FINCEL, E.A.**; WALLACE, E.; MARJANOVIC, M.; COSTANZO, J.P.; LEE, R.E.; Eastern Illinois University, Charleston; Eastern Illinois University, Charleston; Eastern Illinois University, Charleston; Miami University, Miami, OH; Miami University, Miami, OH: Urea: a natural cryoprotectant in the freeze-tolerant wood frog (Rana sylvatica)

The wood frog (Rana sylvatica) is one of only a few vertebrates that are able to withstand the freezing of their body fluids. Various organic osmolytes play a central role in physiological adaptation to freezing and other osmotic stresses in diverse taxa, but in R. sylvatica only one cryoprotectant, glucose, has been identified. The objective of this study was to determine whether urea, an osmolyte that naturally accumulates in the tissues of overwintering R. sylvatica, also functions to limit freeze/thaw injury. Using a force transducer apparatus, isolated gastrocnemius muscles (N = 8) were tested to determine the threshold stimulus, maximum twitch, maximum tetanus twitch, and ½ fatigue time. Subsequently, these muscles were incubated in isotonic Ringer�s solution without or with urea (80 mM) and then frozen for 18 h at �2.5°C. Re-testing the muscles after thawing demonstrated that urea-treated muscles had better performance than controls in two of the four measured parameters. Threshold stimulus was significantly lower and the maximum twitch was significantly higher in the urea-treated group as compared to the controls. Maximum tetanus twitch and ½ fatigue time did not differ significantly between the two groups. Urea concentration in the urea-treated muscles (24.1 ± 6.2 µmol/g muscle) was higher than in control muscles (1.3 ± 0.3 µmol/g muscle), yet within the range found in naturally hibernating frogs. Our findings support the hypothesis that urea plays an important role in freezing adaptation in R. sylvatica and other terrestrially hibernating amphibians. Supported by NSF IOB 0416750.