Ex situ Diet Influences the Bacterial Community Associated with the Skin of Red-Eyed Tree Frogs (Agalychnis callidryas)

Abstract

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.

Dietary Carotenoid Supplementation Enhances the Cutaneous Bacterial Communities of the Critically Endangered Southern Corroboree Frog (Pseudophryne corroboree)

“…However, unlike other vertebrates, very little is known about the specific effects of dietary carotenoids on either the innate or adaptive immune system of amphibians. Results reported by Antwis et al [ 14 ] clearly demonstrate the potential for carotenoid enrichment to enhance the immune function of captive amphibians through their effect on cutaneous bacterial communities. Further research is now required on a diversity of amphibian species in order to ascertain the generality of their findings.…”

“…Frog mass (range = 1.73-2.80 g; mean = 2.18 ± 0.04 g) and snoutvent length (range = 23.46-27.51 mm; mean = 25.52 ± 0.20 mm) did not differ between supplemented and unsupplemented treatments (n = 22 per treatment); t test: weight: t = 1.30, P = 0.20; snoutvent: t = 0.99, P = 0.33). During swab collection, frogs were removed from their individual containers and handled with separate sterile gloves (Skinshield powderfree latex gloves, Livingstone International, Rosebery, NSW, Australia) to prevent cross contamination of cutaneous bacteria [ 14,23 ]. Immediately prior to sampling, frogs were rinsed once with 30mL sterile water to remove transient bacteria [ 6,23,24 ], ensuring minimal handling to prevent loss of cutaneous bacteria.…”

“…Frogs were swabbed (1 swab/frog) by running the natural fibre cotton tip across each of the following surfaces three times: dorsal (anterior to posterior), ventral (anterior to posterior), lateral (left and right sides), front legs from armpit to wrist (left and right sides) and back legs from groin to ankle (left and right sides) (adapted from Shaw et al [ 24 ] and Antwis et al [ 14 ] (Fig. 1 ).…”

“…Using aseptic techniques within a biosafety cabinet (Biological Safety Cabinet Class II, Email Westinghouse Pty Ltd, Australia), swab tips were cut off into a 1.5mL sterile Eppendorf tube (Eppendorf South Pacific Pty Ltd, North Ryde, NSW, Australia) containing 1 mL 0.9 % sterile NaCl and vortexed for 30 s to disassociate bacteria from the tip [ 14 ]. As indicated by a pilot study, dilutions of 10 (undiluted) and 10 (10fold dilution) in 0.9 % sterile NaCl were most appropriate to obtain an optimum amount of bacterial growth suitable for assessing bacterial community composition (i.e.…”

“…A key factor known to affect overall health and disease susceptibility of captive amphibians is nutrition reviewed by [ 13 ]. To date, however, only one study has investigated the effect of host diet on amphibian cutaneous microbial communities [ 14 ], an important component of amphibian immune defence. Antwis et al [ 14 ] investigated the effect of dietary carotenoid enrichment on the cutaneous bacterial communities of captive redeyed tree frogs, Agalychnis callidryas, and found that carotenoids significantly increased bacterial species richness, abundance and overall community composition.…”