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Ex situ amphibian populations are a key component of global amphibian conservation strategy, and optimal husbandry is vital to ex situ conservation success. Animal behavior can be used to inform captive welfare and improve husbandry practices. However, it has been little used for amphibians compared with mammals and birds. The goal of this study was to explore the effect of different colored tank backgrounds on the behavior of the critically endangered Lake Oku clawed frog (Xenopus longipes) in captivity. This was conducted by studying the behavior of a group of 24 captive frogs in 5 groups using established behavioral indicators of presumed stress. Resting and swimming behaviors, established in the literature as linked to acute stress, were recorded under conditions of three background colors and a standard husbandry control of no background. Frog groups were exposed to each background for five days with behavioral frequencies recorded daily from 11:00 until 13:00. Using generalized linear mixed models, we identified a significant effect of background days after the background was changed and the interaction between the two variables on both swimming and resting behavior. The results of this study suggest an initial response of stress to altering the background, modulated by the color of the background, followed by the extinction of the stress response such that by five days after the background change, behaviors were similar to the baseline and indistinguishable between treatments. Overall, this study suggests that frog stress behavior was not differentially directly affected by green, grey, black, or transparent backgrounds but that green and grey backgrounds were associated with the smallest stress response to background change. These colors may therefore be recommended to reduce the impact of stress from disturbance.

Captive insectivore nutrition is challenging due to the differing nutritional profiles of wild and captive diets and an incomplete understanding of both. Ultraviolet B (UVB)-irradiation has recently been explored as a means of improving prey-insect vitamin D3 and Ca content. Although short-term irradiation has been successful in some species, it has been unsuccessful in black field crickets (Gryllus bimaculatus)—a commonly cultured feeder insect. We exposed crickets to UVB irradiation from hatchling to adult stages and measured the vitamin D3 and mineral contents of crickets by sex. We did not detect vitamin D3 (detection limit 0.5 iU/g) or an effect of UVB irradiation on mineral content under either UV+ or UV− conditions. We identified large differences between sexes in Ca, K, Mg and P (females higher) and Cu, Fe, S and Zn (males higher), likely linked to reproductive investment. The differences do not straddle the minimum recommended concentrations of minerals for vertebrate growth and thus may be most relevant to animal nutrition in contexts of particular sensitivity or need. We demonstrate a UV-linked trade-off in cricket performance between individual cricket size and the numbers of crickets produced and characterise the energy costs associated with UVB provision. Our results do not support the use of UVB lighting for G. bimaculatus to improve nutrition but demonstrate previously unreported differences in the nutritional profiles between sexes in this species.

The interest in the welfare of zoo animals, from both the public and the scientific community, has long been biased towards mammals. However, growing evidence of the complex behavioural repertoires of less charismatic animals, such as reptiles, reveals the necessity to better comply with their welfare needs in captivity. Here, we present the effects of an enclosure change towards a more natural habitat in captive Galapagos tortoises (Chelonoidis spp.) held at ZSL London Zoo. Using behavioural observations, we found that the tortoises habituated to their new enclosure in six days. This represents the first quantification of habituation latency to a new enclosure in a reptile model to our knowledge—which is important information to adapt policies governing animal moves. The tortoises expressed time budgets more similar to those of wild individuals after their transition to the new enclosure. Interestingly, the hierarchy between the individuals was inverted and more stable after this change in environment. The tortoises interacted less often, which led to a decrease in the frequency of agonistic encounters. We also found that higher ambient sound volume was associated with increased likelihood of interactions turning into fights. Taken together, our results demonstrate the potential of appropriate enclosure design to improve reptile welfare.

Animal behavior and welfare science can form the basis of zoo animal management. However, even basic behavioral data are lacking for the majority of amphibian species, and species-specific research is required to inform management. Our goal was to develop the first ethogram for the critically endangered frog Xenopus longipes through observation of a captive population of 24 frogs. The ethogram was applied to produce a diurnal activity budget and to measure the behavioral impact of a routine health check where frogs were restrained. In the activity budget, frogs spent the vast majority of time swimming, resting in small amounts of time devoted to feeding, foraging, breathing, and (in males) amplexus. Using linear mixed models, we found no effect of time of day or sex on baseline behavior, other than for breathing, which had a greater duration in females. Linear mixed models indicated significant effects of the health check on duration of swimming, resting, foraging, feeding, and breathing behaviors for all frogs. This indicates a welfare trade-off associated with veterinary monitoring and highlights the importance of non-invasive monitoring where possible, as well as providing candidates for behavioral monitoring of acute stress. This investigation has provided the first behavioral data for this species which can be applied to future research regarding husbandry and management practices.

Many birds, including macaws, are highly visual animals able to detect a wide band of light wavelengths ranging into ultraviolet A, but in captivity, full-spectrum lighting is not universally employed. Where purpose-made bird lighting is used, this is typically made with the provision of ultraviolet B radiation and vitamin D3 synthesis in mind. Limited research in this field suggests behavioural and physiological benefits of broad-spectrum lighting provision, but more work is needed to broaden the taxonomic scope and to investigate its impacts on understudied areas of husbandry, including behavioural management. We compared the duration of time a bonded pair of blue-throated macaws at ZSL London Zoo opted to remain in an inside den after being recalled from an outdoors flight aviary, with and without the presence of artificial lighting in the form of High Output T5 Fluorescent lamps, which are rich in UVA and UVB wavelengths as well as those visible to humans. We hypothesized that the birds would remain inside for longer when T5 lighting was on, as they would be more visually comfortable. Using randomization analyses, we show that, over 54 trials split between winter and spring, the mean duration spent inside after recall increased from 81.04 to 515.13 s with the presence of the lighting unit, which was highly statistically significant. Our results are likely to be explained by much higher visibility of indoor surroundings creating a more hospitable indoor environment for the birds and will have implications for captive macaw management.

Abstract Background The fungal pathogen Batrachochytrium 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. Video Abstract

Visitors to zoos can have positive, neutral, or negative relationships with zoo animals. This makes human–animal interactions (HAIs) an essential component of welfare and an important consideration in species selection for zoo exhibits and in enclosure designs. We measured the effect of visitors on reptiles by comparing open and closed periods during the lockdowns in response to the COVID-19 pandemic in the UK in a low-resolution dataset for thirteen species of reptiles and a high-resolution dataset focussing on just one of these. Scan sampling on thirteen reptile species (two chelonians and eleven squamates) showed species-specific differences in response to the presence/absence of visitors, with most taxa being only weakly affected. High-resolution scan sampling via video footage of an off-show and on-show enclosure was carried out for tokay geckos (Gekko gecko) over the open and closed periods. In this part of the study, tokay geckos were significantly more visible during zoo closure than when visitors were present on-exhibit, but there was no change in off-show animals, indicating the effect of visitors as opposed to other factors, such as seasonality, which applied equally to both on- and off-show animals. The high-resolution study showed that a significant effect was present for tokay geckos, even though the low-resolution suggested that they were more weakly affected than other taxa. Our results indicate that, for cryptic species such as this, more intensive sampling may be required to properly understand visitor effects. Our data do not allow the interpretation of effects on welfare but show that such assessments require a species-specific approach.

Maintaining Gymnophiona in captivity provides opportunities to study the behaviour and life-history of this poorly known Order, and to investigate and provide species-appropriate welfare guidelines, which are currently lacking. This study focuses on the terrestrial caecilian Herpele squalostoma to investigate its sensitivity to disturbances associated with routine husbandry needed for monitoring and maintaining adequate wellbeing in captivity. Fossorial caecilians gradually pollute their environment in captivity with waste products, and substrate must be replaced at intervals; doing so disturbs the animals directly and via destruction of burrow networks. As inappetence is frequently associated with stress in amphibians, the percentage consumption of offered food types, river shrimp (Palaemon varians) and brown crickets (Gryllus assimilis), was measured as an indicator of putative stress following three routine substrate changes up to 297 days post-substrate change. Mean daily variation in substrate temperatures were also recorded in order to account for environmental influences on food consumption, along with nitrogenous waste in tank substrate prior to a substrate change and fresh top soil in order to understand the trade-off between dealing with waste accumulation and disturbing animals. We found a significant negative effect of substrate disturbance on food intake, but no significant effect of prey type. Variations in daily soil temperatures did not have a significant effect on food intake, but mean substrate temperature did. Additionally, substrate nitrogenous waste testing indicated little difference between fresh and tank substrate. In conclusion, this study provides a basis from which to develop further welfare assessment for this and other rarely kept and rarely observed terrestrial caecilian species.

This study compared the resource use of juvenile zoo-living Chinese crocodile lizards, Shinisaurus crocodilurus crocodilurus across three observation windows, spanning nine months, accounting for time of day and lizard age, and under consistent environmental conditions. Lizards showed a significant difference in proportionate resource use, quantified using a modified spread of participation indices between the second and final sampling period, such that with increasing age, resources were more equally utilised. The time of day did not have a significant effect on resource use. Lizards in this study significantly increased their use of water bodies and branches outside the bask zone and decreased their use of the land areas within the bask zones over time. Resource use data suggests the importance of providing enclosures which cater to ontogenetic shifts in captive individuals or within mixed age groupings.

Ensuring high levels of welfare is imperative for modern zoos, but such organisations must also engage visitors in order to successfully spread awareness and raise conservation funds. It is therefore important to understand the responses of animals to visitor interaction to optimise welfare. Often, the opportunity to interact with humans may be enriching for animals, but in other contexts, this interaction may have negative welfare effects. We observed captive female Galápagos giant tortoises (Chelonoidis nigra) to describe aggressive interactions, characterize hierarchy using Elo ratings and assess the impact of visitor interactions. Elo ratings indicated that one individual was dominant over two equally ranked subordinates; aggressive interactions are discussed in this context. We detected significant effects of the presence of visitors and visitor type (keepers, vets or public) within the enclosure on aggression and activity. We suggest that previous miscategorisation of a natural behaviour (the finch response) as an operantly conditioned behaviour, rather than a fixed action pattern, may have triggered aggression. We then document changes made to the management of the animals to mitigate the impacts discovered. This work highlights the importance of empirical evidence in determining optimal management strategies for zoo animals with regards to public interactions and animal welfare.

[This corrects the article DOI: 10.1371/journal.pone.0185151.].

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.

Anthropogenic disturbance via resource acquisition, habitat fragmentation and climate change, amongst other factors, has led to catastrophic global biodiversity losses and species extinctions at an accelerating rate. Amphibians are currently one of the worst affected classes with at least a third of species categorised as being threatened with extinction. At the same time, they are also critically important for many habitats and provide man with a powerful proxy for ecosystem health by acting as a bioindicator group. Whilst the causes of synchronised amphibian losses are varied recent research has begun to highlight a growing role that macroparasites are playing in amphibian declines. However, diagnosing parasite infection in the field can be problematic, principally relying on collection and euthanasia of hosts, followed by necropsy and morphological identification of parasites in situ. The current study developed a non-invasive PCR-based methodology for sensitive detection and identification of parasitic nematode DNA released in the faeces of infected amphibians as egg or tissue fragments (environmental DNA). A DNA extraction protocol optimised for liberation of DNA from resilient parasite eggs was developed alongside the design of a novel, nematode universal, degenerate primer pair, thus avoiding the difficulties of using species specific primers in situations where common parasite species are unknown. Used in conjunction this protocol and primer pair was tested on a wide range of faecal samples from captive and wild amphibians. The primers and protocol were validated and detected infections, including a Railletnema nematode infection in poison dart frogs from ZSL London Zoo and Mantella cowani frogs in the wild. Furthermore, we demonstrate the efficacy of our PCR-based protocol for detecting nematode infection in other hosts, such as the presence of pinworm (Aspiculuris) in two tortoise species and whipworm (Trichuris muris) in mice. Our environmental DNA approach mitigates problems associated with microscopic identification and can be applied to detect nematode parasitoses in wild and captive hosts for infection surveillance and maintenance of healthy populations.