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°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

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°C over a few days before returning them to 4°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°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°C for 24 h, followed by rewarming to 0°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

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

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SUMMARY We 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

Identification and expression of a putative facilitative urea transporter in three species of true frogs (Ranidae): implications for terrestrial adaptation.

Urea transporters (UTs) help mediate the transmembrane movement of urea and therefore are likely important in amphibian osmoregulation. Although UTs contribute to urea reabsorption in anuran excretory organs, little is known about the protein’s distribution and functions in other tissues, and their importance in the evolutionary adaptation of amphibians to their environment remains unclear. To address these questions, we obtained a partial sequence of a putative UT and examined relative abundance of this protein in tissues of the wood frog (Rana sylvatica), leopard frog (R. pipiens), and mink frog (R. septentrionalis), closely related species that are adapted to different habitats. Using immunoblotting techniques, we found the protein to be abundant in the osmoregulatory organs but also present in visceral organs, suggesting that UTs play both osmoregulatory and nonosmoregulatory roles in amphibians. UT abundance seems to relate to the species’ habitat preference, as levels of the protein were higher in the terrestrial R. sylvatica, intermediate in the semiaquatic R. pipiens, and quite low in the aquatic R. septentrionalis. These findings suggest that, in amphibians, UTs are involved in various physiological processes, including solute and water dynamics, and that they have played a role in adaptation to the osmotic challenges of terrestrial environments

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

Abstract 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°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

Physiological Ecology of Overwintering in the Hatchling Painted Turtle: Multiple-Scale Variation in response to Environmental Stress

We integrates 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 analysis. 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°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

Cold-hardiness and evaporative water loss in hatchling turtles.

North American turtles hatch in late summer and spend their first winter either on land or underwater. Adaptations for terrestrial overwintering of hatchlings in northern regions, where winter thermal and hydric regimes are harsh, have not been systematically investigated in many species. We measured intrinsic supercooling capacity, resistance to inoculative freezing, and desiccation resistance in hatchlings of terrestrial and aquatic turtles collected from northern (Terrapene ornata, Chrysemys picta bellii, Kinosternon flavescens, Chelydra serpentina) and southern (Chrysemys picta dorsalis, Trachemys scripta, Sternotherus odoratus, Sternotherus carinatus) locales. Supercooling capacity was estimated from the crystallization temperature of turtles cooled in the absence of external ice nuclei. Mean values ranged from −8.1° to −15.5°C and tended to be lower in terrestrial hibernators. Inoculation resistance was estimated from the crystallization temperature of turtles cooled in a matrix of frozen soil. These values (range of means: −0.8° to −13.6°C) also tended to be lower in the terrestrial hibernators, especially C. picta bellii. Mean rates of evaporative water loss varied markedly among the species (0.9–11.4 mg g−1 d−1) and were lowest in the terrestrial hibernators. Most species tolerated the loss of a modest amount of body water, although half of the sample of S. carinatus died from desiccation. In general, turtles did not regain lost body water from wet soil, and immersion in free water was required for rehydration. Therefore, desiccation resistance may be an important adaptation to terrestrial hibernation. Resistances to inoculative freezing and desiccation were directly correlated, perhaps because they are governed by the same morphological characteristics

Ice nuclei in soil compromise cold hardiness of hatchling painted turtles, Chrysemys picta.

Hatchling painted turtles (Chrysemys picta) commonly overwinter within their natal nests and survive exposure to temperatures as low as -12 degrees C by supercooling. We report that the supercooling capacity of hatchling C. picta is reduced by direct contact with nest soil which, in samples from northwestern and north-central Nebraska, Indiana, and Ontario, contained potent ice nuclei active in the range of -3.5 degrees to -5 degrees C. These nuclei were sensitive to autoclaving and extractable in water. The supercooling capacity of C. picta hatched in native nest soil, or hatched in sterilized vermiculite (which lacks water-extractable nuclei), and subsequently exposed to nest soil, was reduced by ∼10 degrees C relative to control turtles that were hatched and reared in sterilized vermiculite. The effect of these nuclei was potentiated by the presence of environmental moisture, although even transient exposure to dry nest soil markedly reduced supercooling capacity in ∼ 50% of the turtles. Unlike turtle species that hibernate underwater (Sternotherus odoratus, Chelydra serpentina, Apalone spinifera), hatchlings of C. picta exhibited an extraordinary capacity for supercooling (temperature of crystallization, -16 degrees to -20 degrees C) when cooled in isolation from external ice nuclei. However, hatchlings of these four species were equally susceptible to inoculation by suspensions of the ice-nucleating bacterium, Pseudomonas syringae. Indirect evidence suggests that the soil nuclei are associated with such microbes. Nucleating activity was higher in soil collected within nests than in soil collected at the same depth, adjacent to these nests. Differences in the activities of ice nuclei in nesting soils may account for geographic and local variation in winter survival of hatchling C. picta. Our finding that similar agents occur in various other terrestrial habitats in central North America suggests that such nuclei may pose a formidable challenge to the overwintering survival of ectothermic animals that rely on supercooling to withstand frost exposure