Development and validation of two environmental DNA assays for American Eel (Anguilla rostrata)

We developed and validated two species-specific qPCR markers to detect American Eel environmental DNA. Marker validation included assay design, specificity and sensitivity testing, and in vivo laboratory and field experiments. Markers AME1 and AME2 targeted 116 and 129 bp fragments of the mitochondrial NADH dehydrogenase subunit 2 and cytochrome b genes, respectively. Markers were 94%-100% homologous for all 49 aligned American Eel sequences. Specificity tests, with known DNA obtained from 149 individuals spanning 81 fish species, amplified DNA derived from American Eel tissue exclusively. Each marker also had high sensitivity with LOD and LOQ values of 2.8-50 copies. For each marker, pilot testing of American Eel in aquaria at increasing densities (n = 0, 1, 5, 10 eels) showed a significant (p < 0.03) negative relationship between mean qPCR cycle threshold value and number of eels per tank. Our in situ testing of water samples collected from 35 sites on the East Coast (from as far south as Maryland to as far north as Maine) revealed that sites known to contain populations of American Eel (n = 11) were all positive for American Eel DNA, while sites where American Eel were presumed absent (n = 24) failed to amplify American Eel DNA. In three cases, our assays produced positive detections in the lower portion of a watershed but failed to detect American Eel upstream of a presumed impassible barrier in each of the same watersheds (all internal positive controls indicated no evidence of PCR inhibition in our field samples and all negative controls indicated no evidence of contamination). Our encouraging results of in vitro and in situ validation demonstrate the utility of using eDNA as a tool to aid in American Eel conservation efforts.