threadlike, generally over 1 cm in length and usually observable with the naked eye; found on the bottom surface or several centimeters within the bed (Figs. 5c and 10a, b) ....................................................................................................................2 lb. Not elongate, generally under I cm in length and observable only with a lens (Figs. 2c and 11c); found on all exposed surfaces (rocks, on or in plants) as well as on the bottom surface and within the bed ......................................................................................3 2a (1a). Forms usually long (6 cm or more in length), leathery body wall, range in color from light brown to black (Fig. 13a) ........................ Hairworms (phylum Nematomorpha) 2b. Forms usually smaller (under 6 cm in length), body wall fragile, range in color from white to rose, green and yellow (Fig. 5c) ........... Mermithidae (see Section VI.B.) 3a (1b). Head of nematode bearing a stylet (Figs. 8b and 9b) ..........................................................................................................................4 3b. Teeth and other armature may be present, but a stylet is absent (Figs. 7a, b and 8a) ........................................................................16 4a (3a). Stylet knobs usually absent (Fig. 9b); if present, then pharynx lacking a valvated metacorpus; pharynx narrow anterior and thickened posterior at junction with intestine (Fig. 2a); valvated metacorpus absent ...........................................................Dorylaimida 11 4b. Stylet knobs usually present (Fig. 8b); pharynx composed of a valvated metacorpus followed by a slender isthmus and basal glandular bulb leading into the intestine (Figs. 8b, c) ....................................................................................................................................5 5a (4b). Dorsal gland outlet in precorpus; metacorpus moderate in size (Fig. 8b) (less than three-fourths of body width ..............Tylenchida 8 5b. Dorsal gland outlet in metacorpus; metacorpus large (three-fourths of body width or more) (Fig. 8c) ................................................. Aphelenchida .....................................................................................................................................................................................6 6a(5b). Stylet usually with faint or inconspicuous knobs (Fig. 8c); tail tip usually pointed; males lacking bursa and gubernaculum................. Aphelenchoididae ...............................................................................................................................................................................7 6b. Stylet without knobs; tail tip usually rounded; males with a bursa (Fig. 6c) and gubernaculum ........................................................... Aphelenchidae Aphelenchus Bastian, 1865 7a (6a). Tail shape elongate, ﬁliform .................................................................................................................................Seinura Fuchs, 1931 7b Tail shape short, conical ........................................................................................................................Aphelenchoides Fischer, 1894 8a(5b). Head bearing distinct setae (Fig. 7a) .......................................................................................Atylenchidae Atylenchus Cobb, 1913 8b Head without setae ...........................................................................................................................................................................9 9a (8b). Procorpus fused with large, oval metacorpus; cuticle strongly annulated; adult female with cuticular sheath and well-developed stylet (Fig. 8b) ........................................................................................................Criconematidae Hemicycliophora de Man, 1921 9b. Procorpus distinct and narrow before reaching the expanded metacorpus, cuticle not strongly annulated; adult female without cuticular sheath; styles may or may not be well developed.............................................................................................Tylenchidae 10 10a (9b). Large distinct stylet, ovaries paired, amphidelphic (Fig. 6a); tail tapering but not ﬁliform..........................................Hirschmanniella Luc and Goodey, 1963 10b. Stylet small and inconspicuous, ovary single, prodelphic; tail ﬁliform ............................................................Tylenchus Bastian, 1865 11a (4a). Pharynx gradually widens toward basal bulb (Fig. 2a) .....................................................................................................................12 FIGURE 6 (a) Midbody region of a female Pellioditis sp. (Rhabditidae) showing the vagina (arrow) and paired opposite uteri ﬁlled with developing eggs. Note copulation deposit surrounding the vulva opening. (b) Longitudinal striations lining the cuticle of Dorylaimus sp. (Dorylaimidae). (c) Tail of male Pellioditis (Rhabditidae) showing spicule (S), gubernaculum (G), bursa (B), and bursal papillae or rays (arrows). (d) Tail of an aquatic nematode showing caudal gland mucous being emitted from the terminus (arrow). 271 FIGURE 7 (a) Anterior end of an aquatic nematode showing a narrow tube-shaped stoma (arrow) and cuticular bristles or setae. (b) Large oval, cup-shaped stoma of a predatory aquatic nematode. Note dorsal tooth (arrow) on stomal wall. (c) Nematode bearing a medium-sized, circular amphid. (d) Nematode bearing a large, spiral-shaped amphid. 272 FIGURE 8 (a) Pharyngeal region of Pellioditis (Rhabditidae) showing tubular stoma (S), corpus (C), metacorpus (M), isthmus (I), and basal bulb (B). Arrow shows valve in basal bulb. (b) Outer ensheathing cuticle of Hemicycliophora (Hemicycliophoridae). Note long stylet (S) with prominent basal knobs (arrow) and valvated metacorpus (M). (c) Anterior of Aphelenchoides sp. (Aphelenchoididae) showing small stylet lacking knobs (arrow) and large metacorpus (M) with valve. (d) Outstretched ovary (arrow shows tip). (e) Reﬂexed ovary (arrow shows point of reﬂexion). 273 274 George O. Poinar, Jr. 11b. Pharynx abruptly widens to form the basal bulb which contains a valvular chamber (Fig. 8a) ......................................Campydoridae Aulolaimoides Micoletzky, 1915 12a (11a). Head bearing a narrow protrusible mural spear that is symmetrically pointed at tip ..............Nygolaimidae Nygolaimus Cobb, 1913 12b. Head bearing a thick protrusible axial spear that is asymmetrically pointed at tip (sloped on one side) (Fig. 9b) ................................. Dorylaimidae ...................................................................................................................................................................................13 13a (12b). Stomal area bearing distinct teeth or denticles around the tip of the stylet .......................................................................................14 13b. Stomal area lacking distinct teeth or denticles in the stylet tip region ...............................................................................................15 14a (13a). Four large teeth together with mural denticles present ............................................................................Paractinolaimus Meyl, 1957. 14b. Four large teeth only present ........................................................................................................................Actinolaimus Cobb, 1913 15a (13b). Cuticle thick and longitudinally ridged (Fig. 9b) .......................................................................................Dorylaimus Dujardin, 1845 15b. Cuticle smooth ..................................................................................................................................Mesodorylaimus Andrassy, 1959 16a (3b). Amphids minute and borne on the lateral lips, excretory duct cuticularized (canal lined with a ﬁne layer of cuticle); caudal glands absent, conspicuous cephalic setae absent, bursa present or absent ................................................................................................ 17 16b. Amphids usually enlarged and located on the neck region, excretory duct rarely cuticularized (except in Plectidae); head often with conspicuous setae; bursa usually absent. ..........................................................................................................................................21 17a (16a). Pharynx with an expanded median bulb (metacorpus) containing a longitudinal valve and a glandular nonvalvated basal bulb Diplogasteridae ...................................................................................................................................... Rhabditolaimus Fuchs, 1915 17b. Pharynx may or may not contain a metacorpus, but if it does, it does not contain a valve; basal pharyngeal bulb with or without a basal valve........................................................................................................................................................................................18 18a(17b). Pharynx elongate and lacking a valve in the basal bulb or bulb area, slender; slow moving forms (Fig. 9d) ....................Daubayliidae Daubaylia Chitwood and Chitwood, 1934 18b. Pharynx normal in length, with a basal bulb containing a valve (Fig. 8a); stouter, quick-moving forms ...........................................19 19a(18b). Stoma with rhabdions separate, female with a single ovary, bursa absent ..................................................................Cephalobidae 20 19b. Stoma with rhabdions fused, cylindrical (Fig. 8a), female with a single or paired ovaries, bursa usually present (Fig. 6c) .................... Rhabditidae [Most of the genera in this large family are soil forms, some of which appear from time to time in aquatic habitats. Mesorhabditis (Osche, 1952), which has a single ovary and a bursa, occurs ;n sewage bed and run-off water. Pellioditis (Doughtery, 1953), which has paired ovaries also occurs in aquatic habitats.] 20a (19a). Lateral ﬁelds reach to tail tip; female tail rounded .......................................................................................Cephalobus Bastian, 1865 20b. Lateral ﬁelds end at the phasmids; female tail usually pointed...................................................................Eucephalobus Steiner, 1936 21a (16b). Amphids spiral (Fig. 7d), loop-like, stirrup-shaped or circular (Fig. 7c); pharynx usually with a basal bulb (Fig. 8a) .......................22 21b. Amphids pore-like to pocket-like, pharynx usually without a distinct basal bulb .............................................................................41 22a (21a). Ovary usually outstretched (reﬂexed in Prismatolaimus) (Fig. 8d), amphids usually circular (Fig. 7c) but may be slitlike .................... Monhysterida...................................................................................................................................................................................23 22b. Ovaries reﬂexed (Fig. 8e) (except in Odontolaimus which also has circular amphids), amphids variable (including circular)...........25 23a (22a). Amphids slit-like, faint; stoma with denticulated cushions (padded areas covered with minute projections)......................................... Prismatolaimus de Man, 1880 23b. Amphids circular, distinct, stoma lacking denticulated cushions .......................................................................................................24 24a (23b). Stoma shallow; pharynx without distinct basal bulb (as in Fig. 2a) .............................................................Monhystera Bastian, 1865 24b. Stoma elongate; pharynx with distinct basal bulb (as in Fig. 8) ....................................................................Monhystrella Cobb, 1918 25a (22b). Caudal glands and spinneret present (Fig. 2d); stoma usually armed with teeth (Fig. 7b); knobs, bristles (Fig. 7a), or punctations usually present on cuticle .........................................................................................................................................Chromadorida 26 25b. Caudal glands and spinneret present or absent; stoma may or may not be armed with teeth; bristles sometimes present on cuticle,especially around head ..................................................................................................................................................Araeolaimida 33 26a (25a). Amphids circular (Fig. 7c); cuticular punctations minute ..................................................Microlaimidae Microlaimus de Man, 1880 26b. Amphids spiral (Fig. 7d), kidney shaped or circular; cuticular punctations coarse ............................................................................27 27a (26b). Amphids circular (as in Fig. 7c) or spiral (as in Fig. 7d), pharyngeal – intestinal junction large, tri-radiate..............Cyatholaimidae 29 FIGURE 9 (a) Posterior portion of a postparasitic juvenile mermithid (Mermithidae) in the process of molting. Note shedding of third- (3) and fourth-stage (4) cuticles. A, adult. (b) Head of Dorylaimus sp. (Dorylaimidae) showing base of lip region (arrows) and stylet lacking basal knobs. (c) Tail region of a Tobrilus sp. (Tobrilidae) showing spores of a microsporidian parasite ﬁlling the hypodermal tissue (arrows). (d) Adults of Daubaylia (Daubayliidae) inside an aquatic snail. 275 276 George O. Poinar, Jr. 27b. Amphids spiral or kidney shaped, pharyngeal – intestinal junction small, not tri-radiate .........................................Chromadoridae 28 28a (27b). Amphids represented as a broad transverse slit; ﬁve or more preanal genital papillae in male.................Chromadorita Filipjev, 1922 28b. Amphids represented as a ﬂattened, broken ring; no more than three preanal genital papillae in male ................................................. Punctodora Filipjev, 1930 29a (27a). Amphids circular (Fig. 7c) ................................................................................................................................................................30 29b. Amphids spiral (Fig. 7d) ...................................................................................................................................................................31 30a (29a). Stoma cuplike; length shorter than neck width ..................................................................................Prodesmodora Micoletzky, 1923 30b. Stoma tubular (Fig. 7a); length two or more times neck width ...............................................................Odontolaimus de Man, 1880 31a (29b). Stoma tubular (Fig. 7a), there may be three anterior equal teeth in the stoma, but single large dorsal tooth is lacking ......................... Ethmolaimus de Man, 1880 31b. Stoma cup- or funnel-shaped, armed with a prominent dorsal tooth (as in Fig. 7b) much larger than any other teeth......................32 32a (31b). Amphids located at level of stoma, small subventral teeth absent .................................................Paracyatholaimus Micoletzky, 1924 32b. Amphids located posterior to stoma, one or two small subventral teeth present .........................................Achromadora Cobb, 1913 33a (25b). Caudal glands absent; stoma funnel-shaped with separate rhabdions ..................................................................Teratocephalidae 34 33b. Caudal glands present (Fig. 2a) or absent; stoma tubular with rhabdions fused ...............................................................................35 34a (33a). Amphids porelike; cuticular annulation strong (Fig. 8b) ........................................................................Teratocephalus de Man, 1876 34b. Amphids spiral (Fig. 7d); cuticular annulation weak ........................................................................Euteratocephalus Andrassy, 1958 35a (33b). Ovary outstretched (Fig. 8d); amphids circular.............................................................................................................Axonolaimidae Cylindrolaimus de Man, 1880 35b. Ovary reﬂexed (Fig. 8e) ....................................................................................................................................................................36 36a (35b). Pharynx usually with a basal valvated bulb (Figs. 1 and 8a).............................................................................................................37 36b. Pharynx with or without a basal bulb, if bulb present, then valve absent .........................................................................................39 37a (36a). Basal bulb of pharynx lacking valves ................................................................................................................Anonchus Cobb, 1913 37b. Basal bulb of pharynx with valves (Fig. 8a) ......................................................................................................................................38 38a (37b). Pharyngeal basal bulb with postbulbar extension (Fig. 10c) connecting it with the intestine; caudal glands absent .............................. Chronogaster Cobb, 1913 38b. Phayrngeal basal bulb without postbulbar extension, abutting the intestine; caudal galnds present (Fig. 1) ..............................Plectus Bastian, 1865 39a (36b). Basal bulb lacking .......................................................................................................................Bastianiidae Bastiania de Man, 1876 39b. Basal bulb present ................................................................................................................................................Camacolaimidae 40 40a (39b). Amphids circular (Fig. 7c), basal pharyngeal bulb absent .......................................................................Aphanolaimus de Man, 1880 40b. Amphids spiral (Fig. 7d), a slight basal pharyngeal bulb present ...................................................Paraphanolaimus Micoletzky, 1923 41a (21b). Stoma usually oval in outline (Fig. 7b), heavily sclerotized and armed with one or more teeth, setae and bristles absent ..................... Mononchidae 41b. Stoma not in the form of a heavily sclerotized oval cavity, teeth or denticles may be present, setae and bristles may be present (Fig. 7a)............................................................................................................................................................................................42 42a (41b). Cuticle of head double.............................................................................................................................................Oncholaimidae 43 42b. Cuticle of head normal .....................................................................................................................................................................44 43a (42a). Setae or bristles present on tail ...............................................................................................................Oncholaimus Dujardin, 1845 43b. Setae and bristles usually lacking on tail......................................................................................................Mononchulus Cobb, 1918 44a (42b). Stoma heavily sclerotized, cylindrical .................................................................................................................................Ironidae 49 44b. Stoma not heavily sclerotized, funnel-shaped or tubular...................................................................................................................45 45a (44b). Stoma vestigial and unarmed; pharynx base expanded and set off to form a slight, elongate bulb...................................Alaimidae 46 FIGURE 10 Chronogaster troglodytes Poinar and Sarbu, a specialized aquatic cave nematode from Movile Cave in Romania. (a) Anterior end with funnel-shaped mouth cavity. (b) Head showing stirrup-shaped amphid with a circular opening. (c) Basal pharyngeal region showing valve (ﬂeche) in basal bulb and postbulbar extension (arrow) between the basal bulb and intestine. (d) Spermlike bodies (arrow) which occur near the proximal portion of the female gonad, and the absence of males, suggests that C. troglodytes is hermaphroditic. 277 278 George O. Poinar, Jr. 45b. Stoma distinct, or if vestigial then armed with an inconspicuous median tooth; pharynx may be expanded at base, but rarely set off from the remainder in the form of a bulb .........................................................................................................................................47 46a(45a). Amphids porelike, minute ................................................................................................................................Alaimus de Man, 1880 46b. Amphids cup-shaped, distinct......................................................................................................................Amphidelus Thorne, 1939 47a(45b). Base of pharynx expanded to form a valvated bulb; three rodlike thickenings compose posterior part of stoma .................................. Leptolaimidae .........................................................................................................................................Rhabdolaimus de Man, 1880 47b. Pharynx cylindrical, not with basal valvated bulb; stoma a simple tube without rodlike thickenings ..............................Tripylidae 48 48a(47b). Three lips; amphids porelike, minute; stoma cylindrical .....................................................................................Tripyla Bastian, 1865 48b. Six lips; amphids cup-shaped, distinct; stoma funnel-shaped ..........................................................................Tobrilus Andrassy, 1959 49a(44a). Stoma tubular, with two minute teeth at the base; excretory pore opening posterior to head .......................Cryptonchus Cobb, 1913 49b. Stoma tubular, with three anterior hooklike teeth; excretory pore opening in head area ......................................Ironus Bastian, 1865 50a(41a). Pharyngeal-intestinal junction tuberculate; dorsal tooth on stoma pointing posteriorally...............................Anatonchus Cobb, 1916 50b. Pharyngeal-intestinal junction not tuberculate; dorsal tooth on stoma pointing anteriorally ............................................................51 51a (50b). Ventral stomatal ridge with longitudinal row of denticles ..............................................................................Prionchulus Cobb, 1916 51b. Ventral stomatal ridge absent, or if present then unarmed ...........................................................................Mononchus Bastian, 1865 B. Taxonomic Key to Extant Genera of Freshwater Mermithidae The mermithids represent a family of nematodes that have become parasitic on other invertebrates. Over half of the described genera parasitize only aquatic insects. Two genera, Aranimermis and Pheromermis, enter the immature stages of aquatic insects which are later consumed by terrestrial arthropods. However, the free-living stages of these and all other aquatic mermithids (postparasitic juvenile, adults, eggs, and infective juveniles) are found in the aquatic habitat. Only the developing second- and third-stage juveniles parasitize the aquatic stages of insects and other invertebrates; the other stages do not take nourishment. Eggs and infective stage juveniles are microscopic and normally not taken in samples, but the postparasitic juveniles and adults are easily seen and collected. Since the keys are based on adult characters, postparasitic juveniles should be held in water until they molt to the adult stage. One important character in identifying mermithids is the number of hypodermal cords present. These can be determined by cutting thin cross sections with a razor blade by hand, and examining them under the microscope. The hypodermal cords protrude through the muscle ﬁelds in dorsal, ventral, lateral, and often the subdorsal and subventral regions. Only genera known to occur in North America are included. 1a. Adult cuticle with cross-ﬁbers; thick, robust white nematodes usually found along the edges of bogs, springs, and streams (parasites of wasps, ants, and horseﬂies) (Fig. 11b)......................................................................Pheromermis Poinar, Lane, and Thomas, 1976 1b. Adult cuticle without cross-ﬁbers, robust or slender, white, green, pink, brown, or yellow nematodes found in lake, stream, and pond beds...........................................................................................................................................................................................2 2a. With four cephalic papillae..................................................................Pseudomermis de Man, 1903 (syn. Tetramermis Steiner, 1925) 2b. With six cephalic papillae...................................................................................................................................................................3 3a (2b). With single or fused (rare) spicules .....................................................................................................................................................4 3b. With paired, separate spicules ............................................................................................................................................................8 4a (3a). Spicule medium to long, more than twice anal body width ................................................................................................................5 4b. Spicule short, less than twice anal body width....................................................................................................................................6 5a (4a). Mouth terminal; spicules J-shaped; vulval ﬂap present; [parasites of midges (Chironomidae)].......Lanceimermis Artyukhovsky, 1969 5b. Mouth normally shifted ventrally; spicule curved but not J-shaped; vulva ﬂap absent; [parasites of blackﬂies (Simuliidae), midges (Chironomidae), and mayﬂies (Ephemeroptera)]................................................................................Gastromermis Micoletzky, 1923 6a(4b). Spicule shorter than anal body width; amphids small; [parasites of mosquitoes (Culicidae)] ....................Perutilimermis Nickle, 1972 9. Nematoda and Nematomorpha FIGURE 11 (a) A postparasitic juvenile aquatic mermithid emerging from an adult caddis ﬂy, Trichoptera (collected by H. Wolda and submitted by R. Schuster). (b) Adults of Pheromermis pachysoma (Mermithidae) in a California spring bed. These nematodes have emerged from parasitized queen wasps visiting the spring and will undergo molting, mating and oviposition in the spring. (c) Various eggs and juvenile stages of freshwater nematodes developing in a decaying, submerged leaf. 279 280 George O. Poinar, Jr. 6b. Spicule longer than anal body width; amphids medium to large ........................................................................................................7 7a(6b). Tail pointed; eight hypodermal cords; [parasites of midges (Chironomidae) and mosquitoes (Culicidae)] ...Hydromermis Corti, 1902 7b. Tail rounded; six hypodermal cords; [parasites of midges (Chironomidae) and blackﬂies (Simuliidae)] .....Limnomermis Daday, 1911 8a (3b). Vagina straight or nearly so, barrel- or pear-shaped ...........................................................................................................................9 8b. Vagina S-shaped, U-shaped, or elongate with both ends curved........................................................................................................13 9a (8a). Spicules shorter than cloacal body width..........................................................................................................................................10 9b. Spicules equal to or longer than cloacal body width .........................................................................................................................11 10a (9a). Head expanded into a bulblike shape with very thick cuticle; male lacking lateral – dorsal genital papillae; [parasites of midges (Chironomidae)] .....................................................................................................................................Capitomermis Rubtsov, 1968 10b. Head not expanded into a bulblike shape with thick cuticle; male with genital papillae on lateral-dorsal surface; [parasites of biting midges (Ceratopogonidae)] ...................................................................................................................Heleidomermis Rubtsov, 1970 11a(9b). Six hypodermal cords; [parasites of blackﬂies (Simuliidae)] .........................................................................Mesomermis Daday, 1911 11b. Eight hypodermal cords ...................................................................................................................................................................12 12a(11b). Spicules 2 – 4 times anal body width; [parasites of mosquitoes (Culicidae)] ............................................Romanomermis Coman,1961 12b. Spicules 1-2 times anal body width; [parasites of mosquitoes (Culicidae) and midges (Chironomidae)] ............................................... Octomyomermis Johnson, 1963 13a (8b). Eight hypodermal cords ...................................................................................................................................................................16 13b. Six hypodermal cords .......................................................................................................................................................................14 14a(13b). Spicule length less than two times anal diameter; [parasites of midges (Chironomidae) and mosquitoes (Culicidae)] ........................... Strelkovimermis Rubtsov, 1969 14b. Spicule length three or more times cloacal diameter .........................................................................................................................15 15a(14b). Spicules ten or more times body width at cloaca; vagina elongate with bends at both ends; postparasitic juveniles with distinct tail appendage;[parasites of diving beetles (Dytiscidae)]................................................................Drilomermis Poinar and Petersen, 1978 15b. Spicules 3 – 10 times body width at cloaca; vagina elongate with 3 – 6 irregular bends; postparasitic juveniles with indistinct or no tail appendage; [parasites of spiders] ........................................................................................Aranimermis Poinar and Benton, 1986 16a(13a). Vagina elongate, slightly curved; spicules shorter than anal body diameter; [parasites of mosquitoes (Culicidae)]................................ Culicimermis Rubtsov and Isaeva, 1975 16b. Vagina S-shaped, ends distinctly curved; spicules range from shorter than anal body diameter to about 10 times tail diameter .......17 17a(16b). Spicule length equal to or shorter than tail diameter; amphids small; cephalic crown well developed; [parasites of mosquitoes (Culicidae)] ..............................................................................................................................................Empidomermis Poinar, 1977 17b. Spicule length between one and ten times anal diameter; amphids medium – large, cephalic crown absent or only slightly developed; [parasites of blackﬂies (Simuliidae)] ...............................................................................................................Isomermis Coman, 1953 NEMATOMORPHA VII. INTRODUCTION Members of the poorly known phylum Nematomorpha (Gordiacea) constitute a relict group that has no clear or close relationship with any other living forms. Fossil hairworms are rare and controversial.The earlier determination of Gordius tenuiﬁbrosus (Voigt, 1938) as a fossil hairworm (identiﬁed from a 15-mm long fragment of subcuticular tissue in the Eocene brown coals of the Geisel Valley near Halle, Germany) is now considered questionable in light of similar characters found on members of the Mermithidae (Poinar, 1999). And while it has been proposed that fossil palaeoscolecid worms are larval hairworms (Xianguang and Bergstrom, 1994), similarities between these two taxa are only superiﬁcal and provide no evidence for phylogenetic relationship (Poinar, 1999). The only unequivocal record of fossil Nematomorpha are two hairworms, Paleochordodes protus Poinar (1999), emerging from a cockroach in Dominican amber dated at 15 – 45 million years (Fig 20a). However, the Nematomorpha certainly originated considerably earlier (probably in the Early Paleozoic) as an offshoot of now extinct lines. Chitwood (1950) considered that Nematomorpha had the closest ties (albeit distant) with nematodes and rotifers (the Acanthocephala and 9. Nematoda and Nematomorpha Echinodermata were tied for the next closest kin). Using cladistic methods, Schmidt-Rhaesa (1998b) also concluded that nematomorphs are closest to nematodes; however, it is difﬁcult to establish lineages when such basic characters as the number of molts during the 281 growth phase has not been conﬁrmed. The body plan of the Nematomorpha is unique in the animal kingdom and even the sperm are considered aberrant and support the contention that this group separated quite early from the other members of the Aschelminthes and FIGURE 12 (a) Oldest known undisputed fossil nematode, Cretacimermis libani (Poinar Acra and Acra) from 120 – 135 million-year-old Lower Cretaceous Lebanese amber. This mermithid infected and developed in the aquatic larval stage of a midge (Chironomidae) and was carried over into the adult host. (b) Two mermithid nematodes, Heydenius dominicanus Poinar, in 20 – 40 million-year-old Dominican amber. These nematodes probably emerged from an adult mosquito also enclosed in the amber (ﬁgure shows an adjacent craneﬂy adult in the amber; the mosquito could not be included in the photo at this magniﬁcation). 282 George O. Poinar, Jr. evolved independently thereafter (Lora Lami Donin and Cotelli, 1977) (Fig. 15). Some myths surround this group, perhaps associated with the proverbial “Gordian knot” (representing a problem solvable only by drastic action as demonstrated by Alexander the Great cutting the knot that could not be untied, which bound a chariot to a pole at Gordium, the capital of Phrygia, in 333 BC). Another myth, still believed by some today, is suggested by the common name “hairworms” or “horsehair worms” indicating that they were supposed to have arisen from horse hairs that fell into water. This belief was scientiﬁcally disproven by Leidy in 1870, when he observed horse hairs placed in water over a period of many months without “ . . . having had the opportunity of seeing their viviﬁcation.” Adult hairworms have been associated with the digestive and urogenital tract of humans and larval hairworms will burrow into a wide range of invertebrate and vertebrate tissue, including human facial tissue sometimes resulting in orbital tumors (Watson, 1960). A review of the public health aspects of hairworms has been provided by Cappucci (1982). VIII. MORPHOLOGY AND PHYSIOLOGY It is the free-living adults of hairworms that are normally described because these stages are most frequently encountered in sampling. They are dark FIGURE 13 (rarely white), slender worms ranging in length from several centimeters to 1 m and in width from 0.25 to 3 mm (Fig. 13a). The color of most adults varies from yellowish to black; and although the anterior end is generally attenuated, both tips tend to be obtusely rounded or blunt (divided in some males and females). Because the cuticle is normally opaque, it is impossible to examine the internal organs through the body wall. Ultrastructural studies of a North American Gordius sp. revealed some interesting morphological features (Eakin and Brandenburger, 1974). The cuticular structures that are such important taxonomic characters are actually sculpturing on the surface of the thin, superficial epicuticle (Figs. 13b, 16a, 19). This epicuticle is normally criss-crossed by grooves or furrows, leaving small elevations of irregular areas (areoles) between them. The surfaces of the areoles may be smooth (Fig. 19a) or may bear setae (bristles) (Fig. 19b) or cuticular projections (tubercles) (Fig. 19c,d), arranged singly or in clusters. These bristles and tubercles may also occur in the interareolar furrows. Additional observations on the cuticular structure of hairworms include investigations of Paragordius varius by Zapotosky (1971), Parachordodes spp. by Smith (1991), G. aquaticus difficilis by Smith (1994) and Chordodes morgani by Chandler and Wells (1989). Many of these studies involve the use of the scanning electron microscope which is a useful tool for examining details of the surface sculpturing on the epicuticle. (a) A hairworm (Nematomorpha) emerging from its orthopteran host. (b) Transverse section of Gordius sp. C, cuticle; E, epidermis; EC, epicuticle; G, gut; M, muscles; ME, mesenchyme; NC, nerve cord; P, pseudocoel; VNL, ventral neural lamella. [Courtesy of R. M. Eakin and J. L. Brandenburger.] 9. Nematoda and Nematomorpha FIGURE 14 (a) Layer of eggs of Euchordodes nigromaculatus Poinar scraped off a boulder in Cave Stream, Craigieburn forest, New Zealand. Adhering the eggs to submerged substrates is an adaptation of hairworms living in fast ﬂowing streams. (b) Ventral view of tail of Gordius dimorphus Poinar showing bilobed tail with lobes longer than wide, spherical cloacal opening located ventrally and a semicircular postcloacal fold (arrow). The cuticle, beneath the epicuticle, is composed of many crossing layers of cylindrical, nonperiodic collagenous ﬁbers, spirally coiled along the length of the adult (Fig. 16). Beneath the cuticle is the epidermal layer, constructed of a single layer of interdigitated cells. The musculature consists only of overlapping, longitudinal, ﬂat cells; circular muscles are absent. Ultrastructural details of the musculature of G. aquaticus and 283 Nectomena munidae are provided by Schmidt-Rhaesa (1998a). The pseudocoel is nearly ﬁlled with mesenchymal cells containing large clear vacuoles, which give the tissue a foamy appearance. These cells are embedded in a supporting collagenous matrix (Fig. 16b). The mouth is located at the calotte or anterior tip of the body (Fig. 18a). This region is often lighter in color than the rest of the body and contains the nonfunctional pharynx, which, in turn, leads into a degenerate intestine lined with epithelium one cell in thickness. The gut epithelial cells of Gordius sp. contain numerous microvilli that project into the lumen (Eakin and Brandenburger, 1974). In both sexes, the genital ducts empty into the intestine, forming a cloaca lined with cuticle (Fig. 18b). Because adults do not feed, the cloaca is probably used solely for reproductive purposes. All nematomorphs are amphimictic. Males have paired cylindrical testes, each of which connects with the cloaca via a separate sperm duct. Females possess paired ovaries, and the eggs, after passing through separate oviducts, enter the cloaca independently. Adults become sexually mature soon after emerging from their hosts and copulation occurs after the male coils its posterior end around the terminus of the female. The spermatozoa are either deposited as a spermatophore on the female terminus, from where they migrate into the cloaca, or are deposited directly into the female cloaca. Ultrastructural studies of the sperm show that they are composed of a main cell body attached to a nuclear portion containing an elongated nucleus with a helical conﬁguration (Fig. 15a,b) (Lora Lami Donin and Cotelli, 1977) (Schmidt-Rhaesa, 1997). The sperm structure is similar in many aspects to that of mermithid nematodes (Poinar and HessPoinar, 1993). There are unique features, one of which is what has been called a multivesicular complex, actually a number of ﬂattened pockets, each ﬁlled with mucopolysaccharide appearing substances which surround the nuclear portion of the spermatozoan (Fig. 15a,b). The eggs are deposited singly or in clusters which often form elongate strings held together by secretions produced by the antrum (anterior portion of the female cloaca). In certain species that occur in fast ﬂowing waters, ovipositional modiﬁcations occur. While conducting studies on Euchordodes nigromaculatus in a stream in New Zealand, the present author noted that the eggs were attached as a coating to rocks and other submerged debris (Fig. 14a) (Poinar, 1989). This unique manner of oviposition explains how certain hairworms can maintain populations in fastﬂowing streams. Other investigators have noted eggs of Euchordodes attached to twigs and other stationary objects in the water (Bohall et al., 1997) (D.J. Watermolen, personal observation). 284 George O. Poinar, Jr. FIGURE 15 Electron micrographs of the lateral (a) and transverse (b) views of sperm cells of Neochordodes occidentalis (Montgomery). M, main cell body; N, nucleus; G, glycogen deposit surrounding nucleus; O, membraneous organelles forming the multivesicular complex. (Photos courtesy of Roberta Poinar.) The preparasitic stage that hatches from the egg is morphologically quite different from the adult worm and can, therefore, properly be called a larva. [Recall that the term larva implies that some type of metamorphosis occurs before the adult stage.] The larva consists of a presoma or preseptum (featuring an evaginable proboscis armed with cuticular spines) and a body or postseptum containing adult tissue primordia (Figs. 17, 18c,d). Ultrastructural studies of the larvae of Paragordius varius demonstrated quite clearly the nature of the proboscis and spines (Zapotosky, 1974, 1975). It is unfortunate that so few larvae of described nematomorph species are known, since they possess their own speciﬁc characters which could be of taxonomic value, as noted by Poinar and Doelman (1974) and Bohall et al. (1997). 9. Nematoda and Nematomorpha FIGURE 16 (a) Surface view of the areolar pattern on the epicuticle of Neochordodes occidentalis (Montgomery). (b) Raised areoles and intra-areolae furrows on the epicuticle of a Gordius sp. DG, deep groove separating areoles; SG, shallow grove separating areoles. (Courtesy of R. M. Eaken and J. L. Brandenburger.) (c) Cross section through the body wall of a Gordius sp. C, cuticle; E, epidermis; EC. epicuticle; LF, cuticular ﬁbers cut longitudinally; M, muscle plates; XF, cuticular ﬁbers cut crosswise. [Courtesy of R. M. Eaken and J. L. Brandenbruger.] FIGURE 17 285 (a) Figure of the preparasitic larva of Neochordodes occidentalis (Montgomery). P, presoma (preseptum); B, body (postseptum); a, anus; g, globule. g,d, gland duct; i, intestine; i.g, intestinal gland; m, mesenchyme cells; p.g, preintestinal gland; pr, protrusible proboscis; r.m, retractor muscles; st, stylets; sp, circulet of spines; t, tail spines (modiﬁed from Poinar and Doelman, 1974.). (b) Electron micrograph of a larva of N. occidentalis encysted in the gut wall of a tadpole of the tree frog Hyla regilla Baird and Girard. Note that the surrounding cyst is composed of several layers. [Photo courtesy of Roberta Poinar.] FIGURE 18 (a) Anterior end of an adult N. occidentalis. Note the degenerate pharynx (arrow). (b) Ventral view of male tail of N. occidentalis showing the elongate cloacal aperature (arrow). (c) Preparasitic larva of N. occidentalis showing presoma (P), body (B), intestinal gland (C), and stylets (S). (d) Preparasitic larva of N. occidentalis showing extruded secretions from the intestinal gland (arrows), stylet (ST), and spines (SP). 286 9. Nematoda and Nematomorpha FIGURE 19 (a) Surface view of the body wall of Gordius dimorphus Poinar showing the absence of areoles and the presence of parallel criss-cross ﬁbers. (b) Surface view of the areoles of Euchordodes nigromaculatus Poinar with minute spines present, especially in the furrows between the ﬂattened areoles. (c) Surface view of the areoles of Gordionus diblastus (Orley) showing variation in areole size and distribution. (d) Lateral view of the raised areoles of G. diblastus showing size variability. 287 288 George O. Poinar, Jr. IX. DEVELOPMENT AND LIFE HISTORY The four stages in the life history of nematomorphs are the egg, the preparasitic larva that hatches from the egg, the parasitic larva that develops within an invertebrate, and the free-living, aquatic adult. Parasitic larvae of all Nematomorpha mature only within invertebrates, which can be called the developmental or deﬁnitive hosts. A second type of host involved in the life cycle of probably the great majority of hairworms is a transport or paratenic host. The preparasitic larva enters the hemocoel and internal tissues of this host, but does not develop further until the paratenic host is eaten by a scavenger or predator. The paratenic host is usually an invertebrate, but can also be a vertebrate (e.g., tadpoles, ﬁsh, etc.). Poinar and Doelman (1974) noted that when larvae of Neochordodes occidentalis encysted in tadpoles (Hyla regilla), many of the cysts were composed of several overlapping layers as is shown in Figure 17b, in contrast to those normally formed in insects. However, such encysted larvae of N. occidentalis in the gut of tadpole paratenic hosts showed no abnormal features. Three types of life cycles have been described for nematomorphs. The ﬁrst is the direct type where the egg hatches in water and the preparasitic larva is ingested by, and develops in, the deﬁnitive invertebrate host. Such a cycle was demonstrated with Trichoptera larvae (Stenophylax sp.) infected by Gordius sp. (Dorier, 1930). The second type of cycle could be called indirect-free-living, since after the preparasitic larva hatches from the egg, it encysts on submerged leaves or other detritus. As the water dries up (found in forms inhabiting temporary ponds), the deﬁnitive host is infected by ingesting the cysts on the now exposed vegetation. Dorier (1930) indicated this type of development in a Gordius sp. that infected millipedes. For this cycle to be effective, the cysts would have to be resistant to desiccation. Further conﬁrmation of this type of development is needed. The third type of cycle can be called indirect-paratenic where, after hatching, the preparasitic larva is ingested by an invertebrate or vertebrate. Hatching occurs when the mature preparasitic larva forces itself through the wall of the egg (Fig. 20b). The parasite burrows into the tissues of this paratenic host and then encysts, but does not develop further. Only when the paratenic host is eaten by a predator (carabid beetle, praying mantis, dragonﬂy) or omnivore (various Orthoptera) does the parasite initiate development. This type of cycle occurs in the great majority of investigated hairworms, including a Chordodes developing in praying mantids that ingest infected mayﬂy paratenic hosts (Inoue, 1962), a Gordius developing in Dytiscus that feed on infected tadpole (Rana temporaria) paratenic hosts (Blunck, 1922), Euchordodes nigromaculatus developing in stenopelmatids that fed on infected stoneﬂies, mayﬂies or caddisﬂies (Poinar, 1991), Gordius robustus developing in crickets that were fed infected meal worms (Hanelt and Janovy, 1999) and probably Neochordodes occidentalis which readily infects and encysts in mosquitoes and other aquatic insects (Poinar and Doelman, 1974). In fact, it is apparent that this indirect-paratenic type of cycle is the most typical type of development for hairwroms. The ﬁrst two types of development mentioned above need conﬁrmation. There is no evidence that the preparasitic larvae can burrow directly through the outer body wall of either the paratenic or deﬁnitive host. In all cases, they enter by way of mouth and encyst in the midgut (Fig. 20e) or burrow through the midgut and encyst in the tissues of the body cavity. In cases of mass infection as demonstrated in laboratory studies with larvae of N. occidentalis invading mosquito larvae, the tissues may be damaged to such a degree that the host dies (Poinar and Doelman, 1974). In cases where the primary paratenic host is eaten by an insect predator which is not suitable for further parasitic development, the hairworm larvae may be able to re-encyst in the predator. Encysted larvae in dobsonﬂies probably represented cases of secondary paratenic hosts (Poinar, 1991). Host immunity to hairworms has thus far been documented only in the case of larvae entering or remaining in paratenic hosts. The usual type of host reaction to such parasitic larvae is humoral melanization which may occur immediately after entry as in the case of N. occidentalis invading mosquito larvae (Fig. 20c,d) (Poinar and Doelman, 1974) or after the larvae have encysted in the internal tissues of the paratenic host as with hairworm larvae in caddis-ﬂies, mayﬂies, stoneﬂies (Fig. 20e) and especially chironomid larvae (Poinar, 1991). Such melanization reactions often result in the death of invading larvae (Fig. 20d), although in cases where the host reaction occurred after encystment, it is possible that the parasites died of other causes and were then melanized. When parasitic development has been completed and the hairworm is ready to emerge, the host must come into contact with water. This poses no problem for parasites in aquatic hosts, but can present obstacles when the host is a terrestrial arthropod. Although entry into water may occur accidentally, it is more likely that the hosts suddenly have a desire to reach water. This may result from partial desiccation due to the actions of the parasite or be a consequence of some abnormal stimuli from parasite products that affect physiological centers of the host. When the host enters water, the hairworms emerge and either slowly sink or initiate FIGURE 20 (a) Two specimens of Paleochordodes protus Poinar (1999) that were in the process of emerging from their cockroach host in 15 – 45 million-year-old Dominican amber. These represent the ﬁrst unequivocal fossil Nematomorpha. (b) Pre-parasitic larva of Neochordodes occidentalis ready to emerge from its egg. (c) Host reaction showing initial stages of melanization of an invading larva of N. occidentalis that just penetrated into the hemocoel of a fourth stage mosquito larva (experimental infection; see Poinar and Doelman, 1974). (d) Melanized larva of N. occidentalis killed from the host reaction of a fourth stage mosquito larva (experimental infection; see Poinar and Doelman, 1974). (e) Dead, melanized encysted larvae of probably Euchordes nigromaculatus in the intestinal wall of the stoneﬂy, Stenoperla prasina (natural infection; see Poinar, 1991). 289 290 George O. Poinar, Jr. TABLE II List of Host families Reported to contain the Developing Stages of Nematomorphaa Taxon Phylum Annelida Class Hirudinea Order Rhynchobdellida Family Glossiphoniidae Order Pharyngobdellida Family Erpobdellidae Phylum Mollusca Class Gastropoda Subclass Pulmonata Family Lymnaeidae Phylum Arthropoda Class Arachnida Order Scorpionida Family Vaejovidae Order Ambylpygi Family Phrynidae Class Crustacea Order Notostraca Family Apodidae Order Decapoda Family Caridae Class Chilopoda Order Lithobiomorpha Family Lithobiidae Order Scolopendromorpha Family Scolopendridae Class Diplopoda Order Glomerida Family Glomeridae Order Spirobolida Family Spirobolidae Order Julida Family Julidae Class Insecta Order Odonata Family Libellulidae Order Orthoptera Family Acrididae Family Blattidae Family Gryllacrididae Family Gryllidae Family Rhaphidophoridae Family Stenopelmatidae Family Tettigonidae Order Mantodea Family Mantidae Order Phamsatida Family Phasmatidae Order Dermaptera Family Forﬁculidae Order Hemiptera Family Notonectidae Order Neuroptera Family Sialidae Order Lepidoptera Family Saturnidae Order Coleoptera TABLE II (Continued) Taxon Reference Family Carabidae Family Chrysomelidae Family Dytiscidae Family Silphidae Family Tenebrionidae Order Hymenoptera Family Apidae Blunck (1922) Dorier (1930) Blunck (1922) Blunck (1922) Baylis (1944) Reference Leidy (1878) Sawyer (1971) a Chitwood and Chitwood (1937) S. Williams (personal communication) Sciacchitano (1958) Linstow (1878) Linstow (1878) Dorier (1930) Dorier (1930) Dorier (1930) Cooper and Storck (1973) Sahli (1972) Heinze (1937) Blunck (1922) Sciacchitano(1958) Poinar (1989) Montogomery (1907) Poulin (1996) Poinar (1991) Thorne (1940) Sciacchitano (1958) Römer (1895) Sciacchitano (1958) Cury (1946) It is often impossible to determine whether authors are referring to paratenic or developmental hosts in their reports. Questionable references have been omitted. undulating body movements. Males are claimed to be more active than females. As the parasites mature in the host, they change from a light, cream color to a yellowish brown or dark brown color. At the time of emergence, most have already turned their natural color, but in some cases, further darkening occurs after emergence. Since there are so few records of known gordiids from identiﬁed hosts, very little is known about host selection and speciﬁcity. Some hairworms are probably restricted developmentally to certain invertebrate genera, while others may be able to develop in a wide range of hosts. Most deﬁnitive hosts are medium-to-large bodied predaceous or omnivorous arthropods. The great majority of freshwater nematomorphs have been collected from representatives of the insect orders Coleoptera and Orthoptera. Other host groups include myriapods (Diplopoda and Chilopoda), crustaceans, and leeches. A list of invertebrate families known to contain developing stages of hairworms is presented in Table II. The diversity of paratenic hosts is quite great, extending from trematodes (Cort, 1915) to vertebrates. Preparasites will attempt to burrow and encyst into the soft tissues of any cold blooded organism that ingests them. The natural enemies of hairworms are poorly known. Fish ingest the adults and the preparasites are probably fed upon by invertebrate micropredators. A Gordius adult that was brought to nestlings by a parent bird (a tapaculo, Scelorchilus rubecula) in a temperate rain forest in Chile was submitted to the present author by Mary F. Willson (personal correspondence, 1994), so birds can be listed as hairworm predators. Predation by rock bass, brown trout and crayﬁsh has also been documented (Cochran et. al., 1999). Zalmon (1977) Mellanby (1951) X. SAMPLING Camerano (1897) Except for the marine genus Nectonema, all known representatives of the Nematomorpha occur in freshwater. Their habitats range from watering troughs and (Continues) 9. Nematoda and Nematomorpha puddles to rivers, lakes and subterranean streams. Several researchers have commented on their occurrence in the Great Lakes (Watermolen and Haen, 1994; Cochran et al., 1990). No special sampling technique has been developed for hairworms. They are large enough to be seen with the naked eye and can be netted or lifted out of the water by hand. Stream forms often accumulate in slower moving water off to the side of the main current. Holes dug into small streamlets will often catch and hold adults that are being carried downstream and nets can be placed across small streams to collect the unattached adults. Late summer and spring are the best times to look for the free-living adults. Although the long, whitish egg strands of some species can sometimes be spotted in water, the preinfectives are too small to be found and are usually noted only by chance when examining the paratenic hosts. Perhaps dissecting paratenic hosts and searching for encysted larvae is the easiest way to determine if hairworms are present in an aquatic system. The eggs of those species adapted to fast-ﬂowing streams can be noted stuck to the surfaces of rocks and other debris. Growing larvae in developmental hosts can be found throughout the year, but are much less frequent than are encysted larvae in paratenic hosts and are nearly impossible to keep alive if they are removed from their host prior to completion of their development. No methods are available for in vitro development or culture of nematomorphs. May (1919) was able to monitor development by injecting preparasitic larvae of Gordius into the body cavity of long-horned grasshopper hosts (Tettigonidae). However, because of the fragile character of the preparasitic forms and the relatively long period of parasitic development (several months) coupled with the problem of maintaining the hosts, few workers have even bothered to culture hairworms in vivo. XI. IDENTIFICATION Adult hairworms can be preserved in 5‰ formalin or 70% alcohol for identiﬁcation purposes. All diagnostic characters are external features associated with the head, tail, and epicuticular surfaces. The extremi- 291 ties can be removed and mounted in lactophenol or glycerin after dehydration in an alcohol series. For cuticular examination, small slivers of epicuticle can be removed from the body regions with a razor blade and placed in lactophenol or dehydrated in glycerin. The underlying epidermis and muscle tissue can then be scraped away and the cuticular slice mounted (outer surface up) in lactophenol or glycerin. This will expose the areoles and their ornamentation. Some workers prefer to clear the sections in xylene before mounting them. The scanning electron microscope has been used to reveal cuticular details and, although it can be a useful tool, workers should not base descriptions on characters only visible with the SEM since it is not practical for many, including ﬁeld biologists. Besides, adequate diagnostic characters can normally be observed at least with oil immersion objectives (1000 ). In a study comparing the light and scanning electron microscope in differentiating cuticular characters separating Parachordodes lineatus and P. violaceus, Smith (1991) noted that while details were clearer with SEM, the light microscope revealed the essential characters needed to separate the species. Success in large part with the light microscope depends greatly on skill in preparing the sample. Both males and females should be available for accurate identiﬁcation; however, some males can be identiﬁed alone using the following key. The classiﬁcation used here follows that presented by Dorier (1965) in the Traité de Zoologie. It also incorporates the contributions of Heinze (1952) as well as studies on North American forms by May (1919), Leidy (1851), Carvalho (1942), Montgomery (1898a, b, 1899, 1907), Poinar and Doelman (1974), Redlich (1980), Chandler (1985), Chandler and Wells (1989), Smith (1991; 1994), Pennak (1989) and others. A. Taxonomic Key to Extant Genera of Nematomorpha Although some of the genera included have not been reported from North America, it is highly probable that they as well as other still undescribed genera occur in the Nearctic. Those genera with reported North American representatives are marked with an asterisk. 1a. Adults with natatory hairs; marine forms ........................................................................................Nectonematoidea (Rauther, 1930) ....................................................................................................................................................................*Nectonema Verrill, 1879 1b. Adults lacking natatory hairs; freshwater forms ......................................................................................Gordioidea Rauther, 1930 2 2a (1b). Cuticle covered with ridges; male cloaca terminal .....................................................................................Chordodiolus Heinze, 1935 2b. Cuticle not covered with ridges, male cloaca subterminal...................................................................................................................3 3a (2b). Cuticle smooth, lacking aeroles or with ﬂat, smooth areoles; male tail bilobed (Fig. 14b), with a postcloacal fold (Fig. 14b); female tail entire ......................................................................................................................................................Gordiidae May, 1919 4 3b. Cuticle with distinct areoles usually ornamented with bristles or tubercles; male tail entire or if bilobed, then without a postcloacal cresent; female tail entire or trilobed ........................................................................................................Chordodidae May, 1919 5 292 George O. Poinar, Jr. 4a (3a). Lobes of male tail rounded (Fig. 14b); head region not attenuated ...............................................................*Gordius Linneaus, 1766 4b. Lobes of male tail pointed; head region attenuated....................................................................................Acutogordius Heinze, 1952 5a (3b). Male tail entire; female tail entire .......................................................................................................................................................6 5b. Male tail bilobed; female tail entire or trilobed ..................................................................................................................................9 6a (5a). Epicuticle with one type of areole, containing short bristles and tubercles * Neochordodes Carvallo, 1942 ........................................ 6b. Epicuticle with two or more types of areoles ......................................................................................................................................7 7a (6b). Areoles containing prominent tubercles, crowns or papillae; female cloaca terminal ................................* Chordodes Creplin, 1847 7b. Areoles without prominent tubercles, crowns or papillae; female cloaca subterminal........................................................................ 8 8a (7b). Minute spines present on cuticle, especially in furrows between areoles (Fig. 19b); slender, small forms..........................Euchordodes Heinze, 1937 8b. Interareolar furrows without spines; normal-sized forms ...............................................................*Pseudochordodes Carvalho, 1942 9a (5b). Female tail trilobed .............................................................................................................................* Paragordius Camerano, 1897 9b. Female tail entire ..............................................................................................................................................................................10 10a (9b). Areoles arranged in rows separated by ridges ............................................................................................Beatogordius Heinze, 1934 10b. Areoles randomly arranged ..............................................................................................................................................................11 11a (10b). Interareolar furrows large, forming a network around the areoles...........................................................Semigordionus Heinze, 1952 11b. Interareolar furrows normal .............................................................................................................................................................12 12a(11b). A single type of areole present although they may vary in size and be single, biﬁd or triﬁd (Fig. 19c,d)...............................Gordionus G. W. Muller, 1927 12b. Two different types of areoles present ..............................................................................................................................................13 13a(12b). Lobes on male tail longer than wide; adult head clearly ﬂattened; pore canals present on larger areoles in males................................. *Parachordodes Camerano, 1897 13b. Lobes on male tail approximately as long as wide; adult head slightly ﬂattened; pore canals absent or if present, then only in interareolar areas in males ..............................................................................................................................Paragordionus Heinze, 1935 LITERATURE CITED Ayoub, S. M. 1977. Plant Nematology. An agricultural training aid. 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APPENDIX 9.1 Systematic Arrangement of the Nematode Generaa APPENDIX 9.1 (Continued) Phylum Nematoda Class Adenophorea Subclass Chromadorida Order Araeolaimida Suborder Araeolaimina Superfamily Axonolaimoidea Family Axonolaimidae (Cylindrolaimus) Superfamily Leptolaimoidea Family Bastianiidae (Bastiania) Family Leptolaimidae (Rhabdolaimus) Superfamily Camacolaimidae Family Camacolaimidae (Aphanolaimus, Paraphanolaimus) Superfamily Plectoidea Family Chronogasteridae (Chronogaster) Family Plectidae (Anonchus, Plectus) Family Teratocephalidae (Euteratocephalus, Teratocephalus) Order Monhysterida Superfamily Monhysteroidea Family Monhysteridae (Monhystera, Monhystrella, Prismatolaimus) Order Chromadorida Suborder Chromadorina Superfamily Chromadoridea Family Chromadoridae (Chromadorita, Punctodora) Suborder Cyatholaimina Superfamily Cyatholaimoidea Family Cyatholaimidae (Achromadora, Ethmolaimus, Odontolaimus, Paracyatholaimus, Prodesmodora Family Microlaimidae (Microlaimus) Order Enoplida Suborder Enoplina Superfamily Tripyloidea Family Tripylidae (Tobrilus, Tripyla) Family Alaimidae (Alaimus, Amphidelus) Family Ironidae (Cryptonchus, Ironus) Suborder Oncholaimina Superfamily Oncholaimoidea Family Oncholaimidae (Mononchulus, Oncholaimus) Order Dorylaimida Suborder Dorylaimina Superfamily Dorylaimoidea Family Dorylaimidae (Actinolaimus, Dorylaimus, Eudorylaimus, Mesodorylaimus, Paractinolaimus) Family Nygolaimidae (Nygolaimus) Superfamily Leptonchoidea (Continues) Family Campydoridae (Aulolaimoides) Order Mononchida Suborder Mononchina Superfamily Mononchoidea Family Mononchidae (Anatonchus, Mononchus, Prionchulus) Order Mermithida Suborder Mermithina Superfamily Mermithoidea Family Mermithidae (Aranimermis, Capitomermis, Cretacimermis, Culicimermis, Drilomermis, Empidomermis, Gastromermis, Heleidomermis, Heydenius, Hydromermis, Isomermis, Lanceimermis, Limnomermis, Mesomermis, Octomyomermis, Perutilimermis, Pheromermis, Pseudomermis, Romanomermis, Strelkovimermis) Class Secernentea Order Tylenchida Suborder Tylenchina Superfamily Hoplolaimoidea Family Hoplolaimidae (Hirschmaniella) Superfamily Tylenchoidea Family Tylenchidae (Tylenchus) Superfamily Atylenchoidea Family Atylenchidae (Atylenchus) Suborder Criconematina Superfamily Hemicyclcophoroidea Family Hemicycliophoridae (Hemicycliophora) Order Aphelenchida Suborder Aphelenchina Superfamily Aphelenchoidoidea Family Aphelenchoididae (Aphelenchoides, Seinura) Superfamily Aphelenchidae Family Aphelenchidae (Aphelenchus) Order Rhabditida Suborder Diplogasteroidea Superfamily Diplogasteroidea Family Diplogasteridae (Rhabditolaimus) Superfamily Rhabditoidea Family Cephalobidae (Cephalobus, Eucephalobus) Family Rhabditidae (Caenorhabditis, Mesorhabditis, Pellioditis) Family Daubayliidae (Daubaylia)a a The above include all genera cited in this chapter. This Page Intentionally Left Blank 10 MOLLUSCA: GASTROPODA Kenneth M. Brown Department of Biological Sciences Louisiana State University Baton Rouge, Louisiana 70803 I. Introduction II. Anatomy and Physiology A. External and Internal Morphology B. Organ System Function C. Environmental Physiology III. Ecology and Evolution A. Diversity and Distribution B. Reproduction and Life History C. Ecological Interactions I. INTRODUCTION Gastropods are the most diverse class of the phylum Mollusca, with anywhere from 40,000 to 100,000 species, depending on the authority (Bieler, 1992; Ponder and Lindberg, 1997). In North America, there are 14 families, 88 genera and 659 species of freshwater snails (Bogan, 1999). Snails are common organisms along the margins of lakes and streams. They feed on detritus, graze on the periphyton covering of macrophytes or cobble, or even ﬂoat upside down at the water surface, supported by the surface tension, and feed on algae trapped in the same fashion (Fig. 1). In fact, gastropods often control the amount and composition of periphyton in both lotic and lentic environments. They are also the basis of food chains dominated by sport ﬁsh. Predators, by controlling snail populations, may indirectly facilitate algal producers. Freshwater snails also have extensive intraspeciﬁc variation in life histories, productivity, morphology and feeding habits that adapt them to live in uncertain freshwater habitats. There is increasing concern, however, that many riverine gastropod populations in the southeastern United States are currently endangered because of habitat alterations such as impoundments. Ecology and Classiﬁcation of North American Freshwater Invertebrates. 2nd Edition Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved. D. Evolutionary Relationships IV. Collecting and Culturing Freshwater Gastropods V. Identiﬁcation of the Freshwater Gastropods of North America A. Taxonomic Key to Families and Selected Genera of Freshwater Gastropodas Literature Cited Snails are soft bodied, unsegmented animals, with a body organized into a muscular foot, a head, a visceral mass containing most of the organ systems, and a ﬂeshy mantle which secretes the calcareous shell. Gastropods have a univalve shell and possess a ﬁlelike radula used in feeding on the periphyton coverings of rocks or plants. Traditionally, gastropods were divided into three subclasses: Prosobranchia (including 53% of modern species), Opistobranchia (4%), and Pulmonata (43%). Prosobranch snails possess a gill (ctenidium) and a horny (ﬂexible) or calcareous operculum, or “trap door,” which is pulled in after the foot to protect the animal. Pulmonates have secondarily re-invaded freshwaters from the terrestrial habitats used by their ancestors, use a modiﬁed portion of the mantle cavity as a lung, and lack an operculum. Later authors divided the prosobranchs into the ancestral archeogastropods, the mesogastropods, and most-derived neogastropods, with a trend toward reduction in number of gills, auricles, and kidneys. Cox (1960) combined the meso- and neogastropods into the Caenogastropoda. More recent authors have also considered the valvatids to be more closely allied to the opistobranchs and pulmonates than to the prosobranch groups, and they have suggested combining the three 297 298 Kenneth M. Brown II. ANATOMY AND PHYSIOLOGY A. External and Internal Morphology 1. Shell FIGURE 1 An adult Lymnaea stagnalis (length about 5 cm), supported by the surface tension, is foraging upside down at the water surface. former groups into the Heterogastropoda (Kosuge, 1966). Cephalopods are thought to be the closest sister group to the gastropods, and patellid limpets (a marine, intertidal group) are thought to be the most primitive gastropods (Ponder and Lindberg, 1997). More details on the time line of gastropod evolution are given by Bandel (1997). The ﬁrst gastropods are thought to have had coiled shells, and several trends have occurred throughout gastropod evolution (Ponder and Lindberg, 1997). Torsion (a 180° rotation of the gut so that the anus and mantle cavity lie anterior) is a functional homology of all gastropods, although it is secondarily reduced in opistobranchs and pulmonates. Anatomical complexity has also become secondarily simpliﬁed, including the radula, the circulatory system, and digestive anatomy. External fertilization and pelagic larvae are also considered ancestral traits, with internal fertilization, direct development and encapsulated eggs being derived traits. In contrast, respiratory systems, neuro-secretory structures, and life histories have increased in complexity through evolutionary time. A detailed introduction to gastropod biology can be found in Fretter and Graham (1962), and in volumes on the general biology of molluscs edited by Wilbur (1983) or the biology of pulmonates edited by Fretter and Peake (1975, 1978). The structure of the shell is important in systematics. Shells can have a simple conical shape (Fig. 2A), as in the limpets, family Ancylidae, with new shell material secreted at the margin. Second, the shell can be planospiral (with the whorls all in one plane), as in the pulmonate family Planorbidae (Fig. 2B). Whorls may be elevated into a “spire,” as in the pulmonate families Physidae and Lymnaeidae, and in various prosobranch families (Fig. 2 C, D). Russell-Hunter (1983) discusses how new shell material is secreted by the mantle for each of these three basic shapes. If shells are placed with the spire facing away from the observer and the aperture (opening from which the foot extends) upward, shells with the aperture on the left are sinistral (e.g., the physids), whereas those on the right are dextral (e.g., the lymnaeids and prosobranchs). Spiral shells have a central supporting member, the columella, similar to the center support of a spiral staircase. The columella adds to the strength of the shell, and provides an attachment point for the soft parts via the columellar muscle. A spiral shell (Fig. 2D) is convenient for illustrating terminology used in systematics. The pointed end of the shell above the aperture is the apex. Shell length in spiral shells is measured from the apex to the lower tip of the aperture, while the greatest diameter is used in planospiral shells. The spire is separated into a number FIGURE 2 Basic anatomy of the shell, including shell architecture (A, conical; B, planospiral; C and D, spiral, with major shell features indicated in D) and three types of opercula (E, concentric, F, paucispiral, and G multispiral). 10. Mollusca: Gastropoda of whorls by sutures (Fig. 2). The most apical whorl is the nuclear whorl or protoconch, the initial shell of the newly hatched snail or “spat.” The ﬁnal and biggest whorl (representing the most recent shell growth) is the body whorl, ending in the aperture. Whorls may be rounded and sutures deep and well deﬁned (as in a typical lymnaeid shell, Fig. 2D), or they may be ﬂattened and sutures shallow, as in the prosobranch family Pleuroceridae (Fig. 2C). Spiral shells thus have a variety of shapes. If the whorls are ﬂat, the shell is termed coneshaped, while moderately inﬂated whorls produce a “subglobose” shell, and shells that are almost circular are globose; there is a continuum between these three shapes. Shell thickness varies from thin and fragile (as in many pulmonates) to thick and resistant to crushing (as in the prosobranchs). Part of the aperture is often reﬂected over the body whorl at the columella (Fig. 2D) to form an inner lip. If there is a channel between the inner lip and the body whorl, the shell is umbilicate or perforate (the opening of the channel is called the umbilicus and leads up and inward into the columella). Imperforate shells lack an umbilicus. While freshwater shells are not as ornate as their marine relatives (Vermeij and Covich, 1978), shells may have spines, ridges running along the margins of the whorls (called carina), colored bands, or small malleations (hammerings) on the surface. Ridges at right angles to the whorls are called costae, while smaller ridges running spirally along the whorls are lirae. The operculum is useful in classifying prosobranchs. If the growth lines lie completely within each other, the operculum is concentric; whereas, lines arranged in a spiral are termed multispiral or paucispriral (see examples in Fig. 2 E – G). Further discussion on shell sculpture is given in Fretter and Graham (1962) and Burch (1989). The shell is composed of an outer periostracum of organic (mostly protein) composition which may limit shell abrasion or dissolution of shell calcium carbonate by acid waters. Beneath the periostracum is a thick layer of crystalline calcium carbonate with some protein material as a matrix for the calcium crystals (Russell-Hunter, 1978). Calcium carbonate is either absorbed directly from water or is sequestered from food (McMahon, 1983). Snails in calcium-poor waters (5 mg / L, Lodge et al., 1987) expend energy to absorb calcium against a gradient, and many species are therefore limited to calcium-rich habitats. 2. Soft Anatomy The soft parts are separated into: (1) head, (2) foot, (3) visceral mass, and (4) the mantle (Fig. 3). Aquatic pulmonates and prosobranchs possess eyes at the base of their tentacles, unlike terrestrial pulmonates 299 FIGURE 3 Basic internal anatomy (with shell removed) of a planorbid pulmonate (above, after Burch, 1989), and a pleurocerid prosobranch (below, after Pechenik, 1985). whose eyes are at the tips of the tentacles. The muscular foot is provided with both cilia and secretory epithelium to secrete mucus for locomotion, as well as pedal muscles which produce waves of contraction to push the animal forward. The visceral hump includes most of the organs of digestion and reproduction. The mantle covers the visceral mass, and underlays the shell, which it secretes. The anterior mantle, over the head, possesses a mantle cavity, where the gill or ctenidium is located in prosobranchs. Gastropods have ganglia innervating each of these areas. Further information on internal anatomy can be found in Fretter and Graham (1962), Barnes (1987), Pechenik (1985), or Hyman (1967). B. Organ System Function 1. Reproductive System Prosobranchs are usually dioecious, and males use the enlarged right tentacle as a copulatory organ (in the viviparids), or possess a specialized penis or verge (in the hydrobiids, pomatiopsids, and valvatids) or have no copulatory organ (thiarids and pleurocerids). The penis is usually located behind the male’s right tentacle. Many pleurocerids lay clutches of a few eggs, whereas 300 Kenneth M. Brown FIGURE 4 Anatomy of the reproductive system of the monoecious pulmonate Physella (after Duncan, 1975). Sperm are produced in the ovotestis, stored in the seminal vesicle, exit via vas deferens, and are inserted into the vagina of a second individual via the introvertable penis; or sperm may fertilize the same individual’s eggs in the hermaphroditic duct. Most foreign sperm are hydrolyzed in Physa in the bursa copulatrix, but some do fertilize eggs in the fertilization pocket. The albumen gland secretes yolk around the egg, and the oviduct wall secretes both the egg membrane and the jelly and external wall of the egg case. viviparids produce eggs which hatch and develop in a fold of the anterior mantle (the pallial oviduct), and are born free-living. Some viviparids in the genus Campeloma, especially more northern populations, are parthenogenetic (Van Cleave and Altringer, 1937; Vail, 1978; Johnson, 1992). Pulmonates, in contrast, are all monoecious. The basic components of the pulmonate reproductive system are shown in Figure 4. Sperm and eggs are produced in the ovotestis and exit via a common hermaphroditic duct in all pulmonates except ancylids, which have two openings and are thus obligate cross-fertilizers. The albumen gland adds protein and nutrients to the egg. Eggs are either fertilized in the hermaphroditic duct by the same individual’s sperm, or by sperm from another individual near the junction of the hermaphroditic duct and the oviduct. The external egg membranes are then secreted in the oviduct. Eggs are laid in gelatinous egg cases and attached to plants, submersed wood, other snails or rocks. Males have an introvertible penis, and fertilization is internal. Duncan (1975) gives a more detailed discussion of egg and sperm formation, copulation and fertilization, and egg capsule deposition. Although pulmonates are simultaneous hermaphrodites, most species out-cross when possible. Pulmonates that “self” usually mature at later ages and have lower fecundity. For example, Physella heterostropha suffers a reduction of 65% in fecundity when not allowed to out-cross (Wethington and Dillon, 1993). Snails act as males or females based on the amount of stored sperm that they possess (Wethington and Dillon, 1996). Similarly, the African snail Bulinus globosus suffers a 50% reduction in fecundity, and an 18% reduction in hatching rate, under obligate selffertilization, indicating a signiﬁcant genetic load of recessive lethal genes (Jarne et al., 1991). In other physids (Eileen Jokinen, personal communication), the male system is larger in juveniles than in adults, and protandry (a form of sequential hermaphroditism where individuals switch sex from male to female as they age and increase in size) may occur. With the costs to ﬁtness indicated in the preceding paragraph, one might wonder why hermaphroditism occurs in pulmonates. Most pulmonates are slow-moving and go through seasonal “bottlenecks” (precipitous declines in density). The chances of ﬁnding a mate in such situations are small, providing a selective advantage for monoecy. Pulmonates are dispersed passively as spat trapped in mud on birds feet (Boag, 1986 and references therein), and solitary, monoecious immigrants have an obvious advantage. The cost of inbreeding depression is evidently less than not being able to reproduce at all. Parthenogenesis (being able to reproduce without males) may have a similar adaptive value for prosobranch snails that are isolated in small headwater streams (Vail, 1978). 2. Digestive System Food is brought into the mouth by rasping movements of the radula, a ﬁlelike structure (Fig. 5) resting on a cartilage (the odontophore) to which muscles which extend and retract the radula are attached. When the radula is extended, it contacts the substratum, and algal particles are scraped off when retractors pull the radula back into the mouth. The radula may also pulverize food particles by grinding them against the roof of the mouth. A long esophagus leads to the 10. Mollusca: Gastropoda 301 FIGURE 5 Scanning electron microscope photographs of radular teeth of (A) the lymnaeid, Pseudosuccinea columella and (B) the physid, Physella vernalis (from Kesler et al., 1986). Note the lymnaeid has fairly large teeth with few cusps. These teeth, along with large, cropping jaws in the buccal mass, a grinding gizzard equipped with sand, and high cellulase levels allow it to specialize on ﬁlamentous green algae (Kesler et al., 1986). Physella has many, small teeth with long cusps, arranged in chevron-shaped rows, useful for piercing detritus and attached bacteria. stomach, located in the visceral mass. Some gastropods possess a specialized crop where sand grains further abrade food particles. Digestive enzymes are produced by the digestive gland, the hepatopancreas. Considerable digestion also occurs intracellularly in the hepatopancreas. Snails are one of the few animal groups to possess and possibly synthesize cellulases (Kesler, 1983; Kesler et al., 1986), that degrade the algal cell walls. 3. Respiratory and Circulatory Systems The respiratory system differs radically between prosobranchs and pulmonates. Freshwater prosobranchs have a single ctenidium or gill (Aldridge, 1983). The ctenidium, usually in the mantle cavity, has leaﬂike triangular plates richly supplied with blood vessels. Oxygen poor blood passes across the plates in the opposite direction to oxygen-rich water currents (generated by ctenidal cilia). This countercurrent mechanism assures diffusion of oxygen from water into the blood. Pulmonates lost their gill during their intermediate terrestrial phase, and have a vascularized pocket in the mantle used as a lung (hence their name). The opening of the lung is the pneumostome. Pulmonates either rely on surface breathing or have a limited capacity for oxygen transfer across their epithelial tissues (McMahon, 1983). Some physids and lymnaeids ﬁll the mantle pocket with water and use it as a derived gill (Russell-Hunter, 1978). Ancylids and planorbids have re-adapted further to aquatic conditions by using a conical extension of epithelium as a gill, and planorbids also have a respiratory pigment, hemoglobin, which increases the efﬁciency of oxygen transport (McMahon, 1983). 4. The Excretory System Gastropods have a permeable epidermis and are subject to osmotic inﬂow of water from their hypo-osmotic surroundings. Thus, they must pump out excess water in their urine. The gastropod coelom is little more than a small cavity (pericardium) surrounding the heart. The coelomic ﬂuid is largely a ﬁltrate of the blood, containing waste molecules, such as ammonia, that are ﬁltered across the wall of the heart. Additional wastes are actively secreted into the coelom by the walls of the pericardium. The coelomic ﬂuid then enters a metanephridial tubule (the coelomoduct) where selective resorption of salts and further secretion of wastes occurs. The urine is then discharged into the mantle cavity. Freshwater gastropods excrete nitrogen both as ammonia and as urea. Ammonia is adaptive in aquatic environments because, although it is toxic, it is extremely soluble and readily diffuses away. Pulmonates often produce urea which is better in terrestrial situations, or during hibernation or estivation, because it is relatively nontoxic and can be stored in the blood until able to be excreted (McMahon, 1983). Further discussion of excretion can be found in Martin (1983) for gastropods in general and in Machin (1975) for pulmonates. C. Environmental Physiology Pulmonates usually face wider extremes of temperature variation than do most prosobranchs (McMahon, 1983). Most temperate pulmonates, for example, can withstand temperatures near 0°C for several months, whereas tropical pulmonates can withstand 302 Kenneth M. Brown temperatures near 40°C for extended periods. This is undoubtedly adaptive because of the seasonal and diurnal variation in temperature in many pond habitats. Pulmonates better regulate changes in metabolic rate with changing temperatures than do prosobranchs. For example, Q10 values for 18 pulmonate species were less (averaging 2.2) than in 13 prosobranch species (averaging 2.8) (McMahon, 1983). As the average temperature increases, snails grow faster and reproduce at an earlier age, with more generations per year. For example, the subtropical pulmonate Physella cubensis grows slowly and will not mature at 10°C (Thomas and McClintock, 1990), but hatching times and ages at maturity decrease, and growth rates increase as laboratory temperatures are increased to 30°C. Increasing water temperature is also the cue for onset of reproduction in many temperate pulmonates. The ability of pulmonates to reproduce in cold water allows adults to breed early in the spring and juveniles to grow rapidly to adult size before the end of the summer. Pulmonates can secrete a mucus covering over the aperture, called an epiphragm, to retard moisture loss during dry periods (Boss, 1974; Jokinen, 1978). In terms of adaptation to hypoxia, pulmonates: (1) tolerate greater variation in dissolved oxygen than do prosobranchs (possibly because prosobranchs rarely experience hypoxia in the littoral zones of lakes or the fast ﬂowing rivers they are common in); and (2) also regulate oxygen consumption at varying levels of dissolved oxygen better than prosobranchs, which are “oxy-conformers” (Brown et al., 1998). Pulmonates apparently withstand lower oxygen tensions by either TABLE I surface breathing or reliance on anaerobic metabolism (McMahon, 1983). Approximately 45% of freshwater gastropods are restricted to waters with calcium concentrations greater than 25 mg / L, and 95% to levels greater than 3 mg / L. Although it may not take much energy to absorb calcium from water, assuming adequate water hardness, it may be energetically costly to secrete calcium into the shell against an electrochemical gradient. For example, in the northeastern United States (Jokinen, 1987), Aplexa elongata, Helisoma (Planorbella) trivolvis, and all lymnaeids except Fossaria are limited to calciumrich waters. McMahon (1983) and Lodge et al. (1987) discuss the degree of calcium regulation, and the relationship of external calcium level to shell thickness and growth. Shell accretion may lag behind rapid tissue growth in eutrophic habitats, producing thinner shelled individuals (McMahon, 1983). III. ECOLOGY AND EVOLUTION A. Diversity and Distribution Although widespread in Asia, pleurocerids reach their greatest abundance in the rivers and streams of the southeastern United States (Table I). Most are fairly large snails that are quite common in rocky rifﬂes or shoals in both headwaters and large rivers. However, many species have been lost to stream alteration by impoundments, and present-day diversity has decreased dramatically (see Section III.E). Females possess an egg laying sinus on the right side of the foot (Dazo, 1965). The Diversity of Freshwater Snail Families in North Americaa Subclass Family Number of genera Prosobranchia Ampullaridae Bithyniidaec Hydrobiidae Micromelaniidae Neritinidae Pleuroceridae Pomatiopsidae Thiaridaec Valvatidae Viviparidae Acroloxidae Ancylidae Lymnaeidae Physidae Planorbidae 2 1 28 1 1 7 1 2 1 5 1 4 9 4 11 Pulmonata a Data compiled from Burch (1989) and Neves et al. (1998). N, North; E, East; W, West; S, South and U, ubiquitous. c Introduced. b Number of species 4 1 152 1 1 156 6 3 11 29 1 13 58 43 47 Area of greatest diversityb SE NE U SE SE SE SE, W S U E W, NE U N U U 10. Mollusca: Gastropoda Shell anatomy is used to classify genera, although many species show considerable variation in shell characters. The shells of pleurocerids are solid and the aperture may bear a canal anteriorally. The operculum is corneous and paucispiral. Viviparids are worldwide in distribution, and these large snails are fairly diverse (Table I) throughout the eastern states and Canadian provinces. Campeloma, Lioplax, and Tulotoma are endemic to North America. Although the ﬁrst two genera are widespread, Tulotoma was once considered extinct and has only recently been rediscovered in the Coosa River in Alabama, where this ﬁlter feeding, brooding snail is common under rocks in shallow water shoals in large tributaries (Hershler et al., 1990). Viviparus is quite common in rivers and lakes throughout eastern North America. Another large viviparid Cipangopaludina chinensis was introduced to the United States in the 1890’s and has now spread throughout the United States, especially in hard-water lakes with sandy or muddy substrates (Jokinen, 1982). Ampullarids are a tropical, mostly amphibious family with a mantle cavity provided both with a gill and a lung. The two genera present in Florida, Pomacea and Marisa, are quite large snails (50 – 60 mm). Marisa has also been introduced to rivers in central Texas. The Neritinidae are a marine, tropical group. A few species have invaded estuarine and freshwater habitats, for example Neritina reclivata in Florida, Georgia, Alabama, and Mississippi. The paucispiral, calcareous operculum has a pair of projections which lock the operculum against the teeth on the aperture, providing a stronger defense. Of the 20 species of valvatids in the northern hemisphere, 11 are found in North America (Table I), mostly in lakes in northern states. Valvatids are egglaying hermaphrodites, with a single, featherlike gill carried on the left side, and a pallial tentacle carried on the right side of the shell as the animal crawls. Valvatids have small (approximately 5 mm diameter) dextral shells, with a corneous, slightly concave and thin, multispiral operculum. The spire is only slightly elevated, and the shells are sometimes carinated. Hydrobiids are extremely diverse and exist in freshwater, brackish, and marine habitats. They are small but have a thick shell which protects them from ﬁsh predation, and they dominate the gastropod assemblages of many northern lakes (Brown, 1997). There are 103 genera worldwide (Burch, 1989), and they are diverse in North America (Table I). In fact, hydrobiids may be the most diverse group of freshwater snails, as 58 new species in the genus Pyrgulopsis were recently described from small springs in the southwestern 303 United States (Hershler, 1998). Although these populations often reach high densities, many species are endemic to only a few springs, increasing chances of extinction. The genus Fluminicola, which is common in rivers in the northwestern United States, is also quite diverse (Hershler and Frest, 1996). The hydrobiids have small, dextral shells with a paucispiral operculum. Because of the similarity of their shells, the structure of the verge (penis) is used in classiﬁcation. The six North American pomatiopsids are similar in general anatomy to the hydrobiids. Pomatiopsids are, however, amphibious and are often found inhabiting stream banks several meters above the water’s surface, while hydrobiids are truly aquatic (Burch, 1989). Pomatiopsid systematics are discussed by Davis (1979). Thiarid females are parthenogenetic, brooding eggs in a pouch in the neck region, which opens on the right side. The similar-shelled pleurocerids, discussed above, are on the contrary dioecious and oviparous. Ancylids have a worldwide distribution, and all possess a simple cone-shaped shell. In North America, they have reached moderate diversity (Table I). Ancylids have sinistral shells, with the apex inclined slightly to the right, and the gill (pseudobranch) and many of the internal organs opening on the left side of the body. Their streamlined shape allows them to colonize fast-ﬂowing streams, where they are common on rocks or macrophytes, although some species are also common in lentic environments. The family Acroloxidae occurs mainly in Eurasian lakes and ponds. Only one species of Acroloxus occurs in the United States, in Colorado and southeastern Canada. Since the apex in Acroloxus is tipped to the left, the aperture is considered to be dextral, unlike the ancylid limpets. Lymnaeids are worldwide in distribution, and are the most diverse pulmonate group in the northern United States and Canada (Table I). Lymnaeids have broad triangular tentacles, and lay long, sausageshaped egg masses. Lymnaeids have fairly uncomplicated, large teeth on their radula (Fig. 5), which are useful for cropping long strands of ﬁlamentous algae(Kesler et al., 1986). One group of lymnaeids, found along the Paciﬁc coast of North America, has limpet-shaped shells, but they are larger than ancylids. Physids also have a worldwide distribution and are ubiquitous in North America (Table I); few aquatic environments lack physids, because of their ease of introduction. Their shells are small, sinistral, with raised spires. Their tentacles and foot are slender and they have ﬁngerlike mantle extensions (except for the genus Aplexa) and lay soft, crescent-shaped egg masses. Their radular teeth are smaller and more complicated in shape than in the lymnaeids (Fig. 5), and they are 304 Kenneth M. Brown better at harvesting more tightly attached periphyton species like diatoms, or in feeding on detritus (Kesler et al., 1986). Their rate of crawling is much more rapid than most gastropods, and this along with their early age at maturity and high fecundity explains why they are so wide-spread (Brown et al., 1998). Planorbids are widespread and fairly diverse snails (Table I), and range in size from minute (1 mm) to quite large (30 mm) in North America. They possess hemoglobin as a respiratory pigment, sometimes giving the tissue a red hue. Their egg cases are ﬂat and circular, with harder membranes than those of the lymnaeids or physids. Planorbids are considered closely related to the ancylids (McMahon, 1983). B. Reproduction and Life History Freshwater snails are extremely interesting because of the variety of observed life-history patterns. For example, freshwater pulmonates are oviparous (egglaying) hermaphrodites, and are usually annual and semelparous (i.e., they reproduce once and die). On the other hand, almost all prosobranchs are dioecious and are often iteroparous, with perennial life cycles. Prosobranchs can be oviparous or ovoviviparous (RussellHunter, 1978; Calow, 1978, 1983; Brown, 1983). Annual species have essentially a 1 year life cycle, whereas perennials often live and reproduce for 5 years or more. Russell-Hunter (1978) and Calow (1978) have classiﬁed life histories for freshwater gastropods (Fig. 6). At one end are annual adults that reproduce in the spring FIGURE 6 The variety of life cycles in freshwater snails. Curves represent the growth of individual cohorts (in millimeters of shell length), circles represent size at maturity while triangles represent appearance of egg cases in samples. Panel A represents annual semelparity, and B – F represent increasing numbers of cohorts per year, while panel G represents perennial iteroparity (from Calow, 1978). SP, spring; S, summer; F, fall and W, winter. and die (e.g., there is complete replacement of generations). Most pulmonates belong to this group, including species from the genera Lymnaea, Physella, and Aplexa (the original studies are listed in Calow, 1978). In the second category (Fig. 6B), reproduction occurs in both spring and late summer with both cohorts surviving the winter, or (Fig. 6C) where there again is complete replacement of generations. In Figure 6D – F, there are three reproductive intervals, with varying degrees of replacement of generations. These would predominantly be populations in subtropical or tropical environments. Finally, there are populations that can truly be considered perennial and iteroparous (Fig. 6G); most are prosobranchs. For example, the pleurocerid genus Elimia has life cycles lasting from 6 to 11 years in Alabama steams, with several cohorts overlapping at any one time (Richardson et al., 1988; Huryn et al., 1994). Adults reproduce in the spring and summer and are more common in areas of low current velocity, while juveniles are common in the fall in high ﬂow areas (Huryn et al., 1994; Johnson and Brown, 1997). Jokinen (1985) also found Russell-Hunter and Calow’s life-cycle categories useful in describing lifecycle patterns in twelve species of gastropods inhabiting a small Connecticut lake. Four species, Amnicola limosa, Helisoma anceps, Helisoma (Planorbella) companulatum and Laevapex fuscus, had simple, annual patterns. Four species had at least two breeding seasons a year, with varying replacement of cohorts, including Pseudosuccinea collumella, Planorbula armigera, Promenetus exacuous, and Gyraulus deﬂectus. Three species, Fossaria modicella, Gyraulus circumstriatus, and Physella ancillaria, had continuous breeding throughout the ﬁeld season. The prosobranch Campeloma decisum was the only species with a perennial reproductive pattern. Jokinen (1985) suggested that divergence in life history patterns between taxonomically similar species lessened competition, by allowing some species to specialize on detritus (common in the spring), and others periphyton (common in the summer). Marine snails have enormous fecundity, but individual eggs are extremely small. Most marine snails are prosobranchs, and freshwater prosobranchs (their descendants) probably have relatively small eggs as a result. In fact, viviparids and thiarids may have evolved ovoviviparity (and in some cases the production of larger embryos) to cope with the much more unpredictable conditions in freshwaters (Calow, 1978, 1983). Similarly, the loss of the planktonic veliger, and shortening of the developmental period in oviparous freshwater snails has been attributed to the more variable physicochemical conditions (Calow, 1978). Reproductive effort (percent of energy devoted to reproduction) is lower in iteroparous freshwater snails 10. Mollusca: Gastropoda than in semelparous ones (Browne and Russell-Hunter, 1978). For example, pulmonate families are semelparous with relatively high reproductive output (Brown, 1983). Pulmonates also reproduce at smaller sizes and earlier ages, produce more eggs, have larger clutch sizes, greater shell growth rates, shorter life cycles and smaller ﬁnal shell sizes than viviparid prosobranchs. The reduced fecundity but increased parental care found in viviparids (vs semelparous pulmonates) increases offspring survival. Life tables for Viviparus georgianus (Jokinen et al., 1982) and for the pulmonate Lymnaea elodes (Brown et al., 1988) indicate that survival to maturity is indeed much less than 1% in all of the pulmonate populations, but over 40% in the ovoviviparous prosobranch. Prosobranchs are also sexually dimorphic in life-history patterns. Female viviparids live longer (males usually survive for only one reproductive season) and reach larger sizes (Van Cleave and Lederer, 1932; Browne, 1978; Jokinen, 1982; Jokinen et al., 1982. Pace and Szuch, 1985; Brown et al., 1989). Numerous studies have implicated periphyton productivity (Eisenberg, 1966, 1970; Burky, 1971; Hunter, 1975; Browne, 1978; Aldridge, 1982; Brown, 1985) in determining voltinism patterns, growth rates, fecundity, and gastropod secondary production (see review in Russell-Hunter, 1983). Other important factors include water hardness and temperature. For example, populations of Elimia in limestone-substrate streams in Alabama have greater annual production than populations in relatively impermeable slate or sandstone-substrate streams (Huyrn et al., 1995), both because of the higher alkalinity and greater buffering of low temperatures in the winter by ground water. Similarly, populations of Lymnaea stagnalis in Canada often take several seasons to complete their life cycle (Boag and Pearlstone, 1979) while populations are annual in the warmer waters of Iowa (Brown, 1979). Lymnaea peregra in wave-swept habitats in English lakes have r-selected life-history traits (e.g., early reproduction and high reproductive output) in comparison to populations in less-harsh habitats (Calow, 1981). However, other studies of life history variation in molluscs do not agree as well with the predictions of r- and K-theory (see discussion in Burky, 1983).Transplant studies, where individuals from separate populations are reared in a common environment, have usually indicated that environmental effects on life histories are much more important than genetic differences between populations. For example, populations of Lymnaea elodes reared in more productive ponds lay nine times as many eggs, have an annual versus a biennial reproductive cycle, and reach larger individual sizes (Brown, 1985). 305 Although genetic polymorphism has been studied using gel electrophoresis in terrestrial pulmonates and freshwater prosobranchs more than in aquatic pulmonates, freshwater snails still appear to have levels of genetic polymorphism intermediate to terrestrial and marine species (Brown and Richardson, 1988). Terrestrial snails inhabit patchily-distributed microclimates which increase chances for low population densities and self fertilization, resulting in little genetic polymorphism within populations. Freshwater snails decline to low densities because of seasonal bottlenecks and thus may also self. Marine environments are less seasonal, and many marine snails have planktonic larvae, facilitating gene ﬂow and increasing polymorphism. C. Ecological Interactions 1. Habitat and Food Selection, Effects on Producers Slow moving, silty habitats are occupied by pulmonates or detritivorous prosobranchs such as viviparids, whereas fast current areas are dominated by limpets or prosobranch grazers like pleurocerids (Harman, 1972). There is also evidence for habitat selection on the species level. Campeloma decisum is positively rheotactic (moves upstream) and aggregates at any barrier (e.g., logs, rifﬂe zones, etc., Bovbjerg, 1952). Physella integra and Lymnaea emarginata prefer cobble substrates with attached periphyton, while Helisoma anceps and Campeloma rufrum prefer sand (Clampitt, 1973; Brown and Lodge, 1993). Most gastropods in northern Wisconsin lakes, however, prefer periphyton covered cobble over sand or macrophytes (Brown and Lodge, 1993). Cobble, since it offers greater surface area and is present year-round, develops a richer periphyton coating. Substrate selection may even occur on a ﬁner level: gastropods from an English pond prefer periphyton from the macrophytes the snails were found on in the ﬁeld (Lodge, 1986). Snails often move among habitats as well. Migrations occur to deeper water in the fall in lakes and back to the littoral zone in the spring (Cheatum, 1934; Clampitt, 1974; Boag, 1981). Pond pulmonates on the other hand burrow into the substrate with declining temperatures (Boerger, 1975). Freshwater gastropods are herbivores or detritivores, but occasionally ingest carrion (Bovbjerg, 1968) or small invertebrates associated with periphyton (Cuker, 1983). Periphyton is easier to scrape, and contains higher concentrations of nitrogen and other limiting nutrients than macrophyte tissue (Russell-Hunter, 1978; Aldridge, 1983). For example, carbon to nitrogen (e.g., carbohydrate to protein) ratios are below 10.1:1, while macrophytes have ratios of 24.1:1 (McMahon et al., 1974). Algal and diatom remains therefore dominate in the guts of snails (Calow, 1970; 306 TABLE II Kenneth M. Brown Feeding Preferences of Freshwater Snails Feeding type Family References Algivores (scrapers) Ancylidae Lymnaeidae Detritivores or bacterial feeders Neritinidae Pleuroceridae Viviparidae Physidae Planorbidae Viviparidae Bithyniidae Viviparidae Calow (1973a, b, 1975) Bovbjerg (1968, 1975), Brown (1982), Calow (1970), Cuker (1983), Hunter (1980), Kairesalo and Koskimies (1987), Kesler et al. (1986), Lodge, (1986) Jacoby (1985) Aldridge (1982, 1983), Dazo (1965), Goodrich, (1945) Duch (1976), Jokinen et al., (1982) Brown (1982), Kesler et al., (1986), Townsend (1975) Calow (1973b, 1974a, b) Chamberlain (1958), Pace and Szuch, (1985), Reavell (1980) Tashiro (1982), Tashiro and Colman (1982), Brendelberger and Jurgens, (1993) Brown et al. (1989) Filter feeders 1973a, 1975; Calow and Calow, 1975; Reavell, 1980; Kesler et al., 1986; Lodge, 1986; Madsen, 1992). However, gastropods at high densities can exhaust periphyton and then consume macrophytes, suppressing macrophyte species richness (Sheldon, 1987). Feeding preferences for the freshwater gastropod families are summarized in Table II. Lymnaeids are “microherbivores”, scraping algae and diatoms from rocks or macrophytes, but grow more rapidly when animal tissue is in their diet (Bovbjerg, 1968). For example, although Pseudosuccinea columella (a lymnaeid) is an omnivore, it still consumes more algae than the sympatric Physa vernalis (Kesler et al., 1986). The lymnaeid also possesses higher levels of cellulases, as well as a radula and jaws well adapted for cropping algae (Fig. 5), and a gizzard ﬁlled with sand that can macerate food. The physid, a detritivore, lacks these adaptations. Both the limpet family Ancylidae and the prosobranch family Pleuroceridae are also considered to graze on periphyton (Table II). Ancylus ﬂuviatilis selectively grazes diatoms, but the limpet has little effect on periphyton communities, due either to adaptations of algal and diatom species to grazing or to relatively low limpet abundances (Calow, 1973a, b). Aldridge (1983) concluded that pleurocerid grazers feed on periphyton rather than macrophyte tissue again because of higher levels of nitrogen. The prosobranch Bithynia tentaculata both grazes on periphyton and uses its ctenidium to capture phytoplankton that is consolidated into a mucous string which loops from the mantle cavity to the mouth (Brendelberger and Jurgens, 1993). Indeed, ﬁlter feeding may be more efﬁcient than scraping as increasing levels of phytoplankton shade out periphyton, explaining why this species has become so abundant in nutrient rich, eutrophic lakes in New York (Tashiro, 1982; Tashiro and Colman, 1982). Viviparus georgianus is a micro-algivore (Duch, 1976; Jokinen et al., 1982) or a detritivore (Pace and Szuch, 1985). Most viviparids, however, are probably detritivores or utilize bacteria associated with detritus (Table II). As macrophytes decompose, nitrogen levels increase, again increasing their value as food resources. For example, Viviparus reaches extremely high densities (from 151 to 608 individuals m2) in wooded streams in Michigan (Pace and Szuch, 1985) and detritus-rich bayous in Louisiana (up to 1,700 individuals m2, Brown et al., 1989). Both physids (Kesler et al., 1986) and planorbids (Calow, 1973b, 1974a) prefer detritus (Table II). For example, although widely-spread species like Physella gyrina and Helisoma trivolvis did not prefer detritus over periphyton in laboratory experiments, another physid, Aplexa elongata, which is much more common in wooded ponds with a rich detritus food base, preferred detritus (Brown, 1982). Originally, snail algivores were considered indiscriminant grazers, taking all components of the periphyton (Hunter, 1980; Hunter and Russell-Hunter, 1983). However, limpets and planorbids are selective (Calow, 1973a, b), and Lymnaea peregra grazes selectively on ﬁlamentous green algae (Lodge, 1985). Planorbis vortex ingests diatoms in greater quantities than found in the periphyton, but is still predominantly a detritivore. Gastropods have the ability to locate macrophytes through distant chemoreception (Croll, 1983). For example, Lymnaea peregra is positively attracted to Ceratophylum demersum because of dissolved organic materials excreted by the macrophyte (Bronmark, 1985b). Similarly, Potamopyrgus jenkinsi orients toward both plant and animal extracts (Haynes and Taylor, 1984), while Biomphalaria glabrata either orients toward or away from speciﬁc macrophytes (Bouseﬁeld, 1979). Almost all experimental manipulations have indicated snail grazers can decrease periphyton standing crops (Table III, see also review in Bronmark, 1989). For example, Physella at high densities reduces algal 10. Mollusca: Gastropoda 307 TABLE III The Effects of Experimental Manipulations of Gastropods on Periphyton Biomass, Production and Assemblage Structure Decreased algal biomass? Increased algal production? Favored adnate species? Group Genus Prosobranchs Elimia or Juga (N 11) 73% 17% 100% Theodoxus (N 1) Amnicola (N 1) Physella (N 3) Y Y 100% NO N.M. N.M. Y Y 100% Promenetus (N 1) Lymnaea, Physella, Helisoma NO Y NO Y NO Y Pulmonates Studies Mulholland et al. (1983), Steinman et al. (1987), Lamberti et al. (1987), Marks and Lowe (1989), McCormick and Stevenson (1989), Hill and Harvey (1990), Mulholland et al. (1991), Tuchman and Stevenson (1991), Hill et al. (1992), Rosemond et al. (1993) Jacoby (1985) Kesler (1981) Doremus and Harman (1977), Lowe and Hunter (1988), Swamikannu and Hoagland (1989) Doremus and Harman (1977) Hunter (1980) Percentages refer to the number of studies noting an affect. For single studies: Y, effect; NO, no effect, N.M., not measured. biomass by 97%, and richness by 66% (Lowe and Hunter, 1988). In some cases, snails also increase algal production, perhaps by decreasing total biomass and lowering algal competition for light or nutrients, removing senescent cells, or increasing rates of nutrient cycling. Pulmonate gastropods can also alter the quality (e.g., nitrogen-to-carbon ratios and chlorophyll a levels) of periphyton (Hunter, 1980), as can prosobranchs in streams (Steinman et al., 1987). Although low levels of grazing may stimulate production, higher snail densities decrease both biomass and production (McCormick and Stevenson, 1989; Swamikannu and Hoagland, 1989). Grazing may not, however, have as much of an impact on shaded streams, where light may be the primary limiting factor (Hill and Harvey, 1990). Even in unshaded steams there may be little overall effect of grazers, because the loss of the algal over-story to grazing is compensated for by the competitive release and increased growth of adnate algae (Hill and Harvey, 1990). Snail grazers selectively remove larger ﬁlamentous green algae, and leave smaller, adnate species behind (Table III). Under slight gastropod grazing pressure, periphyton assemblages are dominated by ﬁlamentous green algae, but more intensely grazed assemblages are dominated by more tightly adhering or toxic species such as cyanobacteria. Snail grazers may, in fact, indirectly facilitate macrophytes, if periphyton coverings shade and limit macrophyte growth, and snails prefer periphyton. For example, the growth of Ceratophylum demersum increased when gastropod grazers were present (Bronmark, 1985b), although increased growth also occurred when plants were exposed only to snail- conditioned water, indicating increased nutrient recycling was a cause (Underwood, 1991). Similarly, when sunﬁsh depress snail abundances and increase periphyton abundance in ﬁsh enclosures, they may indirectly depress macrophytes (Martin et al., 1992). The molluscivorous tench (a ﬁsh) can have similar cascading effects on European gastropods, periphyton, and macrophytes (Fig. 7). Thus, interactions between gastropod predators, snails, periphyton, and macrophytes may be very complex in natural systems. 2. Factors Regulating Population Size One of the ﬁrst demonstrations of population regulation under ﬁeld conditions was with Lymnaea elodes (Eisenberg, 1966, 1970). When the density of adult snails in pens in a small pond was increased, adult fecundity declined, as did juvenile survival. With addition of a high quality resource, spinach, an increase in the number of eggs per mass occurred. Evidently, the availability of micronutrients in periphyton was the crucial variable (Eisenberg, 1970). Brown (1985) provided additional evidence by transferring juvenile Lymnaea elodes among a series of ponds differing in periphyton productivity. There was an exponential increase in juvenile growth rates with increasing pond productivity, and snails in the most productive pond laid nine times as many eggs as snails in the two less productive ponds. A number of ﬁeld studies also provide indirect evidence for the importance of resource abundance: populations in more eutrophic habitats have more generations a year, more rapid shell growth, and lay more eggs (Burky, 1971; Hunter, 1975; McMahon, 1975; Eversole, 1978). Highly eutrophic sites may however 308 Kenneth M. Brown FIGURE 7 Cascading (also called top-down) effects of a molluscivorous ﬁsh (the tench) on gastropods, periphyton and macrophytes, based on a ﬁeld manipulation (from Bronmark and Vermaat, 1998). Note how snail numbers are depressed by ﬁsh predation (top), and reduced gastropod grazing results in periphyton increases, and macrophytes are shaded by increasing periphyton cover, and decrease. be detrimental to gastropods. For example, gastropod diversity declined over a ﬁfty year period as Lake Oneida, New York, became increasingly eutrophic (Harman and Forney, 1970). In lotic systems, manipulation of periphyton resources and the abundances of pleurocerid grazers have indicated both that food resources can control snail density and size distributions, and that the snails can, in turn, control their food resources (Hill et al., 1992; Rosemond et al., 1993). Although less studied than the role of periphyton, the parasitic larvae of trematode worms may also impact snail population dynamics and evolution (Holmes, 1983). The adult worm produces eggs that are expelled in the feces of the ﬁnal host (a vertebrate) and hatch into an infectious larval stage called a miracidium. Depending on the parasite group, the miracidium either penetrates the epithelium of the snail or is consumed as an egg and hatches within the snail. Once inside the snail host, miracidia asexually produce several stages (redia and sporocysts) which eventually produce thousands of cercaria that infect the ﬁnal host or another intermediate host. Infections either accelerate or decelerate snail growth (Brown, 1978; Anderson and May, 1979; Holmes, 1983, see discussion in Minchella et al., 1985). Immediately after infection or exposure, snail egg production rates increase dramatically (Minchella and Loverde, 1981), evidently reducing the eventual costs to snail ﬁtness. As parasites consume the ovotestis and hepatopancreas, however, both growth and egg production of “patent” snails (those infections in the ﬁnal stage where cercaria are emerging from snails) drop below that of uninfected snails (Minchella et al., 1985). The role of trematodes in controlling snail populations is unclear, as prevalence (percentage of population infected) varies considerably (Brown, 1978; Holmes, 1983). For example, prevalence in Lymnaea elodes in Indiana ponds varied from as low as 4% to as high as 49% (Brown et al., 1988). Prevalences were higher in less productive ponds, evidently because food limitation caused longer snail life cycles that increased chances for snails to be located by miracidia. Life-table models predicted that the number of offspring produced per adult in the next generation declined by 14 to 21% in parasitized populations of L. elodes. Assemblages of trematode larval species within a snail host can themselves be considered communities, with relative abundances and types of biotic interactions dependent on the snail species, the type of ﬁnal host, and the microhabitat the snail occupies. For example, multispecies infections are common and interspeciﬁc competition rare in Physella gyrina, evidently because it is quite mobile and reproduces continually throughout the ﬁeld season, traits which increase chance of infection (Snyder and Esch, 1993). Helisoma anceps is more sedentary and has a simple annual lifehistory pattern; its parasitic trematode community is characterized by fewer multiple infections and a competitive dominance hierarchy among parasites. Regarding microhabitats, trematode larvae that infect snails when eggs are consumed (and that have frogs as ﬁnal hosts) are more common in snails collected from shallow habitats in ponds (Sapp and Esch, 1994); in contrast, while larvae that infect snails as miracidia (and 10. Mollusca: Gastropoda 309 TABLE IV Comparison of Average Standing Stocks, Productivity, and Turn-over Times for Populations of Pulmonate and Prosobranch Snails Reported in Russell-Hunter and Buckley (1983) Subclass Mean biomassa s.e. Mean productionb s.e. Mean turnover timec s.e. Pulmonata Prosobranchia 0.98 4.64 5.71 3.56 98.0 385.3 0.50 (6) 1.80 (4) 2.44 (10) 0.51 (5) 9.46 (10) 33.5 (4) (N) Number of species averaged. a g C / m2. b mg C / m2 per day. c days. have waterfowl as ﬁnal hosts) occur in all microhabitats, but only when the ﬁnal hosts are common. Trematode parasites and their snail hosts are also extremely interesting from a co-evolutionary viewpoint (Holmes, 1983; Minchella et al., 1985). Because invertebrates cannot easily acquire resistance to parasites, frequency-dependent selection may operate to ensure the ﬁtness of any genotype less vulnerable to a particular trematode (Holmes, 1983). Parasites may also cause a shift in investment of resources from costly reproduction to growth and maintenance and, thus, even increase survivorship of infected snails (see also Baudoin, 1975; Minchella et al., 1985). Because trematode larvae evolve surface antigens that mimic those of the snail host, application of one evolutionary model, called the “red queen” hypothesis, suggests that gastropods are constantly evolving new antigen phenotypes merely to stay ahead of their trematode parasites. For example, the New Zealand prosobranch Potamopyrgus has both sexual morphs (able to produce genetically more variable offspring through recombination) and parthenogenetic morphs. The frequency of sexual reproduction is positively correlated with trematode prevalence, as one would expect if sexual morphs have an advantage (Lively, 1987). Sexual morphs are also more common in shallow water, where waterfowl (the ﬁnal hosts) occur (Lively and Jokela, 1995). Infected snails are also more likely to forage during the day than uninfected snails or gravid females, suggesting parasites may modify snail behavior so they are more likely to be consumed by waterfowl, to complete the life cycle (Levri and Lively, 1996). Experimental studies in New Zealand have suggested trematode populations are locally adapted to better infect their own host populations than snails from different lakes (Lively, 1989). Gene ﬂow has not swamped such local adaptation, even though rates of gene ﬂow are greater among parasite populations than among their snail hosts (Dybdhal and Lively, 1996), However, sexual reproduction may not be favored in all snail – trematode systems. In Campeloma, certain trematode metacercaria actually feed on sperm, and parthenogenetic morphs thus have an advantage (Johnson, 1992). 3. Production Ecology Average standing crop biomass, productivity, and turnover times (these terms are explained in the glossary) differ between prosobranchs and pulmonates (Table IV, summarized from Russell-Hunter and Buckley, 1983). Standing crops are greater on the average for prosobranchs than for pulmonates, although prosobranch populations studied to date have not included genera with smaller individuals, such as Amnicola and Valvata. Even if pulmonates have lower standing stocks, their rapid growth and short life cycles still result in higher average production rates and shorter turnover times (Table IV). For example, two pleurocerid grazers in an Alabama stream, Elimia cahawbensis and E. clara, have considerable biomasses of 2 – 5 g ash-free dry mass (AFDM) per square meter, but slow growth rates and long life cycles result in relatively low rates of secondary production (0.5 – 1.5 g AFDM / m2). The combination of high biomass and low production results in a low production to biomass ratio of 0.3 (Richardson et al., 1988). Prosobranch detritivores may, however, have higher production rates. Viviparus subpurpureus and Campeloma decisum, with high densities and short life cycles in Louisiana bayous, have high standing crop biomasses (10 – 20 g AFDM / m2) and production rates (20 – 40 g AFDM / m2 per year) among the highest known for freshwater molluscs (Richardson and Brown, 1989). 4. Ecological Determinants of Distribution Water hardness and pH are often considered major factors determining the distributions of freshwater snails (Boycott, 1936; Macan, 1950; Russell-Hunter, 1978; Okland, 1983; Pip, 1986). For example, in the acid-rain affected lakes of the Adirondack mountains of New York, Jokinen (1991) found that snail diversity declined with declining pH and increasing altitude. 310 Kenneth M. Brown However, in lake districts with adequate calcium (above about 5 mg / L CaCO3), or in the normal (nonacidiﬁed) range of pH, relationships between physico-chemical parameters and gastropod diversity are less clear (Lodge et al., 1987; Jokinen, 1987). For example, species in New York lakes overlap broadly in the ranges of physico-chemical variables in which they occur (Harman and Berg, 1971). Physico-chemical parameters, therefore, set the limits for gastropod distributions, but are not as important in explaining the relative abundance patterns and densities of gastropods in most hard-water, circum-neutral lakes. On a biogeographic scale, a factor determining gastropod distributions is dispersal ability. Studies have indicated diversity increases with the area of lakes and ponds (Lassen, 1975; Browne, 1981; Bronmark, 1985a; Jokinen, 1987). Since immigration rates generally increase and extinction rates decrease with increasing habitat size, larger habitats, all else being equal, usually support more species (MacArthur and Wilson, 1967). Jokinen (1987), following the ideas of Diamond (1975), found that snail species were not distributed randomly with increasing snail diversity. Five snails were “high-S” species (e.g., those that occurred only at the most diverse sites) including Valvata tricarinata, Gyraulus deﬂectus, Laevapex fuscus, and two species of Lyogyrus. Most gastropods were widely distributed across sites (“C – D” tramps in Diamond’s terminology). The species with the greatest dispersal ability (called “super tramps” by Diamond) were found only at low diversity sites; these included the lymnaeid Pseudosuccinea collumella and the limpet Ferrissia fragilis. After dispersal, successful colonization depends on the presence of suitable substrates. For example, a signiﬁcant relationship exists between the number of gastropod species and the number of substrates in lakes and streams in New York, USA (Harman, 1972). In fact, substrate preferences determined in the laboratory are good predictors of the types of ponds where the snails are common (e.g., algivores in open ponds, detritivores in wooded ponds; Brown, 1982). Gastropod diversity is also positively related to macrophyte biomass, probably because macrophytes increase surface area for periphyton colonization (Brown and Lodge, 1993). Disturbance may also determine the assemblage of snails. In temporary ponds, diversity is lowered by frequent drying; and in habitats that go hypoxic, diebacks of macrophyte and gastropod populations will also occur (Lodge and Kelly, 1985; Lodge et al., 1987). Disturbance may also limit some species from areas such as wave-swept shores, littoral zones of reservoirs, etc. Similarly, in streams, current velocity may affect distribution and growth. Adult Elimia avoid high-ﬂow areas, perhaps because increased ﬂow makes movement more difﬁcult and lowers grazing rates or increases metabolic rate, thus lowering adult size (Johnson and Brown, 1997). Juveniles, because they are smaller and can exploit the boundary layer, are actually more common in fast-ﬂowing areas. In large lakes, biotic interactions such as interspeciﬁc competition or predation, are important in determining gastropod diversity and abundance (Lodge et al., 1987). In regards to competition, some evidence suggests it is rare in freshwater snails. For example, pulmonate pond snails in the midwestern USA overlap little on food and habitat dimensions of the niche, although some species do compete (Brown, 1982). Other studies also indicate differences in resource utilization. For example, the European ancylid Ancylus ﬂuviatilis prefers diatoms and is found on the top of cobble where periphyton is abundant; whereas the co-occurring planorbid Planorbis contortus is a detritivore and occurs more often under stones, where detritus accumulates (Calow, 1973a, b, 1974a, b). Similarly, Pseudosuccinea columella and Physa vernalis possess differences in their gut and radulae allowing coexistence in New England ponds (Kesler et al., 1986). Furthermore, niche partitioning may also be facilitated by differences among snails in enzymatic activity (Calow and Calow, 1975; Brendelberger, 1997) or radular morphology (Barnese et al., 1990). However, there is some indirect evidence for competition. First, there are often fewer coexisting congeners in ﬁeld samples than would be expected, based on mathematical simulations (Dillon, 1981, 1987). Second, competition has been inferred from changes in relative abundance of gastropod species through time, that is, by apparent competitive exclusion (Harman, 1968). Third, niche overlap can, in fact, be higher in more diverse snail assemblages found in large lakes. For example, the abundances of gastropod species are often positively associated with high-diversity macrophyte beds in lakes, and experiments show most species have similar preferences for macrophytes (Brown, 1997). Finally, pulmonates are common in ponds or in vegetated areas of lakes, while prosobranchs are rare in ponds and common in lakes and rivers. One explanation could be competitive exclusion of pulmonates from lakes by prosobranchs, or exclusion of prosobranchs from ponds by the same mechanism. However, additional explanations include greater vulnerability of prosobranchs to hypoxic conditions in ponds, poorer dispersal abilities of prosobranchs, or the fact that thinshelled pulmonates are more vulnerable to shell-crushing ﬁsh common in lakes or rivers (Brown et al., 1998). Indeed, Lodge et al. (1987) argue that predators determine the composition of gastropod assemblages in lakes. Indirect evidence includes the fact that gastropods 10. Mollusca: Gastropoda have a number of anti-predator adaptations. These include thick shells to protect against shell-crushing predators (Vermeij and Covich, 1978; Stein et al., 1984, Brown and DeVries, 1985; Saffran and Barton, 1993; Brown, 1998), as well as escape behaviors such as shaking the shell or crawling above the water to protect against shell-invading invertebrate predators (Townsend and McCarthy, 1980; Bronmark and Malmqvist, 1986; Brown and Strouse, 1988; Alexander and Covich, 1991 a, b; Covich et al., 1994). Molluscivores are common in lakes and rivers. For example, the pumpkinseed sunﬁsh, Lepomis gibbosus, and the redear or shell-cracker sunﬁsh, Lepomis microlophus, specialize on gastropod prey and have pharyngeal teeth adapted to crush shells. Crayﬁsh will select snails instead of grazing on macrophytes if given a choice (Covich, 1977), using their mandibles to chip shells back from the aperture and selecting species with thinner shells (Saffran and Barton, 1993; Brown, 1998). Although Osenberg (1989) argued that lentic snail assemblages are limited by food resources, most experimental evidence supports a strong role for predators in determining snail diversity and abundance (Table V). For example, the central mud minnow can signiﬁcantly lower the density of relatively thin-shelled snails in permanent ponds. When ﬁsh densities were manipulated in pens, the number of eggs and juveniles of Lymnaea TABLE V 311 elodes was signiﬁcantly less in the presence of ﬁsh. The small, gape-limited ﬁsh feed on eggs and juveniles and may restrict Lymnaea elodes from permanent habitats such as large marshes or lakes. Pumpkinseed sunﬁsh also strongly prefer large, weak-shelled gastropod species in laboratory experiments, and thin-shelled species also decline dramatically in pumpkinseed enclosures in lakes (see references in Table V). Thus, most thin-shelled pulmonates should occur in lakes only in macrophyte beds, where they have a refuge from visual predators; and sandy areas should be dominated by thicker shelled pulmonates like Helisoma or by prosobranchs. Indeed, gastropod distributions among habitats within Indiana and Wisconsin lakes do follow these patterns (Lodge et al., 1987; Brown and Lodge, 1993; Lodge et al., 1998). In fact, ﬁsh predators may indirectly beneﬁt poorer gastropod competitors by preferentially removing dominant species. Lake trout, for example, preferentially consume larger lymnaeids, releasing valvatids from competition in arctic lakes (Hershey, 1990; Merrick et al., 1991). However, experimental manipulations suggest such indirect effects do not extend through four trophic levels. Piscivores (top predators feeding on ﬁsh) in Swedish lakes cannot depress predators of snails to the extent that interactions cascade down the food web to facilitate snails which negatively impact periphyton (Bronmark and Weisner, 1996). Summary of Experimental Field Manipulations Testing the Role of Snail Predators in Controlling Their Prey Study Prey Predator Conclusions Brown and Devries 1985 Sheldon 1987 Lymnaea elodes assemblage mud minnows decreased abundance of eggs and juveniles of thin-shelled L. elodes sunﬁsh Kesler and Munns 1989 Osenberg 1989 assemblage belastomatid bugs removal of ﬁsh increased snails which decreased macrophytes through herbivory decreased snail abundances in New England pond assemblage littoral sunﬁsh Hershey (1990), Merrick et al. (1991) Martin et al. 1992 Bronmark et al. 1992 Bronmark 1994 Lodge et al. 1994 assemblage lake trout assemblage assemblage assemblage L. microlophus L. macrochiras pumpkinseed (L. gibbosus) Tench O. rusticus Daldorph and Thomas 1995 Bronmark and Weisner 1996 assemblage sticklebacks ﬁsh decreased snails, increased periphyton, decreased macrophytes crayﬁsh decreased snails and macrophytes, had no effect on periphyton ﬁsh decreased thin-shelled snail spp., increased periphyton assemblage piscivores & molluscivores molluscivores did decrease snails, but piscivores did not control molluscivores and increase snails indirectly assemblage Assemblage refers to a natural community of gastropods. ﬁsh controlled only rare, large snail spp., competition between snails considered more important trout removed better competitor (L. elodes), which favored poorer competitor, Valvata ﬁsh decreased snails 10-fold, increased periphyton biomass 2-fold, decreased algal cell size, decreased macrophytes. ﬁsh decreased snails, increased periphyton, favored adnate algae 312 Kenneth M. Brown Invertebrate, shell-invading predators may also limit snail populations or cause shifts in gastropod relative abundances. Although some leeches, such as Nephelopsis obscura, have fairly low feeding rates (Brown and Strouse, 1988), crayﬁsh can consume up to or over a hundred snails per night. The crayﬁsh Orconectes rusticus signiﬁcantly reduced snail abundances in enclosure experiments in Wisconsin lakes (Table V), and an interlake survey indicated that snail abundances were negatively correlated with crayﬁsh catches. Crayﬁsh also affect gastropod habitat selection: even though cobble habitat is preferred by many snails (see earlier discussion), it also provides refugia from ﬁsh predators for crayﬁsh. Crayﬁsh predation overrides the effects of rich food resources (Weber and Lodge, 1990). Crayﬁsh are also quite selective molluscivores. They prefer thinner-shelled gastropods (Saffran and Barton, 1993; Brown, 1998), and often prefer juvenile snails which have shorter handling times and higher proﬁtability unless these more vulnerable prey are protected by macrophytes (Nystrom and Perez, 1998). Crayﬁsh predators also shift size distributions of Physella virgata upwards in Oklahoma streams. This may be due both to size-selective predation and to the diversion of energy of the snail from reproduction to growth in order to reach a size-based refuge from predation (Crowl, 1990; Crowl and Covich, 1990). Interestingly, only snails in vulnerable size classes respond by crawling out of the water after predation by crayﬁsh has occurred on conspeciﬁcs (Alexander and Covich, 1991b). Other invertebrate predators may also be important. For example, belostomatid bugs can eat as many as ﬁve snails per bug per day in the laboratory (Kesler and Munns, 1989). Fish predators may alter snail foraging behavior as well. In experiments, Physella selects refugia in the presence of sunﬁsh predators, only leaving when sufﬁciently starved (Turner, 1997). This behavioral interaction may provide an additional explanation for why periphyton biomass increases when ﬁsh predators are present. For example, use of open habitats by snails decreases with increasing predation risk from sunﬁsh (Fig. 8), with the result that periphyton biomass also increases (Turner, 1997). Interestingly, such indirect facilitation of periphyton does not occur upon experimental addition of crayﬁsh to enclosures, probably because crayﬁsh are also omnivorous and graze on periphyton (Lodge et al., 1994). 5. Suggestions for Further Work Studies of the production ecology of smaller prosobranchs, as well as snail populations in other areas besides the northeastern United States are still needed. Questions still exist on subjects such as: FIGURE 8 Indirect effect of mortality from ﬁsh predation on snail grazing rates. As more crushed snails were added to experimental pools (predation risk perceived by snails, x-axis), snails used refuges more, resulting in reduced use of open areas (left y-axis). Note how periphyton biomass (ashfree dry mass or AFDM, right y-axis) increased in open area as a result of reduced snail grazing (after Turner, 1997). 1. How pulmonates are physiologically adapted to relatively ephemeral or hypoxic aquatic habitats, including studies of differences in metabolic pathways and nitrogen excretion (McMahon, 1983). 2. The relative roles of the physiological and ecological constraints to shell versus tissue growth, given the remarkable intraspeciﬁc variation in shell structure and composition in freshwater gastropods. 3. Physiological bases of feeding preferences (such as relative cellulase activities, etc.). 4. Micro-preferences (e.g., preferences for certain algae or diatom species), for example, whether detritivores are consuming leaves or more nutritious bacteria or fungi. 5. The nutritional quality or toxicity of different food types and their role in preferences by gastropods. Further research on abiotic and biotic factors inﬂuencing snail assemblages is also necessary. Abiotic factors such as pond drying, anoxia, ﬂoods, or macrophyte die-offs could explain much about gastropod population dynamics or species composition. Further work is also necessary to determine whether the allopatric distributions of pulmonates and prosobranchs are due to competition (see discussion in Brown et al., 1998). Further work is needed as well on the relative role of invertebrate predators like leeches, belostomatid bugs, and scyomyzid ﬂy larvae (Eckblad, 1973) in structuring snail assemblages. 10. Mollusca: Gastropoda 313 D. Evolutionary Relationships Marine prosobranchs are ancestral to freshwater prosobranchs and terrestrial and freshwater pulmonates. Freshwater prosobranchs adapted to the dilute osmotic conditions of estuaries and then rivers, and most modern families are widespread because their adaptive radiations predated continental breakups and drift. Davis (1979, 1982) and Clarke (1981) pointed out that prosobranch dispersal is limited in most cases to slow movement of adults along streams and rivers. Such populations are more likely to become isolated, promoting chances of speciation and adaptive radiation. Examples include the dramatic adaptive radiation of hydrobiids (Davis, 1982) and pleurocerids in lotic habitats (Burch, 1989; Lydeard et al., 1997). In contrast, pulmonates, due to their greater rates of passive dispersal as spat on birds and insects, have more widespread distributions. For example, they are widespread in the northern and northeastern states, as well as in Canada (Harman and Berg, 1971; Clarke, 1981). Immigration rates to ponds average 0.8 and can be as high as 9 species per year (Lassen, 1975; Davis, 1982). Pulmonates also predominate in shallow, more ephemeral habitats less than 100 km2 in area and with durations less than 103 years. Populations probably do not exist long enough in such habitats for speciation to occur, explaining why pulmonates are less speciose than prosobranchs (Russell-Hunter, 1983; Clarke, 1981; Davis, 1982). Pulmonates apparently evolved from intertidal prosobranchs that relied less and less on aquatic respiration (Morton, 1955; McMahon, 1983). Modern estuarine pulmonates like Melampus may resemble these ancestral species. The intermediate, terrestrial pulmonates lost the ctenidium, and gave rise both to modern terrestrial pulmonates (order Stylommatophora) and the aquatic pulmonates (order Basommatophora). Figure 9 illustrates a hypothetical phylogeny of aquatic pulmonates based on progressive re-adaptation to aquatic habitats (see discussion in McMahon, 1983). Lymnaeids are in some cases still amphibious (e.g., occurring in temporary wetlands), while physids are intermediate in their adaptation to aquatic habitats (some species come to the surface to breathe, while others have ﬁlled the lung with water and can be found in deeper lakes). Ancylids and planorbids have secondarily acquired more aquatic specializations such as the evolution of secondary gills, and in the planorbids, hemoglobin. Several phylogenetic trees for freshwater snails have been published recently, using both classical morphological and recently developed molecular methods. Jung (1992) used the more traditional characters of shell and FIGURE 9 Hypothetical phylogeny for freshwater pulmonates assuming increased adaptation through time to secondarily-invaded aquatic habitats. The argument is that air-breathing lymnaeids are limited to shallow water, while physids, which in some cases can ﬁll their lung with water and use it as a gill, are found in deeper water, and ancylids and planorbids are the most specialized for an aquatic life style, with secondarily derived epithelial gills and in the planorbids, hemoglobin. soft anatomy to propose a cladogram (Fig. 10) for certain species of planorbids, using 65 characters. He pointed out that systematic studies should not rely on preserved specimens, since ﬁxation alters soft anatomy. In his phylogeny, Biomphalaria is considered more ancestral, and Planorbella trivolvis and Helisoma anceps the most derived species. Remigio and Blair (1997) published a phylogeny for certain lymnaeid species, using populations from both North America and Europe (Fig. 11). They considered molecular methods, such as sequencing ribosomal DNA in mitochondria, superior techniques in comparison to other characters such as shell morphology, which often converge among genera. For example, based on molecular information, they considered karyotypes with 18 chromosomes to be ancestral, and 16 chromosomes to be a derived character, unlike FIGURE 10 Simpliﬁed cladogram for several planorbid species studied by Jung (1992), based on shell and soft anatomy. 314 Kenneth M. Brown FIGURE 12 FIGURE 11 Cladogram for a subgroup of the lymnaeid species found in both North America and Europe, based on molecular data. Simpliﬁed from Remigio and Blair (1997). earlier studies. European and North American stagnicolids (S. elodes and S. palustris) were not considered each other’s closest relatives, contrary to interpretations based on morphology. Stagnicola catascopium, S. emarginata, and S. elodes, based on genetic divergence, could actually be conspeciﬁc taxa. On the other hand, two populations of Lymnaea stagnalis (one in Germany and the other in Italy) were genetically quite divergent, indicating isolation by the geographic barrier of the Alps. The authors propose that lymnaeids evolved in the late Jurassic to early Cretaceous on the common continent Laurasia, and were separated into distinct Palearctic and Nearctic groups at the start of the Cenozoic (65 million years ago) when ancestral species still had 18 chromosomes. The genus Stagnicola was probably extant at that time, explaining why North American and European species are so divergent. Lydeard et al. (1997) used mitochondrial rDNA sequences to study the evolutionary relationships of representative prosobranch snails in the Mobile River Basin in the southeastern United States (Fig. 12). This basin may have once had the most diverse gastropod fauna in the world, with 118 species and nine families. Pleurocerids make up almost two thirds of the surviving fauna. Because of impoundments, the genus Gyrotoma, once found in shallow rifﬂes in the Coosa River, has gone extinct, along with 14 species of Elimia, and 11 species of Leptoxis. Lydeard’s phylogeny considers 14 prosobranch species (with Melanopsis as an out group) and suggests Elimia and Pleurocera are monophyletic genera, but that Leptoxis is paraphyletic. The remaining Elimia species in the Mobile Basin are genetically fairly similar, while the remaining three species of Cladogram for a subgroup of species in several prosobranch genera in the Mobile River Basin, based on molecular data. The greater divergence of leptoxid species suggests a polyphyletic origin for this genus. Simpliﬁed from Lydeard et al. (1997). Leptoxis are quite divergent in their mitochondrial 16S rDNA sequence. Further studies of genetic relationships among pleurocerids found in the Mobile River Basin suggest that earlier species descriptions, relying solely on shell morphology, have overestimated the true diversity of the system. For example, Lydeard et al. (1998), again looking at 16S rDNA sequences, considered Elimia hydei basal to all other elimiads, and Elimia crenatella, E. showalteri, E. fascians, and E. E. caelatura to be true species. The low genetic divergence within another clade led them to question the status of six speces: Elimia carinocostata, E. gerhardtii, E. haysiana, E. alabamiensis, E. olivula, and E. cylindracea. Dillon and Lydeard (1998) used allozyme polymorphism to study relationships among Mobile Basin leptoxids. They argued that Leptoxis praerosa and L. picta were deserving of species status, but that L. taeniata and L. ampla should be subsumed within L. picta. On the other hand, Dillon and Ahlstedt (1997) used allozyme variation to show that Athernia anthonyi and Leptoxis praerosa were different species, although adult shell morphology is similar. Several caveats about the evolution of freshwater snails should be kept in mind (Davis, 1982). First, phylogenetic analyses, and therefore biogeographic studies, are often limited by the poor systematic information available on many groups, as well as the poor fossil record (only shells are preserved, which often show convergence). Second, a number of factors can explain current distributions, as illustrated for hydrobiids and pomatiopsids by Davis (1982): (1) phylogenetic events such as centers of origin and adaptive radiation; (2) past history events such as continental drift and geological alteration of stream and river ﬂow; (3) dispersal powers, and (4) ecological factors (see earlier discussion). 10. Mollusca: Gastropoda E. Conservation Biology of Freshwater Snails Freshwater snails, especially prosobranch species in the southeastern United States, are facing rates of extinction similar to the more widely known case of North American unionid bivalves (Lydeard and Mayden, 1995; Neves et al., 1998; Jenkinson and Todd, 1998). Freshwater snails account for 60% of all North American molluscan species, and 61% of these snails are in the Southeast. Historically, there were 118 species of snails in the Mobile River Basin, 96 species in the Tennessee River, and 36 species each in the Cumberland and Apalachicola River basins. Thirty-eight of the 118 species in the Mobile River Basin are known to be extinct. The decline in diversity in the Mobile River Basin varies from 33 to 84% in different drainages. Nine species of gastropods are currently listed as federally endangered (Bogan, 1999), although over 200 species from 11 families are candidates (69% of which are in the Southeast). In the southeastern states alone, two viviparids, 50 hydrobiids, 83 pleurocerids, nine pulmonates, three ancylids and six planorbid species are considered at risk (Neves et al. 1998). Many of the same factors that have imperiled unionid bivalves are responsible: impoundments which inundate shallow rifﬂes and decrease oxygen and alter water temperature, increased turbidity from agricultural activities, improper sewage treatment, and other pollution sources, to name but a few. Organic enrichment, pesticides and pathogens endanger about a quarter of the impaired river-miles in North American rivers, while excess nutrients and siltation occur, respectively, in 37 and 45% of the impacted river-miles (Neves et al., 1998). At the current time there is little governmental interest in managing imperiled snail populations, although the large, knobbed-shell species, Io ﬂuvialis, has been successfully reintroduced to one river in its original range. 315 Lodge et al. (1994). In coarse sand, Ponar dredges are the best alternative. In cobble, little recourse to direct counts of given areas by visual search is available. One can estimate densities by individually covering the rocks with aluminum foil, and then estimating the area from weight-to-area regressions for the foil. The best sorting technique for gastropods is handsorting. Large adults can be removed visually, while samples are washed through a graded series of sieves (the smallest having a mesh of approximately 0.5 mm) to remove mud but retain smaller snails. Samples are then carefully backwashed from the sieves into shallow water on ﬂat white trays, the vegetation teased apart, and the whole tray examined in a systematic fashion to remove small gastropods and egg cases. Temperate gastropods will grow well at 15 – 20°C, while subtropical species grow better at 20 – 25°C. Any hard substrate with a dense periphyton covering can be added for food, although artiﬁcial foods such as lettuce, cereal, or spinach are sometimes used. Provide food only as needed to avoid fouling containers. Detritivores should be fed leaf litter colonized with bacteria and fungi (i.e., held for at least 2 weeks in a pond or stream). Avoid crowding of snails, as growth and reproduction are sensitive to density. An approximate rule is one snail per liter. Water should be re-circulated through a gravel or charcoal ﬁlter, or at least be changed weekly. Conspeciﬁcs should be paired so that mating can occur. Culturing of “weedy” species like physids is best done at low temperatures to retard egg production, and constant removal of egg cases is necessary to prevent population “explosions”. Adequate lighting (with a 12L:12D cycle) is necessary to promote periphyton growth in aquaria (gro-lites®work well). V. IDENTIFICATION OF THE FRESHWATER GASTROPODS OF NORTH AMERICA IV. COLLECTING AND CULTURING FRESHWATER GASTROPODS A number of sampling techniques exist (reviewed in Russell-Hunter and Buckley, 1983), although some are not quantitative. The least quantitative technique, but one that often gives large numbers of individuals and a good idea of species composition, is sweep netting with a net of 1-mm mesh (Brown, 1979, 1997). In soft sediments or sand, quantitative samples can be collected with an Ekman grab or a corer. When sampling macrophytes, the sampler must collect both plants with attached snails and the substrate with any bottomdwelling species. Examples of such samplers are described in Gerking (1957), Savino and Stein (1982) and The following key is modiﬁed from Burch’s (1989) key for North American freshwater snails. Taxa are keyed down to genera in all families except the diverse hydrobiids. Readers interested in keying specimens down to the species level should consult Burch (1989) or keys addressing regional snail faunas (Harman and Berg, 1971; Jokinen, 1983; Thompson, 1984; Jokinen, 1992). After collecting specimens, ﬁrst relax them with tricaine methane sulfonate, menthol crystals, polypropylene phenoxytol, or sodium nembutol and then preserve them in 70% ethyl alcohol. Preserved specimens are better than dried shells, as possession of an operculum will be one of the determinations necessary. 316 Kenneth M. Brown Collect a number of different-sized specimens, because closely related genera sometime differ only in adult shell size. Sizes reported in the key, following Burch (1989), are small shells less than 10 mm, medium between 11 and 29, and large greater than 30 mm in length. Size bars in keys are in millimeters. Refer to the glossary for shell sculpture terminology like costae, lirae, etc. In some cases, soft tissue characters are used in the key (for example the structure of the verge, or penis, located behind the male’s right tentacle, is important in hydrobiids). The shells of preserved individuals can be carefully crushed and removed, after living snails are relaxed with any of the chemicals mentioned above. Examine the verge under a dissecting microscope. If examination of the radula is necessary, dissect out the buccal mass (a muscular mass behind the mouth that surrounds the radula) from a relaxed specimen and place it in a 10% potassium hydroxide solution to digest the tissue. After several hours, remove any remaining tissue and rinse the radula in 70% alcohol, stain it and mount it on a slide for examination under a compound microscope. However, digestion with strong bases may damage the radula. An alternative technique (Holznagel, 1998) is more involved, but also provides a method to extract DNA for phylogenetic work, and can be used with frozen or alcohol-fixed specimens (but not those preserved in formalin). First prepare stock solutions of NET buffer and Proteinase-K. For the former, add 1 mL pH 8.0 Tris buffer, 2 mL 0.5 M EDTA (ethylene diamine tetra-acetic acid), 1 mL 5M NaCl, and 20 mL of 10% SDS (sodium dodecyl sulfate) solution to 76 mL of deionized water. For the latter, add 20 mg Proteinase-K to 1 mL de-ionized water and store at 20°C until use. Next remove the body from the shell. This is fairly easy in recently thawed or relaxed specimens, but for nonrelaxed specimens the shell may have to be gently cracked and removed from the body. Dissect away the head, and rinse with water. Place the tissue in a 1.5 mL microcentrifuge tube with 500 L of NET buffer and 10 L of Proteinase-K. Cap the tube and place it on a 37°C mixing table for 3 h (for fixed specimens) or 4 days (for dried specimens). The buffer and enzyme may need daily replacement. Observe the radula under a dissecting scope to determine if tissue digestion is complete, and rinse it with de-ionized water several times. The supernatant can be used for extraction of DNA with phenol / chloroform techniques (see Sambrook et al., 1989). Store the radula in 25% ethanol until ready for mounting on a microscope slide. A. Taxonomic Key to Families and Selected Genera of Freshwater Gastropods 1a. Shell an uncoiled cone (limpet, or cap shaped) (Fig. 2) .......................................................................................................................2 1b. Shell coiled .........................................................................................................................................................................................8 2a(1a). Apex nearly central; adult up to 12 mm; Paciﬁc drainage. . . Family Lymnaeidae...............................................................................3 2b. Apex to the right or left of the median line; adult 7 mm or less; dextral or sinistral ...........................................................................4 3a(2a). Apex central (Fig. 13A) ................................................................................................................................................................Lanx 3b. Apex anterior; Columbia river (Fig. 13B) ...............................................................................................................................Fisherola 4a(2b). Shell dextral (apex tips to left when viewed with aperture facing up); Rocky Mountain lakes, northeastern Ontario and northcentral Quebec (Fig. 13C) .................................................Family Acroloxidae ....................................................................Acroloxus 4b. Shell sinistral (apex tips to right when viewed with aperture facing up); throughout North America ..........Family Ancylidae............5 5a(4b). Shell elevated, apex in midline, tinged with pink or red inside and out, radially striate, with a notch-shaped depression in unworn specimens; apertural lip broad and ﬂat. In southeastern rivers (Fig. 13D) ..........................................................................Rhodacmea 5b. Shell height variable; apex in midline or to right, the same color as the rest of the shell, ﬁnely radially striate or smooth; widely distributed in lotic or lentic habitats .......................................................................................................................................................6 6a(5b). Apex with ﬁne radial striae, eroded in older specimens; aperture width variable, open or with a horizontal shelf in the posterior; one lobed, ﬂat pseudobranch; widely distributed in lotic and lentic habitats (Fig. 13E) .................................................................Ferrissia 6b. Shell more depressed; apex without radial striae; aperture ovate to subcircular, always open; two-lobed pseudobranch, the lower elaborately folded; in eastern states and south in lentic habitats .........................................................................................................7 7a(6b). Apex tipped to right, tentacles colorless; In canals in southern Florida and Texas (Fig. 13F)...........................................Hebetancylus 7b. Apex in midline; Tentacles with a black core; East of the Mississippi in lentic habitats, occasionally in south-central streams (Fig. 13G)..............................................................................................................................................................................Laevapex FIGURE 13 Representative limpets and planorbids. (A) The lymnaeid limpet Lanx patelloides (note large size, west coast distribution); (B) lymnaeid limpet Fisherola nutalli; (C) Acoloxus; (D) Ancylid limpet Rhodacmea rhodacme ( hinkezi) (note notched depression and southeastern distribution); (E) ancylid limpet Ferrissia rivularis (note elevated shell, some species possess posterior “shelf” in shell); (F) ancylid limpet Hebetancylus excentricus (note depressed apex to the right of midline, colorless tentacles, and southern distribution); (G) Laevapex fuscus (note obtuse apex near midline of shell, black pigmented tentacles, and widespread distribution in eastern backwaters and southern, slow-ﬂowing streams). (H) Armiger crista (note costae); (I) Planorbula armigera ( jenksii) (note teeth in aperture); (J) Drepanotrema kermatoides; (K) Gyraulus deﬂectus; and (L) Menetus dilatatus (A – L after Burch, 1982). 317 318 Kenneth M. Brown 8a(1b). Shell planospiral (Fig. 2); blood (hemolymph) nearly always red (contains hemoglobin); pseudobranch (false gill) near pneumostome or anus; mantle margin simple ...............................................Family Planorbidae ..............................................................9 8b. Shell with raised spire.......................................................................................................................................................................20 9a(8a). Adult less than 8 mm in diameter .....................................................................................................................................................10 9b. Adult more than 10 mm in diameter ................................................................................................................................................16 10a(9a). Shell with transverse raised ridges (costae). Canada and northern United States. (Fig. 13H)....................................................Armiger 10b. Shell without costae .........................................................................................................................................................................11 11a(10b). Adults 2 mm or less in diameter; Coosa River, Alabama .................................................................................................Neoplanorbis 11b. Adults more than 2 mm in diameter .................................................................................................................................................12 12a(11b). Aperture or body whorl with internal “teeth” or lamellae set back one quarter whorl from aperture (Fig. 13I ...................Planorbula 12b. Aperture or body whorl without teeth..............................................................................................................................................13 13a(12b). Shell extremely ﬂat, multiwhorled, or with numerous, low, close-set spiral ridges (lirae). Florida, Texas and southern Arizona (Fig. 13J) .......................................................................................................................................................................Drepanotrema 13b. Shell not extremely ﬂat or multi whorled, lacking lirae ....................................................................................................................14 14a(13b). Body whorl height equal from one side to the other (Fig. 13K) ..............................................................................................Gyraulus 14b. Body whorl rapidly increases in height toward aperture ...................................................................................................................15 15a(14b) Body whorl rounded (Fig. 13L) ...............................................................................................................................................Menetus 15b. Body whorl with angular edge (carina) (Fig. 14a) ..............................................................................................................Promenetus 16a(9b). Adult greater than 30 mm; operculum present; penis to right of mantle; gelatinous eggs; In Florida, and introduced to rivers in central Texas....................Family Pilidae ......................................(Ampullaridae) (Fig. 18a).................................................... Marisa 16b. Adults less than 30 mm, operculum absent ......................................................................................................................................17 17a(16b). Shell thin, fragile, body whorl relatively depressed; Florida, Texas, Arizona (Fig. 14B) ...................................................Biomphalaria 17b. Shell thicker, solid; body whorl often high........................................................................................................................................18 18a(17b). Body whorl extremely large; Western in distribution (Fig. 14C) ............................................................................................Vorticifex 18b. Body whorl not extremely large .......................................................................................................................................................19 19a(18b). Shell spire (left side) with deep conical depression, with strong carina (Fig. 14D)..................................................................Helisoma 19b. Shell spire (left side) with shallow depression, whorls rounded on at least one side, without carina; aperture lip sometimes ﬂared (Fig. 14E,F) ........................................................................................................................................................................Planorbella 20a(8b). Shell sinistral (aperture to left when shell viewed with spire pointing away from observer); mantle margin may be digitate or lobed..............................Family Physidae.........................................................................................................................................21 20b. Shell dextral (aperture to right) ........................................................................................................................................................24 21a(20a) Mantle edge with ﬁngerlike projections ............................................................................................................................................22 21b. Mantle edge without projections, but may be serrated .....................................................................................................................23 22a(21a). Projections on both sides of mantle .............................................................................................................................................Physa 22b. Projections on parietal side of mantle (Fig. 14G – I) .................................................................................................................Physella 23a(21b). Serrations extend beyond apertural lip, partly overlap shell; Texas (Fig. 14J) .....................................................................Stenophysa 23b. Shell elongate, surface black and glossy, sutures smooth, in wooded ponds in Canada and northern United States (Fig. 14K) ............. ..................................................................................................................................................................................................Aplexa 24a(20b). Aperture with teeth on the parietal inner margin; operculum present and calcareous, paucispiral (Fig. 2); adult length about 20 mm; gill featherlike. Florida and southern Georgia ........................................Family Neritinidae ....................................(Fig. 14I) Neritina 24b. Shell without teeth on parietal wall, operculum present or absent. ...................................................................................................25 FIGURE 14 Representative planorbids, physids, and neritinids. (A) Promenetus exacuous (note ﬂaired body whorl and carina); (B) Biomphalaria glabrata; (C) Vorticifex (Parapholyx) effusa; (D) Helisoma anceps (note strong growth lines and carina); (E) Planorbella (Helisoma) companulata (sometimes called companulatum, note ﬂaired lip of aperture); (F) Planorbella (Helisoma) trivolvis (this species reaches 20 mm in diameter and is extremely common); (G) Physella (Physa) gyrina (very similar and possibly subspecies are P. anatina and P. virgata of the midwest and south, the only physid to reach 20 mm shell length); (H) Physella (Physa) integra (note the more elevated spire than P. gyrina, rarely reaches 10 mm shell length); (I) Physella (Petrophysa) zionis (note the large aperture); (J) Stenophya: (K) Aplexa elongata (note the spindle shape and lustrous almost-black shell); and (I) Neritina reclivata (note markings on shell and teeth on parietal wall). [A – I after Burch, 1982.] 319 320 Kenneth M. Brown 25a. Spire short, operculum multispiral; shell sometimes carinate; external, featherlike gill visible when foot is extended (Fig. 15A, B) Family Valvatidae .....................................................................................................................................................................Valvata 25b. Spire longer ......................................................................................................................................................................................26 26a. Without operculum; shell relatively fragile and easily crushed; respiration by pouch in mantle cavity...............Family Lymnaeidae 27 26b. Shell stronger; operculum and gills present..........................Subclass Prosobranchia ........................................................................33 27(26a). With large, globose body whorl and extremely large aperture (Fig. 15C)....................................................................................Radix 27b. Body whorl more narrow .................................................................................................................................................................28 28a(27b). Shell very narrow and elongate; southern Ontario; north-central United States to Vermont (Fig. 15D) .....................Acella haldemani 28b. Shell not especially narrow ...............................................................................................................................................................29 29a(28b) Shell tear-shaped, transparent and fragile, with large, oval aperture and body whorl, small spire; amphibious. Eastern North America (Fig. 15E)................................................................................................................................................................Pseudosuccinea 29b. Shell not extremely thin or fragile ....................................................................................................................................................30 30a(29b) Adults more than 35 mm in length ...................................................................................................................................................31 30b. Adults less than 35 mm in length .....................................................................................................................................................32 31a(30a). Shell with narrow, pointed spire, moderately fragile; in marshes or streams in north-central and eastern United States, Canada (Fig. 15F).................................................................................................................................................................Lymnaea stagnalis 31b. Shell globose, thick, with relatively wide spire. Great Lakes and St. Lawrence River drainages (Fig. 15G)...........Bulimnea megasoma 32a(30b). Adult shell more than 13 mm, with microscopic spiral striations or malleations; columella with a well-developed twist or plait. Widely distributed in ponds and marshes in northern states or in alpine regions of western states (Fig. 15H) ......................Stagnicola 32b. Adult shell less than 13 mm, spiral sculpture usually absent; columella without a twist or plait; (Fig. 15I)..............................Fossaria 33a(26b). Operculum multispiral or paucispiral (Fig. 2), outer margins not concentric ....................................................................................34 33b. Operculum concentric (center may be paucispiral) ...........................................................................................................................47 34a(33a). Adult less than 7 mm; males possess verge (penis) behind right tentacle ...........................................................................................35 34b. Adults more than 15 mm in length, males lack verge........................................................................................................................40 35a(34a). Spire high, head – foot region divided on each side by longitudinal groove; eyes in prominent swellings on the outer bases of the tentacles; amphibious or terrestrial; crawls with steplike movement; (Fig. 16A) ..............Family Pomatiopsidae....................Pomatiopsis 35b. Shell length variable; head – foot region not divided; eyes at same location but not on prominent swellings; totally aquatic................. Family Hydrobiidae .........................................................................................................................................................................36 36a(35b). Verge simple, no accessory lobes (Fig. 17A)..................................................................................................Subfamily Lithoglyphinae ................................................................................................Lepyrium, Cochliopina, Fluminicola, Antrobia, Clappia, Somatogyrus 36b. Verge with accessory lobes (Fig. 17 B-E)...........................................................................................................................................37 37a(36b). Verge with two arms (mitten shaped, Fig. 17B, C)............................................................................................................................38 37b. Verge with three or more divisions (Fig. 17D, E) ..............................................................................................................................39 38a. Verges with one small and one large lobe with glandular areas ..................................................Subfamily Nymphophilinae (Fig 17B) .....Orygocerus, Birgella, Striobia, Marstonia, Rhapinema, Notogilla, Spilochlamys, Cincinnatia, Pyrgulopsis, Fontelicella, Natricola 38b. Verges with branches of approximately similar diameter...................................................................................................................... Subfamily Amnicolinae (Fig. 17C) .......................................................................................Amnicola, Lyrogyrus, Hauffenia, Horatia 39a. Verges with three accessory lobes branching off main lobe (Fig. 17D)..............................................................Subfamily Hydrobiinae ...................................................................Probythinella, Hoyia, Tryonia, Pyrgophorus, Littoridinops, Aphaostracon, Hyalopyrgus 39b. Verge ending in three similar-sized lobes (Fig. 17E).................Subfamily Fontigentinae ........................................................Fontigens 40a(34b). Mantle edge papillate; parthenogenetic (males absent); females brood young in a pouch posterior to head; Introduced from Florida to Texas ...............................................................................................................Family Thiaridae .................................................41 40b. Mantle edge smooth; dioecious (males present), females oviparous, with egg-laying sinus on the right side of the foot........................ Family Pleuroceridae ........................................................................................................................................................................42 41a(40a). Rounded whorls with spiral grooves and transverse raised lines (costae) (Fig. 16B) ..........................Introduced in Florida to Arizona ...........................................................................................................................................................................................Melanoides 10. Mollusca: Gastropoda FIGURE 15 Representative valvatids and lymnaeids. (A) Valvata sincera; (B) Valvata tricarinata (note carina); (C) Radix (Lymnaea) auricularia (note expanded body whorl); (D) Acella haldemani (note extremely narrow shell); (E) Pseudosuccinea columella (note thin, transparent shell and amphibious habit); (F) Lymnaea stagnalis (note large and fragile shell); (G) Bulimnea megasoma (note thick, large shell); (H) Stagnicola (Lymnaea) elodes (note that malleations sometimes present, and this species is common only in ponds and marshes, rarely lakes); and (I) Fossaria (Lymnaea) humilis (note small size and amphibious habit). [Figures A – I after Burch, 1982.] 321 322 Kenneth M. Brown FIGURE 16 Representative potamiopsids, pleurocerids and bithyniids. (A) Pomatiopsis lapidaria; (B) Melanoides tuberculata (note costae and lirae); (C) Thiara granifera (note tubercules and ﬂattened whorls near apex); (D) Io ﬂuvialis (length of spines variable); (E) Leptoxis praerosa; (F) Lithasia geniculata (note thickened anterior aperture lip and shoulders on whorls); (G) Pleurocera acuta (note acute angle on anterior aperture and relatively ﬂat whorls); (H) Elimia ( Goniobasis, Oxytrema) livescens (note more rounded whorls, although there is tremendous variation in shell sculpture in this genus); and (I) Bithynia tentaculata. [A – I after Burch, 1982.] 10. Mollusca: Gastropoda 323 FIGURE 17 Structure of the verge (penis) in various subfamilies of hydrobiids. (A) Simple, single axis verge of subfamily Lithoglyphinae; (B) glandular crests on verge of subfamily Nymphophilinae; (C) twoducted verge of subfamily Amnicolinae; (D) verge with accessory lobes in subfamily Hydrobiinae; and (E) threeducted verge of subfamily Fontigentinae [A – E after Burch, 1982]. 41b. Whorls ﬂat near spire; spiral rows of nodules on shell (Fig. 16C) .................Introduced to Florida and Texas ...........................Thiara 42a(40b). Large, often with elongated spines and a long, anterior canal on aperture (Fig. 16D); rivers in eastern Tennessee and western Virginia.............................................................................................................................................................................................Io 42b. Shell size and sculpture quite variable; aperture sometimes ends with a short canal .........................................................................43 43a(42b) Shell medium-to-small, a subglobose, or globose cone (Fig. 16E); lateral radular teeth with broad, bluntly rounded median cusps; .... ...............................................................................................................................................................................................Leptoxis 43b. Shell usually a narrow cone; lateral radular teeth with narrow, triangular median cusps..................................................................44 44a(43b). Shell length medium, either cone-shaped or sub-globose, often with spines or nodules; inside margin of the aperture thickened, aperture meets body whorl nearly at right angle (Fig. 16F).............................................................................................................Lithasia 44b. Shell length and sculpture variable, base of aperture either rounded or with a short canal; inner margin of the aperture without thickening ........................................................................................................................................................................................45 45a. Anterior (basal tip of) aperture usually pointed; whorls ﬂat-sided.....................Mississippi, Great Lakes and Hudson River drainages (Fig. 16G) ............................................................................................................................................................................Pleurocera 45b. Anterior aperture and whorls more rounded ....................................................................................................................................46 46a(45b). In river drainages east of the Mississippi River (Fig. 16H) .........................................................................................................Elimia 46b. In river drainages west of the Mississippi ......................................................................................................................................Juga 47a(33b). Adults less than 15 mm; operculum calcareous; from Wisconsin to Pennsylvania and New York (Fig. 16I) ............Family Bithyniidae Bithynia tentaculatum 47b. Adults more than 20 mm (some up to 50 or 60 mm); operculum corneous ......................................................................................48 48a(47b). Globose, length up to 60 mm; penis on right side of mantle; calcareous eggs. Florida ..........Family Pilidae (Ampullariidae) (Fig. 18B) ...............................................................................................................................................................................................Pomacea 48b. Subglobose, right tentacle thicker in males, used as a penis; females ovoviviparous............Throughout the United States and Canada Family Viviparidae ...........................................................................................................................................................................49 49a(48b). Adults over 35 and up to 50 mm; shell relatively thin, whorls not shouldered (Fig. 18C); introduced throughout United States .......... ..................................................................................................................................................................................Cipangopaludina 49b. Shell thick and less than 35 mm .......................................................................................................................................................50 50a(49b). Shell sometimes with one or two spiral rows of nodules; outer margin of aperture concave (when observed from the side); inside (columellar) margin of operculum folded inward; Alabama rivers (Fig. 18D)........................................................................Tulotoma 50b. Shell without nodules; aperture not as above ...................................................................................................................................51 51a(50b). Operculum with spiral center and concentric edge; whorls with a median angle or low ridge (Fig. 18E)..................................Lioplax 51b. Operculum entirely concentric; whorls without angles or ridges ......................................................................................................52 51a(51b). Shell often with colored bands or hirsute in juveniles; aperture nearly circular (Fig. 18F)......................................................Viviparus 51b. Colored bands absent; aperture longer than wide, forms shoulder at junction with body whorl (Fig. 18G).......................Campeloma 324 Kenneth M. Brown FIGURE 18 Representative ampullarids and viviparids. (A) Marisa cornuarietis (note large, planospiral shell, occurs in southern Florida and introduced to rivers in central Texas); (B) Pomacea paludosa (large, ‘Apple’ snail common in southern Florida); (C) Cipangopaludina japonica (a large, introduced, but now common species in North America); (D) Tulotoma magniﬁca (in some southeastern rivers, note tubercules which may be absent in some morphs or species); (E) Lioplax subcarinata; (F) Viviparus georgianus (common, note circular operculum, and bands which may disappear in adults); (G) Campeloma decisum (some times referred to as decisa; note operculum is longer than it is wide, and shouldered junction of aperture and body whorl). [Figures A – G after Burch, 1982.] 10. Mollusca: Gastropoda ACKNOWLEDGMENTS I would like to thank Dr. Jeffery Tamplin for the considerable effort involved in drafting the excellent ﬁgures for the key, and Mr. Sean Keenan for his invaluable help in preparing the chapter. LITERATURE CITED Aldridge, D. W. 1982. 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External Morphology B. Organ-System Function C. Environmental and Comparative Physiology III. Ecology and Evolution A. Diversity and Distribution B. Reproduction and Life History C. Ecological Interactions D. Evolutionary Relationships IV. Collecting, Preparation for Identiﬁcation, and Rearing I. INTRODUCTION North American (NA) freshwater bivalve molluscs (class Bivalvia) fall in the subclasses Paleoheterodonta (Superfamily Unionoidea) and Heterodonta (Superfamilies Corbiculoidea and Dreissenoidea). They have enlarged gills with elongated, ciliated ﬁlaments for suspension feeding on plankton, algae, bacteria, and microdetritus. The mantle tissue underlying and secreting the shell forms a pair of lateral, dorsally connected lobes. Mantle and shell are both single entities. During development, the right and left mantle lobes extend ventrally from the dorsal visceral mass to enfold the body. Each lobe secrets a calcareous shell valve which remains connected by a mid-dorsal isthmus (Allen, 1985). Like all molluscs, the shell valves consist of outer proteinaceous and inner crystalline calcium carbonate elements (Wilbur and Saleuddin, 1983). The lateral mantle lobes secrete shell material marked by a high proportion of crystalline calcium carbonate making them thick, strong and inﬂexible, while the mantle isthmus secretes primarily protein, forming a dorsal elastic hinge ligEcology and Classiﬁcation of North American Freshwater Invertebrates. 2nd Edition Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved. A. Collecting B. Preparation for Identiﬁcation C. Rearing Freshwater Bivalves V. Identiﬁcation of the Freshwater Bivalves of North America A. Taxonomic Key to the Superfamilies of Freshwater Bivalvia B. Taxonomic Key to Genera of Freshwater Corbiculacea C. Taxonomic Key to the Genera of Freshwater Unionoidea Literature Cited ament uniting the calcareous valves (Fig. 1). The hinge ligament is external in all freshwater bivalves. Its elasticity opens the valves while the anterior and posterior shell adductor muscles (Fig. 2) run between the valves and close them in opposition to the hinge ligament which opens them on adductor muscle relaxation. The mantle lobes and shell completely enclose the bivalve body, resulting in cephalic sensory structures becoming vestigial or lost. Instead, external sensory structures are concentrated on the mantle margins where they are exposed to the external environment when the valves open. Compared to other molluscs, the bivalve body is laterally compressed and dorso-ventrally expanded, adapting them for burrowing in sediments, enclosure by shell valves and mantle protecting their soft tissues from abrasion and preventing ﬁne sediments from entering the mantle cavity where they could interfere with gill suspension feeding. These adaptations along with a highly protrusile, muscular, spadelike foot used for burrowing, have made bivalves the most successful infaunal suspension feeders in marine and freshwater habitats. 331 332 Robert F. McMahon and Arthur E. Bogan FIGURE 1 General morphological features of the shells of (A) corbiculoidean, (B) unionoidean, and (C) dreissenoidean freshwater bivalves. The NA bivalve fauna is the most diverse in the world, consisting of approximately 308 extent native and seven introduced taxa (Turgeon et al., 1998). The majority of species fall in the superfamily Unionoidea with 278 native NA and 13 recognized subspecies in 49 genera in the family, Unionidae and ﬁve species in two genera in the family, Margaritiferidae. Many species in this group have unique morphological adaptations and highly endemic, often endangered populations (Neves et al., 1997). In the superfamily, Corbiculoidea, the family Sphaeriidae, has 36 native and four introduced NA species. While falling into only four genera, the Sphaeriidae are more cosmopolitan than unionoideans (note “unionoideans” as used here refers to all species within the superfamily, Unionoidea while “unionid” used later in the chapter refers only to those unionoidean species in the family, Unionidae), several genera and species having pandemic distributions. Corbicula ﬂuminea falls within the Corbiculacea (fam- ily Corbiculidae). This species invaded NA freshwaters in the early 1900s and now extends throughout the coastal and southern United States and Mexico, becoming the dominant benthic species in many habitats (McMahon, 1983a,1999). More recently, two dreissenid species, Dreissena polymorpha (zebra mussel) and Dreissena bugensis (quagga mussel) have invaded North America. D. polymorpha was discovered in Lake St. Clair and Lake Erie in 1988 after introduction in 1986. It now extends throughout the Great Lakes, St. Lawrence River, and the Mississippi River and most of its major tributaries (Mackie and Schloesser, 1996). D. bugensis was ﬁrst found in the Erie-Barge Canal and eastern Lake Erie, NY, in 1991 and has since spread through Lakes Erie and Ontario and the St. Lawrence River (Mills et al., 1996). Both species are likely to have been simultaneously introduced as planktonic veliger larvae released with ballast water from ships entering the Great Lakes from ports on the Bug and 11. Mollusca: Bivalvia 333 FIGURE 2 General external anatomy of the soft tissues of (A) corbiculoidean, (B) unionoidean, and (C) dreissenoidean freshwater bivalves. Dnieper rivers in the Black Sea, Ukraine (Marsden et al., 1996, Mills et al., 1996). but their soft tissue morphologies are relatively similar and, thus, will be discussed in general terms below. A. External Morphology II. ANATOMY AND PHYSIOLOGY NA freshwater bivalves fall into three superfamilies: Unionoidea, Corbiculoidea, and Dreissenoidea. External shell morphology among these groups vary (Fig. 1), 1. Shell Bivalve shells consist of calcium carbonate (CaCO3) crystals embedded in a proteinaceous matrix, both secreted by underlying mantle tissue. In most 334 Robert F. McMahon and Arthur E. Bogan FIGURE 3 A crosssection through the mantle and shell edges of a typical freshwater unionoidean bivalve displaying the anatomic features of the shell, mantle, and mantle edge. Sphaeriids have a complexed cross-lamellar shell structure and lack nacre, but their mantle edge has a similar structure. bivalves, the shell consists of three parts: an outer proteinaceous periostracum secreted from the periostracal groove in the mantle edge, and underlying, calcareous prismatic and nacre layers in which CaCO3 crystals are embedded within an organic matrix (Fig. 3). The periostracum is initially secreted free of the other layers, but soon fuses with the underlying prismatic layer secreted by a portion of the mantle edge just external to the periostracal groove (Fig. 3). The prismatic layer consists of a single layer of elongated CaCO3 crystals oriented 90° to the horizontal shell plane (Fig. 3). The periostracum edge seals the extrapallial space between mantle and shell, allowing CaCO3 concentrations in that space to reach saturation levels required for crystal deposition (Saleuddin and Petit, 1983). The tanned protein of the periostracum is impermeable to water, preventing shell CaCO3 dissolution. Layers of conchiolin (i.e., tanned protein) occurring within unionoidean shells, are suggested to retard shell dissolution in calcium poor freshwaters and are not found in the Corbiculoidea or Dreissenoidea (Kat, 1985). The nacreous layer is continuously secreted by underlying mantle epithelium. It consists of consecutive layers of small CaCO3 crystals deposited parallel to the shell plane embedded in an organic matrix of chitinlike mucopolysaccharide and protein (Machado et al., 1994) (Fig. 3). Microstructure and texture patterns of CaCO3 crystals differ among major molluscan classes and interspeciﬁcally within groups (Hedegaard and Wenk, 1998). Continual secretion of the nacreous layer thick- ens and strengthens the shell, thus accounting for most of its mass. Calcium (Ca2) and bicarbonate (HCO3) necessary for shell CaCO3 crystal deposition are transported from the external medium across the body epithelium into the hemolymph (blood). HCO3 ions are also generated from metabolically released CO2 reacting with hemolymph water (CO2 H2O N H HCO3). These ions are actively transported across the mantle into the extrapallial ﬂuid for CaCO3 deposition into the shell matrix (Wilbur and Saleuddin, 1983). CaCO3 crystal formation requires release of protons (H) (Ca2 HCO3 N CaCO3 H) which must be removed from the extrapallial ﬂuid in order to maintain the high pH required for CaCO3 deposition (extrapallial ﬂuid pH 7.4 – 8.3). It is proposed that H combines with HCO3 to form H2CO3, followed by dissociation into CO2 and H2O which diffuse from the extrapallial ﬂuid into the hemolymph. The enzyme, carbonic anhydrase, which catalyzes this reaction, is present in mantle tissue (Wilbur and Saleuddin, 1983). Extrapallial ﬂuid pH is higher in freshwater than marine bivalves, favoring shell CaCO3 deposition at the lower Ca2 concentrations of the dilute hemolymph of freshwater species (Wilbur and Saleuddin, 1983). CaCO3 and shell matrix precipitate from the extrapallial ﬂuid. Ca2 and HCO3 are actively concentrated in the extrapallial ﬂuid, thus favoring CaCO3 deposition (Wilbur and Saleuddin, 1983). The organic shell matrix separates individual calcareous crystals 11. Mollusca: Bivalvia and binds them with the crystal layers into a uniﬁed structure. It also has crystal-nucleating sites (possibly composed of Ca2-binding polypeptides) on which CaCO3 crystals initially form. CaCO3 crystals grow on these sites to produce a new layer of nacre (Wilbur and Saleuddin, 1983). A minimum of one ATP appears to be required for deposition of two Ca2ions, with additional ATP required for active HCO3 transport (Wilbur and Saleuddin, 1983). Thus, fast-growing bivalves or those with massive shells must devote increased proportions of maintenance energy to shell mineral deposition. Deposition of shell organic matrix and periostracum also requires metabolic energy in the form of four ATP for every peptide bond. Although rarely more than 10% of shell dry mass, the shell organic matrix can account for 30 – 50% of the total dry organic matter (i.e., shell tissue organic matter), requiring up to one-third of the total energy allocated to organic growth (Wilbur and Saleuddin, 1983). Fast-growing, thin-shelled species may devote proportionately less assimilated energy to shell production than slower growing, thick-shelled species, allowing greater energy allocation to tissue growth and reproduction, thereby increasing ﬁtness. However, a thinner, more fragile shell increases probability of predation, lethal desiccation or damage and / or dislodgment from the substrate, reducing ﬁtness. Shell sculpture and shape appear highly correlated with habitat among unionoideans. Species adapted to soft substrates in low-ﬂow habitats have thinner shells, with smaller hinge teeth and greater lateral compression preventing sinking into the substrate while those adapted to faster ﬂowing habitats with harder substrates have, thicker, more inﬂated, more sculptured shells which anchor them more ﬁrmly in the substrate, preventing dislodgment during high water ﬂow (Watters, 1994a). Similarly, concentric sulcations on the shells of Corbicula ﬂuminea increase anchoring capacity in their lotic habitats (McMahon, 1999). Therefore, the ratio of energy allocation to shell versus tissue growth appears to be an adaptive strategy, energetic trade-offs between these two processes being evolved under species-speciﬁc niche selection pressures (see Section III.B). Shell external morphology varies among species. Externally, shells may have concentric or radial corrugations of varying sizes and densities, ridges, pigmented rays or blotches in the periostracum. These features, along with shape of the posterior shell ridge and overall shell shape are major taxonomic characters for distinguishing species (see Section V). Another major external shell feature is the umbo or beak, an anteriorly curving, dorsally projecting structure on each valve, which is the oldest portion of the shell (Fig. 1). 335 Major internal shell features include the hinge and projecting hinge teeth, which interlock to hold the valves in exact juxtaposition, forming the fulcrum on which they open and close, and various mussel scars. These can also be major taxonomic characters (see Section V). In corbiculids, massive conical cardinal teeth form just below the umbos (one in the right and two in the left valve), anterior and posterior of which lie lateral teeth, usually in the form of elongate lamellae (Fig. 1A). In contrast, unionoideans have no true cardinal teeth. Instead, massive, raised, pseudocardinal teeth develop near the umbonal end of the anterior lateral teeth, serving a function similar to cardinal teeth. Elongated, lamellar, posterior lateral teeth extend posteriorly from the umbos (Fig. 1A). Among the unionoideans, hinge teeth are vestigial or lost in the tribe Anodontini. The superfamily, Dreissenoidea, is characterized by a mussel-type shell without teeth in which the anterior end is greatly reduced and posterior end expanded. Thus, the umbos lie at the anterior end making the shell decidedly, anteriorly pointed (Fig. 1C). The nacre of freshwater bivalve shells may have species-speciﬁc colors. Internal-shell muscle scars mark the insertion points of anterior and posterior adductor muscles, anterior and posterior pedal (foot) retractor muscles, pedal protractor muscles and pallial line muscles which attach the mantle margin to the shell (Figs. 2 and 3). Posteriorly, the pallial line may be indented marking the pallial sinus into which inhalant and exhalant siphons are withdrawn during valve closure. The extent of the pallial sinus is directly correlated with size and length of the siphons. 2. Locomotory Structures and Burrowing With the exception of epibenthic dreissenoids which attach to hard substrates with proteinaceous byssal threads, the vast majority of NA freshwater bivalves burrow in sediments. Some species lie on a hard substrate, using the foot to wedge into crevices or under rocks. All species locomote with a muscular, motile, anterio-ventrally directed, protrusile foot (Fig. 2). Bivalve burrowing has been detailed by Trueman (1983) and described for the unionidean, Anodonta cygnea (Wu, 1987). The burrowing cycle begins with relaxation of the adductor muscles, allowing shell valves to open against the substratum by hinge ligament expansion, thus anchoring the shell in place. Contraction of transverse and circular muscles around pedal hemolymph sinuses (i.e., open blood spaces) causes the foot to narrow and lengthen, forcing it anteriorly into the substrate. Once extended, its distal tip is anchored either by expansion with hemolymph or lateral curving into the substrate. Shell adductor muscles then contract, rapidly closing the valves, releasing their hold on the 336 Robert F. McMahon and Arthur E. Bogan substrate. Rapid valve closure expels a jet of water from the mantle cavity, anteriorly through the pedal gape, loosening compacted sediments at the anterior shell margin. Thereafter, alternating contraction of anterior and posterior pedal retractor muscles simultaneously rock the anterior shell margin dorsoventrally and pull it forward into the loosened sediments against the anchored foot tip. Once a new position is achieved, shell adductor muscles relax, re-anchoring the open valves in the substrate, reinitiating the burrowing cycle. NA freshwater bivalves have relatively short siphons (the mantle edges are not fused to form true siphons in unionoideans and siphons are highly reduced or absent in some species of Pisidium), thus, they generally are found either just beneath the sediment surface or with the posterior shell margins just above it. Living near the sediment surface can lead to dislodgment, thus a number of riverine unionoidean taxa have shell and pedal morphologies adapted for rapid reburial (Watters, 1994a). Many juvenile freshwater bivalves crawl considerable distances over the substrate before settlement, holding the shell valves upright on an extended, dorsoventrally ﬂattened foot. Such surface locomotion also occurs among adult sphaeriids (Wu and Trueman, 1984) and in juvenile C. ﬂuminea (observed by the author). Crawling involves extension of the foot, anchoring its tip with mucus and / or a muscular attachment sucker, followed by pedal retractor muscle contraction which pulls the body forward. Pedal surface locomotion is reduced or lost in most adult unionoideans, but is retained to varying degrees in adult sphaeriids, C. ﬂuminea and dreissenids. Adult dreissenids may spontaneously release from the byssus and crawl long distances before re-attachment. Single byssal threads are produced during crawling to prevent dislodgment. Pedal locomotion is particularly common in juvenile and immature dreissenids and allows their escape from locally poor conditions or highly dense mussel clumps (Clarke and McMahon, 1996a). B. Organ-System Function 1. Circulation Bivalves have an ‘open’ circulatory system in which hemolymph is not always enclosed in vessels. Rather, circulatory ﬂuid is carried by vessels from the heart to various parts of the body where it passes into open, spongy hemocoels (i.e., blood sinuses). In the hemocoels, it bathes tissues directly, percolating through them before returning to the heart via the gills. Circulatory ﬂuid in open systems is called “hemolymph.” Bivalves have large hemolymph volumes, accounting for 49 – 55% of total body water (Jones, 1983). The bivalve heart ventricle uniquely surrounds the rectum and pumps oxygenated hemolymph from the gills and mantle via the kidney through anterior and posterior vessels (Jones, 1983, Narain and Singh, 1990) (Fig. 4) which subdivide into smaller vessels to various parts of the body, including pallial arteries to the mantle and visceral arteries to the visceral mass and foot. These secondary arteries further subdivide into many tiny vessels that open into the hemocoels where cellular exchange of nutrients, gases, and wastes occurs. Thereafter, deoxygenated hemolymph is carried from the body tissues and organs to the mantle and gills to be reoxygenated and, thence, to the heart. Evolution of an open circulatory system in molluscs, including bivalves, is associated with coelom reduction. The heart ventricle is surrounded by a coelomic remnant, the “pericardial cavity,” enclosed by the pericardial epithelium or “pericardium.” Other coelomic remnants include spaces comprising the kidneys (‘coelomoducts’) and gonads (Jones, 1983). Like most bivalves, the hemolymph of all freshwater species has no specialized respiratory pigments for O2 transport (Bonaventura and Bonaventura, 1983). Instead, O2 is transported dissolved directly in the hemolymph ﬂuid which has an O2-carrying capacity essentially equivalent to water. The very low metabolic demands and extensive gas exchange surfaces (mantle and gills) of bivalves allow maintenance of a primarily aerobic metabolism in spite of a reduced hemolymph O2-carrying capacity. As proteinaceous respiratory pigments are the primary blood pH buffer in most animals, bivalve blood acid – base balance is dependent on other mechanisms (see Section II.C.3). 2. Gills and Gas Exchange In lamellibranch bivalves, including all NA freshwater species, the gills are expanded beyond the requirements for gas exchange as they are also used for suspension (ﬁlter) feeding (Fig. 2), the main mode of food acquisition for the majority of species (see Section III.C.2). The left and right gills, or ctenidia, consist of an axis which extends anterolaterally along the visceral mass. Many long, thin, inner and outer ﬁlaments extend laterally from the axis. In all NA freshwater bivalves, ﬁlaments are fused together (an evolutionarily advanced condition) and penetrated by a series of pores or ostia (i.e., eulamellibranch condition). From the axis, the ﬁlaments ﬁrst extend ventrally (descending ﬁlament limbs) and then reﬂect dorsally (ascending ﬁlament limbs) to attach distally to the dorsal mantle wall (outer ﬁlaments) or the dorsal side of the visceral mass (inner ﬁlaments), forming two V-shaped, porous curtains called the outer and inner “demibranchs” (Fig. 5). 11. Mollusca: Bivalvia 337 FIGURE 4 General internal anatomy, organs, and organ systems of the soft tissues of (A) corbiculoidean, (B) unionoidean, and (C) dreissenoidean freshwater bivalves. The demibranchs completely separate the mantle cavity into ventral inhalant and dorsal exhalant portions. The descending and ascending ﬁlament limbs are held closely adjacent by tissue bridges called “interlamellar junctions” and enclose an area called a “water tube” or interlamellar space (Fig. 5). Feeding and respiratory currents are sustained by ‘lateral cilia’ on the adjacent external ﬁlament surfaces 338 Robert F. McMahon and Arthur E. Bogan FIGURE 5 The structural features of the gills (ctenidia) of freshwater bivalves. (A) Cross section through the central visceral mass, ctenidia, and mantle of a typical freshwater unionoidean, with high magniﬁcation view showing details of ﬁlaments, ostia, and ciliation. (B) Diagrammatic representation of the respiratory and feeding currents across the ctenidium. (C) Diagrammatic cross-sectional representation of lammellibranch bivalve ctenidia. (Fig. 5). They drive water entering the inhalant mantle cavity via the inhalant siphon or siphonal notch through the ostia between ﬁlaments into the water tubes. Water passes dorsally up the water tubes into the dorsal ‘exhalant mantle cavity,’ called the “suprabranchial cavity” or “epibranchial cavity,” formed above the ctenidial curtain where it ﬂows posteriorly, exiting via the exhalant siphon or siphonal notch (Figs. 2 and 5). While the lateral cilia drive water across the gill, rate of water ﬂow is partially controlled by size of the ostia. In unionoideans, two muscle bands control ostial pore size. Antero-posterior oriented muscle bands are attached to vertically oriented chitinous ﬁlament supporting rods which alternate with rows of ostia. Horizontal muscle contraction pulls adjacent supporting rods together to reduce ostial pore diameter, slowing water ﬂow. A second set of muscle bands run dorso- ventrally between rows of ostia and, when contracted, increase ostial diameter, increasing ﬂow rate. Ostial diameter of unionoidean gills increased 2 – 3 times with external application of the neurotransmitter, serotonin, and external serotonin and dopamine application increased lateral cilia activity, suggesting that gill water ﬂow is ultimately under nervous system control (Gardiner et al., 1991). Similar muscular control of ostial diameter occurs in Dreissena polymorpha where contraction of horizontally oriented muscle bands and spincterlike ostial muscles reduce ostial diameter, slowing water ﬂow. External application of serotonin relaxes these muscles increasing ostial diameter and gill water ﬂow (Medler and Silverman, 1997). 3. Excretion and Osmoregulation Freshwater bivalves, like all freshwater animals, have hemolymph and tissue osmotic concentrations 11. Mollusca: Bivalvia greater than the very dilute freshwater medium, resulting in constant ion loss and water gain. Osmoregulatory problems are compounded by the extensive gill and mantle surfaces of bivalves over which ion and water ﬂuxes occur (Dietz, 1985). To reduce these ﬂuxes, freshwater bivalves have evolved the lowest hemolymph and cell osmotic concentrations of any metazoan, being 20 – 50% of that found in most other freshwater species (Dietz, 1985). In spite of their reduced osmotic concentration, the extensive epithelial surfaces of bivalves still cause water and ion ﬂuxes to be greater than those recorded in other freshwater species, leading to urine clearance rates of 20 – 50 ml/k per hour (Dietz, 1985). In unionoideans, Na is taken up in exchange for outward transport of the metabolic waste cations, H and NH4, and, perhaps, Ca2. Chloride (Cl) ion uptake is in exchange for metabolically produced HCO3 or OH. Active Ca2 uptake also occurs (Burton, 1983). In freshwater snails, the major source of Ca2 is ingested food (McMahon, 1983b), while the relative roles of food and transport in the Ca2 balance of freshwater bivalves are unknown. Sodium ion (Na) uptake does not require presence of Cl which is indicative of separate transport systems for these ions (Burton, 1983); however, freshwater dreissenids appear to co-transport Na and Cl (Horohov et al., 1992). While active transport is the major route by which unionoideans gain ions, exchange diffusion (i.e., transport of an ion linked with diffusion of a second ion species down its concentration gradient) accounts for 67% of Na uptake in C. ﬂuminea. C. ﬂuminea and D. polymorpha have higher ion-transport rates than unionoideans and sphaeriids, reﬂecting their geologically recent penetration of freshwaters (Dietz, 1985, Horohov et al., 1992, Wilcox and Dietz, 1995). C. ﬂuminea has a hemolymph solute concentration that is higher (Dietz, 1985) and D. polymorpha, that is lower than most other freshwater bivalves (Dietz et al., 1996a). The low hemolymph ion-concentration of D. polymorpha occurs in spite of its high rates of active ion uptake, indicating that it is much more ion-permeable than other freshwater bivalves, perhaps due to its recent evolutionary invasion of freshwaters (Dietz et al., 1994, 1995, 1996a, b). In contrast, epithelial ion permeability in C. ﬂuminea is low (Zheng and Dietz, 1998a) and active ion uptake rate high (Zheng and Dietz, 1998b), allowing maintenance of higher hemolymph ion concentrations and tolerance greater ambient osmolarity variation than other freshwater bivalves (Zheng and Dietz, 1998b). Interestingly, exchange diffusion may account for up to 90% of Cl turnover in unionoideans in pond water, but when saltdepleted, active transport dominates Cluptake (Dietz, 1985). Na and Cl uptake can occur over the body 339 epithelia in unionoideans, but the majority occurs over the gills (Dietz, 1985), with the epithelial cells of water canals connecting gill ostia to water tubes being a major site for active ion and osmotic regulation (Kays et al., 1990). Active ion uptake and hemolymph-ion concentrations in unionoideans and, by inference, other freshwater bivalves, may be regulated by neurotransmitter substances released by gill neurons (Dietz et al., 1992). Excess water is eliminated via coelomoducts or kidneys. The heart auricle walls initially ultraﬁlter the blood. Hydrostatic pressure generated by auricle contraction forces blood ﬂuid, ions and small organic molecules through the auricle walls into the pericardial cavity surrounding the heart. Filtration occurs through podocyte cells of the pericardial gland lining the inner auricular surface and, perhaps, through the efferent branchial vein running from the longitudinal kidney vein to the auricles (Martin, 1983) (Fig. 6). Only larger hemolymph protein, lipid and carbohydrate molecules cannot pass the pericardial gland ﬁlter. The ﬁltrate exits the pericardial cavity via left and right “renopericardial openings” in the pericardial wall to enter the “reopericardial canals” leading to the left and right kidneys. Larger organic waste molecules are actively transported into the ﬁltrate by the kidney. In the Unionoidea, the rectal wall is extremely thin where surrounded by the ventricle, suggesting that larger organic waste molecules may be transported through it directly into the rectum in this region (Narain and Singh, 1990). Competed excretory ﬂuid passes via “nephridiopores” into the epibranchial cavity to be carried on exhalant water ﬂow out the exhalant siphon (Figs. 2 and 4A) (Martin, 1983). While little studied in freshwater bivalves, the kidney is the presumed site of major active ion resorption from the ﬁltrate into the hemolymph. Dietz and Byrne (1999) have demonstrated reabsorption of ﬁltrate sulfate ion (SO42) in the kidney of D. polymorpha. As kidney walls appear relatively impermeable to water, active ﬁltrate-ion resorption leads to formation of a dilute excretory ﬂuid facilitating excess water excretion with ﬁltrate osmolarity being 50% that of hemolymph in the unionid, Anodonta cygnea (Martin, 1983). Filtrate-ion reabsorption is less energetically expensive than active-ion uptake from the freshwater medium because ion-concentration gradients between coelomoduct ﬂuid and hemolymph are far less than those between hemolymph and freshwater. Due to rapid water inﬂux, an elevated excretory ﬂuid production is required in freshwater bivalves to maintain osmotic balance, being approximately 0.03 mL/g wet tissue per day in A. cygnea (Martin, 1983). In D. polymorpha excretory ﬂuid production was 2 – 3 mL / g dry 340 Robert F. McMahon and Arthur E. Bogan FIGURE 6 Cross-sectional representation of the anatomic features of the excretory system of a typical freshwater bivalve. Arrows indicate pathways for the excretion of excess water in the hemolymph through the excretory system to be eliminated at the excretory pore (redrawn from Martin, 1983). tissue per day (Dietz and Byrne, 1999) which, when converted to a wet tissue weight value of 0.1 – 0.15 mL / g dry tissue weight per day (based on dry tissue weight being 0.05 wet tissue weight) is 3.3 – 5.0 times greater than that of A. cygnea (Martin, 1983), reﬂecting the high epithelial osmotic permeability of D. polymorpha compared to other freshwater bivalves (Dietz et al., 1996a, b). In spite of renal ion absorption, the excretory ﬂuid of freshwater bivalves has a considerably higher ion concentration than freshwater, making excretion a major avenue of ion loss. Ions lost by excretion and epithelial diffusion are recovered by active transport from the medium across epithelial surfaces, particularly that of the gills (Dietz, 1985, also see Section II.C.6). The main nitrogenous waste product of freshwater bivalves is ammonia (NH3) diffused across external epithelia as dissolved NH3 or ammonium ion (NH4). NH4 can also be actively transported to the external medium. In marine bivalves, 65 – 72% of nitrogen excretion is as ammonia / ammonium ion and 13 – 28% as urea (Florkin and Bricteux-Gregoire, 1972), values likely to be similar in freshwater species. Some freshwater unionoideans appear to be able to detoxify ammonia / ammonium ions by conversion to urea and, perhaps, amino acids and proteins (Mani et al., 1993). 4. Digestion and Assimilation Freshwater bivalves are suspension (ﬁlter) feeders, ﬁltering algae, bacteria and suspended microdetrital particles from water ﬂowing through the gill. Filtered material is transported by gill ciliary tracks to the “labial palps” where it is sorted on corrugated ciliated surfaces into food and nonfood particles before food particles are carried by cilia to the mouth. Some freshwater species may also utilize the foot to feed on sediment organic detrital particles (Reid et al., 1992). Filter and pedal detritus feeding are described in Section III.C.2, while this section is devoted to food digestion and assimilation. 11. Mollusca: Bivalvia The bivalve mouth is a simple opening ﬂanked laterally by left and right pairs of labial palps, whose ciliary tracks deliver ﬁltered food from the gills to the mouth as a constant stream of ﬁne particles. After ingestion, food particles pass through a short, ciliated esophagus where mucus secretion and ciliary action bind them into a mucus string before entering the stomach. The stomach, lying in the anterio-dorsal portion of the visceral mass (Fig. 4), is a complex structure with ciliated sorting surfaces and openings to a number of digestive organs and structures. On its ventral ﬂoor, posterior to the opening of the midgut, is an elongated, evaginated, blind-ending tube, the “style sac.” The distal end of the style sac secretes the “crystalline style,” a long mucopolysaccharide rod that projects from the style sac into the stomach. Style-sac cells secrete digestive enzymes into the style matrix and have cilia which slowly rotate the style. Stomach and style pH ranges from 6.0 – 6.9, acidity depending on phase of digestion (Morton, 1983). The rotating style tip projects against a chitinous plate or “gastric shield” on the dorsal roof of the stomach. The gastric shield is penetrated by microvilli from underlying epithelial cells, believed to release digestive enzymes onto the shield surface (Morton, 1983). Style rotation mixes stomach contents and winds the esophageal mucus food string on to the style tip where slow release of its embedded enzymes begins extracellular digestion. Abrasion of the style tip against the gastric shield, triturates food particles and allows their further digestion by enzymes released from the eroding style matrix and from digestive epithelial cells underlying the shield. After initial trituration and digestion at the gastric shield, food particles released into the stomach may have several fates. Particles are size-sorted. A ciliated ridge, the “typhlosole,” running the length of the midgut returns large particles to the stomach for further digestion and trituration and eventually selects and passes indigestible particles to the hindgut / rectum for egestion. Ciliated surfaces on the posterior “sorting caecum” of the stomach direct sufﬁciently small food particles into tubules of the “digestive diverticulum” for ﬁnal intracellular digestion (Fig. 4). The diverticula have the lowest ﬂuid pH of the gut (Morton, 1983). Larger food particles falling on the caecum are recycled into the stomach for further trituration and enzymatic digestion, thus particles may pass over its ciliated sorting surfaces several times before acceptance for intracellular digestion or rejection into the rectum to form feces. Digestive cells lining the lumina of the tiny, blindending, terminal tubules of the digestive diverticula take up ﬁne food particles by endocytosis into food 341 vacuoles for ﬁnal digestion and assimilation. After completion of intracellular digestion / assimilation, the apical portions of the digestive cells, which contain food vacuoles with undigested wastes and digestive enzymes, are shed into the tubule lumina as “fragmentation spherules” which are returned to the stomach on ciliated rejection pathways. Breakdown of fragmentation spherules in the stomach releases their food vacuole contents, hypothesized to be a major source of stomach acidity and extracellular digestive enzymes (Morton, 1983). Rejected indigestible matter passes through the short hindgut into the rectum for egestion from the anus, opening into the epibranchial cavity on the posterior face of the posterior shell adductor muscle just upstream from the exhalant siphon or opening, allowing feces expulsion on the exhalant current. Undigested particles are consolidated into discrete, dense, fecal pellets by mucus secreted by the hindgut / rectum, preventing their uptake on the inhalant current. The cerebropleural and visceral ganglia release neurohormones which inﬂuence glycogenesis (Joosse and Geraerts, 1983). The vertebrate glycogenic hormones, insulin and adrenalin, have similar effects on unionoideans. Insulin injected into the Indian unionid, Lamellidens corrianus, resulted in declining hemolymph glucose concentration and increase in pedal and digestive diverticular glycogen stores, while adrenaline injection induced glycogen store breakdown and increased hemolymph sugar concentration (Jadhav and Lomte, 1982b). Elevated hemolymph glucose concentrations stimulates gut epithelial cells to produce an insulin-like substance in the unionoideans, Unio pictorum and A. cygnea, which stimulated activity of glucose synthetase, increasing uptake of hemolymph glucose into glycogen stores (Joose and Geraerts, 1983). Cerebropleural ganglionic neurosecretory hormones regulate pedal and digestive diverticular accumulation and release of protein and nonprotein stores in L. corrianus (Jadhav and Lomte, 1983). 5. Reproductive Structures The paired gonads of freshwater bivalves lie close to the digestive diverticula. Among unionoideans, their paired condition is difﬁcult to discern. Unionoidean gonads envelop the lower portions of the intestinal tract and sometimes extend into the proximal portions of the foot (Fig. 4B), while those of sphaeriids, C. ﬂuminea, and freshwater dreissenids lie more dorsally in the visceral mass, extending on either side of the stomach, intestine and digestive diverticula (Fig. 4A, and C; Mackie, 1984; Claudi and Mackie, 1993). A short gonoduct leading from each gonad opens into the epibranchial cavity allowing gamete release on exhalant 342 Robert F. McMahon and Arthur E. Bogan FIGURE 7 Schematic representations of typical reproductive systems of bivalves. (A) The primitive condition in some marine bivalve species: male or female gametes are released from the gonoduct opening into the kidney and passed externally through the excretory pore (shown here is a primitive marine hermaphroditic bivalve; the anatomy is essentially similar in gonochoristic species). (B) Hermaphroditic freshwater corbiculoidean bivalves (Corbicula ﬂuminea and sphaeriids): male and female gametes are passed through the gonoducts into the kidney duct close to the excretory pore from which gametes are shed. (C) Gonochoristic freshwater unionoideans: gametes pass to the outside through a gonoduct and gonopore totally separate from the kidney duct and excretory pore. (After Mackie, 1984.) currents (Fig. 4). Among unionoideans, which are generally gonochoristic except for a few hermaphroditic species of Anodontinae and Ambleminae (Hoeh et al., 1995), the tracts and openings of the renal and reproductive systems are entirely separate, an advanced condition (Fig. 7C). In sphaeriids and C. ﬂuminea, which are hermaphroditic, the gonads have distinct regions in which either male or female acini produce sperm or eggs. In these latter groups and the gonochoristic freshwater dreissenids, ducts carrying eggs or sperm unite into a single gonoduct carrying gametes from each gonad into the distal end of the kidney — allowing gamete discharge into the epibranchial cavity via the nephridial canal and nephridiopore (Fig. 7A) — or directly into the nephridial canal, discharging through a common pore on a papilla in the epibranchial cavity (Fig. 7B; Mackie, 1984; Kraemer et al., 1986). Almost all NA freshwater bivalves are ovoviviparous, brooding developing embryos in specialized gill marsupia. Only the dreisssenids release sperm and eggs externally, leading to development of free swimming larvae (Nichols, 1996). In brooding species, interlamellar spaces are modiﬁed to form gill marsupia (brood chambers). Fully formed juveniles are released from the marsupia via the exhalant siphon in sphaeriids and C. ﬂuminea. In unionoideans, bivalved “glochidia larvae” are released from either the exhalant siphon, specialized gill pores, or ruptures in the ventral margin of the marsupial gills. Unionoidean glochidia parasitize ﬁsh hosts before metamorphosing into free — living juveniles. Interlamellar spaces of the outer demibranch form marsupia in unionoideans except in the subfam- ily, Ambleminae, which form marsupia in both demibranchs (Burch, 1975b). In contrast, marsupia form in the inner demibranchs of sphaeriids and C. ﬂuminea. Among species of the unionid genus, Lampsilis, marsupia develop only in the posterior portions of the outer demibranch from which glochidia are released through small pores directly into the inhalant siphon (for further details, see Section III.B.1). In C. ﬂuminea, interlamellar marsupial brooding spaces have no specialized structures. In sphaeriids, embryos are enclosed in specialized brood chambers evaginated from gill ﬁlaments into the interlamellar space. Among anodontid unionids, the marsupial interlamellar space is divided by septa into separate chambers associated with each ﬁlament. Each space is further subdivided by lateral septa into a central marsupium and inner and outer water tubes transport water from the gill ostia to the epibranchial cavity, an advanced structure not found in other unionoidean groups. In the primitive unionoidean families, Margartiferidae and Ambleminae, the entire outer demibranch forms the marsupium, while among the more advanced tribes, Pleurobemini and Lampsilini, the marsupia are limited to speciﬁc portion of the outer demibranch (Mackie, 1984). Some unionoidean species display sexual dimorphism, generally rare in other freshwater bivalves. In sexually dimorphic species, glochidial incubation distends the female outer marsupial demibranch (Mackie, 1984). Thus, posterior portions of the valves of female lampsilines are inﬂated compared to males, accommodating the expanded posterior marsupia. Less obvious general inﬂation of the female shell occurs in the anodontids (Mackie, 1984). Monoecious unionoideans (some species Anodontinae and Ambleminae) and all sphaeriids are generally simultaneous hermaphrodites, concurrently producing mature eggs and sperm. C. ﬂuminea has an unusual pattern of producing only eggs early after maturity (shell length ⬇6 mm) followed later by sperm production, thereafter, remaining simultaneously hermaphroditic throughout life (Kraemer and Galloway, 1986). Bivalves lack copulatory organs. Except for dreissenids which release both sperm and eggs externally (Nichols, 1996), freshwater bivalves release only sperm externally which is transported on inhalant currents of other individuals to unfertilized eggs in the gill marsupia. Among corbiculids, self-fertilization appears common in sphaeriids, occurring near the conjunction of male and female gonoducts (Mackie, 1984), while, in C. ﬂuminea, embryos frequently occur in the lumina of gametogenic follicles and gonoducts, indicative of self fertilization within the gonad (Kraemer et al., 1986). Self-fertilization makes hermaphroditic species highly invasive (see Section III.B). 11. Mollusca: Bivalvia Temperature appears to be the main reproductive stimulus in most freshwater bivalves. Gametogenesis and fertilization are initiated above and maintained within critical ambient water temperature thresholds. Other environmental factors which may influence reproduction include: neurosecretory hormones, density dependent factors, diurnal rhythms, food availability and parasites. Evidence for neurosecretory and density controls has been demonstrated in sphaeriids (Mackie, 1984). Zebra mussels spawn in response to the neurosecretory hormone, serotonin, and presence of algal extracts (Ram et al., 1996). Evidence of increasing activity and metabolic rate during dark hours in unionoideans (McCorkle et al., 1979; Englund and Heino, 1994a), C. fluminea (McCorkleShiley, 1982) and D. polymorpha (Borcherding, 1992) suggests that spawning activity and gamete / glochidial / juvenile release rates may also display diurnal rhythmicity. Sperm of freshwater bivalves may have ellipsoid or conical nuclei and acrosomes of variable complexity (Mackie, 1984). Unionoidean sperm have rounded or short cylindrical heads considered a primitive condition (Rocha and Azevedo, 1990; Lynn, 1994) while that of Dreissena and Corbicula have elongate heads (Kraemer et al., 1986; Ram et al., 1996). The sperm of C. ﬂuminea is uniquely biﬂagellate (Kraemer et al., 1986). Sperm with elongate heads may be adapted for swimming in gonadal and oviductal ﬂuids more viscous than water and are associated with internal fertilization in gonadal ducts rather in marsupia (Mackie, 1984). However, the elongate-headed sperm of D. polymorpha is an exception to this rule, because it externally fertilizes the egg. D. polymorpha has sperm with a straight head and an oval, bulbous acrosome while D. bugensis sperm has a curved head with a conically shaped acrosome (Denson and Wang 1994). There appears to be speciﬁc site for sperm recognition and entry on the vegetal pole of unionoidean eggs (Truncilla truncata) where the vitelline coat forms a corrugated surface surrounding a truncated cone (Focarelli et al., 1990). The eggs of freshwater bivalves are round and have greater yolk volumes than marine species with planktonic larvae. Only freshwater dreissenids have relatively small eggs (40 – 70 m) associated with their external fertilization and small, free-swimming veliger larva, which grows considerably in the plankton before settlement and metamorphoses to a juvenile (219 – 365 m) (Nichols, 1996). The larger, yolky eggs of all other species contain nourishment supporting development to a more advanced juvenile / glochidium stage. The Sphaeriidae, the smallest adult freshwater bivalves, produce the largest eggs, resulting in very small brood sizes 343 ranging from 6 – 24 per adult in the genus Sphaerium, 1 – 135 per adult in the genus Musculium, and 3.3 – 6.7 per adult in the genus Pisidium (Burky, 1983). Adult Musculium partumeium, only 4.0 mm in shell length (SL), release juveniles of 1.4 mm SL (Hornbach et al., 1980; Way et al., 1980). In contrast, unionoideans and C. ﬂuminea have smaller eggs and release smaller glochidia / juveniles (generally 0.3 mm SL) and have much larger brood sizes (103 – 106 per adult) (Burky, 1983; McMahon, 1999). The small eggs of zebra mussels allow them to have massive fecundities of 106 per adult female (Nichols, 1996) required for successful external fertilization and planktonic larval development. Evolutionary implications of bivalve fecundities are discussed in Section III.B. The bivalve egg is surrounded by a vitelline membrane that is relatively thin in sphaeriids and thicker in unionoideans and C. ﬂuminea (Mackie, 1984). In unionoideans, it remains intact throughout most of embryonic development. It disintegrates during early development in sphaeriids (Heard, 1977), allowing developing embryos to absorb nutrients from brood sacs without embryos and / or from nutrient cells lining the interlamellar spaces of marsupial gills. In dreissenids, the vitelline membrane is lost early to release the freeswimming trochophore larvae which metamorphoses into a planktonic veliger (Nichols, 1996). The vitelline membrane is also lost early to release a free-swimming trochophore retained through development of a juvenile clam in the marsupial gills of C. ﬂuminea (Kraemer and Galloway, 1986). 6. Nervous System and Sense Organs The bivalve head, entirely enclosed within the mantle and shell valves, is not in direct contact with the external environment. Thus, cephalic structures including sense organs have been lost, the head having only a mouth and associated labial palps (Fig. 2). Loss of cephalic sense organs has lead to bivalve nervous system being far less centralized than other advanced molluscan species. A pair of cerebropleural ganglia lateral to the esophagus near the mouth, are interconnected by dorsal, superesophageal commissures (Fig. 8). From these extend two pairs of nerve chords. Paired dorsal nerve chords extend posteriorly through the visceral mass to a pair of visceral ganglia on the anterio-ventral surface of the posterior shell adductor muscle while paired cerebro-pedal nerve chords innervate a pair of pedal ganglia in the foot (Ruppert and Barnes, 1994). The pedal and cerebropedal ganglia exert motor control over the pedal and anterior shell adductor muscles, while motor control of the siphons and posterior shell adductor muscle is affected by the visceral ganglia. Coordination of pedal and valve movements 344 Robert F. McMahon and Arthur E. Bogan FIGURE 8 The anatomic features of the central nervous system of a typical unionoidean freshwater bivalve (central nervous system anatomy is essentially similar in freshwater corbiculoidean and dreissenoidean bivalves). during burrowing and locomotion (see Section II.A.2) resides in the cerebropedal ganglia. Sense organs are concentrated on the mantle edge and in siphonal tissues most directly exposed to the external environment. Mantle edge sense organs are most concentrated on the middle sensory mantle lobe (Fig. 3). Photoreceptor cells detect changes in light intensity associated with shadow reﬂexes, phototaxis and diurnal rhythms; while tentacles and stiffened, immobile stereocilia are associated with tactile mechanoreceptor organs perceiving direct contact displacement (touch) or vibrations. On the siphon margins, such tactile receptors prevent drawing of large particles into the mantle cavity. When these mechanoreceptors are impinged by large particles, the siphons are closed by sphincter muscles and / or rapidly withdraw to prevent particle entry. Stronger mechanical stimuli to the siphons cause the valves to be rapidly adducted, forcing water from the siphons, ejecting any impinging material. Under intense mantle or siphon stimulation, siphons are withdrawn and the valves tightly closed, a common predator defense behavior in all bivalve species. A pair of statocysts lying near or within the pedal ganglia are enervated by commissures from the cerebropedal ganglion (Fig. 8). Statocysts are greatly reduced in sessile marine species (i.e., cemented oysters), suggesting their importance to locomotion and burrowing in free-living freshwater species. They are lined with ciliary mechanoreceptors responding to pressure exerted by a calcareous statolith or series of smaller, granular statoconia held within the statocyst vesicle (Kraemer, 1978). As gravity sensing organs, statocysts detect body orientation and, thus function in geotactic and positioning responses during burrowing and pedal locomotory behavior. Bivalves have a pair of organs called osphradia on the dorsal wall of epibranchial cavity, underlying the visceral ganglion in unionoideans and C. ﬂuminea (Kraemer, 1981). Because they are profusely enervated, and neuronally connected to the visceral ganglion, shell adductor muscles and kidney, they have been considered sense organs, but their sensory function(s) are debated. In gastropods, where osphradia are located on the incurrent side of the ctenidium, they have been considered to be mechanoreceptors, sensing suspended particles in inhalant water ﬂow, or chemoreceptors. However, in bivalves, the epibranchial (exhalant) mantle cavity position of osphradia where they receive only ﬁltered water and their lack of extensive neuronal gill connections appear to preclude either a mechano- or chemoreceptor function. Rather, bivalve osphradia are hypothesized to be light sensing organs that control seasonal activities such as spawning, or which provide sensory input to kidney function, and / or diurnal patterns of shell valve adduction (Kraemer, 1981). C. Environmental and Comparative Physiology 1. Seasonal Cycles Freshwater bivalves display seasonal physiological responses associated with temperature and reproductive cycles. Metabolic rates vary seasonally in freshwater bivalves (for reviews see Burky, 1983; Hornbach, 1985; McMahon, 1996, 1999) with rates being generally greatest in summer and least in winter due to 11. Mollusca: Bivalvia 345 TABLE I Seasonal Variation in the Oxygen Consumption Rates (V̇O2) of Selected Species of Freshwater Bivalves Species and source Sphaerium striatinum Hornbach et al. (1983) Pisidium compressum Way and Wissing (1984) Pisidium variabile Way and Wissing (1984) Pisidium walkeri Burky and Burky (1976) Corbicula ﬂuminea Williams (1985) Dreissena polymorpha Quigley et al. (1992) Pyganodon grandis Huebner (1982) Lampsilis radiata Huebner (1982) a mg dry tissue weight Maximum V̇O2 l O2/h Minimum V̇O2 l O2/h 25 mg 7 mg 2 mg 3 mg 0.9 mg 0.05 mg 3 mg 0.9 mg 0.05 mg 1.3 mg 0.02 mg 348 mg 204 mg 60 mg — 31.5 15.8 8.7 0.71 0.27 0.13 1.13 0.42 0.15 1.11 0.017 430.6 308.2 143.3 7.2 1.40 0.78 0.24 0.04 0.03 0.02 0.22 0.10 0.02 0.85 0.0074 34.4 31.5 25.8 2.1 10 g 5g 5g 2g 4690 2690 2090 810 400 250 170 80 Ratio min:max V̇O2 Q10(Acc.)a Seasonal temperature range (°C) 22.5:1 20.3:1 33.6:1 17.5:1 9.0:1 6.5:1 5.14:1 4.20:1 7.5:1 1.3:1 2.3:1 12.5:1 9.8:1 5.6:1 3.6:1 4.7 4.5 5.8 — — — — — — 1.1 1.4 3.2 2.8 2.2 2.3 2 – 22 2 – 22 2 – 22 — — — — — — 1 – 26 1 – 26 7 – 29 7 – 29 7 – 29 6 – 21 11.7:1 10.8:1 12.3:1 10.1:1 2.7 2.6 2.8 2.6 6 – 31 6 – 31 6 – 31 6 – 31 Q10(Acc.) is the respiratory Q10 value computed from a change in the V̇O2 value recorded for individuals ﬁeld acclimated to their respective seasonal maximum and minimum temperatures. temperature effects (Burky, 1983; Hornbach, 1985). Annual variation in metabolic rate can be extensive (Table I). Maximal summer O2 consumption rates (V̇O2) may be 20 – 33 times minimal winter rates over a seasonal range of 2 – 22°C in Sphaerium stratinum (Hornbach et al., 1983) or as little as 1.3 times minimal winter rates in Pisidium walkeri (1 – 26°C) (Burky and Burky, 1976) (Table I). Immediate temperature increases induce a corresponding metabolic rate increase in ectothermic animals such as bivalves. Acute, temperature-induced variations in metabolic rate or V̇O2 (or in any rate function) are represented by Q10 values, the factor by which a rate function changes with a 10°C temperature increase, computed over any temperature range as: Q10 冢 Rate Rate 冣 2 10/[T2 T1] 1 where Rate1 is the rate at lower temperature, Rate2 the rate at higher temperature, T1 the lower temperature (°C), and T2 the higher temperature (°C). Q10 for metabolic rate in the majority of ectotherms is 2 – 3, essentially that of chemical reactions. Thus, Q10 values outside this range indicate active metabolic regulation, values 1.5 suggesting active metabolic suppression and, 3.5, active metabolic stimulation with temperature change. Freshwater bivalve Q10 values are highly variable within and among species, ranging from 0.2 to 14.8 among 20 species of sphaeriids (Hornbach, 1985) and from 1.2 to 3.72 among three species of sphaeriids (Alimov, 1975). Q10 ranged from 1.5 to 4.1 for D. polymorpha over 10 – 30°C depending on prior acclimation temperature and temperature range of determination (Alimov, 1975; McMahon, 1996). For 10°C acclimated individuals of C. ﬂuminea, respiratory Q10 was 2.1 over 5 – 30°C (McMahon, 1979a). Among unionoideans, in Lampsilis siliquoidea, Q10 ranged from 1.88 to 4.98 and in Pyganodon grandis, from 1.27 to 10.35 (Huebner, 1982). While numerous environmental and physiological factors may affect the Q10 values of freshwater bivalves, no general patterns emerge. Rather, metabolic response to temperature appears to have evolved under species-speciﬁc microhabitat selection pressures (Hornbach, 1985). Temperature effects could lead to massive seasonal metabolic ﬂuctuations; rates being suboptimal during colder months and supraoptimal during warmer months. Thus, many ectothermic species display metabolic temperature acclimation, involving compensatory adjustment of metabolic rate to a new temperature regime over periods of a few days to several weeks. Typically, metabolic rates are adjusted upward upon acclimation to colder temperatures and downward upon acclimation to warmer temperatures which 346 Robert F. McMahon and Arthur E. Bogan dampens metabolic ﬂuctuation with seasonal temperature change, allowing year-round maintenance of near optimal metabolic rates. For most species, such “typical” seasonal metabolic acclimation is partial, with metabolic rates not returning to an absolute optimal level in a new temperature regime. The degree of metabolic temperature compensation can be detected by comparing Q10 values of V̇O2 in instantaneous response to acute temperature change with those measured after acclimation [Acclimation Q10 or Q10(Acc.)]. If Q10(Acc.) approximates 1.0, metabolic temperature compensation is nearly perfect with V̇O2 regulated near an optimal level throughout the year. If Q10(Acc.) is less than 2.0 or considerably less than acute Q10, acclimation is partial, with the metabolic rate approaching, but not reaching, the optimal level. If Q10(Acc.) is 2 – 3 or equivalent to the acute Q10, the species is incapable of temperature acclimation. If Q10(Acc.) is greater than 3.0 or acute Q10, inverse (reverse) acclimation is displayed in which acclimation to a colder temperature further depresses metabolic rate, and further stimulates it on acclimation to a warmer temperature. Three of the above acclimation patterns occur in freshwater bivalves: (1) no capacity for acclimation (i.e., Q10(Acc.) equivalent to acute Q10) is displayed by the unionids P. grandis and L. radiata (Huebner, 1982) and D. polymorpha (Quigley et al., 1992; McMahon, 1996); (2) partial acclimation, by Pisidium walkeri where Q10(Acc.) is considerably less than acute Q10 (Burky and Burky, 1976); and (3) reverse acclimation (i.e., Q10(Acc.) is greater than acute Q10) displayed by Sphaerium striatinum (Hornbach et al., 1983) and C. ﬂuminea (Williams and McMahon, unpublished data) (Table I). The adaptive advantage of reverse acclimation has been questioned because it results in massive seasonal swings in metabolic rates, but among freshwater bivalves it may reduce utilization of energy stores during nonfeeding, overwintering periods (Burky, 1983). V̇O2 is also related to individual size or biomass in all animals as follows: V̇O2 aMb where M is the individual biomass, and a and b are constants. It can be rewritten as a linear regression in which V̇O2 and M are transformed into logarithmic values: Log10 V̇O2 a b(Log10 M) where a and b are the Y-intercept (i.e., V̇O2 at Log10 M 0 or M 1) and slope (i.e., increase in Log10 V̇O2 per unit increase in Log10 M), respectively. Thus, a measures the relative magnitude of V̇O2 and b, the rate of increase in V̇O2 with increasing biomass. If b 1, V̇O2 increases in direct proportion to M. If b is 1, V̇O2 increases at a proportionately greater rate than M, and if b is 1, V̇O2 increases at a proportionately slower rate than M. Thus, b values 1 indicate that weight-speciﬁc V̇O2 (i.e., V̇O2 per unit body mass) decreases with increasing body mass while b 1 indicates that it increases with increasing mass. Conventional wisdom suggests that b values range from 0.5 to 0.8 such that weight speciﬁc V̇O2 decreases with increasing body mass. While generally true for vertebrates, it is less characteristic of invertebrates, and particularly of molluscs, including freshwater bivalves. Among 14 species of sphaeriids, b ranged from 0.12 to 1.45 (Hornbach, 1985). Limited data suggest that unionoideans have more typical b values, being 0.9 for L. radiata, and 0.77 for P. grandis (Huebner, 1982), with a b value range of 0.71 – 0.75 being reported for a number of unionoideans (Alimov, 1975). A b value of 0.63 is reported for D. polymorpha (Alimov, 1975). For C. ﬂuminea, b ranged from 1.64 – 1.66 depending on season, temperature and individual condition (Williams and McMahon, unpublished data). Alimov (1975) estimated an average b of 0.73 for this species. The b value changes with season in some species (Hornbach et al., 1983; Way and Wissing, 1984), but remains constant in others (Burky and Burky, 1976; Huebner, 1982) and can also vary with reproductive condition, reported to increase in some brooding adult sphaeriids (Way and Wissing, 1982) but not in others (Burky, 1983; Hornbach, 1985). Metabolic rate may also vary with physiologic state, increasing in starved individuals of C. ﬂuminea (Williams and McMahon, unpublished data) and declining in Musculium partumieum during estivation or habitat drying (Way et al., 1981). Comparison of a values among species of Pisidium indicated that weight-speciﬁc V̇O2 in this group (mean a 0.399) is about 1 / 3 that of species of Musculium (mean a 1.605) or Sphaerium (mean a 1.439) (Hornbach, 1985). Reduced metabolic rate in pisidiids may reﬂect their reduced relative gill surface areas (Hornbach, 1985) and hypoxic interstitial suspension feeding habitats (Lopez and Holopainen, 1987), reduced metabolic demand of profundal pisidiids perhaps accounting for their high tolerance of hypoxia (Burky, 1983; Holopainen, 1987). Annually, a in C. ﬂuminea varied from 0.12 to 1.43 (mean 0.72) (Williams and McMahon, unpublished data) while overall a for D. polymorpha was 0.140 (Alimov, 1975). The annual range in a for P. grandis was 0.13 to 1.098 (mean 0.563) and, for L. radiata, 0.403 to 1.331 (mean 0.800) (Huebner, 1982) while Alimov (1975) reported a to range from 0.016 to 0.096 for a number of unionoideans. The elevated a of C. ﬂuminea and D. polymorpha relative to unionideans and sphaeriids (range 0.399–1.605) indicates that both species have higher V̇O2 and metabolic rates than other freshwater bivalves. In contrast, the low a values among unionideans reﬂect their relatively low metabolic rates compared to other freshwater bivalve groups. 11. Mollusca: Bivalvia In bivalves, shell production and tissue growth account for a large proportion of metabolic demand, requiring greater than 20% of total metabolic expenditure in young marine blue mussels, Mytilus edulis (Hawkins et al., 1989). Thus, unionoideans with the slowest growth rates among freshwater bivalves (see section III.B.2) also have the lowest metabolic rates, while the fast-growing C. ﬂuminea and D. polymorpha have the highest metabolic rates (McMahon, 1996, 1999). In some sphaeriids, V̇O2 is inﬂuenced by growth and reproductive cycles: maximal metabolic rates occurring during periods of peak adult and brooded juvenile growth (Burky and Burky 1976; Hornbach et al., 1983; Way et al., 1981; Way and Wissing, 1984), perhaps as a result of the elevated metabolic demands associated with tissue growth (Hawkins et al., 1989), elevated V̇O2 of brooded developmental stages, and the energetic costs of providing maternal metabolites to brooded juveniles (Mackie, 1984). In contrast, metabolic rates in C. ﬂuminea (Williams and McMahon, unpublished data) and unionoideans (Huebner, 1982) were unaffected by embryo brooding, perhaps because these species do not provide maternal nourishment to brooded embryos. 347 Filtration rates also vary seasonally and with abiotic conditions in sphaeriids. In S. striatinum (Hornbach et al., 1984b) and M. partumeium (Burky et al., 1985a), ﬁltration rates decreased with increased particle concentration and decreased temperature. Maximal ﬁltration rates occurred during warmer summer months and peaked during reproduction. In M. partumeium, ﬁltration rate and V̇O2 declined in aestivating individuals prior to summer habitat drying (Way et al., 1981; Burky, 1983). Filtration rate is directly correlated with temperature in D. polymorpha, being maximal in summer (MacIsaac, 1996) while it appears to be relatively temperature independent in ﬁeld-acclimated specimens of C. ﬂuminea (Long and McMahon, unpublished data). Freshwater bivalves also display distinct seasonal cycles in tissue biochemical content, primarily related to reproductive cycle. Protein, glycogen and lipid contents are maximal during gonad development and gametogenesis in the freshwater unionid, Lamellidens corrianus, and are minimal during glochidial release (Fig. 9A); a pattern repeated in protein and lipid contents of individual tissues (Fig. 9B – D) (Jadhav and Lomte, 1982a). Similarly, overwintering, nonreproductive individuals of C. ﬂuminea had twice the biomass and greater nonproteinaceous energy stores than FIGURE 9 Seasonal variation in the protein, lipid, and glycogen contents of the wholebody and various tissues of the freshwater unionoidean mussel, Lamellidens corrianus, relative to the reproductive cycle. All organic contents are expressed as percentages of wet tissue weight. (A) Annual vartiation in whole-body contents of proteins (open histgrams), glycogen (solid histograms), and lipid (cross-hatched histograms). Remaining ﬁgures represent levels of protein (B), lipid (C) or glycogen (D) in the mantle (open histograms), digestive diverticulum (cross-hatched histograms), gonad (solid histograms), and foot (stippled histograms). Horizontal bars at the top of each ﬁgure represent reproductive cycles, indicating periods during which either gonads develop or glochidia are released. Gonad development is associated with increases in organic content, and glochidial release, with decreases in organic content of the whole body and various tissues. (From the data of Jadhav and Lomte, 1982a.) 348 Robert F. McMahon and Arthur E. Bogan summer reproductive individuals (Williams and McMahon, 1989). Thus, reproductive effort appears to require massive mobilization of organic energy stores from somatic tissues to support gonad development and gametogenesis in freshwater bivalves. In the sphaeriids, Sphaerium corneum and Pisidium amnicum, increase in tissue glycogen content after fall reproduction was associated with a two-to-threefold increase in anoxia tolerance of winter- over summer-conditioned individuals, the low glycogen contents of summer-conditioned individuals apparently reducing their capacity for anaerobic metabolism. Thus, fall accumulation of glycogen stores not only supported spring gametogenesis, but also provided anaerobic substrate for survival of winter anoxia induced by ice cover (Holopainen, 1987). 2. Diurnal Cycles Freshwater bivalves display diurnal cycles of metabolic activity. Active Na uptake peaked during dark hours in C. ﬂuminea (McCorkle and Shiley, 1982) and the unionid, Toxolasma texasiensis (Graves and Dietz, 1980). This rhythmicity was lost in constant light, suggesting that ion uptake rates were driven by exogenous changes in light intensity. Such diurnal ion transport rhythms appear closely linked to activity rhythms. In the unioniodeans, Ligumia subrostrata, (McCorkle et al., 1979), and European Anodonta anatina and Unio tumidus (Englund and Heino, 1994a) valve gaping activity peaks during dark periods. Rhythmic gaping in L. subrostrata was lost in constant light, suggesting this behavior is also driven by exogenous variation in light intensity (McCorkle et al., 1979). Similarly, diurnal O2 consumption rate rhythms in L. subrostrata appeared driven by light intensity, declining with increase in, and increasing with decrease in light intensity, however, it also had an endogenous component, persisting for 14 days in constant light (McCorkle et al., 1979). Thus, at least some freshwater bivalves may have diurnal activity rhythms, being most active during dark hours. Such activity rhythms may reﬂect diurnal feeding and vertical migration cycles, individuals feeding at the sediment surface at night and retreating below it during daylight to avoid visual ﬁsh and bird predators. Interestingly, diurnal valve movements do not occur in D. polymorpha (Walz, 1978a, b), whose byssal attachment prevents burrowing; however, others have reported diurnal valve movement in this species (Borcherding, 1992). 3. Other Factors Affecting Metabolic Rates Bivalve V̇O2 can be suppressed by pollutants such as heavy metals, ammonia, and cyanide, which degrade metabolic functioning (Lomte and Jadhav, 1982a) lead- ing to extirpation (Starrett, 1971; Williams et al., 1992) or reduced growth rates (Grapentine, 1992). Increased levels of suspended solids impaired V̇O2 and induced starvation in three unionoidean species, indicating interference with gill respiratory and ﬁlterfeeding currents (Aldridge et al., 1987). It also suppresses V̇O2 (Alexander et al., 1994) and ﬁltration rate in D. polymorpha (Lei et al., 1996). Similary, high particle concentrations depress ﬁltration rates in C. ﬂuminea (Way et al., 1990a). Bivalve metabolic rates may also be density dependent. V̇O2 declined with increased density in the unionid, Elliptio complanata. V̇O2 was three times greater in singly held individuals relative to groups of seven or more, suggesting release of a pheromone suppressing metabolic rates of nearby individuals (Paterson, 1983). Unionoideans and sphaeriids display varying degrees of respiratory regulation when subjected to progressive hypoxia (Burky, 1983). Both D. polymorpha (McMahon, 1996) and C. ﬂuminea (McMahon, 1979a) are highly O2 dependent, their V̇O2 declining proportionately with declining partial pressure of O2 (PO2). Such species are generally relatively intolerant of prolonged hypoxia and restricted to well-oxygenated habitats. In contrast, other freshwater bivalve species are oxygen independent and regulate V̇O2 at relatively constant levels with progressive hypoxia until a critical V̇O2 is reached below which V̇O2 declines proportionately with further decline in PO2. Such species can inhabit waters periodically subjected to prolonged hypoxia. Thus, the sphaeriids, Sphaerium simile and Pisidium casertanum, from hypoxic profundal habitats are relatively O2 independent (Burky, 1983) while the shallow temporary pond species, Musculium partumeium, is relatively poor O2 regulator (Hornbach, 1991). The Austrailian riverine unionoidean, Alathyria jacksoni, rarely experiencing hypoxia, is a relatively poor O2 regulator while a periodically hypoxic, Australian, pond species, Velesunio ambiguus, was a strong oxygen regulator (Sheldon and Walker, 1989). Similarly, the lentic unionioidean, A. cygnea regulated V̇O2 at a PO2 as low as 14.3 Torr (0.9% of full air O2 staturation), maintaining near constant hemolymph O2 concentrations during progressive hypoxia by increasing gill ventilation (Massabuau et al., 1991) while maintaining a near-constant heart rate (Michaelidis and Anthanasiadou, 1994). The unioniodeans, Elliptio complanata and Pyganodon grandis, from a small, Canadian, euthrophic lake, were extreme O2 regulators, maintaining near constant V̇O2 down to 1 mg O2/L (PO2 ⬇18 Torr, 11.3% of full air O2 saturation) (Fig. 10) (Lewis, 1984). Their capacity for extreme V̇O2 regulation is adaptive, as winter ice cover made the lake severely hypoxic and overwintering individuals burrow deeply into hypoxic sediments (Lewis, 1984). In contrast, the 11. Mollusca: Bivalvia 349 FIGURE 10 Respiratory responses of the freshwater unionoidean mussels, Elliptio complanata and Pyganodon grandis to ambient O2 concentrations declining from near full-air saturation (8 – 9 mg O2 /l, lower horizontal axis, PO2 140 – 160 torr or mg Hg, upper horizontal axis) to the concentration at which O2 uptake ceases. Respiratory responses of four individuals of (A) E. complanata, and (B) four individuals of P. grandis. Both species maintained normal O2 uptake rates at a PO2 as low as 15 – 20 torr (9 – 13 percent of full-air O2 saturation) suggesting elevated capacity for O2 regulation of oxygen uptake. (Redrawn from Lewis, 1984.) V̇O2 of tropical and semitropical species not experiencing winter hypoxia tends to be more O2 dependent (McMahon, 1979a; Das and Venkatachari, 1984), the V̇O2 of the subtropical species, C. ﬂuminea, declining to very low levels with just a 30% decline in PO2 below full air O2 saturation levels (McMahon, 1979a). Some freshwater bivalves are very tolerant of extreme hypoxia or even anoxia allowing survival in hypoxic conditions below the thermocline of stratiﬁed lakes or above reducing substrates (Butts and Sparks, 1982; Belanger, 1991). Profundal sphaeriid species tolerate extreme hypoxia and anoxia throughout summer lake stratiﬁcation (Holopainen and Jonasson, 1983), surviving 4.5 to 200 days of anoxia, depending on season and temperature (Holopainen, 1987). The unionoidean A. cygnea survived 22 days of anoxia (Zs.-Nagy et al., 1982). In contrast, other freshwater bivalves are highly intolerant of hypoxia / anoxia, including D. polymorpha and C. ﬂuminea, restricting them to well-oxygenated waters (Efﬂer and Siegfried, 1994; Johnson and McMahon, 1998; Matthews and McMahon, 1999). Byssal thread production was inhib- ited in D. polymorpha below a PO2 of 40 Torr (25% of full air O2 saturation) (Clarke and McMahon, 1996b). Individual size also affects tolerance of anoxia / hypoxia. Juveniles of the unionoidean, Elliptio complanata, are less hypoxia-tolerant than adults (Sparks and Strayer, 1998). In contrast, anoxia and hypoxia tolerance decreases with increased size in C. ﬂuminea and D. polymorpha (Johnson and McMahon, 1998, Matthews and McMahon, 1999). The general trend for decreased juvenile hypoxia tolerance among freshwater bivalves may restrict some species to well-oxygenated habitats even though the adults may be hypoxiatolerant (Sparks and Strayer, 1998). Under anoxia, bivalves rely on anaerobic metabolic pathways which are not those of glycolysis; but, instead, involve simultaneous catabolism of glycogen and aspartate or other amino acids to yield the end products, alanine and succinate. Succinate can be further degraded into volatile fatty acids such as proprionate or acetate (Zs.-Nagy et al., 1982; de Zwaan, 1983; van den Thillart and de Vries, 1985). Anoxic for 6 days, A. cygnea maintained 52 – 94% of aerobic ATP levels, 350 Robert F. McMahon and Arthur E. Bogan higher than could occur by typical glycolytic pathways, due to its anaerobic oxidation of succinate (Zs.-Nagy et al., 1982). These alternative anaerobic pathways are more efﬁcient than glycolysis (2 mol of ATP preduced per mole of glucose catabolized), producing 4.71 – 6.43 mol of ATP per mole of glucose catabolized (de Zwaan, 1983). Indeed, typical glycolytic pathway enzyme activities are reduced in A. cygnea during hypoxia (Michaelidis and Athanasiadou, 1994) which favors metabolite catabolism by alternative pathways and allows greater tolerance of hypoxia / anoxia than less efﬁcient glycolysis. Further, the higher ATP yields of the alternative pathways allow excretion of anaerobic endproducts, rather than retention of acidic lactate to deleterious levels as occurs in species dependent on glycolysis (de Zwaan, 1983). During anerobiosis, buildup of acidic end products in bivalve tissues and hemolymph can cause considerable tissue and hemolymph acidosis (i.e., decrease in pH). Without respiratory pigments, freshwater bivalve hemolymph has little inherent buffering capacity, thus bivalves mobilize shell calcium carbonate into the hemolymph through the mantle epithelium (Machado et al., 1990) to buffer acidosis (Heming et al., 1988; Byrne and McMahon, 1994; Burnett, 1997). Thus, when anaerobic, pallial ﬂuid pH of the unionoidean Margaritifera margaritifera remained highly constant, but its Ca2 concentration increased (Heming et al., 1988). Similarly, blood Ca2 levels rose eightfold in L. subrostrata (Dietz, 1974) and nearly ﬁvefold in C. ﬂuminea (Byrne et al., 1991a) under anoxia induced by emersion in a pure N2 atmosphere. A three-fold increase in hemolymph Ca2 concentration occurred in Pyganodon grandis made anoxic by 6 days air emersion (Byrne and McMahon, 1991). The gills of unionoideans (Steffens et al.1985) harbor extensive extracellular calcium phosphate concretions that could buffer hemolymph pH. However, their mass increases during prolonged hypoxia and is inversely related to hemolymph pH and directly to blood Ca2 concentration, suggesting that Ca2 released from the shell during hypoxia is sequestered in gill concretions to prevent its loss by excretion or diffusion. On return to normoxia, release and redeposition in the shell of concretion Ca2 is likely to be much less energetically demanding than replacement of lost shell Ca2 from the dilute freshwater medium (Silverman et al., 1983). The enzyme, carbonic anhydrase, is bound to these concretions, facilitating exchange of concretion Ca2 (uptake or release) with the hemolymph (Istin and Girard, 1970a, b). 4. Desiccation Resistance Body water content in freshwater bivalves can vary greatly seasonally and during drought periods. Thus, many species have evolved adaptations allowing tolerance of prolonged emersion and desiccation stress (Cáceres, 1997). Freshwater bivalves may be emersed for weeks or months during seasonal or unpredictable droughts (Byrne and McMahon, 1994). Lack of mobility leaves individuals stranded in air as water levels recede, while others occur in habitats that dry completely. Unlike other freshwater invertebrates (Cáceres, 1997), bivalves have no obvious structures for maintenance of aerial gas exchange when emerged, However, they have behavioral and physiological adaptations that maintain aerobic metabolism while minimizing water loss rates (Byrne and McMahon, 1994). Survival of emersion in freshwater bivalves is correlated with capacity to control water loss. Among freshwater species, C. ﬂuminea and D. polymorpha are highly emersion intolerant, tolerating only 8 – 36 days (McMahon, 1979b) or 3 – 27 days emersion (McMahon et al., 1993), respectively, depending on temperature and relative humidity. Dreissena bugensis is even less emersion tolerant than D. polymorpha (Ussary and McMahon, 1994; Ricciardi et al.,1995). In these species and unionoideans, death occurs in most cases at a critical threshold of water loss regardless of temperature or relative humidity; thus, rate of water loss dictates emersion tolerance times (McMahon, 1979b; McMahon et al., 1994; Byrne and McMahon, 1994; Ricciardi et al., 1995). In contrast, sphaeriids inhabiting temporary ponds survive emersion for several months during summer drying (Burky, 1983). In some species, juveniles and adults survive emersion (Collins, 1967; McKee and Mackie, 1980), while in others, only juveniles survive (McKee and Mackie, 1980; Way et al., 1981). Sphaeriids burrow into sediments prior to emersion (Burky, 1983) as do some unionoideans (Cáceres, 1997). V̇O2 declines in both emersed, aestivating M. partumeium (Way et al., 1981) and emersed individuals of C. ﬂuminea (McMahon and Williams, 1984; Byrne et al., 1990). Reduction in metabolic demand in emersed individuals allows long-term maintenance on limited energy reserves. Freshwater bivalves have a number of mechanisms for maintaining gas exchange while emersed. Sphaerium occidentale is emersed for several months in its ephemeral pond habitats. In air, its V̇O2 is 20% that of aquatic rates. Gas exchange occurs across specialized pyramidal cells extending through shell punctae which allows continuous valve closure, minimizing water loss (Collins, 1967). In C. ﬂuminea, aerial V̇O2 is 21% that of aquatic rates (McMahon and Williams, 1984). Emersed specimens periodically gape and expose mantle tissue margins cemented together with mucus (Byrne et al., 1988) during which high rates of aerial V̇O2 are sustained while no O2 uptake occurs during valve closure (McMahon and Williams, 1984). 11. Mollusca: Bivalvia Bursts of metabolic heat production occur during mantle edge exposure, associated with aerial oxygen consumption and apparent repayment of an O2 debt accumulated during intervening anaerobic periods of valve closure (Byrne et al., 1990). Thus, periodic mantle margin exposure allows maintenance of aerial gas exchange while greatly reducing the tissue surface area exposed and duration of exposure, greatly reducing water loss rates (Byrne and McMahon, 1994). Frequency and duration of mantle edge exposure is reduced in C. ﬂuminea with increased temperature, decreased relative humidity and increasing duration of emersion, suggesting that increased desiccation pressure leads to a greater reliance on anaerobic metabolism to slow evaporative water loss (Byrne et al., 1988). Emersed unionoideans periodically expose their mantle edges (Byrne and McMahon, 1994). Emersed specimens of Ligumia subrostata periodically expose mantle edges and maintain aerial V̇O2 at 21 – 23% of aquatic rate (Dietz, 1974). Periodic mantle edge exposure also occurs in emersed specimens of M. margaritifera (Heming et al., 1988), Pyganodon grandis (Byrne and McMahon, 1991), Pyganodon grandis, Toxolasma parvus and Uniomersus tetralasmus (Byrne and McMahon, 1994). P. grandis has a thin shell whose margins do not completely seal when closed and a high frequency of mantle edge exposure when emersed (25 – 90% of emersion time), resulting in rapid water loss and poor emersion tolerance (2 – 32 days). It avoids desiccation by rapid down-shore migration during reductions in habitat water levels (Byrne and McMahon, 1994). More emersion-tolerant species like T. parvus and U. tetralasmus have thicker, tightly sealing shells and their frequency and duration of mantle edge exposure is reduced with increasing duration of emersion and decreasing relative humidity, conserving water as desiccation pressure increases. T. parvus continuously closes the valves in latter stages of emersion, allowing it to survive emersion up to 145 days (Byrne and McMahon, 1994). U. tetralasmus is found in small, variable-level, lentic habitats and is highly emersion tolerant. In air, it occludes the siphons with viscous mucus, preventing direct atmospheric exposure of inner mantle tissues. Its frequency and duration of mantle edge exposure is quite low compared to other species and it ceases mantle edge exposure in the latter stages of emersion, making water loss rates lower than recorded for other freshwater bivalves and allowing it to survive emersion for up to 578 days (Byrne and McMahon, 1994). In air, unionoideans and C. ﬂuminea utilize shell Ca2 to buffer accumulating HCO3 (Byrne and McMahon, 1994) manifested by accumulation of Ca2 and HCO3 in mantle cavity ﬂuids of emersed M. margaritifera (Hemming et al., 1998) and hemolymph of 351 emersed C. ﬂuminea (Byrne et al., 1991a) and P. grandis (Byrne and McMahon, 1991). Mantle edge exposure allows release of CO2 generated by metabolic and shell-buffering processes in both C. ﬂuminea (Byrne et al., 1991a) and unionoideans (Byrne and McMahon, 1991). In emersed C. ﬂuminea there is little evidence of O2 debt payment after re-immersion, suggesting that this and other freshwater bivalves may remain primarily aerobic while emersed (Byrne et al., 1990). The Unionoidea include the most emersion-tolerant NA freshwater bivalve species (for a review, see Byrne and McMahon, 1994). Their capacity to tolerate prolonged emersion and / or migrate vertically with changing water levels (White, 1979) may partially account for their dominance in larger NA river drainages subject to extensive seasonal water-level ﬂuctuations. Unionoidean growth, reproduction and other lifehistory phenomena may be partially driven by seasonal water-level variation. Thus, anthropomorphic impoundment / regulation of ﬂow rates and levels within these drainages could be contributing to the present decline of their unionoidean populations and species diversity (Williams et al., 1992). The mode of nitrogen excretion or detoxiﬁcation during emersion is unresolved for freshwater bivalves. Ammonium ion, NH4, is the major nitrogenous excretory product of aquatic molluscs (Bishop et al., 1983). Due to its toxicity, NH4 is generally not accumulated in emersed molluscs even though its high solubility precludes release as ammonia gas (NH3). When emersed, aquatic snails detoxify NH4 by conversion to urea or uric acid, to be excreted on re-immersion. However, most bivalves cannot convert NH4 to urea or uric acid (Bishop et al., 1984). Without the capacity to detoxiﬁy NH4, how do freshwater bivalves tolerate emersion? Corbicula ﬂuminea, unlike intertidal bivalves (Bishop et al., 1983), does not catabolize amino acids during emersion, precluding NH4 formation (Byrne et al. 1991b). Similarly, the unionideans, Lamellidens corrianus and L. marginalis, depend almost exclusively on carbohydrate metabolism while emersed (Lomte and Jadhav, 1982c; Sahib et al., 1983). Interestingly, some unionoideans may be capable of converting ammonia to urea or other protective metabolites (Summathi and Chetty, 1990; Mani et al., 1993) which may partially account for their extensive emersion tolerance. Tolerance of emersion may also be associated with physiological and biochemical alterations in emersed individuals. Thus, individuals of Sphaerium occidentale and Musculium securis from an emersed population were more emersion tolerant than individuals from an immersed, active population (McKee and Mackie, 1980), suggesting that gradual emersion may induce emersion resistant biochemical and physiological 352 Robert F. McMahon and Arthur E. Bogan alterations such as a shift to carbohydrate dominated catabolism and reduced metabolic demand. Such biochemical and physiological compensation may be mediated by neurosecretory hormones (Lomte and Jadhav, 1981a). There have been almost no studies of freeze tolerance in freshwater bivalves even though many species occupy shallow, temperate habitats in which winter water-level reduction could emerse individuals in subfreezing air. Marine intertidal mytilid mussels tolerate exposure to subfreezing conditions by allowing freezing of hemolymph and interstitial ﬂuids while preventing cell freezing, an adaptation manifested by their lack of hemolymph supercooling during freezing (Aarset, 1982). In contrast, D. polymorpha is intolerant of emersion below 3°C (Clarke et al., 1993) and displays hemolymph supercooling (Paulkstis et al., 1996). Poor freeze tolerance in D. polymorpha may reﬂect its tendency to settle at depths of 1 m, preventing winter emersion (Clarke et al., 1993). Freeze sensitivity / tolerance in other freshwater bivalves (particularly shallow water species) is ripe for further study. 5. Gill Calcium Phosphate Concretions in Unionoideans Dense calcium phosphate [Ca3(PO4)2] concretions occur in unionoidean tissues. Mantel concretions may provide Ca2 for shell deposition (Davis et al., 1982; Jones and Davis, 1982; Istin and Girard, 1970a). The mantle contains high concentrations of the enzyme, carbonic anhydrase which may be bound to the concretions (Istin and Girard, 1970b). This enzyme catalyzes the reaction of CO2 and H2O to form bicarbonate ion (HCO3), suggesting that it facilitates mobilization of concretion Ca2. Dense extracellular calcium phosphate concretions (diameter 1 – 3 m) also occur in unionoidean gills (Silverman et al., 1983, 1988; Steffens et al., 1985). They develop from amorphous material initially concentrated in membrane bound vesicles in gill connective tissue cells (Silverman et al., 1989). Concretions account for up to 60% of gill dry weight in some species (Silverman et al., 1985). They are most dense along parallel nerve tracts oriented at 90° to the gill ﬁlaments (Silverman et al., 1983; Steffens et al., 1985). They are 25% protein by dry weight. One of the proteins is similar to vertebrate, calcium-binding calmodulin. This protein is most abundant in protein granules of concretion-forming cells prior to concretion mineral deposition, suggesting that it acts as site for calcium phosphate deposition (Silverman et al., 1988). Besides storing shell Ca2 released to the hemolymph to buffer respiratory acidosis (see Section II.C.3), gill concretions provide a ready source of maternal Ca for shell deposition in brooded glochidia (Silverman et al., 1985, 1987). Thus, gill concretion mass in brooding individuals of L. subrostrata and P. grandis was only 47 and 70% that of nonbrooding individuals, respectively (Silverman et al., 1985). 45Ca tracer studies indicate that 90% of glochidial shell Ca was of maternal origin in P. grandis, the most likely source being gill concretions, with nonmineralized Ca accounting for only 8% of glochidial Ca in L. subrostrata (Silverman et al., 1987). 6. Water and Salt Balance In hypo osmotic freshwater, bivalves lose ions and gain water from their hyperosmotic tissues and hemolymph. Excess body water is excreted as a ﬂuid hypo-osmotic to tissues and hemolymph via the kidneys while ions lost in excretion and over surface epithelia are actively recovered over the gills and epithelial tissues as described in Section II.B.3 (reviewed by Deaton and Greenberg, 1991). Relatively salinity tolerant C. ﬂuminea survives exposure to a salinity of 10 – 14 ppt (Morton and Tong, 1985), above which it remains isosmotic to the medium (Gainey and Greenberg, 1977). Unionoideans and D. polymorpha have lower hemolymph osmolarities than C. ﬂuminea (Dietz et al., 1996a) and generally lose capacity for osmotic and volume regulation above 3 – 4 ppt (Hiscock, 1953; Wilcox and Dietz, 1998). C. ﬂuminea regulates ﬂuid volume by actively increasing hemolymph free amino acid concentration in hyperosmotic media (Gainey and Greenberg, 1977; Matsushima et al., 1982), preventing water loss by active equilibration of hemolymph and medium osmolarity. Such volume regulation does not occur in other freshwater bivalves because their low tissue osmolarities limit the free amino acid pool (Yamada and Matsushima, 1992; Dietz et al., 1996a,b, 1998). Volume regulation may have been lost in unionoideans and D. polymorpha, because they are not exposed to hyperosmotic conditions in freshwater. Instead, both groups retain only limited cell volume regulation through regulation of intracellular inorganic ion concentrations (Dietz et al., 1996a, b, 1998). The elevated osmotic concentration and free amino acid pool volume regulation of C. ﬂuminea reﬂects its recent evolution from an estuarine ancestor (Dietz, 1985) in which such adaptations are common (Deaton and Greenberg, 1991). Both unionoideans and C. ﬂuminea respond to maintenance in very dilute media by increasing active Na uptake rate to maintain hemolymph ion concentration (Dietz, 1985). The activity of (Na and K)-activated ATPase, an enzyme required for active Na /K transport, increased in mantle and kidney tissues of saltdepleted C. ﬂuminea, suggesting activation of Na transport. This response did not occur in the salt-depleted 11. Mollusca: Bivalvia Lampsilis straminca claibornesis, suggesting that active ion uptake regulation has been lost in unionoideans with a longer freshwater fossil history than C. ﬂuminea (Deaton, 1982). In C. ﬂuminea Cl is the major blood ion, maintained by elevated active epithelial Cl uptake. In contrast, in the unionoidean, Toxolasma texasiensis, Cl and HCO3 are equivalent hemolymph anionic components. Because HCO3 can be readily mobilized by respiratory and metabolic processes, unionoideans may depend to a greater extent on active Na relative to Cl uptake to maintain hemolymph osmotic balance (Byrne and Dietz, 1997). The anionic hemolymph component of D. polymorpha, like that of C. ﬂuminea, is dominated by Cl, but this species actively absorbs equal levels of Na and Cl to maintain ionic stasis, suggesting co-transport of these ions (Horohov et al., 1992). Retention of Cl as the main hemolymph anion in C. ﬂuminea and D. polymorpha reﬂects their recent evolution from estuarine ancestors (Deaton and Greenberg, 1991; Byrne and Dietz, 1997; Horohov et al., 1992), while the greater dependence of C. ﬂuminea on Cl uptake for ionic regulation and its increased hemolymph osmotic concentration (Dietz et al., 1994) suggests that it entered freshwater more recently than D. polymorpha. Interestingly, although D. polymorpha can hyperosmotically regulate hemolymph ion concentrations in dilute freshwaters, it is less tolerant of elevated medium ion concentrations than other bivalves restricting its penetration of estuarine habitats (Horohov et al., 1992; Dietz et al., 1994, 1998; Wilcox and Dietz, 1998). In the Asian unionoidean, Anodonta woodiana, mantle cavity water osmolarity at 34 mosmol/L was 76% that of the hemolymph (45 mosmol/L) with pallial ﬂuid Na, K, and Cl concentrations being 71, 76 and 72% those of the hemolymph, respectively. Maintenance of elevated mantle water ion concentrations suggests that it acts as an osmotic buffer, reducing the gradient for, and, thus, the rate of diffusive ion loss to the dilute freshwater medium (Matsushima and Kado, 1982). However, loss of a hyperosmotic mantle ﬂuid to the external medium through the exhalant siphon could also be a major route for ion loss. In unionoideans and C. ﬂuminea, the enzyme, carbonic anhydrase (CA), which catalyzes formation of carbonic acid (H2CO3) from water and carbon dioxide, occurs in gill and mantle tissues (Henery and Saintsing, 1983). H2CO3 degrades into H and CO3 (bicarbonate ion) which are counter ions for active Na and Cl uptake (Horohov et al., 1992; Dietz et al., 1994; Byrne and Dietz, 1997). Gill and mantle CA activity increases when freshwater bivalves are held in extremely dilute media while CA inhibition by actazolamide results in reduced Na and Cl uptake rates, strong evidence of the role of CA in ion regulation (Henery and Saintsing, 1983). 353 Freshwater bivalve hemolymph osmotic concentrations are the lowest recorded for multicellular invertebrates (Dietz, 1985; Deaton and Greenberg, 1991). Among unionoideans, hemolymph osmolarity for the European species, Anodonta cygnea, was 40 – 50 mosmol/L or 4 – 5% that of seawater. D. polymorpha has the lowest hemolymph concentration of all freshwater bivalves (30 – 36 mosmol/L or 3.0 – 3.6% that of seawater) (Dietz et al., 1994), while C. ﬂuminea has the highest value at 65 mosmol /L (6.5% that of seawater) (Zheng and Dietz, 1998a). That for other freshwater invertebrates ranges from 100 to 400 mosmol/L or 10 – 40% that of seawater (Burky, 1983). Low hemolymph osmotic concentrations in freshwater bivalves reduce the gradient for transepithelial osmosis and ion diffusion across their extensive mantle and gill epithelial surfaces to that which can be balanced by water excretion and active ion uptake at energetically feasible levels (Burton, 1983). Hydrostatic pressure generated by heart beat in A. cygena allows ﬁltration of enough hemolymph plasma into the pericardial space to account for urine production and is twice that of the marine clam, Mya arenaria, suggesting that freshwater bivalves can excrete water at higher rates than less hypoosmotically stressed estuarine species (Jones and Peggs, 1983). Exposure of Pyganodon sp. to a very dilute medium induced formation of extensive extracellular membrane spaces in the deep infoldings of kidney epithelial cells, perhaps increasing surface area for active ion uptake from excretory ﬂuids producing a more dilute urine and / or allowing increased excretion of excess water (Khan et al., 1986). Increasing osmotic gradients for water uptake from marine through estuarine into freshwater habitats required that the external epithelia of bivalves became increasing impermeable to water with their evolutionary transition through these environments. Thus, increased epithelial osmo-resistance characterizes all freshwater bivalves (Deaton and Greenberg, 1991; Dietz, 1985; Dietz et al., 1994; Byrne and Dietz, 1997; Zheng and Dietz, 1998a). Among freshwater bivalves, C. ﬂuminea is least osmotically permeable (Zheng and Dietz, 1998a) and D. polymorpha most permeable (Dietz et al., 1995), while unionoideans are of intermediate permeability (Dietz et al., 1996a, b; Zheng and Dietz, 1998a). The “osmotically tight” epithelium of C. ﬂuminea allows it to maintain higher hemolymph osmolarity than other freshwater bivalves while producing normal excretory ﬂuid volumes (Zheng and Dietz, 1998a). In contrast, “osmotically leaky” epithelium in D. polymorpha results in very elevated excretory ﬂuid production (Dietz et al., 1995), requiring extremely high rates of epithelial ion uptake to replace ions lost in voluminous excretory ﬂuids (Dietz and Byrne, 1997). Thus, the main 354 Robert F. McMahon and Arthur E. Bogan osmoregulatory adaptations of C. ﬂuminea and D. polymorpha are quite different, the former being reduced epithelial permeability, and that of the latter, increased water excretion and active epithelial ion uptake. Hormones regulate osmotic control in freshwater bivalves. Cyclic AMP (cAMP) stimulates active Na uptake by unionoideans, while prostglandins inhibit it. In contrast, prostglandin inhibitors stimulate N uptake (Dietz et al., 1982; Graves and Dietz, 1982; Saintsing and Dietz, 1983). Serotonin stimulates tissue accumulation of cAMP, increasing active Na uptake. Thus, an antogonistic relationship between serotonin and prostaglandins modulates adenylate cyclase-catalyzed cAMP stimulation of active Na uptake. Not surprisingly, high concentrations of serotonin occur in unionoidean gill nerve tracts (Dietz, 1985; Dietz et al., 1992). Gill concentrations of the neurotransmitters, dopamine and norepinephrine, greatly declined in unionoideans salt-depleted in extremely dilute mediums, while serotonin was regulated at near-normal levels, suggesting that serotonin regulates Na uptake for ionic balance. The circadian rhythms of Na uptake in freshwater clams (Graves and Dietz, 1980; McCorkleShiley, 1982) may be mediated by an antagonistic serotonin / prostaglandin hormonal system (Dietz, 1985). When cerebropleural or visceral ganglia were ablated, individuals of the Indian unionoidean, Lamellidens corrianus, rapidly lost osmoregulatory capacity which was restored by injection of ganglia extracts, indicating that ganglion neurosectory hormones are involved in water balance (Lomte and Jadhav, 1981b). The afﬁnity of the pedal ganglion of A. cygena for monoamines controlling ion / water balance is temperature dependent indicative of a seasonal component to osmoregulation (Hiripi et al., 1982). III. ECOLOGY AND EVOLUTION A. Diversity and Distribution North American freshwater bivalves distributions, particularly for unionoideans, have been well described. Species distribution maps for unionoideans and sphaeriids exist for Canada (Clarke, 1973), the United States (LaRocque, 1967a) and for the whole of North American (Burch, 1975a, b; Parmalee and Bogan, 1998). LaRocque (1967b) describes living and Pleistocene fossil assemblages at speciﬁc NA localities. There is also a massive literature, too numerous to cite here, describing species occurrences or species assemblages in various NA drainage systems. Native (non-introduced) NA sphaeriid species have broad distributions, often extending from the Atlantic to Paciﬁc coasts. Introduced to NA from southeast Asia in early 1900s (McMahon, 1999), Corbicula ﬂuminea (i.e., light-colored shell morph of Corbicula) has a similarily widespread NA distribution, inhabiting drainages on the west coast of the United States, the southern tier of states, and throughout states east of the Mississippi River, with the exception of the most northern states, and into northern Mexico (McMahon, 1999) (Fig. 11). A second, unidentiﬁed species of Corbicula (i.e., the dark-colored shell morph) is restricted to isolated, spring-fed drainages in southcentral Texas and southern California and Arizona (Fig. 11) (Britton and Morton, 1986). In contrast, NA unionoidean species generally have more restricted distributions. Few species range on both sides of the continental divide and a large number are limited to single drainage systems (Burch, 1975b; LaRocque, 1967a). 1. Dispersal Widespread NA distributions of sphaeriids and Corbicula relative to unionoideans reﬂect fundamental differences in their dispersal capacities. Unionoideans depend primarily on host ﬁsh glochidial transport for dispersal (Kat, 1984), their ranges reﬂecting those of their host ﬁsh species (Haag and Warren, 1998). While host ﬁsh glochidial transport increases probability of dispersal into favorable habitats, as host ﬁsh and adult unionoidean habitat preferences generally coincide (Kat, 1984), it limits the extent of dispersal, leading to highly endemic species. For example, electrophoretic studies of peripheral Nova Scotian populations of unionoideans suggest that they invade new habitats mostly by host ﬁsh dispersal (Kat and Davis, 1984), barriers to host ﬁsh dispersal being barriers to unionid dispersal. Thus, distributions of modern and fossil NA interior basin unionoidean assemblages are limited to areas below major waterfall barriers to ﬁsh host upstream migration in the Lake Champlain drainage system of New York, Vermont, and Quebec (Smith, 1985a) and re-establishment of Anodonta implicata populations in the upper Connecticut River drainage closely followed restoration of its anadromous glochidial clupeid ﬁsh host populations, by construction of ﬁshways past numerous impoundments preventing upstream ﬁsh host dispersal (Smith, 1985b). Vaughn and Taylor (2000) have found that 50% of the variation in unionoidean assemblages is associated with regional distribution and abundances of ﬁshes, indicating that ﬁsh community structure is a determinant of mussel community structure. Sphaeriids and C. ﬂuminea have evolved dispersal mechanisms that make them more invasive than unionoideans, accounting for their more cosmopolitan distributions. Juvenile sphaeriids disperse between 11. Mollusca: Bivalvia 355 FIGURE 11 Distribution of Corbicula in the United States. Hatched area is the distribution of the light-colored morph of Corbicula, Corbicula ﬂuminea. The solid areas are the distribution of the dark-colored morph of Corbicula, yet to be assigned a species designation. drainage systems by clamping shell valves onto limbs of aquatic insects, feathers of water fowl (Burky, 1983), or even limbs of salamanders (Davis and Gilhen, 1982) ﬁrst noted by Darwin (1882). Some sphaeriids survive ingestion and regurgitation by ducks, which commonly feed on them, allowing long-distance dispersal (Burky, 1983). The rapid spread of C. ﬂuminea through NA drainage systems (McMahon, 1999), while partly anthropomorphically mediated, also resulted from its natural dispersal capacities. The long juvenile mucilaginous byssal thread or ﬁlamentous algae on which juveniles settle becomes entangled in the feet or feathers of shore birds or water fowl, making them transport vectors (McMahon, 1999). Its natural dispersal capacity has allowed it to spread into areas of northern Mexico where human-mediated transport is highly unlikely (Hillis and Mayden, 1985), and into southern Britain during interglacial periods (McMahon, 1999). Dreissena polymorpha, byssally attaches to ﬂoating wood or boat and barge hulls, facilitating long distance transport (Mackie and Schloesser, 1996), and to macrophytic vegetation that can be transported across drainages by nesting shore birds and water fowl. Juveniles of C. ﬂuminea can be passively transported long distances downstream suspended in water currents (McMahon, 1999). Water currents also disperse the planktonic veliger of D. polymorpha (Mackie and Schloesser, 1996). Adult C. ﬂuminea can be carried downstream over substratum by water cur- rents (Williams and McMahon, 1986), a process facilitated by production of a mucus dragline from the exhalant siphon (Prezant and Charlermwat, 1984). Passive hydraulic dispersal of juvenile and adult C. ﬂuminea not only accounts for its extraordinary ability to invade downstream portions of drainages after introduction (1999), but also leads to its impingement and fouling of industrial, agricultural, and municipal raw-water systems (1999). Similarly, current-mediated transport of free-swimming veliger and passively suspended juvenile D. polymorpha, and of adults attached to ﬂoating substrates or carried as clumps of individuals over the bottom accounts for its dispersal in European drainage systems after escape from the Caspian Sea (Mackie and Schloesser, 1996). This species rapidly spread downstream throughout the Great Lakes and St. Lawrence River from its original upstream introduction into Lake St. Clair in 1985 – 1986 (Mackie and Schloesser, 1996). It has also been carried throughout most of the Mississippi River and adjoining tributaries both by downstream hydrological transport and upstream by attachment to the hulls of commercial barges (Mackie and Schloesser, 1996) (Fig. 12). Zebra mussels invaded the lower Great Lakes, St. Lawrence River and Erie-Barge Canal by 1990, later invading portions of the upper Great Lakes, the Hudson River, and the Finger Lakes. D. polymorpha entered the Mississippi Drainage from Lake Michigan through the Illinois River and spread 356 Robert F. McMahon and Arthur E. Bogan D. polymorpha, D. bugensis and the majority of unionoideans are gonochoristic, requiring simultaneous introduction of males and females to found a new population, thus reducing their capacity as invaders. 2. Anthropomorphic Impacts FIGURE 12 Distribution of the zebra mussel, Dreissena polymorpha, in North America as of Spring, 2000. downstream to the Mississippi Delta and upstream to La Crosse, Wisconsin. It had invaded all major tributaries of the Mississippi River by 1998 with the exception of the Missouri River, where the ﬁrst specimens were reported upstream of its conﬂuence with the Platte River in 1999. As of early 2000, D. polymorpha occupied drainages in 21 eastern and midwestern U.S. states and in the Canadian provinces of Ontario and Quebec, including 62 conﬁrmed populations in small, isolated inland lakes (Fig. 12). Dreissena bugensis, the second dreissenid species occupying NA inland waters is sympatric with D. polymorpha in Lakes Huron, Erie, and Ontario and the western end of the ErieBarge Canal (New York Sea Grant, 1999). Capacity for downstream transport and byssal attachment has made D. polymorpha a major NA biofouling pest species, recapitulating its history in Europe (Claudi and Mackie, 1993). Juvenile sphaerids are also passively, hydrologically transported downstream (McKillop and Harrison, 1982) which may be an important dispersal mode for many species in this family. In contrast, hydrological transport is extremely rare in unionoideans (Imlay, 1982). As both sphaeriids and C. ﬂuminea are selffertilizing hermaphrodites (see Section II.B.5), a single individual can found a new population. In contrast, The diversity of native NA unionoid bivalves is represented by two of the six recognized families of the Unionoidea: Margaritiferidae with two genera and ﬁve species; and Unionidae with 49 genera, 278 species, 13 subspecies (Turgeon et al., 1998; Johnson, 1998; Williams and Fradkin, 1999). North American unionoidean diversity represents 51 of the approximately 165 unionoidean genera, and between onefourth and one-third of the world’s unionoidean species diversity (Bogan, 1993; Bogan and Woodward, 1992). Neves et al. (1997) documented that 261 of these taxa or 91% of NA unionoidean diversity occurs in the southeastern United States where it is focused in the Mobile Bay, Tennessee and Cumberland River basins. Alabama, with parts of the Mobile Bay and Tennessee River basins, has the greatest unionoidean diversity with 175 taxa followed by Tennessee with 129 taxa. This great diversity of unionoidean bivalves began to be impacted as it began to be described, with European expansion across North America. It was noticed quite early on that the unionoidean fauna was declining (Higgins, 1858). By the turn of the 20th century, people were observing that the unionoidean fauna of entire regions was being decimated or had disappeared. Rhoads (1899) described extirpation of mussels from the Monongahela River at Pittsburgh due to damming and pollution. Ortmann (1909) noted the loss of the freshwater mussels, crayﬁsh and ﬁsh fauna from the upper Ohio River Basin in western Pennsylvania due to acid run-off from coal mines and the complete destruction of the Pigeon River unionoidean fauna in east Tennessee by pollution (Ortmann, 1918). Van der Schalie (1938) warned that the Tennessee Valley Authority’s construction of dams on the Tennessee River could lead to long-term negative impacts on, and the eventual destruction of, its freshwater fauna. Extinction of NA freshwater bivalve species was not reported until Stansbery (1970, 1971) listed 11 presumed extinct taxa. Turgeon et al., (1988) listed 13 taxa, Williams et al., (1993) listed 12% of the unionoid taxa as extinct, and most recently Turgeon et al., (1998) listed 35 taxa as presumed extinct (Table II). As of 1997, 77 NA unionid taxa were endangered, 43 threatened and 73 of special concern with only 70 species listed as currently stable and additional species being added to federal and state lists yearly (Williams et al., 1993; Neves et al., 1997). Massive historical losses of unionoideans have been revealed by comparison of 11. Mollusca: Bivalvia TABLE II 357 Extinct Freshwater Unionoidean Bivalves Species Common name States of occurrence Alasmidonta mccordi Athearn, 1964 Alasmidonta robusta Clarke, 1981 Alasmidonta wrightiana (Walker, 1901) Elliptio nigella (Lea, 1852) Epioblasma arcaeformis (Lea, 1831) Epioblasma biemarginata (Lea, 1857) Epioblasma ﬂexuosa (Raﬁnesque, 1820) Epioblasma ﬂorentina ﬂorentina (Lea, 1857) Epioblasma haysiana (Lea, 1834) Epioblasma lenior (Lea, 1842) Epioblasma lewisii (Walker, 1910) Epioblasma obliquata obliquata (Raﬁnesque, 1820) Epioblasma personata (Say, 1829) Epioblasma propinqua (Lea, 1857) Epioblasma sampsonii (Lea, 1861) Epioblasma stewardsonii (Lea, 1852) Epioblasma torulosa gubernaculum (Reeve, 1865) Epioblasma torulosa torulosa (Raﬁnesque, 1820) Epioblasma turgidula (Lea, 1858) Lampsilis binominata Simpson, 1900 Medionidus mcglameriae van der Schalie, 1939 Pleurobema altum (Conrad, 1854) Pleurobema avellanum Simpson, 1900 Pleurobema bournianum (Lea, 1840) Pleurobema chattanoogaense (Lea, 1858) Pleurobema ﬂavidulum (Lea, 1861) Pleurobema hagleri (Frierson, 1900) Pleurobema hanleyianum (Lea, 1852) Pleurobema johannis (Lea, 1859) Pleurobema murrayense (Lea, 1868) Pleurobema nucleopsis (Conrad, 1849) Pleurobema rubellum (Conrad, 1834) Pleurobema troschelianum (Lea, 1852) Pleurobema verum (Lea, 1861) Quadrula tuberosa (Lea, 1840) Coosa elktoe Carolina elktoe Ochlockonee arcmussel Winged spike Sugarspoon Angled rifﬂeshell Leafshell Yellow blossom Acornshell Narrow catspaw Forkshell Catspaw Round combshell Tennessee rifﬂeshell Wabash rifﬂeshell Cumberland leafshell Green blossom Tubercled blossom Turgid blossom Lined pocketbook Tombigbee moccasinshell Highnut Hazel pigtoe Scioto pigtoe Painted clubshell Yellow pigtoe Brown pigtoe Georgia pigtoe Alabama pigtoe Coosa pigtoe Longnut Warrior pigtoe Alabama clubshell True pigtoe Rough rockshell AL NC, SC FL AL, GA AL, KY, TN AL, KY, TN AL, IL, IN, KY, OH,TN AL, KY, TN AL, KY, TN, VA AL, TN AL, KY, TN AL, IL, IN, KY, OH, TN IL, IN, KY, OH AL, IL, IN, KY, OH,TN IL, IN, KY AL, KY, TN TN, VA AL, IL, IN, KY, OH,TN, WV AL, AR, TN AL, GA AL AL, GA AL OH AL, GA, TN AL AL AL, GA, TN AL AL, GA, TN AL, GA AL, GA, TN AL, GA, TN AL TN, VA (From Williams et al., 1998b.) present species assemblages with those of earlier surveys or with recent fossil assemblages (Neves and Zale, 1982; Parmalee and Klippel, 1982, 1984; Parmalee et al., 1982; Ahlstedt, 1983; Havlik, 1983; Stern, 1983; Hoeh and Trdan, 1984; Miller et al., 1984; Hartﬁeld and Rummel, 1985; Taylor, 1985; Mackie and Topping, 1988; Nalepa and Gauvin, 1988; Starnes and Bogan, 1988; Baily and Green, 1989; Bogan, 1990; Anderson et al., 1991; Counts et al., 1991; Hornbach et al., 1992; Parmalee and Hughes, 1993; Hoke, 1994; Blalock and Sickel, 1996). Extirpations of sphaeriid faunae are far less common, but have occurred (Paloumpnis and Starrett, 1960; Mills et al., 1966). Unionoidean assemblages in Indian middens near the upper Ohio River yielded at least 32 species, while a 1921 survey yielded only 25 of the midden species, and a 1979 survey, only 13 of the midden species in the same area, indicative of massive species extirpation (Taylor and Spurlock, 1982). Similar historical loss of Indian midden species has occurred in the Tennessee River Drainage (Parmalee, 1988). Further evidence of environmental change in the upper Ohio River includes recent establishment of 15 unionid species previously unreported on Indian middens or earlier surveys (Taylor and Spurlock, 1982). Such historical data clearly indicate the toll that anthropomorphic activities are taking on the NA unionoidean fauna (Neves, 1993; Neves et al., 1997). The United States Fish and Wildlife Service began listing unionoideans as threatened and endangered after passage of the Endangered Species Act of 1973. By 1998, 56 taxa were listed as endangered (Turgeon et al., 1998), with about 70% of NA unionoideans at some level of imperilment (Williams et al., 1993). Bogan (1998) reviewed the causes for decline of NA freshwater bivalve diversity and attributed it, and species extinctions, to habitat destruction (loss of both 358 Robert F. McMahon and Arthur E. Bogan unionoidean and host ﬁsh habitat), pollution including acid mine runoff, pesticides, heavy metals, commercial exploitation and introduced species. Bogan (1997) summarized this: “The central cause of this decline and decimation of the freshwater molluscan fauna is the modiﬁcation and destruction of their aquatic habitat, with sedimentation as a leading major factor. Sources of sedimentation include poor agricultural and timbering practices. Damming of major rivers has also had a dramatic impact on this fauna with the loss of unionid obligate host ﬁsh due to changes in local water quality and loss of habitat. In-stream gravel mining, dredging, and canalization have further eliminated stable aquatic habitat. Acidic mine drainage and various point and non-point pollution sources also continue to decimate local aquatic mollusk populations.” Evidence of negative anthropomorphic impacts on unionoidean populations is extensive. The freshwater pearling industry can extirpate entire populations (Laycock, 1983), overﬁshing for pearls being a major factor in the decline of the pearl mussel, Margaritifera magaritifera in Great Britain (Young and Williams, 1983a). Commercial unionid shell ﬁsheries that provide seed pearls for the marine cultured pearl industry negatively impact NA populations (Williams et al., 1993). Impoundments of rivers slow ﬂow and allow accumulation of silt, leading to mussel fauna reductions (Duncan and Thiel, 1983; Parmalee and Klippel, 1984; Stern, 1983; Starnes and Bogan, 1988; Blay, 1990; Williams et al., 1992; Houp, 1993; Parmalee and Hughes, 1993). Impoundments also eliminate glochidial ﬁsh hosts (Mathiak, 1979) or prevent ﬁsh host dispersal of glochidia. Release of cold, hypolimnetic water from impoundments negatively impacts downstream unionoidean populations (Ahlstedt, 1983; Clarke, 1983; Vaughn and Taylor, 1999). Controlled water releases from impoundments lead to major ﬂow-rate oscillations, either scouring the bottom of suitable mussel substrates during high ﬂows or causing lethal aerial exposure during low ﬂows (Miller et al., 1984; Vaughn and Taylor, 1999). Channelization of drainage systems for navigation or ﬂood control is detrimental to unionoideans. Increased ﬂow velocity and propeller wash elevate suspended solids, which interfere with mussel ﬁlter feeding and O2 consumption (Aldridge et al., 1987; Payne and Miller, 1987). It reduces availability of stabilized sediments, sand bars, and low-ﬂow areas, all preferred unionoidean habitats (Payne and Miller, 1989; Strayer and Ralley, 1993; Strayer, 1999a). Pollution adversely affects bivalves. Unionoidean faunas can be negatively impacted or extirpated by industrial pollution (Zeto et al., 1987; Wade et al., 1993), urban wastewater efﬂuents (sewage, silt, pesticides) and resultant eutrophication and hypoxia (Gunning and Suttkus, 1985; St. John, 1982; Neves and Zale, 1982; Arter, 1989; Strayer, 1993), silt and acid discharges from mines (Taylor, 1985; Warren et al., 1984; Anderson et al., 1991) siltation from bank erosion due to deforestation, destruction of riparian zones, and poor agricultural practice (Hartﬁeld, 1993; Williams et al., 1993) or disturbance and silt from river-bed gravel mining (Brown and Curole, 1997). Advent of modern sewage treatment on the Pearl River, Louisiana, allowed re-establishment of ﬁve previously absent unionid species (Gunning and Suttkus, 1985). Nonindigenous species (i.e., “biological pollution”) present a new threat to NA unionoidean taxa. The nonindigenous zebra mussel, D. polymorpha, byssally attaches in great numbers to the exposed, posterior, siphonal shell regions of unionoideans, leading to their slow starvation as the zebra mussels strip suspended food particles from their inhalant current. Thus, zebra mussel infestations have extirpated a number of unionoidean species populations from the lower Great Lakes (Schloesser et al., 1996). 3. Physical Factors Physical factors inﬂuence bivalve distributions. While environmental requirements are species speciﬁc, a number of generalities appear warranted. Sediment type clearly affects distribution patterns. Unionoideans are generally most successful in areas where ﬂow is moderate with a stable substrate of coarse sand or sand – gravel mixtures, and are generally absent from substrates with heavy silt loads and very low water ﬂow (Strayer and Ralley, 1993; Strayer, 1999a). In the Wisconsin and St. Croix rivers, only 7 of 28 unionid species occurred in sand – mud sediments, the majority preferring sand – gravel mixtures. Only three species, Pyganodon grandis, Lampsilis teres, and L. siliquoidea, preferred sand – mud substrates (Stern, 1983). Some unionoidean species select preferred sediment types (Baily, 1989). In rivers subjected to periodic high ﬂows, unionoideans oriented with siphons facing upstream to a greater extent than those in stable ﬂow rivers, a position which presents the narrowest proﬁle to the current, reducing chances of ﬂow-induced dislodgment (Di Maio and Corkum, 1997). The relatively speciﬁc substrate and ﬂow requirements of many unionoidean species may account for their patchy and clumped distributions (Strayer, 1999a). Courser sediments allowing free exchange of interstitial and surface waters may be a requirement for early survival of recently excysted juvenile unionoideans with low pH, hypoxia, and ammonia accumulation in interstitial water being correlated with juvenile mortality in sediments where free water exchange is reduced (Buddensiek et al., 1993). Thus, sediment limitations for juvenile development may result in patchy or clumped adult unionoidean distributions. In contrast, C. ﬂuminea is able to colonize 11. Mollusca: Bivalvia 359 FIGURE 13 Sediment relationships in sphaeriid clam communities from sites along a depth transect in Britannia Bay, Ottawa River, Canada. (A) Mean sphaeriid density (Shannon – Weaver d) values for various sphaeriid communities in relation to mean sediment geometric particle size. (B) Mean sphaeriid density values at various depths. Vertical bars about points are 95 percent conﬁdence limits. Note increase in diversity with decrease in mean sediment particle size and maximization of density at depths less than one meter. (Redrawn from data of Kilgour and Mackie, 1988.) habitats ranging from bare rock through gravel / sand to silt (McMahon, 1999), which has allowed it to invade a wide variety of NA drainage systems. Its optimal habitat is oxygenated sand or gravel – sand (Belanger et al., 1985). In contrast to unionoideans, species diversity in Pisidium increases with decreasing particle size (Fig. 13A), becoming maximal at a mean particle diameter of 0.18 mm (Kilgour and Mackie, 1988). In southeastern Lake Michigan, Pisidium density and diversity were maximal in very ﬁne sand – clay and silt – clay sediments, while peak Sphaerium diversity occurred at somewhat larger particle sizes (Zbeda and White, 1985). Thus, there are differences in substrate preferences among sphaeriids, perhaps associated with sediment organic detritus feeding mechanisms in Pisidium (see Section III.C.2). Apparent differences in substrate preferences may be associated with species-speciﬁc differences in optimal water velocities. Unionoideans are most successful where velocities are low enough to allow sediment stability, but high enough to prevent excessive siltation (Strayer and Ralley, 1993; Strayer, 1999a), making well-oxygenated, coarse sand and sand – gravel beds optimal habitats for riverine species with such habitats being more critical for survival of juvenile unionoideans than adults (Buddensiek et al., 1993). Low or variable velocities allow silt accumulations that make sediments too soft for maintenance of position in adult unionoideans (Lewis and Riebel, 1984; Salmon and Green, 1983) or which interfere with their ﬁlter feeding and gas exchange (Aldridge et al., 1987). In contrast, periodic scouring of substrates during high ﬂows removes substrate and unionoideans, and prevents their successful resettlement (Young and Williams, 1983b). Indeed, unionoidean densities decline in areas of high ﬂow (Way et al., 1990b). Sediment type did not affect burrowing in three lotic unionid species (P. grandis, Elliptio complanata, and Lampsilis siliquoidea) (Lewis and Riebel, 1984), suggesting that it is not involved in substrate preferences, but unionoideans may move to preferred sediments (Baily, 1989). C. ﬂuminea, with its relatively heavy, ridged shell and rapid burrowing ability, is better adapted for life in high current velocities and unstable substrates than most unionoideans (McMahon, 1999). In the Tangipahoa River, Mississippi, it colonizes unstable substrates from which unionoideans are excluded (Miller et al., 1986). In contrast to the majority of unionoideans and C. ﬂuminea, many sphaeriid species occur in small ponds and the profundal portions of large lakes, where water ﬂow is negligible and the substrate has high silt contents and heavy organic loads (Burky, 1983). Preference of some sphaeriids for low-ﬂow, silty habitats may reﬂect interstitial sediment detritus feeding, particularly in Pisidium (Lopez and Holopainen, 1987) (see Section III.C.2). Byssal attachment allows D. polymorpha to inhabit relatively high ﬂow areas compared to other bivalves (pediveliger larvae settle in ﬂows up to 1.5m / sec). It also makes it successful epibenthic species in lentic habitats with a preponderance of hard substrates from which native NA bivalves are generally eliminated (Claudi and Mackie, 1993). 360 Robert F. McMahon and Arthur E. Bogan Water depth affects freshwater bivalve distributions. Most unionoideans prefer shallow habitats less than 4 – 10 m deep (Stone et al., 1982; Salmon and Green, 1983; Machena and Kautsky, 1988; Way et al., 1990b), although some species occur in deeper lotic waters if well oxygenated. C. ﬂuminea is also restricted to shallow, near-shore lentic habitats (McMahon, 1999), as are the majority of Sphaerium and Musculium species (Fig. 13B) (Zbeda and White, 1985; Kilgour and Mackie, 1988). In contrast, some species of Pisidium inhabit profundal regions of lakes (Holopainen and Jonasson, 1983; Kilgour and Mackie, 1988). Depth distributions of D. polymorpha vary between habitats; however, adults rarely occur above 2 m and dense populations extend to 4 – 60 m, but are restricted to well-oxygenated waters above the epilimnion. The quagga mussel, D. bugensis, extends to greater, oxygenated depths than D. polymorpha in the Great Lakes (Mills et al., 1996), but is unlikely to penetrate hypoxic hypolimnetic waters as it is less hypoxia tolerant than D. polymorpha (P. D. Johnson and R. F. McMahon, unpublished data). Recently settled juveniles of D. polymorpha migrate to deeper water (Mackie and Schloesser, 1996), perhaps avoiding wave-induced agitation (Clarke and McMahon, 1996b) or ice scour. Limitation of most lentic bivalves to shallow habitats may reﬂect their poor hypoxia tolerance. In lentic habitats, hypolimnetic waters are often hypoxic. As many species of unionoideans, Sphaerium and Musculium (Burky, 1983), Dreissena (McMahon, 1996) and C. ﬂuminea (McMahon, 1999) cannot maintain normal O2 uptake under severely hypoxic conditions, they are mostly restricted to shallow, well-oxygenated habitats. Juvenile unionids are less hypoxia-tolerant than adults (Dimock and Wright, 1993), preventing colonization of hypoxic habitats. In contrast, many species of Pisidium are extreme V̇O2 regulators ( Burky, 1983) allowing them to inhabit highly hypoxic hypolimnetic habitats (Jonasson, 1984a, b). However, summer hypoxia retards growth and reproduction in profundal Pisidium populations, indicating that hypoxia can deleteriously impact even hypoxia-tolerant species (Halopainen and Jonasson, 1983). Hypoxiaintolerant C. ﬂuminea invaded the profundal regions of a small lake only after artiﬁcial aeration of its hypoxic hypolimnetic waters (McMahon, 1999). Sewageinduced hypoxia in the Pearl River, Louisiana, extirpated its unionoidean fauna (Gunning and Suttkus, 1985). Even hypoxia-tolerant profundal Pisidium communities are extirpated by extreme hypoxia (Jonasson, 1984a). Ambient pH does not greatly limit the distribution of freshwater bivalves. The majority of species prefer waters of pH above 7.0; species diversity declines in acidic habitats (Okland and Kuiper, 1982). However, unionoideans can grow and reproduce over a pH range of 5.6 – 8.3, a pH of less than 4.7 – 5.0 being the absolute lower limit (Fuller, 1974; Okland and Kuiper, 1982; Kat, 1982; Hornbach and Childers, 1987). Low pH may have sublethal effects on unionoideans. It reduces shell thickness (Hinch et al., 1989), tissue cholesterol content (Rao et al., 1987), and hemolymph concentrations of Na, K, and Cl (Pynnönen, 1991; Mäkelä and Oikari, 1992). Unionoidean glochida and juveniles are less low pH tolerant than adults (Huebner and Pynnönen, 1992; Dimock and Wright, 1993), perhaps preventing colonization of mildly acidic waters. In contrast, Pyganodon grandis showed no change in hemolymph Na, K, and Cl concentrations after transfer into an acidic lake (pH 5.9) from an alkaline lake (Malley et al., 1988). Some sphaeriids are relatively insensitive to pH or alkalinity. Species richness, growth and reproduction in sphaeriid faunas of six lowalkalinity lakes was similar to those in higher alkalinity lakes (Rooke and Mackie, 1984a, b; Servos et al., 1985). Musculium partumeium and Pisidium casertanum inhabited acid lakes in New York State (pH 6.0) (Jokinen, 1991). Indeed, maximal laboratory growth and reproduction in Musculium partumeium occurred at pH 5.0, suggesting adaptation to moderately acidic habitats (Hornbach and Childers, 1987). Low-pH habitats generally have low calcium concentrations. Low pH leads to shell dissolution and eventual mortality if shell penetration occurs (Kat, 1982). Sphaeriids occur in waters with Ca concentrations as low as 2 mg Ca / L, while the unionid, Elliptio complanata, occurs at 2.5 mg Ca / L (Rooke and Mackie, 1984a). Among freshwater bivalves, D. polymorpha is the most calciphilous and pH intolerant; adults requiring pH 6.5 and 12 mg Ca / L and veliger larvae requiring pH 7.4 and 24 mg Ca / L for successful development (McMahon, 1996). Freshwater bivalves actively take up Ca2 at medium concentrations as low as 0.5 mM Ca / L (0.02 mg Ca / L, see Section II.A.1), which is far below their minimal ambient Ca2 concentration of 2 – 2.5 mg / L. Thus, the minimum ambient calcium concentration appears to be that at which the rate of calcium uptake and deposition to the shell exceeds that of calcium loss from shell dissolution and diffusion, allowing maintenance of shell integrity and growth. As many factors affect shell deposition and dissolution rates (e.g., temperature, pH, and calcium concentration), the minimal Ca concentration and / or pH tolerated by a species may vary between habitats dependent on interacting biotic and abiotic parameters and are often species-speciﬁc. Low calcium waters usually have low concentrations of other biologically important ions, making them inhos- 11. Mollusca: Bivalvia pitable to bivalves even if Ca concentrations are suitable for shell growth. Temperature inﬂuences bivalve distributions; species have speciﬁc upper and lower limits for survival and reproduction (Burky, 1983). For example, intolerance of 2°C prevents C. ﬂuminea from colonizing drainages in the northcentral United States, which reach 0°C in winter (Fig. 11) (McMahon, 1999), leading to low-temperature winter-population kills on the northern edge of its range (Sickel, 1986). Thus, many northern U.S. C. ﬂuminea populations are restricted to areas receiving heated efﬂuents (McMahon, 1999). In contrast, the maximal temperature for development of D. polymorpha eggs and larva is 24°C and adults do not tolerate temperatures 30°C, preventing this species from colonizing southern and southwestern U.S. waters which exceed 30°C in summer (McMahon, 1996). Indeed, in the most southern U.S. states, D. polymorpha only occurs in the lower Mississippi River which rarely exceeds 30°C (Hernandez et al., 1995). Water-level variation affects bivalve distributions. Declining water levels during droughts or dry periods expose relatively immotile bivalves for weeks or months to air. Restriction of many bivalve populations to shallow waters makes them susceptible to emersion. Many sphaeriid species and some unionoidean taxa are highly tolerant of air exposure, surviving prolonged seasonal emersion in ephemeral or variable level habitats (Burky, 1983; White, 1979; Byrne and McMahon, 1994). These species display unique emersion adaptations (Byrne and McMahon, 1994 and Section II.C.4). Freshwater bivalve distribution is also related to stream size or order. Unionoidean species diversity increased with distance downstream in the Sydenhan River, Ontario (Mackie and Topping, 1988). Similarly, stream size proved to be the only useful predictor of unionoidean species richness of six tested factors (i.e., stream size, stream gradient, hydrologic variablity, Ca concentration, physiographic province and presence / absence of tides) in the Susquehanna, Delaware and Hudson River drainages (Strayer, 1993). The number of unionoidean species in Michigan drainage systems increased proportionately with system size; mainly by species additions (Strayer, 1983) (Fig. 14), but species richness could not be completely accounted for by drainage area size (note the high degree of variation in species richness versus drainage area values in Fig. 14), suggesting that other environmental variables affect unionoidean distribution patterns (Strayer, 1993). In the Ohio River drainage, the number of ﬁsh species was directly related to drainage area, and the number of unionoidean species directly related to the number ﬁsh species, suggesting that 361 FIGURE 14 Unionoidean mussels species richness (total number of species present at a particular site) as a function of stream size measured by its total drainage area in km2 in southeastern Michigan, United States. Numbers next to points indicate the number of observations falling on that point. The relationship between drainage area and mussel species richness was statistically signiﬁcant r 0.68, P 0.001). (Redrawn from Strayer, 1983.) unionoidean diversity is partially dependent on ﬁsh glochidial host species availability (Watters, 1993). Similarly, Margaritifera hembeli populations with the highest juvenile recruitment rates occurred in stream reaches with the highest densities of its ﬁsh host (Johnson and Brown, 1998). Among other variables affecting species richness are stream hydrology, affected by surface geology and soil porosity. Porous soils retain water, buffering runoff, so that streams draining them have constant ﬂows and rarely dry, allowing them to support greater mussel diversity than streams draining soils of poor water-inﬁltration capacity that are prone to ﬂooding – drying cycles. In variable-ﬂow streams, emersion or low oxygen in stagnant pools during dry periods, and bottom scouring and high silt loads during ﬂoods reduce species richness (Strayer, 1983). Indeed, ﬂow stability, high O2, reduced ﬂood risk, and low silt loads of larger streams appear to account for their increased bivalve species richness (Fig. 14) (Strayer, 1983, 1993; Way et al., 1990b; Strayer and Ralley, 1993). However, some unionoidean species, such as Amblema plicata, Pyganodon grandis and Fusconaia ﬂava, are adapted to small, variable ﬂow streams (Strayer, 1983; Di Maio and Corkum, 1995) while other species favor tidally inﬂuenced portions of rivers (Strayer, 1993). The above physical factors may interact to determine distributions of bivalves. The density of the unionoidean, M. hembeli, was affected by stream order, hardness, depth, substrate size and compaction, and 362 Robert F. McMahon and Arthur E. Bogan ﬂow rate, with highest densities occurring in shallow areas of second-order streams, of hardness 8 mg / L, with higher ﬂow rates, and stable sediments with larger particle sizes (Johnson and Brown, 2000). B. Reproduction and Life History North American freshwater bivalves display extraordinary variation in life history and reproductive adaptations. Life-history traits (e.g., those affecting reproduction and survival, including growth, fecundity, life span, age to maturity, and population energetics) have been reviewed for freshwater molluscs (Calow, 1983; Russell-Hunter and Buckley, 1983) and speciﬁcally for freshwater bivalves (Burky, 1983; Mackie, 1984; Mackie and Schloesser, 1996; McMahon, 1999). Most research involves the Sphaeriidae, with less information for unionoideans. Sphaeriids are good subjects for life-history studies, because of their greater abundances, ease of collection and laboratory maintenance, relatively simple hermaphroditic life cycles, ovovivipar- ity, semelparity, release of completely formed miniature adults, and relatively short life spans. In contrast, unionoideans are more difﬁcult subjects because they are gonochoristic, long-lived, interoparous, often rare and difﬁcult to collect, and have life cycles complicated by the parasitic glochidial stage. The life-history traits of C. ﬂuminea and D. polymorpha have been intensely studied due to their invasive nature and economic importance as fouling organisms (Mackie and Schloesser, 1996; Nichols, 1996; McMahon, 1999). 1. Unionoidea The life-history characteristics of freshwater unionoideans are clearly different from those of sphaeriids, C. ﬂuminea or D. polymorpha (Table III). The majority of unionids live in large, stable aquatic habitats which buffer them from periodic catastrophic population reductions, typical of smaller, unstable aquatic environments (see Section III.A). In stable habitats, long-lived adults accumulate in large numbers (Payne and Miller, 1989). A few species inhabit ponds TABLE III Summary of the Life History Characteristics of North American Freshwater Bivalves, Unionoidea, Sphaeriidae, Corbicula ﬂuminea, Driessena polymorpha Life history traita Unionoidea Sphaeriidae Corbicula ﬂuminea Dreissena polymorpha Life span (years) 6 – 100 (Species dependent) 6 – 12 1 – 5 (species dependent) 1–4 4–7 0.17 – 1.0 (1 year in some species) Hermaphroditic 025 – 0.75 0.5 – 2 Hermaphrodictic self-fertilizing Rapid throughout life 35,000 Gonochoristic Age at maturity (years) Reproductive mode Fecundity (young per avg. adult per breeding season) Gonochoristic (few hermaphroditic species) Rapid prior to maturity, slower thereafter 200,000 – 17,000,000 per female Juvenile size at release Very small, 50 – 450 m Slow relative to Unionoideans, C. ﬂuminea or D. polymorpha 3 – 24 (Sphaerium) 2 – 136 (Musculium) 3 – 7 (Pisidium) Large 600 – 4150 m Relative juvenile survivorship Relative adult survivorship Extremely low High High Intermediate Semelparous or iteroparous Highly iteroparous Number of reproductive efforts per year Assimilated energy respired (%) Nonrespired energy allocated to growth (%) Nonrespired energy allocated to reproduction (%) Turnover time in days ( mean standing crop biomass : biomass per day ratio) Habitat stability One Growth rate a Very small 250 m Rapid throughout life 30,000 – 40,000 per female — Semelparous or Iteroparous (species dependent) 1 – 3 (continuous in some species) 21 – 91 (Avg. 45%) Extremely low Low 2 – 41% per year Moderately iteroparous Two (spring and fall) 11 – 42 Extremely small, 40 m Extremely low Intermediate 26 – 88% per year Moderately iteroparous One (2 – 8 months long) — 85.2 – 97.5 65 – 96 (Avg. 81%) 58 – 71 96.1 2.8 – 14.8 4 – 35 (Avg. 19%) 15 4.9 1790 – 2849 27 – 1972 (generally 80) 73 – 91 53 – 869 (habitat dependent) Stable Intermediately stable Unstable Moderately unstable See text for literature citations to data on which this table was based. 11. Mollusca: Bivalvia 363 FIGURE 15 Anatomic features of freshwater bivalve larval stages. (A) the D-shaped juvenile of Corbicula ﬂuminea, the freshwater Asian clam (shell length 200 m). (B) The glochidium larva of unionoideans, which is parasitic on ﬁsh. Depicted is the glochidium of Pyganodon characterized by the presence of paired spined hooks projecting medially from the ventral edges of the shell valves and an attachment thread; not all unionoidean species have glochidia with these characteristics (50– 400 m in diameter depending on species). (C) Lateral and anterior views of the free-swimming, planktonic veliger stage of Dreissena polymorpha, the zebra mussel; the veliger is 40 –290 m in diameter and uses the ciliated velum to swim and feed on phyto- and bacterioplankton. Presence of a foot as shown in this diagram indicates development to the settlement competent pediveliger stage. The juveniles of freshwater sphaeriid species are large and highly developed, having essentially adult features (Fig. 2A) at birth. (Burch, 1975b), but their life-history traits have not been studied. An important aspect of the unionoidean life-history traits is their parasitic glochidium larval stage (Fig. 15B). With the exception of Simpsonaias ambigua, whose glochidial host is the aquatic salamander, Necturus maculosus, all other known NA unionoideans have glochidial ﬁsh hosts (Watters, 1994b). The signiﬁcance of glochidia unionoidean reproduction has been reviewed by Kat (1984) and details of glochidial incubation and development in marsupial brood pouches were described in Section II.B.5. The glochidium has a bivalved shell adducted by a single muscle. Its mantle edge contains sensory hairs. In the genera Unio, Anodonta, Pygauodon, Megalonaias, and Quadrula, a long threadlike structure projects from the mantle beyond the ventral valve margin which may be involved with detection of and / or attachment to, ﬁsh hosts. There are three general forms of glochidia. In the subfamily Anodontinae, “hooked glochidia” occur, with triangular valves from whose ventral edges project an inward curving hinged hook covered with smaller spines (Fig. 15B). On valve closure, the hooks penetrate the skin, scales, or ﬁns of ﬁsh hosts, allowing glochidial attachment and encystment on host external surfaces. The majority of NA unionoideans produce “hookless glochidia” with more rounded valves bearing reinforcing structures and / or small spines or stylets on their ventral margins which generally attach and encyst on ﬁsh gills. “Axe-head glochidia” of the genus Potamilus have a ﬂared ventral valve margin, and near-rectangular valves which may have hooklike structures on each corner. Their host attachment sites are unknown (Kat, 1984). Glochidia initially attach to ﬁsh by clamping (snapping) the valves onto ﬁns, scales, and / or gill ﬁlaments. They are not host-speciﬁc in attachment, attaching to any contacted ﬁsh (Kat, 1984). In contrast, gravid adult unionids (European Anodonta anatina) appear to release glochidia in response to chemicals released by host ﬁsh (Jokela and Palokamgas, 1993). Once 364 Robert F. McMahon and Arthur E. Bogan released, glochidia display ‘snapping’ behavior’ (i.e., host attachment behavior), the valves being rapidly and repeatedly adducted. In M. margaritifera, glochidial valve snapping is stimulated by the presence of mucus, blood, gill tissue, or ﬁns of their brown trout host, but not by water currents or tactile stimulation (Young and Williams, 1984a), suggesting use of chemical cues to detect and attach to host ﬁsh. Glochidia encyst in ﬁsh host tissues within 2 – 36 h of attachment and may or may not grow during encystment, depending on species. Time to juvenile excystment is also species-dependent, ranging from 6 to 160 days, but is reduced at higher temperatures (Zale and Neves, 1982; Kat, 1984). Unsuitable host ﬁsh reject glochidia after encystment (Kat, 1984), ﬁsh blood serum components dictating host suitability (Neves et al., 1985). Since glochidia nonselectively attach to ﬁsh (Kat, 1984; Neves et al., 1985), host suitability appears more dependent on ﬁsh immunity than on glochidial host recognition. Indeed, even suitable host ﬁsh reject glochidia, rejection rate being host ﬁsh species-dependent (Haag et al., 1999). Numbers of M. margaritifera glochidia encysted in a brown trout population declined with time (Young and Williams, 1984b), with laboratory studies revealing that only 5 – 12% of M. margaritifera glochidia successfully excyst from infected host ﬁsh, indicating host rejection of most encysted individuals (Young and Williams, 1984a). Unionoideans have adaptations that increase likelihood of glochidia contact with ﬁsh hosts. Glochidial release occurs annually, but duration of release is speciesdependent with some species having more than one annual release period (Fukuhara and Nagata, 1988; Gordon and Smith, 1990). Cycles of gametogenesis and glochidial release may be controlled by neurosecretory hormones (Nagabhushanam and Lomte, 1981). Tachytictic mussels are short-term breeders, whose glochidial development and release occurs between April and August; shedding of glochidia often corresponding with either migratory periods of anadromous ﬁsh hosts, or nesting periods of their host ﬁsh. Fish hosts of tachytictic unionoideans often construct nests in areas harboring dense unionoidean populations, where ﬁsh host nest construction by fanning away substrates, and ﬁsh host fanning of developing embryos provide optimal conditions for glochidial – host contact. Thus, a high proportion of nest-building ﬁsh species, such as centrarchids, are glochidial hosts for NA unionoideans (Watters, 1994b). In contrast, bradytictic unionoidean species are long-term breeders, retaining developing glochidia in gill marsupia throughout the year, releasing them in summer (Kat, 1984). When released from adult mussels, glochidia are generally bound by mucus into discrete packets, which either dissolve, releasing glochidia, or remain intact as discrete “conglutinates” of various species-speciﬁc forms and colors. Glochidia with attachment threads (Fig. 15B) are released in tangled mucus threads that dissolve relatively rapidly. Many of these glochidia possess hooks and attach to ﬁsh-host external surfaces. Hooked glochidia are larger than other types of glochidia (Bauer, 1994). In some unionoideans, mucilaginous networks of glochidia persist, enhancing host contact by suspending glochidia above the substrate. Unionids with hookless glochidia that attach to ﬁsh gills may release conglutinates that mimic the food items of their ﬁsh hosts. They resemble brightly colored oligochaetes, ﬂatworms, or leeches. Some species hold their wormlike conglutinates partially extruded from the exhalant siphon, making them more obvious to ﬁsh hosts. Some Lamsilis species, such as, Lampsilis perovalis, produce a “supercongultinate” that contains all the glochidia in the marsupial gill and resembles a small ﬁsh. It is tethered to the female’s exhalant siphon by a long, transparent mucus strand (Fig. 16A). In ﬂowing water, it mimics the darting motions of a small ﬁsh, eliciting attacks by host ﬁsh (Haag et al., 1995). Consumption of such conglutinates releases glochidia within the buccal cavity of the ﬁsh followed by transport to gill ﬁlament attachment sites on ventilatory currents. The most unusual unionoidean host food mimicry involves pigmented muscular, posterior mantle edge extensions in female Lampsilis and Villosa. These “mantle ﬂaps’ (Fig. 16B) resemble the small ﬁsh or macroinvertebrate prey of their ﬁsh hosts (Haag and Warren, 1999). Gravid females extend the posterior shell margins well above the substrate and pulsate the projecting mantle ﬂaps to mimic a small, actively swimming ﬁsh or moving macroinvertebrate. When ﬁsh strike these mantle lures, glochidia are forcibly released through posterior pores in the marsupial gill (often projected between the mantle ﬂaps, Fig. 16B) assuring glochidial ingestion and contact with the ﬁsh host gills (Kat, 1984; Haag and Warren, 1999). As the glochidia of some unionoideans do not grow while encysted, their parasitic nature has been questioned. However, in vitro glochidial culture experiments suggest that they absorb organic molecules from ﬁsh tissues and require ﬁsh plasma for development and metamorphosis (Isom and Hudson, 1982), indicative of a true host – parasite relationship. Indeed, glochidial infection damages ﬁsh hosts, especially juvenile ﬁsh (Cunjak and McGladdery, 1991; Panha, 1993). Like other parasites, glochidia are shed in huge numbers to ensure the maximum host contact and attachment. Unionoidean fecundity ranges from 10,000 to 17,000,000 glochidia per female per breeding season (Parker et al., 1984; Young and Williams, 1984b; 365 FIGURE 16 Reproductive adaptations in female Lamsilid unionoideans. (A) Superconglutinate tethered on a mucus strand extending from the exhalant siphon of Lampsilis perovalis. Glochidia are encapsulated in the ﬂattened terminal lure (detailed in the upper portion of the ﬁgure) at the end of the mucus strand which makes darting ﬁshlike movements in water currents, enticing host ﬁsh to strike the lure, thus assuring release of glochidia into its buccal cavity for attachment to the ﬁshes’ gills. (Redrawn from Haag et al., 1995) (B) The modiﬁed, mantle ﬂaps of a female specimen of Lampsilis ovata, which mimic the small ﬁsh prey (note eye spot and lateral linelike pigmentation) of its predatory host ﬁsh species. The posterior portions of the marsupial outer demibranchs are projected from the mantle cavity to lie between the ﬂap lures. When the mantle ﬂap lures are struck by an attacking host ﬁsh, glochidia are released through pores in the marsupial gill ensuring their entry into the Figh’s buccal cavity and attachment to its gills. Superconglutinates and mantle ﬂap lures are characteristic of the unionoidean genus, Lampsilis. 366 Robert F. McMahon and Arthur E. Bogan Paterson, 1985; Paterson and Cameron, 1985; Jansen and Hanson, 1991; Bauer, 1994), brood size increasing exponentially with female size (Janson and Hanson, 1991; Bauer, 1994). Probability for glochidial survival to metamorphosis is extremely small. In a natural population of M. margaritifera, a species that does not produce glochidial conglutinates, only 0.0004% of released glochidia successfully encysted in ﬁsh hosts. Of these, only 5% were not rejected before excystment; and, of those successfully metamorphosing, only 5% established as juveniles in the substrate (Young and Williams, 1984b). Thus, only one in every 100,000,000 shed glochidia became settled juveniles. High glochidial mortality makes the effective fecundity of unionoideans extremely low, which is not unusual for a species adapted to stable habitats. Similarly, only 0.007% of released glochida of Pyganodon grandis encysted in their host ﬁsh (Perca ﬂavescens), however, once encysted, they had relatively high survival, 27% surviving to, and excysting after, two years (Jansen and Hanson, 1991). Based on these data and the fecundity ranges listed in Table III, only 0.002 – 0.17 of the glochidia produced by a female unionoidean would successfully settle as juveniles in sediment. Therefore, the main advantages of the glochidial stage appear to be a directed dispersal by ﬁsh hosts into favorable habitats (Kat, 1984) and utilization of ﬁsh-host energy resources to complete development to a juvenile large enough to effectively compete after settlement in adult habitats (Bauer, 1994). There is a trade-off between glochidial size and number of glochidia released. Species with large glochidia (including toothed glochidia) produce fewer glochidia while species with small glochidia release them in great numbers (Bauer, 1994). Species releasing fewer, larger glochidia, tend to have a greater range of host-ﬁsh species and prefer lower ﬂow habitats (Bauer, 1994). Production of many small glochidia occurs in species with limited ﬁsh hosts and may increase chances of glochidial – host contact, while having a wide range of host-ﬁsh species allows production of fewer, larger glochidia, because chances of glochidium contact with a suitable host are higher. In the highly K-selected, stable habitats of most unionoideans, excysting at a large size makes juvenile mussels more competitive, increasing likelihood of survival to maturity. Thus, small glochidia of unionoideans, such as Margaritifera, remain encysted for longer periods (1000 days), allowing growth to a more competitive size before excystment. In contrast, among species with larger glochidia, the encystment period is much shorter (200 – 300 days) before attainment of competitive juvenile size. The long encystment periods of small glochidia reduce their chances for successful excystment due to increased chances of host mortality and / or rejection by the host’s immune system, selecting for production of greater numbers of glochidia to assure recruitment of enough juveniles to sustain population size (Bauer, 1994). Glochidial utilization of ﬁsh-host energy stores prevents their direct competition with adults for limited food and space resources (Bauer, 1994), as occurs in juvenile C. ﬂuminea and D. polymorpha. Glochidial parasitism also allows female unionoideans to devote relatively small amounts of nonrespired, assimilated energy to reproduction (2.8 – 14.5% of total nonrespired, assimilated energy), leaving the majority for somatic tissue growth (Table V) (Negus, 1966; James, 1985; Paterson, 1985). Allocation of a high proportion of energy to tissue growth is characteristic of species adapted to stable habitats (i.e., K-selected species). As unionoideans are often very long-lived (Margaritifera margaritifera up to 130 – 200 years) and highly iteroparous (6 – 10 reproductive periods throughout life), allocation of the majority of nonrespired energy to growth increases probability of adult survival to future reproductive efforts. Increased growth rate and reduction of reproductive effort increases competitiveness and lowers probability of predation and / or mortality associated with reproductive effort or dislodgment from the substrate during ﬂoods. All these characteristics increase unionoidean ﬁtness in stable habitats (Sibly and Calow, 1986; Bauer, 1994). In the majority of unionoideans, greatest shell growth occurs in immature individuals during the ﬁrst 4 – 6 years of life (Hanson et al., 1988; Payne and Miller, 1989; Harmon and Joy, 1990) (Fig. 17A). Indeed, relative shell growth in young unionids is greater than in sphaeriids or C. ﬂuminea and D. polymorpha (Table III). Unionoidean shell-growth rate declines exponentially with age, but tissue biomass accumulation rate remains constant or increases with age (Fig. 17A, B; see also Haukioja and Hakala, 1978). Thus, early in life, increases in shell size and biomass occur preferentially over tissue accumulation; whereas after maturity (6 years), shell growth slows and tissue accumulates at proportionately higher rates. Delayed maturity in unionoideans (6 – 12 years, Table III) allows allocation of all nonrespired assimilation to growth early in life and, due to an exponential relationship between size and fecundity, leads to reproductive effort being primarily sustained by the oldest, largest individuals (Downing et al., 1993). Growth rates in unionoideans are affected by abiotic conditions. Shell growth of ﬁeld-enclosed specimens P. grandis declined with depth, being greatest 3 m (Hanson et al., 1988). However, in natural populations, depth did not inﬂuence shell growth in P. grandis (Hanson et al., 1988) or A. woodiana (Kiss and Pekli, 1988), suggesting that vertical migration 11. Mollusca: Bivalvia 367 FIGURE 17 The shell and tissue growth of selected species of unionids (Anodonta anatina, open circles; Unio pictorum, open squares; Unio tumidus, open tiangles; Elliptio complanata, solid circles; and Pyganodon cataracta, solid squares). (A) Mean shell-length increase with increasing age over the entire life span of each species. (B) Mean dry tissue-weight increase with increasing age over the entire life span of each species. Note that increase in shell length declines with age, while dry tissue weight increases either linearly or exponentially with age. (Data from Negus, 1966; Paterson, 1985; and Paterson, and Cameron, 1985.) may mask depth inﬂuences on growth in free-living individuals (Hanson et al., 1988). Unionoidean growth rates also vary seasonally with level of precipitation (Timm and Mutvei, 1993) and can be depressed by pollutants (Harman and Joy, 1990). Once mature, large adult unionoideans display high age-speciﬁc survivorship between annual reproductive efforts, being 81 – 86% in 5 – 7 year-old Anodonta anatina (Negus, 1966), greater than 80% in mature M. margaritifera (12 – 90 years) (Bauer, 1983), with a lower value of 15.6% in Pleurobema collina (Hove and Neves, 1994). High adult survivorship, long life spans, and low juvenile survivorship account for the preponderance of large adult individuals in natural unionoidean populations (Bauer, 1983; James, 1985; Negus, 1966; Paterson, 1985; Paterson and Cameron, 1985; Tevesz et al., 1985; Hansen et al., 1988; Huebner et al., 1990; Woody and Holland-Bartels, 1993; Hove and Neves, 1994; Vaughn and Pyron, 1995). Populations dominated by adults are characteristic of stable, highly competitive habitats (Sibly and Calow, 1986). The preponderance of large, long-lived adults in unionoidean populations causes them to have high proportions of standing crop biomass relative to biomass production. The relationship between standing crop biomass and biomass production rate can be expressed as turnover time, computed in days as the average standing crop of a population divided by its mean daily productivity rate (Russell-Hunter and Buckley, 1983). Long-lived unionids, with populations dominated by large adults, have extremely long turnover times ranging from 1790 – 2849 days (computed from data in James, 1985; Negus, 1966; Paterson, 1985; Huebner et al., 1990) compared with sphaeriids (27 – 1972 days), C. ﬂuminea (73 – 91 days), or D. polymorpha (53 – 869 days) (Table III). Such extended turnover times 368 Robert F. McMahon and Arthur E. Bogan characterize long-lived, iteroparous species from stable habitats (Russell-Hunter and Buckley, 1983). In only one aspect do unionoideans deviate from the life-history traits expected of species inhabiting stable habitats and experiencing extensive competition (i.e., K-selected). That is in the production of large numbers of small young (glochidia). However, as described above, this is an adaptation ensuring sufﬁciently high probability of glochidial – host ﬁsh contact. Thus, species producing conglutinates resembling ﬁsh host prey items have greater glochidial – host contact and produce fewer (200,000 – 400,000 glochidia per female) and larger glochidia (Kat, 1984). In contrast, M. margaritifera, which releases very small, dispersed glochidia, has extraordinarily high fecundities (up to 17,000,000 glochidia per female) (Young and Williams, 1984b). Extended life spans, delayed maturity, low effective fecundities, reduced dispersal, high habitat selectivity, poor juvenile survival and extraordinarily long turnover times make unionoidean populations highly susceptible to human perturbations. Because of their life-history traits (particularly long life spans and low effective fecundities), they do not recover rapidly once decimated by pollution or other human- or naturallymediated habitat disturbances (see Section III.A). Successful juvenile settlement appears particularly affected by disturbance, with population structures indicating periods when entire annual generations are not recruited (Bauer, 1983; Negus, 1966; Payne and Miller, 1989). Reduction in population densities may reduce fertilization success in unionoideans. Complete failure of fertilization in Elliptio complanata occurred at densities 10 mussels / m2 with densities 40 mussels / m2 required for 100% of females to be fertilized (Downing et al., 1993). Thus, anthropomorphically inﬂuenced reduction of unionoidean population densities to low levels could prevent further recruitment. Such disturbance-induced lack of juvenile recruitment raises the specter of many NA unionoidean populations being composed of dwindling numbers of long-lived adults destined for extirpation as anthropomorphic disturbances prevent reproduction and / or juvenile recruitment in aging adult populations (for an example, see Vaughn and Pyron, 1995). 2. Sphaeriidae The Sphaeriidae display great intra- and interspeciﬁc life history variation (Holopainen and Hanski, 1986; Mackie, 1984; Way, 1988). Like unionoideans, their lifehistory traits do not fall into suits associated with stable or unstable habitats. Instead, they include the short life spans, early maturity, small adult size, and increased energetic allocation to reproduction associated with adaptation to unstable habitats, and the slow growth rates, low fecundity, and release of large, fully developed young associated with adaptation to stable habitats (Sibly and Calow, 1986) (Table III). Sphaeriids are euryoecic (i.e., have a broad habitat range); some members inhabiting stressful habitats, such as ephemeral ponds, and small, variable ﬂow streams, while others live in stable, profundal lake habitats (Burky, 1983). Here, we generalize the life-history traits of sphaeriids within adaptive and evolutionary frameworks. However, the degree of inter- and intraspeciﬁc life-history variation in this group is such that, for each generality, speciﬁc exceptions can be cited (Mackie, 1979). Central to understanding sphaeriid life-history traits is their viviparous reproduction (Mackie, 1978). All species brood embryos in specialized chambers formed from evaginations of the exhalant side of the inner demibranch gill ﬁlaments. Maternal nutrient material is supplied to embryos supporting their growth and development to release as fully formed miniature adults (see Section II.B.5). Thus, even though adult sphaeriids are the smallest of all NA freshwater bivalves, they release the largest young (Mackie, 1984). Among 13 sphaeriid species, average birth shell length ranged from 0.6 to 4.15 mm (Burky, 1983; Holopainen and Hanski, 1986; Hornbach and Childers, 1986; Hornbach et al., 1982; Mackie and Flippance, 1983a), much larger than unionoidean glochidia (0.05 – 0.45 mm, Bauer, 1994), C. ﬂuminea juveniles (0.25 mm, McMahon, 1999), or D. polymorpha veliger larvae (0.04 – 0.07 mm, Nichols, 1996) (Table III). Based on shell lengths, newborn sphaeriids have (3.4 – 2.1) 105 times greater biomass than newborns of other groups. Ratios of maximum adult shell length : birth shell length in sphaeriids range from 2.8:1 to 5.4:1, suggesting that newborn juveniles have biomass 0.6 – 4.6% of the maximum biomass of adults. There is a signiﬁcant direct relationship between these two parameters, shown in Figure 18. Large offspring size reduces sphaeriid fecundity. Average clutch sizes range from 3 to 24 per young per adult for Sphaerium, 2 to 136 per young per adult for Musculium, and 1.3 to 16 per young per adult for Pisidium (Burky, 1983; Holopainen and Hanski, 1986). Even with reduced fecundity, large juvenile biomass requires allocation of large amounts of nonrespired energy to reproduction (x 19%, Burky, 1983) compared to unionoideans (14.8%), C. ﬂuminea (15%) or D. polymorpha (4.9%) (Table III). As developing juveniles have high metabolic rates (Burky, 1983; Hornbach, 1985; Hornbach et al., 1982) and are supported by adult energy stores (Mackie, 1984), estimates of reproductive costs based on released juvenile biomass may grossly underestimate actual reproductive costs in this group. 11. Mollusca: Bivalvia FIGURE 18 The relationship between shell length (SL) of juveniles at birth and maximal SL of adults for 13 species of sphaeriid freshwater clams. Note that juvenile birth length increases linearly with maximal adult size, suggesting that adult size may limit juvenile birth size in sphaeriids. The solid line represents the best ﬁt of a linear regression relating birth size to maximal adult size as follows: Birth SL 0.0387 0.236 mm (maximal adult SL in mm) (n 13, r 0.935, F 76.3, P 0.0001). (Data from Holopainen and Hanski, 1986; Hornbach and Childers, 1986; Hornbach et al., 1982; and Mackie and Flippance, 1983a.) Life-history hypotheses predict that the low fecundity and large birth size of sphaeriids would maximize ﬁtness in stable habitats. However, the majority of sphaeriids inhabit variable ponds and streams or profundal habitats subject to hypoxia (Burky, 1983; Holopainen and Hanski, 1986). Burky (1983) and Way (1988) have argued that the harsh conditions of such habitats are seasonally predictable, making them, in reality, stable. Thus, sphaeriid species inhabiting them have adaptations that prevent catastrophic population reductions during seasonal episodes of environmental stress (see Section II.C). Thus, sphaeriid populations approach carrying capacity, leading to selection for lifehistory adaptations associated with the intense intraspeciﬁc competition characteristic of life in stable habitats. Seasonal water-level ﬂuctuation and hypoxia often more severely impact smaller individuals. Thus, production of large, well-developed juveniles by sphaeriids may increase their probability of surviving predictable environmental stress. As such stress can cause high adult mortality (Burky et al., 1985b; Holopainen and Jonasson, 1983; Hornbach et al., 1982; Jonasson 1984b), ﬁtness would also be increased 369 by devoting greater proportions of nonrespired assimilation to any one reproductive effort, as chances of adult survival to the next reproduction are low. Thus, sphaeriids devote relatively higher proportions of energy to reproduction than other freshwater bivalves (Burky, 1983) (Table III). This combination of r- selected and K-selected traits, optimizing sphaeriid ﬁtness in habitats with periodic, predictable stress, is a lifehistory strategy called “bet hedging” (Stearns, 1980). The early maturation, characteristic of many sphaeriid species (Burky, 1983; Holopainen and Hanski, 1986) (Table III), is due to the advanced development of newborn juveniles, and allows reproduction before onset of seasonal environmental stress. This trait is particulary displayed by sphaeriid species inhabiting ephemeral ponds and streams. They have rapid growth, early maturity, and reduced numbers of reproductive efforts. Thus, Musculium lacustre, Pisidium clarkeanum, and P. annandalei from temporary drainage furrows in Hong Kong live less than 1 year, and have only one or two life-time reproductive efforts (Morton, 1985, 1986). Similarly, Musculium lacustre, Pisidium casertanum, P. variable, Spharium fabule, and Musculium securis had only 1 – 3 reproductive efforts within life spans of 1 to 2 years (Mackie, 1979) and a lake species, S. japonicum, is univoltine and semelparous (Park and Okino, 1994). An ephemeral pond population of Musculium partumeium lived 1 year, was semelparous, reproduced just prior to pond-drying, and devoted a large proportion of nonrespired assimilation (18%) to reproduction (Burky et al., 1985b). Ephemeral pond populations of M. lacustre were similarly univoltine and semelparous, devoting 19% of nonrespired energy to reproduction (Burky, 1983). A Sphaerium striatinum population, subject to ﬂooding, lived 1 year or less and reproduced biannually (Hornbach et al., 1982), devoting 16.1% of nonrespired assimilation to reproduction (Hornbach et al., 1984b). In contrast, when populations of these species occur in more permanent habitats, they become bivoltine, reproducing in both spring and fall. Spring generations are iteroparous, reproducing in the fall and following spring; whereas fall generations are semelparous, reproducing only in the spring (Mackie, 1979; Burky, 1983; Burky et al., 1985b). Some Pisidium species live in the profundal portions of large, more permanent lentic habitats where they tolerate periodic hypoxia after summer formation of the hypolimnion (see Section II.C.3). As these profundal species are tolerant of hypoxia, such environments are more stable than small ponds and streams, although far less productive, reducing food availability (Holopainen and Hanski, 1986). Thus, profundal sphaeriids display life-history strategies differing from those in small ponds and streams 370 Robert F. McMahon and Arthur E. Bogan (Burky, 1983; Holopainen and Hanski, 1986). Profundal populations have reduced growth rates, longer life spans of 4 to 5 years, delayed maturation often exceeding 1 year, higher levels of iteroparity, and univoltine reproductive patterns (Holopainen and Hanski, 1986), all life-history traits characteristic of more stable habitats (Sibly and Calow, 1986). Interestingly, shallowwater and profundal populations of the same species of Pisidium may display different life-history tactics. Compared to profundal populations, shallow-water populations grow more rapidly, mature earlier, have shorter life spans, and tend toward semelparity (Holopainen and Hanski, 1986), life-history traits associated with unstable habitats. As pisidiids have welldeveloped dispersal capacities, this variation in lifehistory tactics among species’ populations occupying different habitats in unlikely to result from genetic adaptation. Rather, much of this variation appears to be environmentally induced plasticity and is reﬂected in the highly variable turnover times reported for sphaeriids (range 27 – 1972 days, Table III). Freshwater bivalve growth rates are highly dependent on ecosystem productivity. Populations from productive habitats have greater assimilation, allowing allocation of greater absolute amounts of nonassimilated energy to growth (Burky, 1983). Shallow, freshwater habitats are usually highly productive, warm, and rarely O2-limited and, thus, support higher growth rates. Conversely, profundal environments are less productive, cooler, and often O2-limited, leading to lower growth rates. Since sphaeriids mature at a species-speciﬁc size irrespective of growth rate (Burky, 1983; Holopainen and Hanski, 1986), rapid growth leads to early maturity in shallow-water habitats and slow growth to delayed maturity in profundal habitats. Sphaeriids also die at species-speciﬁc terminal sizes whether terminal size is attained rapidly or slowly. As growth rate determines the time required to reach terminal size, fast-growing, shallow-water individuals reach terminal sizes more rapidly (often within less than 1 year) allowing participation in only one or two reproductive efforts, while slow-growing, profundal individuals (Holopainen and Hanski, 1986) reach terminal size more slowly, allowing participation in a greater number of reproductive efforts. Similar interpopulation growth rate and reproductive variation has been recorded in several NA sphaeriid species (Mackie, 1979). The fundamentally ecophenotypic nature this variation has been demonstrated for Pisidium casertanum. When individuals were reciprocally transferred between two populations with different life-history traits or co-reared in the laboratory, the majority of life-history trait differences proved to be environmentally induced. However, electrophoresis revealed ge- netic differences between populations and transfer and laboratory corearing demonstrated a small portion of observed variation to be genetically based (Hornbach and Cox, 1987). Extensive ecophenotypic plasticity may account for the euryoecic nature and cosmopolitan distributions of sphaeriids (see Section III.A). Certainly, capacity to adjust growth rates, maturity, reproductive cycles, life cycles, and energetic allocation patterns to compensate for habitat biotic and abiotic variation enables sphaeriids to have relatively wide niches and, thus broad distributions. 3. Corbicula ﬂuminea The introduced Asian freshwater clam, Corbicula ﬂuminea, unlike unionoideans and sphaeriids, has lifehistory traits adapting it to unstable, unpredictable habitats (McMahon, 1999), making it the most invasive of all NA freshwater bivalves. C. ﬂuminea grows rapidly, in part because it has higher ﬁltration and assimilation rates than other bivalves (Foe and Knight, 1986a; Lauritsen, 1986a; Way et al., 1990a). In a natural population, a relatively small proportion of assimilation (29%) was devoted to respiration (Table V), the majority (71%) being allocated to growth and reproduction (Aldridge and McMahon, 1978). Laboratory studies have also showed that 59 – 78% (Lauritsen, 1986a) or 58 – 89% of assimilation (Foe and Knight, 1986a) was allocated to tissue production. Thus, C. ﬂuminea has among the highest net production efﬁciencies recorded for any freshwater bivalve, reﬂected by its low turnover times, ranging from 73 to 91 days (Table III). The very high proportion of nonrespired assimilation (85 – 95%) devoted to growth in C. fluminea (Aldridge and McMahon, 1978; Long and McMahon, unpublished data), sustains rapid shell growth (shell length 15 – 30 mm in the first year of life, 35 – 50 mm in the terminal third-to-fourth year) (McMahon, 1983a). High growth rates decrease probability of predation, as fish and bird predators feed only on small individuals (Section III.C.3) thus, increasing probability of survival to next reproduction in this moderately iteroparous species. Increase in shell size occurs at the expense of tissue production during summer maximal growth (Long and McMahon, unpublished data), suggesting that larger shells optimize fitness. High growth rates sustain the highest population production rates (10.4 – 14.6 g organic carbon / m2 per year, Aldridge and McMahon, 1978) reported for any freshwater bivalve (Burky, 1983), with dense populations producing 1000 – 4500 g C / m2 per year (McMahon, 1999). Newly released juveniles are small (shell length ⬇250 m), but completely formed, with a characteristically D-shaped shell, adductor muscles, foot, statocysts, 11. Mollusca: Bivalvia gills, and digestive system (Kraemer and Galloway, 1986) (Fig. 15A). Juveniles are denser than water and settle and anchor to sediments or hard surfaces with a single mucilaginous byssal thread. However, they are small enough to be suspended in turbulent water currents and hydrologically transported (McMahon, 1999). A relatively low amount of nonrespired assimilation is allocated to reproduction (5 – 15%; Aldridge and McMahon, 1978; Long and McMahon, unpublished data), equivalent to that of unionids, but less than sphaeriids (19%, Table III). However, the species’ elevated assimilation rates allow higher actual allocation to reproduction than in other species. As the juvenile of C. ﬂuminea is small (organic carbon biomass 0.136 g), fecundity is large, ranging from 97 to 570 juveniles per adult per day during reproductive efforts, yielding an average annual fecundity of 68,678 juveniles per adult per year (McMahon, 1999). Early juvenile survivorship is extremely low and mortality rates remain high throughout life (74 – 98% in the ﬁrst, 59 – 69% in the second, and 93 – 97% in the third year) (McMahon, 1999), causing populations to be dominated by juveniles and immatures. High adult mortality and population dominance by immature individuals characterizes species adapted to unstable habitats (Stearns, 1980). The majority of NA C. ﬂuminea populations have two annual reproductive periods (i.e., are bivoltine) (McMahon, 1999). It is hermaphroditic and capable of self-fertilization (Kraemer and Galloway, 1986; Kraemer et al., 1986) such that single individuals can found new populations. Spermiogenesis occurs only during reproductive periods, but gonads contain mature eggs throughout the year (Kraemer and Galloway, 1986). C. ﬂuminea matures within 3 – 6 months at a shell length of only 6 – 10 mm (Kraemer and Galloway, 1986). Thus, juveniles born in spring can grow to maturity and participate in reproduction the following fall (McMahon, 1999) (Table III). Maximum life span is variable between populations and within populations, ranging from 1 to 4 years (McMahon and Williams, 1986a; McMahon, 1999). Early maturity allows this iteroparous species to participate in 2 – 7 reproductive efforts, depending on life span. A relatively short life span, early maturity, high fecundity, bivoltine reproduction, high growth rates, small juvenile size, and capacity for downstream dispersal make C. ﬂuminea both highly invasive and adapted for life in truly unstable, disturbed lotic habitats subject to unpredictable catastrophic faunal reductions. Its high reproductive potential and growth rates allow it to rapidly attain high densities after invading a new habitat or after catastrophic population declines. Thus, it is successful in NA drainage systems subject to 371 periodic anthropomorphic interference such as channelization, navigational dredging, sand and gravel dredging, commercial and / or recreational boating, and organic and / or chemical pollution, compared to less resilient unionoideans or sphaeriids (McMahon, 1999). Surprisingly, C. ﬂuminea is more susceptible to environmental stresses, such as temperature extremes, hypoxia, emersion, and low pH than most sphaeriids and unionoideans (McMahon, 1999), making it highly susceptible to human disturbance. With only a limited capacity to tolerate unpredictable environmental stress, why is C. ﬂuminea so successful in disturbed habitats? The answer lies in its ability to recover from disturbance-induced catastrophic population crashes much more rapidly than either sphaeriids or unionids. C. ﬂuminea rapidly re-establishes populations even if disturbance has reduced them to a few widely separated individuals, as all individuals are hermaphrodites capable of self-fertilization and have high fecundities. Downstream dispersal of juveniles from viable upstream populations allows rapid re-establishment of decimated populations, the accelerated growth, high fecundity, and relatively short life spans of this species allowing recovery to normal age – size distributions and densities within 2 – 4 years (McMahon, 1999). Biannual (bivoltine) reproduction also increases its probability of surviving catastrophic density reductions, as it prevents loss of an entire generation to chance environmental disturbance (“bet hedging,” Stearns, 1980). Capacity for rapid recolonization allows C. ﬂuminea to sustain populations in substrates subject to periodic ﬂood scouring from which slower growing and late maturing unionoideans are eliminated (Way et al., 1990b, Section III.B.1). Like sphaeriids, NA C. ﬂuminea populations display high interpopulation variation in life-history traits (McMahon, 1999). As there is little or no genetic variation among NA populations (McLeod 1986), this variation must be ecophenotypic. Growth rates increase and time to maturity and life spans decrease in populations from more productive habitats (McMahon, 1999). On the northern border of its NA range, low temperatures reduce growth and reproductive periods, such that populations are univoltine rather than bivoltine. A slow growing C. ﬂuminea population was univoltine and semelparous in an oligotrophic Texas lake (McMahon, unpublished data). Even within populations, life-history tactics vary, dependent on year-toyear variations in temperature and primary productivity (McMahon and Williams, 1986a; Williams and McMahon, 1986). As in sphaeriids, ecophenotypic lifehistory trait variation is adaptive in C. ﬂuminea, allowing colonization of a variety of habitats. Thus, this species is highly euryoecic and highly invasive, making it the single most successful and economically 372 Robert F. McMahon and Arthur E. Bogan costly aquatic animal introduced to North America (McMahon, 1999). 4. Dreissena Polymorpha The zebra mussel, Dreissena polymorpha, is the most recently introduced NA bivalve species (Mackie and Schloesser, 1996). Like C. ﬂuminea, its life-history characteristics (McMahon, 1996; Nichols, 1996) make it highly invasive. Unlike other NA bivalves, it releases sperm and eggs making fertilization completely external. Developments leads to a free-swimming planktonic veliger larva (Fig. 15C) which feeds and grows in the plankton for 8 – 10 days before settlement (Nichols, 1996). The planktonic veliger enhances the zebra mussel dispersal ability. Veligers in the Illinois River traveled at least 306 km downstream before settlement (Stoeckel et al., 1997). Numbers of dispersing veligers can be astounding. Stoeckel et al. (1997) estimated that veligers in the Illinois River reached densitiess 250 veligers / L which resulted in veligers passing a ﬁxed point at a rate as 75 106 per second. Total annual veliger ﬂux was estimated to be 1.935 1014 and 2.131 1014 per year in 1994 and 1995, respectively. Adults byssally attached to ﬂoating objects are also transported downstream. Zebra mussels sexually mature in the ﬁrst or second year of life (ﬁrst year in most NA populations) have life spans of 3 – 5 years in Europe and 2 – 3 years in North America (Mackie and Schloesser, 1996) and, like C. ﬂuminea, sustain high growth rates throughout life (Fig. 19). It is iteroparous and univoltine; an individual participating in 3 – 4 annual reproductive periods within its life span. Reproduction is initiated above 10 – 12°C, but is maximized above 18°C. Spawning is dependent on abiotic and biotic conditions, producing peaks of veliger density within a reproductive season (McMahon, 1996; Nichols, 1996; Ram et al., 1996; Stoeckel, et. al. 1997). The freshly hatched veliger is small (diameter 40 – 70 m), but the pediveliger grows to 180 – 290 m just prior to settlement (Nichols, 1996), leading to a 100 to 400-fold biomass increase during the 8 – 10 day planktonic period. Maximal D. polymorpha adult size ranges from 3.5 to 5 cm depending on growth rate, which, like terminal size, is dependent on habitat primary productivity and temperature. Growth rate of caged mussels in Lake St. Clair, Michigan, initially enclosed at an average shell length (SL) of 4.2 m was 0.095 mm per day over the ﬁrst 150 days reaching a mean SL of 14.3 mm and, thereafter, slowing down to a mean of 0.015 mm per day for the next 182 days reaching a mean SL of 17.0 mm (Bitterman et al., 1994). Growth rates of young mussels in the lower Mississippi River were temperature-dependent, peaking at 0.06 – 0.08 mm per day FIGURE 19 Shell-growth rates in European populations of the zebra mussel, Dreissena polymorpha. Growth rates for North American populations in Lake Erie are similar to or greater than faster growing British populations depicted in this ﬁgure. at ⬇16 – 17°C and declining at higher temperatures (Allen et al., 1999), a result supporting laboratory studies showing that increasing metabolic demands reduce assimilated energy allocation to growth above 15°C (Walz, 1978a). Like C. ﬂuminea, D. polymorpha allocates a very high percentage (96.1%) of nonrespired assimilation to somatic growth, leaving only 3.9% for reproduction (Mackie et al., 1989; Table III); however, Sprung (1991) reports a 30% postspawning reduction in female body mass, indicating higher reproductive energy allocation in some populations. Allocation of a large proportion of nonrespired assimilation to growth allows individuals to rapidly increase in size, making them more competitive and less subject to predation. Zebra mussel veligers settle on the shells of established individuals, forming thick mats or clusters many shells deep, in which competition for space and food must be intense. Thus, rapid postsettlement juvenile growth is highly adaptive as it allows stable byssal attachment to the substrate and positioning of siphons at the mat surface, where food and O2 are most available. In spite of the low levels of energy devoted to reproductive effort by D. polymorpha, its very small eggs make individual fecundity large, ranging from 30,000 to 106 eggs per female (Sprung, 1991; Mackie and Schloesser, 1996). Dreissena polymorpha population densities range from 7000 to 114,000 individuals / m2 and standing crop biomasses from 0.05 – 15 kg / m2 (Claudi and 11. Mollusca: Bivalvia Mackie, 1993). These high densities and biomasses result from the tendency of juveniles to settle on substrates inhabited by adults forming dense mats or clumps many individuals thick. High individual growth rates and population densities lead to very high productivities, estimated to be 0.05 – 15 g C / m2 per year in European populations, and ⬇75 g C / m2 per year in NA Great Lakes populations based on dry tissue mass productivity values in Mackie and Schloesser (1996). The NA productivity value for D. polymorpha is still relatively low compared to values of 1000 – 4500 g C / m2 per year estimated for dense NA C. ﬂuminea populations (McMahon, 1999). Population growth and productivity are highly habitat-dependent in D. polymorpha, making turnover times variable, ranging from low values of 53 days (highly productive) to high values of 869 days (low productivity; Table III). A high growth rate throughout life, elevated fecundity, short life span, and a capacity for adult and larval down-stream dispersal make Dreissena (like Corbicula) a highly invasive species. However, unlike C. ﬂuminea, D. polymorpha populations are restricted to more stable habitats such as larger, permanent lakes and rivers. Its apparent preference for stable habitats is reﬂected by its endemic range in the Caspian Sea and Ural River (large stable habitats), avoidance of shallow, near-shore lentic- and low-ﬂow lotic habitats, relatively long age to maturity (generally at least 1 year of life), iteroparity, gonochorism, and relatively high adult survivorship (26 – 88% per year; Mackie and Schloesser, 1996). Restriction of the original range of D. polymorpha to the Caspian Sea and Ural River also suggests a limited dispersal capacity between drainage systems. Its dispersal through Europe occurred only in the nineteenth century (and continues in western Asia today) as interconnenting canal systems transported it between drainages. Dispersal of D. polymorpha between drainages is primarily by human vectors, including transport of adults attached to boat / barge hulls, ballast water dumping, and transport of veligers through canal systems interconnecting catchments. Adults are poorly tolerant of emersion (McMahon, 1996), precluding extensive natural overland dispersal unless human-mediated. Thus, dispersal of this species between NA catchments has been primarily by human vectors, with larger, permanent, navigable bodies of water being most susceptible (Mackie and Schloesser, 1996). Without anthropomorphic vectors, dispersal of D. polymorpha through NA drainages would almost certainly have proceeded at a much slower pace than recorded for C. ﬂuminea, but human activities have already lead to this species being widely distributed in major NA drainages east of the Mississippi River (Ram and McMahon, 1996; New York Sea Grant, 1999). 373 C. Ecological Interactions 1. Behavioral Ecology Other than borrowing (see Section II.A.2), information on bivalve behavior is sparse. Reviews of molluscan neurobiology and behavior (Willows, 1985, 1986) have no bivalve references. Lack of information, rather than lack of complex and intriguing behaviors, reﬂects difﬁculties in making behavioral observations on predominantly sessile bivalves surrounded by a shell. There are a number of reproductive behaviors in freshwater bivalves, including those ensuring unionioidean glochidial contact with ﬁsh hosts (Section II.B). Adult C. ﬂuminea display downstream dispersal behavior during reproductive periods. While juvenile clams (SL 2 mm) are hydrologically transported throughout the year, immatures (SL 2 – 7 mm) and adults (SL 7 mm) leave the substrate to be passively hydrologically transported over the sediment surface (“rolling”) only prior to reproductive periods (Fig. 20). Dispersing adults have lower dry tissue weights, lower tissue organic carbon-to-nitrogen ratios (Williams and McMahon, 1989), higher levels of ammonia excretion, and reduced molar O2 consumption-to-N2 excretion ratios than those remaining in the substrate (Williams and McMahon, 1985), all indicative of poor nutritional condition. Thus, downstream dispersal allows starving individuals to disperse from areas of low food availability and high intraspeciﬁc competition into areas more nutritionally favorable for reproductive efforts (Williams and McMahon, 1986, 1989). Some adult unionoidean bivalves and C. ﬂuminea display surface locomotory behavior involving the same movements of foot and valves described in Section II.B.2 for burrowing. Unionoidean surface locomotion is fairly common (Imlay, 1982), leaving tracts in sediments 3 – 10-m long (Golightly, 1982). The adaptive signiﬁcance of bivalve surface locomotion is not well understood as it could attract potential predators. However, it may be involved with pedal feeding on organic sediment deposits (see Section III.C.2). Some species, such as Pyganodon grandis, migrate vertically with seasonal changes in water level (White, 1979) to avoid emersion. Other species, such as Uniomerus tetralasmus, C. ﬂuminea, and some sphaeriids, remain in position and suffer prolonged emersion (see Section II.C.4). In Texas, ﬁre ants, Solenopsis invicta, kill emersed bivalves (McMahon, unpublished), suggesting that this introduced insect may be a new threat to unionoideans and sphaeriids as it expands its range in the southeastern U.S. Some sphaeriids, C. ﬂuminea, and D. polymorpha also crawl on hard substrates or macrophytes. Sphaerium corneum holds the valves erect and crawls 374 FIGURE 20 Seasonal variation in downstream dispersal behavior by juvenile, subadult and adult Corbicula ﬂuminea in the water intake canal of a power station. (A) Rate of impingement of adults dispersing downstream onto traveling screens in front of water intake embayments (shell length 10 mm). (B) Rate of retention of subadults (shell length 1 – 7 mm) in a clam trap held on the substrate surface of the intake canal. (C) Juveniles suspended in the water-column (shell length 2 mm). Right vertical axis represents density of juveniles in the intake canal water column (solid circles connected by dashed lines). Left vertical axis is number of juveniles entrained daily with intake water (open circles connected by solid lines). Note that the adult and subadult clams display maximal downstream dispersal behavior during only two periods, March – May and September – November). Juveniles occurred in the water column throughout the year; peak juvenile water column densities occurred during reproductive periods and midwinter periods of low ambient water temperature. (From Williams and McMahon, 1986.) 11. Mollusca: Bivalvia 375 TABLE IV The Effects of Temperature on the Percentage of Time Spent in Various Valve Movement Behaviors in Emersed Specimens of Corbiucla ﬂuminea a Temperature (°C) Time with valves closed (%) Time with mantle edge exposed (%) Time with mantle edge parted or attempting to burrow (%) 15 25 35 29.5 51.2 90.5 65.8 43.5 9.1 4.7 5.3 0.4 a From Byrne et al. (1988). by extending the foot tip, anchoring it with mucus, and pulling the body forward by pedal muscle contraction (Wu and Trueman, 1984). Juvenile C. ﬂuminea crawl on hard subtrates in the same manner, while adults similarly crawl while lying on one of the valves (Cleland and McMahon, unpublished). Small specimens of D. polymorpha are active crawlers and can climb smooth vertical surfaces. They routinely discard their byssus and migrate to a new position for reattachment. Thus, juveniles migrate to deeper waters in the winter to avoid ice scour (Mackie et al., 1989). Freshwater bivalves also respond to external cues. Chief among these is valve closure in response to irritating external stimuli when detected by mantle edge and siphonal sense organs (Section II.B.6). Valve closure seals internal tissues from damage by external irritants. Almost all freshwater bivalves tolerate some degree of anaerobiosis (Section II.C.3). Thus, individuals exposed to irritants may keep the valves shut for relatively long periods until normal external conditions return. Valve closure occurs in response to heavy metals (Doherty et al., 1987), chlorine, and other biocides (Mattice et al., 1982; McMahon and Lutey, 1988), suspended solids (Aldridge et al., 1987), and pesticides (Borcherding, 1992). This ability allows C. ﬂuminea to avoid intermittent exposure to chlorination or other biocides, making chemical control of their fouling difﬁcult (Mattice et al., 1982). Valve closure in immediate response to mantle edge or siphonal tactile stimulation is a predator defense mechanism. Valve closure response in D. polymorpha is used to monitor water pollution in Europe (Borcherding and Volpers, 1994). Freshwater bivalves have adaptive behavioral responses to prolonged emersion. Physiological adaptations were discussed in Section II.C.4. Here, behavioral responses are described in greater detail. When emersed, C. ﬂuminea displays four major behaviors: (1) escape responses involving attempts to burrow; (2) valves gaped widely with mantle edges parted, opening the mantle cavity to the atmosphere; (3) valves narrowly gaped with mantle edges exposed, but cemented to- gether with mucus; and (4) valves clamped shut. Behaviors (1) and (2) are never displayed more than 6% of time in air. Exposure of sealed mantle edges allows aerial gas exchange (Byrne et al., 1988), but results in evaporative water loss. When valves are closed, water loss is minimized; but O2 uptake ceases (Byrne et al., 1988; McMahon and Williams, 1984). As temperature increases (Table IV) or relative humidity decreases, duration of mantle edge exposure decreases (McMahon, 1979b; Byrne et al., 1988 ). Hence, behaviors associated with water loss are reduced in response to increased desiccation pressure. Indeed, relative humidities near zero or temperatures above 30 – 35°C induce continual valve closure (Byrne et al., 1988) (Table IV), reducing water loss, but making individuals anaerobic. Similar behaviors occur in emersed unionoideans (Byrne and McMahon, 1994). Less emersion-tolerant unionoideans, like P. grandis, display high levels of mantle edge exposure (25 – 90% of time emersed) and high rates of water loss. More emersion-tolerant species, like Uniomerus tetralasmus and Toxolasma parvus, have reduced levels of mantle edge exposure behavior when emerged (25% of time emersed). The extremely emersion-tolerant U. tetralasmus plugs the siphon openings with mucus to reduce water loss and spend little time with mantle edges exposed (Byrne and McMahon, 1994). Ability of emersed bivalves to adjust aerial respiratory responses to external dessication pressures is a complex behavior, requiring capacity to sense, integrate, and respond to external temperature and relative humidity levels and internal osmotic concentration. Freshwater bivalves may also have circadian patterns of behavior. Both ion-uptake and O2-consumption rates are greater during dark than light hours in unionoideans and C. ﬂuminea (McCorkle et al., 1979; Graves and Dietz, 1980; McCorkle-Shiley, 1982; Section II.C.2) indicative of circadian-activity patterns in feeding, reproduction, and burrowing. The density of juvenile C. ﬂuminea in the water column increases during dark hours (McMahon, unpublished) suggesting that adults preferentially release juveniles at night. 376 Robert F. McMahon and Arthur E. Bogan Freshwater bivalves may have circadian burrowing cycles, retreating into sediments during light hours to avoid visual predators like ﬁsh and birds. The unionoideans, Anodonta anatina and Unio tumidis, displayed a diurnal activity rhythm with valves open for longer periods at night than during the day (Englund and Heino, 1994a). Similar diurnal valve movement behavior occurs in D. polymorpha (Borcherding, 1992). 2. Filter Feeding a. Particle Capture Most freshwater bivalves feed by ﬁltering suspended material from gill water ﬂow (Section II.B.2) driven by lateral cilia located on each side of the ﬁlaments. Projecting laterally from each ﬁlament’s leading edge are “cirri” made up of partially fused tufts of eulaterofrontal cilia occurring at intervals of 2 – 3.5 m (Figs. 5A and 21). Eulaterofrontal cirri on adjacent ﬁlaments project toward each other forming a stiffened grid on which suspended seston (phytoplankton, bacteria, and ﬁne detritus) is retained from water ﬂow driven between the ﬁlaments by the lateral cilia. Filtering may also involve creation of eddies by cirri in which seston particles settle. Filtered particles range from 1 to 10 m, with D. polymorpha and C. ﬂuminea both able to ﬁlter smaller particles ( 1.0 m) than most other freshwater species (Fig. 22) (Jørgensen et al., 1984; Paterson, 1984; Way et al., 1990a; Silverman et al., 1995). It has been claimed that a mucus net covering the gill is the primary ﬁlter. While the role of mucus in bivalve ﬁltering is debated, there is little doubt that the dense mesh of the cirri could form an effective ﬁlter (Morton, 1983; Way, 1989). Particles are captured at the level of the cirri (Jørgensen, 1996), suggesting their involvement in ﬁltration. However, adjacent cirri are separated by 2 – 3.5 m, which until recently, made their mode of particle capture an enigma because ﬁltered particles were much smaller than the intercirral distance. Two-particle capture mechanisms have been hypothesized: direct physical cirral ﬁltration; or capture by water currents created by gill cilia (Silverman et al., 1996a). Recent confocal microscopic examinations of living gill tissue of D. polymorpha (Silverman et al., 1996a, 1996b) and marine mytilid mussels (Silverman et al., 1999) support direct cirral particle capture as the main feeding mode. The cirrus is composed of a pair of adjacent fused ciliary sheets aligned with inhalant water ﬂow, extending from a single gill epithelial cell (Fig. 21D.b). Thirtyeight-to-42 cilia form the cirrus. Each cilium has a distinct basal “hinge” region (Fig. 21D.c) and a free distal end characterized by reduced numbers of microtubules. Cilia increase in length from the inhalant to the trailing edge of the cirrus (Fig. 21D.b). Cirral cilia move in unison on the hinge. When they reﬂex into the space be- tween adjacent ﬁlaments, the free cirral cilia tips splay out into the inhalant current forming a ﬁlter of submicron dimensions (Fig. 21D.a) on which previously freemoving particles (0.75 m diameter) are trapped. When the cirrus is then ﬂexed back over the leading edge of the ﬁlament, trapped particles are released (Silverman et al., 1996a, 1996b, 1999) (Fig 21D.a), allowing them to entrain in mucus transported either ventrally or dorsally by ﬁlament frontal cilia (Beninger et al., 1997) to specialized, ciliated food grooves on the ventral edges or bases of the demibranches depending on species (Figs. 2 and 5A – C). In the unionoidean, Pyganodon cataracta, particles and mucus transported by ﬁlament frontal cilia become entrained and concentrated in a mucus thread on reaching the ventral food grooves where they are carried anteriorly to the labial palps (Tankersley, 1996) (Figs. 5A and 21). The role of mucus in bivalve food particle transport is being debated Other investigators feel that ciliary-based, ﬂuid-mechanical forces are primarily responsible for particle transport with mucus playing a minor role if any (Jørgensen, 1996). Frontal cirri similar to those of D. polymorpha occur in sphaeriid, unionoidean and corbiculacean bivalves (Fig. 21) (Way et al., 1989) and marine bivalves (Beninger et al., 1997) where they are presumed to similarly function in particle capture. b. Sorting of Filtered Particles Mucus-entrained, ﬁltered particles are carried anteriorly to the labial palps, paired triangular ﬂaps on each side of the mouth (Figs. 2 and 4). The outer labial palp lies against the outer ventral side of the outer demibranch, and the inner palp, against the inner ventral side of the inner demibranch (Fig. 2). Palp epithelial surfaces facing away from the demibranches are smooth, while those adjacent to the demibranch have a series of parallel ridges lying obliquely to a ciliated oral groove formed in the fused dorsal junction of the inner and outer labial palps leading to the mouth (Morton, 1983). In the unionoidean, P. cataracta, the palps transfer the food-laden mucus thread from the ctenidial food groove directly to the mouth for ingestion (Tankersley, 1996). In marine eulammelibranch bivalves, the mucus thread becomes mechanically ﬂuidized on the palp surface by a combination of grinding movements of the opposing palp surfaces, transport across the corrugated palp surface and chemical reduction in mucus viscosity. Fluidization, breaks the mucus thread into a fragmented, ﬂocculent mucus – particle slurry prior to ingestion (Beninger et al., 1997). After ﬂuidization, the corrugated palp surface sorts ﬁltered particles into those accepted for ingestion and those rejected. Generally, smaller particles are carried over the tops of palp corrugations by cilia to be deposited in the oral groove for ingestion. Denser, larger 11. Mollusca: Bivalvia FIGURE 21 Scanning electron micrographs of the gill ciliation of representative freshwater bivalves. (A) Musculium transversum (Sphaeriidae): (a) arrangement of frontal and eulaterofrontal ciliation forming cirri on the leading edge of the gill ﬁlaments in the midgill region; (b) oblique view of a cross-sectional fracture of the gill showing frontal and eulataterofontal cilia forming cirri on the leading edge of the ﬁlament and lateral cilia on the side of the ﬁlament; (c) ciliation of the food groove on the ventral edge of the gill; (d) posterior portion of the outer (foreground) and inner (background) demibranchs of the gill; (e) a frontal cirrus formed from fused cilia emerging between the eulaterofrontal cirri and frontal cilia on the leading edge of a ﬁlament; (f) a frontal cirrus emerging from a band of frontal cilia. (B) Corbicula ﬂuminea (Corbiculidae): (a) leading edge of a gill ﬁlament showing frontal cilia, eluatorofrontal cilia forming cirri, lateral cilia and frontal cirri; (b) highmagniﬁcation view of frontal cilia and eulateralfrontal cirri; (c) oblique view of a longitudinal fracture through the midgill region showing all four types of ciliation, including lateral cilia lining the sides of the gill ﬁliament; (Continues) 377 378 Robert F. McMahon and Arthur E. Bogan FIGURE 21 (Continued) (d) crosssection of the midgill region showing the origin of the frontal cirrus; (e) lower region of the gill showing the presence of less well organized frontal cilia; (f) food groove region at the ventral edge of the inner demibranch; note loss of ciliary organization in food groove, including lack of frontal cirri. (C) Obliquaria reﬂexa (Unionoidea): (a) frontal cilia and eulaterofrontal cilia forming cirri on the leading edges of several gill ﬁlaments (500X); (b) high-power view showing lateral cilia on sides of the gill ﬁlaments between tracts of frontal cilia and eulaterofrontal cilia forming cirri on adjacent ﬁlaments. (D) Dreissena polymorpha (Dreissenoidea): (a) frontal cilia on the leading edge of a gill ﬁlament and some eulaterofrontal cirri reﬂexed into the space between adjacent ﬁlaments with the free, distal ends of their cilia splayed out to form the primary gill ﬁltering mechanism and other cirri reﬂexed over the frontal cilia in a position in which they release particles captured from water ﬂow between adjacent ﬁlaments to frontal cilia of the food groove; (b) detailed structure of a eulaterofrontal cirrus formed from two rows of opposed cilia fused basally, free at their distal tips, with a distinct (Continues) 11. Mollusca: Bivalvia FIGURE 21 (Continued) basal hinge on which the cirral cilia simultaneously ﬂex between the interﬁlament space (food capture) and the frontal cilia on the leading edge of the ﬁlament (food release); (c) transmission electron micrograph showing details of the unique hinge on which cirral cilia ﬂex between the interﬁlament space and the frontal cilia. Note lack of frontal cirri characteristic of the (A) Sphaeriidae, and (B) Corbicula ﬂuminea in (C) unionoideans and (D) Dreissena Label key for ﬁgures A – C : F, frontal cilia; FC, frontal cirrus; L, lateral cilia, LF eulaterofrontal cirri; and FG, food groove. [Photomicrographs supplied by Tony Deneka and Daniel Hornbach (Macalester (Continues) 379 380 Robert F. McMahon and Arthur E. Bogan FIGURE 21 (Continued) College), Carl M. Way (U.S. Army Corps of Engineers), and Harold Silverman and Thomas H. Dietz (Louisiana State University, Baton Rouge).] 11. Mollusca: Bivalvia particles fall between the corrugations where cilia carry them to the ventral edge of the palp to be bound in mucus and released onto the mantle as pseudofeces (pseudofeces are mucus-bound particles that have not been ingested) (Morton, 1983; Beninger et al., 1997). Pseudofeces are carried by mantle cilia to the base of the inhalant siphon (Fig. 2), where they are periodically expelled with water forced from the inhalant siphon by rapid valve adduction (i.e., valve clapping) (Morton, 1983). The palp may also sort medium-sized particles. Smaller medium-sized particles tend to remain longer on corrugation crests than in intervening grooves, thus, reaching the oral groove for ingestion, while larger, medium-sized particles remain longer in the grooves leading to eventual rejection as pseudofeces. Thus, labial palp particle sorting may be primarily by particle size and, perhaps, density. The function of labial palps in particle sorting is being debated, palps appearing to have little if any sorting function in some species (Tankersley, 1996) and extensive sorting functions in others (Beninger et al., 1997). Labial palp function in freshwater bivalves requires further study. The width of palp corrugations and gill ﬁlaments can be adjusted by both muscular activity and distention of gill and palp blood sinuses, thus allowing active particle-size selection (Morton, 1983). When fed natural seston, the percentage of particles retained by the unionoidean, Elliptio complanata, declined linearly over a particle size range of 5 (nearly 100%) to 10m (less than 10% retention), suggesting size selection (Paterson, 1984). Similar size selection for particles occurs in the sphaeriid, Musculium transversum (Way, 1989), and C. ﬂuminea (Way et al., 1990a). Some unionoidean species may select different algal types. In the same river pool, Amblema plicata ingested a greater proportion of green algae, a lower diversity of algal species, and different algal species than Ligumia recta (Bisbee, 1984). Interspeciﬁc differences in particle selection may allow some sympatric species to divide the feeding niche, avoiding competition, however, other unionoideans are nonselective in feeding and have high diet overlap between species. c. Filtering Rate The ﬁltering rate of bivalves can be computed as follows: 冢冣 C0 M In t Ct where M is the volume of suspension ﬁltered, t the time, and Co and Ct the concentrations of ﬁltered particles at time o and t, respectively (Jørgensen et al., 1984). Filtration rates are elevated in C. ﬂuminea compared to other freshwater bivalves. For a 25-mm shell length (SL) specimen, rate ranges from 300 to C 381 2500 L / h depending on temperature and seston concentration (Foe and Knight, 1986b; Lauritsen, 1986b; Way et al., 1990a). The ﬁltering rate of C. ﬂuminea fed natural seston concentrations was not signiﬁcantly correlated with ﬁeld water temperature (Long and McMahon, unpublished) and was independent of temperature between 20 and 30°C in the laboratory (Lauritsen, 1986b), suggesting capacity for temperature compensation of gill ciliary ﬁltering activity. The ﬁltration rate is also somewhat temperature independent in Sphaerium striatinum and Musculium partumeium, reaching maximal values during reproductive periods when water temperatures, were below midsummer maxima (Burky et al., 1981, 1985a; Way et al., 1981). Filtering rates in D. polymorpha are similar to those in C. ﬂuminea (Lei et al., 1996) and 2 – 4 times higher than those in sphaeriids and unionoideans (Kryger and Riisgård, 1988). High particle concentrations reduce ﬁltering rates (Burky et al., 1985a; Way, 1989; Way et al., 1990a; Lei et al., 1996). Filtering rates in S. striatinum (Hornbach et al., 1984a) and M. partumeium (Burky et al., 1985a) were depressed at concentrations below natural seston, making particle ingestion nearly constant above a critical particle concentration. Thus, ﬁltering may have be regulated to control ingestion rate. Particle concentrations within ambient seston concentrations do not affect ﬁltering rate in C. ﬂuminea (Mattice, 1979), ingestion rates increasing directly with concentration. However, ﬁltration rate in C. ﬂuminea declined three-fold with increasing particle concentration between 0.33 and 2.67 l algal volume / L suggesting inhibition at very high concentrations, but increasing algal concentration over this range still resulted in a 3.5-fold rise in ingestion rate (Lauritsen, 1986b). Similar reduction in ﬁltration rate at particle concentrations above natural seston levels occurs in the sphaeriid, M. transversum (Way, 1989). In contrast to C. ﬂuminea, ﬁltration rate of the unionoidean, E. complanta, was depressed by increasing natural seston concentrations (Paterson, 1984). Filtration rate differed among populations of C. ﬂuminea, but ingestion rates were similar, suggesting regulation of the ﬁltration rate to control ingestion rates (Way et al., 1990a). In D. polymorpha, ﬁltration rate was reduced when feeding on artiﬁcial plastic microspheres compared to natural seston. When ﬁltering particles 1.5 m in diameter, ﬁltration rate decreased with decreasing particle concentration below a critical upper limit, and with decreasing temperature. Filtration rate was also affected by prior temperature acclimation (Lei et al., 1996). Such data indicate that the seasonal and environmental ﬁlter-feeding responses of freshwater bivalves are species-speciﬁc and laboratory measurements of ﬁltration rates are likely to be invalid unless carried out at natural seston concentrations and 382 Robert F. McMahon and Arthur E. Bogan sizes and with natural seston rather than artiﬁcial particles. Filter feeding and particle retention rates were depressed in gravid females of the unionoidean, Pyganodon cataracta, in which particle transport was slowed down and the ability to retain small particles (6 m) reduced, when the marsupial gills were distended with glochidia (Tankersley, 1996). Suspended silt can inhibit ﬁltering and consumption rates in freshwater bivalves, perhaps by overwhelming ciliary ﬁltering and sorting mechanisms. High silt concentrations reduced ﬁltering and metabolic rates in three unionoidean species whether exposure was infrequent (once every 3 h) or frequent (once every 0.5 h), suggesting interference with gill ciliary activity. Furthermore, individuals experiencing frequent exposure increased reliance on carbohydrate catabolism (Aldridge et al., 1987), symptomatic of short-term starvation; perhaps, the basis of exclusion of unionoideans from habitats with high silt loads (Adam, 1986). In contrast, natural suspended sediment concentrations did not affect shell or tissue growth in C. ﬂuminea (Foe and Knight, 1985), perhaps, accounting for its ability to colonize high-ﬂow lotic habitats with greater suspended solids than tolerated by most unionoideans (Payne and Miller, 1987; Section III.A.). In contrast, growth in juvenile unionoideans is stimulated by small amounts of suspended silt in their algal food (Hudson and Isom, 1984), perhaps because juveniles (Villosa iris) may feed primarily on sediment interstitial water through the pedal gape, leading to ingestion of silt, microdetritus and bacteria, with little consumption of algae (Yeager et al., 1994). In smaller streams, where phytoplankton productivity may be low, most suspended organic matter is particulate organic detritus or heterotrophic bacteria and fungi (Nelson and Scott, 1962). While experiments are few, at least some freshwater bivalves efﬁciently ﬁlter suspended bacteria. D. polymorpha appears to extract particles of bacteria size (1.0 m in diameter) (Sprung and Rose, 1988, Fig. 22) as can C. ﬂuminea (Silvermann et al., 1995) with D. polymorpha and C. fuminea able to clear the bacterium, Escherichia coli, 30 and 3 times faster than the lotic unoinid, Toxolasma texasiensis, respectively. Increased capacity for bacterial ﬁltration in D. polymorpha appears associated with its enlarged gill with a 100-fold greater density of cirri relative to C. ﬂuminea, that of T. texasiensis has even fewer and smaller cirri than D. polymorpha or C. ﬂuminea (Silverman et al., 1995). Filter-feeding by D. polymorpha may signiﬁcantly reduce bacterioplankton densities in the inner portion of Saginaw Bay, Lake Huron (Cotner et al.,). Musculium transversum (Way, 1989a) and some unionoidean species (Fig. 22) do not efﬁciently ﬁlter bacterial-size particles. Unionoideans appear specialized to ﬁlter larger particles, such as phy- FIGURE 22 Relationship between the percentage of suspended particles retained by the gill ﬁltering mechanism of freshwater bivalves and particle size, as particle diameter in micrometers. Horizontal axis for all ﬁgures is the percentage of total particles retained from suspensions passing over clam gill ﬁltering systems in (A) Dreissena polymorpha, the unionoideans, (B) Unio pictorum, and (C) Anodonta cygnea. Vertical lines about points are standard deviations. Note that all three species efﬁciently retained particles of 2.5 – 8 m diameter, equivalent to most unicellular algae, but only D. polymorpha efﬁciently retained bacteria-sized particles of 1 m diameter. (Redrawn from Jørgensen et al., 1984.) toplankton, detritus, protozoa, and even small zooplankton, such as rotifers (Singh et al., 1991). In contrast, six unionoidean species adapted to lotic habitats where seston was dominated by bacteria and detritus could ﬁlter bacteria at signiﬁcantly higher rates than three species adapted to lentic habitats where seston was dominated by phytoplankton. Lotic species had more complex cirri (25 vs 16 cilia per cirral plate) and greater gill cirral density than lentic species, indicative of a ﬁltering system better adapted for ﬁne particle ﬁltration (Silverman et al., 1997) . Because bacteria and detrital particles may comprise most of the organic matter in some, particularly lotic, habitats, bacterial feeding warrants greater study in freshwater bivalves. d. Sediment Feeding At least some freshwater bivalves may supplement phytoplankton ﬁlter feeding by consuming organic detritus or interstitial bacteria from sediments. The infaunal habit of many Pisidium species suggests dependence on nonplanktonic food sources. Many pisidiids can efﬁciently ﬁlter small interstitial bacteria. They burrow continually through sediments with the shell hinge downward, drawing dense interstitial 11. Mollusca: Bivalvia water and bacteria through parted ventral mantle edges into the mantle cavity to be ﬁltered on the gills (Lopez and Holopainen, 1987). Both Pisidium casertanum and P. conventus feed in this manner, ﬁltering bacteria of smaller diameter (1 m) than most bivalves (Lopez and Holopainen, 1987) (Fig. 22). Filtering of rich interstitial bacterial ﬂora may be associated with the reduced ctenidial surface areas and low ﬁltration and metabolic rates of Pisidium species compared to Sphaerium or Musculium (Hornbach, 1985; Lopez and Holopainen, 1987). Similar interstitial bacteria and detritus feeding occurs in juvenile unionids (Yeager et al., 1994). M. transversum uses its long inhalant siphon to vacuum detrital particles from the sediment surface (Way, 1989). Pedal feeding on sediment organic detritus may be another important food source in some freshwater bivalves. Pedal feeding occurs in marine and estuarine bivalves (Morton, 1983; Reid et al., 1992), but is little studied in freshwater species. Corbicula ﬂuminea draws sediment detrital particles over the ciliated foot epithelium into the mantle cavity, where they accumulate in the food groove of the inner demibranch and are carried to the palps for sorting and ingestion (Way et al.,1990b; Reid et al., 1992). Cellulose particles (40 – 120 m in diameter) soaked in various amino acid solutions and mixed with sediments accumulated in the stomach of pedal-feeding C. ﬂuminea (McMahon, unpublished). Thus, C. ﬂuminea removed sediment organic matter at a rate of 50 mg / clam per day and doubled growth rates when allowed to pedal feed in addition to ﬁlter feeding (Hakenkamp and Palmer, 1999). Similar pedal feeding has been reported in M. transversum (Way, 1989) and in juvenile Villosa iris (Yeager et al., 1994). Thus, pedal deposit feeding may be an important auxiliary feeding mode in freshwater and marine bivalves and may have important impacts on sediment organic dynamics (Hakenkamp and Palmer, 1999). Pedal feeding may be more universal in freshwater species than previously suspected. In a stream S. striatinum population, only 35% of organic carbon assimilation occurred by ﬁlter feeding, leaving 65% to come from sediment detrital sources (Hornbach et al., 1984b), presumably by pedal feeding. Other sphaeriid species reach highest densities in sediments of high organic content (Zbeda and White, 1985) or in habitats receiving organic sewage efﬂuents, suggesting dependence on pedal deposit-feeding (Burky, 1983). Pedal deposit-feeding mechanisms may explain the extensive horizontal sediment locomotion of a number of freshwater bivalves (Section II.D), as it allows pedal contact with organic detritus. 3. Population Regulation a. Abiotic Population Regulation Most of the information regarding regulation of freshwater bivalve 383 populations is anecdotal. Fuller (1974) reviews factors affecting population density and reproductive success. Catastrophic abiotic factors causing reductions in bivalve populations are reviewed in Section III.B. Among these are silt accumulation in sediments of impounded rivers and in the water column during ﬂooding. Silt interferes with gill ﬁlter feeding and gas exchange (Aldridge et al., 1987; Payne and Miller, 1987) causing massive mortality in unionoidean populations (Adam, 1986, Section III.A). In contrast, many sphaeriids thrive in silty sediments (Burky, 1983; Kilgour and Mackie, 1988) associated with their utilization of sediment detrital food sources (Section III.C.2). Shell growth in C. ﬂuminea, a silt tolerant species, can be temporarily inhibited by high turbidity (Fritz and Luz, 1986). Temperature extremes also affect bivalve populations. C. ﬂuminea tolerates 2°C (Mattice, 1979) to 36°C (McMahon and Williams, 1986b). Both cold-induced winter (Blye et al., 1985; Sickel, 1986) and heatinduced summer kills (McMahon and Williams, 1986b) are reported for this species. The incipient upper lethal limit for D. polymorpha is 30°C, preventing its invadsion of many southern U.S. drainage systems (McMahon, 1996). Temperature limits for other NA bivalves are not as well known, but, on average, appear to be broader than either C. ﬂuminea or D. polymorpha (Burky, 1983). Juvenile unionoideans are less temperature tolerant than adults (Dimock and Wright, 1993), suggesting that temperature limitation operates at the juvenile level in this group. Within the tolerated range, temperature may have detrimental effects on reproductive success. Sudden temperature decreases cause abortive glochidial release in unionoideans (Fuller, 1974), and temperatures above 30 – 33°C inhibit reproduction in C. ﬂuminea (McMahon, 1999). Sphaeriid densities were reduced in midsummer by heated efﬂuents (Winnell and Jude, 1982). Heated efﬂuents may also stimulate bivalve growth, inducing early maturity and increasing reproductive effort. They can provide heated refugia in which cold-sensitive species may overwinter as in northernmost NA populations of C. ﬂuminea (McMahon, 1999). Low ambient pH can depauperate or extirpate bivalve populations. Bivalve populations have been extirpated by acid mine drainage (Taylor, 1985; Warren et al., 1984). Highly acidic waters cause shell erosion and eventual death (Kat, 1982). Perhaps of more importance than pH in regulating freshwater bivalve populations are water hardness and alkalinity. Unionoideans do not occur in New York drainage systems with calcium concentrations 8.4 mg Ca / L (Fuller, 1974). In waters of low alkalinity, Ca concentrations may be too low for shell deposition; the lower limit for most freshwater bivalves being 2 – 2.5 mg Ca / L (Okland and Kuiper, 1982; Rooke and Mackie, 384 Robert F. McMahon and Arthur E. Bogan 1984a). Growth and fecundity were suppressed in a Pisidium casertanum population in a low Ca concentration relative to that a higher Ca concentration (Hornbach and Cox, 1987). Waters of low alkalinity have little pH-buffering capacity, subjecting them to seasonal pH variation. This makes them particularly sensitive to acid rain, which detrimentally impacts bivalve faunae (Rooke and Mackie, 1984a). Shell Ca content is related to water Ca concentration. Of 10 Canadian freshwater bivalve species, two sphaeriids and one unionoidean displayed no relationship between shell Ca content and water Ca concentration, shell Ca content decreased with increased water Ca concentration in two sphaeriid species and increased with water Ca concentration in four sphaeriid and two unionoidean species (Mackie and Flippance, 1983b). Lowering of water levels during dry periods induce bivalve mortality by exposing individuals to air. Such emersion caused near 100% mortality in C. ﬂuminea populations (White, 1979) due to its poor desiccation tolerance (Bryne et al., 1988). Many unionoidean species are relatively tolerant of emersion. When sympatric populations of nine unionoidean species, the sphaeriid Musculium transversum, and C. ﬂuminea were emersed for several months, M. transversum and C. ﬂuminea suffered 100% mortality while the unionoideans experienced only 50% mortality, because they either migrated downshore or resisted desiccation (White, 1979). Like C. ﬂuminea, D. polymorpha is relatively emersion intolerant, particularly above 20°C (McMahon, 1996), restricting it to habitats with relatively stable water levels and making it unlikely to develop dense populations in variable level reservoirs. Low environmental O2 concentrations can be detrimental to freshwater bivalves. Continual eutrophication of Lake Estrom, Denmark, so reduced ambient profundal O2 concentrations (0 – 0.2 mg O2 /L for three months) that massive reductions of sphaeriid populations ensued (Jonasson, 1984a, b), including hypoxiatolerant species, such as Pisidium casertanum and P. subtruncatum in which the lower limit for aerobic respiration was 1.7 mg O2 /L (Jonasson, 1984b). Adult unionoideans are relatively hypoxia tolerant. Juveniles are more hypoxia sensitive, those of Utterbackia imbecillis and Pyganodon cataracta tolerating anoxia for less than 24 h (Dimock and Wright, 1993). Thus, restriction of unionoidean species to well oxygenated waters may be a juvenile rather than adult constraint. Pollution is also detrimental to freshwater bivalves (reviewed by Fuller, 1974) including chemical wastes (Zeto et al.,1987), asbestos (Belanger et al.,1986a), organic sewage efﬂuents (Gunning and Sutkkus, 1985; Neves and Zale, 1982; St, John, 1982), heavy metals (Belanger et al., 1986b; Fuller, 1974; Lomte and Jabhav, 1982a; Grapentine, 1992), chlorine and paper mill efﬂuents (Fuller, 1974), acid mine drainage (Taylor, 1985; Warren et al., 1984) and PCBs (Grapentine, 1992). Potassium ions, even in low concentration (4 – 7 mg K /liter), can be lethal to freshwater bivalves including D. polymorpha (Claudi and Mackie, 1993), excluding bivalves from watersheds where potassium is naturally abundant (Fuller, 1974). b. Biotic Population Regulation There have been a few studies of biotic regulation of freshwater bivalve populations. Freshwater bivalves are host for a number of parasites. They are intermediate hosts for digenetic trematodes (Fuller, 1974). Such infections cause sterility in gastropods, and have been reported to induce sterility in the unionoidean, Anodonta anatina, allowing diversion of the mussel’s energy stores from production of its gametes to production of trematode cercariae (Jokela et al., 1993), reducing the mussel population’s reproductive capacity. Parasitic nematode worms inhabit the guts of unionoideans (Fuller, 1974). The external oligochaete parasite Chaetogaster limnaei resides in the mantle cavities of unionoideans (Fuller, 1974), C. ﬂuminea (Sickel, 1986) and D. polymorpha (Conn et al., 1996). Dreissena polymorpha is host to a wide diversity of parasites (Malloy et al., 1997), but their impacts on this species are unknown. All of these parasites probably contribute to the regulation of freshwater bivalve population densities, but the degree to which they do has received little experimental attention. Water mites of the family Unionicolidae, including Unionicola and Najadicola, are external parasites of unionoideans (Vidrine, 1990). Both mature and preadult mites are parasitic, attaching to gills, mantle, and the visceral epithelium (depending on species) (Mitchell, 1955; Chapter 16). Heavy mite infestations cause portions of the gills to be shed, abortion of developing glochidia, or even death (Fuller, 1974), perhaps making unionicolid mites a major regulator of unionoidean populations. The larval stage of the chironomid, Ablabesmyia janta, parasitizes the unionoidean gill ﬁlaments and may exclude unionicolid mites from the gills they infest (Vidrine, 1990). Disease has been little studied. There is little evidence of viral or bacterial involvement in die-offs of C. ﬂuminea (Sickel, 1986) or unionoideans (Fuller, 1974). In some C. ﬂuminea populations, massive die-offs (particularly of older individuals) occur after reproductive efforts (Aldridge and McMahon, 1978; McMahon and Williams, 1986a; Williams and McMahon, 1986), probably due to reductions in tissue energy reserves of postreproductive individuals (Williams and McMahon, 1989). Such postreproductive morality also occurs in sphaeriids (Burky, 1983). 11. Mollusca: Bivalvia TABLE V List of the Major Fish Predators of Freshwater Bivalvesa Family Genus and species Common name Clupeidae Cyprinidae Catostomidae Alosa sapidissima Cyrinus carpio Ictiobus bubalus Ictiobus niger Minytrema melanops Moxostoma carinatum Morone saxatilis Ictalurus furcatus Ictalurus punctatus Lepomis gulosus Lepomis macrochirus Lepomis microlophus Aplodinotus grunniens Acipenser fulvesens American shad Common carp Smallmouth buffalo Black buffalo Spotted sucker River redhorse Striped bass Blue catﬁsh Channel catﬁsh Warmouth Bluegill Red-ear sunﬁsh Freshwater drum Lake sturgeon Moronide Ictaluridae Centrarchidae Sciaenidae Acipenseridae a Data from Fuller (1974), McMahon (1983a), Robinson and Wellborn (1988), and Sickel (1986). Predation may be the most important regulator of freshwater bivalve populations. Shore birds and ducks feed on sphaeriids and C. ﬂuminea (Dreier, 1977; Paloumpis and Starrett, 1960; Smith et al., 1986; Thompson and Sparks, 1977, 1978) and D. polymorpha (Mazak et al., 1997). C. ﬂuminea densities were 3 – 5 times greater in enclosures excluding diving ducks (Smith et al., 1986). Water fowl feeding also signiﬁcantly reduces D. polymorpha populations (Mackie and Schloesser, 1996). Crayﬁsh feed on small bivalves including C. ﬂuminea (Covich et al.,1981) and D. polymorpha (Mackie et al., 1989), and, in some habitats, may regulate bivalve population densities. Fire ants (Solenopis invicta) prey on clams emersed by receding water levels. In additions, turtles, frogs, and the mudpuppy salamander Necturus maculosus all feed to a limited extent on small or juvenile bivalves (Fuller, 1974). Free-living oligochaetes prey on newly released glochidia (Fuller, 1974). Perhaps the major predator of freshwater bivalves are ﬁsh. A number of NA ﬁsh are molluscivores (Table V). While most molluscivorous ﬁsh feed on small bivalve species or juveniles of larger bivalves (SL 7 mm), several routinely take large adults, including carp (Cyprinus carpio), channel catﬁsh (Ictalurus punctatus) and freshwater drum (Aplodinotus grunniens). The latter species crushes shells with three massive, highly muscular pharyngeal plates, as do carp to a lesser extent. Bivalve prey size may be limited by the dimensions of the shell-crushing apparatus. Thus, 273 – 542-mm long A. grunniens did not take D. polymorpha with SL 11.4 mm, but, below this size, ﬁsh size and mussel size taken were not related. Thus, 385 predation by A. grunniens is unlikely to control D. polymorpha populations (French and Love, 1995). In contrast, channel catﬁsh swallow bivalves intact (J. C. Britton, personal communication). The majority of molluscivorous ﬁsh may limit predation to smaller bivalves because shells of larger individuals are too strong to crack or crush. The shell strength of C. ﬂuminea increases exponentially with size [log10 force in newtons to crack the shell 0.76 2.31 (log10 SL in mm)]; it is 6.5 times stronger than the very thick-shelled estuarine bivalve, Rangia cuneata (Fig. 23) (Kennedy and Blundon, 1983). Shell strength is directly correlated with shell thickness. In specimens with an SL of 20 mm, the 3.0 mm thick shell of the unionoidean, Fusconaia ebena, was more crush resistant than the 1.3 mm thick shell of C. ﬂuminea which, in turn, was more crush resistant than the fragile 0.31 mm thick shell of D. polymorpha. The fragile shell of D. polymorpha makes it prone to predation by diving ducks, crayﬁsh and ﬁsh relative other freshwater bivalves (Miller et al., 1994). Fish predation can deplete freshwater bivalve populations. Sphaeriid diversity and density increased in habitats from which molluscivorous ﬁsh were excluded (Dyduch-Falniowska, 1982). Robinson and Wellborn (1988) reported that, 11 months after settlement, densities of C. ﬂuminea were 29 times greater in enclosures excluding ﬁsh. In contrast, exclusion of ﬁsh and turtles did not increase density or species richness of a combined unoinoidean and sphaeriid community in a cooling reservoir (Thorp and Bergey, 1981). As ﬁsh are intermediate hosts for unionoidean glochidia, the size of ﬁsh host population can inﬂuence mussel reproductive success. Absence of appropriate ﬁsh hosts have caused extirpation of unionoideans in a number of NA aquatic habitats (Fuller, 1974; Kat and Davis, 1984; Smith, 1985a, b; Neves et al., 1997; Vaughn, 1997; Section III. A.) and availability of ﬁsh hosts may regulate recruitment (Johnson and Brown, 1998). Indeed, restoration of host ﬁsh populations produced remarkable recoveries of some endangered unionoidean populations (Smith, 1985b). Thus, anthropomorphic activities reducing ﬁsh host population densities can result in destruction of unionoidean populations, making relationships between unionoideans and their glochidial host ﬁsh an important future management consideration for drainage systems and their ﬁsheries to ensure continued health of their remaining unionoidean fauna (Watters, 1993; Neves, 1997). Mammals prey on freshwater bivalves. Otters, minks, muskrats, and raccoons eat bivalves and may regulate populations of some species (Fuller, 1974). Raccoons and muskrats feed on C. ﬂuminea and may account for reductions of adult clam densities in the shallow, near-shore waters of some Texas rivers, where 386 Robert F. McMahon and Arthur E. Bogan FIGURE 23 Shell strength of Corbicula ﬂuminea. Solid line is the best ﬁt of a geometric mean estimate of a least squares log–log linear regression, relating shell length (SL) to force required to crack the shell (open circles) as follows: Log10 force to crack the shell in newtons 0.76 2.31 (log10 mm SL). Dashed line is the best ﬁt of a similar log–log linear regression for the estuarine wedge calm, Rangia cuneata, as follows: log10 force to crack the shell in newtons 0.736 2.42 (log10 SL in mm). R. cuneata, with the strongest shell of eight tested estuarine bivalve species, has a thicker shell than does C. ﬂuminea. However, as the regression slope values for the two species are nearly equal, the elevated y-intercept for C. ﬂuinea (0.76 compared to 1.73 for R. cuneata) indicates that its shell is approximately an order of magnitude stronger. (Redrawn from Kennedy and Blundon, 1983.) feeding sites are marked by thousands of opened shells (McMahon, unpublished observations). Even wild hogs root out and destroy shallow-water unionoidean beds (Fuller, 1974). Muskrats annually consummed 3% of individuals of Pyganodon grandis in a small Canadian lake, equivalent to 31% of the mussel’s annual tissue production. They preferentially consumed larger mussels (shell length 55 mm), strongly affecting mussel population size – age structure, resulting in a biomass decline of large individuals, reducing the population’s annual reproductive effort (Hanson et al., 1989). c. Competitive Interactions There have been no experimental evaluations of interspeciﬁc or intraspeciﬁc competition among freshwater bivalves; only anecdotal observations. Unionoidean or sphaeriid population declines coincident with establishment of C. ﬂuminea have occurred in NA habitats (McMahon, 1999; Sickel, 1986). C. ﬂuminea may detrimentally impact sphaeriid populations after invasion, but there is little evidence of major impacts of C. ﬂuminea on NA unionoideans (Strayer, 1999b). Thus, C. ﬂuminea had little impact on a Tennessee River unionoidean community, the C. fuminea population declining over time while unionid density remained stable (Miller et al., 1992). Rather, habitats are ﬁrst made inhospitable to indigenous unionoideans by channelization, dredging, over-ﬁshing or other water management practices. C. ﬂuminea is more tolerant of high ﬂows, suspended solids, and silting caused by human activities in drainage systems, allowing it to rapidly colonize such disturbed habitants in which unionoideans have been reduced or extirpated (McMahon, 1999). In contrast, establishment of C. ﬂuminea in drainage systems receiving minimal human interference has little effect on native bivalve populations (McMahon, 1999; Sickel, 1986), suggesting an inability of Corbicula to outcompete native unionoideans in undisturbed habitats. The impacts of C. ﬂuminea on native, NA bivalves remains subject to debate (reviewed by Strayer, 1999b) and should be experimentally addressed. In contrast to C. ﬂuminea, there is clear evidence of direct negative impacts of D. polymorpha on NA unionoidean populations. Dreissena polymorpha byssally attaches to the posterior portions of unionoidean valves projecting above the substrate where they can reach densities which strip seston from the unionoidean’s inhalant current, leading to its slow starvation. Extensive D. polymorpha infestations can cause unionoideans to be dislodged from the substrate, leading to death. D. polymorpha infestation has caused near complete extirpation of unionoideans from the lower Great Lakes and St. Lawrence River (Schloesser et al., 1996; Strayer, 1999b). Interestingly, short-term brooding unionoideans (tribes Amblemini and Pleurobemini) appear less sensitive to zebra mussel impacts than long-term brooders (tribe Lampsilini and subfamily Anodontinae), perhaps due to their greater tissue energy reserves (Strayer, 1999b). Unionoidean populations in shallow (1 m), near-shore waters of western Lake Erie were less subject to D. polymorpha infestation than those at greater depths (Schloesser et al., 1997), perhaps because wave-induced agitation inhibits byssal attachment (Clarke and McMahon, 1996b). Dreissena polymorpha can have indirect impacts on N.A. bivalve communities. After it colonized the lower Hudson River in 1992, sphaeriid and unionoidean populations declined even though D. polymorpha did not attach to their shells. This decline resulted from a massive reduction in phytoplankton 11. Mollusca: Bivalvia density by D. polymorpha feeding, causing starvation of indigenous bivalves (Strayer, 1999b). d. Density-Dependent Mechanisms of Population Regulation Factors directly impacting freshwater bivalve population densities may have secondary impacts associated with reduction in gamete fertilization through reduction of sperm concentration in the water column. Thus, egg fertilization in the marsupial gills of female Elliptio complanata was directly correlated with population density, fertilization failing completely at 10 adults / m2 (Downing et al., 1993). Published experimental investigations of intraspeciﬁc, density-dependent, population regulation mechanisms in freshwater bivalves are also lacking. However, some evidence is available from descriptive ﬁeld studies of C. ﬂuminea and sphaeriids. In C. ﬂuminea, extremely successful recruitment of newly settled juveniles occurred after adult population decimation (McMahon and Williams, 1986a, b); whereas, juvenile settlement is generally far less successful in habitats harboring dense adult populations. High adult density may prevent successful juvenile recruitment, thus preventing extensive juvenile – adult competition. This hypothesis was tested by enclosing adult C. ﬂuminea at different densities in a lake harboring a dense Corbicula population. While adult density had no effects on adult or juvenile growth rates, it affected juvenile settlement. Settlement in enclosures without adults was 8702 juveniles / m2. Maximal settlement (26,996 juveniles / m2) occurred at 329 adults / m2; and was least at the highest adult densities of 659 and 1976 clams / m2, being only 8642 and 3827 juveniles / m2, respectively (McMahon, unpublished data). Thus, adults of C. ﬂuminea may inhibit juvenile settlement. Mackie et al., (1978) found that high adult densities in Musculium securis caused signiﬁcant reduction in the number of juveniles incubated, implying that intraspeciﬁc adult competition regulates reproductive effort. In addition, interspeciﬁc competition between M. securis and M. transversum caused reduction in reproductive capacity in the subdominant species, with species dominance dependent on habitat. While intraspeciﬁc density effects and interspeciﬁc competition may regulate Corbicula and sphaeriid populations, results may not be the same for highly K-selected unionoidean populations which require further study before the effects of thinning of mussel beds by commercial harvesters on juvenile recruitment and adult growth can be assessed. 4. Functional Role in the Ecosystem Bivalves play important roles in freshwater ecosystems (reviewed by Strayer et al., 1999a, b). Because they can achieve high densities and are ﬁlter feeders, they are 387 important consumers of phytoplankton primary productivity. Unionoideans, with ﬁltering rates on the order of 300 mL / g dry tissue/ h (Paterson, 1984), can account for a large proportion of total consumption of phytoplankton productivity. As unionoidean densities range from 15 individuals / m2 (Paterson, 1985) to 28 individuals / m2 (Negus, 1966) and dry tissue standing crop biomasses from 2.14 g / m2 (Paterson, 1985) to 17.07 g / m2 (Negus, 1966), ﬁltering by unionoidean communities could be as high as 15 – 122.9 L / m2 per day. Thus, on an annual basis, a eutrophic lake unionoidean community ﬁltered 79% of total lake volume, removing 92 – 100% of all suspended seston. However, even with such high cropping rates, high phytoplankton reproductive rates resulted in unionoideans reducing seston by only 0.44% (Kasprzak, 1986). While the unionoidean community accounted for only 0.46% of seston removed by all phytoplanktivorous animals in the lake, they made up 85% of its standing crop biomass. Their biomass contained a high proportion of the phosphorus load of the lake, limiting the quantity of phosphorus available for phytoplankton production. Similarly, the unionoidean community of Lake St. Clair ingested 13.5% of the total phosphorus load of the lake from May through October and deposited 64% of consumed phosphorus in the sediments, providing nutrients for rooted macrophytic vegetation and invertebrate deposit feeders (Nalepa et al., 1991). D. polymorphia populations in ﬁve European lakes had an average dry weight biomass 38 times that of submerged macrophytes and average standing crop phosphorus and nitrogen contents 2.94 (range, 0.39 – 7) and 4.21 (range, 0.59 – 9.4) times that of submerged macrophytes, respectively, greatly reducing availability of these growth-limiting inorganic nutrients to the aquatic plant community (Stanczykowska, 1984). In the western basin of Lake Erie, D. polymorpha has become a major factor in phosphorus ﬂux; its uptake of phosphorus and nitrogen potentially limiting phytoplankton growth and its low N:P excretion ratios (20:1) potentially shifting phytoplankton community structure toward cyanobacteria (Arnott and Vanni, 1996). Sphaeriids also have important impacts on lentic phytoplankton communities. The sphaeriid community of an oligotrophic lake comprised only 0.12% of total planktivore biomass and 0.2% of the bivalve biomass, but accounted for 51% of total seston consumption (Kasprzak, 1986). This probably resulted from their relatively high ﬁltration rates and population productivity (Burky et al., 1985a; Hornbach et al., 1984a, b). In smaller lotic and lentic habitats, sphaeriids can consume a major portion of primary productivity. In a canal community dominated by sphaeriids (98% of planktivore biomass), they accounted for 96% of total 388 Robert F. McMahon and Arthur E. Bogan seston consumption (Krasprzak, 1986). A stream population of S. straitinum was estimated to ﬁlter 3.67 g organic C / m2 per year or 0.0004% of seston organic carbon ﬂowing over them (Hornbach et al., 1984b). In contrast, ﬁltration by a pond population of M. partumeium removed 13.8 g C / m2 per year as seston (Burky et al., 1985a). Based on the data of Burky et al., (1985b), standing crop seston levels for the entire pond ranged annually between 46 and 550 g carbon. Average annual seston organic carbon consumption by the M. partumeium community was estimated to be roughly 3.9 g C per day, or 0.7 – 8.5% of total standing crop seston carbon per day. These data indicate that sphaeriid communities crop a signiﬁcant portion of the primary productivity of their small lentic habitats. With extremely high ﬁltering rates and dense populations (McMahon, 1999), C. ﬂuminea is potentially a major consumer of phytoplankton productivity. In the Potomac River, phytoplankton densities and chlorophyll a concentrations declined as it ﬂowed over dense beds of C. ﬂuminea, both measures falling 20 – 75% lower than upstream values; current phytoplankton levels in this river section are considerably lower than those prior to C. ﬂuminea invasion. It was estimated that the C. ﬂuminea population ﬁltered the entire water column in the 3 – 4 day period required for it to pass the river reach where the population was most dense (Cohen et al., 1984). Similar reduction in phytoplankton density of a lotic habitat by C. ﬂuminea has been reported by Leff et al. (1990). Reductions in phytoplankton density and chlorophyll a concentration must have been due to clam ﬁlter feeding, particularly as discharge volume variation, zooplankton feeding, toxic substances, and nutrient limitations were not different from other river sections (Cohen et al. 1984). Similarly, Lauritsen (1986b) estimated that a C. ﬂuminea population of 350 clams / m2 in the Chowan River, NC, at an average depth of 5.25 m and clam ﬁltering rate of 564 – 1010 mL / h, ﬁltered the equivalent of the overlying water column every 1 – 1.6 days. At an average depth of 0.25 m, current ﬂow of 18.5 m / min, and average clam ﬁltering rate of 750 mL / h (Lauritsen, 1986b), the entire water column of the Clear Fork of the Trinity River ﬂowing over a C. ﬂuminea population with an average adult density of 3750 clams / m2 was ﬁltered every 304 m of river reach, or every 16 min (McMahon, unpublished observations). Such massive ﬁltering of the water column by dense bivalve populations could keep seston concentrations at low levels and limit the energy available to other seston-feeding species. Consumption of the majority of primary productivity by dense bivalve populations and the accumulation of that production in large, relatively long-lived, predator-resistant adult clams may not only limit the inorganic nutrients available for primary productivity and the energy available to other primary consumers, but may also divert energy ﬂow from higher trophic levels. Bivalves, by ﬁltering suspended seston, are water clariﬁers and organic-nutrient sinks. When co-cultured with channel catﬁsh, C. ﬂuminea increased ambient water O2 concentrations by reducing seston and turbidity levels (Buttner, 1986). Dense bivalve populations, by removing phytoplankton and other suspended material, increase water clarity, and, by binding suspended sediments into pseudofeces, accelerate sediment deposition rates (Prokopovich, 1969). Dense D. polymorpha populations clarify water which increases light penetration, stimulating the growth of rooted aquatic macrophytes (Mackie and Schloesser, 1996). Such major ecological impacts by D. polymorpha have been observed in several locations in the lower Great Lakes, St. Lawrence River and Hudson River harboring dense mussel populations (reviewed by MacIsaac, 1996). In these habitats, D. polymorpha ﬁltering increases water clarity. In the Hudson River, D. polymorpha ﬁltered the equivalent of the entire water column every 1.2 – 3.6 days (Strayer et al., 1999). Phytoplankton densities are most negatively impacted by D. polymorpha ﬁltering in waters 1.85 m above mussel beds (MacIsaac et al., 1999). Besides phytoplankton and small zooplankton, D. polymorpha ﬁlters suspended clay and silt particles and binds them into pseudofeces deposited in sediments (MacIsaac, 1996; Strayer et al., 1999). Phytoplankton reductions by D. polymorpha have been extensive, ranging from 59 to 91% in portions of the Great Lakes (MacIsaac, 1996) and from 80 to 90% in the lower Hudson River, with mussels less efﬁciently removing diatoms than other smaller algae (Strayer et al., 1999). Filter feeding by D. polymorpha can reduce bacterioplankton densities, but can also stimulate bacterioplankton growth through nutrient or organic carbon excretion (Cotner et. al., 1995). Feeding by dense D. polymorpha populations appears to favor development of cyanobacteria blooms in some NA waters, perhaps due to reduction in N:P ratio, enhanced light penetration, greater buoyancy of cyanobacteria ﬁlaments, and / or chemical or mechanical inhibition of mussel ﬁltering by cyanobacteria (MacIsaac et al., 1996). Zooplankton populations are reduced by D. polymorpha ﬁltering. Small zooplankton such as rotifers and even Dreissena veligers can be directly ﬁltered; rotifer density in western Lake Erie declining by 74% within 5 years of D. polymorpha colonization, while larger zooplankton species that avoid entrainment on the mussel’s inhalant currents were relatively unaffected (MacIsaac, 1996). Similarly, zebra mussel 11. Mollusca: Bivalvia invasion brought about 80 – 90% reductions in rotifers, tintinnids, and copepod naupli densities in the lower Hudson River (Strayer et al., 1999). Suppression of small zooplankton could reduce ﬁsh populations whose larvae utilize them as food and result in density increases of bacterioplankton on which small zooplankton feed (Strayer et al., 1999). Increased water clarity and sediment nutrient levels appear to stimulate increased aquatic macrophyte growth in many areas colonized by D. polymorpha, favoring ﬁsh species associated with them over open-water species (MacIsaac, 1996). Increased macrophyte growth also appeared to favor associated invertebrate species, whose densities increased dramatically in the Hudson River after D. polymorpha colonization while deep-water benthic invertebrates concurrently declined (Strayer et al., 1999). Thus, overall, D. polymorpha populations appear to divert resources from the pelagic zone and deep-water sediments to vegetated shallows and zebra mussel beds (Strayer et al., 1999), leading to increases in benthic invertebrate abundance and diversity due to habitat structure enhancement among accumulated mussel shells and increased food supply, enhancement of physical habitat appearing to have the greatest impact (Botts et al., 1996). Increased bentic invertebrate densities support density increases in ﬁsh populations feeding on them (MacIsaac, 1996; Ricciardi et al., 1997; Stewart et al., 1998; Strayer et al., 1999), These postcolonization impacts of D. polymorpha are extensive, with almost all of 11 measured ecological variables being shifted by 50% after establishment of mussel populations in the lower Hudson River (Strayer et al., 1999). Increased food availability, associated with feeding on D. polymorpha, may also be responsible for postzebra mussel colonization increases in diving-duck ﬂock sizes and staging durations in several portions of Lake Erie (MacIsaac, 1996). As freshwater clams ﬁlter suspended organic detritus and bacteria and consume sediment interstitial bacteria and organic detritus (Section III.D.2), they may be signiﬁcant members of the aquatic decomposer assemblage. Pisidium casertanum and P. corneum feed primarily by ﬁltering sediment bacteria (Lopez and Holopainen, 1987). Hornbach et al. (1982, 1984b) estimated that only 24 – 35% of energy needs of a population of Sphaerium striatinum were met by ﬁlter feeding, the remaining coming from sediment organic detritus. C. ﬂuminea pedally feeds on sediment detritus (Reid et al., 1992), impacting the decomposer community (Hakenkamp and Palmer, 1999). Musculium transversum utilizes its long inhalant siphon to vacuum organic detritus from the sediment surface (Way, 1989). The detritivorous habit of many freshwater bivalve species may divert primary productivity ordinarily lost to respiration 389 of the detritivorous community back into bivalve tissue where it is made re-available to higher trophic levels. The activity of freshwater bivalves may also directly affect habitat physical characteristics. Deposition of calcium in shells may reduce ambient Ca concentrations. In a C. ﬂuminea population of 32 clams / m2, annual ﬁxation of shell CaCO3 was 0.32 kg CaCO3 /m2 per year (Aldridge and McMahon, 1978). At this rate, shell CaCO3 ﬁxation in a dense population of 100,000 clams / m2 (Eng, 1979) could be 50 – 60 kg Ca CO3 /m2 per year. Such rapid removal of calcium to shells accumulating in the sediments could reduce water hardness, particularly in lentic habitats (Huebner et al., 1990). Seasonal cycles of shell growth could induce seasonal cycles in water Ca concentration, being greatest in winter when shell growth is minimal and least in summer when shell growth is maximal (Rooke and Mackie, 1984c). Bivalves can also affect solute ﬂux between sediments and the water column. Unionoidean mussels enhanced release of nitrate and chloride and inhibited CaCO3 release from sediments (Matisoff et al., 1985). Dense sphaeriid populations can be the principal affectors of sediment dissolved O2 demand, in cases reaching levels characteristic of semipolluted and polluted streams (Butts and Sparks, 1982). D. polymorpha and C. ﬂuminea increase sedimentation rates, negatively or positively impact phosphate and nitrate ﬂuxes between sediments and the water column, change the N:P ratio of the water column, clarify water, and change sediment physical make up by accumulation of their shells (Arnott and Vanni, 1996; Strayer et al., 1999). Small bivalves (sphaeriids, juvenile unionoideans, D. polymorpha, and C. ﬂuminea) can be a major food source for third trophic level carnivores, including ﬁsh and crayﬁsh (Section III.C.3). Bivalve ﬂesh has a low caloric content, 3.53 – 5.76 kcal / g ash-free dry weight (Wissing et al., 1982), reﬂecting low lipid:protein ratios. C. ﬂuminea has tissue organic C:N ratios ranging from 4.9 – 6.1:1, indicative of a 51 – 63% of dry weight protein content (Williams and McMahon, 1989). High protein content makes bivalve ﬂesh an excellent food source to sustain predator tissue growth. Many freshwater bivalve populations are highly productive (particularly sphaerids, C. ﬂuminea, and D. polymorpha), rapidly converting primary productivity into tissue energy available to third trophic level carnivores. Productivity values range from 0.019 g C / m2 per year for a Pisidium crassum population to 10.3 g C / m2 per year for a C. ﬂuminea population (Aldridge and McMahon, 1978) and 75 g C / m2 per year for a D. polymorpha population (Mackie and Schloesser, 1996); the average for 11 sphaeriid species and two species of Corbicula was 2.5 g C / m2 per year (Burky, 1983). Other values include 12.8 – 14.6 g C / m2 per 390 Robert F. McMahon and Arthur E. Bogan year for an Arizona C. ﬂuminea canal population (computed from data of Marsh, 1985) and 1.07 g C / m2 per year for a New Brunswick lake unionoidean population of Elliptio complanata (computed from data of Paterson, 1985). Bivalves are generally more efﬁcient at converting assimilated food energy into new tissue than most second trophic level aquatic animals because their sessile, ﬁlter-feeding habits minimize energy expended in food acquisition, allowing efﬁcient transfer of energy from primary production to bivalve predators. Thus, bivalves can be important conduits of energy ﬁxed by phytoplankton photosynthesis to higher trophic levels. However, such trophic energy transfer is mainly through smaller species and juvenile specimens, as large adults are relatively immune to predation (Section 3.C). The measure of efﬁciency of conversion of assimilated energy absorbed across the gut wall into energy ﬁxed in new tissue growth is net growth efﬁciency: % Net Growth Efficiency P A(100) where P is the productivity rate or rate of energy or organic carbon ﬁxation into new tissue growth by an individual or population and A the assimilation rate or rate of energy or organic carbon assimilated by an individual or population. The greater the net growth efﬁciency of a second trophic level species, the more efﬁcient its conversion of assimilated energy into ﬂesh, and, therefore, the greater the potential for energy ﬂow though it to third trophic level predators (reviewed by Russell-Hunter and Buckley, 1983; Holopainen and Hanski, 1986; Hornbach, 1985). Net growth efﬁciencies (Table III), are 9 – 79% in sphaeriids (average 55%) and 58 – 89% in C. ﬂuminea, indicating efﬁcient conversion of assimilated energy into ﬂesh compared to other aquatic second trophic level animals. 5. Bivalves as Biomonitors Freshwater bivalves, particularly unionoideans C. ﬂuminea and D. polymorpha, have characteristics such as long life spans, and growth and reproductive rates sensitive to environmental perturbation (Burky, 1983) that make them good biomonitors (Imlay, 1982). They can be held in ﬁeld enclosures without excessive maintenance. Shell growth is sensitive to environmental variation and / or disturbance (Way and Wissing, 1982; Fritz and Lutz, 1986; Belanger et al., 1986a, b; Way, 1988), and the valves remain as evidence of death, allowing mortality rates to be estimated. Shells can be marked by tags (Rosenthal, 1969; Young and Williams, 1983c), including ﬂoating tags tethered to the shell (Englund and Heino, 1994b), paint, or shell-etching (McMahon and Williams, 1986a). Bivalves can be collected throughout the year and easily shipped alive long distances. Because large species stay in place (exception is C. ﬂuminea), they experience conditions in the monitored environment throughout life (Imlay, 1982). Annual shell-growth increments allows determination of variation in heavymetal pollutant levels over long periods by analysis of levels in successively secreted shell layers (Imlay, 1982). However, there is controversy regarding use of shellgrowth rings to age unionoideans. Growth rings are produced annually in some species (Neves and Moyer, 1988), but not in others (Downing et al., 1992). Large size allows analysis of pollutant levels in single individuals, and the wide geographical ranges of some species (Imlay, 1982) allow comparisons across drainage systems. Table VI indicates the types of pollutants and environmental perturbations monitored by freshwater bivalves. The broad distributions of C. ﬂuminea and D. polymorpha in North America and their dense, readily collectible populations make them excellent biomonitors compared to unionoideans, many species of which have restricted distributions and / or are threatened or endangered (Section III.A). D. Evolutionary Relationships Sphaeriid, dreissenids, corbiculids, and unionoideans represent separate evolutionary invasions of freshwaters. Sphaeriids, dreissenids, and corbiculids fall in the order Veneroida, and unionoideans in the order Unionoida (Allen, 1985). The Veneroida became successful infaunal ﬁlter feeders though evolution of inhalant and exhalant siphons, allowing maintenance of respiratory and feeding currents while burrowed. Their eulamellibranch gills with fused ﬁlaments (Fig. 5) were a clear advancement over the primitive (i.e., ﬁlibranch) condition with separate gill ﬁlaments attached by interlocking cilia (Allen, 1985). The family, Sphaeriidae, in the superfamily, Corbiculacea, with a long freshwater fossil history extending from the Cretaceous (Keen and Dance, 1969) has evolved along two major lines. The ﬁrst, represented by Pisidium, involves adaptation to life in organically rich sediments by ﬁltering interstitial bacteria (Lopez and Holopainen, 1987), making them dominant in profundal habitats. The second, represented by Sphaerium and Musculium, involves adaptation to life in small, shallow, lentic or lotic habitats subject to predictable seasonal perturbation, such as habitat-drying. Their adaptations include estivation during prolonged emergence (Section III.A). Like many of the Veneroida, the byssus is absent in adults of most species. The superfamily Unionoidea, like the Sphaeriidae, has a long freshwater fossil history, extending from at 11. Mollusca: Bivalvia 391 TABLE VI List of some Investigations Involving Utilization of Bivalves to Monitor Effects or Levels of Pollutants in Freshwater Habitats Pollutant monitored Species utilized Literature citations Arsenic Corbicula ﬂuminea Cadmium Eight unionoidean species, Anodonta anatina Anodonta cygnea Corbicula ﬂuminea Chromium Copper Eight unionoidean species Corbicula ﬂuminea Corbicula ﬂuminea Iron Lead Lamellidens marginalis Corbicula ﬂuminea Corbicula ﬂuminea Elder and Mattraw (1984), Price and Knight (1978), Tatem, (1986) Price and Knight (1978) Hemelraad et al. (1985) Hemelraad et al. (1985) Elder and Mattraw (1984), Graney et al. (1984), Price and Knight (1978), Tatem (1986) Price and Knight (1978) Elder and Mattraw (1984), Tatem (1986) Annis and Belanger (1986), Elder and Mattraw (1984), Price and Knight (1978), Tatem (1986) Hameed and Raj (1990) Tatem (1986) Annis and Belanger (1986), Elder and Mattraw (1984), Price and Knight (1978), Tatem (1986) Hameed and Raj (1990) Price and Knight (1978) Elder and Mattraw (1984), Tatem (1986) Hameed and Raj (1990) Elder and Mattraw (1984), Price and Knight (1978) Hameed and Raj (1990) Price and Knight (1978) Herwig et al. (1985) Belanger et al. (1986b), Elder and Mattraw (1984), Foe and Knight (1986c) Hameed and Raj (1990) Belanger et al. (1986a, 1987) Elder and Mattraw (1984), Tatem (1986) Elder and Mattraw (1984), Tatem (1986) Pugsely et al. (1985) Fisher et al.1993 Borcherding (1992), Fisher et al. (1993) Elder and Mattraw (1984), Hartley and Johnston (1983), Tatem (1986) Foe and Knight (1986c), Horne and MacIntosh (1979), Weber (1973) Dreier and Tranquilli (1981), Ferris et al. (1988), Foe and Knight (1987), McMahon and Williams (1986b) Borcherding and Volpers (1994) Englund and Heino (1994a) Englund and Heino (1994a) Manganese Mercury Nickel Tin Zinc Lamellidens marginalis Eight unionoidean species Corbicula ﬂuminea Lamellidens marginalis Corbicula ﬂuminea Lamellidens marginalis Eight unionoidean species Anodonta sp. Corbicula ﬂuminea Pentachlorophenol (PCP) Pesticides Lamellidens marginalis Corbicula ﬂuminea Lampsilis siliquiodea Corbicula ﬂuminea Lampsilis siliquoidea Dreissena polymorpha Dreissena. Polymorpha Corbicula ﬂuminea Sewage efﬂuents Corbicula ﬂuminea Power station efﬂuents Corbicula ﬂuminea Natural waters Dreissena polymorpha Unio tumidus Anodonta anatina Asbestos Octachlorostyrene Polychlorinated Biphenols (PCBs) least the Triassic (Haas, 1969). A long fossil history and tendency for reproductive isolation due to gonochorism and the parasitic glochidial stage (Section III.A) which allows sympatric speciation via new glochidial ﬁsh host acquisition (Graf, 1997) has led to extensive radiation, the Unionoidea being represented worldwide by 150 genera and a great number of species (Allen, 1985). Their origin remains unclear. Similarity of shell structure suggests a relationship to marine fossil species in the order Trigonoida (Allen, 1985). Like the majority of sphaeriids, adult unionoideans lack a byssus, which would be of no use in their infaunal habitats. They dominant the shallow regions of larger, relatively stable lentic and lotic habitats, particularly in stable sand – gravel sediments (Section III.A). The division of the superfamily, Unionoidea, into the presumed more primitive family, Margaritiferidae, and the more derived family, Unionidae, has been partially supported by mitochondrial DNA analysis of NA species. This study also supported the monophyly of the NA unionoidean subfamilies Anodontinae and Ambleminae and tribe Pleuorblemini, but provided only weak support for the 392 Robert F. McMahon and Arthur E. Bogan tribe Lampsilini (Lydeard et al., 1996). Further molecular studies are needed to elucidate the complex phylogeny of the Unionoidea, including analysis of species outside of North America. The Sphaeriidae and Unionoidea, with long freshwater fossil histories, have evolved distinct niches. While both occur in stable habitats, the majority of sphaeriids inhabit smaller ponds and streams or the profundal regions of lakes, and the majority of unionoideans inhabit stable sediments of shallow portions of larger rivers and lakes (Sections III.A and III.B). C. ﬂuminea entered freshwater only in recent (Pleistocene) times (Keen and Casey, 1969), making it unlikely to complete effectively with sphaeriids or unionoideans whose long fossil histories have allowed them to become highly adapted to their preferred stable freshwater habitats. Indeed, C. ﬂuminea does not appear to seriously impact native NA bivalves in undisturbed freshwaters (McMahon, 1999; Section C.3). Rather, it is adapted for life in unstable habitats — subject to periodic catastrophic perturbation, particularly ﬂood-induced sediment disturbance — which are generally unsuitable for sphaeriids or unionoideans (Section III.A). Among its adaptations to high-ﬂow habitats are: (1) capacity for rapid burrowing; (2) a strong, heavy, concentrically sculptured, inﬂated shell; and (3) juvenile retention of a byssal thread — all of which allow maintenance of position in sediments (Vermeij and Dudley, 1985). Additionally, its high fecundity, elevated growth rate, early maturity, and extensive capacity for dispersal (Section III.B) allow it to colonize habitats from which other bivalves have been extirpated and rapidly recover from catastrophic population reductions. Thus, C. ﬂuminea evolved a niche in unstable, disturbed habitats not utilized by sphaeriids or unionoideans, having evolved from an estuarine Corbicula ancestor inhabiting similar environments in the upper, near-freshwater portions of estuaries (Morton, 1982). The superfamily Dreissenacea, containing D. polymorpha, while superﬁcially resembling marine mytilacean mussels (order Mytiloida), is placed in the Veneroida because of its advanced eulamellibranch ctenidium. Adult dreissenaceans retain an attachment byssus, considered a primitive condition. In contrast, the mytiliform shell with its reduction of the anterior valves and anterior adductor muscle, is a derived adaptation to an epibenthic niche characterized by byssal attachment to hard surfaces (Allen, 1985; Mackie and Schloesser, 1996). D. polymorpha probably evolved from an estuarine dreissenacean ancestor of the genus Mytilopsis. As with C. ﬂuminea, its fossil record indicates a recent, Pleistocene introduction to freshwaters (Mackie et al., 1989). Also, like C. ﬂuminea, D. polymorpha appears to have successfully colonized freshwaters because its niche (i.e., an epibenthic species attached to hard substrates) is not occupied by infaunal, burrowing sphaeriids or unionoideans, thus minimizing competition with these more advanced groups. Freshwater bivalves have characteristics that are uncommon in comparable marine species. Most shallow-burrowing marine species are gonochoristic with external fertilization and free-swimming planktonic veligers. A planktonic veliger is nonadaptive in lotic freshwater habitats, as it would be carried downstream before settlement, eliminating upstream populations. Thus, the planktonic veliger stage is suppressed in sphaeriids, which brood embryos in gill marsupia and release large, fully formed juveniles ready to take up life in the sediment. In unionids, eggs are retained in gill marsupia and hatch into the glochidium, whose parasitism of ﬁsh hosts allows upstream as well as downstream dispersal and permits release of well-developed juveniles into favorable habitats (Section III.B). Even the recently evolved C. ﬂuminea has suppressed the planktonic veliger stage of its immediate estuarine ancestors (Morton, 1982), producing a small, but fully formed juvenile whose byssal thread allows immediate settlement and attachment to the subtrate. Retention of external fertilization and a free-swimming veliger in D. polymorpha are primitive characteristics reﬂecting its recent evolution from an estuarine ancestor. Downstream veliger dispersal would preclude D. polymorpha from establishing populations in high-ﬂow lotic habitats, unless upstream impoundments provide replacement stock. In fact, in Europe and Asia, this species is most successful in large, lentic or low-ﬂow lotic habitats such as canals or large rivers (Mackie et al., 1989). Serial impoundment of rivers has provided lentic refugia for this species in otherwise lotic drainages, facilitating its invasion of NA waters (Mackie and Schloesser, 1996). Another common adaptation in freshwater species is hermapharoditism with a capacity for self-fertilization. Hermaphroditism makes all individuals in a population reproductive, allowing rapid population expansion during favorable conditions. Hermaphroditism allows sphaeriids to re-establish populations after predictable seasonal perturbation and C. ﬂuminea, along with its high fecundity, to rapidly re-establish populations after catastrophic habitat disturbance. It makes both sphaeriids and C. ﬂuminea invasive, as the introduction of a single individual can found a new population. In contrast, the gonochorism of most unionoideans and D. polymorpha limits their invasive capacity, reﬂected by their stable, relatively undisturbed habitats. Hermaphroditism occurs in only seven species within three subfamilies of NA Unionoidea (Hoeh et al., 1995) with widely different habitats and reproductive traits. Selection pressures for evolution of hermaphroditism within these species have not been elucidated. 11. Mollusca: Bivalvia Finally, unionoideans and sphaeriids have different shell morphologies relative to comparable shallowburrowing marine bivalves. Freshwater species generally do not have denticulated or crenulated inner-valve margins, extensive radial or concentric shell ridges, tightly sealing valve margins, well-developed hinge teeth, overlapping shell margins, or uniformally thick, inﬂated, strong shells to the degree displayed by shallow-burrowing marine species (Vermeij and Dudley, 1985). These shell characteristics allow shallowburrowing marine species to maintain position in unstable substrates and / or resist shell cracking or boring by large predators common in marine habitats. Lack of such structures in unionoideans and sphaeriids reﬂects their preference for stable sediments and the general absence of effective predators on adult freshwater bivalves; certainly there are no shell-boring predators of NA freshwater species. Indeed, the shells of freshwater unionoideans display considerably less nonlethal, predator-induced damage than shallow-burrowing marine species (Vermeij and Dudley, 1985). While retention by C. ﬂuminea of a thick, extremely strong shell (Kennedy and Blundon, 1983; Miller et al., 1992), lacking pedal and siphonal gapes, with a well-developed hinge, shell inﬂation, and concentric ornamentation, may represent a primitive condition, it allows this species to inhabit sediments too unstable to support unionoideans or sphaeriids. Such primitive characteristics reﬂect similar adaptations in the immediate estuarine ancestor of this recently evolved freshwater species. In contrast, the thin, fragile shell of D. polymorpha (Miller et al., 1992) may have evolved due to byssal attachment of the species to hard substrates where the shell is not subject to damage from unstable sediments. IV. COLLECTING, PREPARATION FOR IDENTIFICATION, AND REARING A. Collecting Small sphaeriid clams are best collected by removing sediments containing clams with: (1) a trowel or shovel (shallow water); and (2) a long-handled dip net or shell scoop with a mesh of 0.35 mm (moderate depths) or by Ekman, Ponar, or Peterson dredges (deeper water), the heavier Ponar and Peterson dredges being better in lotic habitats. Drag dredges must have sediment catch bags of small mesh (1 mm or less). Use of scuba is also an effective means of collecting sphaeriids. Larger sphaeriids species can be separated from ﬁne sediments with a 1-mm mesh sieve, but a 0.35-mesh is required for smaller specimens. Fragile sphaeriids should be separated from sediments by gentle vertical agitation of the sieve at the water surface to prevent shell damage. 393 Coarse material should be removed prior to sieve agitation to avoid shell breakage. Exceptionally small, fragile species can be collected by washing small quantities of sediment into settlement pans, specimens being revealed after sediments settle and the water clears. Ekman, Ponar, or Peterson dredges are best for quantitative samples of sphaeriids, because they remove sediments from under speciﬁc sediment surface areas. Quadrat frames can be placed on the bottom (Miller and Payne, 1988) and all surface sediments within the frame collected for sorting. Such frames can be utilized in shallow or deeper waters (the latter in conjunction with scuba equipment). Drag dredging at speciﬁed speeds for known intervals allows partial sample quantiﬁcation. Core sampling devices consisting of tubes driven into the substrate to speciﬁc depths can yield both accurate estimates of sphaeriid density and sediment depth distributions. Core samplers sample only a small surface area, thus are best utilized with dense populations and / or repetitive sampling. Low densities of some unionoidean populations make their collection difﬁcult. Where populations are dense, shoveling sediments through a sieve allows collection of a broad range of sizes and age classes (Miller and Payne, 1988). As unionoideans are larger than sphaeriids, sieve mesh sizes of 0.5 – 1 cm may be appropriate unless recently settled juveniles must be collected. In less dense, shallow-water populations, hand-picking using a glass-bottomed bucket to locate specimens can often sufﬁce. In turbid waters and / or soft sediments (sand or mud), shell rakes may be utilized. Hand-picking and shell rakes can select larger, older individuals. In shallow, turbid water, using hands and ﬁngers to systematically feel for mussels buried in soft sediments can be effective, but selects larger individuals and can lead to cut ﬁngers. On rocky or boulder bottoms, unionoideans generally accumulate in crevices or near downstream bases of large rocks. Here, only hand-picking (in conjunction with scuba techniques in deeper water) is effective. Unionoideans may also be collected by drag or brail dredges. Mussel brails (often used by commercial shellers) consist of a bar with attached lines terminating in blunt-tipped gang hooks. When dragged over unionid beds, mussels clamp the valves onto the hooks allowing them to be brought to the surface. Quantitative sampling of unionoideans can be accomplished with quadrats, along with scuba in deeper waters (Isom and Gooch, 1986; Miller and Payne, 1988; Waller et al., 1993). Only heavy Ponar and Peterson dredges bite deeply enough into the substrate to take large unionoidean species. In sparse populations, errors in density estimates can result from the small surface areas that these dredges sample, requiring numerous samples to improve accuracy. Drag dredges sample larger areas and may be partially quantitative, 394 Robert F. McMahon and Arthur E. Bogan but generally do not provide accurate estimates. A skimmer dredge collected 62.3% of mussels in its path, but resulted in 10% mortality of thin-shelled species (Miller et al., 1989) Unionoideans may also be collected during natural or planned water level drawdowns (White, 1979) by collecting either emersed individuals or those migrating downshore and accumulating at the water’s edge. In a comparative study of sampling, quadrat sampling provided the most accurate analysis of a unionoidean community, but was difﬁcult and expensive to carry out in deeper waters, where systematic sampling by scuba produced the best results (Isom and Gooch, 1986). In a comparison of the accuracy of quadrat and qualitative timed searches for unionoideans, it was found that timed searches overestimated large species, highly sculptured species and those whose shells protruded from the substrate, while underestimating buried, small, smooth-shelled species. In contrast, quadrat sampling tended to underestimate rare species and total number of species unless a large number of quadrats were sampled (Vaughn et al., 1997). Extensive sampling may be required to ﬁnd rare species, particularly if their population densities need to be assessed; thus, time and funding limitations may prevent accurate assessment of presence / absence and population densities of rare unionoideans (Kovalak et al., 1986; Vaughn et al., 1997). Accurate sampling of rare unionoidean species is required for their effective management and conservation. As many NA unionoidean species are endangered, one should ascertain the status of any species before permanently removing specimens from their natural habitats. Take only dead shells or, if absolutely necessary, a few living specimens for identiﬁcation. C. ﬂuminea is easily collected because it occurs in high densities, prefers shallow waters, and is easily identiﬁed. Individuals can be separated from sediments with a 1-mm mesh sieve. Qualitative samples are best obtained by shovel or drag dredge (Williams and McMahon, 1986) and quantitative samples by quadrat frame (Miller and Payne, 1988), or Peterson (Aldridge and McMahon, 1978) or Ekman dredges (Williams and McMahon, 1986). In rock or gravel substrates, C. ﬂuminea are best taken by hand, hand trowel, or shovel; collected sediments should be passed though a coarse mesh sieve to separate specimens. In fast-ﬂowing streams, specimens of C. ﬂuminea accumulate in crevices or behind the downstream sides of large rocks. D. polymorpha is best collected by scuba with manual removal, using quadrat frames for quantiﬁcation, because of its byssal attachment to hard surfaces and its preference for deeper waters (1 – 2 m). Ripping of individuals from the byssus damages their tissues, thus the byssus should be cut with a knife or sharp trowel before removal. Juveniles can be collected after they have settled on settlement blocks or submerged buoys set out during the reproductive season. Ekman, Ponar, Peterson, and drag dredges are generally not suitable for collection of attached epibenthic species such as D. polymorpha, but they can be used for populations on soft or semisoft benthic substrates (Hunter and Baily, 1992). Juveniles of sphaeriids and C. ﬂuminea can be surgically removed from brood sacs or collected from sediments after release with a ﬁne mesh sieve (mesh size 0.35 mm). Juvenile C. ﬂuminea can also be obtained by release from freshly collected, gravid adults left in water for 12 – 24 h (Aldridge and McMahon, 1978). Planktonic C. ﬂuminea juveniles and D. polymorpha veligers may be taken with a zooplankton net towed behind a boat or held in current ﬂow. Their densities may be assessed by passing known volumes of water through a zooplankton net. Plankton net mesh size for collection of C. ﬂuminea juveniles should be 200 m ⬃40 m for all planktonic stages of D. polymorand pha, including the trochophore. Recently settled juveniles of unionoideans may be sieved from sediments. Glochidia can be surgically removed from demibranchs of gravid females or encysted glochicia taken from the ﬁns, pharyngeal cavity, or gills of their ﬁsh hosts (for a list of unionoidean ﬁsh hosts, see Watters, 1994b). B. Preparation for Identiﬁcation To preserve bivalves, larger individuals should ﬁrst be narcotized or relaxed, allowing tissues to be preserved in a lifelike state and preservatives to penetrate tissues through gaped shell valves. Live bivalves placed directly in ﬁxatives clamp the valves, which prevents preservative penetration. There are a number of bivalve relaxing agents (Coney, 1993; Araujo et al., 1995), including alcoholized water (either 3% ethyl alcohol by volume with water or 70% ethyl alcohol added slowly, drop by drop, to the medium until bivalves gape), chloroform added slowly to the medium, methol crystals (one level teaspoon per liter, scattered on the water surface), propylene phenoxetol and phenoxetol BPC (5 mL of product emulsed with 15 – 20 mL of water, added to water containing bivalves or introduction of a droplet equal to 1% of holding water volume), phenobarbitol added in small amounts to the holding medium, magnesium sulfate (introduced into holding medium over a period of several hours to form a 20 – 30% solution by weight), magnesium chloride (7.5% solution by weight), and urethane. None of these agents works equally well with all species. Laboratory tests of narcotizing agents against 11. Mollusca: Bivalvia C. ﬂuminea revealed propylene phenoxetol to be the only agent capable of relaxing this species for experimental surgery, and allowing recovery on return to fresh medium (Kropf-Gomez and McMahon, unpublished). Heating bivalves to 50°C for 30 – 60 min causes most species to relax and gape widely, but is lethal. In larger specimens, wooden pegs or portions of matchsticks forced between the valves prior to ﬁxation allows preservative penetration of tissues. The best long-term preservative for freshwater bivalves is 70% ethyl alcohol (by volume with water). Specimens may be initially ﬁxed in 5 – 10% formaldehyde solutions (by volume with 40% formaldehyde solutions) for 3 – 7 days. But formaldehyde is acidic and dissolves the calcareous portion of shells unless pHneutralized by the addition of powdered calcium carbonate (CaCO3) to make a saturated solution, 5 g of powdered sodium bicarbonate (NaHCO3) per liter, or 1.65 g of potassium dihydrogen orthophosphate and 7.75 g disodium hydrogen orthophosphate per liter (Smith and Kershaw, 1979). After 3 – 7 days in formaldehyde, specimens should be transferred to 70% ethyl alcohol for permanent preservation. Addition of 1 – 3% glycerin (by volume) to alcohol preservatives keeps tissues soft and pliable (Smith and Kershaw, 1979). Smaller species (shell length 15 mm) generally do not require relaxation. Tissues to be utilized in microscopy should be preserved in gluteraldehyde or Bouin’s solution. Shells can be cleaned with a mild soap solution and soft brush. Organic material can be digested from shell surfaces by immersion in a dilute (3% by weight with water) solution of sodium or potassium hydroxide at 70 – 80°C, thereafter, removing remaining organic matter with a soft brush. For dry-keeping, the shell periostracal surface should be varnished or covered with petroleum jelly to prevent drying, cracking, and / or peeling. Numbers identifying collection and specimen can be marked on the inner shell surface with India ink. In order to remove soft parts from living bivalves, immerse them in water at 60°C and remove tissues after valves fully gape. Separated ﬂesh can be ﬁxed in 70% alcohol. For fragile sphaeriids, ﬂesh is best removed with the tip of a ﬁne needle, manipulating specimens with a ﬁne brush. Shells should be dried in air at room temperature, not in an oven; heat causes shells to crack and their periostracum to crack and peel. Both soft tissue and shell characteristics are utilized in the identiﬁcation of freshwater bivalves, so both must be preserved for species identiﬁcation. For unionoideans, C. ﬂuminea, and D. polymorpha, most diagnostic taxonomic characteristics can be seen by eye or with a 10 hand lens. For sphaeriids, a dissecting microscope with at least 10 – 30 power or a com- 395 pound microscope is required (Ellis, 1978). Anatomical details are best observed on dry shells or soft tissues immersed in water. Identiﬁcation of recently released juvenile bivalves is difﬁcult and may require preparation of stained slide whole mounts. Glochidia are best identiﬁed by removal from a gravid adult of an identiﬁed species as are juvenile sphaeriids. Only the juveniles of C. ﬂuminea and larval stages of D. polymorpha are routinely found in the plankton. The juvenile of C. ﬂuminea (Fig. 15A) is easily recognizable, and can be readily separated from the pediveliger of D. polymorpha by the presence of a fully formed foot, lack of a velum and a D-shaped shell while the foot is formed only in the pediveliger stage of D. polymorpha which has an umbonal shell (Nichols and Black, 1994). The planktonic veliger of D. polymorpha is clearly distinguishable by its ciliated velum, lack of a foot and D-shaped shell (Nichols and Black, 1994; Fig. 15C). The glochidia of many unionoidean species have speciﬁc ﬁsh species hosts (Watters, 1994b), whose identiﬁcation will assist glochidial identiﬁcation. Identiﬁcation of glochidia without knowledge of the host ﬁsh or species of origin is extremely difﬁcult, characters such as presence / absence of attachment hooks or threads and glochidial size may allow assignment to a family or subfamily, but may be unreliable even at these higher taxonomic levels (Bauer, 1994). C. Rearing Freshwater Bivalves For artiﬁcial rearing of freshwater bivalves, water in holding tanks should be temperature-regulated. Adequate aeration, ﬁltration, and ammonia removal systems are required, as some species have low hypoxia and ammonia tolerances (Byrne et al. 1991a, 1991b). As unionoideans and C. ﬂuminea are ﬁlter phytoplankton, they require a constant supply of ﬁlterable food to remain healthy and growing. The best artiﬁcial food appears to be algal cultures. When fed monoalgal cultures of the green algae, Ankistrodesmus and Chlorella vulgaris or the cyanobacteruim, Anabaena oscillariodes, assimilation efﬁciencies in C. ﬂuminea were 47 – 57% and net growth efﬁciencies, 59 – 78%, making them excellent food sources for this species (Lauritsen, 1986a). Artiﬁcial diets do not appear to be as successful as algal cultures in maintaining C. ﬂuminea growth. When fed either ground nine-grain cereal, rice ﬂour, rye, bran, brewers’ yeast, or artiﬁcial trout food, small C. ﬂuminea (5 – 8 mm SL) starved on all but nine-grain cereal, the latter supporting little tissue growth. Supplementing these grain diets with live green algae (Ankistrodesmus sp.) greatly enhanced tissue growth, but the greatest growth occurred on pure Ankistrodesmus cultures (Foe 396 Robert F. McMahon and Arthur E. Bogan and Knight, 1986b). C. ﬂuminea fed mixed cultures of the green algae Pedinomonas sp., Ankistrodesmus sp., Chlamydomonas sp., Chorella sp., Scenedesmus sp., and Selenastrum sp. had maximal growth when mixtures did not include Selenastrum, which was toxic to this species. Greatest tissue growth occurred in clams fed mixed cultures of all ﬁve remaining algal species. Growth declined with number of algal species in feeding cultures; feeding with two algal species resulting in starvation (Foe and Knight, 1986b). Thus, artiﬁcial bivalve culture appears to be best supported on mixed algal diets, but certain toxic algal species must be avoided. Temperature also affects bivalve growth rate. When 5 – 8 mm SL specimens of C. ﬂuminea were fed mixed algal cultures of Chamydomonas, Chlorella, and Ankistrodesmus at 105 cells / mL, assimilation efﬁciencies were maximal at 16 and 20°C (48 – 51%). Tissue growth was maximal at 18 – 20°C and became negative (tissue loss) at 30°C (Foe and Knight, 1986a), suggesting 18 – 20°C to be an ideal culture temperature for this species and, perhaps, other NA bivalves. In algaefed cultures of D. polymorpha, growth was maximal at 15°C and suppressed at 20°C (Walz, 1978b). However, tissue growth in a natural population of C. ﬂuminea increased up to 30°C (McMahon and Williams, 1986a), indicating that artiﬁcial culture systems are not equivalent to ﬁeld conditions in supporting bivalve growth. In this regard, excellent tissue growth in C. ﬂuminea was supported by algal cultures produced by several days’ exposure to sunlight of water taken from the natural habitat of the clam to increase its algal concentration (Foe and Knight, 1985). Thus, ideal culture conditions for unionoideans and C. ﬂuminea would appear to be a 20°C holding temperature and feeding with natural algal assemblages whose growth has been promoted with inorganic nutrients and exposure to sunlight; maximal growth being achieved in D. polymorpha under the same conditions at 15°C. Specimens of C. ﬂuminea, D. polymorpha and unionoideans may be held for long periods in the laboratory without feeding. C. ﬂuminea survived 154 days of starvation at room temperature (22 – 24°C) while sustaining tissue weight losses ranging from 41 to 71% (Cleland et al., 1986). Similarly, I have held unionoideans in the laboratory for many months without feeding. Samples of D. polymorpha experienced 100% mortality after being held in the laboratory without feeding for 166, 514, and 945 days at 25, 15, and 5°C, respectively (Chase-Off and McMahon, unpublished data). Thus, maintenance at low temperature (10°C) greatly prolongs the time bivalves may be held in good condition without feeding. C. ﬂuminea has never been reared successfully to maturity or carried through a reproductive cycle in artiﬁcial culture, although ﬁeld-collected, nongravid adults released juveniles after four months in laboratory culture (King et al., 1986). The glochidium stage makes unionoideans difﬁcult to rear in the laboratory as it requires encystment in a ﬁsh host for successful juvenile metamorphosis, but it can be accomplished (Young and Williams, 1984a). Application of an immunosuppressant agent has allowed glochidial transformation to juveniles on nonhost ﬁsh where they would otherwise be rejected by the immune system of the host ﬁsh (Kirk and Layzer, 1997). Glochidia of several unionoidean species have been transformed into juveniles in vitro in a culture medium containing physiologic salts, amino acids, glucose, vitamins, antibiotics, and host ﬁsh plasma (Isom and Hudson, 1982). Juvenile utterbackia imbecillis and Epioblasma triquetra have been successfully cultured in a medium of river water exposed to sunlight for 1 – 4 days to enhance algal concentration. Addition of silt enhanced juvenile growth in both species, while feeding artiﬁcial, mixed cultures of three algal species resulted in starvation (Hudson and Isom, 1984), an observation supported by recent studies suggesting that juvenile unionoideans feed primarily on interstitial water drawn from sediments through the pedal gape, such that silt and associated microdetritus and bacteria may be their main ingested food source (Yeager et al., 1994). Many sphaeriid species can be easily maintained in simple artiﬁcial culture systems. Ease of artiﬁcial culture in this group may relate to their feeding on sediment organic detritus (Burky et al., 1985b; Hornbach et al., 1984b) and interstitial bacteria (Lopez and Holopainen, 1987), making use of algal cultures as a food source unnecessary. Hornbach and Childers (1987) maintained Musculium partumeium through successful reproduction in beakers with 325 mL of ﬁltered river water, without sediments, on a diet of 0.1 mg of ﬁnely ground Tetra Min© ﬁsh food/clam per day. The ﬁrst generation in this simple culture system survived 380 – 500 days, a life span equivalent to the natural population (Hornbach et al., 1980). Similarly, Rooke and Mackie (1984c) maintained 20 adult Pisidium casertanum in a 6-L aquaria with sediments for 35 weeks without feeding, suggesting that individuals fed on sediment bacteria or organic deposits. Live molluscs rapidly remove dissolved calcium from culture media (Rooke and Mackie, 1984c), thus calcium levels in holding media should be augmented by the addition of CaCO3. The ease with which sphaeriids can be artiﬁcially cultured makes them ideal for laboratory microcosm experiments. Ideal culture conditions appear to include provision of natural sediments, a source of calcium (i.e., ground CaCO3), and ﬁnely ground food of reasonable protein content, such as aquarium ﬁsh food or brewers’ yeast, which may be directly assimilated by clams or support the 11. Mollusca: Bivalvia growth of interstitial bacteria upon which they feed (Lopez and Holopainen, 1987). V. IDENTIFICATION OF THE FRESHWATER BIVALVES OF NORTH AMERICA A. Taxonomic Key to the Superfamilies of Freshwater Bivalvia There are ﬁve bivalve superfamilies with freshwater representatives in North America. Of these the Unionoidea, Corbiculoidea, and Dreissenoidea contain 397 the true freshwater species and comprise the vast majority of freshwater bivalve fauna. The remaining two superfamilies, Cyrenoidea and Mactroidea, each contain one brackish water species that can extend into freshwater coastal drainages and so are included here. In North America, the Dreissenacea is represented by two introduced freshwater species and an estuarine species. The Corbiculoidea includes 36 native and ﬁve introduced species in six genera; the Unionoidea are composed of 278 native species and 13 subspecies in 49 genera (Turgeon et al., 1998). Separate taxonomic keys are provided here for the latter two supperfamilies. 1a. Shell hinge ligament is external ..........................................................................................................................................................2 1b. Shell hinge ligament is internal ...........................................................................................................................................................4 2a(1a). Shell with lateral teeth extending anterior and posterior of true cardinal teeth (Fig. 1), shells of adults generally small (25 mm in shell length, shell thin and fragile; exceptions are the genera Polymesoda and Corbicula)..............................................superfamily Corbiculoidea [See Section V.B.] 2b. Shell without lateral teeth extending anterior and posterior of cardinal teeth .....................................................................................3 3a(2b). Shell hinge with two cardinal teeth and without lateral teeth; shell thin and fragile, 12 – 15 mm long with small umbos; Cyrenoida ﬂoridana (Dall) (extends from brackish into coastal freshwater drainages in Florida) .....................................superfamily Cyrenoidea 3b. Shell without true cardinal teeth, when present, lateral teeth only occur posterior to usually well-developed pseudocardinal teeth (Fig. 1), pseudocardial teeth absent or vestigial in some species; Shells of adults are generally large (25 mm in shell length) ................................................................................................................................superfamily Unionoidea [See Section V.C.] 4a(1b). Hinge with anterior and posterior latoral teeth on either side of cardinals; Shell massive, adults 25 – 60 mm shell length, obliquely ovate; Rangia cuneata (Gray) (extends from brackish into coastal freshwater drainages from Delaware to Florida to Veracruz, Mexico) ..........................................................................................................................................................superfamily Mactroidea 4b. Hinge without teeth; shell mytiloid in shape, anterior end reduced and pointed, hinge at anterior end, posterior portion of shell expanded, anterior adductor muscle attached to internal apical shell septum, attached to hard substrates by byssal threads .....................................................................................................................................................superfamily Dreissenoidea 5 5a(4b). Periostracum bluish brown to tan without a series of dorsoventrally oriented black zigzag markings, anterior end hooked sharply ventrally, ventral shell margins not distinctly ﬂattened over entire ventral side of valves; restricted to brackish water habitats. Mytilopsis leucophaeata (Conrad) (extends into coastal freshwater drainages from New York to Florida to Texas and Mexico) ............................................................................................................................................................................................Mytilopsis 5b. Periostracum light tan and often marked with a distinct series of black vertical zigzag markings; anterior portion of shell not ventrally hooked, ventral shell margins can be ﬂattened, restricted to freshwaters, Dreissena polymorpha (Pallas) (Fig. 24); (a European species introduced into the Great Lakes in Lake St. Clair and present by December 2000 throughout the Great Lakes, the St. Lawrence River and the Mississippi Drainage. and Dreissona bugensis Andrusov now in lakes Erie and Ontario, the Erie-Barge Canal and the St. Lawrence River .........................................................................................................................................Dreissena FIGURE 24 The external morphology of the shell of the zebra mussel, Dreissena polymorpha. 398 Robert F. McMahon and Arthur E. Bogan B. Taxonomic Key to the Genera of Freshwater Corbiculacea This key is based on the excellent species key for North American freshwater Corbiculoidea by Burch (1975a) with additional material from Clarke (1973) for the Canadian Interior Basin, and Mackie et. al. (1980) for the Great Lakes. The Corbiculoidea have ovate, subovate, or trigonal shells with lateral hinge teeth anterior and posterior to the cardinal teeth. All North American species expect Corbicula ﬂuminea are in the family Sphaeriidae. The family designation Pisidiidae has also been commonly applied to this group, but the International Commission of Zoological Nomenclature (ICZN) placed the Spheariidae (Name number 573) on the Ofﬁcal List of Family Names (Opinion 1331) in 1985; hence, Sphaeriidae is used as the family designation in this key and the rest of the chapter. In North America, the Sphaeriidae comprise the dominant bivalve fauna in small, often ephemeral ponds, lakes, and streams, the profundal portions of lakes and in silty substrates. Identiﬁcation is generally based on shell morphology, but requires, in some cases, soft tissue morphology. 1a. Shells large (maximum adult shell length 25 mm), thick, lateral teeth serrated .................................................family Corbiculida 2 1b. Shells generally small (maximum shell length 25 mm), thin, lateral teeth smooth ..............................................family Sphaeriidae 4 2a(1a). Maximum adult shell length generally 50 mm, shell ornamented by distinct, concentric sulcations, anterior and posterior lateral teeth with many ﬁne serrations, simultaneous hermaphrodites, massive numbers of small (length 0.3 mm) developmental stages (1000) incubated directly in inner demibranchs, released juveniles (5 mm SL) anchor to substratum with a single mucilaginous byssal thread (Fig. 25)........................................................................................................................................................Corbicula 3 FIGURE 25 External morphology of the shell valves of the light-colored shell morph (Corbicula ﬂuminea) and dark-colored shell morph (Corbicula sp.) of the North American Corbicula species complex. (A) Right and left valves of Corbicula sp. (dark-colored morph). (B) Right and left valves of C. ﬂuminea (light-colored morph). (C) Anterior view of the shell valves of C. ﬂuminea (light-colored morph). (D) Anterior view of the shell valves of Corbicula sp. (dark-colored morph). Note the distinguishing shell characteristics of these two species. C. ﬂuminea, the light-colored morph, which is widely distributed in North America (Fig. 11), has a more nearly trigonal shell, taller umbos, a greater relative shell width and more widely spaced concentric sulcations than does Corbicula sp., the dark-colored morph, that is limited to spring-fed, alkaline, lotic habitats in the southwestern United States (Fig. 11). The dark-colored shell morph also has a dark olive green-to-black periostracum and deep royal blue nacre, while the light-colored shell morph has a yellow green-to-light brown periostracum and white-to-light blue or light purple nacre. 11. Mollusca: Bivalvia 399 2b. Maximum adult shell length generally 50 mm, shell ornamentation of many ﬁne, closely spaced concentric striations, embryos not incubated in demibranchs, dioecious, periostracum deep brown in color, three cardinal teeth, estuarine, restricted to brackish waters in the tidal portions of rivers. Polymesoda caroliniana (Bosc) (Virginia to northern Florida to Texas)...............................Polymesoda 3a(2a). Shell nacre white with light blue, rose, or purple highlights, particularly at shell margin, muscle scars of same color intensity as rest of nacre, periostracum yellow to yellow-green or brown with outer margins always yellow or yellow-green in healthy, growing specimens, shell trigonal to ovate, umbos inﬂated and distinctly raised above dorsal shell margin, shell length : shell height ratio ⬇1.06, shell length : shell width ratio ⬇1.47, shell height : shell width ratio ⬇ 1.38, concentric shell sulcations widely spaced, 1.5 sulcations/mm shell height (Hillis and Patton, 1982); introduced in the early 1900s, it has spread throughout drainage systems of the United States and coastal northern Mexico (Fig. 11), the “light-colored shell morph” of Corbicula or Asian clam (Fig. 25B, C).Corbicula ﬂuminea (Müller) 3b. Shell nacre uniformly royal blue to deep purple over entire internal surface, muscle scars more darkly pigmented than rest of nacre, periostracum dark olive green to black, edges of valves in healthy, growing specimens not yellow or yellow-green, shell more ovate and laterally compressed with umbos less inﬂated and less distinctly raised above the dorsal shell margin than in C. ﬂuminea, shell length : height ratio ⬇1.15, shell length : width ratio ⬇ 1.65, shell height : width ratio ⬇ 1.43, concentric shell sulcations narrowly spaced, particularly at umbos, 2.1 sulcations/mm shell height (Hillis and Patton, 1982); introduced, distribution limited to highly oligotrophic, permanent, spring-fed, calcium carbonate-rich streams in the southwestern United States (Britton and Morton, 1986); (Fig. 11). Called the dark-colored shell morph of Corbicula, its taxonomic status is uncertain, electrophoretic (Hills and Patton, 1982; McLeod, 1986) and physiological evidence (Cleland et al., 1986) suggest it to be distinct from C. ﬂuminea (Fig. 25A, D) ........................................................................................................................................................................................Corbicula sp. 4a(1b). Both inhalant and exhalant mantle cavity siphons present and well developed, umbos lie anterior of center......................................5 4b. Only exhalant mantle cavity siphon present, inhalant siphon either absent or formed as a slit in the posterior-ventral mantle edges, umbos posterior of center, generally small, shell length 0.5 – 12 mm, embryos in inner demibranch held in thick-walled sacs, each with individual chambers for embryos, no byssal gland, 24 species widely distributed in North Amercia; for species identiﬁcations and distributions see Burch (1975a) (Fig. 26A) .......................................................................................subfamily Pisidiinae Pisidium FIGURE 26 Diagrams of the external morphology of the left shell valve of species representative of the North American genera of the freshwater bivalve family, Sphaeriidae. Size scaling bar is 1-mm long. 400 Robert F. McMahon and Arthur E. Bogan 5a(4a). Inhalant and exhalant mantle cavity siphons partially fused, embryos incubated in inner demibranchs in thin-walled longitudinal pouches, no byssal gland, shell with two cardinal teeth in each valve, without external mottling....................subfamily Sphaeriinae 6 5b. Inhalant and exhalant siphons not fused, embryos develop in individual chambers formed between inner and outer lamellae of inner demibranchs, functional byssal gland present, only one cardinal tooth in each shell valve, with external, mottled pigmentation, Eupera cubensis (Prime) (Atlantic coastal plain drainages from southern Texas to North Carolina, Caribbean Islands) (Fig. 26B) ...... .................................................................................................................................................................subfamily Euperinae Eupera 6a(5a). Shell sculptured with relatively coarse or widely spaced striae ( 8 striae/mm in middle of shell), shell relatively massive and strong, Sphaerium simile (Say) (Southern Canada from New Brunswick to British Columbia, south from Virginia to Wyoming, S. striatinum (Lamarck) (Canada from New Brunswick to the upper Yukon River, throughout the United States, Mexico, and Central America), S. fabale (Prime) (Southern Ontario to Georgia and Alabama) (Fig. 26C) ...........................................................Sphaerium 6b. Shell relatively thin, often fragile, with many ﬁne, narrowly spaced striae (12 striae/mm in middle of shell) ...................................7 7a(6b). Shell of adults 8 mm in length .........................................................................................................................................................8 7b. Shell of adults 8 mm in length .........................................................................................................................................................9 8a(7a). Posterior valve margin at near right angle to dorsal margin, shells roughly rhomboidal, umbos large and distinctly elevated above dorsal shell margin, Musculium partumeium (Say) (United States and southern Canada), M. transversum (Say) (Canada and the United States east of the continental divide, extending into Mexico), M. securis (Prime) (Nova Scotia to British Columbia southwestern Northwest Territories in Canada, United States except for southwest) (Fig. 26D)..................................................Musculium 8b. Posterior and dorsal margins rounded or forming an obtuse angle, shells ovate, Sphaerium corneum (Linnaeus) (introduced from Europe, localities in southern Ontario and Lakes Champlain and Erie), S. nitidum Westerlund (distribution is holarctic, northern Canada to northern United States), S. occidentale (Prime) (Canada from New Brunswick to southeastern Manitoba, northern United States south to Florida, west to Utah and Colorado) (Fig. 26 E, F)...........................................................................Sphaerium 9a(7b). Umbos large, distinctly elevated above the dorsal shell margin........................................................................................................10. 9b. Umbos small, indistinctly elevated above the dorsal shell margin, Sphaerium corneum (Linnaeus), (introduced, localities in Ontario, Lakes Champlain and Erie) (Fig. 26E) .................................................................................................................................Sphaerium 10a(9a). Shell rounded, umbos not prominent, Sphaerium occidentale (Prime) (New Brunswick to southeastern Manitoba, northern United States south to Florida in the east and Utah and Colorado in the west) (Fig. 26F) ...............................................................Sphaerium 10b. Posterior end of shell truncate, shell rhomboidal, umbos prominent, Musculium lacustre (Müller) (From treeline in Canada south throughout all but southwestern United States into central America) (Fig. 26G).................................................................Musculium C. Taxonomic Key to the Genera of Freshwater Unionoidea The Unionoidea make up the large bivalve fauna (shell length 25mm) of permanent freshwater lakes, rivers, and ponds. North America has the richest and most diverse unionoidean fauna in the world, including 278 species and 13 recognized subspecies in 49 genera in the Unionidae and ﬁve species in two genera in the Margaritiferidae. The taxonomy used here follows Turgeon et al. (1998) with the addition of Johnson (1998), and Williams and Fradkin (1999). Unionoidean taxonomy remains very uncertain because intraspeciﬁc and interpopulation variation often makes identiﬁcation and systematics difﬁcult. Unionoidean bivalve shells lack true cardinal teeth and, when present, lateral teeth occur only posterior to pseudocardinal teeth (Fig. 1). Figure 27 displays shell-shape outlines and external shell ornamentations referred to in these taxonomic keys. The key in the ﬁrst edition of this chapter, as well as many of the major keys to freshwater bivalves (Walker, 1918; Clench, 1959; Burch, 1975b; Clarke, 1973), relied on the integrated use of important anatomical structures and shell characters to identify unionoidean bivalves. However, most ﬁeld biologists do not have the luxury of having a fully gravid female in hand for identiﬁcation, but more likely, a dead shell or valve. Several colleagues have commented that one can only use a key containing anatomical characters to key out a shell when you already know what the animal is. Several state keys (e.g., Parmalee, 1967; Watters, 1995; Oesch, 1984; Strayer and Jirka, 1997) have provided keys to shells of species occurring within the political boundaries of a particular state. North America is considered to consist of those river systems and lakes north of, and including, the Rio Grande Basin and the rest of the boundary with Mexico. This key is artiﬁcial and the key is divided into four sections corresponding to geographical provinces to facilitate identiﬁcation. The four sections are: Gulf Coast, including Florida; Mississippi River Basin, including the Interior Basin of Canada; the area west of the Rocky Mountain divide; and the Atlantic Coast. These subdivisions are a simpliﬁcation of the 12 faunal provinces recognized by Parmalee and Bogan (1998). Table VII lists 11. Mollusca: Bivalvia 401 FIGURE 27 Illustrations of the diagnostic shell features or characters used for taxonomic identiﬁcation in Section V.C. Shell-shape descriptions: (A) rhomboidal; (B) triangular or trigonal; (C) round; (D) quadrate; (E and F) oval or ovoid; and (G) elliptical. Posterior shell-ridge morphology: (H) posterior ridge convex; and (I) posterior ridge concave. Concentric ridge structures of umbos: (J) single-looped concentric ridges; (K) double-looped concentric ridges; (L); coarse concentric ridges; and (M) ﬁne concentric ridges. (Redrawn from Burch, 1975b.) the genera found in each of these four sections. This geographical approach allows a biologist to focus only on those genera actually occurring in their region and ignores the rest of the diversity (e.g., west of the Rocky Mountains has only three genera: one Margaritiferidae and two Unionidae). We have attempted to provide a logical and clear key to the unionoidean genera of North America based solely on shell characters. The valve or pair of valves to be identiﬁed is assumed for the purposes of this key to be adults. Identiﬁcation of very small juvenile shells and small adult specimens can be difﬁcult. Landmarks, structures and shell shape are based on complete shells. We have attempted to use obvious characters, but many of them require familiarity with unionoidean shell morphology and the range of varia- tion in each of these characters. Strayer and Jirka (1997; p. 28) stated the problem most clearly when they said, “Keys based on shell characters are inevitably ﬁlled with vague, subjective terms, are frustrating for beginners to use, and misidentify many shells.” They have ﬁnally put into print what most malacologists have said: “Users should know that if they rely solely on this key, they will misidentify many shells.” (Strayer and Jirka, 1997; p. 28). There is no substitute for comparing the specimen to be identiﬁed with other identiﬁed specimens in a museum collection or specimens that have had their identiﬁcation veriﬁed by a specialist. This key is an introduction to the genera of North American unionoideans. Each couplet which contains a genus, also lists some of the species in that genus which should key out to that cou- 402 Robert F. McMahon and Arthur E. Bogan TABLE VII List of Unionoidean Genera by Geographic Area Used in the Key Margaritiferidae Paciﬁc coast Atlantic coast Gulf coast Margaritifera Margaritifera Margaritifera Unionidae Alasmidonta Anodonta Anodonta Anodontoides Alasmidonta Amblema Anodonta Anodontoides Arcidens Interior basin Cumberlandia Actinonaias Alasmidonta Amblema Anodonta Anodontoides Arcidens Arkansia Cyclonaias Cyprogenia Cyrtonaias Disconaias Ellipsaria Elliptio Elliptoideus Epioblasma Fusconaia Glebula Elliptio Fusconaia Dromus Ellipsaria Elliptio Epioblasma Fusconaia Gonidea Lampsilis Lasmigona Lampsilis Lasmigona Leptodea Lexingtonia Ligumia Leptodea Ligumia Medionidus Megalonaias Obliquaria Obovaria Plectomerus Pleurobema Uniomerus Utterbackia Pleurobema Popenaias Potamilus Ptychobranchus Pyganodon Quadrula Quincuncina Strophitus Toxolasma Tritogonia Truncilla Uniomerus Utterbackia Villosa 18 Villosa 37 Pyganodon Strophitus Toxolasma Total 3 plet. This list of species is not complete, but is representative of the species in the genus with the listed set of characters. Two genera are a source of much confusion, Elliptio and Pleurobema. The South Atlantic Slope Elliptio species complex has yet to be worked out. Johnson (1970) lumped a considerable number of taxa, Hemistena Lampsilis Lasmigona Lemiox Leptodea Lexingtonia Ligumia Medionidus Megalonaias Obliquaria Obovaria Pegias Plectomerus Plethobasus Pleurobema Potamilus Ptychobranchus Pyganodon Quadrula Simpsonaias Strophitus Toxolasma Tritogonia Truncilla Uniomerus Utterbackia Venustaconcha Villosa 43 which may, in fact, be valid species. The Gulf Coast Pleurobema species complex is the other major area of confusion. There are a large number of named taxa, but it is not clear at the present time which is a valid species and which is part of a cline. This group is still in need of further work. We would recommend that, to identify 11. Mollusca: Bivalvia specimens to the species level, the user move to a key for the state or province in which he/she is working. These volumes will give detailed descriptions, keys and ﬁgures of the species occurring in your local area (e.g., Florida: Clench and Turner, 1956; Johnson, 1972; Tennessee: 403 Parmalee and Bogan, 1998; Louisiana: Vidrine, 1993; New York: Strayer and Jirka, 1997; Illinois: Parmalee, 1967; Missouri: Oesch, 1984; and Ohio: Watters, 1995). A detailed list by state of freshwater bivalve literature can be found in Williams et al. (1993). 1a. Origin of shell is from west of the Rocky Mountains ...................................................................................................................4 1b. Shell is not from this area .............................................................................................................................................................2 2a. (1b) Origin of shell is from the Atlantic Coast of North America ........................................................................................................6 2b. Shell is not from this area .............................................................................................................................................................3 3a. (2b) Origin of shell is from the Mississippi River Basin......................................................................................................................26 3b. Origin of shell is from the Gulf Coast of North America............................................................................................................89 West of the Rocky Mountains 4a. (1a) Shell has lateral muscle scars [small pits for the attachment of mantle muscles to the shell], typically dark periostracum, relatively thick shell with pseudocardinal and lateral teeth .............................................................Margaritifera falcata (Fig. 11.28B) 4b. Shell lacks lateral muscle scars, periostracum typically not dark and hinge teeth reduced or absent .............................................5 5a. (4b) Shell has a very strongly developed posterior ridge, reduced hinge teeth, occurs from British Columbia to central California, east to Nevada and Idaho .........................................................................................................................Gonidea angulata (Fig. 28U) 5b. Shell lacks the sharp posterior ridge, lacks any evidence of hinge teeth .....................................Anodonta [in part] [A. beringiana, A. californiensis, A. dejecta, A. kennerlyi, A. nuttalliana, A. oregonensis] (Fig. 28F) Atlantic Coast 6a. (2a) Shell has lateral muscle scars, typically dark periostracum, relatively thick shell with pseudocardinal teeth and lateral teeth ......... ............................................................................................................................................Margaritifera margaritifera (Fig. 28B) 6b. Shell lacks lateral muscle scars, periostracum may not be dark, hinge teeth present or absent ......................................................7 7a. (6b) Shell with hinge teeth absent or greatly reduced ...........................................................................................................................8 7b. Shell with pseudocardinal teeth present, with or without lateral teeth ........................................................................................12 8a. (7a) Umbo not projecting above the hinge-line ...........................................................Utterbackia [in part] [U. imbecillis] (Fig. 28AW) 8b. Umbo projecting above the hinge-line ..........................................................................................................................................9 9a. (8b) Beak sculpture double looped (Fig. 27), shell uniformly thin..................................Pyganodon [in part] [P. cataracta] (Fig. 28AN) 9b. Beak sculpture consists of concentric bars ..................................................................................................................................10 10a. (9b) Nacre usually orange in the beak cavity, pseudocardinal tooth area represented by a thickening near the umbo, ventral shell margin uniform thickness .......................................................................................Strophitus [in part] [S. undulatus] (Fig. 28AR) 10b. Nacre bluish or white, hinge plate uniformly thin, teeth or swellings absent, ............................................................................11 11a. (10b) Ventral margin with a prominent thickened area along the anterior ventral magin below the pallial line, Anodonta [in part] [A. implicata] (Fig. 28F) 11b. Ventral margin not as above, shell elongate, Anodontoides [in part] [A. ferussacianus, restricted to the Susquehanna and Hudson River basins] (Fig. 28G) 12a. (7b). Shell with lateral teeth absent or reduced, neither functional nor interlocking ..............................................Alasmidonta [in part] [A. varicosa, A. marginata susquehannae, A. undulata] (Fig. 28D) 12b. Shell truncated, with well-developed lateral teeth .......................................................................................................................13 13a. (12b) Right valve with two lateral teeth, small, rare ......................................................Alasmidonta [in part] [A. heterodon] (Fig. 28D) 13b. Right valve with one lateral tooth ..............................................................................................................................................14 14a. (13b) Shell with spines on the umbo and down on to the disk of the shell ...........................................................................................15 14b. Shell lacks any evidence of spines ...............................................................................................................................................16 15a. (14a) Shell thick and may be large .............................Elliptio [in part] [shell from the Altamaha River Basin, Georgia, Elliptio spinosa, or the Tar/Neuse River Basin, North Carolina Elliptio steinstansana] (Fig. 28P) 15b. Shell small and from the James River Basin, Virginia .....................................................................Pleurobema collina (Fig. 28AJ) 404 Robert F. McMahon and Arthur E. Bogan 405 FIGURE 28 Diagrams of the external morphology mostly of right valves of the shell of species representative of the North American genera of the freshwater bivalve superfamily, Unionoidea (Figs. 28A – AY). Figures B – E, G – K, N, P, R – AC, AE – AG, AI, AJ, AL – AQ, AR – AU, reprinted with permission of Mrs. W.T. Edmondson from W.T. Edmondson (1959); Figures Q, AW, reprinted with permission of J.B. Burch from Burch (1973); Figures A, F, L, M, AD, AH, AK, AX, AY, reprinted with permission from the Treatise on Invertebrate Paleontology, courtesy of The Geological Society of America and the University of Kansas, © 1969. (Continues) 406 Robert F. McMahon and Arthur E. Bogan FIGURE 28 (Continued) 11. Mollusca: Bivalvia FIGURE 28 (Continued) 407 408 Robert F. McMahon and Arthur E. Bogan 16a. (14b) Hinge line in left valve with an additional small interdental or accessory tooth, giving the appearance of three pseudocardinal teeth, shell more or less compressed, shell shape rhomboid, periostracum dark green with numerous green rays, beak sculpture consists of prominent bars ...................................................Lasmigona [in part][L. decorata, L. robusta, L. subviridis](Fig. 28X) 16b. Left valve without extra interdental tooth ..................................................................................................................................17 17a. (16b) Shell shape rectangular to broadly triangular .............................................................................................................................18 17b. Shell shape oval, round or rhomboid..........................................................................................................................................19 18a. (17a) Shell is from the James River Basin, Virginia .............................................................................Lexingtonia subplana (Fig. 28AA) 18b. Shell is from an area extending from the Roanoke River Basin south to the headwaters of the Savannah River Basin.................... ...........................................................................................................................................................Fusconaia masoni (Fig. 28S) 19a. (17b) Shell shape rhomboid or rectangular ..........................................................................................................................................20 19b. Shell shape oval or round ...........................................................................................................................................................23 20a. (19a) Shell usually more than twice as long as high .............................................................................................................................21 20b. Shell usually less than twice as long as high................................................................................................................................25 21a. (20a) Nacre color white, shell inﬂated, ...............................................................................................................................................22 21b. Nacre color typically some shade of purple, but ranges from white to salmon to purple .......................................Elliptio [in part] [this genus contains basically three shell shapes, narrow and elongate: the Elliptio lanceolata complex; rectangular with various degrees of inﬂation: the Elliptio complanata complex; those shells with short shell length, not too tall and inﬂated: the Elliptio icterina complex] (Fig. 28P) 22a. (21a) Periostracum unrayed, shell thick, posterior end angled, periostracum mat or fuzzy, rectangular in shell shape, Uniomerus caroliniana (Fig. 28 AV) 22b. Periostracum rayed in juveniles, posterior end tapered to a point in middle of posterior margin, periostracum not mat, Ligumia nasuta (Fig. 28AB) 23a. (19b) Adult shell typically 40 mm in length, with a fuzzy or mat textured dark periostracum ...............................Toxolasma [in part] [T. pullus] (Fig. 28AS) 23b. Adult shell 40 mm in length, lacking the pronounced fuzzy periostracum ...............................................................................24 24a. (23b) Shell shape oval to elongate oval, periostracum very shiny to mat with rays ...................................Lampsilis [in part] [L. cariosa, L. dolabraeformis, L. radiata, L. splendida] (Fig. 28W) 24b. Shell shape oval, periostracum dull yellow, without rays or with ﬁne rays all over the shell, found in or near tidewater, nacre often a salmon color .......................................................................................................Leptodea [in part] [L. ochracea] (Fig. 28Z) 25a. (20b) Periostracum light colored with numerous green rays, shell relatively thin, oval to elongate oval, bladelike pseudocardinal teeth .......................................................................................................Villosa [in part] [V. delumbis, V. villosa, V. vibex] (Fig. 28AY) 25b. Periostracum dark to black, shell thick, no green rays, shell shape oval to round...................................................Villosa [in part] [V. constricta] (Fig. 28AY) Mississippi River Basin 26a. (3a) Shell with lateral muscle scars on the inside center of the valve ............................................Cumberlandia monodonta (Fig. 28A) 26b. Shell lacking any evidence of lateral muscle scars .......................................................................................................................27 27a. (26b) Shell not as above, lacking lateral teeth or all evidence of any hinge teeth ..................................................................................28 27b. Shell with both pseudocardinal and lateral teeth present ............................................................................................................34 28a. (27a) Shell with pseudocardinal teeth, but lacking lateral teeth ...........................................................................................................29 28b. Shell with greatly reduced or totally lacking both pseudocardinal and lateral teeth ....................................................................30 29a. (28a) Shell 25 mm, typically periostracum eroded off with periostracum restricted to narrow strip on shell margin, beak sculpture heavy bars extending down on the disk of the shell, restricted to the upper Cumberland and upper Tennessee River basins .......... ...............................................................................................................................................................Pegias fabula (Fig. 28AG) 29b. Shell not small, periostracum not eroded or erosion restricted to the umbo, beak sculpture consists of heavy bars, widespread in the Mississippi River Basin..................................................................Lasmigona [in part] [L. costata, L. complanata] (Fig. 28X) 30a. (28b) Shell with pseudocardinal tooth consisting of a slight swelling or knob, lateral tooth consisting of a rounded ridge, beak cavity copper colored, umbo centrally located ......................................................................................Strophitus undulatus (Fig. 28AR) 30b. Hinge teeth completely absent ....................................................................................................................................................31 31a. (30b) Umbonal area of shell projects above the plane of the hinge line ................................................................................................32 31b. Umbo level with hinge line or below the level of the hinge line ..................................................................................................33 11. Mollusca: Bivalvia 409 32a. (31a) Umbo elevated well above the hinge line, shell shape variable, ranging from oval, elliptical to rhomboid, beak sculpture consists of double loopedridges (Fig. 27) with projections on the bottom of the loop ...........Pyganodon [in part] [P. grandis] (Fig. 28AN) 32b. Umbo projecting only slightly above the hinge line, shell elongated, inﬂated, beak sculpture consists of 3 or 4 ﬁne sharp concentric ridges ...........................................................................................................................Anodontoides ferussacianus (Fig. 28G) 33a. (31b) Shell shape round to circular, compressed, beak sculpture consists of 4 – 5 pairs of broken irregular double looped ridges............. ............................................................................................................................Anodonta [in part] [A. suborbiculata] (Fig. 28F) 33b. Shell shape elongate, inﬂated, dorsal and ventral margins nearly straight and parallel, beak sculpture consists of 5 – 6 ﬁne irregular concentric ridges ............................................................................................Utterbackia [in part] [U. imbecillis] (Fig. 28AW) 34a. (27b) Shell with sculpture on the external shell surface........................................................................................................................35 34b. Shell with no plications, undulations, ridges, pustules or nodules...............................................................................................55 35a. (34a) Shell with prominent pustules or nodules and/or undulations, plications or ridges .....................................................................36 35b. Shell with only prominent, well-deﬁned pustules, knobs or nodules ...........................................................................................46 36a. (35a) Shell with undulations, ridges, or plications, no pustules or nodules on the surface....................................................................37 36b. Shell with undulations, ridges or plications and pustules on the disk or umbonal area ...............................................................44 37a. (36a) Shell with undulations, plications, ridges restricted to the posterior slope .................................................................................38 37b. Shell with undulations, plications, ridges not restricted to the posterior slope ............................................................................41 38a. (37a) Shell shape rectangular to oval, slablike, compressed, left valve with an accessory dentacle posterior to pseudocardinal teeth ....... .....................................................................................................................................Lasmigona [in part] [L. costata](Fig. 28X) 38b. Shell shape more elongate, lacks the accessory dentacle..............................................................................................................39 39a. (38b) Shell elongate, inﬂated with a blue to blue-green nacre color, restricted to the Tennessee and Cumberland River basins ................ ..........................................................................................................................Medionidus [in part] [M. conradicus] (Fig. 28AC) 39b. Shell elongate, inﬂated, nacre not as above.................................................................................................................................40 40a. (39b) Shell thin, posterior slope steeply angled to moderately angled ............................Alasmidonta [in part] [A. marginata] (Fig. 28D) 40b. Shell thicker, posterior slope rounded, posterior ridge rounded, restricted to the Tennessee and Cumberland River Basins ............ ....................................................................................................................Ptychobranchus [in part] [P. subtentum] (Fig. 28AM) 41a. (37b) Shell shape rectangular, with a distinct posterior ridge, purple nacre, open and shallow beak cavity .............................................. ...........................................................................................................................................Plectomerus dombeyanus (Fig. 28AH) 41b. Shell without a distinct posterior ridge, typically white nacre .....................................................................................................42 42a. (41b) Shell thick, compressed, maximum shell length 50 mm, low plications or wrinkles restricted to the posterior slope and posterior ventral margin of the shell, this species restricted to the Tennessee River Basin..............................Lemiox rimosus (Fig. 28Y) 42b. Shell not as above.......................................................................................................................................................................43 43a. (42b) Shell variably inﬂated, shell shape round to quadrate, heavy pseudocardinal teeth, deep compressed beak cavity, sculpture consists of typically three well-developed ridges or undulations running across the shell from anterior of the umbo to the posterior ridge, widespread in the Mississippi River Basin..................................................................................Amblema plicata (Fig. 28E) 43b. Shell somewhat inﬂated, shell shape quadrate, or approaching oval, pseudocardinal teeth curved, heavy sculpturing on the posterior half of the shell, species is restricted to the Red River Drainage in Oklahoma and the Ouachita River Drainage in Arkansas .............................................................................................................................................Arkansia wheeleri (Fig. 28I) 44a. (36b) Shell elongate with a well-developed diagonal posterior ridge .....................................................Tritogonia verrucosa (Fig. 28AT) 44b. Shell shape not elongate, lacking well-deﬁned posterior ridge.....................................................................................................45 45a. (44b) Well-developed plications running across the shell and pustules on the disk and umbonal area ..................................Megalonaias nervosa (Fig. 28AD) 45b. Plications not well developed and wavy, not straight, plications on posterior slope, two rows of pustules or knobs on umbo........ ......................................................................................................................................................Arcidens confragosa (Fig. 28H) 46a. (35b) Pustules distributed across most of the shell ...............................................................................................................................47 46b. Pustules restricted to a single or double row...............................................................................................................................50 47a. (46a) Pustules covering most of the shell, purple nacre, beak cavity deep and compressed .................. Cyclonaias tuberculata (Fig. 28J) 47b. Pustules covering most of the shell, nacre color white to pink not purple ...................................................................................48 48a. (47b) Pustules consistently small, with a shallow sulcus down the center of the disk, nacre white ...........................Cyprogenia [in part] [C. stegaria in the Ohio, Tennessee and Cumberland rivers; C. aberti in Arkansas, Oklahoma] (Fig. 28K) 48b. Pustules generally larger, and often of variable size and shape ....................................................................................................49 410 Robert F. McMahon and Arthur E. Bogan 49a. (48b) Disk of the shell usually with a broad green stripe running down the disk of the shell with a variable number of pustules, varies from a very few to covering most of the shell, nacre color mostly white, beak cavity deep and open not compressed .................... ...............................................................................................................................Quadrula [in part] [Q. pustulosa] (Fig. 28AO) 49b. Disk of shell without a broad green stripe, consistently covered with a large number of pustules, nacre color often pink, salmon or white, beak cavity open and shallow..............................................................Plethobasus [in part] [P. cooperianus] (Fig. 28AI) 50a. (46b) Pustules forming two distinct rows.............................................................................................................................................51 50b. Pustules or nodules forming a single row....................................................................................................................................52 51a. (50a) Two rows of pustules with one row on the posterior ridge, shell thick ......Quadrula [in part], [Q apiculata, this species has small pustules in the sulcus between the two rows of pustules; Q. nodulata, this species lacks a sulcus between the rows of pustules and may only have very few pustules; Q. quadrula; Q. fragosa, this species has a well-developed wing posterior to the umbo] (Fig. 28AO) 51b. Radiating row of knobs, shell inﬂated, thin to relatively thick, umbo high and full, pseudocardinal teeth compressed, not curved; beak sculpture consists of irregular nodules forming two loops which continue down and across most of the shell in two radiating rows, [mostly in small and juvenile shells] pustules fuse, become elongate and form plications, plications on the posterior slope of adults .......................................................................................................................Arcidens confragosus (Fig. 28H) 52a. (50b) The single row of pustules or knobs restricted to the posterior ridge .................................................................Quadrula [in part] [Q. cylindrica, with pustules on the posterior ridge while Q. cylindrica strigillata of the upper Tennessee River Basin has small pustules over most of the shell as well as pustules on the posterior ridge.] (Fig. 28AO) 52b. The single row of pustules or knobs forming a single row down the center of the disk...............................................................53 53a. (52b) Pustules large, located in a row down the center of the shell and limited to about three pustules on each valve ............................. .......................................................................................................................................................Obliquaria reﬂexa (Fig. 28AE) 53b. Pustules more numerous, but still in a centrally located row ......................................................................................................54 54a. (53b) Pustules usually wider than tall, shell periostracum a waxy yellow color .......................................................Plethobasus [in part] [P. cicatricosus, P. cyphyus] (Fig. 28AI) 54b. Pustules rather small, running down the centerline of the disk, but interrupted by a well-deﬁned, raised ridge running around the shell like a growth line, restricted to the Tennessee and Cumberland River drainages ....................Dromus dromas (Fig. 28N) 55a. (34b) Shell with a well-developed dorsal wing projecting above the hinge line, the wing usually posterior of the umbo, but some species may also have an anterior wing [some older shells, such as in Leptodea fragilis, maybe lacking the dorsal wing] ...........56 55b. Shell without a well-developed dorsal wing ................................................................................................................................58 56a. (55a) Hinge teeth thin pseudocardinal teeth compressed, thin and not projecting perpendicular to the axis of the hinge line .................. ...................................................................................................................Leptodea [in part] [L. fragilis, L. leptodon] (Fig. 28Z) 56b. Well-developed dorsal wing ........................................................................................................................................................57 57a. (56b) White nacre, thick-shelled ....................................................................................Lasmigona [in part] [L. complanata] (Fig. 28X) 57b. Purple nacre, thin-to-thick shelled, may be inﬂated.................................Potamilus [in part] [P. alatus, P. purpuratus] (Fig. 28AL) 58a. (55b) Shell shape round .......................................................................................................................................................................59 58b. Shell shape not round .................................................................................................................................................................68 59a. (58a) Shell compressed, shell shape round ...........................................................................................................................................60 59b. Shell not compressed, but more inﬂated, shell shape round, oval................................................................................................61 60a. (59a) Shell compressed, shape nearly circular, without lateral teeth ...............................Lasmigona [in part] [L. complanata] (Fig. 28X) 60b. Shell compressed, with lateral teeth, steep posterior slope, shell thick with well-developed hinge teeth, periostracum light yellow with interrupted rows of green chevrons ..........................................................................................Ellipsaria lineolata (Fig. 28O) 61a. (59b) Umbo central or nearly central in the dorsal margin...................................................................................................................62 61b. Umbo anterior of the center of the shell .....................................................................................................................................65 62a. (61a) Broad green ray extending from the umbo down only a short distance onto the disk of the shell ......................Quadrula [in part] [Q. pustulosa without pustules] (Fig. 28AO) 62b. No broad green ray ...................................................................................................................................................................63 63a. (62b) Shell with a raised ridge one quarter of the distance from the umbo running around the shell parallel with the growth lines. Restricted to the Tennessee and Cumberland River basins .......................................................................Dromus dromas (Fig. 28N) 63b. Shell without a raised ridge ........................................................................................................................................................64 11. Mollusca: Bivalvia 411 64a. (63b) Shell surface smooth, without a broad central sulcus ..............................................................Obovaria [in part] [O. jacksoniana, from the Mississippi River embayment shell somewhat more oval than round; O. retusa, nacre color white; nacre color purple inside the pallial line, white outside of the pallial line; O. subrotunda, shell round, thick shelled, nacre typically white but some populations with deep purple.] (Fig. 28AF) 64b. Shell with broad shallow-to-pronounced sulcus, ﬁne green rays, shallow beak cavity, well-developed hinge teeth, female shells with a swollen, extended or expanded portion of the posterior slope and posterior ventral margin of the shell.............................. Epioblasma [in part] [E. capsaeformis, E. ﬂorentina ﬂorentina, E. ﬂorentina walkeri, E. lewisi, E. ﬂexuosa, E. biemarginata, E. stewardsoni, E. propinqua, E. torulosa torulosa, E. torulosa gubernaculum, E. torulosa biloba, E. sampsoni] (Fig. 28R) 65a. (61b) Beak cavity deep compressed ......................................................................Fusconaia [in part][F. subrotunda, F. ebena] (Fig. 28S) 65b. Beak cavity shallow and open.....................................................................................................................................................66 66a. (65b) Restricted to the Tennessee River Basin, shell with green rays at least on the umbo area with a thick shell, heavy lateral teeth, very shallow sulcus or missing, shallow-to-no beak cavity ..................................................Lexingtonia dolabelloides (Fig. 28AA) 66b. Not restricted to the Tennessee River Basin, but widespread ......................................................................................................67 67a. (66b) Long axis of the main pseudocardinal teeth parallel with the lateral teeth, umbo anterior ..............................................Obovaria [in part, O. olivaria] (Fig. 28AF) 67b. Teeth not as above, shell wedge shaped to triangular or approaching square with or without a central sulcus, nacre white to deep pink ................................................................................................Pleurobema [in part] [P. rubrum, P. sintoxia] (Fig. 28AJ) 68a. (58b) Shell shape not oval....................................................................................................................................................................69 68b. Shell shape oval to oblong ..........................................................................................................................................................85 69a. (68a) Shell shape rectangular, triangular, square with sulcus................................................................................................................70 69b. Shell shape elongate, two-to-four times longer than high............................................................................................................75 70a. (69a) Beak cavity deep.........................................................................................................................................................................71 70b. Beak cavity shallow or broad and open ......................................................................................................................................72 71a. (70a) Shell shape rectangular, with green rays on the umbo .......................................................................................Fusconaia [in part] [F. ﬂava, F. cuneolus, F. cor, F. ozarkensis, F. barnesiana has a shallow beak cavity but belongs with this group] (Fig. 28S) 71b. Shell shape triangular to broadly triangular..........................................Pleurobema [in part] [P. cordatum, P. plenum] (Fig. 28AJ) 72a. (70b) Posterior slope not very sharp or rounded, covered with lines of green chevrons ................................................Truncilla [in part; T. truncata, T. donaciformis] (Fig. 28AU) 72b. Posterior slope very sharp or steep .............................................................................................................................................73 73a. (72b) Shell thin, inﬂated, abruptly truncate posteriorly, posterior slope lighter colored than the rest of the shell ..................................... Alasmidonta [in part] [A. marginata; shell thin, shell shape elongate, posterior slope not a steep sharp angle, headwaters of the Little Tennessee River in western North Carolina, A. raveneliana; streams of the upper Cumberland River Basin, A. atropurpurea] (Fig. 28D) 73b. Shell thin to thick, inﬂated posterior slope steep shell thick, shell shape rectangular...................................................................74 74a. (73b) Females with posterior slope inﬂated into a marsupial swelling, males evenly rounded posterior ridge and not inﬂated posterior ridge, nacre white ........................................................Epioblasma [in part] [E. triquetra wide spread, E. arcaeformis extinct and formerly restricted to the Tennessee and Cumberland River basins.] (Fig. 28R) 74b. Posterior margin not modiﬁed into a marsupial swelling, nacre purple..........................Elliptio [in part] [E. crassidens] (Fig. 28P) 75a. (69b). Thin shelled................................................................................................................................................................................76 75b. Thick shelled ..............................................................................................................................................................................79 76a. (75a) Shell compressed, very thin, periostracum rayed, nacre iridescent except for a purple wash in the open beak cavity ...................... ...............................................................................................................................................................Hemistena lata (Fig. 28V) 76b. Shell inﬂated...............................................................................................................................................................................77 77a. (76b) Hinge teeth thin, periostracum rayless, adult shell length 50 mm ...........................................Simpsonaias ambigua (Fig. 28AQ) 77b. Periostracum usually rayed, adult shell length 50 mm .............................................................................................................78 78a. (77b) Shell hinge thin, usually heavily rayed....................................................Villosa [in part] [V. iris complex, V. taeniata] (Fig. 28AY) 78b. Shell yellow, plain or rayed ........................Lampsilis [in part] [L. teres, L. raﬁnesqueana, L. siliquoidea, L. virescens] (Fig. 28W) 79a. (75b) Shell compressed ........................................................................................................................................................................80 79b. Shell inﬂated...............................................................................................................................................................................82 412 Robert F. McMahon and Arthur E. Bogan 80a. (79a) Adult shell length 40 mm, rayed, compressed, widespread, ...........................................Villosa [in part] [V. fabalis] (Fig. 28AY) 80b. Adult shell length 40 mm ........................................................................................................................................................81 81a. (80b) Shell hinge massive, curved, nacre white, periostracum with interrupted green rays ................................Ptychobranchus [in part] [P. fasciolaris, P. occidentalis] (Fig. 28AM) 81b. Hinge lighter, straight, nacre purple to white, periostracum typically unrayed ..................Elliptio [in part] [E. dilatata] (Fig. 28P) 82a. (79b). Adult shell length 55mm 82b. Adult shell length 55mm 83a. (82a). Periostracum olive green with numerous rays Villosa [in part] [V. trabalis, restricted to the Tennessee River Basin above Muscle Shoals and the upper Cumberland River basin, inﬂated, white nacre, V. perpurpurea, upper Tennessee River Basin, inﬂated, purple nacre] (Fig. 28AY) 83b. Periostracum ranges from greenish to black without rays Toxolasma [in part] [T. lividus, purple nacre, T. parvus white nacre, T. texasiensis white to salmon nacre] (Fig. 28AS) 84a. (82b) Shell inﬂated, periostracum rayless, posterior shell margin comes to a blunt point ventrally, coarse concentric beak sculpture opening posteriorly .......................................................................................Uniomerus [U. tetralasmus, U. declivus] (Fig. 28AV) 84b. Shell inﬂated, periostracum rayed as a juvenile, becoming dark brown to black as an adult, posterior shell margin comes to a point in middle of posterior margin, nacre white to white with purple wash in umbo area ...............................................Ligumia [L. nasuta, L. recta, L. subrostrata] (Fig. 28AB) 85a. (68b) Shell shape oval ..........................................................................................................................................................................86 85b. Shell shape oblong......................................................................................................................................................................87 86a. (85a) Shell shape oval, inﬂated, rayed, thin to thick shell, posterior ridge rounded or sharp, nacre variously white to pink or salmon ..............Lampsilis [in part] [L. cardium, L. fasciola, L. ovata, L. reeviana, L. raﬁnesqueana, L. higginsi, L. abrupta] (Fig. 28W) 86b. Shell shape elongate oval ............................................Villosa [in part] [V. taeniata, interrupted broad green rays, restricted to the Tennessee and Cumberland River basins; in the following three species males are oval while female shells have a truncate posterior margin, V. vanuxemensis, purple nacre, restricted to the Tennessee River Basin and the central Cumberland River Basin, V. ortmanni, restricted to Kentucky, V. lienosa, variously colored nacre] (Fig. 28AY) 87a. (85b) Shell shape oblong, inﬂated to globose, unrayed...................................Potamilus [in part] [S-shaped, very compressed hinge line, very inﬂated and thin shelled P. capax; inﬂated, black shiny periostracum and purple nacre P. purpuratus] (Fig. 28AL) 87b. Shell shape oblong, inﬂated but not globose ...............................................................................................................................88 88a. (87b) Adult shell length 70 mm, shell shape elliptic to oblong, thick shelled, nacre white with a tinge of salmon to coppery color in center of the shell, shallow beak cavity, rayed, with those rays on the posterior slope thick and wavy ....................Venustaconcha ellipsiformis (Fig. 28AX) 88b. Adult shell length 70 mm, shell shape oblong to oval, thin to thick shelled, nacre white to iridescent, beak cavity broad and open, moderately deep, usually rayed but rays on posterior slope not thin or wavy ..............................Actinonaias [A. pectorosa, thin shelled, restricted to the Tennessee and Cumberland River basins; A. ligamentina, widespread and typically thick shelled.] (Fig. 28C) Gulf Coast 89a (3b) Shell with lateral muscle scars in the interior center of the valve ..................................................................Margaritifera [in part] [M. hembeli, M. marrianae] (Fig. 28B) 89b. Shell without lateral muscle scars in the interior center of the valve ...........................................................................................90 90a. (89b) Shell with pseudocardinal and lateral teeth greatly reduced or absent ........................................................................................91 90b. Shell with both pseudocardinal and lateral teeth present ............................................................................................................95 91a. (90a) Pseudocardinal and lateral teeth present, but reduced ................................................................................................................92 91b. Pseudocardinal and lateral teeth completely absent ....................................................................................................................93 92a. (91a) Shell with pseudocardinal tooth consisting of a slight swelling or knob, lateral tooth consisting of a rounded ridge, beak cavity copper colored, umbo centrally located ....................................Strophitus [in part] [S. subvexus, S. connasaugaensis] (Fig. 28AR) 92b. Shell with pseudocardinal tooth thin, reduced and compressed, umbo projecting only slightly above the hinge line, shell elongated, inﬂated, beak sculpture consists of 3 – 4 ﬁne, sharp, concentric ridges ............................................................Anodontoides [in part] [A. radiatus] (Fig. 28G) 93a. (91b) Umbo elevated well above the hinge line, shell shape variable, ranging from oval, elliptic or rhomboid, beak sculpture consists of double looped with projections on the bottom of the loop ...................................Pyganodon [in part] [P. grandis] (Fig. 28AN) 93b. Umbo level ridges with hinge line or below the level of the hinge line ........................................................................................94 94a. (93b) Shell shape round to circular, compressed, beak sculpture consists of 4 – 5 pairs of broken irregular double looped ridges............. ............................................................................................................Anodonta [in part] [A. suborbiculata, A. heardi] (Fig. 28F) 11. Mollusca: Bivalvia 413 94b. Shell shape elongate, inﬂated dorsal and ventral margins nearly straight and parallel, beak sculpture consists of 5 – 6 ﬁne irregular concentric ridges................................................Utterbackia [in part] [U. imbecillis, U. peggyae, U. peninsularis] (Fig. 28AW) 95a. (90b) Shell with sculpture on the external shell surface........................................................................................................................96 95b. Shell without plications, undulations, ridges, pustules or nodules.............................................................................................109 96a. (95a) Shell with pustules or nodules and/or undulations, plications or ridges ......................................................................................97 96b. Shell with only pustules, knobs or nodules ...............................................................................................................................105 97a. (96a) Shell with undulations, ridges, or plications, but not pustules or nodules on the surface ............................................................98 97b. Shell with undulations, ridges or plications and pustules on the disc or umbonal area .............................................................103 98a. (97a) Shell surface with undulations, plications, ridges restricted to the posterior slope ......................................................................99 98b. Shell surface with undulations, plications, ridges not restricted to the posterior slope ..............................................................101 99a. (98a) Shell shape round to oval, slablike, compressed, left valve with an accessory dentacle posterior to pseudocardinal teeth plications occur on dorsal wing ...................................................................................Lasmigona [in part] [L. complanata] (Fig. 28X) 99b. Shell shape more elongate, lacks the accessory dentacle............................................................................................................100 100a. (99b) Shell inﬂated with a blue to blue-green nacre color .................................Medionidus [in part] [M. accutissimus, M. mcglameriae, M. parvulus, M. penicillatus, M. simpsonianus, M. walkeri] (Fig. 28AC) 100b. Shell inﬂated, nacre not as above, shell thin, posterior slope steeply angled to moderately angled ..............................Alasmidonta [in part] [A. triangulata, A. wrightiana] (Fig. 28D) 101a. (98b) Shell shape rectangular, with a distinct posterior ridge, purple nacre, open and shallow beak cavity ...........................Plectomerus dombeyanus (Fig. 28AH) 101b. Shell shape round to rectangular, without a distinct posterior ridge..........................................................................................102 102a. (101b) Shell thick, compressed, low plications or wrinkles on posterior slope and undulations and ridges on the disk of the shell, nacre with a central white area and a peripheral purple band, restricted to the Apalachicola and Ochlockonee River basins .................. ..................................................................................................................................................Elliptoideus sloatianus (Fig. 28Q) 102b. Shell variably inﬂated, shell shape round to quadrate, heavy pseudocardinal teeth, deep compressed beak cavity, sculpture consists of three to ﬁve well-developed ridges or undulations running across the shell from anterior of the umbo to the posterior ridge, nacre color white to iridescent ...........................................Amblema [in part] [A. plicata, A. neislerii, A. elliotti] (Fig. 28E) 103a. (97b) Shell shape elongate, with a well-developed diagonal posterior ridge ..........................................Tritogonia verrucosa (Fig. 28AT) 103b. Shell shape not elongate, lacking well-deﬁned posterior ridge...................................................................................................104 104a (103b). Shell shape round to quadrate, lacking well-deﬁned posterior ridge, has well developed plications running across the shell and pustules on the disk or umbonal area .........................................................................................Megalonaias nervosa (Fig. 28AD) 104b. Plications not well developed and wavy, not straight, plications on posterior slope, two rows of pustules or knobs on umbo........ ......................................................................................................................................................Arcidens confragosa (Fig. 28H) 105a. (96b) Pustules distributed across most of the shell .............................................................................................................................106 105b. Pustules restricted to a single or double row.............................................................................................................................107 106a. (105a). Adult shell length 60 mm, sculpture ﬁne, shell oval to elongate oval, pustules and chevrons...................................Quincuncina [Q. infucata, Q. burkei restricted to the Choctawhatchee River Basin; Q. mitchelli from the Rio Grand, Guadalupe, Colarado and Brazos rivers in Texas] (Fig. 28AP) 106b. Adult shell length not restricted to 60 mm, pustules covering most of the shell, nacre color white to pink, not purple, with a variable number of pustules, from a very few to pustules covering most of the shell, nacre color mostly white, beak cavity deep and open, not compressed...................................................................................Quadrula [in part] [Q. petrina, Q. houstonensis, Q. couchiana, Q. aurea, Q. asperata] (Fig. 28AO) 107a. (105b) Pustules or nodules large, forming a single row down the center of the disk, and limited to about three pustules on each valve ...............................................................................................................................................Obliquaria reﬂexa (Fig. 28AE) 107b. Pustules forming two distinct rows especially on the umbonal area..........................................................................................108 108a. (107b) Two rows of pustules, with one row on the posterior ridge, shell thick ........................Quadrula [in part] [Q apiculata, has small pustules in the sulcus between the two rows of pustules; Q. nodulata lacks a sulcus between the rows of pustules and may only have very few pustules; Q. quadrula, Q. rumphiana] (Fig. 28AO) 108b. Radiating rows of knobs, shell inﬂated, thin to relatively thick, umbo high and full, pseudocardinal teeth compressed, not curved, beak sculpture consists of irregular nodules forming two loops which continue down and across most of the shell in two radiating rows..............................................................................................................................Arcidens confragosus (Fig. 28H) 414 Robert F. McMahon and Arthur E. Bogan 109a. (95b) Shell with a well-developed dorsal wing projecting above the hinge line, the wing usually posterior of the umbo [see Fig. 30] but some species may also have an anterior wing [some older shells such as in Leptodea fragilis may be lacking the dorsal wing] ........................................................................................................................................................................................110 109b. Shell without a well-developed dorsal wing ..............................................................................................................................111 110a. (109a) Pseudocardinal teeth moderately heavy, projecting perpendicular to the axis of the hinge line, purple nacre thin to thick shelled, may be inﬂated ...................................................................................................Potamilus [P. inﬂatus, P. purpuratus] (Fig. 28AL) 110b. Pseudocardinal teeth compressed, thin and not projecting perpendicular to the axis of the hinge line..............................Leptodea [L. fragilis, L. amphichaenus] (Fig. 28Z) 111a. (109b) Shell shape round .....................................................................................................................................................................112 111b. Shell shape not round ...............................................................................................................................................................117 112a. (111a) Shell compressed, shell shape round, with lateral teeth, steep posterior slope, shell thick with well-developed teeth, periostracum light yellow with interrupted rows of green chevrons.......................................................................Ellipsaria lineolata (Fig. 28O) 112b. Shell not compressed, round to oval .........................................................................................................................................113 113a. (112b) Umbo central or nearly central in the dorsal margin.................................................................................................................114 113b. Umbo anterior of the center of the dorsal margin of the shell...................................................................................................116 114a. (113a) Broad green ray extending around the umbo down only a short distance onto the disk of the shell ................................Quadrula [in part] [Q. asperata without pustules] (Fig. 28AO) 114b. No broad green ray ..................................................................................................................................................................115 115a. (114b) Shell surface smooth, without a broad central sulcus.......................Obovaria [in part] [O. jacksoniana, O. unicolor] (Fig. 28AF) 115b. Shell with broad, shallow to pronounced sulcus, ﬁne green rays, shallow beak cavity, well developed hinge teeth, female shells with a swollen, extended or expanded portion of the posterior slope and posterior ventral margin of the shell.............................. ...............................................................................Epioblasma [in part] [E. penita, E. metastriata, E. othcaloogensis] (Fig. 28R) 116a. (113b) Beak cavity deep compressed ........................................................Fusconaia [in part][F. ebena, F. succissa, F. escambia] (Fig. 28S) 116b. Beak cavity shallow and open, shell shape wedge shaped to triangular or approaching square with or without a central sulcus, nacre white to deep pink ........................................................................................Pleurobema [in part] [P. marshalli] (Fig. 28AJ) 117a. (111b) Shell shape not oval..................................................................................................................................................................118 117b. Shell shape oval ........................................................................................................................................................................130 118a. (117a) Shell shape triangular, square to rectangular, with sulcus..........................................................................................................119 118b. Shell shape elongate, tapered posteriorly, two to four times longer than high ...........................................................................123 119a. (118a) Beak cavity deep.......................................................................................................................................................................120 119b. Beak cavity shallow or broad and open ....................................................................................................................................121 120a. (119a) Shell shape rectangular with green rays on the umbo........................................................Fusconaia [in part] [F. cerina] (Fig. 28S) 120b. Shell shape triangular to broadly triangular ...........................................................Pleurobema [in part] [P. taitianum] (Fig. 28AJ) 121a. (119b) Posterior slope rounded, not very steep, covered with lines of green chevrons ....................................................Truncilla [in part] [T. cognata, T. macrodon, T. truncata, T. donaciformis] (Fig. 28AU) 121b. Posterior ridge very sharp, posterior slope steep .......................................................................................................................122 122a. (121b) Shell thin, inﬂated, abruptly truncate posteriorly, posterior slope lighter colored than the rest of the shell ..................................... ...........................................................................................................................Alasmidonta [in part] [A. triangulata] (Fig. 28D) 122b. Shell thin to thick, inﬂated posterior slope steep shell thick, shell shape rectangular, posterior margin not modiﬁed into a marsupial swelling, often tapering posteriorly, nacre varies pale salmon to purple..........................................................Elliptio [in part] [E. crassidens] (Fig. 28P) 123a. (118b). Thin shelled..............................................................................................................................................................................124 123b. Thick shelled ............................................................................................................................................................................126 124a. (123a) Shell compressed, shell shape elongate to almost rectangular, very thin, periostracum rayed, nacre iridescent, white to dull purple, beak cavity shallow, lateral teeth long and curved to straight, pseudocardinal very small, periostracum brown with faint rays as a juvenile, restricted to the Rio Grande Basin, Texas..............................................................Popenaias popei (Fig. 28AK) 124b. Shell inﬂated.............................................................................................................................................................................125 125a. (124b) Hinge thin, periostracum usually heavily rayed ..................................................................Villosa [in part] [V. nebulosa complex, V. villosa, V. vibex] (Fig. 28AY) 11. Mollusca: Bivalvia 415 125b. Hinge not thin, periostracum yellow, plain or rayed ...................................Lampsilis [in part] [L. teres, L. straminea, L. hydiana, L. bracteata] (Fig. 28W) 126a. (123b) Shell compressed ......................................................................................................................................................................127 126b. Shell inﬂated.............................................................................................................................................................................128 127a. (126a) Hinge massive, curved, nacre white, periostracum with interrupted green rays....................................... Ptychobranchus [in part] [P. greeni] (Fig. 28AM) 127b. Hinge thinner, straight, nacre purple to white, periostracum typically unrayed .....................................................Elliptio [in part] [E. arctata, E. arca] (Fig. 28P) 128a. (126b) Rayed as a juvenile becoming dark brown to black as an adult, tapered to a point in middle of posterior margin, nacre white to white with purple wash in umbo area, Ligumia [L. recta, L. subrostrata] (Fig. 28AB) 128b. Shell unrayed 129a. (128b) Adult shell length 50mm, bluntly pointed posterior ventrally, coarse concentric beak sculpture seeming to radiate from a single point, Uniomerus [in part] [U. tetralasmus, U. declivus] (Fig. 28AV) 129b. Adult shell length 50 mm, bluntly rounded or squared off posteriorly, concentric beak sculpture in form of bars or ridges, angled up posteriorly Toxolasma [in part] [T. parvus, T. texasiensis] (Fig. 28AS) 130a. (117b) Shell shape oval ........................................................................................................................................................................131 130b. Shell shape oblong....................................................................................................................................................................133 131a. (130a) Posterior half of the pseudocardinal tooth divided into several parallel vertical ridges (Fig. 29), thick shelled, inﬂated, periostracum dark brown to dark olive ......................................................................................................Glebula rotundata (Fig. 28T) 131b. Pseudocardinal tooth not divided into vertical ridges as above .................................................................................................132 132a. (131b) Shell shape oval, inﬂated, rayed, thin-to-thick shell, posterior ridge rounded or sharp, nacre variously white to pink or salmon ..............................................................................................................Lampsilis [in part] [L. ornata, L. binominata] (Fig. 28W) FIGURE 29 Structure of the posterior pseudocardinal teeth of the right valve of Gelbula rotundata. Note that the posterior pseudocardinal teeth are deeply divided into parallel, vertical, plicate lamellae, a tooth arrangement uniquely characteristic of this species. (Redrawn from Burch, 1975b.) FIGURE 30 Right shell valve of the unionid, Potamilus alatus, showing the thin, extensive dorsal projection of the shell posterior to the umbos forming a “wing” whose presence or absence is a diagnostic characteristic valuable for identiﬁcation of a number of unionid species. (Redrawn from Burch, 1975b.) 416 Robert F. McMahon and Arthur E. Bogan 132b. Shell shape elongate, oval...............................Villosa [in the following two species male shells are oval while female shells have a truncate posterior margin, V. vanuxemensis umbrans, purple nacre, restricted to the upper Coosa River Basin, V. lienosa, variously colored nacre] (Fig. 28AY) 133a. (130b) Shell shape oblong, inﬂated to globose, unrayed........................Potamilus [in part] [P. amphichaenus, P. purpuratus] (Fig. 28AL) 133b. Shell shape oblong, inﬂated, but not globose ............................................................................................................................134 134a. (133b) Shell shape elongate oval, shell relatively thin to thick, compressed, pseudocardinal teeth compressed, thin, lateral teeth long, beak cavity shallow, periostracum tan to brown, with dark brown rays posteriorly, restricted to the Rio Grande Basin in Texas................................................................................................Disconaias [in part] [D. salinasensis, D. conchos] (Fig. 28M) 134b. Shell shape oval, moderately thick shell, inﬂated, beak cavity relatively deep, periostracum dull/shiny, red-brown to brown, juveniles with faint rays, Brazos River to Rio Grande Basin, Texas ..........................................Cyrtonaias tampicoensis (Fig. 28L) ACKNOWLEDGMENTS Alan P. Covich and Caryn C. Vaugh critically reviewed the chapter and made many valuable suggestions for its improvement. Thomas H. Dietz, Daniel Hornbach, Carl M. Way, and Harold Silverman provided the scanning and transmission electron micrographs of bivalve gill ciliation presented in Figure 21. Paul W. Parmalee, Caryn C. Vaughn, Dan Spooner, Valeria Rice and Jeff Garner kindly reviewed and commented on the unionoidean key. Jonathon A. Raine and Chad Rubins provided technical support with the preparation of the illustrations used in Figure 28. Cindy Bogan provided suggestions and encouragement during the drafting of the unionoidean key. Mrs. W. T. Edmondson kindly granted her permission for us to use the ﬁgures from her late husband’s second edition of Ward and Wipple (Edmondson, 1959). LITERATURE CITED Aarset, A. V. 1982. Freezing tolerance in intertidal invertebrates (a review). Comparative Biochemistry and Physiology A73: 571 – 580. Adam, M. E. 1986. The Nile bivalves: how do they avoid silting during the ﬂood? Journal of Molluscan Studies 52:248 – 252. Ahlstedt, S. A. 1983. The Mollusca of the Elk River in Tennessee and Alabama. 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Evolutionary Relationships IV. Collecting, Rearing, and Preparation of Oligochaetes for Identiﬁcation A. Collection B. Rearing Oligochaetes C. Preparation for Identiﬁcation V. Taxonomic Keys for Oligochaeta A. Taxonomic Key to Families of Freshwater Oligochaeta and Aphanoneura B. Taxonomic Key to Genera and Selected Species of Freshwater Lumbriculidae C. Taxonomic Key to Genera and Selected Species of Freshwater Naididae OLIGOCHAETA I. INTRODUCTION TO THE OLIGOCHAETA One family of aquatic Oligochaeta1, the Tubiﬁcidae (sludge worms), has been recognized since the time of Aristotle for its ability to develop dense colonies in 1 Oligochaeta may be a parahyletic group. The term is used here in the absence of a resolution into monophyletic taxa. Ecology and Classiﬁcation of North American Freshwater Invertebrates. 2nd Edition Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved. D. Taxonomic Key to Genera and Selected Species of Freshwater Tubiﬁcidae BRANCHIOBDELLIDAE VI. Introduction to Branchiobdellidae VII. Anatomy and Physiology of Branchiobdellids A. External Morphology B. Organ-System Function C. Environmental Physiology VIII. Ecology and Evolution A. Diversity and Distribution B. Reproduction and Life history C. Ecological Interactions D. Evolutionary Relationships IX. Identiﬁcation of Branchiobdellids A. Collecting, Rearing, and Preparation for Identiﬁcation B. Taxonomic Key to Genera of Freshwater Branchiobdellids Literature Cited Additional Suggested Readings organically polluted waters (Fig. 1). Beginning with the concepts that all sludge worms are pollution-tolerant indicators, recent work has made biologists aware of the wide range of ecological niches occupied by aquatic annelids in general, and oligochaetes and branchiobdellids in particular. They are found in every kind of freshwater and estuarine habitat, not only in organic mud. Specialized forms can be found in groundwater, in oligotrophic lakes and streams, and as commensals or symbionts on crayﬁsh, molluscs, and even tree frogs. 431 432 Ralph O. Brinkhurst, and Stuart R. Gelder II. OLIGOCHAETE ANATOMY AND PHYSIOLOGY A. Anatomy FIGURE 1 Clump of living tubiﬁcids (photograph by P. M. Chapman). Several North American genera prey on other worms or a variety of small invertebrates. The most surprising recent discovery has been the description of a rich diversity of tubiﬁcids at all depths in the oceans of the world. While most students are familiar with the terms Oligochaeta and Clitellata, there is no general agreement about the equivalent rankings of the various annelid groups. Some authors place the arthropods and annelids in a single phylum, but most texts separate them. Within the Annelida, it is possible to classify the Aclitellata (including Polychaeta) and the Clitellata as superclasses or classes. The former arrangement allowed us to refer to the Polychaeta, Oligochaeta, Hirudinea, and Branchiobdellida as classes, whereas the latter would demote these to orders. Relationships among the last three taxa, as well as the monotypic Acanthobdellida, are presently under investigation using cladistic methods, which greatly assist the process of determining sister-group relationships. Results of recent cladistic analyses provide evidence of a sistergroup relationship between lumbriculids and branchiobdellids, which share a unique arrangement of genital organs. As a result, we have returned to an earlier classiﬁcation of branchiobdellids as a taxon of family rank. The Aeolosomatidae are now recognized as a non-clitellate taxon Aphanoneura. Biologists who reject Hennigian methods may well classify the various groups in a different way from those who do not. The various terms will be used here based on the concept of superclass Clitellata with a series of classes, although we strongly suspect that the ranking of the Acanthobdellida will shortly be revised. Oligochaete worms are bilaterally symmetrical, segmented coelomates with four bundles of chaetae on every segment except the ﬁrst. In earthworms, the chaetal “bundles” usually consist of two chaetae each; but in aquatic species there are often several, and there may even be more than a dozen per bundle. In a few exceptional forms, the chaetae are absent in some or even all segments. Aquatic species (superorder Microdrili) are smaller than earthworms (Megadrili) as the name suggest, and they have simple body walls and none of the specialized regions of the digestive tract found in the earthworms. The clitellum is single-layer thick, and is limited to the genital regions in microdriles, and the eggs are large and yolky. In earthworms, secretions from the multilayered clitellum provide an alternative source of food for the eggs, which are small and less yolky. The clitellum is located well behind the gonadal segments. In aquatic species, the male and female ducts are connected to external genital pores located either in the same segment as the gonads they serve, or in the segment immediately behind them. In earthworms, the male ducts are extended back to open well behind the gonadal segments. It is generally thought that there were originally four gonadal segments, two with paired testes, and two with paired ovaries, in X – XIII (Roman numerals are used for segment number by convention). These have been reduced in number in various ways, as gamete storage and sperm transmission through copulation have evolved (see Section III.D). In some entire families and in a few individual species in other families, the gonadal segments may be found much nearer to the anterior end than usual. This may be due to a tendency to regenerate fewer than normal number of anterior segments in the predominantly asexual-reproducing forms in which this happens (family Naididae). This explanation will not sufﬁce for sexually reproducing forms in which the forward shift involves just one segment (family Lumbriculidae). In the family Opistocystidae, the gonads are located behind segment XX in all but the one species discussed here. The arrangement of the various reproductive organs of some typical aquatic oligochaetes is illustrated in Figure 2. The reproductive system and chaetae of one side are illustrated, but in life these animals are bilaterally symmetrical. The anatomy of the reproductive system is easy to understand if the stages in reproduction from sperm development to egg hatching are described. The mother cells of the sperm leave the testes to ﬂoat free in the coelom. The amount of space 12. Annelida: Oligochaeta, Including Branchiobdellidae 433 FIGURE 2 Reproductive systems and external characteristics of three major families of Oligochaeta. The worms are shown with the anterior to the left, and only the structures of the left side are visible. The dorsal and ventral chaetae are illustrated. In the Naididae, an example with dorsal chaetae beginning in VI is shown. Segments are identiﬁed with Roman numerals by convention (septa would be VII/VIII, VIII/IX, etc.). a, Atrium; e, eye spot; o, ovary; pr, prostomium; pt, prostate; sp, spermatheca; t, testis; v, vas deferens; genital pores. available to the developing sperm masses, or morulae, is increased by the development of outpushings of the testicular segments, slightly anteriad but more extensively posteriad through several segments. These are called sperm sacs. The sperm eventually cluster around large funnels suspended on the posterior walls of the testicular segment, sometimes well within the sperm sacs. These sperm funnels lead into vasa deferentia that originally drained via the male pores, located near the ventral chaetae of the post-testicular segment. Various structures have evolved, presumably at the point where the vasa deferentia contact the original body wall. Inversion of the body wall has led to the evolution of atria. These muscular bodies were originally lined with ectodermal cells, many of which had a glandular function. They presumably fed and lubricated the sperm stored in the atria ready for copulation. The glandular cells are thought to have migrated inward, through the muscle layers, so that their cell bodies now lie in the coelom. This resulted in both an increase in the space available to the glandular cell bodies and an increase in the space available for sperm within the atria. The 434 Ralph O. Brinkhurst, and Stuart R. Gelder ways in which the gland cells have migrated through the muscles of the atria to form the prostate glands vary, and this provides a very useful taxonomic feature (though not one that can be seen without some effort). The term prostate gland is used for very different structures in earthworms and branchiobdellids. In a further evolutionary development, another section of the body wall becomes part of the male reproductive structure between the atria and the male pores. Various types of penes are found in what are considered to be the more advanced groups (family Tubiﬁcidae, family Lumbriculidae) and have evolved quite independently several times. In some penes, the normal cuticular layer over the epidermis has become hypertrophied to form cuticular penis sheaths. Sometimes this cuticular layer is hardly noticeable, but the cuticular tubes may become thick and rigid with soft penes free within them. Cuticular penis sheaths are said to be present if the cuticle over the penes can be seen in cleared, whole-mounted worms and has a consistent shape. In some species, basement membranes or cuticular lining of eversible penes appear on whole mounts, but these are usually irregular in form. These are called “apparent penis sheaths.” When worms copulate, sperm is passed to the partner and is usually deposited in saclike spermathecae. There may be one or more pairs of these structures located in or near the gonadal segments, commonly in front of the testicular segments, or in them. The number and position of the spermathecae may vary within the Lumbriculidae, but there is usually one pair in the single testicular segment (families Tubiﬁcidae, and Naididae), or separated from the other genitalia in V (family Enchytraeidae). In a few species, spermathacae may be present in some specimens but not others, or they may be missing altogether. In Bothrioneurum (Tubiﬁcidae), the sperm are placed in spermatophores that are attached externally to the body wall of the partner, usually near the gonopores, during copulation. In members of the tubiﬁcid subfamily Tubiﬁcinae, two types of sperm are present, which come together in organized masses called spermatozeugmata. These can be recognized within the spermathecae of mated worms. Other forms of spermatozeugmata are found in other subfamilies. After a worm has copulated and has sperm in its spermathecae, the clitellum secretes a cocoon. The eggs have already developed in the coelomic space of the ovarian segment. This segment is extended posteriad into egg sacs. As the sperm sacs already displace the septa of several segments behind the testes, the egg sacs and their contained eggs are only detectable in segments beyond the sperm sacs. The egg and sperm sacs have been omitted from Figure 2 because it becomes confusing to follow a whole series of ﬁngerlike protrusions running through several segments, all contained within each other. [Note that egg and sperm sacs of the Acanthobdellida and Hirudinea are not contained within each other, but lie parallel to each other. They are paired, bilateral structures just like the rest of the reproductive system.] Eggs are deposited into the cocoon via the female ducts. These consist of nothing more than ciliated funnels on the posterior walls of the ovarian segment that open into the intersegmental furrow. In a few primitive forms, the female ducts actually penetrate the postovarian segment and open on its anterior surface. It is assumed that fertilization occurs in the cocoon. The basic position for the spermathecal, male, and female pores is in the longitudinal line of the ventral (ventrolateral) chaetal bundles, but there is some variation in this due to midventral fusion, the development of median copulatory bursae, or the migration of the spermathecal pores to a more dorsal position. While the spermathecal pores may open near the anterior furrow of the segment they occupy, all the genital pores commonly lie close to the ventral chaetae. The worm ﬁnally releases the cocoon, which formed as a ring of material around it, secreted (presumably) by the clitellum. The ends of the cocoon close, and the fertilized eggs develop inside. Small worms eventually escape through the openings at the ends of the cocoons and are recognizable as immature specimens, which can be identiﬁed if they happen to belong to one of the species with characteristic chaetae. The various reproductive organs and the modiﬁed chaetae that may develop close to the genital pores can best be found by looking carefully in the correct segments. When the keys mention genital structures, these will only be found on mature specimens. An immature aquatic oligochaete appears to consist of very little more than the body wall with its chaetae and a simple tubular gut running through the coelom. Very few specialized anatomical features have been added to the basic vermiform shape, unlike the situation in the Polychaeta. That is why it is generally useless to illustrate the general habit of oligochaetes; useful illustrations are those of the chaetae and reproductive systems. In contrast to immature specimens, a mature worm will have an external clitellum, though this is not as prominent as that of an earthworm, and the egg and sperm sacs are full and, therefore, visible. The worm is often somewhat distended in the genital region, and the reproductive organs obscure the normal view of the gut. As most worms burrow through soft substrates and extract their food by ingesting them, the range of externally visible anatomical adaptations are few because this habit imposes severe limitations on body shape. The prostomium lies in front of the mouth, and 12. Annelida: Oligochaeta, Including Branchiobdellidae while its body cavity is clearly coelomic, it is considered to be presegmental. The prostomium may bear a median anterior prolongation, the proboscis. In Bothrioneurum, there is a median dorsal pocket containing sensory cells, the prostomial pit (Fig. 3). Segment numbering begins with the peristomium, the small segment around the mouth. In aquatic species, the peristomium is completely separate from the prostomium. It is also presegmental, and bears no chaetae. This is important to remember when using cleared, whole-mounted specimens as it is often easier to locate speciﬁc segments by counting chaetal bundles (which begin in Segment II). The chaetae vary in form as well as number. This variation provides very useful key characters, but there is increasing evidence that the ﬁne details of chaetal form, or even the presence or absence of a particular type of chaetae, such as hairs, can be affected by quite simple environmental variables such as pH or conductivity (Chapman and Brinkhurst, 1987). The Enchytraeidae have simple-pointed chaetae, which may be straight or sigmoid. The chaetae in a bundle may vary in size (Fig. 4). Hair chaetae are elongate, simple shafts. They are found only in the dorsal bundles of some species of Naididae, Tubiﬁcidae, and in Crustipellis (Opistocystidae). They are present in both dorsal and ventral bundles in the Aeolosomatidae (Aphanoneura, Aclitellata), this being one of a series FIGURE 4 FIGURE 3 435 Prostomial pit, Bothrioneurum (photograph by P. M. Chapman). of fundamental differences between these minute freshwater worms and true oligochaetes. Hair chaetae are termed hispid or serrate when they bear a series of ﬁne lateral hairs. This condition may vary within a Representative chaetae. From the left, top row: lumbriculid biﬁd with reduced upper tooth, head end, and entire; small dorsal and large ventral sickle-shaped chaetae of Haplotaxis; three types of bundles from the Enchytraeidae. Bottom row: two pectinates, a palmate, and a biﬁd dorsal chaeta, Tubiﬁcidae; six types of dorsal needles, Naididae. 436 Ralph O. Brinkhurst, and Stuart R. Gelder species depending on seasonal or environmental variables, but it is still used as a taxonomic character for many naidids for which the limits of intraspeciﬁc variability have yet to be established. In a few naidids, the lateral hairs may be especially prominent along one side of the hair chaeta, and these are called plumose hairs from their featherlike appearance. In one or two instances, minute biﬁd tips have been observed at the ectal end of hair chaetae, indicating their probable origin from the much more ubiquitous biﬁd chaetae. This biﬁd form, with the ectal end divided into two teeth, is characteristic of aquatic as opposed to terrestrial oligochaetes, although the functional reason for this is unknown. Biﬁd chaetae may be the only type present in all bundles, and the ventral bundles usually contain nothing else. Some simple-pointed chaetae may be found in a few anterior ventral bundles (in the tubiﬁcid genus Spirosperma, for instance), or they may accompany the hair chaetae in the dorsal bundles in some Naididae. A common combination in the dorsal bundles is hair chaetae accompanied by pectinate chaetae in, at least, the preclitellar bundles. Pectinate chaetae are usually of the same general form as the biﬁds for the particular taxon, but the space between the two teeth is ﬁlled with either a comb of thin teeth or, more rarely, a ridged web. In a few naidids and tubiﬁcids, these webbed chaetae may be expanded as palmate chaetae. In general, the dorsal chaetae accompanying the hair chaetae in the majority of naidid species differ markedly from the ventral chaetae. For this reason, they have come to be termed needles, whether they are palmate, pectinate, biﬁd, or simplepointed. Some authors like to substitute the terms fascicle, capilliform, bifurcate, and crotchet for bundle, hair, biﬁd, and chaeta, respectively. They also use the terms distal and proximal for upper and lower tooth, the designations used in describing the variations in the length and thickness of the teeth, often a useful characteristic. [Anglo-Saxon terms are more succinct, and are used internationally, but the longer Latin terms are more readily translated into French and Spanish.] The normal ventral chaetae may be replaced by specialized genital chaetae, usually beside the spermathecal or penial pores. These are illustrated where appropriate in the keys. Genital chaetae do not vary much in form in the Naididae, where there are often two or three blunt penial chaetae in each bundle beside the male pore. These chaetae are quite long proximally, but the distal end beyond the nodulus (the swelling on the chaeta at the point where it emerges from the chaetal sac) is short. Penial chaetae are usually somewhat long and thicker than the normal ventral chaetae. Penial chaetae, like those of the Naididae, may be found in the Rhyacodrilinae, a subfamily of Tubiﬁcidae, though there are usually several to a bundle. They are usually arranged with the outer ends close together, the inner ends spread out. In Tubiﬁcinae, the ventral chaetae in the penial segment are usually lost but the spermathecal chaetae are often modiﬁed, with elaborate distal ends shaped like trowels. They may be used to introduce the modiﬁed spermatozeugmata into the spermathecae during copulation. The functions of both these and the penial chaetae as claspers are based on speculation because aquatic oligochaetes have rarely been observed mating. Spermathecal chaetae are often associated with special glands. Where these are large, and there is a great deal of competition for space in the genital segments, the spermathecal chaetae and glands lie immediately in front of the spermathecal segment (Rhizodrilus, Tubiﬁcidae). In general, the number and diversity of chaetae is reduced toward the posterior end of the worm, and modiﬁed teeth on the biﬁds commonly become “normal.” There are a few exceptions. The upper teeth of the chaetae of Amphichaeta (Naididae) get longer posteriorly, and the posterior segments of Telmatodrilus vejdovskyi (Tubiﬁcidae) bear chaetae with almost brushlike tips (see illustrations in the key). A few other anatomical features are used in aquatic oligochaete taxonomy. External gills are found in a few taxa. The posterior segments of the tubiﬁcid Branchiura bear single dorsal and ventral gill ﬁlaments. These are longest in the median segments of the gill-bearing region, very short at each end of the row. Dero (Naididae) has gills at the posterior end of the body, usually enclosed in a gill chamber (branchial fossa). These are best observed in living specimens. All aquatic oligochaetes have red blood pigments which aid oxygen uptake and transport. In most species, respiration takes place through the posterior body wall or even within the pre-anal part of the intestine. Water may be pumped in and out of the anus for this purpose. Most aquatic oligochaetes protrude the tail end out of the sediment into the water and may wave the tail to create enough disturbance to maintain high concentrations of oxygen around the tail. The body wall of posterior segments in many lumbriculids is penetrated by special branching blood vessels, presumably to improve respiration. External sense organs are mostly limited to ultrastructural features of the body wall (Jamieson, 1981). These may become prominent in those species that protect the body wall with secretions that trap bacteria and foreign matter in an external crust. Sensory papillae can be seen protruding through such layers in living specimens, but they are usually retracted in ﬁxed material. This has led to arguments about their presence or absence in certain species, but we may assume that any 12. Annelida: Oligochaeta, Including Branchiobdellidae worm with its normal body-wall sensory system denied access to information by being encrusted has developed localized retractile sensory papillae. It is also easy to understand why the necessarily sensitive prostomium and ﬁrst two or three segments may be naked in taxa that have protected body walls, and why the anterior end is often retractile within the rest of the protected body. Simple pigmented eye spots are located on the lateral surface of segment I in a number of naidids, but may be present or absent in various specimens of the same species. The majority of aquatic oligochaetes feed by ingesting sediment, extracting nutrition from the organic matter and especially the bacteria, which comprise less than 10% of it. They feed continuously in a conveyorbelt fashion, often ingesting recolonized feces. Food is obtained by everting the roof of the pharynx through the mouth. Glandular cells associated with the pharynx have evolved into pharyngeal or septal glands, in which the cell bodies lie in the coelomic cavities of several anterior segments, attached to the posterior septa. Intercellular canals run forward from these cell bodies along the lateral sides of the gut to open dorsally on the pharyngeal pad, which is one of the characteristics of worms of this group; this feature is absent in the Aeolosomatidae. The pharyngeal glands are used in taxonomy of the Enchytraeidae, but not in other families. The pharyngeal glands are lost in predatory oligochaetes, but traces of the dorsal pad can still be found in some (Chaetogaster, Naididae) although it has been lost in others (Phagodrilus, Lumbriculidae, and Haplotaxis, Haplotaxidae). B. Physiology The primary emphasis of studies on worm physiology has been on respiration, as it provides a simple model of the value of red blood pigment function and anaerobic metabolism. A good review of respiration studies with reference to their use in determining production in worms was provided by Bagheri and McLusky (1984), who studied the estuarine tubiﬁcid Tubiﬁcoides benedii (benedeni). Oxygen consumption varied from 0.01 to 0.5 L / mg / h, but while there were small, apparent linear increases in rate with temperature, these were not statistically signiﬁcant. These results are among the lowest reported for worms, suggesting that the experimental technique is not creating stress in the test animals. The response of respiration levels to stress has been suggested as a tool in pollution biology (Brinkhurst et al., 1983). Most tubiﬁcids respire at a more or less constant rate as the external oxygen concentration is lowered. Once a critical value is reached, respiration rate falls dramatically. Stress can 437 elevate or depress the constant respiration rate or it may shift the critical value. It may also cause a reduction in, or a total loss of, respiratory control above the critical value. In some instances, respiratory control actually increased in the face of supposed stress factors. A unique phenomenon is that mixing certain tubiﬁcid species together will actually reduce their respiration rates by a signiﬁcant amount (Chapman et al., 1982a). This result was anticipated by earlier studies of worm production using monocultures and mixed cultures of three tubiﬁcid species. In those studies with mixed populations (summarized in Brinkhurst and Austin, 1978), growth rates increased when respiration decreased, as did assimilation efﬁciencies. These experiments seemed to provide an explanation for the way in which tubiﬁcids live in small clumps of mixed species. Some worms preferred the recolonized feces of another species as food and sought the company of that species. Even when they were only in water from monocultures of the appropriate second species, worms moved less, ate more, and (in the presence of living worms) ingested more of their preferred food. This caused a shift in all of the variables measured in production work based on estimating caloric or carbon budgets. The work provided an unusual example of the value of multi-species ﬂocks. Freshwater oligochaetes, such as the pollutiontolerant tubiﬁcids Tubifex tubifex and Limnodrilus hoffmeisteri, can withstand exposure to 10 ppt salinity, but others can only survive at 5 ppt (Chapman et al., 1982b, c). A low level of salinity actually seems to improve the ability of freshwater worms to withstand stress factors. It is less surprising that the provision of sediment improves stress resistance. Estuarine species may withstand a very wide range of environmental stress, including extreme salinity values (Paranais, Naididae; Monopylephorus, Tubiﬁcidae). It would be a mistake to believe that these animals are exposed to a wide range of salinities due to daily tidal excursions, however. In muddy situations in rivers such as the Fraser in British Columbia, the “interstitial” salinity goes through a seasonal cycle related to the capture and release of snow in the headwaters. As temperatures increase, the ﬂow of freshwater increases and tidal seawater incursions are limited to the outer estuary. All of the benthic communities move slowly up and down the estuary in response to this annual rhythm of interstitial salinity, maintaining themselves at what are presumably preferred salinity ranges for each species. It is selection for these optimal salinities (that remain quite constant within the mud on short-term basis) that causes the sequence of species observed along the salinity gradient in estuaries (Chapman, 1981; Chapman and Brinkhurst, 1981). 438 Ralph O. Brinkhurst, and Stuart R. Gelder While worms in cocoons may be able to survive desiccation, there is no experimental evidence of this. Adult Tubifex tubifex (Tubiﬁcidae) are able to form cysts for adult worms that can survive unmoistened for 14 days, or for as long as 70 days with occasional dampening. Once the worms have been moistened, they may emerge in 20 h (Kaster and Bushnell, 1981). The lumbriculid Lumbriculus variegatus can also encyst, but it only survived for 4 days under experimental conditions. In both instances, the covering of foreign matter, which helps to disguise the cyst, is also essential to the survival of the worms. There are records of a handful of other lumbriculids undergoing ﬁssion in cysts, but no physiological work has been done. Lumbriculus also forms a different type of cyst to withstand freezing (Olsson, 1981). Specimens of the tubiﬁcid Limnodrilus profundicola were collected by R. Brinkhurst at a height of ca. 4000 m on Mount Evans, Colorado. After being accidentally frozen into a solid block of ice, the worms emerged quite unscathed when thawed the next day. As so many aquatic oligochaetes, especially the Naididae, are cosmopolitan or at least very widely distributed, one would expect more evidence of adaptations to counter desiccation. Dispersal mechanisms of oligochaetes are still a matter for speculation. The development of eye spots is unique to the Naididae, a family of small, delicate worms, some of which can make rudimentary swimming movements. Other naidid species, while not free-living, occupy tubes attached to aquatic plants and may protrude the anterior end. The majority of aquatic oligochaetes avoid the light by keeping the head end buried in their food supply, the sediment. Most aquatic worms have quite sensitive tails and have a rapid withdrawal reﬂex based on a mechanosensory detection system that is responsive to the approach of potential predators (Zoran and Drewes, 1987). Branchiura has the fastest escape reaction reported for any invertebrate. This speed is associated with the development of the lateral giant nerve ﬁbers in the ventral nerve cord. These are responsible for rapid forward transmission of sensory information. The median giant nerve ﬁber transmits posteriad from the head end. In Lumbriculus, which lives in shallow littoral habitats, the body is extended up to the air – water interface, with the tail lying horizontally in it. Not surprisingly, these worms have what Drewes and Fourtner (1988) termed hindsight or light-sensitive tails, and they respond to passing shadows. Light can be damaging to worms. A great deal of work has been done in the laboratory on the effect of toxic substances on worms, summarized by Chapman and Brinkhurst (1984) and Wiederholm et al. (1987). III. ECOLOGY AND EVOLUTION OF OLIGOCHAETA A. Diversity and Distribution Many aquatic oligochaetes are cosmopolitan, or at least widely distributed. This is especially true of the Naididae, but even here signiﬁcant patterns are beginning to emerge. The supposedly primitive naidid subfamily Stylarinae is scarce in the southern United States, such genera as Stylaria, Arcteonais, Ripistes, and Vejdovskyella being common in northern locations. Dero and Allonais species (members of the Naidinae) are well represented in subtropical and tropical regions. There are other north – south distribution patterns. The tubiﬁcid Arctodrilus wulikensis is found in Alaska, as are several lumbriculid species that are part of the Paciﬁc Rim fauna mentioned later. Phallodrilus hallae, a tubiﬁcid limited to Lake Superior, is related to a large number of marine taxa in the subfamily Phallodrilinae. Unlike the northern forms, tubiﬁcid species restricted to southern or southeastern localities belong to genera that are otherwise more widely distributed. Among the tubiﬁcids and lumbriculids, some taxa are limited to the western United States and Canada. Some of these are related to or are the same as Asian Paciﬁc Rim counterparts, suggesting an evolutionary distribution pattern. Several tubiﬁcids are known only from Lake Tahoe, which may prove to be home to a number of endemic species, though some forms ﬁrst described there have since been found elsewhere, mostly still in California. The apparent limitation of some species to the eastern United States may simply reﬂect a greater frequency of caves or more interest in cave faunae in those areas. At least six lumbriculid species fall into this category, but there are also some eastern surface water tubiﬁcid species. The groundwater tubiﬁcid Rhyacodrilus falciformis is a European species now known to be widespread in such habitats on Vancouver Island, British Columbia, and in Illinois, and there is a lumbriculid known from a well in California; so there may be as rich a western groundwater fauna (awaiting description) as there is in eastern North America. Many groundwater species have very limited distributions, although the distinctions between species in genera such as Trichodrilus are often based on minute differences. This may mean that biologists look more carefully for differences where they anticipate that they may exist. The reasons for these geographic patterns have not been studied, partly because they have only been recognized for a few years. There may be ecological reasons for the distribution of some taxa. Species of Aulodrilus (Tubiﬁcidae) may be widely distributed, but they appear to reproduce asexually in the more temperate 12. Annelida: Oligochaeta, Including Branchiobdellidae zones . This may suggest a recent spread from warmer areas where they do breed sexually. Some species can be shown to be recent introductions by humans. The North American Limnodrilus cervix has been introduced to Britain and Sweden via ports and canals (Milbrink, 1983). It is possible that the diverse collection of Potamothrix (Tubiﬁcidae) species in the St. Lawrence – Great Lakes represents a series of introductions of common European species into North America. This is more certain for Psammoryctides barbatus (Tubiﬁcidae), a very recent discovery in the St. Lawrence River in Quebec (Vincent et al., 1978), as well as the estuarine and coastal species Tubiﬁcoides benedii found recently by the author in Vancouver Harbor, British Columbia. One of the most common tropical tubiﬁcids, Branchiura sowerbyi, was ﬁrst described from a Victorian greenhouse in Britain, where it had been introduced along with the tropical plants on display. It has now become adapted to temperate waters, though it still thrives best in reservoirs and canals, especially where the temperature is elevated. It has apparently spread all across North America since it was ﬁrst recorded in Ohio in 1931; but, this could simply reﬂect a gradual spread of awareness of this easily recognized species (Brinkhurst, 1965). Some of these regional distributions are mentioned in the keys, but the reader should always be prepared for the unusual. Niche discrimination in aquatic oligochaetes is less obvious than zoogeographic patterns. The majority of these worms are adapted to live in sediments ranging from sand to mud. They can be found in pockets of such sediments in stony habitats as well as in lowland rivers, lakes, and ponds where soft substrates are the norm. There is none of the usual distinction between lacustrine and riverine species. The best guess that can be made about niche discrimination is that the precise nature of the organic matter in sediments, and the microﬂora it supports, determines the outcome of interspeciﬁc competition. The ecophysiological work on multispeciﬁc ﬂocks described earlier is probably applicable to most situations and not limited to the Tubifex – Limnodrilus – Quistadrilus association in which it was initially described. Milbrink (1993) obtained similar results with T. tubifex and Potamothrix moldaviensis. In most habitats, between 5 and 15 species can be found. There are some special adaptations, such as the groundwater fauna already mentioned. Several genera of the Naididae are no longer restricted to life in the sediment, but may be found among aquatic weeds in quiet rivers and ponds. Lumbriculus lives among vegetation in pond and stream margins. Dero (Allodero) species are symbionts of frogs, which, once released from their host come to resemble specimens of Dero (Aulophorus), rendering the 439 taxonomic distinction suspect. The predators Haplotaxis and Phagodrilus are found in springs, seepages, and groundwater, but Chaetogaster is more ubiquitous. Estuarine species form another group, but a number of species are restricted to tidal freshwater where there is little, if any, detectable saltwater. None of these exhibit any marked anatomical adaptations to their way of life, with the exception of the pharyngeal structures of the predators and loss of chaetae and gills in Allodero. The signiﬁcance of food in the ecological specialization of the aquatic oligochaetes, especially the tubiﬁcids, is demonstrated by the sequence of species groups that inhabit progressively more organically polluted stretches of rivers or more eutrophic lakes. The Lumbriculidae and some naidid genera such as Pristina and Pristinella are found in streams with low organic matter in the sediments, and they may be accompanied by tubiﬁcids belonging to the Rhyacodrilinae in particular (Rhyacodrilus, Bothrioneurum, and Rhizodrilus). Tubiﬁcinae, such as Potamothrix and Aulodrilus, can be found in situations with a reasonable supply of organic matter but also a good oxygen supply, although Tubifex tubifex, several Limnodrilus species, and Quistadrilus are found in areas where the oxygen supply can become reduced. This basic pattern has been expanded, quantiﬁed, and recognized in Europe as well as North America (e.g., Lang, 1984). At ﬁrst, this type of work followed the much earlier pattern of establishing lists of chironomid midge species characteristic of lake types (reviewed in Brinkhurst, 1974). The maximum value in identifying aquatic oligochaetes in pollution studies today is to add information for use in data matrices to be analyzed by multivariate statistics, such as cluster analyses. There are relatively few invertebrate species in freshwater muddy habitats, in contrast to stony streams (where there are far more insects) or marine habitats (where there are many more muddwelling taxa). The taxonomic identites of the communities from each sample site are retained in such analyses, whereas they are lost in simpler diversity models. B. Reproduction and Life History Field studies on reproduction in the Naididae were reviewed by Loden (1981). Asexual reproduction, (usually by paratomy), predominates, but sexual reproduction apparently enables populations to persist through periods of unsuitable conditions. Abundance varies seasonally depending on the geographic region, with spring, summer, or winter maxima being produced asexually. Most species apparently cease feeding as they mature because the gut degenerates, and the mouth may even become sealed. According to Loden, 440 Ralph O. Brinkhurst, and Stuart R. Gelder some species incorporate the anterior part of the worm in the cocoon, but Lochhead and Learner (1984) disputed this in describing a life history for Nais. They reported that worms from this and other naidid genera are able to shed their cocoons. According to these investigators, the adult worms are more resistant to environmental stress than the cocoons. All cocoons of aquatic oligochaetes may be disguised with mud particles, or may be placed out of reach of predators (Newrlka and Mutayoba, 1987). Most tubiﬁcids reproduce sexually, but there are several parthenogenetic species. The latter often lack spermathecae and the testes may also be reduced or absent. The vasa deferentia may be reduced to solid strands in these species, but the atria are usually retained. A few other species seem able to reproduce asexually by ﬁssion. The results of various ﬁeld studies produce conﬂicting interpretations of the life histories of such common genera as Limnodrilus. There is less disagreement about events in less adaptable species found in more restricted habitats. The worms may mature a few weeks after hatching if temperature and food supplies are adequate, and they may breed more than once in a season. Alternative studies suggest that many species mature in their second year. After breeding, these forms resorb the gonads and gonoducts and would be recognized as immature specimens. The next year they develop a new set of reproductive organs and breed again, after which most species die. Some published examples are the studies of Block et al. (1982) and Lazim and Learner (1986a, b) for Limnodrilus species, Christensen (1984) on asexual reproduction, and Poddubnaya (1984) on parthenogenesis. Because of the problems encountered in interpreting the results of ﬁeld studies on life histories, considerable effort has been put into the development of laboratory cohort studies (Bonacina et al., 1987). In general, oligochaete life histories are probably not as precisely tuned to environmental variables like day length as they are in more highly evolved forms such as insects. This may be attributable to simpler neurosecretory systems in worms, but there is no evidence to support this idea. Food and temperature may cause considerable local variation in life cycles, especially in the opportunistic species like Limnodrilus hoffmeisteri. Unfortunately, these abundant, cosmopolitan species are the most convenient laboratory animals, but they are so adaptable and perhaps atypical that comparable work should be done with species found in more restricted habitats. C. Ecological Interactions There is some published work on the food and feeding behavior of the Naididae, but a much larger literature exists on the relationship between tubiﬁcids (and one lubriculid species Stylodrilus heringianus) and the sediments that they ingest (and therefore defecate) continuously. Algae and other epiphytic material are the main food of many naidids, but Nais feeds on heterotrophic, aerobic bacteria like the tubiﬁcids. There is no specialized gut ﬂora in either group (Brinkhurst and Chua, 1969; Harper et al., 1981). There is no evidence of selective ingestion or digestion of speciﬁc bacteria in Nais, but selective digestion is important in tubiﬁcids. Nais elinguis will select algal ﬁlaments in preference to monoﬁlament nylon thread and prefers some algae to others or to glass beads (Bowker et al., 1985). Algae also form part of the food supply of the tubiﬁcids Limnodrilus claparedeianus and Rhyacodrilus sodalis and the lumbriculid Lumbriculus variegatus. Studies of defecation rate and the chemical and bacterial content of feces in tubiﬁcids have usually exploited the fact that these worms will continue to feed with their heads in the sediment and their tails in the water column, even when such colonies in small containers are inverted. If a layer of cotton or ﬁne netting is used to prevent the sediment drifting down past the worms into the collecting funnel placed beneath the culture, it is easy to determine the amount and the chemical content of the feces. If the feces are collected over very short time periods, the bacterial ﬂora of the feces can be studied. Similar analyses of the sediment provided as food make it possible to study the effect of passage through the gut on the organic matter content and bacterial ﬂora (Brinkhurst and Austin, 1978). Fecal samples can also be collected from cultures oriented normally, but the feces then have to be collected from the artiﬁcial sediment surface instead of being allowed to drop into a container. Both methods are potentially liable to error and cultures must be handled very carefully. The behavior of the worms is affected by changes in temperature and light and by physical disturbance. The results derived from cultures maintained in both orientations have usually shown that worms are not affected by being inverted, and that, if either method is subject to error in determining the amount of fecal material produced, these errors cancel out. Only Kaster et al. (1984) suggested that worms in the normal position produce more feces (45 – 110%) than those in inverted cultures. Such obvious differences would have been detectable in earlier studies had they existed. Defecation rate seems to be very difﬁcult to determine. The nutritional value of the food provided may decline as the experiment proceeds, and this may cause an increase in the amount of sediment processed in order to feed the colony. Experimental results are affected by periods of acclimation to the artiﬁcial sediments, which may be as 12. Annelida: Oligochaeta, Including Branchiobdellidae long as a month. Two supposedly identical cultures maintained side by side may behave quite differently (Appleby and Brinkhurst, 1970). Defecation rates may be used in studies of worm production when associated with estimates of organic matter content and caloric value of the food and feces. They may also be used in studies of sedimentation and bioperturbation of sedimentary layers in freshwater ecosystems. The lumbriculid Stylodrilus heringianus builds up large populations in many regions of the Great Lakes, where it behaves in the same way as a tubiﬁcid. Krezoski et al. (1984) used gamma spectroscopy methods to measure the rates at which a layer of sediment (labeled with radioactive cesium) and overlying water (labeled with radioactive sodium) were transported by the burrowing activity of worms and other benthos. The results of these and similar studies all support the idea that the superﬁcial sediment layers in lakes are thoroughly mixed by benthic organisms, and that solute transport across the mud – water interface is greatly enhanced by them. Worms are probably the most important animals involved in this because of the depths to which they penetrate the sediment, the large populations that they build up, and their ceaseless activity. Gardner et al. (1981) showed that invertebrates could be responsible for most of the phosphorus released from aerobic sediments in Lake Michigan. Chatarpaul et al. (1979) showed that worms accelerated nitrogen loss from coarse stream sediments, indicating their importance in rivers as well as lakes. There is now a growing realization that the sediments are not just the ﬁnal resting place for organic matter and contaminants, but that they may become a source for them (Karlckhoff and Morris, 1985). D. Evolutionary Relationships There are contradictory ideas about the evolution of any biological group, many of which are due to fundamental differences in the methods used. There are strongly held opinions about the way in which relationships should be determined, even among those who employ cladistic methods and those who agree with at least the basic concepts of Hennig. The following account is a personal one, based on experience with most families of aquatic oligochaetes, an exposure to marine and freshwater worms worldwide, and some familiarity with cladistics. This is not said in order to validate this particular view, but to help the reader identify the biases it inevitably includes. At least it should not suffer from the geographic or taxonomic parochialism that tends to cause taxonomic inﬂation — the tendency to elevate the taxa in which we are most interested to the highest level possible. 441 We should perhaps consider what the original worm looked like, rather than asking whether polychaetes or oligochaetes came ﬁrst. It seems quite reasonable to suppose that an animal looking a lot like a very simple earthworm, but with a reproductive system that released eggs and sperm freely into the environment, provides a common ancestor to both groups (Fig. 5). There has been some support for this idea among polychaete biologists, but, in general, they seem to have difﬁculty deciding on relationships above the family level, and on what constitutes a primitive worm. The “simpliﬁed earthworm” with two chaetae per bundle and no parapodia is adopted following the ideas of Clark (1964) regarding the evolution of segments and coelom. There is, of course, no reason to suppose that every group evolved in the ocean. The teleost ﬁsh provide a good example of a group that may well have undergone a very large diversiﬁcation in freshwater before re-invading the ocean, so that the existence of a large number of marine Tubiﬁcidae is not prima facie evidence for their antiquity, even within that family, though the possibility should not be excluded. All possible combinations of the reproductive system segmental arrangement within the oligochaetes (and other Clitellata) can be derived from a worm with four genital segments, two with paired testes in each, followed by two with paired ovaries. The original position of this series, termed GIGIV (Brinkhurst, 1982), seems to have been in X – XIII. The organisms with this arrangement that exit today are found in the Haplotaxidae, with only one or two exceptions that are thought to be primitive members of groups descended directly from that family (Fig. 6). There are some exceptions that have been explained elsewhere, but only the barest outline will be presented here. [Challenges to this largely Austral group as being ancestral come from the fact that Haplotaxis itself is a highly modiﬁed genus of predatory worms (see the taxonomic key).] It is then thought that a number of families have quite independently lost the anterior testes (GI) and the posterior ovaries (GIV), making copulation easier (there are fewer pores to bring into contact with each other). This happens because the development of egg and sperm sacs enables more gametes to be stored in the coelom from a single pair of gonads than could be achieved in a single segment when the sacs were small or undeveloped. Similarly, the evolution of multiple chaetae per bundle seems to have arisen independently a number of times, most notably in the Enchytraeidae, Tubiﬁcidae, and Naididae. In the former, most species have two chaetae per bundle, a few “specialists” have no chaetae in some or all bundles (another common but independent evolutionary step found in several families), but some have several chaetae. These are all simple-pointed, however, which seems to be correlated 442 Ralph O. Brinkhurst, and Stuart R. Gelder FIGURE 5 Phylogenetic tree of the Annelida. This diagram represents an outline of the major (potential) monophylies and the apomorphic character states that deﬁne them (solid bars across lines). Note that the Branchiobdellidae is shown as included within the Oligochaeta, which may also be true for the Hirudinea according to recent molecular evidence. Aphanoneura is expected to be eliminated as the Aeolosomatidae become placed within the Polychaeta in future publication. Phylogenetic tree of the Oligochaeta. While the Hirudinea (Acanthobdellida Euhirudinea) are said to be related to the Lumbriculida according to molecular analyses, they do not have semiprosoporous male ducts (possibly plesioporous and prosoporus respectively, a single pair of male ducts in Acanthobdella, testes within sperm sacs conﬂuent with the male pores with no male funnels in Euhirudinea). The form of the male ducts in Acanthobdella and Euhirudinea could have arisen independently from a semiprosoporous condition, in which case they could form a monophyly or monophylies within the Lumbriculida, but the molecular and anatomical schemes need to be resolved and hence the Hirudinea are excluded from this ﬁgure. FIGURE 6 12. Annelida: Oligochaeta, Including Branchiobdellidae with the fact that most members of this family are at best only semiaquatic if not totally terrestrial. Chaetae with biﬁd tips are a hallmark of aquatic oligochaetes, as are more complex pectinate and palmate chaetae and their derivatives, hair chaetae. The hair chaetae of the Phreodrilidae (a small, Southern Hemisphere family) seem to have evolved independently of those of the Naididae and Tubiﬁcidae. These are the two most closely related families of aquatic oligochaetes, and they are apparently the most highly evolved in many respects. The Naididae exhibit several adaptations to an aquatic life as opposed to one restricted to burrowing in moist sediments. They have simple eyes, a few have specialized chaetae in a few anterior segments, and some can swim in a very simple way. The predominance of asexual reproduction enables them to exploit a more changeable environment than that available in the sediments, and in some as yet unidentiﬁed way has enabled most of them to become very widely distributed. Their diet now includes algae as well as bacteria, and we can assume that they can absorb macromolecules through the body wall as food (as do the freshwater and marine tubiﬁcids). The male reproductive system has become progressively more complex in these groups. In the Enchytraeidae, the male ducts may terminate in what appear to be more glandular than muscular structures, which are not yet identiﬁable as atria. The tubiﬁcids have muscular atria and the higher forms have stalked prostate glands, elaborate penes, and either penis sheaths, penial chaetae, or spermathecal chaetae. The Lumbriculidae are more of a puzzle. They have usually retained both testicular segments, but have lost the second ovaries (GIV). Many advanced forms secondarily loose GI, and a few parthenogenetic forms have an irregular, asymmetrical, or even exaggerated number of gonads. The chaetae are restricted to two per bundle, but these are usually biﬁd rather than simple-pointed as postulated in the plesiomorphic condition. Some biologists see the paired chaetae as evidence of a close relationship between the lumbriculids and the earthworms, suggesting that the latter are the derivatives of these aquatic forms. This puts an enormous weight on just one character, but these people also regard hair chaetae as being retained from a polychaete ancestor. The difﬁculty with relating the lumbriculids to the earthworms is that the lumbriculids (and branchiobdellidans) have a unique way of reducing the number of male pores. They have their atria in GII and both pairs of male ducts drain into them. In Lumbriculids, the prostate glands consist of a diffuse covering of gland cells and none of them have developed stalked prostates. Cuticular penis sheaths are very rare; genital chaetae are limited to a single species. Too much has been made of the apparent diversity of reproductive 443 segments in this family. While the variation is greater than that of other families, the most common form does seem to be the ancestral one (Brinkhurst, 1989). While the aquatic oligochaetes closest to their presumed ancestors seem to be found in Australia and New Zealand, not unlike many other freshwater invertebrate groups, other families are widely distributed. According to a cladistic analysis of some Enchytraeidae (Coates, 1989), South America seemed to contain the basal forms. In the Naididae, it is the northern Stylarinae that appear to the oldest (Nemec and Brinkhurst, 1987). The Lumbriculidae inhabit the Northern Hemisphere, with a very high proportion of them being limited to, or extending eastward from, Lake Baikal in Siberia. The European Cookidrilus seems to most resemble the presumed ancestor, with the widespread genera Stylodrilus and Trichodrilus being quite generalized (Brinkhurst, 1989). There has yet to be a detailed analysis of the more cosmopolitan Tubiﬁcidae, but there is a greater diversity of genera in the Northern Hemisphere, with very few restricted to the Southern Hemisphere (as Antipodrilus) and very few adapted to the subtropical and tropical regions, apart from Branchiura and, perhaps, Aulodrilus. We are currently trying to assess the relationships between the clitellate taxa usually termed Oligochaeta, Hirudinea, and Acanthobdellida. In a preliminary revision (Brinkhurst and Gelder, 1989), it was shown that the supposed link between the predatory lumbriculid worm Agriodrilus from Lake Baikal (Russia) and the non oligochaete taxa in this group is purely circumstantial, made even clearer by the recent discovery of a parallel evolution in the North American Phagodrilus. Without presenting all of the data here, it now looks as if the branchiobdellids and Lumbriculidae share a common ancestor, both having two pairs of male ducts draining through atria in GII, unless this character proves to be a convergence. Hence, the former taxon is referred to as Branchiobdellidae in this chapter. It remains to be seen if the male ducts of the Hirudinea and the Acanthobdellida are at all related to what we might call the semiprosopore state of the male ducts in the lumbriculids (only one pair of male ducts are now thought to be in their testicular segment in the ancestor of the family according to a cladistic analysis). The extreme modiﬁcation of the male ducts in the leeches, in which the testes lie in sacs behind the two successive segments bearing male and female pores, is considered to be derived from a system with the testes in front of the ovaries, as in the oligochaetes. With the loss of the coelom, the testes are supposed to be transferred into the surviving sperm sacs as a series of small gonads replacing a single original pair. Complex male ducts have been developed, but these are in the form of a 444 Ralph O. Brinkhurst, and Stuart R. Gelder continuous tube in which the sperm are shed internally. These ducts need to be reexamined for any trace of relict sperm funnels, to see if there is any evidence that they were ever prosoporous like the lumbriculids. The acanthobdellids, represented by one species with one probable synonym, have four pairs of chaetae in a few anterior segments including the oral segment. There is no trace of a prostomium, or peristomium, and perhaps some anterior segments (Brinkhurst, 1999). Acanthobdella also retains or possibly recovers the coelom (which is lost in leeches), but the testes still lie within the sperm ducts. Two intriguing aspects are the pattern of eyes and the unique sperm-receiving device shared by this genus and the piscicolid leeches. Other revised information about the branchiobdellids, discussed later, also weakens the argument for a relationship between them and the leech groups. That argument was based on a very superﬁcial examination of the detailed facts available. Textbook accounts in this and many other areas of supposed evolutionary relationships should be read with caution, especially if they give no clear indication of the theoretical basis on which the opinions are based. IV. COLLECTING, REARING, AND PREPARATION OF OLIGOCHAETES FOR IDENTIFICATION A. Collection Oligochaetes may be collected using any of the standard array of samplers from dip nets to grabs and core tubes. Collectors speciﬁcally seeking worms can use reﬁned histological ﬁxatives in the ﬁeld. A great deal of material is collected in the course of ecological surveys where time does not permit such care. Strictly quantitative samples from soft sediments are best obtained with narrow core tubes without core retainers. Using core tube lengths appropriate to the type of substrate can save a great deal of frustration, but most people seem to try to work with a single set of tubes. Soft sediments can be washed through screens and sorted in the laboratory, preferable after being preserved in their entirety in the ﬁeld. If cores are used, the volume to be carried back from the ﬁeld may be quite small. Field sorting is relatively inaccurate, and small specimens may well be missed. The best preservative for bulk samples is 10% buffered formalin (4% formaldehyde solution), but is easier to carry 40% in the ﬁeld and add this to an aqueous sample to approximate 10% in the whole sample. Addition of rose bengal or phloxine B in very small amounts aids sorting, and the stain can be washed out of the worms when they are transferred to 70% alcohol for storage. The use of larger amounts of stain creates problems in identifying worms. Sorting should be done with stereomicroscopes under good lighting. Care should be taken to avoid diluting the alcohol when transferring worms into the storage vial. Many North American collections are sent for identiﬁcation with the worms half decomposed because bulk samples were preserved in alcohol, not formalin, or the original alcohol has not been drained and replaced with 70%. Storage can be quite permanent in patent lip vials with neoprene stoppers. The stoppers tend to swell and seal the vial against alcohol loss. We have some in our laboratories that have not needed attention for 20 years. If these vials and stoppers are not available, vials must be sealed with tape, wax, or paraﬁlm. Some neoprene stoppers dry and crack with age, and all collections should be curated to maintain their integrity. Museum quality material should be donated to institutions that can provide curation. If the collector is not taking quantitative samples for ecological or environmental studies and is working in a remote or unusual location, then it may be worth the effort of sorting, ﬁxing and preserving some specimens while in the ﬁeld if they are readily visible. Putting the sample in an enamel dish and allowing everything to settle will reveal even small worms as they begin to move. Carrying whole samples back to the laboratory to sort them while the worms are alive can cause selective mortalities, as some species surviving elevated temperatures and lowered oxygen concentration better than others (and the latter always seem to be the more interesting species from a taxonomic viewpoint). Live worms can be sorted from sediment and detritus by spreading clean sand over the sample or putting the sample on a screen set over clean water. The worms will then actively migrate into the sand or water. This can be useful in samples full of plant fragments. Samples taken with dip nets or other methods that include coarse substrates can be processed in the ﬁeld to reduce the volume and weight of the sample. Repeated, careful elutriation into a screen, screen bucket, or back through the original dip net can be used, imitating a gold-panning technique. The amount of vigor used can be selected in order to separate enough of the ﬁne fraction including all the specimens from the heavy material. The coarse residues should, of course, be carefully checked for large, heavy, or attached organisms, or specimens trapped in empty shells and other unusual situations. B. Rearing Oligochaetes Culture of worms can be as simple as placing the substrate and the worms in an aerated aquarium, preferable where temperatures can be kept at appropri- 12. Annelida: Oligochaeta, Including Branchiobdellidae ate levels, usually between 4 – 10°C depending on the original habitat. Small cultures used for laboratory experiments can be maintained in shallow vessels half ﬁlled with sterilized lake sand. Filtered lake water is added every two weeks and frozen lettuce strips are buried in the sand to provide bacteria as food. Mass culture to provide ﬁsh food can utilize manure to feed the worms. Marian and Pandian (1984) described raising 7.5-mg Tubifex tubifex every 42 days on a mixture of 75% cow dung and 25% ﬁne sand, maintaining the oxygen level at 3 mg / L. Fresh cow dung is added every four days, and harvesting is supposedly best done at night. Aston (1984) cultured Branchiura sowerbyi at 25°C. Leitz (1987) reviewed attempts to mass culture Lumbriculus variegatus. Production rates of 15 kg / m2 and population doubling times of 11 – 42 days are reported. The use of live worms as ﬁsh food has many advantages over other diets. Most commercially available live worms are now Lumbriculus (called ‘black worms” in commerce) rather than Limnodrilus. C. Preparation for Identiﬁcation Many biologists believe that these animals are hard to identify. It is worth indicating that, as they are hermaphrodites, there is no difﬁculty with keys for males and females, and there are no larval forms. About 60% of the species can be identiﬁed from superﬁcial somatic characters, but the rest require mature specimens. It is, of course, always preferable to conﬁrm identities from mature specimens wherever possible, but it is not essential. Those used to identifying organisms with stereomicroscopes may be bothered by the need to make slide mounts, but simple methods can be used in most instances. The serious taxonomist will want to make more elaborate preparations and may want to section specimens to check details, but this is not necessary for routine identiﬁcation. There are only about 150 freshwater oligochaetes and a small number of branchiobdellidans in North America, and many of these are rare and local. Recent keys have been written with the beginner in mind, and so they emphasize external or readily visible internal characters. Such keys work well if they proceed directly to the speciﬁc level, but it is a little more difﬁcult to write keys to the genera, as preferred in this volume. Generic characteristics are mostly detailed aspects of the soft parts of the male reproductive structures. These may be difﬁcult to see on rapidly made preparations in which the soft parts are dissolved. The keys presented here do not always proceed directly to genera for this reason, but we have managed to write keys involving externally visible characteristics. Some reference to geographic distribution is made in the keys, but this has been done only where the 445 information available seems reliable. Fully illustrated keys with supporting descriptions of all the species are available elsewhere (Kathman and Brinkhurst, 1998). Once the worms are preserved in alcohol, they must be prepared for study with a compound microscope. Examination of specimens from remote or unusual locations should begin with a study of the external characteristics, such as position of the gonopores and other obvious features. However, for routine identiﬁcations of specimens from ordinary localities, very little beneﬁt can be derived from this step in the process. In this case, the worms can either be immersed in a temporary mounting medium by replacing the alcohol with Amman’s lactophenol, or they may be washed with water and then placed in separate drops of lactophenol on slides. It is usually possible to mount live worms under a single coverslip (with two coverslips per slide) unless the worms are very large. These wet mounts should be set aside for 1 – 2 days on stacking trays. Amman’s medium is quite corrosive, so it should be handled with care and any spillage should be wiped off microscope stages immediately. It consists of 20% phenol, 20% lactic acid, 20% water, and 40% glycerine. It is best stored in dark bottles; however, it is hygroscopic and does not have a good shelf life. Somewhat more permanent mounts can be made with media such as CMCP and polyvinyl lactophenol. Such slides should usually be sealed with a ring of material (Glyceel, nail polish, etc.) around the edges of the coverslips to prevent the media from drying up. None of these methods produce satisfactory permanent slides, nor are they adequate for working with the Enchytraeidae or the marine Tubiﬁcidae, in which internal anatomy must be seen. For this type of material, permanent whole mounts using stained and dehydrated worms are mounted in Canada Balsam or one of its more recent substitutes. To prevent worms from becoming brittle, they should be cleared in methyl salicylate rather than xylene. This will enable advanced students to try bisecting (sagitally) the separated genital region of each worm or even dissecting out the male ducts when necessary. This should be limited to identiﬁcation of new taxa to the generic level, when details of the male reproductive system must be established. Serial sections (usually sagital longitudinal) are also employed in taxonomic work, but not in routine identiﬁcation of well-known species. It is important to be knowledgeable about the expected locations of the various organs before attempting dissection or interpretation of serial sections. The procedure for examining a worm on a slide is as follows. Check the prostomium for a proboscis or sense organ. Next, examine the ventral chaetae of the ﬁrst two or three bundles and determine the form of the 446 Ralph O. Brinkhurst, and Stuart R. Gelder dorsal bundles and where they begin (usually in II or between IV and VI). Establish the number and form of these chaetae (biﬁd, pectinate, hair chaetae, etc.). Determine the relative lengths of the teeth. As these are illustrated by taxonomists with the ectal or outer end of the whole chaeta toward the top edge or corner of the ﬁgure, the upper tooth is always drawn lying above the lower tooth. When viewing a whole-mounted specimen, the chaetae often lie ﬂat on the body wall and so no distinction of position can be made; therefore, always visualize a chaeta in the upright position. Care should be taken to examine several chaetae from an exactly lateral aspect, because slight deviations can produce apparent distortion of the relative lengths of the teeth. Measurements often betray the bias of the eye of the observer and are recommended where an eyepiece micrometer scale is available. The worm should then be searched for genital characters. Carefully check the appropriate segments (usually X – XI in a tubiﬁcid, for example) to see if the ventral chaetae are modiﬁed. If the dorsal and ventral chaetae are alike, make sure that you have examined at least three bundles in those segments to ensure that a ventral bundle has been examined. Check the penial segment to see if there is a penis sheath (they should be paired, of course) even if it is thin and inconspicuous. Check the posterior chaetae and the body itself for gills or any other special features. Then proceed to check the key. If the worm has distinct features, [like a single dorsal hair with single simple needles (these beginning in V) and the anterior ventral (II – V) and posterior ventral (VI onward) bundles have chaetae that differ in length, width, and shape], it will rapidly become possible to skip the key altogether and proceed straight to Nais or Dero depending on the presence of gills. Many such easy identiﬁcations to major groups or to genera can be made with a little practice. Identiﬁcations will require a properly aligned microscope, as chaetae and penis sheaths have refractive indices that do not differ much from the background, and too much light makes them impossible to see. Oil immersion lenses must be used to see pectinations on chaetae in tubiﬁcids (although with practice they can be identiﬁed without), and must also be used to determine the form of the needles (dorsal chaetae) in naidids. V. TAXONOMIC KEYS FOR OLIGOCHAETA A. Taxonomic Key to Families of Freshwater Oligochaeta and Aphanoneura 1a. Minute worms, 1 – 2 mm or chains of animals up to 10 mm; hair chaetae in both dorsal and ventral bundles; worms move in a gliding motion using cilia; body wall with colored or refractile epidermal glands; no eversible pharyngeal pad; no clitellum, only ventral copulatory glands in rare mature specimens; nervous system ladderlike (Fig. 7)..............................................................Aphanoneura .........................................................................................Aeolosomatidae [Key to world species in Brinkhurst and Jamieson (1971)] 1b. Larger worms; hair chaetae restricted to dorsal bundles or absent; worms move by alternate contraction of longitudinal and circular muscles; no colored epidermal glands; eversible pharyngeal roof present (except in aquatic earthworms); clitellum present in mature worms; nervous system fused ventrally ..........................................................................................................Oligochaeta .................2 2a(1b). Ventral chaetae large, sickle-shaped, single (i.e., two per segment) dorsal chaetae small, straight, often missing from some or all segments; prostomium very long, furrowed, no proboscis; mouth large, muscular pharynx present; body elongate, slender, resembling a gordian worm (Fig. 8)........................................................................................................................Haplotaxidae............Haplotaxis [Worms in this genus resemble the European H. gordioides (Hartman, 1821), but mature specimens have yet to be described from North America. There may be several species (now regarded as synonyms) differing by the number and distribution of dorsal chaetae.] 2b. All chaetae paired, or multiple in a bundle, unless partially or totally absent; prostomium short, often conical, without a transverse furrow, but with or without a proboscis; body form very rarely elongate and slender ........................................................................3 3a(2b). Chaetae all paired from II or, if more numerous, all simple-pointed ...................................................................................................4 3b. Chaetae more than two per bundle, usually biﬁd, sometimes with pectinate and hair chaetae dorsally, simple-pointed chaetae rare (limited to a few anterior ventral bundles, or single needles with the hairs dorsally)...........................................................................7 4a(3a). Chaetae paired, biﬁd, with small to rudimentary upper teeth (Fig. 9..........................Lumbriculidae (in part)..................(Section V.B) 4b. Chaetae simple-pointed ......................................................................................................................................................................5 5a(4b). Thick-bodied worms with clitellum several segments behind the gonopores (XII – XIV) ....................................................................... .......................................................................................................................Megadrili [Mostly terrestrial, but some aquatic species. Most aquatic species are members of the Sparganophilidae (Sparganophilus) or the Lumbricidae (Eiseniella).] 5b. Thin-bodied worms with clitellum one cell layer thick and in region of gonopores (X – XII or further forward) ................................6 12. Annelida: Oligochaeta, Including Branchiobdellidae 447 FIGURE 7 Aeolosoma sp. Note ciliated prostomium, lack of septa, simple pharynx, dorsal and ventral hair chaetae, and colored body wall inclusions (irregular circles). FIGURE 8 Haplotaxis sp. Whole specimens can be even longer in relation to the breadth. FIGURE 9 Biﬁd chaetae, Lumbriculidae. FIGURE 10 Enchytraeid chaetae. These may be straight, curved, with or without noduale, and may differ in length within a bundle. All chaetae are usually identical. The anterior end of Barbidrilus (below) have ventral bundles of strange rodlike chaetae with biﬁd tips only in II and III, shown on the right. 6a(5b). Larger worms (difﬁcult to mount under a coverslip) with sigmoid, nodulate chaetae; proboscis present or absent; spermathecae in or adjacent to the gonadal segments, male pores between VIII and X................................Lumbriculidae (in part) ..........(Section V.B) 6b. Smaller worms with chaetae often straight, commonly not nodulate, sometimes missing from some if not all segments (in Barbidrilus Loden and Locy, 1981 with uniquely forked chaetae in II – III ventrally, others missing). No proboscis. Spermathecae open in V, male pores in XII (Figs. 2 and 10) ..............................................................................................................................Enchytraeidae 7a(3b). Worms (1.7 – 3.0 mm) with posterior end bearing one median and two lateral processes. Genital region with testes in XI, ovaries and atria in XII, spermathecae in XIII (Fig. 11) .............................................................................................................Opistocystidae [The only deﬁnite North American record is for Crustipellis tribranchiata (Harman, 1970), restricted to Louisiana, Mississippi, Florida, and North Carolina. The tail processes make this obvious (Harman and Loden, 1978).] 7b. Posterior end naked, or with gills, or with two lateral processes plus gills, no median process (Fig. 26); spermathecae in the testicular segment, atria in the ovarian segment, these being in X – XI or further forward (Fig. 2)................................................................8 8a(7b). Length usually 1 cm; hair chaetae usually present in dorsal bundles but absent in some genera; hair chaetae commonly associated with simple-pointed or biﬁd chaetae rather than the rarer palmate or pectinate chaetae, these dorsal chaetae (needles) very often differ from the ventrals in form (Fig. 12); dorsal chaetae may begin behind II, often in V or VI, sometimes elsewhere; dorsal chaetae often limited to one or two needles, and one or two hairs per bundle; ventral bundles with numerous biﬁd chaetae, those of II or even II – V often differ in form and thickness from the rest; asexual reproduction by budding forms chains of individuals; mature specimens have spermathecae in IV, V or VI with the male pores one segment behind them (Fig. 2); eye spots may be present; gills may surround the anus (Fig. 26); prostomium may bear a proboscis (Fig. 23.......................................Naididae .............(Section V.C) 8b. Length usually 1 cm, width usually 0.5 – 1.0 mm; when hair chaetae are present dorsally, they are usually accompanied by pectinate chaetae that closely resemble the ventral chaetae, apart from having intermediate teeth (Fig. 13); when hair chaetae are absent dorsally, all bundles usually contain similar biﬁd chaetae; dorsal chaetae begin in II, normally several per bundle; reproduction 448 Ralph O. Brinkhurst, and Stuart R. Gelder FIGURE 11 Crustipellis (Opistocystidae). Tail end to left, head end with proboscis to right, genital segments below (a, atrium, sp, spermatheca, both of left side). FIGURE 12 Various forms of dorsal chaetae, needles, of Naididae. These usually differ from the more normally biﬁd ventrals. FIGURE 13 Dorsal chaetae of Tubiﬁcidae. Pectinates, on the left, differ relatively little from a biﬁd, shown on the right. Ventral bundles usually consist of biﬁds, sometimes with minute pectinations in those species with pectinate dorsals. Pectinates are usually accompanied by hairs in the dorsal bundles, rarely are biﬁd dorsals accompanied by hairs. All chaetae may be biﬁd. normally sexual, rarely by fragmentation; spermathecal pores normally on X, male pores on XI (Fig. 2); no eyes or proboscis; no gills around anus, single dorsal and ventral gill ﬁlaments on some posterior segments of one species (Fig. 30)....................Tubiﬁcidae ........................................................................................................................................................................................(Section V.D) B. Taxonomic Key to Genera and Selected Species of Freshwater Lumbriculidae 1a. Prostomium with a proboscis (Figs. 14 and 15)..................................................................................................................................2 1b. Prostomium without a proboscis ........................................................................................................................................................6 2a(1a). Chaetae biﬁd, at least in anterior segments.........................................................................................................................................3 2b. Chaetae all simple-pointed .................................................................................................................................................................4 3a(2a). Proboscis elongate; chaetae biﬁd in anterior segments (Fig. 14) ..................................................Kincaidiana hexatheca Altman, 1936 [This species is limited to the Paciﬁc Northwest.] 3b. Proboscis short; all chaetae biﬁd, but with colateral teeth (set side by side) (Fig. 15)............................................................................ Rhynchelmis brooksi Holmquist, 1976 .......................................................................................[Known only from northern Alaska.] 4a(2b). Cave-dwelling species with short proboscis .............................................................................Trichodrilus allegheniensis Cook, 1971 [Known only from Tennessee.] 4b. Surface water species with long proboscis ..........................................................................................................................................5 5a(4b). Atria of male reproductive system with spiral muscles (Fig. 16) ...........................................................................Eclipidrilus (in part) [This genus was reviewed by Brinkhurst (1988). The species with probosces are known mostly from the southeastern United States, with one record from Montana.] 5b. Atria without spiral muscles ...............................................................................................................................Rhynchelmis (in part) [Several species distributed from California – Nevada to Alaska and Northwest Territories of Canada. See Fend and Brinkhurst (2000)] 6a(1b). Chaetae biﬁd ......................................................................................................................................................................................7 6b. Chaetae simple-pointed ......................................................................................................................................................................8 7a(6a). Elongate (to 100 mm or more) slender worms, the front end often green in life, the rest dark red to black; elaborately branched blood vessels laterally in the body wall of posterior segments (Fig. 17); reproduces sexually and asexually (fragmentation with or without encystment); no permanent everted penes............................................................................................................Lumbriculus [One widely distributed species, Lumbriculus variegatus (Mller, 1774), inhabits shallow water, where it ﬂoats the tail in the air – water interface. Several other possible taxa, mostly from Alaska, are almost certainly variants of this (see Lumbriculus and Thinodrilus, in Holmquist 1976).] 7b. Short (25 – 40 mm), tapering worms, pale to white color; blood vessels in the posterior lateral body wall with short, unbranched 12. Annelida: Oligochaeta, Including Branchiobdellidae 449 lateral diverticulae; reproduction sexual; permanently everted soft penes on X close together near the midline on mature specimens .. ..............................................................................................................................................Stylodrilus heringianus Claparéde, 1862 [Widespread in the Great Lakes and Canada, possibly a European introduction.] 8a.(6b). Groundwater species ..........................................................................................................................................................................9 8b. Surface water species ........................................................................................................................................................................11 9a(8a). Four pairs of male pores on X, two pairs of spermathecal pores on IX (Fig. 18) ..........................Spelaedrilus multiporus Cook, 1975 [Known only from a cave in Virginia.] 9b. Male pores paired on X, spermathecal pores paired in either IX, XI, or XI and XII .........................................................................10 10a.(9b). Spermathecal pores paired on IX .........................................................................................................................................Stylodrilus [S. californianus Rodriguez, 1996, California; S. wahkeenensis Rodriguez and Coates, 1996, Oregon, and possibly Tennessee and Alabama.] 10b. Spermathecal pores paired in XI or XI and XII....................................................................................................Trichodrilus (in part) [From caves in West Virginia, Illinois, Washington.] FIGURE 14 Proboscis and chaetae of Kincaidiana hexatheca. FIGURE 15 Proboscis of Rhynchelmis brooksi. FIGURE 16 Spiral atrial muscles, Eclipidrilus. FIGURE 17 Blood vessels of the lateral body wall of posterior segments, Lumbriculus. FIGURE 18 Ventral view of segments IX (to left) and X showing unique gonopores of Spelaedrilus (sp, spermathecal pores; male symbol, male pores, 4 and 8 of each, respectively.) FIGURE 19 Reproductive systems of Styloscolex, anterior to left, left side shown. Note testis (t) and atrium in VIII, ovary (o) and female funnel in X, and spermatheca (sp) in XI. FIGURE 20 Reproductive system, Kincaidiana freidris, anterior to left, organs of left side shown. Note testis (t) and atrium (a) in VIII, prostate gland stalks shown as well as male duct, ovary (o), and spermatheca (sp) in IX. 450 Ralph O. Brinkhurst, and Stuart R. Gelder 11a(8b). Male pores paired in VIII .................................................................................................................................................................12 11b. Male pores paired in X.....................................................................................................................................................................13 12a(11a). Spermathecal pores in XI, ovaries in X (Fig. 19) .............................................................Styloscolex opisthothecus Sokol’skaya, 1969 [This mostly Asian species is restricted to northern Alaska.] 12b. Spermathecal pores paired in IX, ovaries in IX (Fig. 20) ....................................................................Kincaidiana freidris Cook, 1966 [This species may not be congeneric with K. hexatheca, but cladistic analyses suggest they are quite closely related. Both are Paciﬁc Northwest species, this one from California.] 13a(11b). Spermathecal pores paired in IX ........................................................................................Stylodrilus sovaliki (Holmquist, 1976) and .........................................................................................................................Tenagodrilus musculus Eckroth and Brinkhurst, 1996 [S. sovaliki is from Alaska, but it closely resembles its European congener S. absoloni (Hrabe, 1970). T. musculus is from Alabama.] 13b. Spermathecal pores either single median in VIII – IX or in IX, or paired in IX .....................................................Eclipidrilus (in part) [One species, (E. frigidus Eisen, 1881) is from Idaho and California; the other three (E. lacustris Verrill, 1871, E. ithys Brinkhurst, 1998, and E. fontanus Wassell, 1984) are from northeastern North America]. C. Taxonomic Key to Genera and Selected Species of Freshwater Naididae 1a. No dorsal chaetae present (in some rare specimens the dorsal chaetae are recovered, but these can be recognized because of the absence of ventral chaetae in III – V and the presence of an enlarged pharynx and reduced prostomium associated with a predatory habit) ...............................................................................................................................................................................Chaetogaster 1b. Dorsal chaetae present (rare in Ophidonais) ......................................................................................................................................2 2a(1b). Dorsal chaetal bundles without hair chaetae ......................................................................................................................................3 2b. Dorsal chaetal bundles with hair chaetae (Fig. 2) ...............................................................................................................................7 3a(2a). Dorsal chaetae begin in II or III ..........................................................................................................................................................4 3b. Dorsal chaetae begin in V or VI .........................................................................................................................................................5 4a(3a). Dorsal chaetae begin in II; no gap between chaetal bundles of III and IV....................................Homochaeta naidina Bretscher, 1896 4b. Dorsal chaetae begin in III; gap between chaetal bundles of III and IV (Fig. 21) ..............................................................Amphichaeta 5a(3b). Dorsal chaetae begin in V; estuarine species ............................................................................................................................Paranais 5b. Dorsal chaetae begin in VI; freshwater species ...................................................................................................................................6 6a(5b). Dorsal chaetae stout, solitary, bluntly simple-pointed or notched at the outer end (Fig. 22) ......Ophidonais serpentina (Müller, 1773) 6b. Dorsal chaetae curved, with biﬁd tips, 2 – 4 per bundle. [See also Piguetiella, in which hair chaetae may be missing in most, if not all, dorsal bundles; some Uncinais specimens are said to have dorsal chaetae in V.] .........................Uncinais uncinata (Orsted, 1842) 7a(2b). With a proboscis on the prostomium (visible as a stump if broken off) (Fig. 23) ................................................................................8 7b. Without a proboscis on the prostomium ..........................................................................................................................................11 8a(7a). Dorsal chaetae begin in II .........................................................................................................................................................Pristina 8b. Dorsal chaetae begin in VI (observe with care as ventral chaetae of IV and / or V may be missing).....................................................9 9a(8b). Dorsal bundles of VI – VIII with 2 – 16 giant hair chaetae (Fig. 24) ...................................................Ripistes parasita (Schmidt, 1874) 9b. No giant hair chaetae .......................................................................................................................................................................10 10a(9b). Ventral chaetae with a characteristic double bend (Fig. 25); dorsal bundles with 1 – 3 hairs and 3 – 4 shorter, hairlike needles ............. ........................................................................................................................................................Stylaria lacustris (Linnaeus, 1767) 10b. Ventral chaetae slightly curved; dorsal bundles with 8 – 18 hairs and 9 – 12 hairlike needles .........Arcteonais lomondi (Martin, 1907) 11a(7b). Dorsal chaetae begin in II or III ........................................................................................................................................................12 11b. Dorsal chaetae begin in IV or further back .......................................................................................................................................14 12a(11a). Body wall covered with foreign matter adhering to glandular secretions .....................................................................Stephensoniana 12b. Body wall naked...............................................................................................................................................................................13 13a(12b). Dorsal chaetae begin in II; hair chaetae in all bundles, none especially thin, either none especially elongate “or” elongate hair chaetae in III .........................................................................................................................................................................Pristinella 12. Annelida: Oligochaeta, Including Branchiobdellidae 451 FIGURE 21 Anterior end (to right), Amphichaeta, showing dorsal chaetae beginning in III, and gap between chaetae of III and IV, particularly pronounced in this species, but noticeable throughout the genus. FIGURE 22 Dorsal chaetae, Ophidonais. These are single, or even absent in many segments. FIGURE 23 Proboscis on the prostomium of a naidid, this one with dorsal chaetae from II, but this varies (see key). FIGURE 24 Dorsal chaetae of Ripistes begin in VI, with giant hairs in three segments. FIGURE 25 Ventral chaeta of Stylaria lacustris. FIGURE 26 Perianal gills of Dero. Dero (Aulophorus) with long palps (right) and Dero (Dero) without palps (left). 13b. Dorsal chaetae either begin in III, hairs of III elongate “or” begin in II, the hairs being longest in midbody, and often missing from a number of segments, and exceptionally thin when present...................................................................................................Bratislavia 14a(11b). Posterior end of the body with a branchial fossa, normally with gills (Fig. 26); some species symbionts in tree frogs, these develop gills once outside their host. ........................................................................................................................................................Dero [There are three subgenera, Allodero containing the symbiotic species, Aulophorus with long palps on each side of the branchial fossa, and Dero that lacks palps. In a strict cladistic sense, Allodero would not be recognized.] 14b. No gills; free-living species ...............................................................................................................................................................15 15a(14b). Dorsal chaetae begin in XVIII – XX, each bundle with a single short hair and a robust needle ............................................................. ................................................................................................................................................Haemonais waldvogeli Bretscher, 1900 15b. Dorsal chaetae begin in V, VI, or VII (note that ventral chaetae of IV and / or V may be missing).....................................................16 16a(15b). Hair chaetae up to 9 per bundle, thick, with long lateral hairs (Fig. 27)..........................................................................Vejdovskyella 16b. Hair chaetae 1 – 3 per bundle at most, thin with or without ﬁne lateral hairs ...................................................................................17 17a(16b). One to three especially long hair chaetae in each dorsal bundle of VI, 1 or 2 ordinary hair chaetae in the remainder (Fig. 28) ............ .............................................................................................................................................Slavina appendiculata (d’Udekem, 1855) 17b. No elongate hair chaetae ..................................................................................................................................................................18 18a(17b). Hair chaetae very short (84 – 120 m), present in only one or two bundles, some with no hair chaetae at all (see 6) ...........Piguetiella 18b. Hair chaetae mostly 1 or 2 per bundle (two species with up to 5), long (180 – 200 m or more), present in all bundles from V or VI . .........................................................................................................................................................................................................19 19a(18b). Characteristic needle chaetae with thick, unequal teeth, sometimes clearly pectinate (Fig. 29); ventral chaetae change form and length slightly between V and VI .............................................................................................................................................Allonais 452 Ralph O. Brinkhurst, and Stuart R. Gelder FIGURE 27 Hair chaetae of Vejdovskyella, detail to right. FIGURE 28 Dorsal bundles of VI in Slavina with elongate hairs. FIGURE 29 Needle chaetae of various Allonais species. 19b. Needle chaetae simple-pointed, biﬁd, or faintly pectinate; either ventral chaetae of II differ from the rest “or” those of II – V differ markedly in length, width, and form from the rest ...........................................................................................................................20 20a(19b). Needle chaetae biﬁd or faintly pectinate; ventral chaetae of II may differ from the rest ..........................................................Specaria 20b. Needle chaetae simple-pointed, biﬁd, or pectinate; ventral chaetae of II – V differ strongly in length, thickness, and form from the rest in the majority of species. .......................................................................................................................................................Nais [Species of Dero with the posterior end of the body missing may key out here because there will be no gills. Compare specimens to those with gills; the anterior ventral chaetae in Dero species have very long upper teeth.] D. Taxonomic Key to Genera and Selected Species of Freshwater Tubiﬁcidae This key is designed to maximize the use of external characters. As it is primarily a key to genera, it is necessary to refer mostly to characters found on mature specimens. Immature specimens can often be identiﬁed on the basis of chaetal form alone, but then the key must proceed directly to the speciﬁc level (Kathman and Brinkhurst, 1998). In this key, there are two genus groups that are difﬁcult to separate on external characters; and where this is true, evidence from distribution patterns is introduced as an additional aid. Keys for worms of this family are traditionally written at the species level. 1a. Single dorsal and ventral gill ﬁlaments on posterior segments (Fig. 30) ........................................Branchiura sowerbyi Beddard, 1892 [Broken anterior fragments may be mistaken for Aulodrilus pluriseta (see 18b).] 1b. No gill ﬁlaments .................................................................................................................................................................................2 2a(1b). Body wall papillate (with projections usually covered with foreign matter attached by secretions) (Fig. 31)......................................... ...............................................................................................................................Telmatodrilus (in part), Spirosperma, Quistadrilus [Telmatodrilus (Alexandrovia) onegensis Hrabe, 1962 is recorded from Alaska; the subgenus is often elevated to generic status, which may prove correct.] 2b. Body wall naked.................................................................................................................................................................................3 3a(2b). Spermathecal chaetae replace normal ventral chaetae in the spermathecal segment, very rarely in adjacent segments also (Fig. 34); with or without modiﬁed penial chaetae (Figs. 39, 41, 42) and cuticular penis sheaths (Fig. 35) ........................................................4 3b. No modiﬁed spermathecal chaetae .....................................................................................................................................................8 4a(3a). Thin, hollow-tipped spermathecal chaetae in X, similar chaetae in XI together with apparent cuticular penis sheaths (Fig. 32)........... ............................................................................................................................................................Haber speciosus (Hrabe, 1931) 12. Annelida: Oligochaeta, Including Branchiobdellidae 453 4b. Spermathecal chaetae not duplicated (or, if so, on VII or VIII and very rarely scattered from VI – XII); with or without cuticular penis sheaths) .....................................................................................................................................................................................5 5a(4b). Spermathecal chaetae broad, spatulate, one each side of IX associated with long tubular glands; ventral chaetae of X similar or absent; several penial chaetae in each ventral bundle of XI with short, knobbed distal ends; male ducts open into large median inversion of the body wall of XI (Fig. 33) ...................................................................................................Rhizodrilus lacteus Smith, 1900 5b. Without this unique set of characters combined .................................................................................................................................6 6a(5b). Spermathecal chaetae much broader and longer than the normal ventral chaetae, distal ends hollow, located on X (or in one instance on VII or VIII or even irregularly between VI and XII); no cuticular penis sheaths .............................................Potamothrix FIGURE 30 Branchiura, showing posterior fan of gill ﬁlaments. FIGURE 31 Papillate body wall of Spirosperma. FIGURE 32 Spermathecal chaeta of Haber (in which penial chaetae similar.) FIGURE 33 Reproductive organs, left side (anterior to left) of Rhizodrilus lacteus. Note spermathecal chaeta and gland (g) in IX, spermatheca (sp) in X, atrium (a) in XI discharging into median chamber that also receives penial chaetae (on right, opposite atrial pore). FIGURE 34 Spermathecal chaeta of Isochaetides. FIGURE 35 Penis sheath of Isochaetides. FIGURE 36 Inverted (left) and everted penis of Psammoryctides. FIGURE 37 Varichaetadrilus, penis (left) with penial sheath on tip (shown pointing down) and penial chaetae, top right and (enlarged) to right. 454 Ralph O. Brinkhurst, and Stuart R. Gelder [Most species are restricted to the Great Lakes basin or extend into adjacent states; only one is widespread. These may be introduced from Europe, where they are characteristic of productive waters.] 6b. Spermathecal chaetae longer than the normal ventral chaetae, slender distally, somewhat like a hypodermic needle (Fig. 34); true or apparent cuticular penis sheaths present.............................................................................................................................................7 7a(6b). True cuticular penis sheaths present, but not much thicker than the normal cuticular layer of the body wall (Fig. 35)......Isochaetides 7b. Apparent penis sheaths present (probably the cuticular linings of eversible penes), look like crumpled cylinders in whole-mounts of most specimens (Fig. 36) .............................................................................................................................................Psammoryctides [One species is a recent introduction to the St. Lawrence River (Quebec) from Europe, another is known only from coastal swamps of the Gulf of Mexico, and a third is known from California and the Great Lakes.] 8a(3b). Penial chaetae present replacing the normal ventral chaetae of XI (Figs. 37, 39, 41, 42) ....................................................................9 8b. Penial chaetae absent, ventral chaetae of XI usually missing in mature specimens ............................................................................12 9a(8a). Penial chaetae biﬁd but with shortened distal ends and elongate proximal ends, somewhat thicker than the normal ventral chaetae; with short penis sheaths on the ends of erectile penes (Fig. 37) .....................................................................Varichaetadrilus (in part) [Distributed form Alaska to California and Alberta (but see also 14).] 9b. Penial chaetae strongly modiﬁed, with knobbed or hooked tops to short distal ends, elongate proximal ends, arranged fanwise with heads close together (as if they function as claspers) “or” large, single and sickle-shaped (Figs. 39, 41, 42) .....................................10 10a(9b). Male pores open into an eversible chamber in XI that contains so-called paratria (Fig. 38) and penial chaetae when present (Fig. 39); sensory pit on the prostomium (Fig. 3); sperm attached to the body wall close to the male pore in external spermatophores (Fig. 40)............................................................................................................................Bothrioneurum vejdovskyanum Stolc, 1888 10b. No median copulatory chamber or sensory pit; sperm loose in spermathecae after copulation.........................................................11 11a(10b). Penial chaetae in XI, 3 – 6 simple-pointed chaetae with hooked tips (Fig. 41); somatic chaetae 3 – 5 biﬁds from II – XX, simplepointed chaetae from XXV – XXX posteriad, no dorsal hair chaetae ............................Phallodrilus hallae Cook and Hiltunen, 1975 11b. Penial chaetae with knobbed tips, or single, sickle-shaped (Fig. 42); no simple-pointed somatic chaetae (except in the ventral bundles of one species, when accompanied by biﬁd chaetae, and the dorsal bundles of that species possess hair chaetae).................... Rhyacodrilus 12a(8b). Cuticular penis sheaths present in XI (Figs. 42 – 45) .........................................................................................................................13 12b. Cuticular penis sheaths absent..........................................................................................................................................................15 13a(12a). Penis sheaths more or less elongate, cylindrical, with penes free within them (Fig. 43); all chaetae biﬁd ...........................Limnodrilus 13b. Penis sheaths annular to conical, attached to the surface of the penis (Figs. 44 and 45) ...................................................................14 14a(13b). Short cuticular penis sheaths, annular to tub-shaped (Fig. 44)..........................Varichaetadrilus (in part), Tubifex, Ilyodrilus (in part) [The Varichaetadrilus species included here are reported from the southeastern United States; I. frantzi Brinkhurst, 1965 is restricted to freshwater at the head of estuaries from British Columbia (Canada) to California.] 14b. Penis sheaths conical (Fig. 45)...........................................................................Tasserkidrilus, Ilyodrilus templetoni (Southern, 1904) 15a(12b). Chaetae simple-pointed anteriorly, behind the clitellum the chaetae have brushlike tips (Fig. 46) ...................................Telmatodrilus vejdovskyi Eisen, 1879 [This species is known from British Columbia (Canada) to California.] 15b. Chaetae biﬁd, or with upper tooth divided, or pectinate to palmate, all with the teeth in a single plane...........................................16 16a(15b). Pharynx and mouth enlarged, prostomium reduced (Fig. 47); chaetae of III broad, curved, with large lower teeth, the rest thinner, straighter, and with teeth more nearly equal; no spermathecae, presumably parthenogenetic ..Teneridrilus mastix (Brinkhurst, 1978) 16b. Pharynx and mouth not enlarged, prostomium well developed; chaetae of III not modiﬁed; with spermathecae ..............................17 17a(16b). Ventral chaetal bundles with up to 4 simple-pointed or biﬁd chaetae with reduced upper teeth. [In this species from Lake Tahoe there are apparently no penial chaetae, although these are usually present in this genus (see 11b).] ..................................................... ......................................................................................................................................Rhyacodrilus brevidentatus Brinkhurst, 1965 17b. No simple-pointed ventral chaetae ...................................................................................................................................................18 18a(17b). Dorsal chaetal bundles with 2 – 4 pectinate-to-palmate chaetae from II – XIV, from there posteriad all dorsal chaetae biﬁd; anterior ventral chaetae 3 – 5 per bundle, biﬁd with the upper teeth thinner than but only a little longer than the lower, but beyond XIV the upper teeth become much longer than the lower..............................................Arctodrilus wulikensis Brinkhurst and Kathman, 1983 [This species is recorded from the Brooks Range, Alaska.] 18b. Chaetae progressively enlarged from III posteriad, becoming very broad with large lower teeth and recurved distal ends, “or” no hair chaetae dorsally and large numbers of biﬁd chaetae with thin, short upper teeth, becoming palmate beyond VI in one species, “or” hair chaetae present but all ventral chaetae again characteristically numerous and with short upper teeth (Fig. 48 .....Aulodrilus 12. Annelida: Oligochaeta, Including Branchiobdellidae FIGURE 38 Paratrium of Bothrioneurum, shown left in situ in everted terminal portion of male duct, detail to right. FIGURE 39 Penial chaetae, Bothrioneurum, detail of tip above, bundle below. FIGURE 40 Spermatophores of Bothrioneurum, attached to body wall around gonopores (left) and detail (right). Note sperm enclosed, with no duct through attachment stalk. FIGURE 41 Penial chaeta, Phallodrilus hallae. FIGURE 42 Penial chaetae, Rhyacodrilus falciformis. FIGURE 43 Penes of various Limnodrilus species, three to right much longer than shown (40 times the length when fully developed). FIGURE 44 Penis sheath of Tubifex tubifex, often thin and difﬁcult to see, but granular surface often a clue. FIGURE 45 Penis sheath of Ilyodrilus, short conical form. The terminal opening often looks torn, and individuals with a second portion as long as that illustrated here may be seen, this second part presumably shed before mating. FIGURE 46 Posterior brushlike chaetae, Telmatodrilus. FIGURE 47 Inverted (above) and everted pharynx of Teneridrilus mastix. FIGURE 48 Ventral chaeta, Aulodrilus. 455 456 Ralph O. Brinkhurst, and Stuart R. Gelder BRANCHIOBDELLIDAE VI. INTRODUCTION TO BRANCHIOBDELLIDAE Branchiobdellids are leechlike, ectosymbionts living primarily on astacoidean crayﬁsh and a few other crustaceans. A single host can be infested with over 1,000 individuals and may include a taxonomic diversity of up to seven species representing ﬁve genera. Branchiobdellids have an Holarctic distribution with a total of 21 genera and 149 species (Gelder, 1996a), of which 15 genera containing 107 species are reported from North America. In spite of being known for almost 250 years, the ecophysiological interactions of this complex ectosymbiotic association remain largely unstudied. Branchiobdellids have for the most part been considered to be a family, or at best a superfamily, until Holt (1965) raised them to an order, Branchiobdellida. He did this to make the taxon independent of, and equal in rank to, both the oligochaetes and leeches. In a review of the various ranks and names given to the branchiobdellids (Gelder, 1996a), it was found that the taxon has reached class rank, Branchiobdellae, but this was an overly inflated rank for the group. Holt (1986) arranged the branchiobdellid genera into five families within the order. This gave rise to the term, used in the first edition, “branchiobdellid” as the com- mon epithet for members of the order, thus leaving branchiobdellid for members of that family. The demotion of the taxon from order to family, and the five families created by Holt to subfamily rank, enables the relative relationships of these taxa to be maintained. There is no single publication dealing with all of the known species of branchiobdellidans, but a detailed bibliography of the taxon was made by Sawyer (1986). Therefore, as this work will deal only with North American genera, citations are included through which species descriptions and further information can be found. VII. ANATOMY AND PHYSIOLOGY OF BRANCHIOBDELLIDS A. External Morphology Adult branchiobdellids range from 0.8 – 10 mm in length. The body in most species is rod-shaped (terete), although some have a characteristic pyriform or ﬂaskshape with either ventral or dorso-ventral ﬂattening. The segment number is constant and, based on the number of paired ganglia, is accepted to be ﬁfteen (XV) as shown by Roman numerals in Figure 49. The external morphology shows a peristomium and three segments forming a head, and the remaining obvious body FIGURE 49 Diagram of the lateral aspect of a hypothetical branchiobdellidan showing anatomic characters used in the taxonomy of this group: a, anus; aa, anterior attachment surface; b, bursa; d, digitiform dorsal appendages; ds, developing sperm; e, esophagus; ga, glandular atrium; i, intestine; j, jaw; l, lip; m, mouth; ma, muscular atrium; n, nephridia; np, nephridial pore; o, ovum; op, oral papillae; pa, posterior attachment surface; pg, prostate gland; ph, pharynx; s, stomach; sl, supernumerary longitudinal muscles; sp, spermatheca; su, sulcus; t, tentacle; vd, vasa deferentia; H, head; P, peristomium; 1-11, body segments; I-XV, segments based on paired ganglia. 12. Annelida: Oligochaeta, Including Branchiobdellidae segments are numbered in Arabic numerals, 1 to 11. The latter nomenclature is the one used in the descriptive literature on branchiobdellids. Segment 11 is modiﬁed into the posterior, disk-shaped attachment organ (sucker), and contains a single pair of ganglia, XV. Peristomial tentacles or lobes may be present on the dorsal lip, and the mouth is surrounded usually by 16 oral papillae. Each body segment is divided into a major anterior and a minor posterior annulus, although a few species have the major annulus further subdivided into two. Dorsal transverse ridges across the major annuli are produced by supernumerary longitudinal muscles (sl) and sometimes support dorsal digitiform or multibranched appendages (d) (Gelder, 1996b). B. Organ System Function The alimentary canal consists of a pair of sclerotized jaws (j) situated in the anterior pharynx followed by one to three pairs of sulci (su), a short esophagus (e), stomach (s), intestine (i), and an anus (a) opening dorso-medially onto segment 10 (Fig. 49). Locomotory movements are leechlike and involve an anterior and a posterior attachment organ (Gelder and Rowe, 1988). The anterior attachment site is on the ventral surface of the ventral lip and is not the mouth as assumed generally. The muscle cells have the nucleus and cytoplasm in the medulla with the contractile ﬁbers in the cortex. The ultrastructure of the cells in three species from the Branchiobdellinae are all round circomyarian in their contractile ﬁber arrangement. A species from each of the Bdellodrilinae and Cambarincolinae have a few round circomyarian ﬁbered cells, but most are “polyplatymyarian” (Valvassori et al., 1994). The pore(s) of the asymmetrically arranged anterior pair of nephridia open to the exterior on segment 3 (np) and the posterior pair are located in segment 8, but open on segment 9. A pair of testes are located in segments 5 and 6, and the sperm develop (ds) in the coelom of those segments (Fig. 49). The ultrastructure of spermatozoa from four species representing the Branchiobdellinae, Bdellodrilinae, and Cambarincolinae showed an unexpected variation in morphology (Ferraguti and Gelder, 1990). Additional studies have conﬁrmed that morphological variations in the spermatozoa occur between genera, but that interspeciﬁc variations are dimensional only (Ferraguti, 2000). Two vasa efferentia, both with a ciliated funnel, merge into a vas deferens and the two vasa deferentia, one from each segment, enter the glandular atrium in segment 6. When present, the prostate gland forms a protuberance or a diverticulum from the glandular atrium. The connecting muscular atrium passes into the penis which is surrounded by a muscu- 457 lar bursa (b). Most penes are either eversible or protrusible, although a few species have a penis that shows varying degrees of reduction. The bursal atrium opens mesad on the ventral surface of segment 6. The terms, “spermiducal gland” ( glandular atrium) and “ejaculatory duct” ( muscular atrium) as used in the First Edition (Brinkhurst and Gelder, 1991) followed Holt (1986). These have been replaced by the terms in parentheses to promote consistency in anatomical terminology with oligochaetologists in general. The spermatheca (sp), when present, is located in segment 5 and consists of a glandular ental region connected to a muscular duct that opens medially onto the ventral surface of the segment. A pair of ovaries in segment 7 produce eggs that mature in the coelom, and are released through a pair of short ducts in the body wall of that segment. The vascular system consists of a dorsal and ventral longitudinal vessels that are connected by four paired, lateral branches in the head and a single pair of branches in segments 1, 7, and 10, respectively. The dorsal vessel becomes a sinus surrounding the gut epithelium from segments 3 – 7. The central nervous system consists of a dorsal brain, circumesophageal connectives and a paired ventral cord extending into the last segment. The head contains four pairs of ganglia with each body segment containing a single pair of ganglia. The nerve cord is displaced laterally in segments 5 and 6 where the spermatheca and male genitalia pass mesad through the ventral body wall. C. Environmental Physiology Most branchiobdellids browse on the epibionts, such as diatoms and ciliates, attached to the host and are opportunistic commensals when fragments of the host’s food are available (Jennings and Gelder, 1979). Other opportunistic food sources include cannibalism and exposed host tissues at injury sites. Contrary to the reputation of these worms, a parasitic regime has been demonstrated only in Branchiobdella hexodonta, and inferred in a few other species. Food is sucked into the mouth and then the jaws close to crop microorganisms from the substrate or fragment large particles. Various mucoid secretions assist the ingestion of food, but the term “salivary gland” as quoted by Sawyer (1986) is inappropriate and was corrected by Gelder and Rowe (1988). Digestion is extracellular in the stomach and intestine with uptake occurring in the cells of the latter region. Low oxygen tensions do not appear to affect branchiobdellids adversely, although some species tolerate higher water temperatures better than others. 458 Ralph O. Brinkhurst, and Stuart R. Gelder VIII. ECOLOGY AND EVOLUTION A. Diversity and Distribution The branchiobdellids are characterized by a lack of anatomical and ecological diversity in comparison with equivalent clitellate taxa. The range of the branchiobdellids in North America extends from southern Canada to Costa Rica (Gelder, 1999). A few species are found distributed throughout this region, but the others have a more limited range (see Section IX.B). The zoogeography of species is becoming more confused and obscure as crayﬁsh and their symbionts are introduced into new areas for aquaculture, live food, research, and teaching purposes. B. Reproduction and Life History Information on chromosome numbers is restricted to three species, Branchiobdella astaci 2n 14, B. kozarovi 2n 12, and B. parasita 2n 10 (Mihailova and Subchev, 1981), and they refute previous reports of n 4 and 8 for the ﬁrst species. Germ cells from the testes are released into the coelom of segments 5 and 6, where spermatogenesis occurs. The mature spermatozoa then congregate at the mouth of the sperm funnels prior to their passage to the penis. Prior to copulation, the muscular bursa is partially or completely everted (Figs. 50 – 53), and comes close to, or contacts with, the epidermis surrounding the recipient’s spermathecal pore. This is followed by the extended penis entering the pore for insemination. Single inseminations have been observed; however, it is not known if reciprocal insemination occurs. Spermatophores on the epidermis of Xironogiton victoriensis have been examined by FIGURE 50 Gelder (1996c). Members of Ellisodrilus and Caridinophila do not have spermatheca so their method for sperm reception and storage is open to speculation. Although the use of spermatophores has been proposed as a solution to their problem, examinations of preserved specimens of Ellisodrilus have found no such packets. The secretions from the clitellar gland cells have been characterized histochemically and compared with those in other clitellate annelids (Gelder and Rowe, 1988). These secretions produce the pedunculate cocoon and nourishment to sustain the embryo while it develops. Branchiobdellid cocoons deposited on live hosts will develop to maturity and hatch out. The hatching rate as observed on live hosts is temperature dependent, and occurs after 10 – 12 days at 20 – 22°C. The embryo is lecithotrophic and then becomes an albuminotrophic cryptolarva. In this latter phase, precocious feeding of the surrounding nutrient ﬂuid starts with ingestion by a transitory pharynx and the digestion yolk-sac epithelium. There are no reports of growth rates or the time a juvenile requires to reach sexual maturity. A few reports have noted that cocoons were deposited on cast exoskeletons of their hosts and the glass walls of their container, but embryos did not develop and emerge. However, Woodhead (1945) is the only person to report that branchiobdellids mated, attached their cocoons to the walls of the stock dishes, and young hatched a few days later. C. Ecological Interactions Branchiobdellids have been reported on freshwater crustaceans, primarily astacoidean crayﬁsh. Although all Diagrammatic section of a withdrawn, eversible penis: b, bursa, p, penis. FIGURE 51 Diagrammatic section of an extended, eversible penis; b, bursa; p, penis. FIGURE 52 Diagrammatic section of a withdrawn, protrusible penis; b, bursa; p, penis; r, retractor muscle. FIGURE 53 Diagrammatic section of an extended, protrusible penis: b, bursa, p, penis; r, retractor muscle. 12. Annelida: Oligochaeta, Including Branchiobdellidae crayﬁsh appear to be potential hosts, some species actively remove any branchiobdellids from their exoskeleton (Gelder and Smith, 1987). Noncrayﬁsh hosts were reported to be acceptable in troglodytic habitats (Holt, 1973b) and in Central America at and beyond the southern limits of the geographical range of the astacoidean crayﬁsh hosts (Gelder, 1999). Branchiobdellids have been reported on the blue crab, Callinectes sp., in low salinity waters adjacent to the Gulf of Mexico (Overstreet, 1983), and so the possibility exists that a marine branchiobdellid will be found. Branchiobdellids also harbor ectosymbionts and parasites. Externally, these include stalked, solitary and colonial ciliate protozoans, and rotifers. Internally, branchiobdellids serve as host to the third juvenile stage of the nematode Dioctophyme renale (giant kidney worm) (Schmidt and Roberts, 1989), and the trematode mesocercaria of Pharyngostomoides procyonis (reviewed in Schell, 1985). D. Evolutionary Relationships The branchiobdellids form a monophyly (Gelder and Brinkhurst, 1990). The leechlike appearance of branchiobdellids has caused some researchers to included them with the leeches. However, the presence of a semiprosopore male ducts (Fig. 6) in both the branchiobdellids and lumbriculid oligochaetes, strongly supports the branchiobdellids returning to family rank as a sister group to the lumbriculids (Brinkhurst, 1999). The anatomical modiﬁcations shown by branchiobdellids, such as constant segment number, loss of chaetae, development of anterior and posterior attachment organs, jaws, and other features, are not considered homologous to similar states in leeches but, instead, reﬂect a specialization to an ectosymbiotic lifestyle as proposed by Stephenson (1930). The relocation of the branchiobdellids back into the oligochaetes is the result of the best interpretation of the available data. Phylogenetic analyses will soon be available using matrices of nucleotide sequences in selected genes from a signiﬁcant number of clitellate species. Whether the new methodologies will provide new phylogenies or conﬁrm some of those already established remains to be seen. 459 IX. IDENTIFICATION OF BRANCHIOBDELLIDS A. Collecting, Rearing, and Preparation for Identiﬁcation Crayﬁsh and other crustacean hosts are collected by netting or baiting a trap (see Chapter 22) and then placed directly into a container with preservative. If branchiobdellids are to be examined it is recommended that the preservative formalin – alcohol – acetic acid (FAA) be used (70 mL ethanol 5 mL formaldehyde (full strength) 1 mL glacial acetic acid 30 mL water). The volume of preservative should be at least three times that of the crustaceans to ensure thorough preservation of the symbionts and host. Branchiobdellids removed from a live host have been maintained in vitro for several months. Microscopical examination of a living branchiobdellid— in water slightly squeezed between slide and cover-glass — will enable all of the anatomical characters to be observed. First examine the specimen from a dorsal or ventral aspect as this enables one to observe the peristomium, jaws and location of the anterior nephridial pore(s). The specimen can then be rolled to show a lateral aspect, allowing the spermatheca and male genitalia to be studied. Live specimens can be relaxed using carbonated drinks (such as club soda) or a 1 – 2% magnesium chloride solution (Delly, 1985), straightened or ﬂattened, and then immediately preserved. Unstained specimens are dehydrated in graded ethanol solutions, cleared in clove oil or oil of wintergreen (methyl salicylate), and mounted in Canada balsam for permanent preparations. Specimens usually curl when preserved on the host, and so these worms can only be examined from the lateral aspect. Large branchiobdellids (4 – 10 mm long) will require microdissection to expose their genitalia. The necessary instruments can be made from fragments of a razor blade and insect (very small) pins glued to the end of cocktail sticks. For techniques to prepare serial sections and to stain specimens, refer to a book on animal microtechiques, such as Humason (1979). Gelder (1996a) compiled a checklist of branchiobdellids and included citations to all papers containing type descriptions. B. Taxonomic Key to Genera of Freshwater Branchiobdellidae 1a. Two anterior nephridial pores ............................................................................................................................................................2 1b. One anterior nephridial pore ..............................................................................................................................................................4 2a(1a). Ventral ﬂattening of body...................................................................................................................................................................3 2b. Dorso-ventral ﬂattening of body (Fig. 54) .........................................................Xironogiton [British Columbia, Oregon, Washington, California, Idaho, Wyoming, and the Appalachian Region from Maine to Georgia.] 460 Ralph O. Brinkhurst, and Stuart R. Gelder FIGURE 54 Ventral view of Xironogiton instabilis, length about 2 mm. FIGURE 55 Lateral view of Pterodrilus alcicornis, length about 1.5 mm. (Redrawn from Holt, 1986b.) FIGURE 56 Ventral view of Triannulata magna, length about 4.5 mm. (Redrawn from Holt, 1974.) FIGURE 57 Lateral view of Bdellodrilus illuminatus, length about 3 mm, lg, lateral glands. FIGURE 58 Lateral view of Ceratodrilus thysanosomus, length about 3 mm. (Redrawn and modiﬁed from Holt, 1960.) 3a(2a). Vasa deferentia entering glandular atrium mesad............................................................................Ankyrodrilus [Tennessee, Virginia] 3b. Vasa deferentia entering glandular atrium entad................................................................................ Xironodrilus [East-central USA] 4a(1b). Vasa deferentia entering glandular atrium mesad ...............................................................................................................................5 4b. Vasa deferentia entering glandular atrium entad.................................................................................................................................7 5a(4a). Body surface with indistinct segments ................................................................................................................................................6 5b. Body surface with distinct segments (Fig. 59) ....................................................................................................Cronodrilus [Georgia] 6a(5a). Lateral segmental glands, segments 1 – 9 (Fig. 55) ...............................................Bdellodrilus [Southern Canada to southern Mexico] 6b. Lateral segmental glands not in segments 1 – 9 ...........................................................Uglukodrilus [California, Oregon, Washington] 7a(4b). Spermatheca present...........................................................................................................................................................................8 7b. Spermatheca absent ..........................................................................................Ellisodrilus [Tennessee, Kentucky, Indiana, Michigan] 8a(7a). Penis protrusible (Figs. 52 and 53) .....................................................................................................................................................9 8b. Penis eversible (Figs. 50 and 51) .......................................................................................................................................................11 9a(8a). Prostate body present ......................................................................................................................................................................10 9b. Prostate body absent (Fig. 60) ..................................................................................................................Magmatodrilus [California] 10a(8b). Dorsal appendages present, range from paired digitiform unit to single transverse ridge on segment 8 (Fig. 56) ................................. Pterodrilus [Eastern United States] 10b. Dorsal appendages absent (Fig. 61) ..........................................................................Cambarincola [Southern Canada to Costa Rica] 11a(9a). Prostate body absent .......................................................................................................................................................................12 11b. Prostate body present ......................................................................................................................................................................13 12a(11a). Two annuli per segment (Fig. 62) ...........................................................................Sathodrilus (in part) [Florida, California, Mexico] 12b. Three annuli per segment (Fig. 57) ................................................................................................Triannulata [Oregon, Washington] 13a(11b). Dorsal appendages present (Fig. 58) ...........................................................Ceratodrilus [Utah, Wyoming, Idaho, Montana, Oregon] 13b. Dorsal appendages absent. [This grouping cannot be resolved further without detailed histological preparation.](see Fig. 63) ............ Sathodrilus (in part) [South Carolina, Georgia, Mexico] (see Fig. 64) ..................Sathodrilus (in part) [Southern Canada to Mexico] Oedipodrilus [Tennessee, Kentucky, Ohio, Pennsylvania, Indiana, Illinois].......................................................Tettodrilus [Tennessee] 12. Annelida: Oligochaeta, Including Branchiobdellidae 461 FIGURE 59 Lateral view of the male genitalia and spermatheca in Cronodrilus ogyguis. (Redrawn and modiﬁed from Holt, 1968a.) FIGURE 60 Lateral view of the male genitalia and spermatheca in Magmatodrilus obscurus. (Redrawn and modiﬁed from Holt, 1967.) FIGURE 61 Lateral view of the male genitalia and spermatheca in Cambarincola fallax. FIGURE 62 Lateral view of the male genitalia and spermatheca in Sathodrilus hortoni. (Redrawn from Holt, 1973a.) FIGURE 63 Lateral view of the male genitalia and spermatheca in Sathodrilus carolinensis. (Redrawn from Holt, 1968a.) FIGURE 64 Lateral view of the male genitalia and spermatheca in Sathodrilus attenuatus. (Redrawn from Holt, 1981.) LITERATURE CITED Appleby, A. G., Brinkhurst, R. O. 1970. Defecation rate of three tubiﬁcid oligochaetes found in the sediment of Toronto Harbour, Ontario. Journal of the Fisheries Research Board of Canada 27:1971 – 1982. Aston, R. J. 1984. The culture of Branchiura sowerbyi (Tubiﬁcidae, Oligochaeta) using cellulose substrate. Aquaculture 40:89 – 94. Bagheri, E. A., McLusky, D. S. 1984. The oxygen consumption of Tubiﬁcoides benedeni (Udekem) in relation to temperature, and its application to production biology. Journal of Experimental Marine Biology 78:187 – 197. Block, E. M., Moreno, G., Goodnight, C. J. 1982. 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An emendation of the genus Triannulata Goodnight 1940, with the assignment of Triannulata montana to Cambrincola Ellis 1912 (Clitellata: Branchiobdellida). Proceedings of the Biological Society of Washington 87:57 – 72. Holt, P. C. 1981. New species of Sathodrilus Holt 1968, (Clitellata: Branchiobdellida) from the Paciﬁc drainage of the United States, with the synonymy of Sathodrilus virgiliae Holt 1977. Proceedings of the Biological Society of Washington 94:848 – 862. Holt, P. C. 1986. Newly established families of the order Branchiobdellida (Annelida: Clitellata) with a synopsis of the genera. Proceedings of the Biological Society of Washington 99:676 – 702. Humason, G. L. 1979. Animal Tissue Techniques. 4th edition Freeman, San Francisco, CA. Jamieson, B. G. M. 1981. The ultrastructure of the Oligochaeta. Academic Press, London. Jennings, J. B., Gelder, S. R. 1979. Gut structure, feeding and digestion in the branchiobdellid oligochaete Cambarincola macrodonta Ellis 1912, an ectosymbiote of the freshwater crayﬁsh Procambarus clarkii. Biological Bulletin 156:300 – 314. Karlckhoff, S. W., Morris, K. R. 1985. Impact of tubiﬁcid oligochaetes on pollution transport in bottom sediments. Environmental Science and Technology 19:51 – 56. 12. Annelida: Oligochaeta, Including Branchiobdellidae Kaster, J. L., Bushnell, J. H. 1981. Cyst formation by Tubifex tubifex (Tubiﬁcidae). Transactions of the American Microscopical Society 100:34 – 41. Kaster, J. L., Val Klump, J., Meyer, J., Krezoski, J., Smith, M. E. 1984. Comparison of defecation rates of Limnodrilus hoffmeisteri Claparéde (Tubiﬁcidae) using two different methods. Hydrobiologia 111:181 – 184. Kathman, R. D., Brinkhurst, R. O. 1998. Guide to the freshwater oligochaetes of North America. Aquatic Resources Center, College Grove, TN. 264 pp. Krezoski, J. R., Robins, J. A., White, D. S. 1984. Dual radiotracer measurement of zoobenthos-mediated solute and particle transport in freshwater sediments. Journal of Geophysical Research 89:7937 – 7947. Lang, C. 1984. Eutrophication of Lakes Leman and Neuchatel (Switzerland) indicated by oligochaete communities. Hydrobiologia 114:131 – 138. Lazim, M. N., Learner, M. A. 1986a. The life-cycle and production of Limnodrilus hoffmeisteri and L. udekemianus (Oligochaeta, Tubiﬁcidae) in the organically enriched Moat-Feeder stream, Cardiff, South Wales. Archiv fur Hydrobiologie (Supplement) 4:200 – 225. Lazim, M. N., Learner, M. A. 1986b. The life-cycle and productivity of Tubifex tubifex (Oligochaeta, Tubiﬁcidae) in the Moat-Feeder stream, Cardiff, South Wales. Holarctic Ecology 9:185 – 192. Leitz, D. M. 1987. Potential for aquatic oligochaetes as live food in commercial aquaculture. Hydrobiologia: 155:309 – 310. Lochhead, G., Learner, M. A. 1984. The cocoon and hatchling of Nais variabilis (Naididae, Oligochaeta). Freshwater Biology 14:189 – 193. Loden, M. S. 1981. Reproductive ecology of Naididae. Hydrobiologia 83:115 – 123. Marian, M. P., Pandian, T. J. 1984. Culture and harvesting techniques for Tubifex tubifex. Aquaculture 42:303 – 315. Mihailova, P. V., Subchev, M. A. 1981. On the karyotype of three species of the family Branchiobdellidae (Annelida: Oligochaeta). Comptes rendus l’Academie bulgare des Sciences 34:265 – 267. Milbrink, G. 1983. An improved environmental index based on the relative abundance of oligochaete species. Hydrobiologia 102:89 – 97. Milbrink, G. 1993. Evidence for mutualistic interactions in freshwater oligochaete communities. Oikos 68: 317 – 322. Moore, J. P. 1895. Pterodrilus, a remarkable discodrilid. Proceedings of the Academy of Natural Sciences of Philadelphia pp. 449 – 454. Nemec, A. F. L., Brinkhurst, R. O. 1987. A comparison of methodological approaches to the subfamilial classiﬁcation of the Naididae (Oligochaeta). Canadian Journal of Zoology 65:691 – 707. Newrlka, P., Mutayoba, S. 1987. Why and where do oligochaetes hide their cocoons? Hydrobiologia 155:171 – 178. Olsson, T. I. 1981. Overwintering of benthic macroinvertebrates in ice and frozen sediment in a North Swedish River. Holarctic Ecology 4:161 – 166. Overstreet, R. M. 1983. Metazoan symbionts of crustaceans. in: Provenzano A. J., Jr., Ed. The biology of Crustacea, pathobiology. Vol. 6, Academic Press, New York, pp. 155 – 250. Poddubnaya, T. L. 1984. Parthenogenesis in Tubiﬁcidae. Hydrobiologia 115:97 – 99. Sawyer, R. T. 1986. Leech biology and behaviour. Clarendon, Oxford. pp. 1065. 463 Schell, S. C. 1985. Handbook of trematodes of North America, North of Mexico. University of Idaho Press, Moscow, ID. Schmidt, G. D., Roberts, L. S. 1989. Foundations of parasitology. Times Mirror / Morsby, Boston, MA. Stephenson, J. 1930. The Oligochaeta. Clarendon Press, Oxford. Valvassori, R., de Eguileor, M., Lanzavecchia, G., Gelder, S. R. 1994. Comparative body wall musculature and muscle ﬁbre ultrastructure in branchiobdellidans (Annelida: Clitellata), and their phylogenetic signiﬁcance. Hydrobiologia 278:189 – 199. Vincent, V., Vaillancourt, G., McMurray, S. 1978. Premiere mention de Psammoryctides barbatus (Grube) (Annelida, Oligochaeta) en Amerique du Nord et note sur sa distribution dans le haut estuaire du Saint-Laurent. Le Naturaliste Canadien 105:77 – 80. Wassell, J. T. 1984. Revision of the lumbriculid oligochaete Eclipidrilus Eisen, 1881 with description of three subgenera and Eclipidrilus (Leptodrilus) fontanus n. subgen. n. sp. from Pennsylvania. Proceedings of the Biological Society of Washington 97:78 – 85. Wiederholm, T., Wiederholm, A. -M., Milbrink, G. 1987. Bulk sediment bioassays with ﬁve species of fresh – water oligochaetes. Water, Air and Soil Pollution 36:131 – 154. Woodhead, A. E. 1945. The life-history cycle of Dioctophyma renale, the giant kidney worm of man and many other mammals. Journal of Parasitology, supplement 31:12. Zoran, M. J., Drewes, C. D. 1987. Rapid escape reﬂexes in aquatic oligochaetes: variations in design and function of evolutionary conserved giant ﬁber systems. Journal of Comparative Physiology (A) 161:729 – 738. ADDITIONAL SUGGESTED READINGS In addition to the literature actually cited in the text, the following publications will provide more speciﬁc information on taxonomy (Brinkhurst and Wetzel, 1984) and data derived from a series of international conferences on aquatic oligochaete biology. Brinkhurst, R. O., Cook, D. G., Eds. 1980. Aquatic Oligochaete biology. Plenum Press, New York. Brinkhurst, R. O., Wetzel, M. J. 1984. Aquatic Oligochaeta of the World: Supplement. A Catalogue of New Freshwater Species, Descriptions, and Revisions. Canadian Technical Report of Hydrography and Ocean Sciences 44:i-v 101 pp. Bonomi, G., Erseus, C., Eds. 1984. Aquatic Oligochaeta. Developments in hydrobiology, No. 24. Junk, Dordrecht, Netherlands. Brinkhurst, R. O., Diaz, R. J., Eds. 1987. Aquatic Oligochaeta. Developments in hydrobiology, No. 40. Junk, Dordrecht, Netherlands. Coates, K. A., Reynoldson, T. B., Reynoldson, T. B., Eds. 1996. Aquatic oligochaete biology. VI. Developments in hydrobiology, No. 115. Junk, Dordrecht, Netherlands. Kaster, J. L. (Ed.). 1989. Aquatic Oligochaeta. Developments in Hydrobiology, No. 51. Junk, Dordrecht, Netherlands. Healy, B., Reynoldson, T. B., Coates, K. A., Eds. 1999. Aquatic oligochaete biology. VII. Developments in hydrobiology. Junk, Dordrecht, Netherlands (in press.). Reynoldson, T. B., Coates, K. A., Eds. 1994. Aquatic oligochaete biology. VI. Developments in hydrobiology, No. 95. Junk, Dordrecht, Netherlands. 13 ANNELIDA: EUHIRUDINEA AND ACANTHOBDELLIDAE Ronald W. Davies* Fredric R. Govedich Faculty of Science Monash University Clayton, Victoria 3168 Australia Department of Biological Sciences Monash University Clayton, Victoria 3168 Australia I. Introduction to Euhirudinea and Acanthobdellidae II. Taxonomic Status and Characteristics III. Euhirudinea and Acanthobdellidae A. General Anatomy B. Physiology IV. Ecology A. Diversity B. Foraging Relationships C. Reproduction and Life History D. Dispersal E. Predation F. Behavior G. Population Regulation H. Ecotoxicology I. INTRODUCTION TO EUHIRUDINOIDEA AND ACANTHOBDELLIDA Leeches form an important component of the benthos of most lakes and ponds, and some species commonly occur in the quieter ﬂowing sections of streams and rivers. There are a total of 73 species presently recorded from North America, of which the majority are predators feeding on chironomids, oligochaetes, amphipods, and molluscs. The young of several species feed on zooplankton, but Motobdella montezuma is unique in specializing on planktonic amphipods using mechanoreception to detect its prey. Other species of leeches are temporary sanguivorous (blood feeding) * Present address: Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada T2N 1N4. Ecology and Classiﬁcation of North American Freshwater Invertebrates. 2nd Edition Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved. V. Collection and Rearing A. Collection B. Rearing VI. Identiﬁcation A. Preparation of Specimens B. Important Features for Identiﬁcation C. Family Glossiphoniidae D. Family Piscicolidae E. Family Hirudinidae F. Family Erpobdellidae VII. Taxonomic Keys A. Euhirudinea and Acanthobdella B. Species of Euhirudinea Literature Cited ectoparasites of ﬁsh, turtles, amphibians, crocodilians, water birds, or occasionally humans. The majority of predaceous leeches have an annual or biannual life cycle, breeding once and then dying (semelparity). However, at least two species have been shown to be genetically capable of breeding several times (iteroparity) although exhibiting phenotypic semelparity in the ﬁeld. The majority of sanguivorous leeches are iteroparous showing saltatory growth after the three or more blood meals required to reach mature size. Because of the diversity of physico-chemical conditions encountered in different aquatic ecosystems and temporally within a single habitat, leeches show considerable physiological plasticity. They are able to live in waters with very low salt concentrations as well as waters with salinities that exceed the salinity of seawater. Similarly, some species are capable of surviving in 465 466 Ronald W. Davies and Fredric R. Govedich the absence of oxygen (anoxia) for more than 60 days and in the presence of supersaturated water (hyperoxia) for shorter periods. In many small ponds and lakes, leeches are the top predators and are ideal for studying intra-and interspeciﬁc resource competition, niche overlap, and predator – prey interactions. In larger lakes and rivers, leeches may form an important component of the diet of ﬁsh; hence, they are grown commercially for ﬁsh bait. Sanguivorous leeches are being investigated extensively in relation to the pharmacological properties of their salivary secretions, especially their anticoagulants. The leech-like Acanthobdella peledina, a temporary ectoparasite of salmonids, is the only species of Acanthobdellidae in North America. Its ecology and feeding have not been extensively studied, but are likely to be similar to the Piscicolidae — a family of leeches also ectoparasitic on ﬁsh. II. TAXONOMIC STATUS AND CHARACTERISTICS There is an undisputed close taxonomic afﬁnity between leeches and oligochaetes, although there are differences of opinion about the exact nature of the relationship. This has resulted in leeches being variously classiﬁed as Hirudinea, Hirudinoidea, or Euhirudinea. Purschke et al. (1993) showed that not only did the Branchiobdellae (see Chapter 12), Acanthobdellidae, and Euhirudinea resemble each other, but they were also closely related. Their evolutionary relationship remains unanswered, but the present consensus is to consider them all superorders of the subclass Clitellata. The Clitellata are thought to have evolved in the interstitial environment of lagoons near the tributary outlets and entrance to channels opening into the sea in the Palaeozoic, 500 million years ago. The ﬁrst documented fossil of a leech dates back 150 million years to the Jurassic. However, a large annulate with a circular structure similar to a leech sucker discovered at Waukesha, Wisconsin (Briggs, 1991), conﬁrmed as a leech, will extend their history back 400 million years to the Silurian. Cocoons of extant leeches have been recorded from Holocene sediments but Manum et al. (1991) suggest that ovoid fossils 1.0 – 10.0 mm long with a netted or a feltlike wall fabric from the Lower Cretaceous (150 million years) were very probably leech cocoons. Only a few families of microdrilid oligochaetes can be considered as possible close relatives of the leeches. The naiids are the best candidates as most live in aerated freshwater, which seems to be the original environment of leeches, many of them are predators, they have a reduced and sometimes ﬁxed number of FIGURE 1 Dorsal view of the general body shape of a member of each of the families: (a) Glossiphoniidae; (b) Piscicolidae; (c) Hirudinidae; and (e) Erpobdellidae. segments, they have eyes of very similar structure to those of leeches and at least one genus (Chaetogaster) has epizoic species which move by looping and which like leeches lack a prostomium (Omodeo, 1998). The diagnostic features of leeches are: (1) 32 postoral metameres (Fernandez, 1980, Weisblat et al., 1980) plus a nonsegmental prostomium; (2) a reduced or obliterated coelom; (3) absence in the adult of chaetae and septa; (4) the presence of an anterior (oral) sucker and a usually larger posterior (anal) sucker (Fig. 1); (5) superﬁcial subdivision of the metameres into annuli (Figs. 2 and 29a, b); and (6) a median, unpaired, male genital pore placed anterior to the single median ventral, female genital pore (Figs. 3 and 4a). The characteristics delimiting the Euhirudinea are primarily related to their feeding and feeding behavior indicative that leeches are oligochaetes which at ﬁrst become predators and then, subsequently, on at least two separate occasions some became sanguivorous (Siddall and Burreson, 1995). The phylogenetic position of the Acanthobdellidae has been debated. Because they share some morphological, behavioral, and ecological characteristics more typically attributed to leeches some researchers have included them within the same taxonomic group as the FIGURE 2 Annulation showing (a) leech 3-annulate condition and (b) Acanthobdella peledina 4-annulate condition. 13. Annelida: Euhirudinea and Acanthobdellidae FIGURE 3 Ventral view of the anterior male and posterior female gonopores separated by (a) four annuli and (b) two annuli. leeches (Mann, 1961, Elliott and Mann, 1979; Klemm, 1982, 1985; Sawyer, 1972, 1986). However, recent research shows that the Acanthobdellidae, along with the Branchiobdellidae, are sister groups to the leeches sharing a common ancestor (Siddall and Burreson, 1995, 1996). Acanthobdellidae are temporary ectoparasites of salmonid ﬁshes but, unlike leeches, with ﬁve anterior coelomic compartments separated by distinct septa, each anterior segments bearing two pairs of chaetae (setae) (Fig. 4b). The Acanthobdellidae do not FIGURE 4 Acanthobdella peledina, ventral view of (a) the male and female gonopores with a spermatheca and (b) the anterior view showing the absence of an anterior sucker, the presence of a mouth pore, and ﬁve pairs of hooked chaetae. 467 have an anterior sucker, but a small posterior sucker is formed from four of the 30 segments forming the body. Each segment is superﬁcially subdivided into four annuli (Fig. 2b). Classically, oligochaetes and leeches are placed within the phylum Annelida either in the order Hirudinea, class Clitellata, or in the class Euhirudinea. Soös (1965, 1966a, b, c, 1967, 1968, 1969a, b) placed leeches in the taxonomically undeﬁned Hirudinoidea, but subsequently both Soös (1969b) and Richardson (1969) referred them to the class Hirudinoidea. Correctly, Klemm (1985) and Sawyer (1986) indicated that in accordance with the Zoological Code the sufﬁx “-oidea” should only be added to the stem name of a superfamily and recommended the suppression of the class Hirudinoidea. Davies (1971, and many subsequent references) classiﬁed leeches as Hirudinoidea implicitly as a superfamily of the class Oligochaeta. Until recently, these differences were primarily academic because everyone agreed that as Hirudinea, Euhirudinea or Hirudinoidea, leeches were in the phylum Annelida and closely related to oligochaetes. However, Sawyer (1986) deﬁned the class Hirudinea in a signiﬁcantly different way, with Euhirudinea, Acanthobdellidae, Branchiobdellidae, and Agriodrilidae as subclasses within the phylum Uniramia. Thus, the class Euhirudinea (sensu Sawyer, 1986) required the removal of this class from the phylum Annelida, and its placement into a phylum with the Onychophora, Myriapoda, and Hexapoda. The evidence to support this is scant (Sawyer, 1984, 1986) and contentious (Davies, 1987), and the majority of authors did not follow this recommendation and retained the leeches as Euhirudinea, Hirudinea or Hirudinoidea as a class of Annelida, a view supported by Zrzavy et al. (1998). The term Euhirudinea used in this chapter refers to a class of Annelida containing the leeches and not the Uniramian class deﬁned by Sawyer (1986). Sawyer (1986) combined many of the families and genera described and accepted by previous researchers, including those described in detail by Richardson (1969, 1971, 1975). In addition, he placed the North American members of the genus Dina into Erpobdella leading to further confusion about the relationships within the Erpobdellidae and, particularly, between the genus Erpobdella and Mooreobdella. These changes were not justiﬁed scientiﬁcally nor were complete descriptions of the modiﬁed families and genera given. This has resulted in taxonomic confusion. Leech families and genera described prior to Sawyer (1986) have been retained in this chapter and until a formal revision, including a re-analysis of the higher level relationships of leeches, has been published it is recommended that these names be retained. 468 Ronald W. Davies and Fredric R. Govedich III. EUHIRUDINEA AND ACANTHOBDELLIDAE A. General Anatomy The body of a leech consists of two preoral, nonmetameric segments and 32 postoral metameres designated I through XXXIV, while the Acanthobdellidae have only 29 postoral metameres (designated I through XXIX). Each segment is usually divided externally by superﬁcial furrows into 2 – 16 annuli (Figs. 2 and 29). Segments with the full number of annuli (complete segments) are found in the middle of the body; because this number is generally characteristic of the genus or species, it is referred to in the keys. Annuli features can be most easily seen in the lateral margins of the ventral surface. Moore (1898) numbered the three primary annuli a1, a2, and a3 from the anterior, with annulus a2 (neural annulus) containing the nerve cord ganglion. The neural annulus (a2) is marked externally by transverse rows of cutaneous sensillae (Figs. 2 and 29). Repeated bisection of the primary annuli gives more complex annulation. The number and position of the eyes are also important diagnostic features. In some genera, coalescence of eyes sometimes occurs, but the lobed nature of the eyes usually indicates the original condition (Fig. 5b). Eyespots (oculiform spots) can also occur on the lateral margins of the body and on the posterior sucker (Figs. 5c, 6b, and 19 b, c, d). FIGURE 5 Dorsal view of the head of Batracobdella with a single pair of eyes (a) close together; (b) lobed; and (c) Placobdella hollensis with one pair of eyes followed by pairs of accessory eyes. FIGURE 6 (a) Cystobranchus verrilli; and (b) Cystobranchus meyeri showing the pulsatile vesicles on the urosome and the presence on C. meyeri of oculiform spots on the posterior sucker and the paired lateral oculiform spots on the urosome. Papillae, small protrusible sense organs, are often widely scattered over the dorsal surface. Tubercles, which are larger protrusions that include some of the dermal tissues and muscles, may also be present and can bear papillae. The body of most leeches and acanthobdellids is not divisible into regions. With the genera Illinobdella and Piscicolaria as exceptions, the body of the family Piscicolidae is divided into a narrow anterior trachelosome and a longer and wider posterior urosome (Figs. 1b and 6). Lateral gills are absent, but some genera of Piscicolidae have paired pulsatile vesicles on the neural annuli of the urosome (Figs. 1b and 6a, b). The anterior sucker of leeches may be very prominent (Figs. 1b, 6, 7b) or simple (Figs. 1a, c, d, 7a, c), consisting only of the expanded lips of the mouth. In the acanthobdellids, anterior suckers are entirely absent (Fig. 4b). The posterior sucker of a leech is generally directed ventrally and is wider than the body at the point of attachment to the substrate (Figs. 1, 6, 19, and 22 – 24). The mouth is either a small pore on the edge (Fig. 7a) or center (Fig. 7b) of the ventral surface of the anterior sucker, or is large and occupies the entire cavity (Fig. 7c) of the anterior sucker. In the family Hirudinidae, the buccal cavity (which may or may not 13. Annelida: Euhirudinea and Acanthobdellidae 469 FIGURE 9 Surface view of the jaws of Hirudinidae showing the teeth arranged in: (a) one (monostichodont) or (b) two (distichodont) rows. FIGURE 7 Ventral view of the anterior sucker of: (a) the mouth pore on the anterior rim of the sucker (e.g., Marvinmeyeria, Placobdella, Oligobdella); (b) near the center of the sucker (Piscicolidae, Batracobdella, Helobdella); and (c) the mouth occupying the entire cavity (e.g., Hirudinidae, Erpobdellidae). FIGURE 8 Ventral view of the dissection of the mouth and buccal cavity of: (a) Mollibdella grandis; (b) Percymoorensis marmorata; and (c) Macrobdella decora showing the velum, and the relative size of the jaws in Macrobdella decora and P. marmorata and the absence of jaws in Mollibdella grandis. contain jaws) is separated from the cavity of the anterior sucker by a ﬂap of skin (the velum) (Fig. 8). When present, there are three muscular jaws (two ventrolateral and one dorso-medial) (Fig. 8b, c) bearing teeth arranged in one (monostichodont) (Fig. 9a) or two (distichodont) rows (Fig. 9b). In the Erpobdellidae, the buccal cavity contains three muscular ridges, but in the Glossiphoniidae and Piscicolidae, the pharynx is modiﬁed to form an eversible muscular proboscis. Opening into the pharynx are the salivary glands which secrete some digestive enzymes. In bloodsucking leeches the salivary glands are primarily related to the process of bloodsucking rather than digestion. The components of the salivary secretions and their functions can be summarized as lubrication, proteolytic inhibition, spreading (hyaluronidase), vasodilation, and anticoagulation (e.g., hirudin or hementin) (Sawyer, 1986). The anticoagulants produced by North American sanguivores have not been investigated extensively with work so far concentrating on the European Hirudo medicinalis and the South American Haementaria ghilianii. Seymour et al. (1990) and Krezel et al. (1994) reported the presence and structure of decorsin, a potent inhibitor of platelet aggregation from Macrobdella decora. The pharynx in most erpobdellid leeches leads to a simple tubelike crop that lacks lateral ceca. However, this is not the case in the erpobdellid genus Motobdella which has one or two pairs of simple ceca or postceca located in the posterior portions of the crop (Davies et al. 1985; Govedich et al. 1998). The crop in most leech families is adapted for the storage of ﬂuids and usually has 1 – 11 pairs of lateral ceca. Between the last pair of crop ceca, the intestine leads to the anus (Fig. 10a). Only in the Glossiphoniidae does the anterior portion of the intestine give off four pairs of simple ceca (Fig. 10b). The anus usually opens on the dorsal surface on or near segment XXVII, just anterior to the posterior sucker. In a few species, the anus is displaced anteriorly. Motobdella has one or two pairs of post ceca (Fig. 10c). Digestion and absorption mainly occur in the intestine although the crop is also used in predatory species. 470 Ronald W. Davies and Fredric R. Govedich FIGURE 10 Stylized ventral view of the alimentary canal of (a) Hirudinidae; (b) Glossiphoniidae; (c) Motobdella; and (d) Acanthobdella peledina. In sanguivores, digestion is primarily by enzymes produced by endosymbiotic bacteria. The bacteria involved are species speciﬁc for each species of sanguivorous leech (Jennings and Vanderlande, 1967), e.g. Hirudo medicinalis utilizes Aeromonas hydrophila FIGURE 11 (Pseudomonas hirudinis) (Büsing, 1951, Büsing et al., 1953, Jennings and Vanderlande, 1967). Endopeptidases are absent or rare in leeches, but exopeptidases occur abundantly in predatory species and protease (Zebe et al., 1986) and protease inhibitors have been recorded (Roters and Zebe, 1992a, b). Because of their dependence on endosymbiotic bacteria, digestion in sanguivorous leeches takes weeks or months; but for predatory species, digestion requires just a few days (Davies et al., 1977, 1978, 1981, 1982a; Wrona et al., 1979, 1981). The alimentary canal of acanthobdellids (Fig. 10c) is similar to that of the glossiphoniid leeches. A small eversible proboscis leads into the muscular pharynx, which, in turn, leads into a large tubular crop lacking ceca. The crop leads into the intestine, which bears six pairs of lateral ceca; the anus is located dorsally between segments XXV and XXVI. Digestion is probably similar to sanguivorous leeches with the use of endosymbiotic bacteria. The male and female gonopores are visible in mature leeches on the midline of the ventral surface of segments XI and XII, respectively, and are separated by a species-speciﬁc number of annuli (Figs. 3, 4, and 27). The anterior male gonopore is larger and more easily seen than the female gonopore. Leeches do not have true testes or ovaries, but instead possess testisacs and ovisacs. The testisacs are located posterior to segment XI and are either discrete, paired spherical structures (Fig. 11a) (e.g., Hirudinidae, Glossiphoniidae, and Reproductive systems of: (a) Hirudo medicinalis (Hirudinidae); and (b) Nephelopsis obscura (Erpobdellidae). 13. Annelida: Euhirudinea and Acanthobdellidae Piscicolidae) located intersegmentally or multifollicular columns resembling bunches of grapes lying on either side of the ventral nerve cord (Fig. 11b) (e.g., Erpobdellidae; Singhal and Davies, 1985; Davies et al., 1985, Govedich et al., 1998). Short vasa efferentia connect the testisacs to the vasa deferentia on each side of the body, which run anteriorly to form large coiled epididymis (sperm vesicles). Paired ejaculatory ducts run from the epididymis through the atrial cornua and unite to form a medial atrium consisting of a bulb and eversible penis. There is a single pair of ovisacs which are either small, spherical organs conﬁned to segment XII or elongate, coiled tubes sometimes bent back on themselves such that their blind ends lie close to the female gonopore (Singhal and Davies, 1985; Davies et al., 1985). Two oviducts run from the ovisacs to converge and form the common oviduct leading to the female gonopore (Fig. 11). Leeches are frequently recorded as sequential protandrous hermaphrodites (Van Damme, 1974; Lasserre, 1975). Nephelopsis obscura shows sequential protandry during its ﬁrst bout of gametogenesis, although there is a short transition period when gametogenic stages from both sexes co-occur. In the second cycle of gametogenesis, mature sperm and ova occur simultaneously (Davies and Singhal, 1988) and thus this cycle demonstrates simultaneous hermaphroditism. At the time of egg laying, a cocoon is secreted by the clitellum, a specialized region of epidermis usually posterior to the gonopores. As the cocoon moves toward 471 and passes over the head, fertilized eggs pass into the cocoon from the female gonopore. The reproductive system of Acanthobdellida peledina is basically similar with a pair of elongate testisacs and ovisacs and median unpaired male and female gonopores. The excretory system of leeches and acanthobdellids consists of a maximum of 17 pairs of highly modiﬁed metanephridia opening along the body between segments VII and XXII. Many species do not exhibit the full number of metanephridia, showing reductions in the more anterior metameres and / or the segments containing gonads. In addition to excreting nitrogenous wastes (mainly ammonia), the nephridia help maintain water and salt balance in the body. With the osmotic pressure of the body higher than the surrounding freshwater, there is a tendency for an inward ﬂow of water through the epidermis. The nephridia pass out excess water and help retain inorganic ions. Only about 6% of the salts found in the primary urine are excreted, the rest being reabsorbed (Zerbst-Boroffka, 1975). During acclimation to hypersaline media, several different active and passive processes are responsible for the changes of osmolarity and electrolytic concentrations in the blood of the leech (Nieczaj and Zerbst-Boroffka, 1993). In the leeches and acanthobdellids, the large coelom, which in Annelida is typically divided by intersegmental septa, has to varying degrees been obliterated (Fig. 12). In the Glossiphoniidae (Fig. 12a), Piscicolidae (Fig. 12d), and acanthobdellids, the blood FIGURE 12 Stylized transverse sections of: (a) Glossiphoniidae; (b) Hirudinidae; (c) Erpobdellidae; and (d) Piscicolidae showing the coelomic sinuses, dorsal and ventral blood vessels, and the double ventral nerve cord. 472 Ronald W. Davies and Fredric R. Govedich vascular system consists of: (1) a ventral vessel enclosed with the nerve cord in the ventral lacuna; (2) a dorsal blood vessel enclosed in the dorsal lacuna; and (3) transverse blood vessels connecting the dorsal and ventral vessels in the anterior and posterior parts of the body. In the Hirudinidae (Fig. 12b) and Erpobdellidae (Fig. 12c), the true blood vascular system is completely lost; instead, the blood circulates in the coelomic lacuna system, forming a hemocoel. Oxygen uptake in freshwater leeches and acanthobdellids is through the general body surface, although some species of Piscicolidae have small pulsatile vesicles ﬁlled with coelomic ﬂuid that function as accessory respiratory organs. Most leeches can also ventilate their body surfaces by dorsoventral undulations along the body reminiscent of swimming, but with the posterior sucker ﬁrmly attached. However, Acanthobdella peledina does not ventilate (Dahm, 1962). All leeches and acanthobdellids show typical looping; locomotory movements consisting of body elongation and shortening, with the anterior sucker (leeches only) and posterior sucker serving alternately as points of attachment. Many leech species are also able to swim using dorsoventral undulations. All Erpobdellidae and Hirudinidae are good swimmers, but only a few species of Glossiphoniidae and Piscicolidae are efﬁcient swimmers. Some species (Placobdella hollensis) swim readily as juveniles and adults, but others (Placobdella ornata and Placobdella parasitica) swim only as juveniles (Sawyer, 1981). The erpobdellid genus Motobdella is unique in its swimming ability and specializes in feeding almost exclusively on pelagic amphipods by using mechanoreception to identify and track its prey in the water column (Blinn and Davies, 1990; Blinn et al., 1987, 1988, 1990; Davies et al., 1985). B. Physiology Perhaps the greatest variability in the abiotic environment experienced by leeches occurs in reference to temperature and dissolved oxygen concentrations In stratiﬁed lakes, the epilimnion is generally at or above saturation during the summer, while in the hypolimnion oxygen depletion occurs. Under ice, especially at the substrate – water interface, hypoxia and possibly anoxia occur (Babin and Prepas, 1985; Baird et al., 1987a). Thus, hypoxia or anoxia can occur on a diel basis during the open-water season and on a longterm basis during ice cover. As spring approaches, the water temperature rises above 5°C, snow cover melts and under-ice primary productivity can result in shortterm (1 – 2 days) hyperoxia. Similarly during the summer, long-term hyperoxia can persist for several days. Both anoxia and hyperoxia can cause mortality of vertebrates and invertebrates (Casselman and Harvey, 1975). In Europe, Mann (1956) showed that oxygen uptake by some leech species was proportional to the oxygen concentration of the water (i.e., they were conformers), while other species (Glossiphonia complanata, Helobdella stagnalis) showed a comparatively constant oxygen uptake over a range of dissolved oxygen concentrations of the water (i.e., they were regulators). Nephelopsis obscura and Erpobdella punctata exposed to short-term hypoxia both showed a decline in oxygen uptake with reduced oxygen tension in the water (Wrona and Davies, 1984). When exposed to hypoxic conditions, most leeches, but not Acanthobdella peledina, show ventilation movements with the posterior sucker attached to the substrate and the body thrown into dorsoventral ﬂexions (Dahm, 1962). It is assumed that the primary function of ventilation is respiratory since ventilation increases under conditions of low oxygen availability. While N. obscura and E. punctata ventilate in hypoxic conditions, ventilations also occur under saturated (normoxic) conditions and always cease when oxygen decreases below 20% saturation. This suggests ventilation may have additional roles. Many leeches can withstand anaerobic conditions (Von Brand, 1944), but little research has been done on the ecological effect of anoxia (or hypoxia) on the leeches, with the majority of work related to use of leeches as indicator species in the European saprobic system (reviewed in Sladacek and Kosel, 1984). In ﬂow-through experiments, Davies et al. (1987) showed inter- and intraspeciﬁc differences in the survivorship of N. obscura and E. punctata to anoxic conditions and demonstrated the errors of using static experiments for this type of study. For both species, survivorship decreased with increased water temperature. Large N. obscura had longer survival times ( 50 days) than small individuals (12 days) at 5°C and also at 20°C (8 and 4 days, respectively). Conversely, at 5°C large E. punctata had shorter survival times (25 days) than small individuals ( 50 days), but at 20°C, large individuals survived longer (8 days) than small individuals (2 days). As young (small) N. obscura have a low probability of surviving anoxia at winter temperatures (5°C), it is evident that large N. obscura would be taking less risk if they overwintered rather than hazarding cocoon production and hatching of young up to or close to ice formation. In contrast, large E. punctata, which have a low probability of surviving winter anoxia, would be taking less risk to continue cocoon production later in the season since their offspring can survive winter anoxic conditions. These results correlate well with the patterns of reproduction observed in the ﬁeld (Davies, 1978; Davies et al., 1977). 13. Annelida: Euhirudinea and Acanthobdellidae The metabolic and physiological adaptations of N. obscura to long-term anoxia have been studied under laboratory conditions mimicking winter conditions (Reddy and Davies, 1993a, b; Dratnal et al., 1993). Lactic acid did not accumulate in either large or small individuals over a 40-day period of anoxia; however, succinate and alanine did. Both glycogen and amino acids were utilized as energy reserves during the anoxic period along with stored lipids. Following the 40-day period of anoxia, glycolytic ﬂux and adenosine triphosphate diphosphate (ATP / ADP) were signiﬁcantly reduced in both small and large individuals. Upon a return to normoxia, preanoxic levels of glycogen, malate, succinate, alanine, and aspartate are restored within 24 hs. In addition, the preanoxic energy charge is restored within the ﬁrst 6 hs of normoxia. All small and 98% of large individuals survived the 40-day anoxic period without signiﬁcant losses in body weight; however, activity was reduced when compared to N. obscura not exposed to anoxic conditions (Reddy and Davies, 1993a, b). Physiological adaptations to the effects of severe hypoxia are similar to those observed for anoxic conditions and in N. obscura include decreased growth, enhanced energy allocation of glycogen, triclyglycerols and total lipids. Body weight and size at maturity were not affected by hypoxia; however, the time to maturity was longer for N. obscura under hypoxic conditions (Dratnal et al., 1993). Even less work has been done on the effect of hyperoxia on leeches. Survival of N. obscura and E. punctata exposed to hyperoxia (200, 300%) increased with leech size and decreased with increased temperature. The survivorship time for all size classes of N. obscura and E. punctata greatly exceeded the maximum recorded duration (1 – 2 days) of hyperoxia in the ﬁeld during the spring. However, during the summer (20°C), if hyperoxiain the 200 – 300% range persisted for more than 20 days, medium and large N. obscura would show higher mortality than comparable sizes of E. punctata (Davies and Gates 1991a, b). With very low or undetectable levels of superoxide dismutase in N. obscura, Singhal and Davies (1987) concluded that N. obscura had two possible defenses against hyperoxia. These were to move away from areas of hyperoxia or, as recorded by Singhal and Davies (1987), to secrete large amounts of mucus over the epidermis, which reduces oxygen diffusion into the body and thereby the effects of oxygen toxicity. Davies and Everett (1977) and Davies et al. (1977) recorded intraand interspeciﬁc differences in the timing of seasonal movements between lentic microhabitats of N. obscura and E. punctata. The results from these investigations on hyperoxia show that small leeches are highly intolerant of hyperoxia, especially at higher temperatures, 473 and suggest that their seasonally earlier movement to deeper water is related to avoidance of potentially lethal oxygen conditions. Similarly, the presence of only large leeches in the macrophyte zone, where hyperoxia is more common and more persistent, is compatible with the greater tolerance of the large size classes to hyperoxic conditions. During winter, leeches frequently have to rely on energy stored during periods favorable to energy acquisition although energetic demands can be mitigated by decreased activity. During the winter N. obscura exhibits degrowth (Dratnal and Davies, 1990) and posthatchlingsized individuals that have over wintered are different, from similarly sized individuals hatched in the summer with respect to ecophysiological process, life-history and ﬁtness characteristics (Dratnal and Davies, 1990). The cryoprotective glycerol concentration in winter leeches are signiﬁcantly higher (Reddy and Davies, 1993b), but they have had only half the total lipid concentration energy reserves of similarly sized individuals hatched in the summer. Under optimal feeding conditions, lipid depletion at maturity is fully compensated for in over wintered leeches through increased food ingestion, higher absorption efﬁciency, and the allocation of more energy to lipid accumulation than other growth functions (Reddy et al., 1992). At, or just prior to sexual maturity, both winter and summer leeches show decreases in total protein, glycogen, and total lipid as a result of the increased energetic demand of gametogenesis and maturation. Ultrastructural investigations of the neurons of N. obscura exposed to anoxia and hyperoxia (Singhal et al., 1988) showed a marked reduction in numbers of mitochondria, ribosomes, and neurotransmitter vesicles with swollen and fragmented cristae. These changes are more severe in anoxia than hyperoxia. In most lakes and rivers inhabited by leeches, ionic content and total dissolved salts (TDS) in the water show little temporal variability. However, signiﬁcant geographic variability occurs between lakes and rivers primarily related to the edaphic features of the underlying geological formations. It has been shown that the distribution of leeches is, at least in part, related to TDS of the habitat waters (Mann, 1968; Herrmann, 1970a, b; Scudder and Reynoldson and Davies, 1976). Similarly, in Europe, Mann (1955) showed that the composition of the leech fauna in lentic habitats was a function of water hardness. It has been demonstrated that the relative concentrations of ions in the medium modify the mortality of aquatic animals (Beadle, 1939; Croghan, 1958; Linton et al., 1983a, b). The blood of leeches is relatively concentrated compared to oligochaetes and is hyperosmotic to the water in which they live. Nephelopsis obscura and Helobdella stagnalis were able to maintain themselves 474 Ronald W. Davies and Fredric R. Govedich hyperosmotically in a medium between 15.6 and 59.5 mOsm / L and 47 and 112.7 mOsm / L, respectively, but were conformers at higher medium concentrations. Theromyzon trizonare was a regulator in both hypoand hyperosmotic media. Leeches are also capable of regulating body volume when exposed to varying salinities (Rosca, 1950; Madanmohanrao, 1960; Smiley and Sawyer, 1976; Reynoldson and Davies, 1980; Linton et al. 1982). Nephelopsis obscura was able to maintain its weight in medium from 100 – 207 mosmol / L while E. punctata could only regulate between 58 and 200 mOsm / L. Weight regulation and volume regulation are probably not regulated per se, although these variables change in a predictable fashion with change in the osmotic concentration of the environment. It is more likely that leeches regulate the osmotic pressure of the body ﬂuids; hence, with inﬂuxes and efﬂuxes of water and salts, weight and volume change. Using a three-way factorial experimental design, the effect of water temperature, ionic content, and TDS on the mortality and reproduction of N. obscura and E. punctata were examined (Linton et al., 1983a, b). A strong interaction among temperature, ionic content, and TDS was observed on the mortality of E. punctata, but N. obscura showed uniformly low mortality unaffected by temperature, ionic content, or TDS. For both species, temperature showed the greatest inﬂuence on cocoon production and ionic content showed the least. Cocoon production by E. punctata was reduced in waters with low TDS and the proportion of nonviable cocoons was higher in low TDS than in high TDS. IV. ECOLOGY A. Diversity Leeches are represented in North America by four families. Glossiphoniidae are either predators of macroinvertebrates or temporary ectoparasites of freshwater ﬁsh, turtles, amphibians, or water birds; Piscicolidae are parasites of ﬁshes as well as some Crustacea; Erpobdellidae are primarily predators of macroinvertebrates and zooplankton; and, Hirudinidae are either predators of macroinvertebrates (e.g., oligochaetes, snails) or bloodsucking ectoparasites of freshwater and terrestrial Amphibia and mammals. Sanguivorous (bloodsucking) leeches spend a relatively short period of time taking a blood meal on the host and are more frequently found free living in the benthos. For this reason, host speciﬁcity is not often a very useful feature for identiﬁcation of leeches. While the majority of leeches parasitic on ﬁsh belong to the family Piscicolidae, two species of Glossi- phoniidae (Actinobdella inequiannulata and Placobdella pediculata) show a strong preference for ﬁsh hosts. Placobdella pediculata recorded mainly from the midwestern United States has a high degree of host speciﬁcity for the freshwater drum Apolodinotus grunniens (Bur, 1994). However, other species of glossiphoniids (e.g., P. montifera and Desserobdella phalera) are also occasionally found on ﬁshes. Members of the Erpobdellidae and Hirudinidae have been recorded on ﬁshes, but it is doubtful that any species in these families regularly feeds on these hosts. All leeches require a ﬁrm substrate for attachment and are capable of feeding on body ﬂuids. If a healthy or injured ﬁsh can provide either of these requirements, freshwater leeches will attach themselves. Parasitic leeches generally attach periodically to the ﬁshes, take a blood meal, and then move back into the benthos. Acanthobdellids feed similarly on coldwater ﬁshes (Bur, 1994). B. Foraging Relationships Leeches are either predators, consuming macroinvertebrates in the benthos and plankton or ectoparasitic sanguivores, feeding on vertebrates. The effects of predatory ﬁsh on benthic communities have been widely studied some showing weak or no effects whereas other studies have shown strong effects. Dahl and Greenberg (1997) compared prey consumption by brown trout (Salmo trutta) and the leech Erpobdella octoculata and showed that while one leech consumes much less than one trout, the role of leeches in structuring benthic communities is very similar. Leeches and acanthobdellids are primarily ﬂuid feeders; because, even though some predators ingest whole prey, they quickly evacuate the hard skeletal parts through the mouth or anus after extracting body ﬂuids. Sanguivory occurs in the Hirudinidae, which have three-toothed jaws used to bite and penetrate the host’s skin and in the Piscicolidae, and some Glossiphoniidae, which in contrast, lack jaws and penetrate the host with a proboscis. The acanthobdellid similarly has a short proboscislike specialization of the anterior foregut. Theromyzon are sanguivorous glossiphoniids feeding on waterfowl and require a minimum of three blood meals to reach maturity (Davies, 1984; Wilkialis and Davies, 1980b). In comparison, Placobdella papillifera and Haementaria ghilianii require a minimum of four (Davies and Wilkialis, 1982; Sawyer et al., 1981). The glossiphoniids generally appear to require signiﬁcantly fewer blood meals than Hirudo medicinalis, which takes 10 or more meals to reach maturity (Blair, 1927; Pütter 1907, 1908), although in the laboratory at 26°C, only 4 – 5 blood meals are required. For T. trizonare, 13. Annelida: Euhirudinea and Acanthobdellidae 475 FIGURE 13 Changes in mean weight (0 – 0) and the percentage of survival (0 – 0) for populations of Theromyzon rude exposed to different water temperatures and feeding regimes: (a) fed once, maintained at 20°C; (b) fed once, maintained at 5°C; (c) fed twice, maintained at 20°C; (d) fed twice, maintained at 5°C; (e) fed three times, maintained at 20°C; (f) immature (14.7 mg) collected from the ﬁeld, fed once, maintained at 20°C; and (g) immature (14.7 mg) collected from the ﬁeld, fed once, maintained at 5°C. over 80% of the population takes three meals in the ﬁrst 6 months after hatching (Fig. 13). The remainder of the population overwinters after two meals and take the third in the spring so that all the population reproduces approximately 12 months after hatching. Some individuals that overwinter after three meals decline in weight before spring and require a fourth meal before reproduction commences. When ingesting blood, pathogens such as syphilis, erypsipelas, tetanus, cholera, hepatitis B and human immune deﬁciency virus (HIV) enter the gut and can survive for long periods (Nehili et al., 1994). However, as these pathogens do not penetrate the unicellular leech salivary glands, direct transmission to another host is unlikely. Predatory leeches either suck ﬂuids with a proboscis (Glossiphoniidae) or have a suctorial mouth (Erpobdellidae and Hirudinidae). The range of prey species is normally quite diverse and changes seasonally with prey availability. In some instances, prey selection varies intraspeciﬁcally with body size and / or age class (Davies et al., 1978, 1981, 1988; Wrona et al., 1979, 1981). Because leeches either do not ingest identiﬁable hard parts or quickly evacuate them, the only accurate and efﬁcient method of examining feeding is with serologic techniques. Antisera against prey are produced (Davies, 1969) and then used for a serologic test of the gut contents. The feeding of Nephelopsis obscura and Erpobdella punctata was investigated using speciﬁc rabbit antisera against Cladocera / Copepoda, Chironomidae, Oligochaeta, Amphipoda, and Gastropoda. Apart from the absence of Gastropoda in the diet of E. punctata, there were no signiﬁcant differences at the species level in prey utilization, niche breadth, and evenness (Fig. 14). However, temporal differences in prey utilization were evident, and both intra- and interspeciﬁc resource 476 Ronald W. Davies and Fredric R. Govedich FIGURE 14 Frequency of prey utilization histograms (%) for Nephelopsis obscura and Erpobdella punctata. partitioning occurred as a function of different weight class utilization of prey (Davies et al., 1981; Martin et al., 1994c). Erpobdella punctata has also been recorded as a phoront (Khan and Frick, 1997) on salamanders, with the salamander providing the leech with food particles from its feeding. Similarly, temporal differences in feeding and intraspeciﬁc weight class differences in prey utilization were found in sympatric Glossiphonia complanata and Helobdella stagnalis (Wrona et al., 1981) (Fig. 15). The size of both predators significantly affected feeding success on prey (Martin et al., 1994a, b). In freshwater ecosystems, benthic species have a highly heterogeneous spatial and temporal distribution and invertebrate predators, including leeches, experience variable interfeeding intervals. Smith and Davies (1996a,b) measured the acquisition of energy and its allocation to components of bioenergetic balance throughout the life cycle in three groups of N. obscura fed at low, medium and high frequency. As feeding frequency increased, the total amounts of energy ingested, feces plus mucus produced, somatic and reproductive growth, energy storage (total lipid), and respiration all FIGURE 15 Frequency of prey utilization histograms (%) for Glossiphonia complanata and Helobdella stagnalis (C / C, Cladocera / Copepoda; C, Chironomidae; O, Oligochaeta; A, Amphipoda; G, Gastropoda). 13. Annelida: Euhirudinea and Acanthobdellidae increased. In the medium and high frequency feeding treatments, the relative proportion of growth energy allocated to storage was constant, i.e. the ratio of reserves to structural tissue remains the same. While most predatory leech species utilize a variety of prey, Motobdella montezuma is an exception. The pelagic endemic amphipod Hyalella montezuma comprises nearly 90% of the diet of the endemic M. montezuma in the thermally constant environment of Montezuma Well (Arizona), even though numerous other potential prey are abundant throughout the year (Blinn et al., 1987; Davies et al., 1988). This restricted diet was conﬁrmed by both gut content and serological analyses. The highly stable conditions in Montezuma Well have contributed to the very close relationship between the endemic predator and prey. Using a bioenergetic model McLoughlin et al. (1999) showed that the specialized foraging utilized by M. montezuma is more efﬁcient to the benthic opportunistic foraging strategy typical of most other erpobdellids. However, rather than being an obligate forager on H. montezuma, which is abundant during most of the year, Motobdella montezuma exploits other prey types during the winter, thereby achieving an overall higher rate of energy gain. C. Reproduction and Life History All leeches are hermaphrodites showing protandry or cosexuality (Davies and Singhal, 1988), with reciprocal cross-fertilization as the general rule. Fertilization, which is internal, is accomplished in the majority, of the Glossiphoniidae and all of the Piscicolidae and Erpobdellidae by attaching a spermatophore to the body of the partner. The spermatozoa penetrate the body wall and make their way to the ovisacs via the coelomic sinuses. The clitellar region is the most frequent site for the deposition of spermatophores, but they can be attached to any part of the body of the partner. In some species of Piscicolidae, there is a specialized region for the reception of spermatophores; fertilization is affected only by spermatophores deposited there. In the Hirudinidae, reciprocal internal fertilization is brought about by the insertion of an eversible penis into the vagina of the partner. Once fertilization occurs, the eggs are deposited into a cocoon secreted by the clitellum. It has been widely assumed that the presence of a visible clitellum closely parallels maturation of the female reproductive system, and in nonreproductive leeches the clitellar glands are barely distinguishable from the epithelial cells (Fernandez et al., 1992). The clitellum is especially prominent in Erpobdellidae and has been used by numerous authors to determine sexual maturity. Biernacka and Davies (1995) showed that sexual maturity 477 of Nephelopsis obscura cannot be judged by the presence or absence of an externally visible clitellum. Although a high proportion of the population exhibited a visible clitellum at some time, it was not persistent (lasting no longer than 7 days) and thus could easily be missed in studies with weekly or longer sampling periods. Feeding regime affects the timing of the clitellum development but, regardless of feeding regime, all the leeches exhibited two periods with a visible clitellum. The ﬁrst appearance of a clitellum did not coincide with either spermatogenic or oogenic maturity, but at the second appearance of a clitellum fully mature ova were present. The presence of a visible clitellum is thus not a good indicator of maturity in Nephelopsis obscura and probably not a good indicator in most or all species of Erpobdellidae. The presence of a spermatophore is also not a good indicator of maturity. Singhal et al. (1985) found only 5% of mature N. obscura collected from the ﬁeld had observable spermatophores, and Biernacka and Davies (1995) found that only 4% of the mature size range animals had a spermatophore attached. There can be a considerable delay between copulation and cocoon deposition, e.g. in ﬁeld populations of Helobdella stagnalis copulation occurs in the fall and cocoon deposition takes place in the spring (Davies and Reynoldson, 1976). Erpobdellid cocoons are thick walled, oval, and attached to a ﬁrm substrate (stone, leaf, stem, wood). The cocoons of Piscicolidae and Hirudinidae are only loosely attached to the substrate and are usually spherical. In species of Hirudinids depositing their cocoons in moist habitats out of water, the outer wall of the cocoon is spongy, which is thought to reduce water loss. The cocoons of Glossiphoniidae are very thin-walled (sometimes called egg cases) and are either deposited on the substrate and immediately covered by the body of the parent or are attached to the ventral surface of the parent. In both cases, the hatchlings are attached to the ventral body wall and are carried around by the parent for a long period, which for Theromyzon tessulatum can be 5 months and for T. trizonare, 1 month (Wilkialis and Davies, 1980a). Glossiphonia complanata adults have been found to provide their young with nutrients that are passed through the body wall of the parent into the posterior sucker of the young (De Eguileor et al., 1994). Reductions in body weight and size have been recorded for brooding glossiphoniids; however, these losses have been attributed to reduced feeding potentials and to the energy expended ventilating the young (Calow and Riley, 1982; Milne and Calow, 1990). It is possible that other species which brood attached young may provide nutrients to the developing young; however, further studies need to be conducted on a variety 478 Ronald W. Davies and Fredric R. Govedich of glossiphoniid leech species. A comparison on reproductive output for glossiphoniid that brood eggs and young, and for erpobdellids, that leave their eggs in an encapsulated cocoon, shows that brooding is not metabolically more expensive than encapsulation. There are no extra energy costs incurred in terms of carrying or ventilating broods, but weight loss due to reduction in feeding does occur. These costs appear to be energetically similar to the cost of encapsulation for erpobdellids, and it is suggested that brooding and encapsulation represent alternative evolutionary paths for brood protection. The life cycles of all leeches and acanthobdellids consist of egg, juvenile, and mature hermaphrodite adult, which reproduces and produces more eggs. Adults of predatory species usually reproduce only once before death (i.e., semelparity), although some sanguivorous species reproduce several times over a few years (i.e., iteroparity). Semelparity and iteroparity are generally considered genetically different reproductive strategies determined through evolutionary processes. Studies on the reproductive biology of leeches have shown considerable variation in life history with annual or biennial life cycles in predatory species. Some sanguivorous species (e.g., T. trizonare) live for several years with the duration of the life cycle dependent on the interval between blood meals (Davies, 1984). Baird et al. (1986, 1987b) showed that changes in water temperature, size at reproduction, energy loss during reproduction, and postreproductive feeding of the erpobdellid Nephelopsis obscura signiﬁcantly affected postreproductive mortality. Although genetically iteroparous, N. obscura is like most predatory leeches and some sanguivorous species in being almost always semelparous in the ﬁeld because of high postreproductive mortality. Thus, it is quite possible that the range of life histories reported for various species of leeches falls within the range of environmentally induced variability. Indeed, it is conceivable that all leeches are genetically iteroparous but phenotypically exhibit semelparity under most conditions. This ﬂexibility would ensure the long-term persistence of genotypes in a variable environment, where a more rigid strategy might have a low or zero ﬁtness in some years. Freshwater ecosystems in North America are highly variable in terms of both abiotic (experience oxygen concentration and temperature) and biotic (prey availability) environmental parameters, and Bulmer (1985) suggested that iteroparity is found in variable environments as a bet-hedging or risk-avoidance strategy. The life cycle and life history of comparatively few freshwater leech species in North America have been examined. In Alberta, two generations of N. obscura FIGURE 16 Life cycles of spring and summer hatching Nephelopsis obscura in Alberta, Canada. are produced annually, one in the spring and one in the late summer. The spring generation is the progeny of the heavier individuals from the spring generation of the previous year and from the late-summer generation produced two years previously. The late-summer generation is produced by portions of the previous year’s spring and late summer generations. Thus, each generation produces young after either 12 and 15 months or 12 and 19 months, although each individual reproduced only once (Davies and Everett, 1977) (Fig. 16). Reddy et al. (1992) showed that N. obscura which had overwintered differed physiologically, throughout their life history, from individuals which had not. The ﬁrst generation of N. obscura in early summer is the progeny of the heavier individuals from the early summer generation of the previous year and the second late summer generation is produced by portions of the previous year’s early and late summer generations. The late summer generation has a faster growth rate, higher lipid storage, higher proportion of energy available for growth and metabolism, and earlier valuation of larger maximum size than the early summer generation. However, there were no differences in consumption of prey or respiration between the two generations (Qian and Davies, 1994). As there are no signiﬁcant genetic differences between the early and late summer generations, the differences in physiological ecology and life history traits are attributed to differences in prehistory of the parents. In Minnesota, N. obscura delays its age of reproduction to two years and in so doing attains a larger 13. Annelida: Euhirudinea and Acanthobdellidae body size (Peterson, 1982, 1983). Erpobdella punctata is usually subdominant to N. obscura and reproduces after one year. When E. punctata is dominant, it has a more complex cycle with reproduction after one or two years, at a larger size, over a shorter season (Davies et al., 1977). Helobdella stagnalis can produce either one or two generations per year depending on the water temperature regime (Davies and Reynoldson, 1976). In warmer conditions, the overwintering population reproduces in the spring and dies on completion of brooding. The spring generation grows rapidly and a second generation of young is produced in the summer, which forms the overwintering population. In colder temperature regimes, members of the overwintering population reproduce in the late spring / early summer and die after brooding. After summer growth, this generation forms the next overwintering population. Davies (1978) showed that populations of H. stagnalis from cold water regimes are able to produce two generations per year in appropriate conditions. Several environmental factors have been implicated in affecting the reproduction of leeches, including food availability and water temperature. Water temperature regime can affect the relative reproductive success and growth of species (Davies and Reynoldson, 1976; Davies, 1978; Wrona et al., 1987), as well as depth distribution and seasonal movement (Gates and Davies, 1987). A bioenergetics simulation model of the growth and life history of N. obscura (Linton and Davies, 1987) showed that its growth is more sensitive to prey variation between years than to temperature variation. Davies and Singhal (1988) showed that, in the ﬁeld, N. obscura exhibits sequential protandry in the ﬁrst bout of gametogenesis and simultaneous hermaphroditism in the second. In the laboratory maintained under constant conditions, oogenesis initially follows spermatogenesis but mature ova ﬁrst appeared shortly after Stage 4 of spermatogenesis with the ﬁnal stages of spermatogenesis lagging behind oogenesis (sequential protogyny). However, for about 3 weeks mature spermatozoa and ova co-occur, i.e., simultaneous hermaphroditism occurs. This is quite different from what occurs in the ﬁeld. Clearly, N. obscura shows plasticity in the phenology of the development of hermaphroditism, the differences between the ﬁeld and laboratory resulting from the absence of an environmental cue. This was subsequently shown to be a period of low temperature (3 – 5°C) for a minimum of 7 days between stages 3 and 4 of spermatogenesis. Kalarani et al. (1995) demonstrated the presence of ecdysone in N. obscura and showed correlations between ecdysone concentrations and gonad maturation and between ecdysone concentration and utilization of energy reserves during gametogenesis. Responses to 479 ecdysone varied with both the ecdysone concentration and the duration of exposure, indicating the importance of the sequential changes in ecdysone to the target tissues. Hormonal control of reproduction in leeches using brain homogenates has been shown by a number of authors ( Kulkarni et al., 1980; Hagadorn 1969; Webb, 1980; Webb and Omar, 1981). The life cycle of Acanthobdella peledina is basically annual with young immature individuals ﬁrst occurring on their host in June. These individuals leave the ﬁsh in October on reaching sexual maturity. Reproduction with egg and cocoon production occur off the ﬁsh and have not been observed (Holmquist, 1974). D. Dispersal The dispersal of freshwater leeches and acanthobdellids is generally assumed to be the result of passive transfer from one water body to another by other animals. This seems probable for parasitic sanguivorous species, but less probable for the majority of freshwater leeches which are predators of benthic invertebrates. However, some examples have been recorded with Marrinmeyeria lucida transported by birds (Daborn, 1976), and Khan and Frick (1997) showed that a high proportion of salamanders (Ambystoma maculosum) in South Carolina served as phoretic hosts to Erpobdella punctata. Platt et al. (1993) similarly recorded Helobdella stagnalis, previously found on ﬁsh, phoretic on Ambystoma tigrinum in Indiana. Davies et al. (1982b) examined passive transfer by ducks of two parasitic species (Theromyzon trizonare and Placobdella papillifera) and two predatory species (Helobdella stagnalis and Nephelopsis obscura). Theromyzon trizonare adults were transferred both in the nares, while taking a blood meal, and on the duck’s body under the feathers. Placobdella papillifera were also conveyed on the body of the duck, but adult H. stagnalis and N. obscura were not transported. Adult leeches ingested by ducks were not recovered from the duck feces, but viable N. obscura cocoons were recovered from the feces of fed ducks. Passive dispersal by wind has never been recorded, but the transport of cocoons attached to macrophytes is possible. Davies (1979) showed that Helobdella stagnalis, H. triserialis, Glossiphonia complanata, and Mooreobdella fervida were dispersed to Anticosti Island by sea currents from the Quebec north shore of the Gulf of St. Lawrence. Dispersal by currents in large lakes or through rivers is also highly probable, and leeches have been recorded in the drift (Elliott, 1973). Accidental transport of leeches by humans has not yet been shown in North America, but has been demonstrated in other areas of the world (Sawyer, 480 Ronald W. Davies and Fredric R. Govedich 1986). Active directional dispersion up rivers was recorded for Percymoorensis marmorata by Richardson (1942) and for E. punctata by Sawyer (1970). Erpobdella punctata has been found to be a phoront on the salamander Ambystoma maculatum, and it is likely that as the salamanders move from pond to pond the leeches are transported (Khan and Frick, 1997). Passive dispersal, while unlikely, does appear to be the mechanism by which leeches colonize new ecosystems. The passive transport of individuals between systems can be estimated using genetic markers and the level of genetic similarity between populations can be used to estimate the amount of migration between populations. In a recent study of Erpobdella punctata, the estimated migration rates between populations were found to be extremely low, regardless of the distance between neighboring populations. However, some populations are more closely related to each other than to others indicating the potential for either migrants or founding individuals to be transported between ecosystems (Govedich et al., 1999). Differences in intra- and inter-population genetic structure is primarily determined by gene ﬂow with poorly dispersing species exhibiting high inter- and low intra-population variation. In general, there is greater genetic divergence with increased physical isolation restricting gene ﬂow. Qian and Davies (1996) determined whether genetic differences among three populations of Nephelopsis obscura were the result of differential dispersal or environmental heterogeneity. Two of the populations examined were from sites in Alberta, Canada, geographically close (80 km) to each other while the third population in Utah, was geographically distant (1200 km) from the other sites. Despite the high probability of dispersal between the close sites and the lower probability of dispersal between these two sites and the distant site, one of the sites in Alberta differed at only one locus from the Utah population and both these populations differed from the other Alberta population at ﬁve loci. These genetic differences were correlated with the dissolved oxygen regimes acting as a selective force rather than geographic distance between populations. E. Predation In North America, predators of leeches include ﬁsh, birds, garter snakes, newts, and salamanders (Pearse, 1932; Bartonek and Hickey, 1969a; Bartonek and Murthy, 1970; Bartonek and Trauger, 1975; Bartonek, 1972; Able, 1976; Arnold, 1981; Davies et al., 1982b; Kephart, 1982; Kephart and Arnold, 1982), as well as insects, gastropods, and amphipods (Hobbs and Figueroa, 1958; Pritchard, 1964; Sawyer, 1970; Anderson and Raasveldt, 1974; Blinn et al., 1988). Young and Spelling (1986), Spelling and Young (1987) and Young (1987), reviewing predation on three lentic leech species in Great Britain, recorded a similar range of predators as well as interspeciﬁc leech predation and cannibalism. Leeches lack hard body parts and are thus difﬁcult, if not impossible, to identify from the gut contents of predators. Serology is the most appropriate method for analyzing predation on soft-bodied animals (Davies, 1969), but this approach requires prior knowledge of the range and size of potential predators to be collected from the ﬁeld and tested. In the laboratory, Cywinska and Davies (1989) found that four species of dytiscids, one species of Odonata, two species of hemipterans, and two species of amphipods fed on one or more size classes of N. obscura. In Great Britain, Young and Spelling (1986) also noted that species within these taxa fed on E. octoculata, Glossiphonia complanata, and Helobdella stagnalis in addition to triclads, trichopterans, and megalopterans. It would thus appear that larval and adult coleopterans and nymphal odonates and hemipterans are potentially voracious predators of leeches throughout the Holarctic. In experiments with predation on cocoons, Cywinska and Davies (1989) recorded two methods of cocoon consumption; either the embryos and eggs within the cocoons were fed upon, by piercing holes through the cocoon wall (e.g., Coleoptera larvae and adult Hemiptera), or else the whole cocoon (sometimes, plus attached plant leaf) was consumed (e.g., amphipods and adult Coleoptera). Predation has also been recorded by gastropods on E. punctata cocoons (Sawyer, 1970) and by a mite on Haemopsis sanguisuga cocoons (Bennike, 1943). The only known vertebrate predators of leech cocoons are salamanders (Pearse, 1932) and waterfowl (Davies et al., 1982b). Predation rates on N. obscura in the laboratory were inversely related to size, with individuals 30 mg being consumed only at very low rates by certain coleopterans. Young and Spelling (1986) found a similar trend of increased consumption of leeches by most predators with declining leech weight. This can be explained by the inability of some potential predators to capture and overcome the more powerful larger leeches, rapid satiation of predators with larger prey, or the ability of larger leeches to produce more mucus and so impede the movements of the potential predators. A caveat of these feeding experiments was that predators were starved, had no alternative food available, and had no choice in size of N. obscura prey. In such conditions, the results of the experiments probably showed the maximum potential predation rates and suggested that predation on cocoons and size classes 10 mg might be substantial. Considering the very 13. Annelida: Euhirudinea and Acanthobdellidae high densities of potential predators in many lakes and ponds and the ability of some predators to consume large numbers of leeches and / or their cocoons, the effects of predation in the ﬁeld are potentially signiﬁcant in macrophyte zones. The rapid decline in density of hatchling and small ( 10 mg) N. obscura, observed in the ﬁeld by Davies and Everett (1977), could be explained by predation in the shallow macrophyte zones where cocoons are deposited and hatch. Placement of cocoons by M. montezuma on deeply submerged Potamogeton stems at least 3.5 m below the water surface at the interface of the littoral-pelagic zone in Montezuma Well may be a strategy to reduce predation (Davies et al., 1988). Predation in the water column is almost entirely restricted to ﬁsh, amphibia, and birds. The presence of large numbers of leeches in the gut contents of ﬁsh (particularly from rivers) and the commercial development of leeches for bait in some areas indicate predation of ﬁsh can be at least occasionally heavy. F. Behavior Sawyer (1981) suggested that the behavior of leeches and acanthobdellids could be divided into a number of elementary responses: crawling (vermiform, inchworm); swimming; shortening (fast, slow); searching (head movement, body waving); and an alert posture when the body is extended and held motionless for some time. However, species differ markedly in the frequency of expression of these different responses. Behavior of leeches can be modiﬁed by size (age) and physiological state (starved or fed, and mature or immature) as well as by extrinsic environmental parameters monitored by eyes, sensillae, chemoreceptors, and mechanoreceptors (T-cells). The foraging behaviors of sanguivores and predators are usually different. Sanguivorous leeches in the proximity of a potential host respond to changes in illumination (shadows), vibrations in the water, chemical stimuli, and possibly differences in temperature between the host and the surrounding water. Compared to predatory leeches, the sanguivores are less negatively phototactic and nocturnal, especially when hungry. Theromyzon tessulatum, T. trizonare, Piscicola geometra, and Calliobdella vivida, when hungry, have all been found in the open water ready to attach to a host (Herter, 1929a, b; Meyer and Moore, 1954; Sawyer and Hammond, 1973). Hirudo medicinalis and Theromyzon tessulatum are stimulated to attach to and bite hosts with temperatures of 37 – 40°C (Dickinson and Lent, 1984), and Placobdella costata is attracted from as far as 15 cm to hosts with temperatures of 33 – 35°C (Mannsfeld, 1934). Haementaria ghilianii 481 and Hirudo medicinalis are both sensitive to water disturbances (traveling surface waves) (Sawyer, 1981; Young et al., 1981), and it is likely that most sanguivores respond in a similar manner. Water disturbance is frequently the ﬁrst indication of the presence of a potential host; the leech moves toward the disturbance using cues detected by its sensilla (Friesen and Dedwylder, 1978; Friesen, 1981; Derosa and Friesen 1981). Theromyzon tessulatum and T. trizonare respond to water disturbance in a similar manner, but adult brooding T. tessulatum are unique in that they move toward a potential host even though they never take a blood meal. If the parent T. tessulatum locates a host, it attaches to it and the brooding young leave to seek their ﬁrst blood meal (Wilkialis and Davies, 1980a). Although movements toward a potential host by sanguivorous leeches are not speciﬁc, the next step of determining whether or not the potential host is an appropriate source of blood involves some selectivity. After attachment to the potential host with the posterior sucker, the leech makes a number of searching movements with the anterior portion of the body. If the host is not appropriate, the leech detaches. Attachment to a host does not always occur at an appropriate location. For example, T. trizonare primarily feed in the nares; but if one attaches to the feathers rather than the beak it will remain on the bird for up to 30 min, moving about in search of a suitable location to feed. Host recognition by sanguivores presumably results from chemoreception and / or mechanoreception. Predatory leeches are generally more nocturnal and negatively phototactic than sanguivores (Elliott, 1973; Anholt and Davies, 1987; Davies and Kassera, 1989; Davies et al., 1996). Angstadt and Moore (1997) demonstrated that Nephelopsis obscura swimming is entrained to a 12-h light / 12-h dark cycle and that a circadian rhythm of swimming persists in constant darkness. Davies et al. (1996) tested the hypotheses that foraging behavior responses such as numbers of meals consumed, the number of encounters with prey and feeding time as well as activity (swimming) were higher in the dark, and increased with the size of N. obscura and the length of the starvation period. While swimming activity was longer in the dark, not all the foraging behavior variables followed this pattern. There was an inverse relationship between swimming time and the number of meals consumed and the poor correspondence between swimming time and the number of prey encounters indicates that swimming is not always directly associated with increased foraging and is sometimes related to movements between microhabitats. Thus, increased nocturnal activity is not always indicative of increased foraging activity. 482 Ronald W. Davies and Fredric R. Govedich Anholt (1986) showed that N. obscura reduced its foraging activity when satiated and this was conﬁrmed through direct observations of encounters and feeding rates as a function of satiation. Increasing satiation in N. obscura produces a threshold level above which prey with natural escape responses cannot be successfully captured. Only a small fraction of encountered tubiﬁcids were ingested by starved N. obscura when the prey had a substrate refuge. The loss of prey capture ability with increasing satiation is a behavioral foraging constraint. When its threshold level is reached, further successful foraging on prey with a substrate refuge is impossible. This threshold level varies for different prey types, e.g., Anholt (1986) showed that the tube-dwelling chironomids Chironomus riparius and Glypotendipes paripes were superior prey types compared to tubiﬁcids in promoting leech growth, and concluded that T. tubifex could evade capture by N. obscura more efﬁciently than chironomids. This was conﬁrmed by direct observations which showed escape times by chironomids longer than the escape times of tubiﬁcids. Groups of N. obscura were provided with sufﬁcient prey with either a substrate (MS) or without a substrate (NS) to allow asymptotic food ingestion without prey patch depletion. In both groups of leeches, feeding was not reduced by low prey encounters, but by the predator’s ability to capture more prey. Thus, the signiﬁcantly different feeding rates exhibited by MS and NS leeches resulted from different factors determining the asymptote of food ingestion. In MS leeches this was primarily the foraging constraint threshold, but in NS group it was primarily the gut capacity determining maximal feeding potential (Dratnal et al., 1992). As MS leeches showed a higher growth efﬁciency, the lower food acquisition was compensated for by allocating a greater fraction of energy to growth and / or by higher energy absorption efﬁciency, a result of longer food retention time in a less packed alimentary canal. Thus, the foraging constraint signiﬁcantly affected the internal status of N. obscura with a feedback system between the two. Foraging results in increasing satiation, which reduces and eventually stops foraging, until some level of hunger (decreased satiation) is regained. Oxygen uptake (respiration) increased in fed N. obscura suggesting an increased energetic requirement for the processes directly or indirectly associated with digestion with the resulting energetic trade-off producing a lower overall activity in fed leeches. Although several modifying, behavioral factors apply to N. obscura in the ﬁeld they do not preclude the constraint on foraging by N. obscura affecting its sizespeciﬁc rate of feeding and life-history traits, especially as chironomids and tubiﬁcids form major components of the diet in the ﬁeld (Davies et al., 1978). The existence of mechanisms based on individual size-dependent limitation in food ingestion are of great importance not only for the indeterminate growth of N. obscura but must also strongly modify size-dependent food partitioning within the predator population — an important factor in population regulation. To behave adaptively, animals often give higher priority to some behaviors over others. Misell et al. (1998) looked at the effect of feeding behavior on other behaviors, such as swimming, crawling, and shortening, in Hirudo medicinalis. The priority feeding had, relative to other behaviors, showed that as expected feeding occupies a high position in the hierarchy of behaviors and dominated all the other behaviors tested. A feeding H. medicinalis will ignore strong mechanical stimuli which normally cause vigorous escape responses to continue feeding. Although little is know about the neural basis of feeding in H. medicinalis, the role of serotonin has been studied. Increases in serotonin result in an increase in feeding and vice versa, leading to the hypothesis that serotonin is effectively a feeding hormone. While some predators also show increased activity in response to disturbances in the water, they are generally opportunistic, feeding on prey encountered through either their own locomotion or the movements of the prey. Prey detection by predator leeches has received little attention since Gee (1913) noted that Mooreobdella microstoma responded to vibrations in the water and chemical extracts of its prey by unidirectional swimming and random movements of the anterior part of its body. Davies et al. (1982a) examined the chemosensory detection of prey by N. obscura and showed that without direct tactile contact, N. obscura were unable to detect and react to any of the common prey types tested (i.e., they did not show clinotaxis). Whether or not a particular prey appears in the diet of a predatory leech depends on the probability that it will be encountered, the probability that it will be attacked once encountered, and the probability that an attack will result in capture. Simon and Barnes (1996) concluded that Percymoorensis marmorata use olfactory rather than mechano sensory cues to detect their prey (oligochaetes). Olfactory responses of the medicinal leech (Hirudo medicinalis) are very speciﬁc although arousal and searching for prey are primarily initiated by water movement. The response of Bdellerogatus plumbeus to its main prey Erpobdella punctata are also chemosensory (Riggs, 1980). However, Young et al. (1995) concluded that Helobdella stagnalis, Glossiphonia complanata and Erpobdella octoculata do not depend on chemosensory cues to detect prey and that chemosensory cues are only 13. Annelida: Euhirudinea and Acanthobdellidae important when the leech is in contact with the prey as shown by Davies et al. (1982a). They also concluded that the inability of leeches to detect damaged prey in heterogenous environments was the reason for very high juvenile mortality. An exception to the general rule that the diet of predatory leeches is usually nonspeciﬁc and contains a wide variety of prey is demonstrated by Motobdella montezuma. Blinn et al. (1988) showed that M. montezuma forages selectively on the pelagic amphipod Hyalella montezuma despite the presence of a wide variety of alternate prey. Motobdella montezuma discriminates between two congeneric amphipod prey by mechanoreception, exhibiting a high response to the acoustic vibration signals of the endemic H. montezuma but not to H. azteca. Not only does M. montezuma discriminate between species by mechanoreception, but it also selectively discriminates between size classes, feeding principally on juveniles. Although amphipods form part of the diet of both N. obscura and E. punctata, neither species shows a signiﬁcant response to their acoustic vibration signals (Blinn and Davies, 1989). The unusual feeding behavior exhibited by Motobdella can be linked to the highly specialized sensillae that ring the mouth (Fig. 17). These sensillae are composed of cilia that are in contact with the water where vibrations from potential prey can be detected. The number of cilia and the size of the sensillae have been found to be signiﬁcantly greater in Motobdella than in the related erpobdellid genera Erpobdella and Nephelopsis (Govedich et al., 1998). Substrate type affects leech foraging with Erpobdella octoculata foraging more effectively over cobble substrates than over ﬁne gravel with differences in prey type consumed (Dahl and Greenberg, 1997). When leeches and trout were together, trout foraging was unaffected by leeches but leech foraging was affected by trout. Although one leech may consume much less than one trout at ﬁeld densities, leeches have similar effects as ﬁsh in structuring lotic benthic communities (Dahl, 1998). Some animals form temporary or permanent aggregations or groups which may reduce an individual’s risk of predation, enhance mating or food intake, ameliorate physiological stress, coordinate and enhance dispersal or synchronize development. Leeches commonly form aggregations in the ﬁeld, especially the benthic predatory species. Smith and Davies (1996a, b) examined the effects of different group sizes on acquisition and allocation of energy in Nephelopsis obscura and evaluated the physiological cost and beneﬁts of group living. In terms of growth, asymptotic biomass, ingestion and respiration group sizes larger than one and less than 10 were found to be optimal — a group size 483 within the typical range of groups sizes found on natural stony shores. Oxygen concentrations affect both the growth of N. obscura and its feeding behavior (Davies et al., 1992; Davies and Gates, 1991a, b). Higher rates of feeding occurs in 100 and 300% oxygen saturation regions compared to hypoxic regimes. This is related to the greater proportion of time spent on or in the mud substrate and thus increased prey encounter rate. Since hypoxia and anoxia cause high mortality at summer temperatures, differences in vertical distribution of N. obscura are behavioral adaptations to minimize stress. Seasonal migrations of N. obscura from deep water to shallow water in the spring and to deep water from shallow water in the fall cannot be solely attributed to temperature preferences and it is likely that avoidance of oxygen stress also plays a role. G. Population Regulation On a geological time scale, only a very few freshwater ecosystems are long-lived and even on a shorter time scale of 1 – 20 years, many lentic ecosystems are ephemeral. In ephemeral ecosystems, leeches may not have sufﬁcient time to reach carrying capacity. Evidence for competition between leech species has been presented by Davies et al. (1982c) for Nephelopsis obscura and Erpobdella punctata and by Wrona et al. (1981) for Glossiphonia complanata and Helobdella stagnalis. However, only the studies on competition between N. obscura and E. punctata are comprehensive in relation to the established criteria (Reynoldson and Bellamy, 1970; Williamson, 1972). Reynoldson and Davies (1976, 1980) showed a considerable overlap in the range of water conductances in which N. obscura and E. punctata can regulate their weights. The sympatric distributions of the species, therefore, appear to be related to common salinity tolerances. However, among sympatric populations, there is a strong inverse relationship between the numerical abundances of N. obscura and E. punctata in lentic ecosystems (Davies et al., 1977). Dominance of E. punctata over N. obscura is a rare temporary event and the switch to dominance by N. obscura is rapid. The reproductive strategies of E. punctata vary depending on whether it is dominant or subordinate (Davies et al., 1977). Thus, the biological interactions between N. obscura and E. punctata are amenable to explanation based on competition. The utilization of common food resources by N. obscura and E. punctata has been demonstrated by numerous laboratory and ﬁeld studies (Davies and Everett, 1977; Davies et al., 1978, 1981, 1982c). At the species level, there were no signiﬁcant differences in the FIGURE 17 Photomicrographs (5000 ) showing differences in the size of sensilla and the number of cilia within sensilla of: (a) Motobdella sedonensis; (b) M. montezuma; (c) Erpobdella punctata; (d) Nephelopsis obscura (Govedich et al., 1998). 484 13. Annelida: Euhirudinea and Acanthobdellidae diets of either species between years and the trends in prey utilization were similar each year (Fig. 14). Intraspeciﬁc food resource partitioning, as a function of differential weight class utilization of prey was shown by both species. Such resource partitioning minimizing intraspeciﬁc competition, increases population ﬁtness (Giesel, 1974). The change in feeding ecology of E. punctata with changes in numerical dominance provided further evidence for interspeciﬁc competition. Furthermore, a signiﬁcant decrease in mean niche overlap and niche breadth was found for both species when N. obscura became dominant. The majority of criteria established to determine interspeciﬁc competition have been supported by the data collected on N. obscura and E. punctata; however, the manipulation of food resources has not yet been completed. In British lakes, semelparous leech species show very high (95 – 98%) mortality of juveniles (Young et al., 1995). Many young leeches are inept at capturing prey, and feeding success increases when adult leeches are also present suggesting that mortality of young leeches is high because of their inability to locate damaged prey in an heterogenous environment (Dahl, 1998). H. Ecotoxicology Aquatic ecosystems seem to be sinks for many environmental contaminants such as heavy metals, pesticides and other synthetic or natural pollutants. The impacts of these pollutants are generally only seen when the problems have reached critical end points such as ﬁsh kills or algal blooms. However, more subtle effects, such as changes in growth, reproduction, behavior, physiology or survival of freshwater species, may act as indicators for potential larger scale problems. In the longer term, these subtle chronic effects play a role on health and efﬁciency of freshwater ecosystems. Biomonitoring and ecotoxicological studies utilizing freshwater leeches, especially benthic predatory species, have recently become increasingly more common. Changes in the community are frequently assessed by using bioindicators (species or groups of species for which changes in abundance reﬂects the quality of the environment. Leeches were used by Bendell and McNichol (1991) as indicators of acidiﬁcation on 40 small lakes, many of which had been acidiﬁed by nickel smelting works at Sudbury, Ontario, Canada. Nine species of leeches were recorded from the 20 lakes with pH 5.5. Several species of leech disappeared over a narrow range of pH which suggests that acidity or a single chemical or biological factor correlated with pH is responsible. However, Grantham and Hann (1994) showed that lake pH in the Experimental Lakes Area of Ontario was not a dominant variable, describing only a small amount of the variance in the species – environment relationship. 485 Leeches have shown superior chlorophenol bioconcentrating ability over many other aquatic organisms. Prahacs and Hall (1996) evaluated N. obscura as a potential biomonitor of chlorinated phenolic compounds discharged from bleach pulp mills on the Lower Fraser River, British Columbia, Canada. They found a strong linear correlation between water contaminant concentration (0.1 – 10.0 g / L) and bioconcentration of chloroguaiacols. Bioconcentration was inversely related to pH. As in situ biomonitors, Prahacs et al. (1996) showed that and increase in chlorine dioxide substitution in leeches reduced amounts of chlorinated phenolics accumulated, with a sharp decrease observed at chlorine dioxide levels 90%. An alternative approach to bioindicators is the use of biomarkers, deﬁned as measurable variations in cellular or biochemical components or processes, structures or functions, that provides information on a stress and its effects at the population level. The effects of exposure of adult sexually mature Nephelopsis obscura for 4 – 24 days to 0.0 – 580 g / L cadmium showed that the numbers of ova and spermatozoa per unit biomass were signiﬁcantly reduced with increasing cadmium concentration and exposure time, as were the masses of the testisacs and ovisacs (Davies et al., 1995). Exposure of cocoons to cadmium (0.0 – 4000 g / L) had a signiﬁcant effect on post hatchling survivorship (Wicklum et al., 1997). The effects of chronic exposure to cadmium (0.0 – 50 g / L) were examined in terms of changes in energy acquisition and allocation (somatic growth, reproductive growth, feces plus mucus, active respiration, resting respiration and activity time) (Wicklum and Davies, 1996). Leeches in the high cadmium concentration group had signiﬁcantly lower biomass production, survivorship, and ingestion rate. Assimilation efﬁciency, reproductive investment, resting respiration and active respiration were unaffected by cadmium, but leeches in the high concentration group were signiﬁcantly less active and produced more mucus. Decreased ingestion and increased mucus production in the high concentration of cadmium contributed to a lower growth rate. Although cadmium had no effect on resting or active respiration, there were signiﬁcant effects on total respiration as leeches exposed to high cadmium doses spent less time in active and, thus, in total respiration. Macroinvertebrates are generally good indicators of toxic stress in wetlands; but while phorate (a commonly used pesticide) killed all amphipods and chironomids, snails and leeches were tolerant and could survive heavy doses by behavioral adaptations and / or physiological tolerances (Dieter et al., 1996). Leeches are also not good monitors of mercury (usually as methyl mercury) biomagniﬁcation and are not a dietary risk to waterfowl or ﬁsh (McNicol et al., 1997). Studying polychlorinated biphenyls discharged to a lotic system in Ontario, 486 Ronald W. Davies and Fredric R. Govedich ﬁsh and leeches occupying the top of the food web accumulated more PCBs than organisms lower in the food web, thus indicating that biomagniﬁcation (uptake through ingestion) through trophic transfer and not bioconcentration is the primary mechanism governing contamination (i.e., uptake as a chemical from the water) (Zaranko et al., 1997). V. COLLECTION AND REARING A. Collection Leech and acanthobdellid ectoparasites are sometimes found attached to hosts and are usually easily removed. Most leech species (but not Acanthobdella peledina) are free-living for the majority of their life cycle and are normally attached to a ﬁrm substrate or free swimming, as are the predatory leech species. They can be collected with bottom samplers and dip or plankton nets. Free-living leeches can be sampled quantitatively using colonization chambers, grab samplers, or timed collections from rocks and stones. As a general rule, the plan area of the largest leech collected, or its area of escape response, should not exceed 5% of the sampler area (Green, 1979). The only benthic samplers that even approximately meet these criteria are grab samplers such as the Ekman grab (Ekman, 1911). Another problem faced when quantitatively sampling free-living leeches is that they occur in both the sediment and the vegetation. The Gerking box sampler (Gerking, 1957) is one of the few methods of quantitatively estimating population density, separable into these two components. This technique has, however, a number of technical problems including the difﬁculty of clipping macrophytes close to the substrate, the entrainment of sediments and associated fauna in the macrophyte sample, the invasive inﬂuence of the operator, and the difﬁculty of integrating the macrophyte and sediment samples because the benthic samples are taken from inside the area from which the macrophyte samples are collected. These problems are overcome by using a sampler consisting of a bottom mounted detachable grab connected to a box sampler with levered, spring-loaded jaws for cutting macrophytes (Gates et al. 1987). The sampler is triggered from above the water and simultaneously compartmentalizes the soft sediments in the grab and the phytomacrofauna in the water column above. B. Rearing Leeches are generally easy to maintain in the laboratory provided they are maintained under approximately similar conditions encountered in the ﬁeld. Appropriate dissolved oxygen and temperature regimes are particularly important. Light regime is less critical, but if an ambient light regime cannot be provided, darkened conditions are preferable. Leeches are very susceptible to rapid changes in ionic content, temperature, and dissolved oxygen concentrations. If ﬁeld conditions cannot be provided, they should be slowly acclimated to the new conditions (e.g., temperature changes exceeding 5°C per day will usually cause very high mortality, but a temperature change of 1°C per day within the normal ﬁeld range will usually result in little mortality). Predatory leech species are easily fed in the laboratory. If suitable prey at high densities are provided, most leeches will feed avidly when hungry. For maintenance, ad libitum feeding 1 day per week is sufﬁcient. Most predatory species brought into the laboratory and provided with ambient ﬁeld conditions will feed, grow, and reproduce. The viability of the cocoons produced is sometimes decreased if the parent has been maintained in the laboratory for a long period. It is much more difﬁcult to raise hatchlings produced in the laboratory through to mature size, capable of producing viable cocoons. Each species requires special conditions and there is much research to be done in this area. Conversely, while some sanguivorous leeches are more difﬁcult to feed in the laboratory, reproduction, cocoon production, and hatching are generally easier. Some sanguivores (e.g., Placobdella papillifera) will eat if immersed directly into blood. Many others (e.g., Hirudo medicinalis) will feed through a membrane if blood at an appropriate temperature (30 – 40°C) is on the other side. For these species, the simplest technique is to provide the hungry sanguivore with a sausage made from intestinal epithelium ﬁlled with warmed blood. Other species (e.g., Theromyzon trizonare) have not yet been induced to feed artiﬁcially and must be provided with a live host and given the opportunity to reach an appropriate feeding site (the nares of waterbirds for T. trizonare). However, when suitable sites are available, cocoon production occurs readily. VI. IDENTIFICATION A. Preparation of Specimens Some species can be studied live, but permanently stained slides and serial sections are required for identiﬁcation of a few species. Clearing, without permanent staining, is often required for determining the ocular number and arrangement. Most leeches contract strongly when placed in cold ﬁxative and should be narcotized ﬁrst. Before narcotizing 13. Annelida: Euhirudinea and Acanthobdellidae the specimens, the colors of the dorsal and ventral surfaces should be recorded, as chromatophores are dissolved or altered by most narcotizing agents. Leeches are generally difﬁcult to relax properly, and any of the following three methods are recommended: 1. Add drops of 95% methanol slowly to the water containing the leech, gradually increasing the concentration for about 30 min until movement ceases. When the leech is limp and no longer responds to touch, pass it between the ﬁngers to straighten it and remove excess mucous. 2. Add carbonated water or bubble in CO2 until movement of the leech stops. Straighten the leech out on a slide and slowly add warm 70% ethanol and a few drops of glacial acetic acid until it is covered. 3. Add a drop or two of 6% nembutal until movements stop and then straighten the leech on a slide. For some specimens, slight ﬂattening is occasionally desirable. This is best done between two glass slides with weights added if necessary. After relaxation, the leeches should be ﬁxed for 24 h (depending on size) in 5 or 10% formalin. For histological preparations, Fleming’s or Bouin’s ﬁxatives should be used. Large leeches should be injected with 70% ethanol to ensure preservation of the internal organs. To preserve a leech, wash the ﬁxed specimen with deionized water to remove the formalin and then add either 70% ethanol or 5% buffered formalin. Although formalin preserves the colors longer, ethanol is recommended since it is safer to use and less destructive to soft tissues. Leeches ﬁxed in formalin or Bouin’s or Fleming’s FIGURE 18 487 ﬁxative can be stained in Mayer’s paracarmine, borax carmine, or Hams’ hematoxylin for 12 – 94 h, destained in a 1% HCl – 70% ethanol solution until the leech epidermis is free of stain, and then neutralized in a 1% NH4OH – 70% ethanol solution. Fast green or eosin are routinely used as counterstains. Stained specimens should be dehydrated in progressively higher concentrations of ethanol, cleared in methyl salicylate, and mounted in a neutral pH mounting medium. B. Important Features for Identiﬁcation Identiﬁcation of leeches can be difﬁcult, if not impossible, with poorly preserved specimens. External features such as the following are used whenever possible: annulation; number of eyes and their arrangement; presence or absence of papillae, tubercles, and pulsatile vesicles; relative size of anterior (oral) and posterior (anal) suckers; size and position of the mouth; and, the relative positions of the male and female gonopores. The only notes on coloration included in the following keys are those known to persist after preservation. In a few instances, reference is made to internal anatomical features. These have been kept to a minimum and are used only when positive identiﬁcation is impossible without them. To distinguish between Nephelopsis obscura and the Dina-Mooreobdella complex, the atrial cornua must be examined (Fig. 18). The preserved specimen should be pinned out with the ventral surface up and a transverse incision made across the body three or four annuli) posterior to the male gonopore. Cuts should be made anteriorly up the lateral margins of the body for about 20 annuli) and the posterior edge of the ﬂap lifted forward exposing the inner tissues, which must be cleared to expose the atrium. To help (a) Spirally curved atrial cornua of Nephelopsis obscura; and (b) the simply curved atrial cornua of Dina / Mooreobdella. 488 Ronald W. Davies and Fredric R. Govedich identify Desserobdella the salivary gland can be examined in specimens cleared with clove oil. To examine the velum and jaws, the specimen should be pinned out and a median ventral incision made from the lower lip of the anterior sucker back far enough for the margins to be pinned out to expose the inner surface of the pharynx (Fig. 8). Details of the teeth (Fig. 9) can only be seen by removal of a jaw and making a temporary mount on a microscope slide. The length of leeches and acanthobdellids is dependent on species, age, physiological state, and the degree of relaxation. The measurements presented in the keys are maximum total lengths of relaxed individuals. C. Family Glossiphoniidae Confusion exists in the systematics of these genera, and many species have undergone considerable synonymy. Although some taxonomists consider that the genus Haementeria (De Filippi, 1849) has priority over Placobdella (Blanchard, 1893), the latter name is widely recognized and accepted as a genus in North America and is well represented with nine species. Lukin (1976) recognized important differences between Glossiphonia complanata and G. heteroclita, and suggested that the latter species be placed in the subgenus Alboglossiphonia elevated to generic level by Klemm (1982). There has been considerable confusion among the species assigned to the genera Batracobdella Viguier and Placobdella Blanchard. Barta and Sawyer (1990) erected a new genus Desserobdella, differentiated by the presence of two pairs of coalesced eyes and one pair of diffuse salivary glands compared to the two pairs of pairs of compact salivary glands in Placobdella and a single pair of eyes in Batracobdella. Placobdella species primarily specialize on reptilian and crocodilian hosts whereas species of Desserobdella utilize amphibian or piscine hosts. Reexamination of type specimens by Barta and Sawyer (1990) and Jones and Woo (1990) assigned Placobdella (Batracobdella) picta, Placobdella (Batracobdella) phalera, Batracobdella cryptobranchii and Batracobdella (Placobdella) michiganensis to the genus Desserobdella. Placobdella parasitica and P. ornata have been conﬁrmed as members of the genus Placobdella (Moser and Desser, 1996), but similar re-examination of the number and structure of the salivary glands is still needed for P. hollensis, P. pediculata, P. montifera, P. nuchalis, P. translucens, P. mulilineata, and P. papillifera. The single and questionable identiﬁcation of the European species Batracobdella paludosa by Pawlowski (1948) from Nova Scotia, Canada is the only record of this genus from North America. Theromyzon tessulatum is characteristically a European species, but its presence in North America has been well documented (Davies, 1971, 1973, 1991; Klemm, 1982; Sawyer, 1986; Oosthuizen and Davies, 1993). The ﬁrst species of Theromyzon reported from North America, described by Baird (1869) as Glossiphonia rudis, was based on a brief nondiagnostic description. Because Moore and Meyer (1951) and Meyer and Moore (1954) collected specimens from the same locality (Great Bear Lake) they assumed their specimens with the male and female gonopores separated by three annuli were Theromyzon rude. Thereafter, all specimens of Theromyzon collected in North America with three annuli between the gonopores have been identiﬁed as T. rude. However, examination and dissection of the syntypes (Oosthuizen and Davies, 1992) showed that one specimen was Placobdella ornata and the second, which became the holotype of Glossiphonia rudis, had only two annuli separating the gonopores. Specimens with three annuli between the gonopores were subsequently shown to belong to a new species T. trizonare and all specimens previously identiﬁed as T. rude are, in fact, T. trizonare. In North America, there are three species with two annuli between the gonopores (Oosthuizen and Davies, 1993): T. maculosum, T. bifarium, and T. rude. Theromyzon maculosum, characteristically a European species, is easily distinguished from T. bifarium and T. rude by the presence of two independent oviducts with two female pores compared to one female pore in the latter two species. Theromyzon bifarium is distinguished from T. rude by the presence of a cylindrical male atrium, paired dorso-lateral male ducts which enter the atrium separately. Although T. sexoculatum (Moore, 1898) and T. biannulatum (Klemm, 1977) have been described from North America with two annuli between the gonopores they are not valid species. The genus Actinobdella was at ﬁrst erroneously placed in the family Piscicolidae by Moore (1901), who was misled by the unusual annulation of Actinobdella inequiannulata. Complete segments of Actinobdella are 3-annulate (a1, a2, a3), which are further divided into six unequal annuli (b1, b2, b3, b4, b5, b6). D. Family Piscicolidae Although the Family Piscicolidae has been shown to be paraphyletic (Siddall and Burreson, 1995) it has been retained for convenience and is based on plesiomorphic characteristics and parasitism of ﬁsh. Some workers have suggested that Piscicola geometra and P. milneri are synonymous, although P. milneri has 10 – 12 punctiform eyespots, whereas P. geometra has 12 – 14 punctiform eyespots and dark pigmented rays on the posterior sucker (Fig. 19c, d). Klemm (1977) indicated that the number of punctiform 13. Annelida: Euhirudinea and Acanthobdellidae 489 relatively simple to combine the records for Illinobdella with those for Myzobdella. However, if supporting data are not forthcoming, subsuming Illinobdella with Myzobdella will result in an irretrievable loss of information. E. Family Hirudinidae Richardson (1969, 1971) divided the North American Haemopisoid leeches into the genera Mollibdella, Bdellarogatis, and Percymoorensis, rather than placing them all in the genus Haemopis. This was accepted by Soös (1969b), Davies (1971 – 1973), and Klemm (1972a), and is accepted here. Sawyer and Shelley (1976) and Sawyer (1986), for unspeciﬁed reasons, recombined them all back into the genus Haemopis, a view also adopted by Klemm (1977, 1982, 1985). FIGURE 19 Posterior suckers of: (a) Piscicola punctata without oculiform spots; (b) Piscicola salmositica with 8 – 10 crescentiform oculiform spots; Piscicola geometra with 12 – 14 oculiform spots separated by pigmented rays; and (c) Piscicola Milnera with 10 – 12 punctiform oculiform spots on posterior sucker. eyespots on the posterior sucker and the presence or absence of the pigmented rays varies with the age of the specimens. As the gonopores of Piscicola milneri are separated by two annuli, and by three annuli in P. geometra, they are here considered distinct species. The genera Myzobdella and Illinobdella are very similar (Meyer, 1940; Daniels and Sawyer, 1973), but their relationship needs to be thoroughly investigated before the synonymy suggested by Daniels and Sawyer (1973) and Sawyer (1986) can be fully accepted. In the present chapter, a conservative approach has been used. If later evidence supports the propositions of Daniels and Sawyer (1973) and Sawyer (1986), then it will be F. Family Erpobdellidae There is some disagreement as to whether Dina and Mooreobdella are distinct genera (Moore, 1959) or subgenera of the genus Dina (Soös 1968). The diagnostic feature is whether the vasa deferentia form preatrial loops extending to ganglion XI (Dina) or do not have preatrial loops (Mooreobdella). This is difﬁcult to determine, and thus in this key, the genera (or subgenera) are lumped together in a Dina – Mooreobdella complex, separated on external features. Although placed in the genus Erpobdella by Davies et al. (1985), Motobdella montezuma has several morphological traits which distinguish it from Erpobdella including posterior and crop ceca, large sensilla and the location of the anus next to the posterior sucker. Motobdella sedonensis can readily be distinguished from M. montezuma in having fewer papillae (10 – 14 vs 14 – 18) and in having the papillae on the dorsal surface not extending round the body as M. montezuma. VII. TAXONOMIC KEYS A. Euhirudinea and Acanthobdella 1a. Body divided into 29 postoral segments; segments subdivided superﬁcially into four annuli (a1, a2, b5, b6) (Fig. 2b): no anterior sucker, mouth on ventral surface of segment III; no jaws; posterior suckering of four segments: two pairs of chaetae on ﬁve consecutive anterior segments; distal ends of chaetae bent to form hooks (Fig. 4b); chaetae absent from remainder of the body; paired ventral male gonopores: single median ventral male gonopore; (Fig. 4a) length 22 mm; ectoparasite of salmonids.................................... Acanthobdella peledina Grube, 1851 1b. Body divided into 32 postoral segments; segments subdivided superﬁcially into 3 – 16 annuli (Figs. 2a and 31), anterior sucker present consists of four segments: mouth on ventral surface of anterior sucker (Fig. 7); jaws present or absent; posterior sucker consists of seven segments; chaetae absent from entire body; median ventral unpaired male and female gonopores (Figs. 3 and 29) ..........................................................................................................................................................................................Euhirudinea 490 Ronald W. Davies and Fredric R. Govedich B. Species of Euhirudinea 1a. Mouth a small pore on ventral surface of anterior sucker through which a muscular pharyngeal proboscis can be protruded (Fig. 7a, b); no jaws or teeth......................................................................................................................................................................... Rhynchobdellida ...............................................................................................................................................................................2 1b. Mouth large, occupying the entire cavity of anterior sucker (Fig. 7c); no protrusible proboscis; jaws with teeth either present or absent (Fig. 8).................................................................................................................Arhynchobdellida........................................3 2a(1a) Body ﬂattened dorso-ventrally and much wider than head (Fig. 1a) (except Placobdella montifera and P. nuchalis); body not cylindrical (except for Helobdella elongata which is subcylindrical); not differentiated into two body regions: anterior sucker ventral, more or less fused to body and narrower than body; body never divided into anterior trachelosome and posterior urosome; eggs in membranous cocoons and young brooded on ventral surface of parent; one, two, three or four pairs of eyes; no oculiform eye spots on posterior sucker; segments 3-annulate (a1, a2, a3) (Fig. 2a) (except Oligobdella biannulata which is 2-annulate)........................... Family Glossiphoniidae ......................................................................................................................................................................4 2b. Body cylindrical and usually long and narrow; body sometimes divided into a narrow anterior trachelosome and a wider posterior urosome (Illinobdella and Piscicolaria) (Figs. 1b, 6); anterior sucker expanded and distinct from body: zero, one, or two pairs of eyes; pulsatile vesicles along the lateral margins present (Piscicola and Cystobranchus) or absent; seven or more annuli per segment (except Piscicolaria reducta which is 3-annulate); oculiform eye spots sometimes present on posterior sucker (Figs. 6b, 19b – d); no brooding of cocoons or young ...................................................................................................................................................Family Piscicolidae.......................................................................................................................................................................................37 3a(1b) Five pairs of eyes arranged in an arch on segments II – VI with the third and fourth pairs of eyes separated by one annulus (Fig. 20a); body elongate (Fig. 1c); jaws with teeth either present or absent; nine or ten pairs of testisacs arranged metamerically (Fig. 11a); pharynx short ...........................................................................................................................................................Family Hirudinidae......................................................................................................................................................................................49 3b. Zero, three, or four pairs of eyes in separate labial and buccal groups (Fig. 25b); body elongate (Fig. 1d); no jaws; testisacs small and numerous (Fig. 11b); pharynx about one-third of body length .....................................................................Family Erpobdellidae .........................................................................................................................................................................................................62 4a(2a) Posterior sucker conspicuous with a marginal circle of 30 – 60 glands and retractile papillae, their positions being indicated dorsally by faint radiating ridges (Fig. 21) ......................................................................................................................................................... Actinobdella .......................................................................................................................................................................................5 4b. Posterior sucker without a marginal circle of glands or retractile papillae ..........................................................................................6 5a(4a) Posterior sucker on short, distinct pedicel with 29 – 31 digitate processes on rim (Fig. 21); somites 3- or 6-annulate; dorsal papillae in 1 – 5 longitudinal rows; length 22 mm ............................................................................Actinobdella inequiannulata Moore, 1901 5b. Posterior sucker on short distinct pedicel with about 60 digitate processes on rim; somites six-annulate with b3 and b5 the largest and most conspicuous; length 11 mm; dorsal papillae in ﬁve longitudinal rows .........................Actinobdella annectens Moore, 1906 6a(4b) Zero, one, or two pairs of eyes; a series of paired accessory eyes sometimes present along body (Fig. 5c) ..........................................7 6b. Three or four pairs of eyes ...............................................................................................................................................................30 7a(6a) Mouth apical or subapical on rim of anterior sucker (Fig. 7a); zero ,one or two pairs of eyes ............................................................8 FIGURE 20 Arrangement of eyes in: (a) Hirudinidae; and (b) Erpobdellidae. FIGURE 21 Lateral view of the posterior sucker of Actinobdella inequiannulata showing the pedicel and the retractile papillae around the margin. 13. Annelida: Euhirudinea and Acanthobdellidae 491 7b. Mouth within anterior sucker and clearly not on rim (Fig. 7b); one or two pair of eyes; gonopores separated by one or two annuli (Fig. 3b) ...........................................................................................................................................................................................18 8a(7a) Male and female gonopores united in common bursal pore; one pair of eyes well separated; body smooth without papillae; six pairs of crop ceca; length 22 m..............................................................................................................Marvinmeyeria lucida Moore, 1954 8b. Male and female gonopores separated by two annuli (Fig. 3b); zero or one pair of eyes (Fig. 5a) [Placobdella hollensis has several pairs of accessory eyes on the neck (Fig. 5c)]; eyes close together or conﬂuent (Fig. 5b) (except Placobdella montifera and P. nuchalis which have eyes well separated); body usually papillated; seven pairs of crop ceca ................................................................ Placobdella / Oligobdella ....................................................................................................................................................................9 9a(8b) One small pair of eyes on segment II and one pair of larger sometimes coalesced eyes; no supplementary eyes ................................10 9b. One pair of small eyes on segment II and a larger sometimes coalesced pair of eyes on segment III followed by an indeﬁnite number of pairs of accessory eyes (Fig. 5c); tubercles large and rough; length 30 mm...........................Placobdella hollensis (Whitman, 1892) 10a(9a) Anus between segments XXIII and XXIV with the 16 postanal annuli forming a slender stalk (pedicel) which bears the posterior sucker; no papillae; length 35 mm .....................................................................................Placobdella pediculata (Hemingway, 1908) 10b. Anus close to the posterior sucker; no pedicel ..................................................................................................................................11 11a(10b) Margins of posterior sucker denticulate (Fig. 22) head expanded and discoid and set off from body by a narrow neck (Fig. 23) .....12 11b. Margins of posterior sucker not denticulate .....................................................................................................................................13 12a(11a) Dorsum with three prominent tuberculate keels or ridges (Fig. 23); length 16 mm.............................................................Placobdella montifera Moore, 1906 12b. Dorsum smooth; no keels or ridges; length 25 mm ......................................................Placobdella nuchalis Sawyer and Shelley, 1976 13a(11b) Medium longitudinal row of tubercles on dorsal surface are large and conspicuous; tubercles bear several papillae giving a rough, warty appearance; ventrum unstriped; length 40 mm.......................................................................Placobdella ornata (Verrill, 1872) 13b. Dorsal papillae, if present, simple cones with smooth domes ...........................................................................................................14 14a(13b) Dorsum smooth; dorsal papillae inconspicuous or absent ................................................................................................................15 14b. Dorsal papillae small and conical but distinct ..................................................................................................................................17 15a(14a) Dorsum smooth; no papillae or tubercles; dorsum with conspicuous white genital and anal patches; one or more medial white patches; white bar on neck ...............................................................................................................................................................16 15b. Dorsal papillae inconspicuous or absent; when present, papillae arranged in ﬁve longitudinal rows; ventral surface with 11 – 12 blue, brown, or green stripes; annulus a3 in middle of body without distinct cross furrow; length 65 mm ........................................... Placobdella parasitica (Say, 1824) FIGURE 22 Posterior sucker of Placobdella montifera showing the denticulate margins. FIGURE 23 Placobdella montifera showing the expanded discoid head and dorsum with three prominent tuberculate dorsal ridges (keels). 492 Ronald W. Davies and Fredric R. Govedich 16a(15a) 3-annulate (Fig. 2a); posterior sucker not conspicuous; length 11 mm .............................................................Placobdella translucens Sawyer and Shelley, 1976 16b. 2-annulate; posterior sucker large; length 7 mm ...............................................................................................Oligobdella biannulata (Moore, 1900) 17a(14b) Dorsum with 5 – 7 longitudinal rows of small papillae; papillae on posterior sucker; annulus a3 in middle of body with distinct cross furrow; length 45 mm ................................................................................................................Placobdella papillifera (Verrill, 1872) 17b. Dorsum with few or numerous small papillae in ﬁve longitudinal rows; length 50 mm ..................................Placobdella multilineata Moore, 1953 18a(7b) One or two pairs of eyes; if only one part of eyes present, these are close together or lobed indicating coalescence (Fig. 5a, b); gonopores separated by two annuli (Fig. 3b)............................................................................................................................................19 18b. One pair of eyes which are well separated (Fig. 24); gonopores separated by one annulus .............................................Helobdella 23 19a(18a) One large pair of eyes on segment IV sometimes coalesced (Fig. 5a, b); seven pairs of crop ceca; papillae absent; length 20 mm ...............................................................................................................................................Batracobdella paludosa (Carena, 1824) 19b. One pair of eyes on somite II and a larger pair of eyes on segment III eyes sometimes conﬂuent (Fig. 5b); one pair of diffuse salivary glands; two annuli separating gonopores (Fig. 3b) ............................................................................................................................... Desserobdella ...................................................................................................................................................................................20 FIGURE 24 Helobdella stagnalis (or H. california) showing the chitinous scute (nuchal plate) on the dorsal surface and the single pair of eyes. FIGURE 25 Dorsal view of: (a) Theromyzon trizonare; (b) Glossiphonia complanata; and (c) Alboglossiphonia heteroclita showing the arrangement of the eyes. FIGURE 26 (a) Myzobdella lugubris; and (b) Piscicolaria reducta showing the trachelosome and urosome without pulsatile vesicles, the relative sizes of the eyes, and the weakly developed posterior sucker. 13. Annelida: Euhirudinea and Acanthobdellidae 493 20a(19b) Dorsum with white genital and anal patches, one or more medial white patches and a white bar on neck; no papillae on dorsum; ﬁve longitudinal rows of white prominences surrounded by yellowish dots, equidistant longitudinally and transversely; body very ﬂattened; length 10 mm .....................................................................................................Desserobdella michiganensis Sawyer, 1972 20b. Dorsum without white patches and bar on neck ..............................................................................................................................21 21a(20b) Posterior sucker separated from body on a short pedicel; dorsum smooth; eight rows of inconspicuous sensillae on dorsal annuli; only recorded parasitism on Cryptobranchus alleganiensis; length 17 mm .....................................................................Batracobdella (Desserobdella) cryptobranchii Johnson and Klemm, 1977 21b. Posterior sucker not on pedicel; posterior sucker small; low small papillae present; 3-annulate (Fig. 3a); length 25 mm ..................22 22a(21b) Dorsal papillae present; lateral metameric and dorsal color markings; 60 radiating papillaeon rim of posterior sucker; feeds mainly on ﬁsh; length 10 mm .....................................................................................................................Deserobdella phalera (Graf, 1899) 22b. No dorsal papillae; no lateral metameric and dorsal colour markings; no papillae on rim of posterior sucker; feeds mainly on amphibians; length 5 mm ....................................................................................................................Desserobdella picta (Verrill, 1872) 23a(19b) Brown, horny, chitinous scute (nuchal plate) on the dorsal surface of segment VIII (Fig. 24) ...........................................................24 23b. Without a nuchal plate .....................................................................................................................................................................25 24a(23a) Pigment pattern arranged in longitudinal stripes on dorsum; six branched pairs of crop ceca; posterior sucker pigmented on dorsum; length 18 mm ...................................................................................................................Helobdella california Kutschera, 1988 24b. No longitudinal stripes on dorsum; six unbranched pairs of crop ceca, the last pair posteriorly; posterior sucker unpigmented; length 14 mm ............................................................................................................................Helobdella stagnalis (Linnaeus, 1758) 25a(23b) Dorsal surface smooth......................................................................................................................................................................26 25b. Dorsal surface with 3 – 7 longitudinal series of papillae, or with scattered papillae...........................................................................28 26a(25a) Body unpigmented and translucent; body rounded and subcylindrical; lateral margins almost parallel; posterior sucker small and terminal; one pair of crop ceca; length 25 mm ...............................................................................Helobdella elongata (Castle, 1900) 26b. Body pigmented, with or without longitudinal or transverse bands; body ﬂat with posterior wider than tapering anterior; six pairs of crop ceca ......................................................................................................................................................................................27 27a(26b) Dorsum without transverse pigmentation; dorsum with six prominent longitudinal white stripes alternating with brown stripes; length 14 mm ......................................................................................................................................Helobdella fusca (Castle, 1900) 27b. Dorsum with transverse brown interrupted stripes alternating with irregular white banks; length 10 mm ........................................... Helobdella transversa Sawyer, 1972 28a(25b) Dorsal surface with 5 – 9 longitudinal rows of papillae; papillae large, conspicuous, and rounded; lightly or unpigmented; length 14 mm ...............................................................................................................................................Helobdella papillata (Moore, 1906) 28b. Dorsal surface with three or fewer incomplete series of small papillae or with scattered papillae; length 25 – 30 mm .......................29 29a(28b) Ratio of body width to body length 0.26 0.02 SD. Dorsal surface with longitudinal strips many of which are interrupted by circular zones of unpigmented skin. Five pairs of crop caeca which have numerous secondary diverticula with a bumpy outline ....................................................................................................................................Helobdella triserialis (Blanchard, 1849) 29b. Ratio of body width to body length 0.37 0.01 SD. Dorsal surface with ﬁve narrow longitudinal stripes which extend unbroken along the body. Five pairs of crop ceca which are bilobed and smooth in outline ...................................................Helobdella robusta Shankland, Martindale, Nardelli-Haeﬁnger, Baxter and Price, 1992 30a(6b) Four pairs of eyes on paramedian lines of segments II – V (Fig. 25a); body very soft.....................................Theromyzon................31 30b. Three pairs of eyes (Fig. 25b, c) (coalesced eyes sometimes occur, but the lobed nature indicates the original condition); body ﬁrm ..................................................................................................................................................................................................35 31a(30a) Gonopores separated by two annuli .................................................................................................................................................33 31b. Gonopores separated by three or four annuli ...................................................................................................................................32 32a(31b) Gonopores separated by three annuli cocoons attached to ventral body wall, two female pores.......................................Theromyzon trizonare Davies and Oosthuizen, 1992 32b. Gonopores separated by four annuli cocoons attached directly to substrate, single female pore .......................................Theromyzon tessulatum (Müller, 1776) 33a(31a) Two female pores ..................................................................................................................Theromyzon maculosum (Rathke, 1862) 33b. One female pore ...............................................................................................................................................................................34 494 Ronald W. Davies and Fredric R. Govedich 34a(33b) Female atrium cylindrical ....................................................................................Theromyzon bifarium Oosthuizen and Davies, 1993 34b. Female atrium spherical ......................................................................................................................Theromyzon rude (Baird, 1869) 35a(30b) First pair of eyes closer together than succeeding two pairs, i.e., eyes arranged in triangular pattern (Fig. 25c); no papillae; male and female ducts open into a common gonopore; little pigmentation; generally amber colored; length 10 mm .................................... Alboglossiphonia heteroclita (Linnaeus, 1761) 35b. Eyes equidistant in two paramedian rows (Fig. 25b) ........................................................................................................................36 36a(35b) Dorsum sometimes with papillae on annulus a2 in six longitudinal rows; pair of paramedial stripes on dorsum and ventrum; six pairs of crop ceca; gonopores separated by two annuli (Fig. 3b); length 25 mm................Glossiphonia complanata (Linnaeus, 1758) 36b. Dorsum with large, distinct papillae on annuli a2 and a3; dorsum with numerous, irregularly shaped whitish spots; seven pairs of crop ceca; length 25 mm ............................................................................................................Boreobdella verrucata (Müller, 1844) 37a(2b) Posterior sucker ﬂattened, as wide or wider than the widest part of body (Fig. 1b); pulsatile vesicles on lateral margins of neural annuli of urosome (Fig. 6a, b); zero or two pairs of eyes ......................................................................................................................38 37b. Posterior sucker concave, weakly developed, and narrower than widest part of the body; no pulsatile vesicles (Fig. 26); zero or one pair of eyes .......................................................................................................................................................................................45 38a(37a) Body divided into anterior trachelosome and posterior urosome; 11 pairs of pulsatile vesicles not very apparent in preserved specimens; each pulsatile vesicle covers two annuli; body cylindrical or sometimes slightly ﬂattened; two pairs of eyes; both anterior and posterior suckers wider than body; oculiform spots present on posterior sucker (Fig. 19b, c, d) )(except Piscicola punctata) (Fig. 19a): 14-annulate (except Piscicola punctata which is 3-annulate) ...............................................................Piscicola......................39 38b. Body divided into distinct anterior trachelosome and posterior urosome; 11 pairs of large and distinct pulsatile vesicles easily seen in preserved and live specimens (Figs. 1b and 6); each pulsatile vesicle covers four annuli; well-developed anterior and posterior suckers; no papillae; zero or two pairs of eyes (Fig. 6); oculiform spots on posterior sucker present or absent (Fig. 9a); 7-annulate; length 80 mm .......................................................................................................................Cystobranchus.....................................42 39a(38a) Posterior sucker with 8 – 14 oculiform spots (Fig. 19b, c, d) .............................................................................................................40 39b. Posterior sucker without oculiform spots (Fig. 19a); 3-annulate (Fig. 2a); two pairs of crescent-shaped eyes: gonopores separated by three or four annuli; length 16 mm ...................................................................................................Piscicola punctata (Verrill, 1872) 40a(39a) Posterior sucker with 10 – 14 punctiform oculiform spots (Fig. 19c, d).............................................................................................41 40b. Eight to ten crescent-shaped oculiform spots on the posterior sucker (Fig. 19b); gonopores separated by two annuli; 14-annulate length 31 mm...................................................................................................................................Piscicola salmositica Meyer, 1946 41a(40a) With 10 – 12 (usually 10) punctiform oculiform spots on posterior sucker; dark rays absent from posterior sucker (Fig. 19c): gonopores separated by two annuli (Fig. 3a); anterior pair of eyes like heavy dashes twice as long as wide; length 24 mm Piscicola milneri (Verrill, 1874) 41b. With 12 – 14 punctiform, oculiform spots on posterior sucker, separated by an equal number of dark pigmented rays (Fig. 19d); gonopores separated by three annuli; anterior pair of eyes like ﬁne dashes ﬁve times as long as wide; length 30 mm ..Piscicola geometra (Linnaeus, 1758) 42a(38b) With oculiform spots on posterior sucker.........................................................................................................................................43 42b. Without oculiform spots on posterior sucker....................................................................................................................................44 43a(42a) Eight oculiform spots on posterior sucker; two rows of 12 lateral oculiform spots on each side of body (Fig. 6b); two pairs of eyes; length 7 mm ..............................................................................................................Cystobranchus meyeri Hayunga and Grey, 1976 43b. Ten oculiform spots on posterior sucker; no lateral ocelli; two pairs of eyes; length 15 mm..........................Cystobranchus virginicus Hoffman, 1964 44a(42b) Two pairs of eyes; the ﬁrst pair forming conspicuous dashes at 45° to the longitudinal axis; the second part of eyes ovoid (Fig. 6a); gonopores separated by two annuli (Fig. 3b); length 30 m ............................................................Cystobranchus verrilli Meyer, 1940 44b. No eyes; length 30 mm......................................................................................................Cystobranchus mammillatus (Malm, 1863) 45a(37b) Body divided into small trachelosome and larger urosome (Fig. 26b); length-to-width ratio 3 – 6:1; one pair of eyes; 12- or 14-annulate ....................................................................................................................................................Myzobdella lugubris Leidy, 1851 45b. Body not divided into trachelosome and urosome (Fig. 26a); zero or one pair of eyes......................................................................46 46a(45b) Length-to-width ratio 10:1; zero or one pair of eyes in posterior half of the head; 12- or 14-annulate.......................Illinobdella 47 46b. Length-to-width ratio 4 – 5:1 (Fig. 26b); one pair of eyes; 3-annulate (Fig. 2a); dorsum with six black / brown longitudinal stripes; length 8 mm ......................................................................................................................................Piscicolaria reducta Meyer, 1940 13. Annelida: Euhirudinea and Acanthobdellidae 495 47a(46a) Length-to-width ratio about 10:1; 14-annulate ......................................................................................Illinobdella alba Meyer, 1940 47b. Length-to-width ratio 15:1 ............................................................................................................................................................48 48a(47b) Length-to-width ratio 15:1; one pair of eyes (sometimes absent); anus 15 annuli anterior to posterior sucker; 14-annulate; posterior sucker little more than concavity of the posterior body ...............................................................Illinobdella richardsoni Meyer, 1940 48b. Length-to-width-ratio 18:1; one pair of eyes; anus ten annuli or less anterior to posterior sucker; 12-annulate; posterior sucker half as wide as posterior body ................................................................................................................Illinobdella elongata Meyer, 1940 49a(3a) With copulatory gland pores on the ventral surface, 10 or 11 annuli posterior to the male gonopores (Figs. 27 and 28) ..................... Macrobdella .....................................................................................................................................................................................50 49b Ventral copulatory gland pairs absent ..............................................................................................................................................53 50a(49a) With 24 copulatory gland pores (four rows with six gland pores each) (Fig. 28a) on raised pads; dorsum with red / orange spots; two to two-and-a-half annuli between gonopores; length 150 mm ............................................Macrobdella sestertia Whitman, 1886 50b. With four, six, or eight copulatory gland pores (Fig. 28); with or without red / orange spots on dorsum ..........................................51 51a(50b) Eight copulatory gland pores (Fig. 28b) (2 rows of 4); two annuli between gonopores; dorsum without red / orange spots; length 150 mm ...........................................................................................................................................Macrobdella ditetra Moore, 1953 51b. Four or six copulatory gland pores; dorsum with red / orange spots .................................................................................................52 52a(51b) Four copulatory gland pores (Fig. 27) (two rows of two); ﬁve to ﬁve and-a-half annuli between gonopores; jaws well developed with about 65 monostichodont acute teeth (Fig. 9a); 10 pairs of testisacs; ventrum red / orange; length 150 mm ................................. Macrobdella decora (Say, 1824) 52b. Six copulatory gland pores (Fig. 28c) (three rows of two); four or ﬁve annuli between gonopores; ventrum yellow / grey; length 150 mm ......................................................................................................................................Macrobdella diplotertia Meyer, 1975 53a(49b) Glandular area around gonopores; gonopores separated by three or four annuli ................................................................................. Philobdella .......................................................................................................................................................................................54 53b. No glandular area around gonopores; gonopores separated by 5 – 7 annuli......................................................................................55 54a(53a) 20 – 26 distichodont teeth per jaw (Fig. 9b); lateral margins of dorsum without discrete spots; length 85 mm....................Philobdella ﬂoridana (Verrill, 1874) 54b. 35 – 48 distichodont teeth per jaw; seven pairs of testisacs; irregular black spots on margins of dorsum; length 85 mm....................... Philobdella gracilis Moore, 1901 55a(53b) Jaws absent (Fig. 8a) ........................................................................................................................................................................56 55b. Jaws present (Fig. 8b, c) and denticulate (Fig. 9) ..............................................................................................................................57 FIGURE 27 Ventral view of the male and female gonopores of Macrobdella decora. FIGURE 28 Diagrammatic representation of the relative slopes and positions of the male and female gonopores and copulatory glands of: (a) Macrobdella sestertia; (b) Macrobdella ditetra; and (c) Macrobdella diplotertia. 496 Ronald W. Davies and Fredric R. Govedich 56a(55a) Lower surface of velum smooth (Fig. 8a); gonopores in the furrows between the annuli and separated by ﬁve annuli; pharynx with 12 internal ridges; length 300 mm...................................................................................................Mollibdella grandis (Verrill, 1874) 56b. Lower surface of velum papillate; gonopores in the middle of annuli and separated by ﬁve annuli; pharynx with 15 internal ridges ................................................................................................................................................Bdellarogatis plumbeus (Moore, 1912) 57a(55b) Jaws small and retractable into narrow-mouthed tubular pits; 9 – 25 distichodont (Fig. 9b) teeth per jaw............................................ Percymoorensis ................................................................................................................................................................................58 57b. Jaws large; 35 – 100 acute monostichodont (Fig. 9a) teeth; length 100 mm ...................................Hirudo medicinalis Linnaeus, 1758 58a(57a) Gonopores separated by ﬁve to ﬁve-and-a-half annuli; female gonopore small.................................................................................59 58b. Gonopores separated by six and-a-half to seven annuli; female gonopore large and conical length 200 mm ........................................ Percymoorensis septagon Sawyer and Shelley, 1976 59a(58a) Dorsum with median black stripe.....................................................................................................................................................60 59b. Dorsum with irregular, scattered black spots ....................................................................................................................................61 60a(59a) Dorsum uniformly black or slate gray; posterior sucker narrower than body; jaws with 20 – 25 pairs of teeth; length 250 mm ........... Percymoorensis lateralis (Say, 1824) 60b. Dorsum brown / green; posterior sucker as wide as body; 9 – 14 pairs teeth ................................Percymoorensis kingi (Mather, 1954) 61a(59b) Jaws with 10 – 12 teeth; posterior sucker about 75% of maximal body width and attached by very short pedicel; length 75 mm ........ ...................................................................................................................................Percymoorensis lateromaculata (Mather, 1963) 61b. Jaws with 12 – 16 pairs of teeth; posterior sucker about half maximum body width; length 150 mm...........................Percymoorensis marmorata (Say, 1824) 62a(3b) Somites 5-annulate (b1, b2, a2, b5, b6) (Fig. 29a) with all annuli equal in length; three pairs of eyes; gonopores separated by two annuli (Fig. 3b); length 100 mm ...........................................................................................................................................................63 62b. Somites six or seven-annulate (Fig. 29b); annuli of unequal length units will be either subdivided or longer than the others; in any group of six consecutive annuli at least one annulus narrower or wider than the others; three or four pairs of eyes ........................65 63a(62a) Eyes all similar in size; annuli not raised on dorsum and without papillae; anus located three or four segments anterior to posterior sucker; mouth small; gonopores in furrows separated by 2 – 5 annuli .............................................Erpobdella punctata (Leidy, 1870) 63b. Eyes differ in size with the second and third pairs smaller than the anterior pair; each annulus raised on dorsum with 10 – 18 small white-tipped papillae; anus located at base of posterior sucker; one or two pairs of crop ceca gonopores in furrows separate by two annuli...............................................................................................................................................Motobdella..............................64 64a(63b) 14 – 18 small, white-tipped papillae in a ring about each annulus; mouth very large (4.0 mm); two pairs of crop ceca nephridiopores not visible, internal surface of posterior sucker pigmented ...........................Motobdella montezuma Davies, Singhal and Blinn, 1985 FIGURE 29 (a) Annulation of Erpobdella with all annuli (b1, b2, a2, b5, b6) equal in length; and (b) Nephelopsis, Dina, or Mooreobdella with the annuli (b1, b2, a2, b5, c11, c12) of unequal lengths. 13. Annelida: Euhirudinea and Acanthobdellidae 497 64b. 10 – 14 small, white-tipped papillae on dorsal surface only of each annulus; mouth 1.1 mm in diameter; nephridiopores visible as small white spots; internal surface of posterior sucker not pigmented; one or two pars of crop ceca.................Motobdella sedonensis Govedich, Blinn, Keim and Davies, 1998 65a(62b) Four pairs of eyes, two labial and two buccal of similar size ............................................................................................................66 65b. Zero or three pairs of eyes ....................................................................................................................................Dina / Mooreobdella .........................................................................................................................................................................................................68 66a(65a) Two annuli between gonopores (Fig. 3a); anus one segment anterior to poster sucker; atrial cornua spirally coiled like the horn of a ram (Fig. 18a); length 100 mm ........................................................................................................Nephelopsis obscura Verrill, 1872 66b. Three or more annuli between gonopores; atrial cornua simply curved (Fig.18b) .........................................................................Dina .........................................................................................................................................................................................................67 67a(66b) Three-and-a-half to four annuli between gonopores; body heavily blotched with a median stripe; anus large, opening on a conical tubercle; length 60 mm..............................................................................................................Dina dubia (Moore and Meyer, 1951) 67b. Three to three-and-a-half annuli between gonopores; nearly pigmentless or with a few dark spots; anus small, not on tubercle; length 30 mm ................................................................................................................................................Dina parva Moore, 1912 68a(65b) Dorsum lacking scattered black pigment ..........................................................................................................................................69 68b. Dorsum with scattered black pigment; two annuli between gonopores (Fig. 3a); length 55 mm .....................................Mooreobdella melanostoma Sawyer and Shelley, 1976 69a(68a) Two to two-and-a-half annuli between gonopores............................................................................................................................70 69b. Three to four-and-a-half annuli between gonopores .........................................................................................................................72 70a(69a) Male gonopore surrounded by circle of papillae; zero or three pairs of eyes; two annuli between gonopores (Fig. 3a); length 15 mm Dina anoculata Moore, 1898 70b. No papillae around male gonopore; three (rarely four) pairs of eyes ................................................................................................71 71a(70b) Two annuli between gonopores (Fig. 3a); three (rarely four) pairs of eyes; length 50mm................................................Mooreobdella fervida Verrill, 1872 71b. Either 2 or 2 1/2 annuli between gonopores; three pairs of eyes; length 30 mm.............................Mooreobdella bucera Moore, 1949 72a(69b) Three pairs of eyes; gonopores separated by three annuli; length 50 mm ............................Mooreobdella microstoma (Moore, 1901) 72b. Gonopores separated by 4 – 4 1/2 annuli; three pairs of eyes; length 40 mm.............Mooreobdella tetragon Sawyer and Shelley, 1976 ACKNOWLEDGMENTS It is a pleasure to acknowledge the technical assistance and patience of Mrs Thelma Thompson in the preparation of this chapter. We would also like to acknowledge Dr. Bonnie Bain for her assistance in proofreading and reviewing this chapter and key. VIII. LITERATURE CITED Able, K. W. 1976. Cleaning behaviour in the cyprinodont ﬁshes: Fundus majalis, Cyprinodon variegatus and Lucania parva. Chesapeake Science 17:35 – 39. Anderson, R. S., Raasveldt. L.G 1974. Gammarus predation and the possible effects of Gammarus and Chaoborus feeding on the zooplankton composition in some small lakes and ponds in western Canada. Canadian Wildlife Service Occasional Paper No. 18. Angstadt, J. D., Moore. W.H. 1997. A circadian rhythm of swimming behavior in a predatory leech of the Family Erpobdellidae. The American Midland Naturalist 137:165 – 172. 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Wood Department of Biological Sciences Wright State University Dayton, Ohio 45435 I. Introduction II. Anatomy and Physiology A. External Morphology B. Organ System Function C. Environmental Physiology III. Ecology and Evolution A. Diversity and Distribution B. Reproduction and Life History C. Ecological Interactions D. Evolutionary Relationships IV. Entoprocta V. Study Methods VI. Taxonomic Key to North American Freshwater Bryozoans Literature Cited I. INTRODUCTION II. ANATOMY AND PHYSIOLOGY Bryozoans are among the most commonly encountered animals that attach to submerged surfaces in fresh water. During warm months of the year they are found in almost any lake or stream where there are suitable attachment sites. The variety of forms ranges from wisps of stringy material to massive growths weighing several kilograms. In the days before sand ﬁltration, bryozoans were notorious for clogging the distribution pipelines of public water systems. Today they still foul water intake lines, irrigation nozzles, and wastewater treatment systems. In general, bryozoans are sessile, modular invertebrates with ciliated tentacles that capture suspended food particles. Historically they have included what are now recognized as two very distinct and unrelated phyla: Ectoprocta and Entoprocta. Within the ectoproct bryozoans are two major classes: Phylactolaemata is an exclusively freshwater group and the main focus of this chapter; Gymnolaemata is a vastly larger, polyphyletic collection of species, mostly marine except for a few species in the subclass Ctenostomata which occur in fresh or brackish water. In this chapter ctenostomes are included in the general discussion. Entoproct bryozoans, phylogenetically distant from ectoprocts, are treated in a separate section, but still included in this chapter more for reasons of tradition than systematics. A. External Morphology Ecology and Classiﬁcation of North American Freshwater Invertebrates. 2nd Edition Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved. 1. Zooids and Lophophores The phylactolaemate bryozoan colony is composed of identical zooids fused seamlessly into a single structure (Fig. 1). Major anatomical features are shown in Figure 2. Each zooid in the colony has two basic parts: an organ system, or polypide, which can be partially protruded as a unit; and a body wall which can enclose the entire polypide and which separates the colony’s interior from the surrounding water. These parts are joined in different places by three structures: a tentacular sheath, prominent retractor muscles, and a slender funiculus loosely running from the gut caecum to a point on the interior of the body wall. The polypide bears a prominent lophophore composed of ciliated tentacles arranged around a central mouth. In Fredericella species, the tentacles simply encircle the mouth (Fig. 3). In all other phylactolaemates the lophophore extends dorsally in a bilateral pair of arms, forming a U-shaped structure with an outer row of long tentacles and an inner row of shorter ones (Fig. 4). In most species, each tentacle bears one medial and two lateral tracts of cilia which beat in metachronal waves. Tentacles are loosely joined near their base by an intertentacular membrane, effectively forming a groove 505 506 Timothy S. Wood FIGURE 1 Two zooids of a tubular bryozoan colony, one retracted and the other extended in a feeding position. Scale bar 0.5 mm. between rows of tentacles. Two such grooves converge from the lophophore arms toward the mouth. Adjacent to mouth is the epistome, a small, heavily ciliated lobe which may function in food selection. In gymnolaemate (ctenostome) bryozoans the zooids are tubular and rather delicate looking, with small, conical lophophores (Fig. 8a – d). The body wall is thin and transparent. Colonies are diffuse, consisting of creeping chains of zooids sometimes separated by narrow pseudostolons. 2. Statoblasts A conspicuous feature of phylactolaemate bryozoans is the asexual production of encapsulated dormant buds, called statoblasts. The various distinctive types are widely used for species identiﬁcation. Most species produce free statoblast that can be released from the colony as disseminule, each bearing a periph- eral band of sclerotized chambers normally ﬁlled with a gas for buoyancy. In most species, the free statoblasts (often called ﬂoatoblasts) acquire their buoyant gas in a late developmental stage within the colony, while in a few other species the ﬂoatoblast must be dried or frozen before becoming buoyant. Many species with self-inﬂating ﬂoatoblasts also produce specialized sessile statoblasts (“sessoblasts”) cemented to the substrate upon which colonies grow, and bearing a distinctive lamella around the rim. A third type of statoblast, the piptoblast, is a simple, beanlike structure known only in Fredericella species, lacking specialized external structures and held ﬁrmly within the tubular branches of the colony. Mukai (1982) provides an excellent overview of statoblast development. All statoblasts are composed of paired valves joined at an equatorial suture. In most ﬂoatoblasts the valves have two structural layers (Fig. 5a). An inner capsule contains germinal tissue and food reserves; the outer periblast covers the capsule completely and includes the peripheral annulus and a central fenestra. In most species, the so-called dorsal (or cystigenic) valve generally has a smaller fenestra and correspondingly wider annulus than the ventral (deutoplasmic) valve. At least one species forms leptoblasts in which the capsule is lacking and a fully formed zooid is enclosed by the periblast only Sessoblasts have a similar arrangement of inner capsule and outer periblast. The thin peripheral lamella is considered homologous with the ﬂoatoblast annulus, although it is never inﬂated. The basal valve provides a large, irregular ring which adheres tightly to the substrate. Gymnolaemate bryozoans do not produce statoblasts. Instead, many of the species restricted to fresh or brackish water form special thick-walled hibernacula. These irregularly shaped bodies are integrated into the colony structure and attached ﬁrmly to the substrate, remaining long after the colony disintegrates. Like statoblasts, hibernacula survive desiccation, changes of salinity, and other suboptimal conditions, although the limits of their resistance have not been documented. B. Organ-System Function 1. Coelom, Neural System, and Body Wall Most ectoproct colonies have a spacious coelom shared by all zooids. A clear coelomic ﬂuid is circulated by cilia on the peritoneum. The main gastric coelom communicates through an incomplete diaphragm with the coelom of the lophophore, which extends fully into every tentacle. A small nerve ganglion is located on the diaphragm at the base of the epistome between the mouth and the 14. Bryozoans FIGURE 2 507 Schematic longitudinal section of a generalized colony (based on Mukai, 1982). anus. A single nerve tract extends to each of the paired arms of the lophophore, with nerves branching off to each tentacle. Other nerves from the ganglion innervate the epistome, the tentacle sheath, and the digestive tract. There is no nervous communication between zooids. The body wall is composed of living tissues, collectively called the endocyst, together with a nonliving outer ectocyst (Fig. 2). Lining the main coelom is a peritoneum bearing scattered tracts of cilia. Behind the peritoneum lies a thin, longitudinal muscle layer followed by a basement membrane. Overlying this is a thin layer of circular muscles and a single layer of epidermis. Near the apex of the zooid, the endocyst folds inward to form a ﬂat pocket, the vestibule, then joins the FIGURE 3 Portion of a Fredericella colony showing characteristic circular outline of the lophophore. Scale bar 2 mm. 508 Timothy S. Wood FIGURE 4 Portion of a Lophopodella colony showing the typical horseshoe-shaped lophophore. Scale bar 2 mm. lophophore as an eversible tentacle sheath. When the polypide is fully retracted, a well-deﬁned opening, or oriﬁce, remains at the zooid tip. A pore in the vestibular body wall of many species is normally closed except to permit the release of ﬂoatoblasts from living colonies. In some tubular species, a portion of the body wall grows inwardly to form a kind of sclerotized, reinforc- FIGURE 5 Sclerotized components of generalized phylactolaemate statoblasts in exploded view. (a) Floatoblast; (b) sessoblast. ing ring or incomplete septum. Those branches of tubular colonies that are attached to a substrate may have a raised line, or keel, running along the outer surface. The composition of the ectocyst is highly variable among species. In most tubular colonies it is sclerotized and remains intact for a time after living parts have died. In at least two species, the ectocyst is composed largely of chitinous microﬁbrils with random orientation. A thin, proximal electron-dense layer is overlain by a thicker stratum of looser ﬁbrils (Goethals et al., 1984). Many organic and inorganic particles adhere to this outer coat, including bacteria which also penetrate the interior of the loose layer. The ectocyst in these species varies from thin and ﬂexible to leathery, opaque, and brittle. With age the ectocyst may darken as the the chitin is thickened and tanned. Thus, the growing tips of the zooids are often distinctly lighter in color than the rest of the colony. Rao et al. (1978) found that with increasing age a second, separate sclerotized layer can form in the inner body wall of Plumatella casmiana. Mukai et al. (1984) have shown that the body wall breaks down when contact is made between adhesive pads from germinating statoblasts of the same species. The resulting colonies are conﬂuent, possibly allowing a sort of outcrossing when sperm are circulated within the common coelom. In nontubular phylactolaemates the ectocyst is gelatinous and often restricted to certain parts of the 14. Bryozoans colony. Mukai and Oda (1980) reported many vacuolar cells, apparently with a secretory function, in the epidermis of these species. The massive jellylike substance produced by Pectinatella magniﬁca is more than 99% water and contains partially denatured protein along with some chitin, calcium, and sodium chloride (Morse, 1930). 2. Feeding Mechanics The lophophore is a complex feeding organ capable of capturing a wide variety of food particles. In contrast with marine bryozoans, the lophophore of most freshwater species is large and powerful, bearing many closely positioned tentacles. Such an organ may be particularly well suited to lentic habitats; certainly those species with the largest lophophores seem to grow best in quiet water. In species where lophophores are tightly crowded (e.g., Cristatella or Pectinatella) the processed water exits along deﬁned excurrent channels. This results in a signiﬁcant feeding efﬁciency, according to the model proposed by Eckman and Okamura (1998). In a study using Plumatella repens, the ingestion rate per zooid was higher for large colonies than for small ones, and also varied directly with ambient water current (Okamura and Doolan, 1993). Gilmour (1978) showed that differential beating of the lateral and frontal cilia sort particles by density, rejecting heavier, inedible materials before they reach the epistome. Kaminski (1991a) made a distinction between ciliary feeding, which brings in nannoplankton (5 – 10 m), and tentacular feeding in which the tentacles orient segments of ﬁlamentous algae for ingestion. Flicking movements of individual tentacles sometimes throw particles toward the mouth; at other times the tips of tentacles are brought together to prevent the escape of an active protozoan or rotifer. Exactly how the lophophore captures minute particles is not entirely clear. In U-shaped lophophores the membrane joining the base of all tentacles creates an intertentacular groove of relatively still water. Bullivant (1968) suggested that particles accelerating toward the lophophore are impelled the ﬁnal short distance into this groove by their own momentum, while the water is deﬂected abruptly to the sides. This would be consistent with the observation that species with the most powerful lophophores are able to capture the smallest particles (Kaminski, 1984). Food particles entering the mouth collect momentarily in a short, ciliated pharynx, then are swallowed through a narrow esophagus to the Y-shaped stomach. A short unciliated cardia leads to the long, cylindrical cecum, where particles are churned by rhythmic peristaltic contractions. In well-fed colonies waste products collect in brownish, longitudinal bands, giving the ce- 509 cum a striped appearance. A short proximal part of the cecum, the pylorus, leads through a narrow sphincter to a straight intestine. Here processed particles are consolidated, infused with mucus, and ejected as a fecal pellet through the anus, which lies just outside the whorl of tentacles. The bundle of eliminated material is too large to be re-ingested by neighboring zooids, and it falls away from the colony. 3. Reproductive Systems and Larvae Phylactolaemate bryozoans are sexually active during a single brief period of the year. Sperm develop in conspicuous masses on the funiculus of certain zooids, from which they later detach to circulate passively throughout the colony coelom. Egg clusters develop on the peritoneum ventrally within the zooid. There is now direct evidence for outcrossing in populations of Cristatella mucedo based on RADP assays (Jones et al., 1994), but the mechanism for gamete transfer is unknown. Sperm motility has never been reported, nor have sperm even been observed leaving one colony or entering another. Self-fertilization thus appears likely in most species except in instances where two colonies have become conﬂuent. Assuming that fertilization occurs in the coelom, the zygote somehow migrates to a special sac formed as an ingrowth of the body wall. The embryo develops into a specialized free-swimming structure usually called a “larva,” although technically a motile colony. Embryology of the planktonic larva and the steps leading to its metamorphosis are summarized by Hyman (1959). Fine structure is described by Franzén and Sensenbaugh (1983). The larva is composed of a heavily ciliated outer mantle and an inner pear-shaped mass (Fig. 6a). The inner parts include one to four fully formed polypides together with their funiculi and associated musculature. Released from the colony usually after dark, the larva swims with its aboral pole forward, which contains special gland cells and a neural center. Settling normally occurs within an hour (Fig. 6b – d). Among most Gymnolaemata, sexual reproduction leads to the development of small larvae totally unlike those of Phylactolaemata. Settling and metamorphosis occur shortly after their release, since the larvae have no feeding or digestive organs. The larva of Paludicella and its embryological development were described brieﬂy by Braem (1896). However, virtually nothing is known of larval ecology or behavior among freshwater ctenostome bryozoans. All bryozoan zooids are capable of budding new zooids. In the Phylactolaemata, buds arise from a speciﬁc site on the midventral wall of the parental zooid (Figs. 2 and 6a). The development of a new zooid is accompanied by the appearance of new bud primordia, 510 Timothy S. Wood FIGURE 6 Settling and transformation of a larva. Scale bar for all ﬁgures 500 m. (a) Swimming larva containing two fully developed zooids; the more tapered end is the oral pole; (b) the mantle peels back immediately after the larva attaches to a glass substrate; (c) constricted mantle appears as a dark mass; the two zooids are now clearly visible, each with its own duplicate bud; (d) the mantle is drawn into the body cavity, lophophores are extended, and the two zooids begin feeding (from Zimmerman, 1979). which may or may not develop further. Brien (1953) showed that every zooid bears two bud primordia: a “main bud,” which forms the ﬁrst daughter zooid, and an “adventitious bud” between the main bud and parental zooid, which becomes the second daughter zooid. In addition, each main bud itself has a small “duplicate bud” primordium on its ventral side. In the process of budding, duplicate and adventitious buds become main buds, and new duplicate and adventitious buds are formed. These relations have been nicely clariﬁed by Mukai et al. (1987). In ctenostomes the new zooidal bud originates from an outswelling of the body wall of a parental zooid. An interior wall then grows across the base of the bud to separate it from the parental body cavity. Most often, a new bud develops at the distal end of a lineal series of zooids, and as it grows a wall forms at the tip to separate it from the body cavity of the next distal bud in the series. In addition to asexual budding, certain phylactolaemate species increase their numbers signiﬁcantly by an active ﬁssion of the colony. This is a common occurrence among the globular colonies of Cristatella, Pectinatella, Lophopodella, Lophopus, and Asajirella. The products of ﬁssion glide slowly apart along the substrate by a means that is not fully understood. C. Environmental Physiology Little is known about the digestive physiology of phylactolaemate bryozoans, except that the esophagus environment is alkaline, the stomach is acidic, and the intestinal pH is neutral (Marcus, 1926). Passage time through the gut varies from one to 24 hs depending on the ingestion rate. Zones of basophilic and acidophilic cells line the cecum, but there is no agreement among investigators on their function. Statoblasts have been the subject of considerable study. The period of obligate dormancy imposed on most statoblasts serves to maintain populations through periods of unfavorable environmental conditions. Those statoblasts not ﬁxed to immobile substrates may also function in passive dispersal, carried either by water currents or by migrating animals. Brown (1933) demonstrated viability of Plumatella statoblasts after they had passed in separate trials through the digestive tracts of a salamander, frog, turtle, and duck. Like statoblasts, the hibernacula of gymnolaemates maintain the populations during periods of unfavorable conditions. Unlike most statoblasts, however, they are an integral part of the colony, can never be released freely into the water and thus would not normally function as independent disseminules. The resistance of statoblasts to environmental stress has been reviewed by Bushnell and Rao (1974). Most Plumatella and Fredericella statoblasts survive desiccation or freezing for periods of 1 to 2 years. Some Plumatella ﬂoatoblasts can germinate after more than 4 years in cold water. Lophopodella carteri statoblasts have remained viable during dry storage for over 6 years. Other less hardy statoblasts include those of Pectinatella magniﬁca, which do not withstand desiccation and survive only brief periods of freezing temperatures. Statoblasts stored in water at a favorable temperature eventually germinate after a variable dormant period. However, when that period is prolonged by continuous exposure to cold, dry, anaerobic, or other unfavorable conditions, a near simultaneous germination can be achieved when a suitable environment is 14. Bryozoans restored. In some species, light is an important factor for germination (Oda, 1980; Richelle et al., 1994). Ultrastructure of the statoblast suture and its possible relation to germination has been studied by Bushnell and Rao (1974) and Rao and Bushnell (1979). The budgeting of energy resources for colony growth and reproduction is poorly understood. In many cases, gametogenesis precedes statoblast production, but the two processes may also operate concurrently. The ﬁrst zooid to germinate from a statoblast never forms statoblasts itself, but it may participate in gametogenesis. In laboratory colonies of Plumatella emarginata, Mukai and Kobayashi (1988) found sexual activity only in certain colonies, which varied in size from as few as 10 zooids. Only those colonies with the most vigorous development of testes eventually formed mature larvae. The regulation of sessoblast development in Plumatellidae has not yet been clariﬁed. Apparently, statoblast primordia can differentiate into either ﬂoatoblasts or sessoblasts, and this differentiation becomes evident during the late epidermal-disk stage (Mukai and Kobayashi, 1988). In natural populations, Wood (1973) noted that most sessoblasts were formed either early or late in the season, leading him to suppose that this was a response to suboptimal conditions. However, laboratory-reared Plumatella colonies frequently form both ﬂoatoblasts and sessoblasts under apparently favorable growing conditions. III. ECOLOGY AND EVOLUTION A. Diversity and Distribution Freshwater bryozoans are generally restricted to relatively warm water, ﬂourishing at 15 – 28°C. However, Cristatella mucedo has been found at 6°C (Bushnell, 1966), and Lophopus crystallinus survives at 0°C for brief periods (Marcus, 1934). Fredericella indica lives through the winter in most of North America, budding new zooids at 3°C and producing piptoblasts above 8°C. All of these species also grow well at higher temperatures. Only Stephanella hina has never been found above 17°C and is probably restricted to cool water (Smith, 1989a). Temperatures as high as 37°C were recorded for living Plumatella nitens and P. fruticosa (Bushnell, 1966). Shrivastava and Rao (1985) noted that Plumatella emarginata in India shows only meager growth at 34°C. Phylactolaemates tolerate a wide range of pH, but they favor slightly alkaline water. This was the conclusion of Tenney and Woolcott (1966) in a North Carolina study where pH ranged from 6.3 to 8.0. A Fred- 511 ericella species (probably F. indica) has been collected in acidic conditions as low as pH 4.9 (Everitt, 1975), Plumatella fruticosa at pH 5.7 (Bushnell, 1966), and both P. repens and Hyalinella punctata at pH 6.3 (Tenney and Woolcott, 1966). Most species occur in both still and running water, but Plumatella emarginata and Fredericella indica grow especially well in lotic habitats. Among those species associated with still waters are Plumatella nitens, P. rugosa, P. recluse, Lophopus crystallinus, Hyalinella punctata, and P. orbisperma. Many common species tolerate turbid water, but Pectinatella magniﬁca does not, possibly because its large colonies are necessarily more exposed to settling particles (Cooper and Burris, 1984). The reputation of bryozoans as clean water species is not entirely warranted. Henry et al. (1989) found Fredericella sultana, Plumatella emarginata, and P. fungosa thriving in high concentrations of heavy metals and PCBs, although none of these had accumulated in the tissues. Bushnell (1974) also recognized these species to be particularly tolerant of extreme contamination from sewage and industrial wastes. More recently, Plumatella vaihiriae has been noted as an important fouling species in secondary clariﬁers and tertiary sand ﬁlters of wastewater treatment plants (Wood and Marsh, 1999). According to Sládeček (1980), both Plumatella repens and P. fungosa survive at only 30% saturation of dissolved oxygen. Many species, such as Lophopodella carteri ﬂourish both in the laboratory and the ﬁeld when supplied with large quantities of suspended organic particles. In Europe, the distribution of Plumatella fungosa is correlated with nutrient-enriched water (Job, 1976). Pectinatella magniﬁca in the United States, Japan, and Korea become luxuriant in areas that are visibly eutrophic. One clearly limiting factor for all species is the availability of suitable surface on which to grow. Almost any solid, biologically inactive material is acceptable, including rocks, glass, plastics, automobile tires, and aged wood. Surfaces that are seldom successfully colonized include newly dead wood, corroded metal, and oily or tarred materials. Aquatic plants are common substrates, although in a detailed survey of bryozoan – plant relations, Bushnell (1966) found little evidence for species speciﬁcity. Among large, ﬂoating leafed plants, the lotus lily, Nelumbo lutea, supports heavy growths of bryozoans, while other lilies, such as Nymphaea or Nuphar, are seldom colonized. Free ﬂoatoblasts, of course, are passive carriers of living material to new substrates. Swimming larvae, on the other hand, actively select attachment sites. Hubschman (1970) demonstrated particle size discrimination in larvae of Pectinatella magniﬁca, concluding that 512 Timothy S. Wood rock particles smaller than 1-mm diameter were avoided. Additional selection criteria are likely. Curry et al. (1981) found evidence for selective settling of Paludicella articulata larvae on mussel shells. In general, freshwater bryozoan species are widely distributed but variable in local abundance. Some species appear to be distributed as metapopulations with regular cycles of local extinction followed by new immigration (Okamura, 1997). Two of our most common species, Fredericella indica and Plumatella reticulata, are rare in Europe (Massard et al., 1992; Massard and Geimer, 1996); similarly, the abundant European Plumatella fungosa is seldom found on our continent. A near worldwide distribution is enjoyed by Plumatella casmiana, Lophopodella carteri, and Paludicella articulata. However, the old notion that many other species have a cosmopolitan distribution was apparently based on faulty identiﬁcation. Cristatella mucedo is restricted to holarctic regions, while Fredericella browni and Pottsiella erecta are most common where winters are mild. About 25% of North American species are considered rare, and at least one, Lophopus crystallinus, is seriously endangered. Some species are known to be expanding their range: Pectinatella magniﬁca jumped from North America to Europe and Japan and has recently invaded Korea; Lophopodella carteri is increasingly common since its introduction to North America in the 1930s; and the once scarce Plumatella vaihiriae is popping up in lakes and wastewater treatment facilities across the continent. Species believed to be endemic to North America now include Plumatella nitens (Wood, 1996), P. orbisperma (Ricciardi and Wood, 1992), P. recluse (Smith, 1992) and the tiny ctenostome, Sineportella forbesi (Wood and Marsh, 1996). All known North American species have been reported east of the Mississippi River and north of the 39th parallel. Nineteen of the 24 species occur in states bordering the Great Lakes and an additional two species are described only from New England. Only the brackish Victorella pavida has not been found further north than Chesapeake Bay. Relatively little is known about freshwater bryozoans west of Ontario and the Mississippi River. Most states and provinces have no published records of bryozoans. However, various reports show Colorado and Utah with natural populations of “Plumatella repens”, P. casmiana, P. emarginata, P. fruticosa, P. fungosa, Fredericella indica, and Cristatella mucedo. Other western sightings show Cristatella mucedo in British Columbia (Reynolds, 1976), and “Plumatella repens” in Arizona and Nevada (Wilde et al., 1981; Dehdashti and Blinn, 1986). Pectinatella magniﬁca appears to be well established in eastern Texas (Neck and Fullington, 1983) and in the Paciﬁc Northwest (personal commu- nications). In Nebraska, the most abundant species appears to be Plumatella vaihiriae (Wood, unpublished). The most recent regional surveys are those of Bushnell (1965a – c) in Michigan, Wood (1989) in Ohio, Smith (1989b) in Massachusetts, Ricciardi and Reiswig (1994) in eastern Canada, and Marsh and Wood in Illinois (in preparation). Each of these has revealed signiﬁcant new species records for North America. B. Reproduction and Life History In most parts of the United States, bryozoan populations have two or more generations during the growing season. Only Fredericella indica typically is found throughout the year living even under a cover of ice. Populations of other species are normally maintained during unfavorable seasons by dormant statoblasts. An unusual exception to this pattern was reported by Dehdashti and Blinn (1986) in a population of “Plumatella repens” inhabiting an Arizona cave, where nearly constant conditions permitted uninterrupted, active growth. Winter dormancy in statoblasts is broken by conditions favorable to colony growth. In temperate climates the principal triggering factor appears to be temperature. Viable statoblasts often germinate within a few days of each other when the water temperature rises above 8°C. In those regions where winter water temperature never approaches freezing, the time of germination may be more variable. The single zooid that emerges from a statoblast is termed the ancestrula. It eventually buds one-to-ﬁve new zooids, each similarly capable of multiple zooid budding. Colonies grow rapidly as water temperature rises. Bushnell (1966) described naturally occurring colonies of Plumatella nitens (identiﬁed at the time as P. repens) which tripled and quadrupled in size within one week. Doubling times of ﬁve days have been reported for both Plumatella casmiana and Fredericella indica (Wood, 1973). In species of Plumatella, Fredericella, and Lophopodella statoblasts may appear in colonies having fewer than ﬁve zooids; in most other species they are not formed until colonies are much larger. Among natural populations, a second generation usually arises either from the statoblasts or larvae of early spring colonies, or even from fragments of colonies surviving from the ﬁrst period of growth. Third generation colonies also are known, but any statoblasts they produce normally remain in diapause until the following spring. Plumatella casmiana is unique in releasing thinwalled statoblasts which germinate immediately, so there may be many overlapping generations throughout the growing season. 14. Bryozoans Gametogenesis, when it occurs, is normally encountered in the ﬁrst spring generation of colonies, with larvae released 2 – 4 weeks after fertilization. Sperm masses develop on the funiculus as ova appear on the peritoneum. The sperm later break free to circulate in the coelom for 1 to 2 weeks. This is followed by larval development and eventual release. There appears to be little overlap in larval release dates among closely related species living in the same area. In two Ohio lakes, freeswimming larvae of Plumatella rugosa appeared throughout June; P. casmiana larvae were released during July; then came Hyalinella punctata in late July – early August, followed by Plumatella emarginata in late August – early September (Zimmerman, 1979). Sexual activity in phylactolaemates is often reported as rare even in such careful studies as that of Wöss (1996). Among the Plumatellidae, with a proliﬁc production of asexual statoblasts, sexual reproduction would seem a relatively unimportant means for recruitment. However, in Pectinatella magniﬁca and Hyalinella punctata, with only one statoblast generation per year, sexually produced larvae play a signiﬁcant role in establishing new colonies (Hubschman, 1970; Wöss, 1999). Likewise, in Fredericella species, because of their ﬁxed statoblasts, larvae are assumed to be instrumental for both population growth and dispersal. C. Ecological Interactions The constant ﬂow of water through bryozoan lophophores creates a favorable environment for the microscopic aufwuchs community. Protozoans, rotifers, gastrotrichs, microcrustaceans, and other small animals congregate especially on and around branching tubular colonies of bryozoans. Flatworms, oligochaetes, snails, orbatid mites, crayﬁsh, and such insect larvae as caddisﬂies and midges occasionally graze on the living zooids. Predation is seldom extensive. Chironomid larvae enter old tubes of Plumatella repens, hastening their disintegration, and occasionally damaging living portions of the colony. Some workers consider such damage to be accidental rather than the result of direct feeding, while others regard chironomid larvae as important predators. Colonies of P. casmiana escape such harm, possibly because larvae cannot ﬁt themselves inside the narrow, branching tubules, but instead build detritus tubes alongside the exterior colony wall where they cause little harm. Pectinatella magniﬁca is often a host to chironomid larvae, which ﬁnd shelter by burrowing into the gelatinous base close to the substrate and causing no apparent damage. One of these, Tendipes pectinatellae, is reported to be speciﬁcally commensal on P. magniﬁca (Dendy and Sublette, 1959). 513 Myxozoan and microsporidian parasites have long been identiﬁed with freshwater bryozoan hosts. These have been recently conﬁrmed and further described from Cristatella mucedo by Okamura (1996) and Canning et al. (1996, 1997). Myxozoans are easily seen with light microscopy when they circulate in the coelomic ﬂuid. Infected colonies lose their normal vigor and soon die. Buddenbrockia, an active, wormlike parasite, has been described from the coelom of several plumatellids (e.g., Schröder, 1912; Marcus, 1941). Easily detected through the clear body wall of Hyalinella punctata, for example, it seems to cause little distress to the hosts as long as food remains abundant to the host. Life cycle details of any of these parasites are so far unknown. Extensive predation by ﬁsh has not been veriﬁed. Walburg et al. (1971) lists “bryozoans” among the stomach contents of six reservoir ﬁsh species, but provides no details. When Dendy (1963) observed bluegills biting off pieces of Plumatella sp., he believed the ﬁsh were doing so only to obtain insect larvae associated with the colonies. A homogenate of Lophopodella carteri tissues is highly toxic to ﬁsh (Meacham and Woolcott, 1968), and attempted feeding by ﬁsh on this or any other gelatinous species has never been observed. Freshwater prawns will graze on colonies of Plumatella vaihiriae, but only when no other food is available (Bailey-Brock and Hayward, 1984), suggesting either a lack of nutritional value or a repellant chemistry of these colonies. As sessile suspension feeders, bryozoans handle a wide variety of food. Richelle et al. (1994) clearly demonstrated the ability of Plumatella fungosa to thrive on a diet of suspended bacteria. Other ingested particles include diatoms, desmids, green algae, cyanobacteria, dinoﬂagellates, rotifers, small nematodes, protozoa, and even microcrustaceans, along with bits of detritus and inorganic materials. In a comparative study of three species, Kaminski (1984) found that 95% of ingested particles were under 5 m in diameter. Plumatella repens, with its wide mouth and strong gut musculature, ingested larger organisms than did either Cristatella mucedo or Plumatella fruticosa. These included rotifers (Keratella sp.), colonial green algae, and cyanobacteria as large as 75 m. In general, organisms with long body extensions were able to avoid ingestion by bryozoans, while small, rounded shapes were taken most easily. Analysis of stomach contents alone does not reveal the important sources of bryozoan nutrition. Rotifers and green algae have been known to pass through the gut completely unharmed (Hyman, 1959) although there are reports that a large portion of ingested organisms are variously damaged (Rüsche, 1938; Kaminski, 1991a). 514 Timothy S. Wood The quantity of seston removed from a 460-ha eutrophic lake by Plumatella fungosa is estimated at 15 metric tons per year (Kaminski, 1991b). At the same time, 8.8 tons of fecal pellets are deposited in the sediments, about twice the amount contributed by ﬁsh or waterfowl, but less than that deposited by molluscs. D. Evolutionary Relationships Ectoprocta is one of three animal phyla collectively known as lophophorates. The other two, Brachiopoda and Phoronida, are represented by a small number of solitary, mostly sessile, marine animals. For all of these, the main unifying feature is a lophophore with hollow tentacles containing an extension of the coelom, and with cilia beating in metachronal waves to bring water and suspended food toward the mouth. As a group, the lophophorates show no clear afﬁnity with any other invertebrates. The semblance of radial cleavage, mesoderm formation, and other elements of morphology and development in branchiopods appears distinctly deuterostome (Zimmer, 1973; Emig, 1984). However, recent analysis of 18S rDNA sequence data suggest that lophophorates are protostomes (Halanych et al., 1995), a conclusion supported by other biochemical and morphological studies (e.g., Willmer, 1990; Gutmann, 1978). Structural similarities have been noted between ectoprocts and the wormlike sipunculids. The anterior introvert of a sipunculid is protruded and withdrawn in a manner very similar to that of the ectoproct polypide, using coelomic pressure and retractor muscles. The sipunculid introvert ends in a mouth surrounded by hollow, ciliated, tentacular outgrowths. The gut is Ushaped, and the anus is situated near the base of the introvert. In fact, nucleotide sequence data suggest that sipunculids and gymnolaemate bryozoans may be closely related, as are the brachiopods and phoronids (Mackey et al., 1996; Cohen and Gawthrop, 1996). However, the same data indicate that lophophorates, in general, and ectoprocts, in particular, are not monophyletic assemblages. The gymnolaemate, Alcyonidium gelatinosum and the phylactolaemate, Plumatella repens appear to be neither closely related to each other nor to the brachiopod – phoronid grouping. Nevertheless, it is reasonable so far to propose an ancestral line that led directly to phoronids and phylactolaemate ectoprocts. Only these two groups have a crescentic lophophore and an epistome. They also both produce new buds from a region on the oral (ventral) side of the adult, while in other ectoprocts budding is in an anal direction (Jebram, 1973). Many phoronids produce special, fat bodies for energy storage, which are strikingly similar to early developmental stages of phylactolaemate statoblasts. While the larvae, in general, are quite different, the phoronid, Phoronis ovalis, has a unique, ciliated actinotroch (Silén, 1954) that appears similar to the phylactolaemate “larva.” Signiﬁcantly, P. ovalis also forms colonies which resemble somewhat those of the phylactolaemate family Fredericellidae. Phoronids and gymnolaemate ectoprocts are probably more distantly related, as reﬂected especially by their different embryology and larval metamorphosis. The phoronid actinotroch larva, for example, has a distinct coelom, the larval gut is retained in the adult, and the lophophore develops from the metatroch ring of cilia. By contrast, the gymnolaemate cyphonautes larvae has no larval coelom, the larval gut is not retained, and tentacles develop from the episphere region. Within the class Phylactolaemata, similarities in the mode of colony growth and statoblast morphology have long been regarded as the basis for evolutionary relationships. Fredericella species are thought to exhibit primitive features with their simple statoblasts and an open, dendritic pattern of colony branching. The circular outline of the lophophore, associated with a relatively small number of tentacles, may also be a primitive character. The evolutionary trends include a shift toward greater compactness of the colony and the accomodation of increasing numbers of tentacles on the lophophore. Statoblasts serve the two seemingly conﬂicting roles of dispersal and of retaining a position on proven favorable substrate. Among the Plumatellidae and Stephanellidae all species retain a basically tubular design. The two statoblast functions are served by distinctly different statoblast types: ﬂoatoblasts and sessoblasts. In Lophopodidae, Pectinatellidae, and Cristatellidae the colonies are gelatinous and globular, with no trace of branching. This development is coupled with a larger lophophore and a single remarkable statoblast design that incorporates a buoyant ring with marginal hooks. These statoblasts function both as an anchor and a means for dispersal. Radiating spines and hooks were apparently acquired independently by all three families in an interesting case of parallel evolution. Hyalinella punctata, normally classiﬁed with the Plumatellidae, shares many features with the Lophopodidae, and may actually link the two groups. Such features include a gelatinous colony wall, the absence of a sessoblast, and a large ﬂoatoblast which, like those of lophopodids, is initially nonbuoyant upon release. Fossil statoblasts resembling those of recent Plumatella, Hyalinella, and Stephanella species appear in rock strata from the Late Permian (Vinogradov, 1996), demonstrating a remarkable stability in these taxa. Fu- 14. Bryozoans ture fossil discovery and analysis could well provide key information on phylactolaemate systematics. The freshwater Gymnolaemata, all ctenostomes, are members of an ancient marine group. They share with the Cheilostome bryozoans many features expressed in the development of both larvae and colonies. At this point, however, phylogenetic relationships are still speculative. IV. ENTOPROCTA Entoprocta is a small group of about 60 species distinct in almost every way from the Ectoprocta but historically included with them under the name “bryozoan.” Both groups are sessile with ciliated tentacles and an incomplete separation of budded zooids, but the similarities stop there. The only known freshwater species, Urnatella gracilis, was discovered in North America in 1851. Two other species have been proposed elsewhere based mainly on number of stalk segments and morphology of the basal plate. Emscher- FIGURE 7 515 mann (1965) considers all of these synonymous with U. gracilis. Although normally classiﬁed as Urnatellidae, the genus is similar to the marine species Barentsia and may be united with it in the family Pedicellinidae. The zooid is a bulbous calyx borne on a ﬂexible, segmented stalk measuring up to 5-mm long (Fig. 7). Several such stalks, either solitary or sparingly branched, may arise from a basal plate. The calyx bears a single whorl of 8 – 16 uniformly short, ciliated tentacles. The area of the calyx enclosed by the tentacles, called an atrium or vestibule, includes both the mouth and the anus. When the zooid is disturbed, the tentacles fold over the vestibule and are covered by a tentacular membrane. Internal organs include a fully ciliated digestive tract and a large medial ganglion. A number of excretory ﬂame bulbs communicate through short ducts to a common nephridiopore; additional ﬂame bulbs occur in the stalk. The body cavity is a pseudocoel ﬁlled with loose mesenchyme and extending into each tentacle. The calyx is deciduous, dropping off at the onset of cold temperatures or other unfavorable conditions. The Small entoproct colony (Urnatella gracilis) showing external structures. 516 Timothy S. Wood basal segments of stalks, containing food and germinal tissue, can survive the winter much like ectoproct statoblasts, forming new calyxes when the water temperature returns to around 15°C. New zooids develop by budding from the stalk, as illustrated particularly well by Oda (1982). It is reported that young colonies can disperse locally by crawling over the substrate (Protosov, 1980). Sexual reproduction leads to the development of swimming larvae, presumably similar to those of other Pedicellinidae; details are unknown. Like other bryozoans, Urnatella gracilis is a suspension feeder, consuming organic particles, unicellular algae, and protozoans. Two especially important foods are the diatom Melosira and two green algae species, Pediastrum duplex and P. simplex (Weise, 1961). Urnatella gracilis is known from every continent except Antarctica and Australia. In North America its reported distribution ranges from the east to the west coast and from Florida, Louisiana, and Texas to as far north as Michigan. Zooids attach to almost any substrate, including rocks, sticks, aquatic plants, bivalve shells, ectoprocts, and such debris as nails, beverage cans, and lead ﬁshing weights (Eng, 1977). Individuals occur most frequently in ﬂowing water or in shallow areas of large lakes where there is extensive water movement. Tolerance to a wide range of chemical and physical conditions has been noted most recently by Hull et al. (1980), and Cusak and McCullough (1985). In some areas, especially on new substrate, entoprocts may comprise a sizeable fraction of the macroinvertebrate community. Stalk densities of over 225,000 / m2 and an average biomass of 868 mg / m2 (dry weight) were reported from a seven month old artiﬁcial stream in Mississippi (King et al., 1988). Entoproct phylogeny is even less clear than that of the Ectoprocta. Any similarities to ectoproct bryozoans are clearly the result of convergent evolution rather than common ancestry. A close comparison of entoproct tentacles and the ectoproct lophophore reveals little in common despite their outwardly similar structure and function. Body cavities of the two groups are likewise irreconcilable: a pseudocoel on one hand and a true coelom on the other. Analysis of 18S r-RNA data support a wide separation between entoprocts and ectoprocts, placing the entoprocts instead among the pseudocoelomates (Mackey et al., 1996). Nevertheless, puzzles such as this are expected in animals so highly modiﬁed by sessile life style and modular architecture, and for now the question of entoproct origins remains open. V. STUDY METHODS Bryozoans are normally found in areas of shallow water where there is a suitable ﬁrm substrate. Although most species are plainly visible to the unaided eye, a 10 Coddington-type magniﬁer is useful for ﬁeld identiﬁcation. Many species can be detected by examining the edges of ﬂoating objects for their free statoblasts. Some gelatinous colonies can be nudged gently from the substrate with little damage, but with other species it is better to collect pieces of substrate with colonies attached. Basic equipment for this includes a sturdy knife for organic substrates and a cold chisel and hammer for rocks. In lakes, where colonies are elusive, statoblasts can often be retrieved from sediments using a stack of standard seives with mesh openings of 1.0 mm, 500, and 150 m (Jones et al., 1998). Swimming larvae are most easily detected by placing large colonies in a shallow tray of water. If larvae are present, they will generally be released with gentle prodding, or will emerge spontaneously during the nighttime hours. They are best seen against a dark background. Before their release from the colony, zooids with larvae appear swollen at the tip. Bryozoans are not difﬁcult to rear in the laboratory, but the substrate on which they attach must always be inverted so that fecal material and other settling debris will not accumulate around the zooids. Most species can be kept at room temperature. At lower temperatures there are fewer problems with fouling organisms, but colony growth is slow. Food appears to be the most important variable for success with laboratory-reared bryozoans. Oda (1980) maintained colonies of Lophopodella carteri on pure cultures of Chlamydomonas reinhardtii. Wayss (1968) kept Plumatella repens for three years using a variety of unicellular green algae in 1:1 Knop’s solution and soil extract. Others have achieved limited short-term success using mixed protozoan cultures, pet ﬁsh food, and ﬁne detritus. One reliable and trouble-free source of food is simply the suspended organic particles circulated through a dark rearing tank from a large, well-lit aquarium in which active ﬁsh are maintained (Wood, 1996). If specimens are to be preserved, they should be anaesthetized before ﬁxing so the lophophores will remain extended. The most convenient method is to conﬁne colonies to a small covered dish of water with thin wafers of menthol ﬂoating on the surface. The menthol diffuses slowly into the water and relaxes the zooids within an hour or two. Lophophores of anaesthetized zooids can be hardened with drops of full-strength formalin. The colony is then ﬁxed and preserved in 70% ethyl alcohol. Conﬁrmed identiﬁcation of most species requires the presence of statoblasts inside the colony. Statoblast dimensions and surface topography are species-speciﬁc. In specimens stored for an appreciable time, any gas in the ﬂoatoblast annulus is replaced by liquid, making 14. Bryozoans the entire capsule clearly visible through the periblast. In this case, it is important not to mistake capsule dimensions for those of the fenestra. Length and width of sessoblasts should be measured from the base of the lamella, not from its outer edge. Surface features of the ﬂoatoblast fenestra can often be seen in isolated valves using ordinary light microscopy. However, features of the annulus require the use of a scanning electron microscope. The most common surface pattern in the ﬂoatoblast annulus shows the sclerotized cells in relief, like cobblestones, a condition called “paved” (Fig. 12a). The annulus may also be reticulated (Fig. 12b) or adorned with tiny nodules (Fig. 12d). In various species the ﬂoatoblast fenestra may be smooth (Fig. 12a), reticulated (Fig. 12b), tuberculated, or bearing a reticulum in which each cell contains a single interstitial tubercle (Fig. 12d). Other diagnostic features of the ﬂoatoblast include polar grooves on the dorsal valve (Fig. 11b) and appearance of the intact suture (Fig. 12c). All recent species descriptions include statoblast details such as these. For detailed examination of a statoblast using light microscopy, it is useful to ﬁrst separate the component parts by placing it for about 1 min in a hot solution of potassium hydroxide. Then transfer it to distilled water where the valves should separate spontaneously. Use ﬁne insect pins (size 000) to assist in this process and to 517 tease away the yolky contents. I ﬁnd it convenient to arrange these parts under the microscope and photograph them for my working records. To prepare statoblasts for scanning electron microscopy it is ﬁrst necessary to remove manually the thin membrane that often adheres tightly to the surface. If subsequent cleaning is necessary, ultrasonic treatment is too harsh. Instead, place the statoblasts in a small Eppendorf vial with a 0.05 M sodium hexametaphosphate, then hold the vial against the shaft of an electric vibrating blade shaver. Wash the statoblasts in several changes of distilled water. Freeze-drying helps prevents distortion of the fenestra. Sputtering is advisable but not essential. Well over half the recognized phylactolaemate species, are represented by type specimens in major museums, including all species named since the 1940s (Wood, 1999). Even long neglected and desiccated material remains valuable for morphological study of statoblasts. Certain techniques for subcellular study of freshwater bryozoans have proven particularly useful. These include analysis by karyotype (Backus and Mukai, 1987), by randomly ampliﬁed polymorphic DNA (Okamura et al., 1993), 18S ribosomal DNA (Halanych et al., 1995), and the identiﬁcation of polymorphic microsatillite loci (Freeland et al., 1999). VI. TAXONOMIC KEY TO NORTH AMERICAN FRESHWATER BRYOZOANS 1. Zooid composed of bulbous body on externally segmented stalk; tentacles folding individually toward center when zooid is disturbed; statoblasts absent (Fig. 7). Phylum Entoprocta . . . Urnatella gracilis Leidy, 1851. 1. Zooid without externally segmented stalk; tentacles withdrawn together when zooid is disturbed; statoblasts may be present. Phylum Ectoprocta...................................................................................................................................................................................2 2(1). Colony composed of branching tubules, body wall transparent-to-opaque; statoblasts (if present) with smooth margins; tentacles fewer than 65 .....................................................................................................................................................................................3 2. Colony globular and either lobed or entire in outline; body wall transparent; statoblasts with peripheral spines, hooks, or pointed extensions; sessoblasts absent; tentacles more than 65 .....................................................................................................................25 3(2). Extended lophophore circular in outline; statoblasts (if present) piptoblasts only (Fig. 9a, b); tentacles fewer than 25.......................4 3. Extended lophophore U-shaped in outline; statoblasts either ﬂoatoblasts or sessoblasts (or both); tentacles more than 25 ..............10 4(3). Statoblasts never formed; ectocyst stiff, shiny, and transparent; tentacles fewer than 20; individual zooids clearly demarcated by internal septa; oriﬁce appears quadrangular when lophophore is withdrawn. Gymnolaemata, Ctenostomata.......................................5 4. Statoblasts formed, especially in zooids attached to substrate, but possibly missing in some specimens; ectocyst not stiff and shiny; tentacles more than 19. Phylactolaemata, Fredericellidae, Fredericella ...............................................................................................8 5(4). Each zooid includes a uniformly narrow, sinuous, stolonlike tubule by which it is joined to a parental zooid ....................................6 5. Stolonlike tubules absent, zooids branch from each other at nearly right angles; widely distributed in North America and worldwide. (Fig. 8b). Paludicellidae. Paludicella articulata (Ehrenberg, 1831). 6(5). Tentacles numbering exactly 8. Victorellidae ......................................................................................................................................7 6 Tentacles more than 15; zooids ranging from straight and erect-to-bulbous and recumbant. (Fig. 8c). Known throughout eastern North America, especially south of the 40th parallel. Pottsiellidae. Pottsiella erecta (Potts, 1884). 518 Timothy S. Wood Gymnolaemate colonies occurring in fresh water. Scale bar for all ﬁgures 1 mm. (a) Sineportella forbesi; (b) Paludicella articulata; (c) Pottsiella erecta; and (d) Victorella pavida. FIGURE 8 7(6) Erect portion of zooid 0.6 – 1.6 mm long and only slightly contracile (Fig. 8d); occurring mainly in brackish water; reported in North America south from Chesapeake Bay and in southeastern Louisiana. Victorella pavida (Kent, 1870). 7 Erect portion of zooid never more than 0.3 mm long and highly contractile (Fig. 8a), known only from a single site in east central Illinois. Sineportella forbesi Wood and Marsh, 1996. 8(4) Piptoblast surface densely pitted or minutely roughened; appearing dull and granular when dry........................................................9 8 Piptoblast surface appearing mirror-smooth and shiny when dry. Common in Europe, conﬁrmed in North America only from a single site in Curry County, Oregon. Fredericella sultana (Blumenbach, 1779). 9(8) Piptoblast broadly oval to round (Fig. 9b), often more than one per zooid; valves covered by a minutely wrinkled mantle which is easily removed by 5-min immersion in concentrated KOH solution to reveal smooth sclerotized surface. Reported throughout North, Central and South America. Fredericella browni (Rogick, 1945). 9 Piptoblast oval-to-elongate (Fig. 9a), seldom more than one per zooid; surface uniformly pitted and unaffected by KOH. Common throughout North America, also scattered sites in Europe and Asia; probably includes several species not yet distinguished. (Fig. 13a). Fredericella indica Annandale, 1909. 10(3) Floatoblasts circular or nearly so, with little difference in appearance between dorsal and ventral sides; colony wall thick, soft, transparent. Uncommon...................................................................................................................................................................11 10 Floatoblasts oval-or-oblong; fenestrae of two valves distinctly different in size; colony wall not necessarily thick or transparent. Abundant and widespread. Plumatellidae 13 11(10) Zooids erect, extending up to 4 mm. but collapsing when removed from water; polypide and lophophore relatively small; ﬂoatoblast fully reticulate and lacking tubercles (Fig 9m). Conﬁrmed in North America only from several sites in Massachusetts, although ﬂoatoblasts reported from Oregon. Stephanellidae. Stephanella hina Oka 1908. 14. Bryozoans 519 Free statoblasts from the families Plumatellidae and Fredericellidae. Scale bar for all ﬁgures 250 m. (a) Fredericella indica; (b) Fredericella browni; (c) Plumatella casmiana (capsuled ﬂoatoblast); (d) Plumatella casmiana (leptoblast); (e) Plumatella repens; (f) Plumatella fruticosa; (g) Plumatella reticulata; (h) Plumatella emarginata; (i) Hyalinella punctata; (j) Plumatella vaihiriae; (k) Plumatella orbisperma; (l) Plumatella bushnelli; and (m) Stephanella hina [a – e and g – i modiﬁed from Wood, 1989; j based on Rogick and Brown, 1942; k and m based on Bushnell, 1965b]. FIGURE 9 11 Zooids not as above, ﬂoatoblast surface variable. Plumatellidae ......................................................................................................12 12(11) Floatoblast fenestra tuberculated, annulus adorned with minute nodules (Figs. 12d and 9k), sessoblast circular or nearly so; thick walls of large colonies fusing into a ﬁrm, transparent matrix. Known only from a few sites in Michigan and the northern Great Lakes. Plumatella orbisperma (Kellicott, 1882). 12 Floatoblast fully reticulated and without tubercles; sessoblast long oval-to-rectangular. Known only from small ponds in forested sites of New England. Plumatella recluse Smith, 1992. 13(10) Colony wall thick, soft, and transparent; colony entirely adherant to substrate; zooids forming low mounds when polypides are retracted (Fig. 13d); ﬂoatoblast width greater than 300 m, length over 400 m (Fig. 9i), dark and seldom buoyant upon release from colony, sessoblasts never formed. Widely distributed. Hyalinella punctata (Hancock, 1850). 13 Floatoblast and colony not as above, or sessoblasts present .............................................................................................................14 14(13) Width of ﬂoatoblast dorsal annulus at poles never less than length of dorsal fenestra (Figs. 14.9g, h); internal septa frequent.........15 14. Width of ﬂoatoblast dorsal annulus at poles less than length of dorsal fenestra (Fig. 9e, f, j, l); internal septa present or absent. .....16 15(14). Floatoblast dorsal valve nearly ﬂat, long edge curved; suture between valves visible in dorsal view (Figs. 5a, 9h, and 12a); sessoblast surface uniformly granular, tentacles about 40. Internal septa perpendicular to colony wall; mouth region often red. Common and widely distributed, especially in ﬂowing water. Plumatella emarginata Allman, 1844. 15. Floatoblast valves almost equally convex, long edge relatively straight (Fig. 9g), sessoblast surface roughened by network of raised lines; tentacles about 32; internal septa slightly oblique; mouth region never red. Common throughout North America and south at least to Panama, also reported from Israel. Plumatella reticulata Wood, 1988. 520 Timothy S. Wood FIGURE 10 Statoblasts in the families Lophopodidae, Pectinatellidae, and Cristatellidae. Scale bar for all ﬁgures 500 m. (a) Pectinatella magniﬁca; (b) Cristatella magniﬁca; (c) Lophopodella carteri; (d) Lophopus crystallinus [a – c modiﬁed from Wood, 1989]. 16(14). Floatolast and sessoblast length not more than twice width .............................................................................................................17 16. Floatoblast and sessoblast length more than twice width (Fig. 9f); colonies with thin branches largely free of substrate. Widely distributed, especially in northern, oligotrophic habitats. Plumatella fruticosa Allman, 1844. 17(16) Floatoblast length-to-width greater than 1.6 (Fig. 9c, d); colony composed of short, profusely branched tubes always closely adherent to substrate (Fig. 13b), tubes becoming erect and fused when crowded; ﬂoatoblast dorsal fenestra lacking prominent tubercles; Scanning electron micrographs showing ﬂoatoblast features. Scale bar mm. (a) Lateral view; (b) frontal view of dorsal valve. cp, central prominance; da, dorsal annulus; df, dorsal fenestra; pg, polar groove; su, suture; and vf, ventral fenestra. Scale bar 100 m. FIGURE 11 14. Bryozoans 521 delicate thin-walled ﬂoatoblast sometimes present (Fig. 9d); sessoblast lamella relatively narrow. Common and widely distributed. Plumatella casmiana (Oka, 1907). 17 Floatoblast length to width less than 1.6 (Fig. 9e,j,l); colony branches not particularly short, colony or statoblasts not as above ....18 18(17) Floatoblast distinctly tapered at the poles, with strongly reticulated fenestrae (Figs. 9j, 12b); dorsal annulus bulging around inner capsule to form a distinct shoulder; sessoblast surface densely pitted and without tubercles. Uncommon but often locally abundant, ranging throughout the United States. Plumatella vaihiriae (Hastings, 1929). 18 Floatoblast rounded at poles, fenestra not strongly reticulated, annulus extending evenly from suture to fenestra without bulging around capsule; sessoblast surface densely tuberculate .....................................................................................................................19 19(18) Floatoblast lateral edges relatively straight (Fig. 9l); dorsal fenestra with prominent tubercles annulus with dense nodules visible by SEM; sessoblast lamella relatively wide. Known only from a single site on a North Carolina barrier island. Plumatella bushnelli Wood, 2001. 19 Floatoblast lateral edges evenly curved (Fig. 9e); dorsal fenestra with weak tubercles and slight reticulation; common and widespread ..............................................................................................................................................................................................20 20(19) Floatoblast dorsal fenestra small, not more than half the diameter of ventral fenestra; ventral annulus markedly wider at poles than at sides .............................................................................................................................................................................................21 20 Floatoblast dorsal fenestra large, considerably more than half the diameter of ventral fenestra (Fig. 11b); ventral annulus uniformly narrow. Known from Massachusetts to Wisconsin, mostly north of the 41st parallel. Plumatella nitens Wood, 1996. FIGURE 12 Scanning electron micrographs showing ﬂoatoblast surface morphology. Scale bar mm. (a) Plumatella emarginata dorsal valve with “paved” annulus and smooth fenestra; (b) Plumatella vaihiriae with reticulated fenestra and annulus; (c) Hyalinella punctata with paved annulus and suture as a raised cord; and (d) Plumatella orbisperma with reticulation and “interstitial” tuibercles on the annulus, small nodules on fenestra. 522 Timothy S. Wood 21(20) Colony tubules seldom fused together; scanning electron microscopy (SEM) reveals no tubercles on ﬂoatoblast annulus, although tiny nodules may be present, internal septa are uncommon. .............................................................................................................22 21 Large colony forming tight masses of fused tubules without free branches; SEM reveals tubercles on ﬂoatoblast annulus; many internal septa; occurring in highly eutrophic water; abundant in Europe, but generally uncommon in North America. Plumatella fungosa (Pallas, 1768). 22(21) SEM shows ﬂoatoblast annulus surface smooth except for tiny, rash-like nodules............................................................................23 22 SEM shows ﬂoatoblast annulus surface roughened by concave cell walls, nodules absent; species common and widespread in North America, especially in lentic habitats (Fig. 13c). Plumatella rugosa (Wood, Geimer and Massard 1998). 23(22) SEM shows nodules only on ﬂoatoblast annulus ..............................................................................................................................24 23 SEM shows nodules abundant on both ﬂoatoblast annulus and fenestra; known from Illinois, Ohio, and western New York. Plumatella nodulosa Wood, 2001. 24(23) Floatoblast suture deﬁned by a lumpy, double cord; known only from Illinois. Plumatella similirepens Wood, 2001. 24 Floatoblast suture marked by two crooked rows of large, toothlike tubercles; known from Europe and New Zealand, but not yet documented from North America. Plumatella repens (Linnaeus, 1758) 25(2) Mouth region with red pigmentation; prominent pair of white spots at end of each arm of lophophore; statoblasts round with hooked spines radiating from outer margin of annulus (Fig. 10a). Colony gelatinous and slimy, ranging from a ﬂat sheet to footballsized mass. (Fig. 13f). Common and widely distributed. Pectinatellidae. Pectinatella magniﬁca (Leidy, 1851) FIGURE 13 Various colony forms in Phylactolaemata. (a) Fredericella indica growing on a piece of wood, showing many free branches, scale bar 5 cm; (b) Plumatella casmiana, with zooids attached throughout their length to the substrate or to each other, scale bar 2 mm; c) Plumatella rugosa growing on the inner surface of a mussel valve, scale bar 2 cm; (d) Hyalinella punctata, showing both densely packed and freeranging growth, all zooids ﬁrmly attached to the substrate, scale bar 5 mm; (e) Cristatella mucedo, showing a single statoblast inside, scale bar 5 mm; (f) Pectinatella magniﬁca growing on the underside of a small log, scale bar 2 cm [b from Rogick, 1941; c from Rogick and Brown, 1942; c– f from Wood, 1989]. 14. Bryozoans 523 25 Mouth region without red pigmentation, lophophore lacking pair of white spots ............................................................................26 26(25) Colony not distinctly linear, statoblasts oblong, not round...............................................................................................................27 26 Colony linear, often longer than 2 cm (Fig. 13e); statoblasts round with wiry, hooked spines radiating beyond periphery from margin of fenestrae of both valves (Fig. 10b). Occuring mainly in oligotrophic waters. Cristatellidae. Cristatella mucedo Cuvier, 1798. 27(26) Statoblast with series of small hooks localized along the polar margins (Fig. 10c). Uncommon, but can be locally abundant (Fig. 4). Lophopodella carteri (Hyatt, 1866). 27 Statoblast tapering to a single point at each pole (Fig. 10d); extremely rare worldwide, and possibly endangered. 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Acta Zoologica 31:215 – 257. Sládeček, V. 1980. Indicator value of freshwater Bryozoa. Acta Hydrochimica and Hydrobiologica 8:273 – 276. Smith, D. G. 1989a. On Stephanella hina Oka (Ectoprocta: Phylactolaemata) in North America, with notes on its morphology and systematics. Journal of the American Benthological Society 7:253–259. Smith, D. G. 1989b. Keys to the freshwater macroinvertebrates of Massachusetts. No. 4: Benthic colonial phyla, including the Cnidaria, Entoprocta, and Ectoprocta (colonial hydrozoans, moss animals). Massachusetts Division of Water Pollution Control. Westborough, MA. Smith, D.G. 1992. A new freshwater mos animal in the genus Plumatella (Ectoprocta: Phylactolaemata) from New England (U.S.A.). Canadian Journal of Zoology 70:2192 – 2201. Tenney, W. R., Woolcott, W. S. 1966. The occurrence and ecology of freshwater bryozoans in the headwaters of the Tennessee, Savannah, and Saluda River systems. Transactions of the American Microscopical Association 85:241 – 245. Vinogradov, A. V. 1996. New fossil freshwater bryozoans from the Asiatic part of Russia and Kazakhstan. Paleontological Journal 30(3):284 – 292. Walburg et al. 1971. in Hall, Gordon E. Ed., Reservoir ﬁsheries and limnology. Special Publication #8. American Fisheries Society, Washington, DC, pp. 449 – 467. 525 Wayss, K. 1968. Quantitative Untersuchungen über Wachstum und Regeneration bei Plumatella repens (L.). Zoologische Jahrbücher. Abteilung für Anatomie und Ontogenie der Tiere 85:1 – 50. Weise, J. G. 1961. The ecology of Urnatella gracilis Leidy: Phylum Entoprocta. Limnology and Oceanography 6:228 – 230. Wilde, G. A., Burke, T. A., Keenan, C. 1981. Bryozoa from Lake Mead and Lake Mohave, Nevada-Arizona. The Southwestern Naturalist 26:201. Willmer, P. 1990. Invertebrate relationships. Cambridge University Press, Cambridge. Wood, T. S. 1973. Colony development in species of Plumatella and Fredericella (Ectoprocta: Phylactolaemata), in: Boardman, R. S., Cheetham, A., Oliver, J. 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Water Research 33(3):609 – 614. Wöss, E. 1996. Life history variation in freshwater bryozoans, in: Gordon, D., Smith, A., Grant-Mackie, J. Eds., Bryozoans in space and time. National Institute of Water & Atmospheric Research, Wellington, New Zealand, pp. 391 – 399. Wöss, E. 1999. Colonization and development of freshwater bryozoan communities on artiﬁcial substrtes in the Laxenburg Pond (Lower Austria). Proceedings of the 11th International Bryozoology Association Conference, 1999 (in press). Zimmer, R. L. 1973. Morphological and developmental afﬁnities of the lophophorates. in: Larwood, G. P., Ed., Living and fossil Bryozoa. Academic Press, London, pp. 593 – 599. Zimmerman, M. 1979. Larval release and settlement behavior in three species of plumatellid Bryozoa (Ectoprocta). M.S. Thesis, Wright State University, Dayton, OH. This Page Intentionally Left Blank 15 TARDIGRADA Diane R. Nelson Department of Biological Sciences East Tennessee State University Johnson City, Tennessee 37614 I. Introduction II. Anatomy A. Cuticle B. Claws C. Digestive System D. Other Anatomical Systems III. Physiology (Latent States) IV. Reproduction and Development A. Reproductive Modes B. Reproductive Apparatus C. Mating and Fertilization D. Eggs E. Parthenogenesis F. Hermaphroditism G. Development I. INTRODUCTION Since 1773, when J. A. E. Goeze reported the ﬁrst observation of a “Kleiner Wasser Bär,” tardigrades have commonly been called “water bears” (Goeze, 1773). Their bearlike appearance, legs with claws, and slow lumbering gait formed the basis for the descriptive name. In 1776, the naturalist Lazzaro Spallanzani introduced the name “il Tardigrado” (slow-stepper) to describe the slow, tortoiselike movement of the animal (Spallanzani, 1776). The systematic position of the Tardigrada, the taxon name assigned to the group by Doyère (1840), has ranged from inclusion within Infusoria to the Annelida to the Arthropoda (Acarina, Crustacea, Insecta). Tardigrada was acknowledged as a phylum by Ramazzotti (1962) in his ﬁrst monograph, conﬁrming the position of other workers (Richters, 1926). The phylogenetic afﬁnities of the phylum, based on morphological studies, were clariﬁed by recent molecular studies with 18S rRNA that showed tardigrades to be a sister group of the arthropods (Garey et al., Ecology and Classiﬁcation of North American Freshwater Invertebrates. 2nd Edition Text Copyright © 2001 by Academic Press. Figures Copyright © 2001 by Diane R. Nelson All rights of reproduction in any form reserved. V. Ecology A. Molting, Life History, and Cyclomorphosis B. Habitats C. Population Density D. Distribution E. Dissemination VI. Techniques for Collection, Extraction, and Microscopy VII. Identiﬁcation A. Taxonomic Key to Genera of Freshwater and Terrestrial Tardigrada Literature Cited 1996; Giribet et al., 1996; Garey et al., 1999), although one study was inconclusive (Moon and Kim, 1996). The Ecdysozoa was recently established as a new clade containing the molting animals: arthropods, tardigrades, onychophorans, nematodes, nematomorphs, kinorhynchs, and priapulids (Aguinaldo et al., 1997). This phylogenetic analysis, which showed no support for the Articulata (a clade of segmented animals uniting annelids and arthropods), offers an explanation for morphological similarities of tardigrades to both the onychophoran-arthropod and the aschelminth complex. Discussion on phylogenetic relationships of tardigrades and arthropods has been reviewed by Nielsen (1995) and Dewel and Dewel (1997). Thus far, about 800 species of tardigrades have been described from marine, freshwater, and terrestrial habitats; more species are expected as new areas are investigated (McInnes, 1994; Kinchin, 1994; McInnes and Pugh, 1998). Tardigrades are hydrophilous micrometazoans with a bilaterally symmetrical body and four pairs of lobopodous legs usually terminating in claws (Figs. 1 527 528 Diane R. Nelson FIGURE 2 Scanning electron micrographs of a eutardigrade. Macrobiotus tonollii. [From Nelson, 1982a.] FIGURE 1 Scanning electron micrographs of a heterotardigrade. Echiniscus spiniger. a, Cirrus A; b, buccal cirrus; c, clava; p, cephalic papilla. [From Nelson, 1982a.] and 2). Generally convex on the dorsal side and ﬂattened on the ventral side, the body is indistinctly divided into a head (cephalic) segment, three trunk segments each bearing a pair of legs, and a caudal segment with the fourth pair of legs directed posteriorly. Although tardigrades range in body length from 50 m in juveniles to 1200 m in adults (both excluding the last pair of legs), mature adults average 250 – 500 m with a few species exceeding 500 m. Despite their overall abundance and cosmopolitan distribution, the Tardigrada have been relatively neglected by invertebrate zoologists. Frequently categorized as one of the “minor phyla,” they are more appropriately termed one of the “lesser-known phyla.” Because of difﬁculties in collecting and culturing the organisms and their apparent lack of economic importance to humans, our knowledge of tardigrades has expanded slowly since their discovery over 225 years ago. The lack of basic biological information has hindered the growth of interdisciplinary studies, which are currently being developed. Today, new insights into the biology of the Tardigrada are evolving from more comprehensive, contemporary investigations utilizing molecular techniques as well as transmission and scanning electron microscopy, phase- and interference-contrast microscopy, and histochemical and cytological techniques. Tardigrades provide a vast array of opportunities for classical and contemporary biological investigations. Within the last decade, interest and activity in tardigrade research has intensiﬁed. II. ANATOMY A. Cuticle The cuticle, which is highly permeable to water, covers the body surface and lines the foregut and hindgut. [See Dewel et al., 1993 for detailed review.] 15. Tardigrada TABLE I 529 Subdivision of the Phylum Tardigrada with Habitat Classiﬁcationsa Taxon Habitat I. Class Heterotardigrada A. Order Arthrotardigrada B. Order Echiniscoidea 1. Family Echiniscoididae 2. Family Oreellidae 3. Family Carphanidae 4. Family Echiniscidae II. Class Mesotardigrada A. Order Thermozodia 1. Family Thermozodiidae III. Class Eutardigrada A. Order Parachela 1. Family Macrobiotidae 2. Family Calohypsibiidae 3. Family Eohypsibiidae 4. Family Hypsibiidae 5. Family Necopinatidae 6. Incertae sedis B. Order Apochela 1. Family Milnesiidae Marine, except Styraconyx hallasi (freshwater) Marine, freshwater, terrestrial Marine; genera Echiniscoides and Anisonyches Terrestrial; genus Oreella Freshwater; genus Carphania Terrestrial, except for a few species of Echiniscus, Hypechiniscus, and Pseudechiniscus occasionally in freshwater; 12 genera Hot spring; genus Thermozodium Terrestrial and freshwater; genera Adorybiotus, Calcarobiotus, Dactylobiotus, Macrobiotus, Macroversum, Microbiotus, Minibiotus, Murrayon, Pseudodiphascon, Pseudohexapodibius, Richtersius, Xerobiotus Terrestrial; genera Calohypsibius, Hexapodibius, Haplomacrobiotus, Parhexapodibius, Haplohexapodibius Terrestrial and freshwater; genera Eohypsibius, Amphibolus Marine; genus Halobiotus and a few Isohypsibius, Ramajendas. Terrestrial and freshwater; genera Acutuneus Astatumen, Diphascon, Doryphoribius, Eremobiotus, Hebesuncus, Hypsibius, Isohypsibius, Itaquascon, Fujiscon, Mesocrista, Microhypsibius, Mixibius, Paradiphascon, Parascon, Platicrista, Pseudobiotus, Ramajendas, Ramazzottius, Thulinia Terrestrial; genus Necopinatum Terrestrial; genus Apodibius Terrestrial; Milnesium and Limmenius a Additional references for table, not cited speciﬁcally in text: Binda, 1984; Binda and Pilato, 1986; Pilato and Beasley, 1987; Pilato and Binda, 1987a, b, 1989; Biserov, 1992; Dastych, 1992; Pilato and Binda, 1997. Secreted by the underlying epidermis ( hypodermis), the cuticle consists of several layers which may be further divided into: (1) an outer complex epicuticle ( exocuticle); (2) an intracuticle ( mesocuticle), which is not present in all species; and (3) an inner procuticle ( endocuticle), which contains chitin (Greven and Peters, 1986; Greven and Greven, 1987; Dewel et al., 1993). Although most freshwater tardigrades are white or colorless, some terrestrial species exhibit brown, yellow, orange, pink, red, or green coloration due to intestinal contents, body cavity cells and granules, or pigmentation in the epidermis and cuticle (Ramazzotti and Maucci, 1983). Based on cuticular structures, three classes, formerly considered orders, can be distinguished (Table I). The class Heterotardigrada (Figs. 1 and 3) includes mainly the marine and armored terrestrial tardigrades, while the class Eutardigrada (Figs. 2 and 4) primarily encompasses freshwater and other terrestrial species. A third class, Mesotardigrada (Rahm, 1937) is based on a single species, Thermozodium esakii, from a hot spring in Japan; it is difﬁcult to evaluate the validity of this class due to the lack of type specimens and to the destruction of the type locality by an earthquake (Ramazzotti and Maucci, 1983). Heterotardigrades (Fig. 1) are characterized by paired cephalic sensory appendages, which vary considerably in size and shape: internal buccal cirri, cephalic papillae, external buccal cirri, clavae, and lateral cirri (cirri A). Lateral cirri A mark the junction of the cephalic and scapular plates. Some marine species also have a median cirrus, particularly in the most primitive order, Arthrotardigrada. In the class Eutardigrada (Fig. 2), cephalic sensory structures, which may (Dewel et al., 1993) or may not (Schuster et al., 1980) be homologous to those in heterotardigrades, are present only in the order Apochela; Milnesium has two lateral papillae and six peribuccal (oral) papillae, and Lemmenius has two lateral papillae. Other eutardigrades (order Parachela) lack external sensory appendages but have sensory regions on the head (Walz, 1978). The cuticle and its processes are taxonomically important in identifying genera and species and also form the basis for separating tardigrades into two large groups. The “armored” tardigrades are heterotardigrades 530 Diane R. Nelson FIGURE 3 Heterotardigrade internal anatomy (Bryodelphax parvulus, female); lateral view (above) and ventral view (below). [From Nelson, 1982a; drawing by R. P. Higgins.] that have a thickened dorsal cuticle divided into individual plates, which have a species-speciﬁc pattern of sculpture. Armored tardigrades include some marine species (families Renaudarctidae and Stygarctidae, order Arthrotardigrada) and many terrestrial species (family Echiniscidae, order Echiniscoidea). Kristensen (1987) revised the genera of Echiniscidae, adding four new genera to the existing eight. Of these, some species in the genus Pseudechiniscus are found in freshwater, but the dorsal plates are nonsclerotized. The “naked” or unarmored tardigrades comprise the freshwater and terrestrial eutardigrades and most marine heterotardigrades, as well as the heterotardigrade families Oreellidae (terrestrial) and Carphanidae (freshwater) (Binda and Kristensen, 1986; Kristensen, 1987). Unarmored tardigrades have a thin, smooth cuticle or a sculptured one with pores, granulation, reticulation, tubercles, papillae, or spines, but lack plates. B. Claws In addition to cuticular structures, the size, shape and number of claws (Figs. 5 and 6) are important in FIGURE 4 Eutardigrade internal anatomy (Macrobiotus hufelandi, female); lateral view (above) and dorsal view (below). [From Nelson, 1982a; drawing by R. P. Higgins.] tardigrade systematics and have been used for revision of the eutardigrades (Thulin, 1928; Marcus, 1929; Pilato, 1969, 1982; Bertolani and Kristensen, 1987; Kristensen and Higgins, 1984a, b; Ramazzotti and Maucci, 1983; Bertolani and Biserov, 1996). In the heterotardigrade family Echiniscidae, each leg terminates in four single claws in the adult and juvenile or two claws in the larva. Frequently, the two internal claws have a ventral spur, and occasionally one or more spurs may be present on the external claw near the base (Kristensen, 1987; Ramazzotti and Maucci, 1983). In the heterotardigrade family Carphanidae, the freshwater genus Carphania has two claws on legs I – III and only one claw on reduced leg IV; all claws are ﬂexible and have two basal spurs (Binda and Kristensen, 1986). Eutardigrades usually possess two double claws on each leg, arranged as external and internal claws (Schuster et al., 1980; Bertolani, 1982a; Bertolani and Pilato, 1988; Bertolani and Biserov, 1996). Each double claw consists of a basal tract and a long primary branch with two accessory points and a secondary branch without accessory points. In the order Apochela 15. Tardigrada FIGURE 5 Claw types. All claws are shown in ventral view of the fourth right leg. (A) Echiniscus; (B) Carphania; (C) Milnesium, Limmenius; (D) Necopinatum; (E) Macrobiotus, Macroversum, Murrayon, Pseudohexapodibius, Richtersius, Adorybiotus, Calcarobiotus, Xerobiotus; (F) Pseudodiphascon; (G) Dactylobiotus; (H) Minibiotus; (I) Isohypsibius, Thulinia, Doryphoribius; (J) Pseudobiotus; (K) Hypsibius, Mixibius, Platicrista, Mesocrista, Hebesuncus, Diphascon, Itaquascon, Astatumen, Fujiscon, Paradiphascon, Ramajendas; (L) Ramazzottius; (M) Calohypsibius, Microhypsibius; (N) Hexapodibius; (O) Haplomacrobiotus; (P) Eohypsibius, Amphibolus. a, Accessory point; b, basal tract; cb, cuticular bar; e, external claw; i, internal claw; lu, lunule; pb, primary branch; s, spur; sb, secondary branch. [Redrawn from Nelson and Higgins, 1990.] (family Milnesiidae), the primary branch is long, thin, and well separated from the stout, short secondary branch which bears hooks or spurs. In the order Parachela, the secondary branch and the primary branch arise from the common basal tract. The sequence of claw branches, with respect to the midline of extended legs, in some genera is alternate (2 – 1 – 2 – 1) (i.e., secondary – primary – secondary – primary). In other genera, the two primary branches are next to one another (2-1-1-2) (i.e., secondary – primary – primary – secondary). In some eutardigrades (e.g., Macrobiotus), a cuticular thickening called a lunule surrounds the base of each double claw. Lunules vary in size and shape; they may be smooth or dentate. In several genera, one or two cuticular bars may be present on the legs, separate from the claws but located between the double claws, just below the base of the claws, and / or along the sides of the internal claws. Six major claw types have been identiﬁed based on the genera Dactylobiotus, Macrobiotus, Calohypsibius, Hypsibius, Isohypsibius, and Eohypsibius. Additional genera have been erected based on variations in 531 FIGURE 6 Scanning electron micrographs of eutardigrade claws. (A) Pseudobiotus; (B) Hypsibius; prim, primary branch; sec, secondary branch; (C) Itaquascon, ap, accessory point; cb, cuticular bar; (D) Milnesium. [From Schuster et al., 1980.] these basic claw types. Claw reduction, particularly in the hind claws, has evolved independently in the eutardigrades, especially those dwelling in soil habitats (Bertolani and Biserov, 1996). In Dactylobiotus-type claws, the two large double claws on each leg are similar in size and shape, with a 2-1-1-2 sequence, lack lunules, and are connected at the base by a cuticular band. The secondary branch is much shorter than the primary and arises from a very short basal tract. The Macrobiotus-type claws are similar in size and shape, with a distinct peduncle (a septum separating the basal tract from the rest of the claw), and have a 2-1-1-2 sequence, but the claws usually possess lunules and are not connected at the base. A similar genus, Calcarobiotus, has spurs on the bases of the claws (Dastych, 1993a). In the genus Murrayon, separated from Macrobiotus by Bertolani and Pilato (1988), the claws are V-shaped and have a quadrate peduncle (Guidetti, 1998). Macroversum is separated from Macrobiotus by having secondary branch much shorter than the primary branch, and the lunules are connected by a cuticular bar (Pilato and Catanzaro, 1988; Bertolani and Pilato, 1988). The Calohypsibius-type claws are small and similar in size and shape but exhibit the alternate (2-1-2-1) sequence; the primary branch is rigidly attached to the secondary branch. The Hypsibius-type claws are usually dissimilar in size and 532 Diane R. Nelson shape, with the external double claw longer and more slender than the internal claw. The primary branch of the external double claw is inserted on the basal tract, which is continuous with the secondary branch, forming a ﬂexible junction. The Isohypsibius-type claws resemble those of Hypsibius; however, they are usually similar in size and shape. To distinguish the two types, the secondary branch of the external claw from one of the ﬁrst three pairs of legs should be observed in proﬁle. In Hypsibius-type claws, the secondary branch and basal tract run in a continuous arc or curve; whereas, in Isohypsibius-type claws, the secondary branch forms a right angle with the basal tract. The genus Ramajendas was separated from Hypsibius by having very long primary branches (Pilato and Binda, 1990). The Eohypsibius-type claws are divided into three distinct parts: the base, the secondary branch, and the primary branch. The branches are usually arranged in a 2-1-2-1 sequence; however, the internal claw can rotate 180°, resulting in a 2-1-1-2 sequence. The external and internal claws are similar in size, but the angle between the primary and secondary branches is about 45° on the external claw and about 80° on the internal claw. C. Digestive System 1. General Anatomy The digestive system consists of a foregut, a midgut, and a hindgut (Figs. 3 and 4). [For a detailed review, see Dewel et al., 1993.] Both the foregut (buccal apparatus and esophagus) and the hindgut have a cuticular lining. The hindgut is divided into an anterior hindgut (rectum) and a true cloaca in eutardigrades; the opening is a ventral transverse slit just anterior to the fourth pair of legs. In heterotardigrades, however, reproductive and digestive systems are separate, the hindgut is a rectum, and the gonopore opens anterior to the anus, which is a longitudinal slit between or just anterior to the fourth pair of legs. In the heterotardigrade family Echiniscidae, ﬁrst instar individuals have a mouth but no gonopore or anus; second instars have a mouth and anus but no gonopore; subsequent instars have all three openings. 2. Buccal Apparatus The buccal apparatus is a complex structure with considerable taxonomic signiﬁcance in the eutardigrades (Pilato, 1972, 1982; Schuster et al., 1980). Basically, it consists of a buccal tube, a muscular sucking pharynx, and a pair of piercing stylets that can be extended through the mouth opening (Figs. 3,4,7,8). The position of the mouth may be terminal (as in Macrobiotus and FIGURE 7 Buccal apparatus. (A) Heterotardigrada, Echiniscus (ventral); (B) Eutardigrada, Macrobiotus (ventral); (C) Eutardigrada, Macrobiotus (lateral); (D) Carphania; (E) Macrobiotus, Macroversum, Murrayon, Dactylobiotus, Calcarobiotus, Pseudohexapodibius, Xerobiotus; (F) Adorybiotus, Richtersius; (G) Pseudodiphascon; (H) Minibiotus; (I) Hypsibius, Isohypsibius, Ramazzottius, Ramajendas, Mixibius, Necopinatum; (J) Doryphoribius, Apodibius; (K) Pseudobiotus; (L) Thulinia; (M) Diphascon, Platicrista, Mesocrista, Hebesuncus, Paradiphascon; (N) Itaquascon; (O) Milnesium; (P) Limmenius; (Q) Calohypsibius, Microhypsibius; (R) Hexapodibius, Haplomacrobiotus, (S) Eohypsibius; and (T) Amphibolus. a, Apophysis; bc, buccal cavity; bl, buccal lamellae; br, buccal ring; bt, buccal tube; cp, cephalic papilla; d, droplike thickening; f, furca; ma, macroplacoid; mic, microplacoid; p, placoid; pb, pharyngeal bulb; pp, peribuccal papilla; s, septulum; sp, stylet support; ss, stylet sheath, st, stylet; and vl, ventral lamina. [Redrawn from Nelson and Higgins, 1990.] Milnesium) or subterminal (as in Hypsibius). In some eutardigrades, cuticular structures surround the mouth such as elongate peribuccal papillae (6 in Milnesium), ﬂattened peribuccal papulae (10 in Minibiotus and Haplomacrobiotus and 6 in Calohypsibius), or lobes (6 in Isohypsibius and others) (Schuster et al., 1980). The buccal tube begins with a buccal ring, which bears lamellae in some genera. The number of lamellae varies with the genus; e.g., there are 10 in Macrobiotus, 12 in Thulinia, 14 in Amphibolus, and about 30 in Pseudobiotus. In Milnesium, the mouth is surrounded 15. Tardigrada 533 FIGURE 8 Scanning electron micrographs of eutardigrade buccal openings. (A) Dactylobiotus, mu, buccal mucrones; bl, buccal lamella; (B) Minibiotus, buccal opening and papulae; (C) Haplomacrobiotus, pbpu, peribuccal papulae; (D) Isohypsibius, pbl, peribuccal lobe; (E) Pseudobiotus, buccal opening and buccal lamellae; and (F) Milnesium, cp, cephalic (lateral) papilla; pbbl, peribuccal (oral) papilla. [From Schuster et al., 1980.] by six triangular lamellae, which close the opening. The interior of the buccal cavity may have anterior and posterior rows of small cuticular teeth (mucrones) followed by dorsal and ventral transverse ridges (also called crests or bafﬂes), which have signiﬁcant taxonomic value as species-speciﬁc characteristics (Pilato, 1972; Schuster et al., 1980). Most eutardi- grades have a completely rigid buccal tube. However, in some genera, (the Diphascon assemblage, Pseudodiphascon, Itaquascon), there is a rigid anterior buccal tube between the buccal opening and the stylet supports, and a ﬂexible, spiral-walled posterior pharyngeal tube from just below the stylet supports to the pharynx. In a similar genus, Astatumen, the rigid portion of 534 Diane R. Nelson the buccal tube is only as long as the anterior apophyses, the pharyngeal tube is spiraled, and stylet supports are lacking (Pilato, 1997). In Macrobiotus and other genera, there is a median ventral lamina ( buccal tube support, reinforcement rod) that extends from the buccal ring to the midregion of the tube. Four major types of buccal apparatus have been described based on the genera Macrobiotus, Hypsibius, Diphascon, and Pseudodiphascon. The Macrobiotustype has a rigid buccal tube with a ventral lamina (reinforcement rod), whereas the Hypsibius-type has a rigid buccal tube but lacks a ventral lamina. The Diphascontype has a rigid anterior buccal tube and a ﬂexible, annulated posterior pharyngeal tube; it also lacks a ventral lamina. The Pseudodiphascon-type has a buccal tube like Diphascon, but also has a ventral lamina. More types may be identiﬁed in the future as additional genera are described (Pilato and Binda, 1990, 1996). Although the buccal apparatus was used by Schuster et al. (1980) to revise the families of eutardigrades, Ramazzotti and Maucci (1983) and subsequent authors have basically followed Pilato’s classiﬁcation based on claw structure. The stylet mechanism consists of two protrusible stylets, stylet sheaths, stylet supports, and associated muscles (Schuster et al., 1980). The stylets are paired, piercing structures lateral to the buccal tube. Composed of cuticular and calciﬁed substances that dissolve in some mounting media, the stylets lie in the lumen of the salivary glands, which are dorsolateral to the pharynx. The salivary epithelium secretes the new buccal tube, stylets, and stylet supports after the tardigrade molts. The stylets and salivary secretions enter the anterior end of the buccal tube through stylet sheaths on each side. Protractor and retractor muscles operate each stylet and are attached to the furca (condyle), the thickened base at the posterior end of each stylet. Protractor muscles extend from the furca to the buccal tube; retractor muscles insert on the pharynx. Some genera have anterior apophyses for the insertion of the stylet protractor muscles on the buccal tube. In Hypsibius, the dorsal and ventral apophyses are hook-shaped; in Isohypsibius and other genera, they form dorsal and ventral crests. Pilato (1987) divided the genus Diphascon into four genera based on differences in the shape of the apophyses and other characteristics of the buccal – pharyngeal apparatus. The stylet support is a ﬂexible lateral extension that attaches the furca of the stylet to the buccal tube, but it is poorly developed in the genus Itaquascon and absent in Astatumen (Pilato, 1997). In the heterotardigrades, the long, thin stylets may have delicate stylet supports or may be attached to the pharynx by a muscular band. In general, the buccal apparatus in heterotardigrades is of less taxonomic importance than in eutardigrades (Kristensen, 1987). The buccal tube enters the muscular, tripartite pharyngeal bulb ( sucking pharynx, myoepithelial bulb) (Eibye-Jacobsen, 1996), which, in most eutardigrades, is lined with three double rows of cuticular thickenings called placoids. The placoids alternate in position with three equal cuticular apophyses, which are posterior to the end of the buccal tube. The larger, anterior placoids, called macroplacoids, are present in either two or three transverse rows. Smaller, posterior placoids are termed microplacoids; when present, they are arranged in a single row. In some species of the genus Diphascon, a medial septulum is present posterior to and alternating in position with the placoids, in the same plane of focus as the buccal tube apophyses. The number, size, and shape of the placoids are important species characters in the eutardigrades; however, placoids are reduced or absent in some genera (e.g., Itaquascon, Astatumen, Milnesium, and Limmenius). 3. Esophagus, Midgut, and Hindgut The pharyngeal bulb is followed by a short, tripartite esophagus, which is also lined with cuticle. A mucoid secretory substance is produced by esophageal cells, but its function is unknown. The midgut, the largest section of the digestive tract, is derived from endoderm and is not lined with cuticle. There are signiﬁcant differences between eutardigrades and echiniscids in the ultrastructure of the midgut epithelium (Dewel et al., 1993). Eutardigrades have a straight midgut, and three excretory glands empty into the midgut – hindgut junction; in contrast, heterotardigrades have no excretory glands at the junction of the midgut and hindgut. Food is digested in the midgut, which has a convoluted single layer of large epithelial cells joined by deep continuous junctions. The intestinal content varies in color depending on the type of food consumed, the state of the digestive process, and the nutritional status of the animal. The hindgut, which is lined with cuticle, receives the contents of the midgut and is specialized for osmoregulation. The anterior hindgut or rectum probably functions in modiﬁcation of the urine from the excretory glands ( Malpighian tubules) as well as material from the midgut. In eutardigrades, the genital ducts empty into the posterior hindgut, which is therefore a true cloaca. In heterotardigrades, the anus and gonopore are separate openings. D. Other Anatomical Systems Reviews of the morphology of other tardigrade systems are found in Greven (1980), Bertolani (1982a), Nelson (1982a), Ramazzotti and Maucci (1983), Nelson and Higgins (1990), Dewel et al. (1993), and Kinchin (1994). 15. Tardigrada The nervous system consists of a dorsal lobed brain ( cerebral ganglion, supraesophageal mass), a circumesophageal connective around the buccal tube ( ring neuropil), a “subesophageal ganglion”, a circumpharyngeal connective around the pharynx ( posterior neuropil), and four ventral ganglia joined by paired nerve tracts (Figs. 3 and 4) (Dewel and Dewel, 1996, 1997; Wiederhöft and Greven, 1996). Many eutardigrades and some heterotardigrades have a pair of eye spots, cup-shaped structures with pigment granules, embedded in the dorsolateral lobes of the brain (Dewel et al., 1993). Walz (1978) described four sensory areas that function as chemoreceptors or mechanoreceptors in the anterior head region of the eutardigrade Macrobiotus: the anteriolateral sensory ﬁeld, the circumoral ﬁeld, the suboral sensory region, and the pharyngeal organ. The cephalic appendages and buccal ventroposterior plates (third pair of clavae) in heterotardigrades also function as sense organs (Kristensen and Higgins, 1984a, 1989; Dewel et al. 1993). Tardigrade musculature has been studied in detail in only a few species. [See Dewel et al., 1993 for a detailed review.] The muscular system consists of dorsal and ventral longitudinal ﬁbers, transverse muscles for movement of the legs, stylet muscles, pharyngeal and intestinal muscles, and muscles for defecation and deposition of eggs. Muscles are attached to the cuticle by protrusions (apodemes) that are reformed at each molt. The somatic muscles are single elongate cells that insert on the body wall. Those of eutardigrades are intermediate between smooth and obliquely striated muscles (Walz, 1974, 1975) or cross-striated in the pharyngeal bulb (Eibye-Jacobsen, 1996), whereas those of marine arthrotardigrades are cross-striated (Kristensen, 1978). According to Ramazzotti and Maucci (1983), excretion in tardigrades probably occurs in four ways: (1) through the salivary glands at molting; (2) through shedding the cuticle containing accumulated excretory granules; (3) through the wall of the midgut; and (4) through excretory glands. Eutardigrades possess three excretory glands ( Malpighian tubules); one dorsal and two lateral glands empty into the junction between the midgut and hindgut. These function as excretory and / or osmoregulatory structures in addition to the hindgut (rectum). The glands of the marine eutardigrade Halobiotus crispae are unusually large, reﬂecting its secondary adaptation from freshwater to seawater, where it inhabits the subtidal zone and is subject to marked changes in salinity. In echiniscids, excretory and / or secretory organs lie above the ventro-medial body wall at the level of the second and third pair of legs (Dewel et al., 1992). Each is comprised of one medial and two lateral cells enclosing a pair of convoluted 535 ducts. They probably transport material in the body cavity ﬂuid to the cuticle. Associated with miniaturization, specialized respiratory and circulatory systems are absent. Respiration occurs through the cuticle. Circulation is accomplished by the movement of ﬂuid and coelomocytes in the body cavity (hemocoel) when the animal moves. III. PHYSIOLOGY (LATENT STATES) All tardigrades are aquatic, regardless of their speciﬁc habitat, since they require a ﬁlm of water surrounding the body to be active. They are capable, however, of entering a latent state (cryptobiosis) when environmental conditions are unfavorable. Crowe (1975) identiﬁed ﬁve types of latency: encystment, anoxybiosis, cryobiosis, osmobiosis, and anhydrobiosis. When a tardigrade is in a latent state, metabolism, growth, reproduction, and senescence are reduced or cease temporarily, and resistance to environmental extremes such as cold, heat, drought, chemicals, and ionizing radiation is increased. Latent states thus have a signiﬁcant impact on the ecological role of the organism. (See review by Wright et al., 1992; Kinchin, 1994.) Encystment commonly occurs in freshwater tardigrades living in permanent pools, although cysts can also be formed by tardigrades inhabiting soil and moss. The conditions for encystment are not fully known; however, We˛glarska (1957) determined that a gradual worsening of the environment is one inducing factor for encystment in the freshwater tardigrade Dactylobiotus dispar. Specimens encysted regularly within 12 h if placed in water containing cyanophytes or decomposing leaves, but a “swift worsening” of the environment caused the animals to go into an asphyxial state (anoxybiosis). Before encystment, the tardigrade ingests large amounts of food for storage in body cavity cells (coelomocytes). Proceeding through stages similar to molting, the animal ﬁrst ejects the buccal apparatus and enters the simplex stage. After this process, the gut is emptied by defecation, the hindgut lining is shed, and the organism contracts within the cuticle, becoming immobile and barrel-shaped. A cyst membrane is formed and gradually becomes smooth and dark, replacing the old cuticle which sloughs off. The cyst is encased in strong walls, often opaque and encrusted with debris. Metabolism is very reduced in the cysts. Before emergence from the cyst when environmental conditions improve, a new cuticle is produced, including buccal apparatus and claws. Encystment is not obligatory in the life cycle of a tardigrade, and reasons for emergence are not fully known. The cysts are not able to withstand high temperatures, as in anhydrobiosis, because of the high 536 Diane R. Nelson water content; however, the cysts can survive for over a year in nature without totally depleting food reserves. Anoxybiosis, or asphyxia, is a cryptobiotic state induced by low oxygen tensions in the environmental water. The tardigrade becomes immobile, transparent, rigid, and extended due to water absorption resulting from loss of osmotic control. The asphyxial state is temperature dependent; the time required for inducement varies from a few hours to several days. Some species can survive up to 5 days in anoxybiosis; however, strictly aquatic species live only from a few hours to 3 days in the asphyxial state. Prior to this time limit, specimens can be revived by the addition of oxygen to the water; return to the active state requires a few minutes to several hours. The metabolic rate of tardigrades in anoxybiosis is unknown. Induced by low temperatures, cryobiosis is a type of cryptobiosis that enables tardigrades to survive freezing and thawing, allowing limno-terrestrial tardigrades to be common in polar regions (Kristensen, 1982; De Smet and Van Rompu, 1994; Sømme, 1996). Slow cooling rates and the formation of barrel-shaped tuns enhance survival, and membrane protectants such as trehalose stabilize membrane structure (Rudolph and Crowe, 1985). [See Westh and Kristensen, 1992; Westh et al., 1991; Ramløv and Westh, 1992, for detailed information.] Osmobiosis is a form of cryptobiosis that results from elevated osmotic pressures and decreased water activity at the surface of the animal. Some species can tolerate variations in salinity, especially intertidal marine species and euryhaline limno-terrestrial species. However, placed in salt solutions of various ionic strengths, most freshwater and terrestrial tardigrades form contracted tuns (Wright et al., 1992). The marine tardigrade Echiniscoides sigismundi becomes turgid in freshwater but can survive up to 3 days. Activity resumes when the animals are returned to water with the former osmotic concentrations. Anhydrobiosis (formerly called anabiosis) is a type of cryptobiosis induced by evaporative water loss from terrestrial eutardigrades and echiniscids. [See Wright, 1989a, b; Wright et al., 1992; Crowe et al., 1992; Kinchin, 1994; Sømme, 1996 for reviews.] Nearly all marine and true freshwater tardigrades lack the ability to survive dehydration and undergo cryptobiosis, but experimental evidence is sparse. As the environmental water surrounding the animal evaporates, the tardigrade contracts, retracting the head and legs and becoming the characteristic barrel-shaped tun. The rate of desiccation must be slow to ensure survival and return to active life with the addition of water. Survival of dehydration is correlated with synthesis of trehalose, a disaccharide of glucose, during dehydration or its degradation following rehydration. Trehalose alters the physical properties of membrane phospholipids and stabilizes dry membranes in anhydrobiotic organisms (Crowe et al., 1984; Westh and Ramløv, 1991). In the anhydrobiotic state, the tardigrade is highly resistant to extreme environmental factors (temperature, radiation, chemicals). Anhydrobiotic animals have remained viable on herbarium specimens for as long as 120 years. The eggs of terrestrial tardigrades can also withstand desiccation, but little is known about the eggs of the distinctly aquatic species. The life span of anhydrobiotic tardigrades can be greatly extended by going in and out of this cryptobiotic state, although frequent dehydration and rehydration results in reduced viability due to the depletion of lipid reserves, which can be converted to the membrane protectants glycerol and trehalose (Westh and Ramløv, 1991). In the anhydrobiotic state, tardigrades can also withstand extraordinarily high hydrostatic pressure, up to 600 MPa, in perﬂuorcarbon (Seki and Toyoshima, 1998). IV. REPRODUCTION AND DEVELOPMENT A. Reproductive Modes Sexual reproduction and parthenogenesis are reproductive modes exhibited in the Tardigrada (Ramazzotti and Maucci, 1983; Nelson, 1982a; Bertolani, 1982b, 1983a, b, 1987a, 1990, 1992, 1994; Rebecchi and Bertolani, 1988, 1994; Kinchin, 1994; Bertolani and Rebecchi, 1999). Marine tardigrades are bisexual (gonochoristic) with sexual dimorphism evident in the gonopore structure; hermaphroditic species are very rare, and parthenogenesis is absent (Bertolani, 1994). Nonmarine species, especially the eutardigrades, are usually gonochoristic (dioecious) with unisexual or bisexual populations, but external sexual dimorphism is rarely observed. Males are generally smaller than females; however, mature males may be larger than immature females in the same population. Males may also exhibit modiﬁcations of the claws (Rebecchi and Nelson, 1998), especially on the ﬁrst pair of legs, such as in Milnesium and the freshwater Pseudobiotus megalonyx and P. kathmanae (formerly P. augusti, in Kathman and Nelson, 1987, see revision in Nelson et al., 1999). These secondary sexual characteristics arise during the molt preceding sexual maturity (Bertolani, 1992; Rebecchi and Nelson, 1998). In heterotardigrades, additional sexual dimorphism may be exhibited in the length of the cephalic appendages, especially the clavae (Kinchin, 1994; Claxton, 1996). Meiotic maturation of the gametes occurs in amphimictic tardigrades. In three species of gonochoristic freshwater and terrestrial tardigrades, all of the females were iteroparous, but males were either iteroparous or semelparous. In nonmarine tardigrades, unisexual populations 15. Tardigrada are the most common type, and parthenogenesis is the mode of reproduction, often associated with polyploidy and with ameiotic maturation of the oocyte. Simultaneous hermaphroditism is sporadic but occurs in freshwater and terrestrial eutardigrades (Bertolani, 1994). B. Reproductive Apparatus A single (unpaired) gonad is present in both males and females (Figs. 3 and 4). This saclike structure is dorsal to the intestine and attached to the body wall by two anterior ligaments (small muscles, Dewel et al., 1993) in adult eutardigrades or by a single median ligament (basement membrane, Kristensen, 1979) in heterotardigrades (Nelson 1982a, Dewel et al., 1993). The morphology of the gonad varies with age, sex, species, and the reproductive activity of the tardigrade. The male tardigrade has two sperm ducts (vasa deferentia) that open into the hindgut (cloaca); the female has only one oviduct on either the right side or the left side of the intestine that opens into the hindgut. A small seminal receptacle (with internal epithelial spermatheca), which opens ventrally into the hindgut next to the oviduct opening, is present in only a few species of Macrobiotus, Xerobiotus, Ramazzottius, and Hypsibius (Marcus, 1929; Bertolani, 1983a; Rebecchi and Bertolani, 1988; Rebecchi, 1997). In eutardigrades, the cloacal opening is a ventral transverse slit anterior to the fourth pair of legs. In heterotardigrades, the preanal gonopore is rosette-shaped in females but is a protruding, round or oval-shaped aperture in males; only a few marine species have a postanal gonopore (Kinchin, 1994). Paired external cuticular seminal receptacles (“annex glands”) lateral to the gonopore are present in females in many species of marine heterotardigrades, but in only a few eutardigrades. In some species of both heterotardigrades and eutardigrades, males have seminal vesicles. (See Dewel et al., 1993 and Kinchin, 1994 for a review.) 537 hufelandi, the nutcracker-shaped motile spermatozoa with a helical nucleus, kidney-shaped midpiece, and long tail are associated with internal fertilization; the female has a spermatheca that contains straight, nonmotile spermatozoa with a cylindrical midpiece and a very short tail (Rebecchi, 1997). In the freshwater eutardigrade Isohypsibius granulifer, transfer of modiﬁed sperm occurs by copulation, and spermatozoa are found in the ovary of the female (Wolburg-Buchholz and Greven, 1979). In some heterotardigrades, the spermatozoa are less specialized, the female has no seminal receptacle, and fertilization is external (Kristensen, 1979). Rebecchi et al., (1999) reviewed ultrastructural differences in eutardigrade spermatozoa, which are considered highly specialized. Male gametes of eutardigrades are 25 – 100 m in length, usually have an elongated head with a coiled piece (acrosome or nucleus), a midpiece, and a tail with a tuft of 9 – 11 ﬁne ﬁlaments. Heterotardigrade spermatozoa, considered primitive, are shorter (14 – 20 m), have a globose head, lack a well-deﬁned midpiece, and have a tapering ﬂagellum. In eutardigrades, spermatozoan morphology can be correlated with scleriﬁed structure of adults and eggs, thus providing valuable taxonomic characters (Guidi and Rebecchi, 1996). Mating and fertilization have been observed in only a few species of tardigrades (Ramazzotti, 1972; Nelson, 1982a; Bertolani, 1987a, 1992). In some freshwater species, one or more males deposit sperm into the cloacal opening of the old cuticle of the female prior to, or during, a molt. External fertilization occurs as the eggs are deposited in the old cuticle. In the eutardigrade Pseudobiotus megalonyx, diploid males were able to locate mates and to distinguish between diploid amphimictic individuals and triploid parthenogenetic ones (Bertolani, 1992). In heterotardigrades, cephalic sensory structures may play a role in mating. D. Eggs C. Mating and Fertilization Tardigrade spermatozoa have been described in some species (Kristensen, 1979; Baccetti, 1987; Bertolani, 1983b; Rebecchi and Guidi, 1991, 1995; Guidi and Rebecchi, 1996; Rebecchi, 1997; Rebecchi et al., 1999). Henneke (1911) ﬁrst observed the presence of two kinds of sperm in the freshwater tardigrade Pseudobiotus macronyx. The ultrastructure of the spermatozoa of Macrobiotus hufelandi was ﬁrst investigated by Baccetti et al. (1971). Other workers described the morphology of spermatozoa and correlated morphology with a specialized mode of sperm transfer (Wolburg-Buchholz and Greven, 1979; Rebecchi and Guidi, 1991, 1995; Guidi and Rebecchi, 1996; Rebecchi, 1997). For example, in Xerobiotus pseudo- Eggs are often essential for the identiﬁcation of freshwater and terrestrial species, especially in some genera of eutardigrades (Fig. 9). Ornamented eggs, such as those characteristic of Macrobiotus, Murrayon, Minibiotus, Dactylobiotus, Amphibolus, Eohypsibius, Ramazzottius, Hebesuncus, and some Hypsibius species, as well as the heterotardigrade Oreella, are usually deposited freely and singly or in small groups. The ornamentation may include pores, reticulations, and processes. The different types of egg ornamentations and their taxonomic signiﬁcance have been discussed by various authors (Grigarick et al., 1973; Toftner et al., 1975; Greven, 1980; Ramazzotti and Maucci, 1983; Biserov, 1990a, b; Bertolani and Rebecchi, 1993; Bertolani et al., 1996). In some genera, 538 Diane R. Nelson smooth oval eggs with a thin shell are usually deposited in the molted cuticle. These smooth eggs are typical of the armored heterotardigrades, most eutardigrades in the genera Pseudobiotus, Doryphoribius, Hypsibius, Isohypsibius, Diphascon, Platicrista, Itaquascon, and Milnesium, and only a few of the Macrobiotus species. Some aquatic tardigrades deposit eggs inside the empty exoskeletons of cladocerans, ostracods, or insects. The aquatic eutardigrades Isohypsibius annulatus and two species of Pseudobiotus carry the exuvium containing eggs attached to the body for some time (Bertolani, 1982a; Rebecchi and Nelson, 1998). This is the only known example of parental care among tardigrades. The method of egg deposition is unknown for many species, especially in marine tardigrades. Echiniscus, Bryochoerus, Parechiniscus, and many Pseudechiniscus species; males were rare in Bryodelphax, Testechiniscus, Cornechiniscus, and Mopsechiniscus but common (50% of the population) in Oreella, Hypechiniscus, Proechiniscus, and Antechiniscus. In the absence of males in a tardigrade population, parthenogenesis was assumed to be the mode of reproduction. Dastych (1987a), however, reported the presence of males in four species of the genus Echiniscus. Claxton (1996) reported males to be common in a disproportionately large number of species (5 of the 8 species) of Echiniscus found in Australia; males and females were differentiated on the basis of gonopore morphology, the size of the males, and the length of the clavae. Grigarick et al. (1975) reported two “types” of gonopores and published the ﬁrst scanning electron micrograph of a male gonopore of a heterotardigrade. With regard to this report, Nelson (1982a) stated, “If males were indeed present, this would be the ﬁrst observation of males in any Echiniscus species . . . ” Based on the presence of ventral plates, Dastych (1987a) placed the species studied by Grigarick et al. (1975) in Kristensen’s (1987) new genus Testechiniscus, in which males are rare. Polyploidy is often associated with parthenogenesis and is frequently found in eutardigrades in various genera that inhabit different microhabitats (Bertolani, 1975, 1982b; Nelson, 1982a). Meiotic (automictic) parthenogenesis occurs in diploid biotypes of the freshwater eutardigrades Dactylobiotus parthenogenesis and Hypsibius dujardini. Ameiotic (apomictic) parthenogenesis occurs in triploid and tetraploid biotypes of some freshwater and terrestrial eutardigrades (Macrobious, Xerobiotus, Pseudobiotus). According to Pilato (1979), “ . . . many species in which males do occur are actually an association of amphigonic with one or more parthenogenetic, generally polyploid and reproductively isolated strains, often distinguishable by their mechanism of egg deposition.” Since parthenogenesis may be advantageous for invading new habitats, the evolution of parthenogenesis may be correlated with the evolution of cryptobiosis and the invasion of the terrestrial habitat (Pilato, 1979; Bertolani, 1987a; Rebecchi and Bertolani, 1988; Bertolani et al., 1990; Wright et al., 1992). E. Parthenogenesis F. Hermaphroditism Parthenogenesis, a common mode of reproduction in nonmarine tardigrades, is correlated with the ability to undergo cryptobiosis (Bertolani, 1982b; 1994). According to Kristensen (1987), in echiniscid heterotardigrades, males had not been previously recorded in Most eutardigrade populations are unisexual (thelytokus) although some are bisexual. Hermaphroditism is rare in eutardigrades and known only in one marine heterotardigrade (Bertolani, 1987a, 1994). In hermaphrodites, a single ovotestis with only one gonoduct is FIGURE 9 Scanning electron micrographs of Macrobiotus eggs. (A) Macrobiotus areolatus; (B) Macrobiotus hufelandi group; and (C) Macrobiotus tonollii. 15. Tardigrada present. Both mature and almost mature spermatozoa and oocytes may be found in a single individual, indicating the possibility of self-fertilization. Cyclic maturation probably occurs, with the initial prevalence of one type of gamete and then another. Hermaphroditic eutardigrades include Parhexapodibius pilatoi from grasslands, Macrobiotus joannae from moss, Amphibolus weglarskae from leaf litter, and four species of Isohypsibius from freshwater (Bertolani and Manicardi, 1986). G. Development Developmental time, from deposition of the egg to hatching, varies considerably within and between species and with environmental conditions. A minimum of 5 to a maximum of 40 days have been required for egg development under experimental conditions (Marcus, 1928, 1929). Eggs can be found at any time of the year in mosses, lichens, and soil; however, few observations of eggs of freshwater tardigrades have been reported (Ramazzotti and Maucci, 1983). Recently the embryology of the marine eutardigrade Halobiotus crispae and the intertidal heterotardigrade Echiniscoides sigismundi were investigated (Eibye-Jacobsen, 1996, 1996/97, 1997). Cleavage was total, equal, and asynchronous; however, the cleavage pattern and mode of mesoderm formation could not be deﬁnitely determined. Normal development of H. crispae at 8 °C was estimated to be 15 – 16 days. When embryonic development is complete, the immature tardigrade emerges from the egg by the action of stylets or hindlegs or by an increase in hydrostatic pressure created by pumping ﬂuid into the gut with the pharynx (Ramazzotti and Maucci, 1983; Eibye-Jacobsen, 1996/97, 1997). Postembryonic development occurs through several successive molts, primarily by the growth of the individual cells rather than by cell division. Although tardigrades have been considered cell constant, Bertolani (1982b) observed mitosis in eutardigrades. In eutardigrades, development is direct and the ﬁrst-instar animals (juveniles) are similar to the adults but have immature gonads and slight differences in the claws and buccal apparatus. Bertolani et al. (1984) concluded that heterotardigrades undergo indirect development characterized by two larval stages. The ﬁrst larval stage lacks an anus and a gonopore and has two claws less on each leg than the adult. The second larval stage (sometimes called a juvenile) has an anus and the same number of claws as the adult but the gonopore is rudimentary or lacking. The number of ﬁlamentous and spinous cuticular appendages generally increases with age (but decreases in Mopsechiniscus), although 539 variations may also occur (Grigarick et al., 1975; Ramazzotti and Maucci, 1983; Nelson 1982a; Claxton 1996). V. ECOLOGY A. Molting, Life History, and Cyclomorphosis Molting, which usually requires 5 – 10 days, occurs periodically throughout the life of the tardigrade (Walz, 1982). The entire cuticular lining of the foregut, including the stylets and stylet supports, is ejected through the expanded buccal opening. The mouth opening closes and the animal cannot feed. This is the “simplex” stage, characterized by the absence of the buccal apparatus. The salivary glands reform the cuticular structures of the buccal tube, stylets, and stylet supports; the esophagus regenerates its own cuticular lining. Concomitantly, new body cuticle, including the hindgut lining, is synthesized by the underlying epidermis and the new claws are produced by claw glands in the legs (Walz, 1982). During the molt, the old body cuticle is shed, including the claws and the lining of the hindgut (Fig. 10). Generally, body length increases with each molt until maximum size is attained, although lack of food can result in a decrease in size. Although growth is more rapid during the earlier molts, molting and growth continue even after sexual maturity (Nelson 1982a). Defecation and oviposition may also be associated with molting and changes in the ﬂuid pressure in the body cavity. Life histories of certain tardigrade species have been reviewed by Walz (1982), Nelson (1982a), Ramazzotti and Maucci (1983), and Kinchin (1994). Based on frequency distributions of body length and FIGURE 10 Scanning electron Kathmanae undergoing a molt. micrograph of Pseudobiotus 540 Diane R. Nelson buccal length, the number of molts has been estimated to range from 4 to 12. Problems inherent in the method have been discussed by Baumann (1961), Morgan (1977), Ramazzotti and Maucci (1983), and Kinchin (1994). Considerable variation and overlapping of the stages may occur within a species. Sexual maturity is reached with the second or third molt, and egg production continues throughout life, although molting can occur without egg production. The lifespan of active tardigrades (excluding cryptobiotic periods) has been estimated to be from 3 – 30 months (Higgins, 1959; Franceschi et al., 1962/63; Pollock, 1970; Ramazzotti and Maucci, 1983). The total lifespan, from hatching until death, may be greatly extended by periods of latent life (encystment and anhydrobiosis). Since aquatic tardigrades have little or no ability to undergo anhydrobiosis, the period of active life generally corresponds to that of total life. Ramazzotti and Maucci (1983) concluded that freshwater species of Macrobiotus and Hypsibius live about 1 – 2 yr.; whereas moss-inhabiting species of the same genera average 4 – 12 yr. Shorter lifespans of 3 – 4 months were proposed by Pollock (1970) for marine species and Franceschi et al. (1962/63) for moss-inhabiting species. Morgan (1977) estimated a lifespan of 3 – 7 months for Macrobiotus hufelandi and up to 3 months for Echiniscus testudo in roof mosses. Cyclomorphosis, an annual cycle of morphological change in individuals, has been documented in the marine eutardigrade Halobiotus crispae (Kristensen 1982). A sexually mature summer morph alternates with a sexually immature winter morph that tolerates freezing temperatures and low salinities. Cyclomorphosis may complicate identiﬁcations, especially since the distribution of the phenomenon is unknown. B. Habitats Although all active individuals require water, the environments in which tardigrades live are generally divided into: (1) marine and brackish water; (2) freshwater; and (3) terrestrial habitats. The distinction between freshwater and terrestrial species is sometimes unclear because some tardigrades can live in a wide range of habitats. With few exceptions, marine tardigrades belong to the class Heterotardigrada, either in the order Arthrotardigrada or the order Echiniscoidea (Table I). In the class Eutardigrada, only ﬁve species of the genus Halobiotus (formerly Isohypsibius) and a few species of Isohypsibius and Ramajendas are marine (Crisp and Kristensen, 1983; Pilato and Binda, 1990). Although relatively few studies on marine tardigrades have been conducted, they indicate high morphological diversity, with most of the genera having one or two species (Renaud-Mornant, 1982, 1987; Grimaldi De Zio et al., 1983; Villora-Moreno and De Zio Grimaldi, 1996). Marine tardigrades inhabit the intertidal zone and shallow waters off the continental shelf and as well as bathyl and abyssal depths, including manganese nodules in the deep sea of the eastern South Paciﬁc (Bussau, 1992). Three major ecological groups of marine tardigrades are recognized: 1. a small number of species on intertidal algae or other substrates (e.g., Echiniscoides on barnacles), including facultative and obligatory invertebrate ectoparasites (Tetrakentron on a sea cucumber) and commensals (e.g., Actinarctus, Pleocola); 2. a larger group of species inhabiting the interstitial biotype, particularly in the top few centimeters of sand near the low tide mark (e.g., Batillipes); and 3. a highly diversiﬁed group of abyssal and deepsea ooze dwellers (e.g., Coronarctus). Hydrophilous tardigrades are “distinctly aquatic” species that live only in permanent freshwater habitats. Although they are occasionally found in plankton samples, they are benthic organisms that crawl on the surfaces of aquatic plants or in the interstitial spaces of sandy sediments in ponds, lakes, rivers, and streams (Schuster et al., 1977; Wainberg and Hummon, 1981; Kathman and Nelson, 1987; Nelson et al., 1987; Van Rompu and De Smet, 1988, 1991; Van Rompu et al., 1991a, b, 1992; Strayer et al., 1994). Most species live in the littoral zone; however, some individuals have been collected from lakes up to 150 m deep (Ramazzotti and Maucci, 1983). Benthic algal mats in maritime Antarctic lakes and pools are productive habitats for tardigrades (McInnes, 1995; Everitt, 1981; McInnes and Ellis-Evans, 1987, 1990). Cryoconite holes in glaciers, formed when heat is absorbed by surface accumulation of dark dust, also provide a specialized habitat for rotifers and tardigrades, which feed on the microﬂora living there (Dastych, 1993b; De Smet and Van Rompu, 1994; Sømme, 1996). Hygrophilous tardigrades, which inhabit moist mosses, and eurytopic species, which live in a wide range of moisture conditions, are often found in aquatic habitats. In the Heterotardigrada, Styraconyx hallasi is the only freshwater arthrotardigrade. In the order Echiniscoidea, Carphania ﬂuviatilis in the family Carphanidae (Binda and Kristensen, 1986), and a few species of Echiniscus, Hypechiniscus, and Pseudechiniscus in the family Echiniscidae are occasionally limnic (Kristensen, 1987). The single specimen of Echinursellus longiunguis, originally described as an arthrotardigrade from the shores of a Chilean lake, is a cyst or cyclomorphic 15. Tardigrada FIGURE 11 Scanning electron micrograph of an aquatic tardigrade, Pseudobiotus Kathmanae. stage of a eutardigrade in the genus Pseudobiotus, and is not the missing link between Arthrotardigrada and Echiniscoidea as originally reported (Kristensen, 1987). The status of the mesotardigrade Thermozodium esakii from a Japanese hot spring is dubious. The class Eutardigrada contains both terrestrial and freshwater species (Table I). Three genera (Dactylobiotus, Pseudobiotus, and Thulinia) are exclusively freshwater (Fig. 11). Other genera have one or more species that are typically aquatic, i.e., Macrobiotus, Macroversum, Mixibius, Murrayon, Amphibolus, Doryphoribius, Ramajendas, Eohypsibius, Isohypsibius, Hypsibius, and the Diphascon group (Bertolani, 1982a; Bertolani and Pilato, 1988; Pilato and Binda, 1990; Pilato, 1992). Several species of the genus Hypsibius ( e.g., H. dujardini and H. convergens) are not obligate freshwater organisms but are often found in this habitat. Milnesium tardigradum and Macrobiotus hufelandi are eurytopic terrestrial species that may also inhabit freshwater. The tardigrade component of the psammon community in lakes and river banks is poorly known, but it consists primarily of hydrophilous and hygrophilous species that show a distinct seasonality in population densities, with peak periods mainly in the winter and spring (Ramazzotti and Maucci, 1983). The largest number of tardigrade species are terrestrial and live in moist habitats, such as in soil and leaf litter or among mosses, lichens, liverworts, and cushionshaped ﬂowering plants (Ramazzotti and Maucci, 1983; Kinchin, 1994). Arctic and Antarctic species also live in terrestrial moss turfs (Dastych, 1984; Bertolani and Kristensen, 1987; Usher and Dastych, 1987; Maucci, 1996; Miller et al., 1996). Most terrestrial tardigrades inhabit the moss environment, which can be divided into three groups: wet, moist, and dry mosses (Mihelčič, 1954 / 55; Ramazzotti and Maucci, 1983). 541 Wet mosses such as submerged and emergent species in lakes, ponds, and streams never dehydrate completely. In contrast, moist and dry mosses living on tree trunks, rocks, walls, roofs, and soil are subject to total desiccation for various periods of time and are dependent on atmospheric precipitation for moisture. The typical terrestrial habitat of a tardigrade has: (1) sufﬁcient aeration (tardigrades are hypersensitive to low oxygen levels); (2) alternate periods of wet and dry conditions; and (3) suitable and sufﬁcient food. Tardigrades are less likely to inhabit impermeable clay soils or dense cushion mosses with thick cell walls. A distinct insular distribution of tardigrades within patches of mosses and soil has been frequently noted (Ramazzotti and Maucci, 1983). Lichens and liverworts are inhabited by a community usually similar to that in mosses. High species diversity and large populations of eutardigrades are also common in soil and the upper layer of leaf litter; however, heterotardigrades are rare or absent (Fleeger and Hummon, 1975; Manicardi and Bertolani, 1987; Bertolani and Rebecchi, 1996; Guidetti, 1998; Guidetti et al., in press). Interstitial eutardigrades in inorganic soils are characterized by very short legs with small claws and a reduction or absence of the hind claws (Bertolani and Biserov, 1996). The tardigrade community in leaf litter from beech forests contains some species found in freshwater, but not those characterized by very long claws (e.g., Dactylobiotus, Pseudobiotus). There is also some overlap with the turf community, except for turf species with very short claws. In contrast, the litter community is signiﬁcantly different from that of mosses and lichens on beech trees in the same area (Guidetti et al., 1999.) C. Population Density Population densities of tardigrades are highly variable; however, neither minimal nor optimal conditions for population growth are known. [See review in Kinchin, 1994.] Changes in tardigrade population densities have been correlated with a variety of environmental conditions, including temperature and moisture (Franceschi et al., 1962 / 63; Morgan, 1977; Briones et al., 1997), air pollution (Steiner, 1994a – c, 1995), and food availability (Hallas and Yeates, 1972). Other factors such as competition, predation, and parasitism may also play a role. Predators include nematodes, other tardigrades, mites, spiders, springtails, and insect larvae; parasitic protozoa and fungi often infect tardigrade populations (Ramazzotti and Maucci, 1983). Considerable variation in both population density and species diversity occurs in adjacent, seemingly identical, microhabitats. Usually 2 – 6 species are present, but 10 or more species have been found in a single sample. 542 Diane R. Nelson In general, patchiness is a common characteristic of tardigrade populations. Populations of tardigrades from soil and leaf litter habitats range from 5 to 40 individuals / cm2. [See Nelson and Higgins, 1990 for review and Guidetti et al., 1999.] Similarly, population densities in mosses and other terrestrial habitats vary widely, from 0 to 50 – 200 individuals / cm2. (For review, see Ramazzotti and Maucci, 1983; Kinchin, 1994.) Large populations of both heterotardigrades and eutardigrades are often found in thin, spreading mosses growing in open, exposed places; in contrast, very few heterotardigrades, but many eutardigrades, can be collected in mosses from moist shady areas. Studies of the population dynamics of freshwater tardigrades are less common than those of terrestrial habitats (Schuster et al., 1977; Everitt, 1981; Wainberg and Hummon, 1981; Kathman and Nelson, 1987; Nelson et al., 1987; Strayer et al., 1994). Ramazzotti and Maucci (1983) and Nelson et al. (1987) reported that freshwater eutardigrades often reach a very high population density in the spring, whereas Schuster et al. (1977) and Wainberg and Hummon (1981) noted population peaks in the fall. Kathman and Nelson (1987) stated, “Several previous life-history studies indicated wide variation in total numbers of animals obtained from month to month, but few studies extended beyond 12 months or presented data on replicates, means, or standard errors for determining the validity of these variations. Unless there are recurring patterns over several years, abundance estimates may be invalid if adequate numbers of quantitative replicates are not obtained, as our data show that within-period variation was often greater than between-period variation.” D. Distribution Due to the paucity of data and the uncertainty of identiﬁcations, the biogeography of the Tardigrada remains relatively unknown (McInnes, 1994). As a phylum, tardigrades have a worldwide distribution. Many species with broad ecological requirements are considered to be cosmopolitan, whereas others with more narrow tolerances are rare or endemic. Cluster analysis of the distribution of extant fauna showed that limnoterrestrial tardigrades have limited generic and familial distribution ranges deﬁned by biogeographical provinces and major geological events (McInnes and Pugh, 1998; Pugh and McInnes, 1998). Distinct Laurasian and Gondwanan familial clusters correlate with the Triassic disintegration of Pangaea, while separate Antarctic, Australian and New Zealand familial / generic clusters relate to the subsequent Jurassic / Cretaceous disintegration of Gondwana. A “Nearctic-Arctic” biogeographic cluster is distinct from that of “Northern and Alpine Europe”. In general, moss-inhabiting tardigrades are more common in polar and temperate zones than in the tropics. More data are required, however, before deﬁnitive biogeographical distributions of the phylum can be determined. On a more localized scale, signiﬁcant differences in altitudinal distributions of terrestrial tardigrades have been reported by Nelson (1975), Beasley (1988), Dastych (1987b, 1988), Manicardi and Bertolani (1987), Ramazzotti and Maucci (1983), and Guidetti et al. (1999). In contrast, Kathman and Cross (1991) found that the distribution of tardigrades at any altitude is primarily determined by abiotic factors, such as temperature and humidity, which affect the microhabitats at a particular altitude. Physical and chemical monitoring of pollutants in environmental surveys have been used to indicate air and water quality; however, ecological monitoring has not been extensively developed. Terrestrial invertebrates in mosses and soils, as well as aquatic meiofauna, may be suitable as a monitoring system to indicate environmental quality. Steiner (1994a – c, 1995) investigated the inﬂuence of air pollution on moss-dwelling nematodes and tardigrades along an urban – rural gradient near Zürich, Switzerland. Although the abundance of tardigrades varied independently of SO2 and NO2, the number of tardigrade species decreased with increasing levels of SO2. Fumigation experiments with SO2 indicated that the effects were dose-dependent and taxon speciﬁc. The tardigrade Macrobiotus persimilis was typical of sites with relatively high levels of NO2. E. Dissemination Eggs, cysts, and barrel-shaped anhydrobiotic specimens of terrestrial tardigrades are passively dispersed predominantly by wind, although rain, ﬂoodwaters, and melting snow can transport active as well as cryptobiotic forms (Ramazzotti and Maucci, 1983). Other animals, associated with terrestrial communities, e.g., birds, snails, isopods, mites, centipedes, millipedes, and insects, can also assist in the dispersal of tardigrades. Active dissemination by crawling is limited by the necessity for a constant ﬁlm of water covering the body of the tardigrade, and by its size and speed of movement. Little information is available on dispersal of the distinctly aquatic species, which probably undergo limited or no cryptobiosis. Transport of eggs or encysted forms from dry temporary ponds is possible. Active tardigrades may be distributed through river systems, particularly during periods of heavy rainfall and ﬂooding. The mechanism for re-population of aquatic habitats after the summer disappearance of tardigrades or 15. Tardigrada after habitat disturbances has not been investigated (Nelson et al., 1987). VI. TECHNIQUES FOR COLLECTION, EXTRACTION, AND MICROSCOPY Qualitative collections of tardigrades from aquatic habitats with sandy bottoms can be made by stirring the sand in a container of water and decanting immediately after the sand settles (Schuster et al., 1977). The decanted water is passed through a sieve (US Standard No. 325) with pores of about 44 m, and the specimens are rinsed from the screen to a sample jar. Tardigrades in large volumes of water can be preserved by the addition of formalin (5%) or gluteraldehyde. A modiﬁed Boisseau apparatus has also been adapted for tardigrade extraction (Kinchin, 1994). Modiﬁcations of techniques to extract soil nematodes by centrifugation in water, sucrose, or Ludox® have been used to remove tardigrades from aquatic and terrestrial habitats (Hallas, 1975; Hallas and Yeates, 1972; Bertolani, 1982a). A watery suspension of the sample (sediment or vegetation) is prepared and ﬁltered through two stacked sieves with mesh openings of 500 m or greater (top sieve) and 44 m (bottom sieve). The sediment remaining on the ﬁne mesh sieve is centrifuged with water at about 3000 rpm for 5 mins. The supernatant is then poured through the ﬁne mesh sieve, and the remaining sediment is centrifuged with a sucrose solution (sp. gr. 1.18) at about 5000 rpm for 1 min. Ludox® (DuPont product, colloidal silica, Sigma – Aldrich Chemical Co.) can be used in place of sucrose; Ludox AM® is useful for extracting tardigrades from terrestrial / freshwater environment, whereas Ludox TM® is more effective with marine tardigrades (McInnes, personal communication). The supernatant is immediately rinsed through the ﬁne mesh sieve with running tap water, and the residue is washed into a petri dish for examination under a dissecting microscope. Collections of tardigrades from vegetation can be made by removing the plant (moss, lichen, liverwort, etc.) from the substrate (tree, rock, soil) and placing the sample in a small paper bag for dry storage until processing. The sample is then placed in a container of water for several hours to reverse anhydrobiosis. The sample is agitated and squeezed to remove specimens, eggs, and cysts. After the excess water is decanted, the remaining sediment containing the live tardigrades can be examined with a dissecting microscope at a magniﬁcation of 30 or greater. The sample may also be washed through sieves to remove particles smaller than 50 m or larger than 1200 m. Boiling water or boiling alcohol (85% or higher) added to specimens in small 543 amounts of water is adequate for tardigrade ﬁxation, but alcohol is recommended to preserve specimens to be mounted later. R. D. Kathman (personal communication) found that mounting the live tardigrade directly in Hoyer’s medium was more efﬁcient, however longterm preservation was not as satisfactory. Core samples of soil and leaf litter can also be placed in paper bags for dry storage and processed according to the traditional procedure for vegetation. The sample is thoroughly soaked in water for several hours and examined in small quantities of thin aqueous suspensions since eggs and cysts are easily overlooked. The technique is extremely laborious and time-consuming; however, centrifugation with sucrose or Ludox®, as described above for aquatic samples, will reduce the time required for extraction of tardigrades from the substrate. After ﬁxation, individual specimens are mounted on microscope slides and observed with phase contrast and / or differential interference contrast (DIC) microscopy. Hoyer’s mounting medium (distilled water, 50 ml; gum arabic, 30 g; chloral hydrate, 150 g; glycerol, 20 ml) produces cleared, distended specimens, but the coverslip must be sealed (e.g., with epoxy paint) to ensure permanence. The amount of chloral hydrate may be reduced to decrease the extent of clearing of specimens, and potassium iodide (KI, 1 g) may be added to stain specimens slightly to increase resolution of morphological structures. CMC® and CMCP® media are useful for temporary mounts; however, excessive stain may obscure important structures, and the specimens may clear completely after a period of time. Other media used include Faure’s medium and polyvinyl lactophenol. More permanent mounts with balsam or synthetic resins are not as useful for phase microscopy. Kristensen and Higgins (1984a) recommend transferring animals with a drop of 2% formalin to slides and covering with a coverslip. The formalin preparation is infused with a 10% solution of glycerin and 90% ethyl alcohol and allowed to evaporate to glycerin over several days. Then the coverslip is sealed with Murrayite. For scanning electron microscopy (SEM), ﬁxed tardigrades are dehydrated to absolute alcohol, transferred to an intermediary liquid, such as amyl acetate, and dried by the critical point method (Grigarick et al., 1975). Individuals are mounted on SEM stubs and coated with 200 – 300 nm of gold or gold – palladium. Tardigrades can also be purchased commercially from Carolina Biological Supply Company (Burlington, NC) or Ward’s (Rochester, NY). Specimens from Carolina, labeled “Milnesium” or “Milnesium and other species” and shipped in the anhydrobiotic state, may contain a mixture of Macrobiotus, Milnesium, and / or Echiniscus. Cultures of aquatic specimens shipped in the active state from Ward’s, labeled 544 Diane R. Nelson “Hypsibius (water bear)”, were actually Thulinia in the samples personally examined. Caution should be exercised in relying on a company’s identiﬁcations of specialized groups of invertebrates. VII. IDENTIFICATION Identiﬁcation of species in the phylum Tardigrada is based primarily on the morphology of the cuticle, claws, buccal apparatus, and eggs, which have been discussed in previous sections. Early European workers described the majority of tardigrade species from temporary wet mounts of live specimens observed with the light microscope, often resulting in inadequate, incorrect, or incomplete descriptions and illustrations. Morphology, systematics, and natural history were focal points of their studies. Early monographs and other papers by Thulin (1928) and Marcus (1929, 1936) in German and Cuénot (1932) in French formed the basis of tardigrade systematics. Ramazzotti (1962, 1972) published a monograph, in Italian, which compiled keys and illustrated diagnoses of the species, thorough sections on morphology and biology, and an extensive bibliography; a supplement was produced in 1974. The last revision of this comprehensive monograph (Ramazzotti and Maucci, 1983) incorporated parts of the taxonomic revisions by Pilato (1969, 1982) and Schuster et al. (1980). An English translation of this important monograph is available from Dr. Clark Beasley, Department of Biology, McMurry University, Abilene, TX. Useful references in English include those by Morgan and King (1976), Nelson (1982a), Dewel et al. (1993), and Kinchin (1994) and the international symposia volumes (Higgins, 1975; We˛glarska, 1979; Nelson, 1982b; Bertolani, 1987b; McInnes and Norman, 1996; and Greven, 1999). Kinchin (1994) reviewed the systematics, morphology, and ecology of tardigrades and presented a guide to common species. He synthesized classiﬁcations from various authors, dividing tardigrades artiﬁcially into “marine heterotardigrades, marine eutardigrades, limno-terrestrial heterotardigrades, and limno-terrestrial eutardigrades.” Currently, claw structure, considered more conservative than the bucco-pharyngeal apparatus, is the major character for distinguishing genera and families within the eutardigrades (Bertolani and Kristensen, 1987). Figures 5 – 8 illustrate the claw types and the buccal apparatus of the major genera of eutardigrades, basically according to the classiﬁcation system of Ramazzotti and Maucci (1983), but with the inclusion of additional genera. The ﬁgures illustrate the range of variation in morphological structures that are considered of taxonomic importance in deﬁning genera and also the need for further analysis. The following key includes common genera previously found and likely to be found in freshwater and terrestrial habitats in North America. Positive identiﬁcation with the key may be difﬁcult because of inadequate and often vague generic descriptions, additions of new genera (often formed by splitting current genera), and the need for further revision of tardigrade systematics. A. Taxonomic Key to Common Genera of Freshwater and Terrestrial Tardigrada 1a. Lateral cirri A present ........................................................................................................................................................................2 1b. Lateral cirri A absent..........................................................................................................................................................................4 2a(1a). Cuticle without dorsal plates..............................................................................................................................................................3 2b. Cuticle with complete or partial dorsal plates; terrestrial ...Echiniscidae [Bryochoerus, Bryodelphax, Cornechiniscus, Echiniscus, Hypechiniscus, Mopsechiniscus, Parechiniscus, Pseudechiniscus (see Kristensen, 1987 for revision).] 3a(2a). Body indistinctly divided into eight segments; clavae present; four claws on all legs in adults; terrestrial..................................Oreella 3b. Body eutardigrade-like in shape; clavae absent; two claws on legs I – III, one claw on legs IV; freshwater ............................Carphania 4a(1b). Head with cephalic papillae ...............................................................................................................................................................5 4b. Head without cephalic papillae ..........................................................................................................................................................6 5a(4a). Head with six peribuccal papillae and two lateral papillae present; terrestrial, occasionally in freshwater...........................Milnesium 5b. Head without peribuccal papillae; two lateral papillae present; terrestrial ..........................................................................Limmenius 6a(4b). Claws absent on all legs; legs very short; terrestrial (Binda, 1984)........................................................................................Apodibius 6b. Claws present on at least one pair of legs ...........................................................................................................................................7 7a(6b). Single unbranched claws without secondary branches........................................................................................................................8 7b. Double claws, with primary and secondary branches .........................................................................................................................9 15. Tardigrada 545 8a(7a). One unbranched conical claw on leg I, no claws on legs II – IV; terrestrial.......................................................................Necopinatum 8b. Two unbranched claws on legs I – III, small basal spur-like claws on leg IV; terrestrial ............................................Haplomacrobiotus 9a(7b). Two double claws on each leg, similar in size and shape and symmetrical with respect to the median plane of the leg; claw branch sequence is 2-1-1-2 (secondary, primary, secondary, primary) ..........................................................................................................10 9b. Two double claws on each leg usually dissimilar in size and shape and asymmetrical with respect to the median plane of the leg; claw branch sequence is 2-1-2-1 (secondary, primary, secondary, primary) ......................................................................................14 10a(9a). Buccal tube of distinctly greater length between stylet supports and placoids than between stylet supports and buccal opening; posterior part of tube of spiral composition; terrestrial ..................................................................................................Pseudodiphascon 10b. Buccal tube between stylet supports and placoids not longer than distance between stylet supports and buccal opening; tube without spiral composition ...........................................................................................................................................................................11 11a(10b). Stylet supports attach near middle of buccal tube; with 10 peribuccal papulae (not lamellae); terrestrial ...........................Minibiotus 11b. Stylet supports attach in posterior half of buccal tube .....................................................................................................................12 12a(11b). Branches of claws at right angles so that tips are remote; two claws structurally connected at base; claws without lunules; cuticle without pores; freshwater ..............................................................................................................................................Dactylobiotus 12b. Both branches of claw follow similar axis so that tips are close together; claws not structurally connected at base; claws with lunules; cuticle with pores ........................................................................................................................................................13 13a(12b). Ventral lamina (reinforcement rod, buccal tube support) on buccal tube well developed; no anterior crest on buccal tube; 10 peribuccal lamellae; terrestrial or freshwater ...................................................................................................................Macrobiotus 13b. Ventral lamina narrow or absent; unequal dorsal and ventral crests on buccal tube; lamellae unknown; terrestrial .........Adorybiotus 14a(9b). Buccal tube with median longitudinal anteroventral lamina (ventral lamina or buccal tube support) ..............................................15 14b. Buccal tube without median longitudinal anteroventral lamina (ventral lamina or buccal tube support) .........................................16 15a(14a). Legs IV reduced or absent, with no claws or one or two minute double claws; terrestrial...............................................Hexapodibius 15b. Legs IV normally developed; terrestrial and freshwater ................................................................................................Doryphoribius 16a(14b). Claw of Calohypsibius-type, primary and secondary branches rigidly connected, claws equal in size ..............................................17 16b. Claw with two or three distinct parts ..............................................................................................................................................18 17a(16a). Cuticle sculptured, two transverse rows of macroplacoids; terrestrial ............................................................................Calohypsibius 17b. Cuticle smooth; three transverse rows of macroplacoids; terrestrial ............................................................................Microhypsibius 18a(16b). Each double claw with three distinct parts ......................................................................................................................................19 18b. Each double claw with two distinct parts ........................................................................................................................................20 19a(18a). Posterior part of buccal tube of spiral composition; 14 (16?) lamellae present; terrestrial ................................................Eohypsibius 19b. Buccal tube rigid, without spiral composition; 14 lamellae present; terrestrial or freshwater ............................................Amphibolus 20a(18b). Buccal tube rigid, without posterior part of spiral composition .......................................................................................................21 20b. Buccal tube with posterior part of spiral composition .....................................................................................................................25 21a(20a). Internal and external claws of dissimilar size and shape, with secondary branch continuous with the base; no lunules; no peribuccal lamellae . ..........................................................................................................................................................................................22 21b. Internal and external claws of dissimilar size and shape, with secondary branch forming right angle with base; lunules and / or peribuccal lamellae present or absent ..................................................................................................................................23 22a(21a). Claws of oberhaeuseri-type: Hypsibius-type with internal and external claws of very dissimilar size and shape, with primary branch of external claw very long and narrow, with very thin ﬂexible connection to upper basal portion; elliptical dorsolateral sense organs on head; terrestrial (Binda and Pilato, 1986) ..................................................................................................................Ramazzottius 22b. Claws of dujardini-type: Hypsibius-type with external claw slightly larger than internal claw, with primary branch attached to median basal portion of claw; no sense organs on head; terrestrial or freshwater......................................................................Hypsibius 23a(21b). Dorsal and ventral crests on buccal tube absent; peribuccal lunules rarely present; cuticle smooth or with granulations and / or gibbosites; terrestrial or freshwater .......................................................................................................................................Isohypsibius 23b. Dorsal and ventral crests on buccal tube present .............................................................................................................................24 24a(23b). With 12 peribuccal lamellae; freshwater .................................................................................................................................Thulinia 546 Diane R. Nelson 24b. With 30 peribuccal lamellae; freshwater..........................................................................................................................Pseudobiotus 25a(20b). Pharynx with placoids and stylet supports reduced or absent terrestrial, freshwater ...........................................................Itaquascon 25b. Pharynx with normal placoids; buccal tube with obvious stylet supports; terrestrial or freshwater; (formerly Diphascon; revised in Pilato, 1987) ....................................................................................................................................................................................26 26a(25b). Apophyses for insertion of stylet muscles in shape of a “hook” .......................................................................................................27 26b. Apophyses for insertion of stylet muscles in shape of wide, ﬂat “ridges” .........................................................................................28 27a(26a). Stylet apophyses in the shape of a semilunar hook; pharyngeal tube longer than the buccal tube; with (subgenus Diphascon) or without (subgenus Adropion) droplike thickening on buccal below insertion of stylet supports..........................................Diphascon 27b. Stylet apophyses in the shape of a blunt hook; pharyngeal tube shorter than the buccal tube ............................................Hebesuncus 28a(26b). Stylet furcae with posteriolateral processes thickened at apices ...........................................................................................Mesocrista 28b. Stylet furcae with posteriolateral processes spoon-like and tapering at their apices .............................................................Platicrista LITERATURE CITED Aguinaldo, A. M., Turbeville, J. M., Linford, L. S., Rivera, M. C., Garey, J. R., Raff, R., Lake, J. A. 1997. Evidence for a clade of nematodes, arthropods, and other moulting animals. Nature 387:489 – 493. 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Cook Biodiversity Section ECORC, Central Experimental Farm Agriculture and Agri-Food Canada Ottawa, Ontario, Canada K1A 0C6 7836 North Invergordon Place Paradise Valley, Arizona 85253 Bruce P. Smith Biology Department, Ithaca College Ithaca, New York 14850 I. Introduction to Arachnids II. Water Mites (Hydrachnida) A. Introduction B. External Morphology and Internal Anatomy C. Life History D. Habitats and Communities E. Ecology F. Collecting, Rearing and Preparation for Study I. INTRODUCTION TO ARACHNIDS Members of the class Arachnida are the most familiar representatives of the arthropodan subphylum Chelicerata, an ancient lineage characterized by distinctive mouthparts with two pairs of appendages, the anterior chelicerae and posterior pedipalps, and four pairs of walking legs. Chelicerates ﬁrst appear deﬁnitively in the fossil record as giant water scorpions (Merostomata, subclass Eurypterida) in freshwater deposits from the Silurian. By the Devonian, eurypterids had apparently disappeared but other chelicerate lineages were proliferating, such as horseshoe crabs (Merostomata, subclass Xiphosura) in marine environments, and early derivative arachnid groups, including mites, on land. Arachnids are the only group of chelicerates exhibiting extensive modern diversity. The higher classiﬁcation is still being debated, but arachnids Ecology and Classiﬁcation of North American Freshwater Invertebrates. 2nd Edition Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved. G. Taxonomic Keys to Genera of Water Mites in North America III. Other Arachnids in Freshwater Habitats A. Other Mites (Acari) B. Spiders Acknowledgments Literature Cited are usually considered to be a taxonomic class comprising 11 extant subclasses, mostly terrestrial groups of highly specialized predators of other arthropods. Only two subclasses of modern arachnids are represented in freshwater habitats, mites (Acari) which have diversiﬁed extensively to become arguably the most ubiquitous and adaptable clade of arthropods on Earth, and spiders (Araneae) which have maintained a conservative way of life as essentially terrestrial predators. Mites belonging to several unrelated groups are commonly found in freshwater habitats. The true water mites comprise a taxon of actinedid acariform mites which we refer to here as Hydrachnida (Hydrachnellae, Hydracarina, or Hydrachnidia of other authors). Hydrachnida have ﬂourished to become by far the most numerous, diverse, and ecologically important group of freshwater arachnids. Various families in certain suborders of Acariformes (namely, Actinedida, 551 552 Ian M. Smith, David R. Cook, Bruce P. Smith Oribatida and Acaridida) and Parasitiformes (namely, Gamasida) have independently invaded freshwater and become adapted for living there. Compared to Hydrachnida, these groups are less diverse taxonomically, usually less abundant, and relatively conservative in morphology and habits. We summarize diagnostic and ecological information for these mites and for the remarkable spiders of the genus Dolomedes at the end of this chapter. II. WATER MITES (HYDRACHNIDA) A. Introduction Water mites are among the most abundant and diverse benthic arthropods in many habitats. One square meter area of substrate from littoral weed beds in eutrophic lakes may contain as many as 2000 deutonymphs and adults representing up to 75 species in 25 or more genera. Comparable samples from an equivalent area of substrate in rocky rifﬂes of streams often yield over 5000 individuals of more than 50 species in over 30 genera (including both benthic and hyporheic forms). Water mites have co-evolved with some of the dominant insect groups in freshwater ecosystems, especially nematocerous Diptera, and typically interact intimately with these insects at all stages of their life histories. We have made numerous additions and reﬁnements to the information included in the ﬁrst edition of this chapter (Smith and Cook, 1991), and some extensive revisions. We have tried to avoid unnecessary use of specialized acarological terms, and provide a glossary of essential terminology needed for describing morphological and anatomical features. Those wishing to consult a more comprehensive account of mite structure and function are referred to the appropriate sections of the books by Cook (1974) and Krantz (1978). 1. General Relationships Hydrachnida, along with the enigmatic interstitial Stygothrombidioidea and terrestrial Calyptostomatoidea, Trombidioidea, and Erythraeoidea, belong to a remarkably diverse natural group of actinedid acariform mites, the Parasitengona. The complex and essentially holometabolous type of development of this group is unique among Acari. Typically, after emerging from the egg membrane, the hexapod larva seeks out an appropriate host, and becomes an ectoparasite which is passively transported while feeding on host ﬂuids. When fully engorged the larva transforms to the quiescent protonymph (or nymphochrysalis). Radical structural reorganization occurs during this stage, giving rise to the active deutonymph which resembles the adult in being octopod and typically predaceous, but is sexually immature and exhibits incomplete sclerotization and chaetotaxy. The deutonymph feeds and grows in size before entering another quiescent stage, the tritonymph (or imagochrysalis). After completion of metamorphosis during this stage, the mature adult emerges (see Fig. 1). Larval water mites can be distinguished morphologically from those of other parasitengones by having two setae, rather than one, on the genu of the pedipalp. Deutonymphal and adult water mites differ from all other Acari in having glandularia on the idiosoma. 2. Origin Water mites evolved from terrestrial stock, and hypotheses on their origin usually presume an ancestral terrestrial parasitengone (Mitchell, 1957a; Davids and Belier, 1979). A recently proposed alternative hypothesis (Wiggins et al., 1980; Smith and Oliver, 1986) suggests that the primitive parasitengone stock may have been water mites resembling certain extant Hydryphantoidea. According to this postulate, water mites diverged from terrestrial ancestors with direct development, perhaps resembling extant Anystoidea, while evolving the basic parasitengone life-history pattern as a set of adaptations for exploiting spatially and temporally intermittent aquatic habitats. The fossil record provides little insight into the evolutionary history of water mites. The only reported fossils that undoubtedly are water mites (Cook, 1957; Poinar, 1985) are larval specimens from Tertiary deposits representing highly derived taxa. However, distributional data and host associations indicate that the group originated no later than the Triassic or Jurassic period. Available morphological and behavioral data suggest that extant water mites are monophyletic (Mitchell, 1957a; Barr, 1972; Cook, 1974; Smith and Oliver, 1976, 1986), and that the major phyletic lineages of water mites, and possibly all Parasitengona, were derived from hydryphantoidlike ancestors (Mitchell, 1957a; Smith and Oliver, 1986). 3. Diversity and Classiﬁcation Well over 5000 species of water mites are currently recognized worldwide, representing more than 300 genera and subgenera in over 100 families and subfamilies (Viets, 1987). Acarologists usually consider water mites to be a taxon of intermediate rank between superfamily and suborder. Interestingly, the group appears to rival several orders of aquatic insects in diversity, and is comparable to some of them in age. Most of 16. Water Mites (Hydrachnida) and Other Arachnids FIGURE 1 553 Diagrammatic illustration of a generalized water mite history. the genera and families are reasonably stable and appear to approximate closely holophyletic lineages. The families are conservatively grouped into seven superfamilies. Three of these — Hydrovolzioidea, Hydrachnoidea, and Eylaoidea — probably represent natural groupings. The others — Hydryphantoidea, Lebertioidea, Hygrobatoidea and Arrenuroidea — are all either paraphyletic or polyphyletic assemblages subject to future revision. A comprehensive, detailed cladistic analysis of extant fauna is required to permit construction of more stable and informative superfamily groupings. The phylogenetic studies that prove most useful in understanding water mite evolution and developing a more natural classiﬁcation provide syntheses of information based on the morphology of all instars, behav- ioral attributes, and life-history data (Mitchell, 1957a; Cook, 1974; Smith, 1976a; Smith and Oliver, 1976, 1986). The over 1500 species currently estimated to occur in North America, north of Mexico, represent 136 genera in 69 subfamilies, 42 families, and all seven superfamilies. The fauna remains incompletely known at the generic level, and revision of the family level classiﬁcation is ongoing (see Smith, 1990b; Cook et al., 1998). Species of over 25 new or unreported genera have been discovered in North America since the publication of Cook’s (1974) global review, an average of about one each year (see Smith, 1976b, 1979, 1983b, c, d, g, 1989a – c, 1990a, 1991b, d, 1992b, 1998; Smith and Cook, 1994, 1996, 1997, 1998a, b, 1999a, b; Gerecke 554 Ian M. Smith, David R. Cook, Bruce P. Smith et al., 1999). Almost half of the species expected to occur in North America are not yet named, and many described species are known from only a few preserved specimens in collections. The most diverse genera, such as Sperchon, Torrenticola and Aturus in lotic habitats and Piona and Arrenurus in lentic habitats, contain 100 or more nearctic species each (probably more than 250 species in the case of Arrenurus). Additional taxa await discovery throughout the continent, especially in springs and interstitial habitats. The least explored areas, such as southern Appalachia and the boreal and arctic regions of northern Canada and Alaska, undoubtedly will yield some unexpected taxa when thoroughly studied. inhabiting North America belong to these genera, and descended from ancestors that either were present on this continent when it separated from Eurasia during the early Tertiary or immigrated from Eurasia with their hosts via land bridges later in that Period. 4. Zoogeography iv. Genera of North American origin Twenty-four genera are currently known only from North America. Some are probably endemics of recent origin while others have Tertiary relict distributions, suggesting that they originated in Laurasia and may have undiscovered members inhabiting poorly known areas of the Palearctic or Oriental Regions. Distributions of water mite genera within North America were dramatically inﬂuenced by climatic cooling and glaciation during the late Pliocene and Pleistocene. Genera which show evidence of recent adaptive radiation have re-established pan-continental distributions, while those with relatively few species have remained within more limited areas. Many species groups came to be restricted to either temperate or boreal areas of the continent, as reﬂected to some extent in the distributions of genera (see Table I). As a result, typical “Tertiary-relict” distributions in, or adjacent to, unglaciated refugia such as coastal California and Oregon, the Ozark Plateau, and parts of the Appalachian and Rocky Mountains are exhibited by some genera (Smith, 1989d, e, 1991c, 1992a, b) . Other genera (e.g., Teutonia, Midea, Acalyptonotus) have characteristic “boreal” or “arctic-alpine” distributions at higher latitudes and elevations. Distributional limitations are frequently correlated with habitat specializations. For example, many interstitial genera have Tertiary-relict distributions because their habitats were destroyed in most northern regions of the continent by Pleistocene glaciation and will require a long period of climatic stability to become re-established there. The sister species of many nearctic Tertiary relicts now inhabit similar areas in temperate Asia, resulting in strikingly discontinuous distributions for certain genera. In contrast, species adapted to cold stenothermic pools inhabit subarctic areas, high mountains near permanent ice-ﬁelds, and temperate lowlands where groundwater springs create similar conditions. The sister-species of many stenophilic nearctic a. Global Patterns Water mites occur throughout the world, except Antarctica (Cook, 1974). All superfamilies, except Hydrovolzioidea, as well as many early derivative families and subfamilies, are richly represented in all zoogeographic regions. Knowledge of global distribution patterns of water mite taxa has been substantially improved by recent studies of the fauna of austral regions, especially those by K.O. Viets and D.R. Cook. The basic patterns can be explained as the result of vicariance due to plate tectonics (Cook, 1986, 1988; Smith and Cook, 1999c). Dispersal (along with hosts) between adjacent land masses (e.g., Southeast Asia and Australia) or across continents (e.g., Africa) significantly altered these patterns as more recent groups progressively displaced ancient ones. b. Nearctic Patterns Four major groups of genera can be recognized among extant North American water mites based on their apparent origins, reﬂecting successive historical stages in the development of the modern fauna (see Table I). i. Genera of Pangean origin Twenty-three earlyderivative, cosmopolitan genera exhibiting substantial endemism in both the northern and southern hemispheres apparently originated in Pangea, and were represented in both Laurasia and Gondwanaland at the time of their separation during the Jurassic. ii. Genera of Laurasian origin Seventy-two genera with predominantly northern hemispheric distributions, exhibiting comparable diversity and endemism in the Nearctic and Palearctic, probably originated in Laurasia. The genera of Pangean and Laurasian origin probably were distributed throughout Laurasia during the late Cretaceous. The vast majority of species currently iii. Genera of Gondwanan origin Seventeen genera with predominantly southern hemispheric distributions, exhibiting substantial diversity and endemism in the Neotropics (Cook, 1980), are probably of Gondwanan or South American origin. Members of the Gondwanan taxa invaded southern North America from Central and South America after the Panamanian isthmus appeared during the Pliocene. 16. Water Mites (Hydrachnida) and Other Arachnids water mites live in northern Eurasia, yielding essentially circumpolar or boreal holarctic distribution patterns for these genera. B. External Morphology and Internal Anatomy Water mites exhibit the characteristic acarine body plan comprising the gnathosoma, or mouth region, and the idiosoma, or body proper, representing the fused cephalothorax and abdomen. The standard morphological terminology proposed by Greandjean (see Robaux, 1974) has been widely adopted by most authors dealing with prostigmatic mites, but until recently little effort was made to apply this terminology to water mites. The extreme plasticity of the adult exoskeleton during water mite evolution has obscured many homologies with other Prostigmata, and water mite workers have developed a highly useful set of peculiar morphological terms for external structures of this instar. Early taxonomic studies of larval water mites generated an additional set of special morphological terms for the exoskeleton of this strongly heteromorphic instar, and we used a modiﬁed version of this terminology in the 1st edition. However, it has become evident that the relatively plesiotypical body plan of larvae permits much of the standard idiosomal terminology of Grandjean to be used with conﬁdence. We employ it here and indicate where appropriate the equivalence of the various terms in common use. 1. Morphology of Larvae The short gnathosoma (Fig. 2) bears the stocky pedipalps which have ﬁve free segments (trochanter, femur, genu, tibia, tarsus) that ﬂex ventrally. The tarsus of the pedipalp is relatively long and cylindrical in some early derivative genera (eg: Wandesia, Thyas) (Figs. 18, 22), but is typically reduced to a dome- or button-shaped pad in most derivative groups (Figs. 76, 182). A highly modiﬁed thick, curved seta is present dorsally at the end of the tibia (Fig. 2), the homolog of the tibial “claw” which characterizes the pedipalp of most terrestrial Anystoidea, Parasitengona and related groups. The paired chelicerae (Fig. 3), each consisting of a cylindrical basal segment and a movable terminal claw, lie between the pedipalps. The idiosoma is plesiotypically mainly unsclerotized, as in extant Hydryphantoidea. The dorsal integument (Fig. 27) bears a medial eye-spot, two pairs of lenslike lateral eyes, four pairs of lyriﬁssures and the following complement of setae: four pairs on the propodosoma, two pairs of verticils, the internals (vi) or anteromedials (AM) and externals (ve) or anterolaterals (AL), and two pairs of scapulars, the internals (si) or sensillae (SS), and externals (se) or posterolaterals (PL); eight pairs on the hysterosoma, three pairs in the 555 c-row (c1, c2 and c3), two pairs in the d-row (d1 and d2), two pairs in the e-row, (e1 and e2) and one pair from the f-row (f1). Ventrally (Fig. 28) the integument bears the paired coxal plates, paired urstigmata (Ur) laterally between plates I and II, the excretory pore (EP), one pair of lyriﬁssures and the following complement of setae: three-to-six pairs on the coxal plates, two in the 1-row (1a and 1b), zero, one or two in the 2-row (2b usually present, 2a and 2c usually absent) and one or two in the 3-row (3a always present, 3b usually absent), six pairs on the hysterosoma, one pair of preanals (pa), two pairs associated with the excretory pore (ps1 and ps2), one pair from the f-row (f1) and two pairs in the h-row (h1 and h2). The size, shape, and position of these dorsal and ventral structures, and the degree of fusion of the sclerites associated with them, provide useful taxonomic characters. The legs (Fig. 21) are inserted laterally on the coxal plates, and in the plesiotypical condition have six movable segments, namely trochanter (Tr), basifemur (BFe), telofemur (TFe), genu (Ge), tibia (Ti), and tarsus (Ta), that articulate to permit ventral ﬂexion. The segments have characteristic complements of setae and solenidia (as in Figs. 6 – 8). The tarsi bear paired claws and clawlike empodia terminally. The soft-bodied condition, retained in all known larval Hydryphantoidea, apparently provides adequate support for the muscles used in locomotion on the surface ﬁlm. In all other major lineages, the dorsum (Figs. 3, 37, 56, 81, 144, 171) bears an extensive plate which incorporates the bases of the propodosomal setae and, in some cases, one or more hysterosomal pairs. Similarly, on the venter (Figs. 4, 38, 58, 83, 145, 172) the coxal plates are enlarged and variously fused, and the excretory pore plate (Figs. 5, 75, 109, 148, 176) is expanded to incorporate the bases of both pairs of excretory pore plate setae. The coxal plates and excretory pore plate may also bear various combinations of ventral setae. Dorsal plates, and expanded coxal and excretory pore plates, developed independently in Hydrovolzioidea and in most Eylaoidea to provide support for muscles used in running and crawling on the surface ﬁlm, and again in both Hydrachnoidea and the ancestral stock of the three “higher” superfamilies to anchor muscles used in swimming or crawling under water. In Hygrobatoidea, fused coxal plates II III often have conspicuous lateral coxal apodemes (LCA), medial coxal apodemes (MCA), and transverse muscle attachment scars (TMAS) (Fig. 133). Larvae of Hydryphantoidea and Eylaoidea retain six movable leg segments (Figs. 21, 40), but those of all other groups have the basifemoral and telofemoral segments fused (Figs. 6 – 8, 60). Larval Hydryphantoidea also retain the most plesiotypical complement of setae 556 Ian M. Smith, David R. Cook, Bruce P. Smith on the segments of the legs. Larvae of each of the more derivative groups exhibit characteristic leg chaetotaxies that reﬂect reductions from the plesiotypical complement. Certain ventral setae on the genua, tibiae and tarsi are elongate and plumose in larvae adapted for swimming (Fig. 149). 2. Morphology of Deutonymphs and Adults The gnathosoma consists of the capitulum (or gnathosomal base) and associated appendages, namely the chelicerae and pedipalps (Fig. 200). The capitulum is plesiotypically a simple, short channel, derived from extensions of the pedipalpal coxae, leading to the oesophagus (Mitchell, 1962). A protrusible tube of integument connecting the capitulum to the idiosoma has developed independently in several distantly related genera (e.g., Rhyncholimnochares, Clathrosperchon, Geayia). The paired pedipalps (Fig. 200, 227), inserted on the capitulum, have both tactile and raptorial functions. In the plesiomorphic condition, the pedipalps have ﬁve movable segments, namely trochanter, femur, genu, tibia and tarsus, that are essentially cylindrical and articulate to allow ventral ﬂexion. The tibia bears a thick, bladelike, dorsal seta distally in many ancient genera (e.g., Hydryphantes, Tartarothyas, Pseudohydryphantes). As in larvae, this seta is the homolog of the tibial “claw” of terrestrial relatives, and it often makes the pedipalps appear chelate. In derivative groups, other setae along with various denticles and tubercles may be elaborated to enhance the raptorial function of the pedipalps. Segmentation of the pedipalps is reduced by fusion in a few genera. A modiﬁcation that has developed independently in various groups of Arrenuroidea is the so-called uncate condition. In these genera, the tibia is expanded and produced ventrally to oppose the tarsus, permitting the mites to grasp and hold slender appendages of prey organisms securely. The pedipalps are highly modiﬁed in some interstitial genera (e.g., Fig. 426), presumably to facilitate prey capture in conﬁned hyporheic spaces. The paired chelicerae lie in longitudinal grooves between the pedipalps on the dorsal surface of the capitulum. Plesiotypically they consist of a cylindrical basal segment bearing a movable terminal claw. This cheliceral structure is designed for tearing the integument of prey organisms, and is retained in nearly all derivative groups. Hydrachnidae are unique in having unsegmented and stilletoform chelicerae, an obvious adaptation for piercing the insect eggs upon which they feed. The chelicerae are separate in all groups except Limnocharidae and Eylaidae where they are fused medially. The most comprehensive comparative studies of water mite mouthparts were published by Motas (1928) and Mitchell (1962). The idiosoma, or body proper, is plesiotypically round or ovoid in outline, slightly ﬂattened dorsoventrally, and mostly unsclerotized, as in certain extant members of ancient genera such as Hydryphantes, Tartarothyas and Pseudohydryphantes. The dorsal integument (Figs. 224, 256) bears an unpaired medial eye, paired lateral eyes that are usually enclosed in capsules, paired preocular and postocular setae, and longitudinal series of paired glandularia (six dorsoglandularia, ﬁve lateroglandularia), muscle attachment sites (ﬁve dorsocentralia, four dorsolateralia), and lyriﬁssures (ﬁve). Ventrally (Fig. 223) the integument bears the paired coxal plates (fused into anterior and posterior groups on each side), the genital ﬁeld (comprising the gonopore, three pairs of acetabula, and paired genital valves), ﬁve pairs of ventroglandularia (including coxoglandularia I between the anterior and posterior coxal groups and coxoglandularia II behind the posterior groups) and the excretory pore. As in larvae, these idiosomal structures provide a wealth of useful taxonomic characters. The legs (Figs. 200, 325) are inserted laterally on the coxae, and plesiotypically articulate on a vertical major axis and have six movable segments that articulate to permit ventral ﬂexion. The segments are essentially cylindrical and have variable complements of setae. Though chaetotaxy of the legs provides a variety of taxonomic characters in deutonymphs and adults, the expression and position of individual setae are highly variable within taxa. Consequently, the rigorous analysis of chaetotactic patterns that proves so useful in the case of larvae is not practicable for later instars. The leg tarsi plesiotypically bear paired claws terminally. The plesiotypical soft-bodied condition is retained in early derivative genera adapted for walking on the substrate (e.g., Thyas) or swimming in shallow habitats such as seepage pools or temporary pools (e.g., Hydryphantes, Pseudohydryphantes). Walking forms have relatively short, stocky leg segments and setae, whereas swimmers have longer segments bearing fringes of slender swimming setae. As pointed out by Mitchell (1957a, b, 1958, 1964b), the soft integument of these mites permits the highly precise local control of body shape and internal pressure that is needed to produce the walking and swimming movements of the legs. 3. Evolution of Adult Exoskeleton The major lineages of water mites apparently differentiated from ancestral stock that resembled extant soft-bodied Hydryphantoidea (Mitchell, 1957a, b; Smith and Oliver, 1986), primarily through development of apotypical larval behavioral patterns and life histories. Hydrachnidae and Eylaidae became specialized for exploiting standing water habitats, especially 16. Water Mites (Hydrachnida) and Other Arachnids temporary pools, whereas Hydryphantoidea, Lebertioidea, Hygrobatoidea, and Arrenuroidea ultimately diversiﬁed and radiated in a wide range of habitats. In each of these four groups, and presumably in Hydrovolzioidea, sclerotization of the integument came about as soft-bodied ancestral stock began to invade new habitats and diverge. Adaptation to habitats such as seepage areas and springs or substrates in streams required a change in locomotor habits from walking or swimming. Mites adapting to moss mats and wet litter habitats developed hydrophilic integument which draws a ﬁlm of water over the dorsal surface, creating sufﬁcient downward force to press the body to the substrate and prevent efﬁcient walking (Mitchell, 1960). Groups invading streams evolved a wedge-shaped body designed for negotiating conﬁned spaces to avoid exposure to water turbulence and strong currents. Locomotion in both of these types of habitats necessitated evolution of a crawling gait. This change involved a shift in orientation of the major axis of the legs, shortening and thickening of leg segments and setae, enlargement of tarsal claws, and development of stronger, more massive muscles to control leg movements. Expansion of coxal plates and sclerites associated with glandularia, setal bases, and the genital ﬁeld, along with sclerotization of the dorso — and laterocentralia, occurred to provide rigid exoskeletal support for these muscles. Fusion of these sclerotized areas led to development of complete dorsal and ventral shields in adults of certain groups. Multiple invasions of helocrene, rheocrene, and lotic habitats gave rise to a diverse array of dorsoventrally ﬂattened, sclerotized, crawling groups of Hydryphantoidea, Lebertioidea, Hygrobatoidea and Arrenuroidea. During the early evolution of each of these lineages certain groups became adapted for exploiting interstitial habitats in subterranean waters and the hyporheic zone of rheocrenes and streams. These mites tended to lose eyes and integumental pigmentation, and required further stream-lining of the body to facilitate locomotion in interstitial spaces. Consequently, soft-bodied forms adopted a vermiform shape, while sclerotized mites became extremely compressed laterally or dorsoventrally. The crawling mode of locomotion became secondarily modiﬁed in several groups of wellsclerotized interstitial genera (e.g., Neomamersa, Frontipodopsis, Chappuisides) to permit rapid and agile running in hyporheic and ground water habitats. By late Pangean times, evolution within water mites apparently had produced basic communities of essentially soft-bodied species living in temporary and permanent standing water, and partly to fully sclerotized species living in emergent groundwater, lotic, and interstitial habitats. Subsequent phylogeny of 557 Hydryphantoidea, Lebertioidea, Hygrobatoidea, and Arrenuroidea during late Mesozoic, Tertiary, and Quaternary times appears to have followed a recurring pattern of extended periods of gradual evolution leading to specialization within particular habitats, punctuated by episodes of relatively rapid and dramatic diversiﬁcation. Divergence of many of the clades that originated during this time appears to have been correlated with successful invasion of new habitats. Repeated habitat diversiﬁcation within the major clades of these four superfamilies, in response to opportunities provided by the explosive evolution of their primary host groups and by prolonged geological and climatic instability, resulted in several parallel and convergent trends in body sclerotization. Members of different clades developed superﬁcially similar sclerite arrangements in adapting to lotic or interstitial habitats (e.g., species of Diamphidaxona and certain Axonopsinae); others underwent homoplastic reduction or loss of sclerites in secondarily invading lentic habitats (e.g., species of Forelia and Piona). Finally, some groups retained extensive sclerotization while becoming adapted for swimming in standing water (e.g., certain species of Axonopsis and Mideopsis). Consequently, modern communities are highly heterogeneous phylogenetically and morphologically, with each monophyletic component exhibiting unique exoskeletal adaptations for living in the particular habitat. Evolutionary trends involving other readily observed and taxonomically useful characters — such as position and number of genital acetabula, positions of glandularia and lyriﬁssures, number and arrangement of setae on the body plates, and modiﬁcation of claws on the tarsi of the legs — are still not adequately explained. For example, although the genital acetabula are plesiotypically borne in the gonopore in most Hydryphantoidea and Lebertioidea, they are either fused with the genital ﬂaps or incorporated into acetabular plates ﬂanking the gonopore in most taxa of other superfamilies. In addition, the number of acetabula has proliferated from the plesiotypical complement of three pairs several times independently in each of the six largest superfamilies. Alberti (1977, 1979) has recently proposed that acetabula in water mites may function as an osmoregulatory chloride epithelium, and Barr (1982) concluded that analysis of morphological data for early derivative taxa supports this hypothesis. However, until the function of acetabula is clearly understood on the basis of well-designed experiments, the signiﬁcance of the trend to increasing number of acetabula will remain speculative. The often striking and elaborate color patterns of adult water mites present an intriguing enigma. Members of most ancient clades (e.g., Hydrachnoidea, Ey- 558 Ian M. Smith, David R. Cook, Bruce P. Smith laoidea, Hydryphantoidea) have colorless integument but are red, like terrestrial parasitengones, due to the presence of pigment granules which appear to be distributed throughout the body. Members of other superfamilies, except for taxa in interstitial habitats, exhibit highly distinctive patterns resulting from symmetrically arranged concentrations of pigment granules of various colors. In soft bodied taxa the pigments are located beneath the integument, but in sclerotized groups they are incorporated into the plates. Members of well-sclerotized taxa of Lebertioidea, Hygrobatoidea, and Arrenuroidea may be various shades of red, orange, yellow, green, or blue, and the dorsal shield frequently exhibits an intricate pattern combining several contrasting colors. The dorsal patterns of these mites can readily be interpreted as disruptive camouﬂage, but the adaptive value of their bright colors is more difﬁcult to understand. The evolution of a wide range of highly colored pigments in these taxa certainly suggests that distinctive coloration confers some selective advantage. Though protection from predation may be part of the explanation, it is tempting to speculate that these mites, despite their apparently simple eye structure (see below), may detect and use color as one of several cues for recognizing conspeciﬁc individuals. Research is needed to determine the extent of their ability to detect and respond to the often subtle differences in color that distinguish closely related taxa. 4. Internal Anatomy The organ systems of water mites occupy the hemocoel, bathed by hemolymph which is circulated by movements of the body musculature (Schmid, 1935; Bader, 1938; Mitchell, 1964b). As is typical for arthropods, there is no closed circulatory system. a. Digestive System Digestion of food materials begins pre-orally, and only ﬂuids are ingested. Food is drawn into the mouth (or buccal cavity) by the muscular pharynx, then passed through the tubular esophagus to the lobed midgut (Bader, 1938) where digestion and absorption occur. Undigested material accumulates as insoluble particles in a posterodorsal lobe of the midgut. All lobes of the midgut end blindly, and there is no connection between the gut and the excretory pore. b. Excretory System This system consists of a large, thin-walled excretory tubule, apparently derived from the primitive hindgut, that lies dorsal to, and in close contact with, the midgut. Waste products are absorbed from hemolymph, and stored in the excretory tubule as insoluble, whitish or yellowish crystals of unknown chemical composition. When ﬁlled with crys- talline material the excretory tubule may be visible through the dorsal integument as a “T”- or “Y”shaped structure. The excretory tubule connects ventrally with the excretory pore, and is evacuated periodically by pressure generated through movements of the body muscles. Osmoregulation is apparently accomplished by the urstigmata in larvae, and by the genital acetabula in deutonymphs and adults (Barr, 1982). Both of these structures have porous caps which apparently provide an ample surface area of chloride epithelium for maintaining water balance. c. Respiratory System Many groups of water mites retain paired stigmata located between the bases of the chelicerae in all active instars. Plesiotypically, the stigmata lead to tracheal trunks which anastomose repeatedly into tracheolar tubules extending to all parts of the body. However, they appear to be nonfunctional in deutonymphs and adults, and respiration occurs by diffusion through the integument. In adults of many large species inhabiting standing water, a network of closed tubes of tracheolar dimensions lying beneath the integument transports gases to and from the tracheae that lead to internal organs (Mitchell, 1972). Heavily sclerotized mites have the body plates well supplied with regularly arranged “pores” that permit diffusion of gases between the tracheolar loops and surrounding water through areas of thin integument. In some species, there are regions of tracheal anastomosis lateral to the brain (Wiles, 1984). d. Neural System The so-called brain, a fused, undifferentiated central ganglionic mass, surrounds the oesophagus. Nerve trunks dorsal to the oesophagus lead to the anterior sense organs and mouthparts, while ventral trunks lead to the legs and genital region. The lateral eyes appear to be the primary lightsensing structures in water mites. There are typically two pairs, with the eyes of each side located close together and often enclosed in lenslike capsules that may lie above or beneath the integument. In postlarval instars of Eylaoidea, the eyes are borne on a medial sclerite. Many Hydryphantoidea also have a medial eye spot between the lateral eyes, but this structure is absent in most members of the other superfamilies. The eyes seem to function as ocelli, permitting the mites to detect the intensity and direction, and, at least in some taxa, the wavelength of incident light. Experiments with species of Unionicola have demonstrated that some species respond differentially to various wavelengths (Dimock and Davids, 1985) and that the response of the mites can be inﬂuenced by chemicals produced and released by their mollusc hosts (Roberts 16. Water Mites (Hydrachnida) and Other Arachnids et al., 1978). Retinal development, though rudimentary, has been reported for a variety of taxa (Lang, 1905). Members of the various taxa have highly characteristic complements of setae on the idiosoma and appendages. Setae are the main tactile receptors, and many of them have also assumed secondary functions in locomotion, feeding, or mating. All water mites have specialized tactile organs, called glandularia, distributed in pairs on both the dorsum and venter of the body. Each glandularium consists of a small, gobletshaped sac, or “gland”, with a tiny external opening, and an associated seta. The “gland” contains a milky, viscous ﬂuid which is ejected abruptly when the seta in stimulated. This substance quickly stiffens to a sticky gel after it comes in contact with water, and apparently evolved as a deterrent to attackers. In males of Arrenurus, however, material secreted by the glandularia seems to function also in cementing the body of the female in place during copulation. The ﬁve pairs of lyriﬁssures, which are arranged in series on the dorsum and venter of the body are thought to be proprioceptors (Krantz, 1978). In addition, the distal segments of the pedipalps and legs are well supplied with a variety of specialized setiform structures, apparently derived from outgrowths of the integument, which function as chemoreceptors. The most conspicuous of these organs are the solenidia which adorn the dorsal surfaces of the genua, tibiae, and tarsi of the legs, and the tarsi of the pedipalps. e. Reproductive System Males have paired testes and vasa deferentia which lead to an elaborate ejaculatory complex consisting of a series of membranous chambers attached to a sclerotized framework (Barr, 1972). Positioned immediately above the genital ﬁeld, the ejaculatory complex functions as a syringelike organ for compacting masses of spermatozoa, assembling spermatophores, and expelling them from the genital tract through the gonopore. Females have paired ovaries that are more or less fused, and paired oviducts that also fuse to form a single duct leading to the genital chamber within the gonopore. Paired spermathecae, for storing spermatozoa picked up in spermatophores, ﬂank the genital chamber and are connected to it by short ducts. C. Life History The basic life-history pattern of water mites (Fig. 1) was ﬁrst correctly inferred by Wesenburg-Lund (1918) and has been conﬁrmed by subsequent studies on a wide range of taxa (see Smith and Oliver, 1986, for a summary of relevant literature). The ﬁrst well- 559 documented life-history studies were of palearctic species, especially members of the genus Arrenurus, investigated by Münchberg (1935a, b). Subsequently, Mitchell (1959, 1964a) dealt intensively with a number of nearctic species of Arrenurus, and Böttger (1962, 1972a, b) described life cycles of representative palearctic species of Hydrachna, Limnochares, Eylais, Limnesia, Unionicola, Piona, and Arrenurus in considerable detail. A meticulously comprehensive account of the life history of Hydrodroma despiciens (Müller) (family Hydrodromidae) was published by Meyer (1985). Water mites in temperate latitudes typically live for just over one year, most of which is spent in the deutonymphal and adult stages. Most species are univoltine, with long-lived female adults producing multiple clutches of eggs. This tactic apparently minimizes the risks associated with the parasitic larval stage. In temperate latitudes, hosts are usually seasonally limited and parasitism occurs mainly in late spring and early summer. Larvae of most taxa spend no more than several days on their hosts and engorgement results in modest growth. Deutonymphal water mites typically feed and grow throughout the summer and adults appear in late summer or early fall and mate almost immediately. Inseminated females remain in obligative reproductive diapause until the following spring. This type of life history occurs in a wide range of water mite clades associated with host groups that are holometabolous and have aerial imagoes. Several derivative life-history strategies occur among water mites, representing variations on this basic pattern (Smith and Oliver, 1986; Smith, 1988, 1999). One of these involves elimination of larval feeding and host association altogether, thus avoiding risk of not encountering a host but foregoing host-mediated dispersal (Smith, 1998). This has happened at least 20 different times in unrelated genera representing a wide diversity of families. Comparison of closely related species pairs with, and without, larval parasitism shows that loss of larval parasitism correlates with accelerated maturity and metamorphosis to adult at a smaller body size (Smith, 1998). Removal of the seasonal limitations of host association in species with nonparasitic larvae appears to favor a shift to a multivoltine life history. Several clades exhibit a third strategy by greatly extending the association with hosts, in some cases for several months, through the larval and protonymphal stages. Species in these groups typically undergo remarkable growth during the larvae stage, increasing in volume by up to 700 times their original size (Davids, 1973). Growth of these mites as deutonymphs is much more modest, and this stage often lasts only a few days or weeks. Extended larval associa- 560 Ian M. Smith, David R. Cook, Bruce P. Smith tion with hosts is exhibited by all species of Hydrachnoidea and Eylaioidea (excluding Limnochares), and also by atypical members of certain other taxa, such as Hydryphantes tenuabilis Marshall (Lanciani, 1971a). All known examples of extended association involve larval parasites of aquatic Hemiptera and Coleoptera, presumably reﬂecting the longevity, large size and mainly aquatic existence of these hosts. This type of life history allows at least some species to adopt multivoltine life cycles (Davids, 1973). There are interesting variations on these major life history patterns among species within genera (Lanciani, 1971b; Cook et al., 1989). 1. Egg Eggs are typically laid in masses in a gelatinous matrix and attached to plants, wood particles, or stones, but females of some species, such as Midea expansa Marshall, may scatter them individually on the substrate. Females of the genus Hydrachna use a uniquely developed, elongate ovipositor to lay eggs individually in stems of aquatic plants, and those of certain species of Unionicola use short stylets to oviposit in the tissues of sponges or mussels. The most comprehensive morphological study of water mite eggs was published by Sokolow (1977). Detailed accounts of oviposition behavior have been recorded by Crowell (1960), Ellis-Adam and Davids (1970) and Davids (1973). Egg production varies greatly among species. For example, females of Eylais discreta Koenike may produce individual clutches of 1000 – 2000 eggs, with one individual able to produce over 13,000 eggs in a 3month period (Davids, 1973). On the other extreme, species of Thyas, Hygrobates, Piona and Arrenurus that forego larval feeding typically produce 2 – 5 eggs per clutch (Smith, 1998). Most water mite species typically produce about a dozen-to-several hundred eggs in a clutch. There is an evident trade-off between clutch size and egg size, probably related to risks to the larval instar and the proportion of total growth that occurs during this stage. An analysis of this trade-off in a group of 27 species of Arrenurus resulted in two evolutionary trajectories deﬁned by egg size, clutch size, and female size: one representing species parasitic on adult nematocerous ﬂies and the other species parasitizing adult Odonata (Cook et al., 1989). Large, communal clutches of eggs have been observed in several situations. Patches of eggs of Eylais euryhalina Smith in western Canada frequently cover areas of more than 100 cm2 on plant or rock substrates. Females of Arrenurus pseudosuperior Cook produce communal egg masses entirely covering sub- merged oak leaves in central Canadian lakes. The stimulus to lay eggs communally may stem from a shortage of oviposition sites in nature, although there may be selective advantage if either eggs are distasteful or their sheer numbers exceed what local predators can consume. Within the egg membrane, water mites pass through a transitory prelarval instar (Meyer, 1985) and then usually develop rapidly and directly into larvae. Fully formed individuals can be observed moving within the egg membrane just prior to emerging 1 – 3 weeks after oviposition. Arrested larval development has been reported in certain species of Unionicola (Mitchell, 1955), Teutonia (Smith, 1982), Utaxatax (Smith, 1982) and Laversia whose larvae do not become active and emerge until at least six months after eggs are laid. Female mites presumably possess adaptations for selecting appropriate oviposition sites. 2. Larva The vast majority of water mite species are parasitic on imaginal insects as larvae (reviewed by Smith and Oliver, 1976, 1986, and summarized in Table I), with the host providing nutrition as well as dispersal. Growth on the host is modest in many clades, but apparently essential for development as there are no records of purely phoretic associations. Mitchell (1966, 1969a) theorized that the probability of success for larval water mites can be calculated as the product of the probabilities of discovering a host, attaching at an appropriate site on the host, completing engorgement on the host and detaching from the host in a suitable habitat. The risk of failure can be high. Collins (1975) estimated that more than 75% of larval Wandesia (Partnuniella) thermalis Viets fail to locate a host in a system in which the distribution of hosts is extremely clustered and unpredictable (Collins et al., 1976). a. Evolution of Parasitic Associations i. Host selection After emerging, larvae must quickly locate hosts that both provide adequate sites for attachment and feeding and regularly visit habitats that are suitable for postlarval development. Insect hosts provide water mites with both the source of nutrition necessary for larval growth, and their primary dispersal mechanism. Host associations of larval water mites were reviewed by Smith and Oliver (1976, 1986), and are summarized in Table I. Larvae of Hydrovolzia, and most genera of Eylaoidea and Hydryphantoidea, rise to search for potential hosts on the surface ﬁlm, exhibiting plesiotypical terrestrial behavior. In contrast, larvae of 16. Water Mites (Hydrachnida) and Other Arachnids Acherontacarus, Rhyncholimnochares, Wandesia, Hydrachna, Lebertioidea, Hygrobatoidea, and Arrenuroidea begin to swim in the water column or crawl on the substrate as fully adapted aquatic organisms. This apotypical behavior developed independently several times, within the superfamilies Hydrovolzioidea, Eylaioidea and Hydryphantoidea, in ancestral Hydrachnidae, and again in ancestors of the other three superfamilies. The development of fully aquatic larvae can best be understood as an adaptation enhancing the probability of success in locating hosts and, in the higher water mites, facilitating preparasitic attendance of hosts during their ﬁnal preadult instar. Surface dwelling larvae are essentially terrestrial, and typically can locate hosts only when they visit or pass through the surface ﬁlm. Larvae of Limnochares americana Lundblad are unusual in being able to search for hosts up to 50 cm. away from the water surface. Opportunities of these mites to contact hosts are transitory events occurring irregularly in space and time, and are strongly inﬂuenced by speciﬁc aspects of behavior within a host population. For example, parasitic larvae of Thyas barbigera Viets are found only on parous female mosquitoes and those of Limnochares americana are found predominantly on territorial male dragonﬂies (Smith and Cook, 1991; Smith, 1999). Some water mites with terrestrial larvae overcome the risks of failure by utilizing a relatively broad range of host organisms and producing large numbers of eggs. For example, larvae of species of Hydrovolzia and certain genera of Hydryphantidae apparently exploit a wide range of hosts, including insects that are only casual visitors to the habitat of the mites. This strategy presumably results in considerable wastage, as larvae attaching to hosts that do not return to suitable mite habitats will die. Larvae of most Eylaoidea (Eylais, Piersigia, Neolimnochares, some Limnochares) and various Hydryphantidae have developed strong speciﬁcity for particular groups of insect hosts that live on the surface ﬁlm (e.g., Hydrometridae, Gerridae), regularly visit it to replenish air supplies (e.g., aquatic Hemiptera and Coleoptera), or pass through it just before ecdysis and regularly revisit it after emergence (e.g., Odonata, Trichoptera, Diptera). This strategy improves the probability that a larva ﬁnding a host will be returned to an appropriate habitat, but limits the availability of potential hosts. These mites apparently compensate for the high risk of failure in ﬁnding hosts by producing many hundred eggs per female. Species with terrestrial larvae are generally limited to relatively shallow water microhabitats, given that these larvae must swim or crawl to the surface in order to search for potential hosts. Enclosure experiments by 561 one of us (B.P. Smith) indicate that the likelihood of water striders of the genus Gerris being parasitized by larval Limnochares aquatica Linnaeus is strongly dependent on water depth. Differences in rates of infestation are substantial, even between insects caged over 0.5 m of water and those caged over 1.5 m. In contrast, aquatic larvae have access to potential hosts throughout their aquatic existence and can actively seek them in the water column or substrate. Larvae of Hydrachnidae exploit virtually the same range of hosts as Eylaidae, but locate them opportunistically beneath the surface ﬁlm with most species exhibiting no evidence of preparasitic attendance. Members of certain Anisitsiellidae and Teutoniidae (Lebertioidea), as well as Krendowskiidae and Arrenuridae, apparently also seek their hosts above the substrate, but have evolved preparsitic attendance of hosts during their penultimate instar prior to ecdysis. Their larvae parasitize tanypodine Chironomidae, Culicidae, Ceratopogonidae, Chaoboridae and Odonata, all groups that have the ﬁnal aquatic instar active in the water column below the surface ﬁlm. Mites locating their hosts in the water column apparently exploit them more efﬁciently than do those with terrestrial larvae, as their females tend to lay smaller, though still substantial, numbers of eggs. For example, females of Hydrachna conjecta (Koenike) lay only about 10% as many eggs as those of Eylais discreta Koenike, yet these species co-occur in nature and parasitize the same species of corixids (Davids, 1973). Larvae of Rhyncholimnochares and Wandesia are also aquatic and apparently independently evolved adaptations to locate their hosts in the substrate. Larvae of Rhyncolimnochares are subelytral parasites of adult elmids that remain in water. Those of Wandesia attend nymphal stoneﬂies before transferring to the emerging adults and becoming parasites. Interestingly, larval stygothrombidiids exhibit similar behavior in exploiting the same hosts. Larvae of the remaining families of Lebertioidea, Hygrobatoidea, and Arrenuroidea locate hosts as ﬁnal instar larvae or pupae in cases or burrows in the substrate. The hosts, various Chironomidae or Trichoptera, construct cases or retreats where they remain during prepupal and pupal stages. These mites have developed suites of larval adaptations to exploit this sedentary behavior, greatly increasing their probability of successfully locating and associating with appropriate hosts. Larvae in all of these clades attend the host during the preparasitic phase, clinging to the integument of the pupa until it rises to the surface. They typically then transfer to the imago as it emerges, embed their chelicerae, and enter the parasitic phase. Individual females in these groups lay relatively small numbers of eggs (often less than 10), indicating that the 562 Ian M. Smith, David R. Cook, Bruce P. Smith larvae efﬁciently ﬁnd hosts with a high probability of returning to a suitable habitat. The development of fully aquatic larvae had important consequences each time it occurred during water mite evolution. In particular, it was a crucial preadaptation for ancestors of the various groups of Lebertioidea, Hygrobatoidea, and Arrenuroidea that became specialized in exploiting nematocerous Diptera as hosts. The origin and evolution of modern genera and families of these mites apparently followed the explosive radiation of the Nematocera, and especially Chironomidae, during the Jurassic and early Cretaceous. Reﬁnement of their elegant adaptations for parasitizing these ﬂies permitted the so-called higher water mites to co-evolve with them, and to exploit their potential for dispersal, invasion of new habitats, and rapid evolution. ii. Site selection Larvae of many subfamilies of water mites exhibit strong selectivity for attaching to particular sites on the body of the host (Smith and Oliver, 1976, 1986; Oliver and Smith, 1980), as shown in Table I. These preferences appear to illustrate another adaptive process that has permitted mites to coevolve successfully with nematocerous ﬂies by minimizing interference with the dispersal potential of the host. Larvae of Hydrovolzia and certain genera of Hydryphantidae that parasitize Hemiptera, Plecoptera and Trichoptera (e.g., Protzia, Wandesia), seem to attach rather indiscriminately to various parts of the host’s body. Those of all other groups are more selective. Larval Eylais are essentially terrestrial organisms and select attachment sites bathed by the host’s air supplies, under the elytra of aquatic bugs and beetles (Fig. 427). Signiﬁcantly, the fully aquatic larvae of Hydrachna are able to utilize a much wider range of sites on the same hosts (Fig. 428). Larval hydryphantoid mites that parasitize Diptera tend to attach to thoracic sites, exhibiting apparently plesiotypical behavior. Larvae of many early derivative groups of Lebertioidea, and most Arrenuroidea parasitizing Diptera, also prefer thoracic sites. These larvae tend to be relatively large, and only a few individuals can share a single host. When more than one of these larvae attach to the same part of the host’s thorax they are usually symmetrically arranged on either side of the body. Larvae of some species of Sperchon, Lebertia, Oxus and Torrenticola can use the anterior abdominal segments of hosts when thoracic sites are already occupied. In virtually all Hygrobatoidea (except Limnesiidae which may not be closely related to other taxa in the superfamily) and a few families of Arrenuroidea (eg., Mideidae, Momoniidae) larvae attach exclusively to ab- dominal sites on their hosts, usually nematocerous ﬂies but in some cases caddis ﬂies. This apotypical behavior correlates with a marked trend to smaller size, allowing several larvae to share each attachment site on a host. These small larvae occupying abdominal attachment sites can exploit even the smallest species of Chironomidae as hosts without preventing them from ﬂying and dispersing (Fig. 430). The relatively small larvae of various species of Arrenurus (sensu stricto) show preferences for either thoracic or abdominal sites on odonate hosts (Fig. 429). The duration of the parasitic phase of larval development varies considerably. Larvae of certain species in early derivative genera such as Limnochares and Hydryphantes retain their mobility and can transfer from one instar to the next of the same host individual. Larval Limnochares aquatica engorge gradually by feeding on successive host instars, but larvae of Hydryphantes tenuabilis exhibit true preparasitic attendance and delay feeding until the host reaches adulthood. This stategy of tracking hemimetabolous hosts through several molts may reﬂect the plesiotypical condition in water mites. Larval Hydrachna and Eylais remain attached to the same adult bug or beetle throughout the parasitic phase, for several weeks or more. Larvae of species in these families that are adapted to temporary pools often remain on hosts throughout the dry phase of the habitat, and only engorge after a period of arrested development that may last as long as 10 months. Larvae of species parasitizing nematocerous Diptera or other aerial insects typically engorge rapidly and mature within several days. Fully fed larvae enter the quiescent protonymph stage. The growth and dispersal functions of the larva have both strongly inﬂuenced the evolution of host associated behavior in water mites, with different results in the various major clades. Larvae of early derivative genera such as Hydrachna, Eylais, and certain Hydryphantidae grow substantially, increasing several hundred-fold in size in some cases, while feeding on their relatively long-lived hosts. Larvae of many relatively recently evolved groups associated with shortlived insects, especially nematocerous Diptera, still engorge on ﬂuids from the host but increase more modestly in size in the process. The primary strategic role of the larval instar in the life history of most Hydryphantoidea, Lebertioidea, Hygrobatoidea and Arrenuroidea appears to be dispersal. Most feeding and growth occur during the deutonymph and adult stages in these mites. The larva may be suppressed as an active instar in a few unrelated species in widely divergent genera (e.g., Thyas, Lebertia, Limnesia, Hygrobates, Piona, Axonopsis and Arrenurus), apparently permitting the mites to accelerate development and maximize ex- 16. Water Mites (Hydrachnida) and Other Arachnids ploitation of optimal conditions (see Smith and Oliver, 1986; Smith, 1998). In these cases, larvae either remain within egg membranes or emerge for a brief period of activity before entering the protonymph stage. Species known to forego a parasitic phase appear to be isolated and ephemeral evolutionary experiments (Smith and Oliver, 1986; Smith, 1998). Recent studies have detected relatively high levels of morphological divergence and lower levels of heterozygosity within populations of apparently recently diverged species lacking parasitism when compared with closely related species that retain parasitic larvae (Bohonak, 1998). We conclude that although elimination of parasitism confers short-term advantage in larval survival and potential for population growth, host-mediated dispersal is essential for long-term persistence of a lineage. b. Ecological Aspects i. Emergence of larvae In permanent water habitats at north temperate latitudes, most species of water mites overwinter primarily as inseminated females and there appears to be a necessary time interval in the spring for oviposition and development of larvae to proceed before parasitism can commence. Consequently, aquatic insect species whose adults emerge in early spring are rarely parasitized by water mites, even within groups that are otherwise suitable hosts. Interestingly, some of these early emerging species show high susceptibility to parasitism under laboratory conditions. For example, although only a small proportion of Aedes communis and Aedes punctor mosquitoes carry larval Arrenurus angustilimbatus and Arrenurus kenki in nature, they prove to be more susceptible to parasitism in choice experiments than the species typically utilized as hosts by these mites in the ﬁeld (Smith and McIver, 1984a, b). A few mite species that apparently overwinter as eggs or larvae are adapted to exploit hosts that emerge in early spring. For example, larvae of Huitfeldtia rectipes Thor are found regularly on imagoes of early season species of Chironomus emerging from oligotrophic lakes in eastern Canada throughout the ice-free period beginning in mid-April. Available evidence suggests only loose synchrony between life cycles of mites and their host species, even when host speciﬁcity is strong (Smith and McIver, 1984a, c; Smith and Cook, 1991). In the case of Coquillettidia perturbans mosquitoes in Florida parasitized by larval Arrenurus danbyensis and Arrenurus delawarensis, peak infestation did not closely correlate with peak abundance of hosts and there was no close seasonal correspondence between population densities of the two mite species although both are dependent on the same host (Lanciani and McLaughlin, 1989). 563 ii. Seeking and locating hosts After emerging, larvae of species with a parasitic phase immediately begin host-seeking. The possible period of survival for free-living larvae in the preparasitic phase ranges from 4 days to 6 weeks (Smith, 1988), but is about 1 week for most species. Larval Arrenurus remain active and exhibit preparasitic attendance over a period of 2 weeks in the laboratory, but have the greatest chance to locate and infect a host during the ﬁrst few days. Larvae older than 1 week rarely successfully attach to hosts, although those of some cold-adapted species apparently can remain infective for longer periods. Some authors have suggested that water mite larvae ﬁnd hosts by accidental contact, but close observation of both terrestrial and aquatic larvae reveals noticeable behavioral changes when in proximity to hosts. Terrestrial larvae seem to use visual, tactile and chemical cues to locate hosts at the surface ﬁlm. Those of several Hydryphantidae have the hind legs modiﬁed for jumping and leap toward objects positioned above them, an obvious adaptation for encountering hosts on the surface ﬁlm. Larval Limnochares aquatica exhibit questing behavior when within several millimeters of water striders, involving hesitation and reaching upward with the ﬁrst pair of legs. Details of the sensory mechanisms used by aquatic larvae to locate hosts remain unclear, but there is evidence that both chemical and tactile cues are important (Böttger, 1972b). Larvae of Hydrachna often extend the gnathosoma when approaching a host before actual contact is made. Larval Arrenurus are attracted to mosquito pupae and slow their swimming just prior to contact (Smith and McIver, 1984b). These larvae swim in tight circles, as if attempting to orient to some cue, before making a direct approach (Page and B.P. Smith, unpublished data). iii. Recognizing host Larvae of water mite species are typically able to exploit a range of host species. Some species with aerial larvae have remarkably broad host spectra encompassing several orders of insects, whereas species with preparasitic attendance will normally be limited to species within a subfamily or family of host insects. Commonly, one or two host species carry the majority of parasitic larvae of a given mite species in a particular habitat. Although co-occurrence in space and time set the absolute boundaries for host utilization, larval mites also actively select among potential host species. In laboratory experiments, larval mites of diverse genera, such as Hydrachna, Eylais, Limnochares and Arrenurus exhibit preferences when exposed simultaneously to hosts of various species (Smith, 1977; Smith and McIver, 1984b). Choice of host by mite larvae appears to be inﬂuenced by differences in the intensity of cues, 564 Ian M. Smith, David R. Cook, Bruce P. Smith and larval mites favor certain individuals within a host population while rejecting others (Smith and McIver, 1984b). In some instances, hosts react strongly to the presence of mite larvae with avoidance behavior or vigorous grooming activity (Forbes and Baker, 1990; Smith and McIver, 1984b), and there may be differences in intensity of parasite avoidance among host species and individuals. Strong bias for hosts of a speciﬁc gender or age class is evident in many water mite species with terrestrial larvae, apparently resulting from differences in the likelihood of exposure to mites of males and females of various ages within host populations that are determined by their behavior (Smith, 1999). Preparasitic attendance of hosts by aquatic larvae prevents gender based behavioral differences in exposure to parasitism and synchronizes initiation of larval engorgement with emergence of adult hosts. In these mites, infestation rates of male and female hosts of a given species at a particular time may differ in nature, but partitioning the data by date usually removes any apparent pattern. Gender-based selection of hosts among colonizing mites was demonstrated by Lanciani (1988). He found that larval Arrenurus exhibited signiﬁcant but small preference for female pupae of Anopheles crucians, given equivalent exposure to both genders of host pupae, and adjusting for any sexrelated differences in body size. Among mosquitoes, dragonﬂies and some other host groups, the likelihood of an adult parasitized by mite larvae returning to water is strongly inﬂuenced by its sex, and this should result in selective pressure for larval mites to recognize the gender of potential hosts. iv. Infecting host After encountering a potential host, terrestrial larvae locate an appropriate attachment site and begin to feed. Species with larvae that exhibit preparasitic attendance must transfer from the penultimate instar of the host to the imago during ecdysis. Larval Arrenurus cling passively to the preimaginal host instar, becoming active only when it begins to molt. Interestingly, many larvae of species parasitizing mosquitoes appear to have difﬁculty in locating the split in the pupal exuvia and some that do ﬁnd it are unable to penetrate the surface ﬁlm quickly enough to reach the adult mosquito as it emerges. As a result, between 30 and 50% of preparasitic larvae fail to infect the adult host (Smith and McIver, 1984b). Larvae of Arrenurus species parasitic on odonates are carried out of the water on the penultimate instar of the host and almost all of them successfully move from the larval exuvia onto the adult odonate (Mitchell, 1969b). Larvae of species of the genera Atractides, Hygrobates and Unionicola pierce the pupal cuticle of their chironomid hosts and embed their chelicerae in the pharate adult. This behavior virtually ensures successful transfer to the adult because the larvae are passively pulled through the pupal exuvia as the host emerges (Böttger 1972b). Little is known about the mechanisms by which mite larvae select sites on hosts, and virtually all available information pertains to a few species of the genus Arrenurus. Site selection of larval Arrenurus on odonates appears to be largely a function of timing (Mitchell, 1961). Progress of the host ecdysis at the point when mite larvae become active determines where the mites ﬁrst come in contact with the host, and they typically attach close to that point. Larvae experimentally removed from the host and placed elsewhere on the body readily attached there. In contrast, there appears to be little correlation between the process of host ecdysis and site selection in larval Arrenurus parasitizing mosquitoes. For example, larval Arrenurus kenki almost always attach between the head and thorax when infesting adult Aedes excrucians but to the abdomen when attacking Aedes cinereus, without reference to the time taken by the host during ecdysis. v. Attaching to host and engorgement Larval mites attach to hosts by using their pedipalps to stabilize the body and produce the leverage needed to pierce the cuticle of the host with their chelicerae. Larvae of some species of Arrenurus secrete a substance which solidiﬁes and apparently helps to cement the mite to the host (Åbro, 1984), but this has not been observed in other cases (see Redmond and Lanciani, 1982). After attachment, larvae of several genera (e.g., Hydrachna, Rhyncholimnochares, and Arrenurus) are known to form a feeding tube, or stylostome, in the tissues of the host (see Smith, 1988), and these structures are probably produced by larvae of species belonging to many other genera. Gross structure of stylostomes appears to be consistent for species within a genus, but differs greatly among genera. For example, stylostomes of larval Hydrachna (Davids, 1973) and Rhyncholimnochares are multibranched and open-ended, those of larval Arrenurus are single blind tubes (Åbro, 1979; Redmond and Hochberg, 1981) and the known example of a stylostome of a pionid appears to be a single, open-ended tube (Gledhill, 1985). Details of stylostome morphology appear to be constant for a given species of mite regardless of host but differ among mite species, leading to the conclusion that they are produced by larval mites rather than their hosts (Lanciani and Smith, 1989). Stylostomes are acellular, consisting of acid mucopolysaccharides, and have no perforations other than a terminal pore, when present. The mechanism of stylostome function is not well understood. 16. Water Mites (Hydrachnida) and Other Arachnids Davids (1973) argues that the stylostomes of larval Hydrachna are formed by coagulation of host hemolymph in reaction to mite saliva, and that the mites imbibe hemolymph from the host. Stylostomes of Arrenurus larvae are typically surrounded by a ﬂuid-ﬁlled abscess, and it has been suggested that the mites feed upon liqueﬁed tissues in much the same way as larval chiggers do (Pﬂügfelder, 1970; Åbro, 1982, 1984). It is possible that the stylostomes of larvae in these remotely related clades are non-homologous. Duration of the period of larval engorgement varies considerably among water mite taxa. Larvae of Limnochares aquatica and Hydryphantes tenuabilis require 6 – 13 days to engorge (Böttger, 1972a; Lanciani, 1971a; Smith, 1989), but those of various species of Hydrachna and Eylais may spend from 2 weeks to 10 months on the host (e.g., Davids, 1973; Smith, 1977, 1988; Wiggins et al., 1980). Larvae of some species of Eylais can complete engorgement within 2 weeks at the time of year when female hosts are producing eggs, but apparently diapause in a partially engorged state at other times of the year (Smith, 1988). Presumably, growth rate of larvae in these groups is generally dependent on physiological condition of the host. Larvae of more recently evolved groups associated with shortlived insects, especially nematocerous Diptera, require shorter times for engorgement. Those of pionid species inhabiting temporary pools, such as Piona napio and Tiphys americanus, can engorge in as little as 24 h on their chironomid hosts. Larval Arrenurus feeding on mosquitoes take approximately 6 days for full engorgement (Mullen, 1974). Larval growth during engorgement is also highly variable. Larvae parasitic on nematocerous ﬂies increase only modestly in size. For example, larvae of Limnesia maculata and Unionicola crassipes expand to between 3 and 4.3 times their original volume on their chironomid host and those of Arrenurus species feeding on mosquitoes increase in volume about 16-fold (Münchberg, 1938; Böttger, 1972b). Larvae of other species of Arrenurus parasitic on odonates augment their volume 80 to 90 times, and larval Hydrachna and Eylais grow substantially, increasing up to 600-fold in size in some cases (Davids, 1973; Fig. 427). Larvae parasitizing long-lived hosts for extended periods risk premature detachment when the host molts. Caste exuvia of nepid and belastomatid bugs are commonly found with larval and protonymphal Hydrachna still attached. Larvae of Limnochares aquatica retain their mobility and can transfer from one host instar to the next to continue engorgement. vi. Detaching from host Following engorgement, mite larvae must detach from the host and return to an 565 aquatic habitat suitable for postlarval development. Larvae of various taxa may use host behavior, along with mechanical, visual, and chemical stimuli, as cues to initiate detachment (Böttger, 1972a, b). Environmental cues (such as moisture) may be sufﬁcient to induce detachment in some species, but physiological cues from the host are essential in others (Smith and Laughland, 1990). Larvae of the Eurasian species Arrenurus cuspidator parasitic on the damselﬂy, Coenagrion puella require two simultaneous cues to stimulate detachment, high humidity and either mating or oviposition behavior by the host (Rolff and Martens, 1997). Presumably, the mite larvae can detect hormones or some other physiological correlate with reproductive behavior in the host. Engorged larval mites risk detachment from hosts into unsuitable habitats. Fully fed larvae with limited mobility falling on dry ground quickly perish, and those introduced into unsuitable aquatic habitats may fail to survive or reproduce. At least some species of Arrenurus parasitic on odonates are regularly introduced into lakes that apparently cannot support their reproduction (Mitchell, 1964a, 1969b). A large proportion of Cenocorixa biﬁda waterboatmen living in inland saline lakes of central British Columbia are parasitized by nonresident species of Eylais and Hydrachna by late autumn (Smith, 1977). Fully fed larvae are capable of only limited movement, and those successfully re-entering water typically seek out plant material or detritus, attach themselves by their chelicerae and become quiescent. Engorged larvae of Piona and Arrenurus may attempt to swim or crawl for up to 2 days before transforming to protonymphs . 3. Protonymph (Nymphochrysalis) Engorged larvae of all groups of water mites enter a quiescent protonymph stage during which larval tissues are resorbed and reorganized and the deutonymph develops. After a few days fully formed deutonymphs emerge from the larval skins and become active. Mites which parasitize large, long-lived insects, including Hydrachna, most Eylaoidea, and certain Hydryphantidae, apotypically pass through the protonymphal stage while still attached to the host, within the larval integument. This trait is correlated with extreme growth during the parasitic phase which renders engorged larvae incapable of locomotion. Along with other adaptations, extended attachment to the host allows many of these mites to exploit periodically temporary habitats efﬁciently. Members of Arrenurus planus Marshall and A. ventropetiolatus Lavers survive for up to 10 months of the year in the dry basins of vernal temporary pools in 566 Ian M. Smith, David R. Cook, Bruce P. Smith eastern and western North America, respectively, as diapausing protonymphs and pharate deutonymphs. These mites require a period of desiccation, and probably cold temperatures, to release them from diapause (Münchberg, 1937; Wiggins et al., 1980). 4. Deutonymph In all known species the deutonymphal instar is active and predaceous and resembles the adult, but is sexually immature. Deutonymphs typically exhibit relatively undeveloped idiosomal sclerites and chaetotaxy compared to adults. They have only a rudimentary (or provisional) genital ﬁeld bearing an incomplete complement of acetabula. They feed voraciously, often preying upon immature instars of the same taxa of insects they parasitized as larvae (see Table I). The deutonymphal stage varies in duration from a few days or weeks in early derivative groups such as Hydrachnidae, Eylaoidea and certain Hydryphantidae, to several months in many groups of Lebertioidea, Hygrobatoidea and Arrenuroidea. Deutonymphs of some hygrobatoid families are especially long-lived. Those of Pionidae typically live for many months through summer and the following winter, and this trait has pre-adapted certain species to exploit annually temporary pools. In these pionids, deutonymphs are able to endure the dry phase of the cycle in damp retreats in the substrate (Smith, 1976a; Wiggins et al., 1980). The deutonymph is the primary growth instar in most groups of water mites, and body size increases dramatically during this stage. On attaining adult size deutonymphs prepare to transform to tritonymphs by embedding their chelicerae in plant tissue or soft detritus and becoming inactive. 5. Tritonymph (Imagochrysalis) During this stage further structural reorganization occurs to produce the adult. This ﬁnal metamorphosis is rapid, and adults typically emerge within a few days. Mature, fully fed deutonymphs anchor themselves to living or decaying plant material using the legs and mouthparts as they approach transformation to the tritonymph stage and become quiescent. Tritonymphs of several species of Pionidae inhabiting vernal temporary pools are regularly found attached to aquatic mosses in clusters, with each mite attached at a leaf axil. Conspicuous clumps of up to 500 tritonymphs comprising several species of the genera Tiphys and Piona can be observed in moss mats at the edges of these pools as the ice is melting in early spring. The signiﬁcance of this clustering is not known. The tritonymphal instar remains enclosed by the deutonymphal exoskeleton, and the pharate adult may often be seen forming within it. Mites of various species of Arrenurus typi- cally spend 4 to 5 days in the tritonymph stage under laboratory conditions at 20°C. Water mites appear to be particularly vulnerable to poor water quality during the tritonymph stage, perhaps because the metamorphosing adults must respire by diffusion across three levels of cuticle. For example, when dead and decaying prey are present in laboratory cultures, tritonymphs of Arrenurus may take up to 17 days to complete development and mortality rates of this instar may exceed 50%, despite minimal mortality among deutonymphs and adults living in the same container. 6. Adult a. Sclerotization of Exoskeleton Adults emerge from the deutonymphal integument in teneral condition with soft, pliable, and colorless sclerites. They immediately become active, crawling or swimming about while sclerotization of the body is completed and distinctive color patterns become evident. Adults of the various groups display a wide range of body shapes and arrangements of idiosomal sclerites, primarily as adaptations for living in aquatic habitats of varying depth, turbulence, temperature, and substrate type. Shortly after emergence, both males and females mature sexually and begin to mate. In many taxa, males tend to emerge and become mature a few days earlier than conspeciﬁc females, and are ready to mate as soon as females become active in the habitat. Adults of certain Limnochares (Böttger, 1972a), and possibly some Arrenurus (Imamura, 1952), undergo supernumerary molts. b. Mating and Spermatophore Transfer The adult is a highly specialized reproductive instar in water mites, as clearly evidenced by the remarkable behavioral modiﬁcations, and associated morphological adaptations, that have developed in various groups to facilitate successful spermatophore transfer (Smith and Oliver, 1986; Proctor, 1992a). Water mites exhibit a greater variety of spermatophore transfer methods than any other group of arachnids (Proctor, 1991b). Water mite genera can be grouped into categories based on degree of interaction between males and females during spermatophore transfer (Proctor, 1992b). i. Dissociation during transfer The plesiotypical behavior pattern in water mites, complete dissociation involving no chemical communication or physical contact between the sexes, has been observed in species of a wide variety of early derivative genera (see Proctor, 1992a). In these species, males deposit stalked spermatophores on the substrate for subsequent retrieval by conspeciﬁc females, and those of some species have been observed to deposit large numbers when isolated 16. Water Mites (Hydrachnida) and Other Arachnids in small containers of water. After ﬁnding the spermatophores, perhaps using pheromonal cues, females pick them up in the gonopore. An apotypical variant of this, incomplete dissociation requiring chemical or physical cues from females to that stimulate males for depositing spermatophores, occurs in several unrelated genera (Proctor, 1992a). Dissociation during spermatophore transfer is widespread in actinedid mites and probably occurs in most groups of water mites with unmodiﬁed males. ii. Association during transfer A more interactive type of mating, pairing of males and females for indirect spermatophore transfer, has been observed in several species (see Proctor, 1992b). In these cases, males deposit spermatophores on the substrate, then actively assist females to ﬁnd them and pick them up. Males of some Eylais lead receptive females in a circular dance, stopping repeatedly to deposit spermatophores at the same location on the circumference of the route. Halfway through each revolution of the dance, the female pauses with the male over his mass of spermatophores and picks up one or more of them in her gonopore (Lanciani, 1972). Males of Neumania papillator apparently exploit the hunting behavior of females to attract them to spermatophores (Proctor, 1991a, 1992a, c). Upon encountering a female, males vibrate their legs rapidly, simulating the stimuli produced by copepod prey, until she responds with characteristic ambush posture and aggression. The males then turn away, lay down a spermatophore net and rapidly fan water to create a current over the net toward the female, presumably directing pheromones toward her. A receptive female then ceases aggression, approaches the spermatophore net, and begins to collect spermatophores. Males of some species of Unionicola also use leg-trembling to generate vibrational cues to attract females to spermatophores (Proctor, 1992a, c). iii. Copulation during transfer Pairing with direct spermatophore transfer has evolved independently in many water mite lineages. Active transfer of spermatophores to females is accomplished either by modiﬁed appendages or idiosomal sclerites of males or by direct gonopore-to-gonopore contact. Proctor (1992b), following Hinton (1964), considers both of these options as examples of copulation. In Unionicola intermedia, a species that inhabits the mantle cavity of freshwater mussels, males retrieve their own spermatophores and carry them between the genua or tibiae of their hind legs while seeking receptive females. On ﬁnding a female, the male crawls beneath her and places his spermatophores in her gonopore (Hevers, 1978). Males of many groups of 567 Hygrobatoidea produce unstalked spermatophores which they carry between modiﬁed claws on the tarsi of their ﬂexed third pair of legs before transferring them directly into the gonopore of the female. This type of mating has been observed in various species of Feltria (Motas, 1928), Forelia (Uchida, 1940), Tiphys (Viets, 1914; Mitchell, 1957c), Piona (Koenike, 1891; Mitchell, 1957c; Böttger, 1962; Smith, 1976a) and Brachypoda (Motas, 1928; Halik, 1955). In each case, the male uses specially modiﬁed appendages, usually the fourth pair of legs, to hold the female in a characteristic posture so that he can transfer spermatophores to her gonopore by simply extending his third pair of legs. This mating behavior probably occurs in all water mites whose males have highly modiﬁed leg segments but no elaborate caudal development. A variety of complex modes of direct spermatophore transfer occur in the genus Arrenurus. Males of the various species have the idiosoma modiﬁed posteriorly to form a characteristically shaped cauda (Figs. 419, 420). A receptive female mounts a male, positioning herself so that her gonopore rests over the back edge of his cauda. Secretions from glandularia of the male temporarily cement the pair together, and the male then deposits one or more stalked spermatophores on the substrate. In some species, the male then rocks his body down and forward to bring the gonopore of the female in contact with the spermatophore (Lundblad, 1929; Böttger and Schaller, 1961; Böttger, 1962). In others, the male uses his petiole, a cup-shaped posterior appendage of the cauda, to scoop up spermatophores into masses which he then inserts into the gonopore of the female (Böttger, 1965). Variations of this behavioral pattern occur throughout the many species of Arrenurus, along with the multitude of different modiﬁcations in caudal morphology that characterize the males. This type of mating probably can be inferred in all groups of water mites with elaborate caudal development in males. Finally, direct transfer of spermatophores from gonopore to gonopore during copulation occurs in certain species of Eylais (Böttger, 1962; Lanciani, 1972) and in the arrenuroid genera Midea (Lundblad, 1929) and Nudomideopsis (I.M. Smith, unpublished data). In some of these cases, males either have the gonopore protruding from the venter on a cone-shaped projection (Eylais) or are equipped with winglike appendages ﬂanking the gonopore (Midea) to facilitate transfer. Clearly, copulation during spermatophore transfer has evolved many times from antecedent conditions involving association with complex sexual interaction. As deﬁned by Proctor (1991b), copulation has developed independently no fewer than 91 times within the water 568 Ian M. Smith, David R. Cook, Bruce P. Smith mites, including at least three times within a single family, Pionidae (Mitchell, 1957c; Smith, 1976a). Direct transfer from gonopore to gonopore certainly developed independently in Eylais and Arrenuroidea. The often profound modiﬁcation of the male idiosoma and appendages that is associated with diverse modes of spermatophore transfer emphasizes the highly specialized mating function of the adult instar in water mites. Males in many groups mate and die within a few days of emerging. Those of some groups, such as Arrenurus, may live for several weeks. Males of some genera of Foreliinae and Tiphyinae are so modiﬁed morphologically that they are unable to move about easily, and are restricted to crawling slowly over the substrate using the anterior pairs of legs. Sex pheromones appear to mediate pairing in at least some species of water mites. Males of Limnesia undulata increase spermatophore deposition when females are present, and produce comparable numbers when placed in containers from which females have recently been removed (Proctor, 1992a). The vigorous fanning of water over spermatophore nets toward females by males of Neumania papillator strongly suggests chemical communication (Proctor, 1991a). Females of various species of Arrenurus produce male-arrestant / attractant pheromones that can be extracted from water (B.P. Smith and Hagman, unpublished data). Unlike insect pheromones which are often species-speciﬁc, the pheromones of Arrenurus species often elicit response in several closely related and cooccurrent species (B.P. Smith and Florentino, unpublished data). There is also evidence that males of some species of Arrenurus produce pheromones attractive to females (Baker, 1996). Males appear to be much less common than females in many genera of water mites (Meyer and Schwoerbel, 1981; Proctor, 1997). This may in part be due to the shorter life span of males. Biased sex ratios can also result from protandry which has been demonstrated in at least some water mite species (Proctor, 1989, 1992b). Females of Arrenurus manubriator produce strongly gender-biased clutches (Proctor, 1997; B.P. Smith, unpublished data). The bias for males or females appears to be consistent among successive clutches of eggs for a given female and appears to be a trait largely inherited from the maternal parent (B.P. Smith, unpublished data). Water mites appear to be diplo-diploid, with no recognizable sex chromosomes (Sokolow, 1954). Mated females may live for many months, continuing to feed while they produce several clutches of eggs. In many groups, mating occurs in late summer and fertilization is delayed while females overwinter. These females lay their eggs after they have been fertilized early in the following spring or early summer. In contrast, females of species inhabiting temporary pools must lay their eggs within a few days of mating to ensure that their offspring reach a life-history stage capable of avoiding or withstanding the dry period before water disappears from the habitat (Smith, 1976a; Wiggins et al., 1980). Preliminary evidence suggests that the ﬁrst male to mate with a female has almost total sperm precedence over subsequent mates. Given that sperm is regularly stored for many months over winter, there must be substantial selective pressure for behavioral and phenological adaptations to gain early access to females. Guarding of tritonymphs (presumably females) by young males has been observed in Arrenurus planus. Males do not seem to discriminate between virginal and previously mated females. Males of Neumania papillator attempt to mate with all females they encounter. They eventually produce signiﬁcantly greater numbers of spermatophores for virginal females, but this appears to be the result of avoidance behavior by inseminated females rather than discrimination by males (Proctor, 1991a). Males of Arrenurus manubriator choose larger females over smaller ones, perhaps selecting for greater egg production capacity (Proctor and Smith, 1994; Smith and Bovenzi, unpublished data). D. Habitats and Communities Throughout water mite evolution, successful invasion and exploitation of new habitats has depended on the development of compatible adaptive strategies for the various instars. Larval traits tend to promote parasitism and dispersal on hosts, while those of deutonymphs and adults favor feeding, growth and reproduction in water. Water mite species are regularly provided with opportunities to colonize new habitats by the passive transport of their larvae on hosts, and this has been a primary mechanism promoting speciation and divergence over time. Nevertheless, in modern communities most species and many monophyletic groups representing genus or family level taxa are restricted to one or a few similar types of habitats. The strong correlation between certain clades and habitats suggests that conservative factors such as adaptive requirements for locating hosts, prey, mates, and oviposition sites tend to constrain adaptive radiation. It also indicates that successful invasion of new habitats usually resulted in the rapid evolution of signiﬁcant new life-history, behavioral and morphological traits. The communities living in the major types of freshwater habitats in North America are outlined below and in Table I. 16. Water Mites (Hydrachnida) and Other Arachnids 1. Springs (Including Seepage Areas) Species of 47 genera live in moss and wet detritus associated with rheocrenes and helocrenes. Members of ancient clades that may well have originated in this type of habitat are soft-bodied (e.g., Tartarothyas), or partly to fully sclerotized (e.g., Thyas, Panisopsis). Adults of derivative groups that apparently invaded spring and seepage habitats from ﬂowing water more recently have entire dorsal and ventral shields (e.g., Paramideopsis — Fig. 425, certain Mideopsis, Laversia). Most spring inhabiting species are coldadapted stenophiles, but some Wandesia (subgenus Partnuniella) and all Thermacarus live only in hot springs. Over 115 species in 57 genera and 25 families have been identiﬁed from spring habitats in Canada including interstitial taxa (Smith, 1991a). 2. Rifﬂe Habitats Species of 49 genera live on the substrate in rapidly ﬂowing areas of streams and rivers. Diverse communities from a single location may contain up to 50 species in 30 genera. Members of a few early derivative taxa are soft-bodied (e.g., Protzia), but adults of most groups are both strongly ﬂattened and well sclerotized (e.g., Kongsbergia — Fig. 421, Aturus — Fig. 422). Most rheophilic species are cold- or cool-adapted stenophiles. 3. Interstitial Habitats Species of 55 genera live in sand and gravel deposits to depths of 1 m or more, mainly in the hyporheic zone of streams and rheocrenes. Deutonymphs and adults crawl through the spaces between particles, often with considerable speed and agility. Members of most early derivative taxa are soft-bodied (e.g., Rhyncholimnochares) and some are also vermiform (e.g., Wandesia). In adults of more recently derived groups, the dorsum and venter are partly covered by various plates or small shields (e.g., Clathrosperchon, Omartacarus, Atractides), or are well sclerotized with either entire dorsal and ventral shields or greatly expanded coxal plates (e.g., Frontipodopsis, Aturus — Fig. 422, Uchidastygacarus — Fig. 424, Arenohydracarus). Mites adapted to interstitial habitats have reduced eyes and lack pigmentation of the integument. 4. Stenothermic Pools Species of 47 genera live in silty substrates in spring-fed pools, fen pools and depositional areas of streams. Members of ancient taxa are soft-bodied (e.g., Pseudohydryphantes), while adults of early derivative clades exhibit various degrees of sclerotization ranging from slight enlargement of dorsal and ventral plates (e.g., Teutonia, Wettina, Forelia) to development of en- 569 tire dorsal and ventral shields (e.g., Momonia, Geayia). Adults of certain taxa which invaded pools relatively recently from lotic and hyporheic habitats have retained dorsal and ventral shields, though they have become secondarily adapted for swimming (e.g., Axonopsis, Mideopsis). Certain groups inhabiting depositional areas of streams (e.g., Teutonia, Oxus, Wettina) include the fastest and most agile swimmers among the water mites. Most inhabitants of pools are cooladapted stenophiles. 5. Lakes (Including Permanent Ponds, Marshes, Swamps and Bogs) Species of 39 genera inhabit large bodies of standing water. Members of ancient and early derivative genera are generally soft-bodied (e.g., Hydrodroma, Limnesia, Hygrobates, Huitfeldtia). Adults of many genera in more recently evolved clades have extensive dorsal and ventral shields (e.g., Koenikea — Fig. 423), in at least some cases because the group has secondarily invaded standing water from lotic or hyporheic habitats (e.g., Axonopsis, Mideopsis). Most species are strong swimmers and have fringes of long, slender setae on the genua and tibiae of the second, third, and fourth pairs of legs (Fig. 290). These swimming setae help to propel the mites as they move about at the surface of the substrate or among plants. Some species of Unionicola and Piona have especially long swimming setae, and are essentially pelagic or planktonic (Riessen, 1982). A few species which lack swimming setae either walk or crawl actively on plants and detritus, reﬂecting recent rheobiontic ancestry (e.g., Sperchon, Torrenticola, certain Mideopsis, Neoacarus). Members of Tyrrellia are unusual in that they inhabit wet litter at the edges of permanent bodies of water, including lakes, where they crawl about at the surface ﬁlm. Many species of Unionicola and the only known species of Najadicola are obligate parasites or commensals in the mantle cavity of molluscs in both lentic and lotic habitats (Mitchell, 1955; Simmons and Smith, 1984; Gledhill, 1985; Vidrine, 1986). More than 25 species in each of the remarkable genera Arrenurus and Piona often occur together in a single small lake. Mitchell (1964a, 1969a) concluded that behavioral ﬂexibility exhibited by larval Arrenurus in selecting and exploiting their odonate hosts is the main factor permitting such high levels of sympatry, and that competition between deutonymphs and adults of the various species is probably not signiﬁcant in natural communities. Most limnophilic taxa tolerate a broad range of temperatures which they encounter in weed beds and silty substrates in the littoral and sublittoral zones. However, a few are cold-adapted stenophiles restricted 570 Ian M. Smith, David R. Cook, Bruce P. Smith to either arctic-alpine glacial lakes (e.g., Acalyptonotus) or the profundal zone of oligotrophic lakes (e.g., Huitfeldtia, Laversia). Certain species of some genera (e.g., Limnochares, Hydrodroma, Limnesia, Piona and Arrenurus) are highly tolerant of extreme thermal and chemical regimes, and are able to inhabit desert sloughs, alkaline lakes or acid bogs. The water mite fauna of wetland habitats in Canada was treated by I.M. Smith (1987). 6. Temporary Pools Species of 13 genera live in annually temporary pools. Members of ancient genera that may have originated in this type of habitat range from soft-bodied walkers (e.g., Thyas) and swimmers (e.g., Eylais) to mites with unique arrangements of integumental plates that either crawl (e.g., Piersigia) or swim awkwardly (e.g., Hydrachna, Hydryphantes). Species of derivative groups that apparently invaded temporary pools secondarily tend to be relatively good swimmers (eg:Tiphys, Piona, Arrenurus). Water mites inhabiting temporary pools are adapted either to avoid (e.g., species of Hydrachna and Eylais) or to endure the dry phase of the annual cycle (Wiggins et al., 1980). E. Ecology The parasitic larvae and predaceous deutonymphs and adults of water mites have direct and almost certainly signiﬁcant effects on the size and structure of insect populations in many habitats (Smith, 1983, 1988; Lanciani, 1983; Proctor and Pritchard, 1989). Unfortunately, their impact has rarely been measured accurately because of the routine neglect of mites in ecological studies of freshwater communities. Due to their small size and often cryptic habits, mites are usually absent or seriously under-represented in samples that are collected using standard techniques for capturing insects and crustaceans. Most entomologists are unfamiliar with mites, and tend either to disregard them as too poorly known ecologically and difﬁcult taxonomically or to lump them together in a meaningless way, when conducting community studies. Failure to include realistic assessment of the roles played by water mites often results in seriously ﬂawed analyses of the structure and dynamics of freshwater communities. 1. Impact as Parasites a. Effect on Individual Hosts The impact of larval water mites on host insects was reviewed by Lanciani (1983) and B.P. Smith (1988). Numerous laboratory studies have demonstrated reduced survival or longevity of various insect hosts parasitized by larval water mites, including mosquitoes (Lanciani and Boyt, 1977; Lanciani, 1987), ceratopogonid midges (Lanciani, 1986a), chironomid midges (Weiberg and Edwards, 1997), juvenile water striders (Smith, 1989), backswimmers (Lanciani, 1982) and damselﬂies (Forbes and Baker, 1990, 1991; Leung and Forbes, 1997). Lanciani (1983) concluded that the ratio of larval mite weight-to-host weight is a good indicator of the probable impact of parasitism on the host. Studies of parasite-induced mortality in ﬁeld populations have not been conclusive (Robinson, 1983; Åbro, 1990a; Smith, 1999). There is evidence of occasional crashes of host populations related to parasitism by mites (Smith, 1988). Water mite parasitism reduces egg production of insect hosts. The most pronounced effects have been reported for Hemiptera infested with larval Hydrachna and Eylais (Davids, 1973; Smith, 1977), but parasitized nematocerous ﬂies also show lowered fecundity (Smith and McIver, 1984c; Lanciani and Boyt, 1977). Development of juvenile instars is retarded in backswimmers (Notonectidae) parasitized by larvae of Hydrachna virella (Lanciani and May, 1982) and in water striders (Gerridae) attacked by Limnochares aquatica (Smith, 1989). Mite parasitism inﬂuences frequency of foraging, intensity of territorial behavior and likelihood of mating in males of the damselﬂy Enallagma ebrium (Forbes, 1991). Mite parasitism apparently reduces the capacity of the damselﬂy Nehalennia speciosa to ﬂy and disperse (Reinhardt, 1996). The corixid Cymatia bonsdorﬁ increases feeding activity when parasitized by larvae of Hydrachna conjecta (Reilly and McCarthy, 1991). Some hosts exhibit strong avoidance behavior in the presence of larval water mites, involving grooming behavior to dislodge them. Larval Ischnura verticalis typically respond to ﬁsh by reducing movement, but in the presence of both ﬁsh and preparasitic larvae of Arrenurus pseudosuperior exhibit unrestrained grooming and mite-avoidance behavior despite the increased risk of detection by predators (Baker and Smith, 1997). b. Effect on Population Structure of Hosts Larval water mites regularly parasitize 20 – 50% of adults in natural populations of aquatic insects in such diverse families as Corixidae (Hemiptera), Dytiscidae (Coleoptera), Libellulidae (Odonata), Culicidae, and Chironomidae (Diptera). Almost all individuals are parasitized in some host populations, and some may carry remarkably high mite loads. We have seen examples of over 1000 larvae of Arrenurus parasitizing a dragonﬂy (Erythemus simplicicolis), over 350 larvae of Limnochares aquatica on a water strider (Gerris comatus), over 50 larvae of Arrenurus on a ceratopogonid 16. Water Mites (Hydrachnida) and Other Arachnids midge (Bezzia sp.) and over 40 larvae of Arrenurus danbyensis on a mosquito (Coquillettidia perturbans). Individual chironomid midges emerging from mesotrophic lakes in the Great Lakes Basin often carry 20 or more mite larvae representing as many as seven different genera. The distribution of parasitic larval water mites typically follows a clustered pattern within a host population, with a few host individuals heavily infested and most carrying few or no mites (e.g., Smith and McIver, 1984a; Smith, 1988). Under natural conditions, clustering may reﬂect differences in the temporal and spatial distributions of host-seeking larval mites and their potential hosts. However, clustering also occurs in laboratory experiments involving synchronous exposure of insects to mites, where duration of exposure and spatial heterogeneity are controlled. This suggests either initial differential susceptibility to parasitism among host individuals or facilitation of additional loading on hosts that have become parasitized. Intensity of infestation by larval Arrenurus appears to be negatively correlated with body size and condition (i.e., ratio of mass to body size) of their damselﬂy hosts in at least some cases (Forbes and Baker, 1991; Leung and Forbes, 1997). This may be a result of more vigorous avoidance behavior by larger and more robust potential hosts (Smith and McIver, 1984b; Forbes and Baker, 1990). Larval Arrenurus have greater intensity of parasitism on damselﬂies with asymmetry in forewing length, but it is not clear whether increased infestation is a response to or a cause of this asymmetry (Bonn et al., 1996). Behavioral subtleties can increase vulnerability of hosts of certain gender or age classes to colonizing larval mites (Smith and Cook, 1991), with resulting differentials in parasite loads (Smith, 1988, 1999). Observed differences in parasite loads among sex and age classes in natural populations can prove useful for inferring the behavior of the host and parasite and the age structure of the host population (Smith, 1999). c. Effect on Other Mites Sharing the Same Host Clustering of parasitic larvae on certain host individuals results in intraspeciﬁc competition between mites on those hosts that can affect their growth and survival (Lanciani, 1976, 1984, 1986b), and leaves many hosts underutilised. Consequently, clustering apparently both invokes density-dependent control of mite populations and keeps them below carrying capacity, effectively precluding interspeciﬁc competition. Parasitism of a host individual by larvae of more than one species of water mite occurs more commonly than would be expected by chance (Stechmann, 1980; Smith and McIver, 1984a; Lanciani and McLaughlin, 571 1989). In addition, many species of water mites exploit a variety of related host species. As a result, parasitic associations often involve complex assemblages with considerable overlap in exploitation of host resources, including use of similar attachment sites (Kouwets and Davids, 1984). Interspeciﬁc competition appears to be negligible in these assemblages. Consequently, assuming that larval success is the limiting factor determining population size in water mite species, there is probably little competition for food resources in communities of deutonymphs and adults. This may help to explain the remarkably high levels of species richness in many water mite communities. 2. Impact as Predators The feeding habits of deutonymphal and adult water mites were reviewed by Böttger (1970), Gledhill (1985) and Proctor and Pritchard (1989). Data for species inhabiting Canadian wetlands were reviewed by I.M. Smith (1987). Deutonymphs and adults of free-living species are voracious predators on a wide range of small aquatic organisms. Some species at least occasionally scavenge on recently killed organisms. Most species exhibit marked preferences for prey of a particular size with particular morphological and behavioral attributes. Species of Hydrachna, Thyas, Hydryphantes and Hydrodroma are specialized predators of arthropod eggs (Davids, 1973; Mullen, 1975; Lanciani, 1978, 1980; Wiles, 1982; Meyer, 1985). Many members of Eylais and Piersigia and the large genus Arrenurus appear to depend on ostracod crustaceans as prey (although some Eylais and Arrenurus feed on cladocerans), adults of Neumania ambush copepods, pelagic and planktonic species of Unionicola and Piona are highly efﬁcient predators of cladoceran crustaceans and species of many genera of Lebertioidea, Hygrobatoidea and Arrenuroidea prey upon larval Chironomidae (Böttger, 1970; Proctor, 1991b; Proctor and Pritchard, 1989, 1990; also see Table I). Some species of Limnesia and Piona appear to be more opportunistic in their choice of prey, and frequently even attack other mites in crowded containers (Elton, 1922). Parasitism of freshwater molluscs during the deutonymphal and adult stages has evolved at least twice in the superfamily Hygrobatoidea. Many species of Unionicola inhabit the mantle cavity of mussels and snails (Mitchell, 1955; Vidrine, 1986) and the pionid Najadicola ingens has similar habits (Simmons and Smith, 1984). Deutonymphs and adults frequently prey upon the immature instars of the same species of insects that they parasitize as larvae (Davids, 1973; Wiles, 1982; Proctor and Pritchard, 1989). Preliminary indications suggest that many species of mites are specialized to exploit one group of insects throughout their life history, 572 Ian M. Smith, David R. Cook, Bruce P. Smith a strategy that would presumably increase the probability of both spatial and temporal co-occurrence of the mites and their preferred hosts and prey. Some water mites exhibit feeding rates in experimental studies that are high enough to be considered as potentially important inﬂuences on the size and structure of prey populations in nature (Paterson, 1970; Mullen, 1975; Lanciani, 1979; Wiles, 1982). Gliwicz and Biesiadka (1975) reported that individuals of Piona limnetica consume 10 – 20 cladocerans per day in a neotropical lake, and estimated that members of this species could reduce the average standing crop of prey by 50% each week. Adults of Hygrobates nigromaculatus, with reported population densities as high as 1000 individuals / m2, apparently consumed an average of 14,500 larvae of the chironomid Cladotanytarsus mancus per square meter for two consecutive years in a European lake, accounting for a 50% mortality rate in the prey population (Ten Winkel et al., 1989). Tactile cues appear to be of primary importance to predatory water mites (Böttger, 1970; Davids et al., 1981). Hunting mites often ignore stationary potential prey even at close range, but immediately attack and kill individuals of the same prey species after contact is made. Adults of species of Piona typically swim in wide arcs until colliding with potential prey. Upon contact, the mites attack aggressively, and if initially unsuccessful, swim in tight spirals for several seconds until the prey is encountered again (B.P. Smith, personal observation). Several species of Neumania and Unionicola orient toward vibrations in the water as a cue to locate potential prey (Proctor and Pritchard, 1990). Water mites also use various cues to promote efﬁcient prey capture. Adults of Hydrodroma despiciens seem to respond to chemicals in the gelatinous coating of the insect eggs on which they feed (Wiles, 1982). Adults of a number of species show behavioral responses to chemical cues from prey organisms, and Baker (1996) has demonstrated how the mites detect these stimuli. Adults of both free living and parasitic species of Unionicola react to host extracts suggesting that they use chemical cues to initiate regional searching (Proctor and Pritchard, 1989; Roberts et al., 1978; Dimock and LaRochelle, 1980; LaRochelle and Dimock, 1981). 3. Importance as Prey Water mites are typically under represented in the stomach contents of predatory freshwater vertebrates (Modlin and Gannon, 1973; Pieczynski, 1976; Eriksson et al., 1980). Deutonymphs and adults at least occasionally form a signiﬁcant part of the diet of ﬁsh and turtles (Marshall, 1933, 1940; Knight, 1989), and we have seen examples of stomach contents from individ- ual brook trout and coregonids that consisted exclusively of several hundred adults of one species of Piona and Hygrobates, respectively. It appears that ﬁsh can develop narrowly focused search images for water mites in exceptional cases. Adults of many species of water mites eject viscous, sticky ﬂuid from the glandularia when handled roughly, apparently as a deterrent to predation. Experiments have shown that red-colored water mites are distasteful to ﬁsh which quickly learn to reject them as potential prey (Elton, 1922; Kerfoot, 1982). Kerfoot (1982) concluded that red pigmentation in water mites is aposematic coloration. However, most red water mites live in temporary pools and seepage areas where ﬁsh are absent. Terrestrial anystoid and parasitengone mites, and members of many early derivative water mite taxa, are predominantly red or orange suggesting that suffusion of the body with red carotenoid pigments is plesiotypical for the entire lineage. This type of pigmentation may have initially evolved in these mites as protection from ultraviolet wavelengths in sunlight (Garga, 1996). However, it is noteworthy that few members of the derivative water mite superfamilies Lebertioidea, Hygrobatoidea of Arrenuroidea have retained the plesiotypical condition. Most species in these groups are not red, and the relatively small number that are have acquired this coloration secondarily, as explained above. Interestingly, most red species in these groups also inhabit vernal temporary pools or springs. Few studies have examined invertebrate predation on water mites. Elton (1922) reported an instance of predation by an adult dytiscid beetle on a species of Hydrachna. Two authors have reported positive associations between ﬁsh and the mite Piona carnea and suggested both that ﬁsh prefer insects to mites as prey and that predaceous insects, including hemipterans, larval odonates and larval chaoborid midges, feed on the mites (Eriksson et al., 1980; Punčochér and Hrbáček 1991). 4. Potential as Indicators of Environmental Quality Species of water mites are specialized to exploit narrow ranges of physical and chemical regimes, as well as the particular biological attributes (including physico-chemical constraints) of the organisms they parasitize and prey upon. Consequently, water mites should be exceptionally sensitive indicators of habitat conditions and the impact of environmental changes on freshwater communities. Preliminary studies of physico-chemical and pollution ecology of the relatively well-known fauna of Europe have demonstrated that water mites are excellent indicators of habitat quality (Schwoerbel, 1959; Pieczynski, 1976; Biesiadka, 1979; Kowalik and Biesiadka, 1981; Kowalik, 16. Water Mites (Hydrachnida) and Other Arachnids 1981; Bagge and Merilainen, 1985; Cicolani and di Sabatino, 1991; Steenbergen, 1993). Gerecke and Schwoerbel (1991) analyzed changes in water mite communities in the Danube River between 1959 and 1984 and concluded that they were highly correlated with increased levels of organic pollution. One of the species they studied, Hygrobates ﬂuviatilis, appears to be relatively resistant to pollution and increased in dominance during the period of declining water quality. The results of these studies, along with our own observations in sampling a wide variety of habitats in North America and elsewhere, lead us to conclude that water mite diversity is dramatically reduced in habitats that have been degraded by chemical pollution or physical disturbance. There have been few laboratory studies of the tolerances of water mites to environmental variables. Deutonymphs of Arrenurus manubriator have proven to be more susceptible than other life-history stages to iron in solution, probably resulting from the major contribution of this instar to growth of the mites (Rousch et al., 1997). Experiments to assess differential susceptibility among life-history instars may provide valuable insight into the mechanisms responsible for observed changes in species populations in stressed habitats. Progress toward the establishment of essential baseline documentation on water mite communities in North America, however, continues to be hampered by lack of sufﬁcient systematic and ecological information at the species level, and by inadequate sampling and extraction procedures. Application of potentially useful information on water mites in assessing and monitoring the well-being of freshwater habitats and communities demands clearer understanding of the individual niche requirements and trophic relationships of species. This can be achieved by improving systematic knowledge of the group, and by developing more appropriate collecting, observing, and rearing techniques. F. Collecting, Rearing, and Preparation for Study These subjects were treated comprehensively by Barr (1973), and we discuss here only those aspects needing further emphasis or reﬁnement. 1. Collecting and Extracting Techniques Thus far, ﬁeld work on water mites in North America has focussed largely on qualitative sampling to obtain specimens required for systematic or life history studies, and the methods outlined below were developed primarily for these purposes. Efforts to modify these procedures for rigorous and cost-effective quanti- 573 tative sampling are needed, particularly those aimed at capitalizing on behavioral traits to simplify extraction of mites from detritus and silt. a. Deutonymphs and Adults The best results are achieved, in most habitats and for virtually all types of substrates, using a procedure that requires a strong net with a wide opening (25 – 40 cm diameter) and ﬁne mesh size (250 m), a supply of strong, clear polyethylene bags (4 – 5 kg capacity), a set of standard sieves of various mesh size (ranging from 250 m to 2 mm), six large, leak proof polyethylene containers (1 L), an ice chest, six rectangular, white photographic trays (30 cm 40 cm), a ﬂashlight, a set of eye droppers with a range of different sized tip openings, and a supply of small, leak proof polyethylene containers (50 mL). Once the desired habitat or microhabitat has been selected, the substrate must be thoroughly stirred. In ﬂowing water, the net should be positioned and secured so that the current will carry dislodged organisms and particles of substrate into it. In lentic habitats, it must be swept back and forth through the column of mixed water and substrate to pick up the organisms. In very shallow habitats, such as seepage areas, small rheocrenes, fens, and the edges of pools and ponds, the substrate must be gathered and stirred by hand. The net is worked beneath the edge of the wet moss, leaf litter, or detritus that is to be sampled so that mats of wet substrate can be separated and rinsed over the opening. Where there is sufﬁcient surface water, successive handfuls of loose material are picked or scooped up with the aid of a small trowel, thoroughly picked apart and washed in the net. Larger pieces of detritus are discarded after careful washing. In seepage areas, spring edges, fens, and bog margin habitats where there is often an extensive carpet of wet moss and associated plants, mites can also be collected by methodically and vigorously treading down the plants while dragging the net along to scoop up the detritus, silt, and organisms that become temporarily dislodged as water is forced up through the disturbed layer of vegetation. In either case, when a mixture of ﬁne silt, plant fragments and organisms begins to ﬁll the bottom of the net, it is transferred to a plastic bag containing a small amount of water. This process is repeated until the bag is approximately half full of material. The bag is then nearly ﬁlled with clean water and the contents stirred gently but thoroughly to allow heavy inorganic material such as gravel and sand to settle to the bottom. The contents of the bag are then poured carefully through a set of sieves so that gravel and sand remain in the bag. This operation is repeated until all loose organic material is in the sieves. We ﬁnd that a pair of upper and lower sieves with mesh sizes of 1.4 mm and 250 m, respectively, 574 Ian M. Smith, David R. Cook, Bruce P. Smith yields good results. The coarse material in the upper sieve is thoroughly stirred and rinsed with clean water. The ﬁne silt and small organisms accumulating in the lower sieve are regularly transferred to a large polyethylene container with enough water to just cover the surface of the silt. When the container is about half full of ﬁne silt it is closed and placed in an ice chest to be transported back to the laboratory or sorting area. In situations where there is insufﬁcient surface water to permit washing and rinsing of samples on site, accumulations of unsorted substrate material must be gathered, stored in plastic bags and transported on ice to a source of clean water for subsequent processing. With few modiﬁcations, this procedure can be used effectively in other types of habitats. Where the water column is between 25 cm and 1 m in depth and the bottom is ﬁrm enough to permit wading, the feet, hands, and net can be used to agitate the substrate (with the aid of a spade when necessary). In stream rifﬂes with a heterogeneous substrate of rocks, gravel, sand, and detritus, vigorous and persistent agitation is usually necessary to dislodge the large number and variety of mites that cling tenaciously to rocks or burrow in the silt under larger stones. We get good results by securing the net approximately 1 – 2 m downstream, and then systematically working through the substrate to loosen and thoroughly rinse all particles so that organisms are carried into the net. Where well-developed interstitial habitats occur in a stream bed, this approach can be extended to yield rich collections of mites including both surface and hyporheic species. This is accomplished by simply digging down through the gravel and sand beneath the rifﬂe zone, removing all large stones, to allow the current to ﬂush out the pockets of interstitial silty detritus and the organisms that live there. By digging out an area of substrate approximately 1 m2 to a depth of 0.5 m, we are often able to recover good numbers of many hyporheic species. This technique can often be used to sample the hyporheic zone in habitats where the standard Karaman – Chappuis method (Chappuis, 1942; Gledhill, 1982) is not practicable. Quantitative samples of hyporheic arthropods comprising large and diverse collections of water mites have recently been obtained in desert streams in Arizona by driving polyvinyl chloride tubing into the substrate to various depths and using a mechanical pump to evacuate the resulting wells periodically. In littoral lentic habitats such as depositional pools in streams, ponds, and sheltered bays in lakes, the feet can often be used to thoroughly stir up the top few centimeters of sediment and any rooted aquatic plants that may be present. A net is then repeatedly swept back and forth through the entire volume of disturbed water column to ensure that both swimming and crawling species are captured. As some mites in lentic habitats are too large to pass readily through 1.4-mm mesh, we recommend use of an upper sieve with 2.0-mm mesh when sampling ponds and lakes. The procedure outlined here can also be used by scuba divers and a boat operator to collect mites and insects from the sublittoral and profundal zones of lakes down to depths of 30 m or more, at a much higher level of efﬁciency than is possible with conventional grabs or dredges. In the sorting area, a number of white photographic trays are set up on tables and ﬁlled to a depth of 2 – 4 cm with clean water. Once the water has settled, the contents of one polyethylene container are carefully decanted into each tray, so that the silt spreads out in the center of the tray but does not reach the edges. As the mites become active and begin to move around in the trays, they are picked up individually using an eye dropper and transferred to small polyethylene containers. Many swimming species are strongly positively phototropic and can be recovered efﬁciently from trays kept in bright light. However, many crawling mites and a considerable number of swimming species are moderately positively or negatively phototropic and become highly active only at low levels of ambient light. The effectiveness and rate of recovering mites from all types of habitats can be substantially increased by reducing ambient lighting to low levels indoors or waiting until after sunset outdoors, and then scanning the trays systematically and periodically with a ﬂashlight beam. An experienced collector can then spot the mites as they move across a contrasting background provided by either the darkly colored silt or the white bottom of the tray. Mites remain active and continue to emerge from silt in the trays for up to 72 h, as deoxygenation of the water proceeds at room temperature. For this reason, it is suggested that, whenever possible, mites should be extracted from samples in situations that permit periodic observation of undisturbed trays over a period of at least 4 – 6 h with controlled lighting. Under these conditions, mites tend to congregate around the edges of the trays, especially in the corners, where they can be easily spotted and picked out. Although water mites frequently are found in samples collected for quantitative analysis in ecological studies, they are almost always underrepresented in terms of both numbers and diversity. This is often because collecting devices such as dredges or sieves are designed to capture organisms that are larger or more sedentary than mites. Due to their small size and ability to cling tenaciously to substrates, mites in lotic habitats can often resist casual efforts to dislodge them or can simply pass through nets with coarse mesh. By running or swimming, mites in hyporheic or lentic habitats can 16. Water Mites (Hydrachnida) and Other Arachnids avoid capture by traps that are designed to sample instantaneously small areas of superﬁcial substrates. Mite specimens are frequently overlooked when preserved samples are sorted because extraction techniques concentrate on larger organisms or depend on ﬂotation methods which we have found ill-suited for separating water mites efﬁciently from samples of detritus. Water mites can be collected from deeper water with underwater light traps using either battery-powered incandescent bulbs (Hungerford et al., 1955; Pieczynski, 1962, 1969; Pieczynski and Kajak, 1965) or chemoluminescent tubes (Barr, 1979) as light sources. Barr (1979) recommends use of small, portable light traps for qualitative sampling in lentic habitats, and provides a useful assessment of their utility for collecting water mites for various purposes, including quantitative analysis. Recently, a promising technique has been developed for extracting water mites and other aquatic arthropods quantitatively from samples of substrate by inducing them to respond actively to a temperature gradient (Fairchild et al., 1987). This method has yielded very good results for samples of baled sphagnum from bog ponds, and could probably be adapted for use with any type of substrate inhabited by mites. b. Larvae Free-living larvae are often collected from aquatic habitats along with deutonymphs and adults, but these specimens must be preserved in order to be identiﬁed and often cannot be reliably associated with other instars of the same species. Parasitic larvae can be recovered from insect hosts collected at or near the parental habitats. Aquatic hosts are usually captured using a dip net, whereas aerial adults can be trapped as they emerge from water, netted while swarming (especially over water at dusk), swept from vegetation, or attracted to lights at night. 2. Rearing Larvae needed for taxonomic or life history studies can be reared from females using the techniques described by Prasad and Cook (1972), Barr (1973), and Smith (1976a). Fertilized females of many species can be collected in spring and early summer. Females needed for rearing should be placed individually in small dishes or vials containing water and a few fragments of moss or wood as a substrate. The containers should then be covered and placed in a shaded, wellventilated location where the temperature can be maintained near levels experienced in the natural habitat of the mites. Mites from cold, stenothermic habitats must be refrigerated. The rearing containers should be checked periodically for the presence of egg masses. Females should be preserved immediately after they have 575 laid their eggs to ensure that good-quality specimens are available for conﬁrming species identiﬁcations. Larvae should be either preserved or slide-mounted directly as they emerge. Species-level treatment of larvae in certain families can be found in Prasad and Cook (1972), Wainstein (1980) and various papers by Smith (1976a, 1978, 1979, 1982, 1983a – f, 1984, 1989d, 1992a). 3. Preservation and Preparation for Study Deutonymphal and adult mites must be preserved in modiﬁed Koenike’s solution (or GAW), consisting of 5 parts glycerin, 4 parts water, and 1 part glacial acetic acid, by volume (see Barr, 1973), so that they can be properly cleared, dissected, and slide-mounted for identiﬁcation and study. Specimens preserved in alcohol or other hygroscopic agents become so distorted and brittle that subsequent preparation is difﬁcult or impossible. Deutonymphs and adults must be cleared in either acetic corrosive or 10% KOH, dissected, and mounted in glycerine jelly, following the techniques described by Barr (1973) and Cook (1974). Reared larvae are often slide-mounted directly, but can be preserved in 70% alcohol for subsequent mounting without being seriously damaged. Parasitic larvae should be preserved with the host in 70% alcohol, although larvae removed from hosts that have been pinned and dried often can be slide-mounted successfully. Before larvae are removed from hosts, the attachment sites should be noted and recorded. Larvae should be mounted whole in Hoyer’s medium which also acts as an efﬁcient clearing agent for these small specimens (see Barr, 1973). G. Taxonomic Keys to Genera of Water Mites in North America The following keys for larvae and adults permit identiﬁcation of specimens to family, subfamily and genus levels. They are intended for use by nonspecialists and use characters that should be obvious on slidemounted specimens to all careful observers. As a result, the keys are intentionally artiﬁcial in that genera do not necessarily key out with others belonging to the same family. The larval key does not include all taxa that appear in the adult key, as larvae of many genera remain unknown (see Table I). Knowledge of larvae in a number of other genera is based on only a few species. Consequently, the key for larvae will be subject to considerable revision and reﬁnement as larvae of more species are reared and described. We include the genus Stygothrombium (superfamily Styothrombioidea) in the keys and in Table 1 because these mites are frequently collected with hydrachnids and closely resemble them in morphology, behavior and ecology. 576 Ian M. Smith, David R. Cook, Bruce P. Smith The keys presented here are designed for use with a minimum of specimen preparation. Larvae should be whole-mounted on slides. The legs should be spread out from the body as much as possible by gently pressing down on the coverslip, as the chaetotaxy of leg segments is frequently used as a key character. Adults require clearing and some dissection prior to slide mounting. The mouth parts (capitulum and appendages) should be removed to permit examination of the pedipalps in medial and lateral view. Dorsal and ventral shields, when present, should be separated to allow clear viewing of certain key characters. We follow the convention for numbering the setiform structures on the leg segments of larvae that was established by Prasad and Cook (1972) and modiﬁed slightly by I.M. Smith (1976a) (see Figs. 6 – 8). The setae are numbered starting proximally on the posterolateral surface, proceeding distally along the dorsal half of the segment to the end, then returning proximally along the ventral half of the segment. Exceptions are made in the cases of solenidia (setiform chemoreceptors, see Figs. 31, 32, arrows) which are numbered ﬁrst, that is as “s1” on the genua, tibia III, and the tarsi, and as “s1” and “s2” on tibiae I and II, and tarsal eupathids (specialized setae, see Fig. 77, arrow) which are numbered second, that is as “2” on the tarsi. Where it has been necessary to use leg chaetotaxy in the key, the number of solenidia present on a segment is indicated in brackets (e.g. “2s”) immediately following the information on the number of setae, so that all setiform structures can be accounted for. We usually have omitted the empodia and claws from the illustrations of the legs of larvae so that the number and positions of distal setae on the tarsi can be seen clearly. The names of the segments of the appendages are consistently abbreviated as follows:trochanter, Tr; femur, Fe (basifemur, BFe; telofemur, TFe); genu, Ge; tibia, Ti; and tarsus, Ta. 1. Taxonomic Key to Genera for Known Larval Water Mites 1a. Legs with six movable segments, with basifemur (BFe) and telofemur (TFe) separated (Figs. 9, 21, 40, 48) .......................................2 1b. Legs with ﬁve movable segments, with basifemur and telofemur fused (Figs. 6 – 8, 57, 60, 67) ........................................................15 2a(1a). Gnathosoma with elaborate camerostome enclosing chelicerae, with pedipalps inserted ventrally (Figs. 11, 13, 14) ; dorsal plate present and bearing only two pairs of setae (verticils — ve and vi) near anterior edge (Figs. 11 and 13); coxal plates III located posteriorly on idiosoma with insertions of legs III located at posterolateral edges (posterior to level of excretory pore) and legs III directed posteriorly (Figs. 12, 15) .........................................................................................................................................Hydrovolzioidea 3 2b. Gnathosoma lacking elaborate camerostome; dorsal plate absent (Figs. 30, 36), or present and bearing more than two pairs of setae with verticils (ve and vi) anteriorly and at least internal scapulars (si) in posterior half near edge (Figs. 27, 29, 33); coxal plates III located near midlength of idiosoma with insertions of legs III located anterolaterally (anterior to level of excretory pore) and legs III directed laterally (Fig. 28) ..................................................................................................................................................................4 3a(2a). Dorsal plate relatively small (Fig. 11); venter with rows of numerous small urstigmata borne between coxal plates I and II (Fig. 12, arrow); coxal plates I and II separated from one another on each side and from opposite members medially (Fig. 12); pedipalps massive, with tibia (Ti) bearing four long, thick, curved setae (Fig. 10); setae c3 often foliate (Fig. 11)................................................ ................................................................................................................................................................Hydrovolziidae Hydrovolzia 3b. Dorsal plate relatively large (Fig. 13); venter with urstigmata either inconspicuous or absent (Fig. 15); coxal plates I and II fused together to form single anterior plate (Fig. 15); pedipalps moderate in size, with tibia (Ti) bearing two slightly thickened, straight setae (Fig. 14) .....................................................................................................................................Acherontacaridae Acherontacarus 4a(2b). Dorsal plate absent (Figs. 30, 36), or small, covering less than one-third length of idiosoma and usually with internal scapular setae (si) at posterior edge (Figs. 27, 29, 33); lateral eyes borne on separate platelets (Figs. 27, 29, 30, 36); leg tarsi with unmodiﬁed claws and empodium (emp) (Fig. 26)...............................................................................................................................Hydryphantoidea 5 4b. Dorsal plate large, covering well over one-third length of idiosoma and with internal scapular setae (si) near midlength (Figs. 37, 42, 46, 51); lateral eyes borne on single eye plates (Figs. 37, 42, 46); leg tarsi with claws modiﬁed or reduced (Figs. 41, 45, 50) ........ ........................................................................................................................................................................................Eylaoidea 11 5a(4a). Dorsal plate roughly quadrangular, bearing four pairs of setae (ve, vi, se, si) (Figs. 27, 33), or three pairs with external scapulars (se) borne on separate platelets near posterolateral angles of plate ...........................................................................................................6 5b. Dorsal plate triangular, fragmented, or absent, bearing fewer than three pairs of setae (Figs. 29, 30, 36)...........................................9 6a(5a). Claw (movable digit) of chelicerae over one-half length of basal segment (Fig. 17); excretory pore setae (ps1 and ps2) and their alveoli absent (Fig. 16) ..................................................................................................................................Thermacaridae Thermacarus 6b. Claw (movable digit) of chelicerae less than one-third length of basal segment (Fig. 25); excretory pore setae (ps1 and ps2) or at least their alveoli, present (Figs. 24, 28, 34) ...............................................................................................Hydryphantidae (in part) 7 7a(6b). Coxal plates II lacking setae (2b absent) (Fig. 20); urstigmata relatively large (Fig. 20, arrow); pedipalp tibia with terminal clawlike seta deeply bifurcate (Fig. 18, arrow) and tarsus with only one thickened seta; excretory pore plate absent but setae ps1 and ps2 present (Fig. 20)............................................................................................................................................... Wandesiinae Wandesia 16. Water Mites (Hydrachnida) and Other Arachnids 577 7b. Coxal plates II bearing setae 2b (Fig. 28); urstigmata relatively small (Fig. 28, arrow); pedipalp tibia with terminal clawlike seta only slightly bifurcate or undivided, and tarsus with two thickened setae (Fig. 19, arrows); excretory pore plate present; setae ps1 and ps2 present or absent, but their alveoli always present (Fig. 28) ..................................................................................................8 8a(7b). Medial eye present (Fig. 27, arrow) ........................................Thyadinae Panisus, Panisopsis, Thyas, Thyasides, Euthyas, Zschokkea 8b. Medial eye reduced (Fig. 33) ................................................................................................................Tartarothyadinae Tartarothyas 9a(5b). Dorsal plate triangular, bearing scapular setae (se and si), with verticil setae (ve and vi) borne on small platelets ﬂanking apex of plate (Fig. 29); excretory pore setae present, with ps1 borne on plate (Fig. 23) and ps2 borne on small separate platelets; basal segment of chelicerae massive and longitudinally striate (Fig. 25) ....................Hydryphantidae (in part) Hydryphantinae Hydryphantes 9b. Dorsal plate fragmented or absent, with verticil and scapular setae borne on paired platelets (Figs. 30, 36); excretory pore setae reduced to vestiges or absent, but their alveoli present (Figs. 24, 34); basal segment of chelicerae relatively slender and smooth (Fig. 27) ...........................................................................................................................................................................................10 10a(9b). Medial eye present (Fig. 30, arrow); excretory pore plate well sclerotized and nearly triangular (Fig. 24); pedipalp tarsus with all setae slender; solenidia on leg tarsi slender (Fig. 31, arrow) .............................................Hydryphantidae (in part) Protziinae Protzia 10b. Medial eye absent (Fig. 36); excretory pore plate poorly sclerotized and oblong (Fig. 34); pedipalp tarsus with two thickened, bladelike setae (Fig. 35, arrows); solenidia on tarsi of legs I and II very thick (Fig. 32, arrow) .......................Hydrodromidae Hydrodroma 11a(4b). Coxal plates I, II, and III on each side bearing two, two, and two setae, respectively (Figs. 38, 43); tarsi of legs bearing paired claws and clawlike empodium (emp)(Fig. 41) ............................................................................................................................................12 11b. Coxal plates I, II, and III on each side bearing two or one, one, and one setae, respectively (Figs. 44, 53); tarsi of legs bearing two dissimilar clawlike structures terminally (Fig. 45, 50) ..............................................................................................Limnocharidae 13 12a(11a). Dorsum of idiosoma nearly covered by single, elongate dorsal plate in unengorged larvae, bearing seven pairs of setae (or their alveoli) (Fig. 37); coxal plates with setae all simple (Fig. 38); excretory pore plate elongate (Fig. 39) .................................Eylaidae Eylais 12b. Dorsum of idiosoma with three separate plates bearing ﬁve, one, and one pairs of setae respectively (Fig. 42); coxal plates with setae 1a, 1b, 2b and 3b blunt and conical in shape (Fig. 43); excretory pore plate obcordate (Fig. 43) .........................Piersigiidae Piersigia 13a(11b). Dorsal plate covering no more than anterior half of idiosomal dorsum, bearing four pairs of setae (verticils and scapulars) (Figs. 46, 51); coxal plates I bearing two pairs of setae, 1a and 1b (Fig. 44); excretory pore plate diamond-shaped (Fig. 47) or nearly oval (Fig. 49) ...................................................................................................................................................................Limnocharinae 14 13b. Dorsal plate covering entire idiosomal dorsum in unengorged larvae, bearing seven pairs of setae, including verticils, scapulars and three pairs of hysterosomal setae (Fig. 52); coxal plates I bearing one pair of setae (Fig. 53); excretory pore plate pyriform (Fig. 53) .......................................................................................................................................Rhyncholimnocharinae Rhyncholimnochares 14a(13a). Dorsal plate (Fig. 46) ﬁnely punctate; excretory pore plate relatively small, little larger than excretory pore (Fig. 47); leg tarsi bearing two similar clawlike structures with empodium and single ambulacral claw both slender (Fig. 45) ............................Limnochares 14b. Dorsal plate (Fig. 51) strongly striate; excretory pore plate relatively large, much larger than excretory pore (Fig. 49); leg tarsi bearing two dissimilar clawlike structures, with empodium wider than single ambulacral claw (Fig. 50)...........................Neolimnochares 15a(1b). Dorsal plate bearing three pairs of setae (external verticils, scapulars) and an unpaired anteromedial seta (probably representing internal verticils) (Fig. 59, arrow); urstigmata (Ur) stalked (Figs. 59, 61); pedipalp tibiae with terminal clawlike seta four-pronged (Fig. 63, arrow), leg tarsi bearing paired pectinate claws and simple empodium (Fig. 60) .................................................................... .................................................................................................................Stygothrombidioidea Stygothrombidiidae Stygothrombium 15b. Dorsal plate bearing at least four pairs of setae (verticils and scapulars), and in some cases one or more additional pairs of hysterosomal setae, but no unpaired setae (Figs. 56, 64, 68); urstigmata sessile (Figs. 58, 65); pedipalp tibiae with terminal clawlike seta undivided (Fig. 66, arrow), or deeply bisected (Figs. 55, 71, arrows); leg tarsi bearing paired simple claws (Fig. 175) or lacking claws (Fig. 54), but always bearing a simple empodium ...................................................................................................................16 16a(15b). Dorsal plate bearing eight pairs of setae, including verticils, scapulars, c3, and three additional pairs of hysterosomal setae (Fig. 56); leg tarsi lacking paired claws (Fig. 54); excretory pore plate tiny, and bearing neither setae nor their alveoli (Fig. 58, arrow).............. ...........................................................................................................................................Hydrachnoidea Hydrachnidae Hydrachna 16b. Dorsal plate bearing four-to-six pairs of setae, including only the verticils and scapulars (Fig. 64) or, in some cases, the verticils, scapulars and one or two pairs of hysterosomal setae (Fig. 68); leg tarsi bearing paired claws (Fig. 143, 175); excretory pore plate small to large, always bearing setae ps1 and ps2 or at least their alveoli (Figs. 62, 80, 104, 109, 113 – 114, 172, 176, 185, 193) ....17 17a(16b). Coxal plates I, II, and III on each side all separate (Figs. 65, 69, 73, 172, 179, 180, 186), or all fused (Fig. 185) and plates of two sides fused medially and dorsal plate round and bearing setae c1 laterally (Fig. 184) .......................................................................18 17b. Coxal plates I and II on each side separate and plates II and III fused at least medially (Figs. 83, 106, 145, 157), or all plates on each side fused (Figs. 92, 100) but plates of two sides separate medially and dorsal plate elliptical and not bearing setae c1 (Figs. 91, 101, 144, 150, 153, 168) .................................................................................................................................................................34 578 Ian M. Smith, David R. Cook, Bruce P. Smith 18a(17a). Tarsi of legs III bearing 12 or more setae, including Ta8 (Fig. 8, arrow); dorsal plate usually elongate and elliptical and bearing only four pairs of setae (verticils and scapulars) (Figs. 3, 64, 74); when dorsal plate round and bearing ﬁve pairs of setae, including c1 laterally (Fig. 68), then coxal plates III bearing setae pa and h2 in addition to 3a (Fig. 69) and all setae on pedipalp tarsi short and bladelike (Fig. 71) ..........................................................................................................................................Lebertioidea (in part) 19 18b. Tarsi of legs III usually bearing 11 or fewer setae, lacking at least Ta8 (Figs. 174, 181, 196, 198); dorsal plate usually nearly round (Figs. 178, 184, 188, 190, 191), rarely elongate and elliptical (Fig. 171); when tarsi of legs III bearing 12 setae then dorsal plate round and bearing ﬁve pairs of setae, including c1 laterally (Fig. 195) and coxal plates III bearing only setae 3a (Fig. 193) and at least one seta on pedipalp tarsi long and whip-like (similar to Fig. 177) .....................................................................Arrenuroidea 23 19a(18a). Pedipalp tarsi with no setae as long as pedipalp (Fig. 71); tarsi of legs I, II, and III bearing 13 or 14 (1s), 13 or 14 (1s), and 12 setae respectively, lacking Ta15 ...................................................................................................................Anisitsiellidae (in part) 20 19b. Pedipalp tarsi with at least one seta as long as pedipalp (Figs. 2, 66); tarsi of legs I, II, and III bearing 15 (1s), 15 (1s), and 13 or 14 setae respectively, including Ta15 (Figs. 6 – 8, 67)........................................................................................................................21 20a(19a). Dorsal plate bearing six pairs of setae, including verticils, scapulars and both c1 and e1 on lateral edge, and with setae si long, thick, and located near midlength (Fig. 68); coxal plates III bearing three pairs of setae, including 3a and both pa and h2 near posterior edges (Fig. 69) ..........................................................................................................................................................Bandakiopsis 20b. Dorsal plate bearing four pairs of setae (verticils and scapulars), with setae si short, slender, and located in anterior third of plate (Fig. 70); coxal plates III bearing only setae 3a (Fig. 72) ........................................................................................................Utaxatax 21a(19b). Dorsal plate longitudinally striate (Figs. 3, 64); coxal plates III bearing only setae 3a (Figs. 4, 65); excretory pore plate small and usually quadrangular (Figs. 5, 65) ............................................................................................................................Sperchontidae 22 21b. Dorsal plate reticulate (Fig. 74); coxal plates III bearing two pairs of setae, 3a and supernumerary setae 3b (Fig. 73); excretory pore plate relatively large and diamond-shaped (Fig. 73) ............................................................................................Teutoniidae Teutonia 22a(21a). Coxal plates II bearing one pair of setae, 2b, posterolaterally (Fig. 65) .................................................................................Sperchon 22b. Coxal plates II lacking setae (Fig. 4)...............................................................................................................................Sperchonopsis 23a(18b). Coxal plates I, II, and III on each side all fused and coxal plates of two sides fused medially, with setae 1a, 2b and 3a reduced to vestiges (Fig. 185); pedipalp tarsi with no setae as long as pedipalp (Fig. 182); dorsal plate with setae si reduced to vestiges (Fig. 184) ....................................................................................................................................................Acalyptonotidae Acalyptonotus 23b. Coxal plates I, II, and III on each side all separate and coxal plates of two sides separate (Figs. 172, 186, 189, 192, 193); pedipalp tarsi with at least one seta as long as pedipalp (Fig. 177) .................................................................................................................24 24a(23b). Idiosoma moderately ﬂattened dorsoventrally; gnathosoma projecting beyond anterior edge of dorsal plate, entirely exposed in dorsal view (Figs. 171, 195); excretory pore plate variously shaped (Figs. 172, 193, 197) but usually not attenuate anteriorly; pedipalp tarsi with long, thick, distal seta straight or only slightly bowed basally; leg genua with setae Ge5 borne on tubercles that usually are prominent (Fig. 173, arrow) .......................................................................................................................................................25 24b. Idiosoma extremely ﬂattened dorsoventrally; gnathosoma recessed beneath anterior edge of dorsal plate, partially or entirely concealed in dorsal view (in unmounted specimens) (Fig. 188); excretory pore plate triangular with anterior apex attenuate (Figs. 176, 192); pedipalp tarsi with long, thick, distal seta strongly bowed, and usually lobed, basally (Figs. 177, 183, arrows); leg genua with setae Ge5 not borne on tubercles......................................................................................................................................................29 25a(24a). Dorsal plate bearing four pairs of setae (verticils and scapulars), with setae c1 on lateral membranous integument (Fig. 171); setae c2 and d2 thick and long relative to other hysterosomal setae (Fig. 171); excretory pore setae ps1 and ps2 absent, represented by their alveoli on excretory pore plate (Fig. 172); tibiae of legs I bearing eight setae (2s), including Ti11; leg tarsi lacking setae Ta14 (Figs. 173, 175) ...........................................................................................................................................................Momoniidae 26 25b. Dorsal plate bearing ﬁve pairs of setae, including the verticils, scapulars and setae c1 laterally (Fig. 195); setae c2 and d2 similar to other hysterosomal setae (Fig. 195); excretory pore setae ps1 and ps2 present on plate (Fig. 193, 197), tibiae of legs I bearing seven setae (2s), lacking Ti11; leg tarsi bearing setae Ta14 (Figs. 196, 198, arrows) ...............................................................................27 26a(25a). Legs with setae Tr1, Ge5, Ti9 and Ti11 much longer than respective segments; setae IITi9, IITi11, IIIGe5, IIITi9 and IIITi11 plumose (Fig. 173) ...........................................................................................................................................Momoniinae Momonia 26b. Legs with setae Tr1, Ge5, Ti9 and Ti11 shorter than respective segments; setae IITi9, IITi11, IIIGe5, IIITi9 and IIITi11 bladelike (Fig. 175) ........................................................................................................................................Stygomomoniinae Stygomomonia 27a(25b). Tarsi of legs III bearing nine setae, lacking Ta12 (Fig. 198); tarsi of legs II and III with setae Ta4 and Ta6 long (usually as long as respective segments) (Figs. 198); coxal plates I and II with conspicuous denticulate projections on posterior edges (Figs. 197, 199, arrows), coxal plates III with transverse muscle attachment scars (TMAS), excretory pore plate subtriangular (Figs. 197, 199) ......... ..............................................................................................................................................................................Krendowskiidae 28 27b. Tarsi of legs III bearing at least 11 setae, including Ta12 (Fig. 196); tarsi of legs II and III with setae Ta4 and Ta6 usually short (conspicuously shorter than respective segments) (Fig. 196); coxal plates I and II lacking denticulate projections on posterior edges, 16. Water Mites (Hydrachnida) and Other Arachnids 579 coxal plates III lacking transverse muscle attachment scars (TMAS), excretory pore plate variously shaped, but rarely triangular (Fig. 193) ..........................................................................................................................................................Arrenuridae Arrenurus 28a(27a). Coxal plates I and II each with row of denticulate projections along whole posterior edge (Fig. 197, arrows); TMAS weakly developed and short (Fig. 197); tubercles bearing setae f2 large, prominent, and medially attenuate (Fig. 197) ......................Krendowskia 28b. Coxal plates I and II each bearing only few, denticulate projections in medial third of posterior edge (Fig. 199); TMAS strongly developed and long (Fig. 199); tubercles bearing setae f2 not unusually prominent (Fig. 199) ......................................................Geayia 29a(24b). Dorsal plate bearing ﬁve pairs of setae, including verticils, scapulars and setae c1 laterally (Fig. 188) .............Mideopsidae Mideopsis 29b. Dorsal plate bearing four pairs of setae (verticils and scapulars), with setae c1 on lateral membranous integument (Figs. 178, 190) .........................................................................................................................................................................................................30 30a(29b). Tibiae of legs II and III bearing nine setae (2s and 1s, respectively), including both Ti10 and Ti11 (Figs. 174, 181) ...................31 30b. Tibiae of legs II and III bearing fewer than nine setae (2s and 1s, respectively), lacking either Ti10 or Ti11, or both (Fig. 187, 194) .................................................................................................................................................................................................32 31a(30a). Pedipalp tarsi with long, thick, distal seta bowed, but not deeply lobed or fringed basally, and with most medial seta moderately thick, but not fringed (Fig. 183); legs II and III with setae Ge5, Ti9 and Ti11 much longer than respective segments and plumose, and setae Ta4 and Ta6 much longer than respective segments (Fig. 181).....................................................................Mideidae Midea 31b. Pedipalp tarsi with long, thick, distal seta bowed, deeply lobed and fringed basally, and most medial seta thick and fringed (Fig. 177); legs II and III with setae Ge5, Ti9 and Ti11 shorter than respective segments and simple, and setae Ta4 and Ta6 shorter than respective segments (Fig. 174) ...............................................................................Nudomideopsidae Nudomideopsis, Paramideopsis 32a(30b). Tibiae of legs II bearing seven setae (2s), lacking Ti10 and Ti11 (Fig. 194); tibiae of legs III bearing eight setae (1s), lacking Ti10 .............................................................................................................................................................................Laversiidae Laversia 32b. Tibiae of legs II and III bearing eight setae (2s and 1s respectively), including Ti10, but lacking Ti11 (Fig. 187) ........................33 33a(32b). Dorsum with setae si located well in anterior half of plate and setae c1 located in lateral integument near midlength of plate, posterior to level of setae si (Fig. 191) ..............................................................................................Neoacaridae Neoacarus, Volsellacarus 33b. Dorsum with setae si near midlength of plate and setae c1 located in lateral integument near setae c3, anterior to level of setae si (Fig. 190) ...............................................................................Athienemanniidae Athienemanniinae Chelomideopsis, Platyhydracarus 34a(17b). Coxal plates III bearing setae 3a and at least one other pair of setae (pa or 3b) and excretory pore plate bearing only setae ps1 and ps2 (Figs. 83, 86, 90, 92) ..............................................................................................................................Lebertioidea (in part) 35 34b. Coxal plates III usually bearing only one pair of setae, 3a (Figs. 80, 97, 100, 145, 152), when coxal plates III also bearing setae pa, then excretory pore plate bearing setae h2 in addition to ps1 and ps2 (Figs. 104, 159 – 163, 166) ...................................................38 35a(34a). Coxal plates III truncate posteriorly, bearing three pairs of setae, including 3a and both pa and h2 on posterior edges (Fig. 83); tibiae of legs I, II, and III bearing nine setae (2s, 2s, and 1s, respectively), including both Ti9 and Ti10 (Fig. 82); tarsi of legs I and II bearing 14 setae (1s), including Ta14 and Ta15 (Fig. 82); cheliceral bases separate (Fig. 81, arrow) ....................................... ...........................................................................................................................................................Lebertiidae Lebertia, Estelloxus 35b. Coxal plates III rounded or pointed posteriorly, bearing two pairs of setae, including setae 3a and either 3b laterally or pa posteromedially (Figs. 86, 90, 92); tibiae of legs I, II, and III bearing eight setae (2s, 2s, and 1s, respectively), lacking Ti9 or Ti10 (Figs. 85, 88, 89); tarsi of legs I and II bearing 13 setae (1s), lacking Ta15 (Fig. 88), or 12 setae (1s), lacking both Ta15 and Ta14 (Fig. 85); cheliceral bases fused (Figs. 84, 87, 91) ....................................................................................................................36 36a(35b). Dorsal plate bearing ﬁve pairs of setae including verticils, scapulars and setae c1 laterally; integument beneath posterior edge of dorsal plate intricately folded (Fig. 84, arrow); coxal plates I elongate, extending posteriorly nearly to level of excretory pore plate; coxal plates III bearing setae 3b laterally (Fig. 86); tarsi of legs I and II bearing 12 setae (1s), lacking Ta14 (Fig. 85) ....................... ....................................................................................................................................................................Oxidae Oxus, Frontipoda 36b. Dorsal plate bearing four pairs of setae (verticils and scapulars), or ﬁve pairs including verticils, scapulars and setae d1 laterally; integument beneath posterior edge of dorsal plate not intricately folded (Figs. 87, 91); coxal plates I not elongate, extending posteriorly only to level of insertion of legs III (Fig. 90), or fused with plates II (Figs. 92); plates III bearing setae pa posteromedially (Figs. 90, 92); tarsi of legs I and II bearing 13 setae (1s), including Ta14 (Fig. 88) ........................................................Torrenticolidae 37 37a(36b). Dorsal plate bearing ﬁve pairs of setae including verticils, scapulars and setae d1 laterally, and with setae se long and thick (Fig. 87); coxal plates I clearly delineated by complete suture lines, and plates II and III fused with suture line incomplete medially (Fig. 90); excretory pore plate with convex projection posteromedially (Fig. 90); tibiae of all legs lacking setae Ti10, genua of legs III bearing four setae (1s) including Ge3, and tarsi of legs III bearing 11 setae, lacking Ta10 (Fig. 89)...................Testudacarinae Testudacarus 37b. Dorsal plate bearing only four pairs of setae (verticils and scapulars), and with setae se relatively short and slender (Fig. 91); coxal plates of each side all fused, with suture lines obliterated except laterally (Fig. 92); excretory pore plate without projection posteromedially (Fig. 92); tibiae of all legs lacking setae Ti9, genua of legs III bearing three setae (1s), lacking Ge3, tarsi of legs III bearing 12 setae, including Ta10 (Fig. 93).............................................................................................................Torrenticolinae Torrenticola 580 Ian M. Smith, David R. Cook, Bruce P. Smith 38a(34b). Cheliceral bases separate (Fig. 78, arrow); tarsi of legs III bearing 12 setae, including Ta9 (Fig. 79, arrow); excretory pore plate very large (equal in width to coxal plates of one side), bearing setae ps1 and ps2 anteromedially and occasionally also setae h2 at posterolateral angles (Figs. 75, 80), and coxal plates I separate from posterior coxal groups, and coxal plates III bearing only setae 3a (Fig. 80)......................................................................Lebertioidea (in part) Anisitsiellidae (in part) Anisitsiellinae (in part) Bandakia 38b. Cheliceral bases fused (Figs. 101, 107, 108, 144, 150); tarsi of legs III usually bearing 11 or fewer setae, lacking Ta9 (Fig. 132) (exception Limnesiidae); excretory pore plate small to very large, when equal in width to coxal plates of one side (measured from midline to insertion of leg III) (Figs. 100, 103, 104, 152, 164) then either excretory pore located near posterior edge of plate (Fig. 152, arrow), or coxal plates I fused to posterior coxal groups (Fig. 100), or coxal plates III bearing setae pa in addition to setae 3a (Figs. 164) .........................................................................................................................................................................Hygrobatoidea 39 39a(38b). Two pairs of urstigmata borne distally between coxal plates I and II (Figs. 97, arrows A; Fig. 98, arrows); tibiae of legs I and II bearing seven or eight setae (2s), including only three ventral setae, lacking either Ti8 and Ti9 or Ti10 and Ti11 (Figs. 95, 99) ....... .....................................................................................................................................................................................Limnesiidae 40 39b. One pair of urstigmata borne distally between coxal plates I and II (Figs. 100, arrow, 106, 111, 133, 164); tibiae of legs I and II usually bearing nine or more setae (2s), including ﬁve ventral setae (Ti7, Ti8, Ti9, Ti10, and Ti11) (Figs. 102, 141, 149) (exception Feltriidae)..................................................................................................................................................................................41 40a(39a). Coxal plates I and II completely separate (Fig. 98); tarsi of legs III bearing nine setae, lacking Ta9, Ta13 and Ta14 (Fig. 99); dorsal plate extending laterally well beyond level of eye plates (contrast with Fig. 94)....................................................Tyrrelliinae Tyrrellia 40b. Coxal plates I and II on each side fused, with suture lines obliterated medially (Fig. 97, arrow B); tarsi of legs III bearing 12 setae, including Ta9, Ta13 and Ta14 (Fig. 96); dorsal plate narrow, usually conﬁned to region between eye plates (Fig. 94) ......................... ...........................................................................................................................................................................Limnesiinae Limnesia 41a(39b). Coxal plates I, II, and III on each side all fused (Fig. 100)..........................................................................................Hygrobatidae 42 41b. Coxal plates I separate from posterior coxal group on each side (Figs. 106, 111, 145, 152, 169) ....................................................43 42a(41a). Excretory pore plate bearing three pairs of setae, including ps1 and ps2 medially and h2 near anterolateral angles, with setae pa on posterior edges of coxal plates III (Fig. 104) .......................................................................................................................Hygrobates 42b. Excretory pore plate bearing four pairs of setae, including ps1 and ps2 medially, pa near anterior edge, and h2 near lateral angles (Figs. 100, 103) ....................................................................................................................................................................Atractides 43a(41b). Tarsi of legs I and II bearing 10 setae (1s), lacking Ta14, and either Ta12 (Fig. 105) or Ta9 (Figs. 112, 116) ................................44 43b. Tarsi of legs I and II bearing 11 or more setae (1s), including Ta9 (Figs. 128, 170, arrows; Figs. 147, 151, arrows A) ..................47 44a(43a). Dorsal and coxal plates, and leg sclerites, conspicuously longitudinally striate (as in Fig. 107); coxal plates III rounded posteriorly (Fig. 106); tibiae of legs I bearing seven setae (2s), lacking Ti10 and Ti11 (Fig. 105) ..............................................Feltriidae Feltria 44b. Dorsal and coxal plates reticulate (Fig. 108); coxal plates III bearing pointed projections posteriorly (Fig. 111, arrow); tibiae of legs I bearing 9 setae (2s), including Ti10 and Ti11 (Fig. 112, 116) ..............................................................................Unionicolidae 45 45a(44b). Tarsi of legs with empodium undivided (Fig. 115) .......................................................................................................Pionatacinae 46 45b. Tarsi of legs with empodium bifurcate (Fig. 110, arrow) .............................................................................Unionicolinae Unionicola 46a(45a). Excretory pore plate usually oval in shape with excretory pore borne near anterior edge (Fig. 114) ....................................Neumania 46b. Excretory pore plate obcordate with excretory pore borne near middle of plate (Fig. 113)....................................................Koenikea 47a(43b). Tibiae of all legs bearing seven setae (2s, 2s, and 1s respectively), lacking Ti9 and Ti11 (Fig. 170) ..................Aturidae (in part) ...................................................................................................................................................................................Aturinae Aturus 47b. Tibiae of all legs bearing eight or more setae (2s, 2s, and 1s, respectively), including Ti9 and Ti11 (Fig. 151).........................48 48a(47b). Coxal plates III with pointed or lobed projections posteriorly, bearing two pairs of setae, 3a anteromedially and pa posteromedially (Figs. 156, 164); excretory pore plate large, bearing three pairs of setae, ps1 and ps2, and h2 posterolaterally (Figs. 159 – 163, 166) .........................................................................................................................................Aturidae (in part) Axonopsinae (in part) 49 48b. Coxal plates III without, or with small, projections posteriorly, bearing only setae 3a (Figs. 117, 145, 152, 154); excretory pore plate small or large, bearing only setae ps1 and ps2 (Figs. 118, 123, 135, 138, 142, 146)................................................................52 49a(48a). Tarsi of legs I and II bearing 12 setae, including setae Ta8 (Fig. 158); excretory pore plate with setae ps1 at or near anterior edge (Figs. 159, 160) ................................................................................................................................................................................50 49b. Tarsi of legs I and II bearing 11 setae, lacking setae Ta8 (Fig. 167); excretory pore plate with setae ps1 near anterior edge to posterior to midlength (Figs. 162, 163, 166) ............................................................................................................................................51 50a(49a). Excretory pore plate subtriangular (Fig. 159) with acutely rounded posterolateral angles, with setae ps2 anterior to midlength between setae ps1 and h2; dorsal plate not reticulate; coxal plates I weakly fused medially with plates II, and with setae 1a long, extending posteriorly beyond level of setae 3a (Fig. 157) ......................................................................................................Estellacarus 16. Water Mites (Hydrachnida) and Other Arachnids 581 50b. Excretory pore plate obcordate (Fig. 160) or nearly quadrangular (Fig. 161), with broadly rounded posterolateral angles, and with setae ps2 posterior to midlength, not between setae ps1 and h2; dorsal plate reticulate; coxal plates I separate from plates II, and with setae 1a relatively short, not extending posteriorly beyond level of setae 3a (Fig. 156) ...........................................Woolastookia 51a(49b). Suture line between coxal plates II and III long, extending medially to near base of setae 3a (Fig. 164); excretory pore plate either moderately large and subtriangular (Fig. 162) or very large and subquadrangular (Fig. 163) ............................................Brachypoda 51b. Suture line between coxal plates II and III short, not extending medially to region of base of setae 3a (Fig. 165); excretory pore plate moderately large and broadly elliptical or subquadrangular (Fig. 166) ................................................................................Axonopsis 52a(48b). Coxal plates III with small projections posteriorly (Fig. 117, arrow); excretory pore plate small, little larger than combined area of excretory pore and bases of setae ps1 and ps2 (Fig. 118)........................................................................................Wettinidae Wettina 52b. Coxal plates III without projections posteriorly (Figs. 124, 133, 137, 140, 145, 152, 154); excretory pore plate considerably larger than combined area of excretory pore and bases of setae ps1 and ps2 (Figs. 123, 125, 129, 138, 142, 152) ....................................53 53a(52b). Tarsi of legs I bearing 13 setae (1s), including Ta8 (Figs. 147, 151, arrows B); excretory pore plate large, and usually triangular or obcordate (Figs. 138, 139, 148, 152) ...............................................................................................................................................54 53b. Tarsi of legs I bearing 12 setae (1s), lacking Ta8 (Fig. 128); excretory pore plate small to large, and variously shaped (Figs. 119, 121, 123, 125, 134, 135) .................................................................................................................................................................58 54a(53a). Excretory pore plate with setae ps1 close together adjacent to excretory pore in posterior half of plate (Figs. 138, 139, 142, 146, 148) ....................................................................................................................................................................Pionidae (in part) 55 54b. Excretory pore plate with setae ps1 widely separated and removed from excretory pore in anterior half of plate (Fig. 154, arrow) ...........................................................................................................................................................................Aturidae (in part) 57 55a(54a). Tarsi of legs II bearing 13 setae (1s), including Ta8 (Fig. 141); tibiae of legs with setae Ti9 and Ti11 simple (Fig. 141) .................... .....................................................................................................................................................................Najadicolinae Najadicola 55b. Tarsi of legs II usually bearing 12 setae (1s), lacking Ta8 (Fig. 149), when bearing 13 setae then tibiae of legs with Ti9 and Ti11 long and plumose (as in Fig. 149) .....................................................................................................................................Pioninae 56 56a(55b). Femora of legs II with Fe5 shorter than segment; excretory pore plate usually much less than twice as wide as long, when nearly twice as wide as long then plate is concave or transverse anteromedially (Fig. 146) ....................................................................Piona 56b. Femora of legs II with Fe5 longer than segment; excretory pore plate twice as wide as long and convex anteromedially (Fig. 139) ........................................................................................................................................................................................Nautarachna 57a(54b). Excretory pore plate broadly obcordate (Fig. 152) ..................................................................................Axonopsinae (in part) Ljania 57b. Excretory pore plate triangular (Fig. 154) ......................................................................................................................Albiinae Albia 58a(53b). Excretory pore plate small and subtriangular, with setae ps2 borne near posterolateral angles of plate (Fig. 155); suture lines between coxal plates II and III parallel to anterior edge of plate II, coxal plates without distinct lateral coxal apodemes and coxal plates III lacking medial coxal apodemes and transverse muscle attachment scars (Fig. 155) ....................................Aturidae (in part) ................................................................................................................................................Axonopsinae (in part) Neobrachypoda 58b. Excretory pore plate variously shaped, when subtriangular with setae ps2 borne near posterolateral angles of plate then suture lines between coxal plates II and III terminating in distinct lateral coxal apodemes (LCA) that are usually nearly transverse and coxal plates III bearing at least medial coxal apodemes (MCA) (Figs. 120, 124, 126, 129, 133, 136)...........................Pionidae (in part) 59 59a(58b). Suture line between coxal plates II and III on each side extending medially beyond lateral coxal apodeme nearly to midline (Fig. 120, arrow); excretory pore borne on elevated projection which extends to or beyond posterior edge of plate (Figs. 119, 121) ......................................................................................................................................................Hydrochoreutinae Hydrochoreutes 59b. Suture line between coxal plates II and III on each side extending medially only as far as lateral coxal apodeme (Figs. 122, 124, 126, 129, 133, 136); excretory pore usually not borne on elevated projection, when on small projection this does not extend to posterior edge of plate .....................................................................................................................................................................................60 60a(59b). Trochanters of legs III bearing two setae, including supernumerary dorsal seta (Fig. 131, arrow), or tibiae of legs II and III bearing 11 or 12 setae (2s and 1s, respectively), including two supernumerary posteroventral setae (Fig. 132, arrows), or coxal plates III lacking transverse muscle attachment scars and excretory pore plate with setae ps2 near posterior edge of plate (Fig. 136) .......................................................................................................................................................................................Foreliinae 61 60b. Trochanters of legs III bearing only one seta; tibiae of legs II and III bearing only nine setae (2s and 1s, respectively) (as in Figs. 141, 149); coxal plates III usually with transverse muscle attachment scars (Figs. 124, 126, 133), when coxal plates lacking these scars (as in Fig. 122) then excretory pore plate with setae ps2 removed from posterior edge of plate (Fig. 123)...............................62 61a(60a). Trochanters of legs III bearing two setae, including supernumerary dorsal seta (Fig. 131, arrow) ....................................Pseudofeltria 61b. Trochanters of legs III bearing one seta......................................................................................................................................Forelia 582 Ian M. Smith, David R. Cook, Bruce P. Smith 62a(60b). Excretory pore plate transversely elliptical or semicircular (Fig. 123) ...........................................................Huitfeldtiinae Huitfeldtia 62b. Excretory pore plate broadly elliptical, subcircular, or subtriangular ...............................................................................Tiphyinae 63 63a(62b). Excretory pore plate three times as wide as long and broadly elliptical or dumbbell shaped (Figs. 124, 125) .......................Neotiphys 63b. Excretory pore plate less than twice as wide as long and subcircular (Figs. 127, 130) to subtriangular.....................Pionopsis, Tiphys FIGURES 2–5 Sperchonopsis ecphyma Prasad and Cook (Sperchontidae), larva. Fig. 2, Venter of gnathosoma; Fig. 3, dorsum of idiosoma and gnathosoma; Fig. 4, venter of idiosoma; Fig. 5, excretory pore plate. See text for nomenclature and abbreviations of setae. 16. Water Mites (Hydrachnida) and Other Arachnids FIGURES 6–8 Sperchonopsis ecphyma Prasad and Cook (Sperchontidae), larva, anterolateral views of legs. Fig. 6, Leg I; Fig 7, Leg. II; and Fig. 8, Leg III. Fe, Femur; Ge, genu; Ta, tarsus; Ti, tibia; Tr, trochanter. 583 584 Ian M. Smith, David R. Cook, Bruce P. Smith FIGURES 9–12 Hydrovolzia gerhardi Mitchell (Hydrovolziidae), larva. Fig. 9, Leg I, anterolateral view; Fig. 10, distal segments of pedipalp; Fig. 11, dorsum of idiosoma and gnathosoma; Fig 12, venter of idiosoma. FIGURES 13–15 Acherontacarus sp. (Acherontacaridae), larva. Fig. 13, Dorsum of idiosoma and gnathosoma; Fig. 14, venter of gnathosoma; Fig. 15, venter of idiosoma. 16. Water Mites (Hydrachnida) and Other Arachnids FIGURES 16–17 Thermacarus nevadensis Marshall (Thermacaridae), larva. Fig. 16, Excretory pore plate; Fig 17, chelicera. FIGURE 18, 20 Wandesia sp. (Wandesiinae), larva. Fig. 18, Tibia and tarsus of pedipalp; Fig. 20, venter of idiosoma and gnathosoma. FIGURES 19, 21 – 22, 26 – 28 Thyas stolli Koenike (Thyadinae), larva. Fig. 19, Tarsus of pedipalp; Fig. 21, leg I; Fig. 22, pedipalp; Fig. 26, claws and empodium; Fig. 27, dorsum of idiosoma and gnathosoma; Fig. 28, venter of idiosoma and gnathosoma. FIGURES 23, 25, 29 Hydryphantes ruber (de Geer) (Hydryphantinae), larva. Fig. 23, Excretory pore plate; Fig. 25, chelicerae; Fig. 29, dorsum of idiosoma and gnathosoma. FIGURE 24 Protzia sp. (Protziinae), larva, excretory pore plate. 585 586 Ian M. Smith, David R. Cook, Bruce P. Smith FIGURES 30 – 31 Protzia sp. (Protziinae), larva. Fig. 30, Prodorsal region of idiosoma; Fig. 31, tibia and tarsus of leg I. FIGURES 32, 34 – 36 Hydrodroma despiciens (Müller) (Hydrodromidae), larva. Fig. 32, Tarsus of leg I; Fig. 34, excretory pore plate; Fig. 35, tarsus of pedipalp; Fig. 36, dorsum of idiosoma and gnathosoma. FIGURE 33 Tartarothyas sp. (Tartarothyadinae), larva, dorsal plate. FIGURES 37 – 41 Eylais major Lanciani (Eylaidae), larva. Fig. 37, Dorsum of idiosoma and gnathosoma; Fig. 38, venter of idiosoma and gnathosoma; Fig. 39, excretory pore plate; Fig. 40, leg I; Fig. 41, claws and empodium of leg I. 16. Water Mites (Hydrachnida) and Other Arachnids FIGURES 42 – 43 Piersigia sp. (Piersigiidae), larva. Fig. 42, Dorsum of idiosoma; Fig. 43, venter of idiosoma and gnathosoma. FIGURES 44 – 48 Limnochares americana Lundblad (Limnocharinae), larva. Fig. 44, venter of idiosoma and gnathosoma; Fig. 45, claw and empodium of leg I; Fig. 46, dorsum of idiosoma and gnathosoma; Fig. 47, excretory pore plate; Fig. 48, leg I. FIGURES 49 – 51 Neolimnochares sp. (Limnocharinae), larva. Fig. 49, Excretory pore plate; Fig. 50, claw and empodium of leg I; Fig. 51, dorsal plate. 587 588 Ian M. Smith, David R. Cook, Bruce P. Smith FIGURES 52 – 53 Rhyncholimnochares kittatinniana Habeeb (Rhyncholimnocharinae), larva. Fig. 52, Dorsum of idiosoma and gnathosoma; Fig. 53, venter of idiosoma. FIGURES 54 – 58 Hydrachna magniscutata Marshall (Hydrachnidae), larva. Fig. 54, Empodium of leg I; Fig. 55, distal segment of pedipalp; Fig. 56, dorsum of idiosoma and gnathosoma; Fig. 57, leg I; Fig. 58, venter of idiosoma and gnathosoma. 16. Water Mites (Hydrachnida) and Other Arachnids FIGURES 59 – 61, 63 Stygothrombium sp. (Stygothrombidiidae), larva. Fig. 59, Dorsum of idiosoma and gnathosoma; Fig. 60, leg I; Fig. 61, venter of idiosoma; Fig. 63, tibia and tarsus of pedipalp. FIGURES 62, 64 – 67 Sperchon glandulosus Koenike (Sperchontidae), larva. Fig. 62, Excretory pore plate; Fig. 64, dorsum of idiosoma and gnathosoma; Fig. 65, venter of idiosoma; Fig. 66, pedipalp; Fig. 67, leg I. 589 590 Ian M. Smith, David R. Cook, Bruce P. Smith FIGURES 68 – 69, 71 Bandakiopsis fonticola Smith (Anisitsiellidae), larva. Fig. 68, Dorsum of idiosoma and gnathosoma; Fig. 69, venter of idiosoma; Fig. 71, pedipalp. FIGURES 70,72 Utaxatax newelli Habeeb (Anisitsiellidae), larva. Fig. 70, dorsum of idiosoma and gnathosoma; Fig. 72, venter of idiosoma. FIGURES 73, 74 Teutonia lunata Marshall (Teutoniidae), larva. Fig. 73, Venter of idiosoma; Fig. 74, dorsum of idiosoma and gnathosoma. 16. Water Mites (Hydrachnida) and Other Arachnids FIGURES 75 – 80 Bandakia phreatica Cook (Anisitsiellidae), larva. Fig. 75, Excretory pore plate; Fig. 76, pedipalp; Fig. 77, tibia and tarsus of leg I; Fig. 78, dorsum of idiosoma and gnathosoma; Fig. 79, tibia and tarsus of leg III. Fig. 80, venter of idiosoma. FIGURES 81 – 83 Lebertia sp. (Lebertiidae), larva. Fig. 81, Dorsum of idiosoma and gnathosoma; Fig. 82, tibia and tarsus of leg I; Fig. 83, venter of idiosoma. 591 592 Ian M. Smith, David R. Cook, Bruce P. Smith FIGURES 84 – 86 Oxus elongatus Marshall (Oxidae), larva. Fig. 84, Dorsum of idiosoma and gnathosoma; Fig. 85, tibia and tarsus of leg I; Fig. 86, venter of idiosoma. FIGURES 87 – 90 Testudacarus sp. (Testudacarinae), larva. Fig. 87, Dorsum of idiosoma and gnathosoma; Fig. 88, tibia and tarsus of leg I; Fig. 89, distal segments of leg III; Fig. 90, venter of idiosoma. 16. Water Mites (Hydrachnida) and Other Arachnids FIGURES 91 – 93 Torrenticola sp. (Torrenticolidae), larva. Fig. 91, dorsum of idiosoma and gnathosoma; Fig. 92, venter of idiosoma; Fig. 93, distal segments of leg III. FIGURES 94 – 97 Limnesia marshalliana Lundblad (Limnesiinae), larva. Fig. 94, dorsum of idiosoma and gnathosoma; Fig. 95, tibia and tarsus of leg II; Fig. 96, tibia and tarsus of leg III; Fig. 97, venter of idiosoma and gnathosoma. 593 594 Ian M. Smith, David R. Cook, Bruce P. Smith FIGURES 98 – 99 Tyrrellia sp. (Tyrrelliinae), larva. Fig. 98, Venter of idiosoma; Fig. 99, tibia and tarsus of leg III. FIGURES 100 – 101, 103 Atractides grouti Habeeb (Hygrobatidae), larva. Fig. 100, Venter of idiosoma and gnathosoma; Fig. 101, dorsum of idiosoma and gnathosoma; Fig. 103, excretory pore plate. FIGURE 102 Hygrobates sp. (Hygrobatidae), larva, tibia and tarsus of leg II. FIGURE 104 Hygrobates neocalliger Habeeb (Hygrobatidae), larva, excretory pore plate region. FIGURES 105 – 107 Feltria sp. (Feltriidae), larva. Fig. 105, Tibia and tarsus of leg I; Fig. 106, venter of idiosoma; Fig. 107, dorsum of idiosoma and gnathosoma. 16. Water Mites (Hydrachnida) and Other Arachnids FIGURES 108 – 112 Unionicola sp. (Unionicolinae), larva. Fig. 108, Dorsum of idiosoma and gnathosoma; Fig. 109, excretory pore plate; Fig. 110, claws and empodium of leg I; Fig. 111, venter of idiosoma and gnathosoma; Fig. 112, tibia and tarsus of leg I. FIGURE 113 Koenikea marshallae Viets (Pionatacinae), larva, excretory pore plate. FIGURES 114 – 116 Neumania punctata Marshall (Pionatacinae), larva. Fig. 114, excretory pore plate; Fig. 115, claws and empodium of leg I; Fig. 116, tibia and tarsus of leg I. 595 596 Ian M. Smith, David R. Cook, Bruce P. Smith FIGURES 117 – 118 Wettina ontario Smith (Wettinidae), larva. Fig. 117, Venter of idiosoma; Fig. 118, excretory pore plate. FIGURES 119 – 120 Hydrochoreutes intermedius Cook (Hydrochoreutinae), larva. Fig. 119, Excretory pore plate; Fig. 120, venter of idiosoma and gnathosoma. FIGURE 121 Hydrochoreutes minor Cook (Hydrochoreutinae), larva, excretory pore plate. FIGURES 122 – 123 Huitfeldtia rectipes Thor (Huitfeldtiinae), larva. Fig. 122, Venter of idiosoma; Fig. 123, excretory pore plate. FIGURES 124 – 125 Neotiphys pionoidellus (Habeeb) (Tiphyinae), larva. Fig. 124, Venter of idiosoma; Fig. 125, excretory pore plate. 16. Water Mites (Hydrachnida) and Other Arachnids FIGURES 126 – 127 Tiphys americanus (Marshall) (Tiphyinae), larva. Fig. 126, Venter of idiosoma and gnathosoma; Fig. 127, excretory pore plate. FIGURES 128, 130 Tiphys ornatus (Koch) (Tiphyinae), larva. Fig. 128, Tibia and tarsus of leg I; Fig. 130, excretory pore plate. FIGURES 129, 131 Pseudofeltria multipora Cook (Foreliinae), larva. Fig. 129, Venter of idiosoma; Fig. 131, trochanter of leg III. FIGURES 132 – 134 Forelia ovalis Marshall (Foreliinae), larva. Fig. 132, Tibia and tarsus of leg III; Fig. 133, venter of idiosoma and gnathosoma; Fig. 134, excretory pore plate. FIGURES 135, 136 Forelia onondaga Habeeb (Foreliinae), larva. F ig. 135, excretory pore plate; Fig. 136, venter of idiosoma. 597 598 Ian M. Smith, David R. Cook, Bruce P. Smith FIGURES 137 – 138, 141 Najadicola ingens Koenike (Najadicolinae), larva. Fig. 137, Venter of idiosoma; Fig. 138, excretory pore plate; Fig. 141, tibia and tarsus of leg II. FIGURES 139, 140 Nautarachna muskoka Smith (Pioninae), larva. Fig. 139, Excretory pore plate; Fig. 140, venter of idiosoma. FIGURES 142, 143 Piona carnea (Koch) (Pioninae), larva. Fig. 142, excretory pore plate; Fig. 143, claws and empodium of leg III. FIGURES 144, 145, 148 Piona interrupta Marshall (Pioninae), larva. Fig. 144, dorsum of idiosoma and gnathosoma; Fig. 145, venter of idiosoma and gnathosoma; Fig. 148, excretory pore plate. FIGURE 146 Piona constricta (Wolcott) (Pioninae), larva, excretory pore plate. FIGURES 147, 149 Piona mitchelli Cook (Pioninae), larva. Fig. 147, tarsus of leg I; Fig. 149, tibia and tarsus of leg II. 16. Water Mites (Hydrachnida) and Other Arachnids FIGURES 150 – 152 Ljania bipapillata Thor (Axonopsinae), larva. Fig. 150, Dorsum of idiosoma and gnathosoma; Fig. 151, tibia and tarsus of leg I; Fig. 152, venter of idiosoma. FIGURES 153–154 Albia neogaea Habeeb (Albiinae), larva. Fig. 153, Dorsum of idiosoma and gnathosoma; Fig. 154, venter of idiosoma. FIGURE 155 Neobrachypoda ekmani (Walter) (Axonopsinae), larva, venter of idiosoma. 599 600 Ian M. Smith, David R. Cook, Bruce P. Smith FIGURES 156,160 Woolastookia setosipes Habeeb (Axonopsinae), larva. Fig. 156, Venter of idiosoma; Fig. 160, excretory pore plate. FIGURES 157–159 Estellacarus unguitarsus (Habeeb) (Axonopsinae), larva. Fig. 157, Venter of idiosoma, Fig. 158, tibia and tarsus of leg I; Fig. 159, excretory pore plate. FIGURE 161 Woolastookia pilositarsa (Habeeb) (Axonopsinae), larva, excretory pore plate. FIGURE 162 Brachypoda cornipes Habeeb (Axonopsinae), larva, excretory pore plate. FIGURE 163 Brachypoda setosicauda Habeeb (Axonopsinae), larva, excretory pore plate. 16. Water Mites (Hydrachnida) and Other Arachnids FIGURE 164 Brachypoda setosicauda Habeeb (Axonopsinae), larva, venter of idiosoma. FIGURES 165–167 Axonopsis setoniensis Habeeb (Axonopsinae), larva. Fig. 165, venter of idiosoma; Fig. 166, excretory pore plate; Fig. 167, tibia and tarsus of leg I. FIGURES 168–170 Aturus sp. (Aturinae), larva. Fig. 168, dorsum of idiosoma and gnathosoma; Fig. 169, venter of idiosoma; Fig. 170, tibia and tarsus of leg I. 601 602 Ian M. Smith, David R. Cook, Bruce P. Smith FIGURES 171 – 172, 175 Stygomomonia mitchelli Smith (Stygomomoniinae), larva. Fig. 171, dorsum of idiosoma and gnathosoma; Fig. 172, venter of idiosoma; Fig. 175, distal segments of leg III. FIGURE 173 Momonia campylotibia Smith (Momoniinae), larva, distal segments of leg III. FIGURES 174, 177 Paramideopsis susanae Smith (Nudomideopsidae), larva. Fig. 174, tibia and tarsus of leg III; Fig. 177, pedipalp. FIGURE 176 Nudomideopsis magnacetabula (Smith), larva, excretory pore plate. 16. Water Mites (Hydrachnida) and Other Arachnids FFIGURES 178 – 179 Nudomideopsis magnacetabula (Smith) (Nudomideopsidae), larva. Fig. 178, dorsum of idiosoma and gnathosoma; Fig. 179, venter of idiosoma. FIGURES 180–181, 183 Midea expansa Marshall (Mideidae), larva. Fig. 180, venter of idiosoma; Fig. 181, tibia and tarsus of leg III; Fig. 183, venter of gnathosoma. FIGURES 182, 184–185 Acalyptonotus neoviolaceus Smith (Acalyptonotidae), larva. Fig. 182, pedipalp; Fig. 184, dorsum of idiosoma and gnathosoma; Fig. 185, venter of idiosoma. 603 604 Ian M. Smith, David R. Cook, Bruce P. Smith FIGURES 186, 188 Mideopsis borealis Habeeb (Mideopsinae), larva. Fig. 186, venter of idiosoma; Fig. 188, dorsum of idiosoma and gnathosoma. Figures 187, 189–190 Platyhydracarus juliani Smith (Athienemanniinae), larva. Fig. 187, tibia and tarsus of leg II; Fig. 189, venter of idiosoma; Fig. 190, dorsum of idiosoma and gnathosoma. FIGURE 191 Neoacarus occidentalis Cook (Neoacaridae), larva, dorsum of idiosoma and gnathosoma. FIGURE 192 Laversia berulophila Cook (Larversiidae), larva, venter of idiosoma. 16. Water Mites (Hydrachnida) and Other Arachnids FIGURES 193, 195 – 196 Arrenurus planus Marshall (Arrenurinae), larva. Fig. 193, venter of idiosoma; Fig. 195, dorsum of idiosoma and gnathosoma; Fig. 196, tibia and tarsus of leg III. FIGURE 194 Laversia berulophila Cook (Laversiidae), larva, tibia and tarsus of leg II. FIGURES 197–198 Krendowskia similis Viets (Krendowskiidae), larva. Fig. 197, venter of idiosoma; Fig. 198, tibia and tarsus of leg III. 605 606 Ian M. Smith, David R. Cook, Bruce P. Smith 2. Taxonomic Key to Genera for Adult Water Mites 1a. Legs with tarsi bearing empodium and paired claws (Fig. 201); pedipalps with tarsus bearing single, long terminal seta (Fig. 202, arrow); idiosoma soft and elongate.......................................................Stygothrombidioidea Stygothrombidiidae Stygothrombium 1b. Legs with tarsi bearing paired claws, but lacking empodium; pedipalps with tarsus lacking single long terminal seta as above; idiosoma various ............................................................................................................................................................................2 2a(1b). Genital ﬁeld with movable genital ﬂaps, but with genital acetabula borne on coxal plates (Fig. 205, arrow) ......Hydrovolzioidea 3 2b. Genital ﬁeld with or without movable genital ﬂaps; when genital ﬂaps present, genital acetabula borne on them (Fig. 200) or beneath them (Fig. 283) .....................................................................................................................................................................4 3a(2a). Dorsal shield with large central plate surrounded by smaller unpaired plate anteriorly and numerous pairs of small similar platelets laterally and posteriorly (Fig. 204); glandularia represented by setae, with gland portions absent ...................................... ....................................................................................................................................................Acherontacaridae Acherontacarus 3b. Dorsal shield with large central plate surrounded by smaller unpaired plate anteriorly and several pairs of small disimilar plates laterally and posteriorly (Fig. 206); glandularia divided into gland and seta bearing sclerites (Fig. 203)........................................... ............................................................................................................................................................Hydrovolziidae Hydrovolzia 4a(2b). Idiosoma nearly spherical (Fig. 207); pedipalps with tibia much shorter than genu and bearing a distidorsal projection extending well beyond insertion of tarsus (Fig. 209); chelicerae 1-segmented (Fig. 208)................................................................................... .......................................................................................................................................Hydrachnoidea Hydrachnidae Hydrachna 4b. Idiosoma variously shaped, but rarely spherical; pedipalps with tibia usually longer than genu, when shorter then lacking a distidorsal projection; chelicera 2-segmented (Fig. 316) .................................................................................................................5 5a(4b). Lateral eye capsules borne on medial prodorsal plate (Figs. 210, 211, 212) or, in one family, on moderately large, rugose, paired platelets associated with complex of six prodorsal sclerites (Fig. 213)............................................................................Eylaoidea 6 5b. Lateral eye capsules, when present, relatively small and not borne on prodorsal plate (Figs. 220, 224)........................................11 6a(5a). Dorsum with lateral eyes borne on lateral platelets separated by a medial triangular platelet (Fig. 213, arrows); idiosoma bearing 26 pairs of glandularia on large, rugose platelets (Figs. 213, 214); gnathosoma with capitulum wider than long and pedipalps with only one movable segment which ﬂexes medially to oppose anetrior edge of capitulum (Fig. 214)........................................... .............................................................................................................................Apheviderulicidae Apheviderulix (deutonymphs) 6b. Dorsum with lateral eyes borne on a medial prodorsal plate (Fig. 210, 211, 212); idiosoma bearing fewer than 26 pairs of glandularia on small sclerites; gnathosoma with capitulum longer than wide and pedipalps with 3 to 5 movable segments which ﬂex ventrally .........................................................................................................................................................................................7 7a(6b). Lenses of lateral eyes well separated from each other on their respective sides and borne laterally on entire prodorsal plate bearing one pair of setae (Fig. 210) .................................................................................................................................Eylaidae Eylais 7b. Lenses of lateral eyes relatively close together on their respective sides and borne laterally on smooth, entire prodorsal plate bearing four pairs of setae (Figs. 211, 212) ...........................................................................................................................................8 8a(7b). Prodorsal plate approximately as wide as long (Fig. 212); genital acetabula surrounded by acetabular plates.................................. .........................................................................................................................................................................Piersigiidae Piersigia 8b. Prodorsal plate much longer than wide (Fig. 211); genital acetabula scattered in ventral integument ....................Limnocharidae 9 9a(8b). Gnathosoma protrusible; pedipalps three segmented, with tarsus very small and inserted dorsally on tibia (Fig. 215, arrow) ...................................................................................................................................Rhyncholimnocharinae Rhyncholimnochares 9b. Gnathosoma not protrusible; pedipalps 4- or 5-segmented, with tarsus inserted at distal end of tibia (Figs. 216, 217) .................... ............................................................................................................................................................................Limnocharinae 10 10a(9b). Pedipalps 4-segmented (Fig. 217)............................................................................................................................Neolimnochares 10b. Pedipalps 5-segmented (Fig. 216) .................................................................................................................................Limnochares 11a(5b). Dorsum with a series of reticulate platelets as shown in Figure 218 ................................................................................................ ..........................................................................................................Rhynchohydracaridae Clathrosperchoninae Clathrosperchon 11b. Dorsum various, but not with an arrangement of platelets as shown in Figure 218......................................................................12 12a(11b). Pedipalps chelate, with dorsodistal portion of tibia extending well beyond insertion of tarsus (Figs. 221, 227, arrows); capitulum without anchoral process (as in Fig. 342, arrow)..........................................................................................................................13 12b. Pedipalps not chelate, when appearing chelate (as in some Tiphyinae) (Fig. 332) then capitulum with well-developed anchoral process (Fig. 330, arrow A) ..........................................................................................................................................................38 13a(12a). Pedipalps with dorsodistal extension of tibia relatively long (Fig. 221, arrow)...................................Hydrodromidae Hydrodroma 16. Water Mites (Hydrachnida) and Other Arachnids 607 13b. Pedipalps with dorsodistal extension of tibia relatively short (Fig. 227, arrow) .................................................Hydryphantidae 14 14a(13b) Legs with swimming setae on distal segments...............................................................................................................................15 14b. Legs without swimming setae ......................................................................................................................................................16 15a(14a). Dorsum with medial eye plate between lateral eyes bearing a pigmented medial eye and two pairs of setae (Figs. 219, 220) ........... ........................................................................................................................................................Hydryphantinae Hydryphantes 15b. Dorsum without medial eye plate between lateral eyes ................................................Pseudohydryphantinae Pseudohydryphantes 16a(14b). Third and fourth coxal plates with medial margins extensive and close together, not separated by genital ﬁeld (Fig. 223); lateral eyes in capsules; body not elongated......................................................................................................Cowichaniinae Cowichania 16b. Posterior coxal groups usually well separated from each other, with genital ﬁeld extending between them (Fig. 237), when these coxal plates close together then lateral eyes not in capsules and body greatly elongated (Fig. 226) ..............................................17 17a(16b). Lateral eyes in capsules (Fig. 224, arrow).....................................................................................................................................18 17b. Lateral eyes, when present, not in capsules...................................................................................................................................37 18a(17a). Dorsum without dorsalia (Figs. 224, 230)....................................................................................................................................19 18b. Dorsum with dorsalia of various sizes and arrangement (Figs. 232, 234, 245, 256) .....................................................................22 19a(18a). Genital ﬁeld bearing three pairs of acetabula ................................................................................................Thyadinae (in part) 20 19b. Genital ﬁeld bearing numerous acetabula (Fig. 228) ....................................................................................................Protziinae 21 20a(19a) Dorsum bearing well-developed medial eye containing two dots of pigment (Fig. 225); genital ﬁeld with setiferous extensions of genital ﬂaps extending anterior to ﬁrst pair of acetabula (Fig. 229, arrow)...................................................................Notopanisus 20b. Dorsum lacking medial eye (Fig. 230); genital ﬁeld without setiferous extensions of genital ﬂaps extending anterior to ﬁrst pair of acetabula (Fig. 231)......................................................................................................................................................Albertathyas 21a(19b). Legs with tarsal claws bearing palmately arranged clawlets (Fig. 222) ..................................................................................Protzia 21b. Legs with tarsal claws simple ............................................................................................................................................Partnunia 22a(18b). Dorsum with all dorsalia peripheral in position (Fig. 232, arrow) .........................................................Cyclothyadinae Cyclothyas 22b. Dorsum with some dorsalia more medial in position (Figs. 245, 256)...........................................................Thyadinae (in part) 23 23a(22b). Dorsum with single, large anterior plate, but without other dorsalia (Fig. 233) ..........................................................Siskiyouthyas 23b. Dorsum with or without single, large anterior plate, but always bearing other paired dorsalia ....................................................24 24a(23b). Dorsum with large anterior plate, and with two medial and four lateral pairs of platelets (Fig. 234).............................Trichothyas 24b. Dorsum with dorsalia variously developed and fused, but not as above .......................................................................................25 25a(24b). Dorsum with medial eye borne on spindle-shaped sclerite (Fig. 235, arrow); genital ﬁeld with second pair of acetabula located about halfway between ﬁrst and third pairs (Figs. 236, 237) ...............................................................................................Euthyas 25b. Dorsum with medial eye not borne on spindle-shaped sclerite; genital ﬁeld with second pair of acetabula usually located in posterior half of gonopore ...................................................................................................................................................................26 26a(25b). Dorsum with dorsalia fused into single dorsal shield (i.e., unsclerotized integument surrounds dorsoglandularia, but does not occur between dorsalia) (Figs. 238, 240, 241)..................................................................................................................................27 26b. Dorsum with dorsalia not fused into single dorsal shield (i.e., unsclerotized integument separates dorsalia) (Figs. 242, 245).......29 27a(26a). Genital ﬁeld with ﬂaps not extending anterior to ﬁrst pair of acetabula, and second pair of acetabula located beside third pair (Fig. 239) ..........................................................................................................................................................................Thyopsis 27b. Genital ﬁeld with ﬂaps extending anterior to ﬁrst pair of acetabula, and second pair of acetabula located well anterior to third pair (Figs. 244, 246).....................................................................................................................................................................28 28a(27b). Dorsal shield well sclerotized, with some of thickened areas indicating medial dorsalia and adjacent lateral dorsalia fused on each side (Fig. 240, arrows) (edges of dorsalia obscured by secondary sclerotization in mature specimens); genital ﬁeld with setiferous projections of genital ﬂaps medial to third pair of acetabula relatively small (Fig. 244) ..................................................Thyopsella 28b. Dorsal shield weakly sclerotized, with thickened areas indicating medial and lateral dorsalia separate (Fig. 241); genital ﬁeld with setiferous projections of genital ﬂaps medial to third pair of acetabula relatively large (Fig. 246).................................Thyopsoides 29a(26b). Dorsum with most posterior pair of dorsalia fused into single large plate (Figs. 242, 243, arrows)..............................................30 29b. Dorsum with most posterior pair of dorsalia separated medially (Figs. 245, 248, 250) ................................................................31 608 Ian M. Smith, David R. Cook, Bruce P. Smith 30a(29a). Dorsum with central region bearing three pairs of dorsalia that are separated medially (Fig. 242) .......................................Panisus 30b. Dorsum with central region bearing two pairs of dorsalia that are fused medially (Fig. 243)....................................Columbiathyas 31a(29b). Genital ﬁeld with well sclerotized extensions of genital ﬂaps extending anterior to ﬁrst pair of acetabula (Figs. 247, 249, arrows).........................................................................................................................................................................................32 31b. Genital ﬁeld without sclerotized extensions of genital ﬂaps anterior to ﬁrst pair of acetabula (Fig. 253) ......................................34 32a(31a). Dorsum with lateral eye capsules raised and located dorsally on idiosoma (Fig. 245, arrow A) and with medial eye well developed (Fig. 245, arrow B); genital ﬁeld with setae on anterior extensions of genital ﬂaps relatively thick (Fig. 247, arrow) ........................................................................................................................................................................................Panisopsis 32b. Dorsum with lateral eye capsules relatively ﬂat and located at anterior edge of idiosoma (Fig. 248, arrow) and with medial eye indistinct or absent (Figs. 248); genital ﬁeld with setae on anterior extension of genital ﬂaps relatively slender (Fig. 249, arrow) ..........................................................................................................................................................................................33 33a(32b). Dorsum with four pairs of medial dorsalia posterior to anteromedial plate (Fig. 248).................................................Parathyasella 33b. Dorsum with ﬁve pairs of medial dorsalia posterior to anteromedial plate (Fig. 251)........................................................Thyasella 34a(31b). Dorsum with dorsalia large and occupying most of the dorsal area of the idiosoma (Fig. 250) ................................Amerothyasella 34b. Dorsum with dorsalia small and occupying only a small part of the dorsal area of the idiosoma (Figs. 252, 256)........................35 35a(34b). Dorsum with medial eye bearing two pigment dots and usually borne on small platelet formed by fused pre — and postocularia (Fig. 252, arrow)...............................................................................................................................................................Thyasides 35b. Dorsum with medial eye unpigmented or with dispersed pigment, and not borne on small platelet (Fig. 256) .............................35 36a(35b). Genital ﬁeld with posterior two pairs of acetabula not incorporated into edges of genital ﬂaps (Fig. 253)..............................Thyas 36b. Genital ﬁeld with posterior two pairs of acetabula fused with edges of genital ﬂaps (Fig. 257) .......................................Zschokkea 37a(17b). Body not greatly elongated; genital ﬂaps well developed (Fig. 254, arrow).......................................Tartarothyadinae Tartarothyas 37b. Body greatly elongated (Fig. 226); genital ﬂaps poorly developed or absent..................................................Wandesiinae Wandesia 38a(12b). Femur of pedipalp with two long medial setae (Fig. 255, arrow); occuring only in hot springs ...........Thermacaridae Thermacarus 38b. Femur of pedipalp usually without two medial setae and never as illustrated in Figure 255; not occurring in hot springs............39 39a(38b). Genital ﬁeld with movable genital ﬂaps ﬂanking gonopore and either partially or completely covering gonopore when closed; genital acetabula lying free in gonopore (not on ﬂaps or acetabular plates) (Figs. 259, 277, 283) ...........................Lebertioidea 40 39b. Genital ﬁeld usually without movable genital ﬂaps, when ﬂaps present then acetabula lie on ﬂaps rather than in gonopore (Fig. 200) .....................................................................................................................................................................................59 40a(39a). Fourth coxal plates with medial edges reduced to narrow angles and bearing a pair of glandularia (Fig. 264, arrow); tarsus of pedipalp bearing pad-shaped setae and appearing spatulate distally (Fig. 263, arrow) ............................Rutripalpidae Rutripalpus 40b. Fourth coxal plates with medial edges usually extensive, when reduced to narrow angles then lacking glandularia as above.......41 41a(40b). Dorsal and ventral shields present; dorsal shield usually comprising a large plate and a series of smaller platelets (Figs. 273, 276, 278); genital ﬁeld with gonopore usually bearing six pairs of acetabula (Fig. 279), when only three pairs of genital acetabula present then dorsal shield with complete ring of marginal platelets (Fig. 273); one known North American species has platelets fused into an entire dorsal shield, but six pairs of genital acetabula are diagnostic .......................................................Torrenticolidae 42 41b. Dorsal and ventral shields present or absent; when present, dorsal shield entire, without smaller platelets; genital ﬁeld with gonopore bearing three pairs of acetabula............................................................................................................................................46 42a(41a). Dorsal shield with single anteromedial platelet and series of small marginal platelets (Fig. 273); genital ﬁeld with gonopore bearing three pairs of genital acetabula ......................................................................................................Testudacarinae Testudacarus 42b. Dorsal shield with one or two pairs of anterior platelets (Figs. 276, 278) (in one species platelets are fused with large central plate); genital ﬁeld with gonopore bearing six pairs of acetabula.........................................................................Torrenticolinae 43 43a(41b). Pedipalps 4-segmented (Fig. 275)................................................................................................................................Neoatractides 43b. Pedipalps 5-segmented (as in Fig. 274) ........................................................................................................................................44 44a(43b). Gnathosoma protrusible, capitulum long and slender(Fig. 280)..........................................................................Pseudotorrenticola 44b. Gnathosoma not protrusible, capitulum relatively short and stocky (Fig. 281).............................................................................45 45a(44b). Capitulum with short proximodorsal projections (Fig. 281, arrow) .............................................................................Torrenticola 45b. Capitulum with long proximodorsal projections (Fig. 282, arrow) .........................................................................Monoatractides 16. Water Mites (Hydrachnida) and Other Arachnids 609 46a(41b). Fourth coxal plates bearing glandularia (Fig. 265, arrow)...............................................................................Teutoniidae Teutonia 46b. Fourth coxal plates lacking glandularia (Figs. 271, 283) ..............................................................................................................47 47a(46b). Venter without medial suture line separating coxal plates (Fig. 289); all legs inserted near anterior end of idiosoma (Fig. 289) ........................................................................................................................................................................................Oxidae 48 47b. Venter with medial suture line separating coxal plates of either side (Fig. 283, arrow A); fourth pair of legs inserted laterally near middle of idiosoma (Fig. 283, arrow C) .......................................................................................................................................49 48a(47a). Idiosoma strongly compressed laterally with coxal plates extending onto dorsal surface leaving only narrow medial unsclerotized strip of soft integument that usually bears small sclerites (Fig. 291); sexual dimorphism not pronounced ......................Frontipoda 48b. Idiosoma ovoid or moderately compressed laterally, without small sclerites in dorsal integument (Fig. 288); sexual dimorphism usually not pronounced, but in some species coxal plates extend more dorsally in males .......................................................Oxus 49a(47b). Venter with suture lines between second and third coxal plates incomplete (Fig. 283, arrow B), but touching the genital ﬁeld area posteriorly; genu of pedipalp usually with at least ﬁve long setae on medial surface (Fig. 284), but, in one known species, with four long setae on dorsal surface ...............................................................................................................................Lebertiidae 50 49b. Venter with suture lines between second and third coxal plates complete (Fig. 268) and these coxal plates often completely separated on their respective sides (Fig. 259); genu of pedipalp without long setae ......................................................................52 50a(49a). Idiosoma strongly compressed laterally (Fig. 285)............................................................................................................Estelloxus 50b. Idiosoma ovoid or dorsoventrally ﬂattened ..................................................................................................................................51 51a(50b). Dorsum covered by complete dorsal shield (Fig. 286); pedipalps with femur bearing several long setae dorsally and genu bearing four long setae dorsally (Fig. 287)................................................................................................................................Scutolebertia 51b. Dorsum usually covered by soft integument, rarely covered by dorsal shield; pedipalps with femur bearing few short setae and genu bearing ﬁve or six long setae on medial surface (Fig. 284) ..........................................................................................Lebertia 52a(49b). Dorsum and venter covered by complete shields...................................................................................................Anisitsiellidae 53 52b. Dorsum and venter with sclerites variously developed, but complete dorsal and ventral shields never both present ......................... ..............................................................................................................................................................................Sperchontidae 58 53a(52a). Tarsi of fourth pair of legs lacking claws (Fig. 267)....................................................................................................Mamersellides 53b. Tarsi of fourth pair of legs bearing claws (Fig. 266) .....................................................................................................................54 54a(53b). Fourth pair of legs inserted at anterolateral angles of fourth coxal plates, just posterior to insertions of third pair of legs (Fig. 268, arrow) ......................................................................................................................................................................Utaxatax 54b. Fourth pair of legs inserted near posterolateral angles of fourth coxal plates, well posterior to insertions of third pair of legs (Fig. 271, arrow A) ......................................................................................................................................................................55 55a(54b). Accessory glandularia (“Glandula Limnesiae”) located in concavities of anteromedial edges of fourth coxal plates (Fig. 269, arrow) .........................................................................................................................................................................Bandakiopsis 55b. Accessory glandularia located on ﬁrst or third coxal plates ..........................................................................................................56 56a(55b). Accessory glandularia located near anterior angles of ﬁrst coxal plates (Fig. 270, arrow)..............................................Cookacarus 56b. Accessory glandularia located on third coxal plates .....................................................................................................................57 57a(56b). Accessory glandularia located near medial edges of third coxal plates (Fig. 271, arrow B); fourth coxal plates without or with only small projections associated with insertions of fourth pair of legs..............................................................................Bandakia 57b. Accessory glandularia located near lateral edges of third coxal plates (Fig. 272, arrow A); fourth coxal plates with large projections associated with insertions of fourth pair of legs (Fig. 272, arrow B) ..................................................................Oregonacarus 58a(52b). Idiosoma with glandularia borne on smooth platelets that are usually small and ﬂat, rarely enlarged and raised (Fig. 258); pedipalps with tibia usually bearing two peglike setae ventrally (Fig. 260, arrows)............................................................Sperchon 58b. Idiosoma with glandularia borne on tuberculate or papillate platelets that are enlarged and raised (Fig. 261); pedipalps with tibia lacking peg-like setae ventrally (Fig. 262)...................................................................................................................Sperchonopsis 59a(39b). All coxal plates grouped together, with medial edges of anterior coxal groups noticeably longer than those of posterior coxal groups (Fig. 292); fourth coxal plates rounded posteriorly and suture lines between third and fourth plates extending posterolaterally (Fig. 292)...................................................................................................................................Omartacaridae Omartacarus 610 Ian M. Smith, David R. Cook, Bruce P. Smith 59b. Coxal plates not as described and illustrated above......................................................................................................................60 60a(59b). Pedipalps with femur usually bearing single ventral seta (Fig. 296, 306, arrows), when this seta absent then tarsi of pedipalps with curved terminal seta (Fig. 297, arrow); genital ﬁeld with movable genital ﬂaps which cover gonopore when closed in females (Fig. 200) and some males ........................................................................................................................................Limnesiidae 61 60b. Pedipalps with femur lacking ventral setae or, rarely, bearing three setae (Fig. 414, arrow); genital ﬁeld without movable genital ﬂaps which cover gonopore when closed......................................................................................................................................69 61a(60a). Pedipalps with tibia bearing ﬁve or more long, pointed ventral tubercles and a long medial seta (Fig. 296); idiosoma lacking dorsal and ventral shields, although dorsal plates present ...............................................................Kawamuracarinae Kawamuracarus 61b. Pedipalps with tibia bearing four long, pointed tubercles (Fig. 295) or lacking tubercles (Fig. 298), and lacking a long medial seta; idiosoma with dorsal and ventral shields..............................................................................................................................62 62a(61b). Fourth pair of legs with tarsus lacking claws................................................................................................................................63 62b. Fourth pair of legs with tarsus bearing claws ...............................................................................................................................67 63a(62a). Genital ﬁeld with genital ﬂaps on each side of gonopore divided into anterior and posterior sclerites (Fig. 299); dorsum with large, transversely divided dorsal shield (Fig. 294) ............................................................................................Neomamersiinae 64 63b. Genital ﬁeld with genital ﬂaps of females (and acetabular plates of males) entire on each side (Fig. 200); dorsum usually without dorsal shield, but rarely with entire (undivided) shield..............................................................................................Limnesiinae 66 64a(63a). Ventral shield with sclerotized bridge forming dorsal side of camerostome incomplete (Fig. 304, arrow A); venter with posterior region bearing several small platelets (Fig. 304, arrow B) or at least a small excretory pore platelet separate from ventral shield; genital ﬁeld of both sexes with middle pair or group of acetabula borne on posterior sclerites of genital ﬂaps (Fig. 300); pedipalps with femur and tibia relatively long and slender and tibia lacking ventral tubercles (Fig. 298)........................................Meramecia 64b. Ventral shield with sclerotized bridge forming dorsal side of camerostome complete (Fig. 302, arrow A); ventral shield entire posteriorly (Figs. 302, 303); genital ﬁeld of males with middle pair or group of acetabula borne on anterior sclerites of genital ﬂaps (Fig. 301), or on pair of separate, small, medial sclerites (Fig. 299); pedipalps not as above ........................................................65 65a(64b). Pedipalps with femur bearing ventral seta, tibia bearing four long ventral tubercles and tarsus lacking long, curved distal seta (Fig. 295).....................................................................................................................................................................Neomamersa 65b. Pedipalps with femur lacking ventral seta, tibia lacking ventral tubercles and tarsus bearing long, curved distal seta (Fig. 297, arrow) .........................................................................................................................................................................Arizonacarus 66a(65b). Genital ﬁeld of males immediately ﬂanked by pair of glandularia (Fig. 305).............................................................Centrolimnesia 66b. Genital ﬁeld of males not immediately ﬂanked by pair of glandularia ................................................................................Limnesia 67a(62b). Dorsum nearly covered by two large plates (Fig. 307); pedipalps with femur bearing ventral seta inserted directly on surface of segment (as in Fig. 296, arrow)........................................................................................................Protolimnesiinae Protolimnesia 67b. Dorsum entirely covered by dorsal shield (Fig. 310) or less than half covered by small platelets (Fig. 311); pedipalps with femur bearing ventral seta inserted on prominent tubercle (Fig. 306, arrow) .......................................................................Tyrrelliinae 68 68a(67b). Third coxal plates with pair of accessory glandularia (“Glandula Limnesiae”) (Fig. 308, arrow); genital ﬁeld with numerous small acetabula (Fig. 308)..............................................................................................................................................Neotyrrellia 68b. Third coxal plates lacking glandularia (Fig. 309); genital ﬁeld with 3 – 7 pairs of relatively large acetabula (Fig. 309) ........Tyrrellia 69a(60b). Integument between fourth coxal plates and genital ﬁeld with two pairs of glandularia arranged more or less in a row, and either free (Fig. 312, arrows) or variously fused with other ventral sclerites; dorsal shield absent, or present and bearing fewer than ﬁve pairs of glandularia.................................................................................................................................................Feltriidae Feltria 69b. Integument between fourth coxal plates and genital ﬁeld usually without two pairs of glandularia arranged in a row, when glandularia in this position (some Koenikea) then dorsal shield bearing six pairs of glandularia (Fig. 322) ........................................70 70a(69b). Idiosoma strongly compressed laterally and much higher than wide (Fig. 314); fourth legs with all segments ﬂattened (Fig. 313) .....................................................................................................................................................Frontipodopsidae Frontipodopsis 70b. Idiosoma not strongly compressed laterally..................................................................................................................................71 71a(70b). Both dorsal and ventral shields present; ﬁrst pair of legs with tibia lacking two thickened setae and a downturned seta distoventrally (ie. not as shown in Fig. 321) ................................................................................................................................72 71b. Either dorsal or ventral shield, or both, usually absent, when both shields present (males of some Atractides) then ﬁrst pair of legs with tarsus bowed and tibia bearing two thickened setae and a downturned seta distoventrally (Fig. 321) .........................125 16. Water Mites (Hydrachnida) and Other Arachnids 611 72a(71a). First leg with tarsus much shorter than tibia, and claws dorsal in position and ﬂexing proximally (Fig. 381, arrow) ....................... ................................................................................................................................................................................Momoniidae 73 72b. First leg with distal segments not modiﬁed as described and illustrated above; claws terminal in position and ﬂexing ventrally...75 73a(72a). Dorsal shield composed of large plate surrounded by numerous smaller platelets (Fig. 380) ......Cyclomomoniinae Cyclomomonia 73b. Dorsal shield entire or comprising large anterior and posterior plates, but not surrounded by smaller platelets (ie. not as in Fig. 380) .............................................................................................................................................................................................74 74a(73b). Venter with pair of glandularia located close to anterior end of genital ﬁeld (Fig. 382, arrow); genital ﬁeld of males with acetabula borne in gonopore..................................................................................................................................Momoniinae Momonia 74b. Venter without glandularia near anterior end of genital ﬁeld (Fig. 383); genital ﬁeld of males with acetabula borne on plates ﬂanking gonopore (Fig. 383)........................................................................................................Stygomomoniinae Stygomomonia 75a(72b). Dorsal shield large and transversely divided (somewhat as in Fig. 294); suture lines between third and fourth coxal plates noticeably looped around a pair of glandularia (Fig. 293, arrow) ................................................Hygrobatidae (in part) Diamphidaxona 75b. Dorsal shield usually entire, never transversely divided as above; suture lines between third and fourth coxal plates not looped as above ...........................................................................................................................................................................................76 76a(75b). Venter with outer edges of coxal plates forming a smooth arc which does not extend beyond edge of idiosoma (Fig. 389, arrow); pedipalps highly modiﬁed (Fig. 390)...............................................Chappuisididae (in part) Uchidastygacarinae Uchidastygacarus 76b. Venter with outer edges of coxal plates not forming a smooth arc; pedipalps not as described and illustrated above ...................77 77a(76b). Venter with pair of glandularia located near midline at junction of third and fourth coxal plates (Fig. 388, arrow)......................... ....................................................................................................................Chappuisididae (in part) Chappuisidinae Chappuisides 77b. Venter without glandularia in position described and illustrated above ........................................................................................78 78a(77b). Ventral shield with suture lines between third and fourth coxal plates oblique, extending posteromedially to genital ﬁeld region and well separated from each other medially (Fig. 395, arrow); genital acetabula borne in single rows on each side, either in gonopore (males) or on slender plates ﬂanking gonopore (females) (Fig. 395) .........................................................Neoacaridae 79 78b. Ventral shield not with above combination of characters .............................................................................................................80 79a(78a). Pedipalps with tibia moderately produced distoventrally to oppose tarsus (Fig. 396) ......................................................Neoacarus 79b. Pedipalps with tibia and tarsus highly modiﬁed to form chelate appendage (Fig. 397) .................................................Volsellacarus 80a(78b). Ventral shield with coxoglandularia I shifted far forward in coxal plates II, near suture lines between ﬁrst and second coxal plates (Figs. 384 – 387, arrows)...............................................................................................................................................................81 80b. Ventral shield with coxoglandularia I located between second and third coxal plates (Fig. 407, arrow) or in posterior edges of second coxal plates (Fig. 406, arrow A) .......................................................................................................................................84 81a(80a). Ventral shield with genital ﬁeld projecting between, and widely separating, fourth coxal plates (Fig. 384) .............Mideidae Midea 81b. Ventral shield with genital ﬁeld located posterior to fourth coxal plates (Fig. 386) .........................................Nudomideopsidae 82 82a(81b). Genital ﬁeld with three pairs of acetabula (Fig. 385)................................................................................................Nudomideopsis 82b. Genital ﬁeld with more than three pairs of acetabula ...................................................................................................................83 83a(82b). Genital ﬁeld with four-to-six pairs of acetabula arranged in single rows (Fig. 386)....................................................Paramideopsis 83b. Genital ﬁeld with more than six pairs of acetabula arranged in several rows (Fig. 387) .............................................Neomideopsis 84a(80b). Pedipalps distinctly uncate (Fig. 401; Fig. 399, arrow A; Fig. 416, arrow) ...................................................................................85 84b. Pedipalps not distinctly uncate, with tibia bearing either slight distoventral projection (Fig. 417, arrow), or distinct ventral projection near middle of segment (Fig. 405), or no ventral projection..............................................................................................91 85a(84a). Ventral shield with fourth coxal plates bearing pair of glandularia (Fig. 413, arrow); genital ﬁeld extending between fourth coxal plates, widely separating them medially, and usually bearing three or four pairs of genital acetabula (but with ﬁve or six pairs in a few species) ........................................................................................................................................................Krendowskiidae 86 85b. Ventral shield with fourth coxal plates lacking glandularia; genital ﬁeld extending at most only slightly between fourth coxal plates, not separating them medially, and bearing numerous acetabula (Fig. 402) ........................................................................87 86a(85a). Gnathosoma protrusible........................................................................................................................................................Geayia 612 Ian M. Smith, David R. Cook, Bruce P. Smith 86b. Gnathosoma not protrusible........................................................................................................................................Krendowskia 87a(85b). Genital ﬁeld with some or all acetabula lying free in gonopore (Fig. 402), or with acetabula lying on plates which ﬂank gonopore, but are not fused with ventral shield (similar to Fig. 384); capitulum with a pair of long ventral setae (Fig. 401, arrow); tibia of pedipalp rotated relative to genu (up to 90°).......................................................................................Athienemanniidae 88 87b. Genital ﬁeld with acetabula borne on acetabular plates incorporated into ventral shield, never in gonopore (Fig. 415); capitulum with all setae short; tibia of pedipalp not greatly rotated relative to genu (Fig. 416); posterior end of idiosoma variously (and often highly) modiﬁed in males (Figs. 419, 420)...........................................................................Arrenuridae Arrenurinae Arrenurus 88a(87a). Pedipalp with genu bearing pronounced ventral projection (Fig. 399, arrow B); genital ﬁeld with acetabula in one or two rows on each side of gonopore (Fig. 398)..................................................................................................Stygameracarinae Stygameracarus 88b. Pedipalp with genu lacking pronounced ventral projection (Figs. 401); genital ﬁeld with acetabula in one to many rows on each side of gonopore (Figs. 402 – 404) ...................................................................................................................Athienemanniinae 89 89a(88b). Venter with coxal plates I extending anteriorly beyond edge of ventral shield (Fig. 404); genital ﬁeld of males with acetabula borne in one or two rows on each side of gonopore (Fig. 404) ..............................................................................Chelohydracarus 89b. Venter with coxal plates I not extending anteriorly beyond edge of ventral shield (Fig. 402); genital ﬁeld of males with acetabula borne in three or more rows on each side of gonopore (Figs. 400, 402) .......................................................................................90 90a(89b). Genital ﬁeld of males with all acetabula borne in gonopore (Fig. 402) ....................................................................Chelomideopsis 90b. Genital ﬁeld of males with acetabula borne both in gonopore and on acetabular plates ﬂanking gonopore (Fig. 400) ...............................................................................................................................................................................Platyhydracarus 91a(84b). Fourth pair of legs with tarsus indented or curved, but not greatly expanded, and bearing short peglike setae (Fig. 339) ..........................................................................................................................................Pionidae (in part) Foreliinae (in part) 92 91b. Fourth pair of legs with tarsus not as described and illustrated above ..........................................................................................94 92a(91a). Third pair of legs with tarsus and claws similar in shape to those of ﬁrst two pairs of legs ..............................Pseudofeltria (males) 92b. Third pair of legs with tarsus and claws modiﬁed, different from those on ﬁrst two pairs of legs (Fig. 340).................................93 93a(92b). Genital ﬁeld with three pairs of acetabula (Fig. 336) ................................................................Pionacercus (males of some species) 93b. Genital ﬁeld with more than three pairs of acetabula (Fig. 338) .......................................................Forelia (males of some species) 94a(91b). Fourth pair of legs with genu notched dorsally and bearing short peglike setae in notch (Fig. 343).................................................. ............................................................................................................................................Pionidae (in part) Pioninae (in part) 95 94b. Fourth pair of legs with genu not notched as described and illustrated above ..............................................................................96 95a(94a). Capitulum with anchoral process (Fig. 330, arrow A)........................................................................Piona (males of some species) 95b. Capitulum lacking anchoral process (Fig. 341 and as shown in Fig. 342, arrow).............................................Nautarachna (males) 96a(94b). Pedipalps with segments short and partially fused, femur with three ventral setae (Fig. 414, arrow)................................................ ............................................................................................................................................Bogatiidae Horreolaninae Horreolanus 96b. Pedipalps not as described and illustrated above ..........................................................................................................................97 97a(96b). Genital ﬁeld with three or four pairs of acetabula ........................................................................................................................98 97b. Genital ﬁeld with more than four pairs of acetabula ..................................................................................................................102 98a(97a). Ventral shield completely surrounding gonopore; genital ﬁeld with acetabula borne in gonopore (Fig. 407) or in pair of straight or slightly curved rows on ventral shield ﬂanking gonopore, when rows of acetabula slightly curved then genital ﬁeld located well anterior to posterior edge of idiosoma (Fig. 394) .........................................................................................................................99 98b. Ventral shield not completely surrounding gonopore posteriorly (females of most genera) or completely surrounding gonopore and with genital acetabula usually arranged in pronounced triangle or arc (males of all genera and females of one genus) (Fig. 368), when acetabula arranged in weak arc then genital ﬁeld located at extreme posterior edge of ventral shield ......................112 99a(98a). Dorsal shield with four pairs of glandularia including pair at anterior edge (Fig. 393, arrow); ventral shield with suture lines between third and fourth coxal plates oriented at right angles to long axis of body where they meet midline (Fig. 394, arrow) ...............................................................................................................................Chappuisididae (in part) Morimotacarinae 100 16. Water Mites (Hydrachnida) and Other Arachnids 613 99b. Dorsal shield with three pairs of glandularia (as in Fig. 411); ventral shield with suture lines between third and fourth coxal plates oriented at oblique angle to long axis of body where they meet midline...........................................................................101 100a(99a). Pedipalps with ﬁve segments (Fig. 391) ....................................................................................................................Morimotacarus 100b. Pedipalps with four segments, including fused tibiotarsus (Fig. 392) .................................................................................Yachatsia 101a(99b). Pedipalps with base of tarsus only about half as high as distal end of tibia, and inserted distidorsally on tibia (Fig. 417); ventral shield with insertions of fourth pair of legs covered by projections of fourth coxal plates (Fig. 418, arrow) .................................... ...................................................................................................................................Acalyptonotidae (in part) Paenecalyptonotus 101b. Pedipalps with base of tarsus approximately equal in height to distal end of tibia, and inserted distally on tibia (Fig. 405); ventral shield with insertions of fourth pair of legs not covered by projections of fourth coxal plates (Fig. 407).......................................... ...............................................................................................................................................Mideopsidae Mideopsinae Mideopsis 102a(97b). Dorsal shield bearing six pairs of glandularia, with three pairs grouped close together in an arc or triangle on each side (Fig. 322, circled area) ................................................................................................Unionicolidae (in part) Pionatacinae (in part) Koenikea 102b. Dorsal shield not as described and illustrated above...................................................................................................................103 103a(102b). Gonopore located near middle of ventral shield (Fig. 406) .........................................................................................................104 103b. Gonopore located near posterior end of ventral shield ...............................................................................................................105 104a(103a). Ventral shield incorporating excretory pore (Fig. 406, arrow B) .......................................................................Laversiidae Laversia 104b. Ventral shield not incorporating excretory pore...........Pionidae (in part) Pioninae (in part) Nautarachna (females of some species) 105a(103b). Ventral shield with ﬁrst coxal plates projecting beyond anterior edge of ventral shield (Figs. 377, 410, 412) .............................106 105b. Ventral shield with ﬁrst coxal plates not projecting beyond anterior edge of ventral shield (Fig. 357) ........................................123 106a(105a). Pedipalps with femur and tibia bearing distinct ventral projections (Fig. 409, arrows); third and fourth pair of legs with welldeveloped swimming setae (Fig. 408).................................................................................................Amoenacaridae Amoenacarus 106b. Pedipalps with femur and tibia usually lacking ventral projections, when bearing projections then third and fourth pair of legs lack swimming setae...................................................................................................................................................................107 107a(106b). Dorsal shield bearing three pairs of glandularia peripherally (Fig. 411) ..................................Arenohydracaridae Arenohydracarus 107b. Dorsal shield bearing one to several pairs of glandularia, with at least one pair located well medial to edge (Fig. 376).................... ........................................................................................................................................................Aturidae (in part) Aturinae 108 108a(107b). Dorsal shield bearing only one pair of glandularia (Fig. 379); pedipalps with segments extremely long and slender (Fig. 378) ......................................................................................................................................................................................Bharatalbia 108b. Dorsal shield bearing three or more pairs of glandularia (Fig. 371); pedipalps with segments relatively short and stocky (Fig. 373) ...................................................................................................................................................................................................109 109a(108b). Ventral shield bearing pronounced projections associated with openings of insertions of fourth legs (Figs. 374, 375)................110 109b. Ventral shield lacking projections associated with openings of insertions of fourth legs (Fig. 372, 377) .....................................111 110a(109a). Ventral shield with fourth coxal plates bearing pair of conspicuous glandularia medially (Fig. 374, arrow) ....................Neoaturus 110b. Ventral shield with fourth coxal plates lacking glandularia (Fig. 375)...........................................................................Kongsbergia 111a(109b). Ventral shield with genital acetabula in more than one row and restricted to region immediately lateral to gonopore near posterior edge of idiosoma (Fig. 372).........................................................................................................................Phreatobrachypoda 111b. Ventral shield with genital acetabula in single row and distributed along posterolateral edge of shield between gonopore and insertions of fourth legs (Fig. 377) ............................................................................................................................................Aturus 112a(98b). Dorsal shield comprising large central plate completely surrounded by ring of small, paired platelets (Fig. 347) ............................. ....................................................................................................................................................Lethaxonidae (in part) Lethaxona 112b. Dorsal shield comprising single large plate (although smaller posterior platelet bearing excretory pore may be present) (Fig. 365) ...................................................................................................................................Aturidae (in part) Axonopsinae (in part) 113 113a(112b). Ventral shield with posterior edges of fourth coxal plates clearly indicated by suture lines that are indented posteromedially to accommodate posterior coxoglandularia (Fig. 348, arrow) ....................................................................................................Ljania 113b. Ventral shield with posterior edges of fourth coxal plates usually faintly indicated or obliterated by fusion with shield (Fig. 368) and never indented posteromedially to accommodate posterior coxoglandularia ......................................................................114 614 Ian M. Smith, David R. Cook, Bruce P. Smith 114a(113b). Fourth coxal plates with large projections associated with insertions of fourth pair of legs (Fig. 350, arrow A; Fig. 354, arrow) ...................................................................................................................................................................................................115 114b. Fourth coxal plates with small projections or none associated with insertions of fourth pair of legs (Fig. 369) ..........................116 115a(114a). Fourth coxal plates with pair of closely spaced glandularia in females (Fig. 349, arrow) and with looped suture lines extending anteriorly in males (Fig. 350, arrow B) ...........................................................................................................................Stygalbiella 115b. Fourth coxal plates with glandularia widely spaced in females (Fig. 351) and without looped suture lines in males (Fig. 352) .....................................................................................................................................................................................Axonopsella 116a(114b). Ventral shield lacking ridges in region anterolateral to insertions of fourth pair of legs (Figs. 355, 356) ....................................117 116b. Ventral shield bearing well-developed ridges extending anterolaterally from region of insertion of fourth pair of legs (Fig. 368, arrow) ........................................................................................................................................................................................118 117a(116a). Ventral shield with anterior edges of ﬁrst coxal plates extending to or beyond anterior edge of ventral shield (Fig. 355); genital ﬁeld with three pairs of acetabula (Fig. 355) ....................................................................................................................Albaxona 117b. Ventral shield with anterior edges of ﬁrst coxal plates not extending to anterior edge of ventral shield (Fig. 356); genital ﬁeld with four pairs of acetabula (Fig. 356) ........................................................................................................................................Javalbia 118a(116b). Pedipalps with femur bearing spinelike projection in proximal three-quarters of ventral surface (Fig. 370); genital ﬁeld of females separated from ventral shield (Fig. 358) .....................................................................................................................................119 118b. Pedipalps with femur lacking spinelike projection ventrally, although rounded projection may be present at distiventral extremity (Fig. 366); genital ﬁeld of females separated from or fused with ventral shield...........................................................................121 119a(118a). Genital ﬁeld bearing four pairs of acetabula (Fig. 369); fourth pair of legs of males not highly modiﬁed ................Neobrachypoda 119b. Genital ﬁeld bearing three pairs of acetabula (Fig. 358); fourth pair of legs of males highly modiﬁed .......................................120 120a(119b). Fourth coxal plates bearing ridges extending posteriorly from region of insertions of fourth pair of legs (Fig. 358); fourth pair of legs of males with tarsus expanded distally and ﬂattened (Fig. 362) ..............................................................................Estellacarus 120b. Fourth coxal plates lacking ridges extending posteriorly from region of insertions of fourth pair of legs (Fig. 359); fourth pair of legs of males with tibia expanded and curved and tarsus slightly bowed and with enlarged claws (Fig. 363) ................Brachypoda 121a(118b). Fourth coxal plates of males bearing prominent ridges extending posteriorly from region of insertions of fourth pair of legs (Fig. 364, arrow); fourth pair of legs of males with tibia and tarsus thicker and stockier than those of females (Fig. 367); genital ﬁeld of females separated from ventral shield ....................................................................................................................Woolastookia 121b. Fourth coxal plates of males lacking prominent ridges extending posteriorly from region of insertions of fourth pair of legs (as in Fig. 360), although short, faint ridges may be evident in some species (Fig. 368); fourth pair of legs of males similar to those of females, either ﬂattened or unmodiﬁed; genital ﬁeld of females fused with ventral shield (Fig. 360) ...........................................122 122a(121b). Fourth pair of legs with segments ﬂattened and with telofemur relatively short and greatly expanded distally (Fig. 361)................. ..................................................................................................................................................................................Erebaxonopsis 122b. Fourth pair of legs with segments nearly cylindrical and with telofemur similar to other segments .................................Axonopsis 123a(105b). Fourth coxal plates usually with projections partially covering insertions of fourth pair of legs, when projections absent then genital ﬁeld with acetabula arranged in single rows extending laterally from gonopore in ventral shield (Fig. 346) ........................124 123b. Fourth coxal plates lacking projections associated with insertions of fourth pair of legs and genital ﬁeld with acetabula arranged in several rows on acetabular plates (Fig. 357) ..............................................................................Aturidae (in part) Albiinae Albia 124a(123a). Dorsal shield entire (Fig. 353); fourth coxal plates with projections associated with insertions of fourth pair of legs (Fig. 354, arrow).............................................................................................................Aturidae (in part) Axonopsinae (in part) Submiraxona 124b. Dorsal shield comprising large central plate completely surrounded by ring of small, paired platelets (Fig. 345); fourth coxal plates lacking projections associated with insertions of fourth pair of legs (Fig. 346)...............Lethaxonidae (in part) Lethaxonella 125a(71b). Fourth coxal plates bearing glandularia (Fig. 319, arrow)......................................................................Hygrobatidae (in part) 126 125b. Fourth coxal plates lacking glandularia......................................................................................................................................129 126a(125a). Gnathosoma with capitulum broadly fused with ﬁrst coxal plates (Fig. 315) .............................................................................127 126b. Gnathosoma with capitulum separate from ﬁrst coxal plates (Fig. 320) .....................................................................................128 127a(126a). First pair of legs with tarsus straight (Fig. 317) ..............................................................................................................Hygrobates 127b. First pair of legs with tarsus bowed (Fig. 318) .................................................................................................................Mesobates 128a(126b). First coxal plates fused medially (Fig. 319); ﬁrst pair of legs with distal segments modiﬁed as in Fig. 321........................Atractides 16. Water Mites (Hydrachnida) and Other Arachnids 615 128b. First coxal plates separate medially (Fig. 320); ﬁrst pair of legs with distal segments not modiﬁed ...............................Corticacarus 129a(125b). Venter with apodemes of anterior coxal groups extending to approximately middle of fourth coxal plates (Fig. 323, arrow) .................................................................................................................Unionicolidae (in part) Pionatacinae (in part) Neumania 129b. Venter with apodemes of anterior coxal groups not extending to middle of fourth coxal plates .................................................130 130a(129b). Genital ﬁeld surrounded by well developed ventral shield (Fig. 418) .................................Acalyptonotidae (in part) Acalyptonotus 130b. Genital ﬁeld not surrounded by ventral shield ............................................................................................................................131 131a(130b). Fourth coxal plates with medial edges reduced to angles (Fig. 338, arrow) ................................................................................132 131b. Fourth coxal plates with medial edges not reduced to angles (Fig. 344, arrow) ..........................................................................135 132a(131a). First pair of legs with tarsal claw socket large, occupying much more than one half dorsal surface of segment (Fig. 326, arrow); genital ﬁeld with three or four pairs of acetabula; fourth pair of legs with tarsus unmodiﬁed in males...............Wettinidae Wettina 132b. First pair of legs with tarsal claw socket smaller, occupying one half or less of dorsal surface of segment; genital ﬁeld with three or more pairs of acetabula; fourth pair of legs with tarsus indented or bowed and bearing short peglike setae in males (Fig. 339) ........................................................................................................................................Pionidae (in part) Foreliinae (in part) 133 133a(132b). Genital ﬁeld with three pairs of acetabula (Fig. 336) .....................................................................................................Pionacercus 133b. Genital ﬁeld with more than three pairs of acetabula (Figs. 337, 338) .......................................................................................134 134a(133b). Legs with swimming setae on distal segments ................................................Forelia (females of all species, males of some species) 134b. Legs without swimming setae on distal segments ..........................................................................................Pseudofeltria (females) 135a(131b). Capitulum without anchoral process (Fig. 342, arrow); symbionts of clams.................Pionidae (in part) Najadicolinae Najadicola 135b. Capitulum with anchoral process (Fig. 330, arrow A); symbionts of clams or free living ...........................................................136 136a(135b). Pedipalps with segments relatively long and slender (Fig. 329); genital ﬁeld with three pairs of acetabula; males with petiole posterior to genital ﬁeld (Fig. 327); and with genu of third pair of legs modiﬁed (Fig. 328)................................................................... ......................................................................................................................Pionidae (in part) Hydrochoreutinae Hydrochoreutes 136b. Pedipalps with segments relatively short and stocky; genital ﬁeld with three to many pairs of acetabula; males lacking petiole posterior to genital ﬁeld and with genu of third pair of legs unmodiﬁed.....................................................................................137 137a(136b). Venter with suture lines between third and fourth coxal plates extending only a short distance toward midline (Fig. 324, arrow A); with a pair of glandularia present at, and usually fused with, posteromedial angles of fourth coxal plates (Fig. 324, arrow B); ﬁrst pair of legs with long movable setae in free-living species (Fig. 325); symbionts of clams or sponges, or free living ................. ............................................................................................................................Unionicolidae (in part) Unionicolinae Unionicola 137b. Venter with suture lines between third and fourth coxal plates extending to (or nearly to) medial margins of coxal plates (as in Fig. 330, usually longer than shown in Fig. 344); without glandularia at posteromedial angles of fourth coxal plates; ﬁrst pair of legs lacking long, movable setae; free living................................................................................................................................138 138a(137b). Genital ﬁeld usually with three, but rarely up to six, pairs of acetabula ........................................Pionidae (in part) Tiphyinae 139 138b. Genital ﬁeld with seven or more pairs of acetabula ...................................................................................................................142 139a(138a). Fourth pair of legs unmodiﬁed ............................................................................Tiphyinae (females) Neotiphys, Pionopsis, Tiphys 139b. Fourth pair of legs with segments modiﬁed....................................................................................................Tiphyinae (males) 140 140a(139b). Fourth pair of legs with tibia bearing long, thick, curved seta dorsally (Fig. 333, arrow) and with genu only slightly expanded .........................................................................................................................................................................................Neotiphys 140b. Fourth pair of legs with tibia lacking long, thick, curved seta dorsally and with genu slightly or greatly expanded....................141 141a(140b). Fourth pair of legs with genu greatly expanded and bearing numerous long setae with enlarged bases (Fig. 335)..................Tiphys 141b. Fourth pair of legs with genu slightly expanded and bearing short setae with normal bases (Fig. 334)..............................Pionopsis 142a(138b). Pedipalps with genu bearing long seta medially (Fig. 331, arrow); venter with second and third coxal plates each with pair of thickened setae (Fig. 330, arrows B); legs of males unmodiﬁed.....................................Pionidae (in part) Huitfeldtiinae Huitfeldtia 142b. Pedipalps with genu lacking long seta medially; venter with second and third coxal plates lacking thick setae (Fig. 344); fourth pair of legs in males with genu notched and with peglike setae associated with notch (Fig. 343)...................................................... ..........................................................................................................................................Pionidae (in part) Pioninae (in part) 143 143a(142b). Capitulum with anchoral process (as in Fig. 330, arrow A) ..................................................Piona (females, males of most species) 143b. Capitulum without anchoral process (Fig. 341 and as in Fig. 342, arrow) ...........................Nautarachna (females of some species) 616 Ian M. Smith, David R. Cook, Bruce P. Smith FIGURE 200 Limnesia sp. (Limnesiinae), adult female, ventral view. 16. Water Mites (Hydrachnida) and Other Arachnids FIGURES 201, 202 Stygothrombium sp. (Stygothrombidiidae) adult. Fig. 201, claws and empodium of leg I; Fig. 202, pedipalp. FIGURES 203, 205–206 Hydrovolzia marshallae Cook (Hydrovolziinae), adult. Fig. 203, glandularium; Fig. 205, coxal plate III ; Fig. 206, dorsum of idiosoma. FIGURE 204 Acherontacarus sp. (Acherontacarinae), adult, dorsum of idiosoma. FIGURES 207–209 Hydrachna sp. (Hydrachnidae), adult. Fig. 207, dorsal view of idiosoma; Fig. 208, capitulum and chelicera; Fig. 209, pedipalp. 617 618 Ian M. Smith, David R. Cook, Bruce P. Smith FIGURE 210 Eylais sp. (Eylaidae), adult, eye plate. FIGURES 211, 216 Limnochares sp. (Limnocharinae), adult. Fig. 211, eye plate; Fig. 216, pedipalp. FIGURE 212 Piersigia limnophila Protz (Protziidae), adult, eye plate. FIGURES 213–214 Apheviderulix santana Gerecke, Smith and Cook. (Apheviderulicidae), deutonymph. Fig. 213 dorsum of idiosoma; Fig. 214, venter of idiosoma and gnathosoma. FIGURE 215 Rhyncholimnochares sp. (Rhyncholimnocharinae), adult, pedipalp. FIGURE 217 Neolimnochares sp. (Limnocharinae), adult, pedipalp. FIGURE 218 Clathrosperchon sp. (Clathrosperchontinae), adult, dorsum of idiosoma. 16. Water Mites (Hydrachnida) and Other Arachnids FIGURE 219 Hydryphantes sp. (Hydryphantinae), adult, eye plate. FIGURE 220 Hydryphantes ruber (de Geer) (Hydryphantinae), adult, dorsum of idiosoma. FIGURE 221 Hydrodroma sp. (Hydrodromidae), adult, pedipalp. FIGURES 222, 228 Protzia sp. (Protziinae), adults. Fig. 222, claws of leg I; FIG. 228, genital ﬁeld. FIGURE 223 Cowichania interstitialis Smith (Cowichaniinae), adult female, venter of idiosoma. FIGURE 224 Partnunia steinmanni Walter (Protziinae), adult, dorsum of idiosoma. FIGURES 225, 229 Notopanisus sp. (Thyadinae), adult. Fig. 225, medial eye; Fig. 229, genital ﬁeld. FIGURES 226–227 Wandesia sp. (Wandesiinae), adult; Fig. 226, venter of idiosoma and gnathosoma; Fig. 227 pedipalp. 619 620 Ian M. Smith, David R. Cook, Bruce P. Smith FIGURES 230, 231 Albertathyas montana Smith and Cook (Thyadinae), adult. Fig. 230, Dorsum of idiosoma; Fig. 231, genital ﬁeld. FIGURE 232 Cyclothyas siskiyouensis Smith (Cyclothyadinae), adult, dorsum of idiosoma. FIGURE 233 Siskiyouthyas rivularis Smith and Cook (Thyadinae), adult, dorsum of idiosoma. FIGURE 234 Trichothyas muscicola Mitchell (Thyadinae), adult, dorsum of idiosoma. FIGURE 235 Euthyas truncata (Neumann) (Thyadinae), adult, dorsum of idiosoma. FIGURE 236, 237 Euthyas mitchelli Cook (Thyadinae), adult. Fig. 236, Genital ﬁeld; Fig. 237, venter of idiosoma. 16. Water Mites (Hydrachnida) and Other Arachnids FIGURES 238, 239 Thyopsis cancellata (Protz) (Thyadinae), adult. Fig. 238, Dorsal shield; Fig. 239, genital ﬁeld. FIGURE 240 Thyopsella occidentalis Cook (Thyadinae), adult, dorsal shield. FIGURES 241, 246 Thyopsoides plana (Habeeb) (Thyadinae), adult. Fig. 241, Dorsum of idiosoma; Fig. 246, genital ﬁeld. FIGURE 244 Thyopsella dictyophora Cook (Thyadinae), adult, genital ﬁeld. FIGURE 242 Panisus condensatus Habeeb (Thyadinae), adult, dorsum of idiosoma. FIGURE 243 Columbiathyas crenicola Smith and Cook (Thyadinae), adult, dorsum of idiosoma. FIGURES 245, 247 Panisopsis setipes (Viets) (Thyadinae), adult. Fig. 245, dorsum of idiosoma; Fig. 247, genital ﬁeld. 621 622 Ian M. Smith, David R. Cook, Bruce P. Smith FIGURES 248, 249 Parathyasella heatherensis Smith and Cook (Thyadinae), adult. Fig. 248, Dorsum of idiosoma; Fig. 249, genital ﬁeld. FIGURE 250 Amerothyasella appalachiana Smith and Cook (Thyadinae), adult, dorsum of idiosoma. FIGURE 251 Thyasella sp. (Thyadinae), adult, dorsum of idiosoma. FIGURE 252 Thyasides sp. (Thyadinae), adult, dorsum of idiosoma. FIGURE 253 Thyas rivalis Koenike (Thyadinae), adult, genital ﬁeld. FIGURE 254 Tartarothyas sp. (Tartarothyadinae), adult, genital ﬁeld. FIGURE 255 Thermacarus nevadensis Marshall (Thermacaridae), adult, pedipalp. FIGURE 256 Thyas stolli Koenike (Thyadinae),adult, dorsum of idiosoma. FIGURE 257 Zschokkea sp. (Thyadinae), adult, genital ﬁeld. 16. Water Mites (Hydrachnida) and Other Arachnids FIGURES 258 – 260 Sperchon spp. (Sperchontidae), adults. Fig. 258, Dorsum of idiosoma; Fig. 259, venter of idiosoma; Fig. 260, pedipalp. FIGURES 261, 262 Sperchonopsis nova Prasad and Cook (Sperchontidae), adult. Fig. 261, Dorsum of idiosoma; Fig. 262, pedipalp. FIGURES 263, 264 Rutripalpus spp. (Rutripalpidae), adult females. Fig. 263, Pedipalp; Fig. 264, venter of idiosoma. FIGURE 265 Teutonia sp. (Teutoniidae), adult, venter of idiosoma. 623 624 Ian M. Smith, David R. Cook, Bruce P. Smith FIGURES 266, 268 Utaxatax ovalis Cook (Anisitsiellinae), adult. Fig. 266, Tarsus of leg IV; Fig. 268, male, ventral shield. FIGURE 267 Mamersellides sp. (Anisitsiellinae), adult, leg. IV. FIGURE 269 Bandakiopsis fonticola Smith (Anisitsiellinae), adult, ventral shield. FIGURE 270 Cookacarus columbiensis Barr (Anisitsiellinae), adult male, ventral shield. FIGURE 271 Bandakia phreatica Cook (Anisitsiellinae), adult male, ventral shield. FIGURE 272 Oregonacarus rivulicolus Smith (Anisitsiellinae), adult male, ventral shield. 16. Water Mites (Hydrachnida) and Other Arachnids FIGURES 273, 274 Testudacarus americanus Marshall (Testudacarinae), adults. Fig. 273, Dorsal shield; Fig. 274, pedipalp. FIGURES 275 – 277 Neoatractides sp. (Neoatractidinae), adults. Fig. 275, Pedipalp; Fig. 276, dorsal shield; Fig. 277, ventral shield. FIGURES 278, 279, 281 Torrenticola sp. (Torrenticolinae), adults. Fig. 278, Dorsal shield; Fig. 279, ventral shield; Fig. 281, gnathosoma. FIGURE 280 Pseudotorrenticola chiricahua Smith (Torrenticolinae), adult, gnathosoma. FIGURE 282 Monoatractides sp. (Torrenticolinae), adult, gnathosoma. 625 626 Ian M. Smith, David R. Cook, Bruce P. Smith FIGURES 283, 284 Lebertia sp. (Lebertiidae), adults. Fig. 283, Venter of idiosoma; Fig. 284, pedipalp. FIGURE 285 Estelloxus montanus Habeeb (Lebertiidae) adult, venter of idiosoma. FIGURES 286, 287 Scutolebertia trinitensis Smith (Lebertiidae), adults. Fig. 286, Dorsum of idiosoma; Fig. 287, pedipalp. FIGURES 288–290 Oxus spp. (Oxidae), adults. Fig. 288, Dorsum of idiosoma; Fig. 289, venter of idiosoma; Fig. 290, tibia and tarsus of leg IV. FIGURE 291 Frontipoda americana Marshall (Oxidae), adult male, dorsum of idiosoma. 16. Water Mites (Hydrachnida) and Other Arachnids FIGURE 292 Omartacarus sp. (Omartacaridae), adult female, venter of idiosoma. FIGURE 293 Diamphidaxona sp. (Hygrobatidae), adult male, venter of idiosoma. FIGURE 294 Neomamersa chihuahua Smith and Cook (Neomamersinae), adult female, dorsum of idiosoma. FIGURES 295, 299, 301 Neomamersa spp. (Neomamersinae), adult males. Fig. 295, Pedipalp; Figs, 299, 301, genital ﬁelds. FIGURE 296 Kawamuracarus sp. (Kawamuracarinae), adult, pedipalp. FIGURES 297, 303 Arizonacarus chiricahuensis Smith and Cook (Neomamersinae), adult females. Fig. 297, Pedipalp; Fig. 303, venter of idiosoma. FIGURES 298, 300, 304 Meramecia occidentalis Smith and Cook (Neomamersinae), adult females. Fig. 298, Pedipalp; Fig. 300, genital ﬁeld; Fig. 304, venter of idiosoma. FIGURE 302 Neomamersa californica Smith and Cook (Neomamersinae), adult female, venter of idiosoma. 627 628 Ian M. Smith, David R. Cook, Bruce P. Smith FIGURE 305 Centrolimnesia bondi Lundblad (Limnesiinae), adult male, genital ﬁeld. FIGURES 306, 309 – 311 Tyrrellia spp. (Tyrelliinae), adults. Fig. 306, Pedipalp; Fig. 309, male, venter of idiosoma; Fig. 310, male, dorsal shield; Fig. 311, male, dorsum of idiosoma. FIGURE 307 Protolimnesia sp. (Protolimnesiinae), adult, dorsum of idiosoma. FIGURE 308 Neotyrrellia anitahoffmannae Smith and Cook (Tyrrellinae), adult female, venter of idiosoma. FIGURE 312 Feltria exilis Cook (Feltriidae), adult female, venter of idiosoma. FIGURES 313, 314 Frontipodopsis sp. (Frontipodopsidae), adult. Fig. 313, Leg IV; Fig. 314, lateral view of idiosoma. 16. Water Mites (Hydrachnida) and Other Arachnids FIGURES 315 – 317 Hygrobates spp. (Hygrobatidae), adults. Fig. 315 male, venter of idiosoma; Fig. 316 , Chelicera; Fig. 317, tibia and tarsus of leg I. FIGURE 318 Mesobates sp. (Hygrobatidae), adult, tibia and tarsus of leg I. FIGURES 319, 321 Atractides sp. (Hygrobatidae), adults. Fig. 319, Male, venter of idiosoma; Fig. 321, tibia and tarsus of leg I. FIGURE 320 Corticacarus sp. (Hygrobatidae), adult female, venter of idiosoma. FIGURE 322 Koenikea sp. (Pionatacinae), adult dorsal shield. FIGURE 323 Neumania sp. (Pionatacinae), adult male, venter of idiosoma. FIGURES 324, 325 Unionicola sp. (Unionicolinae), adults. Fig. 324, female, venter of idiosoma; Fig. 325, leg I. 629 630 Ian M. Smith, David R. Cook, Bruce P. Smith FIGURE 326 Wettina octopora Cook (Wettinidae), adult, leg I. FIGURES 327 – 329 Hydrochoreutes intermedius Cook (Hydrochoreutinae), adults. Fig. 327, Male, genital ﬁeld; Fig. 328, male, genu of leg III; Fig. 329, pedipalp. FIGURES 330, 331 Huitfeldtia rectipes (Huitfeldtiinae), adults. Fig. 330, female, venter of idiosoma; Fig. 331, pedipalp. FIGURES 332, 335 Tiphys spp. (Tiphyinae), adults. Fig. 332, pedipalp; Fig. 335, male, leg IV. FIGURE 333 Neotiphys pionoidellus (Habeeb) (Tiphyinae), adult male, tibia of leg IV. FIGURE 334 Pionopsis paludis Habeeb (Tiphyinae), adult male, leg IV. 16. Water Mites (Hydrachnida) and Other Arachnids FIGURE 336 Pionacercus sp. (Foreliinae), adult male, venter of idiosoma. FIGURE 337 Pseudofeltria multipora Cook (Foreliinae), adult male, ventral shield. FIGURES 338 – 340 Forelia ﬂoridensis Cook (Foreliinae), adult male. Fig. 338, Venter of idiosoma; Fig. 339, distal segments of leg IV; Fig. 340, tibia and tarsus of leg III. FIGURE 341 Nautarachna queticoensis Smith (Pioninae), adult female, gnathosoma. FIGURE 342 Najadicola ingens Koenike (Najadicolinae), adult male, venter of idiosoma. FIGURES 343, 344 Piona spp. (Pioninae), adult males. Fig. 343, Genu of leg IV; Fig. 344, venter of idiosoma. 631 632 Ian M. Smith, David R. Cook, Bruce P. Smith FIGURES 345, 346 Lethaxonella parva Cook (Lethaxonidae), adult female. Fig. 345, Dorsal plate; Fig. 346, ventral plate. FIGURE 347 Lethaxona sp. (Lethaxonidae), adult, dorsal shield. FIGURE 348 Ljania michiganensis Cook (Axonopsinae), adult male, ventral shield. FIGURES 349, 350 Stygalbiella yavapai Smith and Cook (Axonopsinae), adults. Fig. 349, Female, ventral shield; Fig. 350, male, ventral shield. FIGURES 351, 352 Axonopsella bakeri Smith and Cook (Axonopsinae), adults. Fig. 351, female, ventral shield; Fig. 352, male, ventral shield. 16. Water Mites (Hydrachnida) and Other Arachnids FIGURES 353, 354 Submiraxona gilana (Habeeb) (Axonopsinae), adults. Fig. 353, Dorsal shield; Fig. 354, male, ventral shield. FIGURE 355 Albaxona nearctica Cook (Axonopsinae), adult male, ventral shield. FIGURE 356 Javalbia sp. (Axonopsinae), adult female, ventral shield. FIGURE 357 Albia sp. (Albiinae), adult male, ventral shield. FIGURE 358 Estellacarus unguitarsus (Habeeb) (Axonopsinae), adult female, ventral shield. FIGURE 359 Brachypoda oakcreekensis Habeeb (Axonopsinae), adult female, ventral shield. FIGURES 360, 361 Erebaxonopsis nearctica Cook (Axonopsinae), adults. Fig. 360, female,ventral shield; Fig. 361, male, leg IV. 633 634 Ian M. Smith, David R. Cook, Bruce P. Smith FIGURE 362 Estellacarus unguitarsus (Habeeb) (Axonopsinae), adult male, distal segments of leg IV. FIGURE 363 Brachypoda oakcreekensis Habeeb (Axonopsinae), adult male, distal segments of leg IV. FIGURES 364 – 367 Woolastookia pilositarsa (Habeeb) (Axonopsinae), adults. Fig. 364, Male, venter of idiosoma; Fig. 365, female, pedipalp; Fig. 366, female, dorsal shield; Fig. 367, male, distal segments of leg IV. FIGURE 368 Axonopsis beltista Cook (Axonopsinae), adult male, ventral shield. FIGURES 369, 370 Neobrachypoda ekmani (Walter) (Axonopsinae), adults. Fig. 369, Female, venter of idiosoma; Fig. 370, male, pedipalp. 16. Water Mites (Hydrachnida) and Other Arachnids FIGURES 371, 372 Phreatobrachypoda multipora Cook (Aturinae), adult male. Fig. 371, Dorsum of idiosoma; Fig. 372, venter of idiosoma. FIGURE 373 Phreatobrachypoda oregonensis Smith (Aturinae), adult male, pedipalp. FIGURE 374 Neoaturus sp. (Aturinae), adult female, venter of idiosoma. FIGURES 375, 376 Kongsbergia spp. (Aturinae), adult males. Fig. 375, Venter of idiosoma and fourth leg; Fig. 376, dorsal shield. FIGURE 377 Aturus sp. (Aturinae), adult female, venter of idiosoma. FIGURES 378, 379 Bharatalbia spp. (Aturinae), adults. Fig. 378, B. cooki Smith, female, dorsum of idiosoma; Fig. 379, B. surensis Smith, male, pedipalp. 635 636 Ian M. Smith, David R. Cook, Bruce P. Smith FIGURE 380 Cyclomomonia andrewi Smith (Cyclomomoniinae), adult, dorsal shield. FIGURE 381 Stygomomonia riparia Habeeb (Stygomomoniinae), adult, distal segments of leg I. FIGURE 382 Momonia projecta Cook (Momoniinae), adult female, ventral shield. FIGURE 383 Stygomomonia mitchelli Smith (Stygomomoniinae), adult male, ventral shield. FIGURE 384 Midea expansa Marshall (Mideidae), adult female, ventral shield. FIGURE 385 Nudomideopsis magnecetabula (Smith) (Nudomideopsidae), adult male, ventral shield. FIGURE 386 Paramideopsis susanae Smith (Nudomideopsidae), adult male, ventral shield. FIGURE 387 Neomideopsis suislawensis Smith (Nudomideopsidae), adult male, ventral shield. 16. Water Mites (Hydrachnida) and Other Arachnids FIGURE 388 Chappuisides eremitus Cook (Chappuisidinae), adult male, ventral shield. FIGURE 389 Uchidastygacarus ovalis Cook (Uchidastygacarinae), adult female, ventral shield and legs. FIGURE 390 Uchidastygacarus acadiensis Smith (Uchidastygacarinae), adult, pedipalp. FIGURE 391 Morimotacarus nearcticus Smith (Morimotacarinae), adult, pedipalp. FIGURES 392 – 394 Yachatsia mideopsoides Cook (Morimotacarinae), adults. Fig. 392, Pedipalp; Fig. 393, dorsal shield; Fig. 394, female, ventral shield. FIGURES 395, 397 Volsellacarus sabulonus Cook (Neoacaridae), adults. Fig. 395, female, ventral shield; Fig. 397, gnathosoma. FIGURE 396 Neoacarus minimus Cook (Neoacaridae), adult, pedipalp. 637 638 Ian M. Smith, David R. Cook, Bruce P. Smith FIGURES 398, 399 Stygameracarus cooki Smith (Stygameracarinae), adults. Fig. 398, Male, ventral shield; Fig. 399, pedipalp. FIGURE 400 Platyhydracarus juliani Smith (Athienemanniinae), adult male, genital ﬁeld. FIGURES 401, 402 Chelomideopsis besselingi (Cook) (Athienemanniinae), adults. Fig. 401, Gnathosoma; Fig. 402, adult male, ventral shield. FIGURES 403, 404 Chelohydracarus navarrensis Smith (Athienemanniinae), adult male. Fig. 403, Genital ﬁeld; Fig. 404, ventral shield. FIGURES 405, 407 Mideopsis sp. (Mideopsinae), adults. Fig. 405, Pedipalp; Fig. 407, female, ventral shield. FIGURE 406 Laversia berulophila Cook (Laversiidae), adult female, ventral shield. 16. Water Mites (Hydrachnida) and Other Arachnids FIGURES 408 – 410 Amoenacarus dixiensis Smith and Cook (Amoenacaridae), adults. Fig. 408, Male, leg IV; Fig. 409, pedipalp; Fig. 410, male, ventral shield. FIGURES 411, 412 Arenohydracarus minimus Cook (Arenohydracaridae), adults. Fig. 411, dorsal shield; Fig. 412, female, ventral shield. FIGURE 413 Geayia sp. (Krendowskiidae), adult female, ventral shield. FIGURE 414 Horreolanus orphanus Mitchell (Horreolaninae), adult, pedipalp. FIGURES 415, 416 Arrenurus sp. (Arrenurinae), adults. Fig. 415, Female, ventral shield. Fig. 416, pedipalp. FIGURES 417, 418 Acalyptonotus neoviolaceus Smith (Acalyptonotidae), adults. Fig. 417, Pedipalp; Fig. 418, female, ventral shield. 639 640 Ian M. Smith, David R. Cook, Bruce P. Smith FIGURES 419 – 422 Scanning electron micrographs of adult water mites. Fig. 419 Arrenurus ﬁssicornis Marshall (Arrenuruidae), male; Fig. 420 Arrenurus (Megaluracarus) pseudocylindratus Marshall (Arrenuridae), male; Fig. 421 Kongsbergia sp. (Aturidae), female; Fig. 422 Aturus sp. (Aturidae), male. 16. Water Mites (Hydrachnida) and Other Arachnids FIGURES 423 – 426 Scanning electron micrographs of adult water mites. FIGURE. 423 Koenikea wolcotti Viets (Unionicolidae), female; FIGURE 424 Uchidastygacarus acadiensis Smith (Chappuisididae), female; FIGURE 425 Paramideopsis susanae Smith (Nudomideopsidae), female; FIGURE 426 Volsellacarus sabulonus Cook (Neoacaridae), female. 641 642 Ian M. Smith, David R. Cook, Bruce P. Smith FIGURES 427 – 430 Photographs of parasitic larval water mites attached to host insects. FIGURE 427 Larvae of Eylais sp. attached to the subelytral abdominal integument of adult waterboatmen (Hemiptera: Corrixidae) illustrating larval size before and after engorgement; FIGURE 428 larvae of Hydrachna sp. (Hydrachnidae) attached to the thorax and elytra of a giant water bug (Hemiptera:Belostomatidae); Fig. 429 larvae of Arrenurus sp. (Arrenuridae) attached to the abdominal segments of a damselﬂy (Odonata:Zygoptera); FIGURES 430 larvae of Feltria sp. (Feltriidae) and Aturus sp. (Aturidae) attached to the abdominal segments of a chironomid midge (Diptera:Chironomidae). TABLE I Summary of Some Important Zoogeographic and Ecological Characteristics of North American Water Mite Genera Ecology Zoogeography Taxa Stygothrombidioidea Stygothrombidiidae Stygothrombium Hydrovolzioidea Hydrovolziidae Hydrovolzia Acherontacaridae Acherontacarus Hydrachnoidea Hydrachnidae Hydrachna Eylaoidea Limnocharidae Limnocharinae Limnochares Neolimnochares Rhyncholimnocharinae Rhyncholimnochares Eylaidae Eylais Piersigiidae Piersigia Apheviderulicidae Apheviderulix Hydryphantoidea Hydryphantidae Hydryphantinae Hydryphantes 643 Thyadinae Albertathyas Amerothyasella Columbiathyas Larvae Deutonymphs and Adults Adults Origin North American distribution Type Order of hosts Attachment site Habitat type Mode of locomotion Prey Spermatophore transfer mode Laurasian Temperate, boreal Aquatic Plecoptera Thorax, abdomen Interstitial Crawling Unknown Unknown Laurasian Temperate, boreal Terrestrial Hemiptera, Diptera Thorax, abdomen Springs, rifﬂes Crawling Unknown Unknown Laurasian Temperate Aquatic Unknown Unknown Interstitial Crawling Unknown Unknown Pangean Throughout Aquatic Coleoptera, Hermiptera Thorax, abdomen Lakes, temporary pools Swimming Insect eggs Dissociation Pangean Temperate, boreal Terrestrial Thorax, abdomen Terrestrial Thorax, abdomen Crawling, swimming Crawling UnKnown Temperate Rifﬂes, pools, lakes Pools Diptera larvae Pangean Hemiptera, Odonata Hemiptera Unknown Unknown Gondwanan Temperate Aquatic Coleoptera Abdomen Rifﬂes, interstitial Crawling Unknown Unknown Pangean Throughout Terrestrial Coleoptera, Hemiptera Thorax, abdomen Pools, lakes, temporary pools Swimming Ostracoda, Cladocera Dissociation, association, copulation Laurasian Temperate, boreal Terrestrial Coleoptera Abdomen Springs, interstitial, temporary pools Crawling Ostracoda Unknown Laurasian Temperate Terrestrial Coleoptera Abdomen Springs Crawling Unknown Unknown Pangean Throughout Terrestrial Hemiptera, Odonata, Diptera Thorax, abdomen Lakes, temporary pools Swimming Diptera eggs Dissociation North American North American North American Temperate Temperate Temperate Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Rifﬂes Rifﬂes Springs Crawling Crawling Crawling Unknown Unknown Unknown Unknown Unknown Unknown (continues) 644 TABLE I (Continued) Ecology Zoogeography Larvae Deutonymphs and Adults Origin North American distribution Type Order of hosts Attachment site Habitat type Euthyas Laurasian Temperate Terrestrial Diptera Thorax Notopanisus Panisus Pangean Laurasian Temperate Temperate, boreal Unknown Terrestrial Unknown Thorax Panisopsis Parathyasella Siskiyouthyas Thyas Laurasian Laurasian North American Laurasian Temperate, boreal Temperate, boreal Temperate Temperate, boreal Terrestrial Unknown Unknown Terrestrial Thyasella Thyasides Thyopsella Laurasian Laurasian North American Temperate Boreal Temperate, boreal Unknown Terrestrial Terrestrial Thyopsis Laurasian Temperate Terrestrial Unknown Diptera, Hymenoptera Diptera Unknown Unknown Collembola, Diptera, Trichoptera Unknown Diptera Diptera, Trichoptera Diptera Rifﬂes, temporary pools Springs, rifﬂes Springs, rifﬂes Thyopsoides Trichothyas North American Laurasian Temperate Temperate Unknown Terrestrial Unknown Diptera Laurasian Boreal, arctic Unknown Unknown Laurasian Laurasian Temperate Temperate, boreal Terrestrial Terrestrial Plecoptera Diptera, Trichoptera Pangean Temperate Aquatic Pangean Temperate Laurasian Taxa Zschokkea Protziinae Partnunia Protzia Wandesiinae Wandesia Tartarothyadinae Tartarothyas Cyclothyadinae Cyclothyas Cowichaniinae Cowichania Hydrodromidae Hydrodroma Rhynchohydracaridae Clathrosperchoninae Clathrosperchon Adults Mode of locomotion Prey Spermatophore transfer mode Walking Insect eggs Unknown Crawling Crawling Unknown Unknown Unknown Unknown Unknown Unknown Unknown Thorax Springs, rifﬂes Springs, rifﬂes Rifﬂes Springs, rifﬂes, temporary pools Crawling Crawling Unknown Walking Unknown Unknown Unknown Insect eggs Unknown Unknown Unknown Dissociation Unknown Thorax Thorax, abdomen Springs, interstitial Temporary pools Springs, rifﬂes Walking Walking Crawling Unknown Unknown Unknown Unknown Unknown Unknown Thorax Springs, temporary pools Rifﬂes Springs Crawling Unknown Unknown Crawling Walking Unknown Unknown Unknown Unknown Temporary pools Walking Unknown Unknown Thorax, abdomen Thorax, abdomen Rifﬂes Rifﬂes Walking Walking Unknown Unknown Unknown Unknown Plecoptera Thorax Springs, interstitial Crawling Unknown Unknown Terrestrial Unknown Unknown Springs, rifﬂes Walking, crawling Unknown Unknown Temperate (W) Unknown Unknown Unknown Springs, interstitial Crawling Unknown Unknown North American Temperate (W) Unknown Unknown Unknown Interstitial Crawling Unknown Unknown Pangean Throughout Terrestrial Diptera Thorax Rifﬂes, pools, lakes Swimming Insect eggs Dissociation Gondwanan Temperate Unknown Unknown Unknown Rifﬂes, interstitial Crawling Unknown Unknown Unknown Head, thorax, abdomen Unknown Thermacaridae Thermacarus Pangean Temperate Terrestrial Anura (Amphibia) Body dorsum Hot springs Crawling Diptera larvae Unknown Lebertioidea Sperchontidae Sperchontinae Sperchon Laurasian Throughout Aquatic Thorax, abdomen Diptera larvae Unknown Temperate, boreal Aquatic Thorax Springs, rifﬂes, pools, lakes Rifﬂes, pools, lakes Crawling Laurasian Diptera, Trichoptera Diptera Crawling Unknown Unknown Laurasian Boreal Aquatic Diptera Abdomen Springs, pools Crawling swimming Diptera larvae Unknown Laurasian Boreal (E) Unknown Unknown Unknown Springs, pools Crawling Unknown Unknown Laurasian Temperate, boreal Aquatic Diptera Thorax Crawling Unknown Unknown Bandakiopsis Cookacarus Mamersellides Oregonacarus Utaxatax Lebertiidae Estelloxus Lebertia Laurasian North American Gondwanan North American Laurasian Temperate Temperate Temperate (SE) Temperate Temperate Aquatic Unknown Unknown Unknown Aquatic Diptera Unknown Unknown Unknown Diptera Thorax, abdomen Unknown Unknown Unknown Thorax, abdomen Springs, rifﬂes, interstitial Springs Springs, interstitial Pools Springs Springs, interstitial Crawling Crawling Swimming Crawling Crawling Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown North American Laurasian Temperate (W) Throughout Aquatic Aquatic Diptera Diptera Thorax Thorax Unknown Dissociation North American Temperate (SW) Unknown Unknown Unknown Walking Walking, swimming Crawling Unknown Diptera larvae Scutolebertia Oxidae Frontipoda Oxus Torrenticolidae Neoatractidinae Neoatractides Testudacarinae Testudacarus Torrenticolinae Monatractides Pseudotorrenticola Torrenticola Springs, rifﬂes Springs, rifﬂes, pools, lakes Springs Unknown Unknown Pangean Pangean Temperate, boreal Temperate, boreal Aquatic Aquatic Diptera Diptera Thorax Thorax, abdomen Pools, lakes Pools, lakes Swimming Swimming Diptera larvae Diptera larvae Unknown Unknown Gondwanan Temperate (SW) Unknown Unknown Unknown Rifﬂes Crawling Unknown Unknown Laurasian Temperate, boreal Aquatic Diptera Thorax Rifﬂes, interstitial Crawling Diptera larvae Unknown Laurasian Laurasian Laurasian Temperate Temperate Temperate, boreal Aquatic Unknown Aquatic Diptera Unknown Diptera Thorax Unknown Thorax Rifﬂes, pools, lakes Interstitial Rifﬂes, interstitial, pools, lakes Crawling Crawling Crawling Diptera larvae Unknown Diptera larvae Unknown Unknown Unknown Hygrobatoidea Limnesiidae Kawamuracarinae Kawamuracarus Laurasian Temperate (SW) Unknown Unknown Unknown Interstitial Crawling, walking Unknown Unknown Limnesiinae Centrolimnesia South American Temperate Unknown Unknown Unknown Pools Swimming Unknown Unknown Sperchonopsis Teutoniidae Teutonia Rutripalpidae Rutripalpus Anisitsiellidae Anisitsiellinae Bandakia 645 (continues) 646 TABLE I (Continued) Ecology Zoogeography Larvae Deutonymphs and Adults Adults Origin North American distribution Type Order of hosts Attachment site Habitat type Mode of locomotion Pangean Temperate, boreal Aquatic Diptera Thorax Rifﬂes, interstitial, pools, lakes Crawling, swimming Copepoda, cladocera, insect eggs and larvae Unknown Neomamersinae Arizonacarus North American Temperate (SW) Unknown Unknown Unknown Interstitial Unknown Unknown Meramecia South American Temperate Unknown Unknown Unknown Interstitial Unknown Unknown Neomamersa South American Temperate Unknown Unknown Unknown Interstitial Crawling, walking Crawling, walking Crawling, walking Unknown Unknown South American Temperate (SW) Unknown Unknown Unknown Interstitial Walking Unknown Unknown South American South American Temperate Temperate, boreal Unknown Aquatic Unknown Diptera Unknown Abdomen Rifﬂes Springs, pools, lakes Crawling Crawling Unknown Diptera eggs and larvae Unknown Unknown Gondwanan Temperate (SW) Unknown Unknown Unknown Interstitial Crawling Unknown Unknown Laurasian Temperate, boreal Aquatic Diptera Abdomen Crawling, walking Temperate Temperate Boreal, temperate Unknown Unknown Aquatic Unknown Unknown Abdomen Laurasian Boreal Unknown Unknown Unknown Diptera, Trichoptera Unknown Unknown Crawling Walking Crawling, swimming Crawling Diptera larvae, Ostracoda, Cladocera Unknown Unknown Diptera larvae, Ostracoda Unknown Unknown Gondwanan Gondwanan Laurasian Springs, rifﬂes, interstitial, pools, lakes Rifﬂes Interstitial Springs, rifﬂes, pools, lakes Rifﬂes, pools Unknown Pangean Temperate Aquatic Abdomen Pools, lakes Swimming Diptera larvae Unknown Neumania Pangean Temperate, boreal Aquatic Diptera, Trichoptera Diptera Abdomen Pools, lakes Swimming Diptera larvae, Copepoda Dissociation, association Unionicolinae Unionicola Pangean Temperate, boreal Aquatic Diptera, Trichoptera Abdomen, legs Pools, lakes, mollusc parasites Crawling, swimming Copepoda, Cladocera, Diptera larvae, mollusc tissue Dissociation, association, copulation Taxa Limnesia Protolimnesiinae Protolimnesia Tyrrelliinae Neotyrrellia Tyrrellia Omartacaridae Omartacarinae Omartacarus Hygrobatidae Atractides Corticacarus Diamphidaxona Hygrobates Mesobates Unionicolidae Pionatacinae Koenikea Prey Spermatophore transfer mode Unknown Unknown Dissociation Feltriidae Feltria Laurasian Temperate, boreal Aquatic Diptera Abdomen Springs, rifﬂes, interstitial Crawling Diptera larvae Copulation Laurasian Temperate, boreal Aquatic Diptera Abdomen Pools, lakes Swimming Unknown Dissociation Laurasian North American Temperate (W) Temperate (SW) Unknown Unknown Unknown Unknown Unknown Unknown Interstitial Interstitial Walking Walking Unknown Unknown Unknown Unknown Pangean Temperate (W) Unknown Unknown Unknown Interstitial Walking, swimming Unknown Unknown Laurasian Temperate, boreal Aquatic Diptera Abdomen Pools, lakes Diptera larvae Copulation Laurasian Boreal Aquatic Diptera Abdomen Springs, pools Unknown Copulation Laurasian Temperate Aquatic Diptera Abdomen Springs, interstitial Crawling, swimming Crawling, swimming Crawling Diptera larvae Copulation Laurasian Boreal Aquatic Diptera Abdomen Lakes (profundal) Swimming Cladocera Unknown Laurasian Temperate, boreal Aquatic Diptera Abdomen Pools, lakes Swimming Cladocera Unknown Laurasian Temperate Aquatic Diptera Abdomen Lakes (mollusc parasite) Crawling Mollusc tissue Unknown Laurasian Throughout Aquatic Diptera Abdomen Copulation Throughout Aquatic Diptera Abdomen Crawling, swimming Crawling, swimming Diptera larvae Laurasian Springs, rifﬂes, pools Pools, lakes, temporary pools Copepoda, Cladocera, Diptera larvae Copulation North American Laurasian Boreal, temperate Boreal Aquatic Aquatic Diptera Diptera Abdomen Abdomen Pools Pools, temporary pools Swimming Swimming Copulation Copulation Tiphys Laurasian Throughout Aquatic Diptera Abdomen Pools, lakes, temporary pools Swimming Unknown Ostracoda, Cladocera, Copepoda Diptera larvae, Ostracoda, Cladocera, Copepoda Aturidae Albiinae Albia Aturinae Aturus Laurasian Temperate Aquatic Trichoptera Abdomen Pools, lakes Swimming Unknown Unknown Laurasian Temperate Aquatic Diptera Abdomen Crawling Diptera larvae Copulation Laurasian Laurasian South American Temperate (W) Temperate Temperate Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Springs, rifﬂes, interstitial, pools Interstitial Rifﬂes Rifﬂes Walking Crawling Crawling Unknown Unknown Unknown Unknown Copulation Unknown Wettinidae Wettina Lethaxonidae Lethaxona Lethaxonella Frontipodopsidae Frontipodopsis Pionidae Foreliinae Forelia Pionacercus Pseudofeltria Huitfeldtiinae Huitfeldtia Hydrochoreutinae Hydrochoreutes Najadicolinae Najadicola Pioninae Nautarachna Piona Tiphyinae Neotiphys Pionopsis 647 Bharatalbia Kongsbergia Neoaturus Copulation (continues) 648 TABLE I (Continued) Ecology Zoogeography Larvae Deutonymphs and Adults Adults Origin North American distribution Type Order of hosts Attachment site Habitat type Mode of locomotion Prey Spermatophore transfer mode Laurasian Temperate Unknown Unknown Unknown Interstitial Walking Unknown Unknown Laurasian Gondwanan Laurasian Temperate (E) Temperate (E) Temperate, boreal Unknown Unknown Aquatic Unknown Unknown Diptera Unknown Unknown Abdomen Unknown Unknown Unknown Laurasian Laurasian North American Laurasian Laurasian Laurasian Gondwanan Gondwanan Temperate, boreal Temperate Boreal Temperate Temperate, boreal Boreal Temperate (W) Temperate (W) Aquatic Unknown Aquatic Unknown Aquatic Aquatic Unknown Unknown Diptera Unknown Diptera Unknown Diptera Unknown Unknown Unknown Abdomen Unknown Abdomen Unknown Abdomen Unknown Unknown Unknown Diptera larvae Unknown Diptera larvae Unknown Diptera larvae Unknown Unknown Unknown Copulation Unknown Copulation Unknown Unknown Unknown Unknown Unknown Woolastookia Laurasian Temperate, boreal Aquatic Diptera Abdomen Rifﬂes, pools, lakes Walking Walking Walking, swimming Swimming Walking Swimming Walking Walking Walking Walking Walking, swimming Walking, swimming Unknown Unknown Diptera larvae Brachypoda Erebaxonopsis Estellacarus Javalbia Ljania Neobrachypoda Stygalbiella Submiraxona Interstitial Interstitial Rifﬂes, interstitial, pools, lakes Pools Interstitial Pools, lakes Interstitial Rifﬂes Springs, pools Interstitial Rifﬂes, pools Diptera larvae Copulation Laurasian Throughout Aquatic Diptera Abdomen Pools, lakes Swimming Diptera larvae Copulation North American Temperate (W) Unknown Unknown Unknown Interstitial Walking Unknown Unknown Laurasian Temperate Aquatic Trichoptera Thorax, abdomen Pools, lakes Swimming Diptera larvae Unknown Laurasian Temperate Aquatic Trichoptera Abdomen Interstitial Walking Unknown Unknown North American Pangean Pangean Temperate (W) Temperate, boreal Temperate, boreal Unknown Aquatic Aquatic Unknown Diptera Diptera Unknown Thorax Thorax Springs Springs, interstitial Springs, interstitial Crawling Crawling Crawling Unknown Unknown Unknown Unknown Unknown Unknown Laurasian Temperate, boreal Aquatic Diptera Thorax Springs, rifﬂes, interstitial, pools, lakes Crawling, walking, swimming Diptera larvae Unknown Taxa Phreatobrachypoda Axonopsinae Albaxona Axonopsella Axonopsis Arrenuroidea Mideidae Midea Momoniidae Cyclomomoniinae Cyclomomonia Momoniinae Momonia Stygomomoniinae Stygomomonia Nudomideopsidae Neomideopsis Nudomideopsis Paramideopsis Mideopsidae Mideopsinae Mideopsis Chappuisididae Chappuisidinae Chappuisides Morimotacarinae Morimotacarus Yachatsia Uchidastygacarinae Uchidastygacarus Neoacaridae Neoacarus Volsellacarus Bogatiidae Horreolaninae Horreolanus Acalyptonotidae Paenecalyptonotus Acalyptonotus Athienemanniidae Athienemanniinae Chelohydracarus Chelomideopsis Platyhydracarus Stygameracarinae Stygameracarus Amoenacaridae Amoenacarus Arenohydracaridae Arenohydracarus Laversiidae Laversia Krendowskiidae Geayia Krendowskia Arrenuridae Arrenurinae Arrenurus Laurasian Temperate Unknown Unknown Unknown Interstitial Walking Unknown Unknown Laurasian Laurasian Temperate (W) Temperate (W) Unknown Unknown Unknown Unknown Unknown Unknown Interstitial Interstitial Walking Walking Unknown Unknown Unknown Unknown Laurasian Temperate Unknown Unknown Unknown Interstitial Walking Unknown Unknown Laurasian Temperate, boreal Aquatic Diptera Thorax Walking Unknown Unknown North American Temperate Aquatic Diptera Thorax Rifﬂes, interstitial, lakes Rifﬂes, interstitial Walking Unknown Unknown North American Temperate Unknown Unknown Unknown Interstitial Walking Unknown Unknown North American Laurasian Boreal Boreal Aquatic Aquatic Unknown Diptera Unknown Thorax Springs Springs, pools, lakes Crawling Walking Unknown Unknown Unknown Unknown Laurasian Laurasian North American Temperate (W) Temperate, boreal Temperate (W) Unknown Aquatic Aquatic Unknown Diptera Diptera Unknown Thorax Thorax Interstitial Springs, rifﬂes Springs, rifﬂes, interstitial, pools Crawling Crawling Crawling Unknown Diptera larvae Unknown Unknown Unknown Unknown North American Temperate Unknown Unknown Unknown Interstitial Walking Unknown Unknown North American Temperate (E) Unknown Unknown Unknown Pools Swimming Unknown Unknown Gondwanan Temperate (SW) Unknown Unknown Unknown Interstitial Crawling Unknown Unknown North American Temperate, boreal Aquatic Diptera Thorax Springs, rifﬂes, lakes (profundal) Crawling Unknown Unknown Gondwanan Pangean Temperate Temperate Aquatic Aquatic Diptera Diptera Abdomen Abdomen Pools, lakes Pools, lakes Swimming Swimming Unknown Unknown Unknown Unknown Pangean Throughout Aquatic Diptera, odonata Thorax, abdomen Springs, rifﬂes, interstitial, pools, lakes, temporary pools Walking, swimming Ostracoda, Cladocera, Copepoda, Insect larvae Copulation 649 650 Ian M. Smith, David R. Cook, Bruce P. Smith III. OTHER ARACHNIDS IN FRESHWATER HABITATS A. Other Mites (ACARI) 1. Acariformes a. Other Actinedida The mainly marine family Halacaridae is represented in both benthic and interstitial freshwater habitats in North America by species of the holarctic genera Porohalacarus, Lobohalacarus, Stygohalacarus, Soldanellonyx and Porolohmanella (Fig. 431). Halacarid mites have stilettoform chelicerae and 5-segmented pedipalps. Adults usually have four characteristic dorsal plates on the idiosoma and claws on the tarsi of the legs. All instars have a sprawling gait and crawl slowly on the substrate. Halacarid feeding habits are generally poorly understood, but predaceous, parasitic and algivorous species are known. Members of some species may be extremely abundant in mats of vegetation and detritus in ponds and small lakes. There are no comprehensive references for North American freshwater halacarid mites. All known taxa of freshwater Halacaridae were reviewed by K. H. Viets (1956) and K. O. Viets (1987) and species of the German fauna, including all of the holarctic genera, were treated by K.H. Viets (1936). Homocaligidae is a small family of uncommon species inhabiting wet vegetation and detritus at the margin of ponds. Adults are well sclerotized and have prominent hemispherical dorsal shields and long, protruding cheliceral stylets. These mites frequently venture beneath the surface, apparently aided by accessory respiratory sacs and tubes. They have long legs and crawl slowly on the substrate, but are thought to be predaceous (Krantz 1978). Homocaligid taxa, including the North American species Homocaligus muscorum Habeeb, were reviewed by Wood (1969). b. Oribatida The vast majority of oribatid mite taxa are strictly terrestrial, but members of certain genera of the families Camisiidae, Trhypochthoniidae, Malaconothridae, Hydrozetidae, Limnozetidae (Fig. 432), Ameronothridae, Tegeocranellidae and Zetomimidae are found only in freshwater habitats ranging from springs and streams to large lakes. Oribatid mites have chelate chelicerae and 5-segmented pedipalps. Adults are variously sclerotized, and usually have distinctively shaped prodorsal bothridia and claws on the tarsi of the legs. All active instars of oribatid mites crawl slowly on the substrate, feeding on both living and dead plant and fungal material. The feeding habits of a species of Hydrozetes (Hydrozetidae) were investigated by Baker (1985). Members of some species this genus may be very numerous in mats of plant material and detritus. Freshwater genera of oribatid mites can be identiﬁed using the keys published by Balogh and Balogh (1992a, b). Palearctic taxa, many of which are holarctic, were treated by Balogh and Mahunka (1983). There is no comprehensive reference for North American freshwater oribatid taxa, but systematic treatments have recently been published for the genera Mucronothrus (Trhypochthoniidae) (Norton, Behan-Pelletier and Wang, 1996), Limnozetes (Limnozetidae) (BehanPelletier, 1989) and Tegeocranellus (Tegeocranellidae) (Behan-Pelletier, 1997). c. Acaridida Most acaridid mites are terrestrial saprophages, phytophages, fungivores or parasites, but certain free-living species of the families Histiostomatidae and Hyadesiidae inhabit freshwater. Adult acaridids are generally soft-bodied, have 2-segmented pedipalps and lack both prodorsal trichobothria and claws on the tarsi of the legs. Freshwater species are slowmoving crawlers typically associated with algal mats or decaying detritus, although some have been reported from water reservoirs in pitcher plants. Some species of histiostomatid mites are reportedly predaceous on microorganisms or oligochaete worms. Families of freshwater Acaridida can be identiﬁed using the key published by Krantz (1978). 2. Parasitiformes Nearly all parasitiform taxa are strictly terrestrial, but members of a few genera of the gamasid family Ascidae, subfamily Platyseiinae (Fig. 433), live exclusively on wet plants and detritus and on the water surface of marginal freshwater habitats. Platyseiinae have chelate chelicerae and adults are long-legged mites with large dorsal and ventral shields and conspicuous peritremes laterally on the idiosoma. Active instars of these mites walk about on the surface ﬁlm, apparently feeding on ﬂoating egg masses of nematocerous ﬂies, especially mosquitoes. Adult females of Platyseiinae regularly disperse between aquatic habitats as phoretic associates of adult craneﬂies (Tipulidae). B. Spiders There are no truly aquatic spiders in North America, but many species of nurseryweb spiders (family Pisauridae) are associated with aquatic habitats (Carico, 1973). Species of Dolomedes (Fig. 434), the genus with the greatest afﬁnity for water, are large, long-legged spiders with ﬂattened, mottled bodies. They are often called ﬁshing spiders, dock spiders or raft spiders because they venture onto water to hunt and capture prey at or just below the surface ﬁlm, and can dive below the surface to escape potential preda- 16. Water Mites (Hydrachnida) and Other Arachnids tors (Dondale and Redner, 1990). Members of some species, such as Dolomedes tenebrosus, wander considerable distances from water (Carico, 1973; Dondale and Redner, 1990), and even those of the most aquatic North American species, Dolomedes triton, apparently spend a signiﬁcant part of their lives away from water (Zimmermann and Spence, 1992, 1998). The systematics and distribution of the nine described nearctic species of Dolomedes were thoroughly reviewed by Carico (1973) and the ﬁve species occurring in Canada and Alaska were treated in detail by Dondale and Redner (1990). Given the availability of these relatively recent and comprehensive taxonomic treatments, and in keeping with the level of coverage for other groups in this chapter, we do not include a key for the species of Dolomedes or the extensive morphological information that would be required to support it. The following brief summary of information is intended as an introduction to the literature on the biology and ecology of these spiders. Members of Dolomedes are typically found on rocks or wood at the edge of water or on emergent or ﬂoating vegetation. They move across the surface ﬁlm using a rowing gait and can dive beneath the surface to remain submerged for as long as 30 min (Dondale and Redner, 1990). When hunting, they typically remain motionless with the anterior 2 – 3 pairs of legs resting on the water surface. In this posture, they are able to detect vibrations and chemical stimuli indicating the direction and proximity of potential prey (Roland and Rovner, 1983). They apparently do not use visual stimuli to locate prey (Bleckmann and Lotz, 1987). Their primary prey are adults of terrestrial and aquatic insects that have fallen on the water, including Hemiptera, Diptera and Odonata (Zimmermann and Spence, 1989), but they also capture and consume small ﬁsh, tadpoles, and even small frogs, using their venom to immobilize prey as much as four times their own weight within a few minutes (Comstock, 1912; Bleckmann and Lotz, 1987; Dondale and Redner, 1990). Cannibalism apparently accounts for up to 5% of the prey of female D. triton (Zimmermann and Spence, 1989, 1992, 1998). Certain species of Dolomedes show habitat preferences that seem to be based on exposure and vegetation type. For example, members of D. scriptus are most common along larger, fast-ﬂowing streams, with exposed rocky shorelines, those of D. vittatus prefer edges of smaller and more shaded streams and individuals of D. triton and D. striatus frequent margins of ponds, lakes, and calm backwaters of streams, including ﬂoating and emergent vegetation. The various species have cryptic color patterns correlated with their habitat preferences:those commonly found on rocks or 651 logs are predominately mottled with gray and brown while those frequenting emergent vegetation have conspicuous longitudinal stripes (Carico, 1973; Dondale and Redner, 1990). Species of Dolomedes exhibit complex reproductive behavior. Females of Dolomedes triton mate only once, and because males of this species cannot distinguish between mated and unmated females they are at risk of sexual cannibalism. Males mature 5 – 10 days before females, increasing their probability of ﬁnding immature adult females and their likelihood of surviving multiple matings (Zimmermann and Spence, 1989, 1992). Males signal in the presence of females by waving the legs, drumming with the pedipalps, and twitching the body, to generate distinctive, regular, low-frequency waves in the water surface that differ from those produced by prey (Roland and Rovner, 1983; Bleckmann and Bender, 1987; Bleckmann and Lotz, 1987). They apparently also respond to pheromonal cues, because their courtship displays can be induced by water that has been in contact with conspeciﬁc females (Roland and Rovner, 1983). Sperm is transferred to receptive females by inserting the embolus, a specialized intromittent organ at the tip of the male’s pedipalp, into a bursa copulatrix in the epigynum of the female (Carico, 1973; Roland and Rovner, 1983). Gravid females of Dolomedes produce an egg sac consisting of a silk-wrapped cluster of between 250 and 1500 eggs (Comstock, 1912; Carico, 1973; Zimmermann and Spence, 1992; Spence et al., 1996), and carry it beneath their body using the chelicerae and a silken dragline until the eggs are ready to hatch (Dondale and Redner, 1990). This parental care apparently is necessary for successful hatching (Zimmermann and Spence, 1992). Shortly before the eggs begin to hatch, the female spins a nursery web, consisting of a tentlike structure with the egg sac suspended within it (Dondale and Redner, 1990). These webs are typically in low plant growth or among rocks and are vigorously defended by females against potential predators. Newly emerged juvenile spiders spend 3– 7 days in the web, and disperse some time after their ﬁrst molt either by ballooning or walking (Zimmermann and Spence, 1992, 1998). In central Alberta, Canada, members of Dolomedes triton have approximately 12 juvenile instars but older individuals may experience supernumerary molts if exposed to warmer temperatures in autumn, possibly to delay maturation until spring. Duration of instars 1 – 9 is the same for both sexes, but duration of instars 10 – 12 is signiﬁcantly longer for females, resulting in the protandry mentioned above (Zimmermann and Spence, 1998). At this latitude, D. triton is semivoltine, overwintering as instars 3 – 5 and 652 Ian M. Smith, David R. Cook, Bruce P. Smith FIGURES 431 – 434 Scanning electron micrographs of adult mites. Fig. 431 Porolohmannella sp. (Halacaridae); Fig. 432 Hydrozetes sp. (Hydrozetidae); Fig. 433 Cheiroseius sp. (Ascidae). Fig. 434 Photograph of adult female Dolomedes sp. (Pisauridae). 16. Water Mites (Hydrachnida) and Other Arachnids 9 – 11, with the ﬁrst adults appearing in mid-May. Males have an estimated average adult survival time of 9 – 13 days and disappear by the end of June, female adults apparently live for 25 – 31 days and disappear by late August or early September (Zimmermann and Spence, 1992, 1998). Disappearance of males apparently reﬂects the inﬂuence of cannibalism by females (Zimmermann and Spence, 1992). Overwintering is facultative, with the later instars undergoing quiescence based upon temperature control but the earlier instars having dormancy determined by an interaction of photoperiod and temperature cues (Zimmermann and Spence, 1998). Cannibalism is an important cause of mortality in Dolomedes populations. Interguild predation appears to be a strong selective force on juveniles of D. triton, and sexual cannibalism is correlated with the short seasonal occurrence of males of this species (Zimmermann and Spence, 1989, 1992, 1998). Members of Dolomedes are also eaten by birds (Carico, 1973) and attacked by predatory pompilid and sphecoid wasps (Carico, 1973; Dondale and Redner, 1990). Small- to intermediate-sized juveniles of these spiders are eaten by both water striders (Gerridae) and backswimmers (Notonectidae) (Zimmermann and Spence, 1998). The egg cocoons of Dolomedes are susceptible to infestation by hymenopterous parasitoids (Carico, 1973). ACKNOWLEDGMENTS We thank Michelle MacKenzie of the Biodiversity Section of Agriculture and Agri-food Canada for her expert help in assembling the plates of illustrations using computer-graphics software and for preparing many of the water mite specimens examined during preparation and testing of the keys. We also thank Drs. Valerie Behan-Pelletier, Evert Lindquist and Charles Dondale of the Biodiversity Section of Agriculture and Agri-food Canada for providing illustrations and helpful comments for the sections dealing with oribatid mites, ascid mites and spiders, respectively. LITERATURE CITED Åbro, A. 1979. Attachment and feeding devices of water-mite larvae (Arrenurus spp.) parasitic on damselﬂies (Odonata, Zygoptera). Zoologica Scripta 8:221 – 34. Åbro, A. 1982. The effects of parasitic water mite larvae (Arrenurus spp.) on zygopteran imagoes (Odonata). Journal of Invertebrate Pathology 39:373 – 81. Åbro, A. 1984. The initial stylostome formation by parasitic larvae of the watermite genus Arrenurus on zygopteran imagines. Acarologia (Paris) 25:33 – 46. 653 Åbro, A. 1990. The impact of parasites in adult populations of Zygoptera. Odonatologica 19:223 – 234. Alberti, G. 1977. Zur Feinstruktur und Funktion der Genitalnäpfe von Hydrodroma despiciens (Hydrachellae, Acari). Zoomorphologie 87:155 – 164. 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Smith, I. M. 1984. Larvae of water mites of some genera of Aturidae (Prostigmata: Hygrobatoidea) in North America with comments on phylogeny and classiﬁcation of the family. Canadian Entomologist 116:307 – 374. Smith, I. M. 1987. Water mites of peatlands and marshes in Canada. Pages 3l – 46 in Rosenberg, D. M., Danks, H. V., Eds. Aquatic insects of peatlands and marshes in Canada. Memoirs of the Entomological Society of Canada l40; l74 pp. Smith, I. M. 1989a. Description of deutonymphs and adults of Oregonacarus rivulicolus gen. nov., sp. nov. (Acari: Lebertioidea: Anisitsiellidae). Canadian Entomologist 121:533 – 541. Smith, I. M. 1989b. North American water mites of the family Momoniidae Viets (Acari: Arrenuroidea). I. Description of adults of Cyclomomonia andrewi gen. nov., sp. nov., and key to world genera and subgenera. Canadian Entomologist 121:543 – 549. Smith, I. M. 1989c. Description of two new species of Platyhydracarus gen. nov. from western North America, with remarks on classiﬁcation of Athienemanniidae (Acari: Parasitengona: Arrenuroidea). Canadian Entomologist 121:709 – 726. 658 Ian M. Smith, David R. Cook, Bruce P. Smith Smith, I. M. 1989d. North American water mites of the family Momoniidae Viets (Acari: Arrenuroidea). II. Revision of species of Momonia Halbert, 1906. Canadian Entomologist 121:965 – 987. Smith, I. M. 1989e. North American water mites of the family Momoniidae Viets (Acari: Arrenuroidea). III. Revision of species of Stygomomonia Szalay, 1943, subgenus Allomomonia Cook, 1968. Canadian Entomologist 121:989 – 1025. Smith, I. M. 1990a. Description of two new species of Stygameracarus gen. nov. from North America, and proposal of Stygameracarinae subfam. nov. (Acari: Arrenuroidea: Athienemanniidae). Canadian Entomologist 122:181 – 190. Smith, I. M. 1990b. Proposal of Nudomideopsidae fam. nov. (Acari: Arrenuroidea) with a review of North American taxa and description of a new subgenus and species of Nudomideopsis Szalay, 1945. Canadian Entomologist 122:229 – 252. Smith, I. M. 1991a. Water mites (Acari: Parasitengona: Hydrachnida) of spring habitats in Canada. Pages 141 – 167 in: Williams, D. D., Danks, H. V., Eds. Arthropods of springs, with particular reference to Canada. Memoirs of the Entomological Society of Canada 155; 217 pp. Smith, I. M. 1991b. North American water mites of the genera Phreatobrachypoda Cook and Bharatalbia Cook (Acari: Hygrobatoidea: Aturinae). Canadian Entomologist 123:465 – 499. Smith, I. M. 1991c. North American water mites of the family Momoniidae Viets (Acari: Arrenuroidea). IV. Revision of species of Stygomomonia (sensu stricto) Szalay, 1943. Canadian Entomologist 123:501 – 558. Smith, I. M. 1991d. Descriptions of new species representing new or unreported genera of Lebertioidea (Acari: Hydrachnida) from North America. Canadian Entomologist 123:811 – 825. Smith, I. M. 1992a. North American species of the genus Chelomideopsis Romijn (Acari: Arrenuroidea: Athienemanniidae). Canadian Entomologist 124:451 – 490. Smith, I. M. 1992b. North American water mites of the family Chappuisididae Motas and Tanasachi (Acari: Arrenuroidea). Canadian Entomologist 124:637 – 723. Smith, I. M. 1998. Chelohydracarus navarrensis gen. nov., sp. nov. (Acari: Hydrachnida: Athienemanniidae). International Journal of Acarology 24:327 – 330. Smith, I. M., Cook, D. R. 1991. Water mites. Chapter 16, Pages 523 – 592 in Thorp, J., Covich, A., Eds. Ecology and classiﬁcation of North American freshwater invertebrates. Academic Press, San Diego, CA, 911 pp. Smith, I. M., Cook, D. R. 1994. North American species of Neomamersinae Lundblad (Acari: Hydrachnida: Limnesiidae). Canadian Entomologist 126:1131 – 1184. Smith, I. M., Cook, D. R. 1996. New and unreported species of Neotyrrellia, Protolimnesia (Protolimnesella) and Centrolimnesia (Acari: Hydrachnida: Limnesiidae) from the United States. Anales del Instituto de Biología, Universidad Nacional Autónoma de México, Serie Zoologia 67:265 – 277. Smith, I. M., Cook, D. R. 1997. Description of Amoenacarus dixiensis gen. nov., sp. nov., and proposal of Amoenacaridae fam. nov. (Acari: Hydrachnida: Arrenuroidea). International Journal of Acarology 23:107 – 112. Smith, I. M., Cook, D. R. 1998a. The Axonopsella-like water mites of the United States (Acari: Hydrachnida: Aturidae). International Journal of Acarology. 24:311 – 325. Smith, I. M., Cook, D. R. 1998b. Three new species of water mites representing new genera of Thyadinae from North America (Acari: Hydrachnida: Hydryphantidae). International Journal of Acarology. 24:331 – 339. Smith, I. M., Cook, D. R. 1999a. North American species of Tartarothyas Viets (Acari: Hydrachnida: Hydryphantidae). International Journal of Acarology. 25:37 – 42. Smith, I. M., Cook, D. R. 1999b. North American species of Parathyasella stat. nov. and Amerothyasella gen. nov. (Acari: Hydrachnida: Hydryphantidae). International Journal of Acarology. 25:43 – 50. Smith, I. M., Cook, D. R. 1999c. An assessment of global distribution patterns in water mites (Acari: Hydrachnida). Pages 109 – 124 in: Needham, G. R., Mitchell, R., Horn, D. J., Welbourn, W. C., Eds. Acarology IX: Volume 2, Symposia. Ohio Biological Survey, Columbus, OH, xvii 507 pp. Smith, I. M., Oliver, D. R. 1976. The parasitic associations of larval water mites with imaginal aquatic insects, especially Chironomidae. Canadian Entomologist 108:1427 – 1442. Smith, I. M., Oliver, D. R. 1986. Review of parasitic associations of larval water mites (Acari: Parasitengona: Hydrachnida) with insect hosts. Canadian Entomologist 118:407 – 472. Sokolow, I. I. 1954. Khromosomnye kompleksy kleshchei i ikh zhachenie dlia sistematiki i ﬁlogenii. Trudy Leningradskogo Obschchestva Estestvois-pytatelei 72:124 – 159. Sokolow, I. I. 1977. The protective envelopes in the eggs of Hydrachnellae. Zoologischer Anzeiger 198:36 – 42. Spence, J. R., Zimmermann, M., Wojcicki, J. P. 1996. Effects of food limitation and sexual cannibalism on reproductive output of the nursery web spider Dolomedes triton (Araneae: Pisauridae). Oikos 75:273 – 382. Stechmann, D.-H. 1980. Zum Wirtskreis syntopischer ArrenurusArten (Hydrachnellae, Acari) mit parasitischer Entwicklung an Nematocera (Diptera). Zeitschrift für Parasitenkunde 62:267 – 283. Steenbergen, H. A. 1993. Macrofauna-atlas of North-Holland: distribution maps and responses to environmental factors of aquatic invertebrates. Cip-gegevens Koninkliijke Bibliotheek, Den Haag, 651 pp. Ten Winkel, E. H., Davids, C. 1985. Bioturbation by cyprinid ﬁsh affecting the food availability for predatory water mites. Oecologia 67:218 – 219. Ten Winkel, E. H., Davids, C. 1987. Chironomid larvae and their food web relations in the littoral zone of Lake Maarsseveen. Kaal Boek, Amsterdam. 145 pp. Ten Winkel, E. H., Davids, C., de Nobel, J. G. 1989. Food and feeding strategies of water mites of the genus Hygrobates and the impact of their predation on the larval population of the chironomid Cladotanytarsus mancus (Walker) in Lake Maarsseveen. Netherlands Journal of Zoology 39:246 – 263. Uchida, T. 1940. Über die Begattungsakt bei Forelia variegator. Annotationes Zoologicae Japonenses 19:280 – 282. Vidrine, M. F. 1986. Revision of the Unionicolinae (Acari: Unionicolidae). International Journal of Acarology 12:233 – 243. Viets, K. H. 1914. Über die Begattungsvorgange bei Acercus-Arten. Internationale Revue gesamten Hydrobiologie und Hydrographie, Biological Supplement 6:1 – 10. Viets, K. H. 1936. Wassermilben oder Hydracarina (Hydrachnellae und Halacaridae). Die Tierwelt Deutschlands 31:1 – 288. 32:289 – 574. Viets, K. H. 1956. Die Milben des Sußwassers und des Meeres (Hydrachnellae und Halacaridae, Acari), Teil 2 / 3. Fischer Verlag, Jena. 870 pp. Viets, K. O. 1987. Die Milben des Sußwassers (Hydrachnellae und Halacaridae [part.], Acari). 2. Katalog. Sonderbande Naturwissenschaftlichen Vereins Hamburg 8; 1012 pp. Wainstein, B. A. 1980. Opredelitel lichinok vodjanych kleshchei. (Key to larval water mites). Institut Biologii Vnutrennikh Vod, Akademiia Nauk SSSR, 238 pp. Weiberg, M, Edwards, D. D. 1997. Survival and reproductive output of Chironomus tentans (Diptera: Chironomidae) in response to parasitism by larval Unionicola foili (Acari: Unionicolidae). Journal of Parasitology 83:173 – 175. 16. Water Mites (Hydrachnida) and Other Arachnids Wesenburg-Lund, C. J. 1918. Contributions to the knowledge of the postembryonal development of the Hydracarina. Videnskabelige Meddelelser fra Dansk Naturhistorisk Forening 70:5 – 57. Wiggins, G. B., Mackay, R. J., Smith, I. M. 1980. Evolutionary and ecological strategies of animals in annual temporary pools. Archiv für Hydrobiologie, Supplement 58:97 – 206. Wiles, P. R. 1982. A note on the watermite Hydrodroma despiciens feeding on chironomid egg masses. Freshwater Biology 12:83 – 87. Wiles, P. R. 1984. Watermite respiratory systems. Acarologia 25:27 – 31. Wood, T. G. 1969. The Homocaligidae, a new family of mites (Acari: Raphignathoidea), including a description of a new species from Malaya and the British Solomon Islands. Acarologia 11:711 – 729. 659 Zimmermann, M., Spence, J. R. 1989. Prey use of the ﬁshing spider Dolomedes triton (Pisuaridae: Araneae) an important predator of the neuston community. Oecologia 80:187 – 194. Zimmermann, M., Spence, J. R. 1992. Adult population dynamics and reproductive effort of the ﬁshing spider Dolomedes triton (Araneae, Pisauridae) in central Alberta. Canadian Journal of Zoology 70:2224 – 2233. Zimmermann, M., Spence, J. R. 1998. Phenology and life-cycle regulation of the ﬁshing spider Dolomedes triton Walckenaer (Araneae, Pisauridae) in central Alberta. Canadian Journal of Zoology 76:295 – 309. This Page Intentionally Left Blank 17 DIVERSITY AND CLASSIFICATION OF INSECTS AND COLLEMBOLA1 William L. Hilsenhoff Department of Entomology University of Wisconsin Madison, Wisconsin 53706 I. Introduction A. General Morphology of Aquatic Insects B. Common Techniques for Studying Freshwater Insects II. Aquatic Orders of Insects A. Ephemeroptera — Mayﬂies B. Odonata — Dragonﬂies and Damselﬂies C. Plecoptera — Stoneﬂies D. Trichoptera — Caddisﬂies E. Megaloptera — Fishﬂies and Alderﬂies III. Partially Aquatic Orders of Insects A. Aquatic Heteroptera — Aquatic and Semiaquatic Bugs B. Aquatic Neuroptera — Spongillaﬂies C. Aquatic Lepidoptera — Aquatic Caterpillars D. Aquatic Coleoptera — Water Beetles E. Aquatic Diptera — Flies and Midges IV. Semiaquatic Collembola — Springtails V. Identiﬁcation of the Freshwater Insects and Collembola I. INTRODUCTION This chapter includes orders and families in which one or more life stages are truly aquatic and adapted for survival under or on the water surface. Families that live on or burrow into emergent aquatic vegetation, internal parasites of aquatic animals, and riparian families that are closely associated with water but do 1 Much of the material on Collembola was contributed by Barbara L. Peckarsky, Department of Entomology, Cornell University. The author and editors greatly appreciate her generous contribution of this material. Ecology and Classiﬁcation of North American Freshwater Invertebrates. 2nd Edition Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved. A. Taxonomic Key to Orders of Freshwater Insects B. Taxonomic Key to Families of Freshwater Ephemeroptera Larvae C. Taxonomic Key to Families of Freshwater Odonata Larvae D. Taxonomic Key to Families of Freshwater Plecoptera Larvae E. Taxonomic Key to Families of Freshwater Trichoptera Larvae F. Taxonomic Key to Families of Freshwater Megaloptera Larvae G. Taxonomic Key to Adults of Aquatic, Semiaquatic, and Riparian Heteroptera H. Taxonomic Key to Families of Freshwater Coleoptera I. Taxonomic Key to Families of Freshwater Diptera Larvae J. Taxonomic Key to Families of Semiaquatic Collembola Literature Cited not inhabit it are not included. Terrestrial stages of aquatic families are discussed brieﬂy, but only as they relate to the general life history of the family. In addition, brief mention is made of another arthropod group, the semiaquatic springtails (class Entognatha, order Collembola) (see Sections IV and V.J.) There are 10 orders of insects that contain aquatic species. Five of them (Ephemeroptera, Odonata, Plecoptera, Trichoptera, and Megaloptera) are “aquatic orders” in which almost all species have aquatic larvae. The remaining ﬁve orders (Heteroptera, Coleoptera, Diptera, Lepidoptera, and Neuroptera) are “partially 661 662 W. L. Hilsenhoff aquatic orders” in which most species are terrestrial, but in which there are species or entire families that have one or more life stages adapted for living in an aquatic environment. Three aquatic orders (Ephemeroptera, Odonata, and Plecoptera) have a hemimetabolous life cycle, which includes three developmental stages: egg; larva; and adult. The term “larva” is being used instead of “nymph” or “naiad,” because in these orders the immature developmental stage is adapted for survival in aquatic environments and does not resemble the terrestrial adult, except in Plecoptera. The other two aquatic orders (Trichoptera and Megaloptera) have a holometabolous life cycle, which includes four developmental stages: egg; larva; pupa; and adult. In these orders the larva bears no resemblance to the adult. Four of the ﬁve partially aquatic orders (Coleoptera, Diptera, Lepidoptera, and Neuroptera) also have holometabolous life cycles and larvae that do not resemble the adult. The ﬁfth order, Heteroptera (Hemiptera), has a paurometabolous life cycle, which includes three developmental stages: egg, nymph, and adult. In this order, nymphs inhabit the same habitat as adults and resemble them in many respects, differing mostly by being smaller and less sclerotized, having wing-pads instead of wings, and lacking genital structures FIGURE 1 Plecoptera larva (Perlidae); Acroneuria larva (dorsal) showing various structures (thoracic gills and distal segments of legs and cerci not shown). A. General Morphology of Aquatic Insects Morphological terms used for identiﬁcation of aquatic insects vary somewhat from order to order, but several terms are commonly used for all orders. A Plecoptera larva (Figs. 1 and 2) is used to illustrate these terms. Insects have three body regions: head, thorax, and abdomen. The anterior region is the head, which dorsally (Fig. 1) has a pair of antennae, a pair of compound eyes, and often three ocelli or simple eyes. On the underside of the head are several mouthparts (Fig. 2), including a labrum or upper lip, a pair of mandibles, a pair of maxillae, and a labium or lower lip. The mandibles usually are heavily sclerotized, toothlike structures. The maxillae and labium are usually multisegmented and frequently have a pair of elongate, segmented structures called palps. The thorax is immediately posterior to the head and consists of three large segments (Fig. 1): a prothorax (anterior); a mesothorax; and a metathorax (posterior). The dorsal sclerite of each segment is referred to as a notum, and the ventral sclerite as a sternum. Each segment has a pair of legs that are divided into ﬁve parts (Fig. 2): a coxa, trochanter, femur, tibia, and tarsus. The femur and tibia are usually elongate and the tarsus has one-to-ﬁve segments, depending on the FIGURE 2 Plecoptera larva (Perlidae); head and prothorax (ventral) of Acroneuria larva showing mouthparts and structures of a leg (right leg and distal segments of antennae not shown). 17. Diversity and Classiﬁcation of Insects and Collembola order. Each tarsus has one or two tarsal claws. Preﬁxes pro-, meso- and meta-refer to the respective thoracic segments. Thus, a pronotum would be on the prothorax and a mesofemur would be on the mesothorax. In hemimetabolous and paurometabolous insect larvae or nymphs, wing-pads (developing wings) are usually present dorsally on the meso- and metathorax. The abdomen is the posterior body region and may have as many as 10 visible segments, which are numbered posteriorly from the thorax. The dorsal sclerite of each segment is called a tergum, the ventral sclerite a sternum, and the lateral sclerite, when present, a pleuron. Terminal abdominal appendages vary widely or may be absent; cerci are present in Plecoptera and several other orders. In some orders, gills of various types may be present on the abdomen, thorax, or even on the head. A taxonomic key to orders of aquatic insects is provided in Section V.A. Family level keys are also provided for aquatic larvae and aquatic adults in Sections V.B – I.; keys to eggs, Heteroptera nymphs, pupae, and terrestrial stages of aquatic species are not included. The most recent keys to genera of aquatic insects in North America appear in Merritt and Cummins (1996). Regional keys to genera or species of aquatic insects include those in Usinger (1956), Brigham et al. (1982), Peckarsky et al. (1990), Clifford (1991), and Hilsenhoff (1995c). The most recent keys to aquatic stages of North American species are listed along with regional species keys in tables that appear in the discussion of each order; reliable species keys do not exist for many families and genera. Regional keys are important because they are easier to use and may be more recent than North American keys. Subsequent descriptions of new species are not listed, but should be consulted when species are being identiﬁed. References to recent revisions and descriptions of new species may be found in Merritt and Cummins (1996), bibliographies such as those published annually by the North American Benthological Society, and through computer searches of data bases such as Zoological Record and Dissertation Abstracts Online. B. Common Techniques for Studying Freshwater Insects 1. Qualitative Sampling and Rearing of Aquatic Insects A D-frame or rectangular aquatic net with a mesh size of 1 mm or less is recommended for sampling lotic habitats. Shallow, rock or gravel rifﬂes are sampled by placing the net ﬁrmly against the bottom and disturbing the substrate immediately upstream from the net with 663 one’s feet, allowing insects and debris washed into the water to be carried into the net. This is commonly called a “kick sample.” The contents of the net are emptied into a shallow white pan containing 2 to 3 cm of water, taking care not to collect so much debris that it completely obscures the bottom of the pan. Insects that swim or crawl from the debris are most easily collected with a curved forceps and placed into a collecting jar containing 70% ethanol or Kahle’s ﬂuid (15 parts 95% ethanol, 30 parts distilled water, 6 parts formalin, and 1 part glacial acetic acid). The empty net should be examined for insects that remain clinging to it. Additional samples can be collected with a net by pushing it under the stream bank or through silt and debris in pools and rinsing excess silt from the net before placing the sample in the pan. It is also important to remove pieces of decaying wood from the stream and to collect insects that crawl from the wood as it dries. Additional insects may be collected by examining stones that are temporarily removed from the stream bed. Deep areas of larger streams can be sampled by using grabs or dredges. Ponar or Ekman grabs are most commonly used, but the latter can be used only in soft sediments. Shallow lentic habitats, such as ponds, marshes, swamps, and vegetated lake margins, are also best sampled with a ﬂat-bottomed aquatic net. This is accomplished by pushing, pulling, or sweeping the net through the vegetation or debris, rinsing excess silt from the debris, and processing as described above. Additional larger collections can be processed by placing them on a half-inch (13 mm) mesh screen placed over a large white pan and allowing at least 15 min for insects to crawl from the debris and drop into the pan. This method is especially effective for collecting adult Coleoptera and Heteroptera, as is the use of bottle traps (Hilsenhoff, 1987). Wave-swept shorelines of lakes can be sampled by disturbing the sand or gravel bottom with one’s feet and immediately collecting suspended material with a net. Removal and examination of decaying wood or stones from lake shores should not be overlooked, because these substrates often harbor species that normally will not be collected with a net. Deeper waters of lakes must be sampled with grabs, dredges, or corers. Special habitats such as tree holes or pitcher plants are best sampled with a large syringe or poultry baster. Appropriate sampling methods are discussed under each order. Since immature stages in many families of aquatic insects cannot be identiﬁed to species, it is often necessary to rear them to the adult stage for identiﬁcation. This can be readily accomplished in most orders, but the presence of a terrestrial pupal stage in Coleoptera, Megaloptera, Neuroptera, and some Diptera makes 664 W. L. Hilsenhoff rearing of larvae in these orders more difﬁcult. Chapters on qualitative and quantitative sampling techniques and rearing procedures appear in Merritt and Cummins (1996), Downing and Rigler (1984), McCafferty (1981), and Usinger (1956). 2. Preserving and Storing Aquatic Insects Aquatic insects can most conveniently be killed and preserved by placing them in a jar containing a liquid preservative. Many preservatives have been recommended and used, with 70 – 80% ethanol or isopropanol, or Kahle’s ﬂuid being most popular. The latter penetrates insect larvae more rapidly and preserves some colors better, but should be replaced with 70% ethanol after one or two days. For permanent storage, 70% ethanol containing 3% glycerine is recommended, especially for larvae. The glycerine serves to preserve larvae if the alcohol evaporates due to a poorly ﬁtting stopper. Most aquatic entomologists store specimens in patent-lip vials with neoprene stoppers. Screw-cap vials and cork-stoppered shell vials should be avoided because of gradual evaporation of the alcohol, although screw-cap vials with bevelled polyethyene inserts are satisfactory. One-dram shell vials with polyethylene stoppers, — commonly used for liquid scintillation studies, — are an inexpensive substitute for neoprene stoppered vials. Initially, quality control of these polyethylene-stoppered vials was poor and many leaked, but those purchased in recent years do not leak and retain ﬂuid as well as neoprene-stoppered vials. They have a wider opening than patent-lip or screw-cap vials, which facilitates removal of insects. Because they are optically clear, insects that are stored in these vials can be examined and often identiﬁed under a microscope while still in the vial, if the vial is only half-ﬁlled with alcohol so that a meniscus or bubble does not interfere with viewing. Adult aquatic Coleoptera and Heteroptera are normally preserved on pins, but study collections can be preserved in liquid, with several of the same species kept in a single vial to reduce storage space and curating time. Adult Odonata are preserved on pins or in envelopes. Adults of other orders also may be pinned, but are most frequently preserved in alcohol. Storage in liquid also facilitates removal of genitalia for study, which is important for identiﬁcation of many species. For examination under a dissecting microscope, specimens stored in liquid should be placed in a Syracuse watch glass or similar container and completely covered with 95% alcohol. If 70% alcohol is used, insects may ﬂoat and the alcohol will become cloudy as it evaporates. If insects are not completely covered with alcohol, a meniscus will cause distortions. A layer of very small glass beads or silica sand in the bottom of the watch glass permits insects to be imbedded for viewing from any angle. 3. An Introduction to Biological Literature A discussion of the general biology and habitat of insects in each order and family is provided in Section II, but it is often difﬁcult to make generalizations at the family level. Literature on biology of the numerous genera and species is voluminous. References to biological literature pertaining to life histories and behavior of species in each genus can be found in Merritt and Cummins (1996) and in Current and Selected Bibliographies on Benthic Biology that is published each year by the North American Benthological Society. Several review articles treat speciﬁc aspects of biology such as drift of stream insects (Waters, 1972), predator – prey relationships (Bay, 1974), detritus processing (Anderson and Sedell, 1979), and ﬁlter feeding (Wallace and Merritt, 1980). II. AQUATIC ORDERS OF INSECTS A. Ephemeroptera — Mayﬂies With only about 675 species in 20 families known from North America, this is a relatively small hemimetabolous order, but it is widespread and abundant in most regions. Larvae of all species are aquatic, and while the great majority inhabit streams, several inhabit a variety of permanent and temporary lentic habitats. Ephemeroptera larvae can be found in streams of all sizes and water temperatures, and even occur in temporary streams. In lakes, they occur among vegetation in littoral areas and sometimes also may be found on the bottom in deep water or along waveswept shores. Frequently, they occur in vernal or permanent ponds and marshes. Larvae of the various families often differ so greatly from one another that they can be easily identiﬁed to family in the ﬁeld. They are frequently an important source of food for ﬁsh in streams. Because of the wide range of dissolved oxygen requirements among the species, they are very important in biological monitoring of streams. Some species that develop in lakes or large rivers may be sufﬁciently abundant that emerging subimagos (pre-adults) and adults create severe nuisance problems. Except in warm waters of the southern United States, most species are univoltine, with eggs either hatching shortly after being laid and larvae developing slowly over a 1-year period (slow seasonal life cycle), or with eggs diapausing, followed by rapid development of the larvae after hatching several months later (fast seasonal life cycle). Other species are bivoltine, 17. Diversity and Classiﬁcation of Insects and Collembola with rapid larval development and with eggs of the second generation diapausing over the winter. A few species are multivoltine or semivoltine, with multivoltinism being especially prevalent in the South. The number of larval instars varies widely among species, and even within a species, with 12 or more having been recorded for species that have been studied. After completion of larval development, the larva either crawls from the water or swims or ﬂoats to the surface where the subimago emerges. This winged subadult or “dun” stage is unique among insects. Subimagos of most species ﬂy to nearby vegetation or supports where they spend about a day before molting into the adult (imago) stage. A few species molt almost immediately to the adult, sometimes while in ﬂight. Since most subimagos are sexually mature, some females never molt to the adult. Adult mayflies do not feed, and generally live only a few days. A few may live 2 weeks or more, and some live only an hour or two. Mating normally takes place when adult males form an aerial swarm to attract females and seize them when they enter the swarm. In some species, adult males mate with subimago females as they emerge from the water. Eggs are laid mostly on the water surface by females that repeatedly touch the water with their abdomen while in flight or by females alighting briefly on the surface to oviposit. Females of some species enter the water on a substrate to oviposit on substrates beneath the water. Most females die on the water surface upon completion of oviposition. Mayfly larvae can be readily distinguished from other aquatic insects by their long, filamentous caudal filaments and the presence of lateral or ventrolateral gills on most of the first seven abdominal segments (Fig. 29). Usually three caudal filaments are present (a pair of cerci and a median filament); in a few species the median filament is so reduced that only cerci are visible. Larvae most closely resemble those of Plecoptera, which always have only cerci, lack gills on the middle abdominal segments, and have two tarsal claws (Figs. 44 and 45). Ephemeroptera larvae have compound eyes, ocelli, filamentous antennae, and 1-segmented tarsi with a single claw (Figs. 9 and 29). Subimagos and adults differ greatly from the larvae, having one or two pairs of wings that they hold vertically above the body when at rest, very large compound eyes, and no gills or functional mouthparts. In the subimago, unmarked areas of the wings are opaque; in adults, these areas are transparent. While mayﬂy larvae mostly crawl on the substrate, many are able and rapid swimmers. They swim by moving their abdomen and caudal ﬁlaments up and 665 down rather than horizontally as stoneﬂy do. Their gills aid signiﬁcantly in the uptake of oxygen, and larvae of most species are capable of moving their gills to increase water circulation. This enables larvae of some species to inhabit lentic habitats or lotic habitats that are less rich in oxygen. Almost all mayﬂy larvae are herbivores or detritivores; larvae of a few species are known to be predators on other invertebrates. Subimagos and adults do not feed. Larvae may be readily collected with a net by techniques already described. Those that inhabit deep water must be collected with grabs, and those in shallow sand or silt areas can only be obtained by sieving these substrates. Subimagos and adults may be collected by sweeping stream-side vegetation, and large numbers are often collected at lights. Since the ﬁrst edition (Thorp and Covich, 1991), there have been several changes in the higher classiﬁcation of Ephemeroptera, with the addition of ﬁve new families (McCafferty, 1991). The order was divided into three suborders by McCafferty (1991). Rectrachaeta, Setisura, and Pisciforma, which replace the former suborders Pannota and Schistonota. Although new species and genera are still being discovered, recent revisions have often synonymized species, sometimes resulting in a reduction of the number of known species. In general, larvae are poorly known and cannot be reliably identiﬁed to species in most families. Early books by Needham et al. (1935), Burks (1953), and Berner (1959) provided an essential background for more recent studies. The book by Edmunds et al. (1976) provides generic keys to adults and larvae as well as information on collecting, rearing, and mayﬂy biology; generic keys to adults and larvae were updated by G. F. Edmunds, Jr. and R. D. Waltz in Merritt and Cummins (1996). The biogeography and evolution of Ephemeroptera was reviewed by Edmunds (1972), and Brittain (1982) reviewed the biology of this order. A key to the families of freshwater Ephemeroptera larvae is presented in Section V.B. and references to species keys for identiﬁcation of Ephemeroptera larvae are provided in Table I. Information on the biology, habitat, and identiﬁcation of each family is presented next. Suborders and families are arranged alphabetically in this and subsequent orders. 1. Families of Ephemeroptera — Suborder Pisciforma a. Acanthametropodidae (2 Genera, 2 Species) This family was formerly included in Siphlonuridae. The large larvae inhabit sandy bottoms of medium-to-large rivers where they feed primarily on larval Chironomidae. The rare and difﬁcult-to-collect larvae are recognized by their bowed tibiae and tarsi and very long, slender claws. One species occurs from Saskatchewan 666 W. L. Hilsenhoff TABLE I Literature References to Species Keys for Identiﬁcation of Ephemeroptera Larvae to Utah, the other from Wisconsin to South Carolina. Life cycles are probably one year or longer. Regional keys to larvae that include more than one family California — Day (1956) Florida — Berner and Pescador (1988) Illinois — Burks (1953) North and South Carolina — Unzicker and Carlson (1982) United States — (Ephemeroidea) McCafferty (1975) Keys to larvae listed by family Ameletidae — (Alberta) Zloty and Pritchard (1997) Ametropodidae — Allen and Edmunds (1976) Baetidae Acentrella — McCafferty et al. (1994) Americabaetis — Waltz and McCafferty (1999) Baetis, Acerpenna, Diphetor, Labiobaetis — Morihara and McCafferty (1979); (Wisconsin) Bergman and Hilsenhoff (1978) Baetodes — McCafferty and Provonsha (1993) Callibaetis — Check (1982) Camelobaetidius — Lugo-Ortiz and McCafferty (1995); Wieresma (1998) Cloeodes — Waltz and McCafferty (1987a) Fallceon — Lugo-Ortiz et al. (1994) Labiobaetis — McCafferty and Waltz (1995) Pseudocentroptiloides — Waltz and McCafferty (1989); Wiersema and McCafferty (1998) Baetiscidae — Pescador and Berner (1981); (Wisconsin) Hilsenhoff (1984a) Behningiidae — Hubbard (1994) Caenidae Brachycercus — (Northeast) Burian et al. (1997) Brachycercus and Cercobrachys — Soldan (1984) Caenis — Provonsha (1990) Ephemerellidae Attenella — Allen and Edmunds (1961b) Caudatella — Allen and Edmunds (1961a) Drunella — Allen and Edmunds (1962b) Ephemerella — Allen and Edmunds (1965) Eurylophella — Allen and Edmunds (1963b); Funk and Sweeney (1994) Serratella — Allen and Edmunds (1963a) Timpanoga — Allen and Edmunds (1959); Allen and Edmunds (1962a); McCafferty (1977) Heptageniidae — (Wisconsin) Flowers and Hilsenhoff (1975) Heptagenia — (Rocky Mountains) Bednarik and Edmunds (1980) Macdunnoa — Flowers (1982) Stenonema — Bednarik and McCafferty (1979) Isonychiidae — Kondratieff and Voshell (1984) Leptohyphidae Leptohyphes — Allen (1978); (southwestern United States) Allen and Murvosh (1987) Leptophlebiidae Neochoroterpes — Henry (1993) Paraleptophlebia — (Oregon) Lehmkuhl and Anderson (1971) Thraulodes — Allen and Brusca (1978) Traverella — Allen (1973) Neoephemeridae — Berner (1956); Bae and McCafferty (1998) Oligoneuriidae Homoeoneuria — Pescador and Peters (1980) Potamanthidae — Bae and McCafferty (1991) b. Ameletidae (1 Genus, 32 Species) This family also was formerly included in Siphlonuridae. Larvae are excellent swimmers that inhabit small, rapid streams where they are mostly found clinging to vegetation or debris. They are recognized by their small, oval, lamellate gills that have a sclerotized mesal or lateral band (Fig. 30), short antennae, and lack of posterolateral spines on the apical abdominal segment. Life cycles are probably mostly univoltine, with larvae overwintering, and emergence from February to September, depending on latitude. c. Ametropodidae (1 Genus, 3 Species) Larvae are relatively large and ﬂat, with very long meso- and metathoracic claws and shorter prothoracic claws with long spines (Figs. 21 and 22). They partially burrow into sand in eddies of medium-to-large streams in western and north-central North America, and are generally rare. Larvae have a unique method for feeding on suspended algae and detritus, which they gather with their prothoracic legs from a vortex created in a pit that they construct in the sand. Their life cycle is 1 year, with a relatively long emergence period in late spring or early summer. d. Baetidae (18 Genera, 146 Species) Larvae are relatively small, active swimmers, with lamellate abdominal gills (Fig. 29). They differ from larvae of other species with lamellate gills on most of the ﬁrst seven abdominal segments in having relatively long antennae (mostly twice the width of head), usually no pronounced posterolateral spines on abdominal segment 9, and a median caudal ﬁlament that is often shorter than the cerci, or vestigial (Fig. 29). Baetidae is a widespread and abundant family that occurs in a variety of streams, and also in permanent and temporary ponds or littoral zones of lakes. Lotic species are sometimes univoltine, but more frequently bivoltine; most species overwinter as diapausing eggs. Emergence of various cohorts and species occurs from early spring to late autumn. Lentic species are often multivoltine. Recent studies of larvae (Waltz and McCafferty, 1987a – c, 1989; McCafferty et al., 1994; McCafferty and Waltz, 1995; Lugo-Ortiz and McCafferty, 1998; Lugo-Ortiz et al., 1999) have led to changes in the placement of species within genera. Northern populations of some species are parthenogenetic. e. Metretopodidae (2 Genera, 9 Species) Larvae reach a relatively large size and have lamellate gills, very long meso- and metatarsal claws (Fig. 21), and 17. Diversity and Classiﬁcation of Insects and Collembola shorter protarsal claws that are distinctively biﬁd (Fig. 20). They are excellent swimmers and may be found along banks and among vegetation in small-to-large streams and occasionally along shores of lakes. They occur in Canada, the upper Midwest, and in the East. Their life cycle is 1 year, with emergence in the spring or summer, depending on the species. f. Pseudironidae (1 Genus, 1 Species) This family was formerly included in Heptageniidae. Larvae are apparently rare in sand bottoms of larger streams where they prey on larvae of Chironomidae. Larvae have a somewhat ﬂattened head with dorsal eyes and antennae, as in Heptageniidae. They can be recognized by their long slender legs and claws and their unique gill lamellae (Fig. 19), which have a ﬁbrilliform basal tuft and a ventral ﬁngerlike projection. Life cycles are probably univoltine. g. Siphlonuridae (4 Genera, 26 Species) Most larvae are relatively large with lamellate gills (Fig. 28). They most closely resemble larvae of Baetidae, but larvae are usually larger, have short antennae, and pronounced spines on the posterolateral angles of the ninth abdominal segment. Larvae are mostly herbivores – detritivores, but those of some species include insects in their diet. Larvae occur in vernal forest pools, stream-side pools, marshes, and swamps. Those of some species inhabit streams, but move into small pools just before emergence. Most, if not all, species are probably univoltine. 2. Families of Ephemeroptera — Suborder Rectrachaeta a. Baetiscidae (1 Genus, 12 Species) Larvae are distinctive, having a mesonotal carapace that covers most of the abdomen and all abdominal gills (Fig. 9). They occur throughout eastern and central North America, and are rare or absent in the West. Larvae partially burrow into silty margins or eddies of streams and clean lakes, especially where the primary substrate is sand. All species probably are univoltine and overwinter as larvae. Larvae crawl from the water onto a substrate to emerge, with emergence occurring in spring or early summer. b. Behningiidae (1 Genus, 1 Species) This family occurs mostly in the extreme southeastern United States, with a single record from northwestern Wisconsin. The unusual larvae burrow in the clean shifting sands of larger streams with a fairly rapid current. The relatively large larvae are unique, possessing crowns of bristles on the head and pronotum (Fig. 10), having ventrolateral ﬁlamentous gills, and lacking claws on their legs, 667 which are highly modiﬁed for digging. The life cycle is 2 years, with a very long diapausing egg stage followed by slow larval growth and emergence in late April and early May. Unlike most other mayﬂies, larvae are predators, feeding mostly on larvae of Chironomidae. c. Caenidae (4 Genera, 24 Species) The small larvae are easily recognized by their nearly square operculate gills that are not mesally fused (Fig. 14). They are widespread and common in a wide variety of lotic and lentic habitats, including streams of all sizes, spring seeps, marshes, swamps, ponds, and lakes. Here, they frequent the sediments and often are partially covered with silt. Larvae are generally more tolerant of low levels of dissolved oxygen than those in any other mayﬂy family. Life cycles are poorly known. In the North, most species are probably univoltine with overlapping cohorts; in the South, two or more generations per year are likely. Subimagos emerge at the surface, ﬂy to a support, and molt immediately to adult. Adults, which live only a few hours, swarm and mate shortly after ecdysis. d. Ephemerellidae (8 Genera, 98 Species) Larvae are recognized by the absence of gills on abdominal segment 2, and lamellate or operculate gills on segments 3- or 4 – 7 (Fig. 15). Larvae of most species inhabit clean streams, where they are often abundant in leaf litter, eddies, or near the banks. Those of a few species may persist in organically enriched streams, and others may be found on wave-swept lake shores. While most larvae are herbivores – detritivores, larvae of a few species are omnivorous, also feeding on small invertebrates. Most species are univoltine, with a spring emergence period and a summer egg diapause, or with emergence in summer and no egg diapause. Subimagos emerge from the surface or from protruding substrates. e. Ephemeridae (4 Genera, 17 Species) Larvae are recognized by their relatively large size, ﬁlamentous gills that extend upward over their back (Fig. 4), mandibular tusks that curve upward and outward, and metatibiae with an acutely pointed apex (Fig. 8). Larvae burrow into sand or eddies in rifﬂe areas of smallto-medium-sized streams or inhabit silt bottoms of medium-to-large streams; they also inhabit sandy or silt substrates in relatively clean lakes. In lakes and large rivers, they may occur in such tremendous numbers that synchronized emergences of subimagos create severe nuisance problems. Most species are apparently univoltine with emergence mostly in the summer, but 2-year life cycles are known to occur in Canada. Larvae are primarily ﬁlter feeders, which circulate water 668 W. L. Hilsenhoff through their burrows; they also eat algae and detritus on the substrate surface. Species previously included in Palingeniidae are again included in this family. f. Leptohyphidae (2 Genera, 44 Species) This family was previously named Tricorythidae. Larvae are recognized by their triangular (Fig. 11) or oval (Fig. 12) operculate gills on abdominal segment 2, which are well separated distally, and by the absence of fringed margins on other gills as found in Caenidae and Neoephemeridae. Larvae are widespread, inhabiting detritus, silt, and gravel in streams of all sizes; larvae of some species are quite resistant to lowered levels of dissolved oxygen. Life cycles are univoltine to multivoltine, with eggs often diapausing over winter and emergence occurring in spring and summer after rapid development of each generation. g. Leptophlebiidae (10 Genera, 82 Species) The medium-sized larvae characteristically have gills on segments 2 – 6 that are forked, bilamellate and pointed apically, or terminating in ﬁlaments (Figs. 25 – 27). The gills differ markedly from the lamellate, operculate, or fringed gills found in other families. Larvae inhabit slow or fast currents in a variety of streams where they are found among debris, on rocks or wood, or in gravel. While predominantly lotic, larvae of at least one species migrate upstream in spring and leave the stream to enter adjacent ponds or marshes prior to emergence. Most species are univoltine, with eggs diapausing over winter or summer and adults emerging in spring, summer, or autumn; bivoltine and multivoltine life cycles occur in the South. h. Neoephemeridae (1 Genus, 4 Species) This relatively uncommon family is conﬁned to the East. Larvae have square operculate gills and resemble Caenidae, but the gills are fused mesally (Fig. 13), larvae grow to a larger size than Caenidae larvae, and have metathoracic wing-pads, which Caenidae larvae lack. Larvae cling to vegetation, debris, or undersides of rocks in slow to fairly rapid streams. They apparently are univoltine with a slow seasonal development and emergence in spring or early summer, depending on latitude. i. Polymitarcyidae (4 Genera, 8 Species) Larvae in this small family of burrowing mayﬂies differ from other burrowing mayﬂies by having mandibular tusks that are curved inward and downward and are covered with small spines. Unlike Ephemeridae larvae, the metatibiae are rounded apically (Fig. 7). They inhabit medium-to-large streams and lakes, burrowing into silt, clay, or silt – gravel substrates; frequently they occur in rifﬂes. Larvae circulate water through their burrows and ﬁlter out algae and detritus upon which they feed; they also graze on algae and detritus from the substrate. All species apparently are univoltine, with synchronized emergences during the summer months. In some species there is a prolonged winter egg diapause. j. Potamanthidae (1 Genus, 5 Species) While having mandibular tusks and ﬁlamentous gills like other burrowing mayﬂy larvae, gills of Potamanthidae larvae are horizontal instead of arched over the back, and the legs also are spread more horizontally and not modiﬁed for digging (Fig. 6). They occur in medium-to-large streams where they most often sprawl on gravel and sand in shallow runs. They have a 1-year life cycle with emergence during the summer months. 3. Families of Ephemeroptera — Suborder Setisura a. Arthropleidae (1 Genus, 1 Species) This family was formerly included in Heptageniidae. Larvae have dorsal antennae and eyes and were previously placed in the family Heptageniidae, but they differ in having extremely long maxillary palps that curve around both sides of the head and have long setae (Fig. 18). Larvae inhabit shallow ponds, bogs, or channels with almost no ﬂow. Eggs hatch in spring and larvae develop rapidly with emergence in late spring. The life cycle is univoltine with a long egg diapause. Larvae use their long maxillary palps to strain plankton from the water for food. They are generally uncommon, occurring in northeastern North America. b. Heptageniidae (14 Genera, 135 Species) The distinctive larvae are readily identiﬁed by their ﬂattened appearance and the dorsal location of their eyes and antennae (Fig. 16). Larvae in some genera lack the median caudal ﬁlament. They are widespread and abundant in streams; some also occur on wave-swept shorelines of lakes. Larvae inhabit rocks, wood, debris, and other substrates to which they cling. Most species are probably univoltine, but a few may be bivoltine, especially in the South. Emergence occurs from spring to autumn, depending on the species, with most emerging in the summer. c. Isonychiidae (1 Genus, 17 Species) This family was formerly included in Oligoneuriidae. The large larvae are strong swimmers and fairly common in rapidly ﬂowing streams where they cling to rocks or debris. They are recognized by the double row of long setae on the inner margins of the profemurs and protibiae (Fig. 23), which they use to gather algae and small insects from the current. They also have gill tufts at the base of the procoxae (Fig. 23) and bilamellate gills with a ﬁbrilliform tuft, which separate them from 17. Diversity and Classiﬁcation of Insects and Collembola Oligoneuriidae. Life cycles are mostly bivoltine or univoltine, depending on the latitude. d. Oligoneuriidae 2 Genera, 8 Species) Larvae have two rows of long setae on the inner margin of the prothoracic legs, which distinguish them from larvae of other mayﬂies, except those of Isonychiidae. Gills are small and either rounded or elongate (Fig. 24), quite unlike in larvae of Isonychiidae. They use the setae on their prothoracic legs to ﬁlter diatoms and other algae from the current. Larvae are found either in sandy bottoms of streams or on sticks or rocks. Depending on latitude and species, life cycles are univoltine to multivoltine with emergence during the summer months. B. Odonata — Dragonﬂies and Damselﬁes This relatively small hemimetabolous order has two distinctive suborders in North America: Anisoptera (dragonﬂies), and Zygoptera (damselﬂies). Only about 434 species are known from North America. Larvae of all species are aquatic, with about twothirds of them living in lentic habits and one-third in lotic environments. Larvae of lotic species occur in all types of permanent stream habitats, including gravel and rock rifﬂes, debris along banks, bank vegetation, soft sediments, and sand. Those of lentic species inhabit permanent and temporary ponds, marshes, swamps, and littoral and shoreline areas of lakes. Because all larvae attain a relatively large size and are predators, mostly on other insects, they are often important apex predators in the invertebrate community. Adults are also predators, feeding on mosquitoes and other ﬂying insects, and thus Odonata are regarded as highly beneﬁcial insects. Life cycles are relatively long, mostly 1 year in Zygoptera, and 1 – 6 years in Anisoptera. Slow seasonal developmental cycles predominate, but fast seasonal cycles occur in both suborders. There are normally 11 or 12 larval instars, but the number may vary somewhat between species or even within a species. When larval development is complete, the larva crawls from the water onto emergent vegetation or onto the bank, where ecdysis occurs. Larvae of some species may crawl several meters from the shore to emerge. Adults may live for several weeks. Newly emerged “teneral” adults are feeble ﬂiers for a day or two and vulnerable to predation. After the teneral stage has passed, adults often disperse widely into ﬁelds and other habitats to feed for a week or more on ﬂying insects. They then return to breeding sites where males patrol selected areas, chasing away rival males and other species. Before mating, the male transfers sperm from primary genitalia at the tip of the abdomen to a secondary genital organ on 669 the venter of abdominal segment 2. Using claspers on the terminal segment, a male will seize a female of the same species that ﬂies into his territory, grasping her either by the pronotum (Zygoptera) or by the head (Anisoptera). The female then loops the tip of her abdomen forward and upward to receive sperm from the penis on the second abdominal sternum of the male. Eggs are laid singly in plant tissues (endophytic oviposition) above, at, or below the water surface, or they may be laid singly or in masses on plants, debris, or other substrates, or randomly on the water surface (exophytic oviposition). Oviposition is endophytic in all Zygoptera and in Aeshnidae and Petaluridae; it is exophytic in other families. In many species, especially species of Zygoptera and Libellulidae, the male continues to grasp the female while she is ovipositing, protecting her from other males. Eggs hatch after an incubation period of one to several weeks, or after a long diapause. The ﬁrst larval instar is a prolarval stage that lasts only a few minutes to several hours. Prolarvae lack the well-developed appendages of other larval instars. Odonata larvae can be readily distinguished from all other aquatic insects by their elongate, hinged labium that has been modiﬁed for seizing and grasping prey (Figs. 32 – 34). They have conspicuous compound eyes and relatively short ﬁlamentous antennae (Fig. 43). In Zygoptera, the abdomen is elongate and thin, terminating in three long, vertically oriented, caudal lamellae (Fig. 31). In Anisoptera, the abdomen is relatively broad and terminates in three triangular-shaped, pointed appendages (Fig. 44), the upper one being called the “epiproct” and the lower ones “paraprocts.” In between them are triangular-shaped cerci, which usually are distinctly shorter than the epiproct or paraprocts. Adults do not resemble larvae, being much more elongate, having conspicuous genitalia, two pairs of elongate net-veined wings, and much larger compound eyes. Like Ephemeroptera, the primitive wings of Odonata cannot be folded. Wings of Anisoptera are held horizontally to the side when at rest; those of Zygoptera are held vertically just above or slightly to the side of the abdomen. Odonata larvae move about on the substrates by crawling; some actively stalk prey. Zygoptera larvae may swim by moving their abdomen and caudal lamellae from side to side. Anisoptera larvae can move rapidly when disturbed by expelling water from a rectal chamber. There are tracheal gills within the rectal chamber, and movement of water in, and out of, the chamber aids in respiration of Anisoptera larvae. In Zygoptera, respiration is greatly enhanced by the caudal lamellae, enabling larvae of most species to live in water with relatively low levels of dissolved oxygen. Most larvae can be easily collected with an aquatic net 670 W. L. Hilsenhoff using techniques described earlier. Adults are collected with large aerial nets after being sighted. Zygoptera and the Anisoptera adults that alight on vegetation are relatively easy to capture, but many large Anisoptera adults are extremely difﬁcult to capture with a net. Most larvae of North American Odonata can be identiﬁed to species by using keys and descriptions provided by Walker (1953, 1958) and Walker and Corbet (1975), Needham and Westfall (1955), and Westfall and May (1996). Most keys to Anisoptera larvae, however, are old, and problems often arise when using them. A revised edition of Dragonﬂies of North America (Needham et al., 2000) will contain new larval and adult keys. Because Odonata are relatively homogeneous, differences between families, genera, and species are not as obvious as in most other orders. Larvae of Corduliidae cannot be readily separated from those of Libellulidae without identifying them to genus. There is also considerable disagreement about the status of various genera among odonatologists as well as some disagreement about family status. But, at the species level, North American Odonata are better known as larvae and adults than aquatic insects in any other order. This is perhaps because they have attracted the interest of many amateur odonatologists who have made signiﬁcant contributions to our knowledge. Books by Corbet (1962) and Miller (1987), and a review article by Corbet (1980), summarize knowledge about the biology of dragonﬂies. References to species keys for the identiﬁcation of Odonata larvae are provided in Table II, and a key to families of freshwater Odonata appears in Section V.C. Information on the biology, habitat, and identiﬁcation of dragonﬂy and damselﬂy larvae in each family is presented in the following. 1. Families of Odonata — Suborder Anisoptera (Dragonﬂies) a. Aeshnidae (11 Genera, 39 Species) Larvae are large, with elongate tapered abdomens and can be distinguished from other Anisoptera by their elongate shape, ﬂat prementum, and thin six- or seven-segmented antennae (Fig. 37). Most inhabit lentic habitats, especially weedy permanent ponds, marshes, and littoral areas of lakes. Here, they move about on the vegetation and stalk prey composed of invertebrates and small vertebrates. Larvae of a few species are lotic and inhabit either slower areas of streams or rifﬂes. Life cycles of 2 – 4 years are likely for most species, especially in the North. One species, Anax junius, which does not overwinter in the North, migrates south in autumn and returns in early spring to oviposit. Larvae of this species develop more rapidly than other Aeshnidae and are bivoltine in the South, but rarely complete two TABLE II Literature References to Species Keys for Identiﬁcation of Odonata Larvae Regional keys to larvae that include more than one family Canada and Alaska — (Zygoptera) Walker (1953); (Anisoptera) Walker (1958); (Anisoptera) Walker and Corbet (1975) California — Smith and Pritchard (1956) Florida — (Zygoptera) Daigle (1991b); (Anisoptera) Daigle (1992) North America — (Anisoptera) Needham and Westfall (1955); Needham et al. (2000); (Zygoptera) Westfall and May (1996) North and South Carolina — Huggins and Brigham (1982) Southeastern United States — (lotic Anisoptera) Louton (1982) Texas — Young and Bayer (1979) Utah — (Anisoptera) Musser (1962) Keys to larvae listed by family Aeshnidae Gomphaeschna — Dunkle (1977) Coenagrionidae Enallagma — (West Coast) Garrison (1984) Ischnura — (California) Garrison (1981) Corduliidae Somatochlora — Daigle (1991a) Gomphidae Dromogomphus — Westfall and Tennessen (1979) Lanthus — Carle (1980) Ophiogomphus — Carle (1992) Progomphus — Tennesson (1993) Libellulidae Ladona — Benneﬁeld (1965) Sympetrum — Tai (1967) generations in the northern United States. Flight periods of most species are during the summer months. b. Cordulegastridae (1 Genus, 8 Species) The large, hairy, somewhat elongate larvae with small anterolateral eyes can be easily distinguished from other Anisoptera by the jagged teeth on the palpal lobes of their spoon-shaped prementum (Fig. 39). They inhabit small streams, where they lie partially concealed in silt to ambush prey, mostly at the upstream edge of pools. A 3 – 5-year life cycle is likely for most species, with adults ﬂying from spring into early summer. Eggs are laid in the substrate of shallow rifﬂes. c. Corduliidae (7 Genera, 49 Species) Larvae are short and broad, with a spoon-shaped prementum, and cannot be readily separated from those of Libellulidae. In most, the palpal lobes are more deeply scalloped (Figs. 40 and 41) than in Libellulidae (Fig. 42), but larvae in one genus of Libellulidae also have deeply scalloped palpal lobes. Larvae are mostly lentic, with 2 – 4-year life cycles probably predominating in the North and shorter cycles in the South. They commonly occur in marshes, swamps, cool ponds, and littoral areas of lakes. However, several lotic species have larvae that inhabit debris in streams of all sizes. Synchronized 17. Diversity and Classiﬁcation of Insects and Collembola emergences may lead to formation of exceptionally large swarms. Adult ﬂight periods are mostly from May into early July. d. Gomphidae (15 Genera, 97 Species) As in larvae of Aeshnidae and Petaluridae, the prementum is ﬂat, but gomphid larvae have relatively shorter and broader abdomens. Their broad, four-segmented antennae are diagnostic (Fig. 36). Larvae of most species inhabit streams, where they may be found in rifﬂes or partially buried in silt or sand in slower reaches. Those of other species inhabit lentic habitats, especially small permanent ponds and littoral areas of lakes, where they lie concealed in the substrate to ambush prey. Life cycles are long, usually 2 – 4 years, depending on water temperature and food. Most species have ﬂight periods in late spring and summer; in some species eggs may diapause over the winter. e. Libellulidae (26 Genera, 105 Species) The short, broad larvae closely resemble those of Corduliidae, but in almost all genera the crenulations on the palpal lobe of the mentum are extremely shallow (Fig. 42). Larvae occur in a variety of permanent and temporary lentic habitats where they crawl about in the vegetation and debris; occasionally they are found in vegetation along margins of streams. Littoral areas of lakes, permanent ponds, vernal ponds and marshes, cattail marshes, sphagnum swamps, and bogs all are habitats for larvae of a great variety of species. Many species are univoltine, with some having eggs that diapause. Other species may require two or more years to complete larval development. Flight periods range from spring through autumn, depending on the species. f. Macromiidae (2 Genera, 9 Species) Larvae are readily recognized by their relatively broad abdomens, extremcly long legs, and the presence of a frontal horn that projects forward between the eyes (Fig. 43). They are found among debris in the current of medium-tolarge streams or in margins of lakes exposed to wave action. Life cycles of 2 – 4 years are likely. Flight periods extend from March through August, depending on the species. g. Petaluridae (2 Genera, 2 Species) Both North American species are rare; neither has been found in the central part of the continent. Larvae of one semiterrestrial species inhabit bog seepages at high elevations in the western mountains; those of the other species have been found in bogs and seeps in eastern forests. The relatively broad larvae have a ﬂat prementum, relatively broad six-or seven-segmented antennae that are hairy (Fig. 38), and patches of black bristles on the 671 posterior abdominal terga. Because of their cold habitat, up to 6 years may be required to complete larval development. The ﬂight period ranges from early spring in Florida to summer farther north. 2. Families of Odonata — Suborder Zygoptera (Damselﬂies) a. Calopterygidae (2 Genera, 8 Species) The large larvae are readily distinguished from other Zygoptera by their very elongate ﬁrst antennal segment (Fig. 31). All species are lotic, the larvae inhabiting permanent streams of all sizes, where they are found mostly among bank vegetation and accumulations of debris. Species are univoltine or semivoltine, with ﬂight periods from late May into October. b. Coenagrionidae (13 Genera, 96 Species) The relatively small larvae differ from those of Calopterygidae in having a short basal antennal segment, and differ from those of Lestidae in not having the base of the prementum greatly narrowed (Fig. 34). They are predominantly lentic, although species in one genus (Argia) inhabit rifﬂes of streams and a few species in other genera occur along banks of streams. Larvae of lentic species are found mostly in permanent ponds, marshes, swamps, and littoral areas of lakes; occasionally, they occur among vegetation in parts of streams with little or no current. They crawl about on the vegetation and ambush or stalk prey, which consists mostly of small invertebrates. Most species are univoltine, some having eggs that diapause over the winter; a few species are bivoltine. Adults ﬂy from spring to autumn, depending on the species. c. Lestidae (2 Genera, 18 Species) The long, thin larvae with their long, parallel-sided caudal lamellae are distinctive. They differ from all other Zygoptera in having a prementum that is greatly narrowed and elongate basally (Fig. 33). Larvae of one genus (Archilestes) with 2 species are lotic. Larvae of other species (Lestes) are lentic, only occasionally being found among emergent vegetation along slow streams. They commonly inhabit vegetation in both permanent and vernal ponds and marshes and have a 1-year life cycle. Eggs are laid in vegetation above the water and often diapause until autumn or the following spring. Eggs hatch when the now dead vegetation in which they were laid falls into the water, or in vernal ponds, when water ﬂoods the dead vegetation. Adults ﬂy from late spring through summer. d. Protoneuridae (2 Genera, 2 Species) This tropical family is found only in Texas where larvae inhabit streams. Larvae are similar to those of Coenagrionidae, 672 W. L. Hilsenhoff but their caudal lamellae are thick in the basal half and thin and leaﬂike in the distal half (Fig. 35). C. Plecoptera — Stoneﬂies In this relatively small hemimetabolous order at least 614 species are known from North America, and it is likely that many additional species will be described as the taxonomy of various families and genera is studied in greater detail and mountain streams are collected more thoroughly. The order is poorly represented in middle North America; species richness is greatest in mountain areas where fast, cold streams abound. Larvae of all species are aquatic. Almost all of them inhabit streams, but those of a few species have adapted to living in cold oligotrophic lakes. In the West, larvae of species in at least two genera inhabit the hyporheic zone of certain streams to depths of 10 m or more. Stoneﬂy larvae are an important part of the stream ecosystem. They provide food for ﬁsh and invertebrate predators, and often are top predators in the invertebrate food chain. They are not sufﬁciently abundant to create nuisance problems, and because larvae of most species are intolerant of lowered levels of dissolved oxygen they play an important role in biological monitoring of streams. Most stoneﬂies are univoltine, but several larger species require 2 or 3 years to complete development. Species with 2 or 3 year life cycles in the North may be univoltine in the South. Univoltine species often have fast seasonal life cycles, with eggs or small larvae diapausing during the summer and rapidly developing in autumn, winter or spring. Many species, however, have a slow seasonal life cycle with eggs hatching within a few weeks and larval development progressing slowly throughout the year. Species that have a slow seasonal life cycle in the North may have a bivoltine, fast seasonal life cycle in the South. The number of larval instars varies with the species and even among individuals within a species, with 12 – 23 or more instars having been recorded for species that have been studied. After completing development, larvae crawl from the water onto a convenient substrate where ecdysis occurs. Adults then crawl or ﬂy to nearby vegetation or other structures to ﬁnd a mate. In most species this is accomplished by drumming with their abdomen; males initiate the drumming signal and females respond. After repeated drumming and crawling, males ﬁnd a female and mate. When eggs have matured, the female extrudes them into a mass on the tip of her abdomen. Females of some species ﬂy over the water and land brieﬂy on its surface to deposit their eggs; others jettison their egg mass while ﬂying above the water. In still other species, females crawl into the water on a protruding substrate to oviposit beneath the surface. In most species the female will lay multiple egg masses. Stoneﬂy larvae can be distinguished from most other aquatic insects by their two long ﬁlamentous cerci and generally elongate or ﬂattened appearance (Figs. 45, 48, 49, 54, 56, and 58). They have relatively long antennae, distinct compound eyes, two or three ocelli, chewing mouthparts, two pairs of thoracic wingpads, and three-segmented tarsi with two claws on each tarsus (Figs. 1 and 2). They are likely to be confused only with Ephemeroptera larvae, which also have long cerci, but Ephemeroptera larvae usually have three caudal ﬁlaments instead of two, have only one tarsal claw, and have gills on the middle abdominal segments. Gills on Plecoptera larvae, if present, are on the head, thorax, and / or basal or apical segments of the abdomen. Adult stoneﬂies resemble their larvae, the most noticeable difference being the presence of two pairs of net-veined wings that fold ﬂat over the abdomen; a few species are brachypterous or apterous. Genitalia are well developed in adults and gills are reduced to remnants in species that have them as larvae. Stoneﬂy larvae generally crawl on the substrate; when forced to swim, most do so weakly by moving their abdomen from side to side. Oxygen for respiration is obtained from the water through the cuticle, with thoracic and, sometimes, abdominal gills aiding oxygen uptake in most larger species. Because they generally lack extensive gills, most Plecoptera larvae occur only in oxygen-rich water. Larval feeding habits vary among families and also among species within a family or genus. As in many other aquatic insects, larval feeding habits within a species will vary with developmental stage and availability of food. Adult stoneﬂies are relatively short-lived; most live a few days to 2 weeks, some a little longer. Several species feed as adults, mostly on algae, lichens, and riparian leaves or ﬂowers. Larvae can be readily collected with an aquatic net by using standard sampling techniques. Adults of most species can be collected by using a net to sweep bank vegetation or by examining rocks, tree trunks, and bridges in the vicinity of streams. Adults of some species, however, move high into trees and can be collected only by using nets with very long handles or by using sticky traps. Several species of adults are attracted to lights, while others are rarely found at lights. Through the years there have been many changes in the higher classiﬁcation of Plecoptera, but in the last two decades it has remained quite stable. The order is divided into two suborders, Euholognatha and Systellognatha. The former incudes four families (Capniidae, 17. Diversity and Classiﬁcation of Insects and Collembola Leuctridae, Nemouridae, and Taeniopterygidae) that previously were in the family Nemouridae; larvae in all of these families are primarily herbivores – detritivores. Larvae of two families of Systellognatha (Perlidae and Chloroperlidae) are predators, in two other families (Peltoperlidae and Pteronarcyidae) they are herbivores – detritivores, and in the remaining family (Perlodidae) feeding habits vary with the species and larval age. The biology of Plecoptera was reviewed by Hynes (1976) and a recent book by Stewart and Stark (1988) discusses classiﬁcation, phylogeny, biogeography, ecology, and behavior of Plecoptera. This book includes keys and illustrations for larvae in all North American genera, notes on their biology, and the known distribution of North American species. Species in some genera can be identiﬁed as mature larvae as well as adults; in other genera identiﬁcation of larvae is not possible because larvae of several species remain unknown. About 55% of North American Plecoptera species are known in the larval stage. Information on the biology, habitat, and identiﬁcation of larvae in each family is presented next. References to species keys for the identiﬁcation of stoneﬂy larvae are provided in Table III and a key to families of freshwater Plecoptera larvae appears in Section V.D. 1. Families of Plecoptera – Suborder Euholognatha a. Capniidae (10 Genera, 151 Species) Capniidae are “winter stoneﬂies.” Larvae are generally small, dark colored, and elongate (Fig. 56). They closely resemble larvae of Leuctridae, but can be recognized by their generally shorter and broader metathoracic wingpads, which are nearly as widely separated as the mesothoracic wind-pads (Fig. 56), and by posterior abdominal terga that are usually widened and fringed posteriorly. Larvae inhabit streams of all sizes. They are especially abundant in smaller streams and spring seeps; some even inhabit streams that become dry in the summer. Larvae in two genera occur in cold oligotrophic lakes. Life cycles are univoltine, with emergence in late winter or early spring. Eggs hatch a few weeks after being laid, and after some development, larvae of most species diapause. Larval development resumes in autumn, with some species developing most rapidly in late autumn and winter. A few species have a slow seasonal life cycle with steady development from late spring through autumn. Larvae are mostly detritivores that feed on allochthonous debris, especially decaying leaves. In the North, several species may emerge on warm days while streams are still ice covered; adults often can be found crawling on the snow. Adults of some species are apterous or brachypterous. 673 TABLE III Literature References to Species Keys for Identiﬁcation of Plecoptera Larvae Regional keys to larvae that include more than one family British Columbia (southwest) — Ricker (1943) Louisiana — Stewart et al. (1976) Minnesota — Harden and Mickel (1952) North and South Carolina — Unzicker and McCaskill (1982) Northeastern North America — Hitchcock (1974) Ozark and Ouachita mountains — Poulton and Stewart (1991) Pennsylvania — Surdick and Kim (1976) Rocky Mountains — Baumann et al. (1977) Saskatchewan — Dosdall and Lehmkuhl (1979) Texas — Szczytko and Stewart (1977) Keys to larvae listed by family Capniidae — (eastern Canada) Harper and Hynes (1971b) Leuctridae — (eastern Canada) Harper and Hynes (1971a) Nemouridae — (eastern Canada) Harper and Hynes (1971d) Peltoperlidae Soliperla — Stark (1983) Perlidae Agnetina — Stark (1986) Hesperoperla — Baumann and Stark (1980) Paragnetina — Stark and Szczytko (1981) Perlinella — Kondratieff et al. (1988) Perlodidae — (Wisconsin) Hilsenhoff and Billmyer (1973) Diploperla — Kondratieff et al. (1981) Helopicus — Stark and Ray (1983) Hydroperla — Ray and Stark (1981); Ricker (1952) Isogenoides — Ricker (1952) Isoperla — (western North America) Szczytko and Stewart (1979) Malirekus — Stark and Szczytko (1988) Setvena — Stewart and Stanger (1985) Pteronarcyidae — (West Virginia) Tarter et al. (1975) Taeniopterygidae — Harper and Hynes (1971c) Taeniopteryx — Fullington and Stewart (1980) b. Leuctridae (7 Genera, 55 Species) The relatively small, elongate larvae are similar to those of Capniidae, but have metathoracic wing-pads that are similar in shape, but distinctly closer together than those on the mesothorax (Fig. 58). Larvae also resemble those of Chloroperlidae, which have shorter cerci and meso-and metathoracic wing-pads that are equally far apart (Fig. 49). Larvae are found mostly among gravel in small, cool, permanent streams where they feed on decaying allochthonous material; one genus occurs in temporary streams. Most species are univoltine with emergence from spring to autumn, depending on the species. A semivoltine life cycle occurs in at least one northern species. c. Nemouridae (12 Genera, 72 Species) The small, relatively dark-colored larvae are short and broad, with divergent metathoracic wing-pads in more mature larvae and metathoracic legs that reach or exceed the apex of the abdomen (Fig. 54). Most nemourids are 674 W. L. Hilsenhoff univoltine, with emergence from spring to autumn, depending on the species. Fast seasonal life cycles predominate, with an extended egg diapause in some species. At least one species is semivoltine. Larvae are detrital feeders that inhabit debris and soft sediments in smaller streams and spring seeps; they are often the only stoneﬂy larvae in spring seeps and runs. Larvae in at least one genus inhabit temporary streams, and those of another species have been collected from Arctic lakes. d. Taeniopterygidae (6 Genera, 35 Species) Like Capniidae, these are univoltine “winter stoneﬂies” that emerge in late winter or very early spring and probably spend the summer as diapausing larvae. In size and shape, larvae rsemble those of Perlodidae (Fig. 48), having divergent metathoracic wingpads and elongate abdomens, but they are not as heavily patterned as most Perlodidae, they move much more slowly, and they swim very inefﬁciently. Larvae are herbivores – detritivores that occur in permanent streams of all sizes. They are frequently found among bank vegetation. 2. Families of Plecoptera — Suborder Systellognatha a. Chloroperlidae (13 Genera, 77 Species) Larvae are generally small, elongate, rounded in cross section, and have short cerci (Fig. 49). They are similar to larvae of Capniidae and Leuctridae and are difﬁcult to distinguish from the larvae of Leuctridae in the ﬁeld. They are not likely to be confused with Capniidae, however, because late-instar larvae of Chloroperlidae are present in late spring and summer, while those of Capniidae occur in late autumn, winter, and early spring. Larval mouthparts (Fig. 46) differ greatly from those of Capniidae and Leuctridae (Fig. 47), reﬂecting the carnivorous feeding habits of Chloroperlidae in later larval instars. Larvae of Chloroperlidae inhabit mostly gravel bottoms of cold, small- to medium-sized streams. Life histories are poorly known. Univoltine and semivoltine fast seasonal life cycles have been documented, with emergence in late spring or early summer. b. Peltoperlidae (6 Genera, 20 Species) The broad, ﬂat, roachlike appearance of the medium-sized larvae is distinctive (Fig. 45). They are often abundant in mountain streams of the eastern, southern, and western United States, but are absent from central North America. Larvae inhabit smaller streams, where they feed on decaying leaves, detritus, and associated microorganisms. Many species are semivoltine; some may be univoltine. Adults emerge in late spring or early summer. c. Perlidae (15 Genera, 72 Species) Larvae are relatively large, broad, stoneﬂies (Fig. 1) that are readily recognized by the presence of branched ﬁlamentous gills at the base of each leg (Fig. 2) and the absence of gills on abdominal sterna. They are widespread and common, inhabiting permanent streams of all sizes where they prey on other macroinvertebrates. They are often the dominant insect predators, although earlyinstar larvae may feed on detritus and algae. Most larvae are found in fast water, especially among debris in rifﬂes. Species in most genera have a 2 or 3 year slow seasonal life cycle in northern regions; univoltine life cycles may occur in the South. One common genus is univoltine with a fast seasonal life cycle. Emergence in most species is during the summer months. d. Perlodidae (30 Genera, 122 Species) Larvae are mostly small- to medium-sized stoneﬂies, which usually have distinctive patterns dorsally. They lack the thoracic gills of the similarly shaped and patterned larvae of Perlidae. The metathoracic wing-pads are divergent and the abdomen is rather elongate (Fig. 48). While larvae of some species of Perlodidae are herbivores – detritivores throughout their larval development, most species are predominantly predators. They develop in all sizes and types of streams; some even develop in cold oligotrophic lakes. All species are apparently univoltine, with emergence in spring. Eggs of many species diapause during the summer and hatch in autumn, but in other species eggs hatch within a few weeks after being laid. e. Pteronarcyidae (2 Genera, 10 Species) These large stoneﬂies require 1 – 4 years to complete development. They have a slow seasonal life cycle, with univoltine life cycles occurring only in the south. Larvae are elongate, and can be readily recognized by the branched ﬁlamentous gills at the base of thoracic legs and on the ﬁrst two or three abdominal sterna. They feed mostly on coarse particulate organic matter and inhabit wood or accumulations of debris in small- to medium-sized permanent streams with moderate-torapid currents. Emergence occurs in spring or early summer, being earliest in the south. D. Trichoptera – Caddisﬂies Trichoptera is a rather large holometabolous order. Larvae and pupae of all species are aquatic, with one or two exceptions, and represent an important part of the insect fauna of most streams. About 1400 species are known from North America. Most larvae occur in lotic habitats, but those of some species in half of the families inhabit lentic habitats. Adults are terrestrial, mothlike insects, that sometimes are attracted to lights in large numbers. Most have very long ﬁliform antennae, and their wings, which they fold rooﬂike over their body, are covered with hairlike setae. Eggs of 17. Diversity and Classiﬁcation of Insects and Collembola most species are laid in masses in the water; adults of a few species lay their eggs on vegetation above the water or in moist soil of temporary ponds that will eventually be ﬂooded. Most species are univoltine, often with rather synchronized emergences; however, in some species different cohorts emerge throughout the warm weather period. A few species are bivoltine, with the tendency for bivoltinism increasing farther south, and several species, especially those that develop in coldwater streams, are known to be semivoltine, especially in the northern part of their range. Adult caddisﬂies can be identiﬁed to species by using the many keys and descriptions that have been published, but larval taxonomy lags, with about half of the described species still unknown in the larval stage. This makes accurate identiﬁcation of larvae to species impossible in most genera, and larvae of four genera remain unknown. Wiggins (1996) published excellent keys, descriptions, and illustrations for larvae of known genera. Ross (1944) developed species keys for larvae in many genera that occur in the central United States as well as keys to adults, but in most of these genera there are species for which larvae are unknown, so his larval keys must be used with caution. Trichoptera is divided into three suborders, Annulipalpia, Integripalpia, and Spicipalpia (Wiggins and Wichard, 1989; Frania and Wiggins, 1995). This parallels the division of the order into three superfamilies as used by Wiggins (1977) and others, and to some extent the classiﬁcation proposed by Weaver and Morse (1986). In the Annulipalpia, larvae use silk from their labial glands to construct retreats and nets, which they use to ﬁlter or gather food such as algae, detritus, or macroinvertebrates. Larvae pupate within their retreat in a silk cocoon that has small openings at each end through which water circulates. In the suborder Spicipalpia, larvae in the four families are either free-living or construct portable cases that are barrel-shaped, purselike, or saddle-shaped. All larvae pupate in a completely closed cocoon of parchmentlike silk, differing from other caddisﬂies that have openings in their pupal case to allow circulation of water. Free-living larvae (Rhyacophilidae and Hydrobiosidae) are mostly predators on other arthropods. Larvae that build cases (Glossosomatiae and Hydroptilidae) are mostly herbivores that feed on periphyton. In the suborder Integripalpia, all larvae use silk from their labial glands to construct portable tubular cases of plant or mineral materials. Cases vary widely in size and shape, and are often sufﬁciently distinctive to be employed in generic and species identiﬁcation. Integripalpia larvae are mostly herbivores or detritivores, but in some families (Brachycentridae, Leptoceridae, Limnephilidae, Molannidae, Odontoceridae, and Phryganeidae) omni- 675 vores or predators can be found. Larvae in eight families of Integripalpia have a prosternal horn, that larvae in other families lack. All larvae pupate within their last larval case after it has been attached to a substrate and closed at both ends. Small openings at each end permit water circulation. Trichoptera larvae have a pair of simple eyes, chewing mouthparts, antennae that are so short that they often are difﬁcult to see, three pairs of thoracic legs with a single tarsal segment and tarsal claw, and a pair of ﬂeshy prolegs on the last abdominal segment (Figs. 66 and 67). Many larvae have single or branched gills on the abdominal segments. Most species have ﬁve larval instars, and after completion of larval development a pupal case is constructed, either by sealing off the open end of the portable case (Integripalpia) or by constructing a case and cementing it to the substrate (Annulipalpia and Spicipalpia). The larva then ceases feeding and becomes quiescent with its appendages pressed against its body. This is the prepupal stage, and may last a few days to several weeks. The pupa is of the exarate type, with appendages free from the body. Pupae in most families have well-developed mandibles that the pharate adults (adult within the pupal integument) use to cut their way out of the case just prior to emergence. After a pharate adult leaves the pupal case, it swims to the water surface where eclosion takes place or, more often, it swims to the surface and then rapidly to shore where it crawls out of the water onto some object to emerge. Eclosion is rapid. Trichoptera larvae have many adaptations to their aquatic environment. Respiration in caddisﬂy larvae is through the integument, and in many families this is aided by the presence of abdominal gills. Additionally, especially in Integripalpia, larvae are able to increase respiration by undulating their body within their tubular case to increase the ﬂow of water and dissolved oxygen through the case. Larval cases and retreats also serve to protect larvae from predators, acting either as a physical barrier or serving to camouﬂage the larva. Cases may also protect larvae from their environment. Hard cases protect those that live in sand and gravel from abrasion, and streamlined cases or nets allow others to inhabit substrates in very swift currents. Netspinning larvae (Annulipalpia) use their nets to strain algae and other food material from the current in streams. Some caddisﬂy larvae (Leptoceridae) have fringes of setae on their legs that allow them to swim. Others (some Brachycentridae) have fringes of setae on their legs to permit them to ﬁlter food particles from the current. Most caddisﬂy larvae can be readily collected from lotic and lentic habitats with an aquatic net by using methods already described. Debris in the pan should be 676 W. L. Hilsenhoff examined carefully as many larvae have cases that blend in with the debris. In streams it is important to also examine large substrates such as wood and rocks, because larvae of many species attach themselves ﬁrmly to these substrates or inhabit moss or algae that grows on them. Species of larvae that burrow into decaying wood can be collected as they crawl from retreats when the wood is removed from a stream and allowed to dry. Larvae that inhabit sand or silt substrates are best collected by sieving these substrates through a net or strainer, or by closely watching sand substrates and collecting larvae that expose themselves by moving. Larvae of extremely small species are often overlooked because they remain attached to the substrate and are too small to see. Microscopic examination of macrophytes and other substrates returned to the laboratory may yield many small larvae, especially those of Hydroptilidae. In the ﬁeld a hand lens is helpful in locating very small specimens. Caddisﬂies are a very important part of the stream community; in some streams they dominate the insect biomass. Many species of ﬁsh feed on the larvae and emerging adults. Because of their general abundance and diversity in streams, and the wide variation in tolerance to pollution among larvae of various species, they are very important in biological monitoring. Occasionally, extremely large numbers of adults emerge, and because they are attracted to lights they may create severe problems. These include contamination of products in factories, annoyance of people in restaurants and elsewhere, and allergic reactions in people who are sensitive to them. Larvae of many species have never been associated with adults. Larvae of these species must be reared, associated with adults, and described before we will be able to develop reliable keys to larvae at the species level. Such keys are needed to enable us to complete needed studies of the life history, ecology, and behavior of the various species. Relatively little is presently known about the life history and ecology of most North American species. Most of what is known has resulted from studies within a single stream where the fauna is limited and thoroughly known because larvae have been associated with adults. Wiggins (1996) summarized knowledge of the biology, ecology, morphology, and distribution of larvae in each genus. Information on the biology and habitat, and a brief description of the larva in each caddisﬂy family follows. A key to families of freshwater Trichoptera larvae appears in Section V.F, and references to species keys for the identiﬁcation of larvae are provided in Table IV. 1. Families of Trichoptera — Suborder Annulipalpia a. Dipseudopsidae (1 Genus, 5 Species) The only genus Phylocentropus, was sometimes included in the TABLE IV Literature References to Species Keys for Identiﬁcation of Trichoptera Larvae Regional keys to larvae that include more than one family Florida — Pescador et al. (1995) Illinois — (many incomplete) Ross (1944) North and South Carolina — Unzicker et al. (1982) Keys to larvae listed by family Apataniidae Apatania — Chen (1992) Brachycentridae — (Wisconsin) Hilsenhoff (1985) Brachycentrus — Flint (1984) Micrasema — Chapin (1978) Goeridae Goera — (eastern United States) Flint (1960); Wiggins (1996) Goerita — Parker (1998) other genera — Wiggins (1973) Hydropsychidae — (Wisconsin) Schmude and Hilsenhoff (1986) Arctopsychinae — Flint (1961); (western North America) Givens and Smith (1980) Ceratopsyche (as Hydropsyche) — Schefter and Wiggins (1986) Hydropsyche (including Ceratopsyche) — (eastern and central North America) Schuster and Etnier (1978) Lepidostomatidae — Weaver (1988) Leptoceridae Ceraclea — Resh (1976) Mystacides — Yamamoto and Wiggins (1964) Nectopsyche — Haddock (1977) Oecetis — Floyd (1995) Setodes — Nations (1994) Triaenodes and Ylodes — Glover (1996) Limnephilidae — (eastern United States) Flint (1960) Desmona — Wiggins and Wisseman (1990) Dicosmoecus — Wiggins and Richardson (1982) Hesperophylax — Parker and Wiggins (1985) Pycnopshche — Wojtowicz (1982) Molannidae Molanna — Sherberger and Wallace (1971) Odontoceridae Psilotreta — Parker and Wiggins (1987) Philopotamidae — Ross (1944) Phryganeidae — Wiggins (1960) Polycentropodidae Paranyctiophylax — (as Nyctiophylax) Flint (1964); Wiggins (1996) Psychomyiidae Psychomyia — Flint (1964) Rhyacophilidae — (eastern United States) Flint (1962); (Pennsylvania) Weaver and Sykora (1979) generally predaceous family Polycentropodidae. Larvae differ from those of Polycentropodidae by having broad, ﬂattened, setose tarsi (Fig. 72) and short, broad mandibles with mesal brushes. These are adaptations for their feeding on detritus, which they collect with a net placed within tubes that they construct in sand and silt along margins of sandy streams and lakes. The capture net and feeding habits were described by Wallace et al. (1976). All North American species 17. Diversity and Classiﬁcation of Insects and Collembola occur east of the Great Plains. The life cycle is probably 1 year, with cohorts emerging from May through August. b. Ecnomidae (1 Genus, 2 Species) Larvae of this Neotropical family were collected from a small, cold, rapid stream in the “hills” section of Texas (Waltz and McCafferty, 1983). A second species was described by Bowles (1995). These represent the only records of this family north of Mexico. While previously considered a subfamily of Polycentropodidae, larvae have sclerotized meso- and metanota as do larvae of Hydroptilidae and Hydropsychidae. c. Hydropsychidae (11 Genera, 158 Species) Larvae in this very large and important family are easily recognized by the numerous, branched ﬁlamentous gills that occur ventrally on their abdomen. They frequently represent a large segment of the macroinvertebrate fauna in streams of all sizes, currents, and temperatures. Here, they build retreats on rocks and other larger substrates, in leafpacks, or in moss. Larvae of some species also tunnel into decaying wood and others may occur on wave-swept lake shores. Larvae of most species are omnivorous, feeding primarily on algae, crustacea, and insects collected by their capture nets; those of a few species tend to eat mostly algae and diatoms and others are mostly predaceous, especially in later instars. Life cycles vary from bivoltine to semivoltine, depending very much on stream temperature, with the majority of species probably being univoltine. Because of their widespread abundance and the great variation in the tolerance of species to organic pollution, larvae are very important in biological monitoring of streams. The subfamily Arctopsychinae is generally recognized in Europe as a separate family, and in North America this family has been recognized by Schmid (1968) and Nimmo (1987), but Wiggins (1981) presented compelling arguments for retention of subfamily status. Capture nets of Arctopsychinae larvae are generally larger than those of other Hydropsychidae and larvae are predominantly predators in the later instars. Larvae differ from those of other Hydropsychidae by having an elongate, posteriorly narrowed gula that completely separates the genae. d. Philopotamidae (3 Genera, 42 Species) Larvae live in silken retreats, usually on the bottom of rocks in warm-to-cold streams of all sizes. Their unmarked head with an unsclerotized T-shaped labrum is distinctive (Fig. 70) and readily separates them from other net spinning larvae with unsclerotized meso- and metanota. Larvae feed on algae and detritus that they capture 677 in their silken nets, which have a smaller mesh size than those of other net spinning caddisﬂies. Most, if not all species, are probably univoltine, except in the south, where two or more generations may occur. Cohorts of most species emerge throughout the spring and summer months; one species also emerges in fall and winter. e. Polycentropodidae (6 Genera, 71 Species) While larvae of most species live in streams, they inhabit a wide variety of other habitats, including littoral areas of lakes and temporary ponds. Larvae can be recognized by their pointed protrochantin (Fig. 71), unmodiﬁed tarsi, and the presence of dark or light spots (muscle scars) on the heads of most species. Reliable keys to larvae do not exist, because larvae of many species remain undescribed. Most larvae have a waxy cuticle that tends to repel water, giving the impression when collected that they are terrestrial. Larvae of a few species build trumpet-shaped capture nets similar to other Annulipalpia and are primarily herbivores, but those of most species are predators and have tubular retreats that they sometimes use to detect prey. These tubular retreats also function as an aid to respiration, with larvae circulating water through them by undulating their body. This is especially important for species that live in lentic habitats that occasionally may be deﬁcient in oxygen. Life cycles are probably one year in most species, with emergence during the summer months. f. Psychomyiidae (4 Genera, 17 Species) Larvae live in streams, frequently on or in wood; they also occur on rocks, which they cover with silken retreats that incorporate sand or detritus. Larvae can be readily separated from those of other families without sclerites on the meso- and metanotum by their hatchet-shaped protrochantin (Fig. 69). They graze food from the vicinity of their retreats, mostly consuming detritus, fungi, and periphyton. Most species are probably univoltine, with emergence in late spring and summer. g. Xiphocentronidae (2 Genera, 2 Species) Sometimes considered as part of Psychomyiidae, the only described larvae of Xiphocentronidae north of Mexico differ from Psychomyiidae larvae and those in other families by having their tarsi and tibiae fused into a single segment on all legs (Fig. 68). Larvae are known in North America from southern Texas, where one species was collected from a spring run and described by Edwards (1961), and from northern Arizona, where larvae in a second genus were described by Moulton and Stewart (1997). 678 W. L. Hilsenhoff 2. Families of Trichoptera — Suborder Integripalpia a. Apataniidae (5 Genera, 33 Species) Until recently this family was included in Limnephilidae; the larvae are quite diverse morphologically. All are rather small (12 mm) and live in cornucopia-shaped cases of small rock fragments. They occur in small cold-water streams or seeps, and occasionally in cold lakes where they feed mostly on algae. Most larvae differ from other Limnephilidae by having mandibles with uniform scraper blades; those with toothed mandibles have 25 or more setae on the anterior of the metanotum between the sclerites. b. Beraeidae (1 Genus, 3 Species) The gently curved and tapered case is made of ﬁne sand. Larvae are small and can be recognized by a transverse ridge on the pronotum that curves forward laterally to form rounded lobes (Fig. 77). Larvae, which are known for only two North American species (Wiggins, 1996), inhabit muck margins of spring seeps. Adults have been collected only in southeastern and northeastern North America, where they are rare. A species found in Ontario has been reported to be semivoltine. c. Brachycentridae (5 Genera, 34 Species) The larval case is tapered, of vegetable matter or sand grains, and square or round in cross section. Larvae are distinctive, having their pronotum divided by a distinct furrow (Fig. 76), with the area in front of the furrow depressed. They have no dorsal or lateral humps on the ﬁrst abdominal segment, which is unique among Integripalpia. Larvae in three of the genera are monotypic in North America; most others can be identiﬁed by keys listed in Table IV. Most species are univoltine, but some that inhabit cold northern streams are semivoltine. Larvae of all species inhabit streams, especially cold spring-fed streams, where they live in moss or other vegetation, or attach their cases to substrates in the current. The mostly herbivorous larvae graze on periphyton and other vegetation; some also consume small insects and crustaceans. Some are primarily ﬁlter feeders that use their elongate meso- and metathoracic legs to gather food from the current. d. Calamoceratidae (3 Genera, 5 Species) Cases are of leaf pieces or hollowed sticks. Larvae, which may reach a relatively large size, can be recognized by the projecting, divergent, anterolateral corners of the pronotum and a row of about 16 long setae dorsally on the labrum (Fig. 79). Most species can be identiﬁed as larvae by using descriptions and distributions reported in Wiggins (1996). Larvae inhabit pool areas of cool streams in the west or east, where they feed mostly on detritus. Most species probably have a 2-year life cycle. e. Goeridae (4 Genera, 11 Species) Larvae resemble those of Limnephilidae, but differ in their unique modiﬁcation of the mesepisternum, which is formed into an anteriorly projecting elongate process (Fig. 83) or a rounded spiny prominece. Since there are only a limited number of species, it is often possible to identify larvae to species using publications listed in Table IV. The sand cases are either cornucopia-shaped or tubular with ﬂanges of pebbles. Life cycles are probably univoltine or semivoltine. Larvae inhabit cobbles and other substrates in the current of small cold streams; larvae of one monotypic genus occur in the muck of spring seeps. They feed on algae, vascular plants, or detritus. f. Helicopsychidae (1 Genus, 4 Species) The snail like case, which larvae never abandon, is unique (Fig. 59). Only the larva of Helicopsyche borealis, which is widespread in North America, is known; larvae of the three western species remain unknown. The normally lotic larvae may also be found along margins of lakes, often in rather deep water. Larvae inhabit rocks or wood and scrape periphyton and detritus from the substrate; early instars may burrow into the bed of sandy streams. They can tolerate very warm water, and have been collected from thermal springs with temperatures as high as 34°C. They are probably univoltine, with emergence of cohorts throughout the summer and a prolonged winter diapause of eggs in the North. g. Lepidostomatidae (2 Genera, 82 Species) Cases are highly variable, being made of sand or bits of vegetation and square or round in cross section. The location of the antenna very close to the eye (Fig. 78) readily identiﬁes all larvae. Larvae are primarily detritivores and are found among detritus on a variety of substrates. While larvae of most species inhabit cold streams or springs, some occur in lakes and may be found at a considerable distance from the shore. Species that have been studied are univoltine, with spatial or temporal separation of different species that occur in the same stream. h. Leptoceridae (8 Genera, 113 Species) Larvae construct a wide variety of cases. In some genera they have elongate cases of vegetation, vegetation and sand, or of pure silk, and are adapted for swimming by having fringes of long setae on their legs. Larvae in other genera have shorter, stouter cases that are often cornucopia-shaped and constructed of vegetable or mineral material; these larvae are unable to swim. Most larvae 17. Diversity and Classiﬁcation of Insects and Collembola can be recognized by their relatively long antennae (Fig. 63), which are much longer than those of other caddisﬂy larvae, and also by their elongate metathoracic legs, which are distinctly longer than their other legs. Larvae inhabit a variety of permanent lotic and lentic habitats; those in lotic habitats tend to be in slower water and those in lakes may be found far from shore as well as in the littoral zone. They are generally omnivorous in their feeding habits, but some are primarily predators and a few feed exclusively on freshwater sponges. Most species are probably univoltine. i. Limnephilidae (39 Genera, 243 Species) Case construction in this large and diverse family varies widely, with vegetable or mineral material incorporated into a variety of shapes and sizes. Larvae also vary greatly in size and structure, making them difﬁcult to characterize. All have a prosternal horn and antennae that are located about halfway between each eye and mandible (Fig. 80). Most have numerous setae on the ﬁrst abdominal segment, and sclerites at SA-1 of the metanotum (Fig. 66). Many also have chloride epithelia ventrally or dorsally on abdominal segments. While species in some genera can be identiﬁed by keys listed below, accurate identiﬁcation of larvae is not possible in most genera because larvae of too many species remain unknown. Most species are univoltine, but a few are semivoltine. Larvae of most species inhabit a wide range of lotic habitats, from springs and small streams to large rivers; others inhabit lakes, and several are found in permanent or temporary ponds. In these habitats larvae can be found on vegetation, rocks, or detritus, or in gravel, sand, or soft sediments. Most larvae are herbivores or detritivores, and are important in processing large particulate organic matter; larvae of one species are known to be carnivores. j. Molannidae (2 Genera, 7 Species) Larvae construct characteristic sand cases with lateral ﬂanges and a hood over the anterior opening. They also have long metathoracic legs with either very short or threadlike claws. Larvae live in sandy or silty substrates of lakes or streams where currents are reduced. Here they feed on algae, vascular plants, or even invertebrates. Most species are probably univoltine. k. Odontoceridae (6 Genera, 13 Species) Cases are typically cornucopia-shaped and made of sand grains and tiny stones; they are hard and difﬁcult to crush. Like Sericostomatidae, which they resemble, larvae lack a prosternal horn and have their eyes located close to the mandibles, but their protrochantin is small and not hook-shaped (Fig. 82). Species of larvae in the largest genus (Psilotreta) can be identiﬁed (Table IV); 679 species in monotypic genera can be identiﬁed by using the key preposed by Wiggins (1996). Larvae inhabit small, cold streams or springs where they are found in sandy substrates. They are probably omnivorous, feeding on algae, vascular plants, and invertebrates. Most species are probably semivoltine in the north and univoltine in the south. l. Phryganeidae (10 Genera, 28 Species) These large caddisﬂies have distinctive cases made mostly of pieces of vegetation that are spirally wound or in concentric rings. When disturbed, they readily abandon their cases, but may re-enter them. Larvae are distinctive, generally having a boldly striped head that is more prognathous than other Integripalpia (Fig. 65). They also have a prominent prosternal horn and lack signiﬁcant sclerotization of the mesonotum. Identiﬁcation of larvae to species is not possible in some genera. Larvae may be found among vegetation and detritus along streams of all sizes, in marshes, in temporary and permanent ponds, and even in lakes where they may occur far from shore. While many are mostly predators, vegetation is also consumed, especially by early instars. Life cycles are probably 1 year. m. Rossianidae (2 Genera, 2 Species) This western North American family was erected for two genera previously placed in Limnephilidae and Goeridae. Larvae live in organic muck of spring seeps or in small, cold, mountain streams where they feed on plants and detritus. Their curved cases are made of tiny stones. Larvae are distinctive among Integripalpia because their head, pronotum, and mesepisternum are modiﬁed by prominent surface sculpturing. n. Sericostomatidae (3 Genera, 15 Species) Cases are typically cornucopia-shaped and made of sand and small pebbles, but are not hard as in Odontoceridae. Larvae can be recognized by the pointed anterolateral angles of their pronotum, a hook-shaped protrochantin (Fig. 81), and broad, ﬂat profemur. No key has been published for separating larvae of Agarodes, the largest genus. Larvae live in sandy substrates of streams, springs, and even lake margins, where they feed primarily on plant detritus. Most species are probably univoltine. o. Uenoidae (5 Genera, 46 Species) The cornucopiashaped cases of sand grains are either extremely long and thin or short and stout, depending on the genus. Larvae occur in streams and springs. Until recently they were considered part of Limnephilidae. Larvae differ from Limnephilidae by having a mesally notched anterior of the mesonotum (Fig. 84) and untoothed scraping 680 W. L. Hilsenhoff mandibles, which they use to scrape diatoms and organic detritus from rock surfaces. There are several species in each of the genera, and keys to species of larvae have not yet been developed. Two genera (Neothremma and Farula) occur in the western mountains; Neophylax, a species widespread in North America, was recently added to this family (Vineyard and Wiggins, 1988). ognized by their unsclerotized meso- and metanotum, sclerotized ninth abdominal tergum, and elongate anal prolegs (Fig. 74). Life cycles are probably univoltine for most species, but may be semivoltine in some. Larvae of most species are active predators, but those of a few species feed on living and dead vascular plant tissue and algae. 3. Families of Trichoptera — Suborder Spicipalpia a. Glossosomatidae (6 Genera, 76 Species) Larvae build unique turtlelike cases of sand and pebbles, but readily vacate them when collected. They can be separated from other Spicipalpia with unsclerotized mesoand metanota by their cases and their short anal prolegs with short claws (Fig. 73). Larvae can be identiﬁed to species only by associating them with adults. All species inhabit the upper surfaces of rocks or other large substrates in clean, cool streams or springs. Here they feed on diatoms and other algae that they scrape from the substrate. Most species probably have a oneyear life cycle, often with many cohorts that emerge throughout the spring and summer. b. Hydrobiosidae (1 Genus, 3 Species) Although sometimes included in Rhyacophilidae, the free-living larvae are unique in having chelate prothoracic legs (Fig. 75), which are suited to their predatory habits. They occur in cool streams in Texas, Arizona, and Nevada. The larva of one species was described by Edwards and Arnold (1961). c. Hydroptilidae (16 Genera, 250 Species) Larvae are characterized by their completely sclerotized thoracic nota, lack of branched ventral gills, and very small size. They are frequently called “microcaddisﬂies.” The ﬁrst four larval instars are free-living, have no case, and have a normal-size abdomen; in the last instar the abdomen is greatly expanded and larvae have a case that is purse-shaped or barrel-shaped and sometimes attached to the substrate (Figs. 61, 62). Most species pass through the ﬁrst four instars rapidly, and it is usually not possible to identify these early instars to genus. Larvae in the last instar can be identiﬁed to genus, but usually not to species. Larvae may be found in a wide variety of lotic and lentic habitats where they feed mostly on algae and other plant material. They are probably univoltine, or bivoltine in warmer habitats, but generally very little is known about life histories of the various species. d. Rhyacophilidae (2 Genera, 128 Species) The larvae live in cool or cold streams, have no case, and do not build retreats. Most species occur in mountainous regions where cold, fast streams abound. They are rec- E. Megaloptera — Fishﬂies and Alderﬂies This very small holometabolous order contains two aquatic families that once were included in the order Neuroptera. The aquatic larvae are distinctive, having seven or eight pairs of lateral ﬁlaments and large, conspicuous mandibles (Figs. 85, 86). They can be confused only with some aquatic Coleoptera larvae that have lateral ﬁlaments. Larvae often attain a very large size, and because all are predators, they may exert signiﬁcant pressure on populations of macroinvertebrates that live in the same habitat. Studies indicate that there are usually 10 or 11 larval instars, and that life cycles range from 1 – 4 years, depending on the species, climate, and habitat. Larvae are aquatic; all other stages are terrestrial. Eggs are laid in masses on objects above the water, and pupation takes place in cells on land. The terrestrial adults are short-lived and generally secretive, but because of their large size and often conspicuous mandibles, they attract attention when discovered. Adults are weak ﬂiers, but are often attracted to lights. They fold their net-veined wings rooﬂike over their abdomen when at rest. Larvae of most species occur only in relatively oxygen-rich environments, because they usually obtain oxygen through their integument directly from the water in which they live. Their lateral ﬁlaments greatly increase surface area, allowing them to do this. Species that inhabit lentic habitats, which may at times be low in oxygen, have long caudal respiratory tubes that they use to obtain oxygen from air at the surface. All larvae have spiracles and are capable of living out of water in moist areas; larvae of some species survive in temporary streams. Larvae can be readily collected with an aquatic net by using standard sampling procedures. However, greater numbers of stream species can often be collected by dislodging larger rocks. Rearing of larvae is difﬁcult because of long life cycles, cannibalism, and the need to supply prey species and a terrestrial environment for pupation. Species of adults are well known, and as a result of recent studies larvae of most species can also be identiﬁed. References to species keys for the identiﬁcation of larvae are provided in Table V. Information on the biology, habitat, and identiﬁcation of larvae in the two families is presented next, and a 17. Diversity and Classiﬁcation of Insects and Collembola TABLE V Literature References to Species Keys for Identiﬁcation of Megaloptera Larvae Regional keys to larvae that include more than one family North and South Carolina — Brigham (1982a) Keys to larvae listed by family Corydalidae — (Mississippi) Stark and Lago (1983) Chauliodes — Cuyler (1958) Nigronia — Neunzig (1966) Sialidae — (eastern North America) Canterbury (1978) key to families of freshwater Megaloptera larvae appears in Section V.F. 1. Families of Megaloptera a. Corydalidae (7 Genera, 22 Species) The very large size (20 – 90 mm), lateral ﬁlaments, and large mandibles of late instar “ﬁshﬂy” or “Dobsonﬂy” larvae are distinctive (Fig. 86), but smaller larvae resemble those of Gyrinidae and two other Coleoptera genera. Larvae can be distinguished from Coleoptera larvae by their pair of terminal prolegs, each with two hooks (Fig. 86). Larvae in most genera inhabit streams or spring seeps, which have a relatively high dissolved oxygen content. Because these habitats are generally cold, life cycles of two or more years are likely for most species, with different cohorts and extended emergence periods in some species. The habitat of Chauliodes larvae differs from that of other genera. They inhabit shallow lentic habitats or vegetated margins of streams and are univoltine, with emergence in late spring or early summer. They also are known to consume signiﬁcant amounts of detritus in addition to prey, especially during the winter. b. Sialidae (1 Genus, 24 Species) The lateral ﬁlaments and long, single tail ﬁlament at once separate “alderﬂy” larvae from all other aquatic insects (Fig. 85). Larvae live mostly in depositional sediments of streams, permanent ponds, and lakes. Species that live in lakes may inhabit areas that are several hundred meters from shore, necessitating a considerable migration to land for pupation. Most species are univoltine, but in northern regions species that live in streams or lakes are often semivoltine. III. PARTIALLY AQUATIC ORDERS OF INSECTS A. Aquatic Heteroptera — Aquatic and Semiaquatic Bugs Because all aquatic and semiaquatic bugs are Heteroptera and none are Homoptera, the catalog by Henry and Froeschner (1988) will be followed and this 681 order of insects will be referred to as Heteroptera rather than Hemiptera, or Hemiptera: suborder Heteroptera. The choice of names for this group of insects has created much controversy; pros and cons are discussed by Henry and Froeschner (1988). Heteroptera is a medium-sized paurometabolous order that is primarily terrestrial, but about 8.5% of the species are aquatic, with adults and nymphs either living in water (220 species) or on its surface (106 species). It is divided into seven suborders, only two of which have aquatic species. Species with adults and nymphs that walk on the water surface are in the suborder Gerromorpha, and are referred to as “semiaquatic Heteroptera.” Species with adults and nymphs that live in the water belong to the suborder Nepomorpha. These are referred to as “aquatic Heteroptera,” because they spend their entire life under water, except during dispersal ﬂights. Flightless species of Nepomorpha are the most aquatic of all insects because they rarely, if ever, leave the water. There are six families of Nepomorpha in which all species are aquatic, and ﬁve families of Gerromorpha in which all species are semiaquatic or occasionally riparian. There are also four families of Heteroptera with riparian adults and nymphs; these have been included in the key because they occasionally are collected along with aquatic species. These are Gelastocoridae and Ochteridae (Nepomorpha), Macroveliidae (Gerromorpha), and Saldidae (Leptopodomorpha). Most species of aquatic and semiaquatic Heteroptera breed in lentic habitats, but there are also several lotic species, and adults of many lentic species ﬂy to streams in the north to overwinter. Adults and nymphs of most species are predators, and their presence can signiﬁcantly inﬂuence populations of other insects, especially in lentic habitats. They are known to have an impact on populations of mosquito larvae, and thus can beneﬁt man. Adults of larger species, however, may prey on small ﬁsh and create problems in ﬁsh-rearing ponds. Those of some species also may bite people, and occasionally they become a nuisance when attracted by lights to swimming pools. The vast numbers of corixid adults that overwinter in northern streams are an important source of food for some ﬁsh, and dried corixids are sold as food for pet turtles and tropical ﬁsh. Most other Heteroptera are usually avoided by ﬁsh, probably because of secretions from their scent glands. Generally, however, aquatic and semiaquatic Heteroptera have little impact on humans. Heteroptera adults can be distinguished from other aquatic insects by their mesothoracic wings, or hemelytra, which are hardened in the basal half and membranous apically (Fig. 87), and by sucking mouthparts that are formed into a tube or “rostrum” (Figs. 88, 90). 682 W. L. Hilsenhoff There are normally ﬁve nymphal instars; a few species have only four. Nymphs resemble adults, except that they lack wings and genitalia, and are distinctly less sclerotized. They inhabit the same habitat as adults and are often associated with them. Aquatic and semiaquatic Heteroptera have been rather thoroughly studied, and adults of all North American species can be readily identiﬁed. Families and genera are quite heterogeneous, making it possible to recognize families, many genera, and some species in the ﬁeld. The nymphs, however, have not been well-studied except for Gerridae, and few can be identiﬁed to species except regionally or through association with adults. Some cannot even be identiﬁed to genus. Since the pioneering works of H.B. Hungerford and others 45 – 80 years ago, there have been relatively few additions to the known fauna in North America. References to species keys to adults are provided in Table VI. Information on the biology, habitat, and identiﬁcation of each family is presented next, along with information about suborders. A key to families of adults of aquatic, semiaquatic, and riparian Heteroptera appears in Section V.G. 1. Families of Semiaquatic Heteroptera — Suborder Gerromorpha Adults of semiaquatic species reach peak abundance in September or October, depending on latitude, after which most of them ﬁnd protected terrestrial sites in which to spend the winter; adults of species that overwinter as eggs die. Species that overwinter as adults are bivoltine or multivoltine; those that overwinter as eggs usually are univoltine, but may be bivoltine in the south. Eggs of semiaquatic species are laid on substrates at the water surface. Nymphs and adults feed mostly on invertebrates of an appropriate size in the surface ﬁlm. Gerridae, and probably most other Gerromorpha, have sensillae on their femurs and trochanters that detect vibrations in the surface ﬁlm, enabling them to ﬁnd their prey. In most semiaquatic Heteroptera apterous and brachypterous adults are common. Apterous adults can be distinguished from nymphs by the presence of genitalia, greater sclerotization of the body, and by having two tarsal segments instead of one on most legs. Semiaquatic Heteroptera adults and nymphs are adapted for walking on water by having hydrofuge hairs on their tarsi, and in the larger and heavier Gerridae and Veliidae, by additionally having preapical claws. They are most readily collected by sighting them on the water surface and sweeping them from the surface ﬁlm with an aquatic net. a. Gerridae (8 Genera, 44 Species) All of the largest adults of semiaquatic Heteroptera are species of Gerri- TABLE VI Literature References to Species Keys for Identiﬁcation of Heteroptera Adults Regional keys to adults that include more than one family Alberta, Saskatchewan, and Manitoba — Brooks and Kelton (1967) California — Menke (1979a) Florida — (northern) Herring (1951); Chapman (1958) Minnesota — Bennett and Cook (1981) Mississippi — Wilson (1958) Missouri — Froeschner (1949, 1962) Montana — Roemhild (1976) North and South Carolina — Sanderson (1982) Oklahoma — Schaefer and Drew (1968) Virginia — Bobb (1974) Wisconsin — Hilsenhoff (1984b, 1986) Keys to adults listed by family Belostomatidae – Menke (1979b); (Louisiana) Gonsoulin (1973c) Corixidae – Hungerford (1948); (Oregon and Washington) Stonedahl and Lattin (1986) Cenocorixa – Jansson (1972) Hesperocorixa – Dunn (1979) Trichocorixa – Sailer (1948) Gerridae – (Arkansas) Kittle (1980); (British Columbia) Scudder (1971); (Louisiana) Gonsoulin (1974); (Oregon and Washington) Stonedahl and Lattin (1982); (Ontario) Cheng and Fernando (1970) Aquarius – Andersen (1990) Gerrinae – Drake and Harris (1934); Kuitert (1942) Gerris – Andersen (1993); (Connecticut — also nymphs) Calabrese (1974) Limnoporus – Andersen and Spence (1992) Metrobates – Anderson (1932) Rheumatobates – Hungerford (1954) Trepobates – Kittle (1977) Hebridae – Porter (1950) Hydrometridae – Hungerford and Evans (1934); (Louisiana) Gonsoulin (1973a) Mesoveliidae – Polhemus and Chapman (1979) Naucoridae – (Texas) Davis (1996); (Louisiana) Gonsoulin (1973b) Ambrysus – LaRivers (1951) Pelocoris – LaRivers (1948); (Texas) Sites and Polhemus (1995) Nepidae – Sites and Polhemus (1994); (Louisiana) Gonsoulin (1975) Notonectidae – (Arizona) Zalom (1977); (British Columbia) Scudder (1965); (West Coast nymphs) Voigt and Garcia (1976) Buenoa – Truxal (1953) Notonecta – Hungerford (1933); (New England) Hutchinson (1945) Veliidae – Smith and Polhemus (1978); Smith (1980) Rhagovelia – Polhemus (1997) dae, but the family has many diverse sizes, forms, and life histories. Adult “water-striders” are distinguished by their relatively large size, preapical tarsal claws (Fig. 97), and elongate metafemurs that extend well past the tip of the short abdomen. Apterous and brachypterous forms are common. Although found in all types of aquatic habitats, adults and nymphs of most species prefer quiet areas where there is minimal wave action and current. Adults and nymphs of lotic species inhabit 17. Diversity and Classiﬁcation of Insects and Collembola streams of all sizes; those of lentic species inhabit swamps, marshes, permanent and temporary ponds, littoral areas of lakes, and even the ocean. However, adults of most lotic species will occasionally inhabit lentic habitats and adults of lentic species may occasionally be found along margins of lotic habitats. Most are usually associated with emergent vegetation. Univoltine or bivoltine life cycles prevail, with some species overwintering as adults and some univoltine species overwintering as eggs. All water striders are predaceous. They feed on terrestrial insects and other invertebrates that become trapped in the surface ﬁlm or aquatic insects that live just beneath the surface. b. Hebridae (3 Genera, 15 Species) Because of their very small size (2.5 mm), “velvet water bugs” are likely to be confused only with very small species of Veliidae. When on the water, Hebridae adults and nymphs move much more slowly than those of similarsized Veliidae, and upon close examination Hebridae adults and nymphs have a rostral sulcus and apical claws, which those of Veliidae lack. Adults and nymphs inhabit very shallow, sheltered shoreline areas of lentic habitats, including swamps, marshes, ponds, lakes, and river sloughs. Those of some species are occasionally or even frequently riparian. Apterous adults are common in some species. Adults can be found from spring to autumn, suggesting that they are bivoltine or multivoltine and overwinter as adults. c. Hydrometridae (1 Genus, 9 Species) The small (8 – 11 mm), delicate, sticklike “marsh-treaders” or “water-measurers” with their elongate head (Fig. 99) are unlike any other aquatic insect. Both macropterous and brachypterous adults are common. They walk on vegetation and the surface ﬁlm of permanent habitats that lack wave action and current, including marshes, swamps, and vegetated margins of lakes, ponds, and slow streams. Here they feed by using their barbed rostrum to spear small macroinvertebrates in the surface ﬁlm. They most often are found where ﬁsh are absent. Hydrometridae are multivoltine and overwinter as adults. d. Mesoveliidae (1 Genus, 3 Species) “Water-treaders” become abundant in late summer in sheltered lentic habitats and margins of lotic habitats that have algae and duckweed along with emergent vegetation. Their relatively small size (2 – 4 mm) and the presence of black spines on the legs is distinctive. Apterous adults predominate, but winged adults are not uncommon, especially in autumn. They prey on small terrestrial and aquatic animals in the surface ﬁlm. Adults and nymphs are killed by freezing temperatures, and in the 683 north mesoveliids overwinter as eggs. All species are probably multivoltine. e. Veliidae (5 Genera, 35 Species) “Broad-shouldered” or “short-legged water-striders” are often abundant in a wide variety of aquatic habitats. Some inhabit streams, living in the vicinity of rifﬂes or under the banks; others inhabit spring ponds, swamps, marshes, ponds, and margins of lakes. Adults can be distinguished from other semiaquatic Heteroptera, except Gerridae, by their preapical tarsal claws. They differ from Gerridae in their generally smaller size and relatively shorter legs, the metafemurs not extending past the tip of the abdomen. Apterous adults predominate, with winged forms rare or absent in the various species. Most species are multivoltine and overwinter as adults, some along margins of streams or springs instead of in terrestrial habitals. A few species overwinter as eggs and are univoltine or bivoltine. 2. Families of Aquatic Heteroptera — Suborder Nepomorpha Most species are univoltine in northern North America; a few are bivoltine. Farther south, bivoltine life cycles probably predominate. Adults reach peak abundance in October or November, and overwinter in aquatic habitats. In areas where ponds and lakes freeze, adults of species that typically breed in shallow lentic habitats usually ﬂy to deep lentic habitats or larger streams to overwinter. In late April or early May, they return to their breeding sites to mate and oviposit. Eggs are laid on vegetation or other substrates, usually in the water. Some univoltine species diapause over the winter as eggs. Adults and nymphs of most species are adapted for swimming by having fringes of long setae on their legs, especially the metathoracic legs. They obtain oxygen from air stores held under the hemelytra and from a bubble held ventrally on hydrofuge hairs, which may act as a physical gill or plastron. Air stores are renewed at the surface through air straps in Belostomatidae, air tubes in Nepidae, the pronotum in Corixidae, and the apex of the abdomen in Notonectidae and Naucoridae. In Corixidae, Notonectidae, Pleidae, and Naucoridae the ventral bubble is large in relation to their size and acts as a physical gill, with oxygen from the water diffusing into the bubble and carbon dioxide from respiration diffusing out of it. This allows prolonged submergence in oxygen-rich water. Adults and nymphs of almost all species are carnivores. They feed mostly on small invertebrates and occasionally on vertebrates such as minnows and tadpoles. The largest family, Corixidae, is an exception, with adults and nymphs of most species being primarily detritivores. 684 W. L. Hilsenhoff Nepomorpha adults and nymphs are best collected with an aquatic net along with debris. The contents of the net are then spread on a half-inch (13 mm) mesh screen over a large white pan, and insects are allowed to crawl from the debris and fall into the pan. Adults and nymphs of many species feign death and will crawl from the debris only if left undisturbed for several minutes. Additional specimens in all families can often be collected with bottle traps (Hilsenhoff, 1987). In northern regions, exceptionally large numbers of adults of both lotic and lentic species can be collected in autumn from along banks of large streams where the current is slow, but adults of some lentic species do not ﬂy to streams and will be missed. Adults of many species are attracted to lights, and may be collected with light traps. permanent ponds and littoral areas of small lakes, but those of some species are strictly lotic and can be found only along margins of streams or in spring ponds and seeps. In northern latitudes, adults of most lentic species ﬂy to larger streams to overwinter, and in October and November thousands can often be collected from slow, shallow areas of streams. Adults disperse widely, rapidly invading temporary ponds, and on warm nights they are readily attracted to lights. Eggs are laid on underwater structures in such great numbers that they are harvested for food in some tropical countries. Most northern species are probably univoltine, with overwintering adults returning to breeding sites in spring to mate and oviposit. In the south, two or more generations per year are likely. There is evidence that at least one species overwinters as an egg. a. Belostomatidae (3 Genera, 19 Species) “Giant water bugs” are easily recognized by their large size and a pair of short, straplike, posterior respiratory appendages (Fig. 92) that they use to obtain air at the water surface. Adults and nymphs of two genera (Belostoma and Lethocerus) inhabit permanent lentic habitats, especially weedy ponds, margins of lakes, and marshes, and are important as apex invertebrate predators. Those of the third genus (Abedus) occur in streams among aquatic plants or under rocks in rifﬂes. All adults are powerful predators that will capture and kill anything they can subdue, including ﬁsh, frogs, tadpoles, and other insects. Eggs are laid in large masses above the water on vegetation (Lethocerus) or on the backs of the males, which brood the eggs (Abedus and Belostoma). Belostomatids are univoltine or bivoltine, depending on the latitude, with adults of lentic species in northern regions ﬂying in autumn to larger streams or deep lentic habitats where they overwinter. They return to their breeding sites in early May. Adults frequently ﬂy to lights and are sometimes called “electric light bugs.” c. Naucoridae (4 Genera, 22 Species) “Creeping water bugs” are most common in the southern United States and are rarely found as far north as Canada. They are dorsoventrally ﬂattened, have greatly expanded profemurs, and have a rounded appearance when viewed from above, with margins of the head, pronotum and elytra being continuous (Fig. 93). They are usually associated with lotic habitats, living in streams, spring ponds, or impoundments. Eggs are glued to vegetation or mineral substrates, depending on the species. Most species probably are univoltine and overwinter as adults in deeper areas of their breeding habitat. Because adults carry a large plastron and generally live in well-oxygenated water, they rarely need to surface for air. Although adults of many species have fully developed wings, ﬂight rarely has been observed. b. Corixidae (18 Genera, 129 Species) As the most abundant aquatic Heteroptera, “water boatmen” are found in most permanent aquatic habitats and frequently invade temporary ones as well. Adults most closely resemble those of Notonectidae, but are dorsoventrally ﬂattened (Fig. 87) and have a short, broad, unsegmented, triangular rostrum and widened, scoop-shaped protarsi (palae) (Fig. 88). Unlike other aquatic and semiaquatic Heteroptera, which are predators, corixid adults and nymphs feed primarily on detritus, algae, protozoans, and other extremely small animals. Adults of a few species, however, will capture and eat larger insects, such as mosquito larvae. Adults and nymphs of most species are lentic, inhabiting d. Nepidae (3 Genera, 13 Species) “Water scorpions” can be readily recognized by their long apical breathing tube (Fig. 91). They breed in permanent lentic habitats, especially shallow areas with much vegetation. Here they often remain with their breathing tube above the surface waiting to ambush invertebrates or small vertebrates, but they are capable of remaining completely submerged, using air stores under their hemelytra for respiration. Unlike other aquatic Heteroptera, adults and nymphs are very poor swimmers and usually cling to vegetation or other substrates. Most species are univoltine in northern latitudes and probably bivoltine in the south. They overwinter as adults, which in the north usually ﬂy to deeper lentic or lotic habitats in autumn and return to breeding sites in late April or early May. Eggs that are laid on supports at the water surface have respiratory horns that protrude from the water. When ﬂooded, the eggs respire through a plastron. 17. Diversity and Classiﬁcation of Insects and Collembola e. Notonectidae (3 Genera, 32 Species) “Backswimmers,” as their name implies, swim ventral side up, but orient themselves dorsal side up when not in the water. They most resemble Corixidae, but are deepbodied, not at all ﬂattened (Fig. 95), and have an elongate, segmented rostrum (Fig. 90). Adults of most species breed in littoral areas of lakes and permanent ponds, others in stream pools; some species are transients in temporary ponds. Adults and nymphs of two genera, Buenoa and Notonecta, can inhabit relatively deep water and remain submerged for long periods because they have ventral channels to retain a plastron. Notonecta adults and nymphs typically hang from the surface ﬁlm, while Buenoa adults and nymphs have hemoglobin, which enables them to maintain neutral buoyancy and remain planktonic. Adults and nymphs in both genera are active predators that pursue or ambush other invertebrates or small vertebrates. A third genus, Martarega, occurs only in extreme western United States, where adults and nymphs inhabit eddies of streams and ambush prey carried by the current. Several species overwinter as diapausing eggs and have a univoltine life cycle with nymphal development in late spring and summer. Others have univoltine or bivoltine life cycles, with overwintering adults that mate and lay eggs in the spring. In the north, adults of a few species may overwinter in larger streams and return to lentic habitats in spring. f. Pleidae (2 Genera, 5 Species) “Pygmy backswimmers” are very small (3 mm), convex insects that swim upside down. Adults do not have typical hemelytra, but instead their mesothoracic wings are shelllike (Fig. 94) and resemble elytra of Coleoptera. Their metathoracic wings are vestigial and they cannot ﬂy. Adults and nymphs inhabit vegetation, mostly in permanent ponds, but also may be found in littoral areas of lakes, backwaters of streams, and swamps. If their habitat dries because of drought, they can survive for several months in the moist soil. Pleids are widespread and often abundant, except in the Paciﬁc Coast region where they do not occur. Although adults and nymphs can swim quite well, they mostly crawl about on vegetation and feed on small invertebrates. Because of their small body size and relatively large plastron, they are able to remain submerged for long periods. Life cycles are bivoltine or multivoltine, but their life history is poorly known. B. Aquatic Neuroptera — Spongillaﬂies Neuroptera is a fairly large terrestrial order, but larvae in only one family are aquatic. They live in and feed upon several genera of freshwater sponges 685 (Spongillidae) that occur in permanent lotic or lentic habitats. Taxonomic keys to species of larvae can be found in the following publications: Parﬁn and Gurney (1956), Poirrier and Arceneaux (1972), and for North and South Carolinas in Brigham (1982b). Information on the biology, habitat, and identiﬁcation of larvae is presented next. 1. Families of Aquatic Neuroptera a. Sisyridae (2 Genera, 6 Species) The small (8 mm) aquatic larvae are distinctive. They have sucking mouthparts in the form of a pair of long stylets and are covered with long, spinose setae (Fig. 102). Most species are probably multivoltine, with larvae having only three instars. Other life stages are terrestrial. Eggs are usually laid in very small masses, mostly in crevices of vegetation or supports above aquatic habitats that support freshwater sponges. After an incubation period of about 1 week, the eggs hatch and larvae drop into the water where they swim or are carried by currents until they encounter a suitable species of sponge. After completing development on the sponge, they swim or crawl to shore, crawl out of the water, and ﬁnd a suitable pupation site in a crack or crevice or on a ﬂat surface that may be several meters from the water. Here, they spin a double-walled cocoon in which they pupate, the outer wall having a distinctive meshlike construction. After several days, pharate adults use pupal mandibles to cut their way out of the cocoon and emerge. Adults feed on nectar and apparently live no more than a few weeks. Most species overwinter as larvae on sponges, but some may overwinter as prepupae in the pupal cocoon. Larvae have no special adaptations for the aquatic environment. They obtain oxygen from the water through their cuticle and swim by repeatedly arching their abdomen forward while in a vertical position and snapping it back. Larvae are rarely collected by standard sampling techniques. Substantial numbers can be collected only by collecting sponges and removing larvae that crawl from them as they dry. Rearing has been accomplished, but a suitable colony of freshwater sponges must be maintained and a terrestrial habitat for pupation must be provided. C. Aquatic Lepidoptera — Aquatic Caterpillars Larvae in several genera of this large, primarily terrestrial order, are associated with aquatic habitats. Some feed on emergent parts of aquatic macrophytes and others mine stems of these plants. Still others feed on submerged parts of plants attached to other substrates, and only these will be considered here. The 17 aquatic genera in which aquatic larvae occur are all in 686 W. L. Hilsenhoff the family Pyralidae, and almost all are in the subfamily Nymphulinae. Aquatic caterpillars have three pairs of thoracic legs and pairs of short prolegs ringed with hooklike crochets on abdominal segments 3 – 7 (Fig. 103). They are not likely to be confused with any other aquatic insect larvae, but often it is not possible to distinguish them from Lepidoptera that are inadvertently collected from emergent vegetation. Only those that have numerous ﬁlamentous gills covering their body and those that live in portable cases made from the aquatic vegetation that they inhabit, can be recognized with certainty as being aquatic. Larvae that live in cases are so well camouﬂaged that they often escape notice, even after capture with a net. Some species may become sufﬁciently abundant to have an impact on aquatic plant communities. While adults have been well studied and larvae of many of the species have been reared and described, reliable keys to species of larvae have not been constructed because larvae of many species remain unknown. Larvae inhabit a wide variety of permanent aquatic habitats. Some inhabit rocks in rapid water of larger streams where they feed on diatoms and other algae. Others inhabit aquatic macrophytes or duckweed (Lemna) in ponds or along margins of streams and lakes. Here, they feed on the plants they inhabit and make cases from them. Respiration is cutaneous, and is enhanced by numerous ﬁlamentous gills in larvae that inhabit rocks in streams and also in some lentic casebuilders. Larvae of at least one species have hydrofuge hairs enabling them to maintain a plastron that is used as a physical gill. Pupation of aquatic species is in a cocoon, usually in the larval habitat. Emergence occurs after pupae swim or crawl to the surface of the water; legs of species that swim have long swimming hairs. Adults are generally nondescript small moths that hold their wings rooﬂike over their body and remain in the vicinity of their larval habitat. Adults of stream species crawl under the water to oviposit on rocks, while those of most lentic species deposit rows of eggs just below the water surface on the preferred food plant. Most species are univoltine or bivoltine and have ﬁve larval instars. D. Aquatic Coleoptera — Water Beetles Only about 3% of Coleoptera species have an aquatic stage, but because this is the largest insect order they represent a signiﬁcant segment of the aquatic insect fauna. About 1450 aquatic species in 19 families have been found in North America. There are also several species of riparian Coleoptera that occasionally may be collected while sampling margins of aquatic habitats. In eight families both larvae and adults are aquatic in almost all species, and in nine others either all larvae or all adults are aquatic. A few families with mostly terrestrial species also have some species with larvae and/or adults that are adapted for living in the aquatic environment. Three suborders of Coleoptera have aquatic representatives. Adephaga is represented by ﬁve families in which all species are aquatic both as larvae and adults. These families are often referred to as the “Hydradephaga”. The suborder Myxophaga is represented in North America by a single aquatic species, Hydroscapha natans, in which both larvae and adults are aquatic. Several families of Polyphaga have some species with one or more aquatic stages, but only in a few families do most species have a tie to the aquatic environment. Most aquatic beetles can be collected with an aquatic net by using standard collecting techniques. However, larger Dytiscidae and Hydrophilidae are ineffectively sampled with nets, and bottle traps must be relied on to capture substantial numbers of these larger beetles and also species that are nocturnal (Hilsenhoff, 1987, 1991). Adult Gyrinidae, which mostly swim on the surface in deeper water, are seldom captured without special effort. In late summer, large schools often appear in shallow bays of lakes and streams, and by herding them toward shore and sweeping through the school, large numbers often can be captured. In the north, where ponds and lakes freeze, lentic as well as lotic gyrinids congregate in streams in the fall to overwinter. Here, very large numbers of mixed species can be collected with a net from undercut banks where there is a current and the water is at least 0.5 m deep. Some overwintering lentic species of Dytiscidae, Haliplidae, and Hydrophilidae also may be found along stream banks in autumn and early spring. Large numbers of Hydrophilidae, Helphoridae, and Hydrochidae adults can often be collected by black-light traps on warm nights in spring and summer. Adult water beetles range from 1 – 40 mm in length and vary greatly in structure. All are characterized by chewing or biting mouthparts and shell-like mesothoracic wings that are called elytra (Fig. 108). They have functional spiracles and rely on atmospheric oxygen for respiration. Adult Adephaga carry a bubble of air under their elytra, which is renewed at the surface by swimming or crawling (Amphizoidae) to the surface and breaking the surface ﬁlm with the tip of the abdomen. This air supply must be frequently renewed, except when water temperatures are cold and metabolism is slowed, or when the water is well oxygenated. Hydroscaphidae adults (Myxophaga) crawl to the surface to renew their air supply. Elmidae and Dryopidae adults (Polyphaga) usually live in oxygen-rich water and maintain a plastron of air that is carried on hydrofuge hairs. The plastron acts as a physical gill, with 687 17. Diversity and Classiﬁcation of Insects and Collembola oxygen for respiration diffusing into it from the water, and carbon dioxide from respiration diffusing out into the water. Other Polyphaga adults (Helophoridae, Hydrochidae, Hydrophilidae, Hydraenidae, and Curculionidae) also maintain an air supply on hydrofuge hairs, but must renew the air by swimming (most Hydrophilidae) or crawling (Curculionidae, Hydraenidae, Helophoridae, Hydrochidae, and some Hydrophilidae) to the surface when the water contains too little oxygen for plastron respiration. Unlike Adephaga, adult Polyphaga break the surface ﬁlm with their antennae. Larvae of aquatic Coleoptera vary greatly in size and general morphology. All have three pairs of thoracic legs, chewing or biting mouthparts, and are relatively well sclerotized, especially in the later instars. Some have lateral ﬁlaments and resemble larvae of Megaloptera. Most larvae of aquatic Coleoptera rely on cutaneous respiration, which may be enhanced by gills, although some larger Dytiscidae and Hydrophilidae larvae obtain oxygen at the water surface through functional posterior spiracles. Larvae of Chrysomellidae and Noteridae are able to utilize oxygen from plant tissues, and by this mechanism are able to pupate under water. It is also likely that larvae and adults of Celina (Dytiscidae) obtain oxygen from plants (Spangler 1973; Hilsenhoff, 1994). Most Coleoptera larvae lack spiracles in the early instars, but are equipped with a full complement of spiracles in the ﬁnal instar, which allows them to leave the water and pupate in cells on land. Adephaga adults (except Amphizoidae) and many Hydrophilidae adults are adapted for swimming by having long setae or swimming hairs on at least their metathoracic legs or by having greatly ﬂattened legs (Gyrinidae). Some larvae of Dytiscidae are also adapted for swimming by having long setae on their legs, but larvae of most aquatic Coleoptera crawl on vegetation or other substrates. Gyrinidae larvae, which mostly crawl on vegetation, can swim rapidly with an undulating motion when disturbed. Much of the taxonomy of North American water beetles was completed more than 50 years ago, but some genera need revision and several species remain undiscovered. Adults of almost all species can be reliably identiﬁed, but their distribution remains poorly deﬁned in North America because in many areas aquatic beetles have not been adequately studied. Unlike adults, very few larvae can be identiﬁed to species and a few cannot even be identiﬁed to genus. In most genera, larvae of too few species are known to permit development of reliable keys. Fortunately, the European fauna is much better known, and the European literature gives us insight into probable life cycles, larval and adult habitats, overwintering sites, feeding habits, and larval taxonomy. Development of keys and descriptions for aquatic Coleoptera larvae remains a pressing need. To accomplish this, larva – adult associations must be obtained through laboratory rearing. The most promising method for doing this is to capture mated females, or male – female pairs, allow females to oviposit, and then rear larvae that hatch from the eggs. By using this ex-ovo method (Alarie et al., 1989), all larval instars can be associated with the adult. Because of our inability to identify larvae, life histories of most North American species remain poorly known. This includes our knowledge of adult habits, especially such things as feeding, dispersal, overwintering, and estivation. From the occurrence of teneral adults and collection records for adults and larvae (if identiﬁed), life cycles and overwintering sites have been postulated for many species of Wisconsin Dytiscidae, Helophoridae, Hydrochidae, and Hydrophilidae (Hilsenhoff (1992, 1993 a-c, 1994, 1995a,b,d). Much research is needed before we will understand the bionomics of the many species of aquatic Coleoptera in North America. References to species keys for the identiﬁcation of Coleoptera adults and larvae are provided in Table VII. Keys to families of freshwater Coleoptera adults and TABLE VII Literature References to Species Keys for Identiﬁcation of Coleoptera Adults and Larvae Regional keys to adults that include more than one family California – Leech and Chandler (1956) Florida – Young (1954); Epler (1996) Maine – (Adephaga and Hydrophilidae) Malcolm (1971) North Dakota – Gordon and Post (1965) North and South Carolina — Brigham (1982c) Paciﬁc Northwest — Hatch (1953, 1965) Keys to adults listed by family or superfamily Amphizoidae — Kavanaugh (1986) Dryopoidea — Brown (1972); (Louisiana) Barr and Chapin (1988); (Wisconsin) Hilsenhoff and Schmude (1992) Dytiscidae — (Alberta) Larson (1975); (Canada and Alaska) Larson et al. (2001); (Virginia) Michael and Matta (1977); (Utah) Anderson (1962); (Wisconsin) Hilsenhoff (1992, 1993a, b, c, 1994, 1995a) Acilius — Hilsenhoff (1975) Agabus — Fall (1922); Larson (1989, 1991, 1994, 1996, 1997); Larson and Wolfe (1998); (Paciﬁc Coast) Leech (1938) Celina — Young (1979a) Colymbetes — Zimmerman (1981) Copelatus — Young (1963a) Coptotomus — Hilsenhoff (1980) Desmopachria — Young (1981a, 1981b) Dytiscus — Roughley (1990) Graphoderus — Wallis (1939) Heterosternuta — Matta and Wolfe (1981) Hydaticus — Roughley and Pengelly (1981) Hydroporus — Gordon (1969, 1981) (Continues) 688 TABLE VII W. L. Hilsenhoff (Continued) Hydrovatus — Bistrom (1996), Young (1956, 1963b) Hygrotus — Anderson (1970, 1975, 1983) Ilybius — Larson (1987) Laccophilus — Zimmerman (1970) Laccornis — Wolfe and Roughley (1990) Liodessus — Larson and Roughley (1990); Miller (1998) Lioporeus — (as Falloporus) Wolfe and Matta (1981) Matus — Young (1953) Nebrioporus — Shirt and Angus (1992); (as Deronectes) Zimmerman and A.H. Smith (1975) Neoporus — (as Hydroporus) Fall (1923); Wolfe (1984) Oreodytes — Zimmerman (1985); Alarie (1993) Rhantus — Zimmerman and R. L. Smith (1975) Sanﬁlippodytes — (as Hydroporus) Fall (1923) Stictotarsus — (as Deronectes) Zimmerman and A. H. Smith (1975) Thermonectus — McWilliams (1969) Elmidae Dubiraphia — Hilsenhoff (1973) Optioservus — White (1978) Stenelmis — Schmude (1992), (1999) Gyrinidae — (Minnesota) Ferkinhoff and Gundersen (1983); (Quebec) Morrissette (1979); (Wisconsin) Hilsenhoff (1990) Dineutus — Hatch (1930); Wood (1962) Gyrinus — Oygur and Wolfe (1991) Haliplidae — (Minnesota) Gundersen and Otremba (1988); (Virginia) Matta (1976); (Wisconsin) Hilsenhoff and Brigham (1978) Haliplus — Wallis (1933) Peltodytes — Roberts (1913) Helophoridae — Smetana (1985); (Paciﬁc Northwest) McCorkle (1965) Hydraenidae — Perkins (1980) Hydrochidae — Hellman (1975) Hydrophilidae (includes Helophoridae and Hydrochidae) — (Canada and Alaska) Smetana (1988); (Illinois) Wooldridge (1967); (Michigan) Willson (1967); (Mississippi) Testa and Lago (1994); (Nevada) LaRivers (1954); (Paciﬁc Northwest) Miller (1965); (Virginia) Matta (1974); (Wisconsin) Hilsenhoff (1995b, d) Anacaena — (Europe) Berge Henegouwen (1986) Berosus — Van Tassell (1966) Crenitis — Winters (1926) Cymbiodyta — Smetana (1974) Enochrus — Gundersen (1978) Hydrochara — Smetana (1980) Laccobius — Cheary (1971); Gentili (1985, 1986) Paracymus — Wooldridge (1966) Tropisternus — Spangler (1960) Noteridae Hydrocanthus — Young (1985) Suphisellus — Young (1979b) Keys to larvae listed by family Chrysomelidae — (Michigan) Hoffman (1939) Dytiscidae — (Wisconsin) Hilsenhoff (1992, 1993a, b) Heterosternuta — Alarie (1992) Hydroporus — Alarie (1991) Hygrotus — Alarie et al. (1990) Oreodytes — Alarie (1997) Ptilodactylidae Anchytarsus — Stribling (1986) larvae appear in Sections V.H.1 and V.H.2. Information on suborders and the biology, habitat, and identiﬁcation of adults and larvae in each family is presented in the following. 1. Families of Aquatic Coleoptera — Suborder Adephaga In the ﬁve aquatic families of this suborder (Hydradephaga), both larvae and adults of all species are aquatic. Eggs are laid singly or in masses, mostly on objects beneath the water surface. Larvae have three instars, and except for at least some Noteridae, pupation takes place in terrestrial cells near the larval development site. Almost all ﬁrst and second instar larvae lack spiracles and rely on cutaneous respiration for oxygen. A few have functional posterior spiracles that they use to obtain oxygen at the water surface. Third instar larvae, which have functional spiracles on abdominal segments and the mesothorax, also rely mostly on cutaneous respiration until they leave the water to pupate. Life cycles are typically univoltine, although bivoltine and semivoltine life cycles have been recorded for some species. Some species complete larval development rapidly, requiring only a few weeks; others require several weeks, and those with semivoltine life cycles may need almost two years to complete larval development. Adults are long-lived, with records of unmated beetles living for several years, but typically most adults live several months and die after their ﬁnal mating and oviposition, which is usually in spring. a. Amphizoidae (1 Genus, 3 Species) “Trout-stream beetles” are restricted to mountain streams in northwestern North America. Here, both larvae and adults can be found near the water edge, crawling on plant roots, wood, or debris at or just beneath the water surface. Larvae are characterized by short, broad urogomphi and ﬂattened lateral projections on each segment (Fig. 120). Adults are relatively large (12 – 14 mm long) and broad, with a narrow thorax and no swimming hairs on their legs (Fig. 108). Larvae cannot swim and adults swim very poorly; both usually remain in contact with the substrate. Third instar larvae frequently stay in the splash zone and enter the water only to capture prey. Both larvae and adults feed almost exclusively on Plecoptera larvae. Because their habitat is well-oxygenated, adults may remain submerged for prolonged periods, their air bubble acting as a physical gill. The life cycle is 1 year, with eggs laid on debris in the splash zone in late summer and pupation occurring the following summer, probably in cells in the stream bank. b. Dytiscidae (48 Genera, 600 Species) The “predaceous diving beetles” are the largest family of 17. Diversity and Classiﬁcation of Insects and Collembola water beetles, with more than 550 species distributed throughout North America. Adults range from 2 to 40 mm in length and are characterized by their ovate appearance, rounded sternum and dorsum, and by a usually spear-shaped prosternal process that projects back to the mesocoxae (Fig. 106). The beetles swim by moving both metathoracic legs backward simultaneously. Larvae are generally elongate, and range from 3 to 60 mm in length at maturity. Most have paired terminal appendages (urogomphi) of various lengths (Fig. 122). They have relatively elongate legs, which in several genera are modiﬁed by long hairs for swimming. Adults and larvae of most species are lentic, inhabiting all types of permanent and temporary habitats, and showing a preference for the shallow, vegetated margins of ponds, marshes, bogs, and swamps. They are most abundant in habitats that do not contain signiﬁcant numbers of insectivorous ﬁsh. Most species breed in well-deﬁned habitats, but adults may ﬂy to other habitats to feed or overwinter. Adults and larvae of lotic species are found mostly under stream banks and tend to remain in the habitat in which they breed. Typically adults overwinter, mate in the spring, oviposit, and die. Eggs are laid in or on vegetation beneath the water surface, or on other objects below or just above the surface. The larvae develop over a period of a few to several weeks, then leave the aquatic habitat to pupate in cells they construct in the soil of protected areas nearby. The pupal stage lasts 5 – 14 days, after which the adult emerges and usually re-enters the aquatic habitat. Most species are univoltine, but bivoltine life cycles occur, especially in the south, and there is evidence northern species that breed in cold-water habitats are semivoltine. However, life histories of many species remain poorly known, and there are variations in the life cycle described above. There is ample evidence for estivation of adults during dry periods in the summer and also evidence that several northern species overwinter as diapausing eggs. Many lotic species and a few lentic species overwinter as larvae, even in northern areas that freeze. While most overwintering adults are found in ponds, lakes, or streams, some pass the winter in terrestrial habitats and enter their breeding sites late in the spring. Both adults and larvae are predators, feeding primarily on other invertebrates and small vertebrates. Adults of most species are also scavengers. c. Gyrinidae (4 Genera, 60 Species) Adult “whirligig beetles” are small- to medium-sized beetles (3.5 – 14.0 mm long). They are widespread and often abundant. Most frequently, they are seen resting or swimming in groups of a few to several hundred on the water surface in areas protected from the wind, especially in late 689 summer. However, they can, and often do, swim under the surface. Their greatly ﬂattened legs and two pairs of eyes are distinctive. Larvae are also distinctive, having fringed projections on each abdominal segment posteriorly (Fig. 119). They swim with a vertical undulating motion when disturbed. While adults and larvae of most species are lentic, those of several species are lotic, and adults of most lentic species frequent larger streams in autumn, or in late summer when it is dry. In streams, adult gyrinids will often escape the current by crawling onto vegetation just above the water. In northern regions, where ponds and lakes freeze, adults of most lentic species ﬂy to larger streams or lakes to overwinter. They return to breeding sites in early spring, as soon as air temperatures have warmed enough to permit ﬂight. All species tend to inhabit deeper water than other Hydradephaga, with larvae being found mostly among submerged vegetation in large ponds or rivers, or in littoral zones of lakes and impoundments. Larvae are predators, feeding mostly on other invertebrates, while adults are scavengers on dead animals or predators of small invertebrates in the surface ﬁlm. Most species are probably univoltine, with adults overwintering and larvae developing during the summer. Eggs are laid mostly on submerged aquatic vegetation. Pupation is in cocoons on emergent vegetation, or on terrestrial vegetation adjacent to the larval habitat. d. Haliplidae (4 Genera, 70 Species) “Crawling water beetles” are small (2.5 – 5.0 mm long) yellowish to orangish beetles with black blotches and spots, apically narrowed elytra, and a narrowed thorax. Their large metacoxal plates are distinctive (Fig. 105). They are often abundant in shallow lentic and lotic vegetation-choked habitats. In spite of their common name, they have fringed tarsi and tibiae and are able swimmers. The larvae, which crawl about on vegetation, are either elongate with a long terminal segment (Fig. 123), or have numerous long dorsal projections (Fig. 124). Life cycles of most species are probably univoltine, with eggs being deposited on or in ﬁlamentous algae, or other vegetation. Both larvae and adults are herbivores, feeding on algae or macrophytes. Pupation takes place in cells in moist soil near the larval development site. Most species overwinter in the water as adults, but some adults and larvae are known to overwinter in terrestrial sites adjacent to the water. e. Noteridae (5 Genera, 18 Species) This small family, frequently referred to as “burrowing water beetles,” is generally southern in its distribution and rarely found as far north as Canada or in the west. Adults are small (1.2 – 5.5 mm long) and resemble Dytiscidae, but 690 W. L. Hilsenhoff are usually more attenuate apically than most Dytiscidae. They have a ﬂat, V-shaped metasternal plate and a truncate, instead of a spear-shaped or rounded, prosternal process (Fig. 111). Like Dytiscidae, adults are mostly predators that live in close association with aquatic vegetation. The larvae, which are poorly known, have short legs, short urogomphi, and telescoping body segments (Fig. 121). Their morphology suggests that they are omnivores, which live among roots of aquatic vegetation. Larvae of a European species obtain oxygen from plant tissues and pupate in a cocoon on the plant roots, the pupae also obtaining oxygen from the plant. Life cycles are probably one year, with adults overwintering in the water. 2. Families of Aquatic Coleoptera — Suborder Myxophaga a. Hydroscaphidae (1 Genus, 1 Species) The only North American species occurs from the western United States, north to Idaho. The fusiform shape, truncate elytra, and very small size (1.5 mm) of adult “skiff beetles” is distinctive (Fig. 118). Larvae and adults are found most commonly on algae over which a thin ﬁlm of water ﬂows, but adults have also been collected in deeper water. Both adults and larvae are herbivores, feeding on algae. They tolerate a wide range of water temperatures, and have been found in thermal springs as warm as 46°C and in cold streams that frequently freeze. Females lay single, well-developed eggs on algae, and pupation also takes place on algae at the air – water interface. Larval respiration is aided by distinctive pairs of balloon – like spiracular gills on the prothorax, and ﬁrst and eighth abdominal segments. Adults, which may remain submerged for long periods, retain an air bubble that acts as a physical gill. 3. Families of Aquatic Coleoptera — Suborder Polyphaga There are four families in which both aquatic adults and larvae are found. In Elmidae, adults and larvae of almost all species are aquatic, while in Hydrophilidae most species have aquatic adults and many also have aquatic larvae. Curculionidae has several species with aquatic adults and a few also with aquatic larvae, while Chrysomelidae has several species with aquatic larvae and two also with aquatic adults. In Dryopidae, Hydraenidae, Helophoridae, and Hydrochidae all adults are aquatic, and in Lutrochidae, Psephenidae, Ptilodactylidae, and Scirtidae almost all larvae are aquatic. Larvae in at least one species of Lampyridae are also aquatic. Additional families of Polyphaga have species that are associated with water, but not adapted for living in it, and these families will not be treated here. Five families (Dryopidae, Elmidae, Lutrochidae, Psephenidae, and Ptilodactylidae) form a natural group of beetles in which almost all species have at least one aquatic stage. They formerly were placed in the superfamily Dryopoidea and will be discussed ﬁrst (Section 3.a – e), although they now are included in a larger superfamily (Byrrhoidea) (Lawrence and Britton, 1991). They differ from most other aquatic beetles by generally having more larval instars, longer life cycles, and longer adult lives in species with aquatic adults. Aquatic adults and larvae are unable to swim, which conﬁnes them to well-oxygenated habitats. Aquatic larvae obtain oxygen from the water through their integument, usually with the aid of gills. Aquatic adults have a permanent air bubble (plastron) that is carried on hydrofuge hairs and acts as a physical gill. Terrestrial adults of species with aquatic larvae typically inhabit the splash zone on rocks that project from their aquatic habitats, and they also use a plastron to remain submerged for relatively long periods when ovipositing. In Dryopoidea that have been studied, ﬁve-to eight-larval instars have been recorded, but there is disagreement about the actual number of larval instars, with six being suggested for many species. In Adephaga, Helophoridae, Hydrochidae, Hydrophilidae, and Hydraenidae there are only three larval instars. Larval development probably takes about 2 years for most species of “Dryopoidea”, with 1 – 3-year development times having been suggested for various species. The shorter times are for species in warmer and more southern streams. Aquatic adults typically live a year or more, and some may live several years; terrestrial adults of species with aquatic larvae typically live only a few days to a week. To date, studies of life histories have been mostly on Elmidae and Psephenidae; additional studies are needed. Brown (1987) reviewed the biology of these rifﬂe beetles. Larvae of “Dryopoidea” are poorly known, and identiﬁcation beyond genus is usually not possible. Brown (1972) summarized what was known about the distribution and taxonomy of Dryopoidea in the United States, and provided species keys for adults and generic keys for larvae. Since his publication, the aquatic Limnichidae have been recognized as a separate family, Lutrochidae. Several new species have been discovered recently, and revisions are needed for some genera. a. Dryopidae (5 Genera, 14 Species) In Helichus, the most common and widespread genus of “long-toed water beetles,” only adults are aquatic, living among debris and on decaying wood in streams. Adults in other genera are also probably aquatic, with those in two genera being lotic and those in the other two genera lentic. Larvae and adults resemble those of other 17. Diversity and Classiﬁcation of Insects and Collembola Dryopoidea. Adults are larger than the more abundant Elmidae and have a pectinate antenna (Fig. 116). Larvae are elongate and lack gills in the operculate caudal chamber. Little is known about life histories of species in this family. b. Elmidae 27 Genera, 101 Species) Commonly referred to as “rifﬂe beetles,” Elmidae are widespread and often abundant. Both larvae and adults are usually aquatic and often occur together; in a few species, adults are riparian. Adults are less than 4.5 mm long, smaller than Dryopidae adults, and have ﬁliform or slightly clubbed antennae (Fig. 117). The sclerotized larvae are elongate, rounded in cross section, and have a ventral caudal operculum that closes a chamber containing hooks and numerous ﬁlamentous gills (Fig. 132). Both adults and larvae are found mostly in streams, where they inhabit a variety of substrates, including gravel rifﬂes, algae laden rocks, aquatic macrophytes, and decaying wood. Adults and larvae of some species may also occur on these same substrates in spring ponds or along wave-swept shores of lakes. Larvae and adults are herbivores – detritivores, feeding on algae, decaying wood, and detritus. When larvae complete their development they leave the water and pupate in cells in protected areas on the adjacent shore. Upon emergence, adults disperse widely and frequently are captured in light-traps. Once they ﬁnd a suitable aquatic habitat, they rarely if ever ﬂy again, but stream species may move downstream by drifting in the current. Most adults probably live a year or more, and a semivoltine life cycle seems probable for most species in the north. c. Lutrochidae (1 Genus, 3 Species) Larvae of Lutrochidae occur on calcareous encrustations in hardwater streams in eastern, central, and western United States. They closely resemble larvae of Elmidae, but the last abdominal tergum is rounded apically (Fig. 134) instead of being emarginate (Fig. 133), and thoracic sterna are membranous. They apparently feed on periphyton, but little is known about the bionomics of this family. Adults are riparian, inhabiting splash zones of larger rocks that project from the aquatic habitat. d. Psephenidae (5 Genera, 14 Species) “Water pennies” are ﬂat, rounded larvae (Fig. 128) that occur on rocks and wood in streams from Texas and Arizona north to British Columbia, and east of the Great Plains; they may also be found on wave-swept shores of lakes. In two genera (Psephenus and Eubrianax) larvae lack the ventral caudal operculum of other aquatic dryopoid larvae, but have ventral abdominal gills that aid in respiration. The remaining genera are in the subfamily Eu- 691 briinae, which is sometimes considered a separate family. Their larvae lack exposed ventral gills, possess a ventral caudal operculum, and have the lateral margins of each segment distinctly separated (Fig. 127). Pupation of most species occurs above the water line on rocks near the larval habitat, but at least one species is known to pupate under water. Adults are riparian, typically being found in splash areas of projecting rocks. Most species probably have life cycles of 1 – 2 years. e. Ptilodactylidae (3 Genera, 3 Species) Larvae have been collected from streams and springs in California and Nevada, and from Georgia to New York, but not in central North America. They are elongate, lack a ventral caudal operculum, and have gills either on the ﬁrst seven abdominal sterna or on the ninth sternum (Fig. 131). Very little is known about their life histories. Adults of aquatic species are riparian. f. Chrysomellidae (6 Genera, about 70 Species) In this very large terrestrial family there are several species in which larvae and adults feed on emergent aquatic vegetation, but there is only one subfamily (Donaciinae) in which aquatic larvae feed on submerged portions of aquatic plants. They are adapted for underwater existence by having a pair of caudal spiracular spines that they insert into plant tissues to obtain oxygen, and they can be recognized by these conspicuous spines (Fig. 130). Larvae pupate on underwater portions of plants, with pupae also obtaining oxygen from plants for respiration. Adults of at least two species are also aquatic, feeding on plant stems. g. Curculionidae (20 Genera, about 150 Species) In this very large terrestrial family, adults of species in several genera feed on aquatic plants, both above and below the water surface, and there is no easy way to distinguish truly aquatic species. Adult Curculionidae are readily distinguished by their unique head, which is elongated into a snout, and by their geniculate antennae (Fig. 104). Larvae are grublike, without distinct legs; most mine leaves and other plant tissues and are not aquatic. Aquatic adults crawl on underwater portions of plants and carry with them a plastron for respiration; a few species are capable of swimming. Most aquatic species are nocturnal, and difﬁcult to ﬁnd during the day, when they apparently hide in the bottom substrate. Life histories are poorly known. h. Hydraenidae (3 Genera. 82 Species) Adult “minute moss beetles” are aquatic, living mostly in extremely shallow water along margins of streams, but also occurring along margins of lentic habitats. Adults resemble Hydrophilidae, and once were considered 692 W. L. Hilsenhoff part of that family. They are much smaller (2 mm long) than most Hydrophilidae and have ﬁve segments in the antennal club instead of three. Larvae are riparian, developing and pupating in moist soil adjacent to the water. Adults cannot swim, and ﬂoat upside down in the surface ﬁlm when dislodged from the substrate on which they live. Because of their very small size they are easily overlooked. Their life history and feeding habits are poorly known. i. Helophoridae (1 Genus, 41 Species) Helophorus was previously included in the family Hydrophilidae, but a study by Archangelsky (1998) convinced me to include it in a separate family. Adults are aquatic, but are poorly adapted for swimming and mostly crawl about on aquatic vegetation in a variety of lentic habitats or along margins of streams. Larvae inhabit riparian habitats adjacent to aquatic habitats where adults are found. Adults are small, elongate beetles that are readily recognized by the seven longitudinal grooves and six raised areas on the pronotum (Fig. 114). The univoltine life cycles are variable, with adults or eggs overwintering in riparian habitats, depending on the species (Hilsenhoff, 1995b). j. Hydrochidae (1 Genus, 19 Species) Hydrochus also was included in Hydrophilidae, but is now considered to be in a separate family (Archangelsky, 1998). Like Helophoridae, adults are aquatic, but swim poorly and mostly crawl about on the aquatic vegetation of shallow lentic habitats. Occasionally they occur along margins of streams. Life cycles are univoltine, with adults overwintering in terrestrial habitats, ﬂying back to aquatic habitats in spring, and ovipositing in adjacent riparian habitats where the larvae develop. Adults are readily identiﬁed by their small size and a very narrow pronotum that is covered with small granules and / or punctures (Fig. 115). k. Hydrophilidae (19 Genera, 160 Species) Adults and larvae of this large and abundant family mostly inhabit shallow lentic habitats, although a few species are found in deep water and others are strictly lotic. In several genera only adults are aquatic, and in one subfamily (Sphaeridiinae) none of the life stages are aquatic. While commonly called “water scavenger beetles,” all aquatic larvae are predators and adults feed on algae and perhaps small animals, as well as scavenging. Adults range in size from 1.5 to 40 mm and somewhat resemble Dytiscidae, but are ﬂat ventrally, have clubshaped antennae (Fig. 113), and very long maxillary palps. The predatory larvae have large, conspicuous mandibles and relatively long antennae and maxillary palps (Fig. 125). Adults in many genera mostly crawl on aquatic vegetation and are poorly adapted for swimming. In genera with adults that swim, beetles have fringes of long setae on their meso- and metathoracic legs, which they move alternately instead of in unison as in the Dytiscidae. While adults of most species can be readily collected with an aquatic net, those of larger species that swim rapidly are most effectively collected with bottle traps. Most species are univoltine, with adults being the normal overwintering stage. Adults of several species overwinter in terrestrial habitats, while others that inhabit shallow lentic habitats may ﬂy to streams or larger lentic habitats in late summer and autumn to overwinter. l. Lampyridae Larvae of at least one species of “ﬁreﬂy” have been collected from shallow lentic and semilotic habitats. They are readily recognized by their ﬂat, platelike thoracic nota that mostly or completely conceal the head from above, and by gills ventrally on the last abdominal segment (Fig. 129). Larvae are predaceous, feeding on snails or other insects. m. Scirtidae (8 Genera, 35 Species) Although adults are terrestrial, larvae of “marsh beetles” are aquatic and often common in marshes, swamps, margins of shallow ponds, tree holes, and a variety of other lentic habitats where they crawl about on the aquatic vegetation. Their elongate ﬁliform antennae are unique among aquatic Coleoptera larvae (Fig. 126). They are apparently detritivores, with little being known about their life history. E. Aquatic Diptera — Flies and Midges Diptera is a large, mostly terrestrial order, but larvae and pupae of numerous species are aquatic, and it is the dominant order of insects in the aquatic environment. Diptera larvae inhabit all types of aquatic environments, and because of very short development periods in many species, they are able to utilize ephemeral aquatic habitats as well as permanent ones. About 40% of all species of aquatic insects belong to this order, and at least a third of the aquatic Diptera species are in one family, Chironomidae. It is impossible to accurately estimate the number of species of aquatic Diptera, because in most families only terrestrial adults can be identiﬁed and in many families it is not possible to know which species have aquatic larvae. It is also difﬁcult to estimate the relatively large number of aquatic species that probably remain undiscovered and undescribed. The order is divided into two suborders, Nematocera and Brachycera (Agriculture Canada, 1981), with 17. Diversity and Classiﬁcation of Insects and Collembola Nematocera dominating the aquatic fauna. Formerly a third suborder, Cyclorrhapha, was recognized; it is now included in Brachycera as the infraorder Muscomorpha. In Nematocera there are several families in which all or almost all species have aquatic larvae and pupae. In Brachycera most families that have species with aquatic larvae also have large numbers of terrestrial or riparian species. Larvae of the infraorder Muscomorpha are often referred to as cyclorrhaphous Brachycera, with the remaining Brachycera being called orthorrhaphous Brachycera. Diptera larvae lack segmented thoracic legs, and thus can be readily distinguished from larvae of other aquatic insects. In Nematocera larvae, a completely sclerotized, relatively round head capsule is present (except in most Tipulidae), and the mandibles, which usually have subapical teeth, move laterally. In Brachycera larvae the head is reduced to an internal skeleton (cyclorrhaphous Brachycera) or poorly formed and not rounded (orthorrhaphous Brachycera), and the mandibles (mouth hooks) move vertically and lack subapical teeth. Diptera larvae have many adaptations that allow them to live in a wide variety of environments. Those that live in oxygen-rich environments usually rely on cutaneous respiration, which may be aided by gills that increase the surface area. In Chironomidae, larvae of some species have a hemoglobinlike pigment that aids in oxygen storage. Larvae in many families obtain oxygen at the water surface, aided by short or elongate caudal respiratory siphons that reach to the surface and may be retractile. Other larvae have fringes of hairs surrounding caudal spiracles that allow them to ﬂoat in the surface ﬁlm with their caudal spiracles exposed to the air. Some larvae repeatedly swim to the surface to obtain oxygen (Culicidae). Other larval adaptations include papillae for regulation of salt concentration, sucker disks to anchor lotic forms in rapid currents, and prolegs, pseudopods, or creeping welts on various segments to aid in locomotion. Pupae of Nematocera often have anterior respiratory horns that enable them to obtain oxygen at the surface; others rely on cutaneous respiration. A few swim actively to the surface (Culicidae, Chaoboridae) or pupate just above the water line (Dixidae). Most orthorrhaphous Brachycera pupate on land. Cyclorrhaphous Brachycera and Stratiomyidae pupate in a puparium, which may be terrestrial or ﬂoat at the water surface where oxygen is plentiful. Aquatic Diptera larvae can be readily captured with an aquatic net as described earlier, and when placed in a pan with some water will usually swim or crawl free of the debris, allowing them to be collected. Active Nematocera larvae are difﬁcult to capture with 693 forceps, and the use of a small loop (30-mm diameter) of nylon mesh is often advantageous. Such a loop can be made by bending the end of a piece of thin wire into a loop at a right angle to the remainder of the wire (the handle) and gluing a piece of nylon stocking to the loop. Diptera larvae are relatively easy to rear because most have short life cycles and pupate in the water. Lentic species, which require little or no aeration, are especially easy to rear. Diptera larvae are not only a very important part of almost all aquatic ecosystems, but adults in many aquatic families are important because they transmit disease or create severe nuisance problems. Culicidae (mosquitoes) are the most important group from a public health standpoint because adults transmit diseases of humans, birds, and other animals. Adults of this family, along with those of Tabanidae (horseﬂies and deerﬂies), Simuliidae (blackﬂies), and Ceratopogonidae (biting midges) may create severe nuisance problems because they bite humans and other animals. Nonbiting midges in the families Chironomidae and Chaoboridae may emerge in such tremendous numbers from larger lakes that they damage property and create public health problems by causing severe allergic reactions in some people. Much research is needed on aquatic Diptera in North America. Many species remain to be described, and larvae of most described species remain unknown. Only in families that are of public health importance (Culicidae, Tabanidae, Simuliidae) or useful as a biological control (Sciomyzidae) are larvae well enough known that reliable keys to most species have been developed. Even in these families there are taxonomic problems, especially with separation of sibling species. Great strides in larvae taxonomy have been made in recent years, especially in the largest family, Chironomidae, but until much more is accomplished it will not be possible for us to fully understand the role of Diptera larvae in aquatic ecosystems. The two-volume Manual of Nearctic Diptera (1981, 1987) by Agriculture Canada provides generic keys as well as information about morphology, biology, behavior, classiﬁcation, and distribution. References to species keys for the identiﬁcation of larvae are provided in Table VIII and keys to families of freshwater Diptera larvae appear in Sections V.I. Information on suborders, and the biology, habitat, adaptations, and identiﬁcation of larvae in each family is presented next. 1. Families of Aquatic Diptera – Suborder Nematocera More than half of the families in this suborder have aquatic representatives, and in 12 families all or 694 W. L. Hilsenhoff TABLE VIII Literature References to Species Keys for Identiﬁcation of Diptera Larvae Regional keys to larvae that include more than one family North and South Carolina — Webb and Brigham (1982) Keys to larvae listed by family Blephariceridae Blepharicera — Hogue and Georgian (1986) Ceratopogonidae — Glukhova (1977) Chaoboridae — Cook (1956) Chaoborus — Saether (1970) Chaoborus — (Schadonophasma) Borkent (1979) Chironomidae — references since Simpson (1982) and Wiederholm (1983); (Florida) Epler (1995) Brillia — Oliver and Rousell (1983) Cricotopus — Simpson et al. (1983) Dicrotendipes — Epler (1987) Endochironomus, Endotribelos, Synendotendipes, Tribelos — Grodhaus (1987) Eukiefferiella, Tvetenia — Bode (1983) Guttipelopia — Bilyj (1988) Pagastia — Oliver and Roussell (1982) Paraphaenocladius — Saether and Wang (1995) Polypedilum — Boesel (1985); Maschwitz and Cook (1999) Stenochironomus — Borkent (1984) Tanypodinae — (eastern United States) Roback 1985, 1986a, 1986b, 1987) Corethrellidae — Cook (1956) Culicidae — Carpenter and LaCasse (1955); Darsie and Ward (1981); (Canada) Wood et al. (1979); several regional keys published prior to 1981; (Florida) Breeland and Loyless (1982) Deuterophlebiidae — Courtney (1990) Nymphomyiidae — Courtney (1994) Sciomyzidae Dictya — Valley and Berg (1977) Elgiva — Knutson and Berg (1964) Hoplodictya — Neff and Berg (1962) Pherbella — Bratt et al. (1969) Sciomyza — Foote (1959) Sepedon — Neff and Berg (1966) Tetanocera — Foote (1961) Simuliidae — (Alabama) Stone and Snoddy (1969); (Alberta) Currie (1986); (Colorado) Peterson and Kondratieff (1995); (Michigan) Merritt et al. (1978); (New York) Stone and Jamnback (1955); (Ontario) Wood et al. (1963); (Pennsylvania) Adler and Kim (1986); (southeastern United States) Snoddy and Noblet (1976) Ectemnia — Moulton and Adler (1997) Gymnopais, Twinnia — Wood (1978) Prosimulium — (Canada and Alaska) Peterson (1970) Simulium — Moulton and Adler (1995); (southern California) Hall (1974) Tabanidae — (Arizona) Burger (1977); (eastern North America) Goodwin (1987); Teskey (1969); Teskey and Burger (1976); (Illinois) Pechuman et al. (1983); (Louisiana) Tidwell (1973) Atylotus — Teskey (1983) Tanyderidae — (Alberta) Exner and Craig (1976) Thaumaleidae Thaumalea — (Idaho and California) Gillespie et al. (1994) almost all species have aquatic larvae and usually also aquatic pupae. Unless noted otherwise, larvae have four instars, respiration is through the integument, and pupation is in the larval habitat. a. Blephariceridae (5 Genera, 28 Species) The distinctive ﬂattened larvae of “net-winged midges” have seven apparent body segments and a sucker disk ventrally on the ﬁrst six segments (Fig. 135). They use their sucker disks to cling to rocks in very fast water of western and eastern mountain streams, and also streams in the northern United States and Canada. Here, they feed on diatoms and other algae. Larvae move forward slowly with an undulating motion, or sideways by alternately moving anterior and posterior sucker disks. Pupation occurs in cracks and depressions of rocks near the water surface. Adults live 1 – 2 weeks, with some females being predators on other midges. Eggs are laid just above the water on rocks, usually when water levels are receding. They hatch when ﬂooded by higher water levels. b. Ceratopogonidae (20 Aquatic genera, 501 Species) Aquatic larvae of “biting midges” typically are small, elongate, and without prolegs (Fig. 151). While many species are riparian or live in moist terrestrial habitats, a large number of species occur in aquatic habitats that include tree holes, marshes, swamps, ponds, all areas of lakes, and streams. Most larvae are able swimmers and move about with a serpentine swimming motion. They are poorly known and most have not been associated with adults, making it difﬁcult even to know which species develop in aquatic habitats. Reliable keys to larvae of North American genera have not been developed, although a key to larvae in the U.S.S.R. (Glukhova, 1977) includes most genera found in North America. Most larvae are carnivores; others are herbivores or detritivores. Pupae of aquatic species hang in the surface ﬁlm by their respiratory horns. Adults of a few aquatic species bite people and may become annoying pests in some areas; most others feed on small insects. Kettle (1977) reviewed the biology and ecology of blood sucking Ceratopogonidae. c. Chaoboridae (3 Genera, 14 Species) Most larvae are nearly transparent, except for hydrostatic organs, causing them to be called “phantom midge larvae.” They are unique in that their enlarged antennae have been modiﬁed for capturing prey such as insect larvae and small crustaceans (Fig. 148). They occur in a wide variety of lentic habitats, and are especially abundant in the profundal or sublittoral zones of some lakes. In lakes, third and fourth instar larvae rest in the bottom mud during daylight hours and prey on zooplankton in 17. Diversity and Classiﬁcation of Insects and Collembola the limnetic area at night; earlier instars remain limnetic. They are the only insects frequently found in the limnetic area of lakes. Larvae also occur in permanent ponds, spring ponds, vernal ponds, and margins of swamps. Adults do not feed, but their synchronized emergences may create severe nuisance problems around large lakes because adults are highly attracted to lights. Life cycles are univoltine to multivoltine, depending on species, climate, and habitat. Most larvae can be identiﬁed to species. d. Chironomidae (208 Genera, 2000 Species) Chironomidae is by far the largest family of aquatic insects, with the number of aquatic species surpassing that in any other order. The larvae, which are recognized because they usually have anterior and posterior pairs of prolegs (Fig. 158), are diverse in form and size. They inhabit all types of permanent and temporary aquatic habitats, and a few species inhabit semiaquatic or terrestrial habitats. Larvae are often the dominant insects in the profundal and sublittoral zones of lakes, and consequently adults are sometimes called “lake ﬂies,” although they are most often referred to as “midges.” In larger lakes, adults of some species may emerge in such tremendous numbers that they create nuisance problems along their shores. The short-lived adults cause allergies in some people, invade factories, spot the paint on houses, and accumulate in large, odorous piles. Larvae are an extremely important part of aquatic food chains, serving as prey for many other insects and food for most species of ﬁsh. Life cycles are highly variable, with many species being univoltine and many others being bivoltine or multivoltine. In the north, life cycles are longer, with some arctic species requiring several years to complete development. Feeding habits also vary widely, with herbivores, detritivores and carnivores all commonly represented. Many larvae, especially predators, are free living, but those of most species construct loose cases of substrate cemented together with salivary secretions. Most are herbivores and detritivores that graze on ﬁne particles on the substrate, but some are ﬁlter feeders, which construct webs to ﬁlter water that they circulate through their case or retreat. Larvae of most species are quite tolerant of lowered levels of dissolved oxygen; some can survive in areas where oxygen levels are so low that oxygen cannot be detected. Such species are usually red and contain a hemoglobinlike pigment that retains oxygen. These “blood worms” may become abundant in sewage lagoons or organically polluted areas of lakes or streams. The pupal stage is short, with pupae of most species being quiescent. After completing development, the pupa swims to the surface of the water where 695 emergence occurs. Adults do not feed, and generally live no more than 2 weeks. The biology of the family was recently reviewed by Pinder (1986). In recent years many new species have been named and great progress has been made in rearing and identifying larvae and pupae, but larvae of too many species remain unknown to permit reliable species keys to be constructed for larvae in most genera. A bibliography of taxonomic literature was compiled by Simpson (1982), and in 1983 Wiederholm keyed larvae to genus and species groups, provided comments on identiﬁcation, ecology, and distribution, and also listed known species keys. Only keys to species or species groups that were not included by Simpson or Wiederholm are listed in Table VIII. e. Corethrellidae (2 Genera, 5 Species) Until recently, this was a subfamily of Chaoboridae. Larvae occur in small pools, tree holes, or artiﬁcial containers in the southeastern United States north to New Jersey and west to California. Like Chaoboridae larvae, they have prehensile antennae, but the antennae are very close together basally and not wide apart as in Chaoboridae. Little is known about their biology. f. Culicidae (13 Genera, 172 Species) Because they bite people and transmit disease, “mosquitoes” have been studied more thoroughly than any other family of aquatic Diptera. All species can be identiﬁed as adults or larvae, and life histories and habitats of most species have been documented. Larvae are readily recognized by their swollen thoracic area (Fig. 147), the presence of a caudal respiratory tube (siphon) in most genera, and their characteristic ﬂip-ﬂop swimming motion. Because of this swimming motion larvae are commonly called “wrigglers.” Larvae occur in a variety of shallow lentic habitats that include tree holes, artiﬁcial containers, catch basins, pitcher plants, swamps, shallow temporary or permanent ponds and marshes, and heavily vegetated margins of lakes and streams. They are not found in moving water or water subjected to wave action. Larvae obtain oxygen at the water surface through a caudal siphon, which is very abbreviated in Anopheles (Fig. 147). Larvae of Coquillettidia and Mansonia attach their spinelike siphon to aquatic plants to obtain oxygen. Most larvae are active swimmers, and feed on detritus and microorganisms; larvae in two genera are predators, mostly on other mosquito larvae. Pupae are also active swimmers, and obtain oxygen at the surface through thoracic respiratory horns. Many species are univoltine, with eggs that diapause over the summer, autumn, and winter, and larval development and emergence occurring in the spring. Many others are bivoltine or multivoltine. Although 696 W. L. Hilsenhoff most species overwinter as eggs, a few overwinter as larvae and several species overwinter as adults. Most female adults obtain blood from mammals, birds, reptiles, or amphibians, depending on the species, and may live several weeks or months. Male mosquitoes do not feed on vertebrates, instead they obtain nectar from plants. Culicidae larvae often dominate the insect fauna of temporary ponds and marshes, especially those that ﬂood in spring and summer. g. Deuterophlebiidae (1 Genus, 6 Species) The unique larvae of “mountain midges” are less than 6 mm long and readily recognized by their elongate, branched antennae and the presence of seven pairs of stout ventrolateral prolegs on the abdomen (Fig. 153). They are found in rapid currents of western mountain streams. Here, they inhabit the upper surface of lightcolored, smooth rocks that have cracks or depressions, and are usually at or near the water surface. Pupation is in the same habitat, usually in depressions in darkercolored rocks. In cold water at higher elevations, there is one generation per year, while at lower elevations there may be several generations. Eggs within the pupa are mature, and when adults emerge, mating and oviposition occur almost immediately. Adults live only an hour or two. h. Dixidae (3 Genera, 42 Species) Larvae of “dixid midges” are found at or just below the water surface in lentic habitats or densely vegetated margins of streams. Here, they characteristically lie bent in a U-shape, often resting on vegetation. They can be recognized by pairs of prolegs on the ﬁrst one or two abdominal segments (Fig. 154). Larvae obtain oxygen from the air through caudal spiracles and feed on microorganisms and detritus in the surface ﬁlm. Pupation occurs just above the water surface on vegetation. Life histories are poorly known; most species are probably multivoltine. i. Nymphomyiidae (1 Genus, 2 Species) The small, slender larvae are recognized by pairs of ventrally projecting, slender, two segmented prolegs on abdominal segments 1 – 7 and 9 (Fig. 152). They are rare, and in North America have been found only in Quebec, New Brunswick, and Maine. Larvae occur mostly on mosscovered rocks in small, rapid, cold, spring-fed streams, where they apparently feed on diatoms and other algae. Species are probably bivoltine, with emergences in spring and late summer. j. Psychodidae (6 Aquatic genera, 67 Species) Most species of “moth ﬂies” develop in semiaquatic or moist terrestrial habitats, but larvae of several species occur in aquatic habitats. Aquatic larvae can be recognized by their small size and secondary annulations on thoracic and abdominal segments, which often also have sclerotized dorsal plates on many annuli (Fig. 150). In one aquatic genus (Maruina), the larvae have ventral suckerlike disks. Aquatic larvae occur among vegetation and debris in shallow lentic habitats and along margins of streams, and are frequently associated with organically polluted water. They even occur in sink and ﬂoor drains. Larvae feed near the surface on microorganisms and detritus. Life histories of aquatic species are not well known; most are probably multivoltine, especially in warm-water habitats. Larvae cannot be identiﬁed to species. k. Ptychopteridae (3 Genera, 16 Species) Larvae of “phantom crane ﬂies” are easily recognized by their elongate caudal respiratory siphon and the presence of pairs of prolegs on the ﬁrst three abdominal segments (Fig. 155). They live in very shallow water along margins of lentic or lotic habitats where there is an accumulation of detritus. Here, they obtain oxygen at the surface through their caudal respiratory siphon and probably feed on detritus and associated microorganisms. Their life history is poorly known because they are generlly uncommon. l. Simuliidae (11 Genera, 143 Species) “Blackﬂy” larvae are uniquely shaped, having a swollen abdomen that they attach to the substrate with a caudal sucker (Fig. 156). They also have a relatively large head, and a single ventral proleg on the thorax. They inhabit a wide variety of lotic habitats where they are often abundant on rocks, submerged wood, or vegetation in fast to slow currents, each species usually having rather speciﬁc ecological requirements. Most larvae are ﬁlter feeders, using labral fans to ﬁlter diatoms and other food from the current. Larvae without labral fans feed by grazing on the substrate around them. Many species are univoltine, while others are bivoltine or multivoltine. Species overwinter as either eggs or larvae, with larvae of some species growing signiﬁcantly during the winter months. Unlike most other Nematocera, which have only four larval instars, simuliid larvae have six or sometimes seven instars. Pupation occurs at the site of larval attachment, with pupae having a pair of highly branched thoracic gills that aid in respiration. Females usually require a blood meal for maturation of eggs, and various species may be serious pests, biting humans, livestock, and poultry. They also transmit pathogenic organisms such as ﬁlarial nematodes, various viruses, and avian protozoan parasites. m. Tanyderidae (2 Genera, 4 Species) The elongate larvae of “primitive crane ﬂies”, which may reach a 17. Diversity and Classiﬁcation of Insects and Collembola length of 18 mm, are rare in eastern and western North America and have not been found in central North America. They resemble chironomid larvae, but lack anterior prolegs and have three pairs of long caudal ﬁlaments (Fig. 157). Larvae inhabit the shallow sand, silt, or gravel margins of large streams, but, because they are rare, their life history and feeding habits remain virtually unknown. n. Thaumaleidae (2 Genera, 8 Species) The very rare “solitary midge” larvae resemble chironomid larvae and reach a length of 12 mm, but have a single, broad anterior and posterior proleg instead of paired prolegs (Fig. 159). They also have dorsally sclerotized segments and a pair of short, stalked spiracles dorsolaterally on the prothorax. Larvae inhabit vertical surfaces of rocks in cold, shady, mountain streams, where the water ﬁlm is thin enough so as not to cover them completely. Here, they feed mostly on diatoms. Almost nothing is known about their life history. o. Tipulidae (20 Aquatic Genera, about 240 Species) Larvae of “crane ﬂies” differ from those of other Nematocera by having the posterior portion of the head capsule incompletely sclerotized (Figs. 136 and 137) and retracted into the thorax. In this very large family, the vast majority of species are not aquatic, but there are several genera with species that have aquatic larvae, and these species are often widespread and abundant. Because larvae of fewer than 10% of the species have been described, it is difﬁcult to know which species have aquatic larvae. While most aquatic larvae are found in lotic habitats, larvae in a few genera inhabit shallow lentic habitats. Larvae of most aquatic species have functional caudal spiracles that they may utilize to obtain oxygen at the water surface, but those that live in well-aerated streams probably obtain most of their oxygen from the water through their cuticle. Larvae in some genera are very large. Feeding habits vary widely, with omnivores, herbivores, detritivores and carnivores all represented. Most species are univoltine or bivoltine, but some that live in very cold water may be semivoltine. Pupation most often occurs in riparian habitats adjacent to the larval habitat, but some species pupate in shallow water and have thoracic respiratory horns through which they breathe. Adults are short-lived. The biology of Tipulidae was reviewed by Pritchard (1983). 697 structures are greatly reduced and not rounded and sclerotized as in Nematocera (orthorrhaphous Brachycera). Only in Stratiomyidae is there any signiﬁcant head structure, but heads of Stratiomyidae larvae are angulate (Fig. 138), partially retracted into the thorax, and quite unlike heads of Nematocera. Mandibles of Brachycera larvae move vertically, not horizontally as in Nematocera. They lack subapical teeth and are usually referred to as “mouth hooks.” The majority of species in all families, except Athericidae and Sciomyzidae, have terrestrial or semiaquatic larvae, and it usually is not possible to know which species have aquatic larvae because larvae of most species of Brachycera remain unknown. Furthermore, many genera that are known to have aquatic larvae, also have terrestrial or semiaquatic larvae, so all species within a genus cannot be assumed to be aquatic because larvae of one or more species have been found to be aquatic. Because most larvae cannot be identiﬁed, life histories are poorly known. a. Athericidae (2 Genera, 4 Species) Aquatic larvae of Atherix are distinctive, with pairs of ventral abdominal prolegs on the ﬁrst seven abdominal segments and with a single ventral proleg and a pair of fringed caudal projections on the eighth segment (Fig. 144). Another genus (Suragina) occurs in Texas; larvae were described by Webb (1994) and are similar to those of Atherix. Atherix larvae commonly occur in rifﬂes or among vegetation in streams where they feed mostly on insect larvae. They pupate in moist soil along the edge of the stream. Adults are known to drink water and perhaps feed on nectar; adult females of Suragina suck blood from humans and cattle. Eggs of Atherix are laid on vegetation or supports above the stream, with several females often contributing eggs to form a single, large mass. Larvae drop into the stream upon hatching. A 1-year life cycle is likely for most species. 2. Families of Aquatic Diptera — Suborder Brachycera b. Dolicopodidae (about 8 Aquatic Genera) The “long-legged ﬂies” are mostly terrestrial, but larvae of a few species in eight genera are known to develop in a wide variety of lotic and lentic habitats. The taxonomy of larvae is so poorly known that identiﬁcation to genus is not reliable and it is not possible to clearly deﬁne which species are aquatic. Aquatic larvae differ from other Brachycera by having a posterior spiracular pit that is surrounded by four lobes (Fig. 143). They are predaceous, feeding mostly on other insect larvae. Pupae differ from other orthorrhaphous Brachycera by having thoracic respiratory horns. In larvae of Brachycera, either the head and external head structures are replaced by an internal cephalopharyngeal skeleton (cyclorrhaphous Brachycera), or head c. Empididae (about 8 Aquatic Genera) The small (7 mm) larvae of “dance ﬂies” are too poorly known 698 W. L. Hilsenhoff to estimate the number of aquatic species. Larvae of most species are terrestrial or semiaquatic. Most aquatic larvae can be recognized because they have seven or eight pairs of abdominal prolegs and a pair of caudal respiratory appendages (Fig. 145), which are not elongate and fringed as in Athericidae. Larvae are predaceous on smaller animals, and most aquatic larvae live in rapid water of streams. Because larval taxonomy is so poorly known, almost nothing is known about the life history of aquatic species. d. Ephydridae (68 Genera) Species in most genera have semiaquatic larvae. Some genera, however, have aquatic species with larvae that live in vegetation or detritus in shallow habitats near shore, in algal mats, or in unusual habitats such as hot springs and pools, and ponds containing oil, as well as saline and alkaline marshes, ponds and lakes, where larvae dominate the insect fauna. Larvae of other species are terrestrial or mine leaves of aquatic plants. “Shore ﬂy” larvae are less than 12 mm long and usually have a pair of very short to elongate caudal respiratory tubes that frequently have a black sclerotized tip (Fig. 140). Many also have abdominal prolegs. Most larvae feed on bluegreen and other algae, others feed on detritus, some are predators. Aquatic species pupate in a puparium that usually ﬂoats on the water surface. Eggs are laid singly at the surface of the aquatic habitat. Although the family has received a great deal of attention, larval taxonomy is still poorly known and identiﬁcation of larvae, even at the generic level, is difﬁcult or impossible. Foote (1995) reviewed their biology. e. Muscidae (about 8 Aquatic Genera) In this extremely large terrestrial family, larvae of several aquatic species live in streams, marshes, or ponds; they are mostly predators. Larvae are poorly known, but most have a pair of very short caudal respiratory tubes, a pair of ventral prolegs on the last abdominal segment that are longer than the respiratory tubes, and often creeping welts ventrally on other abdominal segments (Fig. 146). Eggs are laid in algae or debris, and larvae usually remain near the surface. Pupation is in a puparium, which remains at or near the surface. A pair of respiratory siphons that are thrust through the fourth segment of the puparium penetrate the water surface. f. Sciomyzidae (19 Genera, 175 Species) “Marsh ﬂy” larvae are predators or parasitoids of snails, slugs, or ﬁngernail clams, and only those species that attack land snails or slugs are not aquatic. They generally have a very wrinkled appearance, with girdles of pseudopods on abdominal segments. The abdomen ter- minates in a spiracular disk surrounded by eight to ten lobes and contains a pair of spiracles, each surrounded by numerous palmate hairs (Fig. 141). Larvae are found in lentic habitats and margins of lotic habitats inhabited by their prey. Here, they usually remain near the surface with their caudal spiracles at the surface. They swallow a large air bubble to keep themselves aﬂoat, and are usually buoyant enough to keep their prey at the surface. Larvae pupate in a puparium that either remains in the snail shell or ﬂoats at the surface. Eggs are laid on emergent vegetation; newly hatched larvae drop into the water and actively search for prey. Adults of parasitoids oviposit directly on the shell of a host snail. Species may be univoltine or multivoltine. Because snails are intermediate hosts of liver ﬂukes that infect cattle and of schistosomes that attack humans, sciomyzid larvae have been employed as a biological control for snails. For this reason they have been extensively studied and larvae in several genera can be identiﬁed to species. Berg and Knudson (1978) reviewed the biology and systematics. g. Stratiomyidae (about 10 Aquatic Genera) Although most “soldier ﬂy” larvae are terrestrial or semiaquatic, those of many species inhabit shallow, vegetated, lentic habitats. While often quite abundant, larvae are so poorly known that identiﬁcation at even the generic level is not always reliable. The relatively ﬂat larvae are easily recognized by their mostly visible truncate head (Fig. 138), caudal spiracles surrounded by long hydrofuge hairs, and calcium carbonate crystals that cover their integument. Larvae hang suspended in the surface ﬁlm by hydrofuge hairs that surround the spiracular disk, and use their palps to move about. They may at times leave the water and inhabit moist shoreline debris or plants. They feed on detritus and also algae, differing from larvae of other orthorrhaphous Brachycera, which are predators. Pupation is in a puparium, which may ﬂoat in the aquatic habitat. Adults feed mostly on ﬂowers. Eggs are laid in masses, either on submerged or emergent aquatic plants, or on other objects in the water. h. Syrphidae (about 7 Aquatic Genera) Most “rattailed maggosts” become fairly large and differ from other Brachycera by being broad and blunt anteriorly. Aquatic larvae are easily recognized by their very long, extensile, caudal breathing tube (siphon) (Fig. 139). Most also have ventral prolegs. Larvae inhabit shallow lentic habitats or margins of lotic habitats, especially areas high in decomposing organic matter. Here, they feed on detritus and microorganisms. Larvae of some species occur in tree holes. Because they obtain oxygen from the air through their long respiratory siphon, they 17. Diversity and Classiﬁcation of Insects and Collembola frequently inhabit highly polluted areas such as sewage lagoons. Larvae pupate in a puparium, with the puparium of some species having anterior respiratory horns. Life histories are poorly known, but most species probably have more than one generation per year. Adults are called “ﬂower ﬂies” because they feed on nectar from ﬂowers. Eggs are laid in masses on the debris or vegetation that the larvae inhabit. i. Tabanidae (about 4 Aquatic Genera) Most “deerﬂy” or “horseﬂy” larvae develop in wet soil that may be closely associated with aquatic habitats. Some larvae in four genera are known to be aquatic, and a few in other genera also may be aquatic. Many North American species have been reared and described, and larval keys have been developed, but identiﬁcation is still risky because larvae of some species remain unknown. Larvae are recognized by the absence of distinct prolegs and the presence of a girdle of six or more pseudopods on most abdominal segments (Fig. 142). Aquatic larvae have been collected from stream rifﬂes, shallow stream margins, and shallow vegetated lentic habitats. Most species are probably univoltine, but bivoltine and semivoltine species likely occur. Larvae are predators on other aquatic macroinvertebrates; pupation of aquatic species takes place in semiaquatic areas. Most adult females feed on blood of humans, livestock, and other animals, and are often a persistent nuisance. Eggs are laid in masses on vegetation above the larval habitat. IV. SEMIAQUATIC COLLEMBOLA — SPRINGTAILS Until recently Collembola were regarded as insects, but most people no longer consider them to be in the class Insecta and place them in the class Entognatha or in a subclass of Hexopoda. The order Collembola is only partially aquatic, with the vast majority of the nearly 700 North American species inhabiting moist terrestrial habitats. Species that are collected from aquatic habitats are usually found on the water surface and are semiaquatic. Waltz and McCafferty (1979) reported that 96 species of springtails had been collected at least once from freshwater or coastal marine habitats in North America, but most of these records were believed to be incidental occurrences of terrestrial species. They regarded 10 species as semiaquatic and ﬁve as riparian with a propensity to venture onto the water surface. These 15 species exhibited various degrees of adaptation for the semiaquatic environment and are discussed in detail by Waltz and McCafferty, who also key species most likely to be found in freshwater habitats. Keys and descriptions for all North American 699 species of Collembola are provided by Christiansen and Bellinger (1980). Collembola are small, wingless arthropods, usually much less than 6 mm long. They resemble insects by having a distinct head with one pair of segmented antennae, a three-segmented thorax with three pairs of legs, and a segmented abdomen without legs (Figs. 160 and 161). They differ from insects in having only six abdominal segments and in having their maxillae and mandibles concealed by the wall of the head capsule with which their labium is fused. They also have the tibia and tarsus of each leg fused into a single segment. Except for their smaller size and lack of genitalia, young Collembola closely resemble adults. Springtails can be readily recognized by a cylindrical collophore or ventral tube (Fig. 160 and 161) on the ﬁrst abdominal segment and by the furcula, a midventral appendage on the fourth abdominal segment (Fig. 160 and 161) that is present in most species and in all that are known to be semiaquatic. The furcula consists of a basal piece, the manubrium, and two arms (Fig. 162). The basal segment of each arm is the dens and the apical segment is the mucro. The furcula can be folded forward under the abdomen where it is held in place by the tenaculum, a midventral clasplike structure on the third abdominal segment. When the furcula is released, it springs backward and propels the Collembola several centimeters through the air, hence the common name “springtail.” In most semiaquati species there is a widening of each mucro or lamellae on each mucro. This permits greater contact with the surface ﬁlm, allowing individuals to spring farther when on the water surface. The ecology and biology of Collembola remains very poorly known. Semiaquatic species are most often associated with lentic freshwater habitats; none occur on the ocean and reports from lotic habitats are rare. They feed primarily on algae, detritus, and other organic material in the surface ﬁlm; some may eat bacteria. Adults do not copulate like insects. Instead males deposit stalked or sessile spermatophores, which are gathered by the females; usually no contact between sexes is involved. Eggs are laid in moist habitats; in at least one species they are laid beneath the water surface and newly hatched young are truly aquatic, but once they pass through the surface ﬁlm and come in contact with the air they remain semiaquatic. Eggs usually hatch within a few weeks, except those that diapause over the winter or during periods of dryness. The young reach sexual maturity within a few weeks and molt from three to seven times. In some species the young may enter into diapause. Most adults live a few weeks to a few months, and unlike insects, they continue to 700 W. L. Hilsenhoff molt throughout their adult life, with more than ﬁfty molts reported for one species. Some species are coldhardy and may be active at temperatures near freezing; however, most species overwinter as diapausing eggs. Because of their springing ability Collembola are often difﬁcult to capture. Semiaquatic species may be collected most easily by forcing them to jump into a white pan containing either 95% alcohol with 3% glacial acetic acid, or water with detergent to prevent them from jumping from the pan. Floating sticky traps may also be used to capture them. Christiansen and Bellinger (1980) recommend 70 – 95% isopropanol or 80 – 95% ethanol for preservation, with 1 – 2% glycerine added to prevent accidental desiccation. A taxo- nomic key to families of semiaquatic Collembola appears in Section V.J. V. IDENTIFICATION OF THE FRESHWATER INSECTS AND COLLEMBOLA The following taxonomic keys are separated by class (Entognatha and Insecta) and orders (Insecta only). Collembola, which usually are much less than 6 mm long, can be distinguished from small larval insects by the possession ventrally of a collophore (ventral tube) on the ﬁrst abdominal segment and a furcula on the fourth (Figs. 158 and 159). A. Taxonomic Key to Orders of Freshwater Insects 1a. Thorax with 3 pairs of segmented legs ...............................................................................................................................................3 1b. Thorax without segmented legs ..........................................................................................................................................................2 2a(1b). Mummylike, with developing adult structures; in a case, often silk-cemented and contains vegetable or mineral matter...................... .................................................................................................................................................................................pupae (not keyed) 2b. Not in a case; mobile larvae, mostly with prolegs, pseudopods, or creeping welts on one or more segments (Figs. 145 – 147 and 152 – 159).......................................................................................................................................................................Diptera larvae 3a(1a). With large functional wings, which may be shelllike, or leatherlike at the base ..................................................................................4 3b. Wingless, or with developing wings (wing-pads) or brachypterous wings...........................................................................................6 4(3a). All wings completely membranous, with numerous veins ..................................................ovipositing or terrestrial adults (not keyed) 4b. Mesothoracic wings hardened and shell-like (Figs. 108, 117, and 118), or leatherlike in basal half (Figs. 87 and 93 – 95) .................5 5a(4b). Chewing mouthparts; mesothoracic wings hard, shell-like (Figs. 108, 117, 118) ......................................................Coleoptera adults 5b. Sucking mouthparts formed into a broad or narrow tube (Figs. 88 and 90); mesothoracic wings hardened in basal half (Fig. 87); .......................................................................................................................................................Heteroptera ( Hemiptera) adults 6a(3b). With 2 or 3 long, ﬁlamentous terminal appendages (Figs. 9, 29, 45, 48, 49, 54, 56, and 58) .............................................................7 6b. Terminal appendages absent, or with only 1 or 2 segments ................................................................................................................8 7a(6a). Sides of abdomen with featherlike, platelike, or leaﬂike gills (Figs. 4, 11 – 15, and 25 – 30); usually with 3 tail ﬁlaments, occasionally only 2; tarsi with 1 claw.....................................................................................................................................Ephemeroptera larvae 7b. Gills absent from at least middle abdominal segments (Figs. 45, 48, 49, 54, 56, and 58); 2 tail ﬁlaments; tarsi with 2 claws ............... Plecoptera larvae 8a(6b). Labium formed into an elbowed, extensile grasping organ (Figs. 32 – 34); abdomen terminating in 3 lamellae (Fig. 31) or 5 triangular points (Fig. 44) .......................................................................................................................................................Odonata larvae 8b. With sucking or chewing mouthparts; abdomen not terminating in 3 lamellae or 5 triangular points ................................................9 9a(8b). Mouthparts sucking, formed into a broad or narrow tube (Figs. 88 and 90) or a pair of long stylets (Fig. 102)...............................10 9b. Mouthparts not sucking, not formed into a tube or pair of stylets ...................................................................................................11 10a(9a). Parasitic on sponges; mouthparts a pair of long stylets (Fig. 102); all tarsi with 1 claw (Fig. 102) ...........Neuroptera; Sisyridae larvae 10b. Free-living; mouthparts a broad or narrow tube (Figs. 88 and 90); mesotarsi with 2 claws (Figs. 87 and 93 – 96)................................ ......................................................................................................................................Heteroptera ( Hemiptera) adults or nymphs 11a(9b) Ventral prolegs on abdominal segments 3 – 6, each with a ring of ﬁne hooks (Fig. 103) ..........................Lepidoptera; Pyralidae larvae 11b. Abdomen without ventral prolegs on abdominal segments 3 – 6 that have a ring of hooks ...............................................................12 12a(11b). Antennae extremely small, inconspicuous, one-segmented (Figs. 63, 78, and 80).....................................................Trichoptera larvae 12b. Antennae elongate, with 3 or more segments ...................................................................................................................................13 17. Diversity and Classiﬁcation of Insects and Collembola 701 13a(12b). Without long lateral ﬁlaments (Figs. 120 – 130) ........................................................................................................Coleoptera larvae 13b. With long lateral ﬁlaments (Figs. 85, 86, and 119) ...........................................................................................................................14 14a(13b). A single claw on each tarsus (Fig. 121 – 124) or abdomen terminating in 2 slender ﬁlaments or a median proleg with 4 hooks (Fig. 119) ..........................................................................................................................................................................Coleoptera larvae 14b. Each tarsus with 2 claws; abdomen terminating in a single slender ﬁlament (Fig. 85) or in 2 prolegs, each with 2 hooks (Fig. 86) ..............................................................................................................................................................................Megaloptera larvae B. Taxonomic Key to Families of Freshwater Ephemeroptera Larvae 1a. Mandibles with large forward-projecting tusks (Fig. 3); gills on abdominal segments 2 – 7 with fringed margins (Fig. 4) and projecting laterally or dorsally over abdomen ...............................................................................................................................................2 1b. Mandibles without large tusks; conspicuous fringed gills absent or projecting ventrolaterally............................................................4 2a(1a). Gills dorsal, curving up over abdomen; protibiae fossorial (Fig. 5).....................................................................................................3 2b. Gills lateral, projecting from sides of abdomen; protibiae slender, subcylindrical (Fig. 6); East, Midwest......................Potamanthidae 3a(2a). Apex of metatibiae rounded (Fig. 7); mandibular tusks curved inward and downward apically ..................................Polymitarcyidae 3b. Apex of metatibiae projected into an acute point ventrally (Fig. 8); mandibular tusks curved upward or outward apically ........................................................................................................................................................................................Ephemeridae 4a(1b). Mesonotum modiﬁed into a carapace-like structure that covers the gills on abdominal segments 1 – 6 (Fig. 9) ...................Baetiscidae 4b. Mesonotum not modiﬁed into a carapace, gills exposed.....................................................................................................................5 5a(4b). Head and pronotum with pads of long, dark setae on each side (Fig. 10); fringed gills projecting ventrolaterally; Southeast WI .........................................................................................................................................................................................Behningiidae 5b. Head and pronotum without pads of long, dark setae; gills not fringed and projecting ventrolaterally ..............................................6 6a(5b). Gills on abdominal segment 2 operculate or semi-operculate, covering or partially covering gills on succeeding segments (Figs. 11 – 14)...............................................................................................................................................................................................7 6b. Gills on abdominal segment 2 similar to those on succeeding segments or absent ..............................................................................9 7a(6a). Operculate gills oval or subtriangular and well-separated from each other mesally (Figs. 11 and 12); gills on segments 3 – 6 without fringed margins .............................................................................................................................................................Leptohyphidae 7b. Operculate gills quadrate and meeting along mesal edge (Figs. 13 and 14); gills on segments 3 – 6 with fringed margins ...................8 8a(7b). Operculate gills fused mesally (Fig. 13); East..............................................................................................................Neoephemeridae 8b. Operculate gills not fused mesally, but overlapping (Fig. 14) .................................................................................................Caenidae 9a(6b). Gills absent from abdominal segment 2 and sometimes also from 1 and 3, gills on segments 3 or 4 may be operculate (Fig. 15) .....................................................................................................................................................................................Ephemerellidae 9b. Gills present on abdominal segments 1 or 2 to 7 ..............................................................................................................................10 10a(9b). Head ﬂattened dorsoventrally; eyes and antennae dorsal (Fig. 16); gills a single lamella, often with a ﬁbrilliform tuft .....................11 10b. Head and body not dorsoventrally ﬂattened; eyes, and usually also antennae, along lateral margin of head (Fig. 17) ......................13 11a(10a). Distal maxillary palpomere at least 4 times as long as galea-lacinia and visible from above (Fig. 18); Northeast............Arthropleidae 11b. Distal maxillary palpomere much shorter, usually not visible from above ........................................................................................12 12a(11b). Gills with a ventral ﬁngerlike projection on lamellae (Fig. 19); tarsal claws very long: West, Midwest, Southeast ..........Pseudironidae 12b. Gill lamellae without such a projection; claws not exceptionally long............................................................................Heptageniidae 13a(10b). Protarsal claws much shorter than tibiae and those on other tarsi (Figs. 20 and 22); claws on meso- and metatarsi long and slender, about as long as tibiae (Fig. 21)........................................................................................................................................................14 13b. Claws on all tarsi similar in structure and length..............................................................................................................................15 14a(13a). Protarsal claws biﬁd (Fig. 20); no spinose pad on procoxae; Canada, midwest and, southeast United States ..............Metretopodidae 14b. Protarsal claws simple, with long, slender denticles (Fig. 22); spinose pad present on procoxae; West, Upper MI........Ametropodidae 15a(13b). Prothoracic legs with dense row of long setae along inner surface (Fig. 23)......................................................................................16 15b. Prothoracic legs without dense row of setae along inner surface ......................................................................................................17 702 W. L. Hilsenhoff FIGURE 3 Ephemeridae; head of Pentaqenia (dorsal view) showing mandibular tusk (T). FIGURE 4 Ephemeridae; gills of Hexaqenia. FIGURE 5 Ephemeridae; prothoracic leg of Hexaqenia. FIGURE 6 Potamanthidae; prothoracic leg of Anthopotamus. FIGURE 7 Polymitarcyidae; metatibia and tarsus of Ephoron. FIGURE 8 Ephemeridae; metatibia and tarsus of Ephemera. FIGURE 9 Baetiscidae; Baetisca (dorsal view). FIGURE 10 Behningiidae; head and pronotum of Dolania. FIGURE 11 Leptohyphidae; abdomen of Tricorythodes (dorsal view) showing operculate gill (OG). FIGURE 12 Leptohyphidae; abdomen of Leptohyphes (dorsal view) showing operculate gill (OG). FIGURE 13 Neoephemeridae; abdomen of Neoephemera (dorsal view) showing operculate gill (OG). FIGURE 14 Caenidae; abdomen of Caenis (dorsal view) showing operculate gill (OG). 16a(15a). Gills on abdominal segment 1 dorsolateral; gills lamellate, tapered apically, with a basal ﬁbrilliform tuft; gill tuft at base of procoxae (Fig. 23) ..................................................................................................................................................................Isonychiidae 16b. Gills on abdominal segment 1 ventral; gills on abdominal segments 2 – 7 small, either lanceolate with a posterior fringe (Fig. 24), or rounded and platelike; no gill tuft at base of procoxae; West, midwest and and southeast United States.......................Oligoneuriidae 17a(15b). Gills forked (Fig. 25), or bilamellate and terminating in a point (Fig. 26), or terminating in ﬁlaments (Fig. 27) ..........Leptophlebiidae 17b. Gills single or double lamellae (Figs. 28 and 29) ..............................................................................................................................18 17. Diversity and Classiﬁcation of Insects and Collembola 703 FIGURE 15 Ephemerellidae; abdomen of Eurylophella (dorsal view) showing operculate gill (OG). FIGURE 16 Heptageniidae; head of Stenonema (dorsal view). FIGURE 17 Siphlonuridae; head of Siphlonurus (dorsal view). FIGURE 18 Arthropleidae; head of Arthroplea (dorsal view) showing maxillary palp (MP). FIGURE 19 Pseudironidae; gill lamella of Pseudiron (ventral view). FIGURE 20 Metretopodidae; protarsus and tibia of Siphloplecton. FIGURE 21 Metretopodidae; metatarsus and tibia of Siphloplecton. FIGURE 22 Ametropodidae; protarsus and tibia of Ametropus. FIGURE 23 Isonychiidae; prothoracic leg of Isonychia showing gill (G). FIGURE 24 Oligoneuriidae; gill on abdominal segment 3 of Homoeoneuria. FIGURE 25 Leptophlebiidae; gill on abdominal segment 3 of Paraleptophlebia. FIGURE 26 Leptophlebiidae; gills on abdominal segment 3 of Leptophlebia. FIGURE 27 Leptophlebiidae; gill on abdominal segment 3 of Habrophlebia. FIGURE 28 Siphlonuridae; gills on abdominal segment 2 of Siphlonurus. FIGURE 29 Baetidae; Baetis (dorsal view). FIGURE 30 Ameletidae; gill on abdominal segment 3 of Ameletus. 18a(17b). Tibiae and tarsi bowed; claws very long and slender; metatarsal claws about as long as tarsi; Illinois, Wisconsin, South Carolina, and Georgia .......................................................................................................................................................Acanthametropodidae 18b. Tibiae and tarsi not bowed; claws not as above................................................................................................................................19 19a(18b). Abdominal segments 8 and 9 produced posterolaterally into distinct, ﬂattened spines (Figs. 13 – 15); if spines are weak, antenna more than twice width of head .........................................................................................................................................................20 704 W. L. Hilsenhoff 19b. Abdominal segments 8 and 9 without such spines (Fig. 29), if weak spines are present, antenna less than twice width of head ...............................................................................................................................................................................................Baetidae 20a(19a). Gills single, with sclerotized band on ventral margin and little or no tracheation (Fig. 30); maxillae with crown of pectinate spines ...........................................................................................................................................................................................Ameletidae 20b. Gills with well-developed tracheation (Fig. 28); maxillae without a crown of pectinate spines .......................................Siphlonuridae C. Taxonomic Key to Families of Freshwater Odonata Larvae 1a. Abdomen terminating in 3 caudal lamellae, longest more than half length of abdomen (Fig. 31) .......................suborder Zygoptera 2 1b. Abdomen terminating in 3 stiff, pointed valves and 2 pointed cerci, longest 1 / 3 length of abdomen (Fig. 44) .................................. ..........................................................................................................................................................................suborder Anisoptera 5 2a(1a). Zygoptera: ﬁrst antennal segment as long as, or longer than, remaining segments combined (Fig. 31); prementum with deep, median cleft (Fig. 32) ................................................................................................................................................................Calopterygidae 2b. First antennal segment much shorter than others combined; prementum with at most a very small median cleft (Figs. 33 and 34) ....3 3a(2b). Basal half of prementum greatly narrowed and elongate (Fig. 33); prementum in repose extends back to or past mesocoxae .Lestidae 3b. Basal half of prementum not greatly narrowed (Fig. 34); prementum in repose extends only to procoxae..........................................4 4a(3b). Caudal lamellae divided into a thick basal half and thin, lighter colored distal half (Fig. 35); one dorsal seta on each side of prementum; Texas......................................................................................................................................................................Protoneuridae 4b. Caudal lamellae not distinctly divided as above; usually with 2 or more dorsal setae on each side of prementum .......Coenagrionidae 5a(1b). Anisoptera: Prementum ﬂat or nearly so, without dorsal setae; palpal lobes also ﬂat and usually without stout setae ........................6 5b. Prementum rounded, spoon-shaped, and usually with dorsal setae; palpal lobes also rounded, always with stout setae, and covering face to base of antennae .....................................................................................................................................................................8 6a(5a). Antennae 4-segmented (Fig. 36); pro- and mesotarsi 2-segmented......................................................................................Gomphidae 6b. Antennae 6- or 7-segmented (Figs. 37 and 38); pro- and mesotarsi 3-segmented ................................................................................7 7a(6b). Antennal segments slender, not hairy (Fig. 37) .....................................................................................................................Aeshnidae 7b. Antennal segments short, thick, and hairy (Fig. 38); mountain seeps ..................................................................................Petaluridae 8a(5b). Distal margin of palpal lobes with large, irregular teeth (Fig. 39) ..............................................................................Cordulegastridae 8b. Distal margin of palpal lobes with small, even crenulations (Figs. 40 and 41) or nearly straight (Fig. 42) ..........................................9 9a(8b). Head with a prominent, almost erect, thick frontal process between bases of antennae (Fig. 43); legs very long, apex of each metafemur reaching to or beyond apex of abdominal segment 8; metasternum with broad, median tubercle ............................Macromiidae 9b. Head without a prominent frontal process (except in Neurocordulia molesta); legs shorter, apex of metafemurs usually not reaching apex of abdominal segment 8; metasternum without a median tubercle ...........................................................................................10 10a(9b). Crenulations on palpal lobes very shallow, 1 / 6 to 1 / 10 as long as wide (Fig. 42), or separated by minute notches bearing setae ..................................................................................................................................................................................most Libellulidae 10b. Crenulations on palpal lobes large, at least 1 / 4 as long as wide (Figs. 40 and 41)............................................................................11 11(10b). Lateral spines on abdominal segment 8 as long or longer than middorsal length of segment 9 (Fig. 44).............................Libellulidae Lateral spines on abdominal segment 8 shorter than middorsal length of segment 9..........................................................Corduliidae D. Taxonomic Key to Families of Freshwater Plecoptera Larvae 1a. Conspicuous ﬁnely branched gills present ventrally or laterally on all thoracic segments (Fig. 2) .......................................................2 1b. Gills absent, conﬁned to prosternum, or unbranched .........................................................................................................................3 2a(1a). Finely branched gills on abdominal sterna 1, 2 and sometimes 3 ..................................................................................Pteronarcyidae 2b. Gills absent from ﬁrst 3 visible abdominal sterna.....................................................................................................................Perlidae 3a(1b). Thoracic sterna produced into plates that overlap succeeding segment (Fig. 45); single, double, or forked gills behind meso- and metacoxae; form roach-like; mountain streams ................................................................................................................Peltoperlidae 17. Diversity and Classiﬁcation of Insects and Collembola 705 FIGURE 31 Calopterygidae; Calopteryx (dorsal view). FIGURE 32 Calopterygidae; prementum of Hetaerina (dorsal view). FIGURE 33 Lestidae; prementum of Lestes (dorsal view). FIGURE 34 Coenagrionidae; prementum of Enallagma (dorsal view) showing dorsal setae (DS), palpal lobe (PL), and palpal setae (PS). FIGURE 35 Protoneuridae; caudal lamella of Protoneura (lateral view). FIGURE 36 Gomphidae; antenna of Gomphus. FIGURE 37 Aeshnidae; antenna of Anax. FIGURE 38 Petaluridae; right antenna of Tachopteryx (dorsal view). FIGURE 39 Cordulegastridae; palpal lobe of Cordulegaster (dorsal view). FIGURE 40 Corduliidae; palpal lobe of Neurocordulia (dorsal view). FIGURE 41 Corduliidae; palpal lobe of Cordulia (dorsal view). FIGURE 42 Libellulidae; palpal lobe of Tramea (dorsal view). FIGURE 43 Macromiidae; head of Macromia (dorsal view). FIGURE 44 Libellulidae; abdominal segments 7 – 10 of Pantala (dorsal view) showing epiproct (E), cerci (C), and paraprocts (P). 3b. Thoracic sterna not produced into overlapping plates; coxal gills, if present, segmented and not apically pointed; form usually elongate, not roachlike..............................................................................................................................................................................4 4a(3b). Tips of glossae situated much behind tips of paraglossae (Fig. 46) .....................................................................................................5 4b. Tips of glossae produced nearly as far forward as tips of paraglossae (Fig. 47) ..................................................................................6 706 W. L. Hilsenhoff FIGURE 45 Peltoperlidae: Peltoperla (ventral view) with legs removed beyond coxae. FIGURE 46 Chloroperlidae; labium of Alloperla (ventral view) showing glossae (G) and paraglossae (P). FIGURE 47 Nemouridae; labium of Nemoura (ventral view) showing glossae (G) and paraglossae (P). FIGURE 48 Perlodidae; Isoperla (dorsal view). FIGURE 49 Chloroperlidae; Haploperla (dorsal view). 5a(4a). Cerci almost as long or longer than abdomen; metathoracic wing-pads with inner and outer margins strongly diverging from axis of body (Fig. 48); head and thorax usually with a distinct pattern ...........................................................................................Perlodidae 5b. Cerci distinctly shorter than abdomen; metathoracic wing-pads with inner and outer margins nearly parallel to axis of body (Fig. 49); head and thorax usually not patterned ..................................................................................................................Chloroperlidae 6a(4b). Second tarsal segment (lateral view) about as long as, or longer than ﬁrst (Fig. 50); single segmented gills on inner side of each coxa (Fig. 51), or ninth abdominal sternum greatly produced (Fig. 52)..............................................................................Taeniopterygidae 6b. Second tarsal segment much shorter than ﬁrst (Fig. 53); gills absent from inner side of coxae and ninth sternum not produced ........7 7a(6b). Robust larvae, with extended metathoracic legs reaching to or beyond tip of abdomen; metathoracic wing-pads with inner and outer margins strongly diverging from axis of body (Fig. 54)............................................................................................Nemouridae 17. Diversity and Classiﬁcation of Insects and Collembola 707 FIGURE 50 Taeniopterygidae; tarsal segments (1, 2, 3) of Taeniopteryx (lateral view). FIGURE 51 Taeniopterygidae; mesosternum of Taeniopteryx showing gills (G) at base of coxae. FIGURE 52 Taeniopterygidae; ninth abdominal sternum (9) of Strophopteryx (ventral view). FIGURE 53 Nemouridae; tarsal segments of Nemoura (lateral view). FIGURE 54 Nemouridae; Amphinemura (dorsal view). FIGURE 55 Capniidae; abdomen of Allocapnia (lateral view) showing lateral fold (LF). FIGURE 56 Capniidae; Paracapnia (dorsal view). FIGURE 57 Leuctridae; abdomen of Leuctra (lateral view) showing lateral fold (LF). FIGURE 58 Leuctridae; Leuctra (dorsal view). 7b. Elongate larvae, with extended metathoracic legs reaching well short of tip of abdomen; metathoracic wing-pads with inner and outer margins nearly parallel to axis of body (Figs. 56, and 58) .........................................................................................................8 8a(7b). Abdominal segments 1 – 9 divided by a membranous ventrolateral fold (Fig. 55); metathoracic wing-pads, if present, about same distance apart as mesothoracic wing-pads and often short and broad (Fig. 56) ....................................................................Capniidae 8b. Abdominal segments 7 – 9 not divided by a membranous fold (Fig. 57); metathoracic wing-pads similar in shape to mesothoracic wing-pads, but distinctly closer together (Fig. 58) ................................................................................................................Leuctridae 708 W. L. Hilsenhoff E. Taxonomic Key to Families of Freshwater Trichoptera Larvae 1a. Larvae in spiral case of sand grains or tiny stones that resembles a snail shell (Fig. 59); anal claws with many short teeth forming a comb, not hook-shaped at apex (Fig. 60) ....................................................................................................................Helicopsychidae 1b. Case not like a snail shell or absent; anal claws not forming a comb, and hook-shaped at apex.........................................................2 2a(1b). Each thoracic segment covered with a single dorsal plate, which may have a mesal or transverse fracture line...................................3 2b. Metanotum mostly membranous, having only scattered hairs or small plates, or with 2 or more sclerites..........................................5 3a(2a). Abdomen with rows of branched gills ventrally; no portable case...............................................................................Hydropsychidae 3b. Abdomen without branched gills ventrally; often with a portable case ...............................................................................................4 4a(3b). Anal prolegs very short and with a stout claw; abdominal tergum 9 sclerotized; ﬁfth instar larvae with abdomen much enlarged; in a barrel- or purse like case (Figs. 61, and 62), or in a ﬂat, silk case ................................................................................Hydroptilidae 4b. Anal prolegs elongate with a projecting claw; abdominal tergum 9 entirely membranous; abdomen not enlarged; larvae in sand retreats; southwestern United States .......................................................................................................................................Ecnomidae 5a(2b). Antennae long, at least 6 times as long as wide, and arising near base of mandibles (Fig. 63); or mesonotum membranous, except for a pair of sclerotized, narrow, curved or angled bars (Fig. 64); larvae in a case ............................................................Leptoceridae 5b. Antennae very short, not more than 3 times as long as wide, often inconspicuous and arising at various points; mesonotum never with a pair of sclerotized, narrow, curved or angled bars; with or without case .................................................................................6 6a(5b). Meso- and metanotum entirely membranous, or with only weak sclerites at SA-1 of mesonotum (Fig. 65); pronotum never with an anterolateral projection; larvae with or without a case .......................................................................................................................7 6b. Meso- and metanotum with some conspicuous sclerotized plates (Figs. 83 and 84); pronotum sometimes with an anterolateral projection; larvae in a case (Fig. 66) ......................................................................................................................................................15 7a(6a). Abdominal segment 9 with dorsum entirely membranous; no portable case (Fig. 67).........................................................................8 7b. Abdominal segment 9 bearing a sclerotized dorsal plate; with or without portable case...................................................................12 8a(7a). Tibia and tarsus fused to form a single segment on all legs; mesopleura with a forward projecting lobe (Fig. 68); southwestern United States ............................................................................................................................................................Xiphocentronidae 8b. Tibia and tarsus distinct on all legs; mesopleura without a projecting lobe.........................................................................................9 9a(8b). Protrochantins broad, hatchet-shaped (Fig. 69) .............................................................................................................Psychomyiidae 9b. Protrochantins pointed or poorly developed.....................................................................................................................................10 10a(9b). Protrochantins poorly developed; head without markings; labrum membranous and T-shaped (Fig. 70) .....................Philopotamidae 10b. Protrochantins pointed anteriorly (Fig. 71); head usually with dark markings or dark or light muscle scars; labrum sclerotized and widest near base ...............................................................................................................................................................................11 11a(10b). Tarsi broad and densely pilose (Fig. 72); mandibles short and triangular, each with a large, thick mesal brush; East, Central.............. ....................................................................................................................................................................................Dipseudopsidae 11b. Tarsi with little or no pile and with a large claw (Fig. 67); mandibles elongate........................................................Polycentropodidae 12a(7b). Prosternal horn present (Fig. 80); SA-3 on meso- and metanotum with a small sclerite and a cluster of setae (Fig. 65); case of vegetation, spirally wound or a series of rings and readily abandoned ......................................................................................Phryganeidae 12b. Prosternal horn absent; SA-3 on meso- and metanotum without a sclerite and usually with a single seta; case, if present, of sand and pebbles .............................................................................................................................................................................................13 13a(12b). Anal claws very small, much shorter than elongate sclerite on anal legs (Fig. 73); larvae with turtlelike case of sand and pebbles, which is readily abandoned........................................................................................................................................Glossosomatidae 13b. Anal claws large, as long as elongate sclerite on anal legs (Fig. 74); larvae without a case................................................................14 141(13b). Profemurs with ventral projection to form chelate legs (Fig. 75); southwestern United States........................................Hydrobiosidae 14b. Prothoracic legs not chelate..........................................................................................................................................Rhyacophilidae 15a(6b). Claws of metathoracic legs very small or a slender ﬁlament, those of other legs long; case of sand, usually with lateral ﬂanges ........... ..........................................................................................................................................................................................Molannidae 15b. Claws of metathoracic legs as long as those of mesothoracic legs; case never of sand with lateral ﬂanges ........................................16 16a(15b). Pronotum divided by a sharp furrow across middle, area in front of furrow depressed (Fig. 76); no dorsal or lateral humps on abdominal segment 1 ..................................................................................................................................................Brachycentridae 17. Diversity and Classiﬁcation of Insects and Collembola 709 FIGURE 59 Helicopsychidae; case of Helicopsyche (dorsal view). FIGURE 60 Helicopsychidae; claw on anal proleg of Helicopsyche. FIGURE 61 Hydroptilidae; case of Oxyethira (lateral view). FIGURE 62 Hydroptilidae; case of Hydroptila (lateral view). FIGURE 63 Leptoceridae; head of Oecetis (lateral view) showing antenna (A). FIGURE 64 Leptoceridae; head and thoracic terga of Ceraclea. FIGURE 65 Phryganeidae; head and thoracic terga of Oligostomis showing location of setal areas (SA). FIGURE 66 Limnephilidae; Hesperophylax (lateral view). FIGURE 67 Polycentropodidae; Polycentropus (lateral view). FIGURE 68 Xiphocentronidae; pro- and mesothorax of Xiphocentron (lateral view) showing projecting lobe (L). FIGURE 69 Psychomyiidae; pronotum (P) and protrochantin (T) of Psychomyia. FIGURE 70 Philopotamidae; head of Chimarra (dorsal view) showing labrum (L). FIGURE 71 Polycentropodidae; pronotum (P) and protrochantin (T) of Polycentropus. FIGURE 72 Dipseudopsidae; tarsus of Phylocentropus. 16b. Pronotum with at most a shallow furrow; lateral humps and usually dorsal hump present on abdominal segment 1.......................17 17a(16b). Pronotum divided obliquely by a sharp carina that terminates as a projecting, rounded lobe anterolaterally (Fig. 77); East, local ....... ..............................................................................................................................................................................................Beraeidae 17b. Pronotum without a distinct carina terminating in a rounded anterolateral lobe..............................................................................18 710 W. L. Hilsenhoff FIGURE 73 Glossosomatidae; last abdominal segment of Glossosoma (lateral view) showing anal proleg. FIGURE 74 Rhyacophilidae; last abdominal segment of Rhyacophila (lateral view) showing anal proleg. FIGURE 75 Hydrobiosidae; pronotum and prothoracic leg with chelate femur (F) of Atopsyche (posterolateral view). FIGURE 76 Brachycentridae; pronotum and head of Brachycentrus (lateral view). FIGURE 77 Beraeidae; pronotum of Beraea (dorsal view) showing carina (C). FIGURE 78 Lepidostomatidae; head of Lepidostoma (dorsal view) showing antenna (A). FIGURE 79 Calamoceratidae; labrum of Heteroplectron (dorsal view). FIGURE 80 Limnephilidae; head and prothorax of Platycentropus (lateral view) showing antenna (A) and prosternal horn (H). FIGURE 81 Sericostomatidae; pronotum (P), protrochantin (T), and coxa (C) of Aqarodes. FIGURE 82 Odontoceridae; pronotum (P), protrochantin (T), and coxa (C) of Psilotreta. FIGURE 83 Goeridae; thoracic terga of Goera showing mesepisternum (M). FIGURE 84 Uenoidae; thoracic terga of Neophylax. 17. Diversity and Classiﬁcation of Insects and Collembola 711 18a(17b). Antennae located extremely close to eyes (Fig. 78); median dorsal hump absent from abdominal segment 1............Lepidostomatidae 18b. Antennae midway between eyes and base of mandibles or near base of mandibles; dorsal hump present on abdominal segment 1.......................................................................................................................................................................................................19 19a(18b). Anterolateral angles of pronotum produced and divergent; labrum with transverse dorsal row of about 18 long setae (Fig. 79); case of vegetation; East and West ......................................................................................................................................Calamoceratidae 19b. Anterolateral angles of pronotum if produced, not divergent; labrum without a dorsal row of about 18 long setae; case of various materials ..........................................................................................................................................................................................20 20a(19b). Prosternal horn absent; antennae close to base of mandibles ............................................................................................................21 20b. Prosternal horn present (Fig. 80); antennae about midway between base of mandibles and eye (Fig. 80) .........................................22 21a(20a). Protrochantin large, hook-shaped (Fig. 81); anal prolegs each with about 30 long setae............................................Sericostomatidae 21b. Protrochantins small, not hook-shaped (Fig. 82); anal prolegs each with about 5 long setae.........................................Odontoceridae 22a(20b). Mesepisternum formed anteriorly into a sharp, elongate process (Fig. 83) or a rounded spiny prominence............................Goeridae 22b, Mesepisternum not enlarged anteriorly as above ..............................................................................................................................23 23a(22b). Mesonotum with an anterior mesal notch (Fig. 84); SA-1 of metanotum unsclerotized and with only 1 or 2 setae (Fig. 84). ............... ..............................................................................................................................................................................................Uenoidae 23b. Mesonotum without an anterior notch; SA-1 of metanotum with a sclerotized plate and / or more than 2 setae...............................24 24a(23b). Head, pronotum, and mesepisternum with prominent surface sculpturing: northwestern United States.............................Rossianidae 24b. Head, pronotum, and mesepisternum without prominent surface sculpturing ..................................................................................25 25a(24b). Mandibles with uniform scraper blades or if toothed, SA-1 of metanotum with 25 setae on membrane between sclerites; case cornucopia-shaped and mostly of sand grains; larvae 12 mm long .................................................................................Apataniidae 25b. Mandibles not modiﬁed into scraper blades; case of vegetation or mineral materials, larvae often 12 mm long and with a transverse depression of the pronotum in most species...........................................................................................................Limnephilidae F. Taxonomic Key to Families of Freshwater Megaloptera Larvae 1a. Abdominal segments 1 – 7 with lateral ﬁlaments, last segment with a long, median ﬁlament (Fig. 85) ......................................Sialidae 1b. Abdominal segments 1 – 8 with lateral ﬁlaments, last segment without a median ﬁlament, but with a pair of prolegs, each with a pair of claws (Fig. 86) .......................................................................................................................................................Corydalidae FIGURE 85 Sialidae; Sialis (dorsal view). FIGURE 86 Niqronia (dorsal view). Corydalidae; 712 W. L. Hilsenhoff G. Taxonomic Key to Adults of Aquatic, Semiaquatic, and Riparian Heteroptera 1a. Antennae shorter than head, inserted beneath eyes and (except Ochteridae) not visible from above (Figs. 87, 88, 93, and 95) ............ ......................................................................................................................................................................suborder Nepomorpha 2 1b. Antennae longer than head, inserted in front of eyes and visible from above (Figs. 96 and 99 – 101)..................................................9 2a(1a). Nepomorpha: Rostrum broad, blunt, and triangular, not distinctly segmented (Fig. 88); each front tarsus a 1-segmented scoop fringed with setae (Fig. 89) ...................................................................................................................................................Corixidae 2b. Rostrum cylindrical or cone-shaped, distinctly 3- or 4-segmented (Fig. 90); front tarsi not scooplike.................................................3 3a(2b). Apex of abdomen with a long, slender, tubular respiratory appendage (Fig. 91) .....................................................................Nepidae 3b. Apical respiratory appendages absent, or if present, short and ﬂat (Fig. 92) .......................................................................................4 4a(3b). Meso- and metathoracic legs with fringes of swimming hairs; ocelli absent; aquatic ..........................................................................5 4b. Meso- and metathoracic legs without fringes or swimming hairs; ocelli usually present; riparian.......................................................8 5a(4a). Dorsoventrally ﬂattened, ovate insects; profemurs broad, raptorial (Fig. 93) .....................................................................................6 5b. Elongate or hemispherical insects, not ﬂattened dorsoventrally (Figs. 94, and 95); profemurs slender, similar to other legs. ..............7 6a(5a). Length 18 mm; short, ﬂat, straplike apical respiratory appendages present (Fig. 92); eyes protrude from margin of head................. .....................................................................................................................................................................................Belostomatidae 6b. Length 16 mm; apical respiratory appendages absent; eyes do not protrude from margin of head (Fig. 93) ....................Naucoridae 7a(5b). Hemispherical (Fig. 94); length 3 mm ....................................................................................................................................Pleidae 7b. Elongate (Fig. 95); length 5 mm ...................................................................................................................................Notonectidae 8a(4b). Profemurs broad, raptorial; antennae concealed from above.........................................................................................Gelastocoridae 8b. Profemurs slender, similar to other legs; antennae visible from above..................................................................................Ochteridae 9a(1b). Membrane of hemelytra with 4 or 5 equal-sized cells (Fig. 96); metacoxae large, transverse; riparian .....Leptopodomorpha, Saldidae 9b. Membrane of hemelytra without veins or with dissimilar sized cells; metacoxae small, conical; semiaquatic or riparian...................... ...................................................................................................................................................................suborder Gerromorpha 10 10a(9b). Gerromorpha: Claws of at least protarsi inserted before apex (Fig. 97)............................................................................................11 10b. Claws of all tarsi inserted at apex (Fig. 98) ......................................................................................................................................12 11a(10a). Metafemurs very long, greatly surpassing apex of abdomen ...................................................................................................Gerridae 11b. Metafemurs short, not, or only slightly surpassing apex of abdomen.......................................................................................Veliidae 12a(10b). Head as long as entire thorax, very slender, with eyes set about halfway to base (Fig. 99)............................................Hydrometridae 12b. Head short and stout, eyes near posterior margin ............................................................................................................................13 13a(12b). Head grooved ventrally to receive rostrum; tarsi 2-segmented; 2.5 mm long.......................................................................Hebridae 13b. Head not grooved ventrally; tarsi 3-segmented; 2.5 mm long ........................................................................................................14 14a(13b). Inner margins of eyes converge anteriorly (Fig. 100); femurs with 1 or more dorsal black spines distally ........................Mesoveliidae 14b. Inner margins of eyes rounded (Fig. 101); femurs without black spines; riparian; western United States........................Macroveliidae H. Taxonomic Key to Families of Freshwater Coleoptera 1. Key to Adult Water Beetles 1a. Head formed into a snout or beak anteriorly; antennae geniculate (Fig. 104).................................................................Curculionidae 1b. Head not formed into a beak or snout; antennae not geniculate.........................................................................................................2 2a(1b). Two pairs of eyes, a dorsal and a ventral pair divided by sides of head; meso- and metathoracic legs short, extremely ﬂat, with tarsi folding fanlike.......................................................................................................................................................................Gyrinidae 2b. One pair of eyes; meso- and metathoracic legs not extremely ﬂat, with tarsi not folded fanlike..........................................................3 3a(2b). Metacoxae expanded into large plates that cover 2 or 3 abdominal sterna and base of metafemurs (Fig. 105) ....................Haliplidae 17. Diversity and Classiﬁcation of Insects and Collembola FIGURE 87 Corixidae; Siqara (dorsal view), with right wings extended laterally. FIGURE 88 Corixidae; head and prothorax of Siqara (ventral view) showing rostrum (R) and antenna (A). FIGURE 89 Corixidae; pala of male Siqara. FIGURE 90 Notonectidae; head of Notonecta (ventral view) showing rostrum (R) and antenna (A). FIGURE 91 Nepidae; apex of abdomen of Nepa (dorsal view). FIGURE 92 Belostomatidae; apex of abdomen of Belostoma (dorsal view). FIGURE 93 Naucoridae; Pelocoris (dorsal view). FIGURE 94 Pleidae; Neoplea (lateral view). FIGURE 95 Notonectidae; Notonecta (dorsal view). FIGURE 96 Saldidae; Salda (dorsal view) showing membrane (M). FIGURE 97 Gerridae: protarsus of Gerris. FIGURE 98 Mesoveliidae; protarsus of Mesovelia. FIGURE 99 Hydrometridae; Hydrometra (dorsal view). FIGURE 100 Mesoveliidae; head of Mesovelia (dorsal view). FIGURE 101 Macroveliidae; head of Macrovelia (dorsal view). 713 714 W. L. Hilsenhoff FIGURE 102 Neuroptera Larva (Sisyridae); Climacia (dorsal view). FIGURE 103 (Pyralidae); Nymphula (lateral view). Lepidoptera Larva 3b. Metacoxae not expanded into large plates .........................................................................................................................................4 4a(3b). Prosternum with a postcoxal process that extends posteriorly to mesocoxae (Fig. 106); ﬁrst visible abdominal sternum completely divided by metacoxal process (Fig. 106) .............................................................................................................................................5 4b. Prosternum with postcoxal process absent or short; ﬁrst visible abdominal sternum extending for its entire breadth behind metacoxae (Fig. 107) .................................................................................................................................................................................8 5a(4a). Base of pronotum much narrower than base of elytra (Fig. 108); metatarsi rounded and not fringed with long setae; large 10 mm long; western mountains of the United States...................................................................................................................Amphizoidae 5b. Base of pronotum subequal in width to base of elytra; metatarsi ﬂattened and fringed with long setae or beetles 2 mm long..........6 6a(5b). Anterior of prosternum, its postcoxal process and metasternum in same plane (Fig. 109); pro- and mesotarsi distinctly 5-segmented, segment 4 as long as 3 ........................................................................................................................................................................7 6b. Anterior of prosternum greatly depressed and not in same plane as its postcoxal process and metasternum (Fig. 110); pro- and mesotarsi appear to be 4-segmented .......................................................................................................................Dytiscidae (in part) 7a(6a). Prosternal process pointed or nearly so (Figs. 106); no curved spur or hooked apex on protibiae; 4 mm long ....Dytiscidae (in part) 7b. Prosternal process truncate or rounded apically (Fig. 111); protibiae with curved spur or hooked apex (Fig. 112) or beetles 3 mm long; eastern, central United States .......................................................................................................................................Noteridae 8a(4b). Antennae short, club-shaped, with segment 4 or 6 modiﬁed to form a cupule (Fig. 113); maxillary palps usually longer than antennae .....................................................................................................................................................................................................9 8b. Antennae pectinate or ﬁliform (Figs. 116, 117), usually longer than maxillary palps .......................................................................12 9a(8a). Antennae with 5 segments past cupule; less than 2.5 mm long .........................................................................................Hydraenidae 9b. Antennae with 3 segments past cupule (Fig. 113); 1.5 – 40.0 mm long..............................................................................................10 10a(9b). Pronotum with 7 longitudinal grooves, including marginal grooves (Fig. 114); 2.0 – 7.1 mm long ..................................Helophoridae 10b. Pronotum without 7 longitudinal grooves ........................................................................................................................................11 11a(10b). Pronotum much narrower than elytral base and scutellum very small (Fig. 115); pronotum granular or with large punctures; 2.2 – 5.3 mm long .............................................................................................................................................................Hydrochidae Pronotum not much narrower than base of elytra, or scutellum elongate; pronotum not granulate, large punctures lacking; 1.5 – 42.0 mm long.........................................................................................................................................................Hydrophilidae 17. Diversity and Classiﬁcation of Insects and Collembola 715 FIGURE 104 Curculionidae; head (lateral view). FIGURE 105 Haliplidae; metathorax and abdomen of Haliplus (ventral view) showing metacoxal plate (CP). FIGURE 106 Dytiscidae; Aqabus (ventral view) showing prosternal process (PP), ﬁrst abdominal sternum (A-1), and metacoxal process (MP). FIGURE 107 Hydrophilidae; Tropisternus (ventral view) showing ﬁrst abdominal sternum (A-1). FIGURE 108 Amphizoidae; Amphizoa (dorsal view). FIGURE 109 Dytiscidae; Aqabus (lateral view) showing prosternum (PS), prosternal process (PP), metasternum (MS), procoxa (PC), and mesocoxa (MC). FIGURE 110 Dytiscidae; Hydroporus (lateral view) showing prosternum (PS), prosternal process (PP), metasternum (MS), procoxa (PC), and mesocoxa (MC). 12a(8b). Antennae short with pectinate club (Fig. 116); 5.0 mm long ............................................................................................Dryopidae 12b. Antennae slender, ﬁliform; (Fig. 117); 4.5 mm long ......................................................................................................................13 13a(12b). Extremely small, 1.5 mm long (Fig. 118); all tarsi 3-segmented; southwest United States, north to Idaho ...............Hydroscaphidae 13b. Larger, 1.7 mm long; all tarsi 5-segmented ...........................................................................................................................Elmidae 716 W. L. Hilsenhoff FIGURE 111 Noteridae; thorax of Hydrocanthus (ventral view) showing prosternal process (PP) and metasternal plate (MP). FIGURE 112 Noteridae; prothoracic leg of Hydrocanthus showing profemur (F), tibia (TI) and tarsus (TA). FIGURE 113 Hydrophilidae; antenna of Tropisternus showing cupule (C). FIGURE 114 Helophoridae; pronotum of Helophorus showing longitudinal grooves. FIGURE 115 Hydrochidae; Hydrochus (dorsal view). FIGURE 116 Dryopidae; right antenna and eye of Helichus (dorsal view). FIGURE 117 Elmidae; Stenelmis (dorsal view). FIGURE 118 Hydroscaphidae; Hydroscapha (dorsal view). 2. Key to Larval Water Beetles 1a. Tarsi with 2 claws ..............................................................................................................................................................................2 1b. Tarsi with 1 claw................................................................................................................................................................................5 2a(1a). Abdomen with 4 conspicuous hooks on last segment; abdominal segments with at least 8 pairs of lateral ﬁlaments (Fig. 119) Gyrinidae 2b. No hooks on last abdominal segment; if lateral abdominal ﬁlaments are present, there are only 6 pairs ............................................3 3a(2b). Abdominal and thoracic terga ﬂattened and expanded laterally (Fig. 120); western mountains of the United States........Amphizoidae 3b. Abdominal and thoracic terga not ﬂattened and expanded laterally ...................................................................................................4 4a(3b). Urogomphi shorter than last abdominal segment; legs short, stout, adapted for digging (Fig. 121); mandibles short, adapted for chewing; eastern, central United States .................................................................................................................................Noteridae 4b. Urogomphi usually longer than last abdominal segment (Fig. 122); if shorter, legs are elongate with setal fringe for swimming; mandibles elongate, pointed, for piercing .............................................................................................................................Dytiscidae 5a(1b). Legs distinctly 5-segmented; abdomen terminating in 1 or 2 long ﬁlaments (Figs. 123, and 124) .........................................Haliplidae 5b. Legs apparently 4-segmented; abdomen not terminating in long ﬁlaments .........................................................................................6 6a(5b). Mandibles large, readily visible from above (Fig. 125)...................................................................................................Hydrophilidae 6b. Mandibles not readily visible from above ...........................................................................................................................................7 7a(6b). Antennae long, ﬁliform, as long as head and thorax combined (Fig. 126) ...............................................................................Scirtidae 7b. Antennae much shorter than head and thorax combined ...................................................................................................................8 8a(7b). Body oval and extremely ﬂat; head completely concealed from dorsal view (Figs. 127, and 128) ......................................Psephenidae 8b. Body elongate and round or triangular in cross section; head exposed, except mostly concealed in Lampyridae ................................9 9a(8b). Each thoracic and abdominal segment covered by a ﬂat, platelike sclerite dorsally; prothoracic plate mostly or completely concealing head from above (Fig. 129) ..........................................................................................................................................Lampyridae 17. Diversity and Classiﬁcation of Insects and Collembola 717 FIGURE 119 Gyrinidae; Dineutus (dorsal view). FIGURE 120 Amphizoidae; Amphizoa (dorsal view). FIGURE 121 Noteridae; Hydrocanthus (dorsal view). FIGURE 122 Dytiscidae; Aqabus (dorsal view) showing urogomphi (U). FIGURE 123 Haliplidae; Haliplus (dorsal view). FIGURE 124 Haliplidae; Peltodytes (lateral view). FIGURE 125 Hydrophilidae; Tropisternus (dorsal view). FIGURE 126 Scirtidae; Cyphon (dorsal view). FIGURE 127 Psephenidae; Ectopria (dorsal view). FIGURE 128 Psephenidae; Psephenus (dorsal view). FIGURE 129 Lampyridae (lateral view). FIGURE 130 Chrysomelidae; Donacia (lateral view). FIGURE 131 Ptilodactylidae; last abdominal segment of Anchytarsus (lateral view). FIGURE 132 Elmidae; Stenelmis (lateral view). FIGURE 133 Elmidae; last abdominal tergum of Stenelmis. FIGURE 134 Lutrochidae; last abdominal tergum of Lutrochus. 9b.(8a). Thoracic and abdominal segments not covered by ﬂat, platelike sclerites; head visible from above ..................................................10 10a(9b). All terga rounded and pale; grublike larvae with 2 spines on last abdominal tergum (Fig. 130) ....................................Chrysomelidae 10b. Body elongate and sclerotized; no spines on last abdominal tergum (Figs. 131, and 132).................................................................11 11a(10b). Abdominal sterna with distinct tufts of gills, either on sterna 1 – 7 or on last segment and lacking and operculum (Fig. 131); eastern, United States and California..........................................................................................................................................Ptilodactylidae 718 W. L. Hilsenhoff 11b. Abdominal gills, if present, on last abdominal segment and covered by a ventral operculum (Fig. 132) ...........................................12 12a(11b). Last abdominal tergum biﬁd or notched apically (Fig. 133) .....................................................................................................Elmidae 12b. Last abdominal tergum rounded apically (Fig. 134); East, Southwest. ...............................................................................Lutrochidae I. Taxonomic Key to Families of Freshwater Diptera Larvae 1a. Larvae apparently 7-segmented; ﬁrst 6 segments each with a prominent ventral sucker (Fig. 135) ..........Nematocera, Blephariceridae 1b. Larvae with more than 7 apparent segments; without 6 ventral suckers .............................................................................................2 2a(1b). Head capsule completely sclerotized and fully visible (Figs. 147 – 159); mandibles opposed, moving in a horizontal plane, and usually with two or more apical teeth; body never ﬂattened and with a posterior spiracular chamber margined with long, soft hairs. .....................................................................................................................................................................................Nematocera 12 2b. Sclerotized head capsule absent (Figs 138 – 140, 142, 143, 145, and 146), or incomplete behind (Figs. 136 and 137), and retracted at least partially into thorax; mandibles parallel, without secondary apical teeth, and moving in a vertical plane or in a partially sclerotized retracted head may be opposed and moving in a horizontal plane ....................................................................................3 3a(2b). Mandibles opposed, moving in a horizontal plane, and with two or more apical teeth; larvae truncate anteriorly with head capsule retracted into thorax and incomplete posteriorly (Figs. 136 and 137)...............................................................Nematocera, Tipulidae 3b. Mandibles parallel, moving vertically, and without secondary apical teeth; larvae narrowed anteriorly with head capsule lacking or poorly developed and incompletely sclerotized or body terminating in a long respiratory tube (Fig. 139) or a posterior spiracular chamber margined with long, soft hairs ...........................................................................................................................Brachycera 4 4a(3b). Brachycera: Head mostly visible, truncate in shape (Fig. 138); body somewhat ﬂattened; posterior spiracular chamber margined with long, soft hairs; integument covered with shiny calcium carbonate crystals ............................................................Stratiomyidae 4b. Head mostly retracted into thorax and elongate, or indistinguishable; body nearly circular in cross section; without a posterior spiracular chamber margined with long, soft hairs; integument lacking calcium carbonate crystals....................................................5 5a(4b). Larvae with a partially retractile caudal respiratory tube at least one-half as long as body (Fig. 139) ...................................Syrphidae 5b. Larvae without a long respiratory tube, if a short tube is present, it is divided apically ......................................................................6 6a(5b). Body terminating in a short tube that is divided apically (Fig. 140), or in a pair of spines ..................................................Ephydridae 6b. Body not terminating in a short tube or a pair of spines .....................................................................................................................7 7a(6b). Caudal spiracular disk with palmate hairs and surrounded by 8 – 10 lobes, some of which may be very short (Fig. 141); body wrinkled ...................................................................................................................................................................................Sciomyzidae 7b. Caudal spiracular disk without palmate hairs, if surrounded by lobes, body not wrinkled .................................................................8 8a(7b). Abdomen without distinct prolegs, paired ventral pseudopods, or terminal processes except a single spine, but with a girdle of 6 or more pseudopods on each segment (Fig. 142) ......................................................................................................................Tabanidae 8b. Abdomen with distinct prolegs, paired pseudopods, or paired terminal processes present ..................................................................9 9a(8b). Body terminating in a spiracular pit surrounded by 4 pointed lobes (Fig. 143) ............................................................Dolichopodidae 9b. Body not terminating in a spiracular pit with 4 lobes .......................................................................................................................10 10a(9b). Body terminating in a pair of ciliated, divergent processes (Fig. 144); abdomen with 8 pairs of ventral prolegs .................Athericidae 10b. Terminal processes, if present, not ciliated; abdomen with or without prolegs .................................................................................11 11a(10b). Some external head structure visible, with palps and antennae usually present; abdomen usually with ventral prolegs and elongate, paired, terminal appendages (Fig. 145) or a bulbous segment ..............................................................................................Empididae 11b. No visible external head structure; abdomen often with ventral pseudopods and usually short, paired, terminal appendages (Fig. 146) ......................................................................................................................................................................................Muscidae 12a(2a). Nematocera: Prolegs absent (Figs. 147, 150, and 151) .....................................................................................................................13 12b. Prolegs present at one or both ends of body or on abdominal segments (Figs. 152 – 159).................................................................17 13a(12a). Thoracic segments fused and distinctly thicker than abdomen (Fig. 147) .........................................................................................14 13b. Thoracic segments not fused and about equal to abdomen in diameter (Figs. 150 and 151).............................................................16 14a(13a). Antennae prehensile, with long, strong apical spines (Fig. 148) ........................................................................................................15 17. Diversity and Classiﬁcation of Insects and Collembola 719 FIGURE 135 Blephariceridae; Blepharicera (ventral view). FIGURE 136 Tipulidae; head of Limonia (dorsal view). FIGURE 137 Tipulidae; head of Hexatoma (dorsal view). FIGURE 138 Stratiomyidae; head of Odontomyia (dorsal view). FIGURE 139 Syrphidae; Eristalis (lateral view). FIGURE 140 Ephydridae (lateral view). FIGURE 141 Sciomyzidae; spiracular disc of Sepedon. FIGURE 142 Tabanidae; Chrysops (lateral view). FIGURE 143 Dolicopodidae (lateral view). FIGURE 144 Athericidae; terminal segments of Atherix (dorsal view). FIGURE 145 Empididae (lateral view). FIGURE 146 Muscidae; Limnophora (lateral view). FIGURE 147 Culicidae; Anopheles (dorsal view). FIGURE 148 Chaoboridae; head of Chaoborus (lateral view) showing antenna (A). FIGURE 149 Culicidae; head of Coquillettidia (dorsal view) showing antenna (A). FIGURE 150 Psychodidae; Psychoda (dorsal view). FIGURE 151 Ceratopogonidae; Palpomyia (dorsal view). FIGURE 152 Nymphomyiidae; Palaeodipteron (lateral view). 14b. Antennae not prehensile, lacking long apical spines (Fig. 149)...............................................................................................Culicidae 15a(14a). Antennae close together at base; a row of spinose setae on dorsolateral margin of head ................................................Corethrellidae 15b. Antennae at anterolateral margins of head; head without spinose setae dorsolaterally .....................................................Chaoboridae 720 W. L. Hilsenhoff FIGURE 153 Deuterophlebiidae; Deuterophlebia (dorsal view). FIGURE 154 Dixidae; Dixella (lateral view). FIGURE 155 Ptychopteridae; Ptychoptera (lateral view). FIGURE 156 Simuliidae; Simulium (lateral view). FIGURE 157 Tanyderidae; Protoplasa (lateral view). FIGURE 158 Chironomidae; Chironomus (lateral view). FIGURE 159 Thaumaleidae; Thaumalea (lateral view). 16a(13b). Thoracic and abdominal segments each distinctly divided into 2 or 3 annuli, with sclerotized dorsal plates on some annuli (Fig. 150) Psychodidae 16b. No secondary annulations on abdominal segments (Fig. 151) .....................................................................Ceratopogonidae (in part) 17a(12b). Prolegs on intermediate body segments (Figs. 152 – 155) ..................................................................................................................18 17b. Prolegs on anterior and / or posterior ends of body only (Figs. 156 – 159) .........................................................................................21 18a(17a). Seven or eight pairs of distinct prolegs on abdomen (Figs. 152 and 153)..........................................................................................19 18b. Two or three pairs of weak prolegs on abdomen (Figs. 154 and 155)...............................................................................................20 19a(18a). Eight pairs of slender, ventrally projecting prolegs (Fig. 152); PQ, NB, ME ...............................................................Nymphomyiidae 19b. Seven pairs of stout, ventrolaterally projecting prolegs (Fig. 153); western mountains.............................................Deuterophlebiidae 20a(18b). Paired ventral prolegs on abdominal segment 1 and usually also on segment 2; posterior end of body with 2 pairs of fringed processes (Fig. 154) ..................................................................................................................................................................Dixidae 20b. Paired ventral prolegs on abdominal segments 1, 2, and 3; body terminating in a long respiratory tube (Fig. 155) .......Ptychopteridae 21a(17b). A single proleg present only on prothorax; posterior of abdomen swollen and terminating in a ring of numerous small hooks (Fig. 156) .....................................................................................................................................................................................Simuliidae 21b. Posterior prolegs usually present; posterior of abdomen not swollen and terminating in a ring of hooks .........................................22 22a(21b). Only posterior prolegs present (Fig. 157) .........................................................................................................................................23 22b. Anterior and usually posterior prolegs present (Figs. 158 and 159) ..................................................................................................24 23a(22a). Long ﬁlamentous processes arising from last two abdominal segments and from prolegs (Fig. 157); Eastern and Western United States.................................................................................................................................................................................Tanyderidae 23b. Last two abdominal segments without long ﬁlamentous processes...............................................................Ceratopogonidae (in part) 24a(22b). Body covered with long, strong spines .........................................................................................................Ceratopogonidae (in part) 24b. Body at most covered with setae ......................................................................................................................................................25 25a(24b). At least one pair of prolegs separated distally (Fig. 158); stalked spiracles lacking.........................................................Chironomidae 25b. Prolegs unpaired (Fig. 159); short, stalked spiracles dorsolaterally on the prothorax; mountain streams........................Thaumaleidae J. Taxonomic Key to Families of Semiaquatic Collembola 1a. Body somewhat globular; thorax and abdomen indistinctly segmented (Fig. 160) ...........................................................Sminthuridae 1b. Body elongate; thorax and abdomen distinctly segmented (Fig. 161) .................................................................................................2 17. Diversity and Classiﬁcation of Insects and Collembola 721 FIGURE 160 Sminthuridae (lateral view) showing furcula (F) and collophore (C). FIGURE 161 Poduridae; Podura (lateral view) showing furcula (F) and collophore (C). FIGURE 162 Poduridae; furcula of Podura (dorsal view) showing manubrium (MA), dens (D), and mucro (MU). FIGURE 163 Isotomidae anterior portion (lateral view). [Figures 158 – 161 redrawn from Peckarsky et al. (1990).] 2a(1b). Mouthparts directed downward (Fig. 161); furcula distinctly convergent apically (Fig. 162); ﬁrst abdominal segment with dorsal setae .....................................................................................................................................................................................Poduridae 2b. Mouthparts directed forward (Fig. 163); furcula not distinctly convergent apically; ﬁrst abdominal segment lacking dorsal setae ....... ...........................................................................................................................................................................................Isotomidae LITERATURE CITED Adler, P. H., Kim, K. C. 1986. The blackﬂies of Pennsylvania (Simuliidae, Diptera). 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Physical Constraints on Aquatic Insects C. Lentic Insect Communities D. Lotic Insect Communities E. Saline Habitats F. Specialized Habitats III. Aquatic Insect Life Histories IV. Insect-Mediated Processes A. Detritivory I. INTRODUCTION Aquatic insects are abundant in most freshwater habitats and often exhibit high diversity. They play signiﬁcant roles in freshwater ecosystems, including serving as food items for nearly the full range of vertebrate and invertebrate predators found in aquatic systems. The ecology of aquatic insects has been studied from many perspectives, including species diversity, life-history constraints, community structure, predator – prey interactions, detritivory, grazing, and implications for nutrient dynamics. Considerable information is available on insect responses to a variety of environmental conditions, including factors that operate on landscapelevel scales. Thus, they often are used as indicators of water-quality conditions in both lentic and lotic systems. Longitudinal trends in insect functional feeding groups are an important component of the River Continuum Concept (see Fig. 6 in Chapter 2), a major paradigm in the discipline of stream ecology. Although the ecological literature on aquatic insects is extensive, most of it has been produced within the last 20 years, reﬂecting current interest in the topic. In spite of this extensive body of work, the enormous diversity of Ecology and Classiﬁcation of North American Freshwater Invertebrates. 2nd Edition Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved. V. VI. VII. VIII. IX. B. Grazing C. Predator – Prey Interactions D. Insects as Conduits for Energy Flow in Aquatic Food Webs Secondary Production Effects of Land Use on Aquatic Insect Communities Role of Disturbance Aquatic Insects in Biomonitoring Studies Summary Literature Cited insects combined with their widespread distribution dictates that much remains to be learned. This chapter provides a brief overview of aquatic insect communities in both lentic (standing water) and lotic (ﬂowing water) habitats and describes the role of insects in ecological processes within these ecosystems. The discussion is placed in the context of how physical and life-history factors constrain the distribution and abundance of aquatic insects, thereby altering communities and ecosystem function. II. AQUATIC INSECT COMMUNITIES A. Insect Taxonomic Diversity Insects are the most species-rich and often the most abundant group of substrate-dwelling macroinvertebrates, and have successfully invaded virtually all aquatic habitats. Although there are many specialized habitats, most aquatic insects are found either in lotic or lentic habitats (Table I), and are constrained to one or the other habitat by physiological adaptations (see below). We will follow the classiﬁcation used 733 734 A. E. Hershey and G. A. Lamberti TABLE I List of aquatic and partially aquatic orders of insects, with estimated number of North American species, preferred habitat, typical generation time, feeding mode, and examples of common genera. Order Collembola Estimated no. of NA species Generation time (yr) Primary feeding mode (FFG) Examples of common NA genera Ephemeroptera 600 Water surface; semiaquatic Lotic (lentic) Odonata Anisoptera 300 Lentic lotic 1 Predators Zygoptera Orthoptera 120 40 Lentic lotic Water margin; semiaquatic 1 1 Predators Shredders Plecoptera 6 families 3 families Heteroptera 330 250 410 Lotic (lentic) Lotic (lentic) Lentic lotic 1 1 1 Megaloptera Neuroptera Trichoptera 50 6 1350 Lotic (lentic) Lotic lentic Lotic (lentic) Lepidoptera Coleoptera 770 5000 Lentic (lotic) Lentic lotic; water margin 1 1 1 (some or 1) 1 1 (some 1) Shredders Predators Predators, collectorgatherers Predators Piercers All major FFGs Diptera Tipulidae Culicidae Simuliidae 1500 175 150 Lotic Lentic Lotic 1 1 1 Chironomidae 2000 Lotic lentic 1 Shredders, predators Collector ﬁlterers Collector-ﬁlterers, scrapers All major FFGs Others 3200 Lotic lentic 1 All major FFGs Internal parasites 1 Parasites Hymenoptera 40 Primary (and 2°) habitat 60 1 Collector-gatherers Podura, Sminthurides 1 Collector-gatherers, collector-ﬁlterers, scrapers Baetis, Hexagenia Aeshna, Gomphus, Libellula Argia, Lestes Conocephalus Shredders, scrapers All major FFGs Pteronarcys, Capnia Acroneuria, Isoperla Belostoma, Gerris, Trichocorixa Sialis, Corydalus Sisyra Rhyacophila, Hydropsyche, Glossosoma, Neophylax Petrophila Hydroporus, Gyrinus, Psephenus Tipula, Limonia Aedes, Anopheles, Culex Prosimulium, Simulium Chironomus, Polypedilum, Tanytarsus Chaoborus, Chrysops, Ephydra Apsilops, Trichogramma (NA, North American; FFG, functional feeding group.) by Hilsenhoff in this volume (see Chapter 17). The orders Ephemeroptera (mayﬂies), Odonata (dragonﬂies and damselﬂies), Plecoptera (stoneﬂies), Trichoptera (caddisﬂies), and Megaloptera (ﬁshﬂies and alderﬂies) are considered to be strictly aquatic in their immature stages. Other insect orders are partially aquatic (aquatic group listed in parentheses), including Collembola (semiaquatic springtails), Neuroptera (spongillaﬂies), Heteroptera (aquatic and semiaquatic bugs), Coleoptera (water beetles), Diptera (aquatic ﬂies and midges, especially suborder Nematocera but some Brachycera), and Lepidoptera (aquatic caterpillars). All of these latter groups have important aquatic representatives as immatures. The Coleoptera and Heteroptera have groups with adult as well as larval stages that are aquatic. Even several species of Orthoptera (grasshoppers) are found in association with aquatic habitats (Cantrall and Brusven, 1996). These aquatic insect orders are treated taxonomically in Chapter 17. Species diversity in many of the aquatic insect orders is very high. A pristine stream may contain well over one hundred species of insects, including many that are difﬁcult or impossible to identify in the immature aquatic stages. Ponds, lakes, and wetlands can also exhibit very high diversity, depending on water-quality conditions, littoral development, and substrate characteristics. Due to taxonomic problems associated with this high diversity, the role of aquatic insects in aquatic ecosystems is often difﬁcult to study. To minimize this problem, aquatic insects have been categorized into a smaller number of functional feeding groups (see Merritt and Cummins, 1996a, b), as discussed in more detail later in this chapter. 18. Aquatic Insect Ecology 735 B. Physical Constraints on Aquatic Insects The physical environment of aquatic ecosystems exerts substantial control over the population abundances and hence community composition of insects. Still, it is remarkable that virtually every North American freshwater environment has been colonized by aquatic insects, ranging from alpine lakes to desert hot springs, oligotrophic streams to sewage treatment lagoons, and treeholes to the extreme depths of large lakes. Within these habitats, physical factors of particular importance to aquatic insects include: (1) dissolved oxygen concentration; (2) water temperature; (3) water chemistry; (4) type of substrate; and (5) hydrodynamics. 1. Dissolved Oxygen As aerobic organisms, all insects must obtain sufﬁcient oxygen to drive their metabolic machinery (Eriksen et al., 1996). This presents a particular challenge for aquatic insects because water, even when saturated, contains much less oxygen than do terrestrial environments (a maximum of about 15 ppm oxygen in water compared to over 200,000 ppm in the air). Dissolved oxygen concentrations are highly variable over time and space, and oxygen may be totally lacking from some aquatic habitats (termed “anoxia”). Furthermore, oxygen concentrations in lentic systems generally decrease with depth (Fig. 1A). Because the vast majority of aquatic insects are substrate dwellers, this presents a potential physiological constraint. Most cold-water oligotrophic lakes remain oxygenated throughout the open water season because solubility increases as water temperature decreases. However, even in cold climates, shallow ponds and wetlands can become anoxic due to respiratory oxygen demands under ice where no opportunity exists for oxygen replenishment. Deeper oligotrophic lakes typically have sufﬁcient oxygen stores to meet under-ice metabolic demands. The likelihood of under-ice anoxia increases with productivity due to proportionally increasing respiratory requirements and decreases with depth due to high oxygen storage in the hypolimnion. Thus, seasonal and spatial variation in oxygen concentration greatly restricts the types and diversity of insects found in aquatic environments. No insect lacking access to an oxygen supply can survive for long, but some can cope with short-term anoxia. For example, the chironomid midge Chironomus and related Chironomini (Diptera: Chironomidae) contain a hemoglobinlike pigment (hence the common name “bloodworm”) that allows them to store oxygen and thus survive temporary anoxia. The phantom midge Chaoborus (Diptera: Chaoboridae) is also found in anoxic sediments, but uses a different strategy than do bloodworms. Chaoborus migrates vertically at night FIGURE 1 Oxygen features in lakes and streams. (A) Oxygen and temperature proﬁles in a temperate stratiﬁed lake. (B) Oxygen “sag” in a river receiving a point discharge of municipal waste. BOD, biological oxygen demand; COD, chemical oxygen demand; PPr, primary production. toward the surface both to obtain oxygen and to hunt for prey. During the day, it returns to the deep, dark sediments to evade its own predators. Shallower environments (e.g., ponds and marshes) that experience oxygen depletion contain a more diverse array of insects because they can obtain oxygen in a myriad of ways. Some insects, including many heteropterans, carry an air store (“bubble”) on their body that they occasionally replenish by swimming to the water surface (Fig. 2A). Others maintain a semipermanent connection to the atmosphere (“breathing tube”) as exempliﬁed by mosquito larvae (Diptera: Culicidae) and rat-tailed maggots (Diptera: Syrphidae). Yet other insects, such as some beetle larvae (Coleoptera) and a genus of mosquitoes (Fig. 2B), use specialized spiracles to pierce the vascular tissues of rooted aquatic plants and thus “steal” their oxygen (White and Brigham, 1996). 736 A. E. Hershey and G. A. Lamberti ﬂowing water. For example, perlid stoneﬂies perform “push-ups” when oxygen declines, presumably to move more oxygenated water across their ventral thoracic gills. Burrowing mayﬂies of the genus Hexagenia undulate their body to draw water, and thus food and oxygen, through their burrows. 2. Water Temperature FIGURE 2 Respiratory structures of aquatic insects. (A) Ventral air bubble of the backswimmer Neoplea (Heteroptera: Pleidae). (B) Longitudinal section of postabdominal respiratory siphon of the mosquito larva Taeniorhynchus (Diptera: Culicidae), used to pierce plant air stores. (C) Abdominal ﬁlamentous tracheal gills of the caddisﬂy Limnephilus (Trichoptera: Limnephilidae). [Drawings (A) and (B) from Merritt and Cummins, 1996b; (C) from Wiggins, 1996.] In most unimpacted streams, dissolved oxygen concentrations generally are near saturation and thus oxygen rarely limits insect diversity. We often see the greatest diversity of aquatic insects in small streams, due in part to the abundance of oxygen. However, insects requiring a connection to atmospheric air are typically in low numbers, because ﬂow would quickly displace these taxa far downstream. Thus, most lotic insects obtain their oxygen by diffusion through the body wall, often aided by “tracheal gills,” which are thinly sclerotized evaginations of the body wall designed to increase surface area (Fig. 2C). Most representatives of the insect orders that are wholly aquatic (e.g., Ephemeroptera, Plecoptera, and Trichoptera) possess such tracheal gills in the larval, aquatic state and achieve their highest diversity in ﬂowing waters. Gills, however, rely on abundant dissolved oxygen for diffusion into the body, and thus gilled larvae can experience stress at low oxygen concentrations. Such oxygen “sags” can occur in larger rivers with low turbulence (and therefore low reaeration), especially at night when photosynthesis turns off or where municipal or industrial pollutants deplete oxygen due to high respiratory demand downstream of their input (Fig. 1B). In these cases, the aquatic insect fauna will more closely mimic that found in oxygen-poor standing waters (many dipterans, for example). Some behavioral adaptations allow insects to “weather” periods of low oxygen in Water temperature also imposes constraints on aquatic insects. Temperature directly affects insects by regulating their metabolic rates and thus development from egg to adult, and indirectly by inﬂuencing such things as ﬂuid dynamics, gas saturation constants, and primary production rates (Hauer and Hill, 1996). Fluctuation in water temperature is found both spatially and temporally. For example, stratiﬁed lakes show marked temperature gradients from top to bottom, while rivers can show horizontal (bank to bank) and longitudinal (source to estuary) gradients in temperature. Low- to midorder streams also may exhibit pronounced diel temperature ﬂuctuations, especially on sunny days. Shallow unshaded wetlands may exhibit diel ﬂuctuations of 20° (e.g., Hershey et al., 1995a). Temporal ﬂuctuations can range from subfreezing to tepid in northern wetlands. Thus, temperature variation imposes fundamental constraints on the types of insects that may be present in many aquatic habitats. Some aquatic insects prefer a narrow range of temperature, usually cool water (cold “stenotherms”), whereas others can tolerate a broader range of temperatures (“eurytherms”). Consequently, temperature ﬂuctuation can limit the types of insects found within a habitat. Any signiﬁcant change in water temperature, as may result from thermal pollution or climate change, will likely alter the species composition of aquatic insects. Considerable variation in temperature tolerance occurs within the orders of aquatic insects, and so temperature preferences should be examined on a species-speciﬁc basis. The upper lethal limit for all but the most specialized species is between 30 and 40°C (Pritchard, 1991; Wallace and Anderson, 1996), although most temperate aquatic habitats do not approach such temperatures. However, thermally destabilized habitats, such as cooling water reservoirs, have temperatures which may approach this limit intermittently or seasonally, and abundance and richness of most taxa are negatively affected (see Thorp and Diggins, 1982). Types of insects that can tolerate, and even thrive, in hot water include various beetles and true ﬂies, although species diversity declines with increasing temperature. For example, in a survey of hot springs, Brock (1978) recorded 60 species of Coleoptera at 30°C; at 45°C only two species were found. At the other extreme, many species exhibit 18. Aquatic Insect Ecology growth at temperatures only a few degrees above freezing in both lentic (e.g., Butler, 1982) and lotic systems (e.g., Rosenberg et al., 1977). Aquatic insects have several strategies for surviving subfreezing conditions. The antifreeze compounds produced by some species prevent freezing of bodily ﬂuids to several degrees below 0°C (Danks, 1978; Duffy and Liston, 1985). Other species actually tolerate freezing of extracellular tissues, which provides some protection against intracellular freezing (Danks, 1978; Zachariassen, 1979; Ward and Stanford, 1982). Cocoons and diapausing eggs are important strategies to prevent ice damage (see Wallace and Anderson, 1996). Because water and snow cover provide some degree of thermal buffering, even streams and ponds in the arctic do not drop more than several degrees below freezing. In north temperate regions, where terrestrial temperatures may reach 35°C, stream temperatures typically do not drop below 1°C in pools and 2°C in rifﬂes (Fig. 3; Hershey unpublished data). In arctic ponds that freeze solid, some insect species overwinter several times as larvae (Butler, 1982). Thus, aquatic insects only need mechanisms that provide them with a few degrees of thermal buffering. In some streams, avoidance of ice scour may be a more serious problem for 737 benthic insects. Prior to the formation of surface ice, anchor ice attached to the stream bed may form at night due to cooling associated with back radiation (Barnes, 1928). As the stream warms during the day, anchor ice lifts off the bottom, scouring the stream bed and disturbing resident insects (Hynes, 1970). Temperature, and its variation, can inﬂuence aquatic insect diversity in any freshwater habitat. For streams, the River Continuum Concept (Vannote et al., 1980; see also below) predicts that biological diversity should be highest in midorder (medium-sized) streams because temperature variation is highest, thereby providing the most temperature “niches” for insects. The literature on this topic, however, is mixed. Insect faunal diversity can be high in cold streams, which are thought to be the ancestral habitat for aquatic insects (Hynes, 1970). For example, Anderson and Hanson (1987) cataloged over 325 species of aquatic insects from a cool Oregon stream. On the other hand, Morse et al. (1983) found over 500 species of aquatic insects in a warmer coastal South Carolina stream. From wood snags in the warm Ogeechee River in Georgia, Benke and Jacobi (1994) collected over 20 species of mayﬂies alone, suggesting that aquatic insects can be diverse at a variety of temperatures. 3. Water Chemistry FIGURE 3 Pool and rifﬂe temperature in the French River, Minnesota, which is strongly inﬂuenced by ice. Although air temperatures may drop as low as about 30°C, aquatic insects experience temperatures that are only a few degrees below freezing. Many aspects of water chemistry can restrict the occurrence or abundance of aquatic insects, including pH, salinity, and concentrations of speciﬁc ions or elements. Generally, it is the extremes in any of these parameters that result in change to aquatic insect communities, while levels around the “average” have less direct impact. Low pH, as is found in acidiﬁed lakes and streams of pH 5 (due to acid deposition, mine drainage, organic acids, or poor buffering) can alter community composition such that only acid-tolerant taxa are found (Hynes, 1970). However, it is unclear whether it is the low pH itself that adversely affects insects or some related factor. For example, iron can precipitate under acidic conditions with an associated reduction in oxygen. Acidic waters can also leach metals from soils and rock, especially aluminum, that can increase drift and possibly have toxic effects on aquatic insects (Hall et al., 1987). Regardless, some aquatic insects thrive in acid streams and lakes (examples cited in Hynes, 1970). Salinity gradients that form in coastal estuaries, along saline lakes, and even from runoff after road salting, can affect insects, most of which are salt-intolerant. However, some insects such as brine ﬂies (Diptera: Ephydridae) thrive in warm, saline water, where they have few competitors. Many other chemical features, such as calcium concentration and total ionic strength, have potential importance to 738 A. E. Hershey and G. A. Lamberti aquatic insects, but little is known about the requirements of speciﬁc taxa. 4. Substrate and Flow At the scale of local habitat, substrate and hydrodynamics are probably the most important factors determining the types and abundance of aquatic insects present. Swift currents in streams, wave action along shorelines, wind-generated turbulent mixing, and tidal action all present signiﬁcant hydrodynamic challenges to insects. Most aquatic insects spend at least part, if not most, of their life cycle associated with the substrate, and hydrodynamic forces interact strongly with substrate type to produce the habitat experienced by the insect fauna. Hydrodynamic and substrate inﬂuences on aquatic insects are discussed in more detail in the sections below on lentic and lotic insect communities. Cummins and Merritt (1996) recognized eight categories of aquatic insect habits, primarily based on the substrate or habitat they occupy. The ﬁrst four groups remain associated with the substrate for most of their lives. “Burrowers” and “sprawlers” inhabit ﬁne sediments where they either tunnel into the sediments or remain on top of the sediments, respectively. “Climbers” move along the stems of vascular macrophytes or pieces of detritus. “Clingers” attach themselves to the surfaces of substrates exposed to water movement, such as rocks in streams or along wave-swept lake shores. The ﬁnal four groups spend part, or most, or their lives moving through or on the water. “Swimmers” periodically propel themselves through the water to change location, although they mostly remain attached to a substrate. TABLE II “Divers” split their time among swimming to the surface, diving back through the water, and clinging to submerged objects. “Skaters” are adapted for life on top of the water surface, where they use hydrophobic body parts to move over water. “Planktonic” forms ﬂoat or swim about in open water. C. Lentic Insect Communities Lentic environments, including lakes, ponds, and various types of wetlands offer a broad array of aquatic habitats that have been exploited by a wide variety of insects. As in any community, the insect species that are present reﬂect the physical, chemical, and biotic characteristics of the ecosystem, each of which places major physiological, morphological, and trophic constraints on aquatic insects. Insects exhibit a variety of adaptations to these habitat constraints, which combined have a large impact on the structure of lentic insect communities. In addition, numerous trophic and interspeciﬁc factors interact with the physical and chemical constraints to yield the dynamic and diverse insect communities that are present in lentic systems. 1. Littoral Communities Littoral areas of ponds and lakes are typically better oxygenated, structurally more complex, and afford more abundant and diverse food resources than do profundal sediments of lakes. All of these factors lead to a high diversity of insects. Littoral communities generally have aquatic insect representatives from most of the aquatic orders (Table II), including nearly all of the Aquatic insect communities in lentic habitats Habitat type Habitat characteristics Typical insect community References Littoral High O2, macrophytes, rock, sand, or silt substrates Merritt and Cummins, 1996a, b; Ward, 1992 Eutrophic profundal Low O2, soft mineral and organic sediments Ephemeroptera (esp. Siphlonuridae, Baetidae, Heptageniidae, Caenidae, Leptophlebiidae), Odonata (representatives from most families), Diptera (many families, but esp. diverse Chironomidae), Trichoptera (esp. Phryganeidae, Limnephilidae, Leptoceridae), Plecoptera (some Perlidae and Perlodidae), Hemiptera (most aquatic families), Megaloptera (few genera), Neuroptera (both aquatic genera), Coleoptera (adults and larvae of many families), Hymenoptera (parasitoids only) Chironomidae (esp. Chironomus, Procladius) Oligotrophic profundal Wetlands High O2, soft mineral sediments Variable hydrology, variable O2, organic sediments, macrophytes Chironomidae (esp. Tanytarsini and Orthocladiinae) High diversity of most lentic orders. Diptera and Coleoptera are especially well represented. Hilsenhoff, 1966; Jonasson, 1972; Merritt and Cummins, 1996a, b Saether,1979 Rosenberg and Danks, 1987; Batzer and Wissinger, 1996; Hershey et al., 1998 18. Aquatic Insect Ecology 739 morphological and trophic types as well as the full size range of aquatic species. Large taxa such as dragonﬂies, damselﬂies, many of the lentic mayﬂies, aquatic beetles and bugs, and caddisﬂies are important members of littoral zone communities, and typically are uncommon in profundal zones (Table II). Aquatic insects of littoral zones also show considerable variation in habitat (see Ward, 1992). Many of these species use the macrophytes themselves as habitat, although others are characteristic inhabitants of inorganic and organic sediments and rocks. Macrophyte habitats usually have higher density and species diversity of insects than less structurally complex habitats, including many of the larger taxa mentioned above (Crowder and Cooper, 1982; Gilinsky, 1984; Hershey, 1985a). Many highly mobile species, particularly members of the Coleoptera and Heteroptera, swim in the water column (plankton), or use the upper or lower surface of the air – water interface (epineuston and hyponeuston, respectively). Gyrinid beetles, referred to as whirligig beetles because they swim in whirling swarms on the water surface, are especially well adapted for their habitat; they have dorsal and ventral compound eyes permitting them to see above and below the water surface simultaneously. The burrowing mayﬂy, Hexagenia, is often very abundant in littoral and sublittoral sediments of lakes, but is particularly sensitive to anoxia (Beeton, 1961; Rasmussen, 1988). A few Plecoptera (stoneﬂies) are found in erosional lentic habitats (White and Brigham, 1996), reﬂecting the high oxygen requirements of members of this order. Smaller species, especially representing the dipteran family Chironomidae, are usually very diverse and present in high densities (Thorp and Bergey, 1981; Crowder and Cooper, 1982; Gilinski, 1984; Hershey, 1985a). FIGURE 4 Feeding activity of Chironomus tentans returns P to the water column. At high density this can be a signiﬁcant source of P to the water column. [From Gallepp, 1979.] 2. Profundal Communities 3. Wetland Communities Profundal zones of lakes have quite different communities than do littoral zones (Table II). In both oligotrophic and eutrophic lakes, chironomids most often dominate the insect fauna. However, in eutrophic lakes, where the profundal zone is often anoxic during much of the summer, Chironomus and Procladius may be the only insect genera present (Hilsenhoff, 1966; Saether, 1979). Chironomus feeds on suspended particles by pumping water into either U-shaped or J-shaped burrows, ingesting the particles that impinge on its silken funnel at the opening of the burrow (Walshe, 1947). Feces are deposited on the sediment surface forming mounds. At high density, this feeding mechanism can be important in returning phosphorus to the water column (Fig. 4; Gallepp, 1979). Procladius is a free-living chironomid, most often characterized as a predator (Kajak, 1980; Vodopich and Cowell, 1984), but it also The various types of wetlands also afford a variety of habitats for aquatic insects. Water level is highly variable in wetlands, by deﬁnition, and many wetland types are subjected to periods of desiccation. Desiccation provides a variety of challenges for aquatic insects, and results in somewhat different communities than are found in more permanent lentic ecosystems. Hydroperiod is likely, therefore, to be the most important factor affecting wetland insect communities (Wiggins et al., 1980; Batzer and Wissinger, 1996; Hershey et al., 1998). Insect species in wetland habitats exhibit either desiccation-resistance or drought-avoidance strategies (Wiggins et al., 1980). These strategies do not fall neatly along taxonomic lines. Desiccation resistant insects, especially many Diptera, and some odonates, caddisﬂies, and beetles, deposit drought-resistant eggs or lay eggs in plant stems, which then hatch to ingests a variety of sediment dwelling diatoms and desmids (Hershey, 1986). Although ubiquitous in lentic habitats Procladius also is tolerant of low oxygen conditions. In oligotrophic lakes, the chironomid community in the profundal zone is more diverse, but still depauperate compared to littoral zones. The presence of Chironomus spp. versus Tanytarsus spp. has been considered indicative of lake trophic condition (e.g., Brinkhurst, 1974). An elaborate key to lake typology which recognizes several categories of trophic conditions has been developed based on the occurrence of chironomid genera and species (Saether, 1979), and lake classiﬁcation has a long history based on chironomid assemblages (Brundin, 1949; Brinkhurst, 1974). 740 A. E. Hershey and G. A. Lamberti repopulate the habitat following the return of hydric conditions (Wiggins et al., 1980, reviewed by Batzer and Wissinger, 1996). Insects that avoid desiccation have several different strategies. Some migrate to more permanent habitats to complete one or more generations. Wing polymorphism (i.e., winged and unwinged individuals) associated with this strategy is not uncommon and is particularly well-studied in water striders (Spence and Anderson, 1994) and some beetles (see Wallace and Anderson, 1996). Other insects have long ﬂight periods as adults and thus will spend the drought cycle in the terrestrial environment (Wiggins et al., 1980). This strategy is also used by chironomids and mosquitoes, as well as some dragonﬂies and limnephilid caddisﬂies (Wiggins et al., 1980; Wissinger, 1988). These general strategies interact with life history (see below) such that wetland insect communities exhibit a succession following the drought cycle with dominance shifting from mosquitoes and chironomids to larger taxa such as beetles, odonates, heteropterans, and caddisﬂies (Batzer and Wissinger, 1996). The extreme of this cycle is that prolonged drought results in very low density of insects (Fig. 5; Hershey et al., 1999). Under these conditions the wetland fauna is characterized by high abundance of molluscs and annelids and low abundance and species richness of insects, but it becomes more and more dominated by a rich insect fauna with prolonged inundation (Fig. 6; Hershey et al., 1999). D. Lotic Insect Communities Lotic insect communities are functionally and structurally quite different than lentic communities due to the different physical and chemical challenges of the lotic environment. At a local scale in stream ecosystems, substrate and current velocity are probably the most important physical factors determining the community structure of aquatic insects. The stream substrate has obvious importance because the vast majority of stream insects spend most of their lives attached to substrates. Lotic substrates can be broadly divided into inorganic substrates (geologic material ranging from silt to boulders) and organic substrates (ﬁne organic particles up to logs). The particle size of inorganic matter has a large inﬂuence on insect community FIGURE 5 Prolonged drought in Minnesota prairie wetlands in 1987 – 1990 resulted in communities with signiﬁcantly lower density of insects (measured in 1989 – 1990) than in wetter years of 1991 – 1993. Much of this difference was due to Chironomidae. 18. Aquatic Insect Ecology 741 FIGURE 6 Patterns of insect and noninsect abundances in prairie wetlands over a hydrologic cycle. Insects and cladocerans are abundant during the wet phase, but molluscs and copepods dominate during drought. Because of their longer life cycles, the transition back to insects can be relatively long following the return of hydric conditions. [From Hershey et al., 1999.] structure. Coarser bed materials (e.g., gravel, cobble, and boulders) generally provide more interstitial habitat for insects than do ﬁner sediments (e.g., sand and silt). For example, “water penny” beetles (family Psephenidae), hellgrammite larvae (Megaloptera: Corydalidae), and perlid stoneﬂies (Plecoptera) frequently are found in interstitial spaces on the undersides of rocks. Sedentary ﬁlter-feeding insects require the space between particles both to operate their ﬁltration device (e.g., nets, specialized body parts) and to allow water ﬂow to carry food items to them. Many case-building caddisﬂies (Trichoptera) pupate in dense aggregations on protected faces of cobbles and boulders where interstitial water ﬂow carries the dissolved oxygen needed for metamorphosis (Lamberti and Resh, 1979; Resh et al., 1981), but they are more dispersed in the larval stage. A lower diversity of insects is typically found in ﬁne sediments because the tight packing of particles restricts physical habitat and the trapping of detritus, and can limit the availability of oxygen (Allan, 1995). However, taxa adapted to such habitats (e.g., chironomid midges, oligochaete worms, certain mayﬂies) may be abundant. For example, Soluk (1985) recorded densities of chironomid midges exceeding 80,000 individuals / m2 in the sand substrate of an Alberta river. Organic substrates such as large woody debris or leaf packs often are “hot spots” for invertebrate activity because they provide both substrate and nutritional resources (Anderson and Sedell, 1979). In a survey of Oregon streams, Dudley and Anderson (1982) found 56 invertebrate taxa that were closely associated with wood, and another 129 taxa that were facultative associates of wood. The more specialized moss habitat typically supports a high density of chironomids and other ﬂies (e.g., moth ﬂies, family Psychodidae) and a different assemblage of species than found on adjacent mineral substrates (Glime and Clemons, 1972; Suren and Winterbourn, 1992). Flow affects many aspects of insect biology, including body form, food acquisition, and movement. Taxa found in the swift currents of streams usually are streamlined (e.g., Baetis mayﬂies), dorsoventrally compressed (e.g., heptageniid mayﬂies and psephenid “water penny” beetles), possess suctorial disks (e.g., blepharicerid ﬂies), use rock “ballast” in their cases to resist the ﬂow (e.g., Glossosoma caddisﬂies), or are small enough to ﬁt almost entirely within the sheltered “boundary layer” (e.g., chironomid midges). Many insects take advantage of swift ﬂow by allowing current to transport food items to them. Examples include caddisﬂies of the family Hydropsychidae that construct silken nets to capture suspended food particles, black ﬂies 742 A. E. Hershey and G. A. Lamberti FIGURE 8 Channel cross section of a stream during summer low ﬂow, showing position of hyporheic zone relative to surface water and groundwater. [Modiﬁed slightly from Williams, 1993]. FIGURE 7 The larval blackﬂy is a common ﬁlter feeder in rifﬂe habitats of North American streams. The fan-shaped objects, or cephalic fans, are a modiﬁcation of the mouthparts which collect ﬁne particulate organic matter (FPOM) from the water column. The larva orients itself in the current by forming a silk pad on the rock, then attaches itself to the pad using tiny hooks on the posterior portion of its abdomen. [Photograph courtesy of D. A. Craig.] (Simuliidae) that ﬁlter food with specialized mouthparts (Fig. 7), and Brachycentrus caddisﬂies that capture particles on the setae (hairs) of their front legs (Wallace and Merritt, 1980). Filter-feeding insects also can be found along the wave-swept shores of lakes or on stable substrates (e.g., wood debris) of large rivers, where similar hydrodynamic forces are experienced. Many insects prefer low water movement, such as found in quiescent stream pools and along stream margins. In these habitats, organic matter is deposited by gravity and thus can be “gathered” and ingested. These taxa often are more “wormlike” than swift-water forms. Variation in ﬂow (ﬂoods to desiccation) is the major cause of natural disturbance in streams (see section below on disturbance) and is responsible for large, usually temporary, reductions in macroinvertebrate abundance and diversity (Lamberti et al., 1991). The hyporheic (i.e., interstitial) zone and the undersurfaces of rocks provide refugia during ﬂood events (Fig. 8). Dur- ing drought, the deep hyporheic zone is especially important, but is usually accessible only to smaller invertebrates (typically about 1 – 5 mm in length). The spatial extent of the hyporheic zone varies with local geomorphology. In bedrock-dominated reaches, it is virtually nonexistent; whereas in the Flathead River (Montana), the hyporheic zone may extend tens of meters vertically and up to three kilometers laterally from the river, supporting relatively large stoneﬂies several millimeters in length (Stanford and Ward, 1988). Finally, recolonization by egg-laying adults or by individuals drifting from upstream (Table III) serves to reintroduce taxa that become locally extinct during drought, ﬂood, or other disturbance events (Fisher et al., 1982; Smock, 1996). 1. Functional Feeding Groups Rather that focusing on the species as the ecological unit in streams, aquatic insect ecologists often consider the insect community in terms of functional feeding groups (Cummins, 1973, 1974; Cummins and Klug, 1979; Merritt and Cummins, 1996a). The functional feeding group approach categorizes stream consumers according to their mode of feeding, or functional role, rather than taxonomic groups. From an ecosystem perspective, this approach reduces the number of groups to be considered by 1 – 2 orders of magnitude compared to the species approach. Thus, rather than studying dozens or hundreds of speciﬁc consumers, a smaller number of groups of organisms can be studied collectively from the perspective of their function in the stream ecosystem. This categorization also has utility in other aquatic ecosystems, and has recently been applied to all North American aquatic insects regardless of habitat (Merritt and Cummins, 1996b). 18. Aquatic Insect Ecology TABLE III 743 Mechanisms of insect recolonization of stream reaches Mechanism Time scales Example References Downstream drift Many insects Waters, 1972 Many insects Cairns, 1990 Upstream ﬂight Flight from other watersheds Nocturnal, constant, episodic, dispersal stages of some invertebrates Nocturnal, constant, episodic, dispersal stages of some invertebrates Seasonal, depending on emergence period Seasonal, depending on emergence period Baetis mayﬂies Many insects, Baetis mayﬂies Upstream swimming Seasonal Leptophlebia cupida, Paraleptophlebia mayﬂies Upstream crawling Daily Dicosmoecus caddisﬂies, Juga snails Movement from hyporheic refuge Episodic, seasonal Many early instar insects; overwintering stages of many insects in cold climates; many residents of intermittent streams Hershey et al., 1993 Wallace et al., 1986; Schmidt et al., 1995 Hayden and Clifford, 1974; Bird and Hynes, 1981 Hart and Resh, 1980; L. M. Blair and G. A. Lamberti, unpublished data Sedell et al., 1990; Delucchi, 1989; Hershey unpublished data Drift from tributaries [From Hershey and Lamberti, 1998] The functional feeding groups recognized by Merritt and Cummins (1996a, b) are: (1) “scrapers” ( “grazers”), which remove and consume attached algae and associated periphytic material; (2) “shredders,” which ingest coarse particulate organic matter (CPOM), as decomposing leaf litter, living macrophyte tissue, or dead wood; (3) “predators,” which eat living animals; and (4) “collectors,” which consume decomposing ﬁne particulate organic matter (FPOM). The last group can be further broken down into “collector-gatherers,” which collect FPOM from the sediments, and “collector-ﬁlterers,” which collect FPOM from the water column (Fig. 9). Two other, less common functional feeding groups are the “macrophyte-piercers,” which pierce the tissues of macroalgae and rooted hydrophytes, and “parasites,” which develop on or in aquatic insects, generally killing them. Because groups of insect species can be studied collectively to unravel major avenues of organic matter processing, the functional feeding group approach greatly simpliﬁes the study of insect function in aquatic ecosystems. It also provides a strong basis for comparative studies of the biomass and productivity of consumer groups across ecosystems, whereas it is much more difﬁcult (and often less informative) to make such comparisons on a species-by-species basis. Although implementation of this approach has been an important precedent for the development of other major paradigms of aquatic ecology (e.g., river continuum concept of Vannote et al., 1980), the functional feeding group concept is not without limitations. First, assignment of individual organisms from aquatic samples to a functional feeding group generally requires identiﬁcation of the organism to family (see Chapter 17 of this volume) or genus. This can be a very timeconsuming task, although a shortcut approach using an illustrated key is effective for many taxa (see Merritt and Cummins, 1996a). Second, feeding ecology can vary within a genus, and even within a species depending on habitat or food availability. Thus, published functional group designations for the generic level are not always reliable (Mihuc, 1997). In fact, many aquatic insects are known to change feeding habit with growth and development (Chapman and Demory, 1963; Rader and Ward, 1987). For example, the large limnephilid caddisﬂy Dicosmoecus gilvipes begins larval life as a collector-gatherer, feeds primarily as a scraper of periphyton during the middle instars, and ﬁnally becomes partly predaceous in the ﬁnal instar (Gotceitas and Clifford, 1983; Li and Gregory, 1989). Other insects are omnivorous throughout their lives. Filterfeeding caddisﬂies of the genus Hydropsyche will consume virtually anything that becomes entrapped in their nets, including FPOM, living algae, and small animals (Benke and Wallace, 1980), thus making them potential members of several functional feeding groups. Some orders of aquatic insects, or the aquatic representatives within an order, perform mostly as a single functional feeding group, whereas others show great diversity in feeding (Table I). Generally, the more primitive orders of aquatic insects show more limited diversity in feeding mode. For example, aquatic collembolans are exclusively collector-gatherers, odonates are strictly predaceous, and stoneﬂies (Plecoptera) are either shredders or predators, generally depending on family. Less speciose orders also tend to show less feeding diversity, as one might expect. For example, 744 A. E. Hershey and G. A. Lamberti FIGURE 9 Simpliﬁed food web for a hypothetical North American forested stream, showing major energy inputs and use by different macroinvertebrate functional feeding groups [modiﬁed from Allan, 1995, after Cummins, 1973]. Macroinvertebrate examples are: shredders (Peltoperla stoneﬂy, Tipula craneﬂy, Hydatophylax caddisﬂy); collector-ﬁlterers (Hydropsyche caddisﬂy, Simulium blackﬂy); grazers (Glossosoma caddisﬂy, Juga snail); predators (Calineuria stoneﬂy, Orohermes hellgrammite). Broken lines indicate fate of fecal matter. megalopterans are entirely predaceous, neuopterans pierce either animals or plants, and aquatic hymenopterans are mostly parasites. In contrast, more derived orders, such as Trichoptera, Diptera, and Coloptera, show considerable diversity in feeding mode, in part due to their large number of aquatic species. The Diptera and Trichoptera, in particular, have been enormously successful in freshwater ecosystems. Chironomid midges alone may equal the species diversity of all other insects combined in a typical aquatic ecosystem. In any speciﬁc stream, it is usually possible to ﬁnd caddisﬂies represented in all functional feeding groups, and often dominating the biomass of those groups. Beetles possibly occupy the broadest range of aquatic habitats, ranging from ice ﬁelds to seashores, and can be very speciose. 2. The River Continuum Concept The River Continuum Concept or “RCC” (Vannote et al., 1980; see Fig. 6 in Chapter 2) is an important paradigm in stream ecology and functional feeding groups are a major component of that paradigm. The RCC predicts that small, heavily shaded streams will have high inputs of detritus (“allochthonous” CPOM) from adjacent riparian zones. Due to this abundant detritus, shredders will dominate the macroinvertebrate community. Both collector-ﬁlterers and collectorgatherers also will be abundant because high-quality FPOM will be produced as CPOM becomes fragmented. In medium-sized streams, light inputs increase and thus benthic algal production will increase. The shredders are replaced by scrapers, while collectors remain abundant. In large rivers, benthic algal production and direct riparian inputs decline. Food resources for macroinvertebrates are dominated by FPOM in suspension; collectors dominate the macroinvertebrate community. In all streams, predators comprise a small but fairly stable proportion of the fauna. These predictions were tested by Hawkins and Sedell (1981) for the McKenzie River drainage in Oregon, which consisted of streams ranging in size from the 1st- to 7th-order. They found that functional feeding group distributions were generally consistent with predictions of the RCC (Fig. 10). These longitudinal shifts were more pronounced 18. Aquatic Insect Ecology 745 in six small streams in Oregon, but that absence of canopy increased the abundance of all functional feeding groups possibly because both autotrophic (on-site) and heterotrophic (from upstream) inputs were received. Hence, insect consumers responded to the abundance and composition of the available food resources. This notion of energy transfer via both autotrophic and heterotrophic food webs is an underpinning of the lotic ecosystem model of McIntire and Colby (1978), that postulates a hierarchical organization to stream food webs and a central role for aquatic insects. 3. Insect Drift FIGURE 10 Longitudinal changes in relative abundance of macroinvertebrate functional feeding groups at different seasons within an Oregon, USA, river system. “Collectors” collectorgatherers; “Filterers” collector-ﬁlterers. Stream size increases along the x-axis; DCC, Devilsclub Creek; MACK, Mack Creek; LOKC, Lookout Creek; and MZER, McKenzie River. Note the general conformance to predictions of the River Continuum Concept (see Figure 2.6 in this volume). [Modiﬁed slightly from Hawkins and Sedell, 1981.] than were seasonal ﬂuctuations in community structure in the individual Oregon streams. Not all studies of functional feeding groups have shown the same degree of conformance to the RCC (e.g., Minshall et al., 1985; Winterbourn et al., 1981). Some groups, such as shredders, predictably decline with increasing stream size whereas other groups, such as scrapers, do not ﬁt tightly with RCC expectations. In evaluating conformance to the RCC, abundance and biomass have often been considered as currency. However, some researchers have argued that production rather than biomass is a more appropriate currency as it is a better measure of transfer of energy between trophic levels (Benke, 1993, 1998; Grubaugh et al., 1997). Large rivers, at least in their natural state, likely have more complex energy inputs than implied by the RCC. Thorp and Delong (1994) developed the riverine productivity model that suggests that riparian inputs and benthic primary production are quite important to insect production in large rivers, which is contrary to predictions of the RCC. Their preliminary test of the model in the Ohio River suggested that the shallow edges of large rivers function similarly (and have similar insect communities) as midorder streams, because they receive riparian litterfall and also have substantial periphyton production. Hawkins et al. (1982) found that riparian canopy highly inﬂuenced functional feeding group composition “Drift” is the downstream transport of oranisms in the current (usually expressed as number / per cubic meters), and it is a unique property of stream ecosystems. Because it provides insects with a unidirectional transportation system, it has the potential to be important to the distribution and ecology of stream insects. At any given time, the proportion of fauna in the drift, compared to that on the substrate, is very small (0.01 – 0.5%; Ulfstrand, 1968; Waters 1972). Some insect species and groups have a much greater propensity to drift than others. Mayﬂies, especially Baetis, and larval chironomids are perhaps the most common insect drifters. It was once thought that when an insect entered the drift it was essentially lost from the system. Although estimates of drift distance vary widely, most experimental studies in small to mid-sized streams have shown that insects drift only a short distance (a few meters or less) during any drift event (Allan and Feifarek, 1989; Wilzbach and Cummins, 1989). However, working in a high-order blackwater river with an important but widely dispersed snag habitat, Benke et al. (1986) found that insects drifted between snags, a drift distance on the scale of kilometers. Insects may drift passively or inadvertently if they are dislodged, such as might occur during a high discharge event or in response to an anthropogenic disturbance. However, most experimental studies have suggested that most drift is active, or a behavioral response to some stimulus. This can occur catastrophically due to chemical spills, or on a smaller scale in response to depleted food resources (Kohler, 1985) or the presence of a predator (Peckarsky, 1980). It was once thought that drift was a density-dependent response, such that drifting insects represented a portion of the population that exceeded the carrying capacity of the stream (Waters, 1972). However, empirical studies of drift have yielded little support for density dependent behavior (e.g., Hinterleitner-Anderson et al., 1992). In most streams, drift densities are much higher at night than during daylight hours. Because ﬁsh feed heavily on drifting invertebrates, nighttime drift behavior 746 A. E. Hershey and G. A. Lamberti Using 15N as a tracer, adult Baetis mayﬂies were found to ﬂy approximately 2 km upstream. The ﬁgure shows that although adults have a lower 15N signature than nymphs, those captured from upstream of the 15N dripper are considerably more labeled than background. An integral mixing model showed that this label reﬂected mayﬂies from 2 km downstream (hypothesized to be females) mixing with mayﬂies from upstream (hypothesized to be males). [From Hershey et al., 1993.] FIGURE 12 FIGURE 11 Baetis mayﬂies enter drift actively, not passively, as a result of greater activity on upper surfaces of substrates at night. Kohler (1985) showed that only food abundance and time of day affected drift entry, and only when no food was present was there any drift entry during the day. The ﬁgure shows the results from food abundance experiments. [Reprinted from Kohler, 1985.] provides invertebrates with more protection from ﬁsh predation. Because many macroinvertebrates forage more on the upper surfaces of rocks at night, it was once thought that dislodgement was simply more likely at night. However, Kohler (1985) found that well-fed Baetis foraged only at night on tops of stones while starved nymphs foraged during both day and night, but drifted similarly to fed nymphs. Thus, activity during foraging was not related to drift (i.e., they were not accidentally dislodged) and drift entry was active (Fig. 11). Drift clearly is an important mechanism for invertebrate dispersal, especially for early larval instars. It is also an important means for recolonizing reaches that are impacted by natural or anthropogenic disturbance (see Cairns, 1990). However, if there were no upstream movement of macroinvertebrates, downstream drift could result in a net displacement of populations, net depletion of upstream reaches, and loss of invertebrate-mediated ecosystem functions in depleted areas. The colonization cycle proposed by Müller (1954) states that insects displaced downstream as larvae later ﬂy upstream as adults to oviposit, thereby repopulating depleted areas. Upstream ﬂight has been observed for some species (e.g., Madsen et al., 1973) and quantiﬁed for a Baetis population in arctic Alaska (Fig. 12; Hershey et al., 1993). E. Saline Habitats Insects have enjoyed only limited success in colonizing the marine environment, and only water striders of the genus Halobates, which are surface dwelling, are truly oceanic (Spence and Andersen, 1994). The reasons for this are unclear, but have variously been attributed to physiological stresses associated with respiration and osmoregulation, physical stress of wave action, competition for habitat and resources with established species, and predation (see recent reviews by Ward, 1992; Wallace and Anderson, 1996). Because several species of beetles, true ﬂies, and caddisﬂies have successfully colonized intertidal zones and brackish waters, it seems unlikely that physiological stresses alone have hindered radiation into the marine realm. Similarly, because many insects inhabit wave-swept lake shores and stream rifﬂe habitats, and because there are many low energy marine habitats, physical stress also seems unlikely to be a major hindrance. These considerations suggest that competitive and predatory pressures from marine phyla, which have been established in marine habitats much longer, are the most likely mechanism that has precluded high insect diversity in marine habitats (Usinger, 1956; Ward, 1992; Wallace and Anderson, 1996). Alternatively, Williams and Feltmate (1992) suggested that insects may be in the early stages of invading marine systems, such that they may yet become established there after many more millennia. 18. Aquatic Insect Ecology 747 FIGURE 13 Characteristics of a stream insect community along a natural thermal gradient emanating from a California hot spring, depicting total density, species richness (diversity), and total biomass. [Modiﬁed from Lamberti and Resh, 1985.] F. Specialized Habitats Aquatic insects have invaded many specialized microhabitats. The species that comprise these communities often have morphological, behavioral, and life-history adaptations which are unique to those habitats and reﬂect the remarkable phenotypic plasticity of the Insecta. Some of the better studied examples of these habitats include treeholes, hot springs, pitcher plants, and caves and other subterranean environments (see Ward, 1992). Mosquitoes are particularly well adapted to specialized ephemeral habitats such as treeholes, tires, rain gutters, and other containers, that catch rainwater (Merritt et al., 1992). Filter feeding is the most common mechanism for acquiring food in such habitats, but detrital and bacterial particles may also be gleaned from leaf and bark surfaces, and a few species are predatory (Merritt et al., 1992). In treehole habitats, a combination of leaf litter quality and quantity and stemﬂow nutrients inﬂuence mosquito productivity (Carpenter, 1982, 1983), but ﬁeld experiments by Walker and Merritt (1988) found no evidence that quantity of leaf detritus was limiting. Aquatic insects that inhabit subterranean habitats, including cave pools and streams, underground rivers, and extensive hyporheic zones, share some morphological characteristics. These insects, like other subterranean invertebrates, lack pigment, have reduced or absent compound eyes and ocelli, and possess reduced wings (Ward, 1992). Diptera and Coleoptera are perhaps the best represented among subterranean insects although representatives of other groups also have been reported (see Ward, 1992). The trophic ecology of these insect communities is dependent on organic matter imported from surface ecosystems. Most common among these nutritional sources are ﬂocculated DOM (dissolved organic matter), entrained FPOM, bacteria (which utilize DOM), and bat guano. Geothermal springs are another specialized habitat that has been exploited by some insects. Hot springs tend to have representatives from the Diptera, Coleoptera, and Odonata (see Ward, 1992). In addition to the high temperatures, these habitats also have high mineral content, which may pose toxicity problems for some species. However, Lamberti and Resh (1983a, 1985) showed that thermal effects were more important than chemistry in limiting insect diversity in a northern California stream receiving natural hot spring inputs (Fig. 13). III. AQUATIC INSECT LIFE HISTORIES Aquatic insects show tremendous diversity in their life-history patterns. This diversity includes variation in reproductive and dispersal strategies, life-cycle length, growth rate, developmental strategies, and phenology of the various life-history stages. This great variety serves to separate taxa seasonally and spatially, thus providing the basis for the varied and dynamic nature of aquatic insect community composition. Virtually all aquatic insects have an aerial adult phase. Copulation usually occurs outside of the aquatic 748 A. E. Hershey and G. A. Lamberti environment, and eggs are laid on or in the water for most species. Typically, the larval stage dominates the life cycle; the adult stage is very short and does not always involve feeding. Notable exceptions to this rule are the Odonata (dragonﬂies and damselﬂies), that are long-lived and voracious predators as adults. Dragonﬂies and damselﬂies also have long ﬂight periods, feeding on insect swarms and using this energy for ﬂight, food capture, and reproduction. Swarms of dragonﬂies often are thought to be important for control of mosquitoes and blackﬂies. Many dipterans also feed as adults. Mosquitoes and blackﬂies are notorius pests, but are also vectors of diseases such as malaria, encephalites, dengue, Rift Valley Fever, river blindness, and many others that infect and kill millions of people worldwide (e. g., Renshaw et al., 1996; Jacobs-Lorena and Lemos, 1995; Kidwell and Wattam, 1998). With global warming, the geographic distribution of these diseases is expected to increase (Brown, 1996). For many aquatic insects, the adult phase is often important to dispersal between lentic habitats, and permits upstream oviposition (see Müller, 1954; Hershey et al., 1993), and oviposition between watersheds (Wallace et al., 1986) in lotic habitats. Many aquatic beetles and bugs have extended adult life spans and are aquatic in both larval and adult stages of the life cycle, although dispersal still is accomplished by the adult stage. Dispersal varies tremendously among insects and has been studied thoroughly for only a few taxa. For example, Baetis mayﬂies in a tundra river in arctic Alaska have been shown to ﬂy approximately 2 km upstream to lay eggs (Hershey et al., 1993). Even larval insects can move substantial distances, aside from drift. For example, late-instar larvae of the grazing caddisﬂy Dicosmoecus gilvipes can crawl up to 25 m per day in search of algal food (Hart and Resh, 1980). Nymphs of the lotic mayﬂy, Leptophlebia cupida, move upstream at an average rate of 10 m “per hour” during seasonal migrations between habitats (Hayden and Clifford, 1974). The frequency with which an organism completes its life cycle is referred to as “voltinism.” Depending on environmental constraints, especially temperature and nutrition, as well as evolutionary factors, aquatic insects may be univoltine (one generation per year), multivoltine (more than one generation per year; e.g., bivoltine two per year, trivoltine three per year), semivoltine (2-year life cycle), or merovoltine (3- or more-year life cycle). Generally, larger insects will have longer life cycles than smaller species. For example, in the Kuparuk River in arctic Alaska, the relatively small Baetis mayﬂies are univoltine, but the larger Brachycentrus caddisﬂies are merovoltine (Hershey et al., 1997). Univoltine development is by far the most common, especially in temperate regions (see Wallace and Anderson, 1996). However, even in arctic streams, where the stream bed is frozen for almost nine months, most species are univoltine (Hershey et al., 1997). Arctic lakes have chironomid populations with life cycles ranging from 1 to 4 years, but univoltine development predominates (Hershey, 1985a). One arctic pond species of chironomid has a 7-year life cycle (Butler, 1982). Temperate regions display similar variation. The wood-boring aquatic beetle Lara avara has about a 5-year life cycle, likely due to the low nutritional value of wood (Anderson and Sedell, 1979). At the other extreme, some herbivorous chironomid midges in warm desert streams can complete their life cycle in as little as two weeks, a distinct advantage in desert streams where ﬂoods scour organisms and resources (Fisher and Gray, 1983). Variation in growth rate is also important in distinguishing insect species, separating cohorts, and providing structure to aquatic insect communities. Within physiological constraints, temperature and food are the major factors affecting growth for most taxa. These factors vary widely within, and between, lentic and lotic ecosystems, as well as geographically. In cool climates many aquatic insects have life cycles with synchronized development (Fig. 14). Environmental factors, especially temperature and photoperiod, as well as evolutionary factors, serve as proximal cues that synchronize life cycles (Butler, 1984). For example, many insects will overwinter in the egg stage, and hatch in the spring. Others may overwinter as larvae, emerging in the spring when the appropriate temperature and / or photoperiod cues are present. Although there may be several evolutionary as well as environmental mechanisms contributing to synchronous development, and contributing factors vary among species (see Butler, 1984), the result is that within a species, individuals in a “cohort,” or age class, will emerge during a very short interval, enhancing the probability of mating success. Synchrony occurs through two general paths. Larval hatching and growth may be timed such that all individuals within a cohort are of identical or nearly identical size throughout their development and emerge together. This is common in many caddisﬂies, such as the widely distributed Helicopsyche borealis (Resh et al., 1984) and the large limnephilid Dicosmoecus gilvipes (Lamberti and Resh, 1979). Alternatively, hatching and larval development may not be synchronous but instar-speciﬁc growth rates still may result in synchronized emergence (Lutz, 1974). Within a community, co-occurring species typically exhibit a variety of patterns of voltinism and synchrony. For example, 14 species of dragonﬂies in a temperate pond were found to have several different developmental patterns (Wissinger, 1988). Epitheca cynosura was univotine with synchronous development (Fig. 15A), while one of its congeners, E. princeps, also 18. Aquatic Insect Ecology FIGURE 14 Examples of life cycle synchrony of stream macroinvertebrates in response to temperature and photoperiod cues. In temperate North America, many species overwinter as larvae (left). Larval growth and maturation occurs at approximately the same rate for the entire cohort, such that emergence will also be synchronized. Other species overwinter in the egg stage, then begin larval development in the spring (right). These developmental patterns increase the probability that adults will ﬁnd mates, thus enhancing reproductive success. Larval instars indicated as: F, ﬁnal instar; F-1, penultimate instar; etc; and P, pupae. [From Anderson and Bourne, 1975.] 749 FIGURE 15 Developmental patterns of Epitheca spp and Libellula lydia illustrate that even closely related species from the same habitat can exhibit divergent life-history patterns. (A) Univoltine synchronized development. (B) Semivoltine synchronized development. (C) Semivoltine asynchronous development. [From Wissinger, 1988.] 750 A. E. Hershey and G. A. Lamberti FIGURE 16 The two major forms of insect development seen in aquatic insects. In gradual metamorphosis (left), or hemimetabolous development, immature forms are insectlike. Adult characteristics develop gradually through the larval instars, and wings inﬂate in the ﬁnal molt to the adult form. Insects demonstrating complete metamorphosis (right), or holometabolous development, have several larval instars that are less insectlike. Adult characteristics develop as a result of radical restructuring of the anatomy and physiology during the pupal instar. [From McCafferty, 1981.] was synchronized but was semivoltine (Fig. 15B). Yet another congener, E. simplicollis, was sometimes bivoltine. Asynchronous univoltine development was exhibited by Libellula lydia (Fig. 15C), while other species showed different combinations of synchrony, asynchrony, and mixed voltinism (Wissinger, 1988). Developmentally, most insects can be categorized as either “hemimetabolous” or “holometabolous” (Fig. 16). Collembola, considered “ametabolous,” have a developmental pattern that is distinctive from other insects and is phylogenetically primitive. This has led some entomologists to exclude it from the Insecta (Borror et al., 1989). The insect orders Ephemeroptera, Odonata, Heteroptera, and Plecoptera, are hemimetabolous. The aquatic immature forms, with few exceptions, respire using gills, and although they may differ considerably from adults, they are clearly insectlike (Fig. 16). The immature stages are referred to as nymphs or larvae. A newly hatched larva is referred to as a ﬁrst instar, and larval development proceeds through a series of instars. The number of instars varies widely among families, but variability also may occur at the species level depending on environmental conditions (Borror et al., 1989). Most aquatic insects are holometabolous, which means they undergo complete metamorphosis (Fig. 16). The immature instars are referred to as larvae and do not resemble the adults. Larval appearance is variable, but is usually not insectlike. Commonly larvae are described as wormlike, grublike, or caterpillarlike. A pupal stage that follows the last larval instar is characterized by development of the adult form in a protected habitat or cocoon. The insect does not feed during this stage, and appears to be quiescent. However, metabolic activity is very high as 18. Aquatic Insect Ecology larval tissues are reorganized to form the morphologically and physiologically different adult form. IV. INSECT-MEDIATED PROCESSES Insects are important consumers and important prey items in virtually all aquatic habitats, thus are intimately involved in the ﬂows of matter and energy that occur in these ecosystems. Insects also exert top – down controls on nutrient cycles, primary productivity, decomposition, and translocation of materials, and affect relative abundances of other consumers in lentic as well as lotic ecosystems. Speciﬁc relationships between insects and aquatic resources that are of particular interest include detritivory, grazing, and predation. Processes that integrate one or more of these speciﬁc relationships include secondary production and stream drift. All of these processes are shaped by physical habitat and life-history constraints. A. Detritivory Detritivory, the feeding on decaying organic matter, is a major insect-mediated process in both lentic and lotic systems, although it has received more attention in lotic studies. The relative importance of allochthonous versus autochthonous detritus in aquatic ecosystems is widely variable, and size of the water body is the major determining factor. Consumer resources in small lentic systems and low-order streams are typically dominated by allochthonous inputs, whereas lakes and middleorder streams (see Vannote et al., 1980) are usually dominated by autochthonous production. When detritus is plentiful, however, detritivory by insects is usually important to ecosystem function. Detritus forms a major habitat for aquatic insects in ponds and many wetlands. The coarseness of the detrital substrate provides habitat for large aquatic insects, such as odonate nymphs, which prey on small detritivorous chironomids, mayﬂies, and microcrustaceans. For some large insects, detritus serves as a food source, although availability of detritus seldom limits wetland insect abundance or production, and microbial decomposition appears to be more important than insect-mediated decomposition in many wetlands (Batzer and Wissinger, 1996). Detritus is divided into two major fractions: coarse particulate organic matter (CPOM — that portion 1 mm in size) and ﬁne particulate organic matter (FPOM — the portion that is 1 mm). Low-order streams are strongly inﬂuenced by the input of terrestrial organic matter from adjoining ecosystems. In temperate streams, much of this detritus enters as a seasonal (autumnal) pulse of leaves, which 751 is then processed in the stream by microbial and macroinvertebrate communities, and serves as the major energy source for many stream consumers (Fig. 9). Life cycles of many detritivorous insects are cued to this seasonal pulse of leaves such that they develop through the fall and winter when their food resource also is plentiful. Microbes, which colonize the detritus, form an integral part of the CPOM complex and are nutritionally important to shredders (Anderson and Sedell, 1979; Cummins and Klug, 1979). Dominant shredder taxa vary according to stream type and biogeography. Shredders are found in many of the aquatic insect orders (Table I). Some of the more conspicuous insect shredders are pteronarcid and capniid stoneﬂies, larval craneﬂies, and several families of caddisﬂies. Low-order streams often have 30 – 50% shredders in their macroinvertebrate communities (Fig. 10; see also Fig. 6 in Chapter 2), as was found in Oregon streams (Hawkins and Sedell, 1981), by virtue of their closed canopy and high input of CPOM per unit of stream surface (discussed in Vannote et al., 1980). In a ﬁrst-order mountain stream, Wallace et al. (1997) used an ecosystem-level experiment (litter exclusion by suspending a net over a small stream) to demonstrate that terrestrial litter supported the vast majority of insect secondary production in the stream. Interestingly, insects and other consumers occupying a moss microhabitat in the stream did not respond to litter exclusion, suggesting that moss inhabitants comprised a food web that was independent of that supported by terrestrial detritus (Wallace et al., 1997). In a different study, experimental reduction of shredders in a stream using an insecticide resulted in lower CPOM decomposition (20 – 40% that of a reference stream) and 5- to 15-fold lower FPOM transport (Wallace et al., 1982). Clearly, insects are of critical importance to CPOM processing in streams. Formation of FPOM from CPOM by shredders during their feeding is an important process. FPOM is also formed by several other mechanisms, and is dynamic in stream food webs. All consumers produce feces, which are an important component of FPOM (Shepard and Minshall, 1981; Hershey et al., 1996). Bacteria use DOM, thereby incorporating it into bacterial biomass, which itself constitutes a portion of the FPOM pool (e.g., Meyer, 1990). Bacteria produce extracellular materials, which are sloughed into the FPOM pool (e.g., Wotton, 1988). FPOM is produced by ﬂocculation of DOM due to a variety of physical and chemical processes (Dahm, 1981; Petersen, 1986; Wotton, 1988; Ward et al., 1994). The mass of FPOM usually exceeds that of nonwoody CPOM by about an order of magnitude (Wallace and Grubaugh, 1996) and turns over much more rapidly. Insect collector-ﬁlterers and 752 A. E. Hershey and G. A. Lamberti collector-gatherers that use this resource are important components of the macroinvertebrate community in all streams (Fig. 9; Vannote et al., 1980; Hawkins and Sedell, 1981). However, because FPOM is formed by many processes, it varies widely in quality; some FPOM is highly labile, but much is refractory (Newbold et al., 1983). Its retention in streams is also high, but varies considerably due to physical and / or chemical characteristics of the particles (Miller et al., 1998). The variety of collectors that use FPOM also is very high (Table I). DOM is generally considered to be the exclusive resource of stream microbes, which assimilate labile components of DOM into microbial biomass (see Lock et al., 1984; Meyer, 1990). However, larval blackﬂies also ingest DOM (Fig. 17, and see Wotton, 1976) and FIGURE 17 Ingestion of DOM by larval blackﬂies. Heads and bodies of black ﬂies which had been permitted to feed for 10- or 60-min on 0.2-m ﬁltered 14C-DOM were assayed separately for incorporation of labeled material. After 10 min, similar activity was seen on bodies of live and dead larvae, but higher activity was seen on heads of live larvae. This indicated that activity of live larvae enhanced accumulation of 14C from DOM onto the head. After 60 min, considerably more material was present in the body, indicating that labeled DOM was being packed into the gut during feeding. Each set of bars shows results from a separate chamber (two live chambers and one dead chamber). [From Hershey et al., 1996.] this process can be important to the size and quality of the FPOM pool downstream of blackﬂy aggregations (Hershey et al., 1996). DOM ingestion by blackﬂies includes some labile material, such as bacterial exopolymer (Couch et al., 1996), and thus such feeding may be a nontrivial source of nutrition for these aquatic insects. Although direct DOM ingestion by stream insects is not thoroughly understood, its importance is likely limited to a few taxonomic groups. Large woody debris (LWD) often forms an important pool of detritus in stream ecosystems (Anderson and Sedell, 1979; Bilby and Ward, 1989; Swanson et al., 1982; Van Sickle and Gregory, 1990). Wood may cover over 25% of the beds of small streams in old-growth forests (Anderson and Sedell, 1979). However, only very specialized insects use this material as food, and these likely do not ingest quantities that are signiﬁcant to wood decomposition (e.g., Wallace and Anderson, 1996). LWD is important as macroinvertebrate habitat. Benke et al. (1984) showed that much of the secondary production in a southeastern blackwater stream was associated with woody snags. In addition, the insect community on the snags was distinct from other substrates. LWD also serves as an important mechanism for retaining CPOM and FPOM resources that are used by insects (Benke et al., 1984). In addition, LWD is a major factor deﬁning pool and rifﬂe habitat within a stream, which determines many aspects of macroinvertebrate community structure. Wood itself has low nutritional value, but the microorganisms that coat submerged wood (e.g., algae, bacteria, fungi, protozoans) provide a rich food resource for macroinvertebrates. A few invertebrates (“wood gougers,” a special group of shredders) are adapted to feed directly on the wood and include the craneﬂy larva Lipsothrix and the elmid beetle Lara avara (Anderson and Sedell, 1979; Wallace and Anderson, 1996). These taxa bore into and feed strictly on wood and have very long life cycles (3 – 6 years) as a result of the low nutritive value of their food. Examining a piece of water-logged wood will reveal the tunnels of these borers and often the insects themselves. Wood is also used by some larval caddisﬂies for case construction. Heteroplecton californicum, a caddisﬂy that can be found in shallow pools, hollow out twigs to construct portable cases (Fig. 18). When the larvae move, their cases appear to be moving twigs, and the larvae themselves are not visible. B. Grazing Benthic primary production is a major process in virtually all shallow aquatic ecosystems (Lamberti, 1996). As a consequence, grazing insects functioning as primary consumers can be found in nearly all of those 18. Aquatic Insect Ecology 753 FIGURE 18 Wood cases of the caddisﬂy Heteroplectron californicum next to shredded alder leaves. Note opening chewed by caddisﬂies at upper end of twigs; larva resides inside the twig within a silk-lined tunnel. [Photograph by Jon Speir, reproduced from Anderson, 1976a.] ecosystems except, perhaps, where extreme physical conditions predominate (Lamberti and Moore, 1984). Aquatic primary producers include algae, rooted macrophytes, and bryophytes (mosses and liverworts). Benthic algae frequently are the dominant plants in streams and along rocky, wave-swept littoral zones, whereas rooted macrophytes become more important in the ﬁner sediments of lakes and large rivers where they are exploited for both habitat and food by insects. Fine sediments may also be covered with a layer of algal “epipelon,” which is readily grazed by insects. Mosses can be locally important, especially in streams, and because of their ﬁbrous texture and long-lived nature they provide excellent habitat for a variety of insects (Glime and Clemons, 1972; Suren and Winterbourn, 1992). Where mosses line the rock walls and spray zones of waterfalls, often a specialized insect fauna develops termed “torrenticolous” insects. In the discussion below, we will mostly concern ourselves with grazers of periphyton and macrophytes. Scrapers can be found in six orders of aquatic insects (Table I), and for several of those orders (Ephemeroptera, Trichoptera, and Lepidoptera) algal consumption is a major way of life. Grazers often possess elaborate mechanisms to remove periphyton (attached algae, microbes, and associated organic matter) from surfaces (Lamberti et al., 1987a). Many species of mayﬂies bear stout bristles on the labial or maxillary palps that are effective in removing periphyton from cracks and crevices. These bristles increase in length with successive instars, thereby allowing older larvae to remove larger particles while younger larvae remove smaller particles, effectively partitioning the resource by age. Many caddisﬂy larvae have robust or scoopshaped mandibles for removing or “spooning” periphyton into their mouths. Some limnephilid caddisﬂies, such as Dicosmoecus, also use their tarsal claws to gather algae into small piles for ingestion (Lamberti et al., 1987a). Orthocladius chironomids have stout mandibles lined with 4 – 5 teeth that wear down over time from scraping (Saponis, 1977); fortunately, molting periodically provides them with a new set of teeth. Even presumed shredders, such as craneﬂy larvae (Tipulidae), possess setae on the cutting surfaces of their mandibles that may help to collect algae from leaf surfaces, thereby allowing these insects to function as a facultative grazers (Pritchard, 1983). For shredders, the microorganisms, including algae, that grow on decaying leaves may be the most nutritional component of the leaves (Cummins et al., 1989). Grazing has been the focus of much study in streams (reviewed by Steinman, 1996, and Feminella and Hawkins, 1995, for ﬁeld studies and by Lamberti, 1993, for laboratory stream studies) and to a lesser extent in lakes (reviewed by Lodge, 1991; Newman, 1991). In streams, benthic algae will grow on virtually any substrate exposed to light, including on benthic insects themselves or their cases (e.g., Pringle, 1985; Hershey et al., 1988; Bergey and Resh, 1994). Many streams, however, have a low standing crop of benthic algae, sometimes barely perceptible to the naked eye. In comparison with obvious pools of dead organic matter (e.g., leaves and wood), it is tempting to dismiss the signiﬁcance of algae and thereby 754 A. E. Hershey and G. A. Lamberti grazing. However, a speciﬁc standing crop of algae can support a biomass of herbivores 10–20 greater than its own because of high algal turnover rates (McIntire, 1973), sometimes referred to as an “inverted trophic pyramid.” For example, Mayer and Likens (1987) found that an abundant caddisﬂy (Neophylax aniqua) consumed mostly periphyton in a small, heavily shaded New Hampshire stream whose energy base was previously thought to be almost totally detrital (Fisher and Likens, 1973). Still, periphyton is often limiting to the growth of insect grazers, and competition for algal resources has been demonstrated (e.g., McAuliffe, 1984; Lamberti et al., 1987b). Among the stream insects, caddisﬂies (Trichoptera) and mayﬂies (Ephemeroptera) tend to be highly conspicuous grazers although small chironomid midges (Diptera) may be equally important herbivores due to their ubiquity, high densities, and short generation times (Berg and Hellenthal, 1992). Insect grazers have the ability as individual species or groups of taxa to exert large effects on the abundance, productivity, and community structure of benthic algae. Often, a single species can exert primary control over periphyton. For example, Helicopsyche borealis caddisﬂies consumed 95% of the algal standing crop in a northern California stream (Lamberti and Resh, 1983b). Experimental exclusion of these caddisﬂies produced a mat of ﬁlamentous algae almost 30-mm thick (Fig. 19). In another study, the large caddisﬂy Dicosmoecus gilvipes consumed about 60% of benthic algal production, and FIGURE 19 most of the remaining algae were dislodged and washed away by the grazing activities of these “sloppy” feeders (Lamberti et al., 1987a). Furthermore, these large grazers can have negative effects on other benthic invertebrates, which they “bulldoze” and thereby displace as they harvest periphyton (Hawkins and Furnish, 1987; Lamberti et al., 1992). Tightly evolved associations between plants and herbivores are well documented in terrestrial ecosystems (Crawley, 1983). In aquatic ecosystems, however, there are few examples of host-speciﬁc associations between aquatic insects and benthic algae, for reasons that are still unclear (Gregory, 1983). One possible example is found in North American streams, where a mutualistic relationship may exist between the chironomid midge Cricotopus nostocicola and the blue-green alga Nostoc parmelioides (Brock, 1960). The ﬁrst-instar midge larva ﬁnds and enters a small, globular colony of Nostoc and begins feeding on Nostoc cells, in the process changing the colony morphology to an earlike form about 1 cm in diameter (Fig. 20). These darkgreen colonies often are quite obvious on rocks, and the single larva can be seen clearly within the colony. The midge grows and pupates within the colony, ﬁnally emerging as an adult. The association also may beneﬁt the colony, which has a higher photosynthetic rate when the ﬂy is present (Ward et al., 1985; Dodds, 1989). Amazingly, the midge will reattach the colony if it becomes dislodged from the substrate during a Effects of grazing by the caddisﬂy Helicopsyche borealis on algae in Big Sulphur Creek, California. Elevated but submersed platform in the foreground was used to exclude benthic caddisﬂy larvae; tiles in midground and rocks in background were freely grazed by caddisﬂies at densities 1000 / m2. Direction of water ﬂow is from right to left. (A) Beginning of experiment. (B) After 5 weeks elapsed. Note dense growth of ﬁlamentous algae on ungrazed platform tiles. [From Lamberti and Resh, 1983b.] 18. Aquatic Insect Ecology FIGURE 20 Colonies of the blue-green alga Nostoc parmelioides containing the midge larva Cricotopus nostocicola. Upper panel: Nostoc “ear” form with midge larva in left side of colony; Lower panel: Nostoc colonies attached to a rock. Bar 5 mm. [Photographs by Louis Nelson, reproduced from Ward et al., 1985.] 755 756 A. E. Hershey and G. A. Lamberti disturbance (Brock, 1960; Dodds and Marra, 1989), which doubtless beneﬁts both the colony and the larva. Another example, although probably not mutualistic, is the association between the microcaddis ﬂy Dibusa angata and the freshwater red alga Lemanea australis (Resh and Houp, 1986). Larval instars 1 – 4 of Dibusa are found among the basal holdfasts of Lemanea, where they consume epiphytic diatoms. Fifth-instar larvae construct cases made of Lemanea and consume only Lemanea tissue. There is no known beneﬁt of this association to the alga. Many insects consume living macrophyte tissue, a process that generally has been understudied and underappreciated (see review by Lodge, 1991). Unlike for algae, there appear to be a number of close associations between insects and aquatic macrophytes, perhaps because the large size of rooted plants makes specialization more feasible than for microscopic algae. The major insect orders involved in macrophyte grazing are the Trichoptera, Lepidoptera, Coleoptera, and Diptera. It is perhaps not surprising that rooted hydrophytes are eaten by the more advanced insect orders, as their terrestrial insect relatives are also mainly plant eaters. Indeed, it has been speculated that over evolutionary time terrestrial insects followed their host plants as they gradually invaded the water to eventually become true hydrophytes. Furthermore, parasitoids of those insects may have followed their hosts, as evidenced by the hymenopterans that now parasitize aquatic lepidopterans. Insect grazers can consume up to 100% of the standing stock of macrophytes, although the damage is usually much less than that (Lodge, 1991). It is more common to see the grazing scars of insects, such as beetles and moths, on the surfaces of ﬂoating leaves and other plant structures. Lepidopterans are known to feed on aquatic plants in numerous ways, including leaf mining, stem or root boring, foliage feeding, and ﬂower or seed consumption (Lange, 1996). Coleopterans likewise exhibit a wide variety of feeding modes; leaf beetles (Chrysomelidae) are particularly common herbivores of water lillies, where their larval galleries are obvious on the ﬂoating leaves (Cronin et al., 1998). Even though beetles may not consume large amounts of leaf tissue, their feeding activity can increase nutrient uptake and primary production by the plant and accelerate decoposition (Wallace and O’Hop, 1985). C. Predator – Prey Interactions Many aquatic insects function as predators, and predators are found in nearly all of the major groups of aquatic insects. A few groups, notably the Odonata, aquatic Heteroptera, and Megaloptera, are strictly predators, whereas other groups are more heteroge- neous in their trophic afﬁliations, but may have important subdivisions that are predators. For example, among the Plecoptera, most members of the families Perlidae, Perlodidae, and Chloroperlidae are primarily predators (Stewart and Harper, 1996). Within the Trichoptera, members of the family Rhyacophilidae are free-roaming predators, which may inﬂict particular damage to larval black ﬂy populations (Wheeler, 1994). Many of the sedentary, net-spinning Hydropsychidae are facultative predators (Benke and Wallace, 1980), and some larval Limnephilidae become opportunistic predators in later instars (Anderson, 1976a). Many Coleoptera and Heteroptera function as aquatic predators in both the immature and adult forms. The role of predators in controlling the distribution and abundance of prey is a central question in aquatic ecology, and their role in determining aquatic insect communities also has received considerable attention. Predator and prey taxa are subjected to different physical constraints in various lentic and lotic ecosystems (see above), and these physical constraints interact with the predation process to produce different community types. Because predator – prey interactions do not always occur at the same spatial and temporal scales as abiotic factors that affect distribution and abundance of organisms, it is sometimes difﬁcult to extrapolate predator – prey observations to community composition (see Peckarsky et al., 1997). Predators acquire prey using different behavioral strategies (see Peckarsky, 1982). Many predators, such as stoneﬂies, actively search for their prey (“hunters”). Ambush or “sit-and-wait” predators wait until a prey item is within range before striking. Odonate larvae often use this strategy. Once the prey is captured predators typically either engulf their prey intact, in bites, or may feed only on the internal ﬂuids. Fluid feeders use specialized piercing (haustellate) mouthparts for injecting enzymes into their prey and sucking out the partially digested body. Clearly the relative sizes of predators and prey, as well as the mode of feeding used by the predator, are important mechanical constraints governing predator – prey interactions. Engulﬁng predators are limited to prey small enough to be subdued and swallowed intact (see Hershey, 1987), whereas piercing predators have access to a broader variety of prey sizes that are large enough to be handled and to accommodate the piercing mouthparts. In a synthesis of predator effects in lentic communities, Wellborn et al. (1996) characterized control of community structure as falling along a gradient of physical and biotic control, with two major transitions (Fig. 21). Temporary ponds and lakes provide two extremes along a habitat size and permanence gradient, and two major transition thresholds occur along this perma- 18. Aquatic Insect Ecology 757 FIGURE 21 Aquatic communities are controlled by both biotic and abiotic factors. Two major transitions involving predator control of community structure are conceptualized to occur (Wellborn et al., 1996). A permanence transition reﬂects when the wet phase of the hydroperiod is sufﬁciently long to support longer-lived predatory insects. A predator transition occurs when habitats contain ﬁsh, which then eliminate many predatory insects. [From Wellborn et al., 1996.] nence gradient, which provide the template for interaction of physical factors and predator control of insect communities (Fig. 21; Wellborn et al., 1996). A permanence transition (Fig. 21) along the gradient controls predator presence or absence. In highly ephemeral habitats, most predatory insects are excluded (Batzer and Wissinger, 1996; Wellborn et al., 1996, and see above). As a result, predator – prey interactions are relatively unimportant and the insect community is controlled by physical factors (Wellborn et al., 1996), primarily drought cycles (Hershey et al., 1998, 1999). Once the permanence transition is crossed, predatory insects become abundant and control various aspects of prey community composition (e.g., Batzer and Resh, 1996). These habitats tend to have the highest diversity of insect species (see Batzer and Wissinger, 1996). A predator transition occurs when habitats are large enough and deep enough to support ﬁsh (Fig. 21; Wellborn et al., 1996). Fish are size-selective predators, typically eliminating large predatory taxa, which have a major impact on prey diversity and community structure. For example, in an experimental manipulation of bluegill and pumkinseed sunﬁsh, mean invertebrate size decreased as ﬁsh density increased (Mittlebach, 1988). But ﬁsh may control insect prey density without dramatically affecting community composition (Bohanan and Johnson, 1983). Fish and predatory insects interact to increase the effect on prey compared to when either is present alone (Johnson et al., 1996). But ﬁsh and large predatory insects such as dragonﬂies have different strategies for capturing prey (i.e., pursuit versus ambush). These alternative strategies provide contrasting FIGURE 22 A ﬁsh exclusion experiment conducted in dense macrophytes of a cooling water reservoir in South Carolina did not show signiﬁcant reduction of macroinvertebrates in the areas where no ﬁsh were present. NP, no predatory ﬁsh; and P, predatory ﬁsh present. [Reprinted from Thorp and Bergey, 1981.] selective environments for prey; McPeek et al. (1996) have shown that in lakes where dragonﬂy predation is important, Enallagma damselﬁes have larger caudal lamellae which enhance swimming speed, whereas in ﬁsh lakes, Enallagma caudal lamellae are smaller, associated with a cryptic strategy for predator avoidance. Habitat heterogeneity moderates the effects of ﬁsh predation on insect communities (Gilinsky, 1984; Hershey, 1985); and in very dense macrophytes, ﬁsh are not important in controlling insect abundance or species composition (Fig. 22; Thorp and Bergey, 1981; Crowder and Cooper, 1982). Thorp (1986) concluded that although ﬁsh native to a system may control density or biomass of benthic insects, they have little impact on such community structure characteristics as diversity or species composition. However, historical presence or absence of ﬁsh within a community has a major impact on community structure (Fig. 21; Thorp, 1986; Wellborn et al., 1996). Similar to lentic studies of predation, predator – prey interactions involving stream insects also have been studied from two major perspectives: (1) the potential for their communities to be structured by ﬁsh predation; and (2) as predators interacting with, and possibly controlling, other macroinvertebrates. As in lentic systems, experimental studies of ﬁsh predation have not always led to the same conclusions. In an experimental removal of trout from a rocky mountain stream, Allan (1982) found no difference in macroinvertebrate density or diversity where ﬁsh were 758 A. E. Hershey and G. A. Lamberti FIGURE 23 Simpliﬁed food web found in the Eel River, California (Power, 1990) where cascading effects have been demonstrated. Large ﬁsh control small ﬁsh that feed preferentially on predatory insects. This releases tube-building chironomids from control by insects, and dramatically affects the appearance of the algal mat. [Reprinted from Power, 1990.] removed. Similarly, Reice (1983) found no evidence of trout control of any aspect of the stream insect communities that he studied. However, working in California’s Eel River, Power (1990) has shown that predatory ﬁsh control predatory invertebrates (especially damselﬂies) which, in turn, control the abundance of larval chironomids, affecting both algal biomass and the physical appearance of the algal mat in the river (Fig. 23). Her study is an example of food web control via cascading trophic interactions (sensu Carpenter et al., 1985), which have been well studied for pelagic systems. Although it is not clear that trophic cascades are common in stream ecosystems, it is clear that ﬁsh predation does not control insect abundance in all streams. Regardless of their direct effects on prey density or community structure, ﬁsh have important indirect effects on prey in lotic ecosystems (e.g., Wooster, 1994; Wallace and Webster, 1996). Because of the threat of ﬁsh predation, many insects feed more actively on rock surfaces at night and are more prone to drift at night in search of better food patches (Fig. 11; Kohler, 1985). It appears that stream ﬁshes are usually limited by their macroinvertebrate (especially insect) prey resources. This is suggested by Allen’s paradox (Allen, 1951), but the strongest evidence for this comes from experimental fertilization studies that result in increased ﬁsh production (e.g., Stockner and Shortreed, 1978; Deegan and Peterson, 1992; Richardson, 1993). The role of predatory macroinvertebrates in streams has been studied using a combination of gut analyses, laboratory stream microcosms, and in-stream experiments. Numerous studies of gut contents have yielded a wealth of information on which invertebrate species function as predators and which species or groups serve as their major prey. However, insight into their role in structuring stream communities has come primarily from experimental studies. Although notable exceptions exist (e.g., Power, 1990): invertebrate predators probably do not control the abundances of their prey in most stream ecosystems because stream communities are very open to immigration and emigration of both predators and prey (see Cooper et al., 1990). However, indirect effects appear to be of considerable consequence. Behavioral avoidance of predators by prey may affect prey spatial distribution within the substrate. For example, predatory stoneﬂies affect mayﬂy distributions because mayﬂies will drift to avoid contact with them (Peckarsky and Dodson, 1980). When contact occurs, Baetis may further minimize the threat of predation by projecting their cerci forward in a “scorpionlike” posture that renders them more difﬁcult for the stoneﬂy to handle (Fig. 24; Peckarsky, 1980). Prey growth rates and / or fecundity can also be altered by the presence of predators, even if survivorship is not altered (Peckarsky et al., 1993; Scrimgeour and Culp, 1994), and shredding activity can be reduced (Malmqvist, 1993). Alteration of growth rates may or may not have implications for prey demographics, depending on whether adult fecundity is a function of body size, as it is in Baetis mayﬂies but not in Enallagma damselﬂies (McPeek and Peckarsky, 1998). Prey also respond to predators through morphological adaptation. Ephemerella are armored against stoneﬂy predators (Peckarsky, 1996). In stream habitats where Hydra are very abundant, long hairs on the midge larva Cricotopus sylvestris greatly decreases predation risk relative to short haired species, such as C. bicinctus, as well as to individuals of C. sylvestris whose hairs have been experimentally shortened (Fig. 25; Hershey and Dodson, 1987). A conceptual model by Peckarsky (1996) suggests that resource acquisition modes may account for investment in morphological versus behavioral strategies for predator avoidance. These examples show that predator – prey interactions involving insects are diverse, and variously may alter prey abundances, distributions, and behavior, and may have been important in selecting for morphological adaptations for predator defense. D. Insects as Conduits for Energy Flow in Aquatic Food Webs Insects provide a critical linkage in energy ﬂow from microbial to vertebrate populations in aquatic ecosystems. Food web analyses have been used to understand these linkages, and thereby integrate organic-matter processing with community interactions (e.g., Closs and Lake, 1994; Benke and Wallace, 1997). The goals of food web studies for a particular system are to identify organic matter sources for the various 18. Aquatic Insect Ecology 759 FIGURE 25 Long hairs provide C. sylvestris with protection against Hydra, compared to short haired C. bicinctus or C. sylvestris with experimentally shortened hairs. Hydra is a freshwater cnidarian that preys on chironomids and other aquatic invertebrates. [From Hershey and Dodson, 1987.] FIGURE 24 Mayﬂies exhibit a defensive “scorpion” posture when attacked by predatory stoneﬂies. [From Peckarsky, 1980.] consumers and to elucidate the trophic structure of the web. In most aquatic ecosystems, either three or four trophic levels are present including: (1) primary producers and detritus; (2) primary consumers, including detritivores (shredders and collectors) and grazers (scrapers); (3) secondary consumers (predators); and (4) tertiary consumers (vertebrate predators that consume invertebrate predators). These designations are oversimpliﬁcations because omnivory is common, such that many consumers occupy more than one trophic level (see Lamberti, 1996). In stream ecosystems, the relative importance of the detritivore food chain compared to the grazer food chain is a matter of some debate (Minshall, 1978), but this varies as a function of stream order and riparian conditions (Hawkins and Sedell, 1981; Cummins et al., 1989; Gregory et al., 1991). The food web in any particular aquatic ecosystem will reﬂect the combination of factors altering resource and invertebrate abundance and distribution. Thus, wide variation in food web structure occurs. Gut analyses are often used to delineate trophic structure (e.g., Hershey and Peterson, 1996). However, some food items are difﬁcult or impossible to recognize, while others may be overlooked due to rarity or 760 A. E. Hershey and G. A. Lamberti temporal variability. The gut analysis approach is impractical for studies involving ﬂuid-feeding predators because sclerotized parts are not ingested, thus prey identity cannot be determined. Recently, stable isotopes have been used to study food-web relationships in both lotic and lentic systems (e. g., Fry and Sherr, 1984; Minagawa and Wada, 1984; Peterson and Fry, 1987; Fry, 1991; Peterson et al., 1993; Junger and Planas, 1994; Schuldt and Hershey, 1995; Whitledge and Rabeni, 1997; Vander Zanden and Rasmussen, 1999). One useful aspect of carbon stable isotopes is that the various organic matter sources for consumers often have different relative abundances of the two stable isotopes of carbon, 13C and 12C. Because the relative abundances of these isotopes change only slightly as the organic matter is processed by various consumers, the difference between the ratio 13C to 12 C in a consumer and a standard (or value, expressed as parts per thousand, or per mil) can be used to infer the consumer’s food source. Recently, 15N has been used extensively in enrichment studies of lotic and lentic ecosystems to serve as a tracer of consumer food sources as well as nitrogen-distribution through the system (Kline et al., 1990; Hershey et al., 1993; Wolheim et al., 1999). 15N also is used in trophic studies. 15N and 14 N change as food is processed by consumers because the various metabolic processes use, or fractionate, these isotopes differently. The net result is that with each trophic level, consumers become enriched (by about 3 per mil) in 15N relative to 14N, as 15N is retained in the tissues and 14N is excreted. Thus, consumers in a food web can generally be assigned to a trophic level even if their precise food resource is not known (see Fry, 1991; Peterson et al., 1993). However, the del 15N value of the base of the web may vary between systems, thus should be measured for each system where del 15N is being studied in consumers (Vander Zanden and Rasmussen, 1999). When organic matter sources in a stream are isotopically distinct, a combination of 13C and 15N analyses can yield considerable insight into a stream food web. Such an analysis was performed for the dominant consumers and their food resources of Lookout Creek in Oregon (Fig. 26; Fry, 1991). In Lookout Creek, the 15 N values revealed that macroinvertebrates were trophically distinct from two predatory vertebrates studied, whereas the 13C values showed that consumers relied more heavily on stream algae than on terrestrial detritus. The macroinvertebrate scrapers studied (the snail Juga and the caddisﬂy Dicosmoecus gilvipes) appeared to consume algae, whereas the shredding caddisﬂy Heteroplectron californicum apparently ate terrestrial detritus. The stoneﬂy Calineuria californica, a presumed predator, occupied a trophic level close to FIGURE 26 Stable isotope cross-plot of selected members of Lookout Creek, Oregon, food web. Animals horizontally positioned over other sources (i.e., have similar 13C) are inferred to consume those materials. Animals, displaced vertically 2 – 4 15N units upward are inferred to belong to the next higher trophic level in the food web. [Modiﬁed from Fry, 1991.] primary consumers and thus may have consumed some plant matter along with small invertebrates that were not measured. The endosymbiotic midge larva Cricotopus nostocicola (described earlier) was positioned over its algal host Nostoc parmeliodes. Cutthroat trout, Oncorhynchus clarki, and Paciﬁc giant salamanders, Dicamptodon ensatus, consumed invertebrates, as shown by their vertical positioning over the 13C of their food and a 3 – 4 per mil difference in 15N (Fig. 26). Gut contents of trout revealed a variety of invertebrates whereas salamanders had exclusively snails in their guts (R. Wildman, Oregon State University, personal communication). Use of stable isotopes to trace organic matter sources for insect consumers has been a particularly powerful tool in streams that support runs of anadromous salmon. Paciﬁc salmon die after spawning, and the carcasses provide nutrients that are used by stream algae (e.g., Brickell and Goering, 1970; Mathisen et al., 1988), which are then fed upon by grazers (e.g., Richey et al., 1975; Kline et al., 1990). Salmon carcasses also serve as a high quality source of CPOM for stream shredders (Kline et al., 1990). Stable isotope technology provides a means to quantify the importance of salmon carcasses to the stream food web. Salmon returning to streams to spawn have an isotopic signature that reﬂects their position in the marine food web. They are enriched in 15N, reﬂecting their predation on marine ﬁshes, and enriched in 13C reﬂecting their dependence on marine sources of carbon. By comparing the isotopic signature of algae and insects in reaches 18. Aquatic Insect Ecology 761 TABLE IV 15N and 13C values for food-web components and source materials in a Lake Superior tributary stream 15 Material Upstream Salmon CPOM Hydropsyche Heptageniidae 0.2 3.1 1.6 13 N Downstream Upstream 8.9 4.1 4.0 28.5 29.1 29.2 C Downstream 22.8 28.9 28.9 Salmon spawning occurs in the downstream reach of the river, but not the upstream reach. The stable isotope value shows that salmon-derived N, but not salmon-derived C, is incorporated into collector (Hydropsyche) and especially grazer (Heptageniidae) food-web components. [From Schuldt and Hershey, 1995.] not used by salmon, to reaches where salmon spawn, the proportion of marine-derived N and C (from salmon) in the insect community can be traced. Thus, Kline et al. (1990) showed that at least 90% of the N in periphyton from Sashin Creek, in southeastern Alaska, was derived from spawning salmon or salmon eggs. Herbivores contained 50 – 100% salmon-derived N. In a Lake Superior tributary stream, the 15N signature of heptageniid grazers suggested about a 30% reliance on salmon-derived N from Lake Superior (Table IV; Schuldt and Hershey, 1995). V. SECONDARY PRODUCTION Secondary production is the amount of animal biomass that is produced in a given area over a given time period, usually expressed in g / m2 per year. For a cohort, this measurement integrates both individual growth rate and survivorship of individuals within the cohort (see Benke, 1984, 1996). In a food-web context, secondary production is the summation of the amount of biomass that is available to be consumed by the next trophic level over some period of time. Thus, an estimate of secondary production expresses energy or material ﬂow through the consumer components of the ecosystem, and is useful for identifying major pathways of energy ﬂow in an ecosystem (e.g., Smock and Roeding, 1986; Benke and Wallace, 1997). Estimation of secondary production often provides insights that are not apparent from biomass or abundance measurements alone. This is especially true for organisms of small body size if they exhibit rapid growth rates. For example, Benke (1998) found extremely high secondary production of snag-dwelling chironomids in a high-order blackwater river despite their low biomass. This was due to very high biomass turnover rates of these small-bodied organisms (Benke, 1998). In Sycamore Creek, Arizona, macroinvertebrate secondary production, which was primarily insect production, was also extremely high despite fairly low biomass (Fisher and Gray, 1983). This high rate of production reﬂected a long growing season, small adult size, very rapid life cycles, and abundant (if low quality) FPOM (Fisher and Gray, 1983). But, even for large bodied insects measurement of production may provide surprising insights. O’Hop et al. (1984) showed that production of the detritivorous stoneﬂy Peltoperla maria was similar in a disturbed and undisturbed stream despite more than twofold differences in abundances, reﬂecting faster growth and higher density of larger individuals in the disturbed stream. Although stream secondary production is very often dominated by insects, crustaceans and molluscs may also be quite important (see Waters and Hokenstrom, 1980; Fisher and Gray, 1983). In a recent study of production in a neotropical stream, Ramírez and Pringle (1998) show that insect production is low despite rapid growth rates. They attribute this low production to a combination of high abundance of non-insect macroconsumers and scouring of resources by ﬂooding. Because different habitats vary markedly in abundances and community composition of their insect fauna, calculation of secondary production on a habitat-speciﬁc basis is needed to reveal the spatial complexity of insect-mediated pathways of energy ﬂow in aquatic ecosystems. In addition, failure to consider some habitats may grossly underestimate secondary production. For example, Smock et al. (1992) showed that debris dams were very important compared to other habitats in one low-gradient ﬁrst-order stream, but that the ﬂoodplain was most productive in another stream. In addition, the vertical extent of the hyporheic zone in the two streams accounted for a large difference in secondary production between the two systems (Smock et al., 1992). Snag habitats are very important 762 A. E. Hershey and G. A. Lamberti sites of secondary production in higher order rivers, reﬂecting the success of both mayﬂies and chironomids in utilizing the amorphous detritus associated with that habitat (Benke and Jacobi, 1994; Benke, 1998). Fisheries managers often use secondary production to assess the capacity of a stream reach to support ﬁsh. In many cases, there does not appear to be enough invertebrate production to support the observed ﬁsh production, a problem referred to as Allen’s paradox (Allen, 1951). This may be due to underestimates of the production-to-biomass ratio (P / B) for individual species (Benke, 1996), or perhaps failure to consider the production of chironomid midges (Huryn and Wallace, 1986; Berg and Hellenthal, 1992). Recently, Huryn (1996) has shown that trout can consume a very large proportion of the benthic macroinvertebrates (mostly insects) in a stream. Although conceptually very useful, secondary production is time-consuming and expensive to measure at the community level. The varied techniques for measuring secondary production, reviewed by Benke (1984, 1996), involve sampling efforts that quantify consumer standing stocks at several points in the growing season, at least a trophic level understanding of the various consumers, and some knowledge of life histories of the dominant consumer components (Table V). However, in the process of measurement, considerable insight is gained into the insect community, which sheds light on which consumers play key roles in the various aquatic ecosystem processes. TABLE V Methods for measuring secondary production of stream macroinvertebrates. Modiﬁed from Benke, 1984, 1996, where secondary production methods are reviewed. [Modiﬁed from Hershey and Lamberti, 1998] Method Data needed Comments Other references Allen curve Density (No. / m2) for several dates spanning development of a cohort; mean individual mass for each date [often obtained from length weight regressions (Smock, 1980)]. Density (No. / m2) for several dates spanning development of a cohort; mean individual mass for each date [often obtained from length weight regressions (Smock, 1980)]. Density (No. / m2) for several dates spanning development of a cohort; mean individual biomass for each date [often obtained from length weight regressions (Smock, 1980)]. Growth rate; mean biomass Graphical presentation of density versus mean individual mass. The area under the curve corresponds to the secondary production of a population. This method was developed prior to the widespread availability of electronic calculators and computers. Production (P) between sampling dates is calculated as the product of mean density (N) and change in mean individual mass (W): P NW. Annual production is the summation of these incremental measurements for a year. Production lost between sampling dates is the product of the change in density and the mean individual mass: P WN. Annual production is the sum of all production losses over a year. Production between sampling dates is the product of growth rate and mean biomass, where growth rate is determined in laboratory experiments, or from ﬁeld samples of distinct cohorts, and biomass is determined from ﬁeld samples: P g B. Growth is temperature and size-dependent, so accuracy of this method depends on making measurements under the appropriate conditions. Annual production is: P (gi)(Bi), where the I’s are the production intervals. This method is widely used and does not depend on the presence of distinct cohorts in ﬁeld populations. The mean size – frequency distribution, which is skewed toward smaller size categories due to mortality, represents an average cohort. For populations where distinct cohorts are known, this method gives a similar result as the increment summation method. P WNi, where i number of size classes. This estimate of secondary production needs to corrected for the CPI: (multiply P by 365 / CPI) Allen, 1951 Increment-summation Removal-summation Instantaneous growth Size frequency Mean annual density; size distribution of individuals throughout year; mean mass for each size category; cohort production interval (mean development time from hatching to emergence). Waters, 1977; Waters and Crawford, 1973 Waters, 1977 Ross and Wallace, 1981; Hauer and Benke, 1987 Benke, 1979 18. Aquatic Insect Ecology VI. EFFECTS OF LAND USE ON AQUATIC INSECT COMMUNITIES Because aquatic insects are sensitive to physical and chemical conditions in their environment as well as to top-down and bottom-up biotic controls, their communities also reﬂect changes in land use on both geological and ecological time scales and as well as on multiple spatial scales. For both lotic and lentic ecosystems, nutrient loading from either nonpoint or point sources, changes in hydrology, timber harvesting, construction, and riparian or shoreline development are among the most important land-use alterations that affect insects. Lentic insect responses to land-use disturbances are seen through indirect effects of physical and chemical changes in littoral macrophyte zones and chemical changes in profundal zones. Nutrient loading results from many land-use changes, including nutrients associated with sediments derived from construction and tillage; fertilizer from agriculture, suburban and urban runoff; and wastewater and industrial inputs (Carpenter et al., 1998). These inputs are derived from both inﬂow streams and land uses on lake shorelines themselves. Nutrient inputs enhance macrophyte growth and / or phytoplankton productivity. Since macrophytes support a high diversity of aquatic insects as well as affording refuge from ﬁsh predators (Thorp and Bergey, 1981; Crowder and Cooper, 1982; Gilinsky, 1984; Hershey, 1985, and see Fig. 22;), land-use changes altering macrophytes have strong indirect effects on littoral insects. However, if macrophyte growth becomes too dense, anoxia may be a problem, especially at the sediment / water interface. Low dissolved oxygen dramatically affects insect community structure (see Section II.B). Sedimentation may negatively affect macrophytes, thereby indirectly negatively affecting insect abundance and diversity. Nutrient inputs also enhance phytoplankton production (e. g., Schindler, 1977; Axler and Reuter, 1996), and at high level this often leads to bottom-water anoxia and associated low insect diversity and changes in insect community structure (see Hilsenhoff, 1966; Saether, 1979, and Section II.B). Invertebrate populations in wetlands also are affected by vegetational and water management practices and by water quality and plant physical structure (see Ferenc et al., 1999). However, Wolf et al. (1999) found little direct effect of land use on wetland invertebrates in urban playa wetlands, although they did ﬁnd invertebrate abundances were positively associated with macrophyte growth. Similar to littoral areas of lakes, macrophyte beds respond to land use surrounding the wetlands. Water-level ﬂuctuation in wetlands, often associated with land-use changes, has considerable inﬂuence on invertebrate community structure and the bal- 763 ance between insect and noninsect invertebrates (Anderson et al., 1999; Hershey et al., 1999). Wetland insects and other invertebrates are a major source of food for migratory and resident waterfowl (Swanson, 1984 / 1985; Martin et al., 1951; Tester, 1995), thus wetland loss and associated loss of invertebrate food resources and habitat have had a major impact on wetland bird species (Richardson, 1979). Hynes (1975) ﬁrst articulated the importance of employing a landscape perspective for understanding stream ecosystem structure and function. The realization of this linkage between the stream and its terrestrial setting was a critical development in stream ecosystem theory (Minshall et al., 1985) and aquatic insects have been central to this perspective because of their role in processing allochthonous inputs. This aquatic – terrestrial relationship is well appreciated qualitatively, but poorly understood quantitatively. Local geomorphology, vegetation, and climate determine the quality and quantity of organic matter entering a stream. These processes and disturbance history at a site determine the spatial and temporal patterns of riparian zone characteristics that dictate the physical characteristics of the stream habitat (Gregory et al., 1991). Thus, riparian processes and natural disturbances, or disturbances due to changes in land use, will alter insect habitats in the stream channel. These disturbances operate over temporal scales ranging from geologic alterations of landform, to ecological succession of the riparian forest, as well as over spatial scales spanning watersheds, to local patchiness of algal growth on individual rocks within the channel (Gregory et al., 1991). Aquatic insect abundance and diversity responds to all of these perturbations. Aquatic insect communities reﬂect land use within the watershed because changes in land use alter stream habitat and water quality (Gregory et al., 1987; Chauvet and Décamps, 1989; Merritt and Lawson, 1992). As discussed previously, insect communities are sensitive to predominant sources of organic matter, dictated largely by stream order, local geomorphology, and riparian vegetation (Vannote et al., 1980; Naiman et al., 1987; Naiman and Anderson, 1996). Biogeography (Resh, 1992; Hershey et al., 1995b) and water quality factors (Richards and Minshall, 1992; Lamberti and Berg, 1995) also constrain insect communities. Use of geographic information systems has facilitated landscape-level analyses and land-use studies of stream insect communities. In Minnesota north shore tributaries to Lake Superior, insect community structure and substrate characteristics within a stream are correlated with agricultural and residential land uses in the watersheds (Richards and Host, 1994). The types of alterations seen will depend on the nature and intensity 764 A. E. Hershey and G. A. Lamberti FIGURE 27 Theoretical relationship between macroinvertebrate species diversity and frequency of disturbance (e.g., ﬂoods) in streams ecosystems [redrawn from Ward and Stanford (1983) after Connell (1978)], with examples of macroinvertebrates that can be found under different conditions in North America. Rarely disturbed streams often contain large, long-lived taxa such as (top to bottom): Neohermes hellgrammite, Argia damselﬂy, and Dicosmoecus caddisﬂy. Streams with intermediate levels of disturbance usually contain a diverse fauna including (clockwise from upper left): Calineuria stoneﬂy, Tipula craneﬂy, Drunella mayﬂy, Glossosoma caddisﬂy, Juga snail, Hydropsyche caddisﬂy, and Simulium black ﬂy. Streams that are disturbed frequently may contain small-bodied taxa with short life cycles or high reproductive rates such as (clockwise from upper left): Helicopsyche caddisﬂies, Baetis mayﬂies, and chironomid midges. of the land-use change (Cairns, 1990), but local geomorphology modiﬁes some of the effects of land-use change, as well as constraining land cover and dictating what land uses might occur in a watershed (McIntire and Colby, 1978; Naiman et al., 1993). In an extensive study of a 45-catchment river basin in central Michigan, Richards et al. (1996) found that geology and land use were approximatlely of equal importance in determining invertebrate communities in streams. Land-use changes that alter large woody debris (LWD) have large impacts on insect communities (e.g., Fig. 27). Logging is one of the major such land uses. Logging has greatly altered the size and amount of LWD that enters stream channels (Sullivan et al., 1987). Removal of riparian trees changes inputs of LWD for centuries, altering substrate stability and insect habitat (Naiman and Anderson, 1996); but recent changes in forest practices now offer some protection to the riparian zone from excessive harvest (Gregory and Ashkenas, 1990). Streams that lack LWD sources due to past logging are candidates for rehabilitation, which now emphasizes the input of logs in natural volumes and arrangements to improve stream habitat and to augment the food base. In addition to removing LWD and thereby altering detrital inputs and channel structure, logging also removes vegetative cover within the watershed. This results in increased sedimentation into the stream, reduced inﬁltration which decreases groundwater recharge, and an increased runoff which increases the magnitude of stream discharge change following storm events (e.g., Gregory et al., 1991; Naiman and Anderson, 1996). Other land uses, including urban, agricultural, and industrial development may follow timber harvest. 18. Aquatic Insect Ecology Agriculture has a large effect on sedimentation in a stream reach (Allan et al., 1997), but stream buffers are more important than the whole catchment in determining sediment-related habitat variables in agricultural basins (Richards et al., 1996). Road construction is usually associated with all types of human land use. Road construction often destabilizes hillsides, leading to landslides and sometimes debris ﬂows in stream channels downslope (Lamberti et al., 1991; Swanson et al., 1987), which constitute a major disturbance to the stream ecosystem (see below). These changes in land use dramatically alter watershed hydrology, and hence stream hydrology. As discussed previously, insect communities are strongly affected by stream hydrology, but are also sensitive to the various forms of pollution that result from many land uses (see Richards et al., 1993). Thus, insect communities are intimately linked to land use in the watershed as well as to local conditions in the stream channel. VII. ROLE OF DISTURBANCE A “disturbance” has been deﬁned as any discrete event that disrupts population, community, or ecosystem structure, usually by changing resource abundance or the physical environment (Sousa, 1984; Resh et al., 1988). Many types of natural and human-induced disturbance can disrupt the habitat, resources, or population densities of aquatic insects. Large-scale natural disturbances that can affect insects include: (1) hydrologic events such as ﬂoods, ice, or wave action that scour or remove substrates; (2) desiccation events such as droughts that lead to drying of aquatic habitats; (3) watershed disturbances, such as wildﬁre, that can disrupt nutrient and sediment inputs to streams and lakes for decades; and (4) seasonal events, such as summer or winter oxygen depletion, that can lead to insect mortality and major changes in community structure (see Fig. 27). Naturally, many small-scale disturbances can also affect local insect populations. Human-related disturbances of aquatic insect habitats are at least as important and include nominally: (1) point and nonpoint source pollutants that enter aquatic ecosystems from the land, connected water, or the atmosphere; (2) water withdrawal, diversion, and storage; (3) modiﬁcations of river channel geomorphology and destruction of wetlands; (4) watershed disruptions, such as logging and other land uses; and (5) introduction and establishment of exotic species. All of these mechanisms, singly and in combination, can alter population densities and community structure of insects, sometimes for decades or centuries as in the case of watershed land-use change. In general, however, natural disturbances tend 765 to be episodic and, if the habitat returns to its orginal state, insect communities recover quickly. Human disturbance, by contrast, tends to be chronic and thus insect communities may not recover until the disturbance ceases or the habitat is restored (Cairns, 1990). Variation in ﬂow (ﬂoods to desiccation) is the major cause of natural disturbance in streams and is responsible for large, usually temporary, reductions in insect abundance and diversity (Fisher et al., 1982; Lamberti et al., 1991). Stream insects have evolved various mechanisms to deal with such ﬂow variation, including life-history adjustments to minimize the presence of vulnerable stages during times of peak ﬂows, behavioral movement into more protected hyporheic (interstitial) and lateral habitats during ﬂoods, and high reproductive rates to compensate for losses. Despite these adaptations, severe ﬂood disturbance will still remove large proportions of the insect fauna. During drought, refuge provided by the deep hyporheic zone is especially important, but its accessibility to insects will depend on its grain size. Other insects may survive drought via resistant life stages, such as diapausing eggs or cocoons. Insects can recolonize streams from several sources: (1) egg laying by adults, usually aerial in the case of insects; (2) drift from upstream areas; and (3) movement along the bed from upstream, downstream, hyporheic, or lateral habitats (Smock, 1996). These behaviors are discussed in more detail above (see Section II. D. 3) In general, aquatic insect communities recover rapidly from natural disturbance. Lamberti et al. (1991) studied the response and recovery of macroinvertebrates (mostly insects) in a Cascade Mountain stream that experienced an extreme disturbance. A debris ﬂow, which is an episodic mass movement of sediment and debris through the stream channel (usually triggered by a landslide), devastated about a 1-km reach of Quartz Creek, Oregon, and partially disturbed areas downstream (Fig. 28). More than 99% of the macroinvertebrates were removed by the debris ﬂow. An associated ﬂood alone in an upstream reach removed 90% of the invertebrates, so that in-stream sources of insects clearly were low. Yet, in both the ﬂood-impacted and debris ﬂow-impacted reaches, macroinvertebrate species richness and total abundance recovered within one year of the disturbance to “normal” levels (Fig. 28). In another example, the eruption of Mt. St. Helens (southwest Washington) eliminated the entire biota from entire drainage systems, which included streams, lakes, and wetlands. Many stream channels were completely rerouted by topographic changes. Yet, in the ﬁrst year after the eruption, 98 species of macroinvertebrates (mostly insects) were found in one devastated stream (Anderson, 1992). Five 766 A. E. Hershey and G. A. Lamberti FIGURE 28 Debris ﬂow and its effects in Quartz Creek, Oregon. (A) Upstream reach that was ﬂooded only. (B) Reach most severely impacted by debris ﬂow of February 1986. (C) Downstream of debris ﬂow, showing debris dam and partial impact. (D) Recovery of periphyton chlorophyll a in the three reaches. (E) Recovery of macroinvertebrate density (mostly insects). (F) Recovery of macroinvertebrate species richness. [Modiﬁed from Lamberti et al., 1991.] years after the eruption, 141 species were found in the same stream, although nearly half of these were the ubiquitous chironomid midges. Both of these examples attest to the remarkable resilience of stream insects and other invertebrates to natural disturbance. Episodic natural events contrast, however, with chronic anthropogenic disturbance, such as inputs of chemical and thermal pollutants or prolonged watershed disturbance. Prolonged point-source pollution can have devastating effects on aquatic insects. Wallace and Brady (1971) reported that low concentrations (0.2 ppm) of the insecticide dieldrin in a factory efﬂuent reduced benthic insect densities by over 90% in a South Carolina stream, and that the remaining invertebrates had high tissue concentrations (up to 100 ppm) of dieldrin. Wallace and coworkers later explored the structural and 18. Aquatic Insect Ecology functional signiﬁcance of reduced insect populations in streams by conducting an experimental release of insecticide (methoxychlor) in an Appalachian Mountain stream. Insecticide application resulted in massive drift of insects, and reduction of benthic densities by over 85% (Wallace et al., 1982, 1989). The remaining benthic invertebrates were composed of an impoverished fauna of mostly noninsects (e.g., oligochaetes) and chironomid midge larvae. Furthermore, important ecological processes in the stream were altered. For example, leaf litter decomposition declined drastically because an important shredding caddisﬂy, Lepidostoma, was eliminated by the insecticide treatment (Whiles et al., 1993). When treatment ceased, Lepidostoma recolonized rapidly and leaf litter processing recommenced, again pointing to the resilience of aquatic insect communities. However, human chronic disturbance of aquatic ecosystems and their watersheds tends to preclude recovery of insect communities to predisturbance conditions. Watershed disturbances such as forest logging, agriculture, and urbanization can have long-term impacts on aquatic insect communities by altering energy inputs, thermal regimes, and sediment composition (Gregory et al., 1987; Lamberti and Berg, 1995; Wallace et al., 1988). VIII. AQUATIC INSECTS IN BIOMONITORING STUDIES Biomonitoring studies are used to measure response and recovery of aquatic communities to disturbances, protect biodiversity, evaluate compliance, and improve understanding of the relationship between physical, chemical, and biological components (Gurtz, 1994). Many biomonitoring programs incorporate ﬁsh, macroinvertebrate, and algae components in their studies, although macroinvertebrate studies, especially those focusing on aquatic insects, are widely used by many agencies. The National Water Quality Assessment (NAWQA) program of the USGS seeks to evaluate water quality at spatial scales ranging from local to national (Gurtz, 1994). Such biomonitoring studies are used to assess aquatic ecosystem health because organisms function as sensors of the quality of their environment in several ways that direct measurements of water quality cannot (Loeb, 1994). Insects have been particularly useful in biomonitoring of streams but also are used in lentic studies (Brinkhurst, 1974; Warwick and Tisdale, 1989) to assess changes in lake water quality and productivity. Warwick (1980) also has used insects in paleolimnological studies to reconstruct changes in land use surrounding a lake. Stream macroinvertebrates, especially insects, offer distinct advantages in biomonitoring studies (Stewart 767 and Loar, 1994). Their small size and limited mobility renders them relatively easy to sample. They also are known to be sensitive to a variety of pollutants. For example, insects absorb or transform chemical pollutants, such that direct chemical analyses may not be measuring toxicity in the same way as do insects. Insects also integrate environmental quality over a longer time interval than do static measures of water quality. Thus, biomonitoring incorporates and integrates ecology, life history, physiology, and taxonomy into a management and assessment tool. Although valuable and widely used, there are also a few disadvantages to using aquatic insects in these studies (Stewart and Loar, 1994). Spatial and temporal variation in abundance of aquatic life-history stages must be accounted for. Thus, an appropriate sampling design in a biomonitoring program must stratify this spatial and temporal variability to account for as much of it as possible (Hughes et al., 1994). Stratiﬁcation according to underlying geology is also of critical importance (Minshall et al., 1985; Richards et al., 1996). Furthermore, aquatic insect identiﬁcation is time-consuming and requires trained personnel; errors in taxonomy can lead to erroneous conclusions (see Lenat and Barbour, 1994; Rosenberg and Resh, 1996). Rosenberg and Resh (1996) provided a comprehensive review of current biomonitoring approaches that use aquatic insects. These approaches span a range of scales: (1) biochemical and physiological measurements, including metabolic studies and measures of enzyme activities; (2) individual attributes such as morphological, behavioral, and life-history parameters, or use of “sentinel organisms” that serve as bioaccumulators of toxic materials; (3) population- and assemblagelevel responses, such as using occurrence or abundance of indicator species as a measure of sensitivity to a pollutant; (4) community-level approaches which synthesize many types of data into summary responses to a pollutant; and (5) ecosystem-level scales that assess effects of stressors on processes and function. Of these, community-level approaches are probably in most widespread use, and these may be either qualitative or quantitative. Rapid assessment methods are often based on insect communities, are generally qualitative, but permit assessment of many streams with a reasonable degree of effort (see Lenat and Barbour, 1994). However, quantitative approaches are also very common, and the choice between qualitative and quantitative techniques depends on the objectives of the study. Several different metrics of aquatic insect communities are used (Rosenberg and Resh, 1996). Taxa richness is widely used, but is difﬁcult to compare across studies because different levels of resolution tend to be used by different investigators and for different taxonomic groups. EPT richness, which is the combined 768 A. E. Hershey and G. A. Lamberti an integrated picture of the recent history of an aquatic ecosystem. IX. SUMMARY FIGURE 29 Richness of EPT (Ephemeroptera, Plecoptera, and Trichoptera) taxa is robust to seasonal variation within a site, although it varies longitudinally and between streams. [From Stewart and Loar, 1994.] richness of Ephemeroptera, Plecoptera, and Trichoptera (EPT taxa) takes advantage of the fact that EPT taxa are relatively easy to identify and are pollution-sensitive (Lenat, 1988; Plafkin et al., 1989; Resh et al., 1996). This index varies longitudinally but is robust to seasonal and interannual ﬂuctuations (Fig. 29, Stewart and Loar, 1994). Because chironomids as a group are considered pollution-tolerant and EPT taxa are not, the ratio of EPT / chironomids is another popular communitylevel metric. Diversity and similarity indexes afford the advantage of being readily comparable between sites, and biotic indexes offer a readily interpretable score (e.g., Hilsenhoff, 1982). Functional feeding group measures rely on functional rather than taxonomic information, and in this sense they bridge community- and ecosystem-level approaches to biomonitoring. Rather than relying on a single metric for assessment in biomonitoring studies, multimetric and multivariate approaches often are recommended (ter Braak and Prentice, 1988; Karr, 1994; Lenat and Barbour, 1994; Richards et al., 1996; Rosenberg and Resh, 1996). Multimetric approaches can combine the strengths of individual metrics to minimize the chance of an incorrect assessment, and often several metrics can be calculated from the same data set. Multivariate approaches permit partitioning of variance due to different factors, thus providing more useful information than a single measure. They also are less subjective than are univariate approaches. Resh et al. (1996) provide a useful exercise that demonstrates the utility of several different biomonitoring approaches that use benthic macroinvertebrates. They stress the importance of combining information on both physical habitat quality and macroinvertebrate composition when assessing water quality. A cornerstone of their approach is that macroinvertebrates, especially insects, provide The study of aquatic insect ecology is dynamic, with several hundred new papers being added to the literature each year. While it is not possible to provide a comprehensive treatment of aquatic insect ecology in a single chapter, we provide a broad overview of major topics in aquatic insect ecology. Our speciﬁc goals were to: (1) describe the basic life histories of aquatic insects and their general community structure; (2) outline their role in major ecological processes involving insects that occur in aquatic ecosystems; (3) discuss how the structure of insect communities and the services they perform are affected by disturbance to their aquatic ecosystems and the changing landscape in which they are found; and (4) summarize how their communities can be used by managers to assess aquatic ecosystem health. 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Zachariassen, K. E. 1979. The mechanism of the cryoprotective effect of glyceral in beetles tolerant to freezing. J. Insect Physiol. 25:29 – 32. This Page Intentionally Left Blank 19 INTRODUCTION TO THE SUBPHYLUM CRUSTACEA Alan P. Covich James H. Thorp Department of Fishery and Wildlife Biology Colorado State University Fort Collins, Colorado 80523 Department of Biology Clarkson University Potsdam, New York 13699 I. Introduction II. Anatomy and Physiology of Crustacea A. External Morphology B. Organ System Function C. Environmental Physiology III. Ecology and Evolution of Selected Crustacea A. General Relationships B. Ecological Distributions and Interactions C. Life-History Traits D. Biogeography IV. Collecting, Rearing, and Preparation for Identiﬁcation V. Classiﬁcation of Peracarida and Brachiura A. Taxonomic Key Acknowledgments Literature Cited I. INTRODUCTION Crustaceans are the most diverse of any group of arthropods and currently thrive in a wide array of habitats. Most species occur primarily in aquatic habitats but some are adapted to live on land. The fossil record extends back to the lower Cambrian and throughout most of this long period, crustacean fossils are associated with a broad range of aquatic habitats, especially marine waters (Schram, 1982). Groups of crustaceans adapted to freshwater habitats very early in their evolutionary history (Hutchinson, 1967; Abele, 1982). The fossil record for freshwater species is especially well preserved from the Tertiary for some groups that are clearly related to living forms. Although only about 10% of the nearly 40,000 extant species in the subphylum Crustacea (phylum Arthropoda) occur in the inland pools, lakes, and streams of the world (Table I) (Bowman and Abele, 1982; Banarescu, 1990; Barnes and Harrison, 1992), freshwater crustaceans are extremely important in many ecosystem processes of inland waters. Crustaceans have evolved into several major groups (Fig. 1) that represent distinct modiﬁcations for Ecology and Classiﬁcation of North American Freshwater Invertebrates. 2nd Edition Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved. exploiting resources and performing ecosystem services in many different surface waters and subsurface habitats. Amphipods and isopods, as well as mysids and branchiura are discussed in this chapter; ostracodes are reviewed in Chapter 20. The cladocerans and copepods together constitute the dominant component of most freshwater planktonic assemblages and are discussed in Chapters 21 and 22 respectively. Crustacean zooplankton are major consumers of phytoplankton and they transfer energy from these primary producers up the food web to zooplanktivorous ﬁsh in lakes, ponds, and rivers. Some crustacean zooplankton also consume aggregations formed from dissolved organic matter and other organic particulates. Others are predatory on rotifers, protozoans, other crustaceans, aquatic insects, and ﬁsh eggs and larvae. Some zooplankton vertically migrate and thereby link deep, benthic habitats with open waters of lakes. These migratory species are important in nutrient cycling and transfers of toxins (such as heavy metals and pesticides) from sediments to pelagic foodwebs. Along with many other invertebrates, crustaceans provide hard surfaces as semipermanent, mobile substrates for diverse 777 778 A. P. Covich and J. H. Thorp TABLE I Classiﬁcation of Crustacea and Estimates of Species Numbers in North American Fresh Watersa,b Taxa Subphylum Crustacea Class Cephalocarida Class Branchiopoda [eight orders, including cladocerans] Class Remipdia Class Maxillopoda subclass Branchiura subclass Copepoda Class Ostracoda Class Malacostraca Superorder Pancarida [order Thermosbaenacea] Superorder Peracarida [orders Mysidacea, Amphipoda, Isopoda] Superorder Eucarida [order Decapoda] a b Worldwide percentage found in Freshwaters Approximate number of species in North America Chapter(s) of this book 10% 0% 95% ca.1500 0 200 19 – 23 — 21 0% 15% 90% 15% 50% 10% 50% 0 23 200 420 1 — 19, 22 19 22 20 19, 23 19 10% 300 19 334 23 10% North of Mexico. Classiﬁcation based in part on Bowman and Abele (1982). epibiont species such as ciliated protozoa and sessile rotifers (chapters 3 and 8; Threlkeld et al., 1993). Within inland hypersaline environments, brine shrimp (Chapter 21) are sometimes the only metazoan capable of surviving hyperosmotic conditions. As salinity ﬂuctuates, the crustacean community can rapidly change its species composition and exploit different sources of energy (Galat et al., 1988; Hammer et al., 1990; Stephens, 1990; Wurtsbaugh and Berry, 1990; Williams, 1998). Crustaceans also comprise a signiﬁcant portion of the benthic biomass in many lakes and streams. Some species are abundant in the littoral zone and throughout the oxygenated deeper water where they feed on bacteria and fungi on decaying vegetation or live algae (Gee, 1988; Delong et al., 1993). As with the zooplankton, these benthic consumers recycle nutrients that sustain high levels of microbial productivity. Some species horizontally migrate at night and link the littoral zone with pelagic water. This nocturnal mobility, like vertical migrations, can limit vulnerability of these species to visual predators. In streams, crustaceans also occupy many different habitats ranging from deep, subsurface waters to quiet pools and fast-ﬂowing rifﬂes where they actively consume a wide range of foods (Marchant and Hynes, 1981a, b; Boulton et al., 1998). In caves, unique species of crustaceans have evolved that also feed on many types of resources (Culver et al., 1995; Carpenter, 1999). Similarly, some species are found in wetlands and these feed on many types of detrital foods (Smock and Harlowe, 1983). These diverse benthic species function as herbivores, carnivores, and detritivores while also providing many ecosystem services (Palmer et al., 1997; Boulton et al., 1998; Covich et al., 1999). As discussed below, some of these very productive benthic species live in extreme habitats, from cold arctic waters to hot springs and saline waters where they can dominate benthic communities and reach high densities. One of the most ecologically and taxonomically diverse freshwater crustacean groups north of the Rio Grande are omnivorous crayﬁsh (Decapoda; Chapter 23). Of the more than 500 species and subspecies currently known worldwide, 393 taxa (78%) occur in North America and 346 of these occur in the United States and Canada (Sket, 1999a; Taylor, 2000; see Hobbs, Chapter 23). River drainages in the southeastern United States have the highest diversity and highest concentrations of crayﬁsh species of any region anywhere (Taylor, 2000). These diverse benthic consumers are not only important in nutrient cycling, but they also can change the life histories of some of their prey species that have evolved ways to avoid being eaten (Crowl and Covich, 1990). Crayﬁsh maintain very high densities in many different habitats where they feed on a wide range of living and dead plants and animals. In addition to their crucial roles as consumers and secondary producers in natural aquatic ecosystems, some crustaceans are increasing in importance as a food source for humans. Crayﬁsh have become widely available through aquacultural production, as have the larger “river shrimp” (Macrobrachium) in warm 19. Introduction to the Subphylum Crustacea 779 FIGURE 1 General evolutionary linkages of major groups of freshwater crustaceans. From Bowman and Abele (1982) who stated: “This [classiﬁcation] is not to indicate phylogenetic relationships and should not be so interpreted. The dashed line at the base emphasizes uncertainties concerning the origins of the ﬁve classes and their relationships to each other.” temperate-zone and tropical locations. Benthic amphipods, isopods, ostracodes, and various littoral cladocerans and copepods consume a large amount of living organisms and particulate organic matter. All these crustaceans, in turn, can be signiﬁcant constituents in the diets of many invertebrate predators and bottom-feeding ﬁshes (Dahl, 1998; Wooster, 1998; MacNeil et al., 1999a, b). The present chapter provides a brief introduction to crustacean anatomy and physiology. More detailed information can be obtained from general invertebrate zoology texts (e.g., Barnes, 1987 and from several books on crustaceans, such as those by Schmitt (1965), Schram (1986), Fitzpatrick (1983), McLaughlin (1980), and the 10-volume set edited by Bliss (1982 – 1985) and several co-editors. Following an introduction to 780 A. P. Covich and J. H. Thorp anatomy and physiology (based primarily on information from these sources), this chapter summarizes the ecology and evolution of three orders of crustaceans: Mysidacea, Amphipoda, and Isopoda. Brief reviews of the subclass Branchiura and the class Remipedia are also included. This chapter concludes with a discussion of crustacean biogeography, classiﬁcation, and a key to the genera of the major taxa listed above. Related information is presented on other major crustacean groups in Chapters 20, 21, 22, and 23. II. ANATOMY AND PHYSIOLOGY OF CRUSTACEA A. External Morphology Although many other arthropod groups use a single basic body plan, the crustaceans are anatomically very diverse (Schram, 1986). Once you have seen one insect or spider taxon, you have a good idea of the general body form. The crustaceans, however, have evolved a high diversity of body forms by fusing various segments or by developing highly specialized body segments and appendages. Crustaceans are basically metameric, protostomate coelomates whose bodies are covered by a hard exoskeleton and often divided into three regions, or tagmata: the head (cephalic area), thorax, and abdomen (sometimes the ﬁrst two regions are combined as a cephalothorax). The jointed appendages are primitively biramous and may be present in all three body regions. Mandibles, two pairs of maxillae, and two pairs of antennae are present in all species during at least one life stage. Crustaceans are distinguished from the Insecta, one of the other two large arthropod groups, by the presence of two pairs of antennae. 1. Skeleton All crustaceans have a hard exoskeleton composed of chitin that varies in rigidity at various life-history stages. The name Crustacea is derived from the Latin word for shell and refers to this exoskeleton, which in many taxa is hardened with very small inclusions of calcium carbonate. Although not a true “shell” as is found among the Mollusca, this exoskeleton is very durable. Crustaceans then, like other arthropods, are characterized by a chitinized exoskeleton consisting of a thin proteinaceous stiffened epicuticle and a thick, multilayer procuticle strengthened by calcium carbonate. The skeleton attains its maximum thickness and rigidity in the decapods. Projecting inward are chitinized struts, known as apodemes, which serve as sites for internal attachment of the muscles; they also lend partial protection to some organs. 2. Appendages Crustacean appendages are modiﬁed among species to serve a large variety of purposes, including locomotion (walking and swimming), feeding, grooming, respiration, sensory reception, reproduction, and defense. Consequently, the primitive, generally biramous appendages (terminal exopod and endopod) are often extensively modiﬁed with additional lateral and medial projections. Two extreme forms are recognized among adults (Fig. 2): the leaﬂike or lobed phyllopod appendage (as found among branchiopods) and the unbranched, segmented walking leg, or stenopod (typical of crayﬁsh). The cephalic region contains ﬁve basic types of paired appendages: (1) antennules (ﬁrst antennae), which are uniramous in all crustaceans except the malacostracans; (2) antennae (second antennae); (3) mandibles; (4) maxillules (ﬁrst maxillae); and (5) maxillae (second maxillae). The ﬁrst two pairs generally have a sensory function (aiding some taxa in food location and ﬁltering), whereas the last three pairs normally function in food acquisition, handling, or processing. The number of appendages on the thorax and abdomen vary greatly among large taxonomic groups. Malacostracans (such as decapods and amphipods) generally possess 5 – 8 pairs of thoracic appendages (sometimes called pereiopods) and six pairs of abdominal appendages (pleopods and terminal uropods). Primary abdominal appendages are absent in all nonmalacostracans except Notostraca, although comparable structures may have secondarily evolved. B. Organ System Function 1. Nutrition and Digestion Freshwater crustaceans predominantly employ their antennae, maxillae, or thoracic appendages to ﬁlter or grab algae, bacteria, microzooplankton, or suspended dead organic matter (detrital seston) from the water column. In some habitats, a relatively large number of taxa are primarily carnivores or scavengers (e.g., benthic decapods, certain copepods, and others). Parasitism is rare except among specialized copepods and branchiurans (Yamaguti, 1963; Margolis and Kabata, 1988) that attack ﬁsh and bopyrid isopods, such as Probopyrus which attack freshwater shrimps (Beck, 1980; Collart, 1990; Roman Contreras, 1996). Only one ostracod genus, Entocythere, is known to be parasitic. Cannibalism generally occurs when newly molted individuals are vulnerable, especially among the decapods (Goddard, 1988), amphipods (Dick, 1995; MacNeil et al., 1997, 1999), and isopods (Jormalainen and Shuster, 1997). Using their cephalic or thoracic appendages, crustaceans acquire food and pass it through a ventral 19. Introduction to the Subphylum Crustacea 781 FIGURE 2 Representative crustacean appendanges: (A) a phyllopod appendage of Anostraca; (B) biramous appendage of Anaspidacea (superorder Syncarida); and (C) uniramous stenopod appendage of the Stenopodidea (Decapoda: Pleocyemata). [Redrawn from McLaughlin, 1982]. mouth into a tripartite alimentary tract (see Chapter 23, Fig. 4). Crayﬁsh have sharp mandibles that assist in cutting and shredding their food. Crayﬁsh chelae (“claws”) can be used to grasp larger food items (although they are often used for defense) and their numerous sensilla (sensory hairs) may aid in chemically locating food (Fig. 3). In most crustaceans, food is ground into small, digestible particles by the gastric mill (the posterior end of the cardiac stomach) of the muscular foregut (e.g., Schmitz and Sherrey, 1983). Enzymes (primarily from the hepatopancreas) empty into the short midgut, where food is digested and assimilated. Wastes are conveyed by peristalsis through the long hindgut and out the terminal anus. 2. Circulation and Respiration Crustaceans generally have an open circulatory system consisting of a dorsal, neurogenic heart with a single chamber, one or more arteries (in malacostracans only), and several sinuses for returning blood to the heart. Many noncalanoid copepods (and marine barnacles) lack a heart altogether; ﬂuids are circulated as a result of general body movements. The heart is usually located in the thorax or cephalothorax, but is present in the abdomen in species with abdominal gills (such as isopods). The blood contains low amounts of a vis- cous, copper-based respiratory pigment (hemocyanin), which is dissolved in the hemolymph. Respiration takes place entirely across the body integument in nonmalacostracans, but thoracic and abdominal gills are typical of other freshwater crustaceans. Peracaridans (discussed later in this chapter) feature rudimentary epipodal gills (ﬂattened outgrowths of the coxa). Individual species differ in their rates of oxygen consumption and their ability to regulate their respiration as dissolved oxygen concentrations decrease (Toman and Dall, 1998). Freshwater decapods are distinct in having trichobranchiate gills composed of a central axis (with afferent and efferent blood vessels) bearing numerous ﬁlamentous branches (Fig. 4). 3. Fluid and Solute Balance Paired maxillary and antennal glands (also called green glands) are the principal excretory organs in crustaceans. The “labyrinth” of the antennal gland is also involved in reabsorption of glucose, amino acids, and divalent ions from tubule ﬂuids. Maxillary glands predominate within lower crustaceans, whereas the slightly more complex antennal glands characterize most malacostracans. More than 90% of the nitrogenous wastes are voided as ammonia. 782 A. P. Covich and J. H. Thorp FIGURE 3 Scanning electron micrographs of crayﬁsh chela: (A) view of distal portion of chela (Orconectes propinquus, female, 300 magniﬁcation); (B) Plumose sensillia (feathered hairs) located on the cutting edge of chela (Orconectes virilis, female, 6000 magniﬁcation). [Images provided by J. L. Borash, 1997. See Borash and Moore, 1997 for more information.] 19. Introduction to the Subphylum Crustacea 783 living in inland salt lakes. The brine shrimp Artemia salina, tolerates salinities ranging from 10% seawater to crystallizing brine; it is hypertonic to the medium in dilute waters, but hypotonic in concentrated solutions. 4. Neural Systems FIGURE 4 Structure of decapod gill branches, as shown in transverse (upper) and latteral (lower) views: (A) dendrobranchiate; (B) phyllobranchiate; and (C) trichobranchiate. [From McLaughlin, 1982]. Freshwater crustaceans are relatively competent osmoregulators. They face problems of water inﬂux and salt loss (both primarily across the gills) and, therefore, must release copious quantities of urine that is isosmotic to the hemolymph (a few taxa produce urine that is hyposmotic to the blood). Freshwater branchiopods and some copepods maintain a dilute hemolymph equal to about 200 mOsm (20% of seawater), while the blood of crayﬁsh is more salty (about 350 mOsm; see Mantel and Farmer, 1983). An opposite situation is faced by hyporegulating crustaceans FIGURE 5 The crustacean neural system is comprised of a tripartite brain and paired ventral, ganglionated nerve cords linked by commissures in a ladderlike arrangement. The protocerebrum of the brain normally innervates the eye, sinus gland, frontal organs, and head muscles; the deutocerebrum controls the antennules; the tritocerebrum innervates the antennae and a portion of the alimentary tract. Crustaceans are generally sensitive to light, chemicals, temperature, touch, gravity, pressure, and sound. The eyes of adult malacostracans are compound and frequently mounted on a stalk, or peduncle; but the adult eye of many entomostracans (an older term inclusive of most nonmalacostracans) is not much advanced over the simple cluster of inverse pigment cup ocelli that characterizes the larval (naupliar) eye. 5. Reproduction, Development, and Growth Crustaceans are primarily sexually reproducing, dioecious organisms, but hermaphroditism and parthenogenesis occur sporadically among entomostracans (especially in ostracodes and branchiopods) and a few marine malacostracans. Paired gonads lie above or lateral to the midgut in most species. In mysids, the ovaries are partially fused and linked by a cellular bridge. Paired, or rarely single, reproductive ducts open Structure of the antennal gland of a crayﬁsh. [From Mantel and Farmer, 1983.] 784 A. P. Covich and J. H. Thorp ventrally through simple gonopores (paired or single), elevated papillae, or elaborate copulatory structures (as in decapods). The location of the gonopore varies greatly in entomostracans, whereas in malacostracans, it opens on the sternite or coxae of the sixth thoracic somite in females and at the eighth somite in males. Internal fertilization is the general rule, with males transferring sperm through a penis or specialized trunk appendages known as gonopods (in crayﬁsh these are highly modiﬁed pleopods). Females of most species protect their embryos either by retaining them in an internal brood chamber (e.g., in cladocerans) or external ovisac (e.g., in copepods) or by gluing them to certain appendages (decapods employ their abdominal pleopods). Mysids, isopods, and amphipods safeguard their young until an advanced stage in a ventral brooding shelf, the marsupium. Ovigerous female amphipods are readily recognizable when carrying eggs or young in late developmental stages because of their extended brood pouch and its yellowish to grey coloration. Stream-dwelling isopods apparently release their juveniles from the brood chamber when at risk from ﬁsh predation, but not when exposed to lower risk of predation by salamanders (Sparkes, 1996a,b). The embryogeny of crustaceans includes modiﬁed spiral, total cleavage, and gastrulation by invagination. All taxa possess an initial nauplius stage in their development, but these larvae may be free swimming (e.g., in copepods) or enclosed in an egg (e.g., in decapods, cladocerans, and peracaridans). Direct development (i.e., without any external larval stages) characterizes taxa such as cladocerans and peracaridans. The young individuals appear morphologically identical to miniature adults. In contrast, indirect development with a free nauplius followed by either distinctive stages of metamorphosis or gradual development to an adult is typical of most ostracodes, copepods, decapods (except crayﬁsh), and Branchiopoda (other than cladocerans). Growth throughout the larval, juvenile, or adult stages requires periodic shedding of the older, smaller exoskeleton in a process termed molting or ecdysis. Rapid expansion of the body occurs immediately after the crustacean extracts itself from its old exoskeleton and before the new shell hardens. The degree of expansion varies signiﬁcantly among species, ranging from 8 – 9% in some mysids to 22% in many decapods, to as high as 83% in certain cladocerans (Hartnoll, 1982). Some taxa are characterized by indeterminate growth (e.g., the cladoceran Daphnia and apparently most crayﬁsh species), while others reach a ﬁnite body size in a ﬁxed number of molts (e.g., in ostracodes, copepods, and some isopods). The molt process is controlled by hormones released by several organs. With a decline in production of the molt-inhibiting hormones (normally secreted by the cephalic X-organ and sinus gland complex), the Y-organs are no longer suppressed and can begin secretion of ecdysone, a molt-stimulating hormone. The actual molt requires as little as a few minutes to as long as several hours in aquatic taxa. This period is extremely hazardous; the animal may die from physiological stress or succumb when it is unable to extract a portion of its body from the old exoskeleton. The newly molted individual is vulnerable to rapid colonization by epibionts and ectoparasites. Some species of ciliated protozoans and sessile rotifers are well adapted to quickly recolonize the clean surfaces of newly molted isopods (Cook et al., 1998; Roberts and Chubb, 1998). The temporarily soft shell of a recently molted individual is extremely susceptible to predatory attacks (and cannibalism). Some species may seek shelter prior to molting. The larger and older the individual becomes, the longer it takes for the molting process to be completed, and the longer is the exposure to possible cannibalism and predation. Various environmental factors inﬂuence susceptibility of crustaceans to predators after molting. For example, medium, and large-sized individuals experience lower risks of predation from salamanders when living in waters rich in calcium carbonate (tufa and travertine), because lime deposits on their bodies apparently make them less vulnerable (Ruff and Maier, 2000). Thus, although indeterminate growth typically has limitations imposed by physiological stress, parasite infections, epibiont loads, and predatory pressures, each habitat may vary in how environmental complexity inﬂuence rates of molting and survivorship. C. Environmental Physiology 1. Salinity, Temperature, and pH Tolerances A large number of crustacean species have adapted to living completely in freshwater habitats (Table I) while others spend only portions of their lives in these highly variable habitats (e.g., Mantel and Farmer, 1983; Vernberg and Vernberg, 1983; Holsinger, 1994a; Wollheim and Lovvorn, 1996). Branchiurans are distributed worldwide in both marine systems and freshwater habitats; some species can tolerate a rapid shifting of distributions from salt to freshwater and may even switch hosts from marine to nonmarine ﬁshes. Among the Peracarida, as will be discussed later, some species are conﬁned to coastal inland waters and apparently are not completely adapted to many of the relatively unbuffered, dilute waters found farther inland (Sutcliffe, 1971, 1974; Dormaar and Corey, 1978). Amphipod species differ greatly in their ability to osmoregulate in variable salinities. One estuarine species from the western coast of North America, Corophium spinicorne, occurs typically in tidal fresh 19. Introduction to the Subphylum Crustacea waters (Hutchinson, 1967). This species is reported to have dispersed farther inland and now it occurs in fresh waters at two sites along the Snake River, upstream of Lewiston, Idaho (Lester and Clark, in press). This coastal species apparently was transported upriver by barges and may be widely distributed in the lower reaches of the Snake and Columbia rivers. Some species, such as Hyalella azteca, are found in saline lakes with salinities up to 25 parts per thousand (Hammer et al., 1990). Other amphipod species are sensitive to direct or indirect effects of increased salinity (Wollheim and Lovvorn, 1996). Salt pollution from various sources can increase hypo-osmotic stress and create adverse effects on amphipod metabolism (Koop and Grieshaber, 2000). Temperature is an important regulator of metabolism and growth rates as well as survival. The upper lethal temperature that limits survival varies greatly among different species. Some crustacean species have the highest known temperature tolerance among the aquatic metazoans and can live in hot springs. For example, the ostracode Potamocypris, lives on algalcoated substrates in habitats that range from 30 to 54°C (Wickstrom and Castenholz, 1973), the pancarids Thermosbaena mirabilis survives in waters of 45 – 48°C, and Thermobatynella adami lives in 55°C water (Capart, 1951). Some isopods are found in warm springs of the southwestern United States; for example, Thermosphaeroma thermophilum, an endangered species, and T. subequalum thrive in 34 – 35°C pools (Bowman, 1981; Cole and Bane, 1978; Shuster, 1981, Manuel Molles, personal communication). Other benthic crustaceans, such as the amphipod Hyallela azteca, also live in hot springs (Richard Wiegert and Dean Blinn, personal communication) with temperatures up to 33 – 34°C in Devils Hole, Nevada. In general, individual growth rates of Hyalella azteca and other crustaceans are affected directly and indirectly by water temperatures. Variations in water temperatures often result in seasonal movements by differently aged individuals as they track changing temperatures at different depths. For example, ﬁeld observations demonstrate that 20°C is a threshold for induction and termination of reproductive resting stages of Hyalella azteca. This species moves from warmer, shallow littoral zones into colder, deeper water as individuals mature (Panov and McQueen, 1998). If water depths decrease and water temperatures increase in response to both successional and climatic warming, it is anticipated that benthic species such as Hyalella azteca would likely have increased growth rates and earlier breeding in shallow lakes, ponds and intermittent streams. These shifts in water temperatures and habitat fragmentation could affect intra- and interspeciﬁc competition and gene ﬂow among benthic species, espe- 785 cially during prolonged droughts (Hogg and Williams, 1996; Covich et al., 1997; Hogg et al., 1998a). Other amphipod species require colder, relatively constant waters and are often associated with cold springs. For example, Gammarus minus is rarely found in waters warmer than 15°C and is absent in waters above 20°C (Glazier et al., 1992). Monitoring these cool-water habitats and their amphipod populations will provide important information about the long-term effects of climate on groundwater and surface-water temperatures (Wilhelm and Schindler 2000). The widespread acidiﬁcation of lakes and streams has drawn attention to the importance of pH tolerances of many organisms, especially the crustaceans. Because calciﬁcation of the carapace immediately following molting is highly sensitive to low pH, changes in regional distributions among crustaceans may reﬂect regional levels of acidiﬁcation and natural differences in calcium concentrations (e.g., Nero and Schindler, 1983; Greenaway, 1985; Okland and Okland, 1985; Meyran, 1997, 1998; Meyran et al., 1998). There are both lethal and sublethal effects of increased acidity that alter ion regulation, especially by juveniles in soft-water lakes. Although many studies have emphasized lentic species, there is evidence that lotic species may have a narrower range of tolerance to low pH and that egg mortality may be important for some species. Crustaceans generally are good indicators of changes in pH values in well-deﬁned habitats. For example, a regional study documents that Gammarus minus distributions in 32 springs in central Pennsylvania are limited to waters with pH values of 6 and above, with most populations found in waters near pH 7 and where temporal variation was minimal (Glazier et al., 1992, Glazier and Sparks, 1997). Although crayﬁsh are generally thought to require habitats with calcium concentrations in excess of 2 mg / L, low population densities of some species may be maintained by extracting sufﬁcient calcium from their food. In adult Orconectes virilis, calcium uptake is impaired in habitats below pH 5; survivorship is greatly inﬂuenced by age and molt stage (Chapter 23). 2. Photoreception Sensitivity to small differences in light intensities is extremely well developed in crustaceans. Many groups are able to locate microhabitats where they can optimize growth rates and reproduction (Hutchinson, 1967, 1993). Crustaceans have evolved receptors to detect even slight differences in light intensities, especially in deep, thermally stratiﬁed waters. Marine and freshwater zooplankton exploit thermal gradients by migrating daily and seasonally in both vertical and horizontal patterns to obtain their preferred conditions. The adaptive value of this migration for zooplankton is thought to 786 A. P. Covich and J. H. Thorp be a combination of: (1) optimizing growth through reducing metabolic costs by remaining in cool waters; (2) maximizing growth through grazing at times when algae have the highest food value; and (3) avoiding exposure to visual predators. The relative importance of these variables can change seasonally and shift the exact timing of migration to maximize net energy gain under different conditions. When algal food resources are scarce, zooplankton may ascend “early” from deep cool waters and begin grazing 1 – 2 h before sunset, thereby gaining access to limited food supplies before competitors, but at a higher metabolic cost and risk of predation (Enright, 1977, 1979; Gliwicz, 1986). In arctic lakes and ponds, crustacean zooplankton are exposed to continuous daylight during most of their growing season and do not vertically migrate on a daily basis. However, they maintain phototactic reactions at approximately the same threshold values; their photosensitivity remains similar to temperate species (Buchanan and Haney, 1980). Controversy exists regarding how to evaluate the adaptive signiﬁcance of variable migratory responses to different light intensities in the presence of predators (see chapters 21 and 22). Many decapods are nocturnal and avoid sight-feeding predators by remaining inactive and undercover during the day. They have sensitive photoreception and synchonize their activity cycles within their population and thus increase their nighttime densities and reduce their individual vulnerability to predators. Many species of benthic amphipods and isopods seek dark microhabitats such as beneath leaf litter during daylight to minimize exposure to visual predators. This normal avoidance behavior of bright habitats by amphipods and isopods, however, can be affected by endoparasites. Infected crustacean hosts increase their daytime activity and can thereby increase their vulnerability to predators. This predation often continues the life cycle for the parasite with the predatory ﬁsh or bird serving as a second intermediate host (e.g., Helluy and Holmes, 1990; Maynard et al., 1998). How photoreception and activity cycles of some crustacean species are altered by speciﬁc parasites is an active area of research. 3. Chemoreception Crustaceans also have very well-developed chemosensory systems that allow individuals to locate food and mates while avoiding predators (Ache, 1982; Atema, 1988; Zimmer-Faust, 1989). Crustaceans demonstrate highly speciﬁc responses to pheromones (Dahl et al,. 1970; Dunham, 1978; Dahl et al., 1998; Dahl, 1998; Wooster, 1998, see chapters 20 – 23). Hydrodynamics of stream ﬂow and lake currents clearly mediate the effectiveness of diffuse chemicals, but the persistent unidirectional nature of many signals does provide a basis for orientation and communication. Laboratory and ﬁeld studies demonstrate that chemical communication between crustacean predators such as crayﬁsh and their gastropod prey can inﬂuence prey life histories (Crowl and Covich, 1990). Crustacean prey, such as Gammarus minus, respond differently to chemical cues released from injured conspeciﬁc individuals relative to chemical cues released from other species such as sympatric isopods, Lirceus fontinalis (Wisenden et al.,1999). Gammarus lacustris responds to alarm signals from ﬁsh predators (northern pike, Esox lucius), but not to dragonﬂy larvae (Aeshna eremita), apparently because ﬁsh are more effective predators (Wudkevich et al., 1997). Similarly, in laboratory studies Lirceus fontinalis reduces their activity in the presence of alarm substances from ﬁsh mucus of predatory green sunﬁsh (Lepomis cyanellus) but not from grazing ﬁsh, stonerollers (Campostoma anomalum) (Short and Holomuzki, 1992). Within the boundary layer of stream ﬂow, chemoreception can be highly effective as a means for two-dimensional orientation. For organisms drifting downstream or migrating upstream, chemical cues may be extremely important. The relative size of the organism to the depth of the boundary layer is an important ratio (Dodds, 1990) that inﬂuences effectiveness of chemical communication. During periods of stable-ﬂow regimes in streams, crustaceans may depend on chemical cues as well as other sources of information to regulate daily and seasonal patterns of movement, to select foods or mates, and to avoid predators. As pointed out in chapters 21 and 23, there are recent studies on the effectiveness of chemical communication in crustaceans, and the situation is similar for other groups (Dodson et al., 1994). 4. Mechanoreception The ability to orient into currents (positive rheotaxis) is a common trait in many freshwater crustaceans. These physical, hydrodynamic cues are often associated with chemical cues that stimulate directed movement. Crustaceans have many types of setae, which function as mechanical and chemical sensors (Bush and Laverack, 1982). These external receptors are typically positioned near the antennae and the mandibles. However, there is a wide range of other locations so that information can be obtained from several directions simultaneously. The functional anatomy of these mechano-receptors is well studied in decapods. Behaviors associated with frequent cleaning activities are not as thoroughly studied in freshwater crustaceans as in marine taxa nor are there many studies on behavioral responses to changes in current speed (see Chapter 23 for further discussion). Behavioral studies of copepods have emphasized the importance of mechano-receptors for avoidance of predators (see review in Chapter 22), but relatively little is known for other groups. 19. Introduction to the Subphylum Crustacea 5. Thermoreception In warm waters rates of metabolism increase and the need for dissolved oxygen also increases. However, concentrations of dissolved oxygen decrease in warm waters and low-oxygen stress can cause mortality. This low-oxygen stress can be avoided if individuals move to certain microhabitats (e.g., groundwater seeps and deeper, stratiﬁed waters in summer). Thus, sensing differences in relative temperatures is an important ability for crustaceans to locate optimal microhabitats for growth and reproduction. Many species can orient in thermal gradients and combine information on temperature with simultaneous inputs from photo- and chemoreceptors (Ache, 1982). Freshwater crustaceans have distinct thermal preferences (e.g., Cheper, 1980; Oberlin and Blinn, 1997) and considerable information is available for crayﬁsh (as discussed in Chapter 23). As mentioned previously, the interactions between thermal cues and light intensity are often associated with vertical migrations of zooplankton. In these behavioral responses to light and temperature gradients, there is growing evidence that zooplankton may also use chemical cues to orient themselves and to avoid predators (see chapters 21 and 22). III. ECOLOGY AND EVOLUTION OF SELECTED CRUSTACEA A. General Relationships Evolution within the Crustacea has resulted in a very large number of different species. The fossil record demonstrates that both freshwater and marine crustaceans existed in the early Paleozoic and three main lines evolved from the monophyletic origin of arthropods (Schram, 1982; Emerson and Schram, 1990). Almost all of the crustaceans, that have adapted to freshwater environments, are thought to have evolved directly from marine ancestors without having gone through a phase of terrestrial adaptation (Hutchinson, 1967; Notenboom, 1991; Hutchinson, 1993; Holsinger, 1994a; Lee and Bell, 1999; Strayer, 1999). The class Remipedia is a group of blind crustaceans that occur only in unique coastal (anchialine) cave environments (Yager, 1981, 1987; Schram, 1986; Schram et al., 1986; Felgenhauer et al., 1992; Carpenter, 1999). These crustaceans have a long trunk with 32 unfused segments and each segment bears a pair of similar biramous appendages. Remipedes can tolerate saline waters with very little dissolved oxygen and they apparently feed in overlying, well-oxygenated freshwater lens. This unique ecological salinity range gives them some advantages over other crustaceans living in marine polyhaline coastal cave environments. 787 Remipedes are considered to be primitive crustaceans, and their evolution and biogeography have undergone active study since the relatively recent discovery of living representatives, primarily in the Caribbean region (Bahamas, West Indies, and in karst wells or “cenotes” of Yucatan, Mexico) but also from the Canary Islands. There are known to be fewer than a dozen species in two families. Their structural anatomy is better known (from transmission and scanning electron microscopic studies) than their functional anatomy, because they have not been successfully reared in the laboratory and studied experimentally (Felgenhauer et al., 1992). Other freshwater crustaceans also occur in anchialine caves and other complex hypogean habitats as discussed below (Stock and Iliffe, 1990; Holsinger, 1993, 1994a; Holsinger et al., 1994; Notenboom et al., 1994; Sket, 1996; Danielopol et al., 2000). Macrocrustaceans adapted to caves, artesian wells, and other groundwater habitats include a large number of amphipods, isopods, mysids, and decapods (Chapter 23) that inhabit these isolated inland waters (Holsinger, 1972; Holsinger and Longley, 1980; Abele, 1982; Bousfield, 1983; Barr and Holsinger, 1985; Holsinger, 1986, 1988, 1989, 1991, 1994a, b; Stock, 1995). Although most benthic crustaceans are small and difﬁcult to observe without intensive sampling, there are very large-sized crustaceans living in small and large rivers. Macrobrachium are the largest (20 cm carapace length) freshwater crustaceans in North America and are distributed primarily in coastal rivers along the Gulf of Mexico (Chapter 23). They usually have amphidromous life cycles that limit their populations to coastal areas connected to brackish and marine waters where they spend part of their series of complex larval stages. Although more than 90 species occur in the tropics, there are only six species of these palaemonid shrimp in North America. However, their riverine migrations and long-term survival are vulnerable because of barriers created by impoundments, overharvesting, and water pollution. Only M. ohione is known from more interior rivers and its populations are thought to be declining (Bowles et al., 2000). 1. Branchiura The subclass Branchiura is widely distributed throughout the world and is comprised of about 150 species and four genera that are ectoparasites on ﬁsh. Although there are many freshwater species (Yamaguti, 1963; Cressey, 1972; Margolis and Kabata, 1988), only the genus Argulus is typically found in North American freshwater habitats (McLaughlin, 1980; Abele, 1982; Fitzpatrick, 1983; Schram, 1986). Some 23 North American species are encountered; three are found in western and 19 in eastern North America. 788 A. P. Covich and J. H. Thorp The largest number of species occurs along the Gulf of Mexico. Only a few are very restricted in their geographic ranges, while others are widely distributed; for example, A. maculosus occurs from New York to Michigan and down the length of the Mississippi River to Louisiana. For many years, the Branchiura, or “ﬁsh lice,” were classiﬁed as a group of Copepoda (Wilson, 1944) and earlier as Branchiopoda, but their separation as a distinct taxon is now widely accepted. Unlike copepods, the argulids have a pair of compound eyes. The Branchiura also differ from parasitic copepods in having several other unique structures that will be described. The branchiurans have undergone major modiﬁcations of the mandibles and associated feeding appendages. The mandibles typically form a pair of transversely toothed hooks between the labium and labrum, and in Argulus, the mandibles are incorporated into the specialized proboscis (McLaughlin, 1982). The maxillules are large suckers that are characteristic of adult branchiurans (Fig. 6). In Argulus, a sheathed, hollow spine is used to pierce the skin of the host. Some species are blood suckers while others feed on extracellular ﬂuids or mucous. Once engorged, they can wait 2 – 3 weeks between meals. Some reproductive and larval behaviors are unique to Branchiura. In contrast to copepods, female Branchiura do not carry eggs in external ovisacs but, instead, fasten them in rows to rocks and other objects. The actively swimming larvae attach themselves within FIGURE 6 External anatomy of an ectoparasitic “ﬁsh lice” (Branchiura: Argulus). the gill chambers, mouth, or on the outer surfaces of ﬁshes using small, specialized antennal hooks and maxillules. After 4 – 5 weeks of development as ectoparasites, they become adults that again may actively swim (actually more like somersaulting through the water) to ﬁnd mates and other hosts. Adult females are highly motile in seeking egg-laying sites. They apparently are opportunistic in selecting host species of ﬁsh (Lamarre and Cochran, 1992; Shaﬁr and Oldewage, 1992). The mucous on ﬁsh surfaces does not interfere with attachment and low-level, short-term infection may not directly affect the ﬁsh host (Nolan et al., 2000). 2. Peracarida The superorder Peracarida has a relatively low percentage of taxa living in inland waters. The three orders that comprise the Peracarida are the Amphipoda, Isopoda, and Mysidacea. These orders are commonly known as “scuds” (also “sideswimmers”), “sow bugs,” and “opossum shrimp,” respectively (Figs. 7 – 9). Members of a fourth order, the Thermosbaenacea, with some species living in thermal springs and others in fresh or saline groundwaters, have strong peracarid afﬁnities but are generally considered to belong to the superorder Pancarida. Only one taxon, Monodella texana, lives in North American inland waters (Maguire, 1965; Stock and Longley, 1981). Most carcinologists exclude the Thermosbaenacea from the Peracarida because they brood their eggs dorsally under the carapace rather than in a brood pouch, a unique feature of the peracarids (Schram, 1982 1986). Within the Mysidacea, most of the 780 species are marine; worldwide, only 25 species occur in freshwater, and 18 additional species live in freshwater caves (Abele, 1982; Schmitz, 1992). The Amphipoda is the largest peracarid order with some 6000 known species in more than 100 families (Schmitz, 1992). Amphipods are mostly small (5 – 15 mm in length), free-living benthic organisms although some are pelagic and a few are semiterrestrial. There are three large families, Gammaridae, Crangonyctidae and Hyalellidae and worldwide, there are approximately 800 gammarid amphipods (Abele, 1982; Pennak, 1989). In North America, these amphipods are represented mainly by six families (Table II) and about 175 species that occupy surface waters. Another 10 families of amphipods (composed of 28 genera and approximately 125 – 160 species) occupy subterranean waters (Holsinger, 1986, 1994a). Compared to the Amphipoda, the Isopoda is a much less species-rich order but with endemic species often restricted to speciﬁc cave systems and other subterranean habitats. The Asellidae is the largest isopod family in North America with about 115 aquatic 19. Introduction to the Subphylum Crustacea 789 FIGURE 7 External side view of generalized freshwater gammarid amphipod: 1, head; 2, antenna 1; 3, antenna 2; 4, mouth parts; 5, pereonites 1 – 7; 6, pereopods 1 – 7; 7, pleonites 1 – 3; 8, pleopods 1 – 3; 9, uronites 1 – 3; 10, uropods 1 – 3; and 11, telson (redrawn from Holsinger, 1972). Top view of telsons: 12, Gammarus; and 13, Crangonyx. species, of which approximately 65 are cave-dwelling species. The other families only contain 10 species in surface and six in subsurface waters of North America (Sket, 1999a). The relative scarcity of isopods in many freshwater habitats and their wider occurrence in hypogean waters and thermal springs may be related to their lack of competitive ability and more restricted timing of reproduction relative to amphipods and other benthic invertebrates (Bowman, 1981; Graça et al., FIGURE 8 Diagram of top view of isopod Caecidotea (drawn without setation). 1994a, b). In Europe, Asellus aquaticus generally occurs in the lower reachers of river drainages while Gammarus pulex is more common in the upper reaches, but their competitive interactions and susceptibility to predation are not completely documented. Isopods, especially juveniles, are sensitive to low oxygen concentrations, toxic metals, and organic enrichment of habitats. Ratios of widespread species of isopods and amphipods (such as Asellus and Gammarus) are used to compare regional water-quality conditions (Naylor et al., 1990; Maltby, 1991, 1995; Whitehurst, 1991; Mullis et al., 1996). Examples of widely distributed genera of isopods, such as Caecidotea Lirceus, and Asellus, are well studied and often compared with associated amphipod species in surface and sub-surface waters. The Peracarida share several evolutionary similarities that demonstrate a common lineage. For example, all have modiﬁed the ancestral ﬁrst thoracic leg into a mouth part (the maxilliped) and retain seven pairs of thoracic legs for movement. The ﬁrst two pairs of these thoracic legs (the gnathopods) are specialized for grasping food while the remaining ﬁve pairs of legs (the pereiopods) lack any specialized grasping (chelate) appendages. As mentioned previously, peracarid females carry their eggs and young until a relatively advanced stage of development in a specialized ventral brooding chamber, the marsupium. Another character used for separating the peracarids from most other crustaceans is the highly developed lacinia mobilis, a small toothed process that articulates with the incisor process. A row of spines commonly separates the lacinia mobilis and the molar process (McLaughlin, 1982). Several phylogenetic relationships have been proposed for the 790 A. P. Covich and J. H. Thorp FIGURE 9 Diagram of side view of opossum shrimp, Mysis. evolution of the Peracarida (see Schram, 1982, 1986, for review) based on how the fossil record is interpreted relative to modern morphological criteria. Many workers view the Isopoda and Amphipoda as derived from a common mysidlike stock. Watling (1981) proposed a different arrangement that is more consistent with the known, but incomplete, fossil record (Fig. 10). Most workers agree that the amphipods and mysids are more closely related to each other than to the isopods (Schram, 1982, 1986; Sieg, 1983). Evolutionary relationships among species and subpopulations of widely distributed species are active areas of research (as discussed below) and new molecular techniques are providing insights regarding differences in rates of speciation. For example, studies using molecular time scales based on allozyme analysis suggest that the divergence of several amphipod lineages that gave rise to the three main species of Pontoporeia (P. afﬁnis, P. hoyi, and P. femorata) is likely to have occurred over tens of millions of years. The divergence of Mysis relicta from its likely marine congeners apparently dates from the Tertiary (Vainola and Varvio, 1989; Vainola, 1990). TABLE II Representative Taxa Found in North American Fresh Waters Superorder Peracarida Order Amphipoda Family Crangonyctidae Crangonyx, Stygobromus Family Gammaridae Gammarus minus, G. lacustris Family Hyalellidae Hyalella azteca, H. montezuma Family Pontoporeiidae Monoporeia afﬁnis Order Isopoda Family Asellidae Asellus, Caecidotea, Lirceus Order Mysidacea Family Mysidae Mysis relicta, M. littoralis, Neomysis mercedis, Taphromysis louisiannae FIGURE 10 Alternative phylogenetic interpretations of the Peracarida: (A) widely accepted relationship of Mysidacea as the main stem from which two divergent lines evolved, one to Isopoda and the other to Amphipoda; (B) another view derived from Watling (1981) with independent lines of development for each major group. The orders Cumacea, Tanaidacea, and Spelaeogripacea are marine peracarids, while the order Thermosbaenacea is often considered a pancarid group closely related to the peracarids. [From Schram, 1982.] 19. Introduction to the Subphylum Crustacea B. Ecological Distributions and Interactions 1. Habitats The widespread distribution of Branchiura suggests a lack of any speciﬁc pH or salinity requirements, but detailed studies of these ecological relationships are lacking. Temperature inﬂuences the rate of hatching for eggs of different species with a range of 12 – 30 days being required (Yamaguti, 1963). During the freeswimming stage Argulus may aggregate in a narrow zone of temperature and light conditions where encounters with host ﬁshes can be increased. Argulus has no strict host speciﬁcity and is capable of attaching to both freshwater and marine telosts. Most peracarid taxa that have fully adapted to the physiological and ecological conditions of inland waters share ﬁve ecological similarities in that they: (1) are typically restricted to permanent bodies of water that are relatively cool, clean, and well oxygenated; (2) have distinct behavioral patterns of vertical migration (e.g., among mysids and two species of amphipods; these are similar to daily migration in other groups of crustacean zooplankton, as discussed previously and in chapters 21 and 22); (3) have relatively limited ability to move upstream (positive rheotaxis is well studied in amphipods and isopods) or drift downstream in the current under speciﬁc ecological conditions (as do many of the lotic insects discussed in Chapter 18); (4) obtain much of their energy while feeding on the bottom substrates (even if they also feed while in the nekton on suspended algae and smaller taxa of zooplankton); and (5) serve as important prey to a large number of predatory ﬁshes (also discussed in chapters 21 – 23). Some taxa also have various ways of inﬂicting damage to predatory populations either by being direct parasites on predators or by competing with young stages of those predators for the same prey (such as Mysis feeding on plankton) that would otherwise be potentially available to ﬁsh consumers. In contrast to other crustaceans, most of these peracarid species typically lack a diapause phase and are missing any distinct adaptation for avoiding desiccation. Thus, their chances for passive dispersal (e.g., being carried from one habitat to another by ducks or other migratory animals or being carried by the wind during storms) are low in comparison with cladocerans, copepods, or ostracodes (Hairston and Caceres, 1996; Hairston and Bohonak, 1998; Bohonak, 1999). The peracarids also lack any strong active dispersal ability. They generally do not move upstream against strong current (Olyslager and Williams, 1993) so their migration throughout river-drainage networks is restricted, and populations apparently remain more or less in the same locality for long periods. Nonetheless, 791 as discussed later, some taxa are very widely distributed and their patterns of distribution suggest a long history of slow dispersal and persistence. There are a few very widely distributed amphipod species in surface waters. Some surface-dwelling and subsurface species have been well studied ecologically so that comparisons of population densities and life histories provide important information for their conservation and as indicators of water quality (e.g., Mullis et al., 1996; Mosslacher, 1998; Knapp and Fong, 1999). The most widely distributed and diverse genera of cave-inhabiting amphipods are Stygobromus with approximiately 180 species and Crangonyx with 14 species (Peck, 1998). No doubt, many species remain undescribed in caves, hyporeheic zones, deep lakes, and even in well-studied, shallow-water habitats. The continued discovery of new species, and the recent applications of new techniques for genetic analysis of previously described taxa, suggest that many more genetically distinct populations occur than was ﬁrst expected (Hogg et al., 1998b). Many species of amphipods and isopods are exceptionally well adapted to live in surface waters as well as groundwater habitats and caves (Holsinger, 1972, 1986, 1988; Stanford and Ward, 1988; Fong, 1989; Culver et al., 2000). In cave ecosystems, a single species of amphipod may dominate a relatively simple food web based on ﬁne organic particulates (e.g., Drost and Blinn, 1997). The microbiogeographical distributions and evolution of relatively common species such as Gammarus minus are under intensive study (Kane et al., 1992; Sarbu et al., 1993; Culver et al., 1994; Culver and Fong, 1994; Fong and Culver, 1994; Culver et al., 1995; Kane et al., 1995). As discussed below, other intensive studies have focused on the evolution of subpopulations of Hyalella azteca; some apparently have formed distinct species in certain surface-water habitats (Thomas et al., 1997; McPeek and Wellborn, 1998). Ecologically, most of the amphipods and isopods are photonegative, positively rheotactic, thigmotactic, cold stenotherms (i.e., restricted to relatively constant, cold waters where they avoid bright light by moving into the current and into crevices or under stones, leaves and roots). In complex substrates, they are less exposed to predators such as ﬁsh and crayﬁsh. Typically, where refugia from predation exist, the peracarids occur at high densities in small, permanent, spring-fed streams, seeps, ponds, or sloughs (e.g., Allee, 1914, 1929; Juday and Birge, 1927). In isolated springs their populations sometimes genetically diverge from those in other springs (Gooch and Glazier, 1991; Glazier et al., 1992; Culver et al., 1994; Glazier, 1999). Some species of Gammarus reach densities of thousands of individuals per square meter where detrital 792 A. P. Covich and J. H. Thorp food and cover are abundant and predators are few or absent. A relatively smaller number of isopod species have apparently adapted to live in intermittent or permanent surface waters but distributional data are scarce (Richardson, 1905; Hubricht and Mackin, 1949; Williams, 1970; Ellis, 1971). Certain species are common in subsurface habitats. For example, the subterranean isopod Caecidotea tridentata is relatively abundant in groundwaters of the Konza prairie (Edler and Dodds, 1996). The most diverse genus of cave-inhabiting aquatic isopods is Caecidotea with more than 56 species (Peck, 1998). For most genera of isopods the number of described species found in groundwater habitats and caves of North America ranges from one to ﬁve (Holsinger, 1988; Fong and Culver, 1994; Culver et al., 1995; Peck, 1998). Only a few known species tolerate the low oxygen concentrations found in polluted rivers and groundwaters (Williams, 1970; Strayer et al., 1997; Henry and Danielopol, 1998; Mosslacher, 1998; Hervant et al., 1999; Malard and Hervant, 1999) or the stress of high temperatures (Dadswell, 1974). For example, some isopods (Caecidotea recurvata) survived in cave pools only slightly polluted by septic tank efﬂuents, but were absent from highly polluted pools (Simon and Buikema, 1997). There is concern that any level on increased food input by organic pollution could result in competitive displacement of subsurface species by surface-dwelling species that are better adapted to use higher concentrations of food (Sket, 1999b). The order Mysidacea has only a single family and one very important species, Mysis relicta (Fig. 9), which occurs in cold, deep lakes as well as in shallow brackish ponds along the arctic coasts. A second species, Neomysis mercedis, has a more limited, coastal distribution but plays a signiﬁcant role in some large lakes such as Lake Washington (Murtaugh, 1989). Mysis relicta, the “opossum shrimp,” is not a true shrimp (or decapod as deﬁned in Chapter 23), but is an active swimmer and can make long daily trips up and down the water column of deep, well-oxygenated lakes (Beeton and Bowers, 1982; Shea and Makarewicz, 1989). A third species, Taphromysis louisianae, lives in roadside pools in Louisiana and Texas (Banner, 1953; Pennak, 1989). Mysids have a holarctic distribution; they occur in oligotrophic lakes of northern regions of North America and in northern Europe, but are known to tolerate relatively high temperatures and periods of low oxygen at least for short periods (Dadswell, 1974; Sherman et al., 1987). As will be discussed later, their introduction into many western lakes has created several problems for ﬁsheries managers (Martinez and Bergersen, 1989). Their natural limits of distribution apparently have been inﬂuenced by opportunities for migration during the Pleistocene and possibly by biotic interactions (Dadswell, 1974). 2. Food Resources All three peracaridian orders are similar in the range of foraging behaviors used in obtaining food resources. Juveniles are typically dependent on microbial foods such as algae and bacteria associated with either periphyton or aquatic plants in brightly illuminated habitats. They also consume dead organic matter in forested streams, ponds, and lakes where bacteria and fungi living on the detritus provide essential protein for amphipods and isopods (Barlocher and Kendrick, 1973, 1975; Smock and Stoneburner, 1980; Smock and Harlowe, 1983; Findlay et al., 1984, 1986; MacNeil et al., 1997). Adults are not limited to grazing or detritivory because they broaden their food niche to include larger food items and are predatory (Schwartz, 1992; MacNeil et al., 1999b). Some pelagic or nektoplanktonic amphipods in ﬁshless lakes, ponds, and wetlands consume phytoplankton and zooplankton (Anderson and Raasveldt, 1974; Dehdashti and Blinn, 1991). Adults of well-studied species, such as Gammarus minus, are known to depend primarily on detritus when in surface waters but cave populations of this species are more ﬂexible in their diet (Culver et al., 1995). In addition to the many plant, animal, and detrital foods used by different species of Gammarus, intraguild predation and cannibalism are also thought to be relatively common in surface waters (Dick, 1995; MacNeil et al., 1997, 1999b). Amphipods become opportunistic scavengers, predators, and omnivores, depending on food quality and which foods are most available (Delong et al., 1993). Given their wide range of foods, it is clear that different modes of feeding are used to exploit these different resources (e.g., Crouau, 1989). These generalized consumers can reach high population densities by deriving their energy from many sources. Resource competition among guild members can be intense if access to suspended foods is space-limited, as may occur in soft-bottom streams among ﬁlter-feeding species that require hard surfaces for attachment or perching. Interference competition and aggressive behavior can also occur among species that differ in aggressiveness and mobility (e.g., Dick et al., 1995; Haden et al., 1999). Laboratory and ﬁeld studies of production have focused on juvenile and adult growth rates that result from feeding on different types and qualities of foods at widely different locations (Waters and Hokenstrom, 1980; Marchant and Hynes, 1981a; Sutcliffe et al., 1981; Delong et al., 1993; France, 1992, 1993). Only a 19. Introduction to the Subphylum Crustacea few studies have considered the role of secondary plant chemicals as “defensive” compounds for aquatic plants to minimize effects of grazers. Concentrations of tannins, and other inhibitory chemicals are known to differ among various macrophytes, and some compounds inﬂuence crustacean grazing preferences (e.g., Newman et al., 1990, 1992). Plant foods that contain a high percentage of cellulose are often considered to be of relatively low quality for grazers and detritivores (Newman, 1991). However, some amphipods (Monk, 1977) and mysids (Friesen et al., 1986) have digestive enzymes that hydrolyze cellulose in vitro. These cellulases, however, are apparently conﬁned to degrading small particles in the gut rather than whole plant cell walls that are ingested during grazing. Microbial breakdown of ingested cellulose within the gut is generally the mechanism used for digesting this refractory material and requires development of a complex community of various microorganisms within the gut. Given that crustaceans molt periodically and would need to re-establish this microbial community in their new gut lining, the evolution of cellulases would be an important adaptation for expanding use of detrital food resources. 3. Vertical Migrations and Feeding Amphipods and isopods are often conﬁned to burrowing and feeding on the bottoms of streams and lakes, although species like Monoporeia afﬁnis (Pontoporeia afﬁnis) move from their benthic foraging areas during the day to nektonic, open-water foraging at night (Marzolf, 1965; Winnell and White, 1984; Donner et al., 1987). These actively swimming amphipods consume a wide range of particles while on the sediment surface, although the exact nature of the food is not completely known for those species found in the Laurentian Great Lakes or other large basins (Dermott and Corning, 1988; Lopez and Elmgren, 1989; Nalepa et al., 2000). The seasonal deposition of diatoms and other phytoplankton from the surface waters provides important food resources for bottom-dwelling amphipods (Johnson and Wiederholm, 1989; Hill et al., 1992; Johnson and Wiederholm, 1992; Sly and Christie, 1992; Goedkoop and Johnson, 1994). The vertical movements of amphipods, such as Monoporeia afﬁnis, in deep lakes overlap those of mysids with whom they may share food resources. Mysids provide an intriguing example of vertical migration and complex foraging dynamics (Chess and Stanford, 1998, 1999; Rudstam et al., 1999; Spencer et al., 1999). Mysis relicta regulates nocturnal feeding patterns by responding to changing light conditions (Johannsson et al., 1994). These very fast, active swimmers have well-developed eyes for precise photorecep- 793 tion. They rely on daily changes in intensity and quality of light (that penetrates through the photic zone) to determine when to begin swimming up through the water column from the dimly lit, deeper waters and to begin feeding on small zooplankton and phytoplankton (Morgan et al., 1978; Cooper and Goldman, 1982; Folt et al., 1982). By precisely regulating their travel times, migrating crustaceans reduce their exposure to ﬁsh predators and still obtain the food they need to grow and reproduce. Both Mysis and Monoporeia have potential ﬁsh predators that feed on benthic and open-water habitats so that minimizing exposures to a wide range of predators, while also sometimes competing with them for zooplankton prey, is a complex process (McDonald et al., 1990; Johannsson, 1992; Johannsson et al., 1994; Johannsson, 1995; Wilhelm, 1996). As mentioned previously, those zooplankton that seek deeper, cooler waters during the day not only avoid brightly lit waters and exposure to visual predators, but they also lower metabolic rates while digesting the food that they obtained the previous night (Chipps, 1998; Rudstam et al., 1999). Depending on the temperatures of the upper strata, Mysis migrates faster and closer to the surface than does Monoporeia (Wells, 1960). Because Mysis has less tolerance for low concentrations of dissolved oxygen in the deeper strata than does Monoporeia, it may not spend much time in those deep layers during the later portion of the growing season. Decomposition of organic matter in the deep hypolimnion depletes dissolved oxygen if the upper waters are highly productive and dead matter falls into these deep waters. Of considerable importance is the observation that Mysis can eat Monoporeia (Parker, 1980). The spatial refuge from mysid predation for amphipods then is dependent on the length of time that a lake remains thermally stratiﬁed, its level of productivity, and its bottom water temperature, because these factors determine both the concentration of dissolved oxygen in the deepest strata and the presence of various benthic predators. Because mysids are consumed by a number of game ﬁshes, there was a period of widespread, intentional introduction of Mysis relicta into many deep lakes in the western and northern United States (Martinez and Bergersen, 1989; Spencer et al., 1999) and in western Canada (Lasenby et al., 1986). As often happens with introductions of species into habitats where they do not naturally occur, the foodweb dynamics did not develop as expected. Of the 134 recorded introductions into the lakes of western North America, only 45 became directly established and eight others became indirectly established through passive migrations to other lakes (Martinez and Bergersen, 1989). In some lakes, instead of providing more food for predatory ﬁshes 794 A. P. Covich and J. H. Thorp such as lake trout and coregonids, the mysids ate many of the same zooplankton species that the juvenile ﬁsh had previously consumed (Lasenby et al., 1986). Thus, the mysids began competing with ﬁsh for zooplankton prey (Cooper and Goldman, 1982; Spencer et al., 1991, 1999). In these lakes, the mysids did not migrate high enough into the surface waters where the visually oriented ﬁsh could capture them. In the spring and summer, mysids avoided the warmer, upper, dimly lit surface waters and thus escaped predation; furthermore, they intercepted the upwardly moving zooplankton at midwater depths before the zooplankton could be consumed by ﬁsh. Even more unexpected was the predation by Mysis relicta on newly hatched larval ﬁsh (Seale and Binowski, 1988). An unusual example of ﬁnding food occurs with the amphipod Hyalella montezuma. This species migrates to the surface waters of a large karst sink hole (Montezuma Well, Arizona) and ﬁlters out organic material entrapped on the surface ﬁlm (Cole and Watkins, 1977; Blinn and Johnson, 1982; Blinn et al., 1987; Wagner and Blinn, 1987). Its twilight migration is followed by a predatory leech, which swims up to feed on the dense concentration of amphipods and other prey (Blinn et al., 1988, 1990; Blinn and Davies, 1990; McLoughlin et al., 1999); no ﬁsh predators occur in this sink-hole lake. In contrast, Hyalella azteca, which coexists with H. montezuma in Montezuma Well, does not ﬁlter feed or migrate to the surface. In comparative studies of Devils Hole, Nevada, Dean Blinn and Kevin Wilson (personal communication) observed that H. azteca migrate horizontally from the shoreline to the open-water column at night, which is the reverse of what occurs in Montezuma Well (Blinn et al.,1987). In ﬁshless lakes, ponds, and wetlands, other amphipods, such as Gammarus lacutris and G. pulex, are known to feed in the water column as well as on the bottom (Anderson and Raasveldt, 1974). Other well-studied examples of complex vertical migrations of predatory macro-crustacean feeding on zooplankton occur among species living in very deep lakes and are similar to massive aggregations of Mysis relicta in North American lakes (Lasenby et al., 1986). As discussed below, Macrohectopus branickii in ancient Lake Baikal has dynamics and functions similar to North American mysids (Rudstamet al., 1992; Melnik et al., 1993). 4. Responses to Water Quality The general requirement for habitats with relatively high concentrations of dissolved oxygen by most peracarid crustaceans usually limits them to clean, cold waters especially, during their early life histories. Some species of amphipods are sensitive to changes in temperature, stream discharge, and predators in their choice of microhabitats through their onset of downstream drift responses and ability to move upstream in search of appropriate conditions (Waters and Hokenstrom, 1980; Williams and Moore, 1985). In productive habitats crustaceans can deposit excess energy in the form of lipid which they store internally (Hill et al., 1992; Adare and Lasenby, 1994; Cavaletto et al., 1996; Nalepa et al., 2000). Because they feed on a variety of materials and incorporate chemical constituents from their food into their bodies (often in their lipids), these organisms are important in research on water-quality monitoring (Conlan, 1994; Landrum and Nalepa, 1998; Song and Breslin, 1998, 1999). Many species also move over relatively large areas and, thus, are exposed to a wide array of microhabitats that may contain speciﬁc toxins. The ease of collecting amphipods and isopods from a broad range of habitats and their hardiness in laboratory mesocosms make these crustaceans ideal candidates for monitoring pollution. They are especially sensitive to copper (even from copper pipes in laboratory plumbing) and a number of other toxic heavy metals (Thybaud and LeBrac, 1988; Borgmann and Munawar, 1989). They can be used as “sentinels” of chronic exposure to low concentrations of toxins or as indicators of acute episodes of toxic spills that might otherwise go unmeasured by routine sampling of streams and lakes. Accidental spills of toxins can have major impacts on survival of crustaceans and their ecological roles in maintaining high water quality. Experimental studies demonstrate the importance of detrital processing by benthic crustaceans through comparisons of reference “control” streams with “treated” or disturbed streams (e.g., Newman et al., 1987). Mysids also have become important organisms for monitoring the movement of toxins through foodwebs in large lakes (Evans et al., 1982). The presence or absence of Mysis relicta in different lakes is important for understanding how toxins such as mercury accumulate in top ﬁsh predators because of the major role these crustaceans play in ﬁsh production (Cabana et al., 1994; Branstrator et al., 2000). C. Life-History Traits 1. Longevity Although most small species appear to be “annuals” and may complete their life cycle within a single year, larger amphipod species such as Monoporeia are thought to live for two years or more. Troglobitic species may live even longer (5 – 6 years) in stable habitats with low but relatively continuous inputs of organic detritus as food sources. Long-term data demonstrate 19. Introduction to the Subphylum Crustacea cyclic population oscillations, although control mechanisms for the observed population growth cycles are not clear. Generally, effects of hydrographic and nutrient cycles on pelagic primary productivity and related changes in food quality and quantity on amphipod fecundity and secondary production are well documented (Hynes and Harper, 1972; Siegfried, 1985; Sarvala, 1986; Johnson and Wiederholm, 1989; Dehdashti and Blinn, 1991). Isopods inhabiting freshwater and saline ponds typically have an annual life cycle in contrast to the longer lifespan of four years for many terrestrial isopods (Sastry, 1983). Caecidotea recurvata, a cavedwelling isopod, is known to live at least four years (D. C. Culver, personal communication). 2. Patterns of Reproduction Generally, Peracarida mate at, or shortly after, the time of the female molt (Sastry, 1983). In most amphipods and isopods, females produce only a single brood during the annual life cycle. Seasonal peaks in annual reproduction occur in Gammarus minus populations in spring habitats but this seasonal pattern in less distinct in cave populations (Culver et al., 1995). Some species are known to reproduce continuously and have multiple broods. For example, the subterranean isopod Caecidotea tridentata apparently reproduces throughout the year (Edler and Dodds, 1996). Amphipods, such as Hyalella azteca, can produce multiple broods during an extended breeding season (Cooper, 1965; Strong, 1972). Adult females can reproduce at every stage of molting. They carry their eggs in the marsupium and after fertilization continue to bear their young until they release the fully developed juveniles (ranging from a few to more than 20 per brood). Mateguarding is known to occur in some species with timelimited reproductive cycles (e.g., Jormalainen and Shuster, 1999). Some families of amphipods differ in how mating occurs. The Pontoporeiidae have a pelagic mating system while the Gammaridae generally have a benthic mode (Strong, 1973; Naylor and Adams, 1987; Bousﬁeld, 1989; Elwood and Dick, 1990; Wen, 1993). Several types of broad life-history patterns have been documented for both North American and European species (e.g., Cooper, 1965; Strong, 1972; Gee, 1988; Tadini et al., 1988). In general, the cold-spring faunas have longer life spans, produce fewer eggs, and lack marked seasonality in their reproduction compared to warm-water taxa, which only live one year and produce large numbers of young in the spring. A slowing down of reproduction during late summer is reported in some amphipod populations and may be related to food scarcity or shifts in breeding behavior controlled by other factors. In Gammarus minus the onset of amplexus is apparently induced by a rapid 795 decline in temperature (D. C. Culver, personal communication). In widely distributed, warm-water species such as Hyalella azteca, day length has been ruled out as a controlling variable for triggering the onset of reproduction (Strong, 1972). For cold-water species such as Gammarus lacustris, a period of short days and long nights (typical of winter) is needed to induce reproduction (DeMarch, 1982). In Monoporeia afﬁnis, the boreo-arctic amphipod, constant illumination inhibits gonadal development (Sergestrale, 1970). Peracarid activity patterns on some substrates are also known to be inﬂuenced by the presence of predators (Newman and Waters, 1984; Malmqvist and Sjostrom, 1987; Holomuzki and Short, 1988, 1990; Holomuzki and Hoyle, 1990; Holomuzki and Hatchett, 1994; Wooster, 1998). These behavioral changes may inﬂuence life-history patterns in certain habitats. For example, different populations of the streamdwelling isopod Lirceus fontinalis responded to different risks of predation from banded scuplins (Cottus carolinae) or streamside salamanders (Ambystoma barbouri) by maturing at larger sizes in those habitats where gape-limited salamanders were the dominant predators (Sparkes, 1996a, b). Endoparasites also inﬂuence isopod and amphipod behavior and may make some individuals more vulnerable to predators by increasing their tendency to drift (McCahon et al., 1991; Hechtel et al., 1993, Bakker et al., 1997; Maynard et al., 1998). In some cases the infected individuals are more active during daylight or more visible due to changed coloration. In these ways the parasite increases host vulnerability to predators and can enhance transmission of the parasite from the initial crustacean host to deﬁnitive vertebrate hosts such as ﬁshes and birds that completes the parasite’s life cycle (Bethel and Homes, 1977; Camp and Huizinga, 1979; Bakker et al., 1997). D. Biogeography The distribution and abundance of any species reﬂects a large number of variables that may have interacted over very long periods. Climatic changes have clearly inﬂuenced the stability of aquatic habitats and their availability for colonization by crustaceans for millions of years. The most recent glacial advances and retreats were associated with not only major ﬂuctuations in global temperatures, but also with changing water balances of lakes and river drainage ecosystems. When much of the earth’s water was frozen in massive ice sheets, the levels of many lakes and rivers were much reduced in the temperate zones and throughout the Neotropics. As discussed later in this section, important refugia from thermal extremes and desiccation 796 A. P. Covich and J. H. Thorp have persisted in below-ground habitats such as groundwater-fed caves. The mechanisms of transport and available routes of dispersal for a species are primary factors in limiting access to new habitats and recolonization of old habitats that might have had major climatic disturbances. Physical and chemical barriers to entry can also limit range expansions, as may biological barriers such as the presence of predators or strong competitors already established in adjacent waters (MacNeil et al., 1997; 1999b). Among the Peracarida, there is a pattern of restricted distribution for many species (sometimes to a single lake or spring-fed stream) that has interested biologists, geologists, and geographers. A few species are widespread and seem to tolerate a broad range of ecological parameters. For example, Gammarus lacustris occurs throughout most of the northern and parts of the western United States. Hyalella azteca is present from Canada to South America in the littoral zone of glacial lakes as well as in small ponds and streams. Several species of Crangonyx are frequently found in the southeastern United States as well as in Canada (Bousﬁeld and Holsinger, 1989). Habitat modiﬁcation has changed some amphipod distributions in historic times, such as the likely effects of dam building and regulation of ﬂow regimes (e.g., Beckett et al., 1998). Many species are considered to be relatively rare. The isopod, Thermosphaeroma theromophilum, has been declared legally endangered; it is restricted to a currently protected hot-spring habitat near Socorro, New Mexico (Bowman, 1981). As discussed below some cave species are highly restricted in their distributions and some endemic species occur in only a single cave (Holsinger, 1991; Holsinger, 1994a, b). Recent reviews provide examples of threats to survival of crustacean species (Elliott, 2000; Ricciardi and Rasmussen, 1999; Culver et al., 2000; Master et al., 2000). 1. Groundwaters The greatest number of amphipod species are found in subterranean interstitial habitats, and many are associated with caves or deep wells (Barr and Holsinger, 1985; Holsinger, 1993; 1994a). Bousﬁeld (1983) suggested that more than 1000 hypogean amphipod species will eventually be identiﬁed. J. R. Holsinger (personal communication) completed a worldwide survey and found that 117 genera have subterranean species; approximately 615 species are fully adapted to live in groundwaters. There are about 116 species that live in groundwaters in North America, north of Mexico. Holsinger (1972) estimated that 65 – 70% of the North American amphipod fauna occurs in these groundwater habitats; such species are known generally as “stygobionts.” He pointed out that not all of the stygobiont species are restricted to cave waters per se (true cave-dwelling species are termed “troglobites”), yet they may have similar morphological specializations associated with cave living (e.g., loss of eyes and pigmentation). Some cave-dwelling species are endemic to a single location within one county. Generally, the geographic locations of obligate cavedwelling species are highly restricted and these subterranean communities appear regionally undersaturated. Culver et al. (2000) report that over 50% of all the aquatic obligate cave species and subspecies occur in only 19 counties (out of 3112) that comprise less than 1% of the land area in the 48 contiguous states. Most taxa are concentrated in Hays County, Texas or in a few counties in Florida, Oklahoma, Texas, Virgina, and West Viriginia. This high concentration of subterranean taxa in a relatively few locations makes these populations highly vulnerable to habitat destruction. For example, deforestation and dumping of sawdust (into sink-hole cave from a sawmill operation and leachate into groundwater from sawdust piles) in southwestern Virginia led to listing a cave stream isopod (Lirceus usdagalun) as an endangered species (Jacobs, 1991; Culver et al., 1992). Although many subterranean populations are at risk, only a few have protection through federal status as rare or endangered. Groundwaters are insulated from many of the environmental ﬂuctuations that characterize surface waters, but prolonged droughts or massive ice formations associated with glaciation might eliminate these otherwise “constant” habitats. Until recently, it was thought that the few subterranean amphipods and isopods known from glaciated regions represented postglacial colonization from nonglaciated border regions to the south. Subglacial refugia are now thought likely to have persisted during the last glacial episode (known as the Wisconsin, which ended about 12,000 years ago). For example, Castleguard Cave in Alberta, Canada, is currently inhabited by the asellid isopod Salmasellus steganothrix and the crangonyctid amphipod Stygobromus canadensis. The amphipod is endemic to this single cave and was found about 2 km from the entrance. Eleven of the 100 species described in the genus Stygobromus are known to occur in various localities north of the southernmost boundary of the Wisconsin episode of Pleistocene glaciation (Holsinger et al., 1983). Thus, the action of glacial scour of surface habitats did act as a barrier to northern distributions of many species, but others were adapted to subsurface waters and found refuge. The ﬂuctuations of sea levels associated with Pleistocene glaciations and earlier Tertiary events also created barriers to distributions of Peracarida. Colonization of coastal streams and subterranean habitats by 19. Introduction to the Subphylum Crustacea marine-derived species has resulted in a complex mosaic of biogeographic boundaries. Initially, those organisms that adapted to brackish waters were able to escape most of their marine-based ﬁsh predators and to obtain abundant food resources in freshwater habitats. As shallow Cenozoic marine waters receded, the isolated populations of amphipods and isopods developed distinct adaptations; subsequent speciation produced many new taxa (Holsinger, 1986; Notenboom, 1991). In isolated refugia, these species diversiﬁed over a 70million year period (possibly much longer) with apparently only slow and limited active dispersal. Because many of these underground habitats are deep aquifers, the faunal composition is difﬁcult to sample. The Edwards Aquifer in south-central Texas has been studied intensively (Holsinger and Longley, 1980), and a unique ancient fauna of at least 22 species, including 10 amphipod species, is known to live in this stable groundwater habitat. Abele (1982) concluded that this artesian well in San Marcos, Texas, contains probably the richest cave crustacean fauna in North America. 2. Surface Waters Today, the peracarids occupy many types of surface waters that range widely in physical and chemical characteristics. Historically, the chemical barrier of dilute water was a major isolating mechanism (Vernberg and Vernberg, 1983) in the evolution of crustaceans. In contrast to the frequently isolated subsurface populations and subpopulations, relatively wider geographic distributions and frequent gene ﬂow can occur among surface-dwelling, interbreeding populations in large lakes and ponds interconnected by rivers. Long-distance transport by individuals adhering to mud carried by migratory birds, water fowl, and other animals is considered rare among amphipods but might infrequently provide wider dispersal by those individuals that do survive short-term drying. Some species of isopods are reported to move through subterranean waters and into the surface waters for certain periods (e.g., Minckley, 1961). The importance of hyporheic habitats as spatial and temporal refugia for many types of benthic crustaceans and insects is now recognized as being of great importance, especially in previously glaciated regions where massive unconsolidated rock and sand deposits characterize the ﬂoodplains of large rivers (Stanford and Ward, 1988). 3. Barriers to Dispersal and Population Genetics Neighboring populations can be genetically isolated from one another by many types of physical barriers. Dispersal of individuals to distant headwaters of many fast-ﬂowing streams is limited within a drainage area unless there are subsurface connections among 797 aquifers or many short tributary branches of streams that connect. Gooch (1989) quantiﬁed seven levels of isolation in his studies of Gammarus minus in the Appalachian Mountains of West Virginia and Pennsylvania. The larger the barrier, the more isolated were the populations. He has shown that there is a cline of lower genetic variability along a sequence of northern populations. These populations have apparently recolonized habitats since the last glacial advance and now occupy what was once a periglacial zone or perimeter around the ice sheets (Gooch and Glazier, 1986). The importance of the geomorphological setting in determining how gammarid populations are actively and passively distributed is also clear in other studies of karst populations (e.g., Gooch, 1990; Gooch and Glazier, 1991; Culver and Fong, 1994). In addition to the studies on Gammarus mentioned above, other investigations have focused on Hyalella and these also provide important new information about adaptations among distinct populations and subpopulations. For example, in the glacial terrain of southeastern Michigan’s there is evidence for recent divergence among populations of two ecotypes of Hyalella azteca (Saussure) that differ in terms of adult body size (McPeek and Wellborn, 1998). The largebodied adults from different populations found in lakes interbreed as do the small-bodied adults found in marshes. In contrast, individual adults of different ecotypes do not interbreed. This type of recent divergence may have resulted from enhanced mating success favored through sexual selection and mediated by ﬁshpredation selection in the lake populations of different Hyalella (Wellborn, 1994, 1995, 2000). In Arizona, there are distinct populations of Hyalella azteca living in different lake habitats. One group of populations lives in submerged vegetation while another group clings to roots of emergent vegetation (Thomas et al., 1997). Studies have examined genetic differences and behavioral differences in swimming among different populations of Hyalella azecta from Oregon to Arizona (Thomas et al., 1997, 1998) and suggest that xeric landscapes promote genetic and behavioral divergence among amphipods, presumably because of the long distances among freshwater habitats. Habitat preferences and possible segregation of populations likely occur in other amphipod species living in other structurally complex, stable habitats (e.g., Muller, 1998; Muller et al., 2000) or habitats that vary temporally and spatially such as groundwater-fed cave streams and marshes. Several other studies have considered the importance of habitat structure in the dispersal and population genetics of peracarid crustaceans (Hedgecock et al., 1982; Meyran and Taberlet, 1998; Meyran et al., 1997, 1998). The degree of genetic isolation among 798 A. P. Covich and J. H. Thorp populations distributed within a drainage basin is usually related to topographic complexity and elevational gradients for many types of slow-moving stream invertebrates (Siegismund and Muller, 1991; Muller, 1998; Muller et al., 2000). As discussed above, dispersal by downstream drift and infrequent upstream movements limits gene ﬂow among subpopulations within river drainage networks. Long, high ridges dissect the landscape and form effective barriers to the mixing of genes within and among river networks and associated lake basins. 4. Comparisons Among Ancient Lakes Isolation by topographic structure may also be important for benthic crustaceans in deeply dissected lake basins, as apparently has been the case in the ancient lakes such as Lake Titicaca in the Andes of Bolivia and Peru where some 11 species of Hyalella have evolved. One of the most interesting cases of speciation and adaptive radiation among freshwater invertebrates is the high species richness of amphipods in Lake Baikal, Siberia, the oldest and deepest lake in the world where nearly 2000 metazoan species occur (Brooks, 1950; Martens et al., 1994; Yampolsky et al., 1994; Kozhova and Izmest’va, 1998; Kamaltynov, 1999). Almost 25% of the total worldwide freshwater amphipod fauna known to exist today occurs in Baikal, and nearly all of these species are restricted to this single ancient lake (Kozhov, 1963; Martens, 1997; Martens and Schon, 1999; Martens and Danielopol, 1999). The progenitors of the modern fauna can be traced to much earlier Mesozoic brackish-water basins. Major climatic changes during the Tertiary are thought to have accelerated the amphipod species radiation within the Baikal basin during the last 25 – 30 million years (Kamalynov, 1999). Several endemic species also occur within the Baikal drainage in the Angara and Enisei Rivers (Kamaltynov, 1992). In some cases, the locations of the outlets and inlets of rivers apparently create barriers for dispersal along the lake shore and result in genetic separation of different localized populations (Mashiko et al., 1997). There are at least 250 gammarid species in about 45 genera that have evolved specialized niches in this uniquely deep lake (maximal depth is over 1700 m). Some are armed with sharp cutaneous spines or ridges, and they range in size from 10 to 80 mm. Coloration is highly varied (red, green, yellow, violet, pink, and brown) with complex patterning, which is very unusual among freshwater invertebrates. A phylogenetic analysis of 18 of the endemic genera suggests that parallel evolution of body armament and large body size occurred among different genera during the history of the lake. The ﬁrst lineage was apparently benthic and mostly unarmed; the second lineage was composed of mostly armed taxa and were primarily detritivorous and carnivorous (Sherbakov et al., 1998). The evolutionary ages of the various taxa are being studied using molecular techniques such as analysis of mitrochondrial DNA and allozymes (Mashiko et al., 1997; Ogarkov et al., 1997; Vainola and Kamaltynov, 1999). Because of the combination of low water temperatures and very deep circulation, there is sufﬁcient dissolved oxygen in this lake to allow benthic gammarids to live in several depth zones. Deep-water (1400 m) forms have evolved different modes of surviving high hydrostatic pressure (Brauer et al., 1980a; b). Littoral forms are extremely abundant, reaching densities of 30,000 or more per square meter. One endemic species of gammarid amphipod, Macrohectopus branickii, is a dominant pelagic omnivore. Stable isotopic analyses of N-15 demonstrate that individual consumers change their mode of feeding to consuming more zooplankton than phytoplankton as they grow larger (Yoshii et al., 1999). This relatively large (maximum body length 38 mm) species is a major prey species of several ﬁshes, especially omul, Coregonus autumnalis migratorius and pelagic sculpins. In the more productive bays in the middle basin of Lake Baikal, Macrohectopus forms dense aggregations and migrates vertically from 250 m depths to upper waters at night (Melnik et al., 1993). Using vertical tows of especially designed closing nets and hydroacoustic equipment Rudstam et al (1992) were able to determine day and night distributions of individuals and biomass in Barguzin Bay. They found that these aggregations were dominated by immature females (15 mm long) in late summer and contained some nektobenthic species of amphipods as well. The migratory patterns and apparent avoidance of ﬁsh predators by Macrohectopus in very similar to Mysis migrations in Lake Michigan and other North American deep lakes (Lasenby et al., 1986; Rudstam et al., 1992). Unlike the high species richness of amphipods, the isopods in Baikal are represented by a single genus, Asellus, with only ﬁve species. Generally, the diversiﬁcation of crustaceans reﬂects differences in the response of isolated populations to local and regional conditions of long-term habitat stability, plasticity of life-history characteristics, predator – prey interactions, and access to limited food resources. IV. COLLECTING, REARING, AND PREPARATION FOR IDENTIFICATION Most amphipods and isopods can be collected by sweeping a dip net through the littoral zone of lakes and ponds or in vegetated reaches of streams. Mysids 19. Introduction to the Subphylum Crustacea require a large diameter plankton net that can be used for deep vertical tows. Being fast swimmers, mysids can avoid small nets and are more difﬁcult to collect. Quantitative sampling of amphipods and isopods has relied on drift nets and Surber samplers in fast-ﬂowing streams and Ekman grab sampler in shallow ponds and lakes. Drift nets can entrap organisms that may be moving upstream as well as those drifting downstream (if the net is not positioned off the bottom of the stream), and, thus, interpretation of studies dealing with dispersal and migration can be complicated. The use of cores (e.g., Milstead and Threlkeld, 1986), can be very effective for intensive study of microhabitats. Considerable time can be spent in sieving or sorting specimens; elutriators increase the efﬁciency of separating specimens from the inorganic and organic matrix (Magdych, 1981). Baited traps with various mesh sizes also work well for quantitative sampling of isopods and gammarids that respond to current-dispersed chemicals given off by the bait (e.g., Fitzpatrick, 1983; Allan and Malmqvist, 1989). Sorting of specimens is relatively rapid, but construction of ﬁne-meshed traps is necessary. Use of electroﬁshing equipment may also be useful in stunning larger decapod crustaceans so that they can be swept up more effectively with dip nets. Chemicals have been added to stun or poison animals, but these methods are not very effective and clearly can be very detrimental to the environment. As discussed previously, many species are extremely sensitive to even small concentrations of toxic chemicals and are used to monitor water quality. Several papers provide details on care and feeding of laboratory stocks (e.g., Cooper, 1965; Strong, 1972; DeMarch, 1981a, b; Sutcliffe et al., 1981; Barlocher et al., 1989). Dead leaves conditioned with nutritious microbial growths of bacteria and fungi are sufﬁcient food resources for most species of amphipods and isopods. These can be supplemented with cultures of green algae and diatoms or with boiled lettuce or spinach. As in rearing other taxa, the initial water used for setting up an aquarium must be well aerated and allowed to develop an adequate microbial community before the crustaceans are placed into the tank. Temper- 799 ature ﬂuctuations can be detrimental and some type of aquarium heater is useful to maintain optimal growing conditions. Parasites and diseases are, of course, widespread in natural populations (e.g., Pixell Goodrich, 1934; Brownell, 1970; Laberge and McLaughlin, 1989), and brood stock from previously reared laboratory cultures are less likely to suffer from these outbreaks as the complex life histories of many parasites require a variety of vertebrate and invertebrate hosts to maintain the life cycle. Preparation of specimens for study usually requires rapid ﬁxation and storage of ﬁeld-collected organisms in vials of 60 – 70% ethyl alcohol. The alcohol will remove any pigment from the specimens, so careful notes or photographs will be useful if information on coloration or patterns of body markings are of interest. The long-term storage of specimens requires checking to ensure that the alcohol has not evaporated. Addition of a 5 – 20% solution of glycerin to the vials will protect the specimens from drying out. Glycerin jelly is a solid at typical room temperatures, and it is widely used as a permanent mounting medium. Thus, if the material is ﬁrst stored in dilute glycerin, it is convenient to transfer specimens to slides for study and for preparing reference collections. These techniques are described in detail in several texts (e.g., Pennak, 1989; Peckarsky et al., 1990). V. CLASSIFICATION OF PERACARIDA AND BRANCHIURA The taxonomic key for this chapter is intended to allow the reader to identify major taxa of amphipods, isopods, mysids, and Branchiura to the generic level. This key draws on characteristics used in previously published studies (Edmondson, 1959; Williams, 1970; Cressey, 1972; Holsinger, 1972, 1989, 1994b; Fitzpatrick, 1983; Pennak, 1989; Peckarsky et al., 1990); some of these sources can be used to identify organisms to species level, others are useful for determining biogeographical distributions (e.g., Barnard and Barnard, 1983; Banarescu, 1990). A. Taxonomic Key to Major Freshwater Genera of the Orders Mysidacea, Amphipoda, and Isopoda and the Subclass Branchiura 1a. Four pairs of legs and two prominent, highly modiﬁed suckers on upper ventral surface, specialized ectoparasite on ﬁshes (Fig. 6)..... .................................................................................................................................................Subclass Branchiura, order Arguloida, family Arguilidae .....................................................................................................................................................................Argulus 1b More than four pairs of walking legs (pereiopods) prominent second antennae, compound eyes ..................................................... 2 2a(1b) Five pairs of legs, body ﬂattened laterally (Fig. 7) .................................................................................................order Amphipoda 4 800 A. P. Covich and J. H. Thorp 2b Six or seven pairs of legs, body ﬂattened dorsoventally .................................................................................................................... 3 3a(2b) Seven pairs of legs, posterior legs longer than anterior legs, unstalked eyes (Fig.8) .....................................................order Isopoda 8 3b Six pairs of legs, prominent stalked eyes (Fig. 9) .......................................................................................................order Mysidacea, family Mysidae ...........................................................................................................................................................................Mysis 4a(2a) Amphipoda: antenna 1 shorter than antenna 2 (Fig. 7) .................................................................................................................... 5 4b Antenna 1 longer than antenna 2 (Fig. 7) ......................................................................................................................................... 6 5a (4a) Accessory ﬂagellum present on antenna I with 3 – 5 segments (Fig. 7), telson deeply cleft but not to base ..........family Pontoporeiidae .........................................................................................................................................................................................Monoporeia 5b Accessory ﬂagellum absent on antenna 1, telson entire .................................................family Hyalellidae .............................Hyalella 6a (4b) Accessory ﬂagellum of antenna 1 with 2 – 7 segments (usually 3 or more), telson typically cleft to base ................family Gammaridae ...........................................................................................................................................................................................Gammarus 6b Accessory ﬂagellum of antenna 1 with never more than 2 segments, telson usually not deeply cleft but never to the base.................... .....................................................................................................................................................................family Crangonyctidae 7 7a (6b) Eyes may be present and pigmented, apical margin of telson distinctly cleft........................................................................Crangonyx 7b Eyes absent, apical margin of telson entire or with shallow cleft ......................................................................................Stygobromus 8a (3a) Isopoda: margin of head with a pointed median protuberance (carina) between bases of ﬁrst antennae (Fig. 8), eyes typically present ...........................................................................................................................family Asellidae .............................................Lirceus 8b Anterior margin of head without carina, eyes present or absent...............................................................................family Asellidae 9 9a (8b) Abdomen broader than long, up to 16 mm long...................................................................................................................... Asellus 9b Abdomen not broader than long ....................................................................................................................................... Caecidotea ACKNOWLEDGMENTS We greatly appreciate reviews and comments by David C. Culver and Dean W. Blinn as well as help from John R. Holsinger with several key references. Jennifer L. Borash provided scanning electron images of crayﬁsh chelae. Rebecca Rudman assisted with a thorough proof-reading and many authors provided reprints of their studies. LITERATURE CITED Abele, L. G. 1982. Biogeography, in: Bliss, D. E., Ed., The biology of Crustacea. vol. 1. Systematics, the fossil record and biogeography. Academic Press. New York, pp. 242 – 304. Ache, B. W. 1982. Chemoreception and thermoreception, in: Atwood, H. L., Sandeman, D. C., Eds., The biology of Crustacea. Vol. 3. Academic Press. New York, pp. 369 – 398. Adare, K. I., Lasenby, D. C. 1994. 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Hatching and Seasonality E. Molting F. Competition, Coexistence, and Survival IV. Distribution A. Benthic Swimmers B. Benthic Nonswimmers V. Chemical Habitats A. Chemical Water Types B. Salinity and Solute Composition C. Hydrogen Ion Concentration D. Dissolved Oxygen E. Carbonate VI. Physical Habitat A. Temperature B. Lakes, Ponds, and Streams C. Bromeliads D. Cave and Subterranean Habitats I. INTRODUCTION The class Ostracoda in the Crustacea contains both marine and freshwater forms. The subclass Podocopa has one order, Podocopida in which two of the suborders (Metacopina, Podocopina) contain all of the fresh1 Current address: 621 Auburn Crescent, Burlington, Ontario, Canada L7L 5B3 Ecology and Classiﬁcation of North American Freshwater Invertebrates. 2nd Edition Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved. VII. VIII. IX. X. XI. XII. XIII. XIV. E. Sediment – Water Interface, Particle Size, Food Physiological and Morphological Adaptations A. Eggs B. Resting Stage and Torpidity C. Coloration D. Appendages Behavioral Ecology Foraging Relationships Population Regulation A. Suitability of Habitat B. Toxicity of Herbicides and Pesticides C. Ultraviolet-B Radiation D. Parasitism Collecting and Rearing Techniques A. Collecting and Preservation B. Rearing Techniques Uses of Freshwater Ostracoda Current and Future Research Problems Classiﬁcation of Ostracodes A. Taxonomic Controversies B. Classiﬁcation of the Order Podocopida C. Taxonomic Key to Genera of the Superfamily Cypridoidea D. Taxonomic Key to Genera of the Superfamily Cytheroidea Literature Cited water ostracodes (Bowman and Abele, 1982; Schram, 1986). Ostracodes are the oldest known microfauna. Their preservation as fossils is attributed to the calcitic shell present in most species. The ﬁrst fossil representatives are reported from Cambrian marine sediments (Moore, 1961; Maddocks, 1982). The earliest freshwater ostracode species came from coal-forming swamps, ponds, and streams of early Pennsylvanian age (Benson, 1961). 811 812 L. D. Delorme According to Moore (1961), the Podocopina evolved from Podocopida stock during the Ordovician period, and then branched out into the Cytheroidea and the Cypridoidea during the Devonian period (Maddocks, 1982). The Darwinulidae evolved during the Carboniferous period (Maddocks, 1982). Freshwater ostracodes are free living except the Entocytheridae (Hoff, 1942) which are commensal upon crayﬁsh. This chapter will concentrate on freshwater ostracode genera found in North America. To date there have been 56 genera and about 420 species identiﬁed for this continent. Most references older than 1900 have been deleted from the second edition. Those interested in the older references are referred to the original edition. II. ANATOMY AND PHYSIOLOGY A. External Shell Morphology The visceral mass is completely encased within a chitinous and a low-magnesium calcite exoskeleton (carapace). The only exception is the entocytherid group of ostracodes which lacks calcium in the exoskeleton. The exterior surface of the exoskeleton (cuticle) is made up of chitin (Fig. 1) (Harding, 1964). The outer chitinous coating of the epidermis is the only covering over the calcitic shell. The soft epidermal tissue lining the interior part of the carapace is composed of the inner and outer epidermis. The epidermal cells of the outer lamella (Fig. 1) are larger and more irregular in shape than the inner epidermal cells. Subdermal cells occur between the inner and outer epidermal cells as do the liver, ovaries, and testes. The epidermis is contained within a thin chitinous lining. When the ostracode molts, the complete exoskeleton is shed leaving only the visceral mass and the soft tissue or epidermis. The epidermis is continuous with the visceral mass in the dorsal region of the body. When the ostracode molts, the space between the epidermal cells of the outer and inner layer is empty. The shells are secreted by the cells of the outer lamellae (Turpen and Angell, 1971). The immediate source of calcium and magnesium carbonate is thought to be stored in the body and not obtained directly from the water (Fassbinder, 1912). Fassbinder has also shown that ostracodes fed with crushed calcium carbonate from snail shells developed thicker carapaces with more pronounced marginal ornamentation. He concluded that food is an important source of carbonate. Turpen and Angell (1971), however, found that calcium dissolved in water is the source of calcium for the shell. They used dissolved 45Ca as a tracer (pH 7.4, 23°C) to FIGURE 1 Diagrammatic cross section of the shell and the enclosing dermal layers, vestibule located between the outer lamella and the shell portion of the inner lamella in the anterior portion of the shell. [From Kesling, 1951a, b. Figure redrawn by J. L. Delorme.] show that calcium is not resorbed from the shells prior to molting and that ostracodes do not store calcium in their bodies. Chivas et al., (1985) ran laboratory experiments on the Australian Mytilocypris henricae and found that temperature inﬂuenced the rate of shell calciﬁcation. Roca and Wansard (1997) also found the rate of development to be linked to temperature. Using Herpetocypris brevicaudata they were able to show that below 15°C the Ca content of valves is strongly diminished and the length of time for calciﬁcation was longer (17 days). The optimum temperature was 23°C with the fastest rate of calciﬁcation (3 – 7 days). Bodergat (1978) demonstrated that phosphorus was an integral part of freshwater ostracode shells in all specimens investigated with a microprobe. She further noted a signiﬁcant positive correlation between the amount of shell phosphorus and the habitat temperature of the ostracode. The calcareous shell is the most obvious structure when the organism is viewed under a microscope. The shell is divided into two parts, the outer lamella and the duplicature (Fig. 1). The outer lamella is the major part of the shell; whereas the duplicature is the calciﬁed part of the inner lamella bordering the posterior, ventral, and anterior margin. It forms a part of the free margin and projects toward the center of the carapace. The outer lamella may contain pores through which 20. Ostracoda project setae (Fig. 2), that are sensitive to touch. The duplicature is welded to the outer lamella (Fig. 3) around the margins, by a strip of chitin, which joins with the chitin coating and the chitin lining. This juncture is referred to as the line of concrescence or the distal line of adhesive strip (Fig. 1). Within the chitinous strip, radial or marginal pore canals are found (Fig. 2). These are very ﬁne tubes through which setae pass. The inner free margin of the duplicature is referred to as the inner margin (Fig. 1). When the duplicature extends beyond the line of concrescence toward the center of the shell, a shelf is formed (Fig. 3). The space between the shelf and the outer lamella is referred to as the vestibule (Fig. 1). Several structures are found on the duplicature which are very important in taxonomy down to the species level (Sylvester-Bradley, 1941). Pustules, teeth, or crenulations (Fig. 3) may appear on the outer margin of the duplicature. A combination of septa, lists, and grooves are found on the duplicature. Some of these are shown in Fig. 1 (Kesling, 1951a, b). The shell may contain additional structures on the outer lamella such as lateral depressions called sulci (Fig. 30). Raised areas variously termed alae, knobs (Fig. 30), or pustules (Fig. 4), depending on their shape, size, and orientation, may also be found on the shell surface. The surface of the shell may also be pitted (Fig. 5), punctate, wrinkled (Fig. 6), or have a reticulate surface (Fig. 7). These ancillary structures of the shell are very often used for generic and speciﬁc identiﬁcation (see Sylvester-Bradley and Benson, 1971, for terminology). Some authors, notably Benson (1974, 1981), have discussed the roles of ornamentation, form, function, and architecture of the ostracode shell. Intraspeciﬁc morphological diversity of the shell may be pronounced. Limnocythere itasca has short recurved alae on its lateral surfaces. The individuals identiﬁed from Canada (Delorme, 1971a) and northern Minnesota (Forester et al., 1987) appear to be characteristic of this species. R. M. Forester (personal communication) identiﬁed L. itasca from the Laguna de Texcoco, Mexico, which have very long and delicate recurved spines or alae. Intermediate forms have yet to be recovered. In another example, the caudal process of the posteroventral portion of the right valve of the female Candona caudata (may belong to Fabaeformiscandona; see Meisch, 1996, not conﬁrmed for North America) may be blunt (Delorme, 1978) or sharp ( C. novacaudata, Benson and MacDonald, 1963). Both forms and intermediate morphs are found in Lake Erie. B. Internal Shell Morphology Structures on the internal surface of the shell may also be used in ostracode systematics. Many ostracode 813 muscles are attached directly to the calcareous shells. The chitinous exoskeleton does not provide sufﬁcient rigidity to anchor many of the muscles. The points of attachment of these muscles leave scars as raised or depressed areas on the interior of the shell (Figs. 8-11). The closing or adductor muscles form a distinctive pattern of scars as do the mandibular muscles (Benson, 1967; Benson and MacDonald, 1963; Smith, 1965). Traces of the ovaries and testes can also be seen in the posterior of the shell (see Section II.D). C. Body and Appendage Morphology Ostracodes have their body (visceral mass and appendages) suspended from the dorsal region in an elongate, chitinous pouch. The ostracode has a shortened body with a slight constriction in the midregion that separates the head from the thorax (Kesling, 1951a). There is no abdomen. Instead, the posterior of the body tapers off bluntly and ends in a pair of preanal furcae. The head region contains four pairs of appendages which are used for swimming, walking, and feeding. The thoracic region features two pairs (three pairs according to some authors) of appendages used or adapted for feeding, creeping, and cleaning of the shells (Broodbakker and Danielopol, 1982). There is some debate as to whether the maxilla are cephalic or thoracic appendage (Meisch, 1996) Ancillary cuticular, structures such as setae, claws, and pseudochaetae, found on most limbs, are recognized as important in functional morphology and systematics (Broodbakker and Danielopol, 1982). Appendages are shown in Figs. 9 – 12. The head region is composed of the forehead, the upper lip, and the hypostome. The four pairs of appendages in the head region are the ﬁrst antennae (antennules), second antennae (antennae), mandibles, and maxillae. When the distal four podomeres of the ﬁrst antennae have very long, plumose setae, the ostracode is a good swimmer (e.g., Cypridopsis vidua). The ﬁrst antennae are curved up and backward. If these setae are absent, as in the darwinulids (Fig. 10) and the limnocytherids (Fig. 11), the ostracode cannot swim. The second antennae extend down and curve backward. These appendages are robustly constructed and have a strong claw for walking and climbing. Swimming setae may be present on the ﬁrst podomere of the endopodite. These structures are used for rowing when the ostracode is swimming. In most species, welldeveloped mandibles occur between the upper lip and the hypostome. The dorsal tips of the mandibular palps are attached to the interior of the shell by muscles. The ventral portion of the palps terminate in heavily toothed mandibles; these teeth grind the food before it FIGURE 2 Shell surface of Cyclocypris ampla Furtos showing normal pore canals with funnels (without seta) and without funnels (with seta), and dimples. FIGURE 3 Anterior margin of Cyprinotus glaucus Furtos showing crenulations, duplicature with position of radial pore canals, and calciﬁed inner lamella forming a shelf. FIGURE 4 Anterior margin of Cyprinotus glaucus Furtos with pustules on the right valve, left valve beneath. FIGURE 5 Reticulate exterior structure on Limnocythere itasca Cole. FIGURE 6 Anastomosing structure (lines) on the surface of Cypria turneri, note normal pore canals. FIGURE 7 Reticulate structure with superimposed mamilliary pustules on Paracandona euplectella Brady and Norman. FIGURE 8 Distinctive muscle scar pattern of Darwinula. 814 20. Ostracoda 815 FIGURE 10 Sketch of the internal morphology of a female Darwinula stevensoni (Brady and Robertson) (Darwinulidae) from Kesling. First thoracic leg is referred to as the maxilla or maxillar endopodite and the second and third thoracic legs as the ﬁrst and second thoracic legs in the text. [Figure redrawn by J. L. Delorme.] FIGURE 9 Sketch of the internal morphology of (a) male (b) female Candona suburbana Hoff (Cyprididae) from Kesling. First thoracic leg is referred to as the maxilla or maxillar endopodite and the second and third thoracic legs as the ﬁrst and second thoracic legs in the text. [Figure redrawn by J. L. Delorme.] FIGURE 11 Sketch of the internal morphology of a male Limnocythere sanctipatricii (Brady and Robertson) (Cytheridae) from Kesling. First thoracic leg is referred to as the maxilla or maxillar endopodite and the second and third thoracic legs as the ﬁrst and second thoracic legs in the text. [Figure redrawn by J. L. Delorme.] is ingested. A respiratory (branchial) plate extends from the mandibular palp. On the ventral portion of the head is located a keel-shaped structure called the hypostome which forms the mouth. Rake-shaped organs are situated at the rear of the mouth. They consist of chitin shafts with terminal toothed structures and are used for straining and feeding. The fourth and ﬁnal set of cephalic appendages are the maxillae. The maxillae, located on either side of the hypostome posterior of the mandibles, are made up of several cylindrical masticatory processes (maxillula). The maxillae pass the small particles toward the mouth. What were previously referred to as the ﬁrst thoracic legs of most cyprids are now referred to as part of the maxillae. In other groups, this part of the maxillae (formerly the FIGURE 12 Generalized sketch of the internal morphology of a male entocytherid (Cytheridae) from Hart and Hart. First thoracic leg is referred to as the maxilla or maxillar endopodite and the second and third thoracic legs as the ﬁrst and second thoracic legs in the text. [Figure redrawn by J. L. Delorme.] 816 L. D. Delorme ﬁrst thoracic legs) are modiﬁed in sexual ostracodes into prehensile palps (Fig. 9a) used in grasping the female shell during copulation. There are two pairs of thoracic appendages. The ﬁrst thoracic legs (previously called the second thoracic legs) are robustly developed and used for walking, climbing, and clinging onto surfaces. The second thoracic leg (formerly called the third thoracic legs), or cleaning legs, are the most ﬂexible and well-muscled appendages. The end of these legs, in most ostracodes, are modiﬁed pinchers used to grasp and remove foreign objects from between the shells. In the entocytherid group, the modiﬁed maxillae and the two pairs of thoracic legs (Fig. 12) are modiﬁed terminally as hooks to hold onto the gills of the host crayﬁsh (Hart and Hart, 1974). Morphological diversity can be seen in the maxillae of ostracodes. Most notable are the ﬁrst thoracic legs. These can be modiﬁed for feeding, for use during copulation (prehensile palps), or for walking. The commensal entocytherids have modiﬁed the maxillae and two thoracic legs for grasping onto the gills of the host. Paired hemipenes are found in syngamic species behind the third thoracic legs on the ventral side of the thorax. Paired uterine openings lie behind and inside the vaginal openings of sexual and asexual females. The distal part of the thorax terminates in paired furcae. In some species, these are whip-shaped structures while in others they are well developed and armed at the end with long, serrated claws. Rome (1969) and Triebel (1953) consider the furcal attachment to be an important biocharacter to be used in systematics. Sexual dimorphism is common in ostracodes. Determination of gender in most cases can be determined visually with a microscope. This is done by observing the presence / absence of testes and traces of the testes on the inner surface of the shell, Zenker’s organs (see Section II.D), modiﬁcation of the male maxillae (formerly called the ﬁrst thoracic leg) into prehensile palps, hemipenes, and ovaries. For many species, it is necessary to separate the valves to view these organs. Except in cyprinotids, the male is usually larger than the female. In candonids, only the male has an anteroventral notch on the shell. D. Internal Anatomy and Physiology The circulatory system of freshwater ostracodes lacks both heart and gills. Gaseous exchange is through the entire surface of the body and particularly the membranous inner lamella of the exoskeleton. The respiratory plates of some appendages move and renew oxygenated water past the inner surfaces of the organisms. Large respiratory cells are known to occur in the inner lamella, forming a respiratory epithelium over the valve cavity which forms a blood sinus (Bernecker, 1909). The digestive system of ostracodes consists of the mouth, esophagus, stomach, intestine, rear gut, and anus. The mouth is a large opening with the large teeth of the mandibular palps on either side. A gland, probably a salivary gland, opens into the mouth. The esophagus is very muscular and leads into the distended stomach. The hepatopancreas (liver) is found in the epidermal layers next to the shells and empties into the anterior part of the stomach. Most of the digestion takes place in the stomach where the food is formed into balls, passes from the stomach through the rear gut for absorption of the nutrients, and exits the anus as fecal pellets. Approximately eight excretory glands, including the liver, have been identiﬁed in freshwater ostracodes. McGregor (1967) showed that for Chlamydotheca arcuata some food particles pass through the hepatopancreas, aiding in the digestion of food. Most glands have been studied in considerable detail (Bergold, 1910; Fassbinder, 1912; Schreiber, 1922; Cannon, 1925; Rome, 1947; Kesling, 1951a, 1965). The nervous system of the ostracode is composed of a cerebrum (protocerebrum, deutocerebrum, and tritocerebrum; Turner, 1896; Hanström, 1924; Weygoldt, 1961), circumesophageal ganglion, and a ventral chain of fused ganglia (Kesling, 1951a). The ﬁrst and second antennae, mandibular palps, and maxillae have small ganglia which connect to the central nervous system. The ostracode has a single median eye made up of three optic cups each containing lenses. The number of cells composing a lens is variable (Novikoff, 1908; Rome, 1947). The black opaque eye lies in the anterior part of the body just below the rim of the shells. When the shells are open, the animal can detect shapes and motion; however, when the shells are closed it can only distinguish light intensity. Subterranean-interstitial (hypogean) ostracodes are blind (Danielopol, 1980a). The ovaries are found in the epidermis next to the shell as a continuous sequence of gametes. The ova are fairly large in the last instar of many species, forcing the chitin coating of the epidermis into the space normally occupied by the shell. When the new shell is secreted, a trace of the ovaries is commonly seen as part of the shell’s inner structure in the Candoninae. The testes are similarly placed and appear as traces on the inner and posterior region of the shell in the Candoninae. For some genera (e.g., Cypricercus) the testes form a spiral around the inner edge of the shell rather than being conﬁned to the posterior area. The testes of Cyprididae change into the vasa deferentia as they leave the epidermis and enter the body cavity. The vas deferens joins up with the ejaculation ducts or Zenker’s organs (Fig. 9a). These large organs are made up of longitudinal muscles 20. Ostracoda in the shape of a tube on which are found radiating bristles and are covered in a chitinous sheath. The alternate contraction and expansion of these paired organs forces the sperm into the hemipenes during copulation. Zenker’s organs lie in a nearly horizontal position in the posterior part of the body cavity and are visible in many live specimens. The hemipenes are very complex structurally. The ostracode must rotate them before copulation can take place (Kesling, 1957, 1965; Danielopol, 1969; McGregor and Kesling, 1969a, b). The testes and ovaries in Cytheridae are found next to the intestine, Zenker’s organs are absent in this family. There are no males in the Darwinulidae. For further details on the anatomy and physiology, the reader is referred to Hoff (1942) and Kesling (1951a, 1965). III. LIFE HISTORY Ostracode eggs are constructed in a way that allows them to withstand physical and chemical extremes. The time of hatching may be soon after being laid or staggered over time indicating diapause. Once the egg is hatched, freshwater ostracodes will go through eight molt stages before becoming a mature adult. The length of time the organism lives depends on the species. A. Egg The freshwater ostracode egg is a double-walled sphere of chitin impregnated with calcium carbonate. The space between the two spheres is occupied by a ﬂuid (Wohlgemuth, 1914). These two characteristics of the egg allow it to withstand desiccation and freezing. Ostracodes develop from eggs which may or may not have been fertilized, depending on whether the species is bisexual or asexual. The eggs are laid singly or in masses, most often in the part of the habitat that will ensure an oxygen-rich environment when the eggs hatch (Wohlgemuth, 1914). Most ostracode eggs are white, but some are green (Cypridopsis vidua) or bright orange (Cyprinotus incongruens, Potamocypris smaragdina). All North American freshwater ostracodes are oviparous except several species of Darwinula and the marine brackish water Cyprideis which are ovoviviparous. These taxa brood their eggs in the posterior of the carapace from which the nauplii are released. Brood pouches are more common in marine ostracodes. B. Reproduction Reproduction in freshwater ostracodes may be either sexual or asexual. Depending on the gender, the ostracode has a pair of genital lobes or hemipenes. 817 Zissler (1969a, b, 1970) studied in detail the formation of sperm in Notodromas monacha. A single nucleus is formed by fusing of caryomeres; it is characterized by three granulated particles and by a double membranebounded region. The body then becomes spindle-shaped which includes the nucleus and the “Nebenkern” derivatives. Further development of the spindle-shaped cells takes place by the extension of two winglike structures, “Flügelstruckturen,” on either side of the nucleus. The nucleus and the winglike structures extend throughout the sperm body, including the thread. The winglike structures are interpreted as providing motility to the sperm. Further differentiation of the sperm occurs between the testes and the vas deferens. Lowndes (1935), following the research of other authors publishing between 1854 and 1889, reassessed the morphology and function of the sperm and concluded, “apparently the sperms are now functionless.” Ostracode sperm have the dubious distinction of being the longest of any sperm relative to the host animal (Lowndes, 1935). Moore (1961) indicated that sperm do serve a function because females only lay eggs after copulation. However, Moore did not cite references to support his claim. Havel et al. (1990a) demonstrated that sperm have a function. Populations with males examined electrophoretically show genotypic frequencies in close agreement with those expected in a randomly mating sexual population. Female-to-male sex ratios appear to vary depending on the species. Chaplin (1993) found that for Candonocypris novaezelandiae there are two morphs. For the “little brown morph” the sex ratio changes from 2.8:1 to 6.9:1 over a 27-month period. The conclusion was that the sex ratios show an increase in the abundance of parthenogenetic females. Dezfuli (1996) reports a sex ratio for Cypria reptans of 2:1. Sex ratios of modern North American ostracodes of 2:1 show a female bias (Chaplin et al., 1994). These authors indicate there are no reports that male eggs are larger than female eggs so there is no logical explanation for the excess of females. Based on earlier cytological studies, Bell (1982) concluded that all ostracodes are diploid. White (1973) indicated the diploid chromosome number varies between 12 and 19. Chaplin et al. (1994) provide an up-to-date review of the sex mechanisms for freshwater ostracodes. Although males are the heterogamic sex, some species have XO or XY sex determination systems. A case of multi-X systems is found in Cyprinotus incongruens where the females have six pairs of X chromosomes and the males have six X and a Y chromosome (Chaplin et al., 1994). Further, they report that males of this species produce two types of sperms, one with ﬁve and the other with 818 L. D. Delorme 10 chromosomes. Lécher et al. (1995) found some parthenogenetic ostracodes have “supernumerary chromosomes,” and that they have complex sex chromosome mechanisms with XO, XY, and multiple X’s and Y’s. Three Canadian sexual ostracodes (Cypricercus splendida, C. deltoidea, and C.tincta) possess 22 chromosomes (Turgeon and Hebert, 1995). For C. deltoidea and C. tincta meiotic pairing was seen. These authors further indicate the asexuals, Cypricercus horridus, C. reticulatus, and C. fuscatus, had variable chromosome counts and no evidence of meiotic pairing was observed. Electrophoretic studies by Havel et al. (1990b) show that 10 of 12 asexual ostracodes included clones which were polyploid. They also found that clonal diversity varied among species. For instance, Cyprinotus incongruens averaged 1.2 clones (1.3 clones; Havel and Hebert, 1993), whereas Cypridopsis vidua had 8.3 clones (9 clones; Havel and Hebert, 1993), Cypricercus reticulatus averaged 7.8 clones (Havel and Hebert, 1993) per pond. On a regional basis, high-diversity species indicate a range of 18 – 80 clones, while low diversity species show 1 – 7 clones (Havel and Hebert, 1993). Bell (1982) has indicated that asexuals are more abundant in modern habitats, whereas, sexuals are more abundant in habitats such as glacial lakes. The study of ostracodes from various habitats from the Canadian Arctic to the southern U.S.A. (Havel and Hebert, 1993) to tropical habitats in Jamaica (Little and Hebert, 1994) does not show an inﬂuence of latitude on the prevalence of asexual vs sexual reproduction. Rossi et al. (1998) reported similar results for Europe. Hybridization has been documented between the asexual Cyprinotus incongruens and C. glaucus (Turgeon and Hebert, 1994; Chaplin and Hebert, 1997). Diploid asexual species when fertilized by a sexual species produce an asexual triploid. Suspected hybridization between Cypricercus reticulatus and other sexual cypricercids in Canada has been postulated (Turgeon and Hebert, 1995). Theoretical models predict sexually reproducing species should have selective advantage in unstable environments; however, a study by Chaplin and Ayre (1989) does not indicate a complete association of sexually derived recruitment in unstable environments. Darwinula is thought to be the oldest asexual ostracode. Schön et al. (1998) believe that, because of this, accumulation of mutations should have occurred between alleles “with lineages and between lineages” through parthenogenesis. However, their data shows the opposite; there is “no variability in the nuclear ITSI region” despite analyzing individuals in the geographic range between Finland and South Africa. C. Embryology Embryology of ostracodes has been studied by Woltereck (1898), Schleip (1909), Müller-Cale (1913), and Weygoldt (1960). The small size, double-walled sphere, and textured outer surface of the egg does not allow for direct observation of the cleavage process. It is known, however, that after seven divisions, a 128celled blastula is developed (Kesling, 1951a). Differentiation into ectoderm and endoderm (entoderm of Kesling) occurs after the eighth division. D. Hatching and Seasonality Most species begin hatching in the spring in the temperate zone. The timing of this process is controlled in part by temperature and geography (latitude). Species which live in temporary (vernal) ponds have a shorter life cycle. For example, Cypridopsis vidua develops in about one month (Kesling, 1951a) and during several months for Cypria turneri (Strayer, 1988). Some of these same species can also exist in permanent ponds where several generations per year may develop. For some species [e.g., Candona caudata; Delorme, 1978, 1982 (may belong to Fabaeformiscandona, see Meisch, 1996); not conﬁrmed for North America, Cyprinotus carolinensis McLay, 1978c], hatching is delayed and spread over a much longer period. These apparent “resting eggs” allow the animal to sustain itself in the habitat in spite of stressful periods (such as anoxia and pollution; Delorme, 1978) when the nauplii cannot survive. Eggs produced by some species can be either spring or summer forms. The ostracodes hatched from eggs laid in the spring do not tolerate warm summer temperatures. Ostracodes hatched from eggs laid in the summer will live only in warm water and expire in cold water (Schreiber, 1922). Martens et al. (1985) have noted that eggs of the Australian Mytilocypris henricae would not hatch below a certain temperature. Eggs of freshwater ostracodes can withstand freezing (Kesling, 1951a; Sohn and Kornicker, 1979). As early as 1915, Alm noted that temperatures affected the time of hatching. Ephemeral ponds in which the sediments stay frozen for up to 6 months during the winter produce many nauplii from eggs in the spring. Angell and Hancock (1989) found that eggs of Heterocypris incongruens could withstand freezing and remain viable down to 18°C when either wet or dry. Some authors have hatched ostracode eggs from dry lacustrine sediments kept in their laboratories for periods ranging from a few years to many decades (Sars, 1901; Sharpe, 1918). McLay (1978b) reported the eggs of Cypricercus reticulatus do not hatch until dried. Angell and Hancock 819 20. Ostracoda (1989) found that eggs of Heterocypris incongruens remained viable after drying at 22 and 40°C. The eggs did not remain viable if kept wet and heated to 40°C. The eggs of some species (Cypridopsis vidua, Cyprinotus incongruens, Physocypria sp., and Potamocypris sp.) can pass through the lower intestinal tract of both wild and domestic ducks and remain viable (Proctor, 1964). Kornicker and Sohn (1971) reported that ostracode eggs were still viable after being egested by goldﬁsh and swordtail ﬁsh. In an excellent paper detailing a meticulously carried out experiment, Rossi et al. (1996) have shown that, during its life time, the same individual of Heterocypris incongruens can produce diapausing eggs and eggs that hatch within a few days. Angell and Hancock (1989) categorize eggs that hatch within 10 days of being laid as being subitaneous. Rossi et al. (1991) and Rozzi it al. (1991)have shown the ratio of resting eggs depends on the genotype, temperature and photoperiod. Further they indicate the percentage of dormant eggs increases with temperature and daylight hours in a winter clone and decreases in the summer clone. Also their studies show egg hatching decreases at higher temperature, unaffected by photoperiod, in the winter clone and increases with both temperature and daylight hours in the summer clone. Rossi et al. (1996) found rather high number of ﬁrst hatchlings (neonates) at the end of the experiment for the winter clone when they removed the algal mat to count cluster of eggs. They suggest that bioturbation may play a role in the hatching of eggs under ﬁeld conditions. Rossi and Menozzi (1990) found from laboratory experiments that the dominant clone from the ﬁeld have a better chance of survival at low temperatures and conversely the dominant summer clone survives better at high temperature. This indicates that temperature is most likely responsible for seasonal cycles. Most ostracodes are controlled seasonally in their habitat. Research on seasonality has concentrated on those species living in temporary ponds. For temporary ponds in the temperate zone, early spring collections made after the basin receives water yield few live ostracodes [Hoff, 1943a (Illinois), Ferguson, 1958a (South Carolina)]. In these ponds a regular succession of juveniles can be observed until the pond dries, with some species exhibiting at least two generations per year. A temporary coastal habitat investigated by McLay (1978a, b, c) has ostracodes from the fall through the late spring. Ostracodes were absent through the summer because of habitat desiccation. Freezing had only a marginal effect on a part of the habitat. McLay (1978b) stated the maturation rate for Herpetocypris reptans was 190 days, and 45 days for Cyprinotus carolinensis. In permanent ponds, active species are absent for several months [Ferguson, 1944 (Missouri)]. Juveniles began appearing in early spring and continued until late spring, while adults were present until late fall to early winter. This pattern was repeated for the three species studied by Ferguson: Cypridopsis vidua, Potamocypris smaragdina, and Physocypria pustulosa. Ostracodes that live in ponds or ephemeral bodies of water have a short life cycle. Martens et al. (1985) noted that an Australian species took longer (4 – 5 months) to reach sexual maturity in the winter than in the summer (2 – 3.5 months). Candona subtriangulata, Cytherissa lacustris, and Darwinula stevensoni, that inhabit large lakes where the habitat is considered stable, will have a life cycle of 6 – 24 months (McGregor, 1969; Delorme, 1978; Ranta, 1979). The length of the life cycle depends on the species. Danielopol (1980b) observed that the length of the life cycle for ﬁve species ranged from 126 to 489 days, the latter being for a hypogean ostracode. E. Molting During the eight molt stages, the appendages change in size, shape, and function. A summary of these developments is found in Table I (after Kesling, 1951a). Przibram (1931) determined that crustaceans doubled their weight from one molt to the next. He further concluded that because weight varies directly with volume, that volume would also increase by a factor of two. This led him to believe that a given linear dimension would increase by about the cube root of two (1.26) for each molt. Based on this hypothesis, Kesling (1953) devised a circular slide rule which allows one to determine the variability of any linear dimension, area, or volume of any ostracode molt stage and adult. This slide rule is useful in determining which molt stage (shell or appendage) one is examining. Being able to identify an ostracode as an instar saves time because most instars cannot be identiﬁed to the species level. TABLE I The Development of Appendages with Each Instar of the Freshwater Ostracodea Instar 1 2 3 4 5 6 7 8 9 Antenna I Antenna II Mandible Maxilla Furca Leg I Leg II Leg III Ovaries Testes a () indicates the anlagen of a structure. 820 L. D. Delorme F. Competition, Co-existence, and Survival The study of competition, coexistence, and survival by McLay (1978a – c) showed that, in a small temporary puddle, “competition is mediated via density-dependant mortality and maturation rates.” He found that Herpetocypris reptans was a better competitor because of its longer maturation time (4419 degree days vs Cyprinotus with 2034 degree days) and could better withstand crowding and a shortage of food. His model shows that Herpetocypris was dominant over Cyprinotus carolinensis by a factor 7:1. He also found that competitive exclusion of Cyprinotus by Herpetocypris was prevented by periodic freezing. Freezing was also found to harm the population growth of Herpetocypris more than Cyprinotus because of the longer time required for maturation. Oertli (1995) studied the density of invertebrate of three substrates of a manmade pond. He found that life cycles and substrate controlled density ﬂuctuations. High densities in the summer are related to life cycle and newly hatched individuals. He called the summer effect the “reproduction effect” with high mortality occurring in the winter and spring. For Cypridopsis vidua the highest densities occurred on Chara and Typha, with the lowest densities on terrestrial leaf litter. Rieradevall and Roca (1995) found that substrate and organic matter are important factors controlling the distribution of ostracode species in a karstic spanish lake. They collected high densities in the fall and spring. IV. DISTRIBUTION Ostracodes are found in nearly every conceivable aquatic habitat. These range from temporary and permanent ponds, lakes, intermittent, and permanent streams to ditches and irrigation canals, and fens. Caves and the interstices of aquifers are additional habitats. Some (referred to as terrestrial ostracodes) are found in moist organic mats of fens and in axial cups of certain plants such as bromeliads. North American freshwater and brackish water ostracodes thrive in a full range of salinities from 10 to 74,800 mg / L [high sulfate, low chloride waters] (personal ﬁles). Brackish water forms are common in high sulfate waters (Limnocythere staplini), as well as in chloride waters mixed with freshwater (Cytheromorpha fuscata) in inland lakes (Neale and Delorme, 1985). De Deckker (1981) lists an Australian species found in waters having salinitics greater than 170,000 mg / L. Cyprideis, also high salinity species, are common in southern coastal areas of North America (Benson, 1959), England and the Netherlands (van Harten, 1975; Sywula et al., 1995). Jahn et al. (1996) have found that Cyprideis torosa can oxidize hydrogen sulﬁde to nontoxic thiosulfate and sulﬁte and rid itself of the oxidation products quickly. A. Benthic Swimmers Ostracodes are benthic organisms and are only rarely found in the plankton. They are divided into swimmers and nonswimmers. Most often they swim between aquatic plants. If they are disturbed while swimming, they often lose control and spiral down to the substrate. A few species of the genera Cypria and Physocypria are known to swim up into the water column, as they have been recovered from suspended sediment traps. Another swimmer, Notodromas monacha, has a peculiar habit of turning upside down when approaching the water surface. The venter of this species is ﬂat, enabling the organism to adhere and hang beneath the water surface by surface tension. Marmonier et al. (1994) indicate that plant dwelling species, such as Cyclocypris ovum and Cypridopsis vidua, are more likely to be spherical in shape. B. Benthic Nonswimmers The nonswimmers, or true benthic forms, use their well-developed antennae (without setae) and ﬁrst thoracic legs (previously called the maxillae) to crawl on the sediment – water interface. Some species, such as the limnocytherids, are infaunal, crawling beneath the sediment – water interface between the sediment particles (L. Delorme, personal observations; Benzie, 1989). Ostracodes live in the vents of warm and cold-water springs where groundwater is discharging (Forester, 1991; McKillop et al., 1992). Särkkä et al. (1997) has reported Potamocypris pallida, Candona candida, and Candona reducta made up 21% of a spring meiofauna affected by road de-icing salt and gravel extraction. Ostracodes may live in the fractures of bedrock adjacent to streams, lakes, and pools in caves. Hypogean (subterranean) ostracodes are being recovered more frequently from aquifers. These blind animals live in the interstices of sand aquifers. They are recovered by pumping the aquifer or trenching through it (Danielopol, 1980a). Marmonier et al. (1994) have shown that interstitial ostracodes have a geometric shape (triangular or trapezoidal) which is most suited to its habitat. The same can be said for most limnocytherids which live just below the sediment – water interface, crawling between the sediment interstices (personal observations). Ward et al. (1994) observed the highest concentration of hypogean ostracodes in a well close to the main channel of the Flathead River, Montana, and in wells farthest on the ﬂoodplain but near the Whiteﬁsh River. Rogulj et al. (1994) indicate 20. Ostracoda those hypogean ostracodes (Fabaeformiscandona wegelini) that live in shallow interstitial habits are closely linked to running water rich in organic matter. This concept is supported by observations of Rouch and Danielopol (1997) and Marmonier and Creuzé des Châtelliers (1991). Creuzé des Châtelliers and Marmonier (1993) determined there were no meaningful correlations between abundance of ostracodes and particulate organic matter, alkalinity, calcium, or oxygen content of the interstitial water. Creuzé des Châtelliers and Marmonier (1990) found the interstitial fauna above and below a river rifﬂe shows differing ostracode assemblages and abundances. Above the rifﬂe, where the river bed was degrading, the water velocity was greater and the number of species and specimens was less than below the rifﬂe. Below the rifﬂe the river bed was aggrading and the velocity of the water was less, allowing more species to live in this area both in composition and numbers. Those living in deeper aquifers, prefer a more stable environment with less organic matter (Rogulj et al., 1994). Along the length (320 km) of the Rhône River, Marmonier and Creuzé des Châtelliers (1992) found three different species assemblages tied to the ecological requirements of the member species. Two groups were restricted to the upper and lower ends of the river respectively and one was common to the whole length of the river. 821 B. Salinity and Solute Composition Salinity and solute composition are of importance to freshwater ostracodes. Delorme (1989) has shown preliminary salinity tolerance distributions for some 43 species studied from Canadian habitats (Fig. 13). The sequence of species shown for salinity tolerance shows a gradual increase in the mean salinity value. Furthermore, the range, as shown by minimum and maximum values of salinity tolerance, is a function of the species. For example, Cypridopsis vidua, is a moderately salinetolerant species able to survive in a very broad range of salt concentration. On the other hand, species such as Candona subtriangulata and Cytheromorpha fuscata are much more restricted in their chemical, and thus geographic range. The low end of the range, or pristine, low-salinity (SO4) waters are inhabited by such species as Candona subtriangulata (23 – 93 mg / L), V. CHEMICAL HABITATS A. Chemical Water Types Physical and chemical characteristics of the habitat are important factors in ostracode distribution and abundance. From Canadian habitat-waters, four chemical types are deﬁned based on the predominant equivalent anion or oxyanion. These are bicarbonate, sulfate, chloride, and carbonate (personal ﬁles). A high percentage of genera (61%) are found only in bicarbonate waters. Bicarbonate, after being converted to carbonate, is a primary constituent of the shell. Only 9% of the known genera are found living in sulfate-rich waters. Several genera (30%) have species which have a preference for either of the two types of water. Chloride-dependent species, such as Cytheromorpha fuscata and species of Cyprideis, are more properly tolerant of marine brackish water. C. fuscata are occasionally found in inland lakes (Neale and Delorme, 1985) where brine seepages occur. Such species as Candona rectangulata, Cyprinotus salinus, and Cypris bispinosa are found in lake and pond habitats that may receive sea spray. These species are by no means restricted to chlorideenhanced waters. FIGURE 13 Total dissolved solids range for some Canadian freshwater ostracodes. The solid line represents the mean, the bars represent the minimum and maximum values. [Modiﬁed from Delorme, 1989.] 822 L. D. Delorme Limnocythere verrucosa (22 – 255 mg / L) from the Great Lakes area, and Candona protzi (may belong to Fabaeformiscandona, see Meisch, 1996, not conﬁrmed for North America) (56 – 115 mg / L) of the high arctic. The high end of the range or high-salinity water bodies are inhabited by such species as Limnocythere ceriotuberosa (74 – 2780 mg / L) and L. staplini (122 – 74,840 mg / L). De Deckker (1983a) reported there are 14 North American ostracode species (one marine) that live in athalassic saline waters. Species such as Cyprinotus glaucus may be found in lakes where there is groundwater discharge of saline waters of intermediate ﬂow systems (Delorme, 1972). Forester (1983, 1986) and Forester and Brouwers (1985) have emphasized the importance of water composition over salinity. Examination of lake-water chemistry in North America, where Limnocythere sappaensis and L. staplini live, reveals that solute composition and not salinity is most important for their existence (Forester, 1983; Delorme personal ﬁles). Forester found that L. sappaensis lives in water enriched in Na –HCO3 –CO32 and depleted in Ca2 . L. staplini lives in water enriched in various combinations of Na2 –Mg2 –Ca2 –SO42 –Cland depleted in HCO3. Forester (1987) and De Deckker and Forester (1988) have expanded on the relationship between ostracode occurrences and major dissolved ion chemistry and salinity. They further suggested that relationships between species diversity, carbonate saturation, and relative abundance of dissolved calcium and carbonate ions exist, based on limited data from the United States. Most ostracode species may be placed in one of six or seven hydrochemical groups. The groups have welldeﬁned boundaries based on carbonate saturation, major dissolved ion composition, and salinity. FIGURE 14 pH range for some Canadian freshwater ostracodes. The solid line represents the mean, the bars represent the minimum and maximum values. [Modiﬁed from Delorme 1989.] C. Hydrogen Ion Concentration The preservation of the calcareous exoskeleton for live ostracodes suggests that both the pH of the habitat must be circum-neutral and that a ready source of CaCO3 must be available. Delorme (1989) indicated the “average” pH of Canadian aquatic habitats for ostracodes is between 7 and 9.2 (Fig. 14). For carbonate-enriched lakes, common to groundwater discharge areas of the United States and Mexico, pH values are typically in the range of 9.6 – 10.5 (R. M. Forester, personal communication). The species has an individualized pH range. Several of the species have a very broad pH range such as Cypria ophtalmica (5.2 – 13), Candona candida (5.4 – 13), Cypridopsis vidua (5.2 – 12), Cyclocypris sharpei (5.2 – 11), and Cyclocypris ampla (5 – 12). Cytheromorpha fuscata with its limited distribution in the Manitoba Lakes has a very restrictive range of 8.3 to 8.7 in inland lakes. This may be an artifact of the lakes from which the species was collected. The majority of ostracodes have a larger pH range than C. fuscata. When the epidermal lining covering the shell is ruptured and the water and sediments are poorly buffered, the calcareous shell will dissolve, destroying the animal. Larsen et al. (1996) used a number of statistical method to investigate the relationship between invertebrates to pH in Norwegian river systems. They indicate that a signiﬁcant response occurs in the abundance of species when there is an increase in pH. D. Dissolved Oxygen Dissolved oxygen in the aquatic habitat is an important requirement for survival. The mean requisite 20. Ostracoda 823 oxygen content below its lower tolerance limit of 2.8 mg / L. Its survival was accomplished by having a short life cycle allowing it to produce eggs before the onset of anoxia. This survival mechanism has been in place for some time in Lake Erie as indicated by the presence of fossil shells in the lake sediments (Delorme, 1982). Contrary to C. caudata (may belong to Fabaeformiscandona; see Meisch, 1996, not conﬁrmed for North America), C. subtriangulata and Cytherissa lacustris have become locally extinct in the central basin of Lake Erie because they have a 1-year life cycle and cannot reach sexual maturity as they require a minimum dissolved oxygen content of 5.6 and 3.0 mg / L. Cytherissa lacustris is making a comeback with a reduction in anoxia. Newrkla (1985) found C. lacustris could survive at 1 mg O2 /L in the laboratory on a glass substrate and minimal food (20°C and 20-h exposure). Nine of the 43 species listed in Figure 15 can tolerate near-zero dissolved oxygen (below the detection limit of the Winkler azide method, APHA, 1965) for short periods. Candona decora, C. candida, Cyclocypris ampla, C. sharpei, and Cypria ophtalmica have been recovered from water of zero dissolved oxygen from a small river in early December (L. D. Delorme, unpublished data). The river had been covered by 1 ft of ice, suggesting these waters had been anoxic for some time. Fox and Taylor (1954, 1955) have found by experimentation that some ostracodes survive longer at oxygen levels below air saturation of 21%. E. Carbonate FIGURE 15 Dissolved oxygen range for some Canadian freshwater ostracodes. The solid line represents the mean, the bars represent the minimum and maximum values. [Modiﬁed from Delorme, 1991.] for dissolved oxygen by ostracodes falls within a very narrow margin of 7.3 to 9.5 mg / L (Fig. 15). In general the concentration of dissolved oxygen in the water is broad. Candona subtriangulata has the highest minimum oxygen requirement of 5.6 mg / L. Most surprising in Figure 15 is the high number of species (34 out of 43, in Canadian habitats, L. D. Delorme, unpublished data) which can tolerate low dissolved oxygen concentrations. Those species which require a minimum 3 mg / L dissolved oxygen in the water are Cytherissa lacustris, Cytheromorpha fuscata, Ilyocypris gibba, Limnocythere ceriotuberosa, L. herricki, L. itasca, L. verrucosa, and Potamocypris variegata. Delorme (1978) found that Candona caudata (may belong to Fabaeformiscandona, see Meisch, 1996; not conﬁrmed for North America) could survive in Lake Erie even though the hypolimnion has summer dissolved The effect of excess calcium carbonate and anoxia on Cypridopsis vidua was investigated by Reyment and Brännström (1962). Their results indicated the morphology of the shell is adversely affected (smaller shell dimensions) by excess calcium carbonate and anoxia in the environment. They reiterate the common belief that the major contributor to the size and shape of an ostracode is the environment, with a secondary contributor being the genome of the animal. Van Harten (1975) has shown the effect of salinity on shell size. The largest mean size of Cyprideis torosa was found in nearly freshwater, whereas the smallest mean size was found in waters where the salinity was very high. VI. PHYSICAL HABITAT A. Temperature The water temperature of the aquatic habitat plays an important role in the habitat, seasonal, and geographical distribution of ostracodes. This is not to 824 L. D. Delorme say that other factors such as availability of food, solute composition, turbidity, and energy levels are not important. The temperatures in shallow-water habitats can range from 0 to over 30°C, depending on the latitude and altitude. Species living in this niche must have the ability to tolerate this broad range in one or more life stages. Such species as Candona acutula, C. candida, C. ohioensis, Cyclocypris ampla, C. sharpei, Cypria ophtalmica, and Limnocythere staplini exhibit such a range for bottom water temperature (Fig. 16). The exception to the above are shallow ground water fed streams which may have a nearly constant temperature. Candona subtriangulata has a low mean value of 5.5°C, with a range of 2.6 to 19.2°C (Delorme, 1978). This species is common in Lakes Superior, Huron, and Ontario at considerable depth, where the bottom-water temperature does not vary much. Korschelt (1915) ran several experiments with Cypridopsis vidua and Cypria ophtalmica, in which the species were frozen in ice for several hours. The ice was then allowed to melt; the majority of the specimens survived the freezing experiment. At the other temperature extreme, Cypris balnearia has been described from thermal springs at temperatures of 45 to 50.5°C. Wickstrom and Castenholz (1973) have recovered Potamocypris from an algal – bacterial substrate of a hot spring, in Oregon, at temperatures ranging between 30 and 54°C. Chlamydotheca arcuata has been found in warm springs (Utah, Nevada, Arizona, and Mexico) where water temperature varies between 24 and 39°C (Forester, 1991). Mean annual air temperature assists in explaining the geographic distribution of ostracodes in habitats shallower than 25 m. From Figure 17, 20 out of 43 FIGURE 16 Bottom water or habitat temperature range (°C) for some Canadian freshwater ostracodes. The solid line represents the mean, the bars represent the minimum and maximum values. [Modiﬁed from Delorme, 1991.] FIGURE 17 Mean annual temperature range (°C) for some Canadian freshwater ostracodes. The solid line represents the mean, the bars represent the minimum and maximum values. [Modiﬁed from Delorme, 1991.] 20. Ostracoda species can live in aquatic habitats north of latitude 60°. The minimum values of the range for mean annual air temperature for these species habitats are between 8.5 and 11°C. The species restricted to the arctic and low mean annual air temperatures are Candona paralapponica, C. protzi (may belong to Fabaeformiscandona; see Meisch, 1996, not conﬁrmed for North America), C. anceps, C. pedata, C. mülleri, C. ikpikpukensis, Cyclocypris globosa, Limnocythere liporeticulata, and Tonnacypris glacialis . The habitats for those species living in the midlatitudes have a mean annual air temperature greater than 1.5°C. B. Lakes, Ponds, and Streams Most freshwater ostracode species may be found in more than one habitat (Fig. 18). The pond – lake habitats 825 can be viewed as a continuum from shallow to deep water. Some ponds are temporary in nature and, therefore, harbor a speciﬁc faunal association such as Candona renoensis, Cypricercus deltoidea, Megalocypris alba, and Cyclocypris laevis (Delorme, 1989; King et al., 1996; Wiggins et al., 1980). At the opposite end of the scale are those species adapted or restricted to living in permanent lake habitats (Candona subtriangulata, Cytherissa lacustris, and Cytheromorpha fuscata). A faunal mix may occur between pond-lake and stream habitats. There may be a speciﬁc biotope developed at the mouth of a stream or river where it enters the lake or pond. The delta area is characterized by a combination of ﬂowing and standing water. Many ostracode species are adapted but not restricted to living in this biotope such as Candona acuta, Cyclocypris ampla, C. sharpei, Ilyocypris bradyi, I. gibba and Potamocypris variegata. Large lakes have bottom currents which accommodate species such as Candona caudata (may belong to Fabaeformiscandona; see Meisch, 1996, not conﬁrmed for North America) and C. acuta. None of the species listed by Delorme (1989) are completely restricted to ﬂowing water. Those which are considered as lotic (ﬂuvial) species are Candona acuta, Cypria obesa, Ilyocypris bradyi, I. gibba, and Potamocypris variegata. A special case is the fen. Up to 10 species have been recovered from fen vegetation at various levels down to 20 cm and varying levels of dissolved oxygen and redox potential (Douglas and Healy, 1991). C. Bromeliads Axial cups are formed by the spiral overlap of bromeliad leaves. These cups collect rain water and become inhabited by many kinds of invertebrates. Tressler (1956) described Metacypris maracoensis, recovered from the axial cups of Florida bromeliads. Ostracodes are more commonly found associated with bromeliads in Central and South America, and the Caribbean Islands (Little and Hebert, 1996). D. Cave and Subterranean Habitats FIGURE 18 Percentage probability of ﬁnding a Canadian freshwater ostracode in a lake, pond, or stream. [Modiﬁed from Delorme, 1989.] Ostracodes have been recovered from several caves. Klie (1931) reported Entocythere donnaldsonensis commensal on the crayﬁsh Cambarus from the Donnelson Cave of Indiana. From the Marengo Cave of Indiana, Klie named two new species:Candona marengoensis and C. jeanneli. Hart and Hobbs (1961) have described eight new troglobitic cave species from the eastern United States. Walton and Hobbs (1959) have also described troglobitic ostracodes from Florida caves. Pools in deep sinkholes in limestone terrain (cenotes) of Yucatan, Mexico have a rich and varied 826 L. D. Delorme ostracode fauna. Furtos (1936a) described 23 species from these habitats. More research has been carried out on hypogean ostracodes. Cavernocypris wardi has been recovered from spring and interstitial water of the Flathead River, Montana (Ward et al., 1994), from the South Platt River, Colorado (Danielopol et al., 1994; Marmonier et al., 1989; Marmonier and Ward, 1990 (Candona candida, Cavernocypris wardi, Cypria sp., Eucypris sp., Fabaeformiscandona pennaki, F. wegelini, Ilyocypris sp., Nannocandona faba, Potamocypris sp., Pseudocandona cf. P. albicans, Strandesia canadensis), and from springs in Wyoming, Nevada, and Colorado (Forester, 1991). Cavernocypris subterranea (common in Europe) has been found from a spring near Brush Creek, Idaho (Külköylüoĝlu and Vinyard, 1998). Not all ostracodes found near spring discharges are hypogean (Forester, 1991; McKillop et al., 1992; Ward et al., 1994). E. Sediment – Water Interface, Particle Size, Food The distribution of ostracodes at the sediment – water interface is a function of the availability of food, whether the substrate surface is clearly or poorly deﬁned, the particle size distribution of the top centimeters of the substrate, as well as the time of year. The distribution of ostracodes is patchy. Where there is organic detritus, ostracodes will be abundant, particularly in shallow water. The presence of abundant ﬂoating organic debris just above an ill-deﬁned sediment – water interface, discourages ostracodes from living in this part of the habitat. As detritivores and herbivores, ostracodes require a ready source of particulate organic matter that they can sweep into the mouth region. They also use their mandibular teeth to rasp or gnaw off small particles from large organic particles, living plants, or algae. On predominantly mineral substrates, ostracode densities will be low. As the sediment – water interface becomes deeper and further from the shoreline the number of species decrease. In this niche, the only food available will be the resistant cellulose raining down through the water column, bacteria, and fungi. In the deep troughs located in the eastern end of Lake Superior, only Candona subtriangulata has been recovered from depths of 45 – 305 m. Tressler (1957) identiﬁed Candona decora at a depth of 600 m from Great Slave Lake, Canada. For many limnocytherids, especially Limnocythere ceriotuberosa, and L. staplini, the particle size of the substrate is very important. These ostracodes crawl through the interstices of very ﬁne sand (0.0625 mm) to coarse sand (1.0 mm). In these niches, there must be sufﬁcient food and oxygen for survival. Good porosity and permeability of these sediments allows a freeﬂow of oxygenated waters. Most clay-sized, organic-rich sediments are anoxic and reducing and not suitable for ostracodes to live in the interstices. VII. PHYSIOLOGICAL AND MORPHOLOGICAL ADAPTATIONS Ostracodes live in a wide variety of habitats, and within a given habitat physical and chemical conditions ﬂuctuate. Physiological adaptations can be seen for some of these changes. A. Eggs Dispersal of freshwater ostracode eggs is thought to be passive (Neale and Delorme, 1985; Peck, 1994; Sywula et al., 1995; Little and Hebert, 1996; Malmquist et al., 1997) either in the gut or mud on the feet of birds, particularly for species living in the littoral habitat. Eggs that pass through the gut of ﬁsh and remain viable assist in passive dispersal (Kornicker and Sohn, 1971). For sampling sites that were up to 1000 km apart, Chaplin and Ayre (1997) found no evidence that stream ﬂow was a “mediator of short or long distance gene ﬂow in (the large green morph of) Candonopsis novaezelandiae.” B. Resting Stage and Torpidity The primary resting stage for freshwater ostracodes is the egg. The structure of the egg permits the yolk to survive both desiccation and freezing. The other adaptation for surviving harsh conditions in ostracodes is torpidity (Barclay, 1966; Delorme and Donald, 1969; McLay, 1978a; Horne, 1993). Candona rawsoni has been recovered in the seventh instar from frozen sediments of a pond which had gone dry the previous fall. Placing the sediments in water at room temperature caused rejuvenation of the seventh instar candonids. Survival rate was a function of the moisture content and temperature of the sediments holding the instar at the time of rejuvenation (Delorme and Donald, 1969). The major difference was that sediment containing the seventh instar could have a moisture content as low as 4 – 5%. In the fall or spring, hatching of the eggs and development of the nauplii from the seventh instar may occur. In vernal ponds, torpidity allows sufﬁcient time for the species to become sexually mature and lay eggs. In this way, the species is propagated in temporary bodies of water (Wiggins et al., 1980). The development of the torpid state allows ostracodes to survive the drying of vernal ponds. Horne (1993) found a similar situation for Candona 20. Ostracoda patzcuaro, collected from playa lakes of the south plains of Texas. C. Coloration Coloration of ostracodes is a function of the pigment bodies located in epidermal cells of both the outer and inner layer. Ostracodes such as Cypridopsis, Cypris, Cypricercus, Eucypris, Herpetocypris, Potamocypris, and Tonnacypris are normally green, although Cypridopsis vidua and Cypricercus may be found in shades of brown and purple. These genera are all prevalent in littoral areas of lakes and in ponds where there is abundant vegetation. Green (1962) investigated the green coloration of Eucypris virens and found it to be a bile pigment, biladienes, derived from blue-green algae. Other species, such as Cypricercus horridus and Megalocypris alba, are purplish to reddish-brown in color which allows them to blend better with an organic substratum. Mbahinzireki et al. (1991) found that marked coloration increased predation risk by ﬁsh. The majority of the other species are a dirty white to yellowish-buff color which blends in with the mineral substrata. Candonids, cyprinotids, and limnocytherids fall into the last category. D. Appendages The ﬁrst and second antennae are modiﬁed in swimming ostracodes. Finely feathered swimming setae on the ﬁrst antennae are in constant motion and offer resistance to the water when the ostracode is swimming. Setae on the second antennae are used in a rowing motion. When the swimming setae are absent, the second antennae are used exclusively for walking and climbing. In the male of some species, the maxillae (formerly referred to the ﬁrst thoracic leg) is modiﬁed as a prehensile palp. This changes the appendage from a feeding function to a sexual function. VIII. BEHAVIORAL ECOLOGY Experiments were conducted by Towle (1900) on heliotropism using Cypridopsis vidua. She determined that ostracodes initially moved toward the light and then reversed directions. Ostracodes that swim maintain a sustained motion. The swimming setae of the ﬁrst antennae used in propelling the animal forward must be in constant motion or the animal will sink to the substrate. Observation of the swimming motion, using a binocular microscope, shows the swimming setae moving in a pattern 827 similar to a hand-held egg beater directed upward. Because swimming is energetically quite expensive, ostracodes can only swim short distances (usually between adjacent plants). The entry of a stimulus from a cyprinid ﬁsh tank into a microcosm of Chara fragilis (Uiblein et al. 1994) changed the behaviour pattern. The result was that Cypridopsis vidua responded by spending more time among the plant stems and leaves. IX. FORAGING RELATIONSHIPS The diet of most ostracodes is restricted to algae (phytophilic) and organic detritus. Klugh (1927) reported certain algae as being part of the food supply. Kesling (1951a) indicated diatoms are a part of the ostracode diet as did Strayer (1985) for Cypria turneri. Grant et al. (1983) reported feeding Cyprinotus carolinensis the blue-green algae Nostoc sp. Campbell (1995) calculated that tree pollen made up 8% and organic material 42% of the diet for Australocypris insularis. Tabacchi and Marmonier (1994) used Chara with alterable amounts of periphyton covering the stems as did Roca et al. (1993). When feeding, organic particles are swept into the mouth using the maxillae. The mandibular teeth grind large organic particles into a smaller size before they enter the mouth. Although ostracodes are predominantly herbivores and detritivores, a few have shown carnivorous characteristics (Johansen, 1912). Ostracodes have been observed attacking and eating the soft tissue of certain snails (Deschiens et al., 1953; Deschiens, 1954; Sohn and Kornicker, 1975). Some species (Cypridopsis hartwigi, C. vidua, Cypretta kawatai, and Cyprinotus incongruens) are known from experiments, by the above authors, to eat soft tissue of snails, such as the mantle and antennae, and to crawl into the respiratory organs. Have (1993) used Paramecium sp. as a source of food by Eucypris sp. in experiments on effects of area and patchiness on species richness. Campbell (1995) provides detailed information on the preying habits of Australocypris insularis. These large ostracodes feed on smaller ostracodes, Diacypris compacta and D. dietzi. She also found this large species grouped around drowned bee carcasses, presumably feeding on them, with terrestrial invertebrate detritus making up 8% and insects 3% of the diet. The trophic position of most ostracodes is that of a herbivore and detritivore. This group of benthic animals scavenges and consumes organic matter primarily from the sediment – water interface. Some of this food is fresh, while the remainder is in the process of decaying. During the feeding process, nutrients and the more 828 L. D. Delorme resistant cellulose and silica (diatom frustules) are moved back into the food chain. Ostracodes are, in turn, consumed by higher animals. Predators include bottom dwelling ﬁsh. There are several references to the presence of ostracodes in the gut of ﬁshes. Bigelow (1924), Harding (1962), and Ferguson (1964) found ostracodes in the stomachs of suckers and trout. Stomach contents of brook stickleback contained Cyclocypris ampla, that of yellow perch had Candona ohioensis, and bullheads had Cypridopsis vidua and Physocypria globula (L. D. Delorme, unpublished data). Grifﬁths et al. (1993) report the presence of Ostracoda in the guts of arctic charr, Salvelinus alpinus, and the three-spine stickleback, Gasterosteus aculeatus (Dezfuli, 1996). Bataille and Baldassarre (1993) investigated three prairie potholes (near the Delta Waterfowl Research Station, Manitoba) as to the availability of aquatic organisms (including ostracodes) as high protein food for ducks such as mallards and canvasbacks. They used activity traps to capture nektonic organisms during a 24-h period. The sample was then ﬁltered through a #35 U.S. Standard sieve (500 m). It should be noted that this sieve size would lose most ostracodes except Cypris pubera. Batzer et al. (1993) investigated the feeding habits of mallards and Green-Winged Teals on aquatic invertebrates. The contents of esophagi and habitat locations were studied using D-framed sweep nets (30-cm width, 1-mm mesh size) for nektonic and epiphytic invertebrates. For sediment sampling of the habitat, a bilge-pump intake was placed on the wetland bottom and four pumping repetitions of water and sediment were passed through a 0.3-mm sieve. This sieve size would lose most ostracodes. Ostracodes and other invertebrates were each consumed by only 3 or 4 of the 19 mallards. The relative spacing of bill lamellae of speciﬁc duck species inﬂuenced their selection of different orders of invertebrates (Nudds and Bowlby, 1984; Batzer et al., 1993). Green (1954) noted the oligochaete Chaetogaster diaphanus eats ostracodes as do cyclopoid copepods (Fryer, 1957) and the tanypodine midge (Roback, 1969). Benzie (1989) indicated “a negative association” between a predatory mite and Herpetocypris reptans. Swüste et al. (1973) showed experimentally that the phantom midge Chaoborus ﬂavicans released Cypria immediately after capturing it. This suggests that this ostracode either tastes bad or releases a noxious chemical. Lancaster and Robertson (1995) looked at the preying habits of the polyphagous net-spinning caddisﬂy (Plectrocnemia conspersa) and the alderﬂy (Sialis fuliginosa). They found both ﬂies had very high densities of ostracodes in their guts, probably because both groups are very abundant in leaf litter of slow moving streams. The introduction of ﬁsh into a pond, where larger predators have been absent, quickly reduces the invertebrate populations. Martens and de Moor (1995) found the invertebrate populations that lived in a large predator free habitat were adapted to living in such an environment. The consequence is these habitats will not be repopulated with the same invertebrate populations. X. POPULATION REGULATION A. Suitability of Habitat Abundance of ostracodes at the sediment – water interface is a function of substrates, availability of food, season, and depth of water. Strayer (1985) has provided a useful and current summary of abundance and biomass measurements of ostracodes in lake environments. Bechara (1996) has indicated ﬂoodplain lakes with high ﬂoating plant biomass had a decrease in biomass during high-water periods, Ostracoda and Oligochaeta were dominant. The presence of food clearly has an impact on species richness and abundance. Long periods of warm temperatures down to the bottom of the euphotic zone favor ostracode production. The number of species and specimens recovered from the profundal sediment – water interface is small. For example, the ostracode assemblage in the deep troughs (190 – 365 m) of eastern Lake Superior is limited to Candona subtriangulata. At these depths, during the last week in May and the ﬁrst week in June, the bottom water temperature varied between 2.6 and 4.0°C. Based on Canadian habitats sampled for ostracodes, a maximum of nine genera have been counted from one sample. In these samples, between 13 and 19 species were represented with a maximum of nine species being alive at the time of collection (L. D. Delorme, unpublished data). Rouch and Danielopol (1997) deﬁned high species diversity as “species collections of more than 25 species within an inventoried area, at the local scale (i.e., between 1 and 100-m length), low diversity is 1 – 5 species and medium level diversity is 6 – 24 species.” King et al. (1996) found species richness varied up to 13 species per pond. Baltanás (1992) attempted to make a comparison between samples using species numbers referred to a ﬁxed sampled area. He evaluated three methods for estimating species richness (the number of individual species per sample) using computer simulations. These were: Stout and Vandermeer method based on the species – area relation; Cohen’s method which assumes the log-normal distribution of species abundances; and the jackknife 20. Ostracoda method, a nonparametric procedure with no distributional assumptions. The author found the estimators tested showed a negative bias, with the second-order jackknife method giving the best results with the highest accuracy and with the best precision. B. Toxicity of Herbicides and Pesticides Sanders (1970) tested Cypridopsis vidua and ﬁve other crustaceans against 16 herbicides. Irritability and excitability were the ﬁrst noticeable reactions. Cypridopsis vidua could only be tested at low concentrations because the animal would close its shell when it received the stimulus. This species was the second most sensitive to the herbicides. Landis et al. (1994) found a similar pattern when testing ecological risk assessment of Cyprinotus sp. and other organism in a microcosm against the water soluble fraction of jet fuel A. Dieter et al. (1996) tested several invertebrates in microcosms at the edge of lakes for toxicity to insecticides. They found ostracodes were resistant to phorate. Takamura and Yasuno (1986) found that ostracodes in rice ﬁelds decreased in numbers with a treatment of pesticide mixture of propoxur, thiobencarb, and simetryne. There were similar decreases in ostracodes with a treatment of bentazone. In another rice ﬁeld, they used a mixture of methomyl, kasugamycin-hydrochloride, neo-asozin, thiram, and benomyl with some decrease in ostracode fauna. C. Ultraviolet-B Radiation Hurtubise et al. (1998) subjected a number of invertebrates to ultraviolet-B radiation. They found that Cyprintous incongruens was highly tolerant at a water depth of 4 cm when the UV-B irradiance was 56.1 W / cm. They also contend the carapace protected the animal. Further that dissolved carbon (DOC) attenuated the irradiance. D. Parasitism More studies are now documenting the role freshwater ostracodes play in parasitism. Dezfuli (1996) has detailed the effects of the helminth Neoechinorhynchus rutili on the ostracode Cypris reptans. Between 8.6 and 14.5% of this species were infected, with up to three larvae found in one host (up to six worms; see references in Zelmer and Esch, 1998). The effect of this parasitism is a reduction in the number of eggs contained in females, 103 as compared to 207 in uninfected females. Cypridopsis vidua has been found to be the intermediate host of Neoechinorhynchus prolixus (B. B. Nickol, University of Nebraska, personal communica- 829 tion). Cypridopsis sp. acts as a second intermediate host for the parasite Halipegus occidualis (Zelmer and Esch, 1998). For the complete cycle, eggs from the worm pass through the feces of the green frog (Rana clamitans) and are ingested by the pulmonate snail, Helisoma anceps. While in H. anceps sporocysts and redia stages develop ending in the development of cercariocysts. Feeding activity of Cypridopsis sp. transfers the cercaria body into the hemocoel of the ostracode. Odonate naiiads eat the affected ostracodes and, in turn, are eaten by green frogs. Grifﬁths and Evans (1994) found the infestations of the peritrich Nüchterleinella corneliae on Cypria ophtalmica and Cyclocypris ovum. Infestations where more prevalent during winter and decreased during the summer. The parasite tended to be clustered around the bases of the appendages, particularly the genitalia and the furcae. Bronnvall and Larsson (1994, 1995) described new microsporidian parasites, Flabelliforma ostracodae and Binucleospora elongata in the ostracode Candona sp. Their investigations, based on light microscopic and ultrastructural characteristics found the musculature, the frequent site for the development of microsporidia, was undisturbed. The infection was restricted to the adipose and connective tissues and to the haemocytes which appeared to be ﬁlled with spores. Diarra and Toguebaye (1996) examined sections of ostracodes infected by Nosema stenocypris which revealed that the microsporidium develops particulary in the muscles of Stenocypris major which are destroyed and replaced by the developmental stages of the parasite. XI. COLLECTING AND REARING TECHNIQUES A. Collecting and Preservation Ostracodes have been collected using various kinds of gear such as a Birge cone net (Ferguson, 1958b). Using a net has certain disadvantages when gathering benthic organisms; many true benthic forms are not collected because the net usually does not thoroughly sample substrates which ostracodes live on and in. Bais and Agrawal (1995) collected zooplankton using a plankton net (0.25 mm); however, such an apparatus will only collect the larger ostracode species while missing a few smaller forms and the true benthic fauna. The use of an Ekman dredge or similar device is recommended for a quantitative sample of the benthos (Delorme, 1967). Care should be taken not to allow the dredge to settle too far into the substrate, 2 cm being optimal. The actual depth to which a sampling device should go may be checked out by pushing a short core tube into the sediments to observe the depth to which 830 L. D. Delorme ostracodes have penetrated. Any dredge that is used should have ﬂaps on the top so water will ﬂow through the dredge when it is being lowered. This will prevent a bow wave from preceding the dredge and disturbing the sediment – water interface. When the dredge is retrieved, the ﬂaps will close and prevent the interface and collected organisms from being disturbed and swept away by moving water. The large quantity of sediment (500 cm3) retrieved using a dredge means that a method of concentrating the ostracodes must be used. The sample can be washed through a set of 8-in. sediment sieves (#20 mesh, 0.841 mm; #60 mesh, 0.25 mm; #100 mesh. 0.149 mm) using a gentle stream of water from a sprinkler head. This procedure removes particle size fractions (including instars) ﬁner than ﬁne sand (0.149 mm). The smallest freshwater ostracode is Limnocythere friabilis with a height of 0.23 – 0.31 mm. The adult shells of this species are readily retained by both the #60 and #100 mesh. Large cyprids, such as Cypris pubera, megalocyprids, herpetocyprids, and prionocyprids will be concentrated on the #20 mesh sieve. When the specimens are used to study appendages and other organs, ostracodes can be picked by hand from the washed residue by using a steromicroscope. The specimens should then be placed in 5 – 10% buffered formalin solution. Concentrated formalin, ethanol, and methanol should not be used, as these preservatives will decalcify the shell. It has been suggested that sodium tetraborate (250 cc saturated solution) be added to 20 L of 75% ethanol to prevent calcitic shells from being dissolved. Specimens may be mounted in Hoyer’s solution, glycerin, or polyvinyl lactophenol. However, with the use of the scanning electron microscope (SEM) to study appendages, these mounting media are not necessary. When preservation of the visceral mass or the appendages is not critical, freeze drying is suggested to preserve the specimens for easy storage. The residue from the sieves is transferred to a beaker using a spatula and a small amount of water from a wash bottle. The sediment is then frozen. After freezing, ﬁlter paper or a suitable tissue is placed on the beaker with an elastic band and set into a freeze-drying apparatus. In the freeze-dryer, ice is sublimed by vacuum from the specimens and sediments. This process takes 24 – 48 h, depending on the volume of ice being removed, the number of samples, and the size of the freeze-dryer. The specimens are then handpicked from the sample using a steromicroscope and placed in a separate vial or a micropaleontologic slide. This method has the added advantage of not requiring the more extensive labor and time curating wet samples. The dried specimens are also in excellent condition for further study with an SEM to view and photograph appendages. When a large volume of sediment still remains in the sieves after washing and freeze drying, the ostracodes can be concentrated using acetone (Delorme, 1967). [Acetone should only be used under a safe operating fume hood.] The residue from the #60 and #100 mesh sieves is passed through a 3-in. ﬁlter sieve to ensure the sediment particles are disaggregated. The sieved residue is then sprinkled into a funnel (4-in.) containing acetone. The sediment particles sink and the ostracode carapaces ﬂoat to the surface. Normally, the carapace will trap an air bubble which makes the shell buoyant. The lower tip of the funnel is equipped with a latex tube and a clamp. After the sediment has been sprinkled into the acetone, the clamp is carefully removed from the tube and the “sink” allowed to drain into another funnel lined with rapid-draining ﬁlter paper. Care must be taken not to drain all the acetone from the top funnel, otherwise the ostracodes will be removed. When the sink has been removed, another funnel lined with ﬁlter paper is placed beneath, the clamp removed, and the top funnel rinsed with acetone from a wash bottle. In this way, all supernatant particles (the ﬂoat), including the ostracodes, will be transferred to the lower ﬁlter paper. Individual shells will also be a part of the ﬂoat except Cytherissa, Darwinula, Cytheromorpha,and Limnocythere which do not have an anterior vestibule. The ﬁlter paper containing the ostracode residue is then air- or oven-dried, and the residue transferred to a vial. The shells and carapaces are picked with a very ﬁne wetted sable hair brush and mounted onto a micropaleontological slide. The slide is preglued with the water-soluble gum Tragacanth. The ostracodes are then ready to be identiﬁed and counted. For an accurate count of adult ostracodes, the foregoing method is adequate. If, however, all live instars and adults are required for biomass studies, then the complete sediment parcel will be required. Wet sieving would only assist in separating organisms and particles by size. For biomass studies, it might be preferable to use a short core tube pushed into the sediment some 10 – 15 cm. Nalepa and Robertson (1981) have recovered ostracodes using two mesh sizes, 0.595 and 0.106 mm, for biomass studies. The smallest mesh size required to collect all the ostracodes including instars is not indicated by them, as 0.106 mm will not retain all the instars. Hummon (1981) recommends a sieve size of 0.062 mm to retain all ostracodes for quantitative analyses. This sieve size would miss the ﬁrst three instars of Limnocythere friabilis. B. Rearing Techniques Cosmopolitan ostracodes, such as Cypridopsis vidua and Cyprinotus incongruens, are relatively easy 20. Ostracoda to rear in an aquarium. Collection of water, from the habitat associated with the species to be reared, along with some of the substrate will allow the population to develop. Natural water from the habitat is preferred to distilled or tap water. Habitat water contains the proper mix of major and minor ions for an established solute composition. The pH of the water should be checked periodically and adjusted when necessary. This type of aquarium will probably go “wild” and anoxic after several months. A more pristine culture can be set up by ﬁltering the water from the habitat, placing it in an aquarium with sterilized silt, sand, and clay as a substrate. Cultured algae (Klugh, 1927; Kesling, 1951a; Sanders, 1970; Grant et al., 1983; Havel and Talbot, 1995; Roca et al., 1993; Roca and Wansard, 1997; Zelmer and Esch, 1998) have been used for food as well as fresh leaf lettuce and grated boiled egg which has been allowed to decompose bacterially for a month. Roca and Wansard (1997) also used benthic algae, gastropod pellets, and Spirulina with a high survival rate whereas chopped alfalfa as food with garden compost produced the lowest survival rate. Aquaria should not be placed on a window ledge where high temperatures may destroy the artiﬁcial habitat. XII. USES OF FRESHWATER OSTRACODA Ostracode shells are preserved as fossils. Autecological data bases are available for extant species, and so these crustaceans can be used to reconstruct past chemical and physical habitats. With a suitable time frame, the paleolimnology of a pond or lake may be reconstructed from the fossils extracted from a sediment core. Initially, subjective interpretations were made of past conditions of the lake or pond (Gutentag and Benson, 1962; Benson and MacDonald, 1963; Staplin, 1963a, b; Neale, 1988). As autecological data bases were generated, paleointerpretive models were developed that used autecological data for objective reconstruction of the chemical and physical aspects of the habitat (Anderson et al., 1985; Cvancara et al., 1971; Delorme, 1968, 1969b, 1971b, c, 1982, 1996; Delorme and Zoltai, 1984; Delorme et al., 1977, 1978; Forester et al., 1987; Karrow et al., 1975, 1995; Klassen et al., 1967; McAllister and Harrington, 1969; Smith et al., 1992; Westgate et al., 1987; Westgate and Delorme, 1988; Porter et al., 1999). In some instances, it is possible to use a number of different fossils (ostracodes, molluscs, pollen, and diatoms) to obtain comprehensive paleoenvironmental interpretations (Maher et al., 1998). A summary of the use of Canadian ostracodes in paleolimnology and paleoclimatology is given by Delorme (1989). 831 Forester et al. (1994) have devised two interesting indices which estimate the “TDS ratio” of evaporation to outﬂow of a large lake and a “shore – zone ratio” which gives an indication of the proximity of the shore line to the core site. In each case, ostracode abundances are used in the ratios. Curry (1999) has devised two environmental tolerance indices (ETI) based on 341 modern aquatic environments in the United States. The indices are based on a limited set of autecological data and so at this time cannot be universally applied for North America. Trace element analyses of Mg, Ca, and Sr in ostracode shells have been used for reconstructing past salinity changes in a variety of aquatic habitats (Chivas et al., 1983, 1985, 1986a, b; De Deckker, 1983b, 1988; Lamb et al., 1995). Similar studies are being carried out on North American ostracode shells. Engstrom and Nelson (1991) found that distribution coefﬁcients (KD) for (Mg2 /Ca2)H2O and (Sr2 /Ca2)H2O in shells of Candona rawsoni from cultured samples and from shells collected from Devils Lake, North Dakota have similar ratios. Analyses show excess Mg is incorporated into the shell during early calciﬁcation which is a conﬁrmation of the work done by Chivas et al. (1986a). Hu et al. (1998) did trace-element analyses of ostracode shells from a sediment core of Farewell Lake, Alaska. From the Mg / Ca and Sr / Ca ratios they detailed the climatic reconstruction of the area indicating cold and warm climate periods. A 19-m core from Coldwater Lake, North Dakota documented several century-scale changes in the paleoenvironmental record using 18O, 13C, Mg / Ca, and Sr / Ca ratios (Xia et al., 1997b). A total of seven phases of climate and salinity were identiﬁed. Yu and Ito (1999) using Candona rawsoni shells from a Rice Lake, North Dakota, core determined Mg / Ca ratios showing periods of salinity and aridity. Oxygen isotope studies of calcitic shells are being used to develop paleoclimatic interpretations (von Grafenstein et al., 1992, 1994). Middle Holocene ostracodes of Elk Lake, in Grant County, Minnesota, show an assemblage dominated by Limnocythere staplini, a halophyte. This species is a high-salinity form, yet the oxygen isotope values on shells of Candona rawsoni indicate a decrease by 2 – 3 ppt indicating that ground water mediated the paleoclimatic record. Xia et al. (1997a, c) have determined that calcitic shells of Candona rawsoni formed at temperatures of 15 and 25°C were not in isotopic equilibrium with water, but had a constant offset from equilibrium based on oxygen isotope fractionation of about 2 ppt. With the molting process occurring from spring to fall, the intraannual temperature change would exceed the interannual to century-scale variations expected during lateQuaternary climate shifts. The resulting intraannual signal may exceed the long-term paleoclimatic signals. 832 L. D. Delorme The authors suggest 10 – 15 shells per sample be analyzed to ameliorate the interannual noise. XIII. CURRENT AND FUTURE RESEARCH PROBLEMS The European literature on freshwater ostracodes continues to grow. Unfortunately, the North American literature is not doing as well. There is a real need for a comprehensive review and update of North American systematics. Detailed descriptions of soft and hard part morphology is desperately needed together with detailed drawings and SEM photographs. This information is critical if scientists hope to be able to continue to communicate about the biological units on which research is being conducted. Are we talking about the same species? Speciﬁc research into ostracode systematics needs to be emphasized. This is particularly true of several generic pairs such as Cypricercus and Eucypris, Cyprinotus and Heterocypris, and Megalocypris and Cypriconcha. These groups require a detailed cytogenic and SEM study of shell and appendage morphology of species of the world (see Section XIV.A). Only in this way will we be able to resolve which biocharacters of a certain group should be used in deﬁning a genus. The establishment of a universally acceptable systematic nomenclature is important for studies in ecology and paleolimnology. Continuing studies are required in autecology of ostracodes. Data bases of chemical and physical parameters of ostracode habitats are being expanded (B. B. Curry, R. M. Forester, A. J. Smith, personal communication; L. D. Delorme unpublished). Analyses of these data bases are required to give a sense of the factors which control the species in its environment. The roles of solute composition and salinity of the habitat are an integral part of this analysis. The role of ostracodes in the population dynamics of a pond or lake system is poorly known. Only a few studies have attempted to look at biomass (Bechara, 1996), productivity, densities in communities, positions in the aquatic food chain (as predators or prey), behavioral defense mechanisms, and host – parasite interactions. All these factors and others may affect the success of ostracodes in their habitat and need to be studied in more detail. More scientiﬁc papers have been written since the ﬁrst edition of this book, but more are needed as many more questions are being raised. XIV. CLASSIFICATION OF OSTRACODES Ostracodes are typically identiﬁed from adult specimens. Insufﬁcient information is obtained from the instars (juveniles) to make a proper identiﬁcation to the species level unless a monoculture has been obtained and adults are present. In some instances, identiﬁcation of the instar can be taken to the generic level. The most useful biocharacter in taxonomy and systematics is the shell. A ﬁeld key is offered by Delorme (1967) for some of the North American ostracode species as well as his ﬁve-part series (Delorme, 1970a – d, 1971a). Other useful references are Furtos (1933) and Hoff (1942). A. Taxonomic Controversies A number of controversies exist around the naming of certain genera. These will be discussed separately. In order not to confuse the issue any further, a conservative approach will be taken for the use of generic names. Detailed systematics using the scanning electron microscope will be required, using specimens from the global arena rather than from local areas, in order to resolve the issues. The names of two genera Cypricercus and Eucypris have been commonly used. Broodbakker (1983) has provided a literature review of the status for Cypricercus and Strandesia. There is no mention of the genus Eucypris. Sars (1928) differentiated between Eucypris and Cypricercus on the absence and presence of males respectively. He also noted the peculiar arrangement of the “spermatic vessels” (Sars, 1928, p. 117) in the males of Cypricercus. Furtos (1933) maintained the two genera based on reproduction and length of the furcal ramus (1 / 2 the length of shell, Eucypris; 1 / 2 the length of shell, Cypricercus). Hoff (1942) noted the elevation of genera on the basis of propagation was not an acceptable practice. He opted for one genus based on an extremely long furcal ramus for Cypricercus. The species Eucypris crassa was originally assigned to Cypris by O. F. Müller and later to Eucypris by G. W. Müller (1912). This species has always been referred to the genus Eucypris. Martens (1989) has ascribed Eucypris serrata to the genus Trajancypris, but has not included the North American form (Delorme, 1970a) to the new genus. Broodbakker (1983) erected the genus Bradleystrandesia to include those species of Cypricercus which occur in Europe and North America. This author states “further research is needed to prove if the genus Bradleystrandesia is conﬁned to Europe and North America” (Broodbakker, 1983, p. 329). Martens (in Turgeon and Hebert, 1995) continued to use Cypricercus for the North American species. Bradleystrandesia has not been conﬁrmed for North America, but has been used by Rossi et al. (1998) in referencing work done by Turgeon and Hebert (1995). Cypricercus and Eucypris will be used in the key. The genera Cyprinotus Brady and Heterocypris (Claus) have had a confused nomenclature. Purper and Würdig-Macial (1974) did a literature review of these two genera. They concluded the main difference 20. Ostracoda between the two genera was the dorsal gibbosity of the right valve for Cyprinotus. Both genera are reported as having the anteroventral margin of the right valve with denticles or crenulations. Sars (1928) and Müller (1912) did not consider the gibbosity of the right valve as a valid generic biocharacter and, thus, did not recognize Heterocypris. Hoff (1942) also did not recognize Heterocypris. This morphological biocharacter in Physocypria (Delorme, 1970b) may or may not be present and is not used to split the genus. Cyprinotus is used in the key because it was published ﬁrst in 1886. The genus Cypriconcha was erected by Sars (1926) to incorporate the large cyprinids of North America. Sars (1898) had already established Megalocypris for the large cyprinids of Africa. Delorme (1969a) reviewed the two genera and concluded the similarities were many and discrepancies few. As a result he put the North American cypriconchids into the genus Megalocypris. Martens (1986) and McKenzie (1982, referenced in Martens, 1986) argued for the retention of Cypriconcha for the large cyprinids of North America. Megalocypris is used in the key. A number of genera may be found in the North American ostracode literature, but are not included here. The following generic names are no longer considered valid: Cyclocypria Dobbin, 1941 — Cyclocypris Brady and Norman, 1888 Cypriconcha Sars, 1926 — Megalocypris Sars, 1898 [see Delorme, 1969a] Cytherites Sars, 1926 — Entocythere Marshall, 1903 [see Hoff, 1943b] Pionocypris Brady and Norman, 1896 — Cypridopsis Brady, 1868 Pseudoilyocypris Ferguson, 1967 — Pelocypris Klie, 1939 [see Delorme, 1970d] Spirocypris Sharpe, 1903 — Cypricercus Sars, 1895 [see Hoff, 1942] 833 Several species, as listed below, have been assigned to an incorrect genus; therefore, the generic names will not appear in the key: Candonocypris deeveyi Tressler, 1954 — Megalocypris deeveyi (Tressler) 1954 Candonocypris pugionis Furtos, 1936b — Megalocypris[?] pugionis (Furtos), 1936b [see Furtos, 1936b] Candonocypris sarsi Danforth, 1948 — Megalocypris sarsi (Danforth), 1948 Candonocypris serrato-marginata (Furtos), 1935 — Eucypris serrato-marginata (Furtos), 1935 Erpetocypris barbatus Turner, 1899 — Megalocypris sp. (Turner), 1899 Prionocypris canadensis Sars, 1926 — Strandesia canadensis (Sars), 1926 [see Marmonier and Ward, 1990] Prionocypris glacialis (Sars), 1890 — Tonnacypris glacialis (Sars), [see Grifﬁths et al., 1998] Pseudocandona ikpikpukensis Swain, 1963 — Candona ikpikpukensis (Swain), 1963 [see Delorme, 1970c] Stenocypria longicomosa Furtos, 1933 — Isocypris longicomosa (Furtos), 1933 [see Delorme, 1970a] B. Classiﬁcation of the Order Podocopida The Podocopida1 is the only order that contains both marine and freshwater ostracodes (Table II). Dorsal border of the valve is curved, or if straight, it is much shorter than the total length. No permanent anterior opening is present between the valves. There is no heart; two or three pairs of thoracic legs are present. Within Podocopida are two suborders: Metacopina and Podocopina. As will be described below, the suborder Metacopina contains only one freshwater genus in North America, Darwinula (superfamily Darwinuloidea, family Darwinulidae; Figs. 8 and 10). Its characteristics are given as follows2. Shell surface smooth, vestibules absent, distinctive adductor muscle scars (Figs. 8, 10, 19); ﬁrst antenna composed of six podomeres with strong spikelike setae, swimming setae lacking on second antenna, mandibular exopodite with short palp of three podomeres of which the ﬁrst is wide and has a row of long feathered setae, respiratory plate of mandible small, masticatory process of maxilla short and heavy, respiratory plate of maxilla with numerous feathered setae, maxillae with strong masticatory structure and leglike palp of three podomeres, ﬁrst and second thoracic legs similar in structure and direction, furca lacking, ovaries do not originate between inner and outer epidermis, female carries eggs in posterior of carapace ..............................................................................................................................................................................Darwinula Freshwater and some marine ostracodes are included in the suborder Podocopina. Members of this taxon have two pairs of thoracic legs. The exopodite of the second antenna is represented by at most a scale and a few setae. The furcae are rodlike rather than lamelliform; eyes are present in most forms. Closing muscle scars are discrete and arranged in a distinctive group. Taxonomic keys to genera within two superfamilies of this suborder are given below. C. Taxonomic Key to Genera of the Superfamily Cypridoidea2 Surface of the shell usually smooth, dorsal margin without interlocking teeth. Eyes developed to varying degrees, either separated or fused into a single median eye. The ﬁrst antennae with basal portion of two or 1 2 Modiﬁed after Kesling (1951a). Modiﬁed after Hoff (1942). 834 L. D. Delorme TABLE II Classiﬁcation of the Class Ostracodaa Class Ostracoda Subclass Podocopa Order Podocopida Sars, 1866 Suborder Metacopina Sylvester-Bradley, 1961 Superfamily Darwinuloidea Brady and Norman, 1888 Family Darwinulidae Brady and Norman, 1888 Genus Darwinula Brady and Norman Suborder Podocopina Sars, 1866 Superfamily Cypridoidea Baird, 1845 Family Candoniidae Kaufmann, 1900 Subfamily Candoninae Daday, 1900 Genus Candocyprinotus Delorme Genus Candona Baird Genus Fabaeformiscandona Krstic Genus Nannocandona, Eckman Genus Paracandona, Hartwig Genus Pseudocandona, Kaufmann Family Cyprididae Baird, 1845 Subfamily Cypridinae Baird, 1845 Genus Chlamydotheca Sausseure Genus Cyprinotus Brady Genus Cypris Müller Genus Dolerocypris Kaufmann Genus Herpetocypris Brady and Norman Genus Ilyodromus Sars Genus Isocypris Müller Genus Megalocypris Sars Genus Scottia Brady and Norman Genus Stenocypris Sars Genus Strandesia Stuhlmann Subfamily Eucypridinae Bronstein, 1947 Genus Cypricercus Sars Genus Eucypris Vávra Genus Tonnacypris Diebel and Pietrzeniuk Genus Trajancypris Martens Subfamily Cyclocyprinae Hoff, 1942 Genus Candocypria Furtos Genus Cyclocypris Brady and Norman Genus Cypria Zenker Genus Physocypria Vávra Subfamily Cypridopsinae Kaufmann, 1900 Genus Cavernocypris Hartmann Genus Cypretta Vávra Genus Cypridopsis Brady Genus Potamocypris Brady Genus Sarscypridopsis McKenzie Family Ilyocyprididae Kaufmann, 1900 Subfamily Ilyocyprinae Kaufmann, 1900 Genus Ilyocypris Brady and Norman Genus Pelocypris Klie Family Notodromadidae Kaufmann, 1900 Subfamily Notodromadinae Kaufmann, 1900 Genus Cyprois Zenker Genus Notodromas Lilljeborg Superfamily Cytheroidea Baird, 1850 Family Cytheridae Baird, 1850 Subfamily Cytherideinae Sars, 1928 Genus Cyprideis Jones Family Entocytheridae Hoff, 1942 Subfamily Entocytherinae Hoff, 1942 Genus Ankylocythere Hart (Continues) TABLE II (Continued) Genus Ascetocythere Hart Genus Cymocythere Hart Genus Dactylocythere Hart Genus Donnaldsoncythere Hart Genus Entocythere Marshall Genus Geocythere Hart Genus Harpagocythere Hobbs III Genus Hartocythere Hobbs III Genus Litocythere Hobbs and Walton Genus Lordocythere Hobbs and Hobbs Genus Okriocythere Hart Genus Ornithocythere Hobbs Genus Phymocythere Hobbs and Hart Genus Plectocythere Hobbs III Genus Rhadinocythere Hart Genus Saurocythere Hobbs III Genus Sagittocythere Hart Genus Thermastrocythere Hobbs and Walton Genus Uncinocythere Hart Family Limnocytheridae Klie, 1938 Subfamily Limnocytherinae Klie, 1938 Genus Limnocythere Brady Genus Metacypris Brady and Robertson Family Loxoconchidae Sars, 1928 Subfamily Loxoconchinae Sars, 1928 Genus Cytheromorpha Hirschmann Family Neocytherideidae Puri, 1957 Subfamily Neocytherideidinae Puri, 1957 Genus Cytherissa Sars a The systematic list above is alphabetical (after Bowman and Abele, 1982), but the systematic treatment is not. three podomeres and an endopodite of four or ﬁve podomeres, with swimming setae usually well developed. The second antennae with basal part of two podomeres and an endopodite of three or four podomeres. The exopodite is reduced to a small scalelike appendage bearing at most three setae. Maxillula not pediform, but modiﬁed as a mouth part, with the anterior margin of the base adapted for feeding. The maxillar endopodite forms a small palp in the female, but is enlarged to form prehensile palps in the male. The ﬁrst thoracic leg has an endopodite of three or four podomeres and a strong distal claw. The second leg is bent dorsally and is probably used in cleaning the respiratory surfaces and other parts of the body. The second leg usually has three distal setae, but the distal end may be modiﬁed for grasping. The furca is typically well developed and rod-shaped, but may be reduced to a ﬂagellum or whiplike structure. The gonads are located within the valves of the shell. In the male, a portion of the vas deferens is modiﬁed to form an ejaculatory duct. FIGURE 19 External view of Darwinula stevensoni (Brady and Robertson). FIGURE 20 External view of Cypridopsis vidua (Müller). FIGURE 21 External view of Potamocypris smaragdina (Vávra). FIGURE 22 External view of Cyprois marginata Straus. FIGURE 23 External view of a female Candona rawsoni Tressler, note the overlap of the right valve by the left valve. FIGURE 24 External view of Paracandona euplectella Brady and Norman, note the reticulate structure and the pustules. FIGURE 25 External view of Cypricercus reticulatus (Zaddach). 835 836 L. D. Delorme 1a. Furcal ramus greatly reduced, whip-shaped, without terminal claw (difﬁcult to observe)Cypridopsinae3 ...........................................2 1b. Furcal ramus well developed, bar-shaped, with two terminal or subterminal claws; outer masticatory process of maxilla with six nearly equal setae modiﬁed to form toothed spines ..............................................................................................Notodromadinae4 4 1c. Swimming setae of antennae completely lacking, shell white; outer masticatory process with two or three setae modiﬁed as spines .....................................................................................................................................................................................Candoninae5 5 1d. Swimming setae of ﬁrst antenna present; second thoracic leg distally modiﬁed as seizing apparatus, ultimate podomere beak-like with two well developed bristle-like setae, third setae lacking or hook-like....................................................................Cypridinae6 7 1e. Third thoracic leg with one long reﬂexed seta and two shorter unreﬂexed setae .....................................................Cyclocyprinae7 10 1f. Carapace 1. 0 – 2.5 mm, elliptical to elongated, rarely globular; surface ornamentation absent except for shallow pits; marginal teeth possible on both valves; marginal zone with selvage present or absent, sometimes inwardly displaced in the right valve. First antenna with Rome organ small and undivided; mandibular palp with gamma-seta short, stout and hirsute. Maxillula with third endopodite carrying two, mostly smooth, teeth or bristles. The second process of the maxillula with “c”-seta ............Eucypridinae8 12 1g. Second thoracic leg not bearing chela, last podomere cylindrical and bearing three setae; shell oblong to subrectangular, dorsal margin straight, surface pitted, with one or more transverse sulci ................................................................................Ilyocyprinae9 4 2a(1a). Shells tumid........................................................................................................................................................................................3 2b. Shells crescentic in shape, compressed, right valve higher than left and extending dorsally above left, H / L 0.5, right valve overlaps left valve in venter, usually hairy; swimming setae of second antenna well developed, usually extending to tips of claws or beyond; ultimate podomere of maxillary palp distally wider than long (Fig. 21) ..........................................................................Potamocypris 3a(2a). Shells subovate, H / L 0.5, valves nearly equal, left valve overlaps right valve in venter; ultimate podomere of maxillary palp cylindrical, longer than wide (Fig. 20), swimming setae of second antennae well developed......................................................Cypridopsis 3b. Shells subtriangular, H / L 0.5, right valve overlaps left valve in venter; swimming setae of second antennae well developed .....................................................................................................................................................................................Sarscypridopsis 3c. Shells elongate, 1 mm in length, left valve longer than right valve, H / L 0.5, left valve overlaps right valve in venter; swimming setae of second antenna poorly developed, furcal rami absent in males..........................................................................Cavernocypris 3d. Shells tumid, 1 mm in length, anterior margin of both valves with row of radiating septa; two maxillary spines may be smooth or toothed; ovaries or testes coiled in spiral pattern in posterior of carapace...............................................................................Cypretta 4a(1b). Shell from side short and high, compressed; eyes not widely separated; ﬁrst antenna of ﬁve podomeres; swimming setae almost reach tips of end claws; right and left prehensile palps of male maxillar endopodites differing little, with well-developed respiratory plate; setae on distal end of second thoracic leg modiﬁed for grasping; furca with two claws and two setae (Fig. 22) ..............Cyprois 4b. Shell short with height two-thirds of length, dorsal margin forming elevated arch, ventral surface ﬂattened; eyes well separated; ﬁrst antenna of six apparent podomeres, swimming setae extending beyond tips of terminal claws; palps of maxillae in males not similar, each formed with two podomeres; maxillae without respiratory plate; distal three setae of second thoracic leg nearly equal; furca with terminal seta lacking.................................................................................................................................................Notodromas 5a(1c). Carapace with sides convex; outer masticatory process of maxilla smooth; apical claw of ultimate segment of second thoracic leg well developed, furcal claws weakly serrated .............................................................................................................Candocyprinotus 5b. Shell subrectangular to subtriangular in shape, two special sensory setae present at juncture of fourth and ﬁfth podomeres of male ﬁrst antenna, respiratory plate of maxillae with two or three setae ....................................................................................................6 6a(5b). Male carapace shape and size usually different than female; second thoracic leg with three unequally long setae on last podomere; Zenker’s organs usually with seven wreaths of chitinous spines, openings of ejaculatory duct funnel-shaped (Figs. 9a, 9b, 23) ..............................................................................................................................................................................................Candona 6b. Carapace usually elongate, more rarely triangular in lateral view,distinctly compressed in dorsal view; left valve usually with a posterodorsal lobelike extension of the shell which overlaps right valve; male second antenna dimorphic, penultimate segment with male bristle. Setal group of the 2nd segment of the mandibular palp with three (fabaeformis group) or four (acuminata group) setae, gamma seta of the penultimate segment smooth (not plumose). Protopodite of second thoracic leg with two setae (d1 and dp), setae ‘e’ and ‘f’ of ﬁrst endopodite missing, seta ‘g’ of third endopodite present, terminal endopodite with two long (h2 and h3) and one 3 Modiﬁed after Marmonier et al. (1989), and McKenzie (1977). Modiﬁed after Hoff (1942). 5 Modiﬁed after Hoff, 1942, Furtos (1933) and Meisch (1996). 6 Modiﬁed after Hoff (1942) and Delorme (1970a). 7 Modiﬁed after Hoff (1942) and Furtos (1933). 8 Modiﬁed after Martens (1989). 9 Modiﬁed after Hoff (1942) and Tressler (1949). 4 20. Ostracoda 837 short (h1) setae. Female genital lobe usually with more or less well-developed process directed posteriorly. Hemipenis with ‘M’ process heavily sclerotized, proximal part slightly broader than the distal one.....................................................Fabaeformiscandona 6c. Shell usually with small tubercles and reticulations on surface, shape subrectangular; penultimate segment of second thoracic leg divided, each division with strong distal seta; otherwise similar to Candona (Figs. 7, 24) ...................................................Paracandona 6d. Carapace of medium length (0.8 – 1.2 mm), variously shaped, usually short and stout, more rarely elongate (subrectangular) or triangular in lateral view. Surface of adult valves smooth or pitted, usually with long stiff setae emanating through the shell, left valve overlaps the right valve ventrally. The penultimate endopodite of the second antennae subdivided or not, when subdivided with male bristles (t2 and t3) transformed. Setal group of the second endopodite of mandibular palp with three or ﬁve setae, distal seta of penultimate endopodite smooth (not plumose). Protopodite of second thoracic leg with three setae (d1-d2, dp), distal seta ‘g’ on the penultimate endopodite present, terminal endopodite with one short (h1), and two long setae (h2-3). Zenker’s organ with 5 2 rings of spines. Hemipenis with ‘M’ process ﬂat and proximally only weakly sclerotized ............................................Pseudocandona 7a(1d). Margins of valve usually smooth ........................................................................................................................................................8 7b. Margin of valve with spines or denticles...........................................................................................................................................19 8a(7a). Shells 2.2 mm long ..........................................................................................................................................................................9 8b. Shells 2.2 mm long ........................................................................................................................................................................16 9a(8a). Shell surface sculptured ....................................................................................................................................................................10 9b. Shell surface smooth.........................................................................................................................................................................13 10a(9a). Shell surface reticulate or scabrous especially in juveniles.................................................................................................................11 10b. Shell surface punctate or striated ......................................................................................................................................................12 11a(10a). Outer masticatory process with two long, toothed spinelike setae; furcal ramus greater than half length of valve; testes coiled in anterior portion of each valve, males rare (Fig. 25)................................................................................................................Cypricercus 12a. Outer masticatory process with two long, toothed spinelike setae; furcal ramus less than half length of valve, males rare (Fig. 26) ...............................................................................................................................................................................................Eucypris 12b. Swimming setae of second antenna rudimentary, length of gamma-seta of the mandibular palp is nearly ﬁve times its basal width, second segment of mandibular palp is trapezoidal and the terminal endopodite of the third endopodite bears two serrated teeth, ﬁrst thoracic leg has the setae d2 nearly two times d1; shell elongate and moderately large, compressed and subrectangular to clavate, anterior inner lamellae of left valve with small peg tooth sometimes nearly invisible; furcal ramus smooth ..........................Tonnacypris 12c. Right valve with a frontal selvage inwardly displaced to a varying degree, but never marginal; left valve without selvage, but with large frontal inner list, situated about halfway on the calciﬁed inner lamella ....................................................................Trajancypris 13a(10b). Shell elongate, narrow with scattered punctae; swimming seta of second antenna barely reaching tips of claws, second thoracic leg with well-developed apical claw; rami of furcae dissimilar in width, two end claws strongly pectinate...............................Stenocypris 13b. Shells elongate and compressed, surface longitudinally striated; swimming setae of ﬁrst and second antenna short, furcal ramus terminates in three short claws rather than two ......................................................................................................................Ilyodromus 14. Swimming setae of second antenna strongly developed ....................................................................................................................15 15a(14b). Claws of furca ﬁnely denticulate; shell subrectangular, surface smooth and moderately hairy; characteristic marginal pore canals in anterior duplicature; outer masticatory process of maxilla with two smooth spines; second thoracic leg with well-developed apical claw .......................................................................................................................................................................................Isocypris 15b. Claws of furca strongly developed....................................................................................................................................................16 16a(15b). Shell reniform, densely hairy; maxillae bears well-developed respiratory plate; ﬁrst thoracic leg with one long terminal seta and claw, furca with feathered dorsal seta ........................................................................................................................................Scottia 16b. Shells elongate, dorsum often with prominent dorsal ﬂange, left valve always with conspicuous row of tuberclelike canals removed from the free margin; swimming setae of second antenna extend to tips of terminal claws, third masticatory process of maxilla with two strong spines, furcal ramus at least half as long as valve and with two claws and setae .................................................Strandesia 17a(8b). Shells with anterior ﬂangelike projections which produces large vestibule; both antenna with well-developed swimming setae; outer masticatory process of maxilla with one toothed and one denticulate spine; second thoracic leg with well-developed apical claw; claws of furca ﬁnely denticulate ...................................................................................................................................Chlamydotheca 17b. Shells without anterior ﬂangelike projection.....................................................................................................................................18 18a(17b). Shells narrow and spindle-shaped, vestibules well developed at both extremities; both antennae with well-developed swimming setae; maxilla with outer masticatory process with two smooth spines; second thoracic leg with well-developed apical claw, claws of furca coarsely denticulate .................................................................................................................................................Dolerocypris 838 L. D. Delorme FIGURE 26 External view of Eucypris crassa (Müller). FIGURE 27 External view of Cypris pubera Müller, note the posteroventral spines on the right valve. FIGURE 28 External view of Cyclocypris ampla Furtos, note the pitted surface. FIGURE 29 Internal view of Physocypria pustulosa (Sharpe) showing the denticles on the anteroventral margin. FIGURE 30 Exterior view of Pelocypris alatabulbosa Delorme showing a reticulate surface, sulci, and nodes. 18b. Shells subrectangular ........................................................................................................................................................................19 19a(18b). Shells elongate and large, compressed; swimming setae of second antenna not reaching mid position of terminal claw; spines of outer masticatory process of maxilla toothed; ventral edge of furca denticulate with combs of teeth .............................Herpetocypris 19b. Shells subrectangular to subtrapezoidal; swimming setae on ﬁrst antenna extend to end of ultimate podomere and not to tips of claws of second antenna; outer masticatory process made up of short, smooth bristles; maxillar endopodite of male developed into prehensile palp; second thoracic leg with apical claw not well developed; claws of furca denticulate...............................Megalocypris 20. Ostracoda 839 20a(7b). Shells 2 mm, margin of right or left valve tuberculate and margin of other valve smooth, dorsal margin may be arched or have an obtuse apex, valves commonly unequal, anterolateral surfaces pustulose; outer masticatory process of maxilla with two clawlike setae which may or may not be toothed; furcal claw longer than one-half length of ventral margin of furcal ramus, ramus with length less than twenty times least width (Figs. 3, 4) .....................................................................................................................Cyprinotus 20b. Shells 2 mm, tumid, highly arched, venter ﬂattened, anterior margins denticulate, posteroventral margin of right valve with two to four sharp marginal spines, surface wrinkled to minutely crenulate to punctate; second antenna with well-developed swimming setae; outermost masticatory process of maxilla with two denticulate spines, second thoracic leg with well-developed apical claw, claws of furca strongly denticulate (Fig. 27) ...............................................................................................................................Cypris 21a(1e). Swimming setae of second antenna lacking or rudimentary; shells ovoid, compressed; eyes well developed and fused; ultimate segment of second thoracic leg with two short and one long reﬂexed setae; tips of furcal claws serrate ................................Candocypria 21b. Swimming setae of second antenna well developed ..........................................................................................................................21 22a(21b). Shells tumid, height and width greater than one-half length, surface smooth and usually brown in color; eye well developed; respiratory plate of maxillae with six plumose setae; second thoracic leg consisting of four podomeres, ultimate podomere elongated and more than twice as wide and usually at least one-half as long as penultimate podomere; ultimate podomere of third thoracic leg with three distal setae, unequal, with outermost one very long and reﬂexed (Fig. 28) ........................................................Cyclocypris 22b. Shells compressed .............................................................................................................................................................................23 23a(22b). Shells short and high, occasionally elongate reniform, margins smooth not tuberculate; eyes well developed; second antenna of male with penultimate podomere divided and bearing specialized male setae; ultimate podomere of mandibular palp elongated three times as long as proximal width; palp of maxilla well developed, masticatory process weak; penultimate podomere of second thoracic leg undivided, ultimate podomere short scarcely longer than wide, longest distal seta reﬂexed; terminal and subterminal claws of furca strong, dorsal seta may be rudimentary; Zenker’s organ with seven whorls of chitinous rays, proximal end much inﬂated; hemipene with two terminal lobes only, outer lobe lacking........................................................................................................Cypria 23a(22b). Shells commonly unequal in height or length or both, at least anterior margin of one of the valves tuberculate; otherwise as in genus Cypria (Fig. 29) .................................................................................................................................................................Physocypria 24a(1g). Shells may have large rounded humplike projections on lateral surface, marginal spines and pustules; ﬁrst antenna with some swimming setae shortened and clawlike; swimming setae of second antenna may be shortened, male without special setae; maxillar endopodite of male transformed into prehensile palp, in female clearly leglike; ultimate podomere in second thoracic leg cylindrical with three setae, longest may be reﬂexed, claws well developed; Zenker’s organs with numerous chitinous rods and with spherical inﬂated openings at each end; vasa deferentia twisted ...........................................................................................................Ilyocypris 24b. Shells with pronounced mamilliarylike projections on lateral surface, marginal spines and pustules; second antenna with swimming setae which extend considerably beyond tips of terminal claws; maxillar endopodite with long curved terminal claw; ﬁrst leg with one short and one long seta; furca well developed with dorsal seta longer than terminal claw (Fig. 30) ...............................Pelocypris D. Taxonomic Key to Genera of the Superfamily Cytheroidea10 Shell variable in shape and sculpturing, seldom smooth, usually with reticulations, often with spines, furrows, or tubercles. Shells noncalcareous in the Entocytherinae. Valves nearly equal; often toothlike projections along the hinge. The ﬁrst antennae consist of a base of two podomeres and an endopodite of three or four podomeres. The setae of the ﬁrst antennae are short and stout, often clawlike. The exopodite or the ﬂagellum of the second antennae is represented by a long hollow seta forming a duct carrying secretions from a gland. The endopodite of the second antennae 1a. 10 11 of three podomeres, the long penultimate one may be divided. Swimming setae lacking. Maxillar endopodites and two pairs of thoracic legs similar and all adapted for crawling except in the Entocytherinae modiﬁed for grasping. Furca always greatly reduced. In the male, the hemipenes always present and well developed, the ejaculation duct is absent, a male accessory sense organ consisting of numerous setae on a short base is located between and somewhat medially to the bases of the ﬁrst and second pairs of thoracic legs. The gonads do not lie between the inner and outer lamellae of the valves, but are in the body lateral to the intestine. In some species, the eggs are retained in the shell cavity during development. Free margins of valves without conspicuous pore canals, carapaces non calcareous, reniform or subelliptical in shape, furrows or protuberances may be present; respiratory plate of mandible reduced to two or three setae, masticatory lobes well developed, furca rudimentary, penis strongly curved; North American ostracodes commensal on crayﬁshes and a freshwater crab (Fig. 12) ..............................................................................................................................................................Subfamily Entocytherinae11 3 Modiﬁed after Hoff (1942) and Sars (1928). Modiﬁed after Hoff (1942) and Hart and Hart (1974). 840 L. D. Delorme 1b. Free margins of valves ﬂattened, with many long marginal pore canals, subrectangular, often with protuberances or furrows, strongly reticulate; respiratory plate of mandible well developed, furca usually with two short setae .................................. Subfamily Limnocytherinae12 ........................................................................................................................................................................... 22 1c. Shell variable in shape and sculpturing, seldom smooth, usually with reticulations, spines, furrows, or tubercles, valves nearly equal, often with toothlike projections along hinge, vestibules absent; eyes distinctly separated, antennae well developed, exopodite of second antenna in form of long hollow seta carrying secretions from a gland near base of second antenna, setae of ﬁrst antenna short and stout, often claw like, swimming setae on second antenna lacking, ﬁrst thoracic leg of male prehensile, three pairs of thoracic leg similar, furca greatly reduced, hemipenes well developed, Zenker’s organs absent, testes lying in body lateral to intestine, in some species eggs retained in body cavity during development ............................................................................ Subfamily Cytherideinae13 [Shells distinctly sculptured, sometimes with lateral protuberances, right valve with spine in posteroventral corner]............ Cyprideis 1d. Thoracic legs successively increasing in length................................................................................................................................... 2 2a(1d). Shell short and stout, hinge well developed; mandibular palp with terminal joint very small, respiratory plate well developed; furcal ramus edged at tip with two bristles ..........................................................................................................Subfamily Loxoconchinae13 [Shell short and stout, surface pitted, marginal zones thickened, duplicature narrow; eyes distinctly separated; ultimate segment of ﬁrst antenna articulated and with four robust spines; second antenna with two claw like spines inside penultimate joint; masticatory lobes of maxilla short] .................................................................................................................................................Cytheromorpha 2b. Shells plump, surface generally sculptured; hinge well deﬁned, distinct closing teeth lacking in anterior and posterior of hinge line; mandibular palp slender, respiratory plate not well developed; furcal ramus extremely small, not well deﬁned ............................................................................................................................................................................Neocytherideidinae12 [Shell club-shaped, surface rough and pitted; valves unequal with marginal zone thickened, duplicature narrow, vestibule absent, poorly developed teeth present on hinge; eyes well deﬁned; antennae well developed, ﬁrst antenna armed in front with three clawlike spines; respiratory plate of mandibular palp moderately well developed; maxilla with masticatory lobes short and stout; thoracic legs robustly developed; furcal ramus forming two oval-thickened pieces placed vertically, each provided behind with two very small bristles (Fig. 31)] .................................................................................................................................................Cytherissa 3a(1a). Penis with prostatic and spermatic elements widely separated along much of their lengths ................................................................4 3b. Penis simple; or if two elements recognizable, contiguous along their entire lengths...........................................................................6 4a(3a). Ventral portion of peniferum tapering with tip of penis reaching, or almost reaching apex .............................................Plectocythere 4b. Ventral portion of peniferum usually rounded or with one or more prominences, seldom tapering; if tapering, tip of penis never approaching apex...................................................................................................................................................................................5 5a(4b). Ventral portion of peniferum rounded, without prominences.........................................................................................Phymocythere 5b. Ventral portion of peniferum with one or more prominences ventrally and / or anteriorly...............................................Ascetocythere 6a(3b). Penis directed posteroventrally from base........................................................................................................................Lordocythere 6b. Penis directed anteroventrally from base ............................................................................................................................................7 7a(6b). Finger guard absent............................................................................................................................................................................8 7b. Finger guard present.........................................................................................................................................................................18 8a(7a). Anteroventral portion of peniferum with acute beaklike projection..............................................................................Ornithocythere 8b. Anteroventral portion of peniferum never with beaklike projection ...................................................................................................9 9a(8b). External border of horizontal ramus of clasping apparatus with one or more excrescence ...............................................................10 9b. External border of horizontal ramus of clasping apparatus entire or with few shallow subapical grooves........................................14 10a(9a). Anteroventral portion of peniferum produced ventrally in rounded lobe..........................................................................................11 10b. Anteroventral portion of peniferum never produced ventrally in rounded lobe; or if produced, apex acute or truncate ...................12 11a(10a). Spermatic loop horizontal, peniferum distal to dorsal margin of spermatic loop at least twice as long as portion dorsal to loop, clasping apparatus with external border bearing single tubercle and terminating in fanlike cluster of serrations..............Saurocythere 11b. Spermatic loop vertical, peniferum distal to dorsal margin of spermatic loop much less than twice as long as portion dorsal to loop, clasping apparatus with external border broadly serrate and terminating in annulations ................................................Okriocythere 12a(10b). Anteroventral portion of peniferum never with conspicuous anterodorsally directed projection ....................................Ankylocythere 12 13 Modiﬁed after Hoff (1942). Modiﬁed after Sars (1928). 20. Ostracoda 841 12b. Anteroventral portion of peniferum with conspicuous anterodorsally directed projection ................................................................13 13a(12b). Anteroventral portion of peniferum entire ..........................................................................................................................Geocythere 13b. Anteroventral portion of peniferum biﬁd .........................................................................................................................Hartocythere 14a(9b). Internal border of clasping apparatus with more than three teeth, apical cluster with more than two denticles, vertical ramus straight .............................................................................................................................................................................................15 14b. Internal border of clasping apparatus usually with no more than three teeth, if more than three, with only two apical denticles or vertical ramus strongly convex posteriorly .......................................................................................................................................16 15a(14a). Ventral portion of peniferum tapering to slender tip ....................................................................................................Rhadinocythere 15b. Ventral portion of peniferum never slender nor tapering ...................................................................................................Entocythere 16a(14b). Clasping apparatus not clearly divisible into vertical and horizontal rami, extremities directed at angle of at least 100 degrees ..............................................................................................................................................................................Donnaldsoncythere 16b. Clasping apparatus usually divisible into vertical and horizontal rami, extremities directed at angle of no more than 90 degrees ....17 17a(16b). Penis large, S-shaped or sinuous and with curved posteroventral thickening of peniferum giving forcipate appearance to ventral portion of peniferum ....................................................................................................................................................Thermastrocythere 17b. Penis of moderate size, L-shaped, and never so disposed as to contribute forcipate appearance to ventral portion of peniferum ......................................................................................................................................................................................Uncinocythere 18a(7b). Peniferum with accessory groove (except in Dactylocythere leptophylax where ﬁnger guard always slender and triﬁd) ...................19 18b. Peniferum without accessory groove; ﬁnger guard never slender and triﬁd .......................................................................................20 19a(18a). Posteroventral portion of peniferum terminating in barbed point...................................................................................Sagittocythere 19b. Posteroventral portion of peniferum variable, but never ending in barbed point...........................................................Dactylocythere 20a(18b). Ventral portion of peniferum bulbiform, clasping apparatus never extending so far ventrally as does peniferum.............Cymocythere 20b. Ventral portion of peniferum slender or strongly ﬂattened; clasping apparatus extending ventrally to or beyond ventral extremity of peniferum .........................................................................................................................................................................................21 FIGURE 31 Exterior view of Cytherissa lacustris (Sars) showing coarse reticulate surface and nodes. FIGURE 32 Exterior view of Limnocythere itasca Cole showing reticulate surface, sulci, and alae. 842 L. D. Delorme 21a(20b). Ventral portion of peniferum slender, terminating in small recurved projection...........................................................Harpagocythere 21b. Ventral portion of peniferum ﬂattened and with concave border ........................................................................................Litocythere 22a(1b). Shells subrectangular, reticulate, with sulci, alae or pustules on surface, margins may be denticulate, no vestibules developed; males longer, more inﬂated in posterior region than females; second antenna with three apical claws; respiratory plate of mandibular palp well developed; masticatory lobes of maxilla short; thoracic legs moderately slender; furcal ramus well deﬁned, conical in shape with one terminal and one lateral seta; hemipene with basal part very large and protuberant in front (Figs. 11, 32 .......Limnocythere 22b. Shells short and broad, surface pitted, right valve with hinge line toothed; ﬁrst antenna ﬁve or six segmented, second antenna four segmented, maxilla with three masticatory processes each longer than palp........................................................................Metacypris LITERATURE CITED Alm, G. 1915. Monographie der Schwedischen SüsswasserOstracoden nebst systematischen Besprechungen der Tribus Podocopa. 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Predators on Cladocerans VI. Population Regulation VII. Functional Role in the Ecosystem VIII. Paleolimnology and Molecular Phylogeny A. Paleolimnology B. Cladoceran Fossil Record C. Molecular Phylogeny IX. Toxicology X. Current and Future Research XI. Collecting and Rearing A. Sampling Techniques B. Culture Methods XII. Taxonomic Keys for Cladoceran Genera A. Available Keys and Specimen Preparation B. Taxonomic Key to Families of Freshwater Cladocerans C. Taxonomic Key to Genera of the Family Holopediidae D. Taxonomic Key to Genera of the Family Sididae E. Taxonomic Key to Genera of the Family Chydoridae (North of Mexico) F. Taxonomic Key to Genera of the Family Daphniidae and Moinidae G. Taxonomic Key to Genera of the Family Bosminidae H. Taxonomic Key to Genera of Superfamily Macrothricoidea I. Key to the Orders Onychopoda and Haplopoda OTHER BRANCHIOPODS XIII. Anatomy and Physiology of The Other Branchiopods A. General External and Internal Anatomic Features B. Relevant Physiological Information XIV. Ecology of the Other Branchiopods A. Life History B. Distribution and Biogeography C. Physiological Adaptations D. Behavior E. Foraging Relationships F. Population Regulation G. Functional Role in the Ecosystem H. Toxicology XV. Current and Future Research Problems XVI. Collecting and Rearing Techniques XVII. Taxonomic Keys for Non-Cladoceran Genera of Branchiopods A. Available Keys and Specimen Preparation B. Taxonomic Key to Genera of Non-Cladoceran Freshwater Branchiopoda Literature Cited †Dr. David G. Frey’s contribution to this chapter is greatly appreciated, and all of us regret his death prior to its publication. Ecology and Classiﬁcation of North American Freshwater Invertebrates. 2nd Edition Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved. 849 850 S. I. Dodson and D. G. Frey TABLE I The Families and Present Number of Extant Genera of Class Branchiopoda I. INTRODUCTION1 Branchiopods are small crustaceans that have ﬂattened leaﬂike legs. They occur in all freshwater habitats and can be abundant enough to form conspicuous swarms. Branchiopods are useful for studies of community and population ecology, animal behavior, functional morphology, evolution of life history. They occupy a key position in aquatic communities, both as important herbivores eating algae and bacteria and as major prey items of ﬁsh, birds, backswimmers, and other aquatic predators. Their fossil remains open a window into the past climate and ecology of lakes. The orders of branchiopods (Table I), including eight living and two extinct orders are related to each other only distantly (Fryer, 1987; Kerfoot and Lynch, 1987; Olesen, 1998). The Branchiopoda are a heterogeneous group of crustaceans that share a few characteristics, principally similar thoracic legs called phyllopods, which are ﬂat, edged with setae, not distinctly segmented, and usually appear to be unbranched. While this leg architecture resembles that of some of the earliest crustacean fossils, it is probable that the similarities among the branchiopods is due at least as much to convergent evolution as to common ancestry. Branchiopods also have similar mouthparts. Mandibles are simple unsegmented rods with corrugated grinding inner surfaces. The ﬁrst and second pairs of maxillae are reduced to small scalelike structures, or are absent. Branchiopods have on the last body segment a pair of spines or claws. A controversy exists concerning the evolutionary signiﬁcance of these terminal spines (e.g., Bowman, 1971; Schminke, 1976). Because branchiopods are such a diverse group, this chapter is divided into two major sections. The ﬁrst focuses on the “cladocera,” a group of four orders of small-sized branchiopods (Freyer 1987, Olesen 1998). The second section focuses on the “non-cladoceran orders,” which are less diverse and typically somewhat larger than the cladocera. CLADOCERA Most plankton ecologists now agree that the term “cladocera” has no taxonomic signiﬁcance (because the orders are probably not closely related), although 1 The references in this chapter are helpful starting places for futher reading and research. To ﬁnd our more about a topic of interest, search electronic biological data bases, such as Biological Abstracts and the Zoalogical Record, which can be accessed at many university libraries. One typically ﬁnds a rich mine of publications. If your library lacks the publication, many are available through interlibrary loan programs Order Family Number of genera in world Anomopoda Daphniidaea Moinidaeb Bosminidaec Ilyocryptidaed Macrothricidaee Neothricidaee Acantholeberidaee Ophryoxidaee Chydoridaef Sididaeg Holopediidaeg Podonidae h Polyphemidaeh Cercopagidaeh Leptodoridaeh Artemiidae Branchinectidae Branchipodidae Chirocephalidae Linderiellidae Polyartemiidae Streptocephalidae Thamnocephalidae Cyclestheriidae Cyzcidae Leptestheriidae Limnadiidae Lynceidae Triopsidae 8 2 2 1 10 2 1 2 37 7 1 7 1 2 1 1 1 6 7 2 2 1 1 4 4 3 6 3 2 Ctenopoda Onychopoda Haplopoda Anostracai Spinicaudatai Laevicaudataj Notostracak The “cladocera” include the ﬁrst four orders listed in the table. The “conchostraca” include the orders Spinicaudata and Laevicaudata. a Number of genera of Daphniidae, see Fryer (1991). b For Moinidae, see Goulden (1968) and Fryer (1991). c For Bosminidae, see DeMelo and Hebert (1994). d For Ilyocryptidae, see Smirnov (1992). e The families Macrothricidae, Neothricidae, Acantholeberidae, and Ophryoxidae are discussed in Smirmov (1992) and grouped into the superfamily Macrothricoidea by Dumont and Silva-Briano (1998). f For family Chydoridae; see Smirnov (1974, 1996). Alonso (1996) suggests a name change to Eurycercidae. Because of long usage, I prefer the name “Chydoridae” for the family. g For Sididae and Holopedidae, see Korovchinsky (1992). h For the predaceous cladocerans, see Rivier (1998). i Families and number of genera are based on an annotated update (personal communication) by D. Belk, based on Belk (1982). j For Laevicaudata see Martin and Belk (1988). k For Notostraca, see Fryer (1988). the term is still used for convenience. The cladocera are grouped into four orders (Fryer, 1987), 15 families (including the revisions suggested by Dumont and SilvaBriano, 1998), about 80 genera, and roughly 400 species (Table I). There is some controversy over whether the orders Onychopoda and Haplopoda 21. Cladocera and Other Branchiopoda (which are related predaceous species; Olesen, 1998) are even members of the Branchiopoda (Starobogatov, 1986). The number of genera in each order (Table I) is only approximate, because new genera have been established recently in the families Sididae, Daphniidae, Macrothricidae, and Chydoridae, with certainly more to come. Almost all members of the suborder Radopoda (Dumont and Silva-Briano, 1998) and some members of the Sididae (Table I) are benthic (bottom-dwelling), living on and in various surfaces, particularly aquatic plants (macrophytes), coarse plant detritus (decaying plant material), and organic sediments. They are called “meiobenthos” (Frey, 1988a) because of their small size (mostly 1 mm in total length) and because of their benthic habitat preference. Meiobenthos are often most abundant in the plants and associated sediments of the littoral (or shallow water) zone. Chydorid populations commonly have more than a million animals per square meter of bottom, and twenty or more species can coexist in the same small bit of habitat. Most species in the remaining eight families in Table I are primarily or totally planktonic (inhabiting the open water zone), where their facility for swimming makes them quite independent of surfaces. Besides differing in habitat, planktonic and meiobenthic cladocerans differ also in ecological relationships and in their evolution. Hence, to imply, as students of plankton commonly do, that everything learned about Daphnia or Bosmina applies equally well to all cladocera is often misleading or incorrect. The present chapter seeks to present an even treatment of all cladoceran families. II. ANATOMY AND PHYSIOLOGY A. General Form and Function 1. General Female Form and Function Martin (1992) provides a masterful and detailed description of branchiopod anatomy, with a wealth of drawings and photos. In general, cladocerans (water ﬂeas) are a group of small animals belonging to one of four crustacean orders (Table I). Adults are 0.2 – 18.0 mm long. The genera Daphnia (Fig. 1A) and Pleuroxus (Fig. 1B) exemplify typical cladoceran morphology. Cladocerans do not have a clearly segmented body or appendages, although they do possess segmented second antennae. Obvious features of most cladocerans include a single central compound eye as adults and a transparent clear-to-yellow carapace that is used as a brood chamber. The carapace is attached to the back of the neck and acts like an overcoat, wrapping around 851 part or all of the body, except for the head. In many species, the 4 – 6 pairs of thoracic legs are covered by carapace. Crustaceans differ from other arthropods in having two pairs of antennae. In Cladocera, the ﬁrst pair of antennae (called antennules) are usually onesegmented and small, and have only a chemosensory function. The second pair of antennae (called antennae) are large and used for swimming. Most cladocerans are small transparent animals, whose general shape and jerky swimming accounts for their common name, “water ﬂeas.” Although abundant in most standing freshwater, they are small enough to be easily overlooked. Nevertheless, it is possible to observe their behavior and identify at least the larger species using the unaided eye. For a clear view of their structures, however, it is necessary to use a dissecting microscope for gross features and a compound microscope for ﬁner details. Anatomy is best observed using living animals. At ﬁrst, it is easiest to focus on large and transparent animals, such as an adult Daphnia or Simocephalus. Once structures are located in these large animals, they can also be found in small animals, such as chydorids and bosminids. Figure 1 indicates the appearance and relative arrangement of the major cladoceran structures. For observation of a living cladoceran, one or more individuals can be put in a small volume of water in a Petri dish. If most of the water is then withdrawn, the animals will be held down by the surface tension and be unable to dart out of the microscope ﬁeld of view. For more careful observation, an animal can be anchored to a small dab of clear grease. The grease can either be put on the bottom of a Petri dish or on the tip of a small glass rod. Cladocerans typically are discus-shaped. When dead or trapped by a surface ﬁlm of water, they will lie on their side. The body in side view (Fig. 1) is rounded, the legs are obscured by a transparent shelllike covering (the carapace), and the major pigmented parts are likely to be blue-to-yellow eggs in the brood chamber, the green or yellow gut, and the dark black eye. The location of the head is marked by one or more eyes. The head is usually more or less dome-shaped but can have long, even pointed extensions (compare Fig. 2B and 2C) and an elongated beak may be present (Fig. 1). All species possess a single black compound eye except the chydorids Monospilus and Bryospilus (Fig. 14 and 15) and the blind cave-dwelling species of Alona and Spinalona (Dumont, 1995; Brancelj, 1990, 1992; Ciros-Perez and Gutierrez, 1997). The compound eye is single, the result of fusion of two eyes during embryonic development (Threlkeld, 1979). The black color of the cladoceran eye is due to accessory screening pigments, including melanins, ommochromes, FIGURE 1 (A) The anatomy of a daphniid. Daphnia pulicaria Forbes, 1893, emend. Hrbácek, 1959. Nebish Lake, Vilas Co., Wisconsin, 6 June 1987. Figure drawn by Kandis Elliot (KE). (B) The whole-body anatomy of a chydorid Pleuroxus trigonellus (O.F. Mueller, 1776). Rybinsk Reservoir, USSR. 7 December 1962. Redrawn from Smirnov (1971) (KE); (C) Mandibular articulation in the chydorid subfamily Aloninae. Oxyurella brevicaudis Michael and Frey, 1983. D.G. Frey Sample 5019, Clearwater Lake, Putnam Co., Forida. 3 March 1979; (D) Mandibular articulation in the chydorid subfamily Chydorinae. Pleuroxus aduncus (Jurine, 1820). D. G. Frey sample 3004. Langemosa, Sealand, Denmark. 23 October 1972. 21. Cladocera and Other Branchiopoda 853 FIGURE 2 (A) Male Daphnia pulicaria Forbes, 1893, emend. Hrbáček, 1959. Nebish Lake, Vilas Co., WI, 6 June 1987; (B) Daphnia retrocurva Forbes, 1882. With elongated helmet. Lake Wingra, Dane Co., WI. 5 October 1978; (C) Ephippial female Daphnia pulicaria. Nebish Lake, Vilas Co. WI, 6 June 1997. (KE) FIGURE 3 Male and female chydorid. Alona bicolor Frey, 1965. (A) Male redrawn from Frey (1965); (B) female redrawn from Frey (1965). pteridines, or purines (Shaw and Stowe, 1982). Since the black color fades in several mounting media, such as Hoyer’s, it is unlikely that much melanin is present. The actual photoreceptive pigment is a nearly colorless purple, masked by the accessory pigments. With magniﬁcation of 50 , it is possible to see nearly transparent muscles that are attached to the eye and presumably responsible for the motion. Once these muscles are seen it is easier to ﬁnd other muscles, as for example, those attached between the basal segment of the swimming (second) antennae and the back of the thorax and head. Most cladocerans also have a small black ocellus (simple eye) just posterior to the compound eye. Like other crustaceans, cladocerans show evidence of ﬁve pairs of appendages on the head part of the body: two pairs of antennae, one pair of mandibles, and two pairs of maxillae. At the base of the head, near the carapace margin, is a pair of short cigarlike appendages — the ﬁrst antennae (antennules). These are usually shorter than the head and inconspicuous but may be longer than the 854 S. I. Dodson and D. G. Frey head, as in Moina (Fig. 58) and most macrothricids (Figs. 44 – 57). First antennae are not used for swimming or feeding but are probably chemosensory organs. The head may curve toward and beyond the ﬁrst antennae, forming a beak, as in Daphnia and chydorids (Fig. 1), or the immobile ﬁrst antennae can form a long tusk, as in Bosmina (Fig. 42B). Attached to either side of the body at the posterior margin of the head and partly or entirely protruding from the carapace are the second antennae (one on each side). These large branched and segmented appendages are typically used for swimming or crawling. The mandibles are unbranched and elongated, with a pivot point near the carapace and a darkened grinding surface near the mouth. The ﬁrst pair of maxillae are typically reduced to a small ﬂap with a few curved spines. The second pair of maxillae are missing or represented by a spine. In addition to these appendages on the head, just above the mouth is an upper lip (the labrum), which closes the mouth and secretes mucus. A maxillary gland (nuchal organ, or one or more head pores) opens on the dorsal margin of the head in conchostracans and the cladocerans (Olesen, 1996). It secretes a glue in some forms (Sida, Simocephalus), a large gelatinous capsule over the carapace (Holopedium), or salt in marine or hypersaline branchiopods. The head smoothly joins the rest of the body which is often covered by a carapace. The transparent carapace is often ornamented with a faint geometric pattern in the planktonic species or by striae, spines, pits, or hexagonal meshes in many of the littoral forms. The cladoceran body posterior to the head is comprised of a thorax and an abdomen hanging within the overcoatlike carapace. The body ends in a pair of (postabdominal) claws which can reach out of the carapace. Several species have a stiff extension of the carapace: spines near the base of the second antennae or teeth, spines, or one long tail spine on the posterior end. The chemistry of the carapace is important, since cladocerans tend to have a hydrophobic exoskeleton. Although surrounded by water, many cladocerans are unwetted. If brought into contact with the surface ﬁlm, they become trapped. Their hydrophobic carapace is probably an adaptation which discourages growth of algae and protozoa on the carapace. Cladocerans cannot groom the outside of the carapace, and animals that have lived for a few weeks without molting often bear a thick load of encrusting organisms (Chiavelli et al., 1993; Threlkeld and Willey, 1993) which may diminish their feeding and swimming efﬁciency, reduce their escape speed, and increase their sinking rate (Allen et al. 1993). Some cladocerans, such as Scapholeberis (Fig. 40) and some forms of Daphnia, can be colored black over parts of the body. The pigment is melanin, deposited in the exoskeleton. The melanin is located in the parts of the exoskeleton habitually faced toward the sun: the margins of the carapace in Scapholeberis and the head and back in Daphnia. Experiments have shown that the black pigment provides protection against photodamage in Daphnia (Luecke and O’Brien, 1983), but perhaps little photoprotection in Schapholeberis (Hurtubise and Havel, 1998). It is possible that induction of melanin is associated with a decrease in reproductive potential. It has also been proposed that the dark pigment also increases heating rate and, therefore, allows cladocerans an increased reproductive rate in cold water. However, the small cladoceran body size and the high thermal conductivity of water suggest that this effect is insigniﬁcant. Finally, while melanin strengthens bird feathers and insect exoskeletons, it probably does not have this effect in cladocerans (Dodson, 1984). Melanistic animals brought into the lab lose their pigmentation at the ﬁrst molt, suggesting that stimulation of bright sunlight is needed to induce melanin. Scapholeberis contain the darkest pigmented cladoceran species. Scapholeberis kingii is the only known distasteful cladoceran; it is not even ingested by hydra (Schwartz et al., 1983). Scapholeberis homogenate causes Hydra to writhe and contract, and it induces concerts of tentacular spasms. It is not yet known if Scapholeberis is distasteful to visual predators. If so, the black carapace pigmentation may be adaptive both as protection against photodamage and as a warning display. The animal’s thorax and abdomen can be seen moving within the carapace of living individuals. The thorax holds four or six pairs of legs. These ﬂat leafshaped legs (called “phyllopods”) are well supplied with rows of setae and spines and are used for handling food, ﬁltering, scraping, pumping, creeping, or grasping females. The legs beat rhythmically back and forth at roughly ﬁve beats per second at room temperature. If a small amount of an alga suspension is placed with a pipette near the carapace margins, it is possible to follow the water current which brings the algae to the legs, and to watch algae collecting on the legs and being passed toward the mouth. The thoracic legs probably act as electrostatic ﬁlters (not sieves), collecting algae and other particles that stick to the ﬂat surfaces and combs of setae (e.g., Gerritsen and Porter, 1982; Cheer and Koehl, 1987; Gerritsen et al., 1988). Lampert (1994) showed that the ﬁlter comb size of Daphnia (7 species) increased by as much as 83% when the animals were grown at low food levels. Food caught on the thoracic legs is mixed with mucus to make a mass of food (the bolus), which is moved forward toward the mouth. The postabdominal claw is 21. Cladocera and Other Branchiopoda used to clean the ﬁlters or to reject a bolus containing distasteful algae. Cladocerans thus have the option of accepting or rejecting a mass of algae, but little control over the composition of the bolus (they have difﬁculty making decisions about whether to accept each individual algal or bacterial cell, although Strickler developed videos of Daphnia choosing individual particles). A bolus can be rejected if it contains a toxic alga or if its average “taste” is unacceptable (Richman and Dodson, 1983). If the food bolus is accepted, it is chewed by the mandibles and swallowed. The amount of food ingested depends on the algal concentration, until a concentration is reached at which the legs are operating at full capacity. Beyond this concentration, the amount of food ingested remains constant, or even declines, as the legs become clogged with food (Porter et al., 1982). The mouth is near the margin separating the carapace and the head. On either side of the mouth lies a pair of large but simple jaws (mandibles), each of which is a single rod, pointed toward the outer end. The inner dark chewing surfaces lie just in front of the mouth. The jaws are in nearly constant grinding motion. If the animals have been feeding, the gut will be easy to ﬁnd. The gut runs from the mouth, loops through the head, continues along the body through the thorax and abdomen, and terminates at the anus near the posterior end of the animal. The gut can be divided into three regions. The foregut and hindgut are lined with cuticle, which is continuous with the cuticle that covers the outside of the animal. The midgut (in the thorax) is lined with epithelium elaborated into microvilli and is the site of absorption (Peters, 1987). In the head region of Daphnia are a couple of small sacs (hepatic caeca) attached to the gut. In some species, especially in the chydorids, there is a sac attached to the gut in the abdominal region and none in the head region. A green to brown, rather amorphous material typically ﬁlls the gut (Tappa, 1965) along with undigested algal cells and small animals (Porter, 1973, 1977). Waves of rhythmic (peristaltic) contraction move along the gut from the anus toward the midgut. By feeding a trapped animal two different kinds of food (yeast followed by algae), it is possible to measure the rate of movement of food particles through the gut. Lying along the gut, in the thorax region of wellfed adults, are fat globules and a pair of gonads (Fig. 1). The fat globules are light yellow to orange, and are spread throughout the body cavity in greater or lesser abundance (Tessier and Goulden, 1982). The ovaries resemble a mass of large fat globules, but the maturing eggs are more often slightly green in color (the blue or green colors are due to carotenoids that are combined chemically with proteins). Each time an adult female molts, she extrudes a few new eggs into 855 the brood chamber, as she releases the neonates developed over the previous between-molt period. Developing sperm are large round cells, similar in appearance to the fat globules. These are released through the anus, when the postabdomen is inserted into the female’s brood chamber. When released, the spermatozoa are amoeboid, without a ﬂagellum. In most species they are simple ovals 5 – 20 m in diameter. Spermatozoa of Diaphanosoma are oval, about 60 m in diameter, and those of Sida and Moina are spiny and nearly 100 m long (Weismann, 1880; Wingstrand, 1978). The heart (Fig. 1) is obvious because of its rapid beating in live animals. It is a clear muscular organ lying in the thorax above the gut and just behind the head. With the right light and at least 50 magniﬁcation, one can see the blood cells moving about, especially near the heart and in the bases of the second antennae, and in the beak of Daphnia. The abdomen carries at its tip a pair of claws, which may appear to be single. These claws are used to groom the thoracic legs and can be seen to brush over the thoracic legs as the abdomen is swung forward. Some smaller species, especially those in the family Chydoridae, use the abdomen as a sort of foot to kick along surfaces. The part of the body at the end of the abdomen that bears claws and is posterior to the anus is called the postabdomen. The anus lacks a muscle for closure (sphincter). In live animals, rhythmic (peristaltic) waves of contraction can be seen moving up the hindgut. Most, if not all cladocerans show this “anal drinking.” The hindgut cause a current of water to ﬂow into the anus; the water is absorbed by the columnar cells of the anterior gut. This means that dissolved organic compounds may stay in the gut much longer than the food from which they were derived (Fryer, 1970). “Cyclomorphosis” (a type of phenotypic plasticity) is a change in body shape within a species caused by alternate developmental pathways induced by environmental factors (such as temperature and turbulence) and chemical communication (Dodson, 1989; Larsson and Dodson, 1993). For example, shown in Fig. 2b is a Daphnia with an elongated helmet. This same animal could have offspring with much lower helmets, depending on the chemical signals present during embryogenesis. Animals from the same clone mother, but inﬂuenced by different chemical signals, have such different forms that they have been described as different species (Krueger and Dodson, 1981). The changes in form include elongated heads, tail spines, or in Bosmina, elongated ﬁrst antennae. These elongated body parts are sometimes accompanied by a reduced adult body size. Black and Slobodkin (1987) deﬁne cyclomorphosis as “temporal (seasonal or aseasonal) cyclic morphological 856 S. I. Dodson and D. G. Frey changes that occur within a planktonic population.” Animals generally lose elongation of body parts when cultured in the laboratory for one or two molts. For example, Daphnia galeata mendotae and D. retrocurva both produce higher helmets in warm turbulent laboratory environments. They also produce higher helmets in the presence of two invertebrate predators, Chaoborus larvae and Notonecta adults. Similarly, Daphnia pulicaria produced neck teeth the ﬁrst summer that Chaoborus were present in Lake Lenore, Washington (Luecke and Litt, 1987). The induced morphological changes are generally thought to be advantageous for increasing survivorship in the face of speciﬁc predators: small size protects against ﬁsh and long spines protect against small invertebrate predators (Larsson and Dodson, 1993; Tollrian and Dodson, 1998). 2. Differences between the Sexes The two sexes of cladocerans are morphologically similar. Because most cladocerans reproduce asexually at least part of the time, most individuals will be females. Males (Figs. 2A, 3B) resemble females but are smaller. Cladoceran males have a copulatory hook on the ﬁrst thoracic leg, used for holding on to the female during mating. Macrothricidae and Chydoridae have the largest hooks, and Polyphemus and Leptodora have the smallest hooks (Lilljeborg, 1901). Some cladoceran males have longer ﬁrst antennae than are present in the females (especially Sida, Diaphanosoma, Daphnia, Ceriodaphnia, Moina, Polyphemus, and Leptodora). Daphnia and Ceriodaphnia males have elongated setae on the anterior thoracic legs. Males of Diaphanosoma, Latona, Bythothephes, Podon (Lilljeborg, 1901) and Penilia (Della Croce and Gaino, 1970) have a pair of penes on the postabdomen, as do several species of Alona and Leydigia. 3. Life History and Development Detailed information exists for the life histories of many cladocerans (Lynch, 1980; Frey; 1975, 1982c, 1987b, 1989) and especially for Daphnia (Schwartz, 1984; Lynch, 1989; Threlkeld, 1988). Cladoceran reproduce either sexually or asexually, depending on environmental conditions (Fig. 4). Most species reproduce parthenogenetically most of the time (Hebert, 1978, 1988), and most resting eggs develop into females. Species adapted to temporary habitats, such as Moina species, have resting eggs that can develop into either males or females. Embryogenesis begins, and is usually completed, in the brood chamber, a space between the body and the carapace (Fig. 1, also see Kotov and Boikova, 1998). In the Onychopoda and Haplopoda the carapace does not FIGURE 4 Daphnia reproductive strategy. Adult females can produce three different kinds of offspring, depending on environmental conditions. Diploid (subitaneous) eggs are produced asexually, and develop into females or males. Haploid eggs can also be produced, given the correct chemical signal from the environment. cover the body, but is reduced to a brood chamber on the animal’s back (e.g., Figs. 60 and 61). Cladoceran eggs (Figs. 1, 7, 9B, 13, 27, 56, and 61) are of three types: • diploid “subitaneous” eggs, which develop immediately into young; • resting “eggs,” which come from haploid eggs that are fertilized, develop into early embryos, and then enter a diapause (state of physiological stasis) which is resistant to heating, drying, and freezing; and • pseudosexual resting eggs, which are early diapausing embryos from asexually produced diploid eggs (no fertilization needed). Why do cladoceran species often reproduce both asexually and sexually? Two adaptive advantages of asexual reproduction are that population growth rate is maximized, since all offspring are females, and genetic combinations particularly well adapted to present conditions are not disrupted by sexual recombination (Banta, 1939; Williams, 1975). Two disadvantages to asexual reproduction are that asexual clones are slow or unable to adjust to changing conditions by sexual recombination (and so are restricted to speciﬁc habitat conditions) and that asexual genotypes accumulate mutations (which are typically detrimental). Sexually 21. Cladocera and Other Branchiopoda derived resting eggs represent new genetic combinations and have a chance of both being pre-adapted to new environmental conditions and lacking detrimental mutations. The different adaptive advantages of the sexual and asexual eggs would seem to dictate that only the sexual eggs should diapause, as is often the case. The exception to this is the production of “pseudosexual” resting eggs in obligate asexual clones. Pseudosexual resting eggs are produced without recombination but otherwise resemble sexual diapausing eggs and are enclosed in an ephippium. Pseudosexual eggs are probably a solution for the need to produce resting eggs in ponds (such as in the arctic or alpine zones) that are too cold to allow enough time for two generations of Daphnia (one generation of females out of the resting eggs, which produce males and females, which in turn produce new resting eggs). Pseudosexual eggs can be produced by the females that emerge from resting eggs. “Asexual, immediate reproduction” is achieved via subitaneous eggs, which have a visible yolk center and are often pigmented yellow, blue, or green. Subitaneous eggs develop into neonates inside the mother’s brood chamber. Egg developmental stages can be followed easily because the carapace is transparent (Threlkeld, 1979; Kotov and Boikova, 1998). The neonates are released from the brood chamber as the mother molts. After molting, she may extrude another set of eggs into the brood chamber, depending on her age, nutritional status, and the environmental conditions. Except for Eurycercus and two other genera, the chydorids have a constant clutch size of two; whereas in other cladocera, clutch size increases with body size, so that the smallest species produce one or two eggs per clutch while the largest species can produce hundreds of eggs per clutch (Hann, 1985). Small individuals carry only a few eggs (which are huge relative to the adult, as in Fig. 1D). Reproduction is very expensive for small cladocerans. The small Daphnia lumholtzi typically carry two eggs in the ﬁrst reproductive instar. These eggs account for 30 – 80% of the female’s weight (King and Greenwood, 1992). Large Daphnia individuals (such as Daphnia magna) also allocate a signiﬁcant proportion of their energy intake to reproduction; but because they are larger and produce relatively smaller eggs (not much larger than those of the small species), they can carry dozens or even hundreds of subitaneous eggs. These eggs begin developing as soon as they are extruded into the brood chamber. Egg development time and longevity are proportional to the inverse of temperature to an exponent of about 2.5 (Rigler and Downing, 1984). “Delayed reproduction” is often associated with sexual reproduction (Hebert, 1978, 1988). Males are 857 diploid and produced asexually, just as their sisters are. Production of males is induced by some environmental signal, such as a change in food concentration, a chemical signal produced by crowded Daphnia, or changing (usually decreasing) photoperiod (Ferrari and Hebert, 1982; Hobaek and Larsson, 1990; Kleiven et al., 1992, see Fig. 4). When a population begins reproducing sexually, females produce mixed clutches of males and females. The male to female ratio varies greatly between populations and in different years. Highest sex ratios are typically seen in small ponds, and there is some concern that environmental contaminants may have reduced male production (Dodson and Hanazato, 1995). Sexual reproduction begins when one or two haploid eggs are extruded into a modiﬁed (thickened) carapace (except in the Sididae and the predaceous species) called the ephippium (in Greek “saddle”, see Fig. 2C). The changes in carapace development are induced by either environmental (chemical) signals from crowding or toxic food (Ferrari and Hebert, 1982; Carvalho and Hughes, 1983; Shei et al., 1988; Kleiven et al., 1992) or by environmental contamination (Van Der Hoeven, 1990; Shurin and Dodson, 1997). Ephippia are produced most often in temporary ponds, especially in harsh environments (Lynch, 1983; Weider and Hebert, 1987). Upon fertilization of the haploid egg by the male (Jurine, 1820; Baird, 1850), the resulting zygote (or the diploid pseudosexual egg) undergoes several cell divisions (Banta, 1939; Ojima, 1958) and then enters diapause. These resting “eggs” (actually early embryos) are resistant to freezing and drying and are further protected by the thickened part of the carapace. The ephippium is darkly pigmented with melanin. The rough surface of the carapace may serve as a speciesspeciﬁc recognition cue for the male during mating. The ephippium is shed when the female molts. The ephippium closes around the resting eggs, and most of the rest of the exoskeleton detaches from the ephippium (except for the tail spine in Daphnia). In some populations, resting eggs are not the result of sexual reproduction but are produced asexually (Hebert, 1987; Weider and Hebert, 1987; Innes and Hebert, 1988). These “pseudosexual” resting eggs are not produced by sexual reproduction, but are the result of a gene that suppresses meiosis. They act just like the sexually derived resting eggs, but are genetically identical to their mother. Chydorid and macrothricid ephippia sink and are usually attached to the substrate (Bretschko, 1969; Fryer, 1972). Daphnia ephippia tend to ﬂoat, because of their speciﬁc gravity and the hydrophobic character of the carapace. Dark pigmentation of ephippia may aid dispersal, since ﬁsh, amphibian, and bird predators 858 S. I. Dodson and D. G. Frey can easily see the dark ephippium and the ephippium is not damaged by passage through a predator’s gut (Mellors, 1975). The tail spine and, sometimes, a strip of chitin ventrally at the head end of the carapace (Fryer, 1972) may aid dispersal by allowing the ephippium to stick to fur or feathers of wading animals. Ephippia are small enough to blow about with the wind and resistant enough to remain alive on dry land for years (Moghraby, 1977). “Egg banks” are composed of resting eggs stored in the sediments or watershed. The resting eggs may be years or decades old, and they appear to hatch leisurely over several years. Egg banks provide an important large-scale stabilizing inﬂuence on the genetic structure of a cladoceran population and a hedge against extirpation (Hairston, 1996). To continue development, the diapausing embryos in ephippia require stimuli such as water, long or increasing day lengths, and high oxygen concentrations (Schwartz and Hebert, 1987a). The resting eggs usually develop into females which produce more young asexually. Major exceptions are Daphniopsis and Moina, genera which specialize on short-lived temporary ponds and which produce both males and females from the ephippial eggs (Goulden, 1968; Schwartz and Hebert, 1987b). Molting allows cladocerans to increase in size. As the embryos develop, they assume the appearance of the adult. After release from the brood chamber, the newborn (neonates) can grow only by molting. That is, when sufﬁcient energy has been stored, the animals: (a) reabsorb the inner part of their exoskeleton; (b) begin to manufacture a new exoskeleton below the old; (c) absorb water; and (d) pull themselves out of the remaining outer layers of the old exoskeleton, exiting at the back of the neck. The old exoskeleton (the exuvia) is a transparent ghost image that retains the complexities of the outer surface of the animal, including all the ﬁne setae of the thoracic legs. Exuviae have been used to measure changes in size of various structures as an animal grows from neonate to adult. The energetic cost of molting may be a major constraint on body size in Daphnia (Lynch, 1989). Given a limited rate of food intake, cladocerans may be faced with the choice of allocating energy to molting (to become larger) or allocating energy to reproduction. The developmental stage between molts is called an instar. There is some variation in (and controversy about) how many times cladocerans molt. Neonates molt just twice in chydorids but up to seven times in daphnids before producing eggs of their own. The number of juvenile instars is more or less species-speciﬁc and depends on adult body size, with the smaller species having the smallest number of molts. The rate of molting (instar duration) in Daphnia pulex depends mostly on the temperature, less on food (Lynch, 1989). If animals are poorly fed, they may even lose weight when they molt. An instar lasts from about a day to weeks, depending on the temperature. In laboratory studies, cladocerans can live for months at room temperature. Each time a well-fed juvenile molts, it approximately doubles its volume, and its length increases roughly by a factor of 1.26. Growth slows as cladocerans mature; successive adult instars grow slightly, but energy not used for maintenance is allocated to produce eggs instead of growth (Frey and Hann, 1985). Female cladocerans can molt several times after becoming adults. Chydorid males become mature in the third instar and stop molting, although Daphnia males continue to molt and grow after maturity. 4. Induced Changes in Life History Many cladoceran life-history characteristics include instar duration, number of instars to ﬁrst reproduction, time to ﬁrst reproduction, primiparous and neonate size, and clutch size. These characters have a genetic basis (Spitze, 1995) and show trade offs and correlations that are suggestive of adaptations to the environment and to biological interactions, especially predation. Life-history characteristics also show phenotypic plasticity (Hann, 1984; Schwartz, 1984; Larsson and Dodson, 1993). That is, the same genotype can develop differently, as it is inﬂuenced by temperature, food supply, and chemical signals produced by predators. The patterns of phenotypic response (reaction norms) appear to be, in general, adaptive responses to size-selective predation and food limitation. B. Metabolism The many physiological studies of Daphnia provide a reasonably complete picture of cladoceran metabolic rates (e.g., Peters, 1987). Metabolic rate is measured as the rate at which an animal uses oxygen or produces carbon dioxide. As long as oxygen is present, gas exchange (oxygen in and carbon dioxide out) is not a problem for small animals, because of their high surface-to-volume ratio. Gas exchange probably takes place across the entire surface of the animal, not just on the gill like thoracic legs. Porter et al. (1982) and Peters (1987) found no correlation between rate of leg movements and respiration rate. Animals collected in oxygen-poor habitats (sewage lagoons or temporary pools with decaying vegetation) will often have a general pink-to-red color (Weider and Lampert, 1985). This is due to hemoglobin dissolved in the blood. If a red animal is wounded, it will bleed red blood; and if the blood is examined at high magniﬁcation, the blood cells 21. Cladocera and Other Branchiopoda 859 will appear less pigmented than the surrounding ﬂuid. Animals become clear in a day or so if grown in welloxygenated water, suggesting an energetic cost to maintenance of hemoglobin (Landon and Stasiak, 1983). Metabolic rate is inﬂuenced by a number of factors, including temperature, body size, and food concentration. can be ingested. In the absence of food, Daphnia continue to move their legs, as if the thin gill-like legs were also being used as respiratory organs, requiring ventilation. 1. Temperature Environmental temperature may be a causal factor for body size. For example, Stirling and McQueen (1986) found that the direction of change in environmental temperature was correlated with body size and reproductive strategy in Daphniopsis ephereralis, an inhabitant of cool forest ponds. Annual changes in average body size for the population was not directly related to predators and may have a physiological explanation. Starvation is a problem faced by most cladoceran populations sometime during each year (Threlkeld, 1976, 1988). Because smaller animals have a higher metabolic rate per unit body weight, neonates are the more prone to starvation than adults, if no food is present (Tessier and Goulden, 1987). Eggs can receive a large store of energy-rich fat from the mother, if the mother is well fed. However, this energy store is reduced if the adult is faced with limited food. Similarly, if food is absent, the large species can persist for longer than small species. Even for large adults, a single day without food results in a drop in energy stores, body weight, and respiration rate. At food concentrations above starvation levels, the relationship between food availability, energy allocation, and body size are complex. How do cladocerans allocate the energy gained from limited food input? Models of energy allocation based on physiological principles suggest that energy is used for different purposes at different life stages (for example, only for maintenance and growth in immature animals), and that adults have allocation priorities for maintenance, reproduction, and growth (Bradley et al., 1991). Large animals always have lower weight-speciﬁc respiration rates. However, smaller animals have the higher weight-speciﬁc assimilation at low food concentrations, and large animals have higher rates at high food concentrations (Tessier and Goulden, 1987). This suggests that net growth, estimated by the difference between assimilation rate and respiration rate, is highest for small animals at low food concentrations and highest for large animals at high food concentrations. In a study of three Daphnia species (Tillmann and Lampert, 1984), the large D. magna out-reproduced smaller Daphnia. When food was marginally limiting, the three species had similar reproductive rates; and when food was severely limiting, only the smallest species, D. longispina could still reproduce. Enserink et al. (1996) also found that larger individuals appeared to grow Metabolic rate has a hump-shaped (inverted-U) relationship with temperature. At some low threshold temperature, metabolic rate is just fast enough to maintain growth and reproduction. A rise in temperature increases metabolic rate up to a maximum. Above the temperature of maximum metabolic rate, further increases cause death, either because critical metabolic enzymes are thermally damaged or because respiration rate exceeds the rate of energy intake (Moore et al., 1996). Most cladocerans are unable to live at temperatures above 30°C, and animals native to cold waters may have much lower upper thermal limits. This sensitivity to high temperature can be increased if the cladocerans are feeding on toxic bluegreen algae (Threlkeld, 1986a). Both Ceriodaphnia and Diaphanosoma live naturally in water of 27 – 30°C. When bluegreen algae are present, Ceriodaphnia shows a decline in population growth rate. For short periods of time (15 min), Daphnia can tolerate temperatures up to about 35 – 38°C, depending on the temperature at which they were raised (MacIsaac et al., 1985). 2. Body Size Small animals, cladocerans have relatively fast rates of metabolism. Richman (1958) found that respiration rate of Daphnia is proportional to body length raised to the 2.14th power, a value similar to size-speciﬁc respiration rates reported for many invertebrates (Schmidt-Nielsen, 1984). The range of cladoceran body size is enough to ensure signiﬁcant differences in metabolic rates between the smallest and largest cladocerans. While larger animals require more oxygen and energy in an absolute sense, large animals use less energy and oxygen per unit of weight. 3. Food Concentration As food becomes more concentrated, the animals respire at an increasing rate, probably because of increased metabolic needs: assimilation rate also increases with food concentration (Peters, 1984; Lampert, 1986a). When food is superabundant, the grooming required to clean the ﬁlters on the thoracic legs may also increase the respiration rate (Porter et al., 1982). This phenomenon can lead to starvation at concentrations of food high enough so that the increased respiration requirements exceed the maximum rate at which energy 4. Interaction of Temperature, Body Size, and Food Concentration 860 S. I. Dodson and D. G. Frey fastest at constant low food levels, whereas smaller individuals had the advantage with ﬂuctuating food. When a cladoceran is food limited, it tends to mature at a smaller size and produce smaller (but as many) offspring. The main response of Daphnia pulex to low food levels was a reduction in size-speciﬁc food intake and egg size (Lynch, 1989). Over a wide range, food concentration had no effect on length-weight relationship, instar duration, or weight-speciﬁc investment of energy in reproduction. Population growth rate is linked to body growth rate. Competition among various species of cladocerans may largely depend on which species can grow fastest at a given food level. Thus, small species such as Ceriodaphnia and Bosmina may be able to out-compete, or at least out-grow, large species such as Daphnia pulex at low but not at high food concentrations (Tessier and Goulden, 1987). III. DISTRIBUTION A. Zoogeography Cladocerans are a widespread group occurring in all but the most extreme freshwater habitats (Hutchinson, 1967). While they are more abundant in lakes, ponds, and slow-moving streams and rivers (Thorp et al., 1994), they also occur in quiet water and marginal vegetation in fast-moving streams. Several species probably occur in groundwater, especially river gravels (Dumont, 1987). One chydorid cladoceran has been found in water trapped in mosses and bromeliads that drape trees in the cloud forest of El Yunque, Puerto Rico (Frey, 1980b). One species or another are found from sea level to ponds above tree line, and from arctic to tropical latitudes. In North America, the highest diversity of planktonic species is found in the glaciated midtemperate zone (Tappa, 1965), and the highest diversity of benthic species, especially small-bodied species, is found in the southeastern United States (Crisman, 1980; Kerfoot and Lynch, 1987). Chydoridae, now seems to be mainly an illusory concept (Frey, 1987a), based on an incomplete appreciation of the morphology of seemingly the same taxa in different places and on an unquestioning acceptance of the efﬁciency of passive dispersal of these organisms via their resting eggs as carried by wind, water, birds, insects, and mammals (Frey, 1982a). Recent studies in North America (Frey, 1986a) are demonstrating that the geographical ranges of species of chydorids can be just as narrow and circumscribed as those of animals considered to be much less vagile. Careful genetic analysis (Hebert, 1987) has shown Daphnia species to be more diverse and to have distributions restricted to small portions of North America. In fact, those Daphnia species that are obligate parthenogens are genetically very diverse (Hebert, 1987), nearly as diverse as asexual ostracods (Havel and Hebert, 1989; Chaplin, 1994). Thus, the cladocera of different continents are probably different species (even though many now have the same speciﬁc names), and the distributions of species within a continent depend on the distribution of favorable environmental conditions. Hybrids between cladoceran species are often reported (Hebert, 1985; Hann and Hebert, 1986; Hebert, 1987; Mort and Streit, 1992; Taylor and Hebert, 1993; Spaak, 1995; Colbourne et al., 1997). The evidence has until recently been only morphological, or based on lab crosses. For example, Pleuroxus denticulatus and P. procurvus were crossed in the laboratory to produce one individual which could only reproduce asexually and was morphologically intermediate between its two parents (Shan and Frey, 1983). Recent studies based on molecular characters conﬁrm that hybrids can indeed be formed between similar species. The persistence of clones in nature is due to their ability to reproduce asexually. Species remain distinct, in part, because the hybrids are not known to be able to reproduce sexually in nature. Thus, while cladoceran species are often difﬁcult to distinguish using morphological characters, molecular characters show them to be clearly distinct. C. Biodiversity B. Cladocera and the Species Concept Our understanding of the distribution of cladoceran species depends on how well we can distinguish species. Cladoceran taxonomy is currently undergoing a reanalysis. Because of an almost complete disregard of ﬁne but signiﬁcant details of morphology and a consequent impression that taxa on different continents were the same, many investigators in the past (Forbes, 1925) and present have claimed that many species are cosmopolitan. But cosmopolitanism, at least among the One measure of biodiversity is the number of species, also called “species richness.” Cladoceran species richness in a lake depends on several factors, including water chemistry, lake size, productivity, the number of adjacent lakes, and biological interactions (Dodson et al., 1999). 1. Water Chemistry Carter et al. (1980) found that of 23 pelagic cladoceran species occurring in eastern Canada, eight may 21. Cladocera and Other Branchiopoda have distributions restricted by water chemistry: Ceriodaphnia and a species of Eubosmina tended to occur in hard water, and Polyphemus, Sida, two Daphnia species, and two other Eubosmina species were found mostly in soft water. Salinity is a major factor that limits cladoceran distribution. Cladocerans as a group have a wide range of tolerance to salinity (Potts and Durning, 1980), but individual species are adapted to a relatively narrow range of salinity (Teschner, 1995). Particular species can be found from near-distilled water (Dodson, 1982) to the 3.2% salinity of oceans (Della Croce and Angelino, 1987), to water more saline than sea water. For example, Moina lived in Soap Lake Washington at salinities as high as 39 g / L (Edmondson, 1963). High salinity excludes most cladocerans while providing a refuge for physiologically specialized genera such as some Moina. The cladocerans of Pyramid Lake, Nevada, showed strong mortalities at salinities of 0.6% (Diaphanosoma), 0.7% (Ceriodaphnia), and 1.8% (Moina) (Galat and Robinson, 1983). The marine cladocerans Evadne and Penilia are restricted to coastal and open waters, while Podon is found in less saline coastal and estuarine water (Della Croce and Angelino, 1987). Even within a species, there is evidence of adaptation to average salt conditions. For example, different clones of Daphnia magna, from fresh or brackish ponds, show differences in their level of salt tolerance and do poorly when raised at different salinity levels than their native pond (Weider and Hebert, 1987; Teschner, 1995). Acidity also affects cladoceran distribution. Cladocerans are mostly found in neutral or alkaline water, although a few live in acidiﬁed lakes. Only a few small cladocerans such as Bosmina, Chydorus, Diaphanosoma, small Daphnia, Holopedium, and Polyphemus can tolerate extremely acidic water, occurring in lakes with pH values between 5.0 and 3.8 (Sprules, 1975; Frey, 1982c). Turbidity affects cladoceran distribution, probably because clay particles interfere with cladoceran ﬁlters (Gerritsen and Porter, 1982). A combination of laboratory life table experiments and ﬁeld observations suggested that Moina and Diaphanosoma are adapted to silt-laden water, while Ceriodaphnia and Daphnia (subgenus Daphnia) are not (Threlkeld, 1986b). Some species of Daphnia (Ctenodaphnia) (Dodson, 1985) and several chydorids (such as Graptoleberis) are found in very turbid water or in mud. 2. Lake Size, Productivity, and Adjacent Lakes For pelagic cladoceran species, there are more species in larger lakes, with about 35 species in the largest freshwater lakes. Small ponds and pools (as small as a few liters) may have one or two species. As 861 lake size increases, there are probably more distinct habitats for different species. The highest species richness occurs in mesotrophic lakes with average annual primary productivities of about 100 g C / m2 per year. The mechanism by which productivity affects cladoceran species richness is still unclear. Lakes surrounded by lakes have a higher species richness than isolated lakes (such as Crater Lake in Oregon). Clustered lakes probably act as sources for each other for immigrants (usually via resting eggs) among lakes. Whether a species occurs in a body of water depends partly on the: (a) zoogeographic region, that is on the probability of getting into the water; (b) chemical and physical requirements of the species; (c) food conditions within the water; and (d) predators living in the water. Although it is believed that some cladocerans are capable of long distance dispersal via resting eggs carried by the wind and by feathers and guts of water fowl, we still know little about actual rates of colonization. Inbreeding coefﬁcients for small Daphniopsis populations suggest that populations receive an average of 0.3 migrants per generation (Schwartz and Hebert, 1987b). Studies of Daphnia indicate slightly higher rates of migration of about 0.5 migrants per generation. However, studies of several arctic cladocerans suggested that dispersal rates are quite low, and that pond populations, even when near each other, retain genetic differences (Boileau et al., 1992). 3. Biological Interactions Cladocerans compete for food with other cladocerans and with other herbivores (Threlkeld, 1988). In some cases, this competition is intense enough to result in marked reduction in abundance of other cladocerans and potentially to result in competitive exclusion. Vanni (1986) found that Daphnia could reduce algae concentrations to a level that caused Bosmina to decrease. He suggested competition for food is a cause of the often observed scarcity of small species in lakes inhabited by large cladocerans such as Daphnia. This effect may be especially strong in nutrient-rich lakes, where Daphnia populations can increase rapidly enough to produce a sudden and major reduction in algal abundance. Size selective predation sometimes affects the distribution of cladocerans (Zaret, 1980; Kerfoot and Sih, 1987). In general, large-bodied species tend not to coexist with ﬁsh and small-bodied species can be excluded by small predators such as the larvae of the midge Chaoborus or the backswimmer Notonecta. Thorp (1986) analyzed 18 reports of predator – prey community experiments in freshwater zooplankton. He found that adding predators often reduced the number of individuals of cladocerans present, but that there are 862 S. I. Dodson and D. G. Frey few studies showing an effect on the number or relative abundance of prey species. Over the long term, predators may inﬂuence cladoceran diversity via coevolution, size-selective predation, and restrictions on prey distributions. While food limitation and selective predation are components determining the distribution of zooplankton, it is clear that the two factors are intertwined, that predators inﬂuence competition and vice versa via what are coming to be called “indirect effects” (Cooper and Smith, 1982; Lampert, 1987; Kerfoot and Sih, 1987; and Carpenter, 1988). Fryer (1985) concluded that diversity of chydorids within a lake depends only slightly on dispersal, competition or predation, and more on microhabitat preferences and presence of the appropriate speciﬁc food. 4. Littoral and Meiobenthic Species We know much more about pelagic species richness and distribution than we know about the littoral (or meiobenthic) species, such as the chydorid, macrothricid, and sidid cladocerans. The meiobenthic cladocerans comprise a major part of the diverse littoral community of lakes, which includes such additional metazoans as cyclopoid and harpacticoid copepods, occasionally calanoids as well, ostracods, amphipods, rhizopods, hydra, bryozoans, rotifers, mites, nematodes, oligochaetes, and larval or nymphal stages of midges, corixids, notonectids, beetles, caddisﬂies, mayﬂies, damselﬂies, as well as some other groups. Besides this high diversity of animals, there are the various macrophytes, sessile and substrate-associated algae, and bacteria. Of all the groups of animals, the cladocerans typically are most abundant, at times reaching densities greater than 1 million per square meter of bottom, even under the ice in early winter. The composition of this littoral community and its importance relative to the offshore planktonic and benthic associations varies with the geographic location of the lake and its general characteristics of size, depth, and transparency. There are typically about 8 – 15 species of meiobenthic cladocerans present in a given habitat, sometimes ﬁve or fewer and occasionally 25 – 30 or even more. As with pelagic cladoceran species, the species richness of littoral cladoceran species appears to be correlated with lake size. For example, in a study of 207 water bodies in Yorkshire, England, from small ponds (5000 m2) to small lakes (15,000 m2), Fryer (1985) found more littoral cladoceran species in the larger lakes. Small ponds had a median of about eight species; the larger lakes had about 30 species. Some littoral species are associated only with the sediments and others with macrophytes. Some species occur only in acid, softwater habitats, others in quite strongly alkaline ones. Fryer (1968) showed that the species he studied are diversiﬁed as to food eaten and how this is obtained. One species of chydorid is a scavenger, another scrapes food from the bottom as a snail does, and the third an ectoparasite on hydra. Most of the remaining species use the same common food supply on substrates — organic detritus with its contained algae and bacteria. The structure of the ﬁve pairs of trunk limbs is remarkably uniform among all these species, suggesting little variation in method of getting the food. Most specializations within chydorids seem linked to obtaining food from slightly different microhabitats. Meiobenthic cladocerans occur virtually wherever water is present, from water bodies of “normal” size down to marshes, swamps, pools, puddles, ditches, seasonal water bodies, groundwater, running water of all sizes and nearly all velocities, moist sphagnum, and even in overgrowths and cushions of leafy hepatics in rain and cloud forests, up to several meters above the forest ﬂoor. Most species are sensitive to salinity, although a few occur at concentrations up to 5 – 10% (g / L), sometimes even higher. 5. Conservation Issues Unlike the large branchiopods (see below), conservation issues do not exist for cladocerans. Rare species probably exist, as do opportunities to conserve cladoceran biodiversity. However, rare cladoceran species are probably morphologically similar (or identical) to common closely related (sibling) species. Only a combination of morphological and molecular analysis is likely to identify rare cladoceran species (for an example of a fruitful protocol, see DeMelo and Hebert, 1994, and Hebert and Finston, 1996). As in other areas of conservation, the invasion of exotic species is a concern for North American communities. For example, Daphnia lumholtzi was probably introduced into North America from Africa, along with Nile perch. It is now a major component of lake and reservoir plankton communities in southern North America (Havel, 1995) and occurs in some rivers, including the Ohio (Jack and Thorp, 1995). Besides being introduced along with ﬁsh, cladocerans invasions of new continents are either mysterious (Benzie and Hodges, 1996) or have been connected to heavy (military) equipment (Schrimpf and Steinberg, 1982). IV. BEHAVIOR Cladocerans display a number of behaviors relating to mating, escape from predators, feeding, and movements within lakes. 21. Cladocera and Other Branchiopoda A. Mating Behavior Jurine (1820) provides us with one of the few descriptions of mating behavior in cladocera. In November 1797, in the region of Geneva, he found a pond containing a dense population of Daphnia, which included ardent males. He observed numerous couplings in the laboratory, and reported (translated from the original French): “The male jumps onto the back of the female, which sometimes escapes, but if he is able to seize her with the long ﬁlaments of his ﬁrst legs and secure her with his elongated ﬁrst antennae, he is able to catch hold of her ﬁrmly. He soon scuttles quickly over the surface of her carapace until he has attained the lower margin. When he ﬁnds the position so that the openings of the two carapaces are opposed, he quickly introduces into her carapace the long setae of the ﬁrst antennae and of the ﬁrst thoracic legs, and thereby envelopes and binds, so to speak, the legs of the female. When he is established in this position, he curves his postabdomen forward, putting it out far enough to get that of the female. When she feels that part, she is greatly agitated, and ﬂeeing, carries the male so fast it is hard to follow the amorous couple in the vase that contains them. Finally, this agitation ceases and the female in turn advances her postabdomen in order to touch that of the male. The postabdomens hardly touch before they separate. At the instant of this touch, the male is agitated by spasms which give his legs remarkable vibrations. It is during this contact that, in my opinion, the copulation occurs. The embrace is of variable duration; it rarely is sustained for more than eight or ten minutes. During this interval, the postabdomens were brought together more than once. Copulation terminated, the postabdomen of the male is slowly withdrawn into his carapace, but he is not yet able to withdraw the setae of his ﬁrst antennae and ﬁrst thoracic legs because of the spasms that persist in these parts. When these cease, the separation of the two animals takes place. This is not the only mode of embrace in these animals. I have several times seen the male introduce only one ﬁrst antenna, one ﬁrst leg seta, and one swimming (second) antenna with which he envelopes the setae of the females second and third legs. When the copulation is ﬁnished, he is not able to retrieve his swimming antenna before the spasms which he is experiencing are entirely gone. I noticed other animals that were embraced almost transversely, in such a way as to form a sort of cross. Sometimes other males arrived intending to unite with a female already in an embrace. One then sees them explore the female’s body with a remarkable vivacity, trying, although futilely, to penetrate into the sanctuary of pleasure, by elongating their postabdomen as much as they can into the opening of the carapace. In all cases where copulation had taken place, the postabdomen of the male returned slowly and with shudders to its usual position, frequently interrupted by convulsive movements which stretched it out to re-approach the female, such that it took a minute or two for the postabdomen to be completely at rest again. Males attacked indiscriminately all females they encountered. Despite that, I assume that true copulation took place only with those females that had no eggs in the brood chamber, for with these the act was reiterated and prolonged. For the others, 863 although the preliminaries were the same, they terminated very quickly. Ephippial females pushed aside the males and rendered their attacks in vain.” About 60 years later, Weismann (1880) gave cursory descriptions of mating behavior for Moina, Daphnia, and Bythotrephes, and conjectures on possible mating behavior, based on anatomy, for Daphnella, Latona, Sida, Holopedium, Ceriodaphnia, Scapholeberis, Simocephalus, Bosmina, Pasithea, Macrothrix, Eurycercus, Pericantha (now Pleuroxus) truncata, Chydorus, Podon, Evadne, and Leptodora. These mid-Victorian descriptions lack the enthusiastic attention to behavioral details shown by the pre-Victorian Jurine (1820), but do include a wealth of detail concerning cellular and tissue structure and ontogeny “Mating in Daphnia has been observed by many, although only the external association of the genders: the attachment of the male and the combined swimming about. Indeed, Jurine and after him many others noted that for Daphnia pulex, the ardent (begattungslustigen) males attacked nearly every female, whether they are carrying ephippia or embryos. This is indeed correct as far as it goes, but they always quickly let them go again and ﬁnd a true mate — as far as I can judge — only succeeding with a female that carries a winter (haploid) egg in the oviduct. Sometimes the males are not so ardent, and wait for the correct moment. Thus, on the second of September 1875, I brought several Daphnia pulex males together with a female which carried ripe embryos in the brood chamber and a newly ripe haploid egg in each ovary. The males made no advances during the entire morning. First about 4:30 pm, after the female had delivered her young, a male attached himself on the back of the carapace, and quickly within a few seconds, hitched himself around in front. The female did not defend herself, but swam peacefully and slowly about, also sometimes lying still on the bottom. The male did not let go for more than a quarter of an hour. Sadly, I had to interrupt the observations, and when I was able to take them up an hour later, the mating was already consummated, the male had departed, and in the brood chamber, which had not yet developed an ephippium, lay two fertilized winter eggs . . . .” Weismann (1880) further argued that successful fertilization can occur in Daphnia only with the partners face to face and parallel. The veil of modesty is further drawn over mating behavior by monographers of functional morphology, species, and evolution who largely ignored the topic of mating behavior (e.g., Banta, 1939; Brooks, 1957; Fryer, 1968; Goulden, 1968; Fryer, 1974). However, perhaps inﬂuenced by the renaissance of natural history in the last couple of decades and the general freeing-up of sexual attitudes of the late sixties, Shan (1969) and Smirnov (1971) have produced much more informative descriptions of chydorid mating behavior and functional morphology. The theme of female participation, and even perhaps female choice, introduced by Jurine (1820), is developed further by Shan (1969): “My observations on laboratory stocks of P. denticulatus do not agree with Weismann’s (1880) description. At the 864 S. I. Dodson and D. G. Frey beginning, the male swims vigorously here and there, using his ﬁrst antennae, trying to ﬁnd a female that is receptive, usually an ephippial female. A receptive female often cooperates in the copulation process. Sometimes, the ephippial female he approaches may not be receptive and she may reject him using her postabdomen as he approaches her. At times a male may hook onto a parthenogenetic female and try to copulate with her, or a male may hook onto another male. In one occasion, I saw a parthenogenetic female that had three males attached in a series, one after another! As the male hooks onto the edge of the female’s carapace, he moves vigorously back and forth along the edge, often only on one side of the female. The contact points are the two copulatory hooks [on the ﬁrst thoracic leg] and the tip of his rostrum. He jerks his postabdomen and moves his hooks vigorously while the female does not react to him in any way. This activity goes on for a while, usually a few minutes, possibly even longer than ten minutes. The female may become excited when the male reaches a position where his hooks are at the top of the free posterior edge of the female’s carapace. In this position the male and the female are facing the same direction and have the same dorsal orientation. At this time the female may stop feeding, carry the male with her, and swim away. They swim together, both jerk their postabdomens and strike their antennae. They swim for some time, possibly ten minutes or longer. Finally, they fall onto the bottom, still hooked together, and lie on one side. Both of them stretch and jerk their postabdomens back and forth. At this time the female’s postabdomen is in maximum extension downward and backward, while the male is ﬂexing his forward trying to get as close as possible to the brood pouch of the female. Presumably at this time the sperm are ejected. The mechanism that allows the sperm to get into the brood pouch and fertilize the egg is unclear. It is quite possible that the jerks of the female’s postabdomen create an inward current that helps bring the sperm into the brood pouch.” It is possible that the elongated ﬁrst antennae of male daphniids and moinids are used to feel the roughened carapace of a female (Goulden, 1968; Kokkinn and Williams, 1987). The female’s carapace ornamentation might tell the male whether she is the correct species and that she is producing haploid eggs. As with all aspects of behavior in Daphnia, we know few details of the individual behaviors that result in mating. Instead, cladoceran behavior has been studied mostly at the population level, using plankton nets in lakes. B. Individual Swimming Behavior Video and computer analysis techniques allow the analysis of three-dimensional swimming of individual pelagic cladocerans, especially Daphnia to measure swimming speeds, directions of movement (Dodson and Ramcharan, 1991). The characteristic hopping motion has not yet been satisfactorily characterized. Average swimming speed varies from just enough to maintain position (all pelagic cladocerans sink if quiescent), to sustained average speeds as high as about 25 mm / s (Dodson et al., 1997b). Sinking rate is affected very little by temperature, less so than predicted by changes in water viscosity, suggesting Daphnia have behaviors to maintain constant sinking rate (Gorski and Dodson, 1996). Different clones swim differently, with the fastest swimming characteristic of clones from low-predation habitats. This suggests there is a beneﬁt to fast swimming (perhaps increases ﬁltering rate) that is sacriﬁced in communities with high predation rates (where encounter rates can be minimized by slow swimming). Fish predators pay attention to Daphnia swimming behavior, preferentially taking faster swimming individuals of real prey (O’Keefe et al., 1998) or virtual (computer-simulated) prey (Brewer and Coughlin, 1996). Predaceous cladocerans swim slowly through the water. The slow movement is probably a compromise between swimming fast enough to run into prey and slow enough to avoid attracting the attention of ﬁsh. Browman et al. (1989) describe the swimming movement of Leptodora (Fig. 60): “Leptodora swims randomly [at about 13 mm per sec] through the water column with all ﬁve pairs of thoracic appendages spread to form a ‘feeding basket’ and, seemingly by chance, encounters prey. Shortly after prey make contact with any part of Leptodora’s body, (usually ventral), the abdomen is rapidly pulled forward, clamping itself under the feeding basket so that the telson closes it at the posterior end. The duration of this movement is always the same and we conclude that it is an indiscriminate reﬂex. If the prey is encountered anywhere but a short distance directly in front and slightly below the Leptodora, it is not captured.” C. Diel Vertical Migration One of the earliest observations of cladoceran behavior at the population level is Cuvier’s, 1817, description of Daphnia vertical migration (translated by Bayly, 1986): “In the morning and evening, and even during the day when the sky is overcast, the daphnids usually stay at the surface. But during very hot weather, and when the sun beats ﬁercely down on the pools of stagnant water where they live, they sink down in the water, and stay at a depth of six or eight feet or more; often not a single one can be seen at the surface.” Cladoceran populations have been observed to migrate, on a daily cycle in both fresh and marine waters (Hutchinson, 1967; Bayly, 1986; Dodson, 1990). Distances migrated by pelagic species vary from less than a meter in a small rock pool to hundreds of meters in oceans. In freshwaters, the usual distance is 5 – 20 m. Because lake waters are vertically stratiﬁed, vertical migration takes the animals into very different habitats in terms of temperature, light, food, turbulence, oxygen and salts, and predators (Landon and Stasiak, 1983). The greatest depth of the migration is often set by the 21. Cladocera and Other Branchiopoda bottom of the lake, by the depth to which light penetrates, or by the limit of sufﬁcient oxygen (about 1.0 – 0.5 m␥ⲐL; Weider and Lampert, 1985; Nebeker et al., 1992). Littoral organisms also have a pronounced diel up-and-down migration. This behavior allows 90% of the population below a sample area to be trapped over night in inverted funnels (Whiteside and Williams, 1975). The daily pattern often seems to have the greatest amplitude during the summer and fall months. The larger developmental stages typically migrate the farthest. Populations usually migrate downwards at about dawn and do not return until after dusk. Physiological response to light intensity and duration is a component of vertical migration behavior (e.g., Ringelberg, 1987). Directionality of light source is important for Daphnia to be able to assume normal swimming position; if the light comes equally from every direction, the animal is unable to swim normally. Cladocerans can probably orient in the water using polarized light (Ringelberg, 1987). There is also some evidence that cladoceran swimming speed and direction is inﬂuenced differently by red and blue light. Many ecologists have concluded that, while response to light is an important element in vertical migration, the most important factor is escape from predators (Lampert, 1993; DeMeester et al., 1998; Tollrian and Harvell, 1998). Cladocerans move downward during the day, into the darkness of deep water, where they are relatively safe from visual predators such as ﬁsh. However, deep water also tends to be cold. In order to grow and reproduce as fast as possible, the cladocerans return to warmer water near the surface at night. This story has been elegantly demonstrated by a series of experiments in the large plankton towers at the Max Planck Institut für Limnologie in Plön, Germany (Loose, 1993). When exposed to a combination of light and feeding predators, or even the water from cultures of predators, Daphnia rapidly swim downward. They accomplish the vertical migration not by sinking or gentle swimming, but by rapid directional swimming (Dodson et al., 1997a). Because the prey respond to predator water, the behavior is clearly in response to a chemical signal released by the predator. D. Swarming Behavior Cladocerans sometimes form swarms. Baird (1850) reported: “I have, however, frequently seen large patches of water in different ponds assume a ruddy hue, like the red rust of iron, or as if blood had been mixed with it, and ascertained the cause to be an immense number of the D. pulex. The myriads necessary to produce this effect is really astonishing, and it is extremely 865 interesting to watch their motions. On a sunshiny day, in a large pond, a streak of red, a foot broad, and ten or twelve yards in length, will suddenly appear in a particular spot, and this belt may be seen rapidly changing its position, and in a very short time wheel completely round the pond. Should the mass come near enough the edge to allow the shadow of the observer to fall upon them, or should a dark cloud suddenly obscure the sum, the whole body immediately disappear, rising to the surface again when they have reached beyond the shadow, or as soon as the cloud has passed over.” Swarms and movements similar to those described by Baird (1850), if not as colorful, have been reported for other planktonic and littoral cladocerans (Weismann, 1880). Tessier (1983) described swarms of Holopedium, and reviews reports of other cladocerans, including Ceriodaphnia, Bosmina, Daphnia, Moina, and Sida. Daphnia and Polyphemus sometimes form small swarms of a few hundred animals. Although earlier studies with Ceriodaphnia, Daphnia, and Moina suggested that females in swarms tended to produce ephippial eggs, Crease and Hebert (1983) failed to ﬁnd any evidence for sexual pheromones produced by either sex of swarming Daphnia. Butorina (1986) observed Polyphemus swarms consisting of a few hundred animals in mushroom-shaped swarms about 50 cm deep, 6 – 30 cm in diameter, and with most of the animals in the top 5 cm of the swarm at the surface of the water (Butorina, 1986). The swarms were simple aggregations, with no difference in age structure from the surrounding nonswarming Polyphemus. The swarms are dynamic, with animals arriving and leaving as long as the sun shines. In addition to light, ﬁsh smell is also an environmental signal that appears to induce swarming behavior (Jensen et al., 1998). Compared to controls, Daphnia in ﬁsh water swim more uniformly. Ilyocryptus (Fig. 44) has a reputation for nesting (Williams, 1978). They are often found in dense aggregations, which have been interpreted as the result of young staying in the vicinity of their parent. E. Escape Behavior Cladocerans show various escape behavioral responses to predators (reviewed in Brewer et al., 1998). At least some of the planktonic species have a fastswimming response which takes them away from predators. This is effective against nonvisual predators such as Leptodora (Browman et al., 1989), but may be useless against ﬁsh. Diel vertical migration or small body size are the most effective defenses against large predators, such as ﬁsh, that use vision to locate prey. When caught by a small predator, such as a copepod or insect larva, cladocerans struggle to break free. Bosmina, once it escapes a nonvisual predator, closes up its carapace and sinks without making detectable 866 S. I. Dodson and D. G. Frey hydrodynamic pressure waves (noise) (Kerfoot et al., 1980). Also, Bosmina, like chydorine chydorids, tucks the second antennae under the carapace for further protection when captured. Brewer et al. (1998) measured the three-dimensional escape response of Daphnia. Daphnia of a given size always escaped at the same speed, regardless of origin of the clone, but individuals from lakes with ﬁsh tended to start escaping much sooner than those from ponds lacking ﬁsh. V. WHAT CLADOCERANS EAT AND WHAT EATS CLADOCERANS Planktonic cladocerans, such as Daphnia, are of great importance to the ecology of lakes, affecting the amount of algae, via grazing and nutrient regeneration, and providing food for young-of-the-year ﬁsh (Carpenter, 1987; Kitchell, 1992; Sommer, 1989; Lampert and Sommer, 1997). A. Cladoceran Feeding Cladocerans are discriminating in their choice of food (Porter, 1977). There are two major aspects of foraging relationships: what cladocerans eat, and how they get it. Techniques recently developed allow quantitative measurements of the various aspects of the feeding process: food collection, ingestion, assimilation, and defecation (Peters, 1984). The so-called “herbivorous” cladocerans eat a variety of small particles, from bacteria less than a micrometer long (Porter et al., 1983), through algae, up to ciliates, small rotifers, and copepod nauplii as large as 100 m (Porter, 1973; Burns and Gilbert, 1986; Dodson, 1975; Pace et al., 1994). The most important component of their diet is made up of small algae in the range of 1 to 25 m. Algae larger than about 50 m or algae with spines or in colonies are usually rejected. Also, cyanobacteria in the preferred size range are often toxic and are not eaten if abundant (Porter and Orcutt, 1980). The size of food particles is determined by more factors than the body size of the herbivore. For example, Bosmina, a small ﬁlter feeder, specializes on larger particles than do larger Daphnia (DeMott and Kerfoot, 1982). Chydorids typically feed by crawling along surfaces or through mud and scraping up or ﬁltering food (Fryer, 1968; Smirnov, 1971). Many cladocerans use the carapace to help ﬁlter feed or as a pair of cheeks which conﬁne the feeding current. However, some chydorids also employ the carapace as a suction cup. Negative pressure can be created inside the carapace if water is pumped out, as long as there is a good seal between the carapace and substrate. Second antennae, which are used for swimming by pelagic cladocerans, are used for crawling and jumping in some chydorids. Thus, chydorids have a number of specialized feeding and related locomotion adaptions. For example, Eurycercus, the largest chydorid, climbs on upper surfaces of plants and is able to ﬁlter feed. Alonopsis elongata balances upright on its carapace margins on solid surfaces and scrabbles along using its second antennae, scraping up living and dead organic material with its thoracic legs. Peracantha truncata keeps its second antennae inside the carapace when it crawls and can crawl upsidedown if the surface is rough enough to provide a hold for the claws on the ﬁrst leg, aided by weak negative pressure. Alonella exigua can hang upside-down on aquatic vegetation solely by means of negative pressure. Graptoleberis testudinaria has the most effective pressure chamber and exhibits snail-like behavior. Leydigia leydigi has neither pressure chamber nor scraping spines on the thoracic legs. It pushes itself into soft ooze using its ﬁrst antennae and large spiny postabdomen. Food is sieved from the mud using setae along the margin of the carapace and on the shortened thoracic legs. In Leydigia the respiratory current (which passes over the outside of the ﬁrst two thoracic legs) is separated from the feeding current. While most chydorids are herbivorous, the scraping method of gathering food lends itself to carnivory, and Anchistropus is a predator on hydra, while Pseudochydorus globosus is a scavenger of dead animals. Leptodora, Polyphemus, and Bythotrephes are predaceous cladocerans, eating protozoa, rotifers, planktonic stages of small chironomid larvae, and small crustaceans. Leptodora takes prey between about 0.5 and 1.5 mm and seems to prefer other cladocerans (Karabin, 1974; Browman et al., 1989). The smaller polyphemids take smaller prey and prefer small cladocerans, rotifers and protozoans (Monakov, 1972; Lehman, 1987). Algae is a minor component of the diet of Polyphemus and Bythotrephes. Presumably, the metanauplii of Leptodora eat algae. Adult Leptodora and Onychopoda have a reputation of being vampirelike “ﬂuid feeders” (Cummins, 1969; Karabin 1974). Leptodora (and other predaceous cladocerans) need actual contact with prey before they attempt to capture it with the outspread thoracic legs (Browman et al., 1989). Once the prey is grasped, the Leptodora bites it with the mandibles, sucking up body ﬂuids and perhaps small bits of tissue. Small prey, such as rotifers and protozoans are probably chewed and then swallowed. Edmondson and Litt (1987) report large numbers of whole rotifers (Conochilus) and even whole cyclopod copepods in the gut of Leptodora. 21. Cladocera and Other Branchiopoda B. Predators on Cladocerans Cladocerans are eaten by any freshwater predator that can either swallow them or chew pieces out of their small bodies. A few examples of predators include: common pond insects (Cooper, 1983), especially odonates (Hirvonen and Ranta, 1996) and larvae of the phantom midge Chaoborus (Swift and Federenko, 1975), ﬂatworms (Schwartz et al., 1983), hydra (Havel, 1985), salamanders (Dodson and Dodson, 1971), ﬁsh of all kinds but especially small pelagic forms (O’Brien, 1979) except for the smallest immature ﬁsh (Graham and Sprules, 1992), birds (Dodson and Egger, 1980), predaceous copepods (Dodson, 1974; 1984; Havel, 1985), other predaceous cladocerans (Havel, 1985), and even predaceous plants (Havel, 1985). Examination of the gut contents of minnows, sticklebacks, and small ﬁngerlings of other ﬁshes shows a heavy uptake of littoral cladocerans. The young of percid and centrarchid ﬁshes moving through the macrophyte beds in the littoral zone ﬁnd an abundance of these animals to consume. Small catostomid ﬁshes in streams near Bloomington, Indiana seem to feed almost exclusively on chydorids. Whiteside and his students believe that one of the possible causes of the midsummer population crash of chydorids in lakes of northern Minnesota is predation by early ﬁngerling perch. The small size of the littoral cladocerans is suited to the feeding abilities of small ﬁshes. VI. POPULATION REGULATION Cladoceran populations, especially of the planktonic species, are characterized by hundredfold annual variations in population size. The peak population generally occurs during an algae bloom, which occurs when lakes are mixed in the spring in northern areas or when the rainy season begins in southern areas. Cladoceran populations are typically at low points during cold weather or when edible algae are scarce. Littoral species show similar population dynamics (Whiteside, 1978). What causes the large variations in Daphnia abundance? Until the work of Hall (1964), it was assumed that the only signiﬁcant factor was the availability of edible algae. However, Hall showed that while the increase in Daphnia numbers was due to an algal bloom, the decrease was due to predation rather than starvation. Recent studies suggest that the usual cause of the decline in abundance is often a combination of predation and food limitation (Lampert, 1986b). Daphnia possess enough energy stores to carry them through short-term food shortages, such as the “clear-water 867 phase” that sometimes follows peak Daphnia abundances. However, lowered fecundity during the summer, after the clear-water phase, indicates food limitation. Mortality due to predation is generally sizespeciﬁc, depending on the size of the prey and the size and hunting behavior of the predators (Zaret, 1980). Those cladocerans that can coexist with predators tend to have specialized life histories, morphologies, and behaviors that provide defenses against predation. Even so, mortality due to predation can be an important factor in determining the abundance of zooplankton. Hall (1964) made a great contribution to aquatic ecology by showing that predation, as well as food limitation, limits zooplankton populations. He did this by separating the population growth rate coefﬁcient (r) into a component of birth or recruitment (b) and a component of mortality (d). He used a life table technique which requires that individual animals be cultured in the laboratory to determine rates of mortality in the absence of predation and fecundity at various food levels. This important process of separating mortality and recruitment can be achieved much more easily using Edmondson’s (1960) egg-ratio technique. This technique requires that one know the number of eggs per female and the developmental time of an egg in order to estimate b. Then, r can be estimated from changes in population size, and d is estimated by the difference between b and r. This technique provides good estimates of b when compared to life table estimates (Lynch, 1982), even when the assumptions of the egg-ratio technique (concerning age structure) are not met. The original Edmondson egg-ratio technique has been modiﬁed in various ways (Rigler and Downing, 1984). A comparison of these modiﬁcations with predictions based on life table studies (Gabriel, 1987) suggests that different modiﬁcations are most accurate depending on the population age structure and the frequency with which the population is sampled. In general, a declining population with a high value of b indicates high predation, and a stable or declining population with a low value of d indicates food limitation. Studies on the effects of both food limitation and predation suggest that both regulate populations. The best competitors are generally the most susceptible to predators. In a lake dominated by Holopedium and Daphnia, the Holopedium were mainly limited by the food supply, while Daphnia showed signiﬁcant mortality from ﬁsh during the summer (Tessier, 1986). A combination ﬁeld and laboratory study showed that Holopedium required more food than Daphnia. Thus, when ﬁsh predation was intense and food was abundant, Holopedium increased; but whenever food was scarce, Holopedium declined relative to Daphnia; and when predation was weak and food 868 S. I. Dodson and D. G. Frey abundant, Daphnia increased faster than Holopedium. Vanni (1986), using large plankton enclosures in a eutrophic lake, found that large Daphnia could reduce food levels sufﬁciently to reduce populations of Bosmina and several copepods. The Daphnia in the lake would have been kept in check by ﬁsh predation. Cooper and Smith (1982) found that when ﬁsh are absent, large invertebrate predators such as Buenoa could function in a size-selective manner like ﬁsh and remove the larger Daphnia pulex, which was able to live at lower food concentrations than the smaller Daphnia laevis. Kerfoot and Sih (1987), Carpenter (1987), Sommer (1989), Kitchell (1992), Lampert and Sommer (1997) provide further examples of direct and indirect effects of predators on cladoceran abundance. VII. FUNCTIONAL ROLE IN THE ECOSYSTEM Hrbácek (1958) ﬁrst pointed out the importance of certain ﬁsh in excluding cladocerans from communities. These ideas were extended to the effects of selective grazing of cladocerans on algae to produce a model of a dynamic food chain, in which the number and abundance of species in a community is the result of the interaction of competition, selective predation, and nutrients (Fig. 6). For many aquatic predators, cladocerans are a major diet item. While phosphate concentration in a lake is a major determinant of the kind and abundance of algae, and hence of “water quality,” the phosphate concentration is not a particularly good predictor of water quality (Schindler, 1977). For any given level of phosphate, there is great variation in water quality. Several recent studies suggest that food chain dynamics also determine water quality (Shapiro, 1982; Carpenter, 1985; McQueen, 1986; Carpenter, 1988; Kitchell, 1992). Large species of Daphnia appear to be the most efﬁcient in cleaning up lakes made turbid by small algae. Filter-feeding cladocerans can remove signiﬁcant fractions of the algae from a lake each day (Porter, 1977). Each animal, depending on its size, can clear 1 mL per animal per hour (Porter et al., 1982). For example, large Daphnia pulicaria was scarce in Lake Washington until 1976, when it became common (Edmondson and Litt, 1982; Edmondson, 1991). At the same time the mean summer transparency of the lake doubled, due to grazing by Daphnia on diatoms and other algae. The large size and high feeding rate of the large Daphnia make them especially efﬁcient at cleaning up lakes. The hypothesis of food-chain dynamics is a central hypothesis in ecology, because of the perspective it provides and the potential it has for explaining the diversity of natural systems (Fretwell, 1987). The power of this hypothesis results from its inclusion of both direct effects, such as predation of ﬁsh on zooplankton, and indirect effects, such as the effects of ﬁsh predation on the abundance of algae eaten by zooplankton. In aquatic communities, this hypothesis predicts that the abundance of algae (especially nuisance algae, such as the scum-forming blue green algae) can be kept at low levels by maintaining dense zooplankton populations, which depend on the scarcity of plankton-eating ﬁsh, which in turn are controlled by an abundance of large ﬁsh-eating ﬁsh. Thus, the theory of food-chain dynamics predicts that management of game ﬁsh, such as bass or pike in a lake, will have effects on the abundance of algae and, consequently, various aspects of water quality depending on algae, such as clarity and odor. A key link in this chain are the large algae-eating zooplankton, especially Daphnia, which are common and often abundant, the favorite food of plankton-eating ﬁsh, and able to eat rapidly large quantities of algae (Carpenter, 1985). Smaller common cladocerans, such as Bosmina and Ceriodaphnia, while less susceptible to ﬁsh predation, are also less efﬁcient at cleaning up algae. Meiobenthic (littoral) cladocerans constitute one of the prime converters of organic detritus with its associated microorganisms into particles big enough to be handled by small ﬁshes and predaceous invertebrates. Wetzel (1983) demonstrated that often the littoral community is more important than the open-water community in the annual production of a lake. VIII. PALEOLIMNOLOGY AND MOLECULAR PHYLOGENY A. Paleolimnology In paleolimnology, we seek to interpret how a lake functioned in past time. The sediments accumulate chronologically; and hence from changing representation of kinds of sediments and their sorting coefﬁcients, organic and inorganic chemicals, and morphological remains of plants and animals, we attempt to interpret past conditions in the water bodies and their watersheds. Sometimes the record is exceptional. Where varves (annual layers) are present, changes in response from year to year can be determined. Lake sediments tell fascinating stories about the lake and the watershed. Lake sediments have been used to understand the past climate (from pollen from terrestrial plants in the watershed), lake productivity (from the kinds and amounts of microfossils of lake organisms), and the effects of species invasions into the lake. Lake sediments can also supply important archaeological insights. For example, a study of a small lake 21. Cladocera and Other Branchiopoda in central Italy (Lago di Monte Rosi; Hutchinson et al., 1970) found a close correspondence between the 14C date for a thin layer of charcoal and actual records of Roman road-building activity in the watershed. Remains of animals that are preserved in lake sediments are mostly either silicious (sponges), calcareous (ostracods), or chitinous (cladocerans, midges). All groups of freshwater animals leave at least some remains in sediments (Frey, 1964), but certainly the cladocera are usually best represented, with quantities of remains generally between a few thousand and several hundred-thousand per cubic centimeter of fresh sediment. Once the species pool of a region is known, then virtually all the fragments can be identiﬁed positively as to producing species. Remains of the chydorids and bosminids are preserved best and nearly quantitatively, but all cladoceran taxa leave some remains. Those of the meiobenthic forms become integrated over time (at least a year) and habitat before being incorporated into deepwater sediments. The quantity of these remains varies from place to place, being greater toward shore than in the profundal zone, but the percentage composition by species is the same all over the bottom (Mueller, 1964). The best way to determine what species of cladocera occurs in a water body and in what relative abundance over time is from the animal remains in the sediments. Close-interval stratigraphies are constructed of these remains in the same way as for pollen or diatoms, yielding changes in relative abundance over time of the various taxa (Frey, 1986b). Interpretation has been difﬁcult because of an insufﬁcient knowledge of the ecology of these forms. A few species, such as members of the Chydorus sphaericus complex, are considered to respond positively to eutrophication, sometimes comprising more than 90% of all remains. Others seem associated with bog conditions or changes resulting from acidiﬁcation. Daphnia and Bosmina fossil remains record past predation regimes (Kerfoot, 1981; Kitchell and Carpenter, 1987). What is really necessary is to analyze the community differences between time and place. Cotten (1985) studied cladoceran communities in 46 lakes in eastern Finland, using their remains in surﬁcial sediments. She obtained about 70 different taxa. These and their relative abundances were then ordinated using measured chemical and physical characters of the water bodies and their watersheds. She recognized ﬁve kinds or groups of lakes that were almost completely concurrent with the ﬁve kinds revealed previously by diatom analysis. Thus, the cladocera are just as good responders as diatoms to conditions in a water body and to changes in these conditions over time naturally or in response to human activities. 869 A study of the cladocera present in a lake in Denmark during the last interglacial period (roughly 100,000 years ago) revealed 25 species of chydorids, all of which occur in Denmark today (Frey, 1962). No real differences in morphology were apparent; and moreover, the relative order of abundance of these 25 species in the interglacial was the same as in Denmark today, indicating that the ecology of these forms had not changed either. Furthermore, evidence and suggestions from the meager remains of cladocera known from the present to the Tertiary, Cretaceous, and possibly even the Permian indicate that major evolution in the cladocera had occurred by the late Paleozoic or early Mesozoic, and that the species present today have been stabile for millions of years. Thus, knowledge of the present ecology of species will probably be usable in interpreting relationships in lakes even in the distant past. The presence of a vast record of cladocera in lake sediments is valuable for more than just paleolimnology. Sometimes the presence of a particular species of cladoceran at a place is suspected to be the result of establishment by recent transport there, such as Bythotrephes in the Great Lakes (Sprules et al., 1990). This hypothesis can be checked by sedimentary analysis if desired. The development of the cladoceran community in a lake since late-glacial time, or even earlier in nonglacial lakes, can be followed easily (Goulden, 1966). The species diversity increases over time as the number of taxa in the community increases as conditions become stabilized, tending toward the limit for maximum diversity at any given number of taxa (Goulden, 1969). The distribution of species by their relative abundance in a stable community closely follows the MacArthur broken-stick model. Any major deviation in ﬁt to the expected is considered to have arisen from changes in climate, agriculture, volcanism, or other controlling agents (Frey, 1974, 1976). In lakes with varved sediments one can follow changes in relative abundance of the total cladoceran community on a year-to-year basis and then relate these changes to potential causative agents (Boucherle and Zulig, 1983). Access to the literature of paleolimnology can be obtained from the proceedings of the four international symposia on the subject (Frey, 1969; Klekowski, 1978; Löfﬂer, 1987; Merilainen, 1983; North American Lake Management, 1995). B. Cladoceran Fossil Record Fossils of branchiopods and their relatives are known from the Cambrian Period, about 530 million years ago. Kerfoot and Lynch (1987) summarized data on the earliest fossils of Branchiopods. In the Cambrian 870 S. I. Dodson and D. G. Frey FIGURE 5 (A) Phylogenetic tree, showing the divergence of cladoceran genera during the Mesozoic, and differentiation of the subgroups of Daphnia during the Cretaceous (Lehman et al., 1995); (B) phylogenetic tree showing the species in the three major species groups of the genus Daphnia (Colbourne et al., 1997). 21. Cladocera and Other Branchiopoda Period, animals ﬁrst developed mineralized coverings about 540 million years before the present (mybp). There are branchiopodlike fossils from the Burgess Shale, such as Protocaris and Branchiocaris, but these animals are not considered branchiopods (Gould, 1989). A fossil of an animal that is probably an ancestor of the branchiopods (and distinct from other crustaceans) occurs in Upper Cambrian sediments of Sweden (Walossek, 1995). By the lower Devonian (ca. 410 mybp, when ﬁsh also make their appearance), the larger Branchiopoda are well established as three recognizable modern groups: fairy, tadpole, and clam shrimp. The fairy shrimp are represented by Lepidocaris, an anostracanlike animal, but with a long segmented second antenna and more mouth parts than modern fairy shrimp. These animals are from sediments that indicate marine and estuarine, and perhaps freshwater sediments. Tadpole shrimp fossils are found in marine sediments, and a clam shrimp fossil also appears in marine sediments (this animal is possibly an ancestor of both modern clam shrimps and cladocera). By the Carboniferous (ca. 286 – 360 mybp), tadpole and clam shrimp species are diverse and occur in many habitats (marine, freshwater, streams). Smirnov (1970) described some possible Daphnia-like and chydoridlike fossil from the late Permian (ca. 240 mybp). The Daphnia-like animals are 5 – 8.5 mm long. Smirnov also has good specimens of Daphnia-like fossils from the Jurassic (ca. 225 mybp). By the early Triassic, most tadpole and clam shrimp groups are extinct, and cladocera appear, perhaps as neotenic conchostracans (as suggested by Brooks). The modern continental distribution of cladoceran orders suggests the orders were distinct when the large continent Pangea broke up into Laurasia and Gondwanaland, during the Cretaceous (approximately 100 mybp). Starting with the Oligocene (ca. 38 mybp), there are many good fossils of cladocerans, especially of ephippia from lake deposits such as the freshwater Florrisant beds in Colorado. Similarly, the modern distribution of cladoceran species in North America and Europe suggests that the major about of speciation associated with glaciation was associated with speciation which produced modern species (Brooks, 1957). Continental glaciers have been producing new lakes since about 1.8 mybp. For example, several species, such as Daphnia galeata, retrocurva, catawba, longiremis, are restricted to the glaciated portion of North America (or Europe: D. cuculata). C. Molecular Phylogeny Just as lake sediments contain a record of the history of the lake and its watershed, the nucleic acid 871 (nuclear and mitochondrial) of cladocerans contains a record of the evolutionary history of living individuals. The molecular record is particularly valuable, because the fossil record of most zooplankton groups is poor. Information about phylogeny exists in the nucleic acids of branchiopods. For example, analyses of the ribosomal RNA gene (encoded on mitochondrial DNA) suggest that Daphnia originated (differentiated from similar genera) in the Mesozoic (Lehman et al., 1995; Colbourne et al. 1997; see Fig. 5A). Since then, the genus has evolved into at least three species complexes (Fig. 5B). A recent re-analysis of an Australian daphniid (Daphnia jollyi), suggests it may represent a fourth lineage. It is odd that there are only four complexes of Daphnia species, and that they are all rather similar morphologically, given 200 – 300 million years of evolution. IX. TOXICOLOGY Cladocerans, especially Daphnia, Ceriodaphnia, and Moina have long been used in toxicity tests. Cladocerans are relatively small, have short life spans, reproduce well in the laboratory, and are ecologically important — all desirable characteristics for a toxicity test. For a bioassay (a test using organisms), the animals are grown under optimal conditions in the laboratory (or less often, in enclosures in nature). Some animals are treated with known concentrations of chemicals, and their responses are compared to those of untreated animals. Survival and fecundity (number of offspring) are two standard bioassay endpoints (US EPA, 1994). Animals are exposed to test chemicals for a few hours or days as a test for acute toxicity, and for a generation or more as a test for long-term chronic toxicity. Morphology, behavior, sex ratio, physiology, and developmental time are additional bioassay end points. These end points tend to be more sensitive than survivorship or fecundity, and they are of ecological importance. For example, the insecticide carbaryl induces morphological and swimming behavior changes in Daphnia (Hanazato and Dodson, 1993; Dodson et al., 1995). The surfactant (nonionic detergent) and plasticizer nonylphenol interferes with normal development at levels found in streams and lakes, so that neonates are produced and they survive in the laboratory, but they are unable to swim (Shurin and Dodson, 1997). Predator prey behavior is affected at low-dose and sublethal levels of many environmental pollutants. Carbaryl causes Daphnia to swim erratically and faster than normal (Dodson et al., 1995), which in nature, would increase the predator – prey encounter rate, leading to an increased Daphnia mortality. 872 S. I. Dodson and D. G. Frey FIGURE 6 Food-chain models showing: (A) four trophic levels; (B) three trophic levels; and (C) three consumer trophic levels and one scavenger (decomposer) level. 21. Cladocera and Other Branchiopoda Many aspects of animal development are controlled by hormones. There is concern that industrial and agricultural chemicals may be acting like or interfering with normal hormone systems in both wildlife and humans (Colborn et al., 1996). The cladoceran sex ratio is an endpoint that can be used to test for these chemicals (called “endocrine disruptors). Chemicals that change sex ratio in cladocerans will have ecological consequences, and they may also act as endocrine disruptors for other animals, including humans. It is hypothesized that some chemicals that disrupt hormone systems in vertebrates can also interfere with the system regulating molting in arthropods (Zou and Fingerman, 1997). X. CURRENT AND FUTURE RESEARCH How many species are there of cladocerans? Do cladoceran species need conservation? How do you identify the different species? How closely related are the different morphologically similar species? Are cladocerans (or other branchiopods) even really crustaceans? There is a great deal of taxonomy and phylogeny that remains to be done. This is not just a matter of ﬁnding all the species and naming them, but more fundamentally, of deciding what sort of species concept makes sense with cladocerans. These animals are usually at least facultatively asexual; and in many groups, what seem to be good morphological species are made up of a combination of occasionally sexual and totally asexual populations. Descriptions of species before about 1950 are mostly very inadequate and hence cannot be used to compare populations on two different continents claimed to be the same taxon. To determine that any North American taxon is the same as or different than the taxon of the same name described from elsewhere is difﬁcult. One ﬁrst needs to compare both taxa closely, using large populations containing all instars and reproductive stages. Thus far about 16 pairs of taxa with the same name, from the New and Old World, have been compared (partial summary in Frey, 1986a), with the ﬁnding that almost all of them are full species. Sometimes, as in the Chydorus sphaericus complex, Eurycercus, Ephemeroporus, the Chydorus reticulatus group, the Chydorus faviformis group, and Pleuroxus laevis, the same speciﬁc name was being applied to two or more related but different species in the same geographical region. Consequently, for critical studies in ecology, one must be careful not to transfer the ecological requirements and indications of a species across an ocean or some other distributional barrier, on the assumption that all taxa presently bearing the same name are the same 873 species. This question will be sorted out with a mixture of traditional morphological and new molecular and genetic techniques (e.g., DeMelo and Hebert, 1994; Colbourne et al., 1997), and some new ideas! The study of food-chain dynamics, using sets of whole lakes shows promise for interesting research into population and community ecology. Society has a great desire for clean lakes and lots of ﬁsh production. Cladocerans play crucial roles in both water quality and ﬁsh management, but we need to understand cladoceran biology better before we can meet society’s desires. Food chain dynamics, or “biomanipulation” shows promise of being an inexpensive and safe method for improving both water quality and ﬁsh production. Zooplankton live in a sea of chemical signals (Larsson and Dodson, 1993). The chemicals are produced by predators, food, competitors, and environmental sources. The signals induce changes in development, morphology, reproduction, and behavior. However, none of the chemical signals used between species have been characterized. When their chemical structure is known, it will be possible to do interesting experiments involving induction, and to understand how these signals affect development and behavior, which, in turn, affect the aquatic community and trophic dynamics. Environmental toxicology has long depended on tests of cladoceran survival and fecundity. Future research will continue to use more ecologically relevant endpoints, such as behavior, developmental time, and sex ratio (Dodson et al., 1999). XI. COLLECTING AND REARING A. Sampling Techniques Cladocerans can be collected using a ﬁne mesh net pulled through the water or a sieve through water is poured. A large number of specialized nets have been developed for speciﬁc applications (Bernardi, 1984). The mesh should be around No. 10, or have a pore size of about 90 – 150 m. Nets are most useful for towing in open water. Weedy habitats can be sampled by a plankton net attached to a handle, and with a widemesh (1 cm or so) cover over the opening of the net. Weedy habitats or mud can be sampled by picking up water in a bucket and pouring it through a series of nets or sieves (Downing, 1984). In any case, keep mud out of the sample if possible. Large pieces of water weeds and detritus can be ﬁltered out of a sample with screen having a mesh size of about 1 cm. Filters can be made by cutting the bottom off a plastic bottle. The center of the cap is cut out, and a piece of mesh glued over the hole. When the cap is screwed back onto the 874 S. I. Dodson and D. G. Frey bottle, water can be poured into the inverted bottle, and cladocerans will be caught by the mesh. The captured cladocerans can then be washed into a jar of water previously ﬁltered through the mesh. Estimations of population dynamics or productivity require quantitative samples, which require special gear (Bernardi, 1984; Downing, 1984) and a careful experimental design and statistical analysis of samples (McCauley, 1984; Prepas, 1984). If the sample organisms are to be killed, there are at least two ways to do it (the authors disagree on the best technique). Dodson prefers to wash the animals off the screen of a small sieve using a wash bottle ﬁlled with 95% ethanol, or to drop the sample into a large volume of 95% ethanol. This will kill the animals rapidly enough so there will be little distortion and will preserve the sample indeﬁnitely. It will be necessary to keep the samples tightly capped and to add 70% alcohol from time to time. Although messy, a little glycerine will ensure that the sample never dries up. Frey preferred to kill and preserve animals by adding enough commercial formalin containing 40 g sucrose per liter to make a 5% formalin solution. He felt formalin was better for long-term museum preservation of cladocerans, because of lesser distortion of the specimens over time. For safety reasons, formalin samples are best examined in plain water, following ﬁltration. Specimens can then be replaced in a formalin solution for storage. B. Culture Methods Animals will live in ﬁltered pond water for a few days at least, especially if there are fewer than 50 animals / L. Animals can be transferred to new water using pipettes made of glass tubing and rubber bulbs, or for large quantities, turkey basters are useful. For longterm studies of cladocerans, it is possible to maintain cultures for years. Maintenance of cladoceran cultures can be divided into two processes: maintenance of food for the cladocerans and care of the cultures. 1. Cladoceran Food The simplest to maintain and most reliable bulk food culture for cladocerans appears to be a mixture of green algae and bacteria maintained in a large aquarium. Keep the algae in a glass aquarium as large as possible; a 40-gal aquarium is sufﬁcient. Place the aquarium in a south-facing window. Choose a place that can be kept at least 18 – 25°C; a greenhouse is preferable. Fill the aquarium with ﬁltered lake or pond water, using a ﬁne mesh (ca. 90 m mesh) net, or use tap water aged for 1 day with a little additional ﬁltered lake water as a seed source for the phytoplankton. Add a dozen or so small ﬁsh (bait ﬁsh, guppies, or gold ﬁsh) to the tank and feed the ﬁsh regularly. Aerate the tank. Keep the glass walls scraped clean and siphon off bottom detritus now and then. Regardless of what you start with, the algae species will probably eventually stabilize, and you will have a mixture of small greens (like Chlorella, Selenastrum, and Scenedesmus), along with a few other less common species such as Ankistrodesmus. Keep the algae in a healthy state by removing at least a quarter of the culture every week or so. Use a large-diameter siphon to remove the green algae. Well-equipped laboratories can grow single-species algal cultures purchased from biological supply houses. Grow the algae either in a medium supplied by the supply house, or mix artiﬁcial lake water, such as “Combo” (Kilham et al., 1998). The advantage of Combo is that it has the same salinity as average lake water, and the algae can be added directly to branchiopod cultures. Grow the algae in 2-L clear plastic bottles (soda bottles). For fast growth, aerate the cultures and use bright light. Culture Care: Branchiopods can be captured using plankton nets, dip nets, or by pouring water through ﬁlters of about 100-m mesh. Wash the collected animals into some ﬁltered water from the lake or pond. Bring the animals into the laboratory and let them equilibrate to the laboratory temperature. Many cladocerans tend to be captured by the surface ﬁlm. This happens more often for the smaller lake animals, especially if they are initially in cold water with saturated oxygen, because bubbles form on the carapace as the water warms. In extreme cases of mortality, keep the surviving animals in a narrow-mouth bottle ﬁlled to the top and plugged with cotton pushed below the water surface. In the beginning, put the cultures on a dark substrate and keep them in a room with dim light. After a couple of days, it will be obvious which cultures are going to be vigorous. Be sure to label the cultures (with tape on the jar) as to the source and date of capture. Feed the cultures algae suspension. If you cannot see large objects through the culture jar, the algae is probably too concentrated; dilute it. The mixture should be green but not opaque. As the water in each jar clears, add algal suspension. Do not let the water become clear. Even an overnight bout of clear water will cause some clones to starve. It is also possible to overfeed clones, but starvation seems a much more common occurrence. Continue adding algae suspension until the jar is full. After the jar is full, there are two ways to feed the culture. First, if there are too many animals in the jar, pour out half or so of the culture and reﬁll with algal suspension. Beware of cultures in which the animals concentrate in the top centimeter or so of the water; it is possible to pour out the entire culture! Second, if there are too few cladocerans in the jar, siphon off 21. Cladocera and Other Branchiopoda about half of the water and add algal suspension. Be sure to use ﬁne enough mesh (about 90 m) on the end of the siphon in the jar, so that the animals stay in the jar. Also, be sure to wash the siphon and dry it or dip it into hot water before using it on the next culture. The safest population size for a 1-L jar seems to be about 20 – 50 animals. Having some detritus on the bottom of the jar will not harm planktonic species and is beneﬁcial to meiobenthic species. When there is a thick layer, decant off the culture and discard the detritus layer. Clones, once they are established, can be kept in a cold room or refrigerator at about 4°C. At this temperature, the animals need to be fed only about once every two weeks. Beware — some species die when moved from 20 to 4°C, so do not put all your cultures in the cold at once. XII. TAXONOMIC KEYS FOR CLADOCERAN GENERA A. Available Keys and Specimen Preparation Crustaceans are cladocerans if they have four to six pairs of (thoracic) legs, lack any paired eyes, swim with their second pair of antennae, and have at least the head not covered by a carapace. The identiﬁcation keys are based on a large number of sources, which are referenced in Table 1 and in the individual keys. Dodson is responsible for most of the keys. The key to the Chydoridae family was written by Frey. These keys are designed to allow identiﬁcation of adult females only. Dissection is seldom necessary, but it is a good idea to have several animals available for identiﬁcation, because these small, delicate animals are easily crushed out of recognizable shape. Animals shorter than about 1 mm should be mounted on a slide for highmagniﬁcation viewing; the larger species can often be identiﬁed without being mounted, using a dissecting microscope at about 50 diameters magniﬁcation or less. While live animals trapped in a thin ﬁlm of water can usually be identiﬁed, it may be necessary to kill them with alcohol and mount them on slides. The best mounting medium is one which is more or less permanent and which is soluble in water and alcohol solutions so specimens can be added directly to the medium without tedious dehydration. If you have access to chloral hydrate (currently a “controlled substance”), a good medium is Hoyer’s: 875 Dissolve 30 g of ground Gum Arabic in 150 mL of hot distilled water. Add 200 g of chloral hydrate and 20 g of glycerine. Divide the Hoyer’s into two batches. Let one sit in a warm place until it is as thick as honey, and keep the second batch in a closed jar so it remains thin. You can always add more water to thin either batch. Keep at least some of the thick and thin Hoyer’s in screw-top eye dropper bottles for easy use, and be careful to keep the Hoyer’s off the threads of the bottles. To make a slide, use the eye dropper to put a small streak of dilute Hoyer’s (about a quarter of a drop) toward one end of the slide. Arrange the specimens in this streak. It is wise to place four or so of what you think are the same kind of animal on a slide to allow for comparison and viewing of difﬁcult characters. Delicate specimens can be protected from compression by putting a few pieces of broken cover slips around the specimens. Let the Hoyer’s streak dry on a slide warmer or in a warm place (below 100°C). When the streak is dry, add a drop or two of the thick Hoyer’s and gently lower a cover glass over the Hoyer’s. Put the slide back into a warm place to dry again. Label the slide as to the date and location of the collection! It is important to use thickened Hoyer’s in the last step; otherwise, the Hoyer’s will shrink as it dries, and produce large bubbles in the ﬁnal preparation. On the other hand, if you use thickened Hoyer’s, small bubbles produced when you lower the coverslip will disappear as the medium dries. These slides are semipermanent. If the climate is humid, the Hoyer’s will thin and cover glasses will slip off the slide. If the climate is arid, the Hoyer’s will desiccate enough so that the cover glasses pop off the slide. These problems may be avoided by storing the slides horizontally in a climate that has moderate humidity, or you can ring the slide with nail polish. An alternate mounting method, especially useful for bosminids, chydorids, and macrothricids, is to use polyvinyl lactophenol stained with lignin pink. Temporary slides can be made using glycerol or glycerine jelly. Best of all for museum specimens is possibly Canada Balsam, although this requires the specimens be dehydrated through an alcohol series. B. Taxonomic Key to Families of Freshwater Cladocerans This key to families is followed by keys to genera in each family or group of families. 1a. Both branches (or branch) of second antenna ending in three setae (Fig. 1A) .....................................................................................2 1b. At least one of the second antenna branches ending in four or more setae (Fig. 8) .............................................................................8 FIGURE 7 Holopedium gibberum Zaddach, 1855. Why Not Bog, Vilas Co., Wisconsin. 22 September 1973. (KE) FIGURE 8 Sida crystallina (O.F. Mueller, 1776). Lake Mendota, Dane Co., Wisconsin. 11 September 1987. (KE) FIGURE 9 (A) Diaphanosoma birgei (Kořínek, 1981). Lake Wingra, Dane Co., Wisconsin. 5 October 1978. (KE) (B) Penilia avirostris Dana, 1849. Anteriolateral view. Atlantic Ocean near Florida (29°55.8’ N and 81°07.4°W). 27 October 1987. (KE); (C) P. avirostris. Posteriolateral view. (KE) FIGURE 10 Pseudosida bidentata Herrick. 1884. Bull Frog Pond, S.R.P. area, South Carolina. 27 May 1985. (Coll. DGF #7483). (KE) FIGURE 11 Latona setifera (O.F. Muelller, 1776). Lake Mendota, Dane Co., Wisconsin. 30 June 1987. (KE) FIGURE 12 (A) Latonopsis occidentalis Birge, 1891. Dick’s Pond, S.R.P. area, South Carolina. 28 May 1985. (Coll. DGF #7496). (KE); (B) Sarsilatona serricauda Sars, 1901. Itatiba, Brazil. Figure 91 from Korovchinsky 1992. 21. Cladocera and Other Branchiopoda 877 2a (1a). First antenna composed of two segments (one genus, Ilyocryptus, Fig. 44) ......................................................................Ilyocryptidae 2b. First antenna with one segment ..........................................................................................................................................................3 3a(2a). Second antenna (the swimming antenna) with one branch (Fig. 7) ...................................................................................Holopedidae 3b. Second antenna with two branches ....................................................................................................................................................4 4a(3b). First antennae fused with the rostrum to form two long and pointed tusklike structures (Figs. 41 and 42) ........................Bosminidae 4b. First antennae not fused with the rostrum ..........................................................................................................................................5 5a(4b). One branch of the second antenna with three segments, the other with four (look for a small basal segment, as in Fig. 1) ................6 5b. Both branches of the second antenna with three segments (Suborder Radopoda) ...............................................................................7 6a(5a). First antenna about as long as the width of the head; usually a tooth with two points (biﬁd) on the posterior angle of the postabdomen (near the claw), (Fig. 58)............................................................................................................................................Moinidae 6b. First antenna less than half as long as the width of the head; teeth on postabdomen always single (Figs. 1A, 36 – 40)........Daphniidae 7a(5b). First antenna is attached to the underside of the tip of the head, there is no rostrum (beak) (Figs. 44 – 57)........................Superfamily Macrothricoidea 7b. First antenna is attached to the ventral margin of the head, and is more or less covered with a rostrum (beak), (Figs. 13, 35) ..........................................................................................................................................................................................Chydoridae 8a(1b). Body (thorax and abdomen) covered by the two valves (shells) of the carapace (Figs. 8 – 12)....................................................Sididae 8b. Body not covered with a carapace (which is present in some forms as a saclike brood chamber attached to the back; legs are not covered with the carapace (Figs. 60 – 64).....................................................................................Orders Onychopoda and Haplopoda C. Taxonomic Key to Genera of the Family Holopediidae Hebert and Finston (1996) used results of allozyme analysis to conclude that there are two species in North America, Holopedium gibberum Zaddach, 1855 (Fig. 7) and H. amazonicum Stingelin, 1904, conﬁrming the usage of Korovchinsky (1992). These are planktonic animals, which often occur in soft and even acidic water. H. gibberum is broadly distributed in the cool temperate regions of North America, while H. amazonicum occurs in the southern and eastern portions of the continent. These species are remarkable in that each individual is surrounded (out to about an additional body diameter) by a mass of clear jelly attached to the carapace (easily removed by rough handling). The jelly probably defends Holopedium against an array of invertebrate predators. Adult females are about 0.6 – 2.0 mm long. D. Taxonomic Key to Genera of the Family Sididae 1a. Posterior margin of carapace ﬂared out laterally; a point on either posterior-lateral margin of the carapace; marine (Figs. 9B, C) ..................................................................................................................................................................................................Penilia 1b. Posterior margin of carapace not ﬂared laterally; postero-ventral corners of carapace rounded; freshwater .......................................2 2a(1b). Swimming antenna appears to have three branches, the middle branch is an extension of the basal segment of the upper branch; postabdominal claw with two basal spines (Fig. 11) .................................................................................................................Latona 2b. Swimming antenna clearly has only two branches; postabdominal claw has 2 – 3 basal spines ...........................................................3 3a(2b). Postabdominal claw with about 10 long teeth along the claw (“long” means longer than the width of the claw); a palearctic genus ............................................................................................................................................................................................Limnosida 3b. Postabdominal claw with 2 – 4 long teeth near base............................................................................................................................4 4a(3b). Postabdomen with a row of about 20 individual triangular spines along the margin (the anal margin); no clusters of spines on postabdomen; postabdominal claw with three long teeth and an occasional short fourth tooth near the base of the claw; both branches of swimming antennae with a total of 15 long swimming setae (Fig. 8) ..........................................................................Sida 4b. Postabdomen with spines in clusters or patches, spines in rows are hairlike; swimming antennae with more than 15 long swimming setae ...................................................................................................................................................................................................5 5a(4b). Posterior margin of carapace with setae about 20 – 25% as long as the carapace ...............................................................................4 5b. Posterior or ventral margin of carapace with setae shorter than 10% as long as the carapace ............................................................5 878 S. I. Dodson and D. G. Frey 6a(5a). Swimming antennae branches with a total of 16 long swimming setae; postabdominal claw with two long basal spines (Fig. 11) ...........................................................................................................................................................................................Latonopsis 6b. Swimming antennae branches with a total of 24 long swimming setae; postabdominal claw with three long basal spines (Fig. 12B) ...........................................................................................................................................................................................Sarsilatona 7a.(5b). Swimming antennae with a total of 17 long swimming setae; postabdomen with rows of ﬁne hairlike teeth; postabdominal claw with three long teeth (the most basal tooth may be only about as long as the width of the claw at the base (Fig. 9) Diaphanosoma 7b. Swimming antennae with a total of 20 long swimming setae, postabdomen with clusters of 4 – 6 spinelike teeth; postabdominal claw with two long teeth, and a short basal tooth less than half as long as the width of the claw base (10) ................................Pseudosida [Korovchinsky (1992) has recently revised the Sididae, including the genus Sarsilatona and retaining the changes made by Kořínek (1981) for Diaphanosoma. Diaphanosoma and Penilia are planktonic and about 1.0 mm long; species in the other genera tend to be larger (2 – 4 mm) and are associated with littoral vegetation.] E. Taxonomic Key to Genera of the Family Chydoridae (North of Mexico) 1a. Postabdomen strongly ﬂattened, broad, with a single row of 80 – 150 marginal denticles, resulting in a saw like appearance; anus distal; subfamily Eurycercinae (Fig. 13) ................................................................................................................................Eurycerus [Genus is Holarctic except for isolated populations in southern Brazil – northern Argentina and in South Africa. At least ﬁve species in North America, distributed among three subgenera: one species in the subgenus Eurycerus only within 300 km of the Gulf of Mexico and the Atlantic Ocean as far north as the Pinelands of New Jersey (maximum length ca. 2 mm); two very common species in subgenus Bullatifrons, one in the northern tier of states and northward, the other in the south (length to 2.4 mm); and two species in the subgenus Teretifrons in Newfoundland and the Arctic (length to 6 mm). See Frey (1971, 1975, 1978, 1982d) and Hann (1982).] 1b. Postabdomen thicker, not so broad, and usually with two rows of marginal denticles, mostly fewer than 25 on each side; anus proximal ........................................................................................................................................................................................... 2 2a(1b). Proximal tip of mandible articulate where headshield and shell touch each other (Fig. 1C); usually two or three major headpores on midline, which usually are connected by a double sclerotized ridge, resembling a channel (Fig. 1C); occasionally only one median pore (Monospilus, Euryalona) or none (Notoalona); minor headpores lateral to these; free posterior margin of shell not greatly less than maximum height of animal; typically one, occasionally zero, basal spine(s) on postabdominal claw (Fig. 1C); postabdomen nearly always with lateral fascicles (Fig. 1C), sometimes without (Monospilus). ................................................Subfamily Aloninae 3 [In North America, Aloninae contains the genera Acroperus, Alona, Alonopsis, Bryospilus, Camptocerus, Euryalona, Graptoleberis, Kurzia, Leydigia, Monospilus, Notoalona, Oxyurella, and Rhynchotalona. Olesen (1998) presents evidence that Aloninae is a paraphyletic group.] 2b. Proximal tip of mandible articulates in a special pocket on the headshield, some distance in a dorsal direction from the point of contact between the headshield and shell (Fig. 1D); two major headpores on midline (Fig. 1B, D), completely separated from one another and with no sclerotized ridge connecting them, occasionally only one pore (Dadaya), or only one pore in ﬁrst instar and none in later instars (Ephemeroporus); minor pores most commonly near midline between major pores; free posterior margin of shell usually considerably less than maximum height of animal; typically two basal spines on postabdominal claw (Fig. 1D); articulated lateral fascicles on postabdomen lacking, although Pseudochydorus has a row of fasciclelike groups of setae......................Subfamily Chydorinae ......................................................................................................................................................................................15 [In North America, Chydorinae contains the genera Alonella, Anchistropus, Chydorus, Dadaya, Disparalona, Dunhevedia, Ephemeroporus, Pleuroxus, and Pseudochydorus.] 3a(2a). Only an ocellus present; no compound eye.........................................................................................................................................4 3b. Compound eye and ocellus both present ............................................................................................................................................5 4a(3a). Shells from previous instars ﬁrmly nested, so that instar number of specimen can be counted; headshield transversely trucate posteriorly; single median headpore (Fig. 14) .............................................................................................................................Monospilus [Supposedly Monospilus contains just one species in world, mainly Holarctic and Ethiopian in distribution, with several records from New Zealand as well. Length to 0.5 mm.] 4b. Shells lost molting, so that no nesting of shells occurs; two separated median pores without sclerotized connecting ridge; minor pores located far laterally; very short antennae (Fig. 15) ......................................................................................................Bryospilus [Two species are known from New Zealand, Venezuela, and Puerto Rico, and hence this genus may possibly occur on north of Mexico. Length to 0.35 mm. Found among wet, leafy hepatics of rain forests and cloud forests (Frey, 1980b, d).] 5a(3b). Body strongly compressed from side to side, at times almost wafer thin; post-abdominal claw with a secondary spine midway along concave margin, and usually with a comb of ﬁne setules decreasing in length proximally between it and the basal spine (comb also occurs in Euryalona) ..........................................................................................................................................................................6 5b. Body not so strongly compressed; postabdominal claw having only a basal spine of variable length (except in Euryalona); sometimes none at all (one species of Leydigia) ..........................................................................................................................................9 21. Cladocera and Other Branchiopoda 879 6a(5a). Body strongly keeled, and head also usually strongly keeled ..............................................................................................................7 6b. Body weakly keeled, and head without a keel ....................................................................................................................................8 7a(6a). Postabdomen narrow, elongate, tapered distally, with many (generally about 15 or more) margin denticles (Figs. 1C and 16) ......................................................................................................................................................................................Camptocercus FIGURE 13 Eurycercus (Eurycercus) lamellatus (O.F. Mueller, 1776). Uppsala, Sweden. From Lilljeborg (1901). FIGURE 14 Monospilus dispar Sars, 1862. Yddingesjon, Sweden. From Lilljeborg (1901). FIGURE 15 Bryospilus repens Frey, 1980. El Yunque, Puerto Rico. From Frey (1980b). FIGURE 16 Camptocercus rectirostris Schoedler, 1862. Kristianstad, Sweden. From Lilljeborg (1901). FIGURE 17 Acroperus cf. harpae (Baird, 1834). Eastern North America. From Birge (1918). FIGURE 18 Kurzia longirostris (Daday, 1898). Tammanna wewa, Sri Lanka. From Rajapaksa (1986). FIGURE 19 Alonopsis elongata Sars, 1862. England. From Norman and Brady (1867). FIGURE 20 Rhynchotalona falcata (Sars, 1861). Northern Michigan. From Birge. (1893). FIGURE 21 Graptoleberis testudinaria (Fischer, 1851). Eastern North America. From Birge (1918). 880 S. I. Dodson and D. G. Frey [Nine species recognized in the world in 1971, with lengths from 0.7 to 1.26 mm. Except for C. oklahomensis, the species in North America are completely unknown and undescribed, although certainly more than ﬁve species are present. C. rectirostris, C. liljeborgi, and C. macrurus, which were described from Europe, do not occur in North America at all, even though reported by Brooks (1959).] 7b. Postabdomen shorter and broader, parallel-sided, without any distinct marginal denticles (Fig. 17).....................................Acroperus [Seven species in the world in 1971, with maximal lengths from 0.59 to 0.85 mm. Like Campocercus, the species in Acroperus are not yet adequately described. There is marked conformity in body form and shape and in other morphologic characters, making it very difﬁcult to sort out the species satisfactorily.] 8a(6b). Postabdomen rather slender, elongate, somewhat tapered distally; body high; shell widely open behind (Fig. 18) .....................Kurzia [Five species in world and three in North America, K. latissima being widespread, and K. longirostris and K. polyspina occurring only in the southernmost Gulf states. Length to 0.6 mm.] 8b. Postabdomen broader, elongate, parallel-sided; shell with parallel striae sloping downward posteriorly, and with short longitudinal scratch marks in between (Fig. 19)........................................................................................................................................Alonopsis [Two species in world, the one in North America very similar to the one in Europe (see Kubersky, 1977). Occurs from northern New England into the adjacent Canadian provinces and then into those farther East. Smirnov (1971) transferred the European species to the genus Acroperus, which is not adequately justiﬁed. Maximum length: American 0.91 mm, European 0.85 mm.] 9a(5b). Rostrum long, attenuate, recurved, greatly exceeding antennules in length (Fig. 20).....................................................Rhynchotalona [Only one species listed in world, although there is a second species from North America not yet described. Postabdomen with 2 – 4 stout marginal denticles distally and with an almost continuous row of long, hairlike setae laterally. Length to 0.5 mm. A mud dweller.] 9b. Rostrum rounded, barely or only slightly exceeding antennules in length.........................................................................................10 10a(9b). Seen from above, rostrum very broad, semicircular, wider than body; shell and head strongly reticulated (Fig. 21) ........Graptoleberis [Possibly only one species in world, although the taxonomy is not yet clear. Reported maximum length 0.7 mm. Ventral margin of shell provided with a dense fringe of setae, and generally with two large, sharp, triangular teeth at posterior-ventral corner of shell; postabdomen tapered distally, marginal denticles small, lateral fascicles minute; postabdominal claws small, with minute basal spine. This species functions much like a snail, scraping food off the substrate as it moves along. See Fryer (1968).] 10b. Rostrum more narrowly rounded; shell may be weakly reticulated, but head never is ......................................................................11 11a(10b). Postabdomen elongate and rather narrow; with marginal denticles well developed ..........................................................................12 11b. Postabdomen much less elongate (except in Leydigia); marginal denticles usually well developed, but in some species greatly reduced ............................................................................................................................................................................................13 12a(11a). Dorsal margin of postabdomen straight or slightly convex; marginal denticles very short proximally, increasing in length distally to three very long curved denticles; four median headpores (middle one is divided into two), usually completely separated from one another, or with middle pores joined by a sclerotized ridge (Figs. 21. 1C and 22) ................................................................Oxyurella [Basal spine of postabdominal claw long, slender, attached some distance from base of claw; head and shell generally have a yellowish, hyaline appearance. Five species claimed to occur in the world in 1971. Two species in North America, one being conﬁned to the Gulf states, the other more widely distributed in the East. The common North American species is now separated from the formerly cognate species in Eurasia. See Michael and Frey (1983).] 12b. Dorsal margin of postabdomen distinctly concave; marginal denticles increase in length distal, but never reach large size as in Oxyurella; one median headpore, sometimes completely absent (Fig. 23) ............................................................................Euryalona [A tropical to subtropical genus. Three species in world, the one in America north of Mexico originally described from Sri Lanka. Head high; rostrum broadly rounded, with tip scarcely reaching halfway to ventral margin of shell; comb of setae on proximal half of concave margin of postabdominal claw, increasing in length distally; stout spine on inner distal lobe of trunklimb I, with coarse, rounded tubercles on concave edge. See Daday (1898) and Rajapaksa (1986).] 13a(11b). Postabdomen large, broad, ﬂattened, almost semicircular; armed laterally with long, spinelike setae in groups, those of distal groups projecting far beyond margin of postabdomen; margin of postabdomen with short, ﬁne spinules; three median headpores close together, and with minor pores close-in laterally (Fig. 24) ..........................................................................................................Leydigia [Free posterior margin of shell very long; ocellus as large as compound eye or larger. Twelve species claimed for world in 1971, with maximal lengths from 0.8 to 1.15 mm. Two species are widely distributed in United States, and one tropical species barely gets into Florida.] 13b. Postabdomen smaller, and with shorter setae in lateral fascicles; headpores variable ........................................................................14 14a(13b). Shell strongly sculptured, with longitudinal striae in posterior part and about ﬁve vertical striae anteriorly, roughly parallel to the anterior margin; dorsal edge of postabdomen minutely serrate; about 14 fascicles laterally; no median headpores, but instead there are two comma-shaped thickenings, which presumably contain the minor pores (Fig. 25) ..................................................Notoalona [A genus containing two tropical to subtropical species, only one of which gets into the United States. The headpore conﬁguration is unique among the Chydoridae. Length to 0.44 mm. See Rajapaksa and Fernando (1987a).] 21. Cladocera and Other Branchiopoda 14b. 881 Shell usually not sculptured at all, or if so, then rather weakly; postabdomen usually with well-developed marginal denticles and lateral fascicles; two or three median headpores, most commonly united by a sclerotized ridge, although sometimes completely separated (Fig.26) .......................................................................................................................................................................Alona [This is the largest genus in the Chydoridae, with 52 species claimed in the world in 1971 and with more than 20 species in the United States and Canada. Alona intuitively consists of a number of genera, but these have not yet been deﬁned satisfactorily. FIGURE 22 Oxyruella brevicaudis Michael and Frey, 1983. Skater’s Pond, Monros Co., Indiana. From Michael and Frey (1983). FIGURE 23 Euryalona orientalis (Daday, 1898). Ipiranga, Brazil. From Rajapaksa (1986). FIGURE 24 Leydigia acanthocercoides (Fischer, 1854). Uplandia, Sweden. From Lilljeborg (1901). FIGURE 25 Notoalona freyi Rajapaksa and Fernando, 1987. Homer Lake, Leon Co., Florida. From Rajapaksa and Fernando (1987a). FIGURE 26 Alona bicolor Frey, 1965. Goodwil Pond, Barnstable Co., From Massachusetts. From Frey (1965). FIGURE 27 Dadaya macrops (Daday, 1898). Polonnaruwa, Sri Lanka. From Rajapaksa and Fernando (1982). FIGURE 28 Ephermeroporus acanthodes Frey, 1987. Audobon Park, New Orleans. From Frey (1982b). FIGURE 29 Dunhevedia americana Rajapaksa and Fernando 1987. Glades Co., Florida. From Rajapaksa and Fernando (1987b). 882 S. I. Dodson and D. G. Frey Smirnov (1971) placed the species having just two median headpores in a separate genus, Biapertura. Three of the species in North America, currently named A. afﬁnis, A. intermedia, and A. verrucosa, belong here. Smirnov and Timms (1983) found a much greater proportion of two-pored species in Australia, which do not seem to be closely enough related to be included in the same genus. Length of species in America north of Mexico: two-pored species to 1.05 mm; three-pored species 0.4 – 0.9 mm.] 15a(2b). Headshield not elongated posteriorly; in two-pored species, postpore distance usually considerably less than interpore distance.....16 [Includes the genera Alonella, Dadaya, Disparalona, Dunhevedia, and Ephemeroporus.] 15b. Headshield much elongated posteriorly, extending well beyond the heart to the middle of the back; postpore distance usually greater than interpore distance, sometimes by several-fold (Fig. 1D) ................................................................................................19 [Includes the genera Anchistropus, Chydorus, Pleuroxus, and Pseudochydorus.] 16a(15a). Only one median headpore, or none ................................................................................................................................................17 16b. Two median headpores, well separated and not connected by a sclerotized ridge (Fig. 1D) ..............................................................18 17a(16a). One median headpore in all instars (Fig 27); ocellus larger than eye ........................................................................................Dadaya [A single pantropical species occurring sparingly in the Gulf states. Color dark brown. Length to 0.41 mm.] 17b. One median headpore only in ﬁrst instar; none in later instars (Fig 28); ocellus smaller than eye ................................Ephemeroporus [Postabdomen with long proximal and distal marginal denticles, and with shorter ones in between; labral keel large, with 1 – 4 teeth on anterior margin. A tropical to subtropical genus with many species, few of which have been described to date. See Frey (1982b).] 18a(16b). Postabdomen unique among chydorids, consisting of a much expanded postanal portion, having a series of short spines along dorsal margin and many small clusters of spinules laterally (Fig. 29) ......................................................................................Dunhevedia 18b. Postabdomen of more typical chydorid structure, with pre- and postanal angles and a well-developed series of postanal marginal denticles (Figs. 30 and 31) ............................................................................................................................Alonella and Disparalona [Four species claimed for world, two in the United States, one of which is strictly southern. Length for 0.46 – 0.8 mm.] [The taxon Disparalona rostrata of Europe was formerly in the genus Alonella. Fryer (1986) removed it from this genus because it had a long “sweeping” seta on trunklimb III, which the other species of Alonella were claimed not to posses. Fryer (1971) subsequently transferred Alonella acutirostris to Disparalona, and Smirnov (1971) did the same for Alonella dadayi. Michael and Frey (1984) closely examined a number of species of Alonella and of Disparalona and concluded that the sweeper seta is not an additional seta in a few species but rather is the proximal member of the gnathobasic ﬁlter comb, and that species still in Alonella have the same seta variably developed and not always distinctly separable from the Disparalona species. I know of no characters that will clearly separate the two groups of species, suggesting that the validity of Disparalona is questionable. Disparalona presently contains the species D. acutirostris, D. dadayi, D. leei, all occurring in North America, and Alonella the species A. excisa, A. exigua, A. hamulata, A. nana, and A. pulchella, out of a world total of 12. Maximal lengths of the North American taxa in Disparalona are 0.45 – 0.5 mm, and in Alonella, 0.25- 0.6 (Michael and Frey 1984, Hann and Chengalth 1981).] 19a(15b). Body elongate, somewhat ﬂattened, usually with one or more teeth at the posterior-ventral angle of shell (Figs. 1D and 32) .............................................................................................................................................................................................Pleuroxus [Fifteen species claimed in world, with maximal lengths from 0.4 – 0.8 mm. Seven species in America north of Mexico, of which P. procurvus and P. denticulatus are most frequent and most abundant (Frey 1988c). Lengths from 0.5 – 0.8 mm.] 19b. Body globular, nearly round, seldom with any teeth at posteor-ventral angle ...................................................................................20 20a(19b). Ventral margin of shell anteriorly with hooklike development containing a groove, in which a stout seta with teeth on trunk limb I operates; postabdomen with a cluster of long, slender setae at distal angle (Fig. 33)........................................................Anchistropus [Surface of shell and head reticulated, with meshes having wavy edges. Three species in world, at least two of which are parasitic on Hydra. Lengths 0.35 – 0.46 mm, the North American species being the smallest. See Hyman (1926) and Smirnov (1985).] 20b. Ventral margin of shell without such a hook; marginal setae of shell arise submarginally posteriorly, forming a distinct duplicature (except in Chydorus piger) ...............................................................................................................................................................21 21a(20b). Postabdomen elongate, parallel-sided; postanal portion with about 15 slender marginal denticles; lateral surface with a row of fasciclelike clusters of setae (Fig. 34) ...........................................................................................................................Pseudochydorus [Pseudochydorus presently contains a single species, distributed over much of the world, except possibly in South America. Length to 0.8 mm. It is a scavenger. See Fryer (1968).] 21b. Postabdomen shorter, with a smaller number of marginal denticles, and laterally with crescentic clusters of short spinules, which do not resemble fascicles (Fig. 35) ..............................................................................................................................................Chydorus [A highly complex genus, in which the species are difﬁcult to separate, because they conform so closely to a common morphotype. At least ten species have been found in America north of Mexico. Smirnov (1971) recognized 18 species and many subspecies in the world, ranging in maximum length from 0.32 – 1 mm. The species occurring in North America range in length from 0.41 to 0.78 mm. The type of species of the family, Chydorus sphaericus (sens. str.), quite possibly does not occur in North America at all. See Frey (1980a, 1982a, c, 1987b).] 21. Cladocera and Other Branchiopoda 883 FIGURE 30 Alonella pulchella Herrick 1884. Kremer Lake, Minnesota. From Hann and Chengalath (1981). FIGURE 31 Disparalona leei (Chien 1970) Griffey Lake, Monroe Co., Indiana. From Michael and Frey (1984). FIGURE 32 Pleuroxus procurvus Birge, 1878. Lake Mendota, Dane Co., Wisconsin. 11 September 1987. (KE) FIGURE 33 Anchistropus minor Birge, 1893. Eastern North America. From Birge (1918). FIGURE 34 Pseudochydorus globosus (Baird, 1843). River Sutka, USSR. From Smirnov (1971). FIGURE 35 Chydorus brevlabris Frey, 1980. Salmon Lake, Montana. From Frey (1980a). F. Taxonomic Key to Genera of the Family Daphniidae and Moinidae Fryer (1991) presents a complete and detailed analysis of the functional morphology of genera of the Daphniidae. 1a. First antenna about as long as the width of the head; usually a tooth with two points on the posterior angle of the Bostabdomen (near the claw), (Fig. 58) ........................................................................................................................................Family Moinidae 7 1b. First antenna less than half as long as the width of the head; teeth on postabdomen always single (Figs. 1A, 36 – 40). ...........................................................................................................................................................................Family Daphniidae 2 2a. Ventral margin of carapace rounded and not pigmented ....................................................................................................................3 2b. Ventral margin of carapace straight and usually black........................................................................................................................6 884 S. I. Dodson and D. G. Frey FIGURE 36 Ceridodaphnia cf. quadrangula (O.F. Mueller, 1785). Stewart Lake, Dane Co., Wisconsin. 5 September 1973. (KE) FIGURE 37 Simocephalus (A) S. vetulus Schoedler, 1858. Gardener Pond, UW Arboretum, Dane Co., Wisconsin. 14 May 1987. (KE) (B) S. serrulatus (Koch, 1841). Coliseum Pond, Madison, Dane Co., Wisconsin. 15 May 1987. FIGURE 38 Daphniopsis ephemeralis Schwartz and Hebert 1985. Baseline Pond, Ontario. 19 April 1988. (Coll. P.D.N. Hebert). (KE) FIGURE 39 Megafenestra nasuta (Birge, 1879). Dorsal view. Elk Island National Park, Alberta. 5 August 1978. (KE). Redrawn from Dumont and Pensaert (1983). FIGURE 40 Scapholeberis rammneri Dumont and Pensaert, 1983. Ojibwa, Ontario. 19 April 1988. (Coll. P.D.N. Hebert). (KE) 3a(2a). Adults with a tail spine, at least four times as long as broad and pointed (Figs. 1, 2, 4, 5)......................................................Daphnia 3b. Adults lack a tail spine, or if one is present, it is less than four times as long as broad and rounded ..................................................4 4a(3b). Fourth (distal) segment longer than the third segment on the four-segment branch of the second (swimming) antenna (Fig.38) .........................................................................................................................................................................................Daphniopsis [One species, D. ephemeralis in North America (Schwartz and Herbert 1987c); the genus is reviewed by Hann (1986).] 4b. Fourth segment shorter than the third segment on the four-segment branch of the second antenna ...................................................5 21. Cladocera and Other Branchiopoda 5a(4b). 885 The second segment of the four-segment branch of the second antenna with a spine, the spine about 1 / 4 as long as the second segment (Fig 37) .............................................................................................................................................................Simocephalus [Allozyme studies of Simocephalus suggest there are at least ﬁve species in North America, two more than are recognized using morphological characters (Hann and Herbert 1986). Orlova-Bienkowskaja (1998) recognized at least eight North American species.] 5b. The second segment of the four-segment branch of the second antenna without an apical spine (Fig. 39) .......................Ceriodaphnia [Species of Ceriodaphnia were discussed by Brandlova et al. (1972), but are still poorly know and in need of taxonomic attention.] 6a(2b). Dorsum of headshield with a median oval plate about 20 m in diameter, marked by slightly raised edges (Fig 39) .......Megafenestra 6b. Dorsum of headshield without such a plate (Fig 40) ......................................................................................................Scapholeberis [With the exception of Megafenestra and Scapholeberis (Dumont and Pensaert 1983), and perhaps Daphniopsis (Hann, 1986; Schwartz and Hebert, 1987c), the daphniid genera are in need of revision. Daphnia are being carefully studied by Hebert (Hebert, 1995; Colbourne et al., 1997), who recognizes about 34 species in North America. All of the daphniid genera tend toward the planktonic lifestyle. Megafenestra and Scapholeberis are the least planktonic; they are often found cruising along upside-down, at the surface of small pools. Their carapaces have ﬂattened and turned-in margins (Dumont and Pensaert 1983), which may allow the carapace to serve as a suction cup, as in some chydorids. Simocephalus spends much of its time stuck to aquatic plants, using sticky mucous produced at the back of the neck. Daphniopsis has an extremely restricted habitat; it has been found in North America only in shallow temporary pools in maple forests. Daphnia and Ceriodaphnia are often restricted to open water of lakes and ponds. Scapholeberis and Ceriodaphnia adults are small, reaching about 1 mm, while the other genera produce adults in the range of 1 to 3 mm.] 7a.(1a). Branch of second antenna with four segments appears to have four terminal setae; ocellus (a small black spot just back of the eve) absent in North American species (Fig. 58) ................................................................................................................................Moina 7b. Branch of second antenna with four segments has three setae and a short spine; an ocellus is present (Fig. 59).............Moinodaphnia [These two genera were monographed by Goulden (1968). The species are typically planktonic, but the majority are restricted to small temporary ponds, saline, or alkaline lakes. The habitat is often ephemeral, turbid, and warm. Adult females reach 1 – 2 mm, with one Moina species that is only about 0.5 mm long.] G. Taxonomic Key to Genera of the Family Bosminidae 1a. First antennae attached separately to the head, immobile in female, usually nearly vertical and usually curving somewhat posteriorly (Fig. 42A) ...............................................................................................................................................................................Bosmina (with four subgenera) .........................................................................................................................................................................2 1b. First antennae fused together in basal half, separated and curved outward in distal half (resembling a mermaid), ﬁrmly attached to head (Fig. 41) ...................................................................................................................................................................Bosminopsis 2a(1a). Lateral headpore located within ﬁve pore diameters of the edge of the headshield, just dorsal to the base of the second antenna and at the bottom of the reticulated part of the headshield (Figs. 42C, 43D); teeth of distal pecten (comb of spines) on postabdominal claw as wide as long and resemble an equilateral triangle ..................................................................................................................3 2b. Lateral headpore located more than 20 pore diameters from the edge of the headshield, near the point of articulation of the mandibles, in the reticulated region of the headshield; teeth of distal pecten much longer than wide and resemble needles ................4 3a.(2a). Lateral head pore next to the basal striation at edge of the headshield (Fig. 42C); teeth of the proximal pecten of the postabdominal claw are hairlike, only the largest tooth is perceptibly triangular ........................................................Bosmina (Bosmina) longirostris 3b. Lateral head pore between the two branches of the basal striation; teeth of the proximal pecten of the postabdominal claw are narrow triangles ...................................................................................................................................................Bosmina (Sinobosmina) 4a(2b). Mucro (tail spine) absent in juvenile female (Fig. 43A), or if present, with minute incisions present only along the ventral margin; incisions may be lacking in the adult female; mostly in northern locations ........................................................Bosmina (Eubosmina) 4b. Mucro present in juvenile female, with minute incisions or teeth present only on the dorsal margin (Fig. 42D); mostly in southern locations ......................................................................................................................................................... Bosmina (Neobosmina) [There are just two genera in the family Bosminidae. The key to the subgenera of Bosmina is based on DeMelo and Hebert, (1994). The subgenus B. Bosmina contains the single species B. (B.) longirostris, which until DeMelo and Hebert (1994) had been considered to be very common. However, their revision indicated that B. (B.) longirostris is rare, and that instead most records probably should be for the subgenus Sinobosmina, either B. (S.). leideri or B. (S.) freyi. There are currently four North American species in the subgenus Eubosmina and three North American species of Neobosmina. The subgenera are separated using the shape of the carapace, spines on the postabdominal claw, and the position of the lateral head pores on the head shield (Fig. 42E – G). Bosminids are usually planktonic, with Bosminopsis the least so. Adult bosminids range in size from about 0.3 – 0.6 mm long. Because of their small size, bosminids often are abundant when ﬁsh predation is intense.] FIGURE 41 Bosminopsis deitersi Richard 1895. Singletary Lake, North Carolina. 8 August 1948. (coll. DGF #236. (KE) FIGURE 42 (A) Bosmina (Bosmina) longirostris (O.F. Mueller, 1776). Lake Mendota, Dane Co., Wisconsin. 11 September 1987. (KE); (B) Postabdomen of A (KE); (C) Headshield of A, (KE); (D) Headshield of 20.43A. (KE); (E) Head pore position typical of Bosmina (Sinobosmina), (DeMelo and Hebert 1994); (F) Head pore position typical of Bosmina (Bosmina), (DeMelo and Hebert 1994); (G) Head pore position typical of Eubosmina (Bosmina) and Bosmina (Neobosmina), (DeMelo and Hebert 1994) FIGURE 43 (A) Bosmina (Eubosmina) coregoni (Biard, 1850). Lake Waubesa, Dane Co., Wisconsin, 23 July 1988; (B) Postabdomen of A. (KE); (C) Bosmina (Neobosmina) tubicen Brehm, 1953. Presa Presidente Calles. Aquascalientes, Mexico. 8 October 1980. (KE); (D) Bosmina (Sinobosmina) fatalis Burchardt 1924. Redrawn from Kořínek (1971). (KE) 886 21. Cladocera and Other Branchiopoda H. Key to the Genera of Superfamily Macrothricoidea This key makes extensive use of setae on the second antenna (AII, or swimming antenna). A seta is longer than the segment it sits on, and it resembles a feather, having ﬁne setules. A spine is usually shorter than the segment it sits on, and lacks the setules. The AII has two branches: the dorsal branch with four segments (look for a small segment at the base of one of the branches), and 887 a ventral branch with three segments. For best results, dissect at least one AII off the specimen, and mount it so the branches and setae are spread out. This key is for adult females only, and animals are arranged to facilitate ease of identiﬁcation, not to reﬂect phylogenetic relationships. For further details on morphology, see Smirnov (1992), Dumont and Silva-Briano (1998) and SilvaBriano (1998). For a careful morphological analysis of the macrothricids (and related taxa) see Olesen (1998). 1a. Carapace and head covered with ﬁne hairlike setae (Cactus is South American, Neothrix is Australian; Family Neothricidae) (Fig. 56) .......................................................................................................................................................................Cactus and Neothrix 1b. Carapace not covered with hairlike setae ...........................................................................................................................................2 2a.(1). AII dorsal (four-segmented) branch with no setae except for the three terminal setae.........................................................................3 2b. At least one of the subterminal segments of the AII dorsal branch with a seta (Family Macrothricidae, in part) ................................8 3a.(2). Postabdominal claw with two basal spines, each longer than the width of the claw (Family Ophryoxidae) .......................................4 3b. Postabdominal claw without basal spines, or with small spines much shorter than the width of the claw ..........................................5 4a.(3). The three-segmented branch of AII with a seta at the distal end of each of the two subapical segments (Family Ophryoxidae) (Fig. 45)......................................................................................................................................................................................Ophryoxus 4b. The three-segmented branch of AII with no seta on the two subapical segments (Fig. 46) ..............................................Parophryoxus 5a.(3). The posterio-ventral corner of the carapace with setae about half as long as the carapace height (Family Acantholeberidae) (Fig. 47) .....................................................................................................................................................................................Acantholeberis 5b. The posteroventral corner of the carapace with setae less than 0.1 times as long as the height of the carapace (Family Macrothricidae, in part)........................................................................................................................................................................................6 6a.(5). Dorsal margin of the carapace with a small dorsal protrusion (about the size of the compound eye) .............................Onchobunops 6b. Dorsal margin of the carapace without a rounded protrusion ...........................................................................................................7 7a.(6). Dorsal margin of carapace with a backward-pointing tooth, dorsal margin of ﬁrst antenna with teeth about 0.3 times as long as the width of the antenna a Palearctic genus) (Fig. 48) ...........................................................................................................Drepanothrix 7b. Dorsal margin of carapace without a backward-pointing tooth, margins of ﬁrst antenna smooth or with small teeth less than 0.1 times as long as the width of the antenna (Fig. 53) ...................................................................................................................Bunops 8a.(2). Segments 2 and 3 (counting from the base) of the four-segmented branch of AII with setae ...............................................................9 8b. Only segment 3 of the four-segmented branch of AII with a seta......................................................................................................11 9a.(8). Ventral margin of the carapace with ﬂattened spines (“lanceolate”) that are about twice as long as wide (Fig. 50) .............Lathonura 9b. Ventral margin of the carapace with cylindrical spines that are several times as long as wide...........................................................10 10a.(9). Postabdominal claw with a basal spine that is longer than the width of the claw (an Australasian genus) (Fig. 57).........Pseudomoina 10b. Postabdominal claw without a basal spine (a circum-tropical genus) (Fig. 52) ......................................................................Guernella 11a.(8). Postabdomenal claw with a basal spine that is longer than the width of the claw (this character shown in the drawing in SilvaBriano, 1998, but not in Smirnov, 1992); a single large spine near the dorsal margin of the opening of the anus, the spine nearly as long as the width of the opening (a circum-tropical species) (Fig. 51).................................................................................Grimaldina 11b. Postabdominal claw without a basal spine, and anus opening without long spines ..........................................................................12 12a.(11). Postabdomen with large saw teeth along the margin; ﬁrst antenna with a basal seta and four spinelike setae, all of which are longer than the width of the antenna (Fig. 49) .............................................................................................................................Streblocerus 12b.. Postabdomen with ﬁne hair-like teeth along the margin; ﬁrst antenna with a basal seta and some other arrangement of setae toward the tip...............................................................................................................................................................................................13 13a.(12). First antena with terminal seta that are less than 1 / 4 as long as the antenna, the setae are nearly the same length and thickness, the ﬁrst antenna is cylindrical, not inﬂated toward tip (Fig. 55) .................................................................................................Wlassicsia 13b. First antenna with at least two of the terminal setae that are at least 1 / 3 as long as the antenna, two of the terminal setae are at least 50% longer and thicker than the other setae; the tip of the ﬁrst antenna may be wider at the tip than the base (Fig. 54) ...........................................................................................................................................................................................Macrothrix 888 S. I. Dodson and D. G. Frey FIGURE 44 Ilyocryptus sordidus (Liévin 1848). English lake District. From Fryer (1974). FIGURE 45 Ofryoxus gracilis Sars, 1861. English Lake District. From Fryer (1974). FIGURE 46 Paraphryoxus tubulatus Doolittle, 1909. Anonymous Pond, Maine. From Doolittle (1911). FIGURE 47 Acnatholeberiscurvirostris (O.F. Mueller, 1776). English Lake District. From Fryer (1974). FIGURE 48 Drepanothrix dentata (Eurén, 1861). English Lake District. From Fryer (1974). FIGURE 49 Streblocerus serricaudatus (Fischer 1849). English Lake District. From Fryer (1974). FIGURE 50 Lathonura rectirostris (O. F. Mueller 1776). Near Leningrad, USSR. From Smirnov, after Sergeyev (1971). 21. Cladocera and Other Branchiopoda 889 FIGURE 51 Grimaldina brazzai Richard, 1892. Louisiana. From Birge (1918). FIGURE 52 Guernella raphaelis Richard, 1892. Mayumba, Gabon. From Richard (1892). FIGURE 53 Bunops acutifrons Birge, 1893. Minocqua, Wisconsin. From Merrill (1893). FIGURE 54 Macrothrix rosea (Jurine, 1820). Hook Lake, Dane Co., Wisconsin. 25 October 1972. (KE) FIGURE 55 Wlassicsia kinistinensis Birge, 1910. Kinistino, Manitoba. From Birge (1910). FIGURE 56 Neothrix armata Gurney, 1927. Fig. 491 from Smirnov (1992). FIGURE 57 Pseudomoina lemnae (King, 1853). Fig. 475 from Smirnov (1992). I. Key to the Orders Onychopoda and Haplopoda See Rivier (1998) for details of these predatory cladocerans. 1a. Head cylindrical, more than twice as long as wide, swimming antennae with 36 setae (both branches) (Order Haplopoda, Fig. 60) ............................................................................................................................................................................................Leptodora [Leptodora kindtii (Fig. 60) is the sole species in this group. The adult females are up to 18 mm long. Despite their large size, the animals are so transparent as to be nearly invisible. They are common in the open water of large lakes, and occasionally are found in ponds. L. kindtii are voracious predators on smaller zooplankton.] 890 S. I. Dodson and D. G. Frey FIGURE 58 Moina wierzejskii Richard, 1895. 45 km NE of Las Cruces, New Mexico. 6 July 1984 (KE) FIGURE 59 Moinodaphnia macleayii (King, 1853). Roadside canal, Highlands Co., Florida. 20 July 1960. (Coll. DGF #96). (KE) FIGURE 60 Leptodora kindti (Focke, 1844). Lake Michigan, USA. From Balcer et al. (1984). Drawn by Nancy Korda. FIGURE 61 Bythotrephes cederstroemii Schodler, 1877. Summer female from Lake Karesuando in Norrbotten. From Lilljeborg (1901). Redrawn by KE. FIGURE 62 Polyphemis pediculus (Linnee, 1761). Lake Michigan, USA. From Balcer et al. (1984). Drawn by Nancy Korda. FIGURE 63 Evadne nordmanni (LovÇn, 1836). Summer female from the sea near Dalaro. From Lilljeborg (1901). Redrawn by Cheryl Hughes. FIGURE 64 Podon leuckartii Sars, 1862. Summer female from the sea near Bergen, Norway. From Lilljebory (1901). Redrawn by Cheryl Hughes. 21. Cladocera and Other Branchiopoda 891 1b. Head not obviously cylindrical, swimming antennae with 15 or fewer setae (both branches) (Order Onychopoda) ...........................2 2a (1b). Adult female total length 1 – 2 mm, abdominal process shorter than the body....................................................................................3 2a. Adult female total length greater than 2 mm, abdominal process longer than the body ( Fig. 61, Family Cercopagidae)....................5 3a (2a). Abdominal process slender and about half as long as the body, freshwater species in North America (Family Polyphemidae, Fig. 62) Polyphemus [The single species in North America (Polyphemus pediculus) is often found along the margins of small bog lakes.] 3b. Abdominal process blunt and shorter than half of the body length, estuarine or marine species along the coasts of North America (Family Podonidae) ............................................................................................................................................................................4 4a (3b). Body ends in a short extension of the abdomen, about twice as long as broad (Fig. 63) ...........................................................Evadne 4b. Body ends in a triangular point, but without a distinct abdominal process (Fig. 64) ..................................................................Podon 5a (2a). Swimming antennae with 15 setae (both branches) (Fig. 61) ...........................................................................................Bythotrephes 5b. Swimming antennae with 14 setae (both branches) .............................................................................................................Cercopagis [Bythotrephes longimanus and Cercopagis pengoi are invaders of the Lawrentian Great Lakes, having come from Europe, probably in ballast water. See the Sea Grant web page at http: / /www.ansc.purdue.edu / sgnis / home.htm B. longimanus (probably from a Polish harbor) was ﬁrst seen in Lake Huron in 1984 and C. pengoi (from the Caspian-Black Sea region) appeared in Lake Ontario in 1998. ] OTHER BRANCHIOPODS XIII. ANATOMY AND PHYSIOLOGY OF THE OTHER BRANCHIOPODS A. General External and Internal Anatomic Features Martin (1992) provides a masterful and detailed description of branchiopod morphology, with many drawings and photographs. The non-cladoceran branchiopods are composed of four extant orders (Table I) They differ from cladocerans in a number of ways, including having more pairs of legs and paired compound eyes. The four orders are probably not closely related and are morphologically diverse (McLaughlin, 1980; Fryer, 1987). Members of the ﬁrst order, Anostraca (fairy shrimps), swim with their legs up (Fig. 65), have a pair of large compound eyes, and possess no shell-like covering. Anostracan morphology is described in detail by Linder (1941). Adults are 7 – 100 mm long and have 11, 17, or 19 pairs of thoracic legs along their rather delicate, transparent, and elongate body. The legs are all anterior to two fused genital segments (Fig. 65); hence, they can be called thoracic legs. There are no abdominal legs, but rather a pair of terminal lobes (cercopods, Fig. 67) on the last abdominal segment (telson). The genital segments contain the gonads (which extend into abdominal and thoracic segments). Males have a pair of ventral penes (Figs. 70C and 71). Females possess a medical brood pouch (Fig. 70B). Within the brood pouch are lateral oviducal pouches, a single median ovisac, and shell glands. Males have large and sometimes complex second antennae (Fig. 69A) and sometimes outgrowths from the head (Fig. 66C) or antennae (Fig. 70A). The second antennae are used for grasping females (Fig. 73B) but not for swimming (as in cladocerans and cochostracans). Laevicaudata and Spinicaudata (the two conchostracan orders of clam shrimps, Figs. 75 and 76) swim with their legs down or forward, have a pair of closeset compound eyes (separated, except in Cyclestheria where they are fused), and a carapace that wraps around the entire body (Figs. 75 and 76). Adults are 2 – 16 mm long and have 10 – 32 pairs of thoracic legs covered by the transparent to brown carapace (Fig. 76). In species of Spinicaudata, the carapace closely resembles a clam shell, even having growth lines and umbo (Fig. 81). In Laevicaudata, the carapace is more globular, and only one Siberian) species has even a single growth line. The branched armlike appendages that can be extended from the carapace are the second antennae (Fig. 81); they are used for swimming, as in the cladocera. Notostraca, or tadpole shrimps, are sometimes confused with trilobites by the unwary. These crustaceans swim with their legs down, have a pair of dorsal compound eyes, and possess a broad and ﬂat carapace that covers the head and thorax (Fig. 82). Adults are 10 – 58 mm long and have 35 – 70 pairs of trunk legs partially hidden by their dark green or grown carapace. Immature notostracans have a transparent carapace that is folded along the dorsal midline and encloses the body and legs, making them appear somewhat like a conchostracan. Long ﬁlaments (endites) protruding from either side of the carapace are attached to the ﬁrst pair of thoracic legs (Fig. 82). 892 S. I. Dodson and D. G. Frey FIGURE 65 Polyartemiella hazeni (Murdoch, 1874). Pond #14, 18 July 1973, Point Barrow, Alaska. 18 July 1973. (A) female; (B) male; (C) head of male. (KE) FIGURE 66 Thamnocephalus platyrus Packard, 1879. Pond near Ringold, McPherson Co., Nebraska. 28 June 1972. (Coll. S. Cooper). (KE) (A) Female abdomen from side; (B) male abdomen from below; (C) male head; (D) detail of spines on frontal process. FIGURE 67 Branchinella sublettei Sissom, 1976. (Coll. D. Belk #717). (A) Head of male; (B) male abdomen. (KE) FIGURE 68 Artemia franciscana (Kellogg 1906). Male head. Alkali lake, Owen’s Valley, California. 2 July 1974. (KE) FIGURE 69 Streptocephalus texanus Packard, 1971. Texas. (Coll. F. Wiman). (A) Head of male, second antenna unrolled; (B) Head of male, second antenna coiled. (KE) 21. Cladocera and Other Branchiopoda Endites of the second pair of legs are less ﬁlamentous, and the succeeding legs lack ﬁlaments (Fryer 1988). The ﬁrst pair of legs is used in swimming. Phyllopods posterior to the ﬁrst pair are used for swimming and also walking, digging, and food handling (D. Belk, personal communication; Fryer, 1988). Eggs are carried in brood pouches in the specialized eleventh pair of legs. The anterior lid of the concave brood pouch is derived from the exopodite of the leg and the bottom from the subapical lobe of the leg (Fryer, 1988). The eleventh pair of thoracic legs of males resembles the tenth and twelfth pairs. The gross morphological difference between males and females is that males lack the brood pouch (and ovaries) and tend to be slightly larger than the hermaphroditic “females” (Beaton and Hebert, 1988). Maleless populations are usually (Akita, 1971; Beaton and Hebert, 1988; Longhurst, 1954; Fryer, 1988), but not always (Zaffagnini and Trentini, 1980) composed of hermaphrodites. Zaffagini and Trentini (1980) present evidence that reproduction in maleless populations is by automictic parthenogenesis (i.e., selffertilizing hermaphrodites), not autogamic parthenogenesis as previously suggested (Longhurst, 1954). Beaton and Hebert (1988) found no electrophoretic variation in Lepidurus arcticus and were not able to clarify the role of males in reproduction. The notostracan trunk is not properly divided into thorax and abdomen, because the legs continue past the genital opening on the eleventh segment and there are many more legs than “segments.” The legs gradually decrease in size toward the tail. The body ends in a pair of long thin cercopods (Fig. 82); and in Lepidurus, a posterior extension of the telson (supra-anal plate, Fig. 82A). B. Relevant Physiological Information Branchiopods of the different orders are similar in regard to their physiological adaptations. Thus, the discussion of cladoceran physiology applies in general to the noncladoceran orders as well. Artemia is a model animal for physiologists (e.g., Sorgeloos et al., 1987). Conchostracans, notostracans, and anostracans probably depend more on gills for respiration than do the cladoceran orders (Eriksen and Brown, 1980a). Notostracans and anostracans show the usual correlation between surface area and the rate of oxygen consumption, although in conchostracans, small juveniles have an unusually high VO2 (rate of oxygen uptake), perhaps due to the well-developed gills on the trunk legs. Osmoregulation (maintenance of proper salt concentration in the blood and tissues) is important to 893 these animals, which often live in conditions of extreme or variable salinity. The general mechanisms of osmoregulation are probably similar in all branchiopods (Peters, 1987), but there are many variations on the common theme. Different species, even in the same genus, show differences in internal salt concentrations, degree of osmoregulation, and tolerance of extremes (e.g., Horne, 1966; Broch, 1988). The hemoglobins of Moina (a cladoceran), Cyzicus (Spinicaudata), and Triops (Notostraca) are electrophoretically similar (Horne and Beyenbach, 1974). Further physiological topics, including diapause, respiration, and ion balance are discussed in Section VII. C. XIV. ECOLOGY OF THE OTHER BRANCHIOPODS A. Life History Notostracans begin life as nauplii or metanauplii which hatch out of eggs, often resting eggs. Non-cladoceran branchiopods use a variety of signals to begin development from the egg, including day length, salinity level, temperature, and oxygen concentration. Depending on the species, survival through suboptimal conditions is accomplished by either (or both) diapause (an internal physiological state) or quiescence (slow development resulting from low temperature; Brendonck 1996). There are a dozen or so molts before maturity, and the adults continue molting throughout their life (Fryer. 1988). Anostracans hatch from resting eggs as nauplii or metanauplii. Some species have the egg shell as nauplii (Artemia, Anderson, 1967; Branchinecta, Daborn, 1977a), and some hatch as advanced metanauplii with as many as ten pairs of swimming legs (Eubranchipus, Daborn, 1976). As the metanauplii molt, they add segments and apendages, gradually developing into adults (Fryer, 1983). Adults molt several times. Fertilization normally takes place just after the adult female molts. The brood pouch has a pore through which sperm are injected by one or the other of the penes of a male. [It is not clear how the eggs of Artemiopsis, which lacks a brood-pouch pore, are fertilized.] Fairy shrimp typically carry eggs until they molt. New eggs are released into the ovisac only after mating in all genera studied, except Artemia (Munuswamy and Subramoniam, 1985). Anostracans probably do not store sperm (Wiman, 1979). Most conchostracans hatch from resting eggs as nauplii (Anderson, 1967). The carapace appears in about the third naupliar stage. By successive molts, the juveniles gradually come to resemble adults. 894 S. I. Dodson and D. G. Frey The eggs of most species of large branchiopods enter diapause soon after development commences and become resistant resting eggs (Belk and Cole, 1975). Exceptions include Artemia, in which development may continue in the ovisac and free-swimming nauplii may pass out the brood pouch, and perhaps Cyclestheria hislopi (Spinicaudata), which may exhibit direct larval development similar to cladocera (D. Belk, personal communication). Eggs produced parthenogenetically or sexually have similar diapausing characteristics (Eulimnadia antlei; Belk, 1972). Anostracans, conchostracans, and notostracans of temporary ponds typically have only one generation per wet episode and produce resting eggs. There is some evidence that diapausing eggs of different anotracan species specialize on hatching under speciﬁc rainfall patterns, allowing different species to take advantage of variations in climatic conditions from year to year in the same temporary pond (Donald, 1982; Gallagher, 1996). Thiel (1963) and Belk (1972) raised Triops (Notostraca) and Eulimnadia (Spinicaudata), respectively, in the laboratory without drying the eggs. The eggs of Triops overwinter best if exposed to low oxygen. The eggs of Streptocephalus seali show sensitivity to strong drying conditions. This many limit their distribution, excluding them from ephemeral (temporary) ponds in highly desiccating environments (Belk and Cole, 1975). Artemia can develop directly in permanent lakes. Artemia of Mono Lake, a permanent saline lake in the Great Basin desert of mideastern California, hatch from resting eggs in the spring, resulting in adults that produce a second generation from eggs carried and matured in their brood sacs (Lenz, 1984). The large size achieved by most noncladocerans requires a longer time to first reproduction, which is crucial in ephemeral ponds (Loring et al., 1988). Notostracans (1 – 2 cm long) require 2 – 3 weeks to develop from the egg to mature adults (Rzoska, 1961). Anostracans (1 – 2 cm long) mature in 3 weeks at 15°C (Mossin, 1986) and about 45 days at 4 – 17°C (Modlin, 1982). Successive generations of Artemia are separated by a month or so in permanent lakes (Lenz, 1984). Depending on temperature, Eulimnadia diversa (3 – 4 mm) took 4 – 11 days to develop from the egg to an adult (Belk, 1972). Mature, 1 to 2 mm long cladocerans, which live in ephemeral ponds (e.g., Daphnia, Moina), could produce a clutch of eggs in as few as 2 days at high temperatures (Rzoska, 1961). Thus, cladocerans can mature in the most temporary of ponds, while the larger noncladocerans require ponds that retain water long enough to allow completion of their longer life cyles. These large branchiopods may then disappear as slower-developing predators and competitors become abundant (Sublette and Sublette, 1967; Dodson, 1987; Loring et al., 1988). B. Distribution and Biogeography Because of their vulnerability to ﬁsh predation, the noncladoceran branchiopods typically occur in the absence of ﬁsh (Hartland-Rowe, 1972; Kerfoot and Lynch, 1987). However, in each group, there are a few reports of occurrences in lakes with ﬁsh. Anderson (1974) gives examples of all three groups in lakes. Some anostracans co-occur with ﬁsh in large, deep freshwater lakes where ﬁsh predation is low (Branchinecta in Canada, Anderson 1974; Polyartemia in Sweden, Nilsson and Pejler, 1973). The modern distribution of noncladoceran branchiopods is based on 500 million years of history (Walossek, 1995). A branchiopodlike animal ﬁrst appears in the Upper Cambrian. The major groups of branchiopods date from the Paleozoic, with the exception of cladocerans, which probably diverged from conchostracans in the Mesozoic or Cretaceous, perhaps barely 100 million years ago. Conchostracans are rare north of southern Canada, while anostracans and notostracans are found from arctic islands to Central America. All three groups appear to be especially diverse in the arid southwestern United States. Anostracan diversity in Arizona depends mainly on two general factors: (1) chemical heterogeneity among (mostly temporary) habitats; and (2) thermal variation resulting both from ponds ﬁlling at different seasons and from altitudinal and latitudinal effects (Belk, 1977). It is uncommon to ﬁnd congeneric assemblages within the same pond, although associations of different genera are common (Sublette and Sublette, 1967; Dodson, 1979, 1987, Donald, 1982; Kerfoot and Lynch, 1987); however, the two genera of tadpole shrimp, Triops and Lepidurus, do not appear to co-occur. Conservation ecology is an important issue for the larger branchiopods; hence, a conservation-oriented newsletter, Anostracan News, is devoted entirely to them. As its name suggests, the focus is on fairy shrimp, but it often includes information about large branchiopods in general. Conservation of large branchiopods is an issue because many of the species have restricted ranges with only a few small populations in fragile habitats (such as rock pools in deserts) in danger of loss or modiﬁcation. Several large branchiopod species are listed as endangered species by the U.S. federal government. As careful studies of species continue (for example, Torrentera and Dodson, 1995) ad- 21. Cladocera and Other Branchiopoda ditional species with restricted distribution are being discovered. C. Physiological Adaptations Ponds without ﬁsh usually exhibit characteristics that discourage ﬁsh; that is, the ponds tend to be small, isolated, and often have extreme environments that can be tolerated by branchiopods but not ﬁsh. Physiological factors important to the ecology of large branchiopods include high salinity and low oxygen. Because ﬁshless ponds are typically temporary, large branchiopods also need a strategy, such as diapause, to live through periods when the pond is dry. 1. Salinity and Temperature Some of these habitats are saline, and some species within the anostracan and conchostracan orders show a high tolerance to salinity (Hartland-Rowe, 1966; 1972; D’Agostino, 1980). Artemia salina and Branchinecta campestris are the most tolerant, surviving in waters several times as saline as seawater. Most fairy shrimp, conchostracans, and notostracans tolerate a wide range of salinities; but a few taxa, probably most species of Eubranchipus and some of Streptocephalus and Branchinecta, are restricted to low-salinity water. On the other hand, Artemia are rarely found in water as dilute a seawater (D’Agostino, 1980; Eriksen and Brown, 1980b). The greater salt tolerance of Branchinecta mackini (Broch, 1988) may provide an occasional refuge in time or space from its co-occurring congeneric predator B. gigas. The distributions of Artemia species and populations within species can be limited by their salinity tolerances (Hartland-Rowe, 1972; Bowen et al., 1985; Abreu-Grobois, 1987). The viability of several populations in the A. franciscana (Kellogg) superspecies depended on salinity, the concentration of bicarbonate, and the chloride / sulfate ratio. These differences in salinity requirements between populations may be a factor in the process of speciation. Populations in different types of saline lakes are genetically isolated in nature, because nauplii from one source will die in water from a different type of saline lake (Bowen et al., 1985). Because temporary ponds can often get quite warm, these crustaceans must frequently tolerate high temperatures (Horne, 1971; Hartland-Rowe, 1972; Eriksen and Brown, 1980a – c). Conchostracans do not perform well below 10°C, but they can live for at least a few hours at 30 – 35°C. Fairy shrimp from desert areas tolerate for several hours temperatures as high as 40°C. Several species that tolerate the highest temperatures are eurythermal, in that they can also swim at 895 temperatures near 0°C. Notostracans have thermal tolerances similar to those of fairy shrimp, but there is signiﬁcant variation among taxa. For example, the higher temperature tolerance of Branchinecta mackini provides a temporal refuge from its predator Lepidurus lemmoni (Eriksen and Brown, 1980b). 2. Oxygen In warm climates, the combination of high temperatures and high productivities can lead to low oxygen concentrations during the night, or in the deeper levels of stratiﬁed ponds at any time (Horne, 1971; Loring et al., 1988). Functional hemoglobin has been observed in some notostracans (Triops), conchostracans (Cyzicus), and anostracans (Artemia, Horne and Beyenbach, 1974; Streptocephalus mackini, D. Belk, personal communication). All of the larger branchiopods can regulate their oxygen consumption and live at low oxygen concentrations. In the laboratory, adult conchostracans (Cyzicus californicus) showed the greatest tolerance to low oxygen, surviving down to levels of about 0.6 – 0.7 mg / L (roughly 7 – 9% saturation at 20°C) (Eriksen and Brown, 1980a). Horne (1971) found a natural population of the conchostracan Cyzicus setosa able to tolerate oxygen concentrations of 0.14 mg / L for 2 h with no signiﬁcant mortality. Anostracans (Branchinecta mackini) tolerated oxygen concentrations down to about 1.4 – 2.8 mg / L (roughly 15 – 30% saturation at 20°C) (Eriksen and Brown, 1980b). Notostracans (Lepidurus lemmoni) have a minimum oxygen tolerance similar to anostracans, and show the least ability to regulate oxygen consumption at low oxygen concentrations (Eriksen and Brown, 1980c). Oddly enough, a ﬁeld study of lethal oxygen thresholds of these animals revealed that conchostracans (Eulimnadia inﬂecta) had a lower tolerance than two anostracan species (Eubranchipus moorei and Streptocephalus seali) (Moore and Burn, 1968). The apparently greater tolerance of the anostracans may have been due to the behavioral tendency of anostracans (but not E. inﬂecta) to seek the oxygenated surface of the pond. All three groups show a maximum rate of weight-speciﬁc oxygen consumption at about 25°C (Eriksen and Brown, 1980a – c). The saturation level of oxygen concentration is reduced exponentially by salinity. Seawater holds about 20% less oxygen than freshwater at corresponding temperatures and pressures. More saline waters hold even less oxygen. Broch (1969) suggested that Branchinecta campestris may be limited to waters of lower salinity than Artemia salina, both because of salt tolerances and because Artemia produces hemoglobin, which may be necessary at high concentrations of salt and the correlated low oxygen concentrations. 896 S. I. Dodson and D. G. Frey 3. Diapause In Anostraca, Conchostraca, and Notostraca, the eggs are carried for at least a short period by the adult female. After a few cell divisions, the egg typically enters diapause. This resting egg (actually a developing embryo) has a dark covering (tertiary envelope or shell) and is prepared to survive drying, heat, freezing, and ingestion by birds (Belk and Cole, 1975). Belk (1970) removed the covering of diapausing conchostracan embryos to demonstrate that the shell does not reduce desiccation effects, but may reduce mortality due to abrasion or intense (ultraviolet) sunlight. Signals that break diapause include temperature and oxygen concentration (Hart-Rowe, 1972; Belk and Cole, 1975; Clegge and Conte, 1980; Wiggins et al., 1980; Mossin, 1986). In some anostracans, the ﬁnal signal that breaks diapause may be a combination of high oxygen and an increase in CO2 concentration (Mossin, 1986). In fact, the fastest average hatching time of the European anostracan Siphonophanes grubei occurred at pH of 5.5 (associated with high CO2 concentration). At least one conchostracan, Caenestheria setosa, has a requirement for high temperatures to break diapause; its resting eggs hatch between 20 – 35°C. Hatching in Eulimnadia antelei (Spinicaudata) was inhibited by high salinity and darkness (Belk, 1972). Resistant eggs appear to be an adaptation to temporary habitats and long-distance dispersal (Belk and Cole, 1975). The habit of carrying diapausing eggs probably has two major advantages (Daborn, 1977b). First, the eggs are protected from numerous egg predators, such as other crustaceans and chironomid larvae. Second, the eggs are more likely to be ingested by a bird for long-range dispersal. As with cladocerans and copepods, ingestion of dipausing eggs may be an important means of dispersal. Proctor (1964) raised Artemia, Streptocephalus, Triops, and Cyzicus from feces of mallard ducks, Anas platyrhynchos. Yet, curiously, anostracans show subtle genetic differences among populations, even when close to each other, suggesting that the dispersal rate is actually very low (Boileau et al., 1992). D. Behavior 1. Swimming Behavior Notostracans are detritus feeders and predators, plowing through the superﬁcial sediments to capture anostracans and benthic invertebrates. When feeding, the tadpole shrimp is face down and its many legs moving rhythmically, digging into the sediments, and throwing up a plume of sediment behind the animal. Most anostracans feed on their backs, ﬁltering with rhythmic movements of their legs as they swim through the water. Artemiopsis and Branchinecta also roll over and scrape surfaces with their thoracic legs (Daborn, 1977b). Branchinecta gigas catches and holds smaller anostracans with its spiny phyllopods (Fryer, 1966; Daborn, 1975). Conchostracans swim slowly, spending most of their time skimming along the pond bottom in aquatic vegetation (Martin et al., 1986), or above algal mats (e.g., Sissom, 1980). Notostracans and anostracans will swim away from a person walking up to the edge of a pond; conchostracans show no such response. 2. Mating Behavior Tadpole shrimp are generally considered to be hermaphrodites that need to ﬁnd a mate for exchange of sperm. The immediate goal of notostracan mating behavior is for two individuals to join their eleventh pair of thoracic legs. Male and female gonopores are in the same position at the base of the ﬁrst endopodite on these legs (Akita, 1971), so some bodily contortion is required to deposit spermatozoa into the brood pouch of the partner. Engelmann et al. (1996) reported several populations composed of males and females and suggested that sexual reproduction may be common in notostracans. Most conchostracan species have even numbers of males and females, which are morphologically distinct (Sassaman, 1995). Males have specialized hooks on the ﬁrst (Laevicaudata) or ﬁrst and second (Spinicaudata) pairs of thoracic legs. These hooks are probably used in a similar manner to those of cladocerans; the hooks hold onto the female and position the animals for ventral apposition copulation (Mathias, 1937; Martin and Belk, 1988). In laboratory cultures and in nature, it is common to see a female swimming about with a male holding on to the lower (posterior) margins of her carapace. In some species, females can fertilize themselves. Anostracans typically have two sexes, although in some populations the females reproduce asexually (Browne, 1993; Badaracco et al., 1995). The sexes are dimorphic. Males possess enlarged second antennae, often with long and complex ﬁngerlike appendages. These antennae are used to grasp the female just anterior to her genital segments (just behind the last thoracic leg). Only the second antennae are prehensile. Belk (1984) offered evidence indicating no “holding” role for antennal appendages and suggested none for frontal appendages. Anostracans may swim in tandem for days, especially Artemia and Artemiopsis (Daborn, 1977b). Belk (1984) described the mating behavior of Eubranchipus serratus: “Once a sexually active male Eubranchipus locates another fairy shrimp, he positions himself with his head below the dorsal surface of the other shrimp’s genital segments. From this stationtaking posture (Moore and Ogren, 1962), the male may be able 21. Cladocera and Other Branchiopoda to assess suitability of mates (Wiman, 1981). Assuming the station-taking male is below a female, his next action is to clasp her rapidly just anterior to her genital region and his second antennae. As he grabs her, he extends his antennal appendages, laying them along her back. Holding his body stiff with his phyllopods pressed against the ventral surface of his trunk and ﬂexing only at the neck region, he bats at the female several times with quick ventral movements. As soon as she is grabbed, the female rolls the anterior half of her body into a ball with her head pressed ventrally against her genital region. Her abdomen remains straight. This position is usually held only a second or so. Then the female will either struggle violently and dislodge the male, or she will relax and begin swimming slowly. If the female accepts the male, he too relaxes and begins swimming with her while still clasping her with his second antennae. His antennal appendages remain stretched along her back. The male now attempts intromission, trying from ﬁrst one side, then the other. Once one of his penes is inserted into the female’s ovisac, the male assumes an S-posture. He ceases swimming and his phyllopods make only slight vibration movements. The female may continue to swim or she may lie on the bottom. Disengagement is effected by the female with one or more rapid jerking movements. As soon as copulation terminates, the male resumes normal swimming.” (p. 69) Mating in Streptocephalus mackini was similar to that of E. serratus, except that males took station above rather than below females (Wiman, 1981). Male E. serratus were successful in grasping a female in only about 20% of their attempts (Belk, 1984), whereas S. mackini males mate with “undiscriminating eagerness,” approaching males as well as females and also orienting toward fairy shrimp of different genera (Wiman, 1981). Copulation is terminated by the female, possibly when she releases his penis from her gonopore (Wiman, 1981). Males are sometimes observed being towed along by their penis and one occasionally ﬁnds males with only one functional penis, the other being everted permanently. The spines on anostracan penes may be used to hold the penis inside the gonopore during swimming (Wiman, 1981). The pattern of spines on the penis is a useful taxonomic character (Brendonck, 1995, 1997). Eubranchipus bundyi occurs in April in roadside ditches. Dodson (unpublished) watched four adult males and one adult female that were swimming in several adjacent depressions in a marshy area near Madison, Wisconsin. Each depression was about 20 cm deep, with dead grasses at the bottom. One or two males swam around the edge of each depression, making circles about 20 cm in diameter and keeping at middepth, or about 10 cm most of the time. Although both sexes beat their legs 8 – 10 times / s, males swam at about 1 cm / s, females at 0.1 cm / s. Females swam in long straight lines, changing direction every minute or so with a quick burst of speed. They emerged from grass cover for a few minutes every few hours. A female that came up to the midwater level was followed 897 by a male, who adjusted his swimming rate to hers, and swam with his head a millimeter or so below her uropods. He followed for about 10 s, in, then moved closer. The female escaped with a quick burst of speed, about 10 cm / s for 4 cm. The male showed no further interest. The impression these observations give is that males patrol bits of open water, and females spend most of their time in concealment. When the females desire to mate, they swim upward, ﬁnd a male, mate, and then return to the protection of the bottom. The leklike pattern of male and female fairy shrimp mating behavior may result from their reproductive strategy (Wiman, 1981). Because females product large clutches of eggs while males spend little of their energy budget on gametes, life-history theory predicts that females should show discrimination in mating while males should be active and mate as many females as possible. As a consequence of higher activity levels, males are more vulnerable than females to predators. E. Foraging Relationships In general, anostracans are free-swimming ﬁlter feeders, conchostracans process detritus from surfaces or collect plankton, and notostracans are benthic detritus feeders tending toward carnivory (Kaestner, 1970). There are many records of anostracans eating algae. The ﬁltration mechanisms of the thoracic legs is described by Fryer (1983). Cannon (1933) analyzed the feeding movements of fairy shrimp legs, and assumed they lived mainly on algae. However, Bernice (1971) reported that Streptocephalus dichotomus ate a variety of foods, including large algae (e.g., diatoms and bluegreen bacteria), ciliates, small invertebrates (e.g., rotifers, nematodes, and cladocerans), and crustacean eggs. The diet of Artemia is well known both for natural populations and for laboratory, cultures (D’Agostino, 1980). Artemia feed on coccoid green algae in Mono Lake (Winkler, 1977), and Baird (1850) relayed an account of Artemia catching and eating their own nauplii. Branchinecta gigas, the largest extant anostracan, catches and eats other smaller fairy shrimp, and possibly other small zooplankton and benthos (Fryer, 1966; Daborn, 1975). Several species of Branchinecta and Artemiopsis (Fryer, 1966; Daborn, 1977b) have the habit of rolling over and scraping the pond substrate with their thoracic legs. Several species of Branchinecta have been seen scooping up detritus and then sorting through it. Branchinecta species have a row of short curved spines along the inner distal edge of the legs which are probably used for scraping. Even congeneric species show differences in their feeding behavior. Eubranchipus holmni has spinier legs than E. vernalis and shows more scraping behavior (Modlin, 1982). This 898 S. I. Dodson and D. G. Frey scraping action seems a preadaption for feeding on benthos and catching larger planktonic prey, methods employed by the predaceous B. gigas. Conchostracans feed somewhat like cladocerans, by drawing water into the carapace to remove food particles using the phyllopods (Kaestner, 1970). They probably can remove large particles, such as large algal colonies, small crustaceans, and rotifers. They are able to feed in soft mud. Notostracans feed by plowing along the surface or through muddy sediments. They appear to pump mud posteriorly and probably extract any organic particles. Their diet includes algae, amphibian eggs, smaller crustaceans, insect larvae, anostracans, and tadpoles (Kaestner, 1970; Dodson, 1987). They may be signiﬁcant predators on resting eggs of other aquatic invertebrates such as fairy shrimp and clam shrimp (Daborn, 1977b). Notostracans capture mosquito larvae, which is seen as a beneﬁt to humans, but they are also occasionally pests in rice ﬁelds, eating the young plants off at mud level (Dodson, 1987). Tadpole shrimp, available commercially as dried eggs, can be easily cultured in the laboratory. Horne (1966) cultured Triops in the lab using live fairy shrimp. In the lab, notostracans have been observed to kill and eat goldﬁsh (C. Sassaman, personal communication). F. Population Regulation Cladoceran populations are often controlled by a combination of physical and biological factors, with food limitation and predation being important for several generations. Anostracans, conchostracans, and notostracans typically have one generation each time their habitat appears, in some cases brieﬂy once a year (Belk and Cole, 1975; Loring et al., 1988). A major strategy is to make as many small resistant eggs as possible in the shortest amount of time. These eggs then hatch when the pond ﬁlls again, and the animals typically show rapid growth due to a combination of high temperatures and physiological capabilities specialized for rapid growth (Loring et al., 1988). Population regulation can be accomplished by physiologic factors (e.g., salinity, Broch, 1988). In the laboratory when food is not limiting, Artemia females produced the same total number of eggs with little regard as to whether they are mated early or late in life (Browne, 1982). They accomplished this by having larger clutches closer together when mated late in life. Only at limiting food levels was there a trade-off between adult life span and number of eggs produced. Populations of Artemia in permanent lakes can be reduced by bird predation. Mono Lake, a large saline lake in mideastern California, hosts a dense population of Artemia. The Artemia occupy the epilimnion (about 15 m deep) from early spring to December. Cooper et al. (1984) found that the Artemia population declined rapidly in response to feeding by eared grebes using the lake as a migratory stop in August and September. The Artemia in Mono Lake typically have two generations per year, with a signiﬁcant fraction of the second generation being consumed by grebes. The Artemia population of Mono Lake lives at great enough depths to be protected from predation by most waterfowl, which are surface feeders or shallow divers. However, waterfowl can be important predators of large branchiopods in shallow ponds (Dodson and Egger, 1980), and ﬂamingoes are a major predator of Artemia in shallow tropical ponds and lakes (Hulbert et al., 1986). Anostracans are an important part of the diet of many waterfowl, especially dabbling ducks (Swanson et al., 1985) feeding in midwestern prairie ponds. Artemia in a permanent lake are a signiﬁcant source of food for California gull chicks (Lenz, 1984). G. Functional Role in the Ecosystem Because of their large size and abundance, the large branchiopods living in ephemeral ponds are a major link between primary production and detritus on the one hand and predators (often terrestrial) on the other hand (Loring et al., 1988). Temporary ponds in southwestern North America, especially in physically simple playas or rock pools, provide community ecologists with simple, highly replicated, and easily manipulated natural communities (Dodson, 1987; Loring et al., 1988). These natural ponds are easily mimicked by constructing artiﬁcial ponds for community studies of ephemeral pond branchiopods (Tribbey, 1965). Ephemeral ponds in arid areas are closely linked to the surrounding terrestrial landscape (Loring et al., 1988). The ponds are an important water source for terrestrial animals, which then act as dispersal agents for crustaceans. Primary production both in the ponds and on the pond bottoms when they dry are an important food source for terrestrial animals. The position of Artemia in the food web of a permanent saline lake is discussed by Winkler (1977). H. Toxicology Anostracans, especially species of Artemia and Streptocephalus, are frequently used in tests of the acute or chronic effects of environmental contaminants. The Artemia Reference Center of Ghent is a huge data base, especially for the biology, aquaculture, and toxicology of Artemia. Endpoints of branchiopod 21. Cladocera and Other Branchiopoda bioassays are typically survivorship or fecundity (for example, see Centeno et al., 1993), although more subtle endpoints, such as hatching of cysts (resting eggs) is also used (Crisinel et al., 1994). XV. CURRENT AND FUTURE RESEARCH PROBLEMS Temporary ponds are a chancy habitat to study, because they do not always contain water. However, biologists who are willing to work around the vagaries of these animals and their habitats have an almost unlimited ﬁeld for genetic, ecological, and behavioral studies (e.g., Loring et al., 1988). Areas of great potential include application of molecular techniques to questions of phylogeny and speciation, ﬁeld studies of patterns of life-history evolution, grazing and predator – prey relationships, individual behavior, population dynamics, community structure, and landscape ecology. XVI. COLLECTING AND REARING TECHNIQUES Collecting these animals is easy enough when they are in their adult forms. A large-mesh plankton net, a D-frame aquatic dip net, or even a large-bore pipette is sufﬁcient to catch them. The Eubranchipus illustrated in this chapter were collected with a spoon. A traditional technique is to collect mud from a dried habitat, add water, and rear the animals that hatch from the re- 899 sistant eggs. Rearing is easy. Anostracans and conchostracans eat commercial tropical ﬁsh food. Notostracans can be fed anostracans, earthworms, or dried cladoceran ﬁsh food. Artemia diets are myriad, as are recipes for culture media. The most common culture medium is artiﬁcial seawater, although more specialized media may give better viability (Bowen et al., 1985). XVII. TAXONOMIC KEYS FOR NON-CLADOCERAN GENERA OF BRANCHIOPODS A. Available Keys and Specimen Preparation General keys to North American branchiopod genera can be found in Pennak (1989). More recent revisions of large branchiopod groups include keys for Streptocephalidae (fairy shrimp, Maeda-Martinez et al., 1995) and Lynceidae (clam shrimp, Martin and Belk, 1988). This key applies to those crustaceans that have more than ten pairs of thoracic (or trunk) leaﬂike legs and paired compound eyes. As in the cladoceran orders, the taxonomy of the larger branchiopods in undergoing revision based on studies of morphology (e.g., Brendonck, 1995, 1997, Maeda-Martinez, 1995) and molecular studies (e.g., Wiman, 1979; Browne and Sallee, 1984). The animals in this key can be identiﬁed to genus with a good dissecting microscope, without making dissections. Male or female conchostracans and notostracans may be identiﬁed to genus; males only are keyed for Anostraca. B. Taxonomic Key to Genera of Non-Cladoceran Freshwater Branchiopoda 1a. Body soft and ﬂexible, not covered by a shell (order Anostraca) (for a key to species see Belk, 1975) ................................................3 [Belk and Brtek (1995) prepared a checklist of the global anostracan fauna. They provide a strong argument for conservation of these animals and their habitat.] 1b. Body covered at least in part by a hard shell (Figs. 75, and 82) ..........................................................................................................2 2a(1b). Head and body covered with a clamlike shell that wraps around the legs; the abdomen is enclosed inside the shell (conchostracan orders Laevicaudata and Spinicaudata) (Figs. 75 and 76) .................................................................................................................13 2b. Head and body covered dorsally with a ﬂattened shell; the abdomen is straight and trails out behind the shell (order Nostraca) (Fig. 82) ...........................................................................................................................................................................................20 [Fryer (1988) is an excellent source for notostracan biology and taxonomy.] 3a(1a). Seventeen pairs of thoracic legs (Fig. 65)........................................................................................................................Polyartemiella [Belk and Brtek (1995) list a single species for North America.] 3b. Eleven pairs of thoracic legs ...............................................................................................................................................................4 4a(3b). Abdomen ends in and is bordered by a ﬂattened blade (Fig. 66A, B) .........................................................................Thamnocephalus 4b. Abdomen ends in two blade-shaped appendages (cercopods) (Fig. 67) ...............................................................................................5 5a(4b). Large branched frontal appendage growing from between the second antennae (similar to that of Thamnocephalus, Fig. 66C); when unrolled, frontal appendage is longer than the second antennae .........................................................................Branchinella (in part) [Belk and Sissom (1992) report four species of Branchinella from North America. They suggest that at least two of these species qualify as endangered species, because of habitat destruction.] 900 S. I. Dodson and D. G. Frey FIGURE 70 Eubranchipus hundyi Forbes, 1876. AB Woods Pond, Dane Co., Wisconsin. 27 April 1986. (Coll. K. Parejko). (A) Head of male, antennal appendage unrolled; (B) female; (C) male. (KE) FIGURE 71 Dexteria ﬂoridanus (Dexter, 1953). Alachua Co., Florida. 7 March 1939. From paratypes in US National Museum. (Collection #93537). (KE) FIGURE 72 Artemiopsis stephanssoni (Johansen, 1922). Head of male. (Coll. D. Belk #506). (KE) FIGURE 73 Linderiella occidentalis (Dobbs, 1923). Sulfur Mtn. Pond, Venture Co., California. 2 March 1986. (Coll. S. Copper). (A) Head of male; (B) Male clasping female. (KE) FIGURE 74 Branchinecta coloradensis Packard, 1874. Head of male. Stock Pond in Rock Garden, Grand Co., Utah. 29 October 1986. (KE) 21. Cladocera and Other Branchiopoda 5b. 901 Frontal appendage absent, or if present, unbranched and less than half as long as the basal segment or the second antennae ............6 6a(5b). Lateral spines at the posterior margin of each abdominal segment, spines about 1 / 3 as long as the width of the segment (Fig. 67B) ............................................................................................................................................................Branchinella (in part) 6b. Abdomen without lateral spines .........................................................................................................................................................7 7a(6b). Distal (second) segment of the second antennae a ﬂat triangular blade, the base of the triangle about half of the length (Fig. 68A) ................................................................................................................................................................................................Artemia [Belk and Brtek (1995) list three species for North America. Hontoria and Amat (1992) used a multivariate analysis of 25 different North American populations, and found evidence for only two species. However, studies in the Caribbean (Robert Mayer, personal communication) and Yucatan (Torrentera and Dodson, 1995) suggest the existence of several additional species.] 7b. Distal segment not triangular and thin, instead more or less rounded, but may be ﬂattened toward the tip (do not confuse the distal antennal appendage, which is attached on the basal-medial margin of the second antenna and often carried folded or rolled up) (Fig. 70A) ...........................................................................................................................................................................................8 8a(7b). Antennal appendages present and longer than the basal (ﬁrst) segment of the second antennae .........................................................9 8b. Antennal appendages (projections) on the basal segment, if present, no longer than half of the length of the basal segment of the second antennae ...............................................................................................................................................................................10 9a(8a). Complex (branched) medial process attached at the inner corner of the apex of the ﬁrst segment of the second antennae. The second segment is also attached at the apex of the basal segment, but it is outside (lateral to) the medial process and unbranched (Fig. 69) ....................................................................................................................................................................................Streptocephalus [Maeda-Martinez et al. (1995) give a diagnosis and phylogeny of the thirteen North American species of Streptocephalus.] 9b. Antennal appendage unbranched but with a series of lobes and teeth along at least one margin; antennal appendage attached near the proximal end of the basal segment of the second antenna ..........................................................................................................11 10a(8b). Penis with a stout terminal spine about as long as the eversible (distal) part of the penis; several species have a second antenna with branch-like processes on the second segment (Fig. 70) ....................................................................................................Eubranchipus [Belk and Brtek (1995) list eight species for North America.] 10b. Penis without a terminal spine, distal segment of second antenna without branch-like processes (Fig. 71)..............................Dexteria [According to Belk and Brtek (1995), there is one species in this genus which is closely related to Eubranchipus. The species is known only from its type locality near Gainesville, FL.] 11a(9b). Second segment of the second antenna with one or two sharp projections from the medial surface, about halfway along the segment, and approximately doubling the width of the segment at that point; tip of the second segment tapers to a blunt point (Fig. 72) Artemiopsis [Belk and Brtek (1995) list a single species for North America, restricted to arctic ponds.] 11b. Second segment of second antenna with no projections at middle, but sometimes ﬂattened, curved, or with a knob at the tip.........12 12a(11b). First segment of second antenna with a dorsal-medial projection from the base of the segment about half as long as segment; this is the only projection on the basal segment (Fig 73) ................................................................................................................Linderiella [One species is recognized in North America (Belk and Brtek, 1995), and it is a candidate for endangered species status.] 12b. First segment of the second antenna may be without ornamentation, however, usually there is a projection, bump or patch or row of spines on the medial surface of the segment from about the middle of the segment, or a ventral-medial projection (curving upward) from the base of the segment, or some combination of the above; none of the projections more than half as long a the basal segment (Fig. 74) ....................................................................................................................................................Branchinecta [Belk and Brtek (1995) list 14 species for northern North America, and two from Mexico. Three of the species found in California are candidates for listing as endangered species.] 13a(2a). Dorsal union of the carapace valves a true hinge with a groove (Fig. 75A) (order Laevicaudata) .....................................................19 [Martin and Belk, 1988) provide keys for the family Lynceidae in the Americas.] 13b. Dorsal union of the valves a simple fold (order Spinicaudata) ..........................................................................................................14 14a(13b). Dorsal margin of head with a stalked rounded organ (Fig. 76), and an occipital crest at the posterior end of the dorsal margin of the head .................................................................................................................................................................................................15 14b. Dorsal margin of head with occipital crest only (Fig. 78), may be indistinct.....................................................................................16 15a(14a). Ventral surface of telson at point of articulation of terminal claws with a pine (Fig. 76) ....................................................Eulimnadia [Belk (1989) reviews the North American species of Eulimnadia, recognizing ﬁve of the twelve described species as valid, based on traditional morphologic characters and new information on egg-shell morphology.] 902 S. I. Dodson and D. G. Frey FIGURE 75 Lycneus brachyurus O.F. Mueller, 1785. Dane Co., Wisconsin. (A) Dorsal groove and hinge; (B) lateral view; (C) anterior view showing rostrum shape. (KE) FIGURE 76 Eulimnadia cf. agassizzii Packard, 1874. Hwy 190, Oxaca, Mexico. 12 July 1976. (Coll. P. Mündel #235). (KE) FIGURE 77 Limnadia lenticularis (Linnacus, 1761). Female within shell. Woods Hole, MA. 27 August 1887. (USNM coll. Smith). (KE) FIGURE 78 Leptisthera compleximanus (Packard, 1877). Mexico? (A) Female within shell; (B) Female with one valve of shell removed. (KE) 21. Cladocera and Other Branchiopoda FIGURE 79 Eocyzicus concavus (Mackin, 1939). Proﬁle view of male head. Texas. Copied from Mattox, 1950. FIGURE 80 Caenestheriella cf. setosa (Pearse, 1912). (A) Male within shell; (B) male with one valve removed. (KE) FIGURE 81 Cyzicus californicus (Packard, 1874) Male. Collected east of lordsburg, Hidalgo Co., New Mexico. 13 August 1955. (Coll Lynch). (KE) FIGURE 82 (A) Lepidurus couessii (Packard, 1875). Fish hatchery at Valentine, Cherry Co., Nebraska, Spring 1977. (Coll. D.C. Ashley). (KE) (B) Triops longicaudatus LeConte, 1846. Hwy 385, Philips Co., Colorado. 13 June 1972. (Coll. F. Wiman). (KE) 903 904 S. I. Dodson and D. G. Frey 15b. Ventral surface telson at point of articulation of terminal claws without a spine (Fig. 77).....................................................Limnadia 16a(14b). Rostrum (snout or beak) with a sharp spine (Fig. 78) ........................................................................................................Leptistheria 16b. Rostrum with a spine .......................................................................................................................................................................17 17a(16b). Occipital crest acute, conspicuous ....................................................................................................................................................18 17b. Occipital crest obtuse, inconspicuous (Fig. 79) .....................................................................................................................Eocyzicus 18a(17a). Male and female rostrum terminates in an acute point (Fig. 80) ...................................................................................Caenestheriella 18b. Male rostrum terminates bluntly, anterior margin almost as high as the length of the rostrum (Fig. 81), female rostrum acute .................................................................................................................................................................................................Cyzicus 19a(13a). Ridge running from top of head down center of rostrum unbranched; second thoracic leg of male is similar to the more posterior legs .........................................................................................................................................................................................Lynceus [Martin et al. (1986) provide comparative descriptions of the North American species, and Martin and Belk (1988) review the family Lynceidae in the Americas.] 19b. Head ridge branched toward the rostrum; second thoracic leg of male is modiﬁed different from the more posterior legs .........................................................................................................................................................................................Paralimnetis 20a(2b). Last segment of abdomen (the telson) has a medial extension, the supra-anal plate, between the two long cercopods (Fig. 82A) ............................................................................................................................................................................................Lepidurus [King and Hannert (1998) present molecular evidence of at least ﬁve species of Lepidurus in North America; only four of these have been named.] 20b. Telson is concave between the two cercopods (Fig. 82B) ...........................................................................................................Triops ACKNOWLEDGMENTS Professor Frey, a meiobenthic specialist, contributed the keys for the Bosminidae, Chydoridae, and Macrothricidae and discussion of these families, plus the section on paleolimnology. Professor Dodson wrote the remaining keys and recently revised the key to the macrothricids and related families. Each author modiﬁed the other’s contribution. We are grateful for helpful comments from reviewers, especially James Thorp, Alan Tessier, Brenda Hann, Denton Belk, Carlos Santos-Flores, Henri Dumont, Professor Smirnov, and John Havel. The section on noncladoceran branchiopods could have been written only with the extensive advice of Denton Belk. Many of the ﬁgures for this chapter were drawn by Kandis Elliot (“KE” in the ﬁgure legends). LITERATURE CITED Abreu-Grobois, F. A. 1987. 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Williamson Janet W. Reid Department of Earth and Environmental Sciences Lehigh University Bethlehem, Pennsylvania 18015 Department of Invertebrate Zoology National Museum of Natural History Smithsonian Institution Washington, DC 20560 I. Introduction II. Anatomy and Physiology A. External Morphology B. Internal Anatomy and Physiology III. Ecology and Evolution A. Reproduction and Life History B. Distribution C. Physiological Adaptations D. Behavioral Ecology E. Foraging Relationships F. Population Regulation G. Functional Role in the Ecosystem IV. Current and Future Research Problems V. Collecting and Rearing Techniques A. Collection and Preservation B. Rearing Techniques VI. Identiﬁcation Techniques A. Dissection Techniques for Identiﬁcation B. Characteristics for Distinguishing the Free-Living Freshwater Orders C. Identiﬁcation of the Continental Free-Living Copepoda Literature Cited I. INTRODUCTION The subclass Copepoda H. Milne Edwards, 1840, in the class Crustacea is the largest of the entomostracan classes with over 10,000 known species. Due largely to their high densities in the world oceans, they are estimated to be some of the most abundant metazoans on earth (Hardy, 1970). Copepods are also abundant in terrestrial systems where they may reach densities in excess of 100,000 individuals / m2 in wet organic soils (Reid, 1986). Although of marine origin, copepods may also dominate the fauna of some freshwater systems. For example, in Lake Baikal, which is both the deepest lake in the world and one which contains 20% of the unfrozen freshwater on earth, a single species (Epischura baikalensis) contributes up to 96% of the zooplankton (Huys and Boxshall, 1991). Free-living freshwater copepods can be distinguished from other small aquatic invertebrates by a variety of morphological characteristics. They have a somewhat cylindrical, segmented body with numerous segmented appendages on the head and thorax, and two setose caudal rami on the posterior end of the abEcology and Classiﬁcation of North American Freshwater Invertebrates. 2nd Edition Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved. domen. They possess an exoskeleton, conspicuous ﬁrst antennae, and a single, simple, anterior eye. The deﬁning apomorphy of the Copepoda is the structure of their swimming legs, each pair of which is connected at the base by a “coupler” or “intercoxal sclerite”. The name of the class is from the Greek words kope for oar and podos for foot, derived from the coupler, which apparently increases the energetic efﬁciency of the legs. Here, we follow Huys and Boxshall (1991) in recognizing two infraclasses and 10 orders of copepods. The infraclass Progymnoplea includes the order Platycopioida, while the infraclass Neocopepoda contains the other nine orders in two superorders. The superorder Gymnoplea includes the Calanoida, while the superorder Podoplea includes the remaining eight orders. Three of the 10 orders (Monstrilloida, Siphonostomatoida, and Poecilostomatoida) are primarily parasitic on a variety of ﬁsh and invertebrates, and the great majority are marine. Some poecilostomatoids, such as Ergasilus may have free-living planktonic males and copepodids while the adult females are ectoparasites on ﬁsh. Three other orders (Platycopioida, Misophrioida, and Mormonilloida) are primarily marine free-living 915 916 C. E. Williamson and J. W. Reid species of the benthos or plankton. This chapter will focus on the three orders that contain the widespread, abundant, and primarily free-living copepods (Calanoida, Cyclopoida, Harpacticoida), of which there are over 7750 species. The harpacticoids include approximately 3000 species, 10% of which inhabit freshwaters; calanoids include about 2300 species, 25% of which are freshwater; while there are about 2450 species of cyclopoids, 20% of which are freshwater (Bowman and Abele, 1982). Many cyclopoids are parasitic. The remaining order, Gelyelloida, includes two recently discovered free-living species in the genus Gelyella from subterranean habitats in Europe. Another species has been reported from a single location in North America (Reid, unpublished). Free-living freshwater copepods generally range in size from less than 0.5 to 2.0 mm in length, although some species such as the cyclopoids Macrocylops fuscus and Megacyclops gigas, and calanoids in several genera including Heterocope, Epischura, Limnocalanus, and Hesperodiaptomus can reach lengths of 3 – 5 mm. While the vast majority of freshwater copepods are transparent or a pale gray or brown, colors may range from black to red, orange, pink, purple, green and blue. The brighter colors are often caused by plant pigments such as carotenoids in oil droplets of the copepod. Copepods are common in a variety of aquatic and semiaquatic habitats ranging from moist soils, leafpacks, groundwater, wetlands, and phytotelmata, to lakes, estuaries, and open oceans. Most species are omnivorous to some extent, with foods ranging from detritus and pollen, to phytoplankton, other invertebrates, and even larval ﬁsh. Copepods frequently comprise a major portion of the consumer biomass in these habitats. They play a pivotal role in aquatic food webs both as primary and secondary consumers, and as a major source of food for many larger invertebrates and vertebrates. II. ANATOMY AND PHYSIOLOGY A. External Morphology The generalized copepod body in freshwater systems consists of an elongate, segmented body with an exoskeleton. There is usually a single major articulation that divides the body functionally into an anterior portion, the prosome, and a posterior portion, the urosome (Figs. 1 and 2). In the superorder Podoplea which includes the cyclopoids, harpacticoids, and gelyelloids, the major body articulation (not distinct in the gelyelloids) is between the ﬁfth and sixth thoracic segments (somites of the fourth and ﬁfth legs); while in the superorder Gymnoplea, which includes the calanoids, it is between the sixth thoracic segment (somite of the ﬁfth leg) and the genital segment. The head is fused with the ﬁrst and sometimes second thoracic segment to form the cephalosome. The thorax consists of seven segments, starting with the segment that bears the maxillipeds and ending with the genital segment. The number of segments in the urosome is variable, but usually between three and ﬁve. The prosome thus includes the head and most of the thorax, while the urosome includes the last one or two segments of the thorax and the entire abdomen, except for the terminal caudal rami. Five pairs of jointed appendages occur on the head: the ﬁrst antennae (antennules), second antennae (antennae), mandibles, ﬁrst maxillae (maxillules), and second maxillae (Figs. 1 – 3). The ﬁrst antennae serve many important functions related to reproduction, locomotion, and feeding. They are armed with both chemoreceptors and mechanoreceptors that may aid in the discrimination between mates, prey, and potential predators. The ﬁrst antennae of male copepods are geniculate (have a joint that can bend abruptly) and modiﬁed for grasping the female during copulation (Figs. 1 and 3). In male harpacticoids, cyclopoids, and gelyelloids, both ﬁrst antennae are geniculate, while in male calanoids only the right antenna is geniculate. The second antennae of calanoids, harpacticoids, and gelyelloids are biramous, while those of most freshwater cyclopoids lack an exopod and are thus uniramous. Five or six pairs of well-developed thoracic appendages are generally present. The ﬁrst thoracic segment bears the maxillipeds, while the second through the ﬁfth bear four pairs of morphologically similar, biramous swimming legs which are joined by a coupler. In most freshwater copepods, the somite (or segment) bearing the ﬁrst pair of swimming legs is fused with the head and segment bearing the maxillipeds, forming the cephalosome (Fig. 2). The swimming legs consist of two broad basal segments (the coxa, or coxopod and the basis, or basipod), to which are attached inner (endopod) and outer (exopod) rami of 1 – 3 segments each (Fig. 3). In cyclopoids and harpacticoids the ﬁfth pair of legs is highly reduced. In calanoids the ﬁfth legs are well developed and symmetrical in the female and highly asymmetrical and modiﬁed for grasping females during copulation in the male (Fig. 3). Cyclopoids and harpacticoids have vestigial sixth legs, which are larger in males. In adult gelyelloids the ﬁrst three or four pairs of swimming legs have fused coxae and are highly reduced; the fourth legs are absent in the two European species. The ﬁfth legs are absent, and male gelyelloids have vestigial sixth legs in the form of ﬂaps that cover the genital aperture, while females have no sixth legs. Abdominal appendages are absent in copepods, 22. Copepoda FIGURE 1 Major body types of freshwater copepods: (A) a calanoid, Arctodiaptomus dorsalis male; (B) a cyclopoid Mesocyclops americanus female, grasping a chironomid; (C) epibenthic harpacticoids, Attheyella spinipes, with the male guarding the female; (D) an interstitial harpacticoid, Parastenocaris palmerae, female with egg sac; (E) an undescribed species of gelyelloid, female; and (F) a ﬁrst stage nauplius (NI) of Macrocyclops fuscus [redrawn from Dahms and Fernando, 1994]. 917 918 C. E. Williamson and J. W. Reid FIGURE 2 Generalized body plan of free-living freshwater copepods. Abbreviations: R, rostrum; A1, ﬁrst antenna; A2, second antenna; Md, mandible; Mxl, ﬁrst maxilla, Mx, second maxilla; Mxp, maxilliped; P1 – P4, ﬁrst through fourth swimming legs; P5, ﬁfth leg; P6, sixth leg; GS, genital segment; CR, caudal ramus. The ﬁfth and sixth legs in Gelyelloida, and the sixth legs in Calanoida, are lacking. although the abdomen terminates in two caudal rami that are variously armed with setae and spines. B. Internal Anatomy and Physiology The circulatory system of cyclopoids and harpacticoids consists of a hemocoel with no heart or blood vessels. Blood is circulated by body and gut movements, and respiration occurs through the general body surfaces. Copepods have no gills or other respiratory organs. Calanoids have a more developed circulatory system that includes a dorsal heart with one pair of lateral ostia and one ventral ostium. Blood is carried anteriorly by a short anterior aorta; no other blood vessels are present. The digestive system of copepods includes a mouth, esophagus (foregut), midgut, hindgut, and anus. The midgut may have a saclike diverticulum or cecum, and the anus is located at the posterior end of the urosome. The esophagus and hindgut are chitinized. A variable number of labral glands are located around the mouth. They function in a manner analogous to salivary glands by secreting mucus that mixes with food before entering the esophagus. The overall structure of the alimentary canal and the numbers and types of associated cells may vary with the feeding habits of the species (Musko, 1983). Egested fecal material is surrounded by a peritrophic membrane secreted by cells in the midgut of at least some calanoid and harpacticoid copepods, forming pellets (Gould, 1957; Fahrenbach, 1962). Nitrogenous excretion occurs through antennal glands in naupliar (juvenile) stages, and through maxillary glands in adults. Osmoregulation is accomplished largely by low osmolality of the hemolymph (200 mOsm, Mantel and Farmer, 1983). Some harpacticoids have integumental windows on their dorsal cephalothorax that serve as sites of ion exchange, and aid in osmoregulation (Hosfeld and Schminke, 1997). These windows are fairly common in most freshwater and some estuarine canthocamptids and parastenocaridids, as well as in some species of Halicyclops. They do not appear in marine copepods. With the exception of a few species of harpacticoids (see Sarvala, 1979), reproduction in copepods is sexual. Copulation between male and female followed by union of haploid gametes is required for the formation of a fertile diploid zygote. Females have a paired or unpaired ovary dorsal to the midgut, with paired oviducts extending anteriorly. Diverticuli extend from the oviducts posteriorly to the gonopore on the genital segment. In calanoids the diverticuli join just before entering an atrium. Female calanoids also have a pair of 22. Copepoda FIGURE 3 Copepod body parts and appendages, mainly of a harpacticoid, from anterior to posterior order: R, rostrum; A1, ﬁrst antenna; A2, second antenna; Md, mandible; Mxl, ﬁrst maxilla; Mx, second maxilla; Mxp, maxilliped; P1 – 4, ﬁrst through fourth swimming legs; P5, ﬁfth leg; P6, sixth leg; GS, genital segment; RS, seminal receptacle; and CR, caudal ramus. Other abbreviations: exp, exopod; enp, endopod; and benp, baseopod. Note differences in the structure of the ﬁfth legs of male and female calanoids (lower right and lower left in ﬁgure) showing the modiﬁcation of the male ﬁfth leg for grasping the female during mating. Cyclopoid and harpacticoid ﬁfth legs are smaller, and the basal segment of the harpacticoid P5 is enlarged on the inner margin (middle left of ﬁgure). In the upper right of the ﬁgure the second antennae of two different harpacticoids illustrate the difference in A2 structure with basis and allobasis. 919 920 C. E. Williamson and J. W. Reid seminal receptacles that open into the atrium. Female cyclopoids have a single seminal receptacle which is connected to the gonopores by fertilization ducts. Glandular cells along the terminal end of the oviducts secrete material for the egg shells. Skin glands adjacent to the gonopore secrete the egg-sac membrane. Male copepods have a single dorsal gonad (testis). The vas deferens (one in calanoids, two in cyclopoids) is composed of the seminal vesicle, spermatophoric sac, and ejaculatory duct. Male calanoids have a single gonopore, female calanoids and both male and female cyclopoids and harpacticoids have paired gonopores. Spermatophores are elongate and cylindrical in calanoids and harpacticoids, and more compact and often kidney-shaped in cyclopoids (Fig. 4). The nervous system of copepods is composed of a supraesophageal ganglion (brain) connected to a ventral nerve cord by two large circumesophageal connectives. The ventral nerve cord has several thoracic ganglia. Copepods have a variety of sensory receptors. Freshwater copepods have a simple eye spot that cannot form images; they lack the highly developed compound eyes with lenses characteristic of some marine pontellids (Gophen and Harris, 1981). Most copepods have a frontal organ consisting of a complex of ﬁne sensory hairs and pores located on the anterior tip of their rostrum. The organ, referred to as the sensory pore X organ, includes the organ of Bellonci (Hosfeld, 1996), is connected to the brain by paired nerves and probably functions in chemoreception (Elofsson, 1971; Strickler and Bal, 1973). The structures of several other types of sensory receptors have been investigated in freshwater copepods, and their function established by analogy with other known arthropod receptors. The body surfaces of calanoids and cyclopoids are covered with two types of integumental sensilla:small pegs (sensilla basiconica) and ﬁne hairs (sensilla trichodea). The pegs are thought to be chemoreceptors, while the hairs may serve as both chemoreceptors and mechanoreceptors (Strickler, 1975a). Cyclopoids have rows of small spines located at the joints between the segments of the abdomen, the swimming legs, and the caudal rami. These spines are innervated by proprioceptors that sense the position of the relevant body parts (Strickler, 1975b). The ﬁrst antennae of many cyclopoids and calanoids have setae that are modiﬁed ciliary processes that may serve in mechanoreception (Strickler and Bal, 1973; Friedman, 1980). The mouthparts of diaptomids have numerous contact chemoreceptors, but no mechanoreceptors (Friedman and Strickler, 1975; Friedman, 1980). Experiments in which copepods were fed ﬂavored and unﬂavored polystyrene beads conﬁrm the chemoreceptive abilities of copepods (DeMott, 1986). FIGURE 4 Genital segments with attached spermatophores on females of (A) a calanoid, Arctodiaptomus dorsalis, (B) a harpacticoid, Attheyella spinipes, and (C) a cyclopoid, Metacyclops cushae. Calanoids may bear one to several spermatophores, while harpacticoids usually bear only one, and cyclopoids always bear two. III. ECOLOGY AND EVOLUTION A. Reproduction and Life History Copepods develop from fertilized eggs which hatch into a larval stage called a nauplius (Fig. 1F). There are six naupliar stages (N1 – N6) followed by six copepo- 22. Copepoda did stages (C1 – C6), the last of which is the adult. [Some authors use the term “copepodite” instead of copepodid, but the “ite” ending is more properly used to refer to a part of a structure.] Growth is determinate, and adults do not continue to molt as in cladocerans. The nauplius larva starts out with a somewhat rounded body with three pairs of appendages: the ﬁrst antennae, the second antennae, and the mandibles. During development the body of the nauplius becomes more elongate, additional appendages appear, and existing appendages become more elongate and specialized. A pronounced metamorphosis occurs between the last naupliar and the ﬁrst copepodid instar. Copepodids are morphologically much more similar to the adults, as the body is clearly divided into a prosome and a urosome with caudal rami (Fig. 5). The sexes of adult copepods are dimorphic. The mechanism of sex determination is genetic in some species but not known in others (Wyngaard and Chinappa, 1982). Sexual dimorphism is characterized by differences in the structure of the ﬁrst antennae and the ﬁfth and sixth legs, as well as by the number of urosomal segments and the generally larger size of the females versus the males (see taxonomy section). Sexual dimorphism in body size is more pronounced in the cyclopoids than in the calanoids or harpacticoids. Cyclopoids have a mean female:male body length ratio of 1.44; while for calanoids, this ratio is 1.13 (Gilbert and Williamson, 1983). In some cyclopoids the third and fourth swimming legs are dimorphic, while sexual dimorphism of the swimming legs is most pronounced in harpacticoids where some or all of the ﬁve pairs of swimming legs may be dimorphic. Morphological variation brought about by allometric growth of certain body parts (cyclomorphosis) is subtle and not well understood in copepods. Seasonal variation in adult body size is common, with body size generally being smaller during the warmer summer months; both temperature and food may play a role in this seasonal variation (Reed, 1986; Reed and Aronson, 1989; Lescher-Moutoué, 1996). Some cyclopoids also may have more plumose setae on their caudal rami and swimming legs during the summer (LescherMoutoué, 1996). In a study where 22 different body dimensions were measured, allometric variation was established in Tropocyclops prasinus (Riera and Estrada, 1985). The development time from extrusion to hatching for subitaneous (nondiapausing) eggs is usually between 1 and 5 days. Development time from egg to adult is normally on the order of 1 – 3 weeks, while the adult life span is generally from one to several months. Development times and life spans are much longer at lower temperatures and may vary considerably with 921 species. Some cyclopoids have periods of diapause that may extend their life spans up to 1 – 2 years. Males generally mature more rapidly and have shorter life spans than females. After reaching maturity copepods begin to produce gametes and can mate and produce viable embryos (usually called eggs) within a few days of maturation. Under favorable conditions, a single adult female produces a new clutch of eggs every few days and several hundred eggs over her lifetime. Clutches of 2 – 50 or more eggs may be carried laterally in two egg sacs (cyclopoids), ventrally in a single egg sac (most calanoids and harpacticoids), or scattered freely in the water (certain calanoids such as Limnocalanus and Senecella). Diel periodicities in egg laying have been reported for Mesocyclops ogunnus (Gophen, 1978; the original species determination of M. leuckarti was later corrected to M. ogunnus). Calanoids and harpacticoids may produce either of two types of eggs. The normal mode of reproduction is through subitaneous eggs which hatch within a few days of being extruded. Resting eggs (diapausing eggs) that are capable of extended periods of dormancy may also be produced in many freshwater as well as coastal marine calanoids (Hairston, 1996; Hairston and Van Brunt, 1994). Initial studies on the “egg banks” of diaptomid copepods suggested that these resting eggs could survive several years (De Stasio, 1990), but more recent studies have demonstrated that mean ages of the resting eggs in these egg banks may be several decades, and that some eggs may even remain viable for up to several hundred years (Hairston et al., 1995). Some calanoids, such as Limnocalanus macrurus, may produce only resting eggs in some systems but both resting eggs and subitaneous eggs in other systems (Roff, 1972). Cyclopoids produce only subitaneous eggs, but may enter diapause as copepodid stage II — adult, including fertilized females (Dobrzykowski and Wyngaard, 1993; Naess and Nilssen, 1991). These resting stages may vary from a simple developmental arrest to a fully encysted diapausing stage. In temporary ponds, cyclopoids such as Diacyclops can emerge from diapause within a single day of the appearance of water (Wyngaard et al., 1991). Cyclopoid diapause is considered a true diapause similar to that observed in insects (Elgmork and Nilssen, 1978; Naess and Nilssen, 1991). A few species of harpacticoids may encyst in response to adverse environmental conditions (Sarvala, 1979; Frenzel, 1980). Diapause can be initiated during either the spring and summer or the autumn. The proximate environmental cues that trigger the onset of diapause stages (eggs or copepodids) include overcrowding, photoperiod, and possibly age (Walton, 1985). The ultimate factors that 922 C. E. Williamson and J. W. Reid FIGURE 5 Copepodid and adult stages of calanoid (upper) and cyclopoid (lower) copepods. Secondary sex characteristics are apparent in the preadult stage (ﬁfth copepodid, CV). 22. Copepoda confer a selective advantage on diapausing individuals may be related to the avoidance of adverse environmental conditions, including an increase in predation pressures (Hairston and Olds, 1984; Hairston and Walton, 1986). The amphipod Gammarus lacustris preys on copepod resting eggs in the sediments and may deplete the “egg bank” to the extent that later recovery from adverse conditions such as the presence of ﬁsh predators is not possible (Parker et al., 1996). The termination of diapause may be caused by either endogenous mechanisms, exogenous factors (such as low oxygen concentrations, changes in temperature, light, or physical disturbance), or some combination of these (Elgmork, 1967). Female cyclopoids and harpacticoids can store sperm and thus produce fertile clutches of eggs for extended periods of time in the absence of males (Whitehouse and Lewis, 1973; Hicks and Coull, 1983). Most calanoid copepods, on the other hand, must mate repeatedly in order to continue clutch production. Diaptomids must mate before each clutch is produced (Watras and Haney, 1980; Williamson and Butler, 1987), while Epischura can produce multiple clutches from a single mating (Chow-Fraser and Maly, 1988). Under favorable environmental conditions female diaptomids alternate between gravid (oviducts ﬁlled with mature ova) and nongravid reproductive phases. Females must mate when they are gravid in order to produce viable eggs (Watras and Haney, 1980; Watras, 1983a, b; Buskey, 1998; Lonsdale et al., 1998). Mating behavior is similar for freshwater and marine calanoids (Blades and Youngbluth, 1980; Watras, 1983a; Lonsdale et al., 1998). However, while male marine calanoids can locate females at distances of up to 20 cm with the aid of sex pheromones, diaptomids seem to respond to mates at distances of only a few body lengths (Watras, 1983a). Evidence for sex pheromones in freshwater calanoids is scarce (van Leeuwen and Maly, 1991). Watras (1983a) has described a behavioral sequence for mating in diaptomids wherein the male actively pursues the female (pursuit), grasps the female with his geniculate ﬁrst antenna (precopula 1), grasps the female’s urosome with his chelate right ﬁfth leg (precopula 2), and uses the left ﬁfth leg to attach a cigarshaped spermatophore to the genital segment of the female (copula). The spermatophore is afﬁxed with the aid of a cementlike secretion. During both precopula 2 and copula, the male and female are aligned with their urosomes adjacent and their metasomes extending in opposite directions. It is likely that precopula is accompanied by stroking behavior, as occurs in marine calanoids (Blades and Youngbluth, 1980). The entire mating sequence lasts from one to several minutes. Variations in sexual size dimorphism in calanoids may be important to mating success in some species, but not in others 923 (Grad and Maly, 1988, 1992). Eggs are fertilized upon extrusion from the genital segment. In diaptomids, eggs that are not fertilized are released into the environment and disintegrate rapidly (Watras and Haney, 1980; Williamson and Butler, 1987). Mating behavior is similar in harpacticoid and cyclopoid copepods, but the behaviors of both groups differ somewhat from the reproductive activities of calanoids (Gophen, 1979; Glatzel and Schminke, 1996; Dürbaum, 1995, 1997). Male harpacticoids and cyclopoids are more active swimmers than females, and mates are encountered randomly, with no evidence of sex pheromones. When encountering a female, a male grasps her with both his geniculate antennae; usually her third or fourth legs or urosome are held, but any part of her body will do. A period of high activity follows, during which the pair may jump and turn through the water. While still grasping the female with his ﬁrst antennae, the male works himself into the ﬁnal mating position with his ventral metasome facing the ventral surface of the female’s genital segment so that the two bodies are parallel but facing in opposite directions. The male strokes this area of the female with his swimming legs and, with sharp ﬂexing of the abdomen, deposits a compact spermatophore on her genital segment. Both individuals may remain passive for 5 – 10 s after spermatophore transfer. The entire mating process usually takes from 5 to 20 min (in some cases 2 h or more), after which the male releases the female. In some harpacticoids the male may retain his grasp on the female for up to several hours or even days in what is known as postcopulatory mate guarding (Dürbaum, 1995). During this period the female swims around with the passively attached male presumably preventing access to the female by any other males. Fertilization occurs upon extrusion of the eggs. In some harpacticoids precopulatory mate-guarding has also been observed. This involves the male attaching to copepodid stage females (as early as CI in some species) so that they maximize their chances of being able to mate with a virgin female (Dürbaum, 1997). Interspeciﬁc mating has been observed in some cyclopoid species (Maier, 1995), and hybridization has been reported between two species of Leptodiaptomus (Chen et al., 1997). Feifarek et al. (1983) have shown that there may be a cost to mating for cyclopoid copepods. In a laboratory experiment, female Mesocyclops edax that mated and reproduced throughout their lives exhibited lower survivorship than unmated females. Within the group of females which mated, however, there was no relationship between reproductive output and survival. This suggests that the cost is associated with the mating itself rather than with allocation of resources to reproduction versus survival. 924 C. E. Williamson and J. W. Reid Environmental factors have a great inﬂuence on the life-history characteristics of copepods. Temperature, food availability, and predation are the three factors most often implicated as causes of variations in life-history traits. Adult female body size and interclutch duration are generally inversely related to temperature, while clutch size shows no consistent relationship to temperature (but see Chow-Fraser and Maly, 1991). Clutch size is often positively correlated with female body size (Maly, 1973; Hebert, 1985; Chow-Fraser and Maly, 1991). Food availability has also been demonstrated to be an important factor limiting rate of egg production and body size both in the laboratory and in the ﬁeld (Edmondson, 1964; Elmore, 1983; Jersabek and Schabetsberger, 1995). Some species of diaptomids exhibit high reproduction during winter algal blooms (Vanderploeg et al., 1992a). In some marine copepods ingestion of certain species of dinoﬂagellates can cause production of low-quality sperm that results in poor fertilization and hatching failure of eggs (Ianora et al., 1999). Food limitation can decrease fecundity by either decreasing clutch size, increasing interclutch duration, or both. The highly transparent bodies of many copepods allow one to monitor the presence of ova in the oviducts as well as external eggs carried by females. The proportion of a copepod population that occurs in each of these reproductive phases at one point in time can be used to quantify the relative importance of food and (in calanoids) mate limitation in nature (Fig. 6; Williamson and Butler, 1987). The presence or absence of ova in the oviducts alone are not by themselves, however, a good indicator of reproductive activity (Watras and Haney, 1980). This may be due at least in part to mate limitation and to an increase in the per- FIGURE 6 Four major reproductive phases of clutch-bearing female diaptomid copepods: G, gravid; O, ovigerous; N, neither; B, both. When abiotic environmental conditions are favorable for reproduction, transitions between phases are inﬂuenced by the availability of males and food. Phase B may be absent in species where embryos are released before oocyte maturation. [From Williamson and Butler, 1987.] centage of gravid individuals caused by the need for diaptomids to re-mate before each clutch is produced. Predation may inﬂuence adult body size (Carter et al., 1983), sex ratio (Hairston et al., 1983; Svensson, 1997), body pigmentation, or the timing of diapause events in copepods (Hairston and Olds, 1984; Hairston and Walton, 1986). Some of these life history variations have a deﬁnite genetic component to them and, therefore, represent evolutionary adjustments rather than just simple acclimatory responses (Hairston and Walton, 1986; Wyngaard, 1986). When the invertebrate predator Chaoborus preys upon clutch-carrying diaptomids, the clutches may become detached and thus not be ingested; larger clutches are spared more often than smaller clutches (Svensson, 1996). Life-history events of copepods may also differ among populations. Florida populations of Mesocyclops edax have shorter maturation times than populations of conspeciﬁcs in Michigan (Wyngaard, 1986). These differences seem to be largely genotypic, as evidenced by their persistence through two generations in the laboratory under the same controlled environmental conditions, as well as their persistence during a reciprocal transfer experiment carried out with the two different populations (Wyngaard, 1998). B. Distribution Copepods are found in a wide variety of aquatic environments, ranging from the benthic, littoral, and pelagic waters of lakes and oceans, to swamps, wetlands, marshes, large rivers, temporary ponds, and small puddles. Numerous species are common in interstitial and subterranean systems as well. Copepods are often common in phytotelmata, the small volumes of water that collect in the various parts of plants (Janetzky et al., 1996; Reid and Janetzky, 1996). Copepods are present but less abundant in the ﬂowing waters of streams and rivers. Although most prevalent in aquatic habitats, copepods have been collected from many semiterrestrial habitats such as moist forest soil, leaf litter, and even arboreal mosses (Reid, 1986). Calanoids are primarily planktonic, while cyclopoids and harpacticoids are generally associated with substrates in littoral or benthic habitats, although a few species of cyclopoids are planktonic and may contribute substantially to the zooplankton biomass in many lakes and ponds. Benthic copepods generally do not have pelagic larvae, and dispersal may occur in all developmental stages (Hicks and Coull, 1983). The copepod component of the zooplankton is usually dominated by one or two species of calanoid or cyclopoid copepods. The most common and widely distributed planktonic cyclopoid species in North America 22. Copepoda are probably Mesocyclops edax, Diacyclops thomasi, and Acanthocyclops spp. Species richness in benthic copepods generally shows little or no clear pattern with latitude in the Americas, but the number of genera of Canthocamptidae correlates with latitude (Reid, 1992). The distribution of many copepods seems to be related to temperature. For example, Mesocyclops often dominates in the south and in the warmer months; while in the north and in the cooler months in intermediate climates, Mesocyclops is often replaced by either Cyclops, Diacyclops, or Acanthocyclops. In systems where Mesocyclops does not reach high densities (e.g., Lake Michigan), these other cyclopoids may persist through the summer. The only calanoid genus endemic to North America is Osphranticum. The distribution of planktonic copepods in the northeastern United States indicates that historically some species depended on surface waters along retreating glacial ice fronts for postglacial dispersal; while other species, commonly found in smaller ponds and at high elevations less accessible to glaciers, may have been aided in their dispersal by their capability for diapause (Stemberger, 1995). Glaciation has strong residual effects on the present-day distribution of hyporheic (streambed) cyclopoids. Previously glaciated sites contain fewer species of interstitial specialists and fewer narrowly endemic species, but generalist species have apparently easily reinvaded these areas (Strayer and Reid, 1999). The pH of a system may also inﬂuence the distribution and abundance of copepods, but the effects vary with species. In a study of 20 small lakes in the northeastern United States ranging in pH from 4.5 to 7.2, Mesocyclops edax and Leptodiaptomus minutus were both observed across the full range of pH values, but the former was more abundant at higher pH values while the latter was more abundant at lower pH values (Confer et al., 1983). In this same study Epischura lacustris and Cyclops scutifer were found only at pH values of 5.3 or above. Bioassay experiments showed 100% mortality within 8.5 h for Mesocyclops leuckarti at pH values of 5, or of 9.2 (Ramalingam and Raghunathan, 1982). The distribution and abundance of various copepod species may also vary with lake size, depth, water transparency, color, and the presence of vertebrate and invertebrate predators. The horizontal distribution of copepods may be inﬂuenced by aquatic macrophytes (Gehrs, 1974) or ﬁsh (Carter and Goudie, 1986). In benthic habitats, copepods occur primarily in the top 1 – 2 cm of the sediments, although diapausing animals may be found up to depths of 10 – 20 cm or more. The species richness of copepods in lake sediments is usually between 7 and 25 species, with about equal numbers of harpacticoids and cyclopoids 925 (Strayer, 1985). The Laurentian Great Lakes together harbor 30, mostly benthic species of cyclopoids and 34 harpacticoid species (Hudson et al., 1998). The vertical distribution of copepods within the sediments is primarily dependent upon redox potential, and only a few species can survive anaerobic sediments (Hicks and Coull, 1983). The abundance of harpacticoids in benthic communities tends to increase with larger sediment particle size (Hicks and Coull, 1983). The most common species of harpacticoids are in the cosmopolitan family Canthocamptidae, with the most common North American genera being Attheyella, Bryocamptus, and Canthocamptus. Harpacticoids in the family Parastenocarididae are also abundant in well-oxygenated groundwater and interstitial environments. Common littoral – benthic cyclopoids include Acanthocyclops, Diacyclops, Eucyclops, Macrocyclops, and Paracyclops. C. Physiological Adaptations Copepods face a wide range of ﬂuctuating environmental conditions in littoral, benthic, pelagic, and semiterrestrial habitats that require physiological adaptation. Perhaps the most widespread physiological adaptation of copepods is their ability to respond to seasonally unfavorable environmental conditions by reducing their metabolic rate and entering diapause in either the egg or late copepodid stages (see Section IIIA). Diapause may be induced by changes in temperature (Smyly, 1962) or oxygen concentration (Wierzbicka, 1962). These resting stages are highly resistant to temperature extremes and desiccation. Physiological responses to temperature may vary among species and even sex of the copepod. Surfacedwelling harpacticoid species that are exposed to a greater seasonal variability in food availability and temperature can also better regulate respiration rates with temperature than can species living deeper in the sediments (Gee and Warwick, 1984). The longevity of adult male Megacyclops viridis may be extended under ﬂuctuating versus constant temperature regimes, whereas female longevity is reduced under similar conditions (Smyly, 1980). Copepods are frequently found in the benthic sediments of productive lakes and ponds which become hypoxic or anoxic. While low oxygen concentrations may induce diapause, many copepods still remain active under these rigorous oxygen conditions. A few planktonic species (Mesocyclops edax, Microcyclops varicans) vertically migrate into anoxic hypolimnetic waters for several hours on a daily basis (Williamson and Magnien, 1982; Threlkeld and Dirnbirger, 1986). 926 C. E. Williamson and J. W. Reid Laboratory experiments have demonstrated that adult M. edax can tolerate anoxia for 6 – 12 h at 24.6°C (Woodmansee and Grantham, 1961), while adult M. varicans live for at least 36 h. Cyclopoid nauplii can survive anoxia for less than 1 h at unspeciﬁed temperatures (Chaston, 1969). Benthic cyclopoids may persist for at least four days, and probably indeﬁnitely under hypoxic (25% saturation, 8°C) conditions, but die within 2 – 5 h under anoxic conditions at 10°C (Tinson and Laybourn-Parry, 1985). Females are more tolerant of anoxia than males, and smaller species resist anoxia longer than larger species. A few species of harpacticoids are also known to tolerate anoxia (Hicks and Coull, 1983). In surface waters where oxygen is more plentiful, solar radiation may have lethal effects on copepods (Hairston, 1976; Ringelberg et al., 1984; Williamson et al., 1994). The presence of plant-derived carotenoid pigments such as astaxanthin in the body, low temperature conditions, or nutritional state may reduce these lethal light effects (Hairston, 1976, 1979a – c; Ringelberg, 1980; Ringelberg et al., 1984; Luecke and O’Brien, 1981). The darker color of the pigmented copepods may, however, make them more vulnerable to visually feeding vertebrate predators. Some species seem to have overcome these conﬂicting selective pressures by producing two separate morphs: one which has dark red pigments that increase survivorship under high light conditions, and one with a carotenoid – protein complex that is a pale green in systems where vertebrate predation pressures are high (Hairston, 1976; Luecke and O’Brien, 1981). D. Behavioral Ecology Copepods are active and highly proﬁcient swimmers that exhibit a diversity of behavioral responses to environmental stimuli, ranging from the intensity and wavelength of light to the prospective reward or danger of other approaching organisms. The swimming mode of copepods varies with the species and habitat. Benthic harpacticoids and cyclopoids swim and crawl over substrates in the littoral, benthic, and interstitial habitats. The swimming behavior of planktonic copepods has been investigated in some detail (Rosenthal, 1972; Strickler, 1982) and has been reviewed for cyclopoids (Williamson, 1986). Planktonic cyclopoids use their swimming legs, ﬁrst antennae, and urosome to swim through the water, alternating between an active hop and a passive sink phase. The hop phase lasts about 20 ms and consists of a posteriorly directed thrust of the ﬁrst antennae and a metachronal (sequential, one pair at a time, back to front) thrust of each of the pairs of swimming legs. The hops occur about once every second and during normal swimming will accelerate the copepod to a maximum velocity of about 80 mm / s. The sinking phase is the time between hops during which the copepod remains passive. Many variations on this generalized swimming behavior occur. For example, the hops may have a reduced intensity and increased frequency and appear to be more of a shuttle behavior than a hop and sink. In addition, most cyclopoids swim right-side up, while others such as the small, more herbivorous Tropocyclops, swim upside down. When startled, cyclopoids may exhibit escape responses that consist of continuous, more vigorous hops which may accelerate the copepod up to a velocity of 350 mm / s. Cyclops singularis adults, which are on the order of 2 mm in body length, can move over 10 cm in a single jump — a behavior so pronounced that it allows this copepod to be separated from other species (Einsle, 1996b). The swimming behavior of calanoids is distinctly different from that of cyclopoids. Calanoids spend a majority of their time gliding slowly through the water, propelled by the rapid (20 – 80 Hz) vibration of their feeding appendages. The more carnivorous species, such as Epischura and Limnocalanus, may actively cruise through the water in an upright position (dorsal surface up), while the more herbivorous species tend to hang vertically or at a slight angle in the water with their anterior end up. Every few seconds the gliding motion may be interrupted by a hop that involves the same appendages and motions as observed in cyclopoids. Less frequently, calanoids stop vibrating their appendages and sink passively through the water. Nauplii spend a larger proportion of their time in a stationary position than do adults and copepodids; calanoid nauplii vibrate their appendages for only short periods between passive phases, while cyclopoid nauplii hop less frequently than do copepodids or adults (Gerritsen, 1978). Nauplii sink very slowly, if at all, but can exhibit escape responses which are the fastest recorded for any metazoan relative to body size (364 body lengths / s, 55 mm / s, Williamson and Vanderploeg, 1988). Copepods alter their swimming behavior in response to many diverse environmental stimuli, including the presence of food (Williamson, 1981) and predators (De Stasio, 1993; Ramcharan and Sprules, 1991). Light seems to be a particularly important stimulus in regulating the distribution of copepods. For example, Heterocope septentrionalis exhibits a swarming behavior in which it preferentially aggregates over light-colored substrates (Hebert et al., 1980), and Leptodiaptomus tyrrelli may swarm to densities exceeding 11,000 per liter in response to contrasting substrates (Byron et 22. Copepoda al., 1983). Copepods may also respond to different wavelengths of light in different ways. For example, Hesperodiaptomus nevadensis swims faster in blue light than in red, and individuals that contain large amounts of red carotenoid pigments swim faster than those that do not (Hairston, 1976). Light is also responsible for the distinct “avoidance of shore” (“Uferﬂucht”) exhibited by pelagic copepods (Siebeck, 1969, 1980). The diel vertical migration of planktonic copepods through the water column seems to be driven largely by responses to light, predation, and food limitation (Hutchinson, 1967; Williamson and Magnien, 1982; Neill, 1990). The most commonly observed migration patterns consist of downward migrations of several meters into the deeper, darker strata at dawn, and a similar distance upward into the surface waters at dusk. Reverse migrations downward at dusk have also been noted, particularly in response to invertebrate predation (Neill, 1990; Neill, 1992). Adult females often migrate farther than do either males or juveniles (Burgi et al., 1993; Williamson and Magnien, 1982). The adaptive signiﬁcance of these vertical migrations is thought to be related to the reduction of predation pressures by predators (Zaret and Suffern, 1976; Wright et al., 1980; Neill, 1990, 1992), but vertical migration may also inﬂuence food availability to the predators (Williamson et al., 1989; Williamson, 1993; Makino and Ban, 1998). Changes in vertical overlap between predatory copepods and their prey populations can be quantiﬁed with a predation risk model that couples behavioral responses with ﬁeld distribution and migration patterns of predator and prey (Williamson, 1993). The damaging effects of visible radiation cannot, however, be ruled out as an additional ultimate cause of these migrations. Damaging solar ultraviolet radiation may penetrate to depths of 10 m or more in clear lakes with low dissolved organic carbon concentrations (Morris et al., 1995; Williamson et al., 1996) and thus can cause substantial mortality in copepods that remain in the surface waters throughout the day (Williamson et al., 1994). Interestingly, sublethal concentrations of free cupric ions can reduce the phototactic sensitivity of some estuarine and nearshore marine copepods such as Acartia and Temora (Stearns and Sharp, 1994). Copepods may also respond to changes in the density of food in the environment. Cyclopoid copepods exhibit two distinct types of looping behavior: horizontal and vertical looping (Williamson, 1981). Horizontal loops consist of the copepod swimming in horizontal circles in a normal hop and sink swimming mode. When food densities are high, the frequency of horizontal looping behavior increases and serves to main- 927 tain the copepod in the vicinity of high food densities. Predatory cyclopoids may swim in rapid, tight, vertical loops after contacting individual prey organisms. This behavior aids the copepods in capturing prey and in recovering prey that attempt to escape after an initial attack (Williamson, 1981). Calanoids also alter their swimming behavior in response to the presence of food. Senecella calanoides and Epischura lacustris both increase the frequency of their passive sinking phase in the presence of prey. This behavior may reduce the “noise” levels of the predators and thereby permit them to approach and capture their prey by surprise (Wong and Sprules, 1986). In addition to behavioral responses to food, copepods may also react to predators by altering their behavior. Leptodiaptomus tyrrelli reduces its ﬁltering rate (Folt and Goldman, 1981), and Leptodiaptomus minutus decreases the frequency of its hops (Wong et al., 1986) in the presence of predatory copepods. Similarly, Cyclops vicinus reduces its activity levels when the vertebrate predator Abramis brama (bream) is near (Winﬁeld and Townsend, 1983). These diminished activity levels decrease the ability of both types of predators to detect these small copepods. E. Foraging Relationships The foraging relationships of copepods have received a great deal of attention over the years because of the important intermediate position of copepods between the phytoplankton and ﬁsh in aquatic food chains, and the fact that copepods are so abundant in a wide variety of aquatic ecosystems. Copepods contribute a major portion of the biomass and secondary production in a wide variety of aquatic communities. The importance of copepods in these communities derives largely from their impact on energy and material processing, prey mortality rates, and depression of available resources resulting from foraging relationships. 1. Diet Copepods feed on a wide variety of foods including algae, pollen, detritus, bacteria, rotifers, crustaceans, dipteran larvae including mosquitos, chironomids, and chaoborids, and even larval ﬁsh (Fryer, 1957a, b; Monakov, 1976; Fischer and Moore, 1993; Hartig et al., 1982; Reid, 1989b; Brandl, 1998). Therefore, they occupy three of the four major trophic positions in the food chain: detritivore; herbivore; and carnivore. Most species of calanoids and cyclopoids are omnivorous but selective in their feeding (Adrian, 1991; Williamson and Butler, 1986). Calanoids ingest particles ranging in size from small (a few micrometers) 928 C. E. Williamson and J. W. Reid algae and bacteria to larger algae, microzooplankton, and macrozooplankton (1 mm). Cyclopoids are grasping feeders that generally eat larger foods than calanoids. Their diet may include algae, rotifers, copepods, other crustaceans, oligochaetes, chironomids, nematodes, larval ﬁsh, and a variety of other foods. This creates an interesting situation where cyclopoids can prey on the larvae of their own predators, and may cause signiﬁcant declines in the predator populations (Fischer and Frost, 1997; Fischer and Moore, 1993). Smaller cyclopoids such as Tropocyclops prasinus mexicanus tend to be more herbivorous than larger cyclopoid species (Adrian and Frost, 1992). Gelatinous chlorophytes are usually considered inedible or of low food value for zooplankton, but they can support survival and reproduction in some diaptomids (Stutzman, 1995). Harpacticoids consume microalgae, fungi, protozoa, bacteria, and detritus, and some take larger prey such as nematodes. The algae may include phytoﬂagellates, cyanobacteria, and both epipelic and epiphytic diatoms. Harpacticoids also appear to use dissolved organic matter as a food source (Hicks and Coull, 1983). The microbial communities associated with dead foods may be more important than the foods themselves in the nutrition of harpacticoids. Although carnivory has been reported in harpacticoids, predation seems to be relatively unimportant as a mechanism for obtaining food (Hicks and Coull, 1983). However, Phyllognathopus viguieri is an active predator on soil and plant parasitic nematodes (Lehman and Reid, 1993). The structure of the feeding appendages often correlates with dietary preferences, and both chemoreception and mechanoreception are important in selection of different types of food (Price et al., 1983; DeMott, 1986; Legier-Visser et al., 1986). Diaptomids are highly selective in their feeding and tend to prefer larger, more nutritious algae, or even small invertebrates such as rotifers (Williamson and Butler, 1986; DeMott, 1995a; Vanderploeg, 1994). Selectivity in diaptomids varies with food concentration in a way that is consistent with optimal foraging models (DeMott, 1995b). While diaptomids are capable of ingesting ﬁlamentous and colonial cyanobacteria at substantial rates (Schaffner et al., 1994), they exhibit a distinct avoidance of toxic strains (DeMott and Moxter, 1991); existing data suggest that diaptomids are more sensitive than Daphnia to cyanobacterial toxicity (DeMott et al., 1991). When feeding on ﬁlamentous diatoms or cyanobacteria, diaptomids may bite the ends off, a phenomenon known as “ﬁlament clipping” which may in turn reduce ﬁlament length in natural phytoplankton populations (DeMott and Moxter, 1991; Schaffner et al., 1994; Vanderploeg et al., 1988). Both calanoid and cyclopoid copepods feed on ciliates, although many ciliates have behavioral, morphological, or chemical defenses that reduce their vulnerability to attack, capture, or ingestion (Williamson, 1980; Burns and Gilbert, 1993; Hartmann et al., 1993; Wickham, 1995; Burns and Schallenberg, 1996). Reproduction is enhanced in diaptomids when ciliates are added to algal foods, indicating that the ciliate biomass is assimilated and utilized rather than just ingested (Sanders et al., 1996). Thus diaptomids, which were once thought to use only algal biomass, can also use carbon sources from larger heterotrophs such as rotifers, and from protozoan carbon derived from the microbial component of the foodweb. Diet may vary with the developmental stage, especially in the more carnivorous species. Nauplii and early copepodids are generally more herbivorous. The transition to more predatory feeding occurs in the late copepodid stages in calanoids (Maly and Maly, 1974; Chow-Fraser and Wong, 1986), while in cyclopoids even the earlier copepodid stages may be predatory (Brandl and Fernando, 1986; Williamson, 1986). Adult females are generally larger in size and correspondingly more voracious than adult males or juveniles. Many species are cannibalistic. 2. Feeding Mechanisms The feeding mechanisms of suspension-feeding calanoids appear to be similar for marine and freshwater species. Food is brought in toward the copepod on microcurrents created by rapid vibrations of the second antennae, mandibular palps, ﬁrst maxillae, and maxillipeds (Koehl and Strickler, 1981; Vanderploeg and Paffenhöfer, 1985). Smaller food particles are captured passively and are funneled into the mouth through the setae of the second maxillae (Vanderploeg and Paffenhöfer, 1985; Price and Paffenhöfer, 1986). During passive captures, the vibrations of the feeding appendages continue uninterrupted. Larger food particles are captured actively with an outward ﬂing of the second maxillae followed by an inward squeeze to remove the excess water surrounding the food particle. When microzooplankton such as rotifers and nauplii are brought in on the feeding currents of suspension-feeding diaptomids, the copepods will often attack these small animal prey from a distance with an active thrust response in which the antennae and swimming legs are used to orient and pounce toward the prey (Williamson and Butler, 1986; Williamson, 1987; Williamson and Vanderploeg, 1988). Particles less than 5 m are generally captured passively by diaptomids, while those greater than 50 m are generally captured actively or by attack; in intermediate size ranges, the frequency of active captures increases with increasing 22. Copepoda particle size (Vanderploeg and Paffenhöfer, 1985; Williamson and Vanderploeg, 1988). Many of the larger calanoid species such as Heterocope, Epischura, Limnocalanus, and some of the large species of diaptomids have predatory tendencies and feed on other zooplankton as well as algae. These predators cruise through the water, attack their prey with a pounce, and grasp them with their ﬁrst and second maxillae. If a capture attempt fails, the copepod may swim in a vertical loop to try again to capture the prey (Kerfoot, 1978). Cyclopoid copepods do not create currents to aid their feeding but instead grasp their food directly with their ﬁrst maxillae or, to a lesser extent, with their second maxillae and maxillipeds. These appendages push food between the mandibles. By oscillating rapidly during feeding bouts, the mandibles tear prey into pieces and stuff them into the esophagus (Fryer, 1957a). Cyclopoids detect their prey with the help of mechanoreceptors on their ﬁrst antennae. Prey which generate much disturbance as they swim can be detected at distances of several millimeters. Cyclopoids actively orient and attack prey with considerable precision (Kerfoot, 1978). Larger prey such as cladocerans, copepods, and chironomids may be handled for 30 min or more before ingestion (Fryer, 1957a; Gilbert and Williamson, 1978; Williamson, 1980). Small, generally less active prey such as algae, protozoans, rotifers, and nauplii may be detected only after contact, and are generally eaten whole. Cyclopoid nauplii use their mandibles and second antennae to capture and ingest food (Monakov, 1976). Harpacticoid copepods are primarily surface-feeders that use their mouthparts for scraping food from a diversity of substrates. Suspension-feeding has been reported in two primitive marine families, the Longipediidae and the Canuellidae (Hicks and Coull, 1983). In surface-feeding, the second maxillae and second antennae grasp the prey while the maxillipeds aid in anchoring the copepod to the substrate. Once the food is grasped, the ﬁrst maxillae shred and stuff the food into the vibrating mandibles. The second maxillae and maxillipeds are not used in food handling. Harpacticoid nauplii have modiﬁed second antennae that are employed to grasp, masticate, and push food into the mouth (Fahrenbach, 1962). The mandibles of nauplii serve only for locomotion and not for feeding. Suspension-feeding harpacticoids feed by lying on their dorsal or lateral sides and vibrating their ﬁrst and second maxillae and mandibles. These vibrations create feeding currents that bring water and food in toward the mouth and out posteriorly along the ventral surface of the copepod. Some harpacticoids can use both surface-feeding and suspension-feeding mechanisms (Hicks and Coull, 1983). 929 3. Feeding Rates Feeding rates are usually expressed quantitatively as either clearance rates (expressed as volume of water cleared of food per unit time) or ingestion rates (food items ingested per copepod per unit time). Ingestion rates generally depend upon prey density below a saturation food density referred to as the incipient limiting concentration; above this density there is no further increase in ingestion rate. Clearance rates are density-independent below this saturation food concentration and decrease above it. Feeding rates are calculated based on the exponential equations ﬁrst applied to zooplankton feeding by Gauld (1951). The standard experimental design is to ﬁll a number of experimental vessels with either food alone (controls) or food and copepods (experimentals) and then incubate them for a period of time on a rotating wheel to prevent settling and minimize patchiness of the food and copepods. Feeding rates can then be calculated as follows: Ingestion rate (I) 5 Clearance rate (F) Dct 2 Det TN (1) V(ln Dct ln Det) TN (2) where Dct and Det are the food densities in the control and experimental vessels at the end of the incubation, respectively, T the duration of the experiment, and N the number of copepods per experimental vessel. The average food density to which the copepods are exposed during the experiment is then: d Det Deo V(ln Det ln Deo) (3) where d is the average food density, Det and Deo are the ﬁnal and initial food densities in the experimental vessels, respectively, V the volume of the experimental vessel, and I Fd. An alternative method for estimating feeding rates is to add different numbers of predators to the experimental vessels and use regression analysis to determine clearance rates (Lehman, 1980; Landry and Hassett, 1982). This method assumes that changes in the experimental vessels are proportional to the number of predators per vessel and to the incubation time according to the following equation: ln Det Dct FNT (4) This relationship is derived from the conventional population growth rate equation (see Eq. 5). If Det /T is plotted against N, the slope of the regression relationship is the clearance rate (F), the y intercept is an estimate of ln Deo, and standard regression statistics can be used to determine the statistical signiﬁcance of the 930 C. E. Williamson and J. W. Reid regression relationship as well as the percentage of the variation in prey densities that can be attributed to predation (or, how good was the experiment?). An assumption of both of the above methods of estimating feeding rates is that Dct Deo , or, in other words, that prey birth rates as well as nonpredatory death rates were minimal. Gut content analyses can also be helpful in distinguishing the diet of copepods. Copepods can be squashed between a coverslip and a microscope slide for a crude assessment of gut contents. A more quantitative analysis can be obtained by dissection. A pair of ﬁne minuten needles (insect pins) secured to the tips of applicator sticks or glass pipets can be used to tease the metasomal gut tube out of preserved copepods. The gut is placed in a very small drop of water, covered with a coverslip, and examined under a compound microscope before and after the addition of 5% sodium hypochlorite (commercial bleach) (Williamson, 1984). Clearance rates of predatory calanoids are generally in the range of 80 to 180, but values have been reported as high as 630 mL per predator per day, for Hesperodiaptomus shoshone feeding on calanoid nauplii at 20°C (Maly, 1976) and over 1,000 mL per predator per day, for Heterocope septentrionalis feeding on Daphnia pulex at 15°C (Luecke and O’Brien, 1983). Ingestion rates averaging as high as 18.2 prey per predator per day have been reported for Hesperodiaptomus shoshone feeding on various crustacean prey (Anderson, 1970); while rates of up to 54 prey per predator per day have been observed for Heterocope septentrionalis feeding on Bosmina longirostris at 15°C (O’Brien and Schmidt, 1979). The benthic harpacticoid Attheyella reduced bacterial densities in an Appalachian headwater stream by up to 58%; the greater reductions were observed under lower ﬂow conditions (Perlmutter and Meyer, 1991). The feeding rates of suspension-feeding calanoids vary widely and probably depend largely on dietary preferences and available food. Many investigators have obtained clearance rates of only a few milliliters per copepod per day or less for certain algae, and calanoids are thus often thought to have clearance rates that are much lower than similarly sized planktonic cladocerans (Wetzel, 1983). However, when offered high quality algae as food, diaptomids ﬁlter 19 – 25 milliliters per copepod per day, and weight-speciﬁc rates are comparable to or greater than those of cladocerans (Bogdan and Gilbert, 1984; Vanderploeg et al., 1984; Williamson and Butler, 1986). Although most of the smaller diaptomids have historically been considered to be herbivorous, clearance and ingestion rates of diaptomids on rotifers may be ﬁve to six times greater than those for algae (Williamson and Butler, 1986). The presence of algae reduces the predation rate of suspension-feeding diaptomids (Williamson and Butler, 1986), but does not similarly affect more predatory species such as Epischura (Wong, 1981). In general, diaptomids feed more selectively than Daphnia (Richman and Dodson, 1983; DeMott, 1988, 1989; Vanderploeg, 1994). Cyclopoid ingestion rates are density-dependent and may reach 42 prey per predator per day at prey densities approaching 1000 prey / L (Williamson, 1984, 1986). Clearance rates may exceed 150 mL per predator per day (Williamson, 1983b). Ingestion rates, clearance rates, and prey selectivities may be inﬂuenced by hunger levels of the predator, water temperature, and the size, morphology, behavior, and density of the prey (Williamson, 1980, 1986; Stemberger, 1986; Roche, 1987). F. Population Regulation 1. Factors Controlling Population Density Estimates of the per capita growth rates (r) of copepod populations vary from less than 0.1 per day to as high as 0.4 per day (Allan, 1976; Hicks and Coull, 1983). Doubling times (ln 2 / r) are generally on the order of 1 to 2 weeks but may be less than 2 days under optimal conditions. These high reproductive rates generate pronounced density oscillations in nature. These oscillations, in conjunction with the 12 distinct life-history stages, a small body size, and short generation times, make copepods good subjects for population studies. Several hypotheses have been proposed to explain the observed oscillations in copepod populations. The most compelling of these include food limitation, temperature, and predation. Mate limitation may also be important for diaptomids that must mate before the production of each clutch (Williamson and Butler, 1987). The relative contributions of each of these factors vary between systems and are difﬁcult to separate in nature. The physiological importance of temperature in controlling rates of growth and development has been demonstrated in numerous copepod species. Gamete maturation, egg development, clutch production, and instar development rates are all temperature-dependent (Watras, 1983b; Hicks and Coull, 1983). In addition, temperature may determine the seasonal timing of reproductive activity. A recent review of temperature responses of freshwater zooplankton indicates that many species of copepods are cold-water species potentially sensitive to climate warming (Moore et al., 1996). Within the temperature range that permits reproductive 22. Copepoda activity, food availability may limit the rates of reproduction and development of copepods by prolonging interclutch duration and reducing clutch size (Woodward and White, 1981; Elmore, 1982, 1983). Response to food limitation may be quite rapid in some species (Williamson, et al., 1985). The differential response of the reproductive phases of copepods to food density, mate density, and temperature may permit the analytical separation of food and mate limitation from temperature effects in some diaptomid copepods (Williamson and Butler, 1987). The transitions between reproductive phases are largely regulated by food and mate availability and are independent of temperature; hence, the proportion of adult females in the different phases will reﬂect which factors are limiting (Fig. 6). Food limitation can also be examined with an index that quantiﬁes both lipid storage and reproductive condition (Vanderploeg et al., 1992b). This latter index is similar to that developed for marine copepods (Marshall and Orr, 1952) and freshwater cladocerans (Tessier and Goulden, 1982). Optical – digital methods have also been used to quantify seasonal changes in lipid reserves in diaptomids (Arts and Evans, 1991). They are much simpler than, and compare favorably with, more expensive chemical methods of analysis. The response of copepods to food and mate limitation will vary with species and environmental conditions, and all of these indices must therefore be applied only with an adequate understanding of the reproductive physiology of each species (Jersabek and Schabetsberger, 1995). In many populations the nauplii are the “bottleneck” stage that exhibits the highest mortality rate in nature (Confer and Cooley, 1977; Feller, 1980). The small size of the nauplii relative to copepodids may result in an increased vulnerability to both starvation (due to higher weight-speciﬁc metabolic rates) and invertebrate predation, and consequently higher mortality rates for the nauplii (Williamson et al., 1985). The relative abundance of calanoid versus cyclopoid copepods may change with the trophic status of an ecosystem. Several investigators found that as system productivity rose, the abundance of cyclopoid copepods and cladocerans increased while calanoid abundance either decreased or varied independently (Byron et al., 1984; Pace, 1986; Stemberger and Lazorchak, 1994). Feeding and respiration rate experiments with Daphnia and diaptomids indicated that food quality may also play an important role in the relative abundance of calanoids versus cladocerans (Richman and Dodson, 1983; Lampert and Muck, 1985). Richman and Dodson (1983) suggested that calanoids will predominate when either food quality is low or when food density is either extremely high or extremely low. 931 Lampert and Muck (1985) suggested that diaptomids are better adapted to low, ﬂuctuating food densities than is Daphnia, because the former allocates more energy to storage for survival while the latter puts its resources into reproduction under these food-limiting conditions. There is some evidence that the threshold food level for survival is lower for calanoid than for cyclopoid nauplii, and that this “bottleneck” stage may be important in regulating the relative abundance of these two taxa (Santer, 1994). Enclosure experiments in 450 L tanks indicate that the interactions between cyclopoids and calanoids are complex and may involve predation on calanoids by cyclopoids, and depression of food resources for cyclopoid nauplii by calanoids (Soto and Hurlbert, 1991). Predation may also inﬂuence copepod population densities. Cannibalism is common among the more carnivorous species (Gabriel, 1985), and both vertebrate and invertebrate predators prey on copepods. The inconsistent relationship between calanoid abundance and lake trophic status discussed above may be due to invertebrate predation on diaptomids (Edmondson, 1985), or possibly predation by the more carnivorous cyclopoids on calanoids (Soto and Hurlbert, 1991; Adrian, 1997). Other invertebrate predators, including predatory copepods and larvae of the phantom midge Chaoborus, may inﬂict substantial mortality on copepod populations (Luecke and Litt, 1987). The predatory cladoceran Bythotrephes cederstroemi was introduced into the Laurentian Great Lakes from Europe in the 1980s, and has spread to nearby smaller lakes as well. This exotic species preys on both adult copepods and nauplii; and, while copepods seem to be some of the less-preferred prey (Vanderploeg et al., 1993), some cyclopoids such as Mesocyclops edax and Tropocyclops prasinus mexicanus have either declined or disappeared in lakes when Bythotrephes invades (Yan and Pawson, 1997). Vertebrate predators, including ﬁsh, birds, and larval and adult salamanders, may inﬂuence the distribution and abundance of copepods in lakes (Dodson and Egger, 1980; Zaret, 1980; Carter and Goudie, 1986). Larger species such as Hesperodiaptomus may be excluded from lakes when ﬁsh are introduced (Donald et al., 1994). Vertebrate predators may also be instrumental in the induction of diapause (Hairston and Walton, 1986) or diel vertical migrations (Williamson and Magnien, 1982), and both vertebrate and invertebrate predators may contribute to skewed sex ratios in copepod populations on which they prey (Maly, 1970; Hairston et al., 1983; Blais and Maly, 1993). Heavy organic and nutrient loading and associated reductions in dissolved oxygen may differentially reduce populations of different species of benthic 932 C. E. Williamson and J. W. Reid harpacticoids, as well as reduce the abundance of harpacticoids relative to other meiobenthos (Särkkä, 1992). 2. Methods for Analysis of Populations The instantaneous per capita birth rates, death rates, and growth rates of copepods can be estimated using the egg ratio technique developed by Edmondson (1960) and Edmondson et al. (1962), as modiﬁed by Paloheimo (1974). The growth rate of the population (r) is estimated from the population densities of adult females at time zero (N0) and time t (Nt) with the following equation: r ln Nt ln N0 t (5) The birth rate (b) can be estimated from the egg ratio (E) and the development time (D) from egg to adult as: b ln (E 1) D (6) where E is the number of female eggs in the population (assume a 1:1 sex ratio) divided by the number of adult females in the population. The death rate (d) can then be estimated from: dbr (7) These estimates of r are for the midpoint between the two sampling dates while the estimates of b are for the given sample date. Therefore, when estimating d from Eq. 6, the values of r should be linearly interpolated for the given date (Wyngaard, 1983). These methods assume a constant age structure and continuous egg laying in the populations. Estimates of r with this egg-ratio method are best applied to populations with shorter developmental stages such as those found at higher temperatures. An alternative approach which requires much greater resolution of the developmental stage durations is cohort analysis (Hairston and Twombly, 1985). A careful analysis of the allocation of effort between and within sampling stations as well as between subsamples is critical in order to maximize the accuracy of population estimates (Nie and Vijverberg, 1985). The variance associated with estimates of per capita birth, death, and growth rates can be obtained with either jackknife or bootstrap techniques (Meyer et al., 1986). G. Functional Role in the Ecosystem Copepods make up a major portion of the biomass and productivity of freshwater systems. In Mirror Lake, New Hampshire, copepods contributed about 55% of the total zooplankton biomass over a three year period (Makarewicz and Likens, 1979). Cyclopoid copepods may contribute up to 77% and calanoid copepods up to 49% of the macrozooplankton biomass in some lakes (Pace, 1986). Benthic samples in a second-order stream in North Carolina revealed biomass to be on the order of 22 mg dry mass/ m2, equivalent to densities of 3000 – 18,000 individuals / m2, and cyclopoid densities of about 100 – 1600 individuals / m2 (O’Doherty, 1985, 1988). In the benthic communities of lakes, it is not uncommon to ﬁnd densities of copepods in excess of 10,000 and, occasionally, up to even 70,000 individuals / m2, with a biomass of 50 – 70 mg dry weight individuals / m2 (Strayer, 1985). In addition to their numerical contribution, copepods occupy an important intermediate position in aquatic food chains (Kerfoot and DeMott, 1984). Their generally omnivorous habits make them important in energy transfer up the food chain; they also have the potential to regulate the structure of phytoplankton and zooplankton prey assemblages. By packaging bacterioplankton, phytoplankton, and microzooplankton prey into larger particles (their own bodies) copepods may actually increase food-chain efﬁciency in spite of the energy lost by the additional intermediate trophic level (Confer and Blades, 1975). Copepods may also contribute to phosphorus regeneration in lakes (Bowers, 1986). As predators, copepods may have a substantial impact on their prey populations. Predatory calanoids and cyclopoids are intense, selective predators that can alter the distribution and morphology of their prey. Mesocyclops edax, an important cyclopoid predator in the plankton, may crop up to 24% of its prey populations per day (Brandl and Fernando, 1979; Williamson, 1984). Populations of even some of the smaller suspension-feeding diaptomids have the potential to clear the rotifers from a volume of water equivalent to the entire volume of the lake in a single day (Williamson and Butler, 1986). Cyclops vicinus can prey on the rotifer Synchaeta at rates of up to 37 Synchaeta per day, rates that suggest that this cyclopoid may be responsible for declines in this rotifer in Lake Constance in the spring (Plassmann et al., 1997). Cyclopoid copepods prey selectively on, and may selectively inhibit the growth and reproductive rates of, smaller cladoceran species as well as juveniles within species (Gliwicz, 1994; Gliwicz and Umana, 1994; Santer, 1993). In contrast, only very large Daphnia appear to be susceptible to brood predation by copepods. Eudiaptomus gracilis and Acanthocyclops robustus copepodids have both been observed to enter the brood cavities of Daphnia over 2.25 mm long, where they prey on the eggs (Gliwicz and 22. Copepoda Lampert, 1994; Gliwicz and Stibor, 1993). Some of the large species of Hesperodiaptomus may regulate the relative abundance of different rotifer species and the clonal composition of Daphnia in arctic and alpine systems (Paul et al., 1995; Paul and Schindler, 1994; Wilson and Hebert, 1993). Several calanoid species in the Great Lakes can feed on the larvae of the zebra mussel, Dreissena polymorpha (Liebig and Vanderploeg, 1995). Although smaller prey are generally preferred by predatory copepods, the coincident vertical migration of the copepods with some of the larger cladocerans may cause ingestion rates to be disproportionally greater on these otherwise undesirable prey types (Williamson et al., 1989). Many prey species exhibit elaborate morphological and behavioral defense mechanisms that suggest a strong coevolutionary relationship between copepod predators and their prey. The hard loricas, spines, gelatinous sheaths, and rapid escape responses of many prey species are all effective at reducing predation by copepods (Williamson, 1983a, 1986, 1987; Stemberger, 1985; Roche, 1987). Some of the morphological defenses are nongenetic polymorphisms that are induced by chemical substances released by the copepods (Stemberger and Gilbert, 1984). Copepods are themselves important prey for other copepods (Peacock and Smyly, 1983), Chaoborus larvae (Peacock, 1982; Wyngaard, 1983), and ﬁsh. Adult copepods are preyed on by many planktivorous and benthic-feeding ﬁsh, and the nauplii are particularly important prey for larval ﬁsh (Guma’a, 1978; Hicks and Coull, 1983). Some of the larger cyclopoid copepods such as Mesocyclops and Macrocyclops may serve as important agents of biological control of pest insects such as mosquitos (Marten et al., 1994; Vu et al., 1998). Predation rates on early instar mosquito larvae may be quite high (Brown et al., 1991; Reid, 1989b), and cyclopoids can be cultivated in large quantities for this purpose (Marten et al., 1994; Suárez et al., 1992). Cyclopoids and calanoids are also important intermediate hosts for many parasites, including ﬂukes, nematodes, and tapeworms of ﬁsh, amphibians, birds, and mammals. Some cyclopoids such as Mesocyclops and Thermocyclops may serve as intermediate hosts of the human parasite Dracunculus medinensis (guinea worm) in the tropics. Ingestion of the larvae of this nematode in drinking water can cause serious illness or death in humans in areas of equatorial Africa and India (Steib and Mayer, 1988). The bodies of planktonic and epibenthic copepods provide a substrate for the Vibrio cholerae bacterium that carries cholera (Huq et al., 1983). 933 Recent studies on the stoichiometric relationships in planktonic populations have revealed that nutrient ratios in copepods differ from other crustacean plankton. Since high growth rates require high ribosomal RNA, high speciﬁc growth rates should be tied to a higher percentage of phosphorus in zooplankton (Main et al., 1997). Copepods tend to have a lower percentage of phosphorus in their bodies than many other zooplankters (Sterner and Hessen, 1994). For example, the mean atomic ratios of C:N:P are on the order of 212:39:1 for Acanthodiaptomus and 85:14:1 for Daphnia (Andersen and Hessen, 1991). The implications are that these nutrient ratios will give diaptomids a competitive advantage over cladocerans such as Daphnia during periods of phosphorus limitation, and also inﬂuence the importance of copepods in nutrient cycling as well as a variety of other ecosystem processes (Sterner and Hessen, 1994). Although seasonal changes in zooplankton communities may contribute to temporal variations in levels of toxic trace metals such as cadmium, concentrations are much lower in copepods than in cladoceran zooplankton (Yan et al., 1990). IV. CURRENT AND FUTURE RESEARCH PROBLEMS One of the most striking things about freshwater copepods is how little is known about them in comparison with either cladocerans or the marine copepods. Basic information on things as elementary as morphological characterization, life cycles, reproductive biology, feeding ecology, physiology, and genetics is lacking for most species. This is particularly true for the harpacticoids. Outside of Europe, taxonomic knowledge is inadequate for many species, especially for the non-planktonic species. Information of this sort will increase our understanding of copepods as vectors of diseases such as cholera and dracunculiasis, as well as help us to realize their full potential in biological control of pest insects such as mosquitos (discussed above). In addition to expanding our knowledge of copepods themselves, research on copepods can contribute to our knowledge of more general ecological questions. The small size, amenability to culture, discrete instars, existence of diapause stages, and variability in lifehistory patterns permit many interesting questions in population biology to be addressed. The intense and selective feeding of copepods, as well as their importance as food for higher trophic levels, open the way for studies in foraging behavior, community ecology, and systems ecology. While we are aware that food may limit copepod populations, we know very little about the relative 934 C. E. Williamson and J. W. Reid importance of food in controlling the presence or abundance of different copepod species in nature. We are particularly naive about the interactive effects of environmental variables such as temperature, pH, solar ultraviolet radiation, the presence of alternate food, the hunger level of the predator, predation risk, and various forms of pollution and environmental manipulation on copepods. With ecosystem disturbance being the norm rather than the exception now across the globe (Vitousek, 1994), it is critical that we gain a better understanding of how many of these disturbance-related abiotic and biotic variables affect key organisms such as copepods. Another interesting area of research to pursue concerns the relative abundance of cyclopoid versus calanoid copepods across lakes of different trophic status (discussed in Section III. F.1). The densities of cyclopoid copepods and most other zooplankton groups (ciliates, rotifers, cladocerans) are positively correlated with the primary productivity of lakes. This does not, however, seem to be universally true for calanoid copepods (Pace, 1986). What are the characteristics of calanoids that permit them to respond differently from most other groups to variations in primary productivity? Ecological genetics is another area that is advancing rapidly and is ripe for future study. The presence or absence and timing of chromatin diminution varies among species and may be useful in deciphering phylogenetic and evolutionary relationships among taxa (Einsle, 1996; Leech and Wyngaard, 1996; Dorward and Wyngaard, 1997). Little is known about the endogenous versus exogenous control of the wide range of variations in life-history characteristics observed for copepods in different systems or through different seasons. Many interesting evolutionary questions can be asked about the timing and control of life-history events of copepods due to the genetic isolation of populations and variability in selective pressures acting on these populations among lakes. Of particular interest is the recent ﬁnding that resting eggs may contribute substantially to the evolution and persistence of copepod populations over long periods of time (Hairston et al., 1995; Hairston, 1996), thus potentially permitting copepods to survive bottleneck periods related to both anthropogenic and natural disturbances. Recent ﬁnds in North America of groundwaterrelated copepod species that are closely related to Eurasian forms (e.g., Reid, 1998; Reid et al., 1999) are throwing new light on biogeographical relationships and the nature of evolution in copepods. While some groups seem to be extremely conservative, apparently having diverged little since the pre-Tethyan continental separation, other species evolved more rapidly to take advantage of new niches such as the cenotes of the Yucatan Peninsula (Fiers et al., 1996). V. COLLECTING AND REARING TECHNIQUES A. Collection and Preservation Planktonic copepods can be collected with either integrating samplers, such as a plankton net, or tube samplers (Lewis and Saunders, 1979), or with a variety of point samplers such as Schindler traps and Van Dorn bottles (Schindler, 1969). Sampling devices with narrow openings and pump samplers may elicit avoidance responses in copepods and should be tested for sampling efﬁciency before use. Quantitative samples must be taken at night for species that exhibit nocturnal vertical migrations into the sediments. Benthic copepods and the resting stages of planktonic species can be collected with either Ekman grab or core samplers. Separation of the smaller benthic copepods from the sediments presents difﬁculties. Sieving and sorting are tedious and not very efﬁcient for copepods. Density gradient centrifugation seems to be a more promising method (Strayer, 1985). Copepods can be preserved in a 10% solution of formalin (3.7% formaldehyde) or 70% ethanol. Formalin is less expensive but more toxic, and tends to cause specimens to get brittle over time due to the low pH. Buffering formalin with a saturated solution of borax (sodium tetraborate, Na2B4.1H2O) will improve preservation quality by making the specimens less brittle, but the high pH may also lead to more rapid color loss. A 70% solution of ethanol is actually a better preservative but is also more expensive, bulky, and may create difﬁcult eddies in open counting chambers, making quantiﬁcation extremely difﬁcult. Prior narcotization of copepods in carbonated water may enhance the retention of the gut contents of some species when they are placed in formalin (Gannon and Gannon, 1975) but is not always necessary (Williamson, 1984). The retention of egg clutches may be enhanced by prior narcotization in a 1:10 solution of chloroform: ethanol (Williamson and Butler, 1987). The chilling and addition of 40 g of sucrose / L of formalin commonly employed in the preservation of cladocerans (Prepas, 1978) is not required, but it also has no adverse effect on copepods. The adult stages of copepods can be counted in a Bogorov chamber under a dissecting microscope (Gannon, 1971), while the nauplii are more easily counted in a Sedgwick – Rafter chamber under a compound microscope. Several techniques have been developed for the separation of copepods in samples. Many live copepods can be separated from cladocerans with siphon tubes that 22. Copepoda remove the slower cladocerans but not the evasive copepods; or, one can take advantage of the greater resistance of copepods to pH shock (Bulkowski et al., 1985). Live copepods can be separated from dead copepods by staining with neutral red (Dressel et al., 1972; Flemming and Coughlan, 1978). A stock solution is made with 0.5 g of neutral red powder per liter of distilled water. This stock solution is then added to the sample containing the copepods in a 2:100 ratio for a staining period of up to 1 h before preservation in formalin. The prior addition of 4 g of NaOH per liter of formalin reduces the leaching of the stain out of the copepods for up to 28 weeks. The high pH makes the stain much less visible, however, and glacial acetic acid must be used to titrate the sample back down to an acid pH to regenerate the deep red color before sorting. Techniques have also been developed for the critical examination of the anatomy of copepods. The external anatomy, including sensilla and other delicate integumental organs, can be prepared by critical point drying and plating for examination under a scanning electron microscope, or cleared, stained, and examined under a light microscope (Fleminger, 1973; Friedman and Strickler, 1975). The gonads of some copepods can be selectively stained with fast green for easier visualization (Batchelder, 1986), and techniques are available for the histopathological examination of copepods (Barszcz and Yevich, 1976). 935 Freshwater diaptomids ranging from the smaller Leptodiaptomus minutus to the larger more omnivorous Hesperodiaptomus shoshone can be raised through multiple generations on a diet of Cryptomonas alone. Cyclopoid copepods are generally best reared on a combination of plant and animal foods. The nauplii will survive and grow well on a variety of algae including Cryptomonas and Scenedesmus, while animal food is usually necessary for the growth and survival of late copepodids and adults and for successful reproduction of some species (Jamieson, 1980b; Wyngaard and Chinnappa, 1982; Hansen and Santer, 1995; Hart and Santer, 1994). Algae as well as protozoa and invertebrates may still be an important food source for these older predatory stages (Santer, 1996). In culture, the dinoﬂagellate Ceratium furcoides supports the growth and reproduction of several genera of cyclopoid copepods but not those of the calanoid Eudiaptomus gracilis (Santer, 1996; Hopp et al., 1997). The early copepodid stages may need a mixture of plant and animal foods. Artemia nauplii are the most common and convenient source of animal food for cyclopoids, but calanoid copepods or soft-bodied cladocerans and rotifers may also be used. Protozoans are a good food source, but they may lead to bacterial contamination unless care is taken to rinse and remove the organic medium or use an inorganic medium for culture. Mass rearing of cyclopoids is also possible (Suárez et al., 1992). B. Rearing Techniques Inorganic medium, spring water, or ﬁltered lake water can be used in conjunction with a variety of foods to culture copepods. The utmost care should be taken to avoid glassware contaminated with soaps, acids, formaldehyde, or other toxins. Certain plastic containers may also be unsuitable. An easily rinsed detergent such as 7X®, should be used to clean all glassware thoroughly. [The source of 7X® is ICN Biomedicals, Inc., Costa Mesa, CA; phone:800-854-0530; www.icnpharm.com.] Standard detergents may cause high mortalities. Dilute acids may also be satisfactory for washing glassware (Wyngaard and Chinnappa, 1982), but thorough soaking or extra rinsing is suggested. Water quality may also be enhanced by running distilled water through a mixed-bed resin before use. All glassware should be sterilized between uses to avoid contaminant buildup. Harpacticoids have been cultured on a wide range of artiﬁcial and natural foods, including baker’s yeast, dried ﬁsh food, algae, bacteria, and dried, mashed, or rotting animals, eggs, and plants (Hicks and Coull, 1983). Multiple foods are often more successful than a single food type. VI. IDENTIFICATION TECHNIQUES A. Dissection Techniques for Identiﬁcation Dissection is usually required for positive identiﬁcation of copepods. Small insect pins (minuten nadeln, available from entomological supply houses) can be mounted on the end of wood applicator sticks or melted glass pipets for use in dissection. The dissection should be performed on a glass microscope slide so that a coverslip can be placed over the ﬁnal preparation for examination under a compound microscope. The dissection is most easily performed by placing the copepod on its side in a drop of medium, and placing one pin in the dorsal part of the thorax to hold it down. A second pin is then used to tease off ﬁrst the urosome, then each thoracic segment and its associated pair of legs — one segment at a time, and ﬁnally the ﬁrst antennae. The chance of confusing legs is lessened if the ﬁrst cut is made between the second and third legs, and the posterior part of the animal transferred to a second drop of medium on the same slide. Dissection and handling of the pieces is easier when performed in a drop 936 C. E. Williamson and J. W. Reid of glycerin or other viscous, water-miscible mounting medium such as Hoyer’s or CMC-9. Placing a copepod directly in full strength medium may cause osmotic stress and deformation. This problem can be avoided by placing the copepod in a well slide in a dilute mixture of about one drop of the medium to 5 – 10 drops of water and letting the water evaporate. Gentle warming will speed evaporation. Semi-permanent mounts can be made by sealing a glycerin coverslip with clear ﬁngernail polish. One of several standard mounting media can be used for more permanent mounts. A small amount of neutral red dye in glycerin or a mounting medium with a dye will color the dissected parts so that they are easier to locate after dissection. The dissection can be performed right in the viscous medium, the dissected appendages appropriately arranged in order, and the coverslip put in place. When using a mounting medium other than glycerin it is useful to let the mounting medium dry somewhat after dissection before adding more medium and adding a coverslip. This helps the dissected parts stay put. If the medium is allowed to dry for a week and the edges sealed with ﬁngernail polish or spar varnish, the mount will last indeﬁnitely. For more details on techniques for copepod preparation, dissection, staining, mounting, and preservation, the reader is referred to an unpublished pamphlet prepared by J. W. Reid and available in the C. B. Wilson Copepod Library at the National Museum of Natural History, Smithsonian Institution, Washington, DC. B. Characteristics for Distinguishing the Free-Living Freshwater Orders Copepods are most easily identiﬁed from adult specimens, although information is available for the separation of the immature stages of some species (Comita and Tommerdahl, 1960; Czaika and Robertson, 1968; Katona, 1971; Comita and McNett, 1976; Shih and Maclellan, 1977; Bourguet, 1986; PinelAlloul and Lamoureux, 1988a, b). Growth rates and some external morphological characteristics of copepods can be analyzed by examining the highly transparent molted exoskeletons (exuviae) (Twombly and Burns, 1996). In freshwater habitats, the three major copepod orders can be distinguished by a number of characteristics of the adults; the length of the ﬁrst antennae and taper of the body are perhaps the most useful of these (Fig. 1). The ﬁrst antennae of harpacticoids are very short (5 – 9 segments in the female) and rarely extend past the posterior end of the cephalothorax. In cyclopoids the ﬁrst antennae are intermediate in length (6 – 17 segments in the female), and rarely extend beyond the posterior end of the prosome. In calanoids the ﬁrst antennae have 23 – 25 segments in the female and generally extend well beyond the end of the prosome to the urosome or even the caudal setae. The body of harpacticoids generally shows only a slight taper from the anterior to the posterior, so that the metasome and urosome are of similar widths. In cyclopoids and calanoids the metasome is substantially wider than the urosome. In cyclopoids there is a strong but gradual taper from front to back; while in calanoids there is a more abrupt change in body width where the metasome joins the urosome. The body shape of harpacticoids is quite diverse and varies greatly with their habitat. For example, interstitial species are smaller and more elongate, burrowing species are larger and broader, while epibenthic species are large and variable in their body shape (Hicks and Coull, 1983). The distinguishing features of the gelyelloids (all three species of which are under 0.5 mm in body length) are the absence of the fourth (in the European species) and ﬁfth swimming legs, and the fusion of the coxae of the ﬁrst three pairs of swimming legs; their general body shape is narrow near the midsection (Fig. 1E). Other factors that distinguish the four free-living orders include general habitat preferences and the number of egg sacs. Calanoids are almost exclusively planktonic and either carry a single egg sac medially or release their eggs directly into the water. Cyclopoids are primarily benthic, although several planktonic species are widespread and abundant, and carry two egg sacs attached laterally to their genital segment. Harpacticoids are generally smaller than either calanoids or cyclopoids, are generally benthic or associated with other substrates or particles, and usually carry a single egg sac medially. Wilson and Yeatman (1959) provided a useful table that summarizes other differences among these three orders. Smallest of all are the gelyelloids, which occur exclusively in subterranean karst or stream hyporheic sediments; the nature of their egg sacs is unknown. Adult copepods are sexually dimorphic and sexes can be distinguished on the basis of their relative size, as well as certain primary or secondary sexual characteristics. Females are generally larger than males, and mature gonads, spermatophores, or externally-carried egg sacs are often visible. The subitaneous and resting eggs of diaptomids can be distinguished from each other in preserved samples by the presence of a space between the chorion and the vitelline membrane in the subitaneous eggs, and its absence in resting eggs (Lohner et al., 1990). Adult male copepods have one (calanoids) or two (harpacticoids, cyclopoids, and gelyelloids) geniculate ﬁrst antennae that are modiﬁed for grasping the females during mating (Fig. 1 and 3). After preservation the geniculate antennae of males 22. Copepoda often become sharply bent, or elbowed, so sexes are quite easy to distinguish. In harpacticoids, males can be separated from females by their ﬁrst antennae, which have fewer and broader segments, and often more densely clumped setae. Other characteristics such as morphological differences in the caudal rami and legs 2 – 4 can be used to distinguish male and female harpacticoids in many genera. In addition, female harpacticoids generally have a more highly developed ﬁfth leg, and the sixth leg tends to be tiny. Characteristics that distinguish sex are generally not visible in any of the earlier larval stages and are less conspicuous in stage C5. Adult copepods can be distinguished from immature copepodids either by size, or by the primary or secondary sexual characteristics described above. In adult cyclopoids and harpacticoids the last segment of the urosome is shorter than, or similar in length to, the next to last (penultimate) segment, while in immatures the last urosomal segment has not yet divided and is about twice as long as it is wide (Fig. 5). Characteristics of the genital segment such as a ventral swelling or more angular side proﬁle in the adults may also be helpful in separating adult females from immatures. The larval stages within each order can be differentiated on the basis of body shape and the number and structure of the appendages. Several individuals of each sex and stage should be compared to establish valid criteria for distinguishing the sexes and stages of each species examined. C. Identiﬁcation of the Continental Free-Living Copepoda 1. Introduction Here we provide keys to the orders and genera, followed by tables that list the families, genera, and species of copepods recorded from continental and nearshore oligohaline habitats in North America. Keys and revisions for individual genera are cited in the tables, while some of the broader, more comprehensive keys and taxonomic references are listed in the paragraphs below. The taxonomy and systematics of copepods are active areas of research. In North America, the faunas of ephemeral, subterranean, interstitial, and semiterrestrial habitats are still poorly known. Moreover, reﬁnements of taxonomic methods have led to redeﬁnition of many taxa (Reid, 1998). Investigators should consult with appropriate specialists and archive reference specimens. Dussart and Defaye (1995) provided an introduction to the taxonomy and systematics of the continental Copepoda, with diagnoses and keys to most genera. For North American free-living continental copepods, 937 the most complete and best-illustrated keys are those by Wilson and Yeatman (1959). The keys by Pennak (1989) include most species of Calanoida and some Cyclopoida. Basic general taxonomic references for marine harpacticoids include the works of Coull (1977), Lang (1948, 1965), and Huys et al. (1996). There are several useful keys and checklists for different regions of North America. Works treating calanoids, cyclopoids, and harpacticoids include those of Cole (1959) for Kentucky, Robertson and Gannon (1981) for the Great Lakes, Clamp et al. (1999) for North Carolina, and Chengalath and Shih (1994) for northwestern North America. Lists for calanoids and cyclopoids include those of Harris (1978) for northern Mississippi, Reed (1963) for northern Canada, and Smith and Fernando (1978) for Ontario. Cole (1961, 1963, 1966) summarized records of Diaptomidae (Calanoida) from Arizona, and Robertson (1970, 1972, 1975) listed diaptomids from Oklahoma. Czaika (1982) provided keys and tables for adults and developmental stages of planktonic calanoids and cyclopoids of the Great Lakes. Regional works on Cyclopoida include the key by Torke (1976) for Wisconsin and the checklists by Bunting (1973) for Tennessee and Reid and Marten (1995) for Louisiana and Mississippi. Hudson et al. (1998) provided a checklist of cyclopoids and harpacticoids of the Great Lakes and a key for the cyclopoids. Checklists for Mexico were furnished by SuárezMorales and Reid (1998) and by Reid (1990) for Mexico, Central America, and the Antilles. Suárez-Morales et al. (1996) provided keys and taxonomic discussion for species of the Yucatan Peninsula, as well as a Spanishlanguage general introduction to the study of continental copepods. Two public resources for copepod studies are the C. B. Wilson Copepod Library at the National Museum of Natural History, Smithsonian Institution, Washington DC., and the Monoculus Library at Oldenburg University in Germany. The Wilson Library contains some 10,000 works, indexed by author and species. Its website (http: / /www.nmnh.si.edu / iz / copepod) contains ﬁve databases: (1) a bibliography of all known literature on Copepoda and Branchiura; (2) taxonomic lists of families, genera, and species; (3) a world list of researchers; and (4) the taxonomic “types” of Copepoda and Branchiura held in the National Museum of Natural History. Contact F. D. Ferrari, T. C. Walter, or J. W. Reid for more information. The Monoculus Bibliography Project also holds about 10,000 works, many entered in its database; contact H. K. Schminke, Fachbereich 7-Biologie, Universität Oldenburg, Postfach 2503, D-26111 Oldenburg, Germany for details. 938 C. E. Williamson and J. W. Reid The true continental Calanoida are dominated by the families Centropagidae, Diaptomidae, and Temoridae, and the Cyclopoida by the family Cyclopidae. The continental harpacticoid fauna consists mainly of the families Canthocamptidae, Parastenocarididae, and Phyllognathopodidae, but the primarily marine families Ameiridae, Huntemanniidae, and Laophontidae also contain some true freshwater species. Several additional calanoid, cyclopoid, and harpacticoid families include euryhaline species that may wander into coastal freshwaters, but these are not listed in the tables. Use- ful taxonomic references for euryhaline species include C. B. Wilson (1932) for calanoids and cyclopoids; Walter (1989) for Pseudodiaptomus; Rocha (1991), Rocha and Hakenkamp (1993), Rocha et al. (1998), and M. S. Wilson (1958) for Halicyclops; and Coull (1977), Lang (1965), and Huys et al. (1996) for harpacticoids. 2. Key to Orders and Families of the Free-Living Continental Copepoda of North America [Note that there are 3 or 4 couplets in some parts of this taxonomic key.] 1a. Body constricted, or major body joint between somites bearing 4th and 5th legs; ﬁrst antenna of 6 – 17 segments.........................2 1b. Body constricted between somite bearing 5th leg and genital segment; ﬁrst antenna long, of 23 – 25 segments (Figs. 1A, 2, 5) Order Calanoida, ...............................................................................................................................................................................4 1c. No distinct body articulation between prosome and urosome; ﬁrst antenna with 14 poorly deﬁned segments; swimming legs without couplers (intercoxal sclerites), and joined at base (Fig. 1E) .........................................................................Order Gelyelloida 2a(1a). Second antenna of 3 or 4 segments, not prehensile; ﬁrst antenna of with 6 – 17 segments...............................................................3 2b. Second antenna of 3 segments, with large prehensile terminal claw; ﬁrst antenna of with 6 segments ............................................. Order Poecilostomatoida (Ergasilidae) ................................................................................................................................................. 3a(2a). Fifth leg small, of 1 – 3 short segments, or represented only by setae; segment 1 not enlarged on inner margin (Figs. 1B, 2, 5) ...........................................................................................................................................................................Order Cyclopoida 24 3b. Fifth leg broad, of 1 – 2 segments, segment 1 (baseoendopod) enlarged on inner margin (Figs. 1C,D, 2, 3) .......................................... Order Harpacticoida, .......................................................................................................................................................................44 ORDER CALANOIDA 4a(1b). Caudal ramus with 3 or 5 well-developed setae (plus 1 or 2 shorter, slender setae) ....................................................................5 4b. Caudal ramus with 4 well-developed setae (plus shorter, slender outer and inner setae); legs 1 – 4 endopods of 1, 2, 3, and 3 segments respectively; ﬁrst antenna not geniculate; leg 5 lacking...................................................................Aetideidae Senecella 5a(4b). Caudal ramus with 3 well-developed terminal setae (plus reduced or spiniform outer seta and slender, dorsally placed inner seta) ...................................................................................................................................................................................................6 5b. Caudal ramus with 5 well-developed setae (plus slender, dorsally placed inner seta) ..................................................................7 6a(5a). Caudal ramus , outer seta slender, about as long as ramus; leg 5 and left leg 5 , last segment with long apical spine; urosome symmetrical ........................................................................................................................................Temoridae Heterocope 6b. Caudal ramus , outer seta shorter than ramus, and or spiniform; leg 5 and left leg 5 , last segment without long apical spine; urosome asymmetrical, with various processes on right side ................................................................Temoridae Epischura 7a(5b) Caudal ramus long (more than 3 times longer than wide) ..........................................................................................................8 7b. Caudal ramus short (3 times or less than 3 times longer than wide) .........................................................................................10 8a(7a). Maxillipeds elongate (about 2 times body width) in lateral view; legs 1 – 4, endopods all of 3 segments ............................................9 8b. Maxillipeds not elongate (subequal to body width) in lateral view; legs 1 – 4, endopods of 1, 2, 2, and 2 segments, respectively; leg 5 lacking endopods ....................................................................................................................................Temoridae Eurytemora 8c. Maxillipeds reduced; legs 1 – 4, endopodites all of 2 segments; leg 5 lacking endopodite.............................................................. Acartiidae ....................................................................................................................................................................Acartia sinensis 8d. Maxillipeds not elongate; legs 1 – 4, endopodites all of 3 segments............................................Pseudodiaptomidae Pseudodiaptomus 9a(8a). Caudal ramus with tiny spines on surface; body somite bearing leg 5 with no spines; right leg 5 , exopod segment 2, coxa with, basis without medial process; leg 5 , exopod segment 2 with outer spine, and endopod segment 1 with inner seta ........................... Centropagidae ................................................................................................................................................................Limnocalanus 22. Copepoda 939 9b. Caudal ramus with no surface spines; body somite bearing leg 5 with 1 spine on each posterior corner; right leg 5 , coxa without medial process, and basis with medial process; leg 5 , exopod segment 2 with no outer spine, and endopod segment 1 with no inner seta ......................................................................................................................................................Centropagidae Sinocalanus 10a(7b). Caudal ramus , lengths of setae unequal, fourth seta from outer margin longer and stouter than others; legs 1 – 4, endopods all with 3 segments; leg 5 , endopods 3-segmented, last segments with 6 long setae ........................................................................... Centropagidae ...............................................................................................................................................................Osphranticum 10b. Caudal ramus , lengths of setae nearly equal; leg 1 endopod of 2 segments, legs 2 – 4 endopods of 3 segments; leg 5 , endopods 1- or 2-segmented, last segments with 0 – 2 short setae ..................................................................................Diaptomidae 11 11a(10b). Last thoracic segment with rounded or spiniform dorsal process .................................................................................................12 11b. Last thoracic segment of female with no dorsal process ..................................................................................................................13 12a(11a). Right leg 5 , terminal claw of exopod segment 2 slender, at least 1.5 times longer than segment; leg 5 , endopod with row of ﬁne terminal hairs and 2 long terminal spines ................................................................................................................Mastigodiaptomus 12b. Right leg 5 , terminal claw of exopod segment 2 slender, usually twice or more length of segment; leg 5 , endopod usually with ﬁne terminal hairs only..................................................................................................................................Arctodiaptomus (in part) 12c. Right leg 5 , terminal claw of exopod segment 2 stout and about as long as segment; left leg 5 , terminal process strongly corrugated; leg 5 , endopod with row of terminal hairs and 1 short spine ..........................................................................Sinodiaptomus 13a(11b). Right ﬁrst antenna , terminal segment with end rounded, bearing only setae.................................................................................14 13b. Right ﬁrst antenna , terminal segment with small clawlike process on distal end .................................................Acanthodiaptomus 14a(13a). Left leg 5 , distal process of exopod not distinct, i.e., exopod of 2 segments .................................................................................16 14b. Left leg 5 , distal process of exopod distinct, i.e., exopod of 3 segments........................................................................................15 15a(14b). Right ﬁrst antenna , antepenultimate segment with short stout curved spinous process on distal end; leg 5 , exopod segment 3 absent (with 2 remnant spines inserted directly on segment 2).................................................................................Onychodiaptomus 15b. Right ﬁrst antenna , antepenultimate segment with no process; leg 5 , exopod segment 3 present, distinct..........Nordodiaptomus 16a(14a). Right ﬁrst antenna , antepenultimate segment with distal spinous process.....................................................................................18 16b. Right ﬁrst antenna , antepenultimate segment with at most narrow hyaline lamella......................................................................17 17a(16b). Both ﬁrst antennae and left ﬁrst antenna , with 2 setae on segment 11 and 2 setae on segments 15, 16, and 17 ....................................................................................................................................................................................Mixodiaptomus 17b. Both ﬁrst antennae and left ﬁrst antenna , with 1 seta on each of segments 11 and 15 – 17 .................................Skistodiaptomus 18a(16a). Right ﬁrst antenna with simple, rodlike or dentiform process on antepenultimate segment ..........................................................19 18b. Right ﬁrst antenna with comb-shaped (corrugated) process on antepenultimate segment; leg 5 with segment 2 shorter than segment 1 and with 1 rounded, haired pad; leg 5 , endopod usually with terminal hairs only .........................Arctodiaptomus (in part) 19a(18a). Right ﬁrst antenna , antepenultimate segment with short dentiform process (usually shorter than penultimate segment) ..............21 19b. Right ﬁrst antenna , antepenultimate segment with long dentiform process (usually longer than penultimate segment) ................20 20a(19b). Left leg 5 , segment 2 usually longer than segment 1, bearing two distinctly divided, more or less hairy pads; leg 5 , exopod segment 3 present, distinct ..........................................................................................................................................Hesperodiaptomus 20b. Left leg 5 , segment 2 shorter than segment 1, bearing 2 indistinctly divided pads; leg 5 , exopod segment 3 absent, with only 2 remnant spines, inserted directly on segment ................................................................................................Leptodiaptomus (in part) 21a(19a). Both ﬁrst antennae and left ﬁrst antenna , setae on segments 17, 19, 20, and 22 with normal tapered ends (not hooked); left leg 5 , exopod segment 2 with lateral seta short, not extending past end of segment...........................................................................22 21b. Both ﬁrst antennae and left ﬁrst antenna , setae on segments 17, 19, 20, and 22 with ends stifﬂy hooked; left leg 5 , lateral seta of exopod 2 very long .........................................................................................................................................Aglaodiaptomus 22a(21a). Both ﬁrst antennae and left ﬁrst antenna , segment 11 with 1 seta.............................................................................................23 22b. Both ﬁrst antennae and left ﬁrst antenna , segment 11 with 2 setae..............................................................................Diaptomus 23a(22a). Left leg 5 , exopod segment 2 with both terminal process and inner spine short and stout; leg 5 , exopod segment 3 absent, with only 2 remnant spines, inserted directly on exopod 2 ...................................................................................Leptodiaptomus (in part) 23b. Left leg 5 , exopod segment 2 with both terminal process and inner spine relatively long and slender; leg 5 , exopod segment 3 present and distinct .........................................................................................................................................................Eudiaptomus 940 C. E. Williamson and J. W. Reid ORDER CYCLOPOIDA 24a(3a). Mandibular palp consisting of tiny segment bearing 1 – 3 setae, or mandibular palp entirely absent ..............................Cyclopidae 25 24b. Mandibular palp large, complex, with many plumose setae .........................................................................Oithonidae Limnoithona 25a(24a). Leg 5 completely fused to body somite and bearing 3 setae and or spines; ﬁrst antenna of 9 – 11 (usually 11) segments ..............26 25b. Leg 5 consisting of 1 or 2 distinct segments; ﬁrst antenna of 6 – 17 segments ...............................................................................28 25c. Leg 5 consisting of 3 distinct segments; ﬁrst antenna of 16 segments ..........................................................................Orthocyclops 26a(25a). Leg 5 reduced to narrow plate or knobs, bearing 3 tiny setae; legs 1 – 4, endopods and exopods of 2 or fewer segments .................27 26b. Leg 5 consisting of broad plate, bearing 2 strong inner spines and 1 long outer seta; legs 1 – 4, endopods and exopods all of 3 segments .................................................................................................................................................................................Ectocyclops 27a(26a). Anal operculum large, triangular, with dentate margin; leg 4 coxa, inner corner lacking seta; leg 3 , endopod terminal segment with modiﬁed apical spine Bryocyclops ................................................................................................................................................ 27b. Anal operculum small, quadrate, with smooth margin; leg 4 coxa with seta on inner corner; leg 3 , endopod terminal segment with apical spine simple Stolonicyclops ................................................................................................................................................ 28a(25b). Leg 5 consisting of 1 distinct, broad segment and bearing 1 inner spine and 2 outer setae ...............................................................29 28b. Leg 5 consisting of 1 or 2 distinct, usually narrow segments (proximal segment may be fused to body somite, but remnant seta of this segment always present), segment 2 with 1 – 3 spines and setae (if 3, there is 1 middle spine between 2 setae)...........................33 29a(28a). First antenna of 8, 11, or 12 segments; small species (usually under 1.4 mm long).......................................................................30 29b. First antenna of 17 segments; large robust species (1.7 – 2.9 mm long)........................................................................Homocyclops 30a(29a). First antenna of 12 segments ........................................................................................................................................................31 30b. First antenna of 8 or 11 segments ....................................................................................................................Paracyclops (in part) 31a(30a). Caudal ramus , with tiny spines next to lateral and outermost terminal setae only........................................................................32 31b. Caudal ramus with longitudinal row of tiny spines on outer margin; caudal ramus at least 4 times longer than wide .............................................................................................................................................................................................Eucyclops 32a(31a). First antenna , last 2 segments not more that 2 times longer than wide; caudal ramus about 2.5 times longer than wide ............................................................................................................................................................................Paracyclops (in part) 32b. First antenna , last 2 segments more than 3 times longer than wide; caudal ramus about 3 times longer than wide ........................................................................................................................................................................................Tropocyclops 33a(28b). Leg 5 with 2 distinct segments..........................................................................................................................................................34 33b. Leg 5 with 1 distinct segment ...........................................................................................................................................................40 34a(32a). Leg 5, distal segment narrow, bearing 1 – 2 spines or setae ...............................................................................................................35 34b. Leg 5, distal segment broad, bearing 3 spines and setae ..................................................................................................Macrocyclops 35a(34a). Distal segment of leg 5 bearing 1 apical seta and 1 long inner marginal or subapical spine (length of spine more than 4 times length of segment); ﬁrst antenna always of 17 segments .............................................................................................................................36 35b. Distal segment of leg 5 bearing 1 apical seta and (usually) 1 rather short inner marginal or subapical spine (length of spine not more than twice length of segment); ﬁrst antenna of 17 or fewer segments ...............................................................................................37 36a(35a). Leg 5, inner spine of distal segment inserted about midlength of inner margin of segment; ﬁrst antenna, last segment usually 3 or more times longer than wide ............................................................................................................................................Mesocyclops 36b. Leg 5, inner spine of distal segment inserted subterminally on segment; ﬁrst antenna, last segment about twice longer than wide .....................................................................................................................................................................................Thermocyclops 37a(35b). Leg 5, distal segment with apical seta and (usually) small slender subapical spine on inner margin; caudal ramus without longitudinal dorsal ridge, and inner surface with or without hairs .................................................................................................................38 37b. Leg 5, distal segment with apical seta and large spine (about equal in length to distal segment) attached at middle of inner margin of segment; caudal ramus usually with longitudinal dorsal ridge, and inner margin hairy; ﬁrst antenna of 14 – 17 segments........Cyclops 38a(37a). Leg 5, segment 2 with apical seta and small spine or spur on middle of inner surface, or somewhat longer subapical spine (usually shorter than segment 2); caudal ramus with or without hairs on inner surface .................................................................................39 38b. Leg 5, segment 2 with inner subapical spine longer than segment 2; caudal ramus usually without hairs on inner surface ...........................................................................................................................................................................................Diacyclops 22. Copepoda 941 39a(38a). Leg 5, segment 2 with apical seta and small spine or spur (usually a spur, continuous with segment) at about middle of inner surface; caudal ramus, inner surface hairy; ﬁrst antenna of 17 segments ...............................................................................Megacyclops 39b. Leg 5, segment 2 with apical seta and small spine slightly distal to middle of segment, or almost apical; caudal ramus with or without hairs on inner margin; ﬁrst antenna of 11 – 17 segments ........................................................................................Acanthocyclops 40a(37b). Leg 5, free segment narrow and cylindrical, or only slightly longer than wide, with inner apical spine or seta (if present) inserted near outer apical seta .......................................................................................................................................................................41 40b. Leg 5, free segment very broad, with inner apical spine inserted far from outer apical seta ................................................Apocyclops 41a(40a). Leg 5, free segment narrow, cylindrical, with inner spine (if present) tiny and inserted subterminally or on inner surface of segment; proximal segment completely absent, except for remnant seta..........................................................................................................42 41b. Leg 5, free segment only slightly longer than wide, with slender inner terminal seta inserted next to longer outer terminal seta; proximal segment obvious although continuous with body somite ..........................................................................................................43 41c. Leg 5, free segment only slightly longer than wide, with stout inner apical spine and outer apical seta both inserted terminally; proximal segment completely absent, except for remnant seta..................................................................................................Metacyclops 42a(41a). Legs 1 – 4, couplers and coxa-basis very wide (about 2.5 times wider than long); legs 2 – 4, basis with rows of tiny spines on inner expansion; leg 4 endopod, outer terminal spine tiny, less than 1 /6 length of inner terminal spine ..................................Cryptocyclops 42b. Legs 1 – 4, couplers and coxa-basis less wide (about 2 times wider than long); legs 2 – 4, inner expansion of basis with ﬁne hairs; leg 4 endopod, outer terminal spine at least 1 / 2 length of inner terminal spine ...................................................................Microcyclops 43a(41b). Proximal segment of leg 5 present as tiny knob set close to free segment; legs 1 – 4 with 3- or 2-segmented rami; if exopodites of 2,2,3,3 segments, then exopodite segment 1 with medial (inner) seta................................................................................Rheocyclops 43b. Proximal segment of leg 5 present as large knob set apart from free segment; legs 1 – 4 with endopodites 2-segmented and exopodites of 2,2,3,3 segments; exopodite segment 1 without medial (inner) seta ...................................................................Itocyclops ORDER HARPACTICOIDA 44a(3b). Somite bearing leg 1 completely fused with cephalothorax; maxilliped prehensile (ending in long claw), never ﬂat and leaﬂike .........................................................................................................................................................................................................45 44b. Somite bearing leg 1 not fused to cephalothorax; maxilliped ﬂat and leaﬂike, with several setae.......................................................... Phyllognathopodidae Phyllognathopus ................................................................................................................................................ 45a(44a). Leg 1 endopod prehensile (with dist almost segment long) or not, with 2 or more terminal setae (which may be stout)...................46 45b. Leg 1 endopod prehensile, much longer than exopod, ending in 1 stout claw ......................................Laophontidae Onychocamptus 46a(43a). Leg 1 exopod of 3 segments, middle segment with no spine on outer margin; body vermiform (narrow, cylindrical) .......................47 46b. Leg 1 exopod of 3 segments, middle segment with a spine on outer margin, or leg 1 exopod of only 1 or 2 segments; body stout, usually not vermiform ......................................................................................................................................................................48 47a(46a). Legs 1 and 2, endopods of 2 and 1 segment, respectively ................................................................Parastenocarididae Parastenocaris 47b. Legs 1 and 2, endopods of 3 and 2 segments, respectively .......................................................................Ameiridae Psammonitocrella 48a(46b) Second antenna with basis (Fig. 3) ...................................................................................................................................................49 48b. Second antenna with allobasis (Fig. 3)..............................................................................................................................................51 49a(48a). Leg 5 unsegmented .....................................................................................................................................Tachidiidae Tachidius 49b. Leg 5 of 2 distinct segments, i.e. exopod segment separated from baseoendopod ...................................................Ameiridae 50 50a(49b). Caudal rami short, 1-2 times as broad as long; legs 2 – 4, 1 or more endopod 3-segmented..............................................................65 50b. Caudal rami about 5 times longer than wide; legs 2 – 4, endopod with 2, 2, and 1 segments, respectively.....................Stygonitocrella 51a(48b). Leg 1 with endopod of 2 or 3 segments ............................................................................................................................................52 51b. Leg 1 with endopod of 1 short segment ..............................................................................................Huntemanniidae Huntemannia 52a(51a). First antenna of 6 – 9 segments; leg 1 , endopod segment 1 with inner seta............................................................................53 52b. First antenna of 5 segments; leg 1 , endopod segment 1 without inner seta ...................................Huntemanniidae Nannopus 53a(52a). First antenna of 6 – 9 (usually 8) segments; leg 5 of 2 distinct segments, i.e., exopod separated from baseoendopod ...........................................................................................................................................................................Canthocamptidae 54 53b. First antenna of 6 segments; leg 5 unsegmented ...............................................Canthocamptidae incertae sedis Cletocamptus 942 C. E. Williamson and J. W. Reid 54a(53). Leg 1 , exopod of 3 segments ....................................................................................................................................................55 54b. Leg 1 , exopod of 2 segments.................................................................................................................................Maraenobiotus 55a(54a). Leg 1 , exopod segment 2 without inner seta.............................................................................................................................56 55b. Leg 1 , exopod segment 2 with inner seta ..................................................................................................................................60 56a(55a). Leg 1 , endopod of 2 segments ..................................................................................................................................................57 56b. Leg 1 , endopod of 3 segments ....................................................................................................................Bryocamptus (in part) 57a(56a). Leg 1 , endopod reaches from about midlength to slightly past end of exopod segment 3 .........................................................58 57b. Leg 1 , endopod does not reach end of exopod segment 2 .......................................................................................Epactophanes 57c. Leg 1 , endopod longer than exopod by half of endopod segment 3 or more ............................................................Paracamptus 58a(57a). Legs 2 – 4 , exopod 2 without inner seta ....................................................................................................................................59 58b. Legs 2 – 4 , exopod 2 with inner seta ...........................................................................................................Bryocamptus (in part) 59a(58a). Leg 3 , exopod segment 3 with 1 inner seta; legs 2 – 4 , exopod segment 3 with all normal-sized setae and spines (i.e., inner terminal seta not reduced) .......................................................................................................................................................Moraria 59b. Leg 3 , exopod segment 3 with 2 inner setae; legs 2 – 4 , exopod segment 3, inner terminal seta tiny...................Gulcamptus 60a(55b). Leg 1 , endopod of 3 segments and rostrum small to moderate in size, i.e., not extending past segment 1 of ﬁrst antenna; ﬁrst antenna of 7 – 9 (usually 8) segments ............................................................................................................................................61 60b. Leg 1 , endopod of 2 segments and rostrum small, not extending past segment 1 of ﬁrst antenna; ﬁrst antenna of 7 or 8 segments ................................................................................................................................................................Bryocamptus (in part) 60c. Leg 1 , endopod of 2 or 3 segments and rostrum large, extending past end of segment 1 of ﬁrst antenna; ﬁrst antenna of 6 or 7 segments ............................................................................................................................................................................Mesochra 61a(60a). Leg 5 , next outermost seta of baseoendopod not reduced, usually equal or longer than outermost seta; leg 4 , outer corner of last segment of endopod with distinct spine or seta ..........................................................................................................................62 61b. Leg 5 , next outermost seta of baseoendopod tiny; leg 4 , outer corner of last segment of endopod with spinous process ....................................................................................................................................................................................Canthocamptus 62a(61a). Leg 1 , endopod segment 1 not reaching beyond end of exopod segment 2 ...............................................................................63 62b. Leg 1 , endopod segment 1 reaching midlength of exopod segment 3, or longer ..............................................Attheyella (in part) 63a(62a). Legs 2 – 4 , exopod segment 3 with 3 outer spines (total number of spines and setae 6, 7, and 6 or 7, respectively); leg 5 , baseoendopod with 5 or 6 (rarely 4) setae; leg 5 , baseoendopod usually with 2 setae ..................................................................64 63b. Legs 2 – 4 , exopod segment 3 with 2 outer spines (total number of spines and setae 5, 6, and 6, respectively); leg 5 , baseoendopod with 3 – 4 setae; leg 5 , baseoendopod with no setae ...........................................................................................Elaphoidella 64a(63). Legs 2 and 3 , endopods of 3 segments; or of 2 segments and leg 5 with 5 setae on baseoendopod; leg 2 , endopod segment 2 modiﬁed, bearing 1 or 2 knobs, spinous processes, or notches ...........................................................................Bryocamptus (in part) 64b Legs 2 and 3 , endopods of 2 segments; leg 5 with 6 setae on baseoendopod; leg 2 , endopod unmodiﬁed (may have spines on outer margin)..........................................................................................................................................................Attheyella (in part) 65a(50a). Leg 2-4 , all endopods of 3 segments ................................................................................................................................Nitokra 65b. Leg 2-4 , endopods of 3, 3 and 2 segments respectively ...........................................................................................Nitocrellopsis LITERATURE CITED Adrian, R. 1991. 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J. 1990. Cadmium concentrations of crustacean zooplankton of acidiﬁed and nonacidiﬁed Canadian Shield lakes. Environmental Science and Technology 24:1367 – 1372. Yan, N. D., Pawson, T. W. 1997. Changes in the crustacean zooplankton community of Harp Lake, Canada, following invasion by Bythotrephes cederstroemi. Freshwater Biology 37:409 – 425. Zaret, T. M. 1980. Predation in freshwater communities. Yale Uni. Press, New Haven, CT, 187 pp. Zaret, T. M., Suffern, J. S. 1976. Vertical migration in zooplankton as a predator avoidance mechanism. Limnology and Oceanography 21:804 – 813. Orders and Superorders of Copepods Platycopioida Fosshagen 1985 — Small, hyperbenthic marine species in shallow seas and anchialine caves. Calanoida Sars 1903 — Freshwater and marine, free-swimming. Misophrioida Gurney 1933 — shallow coastal waters, deep sea, deep-water plankton, and anchialine caves. Harpacticoida Sars 1903 — Freshwater and marine, free-living, primarily benthic or attached. Monstrilloida Sars 1903 — nauplii and copepodids are endoparasitic on polychaetes and prosobranch molluscs. Adults are free-swimming, but nonfeeding. Adult females carry eggs on spines. Mormonilloida Boxshall 1979 — only two species known, both widely distributed and abundant in deep oceans (400 – 1500 m depth). They are free-swimming particle-feeders. Gelyelloida Huys 1988 — only three species known, two from Europe (France and Switzerland) and one from the United States (South Carolina). All are freshwater and benthic / subterranean. Cyclopoida Burmeister 1835 — Freshwater and marine, benthic and planktonic. Siphonostomatoida Thorell 1859 — parasitic or associated with ﬁsh or invertebrate hosts; primarily marine, but some freshwater species exist. Body often highly reduced, lacking appendages. Poecilostomatoida Thorell 1859 — Mostly parasitic on, or associated with, a wide variety of ﬁsh and invertebrates. Morphologically the most diverse copepod order; mostly marine. Four families are often abundant in the marine plankton. Some feed visually (Corycaeidae and Sapphirinidae). Includes the family Ergasilidae. Superorder Gymnoplea — Articulation between prosome and urosone is between ﬁfth pedigerous and genital somites. This is the ancestral tagmosis that includes the Platycopioida and Calanoida. Superorder Podoplea — Articulation between prosome and urosome is between fourth and ﬁfth pedigerous somites. This is the derived tagmosis, and it includes the other eight orders. From Huys and Boxshall, 1991. 952 C. E. Williamson and J. W. Reid TABLE II North American Copepods of the Order Calanoida G. O. Sars, 1903 Family Acartiidae G. O. Sars, 1903 Acartia: sinensis Shen and Lee, 1963 (I) Family Aetideidae Giesbrecht, 1892 Senecella: calanoides Juday, 1923 Family Centropagidae Giesbrecht, 1892 Limnocalanus: johanseni Marsh, 1920; macrurus G. O. Sars, 1863. Osphranticum: labronectum S. A. Forbes, 1882. Sinocalanus: doerri (Brehm, 1909) (I) (see Orsi et al., 1983). Family Diaptomidae G. O. Sars, 1903 Acanthodiaptomus: denticornis (Wierzejski, 1887). Aglaodiaptomus: atomicus DeBiase and Taylor, 1997; clavipes (Schacht, 1897); clavipoides M. S. Wilson, 1955; conipedatus (Marsh, 1907); dilobatus M. S. Wilson, 1958; forbesi Light, 1938; kingsburyae Robertson, 1975; leptopus (S. A. Forbes, 1882); lintoni (S. A. Forbes, 1893); marshianus M. S. Wilson, 1953; pseudosanguineus (Turner, 1921); saskatchewanensis M. S. Wilson, 1958; savagei DeBiase and Taylor, 2000; spatulocrenatus (Pearse, 1906); stagnalis (S. A. Forbes, 1882). Arctodiaptomus: arapahoensis (Dodds, 1915); bacillifer (Koelbel, 1884); dorsalis (Marsh, 1907); ﬂoridanus (Marsh, 1926); kurilensis Kiefer, 1937; saltillinus (Brewer, 1898). Key: Reddy, 1994, Reed, 1994. Diaptomus: glacialis Lilljeborg, 1889. Eudiaptomus: gracilis (G. O. Sars, 1863); yukonensis Reed, 1991. Hesperodiaptomus: arcticus (Marsh, 1920); augustaensis (Turner, 1910); breweri M. S. Wilson, 1958; caducus Light, 1938; californiensis Scanlin and Reid, 1996; eiseni (Lilljeborg, 1889); franciscanus (Lilljeborg, 1889); hirsutus M. S. Wilson, 1953; kenai M. S. Wilson, 1953; kiseri (Kincaid, 1953); nevadensis Light 1938; novemdecimus M. S. Wilson, 1953; schefferi M. S. Wilson, 1953; shoshone (S. A. Forbes, 1893); victoriaensis Reed, 1958; wardi (Pearse, 1905); wilsonae Reed, 1958. Leptodiaptomus: ashlandi (Marsh, 1893); assiniboiaensis (Anderson, 1970); coloradensis (Marsh, 1911); connexus Light, 1938; cuauhtemoci (Osorio-Tafall, 1941; synonym assiniboiaensis Anderson, 1970); insularis Kincaid, 1956; judayi (Marsh, 1907); minutus (Lilljeborg, 1889); moorei M. S. Wilson, 1954; novamexicanus (Herrick, 1895); nudus (Marsh, 1904); pribilofensis (Juday and Muttkowski, 1915); sicilis (S. A. Forbes, 1882); siciloides (Lilljeborg, 1889); signicauda (Lilljeborg, 1889); spinicornis Light, 1938; trybomi (Lilljeborg, 1889); tyrrelli (Poppe, 1888). Mastigodiaptomus: albuquerquensis (Herrick, 1895); texensis M. S. Wilson, 1953.Key: Suárez-Morales and Elías-Gutiérrez, 2000. Mixodiaptomus: theeli (Lilljeborg, 1889). Nordodiaptomus: alaskaensis M. S. Wilson, 1951. Onychodiaptomus: birgei (Marsh, 1894); hesperus M. S. Wilson and Light, 1951; louisianensis M. S. Wilson and Moore, 1953; sanguineus (S. A. Forbes, 1876); virginiensis (Marsh, 1915). Sinodiaptomus: sarsi (Rylov, 1923)(I). Key: Reddy, 1994. Skistodiaptomus: bogalusensis M. S. Wilson and Moore, 1953; bogalusensis marii Harris, 1978; carolinensis Yeatman, 1986; mississippiensis (Marsh, 1894); oregonensis (Lilljeborg, 1889); pallidus (Herrick, 1879); pygmaeus (Pearse, 1906); reighardi (Marsh, 1895); sinuatus Kincaid, 1953. Family Temoridae Giesbrecht, 1892 Epischura: ﬂuviatilis Herrick, 1883; lacustris S. A. Forbes, 1882; massachusettsensis Pearse, 1906; nevadensis Lilljeborg, 1889; nordenskioldi Lilljeborg, 1889. Eurytemora: afﬁnis (Poppe, 1880); americana Williams, 1906; arctica M. S. Wilson and Tash, 1966; bilobata Akatova, 1949 (synonym yukonensis M. S. Wilson, 1953); canadensis Marsh, 1920; composita Keiser, 1929; gracilicauda Akatova, 1949. Heterocope: septentrionalis Juday and Muttkowski, 1915. Identiﬁcation is based primarily on males. Species primarily occurring in brackish coastal waters, some of which may occasionally enter fresh water, are not included. Introduced species are indicated by (I). Keys and taxonomic revisions published after 1959 are noted. 22. Copepoda TABLE III 953 North American Copepods of the Order Cyclopoida Burmeister, 1834 Family Cyclopidae Dana, 1853 Acanthocyclops: brevispinosus (Herrick, 1884); capillatus (G. O. Sars, 1863); carolinianus Yeatman, 1944; columbiensis Reid, 1990; exilis (Coker, 1934); montana Reid and Reed in Reid et al., 1991; parasensitivus Reid, 1998; parvulus Strayer, 1989; pennaki Reid, 1992; robustus (G. O. Sars, 1863); venustoides (Coker, 1934); vernalis (Fischer, 1853). Key: Einsle, 1996a. Apocyclops: dimorphus (Kiefer, 1931); panamensis (Marsh, 1913); spartinus Ruber, 1968. Bryocyclops: muscicola (Menzel, 1926). (I) Cryptocyclops: bicolor (G. O. Sars, 1862). Cyclops: canadensis Einsle, 1988; columbianus Lindberg, 1956 (inadequately described species); furcifer (Claus, 1857); kolensis alaskaensis Lindberg, 1956; laurenticus Lindberg, 1956 (inadequately described); scutifer G. O. Sars, 1863; strenuus Fischer, 1851; vicinus Uljanin, 1875. Revisions: Reed (1995), Reed and McIntyre (1995); Key: Einsle, 1996a. Diacyclops: alabamensis Reid, 1992; albus Reid, 1992; bernardi (Petkovski, 1986); bicuspidatus (Claus, 1857); bicuspidatus lubbocki (Brady, 1868); bisetosus (Rehberg, 1880); chrisae Reid, 1992; crassicaudis (G. O. Sars, 1863); crassicaudis var. brachycercus (Kiefer, 1927); dimorphus Reid and Strayer, 1994; harryi Reid, 1992; haueri (Kiefer, 1931); hypnicola (Gurney, 1927); jeanneli (Chappuis, 1929); jeanneli putei (Yeatman, 1943); languidoides (Lilljeborg, 1901) s.l.; languidus (G. O. Sars, 1863); nanus (G. O. Sars, 1863); navus (Herrick, 1882); nearcticus Kiefer, 1934; palustris Reid, 1988; sororum Reid, 1992; thomasi (S. A. Forbes, 1882); yeatmani Reid, 1988; Key: Reid, 1988, emend. Reid et al., 1989. Ectocyclops: phaleratus (Koch, 1838); polyspinosus Harada, 1931; rubescens Brady, 1904. Eucyclops: agilis (Koch, 1838); agilis montanus (Brady, 1878); bondi Kiefer, 1934; conrowae Reid, 1992; elegans (Herrick, 1884; synonym E. neomacruroides Dussart and Fernando, 1990); macruroides denticulatus (Graeter, 1903); prionophorus Kiefer, 1931. Homocyclops: ater (Herrick, 1882). Itocyclops: yezoensis (Ito, 1954) (see Reid and Ishida, 2000). Macrocyclops: albidus (Jurine, 1820); fuscus (Jurine, 1820). Megacyclops: donnaldsoni (Chappuis, 1929); gigas (Claus, 1857); latipes (Lowndes, 1927); magnus (Marsh, 1920); viridis (Jurine, 1820)(I). Key: Einsle, 1996a. Mesocyclops: americanus Dussart, 1985; edax (S. A. Forbes, 1891); longisetus (Thiébaud, 1912); longisetus var. curvatus Dussart, 1987; ruttneri Kiefer, 1981 (I); venezolanus Dussart, 1987. Key: Reid and Reed, 1994. Metacyclops: cushae Reid, 1991; in Reid, 1991a. Microcyclops: pumilis Pennak and Ward, 1985; rubellus (Lilljeborg, 1901); varicans (G. O. Sars, 1862). Orthocyclops: modestus (Herrick, 1883). Paracyclops: canadensis (Willey, 1934); chiltoni (Thomson, 1882); ﬁmbriatus (Fischer, 1853); poppei (Rehberg, 1880); smileyi Strayer, 1989; yeatmani Daggett and Davis, 1974. Key: Karaytug, 1999. Rheocyclops: carolinianus Reid, in Reid et al., 1999; hatchiensis Reid and Strayer in Reid et al., 1999; talladega Reid and Strayer in Reid et al., 1999; virginianus (Reid, 1993). Key: Reid et al., 1999. Stolonicyclops: heggiensis Reid and Spooner, 1998. Thermocyclops: crassus (Fischer, 1853) (I); inversus Kiefer, 1936; parvus Reid, 1989; tenuis (Marsh, 1909). Revision: Reid (1989a); Key: Herbst, 1986. Tropocyclops: extensus Kiefer, 1931; extensus f. longispina Kiefer, 1931; jerseyensis Kiefer, 1931; prasinus (Fischer, 1860); prasinus mexicanus Kiefer, 1938. Key: Reid, 1991b. Family Oithonidae Dana, 1853 Limnoithona: sinensis (Burckhardt, 1912) (I). Identiﬁcation is based primarily on females. Species primarily occurring in brackish coastal waters, some of which may occasionally enter freshwater, are not included. Introduced species are indicated by (I). Keys and taxonomic revisions published after 1959 are noted. 954 C. E. Williamson and J. W. Reid TABLE IV North American Copepods of the Order Harpacticoida G. O. Sars, 1903 Family Ameiridae Monard, 1927 Nitocrellopsis: texana Fiers and lliffe, 2000 Nitokra: hibernica (Brady, 1880) (I); lacustris (Shmankevich, 1875). Psammonitocrella: boultoni Rouch, 1992; longifurcata Rouch, 1992. Stygonitocrella sequoyahi Reid, Hunt, and Stanley, 2001. Family Canthocamptidae Brady, 1880 Attheyella: americana (Herrick, 1884); carolinensis Chappuis, 1932; dentata (Poggenpol, 1874); dogieli (Rylov, 1923); idahoensis (Marsh, 1903); illinoisensis (S. A. Forbes, 1882); nordenskioldi (Lilljeborg, 1902); obatogamensis (Willey, 1925); pilosa Chappuis, 1929; spinipes Reid, 1987; trispinosa (Brady, 1880); ussuriensis Rylov, 1933. Bryocamptus: arcticus (Lilljeborg, 1902); calvus (Brehm, 1927); cuspidatus (Schmeil, 1893); douwei (Willey, 1925); hiatus (Willey, 1925); hiemalis (Pearse, 1905); hutchinsoni Kiefer, 1929; minnesotensis (Herrick, 1884; inadequately described species); minusculus (Willey, 1925); minutus (Claus, 1863); morrisoni (Chappuis, 1929); morrisoni elegans (Chappuis, 1929); newyorkensis (Chappuis, 1927); nivalis (Willey, 1925); pilosus Flössner, 1989; pygmaeus (G. O. Sars, 1862); subarcticus (Willey, 1925); tikchikensis M. S. Wilson, 1958; umiatensis M. S. Wilson, 1958; vejdovskyi (Mrázek, 1893); vejdovskyi f. minutiformis Kiefer, 1934; washingtonensis M. S. Wilson, 1958; zschokkei (Schmeil, 1893); zschokkei alleganiensis Coker, 1934. Canthocamptus: assimilis Kiefer, 1931; oregonensis M. S. Wilson, 1956; robertcokeri M. S. Wilson, 1958; sinuus Coker, 1934; staphylinoides Pearse, 1905; staphylinus (Jurine, 1820) (records of staphylinus in North America are doubtful); vagus Coker and Morgan, 1940. Elaphoidella: amabilis Ishida in: Reid and Ishida, 1993; bidens (Schmeil, 1894); californica M. S. Wilson, 1975; carterae Reid in: Reid and Ishida, 1993; ﬂuviusherbae Bruno and Reid, in Bruno et al., 2000); kodiakensis M. S. Wilson, 1975; marjoryae Bruno and Reid, in Bruno et al., 2000); reedi M. S. Wilson, 1975; shawangunkensis Strayer, 1989; subgracilis (Willey, 1934); tenuicaudis (Herrick, 1884; inadequately described species); wilsonae Hunt, 1979. Key: Reid and Ishida, 1993. Epactophanes: richardi Mrázek, 1893. Gulcamptus: alaskaensis Ishida, 1996 in: Reid and Ishida, 1996; huronensis Reid, 1996 in Reid and Ishida, 1996; laurentiacus (Flössner, 1992). Revision: Reid and Ishida, 1996. Maraenobiotus: brucei (Richard, 1898); canadensis Flössner, 1992; insignipes (Lilljeborg, 1902). Mesochra: alaskana M. S. Wilson, 1958. Moraria: afﬁnis Chappuis, 1927; arctica Flössner, 1989; cristata Chappuis, 1929; duthiei (T. and A. Scott, 1896); laurentica Willey, 1927; mrazeki T. Scott, 1902; virginiana Carter, 1944. Paracamptus: reductus M. S. Wilson, 1956; reggiae M. S. Wilson, 1958. Family Canthocamptidae incertae sedis: Cletocamptus: albuquerquensis (Herrick, 1895); brevicaudata (Herrick, 1895); deitersi (Richard, 1897). Family Huntemanniidae Por, 1986 Huntemannia: lacustris M. S. Wilson, 1958. Nannopus: palustris Brady, 1880. Family Laophontidae T. Scott, 1904 Onychocamptus: mohammed (Blanchard and Richard, 1891). Family Parastenocarididae Chappuis, 1940 Parastenocaris: brevipes Kessler, 1913; delamarei Chappuis in Chappuis and Delamare Deboutteville, 1958; lacustris Chappuis in Chappuis and Delamare Deboutteville, 1958; palmerae Reid, 1992; texana Whitman, 1984; trichelata Reid, 1995. Family Phyllognathopodidae Gurney, 1932 Phyllognathopus: viguieri (Maupas, 1892). Identiﬁcation is based on either sex. Species primarily occurring in brackish coastal waters, some of which may occasionally enter freshwater, are not included. Introduced species are indicated by (I). Keys and taxonomic revisions published after 1959 are noted. TABLE V North American Copepods of the Order Poecilostomatoida Thorell, 1859 Family Ergasilidae Nordmann, 1832 Ergasilus: arthrosis Roberts, 1969; auritus Markevich, 1940; caeruleus C. B. Wilson, 1911; celestis Mueller, 1937; centrarchidarum Wright, 1882; cerastes Roberts, 1969; chautauquaensis Fellows, 1887; clupeidarum Johnson and Rogers, 1972; cotti Kellicott, 1892; cyprinaceus Rogers, 1969; elongatus C. B. Wilson, 1916; japonicus Harada, 1930 (I); lanceolatus C. B. Wilson, 1916; luciopercarum Henderson, 1926; megaceros C. B. Wilson, 1916; nerkae Roberts, 1963; rhinos Burris and Miller, 1972; sieboldi Nordmann, 1832; tenax Roberts, 1965; versicolor C. B. Wilson, 1911; wareaglei Johnson, 1971. Adult females are ectoparasites of ﬁshes, but males and copepodids are planktonic. Identiﬁcation is based on females. Species primarily occurring in brackish coastal waters, some of which may occasionally enter freshwater, are not included. Introduced species are indicated by (I). No keys or taxonomic revisions have been published since 1959. 23 DECAPODA H. H. Hobbs III Department of Biology Wittenberg University Springﬁeld, Ohio 45501 I. Introduction II. Anatomy and Physiology A. External Morphology B. Organ System Function C. Aspects of Decapod Physiology III. Ecology and Evolution A. Diversity, Distribution, and Evolutionary Relationships B. Reproduction and Life History C. Ecological Interactions I. INTRODUCTION The Decapoda (Latreille) represent a very signiﬁcant order that is assigned to the class Malacostraca and encompasses an immense diversity of marine, freshwater, and semiterrestrial crustaceans, with some 10,000 species having been described. The two infraorders treated in this chapter, Caridea and Astacidea, are nearly worldwide in distribution and for purposes of this discussion are represented by freshwater shrimps and crayﬁshes (see Bowman and Abele, 1982 for a detailed classiﬁcation; see also Hart, 1994). These aquatic arthropods have successfully invaded a wide variety of aquatic and semiaquatic habitats, occurring as obligate cave-dwellers [stygobites ( stygobionts)], as stream, lake, pond, and swamp dwellers, and as primary burrowers; a few even invade saline environments. The crayﬁshes and one genus of shrimps (Macrobrachium), in particular, attain the greatest size among the freshwater crustaceans in North America. Summarized in this chapter are the ecology and distribution of shrimps and crayﬁshes in North American freshwaters (treatment generally restricted to the United States). Species richness and niche diversiﬁcation are particularly well demonstrated in aquatic ecosystems of Ecology and Classiﬁcation of North American Freshwater Invertebrates. 2nd Edition Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved. IV. Current and Future Research Problems V. Collecting and Rearing Techniques A. Shrimps B. Crayﬁshes: Astacidae and Cambaridae VI. Identiﬁcation A. Preparation of Specimens B. Taxonomic Key to Genera of Freshwater Decapoda Literature Cited the southeastern United States. That these crustaceans have successfully colonized such diverse habitats is reﬂected in various morphological and physiological adaptations discussed. Life-history patterns are reviewed, particularly with reference to how they are inﬂuenced by environmental conditions. An examination is made of the role of these decapods in the functioning of various aquatic communities. As might be anticipated, a much larger body of data is available for crayﬁshes than for shrimps and is reﬂected in the treatment of these groups. Under each major section of the chapter, a discussion of shrimps appears ﬁrst, followed by that of crayﬁshes. Where data are somewhat limited (particularly with reference to shrimps), both groups are discussed in the same section. II. ANATOMY AND PHYSIOLOGY A. External Morphology Although decapods represent the greatest diversity among orders of crustaceans, all possess a number of common characteristics, including a carapace that encloses the bronchial chamber. In addition, the ﬁrst three 955 956 H. H. Hobbs III pairs of thoracic appendages are modiﬁed in all species as maxillipeds. Carideans (Fig. 1) are easily distinguished from other shrimp groups (e.g., penaeids, stenopodids) by the large second abdominal pleura that overlap those of both the ﬁrst and third somites; moreover, all carideans lack terminal chelae on the third pereiopods (Fig. 1a). Astacideans (Fig. 2) include the crayﬁshes of the northern and southern hemispheres and the marine lobsters; only North American crayﬁshes are treated here. In contrast to the carideans, crayﬁshes are rarely compressed laterally and their ﬁrst three pairs of pereiopods are always chelate. Extensive body segmentation and the presence of jointed appendages on all metameres give most decapods a primitive appearance; yet the nervous, sensory, circulatory, and digestive systems are complex, a necessary requirement for animals of large body size. The body of these freshwater decapods is encased in an exoskeleton consisting of complex polysaccharides hardened with inorganic salts (except at joints, where the cuticle is thin and pliable). The head and thoracic segments are fused to form a large cephalothorax covered by a single shield, the carapace (Figs. 1 and 2), but the six abdominal segments are individually distinct. The anterior portion of the cephalothorax contains a pair of large, stalked eyes, a median rostrum, two pairs of antennae, and a pair of mandibles. The body is composed of somites, each with a pair of ventral, jointed appendages that are serially homologous and basically biramous (Fig. 3), but are modiﬁed for various functions (e.g., sensory, food handling, cleaning, gill-bailing, pinching, walking, copulation, egg attachment and incubation, and swimming). The typical biramous appendage is Yshaped and the base of the Y is attached to the somite. The base is the protopodite (consisting of two joints — coxopodite and basiopodite) that bears a mesial endopodite, typically of ﬁve podomeres, and a lateral exopodite, with few to many segments. B. Organ System Function The digestive system of crayﬁshes is simple and includes the mouth, a short tubular esophagus, the stomach, two large hepatopancreatic glands, a short midgut, and a long tubular intestine extending dorsally through the abdomen to the anus (Fig. 4). The relatively simple, straight gastrointestinal tract has few signiﬁcant areas for storage or microbial degradation although the stomach functions as a storage compartment (cardiac portion), as a masticating structure (gastric mill), and as a ﬁlter for gathering digestible material (pyloric portion). Morphological differences among species in the grinding surfaces of the gastric mill relate to diet (Caine, 1975). For example, the surface is reduced in FIGURE 1 (A) Lateral view of generalized shrimp (after Hobbs and Jass, 1988). Ai, appendix interna; Am, appendix masculina; As, antennal spine; B, basis; Bg, branchiostegal groove; Bs, branchiostegal spine; Cp, carpus; D, dactyl; end, endopod; I. ischium; M, merus; P, propodus; and Sc, scaphocerite. (B) Chela of second pereiopod with apical tufts of setae; Palaemonias ganteri (after Hobbs et al., 1977). 23. Decapoda FIGURE 2 Dorsolateral view of generalized crayﬁsh. Al, areola length; Ar, areola; As, antennal scale; Aw, areola width; B, basis; Bcg, branchiocardiac groove; Bs, branchiostegal spine; C,carpus; Car, rostral carina; Cs, cervical spine; D, dactyl of chela; I, ischium; Lru, lateralramus of uropod; M, merus; Mru, mesial ramus of uropod; Ms, marginal spine; P, propodus-palm of chela; Pl, palm length; Ple, pleuron; Pocl, postorbital carapace length; Pr, postorbital ridge; Ps, postorbital spine; Pw, width of palm; R, rostrum; Soa, suborbital angle; Ter, tergum; and pleopods on ventral side of abdomen. FIGURE 3 Biramous appendages:(a) Palaemonias alabamae, third maxilliiped (after Smalley, 1961); (b) Cambarus (Depressicambarus) strigosus Hobbs, dorsal view of telson and biramous uropod (after Hobbs, 1981); and (c) Procambarus (Ortmannicus) lunzi (Hobbs), third pereiopod. 957 958 H. H. Hobbs III FIGURE 4 Generalized diagram of crayﬁsh, demonstrating internal organization. some cave crayﬁshes that feed on organic silt and expanded in epigean species that utilize macromaterials. The pH of the stomach is relatively alkaline and the enzymes of the gastrointestinal tract and hepatopancreas are particularly effective in the processing of a wide spectrum of food materials. Chymotrypsin and pepsin are lacking, but an alkaline protease, trypsin, cellulase (hepatopancrease origin), lysozyme (muramidase), and probably chitinase and chitobiase are present (Brown, 1995). The circulatory system is an open or lacunar system and consists of a heart, arteries (no veins), and sinuses. The heart is situated in a large middorsal pericardial sinus, and blood in the sinus enters the heart via three pairs of ostia (valves). The nearly colorless blood is pumped into arteries that distribute it to various organs and afferent sinuses leading to the gills, and the blood returns through a series of efferent sinuses that ultimately join to the pericardial sinus. Hemocyanin is the principal blood pigment transporting oxygen. The gills are attached to the thoracic appendages (maxillipeds and pereiopods) and are situated along either side of the thorax in the bronchial (gill) chamber. Lateral extensions of the carapace (branchiostegites) cover the gills (Figs. la, 2, and 7). In crayﬁshes, anterior to the gills, paddlelike second maxillae (scaphognathites, see Burggren and McMahon, 1983) beat back and forth, drawing water over the gill ﬁlaments to ensure gas exchange. The gills of crayﬁshes (trichobranchs composed of unbranched, ﬁngerlike ﬁlaments) and shrimps (phyllobranchs consisting of platelike elements) are arranged in three series: the single podobranchia borne on the coxa, the arthrobranchiae on the articular membrane between the coxa and basis, and the pleurobranchia borne on the pleura. The pleurobranch series is absent in all of the North American crayﬁshes except in members of the genus Pacifastacus, which have a single pair. Considerable water constantly diffuses into the blood through the gill surfaces of freshwater decapods. Osmotic control and excretion are maintained by two large antennal (green) glands situated ventral to the subesophageal ganglia and anterior to the esophagus. These glands have an intimate association with the circulatory system and thus maintain water balance (Flynn and Holliday, 1994). The green gland consists of a complexly folded end sac, a convoluted labyrinth, a nephridial canal, a bladder, and an excretory pore situated at the coxae of the second antennae. Crayﬁshes excrete a quantity of hypotonic urine (some ammonia, urea, uric acid, and amines), which is generally isosmotic with the blood. High ammonia excretion rates have been correlated with low pH values (pH 4.6; Daveikis and Alikhan, 1996). The central nervous system functions as the destination for sensory input, the center of synaptic integration, and the source of motor control and consists of paired supraesophageal and subesophageal ganglia, which are joined, forming the “brain.” This juncture is accomplished by a pair of connectives extending caudally from the subesophageal ganglia to the double ventral nerve cord which has segmental ganglia connected by commissures (see Sandeman et al., 1992 for a proposed common nomenclature for homologous structures of the brain of crayﬁshes, crabs, and spiny lobsters). Of signiﬁcance, 30 – 40% of crayﬁsh brain volume is devoted to processing olfactory input (Mellon et al., 1992). Cuadras (1993) evaluated the eleven categories of secretory organelles (membrane bound carriers) present in the central nervous system of crayﬁshes. The peripheral nervous system consists of nerves and receptor cells that function primarily as conducting 23. Decapoda pathways (see Swerup and Rydqvist, 1992). Atwood and Nguyen (1995) reviewed neural adaptations of crayﬁshes to different activity levels, describing how various species are equipped for a variety of environmental changes. The basal segment of each ﬁrst antenna supports an organ of balance (statocyst). Chemoreceptors are abundant on mouth parts, pereiopods, and antennae; these last appendages also function as tactile organs. Lateral rami of the antennules bear chemoreceptive sensilla called aesthetascs that are innervated by sensory neurons. Tierney and Dunham (1984) and Tierney et al. (1984) suggested that these structures may be receptive to pheromones although there have been conﬂicting results from various studies concerning under what conditions and the location of receptor sites where chemical sensation takes place (see Oh and Dunham, 1991; Dunham and Oh, 1992). Rutherford et al. (1996) reported that in agonistic encounters of conspeciﬁc Form I males (breeding males — see below) of Orconectes (Procericambarus) rusticus (Girard), winners had higher rates of antennal movements than losers. Winners also demonstrated longer periods of “large amplitude depression” (LAD — holding antennules approximately 45° below the horizontal plane) which is used often in aggressive encounters. The researchers postulated that the increased use of crayﬁsh LAD movements may be in response to some “stress pheromone” delivered to them by their opponent’s maxillipeds. Pheromones and other biochemicals are used by crayﬁshes to recognize species, sex, food, the presence of animals that are stressed, disturbed, or physically damaged. These alarm substances are widespread and the detection of these will elicit distinct responses by crayﬁshes (Hazlett, 1994b). Dunham et al. (1997) examined the use of the inner and outer rami of the antennules in mediation of feeding behavior of Cambarus (Cambarus) bartonii bartonii (Fabricius) and found that the several behavior patterns used were dependent on the presence of either inner or outer antennular rami, but not on both. Many setae on the body surface contain sensory neurons and are mechanoreceptors of one kind or another and at least three sensory structures on the chelipeds appear to have chemo- and mechanosensory function (Borash and Moore, 1997). A pair of large compound eyes characterizes most decapods [excluding stygobites (obligate, aquatic cave dwellers)], and reduced eye size in most primary burrowing species); each eye is composed of a number of virtually identical, discrete optical units called ommatidia (“mosaic eyes” — good for detecting movement, but limited resolving power) that are arranged in geometrical array. In addition to compound eyes, a pair of neurons originating in the sixth 959 abdominal ganglion function as extraocular photoreceptors and are responsible for photonegative behavior, but also receive information from mechanoreceptors in the abdomen (see Fernández-de-Miguel and Aréchiga, 1992; Pel and Moss, 1996). Further details of the morphology of decapods, in general, can be examined in numerous invertebrate zoology textbooks (see also Holdich and Reeve, 1988), and the external anatomy of crayﬁshes speciﬁcally is treated well by Crocker and Barr (1968). C. Aspects of Decapod Physiology 1. Respiration The adaptation of organisms to various habitats usually involves physiological, biochemical, and behavioral adjustments. Obviously a dynamic interaction exists between the external environment of an organism and the functioning of its internal machinery. For example, reduced metabolic rates in cave decapods may stem from biochemical and physiological adaptations to greater environmental stability, to low number of predators, or to a severely constrained resource base in terms of quantity and variety (Huppop, 1985), as demonstrated by whole animal respiration studies (see Weingartner, 1977). Also, the reduction of oxygen consumption and energy turnover of gill tissues reported by Dickson and Franz (1980) further substantiate the highly specialized nature of physiological and biochemical adaptations in stygobitic animals. In order for any life form to survive, the variety of factors constituting the ambient environmental complex must not exceed genetically determined tolerance limits for that species. That crayﬁshes and shrimps have radiated into nearly every type of aquatic habitat suggests that these crustaceans have been tolerant and highly adaptable, particularly with regard to respiratory physiology. For example, Wiens and Armitage (1961) demonstrated that, for the crayﬁshes Orconectes (Trisellescens) immunis (Hagen) and 0. (Gremicambarus) nais (Faxon), the effect of body size on oxygen consumption and the effects of both temperature and oxygen saturation on oxygen consumption by them are highly signiﬁcant (inverse relationship between body weight and metabolic rate; direct relationship between temperature and oxygen consumption). They also showed that the cumulative effect of increased temperature and lowered oxygen concentration produces greater stress than either parameter acting independently. 0. immunis has a daily metabolic rate 10% lower than 0. nais, and 0. nais does not regulate as well as does 0. immunis when under stressed conditions. Therefore, the inability of 0. nais to maintain a higher and more nearly regulated metabolic rate (as 960 H. H. Hobbs III FIGURE 5 Cambarus tenebrosus from epigean and hypogean (stygophile) lotic habitats in the midwestern and southeastern United States. does 0. immunis) under such conditions, probably excludes it from roadside ditches where 0. immunis is found. Hence, 0. immunis is better adapted physiologically to live under environmental conditions of periodic high temperatures and low oxygen saturations (see Reiber, 1997). Daveikis and Alikhan (1996) found that Cambarus (Puncticambarus) robustus Girard from an acidic, metal-contaminated lake in Ontario, Canada, had lower oxygen consumption rates than conspeciﬁc crayﬁshes from a circumneutral, uncontaminated, high velocity stream. Nelson and Hooper (1982) showed that the shrimp, Palaemonetes kadiakensis Rathbun, exhibited an acute thermal preference within a temperature range of 6 – 10°C below their maximum tolerance limits of 37 – 40°C. This shrimp was unable to tolerate FIGURE 6 sustained exposure to temperatures greater than 32°C. For additional treatment of metabolism, temperature acclimation, preference, and avoidance in these two groups of crustaceans, see McWhinnie and O’Connor (1967), Crawshaw (1974), Becker et al. (1975), Loring and Hill (1978), Mathur et al. (1982), Layne et al. (1985), and Reiber (1995). Burrowing crayﬁshes are often observed above the water table; many species are capable of remaining out of water for extended periods in the humid environment of their tunnels (Hobbs, 1981; McMahon and Hankinson, 1993, and McMahon and Stuart, 1995). The same is true for many cavernicolous crayﬁshes (stygophiles – Fig. 5, and stygobites – Fig. 6), which, in the saturated air, can move from one pool to another or remain out of the wa- Orconectes pellucidus, a stygobitic crayﬁsh from Kentucky and Tennessee. 23. Decapoda ter for hours. Burrowers and surface-dwelling crayﬁshes are commonly noted at the air – water interface, aerating in response to low oxygen levels; the oxygen concentration in burrow waters is frequently below 2 mg / L (Hobbs, 1981), and pool and ditches often become oxygen-depleted. These species that inhabit such oxygenpoor habitats have evolved a greater branchial volume by adopting several characteristics (Figs. 7 and 8), such as a vaulted and elongated carapace as well as branchiocardiac grooves that are closer together thus narrowing the areola and raising the height of the chamber. Kushlan and Kushlan (1980) observed the shrimp Palaemonetes paludosus (Gibbes) swimming at the surface using oxygen that diffused across it as a means of surviving low oxygen tension in the Everglades. Many species (e.g., the crayﬁsh Procambarus (Girardiella) simulans (Faxon)) are oxygen regulators and increase their ventilation rates in response to reduced oxygen or increased carbon dioxide concentrations. Others [e.g., Pacifastacus (Pacifastacus) l. leniusculus (Dana)] are oxygen conformers, lacking speciﬁc adaptations for regulation of oxygen uptake at low oxygen tensions (see Vernberg, 1983). For further discussion of respiratory physiology in crayﬁshes and shrimps, see 961 FIGURE 7 Semi-diagrammatic cross section through the thoracic region of (a) Cambarus diogenes, a primary burrower living in oxygen-poor groundwaters and (b) Procambarus(Pennides) versutus (Hagen), dwelling in the oxygen-rich lotic environment (after Hobbs, 1976); arrows point to branchiocardiac grooves. Moshiri et al. (1970), Cox and Beauchamp (1982), Nelson and Hooper (1982), and Reiber (1995). 2. Ecdysis Molting (ecdysis), a process critical for growth and seasonal changes in form (dimorphic only in male cambarines), is a recurring crisis in the lives of shrimps and FIGURE 8 Dorsolateral views of (a) Cambarus (Jugicambarus) gentryi Hobbs, a primary burrower (Cheatham County, TN); (b) Procambarus lunzi, found in roadside ditch lentic environments (Hampton County, SC); (c) Cambarus (Jugicambarus) distans Rhoades, a dweller of swift, surface streams (Dade County, GA); and (d) Procambarus (Ortmannicus) enoplosternum Hobbs, occupies sluggish surface streams (Washington County, GA). 962 H. H. Hobbs III crayﬁshes. It involves far more than periods of discontinuous growth facilitated by a simple splitting of the cuticle and secretion of a new exoskeleton. The life of a decapod consists of alternating periods of premolt, molt, postmolt, and intermolt; all of these phases are controlled by hormones (endocrines). Neurosecretory cells occur in the sensory papillae and in the X-organs of the medulla terminalis; both secretary masses are situated in the eye stalk. The sinus gland, also located within the eye stalk, receives secretions from the Xorgans and, in turn, produces hormones that inhibit ecdysis. The Y-organs, a pair of glands in the maxillary somites, secrete hormones derived from dietary cholesterol (three ecdysteroids: ecdysone; 25-deoxyecdysone; and 3-dehydroecdysone) that stimulate molting (under the control of the X-organ). During postmolt and intermolt, the X-organ / sinus gland complex liberates a neuropeptide molt-inhibiting hormone, thus regulating the length of the intermolt period (see Lachaise et al., 1993). Under appropriate external or internal conditions (light, temperature, loss of limbs — see Stoffel and Hubschman, 1974), sinus gland hormones are not released and the Y-organ, no longer suppressed, secretes the molt-initiating hormone. This acts on the epidermis to initiate premolt activities that affect most body parts. Glycogen reserves are increased and minerals (e.g., calcium) are resorbed from the exoskeleton and stored. With the secretion of an enzyme that softens the cuticle at its base, it pulls away from the epidermal cells (apolysis), stimulating the formation of a new epicuticle that is impervious to the molting enzyme. At the termination of premolt, the old cuticle splits, the animal emerges from the old exoskeleton (exuvium) including shedding the cuticle covering the gills (see Andrews and Dillaman, 1993), and the soft body within retains the maximum volume due to imbibing of water by the decapod. Tissues grow rapidly; and during the short postmolt period, the new layer becomes rigid as the stored minerals are deposited in the hardening cuticle. Bendell-Young and Harvey (1991) found that crayﬁshes from acid lakes have carapaces with lower Mg concentrations than those from circumneutral lakes, suggesting that low pH conditions may prevent normal mineralization during molting. After postmolt, the crustacean may enter intermolt; however, complete intermolt probably occurs only in adults, and the rapidly growing (molting) juveniles generally have, at best, a very short period of stability (see Aiken and Waddy, 1992; Wheatly, 1995; Wheatly et al., 1996). 3. Reproductive Physiology The development and function of gonads and the ontogeny of secondary sexual characters in the dioecious decapods are regulated by hormones. The Y-organ inﬂuences the development and maturation of gonads in juveniles. In females, the sinus gland / X-organ complex is important in controlling the ovary. During nonbreeding periods, the sinus gland produces a hormone that inhibits egg development. However, during the breeding season, the blood level of this hormone declines and egg development is subsequently initiated. Development of the testes and male sexual characteristics is controlled by hormones produced in the androgenic gland, a small mass of secretory tissue near the vas deferens (Taketomi et al., 1996). Additional information concerning the reproductive cycle for shrimps and crayﬁshes is presented below. Even though the source and nature of interspeciﬁc and intraspeciﬁc chemicals (pheromones) are unknown for crayﬁshes, studies have shown that these chemical signals play an integral role in the behavior of these decapods, although other studies have not demonstrated this (see review by Bechler, 1995). The role of chemoreception in mediating agonistic behavior, species recognition, and mate location in crayﬁshes has been documented by Hazlett (1985). Discrimination between male and female conspeciﬁcs using chemical cues has been reported by some authors; these pheromones may promote sex recognition and induce mating behavior (see Hazlett, 1985; Dunham and Oh, 1996). Tierney et al. (1984) determined that the site of pheromone reception in Orconectes (Crockerinus) propinquus (Girard) is the lateral antennular ﬂagellum (exopod) bearing the chemoreceptive sensilla (aesthetascs). 4. Other Physiological Adaptations As ectotherms, these decapods are subjected to temperature changes that directly affect metabolic rate, and most species demonstrate various thermal tolerances (most upper tolerances range from 32 to 36°C) and preferences for their aquatic medium. Unfortunately, upper lethal temperatures are known only for a few species (see Cox and Beauchamp, 1982, and references therein). Nelson and Hooper (1982) showed experimentally that Palaemonetes kadiakensis exhibited an acute thermal preference (short-term response) within a temperature range of 6 – 10°C below their maximum tolerance limits. Taylor (1984) determined for three southeastern United States crayﬁshes that both the acute preference and ﬁnal preferendum primarily represented the temperature that they encountered during daily or seasonal activity cycles. He hypothesized that temperature choice does not function in temperature regulation except in the very broadest sense; instead, it serves as an “error detection system,” and cues for behavioral arousal from either seasonal hibernation or from their daily refugia. Clearly, water temperature plays an important role in the ecology of these crustaceans (see Mundahl and Benton, 1990). 23. Decapoda Biological rhythms are recognized in decapods and reviewed by DeCoursey (1983). Not only are organismic cycles pronounced (e.g., crayﬁshes have daily locomotor rhythms) but cellular and physiological rhythms are prominently exhibited (e.g., circadian rhythmicity of heart rate, Pollard and Larimer, 1977). Most seem to be in response to environmental cues (e.g., light, temperature); however, even where rhythmic environmental input may be lacking, circadian clock regulation can be maintained. For instance, Weingartner (1977) demonstrated a circadian activity pattern in the cave crayﬁsh Orconectes (Orconectes) i. inermis Cope from Indiana. In a similar way, the Kentucky stygobite 0. (Orconectes) pellucidus (Tellkampf) has retained a circadian clock regulation of locomotor activity and oxygen consumption in spite of isolation from day – night cycles. Crayﬁshes and shrimps are faced with the problems of constant inﬂux of water by osmosis and loss of salts by diffusion (Flynn and Holliday, 1994). Maintenance of constant body ﬂuid composition is accomplished by active sodium uptake via the gills, production of a copious hypotonic (dilute) urine, and by some reduction in boundary membrane permeability (see Newsom and Davis, 1991; Dickson et al., 1991; Sarver et al., 1994). As indicated above, the green gland is intimately associated with the circulatory system. The chromatophore is an integument cell with branched, radiating, noncontractile processes that are associated with one or more types of pigment granules. These white, red, yellow, blue, brown, or black pigments can ﬂow into the processes or may be conﬁned to the center of the cell. A single chromatophore may possess any number of pigments and is accordingly classiﬁed as mono-, bi-, di-, or polychromatic (see Fig. 9). In shrimps, the number of pigments and the number of chromatophores can change (morphological color change). However, physiological color change FIGURE 9 Female Palaemonetes kadiakensis with eggs (note chromatophores, e.g., small speckles on abdomen). 963 (rapid color adaptation to background resulting from dispersal and concentration of pigments within the chromatophores) is the more common type of color alteration and is controlled by hormones and the central nervous system. Chromatophorotropins are released from both the sinus gland and the central nervous system. The latter elaborates several chromatophorotropins, resulting in pigments either dispersing or concentrating in the chromatophores (darkening or blanching of body color). Color morphs of shrimps and crayﬁshes have been documented in a number of species, such as Macrobrachium rosenbergii (deMan), Orconectes propinquus, Procambarus (Ortmannicus) a. acutus (Girard), Procambarus (Scapulicambarus) paeninsulanus (Faxon). Studies have demonstrated that color variations may be due to genetic differences (e.g., Black, 1975) or they can be environmentally induced (see Fitzpatrick, 1987b; Thacker et al., 1993). Increasing awareness of human impact on the environment has directed attention to the effects of various pollutants on the physiological ecology of crustaceans, including shrimps and crayﬁshes. Accumulation of trace metals occurs through runoff or direct dumping of sewage or other efﬂuents into aquatic systems. Lead has become particularly important because of its relative toxicity and increased environmental contamination via automobile exhaust and highway runoff. Anderson (1978) demonstrated for 0. (Gremicambarus) virilis (Hagen) that exposure to lead resulted in damage to gills, which caused a decrease in the uptake of oxygen; ventilation rates increased as ambient lead concentrations increased. Hubschman (1967) noted that the toxic effect of copper on crayﬁshes is dependent not only on concentration and duration of exposure but also on the age (or size) of the crustacean (see Maranhão et al., 1995). At concentrations above 1 mg / L, respiratory enzymes are inhibited quite rapidly as the mechanism of detoxiﬁcation is apparently overwhelmed. At concentrations below 1 mg / L, copper has a chronic effect on cell maintenance. For example, actively secreting cells such as those of the antennal gland apparently are destroyed because cell repair cannot keep pace with cellular activity (see also Taylor et al., 1995). Cadmium, a highly toxic metal, has been implicated as both a carcinogen and a mutagen and causes a number of cardiovascular problems (all resulting from enzyme dysfunction). Cadmium is bioaccumulated and thus when incorporated in tissues of freshwater decapods can have far-reaching effects within aquatic ecosystems as well as in humans (e.g., Dickson et al., 1982; Thorp and Gloss, 1986; Bendell-Young and Harvey, 1991; Naqvi and Howell, 1993a, b). Vermeer (1972) noted high mercury levels in muscle tissue of 0. virilis from several localities 964 H. H. Hobbs III in Manitoba and Ontario and suggested that this species is a good indicator of mercury contamination in aquatic ecosystems (see also Wright and Welbourn, 1993). Insecticides (Albaugh, 1972), herbicides (Naqvi and Leung, 1983), lampricides, and numerous other chemicals have been shown to have various detrimental effects on freshwater decapods. The bioaccumulation of metals and other toxic or persistent chemicals poses particular problems for longlived cavernicoles (stygobites)(see below, Cooper and Cooper, 1978). Dickson et al. (1979) showed higher concentrations of cadmium and lead in tissues of Orconectes (Orconectes) a. australis (Rhoades) (a stygobite) than in those of Cambarus (Erebicambarus) tenebrosus [a stygophile — this species now inclusive of C. cahni (see Hobbs, 1989), C. laevis, and C. ornatus (see Taylor, 1997)], crayﬁshes occurring syntopically in a Tennessee cave. It is possible that some stygobitic decapod populations could be extirpated not from a single, massive dose of a chemical (though this has occurred; see Bechler, 1983; Hobbs III, 1987), but slowly from long-term exposure to low levels of these metals or other toxic substances. Because the longevity of primary burrowing species is apparently greater than that for nonburrowing taxa living in surface waters, a similar threat from chronic pollution exists for these crustaceans. This is particularly true for crayﬁshes living in roadside ditches that are periodically sprayed with various pesticides (Hubschman, 1967; Dimond et al., 1968). III. ECOLOGY AND EVOLUTION A. Diversity, Distribution, and Evolutionary Relationships Most freshwater decapods of North America occur in one or more of the following habitats: shallow (1 – 2 m depths, although this is probably a function of sampling bias) lentic and lotic waters, epigean and hypogean habitats, oligotrophic to hypereutrophic lakes, ponds, marshes, ditches, steep-gradient headwater streams, low-gradient large rivers, springs, stygean streams and pools, and terrestrial burrows leading to groundwater. Individuals have been found in lakes or springs at depths of 30 – 90 m (stygobitic crayﬁshes in submerged caves in north-central Florida are observed by divers at depths greater than 30 m) but crayﬁshes certainly thrive in shallow, littoral areas. They are most abundant in the central, eastern, and southern regions of the United States, while a few species are found in the Paciﬁc slope drainages; they are rare or absent in the large area of the western Great Plains and the Rocky Mountains (see Taylor et al., 1996). This simply means that American crayﬁshes are crustaceans possessing biological plasticity. They have become established in nearly every type of freshwater habitat, except glacial and thermal efﬂuents, and some can tolerate brackish water. Considerable data suggest that habitat has a channelizing inﬂuence (convergence) on certain morphological aspects of crayﬁshes (Hobbs, 1976). For example, stygobites (a polyphyletic group) are characterized by an albino-sightless combination and attenuated appendages; they are classic K-strategists and demonstrate extreme resource efﬁciency. In contrast, “burrowers” (also polyphyletic) possess short, broad, ﬂattened chelipeds, lack carapace spines, have a reduced abdomen and tail fan (length, width, and height), and possess an enlarged gill chamber (an adaptation to the oxygen-poor burrow habitat — see above) (Fig 7). Water currents have certainly inﬂuenced the body form of crayﬁshes, with those species dwelling in fastest ﬂowing water having depressed (dorsoventrally ﬂattened — most common) or compressed (laterally deﬂated) bodies; these include, for example, Orconectes (Gremicambarus) compressus (Faxon) and Procambarus (Tenuicambarus) tenuis Hobbs. Spination is also typically reduced in such species. Those crayﬁshes inhabiting rifﬂe areas of streams and residing in pebble and cobble substrates (rubble dwellers) are markedly depressed dorsoventrally; examples of such taxa are Cambarus (Jugicambarus) friauﬁ Hobbs and Cambarus ( J.) brachydactylus Hobbs. Several decapods (Infraorder Brachyura — crabs) occur sporadically (naturally or introduced) in coastal brackish or freshwater systems, but are not included in the following discussions, numerical summaries, or key. They are represented by the portunid blue crab, Callinectes sapidus Rathbun, a xanthid mud crab, Rhithropanopeus harrisii (Gould) (found in streams of the Atlantic and Gulf coasts and introduced along the coasts of California and Oregon), and a grapsid river crab, Platychirograpsus spectabilis Rathbun, introduced from eastern Mexico into the Hillsboro River near Tampa, Florida (Powers, 1977). Of curious interest is the occurrence of the grapsid crab, Hemigrapsis estellinensis Creel, in Estelline Salt Spring in Hall County, Texas. This representative of the otherwise marine family Grapsidae is endemic (probably a Pleistocene relict) to the spring which is located 800 km from the sea at an elevation of 531 m (see Reddell, 1994). For further information on cavernicolous crabs, refer to Guinot (1994). 1. Shrimps The atyid shrimps (ﬁngers of chelate pereiopods with apical tufts of setae, Fig. 1B) of North America are restricted to two genera, one with two southeast- 23. Decapoda ern, obligate cave species and the other represented by two species found in coastal streams of California. Freshwater palaemonids (ﬁngers of chelate pereiopods lacking apical tufts of setae, Fig. lA) are a very successful family of shrimps; the genus Palaemonetes contains approximately 17 species in the western hemisphere, and Macrobrachium is assigned about 100 species worldwide (35 in the western hemisphere). These genera are represented in North America by 10 and 12 species, respectively. Shrimps are generally found in the macrophyte-rich littoral zone of lakes or in sluggish, vegetation-choked streams. Within these two families residing in the conﬁnes of the United States, ﬁve shrimps are stygobitic and are very restricted in distribution (two species known to occur only in a single karst locality). The North American stygobitic decapods (inclusive of Bahamas, Barbuda, Belize, Bermuda, Caicos Islands, Canada, Cuba, Dominican Republic, Guatamala, Hispaniola, Honduras, Isla de Pinos, Isla Mona, Jamaica, Mexico, Puerto Rico, and the United States) are represented by 36 shrimps assigned to ﬁve families and 16 genera:Agostocarididae [Agostocaris (2)], Alpheidae [Automate (1), Potamalpheops (1), Yagerocaris (1)], Atyidae [Palaemonias (2), Typhlatya (8)], Hippolytidae [Barbouria (1), Calliasmata (1), Janicea (1), Somersiella (1)], and Palaemonidae [Cryphiops (2), Creaseria (1), Macrobrachium (3), Neopalaemonon (1), Palaemonetes (3), Troglocubanus (6)], Procarididae [Procaris (1)] (Hobbs III, 1998). a. Atyidae North American freshwaters are inhabited by three federally endangered and one probable extinct atyid species:Palaemonias ganteri Hay, P. alabamae Smalley, Snycaris paciﬁca (Holmes), and S. pasadenae (Kingsley), respectively (Hedgpeth, 1968). The ﬁrst two taxa are stygobitic shrimps, the former restricted to Mammoth Cave in Kentucky, and the latter is known only from ﬁve caves (three groundwater basins) in Madison County, northeastern Alabama (Jacobson and Hartﬁeld, 1997; McGregor et al., 1997). Because most atyids currently inhabit warmer southern aquatic ecosystems, it is probable that these two stygobites are thermophilic relicts of a former, more widely distributed common ancestor. Syncaris paciﬁca and S. pasadenae are found in small coastal streams of California. The former is reported from Marin, Napa, and Sonoma counties and the latter is previously known from Los Angeles, San Bernardino, and San Diego (may be erroneous report) counties but is likely extinct due to habitat destruction and over-collecting. b. Palaemonidae Those North American palaemonids that occur within the United States and that 965 FIGURE 10 Geographical distribution of Palaemonetes kadiakensis and P. paludosus. are strictly freshwater inhabitants are represented by 10 species belonging to the genus Palaemonetes (three are stygobitic): Palaemonetes antrorum (Benedict) from wells and caves in Hays and Uvalde counties, Texas (federally endangered), P. holthuisi (Strenth) from a cave in Hays County, Texas (the only known site in the United States where two species of stygobitic shrimps are found together), and P. cummingi Chace from a cave in Alachua County, Florida. Palaemonetes cummingi is listed as “vulnerable” (threatened) on the 1996 IUCN Red List of Threatened Animals (Baillie and Groombridge, 1996). Seven are inhabitants of lentic and sluggish lotic systems, including springs (see Strenth, 1976, 1994; Wu and Brown, 1980) (Fig. 10 — distribution map does not include disjunct populations such as P. paludosus in Tygart Valley River, Randolph County, West Virginia — population probably introduced). In addition, ﬁve species assigned to the widespread genus Macrobrachium (North, Central, and South America, and the West Indies) are found in freshwaters in the middle and lower Mississippi River drainage and in low-lying streams from New Jersey to Texas (Figs. 11 and 12). Macrobrachium acanthurus (Wiegmann) ranges from Georgia to Brazil (also Bahamas) and is found in the United States from Georgia and Florida to Texas along the Gulf coast. Macrobrachium carcinus (Linnaeus) is the largest and most spectacular species and is found from Florida to Texas and south to Brazil and the West Indies. Macrobrachium ohione (Smith) is the only endemic species of the genus in North America and is found from Virginia to Texas and in the lower and middle Mississippi River drainage (Fig. 11). The natural range of M. olfersii (Wiegmann) is from Mexico to Brazil (Chace 966 H. H. Hobbs III FIGURE 11 Geographical distribution of Macrobrachium ohione. Gross reduction in populations throughout its range is attributable to loss of suitable habitat. and Hobbs, 1969); however, it likely has been introduced into the St. Augustine, Florida area (Hedgpeth, 1949). As referred to above, the giant Malaysian prawn, M. rosenbergii, has been introduced into ponds of central Florida, South Carolina, and numerous FIGURE 12 other parts of the world (e.g., Israel, Jamaica) (Smith et al., 1978). Strenth (1976), in a hypothetical discussion of the origin and dispersal of Palaemonetes and Macrobrachium, proposed that freshwater Palaemonetes may have arisen during the late Mesozoic or early Cenozoic and that the widely disjunct species of the genus are largely of monophyletic or of limited polyphyletic origin. He presented salinity tolerance data from laboratory experiments supporting the premise that worldwide dispersal of freshwater Palaemonetes over large expanses of open ocean would have been unlikely. Results of his studies on P. kadiakensis, coupled with those of others for P. paludosus, demonstrate that this group of shrimps cannot osmoregulate in salinities greater than 25 ppt. Thus, the distributional patterns of Palaemonetes and Macrobrachium appear consistent with the theories of plate tectonics and continental drift. He proposed that Palaemonetes preceded Marcobrachium into the Gulf of Mexico area and that Macrobrachium (primarily juveniles), through competitive exclusion, barred Palaemonetes from the middle latitudes. Most of the freshwater species of Palaemonetes are found above or below the 30° latitudes; or, if found between, they always occur at higher elevations or are Geographical distribution of Macrobrachium acanthurus (ﬁlled circles), M .carcinus (ﬁlled squares), and M. olfersi (ﬁlled triangles) (after Hedgpeth, 1949). 23. Decapoda located at considerable distances from the coast. Parameters that appear to limit the further spread of Macrobrachium are current velocity, salinity, temperature tolerances, and anthropogenic perturbations. 2. Crayﬁshes The freshwater astacideans (superfamily Astacoidea) constitute a very successful group and currently are represented by 393 described species and subspecies (4 extinct species), 342 species and subspecies which are restricted (introductions ignored) to North America, north of Mexico (see Hobbs, 1986 for lineages of the major groups in North America). These are assigned to twelve genera and two families (Table I), of which species richness sustains a progressive decline with increasing latitude from 30 – 60°N (France, 1992). The families, Astacidae and Cambaridae, have historically occupied allopatric ranges. The former inhabits the Paciﬁc slope drainages, except for Pacifastacus (Hobbsastacus) gambelii (Girard) which has crossed the divide into the upper Missouri; and the Cambari- TABLE I 967 dae occurs in some Hudson Bay watersheds, the Atlantic, and Gulf drainages from southern Canada to Honduras, certain Paciﬁc slope basins in Mexico, and Cuba (Fig. 13). The three most successful genera of crayﬁshes are Procambarus, Cambarus, and Orconectes (166, 86, 81 species and subspecies, respectively — see below). The coastal plain of the southern states is dominated by Procambarus, whereas members of the genus Cambarus (Fig. 13) are major components of aquatic environments from the Cumberland Plateau to the North and East. West and northwest of the Cumberland Plateau, species of the genus Orconectes are the most commonly encountered crayﬁshes. Hobbs (1989) produced a useful listing of the crayﬁsh faunas by country and state and included references treating species within each state. Two recent publications that discuss decapods in the northeastern United States are those of Smith (1988) and Peckarsky et al. (1990), and the following contemporary publications present data for decapods within various states and Canada:Guiasu et al. (1996) — Ontario, Canada; Deyrup and Franz List of Freshwater Decapod Genera and Subgenera Occurring in the United States Infraorder Family Subfamily Genus Subgenus N. A. U. S. Caridea (Shrimps) Atyidae ———— Palaemonidae ———— ———— Palaemonias* Syncaris Palaemonetes* Macrobrachium ———— ———— ———— ———— Astacidae ———— ———— Cambarellinae Pacifastacus Pacifastacus Cambarellus Cambarellus Cambarellus Barbicambarus Bouchardina Cambarus* Hobbseus Pacifastacus Cambarellus Dirigicambarus Pandicambarus ———— ———— Aviticambarus* Cambarus Depressicambarus Erebicambarus* Exilicambarus Glareocola** Hiaticambarus Jugicambarus* Lacunicambarus Puncticambarus* Tubericambarus Veticambarus Distocambarus Fitzcambarus Creaserinus Fallicambarus ———— ———— 4 2 10 12 28 5 3 9 1 7 1 1 3 7 16 5 1 1 9 23 4 15 1 1 2 3 9 7 4 7 4 2 6 5 17 5 3 0 1 7 1 1 3 7 16 5 1 1 9 23 4 15 1 1 2 3 9 7 4 7 Shrimp Totals Astacidea (Crayﬁshes) Cambaridae Cambarinae Distocambarus Fallicambarus Faxonella Hobbseus (Continues) 968 TABLE I Infraorder H. H. Hobbs III (Continued) Family Subfamily Genus Subgenus N. A. U. S. Orconectes* Billecambarus Buannuliﬁctus Crockerinus Faxonius Gremicambarus Hespericambarus Orconectes* Procericambarus Rhoadesius Tragulicambarus Trisellescens** Acucauda Austrocambarus* Capillicambarus Girardiella Hagenides Leconticambarus* Lonnbergius* Mexicambarus Ortmannicus* Paracambarus Pennides Procambarus Remoticambarus* Scapulicambarus Tenuicambarus Villalobosus ———— 1 6 14 3 5 7 8 25 2 1 9 1 20 3 19 10 14 2 1 56 2 19 1 1 6 1 10 1 393 1 6 14 3 5 7 8 25 2 1 9 1 1 3 18 10 14 2 0 50 0 18 0 1 5 1 0 1 342 Procambarus* Troglocambarus* Crayﬁsh Totals Summary listing: [refer also to Hedgpeth (1949), Strenth (1976), and Hobbs (1989)]. Infraorder Caridea (shrimps) Family Atyidae Genus Palaemonias — 4; Genus Syncaris — 2; Family Palaemonidae Genus Palaemonetes — 10; Genus Macrobrachium — 12; Infraorder Astacidea (crayﬁshes) Family Astacidae Genus Pacifastacus — 2 subgenera and 8 species; Family Cambaridae Subfamily Cambarellinae Genus Cambarellus — 3 subgenera, 17 species; Subfamily Cambarinae Genus Barbicambarus — monotypic; Genus Bouchardina — monotypic; Genus Cambarus — 12 subgenera and 86 species; Genus Distocambarus — 2 subgenera and 5 species; Genus Fallicambarus — 2 subgenera and 16 species; Genus Faxonella — 4 species; Genus Hobbseus — 7 species; Genus Orconectes — 11 subgenera and 81 species and subspecies; Genus Procambarus — 16 subgenera and 166 species and subspecies; only 12 subgenera and 124 species and subspecies in North America north of Mexico [Procambarus (Austrocambarus) primaevus (Packard) is an extinct species known only from tertiary deposits in Wyoming and is not included in these ﬁgures]; Genus Troglocambarus — monotypic. Respective numbers of species in North America (inclusive of Belize, Canada, Cuba, Guatemala, Honduras, Isla de Pinos, Mexico, and the United States) are included. Data are presented only for those genera that are assigned U. S. species (* denotes genera/subgenera having stygobitic species; ** see Bouchard and Bouchard, 1995). 23. Decapoda 969 FIGURE 13 Geographical distribution of North American crayﬁsh genera (not inclusive of introductions) (modiﬁed from Hobbs, 1988). (1994) and Franz et al. (1994) — Florida, Georgia; Page (1985) — Illinois; Page and Mottesi (1995) — Indiana; Martin (1997) — Maine; Pﬂieger (1996) — Missouri; Cooper and Braswell (1995) — North Carolina; Eversole (1995) — South Carolina; Jezerinac et al. (1995) — West Virginia; and Hobbs III and Jass (1988) — Wisconsin. The reader should refer to Taylor et al. (1995) for a more complete listing of publications having state crayﬁsh data. Many epigean crayﬁshes occur abundantly in large interstices that afford protection and concealment (dark thigmotactic cover; Alberstadt et al., 1995), particularly during the daytime hours, such as among stones, weed beds, leaf litter, brush piles, logjams, roots of riparian trees; and they often ﬁnd shelter in cans, tires, or among other anthropogenic debris. The genus Cambarellus in North America is distributed below the Fall Line zone in the Gulf Coastal Plain. Whereas a few species are known only from lotic habitats, most members of this genus, as well as those of Faxonella and Bouchardina, frequent lentic habitats such as ponds, backwaters of streams, and roadside ditches. These small crayﬁshes are successful in littoral waters subject to low oxygen concentrations and elevated temperatures. The 970 H. H. Hobbs III FIGURE 14 Pool habitat for the stygobitic crayﬁsh Orconectes a. australis in Russell Cave, Jackson County, AL. members of the genus Hobbseus also occupy such habitats, but they are more common in very small, shallow streams. The monotypic Bouchardina probably lives in similar habitats since it has been collected from leaf litter and aquatic vegetation in a single stream backwater ditch. The monotypic Barbicambarus is found only under large limestone rocks in the swift currents of large streams in the Green and Barren river basins of the Highland Rim. Of note, introductions of nonnative species into various parts of the world are leading to species displacements among crayﬁshes (see below; Hobbs III et al., 1989; Olson et al., 1991; Clancy, 1997). Hypogean species are represented by burrowing and cave-dwelling crayﬁshes. Primary burrowers, which are found in the genera Cambarus, Distocambarus, Fallicambarus, and Procambarus (Fig. 13), construct tunnels (Fig. 22a, d) in such habitats as seepage areas, along stream banks, in open ﬁelds, and in other low-lying areas. Among the stygobitic species are members of the genera Cambarus, Orconectes, Procambarus, and Troglocambarus; some of these cavernicolous crayﬁshes occupy lotic habitats while others appear to have become adapted to lentic situations. Those species inhabiting caves with active streams generally are found in pooled areas where they have aban- doned their preferred, and now less abundant, cover of cobble-gravel in favor of accumulated allochthonous, food-rich, silty substrates (Hobbs III, 1976) (Fig. 14). Some stygobitic crayﬁshes living in sluggish lotic / lentic systems (e.g., caves developed in the porous Ocala limestone of northcentral Florida) are found near cave entrances where organic debris is available; they are generally positioned on the walls and silty substrates of these submerged solution passages (Hobbs et al., 1977; Hobbs and Franz, 1986). Franz and Lee (1982) showed that the geographical and ecological distributions of the cavernicolous crayﬁshes of Florida are correlated with low or high energy (food) cave systems. One species, Troglocambarus maclanei Hobbs, is often found on walls or clinging pendant to ceilings of submerged caves. The stygobitic crayﬁshes are derived from surface ancestors that entered caves during late Tertiary (Hobbs et al., 1977; see also Hobbs and Franz, 1986; Holsinger, 1988; Hobbs III, 1994, 1999). a. Astacidae A single genus, Pacifastacus, is represented by eight species and subspecies in the Paciﬁc drainages of western North America and headwaters of the Missouri River in Wyoming (Fig. 13). One species, P. (Hobbsastacus) chenoderma (Cope), is extinct, known only from Miocene and Pliocene (?) deposits, 23. Decapoda and is not included in Table I. P. (H.) nigrescens (Stimpson) is probably extinct and P. (H.) fortis (Faxon) populations have declined signiﬁcantly (Light et al., 1995) and it is listed as a Federal Endangered Species. These crayﬁshes are found in lentic and lotic habitats ranging from alpine lakes and streams to streams crossing cold deserts. They occur in California, Idaho, Montana, Nevada, Oregon, Utah, Washington, Wyoming, and British Columbia (Fig. 13). Hobbs (1974) suggested that two evolutionary lines arose from a Mesozoic marine nephropoid ancestral stock. One line (represented today by the genera Astacus, Austropotamobius, and Pacifastacus) moved into the freshwater habitat and maintained the primitive characteristic of absence of cyclic dimorphism in males, and females exhibited a sclerite (annular plate) lacking both a sinus and fossa. A second, less conservative stock also made the transition to freshwaters (represented by extant members of the family Cambaridae). These males evolved cyclic dimorphism and associated characters (e.g., hooks on ischia of certain pereiopods in males), while the females developed an annular sclerite with a sinus or fossa for sperm storage (the annulus ventralis). b. Cambaridae As previously stated, the ancestral stock, in which cyclic dimorphism became established, moved into freshwater habitats and gave rise to the Cambaridae. This family consists of three subfamilies, two of which (Cambarellinae and Cambarinae) occur in North America (Fig. 13). The third, Cambaroidinae, is restricted to eastern Asia-Amur Basin, Korea, and Japan, and is treated only brieﬂy in further discussions. The following scenario is an abbreviation of arguments presented by Hobbs (1969, 1981, 1989) concerning the origin and dispersal of cambarid decapods. Whether or not the Asian Cambaroidinae and the American subfamilies shared a common freshwater ancestor or whether there were separate Asiatic and American invasions of freshwaters may never be known. Hobbs, however, suggested that the ancestral cambarine stock entered freshwaters of the southeastern portion of the North American continent during the late Cretaceous or early Cenozoic eras. Through adaptive radiation, this ancestral Procambarus stock moved through estuarine environments and populated available habitats in many of the streams ﬂowing from the southern and southeastern slopes of the existing continent. Except for rare passive dispersal resulting from stream piracy or from crayﬁshes moving during periods of low salinity from one stream mouth to another, stocks became isolated in their respective drainage systems. Before the close of the Miocene (at least 25 million years ago), some stream-dwellers were able to exist in backwaters of lotic habitats; these forms gave rise to the 971 ancestors of some of the lentic species of Procambarus (e.g., subgenera Ortmannicus and Scapulicambarus). These crayﬁshes moved into an ecological vacuum and were no longer conﬁned to ﬂowing water systems. Pools may have dried, or oxygen concentrations became low, or perhaps individuals simply moved across land from one aquatic habitat to another. Whatever the reason, additional ponds, lakes, ditches, streams, etc., were occupied and thus dispersal into lower elevation habitats occurred; also several stocks penetrated the water table by burrowing. Distocambarus occupied the groundwater niche and probably represents a remnant of a much more widely distributed Piedmont stock. The monotypic Troglocambarus represents an offshoot of the Procambarus stock that moved into the karst groundwaters in northcentral Florida [see below, and Hobbs et al. (1977) for further discussion of the evolution of stygobitic decapods]. The genus Cambarellus probably arose from a primitive ancestral Procambarus stock in the Gulf coastal plain and occupied ephemeral swamps, ponds, and ditches. One of the lotic ancestral procambarid stocks moved upstream, reaching the vicinity of the Cumberland Plateau, where ancestors to two very successful genera, Cambarus and Orconectes, became established. The major evolutionary lines were probably established by the Miocene Epoch, and the ﬁnal stage was set for the reﬁnement of evolutionary patterns begun during the early Cenozoic. Such major features as large rivers, the Appalachian and Ozark mountains, the coastal plain, the Cumberland Plateau, the Nashville Basin, and the Highland Rim inﬂuenced the dispersal of these stocks. Superimposed on these were the effects of Pleistocene glaciation. Representatives of the ancestral Cambarus stock were successful in various habitats and spread through lowland areas, giving rise to the species currently assigned to Fallicambarus. This genus, which probably arose on the west Gulf coastal plain, developed into primary burrowers. Barbicambarus is another descendant of the early Cambarus; it moved into sizeable streams with large substrates, rapid currents, and high oxygen concentrations. Hobbseus also may have evolved from the cambaroid line (Fitzpatrick, 1977), but it is more likely that it arose from the orconectoid line [Hobbs (1969)]. The genus Orconectes probably evolved in the Interior Lowland Plateau province (Kentucky / Tennessee) and radiated virtually in every direction (see Fitzpatrick, 1986, 1987a; Fetzner, 1996). Some orconectoid stock successfully moved into the lowland regions where species arose that are now assigned to the genus Faxonella. This genus also seems to have evolved on the west Gulf coastal plain and today these small, tertiary 972 H. H. Hobbs III burrowers are found in lentic situations (ponds, ditches). They occur in ﬂood plains and, when the water level rises, are fairly common in the shallow ﬂowing water. Bouchardina also is derived from the orconectoid stock and occupies backwater areas. Ancestors of the members of these two genera likely arrived along the margins of the retreating Mississippi embayment during the early Cenozoic. Thirty-eight stygobite crayﬁsh species and subspecies (refer to Table I) belonging to the family Cambaridae are assigned to the four genera:Cambarus (11 species), Orconectes (7 species and subspecies), Procambarus (19 species and subspecies), and the monotypic Troglocambarus. These obligate cave shrimps and crayﬁshes were derived from surface ancestors that made numerous colonizations of karst waters during late Tertiary. Fluvial barriers would generally have had minimal effects on the dispersal of stygobites (see Barr and Holsinger, 1985, and Holsinger, 1988; also see above and Hobbs et al., 1977 and Hobbs III, 1994 for a more detailed discussion of their evolution and dispersal). The profound effects of Pleistocene glaciation on the distribution of organisms should be mentioned. The obliteration of immense drainage systems (e.g., Teays) resulted in total habitat destruction for virtually all organisms. The principal zoogeographical effects were the elimination of populations and entire species from the northern portions of North America, with displacements of species farther south than they had occurred preglacially. Retreat of the glaciers resulted in the establishment of new drainage systems (e.g., the Ohio) which undoubtedly were rapidly invaded (re-invaded) primarily from the south and southeast, though some species may have entered from more than one refugium. (1). Cambarellinae The subfamily Cambarellinae is conﬁned to North and middle America where its members are found in the coastal plain from northern Florida to southern Illinois and Texas (Fig. 13) and, disjunctly, on the Paciﬁc slope and Central Plateau of Mexico. Seventeen species are assigned to three subgenera in the single genus Cambarellus (Fitzpatrick, 1983); however, only eight of them are found north of the Rio Grande (Table I). In the United States, eight species are known from the following states:Alabama, Arkansas, Florida, Illinois, Kentucky, Louisiana, Mississippi, Missouri, Tennessee, and Texas (Fig 13); they may have been introduced by humans into Georgia. (2). Cambarinae The subfamily Cambarinae, which is comprised of 10 genera and 368 species, ranges from New Brunswick across southern Canada to Mexico, Guatemala, Honduras, and Cuba (Fig. 13). Ten genera represented by 326 species occur in North America, north of Mexico (Table I; see Hobbs, 1989 for detailed presentation of genera, subgenera, species, and subspecies of American crayﬁshes). B. Reproduction and Life History 1. Shrimps Like all astacideans, freshwater shrimps have external fertilization, with the ova being fertilized as they are extruded and then attached to the pleopods of females. The two common epigean, freshwater shrimps, Palaemonetes kadiakensis (Fig. 9) and P. paludosus, have similar life histories, both species living approximately one year (Fig. 15). Although many individuals reproduce only once (semelparous), Nielsen and Reynolds (1977) observed ovigerous females of P. kadiakensis from Missouri also bearing mature eggs, indicating at least two broods of young per female (iteroparous); Beck and Cowell (1976) also noted that P. paludosus spawned twice. The length of the breeding season varies with latitude and is generally longer in southern localities. For instance, ovigerous females occur in populations of P. kadiakensis in Illinois and Michigan from April until August, but are present in Louisiana populations from February to October; ovigerous females of P. paludosus occur throughout the year in the Florida Everglades. As a general rule, breeding does not begin until late spring or early summer in more northern areas. Only large females breed early in the season, and they are replaced by smaller females as the reproductive season progresses. Females are larger and usually more abundant than males. Mature (eggbearing) females range from 20 – 49 mm in total length and produce one or two broods (two is particularly representative of populations of P. paludosus in Florida where the growing season is longer). The number of eggs produced per female is highly variable, ranging from 8 to 160, with fecundity generally a linear function of total length. The incubation period depends on water temperature and varies from 12 to 24 days; zoeae larval lengths are approximately 4 mm at hatching and these free-swimming larvae pass through six stages (varies from 3 to 8) in about three weeks. Accounts of larval development and postembryonic growth in these two species are summarized by Hubschman and Rose (1969), Beck and Cowell (1976), Beck (1980), Kushlan and Kushlan (1980), Page (1985), and Hobbs III and Jass (1988). Shrimp usually mature when they attain a total length of 20 mm although this is not necessarily the case. Most shrimp die after reproducing, but some females molt within three days (generally within 24 h) after young are released and can later produce another brood. Generally, adults disappear from the population in late summer or early 23. Decapoda FIGURE 15 973 Generalized life history of Palaemonetes (see text for explanation). fall, having a life span of approximately one year. Marine and brackish water Palaemonetes (e.g., P. intermedius Holthuis, P. pugio Holthuis) usually have smaller and more numerous eggs per female than do freshwater species. The production of fewer but larger eggs may enable the freshwater larvae to hatch at an advanced stage with a corresponding reduction in the number of free-swimming larval stages. Dobkin (1971) described three larval stages in the stygobite, P. cummingi, and studies of atyids consist of observations made on Palaemonias ganteri in Mammoth Cave, Kentucky (Hobbs et al., 1977; Leitheuser et al., 1985); the Alabama cave shrimp, P. alabamae, conducted by Cooper (1975) and McGregor et al. (1997); and Syncaris paciﬁca (Eng, 1981). The study of the life history of Macrobrachium ohione has not received as much attention as have the two taxa of Palaemonetes previously discussed. This species is larger (total length of ovigerous females ranging from 27 to 93 mm) and it lives a maximum of two years (Fig. 16). [Huner (1977) determined that M. ohione at Port Allen, Louisiana lived up to two years.] Fecundity is relatively enormous, the number of eggs varying from 6273 – 24,000 per female (Truesdale and Mermilliod, 1979). Ovigerous females are known only during May in Illinois, from March through September in Louisiana, and during March in Texas. Generally, females are larger than males and only the largest individuals in the population bear eggs. Additional ecological data can be obtained from Truesdale and Mermilliod (1979), Page (1985), and references therein. 2. Crayﬁshes Surprisingly few crayﬁshes have been studied carefully for extended periods. Although considerable data are available for many species (e.g., Penn, 1943; Fielder, 1972; Hamr and Berrill, 1985; Corey, 1990; Mitchell and Smock, 1991; Norrocky, 1991; Johnston and Figiel, 1997; see also Hart and Clark, 1987), most are observations from isolated collections. Astacid lifehistory investigations are limited to Pacifastacus leniusculus leniusculus and Pacifastacus (Pacifastacus) leniusculus trowbridgii (Stimpson) (see references in Mason, 1970a, b; McGriff, 1983a). Life-history studies of the more diverse cambarids have been conducted on only about 20 species. In a general sense, the life history of a crayﬁsh can be divided into several phases (Momot, 1984), each having a slightly different component important to the completion of the life history of a given cohort as well 974 H. H. Hobbs III FIGURE 16 Generalized life history of Macrobrachium ohione (see text for explanation). as to the long-term success of the species. Recently hatched juveniles search lake littoral and stream rifﬂe areas for the best food and shelter available (see Figiel et al., 1991). Rapid growth of young-of-the-year (YOY) allows a partial escape from the devastating effects of predation (Gowing and Momot, 1979). As juveniles grow larger, they abandon littoral and rifﬂe areas for deeper waters, only to return after reaching adult size; this habitat segregation is probably related, in part, to such factors as competition with adults and vulnerability to predators. During postembryonic development, food conversion is expressed in maximum growth. Adulthood represents a conservative phase in which energy is shunted from rapid growth toward a periodic maximization of egg output among several cohorts per year. Rapid growth and shorter lifespan are reported to be more common in crayﬁshes in lower latitudes (Momot, 1984; Hazlett and Rittschof, 1985); however, many “northern” species demonstrate these same characteristics. Unlike the cambarids, astacid males do not exhibit sexual dimorphism. Copulation in Pacifastacus l. trowbridgii coincides with the autumn decline of water temperature and is soon followed by oviposition (late October and early November) (Fig. 17). Females carry 90 – 251 eggs (number directly proportional to mass) on their abdomens, and hatching generally occurs after 7 – 8 months, during April to June. After the second molt, the young leave the mother as 3- or 4-week old juveniles. During the ﬁrst year, individuals may undergo as many as 11 molts and nearly triple in length. Some individuals live for eight years, and a few become sexually mature at age three; the majority, however, become part of the breeding population during the fall of their fourth year. This certainly follows the trend suggested by Momot (1984) that crayﬁshes occurring at higher latitudes and in colder environments usually live longer and mature later. The smallest ovigerous female observed by Mason (1974) was 60 mm total length and the largest 102 mm. This suggests that female astacids lay eggs more than once (iteroparous) and, indeed, may spawn as many as three or four times (Fig. 17). Refer to McGriff (1983a, b) for life-history data for Pacifastacus l. leniusculus. The life history of cambarid crayﬁshes (Fig. 18) is somewhat more variable and complex than is typical of the astacid crayﬁshes. The sexually dimorphic males exhibit two distinct and usually alternating body forms (Taylor, 1985). The change from one form to another occurs among mature males during the semiyearly 23. Decapoda FIGURE 17 FIGURE 18 Generalized life history of astacid crayﬁshes (see text for explanation). Generalized life history of cambarid crayﬁshes (see text for explanation). 975 976 H. H. Hobbs III FIGURE 19 Ventral view of basal portions of left pereiopods with ischia bearing hooks darkened (after Hobbs, 1972). molts. The sexually competent stage (Form I) is ﬁrst attained with the last juvenile molt. This more aggressive breeding form can be distinguished by the sclerotized, amber-colored, and lengthened condition of the terminal elements of at least one of the ﬁrst pleopods. In addition, the ischial spines are more pronounced (Fig. 19), the ﬁrst chelipeds are enlarged (Stein, 1975), and the sperm ducts are full of recently formed spermatids (Word and Hobbs, 1958). In Form II males (the nonbreeding form), however, the terminal elements of the ﬁrst pleopod are not as well differentiated (and never corneous), the ischial hooks are shorter and weaker, and the chelipeds are less robust. The ovarian cycle of female cambarids is only partly known and from only a handful of species but endocrine regulation of ovarian development coupled with abiotic inﬂuences such as temperature and photoperiod are certainly paramount (Kulkarni et al., 1991; Dubé and Portelance, 1992; Daniels et al., 1994). Kulkarni et al. (1991) reviewed the studies devoted to this process and, working with Procambarus (Scapulicambarus) clarkii (Girard), determined that the ovary of this species is directly responsive to both ovary-inhibiting and ovary-stimulating hormones. Although Form I males may be present in a population year round (e.g., in Cambarellus and Procambarus; see also life-history data for numerous species in Hobbs, 1981), generally they are more numerous during the fall and / or spring. Hence, many species demonstrate one or two peak periods of copulation (fall and / or spring) although mating clearly occurs throughout the year. [Mason (1970a), Ameyaw-Akumﬁ (1981), Bechler (1981), Hobbs III and Jass (1988), and Kasuya et al. (1996) provide more detailed observations and literature review of the mating behavior in crayﬁshes.] Because oviposition occurs usually in the spring or early summer, spermatophores (sperm plug) may be carried by the female for as long as six or seven months. During oviposition, the female secretes glair, a translucent, sticky, mucilaginous substance released from “cement glands” located on the ventral side of the abdomen. After eggs are released from the genital pore and simultaneously fertilized, they attach to the abdominal pleopods by a short stalk. Amazingly, it is not known how nonmotile sperm inside a hard, waxy plug fertilize an egg mass! Once the glair hardens, the female is considered to be “in berry.” Typically, larger females of a given species carry more eggs, yet the number of eggs is highly variable within as well as between species (epigean forms carry as many as 600 – 700 eggs). For example, Hobbs (1981, p. 415) reported that females of Procambarus (Ortmannicus) pubescens (Faxon) carried between 89 and 443 eggs. He cited numerous other examples of variation in egg number for Georgia crayﬁshes. Corey (1987a, b) reported on the egg number versus individual size for three species of Orconectes as well as for Cambarus robustus. She indicated that there was a positive linear relationship between the total number of eggs carried per female (fecundity) and the size of the female. The total number of eggs for each was:O. rusticus ranged from 75 to 351, mean 161.8; O. virilis from 29 to 195, mean 139.1; O. propinquus from 26 to 195, mean 68.5; and C. robustus ranged from 25 to 128, mean 64.9. See Stechey and Somers (1995) for fecundity data on O. immunis. Little is known about the fecundity of stygobitic species because ovigerous females or those carrying young have not been observed in most species. For the few taxa studied, however, the eggs were larger (more yolk), but less numerous (27 – 80) (Fig. 20), reﬂecting the constraints of life for these K-strategists in such energy-poor systems. Noblitt and Payne (1995) compared water, lipid, and protein content of the eggs of P. clarkii and P. (Ortmannicus) zonangulus Hobbs and Hobbs and found that water contributes signiﬁcantly to weight changes. They also determined that P. clarkii FIGURE 20 Female Orconectes i. inermis in “berry,” Marengo Cave, Crawford County, IN (note size and number of eggs). 23. Decapoda eggs have signiﬁcantly higher percentages of proteins while P. zonangulus eggs have signiﬁcantly higher percentages of lipids. Procambarus clarkii produces relatively large numbers of small eggs (among the highest recorded for crayﬁshes), whereas P. zonangulus generates relatively few but larger eggs with greater size variation. P. clarkii exhibits a proﬂigate reproductive strategy (well adapted to warm water habitats where nutrient input is continuous) whereas P. zonangulus demonstrates a prudential reproductive strategy, being better adapted to cool water environments where nutrient ﬂow is pulsed (Noblitt and Payne, 1995; Noblitt et al., 1995). Depending on the season of laying and the water temperature, eggs are carried by females for 2 to 20 weeks, during which time rhythmic movements of the pleopods effectively aerate the developing eggs. Corey (1987a, b) noted an inverse relationship between the number of newly released eggs and the current velocity. Incomplete extrusion, failure to attach to pleopods, and fertilization failure are three other factors leading to egg loss at oviposition. Additional losses of eggs and young while being carried on the female result from abrasion, predation, high current velocity, low food availability, high density of females, and insufﬁcient cover for females (see also Figler et al., 1995). After hatching, the ﬁrst juvenile instar attaches to the mother with hooked chelae and a telson “thread” anchored by specialized hammate setae (Price and Payne, 1984). The cephalothorax of this instar is disproportionately large, with immense eyes, a yolk-laden carapace, and an incompletely developed abdomen. Generally after 2 – 7 days of attachment, juveniles molt and grow to a form more closely approximating the adult appearance. This instar loses its posterior connection to the mother and uses only chelae to cling to her abdomen. An additional molt usually occurs within 4 – 12 days, producing a large, third instar crayﬁsh. Although this juvenile hangs onto the mother with its chelae and pereiopods, it may leave the parental pleopods intermittently; subsequent instars are free living. [Refer to Price and Payne (1984) for more details of postembryonic ontogeny and to Mason (1970b) for maternal – offspring behavior.] The adult female commonly molts 2 – 3 weeks after all young have left. By fall, 6 – 10 molts have occurred in the developing juvenile, resulting in a considerably larger and often sexually mature adult. Mating among mature yearlings frequently takes place; however, many individuals do not become sexually active until the following late summer or fall. Most adults can live 2.5 – 3 years, and the majority breed more than once. The longest life span reported for noncave dwelling cambarids is 6 or 7 years for the burrowing crayﬁsh Procambarus (Girardiella) h. hagenianus 977 (Faxon) in eastern Mississippi and western Alabama (Lyle, 1938). Longevity in stygobitic crayﬁshes has been demonstrated to be considerably greater than for epigean forms (Cooper and Cooper, 1978; Hobbs III, 1980; Streever, 1996). C. Ecological Interactions 1. Functional Role in the Ecosystem Crayﬁshes have successfully invaded a wide variety of habitats and play important roles in processing organic matter and in the transformation and ﬂow of energy. They are the largest and longest lived crustaceans in freshwater ecosystems of North America and process spatially and temporally large quantities of organic matter (see Huryn and Wallace, 1987) in most lotic and lentic systems. Although they can function as food specialists, most are opportunistic generalists, feeding virtually at all trophic levels (polytrophic) and serve as efﬁcient shredders and macrograzers (see Huryn and Wallace, 1987; France and Welbourn, 1992). Shrimps, on the other hand, are generally restricted to large, sluggish backwaters of rivers where aquatic vegetation (cover) is dense. They function primarily as grazers and, like crayﬁshes, shrimps are eaten by a myriad of predators (Hobbs III, 1993). Indeed, crayﬁshes are almost the sole source of food for some predators, such as the striped swamp snake, Regina alleni ((Franz, 1977; Fotenot et al., 1993). Naturally, shrimps do not play as important a role as do crayﬁshes in energy ﬂow since they are much more limited ecologically (largely restricted to slowly moving streams or lakes and ponds with dense vegetation). For additional information on food and feeding by these decapods, see below. Clearly crayﬁshes are important ecological constituents in numerous freshwater ecosystems yet few studies have deﬁned the habitat requirements for these organisms. Taylor (1983) determined that the distribution of Procambarus (Pennides) spiculifer (LeConte) was inﬂuenced by water depth. Rabeni (1985) ascertained that the sympatric species 0. luteus and 0. punctimanus in south-central Missouri co-exist by partitioning the physical environment. 0rconectes punctimanus tends to be more restricted in its use of habitats, occurring as YOY in shallow water with macrophyte cover. Both YOY and adults forage on larger-sized substrates and both life stages prefer slow current velocities. This species maintains a size advantage over 0. luteus (larger individuals are dominant and occupy their preferred microhabitat). 0. luteus tolerates a wider range of environmental conditions and exploits habitats unavailable or undesirable to its congener. Additionally, Rabeni (1992) determined that these crayﬁshes are the most energetically important component in the diets of rock 978 H. H. Hobbs III bass and small-mouth bass and that predation accounts for a signiﬁcant percentage of total mortality in these crayﬁshes. Eversole and Foltz (1993) demonstrated that abundance and standing crop biomass of Cambarus (Puncticambarus) chaugaensis Prins and Hobbs and Cambarus (Depressicambarus) latimanus (LeConte) were altered by substrate type, velocity, cover, and water depth. For Procambarus (Leconticambarus) alleni (Faxon) occupying the freshwater marsh habitat mosaic of southern Florida, differences in relative risk of predation, food availability, or a combination of these factors are probably inﬂuencing variances in habitat occupation (Jordan et al., 1996). Symbiotic and parasitic relationships involving crayﬁshes and shrimps have been summarized by Johnson (1977), Beck (1980), France and Graham (1985), Lichtwardt (1986), Keller (1992), Hobbs and Peters (1993), Holt and Opell (1993), Font (1994), and Gelder (1996). These decapods serve as intermediate or deﬁnitive hosts and / or substrates to a wide variety of bacteria, algae, protozoa, fungi, worms, and crustaceans. For instance, the metacercariae of the lung ﬂuke (Paragonimus spp.) invade crayﬁshes and are transmitted to another host when the crustacean is eaten. Paragonimus westermani is an important parasite of humans in Asia; fortunately for North Americans, few human infestations have been noted for P. kellicoti. Ecdysis provides periodic relief from the buildup of various ectocommensals. Crayﬁshes serve as a food resource as well as a pest for humans. A multimillion dollar business centers around “crawﬁsh” production in Louisiana and other parts of the world (Huner and Barr, 1984; Avault, 1993). In addition, certain crayﬁshes are economically important as local seasonal nuisances for farming. Burrowing species can be extremely abundant:Hobbs and Whiteman (1991) reported that Fallicambarus (Fallicambarus) devastator Hobbs and Whiteman (Fig. 21) FIGURE 21 Form I male (breeding stage) of Fallicambarus devastator, a primary burrowing crayﬁsh from Texas. was responsible for constructing immense burrows with as many as 62,676 mounds / ha in the prairie section of eastern Texas! Farm equipment can obviously be damaged, crops destroyed, and earthen dams can be weakened by the activities of these fossorial decapods. 2. Foraging Relationships Shrimps are somewhat opportunistic feeders, foraging on a variety of plant and animal material. The shrimp Palaemonetes paludosus feeds heavily on epiphytic algae on vascular plants. Grazing on diatoms (Fragilaria, Navicula, Stephanodiscus, Gomphonema, Synedra, and Cymbella) appears to be the primary method of food intake for populations in Hillsboro County, Florida (Beck and Cowell, 1976); however, they also forage for immature insects. Barr and Kuehne (1971) reported that the stygobitic shrimp Palaemonias ganteri (Mammoth Cave, Kentucky) strained and ﬁltered pool sediments with mouth parts, suggesting that its food consisted of microorganisms in the silt (sediments contained protozoa; Barr, 1968). However, Lisowski (1983) observed this shrimp exploiting another resource; an individual was observed foraging upside-down on the surface ﬁlm of a pool apparently feeding on ﬂoating material. This foraging behavior was similarly noted by Cooper (1975) for Palaemonias alabamae in Shelta Cave, Alabama, (he also observed the sediment-straining behavior as has this author in Bobcat Cave, Madison County, Alabama). Crayﬁshes play a polytrophic role in most ecosystems and, as such, contribute much to the complexities of energy ﬂow in aquatic systems (see above; see also Lorman and Magnuson, 1978; Momot et al., 1978; Momot, 1984; Hobbs III, 1993). By feeding directly on carrion and organic debris of aquatic and terrestrial origin, these two decapods contribute to the processing of organic matter in aquatic ecosystems. They serve in part as decomposers by breaking down particulate matter, altering the chemical composition of detritus, and releasing fecal material back into the system. Budd et al. (1979) demonstrated that juvenile Cambarus robustus and Orconectes propinquus function as collectors by ﬁlter feeding on algae. In Missouri, YOY populations of O. (Procericambarus) punctimanus (Creaser) possessed a greater percentage of plant detritus, O. (Procericambarus) luteus (Creaser) had a higher percentage of ﬁlamentous algae, and both species had a higher percentage of plant rather than animal matter in their stomachs (Rabeni et al., 1995). Lorman (1980) and Lorman and Magnuson (1978) showed that males of Orconectes rusticus in Wisconsin are more carnivorous than females yet are less carnivorous than juveniles. Others (e.g., Momot et al., 1978) found that all age classes tend to utilize vegetation as a food source. 23. Decapoda They are capable of switching roles from herbivore / carnivore to scavenger / detritivore merely in response to food availability. It is important to recognize that gut contents of crayﬁshes typically reﬂect availability of food type much more readily than they indicate preference for a speciﬁc food item. Food materials constituting the majority of gut contents are not necessarily what crayﬁshes prefer to consume. Although individuals of some species and sizes do forage selectively, many species are quite opportunistic and catholic in their diets. For example, Horns and Magnuson (1981) found that crayﬁshes consume signiﬁcant numbers of Lake Trout eggs in northern Wisconsin when this seasonally restricted food item is available. Savino and Miller (1991) determined that signiﬁcant impact on lake trout egg survival would occur only in lake trout spawning grounds with relatively low egg densities where there was a large crayﬁsh population, and where cobble or large rock substrate was available. Juvenile crayﬁshes are somewhat limited to detritivory and herbivory, as they ﬁlter suspended particulates and grasp coarse particles. Adults actively prey and graze on larger items, yet they too scrape microbes from hard substrates and shred vegetation. The total litter processing by these crustacea was estimated to range from 4 – 6% of the annual litter input in a West Virginia headwater stream (Huryn and Wallace, 1987). Because of this ﬂexibility, crayﬁshes can maintain large population densities despite ﬂuctuations in food resources. Although crayﬁshes forage nearly continuously, the peaks of this behavior occur shortly after sunset and during the night. In addition, for example, this activity for Procambarus clarkii decreased as water temperatures began to cool to around 14°C in Oklahoma (Covich, 1978). In contrast, Price (1981), working in Arkansas, found that both diversity in the diet and length of foraging increased for 0. (Procericambarus) neglectus chaenodactylus Williams as food abundance decreased during the winter months. Another factor signiﬁcantly affecting foraging is the interaction of sympatric species, since dominance could be critical in determining both foraging and reproductive successes. Crayﬁshes feed readily on macrophytes, and they can signiﬁcantly reduce species richness and biomass of aquatic vegetation by grazing selectively on plant species (see Lorman and Magnuson, 1978; Lodge and Lorman, 1987; Feminella and Resh, 1989; Lodge, 1991; Creed, 1994; Lodge et al., 1994; Momot, 1995; Nystrom and Strand, 1996). By moving through the shallow littoral zone of lakes and cutting stems near the bottom, they can wreak havoc even though they actually ingest very little of the vascular plant “harvest” (an example of consumptive and nonconsumptive destruction). In contrast, Saffran and Barton (1993) 979 found that O. propinquus in Georgian Bay (Ontario, Canada) had little impact on vascular plants, but that they inﬂuenced primary productivity of the littoral zone by the consumption of periphyton, charophytes, and invertebrates. Crayﬁshes, as predators (Lorman and Magnuson, 1978; Covich et al., 1981; Covich, 1982; Lodge et al., 1987; Hobbs III, 1993; Lodge and Hill, 1994), apparently attempt to maximize energy gain with minimal energy expenditure. For instance, rates of predation by Procambarus clarkii on thin-shelled snails (Physa sp.) were higher than those on thicker-shelled Helisoma sp. (Covich, 1978). Yet, MacIsaac (1994) showed that O. propinquus attacked medium and large zebra mussels (Dreissena polymorpha) more often than small individuals but that predation was limited primarily to smalland medium-sized mussels. Also, O. rusticus damaged more snails and had a greater weight-speciﬁc ingestion rate on snails than did its congeners in Wisconsin (Olsen et al., 1991). Hazlett (1994a) found that crayﬁshes feeding on zebra mussels did not respond to food odors unless they had prior experiences with that protein source. He also determined that food odors are not produced in sufﬁcient quantities to stimulate feeding by experienced individuals unless the food has been degraded by microbial activity. Results of other studies suggest that crayﬁsh predation reduces snail populations in lakes and streams (e.g., Lodge and Lorman, 1987; Hanson et al., 1990; Weber and Lodge, 1990; Olsen et al., 1991; Lodge et al., 1994; Perry et al., 1997) and also induces shifts in snail life history characteristics (Crowl and Covich, 1990). Clearly these crustaceans have ramifying impacts on the structure and dynamics of aquatic communities (e.g., Hanson et al., 1990). That is, crayﬁshes prey upon organisms occupying various trophic levels and compete for food and other resources at the intra- and interspeciﬁc levels. Thus they are positioned high enough in the trophic structure to inﬂuence numerous interactions among several subsets of the community foodwebs (Rabeni et al., 1995 found that Orconectes spp. accounted for more than half of all consumption by the invertebrate community of Missouri Ozark streams). Foraging behavior is altered in the presence of a predator. Stein (1977) showed that the susceptibilities of the crayﬁsh 0. propinquus to ﬁsh predators reﬂected the size and condition of the potential prey. Life stages with the greatest reproductive potential were those least vulnerable to small-mouth bass predation (berried females and Form I males). Young-of-the-year are particularly susceptible to predation; yet they, like other size classes, must balance predation risk with the beneﬁts of foraging. Thus, juveniles, Form II males, females, and recently molted individuals modify their behaviors 980 H. H. Hobbs III (reducing activity) and microdistribution (seeking substrates affording maximum protection) to minimize risk of predation. The degree of behavioral response appears to correlate positively with vulnerability. The importance of top-down forces on community composition and productivity (e.g., the trophic cascade model — see Carpenter and Kitchell, 1992) has received much attention (e.g., Power, 1992; Rabeni, 1992; Strong, 1992; Carpenter and Kitchell, 1993; Lodge et al., 1994). It is apparent that in lakes, the presence of predatory ﬁshes may decrease crayﬁsh impact on lower trophic levels by reducing crayﬁsh abundance (Lorman and Magnuson, 1978; Covich, 1982; Lodge et al., 1987; DiDonato and Lodge, 1993; Garvey et al., 1994), by nonconsumptively increasing crayﬁsh mortality (Hill and Lodge 1995), and by reducing foraging activity by surviving crayﬁsh (Hill and Lodge, 1994). Hill and Lodge (1995) further suggest that a signiﬁcant component of the impact of top predators on food webs may be the result of sublethal effects. Although several studies have determined the thresholds of detection by crayﬁshes for various stimulatory substances (e.g., Hatt, 1984; Tierney and Atema, 1986; Hazlett, 1990; Ciruna et al., 1995), little effort has been made to examine the role or inﬂuence of chemical stimuli and chemoreception on the foraging behavior of crayﬁshes (see Blake and Hart, 1993; Willman et al., 1994). Studies of the hierarchial structure of food search and feeding in the spiny lobster (e.g., Zimmer-Faust and Case, 1983) suggest that chemosensoryinduced foraging in crayﬁshes may also be geared primarily to obtaining nearby rather than distant food sources. Johannes and Webb (1970) have demonstrated that several species of invertebrates release free amino acids into the environment, and Rittschof (1980) showed that amino acids diffuse rapidly from carrion. Thus, amino acids are ubiquitous in aquatic ecosystems and are readily available for use as chemical feeding cues for crayﬁshes. Of particular note, Hatt (1984) demonstrated that amino acid receptors are present on the pereiopods and antennules of crayﬁshes. Tierney and Atema (1988) suggested that the responsiveness of Orconectes rusticus and 0. virilis to a variety of chemicals (e.g., amino acids) may reﬂect the omnivorous foraging habits of these crustaceans. Stygobitic crayﬁshes and shrimps live in heterotrophic environments where the primary source of energy is from the surface in the form of allochthonous materials carried or washed into the cave, such as vegetative debris and bat guano. In addition, the stygobitic crayﬁsh, Troglocambarus maclanei, also is presumed to be a ﬁlter feeder; however, cannibalism by this obligate cave dweller has been noted (Hobbs et al.,1977). In such resource-depressed systems, these crustaceans tend to congregate in regions of the cave where food is spatially or temporally abundant, and thus are commonly observed in pooled areas of streams among accumulated vegetation. Crayﬁsh densities are particularly high in these microhabitats, and these crustaceans often can be observed foraging over the substrate and feeding on microbe-encrusted debris. Their particularly enhanced foraging efﬁciency reﬂects their improved sensory perception, reduced random movements, diminished body size, and increased metabolic efﬁciencies. Foraging behavior of burrowing crayﬁshes is variable; but generally on warm, humid nights, individuals leave their burrows presumably in search of food (vegetative material). A peculiar phenomenon is noted regularly in Louisiana in which large numbers of crayﬁshes (primarily males) leave their marsh habitat, move over land, and die during the fall. They are generally in poor physical condition, and the cause of this mass “deathmigration” is unknown (Penn, 1943, p. 15). See the following references for additional information concerning burrowing crayﬁshes: Penn (1943), Hobbs (1981), Hobbs III and Jass (1988), and Hobbs and Whiteman (1991). 3. Behavioral Ecology The behavioral and distributional responses of organisms to features of their physical and biotic environment is encompassed within the discipline of behavioral ecology. Behavioral responses to drought (burrowing), orientation to current (positive rheotaxis), light (negative phototrophism), reaction to cover, and substrate preference (cobble – pebble), are examples of responses by decapods to the physical environment (see Gore and Bryant, 1990). These factors, to a large extent, determine the nature of selected habitats. Habitat use is inﬂuenced by food, feeding rate, predators, competitors, and other sources of mortality (see Mather and Stein, 1993). Aggressive behavior, maternal-offspring relationships, and behavioral mechanisms resulting in reproductive isolation of a shrimp or crayﬁsh species are representative patterns related to the biotic features of the environment. a. Feeding See Section III.C. 2. b. Burrowing Atkinson and Taylor (1968) suggest that burrowing is a strategy for minimizing adverse extremes of humidity and temperature and probably for protection against predators; this behavior is certainly well developed among the cambarids. Hobbs (1981) recognized three categories of burrowing crayﬁshes. “Primary burrowers” (e.g., Fallicambarus devastator, Fig. 21) spend most of their lives in burrows, occasionally exiting to forage or to mate. Their burrows consist 23. Decapoda 981 FIGURE 22 Generalized crayﬁsh burrows:(a, d) those of primary burrowers; (b) that of secondary burrower; and (c, e) those of tertiary burrowers (after Hobbs, 1981, p. 32; see also Hobbs and Whiteman, 1991, Fig. 2). of complex tunnels that are often quite removed from open bodies of water (Fig. 22a, d) and some species (e.g., Fallicambarus (Creaserinus) gordoni Fitzpatrick) are found only in pitcher-plant bogs (Johnston and Figiel, 1997). Crayﬁshes tend to be attracted out of their burrows by low light intensities, and this may be considered as an initial response that ultimately gives rise to more complex behavior patterns involving food searching, reproduction, and other social interactions. Higher levels of light initiate a withdrawal response, one that protects them from predators (see Fernándezde-Miguel and Aréchiga 1992). “Secondary burrowers” [e.g., Fallicambarus (Creaserinus) fodiens (Cottle) — see Norrocky 1991] spend much of their life history in tunnels but frequent open water during rainy seasons (Fig. 22b). Their burrows usually feature a single subvertical tube that either slopes gently or descends in an irregular spiral (Hobbs, 1981, p. 32). “Tertiary burrowers” (e.g., Procambarus a. acutus) live in open waters and move into burrows only at speciﬁc periods (Fig. 22c, e). For example, they burrow to brood eggs, to move below the frost line during winter, and to avoid desiccation. Burrows also are refugia and can be defended, and juveniles can exhibit communal living within them (see Hasiotis, 1995). Nonburrowing crayﬁshes will occasionally construct burrows when surface waters disappear (Berrill and Chenoweth, 1982) or burrow into the hyporheos of streams. For further details concerning the burrowing behavior of crayﬁshes, see Grow (1981) and Rogers and Huner (1985). c. Water Currents Water currents certainly inﬂuence life processes of lotic organisms and bar many species from rapidly ﬂowing streams. Maude and Williams (1983) demonstrated that when crayﬁshes are exposed to increases in current velocity, they alter their body posture to counteract the effects of drag and to maintain position. Not only is this a behavioral adaptation resulting in certain species optimally occurring in fast-ﬂowing streams, but it may very well play a role in the widespread distribution of species as well as in the expansion of exotics, such as 0. rusticus, into a variety of habitats following their introduction. d. pH Many temperate crayﬁshes inhabit shallow littoral waters during early spring. In some regions, this time period coincides with the spring pulses of acidic water from melting snow. Because crayﬁshes are capable of extensive movement and can avoid streams acidiﬁed by industrial sulfuric acid or mine drainage (pH 4.0 – 4.5; France, 1985b), they should be able to elude environments acidiﬁed by spring melt pulses. France (1985b) determined that Orconectes virilis can avoid lethally acidic waters, implying that during the spring melt, they could escape mass mortality by seeking refuge in the profundal zone of lakes. Yet, no avoidance was demonstrated to lake water with pH values as low as 5.6, levels that are reported to cause serious reproductive difﬁculties [from decreased aeration of eggs, leading to fungal infections and egg mortality (France, 1985a)]. The failure of this crayﬁsh to avoid sublethal acid waters could, therefore, interfere with recruitment; 982 H. H. Hobbs III for this reason, France suggests that the prolonged survival of 0. virilis in waters receiving acid runoff is doubtful. This may be the case for other crustaceans living in poorly buffered systems but, surprisingly enough, some crayﬁshes [e.g., Procambarus (Ortmannicus) fallax (Hagen) and P. paeninsulanus] reach maximal densities in acid waters. Yet, Tierney and Atema (1986) experimentally showed that acid exposure inhibits behavioral responses (speciﬁcally feeding and grooming; see also Uiska et al., 1994); this could cause acid-stressed populations to decline at pH levels well above those at which lethal physiological effects become apparent. e. Shelter and Aggression Obviously important aspects of the ecology of crayﬁshes and shrimps are their intra- and interspeciﬁc behavioral responses. Although they are nonterritorial, they are aggressive and rely on agonism to procure shelter, food, and mates (Stein, 1975). Most crayﬁshes tend to be solitary and occupy and defend crevices in a coarse substrate, both of which are of survival value. Crevices not only provide shelter from pounding shore waves or from high stream velocities, but they also furnish a refuge that can be defended against predators and competitors (see Blank and Figler, 1996). Considering the generalistic feeding habits of crayﬁshes and the apparent absence of food limitation in most systems, the principal resource bottleneck may be crevice availability. Hobbs III (1976) demonstrated a positive relationship between crayﬁsh abundance and availability and size of cobble substrates. Caine (1978) observed that competition among Florida epigean crayﬁshes is primarily for shelter, and individuals unable to obtain cover succumb to predators. A speciﬁc home range has been demonstrated for a number of species (e.g., Hazlett et al., 1974; Hobbs III, 1980), and dispersion and dispersal often appear related not to population density but to water depth, number of crevices, predators, temperature, etc. Experiments examining intraspeciﬁc aggression of resident maternal (carrying eggs and / or hatchlings) P. clarkii against intrusions by Form I males and nonmaternal female conspeciﬁcs showed that maternal residents won a signiﬁcantly higher proportion of encounters with either sex, thus demonstrating maternal aggression in crayﬁshes (Figler et al., 1995). Kershner and Lodge (1995) showed that O. rusticus’ use of cobble habitat in northern Wisconsin lakes was signiﬁcantly and positively correlated with lakewide predator density, reﬂecting the potential importance of predation risk in establishing crayﬁsh distribution patterns. They suggested that the availability of cobble habitat may inﬂuence the success of the invasions of this crayﬁsh. Additionally, crevices serves as a feature, which, by further partitioning the environment, permits a greater species richness in those habitats. Stein (1975) indicated that the chelipeds of O. propinquus make up 40% of their adult total dry weight. These immense pereiopods are used in sexual encounters with females, for procuring food, and in agonistic confrontations. Rutherford et al. (1995) demonstrated that chela length (but not symmetry) is positively related to ﬁghting ability of O. rusticus. Levenach and Hazlett (1996) found that if the display function of chelae is impaired then the crayﬁsh is prevented from competing successfully with conspeciﬁcs (O. virilis), yet impairing the mechanical function of chelae does not reduce the crayﬁshes’ competitive ability. Since crayﬁshes can autotomize their chelae, Figiel and Miller (1995) investigated the inﬂuence of chela autotomy on growth and survival of P. clarkii. They showed that as population density increased, the frequency of chela loss increased and that injured individuals demonstrated reduced growth and survival rates. Hayes (1975) demonstrated eight innate components of social interaction in the primary burrowing crayﬁsh, Procambarus (Girardiella) gracilis (Bundy): alert, approach, threat, combat, submission, avoidance, escape, and courtship. These also have been observed in other species and Ameyaw-Akumﬁ (1979) summarized the sequence of behaviors demonstrated by interacting crayﬁshes during aggressive encounters (Fig. 23). He proposed that movement of gnathal appendages and pereiopods as well as repeated antennal waving occurs prior to the termination of an agonistic encounter and this behavior may in fact constitute an appeasement display. He further stated that “the use of appeasement signals in mating behavior is, probably, a capitalization by the male as this behavior appears to reduce aggressive tendencies” (Ameyaw-Akumﬁ 1979, p. 41). Copulatory behavior (reviewed in Bechler, 1981) is generally seasonal with the pair assuming several positions, the male mounting the female most commonly (Fig. 24). Mating generally lasts from 10 min to 10 h (e.g., Andrews, 1895; Mason, 1970a). After oviposition, egg fanning, and hatching, maternal – offspring behavior is mediated by pheromones released into the water by the female. These cues are speciesspeciﬁc but not brood-speciﬁc, since juveniles are attracted to conspeciﬁc brooding females. Also, Peck (1985) showed that social interaction is an important factor affecting temperature selection by 0. virilis. Individuals demonstrated behavioral mechanisms, not only to ﬁnd favorable temperature ranges but also to segregate according to social rank. f. Predation Avoidance The presence of predators elicits various behavioral response in crayﬁshes. Young- 23. Decapoda 983 FIGURE 23 Agonistic behavior between two male Orconectes propinquus in the “body up” position; crayﬁsh on right performing “antenna tap” (Bruski and Dunham, 1987) (photograph courtesy of Ann Jane Tierney). of-the-year are particularly susceptible to predation and must balance predation risks with foraging beneﬁts (Butler and Stein, 1985). Stein and Magnuson (1976) and Stein (1977) showed that in the presence of smallmouth bass (Micropterus dolomieui), 0. propinquus foraged less, adopted defensive postures, and sought protective substrates. Ideally, crayﬁshes use crypsis to avoid detection by predators. However, when discovered, crayﬁshes typically backswim seeking shelter in vegetation, beneath rocks or debris, or in burrows. If FIGURE 24 Copulating pair of Orconectes propinquus, Form I male on top (photograph courtesy of Ann Jane Tierney). 984 H. H. Hobbs III escape is not possible, individuals assume and intensify a species speciﬁc defensive posture, the predator response posture (PRP). Hayes (1977) described PRP of crayﬁshes and discussed the evolutionary signiﬁcance of these behavioral patterns in three species of the genus Procambarus. The crayﬁshes studied have similar lateral merus displays which result in a lateral presentation of the chelae, allowing for defensive coverage of the entire carapace and for quick ﬂight by tail-ﬂipping. Also, elongated chelae of these species enable them to sense tactilely the approach of a predator and to keep it at a greater distance. The PRP of the terrestrial (burrowing) species P. gracilis appears to have been modiﬁed for threat from terrestrial predators with attack coming primarily from above. Chelae are held shieldlike in front of the carapace, and the tail is folded tightly beneath the body, allowing for rapid reorientation to the attacks of a shifting predator. This posture frees the tail from use as an escape organ on land. Stein and Magnuson (1976) also suggested that, in addition to their effects on the distribution and behavior of crayﬁshes, predators have an inﬂuence beyond simple predator – prey interactions because of their impact on several trophic levels. 4. Population Regulation The population dynamics of shrimps and crayﬁshes are not well studied except for a few species. Traditionally, the four major features of any demographic model are summarized as natality, mortality, immigration, and emigration. Each of these clearly affects the size and growth of natural populations and all are under the constraints of density-dependent (D-D) and / or densityindependent (D-I) factors. It should be emphasized, however, that conventional D-D and D-I regulatory factors, while present, are too often superceded in importance by the effects on density of human activities (Hobbs and Hall, 1974). Rather than a single factor, it should be emphasized that any of these elements may interact strongly in affecting shrimp and crayﬁsh community composition, population sizes, and production. a. Shrimps A 1-year life history is characteristic of epigean shrimps, whereas cave forms probably live longer. Food resources tend not to be limiting for epigean shrimps, but ﬁsh predation can have signiﬁcant impact on their densities (Nielsen and Reynolds, 1977). The same investigators noted that ﬂuctuating water conditions (seasonal drying and extended high water) also elicit population changes. Fecundity appears to be related to size (length) of female and to time of year. In cave environments of the southeast, amblyopsid ﬁshes (and crayﬁshes) are predators of stygobitic shrimps and crayﬁshes. In these food-limited systems, where shrimp and some crayﬁsh populations are very small, loss of ovigerous females (and thus loss of recruitment of young) can be very signiﬁcant to immediate and particularly long-term stability of population densities. Lisowski (1983) attributed the extreme decrease in density of Palaemonias ganteri from baselevel streams in the Mammoth Cave System, Kentucky, to pollution of local groundwater. The sources of contaminants were human sewage and water from the Green River, which had received inputs of oil brines and hydrocarbons during the 1960s. He also indicated that modiﬁcations of habitat and ﬂooding regimes caused by dams within the Green River drainage adversely affected shrimp by reducing the reproductive success and by increasing predation pressure on them. b. Crayﬁshes Lodge and Hill (1994) reviewed the variables regulating population size and the importance of both D-D and D-I factors for some cool-water crayﬁshes. They summarized the D-I parameters as temperature, calcium concentration, pH, dissolved oxygen, salinity, habitat (substrate particle size and food availability), and water velocity and indicated that the D-D factors are diet, competition (intra- and interspeciﬁc), predation, molting, and ectosymbionts and disease. Momot and others, in a variety of population studies (Momot and Gowing, 1977a, b; Gowing and Momot, 1979; France, 1985a), have shown that various populations of 0. virilis are regulated by several D-D and DI factors, including cannibalism, natural mortality at molting (physiological and mechanical problems), predation by snakes (Godley et al., 1984), ﬁshes (e.g., trout, bass) and some insects (e.g., dragonﬂy) on yearlings, predation on adults by various terrestrial vertebrates (Lodge and Hill, 1994), ﬂooding, temperature, pH (Davies, 1984; Siewert and Buck, 1991; France and Collins, 1993), seiches (Emery, 1970), and other factors. Crayﬁshes are generally characterized by high juvenile mortality and reduced adult mortality (concave survivorship curve). It has been suggested (Momot and Gowing, 1977b) that overall, most changes in biomass and productivity of these populations result from internal density-dependent processes operating on fecundity and mortality rates rather than on growth. In several Michigan lakes during most years studied by Momot, the breeding success of 0. virilis depended mainly on the survival and subsequent fecundity of the 1.5-year age group females. At 2.5 years, females are considered an important strategic reserve. Where natality, mortality, emigration, and immigration rates vary, population sizes will ﬂuctuate, especially where birth recruitment is inadequate. As populations expand, they face potential resource depletion and the possibility of attracting more predators — both 23. Decapoda factors that can limit population size. Predation pressure may cause prey to modify their behavior (e.g., increased burrowing and chelae displays, suppressed foraging, and reduced overall activity). In turn, these antipredatory behavioral patterns act as selective (coevolutionary) forces on the predator, thus operating to modify predator strategies. Stein and Magnuson (1976) and Stein (1977, 1979) have demonstrated that juvenile O. propinquus are the preferred crayﬁsh prey of smallmouth bass and, as such, are rarely found on open, sandy substrates. Adult crayﬁsh (least preferred prey) are abundant in these areas, demonstrating both that preferred prey make distributional shifts to protective habitats and that use of space by nonpreferred prey is regulated much less by predators. Inter- and intraspeciﬁc competition for habitat (refuge) and nutrients (food) affects crayﬁsh populations in a variety of ways. When food or shelter are limiting crayﬁshes interact agressively, suggesting that interference competition may be common (Capelli and Munjal, 1982; Capelli and Hamilton, 1984). Bovbjerg (1956) noted that larger size entrusts a competitive advantage; and during intraspeciﬁc competitive encounters, male crayﬁsh usually dominate female crayﬁsh. Also, others (e.g., Flynn and Hobbs III, 1984; Hazlett et al., 1992; Lodge and Hill, 1994) have suggested that interspeciﬁc competition for habitat and food results in competitive exclusion. Length – frequency distributions have traditionally been used by investigators to show age and growth of individuals in populations (see France et al., 1991 for conditions required to produce reliable results). Growth of crayﬁshes is related directly to the number of molts during a growing season and the growth increment per molt; growth is stepwise rather than continuous. Average growth increments range from 0.41 mm for the dwarf crayﬁsh Faxonella clypeata (Hay)(Black, 1958) to 2.6 mm for Procambarus clarkii (Penn, 1943). The difference in yearly size increment between males and females is due primarily to the frequency of molting and not necessarily to the growth increment at each molt (Hazlett and Rittschof, 1985). However, the increment for individuals living in optimum conditions may well have as much impact as does the number of molts. Female growth is depressed, probably in response to the effects of carrying eggs and young, resulting overall in fewer molts. Conditions that promote rapid juvenile growth rather than adjustments in egg production per se modify any stock recruitment interaction. In more northern climates, the temperature of the water in the shallows, especially in “nursery areas,” is an important determinant of the molting rate in juveniles. Flint (1975) found that a coastal stream population of Pacifastacus lenius- 985 culus had a slightly faster overall growth rate than a subalpine lake population; he ascribed this to higher water temperatures and a longer growing season. Because crayﬁshes molt more frequently as juveniles, any parameter reducing juvenile molt frequency also reduces growth rate; this, in turn, may determine size at maturity, although there is no absolute size at which a crayﬁsh becomes mature. Because fecundity is a function of female size, populations with smaller females should have lower recruitment of YOY. Obviously, a cohort that grows faster and reaches a larger overall size will have a better chance of ensuring the replacement of its population. Huryn and Wallace (1987) demonstrated that long life span (ﬁve years) and slow growth of Cambarus bartonii in a West Virginia headwater stream resulted in low production (see also Roell and Orth, 1992); biomass exceeded production and transpired in a production / biomass ratio (P / B) of 0.58, a value similar to those reported from other temperate crayﬁsh populations (e.g., Momot, 1984). Working with long-term data sets for Orconectes virilis in two Ontario, Canada lakes, Momot and Hauta (1995) suggested that crayﬁshes can have cohort P / B ratios ranging from 1 to 8 and that ﬂuctuations in growth and mortality can alter these ratios signiﬁcantly. Crayﬁshes commonly establish sole occupancy of protective crevices in coarse substrates (when available) which they defend agonistically. A study conducted by Aiken (1968) in Alberta found that both male and female Orconectes virilis move to deeper water in late summer, females preceding males. Severe winter conditions would eliminate a population if it remained in the littoral zone where water freezes to the bottom. Rather than burrow to avoid ice, individuals escape by migrating to deeper water. Young-of-the-year appear to have higher mortality rates during the winter than adults. At spring thaw, both males and females return to shallow water. In streams, crayﬁshes generally burrow into the hyporheic zone to overwinter. Periods of low water obviously affect all members of the aquatic community, yet crayﬁshes can escape desiccation by burrowing into the gravel armor of stream channel beds. Prior to complete loss of an aquatic habitat, other responses by crayﬁshes have been noted. Taylor (1983), studying a lotic population of P. spiculifer during a drought in northeastern Georgia, suggested that population shifts (decrease in adult population densities, change in reproductive timing, increase in number of juveniles) resulted from loss of preferred deep-water sites, emigration, and subsequent increase in predation of larger adults. Population regulation among sympatric species has received considerable attention in the last 15 – 20 years (e.g., Lodge et al., 1986; Lodge and Hill, 1994). Many 986 H. H. Hobbs III North American crayﬁshes occur sympatrically with close relatives, yet hybridization appears to occur relatively infrequently although it may be much more common than previously recognized (see Roush, 1997). One mechanism that may be operating to ensure reproductive isolation is chemical or visual recognition of species during mate selection (Smith, 1988). Chemoethological isolating mechanisms could be effective for sympatric but not allopatric species. In consequence, hybridization of a resident and an exotic species might be more likely than between two distinct but geographically overlapping resident species. However, morphometric analyses indicate a strong likelihood that hybridization does occur between O. rusticus and O. propinquus in northern Wisconsin lakes where the former is an exotic. Lodge and Hill (1994) report that putative hybrids comprise up to 27% of mixed-species populations, and laboratory crosses have shown that viable offspring from matings of these two species are produced but in numbers that are 60% lower than in conspeciﬁc matings (Berrill and Arsenault, 1985). Also, Cesaroni et al. (1992) report a narrow hybrid zone maintained by two closely related undescribed crayﬁshes (Procambarus spp.) cohabiting a cave in northern Chiapas, Mexico. Sequential invaders (natural or introduced) can elicit drastic changes in community structure (Hobbs III et al., 1989), including replacement of native species (Hill et al., 1993; Kershner and Lodge, 1995; Gamradt and Kats, 1996). In recent years, biological invasions have been considered such a major environmental problem that many volumes have been devoted to their study (see Lodge, 1993a). Of particular importance to crayﬁsh biology is the apparent replacement of native crayﬁshes in various parts of North America (and world, Hobbs III et al., 1989) by introductions of nonnative species (e.g., interactions of 0. virilis, 0. propinquus, and 0. rusticus in Wisconsin, Lodge; 1993b; Capelli, 1982; Capelli and Magnuson, 1983; Page, 1985; Hobbs III and Jass, 1988; Momot, 1992; Charlebois and Lamberti, 1996; Taylor and Redmer, 1996). Displacement mechanisms are complex, and the mechanisms regulating shifts and / or replacements of species are not well understood in most instances (Flynn and Hobbs III, 1984). However, ﬁeld and laboratory results indicate that reproductive interference, growth, predation, competition, aggressive dominance, mortality, and interspeciﬁc morphological differences probably serve as mechanisms regulating replacement (Capelli, 1982; Capelli and Munjal, 1982; Butler and Stein, 1985; Lodge et al., 1986; Mather and Stein, 1993, Hill et al., 1993; Hill and Lodge, 1994; Garvey and Stein, 1993; Garvey et al., 1994). Based on data from various lakes in northern Wisconsin, Lodge et al. (1986) and Lodge (1993b) stated that outcomes of such invasions are not necessarily predictable and Lodge et al. (1998) suggested that “best-guess” models of invasions, short of experimental work before or during an invasion, is the best approach. This unpredictability is due to a complex suite of factors such as reproductive success, competition for resources, diel and year to year ﬂuctuations in predation risk, and parasitism. A variety of disturbances are involved in the “ﬁnal” community structure (see Olsen et al., 1991; DiDonato and Lodge, 1993; Garvey and Stein, 1993; Hill and Lodge, 1994). IV. CURRENT AND FUTURE RESEARCH PROBLEMS Even though considerable effort has been directed toward the study of these decapods, with particular emphasis placed historically on taxonomy, systematics, evolution, anatomy, and some areas of physiology, satisfying answers only to a few questions concerning them have been obtained. Barely a handful of functional morphology studies have been conducted. Recent realization of the impact of exotic species introductions (e.g., Orconectes rusticus, Capelli, 1982; Magnuson and Beckel, 1985; Hobbs III and Jass, 1988; Hobbs III et al., 1989; Lodge, 1993a) has initiated numerous studies to determine the underlying mechanisms controlling crayﬁsh species replacements and community changes (Butler and Stein, 1985; France and Welbourn, 1992; Mather and Stein, 1993; Garvey et al., 1994). Our knowledge of life histories of shrimps and crayﬁshes is incomplete, at best. As noted above, only about 20 species have been studied in any detail, and varying quantities of data exist for the remaining taxa. Although much time and energy are required for these research projects, the need is paramount. Detailed studies of ovarian development and oogenesis as well as testis development and production of spermatozoa are sorely needed. Also, much is lacking in our understanding of the dynamics of populations for virtually all species of freshwater decapods. Best known are factors inﬂuencing population dynamics in the crayﬁsh 0. virilis. The Fish and Wildlife Service of the U.S. Department of Interior periodically issues a List of Endangered and Threatened Wildlife. Currently there are seven decapod species placed on the Endangered list, four of which are stygobites. Palaemonias alabamae and P. ganteri are stygobitic shrimps in Alabama and Kentucky, respectively; Cambarus (Jugicambarus) aculabrum Hobbs and Brown is known only from two caves in northwest Arkansas and is the “newest addition” to the 23. Decapoda Federal List (Jacobson, 1996; Elliott, 1997; Boyd, 1997); and Cambarus (Jugicambarus) zophonastes Hobbs and Bedinger is found in an Arkansas cave. The crayﬁshes Orconectes (Crockerinus) shoupi Hobbs and Pacifasticus fortis are restricted to streams in western Tennessee and Shasta County, northeastern California, respectively, and the shrimp Snycaris paciﬁca inhabits streams in central California. Additionally, the Fish and Wildlife Service listed the stygobitic shrimp, Palaemonetes cummingi Chace, as a Threatened species. The 1996 IUCN Red List of Threatened Animals (Baillie and Groombridge, 1996) cited three atyid (2 Palaemonias, 1 Syncaris) and two palaemonid shrimps (Palaemonetes); two astacid (Pacifastacus) and 118 cambarid (3 Camabrellus, 34 Cambarus, 2 Distocambarus, 8 Fallicambarus, 6 Hobbseus, 24 Orconectes, and 41 Procambarus) crayﬁshes as Threatened; and the atyid shrimp Syncaris pasadenae is listed as Extinct. The American Fisheries Society Endangered Species Committee listed all crayﬁshes occurring in the United States and Canada and reviewed the conservation status of all taxa (Taylor et al., 1996). There are state lists as well (e.g., Florida — Deyrup and Franz, 1994). Clearly, it is important that these species and their critical habitats be monitored. The ecology of decapods inhabiting resource limited cave habitats is poorly understood (see Hobbs III, 1992; Brown et al., 1994; Streever, 1996). Further investigations should be conducted to determine the physiological mechanisms that allow the reduction of energy utilization in tissues of stygobites. These should be run in conjunction with studies delineating the levels of environmental ﬂexibility of these forms. Brown (1981), using electrophoretic analysis of 19 biochemical loci of six crayﬁshes, substantiated previous reports of low genetic variability in decapod crustaceans [(also demonstrated for crayﬁshes by Fuller et al. (1989), Crandall (1993), Koppelman and Figg (1995), and Fetzner (1996)]. She attributed the low heterozygosity in these crayﬁshes from the southeastern United States to their limited geographical distribution in a low number of drainage systems and to their high mobility within those watersheds — both are factors allowing crayﬁshes to perceive their habitats as ﬁnegrained. Nevo (1978) proposed that generalists are more genetically variable than habitat specialists among all major taxonomic groups and Brown, on the basis of her ﬁndings, suggested that crayﬁshes should be classiﬁed as habitat specialists. Biochemical investigations of other crayﬁshes occupying a variety of habitats and from different phylogenetic positions should be undertaken to test the latter hypothesis. Also, additional allozyme studies as well as nucleotide sequence data from the ampliﬁed mitochondrial DNA from the 987 16S ribosomal subunit may aid in determining more precisely the relationships of currently recognized genera, subgenera, species groups, species, and subspecies (see Fetzner, 1996; Crandall and Fitzpatrick, 1996). Current hypotheses of taxonomic relationships may not reﬂect actual phylogenetic relationships. Recently, considerable effort has been directed toward the study of pheromones and other chemical substances released into the environment by various decapods. Much is to be learned about the importance of chemical communication in the behavior of aquatic animals and about the impact of these substances on the structure of communities. Basic questions remain unanswered concerning the roles of pheromones and chemical cues in population and community dynamics. Obviously, a tremendous variety of behavioral studies await the ethologist. Although James (1979, see his bibliography) has shown that benthic macro invertebrates are good indicators of various forms of pollution and are useful as test organisms for determining the presence of toxic substances, the importance of the role of freshwater decapods has not been completely demonstrated (Vermeer, 1972; France, 1986). Recent technical advances and syntheses have brought together existing data on the effects and measurement of toxicity in decapods (Hobbs and Hall, 1974; Doughtie and Rao, 1984; Roldman and Shivers, 1987; Wright and Welbourn, 1993). Acidiﬁcation due to acid precipitation and loss of buffering capability in freshwater systems in the northeastern United States and Canada have stimulated research on the effects of increased acidity on organisms (France and Graham, 1985; Tierney and Atema, 1986; Wheatly et al., 1996). Crayﬁshes are highly sensitive to an increase in the hydrogen ion concentration (Wood and Rogano, 1986; Bendell-Young and Harvey, 1991) and although there are inter- and intraspeciﬁc variations in the degree of tolerance (Berrill et al.,1985), France (1984, 1985a) has shown that Orconectes virilis juveniles are particularly sensitive. DiStefano et al. (1991) determined that adult C. b. bartonii and C. robustus are among the most acid-tolerant freshwater organisms they tested. This implies that if populations of these two species would begin to be affected negatively by increasing acidiﬁcation, then it may already be too late for the recovery of most other species in those lotic or lentic habitats (see Clancy, 1997). Additionally, studies are needed in order to determine the impact of toxic materials on spermatozoa, ova, developing embryos, young, and individuals in various molt stages (premolt, postmolt, molt, intermolt). For those species that regularly experience dilute seawater in some portions of their ranges (e.g., Pacifastacus leniusculus and P. clarkii), relatively little is 988 H. H. Hobbs III FIGURE 25 Practice of “ditching” to lower local water table signiﬁcantly (note person for scale) is a common practice (Hillsborough County, FL) with numerous negative environmental impacts. known concerning the identity and character of short and long-term control of the sodium pump in the crustacean gill and antennal gland (Sarver et al., 1994). Much is yet to be discovered about the physiology of the gut, including enzyme systems, digestion and absorption of nutrients, and metabolic fate of absorbed nutrients. As a result of increasing demands by humans for energy, aquatic ecosystems undoubtedly will be subjected to considerable additional waste heat. Because crayﬁshes and shrimps occupy important positions in complex food webs of lentic and lotic ecosystems, precise data concerning temperature preferences, heat tolerances, and thermal responses (e.g., McWhinnie and O’Connor, 1967; Claussen, 1980; Mathur et al., 1982; Taylor, 1984) of these and other organisms will be invaluable in establishing temperature standards such that aquatic communities will be protected. The following is only a partial list of threats to freshwater ecosystems. A thorough understanding of these and other potential sources of perturbations is paramount if we are to attempt to protect freshwater organisms, including shrimps and crayﬁshes. Point and nonpoint agricultural (e.g., pesticides, sediments) and other sources (e.g., municipal runoff and discharge) of pollutants; urban and residential development; heavy recreational use of private and public lands (e.g., horse camps); gem, feldspar, coal, and mica mining; logging and chip mills; paper mills; industry (particularly chemical); impoundment structures (dams) and their opera- tions; highway expansion; dewatering of stream sections (e.g., Hiwassee River), including “ditching” (Fig. 25); disturbances (chemical, physical) to hydrological systems in karst areas; sport ﬁshery (stocking, baitbucket introductions); pig lots; disruption of riparian vegetation; acid rain; and catastrophic spills (highways, trains). Sadly, this is only a brief start to a lengthy list of potential threats to freshwater ecosystems. Much effort is and will continue to be required in order to recover disturbed lotic and lentic habitats and to conserve and preserve existing natural ecosystems (Richter et al., 1997); this includes surface and subsurface water resources. Aside from basic research conducted to recognize contamination and to treat perturbed areas, implementation of new laws, education, and stricter enforcement of existing legislature are necessary strategies of the future. In some parts of the region treated in this book (e.g., Louisiana), shrimp and crayﬁsh production are important economic activities. Considerable work has been directed toward increasing the yields of crayﬁsh farmers and this effort should be continued (Avault, 1993; see also Daniels et al., 1994). As marine shellﬁsh populations are depleted, humans will undoubtedly turn to alternate food sources, including crayﬁshes. In various countries in Europe, these crustaceans are valued as food and extensive stocking of exotic species (extreme caution advised!), and rehabilitation programs for native species are ongoing. Most of the interest in North America is centered on development of an export trade for Europe, yet there is 23. Decapoda potential for an expanded domestic market. Those crayﬁsh species with known economic importance in North America, such as the burrowers Cambarus (Lacunicambarus) diogenes Girard and Failicambarus devastator, the stream and lake-dweller Orconectes rusticus, and the tasty Procambarus clarkii, particularly should be studied in detail. V. COLLECTING AND REARING TECHNIQUES Because decapods are found in such a large diversity of habitats, a variety of techniques should be used in procuring representatives. A. Shrimps 1. Atyidae Collections should not be made for any of the atyid shrimps in North American freshwaters. Populations of the two species of Syncaris and the two cave shrimps assigned to Palaemonias are very small (two may already be extirpated) and should not be disturbed! 2. Palaemonidae Freshwater shrimps occupy various sluggish lotic or lentic environments and are most readily collected with the aid of a sturdy, long-handled, ﬁne-meshed dip net or a small-meshed seine. When a particular locality is choked with vegetation or if the water is greater than 1.5 m deep, the use of ﬁne-meshed wire traps with inverted cones baited with meat can be employed. In clear to low turbidity waters, they may be collected at night with the aid of a headlight and dip net (the rubycolored reﬂection of their eyes makes for easy location of individuals). Shrimps can be maintained in well-aerated aquaria with considerable vegetation; the water should be changed weekly. B. Crayﬁshes: Astacidae and Cambaridae The North American astacids are known primarily from lakes and streams west of the Continental Divide, and methods for collecting these crayﬁshes are identical to those for the cambarid stream / lake-dwellers. In shallow streams that have little vegetation, a smallmeshed seine (2 – 5 m long) can be most useful. Deep sections or pooled areas of streams as well as shallow ponds and the littoral zone of lakes may be sampled by pulling the seine (5 – 8 m length) through the water, taking care to ensure that the weighted margin of the seine remains in contact with the substrates. When electroﬁshing equipment is used, small-meshed seines 989 can collect any organism that is stunned and carried downstream. Use of various wire traps is recommended for vegetation-choked streams, swamps, marshes, ponds, or lakes or when sampling deep waters of lakes. Inverted-cone minnow (funnel) traps with enlarged openings (4 – 5 cm diameter) have proven to be quite effective. A residence time of 1 – 2 nights, using chicken and ﬁsh scraps, liver, canned cat food, etc. as bait, is an adequate and productive duration for this sampling technique but this method suffers from sampling bias (Stuecheli, 1991). A sturdy dip net is very helpful in sampling ditches, mud-bottomed pools, overhanging vegetation, and areas choked with vegetation. This type of net (delta net preferred) can be used to collect individuals from rocky substrates in lakes and streams. Probably most crayﬁshes burrow at least occasionally for one or more reasons. The diameter of the burrow tube and chimney pellet size provide good indicators of the size of the crayﬁsh inhabiting the burrow (Hobbs III and Jass, 1988). One may examine the burrow aperture and assume that the smaller openings contain juveniles; to sample the adult population, concentrate on burrows with larger diameters. [This would not be an appropriate procedure for small burrowing species, such as Cambarellus (Pandicambarus) puer Hobbs, Faxonella clypeata, and Procambarus (Hagenides) pygmaeus Hobbs]. On warm, rainy, humid nights, burrowing crayﬁshes can be collected fairly easily. These crayﬁshes come to the mouths of their burrows and often leave them to forage for food. With the aid of a headlight, they can occasionally be collected in large numbers (see also Norrocky, 1984). Use of scuba equipment to observe and / or collect crayﬁshes in deep streams, pools, ponds, and lakes also is a very useful technique [see France et al. (1991) and Lamontagne and Rasmussen (1993)]. During population studies where long-term data are required for individual crayﬁsh, various marking (tagging) techniques are employed. Because these crustaceans periodically molt and thus lose their exoskeleton, external tags are not used since they interfere with casting off the exuvium or they are lost when the exoskeleton is shed. Consequently, internal tags are recommended [see review in Hobbs III (1980); Weingartner (1977) for colored ﬁliaments; Wiles and Guan (1993) for microchip implants]. Stygobitic decapods generally lack pigments and thus appear white or translucent. In subterranean streams, use of a small aquarium net is generally adequate to capture the readily visible organisms. Hand-grabbing also is a fairly productive means of collecting them once they have been disturbed and are “swimming” in open water. Wire minnow traps can be 990 H. H. Hobbs III used if checked often. [Note: cave decapod populations are usually small and because of reduced biotic potentials in stygobitic forms, yearly recruitment is very low. Thus, care must be taken not to collect many (if any) individuals from any cave locality — at any time!] Because crayﬁshes are aggressive, antisocial, and cannibalistic, rearing and maintaining them can be problematic. On the other hand, because they are scavengers and detritus feeders, they will feed on almost anything organic. Ideally, individuals should be maintained separately, each crayﬁsh in a single container (e.g., 20 8 cm stackable glass bowl) and at room temperature. No aeration is required, provided the water does not become fouled with food, wastes, etc. The bowls should have small gravel substrates, and the water should be sufﬁciently deep to cover the crayﬁsh. If larger containers are used, small cobbles limestone fragments, or broken pieces of ﬂower pots, bricks, or PVC pipe cut to varying sizes can be added for cover (see Alberstadt et al., 1995). Individuals should be fed 2 – 3 times per week, and the water should be changed approximately every other week. Food can range from various aquatic plants (Potamogeton, Elodea), scraps of meat, and earthworms to dried cat food. Continuous illumination will ensure a growth of algae but is not necessary. Shrimps can be raised in much the same manner as described for crayﬁshes except more vegetation and deeper water are required for these crustaceans. Females with eggs should deﬁnitely be kept isolated. When young hatch, they will remain attached to the pleopods of the female for up to several weeks. Most of the young will then leave the mother and the female should be separated from them to prevent parent / offspring cannibalism. As the young undergo molting and growth, some sibling cannibalism will probably occur. By providing cover for the molting young or by dividing them among several containers, this can be reduced signiﬁcantly. On a much grander scale, large tanks or ponds can be utilized for raising crayﬁshes or shrimps. These should be designed for complete draining with sluice gates or standpipes and should be long and narrow rather than square (Figure 26). Water depth should be maintained at about 1 – 1.5 m and food can be variable, but catﬁsh feed or poultry starter are good. For additional information on various aspects of crayﬁsh aquaculture, see Huner and Barr (1984), Avault (1993), and Huner (1995). Upon collection, crayﬁshes and shrimps should be killed in 5% neutral formalin and kept in the solution from 12 h to a week, depending on the size and number of individuals. Specimens should then be washed in running tap water for several hours and transferred to 70 – 75% ethanol. If epizooites (e.g., protozoans, copepods, entocytherids, branchiobdellids) are to be saved, FIGURE 26 Aquaculture ponds for raising crayﬁsh (the blue color morph, Procambarus paeninsulanus) in Hillsborough County, FL. 23. Decapoda 991 the formalin in which the decapods were killed should be ﬁltered, the exoskeleton rinsed, and the rinse water poured through a ﬁne seive. Any symbionts should be preserved in 75% ethanol and carefully labeled (Hart and Hart, 1974). Where possible, crayﬁshes and shrimps should be stored in clamp-top glass jars with gaskets. The organisms should be placed in the jar “anterior end down” with room for adequate 70% ethanol preservative. The specimens should be accompanied by a 100% rag paper label on which data are clearly printed using insoluble black ink. VI. IDENTIFICATION A. Preparation of Specimens Reliable identiﬁcation of freshwater shrimps can be made only if appendages are removed, cleared, and mounted on a microscope slide. The second pleopod of adult males should be extirpated and then heated in a lactic acid – chlorazol Black E stain solution [3 – 5 drops in 1% ethanol (95%) stain solution per 20 mL of lactic acid; a 1% solution of fast green can be substituted] at 150°C for 15 min. The translucent appendage should be transferred to glycerine and then examined. Crayﬁshes can be identiﬁed with the aid of a hand lens or a stereoscope. First-form males are required FIGURE 27 Caudal view of ﬁrst pleopod (gonopod) of ﬁrst-form males (after Hobbs, 1972). (a) Bouchardina robinsoni Hobbs (symmetrical); and (b) Procambarus (Ortmannicus) acutissimus (Girard) (asymmetrical); (pms, proximomesial spur). for positive identiﬁcation, and a crayﬁsh’s left pleopod should be removed and used for working with the key. B. Taxonomic Key to Genera of Freshwater Decapoda Morphological characteristics used in the following key are illustrated in Figures 1, 2, 19, and 27 – 29. First-form males are required to identify crayﬁshes at the generic level. la. Rostrum and abdomen compressed laterally; third pair of pereiopods never bearing chelae ................................infraorder Caridea 2 lb. Rostrum and abdomen compressed dorsoventrally; third pair of pereiopods bearing chelae.............................infraorder Astacidea 5 2a(la). Fingers of chelae of ﬁrst and second pereiopods with apical tufts of long setae (Fig.lb); supraorbital spine on either side of base of rostrum; some pereiopods with exopods....................................................................................................................family Atyidae 3 2b. Fingers of chelae of ﬁrst and second pereiopods without tufts; no supraorbital spines; all pereiopods lacking exopods ....family Palaemonidae .............................................................................................................................................................................................4 FIGURE 28 (a – d, f) Lateral view of left ﬁrst pleopods of ﬁrst-form males (after Hobbs, 1972); (e) caudal view of same. a, Orconectes i. inermis; b, Procambarus (Remoticambarus) pecki Hobbs; c, Orconectes propinquus; d, Cambarus latimanus; e, Faxonella creaseri Walls; and f, Procambarus gracilis. 992 H. H. Hobbs III FIGURE 29 Dorsal view of chelae and carpus of ﬁrst form males (after Hobbs, 1972). a, Fallicambarus fodiens; b, Orconectes rusticus; c, Hobbseus cristatus (Hobbs); Distocambarus (Distocambarus) crockeri Hobbs and Carlson. 3a(2a). Eyes reduced, without pigment; all pereiopods with exopods; stygobitic ..........................................................................Palaemonias 3b. Eyes well developed, pigmented; ﬁfth pereiopod without exopod; epigean ..............................................................................Syncaris 4a(2b). Second pereiopod distinctly longer than ﬁrst; mandibular palp present; branchiostegal spine on carapace situated distant to anterior margin; epigean ..........................................................................................................................................................Macrobrachium 4b. Second pereiopod only slightly longer than ﬁrst; mandibular palp absent; branchiostegal spine on carapace situated near or on anterior margin; epigean or hypogean ...................................................................................................................................Palaemonetes 5a(lb). Males lacking hooks on ischia of all pereiopods; ﬁrst pleopod of male with distal portion rolled to form cylinder; male never demonstrating cyclic dimorphism; female lacking annulus ventralis ...........................................................................family Astacidae Pacifastacus 5b. Males with ischial hooks on one or more of second through fourth pereiopods (Fig. 19); ﬁrst pleopod of male complexly folded with sperm tube opening on one terminal element; male always demonstrating cyclic dimorphism; female with annulus ventralis ............................................................................................................................................................................family Cambaridae 6 6a(5b). Males with hooks on ischia of second and third pereiopods; very small (15 – 33 mm total length) ................subfamily Cambarellinae Cambarellus 6b. Males lacking hooks on ischia of second pereiopods; hooks present on ischia of third, third and fourth, or fourth pereiopod ......................................................................................................................................................................subfamily Cambarinae 7 7a(6b). Mesial surface of ﬂagellum of antennae fringed ...........................................................................................................Barbicambarus 7b. Mesial surface of ﬂagellum of antennae never fringed ........................................................................................................................8 8a(7b). Third maxilliped much enlarged; mesial margin of ischium without teeth...................................................................Troglocambarus 8b. Third maxilliped not conspicuously large; mesial margin of ischium bearing teeth.............................................................................9 9a(8b). Coxa of fourth pereiopod with caudomesial boss (Fig. 19c) .............................................................................................................10 9b. Coxa of fourth pereiopod lacking caudomesial boss ........................................................................................................................15 10a(9a). First pair of pleopods asymmetrical (Fig. 27b) ...................................................................................................Procambarus (in part) 10b. First pair of pleopods symmetrical (Fig. 27a) ...................................................................................................................................11 11a(10b). Well-developed hooks on ischia of third and fourth pereiopods, or terminals of pleopods disposed cephalodistally.........................12 11b. Well-developed hooks on ischia of third pereiopods only, terminals of pleopods never disposed cephalodistally ..............................13 12a(11 a). Shaft of ﬁrst pleopods straight and terminating in two very short elements directed caudodistally or distally (Fig. 28a) ...................... Orconectes (in part) 23. Decapoda 12b. 993 Shaft of ﬁrst pleopods straight or bent caudally; if straight, never terminating other than in two very short elements directed caudodistally or distally (Fig. 28b) ..........................................................................................................................Procambarus (in part) 13a(11 b). Opposable margin of dactyl of chela with angular excision in proximal half (Fig. 29a)..................................................Fallicambarus 13b. Opposable margin of dactyl of chela lacking angular excision in proximal half (Fig 29b) ................................................................14 14a(13b). First pleopod with terminal elements directed distally (Fig. 28c) ...........................................................................Orconectes (in part) 14b. First pleopod with terminal elements directed caudally or, rarely, caudodistally (Fig. 28d)...................................................Cambarus 15a(9b). First pleopod with two terminal elements, one of which at least twice as long as other (Fig. 28e) .........................................Faxonella 15b. First pleopod with two or more terminal elements, never two with one at least as long as other ......................................................16 16a(15b). First pleopod terminating in at least three distinct elements (Fig. 28f) ................................................................Procambarus (in part) 16b. First pleopod terminating in only two distinct elements ...................................................................................................................17 17a(16b). Dorsal surface of chela studded with crowded small tubercles (Fig. 29c ...............................................................................Hobbseus 17b. Dorsal surface of chela with tubercles over lateral half widely scattered at most ..............................................................................18 18a(17b). First pleopod with proximomesial spur (Fig. 27a) ............................................................................................................Bouchardina 18b. First pleopod lacking proximomesial spur ........................................................................................................................................19 19a(18b). Carpus of cheliped slender and longer than mesial margin of palm of chela (Fig 29d); rostrum without marginal spines ..................... Distocambarus 19b. 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The occurrence of a freshwater shrimp Palaemonetes paludosus (Gibbes, 1850) (Crustacea:Palaemonidae) in a warm spring of Wellsville, Colorado. Natural History Inventory of Colorado 5:1 – 10. This Page Intentionally Left Blank Glossary acclimation process of adjustment of a rate function (i.e., respiratory rate) to a change in ambient environmental conditions. acetabular plates sclerites associated with the genital ﬁeld in adults (provisional genital ﬁeld in deutonymphs) of certain mite taxa bearing the genital acetabula. acetabulum (acetabula) knob-like structure with a porous cap associated with the genital ﬁeld in deutonymph and adult mites, thought to function as an osmoregulatory chloride epithelium. Acetabula are strictly or roughly paired, and usually are borne on acetabular plates, but may lie in the gonopore or scattered in the integument ﬂanking the gonopore. acidosis decline in the pH of the blood through accumulation of carbon monoxide or anaerobic endproducts. acrosome a cytoplasmic inclusion in the cell body of a sperm cell. Note: Glossary terms were deﬁned by individual authors in reference to their chapter. Consequently, terms may actually apply to other taxa and have broader deﬁnitions than indicated here. acute sharp angle at the end. adductor muscles in bivalve molluscs, the muscles extending between shell valves, closing the valves on contraction. adductor muscle scars raised areas or scars left on the interior of the ostracode shell by the adductor muscles, generally leave a distinctive pattern of scars. adenal produced in the parenchyma (rhabdoids). adenodactyl auxiliary glandular bulb in the male system of some turbellarian taxa. adhesive strip thin layer of chitin between the duplicature and the outer lamella of the ostracode valve. adnate closely adherent, joined together. adoral zone of membranelles orderly arrangement of three or more compound ciliary organelles (membranelles) that are serially arranged along the left side of the oral area of a ciliate. afferent branchial vessel in Mollusca, the blood vessels bringing deoxygenated blood from the visceral mass, foot, and mantle to the distal ends of the gill ﬁlaments. ala(e) a coarse or ﬁne, wing-like spine, as in the posteriorly directed alae on the lateral surface of the ostracode carapace. 1003 1004 Glossary allelochemic biochemical substance produced by one species and released into the environment that subsequently affects the behavior or physiology of another species. allochthonous substances (or sometimes species) originating outside the immediate habitats (e.g., carbon produced by plants located upstream or in the riparian zone); opposite of autochthonous. allopatric referring to multiple species whose habitat distributions do not overlap. allorecruitive type of growth of colonial rotifers whereby young recruited into the colony come predominantly from other colonies; results in higher genetic diversity of the colony. Originally deﬁned as Type I colony formation; see autorecruitive. allozymes enzyme having a speciﬁc biochemical function, but which differs slightly in its primary structure and in its electrophoretic mobility, from other enzymes coded by different alleles of this gene amictic phase in the life cycle of monogonont rotifers in which reproduction is parthenogenetic only; see mictic and mixis. amoeboid amoeba-like, have no ﬁxed shape, and creeping on surfaces. amphidelphic with two opposite ovaries that normally join at the common vagina. amphids paired sensory organs located on opposite lateral surfaces of nematodes; variable in position (lip or neck region) and shape (pore, slit, circular, spiral or cubshaped). amphimixis union of egg and sperm through ordinary sexual reproduction, with the gametes derived from separate individuals. amphoteric type of female rotifers of the class Monogononta that can produce two types of eggs in the same clutch, one developing into female (diploid) and the other into male (haploid) offspring. anastomosing interconnecting. ancestrula in ectoproct bryozoans, the original, single zooid that emerges from a statoblast, differing from subsequent zooids in the colony by its smaller number of tentacles and inability to form statoblasts. anchoral processes posteriorly directed apodemes of the gnathosoma (capitulum) in deutonymphs and adult mites. angulate having an angle. anhydrobiosis ability of bdelloid rotifers to undergo desiccation and then be revived at some later time; a cryptobiotic (latent) state in tardigrades induced by loss of water through evaporation. annual occurring once per year. annulate ringed; surrounded by a ring of a different color; formed into ring-like segments. annulations transverse striations (trenches) in the cuticle of nematodes. annulus ring; in ectoproct bryozoans, the ring of enlarged chambers, usually gas-ﬁlled, on the periphery of ﬂoatoblast valves. annulus ventralis seminal (spermatophore) receptacle of female crayﬁsh. anoxia lack of dissolved oxygen. anoxybiosis cryptobiotic (latent) state in tardigrades induced by low oxygen tensions. antenna whiplike, paired, generally elongate sensory appendages arising from anterior area of the head (or cephalothorax); 1-2 pairs of antennae are present in arthropods. antennal gland one of pair of complex excretory glands in many decapods with duct opening on antenna; green gland. antennule paired appendage of ﬁrst cephalic somite of crayﬁsh; sensory, often bearing aesthetascs. aperture opening to an anterior cavity, as in the opening of a snail shell from which foot and body protrude. apex that part of any structure opposite the base by which it is attached. apical pertaining to the apex. apodeme internal extension of thickened cuticle or exoskeleton in some arthropods, often functioning as muscle attachment site. apomixis form of parthenogenesis in which meiosis is suppressed and offspring are genetically identical to their mother. apophyses see pharyngeal or buccal apophyses. aposymbiotic describing an organism that would normally have symbionts but the symbionts are not present. apotypical (apomorphic) derived, or modiﬁed, state of a biologic attribute or morphologic character. apterous without wings. areoles segment of hairworm cuticle separated from adjacent segments (areoles) by longitudinal and transverse furrows; may bear granules, bristles, and contain pores. articulation a joint. ascus glandular pocket in some turbellarians (Typhloplanidae). atrium area where two or more passages meet, as in the space in ﬂatworms where male and female systems open. attenuate tapering rapidly or abruptly to a point. aufwuchs German term whose broader North American usage refers to all small, attached (except macrophytes) and free-moving organisms forming a living ﬁlm on aquatic substrates such as rocks, snags, and plants; included are some microinvertebrates, fungi, algae, and bacteria. autochthonous substances (or sometimes species) originating within the immediate habitat, for example, carbon produced by resident phytoplankton; opposite from allochthonous. autogamy self-fertilization phenomenon (not true sexual reproduction) in ciliates involving a single individual; haploid gametic nuclei are formed and two fuse with each other in a single individual; see cytogamy. autorecruitive type of growth of colonial rotifers whereby young produced by a colony tend to remain in that colony; characterized by low genetic diversity of the colony; seeallorecruitive and geminative. autotoky type of reproduction where progeny are produced by a single parent (i.e., hermaphroditism or parthenogenesis). Glossary autotrophy synthesis of organic compounds from carbon dioxide using light as the energy source (in photosynthesis) or inorganic chemical compounds (in chemoautotrophy); contrasts with heterotrophy. axe-head glochidium unionacean (Mollusca) glochidium larva characterized by a distinctly quadrate shell form. axenic culture a bacteria-free culture containing only one species of organism. axoneme longitudinal bundle of microtubular ﬁbers in ﬂagella, cilia, and axopodia. axopodium characteristic feeding pseudopodium of Actinopoda, distinguished by needlelike shape, axonemes, and bidirectional streaming of cytoplasm. bafﬂes dorsal and/or transverse ridges posterior to mucrones on interior of the buccal cavity in some eutardigrades. basal at or pertaining to the base or point of attachment to, or nearest the main body. basal bulb swollen, set-off, basal portion of the pharynx (esphagus) of a nematode. basipod (basipodite) second segment of an arthropod appendage. basis second segment from proximal end of segmented appendage. beak raised portion of dorsal margin of the bivalve shell; oldest portion of the shell. benthos animals living on or near the bottom of an aquatic habitat. bident having two points. biﬁd cleft, or divided into two parts; forked. bilamellate divided into two lamellae or plates. bioturbation disturbance of sediments by an animal. bivoltine having two broods or generations per year. bolus a ball. boss expanded protuberance, as on caudomesial surface of coxa of fourth pereiopod of male crayﬁshes. brachypterous with short or abbreviated wings. bristles variously shaped cuticular projections arising from cuticle. bromeliads common name of a family of plants with cupshaped leaves that can hold water at the base (mostly tropical species, e.g. the pineapple); protozoa, ostracodes, and some other invertebrates regularly inhabit these specialized aquatic microhabitats. brood chamber body space in parent where eggs or embryos develop; space between the body and carapace of a cladoceran, where eggs develop. brosse distinctive “brush” of cilia arising from specialized short kineties which is present on the anterodorsal surface of nondividing individuals of certain gymnostome ciliates. buccal apparatus anterior part of tardigrade foregut, consisting of buccal tube, muscular pharynx, and pair of piercing stylets. buccal apophyses cuticular thickenings at junction of buccal tube for insertion of stylet protractor muscles. buccal ciliature compound ciliary organelles, the bases of which are associated with the oral area of some ciliates. buccal cirri in heterotardigrades, paired sensory appendages, usually short and hair-like, on either side of each cephalic papilla on the cephalic plate. 1005 buccal tube support median ventral lamina, also called a “reinforcement rod,” from the buccal ring to the midregion of the buccal tube in some eutardigrades. bulbous see pharynx. bursa folds of cuticle on the male nematode tail that assist in attaching males to females during mating. bursa (copulatrix) copulatory bursa; receptacle in some female reproductive systems into which donor spermatozoa are deposited. byssus proteinaceous threads produced from a byssal gland at the base of the foot, used to attach juvenile or adult molluscan bivalves to hard surfaces. calcium phosphate concretions small extracellular spherules found in gills of unionacean mussels made of calcium phosphate deposited in consecutive lamellae on a proteinaceous matrix. calotte head region of a Nematomorpha, usually lighter in color than the adjoining body. capitulum sclerotized base of a mite’s gnathosoma. carapace expanded, hard covering of a major body region (usually head and/or thorax of some arthropods). cardate type of trophi (jaw structure) found in rotifers which functions by creating a sucking action. cardinal teeth massive conical projections formed at the center of the shell-hinge plate which interdigitate to form the fulcrum on which the valves open and close. carina sharp, spiral edge or keel on a snail’s shell, usually along the center of the whorl. carpus ﬁfth segment from proximal end of segmented appendage. cauda posterior extension of the idiosoma in adult males of certain mite taxa, functioning as an copulatory adaptation during spermatophore transfer. caudal toward the posterior of an organism. caudal glands glands found in the tail region, as in those of nematodes that secrete mucus. caudal rami bifurcated terminal extension of the abdomen in some arthropods. cellulase enzyme that degrades cellulose of plant cell walls. cement glands glands that secrete material to assist ﬁxation of egg capsules to a substrate, often near female gonopore in turbellarians. centroplast central structure in some heliozoa from which axonemes radiate; synonym: central granule, axoplast; a type of microtubule organizing center. cephalic pertaining to, or toward, the head. cephalic papillae large papillae in head region of nematodes; also, paired sensory appendages on cephalic plate of heterotardigrades, located between internal and external cirri. cercus (cerci) paired appendage of the last abdominal segment. In dragonﬂies, one of two pairs of lateral, terminal, triangular sclerites. cerebropleural ganglion mass of nerve cell bodies near the mouth. chaetae see setae. chaetotaxy number and arrangement of setae on the body and appendages. chela claw or pincer; in crayﬁsh, two opposed distal podomeres (dactyl and propodus) of certain pereiopods. 1006 Glossary chelate presence of pincer-like, exoskeletal or cuticular structures at the end of an appendage. In mites, a condition of the pedipalp in deutonymphs and adults of certain taxa in which tibia bear a dorsodistal seta, often associated with a projecting tubercle that opposes the dorsal surface of the tarsus to form a grasping organ. chelicera in mites, a two-segmented, paired feeding appendage located dorsomedially on the gnathosoma; usually two-segmented, but one-segmented in deutonymphs and adults of Hydrachnidae; the distal segment, or “claw,” functions in piercing and tearing the integument of hosts or prey. chitin a long chain, polymeric glucosamine containing 6% nitrogen; used in exoskeletons of many arthropods and often complexed with protein or inﬁltrated with calcium for strength. chloride epithelia oval patches (as in insects) that differ from normal epithelia and are used for osmoregulation. chrysolaminarin liquid carbohydrate storage form, -1:3linked glucan; characteristic of ﬂagellates in the order Chrysomonadida; usually in one or more large refractile vesicles at the posterior of the cell, optically bluish under strong light; synonym is leucosin. cingulum one of two ciliated rings present in the typical rotiferan corona (apical end); seetrochus. circadian rhythm diurnal or daily activity rhythms. cirrus A in heterotardigrades, paired ﬁlamentous sensory appendage at the anterior lateral margin of the scapular plate, situated slightly dorsal to the clava. cirrus eversible, spiny male duct; may contain ejaculatory, prostatic, or accessory ducts and be enclosed in a bulb or special propulsory sac. cladogram classiﬁcation, usually in the form of a branching diagram, of a group of organisms based on cladistic principles (i.e., tracing the evolution of shared, derived characters from ancestral to derived forms). clasping apparatus in ostracodes, the heavily sclerotized, apparently moveable sclerite articulating with midportion of the peniferum in a socket near the ventral cardo and loop of the spermatic tube of entocytherid hemipenes; includes vertical and horizontal rami. clava in heterotardigrades, a paired sensory appendage, usually short and broad, at the anterior lateral margin of the scapular plate, situated slightly ventral to lateral cirrus A. claw raptorial structures; in all mite instars, true claws are paired, curved structures located terminally on tarsi of the legs. clear-water phase period, usually in spring, when lake water becomes unusually clear due to grazing of herbivorous zooplankton. clutch group of eggs. cocoon structure formed by some species to enclose many egg capsules or embryos. cohort group of equal-aged individuals whose survivorship and fecundity are to be followed throughout the lifespan of the entire group. columellar strong central support around which a snail shell coils. commensal animal or plant that lives on, in, or with another, sharing its resources but neither parasitic on it nor injured by it. concentric in Mollusca, describing the growth lines of a snail operculum that lie entirely within each other (not forming a spiral). conglutinate mass of unionacean (Mollusca) glochidia larval bound by mucus; often resembling prey items of unionacean glochidial ﬁsh hosts (increasing chances of host contact). conic shaped like a cone. conjugation in ciliates, a reciprocal fertilization type of sexual phenomenon occuring only between members of differing mating types and involving temporary or total fusion of the pair. contractile vacuole liquid-ﬁlled organelle which functions as an osmoregulator in the cytoplasm of many protozoa. copepodid larval stage of copepods that follows the ﬁrst stage (the nauplius). copulatory bulb swollen muscular and connective tissue region containing the male copulatory organ and often also the seminal vesicle and/or prostate. copulatory organ terminus of male reproductive systems which delivers spermatozoa into the body of a mate, i.e. an intromittent organ. When not armed with hard structures, yet capable of protrusion, it is simply called a penis (or penis papilla). Copulatory organs may be equipped with hard (sclerotized) structures such as a stylet or cirrus spines. corneous slightly hardened but still pliable (corniﬁed or “horny”) proteinaceous material, as in the operculum of a snail or the modiﬁed terminal elements of ﬁrst pleopods of ﬁrst-form male crayﬁsh. corona apical region of rotifers; usually a ciliated band around the anterior end, characteristically composed of two ciliated rings called the trochus and cingulum; in some forms, ciliation is absent and may be replaced by long setae which surround the rim of a funnel-shaped structure called the infundibulum. cosmopolitanism idea that the same species are found on different continents. costa rib or ridge on a snail shell that lies transverse to (at right angles to) the coiling of the whorl. coxa proximal (closest to body) segment of a segmented appendage. coxal plate in mites, a sclerite representing the modiﬁed basal leg segment, attached to the venter of the idiosoma in all instars. Coxal plates may be expanded and fused with one another to cover large areas of idiosomal integument. coxoglandularia series of paired glandularia associated with the coxal plates of mites. coxopod proximal segment of an arthropod appendage. creeping welt slightly raised, often darkened structure on dipteran larvae. crenulate evenly rounded and often rather deeply curved; having an irregularly wavy or serrate outline; possessing a scalloped margin or rounded tooth. crescent (postanal crescent) crescent-shaped fold between the Glossary cloaca and terminus in male hairworms; usually ornamented with setae or bristles. cross ﬁbers ﬁne criss-cross ﬁbers in the upper cuticular layers in mermithid nematodes. cryobiosis cryptobiotic (latent) state in tardigrades induced by low temperatures. cryptobiosis latent state in which metabolism comes to a reversible halt; also known as “anabiosis” or “abiosis.” crystalline style mucopolysaccharide rod containing digestive enzymes projecting dorsally from the style sac in the ﬂoor of the bivalve stomach where it is secreted; the crystalline style is rotated by cilia lining the style sac against the gastric shield on the dorsal side of the stomach. ctenidium molluscan gill, in bivalves consisting of two demibranchs formed from the reﬂection of right and left gill ﬁlaments upon themselves. cupule cup-shaped segment at the base of the club on some antennae. cuticular bar external cuticular thickening on the leg, near or between the claws in some eutardigrades. cyclomorphosis regular (often seasonal or annual) phenotypic change in body size, spine length, etc., found in successive generations of zooplankton within a single habitat (does not apply to morphological changes shown by a single animal). cyrtos basket-like structure supporting the cytopharynx of Ciliophora in the protistan subphylum Cyrtophora. cytogamy occurrence of autogamy in each member of a pair of temporarily fused ciliates. cytolosomes digestive organelles of protozoa, analogous with food vacuoles, but containing the organelles of the cell, which are being broken down under conditions of starvation; synonymous with autophagic vacuole or autophagic vesicle. cytopharynx cell pharynx, leading from the cytostome to site of food vacuole formation in protozoa. cytoproct cell anus; permanent site for phagocytosis in some protozoa. cytostome cell mouth; permanent site for phagocytosis in some protozoa. dactyl most distal segment of the endopod of segmented appendages; smaller, mesially situated, movable ﬁnger of chela. demibranch portion of the bivalve ctenidium (gill) formed from a set of ﬁlaments reﬂecting upon themselves; in each ctenidium, the inner lateral ﬁlaments form the inner demibranch lying next to the visceral mass and the outer lateral ﬁlaments form the outer demibranch lying next to the mantle. denticles small, delicate, spinelike projections, usually of shell material in numerous taxa; also the small teeth-like processes lining the stoma of some nematodes; small teeth. depressed spire a spire that is not raised above the body whorl of a snail shell. determinate growth describing an organism that ceases to grow in size after reaching a certain size or stage; contrasts with indeterminate growth. 1007 detritus decaying organic material. deutocerebrum portion of a crustacean’s brain controlling antennules and innervating antennae and portions of the alimentary tract. deutonymph second nymphal instar in the generalized mite life cycle and the only active, free-living nymphal instar in the water mite life cycle. dextral snail shells with the aperture on right (when viewed with the apex pointed away from the observer and with the aperture fully visible). diapause period of arrested growth and development; often used to survive harsh environmental conditions. diel daily digestive diverticulum in bivalve molluscs, a digestive organ surrounding the stomach, area in which the ﬁnal intracellular phase of digestion takes place. digitate many ﬁnger-like extensions of a mollusc’s mantle. dioecious individual possessing either a male or a female reproductive system; reproductive systems contained in separate individuals. diploid having twice the number of chromosomes normally occurring in a germ cell, most somatic cells are diploid. discobolocyst extrusome of some ﬂagellated protozoa, especially Chrysomonadida. discus-shaped round and ﬂat disk with a thin edge and a thicker middle. distal toward the end of a structure and away from the central body opposite of proximal. diurnal recurring every day and/or relating to the daytime. diverticulum sac-like structure branching from an internal organ such as the intestine. doliiformis see pharynx. dormancy state of minimal metabolic activity when growth ceases, primarily enabling organisms to survive periods of adverse conditions. dorsal furrow strip of soft integument separating dorsal and ventral shields in highly sclerotized adult mites. dorsalia small platelets on the dorsum of the idiosoma in deutonymphs and adult mites that function as muscle attachment sites. dorsal plate large sclerite covering the prodorsal region of the idiosoma in any mite instar, often expanded to cover part of the opisthosomal dorsum as well. dorsal shield single large sclerite or a series of closely ﬁtting platelets covering the entire dorsum of the idiosoma in deutonymphs and adults of certain mite taxa. dorsocentralia series of paired, medially located dorsalia in mites. dorsoglandularia series of paired glandularia located on the dorsum of the idiosoma in mites. dorsolateralia series of paired, laterally located dorsalia in mites. dorsum ﬂattened area adjacent to the hinge line and set off from the lateral surface of the ostracode carapace. ductus communis in turbellarians, the terminal (or common) part of the female canal distal to the joining of vitelline duct(s) and oviduct(s) in ectolecithal systems. duplicature narrow band of shell material around outer edge of the proximal face of the epidermis, composed of the 1008 Glossary same three layers as the outer lamella; only calciﬁed layer of the proximal covering, lining, or epidermis separated into the list strip, selvage strip, and ﬂange strip of the ostracode shell. ecdysis shedding or molting of an older, smaller exoskeleton in arthropods. ecdysone the principal molt-stimulating hormone (in several forms) of arthropods. ecophenotypic plasticity variation in a species induced by nongenetic environmental factors. ectocyst nonliving outer layer of the body wall of bryozoans; may be sclerotized or gelatinous. ectolecithal pertains to systems in which the yolk is not incorporated into the oocyte, i.e., part of the female gonad is a yolk gland (as in higher Turbellaria). efferent branchial vessel blood vessel in the bivalve gill axis carrying oxygenated hemolymph from gill ﬁlaments to the longitudinal vessel of the kidney and eventually to the heart. ejaculatory complex series of membranous chambers and associated sclerotized framework located distally in the reproductive tract of male adult mites, functioning as a syringe-like organ for compacting masses of spermatozoa, assembling spermatophores, and expelling them from the genital tract through the gonopore. ejaculatory duct (ductus ejaculatorius) terminal part of the male duct in Turbellaria, sometimes eversible; carries spermatozoa and usually prostatic secretions through the copulatory organ. ejectisome extrusome of some ﬂagellated protozoa, especially in Cryptophyta. electromorph organism possessing a set of enzymes with a speciﬁc migration pattern as determined by electrophoretic techniques. electrostatic (biotic) ﬁlter surface of an animal that attracts charged particles, because of static-electricity-like charges on the surface. elytra hardened, shell-like mesothoracic wings of Coleoptera. emarginate notched; with an obtuse, rounded, or quadrate section cut from the margin. empodium medial unpaired, claw-like structure located terminally between the true claws on the tarsi of legs in larvae of all mite taxa, and in deutonymphs and adults of Stygothrombidiidae. encystment latent state in aquatic tardigrades in which resistant cysts are produced under deteriorating environmental conditions. endemic restricted to a particular geographic region. endites projections or processes from the inner margin of the inner branch of a branched appendage. endocrine relating to the system of glands that produce chemical signals (hormones) in the body. endocyst in ectoproct bryozoans, the inner living tissues of the body wall, including epidermis, muscle layers, basement membrane, and peritoneum. endocytosis process by which minute food particles are engulfed by cells into food vacuoles for the ﬁnal stages of digestion, as occurs in terminal tubules of molluscan digestive diverticula. endogenous initiated from within an individual, such as nervous or hormonal cues controlling diurnal rhythmicity in the absence of external cues. endolecithal (entolecithal) pertaining to those systems in which the yolk is incorporated into the oocyte (as occurs in more primitive Turbellaria). endopod mesial, or inner branch of an appendage; mesial ramus of biramous appendage, originating on second segment from base. endosymbiotic describing an organism which currently has internal symbionts. epibranchial cavity in bivalve molluscs, the exhalant mantle cavity formed dorsal to the ctenidium, which carries water leaving the gill to the exhalant siphon; it also receives the openings of the gonad, kidney, and anus. epigean referring to surface (above ground) habitats as opposed to hypogean. epilimnion portion of a lake above the thermocline and located in the limnetic zone. epineuston organisms living on or slightly immersed in the air/water interface; see neuston. ephippium darkened thickened carapace that protects resting eggs in cladocera. estivation general term for a physiologic process whereby animals reduce metabolism and activity levels to survive harsh conditions, such as pond drying for freshwater bivalves. eulaterofrontal cilia in bivalve molluscs, stiffened cilia extending laterally from the inhalant side of a gill ﬁlament to form a mesh with eulaterofrontal cilia of adjacent gill ﬁlaments on which particles are ﬁltered. eupathid specialized, thickened seta of mites located on distal, leg segments in all active instars. euryoecic pertaining to species tolerant of a wide range of habitats or environmental conditions. exchange diffusion transport of ions across epithelia such that diffusion of one ion species down its concentration gradient is coupled with transport of a second ion in the opposite direction. excrescences teeth on internal margins, denticles on the tips, and talons on the external margins of the horizontal ramus of the entocytherid ostracode hemipenes. excretophore large vacuolated cells of the gut epithelium of turbellarians. excretory pore plate in mites, a sclerite bearing the excretory pore in larvae, usually expanded to incorporate the bases of the setae associated with the pore. exogenous initiated from outside individuals, such as changes in light intensity controlling valve-gaping activity of a bivalve. exopod outer branch of a crustacean appendage; lateral ramus of biramous appendage, originating on the second segment from the base. exoskeleton hard external supporting structure, often composed of chitin (with or without calcium carbonate); as in covering of crustaceans. exploitative competition form of competition in which the outcome is determined by differential abilities to harvest a resource and/or by differences in reproductive rates; competition in which one organism does not directly Glossary inhibit another’s access to a limiting resource by behavioral means; compare with interference competition. extrapallial ﬂuid in bivalve molluscs, the ﬂuid ﬁlling the space (extrapallial space) between the mantle and the shell. extrapallial space area between the mantle and shell in molluscs. extrusome organelle of the pellicle of protozoa, which extrudes a substance or structure, usually in response to a stimulus. eye plate in mites, a sclerite bearing one or eyes. facilitation indirect food web effects which span two trophic levels; for example, an increase in the abundance of algae because a predator removes a snail herbivore. facultative an optional process. fascicles small tooth-like bundle of ﬁbers. fenestra in ectoproct bryozoans, the clear central portion of a ﬂoatoblast valve. ﬁle row, as in ﬁle of cilia or setae. ﬁliform thread-like; slender and of equal diameter. ﬁlopodium long, ﬁlamentous pseudopodium of certain Rhizopoda (amoeba), sometimes branching out without anastomoses; may function in both food capture and locomotion. ﬁnger guard extension of the ventral cardo along the side of the dorsal and ventral ﬁngers of the entocytherid ostracode hemipenes. ﬁrst-form male one of two morphological forms of cambarine crayﬁsh; sexually functional male; at least one terminal element of ﬁrst pleopod usually corneous. ﬂagellum multiarticulate, endopodite of antennae in some arthropods; whiplike locomotory structure of many protozoa. ﬂange distal ridge of the contact margin of the duplicature between the list and groove, it may be a part of the outer lamella of the ostracode shell. ﬂange groove that part of the duplicature surface of the ostracode valve between the selvage and ﬂange. ﬂange strip part of the duplicature that forms the ﬂange groove and the ﬂange of the ostracode valve. ﬂoatoblast in ectoproct bryozoans, a type of statoblast having a ring of gas-ﬁlled chambers; in some cases, the ring becomes buoyant only after being dried. follicular organ arranged in several small compartments (follicles), i.e., neither diffuse nor compact. foot a highly muscular locomotory or digging structure. forcipate type of trophi (jaw structure) found in rotifers having an action like forceps in which the trophi are projected from the mouth to grasp prey. fossorial associated with digging or burrowing. fragmentation spherules in Mollusca, the shed tips of digestive cells lining the terminal tubules of digestive diverticulum which are released after intracellular digestion has been completed; they contain undigested material remaining in food vacuoles and are carried on ciliary rejection tracts in the digestive diverticulum back into the stomach where their breakdown releases the undigested contents of their food vacuoles to be egested. frontal glands glands at anterior terminal region of turbellarians (frontal organ in Acoela). 1009 fulcrate type of aberrant trophi (jaw structure) found in rotifers in the order Seisonidae. fulcrum one of seven pieces comprising trophi of rotifers; together with the paired rami, they form the incus subunit. funiculus in ectoproct bryozoans, a tubular strand of tissue joining the blind end of the gut to the inner colony wall; the site of statoblast and sperm formation furca forked posterior end of some gastrotrichs. Also, an appendage-like structure that is the termination of the body proper in cypridid and cytherid ostracodes; it is articulated to the body and may assist in locomotion but is not considered a true thoracic leg. fusiform spindle-shaped; tapering at each end. gastric mill posterior end of the cardiac stomach of crustaceans and some other arthropods where food is ground for later digestion. gastric shield chitinous plate on the dorsal side of the stomach of bivalves (Mollusca) against which the distal tip of the crystalline style rotates. geminative type of growth of colonial rotifers whereby young produced by a colony on any day are all born within a span of a few hours and leave the parent colony together as a free-swimming larval colony, attaching to a new substrate in concert. As a consequence of this phenomenon, the genetic diversity of the colony is low; see autorecruitive and allorecruitive. gena (genae) the cheek; part of the head on each side below the eyes, extending ventrally to the gular suture. genet genetically distinct individual; often used to describe individuals that commonly grow as colonies in which the individual is not easily distinguished as a separate genetic entity; contrasts with ramet. geniculate jointed to permit bending at an abrupt angle; distinctly elbowed. genital bay area between the posterior coxal groups that accommodates the genital ﬁeld in adult mites. genital ﬁeld area of the idiosoma occupied by the gonopore and genital acetabula in adult mites. genital ﬂaps paired movable ﬂaps which close to cover the gonopore in adults of certain mite taxa; they typically also cover the genital acetabula, but in some derivative groups, the acetabula lie on the ﬂaps. genital papillae papillae situated on the surface of the cuticle or on elongate ﬁnger-like projections on the tail of male nematodes. germarium an ovary. germovitellarium organ containing both oogonia and oocytes, i.e., ovary and vitelline cells. gibbosity hump, marked by convexity or swelling, protuberant structure normally found on the dorsal part of an ostracode carapace. gill axis in Mollusca, portion of the ctenidium from which gill ﬁlaments extend and which is attached to the dorsal mantle wall. gill ﬁlaments in bivalve molluscs, long, thin, fused lateral extensions from the gill axis which extend ventrally and then reﬂect dorsally to form the inner and outer demibranchs. glandularium (glandularia) specialized organ located in the 1010 Glossary integument of deutonymphs and adult mites, consisting of a small, goblet-shaped sac, or “gland,” with a tiny external opening, and an associated seta. Glandularia have both a sensory and a secretory function. globose globe-shaped. glochidium bivalved larval stage of unionacean bivalves (Mollusca), generally parasitic on ﬁsh. glossa median terminal (or subterminal) lobe of the labium in some arthropods. glycocalyx “hairy” secreted layer covering the outer cell membrane of many of the “naked” rhizopod amoebae. gnathosoma in mites, anterior region of the body comprising the mouth and associated appendages, the chelicerae and pedipalps. gonochoristic two sexes; males and females in the same population reproducing by cross-fertilization; contrasts with hermaphroditism. gonopore external opening of the reproductive system in adults. green glands paired excretory glands in crustaceans; also known as antennal glands. gubernaculum sclerotized structure that supports and guides the spicules out of the cloacal opening during mating. gula throat sclerite, forming the central part of the head beneath the genae. gullet in ﬂagellated protozoa, anterior invagination from which the ﬂagella emerge. gyttja watery, organic sediment that consists mainly of excretory material from benthic animals. haploid having the number of chromosomes characteristic of the mature germ cell (half the number of the somatic cell). haptocyst extrusome of some predatory Ciliophora (Sectoria) used to capture prey. heliotropism tendency of certain plants or other organisms to turn or bend under the inﬂuence of light, especially sunlight. helocrene seepage area where spring water percolates through a substratum of mosses, leaf litter, and detritus. hemelytra mesothoracic wings of heteropteran insects. hemipenes paired penes. hemocoel internal body cavity formed by the expansion of the vascular system. hemocyanin copper-based respiratory pigment which is dissolved within the hemolymph of crustaceans. hemoglobin iron-containing blood respiratory pigment; found in a few invertebrates living in aquatic habitats with low oxygen levels. hemolymph the circulatory ﬂuid or blood of animals with open circulatory systems. hemolymph sinuses or hemocoels open cavities in which the blood or hemolymph bathes the tissues directly, allowing exchange of gases, ions, nutrients, and wastes. hermaphroditic individual containing both male and female reproductive organs. heterotrophy consuming, rather than synthesizing, organic compounds for metabolic activities; contrasts with autotrophy. hibernaculum thick-walled resting bud occurring in certain gymnolaemate bryozoans species. hindgut portion of intestine lying between midgut and rectum, primarily involved with consolidation of undigested particles into feces. hinge section of dorsal shell valve in bivalve molluscs containing the teeth and forming the fulcrum on which valves open and close. hinge ligament elastic proteinaceous portion of the shell of bivalve molluscs secreted by the mantle isthmus which connects the mineralized shell valves; it is ﬂexed or compressed when the valves are closed and when adductor muscles relax and functions to open the valves by returning to an unﬂexed state. hinge teeth interdigitating mineralized projections of shell in bivalve molluscs on the dorsal margins of the valves that hold the valves in position and serve as the fulcrum on which they open and close. histophages protozoa that feed on the tissue of dead metazoa, as opposed to a parasite feeding on live hosts. holophyletic group monophyletic clade including all species descended from a common ancestor. homoplasy parallel adaptive modiﬁcation of homologous attributes or structures in species of different clades due to similar selective pressures. hooked glochidia glochidia larvae in bivalve molluscs with spiny hooks on their ventral shell margins. hormone chemical signal produced and used inside an individual’s body. hyaline margin clear ectoplasmic material surrounding and/or preceding the granular body mass of some amoebae during locomotion. hybrid result of a mating between organisms with different genotypes; used also for matings between species. hypersaline term generally referring to a water body whose salinity is higher than seawater (35 mg / L). hypodermal cords expansions of the hypodermis between muscle ﬁelds of nematodes. hypogean subterranean; burrow, cave, or groundwater environment. hypolimnion portion of a lake below the thermocline and located in the limnetic zone. hyponeuston organisms living just below the air/water interface of a water body; see neuston. hyporheic zone groundwater present below and to the side of a stream and located close enough to be affected by the movement of stream surface waters (unlike the phreatic zone). hypostome pertaining to the ventral portion of the ostracode head is located a keel-shaped structure which forms the mouth. hypoxia condition of environmental oxygen concentration below air oxygen saturation levels but not necessarily anoxic. hysterosoma region of idiosoma of mites posterior to level of primitive sejugal furrow. idiosoma body proper of mites, comprising the fused cephalothorax and abdomen. imagochrysalis quiescent transitional instar between the deutonymph and adult in the water mite life cycle, representing the suppressed tritonymph. Glossary incudate type of trophi (jaw structure) found in rotifers, which functions by grasping prey with a forceps-like action. incus subunit of a rotifer trophi composed of fulcrum and paired rami. induction change in development or behavior due to receiving an internal or external signal (such as a hormone or kairomone) infauna sediment-dwelling animals; also, but more rarely, animals that burrow into and live inside another organism. infraciliature total assemblage of all kinetosomes and the associated subpellicular microﬁbrular and microtubular structures in ciliate protozoa. infundibulum corona of rotifers of the order Collothecacea. inner lamella thin layer of chitin, which, together with the duplicature, forms a proximal covering of the epidermis in ostracodes; at inner limit, it is folded back on itself to form the body wall of the pouch-shaped body of the animal. instar any developmental stage between molts. interareolar furrows network of transverse and longitudinal trenches separating areoles in hairworms; may bear pores or support bristles and warts. interference competition form of competition in which one organism directly or indirectly interferes with another organism (e.g., by aggression), thus preventing it from obtaining a limiting resource; forms of interference include physical combat, the threat of combat, noxious chemical compounds, etc. interlamellar space cavity formed between ascending and descending gill ﬁlaments in Mollusca; water ﬂowing through the ostia of gill ﬁlaments enters the interlamellar space to be carried dorsally to the epibranchial cavity; also called the water tube or water channel. interstitial microhabitat comprising spaces between particles of submerged sand and gravel in groundwater and hyporheic habitats. intracytoplasmic lamina ﬁlament layer of varying thickness present within the syncytial integument of rotifers and acanthocephalans. ischiopodite third segment from the base of a segmented appendage. isthmus portion of nematode pharynx between the metacorpus and basal bulb, usually surrounded by the nerve ring. In bivalve molluscs, the constricted dorsal section of the mantle separating the mantle into two lateral shell-secreting halves; the isthmus secretes the proteinaceous hinge ligament connecting the valves. iteroparous reproducing repeatedly at multiple times during an organism’s life (cf., semelparous). kairomone chemical signal produced by a predator that affects behavior, development, or life history of another species so as to result in higher relative survival of individuals of that species that react to the signal. karst terrain underlain by fractured and extensively dissolved carbonate rock (e.g., limestone) and typiﬁed by numerous sinkholes, springs, caves, and few surface streams. kinetid kinetosome and its associated pellicular structures in Ciliophora, including the cilium, the elementary repeat- 1011 ing structure of the ciliate cortex; the polykinetid is an organellar complex composed of two or more kinetids. kinetocyst extrusome of Actinopoda, which secretes materials that probably serve to make the surface of an axopod sticky. kinetoplast DNA-rich organelle near the anterior of some ﬂagellated protozoa (Kinetoplastida), associated with their unique large mitchondrion. kinetosome subpellicular structure forming the base of a cilium, but may be without a cilium; it is characterized by nine triplets of microtubules at right angles to the plasma membrane arranged in a circle. kinety single, structurally and functionally integrated (somatic) row of kinetosomes along with their cilia (cilia not necessarily associated with each inetosome), i.e., a line of kinetids. knob in bivalve molluscs, a high rounded major protuberance with the sides joining the rest of the valve at a distinct line and at a steep angle. K-selection selection for life-history characteristics that increase ﬁtness in stable environments where populations reach densities near environmental carrying capacity; associated with high levels of intraspeciﬁc competition. labial palps pair of thin ﬂattened structures extending from either side of the mouth region of bivalve molluscs to lie against the outer surface of the outer demibranch and the inner surface of the inner demibranch; they function to receive ﬁltered particles from the ctenidia and sort them into those accepted for ingestion and those rejected as pseudofeces. lacustrine see lentic. lamellae layers; ﬂattened, leaf-like structures in several phyla, such as the gills of bivalve molluscs and the buccal platelike projections on the mouth (buccal) ring of some eutardigrades. Lansing effect putative negative effect of elevated calcium on survivorship in rotifers in which calcium accumulation in older mothers is passed on to their offspring; seeorthoclone. lateral coxal apodeme conspicuous apodeme indicating the line of fusion between the second and third coxal plates in larvae of certain mite taxa. lateral ﬁeld track usually containing several longitudinal striations running on both sides of the nematode. lateral teeth elongated lamellar hinge teeth in bivalve molluscs forming anterior and posterior to the cardinal teeth or posterior to the pseudocardinal teeth in unionacean bivalves. LC50 concentration of a toxic agent at which 50% of a cohort will perish within a certain predetermined period; also called the median lethal concentration and LD50. lentic standing water environments (e.g., lakes, ponds, wetlands, and both permanent and temporary pools). limnetic open water, deeper areas of a lake or pond and away from the shoreline littoral zone. line of concrescence proximal line of junction of the duplicature and outer lamella of the ostracode valve, the inner border of the chitin adhesive strip. lira rib or ridge running along the whorl of a snail shell in the direction of the coiling. 1012 Glossary list proximal ridge on the contact margin of the duplicature of the ostracode valve, not always present. list strip part of the duplicature from the inner margin to and including the list of the ostracode valve. littoral zone or body of water shallow enough for growth of rooted aquatic vegetation. lobopodium blunt pseudopodium used in feeding and/or locomotion. lophophore organized structure of ciliated tentacles used for capturing suspended food particles from the water, and probably also providing an important surface for gas exchange; occurring in ectoprocts and certain other phyla. lorica thickened body wall of rotifers, organized as a syncytial integument possessing two keratin-like proteins which compose a layer called the intracytoplasmic lamina. A loose-ﬁtting surface covering secreted and/or assembled by protozoa; generally synonymous with test. lotic running water environments (e.g., creeks, rivers, and some springs). lunule crescent or half-moon shaped structure at the base of each double claw in some eutardigrades. lyriﬁssure (lyriform ﬁssure) slender sac-like structures in mites opening through slits in the integument, apparently functioning as proprioceptors, in all active instars. lysosome membrane-bound organelle containing hydrolytic enzymes; in protozoa, they are involved in intracellular digestion. macroinvertebrate organisms larger than microinvertebrates and meiobenthos; retained on coarse sieves with a mesh 2 mm. macronucleus in ciliates, the nucleus active in transcription and thus responsible for the phenotype and regulation of metabolism; see micronucleus. macrophagous in protozoa, a species that consumes large items singly by phagocytosis. macrophyte macroscopic photosynthetic organism growing submersed, ﬂoating, or emergent in water; most are angiosperms but also includes some nonvascular plants and macroalgae. macropterous with fully formed wings. malleate type of trophi (jaw structure) found in certain monogonont rotifers in which the rami are massive and may possess teeth along the inner margin. malleoramate type of trophi (jaw structure) found in monogonont rotifers of the order Flosculariacea, resembling the malleate form, except in number of teeth on the unci. malleus subunit of rotifer trophi composed of an uncus and manubrium. mandible hardened mouthparts (cephalic appendages) in many arthropods that are used to grasp, tear, and push food into the mouth; most anterior gnathal appendage and just behind the antennae. mandibular palp endopodite segment of the mandible. mantle thin extension of the dorsal body wall of molluscs underlying the shell and responsible for its secretion. mantle cavity space formed between the body and surrounding mantle tissues in molluscs; completely surrounds the body in bivalves. manubrium one of seven pieces comprising the trophi of rotifers; a manubrium together with an uncus form the malleus subunit. marsupium space formed for incubation of embryos and larval stages. mastax muscular pharynx of rotifers containing jaws called trophi. maxillae ﬁrst or second pair of mouthparts (cephalic appendages) posterior to the mandibles; crustaceans have two pairs of maxillae, insects have one pair. maxillary glands paired excretory glands of crustaceans. maxilliped one of pair of three sets of gnathal appendages situated immediately posterior to second pair of maxillae. maxillule ﬁrst maxilla. medial coxal apodemes series of short apodemes at the medial edges of coxal plates in some larval mites. medial eye plate (frontal plate) sclerite bearing medial eye in early derivative mite taxa; the medial eye may be absent in highly derived groups. meiobenthos small benthic organisms that pass through a 2 mm mesh but are retained on a 40 – 200 m mesh. melanin black, brown, or red pigment made of polymerized amino acids (tyrosine); used for photoprotection and making structures stronger. merus fourth segment from proximal end of segmented appendage. mesial (mesal) pertaining to the middle; toward the middle. metachronal occurring one after another, in succession. metacorpus swollen median portion of nematode pharynx, located between the procarpus and isthmus. metamere homologous body segment, a somite. metasome portion of the copepod body anterior to the major body articulation. microaerophilic protozoa with aerobic metabolism living in microhabitats with very low oxygen concentrations. micronucleus nuclei of ciliates mediating sexual recombination and reproduction, typically much smaller than the macronucleus; diploid; often multiple. microphagous protozoa that consume relatively small items by phagocytosis, collecting many into a single food vacuole. microplankton plankton that are 20 – 200 m in size. microsome simple membrane-bound organelle containing oxidative enzymes; synonymous with microbody. microvilli microscopic projections from a cell surface that increase the surface area of the cell. mictic (mixis) phase in the life cycle of monogonont rotifers in which reproduction is sexual, resulting in a diapausing embryo called a resting egg; seeamictic. midden refuse heap; commonly, shells of unionacean mussels that had been consumed by humans or other mammals and then left at one site. middle piece structure in sperm cells lying between the head and ﬂagellum containing mitochondria. midgut portion of the intestine between stomach and hindgut; in molluscs, it is characterized by the presence of the typhlosole, the ciliated surfaces of which sort particles into those returned to the stomach and those passed to the hindgut for consolidation into feces and egestion. Glossary mixotroph organism driving energy from both autotrophy and heterotrophy, including those whose autotrophy is via endosymbionts. molting process of shedding the cuticle at periodic intervals to allow expansion of the body in a newly developed and larger exoskeleton. monoecious individual possessing both male and female reproductive systems, at least during part of its life cycle. monophyletic group phylogenetic grouping of species descended from a common ancestor and which are classiﬁed together within a single taxonomic unit; compare with polyphyletic. monopodial describing an unbranched, cylindrical type of pseudopodium. morph variant form within a species. morphotype morphologically distinct form; in rotifers, a form present within a single population whose polymorphic feature(s) (e.g., spines, body size) undergo a seasonal phenotypic change. mouth hook vertically-oriented, mandible-like structure in dipteran larvae. muciferous body variety of mucocyst found in some Phytomonadida. mucocyst extrusome of protozoa responsible for secretion of mucous-like material onto the cell surface. mucrones small cuticular teeth on interior of the buccal cavity in some eutardigrades. multispiral growth rings of an operculum of a snail comprised of many, slowly enlarging spirals. multivoltine having more than two broods or generations per year. muscle scars area marking the former position of attachment of a muscle on the interior of a shell or other support structure, distinguished from the rest of the shell by a surrounding groove or by being higher or depressed. mutualism association between two organisms in which both beneﬁt. nacre or nacreous layer inner shell layer of most molluscs, secreted by the mantle as successive layers of calcium carbonate crystals (absent in the Sphaeriidae). nannoplankton plankton that range from 2 – 20 m in size. nauplius earliest larval stage of many crustaceans. neonate newly-born animal. net growth efﬁciency percentage of assimilated energy represented by that allocated to tissue growth and gamete production. neuston organisms living at or very near the air/water interface of a water body; see hyponeuston, epineuston, and pleuston. niche partitioning differences in use of resources that alleviate interspeciﬁc competition. node on the ostracode carapace, a protuberance smaller than a lobe or knob but larger than a tubercle, may have the form of a half sphere. nonrespired assimilation portion of assimilated energy or food not catabolized for maintenance energy and, therefore, available for production of new tissue or gametes. nymphochrysalis quiescent transitional instar between the 1013 larva and deutonymph in the water mite life cycle, representing the suppressed protonymph. oblique slanting; neither parallel nor perpendicular to a reference point. obtuse blunt angle at the end. ocellus simple eye consisting of a single, bead-like lens, occurring singly or in small groups; distinct from a compound eye. omnivore consuming food at more than one trophic level, e.g., crayﬁsh eat live animals (carnivory) and plants (herbivory) and will also consume dead animals and plants (detritivory). open circulatory system circulatory system characterized by blood not being continually maintained in vessels; rather, the blood bathes the tissues directly in large hemocoel sinuses before returning to the heart. operculate formed as a cover. operculum lid or covering structure, as in some insects. Horny or calcareous disk on the foot of a snail that covers the aperture when foot is withdrawn; used as protection for predators. opsiblastic egg type of resting egg produced by gastrotrichs; see tachyblastic egg. oriﬁce opening; in ectoprocts, the terminal opening in a zooid through which the polypide extends. orthoclone clone of a rotifer species established after many generations of culturing the ith offspring of every generation (seeLansing effect). osmobiosis cryptobiotic (latent) state in tardigrades induced by elevated osmotic pressures. ostia pores; openings penetrating fused gill ﬁlaments in bivalve molluscs with eulamellibranch ctenidia which allow passage of water across the ﬁlaments into the interlamellar space. outer lamella in ostracodes, a hard shell material that covers the outside of the epidermis that secreted it, composed of a thick layer of calcite enclosed between two layers of chitin. ovate (ovoid) somewhat oval in shape. oviduct duct from the ovary, through which ova pass. oviparous females that produce eggs that undergo embryonic development and hatch outside the body of the female; see viviparous and ovoviviparous. ovoviviparous females which produce eggs that undergo embryonic development inside the female and that hatch just before or soon after being released; see oviparous and viviparous. paddles feather-shaped locomotory structures attached anteriorly in Polyarthra rotifers. pallial pertaining to the pallium or mantle cavity of a mollusc. pallial line line of muscle scars on the outer perimeter of the inside of the bivalve mollusc shell, marking points at which pallial muscles attach the mantle to the shell. pallial sinus posterior indentation of the pallial line on the inside of the bivalve mollusc shell, marking the space into which the siphons are withdrawn on valve closure. palmate like the palm of the hand, with ﬁngerlike processes. palpal lobes broad, paired, movable lobes on the distal end of the prementum in odonate insects. 1014 Glossary palustrine relating to wetland habitats. papilla one of many small discrete protuberances (as on the ostracode shell surface) each with steep sides; smaller than tubercles. papillate many lobe-like extensions. paraglossa lateral terminal lobe of the labium. paramylon reserve food material characteristic of euglenid ﬂagellates; occurs in a variety of shapes, usually with a laminated structure. paraphyletic group monophyletic grouping including only some of the species descended from a common ancestor. paratene repeated kinetidal patterns at right angles to the longitudinal axis of a ciliate’s body; superﬁcially gives impression that the ciliary rows run circumferentially rather than longitudinally in the affected part of the body. parietal inside wall of a snail aperture. paroral membrane synonymous with undulating membrane. parthenogenesis sexual reproduction in which egg development occurs without fertilization. Amictic parthenogenesis is sexual reproduction without recombination of genetic material. paucispiral growth lines of a snail operculum, present as a few, rapidly enlarging spirals. pectinate having narrow parallel projections suggestive of the teeth of a comb. pedal referring to the foot. pedal feeding use of the foot in bivalve molluscs to feed on organic detrital deposits in or on sediments; generally involves ciliary tracts on the foot to bring detritus to the palps or gill food grooves. pedal gland cement glands in the foot region of rotifers used to achieve temporary (in pelagic or littoral zooplankton) or permanent (in sessile forms) attachment to a surface. pedicles lateral extensions of the corona of certain bdelloid rotifers. pedipalp feeding appendage; in mites, the pedipalp typically has ﬁve movable segments, located laterally on the gnathosoma in all instars. Pedipalps have both sensory and raptorial functions. peduncle pseudopodium-like cytoplasmic extension emerging from the sulcus in some dinoﬂagellates and used in feeding. Also, general name for a holdfast organelle in diverse groups of free-living protozoa. pelagic see limnetic. pellicle surface layer of protozoa, including the plasma membrane and associated membranous, microﬁbrillar and microtubular structures and organelles, but excluding nonliving external coverings such as tests or loricas. peniferum portion of the entocytherid ostracode copulatory apparatus that bears the penis (usually its distal portion), and its proximal portion forms a hinge on which the entire apparatus may be turned through an arc of 180 degrees about the zygum; includes the dorsal and ventral cardo, costa, spermatic tube, and penis. penultimate next to last. pereiopods thoracic appendages of many crustaceans; usually having a prime locomotory function (walking or swimming) and one or more secondary roles. perennial living for several years. periblast part of a statoblast valve of ecotoprocts that excludes the capsule; in ﬂoatoblasts, the periblast includes both annulus and fenestra. peribuccal lobes ﬂattened cuticular projections surrounding the mouth of some eutardigrades. peribuccal (oral) papillae elongated cuticular projections surrounding the mouth of some eutardigrades. peribuccal papulae short, distinct cuticular projections surrounding the mouth of some eutardigrades. pericardial cavity remnant of the coelomic cavity surrounding the heart ventricle in molluscs. pericardial gland specialized tissue in the wall of the heart auricles of molluscs through which hemolymph ﬂuid is ultraﬁltered into the pericardial space from which it enters the kidney. pericardium epithelial layer lining the pericardial cavity. periostracum proteinaceous outer layer of mollusc shells secreted by the mantle edge. periphyton microalgae growing in a thin layer on mostly vascular plants; sometimes used loosely for microalgae growing on other substances. peristalsis rhythmic waves of contraction which moves material through the gut. peristome area surrounding the cytostome of ciliates, especially where it is specialized relative to the rest of the body surface. peristomium ﬁrst functional segment, usually containing the mouth, in segmented worms. phagocytosis process by which a cell engulfs a particle by extending its plasma membrane to form a vacuole or invagination. phagotrophy nutrition by phagocytosis. pharyngeal apophyses cuticular thickenings at the junction of the buccal tube and lumen of the pharynx, alternating in position with the placoids. pharyngeal intestinal junction; in nematodes, a cellular valve located between the pharynx and intestine; may protrude down into the intestine. pharynx portion of the alimentary tract located between the stoma and intestine, often called the esophagus. In turbellarians, “pharynx simplex” is a mostly short, unfolded tube; “pharynx plicatus” is a protrusible, more or less tubular fold enclosed in the pharynx cavity; “pharynx bulbosus” is a bulb separated from the surrounding tissue by a muscular septum. Pharynx bulbosus is represented by three types: rosulatus, doliiformis, and variabilis. Pharynx rosulatus is mostly round with a more or less vertical axis; pharynx doliiformis is barrel-shaped, anteriorly situated and with a horizontal axis; pharynx variabilis is weakly muscular with variable shape and weakly differentiated septum. phasmids paired sensory organs located laterally in the caudal area of nematodes. phenotypic plasticity expression of different morphologies depending on environmental inﬂuences or signals and expressed by a single genotype. pheromone chemical messenger substance secreted by one individual into the external environment, which elicits a Glossary speciﬁc response in another individual of the same species. photodamage damage caused to molecules due to ultraviolet radiation in sunlight. photoperiod length of the light period during a 24 hour lightdark cycle. photoprotection protection from ultraviolet light, as by a dark pigment that absorbs light. phototaxis locomotion directed by light. phreatic zone ﬂowing groundwater located far enough from surface streams so that water movement in the streams does not inﬂuence movement of the phreatic waters; seehyporheic zone. phyllopod a leaf-like or lobed, biramous appendage in some non-malacostracan crustaceans. phytophilic aquatic herbivores with a preference for consuming algae; also called algivores. phytoplanktivorous pertaining to feeding on phytoplankton (i.e., suspended algae). phytoplankton unicellular and multicellular algae and cyanobacteria suspended in the water column. picoplankton plankton 0.2 – 2 m in size, primarily bacteria. pinocytosis process by which cells ingest dissolved or colloidal material, involving attraction of these substances to cell surfaces and then internalizing them through invagination of the plasma membrane. piptoblast type of statoblast in bryozoans that has no annulus and is not cemented to the colony substrate; formed only in the family Fredericellidae. placoids cuticular thickenings in the lumen of the tardigrade pharynx (pharyngeal bulb); large anterior ones are macroplacoids, small posterior row are microplacoids. plankton eukaryotic and prokaryotic organisms living all or part of their lives in the water column of aquatic ecosystems and at the mercy of water currents. planospiral having shell whorls conﬁned to a single plane; coiled but without an elevated spire. plasma membrane unit or cell which is the outer-most living layer of protozoa. plasmodium multinucleate mass of protoplasm enclosed by a plasma membrane; nonhomologous trophic stage in the life cycle of some protozoa. plastron semipermanent bubble of air through which carbon dioxide from respiration is exchanged with oxygen from the water. pleopod one of ﬁve pairs of appendages on ﬁrst ﬁve segments of a decapod’s abdomen; also called “swimmerets,” or modiﬁed into male gonopods. plesiotypical (plesiomorphic) conservative, or unmodiﬁed, state of a biologic attribute or morphologic character. podomere leg segment of an arthropod. polykinetid see kinetid. polykinety compound ciliary organelle composed of several kineties, e.g., a cirrus. polyphyletic group taxonomic assemblage of species that are not all descended from a common ancestor. polypide retractable portion of a bryozoan zooid comprising the central ganglion and all structures related to feeding and digestion. 1015 polyploid having more than twice the number of chromosomes in somatic cells. polytrophic see omnivore. polyvoltine with several generations during the season; contrasts with univoltine. pore canals in nematomorphs, pores connected to ﬁne tubes in the cuticle; in ostracodes, a passage through the entire valve in which are located sensory hairs to the nerves of the hypodermis. postcloacal crescent see crescent. posterior ridge external ridge on unionacean (Mollusca) shells extending from the umbos posterioventrally to the shell margin. postocularia pair of prominent setae located on the dorsum of the idiosoma posterior to the level of the eyes in deutonymphs and adult mites. prehensile ﬁtted or adapted for grasping, holding, or seizing. prehensile palp appendage modiﬁed to grasp, hold, or seize; generally formed of two podomeres, the proximal one referred to as the prodopus, distal one referred to as the dactylus of some crustaceans such as ostracodes. prementum distal segment of the labium in odonate insects, which bears the palpal lobes. preocularia pair of prominent setae located on the dorsum of the idiosoma anterior to the level of the eyes in deutonymphs and adult mites. preparasitic attendance phoretic association by larvae of some species of water mites with the preimaginal stage of their insect host. prismatic layer molluscan shell layer composed of elongated calcium carbonate crystals oriented at a 90° angle to the surface plane of the shell and lying between the outer periostracum and inner nacreous layer. procorpus anterior portion of the nematode pharynx between the stoma and metacorpus. prodelphic with a single anterior-directed ovary in nematodes. profundal deep zone of a lake below the littoral and open water photic zone; often restricted to benthic habitats but may include pelagic zone. proleg process or appendage that serves the purpose of a leg but is not a true leg. promitosis form of nuclear division wherein the endosomal body in the nucleus divides and forms two polar masses with a spindle between them; characteristic of some naked amoeba of the class Heterolobosea. propodosoma region of a mite’s idiosoma posterior to the level of the primitive sejugal furrow. propodus penultimate segment (sixth from base) of a segmented appendage. proprioceptor sensory receptor that responds to physical or chemical stimuli originating from the organism. prostate cells, glands, or organs in male reproductive systems, releases secretions that mix with sperm. prostaglandins local chemical mediators inducing short-lasting effects in adjacent tissues. prostomium region anterior to mouth in some oligochaete worms. protandry sequential hermaphroditism with the male stage preceding the female stage. 1016 Glossary protocerebrum part of a crustacean’s brain that normally innervates eyes, sinus gland, frontal organs, and head muscles. protoconch shell of newborn snail or “spat.” protonephridia excretory-osmoregulatory system of certain pseudocoelomates, including gastrotrichs and rotifers, comprised of ﬂame cells and tubules. protonymph ﬁrst nymphal instar in the generalized mite life cycle. protopodite basal part of a typical crustacean limb consisting of two, more-or-less consolidated segments and bearing at its distal extremity an exopodite, endopodite, or both. proventriculus large cavity in collothecid rotifers that is an extension of the mastax in which prey are held prior to being passed into the stomach. provisional genital ﬁeld region of the idiosoma occupied by the rudimentary gonopore and genital acetabula in mite deutonymphs. proximal toward the median plane of a body. pseudobranch “false gill” present in ancylid and planorbid snails; conical extension of epithelium used in respiration, analogous to ctenidium of prosobranchs. pseudocardinal teeth in bivalve molluscs, compact shell lamellae on the anterior portion of the hinge plate of unionacean mussel shells which performs function of cardinal teeth. pseudocoelom body cavity not lined with mesodermal tissue, as is a true coelom, and found in gastrotrichs, nematodes, and rotifers. pseudofeces masses of mucous-bound particles rejected from the labial palps of bivalve molluscs before ingestion and release into the mantle cavity to be carried out the siphon on water currents induced by valve clapping (i.e., rapid valve adduction). pseudopodium in protozoa, a dynamic extension of the cytoplasm used in feeding or locomotion; in insects, a soft, foot-like appendage that is less prominent than a proleg. pseudosexual eggs in cladocera, asexually produced eggs that resemble those produced by a sexual process. In rotifers, diploid resting egg supposedly produced in rotifers by parthenogenesis in the absence of males; seemixis. pseudostome aperature of a test or shell of a testate amoeba; pseudopodia protrude from it during locomotion and/or feeding. punctae small pit-like opening of a pore canal on the ostracode shell. Microscopic pores traversing the mineral portion of the shells of sphaeriid bivalve molluscs to open beneath the periostracum; contain extensions of the pyramidal cells. pustule protuberance with a pore in the middle, similar to a volcano with a crater, about the size of a tubercle on the ostracode carapace. pyramidal cells in bivalve molluscs, mantle epithelial cells that extend through shell punctae (pores) to lie just below the periostracum. Q10 difference in metabolic rate over a 10°C temperature range (e.g., Q10 would equal 2 if the respiration rate doubled between 10 and 20°C). Q10 (acc.) factor by which a biologic rate function changes over a 10°C increase in acclimation temperature. quadrate square or rectangular in shape. radial pore canal passage through the adhesive strip, between the duplicature and the outer lamella of the proximal valve edge of an ostracode. ramate type of trophi (jaw structure) found in bdelloid rotifers in which the rami are large and semicircular in shape and unci possess many teeth. ramus one of the seven pieces comprising rotifer trophi; paired rami together with the fulcrum form the incus subunit. receptaculum seminis part of the female reproductive system of turbellarians in which recipient spermatozoa are stored. reniform suggesting a kidney in shape. respiratory plate also referred to as branchial plate, modiﬁed from the exopodite and may contain numerous feathered setae. resting egg thick-walled cyst enclosing a diapausing, diploid embryo, usually resistant to extreme environmental conditions. In rotifers these “eggs” are produced by sexual reproduction. reticulopodium threadlike, repeatedly branching and anastomosing pseudopodium, functioning more often in food gathering than locomotion. retrocerebral organ structure of unknown function in rotifers composed of the unpaired retrocerebral sac and paired subcerebral glands together; possible function as an exocrine gland lubricating the anterior part of the body. retrocerebral sac unpaired glandular structure near the subcerebral gland in the head region of rotifers possessing a forked duct that opens into the corona. rhabdions cuticular segments of the nematode stoma, may be fused and produce an entire stomal wall or separate and exhibit a segmented wall. rhabdos organelle supporting the cytopharynx of some ciliates, especially in the subphylum Rhabdophora; although like a cyrtos, rhabdos is considered evolutionarily the more primitive organelle. rheocrene ﬂowing spring with a substratum of sand and gravel. rheotaxis orientation to current (can be positive or negative). riparian living on the bank of a lake, pond, or stream. rostrum any extended portion of the head end of an invertebrate. r-selection selection for life-history traits increasing ﬁtness in an unstable habitat where catastrophic population reductions prevent high levels of intraspeciﬁc competition. scapular setae second most anterior series of sensory setae on the dorsal surface of the idiosoma of water mites, consisting of an internal pair (si) and an external pair (se). sclerite hardened area of the insect body wall bounded by sutures or membranes. semivoltine having one brood or generation every two years. sanguivorous feeding on blood. scabrous having small, round, raised scales or cones. second-form male one of two morphological forms of male cambarine crayﬁshes; sexually nonfunctional male lacking corneous terminal elements on ﬁrst pleopod (gonopod). Glossary sediment focusing uneven deposition of sediments in a basin so that certain portions of the basin accumulate sediments at a greater rate; usually this occurs in the deepest parts of the basin. sejugal furrow primitive line of demarcation on idiosoma at level between insertions of second and third pairs of legs in all mite instars, but expressed only in certain early derivative taxa. selvage middle and principal ridge of the contact margin of the duplicature of the ostracode valve. selvage groove part of the duplicature surface between the list and selvage of ostracode valve. selvage strip part of the duplicature forming the selvage and selvage groove of the ostracode valve. semelparous breeding only once during an organism’s life; one massive reproductive effort. seminal receptacle (spermatheca) sac-like structure in the female reproductive system that receives and stores spermatozoa. seminal vesicle saclike structure in the male reproductive system that stores spermatozoa. sensillum epithelial sense organ. septulum cuticular thickening in the lumen of the pharynx, posterior to the placoids, in the same plane as the pharyngeal apophyses in some eutardigrades. septum dividing wall or membrane; in ostracodes, a small ridge on the list strip on the duplicature of the valve. serial homologies structures having the same embryonic and evolutionary origin but which differ segmentally (e.g., all appendages of a crayﬁsh, except the ﬁrst antennae, arose from similar embryonic primordia and from a series of similarly formed appendages in an ancient ancestral stock). serotonin chemical messenger stimulating active transport of sodium ions from the medium in freshwater bivalve molluscs. serrate notched or toothed on the edge. sessoblast relatively large statoblast cemented ﬁrmly through the body wall to the substratum; formed only in the bryozoan family Plumatellidae. setae (or chaetae) epidermal, hair-like extensions of the cuticle. sexual dimorphism differences in shape and/or size between females and males of the same species. sheath in nematodes, a cuticle retained from the last molt which may surround the entire body or head region. simplex stage molting stage in tardigrades characterized by the absence of the buccal apparatus. sinistral snail shells with the aperture opening toward the left (when viewed with the apex pointed away from the observer and with the aperture fully visible). siphon in bivalve molluscs, tubular structure formed by fusion of opposite mantle margins directing inhalant (inhalant siphon) and exhalant (exhalant siphon) respiratory and feeding water currents into and out of the mantle cavity. An elongated breathing tube in some insects. solenidion (solenidia) specialized setiform chemosensory structure in mites; solenidia are located on distal segments of the pedipalps and legs in all active instars; though seta-like in appearance, solenidia differ structurally and ontogeneti- 1017 cally from setae and are usually given distinctive designations in chaetotactic formulae. somatic cell cell that become differentiated into tissues, organs; opposed to embryonic germ cell. somite homologous body segment, a metamere. spear protrusible pointed, solid rod-like structure in the mouth region of some nematodes. species richness number of species in a habitat. spermatogenesis formation of sperm. spermatophore packet of spermatozoa enclosed in a membranous capsule, produced by adult male mites and either deposited on the substratum or transferred to the female gonopore. spermatozoan individual haploid cell produced by males for sexual reproduction. sphincter circular muscle forming a ring around a tube, such as the gut, that closes the tube by contracting. spicules internal support and/or protective structures of sponges composed principally of silica or calcium salts. Also, paired sclerotized structures in the tail of the male nematode. spindle snail shell with a thick middle and a tapered point at both the apex and the aperture ends. spinneret in nematodes, a terminal pore out of which passes secretions of the caudal glands. spiracle respiratory opening connected to the tracheal (respiratory) system of insects. standing crop biomass amount of tissue produced per unit area at a set point in time, usually measured as g carbon, Kcal, or ash free dry mass (AFDM) per m2. statoblast in phylactolaemate bryozoans, an asexually produced capsule composed of two convex valves enclosing a mass of yolky material and undifferentiated tissue; capable of germinating a new zooid after obligate dormancy. statoconia in bivalve molluscs, many small mineral inclusions in the cavity of a statocyst (gravity orientation organ). statocyst gravity-orientation organ containing mineral inclusions (statolith or statoconia) in a vesicle lined with sensory cilia. statolith mineral inclusion in the cavity of a statocyst. stenopod unbranched, segmented walking leg in some crustaceans, particularly decapods. stenothermal tolerating little variation in temperature. stigmata paired external openings of the tracheal system, located between bases of the chelicerae in all active instars of mites. stolon in certain ectoproct bryozoans, a cylindrical stem-like structure from which individual zooids grow at intervals. stoma portion of the alimentary tract between the oral opening (mouth) and pharynx. stream order method for classifying streams in which the smallest permanent streams is designated as a “ﬁrst order” stream; two ﬁrst orders streams combine to form a second order stream; a ﬁrst and second in combination are still considered a second order, but when two second order tributaries join, a third order stream results. striations (striae) parallel grooves; also, the spiral, microscopic grooves along the whorls of a snail shell (sometimes incorrectly used to refer to spiral ridges or lirae). 1018 Glossary stygobitic species occur exclusively in hypogean habitat. stygophilous species occurring in both surface and subterranean waters but with a preference for the latter habitat. style sac evagination of the stomach ﬂoor of molluscs, which secretes the crystalline style. stylet sclerotized tube or channel of variable shape and complexity; a hollow, sclerotized, hard, rod-like structure in the mouth region of some nematodes. stylet knobs enlarged protuberances (usually three) at the base of the nematode stylet, usually serve for attachment of muscles that aid in stylet extension. stylostome mucopolysaccharide feeding tube secreted by parasitic larval water mites into the body of their host. subcerebral glands paired glands near the retrocerebral sac in the head region of rotifers with ducts that lead to the corona; seeretrocerebral organ. subitaneous egg one developing and hatching with no arrest in development. sulcus depression or furrow on the lateral surface of the organisms in several phyla (e.g., Ostracoda and Mollusca); in protozoa, a longitudinal groove on the surface of dinoﬂagellates from which the ﬂagella and, if present, the peduncle or other feeding structures emerge. suture region where two structures (e.g., plates or valves) are joined. symbiont one member of a symbiotic relationship. sympatric two or more species whose habitat distributions overlap. syngamy fusion of haploid gametes or gametic cells in sexual reproduction to form a diploid zygote; phenomenon exhibited by some members of all major protozoan taxa except ciliates. tachyblastic egg type of egg produced by gastrotrichs in which development occurs immediately; see opsiblastic egg. tagma or tagmata body regions (especially in arthropods) consisting of metameres grouped or fused to perform similar functions, e.g., head, thorax, and abdomen of a crustacean or insect. test loose surface covering of protozoa, created by the inhabitant with secreted and, sometimes, gathered materials; generally synonymous with lorica, but test is normally applied to Rhizopoda. thermocline zone in a lake of relatively rapid temperature change over a small range of depth; commonly deﬁned as 1°C change per meter of depth. tocopherol (a-tocopherol or vitamin E) organic compound that has been shown to induce mixis (sexual reproduction) in some rotifers and lengthen the lifespan in others. torpidity condition or quality of being dormant or inactive, a temporary loss of all or part of the power of sensation or motion. transverse muscle attachment scar conspicuous muscle attachment scar in the posterior region of the third coxal plates in larvae of certain mite taxa. trichocyst type of extrusome emitting a spindle-shaped, nontoxic projectile. triploid having three times the haploid number of chromosomes. tritocerebrum portion of a crustacean’s brain that principally innervates the antennae and a section of the alimentary tract. tritonymph third nymphal instar in the generalized mite life cycle. trochantin small, forward projecting sclerite at the base of the trochanter. trochus one of two ciliated rings of the typical rotiferan corona; seecingulum. troglobite obligate cave-dweller, often characterized in crustaceans by a reduction in eye structure and a lack of pigments; also, frequently demonstrating K-strategies. troglophile organism living in a cave in a nonobligate relationship; such organisms usually complete their life history in the cave and commonly have conspeciﬁcs surviving outside the cave; contrasts with troglobite. trophi complex set of hard jaws characteristically found in the pharynx (mastax) of rotifers. trophozoite mature, vegetative, feeding, adult stage in the protozoan life cycle; used primarily with reference to parasitic species of nonciliate taxa; synonymous with trophont. tubercles small, rounded knobs on the body surface of an animal, larger than papillae, smaller than nodes. tun barrel-shaped form of a tardigrade in a cryptobiotic (latent) state. turnover time amount of time necessary for the standing crop biomass to be replaced completely in a species. type taxon (or specimen) original individual of a species on which the species name is based; type specimens are kept in museums for comparative purposes and to reduce confusion about scientiﬁc nomenclature. typhlosole invaginated, cilated, particle-sorting structure running the length of the midgut and extending into the ciliated sorting surfaces of the ﬂoor of the stomach and digestive diverticulum in bivalve molluscs. umbo in bivalve molluscs, portion of the shell raised above the dorsal shell margin near the hinge (also called the beak). uncate condition of the pedipalp in deutonymphs and adults of some mites in which the ventral surface of the tibia is expanded and produced distally to oppose the tarsus, forming an efﬁcient grasping organ. uncinate type of trophi (jaw structure) found in rotifers characterized by unci possessing few teeth, usually with one large one and a few small ones. uncus one of the seven pieces comprising rotifer trophi; an uncus together with a manubrium form the malleus subunit. undulating membrane compound ciliary apparatus typically lying on the right side of the buccal cavity of some protozoa; synonomous with paroral membrane. undulipodium swimming organelle of eukaryotic cells, including protozoa, includes both the ﬂagellum and the cilium. univoltine having one brood or generation per year. urogomphus paired, unsegmented terminal appendage of coleopteran larvae. uroid posterior part of moving “naked” lobose amoeba. urosome portion of the copepod body posterior to the major body articulation. Glossary urstigma (urstigmata) knob-like structure with a porous cap located between the ﬁrst and second coxal plates in larval mites; the paired urstigmata are apparently homologous with the genital acetabula of deutonymphs and adults and probably function as an osmoregulatory chloride epithelium. uterus region in female reproductive system of invertebrates that stores egg capsules prior to release. vagina portion of the female reproductive tract connecting the vulva with the uterus or uteri; a female tract used in copulation, distal to entrance of the oviducts. valve one of either the left or right shell of a seed shrimp (Ostracoda) or mussel (Mollusca), generally referred to as left or right valve. In nematodes, the disc or nipplelike structure which may occur in the metacorpus or basal bulb of the pharynx. varve layer of sediment representing one year’s accumulation. vas deferens duct from the testes, through which spermatozoa pass. veliger free-swimming larval stage of molluscs characterized by a ciliated velum used for swimming and feeding; in North American freshwater bivalves, veligers occur only in exotic Dreissena spp. ventral shield single large sclerite or series of closely ﬁtting plates and platelets completely covering the venter of the idiosoma in deutonymphs and adults of certain mite taxa. verge penis or organ that bears the penis. verticil setae most anterior series of sensory setae on the dorsal surface of the idiosoma, consisting of and internal pair (vi) and an external pair (ve). vesicula seminalis enlarged part of the sperm duct or ejaculatory duct holding sperm. vestibule space between the outer lamella and the duplicature in the proximal portion of an ostracode valve. virgate type of trophi (jaw structure) found in rotifers that are modiﬁed for piercing and pumping; generally can be recognized by the long fulcrum and manubria; some are asymmetrically shaped. 1019 vitellarium yolk gland associated with the ovary in bdelloid and monogonont rotifers. vitelline membrane most external membrane of a molluscan egg. viviparous type of reproduction in which the young develop internally and are maintained and nourished by the mother before birth; see oviparous and ovoviviparous. VO2 abbreviation for volume of oxygen consumed per unit time. voltine refers to the number of generations during the year or during the reproductive season (e.g., univoltine, bivoltine, polyvoltine). vulva ventrally located genital opening of the female nematode, usually located in the midbody region. water tube in bivalve molluscs, the channels formed between the descending and ascending limbs of lamellibranch gill ﬁlaments which carry water past gill ostia dorsally to the epibranchial cavity; also form gill marsupial chambers in which eggs and larval stages are incubated through development to juveniles or glochidia. wetlands ecosystems whose soil is saturated for long periods seasonally or continuously; include marshes, swamps, ephemeral ponds, etc. wing in bivalve molluscs, a thin, ﬂattened, dorsally extended portion of the posterior slope occurring on the shell of some unionacean species. YOY (young-of-the-year) juveniles during the period from the last larval stage to adulthood, or one year of agewhichever comes sooner. Zenker’s organ ejaculation ducts, composed of longitudinal muscles in the shape of a tube on which occur radiating bristles and are covered in a chitinous sheath in freshwater ostracodes. zooid one of the physically connected, asexually replicated, morphologic units that comprise a colony; in bryozoans, the zooid includes the polypide and associated musculature as well as all parts of the adjacent body wall. This Page Intentionally Left Blank Subject Index Acanthametropodidae, see Ephemeroptera Acanthobdellidae anatomy annuli, 468 digestive system, 469 – 470 excretory system, 471 eyes, 468 morphology, 467 – 468 papillae, 468 reproductive organs, 470 suckers, 468 – 469 vascular system, 471 – 472 behavior, 481 diagnostic features of leeches, 466 dispersal, 479 – 480 diversity, 474 ecology overview, 10 identiﬁcation, 488 life cycle, 479 locomotion, 472 parasitism, 466 – 467,474 predation, 480 – 481 reproduction, 477 – 479 respiration, 472 taxonomic key, 489 taxonomic status, 466 – 467 Acariformes Acaridida, 649 Actinedida, 648 Oribatida, 648 – 649 Acroloxidae, see Gastropoda Adelphaga, see Coleoptera Aeshinidae, see Odonata Alderﬂy, see Megaloptera Allen curve, secondary production measurement, 758 Allozyme, variants in rotifer populations, 222 – 223 Ameletidae, see Ephemeroptera Ametropodidae, see Ephemeroptera Amphipoda, see Peracarida Amphizoidae, see Coleoptera Ampullaridae, see Gastropoda Ancylidae, see Gastropoda Anisoptera, see Odonata Annelida, see also Acanthobdellidae; Branchiobdellida; Euhirudinea; Oligochaeta controversy in taxonomy, 9 – 10,432, 467 Annuli Acanthobdellidae, 468 Euhirudinea, 468 Annulipalpia, see Trichoptera Anomopoda, see Cladocera Anostraca, see Branchiopoda Antennae cladocera, 847,850 Crustacea, 776 Antennules cladocera, 847,849 – 850 Crustacea, 776 Decapoda, 955 Apataniidae, see Trichoptera Aquatic insect, see Insect, aquatic Arachnida, see also Acariformes; Dolomedes; Hydrachnida; Parisitiformes classiﬁcation, 549 1021 1022 Subject Index Arthropleidae, see Ephemeroptera Arthropoda, see also Crustacea; Hydrachnida; Insects, aquatic aquatic insects, 12 crustaceans, 12 – 16 water mites, 12 Astacidae, see Decapoda Athericidae, see Diptera Atyidae, see Decapoda Baetidae, see Ephemeroptera Baetiscidae, see Ephemeroptera Bdelloidea, see Rotifera Behningiidae, see Ephemeroptera Belostomatidae, see Heteroptera Beraeidae, see Trichoptera Bicarbonate, transport in bivalves, 334 Bicoecids, anatomy, 52 Binomial nomenclature, taxonomic classiﬁcation, 2 Birth rate, equation, 221 Bithynidae, see Gastropoda Bivalvia, see also Unionoidea anatomy gastric shield, 341 gills, 336 – 338 kidney, 339 lateral cilia, 337 – 338 locomotory structures, 335 – 336 mouth, 341 nervous system, 343 – 344 overview, 331 reproductive structures, 341 – 343 sensory organs, 344 shell, 333 – 335 stomach, 341 anoxia response, 349 – 350 behavioral ecology, 373,375 – 376 biomonitor applications, 390 – 391 brooding of embryos, 342 burrowing, 335 – 336,373 calcium requirements, 360 collection, 393 – 394 conservation biology, 357 – 358 culture, 395 – 397 desiccation resistance, 350 – 352 distribution, 332 – 333 dispersal, 354 – 356 pH effects, 360 stream size effects, 361 – 262 substrate effects, 358 – 360 temperature effects, 361 water depth effects, 360 – 361 diurnal cucles, 348 diversity, 332,354,356 – 358 economic importance, 9 ecosystem role, 387 – 390 evolution, 390 – 393 extinction, 356 – 357 feeding ﬁlter feeding particle capture, 376 rate of ﬁltering, 381 – 382 sorting of ﬁltered particles, 376,380 sediment feeding, 382 – 383 ﬁltration rate, season effects, 347 glycogenic hormones, 3431 glycogen stores, season effects, 347 – 348 growth efﬁciency, 390 habitat, 9 hemolymph, 336 ion transport, 334,339 nitrogen excretion, 340,351 osmoregulation, 338 – 340 carbonic anhydrase role, 353 hemolymph osmolarity, 353 hormonal control, 354 ion transport, 352 – 353 salt tolerfance, 352 oxygen consumption biomass relationship, 346 growth and reproduction relationship, 347 hypoxia response, 348 – 349 pollutant effects, 348 seasonal cycles, 344 – 346 population regulation competitive interactions, 386 – 387 density-dependent mechanisms, 387 oxygen, 384 parasites, 384 pH, 383 – 384 pollution, 384 predation, 385 – 386 temperature, 383 water levels, 384 reproduction egg, 343 sperm, 343 temperature regulation, 343 shell synthesis, 331,334 – 335 specimen preparation for identiﬁcation, 394 – 395 subclasses, 331 taxonomic keys Corbiculacea, 398 – 400 superfamilies, 397 Unionoidea, 400 – 403,408 – 415 Blephariceridae, see Diptera Blood, cladocera, 854 – 855 Body wall, bryzoans, 507 – 508 Bosminidae, see Cladocera Brachycentridae, see Trichoptera Brachycera, see Diptera Brain, see Nervous system Branchiobdellida anatomy digestive system, 457 morphology, 456 – 457 nervous system, 457 reproductive organs, 457 vascular system, 457 collection, 459 distribution, 458 diversity, 456,458 evolution, 459 feeding, 457 habitat, 10 life cycle, 458 parasitism and parasites, 458 – 459 reproduction, 458 specimen preparation for identiﬁcation, 459 taxonomic key, 459 – 460 Branchiopoda, see also Crustacea adaptations of noncladocerans diapause, 892 oxygen concentrations, 891 salinity, 891 temperature, 891 anatomic features Anostraca, 887 Laevicaudata, 887 Notostraca, 887,889 Spinicaudata, 887 behavior mating, 892 – 893 swimming, 892 cladocerans, see Cladocera collection of noncladocerans, 895 conservation biology, 890 – 891 culture of noncladocerans, 895 distribution of noncladocerans, 890 ecosystem role of noncladocerans, 894 eggs on noncladocerans, 890 feeding mechanisms, 893 – 894 orders and families, 846 overview, 13 – 14 population regulation of noncladocerans, 894 prospects for research, 895 specimen preparation, 895 taxonomic key, 895,897,900 toxicology of noncladocerans, 894 – 895 Branchiura, see also Crustacea distribution, 783 – 784 larva, 784 morphology, 784 overview, 13 reproduction, 784 taxonomic key, 795 – 796 Bryzoans, see also Entoprocta anatomy body wall, 507 – 508 coelum, 506 ectocyst, 508 – 509 lophophore, 505 – 506,509 nervous system, 506 – 507 statoblast, 506,510,517 zooid, 505 – 506 classiﬁcations, 505 collection, 516 culture, 516 distribution, 511 – 512 diversity, 511 environmental physiology, 510 – 511 evolution, 514 – 515 fecal deposits, 514 feeding diet, 513 mechanism, 509 habitats, 505,511 – 512 larvae, 509 life cycle, 512 Subject Index overview, 10 – 11 parasites, 513 predation, 513 reproduction asexual budding, 509 – 510 ﬁssion, 510 gametogenesis, 513 sexual, 509 specimen preparation for identiﬁcation, 516 – 517 taxonomic key, 517 – 523 Caddisﬂy, see Trichoptera Caenidae, see Ephemeroptera Calamoceratidae, see Trichoptera Calanoida, see Copepoda Calcium carbonate shell synthesis in bivalves, 334 – 335 composition of bivalve shells, 333 – 334 crustacean requirements, 781 Gastropoda requirements, 302 mussel requirements, 27,360 transport in bivalves, 334 Calopterygidae, see Odonata Cambaridae, see Decapoda Capniidae, see Plecoptera Carapace, cladocera, 850 Caterpillar, see Lepidoptera Caves, see Underground aquatic habitats Ceratopogonidae, see Diptera Chaetae, Oligochaeta, 435 – 436,443 Chaoboridae, see Diptera Chironomidae, see Diptera Chloroperlidae, see Plecoptera Choanocyte, sponges, 101 Choanoﬂagellates, anatomy, 51 – 52 Chrysomellidae, see Coleoptera Chrysophytes, anatomy, 52 Chydoridae, see Cladocera Cilia bivalves, 337 – 338 Protozoa, 47 – 48 Ciliophora, anatomy, 54 – 55 Circadian clock, Decapoda, 959 Circulatory system cladoceran heart, 851 Copepoda, 914 Crustacea, 777 Decapoda, 954 Gastropoda, 301 Ostracoda, 812 Cladistics, taxonomic classiﬁcation, 1 Cladocera, see also Crustacea anatomy antennae, 847,850 antennules, 847,849 – 850 carapace, 850 digestive system, 851 eye, 847,849 fat globules, 851 head, 847,850 heart, 851 legs, 850 – 851 maxillae, 850 morphology, 847 reproductive organs, 852 behavior diel vertical migration, 860 – 861 escape, 861 – 862 mating, 859 – 860 swarming, 861 swimming, 860 blood, 854 – 855 collection, 869 – 870 coloration, 850 conservation biology, 858 culture, 870 – 871 cyclomorphosis, 851 – 852 distribution, 856 – 858 ecosystem role, 864 eggs, 853 – 854 feeding herbivores, 862 mechanism, 850 – 851 predators, 862 fossil record, 865,867 life cycle, 852 – 854 metabolic rate, factors affecting body size, 855 food concentration, 855 interaction between factors, 855 – 856 temperature, 855 molting, 854 orders and families, 846 – 847 overview, 13 – 14 paleolimnology applications, 864 – 865 phylogenetic analysis, 867 population regulation, 863 – 864 predation, 863 – 864 prospects for research, 869 reproduction, 852 – 854 species distinction, 856 species diversity effects competitive interactions, 857 – 858 lake size, 857 littoral species, 858 pH, 857 salinity, 857 turbidity, 857 specimen preparation, 871 taxonomic keys Bosminidae, 881 Chydoridae, 874 – 878 Daphniidae, 879 – 881 families, 871,873 Halopediidae, 873 Haplopoda, 885,887 Macrothricoidea, 883 Moinidae, 879 – 881 Onychopoda, 885,887 Sididae, 873 – 874 toxicology, 867,869 Claws, Tardigrada, 530 – 532 Clearance rate, calculation, 925 – 926 Clitellata controversy in taxonomy, 432,466 – 467 evolution, 441 – 442,466 Clitellum, Oligochaeta, 432,434 Cnidaria, anatomy body plan, 136 – 137 nematocysts, 137 – 138,140 overview, 6,136 classiﬁcation, 151 collection, 150 culture, 150 – 151 ecological interactions, 140 feeding, 138 molecular biology, 145 – 146 reproduction, 138 – 140 taxa, 135 – 136 taxonomic key, 152 – 153 Coelum, bryzoans, 506 Coenagrionidae, see Odonata Coleoptera, see also Insect, aquatic Adelphaga Amphizoidae, 684 Dytiscidae, 684 – 685 Gyrinidae, 685 Haliplidae, 685 Noteridae, 685 – 686 overview, 684 aquatic species, 682 collection, 682 literature references to species keys, 688 – 689 morphology adults, 682 larvae, 683 Myxophaga, Hydroscaphidae, 686 Polyphaga Chrysomellidae, 687 Curculionidae, 687 Dryopidae, 686 – 687 Elmidae, 687 Helophoridae, 688 Hydraenidae, 687 – 688 Hydrochidae, 688 Hydrophilidae, 688 Lampyridae, 688 Lutrochidae, 687 overview, 686 Psephenidae, 687 Ptilodactylidae, 687 Scirtidae, 688 respiration, 682 – 683 taxonomic keys adults, 708,710 – 711 larvae, 712 – 714 Collection aquatic insects, 659 Branchiobdellida, 459 bryzoans, 516 cladocera, 869 – 870 Cnidaria, 150 Coleoptera, 682 Collembola, 696 Copepoda, 930 Crustacea, 794 – 795 Decapoda crayﬁsh, 985 – 986 shrimp Atyidae, 985 1023 1024 Subject Index Collection (continued) Palaemonidae, 985 Diptera, 689 Euhirudinea, 486 Gastropoda, 315 Gastrotricha, 188 Hydrachnida deutonymphs and adults, 583 – 584 larvae, 584 – 585 Nematoda extraction, 265 – 266,268 sampling, 265 Nematomorpha, 290 – 291 Nemertea, 176 noncladocerans, 895 Oligochaeta, 444 Ostracoda, 825 – 826 Porifera, 115 Protozoa, 69 – 70 Rotifera, 230 – 231 Tardigrada, 541 Trichoptera larvae, 671 – 672 Turbellaria, 163 Collembola classiﬁcation, 695 collection, 696 life cycle, 695 – 696 morphology, 695 overview, 12 specimen preparation, 696 taxonomic key, 716 – 717 Colpodea, anatomy, 55 Conservation biology aquatic insects biomonitoring studies, 763 – 764 disturbance effects ﬂow impairment, 761 – 762 insecticide spills, 762 – 763 natural versus human disturbances, 761 land use effects on communities agriculture, 761 large woody debris alterations, 760 lentic environments, 759 road construction, 761 streams, 759 – 760 wetlands, 759 Bivalvia biomonitor uses, 390 – 391 conservation biology, 357 – 358 Branchiopoda, 890 – 891 cladocera, 858 Crustacea water quality response and biomonitor use, 790 Decapoda, 982 – 984 Euhirudinea as bioindicator, 485 – 486 Gastropoda, 315 Hydrachnida as bioindicator, 582 – 583 Plecoptera biomonitoring studies, 763 – 764 Trichoptera biomonitoring studies, 763 – 764 Unionoidea, 368 Consumption, equation, 263 Contractile vacuole, Protozoa, 46 – 47 Copepoda, see also Crustacea adaptations oxygen concentration, 921 – 922,927 temperature, 921 anatomy circulatory system, 914 digestive system, 914 head, 912 legs, 912,914 morphology, 911 – 912,914 nervous system, 916 reproductive organs, 914,916 sensory organs, 916 sex differences, 917 thorax, 912 behavior feeding, 92 mating, 919 swimming, 922 – 923 classiﬁcation, 911 – 912 collection, 930 culture, 931 diapause, 917,919 dissection techniques for identiﬁcation, 931 – 932 distribution, 920 – 921 diversity, 911 ecosystem role, 928 – 929 eggs, 917 feeding diet, 923 – 924 mechanisms, 924 – 925 mosquito control, 929 rate calculations, 925 – 926 habitats, 912 identiﬁcation, 932 – 934 life cycle, 916 – 917,919 – 920 nitrogen excretion, 914 overview, 14 – 15 parasitism, 929 population regulation birth rate, 928 competition, 927 death rate, 928 food limitation effects, 920,927 growth rates, 926,928 – 929 predation, 920,927 temperature effects, 926 – 927 prospects for research, 929 – 930 reproduction, 916 – 917,919 sexing, 932 – 933 species Calanoida, 948 Cyclopoida, 949 Harpacticoida, 950 Poecilostomatoida, 950 specimen preparation, 930 – 931 staging, 933 taxonomic keys Calanoida, 934 – 935 Cyclopoida, 936 – 937 Harpacticoida, 937 – 938 behavioral ecology, 373,375 collection, 394 competitive interactions, 386 culture, 395 – 397 density-dependent mechanisms of population regulation, 387 ecosystem role, 388 – 390 environmental stresses, 371 evolution, 392 life cycle, 370 – 372 postreproductive die-offs, 384 reproduction, 370 – 371 specimen preparation for identiﬁcation, 394 – 395 taxonomic key for Corbiculacea, 398 – 400 Cordulegastridae, see Odonata Corduliidae, see Odonata Corethrellidae, see Diptera Corixidae, see Heteroptera Corona, Rotifera, 198 – 199,201 Corydalidae, see Megaloptera distribution, 146 – 147 feeding, 147 growth, 147 life cycle, 147 – 148 morphology, 146 reproduction, 147 Crayﬁsh, see Decapoda Cryptobiosis, Tardigrada encystment, 535 – 536 anoxybiosis, 536 cryobiosis, 536 osmobiosis, 536 anhydrobiosis, 536 Crustacea, see also Branchiopoda; Branchiura; Cladocera; Copepoda; Decapoda; Ostracoda; Peracarida anatomy antennae, 776 antennules, 776 appendages, 776 circulatory system, 777 digestive system, 776 – 777 maxillae, 776 maxillules, 776 morphology, 776 nervous system, 779 reproductive organs, 779 – 780 respiratory system, 777 skeleton, 776 calcium requirements, 781 chemoreception, 782 classiﬁcation, 773 – 774 collection, 794 – 795 culture, 795 distribution ancient lake studies, 794 dispersal barriers, 793 – 794 groundwaters, 792 – 793 historical perspective, 791 – 792 surface waters, 793 ecological impact, 773 – 774 embryogeny, 780 evolution, 773,783 feeding mechanism, 776 – 777 resources, 788 – 789 Subject Index vertical migration, 789 – 790 fossil record, 773 habitats, 780,787 – 788 longevity, 790 – 791 mechanoreception, 782 molting, 780 mysid predation, 789 – 790 nitrogen excretion, 777 osmoregulation, 777,779 photoreception, 781 – 782 population genetics, 793 – 794 specimen preparation, 795 taxonomic key to orders, 795 – 796 thermoreception, 783 tolerances pH, 781 salt, 780 – 781 temperature, 781 water quality response and biomonitor use, 790 Cryptomonads, anatomy, 52 Ctenopoda, see Cladocera Culicidae, see Diptera Culture aquatic insects, 659 – 660 Bivalvia, 395 – 397 bryzoans, 516 cladocera, 870 – 871 Cnidaria, 150 – 151 Copepoda, 931 Crustacea, 795 Decapoda, 986 Euhirudinea, 486 Gastropoda, 315 Gastrotricha, 188 – 189 Hydrachnida, 585 Nematoda, 269 Nemertea, 176 noncladocerans, 895 Oligochaeta, 444 – 445 Ostracoda, 826 – 827 Porifera, 115 Protozoa, 69 – 70 Rotifera, 231 Turbellaria, 163 Curculionidae, see Coleoptera Cuticle Nematoda, 256,258 Nematomorpha, 282 – 283 Tardigrada, 528 – 530 Cyclomorphosis, cladocera, 851 – 852 Cyclopoida, see Copepoda Damselﬂy, see Odonata Daphniidae, see Cladocera Death rate, equation, 222 Decapoda adaptations coloration, 959 temperature, 958 anatomy antenulles, 955 biramous appendages, 952 circulatory system, 954 digestive system, 952,954 eye, 955 gills, 954 morphology, 951 – 952 nervous system, 954 – 955 sensory organs, 955 statocyst, 955 behavior of crayﬁsh burrowing, 974,976 – 977 pH effects, 977 – 978 predation avoidance, 978 – 980 shelter and aggression, 978 water current effects, 977 circadian clock, 959 collection crayﬁsh, 985 – 986 shrimp Atyidae, 985 Palaemonidae, 985 conservation biology, 982 – 984 culture, 986 distribution crayﬁsh Astacidae, 966 – 967 Cambaridae, 967 – 968 overview, 963,965 – 966 shrimp Atyidae, 960 – 961 Palaemonidae, 961 – 963 diversity, 774,960 economic impact of crayﬁsh, 974,985 ecosystem role, 973 – 974 feeding crayﬁsh behavior, 975 – 976 chemoreception, 976 diet, 974 – 975 predation, 975 shrimp, 974 genera and subgenera, 963 – 964 habitats, 973 – 974 life cycle crayﬁsh, 969 – 970,972 – 973 shrimp, 968 – 969 molting, 957 – 958 osmoregulation, 959 overview, 15 – 16 parasitism, 974 population regulation crayﬁsh factors competition, 981 displacent by invaders, 982 growth rates, 981 hybridization, 981 – 982 overview, 980 predation, 980 – 981 shrimp, 980 prospects for research, 982 – 985 reproduction crayﬁsh, 969 – 970,972 – 973 gonads, 958 pheromones, 958,983 shrimp, 968 – 969 respiration, 955 – 957 specimen preparation, 986 – 987 1025 taxonomic key, 987 – 989 toxicology, 959 – 960 Detrivory aquatic insects coarse particulate organic matter processing, 747 DOM ingestion, 748 ﬁne particulate organic matter formation, 747 – 748 large woody debris, 748 Deuterophlebiidae, see Diptera Diapause Copepoda, 917,919 noncladoceran adaptation, 892 Digestion system Acanthobdellidae, 469 – 470 Branchiobdellida, 457 cladocera, 851 Copepoda, 914 Crustacea, 776 – 777 Decapoda, 952,954 Euhirudinea, 469 – 470 Gastropoda, 300 – 301 Gastrotricha, 182 Hydrachnida, 567 Nematomorpha, 283 Ostracoda, 812 Porifera digestion, 102 Tardigrada buccal aparatus, 532 – 534 esophagus, 534 hindgut, 534 midgut, 534 overview, 532 Dinoﬂagellates, anatomy, 52 Diplomonads, anatomy, 52 Dipseudopsidae, see Trichoptera Diptera, see also Insect, aquatic Brachycera Athericidae, 693 Dolicopodidae, 693 Empididae, 693 – 694 Ephydridae, 694 Muscidae, 694 Sciomyzidae, 694 Stratiomyidae, 694 Syrphidae, 694 – 695 Tabanidae, 695 collection, 689 habitats, 688 larva, 689,693 literature references to species keys, 690 Nematocera Blephariceridae, 690 Ceratopogonidae, 690 Chaoboridae, 690 – 691 Chironomidae, 691 Corethrellidae, 691 Culicidae, 691 – 692 Deuterophlebiidae, 692 Dixidae, 692 Nymphomyiidae, 692 Psychodidae, 692 Ptychopteridae, 692 Simuliidae, 692 1026 Subject Index Diptera (continued) Tanyderidae, 692 – 693 Thaumaleidae, 693 Tipulidae, 693 pupa, 689 taxonomic key to larvae, 714 – 716 Discobolocyst, Protozoa, 51 Distribution Bivalvia, 332 – 333 dispersal, 354 – 356 pH effects, 360 stream size effects, 361 – 262 substrate effects, 358 – 360 temperature effects, 361 water depth effects, 360 – 361 Branchiobdellida, 458 Branchiura, 783 – 784 bryzoans, 511 – 512 cladocera, 856 – 858 Copepoda, 920 – 921 Crustacea ancient lake studies, 794 dispersal barriers, 793 – 794 groundwaters, 792 – 793 historical perspective of study, 791 – 792 surface waters, 793 Decapoda crayﬁsh Astacidae, 966 – 967 Cambaridae, 967 – 968 overview, 963,965 – 966 shrimp Atyidae, 960 – 961 Palaemonidae, 961 – 963 Gastropoda Acroloxidae, 302 – 303 Ampullaridae, 302 – 303 Ancylidae, 302 Bithynidae, 302 ecological determinants, 309 – 312 Hydrobiidae, 302 – 303 Lymnaeidae, 302 – 303 Micromelaniidae, 302 Neritinidae, 302 – 303 Physidae, 302 – 304 Planorbidae, 302,304 Pleuroceridae, 302 Pomatiopsidae, 302 – 303 Thiaridae, 302 Valvatidae, 302 Viviparidae, 302 – 303 Gastrotricha, 181,183 – 185 Hydrachnida, 552 Nemertea, 175 Oligochaeta, 438 – 439 Ostracoda benthic nonswimmers, 816 benthic swimmers, 816 Porifera, 106 – 107,117 – 127 Protozoa, 59 Rotifera, 195 – 196,208 Tardigrada, 540 Turbellaria, 156,159 – 160,162 Unionoidea, 402 Diversity Acanthobdellidae, 474 aquatic insects, 729 – 730 Bivalvia, 332,354,356 – 358 Branchiobdellida, 456,458 bryzoans, 511 Copepoda, 911 Decapoda, 774,960 Euhirudinea, 474 Gastropoda, 297,302 Gastrotricha, 183 Hydrachnida, 549 – 551 Oligochaeta, 438 – 439 Porifera, 105 – 106 Protozoa, 58 – 59 Trichoptera, 670 Dixidae, see Diptera Dolicopodidae, see Diptera Dragonﬂy, see Odonata Dryopidae, see Coleoptera Dytiscidae, see Coleoptera Earthworm, see Oligochaeta Ecdysis, see Molting Ecdysteroids, molting stimulation in crayﬁsh, 958 Ecnomidae, see Trichoptera Ecosystem role Bivalvia, 387 – 390 Branchiopoda cladocerans, 864 noncladocerans, 89 Copepoda, 928 – 929 Corbicula ﬂuminea, 388 – 390 Decapoda, 973 – 974 Nemertea, 175 – 176 Odonata, 665 Protozoa, 4 – 5,43 Rotifera, 224 – 225 Trichoptera, 672 Turbellaria, 161 – 162 Unionoidea, 387 Ectocyst, bryzoans, 508 – 509 Egg, see also Reproduction cladocera, 853 – 854 Copepoda, 917 Hydrachnida, 568 – 569 Ostracoda, 813,822 Ejectisome, Protozoa, 48,51 Elmidae, see Coleoptera Empididae, see Diptera Entoprocta, see also Bryzoans anatomy, 515 – 516 distribution, 516 evolution, 516 feeding, 516 species, 515 Ephemerellidae, see Ephemeroptera Ephemeridae, see Ephemeroptera Ephemeroptera, see also Insect, aquatic biomonitoring studies, 763 – 764 feeding, 661 identiﬁcation, 661 life cycle, 660 – 661 literature references to species keys, 662 Pisciforma Acanthametropodidae, 661 – 662 Ameletidae, 662 Ametropodidae, 662 Baetidae, 662 Metretopodidae, 662 – 663 Pseudironidae, 663 Siphlonuridae, 663 reproduction, 661 Retrachaeta Baetiscidae, 663 Behningiidae, 663 Caenidae, 663 Ephemerellidae, 663 Ephemeridae, 663 – 664 Leptohyphidae, 664 Leptophlebiidae, 664 Neoephemeridae, 664 Polymitarcyidae, 664 Potamanthidae, 664 Setisura Arthropleidae, 664 Heptageniidae, 664 Isonychiidae, 664 – 665 Oligoneuriidae, 665 taxonomic key to larvae, 697 – 700 Ephydridae, see Diptera Erpobdellidae, see Euhirudinea Euglenids, anatomy, 52 Euhirudinea anatomy annuli, 468 digestive system, 469 – 470 excretory system, 471 eyes, 468 morphology, 468 papillae, 468 reproductive organs, 470 suckers, 468 – 469,475 vascular system, 471 – 472 collection, 486 culture, 486 dispersal, 479 – 480 diversity, 474 ecology overview, 10 feeding, 465 – 466,474 – 477 foraging behavior oxygen level effects, 483 predators, 481 – 483 sanguivores, 481 hyperoxia tolerance, 473 hypoxia tolerance, 472 – 473 identiﬁcation diagnostic features of leeches, 466,487 – 488 Erpobdellidae, 489 Glossiphoniidae, 488 Hirudinidae, 489 Piscicolidae, 488 – 489 specimen preparation for identiﬁcation, 486 – 487 life cycle, 478 – 479 locomotion, 472 population regulation, 483,485 Subject Index predation, 480 – 481 reproduction, 471,477 – 479 respiration, 472 salt tolerance, 473 – 474 taxonomic key, 489 – 497 taxonomic status, 466 – 467 toxicology and use as bioindicator, 485 – 486 Euholognatha, see Plecoptera Evolution Bivalvia, 390 – 393 Branchiobdellida, 459 bryzoans, 514 – 515 cladocera, 865,867 Clitellata, 441 – 442,466 Crustacea, 773,783 Gastropoda phylogenetic trees, 313 – 314 pulmonates, 313 ribosomal DNA analysis, 313 – 314 Gastrotricha, 187 – 188 Hydrachnida exoskeleton, 564,566 – 567 overview, 550 Nematoda, 261 – 262 Nematomorpha, 280 – 282 Oligochaeta clitellate taxa, 443 – 444 Enchytraeidae, 441 Haplotaxidae, 441 Lumbriculidae, 443 Naididae, 441,443 phylogenetic tree of Annelida, 442 Tubiﬁcidae, 441,443 Ostracoda, 807 – 808,827 Peracarida, 785 – 786 Porifera, 114 – 115 Protozoa, 68 – 69 Sphaeriidae, 390,392 – 393 Tardigrada, 527 Unionoidea, 390 – 393 Excretory system Acanthobdellidae, 471 Euhirudinea, 471 Gastropoda, 301 Gastrotricha, 182 – 183 Hydrachnida, 567 Nematoda, 259 Tardigrada, 535 Extrusome, Protozoa, 48,51 Eye Acanthobdellidae, 468 cladocera, 847,849 Decapoda, 955 Euhirudinea, 468 Oligochaeta spots, 437 – 438 Ostracoda, 812 Turbellaria, 157 Fat globules, cladocera, 851 Feeding, see also Detrivory; Grazing; Predation Bivalvia ﬁlter feeding particle capture, 376 rate of ﬁltering, 381 – 382 sorting of ﬁltered particles, 376,380 sediment feeding, 382 – 383 Branchiobdellida, 457 bryzoans diet, 513 mechanism, 509 cladocerans herbivores, 862 mechanism, 850 – 851 predators, 862 Copepoda diet, 923 – 924 mechanisms, 924 – 925 mosquito control, 929 rate calculations, 925 – 926 Crustacea mechanism, 776 – 777 resources, 788 – 789 vertical migration, 789 – 790 Decapoda crayﬁsh behavior, 975 – 976 chemoreception, 976 diet, 974 – 975 predation, 975 shrimp, 974 Ephemeroptera, 661 Euhirudinea, 465 – 466,474 – 477,481 – 483 Gastropoda, 305 – 307 Gastrotricha, 186 – 187 Hydrachnida diet, 581 foraging behavior, 581 – 582 prey population effects, 581 Nematoda, 261 – 263 Nemertea, 174 noncladoceran mechanisms, 893 – 894 Oligochaeta, 437,440 Ostracoda, 823 – 824 Peracarida, 788 – 790 Plecoptera, 668 – 669 Porifera, 110 – 112 Rotifera, 223 – 224 Turbellaria, 160 – 161 Filosea, anatomy, 54 Filtering rate bivalves, 381 – 382 equation, 381 Fishﬂy, see Megaloptera Flagella, Protozoa, 47 – 48 Flatworms, see Nemertea; Turbellaria Flowing waters, see Lotic environments Food vacuole, Protozoa, 45 – 46 Foot Gastropoda, 299 Rotifera, 197 Gastric shield, Bivalvia, 341 Gastropoda anatomy circulatory system, 301 1027 digestive system, 300 – 301 excretory system, 301 foot, 299 head, 299 overview, 297 reproductive organs, 299 – 300 respiratory system, 301 shell, 298 – 299 visceral mass, 299 calcium requirements, 302 collection, 315 conservation biology, 315 culture, 315 dispersal ability, 310 distribution Acroloxidae, 302 – 303 Ampullaridae, 302 – 303 Ancylidae, 302 Bithynidae, 302 ecological determinants, 309 – 312 Hydrobiidae, 302 – 303 Lymnaeidae, 302 – 303 Micromelaniidae, 302 Neritinidae, 302 – 303 Physidae, 302 – 304 Planorbidae, 302,304 Pleuroceridae, 302 Pomatiopsidae, 302 – 303 Thiaridae, 302 Valvatidae, 302 Viviparidae, 302 – 303 diversity, 297,302 evolution, 298 evolution phylogenetic trees, 313 – 314 pulmonates, 313 ribosomal DNA analysis, 313 – 314 feeding, 305 – 307 habitat, 8 – 9,305 hypoxia adaptation, 302 life cycle, 304 – 305 pH effects, 309 population size factors nutrition, 307 – 308 trematode parasites, 308 – 309 predation, 297,310 – 312 production ecology, 309 prospects for ecology research, 312 reproduction, 299 – 300,304 – 305 specimen preparation for identiﬁcation, 315 – 316 subclasses, 297 – 298 taxonomic key, 316,318,320,323 temperature effects, 301 – 302 Gastrotricha anatomy brain, 182 digestive system, 182 excretory system, 182 – 183 morphology, 182 reproductive organs, 182 classiﬁcation, 181 – 182 collection, 188 culture, 188 – 189 density and biomass, 183 – 185,187 1028 Subject Index Gastrotricha (continued) distribution, 181,183 – 185 diversity, 183 evolution, 187 – 188 feeding, 186 – 187 growth, 187 habitat, 7,183 – 184 life cycle, 185 – 186 predation, 187 preservation of specimens, 188 reproduction, 185 – 186 taxonomic key, 189 – 191 Gemmosclere, sponge spicule, 103 – 104,115 – 116 Gerridae, see Heteroptera Gerromorpha, see Heteroptera Gills Bivalvia, 336 – 338 Decapoda, 954 Oligochaeta, 436 Unionoidea calcium phosphate concretions, 350,352 Glochidium, Unionoidea collection, 394 fecindity, 364,366 ﬁsh parasitism, 363 – 364,368 forms, 363 host food mimicry, 364 host reduction effects, 385 size versus number released, 366 Glossiphoniidae, see Euhirudinea Glossosomatidae, see Trichoptera Goeridae, see Trichoptera Gomphidae, see Odonata Granuloreticulosida, anatomy, 54 Grazing, aquatic insects algae crop, 749 – 750 ecosystem impact, 748 – 749 host-speciﬁc associations, 750,752 macrophyte grazing, 752 species distribution, 749 Growth efﬁciency, percent net growth efﬁciency equation, 390 Growth rate, equation for populations, 221 Gyrinidae, see Coleoptera Habitat aquatic insects lentic communities littoral communities, 734 – 735 profundal communities, 735 wetland communities, 735 – 736 lotic communities drift of insects, 741 – 742 ﬂow effects, 737 – 738 functional feeding groups, 738 – 740 recolonization mechanisms, 738 – 739 river continuum concept, 740 – 741 substrates, 736 – 737 saline habitats, 742 specialized habitats, 743 Bivalvia, 9 bryzoans, 505,511 – 512 Copepoda, 912 Crustacea, 780,787 – 788 Decapoda, 973 – 974 Diptera, 688 ﬂowing waters, see Lotic environments Gastropoda, 8 – 9,305 Gastrotricha, 7,183 – 184 Hydrachnida interstitial habitats, 578 – 579 lakes, 579 rifﬂe habitats, 578 springs, 578 stenothermic pools, 579 temporary pools, 579 inland water percentage of biospheric water, 19 lentic habitats, see Lentic ecosystems Nematoda, 262 – 265 Nemertea, 7,175 Ostracoda bromeliads, 821 carbonate content, 816,819 chemical water types, 817 diversity, 816 lakes, 821 oxygen concentrations, 818 – 819 pH tolerance, 818 population regulation, 824 – 825 salinity, 817 – 818 sediment – water interface, 822 solute composition, 818 streams, 821 temperature, 819 – 821 underground waters, 821 – 822 Sphaeriidae, 369 – 370 Tardigrada, 537 – 539 Turbellaria, 7 underground waters, see Underground aquatic habitats Hairworm, see Nematomorpha Haliplidae, see Coleoptera Halopediidae, see Cladocera Haplopoda, see Cladocera Haptocyst, Protozoa, 51 Harpacticoida, see Copepoda Hatching Nematoda, 260 – 261 Ostracoda, 814 – 815 Hatching rate, equation, 221 Head cladocera, 847,850 Copepoda, 912 Gastropoda, 299 Ostracoda, 809,811 Heart, see Circulatory system Hebridae, see Heteroptera Helicopsychidae, see Trichoptera Heliozoa, anatomy, 54 Helophoridae, see Coleoptera Hemolymph bivalves, 336,353 crustaceans, 777 Heptageniidae, see Ephemeroptera Heteroptera, see also Insect, aquatic aquatic and semiaquatic species, 677 Gerromorpha Gerridae, 678 – 679 Hebridae, 679 Hydrometridae, 679 Mesoveliidae, 679 overview, 678 Veliidae, 679 identiﬁcation, 677 – 678 literature references to species keys, 678 Nepomorpha Belostomatidae, 680 Corixidae, 680 Naucoridae, 680 Nepidae, 680 Notonectidae, 681 overview, 679 – 680 Pleidae, 681 taxonomic key, 708 Hirudinidae, see Euhirudinea Hydra, see also Cnidaria distribution, 141 feeding, 142 – 143 growth, 142,144 habitat, 141 molecular biology, 145 – 146 morphology, 140,145 regeneration and immortality, 143,145 reproduction, 141 – 142 species groups, 151 – 152 substrates, 140 – 141 symbiosis with algae, 143 – 145 toxicology, 145 Hydrachnida anatomy digestive system, 567 excretory system, 567 morphology adults, 560,563 – 564 larva morphology, 560 – 561 nervous system, 567 – 568 reproductive organs, 568 respiratory system, 567 collection deutonymphs and adults, 583 – 584 larvae, 584 – 585 culture, 585 deutonymph stage, 575 diversity, 549 – 551 drawings adult, 605 – 628 larva, 561,562,563,565,571,586 – 604 eggs, 568 – 569 evolution exoskeleton, 564,566 – 567 overview, 550 exoskeleton sclerotization, 576 feeding diet, 581 foraging behavior, 581 – 582 prey population effects, 581 global distribution, 552 habitats interstitial habitats, 578 – 579 lakes, 579 rifﬂe habitats, 578 Subject Index springs, 578 stenothermic pools, 579 temporary pools, 579 life cycle, overview, 568 origins Gondwana, 552 Laurasia, 552 North America, 552,560 Pangea, 552 parasitism of insects by larva attachment and engorgement, 574 – 575 detachment, 575 ecological impact host populations, 580 – 581 individual hosts, 580 intraspeciﬁc effects on same host, 581 emergence of larvae, 572 – 573 host selection, 569 – 570 infection, 574 recognition of host, 573 seeking and locating hosts, 573 site selection, 570 – 572 success rates, 569 population density, 550 predation, 582 protonymph stage, 575 reproduction pheromones, 577 sex ratios, 578 spermatophore transfer association during transfer, 576 – 577 copulation during transfer, 577 – 578 dissociation during transfer, 576 scanning electron micrographs, 629 – 630,632 specimen preparation, 585 taxonomic keys adults, 638 – 648 larvae, 633 – 638 overview, 585 – 586 taxonomic relationships, 550 – 552 toxicology and use as bioindicator, 582 – 583 tritonymph stage, 576 Hydraenidae, see Coleoptera Hydrobiidae, see Gastropoda Hydrobiosidae, see Trichoptera Hydrochidae, see Coleoptera Hydrometridae, see Heteroptera Hydrophilidae, see Coleoptera Hydropsychidae, see Trichoptera Hydroptilidae, see Trichoptera Hydroscaphidae, see Coleoptera Hypodermis, Nematoda, 258 ICZN, see International Code of Zoological Nomenclature Identiﬁcation, see Specimen preparation; Taxonomic keys Increment-summation method, secondary production measurement, 758 Ingestion rate, calculation, 925 Insect, aquatic biomonitoring studies, 763 – 764 collection, 659 culture, 659 – 660 detrivory coarse particulate organic matter processing, 747 detritus fractions, 747 DOM ingestion, 748 ﬁne particulate organic matter formation, 747 – 748 large woody debris, 748 disturbance effects ﬂow impairment, 761 – 762 insecticide spills, 762 – 763 natural versus human disturbances, 761 diversity, 729 – 730 food web gut analysis, 755 – 756 insects as energy ﬂow conduits, 754 – 755 stable isotope studies, 756 – 757 trophic levels, 755 grazing algae crop, 749 – 750 ecosystem impact, 748 – 749 host-speciﬁc associations, 750,752 macrophyte grazing, 752 species distribution, 749 land use effects on communities agriculture, 761 large woody debris alterations, 760 lentic environments, 759 road construction, 761 streams, 759 – 760 wetlands, 759 lentic communities littoral communities, 734 – 735 profundal communities, 735 wetland communities, 735 – 736 life cycles, 657 – 658 diversity, 743 – 744 hemimetabolous versus holometabolous development, 746 – 747 synchronized development, 744,746 voltinism, 744,746 lotic communities drift of insects, 741 – 742 ﬂow effects, 737 – 738 functional feeding groups, 738 – 740 recolonization mechanisms, 738 – 739 river continuum concept, 740 – 741 substrates, 736 – 737 morphology abdomen, 659 head, 658 thorax, 658 – 659 orders, see Coleoptera; Diptera; Ephemeroptera; Heteroptera; Lepidoptera; Megaloptera; Neuroptera; Odonata; Plecoptera; Trichoptera physical constraints 1029 oxygen concentrations in water, 731 – 732 pH, 733 salt tolerance, 733 – 734 substrate and ﬂow, 734 temperature, 732 – 733 predator – prey interactions ﬁsh predation, 753 – 754 insects as predators, 752 – 754 lentic versus lotic environments, 752 – 754 saline habitats, 742 secondary production, 757 – 758 specialized habitats, 743 specimen preparation, 660 taxomic key to orders, 696 – 697 Instantaneous growth method, secondary production measurement, 758 Integripalpia, see Trichoptera International Code of Zoological Nomenclature (ICZN), 2 Internet resources, freshwater invertebrate taxonomy, 3 – 4 Intestine, Nematoda, 258 – 259 Isonychiidae, see Ephemeroptera Isopoda, see Peracarida Jellyﬁsh, see Craspedacusta Kidney, Bivalvia, 339 Kinetoplastids, anatomy, 52 Laevicaudata, see Branchiopoda Lakes, see Lentic ecosystems Lampyridae, see Coleoptera Land use, effects on aquatic insect communities agriculture, 761 large woody debris alterations, 760 lentic environments, 759 road construction, 761 streams, 759 – 760 wetlands, 759 Larva Branchiura, 784 bryzoans, 509 Coleoptera, 683 Diptera, 689,693 Hydrachnida, 560 – 561 Nematomorpha, 284 Odonata, 665 – 666 Plecoptera, 668 Trichoptera adaptations, 671 collection, 671 – 672 pupate, 671 Leech, see Euhirudinea Legs cladocera, 850 – 851 Copepoda, 912,914 Ostracoda, 812 1030 Subject Index Lentic ecosystems ephemeral ponds, 36 insect communities littoral communities, 734 – 735 profundal communities, 735 wetland communities, 735 – 736 lakes abiotic zonation, 33 – 34 biotic zonation littoral and profundal benthos, 35 – 36 neuston, 34 – 35 overview, 34 zooplankton of pelagic and littoral habitats, 35 geomorphology, 33 saline lakes abiotic characteristics, 38 geographic distribution, 38 species diversity, 38 – 39 swamps, 36,38 wetlands, 36 Lepidoptera, see also Insect, aquatic larva, 681 – 682 morphology, 682 Lepidostomatidae, see Trichoptera Leptoceridae, see Trichoptera Leptohyphidae, see Ephemeroptera Leptophlebiidae, see Ephemeroptera Lestidae, see Odonata Libellulidae, see Odonata Life cycle Acanthobdellidae, 479 aquatic insects diversity, 743 – 744 hemimetabolous versus holometabolous development, 746 – 747 synchronized development, 744,746 voltinism, 744,746 Branchiobdellida, 458 bryzoans, 512 cladocera, 852 – 854 Collembola, 695 – 696 Copepoda, 916 – 917,919 – 920 Decapoda crayﬁsh, 969 – 970,972 – 973 shrimp, 968 – 969 Ephemeroptera, 660 – 661 Euhirudinea, 478 – 479 Gastropoda, 304 – 305 Gastrotricha, 185 – 186 Hydrachnida, 568 Nematoda, 7,260 – 261 Nematomorpha, 288,290 Odonata, 665 Oligochaeta, 439 – 440 Ostracoda, 813 – 816 Plecoptera, 668 Porifera dormancy, 107 – 108 growth, 108 – 109 overview, 5,107 Protozoa encystment, 60 senescence and sex, 60 – 61 sexual maturity, 60 Sphaeriidae, 369 – 370 Trichoptera, 671 Turbellaria, 158 – 159 Unionoidea, 362 – 363 Life table, cladocera population regulation, 863 Limnephilidae, see Trichoptera Listomatea, anatomy, 55 Locomotion Bivalvia, 335 – 336 Ostracoda, 823 Rotifera, 196,204 – 205 Turbellaria, 157 – 158 Lophophore, bryzoans, 505 – 506,509 Lotic environments basin morphometry, 20 classiﬁcation of streams, 20 current velocity and effects on invertebrates, 23 ecosystem changes along stream course feeding groups and food resources, 27 – 28 large rivers, 29 – 30 patterns along continuum, 27 – 28 river continuum concept, 27 – 28,30 source of river effects, 28 – 29 insect communities drift of insects, 741 – 742 ﬂow effects, 737 – 738 functional feeding groups, 738 – 740 recolonization mechanisms, 738 – 739 river continuum concept, 740 – 741 substrates, 736 – 737 ionic content, 26 – 27 oxygen concentrations, 19,26 permanence, 19 – 20 pH effects, 27 springs as source, 28 – 29 stream discharge calculation, 20,22 substrate types, 23 – 25 temperature ﬂuctuations and effects on invertebrates, 19,25 – 26 Lotka – Volterra equations, 61 – 62 Lumbriculidae, see Oligochaeta Lutrochidae, see Coleoptera Lymnaeidae, see Gastropoda Macromiidae, see Odonata Macrophyte, grazing by insects, 752 Macrothricoidea, see Cladocera Mastax, Rotifera, 201 Mating, see Reproduction Maxillae cladocera, 850 Crustacea, 776 Ostracoda, 812 Maxillules, Crustacea, 776 Mayﬂy, see Ephemeroptera Megaloptera, see also Insect, aquatic Corydalidae, 677 literature references to species keys, 677 overview, 676 – 677 Sialidae, 677 taxonomic key to larvae, 707 Megasclere, sponge spicule, 102 – 103 Mermithidae, see Nematoda Mesoveliidae, see Heteroptera Metretopodidae, see Ephemeroptera Micromelaniidae, see Gastropoda Microsclere, sponge spicule, 103,117 Midge, see Diptera Moinidae, see Cladocera Molannidae, see Trichoptera Mollusca, see also Bivalvia; Gastropoda classes, 8 – 9 Molting cladocera, 854 Crustacea, 780 Decapoda, 957 – 958 Nematoda, 261 Ostracoda, 815 Monogononta, see Rotifera Mouth, Bivalvia, 341 Mucocyst, Protozoa, 51 Mucus sheath, Rotifera, 227 – 228 Muscidae, see Diptera Musculature Nematoda, 258 Rotifera, 202 – 203 Tardigrada, 535 Myxophaga, see Coleoptera Naididae, see Oligochaeta Naucoridae, see Heteroptera Nematocera, see Diptera Nematocysts, Cnidaria, 137 – 138,140 Nematoda anatomy cuticle, 256,258 excretory system, 259 hypodermis, 258 intestine, 258 – 259 morphology, 256 musculature, 258 nervous system, 259 overview, 8,256 pharynx, 258 reproductive organs, 260 sensory organs, 259 – 260 stoma, 258 collection extraction, 265 – 266,268 sampling, 265 culture, 269 development, 260 feeding, 261 – 263 fossil record, 261 – 262 functional classiﬁcation, 255 habitat, 262 – 265 hatching, 260 – 261 life cycle, 7,260 – 261 molting, 261 predation, 265 resistant stages, 261 respiration, 259 Subject Index specimen preparation for identiﬁcation, 268 – 269 systematic arrangement of genera, 295 taxonomic key Mermithidae, 278,280 non-Mermithidae, 270,274,276,278 toxicology, 265 Nematomorpha anatomy cuticle, 282 – 283 digestive tract, 283 overview, 8,282 reproductive organs, 283 collection, 290 – 291 evolution, 280 – 282 fossil record, 280 larva, 284 life cycle, 288,290 oviposition, 283 parasitism, 282,288,290 specimen preparation for identiﬁcation, 291 taxonomic key, 291 – 292 Nemertea anatomy, 6 – 7,173 behavioral ecology, 175 classiﬁcation, 174 collection, 176 culture, 176 distribution, 175 ecosystem role, 175 – 176 feeding, 174 habitat, 7,175 physiological adaptations, 175 preservation of specimens, 176 prospects for research, 176 reproduction, 174 – 175 taxonomic key, 176 Nemouridae, see Plecoptera Neoephemeridae, see Ephemeroptera Nepidae, see Heteroptera Nepomorpha, see Heteroptera Neritinidae, see Gastropoda Nervous system Bivalvia, 343 – 344 Branchiobdellida, 457 bryzoans, 506 – 507 Copepoda, 916 Crustacea, 779 Decapoda, 954 – 955 Gastrotricha brain, 182 Hydrachnida, 567 – 568 Nematoda, 259 Ostracoda, 812 Rotifera, 203 – 204 Tardigrada, 535 Neuroptera, see also Insect, aquatic diversity, 681 Sisyridae, 681 Nitrogen excretion Bivalvia, 340,351 Copepoda, 914 Crustacea, 777 Noteridae, see Coleoptera Notonectidae, see Heteroptera Notostraca, see Branchiopoda Nymphomyiidae, see Diptera Odonata, see also Insect, aquatic Anisoptera (dragonﬂy) Aeshinidae, 666 Cordulegastridae, 666 Corduliidae, 666 – 667 Gomphidae, 667 Libellulidae, 667 Macromiidae, 667 Petaluridae, 667 ecological impact, 665 larva, 665 – 666 life cycle, 665 literature references to species keys, 666 reproduction, 665 taxonomic key to larvae, 700 Zygoptera (damselﬂy) Calopterygidae, 667 Coenagrionidae, 667 Lestidae, 667 Protoneuridae, 667 – 668 Odontoceridae, see Trichoptera Oligochaeta, see also Annelida anatomy chaetae, 435 – 436,443 clitellum, 432,434 eye spots, 437 – 438 gills, 436 morphology, 432 pharyngeal glands, 437 prostomium, 434 – 435 reproductive organs, 432 – 434 sensory organs, 436 – 437 collection, 444 culture, 444 – 445 defecation rates, 440 – 441 desiccation tolerance, 438 distribution, 438 – 439 diversity, 438 – 439 evolution clitellate taxa, 443 – 444 Enchytraeidae, 441 Haplotaxidae, 441 Lumbriculidae, 443 Naididae, 441,443 phylogenetic tree of Annelida, 442 Tubiﬁcidae, 441,443 feeding, 437,440 habitats, 10,431 – 432,438 life cycle, 439 – 440 reproduction, 434,439 – 440 respiration, 437 salt tolerance, 437 specimen preparation for identiﬁcation, 445 – 446 taxonomic keys families, 446 – 448 Lumbriculidae, 448 – 450 Naididae, 450 – 452 Tubiﬁcidae, 452 – 454 Oligohymenophorea, anatomy, 55 Oligoneuriidae, see Ephemeroptera 1031 Onychopoda, see Cladocera Oscula, sponges, 101 Osmoregulation Bivalvia carbonic anhydrase role, 353 hemolymph osmolarity, 353 hormonal control, 354 ion transport, 352 – 353 salt tolerance, 352 Crustacea, 777,779 Decapoda, 959 Osphradia, bivalves, 344 Ostracoda, see also Crustacea anatomy body, 809 circulatory system, 812 digestive system, 812 eye, 812 head, 809,811 legs, 812 maxillae, 812 nervous system, 812 reproductive organs, 812 – 813 shell morphology external, 808 – 809 internal, 809 applications environmental tolerance, 827 paleolimnology, 827 trace element analysis in shells, 827 – 828 collection, 825 – 826 coloration, 823 controversies in taxonomy, 828 – 829 culture, 826 – 827 distribution benthic nonswimmers, 816 benthic swimmers, 816 egg, 813,822 embryology, 814 feeding, 823 – 824 fossils, 807 – 808,827 habitats bromeliads, 821 carbonate content, 816,819 chemical water types, 817 diversity, 816 lakes, 821 oxygen concentrations, 818 – 819 pH tolerance, 818 population regulation, 824 – 825 salinity, 817 – 818 sediment – water interface, 822 solute composition, 818 streams, 821 temperature, 819 – 821 underground waters, 821 – 822 hatching and seasonality, 814 – 815 intraspeciﬁc competition, 816 life cycle, 813 – 816 locomotion, 823 molting, 815 overview, 13 parasitism, 825 Podocopida, 829 1032 Subject Index Ostracoda (continued) predation, 824 prospects for research, 828 reproduction, 813 – 814 resting stage and torpidity, 822 – 823 specimen preparation, 826 taxonomic keys Cypridoidea, 829 – 830,832 – 835 Cytheroidea, 835 – 838 toxicology, 825 ultraviolet-B tolerance, 825 Oxygen concentration aquatic insect effects, 731 – 732 Bivalvia effects, 349 – 350,384 Copepoda adaptation, 921 – 922,927 Euhirudinea hyperoxia tolerance, 473 hypoxia tolerance, 472 – 473 Gastropoda hypoxia adaptation, 302 noncladoceran adaptation, 891 Protozoa effects, 56 – 57 Rotifera tolerance, 206 Oxygen consumption, bivalves biomass relationship, 346 growth and reproduction relationship, 347 hypoxia response, 348 – 349 pollutant effects, 348 seasonal cycles, 344 – 346 Palaemonidae, see Decapoda Paleolimnology cladocera applications, 864 – 865 Ostracoda applications, 827 Porifera, 114 Papillae Acanthobdellidae, 468 Euhirudinea, 468 Parasitism Acanthobdellidae, 466 – 467,474 Bivalvia parasites, 384 Branchiobdellida parasitism and parasites, 458 – 459 bryzoan parasites, 513 Copepoda, 929 Decapoda, 974 Gastropoda, 308 – 309 Hydrachnida parasitism of insects by larva attachment and engorgement, 574 – 575 detachment, 575 ecological impact host populations, 580 – 581 individual hosts, 580 intraspeciﬁc effects on same host, 581 emergence of larvae, 572 – 573 host selection, 569 – 570 infection, 574 recognition of host, 573 seeking and locating hosts, 573 site selection, 570 – 572 success rates, 569 Nematomorpha, 282,288,290 Ostracoda, 825 Rotifera, 228 – 229 Turbellaria, 161 Unionoidea glochidium on ﬁsh, 363 – 364,368 Parisitiformes, features, 649 Peltoperlidae, see Plecoptera Peracarida, see also Crustacea Amphipoda, 784 evolution, 785 – 786 feeding, 788 – 790 Isopoda, 784 – 785 orders, 784 overview, 12 – 13 predation, 789 – 790 reproduction, 791 taxonomic key, 795 – 796 Perlidae, see Plecoptera Perlodidae, see Plecoptera Petaluridae, see Odonata pH aquatic insect effects, 733 Bivalvia distribution effects, 360 population effects, 383 – 384 cladocera species diversity effects, 857 crayﬁsh effects, 977 – 978 Crustacea tolerance, 781 Gastropoda effects, 309 Rotifera tolerance, 206 Phagocytosis, Porifera, 102 Pharyngeal glands, Oligochaeta, 437 Pharynx, Nematoda, 258 Phenemics, taxonomic classiﬁcation, 1 Philopotamidae, see Trichoptera Phryganeidae, see Trichoptera Phyllopharyngea, anatomy, 55 Phylogeny, see Evolution Physidae, see Gastropoda Piscicolidae, see Euhirudinea Pisciforma, see Ephemeroptera Planorbidae, see Gastropoda Plecoptera, see also Insect, aquatic biomonitoring studies, 763 – 764 Euholognatha Capniidae, 669 Nemouridae, 669 – 670 Taeniopterygidae, 670 feeding, 668 – 669 larva, 668 life cycle, 668 literature references to species keys, 669 Systellognatha Chloroperlidae, 670 Peltoperlidae, 670 Perlidae, 670 Perlodidae, 670 Pteronarcyidae, 670 taxonomic key to larvae, 700 – 703 Pleidae, see Heteroptera Pleuroceridae, see Gastropoda Podocopida, see Ostracoda Poecilostomatoida, see Copepoda Polycentropodidae, see Trichoptera Polymitarcyidae, see Ephemeroptera Polyphaga, see Coleoptera Pomatiopsidae, see Gastropoda Ponds, see Lentic ecosystems Porifera anatomy digestion, 102 external morphology, 98,100 overview, 5,97 – 98 skeleton collagen, 102,104 siliceous spicules, 102 – 104 water processing system, 100 – 102 biochemistry, 105 classiﬁcation by spicules, 117 – 127 collection, 115 culture, 115 distribution, 106 – 107,117 – 127 diversity, 105 – 106 ecology algal symbionts, 110 – 111 competition for substrates, 112 – 113 dispersal among habitats, 109 feeding, 110 – 112 functional role in ecosystems, 114 infaunal organisms, 113 – 114 paleolimnology, 114 predation of sponges, 113 silica effects on skeleton structure, 112 substrate availability, 109 – 110 evolutionary relationships, 114 – 115 life cycle dormancy, 107 – 108 growth, 108 – 109 overview, 5,107 phagocytosis, 102 reproduction asexual, 104 – 105,108 – 109 sexual, 104,108 specimen preparation for identiﬁcation, 115 – 116 taxonomic key, 127 – 130 Potamanthidae, see Ephemeroptera Predation Acanthobdellidae, 480 – 481 aquatic insects ﬁsh predation, 753 – 754 insects as predators, 752 – 754 lentic versus lotic environments, 752 – 754 Bivalvia, 385 – 386 bryzoans, 513 cladocera, 863 – 864 Copepoda, 920,927 crayﬁsh, 978 – 981 Crustacea, 789 – 790 Euhirudinea, 480 – 481 Gastropoda, 297,310 – 312 Gastrotricha, 187 Hydrachnida, 582 Nematoda, 265 Ostracoda, 824 Peracarida, 789 – 790 Porifera, 113 Subject Index Protozoa, 62,67 – 68 Rotifera, 226 – 228 Turbellaria, 161 Prostomatea, anatomy, 55 Prostomium, Oligochaeta, 434 – 435 Protonephridium, Rotifera, 204 Protoneuridae, see Odonata Protozoa anatomy amoebas, 53 – 54 ciliatea, 54 – 55 ﬂagellates, 51 – 52 Heliozoa, 54 cellularity, 45 collection, 69 – 70 culture, 69 – 70 distribution, 59 diversity, 58 – 59 ecology algivores, 65,67 bacterivores, 64 – 65 ecosystem role, 4 – 5,43 epizooic associations with animals, 63 – 64 histophagous ciliates, 67 interspeciﬁc interactions, 61 – 62 intraspeciﬁc interactions, 61 metazoan predators, 67 – 68 nutrient cycling, 68 predatory interactions, 62 symbionts, 62 – 63 environmental physiology contaminants, 57 – 58 energetics, 58 factors affecting distribution, 55 – 56 oxygen, 56 – 57 temperature, 56 evolutionary relationships, 68 – 69 life cycle encystment, 60 senescence and sex, 60 – 61 sexual maturity, 60 organelles cilia, 47 – 48 contractile vacuoles, 46 – 47 extrusomes, 48,51 ﬂagella, 47 – 48 food vacuoles, 45 – 46 phyla, 4,44 – 45 reproduction ﬁssion, 59 generation time, 59 – 60 sexual versus asexual reproduction, 60 taxonomic categories, 44 – 45 taxonomic keys ciliates, 78 – 79,81 – 83,85,87,89 ﬂagellates, 71 functional groups, 70 – 71 overview, 70 pseudopodia protozoa, 71,73 – 78 Psephenidae, see Coleoptera Pseudironidae, see Ephemeroptera Psychodidae, see Diptera Psychomyiidae, see Trichoptera Pteronarcyidae, see Plecoptera Ptilodactylidae, see Coleoptera Ptychopteridae, see Diptera Pupa, Diptera, 689 Q10 value, 345 – 346 Radula, Gastropoda, 300 RCC, see River continuum concept rDNA, see Ribosomal DNA Rearing, see Culture Regeneration, Hydra, 143,145 Removal-summation method, secondary production measurement, 758 Reproduction Acanthobdellidae, 477 – 479 Bivalvia egg, 343 sperm, 343 temperature regulation, 343 Branchiobdellida, 458 Branchiura, 784 bryzoans asexual budding, 509 – 510 ﬁssion, 510 gametogenesis, 513 sexual, 509 cladocera, 852 – 854,859 – 860 Cnidaria, 138 – 140 Copepoda, 916 – 917,919 Decapoda crayﬁsh, 969 – 970,972 – 973 gonads, 958 pheromones, 958,983 shrimp, 968 – 969 Ephemeroptera, 661 Euhirudinea, 471,477 – 479 Gastropoda, 299 – 300,304 – 305 Gastrotricha, 185 – 186 Hydrachnida pheromones, 577 sex ratios, 578 spermatophore transfer association during transfer, 576 – 577 copulation during transfer, 577 – 578 dissociation during transfer, 576 Nematomorpha oviposition, 283 Nemertea, 174 – 175 Odonata, 665 Oligochaeta, 434,439 – 440 Ostracoda, 813 – 814 Peracarida, 791 Porifera asexual, 104 – 105,108 – 109 sexual, 104,108 Protozoa ﬁssion, 59 generation time, 59 – 60 sexual versus asexual reproduction, 60 Rotifera cyclical parthenogenesis, 214 – 215 mating behavior, 217 1033 resting eggs, 215 – 217 Sphaeriidae, 368 – 369 Tardigrada modes, 536 – 537 apparatus, 537 mating and fertilization, 537 Reproductive organs Acanthobdellidae, 470 Bivalvia, 341 – 343 Branchiobdellida, 457 cladocera, 852 Copepoda, 914,916 Crustacea, 779 – 780 Euhirudinea, 470 Gastropoda, 299 – 300 Gastrotricha, 182 Hydrachnida, 568 Nematoda, 260 Nematomorpha, 283 Oligochaeta, 432 – 434 Ostracoda, 812 – 813 Rotifera, 204 Tardigrada, 537 Respiration Acanthobdellidae, 472 Coleoptera, 682 – 683 Decapoda, 955 – 957 Euhirudinea, 472 Nematoda, 259 Oligochaeta, 437 Respiratory system Crustacea, 777 Gastropoda, 301 Hydrachnida, 567 Retrachaeta, see Ephemeroptera Rhabdoids, Turbellaria, 157,162 Rhyacophilidae, see Trichoptera Ribosomal DNA (rDNA), Gastropoda phylogenetic analysis, 313 – 314 River continuum concept (RCC), 740 – 741 Rivers, see Lotic environments Rossianidae, see Trichoptera Rotifera aging and senescence, 217 – 218 anatomy corona, 198 – 199,201 distinguishing features, 195 foot, 197 mastax, 201 morphology, 197 – 198 muscular system, 202 – 203 nervous system, 203 – 204 protonephridium, 204 reproductive organs, 204 spines, 226 – 228 trophi, 201 – 202,234 anhydrobiosis and rehydration, 207 biogeography, 210 – 211 classes, 197,233 clearance rates, 224 – 225 collection, 230 – 231 colonization, 196,211 – 213 competition with other zooplankton, 225 – 226 1034 Subject Index Rotifera (continued) culture, 231 distribution and density, 195 – 196,208 ecology overview, 7 ecosystem role, 224 – 225 feeding behavior, 223 – 224 ﬁsh aquaculture application, 229 – 230 locomotion, 196,204 – 205 mucus sheaths, 227 – 228 parasitism, 228 – 229 phenotypic variation and mechanisms, 207 – 208 polyploidy, 205 population dynamics dynamics, 221 – 222 genetic variation, 222 – 223 life table analysis, 218 – 221 population movements, 208 – 209 predator – prey interactions, 226 – 228 reproduction cyclical parthenogenesis, 214 – 215 mating behavior, 217 resting eggs, 215 – 217 sessile species, 213 – 214 specimen preparation for identiﬁcation, 231 – 233 systematics Bdelloidea, 233 Monogononta, 233 – 234 Seisonidea, 233 taxonomic key, 234 – 237,239 – 245,247 – 248 tolerance oxygen deprivation, 206 pH, 206 salt, 206 temperature, 205 – 206 toxicology, 206 – 207 Roundworm, see Nematoda Salinity aquatic insect effects, 733 – 734 cladocera species diversity effects, 857 Crustacea tolerance, 780 – 781 Euhirudinea tolerance, 473 – 474 noncladoceran adaptation, 891 Oligochaeta tolerance, 437 Rotifera tolerance, 206 Salt tolerance, see Salinity Schizopyrenida, anatomy, 53 – 54 Sciomyzidae, see Diptera Scirtidae, see Coleoptera Seisonidea, see Rotifera Sensory organs Bivalvia, 344 Copepoda, 916 Crustacea, 781 – 783 Decapoda, 955 Nematoda, 259 – 260 Oligochaeta, 436 – 437 Turbellaria, 157 Sericostomatidae, see Trichoptera Setisura, see Ephemeroptera Shell Bivalvia synthesis, 331,334 – 335 Gastropoda, 298 – 299 Ostracoda morphology external, 808 – 809 internal, 809 Unionoidea growth, 366 Shrimp, see Decapoda Sialidae, see Megaloptera Sididae, see Cladocera Simuliidae, see Diptera Siphlonuridae, see Ephemeroptera Sisyridae, see Neuroptera Size frequency method, secondary production measurement, 758 Skeleton Crustacea, 776 Porifera collagen, 102,104 siliceous spicules, 102 – 104 Snail, see Gastropoda Specimen preparation aquatic insects, 660 Bivalvia, 394 – 395 Branchiobdellida, 459 bryzoans, 516 – 517 Collembola, 696 Copepoda, 930 – 931 Crustacea, 795 Decapoda, 986 – 987 Euhirudinea, 486 – 487 Gastropoda, 315 – 316 Hydrachnida, 585 Nematoda, 268 – 269 Nematomorpha, 291 Nemertea, 176 noncladocerans, 895 Oligochaeta, 445 – 446 Ostracoda, 826 Porifera, 115 – 116 Rotifera, 231 – 233 Tardigrada, 541 Turbellaria, 155,163 Sphaeriidae, see also Bivalvia evolution, 390,392 – 393 growth rates, 370 habitat and life history, 369 – 370 life cycle, 368 reproduction, 368 – 369 Spicipalpia, see Trichoptera Spinicaudata, see Branchiopoda Sponges, see Porifera Spongillaﬂy, see Neuroptera Springtail, see Collembola Statoblast, bryzoans, 506,510,517 Statocyst bivalves, 344 Decapoda, 955 Stoma, Nematoda, 258 Stomach, Bivalvia, 341 Stoneﬂy, see Plecoptera Stratiomyidae, see Diptera Streams, see Lotic environments Suckers Acanthobdellidae, 468 – 469 Euhirudinea, 468 – 469,475 Swamps, see Lentic ecosystems Syrphidae, see Diptera Systellognatha, see Plecoptera Systematic zoology, taxonomic classiﬁcation, 2 Tabanidae, see Diptera Taeniopterygidae, see Plecoptera Tanyderidae, see Diptera Tardigrada anatomy claws, 530 – 532 cuticle, 528 – 530 digestive system buccal aparatus, 532 – 534 esophagus, 534 hindgut, 534 midgut, 534 overview, 532 excretory system, 535 morphology, 527 – 528 musculature, 535 nervous system, 535 reproductive organs, 537 collection, 541 cryptobiosis encystment, 535 – 536 anoxybiosis, 536 cryobiosis, 536 osmobiosis, 536 anhydrobiosis, 536 dissemination, 540 distribution, 540 evolution, 527 habitats, 537 – 539 historical perspective of study, 527 overview, 11 population density, 539 – 540 reproduction apparatus, 537 mating and fertilization, 537 modes, 536 – 537 specimen preparation and microscopy, 541 taxonomic key, 542 – 544 Taxonomic keys Acanthobdellidae, 489 Bivalvia Corbiculacea, 398 – 400 superfamilies, 397 Unionoidea, 400 – 403,408 – 415 Branchiobdellida, 459 – 460 Branchiopoda cladocera Bosminidae, 881 Chydoridae, 874 – 878 Daphniidae, 879 – 881 families, 871,873 Halopediidae, 873 Haplopoda, 885,887 Macrothricoidea, 883 Moinidae, 879 – 881 Onychopoda, 885,887 Subject Index Sididae, 873 – 874 noncladocera, 895,897,900 bryzoans, 517 – 523 Cnidaria, 152 – 153 Collembola, 716 – 717 Copepoda Calanoida, 934 – 935 Cyclopoida, 936 – 937 Harpacticoida, 937 – 938 Crustacea orders, 795 – 796 Decapoda, 987 – 989 Euhirudinea, 489 – 497 Gastropoda, 316,318,320,323 Gastrotricha, 189 – 191 Hydrachnida adults, 638 – 648 larvae, 633 – 638 overview, 585 – 586 insects, aquatic Coleoptera adults, 708,710 – 711 larvae, 712 – 714 Diptera larvae, 714 – 716 Ephemeroptera larvae, 697 – 700 Heteroptera, 708 Megaloptera larvae, 707 Odonata larvae, 700 orders, 696 – 697 Plecoptera larvae, 700 – 703 Trichoptera larvae, 704 – 705,707 major taxa of freshwater invertebrates, 16 – 18 Nematoda Mermithidae, 278,280 non-Mermithidae, 270,274,276,278 Nematomorpha, 291 – 292 Nemertea, 176 Oligochaeta families, 446 – 448 Lumbriculidae, 448 – 450 Naididae, 450 – 452 Tubiﬁcidae, 452 – 454 Ostracoda Cypridoidea, 829 – 830,832 – 835 Cytheroidea, 835 – 838 Porifera, 127 – 130 Protozoa ciliates, 78 – 79,81 – 83,85,87,89 ﬂagellates, 71 functional groups, 70 – 71 overview, 70 pseudopodia protozoa, 71,73 – 78 Rotifera, 234 – 237,239 – 245,247 – 248 Tardigrada, 542 – 544 Temperature aquatic insect effects, 732 – 733 Bivalvia distribution effects, 361 population effects, 384 Branchiopoda cladocera metabolic rate effects, 855 noncladoceran adaptation, 891 Copepoda adaptation, 921 population regulation effects, 926 – 927 Crustacea tolerance, 781 Decapoda adaptation, 958 Gastropoda effects, 301 – 302 Protozoa effects, 56 Rotifera tolerance, 205 – 206 Testacealobosia, anatomy, 53 Thaumaleidae, see Diptera Thiaridae, see Gastropoda Thorax, Copepoda, 912 Tipulidae, see Diptera Toxicology cladocera, 867,869 Decapoda, 959 – 960 Euhirudinea, 485 – 486 Hydrachnida, 582 – 583 Nematoda, 265 noncladocerans, 894 – 895 Ostracoda, 825 Rotifera, 206 – 207 Trichoptera, see also Insect, aquatic Annulipalpia Dipseudopsidae, 672 – 673 Ecnomidae, 673 Hydropsychidae, 673 Philopotamidae, 673 Polycentropodidae, 673 Psychomyiidae, 673 Xiphocentronidae, 673 biomonitoring studies, 763 – 764 diversity, 670 ecological impact, 672 identiﬁcation, 671 Integripalpia Apataniidae, 674 Beraeidae, 674 Brachycentridae, 674 Calamoceratidae, 674 Goeridae, 674 Helicopsychidae, 674 Lepidostomatidae, 674 Leptoceridae, 674 – 675 Limnephilidae, 675 Molannidae, 675 Odontoceridae, 675 Phryganeidae, 675 Rossianidae, 675 Sericostomatidae, 675 Uenoidae, 675 – 676 larva adaptations, 671 collection, 671 – 672 pupate, 671 life cycle, 671 literature references to species keys, 672 Spicipalpia Glossosomatidae, 676 Hydrobiosidae, 676 Hydroptilidae, 676 Rhyacophilidae, 676 taxonomic key to larvae, 704 – 705, 707 Trinomen, taxonomic classiﬁcation, 2 Trophi, Rotifera, 201 – 202,234 Tubiﬁcidae, see Oligochaeta 1035 Turbellaria anatomy ciliated epidermis, 157 eyes, 157 morphology, 156 – 157 overview, 6 – 7 rhabdoids, 157,162 sensory organs, 157 behavioral ecology, 160 collection, 163 community ecology, 162 culture, 163 distribution, 156,159 – 160,162 ecosystem role, 161 – 162 endosymbiosis, 162 environmental physiology, 158 feeding, 160 – 161 habitat, 7 life history, 158 – 159 locomotion, 157 – 158 population regulation and density, 161 predation and parasitism, 161 preservation of specimens, 155,163 species list, 179 – 180 status in Animal kingdom, 156 Uenoidae, see Trichoptera Underground aquatic habitats caves food sources, 31 karst topography in United States, 31 life cycle of species, 33 protection, 32 – 33 species distribution, 31 – 32 hyporheic zone, 30 – 31 phreatic zone, 31 Unionoidea, see also Bivalvia conservation biology, 368 distribution, 402 ecosystem role, 387 evolution, 390 – 393 gill calcium phosphate concretions, 350,352 glochidium collection, 394 fecuindity, 364,366 ﬁsh parisitism, 363 – 364,368 forms, 363 host food mimicry, 364 host reduction effects, 385 size versus number released, 366 growth rates, 366 – 367 life cycle, 362 – 363 life span, 366 – 367 shell growth, 366 standing crop biomass rate versus biomass production, 367 taxonomic key, 400 – 403,408 – 415 Valvatidae, see Gastropoda Vascular system Acanthobdellidae, 471 – 472 Branchiobdellida, 457 Euhirudinea, 471 – 472 1036 Subject Index Veliidae, see Heteroptera Visceral mass, Gastropoda, 299 Vitamin E, Rotifera effects longevity, 218 mictic reproduction induction, 215 Viviparidae, see Gastropoda Water beetle, see Coleoptera Water ﬂea, see Cladocera Water mite, see Hydrachnida Web sites, freshwater invertebrate taxonomy, 3 – 4 Wetlands, see Lentic ecosystems Xiphocentronidae, see Trichoptera Zebra mussel, see Dreissena polymorpha Zooid, bryzoans, 505 – 506 Zoological ranks, 3 Zygoptera, see Odonata Taxonomic Index Abedus, 680 Ablabesmyia janta, 384 Abramis brama, 923 Abrochta, 236 Acalyptonotus, 552, 559, 579, 635, 647 neoviolaceus, 602, 628 Acanthamoeba, 73, 74 Acanthobdella, 442, 444 peledina, 17, 466, 467, 470, 471, 472, 479, 486, 489 Acanthocyclops, 921, 937 brevispinosus, 949 capillatus, 949 carolinianus, 949 columbiensis, 949 exilis, 949 montana, 949 parasensitivus, 949 parvulus, 949 pennaki, 949 robustus, 928, 949 venustoides, 949 vernalis, 949 Acanthocystis, 78 turfacea, 77 Acanthodiaptomus, 929, 935 denticornis, 948 Acantholeberis, 883 curvirostris, 884 Acartia, 923 sinensis, 934 Acella haldemani, 320, 321 Acentrella, 662 Acerpenna, 662 Acherontacarus, 553, 563, 569, 606, 633, 638 Achromadora, 265, 276 Acilus, 683 Acineta, 81 limnetis, 79 Acipenser fulvesens, 385 guldenstadti, 149 ruthenus, 148, 149 Acroloxus, 303, 316, 317 Acroneuria, 658, 730 Acroperus, 874, 876 harpae, 875 Actinarctus, 538 Actinobdella, 488, 490 annectens, 490 inequiannulata, 474, 488, 490 Actinobolina, 51, 85 radians, 84 Actinolaimus, 274 Actinomonas, 48 Actinonaias, 412 Actinophrys, 78 sol, 61, 77 Actinosphaerium, 54, 62, 78 eichhorni, 77 Acutogordius, 292 Acyclus, 235 Adineta, 228, 235, 236 Adorybiotus, 529, 531, 532, 543 1037 1038 Taxonomic Index Aedes, 730 cinereus, 574 communis, 572 excrucians, 574 punctor, 572 Aeolosoma, 447 Aeromonas hydrophila, 470 Aeshna, 730 eremita, 782 Agabus, 683 Agarodes, 675 Aglaodiaptomus, 935 atomicus, 948 clavipes, 948 clavpoides, 948 conipedatus, 948 dilobatus, 948 forbesi, 948 kingsburyae, 948 leptopus, 948 lintoni, 948 marshianus, 948 pseudosanguineus, 948 saskatchewanensis, 948 spatulocrenatus, 948 stagnalis, 948 Agneta, 669 Agostocaris, 961 Agriodrilus, 443 Alaimus, 278 Alasmidonta, 402, 403, 409, 411, 413, 414 mccordi, 357 robusta, 357 undulata, 404 wrightiana, 357 Alathyria jacksoni, 348 Albaxona, 558, 646 nearctica, 622 Albertathyas, 553, 640 montana, 609 Albia, 557, 622, 638, 647 neogaea, 598 Alboglossiphonia, 488 heteroclita, 492, 494 Allocapnia, 703 Allodero, 439, 451 Allonais, 438, 451, 452 Alloperla, 702 Alona, 847, 852, 874, 877 acutirostris, 878 afﬁnis, 878 bicolor, 849, 877 intermedia, 878 verrucosa, 878 Alonella, 874, 878 dadayi, 878 excisa, 878 exigua, 862, 878 hamulata, 878 nana, 878 pulchella, 878, 879 Alonopsis, 874, 876 elongata, 862, 875 Alosa sapidissma, 385 Amblema, 402, 413 plicata, 361, 381, 404, 409 Ambrysus, 678 Ambystoma barbouri, 791 maculosum, 479, 480 tigrinum, 479 Ameletus, 699 Amerothyasella, 553, 640 appalachiana, 611 Ametropus, 699 Amnicola, 307, 309, 320 limosa, 304 Amoeba, 5, 43, 53, 54, 65, 74 proteus, 53, 74 Amoenacarus, 559, 645 dixiensis, 628 minimus, 628 Amphibolella, 169 spinulosa, 179 Amphibolus, 529, 531, 532, 533, 539, 543 Amphichaeta, 436, 450 Amphidelus, 278 Amphileptus, 87 claparedi, 86 Amphinemura, 703 Amphisiella, 83 oblonga, 82 Amphizoa, 711, 713 Anabaena afﬁnis, 225 oscillariodes, 395 Anacaena, 684 Anacanthoderma, 191 Anas platyrhynchos, 892 Anatonchus, 278 Anatopynia, 161 Anax, 701 junius, 666 Anchistropus, 140, 862, 874, 878 minor, 879 Anchytarsus, 684, 713 Ancylus ﬂuviatilis, 306, 310 Anheteromeyenia, 130 argyrosperma, 116, 117, 130 ryderi, 106, 117, 118, 130 Ankistrodesmus, 395, 396, 870 Ankylocythere, 830, 836 Ankyrodrilus, 460 Anodonta, 363, 402, 403, 409, 412 anatina, 348, 363, 367, 376, 384, 391 cygnea, 335, 339, 340, 341, 353, 354, 382, 391 grandis, 345 imbecelis, 396 implicata, 354 woodiana, 353, 366 Anodontoides, 402, 404, 412 ferrussacianus, 409 Anonchus, 276 Anopheles, 691, 715, 730 crucians, 573 Anthopotamus, 698 Antiprodrilus, 443 Antrobia, 320 Anuraeopsis, 210 ﬁssa, 205, 217 Apartronemertes, 174 Apatania, 672 Aphanolaimus, 276 Aphaostracon, 320 Aphelenchoides, 263, 270, 273 Aphelenchus, 263, 270 Apheviderulix, 553, 639 santana, 607 Aplexa, 303, 304, 318 elongata, 302, 306, 319 Aplodinotus grunniens, 385 Apocyclops, 937 dimorphus, 949 panamensis, 949 spartinus, 949 Apodibius, 529, 532, 542 Apolodinotus grunniens, 474 Apsilops, 730 Aqabus, 711, 713 Aqarodes, 706 Aquarius, 678 Aranimermis, 278, 280 Arcella, 67, 74 vulgaris, 75 Archilestes, 667 Arcidens, 402 confragosa, 404, 409, 410, 413 Arcteonais, 438 lomondi, 450 Arctodiaptomus, 935 arapahoensis, 948 bacillifer, 948 dorsalis, 913, 916, 948 ﬂoridanus, 948 kurilensis, 948 saltillinus, 948 Arctodrilus wulikensis, 438, 454 Arenohydracarus, 559, 579, 646 Arenotus, 185 Argia, 730 Argulus, 13, 783, 784, 787, 795 maculosus, 784 Arizonacarus, 556, 643 chircahuensis, 616 Arkansia wheeleri, 404, 409 Armiger, 318 crista, 317 Arrenurus, 552, 559, 567 – 569, 572 – 576, 579, 580, 628, 631, 635, 644 angustilimbatus, 572 cuspidator, 575 danbyensis, 573, 580 delawarensis, 573 ﬁssicornis, 629 kenki, 572, 574 manubriator, 578, 582 planus, 575, 578, 604 pseudocylindratus, 629 pseudosuperior, 569, 580 ventropetiolatus, 575 Taxonomic Index Artemia, 150, 229, 889 – 895, 897, 931 franchiscana, 888, 891 salina, 38, 891 Artemiopsis, 889, 892, 893, 897 stephanssoni, 896 Arthroplea, 699 Asajirella, 510 Ascetocythere, 830, 836 Ascomorpha, 202, 204, 247, 248 Ascomorphella volvocicola, 244, 246 Ascophora, 170 elegantissima, 180 Asellus, 163, 785, 786, 794, 796 aquaticus, 785 Askensia, 85 volvox, 84 Aspidiophorous, 189, 190 Aspidisca, 81 costata, 80 Asplanchna, 198, 201 – 205, 215, 219, 223 – 228, 232, 239, 240 brightwelli, 205, 208, 217, 218, 219, 222, 223 girodi, 205, 216, 217, 223, 227 intermedia, 208 priodonta, 209, 222 sieboldi, 204, 208 Asplanchnopus, 201, 227, 239 calyciﬂorus, 203 Astacus, 967 Astatumen, 529, 531, 534 Astylozoon, 81 faurei, 80 Atherix, 693, 715 Athernia anthonyi, 314 Atopsyche, 706 Atractides, 556, 574, 579, 618, 637, 643, 647 grouti, 593 Atrochus, 235 Attenella, 662 Attheyella, 921, 926, 938 americana, 950 carolinensis, 950 dentata, 950 dogieli, 950 idahoensis, 950 illinoisensis, 950 nordenskioldi, 950 obatogamensis, 950 pilosa, 950 spinipes, 913, 950 trispinosa, 950 ussuriensis, 950 Aturus, 552, 557, 578, 579, 600, 624, 631, 637, 646 Atylenchus, 270 Atylotus, 690 Aulodrilus, 438, 443, 454, 455 pluriseta, 452 Aulolaimoides, 274 Aulophorus, 439, 451, 451 Australocypris insularis, 823 Austropotamobius, 967 Automate, 961 Axonopsella, 558, 646 bakeri, 621 Axonopsis, 558, 566, 572, 579, 637, 647 beltista, 623 setoniensis, 600 Baetis, 13, 662, 699, 730, 737, 739, 741, 742, 743, 754, 760 Baetisca, 698 Baetodes, 662 Balanonema, 87 biceps, 86 Bandakia, 555, 636, 642 phreatica, 590, 613 Bandakiopsis, 555, 634, 642 fonticola, 589, 613 Barbicambarus, 963, 964, 966, 967, 988 Barbidrilus, 447 Barbouria, 961 Barentsia, 515 Bastiania, 276 Batillipes, 538 Batracobdella, 468, 469, 488 cryptobranchii, 488, 493 michiganensis, 488 paludosa, 488, 492 Bdellergatis, 489 plumbeus, 482, 496 Bdellodrilus, 460 illuminatus, 460 Beatogordius, 292 Belostoma, 680, 709, 730 Beraea, 706 Berosus, 684 Bezzia, 580 Bharatalbia, 557, 624, 646 cooki, 624 surensis, 624 Biapertura, 878 Bicoeca, 71 lacustris, 72 Binucleospora elongata, 825 Biomphalaria, 313 glabrata, 306, 319 Biomyxa, 77 vagans, 76 Birgea, 240 enantia, 237 Birgella, 320 Bithynia tentaculata, 306, 322, 323 Blepharicera, 690, 715 Blepharisma, 43, 61, 62, 83 lateritium, 84 Bodo, 52, 71 caudatus, 72 Boreobdella verrucata, 494 Bosmina, 847, 850, 851, 856, 857, 859, 861, 862, 864, 865, 881 freyi, 2 longirostris, 2, 225, 881, 882 Bosminopsis, 881 deitersi, 882 1039 Bothrioneurum, 434, 435, 439, 455 vejdovskyanum, 454 Bothrioplana, 157, 164 semperi, 159, 169, 179 Bothromesostoma, 161, 171, 173 personatum, 180 Bouchardina, 963, 964, 965, 966, 968, 989 robinsoni, 987 Brachionus, 210, 215, 217, 219, 223, 224, 227, 228, 243, 244 angularis, 223, 232 bidentata, 227 calyciforus, 205, 207, 208, 216, 218, 219, 220, 222 – 227, 229, 232 caudatus, 208 patulus, 205, 217, 232 plicalitis, 39, 196, 197, 204 – 207, 215 – 219, 223, 229, 232, 243 quadridentatus, 223, 223 rubens, 207, 225, 229, 232 urceolaris, 219, 220, 223, 227 Brachycentrus, 672, 706, 738, 743 Brachycercus, 662 Brachypoda, 558, 577, 637, 646 cornipes, 599 oakcreeksensis, 622, 623 setosicauda, 599, 600 Bradleystrandesia, 828 Bradyscela, 235 Branchinecta, 889, 890, 891, 892, 893, 897 campestris, 891 coloradensis, 896 gigas, 892, 893, 894 mackini, 891 Branchinella, 895, 897 sublettei, 888 Branchiobdella astaci, 458 hexodonta, 457 kozarovi, 458 parasita, 458 Branchiocaris, 867 Branchiura, 10, 436, 438, 443, 453 sowerbyi, 439, 445, 452 Bratislavia, 451 Bresslaua, 89 vorax, 88 Brillia, 690 Bryocamptus, 921, 938 arcticus, 950 calvus, 950 cuspidatus, 950 douwei, 950 hiatus, 950 hiemalis, 950 hutchinsoni, 950 minisculis, 950 minnesotensis, 950 minutus, 950 morrisoni, 950 newyorkensis, 950 nivalis, 950 pilosus, 950 pygmaeus, 950 1040 Taxonomic Index Bryocamptus (continued) subarcticus, 950 tikchikensis, 950 umiatensis, 950 vejdovskyi, 950 washingtonensis, 950 zschokkei, 950 Bryochoerus, 542 Bryocyclops, 936 muscicola, 949 Bryodelphax, 542 parvulus, 530 Bryospilus, 847, 874 Buddenbrockia, 513 Buenoa, 678, 681 Bulimnea megasoma, 320, 321 Bulinus globosus, 300 Bullatifrons, 874 Bunops, 883 acutifrons, 885 Bursaria, 62, 83 truncatella, 84 Byrophyllum, 85 lieberkÅhni, 84 Bythotrephes, 852, 859, 862, 865, 887 cederstroemii, 886, 927 longimanus, 887 Cactus, 883 Caecidotea, 14, 785, 786, 796 recurvata, 788, 791 tridentata, 788, 791 Caenestheria, 900 setosa, 892 Caenestheriella setosa, 899 Caenis, 662, 698 Caenomorpha, 83 medusula, 82 Caenorhabditis briggsae, 263 Calcarobiotus, 529, 531, 532 Calineuria, 740, 760 californica, 756 Calliasmata, 961 Callibaetis, 662 Callinectes, 459 sapidus, 960 Calliobdella vividia, 481 Calohypsibius, 529, 531, 532, 543 Calopteryx, 701 Calposoma, 6, 135, 148, 151, 153 dactyloptera, 148 Calyptotricha pleuronemodies, 86 Cambarellus, 963, 964, 965, 967, 968, 972, 983, 988 puer, 985 Cambarincola, 460 fallax, 461 Cambarus, 821, 963, 964, 966, 967, 968, 983, 989 aculabrum, 982 alleni, 974 bartonii, 955, 981, 983 brachydactylus, 960 cahni, 960 chaugaensis, 974 diogenes, 957, 985 distans, 957 friauﬁ, 960 gentryi, 957 laevis, 960 latimanus, 974, 987 ornatus, 960 robustus, 956, 972, 974, 983 strigosus, 953 tenebrosus, 956, 960 zophonastes, 983 Camelobaetidius, 662 Campanella, 81 umbellaria, 80 Campascus, 76 triqueter, 76 Campbellonemertes, 174 Campeloma, 300, 303, 309, 323 decisum, 304, 305, 309, 324 rufrum, 305 Campostoma anomalum, 782 Camptocercus, 874, 875 liljeborgi, 876 macrurus, 876 oklahomensis, 876 rectirostris, 875, 876 Candocypria, 830, 835 Candocyprinotus, 830, 832 Candona, 14, 830, 832 acuta, 821 acutula, 820 anceps, 821 candida, 816, 818, 819, 820, 822 caudata, 809, 814, 819, 821 decora, 819, 822 ikpikpukensis, 821, 829 jeanneli, 821 marengoensis, 821 mulleri, 821 ohioensis, 820, 824 paralapponica, 821 patzcuaro, 822, 823 pedata, 821 protzi, 818, 821 rawsoni, 822, 827, 831 rectangulata, 817 reducta, 816 renoensis, 821 subtriangulata, 815, 817, 819, 820, 821, 822, 824 suburbana, 811 Candonocypris deeveyi, 829 novaezelandiae, 813, 822 pugionis, 829 sarsi, 829 serrato-marginata, 829 Canthocamptus, 921, 938 assimilis, 950 oregonensis, 950 robertcokeri, 950 sinuus, 950 staphylinoides, 950 staphylinus, 950 vagus, 950 Capitomermis, 280 Capnia, 730 Carchesium, 81 polypinum, 80 Caridinophila, 458 Carphania, 529, 530, 531, 532, 542 ﬂuviatilis, 538 Castrada, 157, 162, 170 hofmanni, 180 lutheri, 180 virginiana, 171, 180 Castrella, 173 grafﬁ, 179 pinguis, 171, 179 Catenula, 157, 163, 165 confusa, 179 lemnae, 158, 165, 179 leptocephala, 165, 179 sekerai, 179 turgida, 179 virginia, 179 Caudatella, 662 Cavernocypris, 830, 832 subterranea, 822 wardi, 822 Celina, 683 Cenocorixa, 678 biﬁda, 575 Centrolimnesia, 555, 643 bondi, 617 Cephaliophora, 229 Cephalobus, 274 Cephalodella, 197, 202, 204, 245, 247 forﬁcula, 232 Cephalothamnium, 52, 71 cyclopum, 71 Ceraclea, 672, 705 Ceratium furcoides, 931 hirudinella, 46 Ceratodrilus, 10, 460 thysanosomus, 460 Ceratophylum demersum, 306, 307 Ceratopsyche, 672 Cercobrachys, 662 Cercomonas, 52, 71, 72 Cercopagis, 887 pengoi, 887 Ceriodaphnia, 852, 855, 856, 857, 857, 859, 861, 864, 867, 881 quadrangula, 880 Chaena, 85 teres, 84 Chaetogaster, 437, 439, 450 diaphanus, 824 limnaei, 384 Chaetonotus, 7, 185, 188, 189, 190 Chaetospira, 82 mülleri, 82 Chaoborus, 14, 34, 205, 225, 690, 715, 730, 731, 852, 857, 863, 920, 927, 929 Taxonomic Index Chaoborus ﬂavicans, 824 Chaos, 74 illinoisense, 73 Chappuisides, 559, 566, 643 eremitus, 626 Chara, 816, 823 fragilis, 823 Chauliodes, 677 Cheiroseius, 632 Chelohydracarus, 559, 644 navarrensis, 627 Chelomideopsis, 559, 644 besselingi, 627 Chilodonella, 55, 78 uncinata, 88 Chilomonas, 71 paramecium, 66 Chimarra, 705 Chironomus, 13, 573, 716, 730, 731, 734, 735 riparus, 482 tentans, 735 Chlamydomonas, 69, 396 reinhardtii, 516 Chlamydomyxa, 77 montana, 76 Chlamydophrys, 75 minor, 75 Chlamydotheca, 830, 833 arcuata, 812, 820 Chlorella, 6, 62, 143, 187, 219, 396, 870 fusca, 229 vulgaris, 395 Chlorohydra, 6, 143, 151 Chordarium, 165 europaeum, 179 Chordodes, 288, 292 morgani, 282 Chordodiolus, 291 Chromadora, 265 Chromadorita, 276 Chromogaster, 247, 276 africana, 264 troglodytes, 260, 264, 277 Chrysops, 715, 730 Chydoris, 143, 857, 859, 874, 878 brevlabris, 879 faviformis, 869 piger, 878 reticulatus, 869 sphaericus, 865, 869, 878 Ciliophrys, 78 infusionum, 77 Cincinnatia, 320 Cinetochilum, 87 margaritaceum, 86 Cipangopaludina, 323 chinensis, 303 japonica, 324 Cladotanytarsus mancus, 581 Clappia, 320 Clathrosperchon, 554, 563, 579, 607, 639 Clathrostoma, 89 viminale, 88 Clathrulina, 77 elegans, 77 Cletocamptus, 937 albuquerquensis, 950 brevicaudata, 950 deitersi, 950 Climacia, 710 Climacostomum, 85 virens, 84 Cloeodes, 662 Cochliopina, 320 Cochliopodium, 74 bilimbosum, 75 Codosiga, 71 botrys, 72 Coenagrion puella, 575 Cohnilembus, 87 pusillus, 86 Coleps, 85 hirtus, 67, 84 Colletheca, 202, 214, 224, 235, 236 cornuta, 200 gracilipes, 213, 214 trilobata, 200 Colpidium, 89 colpoda, 57, 88 Colpoda steinii, 88 Columbiathyas, 553, 640 crenicola, 610 Colurella, 241 Colymbetes, 683 Condylostoma, 85 tardum, 84 Conocephalus, 730 Conochilioides, 212, 237 natans, 216 Conochilus, 211, 212, 213, 215, 227, 228, 237, 862 hippocrepis, 212 unicornis, 211 Cookacarus, 555, 642 columbiensis, 613 Cookidrilus, 443 Copelatus, 683 Coptotomus, 683 Coquillettidia, 691, 715 perturbans, 573, 580 Corbicula, 9, 354, 355, 397, 398, 399 ﬂuminea, 332, 335, 336, 339 – 356, 359 – 363, 366 – 368, 370 – 377, 379, 381 – 392, 394 – 396, 398, 399 Cordulegaster, 701 Cordulia, 701 Cordylophora, 135, 148, 150, 151, 153 Coregonus autumnalis migratorius, 794 Cornechiniscus, 542 Coronarctus, 538 Corophium spinicorne, 780 Corticacarus, 556, 618, 647 Corvomeyenia, 111, 115, 128 carolinensis, 115, 117, 118, 129 everetti, 108, 111, 115, 118, 119, 129 Corvospongilla, 129 becki, 118, 129 novaeterrae, 118, 119, 129 Corydalus, 730 Cothurnia, 81 imberbis, 80 Cottus carolinae, 791 Cowichania, 554, 639 interstitialis, 608 Crangonyx, 785, 786, 787, 792, 796 Craspedacusta, 6, 135, 136, 139, 140, 146 – 148, 150, 151, 152, 153 sinensis, 147 sowberii, 146, 147 Creaseria, 961 Crenitis, 684 Cretacimermis, 262 libani, 262, 281 Cricotopus, 39, 264, 690 bicinctus, 754, 755 nostocicola, 750, 751, 756 sylvestris, 754, 755 Cristatella, 509, 510 magniﬁca, 520 mucedo, 511, 512, 513, 522, 523 Cronodrilus, 460, 461 Crustipellis, 435, 448 tribranchiata, 447 Cryphiops, 961 Cryptocyclops, 937 bicolor, 949 Cryptomonas, 50, 931 Cryptonchus, 278 Cubozoa, 145 Culex, 730 pipiens quinquefasciatus, 269 Culicimermis, 280 Cumberlandia monodonta, 404, 408 Cupelopagis, 199, 204, 224, 235 vorax, 236 Cura, 167 foremanii, 161 Cyclestheria, 887 hislopi, 890 Cyclidium, 87 glaucoma, 86 Cyclocypris, 829, 830, 835 ampla, 810, 818, 819, 820, 821, 824, 834 globosa, 821 laevis, 821 ovum, 816, 825 sharpei, 818, 819, 820, 821 Cyclomomonia, 558, 643 andrewi, 625 Cyclonaias tuberculata, 404, 409 Cyclops, 921, 936 canadensis, 949 columbianus, 949 furcifer, 949 kolensis, 949 laurenticus, 949 scutifer, 921, 949 singularis, 922 strenuus, 949 vicinus, 923, 928, 949 1041 1042 Taxonomic Index Cyclothyas, 554, 640 rivularis, 609 siskiyouensis, 609 Cylindrolaimus, 276 Cymatia bonsdorﬁ, 580 Cymbella, 974 Cymbiodyta, 684 Cymocythere, 830, 837 Cyphoderia, 76 ampulla, 76 Cyphon, 713 Cypretta, 830, 832 kawatai, 823 Cypria, 816, 822, 824, 830, 835 obesa, 821 ophtalmica, 818, 819, 820, 825 reptans, 813 turneri, 810, 814, 823 Cypricercus, 812, 823, 828, 829, 830, 833 deltoidea, 814, 821 fuscatus, 814 horridus, 814, 823 reticulatus, 814, 831 splendida, 814 tincta, 814 Cypriconcha, 828, 829 Cyprideis, 813, 816, 817, 830, 836 torosa, 816, 819 Cypridopsis, 823, 825, 829, 830, 832 hartwigi, 823 vidua, 809, 813 – 820, 823 – 826, 831 Cyprinotus, 816, 825, 828, 829, 830, 835 carolinesis, 814, 815, 816, 823 glaucus, 810, 814, 818 incongruens, 813, 814, 815, 823, 825, 826 salinus, 817 Cyprinus carpio, 385 Cypris, 823, 830, 835 balnearia, 820 bispinosa, 817 pubera, 824, 826, 834 reptans, 825 Cyprogenia, 409 stegaria, 404 Cyprois, 832 marginata, 831 Cyrenoida ﬂoridana, 397 Cyrtolophosis, 87 mucicola, 86 Cyrtonaias, 402, 404 tampicoensis, 416 Cyrtonia, 243 Cystobranchus, 490, 494 mammillatus, 494 meyeri, 468, 494 verrilli, 468, 494 virginicus, 494 Cytherissa, 826, 830, 836 lacustris, 815, 819, 821, 837 Cytheromorpha, 826, 830, 836 fuscata, 816, 817, 818, 819, 821 Cyzicus, 889, 891, 892, 900 californicus, 891, 899 setosa, 891 Dactylobiotus, 529, 531, 532, 533, 539, 543 dispar, 535 Dactylocythere, 830, 837 leptophylax, 837 Dadaya, 874, 878 macrops, 877 Dalyellia, 162, 164, 173 viridis, 158, 179 Daphnella, 859 Daphnia, 13, 15, 64, 150, 205, 226, 780, 847, 850 – 857, 859 – 867, 880, 881, 890, 924, 926 – 929 catawba, 867 cuculata, 867 denticulatus, 859 galeata, 852, 867 jollyi, 867 laevis, 864 longiremis, 867 longispina, 855 lumholtzi, 853, 858 magna, 853, 855, 857 pulex, 226, 854, 856, 859, 861, 864, 926 pulicaria, 848, 849, 852, 864 retrocurva, 849, 852, 867 similis, 39 Daphniopsis, 854, 880, 881 ephereralis, 855, 880 Darwinula, 810, 813, 814, 826, 829, 830 stevensoni, 811, 815, 831 Desydytes, 191 ornatus, 187 Dasyhormus, 165 Daubaylia, 261, 274, 275 Deltopylum, 67 Dendrocoelopsis, 157, 169 Dendrocometes, 81 paradoxus, 79 Dendrosoma, 79 radians, 79 Dero, 436, 438, 439, 446, 451, 452 Desmarella, 71 monoliformis, 72 Desmolaimus, 265 Desmona, 672 Desmopachria, 683 Desserobdella, 488, 492 michiganensis, 493 phalera, 493 picta, 493 Deuterophlebia, 716 Dexteria, 897 ﬂoridanus, 896 Diacyclops, 917, 921, 936 alabamensis, 949 albus, 949 bernardi, 949 bicuspidatus, 949 bisetosus, 949 chrisae, 949 crasscaudis, 949 dimorphus, 949 harryi, 949 haueri, 949 hypnicola, 949 jeanneli, 949 languidoides, 949 languidus, 949 nanus, 949 nearcticus, 949 palustris, 949 sororum, 949 thomasi, 921, 949 yeatmani, 949 Diacypris compacta, 823 dietzi, 823 Diamphidaxona, 556, 566, 616, 643 Diaphanosoma, 851, 852, 857, 874 birgei, 872 Diaptomus, 935 glacialis, 948 connexus, 39 Dibusa angata, 752 Dicamptodon ensatus, 756 Dichaetura, 185, 188, 189, 190 Dicosmoecus, 672, 739, 749, 760 gilvipes, 739, 743, 750, 756 Dicranophorus, 202, 239 Dicrotendipes, 690 Dictya, 690 Didinium, 48, 49, 51, 62, 85 nasutum, 84 Difﬂugia, 67, 74 corona, 75 Dileptus, 62, 85 anser, 86 Dina, 487, 489, 496 anoculata, 497 dubia, 497 parva, 497 Dineutus, 684, 713 Dinobryon, 71 sertularia, 66 Dioctophyme renale, 459 Diphascon, 529, 531, 532, 533, 534, 539, 544 Diphetor, 662 Diploeca, 71 placita, 72 Diplois daviesiae, 241 Diploperla, 669 Diplophyrys, 77 archeri, 76 Discomorphella, 83 pectinata, 82 Disconaias, 402, 416 discus, 404 Discophyra, 81 elongata, 79 Disematostoma, 89 bütschlii, 88 Disparalona, 874, 878 acutirostris, 878 dadayi, 878 leei, 878, 879 rostrata, 878 Taxonomic Index Dissotrocha, 235 Distocambarus, 963, 964, 966, 967, 983, 989 crockeri, 988 Dixella, 716 Dochmiotrema, 173 Dolania, 698 Dolerocypris, 830, 833 Dolomedes, 12, 550, 632, 649 scriptus, 649 striatus, 649 tenebrosus, 649 triton, 649, 650 vittatus, 649 Donacia, 713 Donnaldsoncythere, 830, 837 Dorylaimus, 257, 262, 265, 271, 274, 275 atratus, 263 Doryphoribius, 529, 531, 532, 539, 543 Dosilia, 128 palmeri, 119, 128 radiospiculata, 119, 120, 128 Dracunculus medinensis, 929 Dreissena, 9, 360, 397 bugensis, 332, 343, 350, 356, 360 polymorpha, 113, 332, 338 – 340, 343, 345 – 350, 352 – 356, 358 – 363, 366 – 368, 372 – 373, 375, 376, 378, 381 – 397, 929, 975 Drepanothrix, 883 dentata, 884 Drepanotrema, 318 kermatoides, 317 Drilomermis, 280 Dromogomphus, 666 Dromus dromas, 404, 410 Drunella, 662, 760 Dubiraphia, 684 Dugesia, 6, 163, 167 dorotocephala, 168 polychroa, 158, 168 tahitienseis, 161 tigrina, 157, 159, 168 Dunhevedia, 874, 878 americana, 877 Duosclera, 120 mackayi, 98, 106, 120, 121, 129 Dysnomia triquetra, 396 Dytiscus, 288, 683 Echinamoeba, 74 exudans, 73 Echiniscoides, 529, 538 sigismundi, 536 Echiniscus, 529, 531, 532, 538, 541, 542 spiniger, 528 Echinursellus longiunguis, 538 Eclipidrilus, 448, 449, 450 Ectemnia, 690 Ectocyclops, 936 phaleratus, 949 polyspinosus, 949 rubescens, 949 Ectopria, 713 Eiseniella, 446 Elaphoidella, 938 amabilis, 950 bidens, 950 californica, 950 carterae, 950 kodiakensis, 950 reedi, 950 shawangunkensis, 950 subgracilis, 950 tenuicaudis, 950 wilsonae, 950 Elgiva, 690 Elimia, 304, 305, 307, 310, 314, 323 alabamiensis, 314 caelatura, 314 cahawbensis, 309 carinocastata, 314 crenatella, 314 cylindracea, 314 fascians, 314 gerhardtii, 314 haysiana, 314 hydei, 314 livescens, 322 olivula, 314 showalteri, 314 Ellipsaria, 402 lineolata, 404, 410, 414 Elliptio, 402, 403, 408, 411, 412, 414, 415 complanata, 348, 349, 359, 360, 367, 368, 381, 387, 390, 408 crassidens, 405 dilatata, 412 icterina, 408 lanceolata, 408 nigella, 357 Elliptoideus, 402, 405 sloatanus, 413 Ellisodrilus, 458, 460 Elodea, 213, 214, 231, 986 canadensis, 213 Elodium, 150 Elosa, 244 Empidomermis, 280 Enallagma, 666, 701, 753, 754 clausum, 39 ebrium, 580 Encentrum linnhei, 232 Enchelyodon, 85 elegans, 84 Enchelys, 85 simplex, 84 Endochironomus, 690 Endotribelos, 690 Enochrus, 684 Entocythere, 776, 829, 830, 837 donnaldsonensis, 821 Entosiphon, 71 sulcatum, 66 Eocyzicus, 900 concavus, 899 Eogelyella carolinensis, 934 1043 Eohypsibius, 529, 531, 532, 539, 543 Eothinia, 202 Epactophanes, 938 richardi, 950 Ephemera, 698 Ephemerella, 662, 754 Ephemoroporus, 869, 874, 878 acanthodes, 877 Ephoron, 698 Ephydatia, 117, 130 ﬂuviatilis, 101, 106, 107, 108, 110, 117, 120, 121, 130 millsii, 121, 130 müelleri, 99, 105, 106, 107, 108, 120, 121, 122, 130 Ephydra, 38, 730 Epioblasma, 402, 411, 414 aracaeformis, 357 biemarginita, 357 ﬂexuosa, 357, 405 ﬂorentina, 357 haysiana, 357 lenior, 357 lewisii, 357 obliquata, 357 personata, 357 propinqua, 357 sampsonii, 357 stewardsonii, 357 torulosa, 357 turgidula, 357 Epiphanes, 202, 228, 242, 243 senta, 222 Epischura, 227, 912, 919, 922, 925, 926, 934 baikalensis, 911 ﬂuviatilis, 948 lacustris, 921, 923, 948 massachusettsensis, 948 nevadensis, 948 nordenskioldi, 948 Epistylis, 81 plicatilis, 80 Epitheca, 744 cynosura, 743 princeps, 743 simplicollis, 746 Erebaxonopsis, 558, 647 nearctica, 622 Eremobiotus, 529 Ergasilus arthrosis, 950 auritus, 950 caeruleus, 950 celestis, 950 centrarchidarum, 950 cerastes, 950 chautauquaensis, 950 clupeidarum, 950 cotti, 950 cyprinaceus, 950 elongatus, 950 japonicus, 950 lanceolatus, 950 1044 Taxonomic Index Ergasilus (continued) luciopercarum, 950 megaceros, 950 nerkae, 950 rhinos, 950 sieboldi, 950 tenax, 950 versicolor, 950 wareaglei, 950 Eristalis, 715 Erpetocypris barbatus, 829 Erpobdella, 467, 483, 489, 496 octoculata, 474, 480, 482, 483 punctata, 472 – 476, 479, 480, 482 – 485, 496 Erythemus simplicicolis, 580 Escherichia coli, 382 Esox lucius, 782 Espejoia, 67 Estellacarus, 558, 637, 646 unguitarus, 599, 622, 623 Estelloxus, 555, 636, 642 montanus, 615 Ethmolaimus, 263, 276 Eubosmina, 857, 857, 881, 882 coregoni, 882 Eubranchipus, 15, 889, 891, 895, 897 bundyi, 893 holmni, 893 hundyi, 896 moorei, 891 serratus, 892, 893 vernalis, 893 Eubrianax, 687 Eucephalobus, 274 Euchlanis, 198, 204, 223, 241, 242 dilatata, 205, 209, 217, 232 Euchordodes, 292 nigromaculatus, 283, 287, 288, 289 Euchromadora striata, 263 Eucyclops, 921, 936 agilis, 949 bondi, 949 conrowae, 949 elegans, 949 macruroides denticulatus, 949 neomacruroides, 949 prionophorus, 949 Eucypris, 822, 823, 828, 830, 833 crassa, 834 serrata, 828 serrato-marginata, 829 virens, 823 Eudiaptomus, 935 gracilis, 928, 931, 948 Eudorylaimus andrassyi, 263 Euglena, 63 Euglypha, 76 tuberculata, 76 Eukiefferiella, 690 Eulimnadia, 897 agasszii, 898 antelei, 890, 892 diversa, 890 inﬂecta, 891 Eunapius fragilis, 106, 120, 121, 122, 130 igloviformis, 120 mackayi, 120 Eupera, 400 Euplotes, 56, 62, 81 patella, 58, 80 Euryalona, 874, 876 orientalis, 877 Eurycercus, 853, 859, 862, 869, 874 lamellatus, 875 Eurylophella, 662, 699 Eurytemora, 934 afﬁnis, 948 americana, 948 arctica, 948 bilobata, 948 canadensis, 948 composita, 948 gracilicauda, 948 Euteratocephalus, 276 Euthyas, 554, 633, 640 mitchelli, 609 truncata, 609 Evadne, 857, 859, 887 nordmanni, 886 Eylais, 553, 568, 570 – 577, 579, 580, 581, 607, 631, 634, 639 discreta, 569, 570 eurylhalina, 569 major, 571 Fabaeformiscandona, 809, 814, 818, 819, 821, 830, 833 pennaki, 822 wegelini, 817, 822 Fallceon, 662 Fallicambarus, 963, 964, 966, 967, 983, 989 devastator, 974, 976, 985 fodiens, 977, 988 gordoni, 977 Farula, 676 Faxonella, 963, 964, 965, 989 clypeata, 981, 985 creaseri, 987 Feltria, 557, 577, 593, 631, 637, 643 exilis, 617 Ferrissia, 316 fragilis, 310 rivularis, 317 Filamoeba, 74 nolandi, 73 Filinia, 205, 208, 209, 227, 237 longiseta, 221, 227 Fisherola, 316 nutalli, 317 Flabelliforma ostracodae, 825 Floscularia, 211, 212, 214, 224, 237 conifera, 211, 212, 213, 214, 219, 238 Fluminicola, 303, 320 Fontelicella, 320 Fontigens, 320 Forelia, 557, 566, 577, 579, 638, 645 ﬂoridensis, 620 onondaga, 596 ovalis, 596 Fossaria, 302, 320 humilis, 321 modicella, 304 Fragilaria, 974 Fredericella, 505, 506, 507, 510, 512, 513, 514, 517 browni, 512, 518, 519 indica, 511, 511, 512, 518, 519, 522 sultana, 511, 518 Frontipoda, 555, 636, 641 americana, 615 Frontipodopsis, 557, 566, 579, 617, 643 Frontonia, 67, 89 leucas, 88 Fujiscon, 529, 531 Fusconaia, 402, 411, 414 ﬂava, 361, 405 ebena, 385 masoni, 408 Gammarus, 14, 163, 785, 787, 796 lacustris, 782, 786, 790, 791, 792, 919 minus, 781, 782, 786, 787, 788, 791, 793 pulex, 785, 790 Gasterosteus aculeatus, 824 Gastropus, 247, 248 Gastrostyla, 81 steini, 82 Gatromermis, 278 Geayia, 559, 563, 579, 628, 635, 644 Geocentrophora, 157, 164, 166 cavernicola, 179 sphyrocephala, 179 Geocythere, 830, 837 Gerris, 570, 678, 709, 730 comatus, 580 Giardia, 69 Gieysztoria, 157, 173 Glaucoma, 89 scintillans, 88 Glebula, 402 rotundata, 405, 415 Gleotrichia, 238 Glossiphonia complanata, 472, 476, 477, 479, 480, 482, 483, 488, 492, 494 heteroclita, 488 rudis, 488 Glossosoma, 706, 730, 737, 740, 760 Glypotendipes paripes, 482 Goera, 672, 706 Goerita, 672 Gomphaeschna, 666 Gomphonema, 974 Gomphus, 701, 730 Gonidea, 402 angulata, 403, 405 Gonostomum, 83 afﬁne, 82 Taxonomic Index Gordius, 282, 283, 285, 288, 291, 292 aquaticus, 282, 283 diblastus, 287 dimorphus, 283, 287 robustus, 288 tenuiﬁbrosus, 280 Graphoderus, 683 Graptoleberis, 857, 874, 876 testudinaria, 862, 875 Grimaldina, 883 brazzai, 885 Guernella, 883 raphaelis, 885 Gulcamptus, 938 alakaensis, 950 huronensis, 950 laurentiacus, 950 Guttipelopia, 690 Gymnodinium, 66, 71 Gymnopais, 690 Gyratrix, 157, 173 hermaphroditus, 161, 169, 180 Gyraulus, 318 circumstriatus, 304 deﬂectus, 304, 310, 317 Gyrinus, 684, 730 Gyrodinium, 66, 71 Gyrotoma, 314 Haber, 453 speciosus, 452 Habrophlebia, 699 Habrotrocha, 235 rosa, 207, 231, 232 Haementaria, 488 ghilianii, 469, 474, 481 Haemonais waldvogeli, 451 Haemopsis, 489 sanguisuga, 480 Halicyclops, 914, 934 Halipegus occidualis, 825 Haliplus, 684, 711, 713 Halobates, 742 Halobiotus, 529, 537 crispae, 535 Haltdytes, 191 Halteria, 62, 83 grandinella, 82 Haplohexapodibius, 529 Haplomacrobiotus, 529, 531, 532, 533, 543 Haploperla, 702 Haplotaxis, 435, 437, 439, 441, 446, 447 Haptoglossa, 229 Harpagocythere, 830, 838 Harringia, 239, 240 Hartmanella, 74 vermiformis, 73 Hartocythere, 830, 837 Hastatella, 81 radians, 80 Hauffenia, 320 Hebesuncus, 529, 531, 544 Hebetancylus, 316 excentricus, 317 Heleidomermis, 280 Heleidomermis libani, 262 Helichus, 686, 712 Helicopsyche, 705, 760 borealis, 743, 750 Heliophyra, 81 reideri, 79 Helisoma, 307, 311, 318, 975 anceps, 304, 305, 308, 313, 319, 825 companulatum, 304 trivolvis, 302, 306 Helobdella, 469, 492 california, 493 elongata, 490, 493 fusca, 493 papillata, 493 robusta, 493 stagnalis, 472, 473, 476, 477, 479, 480, 482, 483, 492, 493 transversa, 493 triserialis, 479, 493 Helophorus, 688, 712 Helopicus, 669 Hemicycliophora, 270, 273 Hemigrapsis estellinensis, 960 Hemistena, 402 lata, 405, 411 Heptagenia, 662 Herpetocypris, 815, 816, 830, 834 brevicaudata, 808 reptans, 816, 824 Hesperocorixa, 678 Hesperodiaptomus, 912, 927, 929, 935 arcticus, 948 augustaensis, 948 breweri, 948 caducus, 948 californensis, 948 eiseni, 948 franciscanus, 948 hirsutus, 948 kenai, 948 kiseri, 948 nevadensis, 923, 948 novemdecimus, 948 schefferi, 948 septentrionalis, 926 shoshone, 926, 931, 948 victoriaensis, 948 wardi, 948 Hesperoperla, 669 Hesperophylax, 672, 705 Hetaerina, 701 Heterocope, 912, 925, 934 septentrionalis, 922, 926, 948 Heterocypris, 823, 828, 829 incongruens, 814, 815 Heterolepidoderma, 187, 188, 190 Heteromeyenia, 117, 129 baileyi, 121, 122, 123, 124, 129 latitenta, 122, 123, 124, 129 tentasperma, 122, 123, 124, 129 1045 tubisperma, 122, 123, 124, 129 Heteromita, 52 Heterophrys, 78 myriopoda, 77 Heteroplectron, 706 californicum, 748, 749, 756 Heterosternuta, 683, 684 Hetrophrys, 50 Hexagenia, 698, 730, 732, 735 Hexamita, 52, 57, 71 inﬂata, 72 Hexapodibius, 529, 531, 532, 543 Hexarthra, 203, 205, 227, 236 fennica, 39, 216, 221 Hexatoma, 715 Heydenius dominicanus, 262, 281 Hirschmanniella, 270 Hirudo medicinalis, 469, 470, 474, 481, 482, 486, 496 Histobalantium, 87 natans, 86 Hobbseus, 963, 964, 966, 967, 983, 989 cristatus, 988 Holopedium, 857, 859, 861, 863, 864 amazonicum, 873 gibberum, 872, 873 Holophyra, 85 simplex, 86 Homalozoon vermiculare, 84 Homocaligus muscorum, 648 Homochaeta naidina, 450 Homocyclops, 936 ater, 949 Homoeoneuria, 662, 699 Hoplodictya, 690 Horatia, 320 Horreolanus, 559, 645 orphanus, 628 Hoyia, 320 Huitfeldtia, 557, 579, 638, 648 rectipes, 573, 595, 619 Huntemannia, 937 lacustris, 950 Hyalella, 793, 794, 796 azteca, 483, 781, 786, 787, 790 – 793 montezuma, 477, 483, 786, 790 Hyalinella, 514 orbisperma, 511, 521 punctata, 511, 513, 514, 519, 521 Hyalopyrgus, 320 Hyalosphenia, 74 cuneata, 75 Hydaticus, 683 Hydatophylax, 740 Hydra, 6, 136, 137, 138, 140 – 146, 150, 151, 152, 754, 755, 850 attenuata, 145 braueri, 152, 153 oligactis, 152, 153 viridissima, 151, 153 vulgaris, 152, 153 Hydrachna, 553, 568, 569, 572 – 575, 579, 580 – 582, 606, 631, 634, 639 conjecta, 570, 580 1046 Taxonomic Index Hydrachna (continued) magniscutata, 587 virella, 580 Hydramoeba hydroxena, 140 Hydrocanthus, 684, 712, 713 Hydrochara, 684 Hydrochoreutes, 557, 638, 647 intermedius, 595, 619 minor, 595 Hydrochus, 712 Hydrodroma, 554, 579, 581, 633, 639 despiciens, 568, 571, 581 Hydrolimax, 166 grisea, 157, 179 Hydromermis, 280 Hydrometra, 709 Hydroperla, 669 Hydroporus, 683, 684, 711, 730 Hydropsyche, 672, 730, 739, 740, 757, 760 Hydroptila, 705 Hydroscapha, 712 natans, 682 Hydrovatus, 684 Hydrovolzia, 553, 569, 570, 571, 633, 638 gerhardi, 563 marshallae, 606 Hydrozetes, 632, 648 Hydrozoa, 145 Hydryphantes, 553, 564, 572, 579, 581, 608, 633, 639 ruber, 565, 608 tenuabilis, 568, 572, 574 Hygrobates, 556, 569, 572, 574, 579, 582, 593, 618, 637, 647 ﬂuviatilis, 582 neocalliger, 593 nigromaculatus, 581 Hygrotus, 684 Hyla regilla, 285, 288 Hylodiscus, 74 rubidicundus, 73 Hymanella, 168 retenuova, 168 Hypechiniscus, 529, 538, 542 Hypotrichidium, 83 conicum, 82 Hypsibius, 529, 531, 532, 534, 537, 539, 541, 543 convergens, 539 dujardini, 539 Ichthydium, 188, 189, 190 Ichthyophthirius, 67 Ictalurus furcatus, 385 punctatus, 385 Ictiobus bubalus, 385 niger, 385 Illinobdella, 468, 489, 490, 494 alba, 495 elongata, 495 richardsoni, 495 Ilybius, 684 Ilyocryptus, 861 sordidus, 884 Ilyocypris, 822, 830, 835 bradyi, 821 gibba, 819, 821 Ilyodrilus, 454, 455 Ilyodromus, 830, 833 Io, 323 ﬂuvialis, 315, 322 Ironus, 265, 278 Ischnura, 666 verticalis, 580 Isochaetides, 453, 454 Isocypris, 830, 833 longicomosa, 829 Isogenoides, 669 Isohypsibius, 529, 531, 532, 533, 534, 537, 538, 539, 543 granulifer, 537 Isomermis, 280 Isoperla, 669, 702, 730 Itaquascon, 529, 531, 533, 534, 544 Itocyclops, 937 yezoensis, 949 Itura, 202 bostoniensis, 216 Janicea, 961 Javalbia, 558, 622, 646 Juga, 307, 323, 739, 740, 756, 760 Kawamuracarus, 555, 616, 642 Kellicottia, 201, 243, 244 Kenkia, 168 Keratella, 8, 198, 201, 205, 210, 226, 227, 243, 245 americana, 205, 217 cochlearis, 222, 225, 226, 227, 232, 244, 245 crassa, 210, 222 earlinae, 222 quadrata, 208, 221, 245 slacki, 227 tauracephala, 206 testudo, 205, 220, 226, 227, 232, 245 Kerona, 140 Khawkinea, 5, 71 halli, 66 Kijanebalola, 190, 191 Kincaidiana freidris, 449, 450 hexatheca, 448, 449 Koenikea, 556, 579, 618, 637, 645 marshallae, 594 wolcotti, 630 Kongsbergia, 557, 624, 629, 646 Krendowskia, 559, 635, 644 similis, 604 Krumbachia, 169 hiemalis, 171, 180 minuta, 180 virginiana, 180 Kurzia, 874, 876 latissima, 876 longirostris, 875, 876 Labiobaetis, 662 Laccobius, 684 Laccophilus, 684 Laccornis, 684 Lacinea mobilis, 785 Lacinularia, 211, 212, 214, 227, 237 ﬂosculosa, 199 Lacrymaria, 85 olor, 84 Ladona, 666 Laevapex, 316 fuscus, 304, 310, 317 Lambornella, 67, 68 Lamellidens corrianus, 341, 347, 351, 354 marginalis, 391 Lampsilis, 342, 364, 365, 402, 408, 411, 412, 415 binominata, 357 claibornesis, 353 ovata, 365, 405 perovalis, 364, 365 radiata, 345, 346 siliquoidea, 345, 358, 359 teres, 358 Lanceimermis, 278 Lanthus, 666 Lanx, 316 patelloides, 317 Lara avara, 748 Lasmigona, 402, 408, 409, 410, 413 complanata, 410 costata, 405, 409 Lathonura, 883 rectirostris, 884 Latona, 852, 859, 873 setifera, 872 Latonopsis, 874 occidentalis, 872 Laversia, 559, 569, 578, 579, 636, 645 berulophila, 603, 604, 627 Lebertia, 555, 572, 590, 615, 636, 642 Lecane, 210, 243 inermis, 232 lunaris, 242 quadridentata, 205, 217 Lecythium, 75 hyalinum, 75 Lemabadion, 87 magnum, 88 Lemanea australis, 752 Lemiox, 402, 405 rimosus, 409 Lemna, 682 Lepadella, 210, 241 Lepidocaris, 867 Lepidochaetus, 189, 190 Taxonomic Index Lepidodermella, 188, 189, 190 squamata, 182, 186, 187 triloba, 187 Lepidostoma, 706, 763 Lepidurus, 890, 900 arcticus, 889 couessii, 899 lemmoni, 891 Lepomis cyanellus, 782 gibbosus, 311 gulosus, 385 macrochirus, 385 microlophus, 311, 385 Leptistheria, 900 compleximanus, 898 Leptodea, 402, 408, 410, 414 fragillis, 405, 410, 414 Leptodiaptomus, 919, 935 ashlandi, 948 assiniboiaensis, 948 coloradensis, 948 connexus, 948 insularis, 948 judayi, 948 minutus, 921, 923, 931, 948 moorei, 948 novamexicanus, 948 nudus, 948 pribilofensis, 948 sicilis, 948 siciloides, 948 signicauda, 948 spinicornis, 948 trybomi, 948 tyrrelli, 922, 923, 948 Leptodora, 35, 147, 852, 859, 860, 861, 862, 885 kindtii, 885, 886 Leptohyphes, 662, 698 Leptophlebia, 699 cupida, 739, 743 Leptoxis, 314, 323 ampla, 314 picta, 314 praerosa, 314, 322 taeniata, 314 Lepyrium, 320 Lesquereusia, 74 spiralis, 75 Lestes, 667, 701, 730 Lethaxona, 557, 621, 646 Lethocerus, 680 Leuctra, 703 Lexathonella, 557, 647 parva, 621 Lexingtonia, 402 dolabelloides, 405, 411 subplana, 408 Leydigia, 852, 874, 876 acanthocercoides, 877 leydigi, 862 Libellula, 730 lydia, 745, 746 Lieberkuehnia, 77 wagnerella, 76 Ligumia, 402, 412, 415 recta, 381, 406 subrostrata, 348, 350, 351, 352 Limmenius, 529, 531, 532, 534, 542 Limnadia, 900 lenticuraris, 898 Limnesia, 556, 568, 572, 579, 605, 636, 643 maculata, 575 marshalliana, 592 undulata, 577 Limnias, 211, 214 melicerta, 238 Limnocalanus, 63 Limnocalanus, 912, 917, 922, 925, 934 johanseni, 948 macrurus, 917 Limnochares, 553, 568, 570, 572, 573, 576, 579, 607, 634, 639 americana, 569, 570, 586 aquatica, 570, 572, 573, 574, 575, 580 Limnocodium, 135, 147 victoria, 146 Limnocythere, 826, 830, 838 ceriotuberosa, 818, 819, 822 friabilis, 826 herricki, 819 itasca, 809, 810, 819, 837 liporeticulata, 821 sanctipatricii, 811 sappaensis, 818 staplini, 816, 818, 820, 822, 827 verrucosa, 818, 819 Limnodrilus, 439, 440, 445, 454, 455 cervix, 439 claparedeianus, 440 hoffmeisteri, 437, 440 profundicola, 438 Limnoithona, 936 sinensis, 949 Limnomermis, 280 Limnophora, 715, 678 Limnoruanis, 169 romanae, 159, 171, 180 Limnosida, 873 Limnozetes, 649 Limonia, 715, 730 Limpnephilus, 732 Linderiella, 897 occidentalis, 896 Lindia, 237, 240 Liodessus, 684 Lioplax, 303, 323 subcarinata, 324 Lioporeus, 684 Lipsothrix, 748 Lirceus, 785, 786, 796 fontinalis, 782, 791 usdagalun, 792 Lithasia, 323 geniculata, 322 1047 Lithocolla, 78 globosa, 77 Litocythere, 830, 838 Litonotus, 87 fasciola, 86 Ljania, 558, 638, 646 bipapillata, 598 michiganensis, 621 Lobohalacarus, 648 Lophocharis, 241 Lophopodella, 508, 510, 512 carteri, 510, 511, 512, 513, 516, 520, 523 Lophopus, 510 crystallinus, 511, 512, 520, 523 Lordocythere, 830, 836 Loxocephalus, 87 plagius, 86 Loxodes, 55, 56, 57, 85 magnus, 86 Loxophyllum, 51, 87 helus, 86 Lumbriculus, 438, 439, 445, 448, 449 variegatus, 438, 440, 445, 448 Lutrochus, 713 Lymnaea, 304, 307 elodes, 305, 307, 308, 311 emarginata, 305 peregra, 305, 306 stagnalis, 39, 298, 305, 314, 320, 321 Lynceus, 900 brachyurus, 898 Lyrogyrus, 310, 320 Macdunnoa, 662 Macrobdella, 495 decora, 469, 495 diplotertia, 495 ditetra, 495 sestertia, 495 Macrobiotus, 529, 531, 532, 534, 537, 539, 541, 543 areolatus, 538 hufelandi, 530, 537, 538, 539 persililis, 540 tonollii, 528, 538 Macrobrachium, 774, 783, 951, 961, 962, 963, 964, 988 acanthurus, 961, 962 carcinus, 961, 962 ohione, 783, 961, 962, 969, 970 olfersii, 961 rosenbergii, 959, 962 Macrochaetus, 243, 244 Macrocotyla, 168 Macrocyclops, 921, 929, 936 albidus, 949 fuscus, 912, 913, 949 Macrohectopus, 794 branickii, 790, 794 Macromia, 701 Macrostomum, 6, 157, 161, 166 collistylum, 179 1048 Taxonomic Index Macrostomum (continued) curvistylum, 179 gilberti, 167, 179 glochostylum, 179 lewisi, 179 lineare, 179 norfolkense, 179 ontarioense, 179 phillipsi, 179 reynoldsi, 179 riedeli, 179 ruebushi, 179 sensitivum, 179 sillimani, 179 stirewalti, 179 tennesseense, 179 tuba, 167, 179 virginianum, 179 Macrothrix, 859, 883 rosea, 885 Macrotrachella, 235 quadricornifera, 223 Macrovelia, 709 Macroversum, 529, 531, 532, 539 Magmatodrilus, 460 obscurus, 461 Malirekus, 669 Mamersellides, 555, 613, 642 Mansonia, 691 Maraenobiotus, 938 brucei, 950 canadensis, 950 insignipes, 950 Margaritifera, 366, 402, 412 falcata, 403 hembeli, 361 margaritifera, 350, 351, 358, 364, 366, 367, 368, 403, 404 Marinellina, 181, 182, 185, 188 ﬂagellata, 181, 182 Marisa, 303, 318 cornuarietis, 324 Marstonia, 320 Martarega, 681 Marvinmeyeria, 469 lucida, 479, 491 Maryana, 89 socialis, 88 Mastigodiaptomus, 935 albuquerquensis, 948 texensis, 948 Matus, 684 Mayorella, 74 bigemma, 73 Medionidus, 402, 409, 413 conradicus, 406 mcglameriae, 357 Megacyclops, 937 donnaldsoni, 949 gigas, 912, 949 latipes, 949 magnus, 949 viridis, 921, 949 Megadrili, 446 Megafenestra, 881 nasuta, 880 Megalocypris, 828, 829, 830, 834 alba, 821, 823 deeveyi, 829 novaezelandiae, 813, 822 pugionis, 829 sarsi, 829 Megalonaias, 363, 402 nervosa, 406, 409, 413 Melampus, 313 Melanoides, 320 tuberculata, 322 Melanopsis, 314 Melosira, 516 Menetus dilatatus, 317 Meramecia, 556, 643 occidentalis, 616 Mesobates, 556, 618, 647 Mesochra, 938 alaskana, 950 Mesocrista, 529, 531, 532, 544 Mesocyclops, 227, 921, 929, 936 americanus, 913, 949 edax, 919, 920, 921, 922, 927, 928, 949 leuckarti, 917, 921 longisetus, 949 ogunnus, 917 ruttneri, 949 venezolanus, 949 Mesodinium, 85 pulex, 84 Mesodorylaimus, 274 Mesomermis, 280 Mesorhabditis, 274 Mesostoma, 157, 158, 159, 160, 161, 162, 163, 173 arctica, 161, 180 californicum, 161, 180 columbianum, 180 craci, 169, 172, 180 curvipenis, 180 ehrenbergi, 158, 159, 160, 161, 162, 180 lingua, 158, 159, 160, 162 macroprostatum, 180 platygastricum, 180 vernale, 161, 171, 180 virginianum, 180 Mesovelia, 709 Metacineta, 78 mystacina, 79 Metacyclops, 937 cushae, 916, 949 Metacypris, 830, 838 maracoensis, 821 Metacystis, 85 recurva, 84 Metopus, 83 es, 82 Metrobates, 678 Micractinium, 225 Micrasema, 672 Microbiotus, 529, 532 Microcometes, 77 paludosa, 76 Microcyclops, 937 pumilis, 949 rubellus, 949 varicans, 921, 949 Microdalyellia, 157, 173 abursalis, 179 circobursalis, 179 fairchildi, 179 gilesi, 179 rheesi, 179 rochesteriana, 179 rossi, 167, 179 ruebushi, 179 sillimani, 179 tennesseensis, 167, 179 virginiana, 179 Microgromia, 77 haeckeliana, 76 Microhydra ryderi, 146 Microhypsibius, 529, 531, 543 Microkalyptorhynchus, 173 Microlaimus, 274 Micropterus dolomieui, 979 Microrhynchus virginianus, 180 Microsporidium, 228 Microstomum, 157, 166, 265 lineare, 167, 179 Microthorax, 89 pusillus, 80 Midea, 552, 558, 577, 635, 644 expansa, 568, 602, 625 Mideopsis, 558, 566, 578, 579, 627, 645 borealis, 603 Mikracodides, 243 Milnesium, 529, 531, 532, 533, 534, 536, 539, 541, 542 Minibiotus, 529, 531, 532, 533, 543 Minytrema melanopus, 385 Mixibius, 529, 531, 532, 539 Mixodiaptomus, 935 theeli, 948 Moina, 850, 851, 852, 854, 857, 859, 861, 867, 881, 889, 890 wierzejskii, 886 Moinodaphnia, 881 macleayii, 886 Molanna, 672 Mollibdella, 489 grandis, 469, 496 Momonia, 558, 579, 635, 643 campylotiba, 601 projecta, 625 Monas, 71 Monhystera, 263, 265, 274 grelachii, 263 ocellata, 263 Monhystrella, 274 Monoattractides, 555, 614, 641 Monodella texana, 784 Mononchulus, 276 Mononchus, 262, 278 Taxonomic Index Monoporeia, 789, 790, 796 afﬁnis, 786, 789, 791 Monopylephorus, 437 Monosiga, 48, 71 ovata, 72 Monospilus, 847, 874 dispar, 875 Monostyla, 243 Mooreobdella, 467, 487, 489, 496 bucera, 497 fervida, 479, 497 melanostoma, 497 microstoma, 482, 497 tetragon, 497 Mopeschiniscus, 542 Moraria, 938 afﬁnis, 950 arctica, 950 cristata, 950 duthiei, 950 laurentica, 950 mrazeki, 950 virginiana, 950 Morimotacarus, 559, 645 nearcticus, 626 Morone saxatilis, 385 Motobdella, 469, 470, 472, 483, 496 montezuma, 465, 477, 481, 483, 484, 489, 496 sedonensis, 484, 489, 497 Moxostoma carinatum, 385 Mucronothrus, 649 Multifasciculatum, 81 elegans, 79 Murrayon, 529, 531, 532, 539 Musculium, 343, 360, 362, 368, 383, 390, 400 lacustre, 369 partumeium, 343, 346, 347, 348, 350, 360, 369, 381, 388, 396, 400 securis, 351, 369, 387, 400 transversum, 377, 381, 382, 383, 384, 387, 389, 400 Mya arenaria, 353 Myelostoma, 83 ﬂagellatum, 82 Myostenostomum, 165 tauricum, 166, 179 Myriophyllum, 150, 231 Mysis, 14, 225, 786, 789, 794, 796 littoralis, 786 relicta, 35, 786, 788, 789, 790 Mystacides, 672 Mytilina, 241 Mytilocypris henricae, 808, 814 Mytilopsis, 392, 397 leucophaeata, 397 Mytilus edulis, 347 Myzobdella, 489 lugubris, 492, 494 Naegleria, 73 fowleri, 54 gruberi, 53, 54 Nais, 440, 446, 452 elinguis, 440 Najadicola, 384, 557, 579, 638, 647 ingens, 581, 597, 620 Nannocandona, 830 faba, 822 Nannochloris oculata, 226 Nannopus, 937 palustris, 950 Nassula, 63, 67, 78 ornata, 80 Natricola, 320 Nautarachna, 557, 638, 645, 645, 648 muskoka, 597 queticoensis, 620 Navicula, 67, 974 Nebalia, 233 Nebela, 74 collaris, 75 Nebrioporus, 684 Necopinatum, 529, 531, 532, 542 Nectomena, 290, 291 munidae, 283 Nectopsyche, 672 Necturus maculosus, 363, 385 Nehalennia speciosa, 580 Nelumbo lutea, 511 Nemoura, 702, 703 Neoacarus, 559, 579, 636, 644 minimus, 626 occidentalis, 603 Neoatractides, 555, 614, 641 Neoaturus, 557, 624, 646 Neobosmina, 881, 882 tubicen, 882 Neobrachypoda, 558, 638, 646 ekmani, 598, 623 Neochordodes, 292 occidentalis, 284, 285, 286, 288, 289 Neochoroterpes, 662 Neodasys, 187 Neoechinorhynchus prolixus, 825 rutili, 825 Neoephemera, 698 Neogossea, 190, 191 Neohermes, 760 Neolimnochares, 553, 570, 586, 607, 634, 639 Neomamersa, 556, 566, 616, 643 californica, 616 chihuahua, 616 Neomideopsis, 558, 644 suislawensis, 625 Neomysis mercedis, 786, 788 Neopalaemonon, 961 Neophylax, 676, 706, 730 aniqua, 750 Neoplanorbis, 318 Neoplea, 709, 732 Neoporus, 684 Neothremma, 676 Neothrix, 883 armata, 885 Neotiphys, 557, 638, 648 pionoidellus, 595, 619 Neotyrrellia, 556, 643 anitahoffmannae, 617 Nepa, 709 Nephelopsis, 483, 496 obscura, 312, 470 – 485, 487, 497 Neritina, 318 reclivata, 303, 319 Neumania, 556, 581, 618, 637, 647 papillator, 576, 577, 578 punctata, 594 Neurocordulia, 701 Niqronia, 677, 707 Nitokra, 937 hibernica, 950 lacustris, 950 Nordodiaptomus, 935 alaskaensis, 948 Nosema stenocypris, 825 Nostoc, 264 parmelioides, 750, 751, 756 Notholca, 204, 210, 244, 245 acuminata, 208 squamala, 245 Notoalona, 874, 876 freyi, 877 Notodromas, 830, 832 monacha, 813, 816 Notogilla, 320 Notommata, 201, 202, 215, 245, 247 copeus, 224, 232 Notonecta, 678, 681, 709, 857 Notopanisus, 554, 608, 640 Nüchterleinella corneliae, 825 Nudomideopsis, 558, 577, 635, 644 magnacetabula, 601, 602, 625 Nuphar, 511 Nyctiophylax, 672 Nygolaimus, 274 Nymphaea, 511 Nymphula, 710 Obliquaria, 402 reﬂexa, 378, 406, 410, 413 Obovaria, 402, 406, 411, 414 Ochromonas, 50, 71 variabilis, 66 Octomyomermis, 280 Octotrocha speciosa, 199 Odontolaimus, 274, 276 Odontomyia, 715 Oecetis, 672, 705 Oedipodrilus, 460 Okriocythere, 830, 836 Oligobdella, 469, 491 biannulata, 490, 492 Oligostomis, 705 Olisthanella, 157, 173 truncala, 180 Omartacarus, 556, 579, 616, 642 1049 1050 Taxonomic Index Onchobunops, 883 Oncholaimus, 276 Oncorhynchus clarki, 756 Onychocamptus, 937 mohammed, 950 Onychodiaptomus, 935 birgei, 948 Opercularia, 81 nutans, 80 Ophidonais, 450, 451 serpentina, 450 Ophiogomphus, 666 Ophrydium, 81 eichhorni, 80 Ophryoglena, 59, 67 Ophryoxus, 883 gracilis, 884 Opisthocystis, 173 goettei, 169, 180 Opistomum, 169 pallidum, 158, 167, 180 Optioservus, 684 Orconectes, 963, 964, 966, 967, 968, 972, 983, 988, 989, 989 australis, 960, 966 compressus, 960 immunis, 955, 956, 972 inermis, 32, 959, 972, 987 luteus, 973, 974 nais, 955 neglectus chaenodactylus, 975 pellucidus, 956, 959 propinquus, 778, 958, 959, 972, 974, 975, 978, 979, 981, 982, 987 punctimanus, 973, 974 rusticus, 312, 955, 972, 974, 975, 976, 977, 978, 982, 985, 988 shoupi, 983 testii, 32 virilis, 778, 781, 959, 972, 976, 977, 978, 980, 981, 982, 983 Oreela, 529, 542 Oregonacarus, 555, 642 rivulicolus, 613 Oreodytes, 684 Ornamentula, 185, 191 Ornithocythere, 830, 836 Orohermes, 740 Orthocladius, 749 Orthocyclops, 936 modestus, 949 Ortmannicus, 967 Orygocerus, 320 Oscillatoria, 67 Osphranticum, 921, 935 labronectum, 948 Otomesostoma, 157, 166 auditivum, 160, 179 Oxus, 555, 572, 579, 615, 636, 641 elongatus, 591 Oxyethira, 705 Oxytrcha, 81 fallax, 82 Oxyurella, 874, 876 brevicaudis, 848, 877 Pacifastacus, 954, 963, 964, 966, 967, 983, 988 chenoderma, 966 fortis, 967, 983 gambelii, 963 leniusculus, 265, 957, 969, 970, 981, 983 nigrescens, 967 trowbridgii, 970 Paenecalyptonotus, 559, 645 Pagastia, 690 Palaemonetes, 961, 961, 962, 963, 964, 969, 983, 988 antrorum, 961 cummingi, 961, 969, 983 holthuisi, 961 intermedius, 969 kadiakensis, 956, 958, 959, 961, 962, 968 paludosus, 961, 962, 968 pugio, 969 Palaemonias, 961, 963, 964, 983, 985, 988 alabamae, 953, 961, 969, 974, 982 ganteri, 952, 961, 969, 974, 980, 982 paludosus, 957, 974 Palaeodipteron, 715 Paleochordodes protus, 280, 289 Palpomyia, 715 Paludicella, 509 articulata, 512, 517, 518 Panisopsis, 554, 578, 633, 640 setipes, 610 Panisus, 554, 633, 640 condensatus, 610 Pantala, 701 Paracamptus, 938 reductus, 950 reggiae, 950 Paracandona, 830, 833 euplectella, 810, 831 Paracapnia, 703 Parachordodes, 282, 292 lineatus, 291 violaceus, 291 Paracineta, 78 crenata, 79 Paractinolaimus, 274 Paracyatholaimus, 276 Paracyclops, 921, 936 canadensis, 949 chiltoni, 949 ﬁmbriatus, 949 poppei, 949 smileyi, 949 yeatmani, 949 Paracymus, 684 Paradileptus, 85 robustus, 86 Paradiphascon, 529, 531 Paraeuglypha, 76 reticulata, 76 Paragnetina, 669 Paragonimus, 974 kellicoti, 974 westermani, 974 Paragordius, 292 varius, 282, 284 Paraleptophlebia, 662, 699, 739 Paralimnetis, 900 Paramastix, 51, 71 Paramecium, 43, 45, 48, 49, 51, 60, 62, 63, 89, 823 aurelia, 58, 59, 62 biaurelia, 58 bursaria, 62, 63 caudatum, 88 monoaurelia, 58 Paramideopsis, 558, 578, 635, 644 susanae, 601, 625, 630 Paranais, 437, 450 Paranyctiophylax, 672 Paraphaenocladius, 690 Paraphanolaimus, 265, 276 Paraphysomonas, 46, 67, 71 vestita, 66 Parascon, 529 Parastenocaris, 937 brevipes, 950 delamarei, 950 lacustris, 950 palmerae, 913 palmerae, 950 texana, 950 trichelata, 950 Parathyasella, 554, 640 heatherensis, 611 Parechiniscus, 542 Parhexapodibius, 529 Parophryoxus, 883 tabulatus, 884 Partnuniella, 578 Partuninia, 554, 640 steinmanni, 608 Pasithea, 859 Paulinella, 76 chromataphora, 76 Pectinatella, 509, 510 magniﬁca, 509 – 513, 520, 522 Pediastrum duplex, 516 simplex, 516 Pedinomonas, 396 Pegias, 402, 406 fabula, 408 Pellioditis, 271, 273, 274 Pelocoris, 678, 709 Pelocypris, 829, 830, 835 alatabulbosa, 834 Pelomyxa, 67, 68, 71 palustris, 57, 75 Peltodytes, 684, 713 Peltoperla, 702, 740 Penardia, 75 granulosa, 75 Penardochlamys, 74 arcelloides, 75 Penelia, 852, 857, 873, 874 avirostris, 872 Penilia, 857, 873 Pentaqenia, 698 Taxonomic Index Peranema, 62, 67, 71 trichophorum, 66 Perca ﬂavescens, 366 Percymoorensis, 489, 496 kingi, 496 lateralis, 496 lateromaculata, 496 marmorata, 469, 480, 482, 496 septagon, 496 Pericantha, 859 truncata, 862 Peridinium, 66, 71 Perispira ovum, 63 Perlinella, 669 Perutilimermis, 278 Petalomonas, 71 abcissa, 66 Petrophila, 730 Phaenocoria, 158, 161, 162, 163, 170, 171 agassizi, 180 beauchampi, 159 falciodenticula, 180 highlandense, 180 kepneri, 180 lutheri, 180 typhlops, 158, 161 unipunctata, 158 virginiana, 180 Phagocata, 158, 168 velata, 168 Phagodrilus, 437, 439, 443 Phallodrilus hallae, 438, 454, 455 Pharyngostomoides procyonis, 459 Pheidole xerophila tucsonica, 2 Pherbella, 690 Pheromermis, 278 pachysoma, 260, 279 Philaster, 67 Philasterides, 87 armata, 86 Philobdella, 495 ﬂoridana, 495 gracilis, 495 Philodina, 197, 198, 218, 228, 235 acuticornis, 232 citrina, 217 Phoronis ovalis, 514 Phreatobrachypoda, 558, 646 multipora, 624 oregonensis, 624 Phryganella, 74 nidulus, 75 Phyllognathopus, 937 viguieri, 924, 950 Phylocentropus, 672, 705 Phymocythere, 830, 836 Physa, 300, 318, 975 vernalis, 306 Physella, 300, 304, 306, 307, 312, 318 anatina, 319 ancillaria, 304 cubensis, 302 gyrina, 306, 308, 319 heterostropha, 300 integra, 305, 319 vernalis, 301 virgata, 312, 319 zionis, 319 Physocypria, 830, 835 globula, 824 pustulosa, 815, 834 Piersigia, 553, 570, 579, 581, 586, 634, 639 limnophila, 607 Piguetiella, 451 Pilgramilla, 173 virginiensis, 179 Piona, 552, 557, 566, 568, 569, 572, 575 – 577, 579, 581, 582, 620, 638, 645, 648 carnea, 582, 597 constricta, 597 interrupta, 597 limnetica, 581 mitchelli, 597 napio, 574 Pionacercus, 557, 620, 645, 647 Pionopsis, 557, 638, 648 paludis, 619 Piscicola, 490, 494 geometra, 494 milneri, 494 punctata, 494 salmositica, 494 Piscicolaria, 468, 490 geometra, 481, 488, 489 milneri, 488, 489 punctata, 489 reducta, 490, 492, 494 salmositica, 489 Pisidium, 336, 343, 359, 360, 362, 368, 369, 370, 382, 383, 390, 399 amnicum, 348 annandalei, 369 casertanum, 348, 360, 369, 370, 383, 384, 389, 396 clarkeanum, 369 compressum, 345 conventus, 383 corneum, 389 crassum, 389 subtruncatum, 384 variabile, 345, 369 walkeri, 345, 346 Placobdella, 469, 488 costata, 481 hollensis, 468, 472, 488, 491 montifera, 488, 490, 491 mulilineata, 488, 492 nuchalis, 488, 490, 491 ornata, 472, 488, 491 papillifera, 474, 479, 486, 488, 492 parasticia, 472, 488, 491 pediculata, 474, 488, 491 phalera, 488 picta, 488 translucens, 488, 492 Plagiopyla, 89 nasuta, 88 Plagiopyxis, 74 callida, 75 1051 Planaria, 167 Planobella (Helisoma), 9 Planolineus, 174 Planorbella, 318 trivolvis, 313 Planorbis contortus, 310 vortex, 306 Planorbula, 318 armigera, 304, 317 companulata, 319 trivolvis, 319 Platicrista, 529, 531, 532, 544 Platycentropus, 706 Platychirograpsus spectabilis, 960 Platycola, 81 longicollis, 80 Platyhydracarus, 559, 644 juliani, 603, 627 Plectocythere, 830, 836 Plectomerus, 402, 406 dombeyanus, 409, 413 Plectrocnemia conspersa, 824 Plectus, 256, 263, 265, 276 Pleocola, 538 Plethobasus, 402, 410 cyphyus, 406 Pleurobema, 402, 403, 411, 414 altum, 357 avellanum, 357 bourianum, 357 chattanoogaense, 357 clava, 406 collina, 367 ﬂavidulum, 357 hagleri, 357 hanleyianum, 357 johannis, 357 murrayense, 357 nucleopsis, 357 rubellum, 357 troschelianum, 357 verum, 357 Pleurocera, 314, 323 acuta, 322 Pleuromonas, 71 jaculans, 72 Pleuronema, 87 coronatum, 86 Pleurotrocha, 245 Pleuroxus, 847, 859, 874, 878, 878 aduncus, 848 denticulatus, 856, 878 laevis, 869 procurvus, 856, 878, 879 trigonellus, 848 Plistophora aerospora, 228 Ploesma, 245, 247 Plumatella, 11, 510, 511, 512, 513, 514 bushnelli, 519, 521 casmiana, 508, 512, 513, 519, 521, 522 emarginata, 511, 512, 513, 519, 521 fruticosa, 511, 512, 513, 519, 520 fungosa, 511, 512, 513, 514, 522 nitens, 511, 512, 521 1052 Taxonomic Index Plumatella, (continued) nodulosa, 522 orbisperma, 512, 519 recluse, 511, 512 repens, 509, 511, 512, 513, 516, 519, 522 reticulata, 512, 519 rugosa, 522 simirepens, 522 vaihiriae, 511, 512, 513, 519, 521 Podiceps nigricollis, 39 Podon, 852, 857, 859, 887 leuckartii, 886 Podophyra, 79 ﬁxa, 79 Podura, 717, 730 Polyarthra, 198, 201, 205, 208, 224, 227, 228, 245 dolichoptera, 223 major, 232 remata, 222 vulgaris, 223, 232 Polyartmiella, 890, 895 hazeni, 888 Polycelis, 161, 167 coronata, 168 tenuis, 161 Polycentropus, 13, 705 Polychaos timidum, 53 Polymerurus, 189, 190 Polymesoda, 397, 399 caroliniana, 399 Polypedilum, 690, 730 Polyphemus, 852, 857, 861, 862, 887 pediculus, 886 Polypodium, 6, 135, 149, 150, 151, 152 hydriforme, 140, 148 – 150 Polytoma, 71 Polytomella, 71 citri, 66 Pomacea, 9, 303, 323 paludosa, 324 Pomatiopsis, 320 lapidaria, 322 Pompholyx, 237, 239 Pontoporeia afﬁnis, 786, 789 femorata, 786 hoyi, 786 Popenaias, 402 popei, 406, 414 Porohalacarus, 648 Porolohmannella, 632, 648 Potamalpheops, 961 Potamilus, 363, 402, 410, 412, 414, 415 alatus, 406 purpuratus, 412 Potamocypris, 781, 815, 820, 822, 823, 830, 832 pallida, 816 smaragdina, 813, 815, 831 variegata, 819, 821 Potamogeton, 481, 986 Potamonemertes, 174 Potamopyrgus, 309 jenkinsi, 306 Potamothrix, 439, 453 moldaviensis, 439 Pottsiela erecta, 512, 517, 518 Prionchulus, 278 Prionocypris canadensis, 829 glacialis, 829 Prismatolaimus, 274 Pristina, 439, 450 Pristinella, 439, 450 Proales, 202, 243 gigantea, 202 sordida, 217 werneckii, 243 Probopyrus pandalicola, 776 Probythinella, 320 Procambarus, 963, 964, 966, 967, 968, 972, 980, 982, 983, 988, 989 acutissimus, 987 acutus, 959, 977 clarkii, 972, 973, 975, 981, 984, 985 enoplosternum, 957 fallax, 978 gracilis, 978, 980, 987 hagenianus, 973 lunzi, 953, 957 paeninsulanus, 959, 978, 986 pecki, 987 primaevus, 964 pubescens, 972 pygmaeus, 985 simulans, 957 spiculifer, 973 tenuis, 960 versutus, 957 zonangulus, 972, 973 Procaris, 961 Procladius, 734, 735 Procotyla, 169 ﬂuviatilis, 168 Prodesmodora, 265, 276 Progomphus, 666 Proichthydium, 185, 190 Proichthyoides, 185 Promenetus, 307, 318 exacuous, 304, 319 Prorhynchella, 169 minuta, 171, 180 Prorhynchus, 157, 161, 164, 166 stagnalis, 161, 179 Prorodon, 67, 85 teres, 86 Prosimulium, 690, 730 Prosotoma, 6 Prostoma, 7, 172, 174, 175, 176, 265 asensoriatum, 175 canadiensis, 174, 175, 176 eilhardi, 174, 176 graecense, 174, 176 rubrum, 175 Protilimnesia, 556, 617, 643 Protoascus, 170 wisconsinensis, 180 Protocaris, 867 Protoneura, 701 Protoplasa, 716 Protzia, 554, 565, 571, 578, 608, 633, 640 Psammonitocrella, 937 boultoni, 950 longifurcata, 950 Psammoryctides, 453, 454 barbatus, 439 Psephenus, 687, 713, 730 Pseudechiniscus, 529, 530, 538, 542 Pseudiron, 699 Pseudobiotus, 529, 531, 532, 533, 538, 539, 543 augusti, 536 kathmanae, 536 macronyx, 537 megalonyx, 536, 537 Pseudocandona, 822, 830, 833 albicans, 822 Pseudocentroptiloides, 662 Pseudochordodes, 292 Pseudochydorus, 874, 878 globosus, 862, 879 Pseudodiaptomus, 934, 934 Pseudodifﬂugia, 75 gracilis, 75 Pseudodiphascon, 529, 531, 532, 533, 534, 543 Pseudofeltria, 557, 638, 645, 647 multpora, 596, 620 Pseudohexapodibius, 529, 531, 532 Pseudohydryphantes, 564, 579, 639 Pseudomermis, 278 Pseudomicrothorax, 67, 89 agilis, 80 Pseudomoina, 883 lemnae, 885 Pseudophaenocora, 170 sulﬂophila, 180 Pseudoploesma, 245, 247 Pseudoprorodon, 85 ellipticus, 86 Pseudosida, 874 bidentata, 872 Pseudosuccinea, 320 collumella, 304, 306, 310, 321 Pseudotorrenticola, 555 chiricahua, 614 Psilotreta, 672, 675, 706 Psilotricha, 83 acuminata, 82 Psychoda, 715 Psychomyia, 672, 705 Pternarcys, 730 Pterodrilus, 460 alcicornis, 460 Ptychobranchus, 402, 406, 409, 412, 415 Ptychoptera, 716 Ptygura, 202, 214, 223, 224, 237 barbata, 238 beauchampi, 205, 214 Taxonomic Index mucicola, 238 Punctodora, 276 Pycnopshche, 672 Pyganodon, 363, 402, 403, 408, 412 cataracta, 367, 376, 382, 384 grandis, 348 – 352, 358 – 361, 366, 373, 375, 386, 407 Pyrgulopsis, 303, 320 Pyxicola, 81 afﬁnis, 80 Pyxidicula, 74 operculata, 75 Quadrula, 9, 363, 402, 410, 413, 414 cylindrica, 410 quadrula, 407 tuberosa, 357 Quadrulella, 74 symmetrica, 75 Quincuncina, 402, 407, 413 Quistadrilus, 439, 452 Radiospongilla, 117, 130 cerebellata, 124, 125, 130 crateriformis, 124, 125, 130 Radix, 320 auricularia, 321 Ramajendas, 529, 531, 532, 538, 539 Ramazzottius, 529, 531, 532, 537, 543 Rana clamaitans, 825 temporaria, 288 Rangia cuneata, 385, 386, 397 Raphidiophrys, 78 elegans, 77 Rectocephala, 169 Redudasys, 185 fornerise, 182 Regina alleni, 973 Reticulomyxa, 77 ﬁlosa, 76 Rhabditolaimus, 274 Rhabdolaimus, 278 brachyuris, 263 Rhabdostyla, 81 pyriformis, 80 Rhadinocythere, 830, 837 Rhagovelia, 678 Rhantus, 684 Rhapinema, 320 Rheocyclops, 937 carolinianus, 949 hatchiensis, 949 talladega, 949 virginianus, 949 Rheumatobates, 678 Rhinoglena, 215, 224, 243 fertoensis, 221 Rhithropanopeus harrisii, 960 Rhizodrilus, 436, 439 lacteus, 453 Rhodacmea, 316 rhodacme, 317 Rhyacodrilus, 439, 454 brevidentatus, 454 falciformis, 438, 455 sodalis, 440 Rhyacophila, 706, 730 Rhynchelmis, 448 brooksi, 448, 449 Rhyncholimnochares, 553, 563, 569, 570, 574, 578, 579, 607, 634, 639 kittatinniana, 587 Rhynchomesostoma, 162, 170 rostratum, 171, 180 Rhynchomonas, 71 nasuta, 72 Rhynchoscolex, 157, 158, 162, 165, 172 pegephilum, 159 platypus, 179 simplex, 165, 179 tauricum, 179 Rhynchotalona, 874, 876 falcata, 875 Richtersius, 529, 531, 532 Ripistes, 438, 451 parasita, 450 Romanomermis, 280 culicivorax, 269 Rotaria, 198, 235 Rutripalpus, 555, 612, 641 Saccamoeba, 74 lucens, 73 Saﬁlippodytes, 684 Sagittocythere, 830, 837 Salda, 709 Salmasellus steganothrix, 792 Salmo trutta, 474 Salpingoeca, 71 fusiformis, 72 Salvelinus alpinus, 824 Saprodinium, 83 dentatum, 82 Sarscypridopsis, 830, 832 Sarsilatona, 874 serricaudata, 872 Sathodrilus, 460 attenuatus, 461 carolinensis, 461 hortoni, 461 Saurocythere, 830, 836 Scapholeberis, 34, 143, 850, 859, 881 kingi, 226 rammneri, 880 Scapulicambarus, 967 Scelorchilus rebecula, 290 Scenedesmus, 225, 396, 870, 931 costato-granulatus, 229 Sciomyza, 690 Scottia, 830, 833 Scutolebertia, 555, 642 trinitensis, 615 1053 Scyphidia, 81 physarum, 80 Scyphozoa, 145 Seinura, 270 Seison, 233 Selenastrum, 396, 870 Semigordionus, 292 Senecella, 917, 934 calanoides, 923, 948 Sepedon, 690, 715 Serratella, 662 Setnonema, 699 Setodes, 672 Setopus, 191 dubius, 187 Setvena, 669 Sialis, 707, 730 fuliginosa, 824 Sida, 850, 851, 852, 857, 859, 861, 873 crystallina, 872 Simocephalus, 143, 847, 850, 859, 881 serrulatus, 880 vetulus, 880 Simposonaias, 402 ambigua, 363, 407, 411 Simulium, 26, 690, 716, 730, 740, 760 Sinantherina, 8, 211, 212, 215, 224, 227, 237 socialis, 212, 214, 216, 217, 228, 232 spinosa, 227 Sineportella forbesi, 512, 518 Sinobosmina, 881, 882 fatalis, 882 Sinocalanus, 935 doerri, 948 Sinodiaptomus, 935 sarsi, 948 Siolineus, 174 Siphlonurus, 699 Siphloplecton, 699 Siphonophanes grubei, 892 Siqara, 709 Siskiyouthyas, 554, 640 Sisyra, 730 Skistodiaptomus, 935 bogalusensis, 948 carolinensis, 948 mississippiensis, 948 oregonensis, 948 pallidus, 948 pygmaeus, 948 reighardi, 948 sinuatus, 948 Slavina, 452 appendiculata, 451 Sminthurides, 730 Soldanellonyx, 648 Solenopsis invicta, 373, 385 Soliperda, 669 Somatochlora, 666 Somatogyrus, 320 Somersiella, 961 Sparganophilus, 446 1054 Taxonomic Index Spathidium, 85 spathula, 84 Specaria, 452 Spelaedrilus, 449 multiporus, 449 Sperchon, 552, 555, 572, 579, 612, 635, 642 glandulosus, 588 Sperchonopsis, 555, 635, 642 ecphyma, 561, 562 nova, 612 Sphaerium, 343, 359, 360, 362, 368, 383, 390, 400 corneum, 348, 373, 400 fabule, 369, 400 nitidum, 400 occidentale, 350, 351, 400 simile, 348, 400 striatinum, 345, 346, 347, 369, 381, 383, 388, 389, 400 Sphaeroeca, 71 volvox, 72 Sphaerophyra, 79 magna, 79 Sphagnum, 241, 244 Sphalloplana, 167 Sphenoderia, 76 lenta, 76 Spilochlamys, 320 Spinalona, 847 Spirosperma, 436, 452, 453 Spirostomum, 55, 85 minus, 84 Spirulina, 827 Spongilla, 129 alba, 106, 107, 125, 129 aspinosa, 125, 129 cenota, 125, 126, 129 lacustris, 98, 99, 103 – 114, 117, 126, 128, 129 Spumella, 66 Squalorophyra, 81 macrostyla, 79 Squatinella, 241 Stachyamoeba, 73 lipophora, 73 Stagnicola, 320 catascopium, 314 elodes, 314, 321 emarginata, 314 palustris, 314 Stenelmis, 13, 684, 712, 713 Stenochironomus, 690 Stenocypria longicomosa, 829 Stenocypris, 830, 833 major, 825 Stenonema, 662 Stenoperla prasina, 289 Stenophylax, 288 Stenophysa, 318, 319 Stenostomum, 157, 159, 161, 163, 165 anatirostrum, 179 anops, 179 arevaloi, 179 beauchampi, 166, 179 brevipharyngium, 166, 179 ciliatum, 179 cryptops, 179 glandulosum, 166, 179 grande, 179 kepnere, 179 leucops, 158, 162, 166, 179 mandibulatum, 179 membranosum, 179 occultum, 179 pegiphilum, 179 predatorium, 161, 179 pseudoacetabulum, 179 simplex, 179 temporaneum, 179 tuberculosum, 179 unicolor, 179 uronephrium, 179 ventronephrium, 179 virginianum, 179 Stentor, 5, 55, 65, 83 polymorphous, 84 Stephanella, 514 hina, 511, 518, 519 Stephanoceros, 214, 224, 235 Stephanodiscus, 974 Stephensoniana, 450 Stichotricha, 83 aculeata, 82 Stictotarsus, 684 Stokesia, 87 vernalis, 88 Stolonicyclops, 936 heggiensis, 949 Strandesia, 830, 833 canadensis, 822, 829 Stratospongilla penneyi, 126, 129 Streblocerus, 883 serricaudatus, 884 Strelkovimermis, 280 Streptocephalus, 891, 892, 894, 897 dichotomus, 893 mackini, 891, 893 seali, 890, 891 texanus, 888 Striobia, 320 Strobilidium, 62 Strombidinopsis, 83 setigera, 82 Strombidium, 83 viride, 82 Strombidum, 63 capitatum, 60 Strombidium, 83 gyrans, 82 Strongylidium, 82 crassum, 82 Strongylostoma, 170, 172 elongatum, 180 radiatum, 180 simplex, 171 Strophitus, 402, 403, 412 undulatus, 407, 408 Strophopteryx, 703 Stygalbiella, 558, 646 yavapai, 621 Stygameracarus, 559, 644 cooki, 627 Stygobromus, 786, 787, 792 canadensis, 792 Stygohalacarus, 648 Stygomomonia, 558, 635, 643 mitchelli, 601, 625 riparia, 625 Stygonitocrella, 937 sequoyahi, 950 Stygothrombium, 553, 585, 588, 606, 634, 638 Stylaria, 438 lacustris, 450, 451 Stylochaeta, 7, 191 Stylocometes, 81 digitalis, 79 Stylodrilus, 443, 449 californianus, 449 heringianus, 440, 441, 449 sovaliki, 450 Stylonyhcia, 81 mytilus, 82 Styloscolex, 449 opisthothecus, 450 Styraconyx hallasi, 529, 538 Submiraxona, 558, 647 gilana, 622 Suomina, 165 turgida, 165 Suphisellus, 684 Suragina, 693 Sympetrium, 666 Syncaris, 963, 964, 983, 985, 988 paciﬁca, 961, 969, 983 pasadenae, 961 Synchaeta, 195, 198, 201, 202, 210, 223, 227, 228, 245, 247, 928 cecelia, 232 oblongata, 215, 222, 226 pectinata, 209, 215, 222, 228 Synedra, 974 Synendotendipes, 690 Tachidus, 937 Tachopteryx, 701 Taeniopteryx, 669, 703 Taeniorhynchus, 732 Tanytarsus, 730, 735 Taphromysis louisiannae, 786, 788 Tartarothyas, 554, 564, 571, 578, 611, 633, 641 Tasserkidrilus, 454 Tegeocranellus, 649 Telmatodrilus, 452, 455 onegensis, 452 vejdovskyi, 436, 454 Temora, 923 Tenagodrilus musculus, 450 Tendipes pectinatellae, 513 Taxonomic Index Teneridrilus mastix, 454, 455 Teratocephalus, 276 Teretifrons, 874 Testudacarus, 555, 591, 636, 641 americanus, 614 Testudinella, 237, 239 Tetanocera, 690 Tetrahymena, 43, 45, 48, 51, 57, 60, 61, 62, 65, 67, 89 pyriformis, 45, 58, 88 Tetrakentron, 538 Tetramermis, 278 Tettodrilus, 460 Teutonia, 552, 555, 569, 579, 612, 635, 641 lunata, 589 Thamnocephalus, 895 platyrus, 888 Thaumalea, 690, 716 Thecacineta, 78 cothurniodes, 79 Thecamoeba, 53, 67, 74 sphaeronucleolus, 74 Theodoxus, 307 Theristus, 265 pertenuis, 263 Thermacarus, 555, 578, 633, 641 nevadensis, 565, 611 Thermastrocythere, 830, 837 Thermobatynella adami, 781 Thermocyclops, 929, 936 crassus, 949 inversus, 949 parvus, 949 tenuis, 949 Thermonectus, 684 Thermosbaena mirabilis, 781 Thermosphaeroma subequalum, 781 thermophilum, 781, 792 Thermozodium, 529 esakii, 529, 539 Theromyzon, 474, 488, 493 biannulatum, 488 bifarium, 488, 494 maculosum, 488, 493 rude, 475, 488, 494 sexoculatum, 488 tessulatum, 477, 481, 488, 493 trizonare, 474, 477, 478, 479, 481, 486, 488, 492, 493 Thiara, 323 granifera, 322 Thinodrilus, 448 Thraulodes, 662 Thulinia, 529, 531, 532, 533, 539, 541, 543 Thuricola, 81 folliculata, 80 Thyas, 554, 560, 569, 572, 578, 579, 581, 633, 641 barbigera, 569 rivalis, 611 stolli, 565, 611 Thyasella, 554, 640 Thyasides, 554, 611, 633, 641 Thyopsella, 554, 640 dictyophora, 610 occidentalis, 610 Thyopsis, 554, 640 cancellata, 610 Thyopsoides, 554, 640 plana, 610 Tiguassu, 442 Tillina, 89 magna, 88 Timpanoga, 662 Tintinnidium, 83 ﬂuviatile, 82 Tintinnopsis, 83 cylindricum, 82 Tiphys, 557, 576, 577, 579, 619, 638, 648 americanus, 574, 596 ornatus, 596 Tipula, 730, 740, 760 Tobrilus, 265, 275, 278 gracilis, 263 grandipapellatus, 263 Tokophyra, 81 quadripartita, 63, 64, 79 Tonnacypris, 823, 830, 833 glacialis, 821, 829 Torrenticola, 552, 555, 572, 579, 592, 614, 636, 641 Toxolasma, 402, 408 parvus, 351, 375 texasiensis, 348, 353, 382, 407 Trachelius, 85 ovum, 86 Trachelophyllum, 85 apiculatum, 84 Trajancypris, 828, 830, 833 Tramea, 701 Traverella, 662 Trepobates, 678 Trepomonas, 57 Triannulata, 460 magna, 460 Trianodes, 672 Tribelos, 690 Trichamoeba, 74 cloaca, 73 Trichocera, 244, 246 pusilla, 205, 217 rattus, 202, 224 Trichocorixa, 678, 730 Trichodina, 81 pediculis, 80 Trichodrilus, 443, 449 allegheniensis, 448 Trichogramma, 730 Trichophyra, 79 epistylidis, 79 Trichothyas, 554, 640 muscicola, 609 Trichotria, 243, 244 Tricorythodes, 698 Trilobus, 265 1055 Trinema, 76 enchelys, 76 Triops, 15, 889, 890, 891, 892, 894, 900 longicaudatus, 899 Tripyla, 265, 278 Tritogonia, 402, 407 verrucosa, 409, 413 Troaculus, 229 Trochodrilus, 438 Trochosphaera, 237, 239 Trochospongilla, 120 horrida, 106, 126, 127, 130 leidii, 127, 129 pennsylvanica, 106, 108, 127, 130 Troglocambarus, 964, 966, 967, 968, 988 maclanei, 966, 976 Troglocubanus, 961 Tropisternus, 684, 711, 712, 713 Tropocyclops, 227, 922, 936 extensus, 949 jerseyensis, 949 prasinus, 917, 924, 927, 949 Truncilla, 402, 411, 414 truncata, 343, 407 Trypanosoma, 52 Tubifex, 163, 439, 454 tubifex, 437, 438, 439, 445, 455 Tubiﬁcoides benedii, 437, 439 Tulotoma, 303, 323 magniﬁca, 324 Turaniella, 89 vitrea, 88 Turbanella hyalina, 187 Tvetenia, 690 Tylenchus, 270 Tylocephalus, 263 Typha, 816 Typhlatya, 961 Typhloplana, 162, 164, 170 viridata, 158, 160 Typhloplanella, 170 halleziana, 180 Tyrrellia, 556, 593, 617, 636, 643 Uchidastygacarus, 559, 579, 643 acadiensis, 626, 630 ovalis, 626 Uglukodrilus, 460 Uncinais uncinata, 450 Uncinocythere, 830, 837 Undula, 185, 189, 190 Unio, 363 pictorum, 341, 367, 382 tumidus, 348, 367, 376, 391 Uniomerus, 402, 412, 415 caroliniana, 408 tetralasmus, 351, 373, 375, 407, 412 Unionicola, 384, 556, 567 – 569, 574, 577, 579, 581, 582, 594, 618, 637, 648 crassipes, 575 intermedia, 577 Urceolaria, 81 mitra, 80 1056 Taxonomic Index Urceolus, 71 cyclostomas, 66 Urnatella gracilis, 11, 515, 516, 517 Urocentrum, 87 turbo, 88 Uroglena, 71 americana, 72 Uroleptus, 81 piscis, 82 Uronema, 87 griseolum, 86 Urostyla, 81 grandis, 82 Urotricha, 85 farcta, 84 Urozona, 87 bütschlii, 86 Utaxatax, 555, 569, 634, 642 newelli, 589 ovalis, 613 Utricularia, 231 Utterbackia, 402, 403, 409, 413 imbecillis, 384, 407 Vaginicola, 81 ingenita, 80 Valkampﬁa, 73 avaria, 73 Valvata, 309, 320 sincera, 321 tricarinata, 310, 321 Vampyrella, 75 lateritia, 76 Vanella miroides, 74 Varichaetadrilus, 453, 454, 454 Varsoviella kozminski, 159 Vasicola, 85 ciliata, 84 Vaucheria, 243 Vejdovskyella, 438, 451, 452 Velesunio ambiguus, 348 Venustaconcha, 402 ellipsiformis, 407, 412 Vexillifera telemathalassa, 73 Vibrio cholerae, 929 Victorella pavida, 512, 518 Villosa, 364, 402, 408, 411, 412, 414, 415 constricta, 408 iris, 382, 383 villosa, 407 Viviparus, 303, 306, 323 georgianus, 305, 306, 324 subpurpureus, 309 Volsellacarus, 559, 636, 644 sabulonus, 626, 630 Volvox, 244 Vorticella, 5, 55, 64, 81 campanula, 80 Vorticifex, 318 effusa, 319 Wandesia, 554, 560, 565, 569, 570, 571, 578, 579, 608, 633, 641 thermalis, 569 Wettina, 557, 579, 637, 647 octopora, 619 ontario, 595 Wlassicsia, 883 kinistenensis, 885 Woolastookia, 558, 637, 646 pilositarsa, 599, 623 setosipes, 599 Xerobiotus, 529, 531, 532, 537 pseudohufelandi, 537 Xiphocentron, 705 Xironodrilus, 460 Xironogiton, 459 instabilis, 460 victoriensis, 458 Yachatsia, 559, 645 mideopsoides, 626 Yagerocaris, 961 Ylodes, 672 Zoothamnium, 59, 81 arbuscula, 80 Zschokkea, 554, 611, 633, 641

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Ecology and classification of North American Freshwater Invertebrates

Ecology and classification of North American Freshwater Invertebrates

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