Leptin signaling promotes blood vessel formation in the Xenopus tail during the embryo-larval transition.

The signals that regulate peripheral blood vessel formation during development are still under investigation. The hormone leptin promotes blood vessel formation, adipose tissue establishment and expansion, tumor growth, and wound healing, but the underlying mechanisms for these actions are currently unknown. We investigated whether leptin promotes angiogenesis in the developing tail fin using embryonic transgenic xflk-1:GFP Xenopus laevis, which express a green fluorescent protein on vascular endothelial cells to mark blood vessels. We found that leptin protein is expressed in endothelial cells of developing blood vessels and that leptin treatment via injection increased phosphorylated STAT3 signaling, which is indicative of leptin activation of its receptor, in blood vessels of the larval tail fin. Leptin administration via media increased vessel length, branching, and reconnection with the cardinal vein, while decreased leptin signaling via immunoneutralization had an opposing effect on vessel development. We also observed disorganization of major vessels and microvessels of the tail fin and muscle when leptin signaling was decreased. Reduced leptin signaling lowered mRNA expression of cenpk, gpx1, and mmp9, markers for cell proliferation, antioxidation, and extracellular matrix remodeling/cell migration, respectively, in the developing tail, providing insight into three possible mechanisms underlying leptin's promotion of angiogenesis. Together these results illustrate that leptin levels are correlated with embryonic angiogenesis and that leptin coordinates multiple aspects of blood vessel growth and development, showing that leptin is an important morphogen during embryonic development.

Perinatal hair cortisol concentrations linked to psychological distress and unpredicted birth complications.

Maternal well-being and stress during the perinatal period have been hypothesized to influence birth outcomes and the postnatal development of offspring. In the present study, we explored whether hair cortisol concentration (HCC) was related to symptoms of psychological distress during the perinatal period and with unpredicted birth complications (UBCs). Surveys measuring symptoms of perceived stress, state/trait anxiety, and depression were collected from 53 participants (mean age = 31.1, SD = 4.04; 83% Caucasian, 17% other races) during the third trimester and again at two and six months after birth, 24.5% of which reported UBCs. In a subset of participants, we measured HCC in hair samples collected during the third trimester (27-39 weeks) and six months after birth. Compared to participants reporting normal births, those reporting UBCs had significantly elevated composite stress, anxiety, and depression (SAD) scores two months after birth, but scores decreased by six months postpartum. During the third trimester, HCC was positively associated with reported SAD scores, and HCC was elevated in participants reporting birth complications. Logistic regression showed HCC, but not SAD scores, predicted UBCs (p = 0.023, pseudo R2= 19.7%). Repeated measures MANOVA showed HCC varied over the perinatal period depending on both SAD scores reported at two months postpartum and the experience of UBCs; but when SAD scores reported at six months postpartum were included in the model, the association between HCC and SAD scores and the influence of UBCs was diminished. Although generalizability is limited by our relatively small, homogeneous sample, findings support a positive association between reported psychological distress and HCC during pregnancy and at two months postpartum. We also report a novel finding that chronically elevated cortisol concentrations during pregnancy were related to the risk of UBCs and remain elevated through the early postpartum period, suggesting the importance of monitoring both psychological distress and HCC during the perinatal period.

Testing whether adrenal steroids mediate phenotypic and physiologic effects of elevated salinity on larval tiger salamanders.

Salinity (sodium chloride, NaCl) from anthropogenic sources is a persistent contaminant that negatively affects freshwater taxa. Amphibians can be susceptible to salinity, but some species are innately or adaptively tolerant. Physiological mechanisms mediating tolerance to salinity are still unclear, but changes in osmoregulatory hormones such as corticosterone (CORT) and aldosterone (ALDO) are prime candidates. We exposed larval barred tiger salamanders (Ambystoma mavortium) to environmentally relevant NaCl treatments (<32-4000 mg·L-1 ) for 24 days to test effects on growth, survival, and waterborne CORT responses. Of those sampled, we also quantified waterborne ALDO from a subset. Using a glucocorticoid antagonist (RU486), we also experimentally suppressed CORT signaling of some larvae to determine if CORT mediates effects of salinity. There were no strong differences in survival among salinity treatments, but salinity reduced dry mass, snout-vent length, and body condition while increasing water content of larvae. High survival and sublethal effects demonstrated that salamanders were physiologically challenged but could tolerate the experimental concentrations. CORT signaling did not attenuate sublethal effects of salinity. Baseline and stress-induced (after an acute stressor, shaking) CORT were not influenced by salinity. ALDO was correlated with baseline CORT, suggesting it could be difficult to decouple the roles of CORT and ALDO. Future studies comparing ALDO and CORT responses of adaptively tolerant and previously unexposed populations could be beneficial to understand the roles of these hormones in tolerance to salinity. Nevertheless, our study enhances our understanding of the roles of corticosteroid hormones in mediating effects of a prominent anthropogenic stressor.

Neuroendocrine correlates of juvenile amphibian behaviors across a latitudinal cline.

We assessed the macrogeographic and neuroendocrine correlates of behavioral variation exhibited by juveniles, an important life stage for dispersal, across the expansive range of the wood frog. By rearing animals from eggs in a common garden then using a novel environment test, we uniquely demonstrated differential expression of juvenile behaviors among 16 populations spanning 8° latitude. On the individual level, cluster analysis indicated three major behavior profiles and principal component analysis resolved four unique axes of behavior, including escape, foraging, food intake, feeding efficiency. We found that increased escape behavior was associated with lower adrenocorticotropic hormone (ACTH)-induced circulating corticosterone (CORT) levels, however, foraging and food intake behaviors were not associated with either resting or ACTH-induced CORT. At the population level, the expression of foraging behaviors increased with latitude while food intake behaviors declined with latitude, which raised several hypotheses of eco-evolutionary processes likely driving this variation. Given that these behaviors covary along the same ecological gradient as locally adapted developmental traits, genomic studies in this species could provide deep insights into how HPA/I activity is associated with the eco-evolutionary processes that structure intraspecific variation in morphology and behavior.

Evolutionary conservation of leptin effects on wound healing in vertebrates: Implications for veterinary medicine.

In mammals, the cytokine hormone leptin promotes wound healing by increasing inflammation, cellular recruitment, angiogenic regrowth, and re-epithelialization; however, it is not known whether leptin has conserved actions on wound healing in other vertebrates. Here, we tested the hypothesis that leptin promotes both the quality and speed of wound healing in the South African clawed frog, Xenopus laevis. First, fluorescent immunohistochemistry using a polyclonal antibody specific to Xenopus leptin showed that in juvenile dorsal skin, leptin protein is expressed in the dorsal epidermal layer, as well in blood vessel endothelial cells and sensory nerves that run along the base of the dermis. Injection of recombinant Xenopus leptin (rXleptin) stimulates phosphorylated STAT3 (pSTAT3), indicative of leptin-activated JAK/STAT signaling in the epidermis. Similar to mammals, leptin protein expression increases at the wound site after injury of the epidermis. We then cultured "punch-in-a-punch" full-thickness dorsal skin explants in three doses of rXleptin (0, 10, and 100 ng/ml) and showed that leptin treatment doubled the rate of wound closure after 48 h relative to skin punches cultured without leptin. Food restriction prior to wound explant culture reduced the amount of wound closure, but leptin injection prior to euthanasia rescued closure to similar control levels. Leptin treatment also significantly reduced bacterial infection of these epidermal punches by 48 h in culture. This study shows that leptin is likely an endogenous promoter of wound healing in amphibians. Leptin-based therapies have the potential to expedite healing and reduce the incidence of secondary infections without toxicity issues, the threat of antibiotic resistance, or environmental antibiotic contamination. The conservation of leptin's actions on wound healing also suggests that it may have similar veterinary applications for other exotic species.

Effects of salinity and a glucocorticoid antagonist, RU486, on waterborne aldosterone and corticosterone of northern leopard frog larvae.

Increased salinity is an emerging contaminant of concern for aquatic taxa. For amphibians exposed to salinity, there is scarce information about the physiological effects and changes in osmoregulatory hormones such as corticosterone (CORT) and aldosterone (ALDO). Recent studies have quantified effects of salinity on CORT physiology of amphibians based on waterborne hormone collection methods, but much less is known about ALDO in iono- and osmoregulation of amphibians. We re-assayed waterborne hormone samples from a previous study to investigate effects of salinity (sodium chloride, NaCl) and a glucocorticoid receptor antagonist (RU486) on ALDO of northern leopard frog (Rana pipiens) larvae. We also investigated relationships between ALDO and CORT. Waterborne ALDO marginally decreased with increasing salinity and was, unexpectedly, positively correlated with baseline and stress-induced waterborne CORT. Importantly, ALDO increased when larvae were exposed to RU486, suggesting that RU486 may also suppress mineralocorticoid receptors or that negative feedback of ALDO is mediated through glucocorticoid receptors. Alternatively, CORT increases with RU486 treatment and might be a substrate for ALDO synthesis, which could account for increases in ALDO with RU486 treatment and the correlation between CORT and ALDO. ALDO was negatively correlated with percent water, such that larvae secreting more ALDO retained less water. Although sample sizes were limited and further validation and studies are warranted, our findings expand our understanding of adrenal steroid responses to salinization in amphibians and proposes new hypotheses regarding the co-regulation of ALDO and CORT.

Low occurrence of ranavirus in the Prairie Pothole Region of Montana and North Dakota (USA) contrasts with prior surveys.

Ranaviruses are emerging pathogens that have caused mortality events in amphibians worldwide. Despite the negative effects of ranaviruses on amphibian populations, monitoring efforts are still lacking in many areas, including in the Prairie Pothole Region (PPR) of North America. Some PPR wetlands in Montana and North Dakota (USA) have been contaminated by energy-related saline wastewaters, and increased salinity has been linked to greater severity of ranavirus infections. In 2017, we tested tissues from larvae collected at 7 wetlands that ranged in salinity from 26 to 4103 mg Cl l-1. In 2019, we used environmental DNA (eDNA) to test for ranaviruses in 30 wetlands that ranged in salinity from 26 to 11754 mg Cl l-1. A previous study (2013-2014) found that ranavirus-infected amphibians were common across North Dakota, including in some wetlands near our study area. Overall, only 1 larva tested positive for ranavirus infection, and we did not detect ranavirus in any eDNA samples. There are several potential reasons why we found so little evidence of ranaviruses, including low larval sample sizes, mismatch between sampling and disease occurrence, larger pore size of our eDNA filters, temporal variation in outbreaks, low host abundance, or low occurrence or prevalence of ranaviruses in the wetlands we sampled. We suggest future monitoring efforts be conducted to better understand the occurrence and prevalence of ranaviruses within the PPR.

Corticosterone mediates a growth-survival tradeoff for an amphibian exposed to increased salinity.

Life-history tradeoffs are common across taxa, but growth-survival tradeoffs-usually enhancing survival at a cost to growth-are less frequently investigated. Increased salinity (NaCl) is a prevalent anthropogenic disturbance that may cause a growth-survival tradeoff for larval amphibians. Although physiological mechanisms mediating tradeoffs are seldom investigated, hormones are prime candidates. Corticosterone (CORT) is a steroid hormone that independently influences survival and growth and may provide mechanistic insight into growth-survival tradeoffs. We conducted a 24-day experiment to test effects of salinity (<32-4000 mg/L) on growth, development, survival, CORT responses, and tradeoffs among traits of larval Northern Leopard Frogs (Rana pipiens). We also experimentally suppressed CORT signaling to determine whether CORT signaling mediates effects of salinity and a growth-survival tradeoff. Increased salinity reduced survival, growth, and development. Suppressing CORT signaling in conjunction with salinity reduced survival further but also attenuated the negative effects of salinity on growth, development, and water content. CORT of control larvae increased or was stable with growth and development but decreased with growth and development for those exposed to salinity. Therefore, salinity dysregulated CORT physiology. Across all treatments, larvae that survived had higher CORT than larvae that died. By manipulating CORT signaling, we provide strong evidence that CORT physiology mediates the outcome of a growth-survival tradeoff and enhances survival. To our knowledge, this is the first study to concomitantly measure tradeoffs between growth and survival and experimentally link these changes to CORT physiology. Identifying mechanistic links between stressors and fitness-related outcomes is critical to enhance our understanding of tradeoffs.

Evaluating corticosterone as a biomarker for amphibians exposed to increased salinity and ambient corticosterone.

Physiological biomarkers are commonly used to assess the health of taxa exposed to natural and anthropogenic stressors. Glucocorticoid (GC) hormones are often used as indicators of physiological stress in wildlife because they affect growth, reproduction and survival. Increased salinity from human activities negatively influences amphibians and their corticosterone (CORT; the main amphibian GC) physiology; therefore, CORT could be a useful biomarker. We evaluated whether waterborne CORT could serve as a biomarker of salt stress for three free-living amphibian species that vary in their sensitivity to salinity: boreal chorus frogs (Pseudacris maculata), northern leopard frogs (Rana pipiens) and barred tiger salamanders (Ambystoma mavortium). Across a gradient of contamination from energy-related saline wastewaters, we tested the effects of salinity on baseline and stress-induced waterborne CORT of larvae. Stress-induced, but not baseline, CORT of leopard frogs increased with increasing salinity. Salinity was not associated with baseline or stress-induced CORT of chorus frogs or tiger salamanders. Associations between CORT and salinity were also not related to species-specific sensitivities to salinity. However, we detected background environmental CORT (ambient CORT) in all wetlands and spatial variation was high within and among wetlands. Higher ambient CORT was associated with lower waterborne CORT of larvae in wetlands. Therefore, ambient CORT likely confounded associations between waterborne CORT and salinity in our analysis and possibly influenced physiology of larvae. We hypothesize that larvae may passively take up CORT from their environment and downregulate endogenous CORT. Although effects of some hormones (e.g. oestrogen) and endocrine disruptors on aquatic organisms are well described, studies investigating the occurrence and effects of ambient CORT are limited. We provide suggestions to improve collection methods, reduce variability and avoid confounding effects of ambient CORT. By making changes to methodology, waterborne CORT could still be a promising, non-invasive conservation tool to evaluate effects of salinity on amphibians.

Trans-ovo permethrin exposure affects growth, brain morphology and cardiac development in quail.

Permethrin is a commonly used, highly effective pesticide in poultry agriculture, and has recently been trialed in conservation efforts to protect Galápagos finch hatchlings from an invasive ectoparasite. Although permethrin is considered safe for adults, pesticides can have health consequences when animals are exposed during early life stages. The few studies that have examined permethrin's effects in embryonic chicks and rats have shown hydrocephaly, anencephaly, reduced cellular energy conversion, and disruption of developing heart muscle. To test whether trans-ovo exposure of permethrin affects early development in birds, we exposed Japanese quail (Coturnix japonica) eggs to cotton treated with 1% permethrin that was incorporated into nests in two amounts (0.2, 0.8 g), each with a paired untreated cotton control group. When measured on incubation Day 15, we found permethrin-treated developing birds were smaller and showed signs of microcephaly, although mortality rates were the same. Despite no difference in heart mass, ventricular tissue was less compact, cardiac arteries were reduced and heart rates were slower in permethrin-treated birds. Differences in heart development were also observed at 5 days of incubation, indicating that abnormalities are present from early in cardiac development. Future studies are needed to examine permethrin's effects on developmental pathways and to determine if these effects persist after hatching to affect offspring health. This study provides evidence that permethrin can cross the eggshell to cause non-lethal but adverse effects on embryonic development, and studies should look beyond hatching when monitoring the efficacy of permethrin on wild bird populations.

Stressors, coping, and resources needed during the COVID-19 pandemic in a sample of perinatal women.

BACKGROUND: Psychological stress and coping experienced during pregnancy can have important effects on maternal and infant health, which can also vary by race, ethnicity, and socioeconomic status. Therefore, we assessed stressors, coping behaviors, and resources needed in relation to the COVID-19 pandemic in a sample of 162 perinatal (125 pregnant and 37 postpartum) women in the United States. METHODS: A mixed-methods study captured quantitative responses regarding stressors and coping, along with qualitative responses to open-ended questions regarding stress and resources needed during the COVID-19 pandemic. Logistic and linear regression models were used to analyze differences between pregnant and postpartum participants, as well as differences across key demographic variables. Qualitative content analysis was used to analyze open-ended questions. RESULTS: During the COVID-pandemic, food scarcity and shelter-in-place restrictions made it difficult for pregnant women to find healthy foods. Participants also reported missing prenatal appointments, though many reported using telemedicine to obtain these services. Financial issues were prevalent in our sample and participants had difficulty obtaining childcare. After controlling for demographic variables, pregnant women were less likely to engage in healthy stress-coping behaviors than postpartum women. Lastly, we were able to detect signals of increased stressors induced by the COVID-19 pandemic, and less social support, in perinatal women of racial and ethnic minority, and lower-income status. Qualitative results support our survey findings as participants expressed concerns about their baby contracting COVID-19 while in the hospital, significant others missing the delivery or key obstetric appointments, and wanting support from friends, family, and birthing classes. Financial resources, COVID-19 information and research as it relates to maternal-infant health outcomes, access to safe healthcare, and access to baby supplies (formula, diapers, etc.) emerged as the primary resources needed by participants. CONCLUSIONS: To better support perinatal women's mental health during the COVID-19 pandemic, healthcare providers should engage in conversations regarding access to resources needed to care for newborns, refer patients to counseling services (which can be delivered online/via telephone) and virtual support groups, and consistently screen pregnant women for stressors.

Ecological adaptation drives wood frog population divergence in life history traits.

Phenotypic variation among populations is thought to be generated from spatial heterogeneity in environments that exert selection pressures that overcome the effects of gene flow and genetic drift. Here, we tested for evidence of isolation by distance or by ecology (i.e., ecological adaptation) to generate variation in early life history traits and phenotypic plasticity among 13 wood frog populations spanning 1200 km and 7° latitude. We conducted a common garden experiment and related trait variation to an ecological gradient derived from an ecological niche model (ENM) validated to account for population density variation. Shorter larval periods, smaller body weight, and relative leg lengths were exhibited by populations with colder mean annual temperatures, greater precipitation, and less seasonality in precipitation and higher population density (high-suitability ENM values). After accounting for neutral genetic variation, the QST-FST analysis supported ecological selection as the key process generating population divergence. Further, the relationship between ecology and traits was dependent upon larval density. Specifically, high-suitability/high-density populations in the northern part of the range were better at coping with greater conspecific competition, evidenced by greater postmetamorphic survival and no difference in body weight when reared under stressful conditions of high larval density. Our results support that both climate and competition selection pressures drive clinal variation in larval and metamorphic traits in this species. Range-wide studies like this one are essential for accurate predictions of population's responses to ongoing ecological change.

Drivers of distributions and niches of North American cold-adapted amphibians: evaluating both climate and land use.

Species distribution estimates are often used to understand the niche of a species; however, these are often based solely on climatic predictors. When the influences of biotic factors are ignored, erroneous inferences about range and niche may be made. We aimed to integrate climate data with a unique set of available land cover and land use data for the six cold-adapted amphibians of North America (Ambystoma macrodactylum, Anaxyrus hemiophrys, Anaxyrus boreas, Pseudacris maculata, Rana sylvatica, Rana luteiventris) to determine the relative importance of climate and non-climate drivers through the use of ecological niche models for present-day range estimates. We compared climate-only, land use-only, and combination models of climate and land use, derived from two different model selection techniques, to determine which was most likely to drive current distributions of cold-adapted amphibian species. Land use layers included land cover type, human population, vegetation type, ecoregion, and the overall human footprint. The most supported models included both climate and land use, with climate and human footprint variables having the highest permutation importance and percent contribution. Models that incorporated climate and land use data performed best as measured with AIC and AUC, although qualitatively most underestimated the northern range edge, implying potential sampling bias or locations of reduced habitat quality for these species in the northern area of the ranges. There were small differences in overall combination models dependent on the method of model selection. The overall effect sizes of landscape factors within the combination models were small except for one landscape feature: human footprint, which incorporated multiple aspects of anthropogenic change on the landscape, including human population density, travel access, and agricultural impact. This aspect of the landscape was just as important as climate, and counter to what we expected, the association was mostly positive, with a negative response only occurring at very high levels. This highlights the importance of moving beyond climate only species range estimates as land cover, specifically human impact, may be driving the patterns of species' ranges.

Salinity stress increases the severity of ranavirus epidemics in amphibian populations.

The stress-induced susceptibility hypothesis, which predicts chronic stress weakens immune defences, was proposed to explain increasing infectious disease-related mass mortality and population declines. Previous work characterized wetland salinization as a chronic stressor to larval amphibian populations. Thus, we combined field observations with experimental exposures quantifying epidemiological parameters to test the role of salinity stress in the occurrence of ranavirus-associated mass mortality events. Despite ubiquitous pathogen presence (94%), populations exposed to salt runoff had slightly more frequent ranavirus related mass mortality events, more lethal infections, and 117-times greater pathogen environmental DNA. Experimental exposure to chronic elevated salinity (0.8-1.6 g l-1 Cl-) reduced tolerance to infection, causing greater mortality at lower doses. We found a strong negative relationship between splenocyte proliferation and corticosterone in ranavirus-infected larvae at a moderate elevation of salinity, supporting glucocorticoid-medicated immunosuppression, but not at high salinity. Salinity alone reduced proliferation further at similar corticosterone levels and infection intensities. Finally, larvae raised in elevated salinity had 10 times more intense infections and shed five times as much virus with similar viral decay rates, suggesting increased transmission. Our findings illustrate how a small change in habitat quality leads to more lethal infections and potentially greater transmission efficiency, increasing the severity of ranavirus epidemics.

Endocrine Regulation of Epimorphic Regeneration.

Studies aiming to uncover primary mechanisms of regeneration have predominantly focused on genetic pathways regulating specific stages in the regeneration process: wound healing, blastema formation, and pattern formation. However, studies across organisms show that environmental conditions and the physiological state of the animal can affect the rate or quality of regeneration, and endocrine signals are likely the mediators of these effects. Endocrine signals acting directly on receptors expressed in the tissue or via neuroendocrine pathways can affect regeneration by regulating the immune response to injury, allocation of energetic resources, or by enhancing or inhibiting proliferation and differentiation pathways involved in regeneration. This review discusses the cumulative knowledge in the literature about endocrine regulation of regeneration and its importance in future research to advance biomedical research.

Endocrine regulation of regeneration: Linking global signals to local processes.

Regeneration in amphibians and reptiles has been explored since the early 18th century, giving us a working in vivo model to study epimorphic regeneration in vertebrates. Studies aiming to uncover primary mechanisms of regeneration have predominantly focused on genetic pathways regulating specific stages of the regeneration process: wound healing, blastema formation and growth, and pattern formation. However, studies across organisms show that environmental conditions and physiological state of the animal can affect the rate or quality of regeneration, and endocrine signals are likely the mediators of these effects. Endocrine signals working/acting directly on receptors expressed in the structure or via neuroendocrine pathways can affect regeneration by modulating immune response to injury, allocation of energetic resources, or by enhancing or inhibiting proliferation and differentiation pathways in regenerating tissue. This review discusses the cumulative knowledge known about endocrine regulation of regeneration and important future research directions of interest to both ecological and biomedical research.

Glucococorticoid receptor activation exacerbates aminoglycoside-induced damage to the zebrafish lateral line.

Aminoglycoside antibiotics have potent antibacterial properties but cause hearing loss in up to 25% of patients. These drugs are commonly administered in patients with high glucocorticoid stress hormone levels and can be combined with exogenous glucocorticoid treatment. However, the interaction of stress and aminoglycoside-induced hearing loss has not been fully explored. In this study, we investigated the effect of the glucocorticoid stress hormone cortisol on hair cells in the zebrafish lateral line as an important step toward understanding how physiological stressors modulate hair cell survival. We found that 24-hr cortisol incubation sensitized hair cells to neomycin damage. Pharmacological and genetic manipulation demonstrates that sensitization depended on the action of the glucocorticoid receptor but not the mineralocorticoid receptor. Blocking endogenous cortisol production reduced hair cell susceptibility to neomycin, further evidence that glucocorticoids modulate aminoglycoside ototoxicity. Glucocorticoid transcriptional activity was apparent in lateral line hair cells, suggesting a direct action of cortisol in these aminoglycoside-sensitive cells. Our work shows that the stress hormone cortisol can increase hair cell sensitivity to aminoglycoside damage, which highlights the importance of recognizing stress and the impacts of glucocorticoid signaling in both ototoxicity research and clinical practice.

Critical disease windows shaped by stress exposure alter allocation trade-offs between development and immunity.

Ubiquitous environmental stressors are often thought to alter animal susceptibility to pathogens and contribute to disease emergence. However, duration of exposure to a stressor is likely critical, because while chronic stress is often immunosuppressive, acute stress can temporarily enhance immune function. Furthermore, host susceptibility to stress and disease often varies with ontogeny; increasing during critical developmental windows. How the duration and timing of exposure to stressors interact to shape critical windows and influence disease processes is not well tested. We used ranavirus and larval amphibians as a model system to investigate how physiological stress and pathogenic infection shape development and disease dynamics in vertebrates. Based on a resource allocation model, we designed experiments to test how exposure to stressors may induce resource trade-offs that shape critical windows and disease processes because the neuroendocrine stress axis coordinates developmental remodelling, immune function and energy allocation in larval amphibians. We used wood frog larvae (Lithobates sylvaticus) to investigate how chronic and acute exposure to corticosterone, the dominant amphibian glucocorticoid hormone, mediates development and immune function via splenocyte immunohistochemistry analysis in association with ranavirus infection. Corticosterone treatments affected immune function, as both chronic and acute exposure suppressed splenocyte proliferation, although viral replication rate increased only in the chronic corticosterone treatment. Time to metamorphosis and survival depended on both corticosterone treatment and infection status. In the control and chronic corticosterone treatments, ranavirus infection decreased survival and delayed metamorphosis, although chronic corticosterone exposure accelerated rate of metamorphosis in uninfected larvae. Acute corticosterone exposure accelerated metamorphosis increased survival in infected larvae. Interactions between stress exposure (via glucocorticoid actions) and infection impose resource trade-offs that shape optimal allocation between development and somatic function. As a result, critical disease windows are likely shaped by stress exposure because any conditions that induce changes in differentiation rates will alter the duration and susceptibility of organisms to stressors or disease.

Success stories and emerging themes in conservation physiology.

The potential benefits of physiology for conservation are well established and include greater specificity of management techniques, determination of cause-effect relationships, increased sensitivity of health and disturbance monitoring and greater capacity for predicting future change. While descriptions of the specific avenues in which conservation and physiology can be integrated are readily available and important to the continuing expansion of the discipline of 'conservation physiology', to date there has been no assessment of how the field has specifically contributed to conservation success. However, the goal of conservation physiology is to foster conservation solutions and it is therefore important to assess whether physiological approaches contribute to downstream conservation outcomes and management decisions. Here, we present eight areas of conservation concern, ranging from chemical contamination to invasive species to ecotourism, where physiological approaches have led to beneficial changes in human behaviour, management or policy. We also discuss the shared characteristics of these successes, identifying emerging themes in the discipline. Specifically, we conclude that conservation physiology: (i) goes beyond documenting change to provide solutions; (ii) offers a diversity of physiological metrics beyond glucocorticoids (stress hormones); (iii) includes approaches that are transferable among species, locations and times; (iv) simultaneously allows for human use and benefits to wildlife; and (v) is characterized by successes that can be difficult to find in the primary literature. Overall, we submit that the field of conservation physiology has a strong foundation of achievements characterized by a diversity of conservation issues, taxa, physiological traits, ecosystem types and spatial scales. We hope that these concrete successes will encourage the continued evolution and use of physiological tools within conservation-based research and management plans.

Evaluating environmental DNA-based quantification of ranavirus infection in wood frog populations.

A variety of challenges arise when monitoring wildlife populations for disease. Sampling tissues can be invasive to hosts, and obtaining sufficient sample sizes can be expensive and time-consuming, particularly for rare species and when pathogen prevalence is low. Environmental DNA (eDNA)-based detection of pathogens is an alternative approach to surveillance for aquatic communities that circumvents many of these issues. Ranaviruses are emerging pathogens of ectothermic vertebrates linked to die-offs of amphibian populations. Detecting ranavirus infections is critical, but nonlethal methods have the above issues and are prone to false negatives. We report on the feasibility and effectiveness of eDNA-based ranavirus detection in the field. We compared ranavirus titres in eDNA samples collected from pond water to titres in wood frog (Lithobates sylvaticus; n = 5) tadpoles in sites dominated by this one species (n = 20 pond visits). We examined whether ranavirus DNA can be detected in eDNA from pond water when infections are present in the pond and if viral titres detected in eDNA samples correlate with the prevalence or intensity of ranavirus infections in tadpoles. With three 250 mL water samples, we were able to detect the virus in all visits with infected larvae (0.92 diagnostic sensitivity). Also, we found a strong relationship between the viral eDNA titres and titres in larval tissues. eDNA titres increased prior to observed die-offs and declined afterwards, and were two orders of magnitude higher in ponds with a die-off. Our results suggest that eDNA is useful for detecting ranavirus infections in wildlife and aquaculture.