Are fecal samples an appropriate proxy for amphibian intestinal microbiota?

The intestinal microbiota, an invisible organ supporting a host's survival, has essential roles in metabolism, immunity, growth, and development. Since intestinal microbiota influences a host's biology, application of such data to wildlife conservation has gained interest. There are standard protocols for studying the human intestinal microbiota, but no equivalent for wildlife. A major challenge is sampling the intestinal microbiota in an effective, unbiased way. Fecal samples are a popular proxy for intestinal microbiota because collection is non-invasive and allows for longitudinal sampling. Yet it is unclear whether the fecal microbiota is representative of the intestinal microbiota. In wildlife studies, research on the sampling methodology is limited. In this study focusing on amphibians, we characterize and compare the microbiota (small intestine, large intestine, and feces) of two Hong Kong stream-dwelling frog species: Lesser Spiny Frog (Quasipaa exilispinosa) and Hong Kong Cascade Frog (Amolops hongkongensis). We found that the microbiota of both species are similar at the level of phylum and family, but diverge at the level of genus. When we assessed the performance of fecal microbiota in representing the intestinal microbiota in these two species, we found that (1) the microbiota of the small and large intestine differs significantly, (2) feces are not an appropriate proxy of either intestinal sections, and (3) a set of microbial taxa significantly differs between sample types. Our findings raise caution equating fecal and intestinal microbiota in stream-dwelling frogs. Sampling feces can avoid sacrifice of an animal, but researchers should avoid over-extrapolation and interpret results carefully.