Graduate Program

Biological Sciences

Degree Name

Master of Science (MS)

Semester of Degree Completion

2006

Thesis Director

Marina Marjanovic

Thesis Committee Member

Barbara Lawrence

Thesis Committee Member

Charles Costa

Abstract

The wood frog (Rana sylvatica) is one of only a few vertebrates that are able to withstand 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. Secondly, it was investigated whether hyperuremia is associated with increased osmolyte concentration to counteract any negative effect of urea.

Using a force transducer apparatus, isolated gastrocnemius muscles of wood frog and our control species, northern leopard frog (Rana pipiens), were tested to determine threshold stimulus, maximum twitch, and maximum tetanus twitch. Subsequently, these muscles were incubated in isotonic Ringer's solution without or with urea (40 or 80 mM in wood frog, 80 mM in northern leopard frog) and then frozen for 18 h at -2.5°C. Retesting wood frog muscles after thawing, demonstrated that muscles treated with 80 mM urea had significantly better performance for threshold stimulus and maximum twitch than control muscles. Urea concentrations were increased in urea-treated muscles (24.1 ± 6.2 µmol/g muscle) compared to control muscles (1.3 ± 0.3 µmol/g muscle), yet within the range found in naturally hibernating frogs. Wood frog muscle incubated in 40 mM urea did not exhibit better performance after freezing than their controls, which could be attributed to the small amount of urea incorporated into urea-treated muscles (11.2+2.6 µmol/g muscle). After freezing, urea-treated muscles for northern leopard frog showed significantly better performance than corresponding control muscle only for maximum twitch. These muscle contained similar amounts of urea (23.37 ± 3.46 µmol/g muscle) as wood frog muscles incubated in 80 mM urea. In vivo muscle performance experiments were performed on wood frogs injected with Ringer's solution (control) or 1 M urea in Ringer's. Muscles were tested for the same parameters as in in vitro experiments. After freezing, urea-treated muscles did not exhibit statistically better performance than control muscles. Lack of difference between experimental groups could be the result of short incubation time, resulting in low levels of urea incorporated into urea-treated muscles (7.72 ± 1.05 µmol/g muscle).

Examining seasonal differences in osmolyte concentrations revealed several major osmolytes present in different wood frog tissues. Urea was found to be present in liver, muscle, and plasma samples; however, urea was not found to change seasonally. Lack of seasonal changes was the result of frogs overwintering in a cold room without undergoing dehydration or freezing. While freezing did not occur, wood frogs still accumulated trimethylamine oxide (TMAO) and glycerol phosphorylcholine (GPC) in high concentrations in both liver and muscle. These osmolytes exhibited significant seasonal changes from October to March and were believed to accumulate in anticipation of a freezing event and increased urea levels.

Our findings support the hypothesis that urea plays an important, previously undocumented role in freezing adaptation of R. sylvatica and possibly other terrestrially hibernating amphibians. This study also provides evidence that counteracting osmolytes are accumulating in high enough concentrations in anticipation of freezing to reverse urea's destabilizing effect.

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