Alternative overwintering strategies in an Antarctic midge: Freezing vs. cryoprotective dehydration

Summary: Cryoprotective dehydration is a relatively new addition to our understanding of freeze avoidance strategies employed by polar invertebrates. Although the underlying cellular processes associated with this strategy are similar to those of freeze tolerance, little is known about potential trade-offs of overwintering in these physiological states. This study compares the potential of larvae of the terrestrial midge Belgica antarctica (Diptera, Chironomidae) to overwinter in these two states. As the only insect with the capacity to tolerate freezing and to cryoprotectively dehydrate, it is an ideal model to compare the benefits and costs of these strategies. Compared to summer-acclimated larvae, supercooling points of winter-acclimatized larvae were significantly depressed and were lower than observed minima for their microhabitat temperatures. Thus, if larvae avoid inoculative freezing from environmental ice, they could remain unfrozen via cryoprotective dehydration. Both frozen and cryoprotectively dehydrated larvae readily survived a 32-day exposure to simulated overwintering temperatures. Freezing had little effect on larval body water content and haemolymph osmolality. In contrast, cryoprotective dehydration at -5 °C resulted in a progressive loss of body water, ultimately reducing larval water content by 62%. This level of dehydration corresponded to an increase in haemolymph osmolality to c. 2750 mOsm kg^-1, depressing the haemolymph melting point to -4·9 °C. Freezing and cryoprotective dehydration resulted in distinctly different patterns of glycogen breakdown. Whereas the glycogen content decreased only during the first 14 days in cryoprotectively dehydrated larvae, frozen larvae continued to break down glycogen throughout the 32-day subzero exposure. However, after recovery at 0 °C for 5 days, glycogen levels were similar in these two groups, as were the levels of total lipids. Our results indicate that freezing and cryoprotective dehydration are both effective in promoting winter survival of larvae, with surprisingly few differences in energetic costs. Whether larvae freeze or become cryoprotectively dehydrated ultimately depends on the hydric condition of their microhabitat. The physiological flexibility of B. antarctica to overwinter in these alternative states likely contributed to its range distribution that extends further south than any other free-living insect.