Nonlethal amphibian skin swabbing of cutaneous natural products for HPLC fingerprinting

Small organic molecules found on the skin of amphibians may help impart resistance to pathogens, such as the lethal fungus Batrachochytrium dendrobatidis. The study of these compounds has traditionally required euthanasia of the amphibian, followed by chemical extraction of excised skin. As an alternative method, we report the development and assessment of a non-lethal technique using foam-tipped swabs and HPLC analysis to directly isolate and characterize small molecules found on the skin of amphibians. This protocol was field-tested on Bioko Island, Equatorial Guinea with forty-seven frogs (representing 14 native species). Multiple species (particularly Afrixalus paradorsalis and Didynamipus sjostedti) carried sets of species-specific compounds (i.e., a chromatographic fingerprint). A principal coordinate analysis (PCO) of the commonly occurring compounds detected across all species revealed a significant relationship between chromatographic profile and species for all swab samples.