The primary objective of this Limited Scope project was to quantify the detection of environmental deoxyribonucleic acid (eDNA) from beaked whales on the Navy’s Atlantic Undersea Test and Evaluation Centre (AUTEC) range, using an acoustically assisted sampling design. This included quantification of eDNA by droplet-digital polymerase chain reaction (ddPCR) and sequencing by conventional eDNA barcoding and next-generation eDNA metabarcoding. For this project, an extremely experienced team of co-performers was assembled, including acoustic support from the Navy Undersea Warfare Center and Cornell University, logistic support and local knowledge of the beaked whales population from the Bahamas Marine Mammal Research Organisation (BMMRO), and support of genetic analysis from the Marine Mammal Institute, Oregon State University.

The Technical Approach of the project was to:

1. Conduct small-boat surveys on the AUTEC range during a 12-day field season, to locate beaked whales using the acoustic array;
2. Collect seawater from the proximity of the beaked whales using a serial and a spatial sampling design and filter seawater on location;
3. Extract eDNA from filters and quantify eDNA of beaked whales or other cetacean species using ddPCR;
4. Confirm species identity and novel haplotype variants by re-amplification and conventional sequencing of eDNA barcodes and next-generation sequencing of eDNA metabarcode; and,
5. Relate the quantification of eDNA by ddPCR to the presence or absence of beaked whales detected by acoustic monitoring.

During the 12 days available for field work on the AUTEC range, there were 9 small-boat surveys undertaken on 8 days. Records from the 9 small-boat surveys documented 508 kilometers of survey tracks during a total of 51 hours on the water. There were 7 sightings of singles or small groups, totaling 14 individuals. This included 11 Blainville’s beaked whales (Mesoplodon densirostris), 2 unknown cetaceans and a single sperm whale. Of the 11 individual Blainville’s beaked whales encountered, 10 were identified from photographs and 6 of these were new to the BMMRO catalogue. The 4 known individuals had sighting records dating back to 2016, 2012 and 2005. During the 9 surveys, the team was successful in collecting 56 paired samples of 1 liter (L) each (i.e., 102 L total) within the vicinity of beaked whales. This included 4 spatial design collections (20 paired samples each) in the absence of any visible proximity to the whales and 4 serial design collections (31 paired samples total) in the visible proximity of the whales. An additional 12 samples were included in relevant analyses as positive and negative controls for either field or laboratory handling.

Following cleanup of polymerase chain reaction (PCR) inhibitors, all samples were submitted for ddPCR. The average values of the ddPCR, run in duplicate, were low for most samples, with many having 95% Poisson confidence limits overlapping with zero. Only 12 of the 56 samples exceeded the relaxed threshold of 0.1 copies/μl, considered a likely positive for detection, and only 3 of these met the strict threshold of 0.5 copies/μl. Only one of the 12 likely positive samples was collected in a spatial design. Many of the other samples collected in the 4 spatial designs showed average values of 0.0 copies/μl.

A third run of the ddPCR was reserved from the droplet counter and used, instead, to harvest amplifiable products for subsequent conventional PCR and sequencing. This protocol was intended to help overcome any residual inhibitors and to confirm species identification by conventional eDNA barcoding.To assess the potential to detect further haplotype variants with next-generation sequencing, the 12 likely positive samples were submitted for eDNA metabarcoding on the Illumina MiSeq (MiSeq) as both a series of individual and pooled libraries.

Considering both the conventional sequencing and the MiSeq results, 9 of the 12 samples considered to be likely positives from the ddPCR provided species identification of Blainville’s beaked whales. A review of the sequencing variants provided evidence of 5 haplotypes among the 9 samples. These 5 haplotypes were found in at least two independent runs of the MiSeq or supported by conventional PCR and sequencing. Two of the haplotypes, discussed above, were an exact match to sequences reported previously from the North Atlantic and available on GenBank. The other three haplotypes have not, to the team's knowledge, been reported previously. An evolutionary network of all previously reported haplotypes suggests the potential for detection of population structure from haplotype identity, at least at the oceanic scale, and perhaps within oceans using frequency differences.

The Limited Scope effort confirmed the power of eDNA barcoding and metabarcoding to identify species and intra-specific diversity of beaked whales when sampled in the proximity of the terminal dive. This promises to greatly enhance the potential for genetic sampling of beaked whales and other species that are sensitive to the approach of vessels for biopsy sampling. The characterization of intra-specific diversity, as represented by haplotype variants, also shows promise for resolving stock structure at least at the oceanic scale, and perhaps within oceans using frequency differences. The absence of eDNA detection for the spatial sampling design was disappointing but not surprising. The precision of the acoustic localization was highly variable and weather conditions were often marginal during the spatial sampling, as evidenced by the absence of any visual confirmation during these sampling events.