Rapid cold-hardening increases the freezing tolerance of the Antarctic midge Belgica antarctica

SUMMARY Rapid cold-hardening (RCH) is well known to increase the tolerance of chilling or cold shock in a diverse array of invertebrate systems at both organismal and cellular levels. Here, we report a novel role for RCH by showing that RCH also increases freezing tolerance in an Antarctic midge, Belgica antarctica (Diptera, Chironomidae). The RCH response of B. antarctica was investigated under two distinct physiological states: summer acclimatized and cold acclimated. Summer-acclimatized larvae were less cold tolerant, as indicated by low survival following exposure to -10°C for 24 h; by contrast, nearly all cold-acclimated larvae survived -10°C, and a significant number could survive -15°C. Cold-acclimated larvae had higher supercooling points than summer larvae. To evaluate the RCH response in summer-acclimatized midges, larvae and adults, maintained at 4°C, were transferred to -5°C for 1 h prior to exposures to -10, -15 or -20°C. RCH significantly increased survival of summer-acclimatized larvae frozen at -10°C for 1 h compared with larvae receiving no cold-hardening treatment, but adults, which live for only a week or so in the austral summer, lacked the capacity for RCH. In cold-acclimated larvae, RCH significantly increased freeze tolerance to both -15 and -20°C. Similarly, RCH significantly increased cellular survival of fat body, Malpighian tubules and gut tissue from cold-acclimated larvae frozen at -20°C for 24 h. These results indicate that RCH not only protects against non-freezing injury but also increases freeze tolerance.