Cooperation and cheating orchestrate Vibrio assemblages and polymicrobial synergy in oysters infected with OsHV-1 virus.

Polymicrobial infections threaten the health of humans and animals but remain understudied in natural systems. We recently described the Pacific Oyster Mortality Syndrome (POMS), a polymicrobial disease affecting oyster production worldwide. In the French Atlantic coast, the disease involves coinfection with ostreid herpesvirus 1 (OsHV-1) and virulent Vibrio. However, it is unknown whether consistent Vibrio populations are associated with POMS in different regions, how Vibrio contribute to POMS, and how they interact with OsHV-1 during pathogenesis. By connecting field-based approaches in a Mediterranean ecosystem, laboratory infection assays and functional genomics, we uncovered a web of interdependencies that shape the structure and function of the POMS pathobiota. We show that Vibrio harveyi and Vibrio rotiferianus are predominant in OsHV-1-diseased oysters and that OsHV-1 drives the partition of the Vibrio community observed in the field. However only V. harveyi synergizes with OsHV-1 by promoting mutual growth and accelerating oyster death. V. harveyi shows high-virulence potential and dampens oyster cellular defenses through a type 3 secretion system, making oysters a more favorable niche for microbe colonization. In addition, V. harveyi produces a key siderophore called vibrioferrin. This important resource promotes the growth of V. rotiferianus, which cooccurs with V. harveyi in diseased oysters, and behaves as a cheater by benefiting from V. harveyi metabolite sharing. Our data show that cooperative behaviors contribute to synergy between bacterial and viral coinfecting partners. Additional cheating behaviors further shape the polymicrobial consortium. Controlling cooperative behaviors or countering their effects opens avenues for mitigating polymicrobial diseases.

Epigenetic variations are more substantial than genetic variations in rapid adaptation of oyster to Pacific oyster mortality syndrome.

Disease emergence is accelerating with global changes. Understanding by which mechanisms host populations can rapidly adapt will be crucial for management practices. Pacific oyster mortality syndrome (POMS) imposes a substantial and recurrent selective pressure on oyster populations, and rapid adaptation may arise through genetics and epigenetics. In this study, we used (epi)genome-wide association mapping to show that oysters differentially exposed to POMS displayed genetic and epigenetic signatures of selection. Consistent with higher resistance to POMS, the genes targeted included many genes in several pathways related to immunity. By combining correlation, DNA methylation quantitative trait loci, and variance partitioning, we revealed that a third of phenotypic variation was explained by interactions between the genetic and epigenetic information, ~14% by the genome, and up to 25% by the epigenome alone. Similar to genetically based adaptation, epigenetic mechanisms notably governing immune responses can contribute substantially to the rapid adaptation of hosts to emerging infectious diseases.

A core of functional complementary bacteria infects oysters in Pacific Oyster Mortality Syndrome.

BACKGROUND: The Pacific oyster Crassostrea gigas is one of the main cultivated invertebrate species worldwide. Since 2008, oyster juveniles have been confronted with a lethal syndrome known as the Pacific Oyster Mortality Syndrome (POMS). POMS is a polymicrobial disease initiated by a primary infection with the herpesvirus OsHV-1 µVar that creates an oyster immunocompromised state and evolves towards a secondary fatal bacteremia. RESULTS: In the present article, we describe the implementation of an unprecedented combination of metabarcoding and metatranscriptomic approaches to show that the sequence of events in POMS pathogenesis is conserved across infectious environments. We also identified a core bacterial consortium which, together with OsHV-1 µVar, forms the POMS pathobiota. This bacterial consortium is characterized by high transcriptional activities and complementary metabolic functions to exploit host's resources. A significant metabolic specificity was highlighted at the bacterial genus level, suggesting low competition for nutrients between members of the core bacteria. CONCLUSIONS: Lack of metabolic competition between the core bacteria might favor complementary colonization of host tissues and contribute to the conservation of the POMS pathobiota across distinct infectious environments.

Emergence and clonal expansion of Vibrio aestuarianus lineages pathogenic for oysters in Europe.

Crassostrea gigas oysters represent a significant global food source with 4.7 million tons harvested per year. In 2001, the bacterium Vibrio aestuarianus subsp. francensis emerged as a pathogen that causes adult oyster mortality in France and Ireland. Its impact on oyster aquaculture has increased in Europe since its re-emergence in 2012. To better understand the evolutionary mechanisms leading to the emergence and persistence over time of this pathogen, we conducted a survey of mollusc diseases through national reference laboratories across Europe. We analysed 54 new genomes of Vibrio aestuarianus (Va) isolated from multiple environmental compartments since 2001, in areas with and without bivalve mortalities. We used a combination of comparative genomics and population genetics approaches and show that Va has a classical epidemic population structure from which the pathogenic Va francensis subspecies emerged and clonally expanded. Furthermore, we identified a specific cus-cop-containing island conferring copper resistance to Va francensis whose acquisition may have favoured the emergence of pathogenic lineages adapted and specialized to oysters.

Evaluation of tangential flow filtration coupled to long-read sequencing for ostreid herpesvirus type 1 genome assembly.

Whole-genome sequencing is widely used to better understand the transmission dynamics, the evolution and the emergence of new variants of viral pathogens. This can bring crucial information to stakeholders for disease management. Unfortunately, aquatic virus genomes are usually difficult to characterize because most of these viruses cannot be easily propagated in vitro. Developing methodologies for routine genome sequencing of aquatic viruses is timely given the ongoing threat of disease emergence. This is particularly true for pathogenic viruses infecting species of commercial interest that are widely exchanged between production basins or countries. For example, the ostreid herpesvirus type 1 (OsHV-1) is a Herpesvirus widely associated with mass mortality events of juvenile Pacific oyster Crassostrea gigas. Genomes of Herpesviruses are large and complex with long direct and inverted terminal repeats. In addition, OsHV-1 is unculturable. It therefore accumulates several features that make its genome sequencing and assembly challenging. To overcome these difficulties, we developed a tangential flow filtration (TFF) method to enrich OsHV-1 infective particles from infected host tissues. This virus purification allowed us to extract high molecular weight and high-quality viral DNA that was subjected to Illumina short-read and Nanopore long-read sequencing. Dedicated bioinformatic pipelines were developed to assemble complete OsHV-1 genomes with reads from both sequencing technologies. Nanopore sequencing allowed characterization of new structural variations and major viral isomers while having 99,98 % of nucleotide identity with the Illumina assembled genome. Our study shows that TFF-based purification method, coupled with Nanopore sequencing, is a promising approach to enable in field sequencing of unculturable aquatic DNA virus.

Life history of oysters influences Vibrio parahaemolyticus accumulation in Pacific oysters (Crassostrea gigas).

Vibrio parahaemolyticus infection in humans is associated with raw oyster consumption. Evaluation of V. parahaemolyticus presence in oysters is of most interest because of the economic and public health issues that it represents. To explore V. parahaemolyticus accumulation and depuration in adult Crassostrea gigas, we developed a GFP-tagged V. parahaemolyticus strain (IFVp201-gfp+ ), as well as a rapid and efficient quantification method in C. gigas oysters haemolymph by flow cytometry. Impact of the life history of C. gigas on accumulation and depuration of V. parahaemolyticus IFVp201 was subsequently investigated. We found that naive oysters, i.e. grown in controlled facilities with UV treated seawater, accumulated significantly more IFVp201 than environmental oysters, i.e. grown in intertidal environment. We hypothesized that environmental oysters could have been immune primed, thus could limit V. parahaemolyticus accumulation. Meanwhile, both naive and environmental oysters had similar depuration rates.

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.

Differences in chemical contaminants bioaccumulation and ecotoxicology biomarkers in Mytilus edulis and Mytilus galloprovincialis and their hybrids.

The Mytilus mussels are spread all over the world and many related species coexist in several areas and can produce hybrid offspring. Mussels have been used for decades in national and international programs to monitor chemical contamination in the environment. Differences in bioaccumulation and biotransformation abilities between species and their hybrids should be evaluated to assess the comparability of the results obtained within the international biomonitoring programs. The objective of this study was to characterize bioaccumulation abilities and biomarker responses in Mytilus edulis, Mytilus galloprovincialis and their hybrids via an in situ transplantation experimentation on their progenies. Four mussel groups (M. edulis, M. galloprovincialis and two hybrids batches) issued from genetically characterized parents were transplanted for one year in Charente Maritime (France) to ensure their exposure to identical sources of contamination. The bioaccumulation of several families of contaminants (trace metals, polycyclic aromatic hydrocarbons, polybrominated diphenyl ethers, polychlorinated biphenyls), the response of several biomarkers (DNA strand breaks level, lysosomal membrane stability, metallothionein content, acetylcholine esterase activity) and some physiological parameters (growth, mortality, gonadal development), were analyzed. Differences were observed between species, however they were contaminant-specific. Variations in contaminants levels were observed between progenies, with higher levels of Cu, PBDE, PCB in M. edulis, and higher levels of Cd, Hg, Zn in M galloprovincialis. This study demonstrated that variations in contaminant bioaccumulation and different biomarker responses exist between Mytilus species in the field. Data on species or the presence of hybrid individuals (or introgression) is an important additional parameter to add to biomonitoring programs databases.

Gonadal transcriptomes associated with sex phenotypes provide potential male and female candidate genes of sex determination or early differentiation in Crassostrea gigas, a sequential hermaphrodite mollusc.

BACKGROUND: In the animal kingdom, mollusca is an important phylum of the Lophotrochozoa. However, few studies have investigated the molecular cascade of sex determination/early gonadal differentiation within this phylum. The oyster Crassostrea gigas is a sequential irregular hermaphrodite mollusc of economic, physiological and phylogenetic importance. Although some studies identified genes of its sex-determining/-differentiating pathway, this particular topic remains to be further deepened, in particular with regard to the expression patterns. Indeed, these patterns need to cover the entire period of sex lability and have to be associated to future sex phenotypes, usually impossible to establish in this sequential hermaphrodite. This is why we performed a gonadal RNA-Seq analysis of diploid male and female oysters that have not changed sex for 4 years, sampled during the entire time-window of sex determination/early sex differentiation (stages 0 and 3 of the gametogenetic cycle). This individual long-term monitoring gave us the opportunity to explain the molecular expression patterns in the light of the most statistically likely future sex of each oyster. RESULTS: The differential gene expression analysis of gonadal transcriptomes revealed that 9723 genes were differentially expressed between gametogenetic stages, and 141 between sexes (98 and 43 genes highly expressed in females and males, respectively). Eighty-four genes were both stage- and sex-specific, 57 of them being highly expressed at the time of sex determination/early sex differentiation. These 4 novel genes including Trophoblast glycoprotein-like, Protein PML-like, Protein singed-like and PREDICTED: paramyosin, while being supported by RT-qPCR, displayed sexually dimorphic gene expression patterns. CONCLUSIONS: This gonadal transcriptome analysis, the first one associated with sex phenotypes in C. gigas, revealed 57 genes highly expressed in stage 0 or 3 of gametogenesis and which could be linked to the future sex of the individuals. While further study will be needed to suggest a role for these factors, some could certainly be original potential actors involved in sex determination/early sex differentiation, like paramyosin and could be used to predict the future sex of oysters.

Genomic Diversity of the Ostreid Herpesvirus Type 1 Across Time and Location and Among Host Species.

The mechanisms underlying virus emergence are rarely well understood, making the appearance of outbreaks largely unpredictable. This is particularly true for pathogens with low per-site mutation rates, such as DNA viruses, that do not exhibit a large amount of evolutionary change among genetic sequences sampled at different time points. However, whole-genome sequencing can reveal the accumulation of novel genetic variation between samples, promising to render most, if not all, microbial pathogens measurably evolving and suitable for analytical techniques derived from population genetic theory. Here, we aim to assess the measurability of evolution on epidemiological time scales of the Ostreid herpesvirus 1 (OsHV-1), a double stranded DNA virus of which a new variant, OsHV-1 µVar, emerged in France in 2008, spreading across Europe and causing dramatic economic and ecological damage. We performed phylogenetic analyses of heterochronous (n = 21) OsHV-1 genomes sampled worldwide. Results show sufficient temporal signal in the viral sequences to proceed with phylogenetic molecular clock analyses and they indicate that the genetic diversity seen in these OsHV-1 isolates has arisen within the past three decades. OsHV-1 samples from France and New Zealand did not cluster together suggesting a spatial structuration of the viral populations. The genome-wide study of simple and complex polymorphisms shows that specific genomic regions are deleted in several isolates or accumulate a high number of substitutions. These contrasting and non-random patterns of polymorphism suggest that some genomic regions are affected by strong selective pressures. Interestingly, we also found variant genotypes within all infected individuals. Altogether, these results provide baseline evidence that whole genome sequencing could be used to study population dynamic processes of OsHV-1, and more broadly herpesviruses.

Susceptibility variation to the main pathogens of Crassostrea gigas at the larval, spat and juvenile stages using unselected and selected oysters to OsHV-1 and/or V. aestuarianus.

French commercial hatcheries are massively producing Crassostrea gigas selected for their higher resistance to OsHV-1, and soon should also implement selection for increasing resistance to Vibrio aestuarianus. The first objective of this study was to optimize the breeding programs for dual resistance to OsHV-1 and V. aestuarianus to determine the earliest life stage for which oysters are able to develop disease resistance. Wild stocks and selected families were tested using experimental infections by both pathogens at the larval, spat and juvenile stages. Oyster families could be evaluated for OsHV-1 as soon as the larval stage by a bath method, but this only highlighted the most resistant families; those that showed the highest resistance to V. aestuarianus could be determined using the cohabitation method at the juvenile stage. The second objective of this study was to determine if selection to increase/decrease the resistance to OsHV-1 and V. aestuarianus could have an impact on other major pathogens currently detected in hatchery at the larval stage, and in nursery and field at the spat/juveniles stages (V. coralliilyticus, V. crassostreae, V. tasmaniensis, V. neptunius, V. europaeus, V. harveyi, V. chagasi). No relationship was found between mortality caused by V. aestuarianus/OsHV-1 and the mortality caused by the other virulent bacterial strains tested regardless the stages, except between OsHV-1 and V. tasmaniensis at the juvenile stage. Finally, miscellaneous findings were evidenced such as (1) bath for bacterial challenges was not adapted for spat, (2) the main pathogens at the larval stage were OsHV-1 and V. coralliilyticus using bath, while it was V. coralliilyticus, V. europaeus, and V. neptunius at the juvenile stage by injection, and (4) variation in mortality was observed among families/wild controls for all pathogens at larval and juvenile stages, except for V. harveyi for larvae.

The Pacific Oyster Mortality Syndrome, a Polymicrobial and Multifactorial Disease: State of Knowledge and Future Directions.

The Pacific oyster (Crassostreae gigas) has been introduced from Asia to numerous countries around the world during the 20th century. C. gigas is the main oyster species farmed worldwide and represents more than 98% of oyster production. The severity of disease outbreaks that affect C. gigas, which primarily impact juvenile oysters, has increased dramatically since 2008. The most prevalent disease, Pacific oyster mortality syndrome (POMS), has become panzootic and represents a threat to the oyster industry. Recently, major steps towards understanding POMS have been achieved through integrative molecular approaches. These studies demonstrated that infection by Ostreid herpesvirus type 1 µVar (OsHV-1 µvar) is the first critical step in the infectious process and leads to an immunocompromised state by altering hemocyte physiology. This is followed by dysbiosis of the microbiota, which leads to a secondary colonization by opportunistic bacterial pathogens, which in turn results in oyster death. Host and environmental factors (e.g. oyster genetics and age, temperature, food availability, and microbiota) have been shown to influence POMS permissiveness. However, we still do not understand the mechanisms by which these different factors control disease expression. The present review discusses current knowledge of this polymicrobial and multifactorial disease process and explores the research avenues that must be investigated to fully elucidate the complexity of POMS. These discoveries will help in decision-making and will facilitate the development of tools and applied innovations for the sustainable and integrated management of oyster aquaculture.

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.

Differential Mortality and High Viral Load in Naive Pacific Oyster Families Exposed to OsHV-1 Suggests Tolerance Rather than Resistance to Infection.

Pacific oysters, Crassostrea gigas, are one of the most productive aquaculture species in the world. However, they are threatened by the spread of Ostreid herpesvirus-1 (OsHV-1) and its microvariants (collectively "µvars"), which cause mass mortalities in all life stages of Pacific oysters globally. Breeding programs have been successful in reducing mortality due to OsHV-1 variants following viral outbreaks; however, an OsHV-1-resistant oyster line does not yet exist in the United States (US), and it is unknown how OsHV-1 µvars will affect US oyster populations compared to the current variant, which is similar to the OsHV-1 reference, found in Tomales Bay, CA. The goals of this study were to investigate the resistance of C. gigas juveniles produced by the Molluscan Broodstock Program (MBP) to three variants of OsHV-1: a California reference OsHV-1, an Australian µvar, and a French µvar. This is the first study to directly compare OsHV-1 µvars to a non-µvar. The survival probability of oysters exposed to the French (FRA) or Australian (AUS) µvar was significantly lower (43% and 71%, respectively) than to the reference variant and controls (96%). No oyster family demonstrated resistance to all three OsHV-1 variants, and many surviving oysters contained high copy numbers of viral DNA (mean ~3.53 × 108). These results indicate that the introduction of OsHV-1 µvars could have substantial effects on US Pacific oyster aquaculture if truly resistant lines are not achieved, and highlight the need to consider resistance to infection in addition to survival as traits in breeding programs to reduce the risk of the spread of OsHV-1 variants.

Unraveling concordant and varying responses of oyster species to Ostreid Herpesvirus 1 variants.

The Ostreid herpesvirus 1 (OsHV-1) and variants, particularly the microvariants (µVars), are virulent and economically devastating viruses impacting oysters. Since 2008 OsHV-1 µVars have emerged rapidly having particularly damaging effects on aquaculture industries in Europe, Australia and New Zealand. We conducted field trials in Tomales Bay (TB), California where a non-µVar strain of OsHV-1 is established and demonstrated differential mortality of naturally exposed seed of three stocks of Pacific oyster, Crassostrea gigas, and one stock of Kumamoto oyster, C. sikamea. Oysters exposed in the field experienced differential mortality that ranged from 64 to 99% in Pacific oysters (Tasmania>Midori = Willapa stocks), which was much higher than that of Kumamoto oysters (25%). Injection trials were done using French (FRA) and Australian (AUS) µVars with the same oyster stocks as planted in the field and, in addition, two stocks of the Eastern oyster, C. virginica. No mortality was observed in control oysters. One C. virginica stock suffered ~10% mortality when challenged with both µVars tested. Two Pacific oyster stocks suffered 75 to 90% mortality, while one C. gigas stock had relatively low mortality when challenged with the AUS µVar (~22%) and higher mortality when challenged with the French µVar (~72%). Conversely, C. sikamea suffered lower mortality when challenged with the French µVar (~22%) and higher mortality with the AUS µVar (~44%). All dead oysters had higher viral loads (~1000×) as measured by quantitative PCR relative to those that survived. However, some survivors had high levels of virus, including those from species with lower mortality. Field mortality in TB correlated with laboratory mortality of the FRA µVar (69% correlation) but not with that of the AUS µVar, which also lacked correlation with the FRA µVar. The variation in response to OsHV-1 variant challenges by oyster species and stocks demonstrates the need for empirical assessment of multiple OsHV-1 variants.

Removal of pathogens by ultrafiltration from sea water.

Among water treatment processes, ultrafiltration is known to be efficient for the elimination of micro-organisms (bacteria and viruses). In this study, two pathogens were targeted, a bacterium, Vibrio aestuarianus and a virus, OsHV-1, with the objective to produce high quality water from seawater, in the case of shellfish productions. The retention of those microorganisms by ultrafiltration was evaluated at labscale. In the case of OsHV-1, the protection of oysters was validated by in vivo experiments using oysters spat and larvae, both stages being highly susceptible to the virus. The oysters raised using contaminated seawater which was then subsequently treated by ultrafiltration, had similar mortality to the negative controls. In the case of V. aestuarianus, ultrafiltration allowed a high retention of the bacteria in seawater with concentrations below the detection limits of the 3 analytical methods (flow cytometry, direct seeding and seeding after filtration to 0.22 µm). Thus, the quantity of V. aestuarianus was at least, 400 times inferior to the threshold known to induce mortalities in oysters. Industrial scale experiment on a several months period confirmed the conclusion obtained at lab scale on the Vibrio bacteria retention. Indeed, no bacteria from this genus, potentially harmful for oysters, was detected in permeate and this, whatever the quality of the seawater treated and the bacteria concentration upstream of the membrane. Moreover, the resistance of the process was confirmed with a stability of hydraulic performances over time for two water qualities and even facing an algal bloom. In terms of retention and resistance, ultrafiltration process was validated for the treatment of seawater towards the targeted pathogenic microorganisms, with the aim of biosecuring shellfish productions.

Microbiota Composition and Evenness Predict Survival Rate of Oysters Confronted to Pacific Oyster Mortality Syndrome.

Pacific Oyster Mortality Syndrome (POMS) affects Crassostrea gigas oysters worldwide and causes important economic losses. Disease dynamic was recently deciphered and revealed a multiple and progressive infection caused by the Ostreid herpesvirus OsHV-1 µVar, triggering an immunosuppression followed by microbiota destabilization and bacteraemia by opportunistic bacterial pathogens. However, it remains unknown if microbiota might participate to protect oysters against POMS, and if microbiota characteristics might be predictive of oyster mortalities. To tackle this issue, we transferred full-sib progenies of resistant and susceptible oyster families from hatchery to the field during a period in favor of POMS. After 5 days of transplantation, oysters from each family were either sampled for individual microbiota analyses using 16S rRNA gene-metabarcoding or transferred into facilities to record their survival using controlled condition. As expected, all oysters from susceptible families died, and all oysters from the resistant family survived. Quantification of OsHV-1 and bacteria showed that 5 days of transplantation were long enough to contaminate oysters by POMS, but not for entering the pathogenesis process. Thus, it was possible to compare microbiota characteristics between resistant and susceptible oysters families at the early steps of infection. Strikingly, we found that microbiota evenness and abundances of Cyanobacteria (Subsection III, family I), Mycoplasmataceae, Rhodobacteraceae, and Rhodospirillaceae were significantly different between resistant and susceptible oyster families. We concluded that these microbiota characteristics might predict oyster mortalities.

First comparison of French and Australian OsHV-1 µvars by bath exposure.

Economically devastating mortality events of farmed and wild shellfish due to infectious disease have been reported globally. Currently, one of the most significant disease threats to Pacific oyster Crassostrea gigas culture is the ostreid herpesvirus 1 (OsHV-1), in particular the emerging OsHV-1 microvariant genotypes. OsHV-1 microvariants (OsHV-1 µvars) are spreading globally, and concern is high among growers in areas unaffected by OsHV-1. No study to date has compared the relative virulence among variants. We provide the first challenge study comparing survival of naïve juvenile Pacific oysters exposed to OsHV-1 µvars from Australia (AUS µvar) and France (FRA µvar). Oysters challenged with OsHV-1 µvars had low survival (2.5% exposed to AUS µvar and 10% to FRA µvar), and high viral copy number as compared to control oysters (100% survival and no virus detected). As our study was conducted in a quarantine facility located ~320 km from the ocean, we also compared the virulence of OsHV-1 µvars using artificial seawater made from either facility tap water (3782 µmol kg-1 seawater total alkalinity) or purchased distilled water (2003 µmol kg-1). Although no differences in survival or viral copy number were detected in oysters exposed to seawater made using tap or distilled water, more OsHV-1 was detected in tanks containing the lower-alkalinity seawater, indicating that water quality may be important for virus transmission, as it may influence the duration of viral viability outside of the host.

Differential basal expression of immune genes confers Crassostrea gigas resistance to Pacific oyster mortality syndrome.

BACKGROUND: As a major threat to the oyster industry, Pacific Oyster Mortality Syndrome (POMS) is a polymicrobial disease affecting the main oyster species farmed across the world. POMS affects oyster juveniles and became panzootic this last decade, but POMS resistance in some oyster genotypes has emerged. While we know some genetic loci associated with resistance, the underlying mechanisms remained uncharacterized. So, we developed a comparative transcriptomic approach using basal gene expression profiles between different oyster biparental families with contrasted phenotypes when confronted to POMS (resistant or susceptible). RESULTS: We showed that POMS resistant oysters show differential expression of genes involved in stress responses, protein modifications, maintenance of DNA integrity and repair, and immune and antiviral pathways. We found similarities and clear differences among different molecular pathways in the different resistant families. These results suggest that the resistance process is polygenic and partially varies according to the oyster genotype. CONCLUSIONS: We found differences in basal expression levels of genes related to TLR-NFκB, JAK-STAT and STING-RLR pathways. These differences could explain the best antiviral response, as well as the robustness of resistant oysters when confronted to POMS. As some of these genes represent valuable candidates for selective breeding, we propose future studies should further examine their function.