Thermal analysis of ice and glass transitions in insects that do and do not survive freezing

Some insects rely on the strategy of freeze tolerance for winter survival. During freezing, extracellular body water transitions from the liquid to the solid phase and cells undergo freeze-induced dehydration. Here, we present results of a thermal analysis (from differential scanning calorimetry) of ice fraction dynamics during gradual cooling after inoculative freezing in variously acclimated larvae of two drosophilid flies, Drosophila melanogaster and Chymomyza costata Although the species and variants ranged broadly between 0 and close to 100% survival of freezing, there were relatively small differences in ice fraction dynamics. For instance, the maximum ice fraction (IF_max) ranged between 67.9% and 77.7% total body water (TBW). Chymomyza costata larvae showed statistically significant phenotypic shifts in parameters of ice fraction dynamics (melting point and IF_max) upon entry into diapause, cold acclimation and feeding on a proline-augmented diet. These differences were mostly driven by colligative effects of accumulated proline (ranging between 6 and 487 mmol kg^-1 TBW) and other metabolites. Our data suggest that these colligative effects per se do not represent a sufficient mechanistic explanation for high freeze tolerance observed in diapausing, cold-acclimated C. costata larvae. Instead, we hypothesize that accumulated proline exerts its protective role via a combination of mechanisms. Specifically, we found a tight association between proline-induced stimulation of glass transition in partially frozen body liquids (vitrification) and survival of cryopreservation in liquid nitrogen.