PHYSIOLOGY: AMPHIBIANS
FROGS AND TOADS
Szymanski D. C., Gist D. H., Roth T. L. (2006): Anuran gender identification by fecal steroid analysis. Zoo Biology: Published in affiliation with the American Zoo and Aquarium Association 25: 35-46.
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This study tested the hypothesis that steroid hormone metabolites can be measured in anuran feces and their concentrations used to identify the sex of adults. Fecal samples from American toads, Bufo americanus, and boreal toads, B. boreas boreas, were extracted using ethyl acetate, and the concentrations of estradiol, progesterone and testosterone metabolites were measured by enzyme immunoassays with antibodies commonly used to evaluate steroid hormone concentrations in mammalian species. In American toads, mean testosterone metabolite concentrations (P<0.05) between males (224.3±15.5 ng/g feces) and females (80.7±10.6 ng/g), but estradiol and progesterone metabolite concentrations did not. In contrast, estradiol immunoreactivity differed (P<0.05) between male (19.0±1.8 ng/g) and female (48.3±6.3 ng/g) boreal toads. Progesterone and testosterone metabolite concentrations did not differ. Fecal hormone metabolite analysis offers a promising noninvasive approach to gender identification in anuran amphibians. However, the group of metabolites differentiating gender may not be consistent among species.
Narayan E., Molinia F., Christi K., Morley C., Cockrem J. (2010): Urinary corticosterone metabolite responses to capture, and annual patterns of urinary corticosterone in wild and captive endangered Fijian ground frogs (Platymantis vitiana). Australian Journal of Zoology 58: 189-197.
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This study was based on the development of a non-invasive glucocorticoid enzyme-immunoassay for the assessment of stress in wild and captive endangered Fijian ground frogs (Platymantis vitiana). Enzyme-immunoassays were developed and validated for the first time to non-invasively measure both cortisol and corticosterone metabolites in frog urine. Frog urine showed parallel displacement with corticosterone but not cortisol standards, therefore corticosterone enzyme immunoassays were used to examine stress in wild and captive frogs. Urinary corticosterone metabolite concentrations increased in frog urine (n = 4) at 6 h, 1 day and 2 days after injection with adrenocorticotropic hormone (0.44 μg g–1 bodyweight), indicating that the corticosterone enzyme-immunoassay could detect changes in circulating corticosterone in frogs. Urinary concentrations of corticosterone were measured in wild frogs (n = 18) after capture in the field. The first measurement beyond the initial sample was at 2–3 h. Mean urinary corticosterone concentrations rose after the initial sample and were significantly elevated in samples collected 3–4 h after capture. This is the first demonstration of a urinary corticosterone response to capture in amphibians. Urinary corticosterone metabolite concentrations for all months combined were lower in captive males than in wild males, and differed between vitellogenic, non-vitellogenic and captive females. Concentrations did not differ between captive and wild females. In conclusion, urinary corticosterone enzyme immunoassays can be used in frogs for assessing stress responses to capture and natural stress profiles of both captive and wild populations.
Germano J. M., Molinia F. C., Bishop P. J., Bell B. D., Cree A. (2012): Urinary hormone metabolites identify sex and imply unexpected winter breeding in an endangered, subterranean-nesting frog. General and Comparative Endocrinology 175: 464-472.
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Urinary hormone analysis has proved accurate for identifying sex and breeding periods in dimorphic amphibians with known reproductive cycles. We examined whether these techniques could provide this much needed information for a monomorphic anuran with an unconfirmed mating season in the wild. We analysed urinary estrone conjugate, testosterone, and progesterone metabolites to infer the time of breeding and to identify sex in the endangered Maud Island frog, Leiopelma pakeka. Testosterone metabolites in males and estrone and progesterone metabolites in females were at their peak during winter for both wild and captive frogs. These urinary metabolite patterns were consistent with the high proportion of females exhibiting enlarged ovarian follicles in winter months. Sex identification based on urinary estrone metabolite levels was 94% correct in this monomorphic species, in which the sexes overlap in snout-to-vent length (SVL) for over half of their adult size range and in which no other sexually dimorphic trait is known. The seasonal profiles imply unexpected winter or early spring breeding in L. pakeka. Overall, these results demonstrate use of urinary hormone metabolites for reproductive monitoring and sex identification in one of the world’s most threatened and evolutionarily distinct amphibians.
Graham C. M., Narayan E. J., McCallum H., Hero J. M. (2013): Non-invasive monitoring of glucocorticoid physiology within highland and lowland populations of native Australian Great Barred Frog (Mixophyes fasciolatus). General and Comparative Endocrinology 191: 24-30.
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This study used non-invasive endocrinology to examine baseline corticosterone at different altitudes in a free-living Australian amphibian: the Great Barred Frog (Mixophyes fasciolatus). An enzyme immunoassay (EIA) was performed on urine samples and validated biologically using an adrenocorticotropic hormone (ACTH) challenge. Frogs were injected with ACTH on day 0 and recaptures occurred 1–10 days post injection. Urine samples and body condition measurements were collected from lowland (60 m) and highland (660 m and 790 m) sub-populations of M. fasciolatus in South East Queensland (SEQ), close to their post-breeding period during autumn 2011. We simultaneously sampled these sub-populations for Batrachochytrium dendrobatidis (Bd), a pathogenic fungus responsible for mass mortalities of amphibians worldwide. The ACTH challenge successfully validated the urinary corticosterone EIA in M. fasciolatus, with a peak urinary corticosterone response to ACTH injection on day 2 and a return to baseline levels by day 6. Polymerase chain reaction (PCR) analysis of 50 individuals returned only 1 positive result for Bd. Simple linear regression showed a strong positive relationship between baseline urinary corticosterone concentrations and altitude and no relationship with body condition. We hypothesize that higher baseline corticosterone concentrations within highland sub-populations of male M. fasciolatus could be associated with increased environmental challenge at high altitudes and geographic range limits. Whether this pattern is an indication of chronic stress in highland populations or life-time fitness and survival, warrants future investigation.
Narayan E. J., Cockrem J., Hero J. M. (2013): Changes in serum and urinary corticosterone and testosterone during short-term capture and handling in the cane toad (Rhinella marina). General and Comparative Endocrinology 191: 225-230.
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Non-invasive endocrine monitoring with minimally invasive biological samples, such as urine, is being used widely for conservation biology research on amphibians. Currently, it is unknown how closely urinary measurements correspond with the traditional serum hormone measurements. We compared urinary and serum concentrations of corticosterone (CORT) and testosterone (T) in adult male cane toads (Rhinella marina) using a standard capture and handling (short-term stressor) protocol. Free-living male cane toads were captured and sampled for baseline urine (0 h) with a second urine sample taken at 0.5 h and hourly between 1 and 8 h. A single blood sample was collected from each toad after the final urine sampling and capture handling. The mean serum CORT concentration increased between 0 and 0.5 h, reaching the highest level between 6 and 8 h. The mean urinary CORT concentration increased with a lag-time of 1 h and continued to increase up to 8 h. The mean level of serum T decreased between 0 and 7 h and increased between 7 and 8 h. Mean urinary T concentration decreased with a lag-time of 0.5 h. Urinary T levels did not change between 4 and 8 h. Mean serum T levels reached 50% of the original 0 h value at 1 h while mean serum CORT levels reached 200% of the original 0 h value within 0.5 h. Mean urinary T levels reached 50% of the original 0 h value within 3 h while mean urinary CORT levels reached 200% of the original 0 h value within 3 h. The inter-individual variation in baseline serum and urinary CORT and T levels were highly comparable, suggesting that baseline urine sample provides a reliable indicator of the physiological status of the animal. Overall, the results have demonstrated that urine sampling and standard capture handling protocol provide reliable measures of baseline corticosterone and testosterone, as well as short-term stress hormone responses in amphibians.
Narayan E. J., Hero J. M. (2013): Repeatability of baseline corticosterone and acute stress responses to capture, and patterns of reproductive hormones in vitellogenic and non‐vitellogenic female Fijian ground frog (Platymantis vitiana). Journal of Experimental Zoology Part A: Ecological Genetics and Physiology 319: 471-481.
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The survival of animal species and individuals is largely determined by their ability to express physiological stress responses to predictable and unpredictable environmental challenges. Currently, there is no empirical evidence presenting the stress endocrine responses of female frogs during breeding between different reproductive groups. In this study, non-invasive urine sampling and standard capture and handling protocol were used to quantify baseline and short-term corticosterone stress responses in vitellogenic and non-vitellogenic female Fijian ground frog (Platymantis vitiana) during the annual breeding period. Urinary estrogen and progesterone metabolites were also quantified in the same frog urine samples. Repeated sampling of the female frogs (n = 20) on three occasions enabled repeatability (r) of reproductive and stress hormones to be quantified. All female frogs generated urinary corticosterone responses to the standard capture and handling stressor. Both baseline and short-term corticosterone responses were significantly higher in magnitude in the vitellogenic females in comparison to the non-vitellogenic female frogs. Vitellogenic females also showed significantly higher levels of urinary estrogen and progesterone metabolites in comparison to the non-vitellogenic females. Baseline urinary corticosterone, short-term corticosterone responses, urinary estrogen, and progesterone metabolites were highly repeatable for both female groups. The results highlight the importance of reproductive and stress hormones during the breeding period in female ground frogs. Future studies should determine the role of potential biological stressors (such as interactions with invasive species) that could be mediating the observed differences in stress endocrine responses of the vitellogenic and non-vitellogenic female frogs.
Baugh A. T., Bastien B., Still M. B., Stowell N. (2018): Validation of water-borne steroid hormones in a tropical frog (Physalaemus pustulosus). General and Comparative Endocrinology 261: 67-80.
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Minimally invasive methods for estimating hormone concentrations in wild vertebrates offer the opportunity to repeatedly measure behavior and hormone concentrations within individuals while minimizing experimenter interference during sample collection. We examined three steroid hormones (corticosterone, CORT; 17-β estradiol, E2; progesterone, PROG) in túngara frogs (Physalaemus pustulosus) using non-invasive water-borne methods. Using solid-phase extraction of water samples and liquid extraction of plasma and homogenate samples, coupled with enzyme immunoassays, we complimented the conventional validation approaches (parallelism, recovery determination) with dose-response assays that incorporated pharmacological challenges with adrenocorticotropic hormone (ACTH) and human chorionic gonadotropin (HCG). We also compared steroid concentrations in water to those observed in plasma and whole body homogenates. Lastly, we identified the constituent steroids in each sample type with a panel targeting 30 steroid species using high performance liquid chromatography-mass spectrometry (HPLC-MS). We found that a 60-min water-bath captures physiologically relevant changes in concentrations of CORT, E2 and PROG. Peak levels of water-borne CORT were found at approximately 2 h after ACTH injection. Water-borne CORT and E2 concentrations were positively correlated with their plasma and homogenate equivalents, while water-borne PROG was uncorrelated with homogenate PROG concentrations but negatively correlated with homogenate E2 concentrations. Together, our findings indicate that sampling water-borne hormones presents a non-invasive and biologically informative approach that will be useful for behavioral endocrinologists and conservation physiologists.
Hammond T. T., Au Z. A., Hartman A. C., Richards-Zawacki C. L. (2018): Assay validation and interspecific comparison of salivary glucocorticoids in three amphibian species. Conservation Physiology 6: coy055.
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Amphibians are one of the most threatened groups of species, facing stressors ranging from habitat degradation and pollution to disease and overexploitation. Stress hormones (glucocorticoids, GCs) provide one quantitative metric of stress, and developing non-invasive methods for measuring GCs in amphibians would clarify how diverse environmental stressors impact individual health in this taxonomic group. Saliva is an advantageous matrix for quantifying GCs, as it is sampled less invasively than plasma while still detecting both baseline and acute elevation of GCs within a short timeframe. Little work has employed this method in amphibian species, and it has never been pharmacologically and biologically validated. Here, we conduct analytical, pharmacological and biological validation experiments for measuring salivary corticosterone in three amphibian species: the American bullfrog (Rana catesbeiana), the green frog (Rana clamitans) and the northern leopard frog (Rana pipiens). These species are faced with a broad range of environmental challenges, and in part of its range R. pipiens populations are currently in decline. In addition to demonstrating that this method can be reliably used in multiple amphibian species, we present an examination of intrinsic biological factors (sex, body condition) that may contribute to GC secretion, and a demonstration that saliva can be collected from free-living animals in the field to quantify corticosterone. Our findings suggest that saliva may be useful for less invasively quantifying GCs in many amphibian species.
Forsburg Z. R., Goff C. B., Perkins H. R., Robicheaux J. A., Almond G. F., Gabor C. R. (2019): Validation of water-borne cortisol and corticosterone in tadpoles: recovery rate from an acute stressor, repeatability, and evaluating rearing methods. General and Comparative Endocrinology 281: 145-152.
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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.
Scheun J., Greeff D., Medger K., Ganswindt A. (2019): Validating the use of dermal secretion as a matrix for monitoring glucocorticoid concentrations in African amphibian species. Conservation Physiology 7: coz022.
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The complex interaction between factors leading to amphibian declines is responsible for the inability to develop robust, standardized conservation action plans. Monitoring physiological stress responses in amphibians may provide an ideal tool to assist conservationists in this regard. This study aimed to validate dermal secretions as a robust matrix for monitoring glucocorticoid alterations in an African amphibian, the edible bullfrog (Pyxicephalus edulis). Both a biological (handling) and physiological (ACTH challenge) validation were conducted to determine which of five available enzyme immunoassays (EIAs) tested is most suited for monitoring alterations in dermal glucocorticoid (dGC) concentrations. Additionally, the most optimal body region for monitoring dGC concentrations in P. edulis was identified. To confirm the activation of the hypothalamic–pituitary–interrenal (HPI) axis following ACTH administration, urinary glucocorticoid metabolites (uGCM) were also quantified. The tested corticosterone EIA was the only assay able to monitor alterations in dGC concentrations following the handling event in P. edulis. Further validation during the ACTH challenge supported the corticosterone EIA in this regard. Dermal secretions collected from both the ventral and dorsal regions were suitable for monitoring dGC concentrations in both sexes. A considerable increase in uGCM concentrations following ACTH administration was found, suggesting that the dGC concentrations observed were derived from the HPI axis. This study offers further support for the use of dermal secretions as an important matrix for monitoring physiological stress, and thus general health, in amphibian species.
Freel T., Koutsos E., Minter L. J., Tollefson T. N., Ridgley F., Brown J. L., Smith D., Scott H., Ange-van Heugten K. (2021): Urinary corticosterone concentrations in free-ranging and managed cane toads (Rhinella marina). Journal of Zoo and Wildlife Medicine 52: 1234-1240.
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Approximately 40% of amphibian species are threatened with extinction. The welfare of amphibians maintained under managed care as assurance populations is of vital importance to guard against extinction and provide a viable source population for future reintroduction. To manage amphibian species properly ex situ, it is important to understand how stress levels change over time when animals are removed from the wild and placed into managed environments. Corticosterone was analyzed in urine samples from free-ranging cane toads (Rhinella marina, n = 55) in Miami, FL, and under managed care for 22 (n = 48), 50 (n = 11), 81 (n = 25), or 119 (n = 10) days. Concentrations of corticosterone in free-ranging toad urine averaged 1.74 ± 0.195 ng/ml urine specific gravity (sp. gr.), which was greater (P < 0.05) than other time points (day 22: 0.77 ± 0.114 L; day 81: 0.85 ± 0.191 ng/ml sp. gr.; day 119: 0.58 ± 0.093 ng/ml sp. gr.), except day 50 (0.91 ± 0.274 ng/ml sp. gr.), which was not different from free-ranging or managed care values. Thus, corticosterone was lower in cane toads under managed care compared with those sampled in the wild, suggesting that managed care is not a stressor for this species.
Santymire R. M., Sacerdote-Velat A. B., Gygli A., Keinath D. A., Poo S., Hinkson K. M., McKeag E. M. (2021): Using dermal glucocorticoids to determine the effects of disease and environment on the critically endangered Wyoming toad. Conservation Physiology 9: coab093.
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Amphibian populations are declining worldwide, and increased exposure to environmental stressors, including global climate change and pathogens like Batrachochytrium dendrobatidis (Bd), may be contributing to this decline. Our goal was to use a novel dermal swabbing method to measure glucocorticoid (GC) hormones and investigate the relationship among disease and environmental conditions in the critically endangered Wyoming toad (Anaxyrus baxteri). Our objectives were to (i) validate the use of dermal swabs to measure GCs using an adrenocorticotropic hormone (ACTH) challenge on eight captive toads (4 ACTH: 2 M, 2F and 4 saline as a control: 2 M, 2F), (ii) investigate stress physiology and disease status of toads across six reintroduction sites and (iii) compare dermal cortisol between reintroduced and captive toads. Dermal cortisol peaked immediately after the ACTH and saline injections. Faecal GC metabolites (FGMs) were significantly higher one week after the ACTH injection compared with the week before. Saline-injected toads had no change in FGM over time. Toads were only found in three reintroduction sites and dermal cortisol was similar across sites; however, reintroduced toads had higher dermal cortisol in August compared with June and compared with captive individuals. Bd status did not influence dermal cortisol concentrations. Dermal and faecal hormonal metabolite analyses can be used to study amphibian stress physiology and learn how environmental conditions are impacting population success.
Ruthsatz K., Eterovick P. C., Bartels F., Mausbach J. (2023): Contributions of water-borne corticosterone as one non-invasive biomarker in assessing nitrate pollution stress in tadpoles of Rana temporaria. General and Comparative Endocrinology 331: 114164.
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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.
Tornabene B. J., Hossack B. R., Breuner C. W. (2023): Assay validation of saliva glucocorticoids in Columbia spotted frogs and effects of handling and marking. Conservation Physiology 11: coad078.
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Non-invasive methods are important to the field of conservation physiology to reduce negative effects on organisms being studied. Glucocorticoid (GC) hormones are often used to assess health of individuals, but collection methods can be invasive. Many amphibians are imperiled worldwide, and saliva is a non- or semi-invasive matrix to measure GCs that has been partially validated for only four amphibian species. Validation ensures that assays are reliable and can detect changes in saliva corticosterone (sCORT) after exposure to stressors, but it is also necessary to ensure sCORT concentrations are correlated with plasma concentrations. To help validate the use of saliva in assessing CORT responses in amphibians, we captured uniquely marked Columbia spotted frogs (Rana luteiventris) on sequential days and collected baseline and stress-induced (after handling) samples. For a subset of individuals, we collected and quantified CORT in both saliva and blood samples, which have not been compared for amphibians. We tested several aspects of CORT responses and, by collecting across separate days, measured repeatability of CORT responses across days. We also evaluated whether methods common to amphibian conservation, such as handling alone or handling, clipping a toe and tagging elevated sCORT. Similar to previous studies, we show that sCORT is reliable concerning parallelism, recovery, precision and sensitivity. sCORT was weakly correlated with plasma CORT (R2 = 0.21), and we detected elevations in sCORT after handling, demonstrating biological validation. Toe clipping and tagging did not increase sCORT over handling alone, but repeated handling elevated sCORT for ~72 hours. However, sCORT responses were highly variable and repeatability was low within individuals and among capture sessions, contrary to previous studies with urinary and waterborne CORT. sCORT is a semi-invasive and rapid technique that could be useful to assess effects of anthropogenic change and conservation efforts, but will require careful study design and future validation.
SALAMANDERS AND NEWTS
Gabor C. R., Zabierek K. C., Kim D. S., da Barbiano L. A., Mondelli M. J., Bendik N. F., Davis D. R. (2016): A non-invasive water-borne assay of stress hormones in aquatic salamanders. Copeia 104: 172-181.
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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.
Gabor C. R., Davis D. R., Kim D. S., Zabierek K. C., Bendik N. F. (2018): Urbanization is associated with elevated corticosterone in Jollyville Plateau salamanders. Ecological Indicators 85: 229-235.
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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 = 3 years) and stress responsiveness (n = 2 years) 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.
Millikin A. R., Woodley S. K., Davis D. R., Anderson J. T. (2019): Habitat characteristics in created vernal pools impact spotted salamander water-borne corticosterone levels. Wetlands 39: 803-814.
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Spotted salamanders (Ambystoma maculatum) require vernal pools for breeding habitat. Limited protection and preservation of vernal pools makes suitable habitat creation important. Differences in corticosterone levels, a hormone associated with growth, development, and stress in amphibians, could indicate population health and habitat quality. Our objective was to determine if habitat characteristics in created vernal pools influence corticosterone levels of spotted salamander larvae. In May and June of 2015 and 2016, we sampled water-borne corticosterone levels of larval spotted salamanders in 34 created vernal pools constructed 1–5 years earlier. Using multiple regression, we determined the best model predicting corticosterone levels included larval total length, pool-water temperature, year sampled, and pool diameter. Pool-water pH, depth, and age; percent cover; and predator presence were not significant predictors. Annual variation in corticosterone levels and habitat characteristics, and positive associations with water temperature and salamander body size highlighted the importance of controlling for external influences. The negative association between pool diameter and corticosterone indicated that larvae in larger pools (up to 12.75-m maximum diameter) were less stressed and potentially healthier. These results indicate that pool diameter contributes to habitat quality and may be important when constructing vernal pools for spotted salamanders.
Nagel A. H., Beshel M., DeChant C. J., Huskisson S. M., Campbell M. K., Stoops M. A. (2019): Non-invasive methods to measure inter-renal function in aquatic salamanders – correlating fecal corticosterone to the environmental and physiologic conditions of captive Necturus. Conservation Pysiology 7: coz074.
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This study sought to develop non-invasive techniques to monitor glucocorticoids in captive Necturus as a means to correlate inter-renal gland function in relation to environmental and physiological changes. Six individually housed breeding pairs of captive Necturus beyeri were subjected to seasonal changes in water temperature (30°F temperature differential) to stimulate natural breeding, specifically spermatophore deposition and oviposition. An enzyme immunoassay was validated for the measurement of N. beyeri faecal corticosterone metabolites (fCMs) by exhibiting parallelism and accuracy to the standard curve. Longitudinal (December 2016—October 2017) assessment of fCM concentrations and pattern of excretion from samples collected from the six breeding pairs revealed a seasonal inter-renal effect with higher concentrations (P < 0.05) excreted during months (December–March) of the year associated with breeding activity and when water temperatures were lowest. Males from each pair produced spermatophores starting on 08 December 8 2016 and ending on 05 April 2017. Females from four of the six pairs went on to successfully oviposit eggs in mid-late April 2017. One clutch was fertile, and three were non-fertile. No differences (P > 0.05) were detected in fCM concentrations between pairs in which oviposition did or did not occur. In addition, a novel waterborne corticosterone metabolite (wCM) assay was validated to overcome challenges associated with faecal collection in a group-housed amphibian. An adrenocorticotropic hormone (ACTH) challenge performed in an adult male Necturus maculosus resulted in a 50-fold increase in wCM at 4 h post-injection and marked the first demonstration of a waterborne inter-renal response to ACTH in Necturus. This study not only provides insight into inter-renal function in an aquatic salamander that exhibits marked reproductive seasonality but also confirms utility of fCM and wCM measurements as non-invasive means of assessment.
Bryant A. R., Gabor C. R., Swartz L. K., Wagner R., Cochrane M. M., Lowe W. H. (2022): Differences in corticosterone release rates of larval spring salamanders (Gyrinophilus porphyriticus) in response to native fish presence. Biology 11: 484.
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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.
MULTIPLE SPECIES
Gabor C. R., Bosch J., Fries J. N., Davis D. R. (2013): A non-invasive water-borne hormone assay for amphibians. Amphibia-Reptilia 34: 151-162.
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Anthropogenic disturbances have been implicated in the rapid decline of amphibians. Disturbances, such as disease and poor water quality, might cause changes in the physiology of amphibians resulting in chronic stress, which can result in decreased growth and development as well as immunosuppression. In amphibians, corticosterone (CORT) is the main hormone released in response to stressors. We took the first steps towards validating a new, non-invasive, technique to assay CORT in amphibians using a water-borne collection method previously used only with fish. In validation of this technique, we found a significant positive correlation between release rates of water-borne CORT and levels of CORT in circulating plasma in adults of the San Marcos salamander, Eurycea nana, and the common midwife toad, Alytes obstetricans. These results indicate that water-borne CORT can be used as a proxy for plasma CORT. Additionally, we examined basic background information on the physiological states of these two species. We found that captive-reared salamanders had significantly lower release rates of CORT than field-collected salamanders. Field-collected salamanders had significantly higher CORT release rates 24 h after capture and transfer to the laboratory. For tadpoles, we found that field-collected tadpoles did not have significantly different CORT release rates than those maintained in the laboratory for four months. Our research indicates that this method of water-borne hormone collection should be viable for many species of amphibians; however, further validation via adrenocorticotropic hormone (ACTH) challenges is required. This method can be a useful tool for assessing the physiological state of laboratory and field populations of amphibians and the effects of urbanization, pesticides and diseases. An important benefit of this method is that it allows for repeated measures of the same individuals and can be less stressful than drawing blood.
Santymire R. M., Manjerovic M. B., Sacerdote-Velat A. (2018): A novel method for the measurement of glucocorticoids in dermal secretions of amphibians. Conservation Physiology 6: coy008.
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Abstract
Amphibians have been declining in both diversity and abundance due in large part to habitat degradation and the prevalence of emerging diseases. Although stressors can suppress the immune system, affecting an individual’s health and susceptibility to pathogens, established methods for directly collecting stress hormones are not suitable for rapid field use or for use on threatened and endangered species. To overcome these challenges, we are developing an innovative method to collect and measure amphibian glucocorticoid secretions using non-invasive dermal swabs. We tested this methodology using multiple terrestrial, semi-aquatic and fully aquatic species. We swabbed the dorsal side of each animal six times and then induced a stressor of either hand-restraint, ACTH injection, or saline as a control. We then repeated swab collection immediately after the stressor and at 15, 30, 45, 60, 90 and 120 min intervals. Cortisol enzyme immunoassay detected changes in cortisol post-stressor. We also tested this methodology in the field and were successfully able to detect glucocorticoids from multiple species at varying life stages. When using in the field, capture technique should be considered since it may impact stress levels in certain species. Upon further testing, this novel method may be used to greatly increase our understanding of amphibian health especially as disease and environmental changes continue to impact fragile populations.
Tornabene B. J., Hossack B. R., Crespi E. J., Breuner C. W. (2021): Evaluating corticosterone as a biomarker for amphibians exposed to increased salinity and ambient corticosterone. Conservation Physiology 9: coab049.
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Abstract
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.