Background/Question/Methods Beyond determining species presence/absence, resource managers are often interested in obtaining population-level metrics to make conservation or management decisions. Environmental DNA has demonstrated utility in a variety of systems to detect species presence and community composition but has limitations in its inference to population status and demographics. Recently, environmental RNA (eRNA) has been detected in water samples and shown promise for distinguishing living communities and opening the door to applications for gathering population-level information with non-invasive samples. We conducted an experiment to develop eRNA methods that can be used to distinguish age classes in amphibian species to infer population demographics from environmental samples. We housed different life stages of tiger salamanders (Ambystoma mavortium) and American bullfrogs (Lithobates catesbeianus) and collected 100 mL water samples and comparative tissue vouchers monthly. We tested a suite of potential eRNA gene targets with RT-PCR to find those that could be detected in only a single life stage. Then we collected 500 mL field samples where the presence of specific life stages could be confirmed with traditional methods and applied our developed lab methods to those samples for proof-of-concept application.
Results/Conclusions We were able to successfully detect keratin-associated RNA products from water samples of larval and adult life stages. We found that keratin 6A was most specific to the larval stage of tiger salamanders and that Rana larval keratin was specific to the tadpole stage of American bullfrogs in our laboratory samples. We were then able to apply these methods to field collected samples and detect larval life stages in natural populations. Our work demonstrates that eRNA can be used to distinguish age classes of amphibian species with non-invasive samples. We conclude that with further development of methods and applications, eRNA will be a useful technique to gather information on population status and demographics and answer questions that are not currently tractable using environmental DNA sampling methods.