Free-living amoebae (FLA) are ubiquitous and can be found in many environments including soil, freshwater, and marine environments. They feed on various microorganisms and can play an important role in the food web and its dynamics. We previously described that FLA belonging to the Vannella genus isolated from oyster farms in the Thau lagoon in France are able to establish stable interactions with Vibrionaceae, suggesting they can play a role in pathogen dynamics. To further investigate the ecological interactions between FLA and Vibrionaceae in Mediterranean coastal waters, we conducted monthly sampling for 1 year at three contrasting sites. FLA populations were isolated from water and sediment samples on different bacterial lawns, including Escherichia coli or Vibrio tasmaniensis, Vibrio crassostreae, and Vibrio harveyi. Diversity analysis by v4-18S barcoding revealed distinct communities between fractions and sites. The Vannellidae were found significantly enriched in the water, whereas Paramoebidae were found enriched in the sediments. Additionally, uneven distribution of Vexilliferidae, Vermistellae, and, to a lesser extent, Acanthamoebidae and Subulatomonas contrasted between sampling sites. Selection of grazers on different bacterial lawns revealed that V. tasmaniensis inhibits the growth of most Vannellidae, whereas V. crassostreae inhibits the growth of Paramoebidae. These differences were further confirmed functionally using isolates belonging to each Amoebozoa taxonomic group. Overall, our results highlight the need for more comprehensive studies of the diversity and population dynamics of Amoebozoa in marine environments and indicate that the role of these diversified grazers in shaping Vibrio communities is complex and still poorly characterized.IMPORTANCEAlthough they can play an important role in shaping bacterial communities and as intracellular niches for potential pathogens, the diversity and ecology of free-living amoebae (FLA) in marine environments are still poorly characterized. Very few studies have used systematic approaches to unravel the population dynamics and ecological variations of FLA in different environments. Our study in marine coastal environments highlights that FLA taxonomic groups can harbor habitat preferences and have different predation capacities, suggesting that FLA-bacteria interactions need to be considered at different taxonomic levels to uncover generalist versus specialist adaptations.

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.

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.

Disease emergence is accelerating in response to human activity-induced global changes. Understanding the mechanisms by which host populations can rapidly adapt to this threat will be crucial for developing future management practices. Pacific Oyster Mortality Syndrome (POMS) imposes a substantial and recurrent selective pressure on oyster populations ( Crassostrea gigas) . Rapid adaptation to this disease may arise through both genetic and epigenetic mechanisms. In this study, we used a combination of whole exome capture of bisulfite-converted DNA, next-generation sequencing, and (epi)genome-wide association mapping, to show that natural oyster populations differentially exposed to POMS displayed signatures of selection both in their genome (single nucleotide polymorphisms) and epigenome (CG-context DNA methylation). Consistent with higher resistance to POMS, the genes targeted by genetic and epigenetic variations were mainly related to host immunity. By combining correlation analyses, DNA methylation quantitative trait loci, and variance partitioning, we revealed that a third of the observed phenotypic variation was explained by interactions between the genetic sequence and epigenetic information, ∼14% by the genetic sequence, and up to 25% by the epigenome alone. Thus, as well as genetic adaptation, epigenetic mechanisms governing immune responses contribute significantly to the rapid adaptation of hosts to emerging infectious diseases. ### Competing Interest Statement The authors have declared no competing interest.

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.

A major debate in evolutionary biology is whether virulence is maintained as an adaptive trait and/or evolves to non-virulence. In the environment, virulence traits of non-obligatory parasites are subjected to diverse selective pressures and trade-offs. Here, we focus on a population of Vibrio splendidus that displays moderate virulence for oysters. A MARTX (Multifunctional-autoprocessing repeats-in-toxin) and a type-six secretion system (T6SS) were found to be necessary for virulence toward oysters, while a region (wbe) involved in O-antigen synthesis is necessary for resistance to predation against amoebae. Gene inactivation within the wbe region had major consequences on the O-antigen structure, conferring lower immunogenicity, competitive advantage and increased virulence in oyster experimental infections. Therefore, O-antigen structures that favour resistance to environmental predators result in an increased activation of the oyster immune system and a reduced virulence in that host. These trade-offs likely contribute to maintaining O-antigen diversity in the marine environment by favouring genomic plasticity of the wbe region. The results of this study indicate an evolution of V. splendidus towards moderate virulence as a compromise between fitness in the oyster as a host, and resistance to its predators in the environment.

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.

Surface water concentrations of the synthetic estrogen 17α-ethinylestradiol (EE2) as low as 1 ng/L can cause adverse reproductive effects in fish under acute and chronic exposure conditions, whereas higher concentrations (> 5 ng/L) in acute studies are necessary to elicit adverse effects in freshwater mussels. Prolonged chronic exposures of freshwater mussels to EE2 remain un-evaluated. An extended duration testing paradigm was used to examine reproductive and biochemical (carbohydrate, lipid, protein) effects of EE2 on the unionid mussel, Elliptio complanata, throughout its reproductive cycle. Mussels were exposed to a control and EE2 concentrations (5 and 50 ng/L) in six discrete and sequential 28 d tests, and in one discrete and simultaneous 180 d test, from February through August. Foot protrusion and siphoning behavior were recorded daily, along with conglutinate releases and larval (glochidia) mortality. Gonad, hemolymph, and gonad fluid samples were taken for biochemical and vitellogenin-like protein (Vtg) analysis post-exposure. Female mussels released eggs and conglutinates during the months of April to June, indicating sexual maturation during this time. Conglutinates released in the 5 ng/L treatment in 28 d exposures contained fewer glochidia and more eggs, and increased concentrations of Vtg in hemolymph were observed from April to August in the 5 ng/L treatment during the 180 d exposure. Results indicate that the 180 d test approach, concurrent with the sequence of 28 d tests, enabled a more robust evaluation of mussel behavior and physiology than would have been possible with a single short-term (28 d) test.

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.

Although vibrios are frequently associated with marine organisms mortality outbreaks, knowledge on their ecology and pathogenicity is sparse, thus limiting disease management and prophylactic strategies. Here, we investigated V. aestuarianus infection onset and progression in the wild, taking advantage of a 'claire' pond: a semi-closed system with limited seawater renewal, theoretically more adapted to disease transmission. We showed a positive association of the bacteria with oysters, which can constitute a reservoir for the bacteria in the winter. Moreover, passage through oysters was found to be necessary for experimental disease reproduction as vibrios shedding from diseased oysters have higher infectivity than from in vitro grown. We next developed an experimental 'ecologically realistic' infection model in a mesocosm, allowing infection by natural route. By means of this non-invasive protocol, we analysed the pathogenesis of the bacteria and demonstrated the importance of haemolymph for initial colonization and the septicaemic nature of this disease.

Infectious diseases are mostly explored using reductionist approaches despite repeated evidence showing them to be strongly influenced by numerous interacting host and environmental factors. Many diseases with a complex aetiology therefore remain misunderstood. By developing a holistic approach to tackle the complexity of interactions, we decipher the complex intra-host interactions underlying Pacific oyster mortality syndrome affecting juveniles of Crassostrea gigas, the main oyster species exploited worldwide. Using experimental infections reproducing the natural route of infection and combining thorough molecular analyses of oyster families with contrasted susceptibilities, we demonstrate that the disease is caused by multiple infection with an initial and necessary step of infection of oyster haemocytes by the Ostreid herpesvirus OsHV-1 µVar. Viral replication leads to the host entering an immune-compromised state, evolving towards subsequent bacteraemia by opportunistic bacteria. We propose the application of our integrative approach to decipher other multifactorial diseases that affect non-model species worldwide.

Vibrio species have been associated with recurrent mass mortalities of juvenile oysters Crassostrea gigas threatening oyster farming worldwide. However, knowledge of the ecology of pathogens in affected oyster farming areas remains scarce. Specifically, there are no data regarding (i) the environmental reservoirs of Vibrio populations pathogenic to oysters, (ii) the environmental factors favoring their transmission, and (iii) the influence of oyster farming on the persistence of those pathogens. This knowledge gap limits our capacity to predict and mitigate disease occurrence. To address these issues, we monitored Vibrio species potentially pathogenic to C. gigas in 2013 and 2014 in the Thau Lagoon, a major oyster farming region in the coastal French Mediterranean. Sampling stations were chosen inside and outside oyster farms. Abundance and composition of phyto-, microzoo-, and mesozooplankton communities were measured monthly. The spatial and temporal dynamics of plankton and Vibrio species were compared, and positive correlations between plankton species and vibrios were verified by qPCR on isolated specimens of plankton. Vibrio crassostreae was present in the water column over both years, whereas Vibrio tasmaniensis was mostly found in 2013 and Vibrio aestuarianus was never detected. Moreover, V. tasmaniensis and V. crassostreae were found both as free-living or plankton-attached vibrios 1 month after spring mortalities of the oyster juveniles. Overall, V. crassostreae was associated with temperature and plankton composition, whereas V. tasmaniensis correlated with plankton composition only. The abundance of Vibrio species in the water column was similar inside and outside oyster farms, suggesting important spatial dispersion of pathogens in surrounding areas. Remarkably, a major increase in V. tasmaniensis and V. crassostreae was measured in the sediment of oyster farms during cold months. Thus, a winter reservoir of pathogenic vibrios could contribute to their ecology in this Mediterranean shellfish farming ecosystem.

This study investigated oyster infection dynamics by different strains of Vibrio aestuarianus isolated before and after the apparent re-emergence of this pathogen observed in France in 2011. We conducted experiments to compare minimal infective dose, lethal dose 50 and bacterial shedding for six V. aestuarianus strains. Whatever the strain used, mortality was induced in juvenile oysters by intramuscular injection and reached 90–100% of mortality within 5 days. Moreover, bacterial shedding was comparable among strains and reached its maximum after 20 h (≈10 EXP5 bacteria/mL/animal). Similarly, our first estimations of lethal dose 50 were comparable among strains (minimal infective dose around 0.4 × 10EXP5 bacteria/mL and LD50 around 10EXP5 bacteria/mL) by using seawater containing freshly shed bacteria. These results indicate that, at least with these criteria, despite V. aestuarianus strains genetic diversity, the disease process is similar. The strains isolated after the apparent re-emergence of the bacteria in 2011, do not present a more acute virulence phenotype than the reference strains isolated between 2002 and 2007. Finally, our study provides original and noteworthy data indicating that infected oysters shed bacteria at a level above the threshold of LD50 a few days before they die, meaning that infection is expected to spread in a susceptible population.

Oyster diseases caused by pathogenic vibrios pose a major challenge to the sustainability of oyster farming. In France, since 2012 a disease affecting specifically adult oysters has been associated with the presence of Vibrio aestuarianus. Here, by combining genome comparison, phylogenetic analyses and high-throughput infections of strains isolated before or during the recent outbreaks, we show that virulent strains cluster into two V. aestuarianus lineages independently of the sampling dates. The bacterial lethal dose was not different between strains isolated before or after 2012. Hence, the emergence of a new highly virulent clonal strain is unlikely. Each lineage comprises nearly identical strains, the majority of them being virulent, suggesting that within these phylogenetically coherent virulent lineages a few strains have lost their pathogenicity. Comparative genomics allowed the identification of a single frameshift in a non-virulent strain. This mutation affects the varS gene that codes for a signal transduction histidine-protein kinase. Genetic analyses confirmed that varS is necessary for infection of oysters and for a secreted metalloprotease expression. For the first time in a Vibrio species, we show here that VarS is a key factor of pathogenicity.

Background: Massive mortality outbreaks affecting Pacific oyster (Crassostrea gigas) spat in various countries have been associated with the detection of a herpesvirus called ostreid herpesvirus type 1 (OsHV-1). However, few studies have been performed to understand and follow viral gene expression, as it has been done in vertebrate herpesviruses. In this work, experimental infection trials of C. gigas spat with OsHV-1 were conducted in order to test the susceptibility of several bi-parental oyster families to this virus and to analyze host-pathogen interactions using in vivo transcriptomic approaches.Results: The divergent response of these oyster families in terms of mortality confirmed that susceptibility to OsHV-1 infection has a significant genetic component. Two families with contrasted survival rates were selected. A total of 39 viral genes and five host genes were monitored by real-time PCR. Initial results provided information on (i) the virus cycle of OsHV-1 based on the kinetics of viral DNA replication and transcription and (ii) host defense mechanisms against the virus.Conclusions: In the two selected families, the detected amounts of viral DNA and RNA were significantly different. This result suggests that Pacific oysters are genetically diverse in terms of their susceptibility to OsHV-1 infection. This contrasted susceptibility was associated with dissimilar host gene expression profiles. Moreover, the present study showed a positive correlation between viral DNA amounts and the level of expression of selected oyster genes.

Nine dominant bacterial isolates were obtained from different batches of Crassostrea gigas spat experiencing high mortality rates in a French experimental hatchery/nursery in 2007. Using phenotypic analysis combined with multilocus sequence analysis, the isolates were shown to be genetically close to the Vibrio tubiashii type strain. Based on (1) analyses of the recA gene sequences; (2) the results of DNA-DNA hybridization assays between 07/118 T2 (LMG 27884=CECT 8426), which is a representative strain, and the V. tubiashii type strain (69%); and (3) phenotypic traits, the bacteria were classified in a group close to American V. tubiashii strain. Its virulence (70% of mortalities) and the toxicity of the extracellular products of 07/118 T2 was demonstrated (41% of mortalities).Moreover, a QPCR diagnostic tool targeting the gyrB gene was developed to investigate the epidemiological significance of V. tubiashii in French oyster mortality outbreaks recorded by the national surveillance network. Of the 21 batches originating from hatcheries, only two were positive, whereas V. tubiashii DNA could not be detected in any of the batches of moribund animals collected in field/outdoor facilities.These results demonstrate the existence of a group of virulent V. tubiashii in France that episodically infect C. gigas. (C) 2014 Elsevier Inc. All rights reserved.