Validation of the molecular toolkit using zooplankton samples
This study showed good correlation between counts of decapod and bivalve larvae using microscopy (direct method) and strength of genetic signal (indirect method), with the latter providing greater taxonomic resolution power to the species level. Even though previous studies successfully found a similar correlation, it is unclear how the size of larvae and different genetic markers affect signal strength . In this study, the different assays in genes show different results in COI, 18S and nuclear genes, meaning that gene selection is of great importance.
The samples collected for this study were carried out using a 100µm mesh plankton net. Even though such mesh size is not expected to trap smaller fractions of DNA that may be present in the environment such as gametes or other cells released by adults, there was a risk that some target DNA present in a given sample did not belong to larvae (e.g. eDNA adsorbed to larger particles). While this aspect can be challenging to elucidate, the variation of DNA signal in multiple field replicates (n = 5) and the relative proximity of the locations sampled suggested that DNA of target organisms was not ubiquitous and when detected showed differential strength. While discerning fractions of eDNA in plankton samples is beyond the scope of this study, these results emphasize the importance of field replication as well as the usefulness of providing semi quantitative data (e.g. beyond presence-absence. The possibility to incorporate multiple markers/genes targeting the same taxon (species or genus) makes HT-qPCR systems attractive, as this can prove useful in resolving co-occurring closely related species and possibly provide more reliable estimates of DNA copy numbers and hence relative abundance or biomass. For instance, Mytilus spp and Ensis spp complexes consist of very closely related species in which hybridization might occur. However, single marker approaches, as developed by Inoue et al. (1995) only provide an indication of a genotype but lack the resolution power to clearly distinguish between species. Another benefit of HT-qPCR tool is that some markers can rule out possible false positives. For example, C. pagurus and E. siliqua are species that cannot be quantified and due to a risk of false positives, including markers for P. pellucidus , P. exiguum , L. depurator could potentially make the panel fully reliable for the species tested.
Finally, this toolkit proved useful in screening plankton samples for the DNA of commercially important species and will be useful in the future for better understanding spawning activities in support of local aquaculture shellfish industry.