Survival and energetic costs of repeated cold exposure in the Antarctic midge, Belgica antarctica: A comparison between frozen and supercooled larvae

In this study, we examined the effects of repeated cold exposure (RCE) on the survival, energy content and stress protein expression of larvae of the Antarctic midge, Belgica antarctica (Diptera: Chironomidae). Additionally, we compared results between larvae that were frozen at -5°C in the presence of water during RCE and those that were supercooled at -5°C in a dry environment. Although >95% of larvae survived a single 12 h bout of freezing at -5°C, after five cycles of RCE survival of frozen larvae dropped below 70%. Meanwhile, the survival of control and supercooled larvae was unchanged, remaining around 90% for the duration of the study. At the tissue level, frozen larvae had higher rates of cell mortality in the midgut than control and supercooled larvae. Furthermore, larvae that were frozen during RCE experienced a dramatic reduction in energy reserves; after five cycles, frozen larvae had 25% less lipid, 30% less glycogen and nearly 40% less trehalose than supercooled larvae. Finally, larvae that were frozen during RCE had higher expression of hsp70 than those that were supercooled, indicating a higher degree of protein damage in the frozen group. Results were similar between larvae that had accumulated 60 h of freezing at -5°C over five cycles of RCE and those that were frozen continuously for 60 h, suggesting that the total time spent frozen determines the physiological response. Our results suggest that it is preferable, both from a survival and energetic standpoint, for larvae to seek dry microhabitats where they can avoid inoculative freezing and remain unfrozen during RCE.