Molecular biosecurity surveillance programs increasingly use environmental DNA (eDNA) for detecting marine non-indigenous species (NIS). However, the current molecular detection workflow is cumbersome, prone to errors and delays, and is limited in providing knowledge about eDNA beyond the spatial and temporal extent of the sampling. These limitations can hinder management efforts and restrict the “opportunity window” for a rapid response to new marine NIS incursions. Emerging innovative field-deployable digital droplet PCR (ddPCR) systems offer improved workflow efficiency by autonomously analyzing targeted free-floating extra-cellular eDNA (free-eDNA) signals. Despite their potential, these systems have not been tested in marine environments. Thus, an aquarium study was conducted with three distinct marine NIS: the Mediterranean fanworm Sabella spallanzanii, the ascidian clubbed tunicate Styela clava, and the brown bryozoan Bugula neritina to evaluate the detectability of free-eDNA in seawater. The detectability of targeted free-eDNA was assessed by directly analyzing aquarium water samples using an optimized species-specific ddPCR assay, without filtration or DNA extraction, so-called, “direct-ddPCR”. The results demonstrated the consistent detection of Sabella spallanzanii and Bugula neritina free-eDNA when these organisms were present in high abundance. Once organisms were removed, the free-eDNA signal exponentially declined, noting that free-eDNA persisted between 24-72 hours. Results indicate that organism biomass, specimen characteristics (e.g., stress and viability), and species-specific biological differences may influence free-eDNA detectability. These results are critical for implementing in-situ nucleic acid automated continuous sensing systems for marine biosurveillance, enabling point-of-need detection and rapid management response to biosecurity threats.

The measurement of biodiversity is an integral aspect of life science research. With the establishment of second- and third-generation sequencing technologies, an increasing amount of metabarcoding data is being generated as we seek to describe the extent and patterns of biodiversity in multiple contexts. The reliability and accuracy of taxonomically assigning metabarcoding sequencing data has been shown to be critically influenced by the quality and completeness of reference databases. Custom, curated, eukaryotic reference databases, however, are scarce, as are the software programs for generating them. Here, we present CRABS (Creating Reference databases for Amplicon-Based Sequencing), a software package to create custom reference databases for metabarcoding studies. CRABS includes tools to download sequences from multiple online repositories (i.e., NCBI, BOLD, EMBL, MitoFish), retrieve amplicon regions through in silico PCR analysis and pairwise global alignments, curate the database through multiple filtering parameters (e.g., dereplication, sequence length, sequence quality, unresolved taxonomy), export the reference database in multiple formats for the immediate use in taxonomy assignment software, and investigate the reference database through implemented visualizations for diversity, primer efficiency, reference sequence length, and taxonomic resolution. CRABS is a versatile tool for generating curated reference databases of user-specified genetic markers to aid taxonomy assignment from metabarcoding sequencing data. CRABS is available for download as a conda package and via GitHub (https://github.com/gjeunen/reference_database_creator).

Aquatic environmental DNA (eDNA) surveys have emerged as an alternative method for monitoring complex and vast marine ecosystems. One-to-one comparisons between existing survey techniques and eDNA approaches are essential to determine biases associated with this novel methodology. To date, such direct comparative studies have been scarce in the context of marine eDNA surveys. In this study, we conducted simultaneous baited remote underwater video (BRUV) and eDNA surveys to describe the fish community in Paterson Inlet, Stewart Island/Rakiura, New Zealand. BRUV detected three distinct families of bony fish (Actinopterygii) and four families of cartilaginous fish (Chondrichthyes). Three different eDNA assays, detected 32 (MiFish-U), 42 (MiFish-E), and 23 (16S-Fish) families, spanning the classes of Actinopterygii, Chondrichthyes, Hyperoartia, Mammalia, and Aves. Our direct comparison identified the need for (i) increased sampling, (ii) spatial pooling, and (iii) multiple targeted eDNA assays, to achieve similar detection rates of a given species in eDNA and BRUV monitoring. Diversity, ordination, and indicator species analyses identified distinct eDNA signals between different habitats in our relatively small sampling area, showcasing the high spatial resolution of eDNA approaches in marine habitats. Our results provide valuable insights into the potential biases associated with eDNA monitoring, as well as highlight the power of eDNA for detecting a broad range of taxa beyond traditional observational approaches, including terrestrial, invasive and migratory organisms.