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The Pacific cupped oyster Crassostrea gigas is one of the most 'globalized' marine invertebrates and its production is predominant in many parts of the world including Europe. However, it is threatened by mortality events associated with pathogenic microorganisms such as the virus OsHV-1 and the bacteria Vibrio aestuarianus. C. gigas is also a host for protozoan parasites including haplosporidians. In contrast with Haplosporidium nelsoni previously detected in Europe, H. costale was considered exotic although its presence in French oysters was suggested in the 1980s based on ultrastructural examination. Here, a combination of light and transmission electron microscopy, PCR and sequencing allowed characterizing the presence of the parasite in the context of low mortality events which occurred in 2019 in France. Histological observation revealed the presence of uninucleated, plasmodial and spore stages within the connective tissues of some oysters. Ultrastructural features were similar to H. costale ones in particular the presence of axe-shaped haplosporosomes in spore cytoplasms. Three fragments of the genome including partial small subunit rRNA gene, the ITS-1, 5.8S and ITS-2 array and part of the actin gene were successfully sequenced and grouped with H. costale homologous sequences. This is the first time that the presence of H. costale was confirmed in C. gigas in France. Furthermore, a TaqMan real-time PCR assay was developed and validated [DSe = 92.6% (78.2-99.8) and DSp = 95.5% (92.3-98.6)] to enable the rapid and specific detection of the parasite. The application of the PCR assay on archived samples revealed that the parasite has been present in French oyster populations at least since 2008. Considering the little information available on this parasite, the newly developed TaqMan assay will be very helpful to investigate the temporal and geographic distribution and the life cycle of the parasite in France and more generally in C. gigas geographic range.

Mortality outbreaks of young Pacific oysters

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.

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.

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.

Background Microcell parasites are small intracellular protozoans mostly detected in molluscs and can be associated with mortalities. In 2010 and 2011, strong increases in mortality events were reported in different wild beds of the wedge clam Donax trunculus Linnaeus, along the Atlantic coast of France and the presence of potential pathogens, including microcells, was investigated. Methods Clams collected in different beds showing mortality were examined by histology. Based on histological observations, confirmatory analyses were carried out, including transmission electron microscopy (TEM) and molecular characterization. Results Histological analyses revealed the presence of small protozoans similar to microcell parasites in different tissues of Donax trunculus, particularly in muscular and connective tissues. TEM examination confirmed the intracellular localization of the protozoans. Moreover, the lack of haplosporosomes and mitochondria suggested that the observed parasites belong to the genus Mikrocytos Farley, Wolf & Elston, 1988. Mikrocytos genus-specific PCR and in situ hybridization results supported the microscopic observations. Sequence fragments of the 18S rRNA gene shared 75–83% identity with the different Mikrocytos spp. described previously, including Mikrocytos mackini Farley, Wolf & Elston, 1988 and M. boweri Abbott, Meyer, Lowe, Kim & Johnson, 2014. Phylogenetic analyses confirmed that the microcell parasites observed in Donax trunculus in France belong to the genus Mikrocytos and suggest the existence of two distinct species. Conclusions Based on morphological, ultrastructural, molecular data and host information, the two microcell parasites detected in Donax trunculus belong to the genus Mikrocytos and are distinct from previously described members of this genus. This is the first report of Mikrocytos spp. found in France and infecting the clam Donax trunculus. Mikrocytos veneroïdes n. sp. was detected in different wild beds and Mikrocytos donaxi n. sp. was detected only in Audierne Bay. Electronic supplementary material The online version of this article (10.1186/s13071-018-2692-0) contains supplementary material, which is available to authorized users.

To keep pace with rising opportunities for disease emergence and spread, surveillance in aquaculture must enable the early detection of both known and new pathogens. Conventional surveillance systems (designed to provide proof of disease freedom) may not support detection outside of periodic sampling windows, leaving substantial blind spots to pathogens that emerge in other times and places. To address this problem, we organized an expert panel to envision optimal systems for early disease detection, focusing on Ostreid herpesvirus 1 (OsHV-1), a pathogen of panzootic consequence to oyster industries. The panel followed an integrative group process to identify and weight surveillance system traits perceived as critical to the early detection of OsHV-1. Results offer a road map with fourteen factors to consider when building surveillance systems geared to early detection; factor weights can be used by planners and analysts to compare the relative value of different designs or enhancements. The results were also used to build a simple, but replicable, model estimating the system sensitivity (SSe) of observational surveillance and, in turn, the confidence in disease freedom that negative reporting can provide. Findings suggest that optimally designed observational systems can contribute substantially to both early detection and disease freedom confidence. In contrast, active surveillance as a singular system is likely insufficient for early detection. The strongest systems combined active with observational surveillance and engaged joint industry and government involvement: results suggest that effective partnerships can generate highly sensitive systems, whereas ineffective partnerships may seriously erode early detection capability. Given the costs of routine testing, and the value (via averted losses) of early detection, we conclude that observational surveillance is an important and potentially very effective tool for health management and disease prevention on oyster farms, but one that demands careful planning and participation. This evaluation centered on OsHV-1 detection in farmed oyster populations. However, many of the features likely generalize to other pathogens and settings, with the important caveat that the pathogens need to manifest via morbidity or mortality events in the species, life stages and environments under observation.

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.

Summary Environmental DNA approaches are increasingly used to detect microorganisms in environmental compartments, including water. They show considerable advantages to study non‐cultivable microorganisms like Bonamia ostreae, a protozoan parasite inducing significant mortality in populations of flat oyster Ostrea edulis. Although B. ostreae development within the host has been well described, questions remain about its behaviour in the environment. As B. ostreae transmission is direct, seawater appears as an interesting target to develop early detection tools and improve our understanding of disease transmission mechanisms. In this context, we have developed an eDNA/eRNA approach allowing detecting and quantifying B. ostreae 18S rDNA/rRNA as well as monitoring its presence in seawater by real‐time PCR. B. ostreae DNA could be detected up to 4 days while RNA could be detected up to 30 days, suggesting a higher sensitivity of the eRNA‐based tool. Additionally, more than 90% of shed parasites were no longer detected after 2 days outside the oysters. By allowing B. ostreae detection in seawater, this approach would not only be useful to monitor the presence of the parasite in oyster production areas but also to evaluate the effect of changing environmental factors on parasite survival and transmission., An eDNA/eRNA approach was developed, allowing detecting and quantifying Bonamia ostreae 18S rDNA/rRNA as well as monitoring its presence in seawater by real‐time PCR. B. ostreae DNA and RNA could be detected up to 4 and 30 days respectively, suggesting a higher sensitivity of the eRNA based tool. More than 90% of shed parasites were no longer detected after 2 days outside the oysters.

Macroautophagy is a mechanism that is involved in various cellular processes, including cellular homeostasis and innate immunity. This pathway has been described in organisms ranging in complexity from yeasts to mammals, and recent results indicate that it occurs in the mantle of the Pacific oyster, Crassostrea gigas. However, the autophagy pathway has never been explored in the hemocytes of C. gigas, which are the main effectors of its immune system and thus play a key role in the defence of the Pacific oyster against pathogens. To investigate autophagy in oyster hemocytes, tools currently used to monitor this mechanism in mammals, including flow cytometry, fluorescent microscopy and transmission electron microscopy, were adapted and applied to the hemocytes of the Pacific oyster. Oysters were exposed for 24 and 48 h to either an autophagy inducer (carbamazepine, which increases the production of autophagosomes) or an autophagy inhibitor (ammonium chloride, which prevents the degradation of autophagosomes). Autophagy was monitored in fresh hemocytes withdrawn from the adductor muscles of oysters using a combination of the three aforementioned methods. We successfully labelled autophagosomes and observed them by flow cytometry and fluorescence microscopy, and then used electron microscopy to observe ultrastructural modifications related to autophagy, including the presence of double-membrane-bound vacuoles. Our results demonstrated that autophagy occurs in hemocytes of C. gigas and can be modulated by molecules known to modulate autophagy in other organisms. This study describes an integrated approach that can be applied to investigate autophagy in marine bivalves at the cellular level. Abbreviations: MAP1LC3: microtubule associated protein 1 light chain 3; MCA: multiple correspondence analysis; NH4Cl: ammonium chloride; PI: propidium iodide; TEM: transmission electron microscopy

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.

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 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.

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.

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.

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%.

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.

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.

Since its molecular characterisation, Ostreid herpesvirus 1 (OsHV-1) has been regularly detected in Crassostrea gigas in France. Although its pathogenicity was demonstrated on larval stages, its involvement during mortality outbreaks at the juvenile stage was highly suspected but not evidenced. To investigate mortality outbreaks, the French National Network for Surveillance and Monitoring of Mollusc Health (REPAMO) carried out two surveys in juvenile C. gigas. The first survey lasted from 1998 to 2006 and was an epidemiological inquiry occurring when oyster farmers reported mortality outbreaks. The second survey, a longitudinal one, was set up in 1998 to complete the network observations on OsHV-1. Data analysis showed a specific pattern of mortality outbreaks associated with OsHV-1 detection. Ostreid herpesvirus 1 detection mainly appeared during the summer, suggesting the influence of the seawater temperature on its occurrence. It mostly presented a patchy distribution in the field in contrast to the nursery. Significant relationship between OsHV-1 detection and spat mortality was found, preferentially in sheltered and closed environments. The longitudinal survey confirmed most of the network observations. Although subsequent works particularly epidemiological surveys would be useful to confirm the causal link between the detection of OsHV-1 and the mortality outbreaks in juvenile C. gigas, the role of OsHV-1 in oyster mortality is progressing.

Bonamia ostreae is a protozoan, affiliated to the order Haplosporidia and to the phylum Cercozoa. This parasite is intracellular and infects haemocytes, cells notably involved in oyster defence mechanisms. Bonamiosis due to the parasite B. ostreae is a disease affecting the flat oyster, Ostrea edulis. The strategies used by protozoan parasites to circumvent host defence mechanisms remain largely unknown in marine bivalve molluscs. In the present work, in vitro experiments were carried out in order to study the interactions between haemocytes from O. edulis and purified parasite. B. ostreae. We monitored cellular and molecular responses of oyster haemocytes by light microscopy, flow cytometry and real-time PCR 1, 2, 4 and 8 h p.i. Light microscopy was used to measure parasite phagocytosis by oyster haemocytes. Parasites were observed inside haemocytes 1 h p.i. and the parasite number increased during the time course of the experiment. Moreover, some bi-nucleated and tri-nucleated parasites were found within haemocytes 2 and 4 h p.i., respectively, suggesting that the parasite can divide inside haemocytes. Host responses to B. ostreae were investigated at the cellular and molecular levels using flow cytometry and real-time PCR. Phagocytosis capacity of haemocytes, esterase activity and production of radical oxygen species appeared modulated during the infection with B. ostreae. Expression levels of expressed sequence tags selected in this study showed variations during the experiment as soon as 1 h p.i. An up-regulation of galectin (OeGal), cytochrome p450 (CYP450), lysozyme, omega GST (OGST), super oxide dismutase Cu/Zn (Oe-SOD Cu/Zn) and a down-regulation of the extracellular super oxide dismutase SOD (Oe-EcSOD) were observed in the presence of the parasite. Finally, the open reading frames of both SODs (Oe-SOD Cu/Zn and Oe-EcSOD) were completely sequenced. These findings provide new insights into the cellular and molecular bases of the host-parasite interactions between the flat oyster, O. edulis, and the parasite. B. ostreae. (C) 2011 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Ostreid herpesvirus 1 (OsHV-1) infections have been reported around the world and are associated with high mortalities of the Pacific oyster (Crassostrea gigas). In the summer 2008, abnormal mortality rates ranging from 80% to 100% were reported in France and affected only Pacific oysters. Analyses of oyster samples collected during mortality outbreaks demonstrated a significant detection of OsHV-1 (75% of analysed batches), which appeared stronger than previous years. DNA sequencing based on C and IA regions was carried out on 28 batches of OsHV-1 infected Pacific oysters collected in 2008. Polymorphisms were described in both the C and IA regions and characterized a genotype of OsHV-1 not already reported and termed OsHV-1 microVar. A microsatellite zone present in the C region showed several deletions. Additionally, 44 isolates collected in France and in the USA, from 1995 to 2007 were sequenced and compared to the 2008 sequences. The analyses of 76 sequences showed OsHV-1 microVar detection only in 2008 isolates. These data suggest that OsHV-1 microVar can be assumed as an emergent genotype.

The flat oyster Ostrea edulis L. is widespread along the Italian coasts. In particular, the Manfredonia Gulf (Adriatic Sea) represents an important site where natural beds subsist. Previous monitoring conducted in 1990 by light microscopy and ultrastructural studies revealed the presence of Bonamia-like microcell parasites in some flat oysters: following this observation, a new sampling of O. edulis was carried out at this location in 2007. Of 750 oysters collected, 3 showed the presence of uninucleated microcells (2 to 3 microm diameter) free or inside the haemocyte cytoplasm by cytology and histopathology. Molecular analysis confirmed that the microcells in 2 oysters were B. exitiosa, whereas in the third oyster the microcells were B. ostreae. Moreover, molecular studies were carried out to confirm the existence of Bonamia sp. in archived samples, confirming the presence of B. ostreae in the Manfredonia Gulf since 1990.

Bonamiosis due to the intrahaemocytic protistan parasite Bonamia ostreae is a European endemic disease affecting the flat oyster Ostrea edulis. The parasite has been described in various ecosystems from estuaries to open sea, but no clear correlation has yet been demonstrated between disease development and environmental parameters. In this study, the effect of temperature and salinity on the survival of purified parasites maintained in vitro in seawater was investigated by flow cytometry. Purified parasites were incubated in various seawater media (artificial seawater, natural seawater, seabed borewater) at various temperatures (4, 15 and 25 degrees C) and subjected to a range of salinities from 5 to 45 g l(-1). Parasites were collected after 12, 24 and 48 h of incubation for flow cytometry analyses including estimation of parasite mortality and parasite viability through detection of non-specific esterase activities. Artificial seawater appeared unsuitable for parasite survival, and results for all media showed a significantly lower survival at 25 degrees C compared to 4 degrees C and 15 degrees C. Moreover, high salinities (> or = 35 g l(-1)) favoured parasite survival and detection of esterase activities. Flow cytometry appears to be a suitable technique to investigate survival and activities of unicellular parasites like B. ostreae under varied conditions. Although these results contribute to a better understanding of existing interactions between the parasite B. ostreae and its environment, validation through epidemiological surveys in the field is also needed.

We have identified quantitative trait loci (QTL) in the flat oyster (Ostrea edulis) for resistance to Bonamia ostreae, a parasite responsible for the dramatic reduction in the aquaculture of this species. An F(2) family from a cross between a wild oyster and an individual from a family selected for resistance to bonamiosis was cultured with wild oysters injected with the parasite, leading to 20% cumulative mortality. Selective genotyping of 92 out of a total of 550 F(2) progeny (i.e., 46 heavily infected oysters that died and 46 parasite-free oysters that survived) was performed using 20 microsatellites and 34 amplification fragment length polymorphism primer pairs. Both a two-stage testing strategy and QTL interval mapping methods were used. The two-stage detection strategy had a high power with a low rate of false positives and identified nine and six probable markers linked to genes of resistance and susceptibility, respectively. Parent-specific genetic linkage maps were built for the family, spanning ten linkage groups (n = 10) with an observed genome coverage of 69-84%. Three QTL were identified by interval mapping in the first parental map and two in the second. Good concordance was observed between the results obtained after the two-stage testing strategy and QTL mapping.

Marteilia spp. are paramyxean parasites that affect several bivalve species of economic interest, such as Ostrea edulis and Mytilus galloprovincialis. Certain aspects of Marteilia spp., such as their life cycle and host affinity and infection dynamics, still remain unknown. The 'Delta de l'Ebre' constitutes a natural model for the study of the life cycle of the parasite Marteilia, since uninfected mussels and flat oysters immersed in the bays can become infected. This, along with the geographical and ecological characteristics of the bays, make it a very interesting location to study the Marteilia life cycle. Preliminary results concerning marteiliosis, mainly in mussels, such as prevalence dynamics, infectious periods, host affinity and host intermediate candidates are reported in the present paper. This information will be required for further, more exhaustive, studies in the bays of the Ebre delta.