Corticosterone Release Rates Research Articles

Physiological and behavioral variation by urbanization and climate in an urban-tolerant toad

The distribution of a species is best understood by examining the organism-environment interaction. Climate and anthropogenic habitat degradation, including urbanization, are salient features of the environment that can limit species distributions, especially for ectotherms. Comparative studies of the capacity of individuals to cope with rapid environmental change can help us understand the future success or failure of local populations or even the species. Studies of the glucocorticoid stress response are commonly used to understand how species cope with environmental stressors. Glucocorticoids modulate many aspects of physiological homeostasis including changes in energetic allocation and behavior. In a time of global amphibian decline the Gulf Coast Toad (Incilius nebulifer) is increasing its distribution and abundance. To understand how this species deals with thermal and urban stressors, we studied glucocorticoid regulation, hop performance, and lipid storage in I. nebulifer juveniles across nine populations that differed in average annual temperature and level of imperviousness (as an indication of urbanization). We measured corticosterone release rates at baseline, during agitation stress, and during recovery; then measured locomotor performance and whole-body lipids. We tested if I. nebulifer in hotter temperatures and more urbanized habitats exhibits elevated baseline corticosterone levels and either a reduced corticosterone stress response (“stress resistance” hypothesis) or quick post-stress recovery by negative feedback (“on again, off again” hypothesis). We also tested whether they exhibit reduced fat stores and decreased locomotor performance as costs of dealing with thermal and urban stressors. We found that I. nebulifer showed elevated baseline and agitation (stressed-induced) corticosterone release rates, and higher lipid storage with increasing urbanization. Climate had quadratic effects on these traits, such that populations living at the lowest and highest temperatures had the lowest corticosterone release rates and lipid stores, and the highest hop performance was observed in the least urbanized site at the warmest climate. Additionally, the rate of glucocorticoid recovery after agitation (negative feedback) decreased with increasing temperature and increased with increasing urbanization. These results indicate that I. nebulifer follows the “on again, off again” hypothesis in an adaptive pattern, which may help them cope with environmental change in terms of urbanization and climatic differences.

Traffic Noise Impacts Glucocorticoid Response, Activity, and Growth in Two Species of Tadpoles.

There is a large body of evidence linking increased noise to negative health effects for animals. Anthropogenic noise induces behavioral and physiological reactions across a range of taxa and increased traffic noise affects glucocorticoid (GC) hormones associated with the stress response in amphibians. GCs help to maintain homeostasis while balancing energetic trade-offs between reproduction, growth, and activity. Stressors during early development can impact fitness at later life stages. We measured growth, activity, and GCs in response to high levels of traffic noise in two tadpole species that differ in life history: Acris crepitans and Rana berlandieri. We predicted that earlier exposures to traffic noise will slow down the development and alter the behavior and GC concentrations differently than later exposures. Subjects were initially either exposed to natural levels of traffic noise for 8 days (early exposure) or a white noise control (later exposure), then the treatment was switched. Activity was measured via focal sampling and tadpoles were categorized as active if movement was detected. Tadpoles exposed to white noise initially maintained mass and activity throughout the experiment and early exposure to traffic noise had a greater impact on mass, activity, and GCs. Tadpoles exposed to traffic noise initially lost mass, with A. crepitans regaining mass but not R. berlandieri. When exposed earlier to traffic noise, R. berlandieri increased movement when shifted to the white noise treatment while A. crepitans did not significantly change activity. Acris creptians had higher corticosterone release rates compared to R. berlandieri, and in both species, release rates were higher for tadpoles exposed to noise earlier. The longer-lived R. berlandieri allocated more of their energetic resources into activity, while the shorter-lived A. crepitans allocated energy toward growth. Rana berlandieri and A. crepitans utilized different coping strategies to contend with early exposure to traffic noise, potentially due to differences in life histories. Our findings suggest that these tadpoles employ different coping mechanisms to modulate stress responses in noise-polluted environments, and these mechanisms could influence their fitness later in life. Further study is needed to understand the impact in more sensitive tadpole species.

Stress response of fire salamander larvae differs between habitat types.

The larvae of the European fire salamander (Salamandra salamandra) can inhabit two different habitats: streams and ponds. Streams are characterized by lower predation risks and higher food availability. Thus, ponds are considered a less suitable habitat. To investigate the differential impacts of these two habitats on larval physiology, we measured the stress response of larvae. After successfully validating the measure of water-borne corticosterone release rates in fire salamander larvae, we measured the baseline and stress-induced corticosterone of 64 larvae from ponds and streams in the field. We found that larvae in ponds have a higher baseline and stress-induced corticosterone levels. Additionally, we performed a reciprocal transplant experiment (RTE) and tested whether larvae can adapt their stress responses to changing habitats. After two weeks, we did not find an increase in corticosterone levels when comparing stress-induced corticosterone values with baseline corticosterone values in larvae transferred into ponds, irrespective of their habitat of origin. However, larvae transferred into streams still exhibited an increase in the stress-induced corticosterone response in comparison with the baseline values. These results show that non-invasive hormone measurements can provide information on the habitat quality and potential adaptation and thus emphasize the potential for its use in conservation efforts.

Does stress make males? An experiment on the role of glucocorticoids in anuran sex reversal.

Environmentally sensitive sex determination may help organisms adapt to environmental change but also makes them vulnerable to anthropogenic stressors, with diverse consequences for population dynamics and evolution. The mechanisms translating environmental stimuli to sex are controversial: although several fish experiments supported the mediator role of glucocorticoid hormones, results on some reptiles challenged it. We tested this hypothesis in amphibians by investigating the effect of corticosterone on sex determination in agile frogs (Rana dalmatina). This species is liable to environmental sex reversal whereby genetic females develop into phenotypic males. After exposing tadpoles during sex determination to waterborne corticosterone, the proportion of genetic females with testes or ovotestes increased from 11% to up to 32% at 3 out of 4 concentrations. These differences were not statistically significant except for the group treated with 10 nM corticosterone, and there was no monotonous dose-effect relationship. These findings suggest that corticosterone is unlikely to mediate sex reversal in frogs. Unexpectedly, animals originating from urban habitats had higher sex-reversal and corticosterone-release rates, reduced body mass and development speed, and lower survival compared to individuals collected from woodland habitats. Thus, anthropogenic environments may affect both sex and fitness, and the underlying mechanisms may vary across ectothermic vertebrates.

Noise and light pollution elicit endocrine responses in urban but not forest frogs

Urban areas are characterised by the presence of sensory pollutants, such as anthropogenic noise and artificial light at night (ALAN). Animals can quickly adapt to novel environmental conditions by adjusting their behaviour, which is proximately regulated by endocrine systems. While endocrine responses to sensory pollution have been widely reported, this has not often been linked to changes in behaviour, hampering the understanding of adaptiveness of endocrine responses. Our aim was, therefore, to investigate the effects of urbanisation, specifically urban noise and light pollution, on hormone levels in male urban and forest túngara frogs (Engystomops pustulosus), a species with reported population divergence in behaviour in response to urbanisation. We quantified testosterone and corticosterone release rates in the field and in the lab before and after exposure to urban noise and/or light. We show that urban and forest frogs differ in their endocrine phenotypes under field as well as lab conditions. Moreover, in urban frogs exposure to urban noise and light led, respectively, to an increase in testosterone and decrease in corticosterone, whereas in forest frogs sensory pollutants did not elicit any endocrine response. Our results show that urbanisation, specifically noise and light pollution, can modulate hormone levels in urban and forest populations differentially. The observed endocrine responses are consistent with the observed behavioural changes in urban frogs, providing a proximate explanation for the presumably adaptive behavioural changes in response to urbanisation.

An integrated physiological perspective on anthropogenic stressors in the Gulf coast toad (Incilius nebulifer)

Anthropogenic environmental change, including climate change and urbanization, results in warmer temperatures in both terrestrial and aquatic habitats and changes in community assemblages including invasive species introductions, among many other alterations. Anurans are particularly susceptible to these changes because generally they have a biphasic lifecycle and rely on aquatic and terrestrial habitats for survival. Changes such as warmer water temperature can result in direct and carryover effects, after metamorphosis that decrease fitness. However, Gulf Coast toads (Incilius (Bufo) nebulifer) are expanding their range, including into anthropogenically disturbed areas. We hypothesize that I. nebulifer copes with warmer water, reduced water levels, and invasive species by altering their physiology and/or behavior. Corticosterone is the primary glucocorticoid in amphibians, and it modulates many aspects of physiology and behavior, potentially including lipid storage and hop performance, during unpredictable (stressful) events. As a true toad, I. nebulifer also produces bufadienolide toxins that aid in its antipredator defense and may have tradeoffs with corticosterone. In a fully factorial design, we measured baseline corticosterone levels in tadpoles in response to two treatments: decreased water levels and increased water temperatures. After metamorphosis, we measured the corticosterone profile and other associated responses to exposure to the predatory red imported fire ant (Solenopsis invicta; RIFA). We found that tadpoles had elevated baseline corticosterone release rates when reared in warmer water and reduced water levels. Toadlets also had elevated baseline corticosterone release rates when exposed to any combination of two of the three treatments but when exposed to all three treatments toadlets instead showed elevated magnitude of their stress response. Predator avoidance (as measured by hop performance) was reduced after exposure to RIFA. Tadpoles from warmer water developed more quickly and were smaller in mass after metamorphosis. Toadlets had reduced production of two of the three detected bufadienolides and increased energy storage (lipids) after exposure to warmer water and reduced growth after exposure to reduced water levels. We found direct and carryover effects of common anthropogenic changes in I. nebulifer that may aid in their ability to persist despite these changes.

Does the Glucocorticoid Stress Response Make Toads More Toxic? An Experimental Study on the Regulation of Bufadienolide Toxin Synthesis.

Chemical defense is a crucial component of fitness in many organisms, yet the physiological regulation of defensive toxin synthesis is poorly understood, especially in vertebrates. Bufadienolides, the main defensive compounds of toads, are toxic to many predators and other natural enemies, and their synthesis can be upregulated by stressors, including predation risk, high conspecific density, and pollutants. Thus, higher toxin content may be the consequence of a general endocrine stress response in toads. Therefore, we hypothesized that bufadienolide synthesis may be stimulated by elevated levels of corticosterone (CORT), the main glucocorticoid hormone of amphibians, or by upstream regulators that stimulate CORT production. To test these alternatives, we treated common toad tadpoles with exogenous CORT (exoCORT) or metyrapone (MTP, a CORT-synthesis inhibitor that stimulates upstream regulators of CORT by negative feedback) in the presence or absence of predation cues for 2 or 6 days, and subsequently measured their CORT release rates and bufadienolide content. We found that CORT release rates were elevated by exoCORT, and to a lesser extent also by MTP, regardless of treatment length. Bufadienolide content was significantly decreased by treatment with exoCORT for 6 days but was unaffected by exposure to exoCORT for 2 days or to MTP for either 6 or 2 days. The presence or absence of predation cues affected neither CORT release rate nor bufadienolide content. Our results suggest that changes in bufadienolide synthesis in response to environmental challenges are not driven by CORT but may rather be regulated by upstream hormones of the stress response.

Contributions of water-borne corticosterone as one non-invasive biomarker in assessing nitrate pollution stress in tadpoles of Rana temporaria

Among a multitude of stressors to which wildlife is exposed, environmental pollution is a pervasive one that poses a serious threat. The permeable skin of amphibians is likely to increase direct contact of the body with pollutants, making them a group worth studying to access environmental quality. Consequently, finding reliable and complementary biomarkers that will present detectable and predictable changes in response to pollutants is essential to identify pollution sublethal effects on amphibians and to investigate whether these are in part responsible for population declines. The glucocorticoid hormone corticosterone (CORT), involved in many metabolic functions, is often used to measure the physiological stress response to environmental stressors in amphibians. In this study, we evaluated whether water-borne CORT can serve as a non-invasive biomarker for nitrate pollution stress in the European common frog (Rana temporaria) by comparing the effect of nitrate exposure on hormone release rates and on other physiological downstream biomarkers, i.e., ultimate physiological effects of the stressor. Specifically, we investigated the effect of different nitrate concentrations (0, 10, 50, and 100 mg/L) on water-borne CORT release rates, age, size, and body condition. Exposure to nitrate pollution significantly increased age at metamorphosis and water-borne CORT release rates, and led to reduced mass and body condition, but only at higher nitrate concentrations (i.e., 50 and 100 mg/L). Considering this similar sensitivity to other acknowledged biomarkers, water-borne CORT was a reliable biomarker of physiological stress in R. temporaria exposed to nitrate pollution stress in a controlled single-stressor laboratory approach. Thus, water-borne CORT is a promising method to be included in more holistic approaches. We recommend that such approaches keep testing multiple biomarker combinations, as species are exposed to several stressors likely to interact and produce varied outcomes in different biomarkers in their natural habitats.

Differences in Corticosterone Release Rates of Larval Spring Salamanders (Gyrinophilus porphyriticus) in Response to Native Fish Presence.

Simple SummaryIn amphibians, glucocorticoid hormones play a key role in the response to predation stress. Predators can directly affect prey via injury and death, but they can also have indirect effects due to the activity of glucocorticoids. The regulation of glucocorticoids can differ between populations that have co-evolved with predators and those that have not. We measured glucocorticoids at baseline and in response to a novel stressor in free-living larval salamanders that either live with or without fish predators naturally. We found that salamanders living with fish predators had lower measures of glucocorticoids than those without fish predators. Our study indicates that predator presence alters glucocorticoid regulation, which may allow species to better cope with native and introduced predators.Invasive fish predators are an important factor causing amphibian declines and may have direct and indirect effects on amphibian survival. For example, early non-lethal exposure to these stressors may reduce survival in later life stages, especially in biphasic species. In amphibians, the glucocorticoid hormone corticosterone is released by the hypothalamo–pituitary–interrenal axis (HPI), as an adaptive physiological response to environmental stressors. The corticosterone response (baseline and response to acute stressors) is highly flexible and context dependent, and this variation can allow individuals to alter their phenotype and behavior with environmental changes, ultimately increasing survival. We sampled larvae of the spring salamander (Gyrinophilus porphyriticus) from two streams that each contained predatory brook trout (Slavelinus fontinalis) in the lower reaches and no predatory brook trout in the upper reaches. We measured baseline and stress-induced corticosterone release rates of larvae from the lower and upper reaches using a non-invasive water-borne hormone assay. We hypothesized that corticosterone release rates would differ between larvae from fish-present reaches and larvae from fish-free reaches. We found that baseline and stressor-induced corticosterone release rates were downregulated in larvae from reaches with fish predators. These results indicate that individuals from reaches with predatory trout are responding to fish predators by downregulating corticosterone while maintaining an active HPI axis. This may allow larvae more time to grow before metamorphosing, while also allowing them to physiologically respond to novel stressors. However, prolonged downregulation of corticosterone release rates can impact growth in post-metamorphic individuals.

Artificial light at night (ALAN) affects the stress physiology but not the behavior or growth of Rana berlandieri and Bufo valliceps.

Artificial light at night (ALAN) alters the natural light dark patterns in ecosystems. ALAN can have a suite of effects on community structure and is a driver of evolutionary processes that influences a range of behavioral and physiological traits. Our understanding of possible effects of ALAN across species amphibians is lacking and research is warranted as ALAN could contribute to stress and declines of amphibian populations, particularly in urban areas. We tested the hypothesis that exposure to constant light or pulsed ALAN would physiologically stress Rio Grande leopard frog (Rana berlandieri) and Gulf Coast toad (Bufo valliceps) tadpoles. We reared tadpoles under constant or pulsed (on and off again) ALAN for 14 days and measured corticosterone release rates over time using a non-invasive water-borne hormone protocol. ALAN treatments did not affect behavior or growth. Tadpoles of both species had higher corticosterone (cort) release rates after 14 days of constant light exposure. Leopard frog tadpoles had lower cort release rates after exposure to pulsed ALAN while toad tadpoles had higher cort release rates. These results suggest that short-term exposure to constant or pulsed light at night may contribute to stress in tadpoles but that each species differentially modulated their cort response to ALAN exposure and a subsequent stressor. This flexibility in the upregulation and downregulation of hypothalamic-pituitary-interrenal axis response may indicate an alternative mechanism for diminishing the deleterious effects of chronic stress. Nonetheless, ALAN should be considered in management and conservation plans for amphibians.

Stressed tadpoles mount more efficient glucocorticoid negative feedback in anthropogenic habitats due to phenotypic plasticity

Coping with anthropogenic environmental change is among the greatest challenges faced by wildlife, and endocrine flexibility is a potentially crucial coping mechanism. Animals may adapt to anthropogenic environments by dampening their glucocorticoid stress response, but empirical tests of this hypothesis have provided mixed evidence. An alternative hypothesis is that a non-attenuated stress response and efficient negative feedback are favored in anthropogenic habitats. To test this idea, we non-invasively sampled corticosterone release rates of common toad (Bufo bufo) tadpoles in agricultural, urban, and natural habitats, and quantified their stress response and negative feedback by a standardized stress-and-recovery protocol. We repeated the same sampling with tadpoles raised from eggs from the same ponds in a common-garden experiment to infer if the differences observed between populations in different habitats were due to individual phenotypic plasticity rather than microevolution or transgenerational effects. We found that, compared to tadpoles in natural ponds, urban tadpoles had higher baseline and stressed corticosterone release rates, and tadpoles in agricultural ponds had similar corticosterone release rates but greater stress-induced change, indicating stronger stress responses in both types of anthropogenic habitats. As predicted, tadpoles in both agricultural and urban ponds showed more efficient negative feedback than did tadpoles in natural ponds. Water pollution levels, as indicated by the concentrations of carbamazepine and corticoid-disrupting compounds in pond water, contributed to elevating the stress response regardless of land use. Infection by neither Batrachochytrium dendrobatidis nor Ranavirus was detected in free-living tadpoles. No habitat-related glucocorticoid differences persisted in the common-garden experiment. These results suggest that toad tadpoles in anthropogenic habitats increased their glucocorticoid flexibility via phenotypic plasticity. The coupling of stronger stress response and stronger negative feedback in these habitats supports the importance of rapidly “turning on and off” the stress response as a mechanism for coping with anthropogenic environmental change.

Effects of Agricultural Pollutants on Stress Hormones and Viral Infection in Larval Salamanders

Declines in amphibians are a global problem, influenced by complex local factors. While many factors contribute to these declines, much attention has been focused on the roles of contaminants and pathogens. Throughout eastern South Dakota, row-crop farming has contributed to habitat degradation for many amphibians, often through increases in environmental contaminants. For two years we visited four wetlands (two reference wetlands, two agricultural wetlands) to measure water-borne corticosterone (CORT) release rates and ranavirus in larval Western Tiger Salamander (Ambystoma mavortium). We found that both water-borne CORT release rates and ranavirus infection load were greater in larval salamanders from agricultural wetlands compared to reference wetlands. We also found that water-borne CORT release rates were greater in ranavirus-infected individuals compared to uninfected individuals and that water-borne CORT is positively correlated with ranavirus infection load. Though the causal relationships among contaminants, CORT, and ranavirus infection are difficult to determine, chronically elevated CORT is known to be immunosuppressive and may result in high infection loads. This study further describes the negative effects of crop production on amphibian health, provides the first evidence of ranavirus in South Dakota, and supports the use of water-borne CORT as a biomarker of amphibian population health in row-crop landscapes.

Validation of water-borne cortisol and corticosterone in tadpoles: Recovery rate from an acute stressor, repeatability, and evaluating rearing methods

Amphibian populations are declining globally, so understanding how individuals respond to anthropogenic and environmental stressors may aid conservation efforts. Using a non-invasive water-borne hormone assay, we measured the release rates of two glucocorticoid hormones, corticosterone and cortisol, in Rio Grande Leopard frog, Rana berlandieri, tadpoles. We validated this method pharmacologically and biologically using an adrenocorticotropic hormone (ACTH) challenge, exposure to exogenous corticosterone, and an agitation test. We calculated the repeatability of hormone release rates, the recovery time from an acute stressor, and explored rearing methods for tadpoles. Tadpole corticosterone release rates increased following an ACTH challenge, exposure to exogenous corticosterone, and agitation, validating the use of water-borne hormone methods in this species. After exposure to an acute stressor via agitation, corticosterone release rates began to decline after 2 h and were lowest after 6 h, suggesting a relatively rapid recovery from an acute stressor. Tadpoles reared in groups had higher corticosterone release rates than tadpoles reared individually, and lost mass by Day 7, while tadpoles reared individually did not show a stress response, therefore either rearing method is viable, but have differing physiological costs for tadpoles. Repeatability of corticosterone release rates was moderate to high in R. berlandieri tadpoles, indicating that this species can show a response to selection and potentially respond to rapid environmental change. Our results show that the water-borne hormone assay is a viable way to measure glucocorticoids in this species and is useful in the field of conservation physiology for rare and endangered species.

Roundup™ With Corticosterone Functions as an Infodisruptor to Antipredator Response in Tadpoles

Larval amphibians are frequently exposed to multiple stressors in aquatic environments, so understanding how individual stressors and synergisms of multiple stressors affect amphibians is integral to conservation efforts. Glyphosate, the active ingredient in Roundup, is a common pollutant found in aquatic environments. Exposure to glyphosate induces morphological, behavioral, and physiological changes in tadpoles possibly via infodisruption. Urban aquatic environments may have environmentally relevant levels of glyphosate, as well as higher concentrations of exogenous corticosterone (CORT) than rural areas. Elevated CORT levels also affect the morphology and physiology of tadpoles. Dragonfly larvae are common predators of tadpoles, and tadpoles often show elevated CORT and reduced activity in the presence of dragonfly larvae. We tested the hypothesis that combined effects of exogenous CORT and Roundup exposure would affect the antipredator behavior, morphology, and stress hormone responses of Gulf coast toad, Incilius nebulifer, tadpoles. We exposed tadpoles to one of four treatments: Roundup, CORT, Roundup+CORT, or control, for 7 days. Tadpoles exposed to CORT or Roundup+CORT had elevated CORT release rates. Tadpoles exposed to exogenous CORT had lower tail depth compared to tadpoles in other treatments. Subsequently, we exposed tadpoles to dragonfly diet cues. Tadpoles increased activity after predator cue exposure when they had previously been exposed to Roundup+CORT. Taken together, our results suggest that there may be synergistic effects between Roundup and exogeneous CORT on organismal behavior but not their physiology or morphology. It appears that glyphosate is an infordisruptor, that prevents tadpoles from demonstrating adaptive antipredator responses, which may contribute to population declines.

Cross-Life Stage Effects of Aquatic Larval Density and Terrestrial Moisture on Growth and Corticosterone in the Spotted Salamander

For organisms with complex life cycles, conditions experienced during early life stages may constrain later growth and survival. Conversely, compensatory mechanisms may attenuate negative effects from early life stages. We used the spotted salamander, Ambystoma maculatum, to test how aquatic larval density and terrestrial moisture influence juvenile growth, food intake, evaporative water loss and water reuptake rates, and corticosterone levels. We conducted an outdoor mesocosm experiment to manipulate larval density and transferred metamorphosed salamanders into low and high terrestrial moisture treatments in laboratory terrariums. After the larval stage, high-density salamanders were significantly smaller and had higher corticosterone release rates than those from low-density treatments. Salamanders in the low terrestrial moisture treatment consumed fewer roaches, had lower mass-specific growth rates, higher water reuptake, and higher corticosterone release rates than salamanders in high terrestrial moisture treatments. Across moisture treatments, smaller salamanders had higher mass-specific growth rates than larger salamanders. Our results suggest that salamanders can partially compensate for competition in the larval aquatic habitat with increased growth as juveniles, but this response is dependent on terrestrial habitat quality. Thus, the persistence of early life stage effects can be an important, yet context-dependent, component of amphibian life cycles.

Physiological responses to elevated temperature across the geographic range of a terrestrial salamander.

Widespread species often possess physiological mechanisms for coping with thermal heterogeneity, and uncovering these mechanisms provides insight into species' responses to climate change. The emergence of non-invasive corticosterone (CORT) assays allows us to rapidly assess physiological responses to environmental change on a large scale. We lack, however, a basic understanding of how temperature affects CORT, and whether temperature and CORT interactively affect performance. Here, we examined the effects of elevated temperature on CORT and whole-organism performance in a terrestrial salamander, Plethodon cinereus, across a latitudinal gradient. Using water-borne hormone assays, we found that raising ambient temperature from 15 to 25°C increased CORT release at a similar rate for salamanders from all sites. However, CORT release rates were higher overall in the warmest, southernmost site. Elevated temperatures also affected physiological performance, but the effects differed among sites. Ingestion rate increased in salamanders from the warmer sites but remained the same for those from cooler sites. Mass gain was reduced for most individuals, although this reduction was more dramatic in salamanders from the cooler sites. We also found a temperature-dependent relationship between CORT and food conversion efficiency (i.e. the amount of mass gained per unit food ingested). CORT was negatively related to food conversion efficiency at 25°C but was unrelated at 15°C. Thus, the energetic gains of elevated ingestion rates may be counteracted by elevated CORT release rates experienced by salamanders in warmer environments. By integrating multiple physiological metrics, we highlight the complex relationships between temperature and individual responses to warming climates.

Urbanization is associated with elevated corticosterone in Jollyville Plateau salamanders

Urbanization has the potential to induce major changes in freshwater systems. Expected increases in human populations will likely amplify these changes and lead to the overall degradation of habitat quality within these systems. Such habitat alterations may function as stressors that can affect glucocorticoid stress hormones in freshwater vertebrates. Examining changes in physiological stress may provide early warning indicators of environmental threats and provide insights into the sub-lethal effects of habitat degradation. The threatened, obligate aquatic, Jollyville Plateau salamander (Eurycea tonkawae) is found in urbanized and rural catchments within central Texas and has experienced population declines in heavily urbanized areas. We tested the prediction that salamanders from urbanized sites would have different levels of baseline corticosterone (CORT) and muted or no stress responsiveness (to an external stressor, agitation) compared to salamanders from rural sites. We collected water-borne hormones to measure baseline CORT release rates (n=3years) and stress responsiveness (n=2years) in salamanders inhabiting urbanized and rural sites. We also measured “background” CORT from stream water alone at each visit. For the first two years we found that baseline CORT was higher in urbanized sites but not in the third year. Across years and populations, salamanders showed stress responsiveness, suggesting that, even if physiological stress is higher in urbanized areas, it has not resulted in the impairment of the hypothalamic–pituitary–interrenal axis. Background CORT was higher in urban than in rural streams and was positively correlated with mean baseline CORT of salamanders across populations and years. Our results contribute to the goal of finding early warning indicators of environmental threats by demonstrating a relationship between urbanization and the physiological status of E. tonkawae, using a rapid, non-invasive measure of stress.

The association between male-biased sex ratio and indicators of stress in red-spotted newts

In populations with a male-biased operational sex ratio, coercive mating by males can have fitness consequences for females. One component of reduced fitness for females in populations with a male-biased OSR may be greater activation of the stress response, resulting in higher corticosterone release rates (CORT; a glucocorticoid stress hormone in amphibians). We test the hypothesis that a male-biased sex ratio affects female activity and release rates of CORT and testosterone (T) in male and female red-spotted newts (Notophthalmus viridescens). First, we evaluated if chemical cues from a male-biased sex ratio affect activity and CORT release rates in females. We predicted that females exposed to chemical cues of three males would be less active and have higher CORT release rates than those exposed to chemical cues of one male. Second, we measured CORT release rates of red-spotted newts in field enclosures with either a male-biased or a female-biased sex ratio. We predicted that females in the male-biased treatment would have higher CORT and T release rates than those in a female-biased treatment, owing to higher levels of male harassment. We also predicted that males would have higher CORT and T release rates in male-biased treatments due to higher levels of male-male competition. Females were not less active in response to chemical cues from more males over fewer males, but there was a positive relationship between female activity and CORT when they were exposed to the cues of three males. We also found that females, but not males, in the male-biased sex ratio treatment had higher CORT and T release rates than those in the female-biased treatment. Our results support the hypothesis that a male-biased sex ratio leads to a higher stress response, which may underlie the observed decrease in immune function and body condition in previous work exposing female red-spotted newts to a male-biased sex ratio. This study furthers our understanding of the mechanistic basis for costs associated with a male-biased sex ratio in a pond-breeding amphibian.

Differences in chytridiomycosis infection costs between two amphibian species from Central Europe

Batrachochytrium dendrobatidis (Bd) causes the disease chytridiomycosis associated with amphibian declines. Response and costs of infection varies greatly between species. Bd can induce a stress response in amphibians resulting in elevated corticosterone (CORT). We exposed Bombina variegata and Hyla arborea tadpoles to Bd+ or Bd- Salamandra salamandra larvae and measured CORT release rates, Bd infection loads, and survival through metamorphosis. Tadpoles of both species exposed to Bd+ larvae had elevated CORT release rates compared to tadpoles exposed to Bd- larvae. Bombina variegata appear less resistant to infection than H. arborea, showing higher Bd loads and more infected individuals. Within species, we did not find differences in cost of infection on survival, however more B. variegata tadpoles reached metamorphosis than H. arborea. The differences in resistance may be species specific, owing to higher immunity defenses with H. arborea having higher overall CORT release rates, and differences in antimicrobial peptides, or to differences in Bd strain or other unexplored mechanisms.

A Non-Invasive Water-Borne Assay of Stress Hormones in Aquatic Salamanders

Declines in amphibian populations are due to an array of anthropogenic and natural factors requiring a need to detect and monitor populations that are either in decline or at risk of decline. Physiological biomarkers, such as glucocorticoid stress hormones, present a viable option to monitor stress levels and, thus, the physiological health of vertebrate populations. We performed preliminary experiments necessary for the use of water-borne hormone assays to measure corticosterone (CORT) as an assessment of aspects of chronic stress for the management and conservation of three species of aquatic salamanders in central Texas (Eurycea, Plethodontidae). We examined the time for CORT levels to peak in response to capture and handling in populations of E. nana, E. sosorum, and E. tonkawae. Peak CORT release rates differed among species, with peak releases generally occurring in under 3 h. This highlights the need to obtain a timeline for CORT to mount so that peak rates are captured during the leaching phase. We also examined the responsiveness of the hypothalamic-pituitary-interrenal (HPI) axis using an adrenocorticotropic hormone (ACTH) challenge or exposure to an external stressor (agitation). Eurycea sosorum (one population) and E. tonkawae showed HPI responsiveness. To examine the relationship between field and laboratory CORT release rates, we compared baseline CORT release rates between captive and wild-caught populations of all three species. Once the preliminary studies have been performed, water-borne CORT release rates can be an effective biomarker to monitor whether a population is experiencing chronic stress. Water-borne hormone collection also allows for repeated sampling of individuals over time. Our experiments provide a foundation for future studies relating stress to HPI responsiveness in aquatic Eurycea, which, in turn, will help inform management of both wild and captive populations.