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.