Developing and testing the specificity and sensitivity of eDNA protocols for Shortfin Mako shark (Isurus oxyrinchus) monitoring

Effective conservation relies on knowledge of species biology and ecology of a target species. For marine pelagics, this can be especially difficult to obtain due to the difficulties of working in a marine environment, and the nature of large pelagics species to be evasive, to rarely visit near shore habitats, and to persist as solitary animals or in small groups. In Australia, there is a high demand for biological and ecological information on the Shortfin Mako shark Isurus oxyrinchus, due to recreational fishing importance and its recent formal listing as an Endangered species. This new listing was due to information stating that the I. oxyrinchus global population has decreased by 37% over the last 15 years, with further declines being imminent in the absence of effective conservation and fisheries management. In this study I developed an Environmental DNA (eDNA) assay to assist the future collection of data necessary for guiding management. eDNA assays are an emerging tool for the detection of species presence through the detection of extracellular genetic material in environmental samples (e.g. soil, water, air) which are shed from animals via excretions (e.g. hair, blood, sweat, skin, faeces etc). The technology is revolutionising the field of wildlife monitoring and biosecurity, providing a cost-effective, non-invasive and sensitive alternative to traditional methods aimed at determining species presence/absence. The first aim of this study was to create the technology needed for eDNA surveys of I. oxyrinchus, via the development of a polymerase chain reaction (PCR) assay, and subsequent testing of its sensitivity and species specificity. The second goal was to undertake a laboratory experiment which would provide insights into the temporal decay rates of Mako eDNA (i.e. how long does eDNA persist and remain detectable in the environment). Understanding rates of eDNA decay is critical for determining if positive eDNA detections in the field indicate recent species presence in close proximity to the sample location.
3
Using bioinformatic software, a TaqMan probe (consisting of a primer pair and fluorescently labelled probe) was designed to amplify a 20 base pair region of the mitochondrial cytochrome oxidase 1 (CO1) gene. Subsequent testing of the probe demonstrated effective amplification of I. oxyrinchus DNA, and a high level of species specificity based on tests for cross-amplification against a range of Australian endemic and closely related shark species. Next, an eDNA decay trial was conducted by submerging an I. oxyrinchus specimen in seawater for 20 hours, separating the water into triplicate tubs and monitoring each tubs DNA concentration via PCR under controlled laboratory conditions for 10 days. The trial indicated rapid decay of I. oxyrinchus eDNA over the first 12-24 hours (approximately 85% reduction in detectable DNA material), plateauing at 60-84 hours when 99.8% of the material had deteriorated. These findings are highly consistent with other studies of a similar nature in different marine species. I discuss the implications of these lab-condition based findings and underline the need for future testing to determine the temporal and spatial sensitivity of the assay under field conditions. Overall, this study has provided a valuable new tool for monitoring I. oxyrinchus populations in Australian and international waters. While follow up research is needed to validate the spatio-temporal sensitivity of the assay under field conditions, it is envisaged that the adoption of the eDNA assay by researchers and managers will help to gain information on I. oxyrinchus habitat affiliations, movement patterns and general distribution, to inform future management geared toward conserving this important marine apex predator.