Overwintering adaptations and extreme freeze tolerance in a subarctic population of the wood frog, Rana sylvatica.

The terrestrially hibernating wood frog (Rana sylvatica) is well-known for its iconic freeze tolerance, an overwintering adaptation that has received considerable investigation over the past 35 years. Virtually, all of this research has concerned frogs indigenous to the temperate regions of its broad range within North America. However, recent investigations have shown that frogs of subarctic populations are extremely cold hardy, being capable of surviving freezing for longer periods and at much lower temperatures as compared to conspecifics from temperate regions.

Nitric oxide metabolites in hypoxia, freezing, and hibernation of the wood frog, Rana sylvatica.

Nitric oxide (NO) is a gaseous free radical that in diverse organisms performs many signaling and protective functions, such as vasoregulation, inhibition of apoptosis, antioxidation, and metabolic suppression. Increased availability of NO may be especially important during life-history periods when organisms contend with multiple stresses. We investigated dynamics of the NO metabolites, nitrite (NO) and nitrate (NO), in the blood plasma, heart, liver, and skeletal muscle of the wood frog (Rana sylvatica), an amphibian that endures chronic cold, freezing, hypoxia, dehydration, and extended aphagia during hibernation.

Urea hydrolysis by gut bacteria in a hibernating frog: evidence for urea-nitrogen recycling in Amphibia.

Gut bacteria that produce urease, the enzyme hydrolysing urea, contribute to nitrogen balance in diverse vertebrates, although the presence of this system of urea-nitrogen recycling in Amphibia is as yet unknown. Our studies of the wood frog (), a terrestrial species that accrues urea in winter, documented robust urease activity by enteric symbionts and hence potential to recoup nitrogen from the urea it produces. Ureolytic capacity in hibernating (non-feeding) frogs, whose guts hosted an approximately 33% smaller bacterial population, exceeded that of active (feeding) frogs, possibly due to an inductive effect of high urea on urease expression and/or remodelling of the microbial community.

Enzymatic regulation of seasonal glycogen cycling in the freeze-tolerant wood frog, Rana sylvatica.

Liver glycogen is an important energy store in vertebrates, and in the freeze-tolerant wood frog, Rana sylvatica, this carbohydrate also serves as a major source of the cryoprotectant glucose. We investigated how variation in the levels of the catalytic subunit of protein kinase A (PKAc), glycogen phosphorylase (GP), and glycogen synthase (GS) relates to seasonal glycogen cycling in a temperate (Ohioan) and subarctic (Alaskan) populations of this species. In spring, Ohioan frogs had reduced potential for glycogen synthesis, as evidenced by low GS activity and high PKAc protein levels.

Cryoprotectants and extreme freeze tolerance in a subarctic population of the wood frog.

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.

Hepatocyte responses to in vitro freezing and β-adrenergic stimulation: Insights into the extreme freeze tolerance of subarctic Rana sylvatica.

The wood frog, Rana sylvatica LeConte 1825, is a freeze-tolerant amphibian widely distributed in North America. Subarctic populations of this species can survive experimental freezing to temperatures below -16 °C, whereas temperate populations tolerate freezing only at temperatures above -6 °C. We investigated whether hepatocytes isolated from frogs indigenous to Interior Alaska (subarctic) or southern Ohio (temperate) had distinct characteristics that could contribute to this variation in freeze tolerance capacity.

Effect of physiological stress on expression of glucose transporter 2 in liver of the wood frog, Rana sylvatica.

Glucose transporters (GLUTs) have been implicated in the survival of various physiological stresses in mammals; however, little is known about the role of these proteins in stress tolerance in lower vertebrates. The wood frog (Rana sylvatica), which survives multiple winter-related stresses by copiously mobilizing hepatic glycogen stores, is an interesting subject for the study of glucose transport in amphibians. We examined the effects of several physiological stresses on GLUT2 protein and mRNA levels in the liver of R.

Membrane adaptation in phospholipids and cholesterol in the widely distributed, freeze-tolerant wood frog, Rana sylvatica.

Maintaining proper membrane phase and fluidity is important for preserving membrane structure and function, and by altering membrane lipid composition many organisms can adapt to changing environmental conditions. We compared the phospholipid and cholesterol composition of liver and brain plasma membranes in the freeze-tolerant wood frog, Rana sylvatica, from southern Ohio and Interior Alaska during summer, fall, and winter. We also compared membranes from winter-acclimatized frogs from Ohio that were either acclimated to 0, 4, or 10 °C, or frozen to -2.

Cloning, characterization, and expression of glucose transporter 2 in the freeze-tolerant wood frog, Rana sylvatica.

Background: The essential role of glucose transporter 2 (GLUT2) in glucose homeostasis has been extensively studied in mammals; however, little is known about this important protein in lower vertebrates. The freeze-tolerant wood frog (Rana sylvatica), which copiously mobilizes glucose in response to freezing, represents an excellent system for the study of glucose transport in amphibians.

Methods: GLUT2 was sequenced from northern and southern phenotypes of R.

Enzymatic regulation of glycogenolysis in a subarctic population of the wood frog: implications for extreme freeze tolerance.

The wood frog, Rana sylvatica, from Interior Alaska survives freezing at -16°C, a temperature 10-13°C below that tolerated by its southern conspecifics. We investigated the hepatic freezing response in this northern phenotype to determine if its profound freeze tolerance is associated with an enhanced glucosic cryoprotectant system. Alaskan frogs had a larger liver glycogen reserve that was mobilized faster during early freezing as compared to conspecifics from a cool-temperate region (southern Ohio, USA).

Hibernation physiology, freezing adaptation and extreme freeze tolerance in a northern population of the wood frog.

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±10 μmol ml(-1)) and an unidentified solute (to ~73 μmol ml(-1)). In contrast, frogs from a cool-temperate population (southern Ohio, USA) amassed much less glycogen, had a lower uremia (28±5 μmol ml(-1)) and apparently lacked the unidentified solute.

Avoidance and tolerance of freezing in ectothermic vertebrates.

Ectothermic vertebrates have colonized regions that are seasonally or perpetually cold, and some species, particularly terrestrial hibernators, must cope with temperatures that fall substantially below 0°C. Survival of such excursions depends on either freeze avoidance through supercooling or freeze tolerance. Supercooling, a metastable state in which body fluids remain liquid below the equilibrium freezing/melting point, is promoted by physiological responses that protect against chilling injury and by anatomical and behavioral traits that limit risk of inoculative freezing by environmental ice and ice-nucleating agents.

Seasonal variation and response to osmotic challenge in urea transporter expression in the dehydration- and freeze-tolerant wood frog, Rana sylvatica.

Urea accumulation is a universal response to osmotic challenge in anuran amphibians, and facilitative urea transporters (UTs) seem to play an important role in this process by acting in the osmoregulatory organs to mediate urea retention. Although UTs have been implicated in urea reabsorption in anurans, little is known about the physiological regulation of UT protein abundance. We examined seasonal variation in and effects of osmotic challenge on UT protein and mRNA levels in kidney and urinary bladder of the wood frog (Rana sylvatica), a terrestrial species that tolerates both dehydration and tissue freezing.

Seasonal variation in the hepatoproteome of the dehydration and freeze-tolerant wood frog, Rana sylvatica.

Winter's advent invokes physiological adjustments that permit temperate ectotherms to cope with stresses such as food shortage, water deprivation, hypoxia, and hypothermia. We used liquid chromatography (LC) in combination with tandem mass spectrometry (MS/MS) quantitative isobaric (iTRAQ™) peptide mapping to assess variation in the abundance of hepatic proteins in summer- and winter-acclimatized wood frogs (Rana sylvatica), a northerly-distributed species that tolerates extreme dehydration and tissue freezing during hibernation. Thirty-three unique proteins exhibited strong seasonal lability.

Urea-induced hypometabolism in the hibernating wood frog (Rana sylvatica) is not reflected in isolated mitochondria.

It has long been speculated that urea accumulated during seasonal dormancy contributes to metabolic depression. Recent work suggests urea can indeed act as a metabolic depressant during dormancy in a number of taxonomically diverse species of ectotherms. The mechanisms by which urea exerts its hypometabolic effect are unknown, but potentially stem from inhibition of mitochondrial respiration.

Winter severity and phenology of spring emergence from the nest in freshwater turtles.

Although many species of freshwater turtles emigrate to water soon after hatching, the offspring of some species overwinter on land and move to aquatic habitats in the following spring. Timing of emigration can affect the hatchlings' fitness, but the factors underlying phenology of nest emergence are incompletely understood. We tested the supposition that cold stress imposed during hibernation can influence the timing of nest emergence in three species of turtles in the central USA.

Brief Chilling to Subzero Temperature Increases Cold Hardiness in the Hatchling Painted Turtle (Chrysemys picta).

Although many studies of ectothermic vertebrates have documented compensatory changes in cold hardiness associated with changes of season, much less attention has been paid to adjustment of physiological functions and survival limits following more acute exposure to cold. We investigated the ability of hatchling painted turtles (Chrysemys picta) to increase cold hardiness in response to brief exposure to a subzero temperature. Winter-acclimated turtles were "cold conditioned" by chilling them in the supercooled (unfrozen) state to -7 degrees C over a few days before returning them to 4 degrees C.

Evidence for urea-induced hypometabolism in isolated organs of dormant ectotherms.

Many organisms endure extended periods of dormancy by depressing their metabolism, which effectively prolongs the use of their endogenous energy stores. Though the mechanisms of hypometabolism are varied and incompletely understood, recent work suggests that urea accumulation in autumn and early winter contributes to reduced metabolism of hibernating wood frogs (Rana sylvatica). Urea accumulation during dormancy is a widespread phenomenon, and it has long been presumed that numerous species from diverse taxa benefit from its hypometabolic effect.

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

We tested the hypothesis that urea, an osmolyte accumulated early in hibernation, functions as a cryoprotectant in the freeze-tolerant wood frog, Rana sylvatica. Relative to saline-treated, normouremic (10 micromol ml(-1)) frogs, individuals rendered hyperuremic (70 micromol ml(-1)) by administration of an aqueous urea solution exhibited significantly higher survival (100% versus 64%) following freezing at -4 degrees 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.

Urea production capacity in the wood frog (Rana sylvatica) varies with season and experimentally induced hyperuremia.

Wood frogs (Rana sylvatica) can accumulate substantial amounts of urea during fall and winter. In this study, maximal urea production capacity was examined in R. sylvatica collected at various times of the year and in response to experimental hyperuremia and dehydration.

Physiological ecology of overwintering in hatchling turtles.

Temperate species of turtles hatch from eggs in late summer. The hatchlings of some species leave their natal nest to hibernate elsewhere on land or under water, whereas others usually remain inside the nest until spring; thus, post-hatching behavior strongly influences the hibernation ecology and physiology of this age class. Little is known about the habitats of and environmental conditions affecting aquatic hibernators, although laboratory studies suggest that chronically hypoxic sites are inhospitable to hatchlings.

Metabolic depression induced by urea in organs of the wood frog, Rana sylvatica: effects of season and temperature.

It has long been suspected that urea accumulation plays a key role in the induction or maintenance of metabolic suppression during extended dormancy in animals from diverse taxa. However, little evidence supporting that hypothesis in living systems exists. We measured aerobic metabolism of isolated organs from the wood frog (Rana sylvatica) in the presence or absence of elevated urea at various temperatures using frogs acclimatized to different seasons.

Urea loading enhances postfreeze performance of frog skeletal muscle.

The wood frog (Rana sylvatica) is a terrestrial hibernator that can accumulate urea as an osmoprotectant in autumn and winter. This study tested the hypothesis that elevated urea can also function as a cryoprotectant in this freeze-tolerant species. Performance characteristics (threshold stimulus voltage, maximal isometric twitch and tetanic contraction forces, and (1/2) fatigue time) of isolated gastrocnemius muscles were measured before and after experimental freezing at -1.

Osmotic and metabolic responses to dehydration and urea-loading in a dormant, terrestrially hibernating frog.

Physiological responses to dehydration in amphibians are reasonably well documented, although little work has addressed this problem in hibernating animals. We investigated osmotic and metabolic responses to experimental manipulation of hydration state in the wood frog (Rana sylvatica), a terrestrial hibernator that encounters low environmental water potential during autumn and winter. In winter-conditioned frogs, plasma osmolality varied inversely with body water content (range 69-79%, fresh mass) primarily due to increases in sodium and chloride concentrations, as well as accumulation of glucose and urea.

Oxidative stress and antioxidant capacity of a terrestrially hibernating hatchling turtle.

Hatchlings of the painted turtle, Chrysemys picta, hibernate terrestrially and can survive subfreezing temperatures by supercooling or by tolerating the freezing of their tissues. Whether supercooled or frozen, an ischemic hypoxia develops because tissue perfusion is limited by low temperature and/or freezing. Oxidative stress can occur if hatchlings lack sufficient antioxidant defenses to minimize or prevent damage by reactive oxygen species.