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

Abstract

BackgroundAs 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.AimsHere, 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 MethodsWe 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.ResultsThe 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.

Environmental DNA detection tracks established seasonal occurrence of blacktip sharks (Carcharhinus limbatus) in a semi-enclosed subtropical bay

“…The application of eDNA analysis specifically for sharks and their relatives has recently gained momentum, and studies largely focus on providing proof-of-concept that their eDNA can be detected 10,[20][21][22][23] . In this study we developed a real-time PCR assay that detected C. limbatus eDNA in water samples from Terra Ceia Bay.…”

“…It could also be misleading if eDNA shedding rates vary with size, sex, season, or physiological state. Although complicated due to the requirements and restrictions related to using live sharks for controlled experiments 20 , more fundamental studies investigating (the factors related to) shark eDNA shedding, accumulation, persistence and degradation could greatly contribute to our understanding and interpretation of eDNA results relating to spatiotemporal abundance patterns.…”

“…Given the rarity of shark eDNA targets and the limitations of real-time PCR we suggest that future applications of this and similar assays should use digital droplet PCR (ddPCR) 54 for the high resolution quantification of rare target eDNA. This has recently been trialed for the detection of elasmobranch eDNA 20 , 23 , 55 , but has yet to be implemented for the spatio-temporal tracking of abundance.…”

“…Shark eDNA represents a naturally rare target for sampling given their positions as upper level predators (naturally lower densities compared to species occupying lower trophic levels), coupled with low abundances due to overexploitation in many areas 18 , suggesting a high probability of negative results 17 , 19 . Nevertheless, eDNA analysis has recently been applied for the detection of sharks and their relatives and has proven to have potential for presence/absence and biodiversity studies 10 , 20 – 27 . As has been shown for other marine organisms, there is also growing evidence indicating that the quantity of eDNA may be related not only to species presence, but also to abundance 16 , 21 , 28 – 32 .…”

Swimming against the flow—Environmental DNA can detect bull sharks (Carcharhinus leucas) across a dynamic deltaic interface

“…Determining how YOY bull sharks connect these habitats is critical given the role of mobile links in ecosystem resilience (Lundberg & Moberg, 2003), yet cost‐prohibitive when using traditional techniques such as fisheries‐independent monitoring or passive acoustic telemetry. Therefore, the objective of the current study was to use a newly developed bull shark environmental DNA (eDNA) assay (see Schweiss et al., 2020) to examine the potential for eDNA approaches to detect bull sharks across this highly dynamic deltaic interface.…”