COSTANZO, J.P.; BAKER, P.J.; DINKELACKER, S.A.; LEE, R.E.; Miami Univ., Oxford, OH; Miami Univ., Oxford, OH; Miami Univ., Oxford, OH; Miami Univ., Oxford, OH: Endogenous and exogenous ice-nucleating agents constrain supercooling in the hatchling painted turtlele

Hatchlings of the painted turtle, Chrysemys picta, 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 found that the seasonal increase in supercooling capacity coincided with elimination of incubation substratum (and, in fewer cases, eggshell) that the neonates 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. Neonates, but not winter turtles, produced feces that expressed ice-nucleating activity, indicating that seasonal development of supercooling capacity requires elimination of endogenous ice-nucleating agents (INA). Ingestion of vermiculite or eggshell, which had modest ice-nucleating activity, had no effect on turtle crystallization temperature; whereas, ingestion of nesting soil, which contained potent INA, markedly reduced the supercooling capacity of neonates, and the effect persisted long after the turtles had purged their guts of soil particles. Experiments in which winter turtles were fed INAs 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 we found in turtles overwintering in natural nests, and therefore pose a formidable challenge to the winter survival of hatchling turtles.