1| INTRODUCTION
Recent advances in molecular
technology have boosted the biomonitoring capabilities of DNA-based
approaches, particularly in aquatic environments . Such approaches rely
on the detection and quantification of DNA copies in an environmental
sample, which is used as a method for determining the presence and in
some cases the relative abundance or biomass of target species. Since
DNA can be present in an environmental sample as both organismal and/or
extra-organismal DNA , DNA-based approaches are capable of targeting a
wide range of taxa, ranging from micro- to macro-organism (e.g. ,
encompassing virtually all life-stages from larvae to adults (e.g. .
Even though many challenges still exist , the integration of molecular
approaches to traditional monitoring techniques such as direct capture,
offers an unprecedented opportunity to reduce the cost and increase the
throughput of biomonitoring. The development and application of powerful
and time/cost-effective biomonitoring tools would benefit many areas of
research and applied science, which are usually reliant on
labour-intensive screening methods such as microscopic inspection by
expert taxonomists .
Generally, DNA-based species detection approaches fall under two main
categories: (i) multi-taxa detection, which uses “universal” markers
combined with High Throughput Sequencing (HTS) (also known as
metabarcoding) and (ii) targeted detection, which typically uses
species-specific quantitative real-time PCR (qPCR) assays . Approaches
from both categories rely on the availability of robust reference
databases such as curated repositories of DNA data from taxonomically
identified specimens, ; however while HTS-based methods are more readily
available, though require substantial downstream analytical steps,
qPCR-based methods require an extensive initial validation phase (i.e.
use reference data for assay design), though once established they can
be applied rapidly and cost-effectively. The effectiveness of different
methods has been compared both in terms of detectability, as well as
biomass of target organisms (, with species-specific qPCR methods
showing promising results.
The initial validation phase required to deem an eDNA qPCR assay
reliable can be laborious and demanding in terms of cost and time ,
however recent advances in real-time qPCR instrumentation have received
a boost in integration and throughput capability thanks to the use of
microfluidic and nanotechnologies . This includes platforms that can
enable qPCR sample/assay Medium- to High-Throughput . Thus,
High-Throughout qPCR (HT-qPCR) instruments can play a significant role
in speeding up the validation phase of eDNA qPCR assays as well as
co-screening samples for multiple targets simultaneously.
For the present study, we chose the monitoring of spawning and larval
occurrence/distribution of Irish shellfish species of ecological and
commercial importance as a case study. The Irish shellfish industry
produces approximately 29,000 tonnes with an estimated value in excess
of \euro71 million per annum. The production mainly consists of
mussels (Mytilus spp ) (\euro17 million, 17.100 tonnes) and
oysters (Magallana gigas, Ostrea edulis ) (\euro51 million,
11.000 tonnes). Furthermore, many smaller proportions of clam, crab, and
lobster species are harvested all around the country (\euro3 million,
900 tonnes) (BIM, 2022). These fisheries are, like in my countries, of
national importance as they provide employment in rural maritime
communities where employment is especially required to allow people to
work and live in these areas. The production of many shellfish species
relies on natural processes of recruitment (i.e., larvae from wild
stocks), which can be difficult to predict due to seasonal and
interannual environmental variation. Furthermore, shellfish larvae
(particularly bivalve species) can be difficult to taxonomically
identify during early developmental stages . DNA-based approaches have
the potential to overcome such limitations hence improving the
biomonitoring capability of screening protocols, as demonstrated by an
increasing number of targeted single taxon qPCR studies, including for
oysters (Magallana
gigas, Ostrea edulis ) , mussels (Mytilus spp ) , cockles
(Cerastoderma edule ) , king scallop (Pecten maximus ) ,
razor clams (Ensis spp. ) , common clams (Mya
arenaria ) , and crabs (Necora puber, Cancer pagurus, Carcinas
maenas) . However, to the best of our knowledge, except for a recent
study targeting fish species , High-Throughput multi-species
biomonitoring efforts in aquatic ecosystems are limited to using
metabarcoding approaches , which often are non-specific and are
characterized by uncertain quantitative capability .
Thus, the aim of this study
was to use an HT-qPCR platform to describe the development and
validation of a multi-species biomonitoring tool to aid the screening of
shellfish larvae of ecologic and economic importance in Irish waters.