Development of Three Novel eDNA Assays for Detecting Coastal Sharks

Environmental DNA (eDNA) analysis is an emerging, non-invasive community monitoring tool. This study aimed to determine if eDNA methods can be reliably used in a large brackish, partially mixed estuary by developing and testing three novel eDNA primers, for Sandbar Sharks (Carcharhinus plumbeus), Blacknose Sharks (Carcharhinus acronotus), and Bonnetheads (Sphyrna tiburo). These primers were designed to target 109, 156, and 120 base pair (bp) fragments, respectively, of the highly conserved NAD2 gene in the mitochondrial genome of each species. Primer function was validated through testing against 102 known genomic source samples and 25 filtered water samples from aquaria in which the species were exhibited. A total of 198 water samples were collected alongside active longlines in Winyah Bay, South Carolina, and extracted for eDNA analysis. We created three species-specific eDNA primers for the target species and validated them against 8+ target genomic samples and 14 other local elasmobranch species as negative controls. Detection was successful when applied to aquarium samples gathered from five separate institutions. The presence of other coextracted biological macromolecules from Winyah Bay consistently inhibited PCR detection from the raw water samples, thereby limiting the utility of eDNA in the study system. This study provided evidence that species-specific primers of closely related carcharhinid species can be developed and utilized while also showcasing the challenges of eDNA detection in a highly productive marine environment. Further study in waters of lower organic content, or with advanced techniques is needed to demonstrate the full functionality of the primer