Cryoprotective dehydration and the resistance to inoculative freezing in a polar insect

During winter, larvae of the Antarctic midge, Belgica antarctica (Diptera, Chironomidae), must endure 7-8 months of continuous subzero temperatures, encasement in a matrix of soil and ice, and severely desiccating conditions. This environment, along with the fact that larvae possess a high rate of water loss and are extremely tolerant of desiccation, may promote the use of cryoprotective dehydration as a strategy for winter survival. This study investigates the capacity of larvae to resist inoculative freezing and undergo cryoprotective dehydration at subzero temperatures. Slow cooling to -3^oC in an environment at equilibrium with the vapor pressure of ice reduced larval water content by ~40% and depressed the body fluid melting point more than 3-fold to -2.6^oC. This melting point depression was the result of the concentration of existing solutes (i.e., loss of body water) and the de novo synthesis of osmolytes.  By day 14 of the subzero exposure, larval survival was still >95%, suggesting larvae have the capacity to undergo cryoprotective dehydration. However, under natural conditions the use of cryoprotective dehydration may be constrained by inoculative freezing as result of the intimate contact between the larvae and environmental ice. During slow cooling within a substrate of frozen soil, the ability of larvae to resist inoculative freezing and undergo cryoprotective dehydration was dependent upon the moisture content of the soil. As detected by a reduction of larval water content, the percentage of larvae that resisted inoculative freezing increased with decreasing soil moisture. These results suggest that larvae of the Antarctic midge have the capacity to resist inoculative freezing at relatively low soil moisture contents and likely undergo cryoprotective dehydration when exposed to subzero temperatures during the polar winter.