Development of a Duplex Digital <scp>PCR</scp> and Validation on <scp>eDNA</scp> Water Samples for Monitoring of the Asian Swamp Eel ( <i>Monopterus albus</i> / <i>Javanensis</i> ) and Bullseye Snakehead ( <i>Channa aurolineata</i> / <i>Marulius</i>

Invasive species are a significant threat to a variety of ecosystems in Florida, with freshwater habitats being of particular concern. Two nonnative species of fish with established populations that are priority organisms for monitoring and management programs are the bullseye snakehead (BS; <i>Channa</i> species complex) and Asian swamp eel (ASE; <i>Monopterus</i> species complex). Recent technological advances have seen the emergence of environmental DNA (eDNA) analysis as a useful tool for the detection and monitoring of target organisms and to assess removal efforts. In this study, a duplex digital polymerase chain reaction (dPCR) was developed, optimized, and validated on control samples and field samples from locations with documented occurrences of target organisms. The assay developed, which allows simultaneous detection of both fish species when present in a sample, demonstrated highly efficient amplification for the corresponding target species with individual assays failing to cross-amplify. Under controlled conditions, high levels of eDNA were detected as early as five minutes post-introduction of BS to water. Additionally, field eDNA samples yielded varying levels of positive amplification for both species based on comparison of fluorescence levels to positive controls (both tissue extract and plasmids with appropriate inserts). These data indicate that through careful assay design and stringent parameter optimization, eDNA results obtained for monitoring of these two fish species can be a viable and cost-effective strategy to detect the presence of these species simultaneously as well as to evaluate the success of removal efforts.