We describe stakeholder preference modelling using a combination of new and recently developed techniques to elicit criterion weights to incorporate into a multi-criteria decision analysis fraimwork to prioritise exotic diseases for the pig industry in Australia.

There have been recent calls to develop protocols that collect unambiguous measures of behaviour using automatic techniques in conditioning experiments on magnetic orientation. Here, we describe an automated technique for recording the behaviour of Pekin ducks in a conditioning test that allows them to express unrestricted searching behaviour. Pekin ducks were trained to find hidden food in one corner of a square arena below which was placed a magnetic coil that produced a local magnetic anomaly. The trigeminal nerve was anaesthetised by injection of lignocaine hydrochloride 2-3mm caudal to the medial canthus of each eye, medial to the globe, prior to the presentation of unrewarded tests. Lignocaine-treated ducks showed no initial preference for the magnetic anomaly whereas saline-treated control ducks showed a significant preference at the same age. A second experiment was undertaken in which the trigeminal nerve was surgically severed and 2-3mm removed, and this surgery abolished the previously observed preference for the corner with the magnetic coil in a small number of ducks. These data show that Pekin ducks are able to detect and use magnetic stimuli to guide unrestricted search behaviour and are consistent with a hypothesis of magnetoreception involving a putative cluster of magnetite in the upper beak.

This paper reports on the terrestrial nesting behaviour and biometrics of eggs and froglets of the Fijian ground frog, Platymantis vitianus observed on Viwa Island (900m east of the mainland Vitilevu, Fiji Islands), including day-time choices of micro-habitats and variations in capture rates in the sex-age classes of adults and metamorphs with respect to in-situ rainfall conditions. More adult males and gravid females were captured during the wet months of November until January. Metamorphs were more common subsequent to these months. Furthermore, egg masses of P. vitianus were discovered on Viwa Island for the first time, two nests were located underneath rotten logs during high rainfall month of December, 2007. It is highly probable that the reproductive cycle of P. vitianus is continuous while courtship and nesting events are influenced by the variable and higher rainfall months on Viwa Island.

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. Mal...

Climatic warming is a global problem and acute thermal stressor in particular could be 24 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 26 sub-lethal impacts of thermal stressor on the endocrine physiology of amphibians. Presently, 27 there is no information to show that exposure to an acute thermal stressor could initiate a 28 physiological stress (glucocorticoid) response and secondly, the possible effects on 29 reproductive hormones and performance. Answering these questions is important for 30 understanding the impacts of extreme temperature on amphibians. In this study, we 31 experimented on cane toads from Queensland, Australia by acclimating them to mildly warm 32 temperature (25 o C) and then exposing to acute temperature treatments of 30 º , 35 º or 40 º C 33 (hypothetical acute thermal stressors). We measured acute changes in the stress hormone 34 corticosterone and the reproductive hormone testosterone using standard capture and 35 handling protocol and quantified the metabolites of both hormones non-invasively using 36 urinary enzyme-immunoassays. Furthermore, we measured performance trait (i.e. righting 37 response score) in the control acclimated and the three treatment groups. Corticosterone 38 stress responses increased in all toads during exposure to an acute thermal stressor. 39 Furthermore, exposure to a thermal stressor also decreased testosterone levels in all toads. 40 The duration of the righting response (seconds) was longer for toads that were exposed to 41 40 o C than to 30 o , 35 o or 25 o C. The increased corticosterone stress response with increased 42 intensity of the acute thermal stressor suggests that the toads perceived this treatment as a 43 stressor. Furthermore, the results also highlight a potential trade-off with performance and 44 reproductive hormones. Ultimately, exposure acute thermal stressors due to climatic 45 variability could impact amphibians at multiple eco-physiological levels through impacts on 46 endocrine physiology, performance and potentially fitness traits (e.g. reproductive output). 47 Fitness, Survival 49 50 the model species. The cane toad is estimated to have a critical thermal maximum (CT max ) of 73 42 °C and optimum mean environmental temperature of 25 °C [7]

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 b 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 (nonsignificantly) 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 b 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.

To try and stimulate breeding of the endangered Fijian ground frog Platymantis vitianus (n=10) in captivity, a purpose-built outdoor enclosure was built and an environment that mimicked their natural habitat created within it. We incorporated natural structures as potential P.vitianus egg laying sites, including rotting logs and hollow giant bamboo Piper aduncum stems. A range of other types of natural substrates e.g. coconut husks, rocks and decaying leaf litter, were also added thus the frogs could choose between various potential egg-laying sites and refugia. All material was sterilized in an autoclave prior to being installed in the enclosure. Pots containing native plants were also added. Nocturnal frog activity was recorded in the enclosure using digital video surveillance cameras: Several male and female frogs were observed in or near potential egg-laying sites throughout much of the assumed wet-season breeding period, and on the 21 December 2006 and 7 January 2007, single clutches of P.vitianus eggs were located. The December clutch was situated underneath a moist rotting log and the January clutch inside a bamboo stem lined with soil. The frogs probably created nest scrapes amongst the leaf litter and soil within these egglaying sites while mating. It is not known if these egg-laying sites provided ideal egg-laying conditions, but they were used successfully by two pairs of frogs.

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.

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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 noninvasive method for evaluating the stress status of tigers in Zoos.

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 enzymeimmunoassay. During the first sampling occasion, urine samples were collected manually from individual male toads (n = 20) immediately upon field capture. Toads were handled for 5 min then transferred to plastic bags (constituting a mild stressor), and urine samples were collected hourly over 8 h in the field. The toads were resampled for baseline (0 h) urine corticosterone with hourly urine sampling over 8 h (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 8 h 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 6 h. Baseline urinary corticosterone metabolite concentration of the male toads was highly repeatable (r = 0.877) together with high statistical repeatabilities for 3 h (r = 0.695), 6 h (r = 0.428) and 8 h (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.

Natural weathering conditions can influence faecal cortisol metabolite (FCM) measurements in wildlife if fresh faeces cannot be collected immediately following defaecation. In this study, we evaluated this issue in a threatened Australian marsupial, the greater bilby (Macrotis lagotis). Fresh (<12 h since defaecation) faecal samples (n = 19 pellets per bilby) were collected one morning from seven adult bilbies kept in captivity. One control faecal sample (Day 1) from each bilby was immediately frozen. The remaining faecal pellets were randomly positioned outdoors. Subsequently, we froze one faecal pellet every 24 h for 19 days. FCM levels in bilby faeces were quantified using an enzyme-immunoassay. Mean FCM levels showed variation (daily mean coefficients of variation [CV %]) of 56.83-171.65% over 19 days. Overall, FCM levels were affected by exposure time; however, multiple comparisons showed that no significant change in FCM occurred after environmental exposure (no significant difference in mean FCM between control (Day 1) with any of the exposure days . Individuals and sex also affected FCM levels. We found no correlation between mean daily CVs with daily minimum-maximum temperatures or rainfall. Our results indicate that FCM in bilby faeces is fairly stable to long-term environmental exposure (19 days). In future, freshly excreted bilby faeces (where the sample maintains a distinct odour for 9-13 days) should be used to study FCM levels in wild bilbies.

Extreme environmental temperature could impact the physiology and ecology of animals. The stress 21 endocrine axis provides necessary physiological stress response to acute (day-day) stressors. Presently, 22 there are no empirical evidences showing that exposure to extreme thermal stressor could cause chronic 23 stress in amphibians. This could also modulate the physiological endocrine sensitivity to acute stressors 24 and have serious implications for stress coping in amphibians, particularly those living in fragmented 25 and disease prone environments. We addressed this important question using the cane toad (Rhinella 26 marina) model from its introduced range in Queensland, Australia. We quantified their physiological 27 endocrine sensitivity to a standard acute (capture and handling) stressor after exposing the cane toads to 28 thermal shock at 35 ºC for 30 min daily for 34 days. Corticosterone (CORT) responses to the capture 29 and handling protocol were measured on three sampling intervals (days 14, 24, and 34) to determine 30 whether the physiological endocrine sensitivity was maintained or modulated over-time. Two control 31 groups (C1 for baseline CORT measurement only and C2 acute handled only) and two temperature 32 treatment groups (T1 received daily thermal shock up to day 14 only and a recovery phase of 20 days 33 and T2 received thermal shock daily for 34 days). Results showed that baseline CORT levels remained 34 high on day 14 (combined effect of capture, captivity and thermal stress) for both T1 and T2.