Endogenous and exogenous ice-nucleating agents constrain supercooling in the hatchling painted turtle

Abstract

SUMMARYHatchlings 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 (Tc) 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 Tc 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 Tc, 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 Tc of winter turtles reared on nesting soil (mean ±S.E.M.=-11.6±1.4°C) was approximately 6°C higher than the Tc 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(Tc≈-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.

Overwintering in the nest by hatchling map turtles (Graptemys geographica)

“…The loss of 15% of body mass, on average, during overwintering may be partially or wholly attributable to lipid catabolism (Congdon and Gibbons 1990). Because energy exchange for individual eggs and hatchlings is limited to water, gases, and occasionally materials in the nest soil (Packard et al 2001;Costanzo et al 2003), extended periods spent in the nest require hatchlings to have high levels of energy reserves (Congdon 1989;Congdon and Gibbons 1990;Nagle et al 1998). In general, species whose hatchlings delay emergence from the nest allocate higher amounts of energy storage lipids, such as triacylglycerol, to eggs compared with species whose hatchlings exhibit early emergence (Congdon andGibbons 1985, 1990;Rowe et al 1995;Nagle et al 1998Nagle et al , 2003.…”

Brief Chilling to Subzero Temperature Increases Cold Hardiness in the Hatchling Painted Turtle (Chrysemys picta)

“…Seasonal enhancement of supercooling capacity in hatchling C. picta is facilitated by attenuation or masking of endogenous INAs, as well as elimination of ingested INAs from the gut (Costanzo et al 2003). Judging from their extensive supercooling capacity, our winter-acclimated turtles had already undergone these processes and perhaps were unable to further enhance their supercooling capacity.…”

“…Following Costanzo et al (2003), we assessed the supercooling limit of control ( ) n p 6 and cold-conditioned ( ) turtles by progressively cooling n p 10 them until they spontaneously froze. Turtles, still in clean, dry, 50-mL tubes, were suspended in a refrigerated ethanol bath (Neslab RTE 140, Portsmouth, NH) initially set at 0.0ЊC and cooled to Ϫ20.0ЊC in a linear fashion at Ϫ1.0ЊC h Ϫ1 .…”

To freeze or not to freeze: adaptations for overwintering by hatchlings of the North American painted turtle

“…As baby turtles are breaking out of their egg, they typically ingest quantities of soil and fragments of eggshell (Packard et al, 2000aCostanzo et al, 2003;Packard and Packard, 2003c). Such geophagy is of uncertain function in any animal (Dominy et al, 2004), but the behavior has important consequences for creatures such as hatchling painted turtles.…”

“…Baby painted turtles purge their gut of particulate matter before the start of winter Costanzo et al, 2003;Packard and Packard, 2003c), and the purging is accompanied by a reduction in the population of bacterial nucleators (Fig.·3). However, emptying the gut of particulate matter is not, by itself, sufficient to remove the nucleators.…”