Field validation
Amplification of internal controls (IACs) showed no observable difference in Cq values between field samples and no template controls (data not shown), indicating no potential inhibition issues and confirming the robustness of the protocol when applied to DNA from actual environmental samples. Screening of collected samples using the direct method (e.g. microscopy), revealed the presence of bivalve larvae in all the sampling sites (averaging 308 larvae per m3of seawater processed per site) and of decapods in all sampling sites except for site 3 (ranging from one to seven individual larvae per site) (Table 4 ). Across all samples collected, genetic screening using the developed HT-qPCR panel (indirect method) resulted in 13 positive molecular markers detecting the presence of DNA from seven different species, with Mytilus edulis being detected in all five sites (Figure 4 ). Overall, number of larvae and estimated DNA copy numbers (i.e. cumulatively among all markers) showed similar patterns across the five sites for both bivalve and crustaceans, with sites 1, 2 and 5 showing significantly higher larvae/DNA than sites 3 and 4 (p-values varying from 0.0005***-0.05*, Tukey test)(Table 4). The copy numbers detected for Mytilus spp varied across the three genes used (nuclear PAPM, mitochondrial COI, and cytoplasmic 18S), but were consistent over sites with a nuclear: mitochondrial: cytoplasmic ratio of 1: 16: 78, showing potential to follow expected trends in terms of DNA copies per cell (i.e. 1 nucleus vs many organelles and mitochondria). Cerastoderma edule andaequipecten opercularis were detected in all sites except for Site 3. The copy numbers of these species varied over the different sites, with higher prevalence of A. opercularis in Sites 2 and 5, C. edule in Sites 1 and 2. Mya arenaria was only consistently present in all replicates of Site 1, but sporadically detected in Sites 3 and 4. Ensis magnus/ Phaxas pellucidus was detected in lower presence with most presence in Site 5. The only decapod DNA detected was Carcinus maenas in Site 5, which was the site with the highest decapod larvae count observed using the direct method (Table 4 ). The lack of decapods’ DNA detection in Sites 1, 2 and 4 (where decapod larvae were actually observed), implies that these results are either false negatives due to either being below the limit of detection or simply not present in the DNA samples due to very low numbers, and/or the decapod larvae in these samples are not species included in the current molecular HT-qPCR panel.