Development of Environmental DNA Assay for Screening of Blanchard’s Cricket Frog (Acris blanchardi) Via Laboratory and Field Methods in Oklahoma, USA

Amphibians represent one of the most threatened vertebrate groups, and although monitoring amphibian population dynamics is critical for conservation, most traditional survey efforts depend on time-consuming, often invasive monitoring activities and visual surveys. Screening environmental DNA (eDNA), a non-invasive monitoring technique, has the potential to identify species presence at a site, even in the absence of visual confirmation. In this study, we developed an aquatic eDNA detection protocol for a common and widespread frog species in Oklahoma (Acris blanchardi). We first conducted three laboratory tests to examine assay specificity and sensitivity. Once the primer-probe assay was confirmed to discriminate the target species from others consistently, we then sampled eDNA in four of Oklahoma’s six ecological regions to assess how the variation of abiotic factors impact assay sensitivity. In field testing of over 500 samples, we were able to detect A. blanchardi eDNA at 60% of the waterbodies sampled, at nearly all field sites across all sampled ecoregions. The proportion of negative eDNA assay results in the waterbodies where the target species were visually observed underscore the importance of continuing traditional surveys alongside newer genetic screening techniques to improve species detection and occupancy modeling.