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Four species support recreational and commercial fisheries along the U.S. Atlantic Ocean and the Gulf of Mexico, with the Gulf of Mexico stock being overfished for over three decades. The study presented here is part of a fisheries-independent project initiated to determine an absolute abundance of , to expand biological knowledge of the species and to develop novel tools for fisheries management. Environmental DNA (eDNA) tools aimed at the detection and quantification of target species are starting to emerge in support of marine fisheries surveys. Key to progressing the field is Droplet Digital™ PCR (ddPCR™), a highly sensitive technique with advanced multiplexing and direct quantification capabilities that can provide fisheries scientists with improved interpretation of eDNA data. We developed and validated a novel tetraplex ddPCR™ assay able to detect and distinguish between , , , and from seawater eDNA samples. In order to groundtruth ddPCR™ data, and explore its capacity to provide abundance estimates, we compared ddPCR™ detections and quantifications to abundance data inferred from multiple camera (ROV, S-BRUV, chevron trap) and acoustic (VPS array) gears deployed during a fisheries research gear-calibration cruise. We demonstrated that with eDNA contamination controls and best practice protocols, it is viable to conduct eDNA research as part of a fisheries survey cruise. eDNA sampling was completed in less time than camera gears (15 min 2 h). Both eDNA and camera gears detected the presence of and at both sites and all sampling days, but not and . eDNA concentration data was higher for than at both sites for all sampling days, in line with abundance patterns obtained from camera gears. The highest correlation (r = 0.97) was obtained between the measures of eDNA between gear deployments and ROV. Incorporating eDNA in fisheries surveys would not require additional days at sea and could improve precision in fish detection and abundance. eDNA can be a valuable complement to camera gears deployed in geographic areas or seasons with poor visibility conditions, where fish may be present but cannot be confidently identified to the species level. The high correlation obtained between ROV and eDNA data collected between gear deployments adds to a growing number of studies demonstrating the potential of eDNA as an indicator of abundance for fisheries stock assessments. Time-series data from a carefully designed eDNA survey, that estimates relative abundance, could be used as an index of relative abundance for the stock assessment. To achieve this, investment into follow-up studies with increased sample sizes and spatial and temporal replication would be necessary to allow for year-to-year comparisons and validate the robustness of the correlation observed.

Insight into a species’ native and introduced range is essential in understanding the invasion process. Genetic diversity, propagule pressure and environmental conditions all have been recognised as playing a determinant role in invasion success. Here, we aimed to investigate the genetic diversity and population genetic structure (using the COI mtDNA gene region and 22 nDNA microsatellite markers) of the Asian green mussel Perna viridis within its potential native range in Asia and at introduced locations in the USA and the Caribbean. We also analyse genetic data from vessel intercepts and an incursion. By doing so, we aimed to identify genetic signatures that could allow to track vessel samples to their source and provide further insight into potential high-risk invasive populations or areas. Three top hierarchical clusters were identified using the individual-based Bayesian clustering method in STRUCTURE, corresponding to populations in three world regions: (1) USA and Caribbean, (2) India and (3) Southeast Asia. Within Southeast Asia, additional analysis indicate a shallow genetic differentiation of three subgroups consisting of (3a) Thailand, (3b) Taiwan and Hong-Kong, and (3c) a cluster of Singapore–Indonesia samples. Overall, the population structure found in this study suggests that the markers used could be useful in identifying source populations, particularly between the three mains world regions. Most surprisingly however, this study shows that the genetic diversity of samples collected from vessel intercepts and incursions did not differ significantly from established populations in Southeast Asia. In this region, in addition to the high vessel connectivity and number of P. viridis transported, all sampled populations are likely to pose a comparable risk in terms of genetic diversity. The present work represents the most comprehensive population genetic study of P. viridis, and the first to address the potential genetic introduction risk posed by populations of this species. The information and genetic markers in this study constitute a valuable addition to the tools already used to infer on potential high-risk source populations of P. viridis. They should therefore prove useful for biosecurity surveillance and management actions directed at this species.

Hybridization and genetic introgression following the introduction of exotic species can pose a significant threat to the survival of geographically restricted species. A remnant population of the critically endangered freshwater crayfish Cherax tenuimanus in the upper reaches of the Margaret River in southwestern Australia is under threat following the introduction and spread of its congener Cherax cainii. Here, we examine the extent of hybridization and introgression between the two species using twelve polymorphic microsatellite loci. Our study reveals there are three times more C. cainii than C. tenuimanus at our study site in the upper reaches of the Margaret River. There is also evidence of hybridization and introgression between C. tenuimanus and C. cainii at this site, with F1, F2 and backcrossed individuals identified. While interbreeding was confirmed in this study, our simulations suggest that the levels of introgression are much lower than would be expected under random mating, indicating partial reproductive barriers exist. Nevertheless, it is apparent that hybridization and introgression with C. cainii pose a serious threat to C. tenuimanus and their survival in the wild will require active adaptive management and continued genetic monitoring to evaluate management effectiveness.

Despite being a well-recognized issue, the introduction of non-native marine species is an underestimated topic in highly biodiverse developing countries. The Indonesian archipelago sits at the center of the Coral Triangle, extending over several biogeographic regions with unique benthic communities, where the extent of non-native species presence is largely unknown. It is also at the center of maritime traffic between Australasia and Asia, and therefore at risk of introduction of species that are carried on the hulls and ballast water of vessels. In the present study, we aimed to raise awareness on marine biological invasions across the Indonesian archipelago by fostering discussions among scientists, educating science students, generating media articles for the public and actively involving island communities. We also aimed to test the suitability of a range of methods commonly used in bioinvasion research (settlement arrays, DNA barcoding and environmental DNA metabarcoding, vessel surveys) to establish a baseline for biofouling species and potential threats, while developing much needed capacity for monitoring and application to other areas. We recorded 66 different non-coral specimens in the fouling communities of a remote group of islands of the Indonesian archipelago, represented mainly by the taxa Porifera, Tunicata and Mollusca, which are known to include notorious invasive species. None of the identified species was known to be invasive in the region but several were cryptogenic and/or were considered to have a very broad global distribution range that can potentially include part, or all, of the Indonesian archipelago. One species, the ascidian Didemnum molle, was found to quickly settle and spread on available blank substrates. While settlement plates (monitored every 5 months) proved suitable for early detection of potential incursions of these organisms, benthic surveys, DNA barcoding and eDNA metabarcoding provide valuable complementary baseline biodiversity information. A combination of sampling methods is therefore recommended for similar studies in understudied high biodiversity areas. Results from vessel surveys highlights the importance of civil society education in helping prevent bioinvasions. This study represents the first marine biological invasions baseline, awareness and capacity development training carried in the Indonesian archipelago. The lessons taken from the variety of methods explored in a simultaneously scientific and educational setting should prove useful and motivate similar work in other areas of the world.

The colonial tunicate Didemnum perlucidum has been identified from numerous tropical and temperate locations worldwide, but its native and introduced ranges remain largely unknown. In Australia, D. perlucidum is listed as a target species under the introduced marine pest national monitoring network. In order to investigate the introduced status and potential routes of introduction and dispersal of D. perlucidum, we developed 17 new polymorphic microsatellite markers using 454 shotgun sequencing. Two to six alleles per locus were detected. No evidence of linkage disequilibrium between pairs of loci was identified and 12 of the 17 loci were in Hardy-Weinberg equilibrium.

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Numbers of the critically endangered hairy marron Cherex tenuimanus are declining rapidly in its natural habitat, mainly due to competition with smooth marron C. cainii. To determine whether genetic introgression is occurring between the two species we isolated 13 new polymorphic microsatellite markers from C. cainii using 454 shotgun sequencing. The loci were screened for variation in 20 individuals of C. cainii and eight individuals of C. tenuimanus. There was no evidence of linkage disequilibrium between pairs of loci and only one locus deviated significantly from Hardy–Weinberg equilibrium. The number of alleles per locus ranged from two to 11 across both species.

Marine mussels of the genus Perna include three species: P. canaliculus, P. viridis and P. perna. While P. canaliculus appears to be greatly restricted to its endemic range of New Zealand, P. perna and P. viridis introductions have been recorded outside their native ranges in several regions of the globe. Such introductions have often resulted in significant negative ecological, economic and social impacts. Perna perna and P. viridis are exotic to Australia and are listed under the Australian Government National System for the Prevention and Management of Marine Pest Incursions as high priority species. Rapid detection of marine pests such as Perna species remains fundamental to their effective containment and control. The present study reports on the development and validation of both conventional and real-time PCR assays suited to the rapid identification and discrimination of juvenile and adult specimens of P. viridis, P. canaliculus and P. perna. The development of a sensitive high-throughput real-time PCR assay offers further potential for the efficient detection of the presence of single Perna specimens in mixed populations of native mussel species, and for early detection of larval stages in ballast water and plankton samples. This assay offers considerable advantages over traditional identification methods and represents an important step in developing capacity for efficient identification and management of Perna species incursions in Australian waters.

Introduced Marine Pests (IMP, = non-indigenous marine species) prevention, early detection and risk-based management strategies have become the priority for biosecurity operations worldwide, in recognition of the fact that, once established, the effective management of marine pests can rapidly become cost prohibitive or impractical. In Western Australia (WA), biosecurity management is guided by the “Western Australian Prevention List for Introduced Marine Pests” which is a policy tool that details species or genera as being of high risk to the region. This list forms the basis of management efforts to prevent introduction of these species, monitoring efforts to detect them at an early stage, and rapid response should they be detected. It is therefore essential that the species listed can be rapid and confidently identified and discriminated from native species by a range of government and industry stakeholders. Recognising that identification of these species requires very specialist expertise which may be in short supply and not readily accessible in a regulatory environment, and the fact that much publicly available data is not verifiable or suitable for regulatory enforcement, the WA government commissioned the current project to collate a reference collection of these marine pest specimens. In this work, we thus established collaboration with researchers worldwide in order to source representative specimens of the species listed. Our main objective was to build a reference collection of taxonomically vouchered specimens and subsequently to generate species-specific DNA barcodes suited to supporting their future identification. To date, we were able to obtain specimens of 75 species (representative of all but four of the pests listed) which have been identified by experts and placed with the WA Government Department of Fisheries and, where possible, in accessible museums and institutions in Australasia. The reference collection supports the fast and reliable taxonomic and molecular identification of marine pests in WA and constitutes a valuable resource for training of stakeholders with interest in IMP recognition in Australia. The reference collection is also useful in supporting the development of a variety of DNA-based detection strategies such as real-time PCR and metabarcoding of complex environmental samples (e.g. biofouling communities). The Prevention List is under regular review to ensure its continued relevance and that it remains evidence and risk-based. Similarly, its associated reference collection also remains to some extent a work in progress. In recognition of this fact, this report seeks to provide details of this continually evolving information repository publicly available to the biosecurity management community worldwide.