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.