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 (Rana sylvatica), 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. Furthermore, experimentally augmenting the host's plasma urea increased bacterial urease activity. Bacterial inventories constructed using 16S rRNA sequencing revealed that the assemblages hosted by hibernating and active frogs were equally diverse but markedly differed in community membership and structure. Hibernating frogs hosted a greater relative abundance and richer diversity of genera that possess urease-encoding genes and/or have member taxa that reportedly hydrolyse urea. Bacterial hydrolysis of host-synthesized urea probably permits conservation and repurposing of valuable nitrogen not only in hibernating R. sylvatica but, given urea's universal role in amphibian osmoregulation, also in virtually all Amphibia.

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. sylvatica, as well as the freeze-intolerant Rana pipiens. These proteins were expressed and functionally characterized in Xenopus oocytes. Abundance of GLUT2 in tissues was analyzed using immunoblotting techniques. RESULTS: GLUT2s cloned from these anurans encoded proteins with high sequence homologies to known vertebrate GLUT2s and had similar transport properties, although, notably, transport of the glucose analog 3-O-methyl-d-glucose (3-OMG) was strongly inhibited by 150mM urea. Proteins from all study subjects had similar affinity constants (~12mM) and other kinetic properties; however, GLUT2 abundance in liver was 3.5-fold greater in northern R. sylvatica than in the southern conspecific and R. pipiens. CONCLUSION: Our results indicate that amphibian GLUT2s are structurally and functionally similar to their homologs in other vertebrates, attesting to the conserved nature of this transport protein. The greater abundance of this protein in the northern phenotype of R. sylvatica suggests that these transporters contribute importantly to freezing survival. GENERAL SIGNIFICANCE: This study provides the first functional characterization of any GLUT isoform from an anuran amphibian and novel insights into the role of these proteins in glucose homeostasis and cryoprotectant mobilization in freeze-tolerant vertebrates.

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. Freeze tolerance evolved from responses to fundamental stresses to permit survival of the freezing of a substantial amount of body water under thermal and temporal conditions of ecological relevance. Survival of freezing is promoted by a complex suite of molecular, biochemical and physiological responses that limit cell death from excessive shrinkage, damage to macromolecules and membranes, metabolic perturbation and oxidative stress. Although freeze avoidance and freeze tolerance generally are mutually exclusive strategies, a few species can switch between them, the mode used in a particular instance of chilling depending on prevailing physiological and environmental conditions.

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. 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 48 h) 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.

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. Livers of winter frogs had relatively high levels of proteins involved in cytoprotection, including heat-shock proteins and an antioxidant, and a reduced abundance of proteins involved in cell proliferation, protein synthesis, and mitochondrial function. They also exhibited altered levels of certain metabolic enzymes that participate in the biochemical reorganization associated with aphagia and reliance on energy reserves, as well as the freezing mobilization and post-thaw recovery of glucose, an important cryoprotective solute in freezing adaptation.

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. In each year of the 6-year study, Chrysemys picta emerged in late March and early April and, on average, these hatchlings left their nests 2 weeks earlier than those of Graptemys geographica and 4 weeks earlier than those of Trachemys scripta. Emergence of conspecific hatchlings from different nests usually occurred over 3-7 weeks, but in some years lasted several additional weeks. Relatively few nests had siblings that emerged on the same day (i.e., synchronously); complete emergence of the typical sibling group required 1 to 2 weeks. In winter, subzero cold occurred with regularity in the nests of all species, though C. picta experienced the lowest temperatures owing to their shallower nests. However, for no species did emergence date or length of the emergence period correlate with winter minimum temperature and, at the level of the individual nest, neither did emergence synchrony or duration. Despite encountering lower temperatures, hatchlings of C. picta emigrated from their nests before those of sympatric species, suggesting that the fitness benefits of early emergence may lead to the improvement of cold-hardiness adaptations in northern populations of turtles.

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. Their exceptional freeze tolerance is partly supported by an enhanced cryoprotectant system that uses very high levels of urea and glucose to limit ice formation, regulate metabolism, and protect macromolecules and cellular structures from freezing/thawing stresses. In the weeks before they begin hibernating, northern frogs undertake radical physiological transitions, such as depletion of fat stores and catabolism of muscle protein, that prime the cryoprotectant system by accruing urea and stockpiling glycogen from which glucose is mobilized during freezing. Concentrations of cryoprotectants ultimately achieved in Alaskan frogs when freezing occurs vary among tissues but generally are higher than those of frogs inhabiting milder climates. This review summarizes the molecular, biochemical, and physiological adaptations permitting this northern phenotype to survive the long and harsh winters of the region.

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 (NO2-) and nitrate (NO3-), 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. We found elevated concentrations of NO2- and/or NO3- in the plasma (up to 4.1-fold), heart (3.1-fold), and liver (up to 4.1-fold) of frogs subjected to experimental hypoxia (24 h, 4 °C), and in the liver (up to 3.8-fold) of experimentally frozen frogs (48 h, - 2.5 °C), suggesting that increased NO availability aids in survival of these stresses. During a 38-week period of simulated hibernation, NO2- and/or NO3- increased in the plasma (up to 10.4-fold), heart (up to 3.3-fold), and liver (5.0-fold) during an initial 5-week winter-acclimatization regimen and generally remained elevated thereafter. In hibernation, plasma NO2- was higher in frogs indigenous to Interior Alaska than in conspecifics from a temperate locale (southern Ohio), suggesting that NO availability is matched to the severity of environmental conditions prevailing in winter. The comparatively high NO availability in R. sylvatica, a stress-tolerant species, together with published values for other species, suggest that the NO protection system is of general importance in the stress adaptation of vertebrates.

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. In addition, glycogen levels in spring were the lowest of four seasonal samples, as energy input was likely directed towards metabolism and somatic growth during this period. Near-maximal glycogen levels were reached by mid-summer, and remained unchanged in fall and winter, suggesting that glycogenesis was curtailed during this period. Ohioan frogs had a high potential for glycogenolysis and glycogenesis in winter, as evidenced by large glycogen reserves, high levels of GP and GS proteins, and high GS activity, which likely allows for rapid mobilization of cryoprotectant during freezing and replenishing of glycogen reserves during thawing. Alaskan frogs also achieved a near-maximal liver glycogen concentration by summer and displayed high glycogenic and glycogenolytic potential in winter, but, unlike Ohioan frogs, started replenishing their energy reserves early in spring. We conclude that variation in levels of both glycogenolytic and glycogenic enzymes likely happens in response to seasonal changes in energetic strategies and demands, with winter survival being a key component to understanding the regulation of glycogen cycling in this species.

Survival and physiological responses of hatchling blanding's turtles (Emydoidea blandingii) to submergence in normoxic and hypoxic water under simulated winter conditions.

Overwintering habits of hatchling Blanding's turtles (Emydoidea blandingii) are unknown. To determine whether these turtles are able to survive winter in aquatic habitats, we submerged hatchlings in normoxic (155 mmHg Po2) and hypoxic (6 mmHg Po2) water at 4 degrees C, recording survival times and measuring changes in key physiological variables. For comparison, we simultaneously studied hatchling softshell (Apalone spinifera) and snapping (Chelydra serpentina) turtles, which are known to overwinter in aquatic habitats. In normoxic water, C. serpentina and A. spinifera survived to the termination of the experiment (76 and 77 d, respectively). Approximately one-third of the E. blandingii died during 75 d of normoxic submergence, but the cause of mortality was unclear. In hypoxic water, average survival times were 6 d for A. spinifera, 13 d for E. blandingii, and 19 d for C. serpentina. Mortality during hypoxic submergence was probably caused by metabolic acidosis, which resulted from accumulated lactate. Unlike the case with adult turtles, our hatchlings did not increase plasma calcium and magnesium, nor did they sequester lactate within the shell. Our results suggest that hatchling E. blandingii are not particularly well suited to hibernation in hypoxic aquatic habitats.

Hepatocyte responses to in vitro freezing and ß-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. Following in vitro freezing, cell damage, as assessed from lactate dehydrogenase leakage, was similar between samples from Alaskan and Ohioan frogs. Preincubation of cells in media containing glucose or urea, the two primary cryoprotectants used by R. sylvatica, markedly reduced freezing damage to hepatocytes; however, results suggested that cells of the northern phenotype were comparatively more amenable to cryoprotection by urea. Stimulation of isolated hepatocytes with ß-adrenergic agonists, which simulates the freezing-induced cryoprotectant mobilization response, gave rates of glucose production from endogenous glycogen reserves that were similar between the populations. Our findings suggest that extreme freeze tolerance in subarctic R. sylvatica does not require an enhanced ability of the liver to resist freezing stress or rapidly mobilize cryoprotectant.

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 85.5 ± 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 of freezing/thawing between -4 and 0°C, or -16°C increased the liver's synthesis of glucose and, to a lesser extent, urea. Concomitantly, organs shed up to one-half (skeletal muscle) or two-thirds (liver) of their water, with cryoprotectant in the remaining fluid reaching concentrations as high as 0.2 and 2.1 M, respectively. Freeze/thaw cycling, which was readily survived by winter-conditioned frogs, greatly increased hepatic glycogenolysis and delivery of glucose (but not urea) to skeletal muscle. We conclude that cryoprotectant accrual in anticipation of and in response to freezing have been greatly enhanced and contribute to extreme freeze tolerance in northern R. sylvatica.

Postfreeze reduction of locomotor endurance in the freeze-tolerant wood frog, Rana sylvatica.

Considerable study has focused on the physiological adaptations for freeze tolerance in the wood frog, Rana sylvatica, a northern species that overwinters within the frost zone, but little attention has been paid to the associated costs to organismal performance. Here we report that freezing causes transient impairment of locomotor endurance and adverse changes in exercise physiology that persist for at least 96 h. Wood frogs frozen at -2 degrees C for 36 h exhibited normal behaviors and hydro-osmotic status and near-normal metabolite (glycogen, glucose, and lactate) levels within 24 h after thawing began. However, when exercised to exhaustion on a treadmill, these frogs showed a 40% reduction in endurance as compared to sham-treated (unfrozen) controls, a reduction that persisted for at least 96 h. Previously frozen frogs exhibited higher rates of lactate accumulation during exercise than controls, suggesting that prior freezing forces greater reliance on the glycolytic pathways of energy production to support exercise. Given that this species breeds in late winter, when subzero temperatures are common, freezing may result in reduced fitness by hampering their ability to reach the pond, avoid predators, and successfully obtain mates.

Physiological ecology of overwintering in the hatchling painted turtle: multiple-scale variation in response to environmental stress.

We integrated field and laboratory studies in an investigation of water balance, energy use, and mechanisms of cold-hardiness in hatchling painted turtles (Chrysemys picta) indigenous to west-central Nebraska (Chrysemys picta bellii) and northern Indiana (Chrysemys picta marginata) during the winters of 1999-2000 and 2000-2001. We examined 184 nests, 80 of which provided the hatchlings (n=580) and/or samples of soil used in laboratory analyses. Whereas winter 1999-2000 was relatively dry and mild, the following winter was wet and cold; serendipitously, the contrast illuminated a marked plasticity in physiological response to environmental stress. Physiological and cold-hardiness responses of turtles also varied between study locales, largely owing to differences in precipitation and edaphics and the lower prevailing and minimum nest temperatures (to -13.2 degrees C) encountered by Nebraska turtles. In Nebraska, winter mortality occurred within 12.5% (1999-2000) and 42.3% (2000-2001) of the sampled nests; no turtles died in the Indiana nests. Laboratory studies of the mechanisms of cold-hardiness used by hatchling C. picta showed that resistance to inoculative freezing and capacity for freeze tolerance increased as winter approached. However, the level of inoculation resistance strongly depended on the physical characteristics of nest soil, as well as its moisture content, which varied seasonally. Risk of inoculative freezing (and mortality) was greatest in midwinter when nest temperatures were lowest and soil moisture and activity of constituent organic ice nuclei were highest. Water balance in overwintering hatchlings was closely linked to dynamics of precipitation and soil moisture, whereas energy use and the size of the energy reserve available to hatchlings in spring depended on the winter thermal regime. Acute chilling resulted in hyperglycemia and hyperlactemia, which persisted throughout winter; this response may be cryoprotective. Some physiological characteristics and cold-hardiness attributes varied between years, between study sites, among nests at the same site, and among siblings sharing nests. Such variation may reflect adaptive phenotypic plasticity, maternal or paternal influence on an individual's response to environmental challenge, or a combination of these factors. Some evidence suggests that life-history traits, such as clutch size and body size, have been shaped by constraints imposed by the harsh winter environment.

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 -2.5 °C and sampled before or after thawing. Lipids were extracted from isolated membranes, separated by one-dimensional thin-layer chromatography, and analyzed via densitometry. Liver membranes underwent seasonal changes in phospholipid composition and lipid ratios, including a winter increase in phosphatidylethanolamine, which serves to increase fluidity. However, whereas Ohioan frogs decreased phosphatidylcholine and increased sphingomyelin, Alaskan frogs only decreased phosphatidylserine, indicating that these phenotypes use different adaptive strategies to meet the functional needs of their membranes. Liver membranes showed no seasonal variation in cholesterol abundance, though membranes from Alaskan frogs contained relatively less cholesterol, consistent with the need for greater fluidity in a colder environment. No lipid changed seasonally in brain membranes in either population. In the thermal acclimation experiment, cold exposure induced an increase in phosphatidylethanolamine in liver membranes and a decrease in cholesterol in brain membranes. No changes occurred during freezing and thawing in membranes from either organ. Wood frogs use tissue-specific membrane adaptation of phospholipids and cholesterol to respond to changing environmental factors, particularly temperature, though not with freezing.

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. sylvatica. Using immunoblotting techniques to measure relative GLUT2 abundance, we found that GLUT2 numbers increased in response to organismal freezing, hypoxia exposure, and glucose loading; whereas, experimental dehydration and urea loading did not affect GLUT2 abundance. GLUT2 mRNA levels, assessed using quantitative real-time polymerase chain reaction, changed in accordance with protein abundance for most stresses, indicating that transcriptional regulation of GLUT2 occurs in response to stress. Overall, hepatic GLUT2 seems to be important in stress survival in R. sylvatica and is regulated to meet the physiological need to accumulate glucose.

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). In Alaskan frogs the rapid glucose production in the first hours of freezing was associated with a 7-fold increase in glycogen phosphorylase activity above unfrozen frog levels, and the activity of this enzyme was higher than that of frozen Ohioan frogs. Freezing of Ohioan frogs induced a more modest (4-fold) increase in glycogen phosphorylase activity above unfrozen frog values. Relative to the Ohioan frogs, Alaskan frogs maintained a higher total protein kinase A activity throughout an experimental freezing/thawing time course, and this may have potentiated glycogenolysis during early freezing. We found populational variation in the activity and protein level of protein kinase A which suggested that the Alaskan population had a more efficient form of this enzyme. Alaskan frogs modulated their glycogenolytic response by decreasing the activity of glycogen phosphorylase after cryoprotectant mobilization was well under way, thereby conserving their hepatic glycogen reserve. Ohioan frogs, however, sustained high glycogen phosphorylase activity until early thawing and consumed nearly all their liver glycogen. These unique hepatic responses of Alaskan R. sylvatica likely contribute to this phenotype's exceptional freeze tolerance, which is necessary for their survival in a subarctic climate.

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. Using immunoblotting techniques to measure relative UT abundance, we found that UT numbers varied seasonally, with a low abundance prevailing in the fall and winter, and higher levels occurring in the spring. Experimental dehydration of frogs increased UT protein abundance in the urinary bladder, whereas experimental urea loading decreased the abundance of UTs in kidney and bladder. Experimental freezing, whether or not followed by thawing, had no effect on UT numbers. UT mRNA levels, assessed using quantitative real-time polymerase chain reaction, did not change seasonally nor in response to any of our experimental treatments. These findings suggest that regulation of UTs depends on the nature and severity of the osmotic stress and apparently occurs posttranscriptionally in response to multiple physiological factors. Additionally, UTs seem to be regulated to meet the physiological need to accumulate urea, with UT numbers increasing to facilitate urea reabsorption and decreasing to prevent retention of excess urea.

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. To investigate the phylogenetic prevalence of urea-induced hypometabolism, we studied four species of urea accumulators from the clades Amphibia (Spea bombifrons and Ambystoma tigrinum), Reptilia (Malaclemys terrapin), and Gastropoda (Anguispira alternata), and one amphibian species (R. pipiens) that does not accumulate urea during dormancy. We measured rates of oxygen consumption (VO(2)) of excised organ samples from dormant animals in the presence or absence of physiological concentrations of urea. Three of the four urea-accumulating species had at least one organ whose VO(2) was significantly decreased by urea treatment. However, VO(2) of organs from R. pipiens, the one species tested that does not accumulate urea during dormancy, was not affected by urea treatment. Our results support the hypothesis that urea accumulation can reduce metabolic rate of dormant animals and provide a base for further investigation into the evolution of urea-induced hypometabolism.

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. These turtles fared no better than control animals in resisting freezing when cooled in the presence or absence of ice and exogenous ice nuclei. Survival following tests of freeze tolerance (freezing for about 70 h; minimum body temperature, -3.75 degrees C) was nominally higher in cold-conditioned turtles than in controls (36% vs. 13%, respectively), although the difference was not statistically significant. Of the survivors, cold-conditioned turtles apparently recovered sooner. Turtles subjected to cold shock (supercooling to -13 degrees C for 24 h, followed by rewarming to 0 degrees C) were strongly affected by cold conditioning: all controls died, but 50% of cold-conditioned turtles survived. We investigated potential mechanisms underlying the response to cold conditioning by measuring changes in levels of putative cryoprotectants. Plasma levels of glucose and lactate, but not urea, were higher in cold-conditioned turtles than in controls, although the combined increase in these solutes was only 23 mmol L(-1). Cold conditioning attenuated cold-shock injury to brain cells, as assessed using a vital-dye assay, suggesting a link between protection of the nervous system and cold hardiness at the organismal level.