Dynamics of body water during freezing and thawing in a freeze-tolerant frog (Rana sylvatica)

109

SUMMARYEctotherms overwintering in temperate ecosystems must survive low temperatures while conserving energy to fuel post-winter reproduction. Freeze-tolerant wood frogs, Rana sylvatica, have an active response to the initiation of ice formation that includes mobilising glucose from glycogen and circulating it around the body to act as a cryoprotectant. We used flow-through respirometry to measure CO 2 production (V CO2 ) in real time during cooling, freezing and thawing. CO 2 production increases sharply at three points during freeze-thaw: at +1°C during cooling prior to ice formation (total of ). We interpret these increases in metabolic rate to represent the energetic costs of preparation for freezing, the response to freezing and the re-establishment of homeostasis and repair of damage after thawing, respectively. We assumed that frogs metabolise lipid when unfrozen and that carbohydrate fuels metabolism during cooling, freezing and thawing, and when frozen. We then used microclimate temperature data to predict overwinter energetics of wood frogs. Based on the freezing and melting points we measured, frogs in the field were predicted to experience as many as 23 freeze-thaw cycles in the winter of our microclimate recordings. Overwinter carbohydrate consumption appears to be driven by the frequency of freeze-thaw events, and changes in overwinter climate that affect the frequency of freeze-thaw will influence carbohydrate consumption, but changes that affect mean temperatures and the frequency of winter warm spells will modify lipid consumption. Supplementary material available online at

Section: Discussionmentioning

confidence: 91%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Real-time measurement of metabolic rate during freezing and thawing of the wood frog,Rana sylvatica: implications for overwinter energy use

Sinclair

Stinziano

Williams

et al. 2013

109

“…Because the organs of frozen frogs contained 11.3% (flexor carpi) to 51.4% (heart) less water than the organs of unfrozen controls (ANOVA; P<0.05; see Lee et al, 1992), it was necessary to express osmolyte concentrations as mol·g -1 ·dry·tissue in order to allow comparisons between the groups. Urea concentration did not change with freezing in most organs; however, glucose concentrations were up to 35-fold higher in frozen frogs as compared with unfrozen controls (Table·1).…”

Section: Effect Of Somatic Freezing On Urea and Glucose Levelsmentioning

confidence: 99%

Cryoprotection by urea in a terrestrially hibernating frog

Costanzo

Lee

2005

Self Cite

112

SUMMARY The role of urea as a balancing osmolyte in osmotic adaptation is well known, but this `waste product' also has myriad other functions in diverse taxa. We report that urea plays an important, previously undocumented role in freezing tolerance of the wood frog (Rana sylvatica), a northern woodland species that hibernates terrestrially in sites where dehydration and freezing may occur. Wood frogs inhabiting an outdoor enclosure accumulated urea to 65 mmol l-1 in autumn and early winter, when soil moisture was scarce, but subsequently urea levels fell to ∼2 mmol l-1 as the availability of environmental water increased. Laboratory experiments showed that hibernating R. sylvatica can accumulate at least 90 mmol l-1 urea under relatively dry, warm conditions. During experimental freezing, frogs synthesized glucose but did not accumulate additional urea. Nevertheless, the concentrations of urea and glucose in some tissues were similar. We tested urea's efficacy as a cryoprotectant by measuring lysis and lactate dehydrogenase (LDH) leakage in samples of R. sylvaticaerythrocytes frozen/thawed in the presence of physiological levels of urea or other osmolytes. In conferring protection against freeze/thaw damage, urea was comparable to glycerol and as good as or better than glucose, cryoprotectants found in freeze-tolerant frogs and other animals. Urea treatment also improved the viability of intact tissues frozen in vitro, as demonstrated by post-thaw measures of metabolic activity and LDH leakage. Collectively, our findings suggest that urea functions both as an osmoprotectant and a cryoprotectant in terrestrially hibernating amphibians.

“…During freezing, much of the water inside organs (up to 60%) is translocated to the coelom and lymph sacs, where it freezes innocuously (Lee et al, 1992). After thawing, circulation resumes and organs quickly rehydrate to prefreeze levels (Costanzo et al, 1997a).…”

Section: Physiological Responses To Freezing-thawingmentioning

confidence: 99%

Urea loading enhances freezing survival and postfreeze recovery in a terrestrially hibernating frog

Costanzo

Lee

2008

Self Cite

SUMMARYWe tested the hypothesis that urea, an osmolyte accumulated early in hibernation, functions as a cryoprotectant in the freezetolerant wood frog, Rana sylvatica. Relative to saline-treated, normouremic (10 μmol ml ) by administration of an aqueous urea solution exhibited significantly higher survival (100% versus 64%) following freezing at -4°C, a potentially lethal temperature. Hyperuremic frogs also had lower plasma levels of intracellular proteins (lactate dehydrogenase, creatine kinase, hemoglobin), which presumably escaped from damaged cells, and more quickly recovered neurobehavioral functions following thawing. Experimental freezing-thawing did not alter tissue urea concentrations, but did elevate glucose levels in the blood and organs of all frogs. When measured 24 h after thawing commenced, glucose concentrations were markedly higher in urea-loaded frogs as compared to saline-treated ones, possibly because elevated urea retarded glucose clearance. Like other low-molecular-mass cryoprotectants, urea colligatively reduces both the amount of ice forming within the body and the osmotic dehydration of cells. In addition, by virtue of certain non-colligative properties, it may bestow additional protection from freeze-thaw damage not afforded by glucose.