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.

Seaweeds influence oyster microbiota and disease susceptibility.

A growing awareness of role that microbiota can play in mediating the effects of pathogens on hosts has given rise to the concept of the pathobiome. Recently, we demonstrated that the Pacific oyster mortality syndrome affecting Crassostrea gigas oysters is caused by infection with the Ostreid herpesvirus type 1 (OsHV-1) followed by infection with multiple bacterial taxa. Here we extend the concept of this pathobiome beyond the host species and its bacterial microbiota by investigating how seaweed living in association with oysters influences their response to the disease. We hypothesized that by their mere presence in the environment, different species of seaweeds can positively or negatively influence the risk of disease in oysters by shaping their bacterial microbiota and their immune response. Although seaweed and oysters do not have direct ecological interactions, they are connected by seawater and likely share microbes. To test our hypothesis, oysters were acclimated with green, brown or red algae for 2 weeks and then challenged with OsHV-1. We monitored host survival and pathogen proliferation and performed bacterial microbiota and transcriptome analyses. We found that seaweeds can alter the bacterial microbiota of the host and its response to the disease. More particularly, green algae belonging to the genus Ulva spp. induced bacterial microbiota dysbiosis in oyster and modification of its transcriptional immune response leading to increased susceptibility to the disease. This work provides a better understanding of a marine disease and highlights the importance of considering both macrobiotic and microbiotic interactions for conservation, management and exploitation of marine ecosystems and resources.

Species-specific mechanisms of cytotoxicity toward immune cells determine the successful outcome of Vibrio infections.

Vibrio species cause infectious diseases in humans and animals, but they can also live as commensals within their host tissues. How Vibrio subverts the host defenses to mount a successful infection remains poorly understood, and this knowledge is critical for predicting and managing disease. Here, we have investigated the cellular and molecular mechanisms underpinning infection and colonization of 2 virulent Vibrio species in an ecologically relevant host model, oyster, to study interactions with marine Vibrio species. All Vibrio strains were recognized by the immune system, but only nonvirulent strains were controlled. We showed that virulent strains were cytotoxic to hemocytes, oyster immune cells. By analyzing host and bacterial transcriptional responses to infection, together with Vibrio gene knock-outs, we discovered that Vibrio crassostreae and Vibrio tasmaniensis use distinct mechanisms to cause hemocyte lysis. Whereas V. crassostreae cytotoxicity is dependent on a direct contact with hemocytes and requires an ancestral gene encoding a protein of unknown function, r5.7, V. tasmaniensis cytotoxicity is dependent on phagocytosis and requires intracellular secretion of T6SS effectors. We conclude that proliferation of commensal vibrios is controlled by the host immune system, preventing systemic infections in oysters, whereas the successful infection of virulent strains relies on Vibrio species-specific molecular determinants that converge to compromise host immune cell function, allowing evasion of the host immune system.

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.

Fine-scale temporal dynamics of herpes virus and vibrios in seawater during a polymicrobial infection in the Pacific oyster Crassostrea gigas.

The Pacific oyster Crassostrea gigas is currently being impacted by a polymicrobial disease that involves early viral infection by ostreid herpesvirus-1 (OsHV-1) followed by a secondary bacterial infection leading to death. A widely used method of inducing infection consists of placing specific pathogen-free oysters ('recipients') in cohabitation in the laboratory with diseased oysters that were naturally infected in the field ('donors'). With this method, we evaluated the temporal dynamics of pathogen release in seawater and the cohabitation time necessary for disease transmission and expression. We showed that OsHV-1 and Vibrio spp. in the seawater peaked concomitantly during the first 48 h and decreased thereafter. We found that 1.5 h of cohabitation with donors was enough time to transmit pathogens to recipients and to induce mortality later, reflecting the highly contagious nature of the disease. Finally, mortality of recipients was associated with increasing cohabitation time with donors until reaching a plateau at 20%. This reflects the cumulative effect of exposure to pathogens. The optimal cohabitation time was 5-6 d, the mortality of recipients occurring 1-2 d earlier.

Inefficient immune response is associated with microbial permissiveness in juvenile oysters affected by mass mortalities on field.

Since 2008, juvenile Crassostrea gigas oysters have suffered from massive mortalities in European farming areas. This disease of complex etiology is still incompletely understood. Triggered by an elevated seawater temperature, it has been associated to infections by a herpes virus named OsHV-1 as well as pathogenic vibrios of the Splendidus clade. Ruling out the complexity of the disease, most of our current knowledge has been acquired in controlled experiments. Among the many unsolved questions, it is still ignored what role immunity plays in the capacity oysters have to survive an infectious episode. Here we show that juvenile oysters susceptible to the disease mount an inefficient immune response associated with microbial permissiveness and death. We found that, in contrast to resistant adult oysters having survived an earlier episode of mortality, susceptible juvenile oysters never exposed to infectious episodes died by more than 90% in a field experiment. Susceptible oysters were heavily colonized by OsHV-1 herpes virus as well as bacteria including vibrios potentially pathogenic for oysters, which proliferated in oyster flesh and body fluids during the mortality event. Nonetheless, susceptible oysters were found to sense microbes as indicated by an overexpression of immune receptors and immune signaling pathways. However, they did not express important immune effectors involved in antimicrobial immunity and apoptosis and showed repressed expression of genes involved in ROS and metal homeostasis. This contrasted with resistant oysters, which expressed those important effectors, controlled bacterial and viral colonization and showed 100% survival to the mortality event. Altogether, our results demonstrate that the immune response mounted by susceptible oysters lacks some important immune functions and fails in controlling microbial proliferation. This study opens the way to more holistic studies on the "mass mortality syndrome", which are now required to decipher the sequence of events leading to oyster mortalities and determine the relative weight of pathogens, oyster genetics and oyster-associated microbiota in the disease.

HSP70 from the Antarctic sea urchin Sterechinus neumayeri: molecular characterization and expression in response to heat stress.

BACKGROUND: Heat stress proteins are implicated in stabilizing and refolding denatured proteins in vertebrates and invertebrates. Members of the Hsp70 gene family comprise the cognate heat shock protein (Hsc70) and inducible heat shock protein (Hsp70). However, the cDNA sequence and the expression of Hsp70 in the Antarctic sea urchin are unknown. METHODS: We amplified and cloned a transcript sequence of 1991 bp from the Antarctic sea urchin Sterechinus neumayeri, experimentally exposed to heat stress (5  and 10 °C for 1, 24 and 48 h). RACE-PCR and qPCR were employed to determine Hsp70 gene expression, while western blot and ELISA methods were used to determine protein expression. RESULTS: The sequence obtained from S. neumayeri showed high identity with Hsp70 members. Several Hsp70 family features were identified in the deduced amino acid sequence and they indicate that the isolated Hsp70 is related to the cognate heat shock protein type. The corresponding 70 kDa protein, called Sn-Hsp70, was immune detected in the coelomocytes and the digestive tract of S. neumayeri using a monospecific polyclonal antibody. We showed that S. neumayeri do not respond to acute heat stress by up-regulation of Sn-Hsp70 at transcript and protein level. Furthermore, the Sn-Hsp70 protein expression was not induced in the digestive tract. CONCLUSIONS: Our results provide the first molecular evidence that Sn-Hsp70 is expressed constitutively and is non-induced by heat stress in S. neumayeri.

Distinct immune responses of juvenile and adult oysters (Crassostrea gigas) to viral and bacterial infections.

Since 2008, massive mortality events of Pacific oysters (Crassostrea gigas) have been reported worldwide and these disease events are often associated with Ostreid herpesvirus type 1 (OsHV-1). Epidemiological field studies have also reported oyster age and other pathogens of the Vibrio genus are contributing factors to this syndrome. We undertook a controlled laboratory experiment to simultaneously investigate survival and immunological response of juvenile and adult C. gigas at different time-points post-infection with OsHV-1, Vibrio tasmaniensis LGP32 and V. aestuarianus. Our data corroborates epidemiological studies that juveniles are more susceptible to OsHV-1, whereas adults are more susceptible to Vibrio. We measured the expression of 102 immune-genes by high-throughput RT-qPCR, which revealed oysters have different transcriptional responses to OsHV-1 and Vibrio. The transcriptional response in the early stages of OsHV-1 infection involved genes related to apoptosis and the interferon-pathway. Transcriptional response to Vibrio infection involved antimicrobial peptides, heat shock proteins and galectins. Interestingly, oysters in the later stages of OsHV-1 infection had a transcriptional response that resembled an antibacterial response, which is suggestive of the oyster's microbiome causing secondary infections (dysbiosis-driven pathology). This study provides molecular evidence that oysters can mount distinct immune response to viral and bacterial pathogens and these responses differ depending on the age of the host.

Can selection for resistance to OsHV-1 infection modify susceptibility to Vibrio aestuarianus infection in Crassostrea gigas? First insights from experimental challenges using primary and successive exposures.

Since 2008, the emergent virus OsHV-1µvar has provoked massive mortality events in Crassostrea gigas spat and juveniles in France. Since 2012, mortality driven by the pathogenic bacteria Vibrio aestuarianus has stricken market-sized adults. A hypothesis to explain the sudden increase in mortality observed in France since 2012 is that selective pressure due to recurrent viral infections could have led to a higher susceptibility of adults to Vibrio infection. In our study, two OsHV-1-resistant lines (AS and BS) and their respective controls (AC and BC) were experimentally challenged in the laboratory to determine their level of susceptibility to V. aestuarianus infection. At the juvenile stage, the selected lines exhibited lower mortality (14 and 33%) than the control lines (71 and 80%), suggesting dual-resistance to OsHV-1 and V. aestuarianus in C. gigas. Interestingly, this pattern was not observed at the adult stage, where higher mortality was detected for AS (68%) and BC (62%) than AC (39%) and BS (49%). These results were confirmed by the analysis of the expression of 31 immune-related genes in unchallenged oysters. Differential gene expression discriminated oysters according to their susceptibility to infection at both the juvenile and adult stages, suggesting that resistance to V. aestuarianus infection resulted in complex interactions between the genotype, stage of development and immunity status. Finally, survivors of the V. aestuarianus challenge at the juvenile stage still exhibited significant mortality at the adult stage during a second and third V. aestuarianus challenge, indicating that these survivors were not genetically resistant.

Use of OmpU porins for attachment and invasion of Crassostrea gigas immune cells by the oyster pathogen Vibrio splendidus.

OmpU porins are increasingly recognized as key determinants of pathogenic host Vibrio interactions. Although mechanisms remain incompletely understood, various species, including the human pathogen Vibrio cholera, require OmpU for host colonization and virulence. We have shown previously that OmpU is essential for virulence in the oyster pathogen Vibrio splendidus LGP32. Here, we showed that V. splendidus LGP32 invades the oyster immune cells, the hemocytes, through subversion of host-cell actin cytoskeleton. In this process, OmpU serves as an adhesin/invasin required for ß-integrin recognition and host cell invasion. Furthermore, the major protein of oyster plasma, the extracellular superoxide dismutase Cg-EcSOD, is used as an opsonin mediating the OmpU-promoted phagocytosis through its RGD sequence. Finally, the endocytosed bacteria were found to survive intracellularly, evading the host defense by preventing acidic vacuole formation and limiting reactive oxygen species production. We conclude that (i) V. splendidus is a facultative intracellular pathogen that manipulates host defense mechanisms to enter and survive in host immune cells, and (ii) that OmpU is a major determinant of host cell invasion in Vibrio species, used by V. splendidus LGP32 to attach and invade oyster hemocytes through opsonisation by the oyster plasma Cg-EcSOD.

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.

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 hemocyte gene expression signature correlated with predictive capacity of oysters to survive Vibrio infections.

BACKGROUND: The complex balance between environmental and host factors is an important determinant of susceptibility to infection. Disturbances of this equilibrium may result in multifactorial diseases as illustrated by the summer mortality syndrome, a worldwide and complex phenomenon that affects the oysters, Crassostrea gigas. The summer mortality syndrome reveals a physiological intolerance making this oyster species susceptible to diseases. Exploration of genetic basis governing the oyster resistance or susceptibility to infections is thus a major goal for understanding field mortality events. In this context, we used high-throughput genomic approaches to identify genetic traits that may characterize inherent survival capacities in C. gigas. RESULTS: Using digital gene expression (DGE), we analyzed the transcriptomes of hemocytes (immunocompetent cells) of oysters able or not able to survive infections by Vibrio species shown to be involved in summer mortalities. Hemocytes were nonlethally collected from oysters before Vibrio experimental infection, and two DGE libraries were generated from individuals that survived or did not survive. Exploration of DGE data and microfluidic qPCR analyses at individual level showed an extraordinary polymorphism in gene expressions, but also a set of hemocyte-expressed genes whose basal mRNA levels discriminate oyster capacity to survive infections by the pathogenic V. splendidus LGP32. Finally, we identified a signature of 14 genes that predicted oyster survival capacity. Their expressions are likely driven by distinct transcriptional regulation processes associated or not associated to gene copy number variation (CNV). CONCLUSIONS: We provide here for the first time in oyster a gene expression survival signature that represents a useful tool for understanding mortality events and for assessing genetic traits of interest for disease resistance selection programs.

Early life microbial exposures shape the Crassostrea gigas immune system for lifelong and intergenerational disease protection.

BACKGROUND: The interaction of organisms with their surrounding microbial communities influences many biological processes, a notable example of which is the shaping of the immune system in early life. In the Pacific oyster, Crassostrea gigas, the role of the environmental microbial community on immune system maturation - and, importantly, protection from infectious disease - is still an open question. RESULTS: Here, we demonstrate that early life microbial exposure durably improves oyster survival when challenged with the pathogen causing Pacific oyster mortality syndrome (POMS), both in the exposed generation and in the subsequent one. Combining microbiota, transcriptomic, genetic, and epigenetic analyses, we show that the microbial exposure induced changes in epigenetic marks and a reprogramming of immune gene expression leading to long-term and intergenerational immune protection against POMS. CONCLUSIONS: We anticipate that this protection likely extends to additional pathogens and may prove to be an important new strategy for safeguarding oyster aquaculture efforts from infectious disease. tag the videobyte/videoabstract in this section Video Abstract.

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.

In situ characterisation of pathogen dynamics during a Pacific oyster mortality syndrome episode.

Significant mortality of Crassostrea gigas juveniles is observed systematically every year worldwide. Pacific Oyster Mortality Syndrome (POMS) is caused by Ostreid Herpesvirus 1 (OsHV-1) infection leading to immune suppression, followed by bacteraemia caused by a consortium of opportunistic bacteria. Using an in-situ approach and pelagic chambers, our aim in this study was to identify pathogen dynamics in oyster flesh and in the water column during the course of a mortality episode in the Mediterranean Thau lagoon (France). OsHV-1 concentrations in oyster flesh increased before the first clinical symptoms of the disease appeared, reached maximum concentrations during the moribund phase and the mortality peak. The structure of the bacterial community associated with oyster flesh changed in favour of bacterial genera previously associated with oyster mortality including Vibrio, Arcobacter, Psychrobium, and Psychrilyobacter. During the oyster mortality episode, releases of OsHV-1 and opportunistic bacteria were observed, in succession, in the water surrounding the oyster lanterns. These releases may favour the spread of disease within oyster farms and potentially impact other marine species, thereby reducing marine biodiversity in shellfish farming areas.

Molecular diversity of antimicrobial effectors in the oyster Crassostrea gigas.

BACKGROUND: To gain insight into the molecular diversity of antimicrobial peptides and proteins in the oyster Crassostrea gigas, we characterized and compared the sequence polymorphism of the antimicrobial peptides (AMPs), Cg-Defensins (Cg-Defs) and Cg-Proline Rich peptide (Cg-Prp), and of the bactericidal permeability increasing protein, Cg-BPI. For that, we analyzed genomic and transcript sequences obtained by specific PCR amplification and in silico searches. RESULTS: High diversification among the three antimicrobial effectors was evidenced by this polymorphism survey. On the basis of sequence phylogenies, each AMP aggregates into clearly defined groups of variants and is the product of a multigenic family displaying a variety of gene structures. In contrast, Cg-bpi forms a single group and is encoded by a single gene copy. Moreover, we identified for both AMPs several genetic mechanisms of diversification such as recombination, parallel mutations leading to phylogenetic homoplasy and indel events. In addition, the non synonymous to synonymous substitutions ratio by codon (dN/dS) revealed several negatively and positively selected sites for both AMPs, suggesting that directional selection pressures have shaped their sequence variations. CONCLUSIONS: This study shows for the first time in a mollusc that antimicrobial peptides and proteins have been subject to distinct patterns of diversification and we evidence the existence of different evolutionary routes leading to such sequence variability.

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.

Contribution of Viral Genomic Diversity to Oyster Susceptibility in the Pacific Oyster Mortality Syndrome.

Juvenile Pacific oysters (Crassostrea gigas) are subjected to recurrent episodes of mass mortalities that constitute a threat for the oyster industry. This mortality syndrome named "Pacific Oyster Mortality Syndrome" (POMS) is a polymicrobial disease whose pathogenesis is initiated by a primary infection by a variant of an Ostreid herpes virus named OsHV-1 µVar. The characterization of the OsHV-1 genome during different disease outbreaks occurring in different geographic areas has revealed the existence of a genomic diversity for OsHV-1 µVar. However, the biological significance of this diversity is still poorly understood. To go further in understanding the consequences of OsHV-1 diversity on POMS, we challenged five biparental families of oysters to two different infectious environments on the French coasts (Atlantic and Mediterranean). We observed that the susceptibility to POMS can be different among families within the same environment but also for the same family between the two environments. Viral diversity analysis revealed that Atlantic and Mediterranean POMS are caused by two distinct viral populations. Moreover, we observed that different oyster families are infected by distinct viral populations within a same infectious environment. Altogether these results suggest that the co-evolutionary processes at play between OsHV-1 µVar and oyster populations have selected a viral diversity that could facilitate the infection process and the transmission in oyster populations. These new data must be taken into account in the development of novel selective breeding programs better adapted to the oyster culture environment.

A Sustained Immune Response Supports Long-Term Antiviral Immune Priming in the Pacific Oyster, Crassostrea gigas.

Over the last decade, innate immune priming has been evidenced in many invertebrate phyla. If mechanistic models have been proposed, molecular studies aiming to substantiate these models have remained scarce. We reveal here the transcriptional signature associated with immune priming in the oyster Crassostrea gigas Oysters were fully protected against Ostreid herpesvirus 1 (OsHV-1), a major oyster pathogen, after priming with poly(I·C), which mimics viral double-stranded RNA. Global analysis through RNA sequencing of oyster and viral genes after immune priming and viral infection revealed that poly(I·C) induces a strong antiviral response that impairs OsHV-1 replication. Protection is based on a sustained upregulation of immune genes, notably genes involved in the interferon pathway and apoptosis, which control subsequent viral infection. This persistent antiviral alert state remains active over 4 months and supports antiviral protection in the long term. This acquired resistance mechanism reinforces the molecular foundations of the sustained response model of immune priming. It further opens the way to applications (pseudovaccination) to cope with a recurrent disease that causes dramatic economic losses in the shellfish farming industry worldwide.IMPORTANCE In the last decade, important discoveries have shown that resistance to reinfection can be achieved without a functional adaptive immune system, introducing the concept of innate immune memory in invertebrates. However, this field has been constrained by the limited number of molecular mechanisms evidenced to support these phenomena. Taking advantage of an invertebrate species, the Pacific oyster (Crassostrea gigas), in which we evidenced one of the longest and most effective periods of protection against viral infection observed in an invertebrate, we provide the first comprehensive transcriptomic analysis of antiviral innate immune priming. We show that priming with poly(I·C) induced a massive upregulation of immune-related genes, which control subsequent viral infection, and it was maintained for over 4 months after priming. This acquired resistant mechanism reinforces the molecular foundations of the sustained response model of immune priming. It opens the way to pseudovaccination to prevent the recurrent diseases that currently afflict economically or ecologically important invertebrates.