The field of eDNA is growing exponentially in response to the need for detecting rare and invasive species for management and conservation decisions. Developing technologies and standard protocols within the biosecurity sector must address myriad challenges associated with marine environments, including salinity, temperature, advective and deposition processes, hydrochemistry and pH, and contaminating agents. These approaches must also provide a robust framework that meets the need for biosecurity management decisions regarding threats to human health, environmental resources, and economic interests, especially in areas with limited clean-laboratory resources and experienced personnel. This contribution aims to facilitate dialogue and innovation within this sector by reviewing current approaches for sample collection, post-sampling capture and concentration of eDNA, preservation, and extraction, all through a biosecurity monitoring lens.

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 have 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, inclusion/exclusion filter), export the reference database in multiple formats for immediate use in taxonomy assignment software, and investigate the reference database through implemented visualizations for diversity, primer efficiency, reference sequence length, database completeness 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 can be installed via docker and is available for download as a conda package and via GitHub (https://github.com/gjeunen/reference_database_creator).

Marine nonindigenous species (NIS) are spreading at an alarming rate internationally through anthropogenic activities such as shipping and aquaculture, affecting local biodiversity and negatively impacting the ecosystem and human well‐being. Countries and international organizations have recognized this global threat and have begun implementing biosecurity management programs to ensure early detection, effective surveillance, and mitigation of marine NIS spread. Molecular techniques based on environmental DNA and RNA (eDNA/eRNA), collectively referred to as environmental nucleic acids (eNAs), have become a popular noninvasive tool for detecting NIS and monitoring biodiversity locally and globally. However, uncertainties about eNAs detection probabilities and the location of the source population impede the broad uptake of this tool in marine biosecurity programs. It's been hypothesized that most of these uncertainties can be explained by studying the molecules' dynamics within a marine environment and implementing eNAs distribution models. To contribute to further knowledge development in this area, our study reviews data from 20 recent reports on the degradation mechanisms and fate of eNAs in the marine environment. We classified the critical factors influencing eNAs' persistence that should be considered by biosecurity practitioners, outlining the complex interaction between the molecules' degradation processes and particular environmental conditions. To help guide the parameterization of eNAs distribution models, this review also summarizes and standardizes the marine decay rates of eDNA/eRNA from the literature. Finally, this manuscript outlines guidelines to help calculate accurate decay rates to build appropriate “fit‐for‐purpose” marine biosecurity tools for improved target detectability and greater resolution in assessing biodiversity.

Environmental DNA (eDNA) metabarcoding has shown great promise as an effective, non‐invasive monitoring method for marine biomes. However, long filtration times and the need for state‐of‐the‐art laboratories are restricting sample replication and in situ species detections. Methodological innovations, such as passive filtration and self‐contained DNA extraction protocols, have the potential to alleviate these issues. We explored the implementation of passive sampling and a self‐contained DNA extraction protocol by comparing fish diversity obtained from active filtration (1 L; 0.45 μm cellulose nitrate [CN] filters) to five passive substrates, including 0.45 μm CN filters, 5 μm nylon filters, 0.45 μm positively charged nylon filters, artificial sponges, and fishing net. Fish diversity was then compared between the PDQeX Nucleic Acid Extractor and the conventional Qiagen DNeasy Blood & Tissue protocol. Experiments were conducted in both a controlled mesocosm and in situ at the Portobello Marine Laboratory, New Zealand. No significant differences in fish diversity were observed among active filtration and more porous passive materials (artificial sponges and fishing net) for both the mesocosm and harbor waters. For the in situ comparison, all passive filter membranes detected a significantly lower number of fish species, resulting from partial sample drop‐out. While no significant differences in fish eDNA signal diversity were observed between either DNA extraction methods in the mesocosm, the PDQeX system was less effective at detecting fish for the in situ comparison. Our results demonstrate that a passive sampling approach using porous substrates can be effectively implemented to capture eDNA from seawater, eliminating vacuum filtration processing. The large variation in efficiency observed among the five substrate types, however, warrants further optimization of the passive sampling approach for routine eDNA applications. The PDQeX system can extract high‐abundance DNA in a mesocosm with further optimization to detect low‐abundance eDNA from the marine environment.

Globalization has increased connectivity between countries enhancing the spread of marine nonindigenous species (NIS). The establishment of marine NIS shows substantial negative effects on the structure and functioning of the natural ecosystems by competing for habitats and resources. Ports are often hubs for the spread of NIS via commercial and recreational vessels. Prevention, detection, and mitigation efforts are required to avoid and manage the establishment of NIS in new ecosystems. In this study, metabarcoding approaches targeting the nuclear small‐subunit ribosomal RNA (18S rRNA) gene and mitochondrial cytochrome c oxidase I (COI) gene were used to investigate planktonic and sessile (i.e., biofouling) communities and NIS at four locations in Tahiti, including two marinas and one port with varying anthropogenic impacts, and a relatively pristine site (Manava) used as a control. ASV richness values showed significant differences (18S rRNA gene: p = .023; COI: p < .001) between locations in the plankton samples, with the control site (low impact) having the highest diversity for both genes. ASV richness was also significantly different among locations for the biofouling samples in the COI dataset (p = .002). Community composition differed between locations with spatial patterns appearing stronger for the plankton samples compared with the biofouling samples. Detection of NIS based on selected lists of globally invasive species revealed a wide diversity of potentially invasive taxa especially in the more anthropogenically impacted regions. The use of a multigene approach improved the detection of NIS. This study demonstrates the utility of using a metabarcoding approach to routinely monitor areas most at risk from NIS establishment in Tahiti and other coastal nations. These coastal nations are vulnerable to shipping‐mediated incursions, and baseline information is required for both native diversity and nonindigenous diversity.