Understanding the role of Francisella halioticida in mussel mortalities in France: an integrative approach.

Since 2014, mass mortalities of mussels Mytilus spp. have occurred in production areas on the Atlantic coast of France. The aetiology of these outbreaks remained unknown until the bacterium Francisella halioticida was detected in some mussel mortality cases. This retrospective study was conducted to assess the association between F. halioticida and these mussel mortalities. Mussel batches (n = 45) from the Atlantic coast and English Channel were selected from archived individual samples (n = 863) collected either during or outside of mortality events between 2014 and 2017. All mussels were analysed by real-time PCR assays targeting F. halioticida; in addition, 185 were analysed using histological analysis and 178 by 16S rRNA metabarcoding. F. halioticida DNA was detected by real-time PCR and 16S rRNA metabarcoding in 282 and 34 mussels, respectively. Among these individuals, 82% (real-time PCR analysis) and 76% (16S rRNA metabarcoding analysis) were sampled during a mortality event. Histological analyses showed that moribund individuals had lesions mainly characterized by necrosis, haemocyte infiltration and granulomas. Risk factor analysis showed that mussel batches with more than 20% of PCR-positive individuals were more likely to have been sampled during a mortality event, and positive 16S rRNA metabarcoding batches increased the strength of the association with mortality by 11.6 times. The role of F. halioticida in mussel mortalities was determined by reviewing the available evidence. To this end, a causation criteria grid, tailored to marine diseases and molecular pathogen detection tools, allowed more evidence to be gathered on the causal role of this bacterium in mussel mortalities.

De Novo Transcriptome Assembly and Analysis of the Flat Oyster Pathogenic Protozoa Bonamia Ostreae.

The flat oyster Ostrea edulis is an oyster species native to Europe. It has declined to functional extinction in many areas of the NE Atlantic for several decades. Factors explaining this decline include over-exploitation of natural populations and diseases like bonamiosis, regulated across both the EU and the wider world and caused by the intracellular protozoan parasite Bonamia ostreae. To date, very limited sequence data are available for this Haplosporidian species. We present here the first transcriptome of B. ostreae. As this protozoan is not yet culturable, it remains extremely challenging to obtain high-quality -omic data. Thanks to a specific parasite isolation protocol and a dedicated bioinformatic pipeline, we were able to obtain a high-quality transcriptome for an intracellular marine micro-eukaryote, which will be very helpful to better understand its biology and to consider the development of new relevant diagnostic tools.

Monitoring Autophagy at Cellular and Molecular Level in Crassostrea gigas During an Experimental Ostreid Herpesvirus 1 (OsHV-1) Infection.

Mortality outbreaks of young Pacific oysters, Crassostrea gigas, have seriously affected the oyster-farming economy in several countries around the world. Although the causes of these mortality outbreaks appear complex, a viral agent has been identified as the main factor: a herpesvirus called ostreid herpesvirus 1 (OsHV-1). Autophagy is an important degradation pathway involved in the response to several pathologies including viral diseases. In C. gigas, recent studies indicate that this pathway is conserved and functional in at least haemocytes and the mantle. Furthermore, an experimental infection in combination with compounds known to inhibit or induce autophagy in mammals revealed that autophagy is involved in the response to OsHV-1 infection. In light of these results, the aim of this study was to determine the role of autophagy in the response of the Pacific oyster to infection by virus OsHV-1. For this purpose, an experimental infection in combination with a modulator of autophagy was performed on Pacific oysters known to have intermediate susceptibility to OsHV-1 infection. In haemolymph and the mantle, the autophagy response was monitored by flow cytometry, western blotting, and real-time PCR. At the same time, viral infection was evaluated by quantifying viral DNA and RNA amounts by real-time PCR. Although the results showed activation of autophagy in haemolymph and the mantle 14 hours post infection (after viral replication was initiated), they were also indicative of different regulatory mechanisms of autophagy in the two tissues, thus supporting an important function of autophagy in the response to virus OsHV-1.

First characterization of the parasite Haplosporidium costale in France and development of a real-time PCR assay for its rapid detection in the Pacific oyster, Crassostrea gigas.

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.

Vibrio aestuarianus subsp. cardii subsp. nov., pathogenic to the edible cockles Cerastoderma edule in France, and establishment of Vibrio aestuarianus subsp. aestuarianus subsp. nov. and Vibrio aestuarianus subsp. francensis subsp. nov.

Cockle mortality events have been reported in northern France since 2012. In the present study, we describe and investigate the implication of a potential bacterial causative agent in cockle mortality. Bacteria isolated from five different cockle mortality events were characterized and studied. Using phenotypic analysis combined with DNA-DNA hybridization (DDH) and whole genome sequencing, the isolates were shown to belong to Vibrio aestuarianus, a species regularly detected in France during oyster mortality events. Comparison of the strains from cockles with strains from French oysters and the type strain showed that the strains from cockles were genetically different to those from oysters and also different to the V. aestuarianus type strain. Moreover, the cockle and oyster strains were classified into two different, but close, groups both separated from the type strain by: (1) analyses of the ldh gene sequences; (2) DDH assays between 12/122 3T3T (LMG 31436T=DSM 109723T), a representative cockle strain, 02/041T (CIP 109791T=LMG 24517T) representative oyster strain and V. aestuarianus type strain LMG 7909T; (3) average nucleotide identity values calculated on the genomes; and (4) phenotypic traits. Finally, results of MALDI-TOF analyses also revealed specific peaks discriminating the three representative strains. The toxicity of representative strains of these cockle isolates was demonstrated by experimental infection of hatchery-produced cockles. The data therefore allow us to propose two novel subspecies of Vibrio aestuarianus: Vibrio aestuarianus subsp. cardii subsp. nov. for the cockle strains and Vibrio aestuarianus subsp. francensis subsp. nov. for the Pacific oyster strains, in addition to an emended description of the species Vibrio aestuarianus.

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

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

A new multiplex real-time PCR assay to improve the diagnosis of shellfish regulated parasites of the genus Marteilia and Bonamia.

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® 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 (intra-laboratory 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.

Identification of the autophagy pathway in a mollusk bivalve, Crassostrea gigas.

The Pacific oyster, Crassostrea gigas, is a mollusk bivalve commercially important as a food source. Pacific oysters are subjected to stress and diseases during culture. The autophagy pathway is involved in numerous cellular processes, including responses to starvation, cell death, and microorganism elimination. Autophagy also exists in C. gigas, and plays a role in the immune response against infections. Although this process is well-documented and conserved in most animals, it is still poorly understood in mollusks. To date, no study has provided a complete overview of the molecular mechanism of autophagy in mollusk bivalves. In this study, human and yeast ATG protein sequences and public databases (Uniprot and NCBI) were used to identify protein members of the C. gigas autophagy pathway. A total of 35 autophagy related proteins were found in the Pacific oyster. RACE-PCR was performed on several genes. Using molecular (real-time PCR) and protein-based (western blot and immunohistochemistry) approaches, the expression and localization of ATG12, ATG9, BECN1, MAP1LC3, MTOR, and SQSTM1, was investigated in different tissues of the Pacific oyster. Comparison with human and yeast counterparts demonstrated a high homology with the human autophagy pathway. The results also demonstrated that the key autophagy genes and their protein products were expressed in all the analyzed tissues of C. gigas. This study allows the characterization of the complete C. gigas autophagy pathway for the first time. Abbreviations: ATG: autophagy related; Atg1/ULK: unc-51 like autophagy activating kinase; ATG7: autophagy related 7; ATG9: autophagy related 9; ATG12: autophagy related 12; BECN1: beclin 1; BSA: bovine serum albumin; cDNA: complementary deoxyribonucleic acid; DNA: deoxyribonucleic acid; GABARAP: GABA type A receptor-associated protein; IHC: immunohistochemistry; MAP1LC3/LC3/Atg8: microtubule associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; NCBI: national center for biotechnology information; ORF: open reading frame; PBS: phosphate-buffered saline; PCR: polymerase chain reaction; PtdIns3K: class III phosphatidylinositol 3-kinase; RACE-PCR: rapid amplification of cDNA-ends by polymerase chain reaction; RNA: ribonucleic acid; SQSTM1: sequestosome 1; Uniprot: universal protein resource; WIPI: WD repeat domain, phosphoinositide interacting.

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.

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.

A study of autophagy in hemocytes of the Pacific oyster, Crassostrea gigas.

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.

Descriptions of Mikrocytos veneroïdes n. sp. and Mikrocytos donaxi n. sp. (Ascetosporea: Mikrocytida: Mikrocytiidae), detected during important mortality events of the wedge clam Donax trunculus Linnaeus (Veneroida: Donacidae), in France between 2008 and 2011.

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.

Assessing the health status of farmed mussels (Mytilus galloprovincialis) through histological, microbiological and biomarker analyses.

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.

Contribution of in Vivo Experimental Challenges to Understanding Flat Oyster Ostrea edulis Resistance to Bonamia ostreae.

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.

Haemocytes from Crassostrea gigas and OsHV-1: A promising in vitro system to study host/virus interactions.

Since 2008, mass mortality outbreaks associated with the detection of particular variants of OsHV-1 have been reported in Crassostrea gigas spat and juveniles in several countries. Recent studies have reported information on viral replication during experimental infection. Viral DNA and RNA were also detected in the haemolymph and haemocytes suggesting that the virus could circulate through the circulatory system. However, it is unknown if the virus is free in the haemolymph, passively associated at the surface of haemocytes, or able to infect and replicate inside these cells inducing (or not) virion production. In the present study, we collected haemocytes from the haemolymphatic sinus of the adductor muscle of healthy C. gigas spat and exposed them in vitro to a viral suspension. Results showed that viral RNAs were detectable one hour after contact and the number of virus transcripts increased over time in association with an increase of viral DNA detection. These results suggested that the virus is able to initiate replication rapidly inside haemocytes maintained in vitro. These in vitro trials were also used to carry out a dual transcriptomic study. We analyzed concomitantly the expression of some host immune genes and 15 viral genes. Results showed an up regulation of oyster genes currently studied during OsHV-1 infection. Additionally, transmission electron microscopy examination was carried out and did not allow the detection of viral particles. Moreover, All the results suggested that the in vitro model using haemocytes can be valuable for providing new perspective on virus-oyster interactions.

Haemocytes collected from experimentally infected Pacific oysters, Crassostrea gigas: Detection of ostreid herpesvirus 1 DNA, RNA, and proteins in relation with inhibition of apoptosis.

Recent transcriptomic approaches focused on anti-viral immunity in molluscs lead to the assumption that the innate immune system, such as apoptosis, plays a crucial role against ostreid herpesvirus type 1 (OsHV-1), infecting Pacific cupped oyster, Crassostrea gigas. Apoptosis constitutes a major mechanism of anti-viral response by limiting viral spread and eliminating infected cells. In this way, an OsHV-1 challenge was performed and oysters were monitored at three times post injection to investigate viral infection and host response: 2h (early after viral injection in the adductor muscle), 24h (intermediate time), and 48h (just before first oyster mortality record). Virus infection, associated with high cumulative mortality rates (74% and 100%), was demonstrated in haemocytes by combining several detection techniques such as real-time PCR, real-time RT PCR, immunofluorescence assay, and transmission electron microscopy examination. High viral DNA amounts ranged from 5.46×104 to 3.68×105 DNA copies ng-1 of total DNA, were detected in dead oysters and an increase of viral transcripts was observed from 2, 24, and 48hpi for the five targeted OsHV-1 genes encoding three putative membrane proteins (ORFs 25, 41, and 72), a putative dUTPase (ORF 75), and a putative apoptosis inhibitor (ORF 87). Apoptosis was studied at molecular and cellular levels with an early marker (phosphatidyl-serine externalisation measured by flow cytometry and epifluorescence microscopy) and a later parameter (DNA fragmentation by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay (TUNEL)). The down-regulation of genes encoding proteins involved in the activation of the apoptotic pathway (TNF and caspase 3) and the up-regulation of genes encoding anti-apoptotic proteins (IAP-2, and Bcl-2) suggested an important anti-apoptosis phenomenon in haemocytes from OsHV-1 infected oysters at 24 and 48hpi. Additionally, more phosphatidyl-serines were externalized and more cells with DNA fragmentation were observed in haemocytes collected from artificial seawater injected oysters than in haemocytes collected from OsHV-1 infected oysters at 24 and 48hpi, suggesting an inhibition of the apoptotic process in presence of the virus. In conclusion, this study is the first to focus on C. gigas haemocytes, cells involved in the host immune defense, during an OsHV-1 challenge in controlled conditions by combining various and original approaches to investigate apoptosis at molecular and cellular levels.

Flat oyster follows the apoptosis pathway to defend against the protozoan parasite Bonamia ostreae.

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.

Whole-genome amplification: a useful approach to characterize new genes in unculturable protozoan parasites such as Bonamia exitiosa.

Bonamia exitiosa is 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 the Bonamia genus 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 protozoan B. exitiosa. This is the first protein coding gene described in this species. Molecular analysis revealed that B. exitiosa actin is more similar to Bonamia ostreae actin gene-1. Actin phylogeny placed the Bonamia sp. infected oysters in the same clade where the herein described B. exitiosa actin 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.

Heat shock protein 90 of Bonamia ostreae: characterization and possible correlation with infection of the flat oyster, Ostrea edulis.

In this study, we described the cytosolic HSP90 of Bonamia ostreae, an intracellular parasite of Ostrea edulis hemocytes. The complete open reading frame was assembled by Rapid Amplification cDNA Ends reactions on cDNA of B. ostreae-infected hemocytes. 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 hemocyte invasion. Our results inform the molecular basis that governs B. ostreae-O. edulis interactions.

Characterization of the protozoan parasite Marteilia refringens infecting the dwarf oyster Ostrea stentina in Tunisia.

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.