A chromosome-level genome assembly of Ostrea denselamellosa provides initial insights into its evolution.

The oyster Ostrea denselamellosa is a live-bearing species with a sharp decline in the natural population. Despite recent breakthroughs in long-read sequencing, high quality genomic data are very limited in O. denselamellosa. Here, we carried out the first whole genome sequencing at the chromosome-level in O. denselamellosa. Our studies yielded a 636 Mb assembly with scaffold N50 around 71.80 Mb. 608.3 Mb (95.6% of the assembly) were anchored to 10 chromosomes. A total of 26,412 protein-coding genes were predicted, of which 22,636 (85.7%) were functionally annotated. By comparative genomics, we found that long interspersed nuclear element (LINE) and short interspersed nuclear element (SINE) made up a larger proportion in O. denselamellosa genome than in other oysters'. Moreover, gene family analysis showed some initial insight into its evolution. This high-quality genome of O. denselamellosa provides a valuable genomic resource for studies of evolution, adaption and conservation in oysters.

Epigenetic-associated phenotypic plasticity of the ocean acidification-acclimated edible oyster in the mariculture environment.

For marine invertebrates with a pelagic-benthic life cycle, larval exposure to ocean acidification (OA) can affect adult performance in response to another environmental stressor. This carry-over effect has the potential to alter phenotypic traits. However, the molecular mechanisms that mediate "OA"-triggered carry-over effects have not been explored despite such information being key to improving species fitness and management strategies for aquafarming. This study integrated the genome-wide DNA methylome and transcriptome to examine epigenetic modification-mediated carry-over OA impacts on phenotypic traits of the ecologically and commercially important oyster species Crassostrea hongkongensis under field conditions. Larvae of C. hongkongensis were exposed to control pH 8.0 and low pH 7.4 conditions, mimicking near future OA scenario in their habitat, before being outplanted as post-metamorphic juveniles at two mariculture field sites with contrasting environmental stressors for 9 months. The larval carry-over OA effect was found to have persistent impacts on the growth and survival trade-off traits on the outplanted juveniles, although the beneficial or adverse effect depended on the environmental conditions at the outplanted sites. Site-specific plasticity was demonstrated with a diverse DNA methylation-associated gene expression profile, with signal transduction and the endocrine system being the most common and highly enriched functions. Highly methylated exons prevailed in the key genes related to general metabolic and endocytic responses and these genes are evolutionarily conserved in various marine invertebrates in response to OA. These results suggest that oysters with prior larval exposure history to OA had the ability to trigger rapid local adaptive responses via epigenetic modification to cope with multiple stressors in the field.

Limited impact of a bioeroding sponge, Cliona sp., on Ostrea chilensis from Foveaux Strait, New Zealand.

Bioeroding sponges can cause extensive damage to aquaculture and wild shellfish fisheries. It has been suggested that heavy sponge infestations that reach the inner cavity of oysters may trigger shell repair and lead to adductor detachment. Consequently, energy provision into shell repair could reduce the energy available for other physiological processes and reduce the meat quality of commercially fished oysters. Nevertheless, the impacts of boring sponges on oysters and other shellfish hosts are inconclusive. We studied the interaction between boring sponges and their hosts and examined potential detrimental effects on an economically important oyster species Ostrea chilensis from Foveaux Strait (FS), New Zealand. We investigated the effect of different infestation levels with the bioeroding sponge Cliona sp. on commercial meat quality, condition, reproduction, and disease susceptibility. Meat quality was assessed with an index based on visual assessments used in the FS O. chilensis fishery. Meat condition was assessed with a common oyster condition index, while histological methods were used to assess sex, gonad stage, reproductive capacity, and pathogen presence. Commercial meat quality and condition of O. chilensis were unaffected by sponge infestation. There was no relationship between sex ratio, gonad developmental stage, or gonad index and sponge infestation. Lastly, we found no evidence that sponge infestation affects disease susceptibility in O. chilensis. Our results suggest that O. chilensis in FS is largely unaffected by infestation with Cliona sp. and therefore reinforces the growing body of evidence that the effects of sponge infestation can be highly variable among different host species, environments, and habitats.

Species-Specific N-Glycomes and Methylation Patterns of Oysters Crassostrea gigas and Ostrea edulis and Their Possible Consequences for the Norovirus-HBGA Interaction.

Noroviruses, the major cause of acute viral gastroenteritis, are known to bind to histo-blood group antigens (HBGAs), including ABH groups and Lewis-type epitopes, which decorate the surface of erythrocytes and epithelial cells of their host tissues. The biosynthesis of these antigens is controlled by several glycosyltransferases, the distribution and expression of which varies between tissues and individuals. The use of HBGAs as ligands by viruses is not limited to humans, as many animal species, including oysters, which synthesize similar glycan epitopes that act as a gateway for viruses, become vectors for viral infection in humans. Here, we show that different oyster species synthesize a wide range of N-glycans that share histo-blood A-antigens but differ in the expression of other terminal antigens and in their modification by O-methyl groups. In particular, we show that the N-glycans isolated from Crassostrea gigas and Ostrea edulis exhibit exquisite methylation patterns in their terminal N-acetylgalactosamine and fucose residues in terms of position and number, adding another layer of complexity to the post-translational glycosylation modifications of glycoproteins. Furthermore, modeling of the interactions between norovirus capsid proteins and carbohydrate ligands strongly suggests that methylation has the potential to fine-tune the recognition events of oysters by virus particles.

Ecotoxicology of Trace Elements in European Oyster (Ostrea edulis), Seawater, and Sedimentsacross Boston Harbor, Massachusetts,USA.

European oyster (Ostrea edulis) can be used for biological monitoring of water and sediment quality and serve as a conduit of trace elements to humans via consumption. Trace element concentrations in seawater, sediment, O. edulis edible tissues and shells from Boston Harbor were studied and found to be elevated relative to comparative studies in native ecosystems in the Adriatic Sea and Bay of Biscay. Average edible oyster tissues concentrations (mg/kg) were: arsenic 6, cadmium 1.7, cobalt 3.1, chromium 1.9, copper 153, mercury 0.265, nickel 1.8, lead 3.3, and zinc 2390. Arsenic was elevated in seawater and oyster shells. Mercury was elevated in sediments and oyster tissues. Lead was elevated in suspended sediments. Total Hazard Quotient (THQ) was < 1 but when summed across trace elements, THQ was nominally > 1 for all sites. Further study is warranted to determine mechanisms and spatial extent of bioaccumulation.

Proteomic changes associated with predator-induced morphological defences in oysters.

Inducible prey defences occur when organisms undergo plastic changes in phenotype to reduce predation risk. When predation pressure varies persistently over space or time, such as when predator and prey co-occur over only part of their biogeographic ranges, prey populations can become locally adapted in their inducible defences. In California estuaries, native Olympia oyster (Ostrea lurida) populations have evolved disparate phenotypic responses to an invasive predator, the Atlantic oyster drill (Urosalpinx cinerea). In this study, oysters from an estuary with drills, and oysters from an estuary without drills, were reared for two generations in a laboratory common garden, and subsequently exposed to cues from Atlantic drills. Comparative proteomics was then used to investigate molecular mechanisms underlying conserved and divergent aspects of their inducible defences. Both populations developed smaller, thicker, and harder shells after drill exposure, and these changes in shell phenotype were associated with upregulation of calcium transport proteins that could influence biomineralization. Inducible defences evolve in part because defended phenotypes incur fitness costs when predation risk is low. Immune proteins were downregulated by both oyster populations after exposure to drills, implying a trade-off between biomineralization and immune function. Following drill exposure, oysters from the population that co-occurs with drills grew smaller shells than oysters inhabiting the estuary not yet invaded by the predator. Variation in the response to drills between populations was associated with isoform-specific protein expression. This trend suggests that a stronger inducible defence response evolved in oysters that co-occur with drills through modification of an existing mechanism.

Pseudoalteromonas ostreae sp. nov., a new bacterial species harboured by the flat oyster Ostrea edulis.

Three bacterial strains, named hOe-66T, hOe-124 and hOe-125, were isolated from the haemolymph of different specimens of the flat oyster Ostrea edulis collected in Concarneau bay (Finistère, France). These strains were characterized by a polyphasic approach, including (i) whole genome analyses with 16S rRNA gene sequence alignment and pangenome analysis, determination of the G+C content, average nucleotide identity (ANI), and in silico DNA-DNA hybridization (isDDH), and (ii) fatty acid methyl ester and other phenotypic analyses. Strains hOe-66T, hOe-124 and hOe-125 were closely related to both type strains Pseudoalteromonas rhizosphaerae RA15T and Pseudoalteromonas neustonica PAMC 28425T with less than 93.3% ANI and 52.3% isDDH values. Regarding their phenotypic traits, the three strains were Gram-negative, 1-2 µm rod-shaped, aerobic, motile and non-spore-forming bacteria. Cells grew optimally at 25 °C in 2.5% NaCl and at 7-8 pH. The most abundant fatty acids were summed feature 3 (C16:1 ω7c/C16:1 ω6c), C16:0 and C17:1 ω8c. The strains carried a genome average size of 4.64 Mb and a G+C content of 40.28 mol%. The genetic and phenotypic results suggested that strains hOe-66T, hOe-124 and hOe-125 belong to a new species of the genus Pseudoalteromonas. In this context, we propose the name Pseudoalteromonas ostreae sp. nov. The type strain is hOe-66T (=CECT 30303T=CIP 111911T).

Shotgun analysis to identify differences in protein expression between granulocytes and hyalinocytes of the European flat oyster Ostrea edulis.

Recovery of wild populations of the European flat oyster Ostrea edulis is important for ecosystem health and conservation of this species, because native oyster populations have dramatically declined or disappeared in most European waters. Diseases have contributed to oyster decline and are important constrains for oyster recovery. Understanding oyster immune system should contribute to design effective strategies to fight oyster diseases. Haemocytes play a pivotal role in mollusc immune responses protecting from infection. Two main types of haemocytes, granulocytes and hyalinocytes, are distinguished in O. edulis. A study aiming to explore differential functions between both haemocyte types and, thus, to enrich the knowledge of Ostrea edulis immune system, was performed by comparing the proteome of the two haemolymph cell types, using a shotgun approach through liquid chromatography (LC) coupled to mass spectrometry (MS). Cells from oyster haemolymph were differentially separated by Percoll density gradient centrifugation. Shotgun LC-MS/MS performance allowed the identification of 145 proteins in hyalinocytes and 138 in the proteome of granulocytes. After a comparative analysis, 55 proteins with main roles in defence were identified, from which 28 were representative of granulocytes and 27 of hyalinocytes, plus 11 proteins shared by both cell types. Different proteins involved in signal transduction, apoptosis, oxidative response, processes related with the cytoskeleton and structure, recognition and wound healing were identified as representatives of each haemocyte type. Important signalling pathways in the immune response such as MAPK, Ras and NF-κß seemed to be more relevant for granulocytes, while the Wnt signalling pathway, particularly relevant for wound healing, more relevant in hyalinocytes. The differences in proteins involved in recognition and in cytoskeleton and structure suggest differential specialisation in processes of phagocytosis and internalisation of pathogens between haemocyte types. Apoptosis seemed more active in granulocytes. The differences in proteins involved in oxidative response also suggest different redox processes in each cell type.

What do the terms resistance, tolerance, and resilience mean in the case of Ostrea edulis infected by the haplosporidian parasite Bonamia ostreae.

The decline of the European flat oyster Ostrea edulis represents a loss to European coastal economies both in terms of food security and by affecting the Good Environmental Status of the marine environment as set out by the European Council's Marine Strategy Framework Directive (2008/56/EC). Restoration of O. edulis habitat is being widely discussed across Europe, addressing key challenges such as the devastating impact of the haplosporidian parasite Bonamia ostreae. The use of resistant, tolerant, or resilient oysters as restoration broodstock has been proposed by restoration practitioners, but the definitions and implications of these superficially familiar terms have yet to be defined and agreed by all stakeholders. This opinion piece considers the challenges of differentiating Bonamia resistance, tolerance, and resilience; challenges which impede the adoption of robust definitions. We argue that, disease-resistance is reduced susceptibility to infection by the parasite, or active suppression of the parasites ability to multiply and proliferate. Disease-tolerance is the retention of fitness and an ability to neutralise the virulence of the parasite. Disease-resilience is the ability to recover from illness and, at population level, tolerance could be interpreted as resilience. We concede that further work is required to resolve practical uncertainty in applying these definitions, and argue for a collaboration of experts to achieve consensus. Failure to act now might result in the future dispersal of this disease into new locations and populations, because robust definitions are important components of regulatory mechanisms that underpin marine management.

Differences in proteomic profile between two haemocyte types, granulocytes and hyalinocytes, of the flat oyster Ostrea edulis.

Haemocytes play a dominant role in shellfish immunity, being considered the main defence effector cells in molluscs. These cells are known to be responsible for many functions, including chemotaxis, cellular recognition, attachment, aggregation, shell repair and nutrient transport and digestion. There are two basic cell types of bivalve haemocytes morphologically distinguishable, hyalinocytes and granulocytes; however, functional differences and specific abilities are poorly understood: granulocytes are believed to be more efficient in killing microorganisms, while hyalinocytes are thought to be more specialised in clotting and wound healing. A proteomic approach was implemented to find qualitative differences in the protein profile between granulocytes and hyalinocytes of the European flat oyster, Ostrea edulis, as a way to evaluate functional differences. Oyster haemolymph cells were differentially separated by Percoll® density gradient centrifugation. Granulocyte and hyalinocyte proteins were separated by 2D-PAGE and their protein profiles were analysed and compared with PD Quest software; the protein spots exclusive for each haemocyte type were excised from gels and analysed by MALDI-TOF/TOF with a combination of mass spectrometry (MS) and MS/MS for sequencing and protein identification. A total of 34 proteins were identified, 20 unique to granulocytes and 14 to hyalinocytes. The results suggested differences between the haemocyte types in signal transduction, apoptosis, oxidation reduction processes, cytoskeleton, phagocytosis and pathogen recognition. These results contribute to identify differential roles of each haemocyte type and to better understand the oyster immunity mechanisms, which should help to fight oyster diseases.

Flow cytometric characterization of hemocytes of the flat oyster, Ostrea chilensis.

The flat oyster, Ostrea chilensis, native to New Zealand (NZ) and Chile is considered an important ecological, cultural and fisheries resource. Currently, commercial landings of this species in NZ are restricted due to low population numbers caused by ongoing mortalities resulting from the presence of the haplosporidian parasite, Bonamia exitiosa. More recently, the arrival of B. ostreae in NZ led to major mortalities in farmed stocks. To understand how diseases caused by Bonamia spp. affect this oyster species, a more complete understanding of its biology, physiology and immune system is needed. The present study characterized, for the first time, hemocytes of adult O. chilensis, from the Foveaux Strait, NZ, using flow cytometry (FCM) and histology. Based on the internal complexity of the hemocytes, two main circulating hemocyte populations were identified: granulocytes and hyalinocytes (accounting for ~30% and ~70% of the total circulating hemocyte population, respectively). These were further divided into two sub-populations of each cell type using FCM. A third sub-population of granulocytes was identified using histology. Using FCM, functional and metabolic characteristics were investigated for the two main hemocyte types. Granulocytes showed higher phagocytic capabilities, lysosomal content, neutral lipid content and reactive oxygen species production compared to hyalinocytes, indicating their important role in cellular immune defence in this species. Methods of hemocyte sampling and storage were also investigated and flow cytometric protocols were detailed and verified to allow effective future investigations into the health status of this important species.

Haplosporidian host:parasite interactions.

The host:parasite interactions of the 3 serious haplosporidian pathogens of oysters, on which most information exists, are reviewed. They are Bonamia ostreae in Ostrea spp. and Crassostrea gigas; Bonamia exitiosa in Ostrea spp.; and Haplosporidium nelsoni in Crassostrea spp. Understanding the haemocytic response to pathogens is constrained by lack of information on haematopoiesis, haemocyte identity and development. Basal haplospridians in spot prawns are probably facultative parasites. H. nelsoni and a species infecting Haliotis iris in New Zealand (NZAP), which have large extracellular plasmodia that eject haplosporosomes or their contents, lyse surrounding cells and are essentially extracellular parasites. Bonamia spp. have small plasmodia that are phagocytosed, haplosporosomes are not ejected and they are intracellular obligate parasites. Phagocytosis by haemocytes is followed by formation of a parasitophorous vacuole, blocking of haemocyte lysosomal enzymes and the endolysosomal pathway. Reactive oxygen species (ROS) are blocked by antioxidants, and host cell apoptosis may occur. Unlike susceptible O. edulis, the destruction of B. ostreae by C. gigas may be due to higher haemolymph proteins, higher rates of granulocyte binding and phagocytosis, production of ROS, the presence of plasma ß-glucosidase, antimicrobial peptides and higher levels of haemolymph and haemocyte enzymes. In B.exitiosa infection of Ostrea chilensis, cytoplasmic lipid bodies (LBs) containing lysosomal enzymes accumulate in host granulocytes and in B. exitiosa following phagocytosis. Their genesis and role in innate immunity and inflammation appears to be the same as in vertebrate granulocytes and macrophages, and other invertebrates. If so, they are probably the site of eicosanoid synthesis from arachidonic acid, and elevated numbers of LBs are probably indicative of haemocyte activation. It is probable that the molecular interaction, and role of LBs in the synthesis and storage of eicosanoids from arachidonic acid, is conserved in innate immunity in vertebrates and invertebrates. However, it seems likely that haplosporidians are more diverse than realized, and that there are many variations in host parasite interactions and life cycles.

Is pallial mucus involved in Ostrea edulis defenses against the parasite Bonamia ostreae?

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.

Ephemeral detection of Bonamia exitiosa (Haplosporida) in adult and larval European flat oysters Ostrea edulis in the Solent, United Kingdom.

The haplosporidian parasite Bonamia exitiosa was detected using PCR in four adult and six larval brood samples of the European flat oyster Ostrea edulis from the Solent, UK. This represents the second reported detection of this parasite along the south coast of England. Adult oysters were collected and preserved from seabed populations or restoration broodstock cages between 2015 and 2018. The larvae within brooding adults sampled during 2017 and 2018 were also preserved. Molecular analysis of all samples was performed in 2019. The DNA of B. exitiosa was confirmed to be present within the gill tissue of one oyster within the Portsmouth wild fishery seabed population (n = 48), sampled in November 2015; the congeneric parasite Bonamia ostreae was not detected in this individual. This is the earliest record of B. exitiosa in the Solent. Concurrent presence of both B. ostreae and B. exitiosa, determined by DNA presence, was confirmed in the gill and heart tissue of three mature individuals from broodstock cages sampled in October 2017 (n = 99), two from a location on the River Hamble and one from the Camber Dock in Portsmouth Harbour. B. exitiosa was not detected in the November 2018 broodstock populations. A total of six larval broods were positive for B. exitiosa, with five also positive for B. ostreae. None of the brooding adults were positive for B. exitiosa suggesting that horizontal transmission from the surrounding environment to the brooding larvae is occurring. Further sampling of broodstock populations conducted by the Fish Health Inspectorate at the Centre for Environment, Fisheries and Aquaculture Science in June 2019 did not detect infection of O. edulis by B. exitiosa. These findings together suggest that the pathogen has not currently established in the area.

Bonamia exitiosa in farmed native oysters Ostrea angasi in Australia: optimal epidemiological qPCR cut-point and clinical disease risk factors.

Bonamiosis has developed as a problem in Australian native oysters Ostrea angasi since the parasite Bonamia spp. was first detected in Port Phillip Bay, Victoria, in the early 1990s. At that time, large-scale mortalities in both farmed and wild oysters saw the demise of the pilot native oyster culture industry. More recent attempts to farm the species resulted in subclinical infections that progressed over time to clinical disease. The aim of this work was to establish what environmental factors result in the clinical manifestation of disease; determine the diagnostic sensitivity and diagnostic specificity of histopathological examination and a quantitative polymerase chain reaction (qPCR) test for the diagnosis of B. exitiosa infection in clinically diseased farmed native oysters; and calculate the optimal qPCR threshold cycle (CT) epidemiological cut-point for classification of positive and negative cases. After applying a range of stressors to tank-held oysters, results indicated a 58% increased risk (95% CI: 16%, 99%) of a Bonamia-infected oyster dying if the oyster was held at a higher temperature (p = 0.048). Starving and tumbling oysters, in isolation, was not significantly associated with clinical bonamiosis, but a Bonamia-infected oyster was at the greatest risk of death when increased water temperature was combined with both starvation and increased motion (p = 0.02; odds ratio = 3.47). The diagnostic sensitivity and specificity of the World Organisation for Animal Health qPCR protocol were calculated for increasing CT value cut-points from ≤25 to ≤40, with an optimal cut-point identified at ≤34.5 (specificity: 92.2; 95% posterior credible intervals [PCI]: 76.2, 99.8; Sensitivity: 93.5; 95% PCI: 84.7, 99.1).

Infection dynamics of Bonamia exitiosa on intertidal Ostrea angasi farms.

Bonamia spp. cause epizootics in oysters worldwide. In southern Australia, Bonamia exitiosa Hine, Cochennac and Berthe, 2001 threatens aquaculture of Ostrea angasi Sowerby, 1871. Bonamia spp. infections can display strong seasonality, but seasonal dynamics of B. exitiosa-O. angasi are unknown. Ostrea angasi naïve to B. exitiosa infection were stocked onto farms in three growing regions, and B. exitiosa was monitored seasonally for one year. Environmental parameters we measured did not correlate with B. exitiosa prevalence or infection intensities. Extreme temperatures suggest O. angasi culture systems need development. Bonamia exitiosa prevalence increased over time. After three months, O. angasi had B. exitiosa prevalence of 0.08-0.4, and after one year, the prevalence was 0.57-0.88. At some sites, O. angasi had >0.5 B. exitiosa prevalence in >6 months, but at other sites, >9 months passed before prevalence was >0.5. Bonamia exitiosa infection intensities were low with no seasonal pattern but were affected by the interaction of site, season and oyster meat:shell ratio. Understanding infection and initiating a breeding programme for resistance would provide benefits for O. angasi industry expansion.

Rapid transmission of Bonamia exitiosa by cohabitation causes mortality in Ostrea angasi.

The haplosporidian Bonamia was first detected in Australian shellfish in 1991. Australian isolates in Ostrea angasi Sowerby, 1871 were identified as Bonamia exitiosa Hine, Cochennac and Berthe, 2001, which threatens development of an O. angasi aquaculture industry. European field data suggest that Bonamia ostreae Pichot, Comps, Tigé, Grizel and Rabouin, 1980 infections in Ostrea edulis Linnaeus, 1758 build slowly, but infection dynamics of B. exitiosa in O. angasi are unknown. We investigated B. exitiosa infection in O. angasi by cohabiting uninfected juvenile O. angasi with adults infected with B. exitiosa. Oysters were sampled at 10, 21 and 40 days after cohabitation, and B. exitiosa prevalence and intensity were assessed. Bonamia exitiosa rapidly infected and caused disease in O. angasi. Mortalities began at 12 days, with ˜50% mortality by day 21 and >85% mortality by day 40. Mortalities displayed pathology consistent with clinical B. exitiosa infection. Time to first infection is likely influenced by a combination of parasite infectivity, host exposure and host immune capacity. Host death is not required for transmission, but probably facilitates release of parasites from decaying tissue. Understanding B. exitiosa transmission informs design and interpretation of field studies and aids development of management strategies for oyster aquaculture.

Involvement of apoptosis in the dialogue between the parasite Bonamia ostreae and the flat oyster Ostrea edulis.

The protozoan parasite Bonamia ostreae has been associated with the decline of flat oyster Ostrea edulis populations in some European countries. Control of shellfish diseases mostly relies on prevention measures including transfer restrictions and stock management measures such as breeding programmes. These prevention and mitigation measures require a better understanding of interactions between host and pathogens. Previous in vitro studies allowed identifying apoptosis as a mechanism activated by the flat oyster in response to B. ostreae. However, these experiments also suggested that the parasite is able to regulate apoptosis in order to survive and multiply within hemocytes. By simplifying the conditions of infection, in vitro studies allow identifying most distinct features of the response of the host. In order to appreciate the relative importance of apoptosis in this response at the oyster scale, in vivo trials were carried out by injecting with parasites oysters from two French locations, Quiberon Bay (Brittany) and Diana Lagoon (Corsica). Apoptosis was investigated on pools of hemolymph from oysters collected at early and later times after injection using previously developed tools. Apoptotic cellular activities including intracytoplasmic calcium concentration, mitochondrial membrane potential and phosphatidyl serine externalization were analysed using flow cytometry. Moreover, the expression of flat oyster genes involved in both extrinsic and intrinsic pathways was measured using real time quantitative PCR.

PCR and histology identify new bivalve hosts of Apicomplexan-X (APX), a common parasite of the New Zealand flat oyster Ostrea chilensis.

Apicomplexan-X (APX) is a significant pathogen of the flat oyster Ostrea chilensis in New Zealand. The life cycle and host range of this species are poorly known, with only the zoite stage identified. Here, we report the use of molecular approaches and histology to confirm the presence of APX in samples of green-lipped mussels Perna canaliculus, Mediterranean mussels Mytilus galloprovincialis and hairy mussels Modiolus areolatus collected from widely distributed locations in New Zealand. The prevalence of APX infection estimated by PCR was 22.2% (n = 99) and 50% (n = 30) in cultured green-lipped mussels from Nelson and Coromandel, respectively; 0.8% (n = 258), 3.3% (n = 150) and 35.3% (n = 17) in wild Mediterranean mussels from Nelson, Foveaux Strait and Golden Bay, respectively; and 46.7% (n = 30) in wild hairy mussels from Foveaux Strait. Histology detected all cases of PCR that were positive with APX and appeared to be more sensitive. The prevalence of APX estimated by histology in green-lipped mussels from Coromandel was 60% versus 50% by PCR, and 4.3%, 10.7% and 52.9% by histology versus 0.8%, 3.3% and 35.3% by PCR in wild Mediterranean mussels from Nelson, Foveaux Strait and Golden Bay, respectively. The specific identity of the parasite found in mussels was determined by sequencing PCR products for a portion (676 bp) of the 18S rRNA gene; the resulting sequences were 99-100% similar to APX found in flat oysters. Phylogenetic analyses also confirmed that all isolates from green-lipped, Mediterranean and hairy mussels grouped with APX isolates previously identified from flat oysters. This study indicates the wide geographical distribution of APX and highlights the potentially multi-host specific distribution of the parasite in commercially important bivalve shellfish.

Bonamia in Ostrea angasi: Diagnostic performance, field prevalence and intensity.

Bonamia spp. parasites threaten flat oyster (Ostrea spp.) farming worldwide. Understanding test performance is important for designing surveillance and interpreting diagnostic results. Following a pilot survey which found low Bonamia sp. intensity in farmed Ostrea angasi, we tested further oysters (n = 100-150) from each of three farms for Bonamia sp. using heart smear, histology and qPCR. We used a Bayesian Latent Class Model to assess diagnostic sensitivity (DSe) and specificity (DSp) of these tests individually or in combination, and to assess prevalence. Histology was the best individual test (DSe 0.76, DSp 0.93) compared to quantitative polymerase chain reaction (qPCR) (DSe 0.69, DSp 0.93) and heart smear (DSe 0.61, DSp 0.60). Histology combined with qPCR and defining a positive from either test as an infected case maximized test performance (DSe 0.91, DSp 0.88). Prevalence was higher at two farms in a high-density oyster growing region than at a farm cultivating oysters at lower density. Parasite intensities were lower than in New Zealand and European studies, and this is probably contributed to differences in the performance of test when compared to other studies. Understanding diagnostic test performance in different populations can support the development of improved Bonamia surveillance programs.