Physiological Stress in Rescued Wild Koalas (Phascolarctos cinereus) Being Held in a Rehabilitation Sanctuary: A Pilot Study.

Koalas (Phascolarctos cinereus) are one of Australia's most charismatic native small marsupial species. Unfortunately, populations of koalas are rapidly declining throughout Australia as they continue to face increasing pressure from a changing ecosystem. All wildlife species to some degree will use their hypothalamic-pituitary-adrenal (HPA) axis in response to stress. Depending on the duration of activation, the stress response can lead to either acute or chronic side effects and is modulated through the neuroendocrine stress system with the release of catecholamines and glucocorticoids (e.g., cortisol). It is well known that rehabilitation sanctuaries are inherently stressful for all animals, in particular for rescued wild koalas, as it is an unfamiliar environment where the animals cannot predict or control what will happen to them. In this pilot study, we set out to quantify faecal and fur cortisol metabolites in wild rescued koalas undergoing wildlife rehabilitation. Absolute levels of acute and chronic stress were indexed non-invasively, with faecal samples taken to evaluate acute stress, and fur samples taken to evaluate chronic stress. Sampling occurred sporadically over four months (the start of September 2018 to the end of December 2018), and was performed on three rescued koalas (Maree, Tai, and Solstice) being held at the rehabilitation centre. Results of this study show that between the three koalas, the highest recorded faecal cortisol result was 241 ng/g, and the lowest recorded faecal cortisol result was 4 ng/g, whereas the highest recorded fur cortisol result was 1.75 ng/g, and the lowest recorded fur cortisol result was 0.10 ng/g. Statistically, there was a significant difference between all three koalas and their faecal cortisol responses, as well as their fur cortisol responses. Statistically for Maree and Solstice, there was a significant difference in their faecal cortisol response between days when a stressor was recorded, and days when a stressor was not recorded. However, statistically for Tai, this was not the case, as there was no significant difference in his faecal cortisol response between days when a stressor was recorded, and days when a stressor was not recorded. In summary, the hypothesis that faecal glucocorticoids and fur glucocorticoids between koalas will differ based on individual responses to stressors was true as a whole, but individually, this hypothesis was true for Maree and Solstice, but untrue for Tai. The use of biological samples such as faeces and fur to obtain readings of glucocorticoids is a method of measuring absolute levels of physiological stress that is still evolving for koalas, and there is no current glucocorticoid baseline with which to compare the results of this study; although, measuring faecal and fur glucocorticoids is the first step in understanding how koalas undergoing wildlife rehabilitation respond to stressors.

Are physiological and behavioural responses to stressors displayed concordantly by wild urban rodents?

Understanding wild animal responses to stressors underpins effective wildlife management. In order for responses to stressors to be correctly interpreted, it is critical that measurements are taken on wild animals using minimally invasive techniques. Studies investigating wild animal responses to stressors often measure either a single physiological or behavioural variable, but whether such responses are comparable and concordant remains uncertain. We investigated this question in a pilot study that measured responses of wild-caught urban brown and black rats (Rattus norvegicus, Rattus rattus) to fur-based olfactory cues from a predator, the domestic cat (Felis catus); a novel herbivore, the koala (Phascolarctos cinereus); and a familiar herbivore and competitor, the common brushtail possum (Trichosurus vulpecula). Physiological responses, measured by assaying faecal glucocorticoid metabolites, were compared to behavioural responses observed via video recordings. We found that physiological and behavioural responses to stressors were expressed concordantly. There was no sizeable physiological response observed, and the behavioural response when considered across the night was negligible. However, the behavioural response to the predator and competitor cues changed across the observation period, with activity increasing with increasing hours of exposure. Our results indicate that responses of wild rodents to cues are nuanced, with stress responses modulated by behaviour changes that vary over time according to the severity of the perceived threat as animals gather further information. If the physiological response alone had been assessed, this moderated response may not have been evident, and in terms of wildlife management, vital information would have been lost.

Extreme Competence: Keystone Hosts of Infections.

Individual hosts differ extensively in their competence for parasites, but traditional research has discounted this variation, partly because modeling such heterogeneity is difficult. This discounting has diminished as tools have improved and recognition has grown that some hosts, the extremely competent, can have exceptional impacts on disease dynamics. Most prominent among these hosts are the superspreaders, but other forms of extreme competence (EC) exist and others await discovery; each with potentially strong but distinct implications for disease emergence and spread. Here, we propose a framework for the study and discovery of EC, suitable for different host-parasite systems, which we hope enhances our understanding of how parasites circulate and evolve in host communities.

Testing for Short- and Long-Term Thermal Plasticity in Corticosterone Responses of an Ectothermic Vertebrate.

Phenotypic plasticity, broadly defined as the capacity of one genotype to produce more than one phenotype, is a key mechanism for how animals adapt to environmental (including thermal) variation. Vertebrate glucocorticoid hormones exert broad-scale regulation of physiological, behavioral, and morphological traits that influence fitness under many life-history or environmental contexts. Yet the capacity for vertebrates to demonstrate different types of thermal plasticity, including rapid compensation or longer acclimation in glucocorticoid hormone function, when subject to different environmental temperature regimes remains poorly addressed. Here, we explore whether patterns of urinary corticosterone metabolites respond (i.e., evidence of acclimation) to repeated short-term and sustained long-term temperature exposures in an amphibian, the cane toad (Rhinella marina). In response to three repeated short (30-min) high-temperature (37°C) exposures (at 10-d intervals), toads produced urinary corticosterone metabolite responses of sequentially greater magnitude, relative to controls. However, toads subjected to 4 wk of acclimation to either cool (18°C)- or warm (30°C)-temperature environments did not differ significantly in their urinary corticosterone metabolite responses during exposure to a thermal ramp (18°-36°C). Together, these results indicate that adult toads had different, including limited, capacities for their glucocorticoid responses to demonstrate plasticity to different regimes of environmental temperature variation. We advocate further research as necessary to identify plasticity, or lack thereof, in glucocorticoid physiology, to better understand how vertebrates can regulate organismal responses to environmental variation.

Dose-response relationship of tryptophan with large neutral amino acids, and its impact on physiological responses in the chick model.

Tryptophan (Trp) has been associated with the regulation of several behavioral and physiological processes, through stimulation of serotonergic activity. Tryptophan utilization at the metabolic level is influenced by the competitive carrier system it shares with large neutral amino acids (LNAA). This study was carried out using meat-type chicken as a model, to investigate the dose response effects of Trp/LNAA on fear response (tonic immobility; TI) and hormonal responses, including corticosterone (CORT), serotonin (5-HT), triiodothyronine (T3) and thyroxine (T4). A total of 12 cages (48 birds) were assigned to each of the six experimental groups at 29-42 days of age. Experimental diets were formulated to have incremental levels of Trp/LNAA (0.025, 0.030, 0.035, 0.040, 0.045, and 0.050). The results revealed that, Trp/NAA had no significant effect on growth performance and TI of the birds. However, elevation of Trp/LNAA was concurred with a linear reduction in CORT (P < .0001, r2 = 0.819) and linear increases in 5-HT (P < .0001, r2 = 0.945), T3 (P = .0003, r2 = 0.403) and T4 (P < .0001, r2 = 0.937) levels. In conclusion, the results from the current study demonstrated that, although incremental levels of Trp/LNAA did not affect bird growth performance or fearfulness, it increased 5-HT, T3 and T4, and decreased CORT levels in a linear dose-dependent manner. Manipulation of Trp feeding levels could be applied to manage stressful conditions in birds.

Prevalence and determinants of stereotypic behaviours and physiological stress among tigers and leopards in Indian zoos.

India's charismatic wildlife species are facing immense pressure from anthropogenic-induced environmental perturbations. Zoos play a major role in the conservation of threatened species, but their adaptation in captivity is posing a major challenge globally. Stress from inadequate adaptation could lead to suppression of cognitive functioning and increased display of stereotypic behaviour. It is thus necessary to measure biological traits like behaviour, stress physiology, and contextual factors driving the animals maintained at zoos. In this study, we assessed stereotypic behaviour and stress physiology employing standard behaviour scoring, non-invasive stress monitoring, and their contextual drivers in a sub-population of two large felid species managed in six Indian zoos. The prevalence and intensity of stereotypic behaviours and levels of faecal corticosterone metabolites (FCM) were ascertained among 41 Royal Bengal tigers Panthera tigris tigris and 21 Indian leopards Panthera pardus fusca between April 2014 and March 2015. Behavioural observations showed that tigers spent more time stereotyping (12%) than leopards (7%) during daylight hours. Stress levels assessed using FCM revealed that tigers (23.6 ± 1.62 ng/g) had marginally lower level of corticosterone metabolites than leopards (27.2 ±1.36 ng/g). Stereotypic behaviour increased significantly with FCM level when the effect of heath status was controlled in tigers, and the effects tree cover, stone, den and keeper attitude controlled in leopards. Comparison of stereotypes of tigers with various biological and environmental factors using binary logistic regression revealed that stereotypic prevalence decreased with increased enclosure size, and enclosure enrichments like presence of pools and stones, when managed socially with conspecifics, and with positive keeper attitude, these factors accounting for 43% of variations in stereotypic prevalence among tigers. Stereotype among leopards was significantly absent when associated with increased tree cover and presence of pool, and den in the enclosure, age and among zoo-born than wild-born ones. These factors explain 81% of variations in stereotypic prevalence in them. A comparison of FCM levels with context-dependent factors revealed that stress levels among tigers decreased significantly with enclosure size and with individuals from nil to low, and severity of health issues. These factors explain 64% of variations in FCM levels. In leopards, the presence of stones in the enclosure and keepers with positive attitude resulted in significant decrease in FCM levels, these factors together accounting for 94% of variations. Multiple regressions on selected variables based on Factor Analysis of Mixed Data showed that in tigers the intensity of stereotype decreased significantly with enclosure size, sociality and positive keeper attitude and FCM level with health problems. Similarly, analyses in leopards revealed that intensity of stereotype decreased significantly with tree cover, age and FCM level with positive keeper attitude. Overall, our study suggests that to reduce stereotypes and stress level, tigers in captivity should be managed in larger enclosures enriched with pool, and stones, and in appropriate social conditions with adequate veterinary care. Leopards should be managed in enclosures with dense tree cover, pool, stones and den. Positive keeper attitude plays a crucial role in the welfare of both the species in captivity. Our study is promising and is comparable with their natural behaviour in the wild; for example, tigers require larger natural habitats, while leopards can manage even with smaller isolated patches but with dense vegetation cover.

Evaluating Stress Physiology and Parasite Infection Parameters in the Translocation of Critically Endangered Woylies (Bettongia penicillata).

Translocation can be stressful for wildlife. Stress may be important in fauna translocation because it has been suggested that it can exacerbate the impact of infectious disease on translocated wildlife. However, few studies explore this hypothesis by measuring stress physiology and infection indices in parallel during wildlife translocations. We analysed faecal cortisol metabolite (FCM) concentration and endoparasite parameters (nematodes, coccidians and haemoparasites) in a critically endangered marsupial, the woylie (Bettongia penicillata), 1-3 months prior to translocation, at translocation, and 6 months later. FCM for both translocated and resident woylies was significantly higher after translocation compared to before or at translocation. In addition, body condition decreased with increasing FCM after translocation. These patterns in host condition and physiology may be indicative of translocation stress or stress associated with factors independent of the translocation. Parasite factors also influenced FCM in translocated woylies. When haemoparasites were detected, there was a significant negative relationship between strongyle egg count and FCM. This may reflect the influence of glucocorticoids on the immune response to micro- and macro-parasites. Our results indicate that host physiology and infection patterns can change significantly during translocation, but further investigation is required to determine how these patterns influence translocation success.

One size does not fit all: Monitoring faecal glucocorticoid metabolites in marsupials.

Marsupial research, conservation, and management can benefit greatly from knowledge about glucocorticoid (GC) secretion patterns because GCs influence numerous aspects of physiology and play a crucial role in regulating an animal's response to stressors. Faecal glucocorticoid metabolites (FGM) offer a non-invasive tool for tracking changes in GCs over time. To date, there are relatively few validated assays for marsupials compared with other taxa, and those that have been published generally test only one assay. However, different assays can yield very different signals of adrenal activity. The goal of this study was to compare the performance of five different enzyme immunoassays (EIAs) for monitoring adrenocortical activity via FGM in 13 marsupial species. We monitored FGM response to two types of events: biological stressors (e.g., transport, novel environment) and pharmacological stimulation (ACTH injection). For each individual animal and assay, FGM peaks were identified using the iterative baseline approach. Performance of the EIAs for each species was evaluated by determining (1) the percent of individuals with a detectable peak 0.125-4.5days post-event, and (2) the biological sensitivity of the assay as measured by strength of the post-event response relative to baseline variability (Z-score). Assays were defined as successful if they detected a peak in at least 50% of the individuals and the mean species response had a Z⩾2. By this criterion, at least one assay was successful in 10 of the 13 species, but the best-performing assay varied among species, even those species that were closely related. Furthermore, the ability to confidently assess assay performance was influenced by the experimental protocols used. We discuss the implications of our findings for biological validation studies.

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.

Evaluation of physiological stress in Australian wildlife: Embracing pioneering and current knowledge as a guide to future research directions.

Australia has a rich terrestrial and marine biodiversity and high species endemism. However, the oceanic continent is facing the biodiversity extinction crisis. The primary factors are anthropogenic induced environmental changes, including wildlife habitat destruction through urbanisation and predation by feral animals (e.g. red foxes and feral cats), increased severity of diseases (e.g. chytridiomycosis and chlamydia), and increased occurrence of summer heat waves and bush fires. Stress physiology is a dynamic field of science based on the studies of endocrine system functioning in animals. The primary stress regulator is the hypothalamo-pituitary adrenal (interrenal) axis and glucocorticoids (corticosterone and/or cortisol) provide stress index across vertebrate groups. This review paper focuses on physiological stress assessments in Australian wildlife using examples of amphibians, reptiles, birds and marsupials. I provide a thorough discussion of pioneering studies that have shaped the field of stress physiology in Australian wildlife species. The main findings point towards key aspects of stress endocrinology research, such as quantification of biologically active levels of glucocorticoids, development of species-specific GC assays and applications of stress physiology approaches in field ecology and wildlife conservation programs. Furthermore, I also discuss the importance of chronic stress assessment in wildlife populations. Finally, I provide a conceptual framework presenting key research questions in areas of wildlife stress physiology research. In conclusion, wildlife management programs can immensely benefit from stress physiology assessments to gauge the impact of human interventions on wildlife such as species translocation and feral species eradication.

Sexual dimorphism in baseline urinary corticosterone metabolites and their association with body-condition indices in a peri-urban population of the common Asian toad (Duttaphrynus melanostictus).

Field endocrinology research through the quantification of glucocorticoids or stress hormones in free-living wildlife is crucial for assessing their physiological responses towards pervasive environmental changes. Urinary corticosterone metabolite (UCM) enzyme-immunoassay (EIA) has been validated for numerous amphibian species as a non-invasive measure of physiological stress. Body-condition indices (BCIs) have also been widely used in amphibians as an indirect measure of animal health. Field endocrinology research on amphibian species in Asia is limited. In this study, we validated a UCM EIA in a peri-urban sub-population of the common Asian toad (Duttaphrynus melanostictus) in Pune, Maharashtra, India. We determined the baseline levels of UCMs in male (n=39) and female (n=19) toads. Secondly, we used a standard capture handling protocol to quantify changes in UCMs during short-term captivity. We also determined BCIs in the male and female toads using Fulton's index (K) and residual condition index (RCI). The results showed that mean baseline levels of UCMs were significantly higher in male toads than in females. There was no significant change in mean levels of UCMs of males and females between capture and captivity (0-12h). This highlights plausible habituation of the species to the peri-urban environment. Associations between UCMs with BCIs (K and R) were positive in male toads but negative in females. In conclusion, our UCMs EIA can be applied with BCIs to assess health of the Asian toads. We also suggest that direct fitness parameters such as sperm and oocyte quality, reproductive ecology and immunocompetence measurements should be applied in combination with these conservation physiology tools to quantify the fitness consequences of pervasive environmental changes on native amphibians.

Maximizing the reliability of non-invasive endocrine sampling in the tiger (Panthera tigris): environmental decay and intra-sample variation in faecal glucocorticoid metabolites.

Evaluation of physiological stress in the tiger (Panthera tigris) using faecal cortisol metabolite (FCM) enzyme immunoassays (EIAs) provides a powerful conservation physiology tool for the species. However, it is important to validate non-invasive endocrine sampling techniques in field conditions to ensure that the method provides a reliable parameter of physiological stress in the species. This is because endocrine measurements are highly species specific and FCM concentrations can be influenced by environmental factors. Here, we studied the impact of the decay rate of FCMs and intra-sample variation of FCMs using a previously validated EIA. To determine the decay rate of FCMs, we measured FCMs in freshly deposited tiger faeces (n = 8 tigers and 48 scats) that were randomly exposed to the natural environment (dry conditions with no rainfall) for up to 192 h. To determine intra-sample variation in FCMs, we used 10 scats from 10 tigers, divided each sample into four sections and each section into four sub-sections and measured FCMs in each section and sub-section. The results of this decay-rate experiment showed that FCMs in tiger faeces began to decay after 48 h exposure to the environmental conditions available. Thus, FCMs within freshly deposited tiger faeces are influenced by available environmental conditions. Changes in weather conditions (e.g. increased rainfall and humidity) could influence the stability of FCMs. The results of the intra-sample variation study showed that inter-variation among scats accounted for 52% of the variations in FCMs, while intra-sample variation between sections (32%) was greater than the sub-sample variation (16%). Intra-sample variation can be reduced by homogenizing the entire lyophilized faecal sample prior to the EIA. In conclusion, careful evaluation of decay rate and complete homogenization of faeces prior to EIA analysis will increase the reliability of FCMs as a non-invasive index of physiological stress in the tiger.

The neuro-hormonal control of rapid dynamic skin colour change in an amphibian during amplexus.

Sexual signalling using dynamic skin colouration is a key feature in some vertebrates; however, it is rarely studied in amphibians. Consequently, little is known about the hormonal basis of this interesting biological phenomenon for many species. Male stony creek frogs (Litoria wilcoxii) are known to change dorsal colouration from brown to lemon yellow within minutes. This striking change is faster then what has been seen most amphibians, and could therefore be under neuronal regulation, a factor that is rarely observed in amphibians. In this study, we observed colour changes in wild frogs during amplexus to determine the natural timing of colour change. We also investigated the hypothesis that colour change is mediated by either reproductive or neuro- hormones. This was achieved by injecting frogs with epinephrine, testosterone, saline solution (control 1) or sesame oil (control 2). A non-invasive approach was also used wherein hormones and controls were administered topically. Male frogs turned a vivid yellow within 5 minutes of initiation of amplexus and remained so for 3-5 hours before rapidly fading back to brown. Epinephrine-treated frogs showed a significant colour change from brown to yellow within 5 minutes, however, testosterone-treated frogs did not change colour. Our results provide evidence of the role neuronal regulation plays in colour change systems.

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.

Over-wintering tadpoles of Mixophyes fasciolatus act as reservoir host for Batrachochytrium dendrobatidis.

Batrachochytrium dendrobatidis (Bd), a cutaneous amphibian fungus that causes the lethal disease chytridiomycosis, has been implicated as a cause of many amphibian declines. Bd can tolerate low temperatures with an optimum thermal range from 17-24°C. It has been shown that Bd infection may result in species extinction, avoiding the transmission threshold presented by density dependent transmission theory. Prevalence of Bd during autumn and winter has been shown to be as low as 0% in some species. It is currently unclear how Bd persists in field conditions and what processes result in carry-over between seasons. It has been hypothesised that overwintering tadpoles may host Bd between breeding seasons. The Great Barred Frog (Mixophyes fasciolatus) is a common, stable and widespread species in Queensland, Australia, and is known to carry Bd. Investigation into Bd infection of different life stages of M. fasciolatus during seasonally low prevalence may potentially reveal persistence and carry-over methods between seasons. Metamorphs, juveniles, and adults were swabbed for Bd infection over three months (between March and May, 2011) at 5 sites of varying altitude (66 m-790 m). A total of 93 swabs were analysed using Polymerase Chain Reaction (PCR) real-time analysis. PCR analysis showed 6 positive (1 excluded), 4 equivocal and 83 negative results for infection with Bd. Equivocal results were assumed to be negative using the precautionary principle. The 5 positive results consisted of 4 emerging (Gosner stage 43-45) metamorphs and 1 adult M. fasciolatus. Fisher's exact test on prevalence showed that the prevalence was significantly different between life stages. All positive results were sampled at high altitudes (790 m); however prevalence was not significantly different between altitudes. Infection of emerging metamorphs suggests that individuals were infected as tadpoles. We hypothesise that M. fasciolatus tadpoles carry Bd through seasons. Thus, Mixophyes fasciolatus may act as disease reservoirs at multiple life stages.

Acute thermal stressor increases glucocorticoid response but minimizes testosterone and locomotor performance in the cane toad (Rhinella marina).

Climatic warming is a global problem and acute thermal stressor in particular could be considered as a major stressor for wildlife. Cane toads (Rhinella marina) have expanded their range into warmer regions of Australia and they provide a suitable model species to study the sub-lethal impacts of thermal stressor on the endocrine physiology of amphibians. Presently, there is no information to show that exposure to an acute thermal stressor could initiate a physiological stress (glucocorticoid) response and secondly, the possible effects on reproductive hormones and performance. Answering these questions is important for understanding the impacts of extreme temperature on amphibians. In this study, we experimented on cane toads from Queensland, Australia by acclimating them to mildly warm temperature (25°C) and then exposing to acute temperature treatments of 30°, 35° or 40°C (hypothetical acute thermal stressors). We measured acute changes in the stress hormone corticosterone and the reproductive hormone testosterone using standard capture and handling protocol and quantified the metabolites of both hormones non-invasively using urinary enzyme-immunoassays. Furthermore, we measured performance trait (i.e. righting response score) in the control acclimated and the three treatment groups. Corticosterone stress responses increased in all toads during exposure to an acute thermal stressor. Furthermore, exposure to a thermal stressor also decreased testosterone levels in all toads. The duration of the righting response (seconds) was longer for toads that were exposed to 40°C than to 30°, 35° or 25°C. The increased corticosterone stress response with increased intensity of the acute thermal stressor suggests that the toads perceived this treatment as a stressor. Furthermore, the results also highlight a potential trade-off with performance and reproductive hormones. Ultimately, exposure acute thermal stressors due to climatic variability could impact amphibians at multiple eco-physiological levels through impacts on endocrine physiology, performance and potentially fitness traits (e.g. reproductive output).

Evaluating physiological stress in Sumatran tigers (Panthera tigris ssp. sumatrae) managed in Australian zoos.

Glucocorticoid quantification using non-invasive methods provides a powerful tool for assessing the health and welfare of wildlife in zoo-based programmes. In this study, we provide baseline data on faecal-based glucocorticoid (cortisol) monitoring of Sumatran tigers (Panthera tigris ssp. sumatrae) managed at the Melbourne Zoo in Victoria, Australia. We sampled five tigers daily for 60 days. Faecal cortisol metabolites (FCMs) in tiger faecal extracts were quantified using enzyme immunoassays that were successfully validated using parallelism and accuracy recovery checks. Two female tigers had significantly higher mean FCM levels than the two males and another female, suggesting that females may have higher FCM levels. A significant elevation was noted in the FCM levels for one female 2 days after she was darted and anaesthetized; however, the FCM levels returned to baseline levels within 3 days after the event. Comparative analysis of FCM levels of tigers sampled at Melbourne Zoo with tigers sampled earlier at two other Australian Zoos (Dreamworld Themepark and Australia Zoo) showed that FCM levels varied between zoos. Differences in the enclosure characteristics, timing of sampling, size and composition of groupings and training procedures could all contribute to this variation. Overall, we recommend the use of non-invasive sampling for the assessment of adrenocortical activity of felids managed in zoos in Australia and internationally in order to improve the welfare of these charismatic big cats.

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.

Sight of a predator induces a corticosterone stress response and generates fear in an amphibian.

Amphibians, like other animals, generate corticosterone or cortisol glucocorticoid responses to stimuli perceived to be threatening. It is generally assumed that the corticosterone response of animals to capture and handling reflects the corticosterone response to stimuli such as the sight of a predator that are thought to be natural stressors. Fijian ground frogs (Platymantisvitiana) are preyed upon by the introduced cane toads (Rhinellamarina), and we used ground frogs to test the hypothesis that the sight of a predator will induce a corticosterone stress response in an amphibian. Urinary corticosterone metabolite concentrations increased in male ground frogs exposed to the sight of a toad for 1, 3 or 6 h, whereas corticosterone did not change in frogs exposed to another male ground frog, a ball, or when no stimulus was present in the test compartment. The frogs exposed to a toad initially moved towards the stimulus then moved away, whereas frogs exposed to another frog moved towards the test frog and remained closer to the frog than at the start of the test. Tonic immobility (TI) was measured as an index of fearfulness immediately after the test exposure of the frogs to a stimulus. The duration of TI was longer in frogs exposed to a toad than to another frog or to a ball. The results provide novel evidence that the sight of a predator can induce a corticosterone response and lead to increased fearfulness in amphibians. In addition, they show that endemic frogs can recognise an introduced predator as a threat.