While environmental DNA (eDNA) is becoming increasingly established in biodiversity monitoring of freshwater ecosystems, the use of eDNA surveys in the marine environment is still in its infancy. Here, we use two approaches: targeted quantitative PCR (qPCR) and whole-genome enrichment capture followed by shotgun sequencing in an effort to amplify killer whale DNA from seawater samples. Samples were collected in close proximity to killer whales in inshore and offshore waters, in varying sea conditions and from the surface and subsurface but none returned strongly positive detections of killer whale eDNA. We validated our laboratory methodologies by successfully amplifying a dilution series of a positive control of killer whale DNA. Furthermore, DNA of Atlantic mackerel, which was present at all sites during sampling, was successfully amplified from the same seawater samples, with positive detections found in ten of the eighteen eDNA extracts. We discuss the various eDNA collection and amplification methodologies used and the abiotic and biotic factors that influence eDNA detection. We discuss possible explanations for the lack of positive killer whale detections, potential pitfalls, and the apparent limitations of eDNA for genetic research on cetaceans, particularly in offshore regions. K E Y W O R D S eDNA, environmental DNA, metagenomics, Orcinus orca, PCR, Scomber scombrus, wholegenome enrichment | 317 PINFIELD Et aL. S U PP O RTI N G I N FO R M ATI O N Additional supporting information may be found online in the Supporting Information section at the end of the article. How to cite this article: Pinfield R, Dillane E, Runge AKW, et al. False-negative detections from environmental DNA collected in the presence of large numbers of killer whales (Orcinus orca).
During the construction of a gas pipeline from an offshore gas field in northwest Ireland, a year-round shore-based marine mammal monitoring programme was undertaken. Using 6 yr of data, generalised estimating equations-generalised additive models (GEE-GAMs) were used to investigate if construction-related activity and vessel traffic influenced the occurrence of common dolphin, minke whale, harbour porpoise and grey seal within the area where the pipeline made landfall. Construction-related activity reduced harbour porpoise and minke whale presence, whilst an increase in vessel numbers (independent of construction-related activity) reduced common dolphin presence. All species showed some degree of annual and seasonal variation in occurrence. For common dolphins and harbour porpoises, we found similar seasonal patterns to those reported in broader Irish waters, which tentatively suggests that seasonal patterns persisted irrespective of construction-related activity or vessel traffic, indicating that any impact might have been only short-term. Multiple construction-related activities occurred simultaneously in different areas, and the inter-annual variation may, in part, be an indication of variation in species' response to particular activities, their intensity and their location. However, the precise location of the activities was not regularly recorded, limiting our ability to investigate the fine-scale spatio−temporal impact of the diverse range of construction-related activities. Improved communication and coordination between developers, regulators and scientists will help ensure that monitoring programmes are effective and efficient, to better inform our understanding of potential impacts and to mitigate effectively against them for future developments.
Marine construction works often lead to temporary increases in vessel traffic, which, in addition to the construction activity itself, contribute to underwater ambient noise in the affected area and increase the risk of vessel collision for marine mammals. Using a 3 yr data set of cliff-based observations, we investigated whether the presence/absence of minke whales, bottlenose dolphins and grey seals varied with the overall number and type of vessels present during the construction of an underwater gas pipeline through a bay on the northwest coast of Ireland. Results from binary generalised estimation equations showed a positive relationship between the presence of bottlenose dolphins and the overall number of boats, as well as the number of construction vessels. However, the presence of the 2 taxa with higher hearing sensitivity at low frequencies -minke whales and grey seals -was negatively correlated with the total number of boats and the number of utility vessels (as well as the number of fishing boats in the case of minke whales). While bottlenose dolphins may have been attracted to either the vessels per se or high prey concentrations coinciding with construction activities, both minke whales and grey seals appear to have been displaced by high levels of vessel traffic, most likely due to noise disturbance. Careful consideration of mitigation measures, especially for taxa with low-frequency hearing, is therefore essential in the planning phase of offshore construction activities, which should also take local circumstances into account.
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