Evaluating detection limits of next-generation sequencing for the surveillance and monitoring of international marine pests

Most surveillance programmes for marine invasive species (MIS) require considerable taxonomic expertise, are laborious, and are unable to identify species at larval or juvenile stages. Therefore, marine pests may go undetected at the initial stages of incursions when population densities are low. In this study, we evaluated the ability of the benchtop GS Junior™ 454 pyrosequencing system to detect the presence of MIS in complex sample matrices. An initial in-silico evaluation of the mitochondrial cytochrome c oxidase subunit I (COI) and the nuclear small subunit ribosomal DNA (SSU) genes, found that multiple primer sets (targeting a ca. 400 base pair region) would be required to obtain species level identification within the COI gene. In contrast a single universal primer set was designed to target the V1–V3 region of SSU, allowing simultaneous PCR amplification of a wide taxonomic range of MIS. To evaluate the limits of detection of this method, artificial contrived communities (10 species from 5 taxonomic groups) were created using varying concentrations of known DNA samples and PCR products. Environmental samples (water and sediment) spiked with one or five 160 hr old Asterias amurensis larvae were also examined. Pyrosequencing was able to recover DNA/PCR products of individual species present at greater than 0.64% abundance from all tested contrived communities. Additionally, single A. amurensis larvae were detected from both water and sediment samples despite the co-occurrence of a large array of environmental eukaryotes, indicating an equivalent sensitivity to quantitative PCR. NGS technology has tremendous potential for the early detection of marine invasive species worldwide

A Substrate-Independent Benthic Sampler (SIBS) for Hard and Mixed-Bottom Marine Habitats: A Proof-of-Concept Study

Sea cage fish farms are increasingly situated over hard and mixed substrate habitats for production and waste-dispersion reasons; yet in many cases, these installations are not being effectively managed with respect to benthic impacts due to the lack of a practical sampling method. This study presents the first set of results from a newly developed Substrate Independent Benthic Sampler (SIBS) device that captures the unconsolidated organic and inorganic matter that overlies almost all substrates. The contents of the samples were analyzed using extracted environmental DNA (eDNA) followed by metabarcoding of the bacterial 16S rRNA gene. SIBS microbial assemblages reliably changed with proximity to farm and concurred with visual assessments of impact. Moreover, the approach appeared to be very sensitive with respect to the enrichment gradient, being able to discern influences at distances of 500–1500 m from the impact source. Other spatial differences, due to region and farm, were small in comparison, and the effect of the underlying substrate type was minor. The samples contained sufficient previously described bacterial bioindicator taxa from enriched sediments, such that a meaningful biotic index could be calculated, thereby placing them on a well-established benthic enrichment spectrum with established environmental thresholds. SIBS-derived bacterial data provide a powerful new approach for mapping spatial boundaries of farm effects irrespective of substrate type and topography. More importantly, the tool should also permit quantitative assessment of benthic enrichment levels irrespective of substrate type from depths of at least 100 m. It therefore has the potential to solve the hard-bottom problem that has until now prohibited effective environmental monitoring at mixed and hard-bottom locations.publishedVersio

Beyond taxonomy: Validating functional inference approaches in the context of fish-farm impact assessments

Characterization of microbial assemblages via environmental DNA metabarcoding is increasingly being used in routine monitoring programs due to its sensitivity and cost-effectiveness. Several programs have recently been developed which infer functional profiles from 16S rRNA gene data using hidden-state prediction (HSP) algorithms. These might offer an economic and scalable alternative to shotgun metagenomics. To date, HSP-based methods have seen limited use for benthic marine surveys and their performance in these environments remains unevaluated. In this study, 16S rRNA metabarcoding was applied to sediment samples collected at 0 and ≥1,200 m from Norwegian salmon farms, and three metabolic inference approaches (Paprica, Picrust2 and Tax4Fun2) evaluated against metagenomics and environmental data. While metabarcoding and metagenomics recovered a comparable functional diversity, the taxonomic composition differed between approaches, with genera richness up to 20× higher for metabarcoding. Comparisons between the sensitivity (highest true positive rates) and specificity (lowest true negative rates) of HSP-based programs in detecting functions found in metagenomic data ranged from 0.52 and 0.60 to 0.76 and 0.79, respectively. However, little correlation was observed between the relative abundance of their specific functions. Functional beta-diversity of HSP-based data was strongly associated with that of metagenomics (r ≥ 0.86 for Paprica and Tax4Fun2) and responded similarly to the impact of fish farm activities. Our results demonstrate that although HSP-based metabarcoding approaches provide a slightly different functional profile than metagenomics, partly due to recovering a distinct community, they represent a cost-effective and valuable tool for characterizing and assessing the effects of fish farming on benthic ecosystems.publishedVersio

Unique quantitative Symbiodiniaceae signature of coral colonies revealed through spatio-temporal survey in Moorea

One of the mechanisms of rapid adaptation or acclimatization to environmental changes in corals is through the dynamics of the composition of their associated endosymbiotic Symbiodiniaceae community. The various species of these dinoflagellates are characterized by different biological properties, some of which can confer stress tolerance to the coral host. Compelling evidence indicates that the corals' Symbiodiniaceae community can change via shuffling and/or switching but the ecological relevance and the governance of these processes remain elusive. Using a qPCR approach to follow the dynamics of Symbiodiniaceae genera in tagged colonies of three coral species over a 10-18 month period, we detected putative genus-level switching of algal symbionts, with coral species-specific rates of occurrence. However, the dynamics of the corals' Symbiodiniaceae community composition was not driven by environmental parameters. On the contrary, putative shuffling event were observed in two coral species during anomalous seawater temperatures and nutrient concentrations. Most notably, our results reveal that a suit of permanent Symbiodiniaceae genera is maintained in each colony in a specific range of quantities, giving a unique 'Symbiodiniaceae signature' to the host. This individual signature, together with sporadic symbiont switching may account for the intra-specific differences in resistance and resilience observed during environmental anomalies

Advantages and limitations of environmental DNA/RNA tools for marine biosecurity: Management and surveillance of non-indigenous species

To enable successful management of marine bioinvasions, timely and robust scientific advice is required. This knowledge should inform managers and stakeholders on the magnitude of a pressure (rate of human-mediated introductions), the environmental state of an ecosystem (impacts of non-indigenous species), and the success of management response (prevention, eradication, mitigation). This advice often relies on baseline biodiversity information in the form of measureable parameters (metrics). This can be derived from conventional approaches such as visual surveys, but also by utilizing environmental DNA/RNA-based molecular techniques, which are increasingly being touted as promising tools for assessing biodiversity and detecting rare or invasive species. Depending on the stage of incursion, each approach has merits and limitations. In this review we assess the performance of biosecurity-relevant biodiversity parameters derived from eDNA/eRNA samples and discuss the results in relation to different stages of invasion and management applications. The overall performance of considered methods ranged between 42 and 90% based on defined criteria, with target-specific approaches scoring higher for respective biosecurity applications, followed by eDNA metabarcoding. Caveats are discussed along with avenues which may enhance these techniques and their successful uptake for marine biosecurity surveillance and management. To facilitate and encourage uptake of these techniques, there is a need for an international collaborative framework aimed at unifying molecular sampling and analysis methodologies. Improvement of quantitative capacity and cost-efficiency will also enhance their integration in biosecurity programmes

Advantages and Limitations of Environmental DNA/RNA Tools for Marine Biosecurity: Management and Surveillance of Non-indigenous Species

To enable successful management of marine bioinvasions, timely and robust scientific advice is required. This knowledge should inform managers and stakeholders on the magnitude of a pressure (rate of human-mediated introductions), the environmental state of an ecosystem (impacts of non-indigenous species), and the success of management response (prevention, eradication, mitigation). This advice often relies on baseline biodiversity information in the form of measureable parameters (metrics). This can be derived from conventional approaches such as visual surveys, but also by utilizing environmental DNA/RNA-based molecular techniques, which are increasingly being touted as promising tools for assessing biodiversity and detecting rare or invasive species. Depending on the stage of incursion, each approach has merits and limitations. In this review we assess the performance of biosecurity-relevant biodiversity parameters derived from eDNA/eRNA samples and discuss the results in relation to different stages of invasion and management applications. The overall performance of considered methods ranged between 42 and 90% based on defined criteria, with target-specific approaches scoring higher for respective biosecurity applications, followed by eDNA metabarcoding. Caveats are discussed along with avenues which may enhance these techniques and their successful uptake for marine biosecurity surveillance and management. To facilitate and encourage uptake of these techniques, there is a need for an international collaborative framework aimed at unifying molecular sampling and analysis methodologies. Improvement of quantitative capacity and cost-efficiency will also enhance their integration in biosecurity programmes

Metabarcoding as a tool to enhance marine surveillance of nonindigenous species in tropical harbors: A case study in Tahiti

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.publishedVersio

Assessing the performance and efficiency of environmental DNA/RNA capture methodologies under controlled experimental conditions

Growing interest and affordability of environmental DNA and RNA (eDNA and eRNA) approaches for biodiversity assessments and monitoring of complex ecosystems have led to the emergence of manifold protocols for nucleic acids (NAs) isolation and processing. Although there is no consensus on a standardized workflow, the common practice for water samples is to concentrate NAs via filtration using varying pore size membranes. Using the smallest pore is assumed to be most efficient for NAs capture from a wide range of material (including sub-cellular particles); however, a trade-off must occur between detection of a meaningful molecular signal and cost/time effort when processing samples using fine pore membranes. Comparative studies involving formal efficiency assessments are lacking, which restricts informed decision-making around an optimized sampling approach for applications such as biosurveillance (i.e. detection and monitoring of target taxa—nuisance organisms, endangered and indicator taxa or other species of economic or cultural importance). Here, we present an experimental study using an easily cultured microalgal species (Alexandrium pacificum) to test different filter membranes for capturing NAs in the context of cost/time effort and cell fractions encountered in nature (whole cells, partially lysed and naked NAs). The results showed no statistically significant difference between membrane types for capturing target eDNA signal from intact and partially lysed cell treatments. In terms of time effort and volume processed, higher efficiency ratings were obtained with the larger pore size (5 μm) cellulose membranes. Positively charged nylon demonstrated enhanced capture of naked NAs, and especially eRNA signal, across treatments. Our findings support using coarse pore size filters for adequate capture of target NA signal (from both eDNA and eRNA) with less processing time. The framework presented here can provide a quick and robust feasibility check and comparative assessment of new and existing NA processing technologies, and allows sufficient control over multiple parameters, including physical–chemical water properties, temporal scales, and concentration and type of input material

Global Trends of Benthic Bacterial Diversity and Community Composition Along Organic Enrichment Gradients of Salmon Farms

The analysis of benthic bacterial community structure has emerged as a powerful alternative to traditional microscopy-based taxonomic approaches to monitor aquaculture disturbance in coastal environments. However, local bacterial diversity and community composition vary with season, biogeographic region, hydrology, sediment texture, and aquafarm-specific parameters. Therefore, without an understanding of the inherent variation contained within community complexes, bacterial diversity surveys conducted at individual farms, countries, or specific seasons may not be able to infer global universal pictures of bacterial community diversity and composition at different degrees of aquaculture disturbance. We have analyzed environmental DNA (eDNA) metabarcodes (V3–V4 region of the hypervariable SSU rRNA gene) of 138 samples of different farms located in different major salmon-producing countries. For these samples, we identified universal bacterial core taxa that indicate high, moderate, and low aquaculture impact, regardless of sampling season, sampled country, seafloor substrate type, or local farming and environmental conditions. We also discuss bacterial taxon groups that are specific for individual local conditions. We then link the metabolic properties of the identified bacterial taxon groups to benthic processes, which provides a better understanding of universal benthic ecosystem function(ing) of coastal aquaculture sites. Our results may further guide the continuing development of a practical and generic bacterial eDNA-based environmental monitoring approach.publishedVersio

Wanted dead or alive? Using metabarcoding of environmental DNA and RNA to distinguish living assemblages for biosecurity applications

High-throughput sequencing metabarcoding studies in marine biosecurity have largely focused on targeting environmental DNA (eDNA). DNA can persist extracellularly in the environment, making discrimination of living organisms difficult. In this study, bilge water samples (i.e., water accumulating on-board a vessel during transit) were collected from 15 small recreational and commercial vessels. eDNA and eRNA molecules were co-extracted and the V4 region of the 18S ribosomal RNA gene targeted for metabarcoding. In total, 62.7% of the Operational Taxonomic Units (OTUs) were identified at least once in the corresponding eDNA and eRNA reads, with 19.5% unique to eDNA and 17.7% to eRNA. There were substantial differences in diversity between molecular compartments; 57% of sequences from eDNA-only OTUs belonged to fungi, likely originating from legacy DNA. In contrast, there was a higher percentage of metazoan (50.2%) and ciliate (31.7%) sequences in the eRNA-only OTUs. Our data suggest that the presence of eRNA-only OTUs could be due to increased cellular activities of some rare taxa that were not identified in the eDNA datasets, unusually high numbers of rRNA transcripts in ciliates, and/or artefacts produced during the reverse transcriptase, PCR and sequencing steps. The proportions of eDNA/eRNA shared and unshared OTUs were highly heterogeneous within individual bilge water samples. Multiple factors including boat type and the activities performed on-board, such as washing of scientific equipment, may play a major role in contributing to this variability. For some marine biosecurity applications analysis, eDNA-only data may be sufficient, however there are an increasing number of instances where distinguishing the living portion of a community is essential. For these circumstances, we suggest only including OTUs that are present in both eDNA and eRNA data. OTUs found only in the eRNA data need to be interpreted with caution until further research provides conclusive evidence for their origin