Cosmopolitan Distribution of Endozoicomonas-Like Organisms and Other Intracellular Microcolonies of Bacteria Causing Infection in Marine Mollusks.

Intracellular microcolonies of bacteria (IMC), in some cases developing large extracellular cysts (bacterial aggregates), infecting primarily gill and digestive gland, have been historically reported in a wide diversity of economically important mollusk species worldwide, sometimes associated with severe lesions and mass mortality events. As an effort to characterize those organisms, traditionally named as Rickettsia or Chlamydia-like organisms, 1950 specimens comprising 22 mollusk species were collected over 10 countries and after histology examination, a selection of 99 samples involving 20 species were subjected to 16S rRNA gene amplicon sequencing. Phylogenetic analysis showed Endozoicomonadaceae sequences in all the mollusk species analyzed. Geographical differences in the distribution of Operational Taxonomic Units (OTUs) and a particular OTU associated with pathology in king scallop (OTU_2) were observed. The presence of Endozoicomonadaceae sequences in the IMC was visually confirmed by in situ hybridization (ISH) in eight selected samples. Sequencing data also indicated other symbiotic bacteria. Subsequent phylogenetic analysis of those OTUs revealed a novel microbial diversity associated with molluskan IMC infection distributed among different taxa, including the phylum Spirochetes, the families Anaplasmataceae and Simkaniaceae, the genera Mycoplasma and Francisella, and sulfur-oxidizing endosymbionts. Sequences like Francisella halioticida/philomiragia and Candidatus Brownia rhizoecola were also obtained, however, in the absence of ISH studies, the association between those organisms and the IMCs were not confirmed. The sequences identified in this study will allow for further molecular characterization of the microbial community associated with IMC infection in marine mollusks and their correlation with severity of the lesions to clarify their role as endosymbionts, commensals or true pathogens.

Dynamics of the Pacific oyster pathobiota during mortality episodes in Europe assessed by 16S rRNA gene profiling and a new target enrichment next-generation sequencing strategy.

Infectious agents such as the bacteria Vibrio aestuarianus or Ostreid herpesvirus 1 have been repeatedly associated with dramatic disease outbreaks of Crassostrea gigas beds in Europe. Beside roles played by these pathogens, microbial infections in C. gigas may derive from the contribution of a larger number of microorganisms than previously thought, according to an emerging view supporting the polymicrobial nature of bivalve diseases. In this study, the microbial communities associated with a large number of C. gigas samples collected during recurrent mortality episodes at different European sites were investigated by real-time PCR and 16SrRNA gene-based microbial profiling. A new target enrichment next-generation sequencing protocol for selective capturing of 884 phylogenetic and virulence markers of the potential microbial pathogenic community in oyster tissue was developed allowing high taxonomic resolution analysis of the bivalve pathobiota. Comparative analysis of contrasting C. gigas samples conducted using these methods revealed that oyster experiencing mortality outbreaks displayed signs of microbiota disruption associated with the presence of previously undetected potential pathogenic microbial species mostly belonging to genus Vibrio and Arcobacter. The role of these species and their consortia should be targeted by future studies aiming to shed light on mechanisms underlying polymicrobial infections in C. gigas.

Multibiomarker biomonitoring approach using three bivalve species in the Ebro Delta (Catalonia, Spain).

Bivalves have proved to be useful bioindicators for environmental pollution. In the present study, mussels (Mytilus galloprovincialis), cockles (Cerastoderma edule), and razor shells (Solen marginatus) were collected in the Ebro Delta, an extensive area devoted to rice farming and affected by pesticide pollution, from April to July, the heaviest rice field treatment period. Possible effects of pollution were assessed through biochemical markers (carboxylesterase (CE), antioxidant and neurotoxicity-related enzymes, and lipid peroxidation levels). Data on environmental variables, bivalve reproductive condition, and presence of organic pollutants, marine phycotoxins, pathogens, or histopathological conditions in bivalve's tissues were also evaluated. Although the bioaccumulated pesticides did not explain the patterns observed for biochemical responses, the obtained results point to an effect of environmental pesticide pollution on enzymatic markers, with a prominent contribution of CE to such changes. Mussels and razor shells provided a more sensitive biochemical response to pollution than cockles. Environmental variables, bivalve reproductive condition, and marine phycotoxins did not seem to have a relevant effect on the biomarkers assessed.

Rapid capture and detection of ostreid herpesvirus-1 from Pacific oyster Crassostrea gigas and seawater using magnetic beads.

Ostreid herpesvirus-1 (OsHV-1) has been involved in mass mortality episodes of Pacific oysters Crassostrea gigas throughout the world, causing important economic losses to the aquaculture industry. In the present study, magnetic beads (MBs) coated with an anionic polymer were used to capture viable OsHV-1 from two types of naturally infected matrix: oyster homogenate and seawater. Adsorption of the virus on the MBs and characterisation of the MB-virus conjugates was demonstrated by real-time quantitative PCR (qPCR). To study the infective capacity of the captured virus, MB-virus conjugates were injected in the adductor muscle of naïve spat oysters, using oyster homogenate and seawater without MBs as positive controls, and bare MBs and sterile water as negative controls. Mortalities were induced after injection with MB-virus conjugates and in positive controls, whereas no mortalities were recorded in negative controls. Subsequent OsHV-1 DNA and RNA analysis of the oysters by qPCR and reverse transcription qPCR (RT-qPCR), respectively, confirmed that the virus was the responsible for the mortality event and the ability of the MBs to capture viable viral particles. The capture of viable OsHV-1 using MBs is a rapid and easy isolation method and a promising tool, combined with qPCR, to be applied to OsHV-1 detection in aquaculture facilities.

Application of a competitive real time PCR for detection of Marteilia refringens genotype "O" and "M" in two geographical locations: The Ebro Delta, Spain and the Rhine-Meuse Delta, the Netherlands.

Species belonging to the genus Marteilia are protozoan parasites of bivalves. The species Marteilia refringens, jeopardizing the health of European bivalves, is included on the list of OIE notifiable pathogens. Two genotypes of Marteilia refringens are distinguished: type "O" affecting mainly oysters, and type "M" affecting mainly mussels. Historically, detection of Marteilia species is primarily carried out by histology. In recent years molecular assays are more frequently used for the detection of mollusc pathogens, also in routine monitoring. In the present work, a competitive real-time PCR assay was developed for rapid and sensitive detection of M. refringens and discrimination between "M" and "O" genotypes of M. refringens. The real-time PCR assay was shown to be analytically sensitive and specific and has a high repeatability and efficiency. Subsequent application of the assay on collected bivalves from two geographical locations, the Ebro Delta in Mediterranean Spain and the Rhine-Meuse Delta in the Netherlands resulted in detection of M. refringens type M in Mytilus galloprovincialis and M. refringens type O in Ostrea edulis from Spain. In two O. edulis specimen both M. refringens type O and type M were detected. In the Netherlands M. refringens was not observed in any of the tested Mytilus edulis and O. edulis. The results obtained by real time PCR were in correspondence with the results obtained by histopathology and a substantial agreement with the results obtained by conventional PCR. In conclusion, the developed real time PCR assay facilitates rapid detection and subtyping of M. refringens and could be applied for further studies on epidemiology of the parasite, geographical distribution and host specificity.

A Production Calendar Based on Water Temperature, Spat Size, and Husbandry Practices Reduce OsHV-1 µvar Impact on Cultured Pacific Oyster Crassostrea gigas in the Ebro Delta (Catalonia), Mediterranean Coast of Spain.

Since 2006, the production of Pacific oyster Crassostrea gigas in the Ebro Delta area has dramatically declined from around 800 metric tons (MT) per year to 138 MT in 2011. This decline in production has had a significant socio-economic impact in a region where the shellfish sector is a traditional economic activity for many families. The identified agent responsible for this reduction in C. gigas production was Ostreid Herpesvirus microvar (OsHV-1 µvar), which has been associated with C. gigas spat mortalities in France, and in many other countries. In Spain the episodes of mortality became critical for the regional shellfish production between 2008 until 2014, with mortality percentage up to 100%. In this study, local hatchery C. gigas spat was used as sentinel animals for epidemiological studies and management tests carried out with the aim of reducing oyster mortality in the Ebro Delta area. A production calendar mainly based on water temperature dynamics was designed around an optimal schedule for spat immersion. The immersion calendar included two optimal periods for spat immersion, in summer when temperatures are ≥25°C and at the end of autumn and beginning of winter when they are ≤13°C. Such production planning has reduced mortalities from 80% (in 2014 and previous years) to 2-7.5% in 2015 in cemented oysters. Furthermore, other recommendations related to spat immersion size, culture density and methodology, and cementing calendar, which helped to achieve the results presented, were also recorded and transferred to local producers. This work presents a successfully tested management strategy reducing OsHV-1 µvar impact by designing new field management practices mainly focused on the handling and timing of spat immersion. This approach could be used as a management model in areas presenting similar production practices and environmental characteristics.

Marteilia spp. parasites in bivalves: A revision of recent studies.

Marteilia spp. parasites are Paramyxean organisms that affect several commercial species of molluscs, and thus have a socio-economical impact. These parasites also have an ecological impact on the biodiversity and population dynamics of natural mollusc beds. It has been over forty years since the first description of Marteilia refringens, the first Marteilia species described. Despite four decades of research, the biology, cellular development, as well as the life cycle of Marteilia parasites are not fully understood. In recent years, new studies have reported advances in knowledge of the life cycle, the description of new species, the development of new molecular detection/discrimination tools, new data on biotype geographical distribution, as well as new information on host response and defence mechanisms. Such information is summarized, reported and discussed in the present review in order to facilitate a new overview of the subject. However, numerous knowledge gaps are still unresolved and need to be prioritized in the research and funding institution agendas.

Update of information on perkinsosis in NW Mediterranean coast: Identification of Perkinsus spp. (Protista) in new locations and hosts.

This study addressed perkinsosis in commercially important mollusc species in the western Mediterranean area. Perkinsus olseni was found in Santa Gilla Lagoon (Sardinia) infecting Ruditapes decussatus, Cerastoderma glaucum and Venerupis aurea, in Balearic Islands infecting Venus verrucosa and in Delta de l'Ebre (NE Spain) parasitising Ruditapes philippinarum and R. decussatus. Perkinsus mediterraneus was detected infecting Ostrea edulis from the Gulf of Manfredonia (SE Italy) and Alacant (E Spain), V. verrucosa and Arca noae from Balearic Islands and Chlamys varia from Balearic Islands, Alacant and Delta de l'Ebre.

Nocardiosis in Mediterranean bivalves: first detection of Nocardia crassostreae in a new host Mytilus galloprovincialis and in Ostrea edulis from the Gulf of Naples (Italy).

In this work M. galloprovincialis and O. edulis specimens were surveyed for a pathological study in the Gulf of Naples (Mediterranean sea, Campania Region, southern Italy). Clusters of Nocardia sp.-like cells were observed in histological slides. PCR amplification, sequencing and in situ hybridization were carried out in order to corroborate Nocardia species identification for both hosts. Blast results showed a 99% of maximum identity with Nocardia crassostreae sequences in Genbank. This is the first report of N. crassostreae in the new host M. galloprovincialis and, in a new area, the Mediterranean Sea.

Development of a real-time PCR for detection of the oyster pathogen Nocardia crassostreae based on its homogeneous 16S-23S rRNA intergenic spacer region.

Nocardia crassostreae, the causative agent of Pacific oyster nocardiosis (PON), is a Gram-positive actinomycete bacterium associated with Pacific oyster (Crassostrea gigas) mortalities. Oysters infected with this bacterium have been reported previously from the west coast of North America and Japan. More recently, N. crassostreae was reported in oyster culture areas in the Netherlands. In this study, a sensitive real-time PCR for specific detection of N. crassostreae was developed, and the intra-species divergence of N. crassostreae from different geographical locations was studied. The 16S-23S rRNA intergenic spacer (ITS) region of N. crassostreae was sequenced for a number of infected oysters originating from the Netherlands, Japan and Canada. The sequence analyses showed an absence of genetic variation in the ITS region between N. crassostreae from different geographical locations. Based on these ITS sequences a species-specific and highly sensitive SYBR Green real-time PCR assay was developed to facilitate detection of N. crassostreae in oyster tissue. To evaluate this new detection tool for N. crassostreae a preliminary validation was carried out and real-time PCR results were compared with other detection methods (histology, conventional PCR and bacterial isolation) using field samples from Lake Grevelingen, the Netherlands. The genetic homogeneity in the ITS region between N. crassostreae from different geographical locations might be explained by the recent spread of the organism via the international trade in Pacific oysters for aquaculture purposes. However, the lack of genetic variation could also suggest that N. crassostreae is a genetically monomorphic species.

Phylogenetic relationship of Perkinsus olseni from the Ebro Delta, Spain, to other Perkinsus species, based on ribosomal DNA sequences.

The phylogenetic relationship of Perkinsus olseni originating from the Ebro Delta, Spain, to other Perkinsus spp. was investigated using the nontranscribed spacer (NTS) region and the internal transcribed spacer (ITS) region (including ITS1, 5.8S and ITS2) of the ribosomal DNA sequences. These 2 molecular markers (NTS and ITS) were sequenced from prezoosporangia of Perkinsus sp. originating from Manila clam Ruditapes philippinarum from the Ebro Delta. The sequence of the 5.8S ITS region of the ribosomal RNA gene was 100% similar to that of P. olseni. Higher genetic variability was found for the NTS sequence, with 80.7 to 81.8% similarity to P. olseni. The NTS sequence of a P. olseni isolate previously detected in R. decussatus from the same area was also obtained and showed 81% identity with our isolate. Evidence obtained from phylogenetic analysis of the 5.8S ITS and NTS aligned sequences appears to indicate that P. olseni strains group together according to their host rather than their geographic origins within a well-resolved P. olseni clade.

First record of Perkinsus olseni, a protozoan parasite infecting the commercial clam Ruditapes decussatus in Spanish Mediterranean waters.

We present the first record in Spanish Mediterranean waters of the protozoan parasite Perkinsus olseni infecting the clam Ruditapes decussatus. Perkinsus infection was detected all year around albeit at a low level of infection intensity. Histological analysis, induction of zoospores and in situ hybridisation assay confirmed the presence of Perkinsus sp. The identity of the parasite was determined by species-specific PCR assay in DNA samples obtained from infected clams. Sequencing of amplified fragments showed 100% identity to the ITS region of P. olseni. We confirmed for the first time the presence of P. olseni in Spanish Mediterranean waters.