Delphine Destoumieux-Garzón

Laura Canesi

Daniel Oyanedel

Marie-Agnès Travers

Guillaume M Charrière

Carla Pruzzo

Luigi Vezzulli
email: luigi.vezzulli@unige.it

Luigi Vezzuli Distav

Vibrio-bivalve interactions in health and disease

Summary

In the marine environment, bivalve mollusks constitute habitats for bacteria of the Vibrionaceae family. Vibrios belong to the microbiota of healthy oysters and mussels, which have the ability to concentrate bacteria in their tissues and body fluids, including the hemolymph. Remarkably, these important aquaculture species respond differently to infectious diseases. While oysters are the subject of recurrent mass mortalities at different life stages, mussels appear rather resistant to infections.

Thus, Vibrio species are associated to the main diseases affecting the worldwide oyster production. Here we review the current knowledge on Vibrio-bivalve interaction in oysters (Crassostrea sp.) and mussels (Mytilus sp.). We discuss the transient versus stable associations of vibrios with these bivalves as well as technical issues limiting the precise monitoring of vibrios in health and disease. Based on the current knowledge of oyster/mussel immunity and their interactions with Vibrio species pathogenic for oyster, we discuss how differences in immune effectors could contribute to the higher resistance of mussels to infections. Finally, we review the multiple strategies evolved by pathogenic vibrios to circumvent the potent immune defenses of bivalves and how key virulence mechanisms could have been positively or negatively selected in the marine environment through interactions with predators.

Significance statement

This review highlights the multifaceted nature of Vibrio interactions with mussels and oysters, which show contrasting resistance to infections. Vibrios pathogenic for oyster tend to interact differently with major components of oyster/mussel plasma and with their key immune cells, the hemocytes. Moreover, the potent production of antimicrobial peptides (AMPs) in mussels as opposed to oysters could act on disease outcomes. Thanks to their successful colonization of oysters, different strategies of immune evasion and tolerance/resistance have been evidenced in vibrios pathogenic for oysters. These mechanisms include tolerance to the main cellular defenses of oysters (ROS, AMPs, heavy metals), active dampening of cellular immunity through cytolytic mechanisms (toxins, T6SS) as well as reduced antigenic properties through modifications of LPS, a major and hypervariable component of Vibrio outer membrane. Recent knowledge on the interactions of vibrios with environmental predators (phages, grazers) highlights possible mechanisms of both coincidental and conflicting selection acting on these important determinants of vibrio virulence.

Introduction

Animal-bacteria association has a long evolutionary history that could go as far back as to the origins of multicellularity [START_REF] Mcfall-Ngai | Animals in a bacterial world, a new imperative for the life sciences[END_REF]. The presence of complex host-associated microbial communities is a characteristic shared by most animal species [START_REF] Fraune | Why bacteria matter in animal development and evolution[END_REF] and the type of interactions that they establish with their host can range from mutualistic to pathogenic. The capacity for microbes to colonize a host depends on both host and microbial determinants. On the one hand, healthy metazoans control their microbial environment with physical and chemical barriers, which from early stages of development shape the assemblage of their associated microbiota (for review see [START_REF] Bevins | The potter's wheel: the host's role in sculpting its microbiota[END_REF][START_REF] Mcfall-Ngai | Animals in a bacterial world, a new imperative for the life sciences[END_REF]). On the other hand, microbes have evolve strategies to overcome a series of innate immune mechanisms conserved across metazoans, which control both infections and host-microbiome homeostasis [START_REF] Brennan | Evolutionary origins of toll-like receptor signaling[END_REF].

In marine environments, metazoans constitute habitats for bacteria of the Vibrionaceae family (vibrios). These γ-proteobacteria are ubiquitous in marine and brackish environments representing one of the most abundant culturable fraction of the marine microbial community [START_REF] Ceccarelli | Vibrio species[END_REF]. They possess a high colonization potential partially due to their metabolic versatility and genetic variability [START_REF] Le Roux | Oysters and Vibrios as a model for disease dynamics in wild animals[END_REF]. Their associations with plants, algae, zooplankton and other animals have been widely documented [START_REF] Takemura | Associations and dynamics of Vibrionaceae in the environment, from the genus to the population level[END_REF][START_REF] Le Roux | Eco-evolutionary dynamics linked to horizontal gene transfer in Vibrios[END_REF]. In particular, some species of mollusks have established very intimate associations with Vibrio species, as illustrated by the mutualistic symbiosis of the squid Euprymna scolopes (Cephalopod) and Vibrio fischeri [START_REF] Mcfall-Ngai | Divining the essence of symbiosis: insights from the squid-vibrio model[END_REF].

Bivalves, such as the oyster Crassostrea gigas, were shown to host a diversity of Vibrio populations, defined as members of a same species sharing a common gene pool and habitat [START_REF] Le Roux | Oysters and Vibrios as a model for disease dynamics in wild animals[END_REF], in both health and disease [START_REF] Bruto | Vibrio crassostreae, a benign oyster colonizer turned into a pathogen after plasmid acquisition[END_REF].

Vibrio associations with bivalves can be also neutral. Indeed, as filter feeders straining food particles from the seawater, bivalves accumulate exogenous bacteria, often transiently [START_REF] Froelich | The Interactions of Vibrio vulnificus and the oyster Crassostrea virginica[END_REF]. Moreover, Vibrio species attach to the chitinaceaous surfaces of invertebrates, which provide a nutrient-rich habitat (Dana E. Hunt et al., 2008), but do not always colonize their tissues. However, intimate interactions have also been described. Recently, evidence of co-evolution between vibrios and bivalves has been acquired in natural oyster beds [START_REF] Wegner | Molecular targets for coevolutionary interactions between Pacific oyster larvae and their sympatric vibrios[END_REF].

Parasitic interactions are among the more documented Vibrio-bivalve associations, as they cause major damages to the host [START_REF] Goudenège | A single regulatory gene is sufficient to alter Vibrio aestuarianus pathogenicity in oysters[END_REF][START_REF] Lemire | Populations, not clones, are the unit of vibrio pathogenesis in naturally infected oysters[END_REF][START_REF] Bruto | Ancestral gene acquisition as the key to virulence potential in environmental Vibrio populations[END_REF][START_REF] Dias | Vibrio tapetis displays an original Type IV Secretion system in strains pathogenic for bivalve Molluscs[END_REF], with important consequences on the sustainability of the shellfish farming industry [START_REF] Travers | Bacterial diseases in marine bivalves[END_REF]. A key question for predicting and managing disease is whether Vibrio associations with bivalves result from specific (genome-encoded) abilities or disability to colonize these animal species and how much they depend on environmental constraints. A series of genetic determinants favoring bivalve colonization and disease expression have been identified in vibrios so far [START_REF] Duperthuy | Use of OmpU porins for attachment and invasion of Crassostrea gigas immune cells by the oyster pathogen Vibrio splendidus[END_REF][START_REF] Lemire | Populations, not clones, are the unit of vibrio pathogenesis in naturally infected oysters[END_REF][START_REF] Bruto | Vibrio crassostreae, a benign oyster colonizer turned into a pathogen after plasmid acquisition[END_REF][START_REF] Rubio | Species-specific mechanisms of cytotoxicity toward immune cells determine the successful outcome of Vibrio infections[END_REF]. They will be reviewed here in details. Among environmental factors, temperature is known to significantly affect the Vibrio communities associated with bivalves [START_REF] Alfaro | The complex interactions of Ostreid herpesvirus 1, Vibrio bacteria, environment and host factors in mass mortality outbreaks of Crassostrea gigas[END_REF]. In fact, vibrios are strongly thermodependent and many species thrive in warm water exceeding 17 °C [START_REF] Ceccarelli | Vibrio species[END_REF]. The threat posed by vibrios to the shellfish industry can be thus exacerbated by the impact of climate change, in particular sea surface warming, on their global spread [START_REF] Vezzulli | Climate influence on Vibrio and associated human diseases during the past halfcentury in the coastal North Atlantic[END_REF][START_REF] Baker-Austin | Non-cholera vibrios: The microbial barometer of climate change[END_REF]. Additional effects include the temperature-dependent expression of virulence factors and the modifications of host physiology (reproductive effort, immune status, etc.) [START_REF] Travers | Influence of temperature and spawning effort on Haliotis tuberculata mortalities caused by Vibrio harveyi: an example of emerging vibriosis linked to global warming[END_REF], which in bivalves can indirectly favor colonization by opportunistic vibrios [START_REF] Le Roux | Oysters and Vibrios as a model for disease dynamics in wild animals[END_REF].

In this review, we will focus on the oyster Crassostrea gigas and the mussels Mytilus edulis and Mytilus galloprovincialis, which are bivalves of economic interest and ecological relevance with widely documented interactions with a diversity of vibrios.

We will discuss successively Vibrio associations with bivalves, the key role of bivalve cellular defenses in controlling vibrios, the multiple adaptive strategies of vibrios to their bivalve hosts, and the role of the biotic environment in the selection (both positive and negative) of adapted genotypes.

Vibrios as key components of bivalve microbiota

Vibrios account for a significant proportion of bacteria found in healthy and diseased bivalves, where they can reach concentrations about 100-fold higher than those in ambient water [START_REF] Depaola | Incidence of Vibrio parahaemolyticus in U.S. coastal waters and oysters[END_REF][START_REF] Šolić | The rate of concentration of faecal coliforms in shellfish under different environmental conditions[END_REF][START_REF] Shen | Effect of temperature on uptake and survival of Vibrio parahaemolyticus in oysters (Crassostrea plicatula)[END_REF].

Approaches to study Vibrio communities include both culture-dependent protocols and global DNA sequencing approaches. Whole body homogenates or single tissues (e.g., hemolymph, gut, hepatopancreas) are often analyzed. A large fraction of these bacteria might go undetected using culture-dependent methods due to metabolic requirements and culture conditions (e.g. food source, temperature, generation time). Sequencing-based approaches allow to overcome most of the problems related to cultivation [START_REF] Lokmer | Hemolymph microbiome of Pacific oysters in response to temperature, temperature stress and infection[END_REF][START_REF] Lokmer | Spatial and temporal dynamics of pacific oyster hemolymph microbiota across multiple scales[END_REF]Lokmer, Kuenzel, et al., 2016) and to access to the hidden non culturable vibrios. However, species-level resolution among genetically-related Vibrio species is often poor using the most commonly employed 16S rRNA marker, thereby limiting the fine-scale evaluation of taxonomic heterogeneity within the genus [START_REF] Poretsky | Strengths and limitations of 16S rRNA gene amplicon sequencing in revealing temporal microbial community dynamics[END_REF][START_REF] King | A new high throughput sequencing assay for characterizing the diversity of natural Vibrio communities and its application to a pacific oyster mortality even[END_REF]. Additional phylogenetic markers such as the heat shock protein hsp60 [START_REF] Jesser | Vibrio ecology in the Neuse river estuary, North Carolina,characterized by next-generation amplicon sequencing of the gene encoding heat shock protein 6[END_REF][START_REF] King | A new high throughput sequencing assay for characterizing the diversity of natural Vibrio communities and its application to a pacific oyster mortality even[END_REF] were proposed as alternatives to 16S-barcoding to discriminate members of the Vibrio genus more precisely. Still, one single marker often provides insufficient resolution, due to the high genomic plasticity of vibrios [START_REF] Le Roux | Eco-evolutionary dynamics linked to horizontal gene transfer in Vibrios[END_REF], and culture-dependent methods coupled to Multiple Locus Sequence Analyses (MLSA) or core gene phylogenies are generally required for finest resolution analysis [START_REF] Sawabe | Updating the Vibrio clades defined by multilocus sequence phylogeny: Proposal of eight new clades, and the description of Vibrio tritonius sp[END_REF].

Vibrios associated with bivalves

Over the past 30 years, many studies carried out in different geographical areas have highlighted the complex structure of Vibrio populations commonly associated with bivalves [START_REF] Romalde | New Vibrio species associated to molluscan microbiota: A review[END_REF][START_REF] Chen | Microbiological analysis and microbiota in oyster: a review[END_REF][START_REF] Bruto | Vibrio crassostreae, a benign oyster colonizer turned into a pathogen after plasmid acquisition[END_REF]. Species belonging to the clades Splendidus and Harveyi are among the most frequently found in association with healthy oysters and mussels [START_REF] Romalde | New Vibrio species associated to molluscan microbiota: A review[END_REF][START_REF] Le Roux | Oysters and Vibrios as a model for disease dynamics in wild animals[END_REF]. Their concentration, diversity and dynamics are strongly dependent upon environmental parameters, such as temperature and salinity [START_REF] Romalde | New Vibrio species associated to molluscan microbiota: A review[END_REF].

Most of our knowledge on vibrios associated with bivalves derives from studies focusing on pathological contexts. Indeed, a number of bacterial species, which include many Vibrio species, have been associated with mortality outbreaks of cultivated bivalves (oysters, clams, mussels) [START_REF] Saulnier | A large-scale epidemiological study to identify bacteria pathogenic to Pacific oyster Crassostrea gigas and correlation between virulence and metalloprotease-like activity[END_REF][START_REF] Travers | Bacterial diseases in marine bivalves[END_REF][START_REF] Le Roux | Oysters and Vibrios as a model for disease dynamics in wild animals[END_REF][START_REF] Dubert | New insights into pathogenic vibrios affecting bivalves in hatcheries: Present and future prospects[END_REF]. These Vibrio species can be primary [START_REF] Romalde | Bacteria in molluscs: good and bad guys[END_REF][START_REF] Travers | Bacterial diseases in marine bivalves[END_REF][START_REF] Dubert | New insights into pathogenic vibrios affecting bivalves in hatcheries: Present and future prospects[END_REF]. Closely-related species of the Splendidus clade (e.g., V. splendidus, V. tasmaniensis, V. cyclitrophicus, and V. crassostreae) have been repeatedly isolated from C. gigas juveniles (less than 12 months old) affected by the Pacific Oyster Mortality Syndrome (POMS) triggered by the Ostreid herpesvirus OsHV-1 µVar. [START_REF] Lemire | Populations, not clones, are the unit of vibrio pathogenesis in naturally infected oysters[END_REF][START_REF] Bruto | Vibrio crassostreae, a benign oyster colonizer turned into a pathogen after plasmid acquisition[END_REF][START_REF] De Lorgeril | Immune-suppression by OsHV-1 viral infection causes fatal bacteraemia in Pacific oysters[END_REF]. V. aestuarianus (Anguillarum clade) was found to cause mass mortality of adult C. gigas in France since 2001 and since 2011 in Ireland, Scotland and Spain [START_REF] Garnier | Molecular and phenotypic characterization of Vibrio aestuarianus subsp. francensis subsp. nov., a pathogen of the oyster Crassostrea gigas[END_REF][START_REF] Garcia | Vibrio aestuarianus and Pacific oyster in France: a review of 10 years of surveillance[END_REF][START_REF] Travers | Several strains, one disease: experimental investigation of Vibrio aestuarianus infection parameters in the Pacific oyster, Crassostrea gigas[END_REF] High taxonomic resolution analysis of the bivalve pathobiota applied on healthy and diseased C. gigas samples using a new target enrichment next-generation sequencing approach revealed dominance of primary pathogens such as V.

aestuarianus or OsHV-1 in tissues of diseased C. gigas collected during mass mortality events affecting juveniles and/or adults at different European farming sites [START_REF] Lasa | Dynamics of the Pacific oyster pathobiota during mortality episodes in Europe assessed by 16S rRNA gene profiling and a new target enrichment nextgeneration sequencing strategy[END_REF] [START_REF] Russell | Intrahost genetic diversity of bacterial symbionts exhibits evidence of mixed infections and recombinant haplotypes[END_REF][START_REF] De Lorgeril | Immune-suppression by OsHV-1 viral infection causes fatal bacteraemia in Pacific oysters[END_REF], but the role of the non-virulent microbiota should not be neglected as non-virulent Vibrio strains have the potential to dramatically increase the host damages caused by virulent strains [START_REF] Lemire | Populations, not clones, are the unit of vibrio pathogenesis in naturally infected oysters[END_REF]. The role of these bacterial consortia should be deeply analyzed in future functional studies to shed light on the mechanisms underlying polymicrobial infections in bivalves [START_REF] Lasa | Dynamics of the Pacific oyster pathobiota during mortality episodes in Europe assessed by 16S rRNA gene profiling and a new target enrichment nextgeneration sequencing strategy[END_REF].

In contrast to oyster diseases, less information is available on pathogenic Vibrio diseases affecting mussels (Mytilus spp), which also are other important farmed bivalves. Actually, although mussels can concentrate vibrios in their tissues [START_REF] Stabili | Mytilus galloprovincialis filter feeding on the bacterial community in a Mediterranean coastal area (Northern Ionian Sea, Italy)[END_REF], they are considered particularly resistant to microbial infections, in particular vibrioses [START_REF] Lupo | Revue systématique de la littérature relative aux facteurs de risque de mortalité des moules exploitées en France[END_REF]. They rarely experience mass mortalities [START_REF] Domeneghetti | Mortality occurrence and pathogen detection in Crassostrea gigas and Mytilus galloprovincialis close-growing in shallow waters (Goro lagoon, Italy)[END_REF][START_REF] Romero | Occurrence, seasonality and infectivity of Vibrio strains in natural populations of mussels Mytilus galloprovincialis[END_REF] although recent outbreaks of unknown etiology have been reported [START_REF] Charles | High 29 mortality of mussels in northern Brittany -Evaluation of the involvement of pathogens, pathological conditions and pollutants[END_REF].

Among the rare reported cases of Vibrio infection, V. splendidus-related strains were isolated from diseased Mytilus edulis adults and the isolated strains induced mortalities when injected to mussels [START_REF] Ben Cheikh | First evidence for a Vibrio strain pathogenic to Mytilus edulis altering hemocyte immune capacities[END_REF], 2017). Strains identified as V. splendidus and V. coralliilyticus/neptunius-like caused high mortality rates in larvae of the greenshell mussel Perna canalicula (Mytilidae) [START_REF] Kesarcodi-Watson | Two pathogens of Greenshel mussel larvae, Perna canaliculus: Vibrio splendidus and a V. coralliilyticus/neptunius-like isolate[END_REF]. Moreover, strains of V. coralliilyticus isolated from bleached corals had strong and concentration-dependent immunotoxicity and embryotoxicity in mussel in vitro [START_REF] Balbi | Responses of Mytilus galloprovincialis to challenge with the emerging marine pathogen Vibrio coralliilyticus[END_REF]. Interestingly, the susceptibility of mussels to vibrios of the Splendidus clade (oyster pathogens) was shown to be dependent on host physiology [START_REF] Charles | Experimental infection of Mytilus edulis by two Vibrio splendidus-related strains: Determination of pathogenicity level of strains and influence of the origin and annual cycle of mussels on their sensitivity[END_REF]. Whether or not vibrios are causal agents in mussel mortalities observed in the field remains unclear [START_REF] Charles | High 29 mortality of mussels in northern Brittany -Evaluation of the involvement of pathogens, pathological conditions and pollutants[END_REF].

A recent study investigated possible links between the different susceptibility to Vibrio infection of oysters and mussels and their microbiota [START_REF] Vezzulli | Comparative 16SrDNA gene-based microbiota profiles of the Pacific oyster (Crassostrea gigas) and the mediterranean mussel (Mytilus galloprovincialis) from a shellfish farm (Ligurian Sea, Italy)[END_REF]. Using 16S rRNA gene-based analysis, the authors provided the first comparison of microbiota profiles associated with hemolymph and digestive gland of C. gigas and M.

galloprovincialis co-cultured at the same aquaculture site. Vibrios accounted for a larger fraction of the microbiota in C. gigas compared to M. galloprovincialis, suggesting that oysters might provide better conditions than mussels for survival of these bacteria. More recently, [START_REF] Pierce | Gut microbiomes of the eastern oyster Crassostrea virginica and the blue mussel Mytilus edulis: Temporal variation and the influence of marine aggregate-associated microbial communities[END_REF], by comparing the gut microbiome of the oyster C. virginica and the mussel M. edulis, found that while the two bivalves harbor microbial communities with similar composition, on a functional level, the microbial community varies according to host species and season. The authors suggested that host species intrinsic factors affect the composition of the bivalve microbiota independently of environmental conditions and diet.

In addition to vibrios pathogenic for bivalves, both oysters and mussels can concentrate within their tissues other vibrios pathogenic for humans. Since edible bivalves, especially oysters, are often eaten raw or undercooked, they represent an important vehicle for the transmission of these pathogens to humans. Most relevant Vibrio species associated with seafood-related diseases are V. parahaemolyticus, V.

vulnificus and, to a lesser extent, non-toxigenic V. cholerae [START_REF] Jones | Abundance of Vibrio cholerae, V. vulnificus, and V. parahaemolyticus in oysters (Crassostrea virginica) and clams (Mercenaria mercenaria) from Long Island sound[END_REF][START_REF] Froelich | Vibrio bacteria in raw oysters: managing risks to human health[END_REF]Williams et al., 2017;[START_REF] Tack | Preliminary incidence and trends of infections with pathogens transmitted commonly through food -foodborne diseases active surveillance network, 10 U.S. Sites, 2015-2018[END_REF]. These species, as far as we know, do not affect bivalve health but represent a serious and growing threat to public health [START_REF] Tack | Preliminary incidence and trends of infections with pathogens transmitted commonly through food -foodborne diseases active surveillance network, 10 U.S. Sites, 2015-2018[END_REF].

Stable versus transient associations

It has been debated whether the presence of vibrios in bivalves results from stable associations or from the non-specific uptake of vibrios from food or seawater. In a pioneer study, Polz and collaborators (Dana E Hunt et al., 2008) analyzed Vibrio population structures in mussels and in the water column. Diversity and frequency of populations identified by MLSA was similar in mussels and water samples, suggesting that mussels do not represent a strongly selective habitat for vibrios. However, population structure does not imply absence of selection in mussel, but suggests that selection is balanced by migration and/or adaptation to different environments [START_REF] Preheim | Metapopulation structure of Vibrionaceae among coastal marine invertebrates[END_REF]. More recently, Le Roux and collaborators have deeply analyzed Vibrio associations with oysters and relationships between vibrios in the water column and in oyster tissues [START_REF] Le Roux | Oysters and Vibrios as a model for disease dynamics in wild animals[END_REF][START_REF] Bruto | Vibrio crassostreae, a benign oyster colonizer turned into a pathogen after plasmid acquisition[END_REF]. By comparing the frequency and abundance between Vibrio populations in these two compartments, [START_REF] Bruto | Vibrio crassostreae, a benign oyster colonizer turned into a pathogen after plasmid acquisition[END_REF] showed that several populations were unequally distributed. Among them, V. crassostreae and some V. splendidus populations were positively and preferentially associated with oyster tissues.

Because of habitat preferences related to their metabolic requirements, resistance/tolerance mechanisms and specific colonization determinants, distinct

Vibrio species can colonize diverse tissues in bivalves. Total bacterial 16S has proven that hemolymph, which carries the bivalve immune cells, harbors a microbiota highly distinct from the rest of the tissues (Lokmer, Kuenzel, et al., 2016). Strikingly, vibrios can survive in this immune tissue, which is likely a colonization route toward deeper tissues.

Outside pathological contexts, hemolymph colonization is rapidly controlled by hemocytes, the bivalve immune cells [START_REF] Rubio | Species-specific mechanisms of cytotoxicity toward immune cells determine the successful outcome of Vibrio infections[END_REF]. In contrast, pathogenic vibrios escape from hemocyte control and colonize the connective tissue, which appears as a site of proliferation leading to fatal bacteremia [START_REF] Parizadeh | Ecologically realistic model of infection for exploring the host damage caused by Vibrio aestuarianus[END_REF][START_REF] Rubio | Species-specific mechanisms of cytotoxicity toward immune cells determine the successful outcome of Vibrio infections[END_REF] (Figure 1).

New technologies such as metagenomics-based tools combined with manipulative ecological and physiological approaches are expected to significantly increase our current knowledge on Vibrio association with bivalves and their colonization mechanisms.

Vibrios facing bivalve cellular defenses

Upon host colonization, microbes are recognized by pattern-recognition receptors that initiate cellular-based signal transduction cascades connecting microbe recognition signals to expression and secretion of immunomodulatory cytokines and chemokines [START_REF] Reddick | Bacteria fighting back: How pathogens target and subvert the host innate immune system[END_REF] and contribute to control infections by triggering the expression of immune effectors that modify the microbe habitat.

Effectors of metazoan innate immunity include antimicrobial peptides (AMPs), reactive oxygen species (ROS), nitric oxide (NO) and heavy metals (copper, zinc), present at epithelial/mucosal surfaces, in phagocytes and body fluids, which create a hostile environment for microbes [START_REF] Hood | Nutritional immunity: transition metals at the pathogen-host interface[END_REF][START_REF] Franzenburg | Bacterial colonization of Hydra hatchlings follows a robust temporal pattern[END_REF].

These conserved innate immune mechanisms are key components of the defense of bivalves against infections.

The key role of hemocytes

Bivalves are endowed with a powerful innate immune system, mainly based on the activity of circulating immune cells, the hemocytes, soluble plasma factors, as well as on tissue-mediated immune responses [START_REF] Bachère | The new insights into the oyster antimicrobial defense: Cellular, molecular and genetic view[END_REF][START_REF] Canesi | Chapter 6 -Specificity of innate immunity in bivalves: a lesson from bacteria[END_REF][START_REF] Gerdol | Immunity in Molluscs: Recognition and effector mechanisms, with a focus on bivalvia BT -Advances in Comparative Immunology[END_REF], creating a hostile environment for vibrios [START_REF] Schmitt | The antimicrobial defense of the Pacific oyster, Crassostrea gigas. How diversity may compensate for scarcity in the regulation of resident/pathogenic microflora[END_REF][START_REF] Destoumieux-Garzón | Resistance to antimicrobial peptides in vibrios[END_REF]. Due to the presence of an open circulatory system, bivalve hemocytes are not only circulating in the hemolymph but they also infiltrate tissues.

Hemocytes of both oysters and mussels can kill vibrios through phagocytosis, production of highly reactive ROS and NO, as well as a number of antimicrobial peptides (AMPs) and hydrolytic enzymes [START_REF] Canesi | Bacteria-hemocyte interactions and phagocytosis in marine bivalves[END_REF][START_REF] Bachère | The new insights into the oyster antimicrobial defense: Cellular, molecular and genetic view[END_REF][START_REF] Canesi | Chapter 6 -Specificity of innate immunity in bivalves: a lesson from bacteria[END_REF] (Figure 1). Hemocytes populations (granular, semi-granular or agranular cells) [START_REF] Bachère | Separation of Crassostrea gigas hemocytes by density gradient centrifugation and counterflow centrifugal elutriation[END_REF][START_REF] Chagot | Interactions between Bonamia ostreae (Protozoa: Ascetospora) and hemocytes of Ostrea edulis and Crassostrea gigas (Mollusca: Bivalvia): Entry mechanisms[END_REF] vary according to bivalve species, physiological status, and environmental factors [START_REF] Smith | Chapter 1 -Hematopoiesis and hemocytes in pancrustacean and polluscan models[END_REF]. In C. gigas, specialized populations of semi-granular and granular cells harbor strong phagocytic capacity and produce highly reactive molecules (ROS and NO) in response to V. splendidus; they also express lysozymes and AMPs in agreement with their immunological roles [START_REF] Bachère | The new insights into the oyster antimicrobial defense: Cellular, molecular and genetic view[END_REF][START_REF] Wang | The granulocytes are the main immunocompetent hemocytes in Crassostrea gigas[END_REF]. Similarly, in M.

galloprovincialis, granular and semi-granular cells harbor phagocytic activity, they produce ROS and NO; small hyaline cells produce NO only [START_REF] García-García | Immune responses of mussel hemocyte subpopulations are differentially regulated by enzymes of the PI 3-K, PKC, and ERK kinase families[END_REF]. Enzymes involved in oxidative stress (dual oxidases, NADPH oxidases) and hydrolases including lysozymes, which participate in the killing of phagocytized bacteria by degrading bacterial peptidoglycan [START_REF] Costa | Functional and molecular immune response of Mediterranean mussel (Mytilus galloprovincialis) haemocytes against pathogen-associated molecular patterns and bacteria[END_REF][START_REF] Itoh | Presence and characterization of multiple mantle lysozymes in the Pacific oyster, Crassostrea gigas[END_REF]Xue et al., 2010) are expressed by hemocytes of mussels and oysters [START_REF] Rosa | A hemocyte gene expression signature correlated with predictive capacity of oysters to survive Vibrio infections[END_REF][START_REF] Campos | Shotgun analysis of the marine mussel Mytilus edulis hemolymph proteome and mapping the innate immunity elements[END_REF][START_REF] Wang | The granulocytes are the main immunocompetent hemocytes in Crassostrea gigas[END_REF][START_REF] De Lorgeril | Immune-suppression by OsHV-1 viral infection causes fatal bacteraemia in Pacific oysters[END_REF][START_REF] Jiang | Transcriptomic and quantitative proteomic analyses provide insights into the phagocytic killing of hemocytes in the oyster Crassostrea gigas[END_REF]. The respiratory burst generated by hemocytes is a major microbicidal reaction in bivalves, which also triggers the formation of DNA Extracellular traps (ETosis). In oysters and mussels ETosis contributes to control Vibrio infections [START_REF] Altincicek | Hostderived extracellular nucleic acids enhance innate immune responses, induce coagulation, and prolong survival upon infection in Insects[END_REF][START_REF] Poirier | Antimicrobial histones and DNA traps in invertebrate immunity: evidences in Crassostrea gigas[END_REF][START_REF] Romero | Extracellular traps (ETosis) can be activated through NADPH-dependent and -independent mechanisms in bivalve mollusks[END_REF].

More recently, mechanisms involved in heavy metal homeostasis have been shown

to play a key role in the antibacterial response of oysters [START_REF] Vanhove | Outer membrane vesicles are vehicles for the delivery of Vibrio tasmaniensis virulence factors to oyster immune cells[END_REF][START_REF] Shi | The role of copper and zinc accumulation in defense against bacterial pathogen in the fujian oyster (Crassostrea angulata)[END_REF]) [START_REF] Rubio | Species-specific mechanisms of cytotoxicity toward immune cells determine the successful outcome of Vibrio infections[END_REF]) [START_REF] Shi | The role of copper and zinc accumulation in defense against bacterial pathogen in the fujian oyster (Crassostrea angulata)[END_REF]. Copper/zinc redistribution upon Vibrio infections is accompanied in hemocytes by changes in the expression levels of metallothioneins as well as copper and zinc transporter genes (Ctr1, ATP7A, ZIP1, and Znt2), which across animal species mediate the uptake of heavy metals and their accumulation in intracellular compartments for intracellular killing of phagocytized bacteria [START_REF] Neyrolles | Mycobacteria, metals, and the macrophage[END_REF]. It is worth noting that bivalves have widely differing concentrations of heavy metals in their tissues, with oysters (Crassostrea sp.) showing the highest body burden of copper and zinc [START_REF] Pan | Biodynamics to explain the difference of copper body concentrations in five marine bivalve species[END_REF][START_REF] Shi | The role of copper and zinc accumulation in defense against bacterial pathogen in the fujian oyster (Crassostrea angulata)[END_REF].

Although in mussels the role of heavy metals in the immune response to vibrios has been not investigated in such detail, sequences of ATP7A, Ctr1, ZIP1, Znt1 and Znt2 belonging to evolutionarily conserved metal transport systems are present in the genome of M. galloprovincialis (Gerdol M., pers. comm), which suggests they could play a similar role in mussel defense. So far in Mytilus sp., available data indicate that metal exposure can result in increases or decreases in hemocyte immune parameters, depending on the exposure conditions [START_REF] Parry | Interactive effects of temperature and copper on immunocompetence and disease susceptibility in mussels (Mytilus edulis)[END_REF][START_REF] Höher | Immunomodulating effects of environmentally realistic copper concentrations in Mytilus edulis adapted to naturally low salinities[END_REF]. In this light, the role of copper/zinc homeostasis in mussel immune response deserves further investigation.

A high number of highly diversified AMPs have also been identified in oysters and mussels, in which they are expressed by hemocytes and/or epithelia. Some of them, but not all, have been purified and characterized for their various immune functions.

Remarkably, mussels and oysters show contrasting antimicrobial responses. Indeed, AMP expression is potent in mussel [START_REF] Pallavicini | High sequence variability of myticin transcripts in hemocytes of immune-stimulated mussels suggests ancient host-pathogen interactions[END_REF] as opposed to mild in oysters [START_REF] Schmitt | The antimicrobial defense of the Pacific oyster, Crassostrea gigas. How diversity may compensate for scarcity in the regulation of resident/pathogenic microflora[END_REF]. Both species express highly diversified Csαβdefensins [START_REF] Hubert | A member of the arthropod defensin family from rdible mediterranean mussels (Mytilus galloprovincialis)[END_REF][START_REF] Mitta | Mussel defensins are synthesised and processed in granulocytes then released into the plasma after bacterial challenge[END_REF][START_REF] Gueguen | Characterization of a defensin from the oyster Crassostrea gigas: recombinant production, folding, solution structure, antimicrobial activities, and gene expression[END_REF][START_REF] Schmitt | Molecular diversity of antimicrobial effectors in the oyster Crassostrea gigas[END_REF] and big defensins [START_REF] Rosa | A hemocyte gene expression signature correlated with predictive capacity of oysters to survive Vibrio infections[END_REF][START_REF] Gerdol | Massive gene presence/absence variation in the mussel genome as an adaptive strategy: first evidence of a pan-genome in Metazoa[END_REF][START_REF] Gerdol | Functional insights from the evolutionary diversification of big defensins[END_REF][START_REF] Loth | The Ancestral N-Terminal nomain of Big Defensins drives bacterially triggered assembly into antimicrobial nanonets[END_REF] as well as bactericidal/permeability-increasing proteins [START_REF] Gonzalez | Evidence of a bactericidal permeability increasing protein in an invertebrate, the Crassostrea gigas Cg-BPI[END_REF]. These antimicrobial peptides/proteins are expressed in hemocytes and/or epithelia either constitutively or in response to bacterial challenge [START_REF] Schmitt | The antimicrobial defense of the Pacific oyster, Crassostrea gigas. How diversity may compensate for scarcity in the regulation of resident/pathogenic microflora[END_REF].

Although the multiple mechanisms of action of bivalve AMPs are far from being uncovered, activities against vibrios have been evidenced, ranging from potent (e.g. big-defensins) to weak (e.g. defensins) [START_REF] Duperthuy | The major outer membrane protein OmpU of Vibrio splendidus contributes to host antimicrobial peptide resistance and is required for virulence in the oyster Crassostrea gigas[END_REF]Schmitt, Lorgeril, et al., 2012;[START_REF] Loth | The Ancestral N-Terminal nomain of Big Defensins drives bacterially triggered assembly into antimicrobial nanonets[END_REF]. Remarkably, beyond these shared peptide families, the AMP repertoire of oysters and mussels contains species-specific AMP families. In M.

galloprovincialis, these families include myticins, mytilins and mytimicins, which are also the most expressed AMPs [START_REF] Pallavicini | High sequence variability of myticin transcripts in hemocytes of immune-stimulated mussels suggests ancient host-pathogen interactions[END_REF]. Similarly, in C. gigas, they include proline-rich peptides, molluscidins and a Macrophage expressed gene 1-like (for review see [START_REF] Bachère | The new insights into the oyster antimicrobial defense: Cellular, molecular and genetic view[END_REF]. However, some AMP families like mussel myticins are likely not involved in the response to vibrios but other functions such as antiviral responses [START_REF] Balseiro | Mytilus galloprovincialis Myticin C: A chemotactic molecule with antiviral activity and immunoregulatory properties[END_REF] or wound healing (Rey- [START_REF] Rey-Campos | Transcriptomic analysis reveals the wound healing activity of mussel Myticin C[END_REF].

Environmental factors are known to significantly affect immune responses. Thus, immune parameters of mussels and oysters (total hemocyte counts, proportion of hemocyte populations, immune gene expression) vary with temperature and exposure to pollutants, including heavy metals and nanoparticles [START_REF] Parry | Interactive effects of temperature and copper on immunocompetence and disease susceptibility in mussels (Mytilus edulis)[END_REF][START_REF] Höher | Immunomodulating effects of environmentally realistic copper concentrations in Mytilus edulis adapted to naturally low salinities[END_REF][START_REF] Wendling | Adaptation to enemy shifts: Rapid resistance evolution to local vibrio spp. in invasive pacific oysters[END_REF][START_REF] Canesi | Effects of nanomaterials on marine invertebrates[END_REF][START_REF] Auguste | Immune training after repeated exposure to nanoplastics in the marine bivalve Mytilus[END_REF], which may have important consequences on bivalve susceptibility to pathogens. In addition, recent studies have revealed that previous encounter with pathogens is a life history trait that defines the resistance/tolerance of bivalves to infections (i.e. immune priming). In the oyster C. gigas, priming of the antiviral response conferred long-term protection against the OsHV-1 virus, which was controlled by a sustained antiviral response [START_REF] Lafont | Long-lasting antiviral innate immune priming in the Lophotrochozoan Pacific oyster, Crassostrea gigas[END_REF][START_REF] Lafont | A Sustained Immune Response Supports Long-Term Antiviral Immune Priming in the Pacific Oyster, Crassostrea gigas[END_REF]; moreover a primary stimulation with V. splendidus enhanced immune signaling and conferred higher phagocytosis capacities to oyster hemocytes (Zhang et al., 2014;[START_REF] Wang | The involvement of TLR signaling and anti-bacterial effectors in enhanced immune protection of oysters after Vibrio splendidus pre-exposure[END_REF]. In the mussel M. galloprovincialis priming with vibrios modified immune responses allowing mussels to tolerate the infection (Rey- [START_REF] Rey-Campos | Immune tolerance in Mytilus galloprovincialis hemocytes after repeated contact with Vibrio splendidus[END_REF].

Interestingly, [START_REF] Wendling | Adaptation to enemy shifts: Rapid resistance evolution to local vibrio spp. in invasive pacific oysters[END_REF] showed that in selective environments, i.e.

at elevated temperatures reflecting patterns of disease outbreaks in natural populations, oysters can rapidly adapt to widespread communities of pathogenic vibrios. Oysters showing higher resistance to sympatric vibrios showed higher hemocyte counts and phagocytosis rate.

Responses to vibrios in oysters and mussels

Long term records on mussels (Mytilus spp) and oysters (Crassostrea spp. LGP32, V. crassostreae J2-9 (both from the Splendidus clade), and V. aestuarianus 01/032 (Anguillarum clade) (Labreuche, Lambert, et al., 2006;[START_REF] Balbi | Interactions between Mytilus galloprovincialis hemocytes and the bivalve pathogens Vibrio aestuarianus 01/032 and Vibrio splendidus LGP32[END_REF][START_REF] Green | Distinct immune responses of juvenile and adult oysters (Crassostrea gigas) to viral and bacterial infections[END_REF][START_REF] Vanhove | Copper homeostasis at the host vibrio interface: lessons from intracellular vibrio transcriptomics[END_REF][START_REF] Rubio | Species-specific mechanisms of cytotoxicity toward immune cells determine the successful outcome of Vibrio infections[END_REF].

One important question is whether the host can discriminate commensals from pathogenic strains. A recent study in which vibrios of the Splendidus clade were injected to oysters revealed that all Vibrio strains were recognized by oyster hemocytes independently of their virulence status [START_REF] Rubio | Species-specific mechanisms of cytotoxicity toward immune cells determine the successful outcome of Vibrio infections[END_REF]. They induced three major immune signaling pathways, namely the interleukin 17 (IL-17), tumor necrosis factor (TNF), and Toll-like receptor (TLR) pathways. They also induced the production of AMPs (big defensins), ROS (dual oxidase expression), and heavy metals (metallothioneins and metal transporters). However, pathogenic strains were also shown to manipulate bivalve immune responses for successful colonization. These species-specific mechanisms are detailed below.

-Responses to V. tasmaniensis. V. tasmaniensis LGP32 was isolated from moribund C. gigas oysters by Le Roux and collaborators [START_REF] Gay | Two Vibrio splendidus related strains collaborate to kill Crassostrea gigas: Taxonomy and host alterations[END_REF].

LGP32 interactions with its natural host have been widely documented.

LGP32 is a facultative intracellular pathogen of oyster immune cells [START_REF] Duperthuy | Use of OmpU porins for attachment and invasion of Crassostrea gigas immune cells by the oyster pathogen Vibrio splendidus[END_REF]. In C. gigas,

LGP32 uses the major outer membrane protein OmpU to attach to and enter the hemocytes; opsonization is mediated by the major plasma protein Cg-EcSOD recognized by β-integrins at the hemocyte surface (Figure 2). Consistent with a manipulation of hemocyte phagocytosis, LGP32 infection leads to an altered expression of oyster genes involved in cytoskeleton dynamics [START_REF] Rubio | Species-specific mechanisms of cytotoxicity toward immune cells determine the successful outcome of Vibrio infections[END_REF]. As

LGP32 is recognized by the oyster immune system, it induces antimicrobial defenses based on AMPs (big defensins), ROS (dual oxidase) and heavy metals [START_REF] Rubio | Species-specific mechanisms of cytotoxicity toward immune cells determine the successful outcome of Vibrio infections[END_REF]. However, inside the phagosome, it escapes from host cellular defenses by avoiding acidic vacuole formation and by limiting the production of ROS [START_REF] Duperthuy | Use of OmpU porins for attachment and invasion of Crassostrea gigas immune cells by the oyster pathogen Vibrio splendidus[END_REF]. This is accompanied by a repression of the NADPH-oxidase expression [START_REF] Rubio | Species-specific mechanisms of cytotoxicity toward immune cells determine the successful outcome of Vibrio infections[END_REF] and the activation of mechanisms of resistance/tolerance to antimicrobials (see section 3). Inside the hemocytes, LGP32 induces lysosomal disruption [START_REF] Balbi | Interactions between Mytilus galloprovincialis hemocytes and the bivalve pathogens Vibrio aestuarianus 01/032 and Vibrio splendidus LGP32[END_REF] and causes hemocyte lysis through the intracellular delivery of type 6 secretion system (T6SS) effectors [START_REF] Vanhove | Copper homeostasis at the host vibrio interface: lessons from intracellular vibrio transcriptomics[END_REF][START_REF] Rubio | Species-specific mechanisms of cytotoxicity toward immune cells determine the successful outcome of Vibrio infections[END_REF] (Figure 2).

In mussels, LGP32 shows a lower pathogenic potential and induces lower hemocyte damage than in oysters [START_REF] Balbi | Interactions between Mytilus galloprovincialis hemocytes and the bivalve pathogens Vibrio aestuarianus 01/032 and Vibrio splendidus LGP32[END_REF].

LGP32 is rapidly phagocytized by M.

galloprovincialis hemocytes, although phagocytosis does not require opsonization by plasma components, and it elicits release of hemocyte antibacterial effectors [START_REF] Balbi | Interactions between Mytilus galloprovincialis hemocytes and the bivalve pathogens Vibrio aestuarianus 01/032 and Vibrio splendidus LGP32[END_REF] (Figure 2). When internalized, LGP32 remains viable within intracellular vacuoles, escaping lysosomal degradation. Accordingly, V. splendidus is able to proliferate in mussel hemolymph in vivo. The observed resistance of LGP32 to the bactericidal activity of mussel hemocytes was related to alterations of PI-3 Kinase signaling, leading to impairment of the endo-lysosomal system, which suggests that

LGP32 could interfere with host cell signaling pathways, thereby escaping its bactericidal activity. In agreement, in M. galloprovinciallis infected with LGP32, hemocytes show an altered expression of genes involved in innate immune response, inflammatory response, cell migration and defense responses to bacteria (Rey- [START_REF] Rey-Campos | Immune tolerance in Mytilus galloprovincialis hemocytes after repeated contact with Vibrio splendidus[END_REF]. Although less documented, cellular interactions of LGP32 with M. edulis hemocytes revealed an effect on hemocyte adhesion, phagocytosis and oxidative burst [START_REF] Tanguy | Functional and molecular responses in Mytilus edulis hemocytes exposed to bacteria, Vibrio splendidus[END_REF]. In vitro, hemocytes of M. edulis exposed to

LGP32 showed an altered expression of genes involved in recognition (lectins), immune signaling (TLRs, MyD88), antimicrobial defenses (AMPs, lysosomal hydrolases oxidative stress) and apoptosis [START_REF] Tanguy | The immune response of Mytilus edulis hemocytes exposed to Vibrio splendidus LGP32 strain: A transcriptomic attempt at identifying molecular actors[END_REF].

Overall, studies on LGP32 interactions with bivalves (both mussels and oysters) identify this strain as a facultative intracellular pathogen, which uses host immune cell manipulation as a key feature of its biology. The mechanisms underpinning

LGP32/oyster hemocyte interactions are conserved within the species V.

tasmaniensis and the damages caused to oyster hemocytes might be a consequence of V. tasmaniensis intracellular stages [START_REF] Rubio | Species-specific mechanisms of cytotoxicity toward immune cells determine the successful outcome of Vibrio infections[END_REF]. From our current knowledge, the mechanistic interactions of V. tasmaniensis LGP32 with mussel and oyster hemocytes differ and the damages caused by LGP32 to hemocytes during its intracellular stages appear higher in oyster (Figure 2).

-Responses to V. aestuarianus. V. aestuarianus subsp. francensis 01/032 was isolated from adult moribund oysters by Nicolas and collaborators during a French disease outbreak in 2001 [START_REF] Garnier | Evidence for the involvement of pathogenic bacteria in Summer mortalities of the pacific oyster Crassostrea gigas[END_REF][START_REF] Garnier | Molecular and phenotypic characterization of Vibrio aestuarianus subsp. francensis subsp. nov., a pathogen of the oyster Crassostrea gigas[END_REF]. This strain produces extracellular products (ECPs) containing the main virulence factor of V. aestuarianus: the metalloprotease Vam [START_REF] Labreuche | Vibrio aestuarianus zinc metalloprotease causes lethality in the Pacific oyster Crassostrea gigas and impairs the host cellular immune defenses[END_REF]. In vitro, these ECPs were shown to alter phagocytosis by C. gigas hemocytes, adhesion and ROS production (Labreuche, Soudant, et al., 2006). High doses of bacterial cells also affect hemocyte activities in vivo after a few days (Labreuche, Lambert, et al., 2006). It was therefore proposed that ECPs allow 01/032 to avoid phagocytosis by oyster hemocytes (Figure 2), which likely favors oyster colonization. In contrast, hemocytes of M.

galloprovincialis are able to efficiently kill V. aestuarianus 01/032, both in vitro and in vivo [START_REF] Balbi | Interactions between Mytilus galloprovincialis hemocytes and the bivalve pathogens Vibrio aestuarianus 01/032 and Vibrio splendidus LGP32[END_REF]. The different susceptibilty of 01/032 to the antibacterial activity of oyster and mussel hemolymph was dependent on the presence of the extrapallial-like protein (MgEP), the main protein in Mytilus hemolymph, which increases adhesion and killing of 01/032 by mussel hemocytes [START_REF] Pezzati | Susceptibility of Vibrio aestuarianus 01/032 to the antibacterial activity of Mytilus haemolymph: identification of a serum opsonin involved in mannose-sensitive interactions[END_REF] (Figure 2). Interestingly, upon addition of purified MgEP, oyster hemocytes acquired the capacity to kill V. aestuarianus 01/032. MgEP thus can play a key role in promoting mannose-sensitive interactions of this strain (adhesion and killing) with both mussel and oyster hemocytes, by serving as an opsonin [START_REF] Pezzati | Susceptibility of Vibrio aestuarianus 01/032 to the antibacterial activity of Mytilus haemolymph: identification of a serum opsonin involved in mannose-sensitive interactions[END_REF][START_REF] Canesi | Chapter 6 -Specificity of innate immunity in bivalves: a lesson from bacteria[END_REF]. These data further highlight the importance of soluble plasma factors in mediating hemocyte-Vibrio interactions in different bivalve species, which can play a key role in the pathogenic outcome [START_REF] Pruzzo | Persistence of vibrios in marine bivalves: the role of interactions with haemolymph components[END_REF].

Overall data on V. aestuarianus 01/032 interactions with hemocytes reveal an opposite outcome in oysters and mussels. In C. gigas, where it expresses pathogenic potential, 01/032 is able to avoid phagocytosis, one of the main component of bivalve defenses, whereas in mussels, where it does not cause disease, 01/032 is opsonized by a major plasma component, MgEP, and killed by the hemocytes (Figure 2).

However, V. aestuarianus 01/032 was recently demonstrated to be moderately virulent, i.e. it induces mortalities upon injection of high doses (5x10 7 bacteria/animal) [START_REF] Goudenège | A single regulatory gene is sufficient to alter Vibrio aestuarianus pathogenicity in oysters[END_REF] compared to highly virulent strains (e.g. strain 12_016a

isolated during an oyster mortality episode) that kill at low doses (10 2 bacteria/animal) as well as through immersion procedures [START_REF] Parizadeh | Ecologically realistic model of infection for exploring the host damage caused by Vibrio aestuarianus[END_REF].

Therefore strain 01/032 might be well adapted to oyster as a host but does not possess the complete virulence repertoire that triggers disease. Only a few studies have focused on oyster responses to highly virulent strains of V. aestuarianus. In 2011, de Lorgeril et al provided a first catalog of genes related to cellular / immune functions differentially expressed in individuals surviving an infection with the strain 02/041. Genes differentially expressed in susceptible and resistant oyster lineages included genes involved in recognition (L-rhamnose-binding lectin), cell adhesion (β-Integrin) and antioxidants (Cg-EcSOD) [START_REF] Azéma | Can selection for resistance to OsHV-1 infection modify susceptibility to Vibrio aestuarianus infection in Crassostrea gigas? First insights from experimental challenges using primary and successive exposures[END_REF], in agreement with a manipulation of phagocytosis. Still, a clear picture of the mechanisms allowing V.

aestuarianus to proliferate within oyster hemolymph is still missing, and we still ignore if like strain 01/032, highly virulent strains of V. aestuarianus manipulate oyster hemocytes to achieve successful infections.

-Responses to other vibrios. Although less data is available and no specific comparison has been documented between oyster and mussel responses, there are at least two additional examples that evidence close interactions between pathogenic vibrios and hemocytes. First, V. crassostreae J2-9, which is associated with oyster juvenile mass mortalities (POMS) [START_REF] Lemire | Populations, not clones, are the unit of vibrio pathogenesis in naturally infected oysters[END_REF], tends to dampen several immune defenses in oyster. Like LGP32, it represses the NADPH-mediated production of ROS and C-type lectin-mediated antibacterial immunity [START_REF] Rubio | Species-specific mechanisms of cytotoxicity toward immune cells determine the successful outcome of Vibrio infections[END_REF]. It also represses the expression of C1q domain-containing (C1qDC) proteins, a family of complement-related proteins, which, in oysters, can serve as opsonins that enhance Vibrio phagocytosis [START_REF] Lv | Comparative study of three C1q domain containing proteins from pacific oyster Crassostrea gigas[END_REF]. J2-9 remarkably alters cellular defenses by inducing massive hemocyte lysis through a contact-dependent mechanism [START_REF] Rubio | Species-specific mechanisms of cytotoxicity toward immune cells determine the successful outcome of Vibrio infections[END_REF]. Whether or not the observed repression of immune genes is related to the vibrio-triggered lysis of specific hemocyte populations remains to be clarified. Similar to other pathogenic vibrios, V. splendidus 10/068, which was isolated from moribund mussels in France in 2010 [START_REF] Ben Cheikh | Infection dynamics of a V. splendidus strain pathogenic to Mytilus edulis: In vivo and in vitro interactions with hemocytes[END_REF]

Multifaceted adaptive responses of vibrios to their molluscan hosts

According to natural selection, microbes adapted for growth in metazoan hosts have selected strategies to evade, inhibit or manipulate immune responses (Stubbendieck et al., 2016). Commonly, counter-measures to host innate immunity include active dampening of immune defenses, i.e. immune suppression, escape from host recognition (usually by disguising their immunogenic surface components), or resistance/tolerance to antimicrobials [START_REF] Hornef | Bacterial strategies for overcoming host innate and adaptive immune responses[END_REF][START_REF] Reddick | Bacteria fighting back: How pathogens target and subvert the host innate immune system[END_REF]. This evasion/tolerance scenario applies to species belonging to the resident microbiota of metazoans as well as to new invaders. In Vibrio species infecting or simply colonizing bivalves several of these strategies have been recently identified.

These recent data highlight the multifaceted nature of immune evasion in Vibrio populations positively associated with bivalves.

Resistance/tolerance to host antimicrobials has been evidenced as a key determinant of Vibrio infectivity in oysters. Most data were obtained in the facultative intracellular pathogen V. tasmaniensis [START_REF] Duperthuy | Use of OmpU porins for attachment and invasion of Crassostrea gigas immune cells by the oyster pathogen Vibrio splendidus[END_REF]. In this species, resistance to ROS and to heavy metals was identified as essential. Indeed, both antioxidants (SodA, AhpCF, catalase) and copper efflux machineries (CopA, CusABC) were highly induced upon contact with hemocytes and they determined

Vibrio resistance to intracellular killing [START_REF] Vanhove | Copper homeostasis at the host vibrio interface: lessons from intracellular vibrio transcriptomics[END_REF]. Mechanisms that confer increased resistance to oyster AMPs were also evidenced [START_REF] Destoumieux-Garzón | Resistance to antimicrobial peptides in vibrios[END_REF]. Inside the phagosomes of oyster hemocytes, V. tasmaniensis LGP32 was shown to release outer membrane vesicles that entrap AMPs and increase antimicrobial resistance [START_REF] Vanhove | Outer membrane vesicles are vehicles for the delivery of Vibrio tasmaniensis virulence factors to oyster immune cells[END_REF]. An OmpU-mediated protection against AMPs was also evidenced in V. tasmaniensis LGP32 [START_REF] Duperthuy | The major outer membrane protein OmpU of Vibrio splendidus contributes to host antimicrobial peptide resistance and is required for virulence in the oyster Crassostrea gigas[END_REF], which likely results from the OmpU-mediated induction of the envelope stress response, as demonstrated in V. cholerae [START_REF] Mathur | The Vibrio cholerae ToxR-regulated porin OmpU confers resistance to antimicrobial peptides[END_REF][START_REF] Mathur | Antimicrobial peptides activate the Vibrio cholerae σE regulon through an OmpU-dependent signalling pathway[END_REF]. However, up to now potent mechanisms that modify the electronegativity of the lipopolysaccharides (LPS) and increase resistance to AMPs [START_REF] Hankins | Elucidation of a novel Vibrio cholerae lipid A secondary hydroxy-acyltransferase and its role in innate immune recognition[END_REF][START_REF] Hankins | Amino acid addition to Vibrio cholerae LPS establishes a link between surface remodeling in Gram-positive and Gram-negative bacteria[END_REF][START_REF] Cullen | Antimicrobial peptide resistance mediates resilience of prominent gut commensals during inflammation[END_REF] have not been identified in Vibrio strains associated with bivalves. RND efflux machineries that detoxify Vibrio cells from the host antimicrobials [START_REF] Bina | Vibrio cholerae RND family efflux systems are required for antimicrobial resistance, optimal virulence factor production, and colonization of the infant mouse small intestine[END_REF] are present in the genomes of Vibrio strains colonizing bivalves, but their role has not been studied. Moreover, Vibrio strains positively and negatively associated with oysters could not be discriminated on the basis of their resistance to AMPs (Oyanedel et al., unpublished). Therefore, it is unclear how much resistance to AMPs contributes to colonization success in oysters. This may differ for Vibrio species associated with mussels, which express AMPs in large amounts [START_REF] Mitta | Differential distribution and defence involvement of antimicrobial peptides in mussel[END_REF][START_REF] Pallavicini | High sequence variability of myticin transcripts in hemocytes of immune-stimulated mussels suggests ancient host-pathogen interactions[END_REF], or with oysters, which express AMPs in small amounts (Schmitt, Lorgeril, et al., 2012).

Mechanisms of immune cell lysis needed to escape from hemocyte control were identified in V. tasmaniensis and V. crassostreae [START_REF] Vanhove | Copper homeostasis at the host vibrio interface: lessons from intracellular vibrio transcriptomics[END_REF][START_REF] Piel | Selection of Vibrio crassostreae relies on a plasmid expressing a type 6 secretion system cytotoxic for host immune cells[END_REF][START_REF] Rubio | Species-specific mechanisms of cytotoxicity toward immune cells determine the successful outcome of Vibrio infections[END_REF]. Such an active dampening of oyster cellular defenses was shown to depend on distinct molecular effectors: a chromosome-encoded T6SS active against eukaryotic cells in V. tasmaniensis, and the ancestral R5-7 virulence factor together with a T6SS carried by a plasmid (T6SSpGV) in V. crassostreae (Piel et al., 2019;[START_REF] Rubio | Species-specific mechanisms of cytotoxicity toward immune cells determine the successful outcome of Vibrio infections[END_REF]. Both mechanisms enable virulent Vibrio to escape hemocyte control and cause systemic infections (Figure 1). Recently, strains of V.

splendidus able to colonize oyster tissues were also found to be cytotoxic toward hemocytes [START_REF] Oyanedel | Vibrio splendidus O-antigen structure: a trade-off between virulence to oysters and resistance to grazers[END_REF]. Similar evolution toward cytotoxic phenotypes illustrates the importance for successful colonizers to overcome the major cellular defenses of oysters: they create a less hostile habitat that enables colonization of oyster tissues. Cytotoxicity is therefore an important determinant of strain infectivity, which enables virulence expression when Vibrio strains harbor virulence factors.

Some bacterial species have acquired the capacity to escape from host recognition by becoming less immunogenic or by reducing the host immune responses elicited by the release of microbe-associated molecular patterns such as LPS and peptidoglycan [START_REF] Charroux | Cytosolic and secreted peptidoglycan-degrading enzymes in Drosophila respectively control local and systemic immune responses to microbiota[END_REF]. Such mechanisms of immune evasion remain poorly described in Vibrio species. However, in a recent study of a V. splendidus population associated with oysters, distinct structures of LPS O-antigen were identified. Manipulations of the wbe hypervariable genomic region, which determines the O-antigen structure in V. splendidus [START_REF] Wildschutte | O-antigen diversity and lateral transfer of the wbe region among Vibrio splendidus isolates[END_REF] lead to less immunogenic structures that favored oyster colonization [START_REF] Oyanedel | Vibrio splendidus O-antigen structure: a trade-off between virulence to oysters and resistance to grazers[END_REF].

This identifies immune evasion as a novel adaptive mechanism underpinning colonization in Vibrio associated with bivalves.

Within metazoan hosts, microbes are also engaged in interbacterial competition for survival among the diverse resident microbial communities and against new colonizers [START_REF] Fraune | Bacteria-bacteria interactions within the microbiota of the ancestral metazoan Hydra contribute to fungal resistance[END_REF]. Bacteria have adapted to growth with neighbors (Stubbendieck and Straight, 2016). Tools for competition between bacteria can point to the fight for vital resources, growth inhibition or direct killing of rivals by the delivery of toxins and AMPs such as bacteriocins (Stubbendieck and Straight, 2016). The fight for iron, present in limiting concentrations within metazoan hosts, was evidenced in the transcriptomic activity of vibrios colonizing bivalves with a major induction of siderophore biosynthesis and iron-uptake machineries [START_REF] Vanhove | Copper homeostasis at the host vibrio interface: lessons from intracellular vibrio transcriptomics[END_REF][START_REF] Rubio | Species-specific mechanisms of cytotoxicity toward immune cells determine the successful outcome of Vibrio infections[END_REF]. This likely has major impact on colonization success, as earlier shown in V.

vulnificus infecting mice [START_REF] Arezes | Hepcidin-induced hypoferremia is a critical host defense mechanism against the siderophilic bacterium Vibrio vulnificus[END_REF]. While several studies have evidenced bacteria harboring antimicrobial activity in the hemolymph of bivalves [START_REF] Defer | Antimicrobial peptides in oyster hemolymph: The bacterial connection[END_REF][START_REF] Desriac | Exploring the hologenome concept in marine bivalvia: haemolymph microbiota as a pertinent source of probiotics for aquaculture[END_REF], the production of bacteriocins and resistance to such non-host-derived immune effectors remains a poorly explored aspect of Vibrio infectivity.

However, it was suggested that cooperation exists within Vibrio populations based on bacteriocin production and uptake [START_REF] Cordero | Ecological populations of bacteria act as socially cohesive units of antibiotic production and resistance[END_REF]. Similarly, recent studies have revealed the broad distribution of interbacterial T6SS in Vibrio species, a killing mechanism that uses contact-dependent toxin delivery and toxin-resistance mechanism to inhibit competitors [START_REF] Ho | A view to a kill: the bacterial type 6 secretion system[END_REF]. In Vibrio species pathogenic for oysters such T6SS systems are induced upon host colonization although a formal role in interbacterial competition has not be demonstrated [START_REF] Rubio | Species-specific mechanisms of cytotoxicity toward immune cells determine the successful outcome of Vibrio infections[END_REF]. Such a demonstration was achieved in V. vulnificus, in which two T6SS are involved in inter-/intra-species competition within oyster hosts [START_REF] Hubert | V. vulnificus type VI secretion System (T6SS) interactions in an oyster in vivo model and their impact on Vibrio ecology[END_REF], and in V. fischeri, in which a T6SS controls competitions during squid colonization [START_REF] Speare | Bacterial symbionts use a type VI secretion system to eliminate competitors in their natural host[END_REF].

Altogether, recent advances in Vibrio-bivalve interactions highlight the adaptive potential of bivalve colonizers, which have not only developed strategies to circumvent the potent cellular defenses of their natural hosts but also tools for competition with the abundant microbial communities hosted by bivalves.

The selection of adapted genotypes in bivalves and the aquatic environment

As free-living and non-obligatory parasites, vibrios engage in diverse biotic interactions in the environment outside of their bivalve hosts. Among these biotic interactions they encounter two major groups of bacterial predators, i.e.

bacteriophages and bacterivorous protists (grazers), i.e. ciliates, flagellates and amoebozoae [START_REF] Pernthaler | Predation on prokaryotes in the water column and its ecological implications[END_REF]. As predator-prey interactions are some of the strongest forces driving rapid co-evolution of both partners, often referred to as an arm-race [START_REF] Dawkins | Arms races between and within species[END_REF], predator-prey interactions in bacterial communities significantly influence and shape bacterial adaptive strategies and favor the selection of predation resistance traits.

Grazers can be found in most ecological niches inhabited by bacterial communities, and their predation activity shares similar cellular and molecular processes with their metazoan immune cells counterparts, the phagocytes, i.e. the hemocytes in the case of bivalves. Phagocytosis and intracellular antimicrobial processes are indeed highly conserved from protozoans to mammals [START_REF] Boulais | Molecular characterization of the evolution of phagosomes[END_REF]. Hence, bacterial predation by grazers has often been suggested to favor co-incidental selection of bacterial virulence factors, including in Vibrionaceae [START_REF] Erken | The rise of pathogens: predation as a factor driving the evolution of human pathogens in the environment[END_REF]. A diversity of survival adaptations against predation by grazers can be found in bacteria and classified in two main groups: either pre-ingestional or postingestional adaptations [START_REF] Matz | Off the hook -how bacteria survive protozoan grazing[END_REF]. Pre-ingestional adaptions including high motility, filamentation, surface masking or toxin release are commonly found in extracellular pathogens; whereas post-ingestional adaptations including digestion resistance through vacuolar trafficking inhibition or vacuolar escape, and intracellular toxin release are commonly found for intracellular pathogens and tend to favor bacterial growth [START_REF] Matz | Off the hook -how bacteria survive protozoan grazing[END_REF].

Escape from phagocytosis. Among pre-ingestional adaptations some are widely shared by Vibrionaceae as they are flagellated and highly motile. In addition some of them can form biofilms, as e.g. V. cholerae that produces Vibrio Polysaccharides (VPS) favoring resistance to grazing [START_REF] Henst | Molecular insights into Vibrio choleraea intra-amoebal host-pathogen interactions[END_REF][START_REF] Sun | Relative contributions of Vibrio polysaccharide and quorum sensing to the resistance of Vibrio cholerae to predation by heterotrophic protists[END_REF]. Interestingly the oyster V. tasmaniensis LGP32 has been suggested to form biofilms [START_REF] Vezzulli | Aquatic ecology of the oyster pathogens Vibrio splendidus and Vibrio aestuarianus[END_REF], although a potential role of this process in resistance to grazers remains to be investigated.

Cytotoxic activities.

Toxin/protease release and cytotoxic activities can both play a role as pre-ingestional or post-ingestional adaptations to grazers [START_REF] Matz | Off the hook -how bacteria survive protozoan grazing[END_REF]. Protease secretion is widespread among vibrios and it has been described for most strains pathogenic toward bivalves [START_REF] Binesse | Metalloprotease Vsm is the major determinant of toxicity for extracellular products of Vibrio splendidus[END_REF][START_REF] Labreuche | Vibrio aestuarianus zinc metalloprotease causes lethality in the Pacific oyster Crassostrea gigas and impairs the host cellular immune defenses[END_REF]. Some of these proteases favor resistance to grazing by amoeba and ciliates as shown for the metalloprotease Vsm secreted by V.

tasmaniensis LGP32 [START_REF] Robino | Resistance of the oyster pathogen Vibrio tasmaniensis LGP32 against grazing by Vannella sp. marine amoeba involves Vsm and CopA virulence factors[END_REF] and the protease PrtV secreted by V.

cholerae [START_REF] Vaitkevicius | A Vibrio cholerae protease needed for killing of Caenorhabditis elegans has a role in protection from natural predator grazing[END_REF]. To date in vibrios, they represent some of the best examples of factors under environmental selective pressure that contribute to virulence/toxicity in a metazoan host. V. tasmaniensis and V. crassostreae also harbor cytotoxic capacities that are necessary for their pathogenicity [START_REF] Rubio | Species-specific mechanisms of cytotoxicity toward immune cells determine the successful outcome of Vibrio infections[END_REF], among them MARTX toxins and T6SS components [START_REF] Bruto | Vibrio crassostreae, a benign oyster colonizer turned into a pathogen after plasmid acquisition[END_REF][START_REF] Piel | Selection of Vibrio crassostreae relies on a plasmid expressing a type 6 secretion system cytotoxic for host immune cells[END_REF][START_REF] Rubio | Species-specific mechanisms of cytotoxicity toward immune cells determine the successful outcome of Vibrio infections[END_REF]. However, up to now, none of these systems were shown to protect bivalve pathogens against grazing. The role of MARTX toxins in resistance to grazing was shown for V. vulnificus, in which a MARTX involved in eel infection confers protection against amoeba grazing [START_REF] Lee | MARTX of Vibrio vulnificus biotype 2 is a virulence and survival factor[END_REF]. However, such a role has not yet been investigated in bivalve pathogens. Similarly, the T6SS of V. cholerae has shown cytotoxic activity on the slime mold Dictyostelium discoideum during its actively grazing unicellular life stage [START_REF] Pukatzki | Identification of a conserved bacterial protein secretion system in Vibrio cholerae using the Dictyostelium host model system[END_REF]. However, in the oyster pathogen V. tasmaniensis LGP32, where a T6SS determines cytotoxicity towards oyster hemocytes [START_REF] Rubio | Species-specific mechanisms of cytotoxicity toward immune cells determine the successful outcome of Vibrio infections[END_REF], no effect was found in the interaction with the amoebae Vannella sp. 1411 [START_REF] Robino | Resistance of the oyster pathogen Vibrio tasmaniensis LGP32 against grazing by Vannella sp. marine amoeba involves Vsm and CopA virulence factors[END_REF]. Hence the role of T6SS in biotic interactions outside bivalves remains to be determined.

Resistance to heavy metals. Evidence of post-ingestional adaptations in vibrios are limited to a number of pathogenic strains that adopt intracellular stages and survive intracellular digestion [START_REF] Rosenberg | The Vibrio shiloi/Oculina patagonica Model System of Coral Bleaching[END_REF][START_REF] Ma | Translocation of a Vibrio cholerae type VI secretion effector requires bacterial endocytosis by host cells[END_REF][START_REF] Vidal-Dupiol | Innate immune responses of a scleractinian soral to vibriosis[END_REF][START_REF] Ritchie | Inflammation and disintegration of intestinal villi in an experimental model for Vibrio parahaemolyticus-induced diarrhea[END_REF][START_REF] De Souza Santos | Intracellular Vibrio parahaemolyticus escapes the vacuole and establishes a replicative niche in the cytosol of epithelial cells[END_REF][START_REF] Van Der Henst | An intracellular replication niche for Vibrio cholerae in the amoeba Acanthamoeba castellanii[END_REF], as shown for V. tasmaniensis LGP32 in hemocytes of bivalves [START_REF] Duperthuy | Use of OmpU porins for attachment and invasion of Crassostrea gigas immune cells by the oyster pathogen Vibrio splendidus[END_REF][START_REF] Balbi | Interactions between Mytilus galloprovincialis hemocytes and the bivalve pathogens Vibrio aestuarianus 01/032 and Vibrio splendidus LGP32[END_REF][START_REF] Vanhove | Copper homeostasis at the host vibrio interface: lessons from intracellular vibrio transcriptomics[END_REF][START_REF] Rubio | Species-specific mechanisms of cytotoxicity toward immune cells determine the successful outcome of Vibrio infections[END_REF]. Such properties are rather unusual for pathogenic vibrios and potentially reminiscent of post-ingestional adaptation to grazers predation. Indeed, LGP32 was found resistant to grazing by the free-living amoeba Vannella and some virulence factors, such as the p-ATPase copper efflux pump CopA, were found to play a role in this resistance [START_REF] Robino | Resistance of the oyster pathogen Vibrio tasmaniensis LGP32 against grazing by Vannella sp. marine amoeba involves Vsm and CopA virulence factors[END_REF]. Interestingly the CopA-mediated resistance to copper is also necessary for V.

fischeri colonization in the bobtail squid [START_REF] Brooks | Global discovery of colonization determinants in the squid symbiont Vibrio fischeri[END_REF]. Therefore, copper resistance favoring resistance to amoeba predation is also a key determinant of Vibrio associations with molluscan hosts, both in parasitic and mutualistic interactions.

Adsorption inhibition. In addition to grazers, bacteriophages are an important class of predators which are abundant and highly diverse in marine environments; they contribute to the evolution of bacteria by mediating horizontal gene transfer and genomic rearrangements, as well as by bactericidal selection [START_REF] Dy | Remarkable mechanisms in microbes to resist phage infections[END_REF]. A critical step in their interaction with bacteria is the attachment to cell wall components (membrane receptors, porins, LPS, flagella, pili) thus exerting an important selective pressure on cell wall composition and cell wall plasticity. Hence bacteria adaptation to phage predation can share similarities with pre-ingestional masking strategies to avoid recognition by grazers. Among adaptations to phages that have been described for vibrios, LPS modifications [START_REF] Seed | Phase variable o-antigen biosynthetic genes control expression of the major protective antigen and bacteriophage receptor in Vibrio cholerae O1[END_REF] play a key role and OMVs release can be used as decoy strategy [START_REF] Manning | Contribution of bacterial outer membrane vesicles to innate bacterial defense[END_REF] in a similar manner than OMVs play a role as decoy against AMPs during bivalve immune response [START_REF] Vanhove | Outer membrane vesicles are vehicles for the delivery of Vibrio tasmaniensis virulence factors to oyster immune cells[END_REF]. Interestingly OMVs are involved in V.

cholerae resistance to phages [START_REF] Reyes-Robles | Vibrio cholerae outer membrane vesicles inhibit bacteriophage infection[END_REF] as well as in resistance against the amoeba Acanthamoebae castellanii [START_REF] Valeru | Lack of outer membrane protein A enhances the release of outer membrane vesicles and survival of Vibrio cholerae and Suppresses Viability of Acanthamoeba castellanii[END_REF] The LPS structure under multiple selective pressures. Some virulence traits can be under adverse selection in different biotic interactions and generate a tradeoff equilibrium. In a recent study of a V. splendidus population associated with oysters, we identified distinct structures of LPS O-antigen encoded by the wbe hypervariable genomic region, which determines the O-antigen structure in V. splendidus [START_REF] Wildschutte | O-antigen diversity and lateral transfer of the wbe region among Vibrio splendidus isolates[END_REF]. Interestingly less immunogenic structures of LPS O-antigen that favored oyster colonization led to attenuated resistance to grazing by the Vannella sp. AP1411 free-living marine amoebae, suggesting a tradeoff between grazing resistance and oyster colonization [START_REF] Oyanedel | Vibrio splendidus O-antigen structure: a trade-off between virulence to oysters and resistance to grazers[END_REF]. Such a tradeoff between virulence and resistance to predators was also reported for V. cholerae Oantigen structure, which is under epigenetic control: phase variants of the wbe region defective in producing O-antigen are indeed highly resistant to phage infection, but severely attenuated in terms of virulence [START_REF] Seed | Phase variable o-antigen biosynthetic genes control expression of the major protective antigen and bacteriophage receptor in Vibrio cholerae O1[END_REF]. Altogether these studies highlight the critical role of LPS O-antigen in biotic interactions involving vibrios, strains pathogenic for bivalve being no exception. The extreme variability of this major membrane structure is controlled by genetic or epigenetic determinants favoring environmental persistence but leading to attenuated virulence in bivalves.

Altogether these recent studies show that the multiple biotic interactions of vibrios in the environment can lead to either positive (coincidental selection), neutral (relaxed selection), or negative (conflicting selection) effects on the selection of Vibrio traits required for virulence expression in bivalves [START_REF] Mikonranta | Life history trade-offs and relaxed selection can decrease bBacterial virulence in environmental reservoirs[END_REF] (Figure 1).

Predation could favor the selection of some virulence factors that participate to pathogenicity in bivalve host [START_REF] Adiba | From Grazing resistance to pathogenesis: The coincidental evolution of virulence factors[END_REF]. However, as illustrated in the wellknown intracellular pathogen L. pneumophila, hundreds of virulence factors can interact with a diversity of hosts, without having a similar importance in each interaction [START_REF] Boamah | From many hosts, one accidental pathogen: The diverse protozoan Hosts of Legionella[END_REF][START_REF] Ghosh | Beyond paralogs: The multiple layers of redundancy in bacterial pathogenesis[END_REF][START_REF] Park | Combinatorial selection in amoebal hosts drives the evolution of the human pathogen Legionella pneumophila[END_REF]. Thus, in V. cholerae, some virulence factors involved in the resistance to grazing by A.

castellanii have a minor role in pathogenesis in human (Van der [START_REF] Van Der Henst | Molecular insights into Vibrio cholerae's intra-amoebal host-pathogen interactions[END_REF].

They can also have adverse effects as recently illustrated in a V. splendidus moderately virulent for oysters [START_REF] Oyanedel | Vibrio splendidus O-antigen structure: a trade-off between virulence to oysters and resistance to grazers[END_REF] Vibrios being mostly opportunistic pathogens, they appear to have acquired, on the one hand, "generalist" virulence factors involved in multiple biotic interactions, and on the other hand, "specialist" virulence factors involved in interactions with a particular host, with possible trade-offs on other interactions.

Concluding Remarks

This review highlights the multifaceted nature of Vibrio interactions with mussels and oysters, aquaculture species showing contrasting resistance to infections. While only a limited number of vibrios pathogenic for mussels have been described, the current literature shows that oyster pathogens tend to interact differently with major components of oyster/mussel plasma and with their key immune cells, the hemocytes, which perform phagocytosis and produce highly microbicidal reactive oxygen/nitrogen species and accumulate heavy metals. The potent production of AMPs in mussels as opposed to oysters could be an important factor controlling disease outcomes. Plasma-soluble recognition proteins specific of the bivalve species also appear to have critical role. However, understanding the higher tasmaniensis LGP32 is readily phagocytized by both oyster and mussel hemocytes, but entry pathways differ. Opsonisation by the major plasma protein Cg-EcSOD is only required in oyster, in which it mediates uptake through -integrin recognition.

LGP32 survives intracellularly in both species (see section 3 for mechanisms of

  ) have revealed they experience rare or frequent pathologies, respectively, in particular in relation to Vibrio diseases. Although no data on species-specific comparison are available, the main immune mechanisms and effectors seem to be shared by the hemocytes of different mollusks, as reported in[START_REF] Escoubas | Immunity in Molluscs[END_REF]. Experimental infections with different Vibrio species and strains have revealed how the complex machinery of immune pathways in hemocytes, together with soluble plasma factors, is activated when facing a Vibrio challenge. Here some examples of responses of mussels and oysters to challenge with pathogenic vibrios are summarized. Oyster pathogens have been often used to challenge both species of bivalves. Thus, most data have been collected on the responses to the oyster pathogens V. tasmaniensis

  , was shown to alter different functions of hemocytes including motility, adhesion, ROS production, phagosome maturation and viability. Moreover, its extracellular products inhibited cellular responses such as ROS production[START_REF] Ben Cheikh | First evidence for a Vibrio strain pathogenic to Mytilus edulis altering hemocyte immune capacities[END_REF] through an unknown mechanism.Overall, current insights into Vibrio-bivalve interactions show a key role of hemocytes in controlling infections through phagocytosis and its associated arsenal of antimicrobial compounds (ROS, heavy metals and AMPs). Hemocyte reactions also include ETosis which is triggered by the oxidative burst and could participate in containing vibrios inside the hemolymph compartment. While the cellular defense arsenal is generally comparable in oyster and mussels, it appears to mainly differ in the AMPs expressed by immune cells and tissues and in the major soluble components present in the plasma, which serve as opsonins. Both AMPs and soluble plasma factors and might contribute to the distinct susceptibility of oysters and mussels to infections.

  susceptibility of oysters to infections requires a more systematic comparison of Vibrio interactions with mussels and oysters. Thanks to their successful colonization of oysters, different strategies of immune evasion and tolerance/resistance have been evidenced in vibrios pathogenic for oysters. These mechanisms include tolerance to the main cellular defenses of oysters (ROS, AMPs, heavy metals), active dampening of cellular immunity through cytolytic mechanisms (toxins, T6SS) as well as reduced antigenic properties through modifications of LPS, a major and hypervariable component of Vibrio outer membrane. Recent knowledge on the interactions of vibrios with environmental predators (phages, grazers) highlight possible mechanisms of coincidental selection as well as tradeoffs acting on these important determinants of Vibrio virulence (Figure1).

Figure 1 .

 1 Figure legend.

Figure 2 .

 2 Figure 2. A simplified view of hemocyte-vibrio interactions. The interaction of the

Figure 2

 2 Figure 2

  . Albeit at lower relative abundance, other bacterial species were exclusively identified in infected oysters, particularly Vibrio sp. (V. alginolyticus, V.

	coralliilyticus, V. crassostreae, V. splendidus, V. tasmaniensis) and Arcobacter sp.
	Thorough studies on vibrios pathogenic for oysters have shown that the functional
	unit of pathogenesis can be a clone (e.g. V. aestuarianus), a population (e.g. V.
	crassostreae) or a consortium, and this is further complicated by Vibrio interactions
	with the host microbiota (Le Roux et al., 2016). Recently, de Lorgeril et al. (2018)
	demonstrated that the POMS is caused by multiple infections with an initial and
	necessary step involving OsHV-1 µVar. The virus leads the host to enter an immuno-
	compromised state preceding the invasion by opportunistic pathogenic bacteria
	(including pathogenic vibrios) that kill the oyster. It can be speculated that POMS-
	associated mortalities are caused by multiple species/populations of pathogens
	(e.g. Vibrio, Arcobacter sp.) that collaborate to kill oysters

Acknowledgements

We are grateful to Prof. Marco Gerdol (University of Trieste) for precious genomic data on mussels. This work was supported by the European Union's Horizon 2020

Research and Innovation Program Grant Vivaldi 678589; the CNRS, PEPS Blanc 2016

(project Like Inhospitality); CONICYT PFCHA/Doctorado En El Extranjero Becas

Chile/2016-72170430 to D.O.; AMIBADAPT project funded by LABEX CeMEB. This study is set within the framework of the "Laboratoires d'Excellence (LABEX)" Tulip (ANR-10-LABX-41).

Different abundance and correlational patterns exist between total and presumed pathogenic Vibrio vulnificus and V. parahaemolyticus in shellfish and waters along the North Carolina coast. FEMS Microbiol Ecol 93:. Xue, Q., Hellberg, M.E., Schey, K.L., Itoh, N., Eytan, R.I., Cooper, R.K., and La Peyre, J.F.