Cryoprotectant Production in Freeze-Tolerant Wood Frogs Is Augmented by Multiple Freeze-Thaw Cycles.

Ice nucleation across the skin of wood frogs (Lithobates sylvaticus) rapidly induces endogenous production of glucose, a cryoprotectant necessary for freeze tolerance. In laboratory studies of freeze tolerance, wood frogs are cooled slowly, often at -0.05°C h(-1), to facilitate high cryoprotectant production and survival. Under natural conditions in Alaska, however, wood frogs accumulate maximal tissue glucose concentrations while cooling at much faster rates, -0.35° to -1.6°C h(-1), and in addition undergo multiple successive freeze-thaw cycles before remaining frozen for the winter. We examined whether simulating these ecologically relevant cooling rates and repeated freeze-thaw events in captive wood frogs results in the high glucose concentrations found in naturally frozen wood frogs. We found that over successive freezing and thawing events, glucose concentrations increased stepwise in all measured tissues. Short thawing periods did not result in a statistically significant decline of glucose concentrations. Wood frogs that experienced three freeze-thaw events had fresh weight glucose concentrations that approached values found in tissues of wood frogs frozen in natural conditions. Laboratory wood frogs survive frozen for 2 mo, while wood frogs frozen under natural conditions survive frozen for up to 7 mo at temperatures below -18°C. We hypothesize that repeated freeze-thaw cycles with rapid cooling and warming rates allow for greater survival in Alaskan wood frogs through enhanced cryoprotectant production.