Global stressors and the global decline of amphibians: tipping the stress immunocompetency axis

There is a widespread consensus that the earth is experiencing a mass extinction event and at the forefront are amphibians, the most threatened of all vertebrate taxa. A recent assessment found that nearly one-third (32%, 1,856 species) of the world’s amphibian species are threatened. Amphibians have existed on the earth for over 300 million years, yet in just the last two decades there have been an alarming number of extinctions, nearly 168 species are believed to have gone extinct and at least 2,469 (43%) more have populations that are declining. Infectious diseases have been recognized as one major cause of worldwide amphibian population declines. This could be the result of the appearance of novel pathogens, or it could be that exposure to environmental stressors is increasing the susceptibility of amphibians to opportunistic pathogens. Here I review the potential effects of stressors on disease susceptibility in amphibians and relate this to disease emergence in human and other wildlife populations. I will present a series of case studies that illustrate the role of stress in disease outbreaks that have resulted in amphibian declines. First, I will examine how elevated sea-surface temperatures in the tropical Pacific since the mid-1970s have affected climate over much of the world and could be setting the stage for pathogen-mediated amphibian declines in many regions. Finally, I will discuss how the apparently rapid increase in the prevalence of amphibian limb deformities is linked to the synergistic effects of trematode infection and exposure to chemical contaminants.     

Natural Stressors and Ranavirus Susceptibility in Larval Wood Frogs (Rana sylvatica)

Chronic exposure to stressors has been shown to suppress immune function in vertebrates, making them more susceptible to pathogens. It is less clear, however, whether many natural stressors are immunosuppressive. Moreover, whether stressors make disease more likely or more severe in populations is unclear because animals respond to stressors both behaviorally and physiologically. We tested whether chronic exposure to three natural stressors of wood frog tadpoles—high-densities, predator-cues, and low-food conditions—influence their susceptibility to a lethal ranavirus both individually in laboratory experiments, and collectively in outdoor mesocosms. Prior to virus exposure, we observed elevated corticosterone only in low-food treatments, although other treatments altered rates of growth and development as well as tadpole behavior. None of the treatments, however, increased susceptibility to ranavirus as measured by the proportion of tadpoles that became infected or died, or the time to death compared to controls. In fact, mortality in the mesocosms was actually lower in the high-density treatment even though most individuals became infected, largely because of increased rates of metamorphosis. Overall we find no support for the hypothesis that chronic exposure to common, ecologically relevant challenges necessarily elevates corticosterone levels in a population or leads to more severe ranaviral disease or epidemics. Conditions may, however, conspire to make ranavirus infection more common in metamorphosing amphibians.     

An examination of amphibian sensitivity to environmental contaminants: are amphibians poor canaries?

Nearly two decades ago, the global biodiversity crisis was catapulted to the front pages of newspapers with the recognition of worldwide amphibian declines. Amphibians earned their appellation, 'canaries in a coal mine', because of apparent high sensitivity to human-mediated environmental change. The most frequently cited causes for high susceptibility include permeable skin, a dual aquatic-terrestrial life cycle and a relatively rudimentary immune system. While some researchers have questioned the basis for the canary assertion, there has been no systematic evaluation of amphibian sensitivity to environmental challenges relative to other taxa. Here, we apply a database representing thousands of toxicity tests to compare the responses of amphibians relative to that of other taxonomic groups. The use of standardized methods combined with large numbers of identical challenges enables a particularly powerful test of relative effect size. Overall, we found that amphibians only exhibit moderate relative responses to water-borne toxins. Our findings imply that, as far as chemical contaminants are concerned, amphibians are not particularly sensitive and might more aptly be described as 'miners in a coal mine'. To the extent that amphibian declines have been mediated by chemical contaminants, our findings suggest that population losses and extinctions may have already occurred in a variety of taxa much more sensitive than amphibians.
Ecology Letters (2010) 13: 60–67     

Stable Isotopes Reveal Trophic Partitioning and Trophic Plasticity of a Larval Amphibian Guild

Temporary ponds are highly variable systems where resource availability and community structure change extensively over time, and consequently the food web is highly dynamic. Amphibians play a critical role both as consumers and prey in aquatic communities and yet there is still little information on the trophic status of most amphibians. More importantly, little is known about the extent to which they can alter their trophic ecology in response to changing conditions. We experimentally investigated the effects of increased amphibian density, presence of intraguild competitors, and presence of native and invasive predators (either free or caged) on the trophic status of a Mediterranean amphibian guild, using stable isotopes. We observed variations in δ ¹³C and δ ¹⁵N isotopic values among amphibian species and treatments and differences in their food sources. Macrophytes were the most important food resource for spadefoot toad tadpoles (Pelobates cultripes) and relatively important for all anurans within the guild. High density and presence of P. cultripes tadpoles markedly reduced macrophyte biomass, forcing tadpoles to increase their feeding on detritus, algae and zooplankton, resulting in lower δ ¹³C values. Native dytiscid predators only changed the isotopic signature of newts whereas invasive red swamp crayfish had an enormous impact on environmental conditions and greatly affected the isotopic values of amphibians. Crayfish forced tadpoles to increase detritus ingestion or other resources depleted in δ ¹³C. We found that the opportunistic amphibian feeding was greatly conditioned by intra- and interspecific competition whereas non-consumptive predator effects were negligible. Determining the trophic plasticity of amphibians can help us understand natural and anthropogenic changes in aquatic ecosystems and assess amphibians’ ability to adjust to different environmental conditions.     

Exposure to coal combustion residues during metamorphosis elevates corticosterone content and adversely affects oral morphology, growth, and development in Rana sphenocephala

Coal combustion residues (CCRs) are documented to negatively impact oral morphology, growth, and development in larval amphibians. It is currently unclear what physiological mechanisms may mediate these effects. Corticosterone, a glucocorticoid hormone, is a likely mediator because when administered exogenously it, like CCRs, also negatively influences oral morphology, growth, and development in larval amphibians. In an attempt to identify if corticosterone mediates these effects, we raised larval Southern Leopard Frogs, Rana sphenocephala, on either sand or CCR substrate and documented effects of sediment type on whole body corticosterone, oral morphology, and time to and mass at key metamorphic stages. Coal combustion residue treated tadpoles contained significantly more corticosterone than controls throughout metamorphosis. However, significantly more oral abnormalities occurred early in metamorphosis when differences in corticosterone levels between treatments were minimal. Overall, CCR-treated tadpoles took significantly more time to transition between key stages and gained less mass between stages than controls, but these differences between treatments decreased during later stages when corticosterone differences between treatments were greatest. Our results suggest endogenous increase in corticosterone content and its influence on oral morphology, growth and development is more complex than previously thought.     

A small increase in UV-B increases the susceptibility of tadpoles to predation

Increased ultraviolet-B (UV-B) radiation as a consequence of ozone depletion is one of the many potential drivers of ongoing global amphibian declines. Both alone and in combination with other environmental stressors, UV-B is known to have detrimental effects on the early life stages of amphibians, but our understanding of the fitness consequences of these effects remains superficial. We examined the independent and interactive effects of UV-B and predatory chemical cues (PCC) on a suite of traits of Limnodynastes peronii embryos and tadpoles, and assessed tadpole survival time in a predator environment to evaluate the potential fitness consequences. Exposure to a 3 to 6 per cent increase in UV-B, which is comparable to changes in terrestrial UV-B associated with ozone depletion, had no effect on any of the traits measured, except survival time in a predator environment, which was reduced by 22 to 28 per cent. Exposure to PCC caused tadpoles to hatch earlier, have reduced hatching success, have improved locomotor performance and survive for longer in a predator environment, but had no effect on tadpole survival, behaviour or morphology. Simultaneous exposure to UV-B and PCC resulted in no interactive effects. These findings demonstrate that increased UV-B has the potential to reduce tadpole fitness, while exposure to PCCs improves their fitness.     

Ontogeny of corticotropin-releasing factor effects on locomotion and foraging in the Western spadefoot toad (Spea hammondii)

We investigated the effects of corticotropin-releasing factor (CRF) and corticosterone (CORT) on foraging and locomotion in Western spadefoot toad (Spea hammondii) tadpoles and juveniles to assess the behavioral functions of these hormones throughout development. We administered intracerebroventricular injections of ovine CRF or CRF receptor antagonist alphahelical CRF((9-41)) to tadpoles and juveniles, and observed behavior within 1.5 h after injection. In both premetamorphic (Gosner stage 33) and prometamorphic (Gosner stages 35-37) tadpoles, CRF injections increased locomotion and decreased foraging. Injections of alphahelical CRF((9-41)) reduced locomotion but did not affect foraging in premetamorphic tadpoles, but dramatically increased foraging in prometamorphic tadpoles compared to both placebo and uninjected controls. Similarly, alphahelical CRF((9-41)) injections stimulated food intake and prey-catching behavior in juveniles. These results suggest that in later-staged amphibians, endogenous CRF secretion modulates feeding by exerting a suppressive effect on appetite. By contrast to the inhibitory effect of CRF, 3-h exposure to CORT (500 nM added to the aquarium water) stimulated foraging in prometamorphic tadpoles. These tadpoles also exhibited a CORT-mediated increase in foraging 6 h after CRF injection, which was associated with elevated whole-body CORT content and blocked by glucocorticoid receptor (GR) antagonist (RU486) injections. Thus, exogenous CRF influences locomotion and foraging in both pre- and prometamorphic tadpoles, but endogenous CRF secretion in relatively unstressed animals does not affect foraging until prometamorphic stages. Furthermore, the opposing actions of CRF and CORT on foraging suggest that they are important regulators of energy balance and food intake in amphibians throughout development.     

Effects of an infectious fungus, Batrachochytrium dendrobatidis, on amphibian predator-prey interactions

The effects of parasites and pathogens on host behaviors may be particularly important in predator-prey contexts, since few animal behaviors are more crucial for ensuring immediate survival than the avoidance of lethal predators in nature. We examined the effects of an emerging fungal pathogen of amphibians, Batrachochytrium dendrobatidis, on anti-predator behaviors of tadpoles of four frog species. We also investigated whether amphibian predators consumed infected prey, and whether B. dendrobatidis caused differences in predation rates among prey in laboratory feeding trials. We found differences in anti-predator behaviors among larvae of four amphibian species, and show that infected tadpoles of one species (Anaxyrus boreas) were more active and sought refuge more frequently when exposed to predator chemical cues. Salamander predators consumed infected and uninfected tadpoles of three other prey species at similar rates in feeding trials, and predation risk among prey was unaffected by B. dendrobatidis. Collectively, our results show that even sub-lethal exposure to B. dendrobatidis can alter fundamental anti-predator behaviors in some amphibian prey species, and suggest the unexplored possibility that indiscriminate predation between infected and uninfected prey (i.e., non-selective predation) could increase the prevalence of this widely distributed pathogen in amphibian populations. Because one of the most prominent types of predators in many amphibian systems is salamanders, and because salamanders are susceptible to B. dendrobatidis, our work suggests the importance of considering host susceptibility and behavioral changes that could arise from infection in both predators and prey.     

Widespread occurrence of ranavirus in pond-breeding amphibian populations

Ranaviruses are an emerging threat for many amphibian populations, yet their distribution in amphibian communities and the association of infection with possible stressors and species is not fully understood due to historically sparse surveillance. Agricultural practices that reduce the water quality of amphibian breeding habitats (e.g., cattle access to wetlands) and environmental stressors (e.g., lower temperatures) may contribute to ranavirus emergence. We tested larval amphibians for ranavirus infection across four seasons in farm ponds (n?=?40) located in Tennessee, USA. Cattle at various densities were allowed access to half of the sampled ponds. Ranavirus infections were detected in nine species and in 33 of the sampled ponds (83%), illustrating widespread occurrence of the pathogen. Species within the family Ranidae were the most frequently infected. In 13 of the ponds containing infected individuals, prevalence exceeded 40% during at least one season. Infections were detected in multiple seasons in 20 of the sampled ponds containing infections, suggesting that ranaviruses are relatively persistent in these systems. Cattle had negative effects on water quality (turbidity and ammonia) and there was a positive association between cattle abundance and ranavirus prevalence in the summer. Counter to previous field studies in North America, we found a significant positive association between water temperature and ranavirus prevalence in the fall sampling events. Despite these findings, the influences of cattle and temperature on ranavirus prevalence were not consistent across seasons. As such, the mechanisms driving high ranavirus prevalence across the landscape and over time remain unclear. Given the widespread occurrence of ranaviruses in wild amphibians, we encourage the implementation of surveillance programs to help identify potential drivers of emergence. Sites with high ranavirus prevalence should be monitored annually for outbreaks, and the long-term effects on population size determined.     

Differential migration and an endocrine response to stress in wintering dark-eyed juncos (Junco hyemalis)

The dark-eyed junco (junco hyemalis) exhibits differential migration in autumn that, in general, results in females overwintering south of males, and young within each sex overwintering north of older birds. Individuals overwintering at higher latitudes face less predictable and more challenging environmental conditions. Rapid increases in circulating levels of the energy-regulating glucocorticosteroid, corticosterone, occur in response to environmental stressors. To establish whether the strength of acute corticosterone secretion was correlated with the probability of encountering poor environmental conditions, we compared the corticosterone stress response (e.g. initial plasma concentrations at the time of capture and 30 min later) in dark-eyed juncos overwintering in Mississippi (MS), USA, near the southern limit of their wintering range, with juncos overwintering in New York (NY), USA, near the northern limit of their wintering range. During two winters, 22 males and one female were sampled in NY; 13 males, 12 females and one bird of undetermined sex were sampled in MS. Not unexpectedly, NY birds carried greater fat reserves that resulted in a significantly higher value of energetic condition (mass corrected for wing cord cubed). There was no difference between the two winters sampled at either site, nor was there an effect of sex on patterns of corticosterone secretion in MS birds. With sexes pooled, MS and NY birds had similar baseline corticosterone levels. However, as predicted, NY birds exhibited significantly higher corticosterone concentrations 30 min after capture. These results support the hypothesis that birds wintering in less predictable, more extreme environments show a higher amplitude corticosterone response, which may enable them to adjust their behaviour and physiology more rapidly in response to environmental stressors such as storms. Adrenocortical sensitivity may be a part of the physiological milieu associated with differential migration in juncos; whether it results from endogenous differences in the migratory programmes of individuals or from acclimatization to local environmental conditions remains to be determined.     

The effects of fish on assemblages of amphibians in ponds: a field experiment

1. Bluegill sunfish ( Lepomis macrochirus ) dominate fish assemblages of small lakes and ponds throughout the eastern United States and may play a major role in structuring aquatic communities. We examined the impact of adult bluegill on amphibian density by stocking bluegill at a range of densities into partitions of an experimental pond in which amphibians were free to colonize.
2. Adult bluegill had a major impact on the amphibian assemblage. By the end of the experiment, gray treefrog ( Hyla versicolor ) tadpoles were nine times less abundant, and red-spotted newt ( Notophthalmus viridescens ) adults were three times less abundant in the presence of adult bluegill than in their absence. In contrast, bullfrog ( Rana catesbeiana ) tadpoles tended to increase in the presence of adult bluegill. Adult bluegill also had a negative effect on the abundance of predaceous aquatic insects.
3. There was no indication that interactions among amphibians were significant in determining the above patterns. We suggest that the strong impact of adult bluegill resulted from a combination of direct and indirect effects on amphibian larvae and predaceous aquatic insects.     

The amphibian microbiome: natural range of variation,pathogenic dysbiosis,and role in conservation

Recent research in humans, livestock, and wildlife using high-throughput next-generation sequencing (NGS) has identified that resident microbiota play an essential role in disease resistance, host health, and adaptation to biotic and abiotic stressors. Since amphibians are currently facing population declines and extinctions attributable to anthropogenic pressures and emerging diseases, an understanding of the effects of microbiome dysbiosis and mitigation is a prerequisite for amphibian conservation and disease management. Interest is now growing with regard to understanding the influence of unfavorable environmental conditions on the amphibian microbiome and the effects of dysbiosis on the susceptibility to pathogenic infections. Here, we summarize information on the amphibian microbiome, specifically concerning intrinsic and extrinsic factors that shape the skin and gut microbiome. We explore diverse types of unfavorable environmental perturbations and the ways in which they can impact the microbiota of an individual so that we can better comprehend the consequences of stressors and dysbiosis on pathogen emergence and health. We discuss the role of the microbiome in amphibian conservation and identify gaps of knowledge that need to be filled if we are to achieve a meta-organism conservation approach. NGS studies should be complemented with other high-throughput “-omic” approaches to target microbiome functionality. Understanding the microbiome might be the missing piece in the overall strategy that will help maintain the health of amphibians in a world with highly affected environments and that will prevent/mitigate emerging infectious diseases.     

Effects of nutrient supplementation on host‐pathogen dynamics of the amphibian chytrid fungus: a community approach

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Exogenous and endogenous corticosterone alter feather quality

We investigated how exogenous and endogenous glucocorticoids affect feather replacement in European starlings (Sturnus vulgaris) after approximately 56% of flight feathers were removed. We hypothesized that corticosterone would retard feather regrowth and decrease feather quality. After feather regrowth began, birds were treated with exogenous corticosterone or sham implants, or endogenous corticosterone by applying psychological or physical (food restriction) stressors. Exogenous corticosterone had no impact on feather length and vane area, but rectrices were lighter than controls. Exogenous corticosterone also decreased inter-barb distance for all feathers and increased barbule number for secondaries and rectrices. Although exogenous corticosterone had no affect on rachis tensile strength and stiffness, barbicel hooking strength was reduced. Finally, exogenous corticosterone did not alter the ability of Bacillus licheniformis to degrade feathers or affect the number of feathers that failed to regrow. In contrast, endogenous corticosterone via food restriction resulted in greater inter-barb distances in primaries and secondaries, and acute and chronic stress resulted in greater inter-barb distances in rectrices. Food-restricted birds had significantly fewer barbules in primaries than chronic stress birds and weaker feathers compared to controls. We conclude that, although exogenous and endogenous corticosterone had slightly different effects, some flight feathers grown in the presence of high circulating corticosterone are lighter, potentially weaker, and with altered feather micro-structure.     

Roles of stress hormones in food intake regulation in anuran amphibians throughout the life cycle

Towards understanding the ontogeny of energy balance regulation in vertebrates we analyzed the responses of corticotropin-releasing factor (CRF) and corticosterone to food deprivation in the Western spadefoot toad (Spea hammondii) at three developmental stages: premetamorphic tadpole, prometamorphic tadpole, and juvenile. Corticosterone responses to 5 days of food deprivation varied among developmental stages. Both pre- and prometamorphic tadpoles increased whole-body corticosterone content with food deprivation, but the magnitude of the response of premetamorphic tadpoles was significantly greater. By contrast, juvenile toads decreased plasma corticosterone concentration. Similarly, brain CRF peptide content tended to increase in food-deprived tadpoles but did not change in food-deprived juveniles. Therefore, there is an ontogenetic difference in the way the hypothalamic-pituitary-interrenal (HPI) axis responds to food deprivation in amphibians. In tadpoles, the HPI axis is activated in response to fasting as is seen in birds and mammals, and may be associated with mobilization of stored fuels and increased foraging. Juvenile toads do not respond to food deprivation by activating the HPI axis, but instead pursue a strategy of energy conservation that involves a reduction in plasma corticosterone concentration.     

Cooler temperatures slow the repair of DNA damage in tadpoles exposed to ultraviolet radiation: Implications for amphibian declines at high altitude

Ultraviolet B radiation (UVBR) damages the DNA of exposed cells, causing dimers to form between adjacent pyrimidine nucleotides. These dimers block DNA replication, causing mutations and apoptosis. Most organisms utilize biochemical or biophysical DNA repair strategies to restore DNA structure; however, as with most biological reactions, these processes are likely to be thermally sensitive. Tadpoles exposed to elevated UVBR at low environmental temperatures have significantly higher rates of mortality and developmental deformities compared with tadpoles exposed to the same levels of UVBR at higher environmental temperatures. We hypothesized that low environmental temperatures impair the primary enzymatic (photolyase) DNA repair pathway in amphibians, leading to the accumulation of DNA damage. To test this hypothesis, we compared DNA repair rates and photolyase gene expression patterns in Limnodynastes peronii. Tadpoles were acutely exposed to UVBR for 1 hr at either 20 or 30°C, and we measured DNA damage and photolyase expression levels at intervals following this exposure. Temperature had a significant effect on the rate of DNA repair, with repair at 30°C occurring twice as fast as repair at 20°C. Photolyase gene expression (6‐4 PP and CPD) was significantly upregulated by UVBR exposure, with expression levels increasing within 6 hr of UVBR exposure. CPD expression levels were not significantly affected by temperature, but 6‐4 PP expression was significantly higher in tadpoles in the 30°C treatment within 12 hr of UVBR exposure. These data support the hypothesis that DNA repair rates are thermally sensitive in tadpoles and may explain why enigmatic amphibian declines are higher in montane regions where UVBR levels are naturally elevated and environmental temperatures are lower.     

Risk of predation enhances the lethal effects of UV‐B in amphibians

Amphibian declines are a prominent part of the global biodiversity crisis and have received special consideration because they have occurred relatively recently, on a global scale, and in seemingly pristine habitats where no obvious anthropogenic cause is apparent. Although several causes for declines have been implicated, the isolation of a singular cause has proven elusive. Consequently, it has been hypothesized that complex interactions between multiple environmental stressors, particularly those associated with global change, may be responsible. Increasing ultraviolet‐B radiation (UV‐B) associated with stratospheric ozone depletion is one such stressor that has received considerable attention. UV‐B causes enhanced lethal effects when combined with other factors such as aquatic pH, contaminants, temperature and pathogens, but little is known of how UV‐B interacts with pervasive biological stressors, such as risk of predation. We exposed Limnodynastes peronii tadpoles to UV‐B and predatory chemical cues in a controlled laboratory experiment to determine their independent and interactive effects on survival and morphology. We show that UV‐B and risk of predation interact synergistically to enhance mortality above the additive effects of the independent stressors, and that exposure to UV‐B affects the ability of tadpoles to morphologically respond to predatory chemical cues (i.e. predator‐induced phenotypic plasticity), which has implications for their survival in an environment with predators. This highlights the importance of considering both anthropogenic and naturally occurring stressors when examining the underlying causes of amphibian declines.     

Searching for the physiological mechanism of density dependence: does corticosterone regulate tadpole responses to density?

Density-dependent population regulation is important in many natural populations, and in those populations, high population density is a likely stressor. In amphibians, previous laboratory studies with tadpoles suggest that corticosterone, the main glucocorticoid stress hormone, is one of the key regulators of density-dependent growth. To test this relationship in natural settings, we manipulated wood frog (Rana sylvatica) tadpole density at three levels in outdoor mesocosms and used a capture stress protocol to examine the hormonal stress response. In addition, we used the same capture protocol in six natural ponds (three high density and three low density). In the mesocosms, there was an increase in corticosterone levels in tadpoles following 1 h of confinement at weeks 1, 2, and 5. However, while tadpoles maintained at higher densities were smaller after metamorphosis, density did not alter mean levels of corticosterone obtained during confinement, and baseline levels of corticosterone did not differ between the densities. Similarly, in natural ponds, density did not correlate with baseline corticosterone or mean corticosterone levels obtained during confinement. We suggest that the physiological response to density may vary across the range of natural densities and that the role of corticosterone may be limited to periods of extreme high density, such as during pond-drying events.     

Variation in stress and innate immunity in the tree lizard (<Emphasis Type="Italic">Urosaurus ornatus</Emphasis>) across an urban–rural gradient

The urban environment presents new and different challenges to wildlife, but also potential opportunities depending on the species. As urban encroachment onto native habitats continues, understanding the impact of this expansion on native species is vital to conservation. A key physiological indicator of environmental disturbance is the vertebrate stress response, involving increases in circulating glucocorticoids (i.e. corticosterone), which exert influence on numerous physiological parameters including energy storage, reproduction, and immunity. We examined how urbanization in Phoenix, Arizona influences corticosterone levels, blood parasitism, and innate immunity in populations of tree lizards (Urosaurus ornatus) to determine whether urbanization may be detrimental or beneficial to this species. Both baseline and stress-induced corticosterone concentrations were significantly lower in urban lizards relative to the rural ones, however, the magnitude of the increase in corticosterone with stress did not differ across populations. Urban lizards also had a lower ratio of heterophils to lymphocytes, but elevated overall leukocyte count, as compared to lizards from the natural site. Urban and rural lizards did not differ in their prevalence of the blood parasite, Plasmodium mexicanum. Taken together, these results suggest that urban tree lizards may have suppressed overall corticosterone concentrations possibly from down-regulation as a result of frequent exposure to stressors, or increased access to urban resources. Also, urban lizards may have bolstered immunocompetence possibly from increased immune challenges, such as wounding, in the urban environment, or from greater energetic reserves being available as a result of access to urban resources.     

Urinary corticosterone metabolites and chytridiomycosis disease prevalence in a free-living population of male Stony Creek frogs (Litoria wilcoxii)

The emerging amphibian disease chytridiomycosis, which is caused by the fungal pathogen (Batrachochytrium dendrobatidis, Bd), has caused mass mortalities of native amphibian populations globally. There have been no previous studies on the relationships between stress hormones in free-living amphibians and Bd infections. In this study, we measured urinary corticosterone metabolite concentrations and Bd infections within free-living populations of male Stony Creek frog (Litoria wilcoxii) in Queensland, Australia. Prevalence of Bd zoospores from frog skin swabs was quantified using a real-time quantitative PCR technique. A urinary corticosterone enzyme-immunoassay (EIA) was validated using adrenocorticotropic hormone (ACTH) challenge. Urinary corticosterone concentrations of male frogs increased within 1-2 days after ACTH challenge and returned to baseline levels within 3 days post-ACTH injection. None of the frogs showed any rise in urinary corticosterone after saline injections. Individual male frogs showed either low or high baseline corticosterone concentrations. Male frogs identified as positive for Bd infection had significantly higher baseline urinary corticosterone concentrations in comparison to Bd negative male frogs. Urinary corticosterone EIA provides a reliable indication of stress in this frog species and this non-invasive physiological tool can be used to further assess the dynamics of Bd infections and physiological stress responses in other native amphibians.