Development of highly sensitive environmental DNA methods for the detection of Bull Sharks, Carcharhinus leucas (Müller and Henle, 1839), using Droplet Digital™ PCR

Abstract

Abstract Background As apex and mesopredators, elasmobranchs play a crucial role in maintaining ecosystem function and balance in marine systems. Elasmobranch populations worldwide are in decline as a result of exploitation via direct and indirect fisheries mortalities and habitat degradation; however, a lack of information on distribution, abundance, and population biology for most species hinders their effective management. Environmental DNA analysis has emerged as a cost‐effective and non‐invasive technique to fill some of these data gaps, but often requires the development of species‐specific methodologies. Aims Here, we established eDNA methodology appropriate for targeted species detections of Bull Sharks, Carcharhinus leucas, in estuarine waters in the northern Gulf of Mexico. Materials and Methods We compared different QIAGEN®DNeasy® extraction kit protocols and developed a species‐specific Droplet Digital™ PCR (ddPCR) assay by designing primers and an internal probe to amplify a 237 base pair portion of the ND2 gene in the mitochondrial genome of C. leucas. To validate the developed methods, water samples were collected from known C. leucas habitat and from an ex situ closed environment containing a single C. leucas individual. The effectiveness of the assay in an open environment was then assessed by placing one C. leucas into a flow‐through mesocosm system and water samples were collected every 30 min for 3 hr. Results The developed C. leucas ‐specific assay has the ability to detect target DNA concentrations in a reaction as low as 0.6 copies/μl. DdPCR reactions performed on water samples from known habitat and 30 min after a shark was added to the closed environment contained 1.62 copies/μl and 166.6 copies/μl of target C. leucas eDNA, respectively. Carcharhinus leucas eDNA was detected in the flow‐through system within 30 min, but concentrations remained low and variable throughout the duration of the experiment.

Keywords

• elasmobranch
• Gulf of Mexico
• habitat use
• mitochondrial genome
• threatened species
• water sample