Development and validation of two environmental <scp>DNA</scp> assays for American Eel ( <i>Anguilla rostrata</i> )

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