34 research outputs found

    Temporal and spatial genetic differentiation in the crab Liocarcinus depurator across the Atlantic-Mediterranean transition

    Get PDF
    Spatial genetic studies often require sampling broadly separated areas, difficult to access simultaneously. Although comparing localities surveyed at different time periods might result in spurious genetic differentiation, there is a general believe on the stability of genetic structure through time, particularly if sampled localities are isolated or very distant. By analysing spatial and temporal genetic differentiation of the portunid crab Liocarcinus depurator we assessed the contribution of historical and contemporary processes on population connectivity patterns across three main oceanographic discontinuities along the Atlantic-Mediterranean transition: Gibraltar Strait, Almeria- Oran Front and Ibiza Channel. A partial fragment of the cytochrome oxidase I gene was sequenced in 366 individuals collected from localities at both sides of each discontinuity during three time periods. Although localities showed genetic fluctuations through time, a significant gradient was detected along the coast for all sampling periods. Significant inter-annual differences identified within the Alicante area, north of the Almeria-Oran Front, were associated with shifts in the relative contribution of Atlantic and Mediterranean water masses. The persistence of a clinal pattern in the Atlantic-Mediterranean transition area together with local fluctuations suggests a complex balance of dispersal and selection

    A real-time PCR assay to estimate invertebrate and fish predation on anchovy eggs in the Bay of Biscay

    Get PDF
    In order to investigate the role of predation on eggs and larvae in the recruitment of anchovy (Engraulis encrasicolus), sardine (Sardina pilchardus), sprat (Sprattus sprattus) and 52 macrozooplankton taxa were assayed for anchovy remains in the gut during the 2010 spawning season using a molecular method. This real-time PCR based assay was capable of detecting 0.005 ng of anchovy DNA (roughly 1/100 of a single egg assay) in a reliable way and allowed detecting predation events up to 6 h after ingestion by small zooplankton taxa. A total of 1069 macrozooplankton individuals, 237 sardines and 213 sprats were tested. Both fish species and 32 macrozooplankton taxa showed remains of anchovy DNA within their stomach contents. The two main findings are (1) that the previously neglected macrozooplankton impact in anchovy eggs/larvae mortality is in the same order of magnitude of that due to planktivorous fishes and that, (2) the predation pressure was notably different in the two main spawning centers of Bay of Biscay anchovy. While relatively low mortality rates were recorded at the shelf-break spawning center, a higher predation pressure from both fish and macrozooplankton was exerted at the shelf one.This research was financially supported by the projects ECOGENBAY (MICINN CTM2009-13570-C02-02), funded by the Ministry of Science and Research of the Government of Spain, and BIOMAN, funded by the Department of Economic Development and Competitiveness of the Basque Government and by the European Commissio

    Evaluating detection limits of next-generation sequencing for the surveillance and monitoring of international marine pests

    Get PDF
    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‚ĄĘ 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‚ÄďV3 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

    Development of duplex real-time PCR for the detection of WSSV and PstDV1 in cultivated shrimp

    Get PDF
    BACKGROUND: The White spot syndrome virus (WSSV) and Penaeus stylirostris penstyldensovirus 1 (previously named Infectious hypodermal and hematopoietic necrosis virus-IHHNV) are two of the most important viral pathogens of penaeid shrimp. Different methods have been applied for diagnosis of these viruses, including Real-time PCR (qPCR) assays. A duplex qPCR method allows the simultaneous detection of two viruses in the same sample, which is more cost-effective than assaying for each virus separately. Currently, an assay for the simultaneous detection of the WSSV and the PstDV1 in shrimp is unavailable. The aim of this study was to develop and standardize a duplex qPCR assay for the simultaneous detection of the WSSV and the PstDV1 in clinical samples of diseased L. vannamei. In addition, to evaluate the performance of two qPCR master mixes with regard to the clinical sensitivity of the qPCR assay, as well as, different methods for qPCR results evaluation. RESULTS: The duplex qPCR assay for detecting WSSV and PstDV1 in clinical samples was successfully standardized. No difference in the amplification of the standard curves was observed between the duplex and singleplex assays. Specificities and sensitivities similar to those of the singleplex assays were obtained using the optimized duplex qPCR. The analytical sensitivities of duplex qPCR were two copies of WSSV control plasmid and 20 copies of PstDV1 control plasmid. The standardized duplex qPCR confirmed the presence of viral DNA in 28 from 43 samples tested. There was no difference for WSSV detection using the two kits and the distinct methods for qPCR results evaluation. High clinical sensitivity for PstDV1 was obtained with TaqMan Universal Master Mix associated with relative threshold evaluation. Three cases of simultaneous infection by the WSSV and the PstDV1 were identified with duplex qPCR. CONCLUSION: The standardized duplex qPCR was shown to be a robust, highly sensitive, and feasible diagnostic tool for the simultaneous detection of the WSSV and the PstDV1 in whiteleg shrimp. The use of the TaqMan Universal Master Mix and the relative threshold method of data analysis in our duplex qPCR method provided optimal levels of sensitivity and specificity

    The importance of the pelagic larval phase of the wedge shell Donax trunculus (L.): implications for the management of the fishery

    Get PDF
    The wedge shell, Donax trunculus, inhabits high energy environment of exposed sandy beaches from the Atlantic coast of France to Senegal. Like all Donacidae, it is relative small, flat-shaped with elongated solid shells. It is a highly demanded and valuable species mainly in Algarve, with the dredge fleet increasing the pressure on species stocks. In 1986 the Portuguese Institute for the Ocean and Atmosphere (IPMA) initiated a bivalve survey program to evaluate the stock status of species with economical valuable but always on the adult population. Since then, several managing measures were implemented to guarantee bivalves‚Äô sustainable exploitation. Despite the available information on the abundance and distribution of the wedge shell along the Algarve coast, no information on the larval planktonic phase is available. To fill in this gap, the present study aimed at obtaining, for the first time information on the broodstock condition and on the pelagic phase of the wedge shell in the Algarve coast.A conquilha, Donax trunculus, habita em sedimentos arenosos de praias com algum hidrodinamismo desde a costa atl√Ęntica de Fran√ßa at√© ao Senegal. Tal como outros Donacidae, √© relativamente pequena, achatada com uma concha s√≥lida e alongada. √Č uma esp√©cie com elevado valor econ√≥mico e muito procurada especialmente no Algarve, levando a que exista um aumento da press√£o por parte da frota de arrasto de ganchorra sobre a popula√ß√£o. Em 1986 o Instituto Portugu√™s do Mar e da Atmosfera (IPMA) iniciou um programa de monitoriza√ß√£o dos recursos bivalves de modo a avaliar o estado dos estoques populacionais das esp√©cies com valor econ√≥mico, embora sempre sobre a popula√ß√£o adulta. Desde esse per√≠odo, v√°rias medidas de gest√£o t√™m sido implementadas garantido a sustentabilidade da pesca. Apesar de existir variada informa√ß√£o acerca da popula√ß√£o adulta ao longo da costa algarvia, n√£o existe qualquer informa√ß√£o sobre a fase planct√≥nica desta esp√©cie. Com o intuito de colmatar esta falha, o presente estudo teve como objectivo obter pela primeira vez informa√ß√£o acerca da condi√ß√£o dos progenitores e da fase planct√≥nica da conquilha na costa Algarvia.Funda√ß√£o para a Ci√™ncia e TecnologiaFundo Social Europe

    Morphology of the megalopa of the mud crab, Rhithropanopeus harrisii (Gould, 1841) (Decapoda, Brachyura, Panopeidae), identified by DNA barcode.

    Get PDF
    The morphology of the megalopa stage of the panopeid Rhithropanopeus harrisii is redescribed and illustrated in detail from plankton specimens identified by DNA barcode (16S mtDNA) as previous descriptions do not meet the current standard of brachyuran larval description. Several morphological characters vary widely from those of other panopeid species which could cast some doubt on the species’ placement in the same family. Besides, some anomalous megalopae of R. harrisii were found among specimens reared at the laboratory from zoeae collected in the plankton. These anomalous morphological features are discussed in terms of problems associated with laboratory rearing conditions

    Aplicación de técnicas morfológicas y moleculares en la identificación de la megalopa de decápodos braquiuros de la península ibérica

    Get PDF
    Entre los crust√°ceos dec√°podos, el Infraorden Brachyura Linnaeus, 1758 es el grupo m√°s diverso y de mayor √©xito evolutivo, con aproximadamente 7.000 especies pertenecientes a 98 familias (Tsang et al. 2014). Los braquiuros, com√ļnmente llamados cangrejos, han conquistado casi todos los h√°bitats y numerosos nichos ecol√≥gicos (De Grave et al. 2009; Ahyong et al. 2011). La mayor√≠a de las especies son marinas, aunque tambi√©n existen especies de agua dulce o incluso especies terrestres. El desarrollo larvario de los braquiuros suele constar de dos fases de vida libre y planct√≥nicas (con las escasas excepciones de aquellos con desarrollo directo, principalmente de agua dulce): zoea (con varios estadios) y megalopa (Anger 2006). La megalopa es una fase de transici√≥n entre la zoea planct√≥nica y la fase juvenil y adulta, t√≠picamente bent√≥nicas (Rice 1981). La notable variaci√≥n de morfolog√≠a, comportamiento y h√°bitat entre larvas y adultos representa un gran problema a la hora de identificar las larvas del zooplancton. La morfolog√≠a de las formas larvarias es dif√≠cil de relacionar con la de los adultos y, aunque a veces las larvas se pueden distinguir morfol√≥gicamente, no resulta sencillo atribuirlas a la forma adulta correcta (Bucklin 2010). La falta de datos a priori que relacionen los estadios larvarios con la especie a la que pertenecen, ha ralentizado el avance en el conocimiento de la fase megalopa. De las 140 especies de braquiuros conocidas en la Pen√≠nsula Ib√©rica, solo se dispone de descripciones fiables de la megalopa de 67 especies (< 48%). En la √ļltima d√©cada se han empezado a aplicar nuevas t√©cnicas que minimizan estas limitaciones y/o restricciones, y que permiten avanzar a un mayor ritmo en el conocimiento de la morfolog√≠a larval de los braquiuros y sus aplicaciones, como la filogenia y sistem√°tica moleculares (Ampuero et al. 2010; Spiridonov et al. 2014). Una de estas nuevas t√©cnicas fue presentada en 2003 por el doctor Paul Hebert y colaboradores quienes propusieron la utilizaci√≥n de una regi√≥n peque√Īa del genoma como DNA barcode (c√≥digo de barras gen√©tico), al gen citocromo oxidasa 1 (Cox1) (Hebert et al. 2003). El c√≥digo de barras de ADN ha demostrado ser muy √ļtil tanto para diferenciar especies (Costa et al. 2007) como para la diferenciaci√≥n entre poblaciones de una misma especie (Palero et al. 2008; Garc√≠a-Merch√°n et al. 2012). Adem√°s del Cox1, el gen mitocondrial de la subunidad ribosomal 16S tambi√©n ha demostrado ser una herramienta eficiente en estudios sistem√°ticos de crust√°ceos dec√°podos (Schubart et al. 2000; Ahyong et al. 2007). La aplicaci√≥n de t√©cnicas moleculares para la identificaci√≥n de megalopas en muestras del plancton, nos ha permitido incrementar el n√ļmero de especies para las que se conoce este estadio larval, y que a partir de ahora pueden ser identificadas directamente del plancton en base a su morfolog√≠a (Weeb 2006). Se podr√≠a concluir que una clasificaci√≥n sistem√°tica adecuada, que refleje las relaciones filogen√©ticas entre los diferentes taxa, deber√≠a representar un compendio de todas las fuentes de informaci√≥n disponibles, considerando siempre que existan los datos larvales

    Measuring Fertilization in Populations of Sea Scallop (Placopecten magellanicus): Developing and Testing Methods in the Laboratory and Field

    Get PDF
    Most marine organisms are broadcast spawners, releasing their sperm and eggs into the water column. Methods of measuring in situ fertilization have proven successful with a few model species, which are reviewed in my introductory chapter. However, many commercially exploited species, such as the sea scallop Placopecten magellanicus, have been neglected. Sea scallop populations have greatly increased from fishing closures, but the mechanism behind this response is uncertain, particularly in regard to fertilization. In this dissertation I developed a methodology of measuring fertilization success and spawning events of P. magellanicus, tested it in laboratory and field settings, and developed a novel genetic probe to detect and quantify scallop gametes. Chapter 2 describes laboratory experiments and field results from our development of nylon mesh chambers used to measure fertilization success (percent of eggs fertilized) in situ. In dilution-series experiments, maximum fertilization success occurred at sperm concentrations \u3e107 sperm ml‚Äď1 . Between 8 and 24 h at ambient temperature, egg viability fell to zero. Sperm half-life shortened from 2 h to 9 min when sperm concentrations diluted by 10-fold from 107 cells ml‚Äď1 to 106 cells ml‚Äď1 . Flume trials demonstrated chamber artifacts: fertilization was lower inside the chamber than outside, and the effect was greater at higher flow rates, but chamber orientation to flow had no effect on fertilization. Increasing the numbers of eggs tended to reduce fertilization success. In dockside tests, a 30-fold difference in spawner numbers had a significant effect on fertilization success. In Chapter 3, I analyzed video surveys of scallop aggregations on western Atlantic fishery grounds to determine whether population density, degree of aggregation, and shell size were correlated with fishing closures. Based on these data, I created experimental benthic populations to measure fertilization success in situ. Fertilization success in these experiments did not vary significantly across a 10-fold difference in population density, a result which was inconsistent with the outcome predicted by a current fertilization model. This likely underscores the extreme variability in fertilization success in the field that is not captured by models. In Chapter 4 I developed and tested a genetic probe (Pmag_304F) and primer set (Pmag_282F, Pmag_492R) to detect and quantify P. magellanicus gametes in the water column. I used a TaqMan fluorescent probe and primer set to target the intergenic spacer region (ITS) in the scallop genome. To verify this probe works on scallop gametes, I tested it on replicate sperm dilution series. This method may be applied to field samples to detect and quantify spawning events for this species and other important invertebrates. This dissertation presents empirical data on the relationship between spawner abundance and fertilization success in P. magellanicus, evidence for a possible component Allee effect, some form of compensation at low densities and the development of two methods to detect spawning events in the field. These new tools and data improve our understanding of a previously poorly studied aspect of scallop reproduction, and may provide insight into their resilience to fishing pressure
    corecore