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.11 page(s

High polymorphism in big defensin gene expression reveals presence-absence gene variability (PAV) in the oyster Crassostrea gigas

We report here the first evidence in an invertebrate, the oyster Crassostrea gigas, of a phenomenon of Presence-Absence Variation (PAV) affecting immune-related genes. We previously evidenced an extraordinary interindividual variability in the basal mRNA abundances of oyster immune genes including those coding for a family of antimicrobial peptides, the big defensins (Cg-BigDef). Cg-BigDef is a diverse family composed of three members: Cg-BigDef1 to -3. Here, we show that besides a high polymorphism in Cg-BigDef mRNA expression, not all individual oysters express simultaneously the three Cg-BigDefs. Moreover, in numerous individuals, no expression of Cg-BigDefs could be detected. Further investigation at the genomic level revealed that in individuals in which the transcription of one or all Cg-BigDefs was absent the corresponding Cg-bigdef gene was missing. In our experiments, no correlation was found between Cg-bigdef PAV and oyster capacity to survive Vibrio infections. The discovery of P-A immune genes in oysters leads to reconsider the role that plays the immune system in the individual adaptation to survive environmental, biotic and abiotic stresses

MOESM2 of Distinct immune responses of juvenile and adult oysters (Crassostrea gigas) to viral and bacterial infections

Additional file 2.  List of regulated genes in juveniles and adults in response response to OsHV-1, V. aestuarianus and V . tasmaniensis LGP32. Statistical analysis of qPCR data was performed separately for juvenile and adult oysters. Two-way analysis of variance (ANOVA) was conducted to individually assess expression levels of the 102 target genes using the univariate general linear model (GLM) with post hoc Tukey’s HSD test in IBM SPSS Statistics v 20.0

Exposure to the Neurotoxic Dinoflagellate, Alexandrium catenella, Induces Apoptosis of the Hemocytes of the Oyster, Crassostrea gigas

This study assessed the apoptotic process occurring in the hemocytes of the Pacific oyster, Crassostrea gigas, exposed to Alexandrium catenella, a paralytic shellfish toxins (PSTs) producer. Oysters were experimentally exposed during 48 h to the toxic algae. PSTs accumulation, the expression of 12 key apoptotic-related genes, as well as the variation of the number of hemocytes in apoptosis was measured at time intervals during the experiment. Results show a significant increase of the number of hemocytes in apoptosis after 29 h of exposure. Two pro-apoptotic genes (Bax and Bax-like) implicated in the mitochondrial pathway were significantly upregulated at 21 h followed by the overexpression of two caspase executor genes (caspase-3 and caspase-7) at 29 h, suggesting that the intrinsic pathway was activated. No modulation of the expression of genes implicated in the cell signaling Fas-Associated protein with Death Domain (FADD) and initiation-phase (caspase-2) was observed, suggesting that only the extrinsic pathway was not activated. Moreover, the clear time-dependent upregulation of five (Bcl2, BI-1, IAP1, IAP7B and Hsp70) inhibitors of apoptosis-related genes associated with the return to the initial number of hemocytes in apoptosis at 48 h of exposure suggests the involvement of strong regulatory mechanisms of apoptosis occurring in the hemocytes of the Pacific oyster

A Feedback Mechanism to Control Apoptosis Occurs in the Digestive Gland of the Oyster Crassostrea gigas Exposed to the Paralytic Shellfish Toxins Producer Alexandrium catenella

To better understand the effect of Paralytic Shellfish Toxins (PSTs) accumulation in the digestive gland of the Pacific oyster, Crassostrea gigas, we experimentally exposed individual oysters for 48 h to a PSTs producer, the dinoflagellate Alexandrium catenella. In comparison to the effect of the non-toxic Alexandrium tamarense, on the eight apoptotic related genes tested, Bax and BI.1 were significantly upregulated in oysters exposed 48 h to A. catenella. Among the five detoxification related genes tested, the expression of cytochrome P450 (CYP1A) was shown to be correlated with toxin concentration in the digestive gland of oysters exposed to the toxic dinoflagellate. Beside this, we observed a significant increase in ROS production, a decrease in caspase-3/7 activity and normal percentage of apoptotic cells in this tissue. Taken together, these results suggest a feedback mechanism, which may occur in the digestive gland where BI.1 could play a key role in preventing the induction of apoptosis by PSTs. Moreover, the expression of CYP1A, Bax and BI.1 were found to be significantly correlated to the occurrence of natural toxic events, suggesting that the expression of these genes together could be used as biomarker to assess the biological responses of oysters to stress caused by PSTs

Efficient and long-lasting protection against the pacific oyster mortality syndrome through antiviral immune priming

International audienceThe major economic and environmental consequences of recurring mortalities affecting the Pacific oyster Crassostrea gigas have initiated many research projects aiming at understanding these phenomena. The solutions anticipated to deal with these mortalities are mainly based on mass selection breeding programs but preventive treatments are still lacking. However, over the last decade, studies have been accumulating revealing the adaptive capabilities of innate immunity, the only component of defense mechanisms in invertebrates. Numerous findings have shown that a wide range of invertebrates can develop innate immune memory (also called immune priming) leading to improved survival during a second encounter with a pathogen. In this context, we undertook to study the possibilities of acting against mortalities by stimulating immune capacities of oysters.In the present study, we show that the exposure of oyster juveniles to an immunostimulant (a viral mimic called poly (I: C)) can lead to enhanced survival capacities (up to 100%) following OsHV-1 infection or during a mortality episode in the field. That protection is specific to viral protection as poly(I:C) fails to protect oyster against a pathogenic bacteria. We also show that this priming phenomenon is durable as it can last more than 4 months suggesting for the first time the existence of mechanisms of immune memory in this invertebrate species. Finally, analysis of the molecular pathways underlying that protection using dual RNAseq, revealed that priming was based on the triggering of a strong and sustained antiviral response limiting replication of the virus, thus allowing the protection of oysters on the long term. Altogether these results bring new insights into the oyster capacities to build an innate immune memory, its adaptive capacities and provide a platform to further explore novel strategies to help mitigate disease threats upon marine bivalves

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

Laligation native simplifie l’étude de l’activité etdu mécanismed’action des peptides antimicrobiens à multiples domaines :cas des Big défensines d’huître.

International audienceLesBigdéfensinesd'invertébrésmarinssontdespetitesprotéinesantimicrobiennesdontlastructureparticulièrementoriginaleassocieundomaineN-terminalglobulaireàundomaineC-terminalsemblableauxβ-défensinesdevertébrés.LapremièreBigdéfensineaétédécritechez un chélicérate ancestral, la Limule. Depuis, ces molécules ont été identifiées dansdiverses espèces de mollusques bivalves, notamment les huitres chez lesquelles plusieursformes ont été décrites. Certaines de ces formes, comme la Cg-BigDef1, voient leurexpressiontrèsfortementinduiteenréponseàl'infectionmicrobienne,laissantsupposerunrôle majeur dans la défense antimicrobienne des huitres. Nous avons ici entrepris decaractériserl’activitéantimicrobiennedelaCg-BigDef1etdesesdeuxdomainesstructurauxisolés. Pour cela nous avons synthétisé les deux domaines et la molécule entière parsynthèse en phase solide et ligation native (NCL) en s’appuyant sur une méthodologierécemment développée pour la synthèse du partenaire thioester. Les résultats obtenusmontrent que la Cg-BigDef1 est microbicide sur un large spectre de bactéries et dechampignons filamenteux, incluant des pathogènes des huitres du genre Vibrio. NousobservonségalementdepuissantesactivitésantimicrobiennessurdessouchescliniquesdeStaphylocoques isolées de patients mucoviscidosiques et posant des problèmes demultirésistance aux antibiotiques (MRSA). Ces activités sont stables à salinité élevée.L’activitédesdeuxdomainesséparésestglobalement très faiblepar rapportàlamoléculeentièreetcesdeuxdomainesdéploientdesactivitéssynergiques.Enfin,auxconcentrationsminimales inhibitrices, laCg-BigDef1 induit seulement la perméabilisation desmembranesdesbactériesàGramnégatifalorsquesondomaineglobulaireN-terminalseulperméabiliselesmembranesdesbactériesàGrampositifetdesbactériesà Gramnégatif

The Ancestral N-Terminal Domain of Big Defensins Drives Bacterially Triggered Assembly into Antimicrobial Nanonets

Big defensins, ancestors of β-defensins, are composed of a β-defensin-like C-terminal domain and a globular hydrophobic ancestral N-terminal domain. This unique structure is found in a limited number of phylogenetically distant species, including mollusks, ancestral chelicerates, and early-branching cephalochordates, mostly living in marine environments. One puzzling evolutionary issue concerns the advantage for these species of having maintained a hydrophobic domain lost during evolution toward β-defensins. Using native ligation chemistry, we produced the oyster Crassostrea gigas BigDef1 (Cg-BigDef1) and its separate domains. Cg-BigDef1 showed salt-stable and broad-range bactericidal activity, including against multidrug-resistant human clinical isolates of Staphylococcus aureus. We found that the ancestral N-terminal domain confers salt-stable antimicrobial activity to the β-defensin-like domain, which is otherwise inactive. Moreover, upon contact with bacteria, the N-terminal domain drives Cg-BigDef1 assembly into nanonets that entrap and kill bacteria. We speculate that the hydrophobic N-terminal domain of big defensins has been retained in marine phyla to confer salt-stable interactions with bacterial membranes in environments where electrostatic interactions are impaired. Those remarkable properties open the way to future drug developments when physiological salt concentrations inhibit the antimicrobial activity of vertebrate β-defensins. IMPORTANCE β-Defensins are host defense peptides controlling infections in species ranging from humans to invertebrates. However, the antimicrobial activity of most human β-defensins is impaired at physiological salt concentrations. We explored the properties of big defensins, the β-defensin ancestors, which have been conserved in a number of marine organisms, mainly mollusks. By focusing on a big defensin from oyster (Cg-BigDef1), we showed that the N-terminal domain lost during evolution toward β-defensins confers bactericidal activity to Cg-BigDef1, even at high salt concentrations. Cg-BigDef1 killed multidrug-resistant human clinical isolates of Staphylococcus aureus. Moreover, the ancestral N-terminal domain drove the assembly of the big defensin into nanonets in which bacteria are entrapped and killed. This discovery may explain why the ancestral N-terminal domain has been maintained in diverse marine phyla and creates a new path of discovery to design β-defensin derivatives active at physiological and high salt concentrations

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