Primer set evaluation and sampling method assessment for the monitoring of fish communities in the North-western part of the Mediterranean Sea through eDNA metabarcoding

Environmental DNA (eDNA) metabarcoding appears to be a promising tool to survey fish communities. However, the effectiveness of this method relies on primer set performance and on a robust sampling strategy. While some studies have evaluated the efficiency of several primers for fish detection, it has not yet been assessed in situ for the Mediterranean Sea. In addition, mainly surface waters were sampled and no filter porosity testing was performed. In this pilot study, our aim was to evaluate the ability of six primer sets, targeting 12S rRNA (AcMDB07; MiFish; Tele04) or 16S rRNA (Fish16S; Fish16SFD; Vert16S) loci, to detect fish species in the Mediterranean Sea using a metabarcoding approach. We also assessed the influence of sampling depth and filter pore size (0.45 μm versus 5 μm filters). To achieve this, we developed a novel sampling strategy allowing simultaneous surface and bottom on-site filtration of large water volumes along the same transect. We found that 16S rRNA primer sets enabled more fish taxa to be detected across each taxonomic level. The best combination was Fish16S/Vert16S/AcMDB07, which recovered 95% of the 97 fish species detected in our study. There were highly significant differences in species composition between surface and bottom samples. Filters of 0.45 μm led to the detection of significantly more fish species. Therefore, to maximize fish detection in the studied area, we recommend to filter both surface and bottom waters through 0.45 μm filters and to use a combination of these three primer sets.