Hibernation physiology, freezing adaptation and extreme freeze tolerance in a northern population of the wood frog (original) (raw)
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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 ...
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.
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.
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.
Survival and metabolism of Rana arvalis during freezing
Journal of Comparative Physiology B, 2009
Freeze tolerance and changes in metabolism during freezing were investigated in the moor frog (Rana arvalis) under laboratory conditions. The data show for the Wrst time a well-developed freeze tolerance in juveniles of a European frog capable of surviving a freezing exposure of about 72 h with a Wnal body temperature of ¡3°C. A biochemical analysis showed an increase in liver and muscle glucose in response to freezing (respectively, 14-fold and 4-fold between 4 and ¡1°C). Lactate accumulation was only observed in the liver (4.1 § 0.8 against 16.6 § 2.4 mol g ¡1 fresh weight (FW) between 4 and ¡1°C). The quantiWcation of the respiratory metabolism of frozen frogs showed that the aerobic metabolism persists under freezing conditions (1.4 § 0.7 l O 2 g ¡1 FW h ¡1 at ¡4°C) and decreases with body temperature. After thawing, the oxygen consumption rose rapidly during the Wrst hour (6-fold to 16-fold) and continued to increase for 24 h, but at a lower rate. In early winter, juvenile R. arvalis held in an outdoor enclosure were observed to emerge from ponds and hibernate in the upper soil and litter layers. Temperature recordings in the substratum of the enclosure suggested that the hibernacula of these juvenile frogs provided sheltering from sub-zero air temperatures and reduced the time spent in a frozen state corresponding well with the observed freeze tolerance of the juveniles. This study strongly suggests that freeze tolerance of R. arvalis is an adaptive trait necessary for winter survival.
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.
Time course for cryoprotectant synthesis in the freeze-tolerant chorus frog, Pseudacris triseriata
Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 2000
Increases in liver glycogen phosphorylase activity, along with inhibition of glycogen synthetase and phosphofructokinase-1, are associated with elevated cryoprotectant (glucose) levels during freezing in some freeze-tolerant anurans. In contrast, freeze-tolerant chorus frogs, Pseudacris triseriata, accumulate glucose during freezing but exhibit no increase in phosphorylase activity following 24-h freezing bouts. In the present study, chorus frogs were frozen for 5-and 30-min and 2-and 24-h durations. After freezing, glucose, glycogen, and glycogen phosphorylase and synthetase activities were measured in leg muscle and liver to determine if enzyme activities varied over shorter freezing durations, along with glucose accumulation. Liver and muscle glucose levels rose significantly (5 -12-fold) during freezing. Glycogen showed no significant temporal variation in liver, but in muscle, glycogen was significantly elevated after 24 h of freezing relative to 5 and 30 min-frozen treatments. Hepatic phosphorylase a and total phosphorylase activities, as well as the percent of the enzyme in the active form, showed no significant temporal variation following freezing. Muscle phosphorylase a activity and percent active form increased significantly after 24 h of freezing, suggesting some enhancement of enzyme function following freezing in muscle. However, the significance of this enhanced activity is uncertain because of the concurrent increase in muscle glycogen with freezing. Neither glucose 6-phosphate independent (I) nor total glycogen synthetase activities were reduced in liver or muscle during freezing. Thus, chorus frogs displayed typical cryoprotectant accumulation compared with other freeze-tolerant anurans, but freezing did not significantly alter activities of hepatic enzymes associated with glycogen metabolism.
Freeze-Thaw Effects on Metabolic Enzymes in Wood Frog Organs
Cryobiology, 2001
To determine whether episodes of natural freezing and thawing altered the metabolic makeup of wood frog (Rana sylvatica) organs, the maximal activities of 28 enzymes of intermediary metabolism were assessed in six organs (brain, heart, kidney, liver, skeletal muscle, gut) of control (5°C acclimated), frozen (24 h at Ϫ3°C), and thawed (24 h back at 5°C) frogs. The enzymes assessed represented pathways including glycolysis, gluconeogenesis, amino acid metabolism, fatty acid metabolism, the TCA cycle, and adenylate metabolism. Organ-specific responses seen included (a) the number of enzymes affected by freeze-thaw (1 in gut ranging to 17 in heart), (b) the magnitude and direction of response (most often enzyme activities decreased during freezing and rebounded with thawing but, liver showed freeze-specific increases in several enzymes), and (c) the response to freezing versus thawing (enzyme activities in gut and kidney changed during freezing, whereas most enzymes in skeletal muscle responded to thawing). Overall, the data show that freeze-thaw implements selected changes to the maximal activities of various enzymes of intermediary metabolism and that these may aid organ-specific responses that alter fuel use during freeze-thaw, support cryoprotectant metabolism, and aid organ endurance of freeze-induced ischemia.