Novel tools for early detection of a global aquatic invasive, the zebra mussel Dreissena polymorpha

1. This study presents a species-specific DNA-based marker for detection of the zebra mussel Dreissena polymorpha, recognized as one of the worst invasive species worldwide. 2. The marker was developed in silico and experimentally tested on environmental samples. Gel and capillary electrophoreses for visualization of the PCR products were compared. 3. Marker specificity and sensitivity were assessed in vitro by cross-amplifications and serial dilutions, respectively. The method allows detecting at least 0.7 ng of Dreissena DNA per μL and cross-species amplification was not found in any case. 4. Next-generation sequencing (NGS) metabarcoding (PCR amplification and massive sequencing of a DNA barcode) was used as an independent method for verifying presence of Dreissena DNA molecules in environmental plankton samples collected from the south-eastern Baltic Sea. 5. The consistency between NGS results reporting presence of Dreissena and positive PCR amplification of the marker from the plankton samples confirmed the efficacy of this highly reproducible, fast, cheap and technically easy method

Molecular barcoding confirms the presence of exotic Asian seaweeds (Pachymeniopsis gargiuli and Grateloupia turuturu) in the Cantabrian Sea, Bay of Biscay

Background The introduction of exotic species can have serious consequences for marine ecosystems. On the shores of the Cantabrian Sea (North of Spain) there are no routine examinations of seaweeds that combine molecular and morphological methods for early detection of exotic species making it difficult to assess in the early stages their establishment and expansion processes as a result of anthropogenic activities (e.g., shipping and/or aquaculture). Methods In this work we used both morphological identification and molecular barcoding (COI-5P and rbcL genes) of red algae collected in Asturias, Bay of Biscay (Gijón and Candás harbours) and from the University of Oviedo’s herbarium samples. Results The results confirmed the presence of exotic Asian seaweeds Pachymeniopsis gargiuli and Grateloupia turuturu Yamada on Cantabrian Sea shores. Several individuals of these species were fertile and developing cystocarps when collected, underlining the risk of possible expansion or continued establishment. This study constitutes the first report of the Asian P. gargiuli in this area of the Bay of Biscay. Conclusions Here the presence of the exotic species of the Halymeniales P. gargiuli is confirmed. We hypothesize that this species may have been established some time ago as a cryptic introduction with G. turuturu in Galician shores. The detection of these species on the shores of the Cantabrian Sea is relevant since introductions of Pachymeniopsis species could have been overlooked on other European coasts, probably mixed with G. turuturu and P. lanceolata. Our results confirm one new alien seaweed species that has been detected using molecular methods (COI-5P region and rbcL genes barcoding) on North Atlantic shores: the Asian native P. gargiuli. This demonstrates that routine screening for early detection of exotic algae in the Cantabrian Sea can be used for risk assessment. Genetic barcoding should be done using both rbcL gene and COI-5P regions since, although COI-databases are still poorer in sequences and this inhibits successful outcomes in Grateloupia-related species identifications, it is nonetheless a useful marker for species-level identifications in seaweeds

DNA Barcoding and Phylogeny of Patellids from Asturias (Northern Spain)

The main role for genetics in marine invertebrates is the identification of species and groups of interbreeding individuals. In Asturias exists an ancient culinary tradition of consumption for limpets (Patella s.s.) but there is a lack of studies about these species. We have sampled Asturian marine Patella s.s. specimens and conducted sequencing of the mtDNA COI gene. We have confirmed the presence of four Patella s.s. in the Asturian coasts (P. vulgata, P. depressa, P.aspera, and P. rustica). Our work raises concerns about the current state of the P. vulgata populations in Asturias, where it is exploited, due to its low levels of genetic variation. Our phylogenetic analyses using Bayesian approaches confirmed that patellids belong to four different clades but gives a new version about how these clades are related inside the genus aiming for the necessity of more work to address this issue

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

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

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. Reproductionwas 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

Assessment of parental contributions to fast- and slow-growing progenies in the sea bream Sparus aurata L. using a new multiplex PCR

Molecular tools to assist breeding programs in the gilthead sea bream (Sparus aurata L.) are scarce. A new multiplex PCR technique (OVIDORPLEX), which amplifies nine known microsatellite markers, was developed in this work. This multiplex system showed a high mean heterozygosity (>0.800) and a high mean number of alleles per marker (>14) when tested in two sea bream broodstocks (A: 40 breeders and B: 38 breeders). We tested this multiplex PCR for inferring parentage in a Spanish hatchery that graded the animals by size as part of their management procedure. The progeny of the broodstock were divided into fast- and slow-growth groups. Parentage studies revealed that this management procedure entailed a global reduction of the breeders' representation in progeny and that breeders' contributions were significantly unequal. Due to this, effective sample sizes fell to N ê¿13-14 for fast- and N ê¿18-24 for slow-growth progeny groups. These results imply a 3 to 4% rate of inbreeding per generation in the fast-growth group, which is more important to hatchery managers than the slow group. Not all the progeny were evaluated in this experiment (due to the discarding steps), and thus it is difficult to know if the phenotypic performance showed by the fast-growing progeny will be heritable. However, there were genetic differences between the differentiated growth progeny groups (fast vs. slow, F ST values=0.016 to 0.023; P<0.01). We also identified breeders with significantly different contributions to the fast- (10 breeders) or to the slow- (15 breeders) growth progeny groups. Our results demonstrated that this new multiplex PCR could be useful for quantitative programs (breeding programs, detection of QTL, inbreeding control or reconstruction of fish genealogies) to improve the aquaculture of the gilthead sea bream (S. aurata). © 2011 Elsevier B.V.This work was carried out in collaboration with the fish farm Granja Marina Safor, S.L. (Gandia, Valencia, Spain) and the hatchery Piscicultura Marina Mediterranea, S.L. (Burriana, Castellon, Spain). It was financed by JACUMAR (the PROGENSA project) and the Spanish Ministry of Science and Innovation (MICINN; National Program of Resources and Food and Agriculture Technologies, AGL2006-13411-C03-00, SELECTSPARUS, and AGL2007-64040-C03-00, SELECTBREAM, including European Regional Development Funds). V. Gallego was supported by a FPI scholarship financed by MICINN; C. Garcia-Fernandez was supported by a FPU scholarship financed by MICINN; and I. Mazzeo was supported by a FPI scholarship financed by Generalitat Valenciana. We are indebted to three anonymous referees and the journal editor for valuable comments.Borrell, YJ.; Gallego Albiach, V.; García Fernández, C.; Mazzeo ., I.; Pérez Igualada, LM.; Asturiano Nemesio, JF.; Carleos, CE.... (2011). Assessment of parental contributions to fast- and slow-growing progenies in the sea bream Sparus aurata L. using a new multiplex PCR. Aquaculture. 314(1-4):58-65. https://doi.org/10.1016/j.aquaculture.2011.01.028S58653141-

Panorámica actual de las especies no indígenas marinas en el mar Cantábrico y Atlántico adyacente (NO-N de la península Ibérica): primera aproximación a la Directiva Marco de Estrategias Marinas en la demarcación noroeste

Simposio Ibérico de Estudios de Biología Marina (18º. 2014. Gijón (España))La introducción de especies invasoras no indígenas es un problema cada vez más común a escala mundial, siendo considerado una de mayores amenazas para la conservación de la biodiversidad de todo el planeta. Estas invasiones biológicas causan graves problemas ecológicos y en muchos casos ocasionan también importantes pérdidas a la economía local. La gravedad y el alcance de los impactos generados por las especies invasoras es tal, que en los últimos años ha trascendido del ámbito científico al político-social, siendo considerado como uno de los descriptores de la calidad ambiental en la Directiva Marco de la Estrategia Marina (MSFD 2008/56/EC). A pesar de la envergadura y repercusión de este problema, el estudio de las especies invasoras en aguas de la península Ibérica no ha sido abordado hasta fechas muy recientes, estando en su mayoría centrado en el ámbito continental de los ecosistemas dulceacuícolas y en el ámbito marino de la cuenca mediterránea de la península. Para las costas noratlánticas españolas(las cuales constituyen la demarcación noroeste de la Directiva Marco de la Estrategia Marina) los datos existentes son bastante escasos y sobre todo dispersos, siendo también en muchos casos de afiliación taxonómica dudosa. En este trabajo se presenta la primera revisión actualizada de las especies marinas reportadas como no indígenas (exóticas, no autóctonas, alóctonas o “alien”) ocriptogénicas para el mar Cantábrico y el Atlántico adyacente (costas de Galicia) hasta la actualidad; incluyendo también nuevas adiciones procedentes de los muestreos realizadosen las costas cantábricas y atlántico-gallegas entre los años 2010 y 2014. El área de estudio fue dividida en cuatro zonas (A-D) de Oeste a Este. En esta revisión no se reconocen como alóctonosy/o criptogénicosalgunos taxones previamente reportados como tales, ya que según nuestro criterio se tratan de especies nativas y/o especies que están extendiendo sus rangos de distribución de forma natural, en muchos casos como consecuencia de los fenómenos de calentamiento global. Se listan un total de 287 taxones considerados como no indígenas y/o criptogénicos para el área de estudio, constituyendo estos últimos aproximadamente un tercio del total (92 taxones). Para las taxones alóctonos(195), se ha seguido la clasificación de Zenetoset al. basada en el grado de invasión de las mismas. Las especies consideradas como “casuales” representaron aproximadamente una quinta parte del total (17%), mientras que las especies “establecidas” constituyeron algo menos de la mitad (41%). Finalmente, alrededor de un 6% de los taxones recogidos (17) se categorizaron como “invasores” para las aguas objeto de estudio. En función de las zonas geográficas en las que fue dividida el área de estudio, la gran mayoría de las especies (78%) fueron detectadas para una sola zona; un 21% lo fueron para al menos dos zonas; un 11% para tres y sólo un 6% de las especies fueron detectadas en las cuatro zonas (formando parte de éstas, 10 de las especies consideradas “invasoras”). Taxonómicamente, los filos con un mayor número de especies introducidas son los moluscos y los anélidos, agrupando, cada uno de ellos, un 21% del total de especies; seguidos del grupo de las algas rojas (16%) y de los artrópodos (14%). Otros filos que representan porcentajes mucho menores son los cnidarios (7%), los briozoos (5%), las algas pardas (4%), las ascidias (4%), los poríferos (3%), las algasverdes (2%), los gusanos nemertinos (<1%) y los equinodermos (<1%). El filo con mayor número de especies consideradas como “invasoras” es el grupo las algas rojas con 7 especies

Loci microsatélites como marcadores genéticos para la mejora del rendimiento en acuicultura de especies marinas

Esta tesis se propone como objetivo general comprobar la utilidad de la variación microsatélite en el manejo de poblaciones de tres especies con gran interés económico: el salmón atlántico salmo salar l., el rodaballo scophtalmus maximus, y el camarón blanco litopenaeus schmitti. Los resultados obtenidos obtenidos demuestran que los loci microsatélites son útiles para el manejo de reproductores en las estaciones de cultivo a través del establecimiento de pedigríes si se tienen en cuenta: un análisis previo de variabilidad en los reproductores, la posibilidad de mutaciones y alelos nulos y las relaciones genéticas existentes entre los reproductores. No se encuentra una relación alelos-caracteres de interés comercial en el cultivo, aunque si asociación entre la heterocigosidad enzimática y estos últimos, mientras parece no existir ninguna relación con la heterocigosidad microsatélite. Nuestros resultados apoyan la idea de que las enzimas, a través del control del metabolismo, juegan un papel fundamental en el incremento de la eficacia biológica de los individuos. Finalmente, los loci microsatélites muestran mayores niveles de variabilidad que las enzimas en poblaciones naturales, aunque el reparto de esa variabilidad genética es similar para ambos marcadores en camarón. No obstante, la mayor variabilidad de los microsatélites permite asignar los individuos a sus poblaciones de origen, lo cual sería de gran utilidad en el manejo poblaciones naturales y cultivadas