Reading tea leaves worldwide: Decoupled drivers of initial litter decomposition mass-loss rate and stabilization.

The breakdown of plant material fuels soil functioning and biodiversity. Currently, process understanding of global decomposition patterns and the drivers of such patterns are hampered by the lack of coherent large-scale datasets. We buried 36,000 individual litterbags (tea bags) worldwide and found an overall negative correlation between initial mass-loss rates and stabilization factors of plant-derived carbon, using the Tea Bag Index (TBI). The stabilization factor quantifies the degree to which easy-to-degrade components accumulate during early-stage decomposition (e.g. by environmental limitations). However, agriculture and an interaction between moisture and temperature led to a decoupling between initial mass-loss rates and stabilization, notably in colder locations. Using TBI improved mass-loss estimates of natural litter compared to models that ignored stabilization. Ignoring the transformation of dead plant material to more recalcitrant substances during early-stage decomposition, and the environmental control of this transformation, could overestimate carbon losses during early decomposition in carbon cycle models.

Research priorities for natural ecosystems in a changing global climate.

Climate change poses significant emerging risks to biodiversity, ecosystem function and associated socioecological systems. Adaptation responses must be initiated in parallel with mitigation efforts, but resources are limited. As climate risks are not distributed equally across taxa, ecosystems and processes, strategic prioritization of research that addresses stakeholder-relevant knowledge gaps will accelerate effective uptake into adaptation policy and management action. After a decade of climate change adaptation research within the Australian National Climate Change Adaptation Research Facility, we synthesize the National Adaptation Research Plans for marine, terrestrial and freshwater ecosystems. We identify the key, globally relevant priorities for ongoing research relevant to informing adaptation policy and environmental management aimed at maximizing the resilience of natural ecosystems to climate change. Informed by both global literature and an extensive stakeholder consultation across all ecosystems, sectors and regions in Australia, involving thousands of participants, we suggest 18 priority research topics based on their significance, urgency, technical and economic feasibility, existing knowledge gaps and potential for cobenefits across multiple sectors. These research priorities provide a unified guide for policymakers, funding organizations and researchers to strategically direct resources, maximize stakeholder uptake of resulting knowledge and minimize the impacts of climate change on natural ecosystems. Given the pace of climate change, it is imperative that we inform and accelerate adaptation progress in all regions around the world.

Calling phenology of a diverse amphibian assemblage in response to meteorological conditions.

The strong association between amphibian activity, breeding and recruitment with local environmental conditions raises concerns regarding how changes in climate may affect the persistence of species populations into the future. Additionally, in a highly diverse assemblage of anurans, competition for breeding sites affects the time and duration of activity, as species compete for limited resources such as water. Meteorological conditions are strong drivers of amphibian activity, so we assessed whether temperature, rainfall, atmospheric pressure and humidity were associated with the calling phenology of an assemblage of anurans in South East Queensland, Australia. We performed calling surveys and collected digital recordings at 45 ponds in an area known for high anuran diversity. We performed detection analyses to investigate the influence of 10 meteorological variables in detection of calling activity in 19 amphibian species. Our results suggest four breeding strategies in the assemblage: explosive summer breeders, prolonged breeders, opportunistic breeders and a winter breeder. Classifying these species into associations provides a framework for understanding how species respond to environmental conditions. Explosive breeders (i.e. species demonstrating short and highly synchronised breeding periods) were particularly responsive to temperature. Our findings help elucidate the breeding phenology of frogs and provide valuable information on their mating systems in native Australian forests. This study highlights the difficulties of surveying even common anurans. We highlight the importance of predictability and stability in climate and the vulnerability of species for which reproduction appears to require highly specific environmental cues.

Does physiological response to disease incur cost to reproductive ecology in a sexually dichromatic amphibian species?

It is well known that the disease chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd) has contributed to amphibian declines worldwide. The impact of Bd varies, with some species being more susceptible to infection than others. Recent evidence has shown that Bd can have sub-lethal effects, whereby increases in stress hormones have been associated with infection. Could this increased stress response, which is a physiological adaptation that provides an increased resilience against Bd infection, potentially be a trade-off with important life-history traits such as reproduction? We studied this question in adult male frogs of a non-declining species (Litoria wilcoxii). Frogs were sampled for (1) seasonal hormone (testosterone and corticosterone), color and disease profiles, (2) the relationship between disease infection status and hormone levels or dorsal color, (3) subclinical effects of Bd by investigating disease load and hormone level, and (4) reproductive and stress hormone relationships independent of disease. Testosterone levels and color score varied seasonally (throughout the spring/summer months) while corticosterone levels remained stable. Frogs with high Bd prevalence had significantly higher corticosterone levels and lower testosterone levels compared to uninfected frogs, and no differences in color were observed. There was a significant positive correlation between disease load and corticosterone levels, and a significant negative relationship between disease load and testosterone. Our field data provides novel evidence that increased physiological stress response associated with Bd infection in wild frogs, could suppress reproduction by down-regulating gonadal hormones in amphibians, however the impacts on reproductive output is yet to be established.

Repeated thermal stressor causes chronic elevation of baseline corticosterone and suppresses the physiological endocrine sensitivity to acute stressor in the cane toad (Rhinella marina).

Extreme environmental temperature could impact the physiology and ecology of animals. The stress endocrine axis provides necessary physiological stress response to acute (day-day) stressors. Presently, there are no empirical evidences showing that exposure to extreme thermal stressor could cause chronic stress in amphibians. This could also modulate the physiological endocrine sensitivity to acute stressors and have serious implications for stress coping in amphibians, particularly those living in fragmented and disease prone environments. We addressed this important question using the cane toad (Rhinella marina) model from its introduced range in Queensland, Australia. We quantified their physiological endocrine sensitivity to a standard acute (capture and handling) stressor after exposing the cane toads to thermal shock at 35°C for 30min daily for 34 days. Corticosterone (CORT) responses to the capture and handling protocol were measured on three sampling intervals (days 14, 24, and 34) to determine whether the physiological endocrine sensitivity was maintained or modulated over-time. Two control groups (C1 for baseline CORT measurement only and C2 acute handled only) and two temperature treatment groups (T1 received daily thermal shock up to day 14 only and a recovery phase of 20 days and T2 received thermal shock daily for 34 days). Results showed that baseline CORT levels remained high on day 14 (combined effect of capture, captivity and thermal stress) for both T1 and T2. Furthermore, baseline CORT levels decreased for T1 once the thermal shock was removed after day 14 and returned to baseline by day 29. On the contrary, baseline CORT levels kept on increasing for T2 over the 34 days of daily thermal shocks. Furthermore, the magnitudes of the acute CORT responses or physiological endocrine sensitivity were consistently high for both C1 and T1. However, acute CORT responses for T2 toads were dramatically reduced between days 24 and 34. These novel findings suggest that repeated exposure to extreme thermal stressor could cause chronic stress and consequently suppress the physiological endocrine sensitivity to acute stressors (e.g. pathogenic diseases) in amphibians.

Changes in serum and urinary corticosterone and testosterone during short-term capture and handling in the cane toad (Rhinella marina).

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 (0h) with a second urine sample taken at 0.5h and hourly between 1 and 8h. 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 0.5h, reaching the highest level between 6 and 8h. The mean urinary CORT concentration increased with a lag-time of 1h and continued to increase up to 8h. The mean level of serum T decreased between 0 and 7h and increased between 7 and 8h. Mean urinary T concentration decreased with a lag-time of 0.5h. Urinary T levels did not change between 4 and 8h. Mean serum T levels reached 50% of the original 0h value at 1h while mean serum CORT levels reached 200% of the original 0h value within 0.5h. Mean urinary T levels reached 50% of the original 0h value within 3h while mean urinary CORT levels reached 200% of the original 0h value within 3h. 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.

Non-invasive monitoring of glucocorticoid physiology within highland and lowland populations of native Australian Great Barred Frog (Mixophyes fasciolatus).

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 1-10days post injection. Urine samples and body condition measurements were collected from lowland (60m) and highland (660m and 790m) 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.

Non-invasive evaluation of physiological stress in an iconic Australian marsupial: the Koala (Phascolarctos cinereus).

Koalas (Phascolarctos cinereus) are the only extant representatives of Australia's unique marsupial family Phascolarctidae and were listed as nationally Vulnerable in 2012. Causes of mortality are diverse, although the disease chlamydiosis, dog attacks, collisions with cars, and loss of habitat represent the principal reasons for the continued species decline. Koala breeding facilities in Queensland and New South Wales, Australia have been established for conservation and tourism. Non-invasive monitoring of physiological stress is important for determining the sub-lethal effects of environmental stressors on the well-being, reproduction and survival of Koalas in Zoos and also in the wild. In this study, we developed a faecal cortisol metabolite (FCM) enzyme-immunoassay (EIA) for monitoring physiological stress in Koalas from two established Zoos in Australia and also within a free-living sub-population from Queensland. Biological validation of the FCM EIA was done using an adrenocorticotropic hormone (ACTH) challenge. We discovered excretory lag-times of FCM of 24 h in females (n=2) and 48 h in male (n=2) Koalas in response to the ACTH challenge. FCM levels showed an episodic and delayed peak response lasting up to 9 days post ACTH challenge. This finding should be taken into consideration when designing future experiments to study the impacts of short-term (acute) and chronic stressors on the Koalas. Laboratory validations were done using parallelism and recovery checks (extraction efficiency) of the cortisol standard against pooled Koala faecal extracts. Greater than 99% recovery of the cortisol standard was obtained as well as a parallel displacement curve against Koala faecal extracts. FCM levels of the captive Koalas (n=10 males and 13 females) significantly differed by sex, reproductive condition (lactating versus non-lactating Koalas) and the handling groups. Handled male Koalas had 200% higher FCM levels than their non-handled counterparts, while females were not affected by handling as long they were not undergoing lactation. There was no significant difference in FCM levels between the captive and wild Koalas (n=9 males and 7 females). Overall, these results provide foundation knowledge on non-invasive FCM analysis in this iconic Australian marsupial. Non-invasive stress endocrinology opens up opportunities for evaluating the sub-lethal physiological effects of management activities (including caging, translocation) on the nutritional status, reproductive behaviors and disease status of captive and managed in situ Koala populations.

Faecal cortisol metabolites in Bengal (Panthera tigris tigris) and Sumatran tigers (Panthera tigris sumatrae).

The tiger (Panthera tigris) faces a great risk of extinction as its wild numbers have plummeted due to poaching and habitat destruction so ex-situ conservation programs are becoming ever more necessary. Reliable non-invasive biomarkers of the stress hormone (cortisol) are necessary for assessing the health and welfare of tigers in captivity. To our knowledge, non-invasive stress endocrinology methods have not been tested as widely in tigers. The first aim of this study was to describe and validate a faecal cortisol metabolite enzyme-immmunoassay (FCM EIA) for two tiger sub-species, the Bengal tiger (Panthera tigris tigris) and the Sumatran tiger (Panthera tigris sumatrae). Individual tigers (n=22) were studied in two large Zoos in Queensland, Australia (Dreamworld Theme Park and Australia Zoo). Fresh faecal samples (<12 h old) were collected each morning from both Zoos over a study period of 21 days. Biological validation was conducted separately by collecting feces 5 days before and 5 days after blood was taken from four male and five female tigers. Results showed that mean FCM levels increased by 138% and 285% in the male and female tigers within 1 day after bloods were taken, returning to baseline in 5 days. Laboratory validations of the FCM EIA were done using an extraction efficiency test and parallelism. Results showed >89% recovery of the cortisol standard that was added to tiger faecal extract. We also obtained parallel displacement of the serially diluted cortisol standard against serially diluted tiger faecal extract. Our second aim was to determine whether the FCM levels were significantly different between tiger sub-species and sex. Results showed no significant difference in mean FCM levels between the Bengal and Sumatran tiger sub-species. Mean levels of FCMs were significantly higher in females than in male tigers. Those male and female tigers with reported health issues during the study period expressed higher FCM levels than the reportedly healthy tigers. Interestingly, those tigers that took part in some activity (such as walks, photos, presentations and guest feeds) expressed moderately higher FCM levels at Dreamworld and lower FCM levels at Australia Zoo in comparison to those tigers that did not take part in such activities. These results indicate potential habituation in some tigers for routine activity through specialized training and pre-conditioning. In conclusion, the FCM EIA described in this study provides a reliable non-invasive method for evaluating the stress status of tigers in Zoos.

Repeatability of baseline corticosterone and short-term corticosterone stress responses, and their correlation with testosterone and body condition in a terrestrial breeding anuran (Platymantis vitiana).

Repeatability of physiological response variables, such as the stress hormone corticosterone, across numerous sampling occasions is an important assumption for their use as predictors of behaviour, reproduction and fitness in animals. Very few studies have actually tested this assumption in free-living animals under uncontrolled natural conditions. Non-invasive urine sampling and standard capture handling protocol have enabled the rapid quantification of baseline corticosterone and short-term corticosterone stress responses in anuran amphibians. In this study, established non-invasive methods were used to monitor physiological stress and urinary testosterone levels in male individuals of the terrestrial breeding Fijian ground frog (Platymantis vitiana). Adult male frogs (n = 20) were sampled at nighttime on three repeated occasions at intervals of 14 days during their annual breeding season on Viwa Island, Fiji. All frogs expressed urinary corticosterone metabolite responses to the capture and handling stressor, with some frogs showing consistently higher urinary corticosterone responses than others. Ranks of corticosterone values at 0, 4 and 8 h, and the corrected rank were highly significant (r = 0.75-0.99) between the three repeated sampling occasions. Statistical repeatabilities were high for baseline corticosterone (r = 0.973) and for corticosterone values at 2 h (r = 0.862), 4 h (r = 0.861), 6 h (r = 0.820) and 8 h (r = 0.926), and also for the total (inclusive of baseline corticosterone values) and the corrected integrated responses (index of the acute response) [r = 0.867 and r = 0.870]. Urinary testosterone levels also showed high statistical repeatability (r = 0.78). Furthermore, variation in baseline and short-term corticosterone stress responses was greater between individuals than within individuals. Baseline urinary corticosterone was significantly negatively correlated with the corrected integrated corticosterone response (r = -0.3, p < 0.001) but non-significantly with body-condition (r = -0.04) and baseline urinary testosterone (r = -0.07). In contrast, the corrected integrated corticosterone response was positively correlated (non-significantly) with baseline urinary testosterone (r = +0.04) and body-condition (r = +0.08). Urinary testosterone levels and body-condition were significantly negatively correlated (r = -0.23, p < 0.001). The results suggest that male frogs with higher levels of testosterone could have depleted energy reserve during the breeding period. The acute corticosterone responses help in replenishing energy that is needed for breeding and survival. The results also provide some support to the 'cort-fitness' hypothesis as highlighted by the negative correlation between baseline corticosterone and body-condition. It is most likely that the acute corticosterone response is adaptive and linked positively with reproductive fitness and survival in male anurans.

Are baseline and short-term corticosterone stress responses in free-living amphibians repeatable?

Amphibians respond to environmental stressors by secreting corticosterone, a stress hormone which promotes physiological and behavioral responses. Capture handling can be used to stimulate physiological stress response in amphibians. The use of single blood sampling and presentation of mean data often limits the quantification of within and between individual variation in baseline and short-term corticosterone stress responses in amphibians. It is important for studies of amphibian physiological ecology to determine whether baseline and short-term corticosterone stress responses are consistent or not. We quantified repeatability (r), a statistical measure of consistency, in baseline and short-term corticosterone stress responses to a standard capture and handling stress protocol in free-living adult male cane toads (Rhinella marina). Corticosterone metabolite concentrations were measured entirely non-invasively in male toad urine samples via an enzyme-immunoassay. During the first sampling occasion, urine samples were collected manually from individual male toads (n=20) immediately upon field capture. Toads were handled for 5min then transferred to plastic bags (constituting a mild stressor), and urine samples were collected hourly over 8h in the field. The toads were resampled for baseline (0h) urine corticosterone with hourly urine sampling over 8h (for quantification of the stress induced corticosterone) at 14 day intervals on three consecutive occasions. Within and between sample variations in urinary corticosterone metabolite concentrations were also quantified. All toads expressed a corticosterone stress response over 8h to our standard capture and handling stress protocol. Variations both within and between toads was higher for corrected integrated corticosterone concentrations than corticosterone concentrations at baseline, 3 or 6h. Baseline urinary corticosterone metabolite concentration of the male toads was highly repeatable (r=0.877) together with high statistical repeatabilities for 3h (r=0.695), 6h (r=0.428) and 8h (r=0.775) corticosterone metabolite concentrations, and for the total and corrected integrated corticosterone responses (r=0.807; r=0.743 respectively). This study highlights that baseline and short-term corticosterone stress responses are repeatable in free-living amphibians. Future studies should utilize this non-invasive tool to explore repeatability among seasons and across years, and determine its functional significance in relation to behavioral ecology and reproduction in amphibians generally.

The effect of stress and stress hormones on dynamic colour-change in a sexually dichromatic Australian frog.

Rapid colour changes in vertebrates have fascinated biologists for centuries, herein we demonstrate dynamic colour change in an anuran amphibian, the stony creek frog (Litoria wilcoxii), which turns from brown to bright (lemon) yellow during amplexus. We show this by comparing the colour of baseline (unpaired males) and amplecting (paired) males. We also investigate the possible role of stress and stress hormones on this colour change. Frogs were subjected to four different levels of stressors (handling, toe-clipping, saline injection and adrenocorticotropic hormone [ACTH] injection) and the colour change was measured using digital photography. A comparison of baseline colour and stress hormone (corticosterone) levels was also conducted to give further insight to this topic. From the images, the Red Blue Green (RGB) colour values were calculated, and a principal components analysis (PCA) was used to create a single colour metric (the major axis) as an index of colour in the visible spectrum. A moderate stressor (toe-clipping) led to a significant change in colour (within 10 min) similar to that of amplecting males. Surprisingly, neither a mild stressor (handling and saline injection) nor the maximum stressor (handling and ACTH injection) led to a lightening response. This study confirms that the dynamic male colour change in this species in response to medium stressors adds new knowledge to the understanding of the functional mechanisms of dynamic colour change in amphibians.

Effects of temperature on urinary corticosterone metabolite responses to short-term capture and handling stress in the cane toad (Rhinella marina).

Extreme temperature can cause metabolic, immune and behavioural changes in amphibians. Short-term stress hormonal response via increased secretion of corticosterone enables amphibians to make necessary physiological and behavioural adjustments for coping with stressors. The effect of temperature on short-term corticosterone responses has not been studied in amphibians. In this study, this relationship was evaluated in adult male cane toads (Rhinella marina). We acclimated male toads (n=24 toads per group) at low, medium and high temperature (15, 25 or 35°C) under controlled laboratory conditions for a 14 day period. After thermal acclimation, short-term corticosterone responses were evaluated in the toads subjected to a standard capture and handling stress protocol over a 24h period. Corticosterone metabolites in toad urine were measured via enzyme-immunoassay. During acclimation, mean baseline urinary corticosterone level increased after transfer of the toads from wild into captivity and returned to baseline on day 14 of acclimation for each of the three temperatures. At the end of the 14 days of thermal acclimation period, baseline corticosterone level were highest for toad group at 35°C and lowest at 15°C. All toads generated urinary corticosterone responses to the standard capture and handling stressor for each temperature. Both individual and mean short-term corticosterone responses of the toads were highest at 35°C and lowest at 15°C. Furthermore, Q(10) values (the factor by which the reaction rate increases when the temperature is raised by 10°) were calculated for mean corrected integrated corticosterone responses as follows; (15-35°C) Q(10)=1.51, (15-25°C) Q(10)=1.60; (25-35°C) Q(10)=1.43. Both total and corrected integrated corticosterone responses were highest for toads at 35°C followed by 25°C and lowest for the 15°C toad group. Overall, the results have demonstrated the thermodynamic response of corticosterone secretion to short-term capture and handling stress in an amphibian species.

Urinary corticosterone metabolite responses to capture and handling in two closely related species of free-living Fijian frogs.

Studies of baseline (unstressed) and short-term corticosterone stress responses in free-living amphibians can provide crucial information on the physiological responses of different populations to environmental change. In this study, we compared baseline and urinary corticosterone metabolite responses of free-living adult males and females of two closely related Fijian frogs of the Platymantis genus (Family: Ceratobatrachidae). Fijian ground frogs (Platymantis vitiana) live on the ground while Fijian tree frogs (Platymantis vitiensis) are arboreal. We captured free-living frogs and applied our moderate stress protocol (5 min handling during urine sampling at hourly intervals), with urinary corticosterone metabolite concentrations measured by enzyme-immunoassay. Mean urinary corticosterone metabolite concentrations in male and female Fijian ground frogs increased from 0 to 2 h and continued to increase to peak concentrations 5-6 h after capture. Mean baseline corticosterone concentration was significantly different between sexes (higher in males than females) only for Fijian ground frogs. There was no significant difference between sexes in the integrated corticosterone responses for both species. Mean baseline and urinary corticosterone metabolite responses of Fijian tree frogs were lower than those of Fijian ground frogs. Corticosterone levels increased for 4-5 h in both species and began to decrease again 7 h after initial capture. Corticosterone responses were consistently higher for Fijian ground frogs than Fijian tree frogs. Individuals in both species showed markedly variable corticosterone responses over the 8h duration of the stressor, with some individuals showing low stress responses and others showing high stress responses. The magnitude of the corrected integrated response of the ground frogs was almost twice that of the tree frogs. These differences in baseline and short-term corticosterone stress responses between these two species could be a consequence of ecological differences including micro-habitat, predator interactions and/or competitive interactions with the introduced cane toad (Rhinella marina). Comparisons of corticosterone responses between populations and species provide a valuable tool for measuring the physiological responses of the amphibians to environmental change.

Inverse urinary corticosterone and testosterone metabolite responses to different durations of restraint in the cane toad (Rhinella marina).

Non-invasive measurement of urinary corticosterone and testosterone metabolites in amphibians provides opportunities for endocrine studies of responses to physiological and psychological stressors. Typically, corticosterone metabolite concentrations increase in frog urine within 1-2h of a mild capture and handling stress protocol. However, no study has investigated the effect of duration of manual restraint on the changes in corticosterone and reproductive hormones in amphibians. We quantified urinary corticosterone and testosterone metabolite responses for 8h following various durations of manual restraint (control, 5, 15 or 30min) in adult male cane toads (Rhinella marina) under controlled laboratory conditions. All toads had a corticosterone stress response over 8h to our standard capture and handling stressor. The mean corticosterone stress response was significantly higher after 15 or 30min restraint in comparison to the control (no restraint) or to 5min restraint. Manual restraint for 5, 15 or 30min caused a significant reduction in urinary testosterone concentrations over 8h. We also provide a novel method of quantifying plasticity in corticosterone stress responses in amphibians with respect to restraint duration using the concept of a "reaction norm". The reaction norm, which was calculated as slope of the regression line of integrated corticosterone response against restraint duration, was 9.69 (pg corticosterone/µg creatinineh)/min for male toads. In summary, corticosterone and testosterone responses to restraint are affected by restraint duration in male toads. Glucocorticoid reaction norms can be applied to study the change in physiological stress hormonal response with respect to restraint duration in other amphibian species.

Individual variation and repeatability in urinary corticosterone metabolite responses to capture in the cane toad (Rhinella marina).

Urinary corticosterone metabolite enzyme-immunoassay (EIA) can be used for the non-invasive assessment of baseline levels and corticosterone responses in amphibians. In this study, urinary corticosterone responses of wild male cane toads (Rhinella marina) to confinement and repeated handling were measured to quantify individual variation in corticosterone responses for the first time in an amphibian species. Urine samples were collected at 0 h in the wild, hourly from 2 to 8 h after transfer into captivity, and again at 12 and 24 h in captivity. Toads were then held in captivity and subjected to the same sampling protocol on three occasions at 14 days intervals to quantify variation in corticosterone metabolite responses within and between toads. Baseline and individual corticosterone metabolite responses in male cane toads were generally consistent, with high statistical repeatabilities for 0 h (r=0.630), 6 h (r=0.793), 12 h (r=0.652) and 24 h (r=0.721) corticosterone metabolite concentrations, and for the total and corrected integrated corticosterone responses (r=0.567, p=0.033; r=0.728, p=0.014 respectively). Urinary corticosterone responses appear to be a stable, repeatable trait within individuals. Corticosterone responses in amphibians can be more readily measured when urine rather than plasma samples are collected, and the protocol established in the current study can now be applied to the study of variation in corticosterone responses in other amphibians.

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.

Urinary corticosterone metabolite responses to capture and captivity in the cane toad (Rhinella marina).

Urinary corticosterone metabolite responses to capture have recently been shown for the first time in amphibians, and in the present study urinary corticosterone metabolite responses to capture and to confinement in captivity were measured in adult cane toads (Rhinella marina) in Queensland, Australia. An adrenocorticotropic hormone (ACTH) challenge was used to provide a biological validation for urinary corticosterone metabolite concentrations measured by radioimmunoassay (RIA). Urinary corticosterone metabolite increased 1-2 days after ACTH but not saline injection and then returned to initial values, indicating that the RIA could detect changes in corticosterone secretion in toads. Urinary corticosterone metabolite responses to short-term capture and restraint in plastic bags were first apparent 2h after capture of wild toads. Toads held communally in captivity for 5 days had elevated urinary corticosterone metabolite concentrations. Mean corticosterone concentrations declined significantly after a further 7 days in individual housing chambers. There was no sex difference in urinary corticosterone metabolite responses of toads to ACTH challenge, short-term capture or captivity. The relative amount of variation in the mean corticosterone responses was quantified by calculating coefficients of variation (CV) for each mean corticosterone response. Mean corticosterone at 0 min was more variable for captive toads than wild toads. Furthermore, initial corticosterone concentrations (0 min) were more variable than concentrations during the ACTH challenge, short-term capture and captivity. There was little change in the amount of variation of mean corticosterone levels between male and female toads with increasing time in captivity (12-29 days). This study has shown individual corticosterone responses of amphibians for the first-time, and has provided a novel method for quantifying the relative amount of variation in amphibian corticosterone responses.

Urinary corticosterone responses to capture and toe-clipping in the cane toad (Rhinella marina) indicate that toe-clipping is a stressor for amphibians.

Toe-clipping, the removal of one or more toes, is a common method used to individually mark free-living animals. Whilst this method is widely used in studies of amphibians, the appropriateness of the method, and its potential detrimental effects have been the subject of debate. Here, we provide for the first time, evidence that toe-clipping is a stressor in a wild amphibian. We measured urinary corticosterone responses of male cane toads (Rhinella marina) to capture and handling only, and to toe-clipping under field conditions. Urinary testosterone concentrations and white blood cell proportions were also measured. Urinary corticosterone metabolite concentrations increased 6h after capture and handling only and remained high for 24h; corticosterone returned to baseline levels after 48 h and remained low at 72 h post capture and handling. Corticosterone concentrations in toads subjected to toe-clipping increased at 6h to significantly higher concentrations than after capture and handling only, then decreased more slowly than after capture and handling, and were still elevated (approximately double basal level) 72 h after toe-clipping. Testosterone did not change significantly after capture and handling only, whereas after toe-clipping testosterone decreased at 6h and remained low at 72 h. There were weak short-term effects of toe-clipping compared with capture and handling only on white blood cell proportions. We have clearly shown that toe-clipping is a distinctly stronger stressor than capture and handling alone. This indicates that there is an ethical cost of toe-clipping, and this should be considered when planning studies of amphibians.

Rate-Pressure Product Responses to Static Contractions Performed at Various Altitudes.

Simmonds, Michael J., Surendran Sabapathy, and Jean-Marc Hero. Rate-pressure product responses to static contractions performed at various altitudes. High Alt Med Biol. 22: 166-173, 2021. Background: Adventure tourism has led to an unprecedented number of individuals being exposed to altitude, including those with subclinical cardiometabolic disorders. The disproportionate hemodynamic challenge associated with small-muscle static activities is potentially dangerous at altitude as these may compound the risk for cardiac events. We thus examined the cardiovascular response to, and during recovery from, static exercise performed at altitude. Methods: Eighteen individuals completed this study at three altitudes (sea level; ∼1,500 m; ∼3,000 m) in central Nepal. At each altitude, individuals performed two handgrip contractions for 2 minutes at the same intensity (30% maximal voluntary contraction [MVC]), with two distinct recovery periods: during control recovery was completed quietly at rest, while during ischemic challenge recovery was conducted with a cuff occluding the upper limb. Results: Oxygen saturation decreased during ascent to 1,500 m (-2%) and 3,000 m (-8%), compared with sea level. Handgrip MVC was not affected by altitude, although heart rate at rest (∼70 beat/min), during static exercise (range ∼90-95 beat/min), and during recovery in both conditions (each ∼70 beat/min) was significantly increased by ∼15% at 3,000 m, but not 1,500 m. The magnitude of the muscle metaboreflex during recovery from static exercise was unaffected by altitude; however, the rate-pressure product was significantly elevated by ∼10% during and following static exercise at 3,000 m. Conclusions: A significant increase in the rate-pressure product during static exercise was observed at altitude, which persisted during recovery. Individuals at risk for cardiac events should use awareness of static contractions while at altitude, especially considering that stress induced by static exercise is additive to that of dynamic activities such as hiking.