Alba Ardura

Applications of DNA barcoding to fish landings: authentication and diversity assessment.

Abstract DNA barcoding methodologies are being increasingly applied not only for scientific purposes but also for diverse real-life uses. Fisheries assessment is a potential niche for DNA barcoding, which serves for species authentication and may also be used for estimating within-population genetic diversity of exploited fish. Analysis of single-sequence barcodes has been proposed as a shortcut for measuring diversity in addition to the original purpose of species identification. Here we explore the relative utility of different mitochondrial sequences (12S rDNA, COI, cyt b, and D-Loop) for application as barcodes in fisheries sciences, using as case studies two marine and two freshwater catches of contrasting diversity levels. Ambiguous catch identification from COI and cyt b was observed. In some cases this could be attributed to duplicated names in databases, but in others it could be due to mitochondrial introgression between closely related species that may obscure species assignation from mtDNA. This last problem could be solved using a combination of mitochondrial and nuclear genes. We suggest to simultaneously analyze one conserved and one more polymorphic gene to identify species and assess diversity in fish catches.

Metabarcoding approach for nonindigenous species surveillance in marine coastal waters.

In this study, high-throughput sequencing (HTS) metabarcoding was applied for the surveillance of plankton communities within the southeastern (SE) Baltic Sea coastal zone. These results were compared with those from routine monitoring survey and morphological analyses. Four of five nonindigenous species found in the samples were identified exclusively by metabarcoding. All of them are considered as invasive in the Baltic Sea with reported impact on the ecosystem and biodiversity. This study indicates that, despite some current limitations, HTS metabarcoding can provide information on the presence of exotic species and advantageously complement conventional approaches, only requiring the same monitoring effort as before. Even in the currently immature status of HTS, this combination of HTS metabarcoding and observational records is recommended in the early detection of marine pests and delivery of the environmental status metrics of nonindigenous species.

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.

Universal primers for species authentication of animal foodstuff in a single polymerase chain reaction

BACKGROUND There are many DNA-based systems for detecting animal species present in food and food products, applicable for food quality control and authentication. However, most (if not all) methods require more than one pair of primers and cannot be applied over a wide taxonomic range, e.g. identifying vertebrates and invertebrates with the same primers and protocols. RESULTS A pair of primers is described here that allows in a single polymerase chain reaction the identification of animal species in food and processed (precooked, canned or smoked) food products over a wide taxonomic range. CONCLUSION These primers permit the identification of most animal taxa employed in human nutrition, from invertebrates such as molluscs to higher vertebrates, distinguishing between species of the same genus. The short fragment amplified within the 16S rDNA exhibits phylogenetic value and could be considered universal based on the wide taxonomic range assayed. The primers are easy to use and accessible for laboratories with a modest budget, as well as being valuable for consumer information and to reveal food fraud.

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.

DNA barcoding for assessment of exotic molluscs associated with maritime ports in northern Iberia.

ABSTRACT Ports are gateways for aquatic invasions. New arrivals from maritime traffic and disturbed environmental conditions can promote the settlement of exotic species. Molluscs fall into the most prevalent group of invasive species and can have a tremendous impact on aquatic ecosystems. Here we have investigated exotic molluscs in three ports with different intensities of maritime traffic in the Cantabrian Sea. DNA barcodes were employed to identify the species using BLASTn and BOLD IDS assignment. Deep morphological analysis using diagnostic criteria confirmed BLAST species assignation based on COI and 16S rRNA genes. Results confirmed the usefulness of DNA barcoding for detecting exotic species that are visually similar to native species. Three exotic bivalves were identified: Ostrea stentina (dwarf oyster), the highly invasive Crassostrea gigas (Pacific oyster) and Xenostrobus securis (pygmy mussel). This is the first record of O. stentina in the Bay of Biscay and the second of X. securis in the Cantabrian Sea. Furthermore, we report on the presence of the cryptogenic mussel Mytilaster minimus in the central Cantabrian Sea. These exotic species might have been overlooked due to their phenotypic similarity with co-occurring oyster and mussel species. This study illustrates how combining morphological and DNA taxonomic analysis can help in port and marina biosecurity surveys.

Epigenetic signatures of invasive status in populations of marine invertebrates

Epigenetics, as a DNA signature that affects gene expression and enables rapid reaction of an organism to environmental changes, is likely involved in the process of biological invasions. DNA methylation is an epigenetic mechanism common to plants and animals for regulating gene expression. In this study we show, for the first time in any marine species, significant reduction of global methylation levels during the expansive phase of a pygmy mussel (Xenostrobus securis) recent invasion in Europe (two-year old), while in older introductions such epigenetic signature of invasion was progressively reduced. Decreased methylation was interpreted as a rapid way of increasing phenotypic plasticity that would help invasive populations to thrive. This epigenetic signature of early invasion was stronger than the expected environmental signature of environmental stress in younger populations sampled from ports, otherwise detected in a much older population (>90 year old) of the also invasive tubeworm Ficopomatus enigmaticus established in similar locations. Higher epigenetic than genetic diversity found in X. securis was confirmed from F. enigmaticus samples. As reported for introduced plants and vertebrates, epigenetic variation could compensate for relatively lower genetic variation caused by founder effects. These phenomena were compared with epigenetic mechanisms involved in metastasis, as parallel processes of community (biological invasion) and organism (cancer) invasions.

Beyond Biodiversity: Fish Metagenomes

Biodiversity and intra-specific genetic diversity are interrelated and determine the potential of a community to survive and evolve. Both are considered together in Prokaryote communities treated as metagenomes or ensembles of functional variants beyond species limits. Many factors alter biodiversity in higher Eukaryote communities, and human exploitation can be one of the most important for some groups of plants and animals. For example, fisheries can modify both biodiversity and genetic diversity (intra specific). Intra-specific diversity can be drastically altered by overfishing. Intense fishing pressure on one stock may imply extinction of some genetic variants and subsequent loss of intra-specific diversity. The objective of this study was to apply a metagenome approach to fish communities and explore its value for rapid evaluation of biodiversity and genetic diversity at community level. Here we have applied the metagenome approach employing the Barcoding target gene COI as a model sequence in catch from four very different fish assemblages exploited by fisheries: freshwater communities from the Amazon River and northern Spanish rivers, and marine communities from the Cantabric and Mediterranean seas. Treating all sequences obtained from each regional catch as a biological unit (exploited community) we found that metagenomic diversity indices of the Amazonian catch sample here examined were lower than expected. Reduced diversity could be explained, at least partially, by overexploitation of the fish community that had been independently estimated by other methods. We propose using a metagenome approach for estimating diversity in Eukaryote communities and early evaluating genetic variation losses at multi-species level.

Barcodes of marine invertebrates from north Iberian ports: Native diversity and resistance to biological invasions

Ports are gateways for many marine organisms transported by ships worldwide, especially non-indigenous species (NIS). In this study carried out in North Iberian ports (Cantabrian Sea, Bay of Biscay) we have observed 38% of exotic macroinvertebrates. Four species, namely the barnacle Austrominius modestus, the tubeworm Ficopomatus enigmaticus, the Pacific oyster Crassostrea gigas and the pygmy mussel Xenostrobus securis, exhibited clear signs of invasiveness. A total of 671 barcode (cytochrome oxidase subunit I or 18S rRNA) genes were obtained and confirmed the species status of some cryptic NIS. Negative and significant correlation between diversity estimators of native biota and proportion of NIS suggests biotic resistance in ports. This could be applied to management of port biota for contributing to prevent the settlement of biopollutants in these areas which are very sensitive to biological invasions.

An extremely sensitive nested PCR-RFLP mitochondrial marker for detection and identification of salmonids in eDNA from water samples

Background Salmonids are native from the North Hemisphere but have been introduced for aquaculture and sport fishing in the South Hemisphere and inhabit most rivers and lakes in temperate and cold regions worldwide. Five species are included in the Global Invasive Species Database: rainbow trout Oncorhynchus mykiss, Atlantic salmon Salmo salar, brown trout Salmo trutta, brook trout Salvelinus fontinalis, and lake trout Salvelinus namaycush. In contrast, other salmonids are endangered in their native settings. Methods Here we have developed a method to identify salmonid species directly from water samples, focusing on the Iberian Peninsula as a case study. We have designed nested Salmonidae-specific primers within the 16S rDNA region. From these primers and a PCR-RFLP procedure the target species can be unequivocally identified from DNA extracted from water samples. Results The method was validated in aquarium experiments and in the field with water from watersheds with known salmonid populations. Finally, the method was applied to obtain a global view of the Salmonidae community in Nalón River (north coast of Spain). Discussion This new powerful, very sensitive (identifying the species down to 10 pg DNA/ml water) and economical tool can be applied for monitoring the presence of salmonids in a variety of situations, from checking upstream colonization after removal of river barriers to monitoring potential escapes from fish farms.