New dockside eDNA based protocol to detect the seaweed Asparagopsis armata evaluated by stakeholders.

Early detection of invasive species is crucial to deal effectively with biological invasions in ports, which are hotspots of species introductions. In this study, a simplified end-time PCR methodology conducted on eDNA from water samples was developed for rapid detection of the invasive seaweed Asparagopsis armata (four hours from water collection to result visualization). It was tested dockside in four international Spanish ports in presence of stakeholders, whose feedback was obtained to explore the real applicability of this biotechnology. Although biological invasions were not a main concern for them, results indicate a unanimous approval of the methodology by the stakeholders, having detected the presence of A. armata in three of the ports. Stakeholders suggested further developments for easier application of the tool and multiple species detection, to be adopted for the control of invasive species in ports.

Mitochondrial Variation of Bottlenose Dolphins (Tursiops truncatus) from the Canary Islands Suggests a Key Population for Conservation with High Connectivity within the North-East Atlantic Ocean.

In recent decades, worldwide cetacean species have been protected, but they are still threatened. The bottlenose dolphin (Tursiops truncatus) is a vulnerable keystone species and a useful bioindicator of the health and balance of marine ecosystems in oceans all over the world. The genetic structure of the species is shaped by their niche specialization (along with other factors), leading to the classification of two ecotypes: coastal and pelagic. In this study, the genetic diversity, population structure, and ecotypes of bottlenose dolphins from the Canary Islands were assessed through the analysis of 49 new samples from biopsies and from stranded animals using the 636 bp portion of the mitochondrial control region and 343 individuals from databases (n = 392). The results reveal high genetic diversity in Canarian bottlenose dolphins (Hd = 0.969 and π = 0.0165) and the apparent lack of population genetic structure within this archipelago. High genetic structure (Fst, Φst) was found between the Canary Islands and coastal populations, while little to no structure was found with the pelagic populations. These results suggest that Canarian bottlenose dolphins are part of pelagic ecotype populations in the North Atlantic. The studied Special Areas of Conservation in the Canary Islands may correspond to a hotspot of genetic diversity of the species and could be a strategic area for the conservation of the oceanic ecotype of bottlenose dolphins.

Insights on the Evolutionary History and Genetic Patterns of Octopus vulgaris Cuvier, 1797 in the Northeastern Atlantic Using Mitochondrial DNA.

Octopus vulgaris is one of the most harvested octopus species in the world. In the Iberian Peninsula, there are several small-scale fisheries that have a long-term tradition of harvesting octopus. The Asturias fleet (in Northern Spain) has an internationally recognized MSC label for its exploitation. Of concern, genetic assessments of exploited stocks are currently scarce, which could prevent the implementation of adequate managing strategies. We use two mitochondrial regions (cytochrome oxidase subunit 1 and control region) to analyze the genetic status and evolutionary events that conditioned octopus populations' characteristics in the Northeastern Atlantic. A total of 90 individuals were sampled from three different localities in the Iberian Peninsula as well as a location in Macaronesia. Temporal genetic analyses on Asturias and Algarve populations were also performed. Results indicated the absence of fine spatial genetic structuring but showed the Canary Islands (in Macaronesia) as the most distinct population. Our analyses detected two distinct clades, already described in the literature, but, for the first time, we confirmed the presence of the α-southern haplogroup in the Northern Iberian Peninsula. This result indicates a more continuous cline for the distribution of these two haplogroups than previously reported. Temporal changes in the distribution of both haplogroups in contact zones were also detected.

Bioremediation as a promising strategy for microplastics removal in wastewater treatment plants.

Microplastics (MPs) attract ever-increasing attention due to environmental concerns. Nowadays, they are ubiquitous across ecosystems, and research demonstrates that the origin is mainly terrestrial. Wastewater treatment plants (WWTPs) are a major source of MPs, especially fibres, in water masses. This review is focused on understanding the evolution and fate of microplastics during wastewater treatment processes with the aim of identifying advanced technologies to eliminate microplastics from the water stream. Among them, bioremediation has been highlighted as a promising tool, but confinement of microorganisms inside the WWTP is still a challenge. The potential for MPs bioremediation in WWTPs of higher aquatic eukaryotes, which offer the advantages of low dispersion rates and being easy to contain, is reviewed. Animals, seagrasses and macrophytes are considered, taking into account ecoethical and biological issues. Necessary research and its challenges have been identified.

Lab experience with seafood control at the undergraduate level: Cephalopods as a case study.

The correct labeling of seafood is important to protect nature and the rights of consumers. Given the certainty that the resources of the sea are not inexhaustible, only strict regulations and the implementation of sustainable fishing systems and reliable and traceable marketing systems can help ensure the long-term sustainability of fishery resources. Detecting mislabeling and seafood fraud is a useful resource for improving students' motivation and developing active learning methodologies in higher education. In the present study, we have proposed to the students a lab exercise consisting of exploring 25 different commercial cephalopod products from three major European supermarkets by using DNA barcoding and analyzing the results under the framework of EU and Spanish regulations. The problem is connected with the last theme (traceability) of the Conservation Genetics and Breeding course with the aim of providing students with a practical research lab experience about a real problem before going deeper into more theoretical contents. In this way, they can use the knowledge and the skills they acquired previously to better comprehend and think critically about the problem. Findings from students' answers to a survey revealed that the use of this approach generates useful information for communities, increases curiosity and feelings of benefit, and leads to high levels of satisfaction with lab practices compared with those in other courses. In conclusion, lab exercises focused on seafood control, in addition to being viable, can be used as a tool in classes to improve students' commitment to higher education.

Contrasting seasonal and spatial distribution of native and invasive Codium seaweed revealed by targeting species-specific eDNA.

AIM: Codium fragile, an invasive seaweed, has spread widely during the last century, impacting on local seaweed communities through competition and disturbance. Early detection of C. fragile can help on its control and management. Environmental DNA (eDNA) has proved successful for early detection of aquatic invasive species but its potential use for seaweed remains understudied. We used a species-specific eDNA qPCR approach to investigate the spatial distribution, abundance, and coexistence of the invasive C. fragile and three native Codium species (Codium vermilara, Codium tomentosum, and Codium decorticatum) in the Cantabrian Sea. LOCATION: Bay of Biscay, Northern Atlantic Coast of the Iberian Peninsula; two ports, a beach and a rocky cliff. METHODS: We designed species-specific primers in barcoding regions targeting short fragments of the rbcL gene for the invasive Codium species, and the elongation factor Tu (tufA) gene for the native species, to assess their spatial and seasonal distributions using quantitative real-time PCR in samples collected during summer, autumn, and winter. RESULTS: We found seasonal differences in the presence of the invasive Codium fragile and two of the native Codium species, but did not detect C. decorticatum at any point. Species distribution patterns produced with qPCR targeting species-specific eDNA coincided with the known distribution based on previous conventional sampling, with a seasonal alternance of C. fragile and C. vermilara, and a marked dominance of invasive C. fragile in ports, which are known hotspots for invasive species. MAIN CONCLUSIONS: Our results demonstrate the utility of using eDNA for early detection and monitoring of invasive seaweed. Native and invasive Codium spp. displayed significant seasonal and spatial differentiation that needs to be taken into account in risk management. Regular monitoring of ports and adjacent areas using eDNA should help to assess the potential expansion of invasive Codium and the need for management interventions to avoid the displacement of native seaweed.

Evaluating freshwater macroinvertebrates from eDNA metabarcoding: A river Nalón case study.

Rivers are a vital resource for human wellbeing. To reduce human impact on water bodies, the European Union has established an essential regulatory framework for protection and sustainable management (WFD; 2000/60/EC). In this strategy, reliable and economic bioindicators are a fundamental component. Benthic macroinvertebrates are the group most commonly used as bioindicators through all European countries. However, their conventional assessment currently entails serious cost-efficiency limitations. In this study, we have tested the reliability of metabarcoding as a tool to record river macroinvertebrates using samples from a mock community (in vitro validation) and eDNA extracted for field validation from water from six sites within a north Iberian river (River Nalón, Asturias, Spain). Two markers (V4 region within the nuclear 18S rDNA and a fragment of the mitochondrial COI gene) were amplified and sequenced using an Illumina platform. The molecular technique has proven to be more sensitive than the visual one. A cost-benefit analysis shows that the metabarcoding approach is more expensive than conventional techniques for determining macroinvertebrate communities but requires fewer sampling and identification efforts. Our results suggest metabarcoding is a useful tool for alternative assessment of freshwater quality.

DNA in a bottle-Rapid metabarcoding survey for early alerts of invasive species in ports.

Biota monitoring in ports is increasingly needed for biosecurity reasons and safeguarding marine biodiversity from biological invasion. Present and future international biosecurity directives can be accomplished only if the biota acquired by maritime traffic in ports is controlled. Methodologies for biota inventory are diverse and now rely principally on extensive and labor-intensive sampling along with taxonomic identification by experts. In this study, we employed an extremely simplified environmental DNA (eDNA) sampling methodology from only three 1-L bottles of water per port, followed by metabarcoding (high-throughput sequencing and DNA-based species identification) using 18S rDNA and Cytochrome oxidase I as genetic barcodes. Eight Bay of Biscay ports with available inventory of fouling invertebrates were employed as a case study. Despite minimal sampling efforts, three invasive invertebrates were detected: the barnacle Austrominius modestus, the tubeworm Ficopomatus enigmaticus and the polychaete Polydora triglanda. The same species have been previously found from visual and DNA barcoding (genetic identification of individuals) surveys in the same ports. The current costs of visual surveys, conventional DNA barcoding and this simplified metabarcoding protocol were compared. The results encourage the use of metabarcoding for early biosecurity alerts.

Detection and characterisation of the biopollutant Xenostrobus securis (Lamarck 1819) Asturian population from DNA Barcoding and eBarcoding.

DNA efficiently contributes to detect and understand marine invasions. In 2014 the potential biological pollutant pygmy mussel (Xenostrobus securis) was observed for the first time in the Avilés estuary (Asturias, Bay of Biscay). The goal of this study was to assess the stage of invasion, based on demographic and genetic (DNA Barcoding) characteristics, and to develop a molecular tool for surveying the species in environmental DNA. A total of 130 individuals were analysed for the DNA Barcode cytochrome oxidase I gene in order to determine genetic diversity, population structure, expansion trends, and to inferring introduction hits. Reproduction was evidenced by bimodal size distributions of 1597 mussels. High population genetic variation and genetically distinct clades might suggest multiple introductions from several source populations. Finally, species-specific primers were developed within the DNA barcode for PCR amplification from water samples in order to enabling rapid detection of the species in initial expansion stages.

PCR-based assessment of shellfish traceability and sustainability in international Mediterranean seafood markets.

Two mitochondrial markers (cytochrome oxidase COI and 16S rDNA) were employed for species identification of commercial shellfish from two Mediterranean countries. New COI Barcodes were generated for six species: Pleoticus robustus, Metapenaeopsis barbata, Parapenaeus fissuroides, Hymenopenaeus debilis, Metapenaeus affinis and Sepia aculeata. Biodiversity of the seafood species analyzed was greater in Egypt, with nine crustacean and two cephalopod species found compared with only three crustaceans and three cephalopods in Spain. In total, 17.2% and 15.2% products were mislabeled in Egypt and Spain, respectively. Population decline is a problem for some of the substitute species. Others were exotic and/or invasive in exporters' regions. This study offers the first comparable study of shellfish traceability in these Mediterranean markets. The PCR-based method used in this study proved to be reliable, effective and, therefore, could be employed for routine seafood analysis.

Detecting nuisance species using NGST: Methodology shortcomings and possible application in ballast water monitoring.

Detecting the presence of potential invasive species in ballast water is a priority for preventing their spread into new environments. Next generation sequencing technologies are being increasingly used for exploring and assessing biodiversity from environmental samples. Here we apply high throughput sequencing from DNA extracted from ballast water (BW) samples employing two different platforms, Ion Torrent and 454, and compare the putative species catalogues from the resulting Operational Taxonomic Units (OTU). Water samples were taken from the RV Polastern ballast tank in five different days between the second and the twentieth navigation day. Pronounced decrease of oxygen concentration and increase of temperature occurred in the BW during this time, coincident with a progressively higher proportion of unassigned OTU and short reads indicating DNA degradation. Discrepancy between platforms for species catalogues was consistent with previously published bias in AT-rich sequences for Ion Torrent platform. Some putative species detected from the two platforms increased in frequency during the Polarstern travel, which suggests they were alive and therefore tolerant to adverse conditions. OTU assigned to the highly invasive red alga Polysiphonia have been detected at low but increasing frequency from the two platforms. Although in this moment NGST could not replace current methods of sampling, sorting and individual taxonomic identification of BW biota, it has potential as an exploratory methodology especially for detecting scarce species.