Cardiovascular contributions and energetic costs of thermoregulation in ectothermic vertebrates.

Ectothermic vertebrates use a suite of physiological and behavioral mechanisms to thermoregulate, which result in various thermoregulatory strategies from thermoconformity to thermoregulation. Here, we present a novel synthesis of theoretical and empirical methods to determine cardiovascular contributions to heat transfer in free-living ectothermic vertebrates. We start by identifying the fundamental components of heat transfer and the cardiovascular mechanisms for physiological modulation of heat exchange, and then integrate these components into a single, integrative framework: the cardiovascular heat exchange framework (CHEF). We demonstrate that this framework can identify details of the thermoregulatory strategy in two turtle species, most notably the preponderance of instances where turtles use physiological mechanisms to avoid overheating, suggesting vulnerability to climate change. As modulated physiological contributions to heat flow incur a greater energy demand than relying on unmodulated passive heat transfer, we then asked whether we could characterize the energetic costs of thermoregulation. We measured field metabolic rate (FMR) in free-living turtles and used the CHEF to determine FMR while actively or passively thermoregulating. Comparing an individual's actual FMR to the rate calculated assuming absence of thermoregulation revealed that painted turtles, a partial thermoregulator, elevate their daily energy expenditure (DEE) by about 25%, while box turtles, a thermoconformer, have a DEE that is nearly unchanged as a result of thermoregulation. This integrative framework builds a new paradigm that provides a mechanism to explain correlations between energy demand and thermoregulatory strategy, quantifies the energetic costs of thermoregulation, and identifies the role of cardiovascular contributions to thermoregulation in free-living animals.

Daily energy expenditure in white storks is lower after fledging than in the nest.

Many juvenile birds turn into long-distance migrants within weeks of fledging. This transition involves upheavals in their energy management as major changes in growth and activity occur. Understanding such ontogenetic transitions in energy allocation has been difficult because collecting continuous data on energy costs in wild developing birds was previously largely impossible. Here, we continuously measured heart rate and fine-scale movements of 20 free-living juvenile white storks (Ciconia ciconia) using on-board bio-loggers to explore individual and environmental factors relating to daily mean heart rate. In addition, we explored which specific energy management strategy storks use during these crucial early life stages. We found that daily mean heart rate increased with overall movement activity, and increasing body temperature, but that it decreased with age. Further, we found that during the nestling period, when growth costs are high, activity costs are low, and post-fledging that activity costs are increased while maintenance costs are low, indicating a constraint on overall energy use in both phases. Our observations are consistent with the hypothesis that individuals invested more energy per unit time while still in the nest than after fledging despite the high costs of flight.

PPAR expression, muscle size and metabolic rates across the gray catbird's annual cycle are greatest in preparation for fall migration.

Phenotypic flexibility across the annual cycle allows birds to adjust to fluctuating ecological demands. Varying energetic demands associated with time of year have been demonstrated to drive metabolic and muscle plasticity in birds, but it remains unclear what molecular mechanisms control this flexibility. We sampled gray catbirds at five stages across their annual cycle: tropical overwintering (January), northward spring (late) migration (early May), breeding (mid June), the fall pre-migratory period (early August) and southward fall (early) migration (end September). Across the catbird's annual cycle, cold-induced metabolic rate (V̇O2summit) was highest during migration and lowest during tropical wintering. Flight muscles exhibited significant hypertrophy and/or hyperplasia during fall migratory periods compared with breeding and the fall pre-migratory period. Changes in heart mass were driven by the tropical wintering stage, when heart mass was lowest. Mitochondrial content of the heart and pectoralis remained constant across the annual cycle as quantified by aerobic enzyme activities (CS, CCO), as did lipid catabolic capacity (HOAD). In the pectoralis, transcription factors PPARα, PPARδ and ERRß, coactivators PGC-1α and ß, and genes encoding proteins associated with fat uptake (FABPpm, Plin3) were unexpectedly upregulated in the tropical wintering stage, whereas those involved in fatty acid oxidation (ATGL, LPL, MCAD) were downregulated, suggesting a preference for fat storage over utilization. Transcription factors and coactivators were synchronously upregulated during pre-migration and fall migration periods in the pectoralis but not the heart, suggesting that these pathways are important in preparation for and during early migration to initiate changes to phenotypes that facilitate long-distance migration.

Conserved transcriptional activity and ligand responsiveness of avian PPARs: Potential role in regulating lipid metabolism in mirgratory birds.

Migratory birds undergo metabolic remodeling in tissues, including increased lipid storage in white adipose and fatty acid uptake and oxidation in skeletal muscle, to optimize energy substrate availability and utilization in preparation for long-distance flight. Different tissues undergo gene expression changes in keeping with their specialized functions and driven by tissue specific transcriptional pathways. Peroxisome proliferator-activated receptors (PPARs) are lipid-activated nuclear receptors that regulate metabolic pathways involved in lipid and glucose utilization or storage in mammals. To examine whether PPARs might mediate fatty acid activation of metabolic gene programs that would be relevant during pre-migratory fattening, we used gray catbird as the focal species. PPAR isoforms cloned from catbird share high amino acid identity with mammalian homologs (% vs human): gcPPARα (88.1%), gcPPARδ (87.3%), gcPPARγ (91.2%). We tested whether gcPPARs activated fatty acid (FA) utilization genes using Lpl and Cpt1b gene promoter-luciferase reporters in mammalian cell lines. In C2C12 mouse myocytes gcPPARα was broadly activated by the saturated and unsaturated FAs tested; while gcPPARδ showed highest activation by the mono-unsaturated FA, 18:1 oleic acid (+80%). In CV-1 monkey kidney cells gcPPARγ responded to the poly-unsaturated fatty acid, 20:5 eicosapentaenoic acid (+60%). Moreover, in agreement with their structural conservation, gcPPARs were activated by isoform selective synthetic agonists similar to the respective mammalian isoform. Adenoviral mediated over-expression of PPARα in C2C12 myocytes induced expression of genes involved in fatty acid transport, including Cd36/Fat, as well as Cpt1b, which mediates a key rate limiting step of mitochondrial ß-oxidation. These gene expression changes correlated with increased lipid droplet accumulation in C2C12 myoblasts and differentiated myotubes and enhanced ß-oxidation in myotubes. Collectively, the data predict that the PPARs play a conserved role in gray catbirds to regulate lipid metabolism in target tissues that undergo metabolic remodeling throughout the annual migratory cycle.

Annual life-stage regulation of lipid metabolism and storage and association with PPARs in a migrant species: the gray catbird (Dumetella carolinensis).

The annual cycle of a migrating bird involves metabolically distinct stages of substantial fatty acid storage and periods of increased fatty acid mobilization and utilization, and thus requires a great deal of phenotypic flexibility. Specific mechanisms directing stage transitions of lipid metabolism in migrants are largely unknown. This study characterized the role of the PPARs (peroxisome proliferator-activated receptors) in regulating migratory adiposity of the gray catbird (Dumetella carolinensis). Catbirds increased adipose storage during spring and autumn migration and showed increased rates of basal lipolysis during migration and tropical overwintering. Expression of the PPAR target genes involved in fat uptake and storage, FABPpm and PLIN3, increased during pre-migratory fattening. We found significant correlation between PPARγ and target gene expression in adipose but little evidence that PPARα expression levels drive metabolic regulation in liver during the migratory cycle.

Animal galloping and human hopping: an energetics and biomechanics laboratory exercise.

This laboratory exercise demonstrates fundamental principles of mammalian locomotion. It provides opportunities to interrogate aspects of locomotion from biomechanics to energetics to body size scaling. It has the added benefit of having results with robust signal to noise so that students will have success even if not "meticulous" in attention to detail. First, using respirometry, students measure the energetic cost of hopping at a "preferred" hop frequency. This is followed by hopping at an imposed frequency half of the preferred. By measuring the O2 uptake and work done with each hop, students calculate mechanical efficiency. Lessons learned from this laboratory include 1) that the metabolic cost per hop at half of the preferred frequency is nearly double the cost at the preferred frequency; 2) that when a person is forced to hop at half of their preferred frequency, the mechanical efficiency is nearly that predicted for muscle but is much higher at the preferred frequency; 3) that the preferred hop frequency is strongly body size dependent; and 4) that the hop frequency of a human is nearly identical to the galloping frequency predicted for a quadruped of our size. Together, these exercises demonstrate that humans store and recover elastic recoil potential energy when hopping but that energetic savings are highly frequency dependent. This stride frequency is dependent on body size such that frequency is likely chosen to maximize this function. Finally, by requiring students to make quantitative solutions using appropriate units and dimensions of the physical variables, these exercises sharpen analytic and quantitative skills.

Contrasting Torpor Use by Reproductive Male Common Noctule Bats in the Laboratory and in the Field.

Metabolic processes of animals are often studied in controlled laboratory settings. However, these laboratory settings often do not reflect the animals' natural environment. Thus, results of metabolic measurements from laboratory studies must be cautiously applied to free-ranging animals. Recent technological advances in animal tracking allow detailed eco-physiological studies that reveal when, where, and how physiological measurements from the field differ from those from the laboratory. We investigated the torpor behavior of male common noctule bats (Nyctalus noctula) across different life history stages using two approaches: in controlled laboratory experiments and in the field using calibrated heart rate telemetry. We predicted that non-reproductive males would extensively use torpor to conserve energy, whereas reproductive males would reduce torpor use to promote spermatogenesis. We did not expect differences in torpor use between captive and wild animals as we simulated natural temperature conditions in the laboratory. We found that during the non-reproductive phase, both captive and free-ranging bats used torpor extensively. During reproduction, bats in captivity unexpectedly also used torpor throughout the day, while only free-ranging bats showed the expected reduction in torpor use. Thus, depending on life history stage, torpor behavior in the laboratory was markedly different from the wild. By implementing both approaches and at different life history stages, we were able to better explore the limitations of eco-physiological laboratory studies and make recommendations for when they are an appropriate proxy for natural behavior.

Manipulation of photoperiod induces fat storage, but not fat mobilization in the migratory songbird, Dumetella carolinensis (Gray Catbird).

The annual cycle of migratory birds requires significant phenotypic remodeling. We sought to induce the migratory phenotype in Gray Catbirds by exposing them to a short-day light cycle. While adipose storage was stimulated, exceeding that typically seen in wild birds, other aspects of the migratory phenotype were unchanged. Of particular interest, the rate of lipid export from excised adipose tissue was nearly halved. This is in contrast to wild migratory birds in which lipid export rates are increased. These data suggest that exposure to an altered light cycle only activated the lipid storage program while inhibiting the lipid transport program. The factors governing lipid mobilization and transport remain to be elucidated.

Acclimatization of seasonal energetics in northern cardinals (Cardinalis cardinalis) through plasticity of metabolic rates and ceilings.

Northern cardinals (Cardinalis cardinalis) are faced with energetically expensive seasonal challenges that must be met to ensure survival, including thermoregulation in winter and reproductive activities in summer. Contrary to predictions of life history theory that suggest breeding metabolic rate should be the apex of energetic effort, winter metabolism exceeds that during breeding in several temperate resident bird species. By examining whole-animal, tissue and cellular function, we ask whether seasonal acclimatization is accomplished by coordinated phenotypic plasticity of metabolic systems. We measured summit metabolism (V(O(2),sum)), daily energy expenditure (DEE) and muscle oxidative capacity under both winter (December to January) and breeding (May to June) conditions. We hypothesize that: (1) rates of energy utilization will be highest in the winter, contrary to predictions based on life history theory, and (2) acclimatization of metabolism will occur at multiple levels of organization such that birds operate with a similar metabolic ceiling during different seasons. We measured field metabolic rates using heart rate telemetry and report the first daily patterns in avian field metabolic rate. Patterns of daily energy use differed seasonally, primarily as birds maintain high metabolic rates throughout the winter daylight hours. We found that DEE and V(O(2),sum) were significantly greater and DEE occurred at a higher fraction of maximum metabolic capacity during winter, indicating an elevation of the metabolic ceiling. Surprisingly, there were no significant differences in mass or oxidative capacity of skeletal muscle. These data, highlighting the importance of examining energetic responses to seasonal challenges at multiple levels, clearly reject life history predictions that breeding is the primary energetic challenge for temperate zone residents. Further, they indicate that metabolic ceilings are seasonally flexible as metabolic effort during winter thermoregulation exceeds that of breeding.

Flexible energy-saving strategies in female temperate-zone bats.

Torpor is characterized by an extreme reduction in metabolism and a common energy-saving strategy of heterothermic animals. Torpor is often associated with cold temperatures, but in the last decades, more diverse and flexible forms of torpor have been described. For example, tropical bat species maintain a low metabolism and heart rate at high ambient and body temperatures. We investigated whether bats (Nyctalus noctula) from the cooler temperate European regions also show this form of torpor with metabolic inhibition at high body temperatures, and whether this would be as pronounced in reproductive as in non-reproductive bats. We simultaneously measured metabolic rate, heart rate, and skin temperature in non-reproductive and pregnant females at a range of ambient temperatures. We found that they can decouple metabolic rate and heart rate from body temperature: they maintained an extremely low metabolism and heart rate when exposed to ambient temperatures changing from 0 to 32.5 °C, irrespective of reproductive status. When we simulated natural temperature conditions, all non-reproductive bats used torpor throughout the experiment. Pregnant bats used variable strategies from torpor, to maintaining normothermy, or a combination of both. Even a short torpor bout during the day saved up to 33% of the bats' total energy expenditure. Especially at higher temperatures, heart rate was a much better predictor of metabolic rate than skin temperature. We suggest that the capability to flexibly save energy across a range of ambient temperatures within and between reproductive states may be an important ability of these bats and possibly other temperate-zone heterotherms.

Metabolic rate in common shrews is unaffected by temperature, leading to lower energetic costs through seasonal size reduction.

Small endothermic mammals have high metabolisms, particularly at cold temperatures. In the light of this, some species have evolved a seemingly illogical strategy: they reduce the size of the brain and several organs to become even smaller in winter. To test how this morphological strategy affects energy consumption across seasonally shifting ambient temperatures, we measured oxygen consumption and behaviour in the three seasonal phenotypes of the common shrew (Sorex araneus), which differ in size by about 20%. Body mass was the main driver of oxygen consumption, not the reduction of metabolically expensive brain mass. Against our expectations, we found no change in relative oxygen consumption with low ambient temperature. Thus, smaller body size in winter resulted in significant absolute energy savings. This finding could only partly be explained by an increase of lower cost behaviours in the activity budgets. Our findings highlight that these shrews manage to avoid one of the most fundamental and intuitive rules of ecology, allowing them to subsist with lower resource availability and successfully survive the harsh conditions of winter.

Impaired contractile function and calcium handling in hearts of cardiac-specific calcineurin b1-deficient mice.

To define the necessity of calcineurin (Cn) signaling for cardiac maturation and function, the postnatal phenotype of mice with cardiac-specific targeted ablation of the Cn B1 regulatory subunit (Ppp3r1) gene (csCnb1(-/-) mice) was characterized. csCnb1(-/-) mice develop a lethal cardiomyopathy, characterized by impaired postnatal growth of the heart and combined systolic and diastolic relaxation abnormalities, despite a lack of structural derangements. Notably, the csCnb1(-/-) hearts did not exhibit diastolic dilatation, despite the severe functional phenotype. Myocytes isolated from the mutant mice exhibited reduced rates of contraction/relaxation and abnormalities in calcium transients, consistent with altered sarcoplasmic reticulum loading. Levels of sarco(endo) plasmic reticulum Ca-ATPase 2a (Atp2a2) and phospholamban were normal, but phospholamban phosphorylation was markedly reduced at Ser(16) and Thr(17). In addition, levels of the Na/Ca exchanger (Slc8a1) were modestly reduced. These results define a novel mouse model of cardiac-specific Cn deficiency and demonstrate novel links between Cn signaling, postnatal growth of the heart, pathological ventricular remodeling, and excitation-contraction coupling.

Differential plasticity of membrane fatty acids in northern and southern populations of the eastern newt (Notophthalmus viridescens).

Seasonal changes in membrane composition and metabolic activity allow many temperate ectotherms to contend with changes in body temperature, but few studies have investigated whether the plasticity of these traits has diverged within a single species. Therefore, we studied the effects of thermal acclimation on the membrane fatty acid composition and the activities of cytochrome c oxidase (CCO) and citrate synthase (CS) in the skeletal muscle and liver of eastern newts from Maine and Florida. Newts were acclimated to either 6 °C or 28 °C for 12 weeks prior to experiments. Cold acclimation resulted in a lower saturated fatty acid (SFA) content in the muscle membranes of both populations. SFA content in liver was lower in cold compared to warm-acclimated newts from Florida, but acclimation did not affect SFA content in liver membranes of the Maine population. In liver, cold acclimation resulted in a higher monounsaturated fatty acid (MUFA) content in the Florida population and a higher polyunsaturated fatty acid (PUFA) content in the Maine population. Regardless of acclimation conditions, the muscle and liver membranes of the Maine population had higher SFA and PUFA contents compared to those of the Florida population. MUFA content of muscle and liver membranes was higher in the Florida population compared to the Maine population. The effect of acclimation on CCO and CS activity was tissue-specific. In muscle, CCO and CS activities were higher in cold compared to warm-acclimated newts in both populations, and CS and CCO activities were higher in the Maine compared to the Florida population. In liver, CCO and CS activity were unaffected by acclimation in the Florida population, but activity was lower in cold compared to warm-acclimated Maine newts. These results demonstrate that the phenotypic plasticity of these traits in response to seasonal change has diverged between northern and southern populations.

PGC-1alpha deficiency causes multi-system energy metabolic derangements: muscle dysfunction, abnormal weight control and hepatic steatosis.

The gene encoding the transcriptional coactivator peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) was targeted in mice. PGC-1alpha null (PGC-1alpha(-/-)) mice were viable. However, extensive phenotyping revealed multi-system abnormalities indicative of an abnormal energy metabolic phenotype. The postnatal growth of heart and slow-twitch skeletal muscle, organs with high mitochondrial energy demands, is blunted in PGC-1alpha(-/-) mice. With age, the PGC-1alpha(-/-) mice develop abnormally increased body fat, a phenotype that is more severe in females. Mitochondrial number and respiratory capacity is diminished in slow-twitch skeletal muscle of PGC-1alpha(-/-) mice, leading to reduced muscle performance and exercise capacity. PGC-1alpha(-/-) mice exhibit a modest diminution in cardiac function related largely to abnormal control of heart rate. The PGC-1alpha(-/-) mice were unable to maintain core body temperature following exposure to cold, consistent with an altered thermogenic response. Following short-term starvation, PGC-1alpha(-/-) mice develop hepatic steatosis due to a combination of reduced mitochondrial respiratory capacity and an increased expression of lipogenic genes. Surprisingly, PGC-1alpha(-/-) mice were less susceptible to diet-induced insulin resistance than wild-type controls. Lastly, vacuolar lesions were detected in the central nervous system of PGC-1alpha(-/-) mice. These results demonstrate that PGC-1alpha is necessary for appropriate adaptation to the metabolic and physiologic stressors of postnatal life.

PGC-1alpha coactivates PDK4 gene expression via the orphan nuclear receptor ERRalpha: a mechanism for transcriptional control of muscle glucose metabolism.

The transcriptional coactivator PGC-1alpha is a key regulator of energy metabolism, yet little is known about its role in control of substrate selection. We found that physiological stimuli known to induce PGC-1alpha expression in skeletal muscle coordinately upregulate the expression of pyruvate dehydrogenase kinase 4 (PDK4), a negative regulator of glucose oxidation. Forced expression of PGC-1alpha in C(2)C(12) myotubes induced PDK4 mRNA and protein expression. PGC-1alpha-mediated activation of PDK4 expression was shown to occur at the transcriptional level and was mapped to a putative nuclear receptor binding site. Gel shift assays demonstrated that the PGC-1alpha-responsive element bound the estrogen-related receptor alpha (ERRalpha), a recently identified component of the PGC-1alpha signaling pathway. In addition, PGC-1alpha was shown to activate ERRalpha expression. Chromatin immunoprecipitation assays confirmed that PGC-1alpha and ERRalpha occupied the mPDK4 promoter in C(2)C(12) myotubes. Additionally, transfection studies using ERRalpha-null primary fibroblasts demonstrated that ERRalpha is required for PGC-1alpha-mediated activation of the mPDK4 promoter. As predicted by the effects of PGC-1alpha on PDK4 gene transcription, overexpression of PGC-1alpha in C(2)C(12) myotubes decreased glucose oxidation rates. These results identify the PDK4 gene as a new PGC-1alpha/ERRalpha target and suggest a mechanism whereby PGC-1alpha exerts reciprocal inhibitory influences on glucose catabolism while increasing alternate mitochondrial oxidative pathways in skeletal muscle.

Activity and movement of free-living box turtles are largely independent of ambient and thermal conditions.

BACKGROUND: Ectotherms are assumed to be strongly influenced by the surrounding ambient and environmental conditions for daily activity and movement. As such, ecological and physiological factors contribute to stimuli influencing navigation, extent of movement, and therefore habitat use. Our study focused on the intensity of activity (from acceleration data) and extent of movement (from GPS and thread trailing data) of Eastern box turtles (Terrapene carolina carolina) in a fragmented landscape near their northern population limit. First, we quantified the thermal performance curve of box turtles using activity as a measure of performance. Second, we investigated ecological factors that could influence activity and movement and characterized the movement as extensive (exploration) and intensive (foraging). RESULTS: In contrast to previous lab work investigating effects of temperature on activity, we found no relationship between box turtle activity and temperature in the field. Furthermore, box turtle activity was consistent over a wide range of temperatures. Cluster analysis categorized movement recorded with GPS more as intensive than as extensive, while thread trailing had more movement categorized as extensive than intensive. Box turtle activity was higher during the morning hours and began to decrease as the day progressed. Based on the microclimate conditions tested, we found that box turtle movement was influenced by precipitation and time of day, and activity was most influenced by absolute humidity, ambient temperature, cloud cover, and time of day. CONCLUSIONS: Our model ectotherm in this study, the Eastern box turtle, had activity patterns characteristic of a thermal generalist. Sampling resolution altered the characterization of movement as intensive or extensive movement, possibly altering interpretation. More information on the resolution needed to definitively identify foraging and exploratory behavior in turtles is needed. Activity and movement were nearly independent of environmental conditions, which supports the overall interpretation that turtle performance is that of a broad environmental generalist. Future studies of movement of other turtle and reptile species are needed to determine the generality of these findings.

Intramuscular triglyceride content precedes impaired glucose metabolism without evidence for mitochondrial dysfunction during early development of a diabetic phenotype.

The incidence of type 2 diabetes is highly correlated with obesity; however, there is a lack of research elucidating the temporal progression. Transgenic FVB/N UCP-dta mice, which develop a diabetic phenotype, and their nontransgenic littermates were fed either a high-fat or normal-chow diet and were studied at 6, 9, 12, 15, 18, 21, and 24 weeks of age to test the hypothesis that increased lipid accumulation in skeletal muscle causes mitochondrial dysfunction, leading to the development of insulin resistance. Body composition, intramuscular triglyceride (IMTG) content, glucose metabolism, and mitochondrial function were measured to determine if IMTG drove mitochondrial dysfunction, leading to the development of type 2 diabetes. High-fat-fed transgenic mice had a significantly greater body mass, lipid mass, and IMTG content beginning early in the experiment. Glucose tolerance tests revealed that high-fat-fed transgenic mice developed a significantly insulin resistant response compared with the other 3 groups toward the end of the time course while plasma insulin was elevated very early in the time course. There was no significant difference in several measures of metabolic function throughout the time course. Long-term high-fat feeding in transgenic mice produced increases in IMTG, adiposity, body mass, and plasma insulin accompanied by decreases in glucose metabolism, but did not reveal any deficits in mitochondrial function or regulation during the early stage of the development of type 2 diabetes. It does not appear that lipotoxicity is driving defects in mitochondrial function prior to the onset of insulin resistance.

Thermoregulatory performance and habitat selection of the eastern box turtle (Terrapene carolina carolina).

Environmental conditions may affect individual physiological processes that influence short-term performance and ultimately growth, survival and reproduction. As such, habitats selected by animals must provide suitable and adequate resources. Ectothermic species are highly dependent on climatic conditions and ambient temperatures that dictate body temperature regulation and in turn physiological processes. We investigated the thermoregulatory performance, habitat selection, and movements of an ectothermic vertebrate, the Eastern box turtle (Terrapene carolina carolina) to assess the importance of thermoregulatory physiology in habitat selection. We evaluated the relationship between habitat selection and thermoregulatory performance in Southwest Ohio over two active seasons from May until October. We found that T. carolina selected shaded habitats, including evergreen and deciduous forests, as well as herbaceous grasslands, conformed to the ambient temperatures throughout the active season, although these habitats had temperatures below those expected based on thermal optima of box turtles. Further, we found that movement was not correlated with internal body temperature. Our study shows that thermal conditions are not paramount in habitat selection of box turtles, but that cooler temperatures do not have an effect on the extent of their locomotion.

Thermal Acclimatization in Overwintering Tadpoles of the Green Frog, Lithobates clamitans (Latreille, 1801).

Seasonal acclimatization permits organisms to maintain function in the face of environmental change. Tadpoles of the green frog (Lithobates clamitans) overwinter as tadpoles in much of their range. Because they are active in winter, we hypothesized that green frog tadpoles would display acclimatization of metabolic and locomotor function. We collected tadpoles in Sewanee, Tennessee (35.2°N) in winter and summer. Tadpoles collected during each season were tested at both winter (8°C) and summer (26°C) temperatures. Winter tadpoles were able to maintain swimming performance at both temperatures, whereas swimming performance decreased at cold temperatures in summer tadpoles. There was no evidence for seasonal acclimatization of whole-animal metabolic rate. Although whole-animal metabolic acclimatization was not observed, the activities of cytochrome c oxidase, citrate synthase, and lactate dehydrogenase measured in skeletal muscle homogenates showed higher activity in winter-acclimatized tadpoles indicating compensation for temperature. Further, the composition of muscle membranes of winter tadpoles had less saturated and more monounsaturated fatty acids and a higher ω-3 balance, unsaturation index, and peroxidation index than summer tadpoles. These data indicate that reversible phenotypic plasticity of thermal physiology occurs in larval green frog tadpoles. They appear to compensate for colder temperatures to maintain burst-swimming velocity and the ability to escape predators without the cost of maintaining a constant, higher standard metabolic rate in the winter.

Does the thermal plasticity of metabolic enzymes underlie thermal compensation of locomotor performance in the eastern newt (Notophthalmus viridescens)?

Eastern newts (Notophthalmus viridescens) upregulate the metabolic capacity of skeletal muscle in winter to compensate for thermodynamic effects on metabolism. However, whether this compensation facilitates locomotor performance at low temperature is unknown. Therefore, our aim was to determine if thermal acclimation of metabolic enzymes in muscle benefits locomotion. Eastern newts from southern Ohio were acclimated to cold (5°C, 10:14 L:D) or warm (25°C, 14:10 L:D) conditions for 12 weeks. Following acclimation, we measured the locomotor performance (burst speed and time until exhaustion) and the activities of metabolic enzymes in skeletal muscle at 5-30°C. Creatine kinase (CK) activity in skeletal muscle was higher in cold compared to warm-acclimated newts, and cold-acclimated newts had a higher burst speed at low temperature compared to warm-acclimated newts. At low temperature, time until exhaustion was higher in cold compared to warm-acclimated newts, but the activities of citrate synthase (CS) and cytochrome c oxidase (CCO) in muscle were lower in cold compared to warm-acclimated newts. Together, these results demonstrate that eastern newts compensate for the effects of low temperature on locomotor performance. Whereas thermal compensation of CK activity is correlated with burst locomotion at low temperature, aerobic enzymes in skeletal muscle (CS and CCO) are not linked to compensation of sustained locomotion.