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

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

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

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

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

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

Tri-locus sequence data reject a Gondwanan origin hypothesis for the African/South Pacific crab genus Hymenosoma

Crabs of the family Hymenosomatidae are common in coastal and shelf regions throughout much of the southern hemisphere. One of the genera in the family, Hymenosoma, is represented in Africa and the South Pacific (Australia and New Zealand). This distribution can be explained either by vicariance (presence of the genus on the Gondwanan supercontinent and divergence following its break-up) or more recent transoceanic dispersal from one region to the other. We tested these hypotheses by reconstructing phylogenetic relationships among the seven presently-accepted species in the genus, as well as examining their placement among other hymenosomatid crabs, using sequence data from two nuclear markers (Adenine Nucleotide Transporter [ANT] exon 2 and 18S rDNA) and three mitochondrial markers (COI, 12S and 16S rDNA). The five southern African representatives of the genus were recovered as a monophyletic lineage, and another southern African species, Neorhynchoplax bovis, was identified as their sister taxon. The two species of Hymenosoma from the South Pacific neither clustered with their African congeners, nor with each other, and should therefore both be placed into different genera. Molecular dating supports a post-Gondwanan origin of the Hymenosomatidae. While long-distance dispersal cannot be ruled out to explain the presence of the family Hymenosomatidae on the former Gondwanan land-masses and beyond, the evolutionary history of the African species of Hymenosoma indicates that a third means of speciation may be important in this group: gradual along-coast dispersal from tropical towards temperate regions, with range expansions into formerly inhospitable habitat during warm climatic phases, followed by adaptation and speciation during subsequent cooler phases

Biodiversidade e distribuição de larvas de invertebrados da plataforma Sudeste-Sul do Brasil (21-34 ºS), com ênfase em larvas de Decapoda

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Ecologia, Florianópolis, 2015.A maioria dos invertebrados marinhos produz larvas planctônicas, que permanecem desde minutos até meses no plâncton. Larvas de crustáceos decápodes frequentemente representam uma grande porcentagem das comunidades planctônicas em regiões neríticas. As características ambientais locais são um dos principais fatores que estruturam as comunidades planctônicas. Um dos principais desafios para os ecólogos marinhos é compreender em que medida as assembleias de larvas são influenciadas por fatores físicos e biológicos em diferentes cenários ambientais. No capítulo 1 foi testada a hipótese de que zonas frontais influenciam a distribuição da comunidade de larvas de invertebrados bentônicos ao longo da plataforma continental Sudeste-Sul do Brasil (entre 21 ºS e 34 ºS). Os resultados mostraram que a abundância e composição do meroplâncton são influenciadas pela distância da costa, frentes costeiras, de plataforma e estuarinas, além da ressurgência da Água Central do Atlântico Sul. Decapoda foi o grupo meroplanctônico mais frequente e recebeu maior destaque e resolução taxonômica na descrição da comunidade, originando o capítulo 2. O principal objetivo do capítulo 2 foi identificar os processos oceanográficos que estruturam a comunidade de larvas de decápodes ao longo da plataforma Sudeste-Sul do Brasil. Foi verificado que existe um acoplamento entre a abundância de larvas e a concentração de clorofila, especialmente para larvas de espécies bentônicas. Associações específicas de larvas foram observadas nos cenários ambientais mais relevantes: ambientes costeiros, ambientes oceânicos, e ambientes com predominância de escoamento continental. Dentre as larvas de decápodes bentônicos, caranguejos braquiúros apresentaram maior abundância e diversidade, especialmente próximo à costa. Com o objetivo de contribuir para os avanços na identificação molecular de larvas de decápodes, foi elaborado o capítulo 3. Nesse estudo, Brachyura foi utilizado como o primeiro grupo para investigar a viabilidade de identificação das larvas por DNA barcoding, em um arquipélago costeiro (27 ºS) do Sudeste-Sul do Brasil. A maior parte das larvas foi identificada com sucesso em nível específico, indicando que o uso dessa metodologia em trabalhos futuros de meso e larga escala pode contribuir para a identificação precisa e rápida de larvas de decápodes.Abstract : Most marine invertebrates produce planktonic larvae, which may spend from minutes to months in the plankton. Decapod crustacean larvae often form a large portion of the planktonic community in neritic regions. The local environmental characteristics are one of the main factors which structure the planktonic communities. One of the major challenges for marine ecologists is to understand to which extent larval assemblages are influenced by physical and biological factors under different environmental scenarios. In chapter 1 we tested the hypothesis that frontal zones affect the distribution of the benthic invertebrate larval community along the South Brazil Shelf (between 21 °S and 34 °S). The results showed that the abundance and composition of the meroplankton are influenced by the distance from the coast, coastal, shelf and estuarine fronts, in addition to the upwelling of the South Atlantic Central Water. Decapod was the most frequent meroplanktonic group and received more emphasis and taxonomic resolution in the community description, originating chapter 2. The main goal of chapter 2 was to identify the oceanographic processes that structure the decapod larval community along the South Brazil Shelf. A coupling between chlorophyll concentration and larval abundance, mainly of early larvae of benthic species, was observed. Specific assemblages of larvae were observed in the most relevant environmental scenarios: coastal environments, offshore environments, and areas highly influenced by the continental runoff. Among benthic decapod larvae, brachyuran crabs presented the highest abundance and diversity, especially near the coast. In order to contribute to advances in the molecular identification of decapod larvae we developed chapter 3. In this study, Brachyura was used as the first group to investigate the feasibility of using DNA barcoding for larval identification in a coastal archipelago (27 ºS) of the South Brazil Shelf. The majority of the larvae were successfully identified into species, indicating that the use of this method in future mesoscale and large-scale studies, may contribute to precise and fast identification of decapod larvae

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

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–1 . 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–1 to 106 cells ml–1 . 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

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

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’ 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 atlântica de França até ao Senegal. Tal como outros Donacidae, é relativamente pequena, achatada com uma concha sólida e alongada. É uma espécie com elevado valor económico e muito procurada especialmente no Algarve, levando a que exista um aumento da pressão por parte da frota de arrasto de ganchorra sobre a população. Em 1986 o Instituto Português do Mar e da Atmosfera (IPMA) iniciou um programa de monitorização dos recursos bivalves de modo a avaliar o estado dos estoques populacionais das espécies com valor económico, embora sempre sobre a população adulta. Desde esse período, várias medidas de gestão têm sido implementadas garantido a sustentabilidade da pesca. Apesar de existir variada informação acerca da população adulta ao longo da costa algarvia, não existe qualquer informação sobre a fase planctónica desta espécie. Com o intuito de colmatar esta falha, o presente estudo teve como objectivo obter pela primeira vez informação acerca da condição dos progenitores e da fase planctónica da conquilha na costa Algarvia.Fundação para a Ciência e TecnologiaFundo Social Europe

Development of Molecular Markers for the Identification of Bivalve Molluscan Larvae for Mariculture Applications

Mariculture of Perna viridis (green mussel), Perna indica (brown mussel) and Crassostrea madrasensis (Indian backwater oyster) is getting popular in India but the productivity of this sector is affected by inadequate supply of spats. The natural spat resources are the only viable option available to the bivalve farmers to meet the seed requirements. Early detection of the larvae of the three target species in the plankton samples collected from the coastal waters will be helpful to predict the time of spat settlement which is crucial for the successful spat collection using cultch materials. The present work deals with the development of DNA markers in the form of Species Specific PCR (SSPCR) and Species Specific nested PCR (SSnPCR) for the specific and sensitive detection of the larvae of three target bivalves from unsorted plankton samples. Analysis of the experimental plankton samples using SSnPCR proved that the method can detect even a single veliger larva from a plankton biomass of 40mg. Similarly, SSPCR could be used to detect a minimum of 20 numbers of veliger larve from a plankton biomass of 40 mg. The SSPCR could also be used to assess the numerical density of the target bivalve larvae in coastal water and it was possible to detect approximately 106 larvae in 1000 liters of water. The utility of SSPCR and SSnPCR was also evaluated in the plankton samples collected from the coastal waters. The spat collectors placed in the study area were observed to have rich settlement of the target bivalve larvae showing a positive correlation with the lab results obtained. Screening of the Plankton samples have proved that these methods can be effectively employed to detect the presence of the target bivalve larvae in the coastal waters and it can be used as a ‘spat-fall prediction tool’ to manage the spat collection process more effectively by the bivalve farmers

The future of cephalopod populations, fisheries, culture, and research in Europe

International audienc