2.1. PHASE 1 - DEFINITION OF TARGET TAXA AND ESTABLISHMENT OF REFERENCE DATABASES
The first step was to identify the main purpose of the toolkit and the research questions being addressed. In the case of this study, the goal was to develop a screening tool capable of determining the presence of bivalve and crustacean larvae in zooplankton samples, with particular interest in the Irish shellfish fisheries and aquaculture industry. Thus, a total of 24 species were chosen based on their ecological and economic importance in the context of Irish coastal waters, including seventeen bivalve species and decapod species (Table 1 ). To identify the most suitable genes for molecular species identification, a literature review was conducted for previously published studies including genetic markers designed for taxonomic resolution of selected taxa. In order to identify the final target and off-target species occurring in the geographic region of interest (Irish marine coastal waters), a review of organisms of interest and other closely related species (i.e. same Order) was carried out by consulting the World Register of Marine Species (WoRMS) and Ireland’s National Biodiversity Data Centre databases (August 2022).
A dedicated reference DNA database was created by combining publicly available barcode sequences with new data from locally sourced organisms. Firstly, based on results obtained from the above review of target and off-target species, previously published DNA sequence data of selected genes from all relevant taxa was obtained from the Barcode Of Life Database (BOLD) and GenBank database. Secondly, to fill any data gaps and confirm target DNA sequences of local taxa, DNA barcode sequence data was also obtained from taxonomically identified voucher specimens (e.g. adult individuals obtained from local markets or opportunistically). For this latter step, DNA was isolated from 50 mg of muscle tissue using the Blood and Tissue DNEasy kit (Qiagen), and 658 bp fragments of the COI gene were amplified and sequenced following the protocol described in . Finally, the resulting dedicated reference DNA database was organized in relevant gene-specific alignments created using the mafft alignment plugin as implemented in Geneious Prime 2020.2.1 (Biomatters Ltd).