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Hibernation physiology, freezing adaptation and extreme freeze tolerance in a northern population of the wood frog
Jon P. Costanzo, M. Clara F. do Amaral, Andrew J. Rosendale, Richard E. Lee, Jr


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.



    J.P.C. and M.C.F.A conceived the study; J.P.C., M.C.F.A and A.J.R. designed the experiments and collected the data; J.P.C. analyzed the data and wrote the paper; J.P.C., M.C.F.A, A.J.R. and R.E.L. contributed substantially to interpreting the data and developing the manuscript and take full responsibility for the content of the paper.


    No competing interests declared.


    This work was supported by the National Science Foundation [IOS1022788 to J.P.C.]; and the Portuguese Science and Technology Foundation (Fundação para a Ciência e Tecnologia, Ministério da Educação e Ciência, Portugal) [SFRH/BD/63151/2009 to M.C.F.A.].


    equilibrium freezing/melting point (°C)
    glutamate dehydrogenase
    hepatosomatic index (g dry liver g−1 dry body × 100)
    body temperature (°C)
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