Int. J. Radiat. Biol., Vol. 82, No. 8, August 2006, pp. 587 – 592 Biological effects of anhydrobiosis in an African chironomid, Polypedilum vanderplanki on radiation tolerance MASAHIKO WATANABE1, TETSUYA SAKASHITA2, AKIHIKO FUJITA1, TAKAHIRO KIKAWADA1, DAIKI D. HORIKAWA1, YUICHI NAKAHARA1, SEIICHI WADA2, TOMOO FUNAYAMA2, NOBUYUKI HAMADA2,3, YASUHIKO KOBAYASHI2 & TAKASHI OKUDA1 1 Department of Physiology and Genetic Regulation, National Institute of Agrobiological Sciences (NIAS), Tsukuba, Microbeam Radiation Biology Group, Japan Atomic Energy Agency (JAEA), Takasaki, Gunma, and 3Department of Quantum Biology, Gunma University Graduate School of Medicine, Maebashi, Gunma, Japan 2 (Received 21 December 2005; revised 22 February 2006; accepted 15 June 2006) Abstract Purpose: Anhydrobiotic organisms are known to have an extremely high tolerance against a range of stresses. However, the functional role of anhydrobiosis in radiation tolerance is poorly understood, especially in development following irradiation. The present study aims to evaluate effects of anhydrobiosis on radiation tolerance in an anhydrobiotic insect, Polypedilum vanderplanki. Materials and methods: Larval survival (48 h), anhydrobiotic ability, metamorphosis and reproduction after exposure to 1 – 9000 Gy of gamma-rays at the larval stage were compared between anhydrobiotic (dry) and normal (wet) phases. Results: Wet larvae were killed in a dose-dependent manner at doses higher than 2000 Gy, and all died within 8 h after 4000 Gy exposure. In contrast, dry larvae survived even 5000 Gy, and some of them still tolerated 7000 Gy and were alive at 48 h after rehydration. Moreover, greater radiotolerance of dry larva, compared to wet ones, was demonstrated in terms of metamorphoses. However, anhydrobiosis did not protect against radiation damage in terms of producing viable offspring. Conclusion: These results indicate that anhydrobiosis enhances radiotolerance, resulting in increases of successful metamorphoses. Keywords: Polypedilum vanderplanki, anhydrobiosis, gamma-rays, radiation tolerance, biological effect Introduction Anhydrobiosis is a term referring to the dehydrated biological state, and anhydrobiotic organisms have an extremely high tolerance against various types of stress such as desiccation, temperature extremes and vacuum (Clegg 2001, Watanabe 2006). In addition, anhydrobiotic invertebrates have been shown to have extremely high radiation tolerance: for example, encysted dry embryos of Artemia can hatch with the median inhibitory dose (ID50) of 5000 Gy of gamma-rays (Iwasaki 1964a). A tardigrade, Macrobiotus areolatus, can survive 1 h after around 570,000 R (approximately 5000 Gy, LD50) of X-rays (May et al. 1964). Radiotolerance in the former decreased as water content increased (Iwasaki 1964b) or as embryogenesis progressed (Iwasaki 1964c). In these species, however, it has not been examined whether individuals irradiated at high doses undergo normal metamorphoses and reproduction, and how the anhydrobiotic state affects these events after irradiation. Addressing these questions is important in order to elucidate physiological mechanisms of high radiation tolerance in anhydrobiotic invertebrates. An African chironomid, Polypedilum vanderplanki, is the only insect species capable of anhydrobiosis (Hinton 1951, 1960). These anhydrobiotic larvae can tolerate exposure to extremely high (1038C) and low (72708C) temperatures and submersion in pure ethanol (Hinton 1960), and thus are expected to have high radiation tolerance, like other anhydrobiotic invertebrates. In the present study, we compared Correspondence: Takashi Okuda, Department of Physiology and Genetic Regulation, National Institute of Agrobiological Sciences (NIAS), Ohwashi 1-2, Tsukuba, Ibaraki 305-8634, Japan. E-mail: oku@affrc.go.jp ISSN 0955-3002 print/ISSN 1362-3095 online Ó 2006 Informa UK Ltd. DOI: 10.1080/09553000600876652 588 M. Watanabe et al. short-term larval survival, metamorphosis and reproduction after larval gamma irradiation between anhydrobiotic and hydrated larvae. We also examined whether P. vanderplanki larvae retain the ability to re-enter anhydrobiosis after high doses of gamma-rays. Materials and methods Insect rearing and preparation A stock culture of P. vanderplanki was established using anhydrobiotic larvae collected from rock pools in Nigeria in 2000, and reared for successive generations under controlled light (13 h light: 11 h dark) and temperature (278C). Larvae were reared in a glass container (600 ml) with water (depth 5 – 7 cm) on milk (2%, v/v)-agar (1%, w/v) as food, and sand for their tubular nests (Watanabe et al. 2005). Wet (normally hydrated state) and dry (anhydrobiotic state) larvae at the final larval instar were used for all experiments. The dry samples were prepared as follows: around 70 larvae were put into a mixture of soil (3.6 g) and distilled water (4 ml) in a glass Petri dish (diameter 65 mm, height 20 mm). The dish was sealed with vinyl tapes to inhibit water evaporation, and incubated for 2 days at around 258C. During the pre-incubation, the larvae construct their tubular nest using soil and their saliva. The dish bottom was then placed into a desiccation box (200 6 250 6 300 mm) with 1 kg of silica gel, generating a relative humidity (r.h.) of 55%, where larvae were gradually dehydrated over 3 – 5 days. The dehydrated larvae in the soil nests were stored in the desiccation box at ambient temperatures around 258C until use. Irradiation Approximately 200 dry larvae within their tubular nests were put into a plastic vial (diameter, 15 mm; height, 50 mm) without water, whereas wet larvae were put into comparable vials with 5 ml of distilled water. Both dry and wet samples were irradiated with 1 – 9000 Gy of gamma-rays from a Cobalt-60 source at 60 Gy/min. Control samples were sham-irradiated and manipulated in parallel with the test samples. Larval survival, metamorphosis and reproduction after irradiation Effects of gamma radiation of larvae on subsequent development and reproduction were examined. Wet larvae were transferred into 20 ml of distilled water in a plastic Petri dish (diameter 90 mm, height 20 mm) immediately after irradiation, and survival was examined at 2, 8, 24 and 48 h post-irradiation. Short-term preservation of irradiated dry larvae at least up to 24 h did not affect survival rate (data not shown). Irradiated dry larvae were put into distilled water in similar Petri dishes within 8 h after irradiation, and survival was checked at 2, 8, 24 and 48 h after rehydration. Physiological differences between rehydrated dry larvae and wet larvae were likely to be negligible, because dry larvae usually start to move within 15 min after rehydration, and normally swim 1 h after rehydration (data not shown). Only larvae which moved their pharyngeal plunger and abdomen were counted as survivors. Each cohort was separately reared with water on a milk-agar diet in glass containers, and examined for pupation and emergence as adults over three months. Newly emerged adults of both sexes were transferred to a glass container (2500 ml) with a mesh cover. Mating succeeds only when a number of adult males and females take part in aerial swarming. Sex ratio (males/females) is usually biased to females (0.3 to 0.5). One adult female laid once an egg mass containing many eggs enveloped by gelatinous coats. Virgin females occasionally deposit an infertile egg mass. Each egg mass obtained was separately incubated for 2 days to count hatchlings from the egg mass. Anhydrobiosis ability after irradiation Whether irradiated larvae can successfully re-enter anhydrobiosis was examined. Immediately after 1000 – 7000 Gy of gamma-rays, wet and dry larvae were submerged into distilled water, and then incubated for 24 h. The survivors were further challenged by a series of desiccation stresses: 100% r.h., 76% r.h., and finally 5% r.h., each for 1 day. This procedure allows almost all intact larvae to recover from anhydrobiosis (Watanabe et al., unpublished data). Recovery from anhydrobiosis was examined at 48 h after rehydration. Results Larval survival To evaluate the radiation tolerance of wet and dry larvae, survival at 2 h after gamma-irradiation was analysed. As shown in Figure 1A, most wet larvae survived 2000 Gy of gamma-rays, and dose-dependent killing was observed at doses over 3000 Gy. In contrast, most of the dry larvae irradiated with 5000 Gy recovered from anhydrobiosis. After exposure to 6000 Gy, about half of the dry larvae started to move upon rehydration, whereas all wet ones were motionless. To our great surprise, even 9000 Gy irradiation did not kill all dry larvae immediately. This result clearly indicates that Effects of anhydrobiosis on radiotolerance 589 Figure 1. Effect of gamma irradiation (1000 – 9000 Gy) on percentage survival of dry larvae of P. vanderplanki at 2 h after rehydration and wet larvae at 2 h post-irradiation (A). The time course of survival after irradiation (wet) or rehydration (dry) in larvae irradiated at the dose of 2000 – 7000 Gy (B, dry; C, wet). The number of irradiated larvae ranges from 267 to 300. anhydrobiosis in P. vanderplanki is protective against radiation in terms of larval killing. The temporal kinetics of larval survival were different between dry and wet larvae (Figures 1B and 1C respectively). Survival of dry larvae irradiated with 3000 Gy was about 60% at 48 h after rehydration, nearly identical to the value at 2 h after rehydration of wet larvae irradiated with the same dose. All wet larvae irradiated with 4000 Gy died within 24 h, whereas 40% of their dry counterparts survived after 48 h. These results indicate that the lethal effect induced by gamma-irradiation was temporally arrested or delayed at least at 48 h after irradiation by the anhydrobiotic state, consistent with the previous findings in Artemia (Iwasaki 1964a, Iwasaki et al. 1974) Alternatively, radiation-induced damage might be reduced by anhydrobiosis. Metamorphosis A total of 2000 Gy of gamma-rays caused little lethal damage within 48 h after irradiation in both wet and dry samples (Figures 1B, 1C and 2A). However, this dose inhibited pupation and adult emergence, and the radiation-induced damage became obvious even at lower doses as development progressed (Figures 2B and 2C). From these data, we estimated the ID50 doses for pupation and adult emergence (Table I). The ID50 values were greater than 2000 Gy in the case of larval survival after 48 h, whereas the values were less than 500 Gy in terms of pupation, and less than 200 Gy in the case of adult emergence. Comparing the values of ID50 between wet and dry samples, it is clear that anhydrobiosis reduced damage by gamma-irradiation. Reproduction Newly-emerged adults were allowed to mate in a group and to oviposit. For both wet and dry samples, the rate of ovipositing females and number of progeny per adult female decreased as the radiation dose increased, and 200 Gy of irradiation almost completely inhibited production of viable progeny (Table II). The drastic decrease in fertility was correlated with an increase of completely infertile egg masses in which all eggs were infertile. Unexpectedly, the inhibitory dose for reproductive activity seems slightly higher in wet samples compared to dry ones, suggesting that anhydrobiosis did not reduce radiation damage associated with reproduction. Ability for anhydrobiosis Entry into anhydrobiosis is a complex physiological event accompanied by vigorous gene expression (Watanabe 2006). We examined whether P. vanderplanki larvae maintain the ability to enter anhydrobiosis after receiving a high dose of gamma-rays. At doses below 2000 Gy, anhydrobiotic ability was not affected in both dry and wet larvae (Table III). After 3000 Gy irradiation, wet larvae failed to successfully enter anhydrobiosis, whereas most of the irradiated 590 M. Watanabe et al. Figure 2. The percent of larval survival (A), pupation (B) and adult emergence (C) of dry and wet larvae of P. vanderplanki after gamma irradiation of 1 – 9000 Gy at the larval stage. The data were normalized by the value of non-irradiated control of dry or wet larvae. The number of irradiated larvae ranges from 179 to 256. Table I. Biological effects of gamma irradiation in larvae of P. vanderplanki. ID50 (Gy)a,b State Larval survivalc Pupation Adult emergence Dry Wet 4400 2000 460 160 160 70 a The median inhibitory dose; bThe value was calculated from rates of survival, pupation and adult emergence by regression analysis; c Larval survival was examined 48 h after rehydration. dry larvae were able to do so after rehydration. Moreover, some dry larvae successfully entered anhydrobiosis even after 6000 Gy irradiation. Thus, the effect of radiation on anhydrobiosis ability was similar to that observed for 48 h-larval survival. Discussion Radiotolerance of P. vanderplanki It is known that insects have much higher radiation tolerance than vertebrates and that the critical dose for instant death in insects ranges from several hundred to a few thousand Gy (Hirano 1964, Sparrow et al. 1967, Koyama 2001). We have shown here that wet P. vanderplanki larvae survive for at least 48 h after 2000 Gy of gamma-rays. This radiotolerance markedly increased after entry into an anhydrobiosis state: A number of dehydrated larvae recovered and moved transiently after extremely high doses of gamma-rays, up to 9000 Gy. In general, such extremely high doses of irradiation cause instant death in most animals, with some exceptions. For example, dry encysted embryos of Artemia can hatch after exposure to 5000 Gy of gamma-rays (Iwasaki 1964a), and only a few individuals of a tardigrade, M. areolatus, known as the most radiotolerant animal, can move immediately after 9000 Gy of X-rays (May et al. 1964). Thus, P. vanderplanki also has extreme radiotolerance comparable to or higher than this tardigrade, based on short-term survival after irradiation. To properly evaluate the radiotolerance in P. vanderplanki, it must be determined whether the irradiated larvae complete development and produce viable progeny. Few anhydrobiotic larvae succeeded in adult emergence after about 400 Gy of gamma irradiation, and no female adult produced viable progeny after 200 Gy. The dose for complete sterilization did not differ from that in nonanhydrobiotic insects (Hirano 1964, Koyama 2001). Unexpectedly, this anhydrobiotic species did not show high radiation tolerance based on development and reproduction after irradiation. Thus, animals that have been thought to be highly radiotolerant may not always be so in the strictest sense. Biological effect of anhydrobiotic status in P. vanderplanki Here we showed that anhydrobiosis seems to reduce radiation-induced damage, or delay its effects, in Effects of anhydrobiosis on radiotolerance 591 Table II. Reproductive activity of P. vanderplanki after gamma irradiation from 3 – 200 Gy at the larval stage. State Dose (Gy) The rate of ovipositing females (%)a Dry 0 3 10 30 50 100 58.8 66.9 40.4 28.1* 25.8* 33.3* Wet 0 3 10 30 50 100 200 54.3 60.6 53.3 47.4 29.2* 24.6* 13.9* Number of eggs per egg massb,c Rate of fertilized eggs in an egg mass 0% 0.1 to 70% 70.1 to 100% Number of progeny produced per femaleb,c 111.4 + 3.4 125.3 + 4.4 120.9 + 7.9 96.2 + 5.0* 110.4 + 12.3 90 7.4 16.8 21.1 69.2 78.9 100.0 13.9 13.7 8.8 18.0 5.3 0.0 78.7 69.5 70.2 12.8 15.8 0.0 90.0 + 4.6 88.1 + 5.9 84.6 + 9.1 17.5 + 5.6* 10.6 + 7.4* 0* 118.6 + 2.6 112.1 + 2.9 100.6 + 3.0* 104.0 + 2.8* 91.8 + 4.8* 98.8 + 4.4* 108.4 + 22.1 27.1 17.5 30.1 9.8 60.0 68.2 100.0 7.6 8.8 8.0 7.8 6.6 10.6 0.0 65.4 73.8 61.9 82.4 33.3 21.2 0.0 72.3 + 5.5 84.1 + 4.6 54.8 + 4.1* 82.9 + 4.0 26.5 + 6.1* 24.3 + 5.5* 0* a Asterisks indicate a significant difference at 1% level (w2 test) in comparison with the control one; bMean + SE; cAsterisks indicate a significant difference at 1% level (Mann-Whitney U test) in comparison with the control one. Table III. Effect of gamma irradiation on the ability entering anhydrobiosis in larvae of P. vanderplanki. Successful induction for anhydrobiosis after irradiation (%)a,b,c Dose (Gy) 1000 2000 3000 4000 5000 6000 7000 nd 111 110 121 80 89 33 18 Dry 100.0 100.0 81.6 33.0 23.7 5.6 0.0 x x x y y z z nd Wet 111 87 2 – – – – 89.1 x 92.1 x 0.0 z – – – – a The values were shown by recovery rate 48 h after rehydration; The rate was corrected by that of control dry and wet ones; c Means followed by the same letter are not significantly different (w2 test, p 4 0.01); dNumber of surviving larvae at the set of desiccation (24 h post-rehydration). b P. vanderplanki. The most conspicuous differences between dry and wet larvae are the contents of water and trehalose, i.e., wet larvae contain much more water (81.6% of the body weight) and a smaller amount of trehalose (51.5%), whereas anhydrobiotic larvae lose water down to 2.9% of their body weight and accumulate a large amount of trehalose, around 20% of their dry body weight (Watanabe et al. 2002, 2003). As a general model for the indirect action of radiation, intracellular water frequently produces OH radicals that cause serious damage including DNA breakage (Egami 1990, Sugawara 2004). We assume that the reduced water in dehydrated larvae produce less OH radicals due to radiation, and it seems likely that the abundant trehalose would act as a radical scavenger as suggested by Yoshinaga et al. (1997). Trehalose might also achieve a radioprotective effect, by stabilizing biomolecules by replacing water, or by sugar glass formation under dehydrated conditions (Burke 1986, Crowe et al. 1992, 1998, Sakurai & Inoue 2004). During metamorphosis, insects actively remodel their tissues from developmental to reproductive phases (Riddiford et al. 2003). Based on ID50 values for pupation and adult emergence in P. vanderplanki, more than half of the wet larvae irradiated with less than 70 Gy successfully underwent metamorphosis, indicating that their cells normally proliferated and differentiated into mature cells. At higher doses, gamma irradiation might cause critical DNA damage perhaps in stem and progenitor cells of adult tissues. Dry larvae irradiated with 160 Gy reached the adult stage, suggesting that the anhydrobiotic status protects DNA in regenerative cells from gamma radiation ranging from 70 – 160 Gy. On the other hand, dry larvae irradiated up to 2000 Gy, then hydrated, moved actively for at least 48 h and maintained an ability for anhydrobiosis. At doses over 2000 Gy, the anhydrobiotic status of larvae probably prevented serious damage that could cause instant death from radiation. Contrary to these developmental and metabolic events, anhydrobiosis did not protect against radiation damage in relation to production of viable progeny. The radiation-induced decline in the number of viable progeny seems to be due to an increasing number of completely infertile egg masses. Infertility of whole egg mass will be caused by several reasons such as increasing rate of unmated females, sterilization of sperm and some damage to female reproductive organs. It would be interesting to know the most radiation-sensitive target in reproduction. Further physiological and molecular analyses are needed to evaluate the issue of the protective effect of anhydrobiosis in P. vanderplanki larvae. 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