Accurate Detection and Quantification of Seasonal Abundance of American Bullfrog (Lithobates Catesbeianus) Using ddPCR eDNA Assays

Abstract The invasive American bullfrog (Lithobates catesbeianus) imperils freshwater biodiversity worldwide. Effective management includes rapid detection and response to incipient invasions, as established populations are extremely difficult to eradicate. Although environmental DNA (eDNA) approaches provide a highly sensitive alternative to conventional surveillance techniques, extensive testing is paramount to generate reliable output. Here, we tested and compared the performance of two primer/probe assays to detect and quantify the abundance of bullfrogs in silico and in situ using digital droplet PCR. Although both assays proved to be equally specific and sensitive, one outperformed the other in detection resolution (i.e. distinguishing target and non-target droplets), and hence was selected for further analyses. Mesocosm experiments using density series of larval and juvenile bullfrogs revealed that eDNA concentrations could explain 99% of the larval abundance and biomass. Per individual eDNA emission rates did not differ significantly among life stages, indicating that eDNA concentrations can be used as a reliable proxy to asses bullfrog abundance in natural populations. Seasonal eDNA patterns in three infested ponds showed parallel fluctuations in bullfrog eDNA concentrations. A peak in late summer (August, September or October), coinciding with the breeding season, was followed by continuously low eDNA concentrations in winter and spring. These findings demonstrate that eDNA analyses can be used as a solid and reliable tool to detect the early stages of bullfrog invasions and to quantify temporal changes in abundance that will be useful in coordinating and evaluating large-scale bullfrog eradication programs.