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.

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Environmental DNA (eDNA) methods are providing tools for detecting invasive species in aquatic environments. Targeted qPCR assays applied to eDNA samples promise to overcome limitations of traditional methods, especially for early detection. The European green crab (Carcinus maenas) is considered one of the most successful invasive species globally due to the large range it has invaded and negative impacts on native species, marine habitats, and shellfish industries. We developed, laboratory‐validated, and field‐tested a specific qPCR assay for the detection of green crab from eDNA samples. We also show that the assay can detect green crab in bulk DNA extracted from plankton samples. Assay design, optimization, sensitivity, and specificity testing generally followed the validation pathway recommended by the World Organization for Animal Health for assays used to manage global aquatic animal health and infectious disease. Assay specificity was verified in silico and in vitro by laboratory testing 26 nontarget species, none of which showed potential for amplification. Assay sensitivity was appropriately high, with the limit of detection approaching two gene copies/μl. The assay was field‐tested on eDNA samples collected from filtered seawater at five sites on the Pacific coast of Canada known to harbor green crab based on historical monitoring data; green crab DNA was amplified from all sites. We also present early pilot field testing of the assay done on bulk DNA extracted from plankton samples from four sites from Australia, two sites with and two sites without reported records of green crab presence. Green crab was detected at both sites with known green crab records. Significant inhibition was recorded for some plankton samples but not for eDNA samples. This is the first qPCR assay for detection of European green crab, providing researchers and managers with a valuable new tool to aid early detection and ongoing monitoring.

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Global distribution of platyhelminth parasites and their host specificities are not well known. Our hypothesis was that platyhelminth parasites of large pelagic fishes are common around the world. We analysed molecular variation in three different taxa of platyhelminth parasites infecting four species of tunas: yellowfin tuna (Thunnus albacares, Scombridae) from Western Australia, southern bluefin tuna (Thunnus maccoyii, Scombridae) from South Australia, Pacific bluefin tuna (Thunnus orientalis, Scombridae) from Pacific Mexico and northern bluefin tuna (T. thynnus, Scombridae) from two localities in the Mediterranean (Spain and Croatia). Comparisons of ITS2 and partial 28S rDNA demonstrated two congeneric species of blood flukes (Digenea: Sanguinicolidae) from multiple hosts and localities: Cardicola forsteri from southern bluefin and northern bluefin tunas, and Cardicola sp. from Pacific bluefin and northern bluefin tunas; and a gill fluke, Hexostoma thynni (Polyopisthocotylea: Hexostomatidae), from yellowfin, southern bluefin and northern bluefin tunas. Partial 28S rDNA indicates that a second type of fluke on the gills, Capsala sp. (Monopisthocotylea: Capsalidae), occurs on both southern bluefin and Pacific bluefin tunas. This appears to be the first report of conspecific platyhelminth parasites of teleosts with a wide-ranging geographical distribution that has been confirmed through molecular approaches. Given the brevity of the free-living larval stage of both taxa of flukes on the gills (H. thynni and Capsala sp.), we conclude that the only feasible hypothesis for the cosmopolitan distribution of these flatworms is migrations of host tunas. Host migration also seems likely to be responsible for the widespread occurrence of the two species of blood flukes (Cardicola spp.), although it is also possible that these were translocated recently by the spread of infected intermediate hosts.

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