Ursids evolved early and continuously to be low-protein macronutrient omnivores.

The eight species of bears world-wide consume a wide variety of diets. Some are specialists with extensive anatomical and physiological adaptations necessary to exploit specific foods or environments [e.g., polar bears (Ursus maritimus), giant pandas (Ailuropoda melanoleuca), and sloth bears (Melursus ursinus)], while the rest are generalists. Even though ursids evolved from a high-protein carnivore, we hypothesized that all have become low-protein macronutrient omnivores. While this dietary strategy has already been described for polar bears and brown bears (Ursus arctos), a recent study on giant pandas suggested their macronutrient selection was that of the ancestral high-protein carnivore. Consumption of diets with inappropriate macronutrient profiles has been associated with increased energy expenditure, ill health, failed reproduction, and premature death. Consequently, we conducted feeding and preference trials with giant pandas and sloth bears, a termite and ant-feeding specialist. Both giant pandas and sloth bears branched off from the ursid lineage a million or more years before polar bears and brown bears. We found that giant pandas are low-protein, high-carbohydrate omnivores, whereas sloth bears are low-protein, high-fat omnivores. The preference for low protein diets apparently occurred early in the evolution of ursids and may have been critical to their world-wide spread.

Metabolome-Informed Microbiome Analysis Refines Metadata Classifications and Reveals Unexpected Medication Transfer in Captive Cheetahs.

Even high-quality collection and reporting of study metadata in microbiome studies can lead to various forms of inadvertently missing or mischaracterized information that can alter the interpretation or outcome of the studies, especially with nonmodel organisms. Metabolomic profiling of fecal microbiome samples can provide empirical insight into unanticipated confounding factors that are not possible to obtain even from detailed care records. We illustrate this point using data from cheetahs from the San Diego Zoo Safari Park. The metabolomic characterization indicated that one cheetah had to be moved from the non-antibiotic-exposed group to the antibiotic-exposed group. The detection of the antibiotic in this second cheetah was likely due to grooming interactions with the cheetah that was administered antibiotics. Similarly, because transit time for stool is variable, fecal samples within the first few days of antibiotic prescription do not all contain detected antibiotics, and the microbiome is not yet affected. These insights significantly altered the way the samples were grouped for analysis (antibiotic versus no antibiotic) and the subsequent understanding of the effect of the antibiotics on the cheetah microbiome. Metabolomics also revealed information about numerous other medications and provided unexpected dietary insights that in turn improved our understanding of the molecular patterns on the impact on the community microbial structure. These results suggest that untargeted metabolomic data provide empirical evidence to correct records and aid in the monitoring of the health of nonmodel organisms in captivity, although we also expect that these methods may be appropriate for other social animals, such as cats.IMPORTANCE Metabolome-informed analyses can enhance omics studies by enabling the correct partitioning of samples by identifying hidden confounders inadvertently misrepresented or omitted from carefully curated metadata. We demonstrate here the utility of metabolomics in a study characterizing the microbiome associated with liver disease in cheetahs. Metabolome-informed reinterpretation of metagenome and metabolome profiles factored in an unexpected transfer of antibiotics, preventing misinterpretation of the data. Our work suggests that untargeted metabolomics can be used to verify, augment, and correct sample metadata to support improved grouping of sample data for microbiome analyses, here for nonmodel organisms in captivity. However, the techniques also suggest a path forward for correcting clinical information in microbiome studies more broadly to enable higher-precision analyses.

Tagging frogs with passive integrated transponders causes disruption of the cutaneous bacterial community and proliferation of opportunistic fungi.

Symbiotic bacterial communities play a key role in protecting amphibians from infectious diseases including chytridiomycosis, caused by the pathogenic fungus Batrachochytrium dendrobatidis. Events that lead to the disruption of the bacterial community may have implications for the susceptibility of amphibians to such diseases. Amphibians are often marked both in the wild and in captivity for a variety of reasons, and although existing literature indicates that marking techniques have few negative effects, the response of cutaneous microbial communities has not yet been investigated. Here we determine the effects of passive integrated transponder (PIT) tagging on culturable cutaneous microbial communities of captive Morelet's tree frogs (Agalychnis moreletii) and assess the isolated bacterial strains for anti-B. dendrobatidis activity in vitro. We find that PIT tagging causes a major disruption to the bacterial community associated with the skin of frogs (∼12-fold increase in abundance), as well as a concurrent proliferation in resident fungi (up to ∼200-fold increase). Handling also caused a disruption the bacterial community, although to a lesser extent than PIT tagging. However, the effects of both tagging and handling were temporary, and after 2 weeks, the bacterial communities were similar to their original compositions. We also identify two bacterial strains that inhibit B. dendrobatidis, one of which increased in abundance on PIT-tagged frogs at 1 day postmarking, while the other was unaffected. These results show that PIT tagging has previously unobserved consequences for cutaneous microbial communities of frogs and may be particularly relevant for studies that intend to use PIT tagging to identify individuals involved in trials to develop probiotic treatments.

Ex situ diet influences the bacterial community associated with the skin of red-eyed tree frogs (Agalychnis callidryas).

Amphibians support symbiotic bacterial communities on their skin that protect against a range of infectious pathogens, including the amphibian chytrid fungus. The conditions under which amphibians are maintained in captivity (e.g. diet, substrate, enrichment) in ex situ conservation programmes may affect the composition of the bacterial community. In addition, ex situ amphibian populations may support different bacterial communities in comparison to in situ populations of the same species. This could have implications for the suitability of populations intended for reintroduction, as well as the success of probiotic bacterial inoculations intended to provide amphibians with a bacterial community that resists invasion by the chytrid fungus. We aimed to investigate the effect of a carotenoid-enriched diet on the culturable bacterial community associated with captive red-eyed tree frogs (Agalychnis callidryas) and make comparisons to bacteria isolated from a wild population from the Chiquibul Rainforest in Belize. We successfully showed carotenoid availability influences the overall community composition, species richness and abundance of the bacterial community associated with the skin of captive frogs, with A. callidryas fed a carotenoid-enriched diet supporting a greater species richness and abundance of bacteria than those fed a carotenoid-free diet. Our results suggest that availability of carotenoids in the diet of captive frogs is likely to be beneficial for the bacterial community associated with the skin. We also found wild A. callidryas hosted more than double the number of different bacterial species than captive frogs with very little commonality between species. This suggests frogs in captivity may support a reduced and diverged bacterial community in comparison to wild populations of the same species, which could have particular relevance for ex situ conservation projects.

Effects of visible implanted elastomer marking on physiological traits of frogs.

Amphibians possess innate immune defences, including antimicrobial peptides and symbiotic bacterial communities, that can protect them from infectious diseases, including chytridiomycosis. On-going research is attempting to use amphibian symbiotic bacteria to develop probiotic treatments that can protect hosts from the causative agent of chytridiomycosis, the fungal pathogen Batrachochytrium dendrobatidis. Events that cause disruption of symbiotic bacterial communities or deplete peptide stores could increase the susceptibility of individuals to disease and may have implications for amphibians involved in probiotic trials or time course studies that investigate symbiotic bacterial communities. It has previously been shown that passive integrated transponder tagging of frogs causes a rapid (within 24 h) and major proliferation of micro-organisms on the skin. Here, we show that marking of red-eyed tree frogs (Agalychnis callidryas) with visible elastomer has no effect on adrenal response (represented by faecal glucocorticoid metabolite concentrations) or peptide production, although there was evidence of a slightly greater microbial abundance associated with the skin of marked frogs 2 weeks after tagging. The results indicate that visible elastomer may be a preferable marking technique to passive integrated transponder tagging, particularly in the context of probiotic trials or time course studies that investigate symbiotic bacterial communities. More work is required to determine the effects of different marking techniques on physiological responses of amphibians, whether these physiological responses are consistent across host species and whether such 'non-invasive' marking methods affect the susceptibility of amphibians to infectious pathogens, such as B. dendrobatidis.

Differences in carotenoid accumulation among three feeder-cricket species: implications for carotenoid delivery to captive insectivores.

There are a limited number of feeder-invertebrates available to feed captive insectivores, and many are deficient in certain nutrients. Gut-loading is used to increase the diversity of nutrients present in the captive insectivore diet; however, little is known about delivery of carotenoids via gut-loading. Carotenoids may influence health and reproduction due to their roles in immune and antioxidant systems. We assessed interspecific variation in carotenoid accumulation and retention in three feeder-cricket species (Gryllus bimaculatus, Gryllodes sigillatus and Acheta domesticus) fed one of three diets (wheat-bran, fish-food based formulated diet, and fresh fruit and vegetables). Out of the three species of feeder-cricket in the fish-food-based dietary treatment group, G. bimaculatus had the greatest total carotenoid concentration. All cricket species fed the wheat-bran diet had very low carotenoid concentrations. Species on the fish-food-based diet had intermediate carotenoid concentrations, and those on the fruit and vegetable diet had the highest concentrations. Carotenoid retention was poor across all species. Overall, this study shows that, by providing captive insectivores with G. bimaculatus crickets recently fed a carotenoid-rich diet, the quantity of carotenoids in the diet can be increased.