Effects of urbanization on the population structure of freshwater turtles across the United States.

Landscape-scale alterations that accompany urbanization may negatively affect the population structure of wildlife species such as freshwater turtles. Changes to nesting sites and higher mortality rates due to vehicular collisions and increased predator populations may particularly affect immature turtles and mature female turtles. We hypothesized that the proportions of adult female and immature turtles in a population will negatively correlate with landscape urbanization. As a collaborative effort of the Ecological Research as Education Network (EREN), we sampled freshwater turtle populations in 11 states across the central and eastern United States. Contrary to expectations, we found a significant positive relationship between proportions of mature female painted turtles (Chrysemys picta) and urbanization. We did not detect a relationship between urbanization and proportions of immature turtles. Urbanization may alter the thermal environment of nesting sites such that more females are produced as urbanization increases. Our approach of creating a collaborative network of scientists and students at undergraduate institutions proved valuable in terms of testing our hypothesis over a large spatial scale while also allowing students to gain hands-on experience in conservation science.

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.

Daily thermal fluctuations to a range of subzero temperatures enhance cold hardiness of winter-acclimated turtles.

Although seasonal increases in cold hardiness are well documented for temperate and polar ectotherms, relatively little is known about supplemental increases in cold hardiness during winter. Because many animals are exposed to considerable thermal variation in winter, they may benefit from a quick enhancement of cold tolerance prior to extreme low temperature. Hatchling painted turtles (Chrysemys picta) overwintering in their natal nests experience substantial thermal variation in winter, and recently, it was found that brief subzero chilling of winter-acclimated hatchlings decreases subsequent chilling-induced mortality, increases blood concentrations of glucose and lactate, and protects the brain from cryoinjury. Here, we further characterize that phenomenon, termed 'cold conditioning', by exposing winter-acclimated hatchling turtles to -3.5, -7.0, or -10.5 °C gradually or repeatedly via daily thermal fluctuations over the course of 5 days and assessing their survival of a subsequent cold shock to a discriminating temperature of -12.7 °C. To better understand the physiological response to cold conditioning, we measured changes in glucose and lactate concentrations in the liver, blood, and brain. Cold conditioning significantly increased cold-shock survival, from 9% in reference turtles up to 74% in cold-conditioned turtles, and ecologically relevant daily thermal fluctuations were at least as effective at conferring cryoprotection as was gradual cold conditioning. Cold conditioning increased glucose concentrations, up to 25 µmol g-1, and lactate concentrations, up to 30 µmol g-1, in the liver, blood, and brain. Turtles that were cold conditioned with daily thermal fluctuations accumulated more glucose in the liver, blood, and brain, and had lower brain lactate, than those gradually cold conditioned. Given the thermal variation to which hatchling painted turtles are exposed in winter, we suggest that the supplemental protection conferred by cold conditioning, especially that induced by daily thermal fluctuations, may be important for their overwinter survival. Investigation into the duration of the cold-conditioning induced protection and its occurrence in natural field conditions is needed to better understand its ecological significance. We suggest that future work exploring the underlying mechanisms of cold conditioning should focus on non-colligative effects of glucose, expression of small Hsps, changes in membrane structure, and ion homeostasis.

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.

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.

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. We isolated mitochondria from Rana sylvatica skeletal muscle, an organ that is metabolically responsive to urea, and measured respiration rates in the absence or presence of 80 mmol l(-1) urea in the respiration medium. Because the effect of urea may be influenced by the intracellular milieu, in these experiments we varied substrate (pyruvate or palmitoylcarnitine), temperature (4, 10, or 15°C), and pH (6.8 or 7.4). Oxygen consumption of control and urea-treated mitochondria was sensitive to each of these variables, but neither state 3 nor state 4 respiration was reduced by urea treatment and, to the contrary, urea treatment slightly increased state 4 respiration at higher test temperatures. Although we did not test the efficacy of other incubation times or urea concentrations, the outcome of our experiment intimates that the urea-induced hypometabolism observed in hibernating R. sylvatica results from inhibition of energy-utilizing processes elsewhere in the cell, rather than a direct inhibition of mitochondrial respiration. Future investigation into urea's effects on non-mitochondrial metabolic pathways is necessary to uncover the mechanisms by which urea depresses metabolic rate.

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. The depressive effect of urea on metabolism was not consistent across organs, seasons, or temperatures. None of the organs from summer frogs, which were tested at 20 degrees C, or from winter frogs tested at 4 degrees C were affected by urea treatment. However, liver, stomach, and heart from spring frogs tested at 4 degrees C had significantly lower metabolic rates when treated with urea as compared with control samples. Additionally, when organs from winter frogs were tested at 10 degrees C, metabolism was significantly decreased in urea-treated liver and stomach by approximately 15% and in urea-treated skeletal muscle by approximately 50%. Our results suggest that the presence of urea depresses the metabolism of living organs, and thereby reduces energy expenditure, but its effect varies with temperature and seasonal acclimatization. The impact of our findings may be wide ranging owing to the number of diverse organisms that accumulate urea during dormancy.

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. Decreased hydration was accompanied by a marked reduction in the resting rate of oxygen consumption, which was inversely correlated with plasma osmolality and urea concentration. In a separate experiment, resting rates of oxygen consumption in fully hydrated frogs receiving injections of saline or saline containing urea did not differ initially; however, upon dehydration, metabolic rates decreased sooner in the urea-loaded frogs than in control frogs. Our findings suggest an important role for urea, acting in concert with dehydration, in the metabolic regulation and energy conservation of hibernating R. sylvatica.