Although seasonal increases in cold hardiness are well documented for temperate and polar ectotherms, relatively little is known about supplemental increases in cold hardiness during winter. Because many animals are exposed to considerable thermal variation in winter, they may benefit from a quick enhancement of cold tolerance prior to extreme low temperature. Hatchling painted turtles (Chrysemys picta) overwintering in their natal nests experience substantial thermal variation in winter, and recently, it was found that brief subzero chilling of winter-acclimated hatchlings decreases subsequent chilling-induced mortality, increases blood concentrations of glucose and lactate, and protects the brain from cryoinjury. Here, we further characterize that phenomenon, termed 'cold conditioning', by exposing winter-acclimated hatchling turtles to -3.5, -7.0, or -10.5 °C gradually or repeatedly via daily thermal fluctuations over the course of 5 days and assessing their survival of a subsequent cold shock to a discriminating temperature of -12.7 °C. To better understand the physiological response to cold conditioning, we measured changes in glucose and lactate concentrations in the liver, blood, and brain. Cold conditioning significantly increased cold-shock survival, from 9% in reference turtles up to 74% in cold-conditioned turtles, and ecologically relevant daily thermal fluctuations were at least as effective at conferring cryoprotection as was gradual cold conditioning. Cold conditioning increased glucose concentrations, up to 25 μmol g, and lactate concentrations, up to 30 μmol g, in the liver, blood, and brain. Turtles that were cold conditioned with daily thermal fluctuations accumulated more glucose in the liver, blood, and brain, and had lower brain lactate, than those gradually cold conditioned. Given the thermal variation to which hatchling painted turtles are exposed in winter, we suggest that the supplemental protection conferred by cold conditioning, especially that induced by daily thermal fluctuations, may be important for their overwinter survival. Investigation into the duration of the cold-conditioning induced protection and its occurrence in natural field conditions is needed to better understand its ecological significance. We suggest that future work exploring the underlying mechanisms of cold conditioning should focus on non-colligative effects of glucose, expression of small Hsps, changes in membrane structure, and ion homeostasis.
