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Mountain chicken frogs Leptodactylus fallax are assessed as Critically Endangered on the IUCN Red List due to threats including chytridiomycosis and habitat loss. Ex-situ populations underpin species survival, but captive management is hampered by incomplete species knowledge, including its behavioural biology. In sixteen adult frogs, we investigated enclosure usage, nocturnal activity budgets, and behavioural responses to varying levels of husbandry-related disturbance through instantaneous scan sampling of camera trap footage over forty-two consecutive nights. Enclosure usage was quantified through the application of modified Spread of Participation Indices (mSPI). We present the first detailed activity budgets published for this species and found that broad activity patterns of captive animals corresponded well with their wild conspecifics; taking refuge during the day and emerging at dusk into exposed areas of the enclosure. Some behaviours, especially hunting and bathing, were partially phased throughout the nocturnal period. Enclosure use was not even, with disproportionately large amounts of time spent using nest boxes and deep leaf litter, even at night, and the converse in thin leaf litter and elevated perches. Principal Components Analysis (PCA) identified a bold-shy behavioural grouping (movement and resting opposed to refuge behaviours) that was consistent across husbandry-related disturbance conditions. Randomisation analyses showed that disturbance significantly affected multiple measured behaviour, especially feeding, social interactions and vocalisation; the impact was associated with the degree of disturbance. Novel behaviours were also documented, which are thought to be related to courtship. Our data provide insights into the behaviour of mountain chicken frogs and highlight potential trade-offs between enclosure maintenance, veterinary intervention, and animal welfare. Keywords: Amphibians, behaviour, mSPI, disturbance, welfare, Zoo

Sexual dimorphism is commonly encountered in amphibians. Its presence and its ontogeny in a given species has implications for ecology, conservation and captive husbandry. We monitored changes in length and mass of captive mountain chicken frogs Leptodactylus fallax. Initially, sexes were no different in snout-vent length or mass, but by about 17 months after metamorphosis females became significantly larger and heavier than males. Diverging growth trajectories between male and female frogs appeared to coincide with the development of secondary sexual characters in males, indicating sexual maturity, while female frogs did not produce nests until the following breeding season, suggesting later reproductive maturity. The absence of similar dimorphism in other large leptodactylids suggests that its presence in L. fallax may be linked to its unique and energy-intense reproductive strategy, which involves extended maternal provisioning of their young.

Calcium metabolism in insectivores may be perturbed by insufficient calcium or vitamin D3. Insects may be gut loaded to increase calcium content, and recent research shows that exposure to UVB radiation can increase the vitamin D3 content of some invertebrates. Typical gut loading protocols result in peak calcium content after 24–48 h, while existing evidence with UVB irradiation involves exposure periods of tens of days. We UVB-irradiated fasted black field crickets (Gryllus bimaculatus) while feeding them on high calcium diets for 48 h, then fasted them for a further 24 h, and measured the vitamin D3, calcium, and phosphorus content compared with non-irradiated controls. UVB irradiation had no effect on vitamin D3 (crickets had no detectable levels of vitamin D3 at any point), or on calcium accumulation rates, which approximated existing research. Crickets significantly increased their calcium:phosphorus ratio from 0.17 to approximately 0.4 over 24 h and this did not increase over a further 24 h of feeding. Removal of the food source resulted in loss of all accumulated calcium within 24 h. Our results have implications for managing food sources for captive insectivores and highlight the importance of good feeder preparation and rapid consumption to ensure optimal calcium delivery to predators.

The mountain chicken frog (Leptodactylus fallax) is among the 42 % of amphibians threatened with extinction and is dependent upon ex situ populations to recover in the wild. Amphibian captive husbandry is not fully understood and empirical data are required to optimise protocols for each species in captivity. Calcium metabolism and homeostasis are areas of importance in captive husbandry research and have been identified as a challenge in maintaining ex situ populations of L. fallax. We trialled two frequencies (twice and seven times weekly) of calcium supplementation via dusting of feeder insects in two groups of L. fallax juveniles and measured growth and health effects through morphometrics, radiography, ultrasonography and blood and faecal analysis over 167 days, followed by a further 230 days of monitoring on an intermediate diet informed by the initial dataset. We showed that supplementation treatment did not affect growth or health status as measured through blood analysis, radiography and ultrasonography. More frequent supplementation resulted in significantly more radiopaque endolymphatic sacs and broader skulls. Frogs fed more calcium excreted twice as much calcium in their faeces. The intermediate diet resulted in previously lower supplementation frogs approximating the higher supplementation frogs in morphometrics and calcium stores. Comparison with radiographic data from wild frogs showed that both treatments may still have had narrower skulls than wild animals, but mismatching age class may limit this comparison. Our data may be used to inform dietary supplementation of captive L. fallax as well as other amphibians.

The fire salamander (Salamandra salamandra and its relatives) is of increasing priority for ex situ conservation due to the spread of the fungal pathogen Batrachochytrium salamandrivorans in Europe. In captivity, the species may be maintained on a clinical paper-based or a naturalistic substrate, either of which has its own advantages and disadvantages. However, the impact of these two substrates on bacterial microbiotas within an enclosure and on the salamanders themselves is unknown. To investigate this, we maintained captive fire salamanders on either paper towels or a naturalistic substrate and quantified the culturable microbiotas of both substrates across the one-week lifespan of a paper towel and of the salamanders themselves over a six-month period. We found significant differences in the bacterial communities associated with the two substrates. Over a week-long period, there were major fluctuations in the community composition and abundance on paper towels while on the naturalistic substrate bacterial communities were relatively stable. The skin microbiota of salamanders were indistinguishable at the beginning of the study but after six months differed significantly between the two treatments, although the bacterial morphotypes present remained relatively similar compared with changes between substrates. These data show that husbandry protocols may have a strong influence on the culturable bacterial communities to which captive amphibians are exposed. Nevertheless, the animals were apparently able to maintain their own microbiota to a considerable degree. These findings should be borne in mind when determining husbandry protocols. Given the relative benefits of both types of enclosure, it is possible that a hybrid approach could be used whereby a small amount of naturalistic substrate is provided in a container within an otherwise clinical enclosure, to act as a bacterial reservoir.

Identifying individual animals is important for studying populations and for the optimal management of individual animals in captivity. In the absence of natural markings that discriminate individuals, such identification may require animals to be marked by researchers. Amphibians are challenging subjects to mark due to their small size and sensitive, permeable and frequently shed skin. Visible Implant Elastomer (VIE) has been widely used to mark amphibians, but no long-term study has validated this technique in caecilian amphibians. We anaesthetised and attempted to VIE mark seven Herpele squalostoma and one Microcaecilia unicolor held at ZSL London Zoo. No specimens suffered ill effects of anaesthesia or VIE injection, but mean persistence of marks was 191 days in H. squalostoma suggesting that this marking technique is not suitable for identifying individuals of this species in the long-term. We were unable to inject VIE into the M. unicolor and/or the elastomer was not visible through the darkly pigmented skin. Further research is required to develop methods for long-term marking of a diversity of caecilians.

Dominance is a key component of behaviour in many animal species and is central to social system dynamics, resource acquisition, individual fitness and ultimately reproductive success. We investigated dominance interactions and social behaviours in a group of captive juvenile gidgee skinks (Egernia stokesii). We hypothesised that a dominance hierarchy existed within the group, and that aggressive behaviours would be used to secure limited resources, especially high-value resources. We also hypothesised that body weight would be positively correlated with dominance and aggressive behaviours. We filmed the lizards at 1200 hours for six days a week over the course of eight weeks. We exposed the lizards to three different diets, which consisted of an animal-based diet (crickets), plant-based diet (plants), and a non-feeding control (no food offered). The relative value of these resources to the skinks was established through preference tests. We identified a dominance hierarchy, with dominant individuals exhibiting more aggressive behaviours than subordinates. We found that the frequency of aggressive behaviours was significantly higher in trials where high-valued resources (crickets) were at stake. Furthermore, we found a significant positive correlation between body weight and dominance, bite and chase; larger individuals were ranked higher in the social hierarchy compared to smaller individuals. Our results demonstrate the importance of morphological and behavioural traits in determining a dominance hierarchy in E. stokesii and how dominance can have ecological advantages.

[Background] The fungal pathogenBatrachochytrium dendrobatidis (Bd) threatens amphibian biodiversity and ecosystem stability worldwide. Amphibian skin microbial community structure has been linked to the clinical outcome of Bd infections, yet its overall functional importance is poorly understood. [Methods] Microbiome taxonomic and functional profiles were assessed using high-throughput bacterial 16S rRNA and fungal ITS2 gene sequencing, bacterial shotgun metagenomics and skin mucosal metabolomics. We sampled 56 wild midwife toads (Alytes obstetricans) from montane populations exhibiting Bd epizootic or enzootic disease dynamics. In addition, to assess whether disease-specific microbiome profiles were linked to microbe-mediated protection or Bd-induced perturbation, we performed a laboratory Bd challenge experiment whereby 40 young adult A. obstetricans were exposed to Bd or a control sham infection. We measured temporal changes in the microbiome as well as functional profiles of Bd-exposed and control animals at peak infection. [Results] Microbiome community structure and function differed in wild populations based on infection history and in experimental control versus Bd-exposed animals. Bd exposure in the laboratory resulted in dynamic changes in microbiome community structure and functional differences, with infection clearance in all but one infected animal. Sphingobacterium, Stenotrophomonas and an unclassified Commamonadaceae were associated with wild epizootic dynamics and also had reduced abundance in laboratory Bd-exposed animals that cleared infection, indicating a negative association with Bd resistance. This was further supported by microbe-metabolite integration which identified functionally relevant taxa driving disease outcome, of which Sphingobacterium and Bd were most influential in wild epizootic dynamics. The strong correlation between microbial taxonomic community composition and skin metabolome in the laboratory and field is inconsistent with microbial functional redundancy, indicating that differences in microbial taxonomy drive functional variation. Shotgun metagenomic analyses support these findings, with similar disease-associated patterns in beta diversity. Analysis of differentially abundant bacterial genes and pathways indicated that bacterial environmental sensing and Bd resource competition are likely to be important in driving infection outcomes. [Conclusions] Bd infection drives altered microbiome taxonomic and functional profiles across laboratory and field environments. Our application of multi-omics analyses in experimental and field settings robustly predicts Bd disease dynamics and identifies novel candidate biomarkers of infection. [MediaObject not available: see fulltext.], K.A.B. was funded by a CASE studentship from NERC, NERC Biomolecular Analysis Facility grant (NBAF939) and an E.P. Abraham Junior Research Fellowship from St Hilda’s College, University of Oxford. M.C.F and T.W.J.G. were funded by NERC award NE/E006701/1 and the Biodiversa project RACE: Risk Assessment of Chytridiomycosis to European Amphibian Biodiversity. T.W.J.G was also funded by Research England and NERC NE/S000062/1. D.S.S. and A.L. received funding through the project People, Pollution, and Pathogens financed through the call “Mountains as Sentinels of Change” by the Belmont-Forum (ANR-15-MASC-0001 - P3, DFG-SCHM3059/6-1, NERC-1633948, NSFC-41661144004). D.S.S. holds the AXA Chair for Functional Mountain Ecology funded by the AXA Research Fund through the project GloMEc and M.C.F. is a fellow in the CIFAR ‘Fungal Kingdoms’ Program.

It is vital to provide appropriate nutrition to maintain healthy populations in conservation breeding programs. Knowledge of the wild diet of a species can be used to inform captive diet formulation. The nutritional content of the wild diet of the critically endangered mountain chicken frog (Leptodactylus fallax) is unknown, like that of most amphibians. In this study, we analyzed the nutritional content of food items that comprise 91% of the wild diet of L. fallax, by dry weight of food items, and all food items offered to captive L. fallax at ZSL London Zoo and Jersey Zoo. We subsequently compared the nutritional content of the wild diet and captive diet at ZSL London Zoo consumed by L. fallax. To the authors' knowledge, this is the first study to directly compare the nutritional content of the wild and captive diets of an anuran amphibian. The captive diet at ZSL London Zoo, without dusting of nutritional supplements, was higher in gross energy and crude fat and lower in ash, calcium and calcium:phosphorus ratio than the wild diet. Most of the food items in the captive diets had a high omega-6:omega-3 fatty acid ratio and in the wild diet had a low omega-6:omega-3 fatty acid ratio. We recommend a combination of modifications to the captive diets to better reflect the nutritional content of the wild diet. Nutritional analysis of captive and wild diets is recommended for other species in conservation breeding programs to improve captive husbandry and ultimately fitness.

The IUCN Red List of Threatened Species (Red List) details the extinction risk of the world's species and presents an important biodiversity indicator for conservation policy. Its continued utility relies on it containing up-to-date information on the extinction risk of species. This requires both regular reassessments and the timely assessment of newly described species. We provide an overview of the status of amphibian Red List assessments to highlight the difficulties of keeping assessments updated for species groups with high rates of species description. Since the publication of the IUCN's Global Amphibian Assessment in 2004, description rates of new species and assessment rates were initially similar; yet while the former has remained consistent, the latter has recently sharply declined. Currently 61.3% of amphibian species are either Not Evaluated or have out-of-date assessments. The situation is particularly problematic in countries with the richest amphibian diversity, which typically have the highest rates of amphibian species discovery and face the greatest threats. Efforts to keep the Red List up-to-date are primarily limited by funding, we estimate that an annual investment of US $170,478–$319,290 is needed to have an up-to-date Red List for amphibians. We propose suggestions to increase assessment rates by improving the availability of data relevant to the process: authors of species descriptions or taxonomic revisions should publish information relevant to Red List assessments. Taxonomic journals should suggest inclusion of such information in their author guidelines. We suggest that contributors with significant input into assessments should be rewarded with co-authorship of published assessments.

Staggering food availability through a delivery device is a common way of providing behavioural enrichment as it is usually thought to increase the amount of natural behaviour due to the unpredictability of the food source. Tree-runner lizards ( Plica plica ) are a Neotropical, scansorial, insectivorous species. We provided these lizards with an enrichment device that slowly released insect prey and tested its effect on the activity and frequency of a number of behaviours in comparison with a scatter control (where prey items were broadcast in the enclosure; standard food presentation for captive insectivorous lizards) and a non-feeding control. Both types of food increased activity and counts of several behaviours in comparison with the non-feeding control. However, we found the provision of the behavioural enrichment device led to a significantly lower frequency of almost all analysed behaviours in comparison with scatter control trials, mainly in behaviours associated with activity (unsuccessful strikes (= unsuccessful capture of prey) (p = 0.004), locomotion (p = 0.004), alertness (p = 0.004) and the number of times a boundary in the enclosure was crossed ie. activity (p = 0.05) between scatter and enriched trials. The relative success of the scatter feed in promoting activity and increasing hunting difficulty was likely partly due to the enclosure design, where the complex physical environment contributed to the difficultly in catching the prey. However, when the feeding duration and enclosure use was analysed there was no significant difference between the scatter control and enrichment trails. The results from this study highlight the importance of evaluating enrichment strategies, and the role of complex enclosure design in creating effective enrichment for insectivores, which can contribute to their welfare in captivity.

The genus Rhacophorus Kuhl & Van Hasselt is currently known to contain 92 species of frogs (Frost 2018), distributed across south and south-east Asia. Rhacophorus feae Boulenger is a large member of this genus and has a seemingly expansive range been recorded from southern Yunnan in China, the Karen hills in Myanmar, northern Thailand, northern Laos and northern Vietnam (Chanard et al. 1999; Orlov et al. 2002; Nguyen et al. 2005; Stuart 2005; Yang 2008). It is currently included in the intrageneric R. dennysi group (Dubois 1986) along with R. duboisi Ohler, Marquis, Swan & Grosjean , R. dennysi Blanford, R. dugritei (David), R. minimus Rao, Wilkinson & Liu, R. hungfuensis Liu & Hu, R. dorsoviridis Bourret, R. nigropunctatus Liu, Hu & Yang, and R. smaragdinus (Blyth).

Amphibians are facing an extinction crisis, and conservation breeding pro- grammes are a tool used to prevent imminent species extinctions. Compared to mammals and birds, amphibians are considered ideal candidates for these programmes due to their small body size and low space requirements, high fecundity, applicability of reproductive technologies, short generation time, lack of parental care, hard wired behaviour, low maintenance requirements, relative cost effectiveness of such programmes, the success of several amphibian conservation breeding programmes and because captive husbandry capacity exists. Superficially, these reasons appear sound and conservation breeding has improved the conservation status of several amphibian species, however it is impossible to make generalisations about the biology or geo-political context of an entire class. Many threatened amphibian species fail to meet criteria that are commonly cited as reasons why amphibians are suitable for conservation breeding programmes. There are also limitations associated with maintaining populations of amphibians in the zoo and private sectors, and these could potentially undermine the success of conservation breeding programmes and reintroductions. We recommend that species that have been assessed as high priorities for ex situ conservation action are subsequently individually reassessed to determine their suitability for inclusion in conservation breeding programmes. The limitations and risks of maintaining ex situ populations of amphibians need to be considered from the outset and, where possible, mitigated. This should improve programme success rates and ensure that the limited funds dedicated to ex situ amphibian conservation are allocated to projects which have the greatest chance of success.

The Critically Endangered mountain chicken frog (Leptodactylus fallax) has undergone drastic population decline due to habitat loss, hunting, invasive species, and chytridiomycosis. In response, several partner institutions initiated a conservation breeding program. It is important to maintain the captive population in good health. Therefore the program partners have recommended establishment of protocols for health examination of the species, including body condition assessment. Visual body condition scoring is a useful means to assess body condition in zoo animals for which regular bodyweight measurements are impractical or associated with capture-related stress. In this study, the authors developed a visual body condition score for the mountain chicken frog based on an ordinal categorical scale from 1 to 5 (1 = lowest body condition, 5 = highest body condition) using anatomical features that vary with total body energy reserves. Veterinary staff, animal managers, keepers, researchers, and students subsequently used the body condition score to assign scores to 98 mountain chicken frogs (41 male, 57 female) aged between 8 months and 12 years housed in five zoos in the UK and Jersey between February and March 2016. Body condition scores showed moderate (rho = 0.54; males) to strong (rho = 0.6; females) correlation with the scaled mass index, an objective measure of total energy reserves. The majority of pairwise comparisons between scores showed slight to substantial intra-observer agreement (93.8%) and slight to almost perfect inter-observer agreement (97.2%). Cases of poor agreement were likely due to limited observer experience working with the species.

Many amphibian species are dependent on ex situ conservation interventions for their long-term persistence. However, projects have been jeopardised by husbandry issues involving poor calcium metabolism and nutritional metabolic bone disease (NMBD). Healthy calcium metabolism requires appropriate dietary calcium content and access to vitamin D3 . In many animals, vitamin D3 can be photobiosynthesised in skin exposed to UVB radiation, as well as extracted from the diet, but the extent of vitamin D3 photobiosynthesis in amphibians is poorly known. Additionally, prey insects for captive amphibians are deficient in calcium and calcium content must be artificially increased, but the effects of different levels of augmentation and their interaction with UVB exposure are also little understood. We fed captive fire-bellied toads (Bombina orientalis) with crickets augmented to contain 5% and 10% calcium and housed them with and without UVB exposure. Despite additional dietary vitamin D3 supplementation, we found that toads exposed to UVB radiation exhibited significantly higher serum vitamin D3 levels, indicating that this species may partly rely on photobiosynthesis sources of vitamin D3 . These data are the first to show a direct link between UVB exposure and serum vitamin D3 in an amphibian. We found significant positive effects of UVB exposure and 10% dietary calcium content on skeletal structure, as well as complex interactions between treatments. We also found UVB radiation exposure resulted in more rapid natural coloration acquisition. Together, this indicates that standard calcium plus vitamin D3 supplementation methods may not fully substitute for UVB exposure and for increased feeder insect calcium content. This may have implications for the success of ex situ amphibian conservation, as well as for the welfare of captive amphibians in general. Our data lend support for the provision of UVB radiation for captive, basking amphibians.

The diet of Bubo ascalaphus in Qatar was assessed based on pellets collected from the first known nesting site of the species in the country. The pellets contained a total of 68 prey items, representing 9 different species: 4 mammals, 1 bird, 1 reptile, and at least 3 scorpions. Mammals clearly comprised the major food source (89.7% and 97.7% in frequency and biomass respectively). Our data suggest that Pharaoh Eagle Owls are opportunistic predators that feed on a variety of prey depending on their temporal/spatial availability, which is consistent with previous studies. A literature review clearly suggests that Eagle Owls in arid to semi-arid environments are opportunistic predators with small mammals being their main prey. Predation on migrating Blue-cheeked Bee-eaters Merops persicus supports this hypothesis. Scopus

Xenopus longipes Loumont and Kobel, 1991 is an aquatic polyploid frog endemic to the high altitude crater lake, Lake Oku in North West region, Cameroon (LoumontKobel 1991). The tadpole of X. longipes is currently undescribed. So far, only dead tadpoles have been found at Lake Oku during regular monitoring since 2008 (Doherty-Bone et al. 2013), with specimens too decomposed to make adequate descriptions. Captive breeding provides one opportunity to obtain fresh specimens for description.

Despite the importance of ex situ conservation programmes as highlighted in the Amphibian Conservation Action Plan, there are few empirical studies that examine the influence of captive conditions on the fitness of amphibians, even for basic components of enclosure design such as cover provision. Maintaining the fitness of captive amphibian populations is essential to the success of ex situ conservation projects. Here we examined the impact of plant cover on measures of fitness and behaviour in captive red-eyed tree frogs (Agalychnis callidryas). We found significant effects of plant provision on body size, growth rates and cutaneous bacterial communities that together demonstrate a compelling fitness benefit from cover provision. We also demonstrate a strong behavioural preference for planted rather than non-planted areas. We also assessed the impact of plant provision on the abiotic environment in the enclosure as a potential driver of these behavioural and fitness effects. Together this data provides valuable information regarding enclosure design for a non-model amphibian species and has implications for amphibian populations maintained in captivity for conservation breeding programmes and research. © 2014 Michaels et al.