Application of environmental DNA to detect an endangered marine skate species in the wild

Abstract

Environmental DNA (eDNA) techniques have only recently been applied in the marine environment to detect the presence of marine species. Species-specific primers and probes were designed to detect the eDNA of the endangered Maugean skate (Zearaja maugeana) from as little as 1 L of water collected at depth (10-15 m) in Macquarie Harbour (MH), Tasmania. The identity of the eDNA was confirmed as Z. maugeana by sequencing the qPCR products and aligning these with the target sequence for a 100% match. This result has validated the use of this eDNA technique for detecting a rare species, Z. maugeana, in the wild. Being able to investigate the presence, and possibly the abundance, of Z. maugeana in MH and Bathurst harbour (BH), would be addressing a conservation imperative for the endangered Z. maugeana. For future application of this technique in the field, the rate of decay was determined for Z. maugeana eDNA under ambient dissolved oxygen (DO) levels (55% saturation) and lower DO (20% saturation) levels, revealing that the eDNA can be detected for 4 and 16 hours respectively, after which eDNA concentration drops below the detection threshold of the assay. With the rate of decay being influenced by starting eDNA concentrations, it is recommended that samples be filtered as soon as possible after collection to minimize further loss of eDNA prior to and during sample processing.

Fig 1. Map of sample sites in Macquarie Harbour (MH).

This map shows the location of the two sampling sites used in this study to collect water at depth from Macquarie Harbour (MH), including Table Head (A) and Liberty Point (B). The inset map shows the location of MH on west coast of Tasmania.

Fig 2. Example of species-specific primer and probe design, using mitochondrial sequence (NADH4 region) aligned between two species.

The mitochondrial reference sequence for the Maugean skate (Z. maugeana) is aligned with the same mitochondrial region occurring in the Thornback skate (D. lemprieri), with the penultimate bases (underlined) indicating the differences in bp between the two skate sequences. These differences were used to design the selected forward (qF1) and reverse (qR1) primers and a matching probe (qP1) that is specific to Z. maugeana (product size = 331 bp).

Fig 3. Exponential decay rate of Z. maugeana eDNA concentrations (copies per μl) over time.

For both DO treatments, including 55% saturation DO (---) and 20% saturation DO (—), Z. maugeana eDNA was still detected after 3 days using the assay and primer and probe pairs designed in this study.