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.