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.

Microbiota of the Digestive Glands and Extrapallial Fluids of Clams Evolve Differently Over Time Depending on the Intertidal Position.

The Manila clam (Ruditapes philippinarum) is the second most exploited bivalve in the world but remains threatened by diseases and global changes. Their associated microbiota play a key role in their fitness and acclimation capacities. This study aimed at better understanding the behavior of clam digestive glands and extrapallial fluids microbiota at small, but contrasting spatial and temporal scales. Results showed that environmental variations impacted clam microbiota differently according to the considered tissue. Each clam tissue presented its own microbiota and showed different dynamics according to the intertidal position and sampling period. Extrapallial fluids microbiota was modified more rapidly than digestive glands microbiota, for clams placed on the upper and lower intertidal position, respectively. Clam tissues could be considered as different microhabitats for bacteria as they presented different responses to small-scale temporal and spatial variabilities in natural conditions. These differences underlined a more stringent environmental filter capacity of the digestive glands.

Functional Diversification of Oyster Big Defensins Generates Antimicrobial Specificity and Synergy against Members of the Microbiota.

Big defensins are two-domain antimicrobial peptides (AMPs) that have highly diversified in mollusks. Cg-BigDefs are expressed by immune cells in the oyster Crassostrea gigas, and their expression is dampened during the Pacific Oyster Mortality Syndrome (POMS), which evolves toward fatal bacteremia. We evaluated whether Cg-BigDefs contribute to the control of oyster-associated microbial communities. Two Cg-BigDefs that are representative of molecular diversity within the peptide family, namely Cg-BigDef1 and Cg-BigDef5, were characterized by gene cloning and synthesized by solid-phase peptide synthesis and native chemical ligation. Synthetic peptides were tested for antibacterial activity against a collection of culturable bacteria belonging to the oyster microbiota, characterized by 16S sequencing and MALDI Biotyping. We first tested the potential of Cg-BigDefs to control the oyster microbiota by injecting synthetic Cg-BigDef1 into oyster tissues and analyzing microbiota dynamics over 24 h by 16S metabarcoding. Cg-BigDef1 induced a significant shift in oyster microbiota ß-diversity after 6 h and 24 h, prompting us to investigate antimicrobial activities in vitro against members of the oyster microbiota. Both Cg-BigDef1 and Cg-BigDef5 were active at a high salt concentration (400 mM NaCl) and showed broad spectra of activity against bacteria associated with C. gigas pathologies. Antimicrobial specificity was observed for both molecules at an intra- and inter-genera level. Remarkably, antimicrobial spectra of Cg-BigDef1 and Cg-BigDef5 were complementary, and peptides acted synergistically. Overall, we found that primary sequence diversification of Cg-BigDefs has generated specificity and synergy and extended the spectrum of activity of this peptide family.

m6A Profile Dynamics Indicates Regulation of Oyster Development by m6A-RNA Epitranscriptomes.

The N6-methylation of RNA adenosines (N6-methyladenosine, m6A) is an important regulator of gene expression with critical implications in vertebrate and insect development. However, the developmental significance of epitranscriptomes in lophotrochozoan organisms remains unknown. Using methylated RNA immunoprecipitation sequencing (MeRIP-seq), we generated transcriptome-wide m6A-RNA methylomes covering the whole development of the oyster from oocytes to juveniles. Oyster RNA classes display specific m6A signatures, with messenger RNAs (mRNAs) and long non-coding RNAs (lncRNAs) exhibiting distinct profiles and being highly methylated compared to transposable element (TE) transcripts. Epitranscriptomes are dynamic and correspond to the chronological steps of development (cleavage, gastrulation, organogenesis, and metamorphosis), with minimal mRNA and lncRNA methylation at the morula stage followed by a global increase. mRNA m6A levels are correlated to transcript levels, and shifts in methylation profiles correspond to expression kinetics. Differentially methylated transcripts cluster according to embryo-larval stages and bear the corresponding developmental functions (cell division, signal transduction, morphogenesis, and cell differentiation). The m6A level of TE transcripts is also regulated and peaks during the gastrulation. We demonstrate that m6A-RNA methylomes are dynamic and associated with gene expression regulation during oyster development. The putative epitranscriptome implication in the cleavage, maternal-to-zygotic transition, and cell differentiation in a lophotrochozoan model brings new insights into the control and evolution of developmental processes.

Harsh intertidal environment enhances metabolism and immunity in oyster (Crassostrea gigas) spat.

The Pacific oyster Crassostrea gigas is established in the marine intertidal zone, experiencing rapid and highly dynamic environmental changes throughout the tidal cycle. Depending on the bathymetry, oysters face oxygen deprivation, lack of nutrients, and high changes in temperature during alternation of the cycles of emersion/immersion. Here we showed that intertidal oysters at a bathymetry level of 3 and 5 m delayed by ten days the onset of mortality associated with Pacific Oyster Mortality Syndrome (POMS) as compared to subtidal oysters. Intertidal oysters presented a lower growth but similar energetic reserves to subtidal oysters but induced proteomic changes indicative of a boost in metabolism, inflammation, and innate immunity that may have improved their resistance during infection with the Ostreid herpes virus. Our work highlights that intertidal harsh environmental conditions modify host-pathogen interaction and improve oyster health. This study opens new perspectives on oyster farming for mitigation strategies based on tidal height.

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.

Phage-host coevolution in natural populations.

Coevolution between bacteriophages (phages) and their bacterial hosts occurs through changes in resistance and counter-resistance mechanisms. To assess phage-host evolution in wild populations, we isolated 195 Vibrio crassostreae strains and 243 vibriophages during a 5-month time series from an oyster farm and combined these isolates with existing V. crassostreae and phage isolates. Cross-infection studies of 81,926 host-phage pairs delineated a modular network where phages are best at infecting co-occurring hosts, indicating local adaptation. Successful propagation of phage is restricted by the ability to adsorb to closely related bacteria and further constrained by strain-specific defence systems. These defences are highly diverse and predominantly located on mobile genetic elements, and multiple defences are active within a single genome. We further show that epigenetic and genomic modifications enable phage to adapt to bacterial defences and alter host range. Our findings reveal that the evolution of bacterial defences and phage counter-defences is underpinned by frequent genetic exchanges with, and between, mobile genetic elements.

Genetic diversity and connectivity of the Ostreid herpesvirus 1 populations in France: A first attempt to phylogeographic inference for a marine mollusc disease.

The genetic diversity of viral populations is a key driver of the spatial and temporal diffusion of viruses; yet, studying the diversity of whole genomes from natural populations still remains a challenge. Phylodynamic approaches are commonly used for RNA viruses harboring small genomes but have only rarely been applied to DNA viruses with larger genomes. Here, we used the Pacific oyster mortality syndrome (a disease that affects oyster farms around the world) as a model to study the genetic diversity of its causative agent, the Ostreid herpesvirus 1 (OsHV-1) in the three main French oyster-farming areas. Using ultra-deep sequencing on individual moribund oysters and an innovative combination of bioinformatics tools, we de novo assembled twenty-one OsHV-1 new genomes. Combining quantification of major and minor genetic variations, phylogenetic analysis, and ancestral state reconstruction of discrete traits approaches, we assessed the connectivity of OsHV-1 viral populations between the three oyster-farming areas. Our results suggest that the Marennes-Oléron Bay represents the main source of OsHV-1 diversity, from where the virus has dispersed to other farming areas, a scenario consistent with current practices of oyster transfers in France. We demonstrate that phylodynamic approaches can be applied to aquatic DNA viruses to determine how epidemiological, immunological, and evolutionary processes act and potentially interact to shape their diversity patterns.

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.

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.

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.

A functional m6 A-RNA methylation pathway in the oyster Crassostrea gigas assumes epitranscriptomic regulation of lophotrochozoan development.

N6 -methyladenosine (m6 A) is a prevalent epitranscriptomic mark in eukaryotic RNA, with crucial roles for mammalian and ecdysozoan development. Indeed, m6 A-RNA and the related protein machinery are important for splicing, translation, maternal-to-zygotic transition and cell differentiation. However, to date, the presence of an m6 A-RNA pathway remains unknown in more distant animals, questioning the evolution and significance of the epitranscriptomic regulation. Therefore, we investigated the m6 A-RNA pathway in the oyster Crassostrea gigas, a lophotrochozoan model whose development was demonstrated under strong epigenetic influence. Using mass spectrometry and dot blot assays, we demonstrated that m6 A-RNA is actually present in the oyster and displays variations throughout early oyster development, with the lowest levels at the end of cleavage. In parallel, by in silico analyses, we were able to characterize at the molecular level a complete and conserved putative m6 A machinery. The expression levels of the identified putative m6 A writers, erasers and readers were strongly regulated across oyster development. Finally, RNA pull-down coupled to LC-MS/MS allowed us to prove the actual presence of readers able to bind m6 A-RNA and exhibiting specific developmental patterns. Altogether, our results demonstrate the conservation of a complete m6 A-RNA pathway in the oyster and strongly suggest its implication in early developmental processes including MZT. This first demonstration and characterization of an epitranscriptomic regulation in a lophotrochozoan model, potentially involved in the embryogenesis, bring new insights into our understanding of developmental epigenetic processes and their evolution.

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.

High temperature induces transcriptomic changes in Crassostrea gigas that hinder progress of ostreid herpesvirus (OsHV-1) and promote survival.

Of all environmental factors, seawater temperature plays a decisive role in triggering marine diseases. Like fever in vertebrates, high seawater temperature could modulate the host response to pathogens in ectothermic animals. In France, massive mortality of Pacific oysters, Crassostrea gigas, caused by the ostreid herpesvirus 1 (OsHV-1) is markedly reduced when temperatures exceed 24°C in the field. In the present study we assess how high temperature influences the host response to the pathogen by comparing transcriptomes (RNA sequencing) during the course of experimental infection at 21°C (reference) and 29°C. We show that high temperature induced host physiological processes that are unfavorable to the viral infection. Temperature influenced the expression of transcripts related to the immune process and increased the transcription of genes related to the apoptotic process, synaptic signaling and protein processes at 29°C. Concomitantly, the expression of genes associated with catabolism, metabolite transport, macromolecule synthesis and cell growth remained low from the first stage of infection at 29°C. Moreover, viral entry into the host might have been limited at 29°C by changes in extracellular matrix composition and protein abundance. Overall, these results provide new insights into how environmental factors modulate host-pathogen interactions.

Latitudinal drivers of oyster mortality: deciphering host, pathogen and environmental risk factors.

Diseases pose an ongoing threat to aquaculture, fisheries and conservation of marine species, and determination of risk factors of disease is crucial for management. Our objective was to decipher the effects of host, pathogen and environmental factors on disease-induced mortality of Pacific oysters (Crassostrea gigas) across a latitudinal gradient. We deployed young and adult oysters at 13 sites in France and we monitored survival, pathogens and environmental parameters. The young oysters came from either the wild collection or the hatchery while the adults were from the wild only. We then used Cox regression models to investigate the effect of latitude, site, environmental factors and origin on mortality risk and to extrapolate this mortality risk to the distribution limits of the species in Europe. We found that seawater temperature, food level, sea level atmospheric pressure, rainfall and wind speed were associated with mortality risk. Their effect on hatchery oysters was generally higher than on wild animals, probably reflecting that hatchery oysters were free of Ostreid herpesvirus 1 (OsHV-1) whereas those from the wild were asymptomatic carriers. The risk factors involved in young and adult oyster mortalities were different, reflecting distinct diseases. Mortality risk increases from 0 to 90% with decreasing latitude for young hatchery oysters, but not for young wild oysters or adults. Mortality risk was higher in wild oysters than in hatchery ones at latitude > 47.6°N while this was the opposite at lower latitude. Therefore, latitudinal gradient alters disease-induced mortality risk but interacts with the initial health status of the host and the pathogen involved. Practically, we suggest that mortality can be mitigated by using hatchery oysters in north and wild collected oysters in the south.

The marine intertidal zone shapes oyster and clam digestive bacterial microbiota.

Digestive microbiota provide a wide range of beneficial effects on host physiology and are therefore likely to play a key role in marine intertidal bivalve ability to acclimatize to the intertidal zone. This study investigated the effect of intertidal levels on the digestive bacterial microbiota of oysters (Crassostrea gigas) and clams (Ruditapes philippinarum), two bivalves with different ecological niches. Based on 16S rRNA region sequencing, digestive glands, seawater and sediments harbored specific bacterial communities, dominated by operational taxonomic units assigned to the Mycoplasmatales,Desulfobacterales and Rhodobacterales orders, respectively. Field implantation modified digestive bacterial microbiota of both bivalve species according to their intertidal position. Rhodospirillales and Legionellales abundances increased in oysters and clams from the low intertidal level, respectively. After a 14-day depuration process, these effects were still observed, especially for clams, while digestive bacterial microbiota of oysters were subjected to more short-term environmental changes. Nevertheless, 3.5 months stay on an intertidal zone was enough to leave an environmental footprint on the digestive bacterial microbiota, suggesting the existence of autochthonous bivalve bacteria. When comparing clams from the three intertidal levels, 20% of the bacterial assemblage was shared among the levels and it was dominated by an operational taxonomic unit affiliated to the Mycoplasmataceae and Spirochaetaceae families.

Author Correction: Proteinaceous secretion of bioadhesive produced during crawling and settlement of Crassostrea gigas larvae.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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.