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Abstract 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.

Abstract 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

Abstract 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.

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.

ABSTRACT 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.

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.

Pacific oyster mortality syndrome is a poorly understood cause of mortality in commercially important oyster species. Here, the authors use multiple infection experiments to show that the syndrome is caused by sequential infection by herpesvirus and opportunistic bacteria.

Cultured pearls are the product of grafting and rearing of Pinctada margaritifera pearl oysters in their natural environment. Nucleus rejections and oyster mortality appear to result from bacterial infections or from an inappropriate grafting practice. To reduce the impact of bacterial infections, synthetic antibiotics have been applied during the grafting practice. However, the use of such antibiotics presents a number of problems associated with their incomplete biodegradability, limited efficacy in some cases, and an increased risk of selecting for antimicrobial resistant bacteria. We investigated the application of a marine antimicrobial peptide, tachyplesin, which is present in the Japanese horseshoe crab Tachypleus tridentatus, in combination with two marine bacterial exopolymers as alternative treatment agents. In field studies, the combination treatment resulted in a significant reduction in graft failures vs. untreated controls. The combination of tachyplesin (73 mg/L) with two bacterial exopolysaccharides (0.5% w/w) acting as filming agents, reduces graft-associated bacterial contamination. The survival data were similar to that reported for antibiotic treatments. These data suggest that non-antibiotic treatments of pearl oysters may provide an effective means of improving oyster survival following grafting procedures.

The adaptative bleaching hypothesis (ABH) states that, depending on the symbiotic flexibility of coral hosts (i.e., the ability of corals to “switch” or “shuffle” their algal symbionts), coral bleaching can lead to a change in the composition of their associated Symbiodinium community and, thus, contribute to the coral’s overall survival. In order to determine the flexibility of corals, molecular tools are required to provide accurate species delineations and to detect low levels of coral-associated Symbiodinium. Here, we used highly sensitive quantitative (real-time) PCR (qPCR) technology to analyse five common coral species from Moorea (French Polynesia), previously screened using only traditional molecular methods, to assess the presence of low-abundance (background) Symbiodinium spp. Similar to other studies, each coral species exhibited a strong specificity to a particular clade, irrespective of the environment. In addition, however, each of the five species harboured at least one additional Symbiodinium clade, among clades A–D, at background levels. Unexpectedly, and for the first time in French Polynesia, clade B was detected as a coral symbiont. These results increase the number of known coral-Symbiodinium associations from corals found in French Polynesia, and likely indicate an underestimation of the ability of the corals in this region to associate with and/or “shuffle” different Symbiodinium clades. Altogether our data suggest that corals from French Polynesia may favor a trade-off between optimizing symbioses with a specific Symbiodinium clade(s), maintaining associations with particular background clades that may play a role in the ability of corals to respond to environmental change.

The genomics of economically important marine bivalves is studied to provide better understanding of the molecular mechanisms underlying their different reproductive strategies. The recently available gonad transcriptome of the black-lip pearl oyster Pinctada margaritifera is a novel and powerful resource to study these mechanisms in marine mollusks displaying hermaphroditic features. In this study, RNAseq quantification data of the P. margaritifera gonad transcriptome were analyzed to identify candidate genes in histologically-characterized gonad samples to provide molecular signatures of the female and male sexual pathway in this pearl oyster. Based on the RNAseq data set, stringent expression analysis identified 1,937 contigs that were differentially expressed between the gonad histological categories. From the hierarchical clustering analysis, a new reproduction model is proposed, based on a dual histo-molecular analytical approach. Nine candidate genes were identified as markers of the sexual pathway: 7 for the female pathway and 2 for the male one. Their mRNA levels were assayed by real-time PCR on a new set of gonadic samples. A clustering method revealed four principal expression patterns based on the relative gene expression ratio. A multivariate regression tree realized on these new samples and validated on the previously analyzed RNAseq samples showed that the sexual pathway of P. margaritifera can be predicted by a 3-gene-pair expression ratio model of 4 different genes: pmarg-43476, pmarg-foxl2, pmarg-54338 and pmarg-fem1-like. This 3-gene-pair expression ratio model strongly suggests only the implication of pmarg-foxl2 and pmarg-fem1-like in the sex inversion of P. margaritifera. This work provides the first histo-molecular model of P. margaritifera reproduction and a gene expression signature of its sexual pathway discriminating the male and female pathways. These represent useful tools for understanding and studying sex inversion, sex differentiation and sex determinism in this species and other related species for aquaculture purposes such as genetic selection programs.

Cultured pearls are human creations formed by inserting a nucleus and a small piece of mantle tissue into a living shelled mollusc, usually a pearl oyster. Although many pearl observations intuitively suggest a possible rotation of the nucleated pearl inside the oyster, no experimental demonstration of such a movement has ever been done. This can be explained by the difficulty of observation of such a phenomenon in the tissues of a living animal. To investigate this question of pearl rotation, a magnetometer system was specifically engineered to register magnetic field variations with magnetic sensors from movements of a magnetic nucleus inserted in the pearl oyster. We demonstrated that a continuous movement of the nucleus inside the oyster starts after a minimum of 40 days post-grafting and continues until the pearl harvest. We measured a mean angular speed of 1.27° min−1 calculated for four different oysters. Rotation variability was observed among oysters and may be correlated to pearl shape and defects. Nature's ability to generate so amazingly complex structures like a pearl has delivered one of its secrets.

In this study, we analyzed the combined effect of microalgal concentration and temperature on the shell growth of the bivalve Pinctada margaritifera and the molecular mechanisms underlying this biomineralization process. Shell growth was measured after two months of rearing in experimental conditions, using calcein staining of the calcified structures. Molecular mechanisms were studied though the expression of 11 genes encoding proteins implicated in the biomineralization process, which was assessed in the mantle. We showed that shell growth is influenced by both microalgal concentration and temperature, and that these environmental factors also regulate the expression of most of the genes studied. Gene expression measurement of shell matrix protein thereby appears to be an appropriate indicator for the evaluation of the biomineralization activity in the pearl oyster P. margaritifera under varying environmental conditions. This study provides valuable information on the molecular mechanisms of mollusk shell growth and its environmental control.

Antilipopolysaccharide factors (ALFs) have been described as highly cationic polypeptides with a broad spectrum of potent antimicrobial activities. In addition, ALFs have been shown to recognize LPS, a major component of the Gram-negative bacteria cell wall, through conserved amino acid residues exposed in the four-stranded β-sheet of their three dimensional structure. In penaeid shrimp, ALFs form a diverse family of antimicrobial peptides composed by three main variants, classified as ALF Groups A to C. Here, we identified a novel group of ALFs in shrimp (Group D ALFs), which corresponds to anionic polypeptides in which many residues of the LPS binding site are lacking. Both Group B (cationic) and Group D (anionic) shrimp ALFs were produced in a heterologous expression system. Group D ALFs were found to have impaired LPS-binding activities and only limited antimicrobial activity compared to Group B ALFs. Interestingly, all four ALF groups were shown to be simultaneously expressed in an individual shrimp and to follow different patterns of gene expression in response to a microbial infection. Group B was by far the more expressed of the ALF genes. From our results, nucleotide sequence variations in shrimp ALFs result in functional divergence, with significant differences in LPS-binding and antimicrobial activities. To our knowledge, this is the first functional characterization of the sequence diversity found in the ALF family.

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. [ABSTRACT FROM AUTHOR]