Diversity and Evolution of Frog Visual Opsins: Spectral Tuning and Adaptation to Distinct Light Environments.

Visual systems adapt to different light environments through several avenues including optical changes to the eye and neurological changes in how light signals are processed and interpreted. Spectral sensitivity can evolve via changes to visual pigments housed in the retinal photoreceptors through gene duplication and loss, differential and coexpression, and sequence evolution. Frogs provide an excellent, yet understudied, system for visual evolution research due to their diversity of ecologies (including biphasic aquatic-terrestrial life cycles) that we hypothesize imposed different selective pressures leading to adaptive evolution of the visual system, notably the opsins that encode the protein component of the visual pigments responsible for the first step in visual perception. Here, we analyze the diversity and evolution of visual opsin genes from 93 new eye transcriptomes plus published data for a combined dataset spanning 122 frog species and 34 families. We find that most species express the four visual opsins previously identified in frogs but show evidence for gene loss in two lineages. Further, we present evidence of positive selection in three opsins and shifts in selective pressures associated with differences in habitat and life history, but not activity pattern. We identify substantial novel variation in the visual opsins and, using microspectrophotometry, find highly variable spectral sensitivities, expanding known ranges for all frog visual pigments. Mutations at spectral-tuning sites only partially account for this variation, suggesting that frogs have used tuning pathways that are unique among vertebrates. These results support the hypothesis of adaptive evolution in photoreceptor physiology across the frog tree of life in response to varying environmental and ecological factors and further our growing understanding of vertebrate visual evolution.

POSTMORTEM FINDINGS IN EIGHT SPECIES OF CAPTIVE CAECILIAN (AMPHIBIA: GYMNOPHIONA) OVER A TEN-YEAR PERIOD.

Between July 2007 and June 2017 there were 86 deaths in the populations of eight caecilian species at the Zoological Society of London (ZSL) London Zoo. The mortality rate (deaths per animal-year at risk) ranged from 0.03 in the Congo caecilian (Herpele squalostoma) to 0.85 in Kaup's caecilian (Potomotyphlus kaupii). Among the 73 individuals examined post mortem, no cause of death or primary diagnosis could be established in 35 cases, but of the others the most common cause of death was dermatitis (22 cases). When all significant pathological findings were considered, skin lesions of varying types were again the commonest (56 cases), particularly among the aquatic species: Typhlonectes compressicauda (18 out of 21 cases), T. natans (8/10) and P. kaupii (12/14). Other common findings were poor gut-fill (35 cases), kidney and gastrointestinal lesions (10 cases each), generalized congestion (8 cases) and poor body condition (6 cases). This review adds to the growing body of knowledge regarding the presentations and causes of disease in captive caecilians.

Comparison of the nutritional content of the captive and wild diets of the critically endangered mountain chicken frog (Leptodactylus fallax) to improve its captive husbandry.

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.

Development of a body condition score for the mountain chicken frog (Leptodactylus fallax).

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.

Saccharomyces jurei sp. nov., isolation and genetic identification of a novel yeast species from Quercus robur.

Two strains, D5088T and D5095, representing a novel yeast species belonging to the genus Saccharomyces were isolated from oak tree bark and surrounding soil located at an altitude of 1000 m above sea level in Saint Auban, France. Sequence analyses of the internal transcribed spacer (ITS) region and 26S rRNA D1/D2 domains indicated that the two strains were most closely related to Saccharomyces mikatae and Saccharomyces paradoxus. Genetic hybridization analyses showed that both strains are reproductively isolated from all other Saccharomyces species and, therefore, represent a distinct biological species. The species name Saccharomyces jurei sp. nov. is proposed to accommodate these two strains, with D5088T (=CBS 14759T=NCYC 3947T) designated as the type strain.

The tadpole of the Lake Oku clawed frog Xenopus longipes (Anura; Pipidae).

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 (Loumont & Kobel 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.

Microbiome function predicts amphibian chytridiomycosis disease dynamics.

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.

Documenting Aggression, Dominance and the Impacts of Visitor Interaction on Galápagos Tortoises (Chelonoidis nigra) in a Zoo Setting.

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.

Description and development of the tadpole of Rhacophorus feae (Anura; Rhacophoridae).

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).

Correction: A novel copro-diagnostic molecular method for qualitative detection and identification of parasitic nematodes in amphibians and reptiles.

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

A novel copro-diagnostic molecular method for qualitative detection and identification of parasitic nematodes in amphibians and reptiles.

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.

Impact of plant cover on fitness and behavioural traits of captive red-eyed tree frogs (Agalychnis callidryas).

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.