Drop it all: extraction-free detection of targeted marine species through optimized direct droplet digital PCR.

Molecular biomonitoring programs increasingly use environmental DNA (eDNA) for detecting targeted species such as marine non-indigenous species (NIS) or endangered species. However, the current molecular detection workflow is cumbersome and time-demanding, and thereby can hinder management efforts and restrict the "opportunity window" for rapid management responses. Here, we describe a direct droplet digital PCR (direct-ddPCR) approach to detect species-specific free-floating extra-cellular eDNA (free-eDNA) signals, i.e., detection of species-specific eDNA without the need for filtration or DNA extraction, with seawater samples. This first proof-of-concept aquarium study was conducted with three distinct marine species: 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 analysing aquarium marine water samples using an optimized species-specific ddPCR assay. The results demonstrated the consistent detection of S. spallanzanii and B. 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 h. Results indicate that organism biomass, specimen characteristics (e.g., stress and viability), and species-specific biological differences may influence free-eDNA detectability. This study represents the first step in assessing the feasibility of direct-ddPCR technology for the detection of marine species. Our results provide information that could aid in the development of new technology, such as a field development of ddPCR systems, which could allow for automated continuous monitoring of targeted marine species, enabling point-of-need detection and rapid management responses.

Harnessing environmental DNA to reveal biogeographical patterns of non-indigenous species for improved co-governance of the marine environment in Aotearoa New Zealand.

Aotearoa New Zealand's Northern region is a major gateway for the incursion and establishment of non-indigenous species (NIS) populations due to high numbers of recreational and commercial vessels. This region also holds a unique marine ecosystem, home to many taonga (treasured) species of cultural and economic importance. Regular surveillance, eradication plans and public information sharing are undertaken by local communities and governmental organizations to protect these ecosystems from the impact of NIS. Recently, considerable investments went into environmental DNA (eDNA) research, a promising approach for the early detection of NIS for complementing existing biosecurity systems. We applied eDNA metabarcoding for elucidating bioregional patterns of NIS distributions across a gradient from harbors (NIS hotspots) to open seas (spreading areas). Samples were collected during a research cruise sailing across three Aotearoa New Zealand harbors, Waitemata, Whangarei and Pewhairangi (Bay of Islands), and their adjacent coastal waters. The small-ribosomal subunit (18S rRNA) and mitochondrial cytochrome c oxidase I (COI) genes were screened using the online Pest Alert Tool for automated detection of putative NIS sequences. Using a probabilistic modelling approach, location-dependent occupancies of NIS were investigated and related to the current information on species distribution from biosecurity surveillance programs. This study was collaboratively designed with Maori partners to initiate a model of co-governance within the existing science system.

Pest Alert Tool-a web-based application for flagging species of concern in metabarcoding datasets.

Advances in high-throughput sequencing (HTS) technologies and their increasing affordability have fueled environmental DNA (eDNA) metabarcoding data generation from freshwater, marine and terrestrial ecosystems. Research institutions worldwide progressively employ HTS for biodiversity assessments, new species discovery and ecological trend monitoring. Moreover, even non-scientists can now collect an eDNA sample, send it to a specialized laboratory for analysis and receive in-depth biodiversity record from a sampling site. This offers unprecedented opportunities for biodiversity assessments across wide temporal and spatial scales. The large volume of data produced by metabarcoding also enables incidental detection of species of concern, including non-indigenous and pathogenic organisms. We introduce an online app-Pest Alert Tool-for screening nuclear small subunit 18S ribosomal RNA and mitochondrial cytochrome oxidase subunit I datasets for marine non-indigenous species as well as unwanted and notifiable marine organisms in New Zealand. The output can be filtered by minimum length of the query sequence and identity match. For putative matches, a phylogenetic tree can be generated through the National Center for Biotechnology Information's BLAST Tree View tool, allowing for additional verification of the species of concern detection. The Pest Alert Tool is publicly available at https://pest-alert-tool-prod.azurewebsites.net/.

Assessing the utility of marine filter feeders for environmental DNA (eDNA) biodiversity monitoring.

Aquatic environmental DNA (eDNA) surveys are transforming how marine ecosystems are monitored. The time-consuming preprocessing step of active filtration, however, remains a bottleneck. Hence, new approaches that eliminate the need for active filtration are required. Filter-feeding invertebrates have been proven to collect eDNA, but side-by-side comparative studies to investigate the similarity between aquatic and filter-feeder eDNA signals are essential. Here, we investigated the differences among four eDNA sources (water; bivalve gill-tissue; sponges; and ethanol in which filter-feeding organisms were stored) along a vertically stratified transect in Doubtful Sound, New Zealand using three metabarcoding primer sets targeting fish and vertebrates. Combined, eDNA sources detected 59 vertebrates, while concurrent diver surveys observed eight fish species. There were no significant differences in alpha and beta diversity between water and sponge eDNA and both sources were highly correlated. Vertebrate eDNA was successfully extracted from the ethanol in which sponges were stored, although a reduced number of species were detected. Bivalve gill-tissue dissections, on the other hand, failed to reliably detect eDNA. Overall, our results show that vertebrate eDNA signals obtained from water samples and marine sponges are highly concordant. The strong similarity in eDNA signals demonstrates the potential of marine sponges as an additional tool for eDNA-based marine biodiversity surveys, by enabling the incorporation of larger sample numbers in eDNA surveys, reducing plastic waste, simplifying sample collection, and as a cost-efficient alternative. However, we note the importance to not detrimentally impact marine communities by, for example, nonlethal subsampling, specimen cloning, or using bycatch specimens.

Net overboard: Comparing marine eDNA sampling methodologies at sea to unravel marine biodiversity.

Environmental DNA (eDNA) analyses are powerful for describing marine biodiversity but must be optimized for their effective use in routine monitoring. To maximize eDNA detection probabilities of sparsely distributed populations, water samples are usually concentrated from larger volumes and filtered using fine-pore membranes, often a significant cost-time bottleneck in the workflow. This study aimed to streamline eDNA sampling by investigating plankton net versus bucket sampling, direct versus sequential filtration including self-preserving filters. Biodiversity was assessed using metabarcoding of the small ribosomal subunit (18S rRNA) and mitochondrial cytochrome c oxidase I (COI) genes. Multispecies detection probabilities were estimated for each workflow using a probabilistic occupancy modelling approach. Significant workflow-related differences in biodiversity metrics were reported. Highest amplicon sequence variant (ASV) richness was attained by the bucket sampling combined with self-preserving filters, comprising a large portion of microplankton. Less diversity but more metazoan taxa were captured in the net samples combined with 5 µm pore size filters. Prefiltered 1.2 µm samples yielded few or no unique ASVs. The highest average (~32%) metazoan detection probabilities in the 5 µm pore size net samples confirmed the effectiveness of preconcentration plankton for biodiversity screening. These results contribute to streamlining eDNA sampling protocols for uptake and implementation in marine biodiversity research and surveillance.

crabs-A software program to generate curated reference databases for metabarcoding sequencing data.

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).

An Efficient Tetraplex Surveillance Tool for Salmonid Pathogens.

Fish disease surveillance methods can be complicated and time consuming, which limits their value for timely intervention strategies on aquaculture farms. Novel molecular-based assays using droplet digital Polymerase Chain Reaction (ddPCR) can produce immediate results and enable high sample throughput with the ability to multiplex several targets using different fluorescent dyes. A ddPCR tetraplex assay was developed for priority salmon diseases for farmers in New Zealand including New Zealand Rickettsia-like organism 1 (NZ-RLO1), NZ-RLO2, Tenacibaculum maritimum, and Yersinia ruckeri. The limit of detection in singleplex and tetraplex assays was reached for most targets at 10-9 ng/µl with, respectively, NZ-RLO1 = 0.931 and 0.14 copies/µl, NZ-RLO2 = 0.162 and 0.21 copies/µl, T. maritimum = 0.345 and 0.93 copies/µl, while the limit of detection for Y. ruckeri was 10-8 with 1.0 copies/µl and 0.7 copies/µl. While specificity of primers was demonstrated in previous studies, we detected cross-reactivity of T. maritimum with some strains of Tenacibaculum dicentrarchi and Y. ruckeri with Serratia liquefaciens, respectively. The tetraplex assay was applied as part of a commercial fish disease surveillance program in New Zealand for 1 year to demonstrate the applicability of tetraplex tools for the salmonid aquaculture industry.

Towards reproducible metabarcoding data: Lessons from an international cross-laboratory experiment.

Advances in high-throughput sequencing (HTS) are revolutionizing monitoring in marine environments by enabling rapid, accurate and holistic detection of species within complex biological samples. Research institutions worldwide increasingly employ HTS methods for biodiversity assessments. However, variance in laboratory procedures, analytical workflows and bioinformatic pipelines impede the transferability and comparability of results across research groups. An international experiment was conducted to assess the consistency of metabarcoding results derived from identical samples and primer sets using varying laboratory procedures. Homogenized biofouling samples collected from four coastal locations (Australia, Canada, New Zealand and the USA) were distributed to 12 independent laboratories. Participants were asked to follow one of two HTS library preparation workflows. While DNA extraction, primers and bioinformatic analyses were purposefully standardized to allow comparison, many other technical variables were allowed to vary among laboratories (amplification protocols, type of instrument used, etc.). Despite substantial variation observed in raw results, the primary signal in the data was consistent, with the samples grouping strongly by geographical origin for all data sets. Simple post hoc data clean-up by removing low-quality samples gave the best improvement in sample classification for nuclear 18S rRNA gene data, with an overall 92.81% correct group attribution. For mitochondrial COI gene data, the best classification result (95.58%) was achieved after correction for contamination errors. The identified critical methodological factors that introduced the greatest variability (preservation buffer, sample defrosting, template concentration, DNA polymerase, PCR enhancer) should be of great assistance in standardizing future biodiversity studies using metabarcoding.

Release and degradation of environmental DNA and RNA in a marine system.

Over the last decade, there has been growing interest in the analysis of environmental DNA (eDNA) to infer the presence of organisms in aquatic environments. The efficacy of eDNA/eRNA based tools are highly depend on the turnover rate of the molecule (their release and degradation). Environmental DNA has been shown to persist for days, weeks or years in environmental samples. Environmental RNA (eRNA) is thought to degrade faster than eDNA, however to our knowledge, no experimental studies have explored this. Here we present an aquarium study to investigate eDNA and eRNA shedding rates and degradation for two sessile marine invertebrates. The copy numbers for eDNA and eRNA were assessed using droplet digital PCR targeting the mitochondrial Cytochrome c Oxidase subunit 1 (COI) gene. Environmental RNA persisted after organism removal for much longer than expected with detections for up to 13 h. In contrast, eDNA was detected is samples collected up to 94 h after organism removal. There was no evidence that the decay rates constants for eDNA and eRNA were different (p = 0.6, Kruskal-Wallis tests). Both eDNA and eRNA was detected in biofilms collected at the end of the experiment (day 21). This suggests binding with organic or inorganic compounds or stabilization of these molecules in the biofilm matrix. The finding of the prolonged persistence of eRNA may provide new opportunities for improved biodiversity surveys through reducing false positives caused by legacy DNA and could also facilitate new research on environmental transcriptomics.

A comparison of droplet digital polymerase chain reaction (PCR), quantitative PCR and metabarcoding for species-specific detection in environmental DNA.

Targeted species-specific and community-wide molecular diagnostics tools are being used with increasing frequency to detect invasive or rare species. Few studies have compared the sensitivity and specificity of these approaches. In the present study environmental DNA from 90 filtered seawater and 120 biofouling samples was analyzed with quantitative PCR (qPCR), droplet digital PCR (ddPCR) and metabarcoding targeting the cytochrome c oxidase I (COI) and 18S rRNA genes for the Mediterranean fanworm Sabella spallanzanii. The qPCR analyses detected S. spallanzanii in 53% of water and 85% of biofouling samples. Using ddPCR S. spallanzanii was detected in 61% of water of water and 95% of biofouling samples. There were strong relationships between COI copy numbers determined via qPCR and ddPCR (water R2  = 0.81, p < .001, biofouling R2  = 0.68, p < .001); however, qPCR copy numbers were on average 125-fold lower than those measured using ddPCR. Using metabarcoding there was higher detection in water samples when targeting the COI (40%) compared to 18S rRNA (5.4%). The difference was less pronounced in biofouling samples (25% COI, 29% 18S rRNA). Occupancy modelling showed that although the occupancy estimate was higher for biofouling samples (ψ = 1.0), higher probabilities of detection were derived for water samples. Detection probabilities of ddPCR (1.0) and qPCR (0.93) were nearly double metabarcoding (0.57 to 0.27 marker dependent). Studies that aim to detect specific invasive or rare species in environmental samples should consider using targeted approaches until a detailed understanding of how community and matrix complexity, and primer biases affect metabarcoding data.

Considerations for incorporating real-time PCR assays into routine marine biosecurity surveillance programmes: a case study targeting the Mediterranean fanworm (Sabella spallanzanii) and club tunicate (Styela clava) 1.

Molecular techniques may provide effective tools to enhance marine biosecurity surveillance. Prior to routine implementation, evidence-based consideration of their benefits and limitations is needed. In this study, we assessed the efficiency and practicality of visual diver surveys and real-time PCR assays (targeting DNA and RNA) for detecting two marine invasive species whose infestation levels varied between species and location: Sabella spallanzanii and Styela clava. Filtered water samples (n = 171) were collected in parallel with dive surveys at two locations as part of the New Zealand Marine High Risk Site Surveillance programme: Nelson Harbour (27 sites) and Waitemata Harbour (30 sites). Diver surveys resulted in a greater number of detections compared to real-time PCR: S. clava - 21 versus 5 sites in Nelson, 6 versus 1 in Auckland; S. spallanzanii - 18 versus 10 in Auckland, no detections in Nelson. Occupancy modelling derived detection probabilities for the real-time PCR for S. clava were low (14%), compared to S. spallanzanii (66%). This could be related to abundances, or species-specific differences in DNA shedding. Only one RNA sample was positive, suggesting that most detections were from extracellular DNA or non-viable fragments. While molecular methods cannot yet replace visual observations, this study shows they provide useful complementary information.

The impact of artificial surfaces on marine bacterial and eukaryotic biofouling assemblages: A high-throughput sequencing analysis.

Vessel hulls and underwater infrastructure can be severely impacted by marine biofouling. Knowledge on which abiotic conditions of artificial structures influence bacterial and eukaryotic community composition is limited. In this study, settlement plates with differing surface texture, orientation and copper-based anti-fouling coatings were deployed in a marina. After three months, biofouling samples were collected and bacterial and eukaryotic communities characterised using DNA metabarcoding. The copper anti-fouling coating treatments incurred the most significant compositional changes (p ≤ 0.001) within both domains. Bacterial diversity decreased, with Gammaproteobacteria becoming the dominant phylum. In contrast, protist diversity increased as well as opportunist nematodes and bryozoans; urochordates and molluscs became less abundant. Network analyses displayed complex relationships on untreated plates, while revealing a simpler, but disturbed and unstable community composition on the anti-fouling coated plates. These networks of copper treatments displayed opportunist taxa that appeared as key organisms in structuring the bacterial and eukaryotic communities.