Survival and metabolism of Rana arvalis during freezing (original) (raw)
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Seasonality of Freeze Tolerance in a Subarctic Population of the Wood Frog, Rana sylvatica
International Journal of Zoology, 2014
We compared physiological characteristics and responses to experimental freezing and thawing in winter and spring samples of the wood frog, Rana sylvatica, indigenous to Interior Alaska, USA. Whereas winter frogs can survive freezing at temperatures at least as low as −16 ∘ C, the lower limit of tolerance for spring frogs was between −2.5 ∘ C and −5 ∘ C. Spring frogs had comparatively low levels of the urea in blood plasma, liver, heart, brain, and skeletal muscle, as well as a smaller hepatic reserve of glycogen, which is converted to glucose after freezing begins. Consequently, following freezing (−2.5 ∘ C, 48 h) tissue concentrations of these cryoprotective osmolytes were 44-88% lower than those measured in winter frogs. Spring frogs formed much more ice and incurred extensive cryohemolysis and lactate accrual, indicating that they had suffered marked cell damage and hypoxic stress during freezing. Multiple, interactive stresses, in addition to diminished cryoprotectant levels, contribute to the reduced capacity for freeze tolerance in posthibernal frogs.
Freezing tolerance of the European water frogs: the good, the bad, and the ugly
American Journal of Physiology Regulatory Integrative and Comparative Physiology, 2005
Survival and some physiological responses to freezing were investigated in three European water frogs (Rana lessonae, Rana ridibunda, and their hybridogen Rana esculenta). The three species exhibited different survival times during freezing (from 10 h for R. lessonae to 20 h for R. ridibunda). The time courses of percent water frozen were similar; however, because of the huge differences in body mass among species (from 10 g for Rana lessonae to nearly 100 g for Rana ridibunda), the ice mass accumulation rate varied markedly (from 0.75 Ϯ 0.12 to 1.43 Ϯ 0.11 g ice/h, respectively) and was lowest in the terrestrial hibernator Rana lessonae. The hybrid Rana esculenta exhibited an intermediate response between the two parental species; furthermore, within-species correlation existed between body mass and ice mass accumulation rates, suggesting the occurrence of subpopulations in this species (0.84 Ϯ 0.08 g ice/h for small R. esculenta and 1.78 Ϯ 0.09 g ice/h for large ones). Biochemical analyses showed accumulation of blood glucose and lactate, liver glucose (originating from glycogen), and liver alanine in Rana lessonae and Rana esculenta but not in Rana ridibunda in response to freezing. The variation of freeze tolerance between these three closely related species could bring understanding to the physiological processes involved in the evolution of freeze tolerance in vertebrates. cold hardiness; ice content; osmolality; glucose Address for reprint requests and other correspondence: Y. Voituron, Physiologie des régulations énergétiques, cellulaires et moléculaires (U.M.R. CNRS 5123), Bât.
Survival and metabolic responses to freezing by the water frog (Rana ridibunda
Journal of Experimental Zoology, 2003
We studied the ability of the marsh frog Rana ridibunda to survive freezing exposure and the associated subsequent metabolic variations. This species that typically overwinters under water tolerates the conversion of 55% of its body water into ice. This ice content is attained after a few hours (between 8 and 36 hours depending on the mass of the individual and the environmental temperature) but death occurs at greater than 58% ice. Freezing stimulated a significant increase in blood carnitine and trimethylamine levels (respectively 4.5±2.5 and 0.5±0.2 µmol.l−1 for controls versus 27.0±18.9 and 3.6±4.1 µmol.l−1 after thawing) but these increases had no significant effect on plasma osmolality which was unchanged between control and freeze exposed frogs (252.6±20.3 versus 240.2±25.0 mOsmol.l−1, respectively). Freezing also induced a significant dehydration of heart, liver and muscles (respectively 4.2, 3.2 and 2.8%) but the observed levels are low compared to values found in highly freeze tolerant species. This species could be classified as “partially freeze tolerant” enduring the transformation of a significant part of its body water into ice but not the completion of the exotherm. The existence of freeze tolerance in an aquatic hibernator that does not accumulate cryoprotectant, exhibiting low organ dehydration after freezing and low hypoxia tolerance, raises the possibility that a tolerance of nearly 60% ice within the body is common among anurans. J. Exp. Zool. 299A:118–126, 2003. © 2003 Wiley-Liss, Inc.
Journal of Experimental Biology, 2013
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±10μmolml −1 ) and an unidentified solute (to ~73μmolml −1 ). In contrast, frogs from a cool-temperate population (southern Ohio, USA) amassed much less glycogen, had a lower uremia (28±5μmolml −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 48h) 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.
Wood frog adaptations to overwintering in Alaska: New limits to freezing tolerance
Journal of Experimental Biology, 2014
We investigated the ecological physiology and behavior of free-living wood frogs [Lithobates (Rana) sylvaticus] overwintering in Interior Alaska by tracking animals into natural hibernacula, recording microclimate, and determining frog survival in spring. We measured cryoprotectant (glucose) concentrations and identified the presence of antifreeze glycolipids in tissues from subsamples of naturally freezing frogs. We also recorded the behavior of wood frogs preparing to freeze in artificial hibernacula, and tissue glucose concentrations in captive wood frogs frozen in the laboratory to −2.5°C. Wood frogs in natural hibernacula remained frozen for 193±11 consecutive days and experienced average (October-May) temperatures of −6.3°C and average minimum temperatures of -14.6±2.8°C (range −8.9 to −18.1°C) with 100% survival (N=18). Mean glucose concentrations were 13-fold higher in muscle, 10-fold higher in heart and 3.3-fold higher in liver in naturally freezing compared with laboratory frozen frogs. Antifreeze glycolipid was present in extracts from muscle and internal organs, but not skin, of frozen frogs. Wood frogs in Interior Alaska survive freezing to extreme limits and durations compared with those described in animals collected in southern Canada or the Midwestern United States. We hypothesize that this enhancement of freeze tolerance in Alaskan wood frogs is due to higher cryoprotectant levels that are produced by repeated freezing and thawing cycles experienced under natural conditions during early autumn.
Cryoprotectants and extreme freeze tolerance in a subarctic population of the wood frog
PloS one, 2015
Wood frogs (Rana sylvatica) exhibit marked geographic variation in freeze tolerance, with subarctic populations tolerating experimental freezing to temperatures at least 10-13 degrees Celsius below the lethal limits for conspecifics from more temperate locales. We determined how seasonal responses enhance the cryoprotectant system in these northern frogs, and also investigated their physiological responses to somatic freezing at extreme temperatures. Alaskan frogs collected in late summer had plasma urea levels near 10 μmol ml-1, but this level rose during preparation for winter to 85.5 ± 2.9 μmol ml-1 (mean ± SEM) in frogs that remained fully hydrated, and to 186.9 ± 12.4 μmol ml-1 in frogs held under a restricted moisture regime. An osmolality gap indicated that the plasma of winter-conditioned frogs contained an as yet unidentified osmolyte(s) that contributed about 75 mOsmol kg-1 to total osmotic pressure. Experimental freezing to -8°C, either directly or following three cycles ...
Journal of Experimental Biology, 2013
Ectotherms 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 104±17μlCO 2 frog −1 event −1 ), at the initiation of freezing (565±85μlCO 2 frog −1 freezingevent −1 ) and after the frog has thawed (564±75μlCO 2 frog −1 freezingevent −1 ). 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.
Journal of Experimental Zoology, 2004
It has been hypothesized that freeze-tolerance in anurans evolved from a predisposition for dehydration tolerance. To test this hypothesis, we dehydrated summer/fallcollected and winter acclimated freeze-tolerant chorus frogs and dehydration-tolerant, but freezeintolerant, Woodhouse's and Great Plains toads to 25% and 50% body water loss (BWL). Following treatments, we measured glucose, glycogen, and glycogen phosphorylase and glycogen synthetase (summer/fall only) activities in liver and leg muscle. Hepatic glucose levels were not significantly altered by dehydration in either summer/fall-collected frogs or toads. Conversely, winter acclimated frogs did show an increment (2.9-fold) in hepatic glucose with dehydration, accompanied by a reduction in hepatic glycogen levels. Winter acclimated toads did not mobilize hepatic glucose in response to dehydration. Further, hepatic glycogen and phosphorylase activities did not vary in any consistent manner with dehydration in winter toads. Mean leg muscle glucose values were elevated at 50% BWL relative to other treatments, significantly so compared to 25% BWL for summer/fallcollected frogs. The pattern of hepatic glucose mobilization with dehydration in winter frogs is consistent with that in other freeze-tolerant frog species, and provides additional support for the hypothesis that freezing tolerance evolved from a capacity for dehydration tolerance. However, the lack of hepatic glucose mobilization in response to dehydration in fall frogs suggests that a seasonal component to dehydration-induced regulation of glucose metabolism exists in chorus frogs. Furthermore, the absence of a dehydration-induced mobilization of hepatic glucose at both seasons in toads suggests that this dehydration response is not universal for terrestrial anurans.