Summary Ectothermic vertebrates have colonized regions that are seasonally or perpetually cold, and some species, particularly terrestrial hibernators, must cope with temperatures that fall substantially below 0°C. Survival of such excursions depends on either freeze avoidance through supercooling or freeze tolerance. Supercooling, a metastable state in which body fluids remain liquid below the equilibrium freezing/melting point, is promoted by physiological responses that protect against chilling injury and by anatomical and behavioral traits that limit risk of inoculative freezing by environmental ice and ice-nucleating agents. Freeze tolerance evolved from responses to fundamental stresses to permit survival of the freezing of a substantial amount of body water under thermal and temporal conditions of ecological relevance. Survival of freezing is promoted by a complex suite of molecular, biochemical and physiological responses that limit cell death from excessive shrinkage, damage to macromolecules and membranes, metabolic perturbation and oxidative stress. Although freeze avoidance and freeze tolerance generally are mutually exclusive strategies, a few species can switch between them, the mode used in a particular instance of chilling depending on prevailing physiological and environmental conditions.

SUMMARY Hatchlings of the painted turtle ( Chrysemys picta ) commonly hibernate in their shallow, natal nests. Survival at temperatures below the limit of freeze tolerance (approximately -4°C) apparently depends on their ability to remain supercooled, and, whereas previous studies have reported that supercooling capacity improves markedly with cold acclimation, the mechanistic basis for this change is incompletely understood. We report that the crystallization temperature ( T c ) of recently hatched(summer) turtles acclimated to 22°C and reared on a substratum of vermiculite or nesting soil was approximately 5°C higher than the T c determined for turtles acclimated to 4°C and tested in winter. This increase in supercooling capacity coincided with elimination of substratum (and, in fewer cases, eggshell) that the hatchlings had ingested; however, this association was not necessarily causal because turtles reared on a paper-covered substratum did not ingest exogenous matter but nevertheless showed a similar increase in supercooling capacity. Our results for turtles reared on paper revealed that seasonal development of supercooling capacity fundamentally requires elimination of ice-nucleating agents (INA) of endogenous origin: summer turtles, but not winter turtles, produced feces(perhaps derived from residual yolk) that expressed ice-nucleating activity. Ingestion of vermiculite or eggshell, which had modest ice-nucleating activity, had no effect on the T c , whereas ingestion of nesting soil, which contained two classes of potent INA, markedly reduced the supercooling capacity of summer turtles. This effect persisted long after the turtles had purged their guts of soil particles, because the T c of winter turtles reared on nesting soil (mean ±S.E.M.=-11.6±1.4°C) was approximately 6°C higher than the T c of winter turtles reared on vermiculite or paper. Experiments in which winter turtles were fed INA commonly found in nesting soil showed that water-soluble, organic agents can remain fully active for at least one month. Such INA may account for the limited supercooling capacity( T c ≈-7.5°C) we found in turtles overwintering in natural nests and may therefore pose a formidable challenge to the winter survival of hatchling C. picta.

When free of surface water in air or liquid paraffin, the antarctic nematode Panagrolaimus davidi is freezing intolerant but avoids freezing by supercooling. Survival of long-term exposure is enhanced by sub-zero temperatures compared with controls maintained at 99% relative humidity and 15°C. In water the nematodes are seeded by exogenous ice nucleation and a proportion are freezing tolerant. Ice formation appears to be restricted to the pseudocbel. The degree of freezing tolerance is dependent upon the age of the culture and its thermal history. P. davidi is freezing tolerant when exposed to sub-zero temperatures in water and freezing intolerant when free of surface water and able to supercool. These two strategies are not mutually exclusive as they are often thought to be in arthropods.