Hibernation physiology, freezing adaptation and extreme freeze tolerance in a northern population of the wood frog

Abstract

We investigated hibernation physiology and freeze tolerance in a population of the wood frog, Rana sylvatica, indigenous to Interior Alaska, USA, near the northernmost limit of the species' range. Winter acclimatization responses included a 233% increase in the hepatic glycogen depot that was subsidized by fat body and skeletal muscle catabolism, and a rise in plasma osmolality that reflected accrual of urea (to 106±10 μmol ml(-1)) and an unidentified solute (to ~73 μmol ml(-1)). In contrast, frogs from a cool-temperate population (southern Ohio, USA) amassed much less glycogen, had a lower uremia (28±5 μmol ml(-1)) and apparently lacked the unidentified solute. Alaskan frogs survived freezing at temperatures as low as -16°C, some 10-13°C below those tolerated by southern conspecifics, and endured a 2-month bout of freezing at -4°C. The profound freeze tolerance is presumably due to their high levels of organic osmolytes and bound water, which limits ice formation. Adaptive responses to freezing (-2.5°C for 48 h) and subsequent thawing (4°C) included synthesis of the cryoprotectants urea and glucose, and dehydration of certain tissues. Alaskan frogs differed from Ohioan frogs in retaining a substantial reserve capacity for glucose synthesis, accumulating high levels of cryoprotectants in brain tissue, and remaining hyperglycemic long after thawing. The northern phenotype also incurred less stress during freezing/thawing, as indicated by limited cryohemolysis and lactate accumulation. Post-glacial colonization of high latitudes by R. sylvatica required a substantial increase in freeze tolerance that was at least partly achieved by enhancing their cryoprotectant system.