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There is a growing effort throughout Europe to restore populations of native oysters (Ostrea edulis), with the ecological objective of enhancing ecosystem biodiversity and resilience.The introduced parasite, Bonamia ostreae, caused catastrophic mortalities during the 1980s, furthering the decline of this species, and is now present throughout much of the natural range of O. edulis. It is therefore important that restoration attempts avoid further introduction and spread of this parasite, which can cause lethal infections of O. edulis.This article presents a comprehensive overview of the scale and distribution of current infection, transmission pathways, and preventive measure guidelines, focusing on the seas, inlets, and estuaries of north‐west Europe, where most ecological restoration attempts for the native European oyster have occurred so far.This is critical information for restoration project planning in which the risk of Bonamia infection must be taken into account.

International audience; In bivalves, no clear-cut functional role of microbiota has yet been identified, although many publications suggest that they could be involved in nutrition or immunity of their host. In the context of climate change, integrative approaches at the crossroads of disciplines have been developed to explore the environment-host-pathogen microbiota system. Here, we attempt to synthesize work on (1) the current methodologies to analyse bivalve microbiota, (2) the comparison of microbiota between species, between host compartments and their surrounding habitat, (3) how the bivalve microbiota are governed by environmental factors and host genetics and (4) how host-associated microorganisms act as a buffer against pathogens and/or promote recovery, and could thereby play a role in the prevention of disease or mortalities.

Diseases represent a major threat for the bivalve production industry. Their control relies on biosecurity measures to prevent their introduction and limit their spread. When maintained in hatcheries, nurseries and depuration centers, bivalves can become infected from the surrounding water and might release pathogens through wastewater effluents. A major effort was done in controlling the safety of bivalves for human consumption, but, on the other hand, information regarding the resistance of mollusc pathogens to water treatment is scarce. The effect of ultraviolet exposure was tested on two protozoan parasites of marine bivalves, the non-culturable parasite Bonamia ostreae and Perkinsus olseni culturable in DMEM/HAM´s medium. UV exposure experiments were carried out first at the bench scale and then, for P. olseni, at a larger scale mimicking depuration plants, hatcheries and nurseries conditions. At the bench scale, our study indicated that up to 40% of B. ostreae cells and 85% of P. olseni cells died 24 h and 21 days respectively after an exposure to 94 mJ/cm2 of UV-C. After 40 mJ/cm2 exposure, P. olseni density increased between 15 and 21 days of culture suggesting that the parasite is able to recover from low UV intensity exposure. At large scale, no signs of UV recovery were seen in P. olseni cultures, but, at lower intensity (216–244 mJ/cm2), 15% of the parasites remained alive 21 days post exposure. Finally, a minimum dose of 94 mJ/cm2 seems required to inhibit proliferation of parasites and 450 mJ/cm2 to completely kill all parasites. Taken this into account, a dose higher than 450 mJ/cm2 is suggested to properly treat water to avoid dispersion of bivalve protozoan parasites such as P. olseni.

The aetiological agent Perkinsus olseni is globally recognised as a major threat for shellfish production considering its wide geographical distribution across Asia, Europe, Australia and South America. Another species, Perkinsus chesapeaki, which has never been known to be associated with significant mortality events, was recently detected along French coasts infecting clam populations sporadically in association with P. olseni. Identifying potential cryptic infections affecting Ruditapes philippinarum is essential to develop appropriate host resource management strategies. Here, we developed a molecular method based on duplex real-time quantitative PCR for the simultaneous detection of these two parasites, P. olseni and P. chesapeaki, in the different clam tissues: gills, digestive gland, foot, mantle, adductor muscle and the rest of the soft body. We firstly checked the presence of possible PCR inhibitors in host tissue samples. The qPCR reactions were inhibited depending on the nature of the host organ. The mantle and the rest of the soft body have a high inhibitory effect from threshold of host gDNA concentration of 2 ng.µL−1, the adductor muscle and the foot have an intermediate inhibition of 5 ng.µL−1, and the gills and digestive gland do not show any inhibition of the qPCR reaction even at the highest host gDNA concentration of 20 ng.µL−1. Then, using the gills as a template, the suitability of the molecular technique was checked in comparison with the Ray’s Fluid Thioglycolate Medium methodology recommended by the World Organisation for Animal Health. The duplex qPCR method brought new insights and unveiled cryptic infections as the co-occurrence of P. olseni and P. chesapeaki from in situ tissue samples in contrast to the RFTM diagnosis. The development of this duplex qPCR method is a fundamental work to monitor in situ co-infections that will lead to optimised resource management and conservation strategies to deal with emerging diseases.

The conservation and active restoration of European flat oyster (Ostrea edulis) populations are a major focus of ecological restoration efforts to take advantage of the wide‐ranging ecosystem functions and services this species provides. Accordingly, additional and new demands for seed oysters have arisen. In commercial aquaculture (mariculture), the production of O. edulis is still largely based on natural seed collection. Considering the specific requirements, related to ecological restoration, such as the absence of pathogens and the preservation of high genetic diversity, the current supply is insufficient. Despite the development of breeding and controlled reproduction techniques for this species since the late 1930s, seed production today is mainly based on empirical concepts. Several of the issues that producers still face are already subjects of research; many others are still unanswered or even unaddressed. This review provides a summary of all available knowledge and technologies of O. edulis seed production. Furthermore, it provides a detailed reflection on implications for restoration, future challenges, open questions and it identifies relevant research topics for sustainable seed supply. The study covers the following aspects on (i) biology of the species, (ii) stressors – including pathogens and pollutants, (iii) genetics, (iv) history of production technologies, (v) seed production in polls, (vi) seed production in ponds and (vii) seed production in hatcheries. Future research needs on sex determinism, gametogenesis, cryopreservation, nutrition, selective breeding, pathogens and disease, and the development of reliable protocols for production are highlighted.

Aquaculture including shellfish production is an important food resource worldwide which is particularly vulnerable to infectious diseases. Marteilia refringens, Bonamia ostreae and Bonamia exitiosa are regulated protozoan parasites infecting flat oysters Ostrea edulis that are endemic in Europe. Although some PCR assays have been already developed for their detection, a formal validation to assess the performances of those tools is often lacking. In order to facilitate the diagnosis of flat oyster regulated diseases, we have developed and evaluated a new multiplex Taqman (R) PCR allowing the detection of both M. refringens and Bonamia sp. parasites in one step. First part of this work consisted in assessing analytical sensitivity and specificity of the new PCR assay. Then, diagnostic performances were assessed by testing a panel of field samples with the new real-time PCR and currently recommended conventional PCR methods for the detection of M. refringens and Bonamia sp. Samples were collected from the main flat oyster production sites in France (N = 386 for M. refringens and N = 349 for B. ostreae). In the absence of gold standard, diagnostic sensitivity and specificity of the new PCR were estimated through Bayesian latent class analysis (DSe 87,2% and DSp 98,4% for the detection M. refringens, DSe 77,5% and DSp 98,4% for the detection of Bonamia sp.). Those results suggest equivalent performances for the detection of Bonamia sp. and an improved sensitivity for the detection of M. refringens compared to commonly used conventional protocols. Finally, the new PCR was evaluated in the context of an inter-laboratory comparison study including 17 European laboratories. Results revealed a very good reproducibility with a global accordance (intralaboratory precision) >96% and a global concordance (inter-laboratory precision) >93% for both targets, demonstrating that this new tool is easily transferable to different laboratory settings. This is the first assay designed to detect both Marteilia refringens and Bonamia sp. in a single step and it should allow reducing the number of analysis to monitor both diseases, and where relevant to demonstrate freedom from infection.

17 pages, 3 tables, 4 figures, Bonamia ostreae is an intrahemocytic parasite that has been responsible for severe mortalities in the flat oyster Ostrea edulis since the 1970́s. The Pacific oyster Crassostrea gigas is considered to be resistant to the disease and appears to have mechanisms to avoid infection. Most studies carried out on the invertebrate immune system focus on the role of hemolymph, although mucus, which covers the body surface of molluscs, could also act as a barrier against pathogens. In this study, the in vitro effect of mucus from the oyster species Ostrea edulis and C. gigas on B. ostreae was investigated using flow cytometry. Results showed an increase in esterase activities and mortality rate of parasites exposed to mucus from both oyster species. In order to better understand the potential role of mucus in the defense of the oyster against parasites such as B. ostreae, liquid chromatography and tandem mass spectrometry were used to describe and compare mucus protein composition from both species. In all oyster species, pallial mucus contains a high level of proteins; however, O. edulis mucus produced a variety of proteins that could be involved in the immune response against the parasite, including Cu/Zn extracellular superoxide dismutase, thioxiredoxin, peroxiredon VI, heat shock protein 90 as well as several hydrolases. Conversely, a different set of antioxidant proteins, hydrolases and stress related proteins were identified in mucus from C. gigas. Our results suggest an innate immunity adaptation of oysters to develop a specific response against their respective pathogens. The mucosal protein composition also provides new insights for further investigations into the immune response in oysters, The Région of Poitou Charentes and IFREMER supported this research. This work was also supported by the project POCI-01-0145-FEDER-030232, co-financed by COMPETE 2020, Portugal 2020 and the European Union through the ERDF, and by the FCT through national funds and strategical project UID/Multi/04423/2019

The Gulf of La Spezia (northern Tyrrhenian Sea, Italy) is a commercially important area both as a shipping port and for mussel farming. Recently, there has been increased concern over environmental disturbances caused by anthropogenic activities such as ship traffic and dredging and the effects they have on the health of farmed mussels. This paper reports the results of microbiological and histological analyses, as well as of measurement of several biomarkers which were performed to assess the health status of mussels (Mytilus galloprovincialis) from four rearing sites in the Gulf of La Spezia. Mussels were collected between October 2015 and September 2016 and histological analyses (including gonadal maturation stage), as well as the presence of pathogenic bacteria (Vibrio splendidus clade, V. aestuarianus and V. harveyi), viruses (Herpes virus and ostreid Herpes virus 1) and protozoa (Marteilia spp., in the summer season only) were carried out on a monthly basis. Conversely, biomarker responses in haemocyte/haemolymph (total haemocyte count, haemocyte diameter and volume, lysozyme and lactate dehydrogenase activities in cell-free haemolymph, and micronuclei frequency) and in gills and digestive gland (cortisol-like steroids and lipid peroxidation levels), were evaluated bimonthly. Microbiological data indicated that mussels contain a reservoir of potentially pathogenic bacteria, viruses and protozoa that in certain environmental conditions may cause a weakening of the immune system of animals leading to mortality episodes. The percentage of parasites detected in the mussels was generally low (9.6% for Steinhausia mytilovum, that is 17 samples out of 177 examined females; 3.4% for Proctoeces maculatus; 0.9% for Mytilicola intestinalis and 2% for ciliated protozoa), while symbiont loads were higher (31% for Eugymnanthea inquilina and Urastoma cyprinae). Interestingly, a previously undescribed haplosporidian was detected in a single mussel sample (0.2%) and was confirmed by in situ hybridization. Cells morphologically similar to Perkinsus sp. trophozoites were observed in 0.7% of the mussels analysed; however, infection with Perkinsus spp. could neither be confirmed by ISH nor by PCR. Different pathological aspects, such as host defence responses and regressive/progressive changes were detected in the gills, digestive glands, gonads and mantle. Only one single case of disseminated neoplasia (0.2%) was observed. As for the biomarker evaluation, the MANOVA analysis revealed the statistically significant effect that the variable "sampling site" had on the biological parameter measured, thus suggesting that the multibiomarker approach was able to differentiate the rearing sites.

Invasive species, and especially invasive parasites, represent excellent models to study ecological and evolutionary mechanisms in the wild. To understand these processes, it is crucial to obtain more knowledge on the native range, invasion routes and invasion history of invasive parasites. We investigated the consecutive invasions of two parasitic copepods (Mytilicola intestinalis and Mytilicolaorientalis) by combining an extensive literature survey covering the reported putative native regions and the present-day invaded regions with a global phylogeography of both species. The population genetic analyses based on partial COI sequences revealed significant population differentiation for M. orientalis within the native region in Japan, while introduced populations in North America and Europe could not be distinguished from the native ones. Thus, M. orientalis’ invasion history resembles the genetic structure and recent spread of its principal host, the Pacific oyster, Crassostrea gigas, while M. intestinalis lacks population genetic structure and has an overall low genetic diversity. Therefore, the native origin of M. intestinalis remains unclear. With this study, we demonstrate that even highly related and biologically similar invasive species can differ in their invasion genetics. From this, we conclude that extrapolating invasion genetics dynamics from related invasive taxa may not always be possible.

Marteilia refringens causes marteiliosis in oysters, mussels and other bivalve molluscs. This parasite previously comprised two species, M. refringens and Marteilia maurini, which were synonymized in 2007 and subsequently referred to as M. refringens ‘O-type’ and ‘M-type’. O-type has caused mass mortalities of the flat oyster Ostrea edulis. We used high throughput sequencing and histology to intensively screen flat oysters and mussels (Mytilus edulis) from the UK, Sweden and Norway for infection by both types and to generate multi-gene datasets to clarify their genetic distinctiveness. Mussels from the UK, Norway and Sweden were more frequently polymerase chain reaction (PCR)-positive for M-type (75/849) than oysters (11/542). We did not detect O-type in any northern European samples, and no histology-confirmed Marteilia-infected oysters were found in the UK, Norway and Sweden, even where co-habiting mussels were infected by the M-type. The two genetic lineages within ‘M. refringens’ are robustly distinguishable at species level. We therefore formally define them as separate species: M. refringens (previously O-type) and Marteilia pararefringens sp. nov. (M-type). We designed and tested new Marteilia-specific PCR primers amplifying from the 3’ end of the 18S rRNA gene through to the 5.8S gene, which specifically amplified the target region from both tissue and environmental samples.

This is the first study of the metabolic enzyme activity and energy state of the dwarf oysterOstrea stentina(Payraudeau, 1826) in Tunisia and in the Mediterranean region. The main purpose of this study was to examine the modulation of the physiological status of the oysterO. stentinadepending on the season and the presence of parasiteMarteilia refringens. The prevalence of bonamiosis and marteiliosis were established by PCR.Bonamia exitiosawas detected only in 2.91% of oysters while the prevalence ofM. refringenswas 100% (30 Ind./30) in February and 93.93% (28 Ind./30) in March. A 42–87% mortality rate was reported during the study period. Biochemical analyses were carried out to evaluate the management of the energy resources in regard to the biochemical changes of lipids and carbohydrates. The enzyme activity of pyruvate kinase (PK), citrate synthase (CS) and cytochrome c oxidase (CCO) were measured and the mean level of enzyme activity was respectively 20.31 mU mg−1protein; 12.06 mU mg−1protein and 3.59 mU mg−1protein. Carbohydrate contents ofO. stentinawere very low all year round with an average of 15.18% in dry weight, and lipid contents remain similar (11.77% in dry weight) compared with the values reported for most other temperate bivalves. Enzyme activity significantly decreased over time (P< 0.001). The energy resources of the oysterO. stentinawherein much of the energy was devoted to reproduction seem to affect the defence system.

An understanding of husbandry strategies and any associated risk factors is important for designing management control measures that can reduce mortality in Pacific oysters, Crassostrea gigas, caused by ostreid herpesvirus 1 (OsHV‐1). The type of culture facility can be considered in relation to the potential pathways that could lead to the entry of a pathogen and its survival. In addition, the animal host (e.g. age, physiological state, selective breeding programmes), husbandry procedures (e.g. stocking density), the pathogen itself (e.g. pathogenicity, virulence) and environmental effects (e.g. temperature) represent other relevant interconnected factors. However, all these factors provide valuable background information for outlining the mitigation strategies needed by the industry, as well as in the context of surveillance and biosecurity programmes. These control mechanisms for hatchery or nursery areas are related to movement restrictions, water treatment, virus inactivation, the production calendar and practical farm management decisions. This comprehensive literature review compiles information related to such approaches and also includes the different existing guidelines suggested for control of OsHV‐1. Therefore, the review represents a solid foundation for a more critical appraisal currently being developed to support recommendations for disease management strategies. info:eu-repo/semantics/publishedVersion

International audience; The SAR group (Stramenopila, Alveolata, Rhizaria) is one of the largest clades in the tree of eukaryotes and includes a great number of parasitic lineages. Rhizarian parasites are obligate and have devastating effects on commercially important plants and animals but despite this fact, our knowledge of their biology and evolution is limited. Here, we present rhizarian transcriptomes from all major parasitic lineages in order to elucidate their evolutionary relationships using a phylogenomic approach. Our results suggest that Ascetosporea, parasites of marine invertebrates, are sister to the novel clade Apofilosa. The phytomyxean plant parasites branch sister to the vampyrellid algal ectoparasites in the novel clade Phytorhiza. They also show that Ascetosporea + Apofilosa + Retaria + Filosa + Phytorhiza form a monophyletic clade, although the branching pattern within this clade is difficult to resolve and appears to be model-dependent. Our study does not support the monophyly of the rhizarian parasitic lineages (Endomyxa), suggesting independent origins for rhizarian animal and plant parasites.

Bonamiosis due to the parasite Bonamia ostreae has been associated with massive mortality outbreaks in European flat oyster stocks in Europe. As eradication and treatment are not possible, the control of the disease mainly relies on transfer restriction. Moreover, selection has been applied to produce resistant flat oyster families, which present better survival and lower prevalence than non-selected oysters. In order to better understand the mechanisms involved in resistance to bonamiosis, cellular and molecular responses of 2 oyster groups (selected oysters and wild-type oysters) were analyzed in the context of experimental injection and cohabitation infections. Cellular responses including non-specific esterases detection, ROS production and phagocytosis activity were analyzed by flow cytometry. Four genes homologous to those shown to be involved in immunity were selected (Inhibitor of apotosis OeIAP, Fas ligand OeFas-ligand, Oe-SOD, and OeEc-SOD) and monitored by quantitative reverse-transcription PCR (qRT-PCR). Infected oysters showed higher phagocytosis activity than controls. Infected selected oyster show a lower phagocytosis activity which might be a protection against the parasite infection. The expression of OeIAP and OeFas-ligand gene was significantly increased in selected oysters at 5 days post-injection. OeIAP gene expression appeared to be significantly increased in wild-type oysters at 8 days post-injection. Our results suggest that resistance to bonamiosis partly relies on the ability of the oysters to modulate apoptosis.

SUMMARYBonamia ostreaeis a protozoan parasite that infects the European flat oysterOstrea edulis, causing systemic infections and resulting in massive mortalities in populations of this valuable bivalve species. In this work, we have characterizedB. ostreaeactin genes and used their sequences for a phylogenetic analysis. Design of different primer sets was necessary to amplify the central coding region of actin genes ofB. ostreae. Characterization of the sequences and their amplification in different samples demonstrated the presence of 2 intragenomic actin genes inB. ostreae, without any intron. The phylogenetic analysis placedB. ostreaein a clade withMinchinia tapetis,Minchinia teredinisandHaplosporidium costaleas its closest relatives, and demonstrated that the paralogous actin genes found inBonamiaresulted from a duplication of the original actin gene after theBonamiaorigin.

Although a wide range of viruses have been reported in marine molluscs, most of these reports rely on ultrastructural examination and few of these viruses have been fully characterized. The lack of marine mollusc cell lines restricts virus isolation capacities and subsequent characterization works. Our current knowledge is mostly restricted to viruses affecting farmed species such as oysters Crassostrea gigas, abalone Haliotis diversicolor supertexta or the scallop Chlamys farreri. Molecular approaches which are needed to identify virus affiliation have been carried out for a small number of viruses, most of them belonging to the Herpesviridae and birnaviridae families. These last years, the use of New Generation Sequencing approach has allowed increasing the number of sequenced viral genomes and has improved our capacity to investigate the diversity of viruses infecting marine molluscs. This new information has in turn allowed designing more efficient diagnostic tools. Moreover, the development of experimental infection protocols has answered some questions regarding the pathogenesis of these viruses and their interactions with their hosts. Control and management of viral diseases in molluscs mostly involve active surveillance, implementation of effective bio security measures and development of breeding programs. However factors triggering pathogen development and the life cycle and status of the viruses outside their mollusc hosts still need further investigations.

SUMMARYThe paramyxean parasite Marteilia refringens infects several bivalve species including European flat oysters Ostrea edulis and Mediterranean mussels Mytilus galloprovincialis. Sequence polymorphism allowed definition of three parasite types ‘M’, ‘O’ and ‘C’ preferably detected in oysters, mussels and cockles respectively. Transmission of the infection from infected bivalves to copepods Paracartia grani could be experimentally achieved but assays from copepods to bivalves failed. In order to contribute to the elucidation of the M. refringens life cycle, the dynamics of the infection was investigated in O. edulis, M. galloprovincialis and zooplankton over one year in Diana lagoon, Corsica (France). Flat oysters appeared non-infected while mussels were infected part of the year, showing highest prevalence in summertime. The parasite was detected by PCR in zooplankton particularly after the peak of prevalence in mussels. Several zooplanktonic groups including copepods, Cladocera, Appendicularia, Chaetognatha and Polychaeta appeared PCR positive. However, only the copepod species Paracartia latisetosa showed positive signal by in situ hybridization. Small parasite cells were observed in gonadal tissues of female copepods demonstrating for the first time that a copepod species other than P. grani can be infected with M. refringens. Molecular characterization of the parasite infecting mussels and zooplankton allowed the distinguishing of three Marteilia types in the lagoon.

Among 1280 cultured and wild adult Mediterranean mussels, Mytilus galloprovincialis, collected over a 1-year surveillance period from the Slovene Adriatic Sea, 0.3% were histologically positive for the presence of Marteilia spp. The infection was concentrated in winter. Employing the molecular methods of PCR, cloning, DNA restriction and sequencing, only Marteilia refringens type M was detected in all the infected mussels. Although all life-cycle stages of M. refringens severely infected digestive glands, only sporadic disruption of epithelial cells of digestive tubules and focal destruction of digestive tubules were observed in the infected mussels. This was the first detection of M. refringens in M. galloprovincialis from the Slovene Adriatic Sea with the lowest prevalence reported to date. In addition, our results highlight the need for sequencing to complement the established PCR-RFLP analysis for correct parasite typing.

The yellow clam Amarilladesma mactroides (Deshayes, 1854) is considered a vulnerable species since the mid-1990s. Populations have experienced mass mortalities throughout their entire range of distribution (23–41°S) along exposed sandy beaches of Atlantic South America. Detrimental anthropogenic impacts have further contributed to failure of populations to make a recovery. To determine the factors involved in these events, density prior to a mortality event was calculated and live yellow clams encompassingmost of its geographic range distribution were analyzed histologically to describe parasites and pathologies. Moreover, moribund specimens were analyzed by molecular techniques to test for the presence of the virus OsHV-1. A mortality event was recorded after a maximum density of 127 clams/m2 was attained. No clear pattern was found between the prevalence and intensity of infection and localities, mortality events, or sampling season. Although OsHV-1 was not observed in any of the yellow clams tested, the possibility that another viral agent was implicated cannot be ruled out. The presence of bacteria of the genus Vibrio in combination with stress caused by a relatively high population density is suggested as the likely cause of these episodic mass mortalities. Fil: Vázquez, Nuria Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Centro Nacional Patagónico. Instituto de Biología de Organismos Marinos; Argentina Fil: Fiori, Sandra Marcela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto Argentino de Oceanografía. Universidad Nacional del Sur. Instituto Argentino de Oceanografía; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina Fil: Arzul, Isabelle. Institut Francais de Recherche pour l'Exploitation de la Mer; Francia Fil: Carcedo, Maria Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto Argentino de Oceanografía. Universidad Nacional del Sur. Instituto Argentino de Oceanografía; Argentina Fil: Cremonte, Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Centro Nacional Patagónico. Instituto de Biología de Organismos Marinos; Argentina

The in vitro model Ostrea edulis hemocyte - Bonamia ostreae is interesting to investigate host-parasite interactions at the cellular level. Indeed, this unicellular parasite infects the flat oyster Ostrea edulis and multiplies within hemocytes, the central effectors of oyster defenses. Apoptosis is a mechanism used by many organisms to eliminate infected cells. In order to study the potential involvement of this mechanism in the oyster response to B. ostreae, in vitro experiments were carried out by exposing hemocytes from the naturally susceptible oyster O. edulis and a resistant oyster species Crassostrea gigas to live and heat-inactivated parasites. Hemocyte apoptotic response was measured using a combination of flow cytometry and microscopy analyses. Whatever the host species was, the parasite was engulfed in hemocytes and induced an increase of apoptotic parameters including intracytoplasmic calcium concentration, mitochondrial membrane potential or phosphatidyl-serine externalization as well as ultrastructural modifications. However, the parasite appears more able to infect flat oyster than cupped oyster hemocytes and the apoptotic response was more important against live than dead parasites in the natural host than in C. gigas. Our results suggest that O. edulis specifically responds to B. ostreae by inducing apoptosis of hemocytes.

Marine mollusc production contributes to food and economic security worldwide and provides valuable ecological services, yet diseases threaten these industries and wild populations. Although the infrastructure for mollusc aquaculture health management is well characterized, its foundations are not without flaws. Use of notifiable pathogen lists can leave blind spots with regard to detection of unlisted and emerging pathogens. Increased reliance on molecular tools has come without similar attention to diagnostic validation, raising questions about assay performance, and has been accompanied by a reduced emphasis on microscopic diagnostic expertise that could weaken pathogen detection capabilities. Persistent questions concerning pathogen biology and ecology promote regulatory paralysis that impedes trade and which could weaken biosecurity by driving commerce to surreptitious channels. Solutions that might be pursued to improve shellfish aquaculture health management include the establishment of more broad-based surveillance programmes, wider training and use of general methods like histopathology to ensure alertness to emerging diseases, an increased focus on assay assessment and validation as fundamental to assay development, investment in basic research, and application of risk analyses to improve regulation. A continual sharpening of diagnostic tools and approaches and deepening of scientific knowledge is necessary to manage diseases and promote sustainable molluscan shellfish industries.

Complete and transparent reporting of key elements of diagnostic accuracy studies for infectious diseases in cultured and wild aquatic animals benefits end-users of these tests, enabling the rational design of surveillance programs, the assessment of test results from clinical cases and comparisons of diagnostic test performance. Based on deficiencies in the Standards for Reporting of Diagnostic Accuracy (STARD) guidelines identified in a prior finfish study (Gardner et al. 2014), we adapted the Standards for Reporting of Animal Diagnostic Accuracy Studies-paratuberculosis (STRADAS-paraTB) checklist of 25 reporting items to increase their relevance to finfish, amphibians, molluscs, and crustaceans and provided examples and explanations for each item. The checklist, known as STRADAS-aquatic, was developed and refined by an expert group of 14 transdisciplinary scientists with experience in test evaluation studies using field and experimental samples, in operation of reference laboratories for aquatic animal pathogens, and in development of international aquatic animal health policy. The main changes to the STRADAS-paraTB checklist were to nomenclature related to the species, the addition of guidelines for experimental challenge studies, and the designation of some items as relevant only to experimental studies and ante-mortem tests. We believe that adoption of these guidelines will improve reporting of primary studies of test accuracy for aquatic animal diseases and facilitate assessment of their fitness-for-purpose. Given the importance of diagnostic tests to underpin the Sanitary and Phytosanitary agreement of the World Trade Organization, the principles outlined in this paper should be applied to other World Organisation for Animal Health (OIE)-relevant species.

Bonamiosis due to the parasite Bonamia ostreae has been associated with massive mortality in flat oyster stocks in Europe. Control of the disease currently relies on disease management practices and transfer restriction. Previously, massal selections based on survival to challenge to infection with B. ostreae have been applied to produce flat oyster families with resistant progeny. In an attempt to understand the molecular mechanisms involved in disease resistance, differentially expressed sequence tags between resistant and wild Ostrea edulis haemocytes, both infected with the parasite, were identified using suppression subtractive hybridisation. Expression of seven ESTs has been studied using quantitative reverse-transcriptase PCR. The base-line expression of an extracellular superoxide dismutase, inhibitor of apoptosis (OeIAP), Fas ligand (OeFas-ligand) and Cathepsin B was significantly increased, whilst cyclophilin B appeared significantly decreased in resistant oysters. Considering their great interest for further studies, the open reading frames of the OeFas-ligand and OeIAP were completely sequenced. Highlights ► Bonamia ostreae is an intra-haemocytes parasite. ► Flat oyster Ostrea edulis, is the natural host of B. ostreae. ► In this study we have studied genes expressed in response to the parasite. ► Extracellular superoxide dismutase (OeEcSOD) is up regulated in resistant haemocyte. ► Genes involved in apoptosis is up regulated in resistant haemocytes from flat oyster.

Bonamia ostreae is an intracellular protistan parasite affecting flat oysters Ostrea edulis. It can be detected in juveniles but mortalities mainly affect oysters which are more than 2 years old. The parasite is usually observed inside haemocytes and sometimes free, notably in gill epithelia suggesting a parasite release through this organ. However, the infective form and ways of entry and release remain undetermined. Flat oysters incubate their larvae in their pallial cavity for 8-10 days before releasing them into the water column. Flat oysters in Bay of Quiberon in South Brittany (France) are known to be infected with B. ostreae since 1979 and is the most important area in France for O. edulis spat collection. Flat oysters incubating larvae were sampled in this area during summertime between 2007 and 2009. Both adults and larvae were preserved and assayed by PCR and in situ hybridisation (ISH). PCR tests revealed the presence of parasite DNA in some adults and larvae. Specific labelling could be detected by ISH in gills, digestive system, gonad and mantle in adults and in the epithelium surrounding the visceral cavity of some larvae. Our results demonstrate that larvae can be infected with B. ostreae. Larvae might thus contribute to the spread of the parasite during their planktonic life. In addition, their transfer for aquaculture purpose should be controlled especially when they are exported from infected zones.

Bonamiosis due to the parasite Bonamia ostreae is a disease affecting the flat oyster Ostrea edulis. B. ostreae is a protozoan, affiliated to the order of haplosporidia and to the cercozoan phylum. This parasite is mainly intracellular, infecting haemocytes, cells notably involved in oyster defence mechanisms. Suppression subtractive hybridisation (SSH) was carried out in order to identify oyster genes differentially expressed during an infection of haemocytes with B. ostreae. Forward and reverse banks allowed obtaining 1104 and 1344 clones respectively, among which 391 and 480 clones showed a differential expression between both tested conditions (haemocytes alone versus haemocytes in contact with parasites). ESTs of interest including genes involved in cytoskeleton, respiratory chain, detoxification membrane receptors, and immune system were identified. The open reading frames of two selected genes (galectin and IRF-like) were completely sequenced and characterized. Real time PCR assays were developed to study the relative expression of candidate ESTs during an in vitro infection of haemocytes by live and dead parasites. Haemocyte infection with B. ostreae induced an increased expression of omega glutathione S-transferase (OGST), superoxide dismutase (SOD), tissue inhibitor of metalloproteinase (TIMP), galectin, interferon regulatory factor (IRF-like) and filamin genes.

Bonamia ostreae is an intrahaemocytic protozoan affecting Ostrea edulis. The parasite multiplies within haemocytes without being degraded and involves changes in cellular activities. Studies aiming at better understanding host response to a pathogen at the transcriptome levels are frequently based on the use of real time PCR assays, which require some reference genes. However, very few sequence data is available for Ostrea edulis in public databases. Subtracted cDNA libraries were constructed from the Ostrea edulis haemocytes in order to identify genes involved in host reactions against the parasite and quantitative real time PCR assays were developed to study expression of these genes. In this context, identification of reference genes and study of their relative expression stability were required for quantitative real time PCR normalization. The expression of 5 potential candidate reference genes from Ostrea edulis (ie elongation factor 1 alpha (EF1-α), 60S ribosomal protein L5 (L5), glyceraldehyde 3-phosphate-dehydrogenase (GAPDH), polyubiquitin (Ubiq) and β-actin (ACT)) was studied using RNAs extracted from pools of haemocytes in contact with the parasite Bonamia ostreae and haemocytes alone. Gene expression was quantified by real time PCR and expression stability was analysed with two analytical approaches GeNorm and NormFinder. GAPDH and EF1- were identified as the most stable genes with the GeNorm analysis. Whatever were the tested conditions, EF1- was also found as the most stable gene using Normfinder. The less stable gene was -actin although this gene is commonly used as housekeeping gene in many studies. Our results suggest using GAPDH and EF1- combined as reference genes when studying expression levels in haemocytes of Ostrea edulis. In addition, the complete ORF of these two genes was characterized.

Bonamia ostreae is an intracellular protozoan which is recognized as a cause of mortality in European populations of flat oysters (Ostrea edulis). Based on the recent characterization of actin genes of B. ostreae, specific primers were designed for real-time PCR using SYBR® Green chemistry. Specificity was demonstrated by the unique melting temperature peak observed in positive samples and by the lack of amplification in samples of oysters infected by closely related parasites, including Bonamia exitiosa. A calibration curve using a cloned template was defined to estimate copy number. The assay had a 6 log- dynamic range, mean inter- and intra-assay variation coefficients of

Bonamia ostreae is an intracellular protozoan parasite, infecting haemocytes of the European flat oyster Ostrea edulis. Oyster defence mechanisms mainly rely on haemocytes. In the present study in vitro interactions between parasites and flat oyster haemocytes were investigated using flow cytometry and light microscopy. Haemocyte parameters including: non specific esterase activity, reactive oxygen species (ROS) production and phagocytosis were monitored using flow cytometry after 2 hour cell incubation with live and dead B. ostreae. Two ratios of parasites per haemocyte were tested (5:1 and 10:1), haemocytes alone were used as controls and the experiment was carried out three times. Flow cytometry revealed a decrease of non specific esterase activities and ROS production by haemocytes after incubation with live parasites, while there was little difference in phagocytosis activity when compared with controls. Similarly, dead parasites induced a decrease in haemocyte activities but to a lesser extent compared to live parasites. These results suggest that B. ostreae actively contributes to the modification of haemocyte activities in order to ensure its own intracellular survival.

Paracartia grani (Copepoda) has been identified as a potential intermediate host in the life cycle of Marteilia refringens, a paramyxean parasite infecting flat oysters. However, no intermediate host has yet been identified for Marteilia maurini that infects mussels. A better understanding of the life cycle of these two Marteilia types would clarify their taxonomic relationship and hypothesized co-specificity. For this purpose, experimental infections of copepods, P. grani, were performed using naturally infected flat oysters and mussels. Infection patterns were different depending whether copepods were infected from oysters or mussels. M. maurini did not proliferate in copepods while M. refringens rapidly proliferated in infected copepods. Previously unrecognized developmental stages of M. refringens were found during this study.

8 pages, 4 figures, The life cycle of the species forming the genus Marteilia (Phylum Paramyxea), which are marine protozoan parasites of molluscs, is still poorly known, although there is evidence of the need for intermediate hosts. To date, only the calanoid copepod Acartia (Paracartia) grani has been demonstrated to be involved in the life cycle of the parasite infecting flat oysters growing in ponds with a low biodiversity. However, no complex natural environments, such as bays or estuaries, have been studied for a similar purpose. Therefore, a survey for the presence of the protozoan Marteilia was conducted by PCR on the zooplankton community of a natural ecosystem, the Alfacs and Fangar bays in the Delta de l'Ebre (NW Mediterranean). Identification of zooplankton species found infected allowed two new Marteilia hosts to be proposed: the cyclopoida Oithona sp. and an indeterminate harpaticoida species. This is the first report of Marteilia in zooplankton species sampled from a natural enzootic area. © 2007 Elsevier B.V. All rights reserved, This study was supported by funds from an INIA predoctoral grand provided by the Spanish Minister of Science, the MCYT Project CTM-02575/MAR to M.A., the EU Community Reference Laboratory for Mollusc Diseases and Technology, and the Marie Curie Host Fellowships (n°405865H/P297815F) provided by the European Commission

Herpes-like viral infections have been reported in different bivalve mollusc species throughout the world. High mortalities among hatchery-reared larvae and juveniles of different bivalve species have been associated often with such infections. The diagnosis of herpes-like viruses in bivalve molluscs has been performed traditionally by light and transmission electron microscopy. The genome sequencing of one of these viruses, oyster herpesvirus 1 (OsHV-1), allowed the development of DNA-based diagnostic techniques. The polymerase chain reaction (PCR) has been used for the detection of OsHV-1 DNA in bivalve molluscs at different development stages. In addition, the PCR used for detection of OsHV-1 has also allowed the amplification of DNA from an OsHV-1 variant. The literature on DNA extraction methods, primers, PCR strategies, and confirmatory procedures used for the detection and identification of herpesviruses that infect bivalve molluscs are reviewed. (c) 2006 Elsevier B.V. All rights reserved.

The present study identifies quantitative trait loci (QTLs) in response to an experimental infection with the parasite responsible for bonamiosis, Bonamia ostreae, in two segregating families of the European flat oyster, Ostrea edulis. We first constructed a genetic-linkage map for each studied family and improved the existing genetic-linkage map for the European flat oyster with a set of SNP markers. This latter map now combines the best accuracy and the best estimate of the genome coverage available for an oyster species. Secondly, by comparing the QTLs detected in this study with those previously published for O. edulis in similar experimental conditions, we identified several potential QTLs that were identical between the different families, and also new specific QTLs. We also detected, within the confidence interval of several QTL regions, some previously predicted candidate genes differentially expressed during an infection with B. ostreae, providing new candidate genome regions which should now be studied more specifically.

Apoptosis is a fundamental feature in the development of many organisms and tissue systems. It is also a mechanism of host defense against environmental stress factors or pathogens by contributing to the elimination of infected cells. Hemocytes play a key role in defense mechanisms in invertebrates and previous studies have shown that physical or chemical stress can increase apoptosis in hemocytes in mollusks. However this phenomenon has rarely been investigated in bivalves especially in the flat oyster Ostrea edulis. The apoptotic response of hemocytes from flat oysters, O. edulis, was investigated after exposure to UV and dexamethasone, two agents known to induce apoptosis in vertebrates. Flow cytometry and microscopy were combined to demonstrate that apoptosis occurs in flat oyster hemocytes. Investigated parameters like intracytoplasmic calcium activity, mitochondrial membrane potential and phosphatidyl-serine externalization were significantly modulated in cells exposed to UV whereas dexamethasone only induced an increase of DNA fragmentation. Morphological changes were also observed on UV-treated cells using fluorescence microscopy and transmission electron microscopy. Our results confirm the apoptotic effect of UV on hemocytes of O. edulis and suggest that apoptosis is an important mechanism developed by the flat oyster against stress factors.

SUMMARYBonamia exitiosais an intracellular parasite (Haplosporidia) that has been associated with mass mortalities in oyster populations in the Southern hemisphere. This parasite was recently detected in the Northern hemisphere including Europe. Some representatives of theBonamiagenus have not been well categorized yet due to the lack of genomic information. In the present work, we have applied Whole-Genome Amplification (WGA) technique in order to characterize the actin gene in the unculturable protozoanB. exitiosa. This is the first protein coding gene described in this species. Molecular analysis revealed thatB. exitiosaactin is more similar toBonamia ostreaeactin gene-1. Actin phylogeny placed theBonamiasp. infected oysters in the same clade where the herein describedB. exitiosaactin resolved, offering novel information about the classification of the genus. Our results showed that WGA methodology is a promising and valuable technique to be applied to unculturable protozoans whose genomic material is limited.

An Australian (New South Wales) isolate of Bonamia was characterised at the molecular level by sequencing the 18S-ITS-1 region of the small subunit rRNA operon obtained from flat oysters Ostrea angasi shown to be infected by histological examination. Sequence data alignment with homologous genes from 2 other isolates of Bonamia (New Zealand and France) revealed high levels of nucleotide identity with both isolates. However, the Australian Bonamia is shown to be more closely related to the New Zealand isolate, suggesting the existence of an oceanic subgroup of Bonamia.

Organisms of the genus Bonamia are intracellular protistan parasites of oysters. To date, 4 species have been described (B. ostreae, B. exitiosa, B. perspora and B. roughleyi), al though the status of B. roughleyi is controversial. Introduction especially of B. ostreae and B. exitiosa to naïve host populations has been shown to cause mass mortalities in the past and has had a dramatic impact on oyster production. Both B. ostreae and B. exitiosa are pathogens notifiable to the World Organisation for Animal Health (OIE) and the European Union. Effective management of the disease caused by these pathogens is complicated by the extensive nature of the oyster production process and limited options for disease control of the cultured stocks in open water. This review focuses on the recent advances in research on genetic relationships between Bonamia isolates, geographical distribution, susceptible host species, diagnostics, epizootiology, host− parasite interactions, and disease resistance and control of this globally important genus of oyster pathogens.

Sporadic high mortalities were reported among larval French scallops (Pecten maximus). Electron microscopy of moribund larvae revealed particles with the characteristics of a herpesvirus in association with cellular lesions. PCR and DNA sequencing showed that the virus is a variant of ostreid herpesvirus-1 that has already been described in clams and oysters. This is the first description of a herpesvirus infection of a scallop species. The virus was transmitted successfully from an extract of infected scallop larvae to uninfected scallop or oyster (Crassostrea gigas) larvae, demonstrating that it is able to infect both species. Detection of viral DNA in asymptomatic adult scallops by in situ hybridisation indicates that the herpesvirus may have been transmitted from adults to larvae. It is notable that, unlike most herpesviruses, this virus has a wide host range reflected by its ability to infect several species of marine bivalve.

Concomitant sporadic high mortalities were reported in June 1997 among batches of larval Pacific oyster, Crassostrea gigas, and Manila clam, Ruditapes philippinarum, in a French commercial hatchery. Histological observation showed the presence of cellular abnormalities in affected animals. Electron transmission microscopy revealed the presence of herpes-like virus particles in infected larvae of both bivalve species. Viruses observed in C. gigas and R. philippinarum are closely related with respect to ultrastructure and morphogenesis. They were detected simultaneously in both bivalve species larvae indicating possible interspecific transmission. Moreover, PCR analysis using oyster herpes-like virus specific primers allowed amplification of fragments of expected sizes for both bivalve species and demonstrated the presence of viral DNA. The PCR products obtained for both bivalve species and digested by restriction enzymes displayed the same patterns. These data suggest that the same herpes-like virus may infect larval oysters and clams.

Since 1991, numerous herpesvirus infections associated with high mortality have been reported around the world in various marine bivalve species. In order to determine whether these infections are due to ostreid herpesvirus-1 (OsHV1), a previously characterized pathogen of the Japanese oyster (Crassostrea gigas), PCR analysis was carried out on 30 samples of larvae collected from four bivalve species (C. gigas, Ostrea edulis, Ruditapes decussatus and Ruditapes philippinarum), most exhibiting mortality prior to collection. All samples were shown to be infected by OsHV1. Viral genomes in three samples of C. gigas and three of R. philippinarum that originated from the same hatchery were unusual in bearing a deletion of at least 2·8 kbp in an inverted repeat region. The results demonstrate that OsHV1 is capable of infecting several bivalve species, and this raises the possibility that interspecies transmission may be promoted by intensive rearing in modern hatcheries.

The dynamics of the protozoan parasite Marteilia refringens was studied in Thau lagoon, an important French shellfish site, for 1 year in three potential hosts: the Mediterranean mussel Mytilus galloprovincialis (Mytiliidae), the grooved carpet shell Ruditapes decussatus (Veneriidae) and the copepod Paracartia grani (Acartiidae). Parasite DNA was detected by PCR in R. decussatus. In situ hybridisation showed necrotic cells of M. refringens in the digestive epithelia of some R. decussatus suggesting the non-involvement of this species in the parasite life cycle. In contrast, the detection of M. refringens in mussels using PCR appeared bimodal with two peaks in spring and autumn. Histological observations of PCR-positive mussels revealed the presence of different parasite stages including mature sporangia in spring and autumn. These results suggest that the parasite has two cycles per year in the Thau lagoon and that mussels release parasites into the water column during these two periods. Moreover, PCR detection of the parasite in the copepodid stages of P. grani between June and November supports the hypothesis of the transmission of the parasite from mussels to copepods and conversely. In situ hybridisation performed on copepodites showed labeling in some sections. Unusual M. refringens cells were observed in the digestive tract and the gonad from the third copepodid stage, suggesting that the parasite could infect a copepod by ingestion and be released through the gonad. This hypothesis is supported by the PCR detection of parasite DNA in copepod eggs from PCR-positive females, which suggests that eggs could contribute to the parasite spreading in the water and could allow overwintering of M. refringens. Finally, in order to understand the interactions between mussels and copepods, mussel retention efficiency (number of copepods retained by a mussel) was measured for all P. grani developmental stages. Results showed that all copepod stages could contribute to the transmission of the parasite, especially eggs and nauplii which were retained by up to 90%.

The health status of cultured and wild Mediterranean mussels in the Slovene Sea has so far been unexplored. Initially, 1280 adult Mediterranean mussels (Mytilus galloprovincialis), 960 from a shellfish farm and 320 from natural beds, were collected over a one-year period. Water temperature, oxygenation and salinity were measured at each sampling. Mussels were measured and weighted to calculate the condition index and microscopically examined for the presence of haemocytic neoplasia. Haemocytic neoplasia was detected in 14 mussels (1.1%) with a higher prevalence in cultured mussels. Neoplastic cells singularly infiltrated the connective tissue, in small foci or diffusely. Necrosis and multifocal atrophy of digestive tubules were noticed in mussels with diffuse neoplasia, whereas severe haemocytic infiltration of connective tissue was seen in mussels with single neoplastic cells. Haemocytic neoplasia was more frequently observed in spring and autumn. The average condition index of mussels with haemocytic neoplasia was slightly higher than in healthy ones. This is the first report of haemocytic neoplasia in Mediterranean mussels in the Northern Adriatic Sea. The disease occurs only sporadically and to date no significant impact on the mussel population has been noted.

Marteilia refringens is a protozoan parasite recognized as a significant pathogen of the European flat oyster Ostrea edulis. The life cycle of this species is still poorly known, although there is evidence of the need for intermediate host(s). In the present study, we have used molecular approaches to identify this parasite in samples of the dwarf oyster Ostrea stentina after reports of massive mortality along the Tunisian coasts. In 2009 we evaluated the status of O. stentina from Monastir and checked if there was an infection with M. refringens, using polymerase chain reaction assays. Of the 103 tested O. stentina, 85 were PCR-positive using a Marteilia genus-specific assay. Additional assays were subsequently carried out on some samples collected in 2010 in Monastir and processed for histology, transmission electron microscopy and complementary molecular analyses. PCR was carried out to amplify the IGS and ITS regions. Histological and transmission electron microscopy analyses allowed us to confirm the presence of this parasite in the digestive gland tissue of O. stentina and to characterize it at the ultrastructural level. This is the first record of the occurrence of M. refringens in the oyster O. stentina along the Tunisian coasts.

Prevalence of the protozoan Perkinsus spp. in the gills of the pleasure oyster Crassostrea corteziensis from two estuaries in Nayarit, Mexico, was measured. The protozoan was identified by PCR amplification of the internal transcribed spacer (ITS) region of the rDNA of Perkinsus spp. The pathogen was found in 92% of oysters from Boca de Camichín and 77% of oysters from Pozo Chino. ITS sequences characterized from C. corteziensis showed 96–100% similarity to Perkinsus marinus. The most frequent ITS sequence, (GenBank JQ266236) had 100% identity with the ITS locus of P. marinus from New Jersey, Maryland, South Carolina, and Texas, and the second most frequent observed sequence (GenBank JQ266240) was 100% identical to ITS sequences of P. marinus from New ersey, South Carolina, Louisiana, and Bahía Kino, Sonora, Mexico. The 14 sequences from the non-transcribed spacer (NTS) showed 98% similarity to P. marinus from Texas. The most frequent polymorphism identified was at nucleotide 446 of the ITS region, however, the NTS showed the highest nucleotide diversity, thereby suggesting that this region is suitable for genotype identification. Moreover, the most conserved ITS marker is better for species-specific diagnosis. Both the ITS and NTS sequences of P. marinus obtained from C. corteziensis were grouped in two clades, identifying two allelic variants of P. marinus

SUMMARYAlthough clam populations in France are known to be infected with protozoans of the genus Perkinsus, no molecular characterization was previously performed on these parasites. Considering that several members of this genus have been associated with mortalities of molluscs worldwide, a study was undertaken in order to characterize these parasites in France. For that purpose, clams, Ruditapes philippinarum and R. decussatus, collected from different production areas and found to be infected with Perkinsus sp. in thioglycolate culture medium, were selected for PCR-RFLP tests and sequencing. Perkinsus olseni was detected in all the investigated areas and results also suggested the presence of P. chesapeaki in Leucate, a lagoon on the Mediterranean coast and in Bonne Anse in Charente Maritime, on the Atlantic coast. Clonal cultures from both detected species were produced in order to describe and compare in vitro stages. Differences in size between both Perkinsus spp. were noticed especially for schizonts and zoosporangia. Lastly, in situ hybridization tests allowed confirmation of the presence of both species in the same R. decussatus population and even in same clams. This is the first detection of P. chesapeaki in Ruditapes species and outside North America, which questions its introduction into Europe.

Paracartia grani originates from high latitudes and has progressively been recorded in the Mediterranean Sea since the 1980s. In Thau lagoon, while Acartia clausi and Acartia discaudata present maximum peaks of abundance from January to April and in November and December, both species being perennial over the year, P. grani and Paracartia latisetosa appear in May, develop during summer, in July and August, and slowly decrease in autumn before disappearing from the water column in December. The Acartiidae populations have changed over the years at our monitoring station. Indeed, P. latisetosa was last recorded in August 1983 while P. grani appeared in the lagoon in 1998.

In this study we described the cytosolic HSP90 of Bonamia ostreae, an intracellular parasite of Ostrea edulis haemocytes. The complete open reading frame was assembled by Rapid Amplification cDNA Ends reactions on cDNA of B. ostreae infected haemocytes. HSP90 amplification was corroborated in infected oysters and B. ostreae purified cells. The functionality of the HSP90, studied by inhibitory assays with radicicol, suggests that this protein may play a role in haemocyte invasion. Our results inform the molecular basis that governs B. ostreae-O. edulis interactions.

Farming of the flat oyster Ostrea edulis in Europe is severely constrained by the protozoan Bonamia ostreae. The introduction of the resistant species Crassostrea gigas has been a relief for the farmers, while the pilot programmes to select O. edulis strains resistant to bonamiosis performed in various countries can be seen as a promising strategy to minimise the effects of bonamiosis. However, the physiological bases of this differential susceptibility remain unknown. A search for an explanation of the intra and interspecific differences in oyster susceptibility to bonamiosis was accomplished by comparing some immune parameters among various O. edulis stocks and C gigas. On December 2003, naive and Bonamia-relatively resistant flat oysters from Ireland. Galician flat oysters and Pacific oysters C. gigas were deployed in a Galician area affected by bonamiosis; haemolymph samples were taken in February and May 2004. A new oyster deployment at the same place was carried out on June 2004 and haemolymph sampling was performed on April 2005. On November 2004, new sets of Irish flat oysters and C. gigas were deployed in Ireland and haemolymph sampling was performed in June 2005. Various haemocytic parameters were measured: total and differential haemocyte count, phagocytic ability, respiratory burst (superoxide anion [O-2(-)] and hydrogen peroxide [H2O2]) and nitric oxide [NO] production. The comparison of the parameters was carried out at 3 levels: (1) between O. edulis and C. gigas, (2) among O. edulis stocks with different susceptibility to bonamiosis, and (3) between Bonamia-infected and non infected O. edulis. In addition, haemocyte-B. ostreae in vitro encounters were performed to analyse interspecific differences in the haemocytic respiratory burst, using flow cytometry. Significant differences associated with total and differential haemocyte count, and respiratory burst between O. edulis and C. gigas were detected, which could be linked to differences in susceptibility to bonamiosis between both species. Additionally, significant changes in total and differential haemocyte count, and respiratory burst of O. edulis associated with B. ostreae infection were found. However, no consistent difference in any haemocyte parameter between the O. edulis stocks involved in the study was recorded. (C) 2012 Elsevier Inc. All rights reserved.