Environmental DNA (eDNA) From the Wake of the Whales: Droplet Digital PCR for Detection and Species Identification

Abstract

Genetic sampling for identification of species, subspecies or stock of whales, dolphins and porpoises at sea remains challenging. Most samples have been collected with some form of a biopsy dart requiring a close approach of a vessel while the individual is at the surface. Here we have adopted droplet digital (dd)PCR technology for detection and species identification of cetaceans using environmental (e)DNA collected from seawater. We conducted a series of eDNA sampling experiments during 25 encounters with killer whales, Orcinus orca, in Puget Sound (the Salish Sea). The regular habits of killer whales in these inshore waters allowed us to locate pods and collect seawater, at an initial distance of 200 m and at 15-min intervals, for up to 2 h after the passage of the whales. To optimize detection, we designed a set of oligonucleotide primers and probes to target short fragments of the mitochondrial (mt)DNA control region, with a focus on identification of known killer whale ecotypes. We confirmed the potential to detect eDNA in the wake of the whales for up to 2 h, despite movement of the water mass by several kilometers due to tidal currents. Re-amplification and sequencing of the eDNA barcode confirmed that the ddPCR detection included the "southern resident community" of killer whales, consistent with the calls from hydrophone recordings and visual observations.

False‐negative detections from environmental DNA collected in the presence of large numbers of killer whales (Orcinus orca)

“…In this study, the first of its kind, similar mitochondrial haplotype frequencies were recovered from seawater eDNA compared to tissue samples, expanding the applications of eDNA to encompass population genetics of aquatic organisms (Baker et al, 2018). A study by demonstrated the use of eDNA to provide estimates of genetic diversity in a whale shark (Rhincodon typus) aggregation off Qatar in the Arabian Gulf.…”

“…However, in Iceland, conditions were calm and seawater samples were collected from a RIB following long encounters (1-2 hr) with killer whales, but again Feces are considered one of the highest sources of eDNA in environments (Alberdi et al, 2018;Baker et al, 2018;Klymus, Richter, Chapman, & Paukert, 2015), particularly for marine mammals whose feces are known to float (Gillett, Frasier, Rolland, & White, 2010;Stewart, 2019). However, in Iceland, conditions were calm and seawater samples were collected from a RIB following long encounters (1-2 hr) with killer whales, but again Feces are considered one of the highest sources of eDNA in environments (Alberdi et al, 2018;Baker et al, 2018;Klymus, Richter, Chapman, & Paukert, 2015), particularly for marine mammals whose feces are known to float (Gillett, Frasier, Rolland, & White, 2010;Stewart, 2019).…”

“…When the starting number of DNA templates is small, PCR stochasticity can result in variable amplification success across replicate PCRs (Taberlet et al 1996). This can occur even in those studies that use more sensitive methods such as the digital droplet (dd)PCR used by Baker et al (2018). This can occur even in those studies that use more sensitive methods such as the digital droplet (dd)PCR used by Baker et al (2018).…”

“…While qPCR is considered sensitive to low concentrations of target DNA as was evident in its ability to detect mackerel DNA in the Icelandic eDNA sample and to amplify highly diluted positive controls of killer whale DNA, the lack of killer whale eDNA suggests that ultra-sensitive methods such as ddPCR (Baker et al, 2018;Doi et al, 2015;Nathan, Simmons, Wegleitner, Jerde, & Mahon, 2014) or CRISPR-Cas (Williams et al 2019) may be essential, unless the biomass of the target species and/or environmental conditions allows for detection using less sensitive methods. While qPCR is considered sensitive to low concentrations of target DNA as was evident in its ability to detect mackerel DNA in the Icelandic eDNA sample and to amplify highly diluted positive controls of killer whale DNA, the lack of killer whale eDNA suggests that ultra-sensitive methods such as ddPCR (Baker et al, 2018;Doi et al, 2015;Nathan, Simmons, Wegleitner, Jerde, & Mahon, 2014) or CRISPR-Cas (Williams et al 2019) may be essential, unless the biomass of the target species and/or environmental conditions allows for detection using less sensitive methods.…”

“…Subsequently, many applications have started to emerge, including the use of eDNA to determine species abundance (Pilliod et al, 2013;Thomsen et al, 2016), biomass (Jane et al, 2015;Nevers et al, 2018;Takahara, Minamoto, Yamanaka, Doi, & Kawabata, 2012), and population structure Parsons, Everett, Dahlheim, & Park, 2018). Additionally, eDNA research has developed into sampling in the more challenging marine environment (Baker et al, 2018;Bakker et al, 2017;Foote et al, 2012;Minamoto et al, 2017;Parsons et al, 2018;Sigsgaard et al, 2016;Thomsen, Kielgast, Iversen, Møller et al, 2012;Thomsen et al, 2016;Thomsen & Willerslev, 2015).…”