Monsieur Frédérique Le Roux

Hervé Cottet

Je Voudrais Aussi Remercier Madame

Monsieur Marie-Agnès Travers

Benjamin Morga

Delphine Toubiez

Daniel Oyanedel-Trigo

Laura Chapelet

Yannick Gueguen

Jérémie Vidal-Dupiol

Agnès Delmas

Guillaume M Charrière

Delphine Destoumieux-Garzón

RESISTANCE OF VIBRIO POPULATIONS TO OYSTER ANTIMICROBIAL DEFENSES IS INSUFFICIENT TO EXPLAIN HOST ASSOCIATION

Je n´aurais pas pu réaliser cette thèse sans l´aide des nombreuses personnes qui ont collaboré professionnellement et personnellement mener à terme ce projet. C´est pourquoi que je voudrais exprimer mes sincères remerciements à tous ceux qui m'ont aidé avec leur connaissance, patience, volonté, gentillesse et soutien pendant ces quatre années.

En premier lieu, je voudrais remercier Madame Carmen Amaro et Monsieur François Lallier pour m´avoir fait l´honneur d´accepter d'être les rapporteurs de ce manuscrit. Je voudrais également remercier Madame Sophie Gaudriault, Monsieur François Delavat et Madame Annick Jacq pour avoir accepté de participer au jury de cette thèse.

Merci également aux membres de mon comité de suivi : Madame

TABLES

Elles possèdent une plasticité génomique et phénotypique remarquable, donnant lieu à une diversité de spécialisation de niche, allant des formes de vie libres à des associations mutualistes, commensales ou parasitaires avec des hôtes métazoaires et protozoaires [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]. Les vibrios font partie du microbiote naturel des animaux marins comme les coraux, les éponges et diverses espèces de bivalves.

Même si certaines espèces établissent des symbioses mutualistes à l'exemple de Vibrio fischeri avec les calamars sépiloïdes (Ceccarelli et Colwell, 2014 ;McFall-Ngai, 2014), de nombreuses espèces sont des agents pathogènes, dont certaines bien connues chez l'homme (V. cholerae est l'agent étiologique du cholera), et d'autres sont responsables de vibrioses chez les organismes aquatiques, avec un impact fort sur la pisciculture et la conchyliculture dans le monde (Romalde et Barja, 2010).

L'huître du Pacifique Crassostrea gigas est produite tout le long de la côte française pour la consommation humaine. Depuis 2008, les populations d'huîtres d'élevage subissent des épisodes sévères de mortalité affectant particulièrement les huîtres juvéniles de moins de 18 mois ; les taux de mortalité pouvant atteindre 100% dans les exploitations françaises [START_REF] Segarra | Detection and Description of a Particular Ostreid Herpesvirus 1 Genotype Associated with Massive Mortality Outbreaks of Pacific Oysters, Crassostrea gigas, in France in 2008[END_REF]. Cette maladie est connue sous le nom de syndrome de mortalité des huîtres du Pacifique (POMS). Son complexe étiologie implique l'interaction entre la génétique et l'état physiologique de l'hôte, l'environnement et des agents pathogènes, en particulier des microvariants du virus de l'herpès ostreid OsHV-1 et des bactéries opportunistes, dont des vibrios [START_REF] Samain | Summer mortality of Pacific oyster Crassostrea gigas[END_REF]Petton et al., 2015). Le POMS survient de façon saisonnière, dans une fenêtre de température de l'eau de mer permissive allant de 16 ° C à 24 ° C [START_REF] Pernet | Mass Mortalities of Pacific Oysters Crassostrea gigas Reflect Infectious Diseases and Vary with Farming Practices in the Mediterranean Thau Lagoon, France[END_REF]. Les mortalités suivent une séquence d'événements qui commence par la réplication intense d'OsHV-1 dans l'huître conduisant à un état d'immunosuppression qui bénéficie à des bactéries opportunistes, dont certaines espèces de Vibrio, favorisant une infection secondaire entraînant la mort de l'hôte (de Lorgeril et al., 2018). La question de savoir si ces espèces de Vibrio sont de simples opportunistes ou participent activement au processus infectieux (agents pathogènes opportunistes) n'était pas résolue au démarrage de cette thèse.

Néanmoins, les interactions entre les huîtres et les vibrios ne se limitent pas à des interactions pathogènes. Ainsi, la structure des populations de vibrios présentes dans différentes fractions de la colonne d'eau et dans les tissus de l'huître montre que certaines populations de Vibrio présentent une association préférentielle avec l'huître même en dehors d'un contexte pathologique, avec peu ou pas de chevauchement des populations entre les huîtres malades et saines (Bruto et al., 2017). Ceci suggère que sans l'influence de facteurs de stress majeurs tels que la température (Green et al., 2019) ou d'agents pathogènes tels que OsHV-1 µvar, les huîtres possèdent une capacité remarquable à maintenir l'homéostasie immunitaire avec son microbiote résident [START_REF] Schmitt | Immune Responses in the Pacific Oyster Crassostrea gigas: An Overview with Focus on Summer Mortalities[END_REF], ce qui devrait avoir un impact sur les génomes des groupes bactériens (ici des bactéries du genre Vibrio) capables d'établir une interaction stable avec les huîtres en tant qu'hôte (Chow et al., 2010).

L'objectif de la thèse a été de caractériser les mécanismes sous-jacents à l'association de l'huître C. gigas avec les populations de vibrios en contexte sain et pathologique. Vibrio formant des unités taxonomiquement et fonctionnellement proches, colonisent les huîtres affectée par le POMS (Bruto et al., 2017). Leurs travaux et les autres ont permis l'identification des déterminants de la pathogénicité de populations de vibrios pathogènes [START_REF] Binesse | Metalloprotease Vsm Is the Major Determinant of Toxicity for Extracellular Products of Vibrio splendidus[END_REF][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] Vanhove | Copper Homeostasis at the Host Vibrio Interface: Lessons from Intracellular Vibrio Transcriptomics[END_REF]Bruto et al., 2017Bruto et al., , 2018 ; ;Piel et al., 2019). Néanmoins ces travaux ont aussi révélé qu'en rade de Brest (façade Atlantique), certaines populations de vibrios s'associent préférentiellement aux huîtres au printemps en l'absence de mortalités massives d'huîtres dues au POMS. Par conséquent, nous avons voulu étudier les espèces de Vibrio colonisant les huîtres en dehors du contexte pathologique pour dévoiler les déterminants clés de la colonisation des huîtres immunocompétentes. Pour cela, nous avons bénéficié des populations de vibrios caractérisées par Bruto et al. (2017). Nous nous sommes intéressés en particulier à une population affiliée taxonomiquement à V. splendidus (population #23), isolée d'huitres saines, et phylogénétiquement proche de V. splendidus (population #24) trouvée dans les huitres pendant le POMS. La population #23 présente un potentiel de virulence modéré.

Par une étude de génomique comparative et d'invalidation fonctionnelle de gène, nous avons montré que deux loci, un complexe MARTX (Multifunctional Autoprocessing Repeatsin-Toxin) et un système de sécrétion de type 6 (T6SS) étaient nécessaires à la virulence modérée de cette population. Ces résultats ont été publiés dans l'article 1. Nous avons ainsi découvert que chez V. splendidus, la capacité de colonisation et la virulence étaient limitées par une structure de l'antigène O (qui décore les lipopolysaccharides à la membrane externe de la bactérie). Leur structure est plus facilement reconnue par le système immunitaire de l'huître par rapport à d'autres structures alternatives, avec un effet délétère sur la virulence mais qui confère une résistance aux prédateurs environnementaux que sont les amibes marines. Ainsi, nos résultats suggèrent une évolution de V. splendidus vers une virulence modérée comme compromis entre la « fitness » chez l'huître en tant qu'hôte et la résistance à ses prédateurs dans l'environnement. Les données des résultats complémentaires 1 ont confirmé cette hypothèse dans un fond génétique de Vibrio différent.

Dans les résultats complémentaires 2 (« short letter », en préparation), nous avons utilisé des populations supplémentaires de Vibrio associées positivement et négativement aux huîtres pour interroger si la résistance aux puissantes défenses antimicrobiennes de l'huître est un facteur suffisant pour déterminer la capacité des Vibrio à coloniser l'habitat hostile que représente l'huitre sain. Dans l'ensemble, nos résultats ne permettent pas de discrimer les associations vibrio-huître sur la base du seul caractère de résistance, sans doute parce que les multiples interactions biotiques qui ont lieu dans le milieu aquatique exercent des pressions similaires sur les populations qui ne sont pas associées aux huitres. Néanmoins, en considérant toutes les souches de vibrios indépendamment de leur affiliation taxonomique, les principaux facteurs expliquant l'association aux huîtres étaient : La résistance aux ROS, la virulence et la charge de surface.

SECTION II. LE ROLE DE LA CYTOTOXICITE CHEZ LES VIBRIOS DU CLADE SPLENDIDUS ASSOCIES AU POMS

Les huîtres possèdent une capacité remarquable à maintenir une homéostasie immunitaire en présence des communautés microbiennes abondantes et diversifiées associées à leurs tissus (Schmitt et al., 2012). Les facteurs biotiques et abiotiques pouvant conduire à la perte de cette homéostasie, comme l'infection à OsHV-1 µvar (de Lorgeril et al., 2018), sont des facteurs de risque clés pour l'apparition de mortalités massives. Parmi les bactéries associées à cette dysbiose, les vibrios du clade Splendidus, notamment V. tasmaniensis et V.

crassostreae, sont les plus étudiés. V. tasmaniensis LGP32, souche isolée à l'origine d'un événement de mortalité en 2004, présente un important potentiel de virulence, causant la mort des huîtres en infections expérimentales, par injection (Gay et al. 2004). Notre équipe a montré qu'il s'agit d'un pathogène intracellulaire facultatif cytotoxique pour les hémocytes [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] Vanhove | Copper Homeostasis at the Host Vibrio Interface: Lessons from Intracellular Vibrio Transcriptomics[END_REF], dont sa cytotoxicité dépend de l'étape de phagocytose. Aujourd'hui cette souche est utilisée comme modèle d'interaction hémocytevibrio dans un contexte pathogène [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][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] V-Vanhove | Outer Membrane Vesicles are Vehicles for the Delivery of Vibrio tasmaniensis Virulence Factors to Oyster Immune Cells[END_REF][START_REF] Vanhove | Copper Homeostasis at the Host Vibrio Interface: Lessons from Intracellular Vibrio Transcriptomics[END_REF].

Une autre espèce à fort potentiel de virulence, V. crassostreae, a été caractérisée pour sa capacité à remplacer les communautés commensales de l'huître pendant le POMS. Une protéine extracellulaire de fonction inconnue, codée par le gène r5.7, et un plasmide (pGV1512), sont nécessaires pour la virulence de la souche V. crassostreae J2-9 en infection expérimentale, par injection (Bruto et al., 2017). Toutefois, ces deux déterminants de virulence sont absents chez V. tasmaniensis. Ceci suggère que la virulence de ces deux espèces résulte de différents mécanismes. Au départ de ma thèse, les mécanismes d'interaction de J2-9 avec les hémocytes venaient d'être révélés par Tristan Rubio qui avait mis en évidence une cytotoxicité vis à vis des hémocytes, comme précédemment observé pour V.

tasmaniensis LGP32. Cette cytotoxicité ne dépendait pas toutefois de la phagocytose mais du simple contact avec les hémocytes, indiquant un mécanisme extracellulaire (Rubio, 2018) Dans l'article 2 nous avons ainsi développé une étude intégrative et comparative de l'interaction hôte-microbe de V. crassostreae et V. tasmaniensis avec les huîtres. Nous avons pu décrire les interactions cellulaires et les changements moléculaires qui se produisent chez l'hôte et les bactéries au cours de la phase intra-hôte de l'infection. Ceci a été rendu possible grâce à la mise au point d'un dual RNAseq in vivo. J'ai pu réaliser la validation fonctionnelle des gènes candidats bactériens nouvellement identifiés par l'invalidation de ces gènes (collaboration avec l'équipe de Frédérique Le Roux) et le phénotypage des interactions in vivo et in vitro entre les huîtres ou leurs hémocytes et les mutants bactériens obtenus. Nos données (article 2) nous ont permis de conclure qu'au sein du clade Splendidus, V.

crassostreae et V. tasmaniensis ont acquis des mécanismes spécifiques à l'espèce qui semblent converger vers la suppression des défenses cellulaires des huîtres. Ces phénotypes cytotoxiques sont nécessaires aux deux espèces pour la colonisation des tissus d'huitres conduisant à la mort, et sont caractéristiques des souches virulentes. Ils reposent sur R5.7 chez V. crassostreae, dont l'expression est induite lors de la colonisation, et sur un système de sécrétion de type 6 (T6SS) nouvellement identifié chez V. tasmaniensis. Dans des résultats complémentaires de cette section, j'ai identifié un effecteur associé au T6SSChr1-LGP32 responsable de la cytotoxicité de V. tasmaniensis envers les hemocytes. Cependant, cet effecteur ne semble pas nécessaire pour la virulence in vivo. Ces résultats préliminaires ouvrent la voie à de nouveaux projets sur la caractérisation des effecteurs T6SS impliqués dans la pathogenèse de V. tasmaniensis.

SECTION III. COMPORTEMENT DE TRICHEURS DE VIBRIOS ASSOCIES AUX POMS

Le POMS a été largement caractérisé en rade de Brest, sur le littoral Atlantique français, où des espèces de Vibrio appartenant au clade Splendidus ont été régulièrement trouvées associées aux huîtres moribondes qui contribuent de manière significative à l'infection systémique conduisant à la mort des huîtres (Gay et al., 2004 ;Saulnier et al., 2010 ;[START_REF] Lemire | Populations, not clones, are the unit of vibrio pathogenesis in naturally infected oysters[END_REF]Bruto et al., 2017Bruto et al., , 2018)). Néanmoins, le POMS affecte l'élevage d'huîtres dans le monde entier, dans des environnements aux écologies différentes où des variations dans la composition de la communauté du Vibrio sont attendues (Turner et al., 2009 ;[START_REF] Takemura | Associations and Dynamics of Vibrionaceae in the Environment, From the Genus to the Population Level[END_REF][START_REF] Li | The Role of Vibrio Vulnificus Virulence Factors and Regulators in its Infection-Induced Sepsis[END_REF]. Nous nous sommes donc demandés si différents assemblages microbiens contribuaient au POMS dans différents écosystèmes exploités pour l'ostréiculture, et si oui, qu'elles étaient les fonctions exprimées par les vibrios participant à la pathogenèse dans ces différents environnements. Pour cela, nous avons étudié les communautés de vibrios associés au POMS dans La lagune de Thau. Cette lagune est un écosystème marin méditerranéen avec une activité ostréicole importante qui souffre également depuis 2008 d'épisodes répétés de POMS affectant les hûitres juvéniles avec une forte présence d'OsHV-1 restreinte aux zones d'élevage (20% de la surface totale) [START_REF] Pernet | Mass Mortalities of Pacific Oysters Crassostrea gigas Reflect Infectious Diseases and Vary with Farming Practices in the Mediterranean Thau Lagoon, France[END_REF]. La lagune de Thau a une écologie qui diffère de la rade de Brest, avec notamment une salinité plus élevée et un régime de température qui pendant la moitié des années varie entre 16 et 30 ° C [START_REF] Lopez-Joven | Oyster Farming, Temperature, and Plankton Influence the Dynamics of Pathogenic Vibrios in the Thau Lagoon[END_REF]. rotiferianus, emploierait une stratégie de tricheur pour acquérir le fer nécessaire à sa croissance de manière non coopérative, grâce aux sidérophores produits par V. harveyi. De manière remarquable ces mécanismes ne sont efficaces que pour coloniser l'huître infectée par le virus, avec qui ces espèces exercent une activité synergique qui se traduit par une pathogénèse accélérée. En l'absence de virus, la communauté des vibrios qui pénèterent l'huitre de manière transitoire, n'induisent pas de réponse immunitaire, révélant une tolérance très importante de l'huitre aux communautés bactériennes. CHAPITRE 3. DISCUSSION GENERALE ET PERSPECTIVES FUTURES.

Dans l'état d'homéostasie immunitaire qui caractérise les huîtres saines, des associations préférentielles et positives de populations de vibrios peuvent être maintenues dans les huîtres sans développement de maladie (Schmitt et al., 2012). Cependant, les facteurs abiotiques et biotiques agissant sur l'holobionte (l'huître et son microbiote associé) peuvent déclencher une dysbiose dans laquelle la prolifération d'espèces opportunistes de vibrios entraîne la mort de l'hôte. Dans cette thèse, nous nous sommes intéressés à caractériser ces deux états différents, qui questionnent la tolérance du système immunitaire de l'huître et sa manipulation par les vibrios. La section suivante abordera les principaux résultats de la thèse, qui sont mis en perspective de la littérature disponible. Je décris également un certain nombre de développements futurs découlant de nos conclusions.

DES ARMES SPECIFIQUES AUX ESPECES DE VIBRIO POUR LYSER LES CELLULES IMMUNITAIRES ET AFFAIBLIR LES DEFENSES IMMUNITAIRES DES HUITRES

Nous avons étudié ici des vibrios associés au POMS issus de deux milieux marins côtiers différents qui abritent une activité ostréicole importante, à savoir la rade de Brest sur la côte atlantique française et la lagune de Thau, sur la côte méditerranéenne. Les espèces de vibrios pathogènes pour les huîtres appartiennent au clade Splendidus dans l'Atlantique (Lemire et al., 2015 ;Bruto et al., 2017) et au clade Harveyi dans la lagune de Thau (Oyanedel en préparation). Nous avons constaté que pour l'ensemble des populations virulentes de vibrios que nous avons étudié, celles-ci expriment une cytotoxicité envers les cellules immunitaires des huîtres, les hemocytes (Rubio et al., 2019, Oyanedel en préparation).

Nos résultats de l'article 2, ont montré que les espèces virulentes de vibrios, V. crassostreae et V. tasmaniensis sont caractérisées par des mécanismes spécifiques à l'espèce leur permettant d'affaiblir les défenses cellulaires de l'huître et d'ouvrir la voie à des infections systémiques. Au contraire, les souches non cytotoxiques des mêmes espèces de Vibrio sont rapidement contrôlées par les hémocytes par phagocytose et agglutination cellulaire, empêchant ainsi une infection systémique. Cette capacité à supprimer les défenses cellulaires des huîtres a également été mise en évidence chez V. harveyi (article 3), une espèce qui colonise efficacement les huîtres infectées par OsHV-1 à la fois sur le terrain et dans des expériences au mésocosme. Virulence et cytotoxicité apparaissent donc étroitement liées, probablement parce qu'elles déterminent le succès de la colonisation, comme nous l'avons démontré dans le clade Splendidus [START_REF] Rubio | Speciesspecific mechanisms of cytotoxicity toward immune cells determine the successful outcome of Vibrio infections[END_REF].

L'activité cytotoxique des bactéries pathogènes peut mettre en jeu une diversité de mécanismes et d'effecteurs. De manière générale elle est liée l'activité de toxines qui sont soit exportées par les bactéries dans le milieu extracellulaire, soit injectées ou administrées par voie intracellulaire dans les cellules cibles via des systèmes de sécrétion spécialisés (Ma et al., 2009 ;[START_REF] Osorio | T3SS Effectors in Vibrios: Homology in Sequence, Diversity in Biological Functions?[END_REF]. Comme développé dans l'article 2, les déterminants de la cytotoxicité des vibrios envers les hémocytes ont évolué indépendamment chez V.

crassostreae et V. tasmaniensis. Chez V. crassostreae, la cytotoxicité et la virulence dépendent fortement de la protéine R5.7 (absente de V. tasmaniensis), même si la protéine elle-même n'est pas cytotoxique (Bruto et al., 2018). De plus cette cytotoxicité de V.

crassostreae exprimant R5.7 nécessite un contact physique direct avec les hémocytes [START_REF] Rubio | Speciesspecific mechanisms of cytotoxicity toward immune cells determine the successful outcome of Vibrio infections[END_REF]. Dans le cas de V. tasmaniensis qui se comporte comme un pathogène intracellulaire facultatif des hémocytes d'huître [START_REF] Duperthuy | Use of OmpU Porins for Attachment and Invasion of Crassostrea gigas Immune Cells by the Oyster Pathogen Vibrio splendidus[END_REF], la perte du gène ancestral r5.7, aurait été compensée par l'acquisition d'un T6SSChr1-LGP32 qui délivre des effecteurs putatifs participant à la virulence et la cytotoxicité du V. tasmaniensis. De plus, nous avons potentiellement identifié l'effecteur putatif du T6SSChr1-LGP32 présent dans toutes les souches virulentes de V. tasmaniensis qui est l'orthologue evpP sécrété par le T6SS du pathogène du poisson Edwardsiella tarda [START_REF] Hu | A Disordered Region in the EvpP Protein from the Type VI Secretion System of Edwardsiella Tarda is Essential for EvpC Binding[END_REF]. La délétion d'evpP chez V.

tasmaniensis altère son potentiel de cytotoxicité, cependant, il n'a montré aucune atténuation de la virulence lors d'infections expérimentales (données complémentaires, section II.3). Des expériences de complémentation ectopique sont à prévoir pour valider ces phénotypes.

Chez l'espèce V. harveyi associée aux POMS de la lagune de Thau, qui elle aussi est cytotoxique, la génomique comparative nous a permis d'émettre l'hypothèse que la distribution différentielle d'un système de sécrétion de type III dans le chromosome 1 (T3SS1) et les effecteurs cytotoxiques associés putatifs précédemment identifiés chez V.

parahaemolyticus (Broberg et al., 2010 ;[START_REF] Osorio | T3SS Effectors in Vibrios: Homology in Sequence, Diversity in Biological Functions?[END_REF][START_REF] Wu | Type III Secretion 1 Effector Gene Diversity Among Vibrio Isolates From Coastal Areas in China[END_REF], pourraient contribuer à leur cytotoxicité. Chez V. harveyi et V. jasicida, qui contiennent le deux les gènes structuraux du T3SS, mais présentent une cytotoxicité contrastée (V. jasicida n'est pas cytotoxique), la principale différence que nous avons pu identifier est le nombre plus élevé d'effecteurs cytotoxiques putatifs présents chez V. harveyi (Arginine ADP-ribosyl transférase, VopQ, VopR, VopS et VPA0450) par rapport à V. jasicida (VopQ et VopS). La délétion ou l'invalidation ciblée des éléments structuraux de ce T3SS1 et des effecteurs putatifs associés devraient aider à clarifier leurs rôles respectifs dans la cytotoxicité et la virulence.

Récemment, des méthodes de manipulation génétique de V. harveyi ont été développées avec succès [START_REF] Delavat | Rapid and Efficient Protocol to Introduce Exogenous DNA in Vibrio harveyi and Pseudoalteromonas sp[END_REF], ce qui devrait être envisagé pour de futurs travaux de manipulation de gènes dans des souches Harveyi d'intérêt.

Dans l'ensemble, nos données sur des populations distinctes de Vibrio (V. tasmaniensis, V. crassostreae, V. harveyi) montrent que des armes spécifiques à l'espèce sont utilisées pour supprimer les cellules immunitaires des huîtres au cours du POMS. Cela suggère que V. tasmaniensis, V. crassostreae et V. harveyi sont probablement des agents pathogènes opportunistes et non de simples colonisateurs bénéficiant de l'immunosuppression de l'OsHV-1.

AU DELA DES SOUCHES CYTOTOXIQUES, DES TRICHEURS BENEFICIENT DE L'INFECTION PAR

OsHV-1

En réalisant des études en mésocosmes pour réaliser des infections expérimentales écologiquement réalistes. Les huîtres ont été exposées à de l'eau de mer contenant le virus

OsHV-1 et une communauté Vibrio, au lieu des traditionnelles injections intramusculaires de pathogènes donnés, ce qui nous a permis d'observer de nouveaux comportements parmi les vibrios associés aux POMS. Dans un contexte, polymicrobien, il peut arriver que de multiples interactions hôte-bactéries et bactéries-bactéries modifient les pressions sélectives intra-hôtes agissant sur les colonisateurs potentiels [START_REF] Alizon | Co-infection and Super-infection Models in Evolutionary Epidemiology[END_REF]. Dans l'article 3, nos résultats ont montré que tous les vibrios capables de coloniser les huîtres ne sont pas cytotoxiques, mais que certains d'entre eux peuvent bénéficier de l'activité du microbiote, qui non seulement affaiblir les défenses immunitaires des huîtres mais produire également des « biens publics » utilisables par des tricheurs opportunistes.

C'est ainsi que l'espèce V. rotiferianus semble adopter un comportement de tricheur pour l'acquisition du fer [START_REF] Kramer | Bacterial Siderophores in Community and Host Interactions[END_REF] par absorption du sidérophore vibrioferrine. Cette espèce, qui colonise efficacement les huîtres, n'est pas cytotoxique et elle a perdu le cluster de synthèse de la vibrioferrine présent chez V. harveyi tout en conservant le gène codant pour le récepteur apparenté, ce qui serait un exemple d'évolution des dépendances par perte de gène adaptative, également connu comme l'hypothèse de la reine noire [START_REF] Morris | Black Queen hypothesis[END_REF]. Nous avons confirmé in vitro que la croissance peut être restaurée dans des conditions de limitation du fer par les produits extracellulaires de V. harveyi, un producteur de vibrioferrine. Ce résultat nous amène à proposer qu'en tant que population moyennement virulente et cytotoxique, V. rotiferianus bénéficie de l'état physiologique altéré de l'hôte causé par des populations virulentes comme V. harveyi mais aussi de l'acquisition non coopérative de biens publics tels que les sidérophores pour coloniser l'huître par une stratégie purement opportuniste. Il sera nécessaire de confirmer ces hypothèses en réalisant des expériences supplémentaires avec des combinaisons isolées et spécifiques de populations de vibrios pour répondre à deux questions en suspens : (i) La colonisation de l'hôte par V.

rotiferianus résulte-t-elle d'une stratégie opportuniste qui dépend de la présence de V.

harveyi ? (ii) Quels facteurs expliquent l'exclusion de V. owensii et V. jasicida des huîtres ? Estce dû à des contraintes physiologiques (par exemple des exigences métaboliques) ou via l'exclusion grâce à l'action antagoniste des membres du microbiote de l'hôte ? En étudiant la population de V. splendidus #23 qui est associée aux huîtres en dehors des périodes de mortalités, et qui présente un potentiel de virulence modéré par rapport aux populations de V. crassostreae ou V. splendidus # 24 collectées en période de mortalités estivales, nous avons pu montrer que toutes les souches de Pop #23 étaient cytotoxiques visà-vis des hémocytes in vitro, y compris la souche 4G1_4 considérée comme non virulente (survie des huîtres> 82% six jours après injection). Cette souche non virulente est caractérisée par l'absence de deux loci codants pour des facteurs de virulence potentiels, un complexe MARTX et un T6SS. Dans le cadre de l'article 1 la caractérisation des déterminants de la cytoxicité de V. splendidus n'a pas été ralisée. Cependant, comment il est présent dans toutes les souches de Pop#23, nous pensons que R5.7 pourrait être impliqué dans la cytotoxicité de V. splendidus, comme montré chez V. crassostreae, (Bruto et al., 2017(Bruto et al., , 2018) ) où il agit indépendamment de la présence du T6SSpGV1512 pour provoquer la lyse hémocytaire (Piel et al., 2019) (Wildschutte et al., 2004 ;March et al., 2013).

Les résultats présentés dans l'article 1 indiquent que la sélection pour la résistance à la prédation par modifications de l'antigène O peut être en conflit avec la virulence, contrairement à la sélection coïncidente identifiée dans V. tasmaniensis LGP32 pour des facteurs de virulence chez l'huître également impliqués dans la résistance aux amibes [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].

Le POMS semble sélectionner majoritairement des bactéries aux phénotypes virulents, cytotoxiques, capables de coloniser des huîtres immunodéprimées, à l'exception des tricheurs opportunistes identifiés en milieu méditerranéen (article 3). Étant donné que le virus

OsHV-1 se transmet rapidement parmi les populations d'huîtres sensibles [START_REF] Fleury | Latitudinal drivers of oyster mortality: deciphering host, pathogen and environmental risk factors[END_REF], on s'attend à ce que dans ce contexte, la mort rapide de l'hôte favorise la transmission du

GENERAL INTRODUCTION

Bacteria of the Vibrio genus are ubiquitous inhabitants of aquatic environments that possess a remarkable genomic and phenotypic plasticity, which allows them to undergo into a wide range of niche specialization going from free-living forms to mutualistic, commensal and pathogenic association with plants and animals [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 their association with animals, vibrios are part of the natural microbiota of healthy marine organisms like corals, sponges and diverse species of bivalves as well as intimate mutualistic symbionts of sepiloid squids (Ceccarelli and Colwell, 2014;McFall-Ngai, 2014). Nonetheless, several Vibrio species are well-known human pathogens and causative agents of vibrioses in aquatic organisms with severe impact on fish and shellfish farming worldwide (Romalde and Barja, 2010).

The Pacific Oyster Crassostrea gigas is a valuable product for human consumption that is produced all along the French coast. Since 2008, farmed populations of oysters had suffered from severe episodes of summer mortalities specially affecting juvenile oysters of less than 18 months, reaching mortality rates as high as 100% in French farms [START_REF] Segarra | Detection and Description of a Particular Ostreid Herpesvirus 1 Genotype Associated with Massive Mortality Outbreaks of Pacific Oysters, Crassostrea gigas, in France in 2008[END_REF]. This disease is known as the Pacific Oyster Mortality Syndrome (POMS). It has a complex etiology, which involves the interaction between the host genetics and physiological state, the environment, and pathogens, specifically microvariants of the ostreid herpes virus OsHV-1 and opportunistic bacteria including vibrios (Petton et al., 2015). POMS occur seasonally, during a permissive seawater temperature window ranging from 16°C to 24°C and follows a sequence of events that starts with the intense replication of OsHV-1 within the host leading to an immunosuppressed state that is used for opportunistic bacteria, among them vibrios, to mount a secondary infection that causes the death of the host (de Lorgeril et al., 2018).

Whether these Vibrio species are simple opportunists or actively participate in the infectious process (opportunistic pathogens) has been highly debated.

Nonetheless pathogenicity does not summarize the interaction between the oysters and vibrios. The population structure of the vibrio community present in different fraction of the water column and in the oyster tissues shows that certain vibrio populations show preferential association with the oyster even outside a pathological context, with little or no overlapping in populations between diseased and healthy oysters (Bruto et al., 2017). Without the influence of major stressors such as temperature (Green et al., 2019) or pathogens such as

OsHV-1 µvar, oysters possess a remarkable capacity to maintain immune homeostasis with its resident microbiota [START_REF] Schmitt | Immune Responses in the Pacific Oyster Crassostrea gigas: An Overview with Focus on Summer Mortalities[END_REF], which should have shaped the genomes of the bacterial groups able to establish a stable interaction with oysters as a host (Chow et al., 2010).

The objective of the present thesis was to characterize the mechanisms underlying the association of the oyster C. gigas with vibrio populations in health and disease. For this, we studied vibrio populations that display a preferential association to the oyster tissues but are isolated from contrasted contexts; i.e. the absence of mass mortalities when seawater temperature is below 16°C, and the POMS outbreaks characterized by warmer waters (above 16°C).

In CHAPTER 1 of my thesis, I present a review of (I) the main characteristic of the Vibrio genus, the diversity of association that Vibrio establish in the environment and the mechanisms of colonization with metazoan hosts, (II) the biology and immune defenses of our model host, the oyster C. gigas and (III) the latest knowledge about the POMS, the polymicrobial disease that affects juvenile oysters whose etiology involves the OsHV-1 µvar virus and opportunistic vibrios.

In CHAPTER 2, I present the results obtained during my thesis, divided into three section.

Section I describes a moderately virulent species of V. splendidus associated with healthy oysters. We explore the tradeoffs existing between virulence and environmental persistence in this species (article 1, Env. Microbiol.). We also investigate whether the resistance to oyster antibacterial defenses determines the capacity of vibrios to colonize healthy oysters (short letter, in prep). In section II, we present a comparative study of the mechanism of virulence of V. crassostreae and V. tasmaniensis, two vibrio species associated with mortality outbreaks in the Atlantic marine ecosystem. We show by which means these virulent species subvert the host defenses enabling systemic infection (article 2, PNAS). Finally, in section III, we characterize the Vibrio community associated with POMS in the Mediterranean oysterfarming site whose ecology differs from that of the Atlantic. We identify the strategies of those

Vibrio species that are able to benefit from the OsHV-1 immunosuppressed state of the host, both killers and cheaters, to successfully colonize and infect oysters (article 3, in prep).

CHAPTER 3 presents a general discussion of the results obtained as a well as perspectives to further explore the colonization of the oyster C. gigas by vibrios, considering the diversity of biological and ecological contexts in which this process occurs.

This thesis manuscript ends with a minireview on Vibrio-bivalve interactions (article 4, Env.

Microbiol., ANNEX 1), in which we compare Vibrio interactions with important aquaculture species, the oyster C. gigas and the mussels Mytilus edulis and M. galloprovincialis.

CHAPTER 1. STATE OF THE ART

I. THE GENUS VIBRIO

I.1 VIBRIO BIOLOGY AND DIVERSITY

Members of the family Vibrioneaceae (thereafter vibrios) are natural and ubiquitous inhabitants of temperate marine, freshwater and estuarine environments where they contribute to the carbon cycle, rhizospheric nitrogen fixation and display a wide range of niche specialization that goes from free-living forms to biotic associations establishing mutualistic, commensal and pathogenic association with plants and animals [START_REF] Johnson | Fitness Factors in Vibrios: A Mini-review[END_REF][START_REF] Ceccarelli | Vibrio species[END_REF]. Several Vibrio species are well-known human pathogens causing gastrointestinal diseases and wound infections [START_REF] Osunla | Vibrio Pathogens: A Public Health Concern in Rural Water Resources in Sub-Saharan Africa[END_REF] as well as causative agents of vibrioses in aquatic organisms with severe impact on fish and shellfish farming worldwide [START_REF] Travers | Bacterial Diseases in Marine Bivalves[END_REF][START_REF] A-Abdelaziz | Monitoring of Different Vibrio Species Affecting Marine Fishes in Lake Qarun and Gulf of Suez: Phenotypic and Molecular Characterization[END_REF]. However, vibrios are also part of the natural microbiota of healthy marine organisms like, corals, sponges and diverse species of bivalves [START_REF] Boyd | Post-Genomic Analysis of Members of the Family Vibrionaceae[END_REF]. As for example, the well-known bobtail squid-Vibrio fischeri (now Aliivibrio fischeri) association represents a case of intimate mutualistic symbiosis [START_REF] Nyholm | The Winnowing: Establishing the Squid -Vibrios Symbiosis[END_REF].

Vibrios are small (0.5-0.8 μm wide, 1.4-2.6 μm long) straight or curved rod-shaped gramnegative bacteria, with a single polar flagellum enclosed in a sheath [START_REF] Thompson | Genomic Taxonomy of Vibrios[END_REF] (Figure 1A andB). Median genome size in vibrios is around 5 Mbp and it is distributed in two independently-replicating chromosomes of unequal size. This bipartite genomic structure, which is an exception to the rule in bacterial genomes [START_REF] O-Okada | Vibrios Commonly Possess Two Chromosomes[END_REF], is conserved among vibrios, despite some naturally occurring chromosomes fusion events described in isolates of V. cholerae [START_REF] Chapman | Scanning the Landscape of Genome Architecture of Non-o1 and Non-o139 Vibrio cholerae by Whole Genome Mapping Reveals Extensive Population Genetic Diversity[END_REF]. While the larger chromosome (chromosome 1) usually carries most of the essential genes, the smaller one (chromosome 2) displays adaptive, species-specific genes and it would have been acquired as a mega plasmid before the diversification of Vibrionaceae [START_REF] Reen | The Genomic Code: Inferring Vibrionaceae Niche Specialization[END_REF].

Vibrios use a chemosensory system to sense environmental signals and actively move towards dissolved or particulate organic matter in search for nutritional sources or escape from harmful substances, in a process called chemotaxis [START_REF] Takemura | Associations and Dynamics of Vibrionaceae in the Environment, From the Genus to the Population Level[END_REF][START_REF] Ringgaard | Chemotaxis Arrays in Vibrio Species and their Intracellular Positioning by the ParC/ParP System[END_REF]. Locomotion is mediated by the flagella, which in vibrios correspond to a sodium-driven molecular machine that can spin at up to 1,700 Hz, conferring them a higher speed and motility relative to other bacteria like salmonella, which posses a proton-driven flagellum with spin frequencies around 300 Hz [START_REF] Minamino | The Bacterial Flagellar Motor and its Structural Diversity[END_REF].

They are heterotrophic bacteria with a facultative anaerobic metabolism [START_REF] Gomez-Gil | The Famlily Vibrionaceae[END_REF]. In the marine environment they mostly rely on catabolizing carbohydrates, proteins and lipids and are able to hydrolyze sugars and amino acids, when available [START_REF] Grimes | The Vibrios : Scavengers, Symbionts, and Pathogens from the Sea[END_REF].

The majority of vibrios can degrade the chitin polymer [START_REF] Gooday | Physiology of Microbial Degradation of Chitin and Chitosan[END_REF][START_REF] Keyhani | Physiological Aspects of Chitin Catabolism in Marine Bacteria[END_REF], which is the most abundant biopolymer in the aquatic environment, composed of β- [START_REF] Gay | Screening of Vibrio isolates to develop an experimental infection model in the Pacific oyster Crassostrea gigas[END_REF][START_REF] De Lorgeril | Immune-suppression by OsHV-1 viral infection causes fatal bacteraemia in Pacific oysters[END_REF]-linked N-acetyl-D-glucosamine (Le Roux and [START_REF] Le Roux | Eco-evolutionary Dynamics Linked to Horizontal Gene Transfer in Vibrios[END_REF], as well as, to a lesser extent, algal and plant polysaccharides [START_REF] Takemura | Natural Resource Landscapes of a Marine Bacterium Reveal Distinct Fitness-Determining Genes Across the Genome[END_REF]. As an example of specialization to algal exploitation, V. breoganii, which has lost the ability to degrade chitin, is able to catabolize alginate, laminarin and additional glycans present in the cell wall of brown algae [START_REF] Corzett | Evolution of a Vegetarian Vibrio: Metabolic Specialization of Vibrio breoganii to Macroalgal Substrates[END_REF]. Additionally, the production of extracellular proteases allow them to effectively utilize some abundant animal resources such as collagens, which all vibrios are able to catabolize [START_REF] Grimes | The Vibrios : Scavengers, Symbionts, and Pathogens from the Sea[END_REF].

The study of environmental dynamics of Vibrio species, has mainly focused on species pathogenic to humans. It was shown that salinity and temperature are the two main drivers of Vibrio dynamics [START_REF] Takemura | Associations and Dynamics of Vibrionaceae in the Environment, From the Genus to the Population Level[END_REF]. Vibrios inhabit temperate waters growing in a temperature range that goes from 15 to 40°C with an optimum between 25-30°C [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]. They are mesophilic bacteria and most species are halophilic and require saline medium for growth [START_REF] Thompson | Diversity and dynamics of a North Atlantic coastal Vibrio community[END_REF]. The minimal concentration of Na+ for optimal growth varies between 5 and 700 mM, depending on the species (Farmer et al., 2015). It is estimated that vibrios constitute 10% of the total culturable marine bacteria, thus representing one of the most abundant culturable fraction of marine bacteria [START_REF] Eilers | Culturability and In Situ Abundance of Pelagic Bacteria from the North Sea[END_REF]. In the water column, culture dependent studies estimate an average of 10 3 to 10 6 CFU L -1 while culture independent methods estimate 10 4 to 10 8 16S rRNA copies L -1 representing 1% of the total bacterioplankton in coastal waters [START_REF] Rehnstam | Blooms of Sequence-Specific Culturable Bacteria in the Sea[END_REF].

Vibrios are easily cultured on both selective and non-selective standard media and this had allowed research activities over the pre-molecular era of microbiology [START_REF] Takemura | Associations and Dynamics of Vibrionaceae in the Environment, From the Genus to the Population Level[END_REF] and more recently, DNA sequencing and culture-independent approaches has unraveled the outstanding genetic diversity that characterize environmental vibrios [START_REF] Dryselius | Vibrionaceae, a Versatile Bacterial Family with Evolutionarily Conserved Variability[END_REF].

I.2 VIBRIO TAXONOMIC ORGANIZATION

The Vibrionaceae is a family of γ-proteobacteria that gathers, to date, 155 validly published species distributed among 11 genus: Aliivibrio (6 spp), Catenococcus This represents a wide and diversified family of bacteria belonging to many different closely related species. This limits the interspecies resolution using 16S rRNA gene polymorphism;

hence several faster evolving and highly polymorphic protein-coding genes have been used

in Multi Locus Sequence Analysis (MLSA) to reconstruct the phylogenetic relationships, to assess biodiversity and to infer the evolutionary history of this group (Sawabe et al., 2007;[START_REF] Gabriel | Optimization of Multilocus Sequence Analysis for Identification of Species in the Genus Vibrio[END_REF]. By using a MLSA approach based on the analysis of 9 housekeeping genes (ftsZ, gapA, gyrB, mreB, pyrH, recA, rpoA, topA and the 16S RNA gene) over 58 strains, Sawabe and collaborators defined 14 monophyletic clades and more than 60 vibrio species (Sawabe et al., 2007). This phylogeny was improved further more recently [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] by using 86 vibrio strains, 10 vibrio genomes, and 8 housekeeping genes: eight new clades were identified and 9 others were updated (Figure 1C).

I.3 VIBRIO ECOLOGICAL POPULATIONS: BRINGING TOGETHER GENETIC RELATEDNESS AND ECOLOGICAL FUNCTION

Taxonomic classification of bacterial isolates relies on a polyphasic identification of species that integrates all available phenotypic and genotypic information [START_REF] Gillis | Polyphasic Taxonomy. In Bergey's Manual of Systematics and Archaea and Bacteria[END_REF]. This approach often fails to integrate fine scale information about bacteria-scale potential habitats (Preheim et al., 2011b). Although the physical space that a group of organisms occupies is key information to characterize the ecology of a species, this aspect remains limited for bacteria including vibrios. Pioneering work has coupled ecological sampling with high resolution genotyping, showing that vibrios whose coexist in the water column following different lifestyles (free-living or attached to particles or zooplankton) can be partitioned according to genetic and ecological similarities and thus, define vibrio ecological populations constituted by genotypic clusters sharing temporal or spatial common habitats which are connected by dispersal (Hunt et al., 2008;Preheim et al., 2011b). Due to this connection between habitats by dispersal, a metapopulation framework can be applied to consider possible overlaps or differences of populations isolated from distinct habitats (Preheim et al., 2011a).

Moreover, it has been demonstrated that these ecological populations also represent units of gene flow where the recombination rate is higher within than between populations, which allows adaptive genes to spread in a population-specific way [START_REF] Le Roux | Oysters and Vibrios as a Model for Disease Dynamics in Wild Animals[END_REF]. In the context of vibrios interactions with oysters, this ecological population framework has allowed to study how vibrio populations assemble into the oyster during health and disease and to identify functional units of pathogenesis in vibrios by mapping virulence determinants onto population structure (Lemire et al., 2015;Bruto et al., 2017Bruto et al., , 2018)). Prochlorococcus, and Synechococcus corresponds to relatively small cells with streamlined genomes and low biomass, vibrios are characterized by their lower abundance but high biomass per cell, metabolic diversity and phenotypic plasticity (Leroux and Blokesch, 2018).

Although it was first proposed that the presence of vibrio in the water column was mainly a consequence of their excretion on animal feces [START_REF] Takemura | Associations and Dynamics of Vibrionaceae in the Environment, From the Genus to the Population Level[END_REF], It was later evidenced that their genetic diversity and phenotypic plasticity allow them to adopt a variety of complex associations in the environment; while tight associations with animals do exist for some Vibrio species, other can be loosely associated with metazoans (Preheim et al., 2011a).

I.4.1 FREE-LIVING AND PARTICLE ATTACHED LI FESTYLES

In microcosms, purely dissolved nutrients, derived from algal exudates, are able to sustain the proliferation of vibrios in a free-living state, overcoming competition and predation by grazers [START_REF] Mouriño-Pérez | Growth of Vibrio cholerae O1 in Red Tide Waters off California[END_REF][START_REF] Worden | Trophic Regulation of Vibrio cholerae in Coastal Marine Waters[END_REF][START_REF] E-Eiler | Growth Response of Vibrio cholerae and Other Vibrio spp. to Cyanobacterial Dissolved Organic Matter and Temperature in Brackish Water[END_REF]. Although able to proliferate from dissolved nutrient, vibrios are more frequently found attached to biotic and abiotic surfaces, and this lifestyle has been related to their environmental persistence [START_REF] Reen | The Genomic Code: Inferring Vibrionaceae Niche Specialization[END_REF]. Dead biomass of small planktonic organisms, fecal pellets and polymer aggregates such as marine snows may represent microhabitats for vibrios and provide resources for growth [START_REF] Takemura | Associations and Dynamics of Vibrionaceae in the Environment, From the Genus to the Population Level[END_REF].

Two sympatric ecological populations of V. cyclitrophicus presenting different food-seeking behavior have been shown to favor early stages of differentiation through a gene-specific selective sweep mechanism leading to gradual genome wide differentiation [START_REF] Shapiro | Population Genomics of Early Events in the Ecological Differentiation of Bacteria[END_REF]. Behavioral confirmation defined two distinct populations, the L population, which is specialized in growing attached to large particles, and the S population, which benefits from rapid detection and swarming towards transient patches of dissolved nutrients (Figure 3A).

The recent gain or loss of 2 gene clusters exclusive to the L population related to attachments (msh gene cluster) and biofilm formation (syp gene cluster) have been proposed to explain the behavioral differences between L and S that impose boundaries to previous unrestricted recombination within the parental populations, triggering an ecological differentiation that may lead to speciation [START_REF] Vaninsberghe | How Can Microbial Population Genomics Inform Community Ecology?[END_REF].

As certain vibrio population transiently associate to organic particles and planktonic hosts (both metazoans and protozoans), this can increase the colonization efficiency of filter feeding organisms that show size-dependent retention of particles such as oysters or mussels [START_REF] Duinker | Variability in Particle Retention Efficiency by the Mussel mytilus Edulis[END_REF]Bruto et al., 2017). It has been recently demonstrated that the association of V. vulnificus with artificial marine snow (AMS) can dramatically increase the colonization success of the oyster C. gigas (Figure 3B). While this study was applied to the human pathogen V. vulnificus, this novel mechanism enhancing bivalve colonization may be extended to other vibrio species that are pathogenic for oysters.

ASSOCIATIONS WI TH PHYTO AND ZOOPLANKTON

Vibrios have a numerical dominance on phytoplankton surfaces, as demonstrated by culture-based studies, but direct physical association with specific phytoplankton has only been described for V. cholerae with two microalgal species, the freshwater Rhizoclonium fontanum (Islam et al., 1989) and Anabaena sp, both in freshwater (Islam et al., 1999) and saline condition (Ferdous, 2009).

Associations with zooplankton have also been studied, attachment of V. cholerae to copepods species such Ascartia and Eurytemora has been evidenced while exploring the potential role of these planktonic species as a reservoir of V. cholerae. It has been proposed

a role of the main virulence factors of V. cholerae, the cholera toxin (CT) and the toxin coregulated pilus (TCP) in a mutualistic relationship with copepods, where attachment to the chitinous exoskeleton is mediated by the TCP, providing a growth substrate to V. cholerae, and the increased Cl-secretion and reduced Na+ absorption produced by the CT has an advantageous osmoregulatory effect for the crustacean host moving towards increasing salinities [START_REF] Reen | The Genomic Code: Inferring Vibrionaceae Niche Specialization[END_REF].

Other than copepods, cladocerans have been described also as a possible host for V.

cholerae with the potential to enhance growth when added to the mesocosm. The number of vibrios attached per individual cladoceran was 100 times higher compared to copepods.

Nevertheless, there was no evidence of an enrichment of vibrios attached to cladocerans compared to the water column and therefore the authors suggested that frequent dispersal and no permanent attachment characterize this supported growth of vibrios by cladocerans (Kirschner et al., 2011).

Beyond V. cholerae, in a comprehensive study on the metapopulation structure of Vibronacea among invertebrates (Figure 2A), Preheim et al. (2011) showed that the crustacean zooplanktonic fraction of the water column contained the lesser diverse but more specific assembly of vibrio populations compared to other invertebrates such as crabs and mussels. Interestingly, the authors distinguished dead from alive zooplankton. While vibrios were found associated with both dead and alive specimens, they displayed a higher relative frequency in dead individuals. This suggests a preferential saprophytic lifestyle benefiting from the degradation of the dead host tissues and the chitinaceous carapaces, thanks to the already discussed capacity of vibrios to degrade chitin. Overall, both phyto and zooplankton appear to be able to support vibrio growth in a free-living state by providing dissolved organic carbon but also participating in direct physical interaction with vibrios. Still, for the majority of these associations a clear distinction between transitory or specialized and stable associations is missing.

ASSOCIATION WI TH ANIMALS

Historically, since the first observation of V. cholerae by Filippo Pacini from cholera stools in 1854 and its later isolation by Robert Koch in 1883, vibrios have been mainly studied as vertebrate and invertebrate pathogens [START_REF] Thompson | Diversity and dynamics of a North Atlantic coastal Vibrio community[END_REF]. The four well-known species,

V. cholerae, V. vulnificus, V. parahaemolyticus and V. alginolyticus, have been defined based on their incidence and severity of the disease that they cause to humans; and several other species, such as V. splendidus, V. crassostreae, V. harveyi, V. coralliilyticus, V. aestuarianus, V. anguillarum have been associated with infectious diseases affecting marine organisms like fish, bivalves and corals [START_REF] Le Roux | The Emergence of Vibrio Pathogens in Europe: Ecology, Evolution, and Pathogenesis (Paris, 11-12th March 2015)[END_REF].

In contrast, for some of them an exchange of benefits between vibrios and the host has been suggested to represent true mutualistic relationships. The mutualism of luminescent vibrios with certain squid species has served as models to disentangle the mechanisms underpinning this type of symbiotic association (Figure 4B) [START_REF] Ruby | The Vibrio fischeri-Euprymna Scolopes Light Organ Association: Current Ecological Paradigms[END_REF]. The symbiosis between the V.

fischeri and the bobtail Euprymna scolopes being the most documented one. While not an obligatory relationship, the light organ of the squid, provides a safe and nutritious environment for V. fischeri while the squid uses the light produced by the bacteria in a counter illumination strategy to avoid predators during the night [START_REF] Nyholm | The Winnowing: Establishing the Squid -Vibrios Symbiosis[END_REF]. The establishment, development and maintenance of V. fischeri in the light organ results from an intricate interplay between the symbionts and the host. V. fischeri cells use chemotaxis and aggregate in biofilms in the mucus secreted by the ciliated epithelia of the surface of the light organ. Next, symbionts migrate into the pores and through the ducts that connect to the light organ crypts where V. fischeri is exposed to oxidative stress. Symbionts are able to attenuate the nitric oxide production and induce, through peptidoglycan and lipopolysaccharide signaling, a morphogenesis of the epithelial fields that discourage subsequent colonization (Figure 5A). From there, symbionts go through daily cycles of expulsion and regrowth. The continuous discovery of novel symbiotic determinants in this model has shown that copper efflux is also determinant for V. fischeri resilience in the light organ [START_REF] Brooks | Global Discovery of Colonization Determinants in the Squid Symbiont Vibrio fischeri[END_REF] and that different V. fischeri genotypes can undergo into bacterial competition, which could underlie the observation that the vibrio populations in the light organ are often dominated by a single strain (Figure 5B) [START_REF] Speare | Bacterial Symbionts use a Type VI Secretion System to Eliminate Competitors in their Natural Host[END_REF]. Ngai. 2004). (B) Symbionts V. fischeri are able to compete with conspecifics through a strain-specific Type VI secretion system. Co-incubation of the non-lethal type strain ES114 with different V. fischeri strains isolated from the light organ show a strain-specific killing activity.

Among animals, interactions of vibrios with diverse benthic organisms have been well

documented also, such as interactions with diverse bivalves [START_REF] Lokmer | Hemolymph Microbiome of Pacific Oysters in Response to Temperature, Temperature Stress and Infection[END_REF]Lokmer et al., 2016a), gastropods [START_REF] Sawabe | The Mutual Partnership Between Vibrio halioticoli and Abalones[END_REF] and crustaceans [START_REF] Davis | Incidence of Vibrio Species Associated With Blue Crabs (Callinectes sapidus) Collected From Galveston Bay, Texas[END_REF][START_REF] Sullivan | Effects of Temperature and Salinity on Prevalence and Intensity of Infection of Blue Crabs, Callinectes sapidus, by Vibrio cholerae, V. parahaemolyticus, and V. vulnificus in Louisiana[END_REF]. There are evidences that suggest the stable gut colonization of the herbivorous Haliotis abalone by V. haliotis, based on specificity of occurrence only in Haliotis and a possible role of the alginolytic activity of V. haliotis in the digestion of algal polysaccharides in the abalone [START_REF] Takemura | Associations and Dynamics of Vibrionaceae in the Environment, From the Genus to the Population Level[END_REF]. In contrast other vibrio species such as V. harveyi and V. parahaemolyticus are the etiological agents of severe vibriosis in Haliotis species [START_REF] C-Cardinaud | Vibrio harveyi Adheres to and Penetrates Tissues of the European Abalone Haliotis tuberculata Within the First Hours of Contact[END_REF][START_REF] Lu | Metabolic Responses of Haliotis Diversicolor to Vibrio parahaemolyticus Infection[END_REF]. The same is true for filter feeding organisms like oyster and mussels where vibrios are part of the normal microbiota of healthy individual but also, especially in the case of oysters, are responsible for severe vibriosis in larvae, juveniles and adults [START_REF] H-Hasegawa | The Extracellular Metalloprotease of Vibrio Tubiashii is a Major Virulence Factor for Pacific Oyster (Crassostrea gigas) Larvae[END_REF]Bruto et al., 2017;[START_REF] Lupo | Modeling the Transmission of Vibrio gestuarianus in Pacific Oysters Using Experimental Infection Data[END_REF]. We will see these interactions in further details in a dedicated section below.

ACCIDENTAL INFECTIONS IN HUMANS

Humans can also become hosts for vibrio through the accidental ingestion of contaminated water or seafood, but also by the mere exposure of open wound to contaminated water. The most common disease caused by the 11 recognized species pathogenic for humans are gastroenteritis and wound infections, which can be deadly specially in immunocompromised individuals.

V. CHOLERAE Cholera is the most famous disease caused by any vibrio in humans as it can cause epidemic outbreaks. It is a severe diarrheal disease caused by V. cholerae for which it has been described more than 200 serogroups, but only O1 and O139 serogroups have been responsible for large scale cholera outbreaks. Upon pathogen entrance through ingestion, V.

cholerae colonizes the small intestine thanks to a lipopolysaccharide (LPS) mediated resistance to complement and the expression of the toxin co-regulated pilus (TCP) that promote bacteria-bacteria interaction and the formation of microcolonies on the surface of epithelial cells. Once stablished, the expression of the cholera toxin promotes a massive fluid and electrolyte efflux that causes the severe diarrhea that characterizes the cholera disease (Saha and LaRocque, 2012).

V. PARAHAEMOLYTICUS Acute gastroenteritis caused by V. parahaemolyticus is also linked to the consumption of raw or uncooked fish and shellfish [START_REF] Lee | Vibrio parahaemolyticus Infectious for Both Humans and Edible Mollusk Abalone[END_REF]. The vibrio colonizes the human small intestine presumably through the expression of a series of virulence factors including adhesins, hemolysins (TDH and TRH) and effectors involved in attachment and cytotoxic activity toward the host cells which are secreted through a specialized protein delivery nanomachine called type III secretion system (de Souza Santos and Orth, 2014). V. parahaemolyticus can eventually infect wounds which is related to higher mortality risk due to septicemia. In shrimps,

V. parahaemolyticus is the causative agent of an acute hepatopancreatic disease (AHPND)

which is currently threatening shrimp culture worldwide. The highly virulent strains of V.

parahaemolyticus associated with this disease, express two deadly toxins, PirA and PirB, encoded in the virulence plasmid pVA1, which has been suggested to have been either lost or acquired through horizontal gene transfer [START_REF] Lee | The Opportunistic Marine Pathogen Vibrio parahaemolyticus Becomes Virulent by Acquiring a Plasmid that Expresses a Deadly Toxin[END_REF] and has been identified in other Vibrio species pathogenic for shrimps such as V. campbellii, V. harveyi and V. owensii [START_REF] Peña-Navarro | Molecular Detection of Acute Hepatopancreatic Necrosis Disease (AHPND) in Penaeus vannamei Shrimps in Costa Rica[END_REF].

(
V. VULNIFICUS V. vulnificus is a rarely infecting but highly lethal opportunistic pathogen for humans which naturally inhabits the coastal marine environment and an emergent pathogen for aquaculture species [START_REF] Jones | Vibrio vulnificus: Disease and Pathogenesis[END_REF][START_REF] Le Roux | Eco-evolutionary Dynamics Linked to Horizontal Gene Transfer in Vibrios[END_REF]. Strains are classified based on biochemical, serological and host range characteristics in three different biotypes. Biotypes 1 is mostly associated with disease in humans and commonly found associated from shellfish, biotype 2 associated with disease in eels but also an opportunistic pathogen for humans [START_REF] Amaro | Vibrio vulnificus Biotype 2, Pathogenic for Eels, si Also an Opportunistic Pathogen for Humans[END_REF]) and a third one with strains genetically distinct from biotype 1 and 2 and limited to Israel [START_REF] Bisharat | Clinical, epidemiological, and microbiological features of Vibrio vulnificus biogroup 3 causing outbreaks of wound infection and bacteraemia in Israel[END_REF].

Consumption of contaminated seafood or open wound exposure can lead to severe systemic infection and septic shock linked to the coordinated expression of several putative virulence factors such as capsular polysaccharide, cytotoxic MARTX (Multifunctional-Autoprocessing Repeats-in-ToXin) toxins, hemolysin, the metalloprotease VVP, essential for mucus colonization in fish and siderophores for iron acquisition under limiting conditions, [START_REF] Hernández-Cabanyero | Phylogeny and life cycle of the zoonotic pathogen Vibrio vulnificus[END_REF][START_REF] Li | The Role of Vibrio Vulnificus Virulence Factors and Regulators in its Infection-Induced Sepsis[END_REF]. Survival of V. vulnificus in the human blood is directly linked to elevated iron concentration in the serum due to hematological conditions such as hemochromatosis and thalassemia [START_REF] Strom | Epidemiology and Pathogenesis of Vibrio vulnificus[END_REF]. Despite the number of putative virulence factors identified, the majority of V. vulnificus isolated are non-pathogenic to humans and the factors described above have not been reliably correlated with disease in humans [START_REF] Li | The Role of Vibrio Vulnificus Virulence Factors and Regulators in its Infection-Induced Sepsis[END_REF].

I.4.3 VIBRIO ASSOCIATIONS WI TH BIVALVES IN HEALTH AND DI SEASE

Bivalves are the second largest group in the Phyllum Mollusca and have a fundamental role in ecology marine ecosystem including nutrient recycling, habitat creation and influence over food webs [START_REF] Vaughn | Bivalve Impacts in Freshwater and Marine Ecosystems[END_REF]. They include farmed species of economic importance and represent a unique model to study colonization and disease dynamics in vibrios [START_REF] Le Roux | Oysters and Vibrios as a Model for Disease Dynamics in Wild Animals[END_REF].

Most bivalves adopt a sessile lifestyle in their adult stages and therefore, they cannot escape predation. They take up large amounts of water for feeding and oxygen assimilation purpose, thus are unavoidably submitted to the dynamic of the diverse microbial communities in close proximity [START_REF] Takeuchi | Bivalve-Specific Gene Expansion in the Pearl Oyster Genome: Implications of Adaptation to a Sessile Lifestyle[END_REF]. From this microbe-rich environment, bivalves are colonized by a diverse and rich microbiota composed of species from various genera like Vibrio, Pseudomonas, Acinetobacter, Photobacterium, Moraxella, Aeromonas, Micrococcus and Bacillus [START_REF] Romalde | New Vibrio Species Associated to Molluscan Microbiota: A Review[END_REF]. This composition has been determined by cultureindependent methods based on the sequencing of the 16S rRNA gene in oysters, clams and mussels showing that the gut microbiota possesses a community composition that differs from that of the proximal water column (Lokmer et al., 2016b). In the oyster, where other tissues such as the gills, mantle, muscle and hemolymph have been studied, the latter is characterized by a similar evenness but higher species richness than the solid-tissues. Despite this apparent selectivity imposed by the host to microbial assembly, the existence of a hostspecific microbiota in oysters is still debated as its composition varies according to the seasonal and local variations of the environmental microflora (Schmitt et al., 2012b).

Moreover, depuration in UV-treated water decreases the concentration of some vibrio species like V. vulnificus in oysters [START_REF] Tamplin | Persistence of Vibrio vulnificus in tissues of Gulf Coast oysters, Crassostrea virginica, exposed to seawater disinfected with UV light[END_REF], while other vibrio species resist to this treatment in other edible bivalves [START_REF] Pruzzo | Persistence of Vibrios in Marine Bivalves: The Role of Interactions with Haemolymph Components[END_REF][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], which suggests that they could belong to a persistent microbiota 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]. Paradoxically, vibrios that are able to enter the circulatory system of bivalves, coexist in the invertebrate analog of blood, the hemolymph, with immunocompetent cells and soluble antimicrobial effectors which through complex mechanisms of recognition and regulation, are able to tolerate the presence of a diversity of bacterial groups while limiting the proliferation and infection of potentially pathogenic bacteria [START_REF] Bachère | The New Insights into the Oyster Antimicrobial Defense: Cellular, Molecular and Genetic View[END_REF].

Vibrios include opportunistic pathogens which under certain conditions proliferate and invade deeper tissues causing systemic infection (Lemire et al., 2015;Bruto et al., 2017).

Mortality outbreaks associated with several vibrio species have been registered in cultivated bivalves such as oyster, clams and mussels (Saulnier et al., 2010;[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]Green et al., 2019). Susceptibility to vibrio infection is not homogeneous among bivalves. While oysters are frequently affected by recurrent and seasonal vibriosis, few cases of mortality outbreaks are reported for mussels (Gestal et al., 2008;Watermann et al., 2008) despite that, just like in oysters, vibrios concentrate on their tissues [START_REF] Stabili | Mytilus Galloprovincialis Filter Feeding on the Bacterial Community in a Mediterranean Coastal Area (Northern Ionian Sea, Italy)[END_REF]. More recently a Vibrio strain (10/068) associated with one of these outbreaks was isolated and used to evaluate the response to infection mussel, which previously had been only evaluated with strains pathogenic to the oyster. Vibrio 10/068 was shown to be rapidly internalized by the phagocytes of the mussel but it inhibited defenserelated functions such as ROS production through the secretion of extracellular products that still remain to be characterized [START_REF] Ben Cheikh | First Evidence for a Vibrio strain Pathogenic to Mytilus edulis Altering Hemocyte Immune Capacities[END_REF].

A thorough analysis of vibrio population structure between the oyster tissues and the water column was made by comparing the frequency and abundance of defined vibrio population between these two main compartments in the spring and summer season. This allowed to define first, that some vibrio populations are preferentially associated with the oyster tissues in the spring seasons in absence of juvenile mortalities and second, that the vibrio population structure in both the water column and the oyster is highly defined by the season with V. crassostreae particularly abundant in diseased animals in summer showing a clear correlation of pathogenicity with the presence of a large mobilizable plasmid (Bruto et al., 2017).

I.5 VIBRIO ECO-EVOLUTIONARY DYNAMICS

It is typically assumed that evolution and population dynamics are processes that occur at such different timescales that they are considered as de-coupled, nevertheless growing evidence has shown that rapid changes in allelic frequencies within microbial population can in fact cause noticeable ecological changes establishing an eco-evolutionary dynamics [START_REF] Bohannan | Linking Genetic Change to Community Evolution: Insights From Studies of Bacteria and Bacteriophage[END_REF][START_REF] Sanchez | Feedback Between Population and Evolutionary Dynamics Determines the Fate of Social Microbial Populations[END_REF].

We have discussed so far, the diversity of interactions of vibrios within the environment. In this dynamics, biotic and abiotic factors in the environment shape the evolution of vibrio populations by selecting genomic events (deletion, duplication or horizontal gene transfer), thus generating phenotypic diversity [START_REF] Le Roux | Eco-evolutionary Dynamics Linked to Horizontal Gene Transfer in Vibrios[END_REF].

Among the events that contribute to genomic variation in vibrio, intensive horizontal gene transfer has ben described has a major player in the eco-evolutionary dynamics of vibrios, driving niche adaptation and speciation [START_REF] Le Roux | Eco-evolutionary Dynamics Linked to Horizontal Gene Transfer in Vibrios[END_REF][START_REF] Chibani | Genomic Variation Among Closely Related Vibrio alginolyticus Strains is Located on Mobile Genetic Elements[END_REF].

I.5.1 HORIZONTAL GENE TRANSFER

In an ecological population framework, closely related vibrio strains share a common habitat and gene pool, exchanging genes by horizontal gene transfer at a sufficiently high rate to evolve as gene flow units where gene flow boundaries are strong enough for adaptive genes to spread in a population-specific way [START_REF] Le Roux | Oysters and Vibrios as a Model for Disease Dynamics in Wild Animals[END_REF]. This gene flow boundaries specifically refers to the magnitude of gene flow that occurs between microbial populations and which is mainly determined by the genetic similarity and ecological overlap between the strains that make up those populations [START_REF] Shapiro | Microbial Speciation[END_REF]. Here again, genetics interlinks with ecology as efficiency of homologous recombination of genes acquired by HGT decrease exponentially with sequence divergence but the transfer probability increases with the greater physical contact that characterize strains that share the same habitat [START_REF] Vaninsberghe | How Can Microbial Population Genomics Inform Community Ecology?[END_REF].

Through the study of genome plasticity, it has been demonstrated that the three major and most studied mechanisms of HGT in prokaryotes, namely natural competence for transformation, conjugation and transduction, occur in vibrios. The principal characteristics of these mechanisms are briefly described below.

NATURAL COMPETENCE FOR TRANSFORM ATI ON

This mechanism describes the physiological state in which a DNA-uptake nanomachine enables its uptake from the surroundings. Briefly, it includes a type IV pilus adherence structure, an extendable appendage that binds extracellular DNA, and upon retraction, is thought to open the outer membrane secretin pore from which the extracellular DNA is internalized and pulled into the cytoplasm by a periplasmic DNA-binding protein [START_REF] Blokesch | Natural Competence for Transformation[END_REF]. If the incoming DNA is successfully integrated into the chromosome by homologous recombination the bacterial genome is considered naturally transformed [START_REF] Lo Scrudato | The Regulatory Network of Natural Competence and Transformation of Vibrio cholerae[END_REF][START_REF] Metzger | Regulation of Competence-Mediated Horizontal Gene Transfer in the Natural Habitat of Vibrio cholerae[END_REF][START_REF] Le Roux | Eco-evolutionary Dynamics Linked to Horizontal Gene Transfer in Vibrios[END_REF]. The abundance of the chitinous polysaccharides in the marine environment could favor such HGT events, as chitin can induce the state of natural competence for transformation in several vibrio species (V. cholerae, V. fischeri, V. parahaemolyticus, and V. vulnificus) upon growth on chitin. The characterization of this developmental program in V. cholerae has shown that additional environmental cues such as a high cell density and the absence of catabolite-repressing sugars are necessary for entering the natural competence state [START_REF] Lo Scrudato | The Regulatory Network of Natural Competence and Transformation of Vibrio cholerae[END_REF].

CONJUGATION

Transfer of genetic material by conjugation requires direct cell-to-cell contact and the synthesis of a multiple-protein mating pilus between the donor and recipient bacteria. The acquisition of phage-associated toxins is not restricted to V. cholerae but also occur in other several marine vibrio species such as V. coralliilyticus [START_REF] Weynberg | From Cholera to Corals: Viruses as Drivers of Virulence in a Major Coral Bacterial Pathogen[END_REF], V.

anguillarum [START_REF] Castillo | Comparative Genome Analyses of Vibrio anguillarum Strains Reveal a Link with Pathogenicity Traits[END_REF], V. harveyi [START_REF] Munro | Experimental Bacteriophage-Mediated Virulence in Strains of Vibrio harveyi[END_REF] and V. parahaemolyticus [START_REF] Nasu | A Filamentous Phage Associated with Recent Pandemic Vibrio parahaemolyticus O3:K6 Strains[END_REF] suggesting that prophages harbor diverse genetic elements with a large dissemination potential among the Vibrio group. The acquisition of prophage-associated virulence factors in V. parahaemolyticus named above, specifically refers to the emergence of the pandemic strain O3:K6 which has been attributed to a generalized acquisition of virulence factors by HGT. Analysis of pandemic and pre-pandemic isolates suggested that the founder strain,a nonpathogenic O3:K6 initially acquired a new virulence regulator thought the toxRS operon and subsequently at least seven genomic islands [START_REF] Espejo | Insight Into the Origin and Evolution of the Vibrio parahaemolyticus Pandemic Strain[END_REF] and the 80-kb pathogenicity island Vp-PAI [START_REF] Izutsu | Comparative Genomic Analysis Using Microarray Demonstrates a Strong Correlation Between the Presence of the 80-Kilobase Pathogenicity Island and Pathogenicity in Kanagawa Phenomenon-Positive Vibrio parahemolyticus Strains[END_REF].

SEROCONVERSION BASED ON O-ANTIGEN CLUSTER EXCHANGE

Essential membrane determinants can be affected by horizontal exchange of genes. V.

parahaemolyticus serotype O3:06 underwent rapid changes in the O-antigen (O) and capsular antigen (K) after its transcontinental spreading, resulting in more than 20 serovariants [START_REF] Chen | Genetic Analysis of the Capsule Polysaccharide (K antigen) and Exopolysaccharide Genes in Pandemic Vibrio parahaemolyticus O3:K6[END_REF]. This O and K antigen correspond to two types of polysaccharides that decorate the outer membrane of Gram-negative bacteria. The K or capsular antigen is composed of high molecular weight polysaccharides that form a loosely attached coat outside the bacterial cell contributing to the resistance to phagocytosis and to the complement system [START_REF] Chen | Genetic Analysis of the Capsule Polysaccharide (K antigen) and Exopolysaccharide Genes in Pandemic Vibrio parahaemolyticus O3:K6[END_REF]. In contrast, the O-antigen constitutes the distal component of the lipopolysaccharide and is composed of up to 50 repeating units of two to eight sugars [START_REF] Wildschutte | O-Antigen Diversity and Lateral Transfer of the wbe Region Among Vibrio splendidus Isolates[END_REF]. From the three structural domains of LPS (Lipid A, coreoligosaccharide and O-antigen) the O-antigen is the most variable, with diverse monosaccharides composition, linkage and number of these repeating units (Figure 6B). The high phenotypic diversity observed in the O-antigen structure is the product of the hypervariability of the wb* region which was discovered to encode the products responsible for its synthesis in V.cholerae O1(wbe) and later in the seroconverted O139 strain (wbf) which emerged in 1992 and caused cholera outbreaks in India and Bangladesh and spread through Asia replacing the ancestral O1 El Tor strain in the environment [START_REF] Popovic | Molecular Ubtyping of Toxigenic Vibrio cholerae o139 Causing Epidemic Cholera in India and Bangladesh, 1992-1993[END_REF].

Comparative genomics between vibrios with different O-antigen structures showed that shared genes differ based on mutations, while non-homologous genes are the result of horizontal transfer [START_REF] Wildschutte | O-Antigen Diversity and Lateral Transfer of the wbe Region Among Vibrio splendidus Isolates[END_REF]. The wb* region presents a cassette-like organization with conserved genes (gmhD and rjg) flanking the variable region [START_REF] Sozhamannan | Cloning and Sequencing of the Genes Downstream of the wbf Gene Cluster of Vibrio cholerae Serogroup O139 and Analysis of the Junction Genes in Other Serogroups[END_REF]. Therefore, fragment acquisition by natural transformation, followed by homologous recombination has been proposed as a possible mechanism of lateral transfer of O-antigen synthesis genes occurring in the aquatic environment, since through the induction by chitin, it can mediate the seroconversion of V. cholerae O1 through a serogroup specific gene cluster exchange with the V. cholerae O139 strain [START_REF] Blokesch | Serogroup Conversion of Vibrio cholerae in Aquatic Reservoirs[END_REF]. The acquisition of this gene cluster, which encodes for a novel O-antigen structure, and the ability to synthesize a capsular polysaccharide, confers resistance to bacteriolysis by a O1-specific bacteriophage, suggesting that it simultaneously granted the ability to the O193 strain to infect individuals already immunized with the O1 strain and an advantage for its persistence in aquatic habitat that could explain its rapid environmental dominance after its emergence [START_REF] Blokesch | Serogroup Conversion of Vibrio cholerae in Aquatic Reservoirs[END_REF].

Divergence in O-antigen structure is not restricted to pathogenic species of human health interest, like V. cholerae but it has also been characterized in a collection of coastal V.

splendidus isolates collected from diverse microhabitats [START_REF] Wildschutte | O-Antigen Diversity and Lateral Transfer of the wbe Region Among Vibrio splendidus Isolates[END_REF]. In this work, genetic diversity of the wbe region correlated with phenotypic variation in the O-antigen structure. As the O-antigen structure is at the interphase between the vibrio and its environment, it is therefore subjected to different types of selective pressures characteristic of each of the diverse associations that vibrio establish, ranging from the predation activity of bacteriophages and protist grazers to the reactivity of the innate defenses of metazoan hosts. In consequence, the ability to diversify the O-antigen structure trough HGT may offer fitness advantages in specific environments with possible tradeoffs due to conflicting selective pressures targeting O-antigen structure. As an example, Salmonella enterica is able to epigenetically control the length of its O-antigen conferring resistance to bacteriophage predation at the expense of a transient reduction in pathogenicity [START_REF] Cota | Epigenetic Control of Salmonella enterica O-Antigen Chain Length: A Tradeoff between Virulence and Bacteriophage Resistance[END_REF].

MULTIDRUG RESISTANCE

Mobile genetic elements are also major drivers in the emergence of multidrug resistant strains (MDR). MDR has been identified in more than 99% of recent V. cholerae isolates from stool samples [START_REF] Verma | Genomic Plasticity Associated with Antimicrobial Resistance in Vibrio cholerae[END_REF], identified in environmental non-O1/non-O139 strains [START_REF] Lepuschitz | Phenotypic and Genotypic Antimicrobial Resistance Traits of Vibrio cholerae Non-O1/Non-O139 Isolated From a Large Austrian Lake Frequently Associated With Cases of Human Infection[END_REF]. MDR is also found in Vibrio spp isolated from reared fish [START_REF] Scarano | Antibiotic Resistance of Vibrio Species Isolated from Sparus aurata Reared in Italian Mariculture[END_REF][START_REF] Deng | Horizontal Gene Transfer Contributes to Virulence and Antibiotic Resistance of Vibrio harveyi 345 Based on Complete Genome Sequence Analysis[END_REF][START_REF] Mohamad | Virulence-Associated Genes and Antibiotic Resistance Patterns of Vibrio spp. Isolated from Cultured Marine Fishes in Malaysia[END_REF], and wild and farmed mollusks [START_REF] Banerjee | Trend and Pattern of Antimicrobial Resistance In Molluscan Vibrio species Sourced to Canadian Estuaries[END_REF]. MDR can be achieved through the reduction of the intracellular concentration of the antibiotic, through reduced permeability or active drug efflux, through the alteration or the protection of the target site, or through the direct inactivation of the antibiotic molecule.

These mechanisms can be acquired through vertical gene transfer but also disseminated to more distantly related species through HGT [START_REF] Verma | Genomic Plasticity Associated with Antimicrobial Resistance in Vibrio cholerae[END_REF]. Therefore both, the overuse and misuse of antibiotics for treatment of human infections, but also in the control of diseases in aquaculture, represent a driver for the development of antibiotic resistance in the Vibrio group which is submitted to extensive gene exchange in the environments such as the hatcheries of cultures bivalves [START_REF] Dubert | New Insights into Pathogenic Vibrios Affecting Bivalves in Hatcheries: Present and Future Prospects[END_REF].

In an ecological context, horizontal gene transfer has been also linked to the socially cohesive behavior of ecological populations of Vibrio, where antagonism, due the production of broad-range antibiotics, occur between rather than within populations. The establishment of a network of antibiotic-mediated antagonistic interaction among populations of V. ordalii, V. crassostreae and V. tasmaniensis, showed that within the V. ordalii population collaboration to compete with the other co-occurring populations can occur by the production of broad-range antibiotics only by a subset of the population while the rest of the strains are resistant. As the resistance gene is coded in a different region than the antimicrobial gene it is suggested that only the antibiotic resistant individuals within the populations can acquire the antimicrobial coding gene (Cordero et al., 2012b).

II. COLONIZATION PROCESS IN VIBRIOS, ADAPTABILITY TO THE HOST'S HOSTILE ENVIRONMENT

The transition of vibrios from a free-living or particle-associated state to the colonization of a metazoan capable of non-self-recognition, requires specific adaptations to respond to the new environment. In this process, colonization is a necessary step that precedes proliferation (and eventually infection) of metazoan hosts, which can be aquatic organisms or humans in the case of vibrios. Aside from the well characterized vibrio-squid mutualistic symbiosis, colonization has been mainly characterized in the context of pathogenesis. In C. gigas, studies have mainly focused on factors that determine the virulence potential of strains associated with mortality outbreaks by means of experimental infections by injection.

Nevertheless, high resolution population structure of vibrios has established that certain populations have the ability to transition between environmental and host associations (Bruto et al., 2017), although the mechanisms involved in the transition are mostly unknown.

However, what we know about the colonization process of mutualistic and pathogenic vibrios can serve as a guiding framework to future research on the colonization process of the diverse Vibrio populations that have been recently identified associated with C. gigas.

II.1 RESPONSE TO ENVIRONMENTAL CHANGES

Sensing environmental changes correspond to a key feature that allows vibrios to respond to the dynamic coastal waters where they establish a plethora of different interactions. As examples, responding to environmental and host cues is essential in the transition from an aquatic of V. cholerae to a human habitat and of V. vulnificus to human serum [START_REF] Williams | Transcriptome Sequencing Reveals the Virulence and Environmental Genetic Programs of Vibrio vulnificus Exposed to Host and Estuarine Conditions[END_REF], and the establishment of V. fischeri as a mutualist of the bobtail squid [START_REF] Visick | Two-Component Sensor Required for Normal Symbiotic Colonization of Euprymna scolopes by Vibrio fischeri[END_REF][START_REF] Hussa | Two-Component Response Regulators of Vibrio fischeri: Identification, Mutagenesis, and Characterization[END_REF]. In simple TCSs, a signaling event leads to the activation of a receptor kinase activity and the autophorylation of a specific histidine residue, this phosphoryl group is subsequently transferred to a conserved aspartate residue of the cognate response regulator (RR) which mediates the cellular response, through DNA binding as well as non-DNA binding activities (Figure 7B) [START_REF] Hussa | Two-Component Response Regulators of Vibrio fischeri: Identification, Mutagenesis, and Characterization[END_REF]. In the facultative mutualist V. fischeri a comprehensive identification and functional characterization, by gene disruption, of 39 putative RRs, identified 12 RRs that are linked to colonization success of the squid's light organ [START_REF] Hussa | Two-Component Response Regulators of Vibrio fischeri: Identification, Mutagenesis, and Characterization[END_REF]. Two-component signal transduction systems have been identified in the Vibrio genus sensing nutrient availability, pH, osmolarity, host factors, and the environment, These systems lead to modulation of motility, bioluminescence, as well as biofilm production and the onset of virulence. [START_REF] Hussa | Two-Component Response Regulators of Vibrio fischeri: Identification, Mutagenesis, and Characterization[END_REF]. The two-component system VarA/VarS is known to regulate viru-lence in V. cholerae and luminescent in V. harveyi but also in the oyster associated pathogens V. aestuarianus, and V. tasmaniensis LGP32. In V. aestuarianus a single mutation in the regulator VarS is sufficient to impact the secretion of the Vam metalloprotease impairing pathogenicity [START_REF] Goudenège | A Single Regulatory Gene is Sufficient to Alter Vibrio aestuarianus Pathogenicity in Oysters[END_REF]. In V. tasmaniensis LGP32 the production of two proteases related to strain virulence and toxicity, Vsm and PrtV, is modulated by a complex regulatory network that involves VarA/VasS signaling, both dependent and independent of CsrB.

Members of the

CsrB are conserved multiple-copy noncoding small RNAs that normally are regulated by the VasA/VasS signaling and mediate changes in gene expression [START_REF] Nguyen | csrB Gene Duplication Drives the Evolution of Redundant Regulatory Pathways Controlling Expression of the Major Toxic Secreted Metalloproteases in Vibrio tasmaniensis LGP32[END_REF]. The analysis of QS sensing network in several Vibrio species (V. fischeri, V. harveyi, V. cholerae, V. anguillarum and V. vulnificus) identified a diversity in the type of signal networks and different regulatory responses that reflects the diversity of environmental roles and kinds of associations within the Vibrio group [START_REF] Milton | Quorum Sensing in Vibrios: Complexity for Diversification[END_REF]. Three different species V. fischeri, V. harveyi and V. cholerae can serve as examples for the role of quorum sensing in Vibrio symbiosis and pathogenesis towards both, animal and human hosts.

The phenomenon of quorum sensing was originally described in V. fischeri and referred to as "autoinduction" due to the capacity of culture media supernatants to induce bioluminescence in freshly inoculated V. fischeri of the same strain [START_REF] Ulrich | One-Component Systems Dominate Signal Transduction in Prokaryotes[END_REF].

Bioluminescence induction, along with early and late colonization factors such as motility and biofilm formation, are critical for the Vibrio-squid symbiosis. Its regulation is achieved in V.

fischeri though three QS systems; LuxI/R, AinS/R and LuxS/Q that form a regulation network essential for colonization of the light organ. While paradigmatic in the QS systems described for Gram-negative bacteria, the LuxI/R system is not present in all vibrios [START_REF] Milton | Quorum Sensing in Vibrios: Complexity for Diversification[END_REF].

V. harveyi is also capable of luminescence and is considered as a common pathogen of diverse marine vertebrate and invertebrate organisms such as fishes, crustaceans and mollusks (Kraxberger-Beatty et al., 1990;[START_REF] Balebona | Role of Extracellular Products in the Pathogenicity of Vibrio strains on Cultured Gilt-head Seabream (Sparus aurata)[END_REF]Saeed, 1995;Hispano et al., 1997;Company et al., 1999;Diggles et al., 2000;Alcaide et al., 2001;Tendencia, 2002). Both luminescence and expression of virulence factors linked to its pathogenicity potential are sensitive to cell density-dependent regulations through a three-channel QS network composed of LuxM/N, LuxS/PQ and CqsA/S each one with its own autoinducer (Figure 10).

Interestingly, in the V. campbelli strain ATCC BAA-1116 (originally classified as V. harveyi), pathogenic for the larvae of the brine shrimp Artemia franciscana, it has been shown that in vitro conditions of high cellular density positively regulates bioluminescence production and synthesis of metalloproteases, siderophores, exopolysaccharides but represses expression of the type III secretion system (Detailed in section II.8 Secretion systems) [START_REF] Milton | Quorum Sensing in Vibrios: Complexity for Diversification[END_REF]. CqsA synthases, autoinduciers accumulate in the cytoplasm to be diffused or transported to the periplasmic region and is believed that they bind to their respective sensors which converts LuxN, LuxQ and CqsS into phosphatases that, leading to the dephosphorylation and inactivation of LuxO and LuxR transcription (modified from [START_REF] Milton | Quorum Sensing in Vibrios: Complexity for Diversification[END_REF] In V. cholerae, QS is at the top of the signaling cascade leading to virulence gene expression.

During the transition to a human host downstream gene expression is regulated through four parallel QS systems (CqsS, LuxPQ, CqsR and VpsS) that converge at LuxO for gene regulation.

At a high cellular density that characterizes the passing through the upper digestive track, biofilm production is induced to increase acid resistance. After a substantial bacterial elimination in the upper gut, V. cholerae arrives to the small intestine and detaches from the biofilm and colonizing the intestine in a low cellular density that activates the expression of CT and TCP. Upon successful colonization and increase in population density, QS modulates the repression of virulence factors and the production of the HA protease that leads to detachment from the epithelium and exiting from the host into the environment [START_REF] Milton | Quorum Sensing in Vibrios: Complexity for Diversification[END_REF][START_REF] Watve | Parallel Quorum-Sensing System in Vibrio cholerae Prevents Signal Interference Inside the Host[END_REF].

II.3 ADHESION MOLECULES AND BIOFILM FORMATION

Planktonic vibrio species are able to explore surfaces thanks to the presence of the haemagglutinin pili (MSHA), Type IV pili (TFP) and the flagellum. Upon recognition of a suitable surface, cells attach to it and the loss of flagellum triggers the production of extracellular matrix substances [START_REF] Rodrigues | Relation Between Biofilm and Virulence in Vibrio tapetis: A Transcriptomic Study[END_REF].

Physical adhesion of bacteria to host cells is considered as the essential step of colonization of an encountered organism, specifically for pathogens, a prerequisite for the delivery of effectors and induction of signaling events facilitate pathogen invasion [START_REF] Stones | Against the Tide: The Role of Bacterial Adhesion in Host Colonization[END_REF]. Attachment is achieved through adhesive appendages such as pili and flagella, and molecules such as chitin-binding proteins and capsular polysaccharides which are widespread among vibrios [START_REF] Ceccarelli | Vibrio species[END_REF].

Vibrio tasmaniensis LGP32, which is a facultative intracellular pathogen, uses the OmpU porin as an adhesin/invasin that binds to the RGD motif of the major plasma opsonin Cg-EcSOD to promote phagocytosis and a β-integrin mediated invasion of the hemocytes of the oyster C.

gigas, a key event in the expression of its pathogenicity in the oyster [START_REF] Duperthuy | Use of OmpU Porins for Attachment and Invasion of Crassostrea gigas Immune Cells by the Oyster Pathogen Vibrio splendidus[END_REF].

The adhesion properties and role in pathogenesis of OmpU were also found in V. cholerae

where OmpU binding to fibronectin was investigated in the context of colonization of mice and rabbit intestinal epithelium [START_REF] Sperandio | The OmpU Outer Membrane Protein, A Potential Adherence Factor of Vibrio cholerae[END_REF][START_REF] N-Nakasone | Characterization of Outer Membrane Protein OmpU of Vibrio cholerae O1[END_REF].

Exopolysaccharides are polymeric carbohydrates that are present on the surface of many bacteria, they form an interbacterial matrix that support biofilm formation. QS systems LuxS/LuxPQ and CqsA/CqsS regulate biofilm formation in several vibrio species such as V.

fischeri, V. harveyi, V. anguillarum, V. cholerae, and V. vulnificus [START_REF] Milton | Quorum Sensing in Vibrios: Complexity for Diversification[END_REF]. They are a preferential life-style in bacteria as it facilitates nutrient access and confers protection from predators, shields bacteria from antimicrobials, [START_REF] Yildiz | Vibrio Biofilms: So Much the Same Yet so Different[END_REF]but also promote gene exchange by HGT specially when interbacterial competition ocurr and free DNA is liberated from prey bacterias (Le Roux and Blokesch, 2018).

II.4 METAL HOMEOSTASIS.

Bacteria have a dichotomic relationship with metals: while metal ions are essential for many reactions in the bacterial metabolism, at high concentrations they can become toxic (Z. [START_REF] Ma | Metal Transporters and Metal Sensors: How Coordination Chemistry Controls Bacterial Metal Homeostasis[END_REF]. As this concentration-dependent toxicity of metals is used as a defense mechanism by the innate defenses of metazoans which have the ability to sequester iron in response to infections, bacteria have evolved mechanism to cope with metal concentration variations and maintain homeostasis mainly by transport of metals from in and out of the cell.

In a metal deprivation state, bacteria increase metal intake, mobilize stored metals and switch to metabolic pathway that are independent of the limiting metal. To face metal excess, bacteria respond by the activation of efflux-systems for detoxification [START_REF] Takemura | Associations and Dynamics of Vibrionaceae in the Environment, From the Genus to the Population Level[END_REF].

Zinc, iron and manganese are considered among the three most important transition metals for bacterial metabolisms and therefore extensive literature is available devoted to characterize sensing and response to changes in ion metal status in model organisms such as Bacillus subtilis. Among mutualistic and pathogenic vibrio, copper and iron homeostasis appears as tightly associated with their colonization capacity [START_REF] Brooks | Global Discovery of Colonization Determinants in the Squid Symbiont Vibrio fischeri[END_REF][START_REF] Vanhove | Copper Homeostasis at the Host Vibrio Interface: Lessons from Intracellular Vibrio Transcriptomics[END_REF][START_REF] Kramer | Bacterial Siderophores in Community and Host Interactions[END_REF] COPPER HOMEOSTASIS AND COLONIZATION SUCCESS

In the oyster facultative intracellular pathogen, V. tasmaniensis LGP32, invasion of the hemocytes, the oyster immune cells, depends on copper homeostasis through the P-type ATPase A, CopA, and to a lower extent the CusABC efflux machinery. CopA is essential for

LGP32 intracellular survival and virulence expression [START_REF] Vanhove | Copper Homeostasis at the Host Vibrio Interface: Lessons from Intracellular Vibrio Transcriptomics[END_REF].

Similarly, in V. fischeri, the global discovery of determinants of symbiosis through a transposon insertion strategy, allowed to identify two genes related to copper efflux systems (cusC and copA) as implicated in the colonization success of V. fischeri. Interestingly, oxygen arrives to the crypts of the light organ carried by hemocyanin, a metalloprotein that coordinates copper ions to reversibly bind to oxygen. If this copper coordination is conserved in E.

scolopes, as predicted [START_REF] Brooks | Global Discovery of Colonization Determinants in the Squid Symbiont Vibrio fischeri[END_REF], it could explain the copper presence in the crypts and therefore the relevance of copper detoxification in the persistence of V. fischeri in the light organ.

IRON ACQUISI TION

Iron plays an essential role in bacterial metabolism as a cofactor in the catalysis of redox reactions that are part of vital processes like respiration, DNA synthesis and detoxification of reactive oxygen species. Despite its abundance, many natural environments are characterized by neutral and basic levels of pH at which the predominant oxidized ferric (Fe 3+ ) state of iron is highly insoluble making it not readily bioavailable for bacterial assimilations [START_REF] Kramer | Bacterial Siderophores in Community and Host Interactions[END_REF]. Its dissolved concentration in ocean open waters can reach as low as 20 to 30 pM, well below the 0.1 to 0.5 μM range for standard optimal growth under laboratory conditions [START_REF] P-Payne | Vibrio Iron Transport: Evolutionary Adaptation to Life in Multiple Environments[END_REF]. Moreover, bacteria not only have to face iron scarcity in the environment but also during colonization of metazoan hosts that present levels of freely available iron under the minimum required for bacterial growth, with most of it associated with high affinity iron binding proteins. As iron withholding capacity is induced under infection it is considered as a form of nutritional immunity in response to pathogen invasion [START_REF] Hood | Nutritional Immunity: Transition Metals at the Pathogen-Host Interface[END_REF].

The model for iron acquisition in vibrios is V. cholerae with more than 1% of its genome dedicated to iron transport systems [START_REF] P-Payne | Vibrio Iron Transport: Evolutionary Adaptation to Life in Multiple Environments[END_REF]. While vibrios possess transporters for ferric (Fe 3+ ) or ferrous (Fe 2+ ) iron and also for iron complexes with organic molecules such as citrate or heme, they also synthesize small high-affinity iron chelators, called siderophores, as well as their cognate receptors and transport proteins (Figure 11). The Ferric uptake regulator fur is the transcription factor that represses siderophore synthesis in presence of Fe 2+ (Troxell and Hassan, 2013). Once secreted, siderophores scavenge iron either freely in the extracellular medium or bound to the vibrio surface. Upon iron binding, the ferrisiderophore complex is recognized by its cognate receptor and transported across the outer membrane into the periplasm in an energy-dependent manner which is powered by the coupling of the receptor with the inner membrane complex TonB-ExbB-ExbD. In the periplasm, the iron complex bound to a periplasmic binding protein is transferred to the cytoplasm by less specific membrane permeases. Finally, iron reduction or enzymatic cleavage release iron from the siderophore making it available to the cell. Thus, the successful fight for iron through the synthesis of siderophores is identified as a virulence determinant in vibrios including V. parahaemolyticus, V. harveyi, and V. alginolyticus.

Interestingly, in a community of natural bacterioplankton populations of vibrios the distribution of siderophore was identified as unevenly distributed across isolates. Only clusters of strains associated with animal hosts present a high frequency of siderophore producers, which is expected because of iron-limiting environment, that host represent as part of their infectionlimiting strategies [START_REF] Hood | Nutritional Immunity: Transition Metals at the Pathogen-Host Interface[END_REF]. Despite that siderophore and receptor synthesis genes are often co-localized in large clusters, non-producers showed to evolved by a selective loss of only the biosynthesis genes but retention of the associated receptor allowing a cheating behavior for iron acquisition (Cordero et al., 2012a).

This social interaction based on "public goods" shows to be better supported in larger particle sizes of organic matter that offer a more stable community structure, allowing the emergence of dependence through selective gene loss. Whether this social interaction between producers and cheaters can occur during host colonization remains to be explored.

II.5 EVASION AND RESISTANCE TO PHAGOCYTOSIS

On its way to establishment on either vertebrate or invertebrate hosts, bacteria will inevitably encounter specialized phagocytic cells armed with diverse antimicrobial effectors to kill invaders inside the phagolysosome, where an oxidative and degradative milieu is configured upon microbe internalization.

To evade control by phaogocytes, bacteria have evolved mechanisms to interfere with this process at any step between recognition and phagosome maturation, or even physical scape from the phagosome. Phagocytosis evasion at different steps has been mainly characterized on relevant extra and intracellular human pathogens such as S. aureus, M. In human infection by V. vulnificus and V. cholerae, the pathogens were shown to directly suppress phagocytes through cytotoxicity. Apoptosis of murine macrophages was evidenced to be induced by V. vulnificus although the underlying mechanisms were not explored [START_REF] K-Kashimoto | Vibrio vulnificus Induces Macrophage Apoptosis In Vitro and In Vivo[END_REF] while in a more recent article the secretion of a RTX toxin shown to confer protection from phagocytosis enhancing pathogen survival [START_REF] Lo | RTX Toxin Enhances the Survival of Vibrio vulnificus During Infection by Protecting the Organism from Phagocytosis[END_REF].

Clinical and environmental isolates of V. cholerae contain a T6SS considered among the group of accessory virulent factor whose contribution to cholera disease is not fully understood and roles for environmental fitness are proposed. Vibrio internalization by phagocytic cells is required for the T6SS-dependent secretion of VgrG-1 protein into the cytosol. This VgrG-1 contains an Actin Crosslinking Domain (ACD) that cross-links G-actin causing F-actin depolymerization and leading to an irreversible cytoskeleton compromise and deactivated phagocytic capacity (A. T. [START_REF] Ma | Metal Transporters and Metal Sensors: How Coordination Chemistry Controls Bacterial Metal Homeostasis[END_REF].

In V. anguillarum the O-antigen of the LPS prevents phagocytosis by the skin epithelial cells of the rainbow trout protecting bacteria during the early stages of skin infection [START_REF] Lindell | Lipopolysaccharide O-antigen Prevents Phagocytosis of Vibrio anguillarum by Rainbow Trout (Oncorhynchus mykiss) Skin Epithelial Cells[END_REF].

In C. gigas the extracellular products (ECPs)of V. aestuarianus 01/32 are lethal when injected into oysters and cause immunosuppression by causing morphological changes in hemocytes that inhibit phagocytosis (Labreuche et al., 2006a;Labreuche et al., 2006b). It was demonstrated that the Vam zinc metalloprotease constitutes a major contributor to the impairment of the cellular functions caused by the ECPs of V. aestuarianus [START_REF] Labreuche | Vibrio aestuarianus Zinc Metalloprotease Causes Lethality in the Pacific Oyster Crassostrea gigas and Impairs the Host Cellular Immune Defenses[END_REF].

A more detailed characterization of phagocyte manipulation by vibrios has been produced in the study of V. tasmaniensis LGP32, a facultative intracellular pathogen of juvenile oysters.

After entrance into the circulatory system, LGP32 is opsonized in the oyster hemolymph by the protein Cg-EcSOD which binds to the OmpU porin. The formed complex is recognized by hemocyte β-integrins, which mediate vibrio internalization. Inside the hemocytes LGP32 resist degradation through the a combined inhibition of vacuole acidification, limiting of ROS production, antioxidant activity, copper efflux and resistance to AMPs (Figure 12) [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] V-Vanhove | Outer Membrane Vesicles are Vehicles for the Delivery of Vibrio tasmaniensis Virulence Factors to Oyster Immune Cells[END_REF][START_REF] Vanhove | Copper Homeostasis at the Host Vibrio Interface: Lessons from Intracellular Vibrio Transcriptomics[END_REF]. 4) inhibition of phagosome acidification [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] V-Vanhove | Outer Membrane Vesicles are Vehicles for the Delivery of Vibrio tasmaniensis Virulence Factors to Oyster Immune Cells[END_REF][START_REF] Vanhove | Copper Homeostasis at the Host Vibrio Interface: Lessons from Intracellular Vibrio Transcriptomics[END_REF]. (modified from [START_REF] Duperthuy | Effecteurs moléculaires de lassociation Crassostrea gigas / Vibrio splendidus. Rôle de la porine OmpU dans les mécanismes de résistance et déchappement à la réponse immunitaire de lhôte[END_REF] II.6 RESISTANCE TO HOST ANTIMICROBIALS Several strategies have been described in colonizing vibrios that allows them to cope with the presence of the diverse antimicrobial effectors that are produced by their host to control the proliferation of resident and invasive microbes.

Among bactericidal effectors, antimicrobial peptides are a diverse group that mostly comprises cationic molecules with the common characteristic of being able to interact electrostatically with the bacterial surface and insert into the membrane bilayer forming deleterious pores, binding to essential components of the membrane or translocate into the cytoplasm where they can interfere in vital cellular processes (Schmitt et al., 2012b).

The initial electrostatic interaction of AMPs in gram negative bacteria, like vibrios, is possible thanks to the negatively-charged LPS that constitutes a major and essential component of outer membrane. The hydrophobic domain of LPS named Lipid A (Figure 6) anchors the molecule to the membrane and confers the negative charge to the molecule [START_REF] Steimle | Structure and Function: Lipid A Modifications in Commensals and Pathogens[END_REF]. A strategy used by bacteria to impair the initial interaction with cationic antimicrobial peptides is to lower the net negative charge of the membrane by modifying the Lipid A. In some strains of V. cholerae resistant to AMPs, the VprA/B two component system regulates the substitution of Lipid A with glycine or diglycine residues, altering the bacterial surface charge, upon the sensing of host cues such as bile, mildly acidic pH and cationic antimicrobial peptides. Both the VprA/B and the almEFG operon that encodes genes for glycine substitution are necessary for host colonization [START_REF] Herrera | The Vibrio cholerae VprA-VprB Two-Component System Controls Virulence Through Endotoxin Modification[END_REF]. This strategy is also employed by V. fischeri which is able to sense the acidic pH experienced in the transition from environmental to host association and trigger the substitution of Lipid A with positively charged ethanolamine residues which confer resistance to polymyxin B, a well-known prokaryotic AMP, and increase colonization of V. fischeri in-vivo [START_REF] Schwartzman | Acidic pH Promotes Lipopolysaccharide Modification and Alters Colonization in a Bacteria-Animal Mutualism[END_REF].

Another protective response against AMPs was described in the oyster pathogen V.

tasmaniensis LGP32, which otherwise lacks the genetic determinants to substitute its Lipid A with glycine or diglycine residues. In response to membrane damage, LGP32 secretes ECPs that contain an insoluble fraction consisting of outer membrane vesicles (OMVs). OMV release was observed both in the extracellular milieu and inside the phagosomes (Figure 13).

These OMVs entrap periplasmic content during extrusion from the cell envelope. In LGP32 they contain a vesicular serine protease (Vsp) that participates in the virulence of LGP32, but does not degrade AMPs. Nonetheless the released OMVs are highly protective against AMPs which are entrapped in the membranes of the released vesicles. It is therefore believed that OMVs can confer protection against AMPs produced by the oyster or by the resident microbiota, thereby conferring a colonization advantage to LGP32 [START_REF] V-Vanhove | Outer Membrane Vesicles are Vehicles for the Delivery of Vibrio tasmaniensis Virulence Factors to Oyster Immune Cells[END_REF]. V. cholerae probably has to face ROS during infection, as cholera patients show a decreased level of antioxidant enzymes and an activation of oxidative stress-related enzymes, but also in the environment after escape from the human host (H. Wang et al., 2017). Two LysR-family transcriptional regulators, OxyR1/2 regulate the expression of genes essential for resistance of V. cholerae to oxidative stress. The firstly identified, OxyR1 activate the expression of peroxiredoxin PrxA, DPS (DNA-binding protein from starved cell) and the catalases KatG and KatB, all of them involved on in-host resistance to oxidative damage in response to H2O2. The later identified OxyR2, regulates the expression of the alkylhydroperoxide reductase gene ahpC in a H202-independent way, which directly regulates OxyR1 and allows V. cholerae to better adapt to the low levels of oxidative stress of the environment after host escape (H. Wang et al., 2017).

V. fischeri is exposed to two different types of oxidative stress on its migration towards the crypts of the light organ. Nitric oxide (NO) is present in toxic concentration in the ciliated fields, ducts and the crypts antechambers and the deep crypts present halide peroxidase that can generate bactericidal hypohalous acid [START_REF] Davidson | NO Means "Yes" in the Squid-Vibrio Symbiosis: Nitric oxide (NO) During the Initial Stages of a Beneficial Association[END_REF][START_REF] Nyholm | The Winnowing: Establishing the Squid -Vibrios Symbiosis[END_REF]. The daily exudate that is expelled from the light organ containing bacterial symbionts and host cells was used to characterize the proteomic characteristic linked to this association. The study revealed the presence of detoxification proteins (eg. alkyl hydroperoxide reductase, C22, thioredoxin reductase, hydroperoxidase KatA, FeSOD, thioredoxin-dependent thiol peroxidase, nitric oxide dioxygenase, thioredoxin peroxidase) for ROS and RNS supporting the role of oxidative stress protection among the determinants factors that ensure the symbiotic persistence of V. fischeri in the bob-tail squid [START_REF] Schleicher | Characterizing the Host and Symbiont Proteomes in the Association Between the Bobtail Squid, Euprymna scolopes, and the bacterium, Vibrio fischeri[END_REF].

Vibrios adopting intracellular stages experience strong oxidative stress. This was shown by a transcriptomic analysis of the intracellular stages of the oyster pathogen V. tasmaniensis

LGP32, which survives into the oyster phagocytes. Inside the hemocytes, LGP32 undergoes a significant gene expression reprograming. Among the overexpressed genes, superoxide dismutase [Mn] (sodA) and alkyl hydroperoxide reductase C (ahpC) were associated with oxidative stress resistance. Deletion mutants confirmed that MnSOD (sodA), which catalyzes the conversion of superoxide (O2 -. ) to hydroperoxide (H2O2) and dioxygen (O2), was essential for LGP32 survival in and cytotoxicity to oyster phagocytes [START_REF] Vanhove | Copper Homeostasis at the Host Vibrio Interface: Lessons from Intracellular Vibrio Transcriptomics[END_REF] II.8 SECRETION SYSTEMS Transport of proteins between compartments, into the environment or to other bacteria is an essential function in prokaryotic cells. Dedicated bacterial secretion systems assist this transfer of cargo proteins and are essential for growth of bacteria but also implicated on interbacterial competition or virulence against eukaryotic cells when toxic effectors are the cargo proteins that are delivered through the secretion system. Different classes of secretion systems have been identified that are exclusive of gram-negative bacteria, that assist the translocation of protein substrates across 1, 2 or 3 membranes, depending on the apparatus (Figure 14). Not going further from the model of Vibrio pathogenesis, the cholera toxin is secreted through a type II secretion system (T2SS) along with several degradative enzymes such as chitinases, proteases, lipases/esterases, phospholipases, nucleases, pilin and matrix proteins for biofilm formation [START_REF] Johnson | The Type II Secretion System Delivers Matrix Proteins for Biofilm Formation by Vibrio cholerae[END_REF]. In the transient life-styles of V. cholerae between a mammalian host and the aquatic environment, adhesion to mucins covering epithelial cells and chitinous surfaces is facilitated by the secretion of the N-acetyl-D-glucosamine binding protein (GbpA), therefore, considered a broad-host colonization factor [START_REF] Sikora | Proteins Secreted Via the Type II Secretion System: Smart Strategies of Vibrio cholerae to Maintain Fitness in Different Ecological Niches[END_REF]. The zinc metalloprotein HapA displays proteolytic activity that could facilitate obtaining nutrients in different environmental contexts but also a determinant factor for the detachment of V.

cholerae from the intestinal cell layer and the successful exit from the host [START_REF] Silva | Haemagglutinin/Protease Expression and Mucin Gel Penetration in El Tor Biotype Vibrio cholerae[END_REF].

Expression of T2SS has been, until recently suggested to be constitutive; however, it is now known to be regulated by the high concentration of the bacterial second messenger cyclic diguanylate monophosphate (c-di-GMP), which in turn is regulated by QS and related to high cell density. Remarkably, overexpression of T2SS was not related with higher toxin secretion or biofilm formation [START_REF] Sloup | Cyclic Di-GMP and VpsR Induce the Expression of Type II Secretion in Vibrio cholerae[END_REF]. Taken all together secretion of T2SS effectors appears to strongly contribute to the fitness of V. cholerae in different ecological niches [START_REF] Sikora | Proteins Secreted Via the Type II Secretion System: Smart Strategies of Vibrio cholerae to Maintain Fitness in Different Ecological Niches[END_REF].

Type III secretion (T3SS) systems have been identified in several Vibrio species. First identified in V. parahaemolyticus, two T3SS gene clusters (T3SS1 and T3SS2), one on each chromosome were discovered [START_REF] Makino | Genome Sequence of Vibrio parahaemolyticus: A Pathogenic Mechanism Distinct from that of V cholerae[END_REF], with T3SS1 found in all strains while T3SS2 was restricted to KP-positive strains (hemolysis of blood-agar produced by the presence of the thermostable direct haemolysin). Subsequently T3SS clusters where identified in non-O1/non-O139 and nontoxigenic O1 V. cholerae, V. alginolyticus, V. mimicus, V. harveyi and V. tubiashii. Several cytotoxic effectors have been characterized to be secreted in a T3SS-dependent manner.

Among them, T3SS1 secreted toxins identified are VopQ, VopR, VopS, VP1659 and VPA0450

which have been characterized to induce autophagy, cell rounding, cell lysis and cell death in eukaryotic cells [START_REF] Whitaker | The Vibrio parahaemolyticus ToxRS Regulator is Required for Stress Tolerance and Colonization in a Novel Orogastric Streptomycin-Induced Adult Murine Model[END_REF].

V. campbelli strain BB120 (originally assigned to the species V. harveyi) posseses one T3SS locus, whose expression and transcriptional control has been investigated in vitro using nutrient rich synthetic growth media and in-vivo during infection of gnotobiotic brine shrimp larvae of Artemia franciscana [START_REF] Ruwandeepika | Quorum Sensing Regulation of Virulence Gene Expression in Vibrio harveyi In Vitro and In Vivo During Infection of Gnotobiotic Brine Shrimp Larvae[END_REF]. QS regulation at high cell density was shown to inhibit the expression of T3SS genes in vitro (Figure 10) [START_REF] Henke | Quorum Sensing Regulates Type III Secretion in[END_REF].

T3SS expression is regulated by QS through the master regulators LuxR and AphA. Both are repressors of the expression of T3SS, but while LuxR transcription is upregulated by high cell density, AphA transcription is repressed under this condition and induced al low cellular densities [START_REF] Ruwandeepika | Quorum Sensing Regulation of Virulence Gene Expression in Vibrio harveyi In Vitro and In Vivo During Infection of Gnotobiotic Brine Shrimp Larvae[END_REF], Using an in-vivo model of infection, Ruwandeepike et al., (2011) showed that in-vivo levels of expression at low and high cellular density are more similar between them and <1000 fold higher than in the in vitro conditions, suggesting that additional host cues are integrated in the regulation of the T3SS during infection to only express this energy consuming mechanism when there is a host present. It is proposed that the repression by LuxR at higher cellular densities helps to silence the expression of T3SS inside biofilms in absence of a host and the repression by AphA to silence T3SS expression at low cellular densities when bacteria are in planktonic life style.

The Type 6 secretion system was discovered in the interaction of V. cholerae with the model amoeba Dictyostellium discoideum. D. discoideum is used as a model of eukaryotic cell that mimics mammalian macrophages to study several environmental pathogens that display mechanisms of killing or intracellular resistance to amoeba and mammalian cells. Here, mechanism of cytotoxicity dependent on a cell to cell contact is associated with the presence of gene cluster coding for a new secretion system that was named type 6 secretion system in relation to the previously characterized microbial secretion systems [START_REF] Pukatzki | Identification of a Conserved Bacterial Protein Secretion System in Vibrio cholerae Using the Dictyostelium Host Model System[END_REF].

The T6SS is a syringe-like molecular nanomachine consisting of two main complexes: a membrane-associated complex that include elements homologue to the type IV secretion system and a second complex that structurally resemble a bacteriophage sheath, tube and tail spike proteins that work together to translocate effector proteins across the membranes of the donor cell and through the outer membrane of the recipient cell [START_REF] Russell | Type VI Secretion System Effectors: Poisons with a Purpose[END_REF].

These secreted effector proteins are recruited to the spike-tube complex through the VgrG Since its discovery T6SS-encoding genes have been identified over a fourth of sequenced Gram-negative genomes and while virulence-associated functions have been characterized linked to the secretion of toxic effectors, the discovery of T6SSs in non-pathogenic or even mutualistic bacteria has highlighted roles for this secretion system different from virulence towards eukaryotic hosts [START_REF] Jani | Type VI Secretion: Not Just for Pathogenesis Anymore[END_REF]. Anti-inflammatory (Chow and Mazmanian, 2010), interbacterial cooperation [START_REF] Konovalova | Extracellular Biology of Myxococcus xanthus[END_REF] and competition are among the novel functions associated with T6SS, with the latter possibly been specially relevant, as most of the secreted effectors discovered have antibacterial activity [START_REF] Fridman | A Modular Effector with a DNase Domain and a Marker for T6SS Substrates[END_REF].

Cytotoxic activity towards eukaryotic cells has been mainly attributed to the presence of a Cterminal actin crosslinking domain in the VgrG-1 protein that is required both for T6SSdependent secretion but also secreted through the system by only internalized bacteria. The with the ACD not required for secretion but necessary for the occurrence of T6SS-dependent phenotypes (A. T. [START_REF] Ma | Metal Transporters and Metal Sensors: How Coordination Chemistry Controls Bacterial Metal Homeostasis[END_REF]. Moreover, the induction of T6SS-encoding genes after bacterial uptake by host cells has been observed in several other species such as Burkholderia pseudomallei, B. cenocepacia and Salmonella enterica serovar Typhimurium [START_REF] Jani | Type VI Secretion: Not Just for Pathogenesis Anymore[END_REF].

T6SS-mediated antagonistic interaction between bacteria are linked to the presence of toxinantitoxin or effector-immunity pairs modules constituted by effector proteins reported to mainly target cell wall components (muramidases and glycoside hydrolases), cellular membranes (lipases) or nucleic acids (nucleases) and a cognate immunity protein that prevent self-intoxication of the toxin-producing cell [START_REF] Yang | Effector-Immunity Pairs Provide the T6SS Nanomachine its Offensive and Defensive Capabilities[END_REF].

The relevance of the interbacterial competition in the final step of colonization, that is establishment, has been exemplified above in the examples of V. vulnificus T6SS1-positive strains able to kill T6SS-negative strains within the oyster and the contribution of a T6SS2 to the successful colonization of the light organ by specific strains of V. fischeri.

II.9 OPPORTUNISM, ENVIRONMENTAL INTERACTIONS AND SELECTION OF VIRULENCE TRAITS

As described above, vibrios can use a variety of strategies to overcome host defenses favoring colonization of new hosts. As opportunists, they interact in the environment with a diversity of biotic and abiotic components that could participate to the selection of traits that will provide advantages for colonizing animals.

RESISTANCE TO PHAGES

Bacterial mortality in the environment is rarely caused by a lack of resources, and specially

Vibrios are characterized by having a remarkable ability to survive in oligotrophic conditions and rapidly recover in response to nutrient pulses [START_REF] Pernthaler | Predation on Prokaryotes in the Water Column and its Ecological Implications[END_REF][START_REF] Takemura | Associations and Dynamics of Vibrionaceae in the Environment, From the Genus to the Population Level[END_REF].

Instead, it is considered that activity of bacteriophages and predation by heterotrophic protist are the major causes of mortality in bacterial populations and a key environmental pressure that has promoted the evolution of a number of strategies for resistance to predation, on which it largely depends the survival and persistence of bacteria in the environment [START_REF] Sun | Relative Contributions of Vibrio Polysaccharide and Quorum Sensing to the Resistance of Vibrio cholerae to Predation by Heterotrophic Protists[END_REF].

As reviewed earlier, bacteriophages can contribute to evolution of bacteria by mediating HGT, but also trough bactericidal selection. Membrane receptors, porins, LPS, flagella and pili are components of the cell wall that mediate attachment of bacteriophages to the cell and thus are submitted to important selective preassures [START_REF] Dy | Remarkable Mechanisms in Microbes to Resist Phage Infections[END_REF].

During cholera disease, phages expand in the intestine and are release in stool back in the environment making cholera epidemics self-limiting (Le Roux and Blokesch, 2018), Nevertheless, V. cholerae strains resistant to phage predation have emerged through the modification of the O-antigen structure that serve as receptors for phage adsorption and entrance [START_REF] Knirel | Variations in O-antigen biosynthesis and O-acetylation Associated with Altered Phage Sensitivity in Escherichia coli 4s[END_REF], along with the horizontal acquisition of phage-inducible chromosomal island-like elements that reduces phage replication and accelerates bacterial cell lysis, limiting phage propagation (O'Hara et al., 2017) PREDATION BY ENVIRONMENTAL GRAZERS Grazing activity by phagotrophic protists involves several steps that go from prey encounter, internalization in food vesicles and digestion. In this process, different bacterial species have evolved resistance to predation either by pre-ingestional or post-ingestional strategies to avoid elimination [START_REF] Matz | Off the Hook -How Bacteria Survive Protozoan Grazing[END_REF]. Among pre-ingestional adaptations motility, reduction in size, cell wall structure, morphology, secretion of exopolymers, and cell-cell communication are among the phenotypes described with evidence of providing protection from protist predation. Post-ingestional adaptation include inhibition of vacuolar trafficking and scape from vacuoles together with toxin release.

One remarkable aspect of this predator-prey dynamic is that the phagocytic processes of protist as remained conserved in the immune cells of metazoans at the cellular and molecular level. Therefore, it has been hypothesized that strategies evolved by bacteria to resist protist predation and more precisely survival to intracellular degradation favoring the emergence of intracellular pathogens. Related to our model, C. gigas, it has recently been described that the detoxifying copper p-ATPase CopA [START_REF] Vanhove | Copper Homeostasis at the Host Vibrio Interface: Lessons from Intracellular Vibrio Transcriptomics[END_REF] and the secreted protease Vsm [START_REF] Binesse | Metalloprotease Vsm Is the Major Determinant of Toxicity for Extracellular Products of Vibrio splendidus[END_REF], that are among the virulence factors expressed by the intracellular pathogen of the oyster, V. tasmaniensis LGP32, also confer resistance against grazing by the marine amoeba Vannella sp. This constitute a first insight into an hypothetical environmental selection of virulence factors in the emergence of vibrio pathogenic to the oyster [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]. Use of the newly developed population framework and high-resolution sampling, genetic manipulation tools and readily available NGS technologies, will allow to explore further the contribution of predators in the ecoevolutionary dynamic of vibrio populations.

III. THE PACIFIC OYSTER CRASSOSTREA GIGAS

III.1 THE OYSTER BIOLOGY

The Pacific cupped oyster Crassostrea gigas is a Lophotrochozoan belonging to the phylum Mollusca, class Bivalvia. After a swimming larval stage, juvenile and adult stages are sessile and adopt a benthic life-style showing a settlement preference on firm rocky bottoms of low intertidal to subtidal estuaries, but they can also be found on muddy or sandy bottoms as epibionts of natural beds of other organisms like mussels [START_REF] Diederich | Introduced Pacific Oysters (Crassostrea gigas) in the Northern Wadden Sea: Invasion Accelerated by Warm Summers?[END_REF]. Oyster display a wide range of tolerance for varying environmental conditions such as temperature and salinity [START_REF] Strand | High Tolerance of the Pacific Oyster (Crassostrea gigas, Thunberg) to Low Temperatures[END_REF], particularly adults, which can survive to temperatures between - 2°C and 38°C, and salinity between 15‰ and 41‰ (EFSA. 2015).

The global aquaculture industry has promoted the introduction of this species, originating from Japan, worldwide. It has benefited from remarkable culturing traits like fast growth, important reproductive performances, physiologic adaptations and resistance to diseases like bonamiasis, which affects other oyster farmed species like Ostrea edulis [START_REF] Kennedy | A Survey of the Current Status of the Flat Oyster Ostrea edulis in Strangford Lough, Northern Ireland, with a View to the Restoration of its Oyster Beds[END_REF]. C. gigas was introduced in Europe in the 1970's to mitigate the decrease in stock of the Portuguese oyster Crassostrea angulata caused by an iridovirus [START_REF] Grize | Introduction into France of the Japanese Oyster (Crassostrea gigas)[END_REF]. Similarly C. angulata was previously introduced to France in response to the overexploitation and mortality outbreaks affecting the indigenous flat oyster Ostrea edulis [START_REF] Cognie | Spatial Distribution in a Temperate Coastal Ecosystem of the Wild Stock of the Farmed Oyster Crassostrea gigas (Thunberg)[END_REF]. Despite its subtropical origin, C. gigas has been successfully introduced in regions outside its native range. Nowadays the species shows a near-global distribution and establishes wild populations in the introduction sites due to escape from aquaculture facilities [START_REF] Escapa | The Distribution and Ecological Effects of the Introduced Pacific Oyster Crassostrea gigas (Thunberg, 1793) in Northern Patagonia[END_REF][START_REF] Stagličić | Distribution Patterns of Two Co-Existing Oyster Species in the Northern Adriatic Sea: The Native European Flat Oyster Ostrea edulis and the Non-Native Pacific Oyster Magallana gigas[END_REF].

III.1.1 MORPHOLOGY, ANATOMY AND PHYSIOLOGY

The soft body mass of the oyster is enclosed and protected inside two hinged asymmetric valves composed of calcium carbonate and a proteinaceous matrix, which are joined by the hinge and the adductor muscle [START_REF] Gosling | Morphology of Bivalves[END_REF]. The valves are solid, extensively fluted with the superior valve deeply cupped and concave, and the inferior valve flat or slightly convex. The opening and closing of the shell are controlled by the action of the elastic ligament of the hinge that forces the valves to separate and the contraction of the adductor muscle that brings them together in a hermetic closing, which is essential during periods of emersion (Figure 16A).

The bilateral body of the oyster is divided into 4 sections; the visceral mass, the adductor muscle, the gills and two asymmetric mantle lobes [START_REF] Evseev | The Anatomy of the Pacific Oyster, Crassostrea gigas (Thurnberg)(Bivalvia : Ostreidae)[END_REF] (Figure 13B).

The mantle presents well-developed hemolymph vessels, nerves and muscles, and is responsible for the synthesis of the shell and to perform sensory, particle sorting and oxygen intake functions. The two mantle lobs join at the dorsal zone, they close the hinge and cover the internal body organs enclosing the animal within the shell. The space between these two lobes is called the palleal cavity, a semi-closed space divided by the gills into the entrance and cloacal chamber. The oyster possesses two gills that are curtain-like structures suspended from the ctenidial axis that runs along the dorsal margin of the mantle from the labial palps to the adductor muscle [START_REF] Godfrey | General Anatomy[END_REF]. Each gill is composed of several W-shaped filaments with divided in two V-shaped demibranchs made of one inner descending filament and one outer ascending filament called lamellae. Thanks to the ciliated filaments, the water flow passes from the inhalant chamber ventral to the filament and connected to the inhalant area of the mantle edge to the exhalant chamber, inside each demibranch, which connects with the exhalant area of the mantle edge. From the water current gills assimilate oxygen and move food particles to the labials palps that precede the mouth.

Directly from the mouth, food passes through the esophagus and into the stomach, a twochamber sac-like organ embedded in the digestive gland that connects with the stomach by several ducts that allow a continuous two-way flow where food particles enter the digestive gland for intracellular digestion and absorption and waste leaves to the stomach and towards the intestine to be eliminated as feaces through the anus located next to the adductor muscle.

The gonadal tissue, site of gamete production, is located around the surface of the digestive gland with a structure that varies depending on the specific phase of the reproductive cycle, which is seasonal [START_REF] F-Fabioux | Temperature and Photoperiod Drive Crassostrea gigas Reproductive Internal Clock[END_REF].

The circulatory system of the oyster is considered as semi-open (Schmitt et al., 2012b). The hearth is located inside the pericardic cavity next to the adductor muscle; it pumps the hemolymph through arteries and veins into sinuses that connect through the conjunctive tissue forming the hemocoelic cavity. Here the hemolymph is in direct contact with the cell membrane of the tissues.

The major functions of the hemolymph are related to the transport of dissolved oxygen, in the absence of respiratory pigments, nutrient supply, waste removal and immune defense mainly due to the presence of circulating immune-competent phagocytes, the hemocytes [START_REF] Schmitt | Immune Responses in the Pacific Oyster Crassostrea gigas: An Overview with Focus on Summer Mortalities[END_REF]. oyster showing the organ disposition (modified from Kennedy et al., 1996).

IIII.1.2 REPRODUCTION AND LIFE CYCLE

C. gigas oyster present sequential hermaphroditism, meaning that male and female gametes are produced at different periods of its life. Despite a proposed bias in the primary sex ratio towards males (protandry) has been proposed, female bias (protogyny) can also be found in the literature [START_REF] Broquard | Sex Determination in the Oyster Crassostrea gigas: A large Longitudinal Study of Population Sex Ratios and Individual Sex Changes[END_REF]. Oysters have an annual reproductive cycle where environmental temperature and photoperiod play a determinant role on the regulation of gametogenesis [START_REF] F-Fabioux | Temperature and Photoperiod Drive Crassostrea gigas Reproductive Internal Clock[END_REF] in parallel with the availability and quality of food which fuels this energetically demanding process (Ubertini et al., 2017). As oysters are ectotherm animals (they do not regulate their temperature), the favorable temperature for gamete production is determined by the season [START_REF] F-Fabioux | Temperature and Photoperiod Drive Crassostrea gigas Reproductive Internal Clock[END_REF].

After spawning, gametes are released in the water column where fecundation occurs. The embryo develops into a ciliated trochophore larvae after 6 h and into a D-stage larva at 24h that remains ciliated but has acquired the velum which also participates in mobility. From the 2 nd until the 20 th day, a veliger larva develops, which remains pelagic thanks to the cilia and a full developed velum. Around day 20, the pediveliger larva has developed, starting the transition between the pelagic and benthonic life-style by the development of the foot. The pediveliger larva actively sinks to the bottom and uses the newly developed foot search for a suitable substrate to settle. Once the substrate is selected, the larva attaches and metamorphosis occur involving and internal counterclockwise reorganization of the organs and a switch from aragonite to calcite for shell calcification [START_REF] Baker | Description of Metamorphic Phases in the Oyster Crassostrea virginica and Effects of Hypoxia on Metamorphosis[END_REF][START_REF] Ginger | Larval and Post-Larval Stages of Pacific Oyster (Crassostrea gigas) Are Resistant to Elevated CO2[END_REF]. After metamorphosis the oyster larva enters the juvenile stage until the first spawning cycle that occurs within the first year of age. From there, oysters will develop into adults by only experiencing growth in size (Figure 17). The immune defense of C. gigas, as for all invertebrates, is considered to rely entirely on innate immunity, i.e mechanisms to respond to infection without the development of a lymphocyte mediated immune memory against the re-exposure to a particular antigen [START_REF] Rowley | Invertebrate Immune Systems-Specific, Quasi-Specific, or Nonspecific?[END_REF]. However, oysters are capable of immune memory, a phenomenon known as immune priming, in which prior exposure to a sublethal dose or pathogen-associated material confers protection against a subsequent infection, either within or between generations, the latter being referred to as trans-generational immune priming (Lafont et al., 2017[START_REF] Lafont | Transgenerational Plasticity and Antiviral Immunity in the Pacific Oyster (Crassostrea gigas) Against Ostreid Herpesvirus 1 (OsHV-1)[END_REF][START_REF] Robinson | Fitness Costs Associated with Maternal Immune Priming in the Oyster[END_REF]. This priming capacity, which still requires a mechanistic characterization (the cellular support of the immune memory remains unknown), adds a new complexity layer to the more thoroughly described but not fully understood immune mechanisms and regulatory networks of the oyster defenses.

Functionally oyster immune defenses rely on multiple components, tissues, cellular and molecular effectors. Water intake, which is necessary for feeding and gas exchange, also represents an abundant and continuous source of microorganisms towards exposed epithelial surfaces such as gills and mantle, which are also part of the first line of defense against infection. These epithelia harbor a diverse bacterial community and produce antimicrobial effectors that offer a hostile environment to potential pathogens [START_REF] Bachère | The New Insights into the Oyster Antimicrobial Defense: Cellular, Molecular and Genetic View[END_REF]. Upon breaching of these barriers, bacteria encounter hemocytes, the oyster immune cells that circulate into the hemolymph and infiltrate tissues in response to infection.

In these tissues, both humoral and cellular defenses act together to limit pathogen proliferation and systemic invasion. Given that healthy oysters harbor a microbiota associated with immunocompetent tissues, they must harbor a complex system of recognition and regulations of the immune response to allow the presence of advantageous microorganisms while controlling pathogenic ones in the constant dynamic of maintaining an immune homeostasis. The comprehensive annotation of the oyster genome revealed that the multigene families that make up the immunome are characterized by large-scale gene duplication events and functional divergence, which is suggested to be evolutionary driven by both biotic and abiotic stressors. Such a functional diversification seems determinant to mount highly specific responses towards bacterial, viral or abiotic stressors, mediated by the differential expression of specialized genes within these expanded multigene families (Zhang et al., 2015).

III.1.3.2 EPITHELIA, HEMOLYMPH AND HEMOCYTES

Epithelia of various organs are able to express non-self-recognition receptors and also produce immune effectors with microbicidal activity [START_REF] Bachère | The New Insights into the Oyster Antimicrobial Defense: Cellular, Molecular and Genetic View[END_REF][START_REF] Wang | The Oyster Immunity[END_REF].

For instance, several cDNA sequences identified as peptidoglycan recognition proteins (PGRP) have been identified in the epithelium of the gills, digestive tissue and digestive gland of the oyster [START_REF] Itoh | Distribution of Multiple Peptidoglycan Recognition Proteins in the Tissues of Pacific Oyster, Crassostrea gigas[END_REF] where they specifically binds to peptidoglycan and produce bacterial death by inducing production of ROS, depletion of antioxidant thiols and incrementing the concentration of metal concentration, by overactivating two component systems and exploiting the stress defense response of bacteria (Dziarski et al., 2012, Kashyap et al., 2014). In addition to recognition proteins, epithelial tissues produce antimicrobial effectors such as lysozyme [START_REF] Matsumoto | Cloning of cDNAs and Hybridization Analysis of Lysozymes from Two Oyster Species, Crassostrea gigas and Ostrea edulis[END_REF], Itoh and Takahashi 2007, Xue et al., 2010), hydrolytic enzymes (Itoh andTakahashi 2007, Itoh et al., 2010b) and antimicrobial peptides (summarized in [START_REF] Wang | The Oyster Immunity[END_REF]. The presence of recognition proteins and antimicrobial effectors upport an active role of epithelial surfaces in the defense against invading microorganisms.

Hemolymph, in the immune context, constitutes an important interface of interaction between microorganisms and the immune system [START_REF] Dupont | Oyster Hemolymph is a Complex and Dynamic Ecosystem Hosting Bacteria, Protists and Viruses[END_REF]. Remarkably, it harbors both the hemocytes, i.e the principal cellular mediators of immunity, and an abundant and diverse microbial community [START_REF] Dupont | Oyster Hemolymph is a Complex and Dynamic Ecosystem Hosting Bacteria, Protists and Viruses[END_REF]. Although the hemolymph is devoid of clotting factors, the hemocytes, which display a strong aggregation capacity, perform a reversible cellular cloth in response to wounding (Bachere et al., 1988). The composition of the plasmatic fraction of the hemolymph is dominated by proteins with homologies with a metalloenzyme known as extracellular Superoxide Dismutase (EcSODs).

While it has not been confirmed that all the SOD-related proteins found in the oyster plasma possess a SOD activity, their role in non-self-recognition and opsonization of bacteria have been shown for the Cu/Zn Cg-EcSOD, the major plasma protein found in the oyster [START_REF] Gonzalez | Evidence in Oyster of a Plasma Extracellular Superoxide Dismutase which Binds LPS[END_REF][START_REF] Duperthuy | Use of OmpU Porins for Attachment and Invasion of Crassostrea gigas Immune Cells by the Oyster Pathogen Vibrio splendidus[END_REF].

Hemocytes, the oyster circulating immune cells, are transported by the hemolymph across

the semi-open circulatory system from where they are able to migrate by diapedesis from sinuses through the conjunctive tissue to the surface of, particularly, mantle and gills where they are discarded [START_REF] B-Bachère | Insights into the Anti-microbial Defense of Marine Invertebrates: The Penaeid Shrimps and the Oyster Crassostrea gigas[END_REF][START_REF] Schmitt | Immune Responses in the Pacific Oyster Crassostrea gigas: An Overview with Focus on Summer Mortalities[END_REF]. In addition to their key immune functions described in the following chapters, hemocytes mediate vital functions such as wound and shell repair, gamete reabsorption, nutrient transport, digestion and excretion [START_REF] B-Bachère | Insights into the Anti-microbial Defense of Marine Invertebrates: The Penaeid Shrimps and the Oyster Crassostrea gigas[END_REF]. Upon infection or wounding, hemocytes rapidly migrate to sites of infection through chemotaxis towards a C1q domain containing protein (H. Li et al., 2019). To date there is no consensus on the hematopoietic site and the life-cycle of hemocytes in bivalves has not being established [START_REF] Vogt | Hidden Treasures in Stem Cells of Indeterminately Growing Bilaterian Invertebrates[END_REF]). An origin on the connective tissue (Cheng, 1981), the endothelial cells of vessels and arteries [START_REF] Tirapé | Expression of Immune-Related Genes in the Oyster Crassostrea gigas During Ontogenesis[END_REF] and more recently the tubules of the gill filaments have been proposed for the hemocytes of C. gigas [START_REF] Jemaa | Adult Somatic Progenitor Cells and Hematopoiesis in Oysters[END_REF][START_REF] Li | The Hematopoiesis in Gill and its Role in the Immune Response of Pacific Oyster Crassostrea gigas Against Secondary Challenge with Vibrio splendidus[END_REF]. The proposition of gills as the hematopoietic organ of the oyster is supported by the finding of abundant stem-like cells associated with an irregular folded structure (IFS) in the gill epithelium. These cells were found to be actively going under replication (BrdU-labeled) and positive for the stemness marker SOX-2 and the hemocytespecific enzyme Cu/Zn CgEc-SOD. Associated with this potential hemocyte precursor, the presence of large BruU-labeled cells positives for Cu/Zn CgEc-SOD and the protein filamin specific for hemocytes was considered by the authors as the evidence connecting the replicative activity the stem-like cells and their differentiation into hemocytes. A transcription factors, the Stem Cell Leukemia gene (SCL), having relevant hematopoietic functions in vertebrates, has been found to be highly expressed in hemocytes and necessary for the expression of hemocyte-specific genes [START_REF] Song | Conserved Hemopoietic Transcription Factor Cg-SCL Delineates Hematopoiesis of Pacific Oyster Crassostrea gigas[END_REF].

There is no general consensus in the number of different cell types that compose the cellular fraction of the hemolymph of C. gigas, nonetheless, two clearly differentiable populations of granular and agranular hemocytes are consistently reported in the literature. From this simple categorization two subpopulations of granulocytes (basophils and eosinophils) and three of agranulocytes (blast-like cells, agranular basophils and macrophage-like hyalinocytes) have been proposed. Among them, a particular population behaves as professional phagocytes with strong phagocytic activity [START_REF] Jiang | Functional Characterisation of Phagocytes in the Pacific Oyster Crassostrea gigas[END_REF][START_REF] T-Takahashi | Unique Phagocytic Properties of Hemocytes of Pacific Oyster Crassostrea gigas Against Yeast and Yeast Cell-Wall Derivatives[END_REF]W. Wang et al., 2017). A diversity of MAMPs can be recognized by PRPs and PRRs like peptidoglycan, the major structural polymer of the bacterial cell wall, lipopolysaccharides that extensively cover the outer membrane of Gram-negative bacteria, lipoteichoic acids that are present in the cell wall of gram-positive bacteria, but also proteins like bacterial flagellin, or sugars like β-glucans from the cell wall of fungi. Some nucleic acids can also play a role as MAMPs, like double stranded RNA (dsRNA) involved in the replication cycle of certain viruses [START_REF] Beutler | Innate Immunity: an Overview[END_REF].

The reliance of invertebrates on innate mechanisms of defense does not imply by any means an impaired immunocompetence against infection, compared to vertebrates. Oysters possess a vast repertoire of both PRRs and PRPs with most of them able to perform not only immune recognition but also exert antibacterial activity towards microbes. The functional divergence that characterizes the oyster immunome is suggested to be a source of innovation and adaptability (Zhang et al., 2015).

So far, the list of PRR/Ps identified in the oyster, includes peptidoglycan recognition proteins, carbohydrate binding lectins, Toll-like receptors, Gram-negative binding proteins, scavenger receptors, and fibrinogen-related proteins (FREPs) [START_REF] Wang | The Oyster Immunity[END_REF].

A recent survey of PRR/P using the latest genome version available (V9, size: 581 Mb, ORFs:

26.811 [X. [START_REF] Wang | The Transcriptomic Expression of Pattern Recognition Receptors: Insight into Molecular Recognition of Various Invading Pathogens in Oyster Crassostrea gigas[END_REF]) identified a total of 1084 genes assigned to recognition function distributed in at least 12 gene families. The transcriptomic profile of this set of PRR/P in response to LPS, peptidoglycan, glucan, and poly I:C (which mimics dsRNA) was examined by RNA-seq. Overall, the principal gene families involved in the response to MAMPs were C1q-domain containing proteins (C1qDCs), C-type lectins (CTLs) and FREPs with a dynamic and specific regulation of numerous PRR/Ps in response to the stimulus [START_REF] Wang | The Transcriptomic Expression of Pattern Recognition Receptors: Insight into Molecular Recognition of Various Invading Pathogens in Oyster Crassostrea gigas[END_REF].

C1q-domain containing proteins (C1qDC) have begun to be considered as key players in the immune response of bivalves. The C1q domain is present in the C1 complex that initiates the classical complement pathway in vertebrates by the binding of the C1q domain to antigenantibody complexes [START_REF] Cooper | The Classical Complement Pathway: Activation and Regulation of the First Complement Component[END_REF]. A larger number of proteins containing the globular C1qDC motif are found in metazoans that lack an adaptive immune system, specially species of bivalve mollusks, which can harbor up to 1000 different C1qDC proteins [START_REF] Gerdol | Extensive Tandem Duplication Events Drive the Expansion of the C1q-Domain-Containing Gene Family in Bivalves[END_REF]. This highly expanded gene binds a wide range MAMPs from bacteria, virus and fungi [START_REF] Gerdol | Extensive Tandem Duplication Events Drive the Expansion of the C1q-Domain-Containing Gene Family in Bivalves[END_REF]. Nonetheless, it is not clear whether all the members are involved in immune recognition [START_REF] Gerdol | The Genome of the Pacific Oyster Crassostrea gigas Brings New Insights on the Massive Expansion of the C1q Gene Family in Bivalvia[END_REF] as they have also been associated with the response to environmental pollutants and involved into shell mineralization (Hattan et al., 2001;Liu et al., 2007;Yin et al 2005), which suggests functional diversification following gene expansion in the oyster [START_REF] Gerdol | The Genome of the Pacific Oyster Crassostrea gigas Brings New Insights on the Massive Expansion of the C1q Gene Family in Bivalvia[END_REF][START_REF] Gerdol | Extensive Tandem Duplication Events Drive the Expansion of the C1q-Domain-Containing Gene Family in Bivalves[END_REF].

The oyster C. gigas harbors in its genome more than 300 C1qDC proteins ( Fibrinogen-related proteins (FREPs) contain one or more fibrinogen-like (FBG) domain which display high sequence similarity with the soluble plasma protein fibrinogen involved in blood coagulation in vertebrates. FREPs are widespread in vertebrates, urochordates and invertebrates. Their inducibility and binding activity in organisms like the scallop Argopecten purpuratus and the snail Biomphalaria glabrata has been shown to display specificity to the type of pathogen related stimuli [START_REF] Zhang | Expression Profiling and Binding Properties of Fibrinogen-related Proteins (FREPs), Plasma Proteins from the Schistosome Snail Host Biomphalaria glabrata[END_REF][START_REF] Zhang | A Fibrinogenrelated Protein from Bay Scallop Argopecten irradians Involved in Innate Immunity as Pattern Recognition Receptor[END_REF]. The first report of complete FREPs coding sequence in C. gigas was identified through a cloning strategy: 5 different FREPs-like sequences were amplified using a single pair of primers, suggesting a high sequence diversity of this gene family (Zhang et al., 2012). Genomic studies allowed to found a total of 202 predicted protein identified in the oyster genome [START_REF] Wang | The Transcriptomic Expression of Pattern Recognition Receptors: Insight into Molecular Recognition of Various Invading Pathogens in Oyster Crassostrea gigas[END_REF] ; they were found to be highly expressed in the gill tissue, thus suggesting an important role in the response towards potential microbial invaders.

Toll-like receptors (TLR) are type I membrane proteins, where a transmembrane domain connects an extracellular domain that contains leucine rich repeats (LRR), with a cytoplasmatic domain (TIR) that resembles the Interleukin -1 receptor. TLR are considered main mediators of the innate immunity [START_REF] Satake | Toll-Like Receptors of Deuterostome Invertebrates[END_REF].

They present an important level of expansion with 83 TLR genes predicted to be part of the oyster genome. They are distributed in five groups according to their sequence similarity [START_REF] Wang | The Oyster Immunity[END_REF]. From the large repertoire of predicted TLRs only six have been identify as involved in the immune response based on their induction in response to MAMPs (CgToll-1), their ability to activate NF-κB in transfected HEK293 cells (CgTLR1-4) and their broad recognition capacity towards bacteria, fungi, LPS and PGN (CgTLR-6) [START_REF] Wang | A New Non-Phagocytic TLR6 with Broad Recognition Ligands from Pacific Oyster Crassostrea gigas[END_REF]. Zhang et al., 2014). RLR pathways are involved in the antiviral response that characterized the ostreid-herpes virus 1 infection of juvenile oyster (Lafont et al., 2017); it contributes to the protective effect of polyI:C priming in OsHV-1 infected oysters (Lafont et al., 2020) and falls within the group of immune genes whose higher basal expression has been linked to oyster survival against OsHV-1 infection [START_REF] Lorgeril | Differential basal expression of immune genes confers Crassostrea gigas resistance to Pacific oyster mortality syndrome[END_REF].

Nine PGRPs (peptidoglycan recognition proteins) have been identified in the oyster (Wang et al., 2019). They recognize peptidoglycan, which forms the bacterial cell wall as a mesh-like structure consisting of a carbohydrate backbone cross-linked to peptides. Peptidoglycan monomers are composed of two joined units of N-acetylglucosamine (NAM) and Nacetylmuramic acid with a pentapeptide attached to the NAM unit. This essential bacterial motif is bound by peptidoglycan recognition proteins. Six out of the nine oyster PGRPs are short PGRP type carrying a conserved PGRP/amidase domain in then C-terminus, suggesting a PGN lysis activity [START_REF] Itoh | Distribution of Multiple Peptidoglycan Recognition Proteins in the Tissues of Pacific Oyster, Crassostrea gigas[END_REF]. This amidase activity plays an important role in microbiota tolerance as demonstrated in Drosophila, where PRGP-LB degrades Gramnegative bacteria peptidoglycan to attenuate immune activation (Zaidman et al., 2006).

While all the oyster PGRPs show conservation of the amidase domain, the residues directly involved in PGN binding present a degree of mutation that could translate into a more flexible response towards microbial challenges.

The Gram-Negative Binding Protein (GNBP) are inducible by immune challenge and includes members that bind to Gram-negative bacteria, lipopolysaccharide (LPS) and β-1,3-glucan. Three essential components of the canonical pathway have been identified in C. gigas, the transcription factor Rel/NF-κB itself (Huang et al 2012[START_REF] Montagnani | Cg-Rel, the First Rel/NF-κB Homolog Characterized in a Mollusk, the Pacific Oyster Crassostrea gigas[END_REF]Wu et al., 2007), three IκB proteins (CgIkB1-3) that inhibit NF-kB activation in HEK293 cells, in agreement with their putative ability to sequester Rel proteins in the cytoplasm (Xu et al., 2015;[START_REF] Zhang | A Fibrinogenrelated Protein from Bay Scallop Argopecten irradians Involved in Innate Immunity as Pattern Recognition Receptor[END_REF]. Finally, two IKKα/β and one IκB kinase-like (oIKK) were found that should mediate the inducible degradation of IκB and release of NF-κB along with the unmasking of its nuclear localization signal. Transfection of oIKK successfully promoted the translocation of NF-kB to the nucleus, activity that was dependent on the kinase activity of the oIKK [START_REF] Montagnani | Cg-Rel, the First Rel/NF-κB Homolog Characterized in a Mollusk, the Pacific Oyster Crassostrea gigas[END_REF]. The IKKα/β-2 homologues have been suggested to be involved in the oyster interferon (IFN)-like antiviral pathway as it was found that they are able to interact with the interferon regulatory factor [START_REF] Huang | Oyster Versatile IKKα/βs Are Involved in Toll-Like Receptor and RIG-I-Like Receptor Signaling for Innate Immune Response[END_REF].

Mitogen-Activated Protein Kinases (MAPK) pathway

MAPKs belong to a family of conserved protein kinases that are activated by several PRRs, with the best studied example being TLRs. A p38 MAPK was identified in C. gigas closely related to the ChP38 from C. hongkongensis. Cgp38 is inducible by both LPS and V.

splendidus stimulation and it was shown to regulate positively the expression of proinflammatory cytokines including CgIL17-1,2,3,4,6, and CgTNF [START_REF] Sun | P38 is Involved in Immune Response by Regulating Inflammatory Cytokine Expressions in the Pacific Oyster Crassostrea gigas[END_REF].

The interferon (IFN) pathway

The C. gigas possesses a complex antiviral response that involves several components of the interferon pathway which includes related sensors molecules such as cytoplasmic TLRs, RIG-1 and cGAS, the MyD88 and IkKB signal transducers, IRFs, stimulator of interferon genes (STING), JAK/STAT signaling components and effectors such as OAS, Viperin and Mx. Interestingly, a sustained activation of this pathway in the oyster can be induced by the injection of dsRNA or by its synthetic analog the polyI:C that confers protection against OsHV-1 infection over months. This phenomenon relates to immune priming; see below (Lafont et al., 2017(Lafont et al., , 2020)).

Cytokines

Following the activation of the innate immune system, the response is regulated by the action of several soluble factors which include cytokines and chemokines that fine tune the humoral and cellular response of the host. In the C. gigas genome, six interleukin-17 (CgIL17) [START_REF] Roberts | Rapid Accumulation of an Interleukin 17 Homolog Transcript in Crassostrea Gigas Hemocytes Following Bacterial Exposure[END_REF], one Interferon like protein (CgIFNLP) and 23 tumor necrosis factor homologs have been annotated so far. In vertebrates, IL-17 is a major pro-inflammatory cytokine in the innate and adaptive immunity of vertebrates. It is involved in recruiting neutrophils to the site of infection and it can induce the production of antimicrobial peptides [START_REF] Veldhoen | Interleukin 17 is a Chief Orchestrator of Immunity[END_REF] Hemocytes are very reactive, and they can migrate rapidly towards the site of injury or infection. This allows hemocytes to concentrate in the affected site and through aggregation, phagocytosis and production of defense molecules control either pathogen invasion or tissue injury (Schmitt et al., 2012b;[START_REF] Bachère | The New Insights into the Oyster Antimicrobial Defense: Cellular, Molecular and Genetic View[END_REF].

Phagocytosis.

Phagocytosis is a highly conserved process that has been thoroughly characterized in vertebrates. It is utilized by diverse types of cell to ingest particles usually larger than 0.5 μm (Rosales and Uribe-Querol, 2017). The key events of attachment, formation of pseudopods and phagosome internalization, and finally phagosome maturation into a degradative phagolysosome are conserved in hemocytes and all of them involve a coordinated reorganization of the cytoskeleton membrane trafficking (Underhill and Goodridge, 2012). Six genes, CgIntegrin, CgPI3K, CgRho J, CgMAPKK, CgRab 32, and CgNADPH, putatively involved in phagocytosis are inducible by stimulation with V. splendidus and are responsive to a second exposure by an enhance overexpression (T. Zhang et al., 2014). Furthermore, a member of the Nimrod family receptor was described for its role in hemocyte phagocytosis like in other invertebrates. CgNimC harbors binding capacity towards LPS and Gram-negative bacteria. Moreover, monoclonal antibodies raised against CgNimC significatively reduced the phagocytic index of Gram-negative bacteria [START_REF] Wang | A Novel Phagocytic Receptor (CgNimC) from Pacific Oyster Crassostrea gigas with Lipopolysaccharide and Gram-Negative Bacteria Binding Activity[END_REF].

Certain oyster pathogens like V. aesturianus are able to inhibit hemocyte phagocytosis to evade immune control [START_REF] Labreuche | Effects of Extracellular Products from the Pathogenic Vibrio aestuarianus Strain 01/32 on Lethality and Cellular Immune Responses of the Oyster Crassostrea gigas[END_REF] but others like V. tasmaniensis

LGP32 benefit from phagocytosis to adopt an intracellular stage thanks to several resistance mechanism to phagolysosomal degradation [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] V-Vanhove | Outer Membrane Vesicles are Vehicles for the Delivery of Vibrio tasmaniensis Virulence Factors to Oyster Immune Cells[END_REF][START_REF] Vanhove | Copper Homeostasis at the Host Vibrio Interface: Lessons from Intracellular Vibrio Transcriptomics[END_REF].

Reactive species of oxygen and nitrogen

Reactive oxygen/nitrogen species (ROS/RNS) are normally produced by all aerobic organisms and have important roles in the host physiology including defense due to their toxicity at high concentration that can be employed by the host in bacterial killing. During phagocytosis, transmembrane NADPH oxidases together with Dual oxidases (DUOX) participate in a respiratory burst that will generate highly reactive superoxide (O -2) resulting in a variety of intermediate ROS such as hydrogen peroxide (H2O2), hydroxyl radicals, hypochlorous acid and singlet oxygen that will accumulate in the phagolysosome configuring a highly oxidative environment. The production of RNS is catalyzed by inducible nitric oxide synthases (NO) that use L-arginine and oxygen to produce nitric oxide. Both ROS and RNS can synergize inside the phagolysosome to create a highly oxidative environment for phagocyted microorganisms (X. Zhang et al., 2019).

While valuable for host defense, the toxicity of ROS and RNS has no specificity and can also generate significant damage to host macromolecules such as proteins, lipids and specially DNA (Kohen and Nyska, 2002). One important group of antioxidant enzyme identified in the oyster are Superoxide dismutases (SOD), i.e. metalloenzymes that convert the superoxide into hydrogen peroxide (H2O2) and O2. Two SOD types have been identified and characterized in the oyster according to their metal content, a Manganese and Copper/Zinc SOD (MnSOD, Cu/ZnSOD) which serve as antioxidants but also as PRR as it has been described for the extracellular EcSOD which is able to bind LPS, PGN, and poly (I:C) and promotes the internalization of V. tasmaniensis LGP32 by bridging the vibrio virulence factor OmpU (a major outer membrane protein required to enter oyster hemocytes) with the host CgIntegrin [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] Vanhove | Copper Homeostasis at the Host Vibrio Interface: Lessons from Intracellular Vibrio Transcriptomics[END_REF].

It was reported that the expression of the DUOX genes in hemocytes was associated with survival of oyster to Vibrio infection (de [START_REF] De Lorgeril | Whole Transcriptome Profiling of Successful Immune Response to Vibrio Infections in the Oyster Crassostrea gigas by Digital Gene Expression Analysis[END_REF] and that higher extracellular SOD expression is a trait selected by oyster families resistant to field mortalities [START_REF] Fleury | Microarray Analysis Highlights Immune Response of Pacific Oysters as a Determinant of Resistance to Summer Mortality[END_REF], supporting the contribution of the respiratory burst to the antimicrobial capacity of C.

gigas. However, some pathogenic Vibrios such as V. aesturianus and V. tasmaniensis LGP32 are able to suppress the production of ROS, which contributes to their virulence to the oyster.

The ability of pathogenic vibrios to the oyster to evade ROS activity suggests a major role of ROS in the defenses of the host.

Lysozymes

Lysozymes belong to a family of hydrolases which specific substrate is the peptidoglycan of the bacterial cell wall catalyzing the hydrolysis of the β-1,4 glycosidic bond of the peptidoglycan (Itoh et al., 2010b). In addition to their direct role as bacteriolytic agents they have been described involved in immunomodulation and as important digestive enzymes in some species. So far, three invertebrate-type (i-type) lysozymes have been reported (CGL-1, CGL-2, CGL-3). CGL-2 mRNA is expressed in all oyster tissues except the adductor muscle, while the expression in the digestive gland is restricted to the basophil cell of the gland tubule [START_REF] Matsumoto | Cloning of cDNAs and Hybridization Analysis of Lysozymes from Two Oyster Species, Crassostrea gigas and Ostrea edulis[END_REF]. CGL-1 and 3 were found mainly expressed in the mantle tissue and the recombinant protein showed antibacterial activity in mantle extract of C. gigas (Itoh et al., 2010b)

Antimicrobial peptides

Antimicrobial peptides (AMPs) are evolutionary ancient small proteins (<10kDa) with a broad spectrum of antibacterial and immunomodulatory activities that are ubiquitous among metazoans [START_REF] Spohn | Integrated Evolutionary Analysis Reveals Antimicrobial Peptides with Limited Resistance[END_REF]. Several but not all AMP classes from eukaryotic origin adopt an amphipathic design of clusters of hydrophobic and cationic amino acids (Zasloff, 2002).

They are usually cationic, thereby exploiting the intrinsic negative charge of the outermost bilayer membrane of bacteria to initiate a primary interaction that either leads to membrane permeabilization by pore formation or their seamlessly translocation to their cytoplasm where they can interfere with the bacterial metabolism [START_REF] Spohn | Integrated Evolutionary Analysis Reveals Antimicrobial Peptides with Limited Resistance[END_REF].

The oyster genome contains AMPs families that have been characterized to variable extent.

CSαβ-defensins (characterized by a cysteine stabilized alpha beta motif broadly found in plant and arthropod defensins) and Big Defensins (multidomain defensins mostly found in species of marine invertebrates and cephalochordates) are among the best studied AMPs from C. gigas (Schmitt et al., 2012b)). Members of the CSαβ-defensins are the mantle defensin Cg-Defm (Gueguen et al., 2006) and two hemocyte defensins, Cg-Defh1 and Cg-Defh2 [START_REF] Gonzalez | Evidence of a Bactericidal Permeability Increasing Protein in an Invertebrate, the Crassostrea gigas Cg-BPI[END_REF], which act by inhibiting the biosynthesis of peptidoglycan (Schmitt et al., 2012). Big defensins are multi-domain polypeptides (Schmitt, et al., 2016)

Bactericidal/permeability-increasing protein

Bactericidal permeability-increasing protein (BPI) and lipopolysaccharide (LPS)-binding protein (LBP) are considered to be members of a family of lipid-binding proteins. Vertebrate BPI and LBP bind to the Lipid A moiety of LPS but they also display antagonistic proinflammatory and anti-inflammatory activities upon recognition, respectively [START_REF] Bingle | Meet the Relatives: A Family of BPI-and LBP-Related Proteins[END_REF]. Two homologues of the human BPI (hBPI) have been identified in the oyster (Cg-BPI1 and Cg-BPI2), they are constitutively expressed in various tissues and they are induced in hemocytes after immune challenge presenting different temporal expression profiles. Recombinants Cg-BPI1 and 2 show both antibacterial activities exclusively towards Gram-negative bacteria [START_REF] Gonzalez | Evidence of a Bactericidal Permeability Increasing Protein in an Invertebrate, the Crassostrea gigas Cg-BPI[END_REF][START_REF] Zhang | The Second Bactericidal Permeability Increasing Protein (BPI) and its Revelation of the Gene Duplication in the Pacific Oyster, Crassostrea gigas[END_REF]. Cg-BPI1 acts by permeabilizing the bacterial membranes [START_REF] Gonzalez | Evidence of a Bactericidal Permeability Increasing Protein in an Invertebrate, the Crassostrea gigas Cg-BPI[END_REF].

Diversity of mechanism and synergism

Synergistic activities have been described between different families of oyster AMPs. As a notable example, synthetic proline-rich AMPs identified in hemocytes have poor activity against Gram-positive and no activity towards Gram-negative bacteria, but they act synergistically with Cg-Defs and Cg-BPI against both Gram-positive and Gram-negative bacteria [START_REF] Gueguen | Oyster Hemocytes Express a Proline-Rich Peptide Displaying Synergistic Antimicrobial Activity with a Defensin[END_REF]Schmitt et al., 2012a). The migratory behavior and infiltration response of hemocytes has been proposed to contribute to the colocalization of diverse antimicrobial thus allowing synergy, as different AMPs are expressed in hemocytes and different tissues like mantle, during injury or infection (Schmitt et al., 2012a).

III.1.3.6 IMMUNE PRIMING

Evidences have accumulated over the years supporting the existence of an immune memory in invertebrates where re-exposure to a microbial stimulus generates a faster and more effective immune response relative to naive individuals, which can translate in an increased survival to infection [START_REF] Melillo | Innate Immune Memory in Invertebrate Metazoans: A Critical Appraisal[END_REF]. As the concept of immune memory is paradigmatically linked to the cellular (lymphocytes) and humoral components (antibodies) of adaptive immunity in vertebrates, that are missing in invertebrates, this phenomenon requires to redefine immune memory in a way that is independent of the underlying mechanisms [START_REF] Milutinović | Immune Memory in Invertebrates[END_REF].

Although phenotypic evidences of an immune memory have accumulated across different taxa, the remaining controversy is mainly due to the lack of knowledge about the underlying molecular and cellular mechanisms [START_REF] Hauton | Adaptive Immunity in Invertebrates: A Straw House Without a Mechanistic Foundation[END_REF].

In the oyster C. gigas antiviral immune priming was evidenced in response to dsRNA. This response confers to juvenile oysters a specific and long-lasting protection to juvenile oysters against the infection by the ostreid herpes virus 1 (OsHV-1) by blocking viral replication (Lafont et al., 2017(Lafont et al., , 2020)). This translates into enhanced field survival to the mass mortalities of the Pacific Oyster Mortality Syndrome. The molecular bases of this immune priming phenomenon have been recently studied; results showed that a sustained upregulation of gene expression is triggered after priming that can still be observed after 4 months after priming. This sustained alert state is proposed to allow an effective early immune response towards OsHV-1 (Lafont et al., 2020).

In addition, a maternal transgenerational priming phenomenon has been evidenced in C.

gigas. Upon genitor stimulation with polyI:C, only mothers are able to transfer immunity to the next generation conferring larvae with higher resistance to OsHV-1 infection. This phenomenon could be the result of the maternal provisioning of the eggs with antiviral compounds (Green et al., 2016;[START_REF] Lafont | Transgenerational Plasticity and Antiviral Immunity in the Pacific Oyster (Crassostrea gigas) Against Ostreid Herpesvirus 1 (OsHV-1)[END_REF]. Nevertheless, a tradeoff in maternal offspring protection has been recently identified, where improved larval survival is linked with impaired growth and a higher Vibrio loads compared with trans-generationally unprimed offspring [START_REF] Robinson | Fitness Costs Associated with Maternal Immune Priming in the Oyster[END_REF].

Immune priming by bacterial components has also been studied in the oyster. Although immune functions have been shown to be enhanced upon re-exposure to bacteria, no evidence of enhanced survival of primed oyster has been reported so far making debatable the functional relevance of this primed response against bacterial infection (T. Zhang et al., 2014;[START_REF] Li | The Hematopoiesis in Gill and its Role in the Immune Response of Pacific Oyster Crassostrea gigas Against Secondary Challenge with Vibrio splendidus[END_REF][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].

III.2. THE PACIFIC OYSTER MORTALITY SYNDROME (POMS) AS A MODEL FOR POLYMICROBIAL HOST-MICROBE INTERACTIONS

Over the past twenty years, two main different infectious diseases have affected juvenile and adult C. gigas oysters causing mortality outbreaks on reared oysters. These diseases have been considered to challenge the sustainability of C. gigas farming in France and worldwide [START_REF] Dégremont | Enhancing Resistance to Vibrio aestuarianus in Crassostrea gigas by Selection[END_REF]. Since 2012 adult mortalities have been caused by V. aestuarianus, a bacterial species that is suggested to have re-emerged as an oyster pathogen after a period of low detection in moribund oysters between 2008 and 2012 [START_REF] Goudenège | A Single Regulatory Gene is Sufficient to Alter Vibrio aestuarianus Pathogenicity in Oysters[END_REF].

In contrast, a disease of a higher etiological complexity, able to cause 100% mortality in farmed juveniles has severely affected oyster farming since 2008. This severity is associated with the emergence of a microvariant genotype of the previously identified Ostreid herpesvirus 1 [START_REF] Segarra | Detection and Description of a Particular Ostreid Herpesvirus 1 Genotype Associated with Massive Mortality Outbreaks of Pacific Oysters, Crassostrea gigas, in France in 2008[END_REF]. This microvariant refreed to as OsHV-1 µvar is the etiological agent of the Pacific Oyster Mortality Syndrome (POMS), a polymicrobial disease that integrates complex interaction between the environment, host and pathogens. In this infectious disease OsHV-1 is necessary for the onset while opportunistic bacterial pathogens, notably vibrios, determine its fatal outcome (de Lorgeril et al., 2018;[START_REF] Petton | Fine-Scale Temporal Dynamics of Herpes Virus and Vibrios in Seawater During a Polymicrobial Infection in the Pacific Oyster Crassostrea gigas[END_REF]. isolated from mortality episodes and extensively characterized with respect to its ecology and the evolution and mechanics of its virulence determinants towards the oysters [START_REF] Lemire | Populations, not clones, are the unit of vibrio pathogenesis in naturally infected oysters[END_REF]Bruto et al., 2017Bruto et al., , 2018;;Piel et al., 2019).

III.2.1 RISK FACTORS OF POMS

POMS is a multifactorial disease, not only dependent on multiple microorganisms (viral and bacterial pathogens) but also on distinct environmental and host variables that contribute to the expression and severity of this syndrome. Initial approaches to characterize the disease have relied on direct injection of the different viral and bacterial pathogens into specific pathogen free oysters. Although this methodology allowed primary characterization of the microorganisms involved, it did not correctly represent the natural way of infection neither the polymicrobial complexity of the field. An ecologically realistic infection protocol was therefore developed over the past years by Bruno Petton and collaborators (Petton et al., 2013(Petton et al., ,2015) ) to study the full complexity of the disease. Specific pathogen-free oysters were used to naturally "catch" the disease by deploying them on the field during mortality season.

Once they were naturally infected, they were placed in cohabitation with naive oysters under controlled conditions and the disease dynamics was monitored in the recipient oysters.

This methodology to effectively transfer POMS into the laboratory has allowed characterizing in details several risk factors associated with the development of the syndrome.

A now well-established environmental determinant of POMS is seawater temperature (Petton et al., 2013). Seasonal outbreaks occur in the summer season when seawater temperature is in a 16°C to 24°C range, which is permissive for virus replication and transmission [START_REF] Pernet | Mass Mortalities of Pacific Oysters Crassostrea gigas Reflect Infectious Diseases and Vary with Farming Practices in the Mediterranean Thau Lagoon, France[END_REF]Petton et al., 2013). This factor, which is undoubtedly the main trigger of disease outbreaks in the field, is currently explored for its role on the whole pathosystem (ANR project Decicomp, led by Guillaume Mitta).

Two other key factors controlling disease expression are currently investigated.

The first factor is Oyster age. It was shown to determine the susceptibility of oysters to POMS.

Indeed, resistance is suspected to be acquired between 15 and 25 months (Azema et al., 2017, Petton et al., 2015) The second one is diet restriction, which can influence host or pathogen physiology.

Energetic reserves (carbohydrates and neutral lipids) have already been associated with mortality risk during oyster epizootics (Corporeau et al., 2014, Pernet et al., 2014b, Young et al., 2017). Preliminary results obtained by Petton and collaborators showed a huge influence of oyster diet in the susceptibility to POMS. A starvation of two weeks was sufficient to prevent POMS expression (personal communication).

Beyond these key factors, other possible determinants of the disease have been investigated.

Oysters are exposed to high variations in seawater salinity, driven by warm periods and precipitation, showing a remarkable tolerance range that goes from below 10‰ up to 35‰ (Helm 2005). Although not as strong as temperature as a trigger, salinity could modify oyster susceptibility POMS. Indeed, acclimation of oysters to salinity of 10‰ showed to increase survival rate to 95.8% compared to the 73.2, 43.2 and 61% of oysters acclimated at 15, 25, 35‰ respectively (Fuhrmann et al., 2016). The physiological changes associated with this survival success was later studied, and were shown to be associated with an increased water content, protein level and energetic reserves along with the inhibition of hexokinase activity, involved in glycolysis, and decreased catalase activity [START_REF] Fuhrmann | Metabolism of the Pacific Oyster, Crassostrea gigas, is Influenced by Salinity and Modulates Survival to the Ostreid Herpesvirus OsHV-1[END_REF].

Rearing practices (including densities) also have important consequences on POMS severity.

The level of oyster mortalities also varies among farming sites that present different farming practices. Pernet and collaborator (2012) showed that oysters that are kept in open waters of the Mediterranean Sea during the window of permissive temperatures (17°C to 24°C) did not experienced mortalities and presented a low level of OsHV-1 detection. Contrastingly, oysters kept within the farming area of the Thau Lagoon suffered from severe mortality outbreaks while those maintained also in the lagoon but outside the farming area presented sporadic mortalities that correlated with their connection with the farming sites through water currents.

Within the farming sites poor water renewal, high oyster density and the transfer of asymptomatic virus carriers between sites, have been proposed to have important implications in the mortality risk in farming sites and therefore candidates to be considered in disease management initiatives [START_REF] Pernet | Mass Mortalities of Pacific Oysters Crassostrea gigas Reflect Infectious Diseases and Vary with Farming Practices in the Mediterranean Thau Lagoon, France[END_REF](Pernet et al., , 2014a;;Petton et al., 2015).

III.2.2. OSTREID HERPES VI RUS MICROVARI ANT (OSHV-1 ΜVAR)

The Ostreid herpesvirus type 1 was firstly identified during 1991 in C. gigas larvae affected by mass mortalities in French and New Zeland hatcheries [START_REF] Vigneron | Detection of Ostreid Herpesvirus 1 (OsHV-1) DNA in Seawater by PCR: Influence of Water Parameters in Bioassays[END_REF].

Extracellular viruses are usually enveloped and present a diameter of 100-180 nm. Infected oysters present circular or polygonal empty capsids and nucleocapsids of 70-80 nm of diameter scattered in the nucleus of infected cells while cytoplasmic vesicles contain single or groups of enveloped virions [START_REF] Renault | Malacoherpesviruses of Mollusks[END_REF]. Cryo-electron microscopy was used to reconstruct the icosahedral capsid, revealing the characteristic triangulation number of T=16 of herpesviruses. Isolated virions were used to obtain the first complete genome sequence of the virus which corresponds to the reference type [START_REF] Davison | A Novel Class of Herpesvirus With Bivalve Hosts[END_REF]. Although the overall genome structure and aminoacidic sequences demonstrated a distant relationship with vertebrate herpesviruses, based on morphological features, capsid structure and the presence of a putative terminase gene, it was classified under the name Ostreid herpesvirus 1 representing the first of the 2 species that compose the Malacoherpesviridae family within the order Herpesvirales [START_REF] Davison | A Novel Class of Herpesvirus With Bivalve Hosts[END_REF][START_REF] Renault | Malacoherpesviruses of Mollusks[END_REF].

A more virulent microvariant of the ostreid herpesvirus 1 (OsHV-1 μVar) was identified in 2008 associated with mass mortality events of juvenile oysters in France and defined by a series of mutations relative to the reference type [START_REF] Segarra | Detection and Description of a Particular Ostreid Herpesvirus 1 Genotype Associated with Massive Mortality Outbreaks of Pacific Oysters, Crassostrea gigas, in France in 2008[END_REF].

Recently, Delmotte et al. evidenced distinct populations of OsHV-1 µvar in the French Mediterranean and Atlantic farms [START_REF] Delmotte | Contribution of Viral Genomic Diversity to Oyster Susceptibility in the Pacific Oyster Mortality Syndrome[END_REF]. How specific genetic differences in

OsHV-1 µvar populations influence the outcome of the POMS remains to be elucidated.

III.2.3 BACTERIA

Until recently, the bacterial component of POMS has mainly been studied through cultural approaches. Vibrio species are among the cultural bacteria those that have been more

clearly associated with POMS. Members of the Splendidus clade (e.g., V. tasmaniensis, V.

splendidus, V. cyclitrophicus and V. crassostreae) have been systematically isolated from diseased juvenile oyster during summer mortalities and to lower extent, V. harveyi and V.

aestuarianus, which fall outside the clade (Saulnier et al., 2010;[START_REF] Segarra | Detection and Description of a Particular Ostreid Herpesvirus 1 Genotype Associated with Massive Mortality Outbreaks of Pacific Oysters, Crassostrea gigas, in France in 2008[END_REF]Bruto et al., 2017;[START_REF] Go | Mass Mortalities of Unknown Aetiology in Pacific Oysters Crassostrea gigas in Port Stephens, New South Wales, Australia[END_REF]. Their association to POMS has promoted extensive research to characterize the particular role and contribution of vibrios to mortalities.

Initial approaches demonstrated the pathogenic potential of Vibrio isolates from disease animals and achieved to identify some relevant factors that contribute to virulence in oysters.

From there, in a pioneer work by the group of Frédérique Le Roux and collaborators, which used pathogen free oyster spats, allowed field-based approaches. The authors characterized the population structure of Vibrio species found in naturally infected oysters during mortality season in the Atlantic French coast, and characterized the population structure of vibrios associated with the development of POMS (Lemire et al., 2015). They evidenced the replacement of the resident microbiota of the oyster by a genetically diverse but phylogenetically coherent vibrio population of Vibrio crassostreae that was established as a unit of pathogenesis. The isolation of related non-virulent V. crassostreae strains allowed to confirm, by injection, the previous observation that non-pathogenic strain can act synergistically with pathogenic ones to increase mortality suggesting that a similar phenomenon could occur during the natural development of POMS derived bacterial dysbiosis (Gay et al., 2004;Lemire et al., 2015).

This field-based approach was further applied to characterize, through genomics, simultaneously the population structure of the vibrio community in the oyster tissues and different fractions of the water column at two different dates in the spring season in absence of mortalities and during mortality episodes in the summer season (Bruto et al., 2017). This confirmed the predominance of the V. crassostreae population during mortality season and its almost exclusive association to the oyster tissues, but also evidenced the seasonality of the vibrio population structure between the spring and summer season, the presence of Vibrio populations from the Splendidus clades in healthy oysters during the spring season and a set of populations that are preferentially association to the water column fractions, apparently no founding the oyster as a suitable host for colonization. The mechanisms of association of vibrio populations in absence of disease and alternative colonization determinants other than virulence-related are aspect of vibrio/oyster interaction that have not been explored so far in the oyster. Vibrios have been the unique bacterial group to be studied in detail as one of the etiological agent of POMS, nonetheless other groups such as Arcobacter and Shewanella have been also found associated with mortalities but their particular contribution and potential synergisms with other bacterial communities that participate in the fatal dysbiosis in oyster is still unknown [START_REF] Go | Mass Mortalities of Unknown Aetiology in Pacific Oysters Crassostrea gigas in Port Stephens, New South Wales, Australia[END_REF]de Lorgeril et al., 2018;[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 Next-Generation Sequencing Strategy[END_REF] However, since the elucidation of the complexity of POMS pathogenesis in our laboratory, we know that vibrios associated with POMS colonize oysters whose immunity is altered by the OsHV-1 virus (de Lorgeril et al. 2018). Therefore, analyzing Vibrio species colonizing oysters outside this pathological context is of great importance to unveil key determinants of Vibrio colonization in oysters with an intact immune system. The use of Vibrio species of known ecology was therefore key to undertake this exploration.

As reviewed in the introduction, vibrios are a diverse group of heterotrophic bacteria that are part of planktonic and animal-associated microbial communities. Population genetic coupled to ecological sampling applied to vibrios have shown that they can be grouped in closely related genetic units distributed differentially among predicted habitats but connected by the high degree of dispersal that characterize the marine environment (Hunt et al., 2008;Preheim et al., 2011;Szabo et al., 2013;Shapiro and Polz, 2014).

This population framework has allowed to determine the population structure of vibrios in the oyster tissues and the surrounding seawater as well as how functionally and cohesive vibrio population assembly in the oyster during disease (Bruto et al., 2017). These studies have unraveled that, in the Atlantic marine ecosystem, some vibrio populations do preferentially associate to oysters during the spring season in absence of massive oyster mortalities, whereas the vibrio populations that colonize the oysters during POMS tend to harbor more virulent phenotypes.

The assessment of virulence potential of the vibrio populations characterized by Bruto et al.

(2017) showed that one population taxonomically assigned to V. splendidus (population #23),

that is phylogenetically close to V. splendidus (population #24) found during POMS, displayed a moderate virulence potential and belonged to the microbiota associated with healthy oysters in the spring season.

So far, virulence and cytotoxicity have been characterized as determinant for the ability of vibrios to colonize oysters, but in a pathological context were the host's defenses are disturbed by the OsHV-1 virus and leads to the death of the host.

We were interested in exploring the alternative context of association of vibrios with healthy oysters using V. splendidus population #23.

In article 1, we studied this particular association searching for the molecular determinants of this moderate virulence and possible tradeoffs involved in the maintenance of such phenotype. We discovered that in V. splendidus, certain structures of O-antigen are more easily recognized by the immune system of the host and constrain colonization and virulence, but confer resistance to environmental grazers, thus providing an additional advantage to this vibrio population for environmental persistence. We indeed found that 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. Our results suggest 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. Data in complementary results 1 confirmed this hypothesis in a different Vibrio genetic background.

In complementary results 2 (unpublished results that will be submitted for publication as a short letter, in preparation), we used the same biological material and additional Vibrio populations positively and negatively associated with oysters to ask one fundamental question: Are vibrios able to colonize characterized by resistance traits that explain their capacity to cope with an intact immune system? Overall, our results illustrate that the multiple biotic interaction taking place in the aquatic environment do not allow for discrimination of vibrio oyster-association on the basis of the only resistance trait. When considering all vibrio strains independent of their taxonomic affiliation, the main factors explaining association to oysters were resistance to ROS, virulence and surface charge. Figure S3. Genetic organization of the T6SS cluster in V. splendidus #23. The figure shows gene replacements (in blue) between non-virulent and moderately virulent strains leading to loss/acquisition of a chromosomal T6SS-encoding gene cluster. Genes in green are specifically found in the non-virulent strain 4G1-8. Genes in red are specifically found in virulent strains and contain the T6SS genes. The gene cluster of the virulent strains is present on two contigs (genes GV4G44_380041 -GV4G44_380047 and GV4G44_320001 -GV4G44_320044). From the complete genome of V. tasmaniensis LGP32, which contains the same gene cluster, we found that genes GV4G44_380047 and GV4G44_320001 (hatched red genes) form a single large gene that is splitted in our assemblies due to numerous repeats. et al., 2011, 2012), with one suggested exception i.e. the presence of non-hydroxylated 12:0 fatty acid at the secondary C3' position. As a consequence, no molecular species with Glycine substitution was found in either strain. Moreover, no substitution of phosphate groups with charged amino derivatives such as phosphoethanolamine or aminoarabinose could be inferred from our MS spectra. An additional peak, common to both strains was observed at m/z 1976. 1-1977.4, which could be tentatively attributed to a hepta-acyl Lipid A molecular species (7 FA) carrying an additional 16:0 or 16:1 fatty acid. This molecular species appeared more intense in the virulent 4G4_4 than in the non-virulent 4G1_8. Figure S7. Immune genes that do not respond to early infection by V. splendidus. 4 pools of 10 oysters were sampled 2 h after being injected either with sterile seawater (SSW) or vibrio strains (2x10 7 CFU per animal). RNA was extracted from the 4 pools. For each pool, RT-qPCR was performed on immune genes to determine their expression relative to the elongation factor 1 alpha (EF1-α). The ΔΔ ct method was used (Pffafl., 2001). Data are presented as the mean ± SD. Different letters indicate a significant difference (ANOVA, p < 0.05 and Tukey HSD test). We confirmed that the modification of the O-antigen structure was also determinant in the susceptibility to amoeba grazing in other genetic background within population #23. In the supplemental results of article 1, presented above, we show that the mutation of the wbe region results in highly similar O-antigen profiles in two different mutant strains of population #23 and here we corroborate that this alternative structure is also determinant for the susceptibility to grazing of strain 4D1_8. [START_REF] Reddick | Bacteria Fighting Back: How Pathogens Target and Subvert the Host Innate Immune System[END_REF]. Conversely microbes employ strategies to evade, inhibit or manipulate the innate immune responses. Commonly, counter-measures to host innate immunity include resistance/tolerance to antimicrobials, escape from host recognition (usually by disguising their immunogenic surface components) or active dampening of immune defense, i.e. immune suppression. Those mechanisms have been described and reviewed for a series of human pathogens (Hornef et al., 2002;[START_REF] Reddick | Bacteria Fighting Back: How Pathogens Target and Subvert the Host Innate Immune System[END_REF].

I.2. ARTICLE N°1. VIBRIO SPLENDIDUS O-ANTIGEN STRUCTURE

In the continuum of symbiotic interactions, from mutualisms to pathogenicity, microorganisms have evolved the capacity to establish stable interactions with metazoan hosts by selecting one or several of these strategies to avoid elimination by their host immune system. In marine aquatic environments, vibrios have co-evolved with a diversity of sessile and planktonic metazoans hosts. A thorough analysis of vibrio ecological associations has been recently published by Bruto et al. (2016) in a marine coastal environment used for oyster farming. Some vibrio populations were shown to be positively associated with oysters in health and/or disease, indicating they have evolved the capacity to tolerate their host defenses, which only rely on innate immune mechanisms in the case of mollusks. In contrast, other populations were shown to be negatively-associated with oysters indicating they find better ecological niches in other host species present in phytoplankton and zooplankton, or adopt a free-living style, maybe as a consequence of their inability to tolerate the immune defenses of metazoan hosts present in the ecosystem.

Among vibrios colonizing oysters, one strain referred to as V. tasmaniensis LGP32 (Splendidus clade) adopts an original life-style as it enters oyster immune cell as part of its pathogenic process [START_REF] Duperthuy | Use of OmpU Porins for Attachment and Invasion of Crassostrea gigas Immune Cells by the Oyster Pathogen Vibrio splendidus[END_REF]. As a possible requirement for its intracellular stages, LGP32 was shown to be equipped with mechanisms of resistance to oyster AMPs [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][START_REF] V-Vanhove | Outer Membrane Vesicles are Vehicles for the Delivery of Vibrio tasmaniensis Virulence Factors to Oyster Immune Cells[END_REF]. The most potent mechanism relies on trapping of AMPs through secretion of outer membrane vesicles that bind and sequester AMPs [START_REF] V-Vanhove | Outer Membrane Vesicles are Vehicles for the Delivery of Vibrio tasmaniensis Virulence Factors to Oyster Immune Cells[END_REF].

V. tasmaniensis

LGP32 was also shown to resist ROS and heavy metals present in oyster phagocytes and/or plasma, and these mechanisms of resistance are required for efficient host colonization [START_REF] Vanhove | Copper Homeostasis at the Host Vibrio Interface: Lessons from Intracellular Vibrio Transcriptomics[END_REF]. Thus, resistance/tolerance to antimicrobials is essential for LGP32 to colonize oysters. In other species of vibrios colonizing oysters, some virulence factors were shown to play a role in host colonization. In the species V. crassostreae and V. splendidus (Lemire et al., 2015;Bruto et al., 2018), virulence factors include toxins of the MARTX family as well as proteins of unknown functions such as the R5-7 protein (Bruto et al., 2017(Bruto et al., , 2018)). Still, the relative weight of virulence and resistance to host defenses in favoring colonization of oysters by environmental vibrios remains unknown. Moreover, little attention has been paid until now to non-virulent strains that may use alternative ways of immune evasion like avoiding host recognition to efficiently colonize their host. Exploring the diversity of immune evasion processes along a gradient of virulence in natural populations of vibrios is now needed to understand the multiple factors that govern host colonization and may distinguish the evolution towards commensalism versus virulence among vibrios.

With that objective, we explored the complex interface between vibrios and a metazoan host, the oyster. We used a series of 6 ecological populations of vibrios known to be positively (V. crassotreae, V. splendidus pop23 and pop24) or negatively-associated with oysters (V. breogani, V. orientalis, A. fisheri) although present in the oyster environmental waters. By phenotyping strains for resistance to antimicrobials (AMPs, ROS, and heavy metals) we showed that resistance to oyster antimicrobials is a population-specific trait insufficient to recapitulate host associations. Overall, the main factors explaining association to oysters were resistance to ROS, virulence and surface charge.

Materials and methods

Strains and growth conditions

Vibrio populations used in this part of the study were taxonomically assigned to the species V. crassostreae, V. splendidus, V. breogani, V. orientalis and V. fischeri (Bruto et al., 2017). Strains were grown at 20°C in Zobell broth or agar (4g/L bactopeptone and 1g/L of yeast extract in artificial seawater, pH 7.6) and with agitation at 150 rpm. Modified Zobell broth used for Minimal Inhibitory Concentration tests contained 1.3g/L bactopeptone and 0.3 g/L of yeast extract in artificial seawater, pH 7.6. Saline Peptone Water (SPW) medium agar was used for copper and zinc sensibility assays (bacto peptone 1.5% P7vol Bio-Rad, NaCl 2.92% P/vol Bio-Rad, Agar 1.5% P/vol Bio-Rad, pH 7.2)

Cg-BigDef1 synthesis

The antimicrobial peptide Cg-BigDef1 (10.7 kDa) secreted by oyster hemocytes was produced by chemical synthesis by Agnès Delmas in the Center of Molecular Biophysics (CMB) of the CNRS in Orléans -UPR 4301 according to the recently published procedure (Loth et al., 2020).

Determination of the Minimal Inhibitory Concentration

From stationary phase bacterial cultures, fresh cultures were started with a 1:100 ratio of inoculum in Zobell medium, respectively. Cultures were incubated at 20º and 150 rpm until an OD600 between 0,2 -0,4 corresponding to exponential phase of growth. Cultures in exponential phase were adjusted to a theoretical OD600 :0.001 in modified Zobell medium. Of this bacterial suspension 90 μl were placed into 96 well plates and 10 μl of a serial dilution of CgBigDef-1 were added to each well. Plates were incubated 18 hrs at 20º C with shaking at 150 rpm. After incubation time, plates were read at OD600 to determine the minimal inhibitory concentration of CgBigDef-1 for each strain.

Susceptibility to Copper and Zinc

Bacterial cultures in stationary phase were washed twice with sterile seawater (SSW), by centrifugation at 1500 x g, 20ºC, to eliminate Zobell medium from cultures. After, the OD600 was measured and adjusted to 1 in SSW. After adjusting the OD, 500 μl of each culture were added to Saline Peptone Water (SPW) medium agar plates supplemented with NaCl to a final concentration of 0.5M (bacto peptone 1.5% P7vol Bio-Rad, NaCl 2.92% P/vol Bio-Rad, Agar 1.5% P/vol Bio-Rad, pH 7.2) Cultures were homogeneously spread, and the excess liquid not absorbed was removed.

After, in sterile conditions, air-dried cellulose disc previously soaked for 30 min in a 0.2 μM filtrated 50 mM copper sulfate (CuSO4) or 50 mM Zinc sulfate solution (cupric sulfate pentahydrate, 99%, C-7631 Sigma -Aldrich; Zinc sulfate heptahydrate, 99%, 205986-50g Sigma -Aldrich) were placed per plate. Petri dishes were incubated for 18 h at 20ºC and the size of the inhibition zone was measured for each strain with a digital caliper.

Susceptibility to oxygen reactive species. Bacterial cultures (100ul) in stationary phase were homogeneously plated in Zobell agar. A cellulose disc soaked with 10ul of a solution of cumene hydroperoxide at 160 uM (technical grade 80%, reference 247502-100G Sigma-Aldrich was placed in the center of the petri dish. After 18 hours of incubation at 20ºC, the size of the inhibition zone was measured. The results represent the average of three independent experiments.

Cytochrome C binding assay. Cytochrome C has a net charge of +8 and two different type of interaction characterized in model membranes bearing acid phospholipids like cardiolipin present in mitochondrial and bacterial membranes (Mileykovskaya and Dowhan, 2010). At neutral pH the interaction is electrostatic and dependent on the negative surface charge density and reversible by the addition of competing anionic species like ATP with the A-site of cyt c, by other hand, at acidic pH cyt c bound by its C-site by hydrogen bonds and hydrophobic interaction that cannot be reversed by ATP or salt addition. Stationary phase cultures were wash twice by centrifugation (1500 x g, 20ºC) with 20 mM MOPS buffer supplemented with sucrose for a final osmolarity of 450 mOsm to make it compatible with the hyper saline environment of marine bacteria. After washing, OD600 was measured, cultures were centrifuged and pellet suspended in MOPS sucrose buffer adjusting to an OD600 = 6.

After, 900 μl of bacterial suspension were placed in 1.5 mL polipropilene microcentrifuge tubes. Cytochrome c solution was prepared in MOPS Sucrose buffer to a concentration of 5 mg/mL and added to bacterial suspension for a final concentration of cytochrome c of 0.5 mg/mL. Tubes were incubated 15 minutes at 20ºC and then centrifuged at 6000 x g at room temperature for 15 min, finally 300 ul of supernatant were placed in triplicate in to 96 wells plates and the OD530 was measured in a TECAN micro plate reader. The percentage of cytochrome C bound to bacteria was calculated relative to the OD530 value of a 0.5 mg/mL cytochrome c control solution not exposed to bacteria.

Statistical analyses

Redundancy analysis (hereafter named RDA) was used to investigate the assemblage of the different bacterial strains (positive or negative association to oysters). RDA was performed on AMP resistance, ROS resistance, copper resistance, zinc resistance, surface charge and virulence. Then significant variables (i.e., variables that significantly explained changes in the distribution of strains, oyster versus water-column) were identified using a forward-selection procedure (999 permutations), implemented in the R "vegan" package and in the "rda" and "ordiR2step" functions. For all analyses, the threshold significance level was set at 0.05.

Results

In our work with the collection of vibrio populations characterized by Bruto et al. (2017), we hypothesized that resistance to oyster antimicrobial defenses could be a major explicative factor for the preferential association of certain populations to the oyster tissues, while susceptibility to these antimicrobials could be a common trait of vibrio populations that are excluded from the oyster. For that, we used 30 strains representative of six populations of vibrios that display positive (V. crassostreae, V. splendidus Pop #23 and Pop #24) or negative associations (V. breogani, V. orientalis, A. fisheri) with oyster tissues (Bruto et al., 2017). These included both virulent and non-virulent populations of vibrios (Bruto et al., 2016). Every strain was phenotyped for its resistance to the oyster antimicrobial peptide Cg-BigDef1, ROS (cumene hydroperoxyde), copper and zinc, which all participate in oyster immune cell defenses against pathogenic vibrios [START_REF] Vanhove | Copper Homeostasis at the Host Vibrio Interface: Lessons from Intracellular Vibrio Transcriptomics[END_REF].

Individual resistance phenotypes were found to be population-specific traits presenting little to no intra-population variability (Figure 21A). For some populations, resistance perfectly fitted with our initial hypothesis. Thus, strains of V. splendidus pop23, a population positively associated with oysters were highly resistant to antimicrobial peptides, ROS and heavy metals. By contrast, strains of V. breogani, a population negatively associated with oysters, were among the most susceptible to ROS and heavy metals. However, some other population like the virulent V. splendidus pop24, positively associated with oysters, showed more mosaic traits of resistance (e.g. high resistance to heavy metals and ROS but low resistance to AMPs.

Overall, when considering all vibrio strains independent of their taxonomic affiliation, only resistance to ROS discriminated significantly vibrios according to their known association with oysters (p<0.0001) (Figure 22A). To better estimate the relative weight of resistance to host antimicrobials in the capacity of vibrios to colonize oysters, we included other parameters influencing host-microbe interactions such as strain virulence and surface charge. As a proxy of strain resistance, we used a resistance index defined as the sum of every single resistance trait. For strain virulence, we used the mortality induced by intramuscular injection of vibrios, previously determined (Bruto et al., 2017). Finally, for surface charge we used the strain ability to bind to the cationic protein cytochrome C as a proxy (Saar-Dover et al., 201;Cullen et al., 2015). Only the last two parameters discriminated significantly (p<0.01) vibrio strains according to habitat. By using a general linear model, we showed that vibrio association to oysters are explained at 50.4 % by strain virulence while surface charge explains 12.3 % and resistance to host antimicrobials only 10 % of the association to oysters (Figure 21D). Therefore, the capacity of vibrios to colonize oysters depends on a multiplicity of factors that includes resistance to ROS, virulence factors and surface determinants, which are evocative of distinct strategies of avoidance of the host immune system.

Discussion

Based on an in vitro approach, which obviously does not recapitulate the complexity of in vivo interactions, we found that resistance to oyster innate antimicrobial defenses was insufficient to discriminate environmental vibrios according to their known association with oysters. This result contrasted with the many examples of both commensal and pathogenic interactions between bacteria and metazoan hosts that have been shown to depend on such resistance phenotypes. For instance, resilience of Bacteroidetes in the mammalian gut was shown to depend on resistance to AMPs (Cullen et al., 2015); Mycobacterium tuberculosis survival inside macrophages depends on resistance to copper and zinc (Neyrolles et al., 2015), etc. It also contrasted with our earlier studies of the oyster opportunistic pathogen V. tasmanienis LGP32, which identified diverse mechanisms of resistance to oyster AMPs, ROS, and heavy metals, the later having important consequences on the capacity of

LGP32 to colonize its host [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][START_REF] V-Vanhove | Outer Membrane Vesicles are Vehicles for the Delivery of Vibrio tasmaniensis Virulence Factors to Oyster Immune Cells[END_REF][START_REF] Vanhove | Copper Homeostasis at the Host Vibrio Interface: Lessons from Intracellular Vibrio Transcriptomics[END_REF].

Interestingly, resistance to oyster innate antimicrobial defenses was shown here to be a population-specific trait, and until now only V. tasmaniensis LGP32 has been studied in details for its host-pathogen interactions with oysters. This strain has evolved the capacity to enter and survive inside oyster immune cells, the hemocytes, as a necessary stage in its infectious process [START_REF] Duperthuy | Use of OmpU Porins for Attachment and Invasion of Crassostrea gigas Immune Cells by the Oyster Pathogen Vibrio splendidus[END_REF]. It is therefore likely that resistance to antimicrobials is a trait essential for vibrios adopting hemocyte intraphagosomal stages, the phagosome being the recipient of hydrolytic enzymes, AMPs and heavy metals [START_REF] Bachère | The New Insights into the Oyster Antimicrobial Defense: Cellular, Molecular and Genetic View[END_REF]. Likewise, V. crassostreae was also reported to inhabit hemolymph and gills (Bruto et al., 2017), which are antimicrobial tissues in oysters (Destoumieux-Garzón et al., 2014) and not surprisingly we found it highly resistant to antimicrobials in the present study. But little is still known on the tissue niches of other vibrio-populations associated with oysters such as the V. splendidus pop23 and 24 from this study.

Resistance to antimicrobials may therefore be not as discriminant for vibrio populations having different tissue tropism, for example populations that would colonize tissue poor in antimicrobials (i.e. not colonizing hemolymph nor epithelia). An interesting finding is the contrast between V. splendidus pop23 (associated with oysters outside infectious events) and pop24 (associated with oysters during mortality events) in terms of resistance to Cg-BigDef1.

We recently showed that Cg-BigDef1 is repressed by OsHV-1 virus, which triggers bacterial colonization during infectious episodes (de Lorgeril et al. 2018). It is therefore likely that opportunistic pathogens including strains of pop24 benefit from such a repression of oyster antimicrobial defenses, whereas other resident population like pop23 have intrinsic resistance capacities that help them colonize oyster tissues of healthy oysters. Finally, outside the oystervibrio interactions studied here, we cannot ignore that interactions with other metazoan hosts occur in the water column (e.g. copepods), which share many innate immune effectors with mollusks. By applying similar selective pressures, these interactions have likely shaped resistance phenotypes among populations negatively associated with oysters. Altogether, this illustrates that the multiple biotic interaction taking place in the aquatic environment do not allow for discrimination of vibrio oyster-association on the basis of the only resistance trait.

Individual resistance phenotypes were found to coincide with vibrio population delineation, showing low to no variability within each population. Populations negatively associated with oysters were susceptible to at least one the main antimicrobial tested (Figure 21A). Strains of the algal specialist, V. breogani, were among the most susceptible to ROS and heavy metals.

In contrast, two out of three populations positively associated with oysters, namely V.

splendidus Pop #23 and V. crassostreae, were highly resistant to all antimicrobials tested (CgBigDef-1, ROS, zinc and copper), which may confer a great advantage in colonizing their molluscan host. Only V. splendidus Pop #24, positively associated with oysters, showed high resistance to copper, zinc and ROS but lower resistance to CgBigDef-1). Noteworthy, this population, like V. crassotreae (Lemire et al., 2015), colonizes oysters affected by the POMS (Bruto et al., 2017), a disease in which the OsHV-1 virus suppresses CgBigDef-1 expression (de Lorgeril et al., 2018). Therefore, the resistance profile of vibrio populations positively associated with oysters agrees with their ecology and variations of their niche biochemical landscape (determined by the physiology of their healthy or immune-compromized host). This suggests a role of homeostasis of antimicrobial defenses in shaping the Vibrio communities associated with oysters.

To determine the contribution of Big-defensins in control of the commensal microbiota and opportunistic pathogens like vibrios, is one of the objectives of the doctoral project of Noémie de San Nicolas, that is currently been developed in our team. Through an RNAi approach, they aim to block in vivo the transcription of Big-defensins and evaluate how it affects the stability of the associated microbiota and the success of infection of known opportunistic pathogens, functionally testing the immunosuppression hypothesis.

SECTION II. THE ROLE OF CYTOTOXICITY IN VIBRIO OF THE SPLENDIDUS CLADE ASSOCIATED WITH POMS

II.1. CONTEXT AND OBJECTIVE OF THE STUDY

Oysters possess a remarkable capacity to maintain an immune homeostasis in the presence of the abundant and diverse microbial communities that are associated with its tissues. The biotic and abiotic factors that can lead into the loss of this homeostasis are of particular interest as they are key risk factors for the onset of mass mortalities.

As described earlier, during POMS, the OsHV-1 infection of oysters causes immunosuppression disrupting immune homeostasis and favoring proliferation of pathogenic bacteria ( de Lorgeril et al, 2018).

Within the bacteria associated with this dysbiosis, vibrios from the Splendidus clade, notably V. tasmaniensis and Vibrio crassostreae, are among the most studied.

One strain, V. tasmaniensis LGP32, originally isolated from a mortality event in 2004 causes mortalities when injected into oysters (Gay et al. 2004). It is a facultative intracellular pathogen cytotoxic to hemocytes [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] Vanhove | Copper Homeostasis at the Host Vibrio Interface: Lessons from Intracellular Vibrio Transcriptomics[END_REF]. The sequencing of its genome and the development of molecular tools for genetics investigations (Le Roux et al. 2007, 2009) established this strain as a model of hemocyte-vibrio interaction in a pathogenic context [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][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] V-Vanhove | Outer Membrane Vesicles are Vehicles for the Delivery of Vibrio tasmaniensis Virulence Factors to Oyster Immune Cells[END_REF][START_REF] Vanhove | Copper Homeostasis at the Host Vibrio Interface: Lessons from Intracellular Vibrio Transcriptomics[END_REF].

The other well described species, V. crassostreae, has not been characterized on its specific interactions with hemocytes. Yet, it has been shown that functionally cohesive populations of V. crassostreae replace the commensal communities in the oyster during disease and share virulence as a core function with an extracellular protein of unknown function, encoded by the gene r5.7, and a plasmid (pGV1512), both necessary for full virulence (Bruto et al., 2017). These virulence mechanisms were characterized in the strain V. crassostreae J2-9 isolated by Lemire et al. (2015). Interestingly r5.7 which is widely distributed among virulent populations of the Splendidus clade, and the virulence plasmid pGV1512, characteristic of virulent V. crassostreae strains are absent from V. tasmaniensis, suggesting that virulence in this these two species is achieved through different mechanisms that determine the success of infection.

Puzzled by the varrying association of these two Vibrio spieces to POMS-diseased oysters, we performed a broader study to characterize the pathogenic mechanisms used by V. crassostreae and V. tasmaniensis. We used 4 virulent strains as well a non-virulent strain from each species with which we developed an integrative study of the host-microbe interaction.

We described the cellular interactions and molecular changes that occur in the host and the bacteria during the within-host phase of the infection (in vivo dual RNAseq), along with functional validation of the bacterial candidate genes newly identified (gene knock-down).

In article 2, we present the results of this study. Our data, allowed us to conclude that within the Splendidus clade, V. crassostreae and V. tasmaniensis had acquired species-specific mechanisms that seems to converge in a common objective, which is to suppress the cellular defenses of the oysters using different cytotoxic mechanisms for a successful colonization during pathogenesis. These species-specific mechanisms rely on R5.7 in V. crassostreae and a newly identified T6SS encoded by chromosome 1 in V. tasmaniensis.

In complementrary results, we have identified a T6SS-secreted effector responsible for V. tasmaniensis cytotoxicity toward hemocyte. However, this effector does not seem to be required for virulence in vivo. These preliminary results open the way to new projects on the characterization of T6SS effectors involved in V. tasmaniensis pathogenesis

II.2. ARTICLE N°2. SPECIES-SPECIFIC MECHANISMS OF CYTOTOXICITY TOWARD IMMUNE CELLS DETERMINE THE SUCCESSFUL OUTCOME OF VIBRIO INFECTIONS.

By Rubio, T.*, Oyanedel, D.*, Labreuche, Y., Toulza, E., Luo, X., Bruto, M., Chaparro, C., Torres, M., de Lorgeril, J., Haffner, P., Vidal-Dupiol, J., Lagorce, A., Petton, B., Guillaume, M., Jacq, A., Le Roux, F., Charrière, G.M., Destoumieux-Garzón, D. (2019). LGP32, J2-9 and J2-8 induced 58.7%, 46% and 2.3% of mortalities of oysters respectively. LMG20012T did not induce mortalities. J2-9 and LGP32 conditions are not significantly different (Log-rank p-value=0.560). (B) Mortalities on oyster Decipher family #1. LGP32, J2-9 and J2-8 induced 40.7%, 57.1% and 3.3% of mortalities in oysters, respectively. LMG20012T did not induce mortalities. J2-9 and LGP32 conditions are not significantly different (Log-rank p-value=0.401).

Figure S3 l Only non-virulent vibrios are degraded by hemocyte phagocytosis. Confocal microscopy of hemolymph collected from animals infected with V. crassostrea J2-9 or the non-virulent control J2-8. Juvenile oysters were injected with 1.5 x 10 8 CFU of GFP-expressing vibrios. For each experimental condition, hemolymph was withdrawn from five oysters, at 2, 4, and 8 h after injection. Hemocytes were observed by confocal microscopy on cytospin glass slides after the staining of nuclei and actin cytoskeleton with DAPI and phalloidin-TRITC, respectively. In oysters injected with J2-9, J2-9 cells were mostly extracellular and were detected at every time-point (white arrows). In contrast, the non-virulent J2-8 was massively phagocytized (yellow arrows) as soon as 2 h after infections showing loss of cell interatity at 4 h; they became almost undetectable after 8h in the oyster hemolymph. Scale bar: 10 µm.

Figure S4 l Validation of the RNAseq data using quantitative RT-PCR. Nineteen oyster genes were selected and their expression (relative to the control genes ef1, c23 and RP56) was quantified by qRT-PCR in RNA samples used for each RNAseq condition. Expression data were compared to those obtained using the RNAseq approach (normalized read counts). Fold changes of RNAseq were fully validated by qPCR (R 2 =0.926, p<0.001 linear regression test ). LGP32 and J2-9 transcripts were sorted according to foldchanges, the 200 most-induced genes during oyster colonization were then counted according to functional categories (genes without annotated function were not included). Table S1 l qPCR quantification of vibrios in oyster tissues.

Real-time qPCR was performed on 40ng/μl of DNA on the same samples using for RNAseq. Quantification of vibrios was calculated in number of vibrio genome copy per number of oyster genome copy because of Avogadro number and genome size of oyster and vibrios. These primers have been chosen because they are specific for each vibrio species. The ratio is the mean of 3 independent experiments. LGP32, V. tasmaniensis Gay et al., 2004 LGP32 + pMRB-PLACgfp [START_REF] Vanhove | Copper Homeostasis at the Host Vibrio Interface: Lessons from Intracellular Vibrio Transcriptomics[END_REF] LGP32 ΔvipA T6SS1::pSW23T necessary. Conjugation between E. coli and Vibrio were performed at 30 °C as described previously (8). To label the strains with a fluorochrome, gfp gene was cloned in Apa1/Xho1 sites of the pMRB plasmid known to be stable in Vibrio spp. (9) resulting in a constitutive expression from a Plac promoter. Gene inactivation was performed by cloning about 500 bp of the target gene in pSW23T (10) and selecting on Cm the suicide plasmid integration obtained by a single recombination (11). Mutants were screened for insertion of the suicide vector by PCR using external primers flanking the different targeted genes.

Histology

Groups of 10 spats (1.5-2 cm) were injected with 7.5 x 10 7 CFU of GFP-expressing LGP32 or J2-9. Groups of 5 spats were injected with 7.5 x 10 7 CFU of GFP-expressing LMG20 012T or J2-8 (controls). Two non-injected oysters were used as controls. Spats were distributed in one tank containing 3 L of seawater per condition, at 20 °C. Oysters were sampled and fixed 8 h after injection, in seawater Davidson (20% of formaldehyde, 30% of sterile seawater, 10% of glycerol and 30% of 95% ethanol). Histological sectioning was performed at HISTALIM, Montpellier, France. Briefly, oysters were extracted from the shell, tissues were dehydrated and included in paraffin with an inclusion automaton. Longitudinal sections were immunostained with an anti-GFP primary antibody coupled to alkaline phosphatase (Abcam). Revelation was performed with the PA-Red kit (Roche). Sections were scanned with slide scanner Hamamatsu NANOZOOMER. Hemocytes were observed with the ZEISS Z1 microscope equipped with Zeiss 63X/1.4 Plan-Apo Oil objective.

In vitro cytotoxicity assays

Hemocytes were plated in 96 well-plates (2 x 10 5 cells/well) or in 12 well-plates (5 x 10 5 cells/well) with a Transwell Permeable Support with a pore size of 0.4 µm. After 1 h, plasma was removed and 5 µg/µl Sytox Green (Molecular Probes) diluted in 200µl sterile seawater was added to each well. Vibrios, previously washed and opsonized in plasma for 1 h, were then added to the wells at a MOI of 50:1. Sytox fluorescence was monitored (ƛex 480nm/ƛem 550nm) for 15 h using a TECAN microplate reader. Maximum cytolysis was determined by adding 0.1% Triton X-100 to hemocytes. To determine the role of phagocytosis in the vibrioinduced cytolysis of hemocytes, 5 µg/ml cytochalasin D was added to the wells 30 min before addition of vibrios. Statistical analysis was performed using RM-ANOVA.

Confocal microscopy of interaction between hemocytes and vibrios in vivo

Groups of 15 juvenile oysters (4-5 cm) were injected with 1.5 x 10 8 CFU of GFP-expressing vibrios (either J2-8 or J2-9) a priori washed and resuspended in sterile seawater. Control oysters received an injection of an equal volume of sterile seawater. For each experimental condition, hemolymph was collected from five oysters at 2 h, 4 h and 8 h after injection. Samples were fixed with 4% paraformaldehyde for 15 min and cytospun at 600 g for 10 min onto Superfrost glass slide. Glass slides were then washed with PBS and stained with 0.25 µg/ml DAPI (Sigma) and 1 µg/ml Phalloïdine-TRITC (Sigma) for 30 min at room temperature. Microscopy was performed using Leica DM 2500 confocal microscope with a Leica 63x ACS APO 1.3 objective.

Dual RNA-seq experiments Three independent experimental infections were performed on juvenile oysters from family Decipher#14 as described above. For every independent experiment, 150 oysters (30 animals per condition) were injected with either bacteria (10 7 CFUs of LGP32, LMG20012T, J2-9, J2-8 overnight cultures washed in sterile seawater), or sterile seawater (mock-infected oysters). In addition, both bacterial suspensions and 30 non-treated oysters were collected at time 0. For every experimental condition, 30 oysters were then sampled 8 h after injection (i.e. before mortality occurred). Oysters were extracted from shell and immediately frozen in liquid nitrogen. Pools of tissues from 30 oysters were ground by bead-beating (Retsch, Mixer Mill MM400) with a stainless-steel ball pre-chilled in liquid nitrogen. Both, oyster powder homogenates and bacterial suspensions, were lysed in 1.6 ml Trizol (Life technologies Inc.) and vortexed for 2.5 h at 4 °C. Samples were then stored at -80 °C until RNA extraction.

RNA was extracted from oyster tissues using the Direct-Zol RNA Miniprep kit from Proteigene, and according to the manufacturer's protocol. RNA concentration and purity were checked with a Nanodrop ND-1000 spectrometer (Thermo Scientific, Les Ulis, France; oyster RNA-seq) or a DS-11 Spectrophotometer (DeNovix Inc.; bacterial RNA-seq) and the integrity of the RNA was analyzed by capillary electrophoresis with an Agilent BioAnalyzer 2100. For oyster RNAseq, library construction and sequencing were performed on polyadenylated mRNAs by the Fasteris Company (Switzerland, https://www.fasteris.com). Directional cDNA libraries were constructed and sequenced on an Illumina Hiseq, in paired-end reads of 2 x75 bp. For bacterial RNA-seq, total RNA obtained from oyster tissues was used to prepare non rRNAbacterial RNA enriched libraries. After polyadenylated mRNAs (i.e. host mRNAs) were removed using MICROBEnrich TM Kit (Ambion), cDNA oriented sequencing libraries were prepared starting from 100 ng enriched RNA using Ovation Universal RNA-Seq System kit (NuGEN). Library construction included a depletion step of abundant, non-desired sequences, using a Nugen-customized mixture of primers complementary to RNA sequences to be depleted, viz. oyster nuclear, mitochondrial, and ribosomal and bacterial ribosomal RNA sequences. The libraries were quantified by DeNovix dsDNA Broad Range Assay (DeNovix Inc.) and the quality was monitored using Agilent High Sensitivity DNA kit (Agilent Technologies). For each condition, three biological replicate libraries were prepared. Libraries were sequenced in paired-end mode (2 × 150 bp) by Fasteris on an Illumina HiSeq 4000, to obtain ~ 100 million reads per sample/per run.

Oyster transcriptome analysis

For oyster RNA-seq data, data treatment was carried out using the galaxy instance of the IHPE lab (https://bioinfo.univ-perp.fr) (12). Phred quality scores were checked using the FastX toolkit galaxy interface (http://hannonlab.cshl.edu/fastx_toolkit/) and were higher than 26 for over 90% of the read length for all the sequences. All the reads were thus kept for subsequent analyses. Mapping to the C. gigas reference genome (assembly version V9 ( 13)) was performed using RNAstar (Galaxy Version 2.4.0d-2, ( 14)). HTSeq-count was used to count the number of reads overlapping annotated genes (mode Union) (Galaxy Version v0.6.1) (15).

The resulting files were used as input to determine differential expression for each feature between oysters injected with vibrios and mock-infected oysters using DESeq2 ( 16). Fold changes in gene expression between test animals and controls were considered significant when the adjusted P-value (Padj) for multiple testing with the Benjamini-Hochberg procedure, which controls false discovery rate (FDR), was < 0.05. The same threshold was applied to fold changes in gene expression between mock-infected oysters and naïve controls. Oyster RNAseq data has been made available through the SRA database (BioProject accession number PRJNA515169) under SRA accessions SRR8551076-SRR8551093.

As genes for C. gigas antimicrobial peptides (AMPs) are not annotated in the C. gigas reference genome (assembly version V9, ( 13)), read counts for all AMPs were obtained by alignment to an in-house database of AMP protein sequences using DIAMOND 0.7.9 (17). The AMPs database was prepared using diamond 'makedb' and the reads for each sample/replicate were compared to the database using diamond 'blastx'. Alignments were filtered for the best hit and e-value < 1e-6. Read counts for each AMP were normalized against the total sequence number for each sample/replicate.

Vibrio transcriptome analysis

For bacterial RNA-seq data, all the analyses but the last step (DESeq2) were carried out on a personal computer (Ubuntu 16.04 LTS), on which the necessary tools were executed from a docker container image (https://www.docker.com/why-docker). Raw Illumina® sequencing reads were trimmed by Trimmomatic in paired-end mode (18) to remove sequences corresponding to the NuGen provided adapters, clipping part of reads having a quality score of below 20, and dropping reads of less than 36 nt. The two resulting files containing the forward and reverse reads, respectively, of the paired-end reads, were used as input for Bowtie2 (19) to align the reads on V. tasmaniensis LGP32 or V. crassostrea J2-9 genome, respectively. In the case of LGP32, where the complete genome is available, the sequences of the two chromosomes were concatenated. In the case of J2-9, the genomic sequence corresponds to a concatenation of multiple contigs. In addition, whereas the rRNA operon was present in multiple copies in the genome of LGP32, it was unique in the J2-9 sequence file. We thus artificially duplicated the operon sequence in J2-9 so that reads corresponding to rRNA genes were listed by Bowtie2 as those with multiple alignments, and thus were eliminated as it was the case for LGP32.

As expected, in the case of bacterial RNA-seq samples resulting from oyster infection experiments, the rate of read alignment on the bacterial genome was ~2% at the most, whereas it was more than 98% in the case of the in vitro grown bacteria. To prevent a potential bias induced by this difference in the subsequent computational steps, we introduced a random downsampling step of the aligned reads of the control samples (in vitro growth). This was done using the command "downsampleSam" of the Picard tools (http://broadinstitute.github.io/picard/command-line-overview.html#DownsampleSam) with p= 0.02, leading to a number of aligned reads similar to what was obtained in case of the oyster infection samples. Counts for each annotated feature (CDS + sRNAs) of either V. tasmaniensis LGP32 or V. crassostreae J2-9 were then computed using featureCounts (20).

The resulting files were used as input to determine differential expression for each feature between oyster injected with vibrios and vibrios grown in vitro by DESeq2 (16). DESeq2 was carried out on a Galaxy instance at the INRA migale bioinformatics platform: http://migale.jouy.inra.fr/galaxy/. Genes were considered as differentially expressed between tested conditions and controls when |log2(fold change)| was ≥1 and adjusted Pvalue (Padj) for multiple testing with the Benjamini-Hochberg procedure was < 0.05. Bacterial RNA-seq data has been made available through the SRA database: BioProject accession number PRJNA521688 under SRA accessions SRR8567597-SRR8567602 (Vibrio crassostreae J2-9) and BioProject accession number PRJNA521693 under SRA accessions SRR8573808-SRR8573813 (Vibrio tasmaniensis LGP32).

Functional gene ontology annotations

For oyster genes, Blastx (21) comparison against NR database was performed for each set of genes with a maximum number of target hits of 20 and a minimal e-value of 0.001. XML blast result files were loaded onto Blast2GO ( 22) for GO mapping and annotation of the B2G database. Functional categories corresponding to significantly modulated genes (331 genes), were then determined manually; this was supported by background literature review.

A heatmap of the mean of log2 fold change in three independent experiments was then generated using cluster 3.0 (http://bonsai.hgc.jp/~mdehoon/ software /cluster/software.htm).

For bacterial genes, annotations of differentially expressed genes (fold change ≥ 2 and padj ≤ 0.05) were manually curated using the MicroScope platform ( 23) and the MetaCyc data base (24). Based on these curated annotations, each gene was assigned to one of 32 functional categories (see Figs 5 & S5 for the list of categories).

In addition, with the help of the tools provided by MicroScope, which allows for comparison between genomes based on sequence and synteny conservation; we identified genes which were common to both (J2-9 and LGP32) differential transcriptomes, with the exception of genes of unknown function. This subset of genes is defined as the intersection between the J2-9 and LGP32 differential transcriptomes.

RT-qPCR

RT-qPCR was performed either on the same RNA samples as those used for deep sequencing to validate RNAseq data (Fig. S3), or on specific RNA samples in order to monitor the timecourse of bacterial gene expression in oysters (Fig S6). cDNA was synthetized using M-MLV RT (Invitrogen Inc.) using 1 µg of RNA and random primers (250 ng). Real-time qPCR was performed at the qPHd platform of qPCR in Montpellier using the Light-Cycler 480 System (Roche) and oyster gene specific primers (Table S8). The total Real-time qPCR reaction volume was 1.5 μl with 0.5 μl cDNA (40 ng µl -1 ) and 1 μl LightCycler 480 SYBR Green I Master mix (Roche) containing 0.5 μM PCR primer (Eurogentec SA). Relative expression was calculated using the 2 -ΔΔCq method (Pfaffl, 2001), with normalization to the C. gigas reference genes, namely, Cg-RPL40 (GenBank FP004478), Cg-RPS6 (GenBank HS119070) and Cg-EF1α (GenBank AB123066). For understanding the time-course of the bacterial gene expression, 6phosphofructokinase (VS_2913) and Methyl glyoxal synthase (VS_II1055) genes [START_REF] Vanhove | Copper Homeostasis at the Host Vibrio Interface: Lessons from Intracellular Vibrio Transcriptomics[END_REF] were used as reference genes to monitor the relative expression of vipA1 and vipA2.

5.

Petton

II.3. COMPLEMENTARY RESULTS. ASSESSING THE ROLE OF AN EVPP ORTHOLOG AS A PUTATIVE T6SSCHR1-LGP32 EFFECTOR IN LGP32

Despite the many in vivo, in vitro and in silico global approaches that we have been using up to now, the nature of the effectors enabling V. tasmaniensis cytotoxicity have remained unknown four years after the description of LGP32 cytotoxicity towards hemocytes [START_REF] Vanhove | Copper Homeostasis at the Host Vibrio Interface: Lessons from Intracellular Vibrio Transcriptomics[END_REF], a key determinant of its virulence to oysters. We have shown in article 2 that the T6SS encoded on V. tasmaniensis LGP32 chromosome 1 is essential for V. tasmaniensis virulence. We were therefore interested in characterizing the toxic effectors that are secreted through this system. As mentioned in article 2, the vgrG and paar-encoded domains, which form the tip of the T6SS to which effectors can form complexes and be delivered to the target cell. Moreover, the C-terminal domain of several of these VgrG and PAAR proteins have Cterminal domains with predicted enzymatic activity toxic for prokaryotic and eukaryotic cells (Shneider et al., 2013;Bondage et ai., 2016). However, no potential C-terminal effectors domain could be identified in the VgrG and PAAR proteins found in all virulent strains of V.

tasmaniensis [START_REF] Rubio | Speciesspecific mechanisms of cytotoxicity toward immune cells determine the successful outcome of Vibrio infections[END_REF]. Still, we identified evpP, a non-VgrG non PAAR T6SS effector characterized in the intracellular pathogen Edwardsiella tarda which is involved in cell invasion and inhibition of the host immune response (Chen et al., 2017). The effector EvpP secreted by the T6SS of E. tarda suppresses pyroptosis. The suppression of inflammasome activation and pyroptosis is suggested to be beneficial to E. tarda colonization in the host (Chen et al., 2017). Remarkably, in his thesis, Tristian Rubio showed that inhibition of caspases with z-vad tended to inhibit LGP32 cytotoxicity. As the effector subunit caspase-1 activates a pyroptotic cell-death pathway, it was hypothesized that LGP32-trigerred cell-death is due to pyroptosis (Rubio, 2017).

In order to determine, the potential role of evpP in the cytotoxicity and virulence of LGP32, Romain Guiraud (during his internship) and I constructed an in-frame deletion mutant for the coding sequence of evpP in V. tasmaniensis which we phenotyped both in vitro and in vivo.

Materials and methods

Construction of the suicide vector

An In-frame deletion of the targeted coding sequence was performed by allelic exchanged using the suicide plasmid pSW7848T according to (Le Roux et al. 2007).

Two 500 bp fragments flanking the region to delete were amplified with the following primers.

5'evpPFw GTATCGATAAGCTTGATATCGAATTCCCTTTAGGTAGAAACATTGTG 5'evpPRv GCTAGACCGGTACTGGTGGTTTACAATATCACCACCAAGC 3'evpPFw GCTTGGTGGTGATATTGTAAACCACCAGTACCGGTCTAGC 3'evpPFw CCCCCGGGCTGCAGGAATTCCATGCAGAGATTAGCGTTCC pSWF GAATTCCTGCAGCCCGGGGG psWR GAATTCGATATCAAGCTTATCGATAC
Two independent PCR amplifications were performed using 5'evpPFw + 5'evpPRv to amplify 500 bp upstream evpP and 3'evpPFw + 3'evpPFw for the 500 bp downstream evpP. The pSW7848T linear sequence was amplified by a PCR inside out using the vector DNA and the primers pSWF + psWR with a high-fidelity polymerase (Q5 ® High-Fidelity DNA Polymerase, NEB).

The recombinant plasmid was then assembled using the Gibson assembly master mix (Nex England Biolabs) according to the provider's instructions. Correct insertion of the fragment with the correct size was verified by digestion with the restriction enzyme EcoRI and visualization of the digestion products by gel electrophoresis. Strains E. coli π3813 and E. coli β3914 were used as plasmid hosts for cloning and conjugation of the constructed vector, respectively (Le Roux et al., 2007) Conjugation between E. coli β3914 with V. tasmaniensis LGP32

The donor strain E. coli β3914 carrying the pSW4878T:evpP was grown in LB media supplemented with chloramphenicol 35 ug/mL and 0.5 mM of diaminopimelic acid (DAP)

and the recipient V. tasmaniensis in LB supplemented with 0.5 mM NaCl. Overnight cultures of both strains were diluted at 1:100 in culture media without antibiotic and grown at 30°C to an OD600nm of 0.3. Conjugation was performed by a filter mating procedure described previously (Le Roux et al., 2007) with 1 ml donor and 10ml recipient. Conjugations were performed overnight on filters incubated on LBA + NaCl 0.5N + diaminopimelic acid (DAP) plates at 30°C. Counter-selection the donor E. coli β3914, auxotrophic for DAP, was done by plating on a medium devoid of DAP, supplemented with chloramphenicol 5 ug/mL for selection of vibrio Cm R trans-conjugants, and 1% glucose to suppress the expression of the ccdB toxin. For mutagenesis, Cm R resistant colonies were grown in LB + NaCl 0.5N + glucose 1% up to late logarithmic phase and spread on plates containing 0.2% arabinose to induce the expression of the ccdB toxin and select for vector excision. Colonies susceptible to chloramphenicol, indicative of successful excision of pSW7848. Mutants were screened by PCR using external primers to verify the correct deletion of the target region (Figure 23).

Cytototoxicity assays and Experimental infections

Procedures are identical to materials and methods from article 2

Results

Two evpP deletion mutants were selected after PCR-verification of the correct gene deletion (Figure 23). They were first used on cytotoxicity assays against oyster hemocytes and compared to the wild-type LGP32 (cytotoxic) and the vipA mutant, which harbours a defective T6SS1 machinery and is devoid of cytotoxicity [START_REF] Rubio | Speciesspecific mechanisms of cytotoxicity toward immune cells determine the successful outcome of Vibrio infections[END_REF]. Our results

showed that both evpP mutants lacked cytotoxicity towards oyster hemocytes, similar to the vipA deletion mutant (Figure 24). Therefore, our results strongly suggest that evpP is a key effector of LGP32 T6SS1 cytotoxicity toward hemocytes. As they harbored identical phenotypes in vitro, we next tested one of the two deletion mutants in oyster experimental infection together with the wild-type LGP32 (virulent) and the

LGP32 vipA-i mutant, which is non-virulent in vivo (Rubio et al., 2018). Our assays showed no impairment in virulence of the evpP mutant when injected into oysters (Figure 25) in contrast with the strong effect observed upon inhibition of the assembly of the T6SS Chr1-LGP32 (LGP32 vipA-i). This suggests that evpP is not required for virulence in vivo. These results highlight a dycotomy between cytotoxicity toward hemocytes and virulence, which we had never observed with our previous LGP32 mutants.

Noteworthy, the mortality induced by wild-type LGP32 control was particular high (80% after 24h) in our experimental infection. If the evpP effector is one effector among diverse T6SS

effectors that contribute to virulence, the experiment should be repeated by injecting lower doses to oysters that help revealing more subtle phenotypes. This strategy was used for instance by Piel et al (2019) to reveal the role of T6SS in V. crassostreae cytotoxicity.

If evpP is indeed key for cytotoxicity but not required for virulence, it strongly suggest that other unidentified effectors secreted by this T6SS Chr1-LGP32 are responsible for the virulence of

LGP32. One possibility is that these other effectors, which may be acting on distinct cell types, are not revealed by our in vitro assays.

Further investigation of the evpP mutant, in particular complementation assays, are now needed to confirm our results and determine the role of evpP and/or other T6SS effectors in

LGP32 pathogenesis. It will also be essential to determine whether and how V. tasmaniensis evpP relates to pyroptosis, since in evpP has been described as a pyroptosis suppressesor in E.

tarda.

SECTION III. CHEATING BEHAVIOR IN VIBRIOS ASSOCIATED WITH POMS

III.1. CONTEXT AND OBJECTIVE OF THE STUDY

The Pacific Oyster Mortality Syndrome (POMS) has been extensively characterized in the French Atlantic environment where Vibrio species from the diverse Splendidus clade have been recurrently found associated with moribund oysters as secondary opportunistic colonizers that contribute significantly to the systemic infection leading to oyster death (Gay et al., 2004;Saulnier et al., 2010;Lemire et al., 2015;Bruto et al., 2017Bruto et al., , 2018)). Nonetheless, POMS affect oyster farming worldwide, and is observed in environments with different ecologies where variations in the composition of the Vibrio community are expected. Indeed, composition and abundance of Vibrio communities in the marine ecosystems are highly influenced by planktonic composition and environmental factors such as temperature, salinity, pH and dissolved oxygen (Turner et al., 2009;[START_REF] Takemura | Associations and Dynamics of Vibrionaceae in the Environment, From the Genus to the Population Level[END_REF][START_REF] Li | The Role of Vibrio Vulnificus Virulence Factors and Regulators in its Infection-Induced Sepsis[END_REF].

Moreover, as shown by Bruto et al., (2017) population structure of vibrio communities in the water column suffer local seasonal variations affecting as consequence Vibrio population assembly in oysters.

The Thau Lagoon is a Mediterranean marine ecosystem with significant oyster farming activity that has also suffered since 2008 from repeated episodes of POMS affecting juveniles with a strong presence of OsHV-1 restricted to the farming areas (20% of the total surface) [START_REF] Pernet | Mass Mortalities of Pacific Oysters Crassostrea gigas Reflect Infectious Diseases and Vary with Farming Practices in the Mediterranean Thau Lagoon, France[END_REF]. The Thau lagoon has an ecology that differs from the Atlantic coast, including higher salinity and a temperature regimen that during half of the years ranges between 16-30°C [START_REF] Lopez-Joven | Oyster Farming, Temperature, and Plankton Influence the Dynamics of Pathogenic Vibrios in the Thau Lagoon[END_REF].

With the initial obsjective to characterize the vibrio community associated with POMS in the Mediterranean, we performed a series of Vibrio samplings, starting with a POMS episode during autumn of 2015, confirmed by the presence of OsHV-1 in moribund oysters. We characterized the population structure of vibrios in oysters and the water column and investigated the molecular basis underlying the observed associations.

In article 3 (in preparation), we present the results of the present study. Our data show that in the Thau Lagoon OsHV-1 infection was associated with a particular prevalence of members of the Harveyi clade, with strains taxonomically assigned to Vibrio harveyi and V. rotiferianus species preferentially associated with oyster tissues and V. jasicida and V. owensii species present in the water column excluded from moribund oysters. This preferential association of V. harveyi and V. rotiferianus to oysters could be confirmed experimentally by the reproduction of the polymicrobial infection in mesocosm experiments. The assessment of virulence potential and cytotoxic capacity, comparative genomics and functional assays led us to conclude that two different strategies for colonization were present among these two vibrio species from the Thau Lagoon. On the one hand, the highly virulent V. harveyi suppressed the host cellular defenses through cytotoxic properties, as already observed in the Splendidus clade. On the other hand, we characterized the cheating strategy for iron acquisition of V. rotiferianus through non-cooperative siderophore uptake

Introduction

Since 2008, the worldwide production of Crassostreae gigas oysters has been dramatically

affected by the Pacific Oyster Mortality Syndrome (POMS), whose severity reached up to 100% mortality rates in French farms and expanded worldwide [START_REF] Samain | Summer mortality of Pacific oyster Crassostrea gigas[END_REF]EFSA, 2015;[START_REF] Burge | First comparison of French and Australian OsHV-1 µvars by bath exposure[END_REF] This polymicrobial disease is caused by a Ostreid herpesvirus,

OsHV-1 μVar, and opportunistic bacteria (de Lorgeril et al., 2018) , among which members of the Vibrionaceae family (hereafter vibrios) are the best characterized (Lemire et al., 2015;Petton et al., 2015;Bruto et al., 2017;Piel et al., 2019). Oyster families susceptible to POMS present an overall lower basal expression of immune-related genes [START_REF] Lorgeril | Differential basal expression of immune genes confers Crassostrea gigas resistance to Pacific oyster mortality syndrome[END_REF] and a delayed (inefficient) transcriptomic response to the OsHV-1 μVar infection, compared to resistant families (de Lorgeril et al., 2018;Lafont et al., 2020). Viral replication in susceptible oysters leads to the host entering an immune-compromised state, causing a dysbosis that evolves towards bacteremia [START_REF] Lorgeril | Differential basal expression of immune genes confers Crassostrea gigas resistance to Pacific oyster mortality syndrome[END_REF] Along the French Atlantic coastline, multiple Vibrio species of the highly diverse Splendidus clade (e.g. Vibrio crassostreae, Vibrio splendidus, Vibrio tasmaniensis and Vibrio cyclitrophicus) have been associated with POMS episodes and their virulence potential has been experimentally confirmed by Le Roux and collaborators (Gay et al., 2004;Saulnier et al., 2010;Lemire et al., 2015;Bruto et al., 2017Bruto et al., , 2018) ) The repeated occurrence of the species V.

crassostreae was evidenced during summer mortalities (Lemire et al., 2015;Bruto et al., 2017;de Lorgeril et al., 2018) We recently showed that two virulent species associated with the disease, namely V. crassostreae and V. tasmanienesis, use species-specific cytotoxic properties to escape oyster cellular defenses and cause a systemic infection (Piel et al., 2019;[START_REF] Rubio | Speciesspecific mechanisms of cytotoxicity toward immune cells determine the successful outcome of Vibrio infections[END_REF] Aside from the French Atlantic environment, POMS affect oyster farming in a large number of countries worldwide, which harbor diverse ecology and shellfish farming systems [START_REF] Fuhrmann | Low pH reduced survival of the oyster Crassostrea gigas exposed to the Ostreid herpesvirus 1 by altering the metabolic response of the host[END_REF]. Recently, we showed that POMS is caused by two distinct viral populations of OsHV-1 µvar in French Atlantic and Mediterranean ecosystems [START_REF] Delmotte | Contribution of Viral Genomic Diversity to Oyster Susceptibility in the Pacific Oyster Mortality Syndrome[END_REF]. Still, little is known on the population structure and distribution of vibrios co-occurring with OsHV-1 µvar in these other ecosystems. In addition, when vibrios are found associated with diseased animals, it is often difficult to disentangle vibrios with virulence potential from opportunistic colonizers. The underlying question is the role of Vibrio species in disease expression across environments.

Here, we took advantage of a Mediterranean marine ecosystem, the Thau lagoon (south of France, N 43 251, E 03 391) hosting a significant oyster farming activity (20% of the total surface), whose ecology and farming practices highly differ from the Atlantic (Petton et al., 2013;Pernet et al., 2014b). In this environment, which also suffers from dramatic mortalities of juvenile oysters (Petton et al., 2013;Pernet et al., 2014b), mortality events occur twice a year, in spring and autumn, when the seawater temperature is in the 16-24°C range [START_REF] Lopez-Joven | Oyster Farming, Temperature, and Plankton Influence the Dynamics of Pathogenic Vibrios in the Thau Lagoon[END_REF]. We investigated the Vibrio community associated with the disease in the Thau lagoon by immersing specific pathogen-free juvenile oysters in the lagoon during the autumn 2015 mortalities. The Vibrio population structure showed a strong prevalence of the Harveyi clade in diseased oysters. V. harveyi and V. rotiferianus species were the most prevalent in oyster tissues, whereas V. jasicida and V. owensii were mainly associated with the water column. We confirmed further this association experimentally in a mesocosm experiment:

OsHV-1 μVar enabled stable colonization by V. harveyi and V. rotiferianus but not V. jasicida and V. owensii. Colonization by V. harveyi and V. rotiferianus was facilitated by the virusmediated immune suppression of oysters, as revealed by RNAseq analyses. Comparative genomics and functional assays were finally used to identify determinants of Vibrio colonization. The species V. harveyi was shown to carry virulence potential, being cytotoxic for oyster immune cells. It also produces vibrioferrin, a key siderophore that benefits to the oyster colonizer V. rotiferianus, which behaves as a cheater in this context. Our data unravel two strategies to colonize an oyster in V. harveyi and V. rotiferianus, one is the active dampening of oyster defenses whereas the other one relies on the use of key siderophores produced by the microbiota.

Results and discussion

V. harveyi and V. rotiferianus are two main Vibrio species associated with POMS in the Thau lagoon.

We characterized the population structure of vibrios isolated from juvenile oysters and from the seawater during a mortality outbreak of juvenile oysters that occurred in October 2015 in the Thau lagoon, south of France. The oysters were positive for OsHV-1 μVar detection (Supplementary Table 1), revealing an on-going event of the POMS. A total of 472 bacterial isolates were sampled from oyster tissues and from the size-fractionated water column by plating on TCBS medium, selective for vibrios (Supplementary Table 2). Partial hsp60 sequences were obtained for 448 isolates. Among them, 304 sequences (67.8 %) exhibited ≥ 95% identity with Vibrio type-strain sequences. Below this threshold, isolates were not included in the study. We found a strong association of the Harveyi clade to oyster tissues with 54 among the 55 isolates belonging to the species V. harveyi, V. rotiferianus and V. owensii (Figure 1). Only one isolate from oyster, assigned to V. chagasii (Splendidus clade), fell outside the Harveyi clade. The water column showed a higher diversity of Vibrio species with Harveyirelated strains representing 21% of the total isolates (53/249) with a predominance of V.

jasicida (39/53). The taxonomical assignation and phylogeny of the Harveyi-related strains were confirmed by a multi-locus sequence typing (MLST) based on 4 genes (hsp60, rctB, topA and mreB) (Figure 2A). A total of 104 isolates could be assigned to four main species: V. harveyi (n=63), V. rotiferianus (n=16), V. jasicida (n=15) and V. owensii (n=6), (Figure 2).

Among them, V. harveyi and V. rotiferianus showed a positive association with oyster tissues whereas V. jasicida and V. owensii were almost exclusively associated with the water column (Figure 2A). The dominance of the Harveyi clade in POMS-diseased oysters was confirmed by PCR on three subsequent samplings, within and outside POMS episodes: Harveyi isolates, dominated by the species V. harveyi, were only associated with oysters infected with OsHV-1 µvar (Supplementary Table 1).

V. harveyi strains have been shown to cause disease in aquatic animals including marine fish and crustaceans (Kraxberger-Beatty et al., 1990;Saeed, 1995;Hispano et al., 1997;Diggles et al., 2000;Alcaide et al., 2001;Company et al., 2002;Tendencia, 2002) In agreement with our data, Saulnier et al. isolated a virulent V. harveyi strain (03/082 2T1) in 2003 from C. gigas oyster spat during a mortality episode in the Thau lagoon (Saulnier et al., 2010). More recently,

V. harveyi was associated with mass mortalities of heat-stressed C. gigas in Australia (Green et al., 2019) which agrees with the higher virulence of V. harveyi at high temperature [START_REF] Montánchez | Analysis of Vibrio harveyi adaptation in sea water microcosms at elevated temperature provides insights into the putative mechanisms of its persistence and spread in the time of global warming[END_REF]. However, contrary to our findings, no OsHV-1 could be detected in these diseased oysters (Green et al., 2019), which suggests that oyster colonization by V.

harveyi can be facilitated by stressors (e.g. high temperatures) other than a viral primary infection. The dominance of the Harveyi clade in our study contrasts markedly with known vibrios isolated from POMS-diseased oysters on the Atlantic coast, which mostly belong to the Splendidus clade (Lemire et al., 2015;Bruto et al., 2017). A possible reason for that difference is the preferential growth of V. harveyi in warm waters [START_REF] Zhou | Effect of combined function of temperature and water activity on the growth of Vibrio harveyi[END_REF], such as those found the Thau lagoon (16-30°C) during half of the year (Lopez-Joven et al., 2018)

V. harveyi isolates induce high mortality of juvenile oysters

To determine the virulence potential of the 104 Harveyi-related isolates, we performed experimental infections of juvenile oysters by injecting bacteria in the oyster adductor muscle (Figure 2A). We observed a clear correspondence between virulence (measured as the induced mortality rate) and species delineation. V. harveyi isolates were by far the most virulent with 50% of V. harveyi isolates inducing ≥ 50% mortality after 1 day (50% average mortality, Figure 2B). V. harveyi was statistically (Kruskal-Wallis and Mann-Whitney tests, p < 0.0001) more virulent than V. rotiferianus, V. jasicida and V. owensii species (Figure 2B). V.

3A). In this condition the Vibrio load in tissues of live oysters was 1-2 log higher than in other conditions at 2 day (p < 0.001, Figure 3B). This time point corresponds to an active replication phase for OsHV-1 μVar (Figure S1), earlier shown to trigger dysbiosis and bacteremia of opportunistic pathogens (de Lorgeril et al., 2018). Thus, our results not only confirm that OsHV-1 µvar facilitates Vibrio colonization, but also evidence that interaction between OsHV-1 µvar and the Harveyi clade accelerates oyster death. This interaction, which results in enhanced disease compared to infections with microbes acting alone, is typical of synergies at play in polymicrobial diseases (Murray et al., 2014) OsHV-1 µvar modifies the oyster habitat to facilitate bacterial infection

To unveil the molecular bases of the different disease outcomes, we analyzed on the one hand the transcriptomic responses of oysters that develop the disease when exposed to both

Harveyi and the OsHV-1 µvar virus (HV), and on the other hand, the transcriptomic responses of oysters exposed to Harveyi only (H), which control the infection. Three individuals from each condition were used for RNAseq. A total of 1467 genes were differentially expressed within all the libraries (FDR < 0.05, supplementary Table 3).

Oysters exposed to Harveyi only showed a limited number of differentially expressed genes (DEGs) when compared to the controls (DEGs, FDR < 0.05), with no genes common to all time points (Figure 4A); 21 were shared with the HV conditions (supplementary Table 3). DEGs were homologous to genes encoding proteins involved in interactions between host cells and bacteria (proteoglycans, mucins, agglutinin, von Willebrand factor) and a LPS-induced protein (supplementary Table 3). However, no induction of known immune genes was observed among DEGs. In agreement, we did not observe the induction of genes encoding antimicrobial peptides (AMPs) when performing a targeted alignment of oyster RNAseq reads to an in-house database of AMP sequences (data not shown), contrary to what we observed when we injected bacteria into oyster tissues, thereby breaching natural defenses [START_REF] Rubio | Speciesspecific mechanisms of cytotoxicity toward immune cells determine the successful outcome of Vibrio infections[END_REF]. This suggests that resistance of oyster to colonization by vibrios does not result from an active induction of immune defenses that controls the microbiota, but it rather suggests that healthy oysters constitute a hostile habitat for vibrios [START_REF] Destoumieux-Garzón | Vibrio-bivalve interactions in health and disease[END_REF]. It also indicates that oysters can tolerate transiently high Vibrio loads when acquired through natural routes.

On the opposite, a strong modification of oyster as a habitat for bacterial populations was observed in oysters exposed both OsHV-1 µvar and Harveyi. Significant transcriptomic changes were observed compared to untreated individuals with a total of 1468 DEGs (FDR < 0.05), 142 of which were common to all time points, and 384 DEGs common to 24 and 48 h, representing 59% of common DEGs between these time points (Figure 4A). The transcriptional response increased gradually from 4 to 48 h with a discrete peak at 24 h (Figure 4B). A majority of the DEGs found in HV libraries were up-regulated (85% at HV4; 67% at HV24 and HV48). The response of oysters to this multipathogen exposure was consistent with the response to POMS (de Lorgeril et al., 2018), particularly in the late time-points (24 and 48h).

Only one GO category, namely epithelial to mesenchymal transition, was significantly enriched at 4 h in HV libraries as revealed by a rank-based gene ontology analysis of the DEGs, (RGBOA with a false-discovery rate [FDR] < 0.05) (Figure 4C, Supplementary Table 4)

and represented by two genes, a transcriptional regulator Erg-partial and a hepatocyte growth factor. A major shift in gene expression was observed at 24-48h (only ~20% of common DEGs with the 4h time point), when OsHV-1 µvar and Vibrio proliferated (Figure S1).

At these late time points, significantly enriched GO categories were all related to the immune response (Figure 4C). Recognition of external stimulus, signal transduction, immunity, response to stress, and response to LPS were among the 13 biological processes modulated in diseased oysters. The corresponding 155 genes belong to gene families modulated during POMS in susceptible oysters (Figure 4B, supplementary Table 3) (de Lorgeril et al., 2018;Lafont et al., 2020).

Altogether our transcriptomic data suggest that oyster constitute a hostile habitat for vibrios which can be modified by the immunosuppressive OsHV-1 µvar virus to enable successful colonization.

OsHV-1 μVar favors colonization by V. harveyi and V. rotiferianus only

We next asked whether the immune suppression by the OsHV-1 µvar could similarly facilitate the colonization by the four species of the Harveyi clade found in the oyster environment.

Individuals collected during the time course of the infection were therefore subjected to illumina Miseq sequencing of the rctB polymorphic region which discriminated the four species introduced into the mesocosm (Figure S2). The relative abundance of the 4 species remained stable in the oysters only exposed to the Harveyi community during 48 h, as revealed by rctB barcoding analysis (Figure 5, left panel). On the contrary, in the presence of OsHV-1 µvar, not only the total Vibrio load increased over time (Figure 3B) but there was also a significant enrichment of the species V. harveyi and V. rotiferianus at 48 h, after viral replication occurred (24h) (Figure 5, right panel). Investigations at an individual level revealed that the Vibrio population structure differed from one oyster to another, but V. harveyi and V.

rotiferianus always dominated in relative abundance over the other 3 monitored species in all individuals. By this approach we were able to provide experimental evidence that the OsHV-1 μVar infection is sufficient to reproduce the differential association of Harveyi species found in the field. In this context the virulence potential found in V. jasicida isolates does not seem to be a necessary trait that allows successful colonization through a natural route of infection.

This shows that the capacity to colonize OsHV-1 µvar-infected oysters is highly dependent on bacterial determinants. It also shows that species-specific determinants rather than strainspecific virulence traits define the capacity of V. harveyi and V. rotiferianus to colonize oysters infected by OsHV-1 µvar.

V. harveyi, the strategy of a killer

To gain access to genomic features that might be explanatory for the V. harveyi and V.

rotiferianus colonization abilities, we sequenced 17 Harveyi-related genomes from our collection (4 V. harveyi, 5 V. owensii, 4 V. jasicida and 4 V. rotiferianus isolates) (Supplementary Table 5). Genome comparisons aimed to extract genes exclusively shared by the V. harveyi and V. rotiferianus gave as few as 15 genes, suggesting that speciesspecific determinants might be responsible for the colonization ability of these two species (Supplementary Table 6).

We first focused on V. harveyi, a species in which several virulence factors underlying the pathogenesis have been identified. These factors include adhesins, chitinases, extracellular polysaccharides involved in biofilm formation, haemolysins and proteases, siderophores and iron acquisition systems, Type III Secreting Systems (T3SS), and bacteriophage-mediated virulence (Darshanee [START_REF] Darshanee Ruwandeepika | Pathogenesis, virulence factors and virulence regulation of vibrios belonging to the Harveyi clade[END_REF]. Among the four species from the present study, V. harveyi was by far the species that gathered the greatest number of the virulence factors described so far. In particular, V. harveyi harbored a type 3 secretion system homologous to the V. parahaemolyticus T3SS1 (Supplementary Table 3), also present in V.

jasicida (Supplementary Table 5; Figure S3). In V. parahaemolyticus T3SS1 is implicated in cytotoxicity on human cell lines while T3SS2 appears to act more specifically on intestinal cell lines (Ham and Orth, 2012). Among the effectors characterized as translocated by the T3SS1 (VopQ, VopR, VopS and VPA0450), we identified orthologs for VopQ (lysosome disruption and deacidification), VopR and VPA0450 (Plasma membrane-destabilizing inositol phosphatase (Broberg et al., 2010) as well as an associated cargo chaperone orthologuous to VPA0451, which is needed for translocation of VPA0450 into the host cell membrane [START_REF] Waddell | Identification of VPA0451 as the specific chaperone for the Vibrio parahaemolyticus chromosome 1 type IIIsecreted effector VPA0450[END_REF]. By comparison, in V. jasicida, only two characterized secreted effectors were found, namely VopQ and the pro-apoptotic effector VopS [START_REF] Bhattacharya | Economic development, gender inequality, and demographic outcomes: Evidence from India[END_REF]. In addition, an arginine ADP ribosyltransferase (964 AA, 106kDa) only found in V. harveyi isolates was evidenced (gene 180147 of V. harveyi O-A1). This protein contains a 72 AA motif related to ExoToxin (a two-component protein complex known as AB toxin) like the cholera toxin (and in Clostridium, Bacillus anthracis toxins).

Besides, T6SS systems appeared widespread among our Harveyi strains (Supplementary Table 5). Orthologs of the T6SS1 and T6SS2 of the oyster pathogen V. tasmaniensis LGP32 were found. T6SS1 was present in all strains and T6SS2 only absent from V. rotiferianus. The T6SS1 has been already characterized as necessary for cytotoxicity towards hemocytes and virulence of V. tasmaniensis LGP32 while the role of the T6SS2 remains to be elucidated [START_REF] Rubio | Speciesspecific mechanisms of cytotoxicity toward immune cells determine the successful outcome of Vibrio infections[END_REF]. Similarly, in V. cholerae, a T6SS mediates cytotoxicity to macrophages by inducing actin cross-linking [START_REF] Ma | In vivo actin cross-linking induced by Vibrio cholerae type VI secretion system is associated with intestinal inflammation[END_REF] Two other T6SS were found, the first named T6SS3 was found in all strains and a second one named T6SS4 was found associated with the V. rotiferianus species and to only one strain from the V. harveyi species (Supplementary Table 5).

The cytotoxic potential of V. harveyi was confirmed experimentally by comparing two strains representative of the four Harveyi species from our study. We found that V. harveyi strains exerted a potent cytotoxicity toward hemocytes, the oyster immune cells, 25-35% of which were lysed upon incubation with V. harveyi strains (Figure 6). By comparison, the three other species from the present study were significantly less cytotoxic (7-13% lysed hemocytes), p < 0.001 (One-way ANOVA test). Cytotoxic activtity was earlier described as the hallmark of two pathogenic Vibrio species (Splendidus clade) associated with the POMS in the Atlantic environment [START_REF] Rubio | Speciesspecific mechanisms of cytotoxicity toward immune cells determine the successful outcome of Vibrio infections[END_REF]. From the present study, the Mediterranean V. harveyi isolates resemble the Atlantic V. tasmanienis and V. crassostreae, which are equipped with specific virulence/cytotoxicity factors that are needed to dampen oyster cellular defenses and cause systemic infections [START_REF] Rubio | Speciesspecific mechanisms of cytotoxicity toward immune cells determine the successful outcome of Vibrio infections[END_REF] V. rotiferianus, the strategy of a cheater Unlike V. harveyi, V. rotiferianus (poorly virulent, good oyster colonizer) and V. jasicida (virulent, poor oyster colonizer) were poorly cytotoxic toward oyster immune cells. This indicates (i) that virulence in the Harveyi clade is multifactorial and (ii) that beyond cytotoxicity, other attributes determine the capacity to colonize oysters affected by the POMS. This was further explored by comparative genomics. Iron acquisition systems were a major difference between species (Supplementary Table 5). Indeed, V. harveyi was found to harbor a conserved set of genes responsible for the synthesis of vibrioferrin, a tricarboxylic acid siderophore derived from citric acid and for the synthesis of amphi-enterobactin. In contrast, V. rotiferianus does not exhibit known Vibrionaceae siderophore biosynthetic proteins but it carries the vibrioferrin receptor necessary for the siderophore uptake (Supplementary Table 5, Figure 7A). This receptor is present in both V. harveyi and V.

rotiferianus (good colonizers) together with receptor proteins for aerobactin, enterobactin and deferoxamine B. In contrast, it is absent from the V. owensii and V. jasicida genomes (poor colonizers), suggesting iron acquisition systems could determine the capacity of Harveyi species to colonize oysters. Iron acquisition systems are indeed well-known determinants of vibrio colonization in metazoan hosts as they determine their capacity to grow in iron-limited environments by the use of siderophores that bind poorly soluble iron and make it available to cells via active uptake [START_REF] Arezes | Hepcidininduced hypoferremia is a critical host defense mechanism against the siderophilic bacterium vibrio vulnificus[END_REF]. To investigate the role of vibrioferrin uptake, we compared the growth of V. rotiferianus (non-producer) in the presence/absence of two different doses of V. harveyi (producer) culture supernatant, which contains vibrioferrin. V.

harveyi growth was not impaired in iron depleted media (EDDA used as iron chelator, Figure 7B), in agreement with its ability to produce siderophores. On the contrary, iron depletion significantly affected the growth of V. rotiferianus, p <0,01 (Oneway ANOVA test). This growth defect was rescued in a dose-depended manner by the addition of cell-free culture supernatant from V. harveyi (Figure 7B). This demonstrates that V. rotiferianus is able to acquire important resources (most likely vibrioferrin) produced by V. harveyi, which are needed to grow in iron-poor environments. It also strongly supports the hypothesis that vibrioferrin uptake is a key determinant of Vibrio growth.

While V. rotiferianus has been described as a pathogen for the rainbow trout [START_REF] Austin | Pathogenicity of vibrios to rainbow trout (Oncorhynchus mykiss, Walbaum) and Artemia nauplii[END_REF], the present data indicate that the species does not participate actively to the POMS pathogenesis. Instead, our data highlight a cheating behavior of V. rotiferianus in siderophore utilization. Such a behavior has been reported in vibrios, the proportion of cheaters being higher in vibrios associated with large particles than in free-living vibrios (Cordero et al., 2012).

It is likely that the high bacterial loads that characterize the POMS fatal bacteriemia are very favorable to cheaters like V. rotiferianus, which benefit from the "public goods" (here the siderophores) produced by the dense Vibrio community, or other members of the microbiota, to grow in an iron-limited environment.

In conclusion, the present study shows that (i) healthy oyster constitute a rather hostile habitat that tolerates transiently high vibrio loads but does not allow stable colonization by vibrios. This habitat becomes permissive to specific vibrio species when immune defenses are altered by

OsHV-1 µvar. (ii) The Vibrio community able to colonize OsHV-1 µvar-infected oysters varies according to ecosystems, but contains species that express the same capacity to dampen the oyster cellular defenses using cytotoxic activities. (iii) In the Thau lagoon, V. harveyi (adapted to elevated temperature) is a virulent species associated with POMS that synergizes with OsHV-1 µvar to accelerate oyster death. (iv) The Vibrio community associated with POMS is composed of both opportunistic pathogens, like V. harveyi, and simple opportunists (cheaters), like V. rotiferianus, benefiting from the activity of the pathogens, which dampen the oyster immune defenses to facilitate the colonization and produce vibrioferrin, a siderophore that the cheaters can utilize for their own growth within diseased oysters.

Materials and methods

Oyster and bacterial sampling

In October 2015 (autumn mortality), 30 juvenile oysters (15 spats from Decipher family + 15 spats from France Naissain) and 40L of seawater were collected in the Mediterranean Thau lagoon (Languedoc-Roussillon, France; Supplementary Table 1). In 2016 and 2017, only juvenile oysters (NSI spats) were collected during mortality events (June 2016) or in absence of mortality (June 2016, march 2017, Table S1). OsHV-1 μvar DNA detection in oyster flesh using quantitative PCR as described by Lafont et al. (2017) showed that sampled oysters were infected by OsHV-1 μVar virus (Supplementary Table 1). Seawater was size-filtrated to collect zooplankton, large phytoplankton and organic molecules (60µm pore size filter), smaller organic particles (5 and 1µm) and free-living bacterial cells retained at 0.22µm (Bruto et al., 2017).

Bacterial sampling

Oysters and the large seawater particles fraction were ground (Ultra-Turrax, IKA) and 100µL

were plated on Vibrio selective media (thiosulfate-citrate-bile salts sucrose agar, TCBS, Difco DB, Le pont de Claix, France). The others filters (5, 1 and 0.22 µm) were directly placed on TCBS agar plate and incubated 2 days at 20°C. Around 100 colonies for each sample were randomly picked, re-steaked first on TCBS and then on Zobell agar (4g.l-1 bactopeptone, 1 g.l-1 yeast extract and 15g.l-1 agar in sterile seawater, pH 7.4) and stock cultures were stored at -80 °C in Zobell containing 15% glycerol (v/v). For subsequent molecular analyses, all isolates were grown overnight at 20 °C in liquid Zobell medium and DNA extracted using a DNA extraction kit (Nucleospin tissue, Macherey-Nagen) or using Whathman FTA cards (GE Healthcare).

Population structure analysis

All isolates from October 2015 were genotyped by hsp60 partial gene sequence as described previously [START_REF] Gob | HSP60 gene sequences as universal targets for microbial species identification: Studies with coagulase-negative staphylococci[END_REF]Hunt et al., 2008) using primers Hsp60 R and F generating a total of 448 hsp60 sequences (Supplementary Table 2). The PCR conditions for hsp60 amplification were: 3 min at 95°C followed by 30 cycles of 30 sec at 95°C, 30 sec at 37°C and 1min at 72°C with a final step of 5 min at 72°C. hsp60 sequence ambiguities were corrected using 4peaks

and Seaview software packages and received a taxonomic affiliation if the best blast-hit displays an identity superior to 95% with a type-strain. This resulted in the assignment of 304/448 isolates to 21 different species whereas the others were unknown species (Figure 2).

MLST genotyping

To identify and genotype isolates from October 2015 belonging to the Harveyi clade, 3 additional protein-coding genes have been sequenced (rctB, topA and mreB)

(Supplementary Table 8). Once the 104 Harveyi-related strains identified, the rctB, topA and mreB sequences were amplified using rctB-F/rctB-R, VtopA400F/VtopA1200R and VmreB12F/VmreB999R primers, respectively (Sawabe et al., 2007), (Supplementary Table 8).

For rctB, the PCR program was: 2 min at 95°C followed by 30 cycles of 30 sec at 95°C, 1 min at 53°C and 1.45 min at 72°C with a final step of 5 min at 72°C For topA and mreB, the PCR conditions were: 2 min at 95°C followed by 30 cycles of 30 sec at 95°C, 30 sec at 50°C and 1min at 72°C with a final step of 5 min at 72°C. All PCR were performed using the Gotaq PCR polymerase (Gotaq G2 flexi, Promega) following the manufacturer's instructions. Genes were sequenced using the reverse PCR primer and sequencing was performed at GATC (https://www.gatc-biotech.com/fr).

On the bacterial strains isolated in the 3 other sampling points, the isolates were screened by PCR using rctB-F/rctB-R Vh-mreB-F/Vh-mreB-R primers designed to specifically hybridize with Harveyi-related rctB gene and V. harveyi mreB gene, respectively (Supplementary Table 8).

For both detections, the PCR program was: 2 min at 95°C followed by 30 cycles of 30 sec at 95°C, 1 min at 53°C and 1.45 min at 72°C with a final step of 5 min at 72°C. Following this rational, 143 rctB+ isolates were considered as related to the Harveyi clade and related to V.

harveyi species.

MLST in silico analyses

Sequences for each marker (hsp60, rctB, topA and mreB) were aligned with 8 V. harveyi superclade type strains using Muscle (Edgar, 2004). Alignments were concatenated with Seaview [START_REF] Gouy | Sea view version 4: A multiplatform graphical user interface for sequence alignment and phylogenetic tree building[END_REF]. Phylogenetic trees were reconstructed with RAxML using a GTR model of evolution and Gamma law of rate heterogeneity. All other options were left at default values. Nodes support was assessed based on bootstraps with 100 replicates.

Recombination can have profound effects on phylogenetic reconstruction, especially when the event is recent [START_REF] Posada | The Effect of Recombination on the Accuracy of Phylogeny Estimation[END_REF]. To determine if some markers recombined, each individual tree was manually inspected to search incongruent marker(s) for strains having long branches or low bootstrap support in the MLST tree. Incongruent markers were discarded in the alignment and strains having less than three non-recombinant markers were not considered. The final tree was reconstructed with RAxML as explained above with the filtered alignment.

Virulence studies on juvenile oysters

For injection procedures, bacteria were grown under agitation for 18 h at 20°C in Zobell liquid medium before dilution (with medium) to adjust the optical density (OD600) to 0.7. A volume of 40μL of each isolate was injected intramuscularly to 20 specific pathogen-free (SPF) juvenile C. gigas oysters (Petton et al., 2015). V. crassostreae J2-9 (virulent strain), V.

tasmaniensis LMG20012T (non-virulent strain) and sterile filtered seawater (negative control)

were injected in parallel to pathogen free juvenile oysters. Animals were previously anesthetized in hexahydrate MgCl2 (50 g. L-1, 100 oysters/liter) for 14 to 16 h. Bacteria purity and concentration were checked by plating on Zobell agar. After injection, the juvenile oysters were transferred to aquaria (20 oysters per 1l aquarium) containing 400 mL of aerated seawater at 20 °C and kept under static conditions. Mortality was followed daily (24, 48 and 72hrs) and dead spats were removed from tanks.

Experimental infection in mesocosm

Seawater containing OsHV-1 μVar virions was produced by anesthetizing 90 oysters in hexahydrate MgCl2 (50 g. L-1, 100 oysters/liter) for 1-2 hrs, injecting them in the aductor muscle with 100 μl of a 0.2 μm filtrated viral suspension (10 8 genomic units mL -1 ) and placed them in a 40 L tank for 24 hrs. After virions release, confirmed and quantified by qPCR, and previous to the mortality of donor oysters, contaminated was recover and used to fill the tanks for the different experimental conditions. Tanks with 2L of seawater and 10 oysters were prepared for sampling at different time points and tanks with 3L of seawater with 15 oysters to track the daily mortalities on each condition. Donor oysters were transferred to another tank to confirm mortalities. Clean seawater was used for the experimental conditions in absence of OsHV-1 μVar infection.

Bacterial strains were striked on Zobell agar and incubated at 20°C. Precultures were made by picking single colonies from the agar plates and growth in Zobell broth for 5h at 20°C in a rotary shaker. Zobell broth was seeded with the precultures and incubated for 18h at 20°C with agitation at 150 rpm. Cultures were centrifuged at 1500 x g for 10 min, the supernatant discarded and the pellet resuspended in sterile seawater. The OD600 of the bacterial suspension was measured and adjusted to OD600=1.

After placing the oysters in the tanks, bacterial suspension in seawater adjusted to OD600 = 1 (10 9 CFU/mL) were added to each tank to reach a final concentration of 10 7 CFU/mL. At each sampling time point, the oysters from the respective tank were taken together with a sample of 100 mL of seawater. The oyster flesh was removed from the shell, put into individual aluminum pockets and snap frozen in liquid nitrogen to be stored after at -80°C. Mortalities in the monitoring tanks were recorded daily. 30 mL of seawater was filtered through 0.2um pore size and the filter stored individually at -80°C.Frozen individuals stored at -80°C were put into liquid nitrogen and transferred individually into stainless steel cylinders containing a stainlesssteel ball cooled in liquid nitrogen, the cylinder was shacked at a frequency of 35 cycles/s for 30 s in a Retsch MM400 mixer mill. The pulverized tissue was transfer to a 2mL screw capped tube and stored at -80°C until further processing.

Nucleic acid extraction

Total DNA was extracted with the Nucleospin tissue DNA extraction kit (Machery Nagel, ref:

740952.250) with a modified protocol. Briefly, 20 mg of frozen tissue-powder, a pellet from 1 mL of stationary phase bacterial cultures or a 0.2um filter was added to a 2 mL screw capped tube containing Zirconium beads, lysis buffer and proteinase K and shaked for 12 min at a frequency of 35 cycles/s for in a Retsch MM400 mixer mill at room temperature. The samples were incubated for 1h 30min in a water bath at 56°C, after incubation RNAse A was added and the sample incubated were incubated for 5 min at 20°C and after for 10 min at 70°C. The following steps were carried out as recommended by the protocol provided by the manufacturer. DNA concentration and purity Nanodrop ND-1000 spectrometer (Thermo Scientific)

Total RNA was extracted from 20 mg of frozen tissue-powder using Direct-zol RNA extraction kit (Zymo research). Briefly, 20 mg of pulverized-frozen oyster tissue were resuspended in 1 mL of TRIzol and agitated at 150 rpm for 1.5 hrs. at 4°C. Trizol suspension was then incubated at 20°C for 5 minutes to finish the lysis. Next, 100 ul of chlorophorm were added, mixed by inversion and after 5 minutes tubes were centrifuged at 12.000 x g. The aqueous phase was recovered and the following steps were carried out as recommended by the protocol provided by the manufacturer. RNA concentration and purity were estimated using Nanodrop ND-1000 spectrometer (Thermo Scientific) and their integrity analyzed by capillary electrophoresis on a BioAnalyzer 2100 system (Agilent).

Vibrio genome sequencing

Genomic DNA for vibrio strains was extracted with the NucleoSpin Tissue kit (Macherey-Nagel Inc.). Individual genomic libraries were prepared at the Bio-Environment platform (University of Perpignan) using the nextera XT DNA Library Prep Kit (Illumina) according to the manufacturer's instructions starting from 1 ng of bacterial DNA and purified using AMPure XP beads. The quality of the libraries was checked using High Sensitivity DNA chip (Agilent) on a Bioanalyzer. Pooled libraries were sequenced in 2x150 paired-end mode on a NextSeq 550 (Illumina) resulting in ~100-fold coverage. Reads were assembled de novo using Spades software. Computational prediction of coding sequences together with functional assignments and comparative genomics were performed using the MAGE software at the MicroScope platform [START_REF] Vallenet | MicroScope -An integrated microbial resource for the curation and comparative analysis of genomic and metabolic data[END_REF].

RNA sequencing and transcriptome analysis

RNA-seq library construction and sequencing were performed by the Fasteris Company (Switzerland). Directional cDNA libraries were constructed using the Ovation Complete Prokaryotic RNA-Seq (NuGen) and sequenced on a Novaseq system aiming to 100M reads in paired-end reads of 2 × 50 bp. Data treatment and analysis was performed with tools implemented under a Galaxy instance [START_REF] Goecks | Galaxy: a comprehensive approach for supporting accessible, reproducible, and transparent computational research in the life sciences[END_REF]. All reads were kept for subsequent analysis as Phred scores determined by the Fastq-toolkit were higher than Q30 over 90% of the length for all the sequences. RNAstar (Galaxy Version 2.40d-2 [START_REF] Dobin | STAR: Ultrafast universal RNA-seq aligner[END_REF]) was used to map reads to the C. gigas reference genome (assembly version V9 [Zhang et al., 2012]. The number of reads overlapping annotated genes (mode Union) were determined by HTSeq-count (Galaxy Version v0.61 [START_REF] Anders | HTSeq-A Python framework to work with highthroughput sequencing data[END_REF]). Finally, differential gene expression was analyzed with the DESeq2 package [START_REF] Love | Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2[END_REF]. Fold changes between each time point of the kinetics and the control of un-treated oysters were considered to represent a differential gene expression when the adjusted p-value (Padj) for multiple testing with the Benjamini-Hochberk procedure to control the false discovery rate (FDR), was <0.05. The list of DEGs were represented on a heat map using a hierarchical clustering with uncentered Pearson correlation (MeV 4_9_0 software)

Enrichment analysis

DEGs were functionally annotated according to the database previously used by de Lorgeril et al. (2018). These annotated DEGs were used as input for GO enrichment analysis using an adaptive clustering and a rank-bases statistical analysis (Mann-Whitney U-test combined with adaptive clustering). The R and Perl scripts used can be downloaded To represent the level of enrichment of modulated GO categories, a ratio was calculated for (i) upregulated enriched categories (number of genes significantly overexpressed in the category/total number of genes in this category) or (ii) for downregulated enriched categories, -1 * (number of genes significantly downregulated in this category/total number of genes in this category) and represented on a heat map (MeV 4_9_0 software).

Vibrio quantification

Quantification of total Vibrio in oysters was performed on DNA extracted from 20 mg of frozen tissue powders (see above) by targeting 16s rDNA.

Amplification reactions were carried out in duplicate, in a total volume of 20μl on Mx3000 and Mx3005 Thermocyclers (Agilent). Each well contained 5μl of genomic DNA (at 5 ng/μl), 10μl of the Brillant III Ultra-Fast SyberGreen Master Mix (Agilent), 0.060μl of each primer at 100μM (567F and 680R, table S8, [START_REF] Thompson | Diversity and dynamics of a North Atlantic coastal Vibrio community[END_REF], and distilled water. qPCR cycling conditions were as follows: 3 min at 95C, followed by 40 cycles of amplification at 95°C for 10 s and 60°C for 20 s.

Standard curves of known concentration of Vibrio genomes were generated using the relation between the concentration of DNA and the theoretical copy number of genomes, calculated on the basis of the DNA mass divided by molecular weight of the genome. They were also validated by the limit dilution method, assuming that the dilution at which 1 replicate in 10 was positive corresponds to 1 copy.

Absolute quantification of Vibrio genomes in oyster samples was then estimated by comparing the observed Ct values to known standards from 10 2 to 10 9 copies of Vibrio genomes. The absence of amplification (no Ct), or unquantifiable values (outside the standard range) were labelled as 1 copy and 10 copies respectively.

OsHV-1 µvar detection

Detection and quantification of OsHV-1 µvar DNA in oyster flesh was performed using quantitative PCR grounded oyster flesh (1.5mL) was centrifuged for 10 min at 4°C and 1500rpm and genomic DNA was extracted from 50µL of the supernatant using phenol: The PCR over the total DNA extracted from the sampled oysters (n=60) was carried out with a high fidelity Q5 polymerase in a total volume of 50 μl and a thermal cycler program with an initial step of 98°C for 25 s followed by 35 cycles of 98°C for 10 s, 51°C for 25 s and 72°C for 30 s. Final extension was performed at 72°C for 2 minutes. PCR products were resolved in a 1.5% gel electrophoresis, stained with gel red and visualized in a UV-photo capture system to confirm the presence of the 573 bp fragment.

Libraries were constructed with the 16S Metagenomic Sequencing Library Preparation (ref.

15044223) and sequenced in a MiSeq Illumina system to produce paired end reads 2x300 bp, by the GenSeq platform, University of Montpellier (ISEM), France.

The FROGS pipeline (Find Rapidly OTUs with Galaxy Solution) implemented into a Galaxy instance was used to process sequencing data and define Operation Taxonomic Units and taxonomic affiliations [START_REF] Escudié | FROGS: Find, Rapidly, OTUs with Galaxy Solution[END_REF] Paired reads were merged using FLASH [START_REF] Magoč | FLASH: Fast length adjustment of short reads to improve genome assemblies[END_REF]. Primer/adapter removal and denoising was done by cutadapt [START_REF] Martin | Cutadapt removes adapter sequences from high-throughput sequencing reads[END_REF]. Dereplicated sequences were used to perform de novo clustering using SWARM with an aggregation distance d = 3 [START_REF] Mahé | Swarmv2: Highlyscalable and high-resolution amplicon clustering[END_REF]. Chimera were removed using VSEARCH (de novo chimera detection) [START_REF] Rognes | VSEARCH: A versatile open source tool for metagenomics[END_REF]. OTUs generated were annotated using Blast+ against an in-house database containing the Harveyi rctB sequences to generate an OTU and affiliation table in standard BIOM format. Affiliation and abundance data associated with each cluster was exported and used to estimate the relative abundance of the 4 different Harveyi species on each sample.

In vitro cytotoxicity assays

Hemocytes were plated in 96 well-plates (2 x 105 cells/well) or in 12 well-plates (5 x 105 cells/well). After 1 h, plasma was removed and 5 μg/μl Sytox Green (Molecular Probes) diluted in 200μl sterile seawater was added to each well. Vibrios, previously washed and opsonized in plasma for 1 h, were then added to the wells at a MOI of 50:1. Sytox Green fluorescence was monitored (ƛex 480nm/ƛem 550nm) for 15 h using a TECAN microplate reader. Maximum cytolysis was determined by adding 0.1% Triton X-100 to hemocytes.

Statistical analysis was performed using RM-ANOVA.

Iron depency growth of Harveyi

Growth experiments in iron depleted medium were performed with V. harveyi Th15_O_G11 and V. rotiferianus Th15_O_G10. Isolates were grown overnight at room temperature in minimal medium: 35g/L reef salts, 0.3% (wt/vol) casamino acids and vitamins: 0.1 μg/L vitamin B12, 2μg/L biotin, 5μg/L calcium pantothenate, 2μg/L folic acid, 5 μg/L nicotinamide, 10 μg/L pyridoxin hydrochloride, 5 μg/L riboflavin, 5 mg/L thiamin hydrochloride. Cultures were pelleted (2 min at 10,000 rpm in a microcentrifuge), the supernatant was discarded, and pellets were washed once in reef salts solution (35g/L). Cells were then inoculated (1:1,000) into minimal media with (iron-poor) or without (iron-replete) the iron-specific chelator EDDA (ethylenediamine-N,N′-diacetic acid). Cells were incubated at room temperature and the absorbance at 600 nm measured every hour for 24 h. The filtered V. harveyi Th15_O_G11 cellfree supernatant (3-kDa membrane using an Amicon Ultra centrifugal unit, Millipore) added to culture results from growth in iron-poor conditions (200µM EDDA). breoganii 1C10, V. campbellii CAIM519 T , V. celticus CECT7224 T , V. chagasii LMG21353 T , V. crassostreae

LGP7 T , V. cyclitrophicus LMG21359 T , V. fischeri MJ11, V. fortis Dalian14, V. harveyi NBRC15634 T , V. hemicentroti CECT8714 T , V. jasicida CAIM1864 T , V. lentus CIP107166 T , V. mediterranei NBRC15635 T , V. owensii CAIM1854 T , V. rotiferianus CAIM577 T , V. scophthalmi LMG19158 T , V. shilonii AK1 T .` juvenile oysters and the mortality rate (%) scored after 24h. The effect of the vibrio species on oyster mortality rate was determined using Kruskal-Wallis test (p-value < 0.001). Significant differences between Vibrio species were performed with Mann-Whitney tests using the GraphPad Prism software (p-value < 0.001). V. harveyi (n=63), V. rotiferianus (n=16), V. jasicida (n=15), V. owensii (n=6), NA (not assigned, n=4). Growth of the vibrioferrin producer V. harveyi O_G11 strain in minimal media (V.h.) and in media supplemented with 100 μM EDDA (V.h. EDDA), an iron-specific chelator. OD600 was measured after a 20 h growth. V.h. grew similarly in all media. By contrast, the non-producer V. rotiferianus O_G10 strain (V.r.), which only has specific receptors for vibrioferrin, showed a growth defect in the presence of 100 μM EDDA (V.r EDDA). This defect was completed by the addition of cell-free culture supernatant from V. The overall objective of the present thesis was to characterize the mechanisms underpinning the association of the oyster Crassostrea gigas with vibrio populations in health and disease.

Oysters possess a remarkable ability to maintain an immune homeostasis in presence of an abundant and diverse microbiota, which paradoxically is in close contact with immunocompetent tissues (Schmitt et al. 2012). In this state, preferential and positive associations of vibrio populations can be sustained within oysters without the development of disease. However, abiotic and biotic stressors acting on the oyster holobiont (the oyster and its associated microbiota) can trigger a dysbiosis in which the proliferation of opportunistic vibrio species leads to oyster death. In this thesis we have been interested in characterizing these two different states, which both question the tolerance of the oyster immune system and its manipulation by vibrios. The following section will discuss the thesis main results in perspective with the available literature and the future prospects derived from our conclusions.

CYTOTOXICITY AS A MEAN FOR VIBRIOS TO DAMPEN OF OYSTER IMMUNE DEFENSES

Over the past years, significant attention has been paid to the vibrio populations associated with the Pacific Oyster Mortality Syndrome (POMS) (Lemire et al., 2015;Bruto et al., 2017;Piel et al., 2019). We studied here vibrios associated with POMS isolated from two different marine coastal environments that host important oyster farming activity, namely the bay of Brest on the French Atlantic coast and the Thau Lagoon, on the Mediterranean coast. Vibrio species pathogenic for oysters belong to the the Splendidus clade in the Atlantic (Lemire et al., 2015;Bruto et al., 2017) and to the Harveyi clade in the Thau lagoon (Oyanedel in prep).

Remarkably in both clades, we found that virulent populations of vibrios express cytotoxicity toward oyster immune cells, the hemocytes (Rubio et al., 2019, Oyanedel in prep). Inside a given environment virulent vibrio species vary with time (Bruto et al., 2017). However, our results from article 2, showed that the virulent vibrio species, V. crassostreae and V.

tasmaniensis are characterized by species-specific mechanisms to suppress the cellular defenses and enable systemic infections. On the opposite, non-cytotoxic strains from the same vibrio species are rapidly controlled by hemocytes through phagocytosis and cell clumping, thus preventing systemic infection. This ability to suppress oyster cellular defenses was also evidenced in V. harveyi (article 3), a species that efficiently colonizes OsHV-1 infected oysters both in the field and in mesocosm experiments. Virulence and cytotoxicity therefore appear closely related, probably because they determine colonization success, as we demonstrated in the Splendidus clade [START_REF] Rubio | Speciesspecific mechanisms of cytotoxicity toward immune cells determine the successful outcome of Vibrio infections[END_REF].

VIBRIO SPECIES-SPECIFIC WEAPONS TO SUPPRESS OYSTER IMMUNE CELLS Subversion of the host defenses is achieved by bacteria through several strategies including the avoidance or degradation of antimicrobial peptides, disguising of their MAMPs and manipulation of signaling pathways and vital cellular functions [START_REF] Reddick | Bacteria Fighting Back: How Pathogens Target and Subvert the Host Innate Immune System[END_REF], but also through more aggressive mechanisms that aim directly at the annihilation of the antimicrobial functions of the central mediator of the innate immunity, the hemocytes, achieved through the activity of toxins that are exported by bacteria into the extracellular milieu, or either injected or intracellularly delivered into target cells through specialized secretion systems (A.T. [START_REF] Ma | Metal Transporters and Metal Sensors: How Coordination Chemistry Controls Bacterial Metal Homeostasis[END_REF][START_REF] Osorio | T3SS Effectors in Vibrios: Homology in Sequence, Diversity in Biological Functions?[END_REF].

As developed in article 2, determinants of cytotoxicity towards the hemocytes are speciesspecific and have independently evolved in V. crassostreae and V. tasmaniensis. In V.

crassostreae, we found that cytotoxicity and virulence are highly dependent on the protein R5.7 (absent from V. tasmaniensis); whereas in V. tasmaniensis virulence and cytotoxicity are highly dependent on a type VI secretion system present in chromosome 1 (T6SSChr1-LGP32) [START_REF] Rubio | Speciesspecific mechanisms of cytotoxicity toward immune cells determine the successful outcome of Vibrio infections[END_REF]. Bruto et al. (2018), evidenced that the r5.7 gene is ancestral in the Splendidus clade. The protein itself is not cytotoxic (Bruto et al., 2018); however, to mediate cytotoxicity, we found that R5.7-expressing V. crassostreae require physical contact with hemocytes [START_REF] Rubio | Speciesspecific mechanisms of cytotoxicity toward immune cells determine the successful outcome of Vibrio infections[END_REF]. Still, as an extracellular pathogen, V. crassotreae did not depend on internalization through phagocytosis to express cytotoxicity. Interestingly, we found that upon the loss of the ancestral r5.7 gene, V. tasmaniensis, which is a facultative intracellular pathogen of oyster hemocytes (Duperthuy 2011), has acquired a T6SSChr1-LGP32 that delivers cytotoxic effectors to oyster hemocytes intracellularly upon phagocytosis. These findings shed light on the mechanisms of V. tasmaniensis cytotoxicity and virulence, which both require the OmpUmediated mechanism of phagocyte internalization and depend on the resistance of V.

tasmaniensis to intra-phagolysosomal degradation, as necessary for this facultative intracellular pathogen [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] V-Vanhove | Outer Membrane Vesicles are Vehicles for the Delivery of Vibrio tasmaniensis Virulence Factors to Oyster Immune Cells[END_REF][START_REF] Vanhove | Copper Homeostasis at the Host Vibrio Interface: Lessons from Intracellular Vibrio Transcriptomics[END_REF]. So far, in the search for the putative effectors associated with the set of T6SSs identified on both species we constructed a mutant for the evpP ortholog identified as a potential T6SSChr1-LGP32 effector present in all virulent strains of V. tasmaniensis. EvpP is one of the three proteins secreted by the T6SS and essential for the virulence of the fish pathogen Edwardsiella tarda [START_REF] Hu | A Disordered Region in the EvpP Protein from the Type VI Secretion System of Edwardsiella Tarda is Essential for EvpC Binding[END_REF]. However, the mutant did not show any attenuation in virulence during experimental infections but only lost cytotoxicity potential (complementary data, section II.3).

Whether or not a T6SS also participates in the cytotoxicity of V. crassostreae has been debated. In V. crassostreae, a T6SS is carried on the virulence plasmid called pGV1512, required for virulence. In our assays, we could not evidence any role of pGV1512 in V.

crassoatreae cytotoxicity. However, the team of Frédérique Le Roux recently demonstrated the contribution of the T6SS encoded in pGV1512 to cytotoxicity, by using an alternative cytotoxicity assay that involves the contact of bacteria with the total hemocyte population in suspension (Piel et al., 2019), in contrast with our assay that relies on a monolayer of adherent cells that was standardized for the phagocytosis-dependent cytotoxicity of V. tasmaniensis [START_REF] Vanhove | Copper Homeostasis at the Host Vibrio Interface: Lessons from Intracellular Vibrio Transcriptomics[END_REF]. While no putative secreted effectors have been identified so far based on genomic analysis for the V. crassostreae T6SSpGV1512 the authors showed that T6SS and R5.7 act independently but additively to produce cytotoxicity (Piel et al., 2019).

In the cytotoxic species V. harveyi associated with POMS in the Thau lagoon, we have not been able to produce isogenic mutants so far, preventing us from clarifying the molecular basis of the species virulence and cytotoxicity. However, comparative genomics between populations of the Harveyi clade allowed us to hypothesize that the differential distribution of a type III secretion system in chromosome 1 (T3SS1) and putative associated cytotoxic effectors previously identified in V. parahaemolyticus (Broberg et al., 2010;[START_REF] Osorio | T3SS Effectors in Vibrios: Homology in Sequence, Diversity in Biological Functions?[END_REF][START_REF] Wu | Type III Secretion 1 Effector Gene Diversity Among Vibrio Isolates From Coastal Areas in China[END_REF], could contribute to the contrasted difference in cytotoxicity between V. harveyi and the other three populations from the same clade. Briefly, this T3SS cluster and associated putative effectors are absent from the low cytotoxic V. rotiferianus and V. owensii. In V.

harveyi and V. jasicida, which both contain the T3SS structural genes, but display contrasted cytotoxicity (V. jasicida is not cytotoxic), the main difference that we could identify is the higher number of putative cytotoxic effector present in V. harveyi (Arginin ADP-ribosyl transferase, VopQ, VopR, VopS, and VPA0450) relative to V. jasicida (VopQ and VopS).

Targeted gene deletion or invalidation of structural elements of this T3SS1 and associated putative effectors will help to clarify their respective roles in cytotoxicity and virulence. The available methods for genetic manipulation, by transfer of non-replicative suicide plasmids through conjugation, that we used in article 1 and 2, has been optimized for the Splendidus clade in model strains such as V. tasmaniensis LGP32 and V. crassostreae J2-9, therefore, similar optimization is necessary to be applied to our Harveyi collection. Recently, methods for genetic manipulation of V. harveyi have been successfully developed [START_REF] Delavat | Rapid and Efficient Protocol to Introduce Exogenous DNA in Vibrio harveyi and Pseudoalteromonas sp[END_REF] which should be considered for future work in gene manipulations in Harveyi strains of interest.

Altogether our data on distinct Vibrio populations (V. tasmaniensis, V. crassostreae, V.

harveyi) show that species-specific weapons are used to suppress oyster immune cells during POMS pathogenesis. This suggests that V. tasmaniensis, V. crassostreae, and V. harveyi should be considered as opportunistic pathogens, and not simple colonizers benefiting from OsHV-1 immune suppression.

NOT ONLY VIBRIO KILLERS CAN BENEFIT FROM OSHV-1 INFECTION

In the course of the present thesis, the polymicrobial nature of the POMS was demonstrated in our laboratory ( de Lorgeril et al., 2018). This prompted us to adapt our infection protocols. We therefore used mesocosms as ecologically realistic experimental infections in which oysters were exposed to seawater containing the OsHV-1 virus and a Vibrio community, instead of classical intramuscular injections of given pathogens. This modification of our procedures allowed us to unveil novel behaviors among the vibrios associated with POMS. As we worked in a polymicrobial framework, it can happen that multiple host-bacteria and bacteriabacteria interactions alter the within-host selective pressures acting on potential colonizers [START_REF] Alizon | Co-infection and Super-infection Models in Evolutionary Epidemiology[END_REF]. In article 3, our results showed that not all vibrios able to colonize oysters are cytotoxic, but some of them can benefit from the activity of the microbiota, which not only dampens the oyster immune defenses but also produces "public goods" that can be utilize by opportunistic cheaters.

We evidenced here a cheating behavior for iron acquisition [START_REF] Kramer | Bacterial Siderophores in Community and Host Interactions[END_REF] through vibrioferrin uptake in V. rotiferianus. This species, which colonizes efficiently oysters, is not cytotoxic and it has lost the vibrioferrin synthesis cluster present in V. harveyi while retaining the gene coding for the cognate receptor, an example of evolution of dependencies through adaptive gene loss, also known as the black queen hypothesis [START_REF] Morris | Black Queen hypothesis[END_REF]. We confirmed in vitro that growth can be restored under iron-limiting condition by the cell free extracellular products of V. harveyi, a vibrioferrin producer. This result leads us to propose that, as a moderately virulent and cytotoxic population, V. rotiferianus benefits from the compromised physiological state of the host caused by highly virulent populations like V.

harveyi but also from the non-cooperative acquisition of public goods such as siderophores to colonize the oyster through a purely opportunistic strategy.

It will now be necessary to perform additional mesocosm experiments with isolated and specific combination of populations to answer two pending questions. (i) Does the colonization of the host by V. rotiferianus result of an opportunistic strategy that depends on the presence of V. harveyi? If so, this would confirm that the oyster could serve as a niche for the development of dependency between vibrio populations (here, killers and cheaters). (ii)

What factors explain the exclusion of V. owensii and V. jasicida from oysters? Is it due to intrinsic potential disadvantages (e.g. metaolic requirements) specific or common to both populations or does it result from the interaction with the other two Harveyi populations (as neither V. owensii nor V. jasicida express the vibrioferrin siderophore and cognate receptor synthesis genes, unlike V. harveyi and V. rotiferianus)?

SYNERGISM BETWEEN VIBRIO POPULATIONS AND OSHV-1 VIRUS

We have shown here that two of the Harveyi populations (V. harveyi and V. rotiferianus) are capable, through virulence or public good-exploitation traits, to colonize oysters under POMS.

If vibrios alone did not cause mortality in our mesocosm experiments, they significantly worsened the mortality rate of oysters compared to the OsHV-1 only condition. Such a synergistic effect is a typically expected (Murray et al., 2014) but rarely demonstrated in ecologically-realistic experimental set-ups recapitulating the complexity of polymicrobial diseases. We have shown that OsHV-1 (de Lorgeril et al., 2018) and vibrios (Rubio et ai., 2019, Oyanedel, in prep) are pathogens that respectively manipulate or lyse hemocytes, the oyster immune cells. However, as described in Chapter 1, populations of hemocytes are diverse and still poorly characterized. Understanding synergism will now require to identify the hemocytes populations targeted by both pathogens, and the key functions consequently altered. For that, we propose the use of single cell RNAseq that should be of high interest to address these two specific questions and better understand the cellular and molecular determinants of pathogen synergism.

OSHV-1 IMMUNOSUPPRESSION AND MODIFICATION OF OYSTER AS A HABITAT

We observed here a contrasted dynamic of oyster-vibrio interaction when OsHV-1 was absent from the mesocosm (article 3). In this case, vibrio load only transiently increased in oyster tissues to later decrease by 48h without changes in the relative abundance of each of the four species of vibrio. Moreover, no significant differential gene expression was observed between naive and bacteria-only exposed oysters, as if the oyster immune system did not sense high vibrio loads when acquired through natural infection routes. This contrasts with our In the absence of host transcriptomic changes, the refractiveness of oysters to a stable colonization by exogenous vibrios prompted us to speculate that the oyster represents a is likely that R5.7 plays a key role. In V. crassostreae, another species of Splendidus clade, R5.7

is not sufficient but necessary for full virulence (Bruto et al., 2017(Bruto et al., , 2018) ) and acts independently of the presence of the T6SSpGV1512 to cause hemocyte cytotoxicity (Piel et al., 2019). As R5.7 is present in all the strains of Pop#23, we speculate that it could be responsible for cytotoxicity. While the determinants of Pop#23 cytotoxicity remains to be elucidated, we have identified that cytotoxicity towards hemocytes and the dampening of the cellular defenses of the oysters is also present in vibrios belonging to the microbiota of healthy oysters and not exclusive of highly virulent vibrios that recurrently cause systemic infection in diseased oysters. These findings further support that cytotoxicity is key in vibrios of the Splendidus clade to colonize oysters, but they also indicate that cytotoxicity is not a sufficient indicator of virulence; it rather contributes to modify the oyster towards a less hostile habitat.

TRADE-OFFS ON VIRULENCE ACTING ON VIBRIOS COLONIZING HEALTHY OYSTERS

We were intrigued by the strains of the Pop#23 that displays moderately virulence while carrying virulence factors such as an MARTX and a T6SS4G4_4. Comparative genomic among Pop#23 strains allowed us to identify differences in the gene content of the wbe cluster responsible for the synthesis of the O-antigen of the LPS. This wbe region is a hypervariable locus susceptible to extensive gene exchange through HGT that contributes to structural variation of the O-antigen. Due to its localization in the outer membrane, the O-antigen is subjected to selective pressures by environmental predators, and by the antimicrobial effectors of metazoan hosts (Wildschutte et al., 2004;March et al., 2013).

We demonstrated that while the O-antigen structure shared by moderately virulent strains confers resistance to predation by amoeba, it constrained the colonization efficiency and virulence potential of these strains. This was evidenced by the mutation of the wbe region that on the one hand, lowered the immunogenicity of the strain, increasing its colonization capacity and virulence potential in the oyster, but on the other hand, made it susceptible to predation by amoeba.

These results suggest that the selection for resistance to predation through O-antigen modifications can be in conflicting selection with virulence, in contrast with the coincidental selection identified in 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]. POMS seems to select predominantly highly virulent phenotypes that seem better adapted to benefit from immunosuppressed oysters, with the exception of opportunistic cheaters identified in the Mediterranean environment (article 3). Considering that OsHV-1 rapidly transmits among susceptible populations of oysters, it is expected that in this context, host rapid killing will promote pathogen transmission to proximal permissive individuals ensuring new replicative niches for virulent populations to thrive [START_REF] Fleury | Latitudinal drivers of oyster mortality: deciphering host, pathogen and environmental risk factors[END_REF]. In contrast, healthy juvenile oysters are hostile to new colonizers and therefore the expression of high virulence potential leading to the killing of the host can be in this case maladaptive (Alizon and Michalakis, 2015) as it will eliminate the bacterial niche forcing vibrios to undergo colonization of immunocompetent hosts with an established and stable microbiota. Therefore, the evolution towards a moderate virulence and resistance to predation, exemplified by Pop#23, could result in a higher overall fitness than the selection for higher virulence in this particular context of association with oysters.

OPPORTUNISTIC PATHOGENS OF POMS OUTSIDE THE VIBRIO GENUS Recently, culture independent studies of massive mortality episodes of oysters have allowed to identify that although vibrios are indeed present in diseased animals they are not the only bacterial group that thrive in diseased oysters affected by biotic and abiotic environmental stressors such as the OsHV-1 or high temperatures (de Lorgeril et al., 2018;Green et al., 2019;[START_REF] King | Characterisation of the Pacific Oyster Microbiome During a Summer Mortality Event[END_REF]. Among them, Arcobacter has been recurrently found more represented in diseased oysters and also capable to benefit from the disturbance of the microbiota and physiology of the host caused by a Vibrio injection to dominate the microbiota associated with the hemolymph. Arcobacter is considered as an emerging food and waterborne pathogen capable to cause gastroenteritis in humans. Arcobacter species have been found in human stool samples as well as livestock and raw food from animal origin (Sekhar et al., 2017) including edible mollusks with a general prevalence of 40% with the highest values in surf clams (87.4%) and the lowest in oysters (18%) (Collado et al., 2014). Among Arcobacter spp., antimicrobial and metal resistance together with virulence factors with cytotoxic effects towards mammalian cells have been described (Brückner et al., 2020). Given these characteristics it is likely that Arcobacter could functionally replace Vibrio as secondary opportunist of diseased oysters. Still, to date our knowledge of Arcobacter is very limited and much more investigation would be needed to demonstrate their role in disease expression.

We still ignore whether strains associated with POMS express virulence potential or have an opportunistic behavior through resources acquisition. So far, the ubiquity, ease of isolation and genetic manipulability of vibrios, together with the evidences about their involvement in oyster diseases has driven the research efforts towards this group contributing without question to the understanding of disease of complex etiology such as POMS. Nonetheless, non-targeted culture-independent approximations of the dynamics of the oyster microbiota highlight the importance to consider higher order interactions and other microbial taxon when studying diseases in oysters. With the development of metatranscriptomics applied to POMS by Annick Jacq (CNRS, Orsay, France) in collaboration with Eve Toulza in our laboratory, we expect to learn more on the functional complementarity / cooperation of microbial populations contributing to the fatal outcome of POMS.

similar dampening of host immune defenses and enable systemic infections. In V.

tasmaniensis and V. crassotreae, these mechanisms involve a T6SS and the protein R5.7 of unknown function, respectively. An effector of T6SS-mediated cytotoxicity toward immune cells was identified in V. tasmaniensis.

Finally, by characterizing vibrio populations associated with POMS in a Mediterranean oysterfarming site (article 3), we found that vibrios able to colonize OsHV-1 µvar-infected oysters vary according to ecosystems, but include species that express the same capacity to dampen the oyster cellular defenses using cytotoxic activities. In particular in the Thau lagoon, V. harveyi (adapted to elevated temperature) is a virulent species associated with POMS that synergizes with OsHV-1 µvar to accelerate oyster death. Moreover, we showed that the Vibrio community associated with POMS is composed of both opportunistic pathogens, like V. harveyi, and simple opportunists (cheaters), like V. rotiferianus, benefiting from the activity of the pathogens, which dampen the oyster immune defenses to facilitate the colonization and produce vibrioferrin, a siderophore that the cheaters can utilize for their own growth within diseased oysters.

Article 4 (annexe 1) provides a synthesis of our current understanding of Vibrio-bivalve interactions, which integrates the results of the present thesis and draws hypothesis that may explain the higher virulence of Vibro species in oysters as opposed to mussels

Bases moléculaires et cellulaires des interactions Vibrio / Crassostrea gigas en contexte sain et pathologique.

Les bactéries du genre Vibrio sont ubiquitaires dans les milieux aquatiques. Elles adoptent des modes de vie libres et associés. Elles établissent des symbioses mutualistes, commensales et parasitaires avec de nombreux métazoaires. Chez l'huître Crassostrea gigas saine, un microbiote abondant et diversifié qui comprend de nombreux Vibrio est maintenu sous homéostasie immunitaire. Toutefois, sous l'influence de stress biotiques ou abiotiques une dysbiose se crée favorisant la prolifération d'espèces opportunistes de Vibrio qui peuvent induire la mort des huîtres. C'est notamment le cas lors du syndrome de mortalité des huîtres du Pacifique (POMS) provoqué par un virus immunosuppresseur, OsHV-1 µvar. Nous avons ici caractérisé les interactions Vibrio / C. gigas en contexte sain et pathologique. Nous montrons que chez des populations de V. splendidus associées à des huîtres saines, la virulence et la capacité de colonisation peuvent être contraintes par la sélection de structures de l'antigène O qui sont plus facilement reconnues par le système immunitaire de l'hôte mais qui confèrent une résistance au broutage par des amibes marines. Ce compromis entre la capacité à coloniser son hôte et la persistance environnementale est vraisemblablement à l'origine de la grande diversité structurale observée pour l'antigène O dans cette espèce. En contexte pathologique, nous avons montré que les espèces associées à l'huître sont en grande majorité cytotoxiques pour les cellules immunitaires de leur hôte, et ce, quel que soit l'environnement considéré (Atlantique, Méditerranée). Cette cytotoxicité est un déterminant clé de l'échappement aux puissantes défenses cellulaires de l'huître et du succès infectieux. Nous avons observé que les bases moléculaires de la cytotoxicité sont spécifiques à chaque espèce de Vibrio étudiée. Enfin, au-delà des agents pathogènes opportunistes qui utilisent la cytotoxicité (V. tasmaniensis, V. crassostreae, V. splendidus, V. harveyi), nous avons identifié de simples opportunistes, comme V. rotiferianus, qui non seulement bénéficient de l'activité immunosuppressive des autres agents pathogènes mais également adoptent des stratégies d'acquisition non coopérative de biens publics, tels que les sidérophores produits par la communauté microbienne hébergée par leur hôte.

Cellular and molecular bases of Vibrio interactions with Crassostrea gigas oysters in health and disease

Bacteria of the genus Vibrio are ubiquitous in aquatic environments. They adopt free and associated lifestyles. They establish mutualistic, commensal and parasitic symbioses with numerous metazoa. In the healthy Crassostrea gigas oyster, an abundant and diverse microbiota that includes many Vibrio is maintained under immune homeostasis. However, under the influence of biotic or abiotic stressors a dysbiosis is created favoring the proliferation of opportunistic Vibrio species, which can induce the death of oysters. This occurs during the Pacific Oyster Mortality Syndrome (POMS), which is caused by an immunosuppressive virus, OsHV-1 µvar. We have here characterized the Vibrio / C. gigas interactions in health and disease. We show that in populations of V. splendidus associated with healthy oysters, virulence and colonization capacity can be constrained by the selection of O-antigen structures that are more easily recognized by the host immune system but which confer resistance to grazing by marine amoebae. This trade-off between the ability to colonize its host and environmental persistence is likely the cause of the great structural diversity observed for the O-antigen in this species. In a pathological context, we have shown that the species associated with the oyster are for the most part cytotoxic for the immune cells of their host, regardless of the environment considered (Atlantic, Mediterranean). This cytotoxicity is a key determinant of the escape from the oyster's powerful cellular defenses and determines the infectious success. We observed that the molecular bases of cytotoxicity are specific to each Vibrio species studied. Finally, beyond opportunistic pathogens that use cytotoxicity (V. tasmaniensis, V. crassostreae, V. splendidus, V. harveyi), we have identified simple opportunists, such as V. rotiferianus, which not only benefit from the immunosuppressive activity of other pathogens but also adopt strategies of non-cooperative acquisition of public goods, such as siderophores produced by the microbial community hosted by their host.

FIGURESFigure 1 .

 1 FIGURESFigure 1. Morphological traits and taxonomy of vibrios .....................................................................

Figure 2 .

 2 Figure 2. Metapopulation structure of vibrios. .....................................................................................

Figure 3 .

 3 Figure 3. Free-living and particle-associated dynamics of vibrios. ..................................................

Figure 4 .

 4 Figure 4. Environmental dynamics of vibrios association with biotic surfaces. ..............................

Figure 5 .

 5 Figure 5. The paradigmatic vibrio-squid mutualism. ...........................................................................

Figure 6 .

 6 Figure 6. LPS structure and the O-antigen structural diversity. ..........................................................

Figure 7 .

 7 Figure 7. The three major transmembrane signaling system in bacteria. .......................................

Figure 8 .

 8 Figure 8. One and two component signaling system. .......................................................................

Figure 9 .

 9 Figure 9. Representative quorum sensing autoinducers. ...................................................................

Figure 10 .

 10 Figure 10. Cell-density dependent regulationof cellular functions in V. harveyi. .........................

Figure 11 .

 11 Figure 11. Diversity of V. cholerae iron transport system. ...................................................................

Figure 12 .

 12 Figure 12. Internalization of V. tasmaniensis LGP32 .............................................................................

Figure 13 .

 13 Figure 13. Secretion of outer membrane vesicles by V. tasmaniensis LGP32 ................................

Figure 14 .

 14 Figure 14. Class of secretion systems of Gram-negative bacteria ...................................................

Figure 15 .

 15 Figure 15. Type VI secretion system (T6SS) model of V. cholerae. ...................................................

Figure 16 .

 16 Figure 16. Crassostrea gigas anatomy ..................................................................................................

Figure 17 .

 17 Figure 17. Life cycle of the oyster Crassostrea gigas. .........................................................................

Figure 18 .

 18 Figure 18. Types of circulating hemocytes of C. gigas observed by transmission electron microscopy. ................................................................................................................................................

Figure 19 .

 19 Figure 19. Reconstruction of signaling pathways in C. gigas. ...........................................................

Figure 20 .

 20 Figure 20.Bacterial resistance to grazing by Vanella sp 1411. ........................................................Figure 21. Relative weight of resistance to antimicrobials, virulence and surface charge in vibrio association to oysters. ..................................................................................................................

Figure 22 .

 22 Figure 22. Resistance to ROS, virulence and surface charge discriminate vibrios according to association to oysters ..............................................................................................................................

Figure 23 .

 23 Figure 23. PCR verification of evpP deletion. .....................................................................................

Figure 24 .

 24 Figure 24. Cytotoxic activty of LGP32 ΔevpP. ....................................................................................

Figure 25 .

 25 Figure 25. Contribution of evpP to V. tasmaniensis virulence. ........................................................

CHAPITRE 2 :

 2 RESULTATS SECTION I. DETERMINANTS DE LA COLONISATION ET PERSISTANCE ENVIRONNEMENTALE CHEZ V. SPLENDIDUS ASSOCIES A DES HUITRES SAINES En raison de leurs conséquences importantes sur l'ostréiculture, les associations entre les vibrios et les huîtres ont été principalement étudiées dans le but d'identifier leur rôle en tant qu'agents étiologiques dans les maladies des huîtres. Le travail de notre proche collaboratrice Frédérique Le Roux à l'Ifremer (laboratoire de biologie intégrative des modèles marins, Station Biologique de Roscoff) a révélé que les espèces de Vibrio associées aux POMS expriment un potentiel de virulence contre l'huître, et que des populations virulentes de

  Une série d'échantillonnages a été réalisée dans la lagon de Thau par Philippe Haffner, en commençant par un épisode POMS à l'automne 2015, confirmé par la présence d'OsHV-1 dans les huîtres moribondes. Les échantillonnages suivants ont été réalisés en 2016 pendant et en dehors des périodes de mortalité. En collaboration avec Maxime Bruto, Arnaud Lagorce a caractérisé la structure des populations de vibrios dans les huîtres et la colonne d'eau. Mon travail a consisté à (i) phénotyper les principales espèces retrouvées pour leur capacité à coloniser les huîtres dans des infections écologiquement réalistes (balnéation) mettant en jeu le partenaire viral OsHV-1 µvar et (ii) à déterminer les bases moléculaires sousjacentes aux associations mises en jeu par les espèces colonisatrices. Dans l'article 3 (en préparation), les résultats présentés montrent que dans la lagune de Thau, l'infection par OsHV-1 µvar était associée à une prévalence particulière des membres du clade Harveyi, avec des souches affiliées taxonomiquement aux espèces de Vibrio harveyi et V. rotiferianus préférentiellement associées aux tissus d'huîtres tandis que V. jasicida et V. owensii étaient présents dans la colonne d'eau et exclus des huîtres moribondes. La reproduction de cette infection polymicrobienne en mésocosme et une étude fonctionnelle de cette collection de souches nous ont permis de montrer que seules les espèces V. harveyi et V. rotiferianus colonisaient les huitres infectées par OsHV-1 µvar, reproduisant ainsi en condition expérimentale l'observation de terrain. Lors de cette colonisation, l'hôte manipulé par le virus n'en demeure pas moins un habitat que seules certaines espèces de Vibrio ont la capacité de coloniser. L'analyse des génomes des espèces colonisatrices et des tests fonctionnels de cytotoxicité et de production/acquisition de sidérophores nous permettent de proposer que deux stratégies différentes de colonisation soient présentes parmi ces deux espèces de Vibrio. D'une part, l'espèce V. harveyi au fort potentiel de virulence utilise des propriétés cytotoxiques pour affaiblir les défenses cellulaires de l'huître, comme déjà observé dans le clade Splendidus. D'autre part, l'espèce au faible potentiel de virulence, V.

L

  'IMMUNOSUPPRESSION PAR L'OSHV-1 ET LA MODIFICATION DE L'HUITRE EN TANT QU'HABITAT Dans le cas de l'infection expérimentale en mésocosme, la charge en vibrios n'augmente que de façon transitoire dans les tissus d'huître pour diminuer plus tard de 48 h en absence du virus OsHV-1. De manière surprenante, nous n'avons pas observé d'expression génique différentielle significative entre les huîtres naïves et les huîtres exposées aux vibrios dans ce contexte, contrairement aux fortes réponses transcriptionnelles qui sont induites par l'injection de vibrios virulents ou non virulents (articles 2). Comme si le système immunitaire des huîtres ne détectait pas l'augmentation des charges de vibrios lorsqu'ils sont acquis par des voies naturelles d'infection. La raison pour laquelle les vibrios n'induisent pas la réponse immunitaire dans les configurations d'infection naturelle reste inconnue. Une hypothèse possible est la production par l'hôte de PGRP à activité amidase (PGRP-LB), dont il a été démontré qu'elle supprime la signalisation immunitaire conférant une tolérance au microbiote, chez d'autres invertébrés (Zaidman-Rémy et al., 2006). En l'absence de changements transcriptomiques de l'hôte, la réfractivité des huîtres à une colonisation stable par des vibrios exogènes nous a incités à spéculer que l'huître représentait un habitat hostile à coloniser par des vibrios sans l'effet perturbateur de facteurs supplémentaires tels que l'immunosuppression par OsHV -1. Ceci est étayé par nos données supplémentaires sur la tolérance au cuivre, au zinc et au ROS (chapitre 2, section I, 4) et discuté plus en détail dans notre mini-revue (article 4). Cette hypothèse est en outre étayée par la reprogrammation transcriptionnelle des huîtres en présence d'OsHV-1, qui permet une colonisation stable des vibrios. Pour aider à clarifier la dynamique d'association des vibrios avec les huîtres saines, nous proposons de suivre la localisation histologique des bactéries exprimant la GFP chez les huîtres saines et affectées par le POMS, au cours de la cinétique d'exposition de la même manière que les vibrios ont été localisés par immunohistologie dans l'article 2. LA CYTOTOXICITÉ DE V. SPLENDIDUS COLONISANT DES HUÎTRES SAINES : UN FACTEUR DE COLONISATION PLUTÔT QU'UN DÉTERMINANT DE LA VIRULENCE

  Photobacterium (1 spp), Salinivibrio (6 spp), Thaumaslovibrio (2 spp) and Vibrio (128 spp)

Figure 1 .

 1 Figure 1. Morphological traits and taxonomy of vibrios (A) Light microscopy images of Vibrios species, scale bars are 2 μm. (B) Transmission Electron Microscopy image of negatively stained V. alginolyticus showing the polar flagellum, scale bar 0.5 μm (right), Imaging of a single sheathed polar flagellum of V. alginolyticus (scale bar 0.15 μm) showing the detailed motor structure (scale bar 0.2 μm) (left) (modified from Zhu et al., 2017). (C) Phylogenetic tree of Vibrionaceae based on 86 strains, 10 sequenced genomes and 8 housekeeping genes (ftsZ, gapA,

Figure 2 .

 2 Figure 2. Metapopulation structure of vibrios. (A) Dispersal routes of bacterial populations through food chain interactions proposed by Preheim et al., 2011, and used as a sampling strategy to define and compare the population structure of vibrios across animal host and the water column. (B) Prediction of the population structure based on the ecological sampling showed in (A). Maximum likelihood gene tree constructed by concatenated protein-coding genes used for the MLST genotyping. Phylogenetic tree constructed trough MLST genotyping with 1753 vibrio isolates integrating season of sampling (grey or black outer ring) and predicted habitat categories (colored inner ring). The right panel indicated the distribution of each of the identified populations across seasons and predicted habitats. It highlights the overlap of populations among different habitats. Color code for the predicted habitats and live or death condition of the host is indicated in the "Isolation data" box. Abbreviation : F, fall ; S, Spring ; C, Crab ; M, Mussel ; G, Gill ; S, Stomach Tissue ; SC, Stomach Contents ; H, Hindgut Tissue ; HC, Hindgut Contents ; Z, Zooplankton.

Figure 3 .

 3 Figure 3. Free-living and particle-associated dynamics of vibrios.(A) Model for behavioral adaptations of two sympatric V. cyclitrophicus populations. Both populations respond to nutrient gradient but only the L population carrying key genes linked to adhesion and biofilm-producing genes are able to establish in the particle while the S populations stay around the particle benefiting from the dissolved nutrient patch. In proximity of a new nutrient rich particle, the freeswimming S population is able to readily migrate (Modified from[START_REF] Shapiro | Microbial Speciation[END_REF]. (B) Scanning electron microscopy photograph of V. vulnificus bacteria (in pink) attached to artificial marine snow particles (Modified from[START_REF] Hubert | A Universal Oyster Infection Model Demonstrates that Vibrio vulnificus Type 6 Secretion Systems Have Antibacterial Activity in vivo[END_REF]).

Figure 4 .

 4 Figure 4. Environmental dynamics of vibrios association with biotic surfaces. (A) The wide range of associations of vibrios exemplified with four well described Vibrio species (modified from Reen et al., 2006). (B) SEM image of V. cholerae biofilm formed in chitin derived from crab shells, bar 5μm (modified from Echazarra and Klore. 2019. (C) Migration of Vibrio fischeri (in green) through the pores that connect with the light organ 2-5 hours after the initial aggregation, bar 10 μm (modified from Nyholm and Macfall-Ngai. 2004).

Figure 5 .

 5 Figure 5. The paradigmatic vibrio-squid mutualism. (A) Model of colonization of the light organ of E. scolopes (a) Mucus secretion by E. scolopes in response to bacterial presence (b) Only Gram-negative bacteria aggregate in the mucus (c) Grampositive bacteria exclusion from aggregates (d) Viable and motile V. fischeri migrate through the pores that connect with the light organ (e) Successful colonizers become non-motile and induce morphological changes in the host that inhibits further colonization (Modified from Nyholm and McFall-

Figure 6 .

 6 Figure 6. LPS structure and the O-antigen structural diversity. (A) Basic structure of lipopolysaccharide : At the bottom the Lipid A moiety with the di-glucosamine headgroup indicated by the polygonal structures, the acyl chains represented by zigzag lines, and the negatively charged phosphate groups indicated by red dots. Inner and outer core consisting of repeating units and the O-antigen including a bracket with an "n" that indicates the variable number of repeating units that form the outermost structure of the LPS (modified from Maeshima and Fernandez, 2013. (B) O-antigen profiles, indicating divergence from the general structure show in (A) can be resolved by migration of LPS extracts in SDS-PAGE gels and visualization by silver staining profiles. The image shows the diversity of O-antigen structure associated with different vibrio isolates associated with different marine organisms (modified from Wildschutte et al, 2010).

  Transmembrane signaling systems sense these environmental cues and transduce them into intracellular signals that regulate cellular functions in response to the extracellular stimuli. Onecomponent signaling systems (Figure7A) (OCS), two-component systems (TCS) (Figure7B) and extracytoplasmic function (ECF) sigma factors (σ) (Figure7C) are the three major types of transmembrane signaling systems in bacteria[START_REF] Jung | Bacterial Transmembrane Signalling Systems and their Engineering for Biosensing[END_REF].

Figure 7 .

 7 Figure 7. The three major transmembrane signaling system in bacteria. (A) One-component systems reunite an extracellular sensor input domain and an intracellular signal output binding domain in a single molecule. (B) Two-component systems present a membraneintegrated histidine kinase (HK) and a response regulator (RR). (C) Extracytoplasmic function (ECF) sigma factors (σ) are released from the anti-sigma factor (anti-σ) upon stimulus (modified from Jung et al, 2018).

  coli and Vibrio species[START_REF] Brameyer | DNA-Binding Directs the Localization of a Membrane-Integrated Receptor of the ToxR Family[END_REF]. The periplasmic sensors ToxR/S and TcpP/H regulate the expression of virulence proteins in V. cholerae through the regulation of the AraC-like transcriptional activator ToxT which is coded in the Vibrio pathogenicity island-1, and is induced upon sensing of the intestinal environment[START_REF] Childers | Regulation of Virulence in Vibrio cholerae: The ToxR Regulon[END_REF] (Figure8).

Figure 8 .

 8 Figure 8. One and two component signaling system. (A) Signal transduction in one (upper) and two (lower) component systems. (A) One component system consists of a single protein that possesses an input and output domain but it lacks the His-Asp phosphotransfer domain (grey boxes). Two component system containing the input domain that receives the signal and the output domain located in two different proteins that communicate through the His-Asp phosphotranfer domain (grey bowes) (modified from Schaller et al., 2011) (B) Onecomponent system ToxR/S and TcpP/H (ToxR regulon) controls the expression of virulence proteins in V.cholerae. ToxR and TcpP are inner-membrane proteins that binds to the toxT promoter activating its transcription. In turn ToxT activates the transcription of ctxAB and tcpA-F coding for the cholera toxin and the toxin-regulated pilus, respectively (taken fromHaas et al., 2014)

II. 2

 2 QUORUM SENSING Quorum sensing (QS) is a cellular communication mechanism that allows a wide diversity of bacteria to switch from individual to coordinated population behaviors through collective changes in gene expression in response to cell density (Rutherford 2011). Coordination is achieved through the extracellular accumulation of signaling molecules, referred to as autoinducers (Figure 9), which are produced constitutively by the bacterial populations. Their recognition is mediated by perisplasmic sensors that resemble two component systems with transmembrane histidine kinase and a response regulator domain but with no DNA-binding domain.

Figure 9 .

 9 Figure 9. Representative quorum sensing autoinducers. (A) N-Acyl-L-homoserine lactone (AHL). (B) Autoinducer-2 (AI-2). (C) γ-Butyrolactones. (D) A-factor. (E) p-Coumaryl homoserine lactone (pC-HSL). (F) Indole.

Figure 10 .

 10 Figure 10. Cell-density dependent regulationof cellular functions in V. campbelli BAA-1116. QS is regulated by three two-component signaling systems that work in parallel to regulate virulence and bioluminescence. At low cellular densities, in absence of signaling molecules, LuxN, LuxQ and CqsS madiate the phosphorylation of the σ 54 -dependent activator LuxO, which activates the expression of small regulatory RNAs that contribute to the destabilization of the mRNA encoding LuxR the master regulator. High cellular densities promote the productions of signaling molecules by the LuxM, LuxS and

Figure 11 .

 11 Figure 11. Diversity of V. cholerae iron transport system. Localization of each iron transporting system respect to the cell envelope and the compound transported is shown. The names of the siderophores recognized by the specific transporters are indicated outside the bacterial cell. The TonB-ExbB-ExbD complex located at the inner membrane allows energy transduction to the specific siderophore receptors located at the outer membrane (taken from Payne et al., 2016)

  tuberculosis and L. monocytogenes (Uribe-Quero and Rosales, 2017) while available examples in vibrios remain fragmentary based on different vibrio species infecting different hosts.

Figure 12 .

 12 Figure 12. Internalization of V. tasmaniensis LGP32 V. tasmaniensis LGP32 uses the OmpU porin as an adhesin that facilitates the internalization into hemocytes. (1) The OmpU porin is recognized by the plasmatic protein Cg-EcSOD that will cover the

Figure 13 .

 13 Figure 13. Secretion of outer membrane vesicles by V. tasmaniensis LGP32 Negatively stained LPG32 cultures in logarithmic phase and phagocyted bacteria observed by electron microscopy. (A) Outer membrane vesicles (OMV) produced by LGP32 indicated by black arrows. (B) Detail of OMVs detaching from the outer membrane or (B) from the the polar flagellum (modified from Vanhove et al., 2015)

Figure 14 .

 14 Figure 14. Class of secretion systems of Gram-negative bacteria (taken from Green and Mecsas, 2015)

  and/or PAAR-repeat proteins and incorporated into the Hcp tube. Extracellular signals that trigger the contraction of the sheath cause the ejection of the spike-tube complex carrying the effectors across the donor and target membranes into the recipient cell (Navarro-Garcia et al., 2019).

Figure 15 .

 15 Figure 15. Type VI secretion system (T6SS) model of V. cholerae. Diagram of the extended and contracted state of the T6SS. It is composed of a membrane and baseplate complex; the outer contractile sheath is composed by protein VipA/B and the inner needle complex by the Hcp and VgrV spike. Following the contraction of the VipA/VipB sheath, the VgrG, Hcp and associated toxins are injected into the target cell (Taken from Crisan et al., 2019).

Figure 16 .

 16 Figure 16. Crassostrea gigas anatomy (A) External morphology (modified from galtsoff, 1964). (B) Internal view of the left valve of an adult

Figure 17 .

 17 Figure 17. Life cycle of the oyster Crassostrea gigas. (Taken from Le Roux et al, 2016)

Figure 18 .

 18 Figure 18. Types of circulating hemocytes of C. gigas observed by transmission electron microscopy. (A) Small, probably young hyalinocyte. (B) Hyalinocyte. (C) Large Hyalinocyte. (D) Granulocyte with electron-dense granules. (E) Granulocyte displaying electron lucent granules and numerous cytoplasmic bodies apparently corresponding to phagocytic residues (modified from Bachère et al., 2004).

  Retinoic acid-inducible gene I (RIG-I)-like receptor (RLRs) contribute to the recognition of viral nucleic acids by sensing non-self RNA in the cellular cytoplasm. Search for homologues of the three human RLRs members, RIG-I, MDA5 and LGP2 identified thirteen candidate CgRLRs annotated as RIG-I family members with an unusual diversity of domain architecture. The first RLR identified in C. gigas, CgRIG-I, has the typically conserved domains, i.e. the caspase activation and recruitment domains (CARDs), a helicase domain and a C-terminal regulatory domain (RD) (Y. Y. Zhang et al., 2014). It is exclusively induced by the dsRNA analog polyI:C among a repertoire of other MAMPs and its promoter region contains predicted sites for NF-kB, IRF and STAT binding, sustaining the responsiveness to polyI:C (Y. Y.

  Two β-1,3-glucan binding proteins (βGBP) have been identified in C. gigas, CgβGBP-1 and CgβGBP-2 have been suggested to have different immunological functions based on in vitro investigations using recombinant proteins(Itoh et al., 2010a).Scavenger receptors (SR) are endocytic PRR with the ability to recognize host-modified lowdensity lipoproteins and various bacterial ligands. The Scavenger Receptor Cystein-Rich (SRCR) domain is an ancient and highly conserved domain of typically 110 amino acids in length with six or eight cysteine residues connected by three or four interdomain disulfide bonds. SRCRs are found in SRs but are also common to a large variety of molecules restricted to immune cells[START_REF] Mukhopadhyay | The Role of Scavenger Receptors in Pathogen Recognition and Innate Immunity[END_REF]. In the genome of C. gigas, 71 different predicted SRCRs have been identified. The contribution of SR to the adaptation to multiple stressors have been proposed, as several of them are up-regulated during the Pacific Oyster Mortality Syndrome affecting C. gigas juveniles (Fleury et al., 2010), during hypoxia (David et al., 2005) and during bacterial challenge (McDowelll et al., 2014). III.1.3.4 CELLULAR COMMUNICATI ON The signals recognized by the different receptor described above activate different signaling pathways to mount humoral and cellular responses, which usually depend on the type of stimuli.Toll-like receptors signaling pathwayAlthough details of the TLRs signaling and involvement in the immune response still need to be investigated with greater details in oyster, several components of the signaling pathways coupled to this type of PRR have been identified in the genome of C. gigas, namely MyD88, IRAK, TRAF, ECSIT, IKK and members of the canonical pathway for Nuclear factor-kappaB (NF-κB) activation (see section below). The Myeloid differentiation factor 88 (MyD88) corresponds to a cytosolic adaptor that links, through protein-protein interaction, the IL-1R domain to the IL-1R-associated kinases (IRAK) family kinases, which in turn can activate NF-κB, MAPK and AP-I. Ten MyD88 genes have been annotated with some of them either induced or suppressed by bacterial challenge. A blocking peptide designed against the TIR domain of CgMyD88 impaired NF-kB activation in HEK293 cell transfected with CgTLR1 and CgMyD88 but also inhibit hemocyte degranulation and CgTNF expression in response to V. parahaemolyticus injection(Zhang et al. 2013).

Figure 19 .

 19 Figure 19. Reconstruction of signaling pathways in C. gigas. The presented diagram was constructed based on the identification of conserved sequences in the oyster genome and most interactions between signaling componentes have been inferred from vertebrate and insect literature (taken from Green and Montagnani 2015).

  interferon (IFN) family cytokines are produced upon recognition of MAMPs related to viral infection, such as dsRNA and viral glycoproteins which promotes, through the JAK/STAT signaling, the translocation of interferon regulatory factors (IRFs) and other transcription factors to the cell nucleus (Randall and Goodbourn, 2008; Martins, 2020). The production of interferon in turn leads to the induction of IFN-stimulated genes that stimulate an amplification loop of IFN production and also antiviral mechanisms such as apoptosis or autophagy to limits the viral replication (Sadler and Williams, 2008).

  ; they contain a hydrophobic N-terminal domain and a cationic C-terminal domain. C. gigas possesses a diversity of Big defensins (Gerdol et al., 2020) among which CgBigDef-1 and -2 are specifically expressed in circulating and infiltrating hemocytes, after bacterial challenge (Rosa et al. 2011). CgBigDef-1 was shown to entrap and kill bacteria into elongated fibrils called nanonets induced by contact with bacteria (Loth et al., 2019).

  POMS occur seasonaly, during a permissive seawater temperature window ranging from 16°C to 24°C[START_REF] Pernet | Mass Mortalities of Pacific Oysters Crassostrea gigas Reflect Infectious Diseases and Vary with Farming Practices in the Mediterranean Thau Lagoon, France[END_REF]. Susceptible oysters reared in the intertidal farming sites become infected by the OsHV-1 virus, which starts replicating exponentially during the first 24h. This promotes the shedding of virions into the water column which are then dispersed towards other hosts. Mortalities start around day 3 post infection, following a viral-driven suppression of antimicrobial defenses. The destabilization of the host microbiota and a fatal systemic infection due to the replacement of the commensal bacteria communities with virulent populations (deLorgeril et al., 2018), among which Vibrio crassostreae have been repeatedly

  Figure S1. The rtxACHBDE cluster in V. splendidus #23. A-The figure shows gene replacement between non-virulent and moderately virulent strains leading to loss/acquisition of the MARTX encoding genes cluster. B-MARTX effector domains. The domain abbreviations are: Rtx = Repeats-in toxin; ACD = Actin Cross-linking Domain; ABH = α/ Hydrolase; MCF = Makes Caterpillars Floppy; RRSP = Ras/Rap1-specific endopeptidase; CPD = Cysteine Protease Domain (Satchell, 2015)

Figure S4 .

 S4 Figure S4. Strains of V. splendidus #23 are cytotoxic towards hemocytes. Maximum cytotoxicity exerted by V. splendidus over oyster hemocytes in vitro. A hemocyte monolayer was exposed to vibrios at a MOI of 1:50 and the percentage of cell lysis was monitored by the SYTOX Green assay. (A) shows that the four strains of V. splendidus #23 are cytotoxic towards hemocytes. Data are representative of five independent experiments. (B) shows that the vipA-i mutant derived from V. splendidus 4G4_4 does not show any attenuation of its cytotoxicity towards hemocytes, unlike the vipA1-I mutant derived from V. tasmaniensis LGP32. Data are representative of three independent experiments. Mean values of three technical replicates are displayed  SD. Identical letters indicate a non-significant difference (ANOVA, p < 0.05 and Tukey HSD test).

Figure S5 .

 S5 Figure S5. Lipid A inferred structure does not differ between strains 4G1_8 and 4G4_4. (A) Data show the MALDI-TOF spectra of 4G1_8 and 4G4_4 Lipid A. MALDI-TOF-MS analysis was performed in negative-ion [M-H]-/ linear mode. A major Lipid A molecular species was observed at m/z 1740.1-1740.7, which by analogy with V. cholerae Lipid A could be attributed to a hexa-acyl (6 FA) molecular species (Hankinset al., 2011, 2012), with one suggested exception i.e. the presence of non-hydroxylated 12:0 fatty acid at the secondary C3' position. As a consequence, no molecular species with Glycine substitution was found in either strain. Moreover, no substitution of phosphate groups with charged amino derivatives such as phosphoethanolamine or aminoarabinose could be inferred from our MS spectra. An additional peak, common to both strains was observed at m/z 1976.1-1977.4, which could be tentatively attributed to a hepta-acyl Lipid A molecular species (7 FA) carrying an additional 16:0 or 16:1 fatty acid. This molecular species appeared more intense in the virulent 4G4_4 than in the non-virulent 4G1_8. (B) Lipid A structure from Vibrio cholerae O1 adapted from Hankins et al. (2012), Fig.2. The upper panel shows

  Figure S5. Lipid A inferred structure does not differ between strains 4G1_8 and 4G4_4. (A) Data show the MALDI-TOF spectra of 4G1_8 and 4G4_4 Lipid A. MALDI-TOF-MS analysis was performed in negative-ion [M-H]-/ linear mode. A major Lipid A molecular species was observed at m/z 1740.1-1740.7, which by analogy with V. cholerae Lipid A could be attributed to a hexa-acyl (6 FA) molecular species (Hankinset al., 2011, 2012), with one suggested exception i.e. the presence of non-hydroxylated 12:0 fatty acid at the secondary C3' position. As a consequence, no molecular species with Glycine substitution was found in either strain. Moreover, no substitution of phosphate groups with charged amino derivatives such as phosphoethanolamine or aminoarabinose could be inferred from our MS spectra. An additional peak, common to both strains was observed at m/z 1976.1-1977.4, which could be tentatively attributed to a hepta-acyl Lipid A molecular species (7 FA) carrying an additional 16:0 or 16:1 fatty acid. This molecular species appeared more intense in the virulent 4G4_4 than in the non-virulent 4G1_8. (B) Lipid A structure from Vibrio cholerae O1 adapted from Hankins et al. (2012), Fig.2. The upper panel shows

Figure S6 .

 S6 Figure S6. Inactivation of epsE in strains 4G4_4 and 4D1_8 generates the same O-antigen modifications and cytochrome C binding phenotype. (A) SDS PAGE electrophoresis of LPS extractions from bacterial cultures in stationary phase. (B) Percentage of cytochrome C bound to the bacterial surface of wild type strains and epsE-i isogenic mutants. Data represent mean values of 3 independent experiments  SD. Different letters indicate a significant difference (ANOVA, p < 0.05 and Tukey HSD test).

Figure S8 .

 S8 Figure S8. Strain 4G4-4 do not show killing activity against E. coli ML35p in vitro. Cells were coincubated in agar media for 4 h to allow cell to cell contact and T6SS mediated killing. Bars represent the mean cfu per milliliter of surviving E. coli from two replicates (+ SD). Means were compared by a Student's t-test, ns= p > 0.05

Figure 20 .

 20 Figure 20. Bacterial resistance to grazing by Vanella sp 1411. Ressistance to amoeba grazing was assessed for V. splendidus strains 4D1_8 and epsE-i by measuring the fluorescence of the GFP-expressing bacteria after contact with amoebae over 7 days. The strain V. tasmaniensis LMG20012T and the published mutant 4G4_4 epsE-i were used as a grazing-susceptible and grazing-resistance control, respectively. Results show the mean of three technical replicates  SD. Data are representative of two independent experiments. Different letters indicate a significant difference, p<0.001 (RM-ANOVA).

Figure 21 .

 21 Figure 21. Relative weight of resistance to antimicrobials, virulence and surface charge in vibrio association to oysters. 6 vibrio populations (30 strains) were phenotyped. (a) Resistance to antimicrobials. Minimum Inhibitory Concentration of Cg-Bigdefensin1, inhibition halo of Copper, Zinc and ROS (cumene hydroperoxide). Statistical significance (p<0.05) were determined by ANOVA one-way with a Holm-Sidak's post test and designated by different letters. (b) Relative surface charge of vibrio strains revealed by cytochrome c binding. Statistical significance (p<0.05) determined by non-parametric Mann Whitney and designated by an asterisk. (c) General lineal model generated including antimicrobial resistance, surface charge and virulence data. (c) Virulence in oyster experimental infections. Mortalities were monitored at day 1

Figure 22 .

 22 Figure 22. Resistance to ROS, virulence and surface charge discriminate vibrios according to association to oysters 6 vibrio populations (30 strains) were phenotyped and grouped by preferential habitat (a) Resistance to

*

  Figure S1 l Extracellular secretion products of J2-9 are not cytotoxic to hemocytes. Cytotoxicity of J2-9 was monitored on monolayers of hemocytes by measuring the fluorescence of Sytox green over 15 hours. At time 0, J2-9 cells (overnight culture) were added to hemocyte monolayers at a MOI of 50:1. Alternatively, an overnight culture supernatant of J2-9 devoid of bacteria (secretion products only) was added to hemocytes. The graph shows the time course of cytotoxicity of J2-9 (black diamonds) and the lack of cytotoxocity of J2-9 secretion products (grey diamonds). The error bars represent the standard deviation of technical triplicates. Data are representative of three independent experiments.

Figure S2 l

 S2 Figure S2 l Virulence of V. tasmanienis LGP32 and V. crassotreae J2-9 is independent on oyster genetic background. Kaplan Meier survival curves were generated from two additional families of biparental oysters injected with 7.5 x 10 7 CFU per oyster of LGP32, J2-9, LMG20012T or J2-8. Mortalities were monitored on 2 groups of 20 oysters per family (2 seawater tanks per condition) during 7 days after injection. (A) Mortalities on oyster Decipher family #15.LGP32, J2-9 and J2-8 induced 58.7%, 46% and 2.3% of mortalities of oysters respectively. LMG20012T did not induce mortalities. J2-9 and LGP32 conditions are not significantly different (Log-rank p-value=0.560). (B) Mortalities on oyster Decipher family #1. LGP32, J2-9 and J2-8 induced 40.7%, 57.1% and 3.3% of mortalities in oysters, respectively. LMG20012T did not induce mortalities. J2-9 and LGP32 conditions are not significantly different (Log-rank p-value=0.401).

Figure S5 l

 S5 FigureS5l Antimicrobial peptides respond similarly to virulent and non-virulent strains. Normalized read counts for AMP genes (cg-bigdef1, cg-bigdef2, cg-bpi, cg-defh, cg-defm, cg-prp) are plotted for vibrioinfected oysters (LGP32, LMG20012T, J2-9,J2-8), mock-infected oysters (sterile seawater, SSW) and naive oysters (Ctrl). cg-bigdef3 was absent from oyster family Decipher#14. Statistical analysis was performed using One-way ANOVA (p<0.001)

Figure

  Figure S7 l qPCR monitoring of vipA1 (VS-1332) and vipA2 (VS_II0997) during host colonization. An overnight culture of V. tasmaniensis LGP32 was washed and resuspended in sterile seawater (SSW). Part of the bacterial suspension was immediately frozen for future RNA extraction (Control). In parallel, 300 juvenile oysters (NSI produced at Ifremer Argenton) were injected with the bacterial suspension (6 x 10 7 CFU per animal, 100 animals per tank). At each time point (2h, 8h, 24h), 10 live oysters and 10 dead oysters (for 24h only) were collected from each tank. Oysters were snap frozen in liquid nitrogen and grinded for RNA extraction. vipA1 and vipA2 expression were compared to the geometric mean of two reference genes 6PKF (VS_2913), MGS (VS_II1055). Data correspond to 2 -Ct values and Y-axis are in the log scale. Each dot represents a pool of 10 oysters. Data show that vipA1 expression is frontloaded as opposed to the within-host induction of vipA2. Significance established by a One-way ANOVA with Tukey's multiple comparisons test.

Figure S8 l

 S8 Figure S8 l Phenotyping of V. tasmaniensis LGP32 mutants. A. Growth curves of wild-type LGP32 and isogenic mutants. Cultures were made in LB NaCl 0.5M. OD600 was recorded in triplicate for each strain every 10 minutes after continuous orbital shaking in a TECAN M200 Infinite Pro plate reader, for a 16 hour

Figure S11 l

 S11 Figure S11 l Functional annotation of VgrG in the T6SSs of V. tasmaniensis and V. crassostreae. The different domains are indicated by squared with different motifs. The numbers indicate the beginning/end of the different domains.

Figure S12 l

 S12 Figure S12 l Functional annotation of PAAR in the T6SSs of V. tasmaniensis and V. crassostreae. The different domains are indicated by squared with different motifs. The numbers indicate the beginning/end of the different domains.

Figure S13 l

 S13 Figure S13 l Cytotoxicity of V. crassostreae J5-5 wild-type and isogenic mutant lacking the R5.7 virulence gene. Cytotoxicity was determined on monolayers of hemocytes and monitored by the Sytox green assay. Cells were infected with bacteria at a MOI of 50:1. Maximum of cytotoxicity of vibrios is displayed. Error bars represent the standard deviation of the mean (RM-ANOVA p-value<0.001).

  et al. (2017) MicroScope-an integrated resource for community expertise of gene functions and comparative analysis of microbial genomic and metabolic data. (Translated from eng) Brief Bioinform (in eng). 24. Caspi R, et al. (2018) The MetaCyc database of metabolic pathways and enzymes. (Translated from eng) Nucleic Acids Res 46(D1):D633-D639 (in eng). 25. Vallenet D, et al. (2013) MicroScope--an integrated microbial resource for the curation and comparative analysis of genomic and metabolic data. (Translated from eng) Nucleic Acids Res 41(Database issue):D636-647 (in eng).

Figure 23 .

 23 Figure 23. PCR verification of evpP deletion. 10 Clones susceptible to chloramphenicol after plasmid excision were tested by PCR to verify gene deletion. Primers upstream and dowstrem evpP sequence were used for PCR. Correct deletion of the evpP sequence should produce a fragment of 1082, bp while the wild type one of 1753 bp. 9 of the 10 clones tested corresponded to ΔevpP mutants while only one resulted in the wild type genotype after excision.

Figure 24 .Cell

 24 Figure 24. Cytotoxic activty of LGP32 ΔevpP. Deletion of evpP induces loss of cytotoxicity toward immune cells in V. tasmaniensis LGP32. Cytotoxicity of LGP32 ΔevpP and LPG32 vipA-i as control was determined on monolayers of hemocytes and monitored by the SYTOX Green assay. Cells were infected with bacteria at an MOI of 50:1. Maximum cytotoxicity displayed by vibrios is shown here. Error bars represent the SD of the mean (RM-ANOVA, P < 0.001)

[

  https://github.com/z0on/GO_MWU]. The parameters used for the adaptive clustering were: largest = 0.1, smallest = 10, and clusterCutHeight = 0.25. To combine both the significance of the differential expression and the level of expression into the analysis, the input table of

  continuous measures was generated by assigning the log2 fold-change values to those genes differentially expressed (FDR < 0.05) and a value of "0" to those genes with an FDR >0.05. A category was considered enriched with an FDR < 0.05

  chloroform:isoamyl alcohol (25:24:1) and isopropanol precipitation. All amplification reactions were performed in duplicate using a Roche LightCycler 480 Real-Time thermocycler (qPHD-Montpellier GenomiX platform, Montpellier University). PCR reaction were performed in 6µL containing 3µL of LightCycler 480 SYBR Green I Master mix (Roche), 2µL (0.5µM) of pathogen specific primers and 100 ng of genomic DNA following a qPCR program with an initial denaturation step (95 °C for 10 min) and 40 cycles of denaturation (95 °C, 10 s), hybridization (60 °C, 20 s) and elongation step (72 °C, 25 s). Virus specific primer pairs targeting a region of the OsHV-1 µvar genome predicted to encode a DNA polymerase catalytic subunit (DP) were chosen as previously described: OsHVDPFor/OsHVDPRev (TableS6). Standard curves of known concentration of DP plasmid were generated according to the Applied Biosystems manual of absolute real-time RT-PCR quantification. Absolute quantification of OsHV-1 µvar genome copies in oyster samples was then estimated by comparing the observed crossing point (Cp) values to known plasmid standards from 10 2 to 10 9 copies of DP. Oyster flesh samples exhibiting a viral load superior to 100 unit genome per ng of total DNA (UG/ng) were considered as infected by OsHV-1 µvar.BarcodingLocus-specific PCR primers including the required Illumina overhang adaptors were design to amplify a 573 bp region of the rctB gene in all our Harveyi strains, (rctB-Fw-I: 5). Two random bases, Y and R were added to the end of each of the Illumina overhang adaptors to increase sequencing efficiency has recommended by the GenSeq sequencing platform.

FiguresFigure 1 .

 1 Figures

  Figure 1. Population structure prediction of Vibrionaceae bacteria recovered from different seawater fractions or oyster flesh. The distribution (%) of vibrios isolated from oysters and size-filtered seawater fractions (>63um, 60-5um, 5-1um, 1-0.2um) in October 2015 during autumn mortality was analysed using the hsp60 marker gene. Hsp60 sequences were blasted against sequences of Vibrio and an assignation was proposed if identity ≥ 95%. Altogether, 304 hsp60 sequences display an identity ≥ 95% with E. calviensis DSM14347 T , P. damselae CIP102761 T , V. alginolyticus NBRC15630 T , V. atlanticus CECT7223 T , V.

Figure 2 .

 2 Figure 2. Harveyi clade population structure and virulence. (A) Phylogenetic tree based on hsp60, rctB, topA and mreB marker genes. Sequences were aligned using Muscle and concatenated with Seaview before tree reconstruction using a GTR model of evolution and Gamma law of rate heterogeneity. The inner ring indicates whether a strain was isolated from a specific seawater fraction or from oysters and the outer rings represents the mortality rates at 24hrs post-injection. Asterisks indicate the strains sequenced in this study. (B) Mortality rate following experimental infection with Harveyi-related isolates collected in the Thau lagoon in October 2015. Strains were individually injected into the muscle of 20

Figure 3 .Figure 4 .

 34 Figure 3. Colonization and mortality rate of OsHV-1 infected oysters by bacteria of the Harveyi clade.(A) Oyster mortalities were tracked daily over a 6-day period and Kaplan-Meier survival curves were generated with the data. No mortality was recorded in the untreated condition and for the oysters exposed to Harveyi only, until the last day of monitoring. A significant increase in the mortality rate was observed for oysters exposed to both Harveyi and OsHV-1 (log-rank test, P value = 0.0018). (B) Total Vibrio load was quantified on individual oysters by Vibrio-specific amplification of the 16S rRNA gene by qPCR. Total DNA for Vibrio quantification was extracted from frozen powder of whole oyster flesh. We quantified in average a 35-fold higher Vibrio load in oysters also exposed to OsHV-1 compared to the charge in oysters only exposed to bacteria (Kruskal-Wallis test, P value < 0.001). Total Vibrio load in untreated oysters was quantified only at time point 0

Figure 5 .

 5 Figure 5. Analysis at the vibrio population level of the Vibrio colonizing oysters and OsHV-1 load. Vibrio populations inside oysters were analyzed by performing rctB gene-based barcoding by sequencing a 573 bp region of the rctB gene that allows phylogenetic resolution at the population level of the Harveyi clade. The PCR products were sequenced using Illumina Miseq system and data were used to estimate the relative abundance of each Harveyi population associated with individual oysters. Upper horizontal bars correspond to the average percentage of abundance of each Harveyi population for each time point (N=10 oysters). Lower charts show in vertical bars the relative abundance at the individual scale (left y-axis) together with black dots to represent the OsHV-1 load per individual (right y-axis) expressed has the log10 of the number of genome copies of OsHV-1 per ng of total DNA. Individuals used for RNAseq were H4 #2 #3 #4; H24 #1, #4, #10; H48 #1, #3, #6; HV4 #3 #4 #7; HV24 #2, #3, #4; HV48 #2, #8, #9.

Figure 6 .

 6 Figure 6. Cytotoxicity of Harveyi strains toward oyster immune cells. Cytotoxicity was determined on monolayers of hemocytes and monitored using the Sytox green labelling. Cells were infected with bacteria at a MOI of 50:1. Maximum of cytotoxicity of vibrios is displayed. Error bars represent the standard deviation of the mean, different letters indicate significant variation between conditions (p<0,05, One-way ANOVA with post-hoc Tukey HSD Test).

Figure 7 .

 7 Figure 7. Siderophores distribution and growth of V. harveyi and V. rotiferianus in iron-depleted medium. (A) Known Vibrionaceae siderophore biosynthetic and receptor proteins in V. harveyi and V. rotiferianus. Biosynthetic proteins are indicated in blue and receptor proteins in green. Enterobactin and deferoxamine B are xenosiderophores produced by E. coli and Streptomyces pilosus, respectively. (B)

  Figure S1. Total OsHV-1 μVar quantified on individual oysters by qPCR. Number of copies of OsHV-1 per ng of total DNA were log10(x+1) transformed. Final time points were compared by a Kruskal-Wallis test.Significative differences between conditions are noted with different letters (P value < 0.001)

results in article 2 ,

 2 where strong transcriptional responses were induced by the injection of either virulent or non-virulent vibrios. The reason why vibrios do not induce the immune response in natural infection set-ups remains ignored. One possible hypothesis is the host production of PGRPs with amidase activity (PGRP-LB), which has been shown to suppress immune signaling confering tolerance to the microbiota, in other invertebrates(Zaidman- Rémy et al., 2006).

Table 1

 1 Vibrio sont des -proteobactéries ubiquitaires des milieux aquatiques.

		LIST OF ABBREVIATIONS
	AhpC	Alkyl hydroperoxide reductase C
	AMPs	Antimicrobial Peptides
	BPI	Bactericidal Permeability Increasing Protein
	cDNA	Complementary Permeability Increasing Protein
	CFU	Colony Forming Units
	Cm	Chloramphenicol
	DAMPs	Damage Associated Molecular Patterns
	Dap	Diaminopimelic acid
	DAPI	4´,6-diamidino-2-phenylindole
	DNA	Deoxyribonucleic Acid
	DUOX	Dual Oxidase
	ECP	Extra Cellular Product
	FAO	Food and Agriculture Organisation
	GFP	Green Fluorescent Protein
	IFN	Interferon
	IL-17	Interleukin 17
	Kda	Kilodalton
	LPS	Lipopolysaccharides
	LRR	Leucine-rich repeats
	MARTX	Multifunctional Autoprocessing Repeats-in-Toxin
	MIC	Minimal Inhibitory Concentration
	NETs	Neuthropil Extracellular Traps
	OMV	Outer Membrane Vesicule
	ORF	Open Reading Frame
	OsHV-1	Ostreid HerpesVirus-1
	OTU	Operational Taxonomic Unit
	PAMPs	Pathogen Associated Molecular Patterns
	PGRP	Peptide Glycan Recognition Protein
	PRP	Pattern Recognition Protein
	Prp	Proline Rich Peptide
	PRR	Pattern Recognition Receptor
	q-PCR	Quantitative Polymerase Chain Reaction
	RNA	Acid Ribonucleic
	RNS	Reactive Nitrogen Species
	ROS	Reactive Oxygen Species
	SDS-PAGE	SDS PolyAcrylamide Gel Electrophoresis
	SOD	Super Oxyde Dismutase
	Spc	Spectinomycin
	T2SS	Type 2 Secretion System
	T3SS	Type 3 Secretion System
	T6SS	Type 6 Secretion System
	TLR	Toll Like Receptor
	TNF	Tumor Necrosis Factor
	UDP-GIcNAc	UDP-N-acetylglucosamine
	UDP-MurNAG	UDP-N-acetylmuramyl Acid

. Primers used for amplification of the regions flanking evpP ............................................

  Splendidus pour coloniser les huîtres, mais ils montrent également que la cytotoxicité n'est pas un indicateur suffisant de virulence ; il contribuerait plutôt à rendre l'huitre moins hostile à sa colonisation.TRADE-OFFS AGISSANT SUR LA VIRULENCE DES VIBRIOS COLONISANT DES HUÎTRES SAINESNous avons été intrigués par les souches de V. splendidus Pop #23 qui affichent une virulence modérée tout en portant des facteurs de virulence tels qu'un MARTX et un T6SS4G4_4. Nous avons pu identifier des différences dans le contenu du cluster génique hypervariable wbe, responsable de la synthèse de l'antigène O du LPS, entre les souches moyennement virulentes et non virulentes. En raison de sa localisation dans la membrane externe, l'antigène O est soumis à des pressions sélectives de la part des prédateurs environnementaux et des effecteurs antimicrobiens des hôtes métazoaires

. Nos résultats montrent que la cytotoxicité envers les hémocytes est présente dans les vibrios non virulents appartenant au microbiote des huîtres saines (hors périodes de mortalités). Ces résultats suggèrent que la cytotoxicité est essentielle dans les vibrios du clade

  que, bien que les vibrios soient effectivement présents chez les animaux malades, ils ne sont pas le seul groupe bactérien à se développer dans les huîtres malades affectées par des facteurs de stress environnementaux biotiques et abiotiques tels que l'OsHV-1 ou de hautes températures (deLorgeril et al., 2018 ; Green et al., 2019 ;[START_REF] King | Characterisation of the Pacific Oyster Microbiome During a Summer Mortality Event[END_REF]. Parmi eux, le genre Arcobacter a été régulièrement trouvé enrichi dans les huîtres malades. En outre, Arcobacter bénéficie de la perturbation du microbiote et de la physiologie de l'hôte provoquée par une injection de Vibrio pour dominer le microbiote associé à l'hémolymphe[START_REF] Lokmer | Hemolymph Microbiome of Pacific Oysters in Response to Temperature, Temperature Stress and Infection[END_REF]. Parmi Arcobacter spp., la résistance aux antimicrobiens et aux métaux ainsi que des facteurs de virulence ayant des effets cytotoxiques envers les cellules de mammifères ont été décrites(Brückner et al., 2020). Compte tenu de ces caractéristiques, il est probable qu'Arcobacter pourrait remplacer fonctionnellement Vibrio comme pathogène opportuniste des huîtres malades, cependant nous ignorons toujours si les souches associées aux POMS expriment un potentiel de virulence ou si elles ont un

	nouvelles niches réplicatives dans l'environment en dehors de cet hôte. Ainsi, l'évolution vers
	une virulence modérée et une résistance à la prédation, illustrée par Pop # 23, pourrait se
	traduire par une « fitness » globale plus élevée que celle résultant de la sélection pour la
	virulence chez les populations colonisant des huîtres saines.

virus aux individus permissifs proximaux, assurant ainsi de nouvelles niches réplicatives pour le développement des populations virulentes. Il en va de même pour les populations bactériennes qui colonisent les huitres infectées par le virus, armées pour se répliquer dans l'hôte immunodéprimé. Dans ce cas, la virulence dépendant de la cytotoxicité envers les hémocytes favorise la multiplication et la transmission bactérienne. En revanche, un raisonnement opposé pourrait s'appliquer aux populations qui colonisent les huîtres juvéniles saines. Ces animaux sont en effet hostiles aux bactéries colonisatrices et donc l'expression d'un potentiel de virulence élevé conduisant à la mort de l'hôte peut être dans ce cas mal adapté

(Alizon et Michalakis, 2015)

car elle élimine la niche bactérienne obligeant les vibrios à devoir coloniser de nouveaux hôtes immunocompétents avec un microbiote stable ou de PATHOGÈNES OPPORTUNISTES ASSOCIES AU POMS EN DEHORS DU GENRE VIBRIO Récemment, des études indépendantes sur les mortalités massives de C. gigas ont permis d'identifier comportement opportuniste par acquisition de ressources. Avec le développement de la métatranscriptomique appliquée au POMS par Annick Jacq et a sa collaboration avec notre laboratoire, nous espérons en savoir plus sur la complémentarité fonctionnelle et/ou la coopération des populations microbiennes contribuant à l'issue fatale du POMS. CONCLUSION L'objectif de cette thèse était de caractériser les mécanismes d'interaction entre l'huître Crassostrea gigas et les vibrios dans un contexte sain ou pathologique. Si la plupart des vibrios pénètrent le système circulatoire des huitres de manière transitoire, sans induire de réponse immunitaire, certaines espèces montrent des associations préférentielles. Nos études montrent que si la résistance aux antimicrobiens est un trait partagé par les populations de vibrios associés aux huîtres, elle n'est pas suffisante pour la colonisation d'huîtres saines. En revanche chez les vibrios colonisant les huitres saines, une virulence modérée pourrait être avantageuse. Ainsi, chez certains vibrios, certaines structures de l'antigène O favorisent la résistance aux prédateurs environnementaux mais entraînent une activation accrue du système immunitaire des huîtres qui réduit la virulence et la capacité de colonisation des Vibrio chez cet hôte. En parallèle, en explorant les populations de vibrios associés aux huîtres dans un contexte pathologique, suite à la perturbation de de l'huître par le virus OsHV-1, nous avons montré que la cytotoxicité envers les hémocytes de l'huître était un déterminant clé de la colonisation. Cette cytotoxicité est étroitement (mais pas exclusivement) associée à la virulence et favorise la colonisation des huitres par des vibrios pathogènes capables de lyser les cellules immunitaires de leur hôte. Une autre stratégie basée sur l'utilisation non coopérative des biens publics a également été observée chez une espèce de vibrios capable de coloniser les huitres affectées par le POMS. Il est vraisemblable que la synergie du virus OsHV-1 et des vibrios virulents associés au POMS, en accélérant la mort des huîtres, favorise la transmission des pathogènes à de nouveaux hôtes.

  To further explore the prophage influence in

	Vibrio adaptation, Castillo et al. (2018) characterized prophage-like elements in 1874
	available whole genome sequenced Vibrio strains showing that, indeed, prophage-encoded
	genes potentially conferring virulence, niche adaptations and antibiotic resistance, are not
	restricted to human pathogens but extensively exchanged among the Vibrio genus.

  ToxR family receptors are examples of one-component systems and include major regulators of virulence such as ToxR/S and TcpP/H in V. cholerae, PsaE in Yersinia tuberculosis, WmpR in Pseudoalteromonas tunicate and the pH sensor CadC identified in E.

  one globular head C1q domain (ghC1q) and those who also presenting a collagen region N-terminal to the C1q domain (C1q-like), which is characteristic of the vertebrate C1 complex. According to the study of the expansion of the C1q gene family in C. gigas byGerdol and coll. (2015), most of the C1qDC genes have, to some extent,[START_REF] Lv | Comparative Study of Three C1q Domain Containing Proteins from Pacific Oyster Crassostrea gigas[END_REF][START_REF] Li | A C1qDC (CgC1qDC-6) with a Collagen-Like Domain Mediates Hemocyte Phagocytosis and Migration in Oysters[END_REF]. Finally, a transcriptomic analysis in response to LPS, identified several homologues of the complement system of vertebrates in C. gigas, including a C3 protein which is responsible for the activation of the alternative complement

	a tissue-specific expression. From a total of 337 annotated C1qDC proteins, 156 were
	associated with the digestive gland, 73 to the gills, 5 to hemocytes, 5 to the mantle tissue and
	91 with non-tissue-specific expression. Function-wise, five CgC1qDC that only contain the
	ghC1q motif and one that also carries a collagen-like domain (CgC1qDC-6) are involved in
	either the enhancement of phagocytosis towards V. splendidus, direct antimicrobial activity,
	or cell migration (

Zhang et al., 2015;[START_REF] Gerdol | Extensive Tandem Duplication Events Drive the Expansion of the C1q-Domain-Containing Gene Family in Bivalves[END_REF][START_REF] Wang | The Transcriptomic Expression of Pattern Recognition Receptors: Insight into Molecular Recognition of Various Invading Pathogens in Oyster Crassostrea gigas[END_REF]

, which are mainly categorized into two subgroups: those containing only pathway. This suggests the existence of a multi-component complement system in the oyster (L.

Wang et al., 2017)

.

Glycans are found in several MAMPs, such as glycoproteins, glycolipids, and polysaccharides which are bound with high affinity by lectins that also participate in agglutination, immobilization and complement-mediated opsonization. C-type, P-type, F-type, I-type, galectins, ficolins, and chitinase-like are the seven groups of lectins identified in the oyster

[START_REF] Wang | The Immune Role of C-Type Lectins in Molluscs[END_REF]

.

C-type lectins (CTLs) bind carbohydrates in a calcium dependent manner and contain at least one carbohydrate-recognition domain. Seven different CTLs have been identified in the oyster

[START_REF] Y-Yamaura | Identification and Tissue Expression Analysis of C-type Lectin and Galectin in the Pacific Oyster, Crassostrea gigas[END_REF][START_REF] Jia | Comparative Study of Two Single CRD C-Type Lectins, CgCLec-4 and CgCLec-5, from Pacific Oyster Crassostrea gigas[END_REF]

. Among them, CgCLT-2 possesses only one CRD and the recombinant protein can trigger hemocyte phagocytosis. CgCLT-4 and CgCLT-5 are secreted and intracellular lectins, respectively, both induced by LPS. Both recombinant proteins display a broad MAMP, bacteria-binding and agglutinating ability, but only CgLec-4 exerts antimicrobial activity against bacteria and fungi

[START_REF] Jia | Comparative Study of Two Single CRD C-Type Lectins, CgCLec-4 and CgCLec-5, from Pacific Oyster Crassostrea gigas[END_REF]

.

  . All the six IL-17 homologues found in C. gigas are differentially regulated by MAMPs[START_REF] Li | Genomic Characterization and Expression Analysis of Five Novel IL-17 Genes in the Pacific Oyster, Crassostrea gigas[END_REF][START_REF] Sun | P38 is Involved in Immune Response by Regulating Inflammatory Cytokine Expressions in the Pacific Oyster Crassostrea gigas[END_REF] and most of them have been shown to be responsive to the regulation by the NF-kB/Rel transcription factor, CgRel in hemocytes of oysters injected with LPS (Y.Li et al., 2019). Furthermore, IL17-5 is able to activate NF-kB, CREB and ATF-1 in HEK293T cells and induce IL6 in HuVEC cells[START_REF] X-Xin | CgIL17-5, An Ancient Inflammatory Cytokine in Crassostrea gigas Exhibiting the Heterogeneity Functions Compared with Vertebrate Interleukin17 Molecules[END_REF].

The interferon-like protein (CgIFNLP) and its cognate receptor interact in vitro and both transcripts are induced in response to polyI:C. As CgIFNR-3 is able to activate the JAK/STAT pathway in oyster and CgIFNLP induces phagocytosis and apoptosis in hemocytes, they are likely part of the antiviral response in oysters

[START_REF] Green | Antiviral Immunity in Marine Molluscs[END_REF]

. From the 23 TNF homologues, 15 are oyster-specific and one (rCgTNF-1) regulates apoptosis and antibacterial activities in hemocytes

[START_REF] Sun | The Immunomodulation of a Novel Tumor Necrosis Factor (CgTNF-1) in Oyster Crassostrea gigas[END_REF]

.

III.1.3.5 EFFECTOR MECHANISMS OF OYSTER DEFENSE

Several cellular and humoral responses can be mounted by hemocytes in response to MAMPs or DAMPs recognition.

  . CHAPTER 2. RESULTS SECTION I. COLONIZATION DETERMINANTS AND ENVIRONMENTAL PERSISTENCE IN V. SPLENDIDUS ASSOCIATED WITH HEALTHY OYSTERS I.1. CONTEXT AND OBJECTIVE OF THE STUDY The association between vibrios and oysters have been studied mainly in a pathological context, due to important consequences on oyster farming. Vibrios have been systematically isolated from moribund oysters and studied to identify their potential role as etiological agents of oyster disease. The large amount of knowledge produced by our close collaborator Frédérique Le Roux at Ifremer (laboratoire de biologie integrative des modèles

marins, Station Biologique d Roscoff) has revealed that Vibrio species associated with POMS express virulence potential; this precious biological material enabled the identification of determinants of Vibrio pathogenicity both by her group and our research unit

[START_REF] Binesse | Metalloprotease Vsm Is the Major Determinant of Toxicity for Extracellular Products of Vibrio splendidus[END_REF][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] Vanhove | Copper Homeostasis at the Host Vibrio Interface: Lessons from Intracellular Vibrio Transcriptomics[END_REF] Bruto et al., 2017 Bruto et al., , 2018;; Piel et al., 2019)

.

Table S1 .

 S1 Genes specific to moderately virulent strains of V. splendidus Pop#23. Genes listed below are present in the genomes of 3T8_11, 4D1_8 and 4G4_4 but absent from the 4G1_8 genome.

	GV4G44_v1_40060 GV4G44_v1_90080 GV4G44_v1_190030 GV4G44_v1_320012 GV4G44_v1_480041 GV4G44_v1_760017	_ _ _ _ _ _	putative AraC-family transcriptional regulatory protein Potassium efflux system KefA protein / Small-conductance mechanosensitive channel membrane protein of unknown function conserved protein of unknown function ABC-type uncharacterized transport system component conserved protein of unknown function
	GV4G44_v1_40070 GV4G44_v1_90081 GV4G44_v1_190031 GV4G44_v1_320013 GV4G44_v1_480042 GV4G44_v1_760018	_ _ _ _ _ _	protein of unknown function conserved protein of unknown function exported protein of unknown function conserved protein of unknown function putative Outer membrane protein conserved protein of unknown function
	GV4G44_v1_40162 GV4G44_v1_90082 GV4G44_v1_190032 GV4G44_v1_320015 GV4G44_v1_480043 GV4G44_v1_760019	_ _ _ _ _ _	protein of unknown function conserved protein of unknown function conserved protein of unknown function kinase Agglutination protein putative replication initiation protein, phage-related
	GV4G44_v1_50053 GV4G44_v1_90083 GV4G44_v1_190033 GV4G44_v1_320016 GV4G44_v1_480044 GV4G44_v1_760020	_ _ _ vasJ _ _	conserved protein of unknown function conserved protein of unknown function conserved protein of unknown function Type VI secretion system vasJ conserved protein of unknown function conserved protein of unknown function
	GV4G44_v1_50069 GV4G44_v1_90084 GV4G44_v1_190034 GV4G44_v1_320017 GV4G44_v1_490006 GV4G44_v1_760021	_ _ _ vipA _ _	putative Type IV pilin conserved protein of unknown function conserved protein of unknown function Type VI secretion system vipA conserved protein of unknown function conserved protein of unknown function
	GV4G44_v1_50071 GV4G44_v1_90085 GV4G44_v1_190035 GV4G44_v1_320018 GV4G44_v1_490015 GV4G44_v1_760022	_ _ _ vipB _ _	conserved protein of unknown function conserved protein of unknown function conserved protein of unknown function Type VI secretion system vipB Long-chain fatty acid transport protein conserved protein of unknown function
	GV4G44_v1_50072 GV4G44_v1_90086 GV4G44_v1_190036 GV4G44_v1_320019 GV4G44_v1_510029 GV4G44_v1_760023	_ _ _ vasS _ _	Pili retraction protein PilT putative permease protein conserved protein of unknown function Type VI secretion system vasS conserved protein of unknown function conserved protein of unknown function
	GV4G44_v1_50073 GV4G44_v1_90119 GV4G44_v1_190037 GV4G44_v1_320020 GV4G44_v1_510030 GV4G44_v1_760024	_ _ _ vasA _ _	Pili subunit Site-specific recombinase, phage integrase family (fragment) Site-specific recombinase, phage integrase family Type VI secretion system VasA conserved protein of unknown function conserved protein of unknown function
	GV4G44_v1_50076 GV4G44_v1_90120 GV4G44_v1_190038 GV4G44_v1_320021 GV4G44_v1_520009 GV4G44_v1_760025	_ _ _ vasF _ _	putative alginate lyase protein of unknown function conserved protein of unknown function Type VI secretion system vasF protein of unknown function conserved protein of unknown function
	GV4G44_v1_50145 GV4G44_v1_110106 GV4G44_v1_190039 GV4G44_v1_320022 GV4G44_v1_520014 GV4G44_v1_760026	_ _ _ _ _ _	Methyl-accepting chemotaxis protein I (Serine chemoreceptor protein) conserved protein of unknown function conserved protein of unknown function putative HTH-type transcriptional regulator conserved exported protein of unknown function Integrase
	GV4G44_v1_60017 GV4G44_v1_110108 GV4G44_v1_190041 GV4G44_v1_320023 GV4G44_v1_520015 GV4G44_v1_790009	_ _ _ vasK _ _	conserved protein of unknown function conserved protein of unknown function conserved protein of unknown function Putative Type VI secretion system protein IcmF protein of unknown function protein of unknown function
	GV4G44_v1_60019 GV4G44_v1_110109 GV4G44_v1_190042 GV4G44_v1_320024 GV4G44_v1_520025 GV4G44_v1_790010	_ _ _ vasL _ _	conserved protein of unknown function protein of unknown function conserved protein of unknown function Type VI secretion system vasL exported protein of unknown function protein of unknown function
	GV4G44_v1_60034 GV4G44_v1_110110 GV4G44_v1_190044 GV4G44_v1_320025 GV4G44_v1_580014 GV4G44_v1_800001	_ int _ _ agaR _	protein of unknown function Integrase/recombinase conserved protein of unknown function Type VI secretion system protein of unknown function DNA-binding transcriptional dual regulator Metal-dependent phosphohydrolase, HD subdomain
	GV4G44_v1_60058 GV4G44_v1_120079 GV4G44_v1_190045 GV4G44_v1_320026 GV4G44_v1_580015 GV4G44_v1_800002	_ _ _ _ agaR _	conserved protein of unknown function protein of unknown function protein of unknown function Type VI secretion system forkhead-associated protein DNA-binding transcriptional dual regulator conserved protein of unknown function
	GV4G44_v1_60071 GV4G44_v1_120085 GV4G44_v1_200038 GV4G44_v1_320027 GV4G44_v1_580016 GV4G44_v1_800003	_ _ _ vasD kbaZ _	protein of unknown function Outer membrane protein protein of unknown function Type VI secretion system vasD tagatose 6-phosphate aldolase 1, kbaZ subunit protein of unknown function
	GV4G44_v1_60073 GV4G44_v1_120086 GV4G44_v1_200075 GV4G44_v1_320028 GV4G44_v1_580017 GV4G44_v1_800004	_ _ _ vasE agaS _	conserved protein of unknown function putative Flp pilus assembly protein TadG conserved protein of unknown function Type VI secretion system vasE tagatose-6-phosphate ketose/aldose isomerase conserved protein of unknown function
	GV4G44_v1_60074 GV4G44_v1_120087 GV4G44_v1_200076 GV4G44_v1_320029 GV4G44_v1_580018 GV4G44_v1_800005	_ _ _ vasF agaV _	conserved membrane protein of unknown function conserved protein of unknown function conserved protein of unknown function Type VI Secretion System, VasF N-acetylgalactosamine-specific enzyme IIB component of PTS protein of unknown function
	GV4G44_v1_60075 GV4G44_v1_120088 GV4G44_v1_200077 GV4G44_v1_320030 GV4G44_v1_580019 GV4G44_v1_800006	_ _ _ paar _ _	conserved protein of unknown function putative Flp pilus assembly protein TadE conserved protein of unknown function Type VI secretion system protein with PAAR domain + unknown extension putative N-acetylgalactosamine-specific IID component, PTS system protein of unknown function
	GV4G44_v1_60076 GV4G44_v1_120089 GV4G44_v1_200088 GV4G44_v1_320031* GV4G44_v1_580020 GV4G44_v1_800007	_ _ _ _ agaD _	conserved protein of unknown function putative Flp pilus assembly protein TadD transposase (fragment) Putative bacterial toxin system with endonuclease activity N-acetylgalactosamine-specific enzyme IID component of PTS putative caffeoyl-CoA O-methyltransferase (fragment)
	GV4G44_v1_60078 GV4G44_v1_120090 GV4G44_v1_220015 GV4G44_v1_320032* GV4G44_v1_580021 GV4G44_v1_800008	relE _ _ _ _ _	Toxin of the RelE-RelB toxin-antitoxin system; Qin prophage putative Flp pilus assembly protein TadC protein of unknown function Putative immunity protein Phosphotransferase system, mannose/fructose-specific component IIA putative caffeoyl-CoA O-methyltransferase (fragment)
	GV4G44_v1_60079 GV4G44_v1_120091 GV4G44_v1_230011 GV4G44_v1_320038 GV4G44_v1_580022 GV4G44_v1_800009	_ _ yefM _ manD N-acetylglucosamine-6-phosphate deacetylase Prevent-host-death family protein putative Flp pilus assembly protein TadB antitoxin of the YoeB-YefM toxin-antitoxin system protein of unknown function _ RNA-directed DNA polymerase (fragment)
	GV4G44_v1_60080 GV4G44_v1_120092 GV4G44_v1_230012 GV4G44_v1_320039 GV4G44_v1_580023 GV4G44_v1_800010	_ _ yoeB _ kbaY _	transposase Flp pilus assembly protein toxin of the YoeB-YefM toxin-antitoxin system protein of unknown function tagatose 6-phosphate aldolase 1, kbaY subunit conserved protein of unknown function
	GV4G44_v1_60081 GV4G44_v1_120093 GV4G44_v1_230029 GV4G44_v1_320042 GV4G44_v1_580035 GV4G44_v1_800011	_ _ _ _ _ _	protein of unknown function ATPase involved in chromosome partitioning conserved protein of unknown function conserved protein of unknown function Transcriptional regulator Methyl-accepting chemotaxis protein
	GV4G44_v1_60082 GV4G44_v1_120094 GV4G44_v1_230031 GV4G44_v1_320043 GV4G44_v1_580036 GV4G44_v1_800012	_ _ _ _ _ _	conserved protein of unknown function conserved protein of unknown function ThiJ/PfpI family protein Cytochrome c553 conserved protein of unknown function protein of unknown function
	GV4G44_v1_60087 GV4G44_v1_120095 GV4G44_v1_230032 GV4G44_v1_330027 GV4G44_v1_580037 GV4G44_v1_830008	_ _ _ _ _ _	putative c-di-GMP phosphodiesterase A-related protein Flp pilus assembly protein conserved exported protein of unknown function protein of unknown function conserved exported protein of unknown function Internal scaffolding protein B
	GV4G44_v1_60091 GV4G44_v1_120096 GV4G44_v1_230063 GV4G44_v1_340001 GV4G44_v1_580038 GV4G44_v1_840003	_ _ _ _ _ _	protein of unknown function conserved protein of unknown function putative Phage integrase family protein Putative RTX protein (fragment) protein of unknown function protein of unknown function
	GV4G44_v1_60147 GV4G44_v1_120097 GV4G44_v1_230064 GV4G44_v1_340025 GV4G44_v1_620024 GV4G44_v1_880001	_ _ _ _ pilA _	protein of unknown function conserved protein of unknown function conserved protein of unknown function protein of unknown function Fimbrial protein conserved protein of unknown function
	GV4G44_v1_70001 GV4G44_v1_120098 GV4G44_v1_230065 GV4G44_v1_340035 GV4G44_v1_630001 GV4G44_v1_880002	_ _ _ _ _ _	conserved protein of unknown function putative membrane protein conserved protein of unknown function conserved exported protein of unknown function conserved protein of unknown function Helix-turn-helix family protein
	GV4G44_v1_70002 GV4G44_v1_120100 GV4G44_v1_230066 GV4G44_v1_340044 GV4G44_v1_630004 GV4G44_v1_880003	_ _ _ tnpA _ _	conserved membrane protein of unknown function Transcriptional regulator conserved protein of unknown function transposase Restriction endonuclease family protein conserved protein of unknown function
	GV4G44_v1_70003 GV4G44_v1_120101 GV4G44_v1_230067 GV4G44_v1_340045 GV4G44_v1_630005 GV4G44_v1_920001	_ _ _ _ _ _	conserved exported protein of unknown function conserved exported protein of unknown function conserved protein of unknown function protein of unknown function conserved protein of unknown function conserved protein of unknown function
	GV4G44_v1_70004 GV4G44_v1_130004 GV4G44_v1_230068 GV4G44_v1_340048 GV4G44_v1_630006 GV4G44_v1_990001	_ _ _ _ _ _	conserved protein of unknown function conserved protein of unknown function conserved protein of unknown function conserved membrane protein of unknown function conserved protein of unknown function Acetyltransferase family protein
	GV4G44_v1_70005 GV4G44_v1_130031 GV4G44_v1_230069 GV4G44_v1_360022 GV4G44_v1_630007 GV4G44_v1_1010001	_ _ _ _ _ _	conserved protein of unknown function conserved membrane protein of unknown function conserved protein of unknown function conserved protein of unknown function conserved protein of unknown function transposase (fragment)
	GV4G44_v1_70006 GV4G44_v1_130046 GV4G44_v1_240031 GV4G44_v1_360039 GV4G44_v1_630008 GV4G44_v1_1060001	_ _ _ _ _ _	conserved protein of unknown function protein of unknown function conserved exported protein of unknown function protein of unknown function conserved protein of unknown function conserved membrane protein of unknown function
	GV4G44_v1_70007 GV4G44_v1_140001 GV4G44_v1_240071 GV4G44_v1_370003 GV4G44_v1_630009	vspR _ _ _ _	Transcriptional regulator VspR protein of unknown function Predicted membrane protein conserved protein of unknown function DNA polymerase I
	Locus tag GV4G44_v1_70009 GV4G44_v1_140003 GV4G44_v1_270001 GV4G44_v1_370014 GV4G44_v1_630011 GV4G44_v1_70008 GV4G44_v1_140002 GV4G44_v1_240072 GV4G44_v1_370006 GV4G44_v1_630010 GV4G44_v1_320031 and GV4G44_v1_320032 indicated with an * in the locus tag are genes exclusive of strain 4G4_4 Gene _ transposase (fragment) _ transposase _ conserved protein of unknown function _ putative UDP-galactose phosphate transferase (WeeH) _ conserved protein of unknown function Putative function or gene name _ putative transcriptional regulator _ Transcriptional repressor of sporulation, septation and degradative enzyme _ Zn-dependent protease with chaperone function groES Co-chaperonin GroES _ conserved protein of unknown function
	GV4G44_v1_10002 GV4G44_v1_70010 GV4G44_v1_140004 GV4G44_v1_270021 GV4G44_v1_370034 GV4G44_v1_630012 GV4G44_v1_10024 GV4G44_v1_70011 GV4G44_v1_140005 GV4G44_v1_270067 GV4G44_v1_370035 GV4G44_v1_630013 GV4G44_v1_10046 GV4G44_v1_70039 GV4G44_v1_140006 GV4G44_v1_280014 GV4G44_v1_370036 GV4G44_v1_630014 GV4G44_v1_10050 GV4G44_v1_70040 GV4G44_v1_140007 GV4G44_v1_280018 GV4G44_v1_370037 GV4G44_v1_630015 GV4G44_v1_10056 GV4G44_v1_70041 GV4G44_v1_140061 GV4G44_v1_280044 GV4G44_v1_370038 GV4G44_v1_630016 GV4G44_v1_10057 GV4G44_v1_70042 GV4G44_v1_140064 GV4G44_v1_280045 GV4G44_v1_370040 GV4G44_v1_630017 GV4G44_v1_10058 GV4G44_v1_70043 GV4G44_v1_140068 GV4G44_v1_280059 GV4G44_v1_370041 GV4G44_v1_630018 GV4G44_v1_10132 GV4G44_v1_70044 GV4G44_v1_140069 GV4G44_v1_280060 GV4G44_v1_370042 GV4G44_v1_630019 GV4G44_v1_10136 GV4G44_v1_70158 GV4G44_v1_150005 GV4G44_v1_290030 GV4G44_v1_370043 GV4G44_v1_630020 GV4G44_v1_10152 GV4G44_v1_70159 GV4G44_v1_150025 GV4G44_v1_290047 GV4G44_v1_370044 GV4G44_v1_630021 GV4G44_v1_10162 GV4G44_v1_80003 GV4G44_v1_160049 GV4G44_v1_290060 GV4G44_v1_370045 GV4G44_v1_630022 GV4G44_v1_10163 GV4G44_v1_80004 GV4G44_v1_160073 GV4G44_v1_300029 GV4G44_v1_370046 GV4G44_v1_630023 GV4G44_v1_10207 GV4G44_v1_80005 GV4G44_v1_160084 GV4G44_v1_300030 GV4G44_v1_370047 GV4G44_v1_630026 GV4G44_v1_10262 GV4G44_v1_80036 GV4G44_v1_170011 GV4G44_v1_300031 GV4G44_v1_370048 GV4G44_v1_640021 GV4G44_v1_20013 GV4G44_v1_80037 GV4G44_v1_170086 GV4G44_v1_300032 GV4G44_v1_370049 GV4G44_v1_650021 GV4G44_v1_20017 GV4G44_v1_80038 GV4G44_v1_170087 GV4G44_v1_300033 GV4G44_v1_370051 GV4G44_v1_670015 GV4G44_v1_20041 GV4G44_v1_80039 GV4G44_v1_180039 GV4G44_v1_300034 GV4G44_v1_370052 GV4G44_v1_670027 GV4G44_v1_20042 GV4G44_v1_80074 GV4G44_v1_180040 GV4G44_v1_310021 GV4G44_v1_370053 GV4G44_v1_710001 GV4G44_v1_20043 GV4G44_v1_80091 GV4G44_v1_180041 GV4G44_v1_310022 GV4G44_v1_370054 GV4G44_v1_730016 GV4G44_v1_20080 GV4G44_v1_80099 GV4G44_v1_190001 GV4G44_v1_310023 GV4G44_v1_370055 GV4G44_v1_730017 GV4G44_v1_20081 GV4G44_v1_80100 GV4G44_v1_190015 GV4G44_v1_310024 GV4G44_v1_370056 GV4G44_v1_740016 GV4G44_v1_20082 GV4G44_v1_80101 GV4G44_v1_190016 GV4G44_v1_310025 GV4G44_v1_380016 GV4G44_v1_760001 GV4G44_v1_20083 GV4G44_v1_90039 GV4G44_v1_190017 GV4G44_v1_310026 GV4G44_v1_380046 GV4G44_v1_760002 GV4G44_v1_20084 GV4G44_v1_90069 GV4G44_v1_190018 GV4G44_v1_320001 GV4G44_v1_380047 GV4G44_v1_760003 GV4G44_v1_20249 GV4G44_v1_90070 GV4G44_v1_190020 GV4G44_v1_320002 GV4G44_v1_390025 GV4G44_v1_760004 GV4G44_v1_20250 GV4G44_v1_90071 GV4G44_v1_190021 GV4G44_v1_320003 GV4G44_v1_400018 GV4G44_v1_760005 GV4G44_v1_30001 GV4G44_v1_90072 GV4G44_v1_190022 GV4G44_v1_320004 GV4G44_v1_410007 GV4G44_v1_760006 GV4G44_v1_30002 GV4G44_v1_90073 GV4G44_v1_190023 GV4G44_v1_320005 GV4G44_v1_410014 GV4G44_v1_760007 GV4G44_v1_30019 GV4G44_v1_90074 GV4G44_v1_190024 GV4G44_v1_320006 GV4G44_v1_410015 GV4G44_v1_760008 GV4G44_v1_30127 GV4G44_v1_90075 GV4G44_v1_190025 GV4G44_v1_320007 GV4G44_v1_420016 GV4G44_v1_760009 GV4G44_v1_40019 GV4G44_v1_90076 GV4G44_v1_190026 GV4G44_v1_320008 GV4G44_v1_450042 GV4G44_v1_760013 GV4G44_v1_40020 GV4G44_v1_90077 GV4G44_v1_190027 GV4G44_v1_320009 GV4G44_v1_460001 GV4G44_v1_760014 GV4G44_v1_40059 GV4G44_v1_90078 GV4G44_v1_190028 GV4G44_v1_320010 GV4G44_v1_460004 GV4G44_v1_760015 GV4G44_v1_90079 GV4G44_v1_190029 GV4G44_v1_320011 GV4G44_v1_470021 GV4G44_v1_760016	_ _ _ _ yjbH _ _ _ _ _ _ _ bsn yeiJ _ _ _ _ _ ahcY _ _ ymcD _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ wbcM WbcM protein protein of unknown function transposase conserved exported protein of unknown function putative porin protein of unknown function protein of unknown function transposase (fragment) membrane protein of unknown function protein of unknown function conserved exported protein of unknown function protein of unknown function protein of unknown function putative sodium/proton nucleoside transporter conserved protein of unknown function conserved protein of unknown function putative regulator conserved exported protein of unknown function Extracellular ribonuclease bsn Adenosylhomocysteinase protein of unknown function conserved exported protein of unknown function conserved hypothetical protein; putative inner membrane protein conserved protein of unknown function Unknown Regulatory protein LysR:LysR substrate-binding protein of unknown function conserved exported protein of unknown function Regulator of length of O-antigen component of lipopolysaccharide chains conserved protein of unknown function Unknown Bifunctional 2',3'-cyclic nucleotide 2'-phosphodiesterase/3'-nucleotidase periplasmic protein protein of unknown function conserved exported protein of unknown function conserved protein of unknown function conserved protein of unknown function Radical SAM conserved protein of unknown function protein of unknown function conserved protein of unknown function membrane protein of unknown function conserved protein of unknown function Unknown conserved protein of unknown function conserved protein of unknown function conserved protein of unknown function _ Site-specific recombinase XerD-like Unknown _ _ Transcriptional regulators, LysR family _ protein of unknown function _ protein of unknown function _ putative Glycosyl transferase, group 2 family _ protein of unknown function Outer membrane protein _ _ conserved exported protein of unknown function _ Sensory box/GGDEF family protein _ protein of unknown function _ protein of unknown function _ putative thiol peroxidase (fragment) Unknown _ _ conserved protein of unknown function _ protein of unknown function _ protein of unknown function _ protein of unknown function _ Nucleoside 2-deoxyribosyltransferase ABC-type uncharacterized transport system, periplasmic component _ _ protein of unknown function _ Selenoprotein W-related protein rtxE Leukotoxin translocation ATP-binding protein LktB _ putative Uncharacterized glycosyltransferase HI_1578 _ exported protein of unknown function conserved exported protein of unknown function _ _ Prophage MuSo1 transcriptional regulator Cro/CI family (fragment) _ conserved exported protein of unknown function rtxD RTX toxin transporter _ putative Glycosyl transferase family 11 _ protein of unknown function Alpha-amylase _ _ conserved protein of unknown function _ protein of unknown function rtxB Cyclolysin secretion/processing ATP-binding protein CyaB _ membrane protein of unknown function _ conserved protein of unknown function Two-component system sensory/regulatory protein-like protein (Hybrid family) _ _ membrane protein of unknown function _ conserved membrane protein of unknown function rtxH conserved protein of unknown function _ CDP-4-dehydro-6-deoxy-D-glucose 3-dehydratase (fragment) _ conserved exported protein of unknown function Uncharacterized conserved membrane protein _ _ exported protein of unknown function _ Prophage Lp2 protein 6 rtxC Cytolysin-activating lysine-acyltransferase RtxC fcl bifunctional GDP-fucose synthetase: _ DNA mismatch repair protein MutT protein of unknown function _ _ protein of unknown function _ protein of unknown function rtxA protein of unknown function gmd GDP-D-mannose dehydratase, NAD(P)-binding _ conserved protein of unknown function protein of unknown function _ _ conserved protein of unknown function _ Maltose operon periplasmic protein MalM _ MSHA pilin protein MshA manB Phosphomannomutase _ exported protein of unknown function conserved protein of unknown function _ _ protein of unknown function lamB Maltoporin _ putative MSHA pilin protein MshC cpsB mannose-1-phosphate guanyltransferase _ conserved membrane protein of unknown function protein of unknown function _ _ conserved protein of unknown function _ protein of unknown function _ MSHA pilin protein MshD nudD GDP-mannose mannosyl hydrolase _ conserved protein of unknown function conserved exported protein of unknown function _ _ membrane protein of unknown function yfbR deoxyribonucleoside 5'-monophosphatase _ putative type IV prepilin, MshO _ protein of unknown function _ conserved protein of unknown function Transcriptional regulatory protein _ _ protein of unknown function _ conserved protein of unknown function _ MSHA biogenesis protein MshP _ protein of unknown function _ conserved membrane protein of unknown function putative 17-beta-estradiol 17-dehydrogenase _ _ protein of unknown function _ conserved protein of unknown function _ MSHA biogenesis protein MshQ _ conserved protein of unknown function _ conserved protein of unknown function Lysophospholipase _ _ protein of unknown function _ conserved membrane protein of unknown function _ protein of unknown function _ conserved exported protein of unknown function _ conserved protein of unknown function conserved protein of unknown function _ ectA L-2,4-diaminobutyric acid acetyltransferase bdcA Cyclic-di-GMP-binding biofilm dispersal mediator protein _ protein of unknown function _ protein of unknown function _ conserved protein of unknown function protein of unknown function _ ectB Diaminobutyrate--2-oxoglutarate transaminase _ putative transcriptional regulator, LysR family protein _ conserved protein of unknown function _ conserved protein of unknown function _ conserved protein of unknown function conserved protein of unknown function _ ectC L-ectoine synthase _ conserved protein of unknown function _ conserved exported protein of unknown function _ protein of unknown function _ Bacteriophage P7 related protein Predicted PE--lipooligosaccharide phosphorylethanolaminetransferase _ _ Aspartokinase associated with ectoine biosynthesis _ Cellobiose phosphotransferase system CelC ompA OmpA family protein _ putative capsular polysaccharide biosynthesis protein _ conserved protein of unknown function conserved protein of unknown function _ _ putative sigma-54 interacting response regulator transcription regulator protein _ Transcriptional regulator _ Sigma 54 dependant transcriptional regulator _ conserved protein of unknown function _ conserved protein of unknown function conserved exported protein of unknown function _ _ putative hyperosmotically inducible periplasmic protein _ protein of unknown function vasG Protein ClpV1 _ conserved exported protein of unknown function _ conserved protein of unknown function Outer membrane protein _ _ conserved protein of unknown function _ putative sugar transport protein hcpC hcp _ protein of unknown function _ Outer membrane receptor protein protein of unknown function _ _ conserved exported protein of unknown function _ protein of unknown function vgrG putative Vgr protein _ conserved protein of unknown function _ conserved protein of unknown function putative ferrichrome-iron receptor _ _ conserved protein of unknown function _ conserved exported protein of unknown function _ Protein of unknown function (structural homology with vgrG tip) _ Metallophosphoesterase _ conserved protein of unknown function putative adhesin _ Methylglyoxal synthase _ protein of unknown function _ conserved protein of unknown function _ protein of unknown function _ conserved protein of unknown function

Table S3 .

 S3 Bacterial strains

	4G4_4 *epsE-i I.3. COMPLEMENTARY RESULT 1. ASSESSMENT OF THE SUSCEPTIBILITY TO GRAZING *espE::pSW23T [Cm R ] GV4G44_v1_37004 3 This study
	4G4_4 *epsE-i GFP Ext *rtxA-R GV4G44_v1_300034 TGGCGATCGGCTTCCGCTCG *espE::pSW23T [Cm R ] + pMRB-PLACgfp RtxA protein DUE TO O-ANTIGEN MODIFICATIONS, IN A DIFFERENT GENETIC BACKGROUND OF This study V. splendidus 4G4_4 [Spt R ] LMG20012T GFP pMRB-PLACgfp[Cm R ] Vanhove et al., 2016 from MARTX cluster *vipA-F GV4G44_v1_320017 GtatcgataagcttgatatcgaattcTTCCAG VipA protein from T6SS V. splendidus 4G4_4 THE POPULATION #23	Control of mutagenesis Mutagenesis
			CAACAGGCGATGCG
	Table S5. Oligonucleotides *vipA-R GV4G44_v1_320017 CccccgggctgcaggaattcACGGAAAG	VipA protein from T6SS		V. splendidus 4G4_4	Mutagenesis
			CAGGTACATTCC
	Oligonucleotide Ext *vipA-F	Gene id GV4G44_v1_320017 ACGTGATGGCTCGGTGGCTC Sequence 5'-3'	Description VipA protein from T6SS		Organism V. splendidus 4G4_4	Use Control of mutagenesis
	EFUfw Ext *vipA-R EFUrv *epsE-F	AB122066 GV4G44_v1_320017 TCTGCACCAATGCTTAGTTC GAGCGTGAACGTGGTATCAC Table S4. Plasmids AB122066 ACAGCACAGTCAGCCTGTGA AGGGACTGGCGCAC GV4G44_v1_370043 GtatcgataagcttgatatcgaattcTTGAGG	EF-1α VipA protein from T6SS EF-1α from wbe region Glycosyltransferase		C. gigas V. splendidus 4G4_4 C. gigas V. splendidus 4G4_4	Control of mutagenesis RT-qPCR of host genes RT-qPCR of host genes Mutagenesis
	Plasmid CGI_10025754 F1 CGI_10025754 *epsE-R GV4G44_v1_370043 CccccgggctgcaggaattcCTTGACGGA Description GCCCACGAATCTTGCTGAAC pSW23T oriVR6Kγ ; oriTRP4; [Cm R ] CGI_10025754 R1 CGI_10025754 TTTGGGTAGCGGGTGGAAAG TGAGGGTTACG Ext *epsE-F GV4G44_v1_370043 TCTCTGATGACTGCTCAACTG	Interleukin 17-1 Glycosyltransferase Interleukin 17-1 from wbe region Glycosyltransferase	C.gigas V. splendidus 4G4_4 Reference Demarre et al., 2005 C. gigas V. splendidus 4G4_4	RT-qPCR of host genes Mutagenesis RT-qPCR of host genes Control of mutagenesis
	pSWδrtxA CGI_10021486 pSWδvipA CGI_10021486 GV4G44_v1_370043 TAAATAGACACGAAGGACCC pSW23T; ΔrtxA GV4G44_v1_300034 CTGTCGCTGCAATGTGATCT Interleukin 17 receptor from wbe region pSW23T; ΔvipA GV4G44_v1_320017 TTTCTTGCCATTCGTTTTCC Interleukin 17 receptor Glycosyltransferase from wbe region MyD88like-22_F CGI_10012722 IL-17 R-86 F IL-17 R-86 R Ext *epsE-R CGATTGGATTCCACAGAGGT Myeloid differentiation 4G4_4-370043F GV4G44_v1_370043 TGCTATTGAGGAGGGACTGG Glycosyltransferase	C. gigas This study C. gigas V. splendidus 4G4_4 This study C. gigas V. splendidus 4G4_4	RT-qPCR of host genes Control of mutagenesis RT-qPCR of host genes RT-qPCR of host genes Strain
		pSWδepsE	pSW23T; Δgtf2 GV4G44_v1_370043 primary response 88-like This study from wbe region	identification
	pMRB-PLACgfp MyD88like-22_R CGI_10012722 CCGGAAGTGACACAGCTTTT oriVR6Kγ ; oriTRP4; oriVpB1067 ; Placgfp [Cm R ] Myeloid differentiation 4G4_4-370043R GV4G44_v1_370044 CACCTGAACCCAAAAATCGT Glycosyltransferase primary response 88-like Le Roux et al., 2011 C. gigas V. splendidus 4G4_4 from wbe region	RT-qPCR of host genes Strain identification
	TNF14-07 F 4G1_8-10031F	pMRB-PLACgfp CGI_10028807 TCCGCTAGGAAGTGCCTTTA oriVR6Kγ ; oriTRP4; oriVpB1067 ; Placgfp [Spt R ] Tumor necrosis factor GV4G18_v1_10031 CAGAACTCTCTGGGCATGTG LysR family ligand superfamily transcriptional regulator Le Roux et al., 2011 C. gigas V. splendidus 4G1_8	RT-qPCR of host genes Strain identification
	TNF14-07 R 4G1_8-10031R	CGI_10028807 GV4G18_v1_10032 AAAATCACACCCGACTCGAC GGGCTGTATAGCCTTGCAGA	Tumor necrosis factor LysR family		C. gigas V. splendidus 4G1_8	RT-qPCR of host genes Strain
				ligand superfamily transcriptional regulator	identification
	NF-kB-49 F	CGI_10016949	CCCAAACAGGAGAAGGTCAA	Nuclear factor-kappa B		C. gigas	RT-qPCR of host genes
	NF-kB-49 R	CGI_10016949	TTCTTTTGCTCCATGGCTCT	Nuclear factor-kappa B		C. gigas	RT-qPCR of host genes
	CgLBP F	XM_011416094	AAGTTGTCGACCCGAACACG	Lipopolysaccharide		C. gigas	RT-qPCR of host genes
				binding protein
	CgLBP R	XM_011416094	ACGCTAATAACTGGCACTGG	Lipopolysaccharide		C. gigas	RT-qPCR of host genes
				binding protein
	CgBPI F	AAN84552.1	GATAGAAATAGGAATGGACGG	Bactericidal permeability	C. gigas	RT-qPCR of host genes
				increasing protein
	CgBPI R	AAN84552.1	GTTATAGATCCACGGTGCTCC	Bactericidal permeability	C. gigas	RT-qPCR of host genes
				increasing protein
	CgBigdef-1 F	AEE92772.1	TTCGCCTGCTTCCATAATGG	Big defensin-1			C. gigas	RT-qPCR of host genes
	CgBigdef-1 R	AEE92772.1	GAATAAGGAGTGACCATGAC	Big defensin-1			C. gigas	RT-qPCR of host genes
	*rtxA-F	GV4G44_v1_300034 GtatcgataagcttgatatcgaattcTTCCG	RtxA protein from MARTX	V. splendidus 4G4_4	Mutagenesis
		Strain	Description ATCTAGGAGACGGGC	cluster	Mutated gene	Reference
	E. coli Π3813 GV4G44_v1_300034 CccccgggctgcaggaattcTTCTATTCGG lacIQ, thi1, supE44, endA1, recA1, RtxA protein hsdR17, gyrA462, zei298::Tn10, TTGCTGCCAG from MARTX cluster Exr *rtxA-F *rtxA-R GV4G44_v1_300034 AGCCTAGCCGTCGCGTCAAG RtxA protein		V. splendidus 4G4_4 Le Roux et al., 2007 V. splendidus 4G4_4	Mutagenesis Control of mutagenesis
			DthyA::(erm-pir116) [TcR Erm R] from MARTX cluster
			(F -) RP4-2-Tc::Mu ΔdapA ::(erm-
	E. coli β3914	pir116), gyrA462, zei298::Tn10				Le Roux et al., 2007
			[KmR EmR TcR]
	E. coli SBS3	K12, derived from E. coli D22, F-Ro reverse Trp+, galU129 (short-chain LPS)			Saclay Bacterial strain bank
	E. coli ML-35p	i-, y-, z+ + pBR-322[AmpR]				Lehrer et al., 1988
	GV 2390		3T8_11, V. splendidus Pop #23				Bruto el al., 2017
	GV 2386		4D1_8, V. splendidus Pop #23				Bruto el al., 2017
	4D1_8 *epsE-i	ΔepsE::pSW23T [Cm R ]	GV4D18_v1_12005 7	This study
	GV 2388		4G1_8, V. splendidus Pop #23				Bruto el al., 2017
	GV 2389		4G4_4, V. splendidus Pop #23				Bruto el al., 2017
	4G4_4 *rtxA-i	*rtxA::pSW23T [Cm R ]	GV4G44_v1_30003 4	This study
	4G4_4 *vipA-i	*vipA::pSW23T [Cm R ]	GV4G44_v1_32001 7	This study

Table S3 .

 S3 Plasmids

	Plasmid CGI_10020815 F2	CGI_10020815	Description TGGCCAACATGAACATGTCC	Reference Complement C1q2	C. gigas	qRT-PCR of host genes
	pSW23T CGI_10020815 R2 Table S4. Oligonucleotides CGI_10020815 CGI_10000960 F1 CGI_10000960 Oligonucleotide Gene id CGI_10000960 R1 CGI_10000960	oriVR6K ; oriTRP4; [Cm R ] TGCTCCCACTGTTGTACCAA ACTGGGGAAAGAGGACGGAT Sequence 5'-3' ACACGCTTGGCTCCATGTAA	Complement C1q2 Frizzled-1 Demarre et al. 2005 Description Frizzled-1	C. gigas C. gigas Organism C. gigas	qRT-PCR of host genes qRT-PCR of host genes Use qRT-PCR of host genes
	pSWδVCR9J2v1_740022 CGI_10007949 F1 CGI_10007949 CGI_10020472 F2 CGI_10020472	pSW23T; ΔVCR9J2v1_740022 GGATGACGTGTTTCGTAGGG TGATGGACTCGGCTCAGTTG	This study Β-1,3-galactosyltransferase brn Deleted in malignant brain tumors 1-like	C. gigas C. gigas	qRT-PCR of host genes qRT-PCR of host genes
	pSWδVCR9J2v1_740023 CGI_10007949 R1 CGI_10007949 CGI_10020472 R2 CGI_10020472 pSWδVCR9J2v1_730029 CGI_10010688 F2 CGI_10010688 EFUfw AB122066 CGI_10010688 R2 CGI_10010688 EFUrv AB122066	pSW23T; ΔVCR9J2v1_740023 TCAATGTCTGCCAACAGCTC TCAATGGCGGTTCTCTCACC pSW23T; ΔVCR9J2v1_730029 GACGGAAAGCTGGACACTGA GAGCGTGAACGTGGTATCAC AGGTCATAGGGAGGGGCTTT ACAGCACAGTCAGCCTGTGA	Β-1,3-galactosyltransferase brn Deleted in malignant brain tumors 1-like This study Bone-morphogenetic 1-like EF1 This study Bone-morphogenetic 1-like EF1	C. gigas C. gigas C. gigas C. gigas C. gigas C. gigas	qRT-PCR of host genes qRT-PCR of host genes qRT-PCR of host genes qRT-PCR of host genes qRT-PCR of host genes qRT-PCR of host genes
	pSWδmshM CGI_10000441 F2 C23fw	CGI_10000441 HS119070	pSW23T; ΔmshM VS_0329 CCCAGGGTGCTCAACAATCA AATCTTGCACCGTCATGCAG	This study Krueppel-like factor 5 C23	C. gigas C. gigas	qRT-PCR of host genes qRT-PCR of host genes
	pSWδpilB CGI_10000441 R2 C23rv CGI_10000066 F2 rp56F	CGI_10000441 HS119070 CGI_10000066 FP004478	pSW23T; ΔpilB VS_2548 TGTGTTTTCGTATGTGGCGC AATCAATCTCTGCTGATCTGC TCTGCTCTGATTCGTGTCCA CAGAAGTGCCAGCTGACAGTC	Krueppel-like factor 5 C23 This study Metallothionein RP56	C. gigas C. gigas C. gigas C. gigas	qRT-PCR of host genes qRT-PCR of host genes qRT-PCR of host genes qRT-PCR of host genes
	pSWδrtx CGI_10000066 R2 rp56R	CGI_10000066 FP004478	pSW23T; Δrtx VS_1394 TTGCAGTTTTCCGGTCCAGT AGAAGCAATCTCACACGGAC	This study Metallothionein RP56	C. gigas C. gigas	qRT-PCR of host genes qRT-PCR of host genes
	pSWδvipAT6SS1T CGI_10024165 F2 CGI_10024165 OmpUS3 VS_2494 CGI_10024165 R2 CGI_10024165 OmpUAS2 VS_2494	pSW23T; ΔvipA T6SS1 AGGATGAGACGGTTGCTCAG GTCCTACAGACAGCGATAGC CCAACAACTTTGGCAGAACC GTGGTAAGCCATGATATCGC	Paired bov Pax-8-like isoform X1 Porin OmpU This study Paired bov Pax-8-like isoform X1 Porin OmpU	C. gigas LGP32 C. gigas LGP32	qRT-PCR of host genes qPCR quantification of vibrios qRT-PCR of host genes qPCR quantification of vibrios
	pSWδvipAT6SS2T CGI_10020521 F1 CGI_10020521 R5-2aF VRSK9J2v1_730268 GGATGGGACCAACTACGGTG pSW23T; ΔvipA T6SS2 CCACCTACACTCCAACGGAC	This study Zinc transporter 2-like Protein R5-2	C. gigas J2-9	qRT-PCR of host genes qPCR quantification of vibrios
	pMRB-PLACgfp CGI_10020521R1 R5-2aR CGI_10003627F1 LMG20012TF2	oriVR6Kg ; oriTRP4; oriVpB1067 ; PLAC-gfp [Cm R ] GGACGTGGATAAAGGCTGCT VRSK9J2v1_730268 CGTAGCCCGGAGGAAGAATC CGI_10020521 CGI_10003627 AGATTGCCTGTCCAGTCACG VTAS012-v1-10089 GGCGTTTCTTCACTTGCGAC	Zinc transporter 2-like Protein R5-2 Le Roux et al., 2011 Metalloreductas STEAP4-like Exported protein of unknow function	C. gigas J2-9 C. gigas LMG20	qRT-PCR of host genes qPCR quantification of vibrios qRT-PCR of host genes qPCR quantification of vibrios
	CGI_10003627R1	CGI_10003627	AAACTTCATGGCTCCCTCCG	Metalloreductas STEAP4-like	C. gigas 012T	qRT-PCR of host genes
	CGI_10002304F1 LMG20012TR2	CGI_10002304 VTAS012-v1-10089 TTTGCGGAGAAGTGCTAGGG TGTATCCGGTCGAGAGTGAG	Cyclin-G-associated kinase-like Exported protein of unknow function	C. gigas LMG20	qRT-PCR of host genes qPCR quantification of vibrios
	CGI_10002304R1 CGI_10010647F2 J2-8F2	CGI_10002304 CGI_10010647 VCR8J2v1-840028	TAACCGATTCCAGCTTGCGG TACGTCGCCTTCACAGACAC GAGCTTGAGATGGAGAATCG	Cyclin-G-associated kinase-like Heat shock 70 Anticodon nuclease	C. gigas 012T C. gigas J2-8	qRT-PCR of host genes qRT-PCR of host genes qPCR quantification of vibrios
	CGI_10010647R2 J2-8R2 CGI_10019038F2 ΔmshM-1	CGI_10010647 VCR8J2v1-840028 CGI_10019038 VS_0329	CGGCCAATGTTTCATGTCGG ACCGTTTCTACTTCTGCACC TGGCCACATTGTCCCTTCAG CGGCGTTAGAAATGGGTGAG	Heat shock 70 Anticodon nuclease Arginine deiminase/G1/S-specific cyclin-E1 MSHA biogenesis protein MshM	C. gigas J2-8 C. gigas LGP32	qPCR quantification of vibrios qRT-PCR of host genes qRT-PCR of host genes Mutagenesis
	CGI_10019038 R2 ΔmshM-2 CGI_10025754 F1 ΔpilB-1 CGI_10025754 R1 ΔpilB-2 CGI_10000478 F1 Δrtx-1 CGI_10000478 R1 Δrtx-2	CGI_10019038 VS_0329 CGI_10025754 VS_2548 CGI_10025754 VS_2548 CGI_10000478 VS_1394 CGI_10000478 VS_1394	CGAAGGACCAGTTGAGGAGG CCAAAGAAGAGCGCCAGATC GCCCACGAATCTTGCTGAAC GCCTGACTCAAGAACAAGCC TTTGGGTAGCGGGTGGAAAG GATTGGCGAGCTCATCTTGG GACGACCCTATCCCAACACC TCAAAGTCCATGATGCTGCG ATCTCGCCGATCCTTTTCCC CAAGGTTTGCTTTGCCAACG	Arginine deiminase/G1/S-specific cyclin-E1 MSHA biogenesis protein MshM Interleukin 17 Type IV pilin assembly protein ATPase PilB Interleukin 17 Type IV pilin assembly protein ATPase PilB Neuronal acethylcholine receptor subunit α-6-like Putative hemolysin-type RTX toxin Neuronal acethylcholine receptor subunit α-6-like Putative hemolysin-type RTX toxin	C. gigas LGP32 C. gigas LGP32 C. gigas LGP32 C. gigas LGP32 C. gigas LGP32	Mutagenesis qRT-PCR of host genes Mutagenesis qRT-PCR of host genes Mutagenesis qRT-PCR of host genes Mutagenesis qRT-PCR of host genes qRT-PCR of host genes Mutagenesis
	CGI_10004930 F2 ΔVCR9J2v1_730029-CGI_10004930 R2 1 CGI_10007268 F2 ΔVCR9J2v1_730029-CGI_10007268 R2 2 CGI_10009274 F2 ΔVCR9J2v1_740022-1 CGI_10009274 R2 ΔVCR9J2v1_740022-2 CGI_10000910 F2 CGI_10000910 R2 1 ΔVCR9J2v1_740023-	CGI_10004930 VCR9J2v1_730029 CGI_10004930 CGI_10007268 VCR9J2v1_730029 CGI_10007268 CGI_10009274 VCR9J2v1_740022 CGI_10009274 VCR9J2v1_740022 CGI_10000910 CGI_10000910 VCR9J2v1_740023	AAGTGGTCGAGAAACACCCG CCAATGGTTGGCGCTACTCC CCGGGTCAGACGAACTCTTC AGCAATGCTGGACACAACAG CGCTCTATCTGGGTGATTCG AATGGCGATGAAGCTGTACC AGCTGACAACACTGCCCAAT TGAGTTCGCTTCAGGTCGAG TTACTGGTTTCAGCTGTGCG TAA CCA TTC TAG GAG CGT GC AATGTCACAATGGGAAGCGC GGAGGTGGAATATGCGCTGT AACGCTAACAGGTCGCGTCG	NADPH oxidase 5 Putative NAD(+)-arginine ADP ribosyltransferase NADPH oxidase 5 Zinc transporter ZIP10-like isoform X1 Putative NAD(+)-arginine ADP ribosyltransferase Zinc transporter ZIP10-like isoform X1 Complement C1q tumor necrosis factor-related 7-Toxin secretion: membrane fusion protein (fragment) like Complement C1q tumor necrosis factor-related 7-Toxin secretion: membrane fusion protein (fragment) like Complement C1q4 Complement C1q4 (fragment) Toxin secretion: membrane fusion protein	C. gigas J2-9 C. gigas C. gigas J2-9 C. gigas C. gigas J2-9 C. gigas J2-9 C. gigas C. gigas J2-9	Mutagenesis qRT-PCR of host genes qRT-PCR of host genes Mutagenesis qRT-PCR of host genes qRT-PCR of host genes Mutagenesis qRT-PCR of host genes Mutagenesis qRT-PCR of host genes qRT-PCR of host genes qRT-PCR of host genes Mutagenesis

Table 1 .

 1 Primers used for amplification of the regions flanking evpP Sequence in bold: Homology with pSW7848T for cloning by Gibson assembly Sequence underlined: Ecor1 restriction site Sequence double underline homologies to allow fragment assembly during Gibson reaction

	Primer	Sequence 5'-3'

Table S4 .

 S4 RGBOA analysis

	CGI_10025133 CGI_10017943 CGI_10001123 CGI_10027395 CGI_10018396 CGI_10011332 CGI_10025133 CGI_10018396 CGI_10005003 CHAPTER 3. GENERAL DISCUSSION AND FUTURE 1.73 [Nematostella vectensis] 1.48 28S ribosomal mitochondrial 3.29 Chorion peroxidase 2.67 78 kDa glucose-regulated 3.29 radical S-adenosyl methionine domain-containing 2 -1.59 transient receptor potential cation channel subfamily M member 2-like isoform X2 1.68 [Nematostella vectensis] 3.29 radical S-adenosyl methionine domain-containing 2 1.70 signal transducer and transcription activator isoform X5 [Stomoxys calcitrans]
	CGI_10005650 CGI_10022879 CGI_10014532 CGI_10018396 CGI_10010240 CGI_10021734 CGI_10016583 CGI_10023950 CGI_10025133 CGI_10004787 CGI_10014323 CGI_10016240 CGI_10006027 CGI_10021345 CGI_10025133 CGI_10021171 CGI_10017630 CGI_10016254 PROSPECTS		2.55 2.07 2.95 2.74 3.67 2.40 1.85 1.86 1.68 -1.82 -2.15 -1.88 1.56 -2.17 1.68 1.44 2.10 3.14	ras-related Rab-11A-like deoxynucleoside triphosphate triphosphohydrolase SAMHD1 interferon-induced 44-like [Crassostrea gigas] radical S-adenosyl methionine domain-containing 2 Chorion peroxidase chorion peroxidase-like Aggrecan core galectin-5-partial [Nematostella vectensis] E3 ubiquitin-ligase RNF123-like Transient receptor potential cation channel subfamily M member 3 transient receptor potential cation channel subfamily M member 2-like CREB-binding -like isoform X2 Ras-related C3 botulinum toxin substrate 1 [Nematostella vectensis] interferon regulatory factor 2 signal transducer and activator of transcription 5B [Ceratosolen solmsi marchali] Core-binding factor subunit beta
	CGI_10027441 CGI_10021019 CGI_10028272 CGI_10012829 CGI_10001488 CGI_10015111 CGI_10028827 CGI_10019528 CGI_10027512		2.95 1.67 2.35 1.37 -1.92 -1.64 2.15 2.27 1.87	helicase with zinc finger domain 2-like zinc finger CCCH domain-containing 13-like myeloperoxidase-like [Crassostrea gigas] PX domain-containing kinase E3 ubiquitin-ligase UBR3 potassium voltage-gated channel subfamily A member 1-like isoform X1 [Crassostrea gigas] peptidoglycan recognition SC2 [Drosophila melanogaster] suppressor of cytokine signaling 2-like Toll-like receptor 13
	CGI_10000144 CGI_10025133 CGI_10005474 CGI_10003308 CGI_10028226 CGI_10024636 CGI_10021171 CGI_10010457 CGI_10019528		3.26 1.73 1.83 0.98 1.27 -1.36 1.44 2.00 2.27	helicase with zinc finger domain 2-like isoform X1 [Crassostrea gigas] [Nematostella vectensis] NFX1-type zinc finger-containing 1-like MAP kinase-activated kinase 2 E3 ubiquitin-ligase MGRN1-like isoform X2 Potassium voltage-gated channel Shaw [Trichinella britovi] interferon regulatory factor 2 Ddx58 partial suppressor of cytokine signaling 2-like
	CGI_10004570 CGI_10022879 CGI_10014276 CGI_10027721 CGI_10021171 CGI_10016977 CGI_10020437 CGI_10012998 CGI_10028806		-0.85 2.07 1.87 1.41 1.44 -2.16 -1.85 1.83 2.64	rapamycin-insensitive companion of mTOR-like deoxynucleoside triphosphate triphosphohydrolase SAMHD1 laccase-like [Octopus bimaculoides] proto-oncogene tyrosine-kinase Src isoform X2 interferon regulatory factor 2 sodium calcium exchanger 1-like isoform X1 [Lingula anatina] T-box transcription factor TBX20 Double-stranded RNA-specific adenosine deaminase [Nematostella vectensis]
	Enriched Biological functions at 4 hours CGI_10022075 CGI_10028827 CGI_10016651 CGI_10010457 CGI_10019528 CGI_10027466 CGI_10016154 CGI_10026305 CGI_10000304		1.99 2.20 2.15 2.00 2.27 -2.23 1.60 1.04 -2.64	scaffold salvador-like [Biomphalaria glabrata] peptidoglycan recognition SC2 [Drosophila melanogaster] deleted in malignant brain tumors 1 -like Ddx58 partial suppressor of cytokine signaling 2-like sodium calcium exchanger 2-like isoform X3 [Crassostrea gigas] tumor necrosis factor alpha-induced 3 CCAAT enhancer-binding gamma eukaryotic initiation factor 4A-I
	CGI_10003308 CGI_10016154 CGI_10027957 CGI_10003301 CGI_10005873 CGI_10004070 CGI_10027395 immune system process	0.00303	1.16 1.61 2.36 2.09 2.78 1.60 2.67	MAP kinase-activated kinase 2 tumor necrosis factor alpha-induced 3 ras-related GTP-binding A NFX1-type zinc finger-containing 1-like polyubiquitin 8-like [Acropora digitifera] ATP synthase F(0) complex subunit mitochondrial-like 78 kDa glucose-regulated
	Biological function CGI_10014861 CGI_10027441 CGI_10014532 CGI_10023396 CGI_10005452 CGI_10026671 CGI_10009679 CGI_10003083 intracellular signal transduction	p.adj 0.03371	Log2 FC 1.15 2.95 2.34 1.84 1.54 2.44 -1.66 2.43	Annotation Ubiquitin-conjugating enzyme E2 N helicase with zinc finger domain 2-like interferon-induced 44-like [Crassostrea gigas] NFX1-type zinc finger-containing 1-like E3 ubiquitin-ligase RNF213-like sulfite oxidase-like cilia-and flagella-associated 20 probable ATP-dependent RNA helicase DHX58
	epithelial to mesenchymal transition CGI_10023630 CGI_10000144 CGI_10004922 CGI_10026305 CGI_10027441 CGI_10003777 CGI_10021162 CGI_10021170 CGI_10003083	3.56E-07	1.31 3.26 5.19 1.04 3.14 -1.02 -1.53 4.06 2.43	Prickle 2 helicase with zinc finger domain 2-like isoform X1 [Crassostrea gigas] IL17-5 [Crassostrea gigas] CCAAT enhancer-binding gamma helicase with zinc finger domain 2-like sodium channel 1 brain-like isoform X2 nucleoside diphosphate kinase homolog 5-like Interferon regulatory factor 2 probable ATP-dependent RNA helicase DHX58
	CGI_10005518 CGI_10016921 CGI_10024595 CGI_10012998 CGI_10000144 CGI_10017857 CGI_10027441 CGI_10010459 CGI_10021170		2.26 -2.37 2.11 1.83 3.49 2.25 3.14 1.80 4.06	transcriptional regulator Erg-partial histone-lysine N-methyltransferase EHMT1 Complement C1q 4 Double-stranded RNA-specific adenosine deaminase helicase with zinc finger domain 2-like isoform X1 [Crassostrea gigas] inter-alpha-trypsin inhibitor heavy chain H3-like helicase with zinc finger domain 2-like probable ATP-dependent RNA helicase DDX58 Interferon regulatory factor 2
	CGI_10010444 immune response CGI_10010444 CGI_10023765 CGI_10017480 CGI_10027442 CGI_10026568 CGI_10000144 CGI_10021954 CGI_10010459	0.01748	1.60 2.83 1.83 -2.32 2.73 1.94 3.49 2.24 1.80	Hepatocyte growth factor Hepatocyte growth factor CD109 antigen-like E3 ubiquitin-ligase UHRF1 helicase with zinc finger domain 2-like isoform X1 [Crassostrea gigas] sodium-and chloride-dependent glycine transporter 2-like helicase with zinc finger domain 2-like isoform X1 [Crassostrea gigas] ATP-dependent RNA helicase chloroplastic-like [Parasteatoda tepidariorum] probable ATP-dependent RNA helicase DDX58
	Enriched Biological functions at 24hours CGI_10018396 CGI_10003308 CGI_10017629 CGI_10016804 CGI_10010457 CGI_10025447 CGI_10010444 CGI_10004742 CGI_10021954		2.74 1.16 1.88 2.30 2.00 -1.50 1.60 -2.98 2.24	radical S-adenosyl methionine domain-containing 2 MAP kinase-activated kinase 2 Signal transducer and transcription activator 6 myosin light chain smooth muscle isoform X4 [Monodelphis domestica] Ddx58 partial multidrug resistance-associated 1-like isoform X1 Hepatocyte growth factor zinc finger CCCH domain-containing 11A-like ATP-dependent RNA helicase chloroplastic-like [Parasteatoda tepidariorum]
	Biological function CGI_10027512 CGI_10014861 CGI_10005003 CGI_10024107 CGI_10012998 CGI_10017841 CGI_10003308 CGI_10024393 CGI_10004742	p.adj	Log2 FC 1.83 1.15 1.70 1.13 1.83 2.37 0.98 1.75 -2.98	Annotation Toll-like receptor 13 Ubiquitin-conjugating enzyme E2 N signal transducer and transcription activator isoform X5 [Stomoxys calcitrans] E3 ubiquitin-ligase rififylin Double-stranded RNA-specific adenosine deaminase Excitatory amino acid transporter partial MAP kinase-activated kinase 2 Interferon-induced helicase C domain-containing 1 zinc finger CCCH domain-containing 11A-like
	myeloid cell differentiation CGI_10027721 CGI_10012725 CGI_10001942 Ras protein signal transduction CGI_10017480 CGI_10003810 CGI_10027721 CGI_10024392 CGI_10024393	0.01426 0.00496	1.66 2.70 2.23 -2.32 -2.40 1.41 1.97 1.75	proto-oncogene tyrosine-kinase Src isoform X2 Myeloid differentiation primary response 88 DNA polymerase lambda E3 ubiquitin-ligase UHRF1 sodium-and chloride-dependent glycine transporter 1-like proto-oncogene tyrosine-kinase Src isoform X2 probable ATP-dependent RNA helicase DHX58 Interferon-induced helicase C domain-containing 1
	CGI_10027197 CGI_10026305 CGI_10008714 CGI_10017630 CGI_10023902 CGI_10024107 CGI_10009981 CGI_10010457 CGI_10003270 CGI_10024392		1.97 1.15 -3.16 2.10 1.47 1.13 2.35 2.00 2.36 1.97	60S ribosomal L35 CCAAT enhancer-binding gamma WW domain-containing oxidoreductase signal transducer and activator of transcription 5B [Ceratosolen solmsi marchali] ras guanyl-releasing 3-like isoform X1 [Biomphalaria glabrata] E3 ubiquitin-ligase rififylin sodium-and chloride-dependent glycine transporter 1-like Ddx58 partial interferon regulatory factor 8 probable ATP-dependent RNA helicase DHX58
	CGI_10016254 CGI_10028827 CGI_10027721 CGI_10018396 CGI_10006027 CGI_10023765 CGI_10024262 CGI_10011040 CGI_10005182 CGI_10003270		2.97 2.20 1.66 3.29 1.56 1.83 -1.68 2.21 1.73 2.36	Core-binding factor subunit beta peptidoglycan recognition SC2 [Drosophila melanogaster] proto-oncogene tyrosine-kinase Src isoform X2 radical S-adenosyl methionine domain-containing 2 CREB-binding -like isoform X2 CD109 antigen-like Sodium-dependent noradrenaline transporter Phosphoinositide 3-kinase adapter 1 double-stranded RNA-specific adenosine deaminase-like interferon regulatory factor 8
	CGI_10026305 CGI_10028806 CGI_10024321 CGI_10007774 CGI_10005650 CGI_10003823 CGI_10009522 CGI_10025934 CGI_10017943 CGI_10005182		1.15 2.73 1.88 1.98 2.37 3.06 -1.66 -1.00 1.45 1.73	CCAAT enhancer-binding gamma [Nematostella vectensis] Actin-related 2 3 complex subunit 3 Ubiquitin-like modifier-activating enzyme 1 ras-related Rab-11A-like COMM domain-containing 3 solute carrier family facilitated glucose transporter member 6 [Oreochromis niloticus] receptor for activated C kinase 1 28S ribosomal mitochondrial double-stranded RNA-specific adenosine deaminase-like
	CGI_10006667 CGI_10006667 CGI_10026305 CGI_10003823 cellular response to external stimulus CGI_10012135 CGI_10027842 CGI_10012998 CGI_10014532 CGI_10007096	0.03371	1.18 1.18 1.15 3.06 2.05 2.51 1.83 2.34 0.93	microphthalmia-associated transcription factor isoform X1 microphthalmia-associated transcription factor isoform X1 CCAAT enhancer-binding gamma COMM domain-containing 3 C-type lectin domain family 3 member A Solute carrier family facilitated glucose transporter member 1 Double-stranded RNA-specific adenosine deaminase interferon-induced 44-like [Crassostrea gigas] 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase epsilon-1-like
	intracellular signal transduction immune effector process CGI_10005518 CGI_10012135 CGI_10018604 CGI_10023840 CGI_10009497 CGI_10018396 CGI_10023902 CGI_10021170 CGI_10021170 CGI_10020437 CGI_10023840 CGI_10027957 CGI_10014497 immune response CGI_10024107 CGI_10025133 CGI_10017807 CGI_10010459 CGI_10010459 CGI_10006667 CGI_10025133 CGI_10006027 CGI_10028827 CGI_10018396 CGI_10015817 CGI_10028827 CGI_10003481	0.01426 0.01748 0.00046	1.85 2.05 2.17 -1.88 1.39 3.29 1.47 3.94 3.94 -1.53 -1.88 2.36 3.01 1.13 1.68 1.47 1.67 1.67 1.18 1.68 1.56 2.15 3.29 2.27 2.15 1.27	transcriptional regulator Erg-partial C-type lectin domain family 3 member A Autophagy 5 low affinity immunoglobulin epsilon Fc receptor-like isoform X2 caspase recruitment domain-containing 9-like radical S-adenosyl methionine domain-containing 2 ras guanyl-releasing 3-like isoform X1 [Biomphalaria glabrata] Interferon regulatory factor 2 T-box transcription factor TBX20 low affinity immunoglobulin epsilon Fc receptor-like isoform X2 ras-related GTP-binding A von Willebrand factor type EGF and pentraxin domain-containing 1-like isoform X2 [Crassostrea gigas] E3 ubiquitin-ligase rififylin [Nematostella vectensis] ras-related M-Ras-like Interferon regulatory factor 2 probable ATP-dependent RNA helicase DDX58 probable ATP-dependent RNA helicase DDX58 microphthalmia-associated transcription factor isoform X1 [Nematostella vectensis] CREB-binding -like isoform X2 peptidoglycan recognition SC2 [Drosophila melanogaster] radical S-adenosyl methionine domain-containing 2 phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform-like peptidoglycan recognition SC2 [Drosophila melanogaster] Ras-related Rab-11A
	CGI_10021954 CGI_10003183 CGI_10017629 CGI_10006027 CGI_10027395 CGI_10012515 CGI_10027512 CGI_10005650 CGI_10021171 CGI_10014532		2.19 2.93 2.02 1.56 2.67 -2.40 1.87 2.37 1.44 2.34	ATP-dependent RNA helicase chloroplastic-like [Parasteatoda tepidariorum] deoxynucleoside triphosphate triphosphohydrolase SAMHD1-like Signal transducer and transcription activator 6 CREB-binding -like isoform X2 78 kDa glucose-regulated C-type lectin mannose-binding isoform-like Toll-like receptor 13 ras-related Rab-11A-like interferon regulatory factor 2 interferon-induced 44-like [Crassostrea gigas]
	CGI_10003183 CGI_10003270 CGI_10005003 CGI_10001792 CGI_10012725 CGI_10016583 CGI_10027721 CGI_10021178 CGI_10016154 CGI_10011731		2.93 2.15 1.84 -2.42 2.31 1.85 1.41 1.56 1.60 -1.15	deoxynucleoside triphosphate triphosphohydrolase SAMHD1-like interferon regulatory factor 8 signal transducer and transcription activator isoform X5 [Stomoxys calcitrans] hypothetical protein CGI_10001792 Myeloid differentiation primary response 88 Aggrecan core proto-oncogene tyrosine-kinase Src isoform X2 SH2 domain-containing adapter F tumor necrosis factor alpha-induced 3 serine threonine-kinase pats1
	CGI_10010458 CGI_10005182 CGI_10017630 CGI_10012514 CGI_10021345 CGI_10023950 CGI_10010457 CGI_10005518 CGI_10021162 CGI_10026034		1.75 1.63 2.15 2.00 -2.17 1.86 2.00 2.38 -1.53 2.12	probable ATP-dependent RNA helicase DHX58 double-stranded RNA-specific adenosine deaminase-like signal transducer and activator of transcription 5B [Ceratosolen solmsi marchali] Toll-like receptor 13 Ras-related C3 botulinum toxin substrate 1 galectin-5-partial Ddx58 partial transcriptional regulator Erg-partial nucleoside diphosphate kinase homolog 5-like probable serine threonine-kinase pats1
	CGI_10024392 CGI_10014532 CGI_10016254 CGI_10024612 response to external stimulus CGI_10003308 CGI_10026305 CGI_10011295 CGI_10027441 CGI_10015931	0.03371	2.32 2.95 2.97 2.24 0.98 1.04 -2.82 3.14 2.47	probable ATP-dependent RNA helicase DHX58 interferon-induced 44-like [Crassostrea gigas] Core-binding factor subunit beta hypothetical protein CGI_10024612 MAP kinase-activated kinase 2 CCAAT enhancer-binding gamma calmodulin-A-like isoform X1 helicase with zinc finger domain 2-like ADP-ribosylation factor 1
	CGI_10003270 CGI_10017629 CGI_10027512 CGI_10012722 CGI_10003083 CGI_10006027 CGI_10028827 CGI_10026305 CGI_10000144 CGI_10028634		2.15 2.02 1.83 4.99 2.43 1.56 2.15 1.04 3.49 1.34	interferon regulatory factor 8 Signal transducer and transcription activator 6 Toll-like receptor 13 myeloid differentiation primary response 88-like [Crassostrea gigas] probable ATP-dependent RNA helicase DHX58 CREB-binding -like isoform X2 peptidoglycan recognition SC2 [Drosophila melanogaster] CCAAT enhancer-binding gamma helicase with zinc finger domain 2-like isoform X1 [Crassostrea gigas] Rho-related GTP-binding
	CGI_10005182 CGI_10005003 CGI_10028806 CGI_10026174 CGI_10021170 CGI_10000304 CGI_10028806 CGI_10006667 CGI_10010444 CGI_10019413 CGI_10007096 CGI_10017630 CGI_10000304 CGI_10026493 CGI_10010459 CGI_10018604 CGI_10006667 CGI_10001126 CGI_10003308 CGI_10014971 CGI_10023902 CGI_10018396 CGI_10021136 CGI_10021954 CGI_10012725 CGI_10021863 CGI_10010457 CGI_10018396		1.63 1.84 2.73 -1.93 4.06 -2.64 2.64 1.23 1.60 -2.49 1.06 2.15 -3.03 1.77 1.80 2.17 1.23 1.74 0.98 2.58 1.41 2.74 2.38 2.24 2.31 2.15 2.00 3.29	signal transducer and transcription activator isoform X5 [Stomoxys calcitrans] [Nematostella vectensis] hypothetical protein CGI_10026174 Interferon regulatory factor 2 eukaryotic initiation factor 4A-I [Nematostella vectensis] microphthalmia-associated transcription factor isoform X1 Hepatocyte growth factor ADP-ribosylation factor 2 [Cucumis melo] double-stranded RNA-specific adenosine deaminase-like signal transducer and activator of transcription 5B [Ceratosolen solmsi marchali] eukaryotic initiation factor 4A-I Toll-like receptor 13 probable ATP-dependent RNA helicase DDX58 Autophagy 5 microphthalmia-associated transcription factor isoform X1 Annexin A7 MAP kinase-activated kinase 2 guanine nucleotide-binding G(s) subunit alpha-like 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase epsilon-1-like ras guanyl-releasing 3-like isoform X1 [Biomphalaria glabrata] radical S-adenosyl methionine domain-containing 2 Toll-like receptor 1 ATP-dependent RNA helicase chloroplastic-like [Parasteatoda tepidariorum] Myeloid differentiation primary response 88 Annexin A7 Ddx58 partial radical S-adenosyl methionine domain-containing 2
	CGI_10022146 CGI_10021019 Enriched Biological functions at 48hours CGI_10027512 CGI_10004742 CGI_10027957 regulation of response to stimulus CGI_10007222 CGI_10011040 CGI_10015817 CGI_10017070 CGI_10025133 Biological function CGI_10021345 CGI_10024393 CGI_10021345 CGI_10003083 CGI_10022740 CGI_10012998 CGI_10025133	0.01456 p.adj	-2.15 1.67 1.87 -2.98 2.36 1.32 2.21 2.27 2.02 1.73 Log2 FC -2.17 1.75 -2.17 2.43 -1.79 1.83 1.68	zinc finger CCCH domain-containing 13-like Toll-like receptor 13 zinc finger CCCH domain-containing 11A-like ras-related GTP-binding A ubiquitin-conjugating enzyme E2-17 kDa [Bactrocera oleae] Phosphoinositide 3-kinase adapter 1 phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform-like transmembrane 201-like ras-related RABF2b-like [Crassostrea gigas] [Nematostella vectensis] Annotation Ras-related C3 botulinum toxin substrate 1 Interferon-induced helicase C domain-containing 1 Ras-related C3 botulinum toxin substrate 1 probable ATP-dependent RNA helicase DHX58 CD2-associated [Mus musculus] Double-stranded RNA-specific adenosine deaminase [Nematostella vectensis]
	CGI_10021960 CGI_10022879 Response to stress CGI_10014497 CGI_10024392 CGI_10027395 CGI_10021170 CGI_10012725 CGI_10006027		-1.19 2.07 3.01 1.97 2.67 4.06 2.31 1.56	ras-related Rab-3-like isoform X4 [Crassostrea gigas] deoxynucleoside triphosphate triphosphohydrolase SAMHD1 von Willebrand factor type EGF and pentraxin domain-containing 1-like isoform X2 [Crassostrea gigas] probable ATP-dependent RNA helicase DHX58 78 kDa glucose-regulated Interferon regulatory factor 2 Myeloid differentiation primary response 88 CREB-binding -like isoform X2
	CGI_10003481 CGI_10026305 CGI_10018604 CGI_10028827 CGI_10003270 CGI_10010444 CGI_10012725 immune effector process CGI_10027957 CGI_10021345	0.00052	1.49 1.15 2.17 2.15 2.36 1.60 2.31 2.36 -2.17	Ras-related Rab-11A CCAAT enhancer-binding gamma Autophagy 5 peptidoglycan recognition SC2 [Drosophila melanogaster] interferon regulatory factor 8 Hepatocyte growth factor Myeloid differentiation primary response 88 ras-related GTP-binding A Ras-related C3 botulinum toxin substrate 1
	CGI_10014532 CGI_10024321 CGI_10003083 CGI_10021171 CGI_10005182 myeloid cell differentiation CGI_10010459 CGI_10003083 CGI_10006027 CGI_10005650	0.00197	2.95 1.88 2.43 1.44 1.73 1.80 2.43 1.56 2.37	interferon-induced 44-like [Crassostrea gigas] Actin-related 2 3 complex subunit 3 probable ATP-dependent RNA helicase DHX58 interferon regulatory factor 2 double-stranded RNA-specific adenosine deaminase-like probable ATP-dependent RNA helicase DDX58 probable ATP-dependent RNA helicase DHX58 CREB-binding -like isoform X2 ras-related Rab-11A-like
	CGI_10026034 CGI_10021170 CGI_10019528 CGI_10001246 CGI_10027197 CGI_10021954 CGI_10021170 CGI_10021345 CGI_10021171 CGI_10015931 immune system process CGI_10012725 CGI_10012515 CGI_10026035 CGI_10016254 CGI_10004742 CGI_10010459 CGI_10009679 CGI_10019528 CGI_10006953 CGI_10021170 CGI_10010459 CGI_10012379 CGI_10021893 CGI_10026305 CGI_10024393 CGI_10004742 CGI_10027721 CGI_10027441	0.02906	2.00 4.06 2.27 2.12 1.62 2.24 4.06 -2.17 1.44 2.93 2.31 -2.40 1.23 3.14 -2.98 1.80 -1.66 2.27 1.27 3.94 1.80 2.49 2.06 1.04 1.75 -2.98 1.41 3.14	Interferon regulatory factor 2 suppressor of cytokine signaling 2-like probable E3 ubiquitin-ligase RNF144A 60S ribosomal L35 ATP-dependent RNA helicase chloroplastic-like [Parasteatoda tepidariorum] Interferon regulatory factor 2 Ras-related C3 botulinum toxin substrate 1 interferon regulatory factor 2 probable serine threonine-kinase pats1 Myeloid differentiation primary response 88 C-type lectin mannose-binding isoform-like E3 ubiquitin-ligase MIB1-like isoform X1 Core-binding factor subunit beta zinc finger CCCH domain-containing 11A-like probable ATP-dependent RNA helicase DDX58 cilia-and flagella-associated 20 suppressor of cytokine signaling 2-like ADP-ribosylation factor 1 rho-related BTB domain-containing 2-like [Aplysia californica] Interferon regulatory factor 2 probable ATP-dependent RNA helicase DDX58 Deleted in malignant brain tumors 1 E3 ubiquitin-ligase HERC2 CCAAT enhancer-binding gamma Interferon-induced helicase C domain-containing 1 zinc finger CCCH domain-containing 11A-like proto-oncogene tyrosine-kinase Src isoform X2 helicase with zinc finger domain 2-like
	CGI_10018706 CGI_10010459 CGI_10004742 CGI_10018753 CGI_10000695 CGI_10006667 CGI_10024392 CGI_10024393 CGI_10009497 CGI_10000144 CGI_10028634 CGI_10021954 CGI_10018142 CGI_10008241 CGI_10015483 response to lipopolysaccharide CGI_10003270 CGI_10003270 CGI_10026305 CGI_10004570	0.01619	2.87 1.67 -2.98 3.65 -3.36 1.23 1.97 1.75 1.39 3.49 2.11 2.19 1.80 4.07 1.76 2.36 2.36 1.04 -0.89	probable ATP-dependent RNA helicase DDX58 zinc finger CCCH domain-containing 11A-like deleted in malignant brain tumors 1 -like E3 ubiquitin-ligase MIB2 microphthalmia-associated transcription factor isoform X1 probable ATP-dependent RNA helicase DHX58 Interferon-induced helicase C domain-containing 1 caspase recruitment domain-containing 9-like helicase with zinc finger domain 2-like isoform X1 [Crassostrea gigas] GTP-binding RAD [Crassostrea gigas] Rho-related GTP-binding ATP-dependent RNA helicase chloroplastic-like [Parasteatoda tepidariorum] Transcription factor p65 C-type lectin domain family member A-like E3 ubiquitin-ligase MIB2 interferon regulatory factor 8 interferon regulatory factor 8 CCAAT enhancer-binding gamma rapamycin-insensitive companion of mTOR-like
	CGI_10014971 CGI_10003183 CGI_10024393 CGI_10011916 CGI_10014532 CGI_10003308 CGI_10005182 CGI_10005182 CGI_10022740 CGI_10022075		2.38 2.93 1.75 2.73 2.34 0.98 1.73 1.73 -1.79 1.66	guanine nucleotide-binding G(s) subunit alpha-like deoxynucleoside triphosphate triphosphohydrolase SAMHD1-like Interferon-induced helicase C domain-containing 1 tyrosinase tyr-3 interferon-induced 44-like [Crassostrea gigas] MAP kinase-activated kinase 2 double-stranded RNA-specific adenosine deaminase-like double-stranded RNA-specific adenosine deaminase-like CD2-associated [Mus musculus] scaffold salvador-like [Biomphalaria glabrata]
	CGI_10018396 CGI_10010458 CGI_10003270 CGI_10009318 CGI_10017629 CGI_10006027 CGI_10017943 CGI_10014532 CGI_10005518 CGI_10003308		2.74 1.75 2.36 2.49 1.88 1.56 1.45 2.34 2.38 0.98	radical S-adenosyl methionine domain-containing 2 probable ATP-dependent RNA helicase DHX58 interferon regulatory factor 8 tyrosinase tyr-3 Signal transducer and transcription activator 6 CREB-binding -like isoform X2 28S ribosomal mitochondrial interferon-induced 44-like [Crassostrea gigas] transcriptional regulator Erg-partial MAP kinase-activated kinase 2
	CGI_10015817 CGI_10024392 CGI_10005182 CGI_10007753 CGI_10005003 CGI_10027395 CGI_10027957 CGI_10017629 CGI_10020437 CGI_10010457		2.49 2.32 1.73 2.57 1.70 2.67 2.36 1.88 -1.85 2.00	phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform-like probable ATP-dependent RNA helicase DHX58 double-stranded RNA-specific adenosine deaminase-like tyrosinase tyr-3 signal transducer and transcription activator isoform X5 [Stomoxys calcitrans] 78 kDa glucose-regulated ras-related GTP-binding A Signal transducer and transcription activator 6 T-box transcription factor TBX20 Ddx58 partial
	CGI_10021019 CGI_10005182 CGI_10007793 CGI_10028079 CGI_10018945 transmembrane transport CGI_10018396 CGI_10017630 CGI_10017629 CGI_10003270 CGI_10024233 CGI_10009319 CGI_10017630 CGI_10014532 CGI_10005003 CGI_10006667 CGI_10012998	0.03371	1.67 1.63 3.45 1.87 1.26 3.29 2.10 1.88 2.15 3.90 1.88 2.10 2.34 1.70 1.23 1.83	double-stranded RNA-specific adenosine deaminase-like tyrosinase tyr-3 E3 ubiquitin-ligase AMFR radical S-adenosyl methionine domain-containing 2 signal transducer and activator of transcription 5B [Ceratosolen solmsi marchali] Signal transducer and transcription activator 6 tyrosinase tyr-3 zinc finger CCCH domain-containing 13-like interferon regulatory factor 8 hemagglutinin amebocyte aggregation factor-like tyrosinase tyr-3 signal transducer and activator of transcription 5B [Ceratosolen solmsi marchali] interferon-induced 44-like [Crassostrea gigas] signal transducer and transcription activator isoform X5 [Stomoxys calcitrans] microphthalmia-associated transcription factor isoform X1 Double-stranded RNA-specific adenosine deaminase

Remerciements

son aide en bioinformatique. inhospitality), l´Ifremer (politique de site, projet Diversivib), l'divers projets.

ΔVCR9J2v1_740023-2

VCR9J2v1_740023

GTCAAGGCGTATGGTGTTGG Toxin secretion: membrane fusion protein (fragment)

J2-9 Mutagenesis

ΔvipAT6SS1 Virulent strains used in this study were V. tasmaniensis LGP32 (1), V. crassostreae J2-9 (2). Nonvirulent strains were Vibrio sp.nov. J2-8, close phylogenetic neighbor of the virulent V. crassostreae population (2) as well as the type strain of V. tasmaniensis LMG20 012T [START_REF] Thompson | Vibrio tasmaniensis sp. nov., isolated from Atlantic salmon (Salmo salar L.). (Translated from eng[END_REF]. Other strains, plasmids and primers used or constructed in the present study are described in SI Appendix Tables S2-S3-S4.

Animals

C. gigas diploid oyster spat were used from three families of siblings (referred to as Decipher #1, #14 and #15) ( 4) and a batch of standardized Ifremer spats (NSI) were produced from a pool of 120 genitors (5). Animals were from both sexes. Spats (4 to 6 months old) were used for experimental infections and dual RNA-seq. Adults (18 months old) produced from a pool of 120 genitors (ASI) were used to collect hemolymph for cytotoxicity assays. All oysters were produced at the Ifremer hatchery in Argenton, France. All families have been tested in experimental infections. The oysters of family #14 were chosen for the RNA-seq experiments.

Experimental infections

Experimental infections were performed at 20 °C by adapting a previously described procedure (6). Briefly, oysters were anesthetized for 3 h (7). Groups of 20 juvenile oysters (1.5-2 cm) were then distributed into tanks containing 3 L of seawater, per condition. Stationary phase cultures of bacteria (20 h at 20 °C) were washed by centrifugation (15 min, 1500 g, 20 °C) and resuspended in sterile seawater (SSW) before injecting at 7.5 × 10 7 CFU into oyster adductor muscle. Mortalities were monitored daily for 7 days. Two tanks were monitored per condition. Statistical analyses were performed using non-parametric Kaplan Meier test in order to estimate log-rank values for comparing conditions (GraphPad Prism 6). All experimental infections were performed according to the Ifremer animal care guidelines and policy.

Molecular microbiology

Vibrio isolates were grown at 20 °C in Zobell broth or agar (4 g.l -1 bactopeptone and 1 g.l -1 yeast extract in artificial seawater, pH 7.6), Luria-Bertani (LB) broth or LB-agar (LBA) containing 0.5 M NaCl. Escherichia coli strains were grown at 37°C in LB or on LBA for cloning and conjugation experiments. Chloramphenicol (Cm, 5, 10 or 25μg.ml -1 depending on the strain), thymidine (0.3 mM) and diaminopimelate (0.3 mM) were added as supplements when Specific vibrio quantification by qPCR Genomic DNA was extracted with the DNA kit Promega (Wizard SV Genomic DNA Purification System) on the same oyster samples used for RNA-seq. DNA concentration was measured using a NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific Inc.). Realtime qPCR was performed on a Light-Cycler 480 System (Roche). The total Real-time qPCR reaction volume was 1.5 μl with 0.5 μl cDNA (40 ng µl -1 ) and 1 μl LightCycler 480 SYBR Green I Master mix (Roche) containing 0.5 μM PCR primer (Eurogenetec SA). Strain-specific primers were designed using Primer 3 plus (http://primer3plus.com/), to amplify monocopy genes from specific vibrio genomes (Table S8). Efficiency and specificity were checked on DNA extracted from six different vibrio strains. To quantify oyster genomes, Cg-BPI (GenBank: AY165040) primers were used (Table S8). The vibrio charge in oyster tissues was calculated as a number of vibrio genome copies per number of oyster genome copy considering the following genome sizes: C. gigas (637 Mbp), LGP32 (4.97 Mbp), LMG20012T (4.84 Mbp), J2-9 (5.79 Mbp), J2-8 (5.39 Mbp).

Vibrio genome sequencing and assembly

Genomic DNA for the J0-13, J5-13 and J5-9 strains was extracted with the NucleoSpin Tissue kit (Macherey-Nagel Inc.). Starting from 1 ng DNA, library construction was performed at the Bio-Environnement platform (University of Perpignan, France) with the Nextera XT kit (Illumina, USA) in paired-end mode (2 × 150 bp). Sequencing was carried out using a NextSeq 550 instrument resulting in ~100-fold coverage. Reads were assembled de novo using Spades software. Computational prediction of coding sequences together with functional assignments was performed using the automated annotation pipeline implemented in the MicroScope platform (25). The genome sequences of V. tasmaniensis J0-13, J5-13 and J5-9 were deposited in Genbank with the following accession numbers: SEOP00000000, SESM00000000 and SESL00000000.

Killing or cheating: two strategies to colonize an oyster in Vibrio harveyi and Vibrio rotiferianus Daniel Oyanedel* 1 , Arnaud Lagorce* 1 , Maxime Bruto* 2,3 , Philippe Haffner 1 , Marie-Agnès

Travers 1 , Benjamin Morga 4 , Eve Toulza 1 , Cristian Chaparro 1 , Léa-lou Pimparé 1 , Caroline

Montagnani 1 , Yannick Gueguen 1 , Delphine Toubiez 4 , Marc Leroy 1 , Juliette Pouzadoux 1 , Guillaume Mitta 1 , Frédérique Le Roux 2,3 , Guillaume Charrière 1 , Delphine Destoumieux-Garzón 2B). V. jasicida isolates induced heterogeneous mortality rates with high contrast between isolates, from 0-44% mortality (18% average mortality, Figure 2B). Almost no mortality could be observed following injection of V. owensii and non-classified isolates of the Harveyi clade (Figure 2B). In conclusion, V. harveyi appears as a virulent species that colonizes oysters during a POMS event. Again, this result paralleled previous knowledge on the preferential association of virulent species to diseased oysters during Atlantic POMS episodes (Bruto et al, 2016). Still, the virulence potential does not always correlate with the colonization potential among species of the Harveyi clade. Indeed, V. rotiferianus isolates, that harbors low to moderate virulence, colonized oysters better than the virulent V. jasicida isolates (Figure 2A).

OsHV-1 μvar synergizes with vibrios of the Harveyi clade to kill oysters

We tested experimentally the synergy between species of the Harveyi clade and OsHV-1 µvar to kill oysters. For that, we performed a mesocosm experiment that enables to test infection with different pathogen combinations while using natural infection routes. Thus, oysters susceptible to the POMS were immersed into seawater containing either a reduced representation of the Harveyi phylogenetic and phenotypic diversity found in the field, or

OsHV-1 μvar alone, or both. The bacterial community was composed of virulent isolates of V.

harveyi (mortality rates 28-51%), isolates of V. rotiferianus and V. jasicida with contrasting virulence (mortality rates 0-44%), and non-virulent isolates of V. owensii (mortality rate 0%) (Supplementary Table 3). Mortalities were monitored over 6 days and individual oysters were collected over 2 days, until the first mortalities occurred, to monitor the Vibrio load and population structure over disease development. The load of total Vibrio was monitored in oysters by qPCR as a general approximation to the Vibrio dynamic within oysters.

No mortalities were observed when oysters were immersed in seawater containing only the Harveyi community (Figure 3A), indicating that the community is not sufficient to cause disease by natural routes of infections. A transient increase in Vibrio load was observed from 4h to 24h followed by a decreased at 48h, revealing a control of Vibrio colonization. In contrast, immersion into OsHV-1 µvar-containing seawater was sufficient to induce significant mortalities in juvenile oysters (85% mortality at day 6) with a progressive increase in Vibrio load observed over the first 2 days (Figure 3). The same phenomenon was earlier observed when pathogenic V. crassostreae (Atlantic isolate J2-9) was used to infect oysters in the presence/absence of OsHV-1 µvar (de Lorgeril et al., 2018). Remarkably, mortalities were significantly enhanced (100% at day 4) when the Harveyi community and OsHV-1 µvar were combined into the mesocosm, as revealed by Kaplan-Meier survival curves (p=0.0018, Figure

androgen-induced gene

SH3 and multiple ankyrin repeat domains 3-like isoform X3

leucine-rich repeat and fibronectin type III domain-containing 1 We focused here on population #23 taxonomically assigned to the species V. splendidus Pop#23). This population presented a moderate virulence potential compared to populations of V. crassostreae or the closely related but collected from summer season and carrier of the plasmid pGV1512, population V. splendidus #24. All the strains of Pop#23 were cytotoxic towards hemocytes in vitro including the strain 4G1_4 considered non-virulent (oyster survival >82% six days after injection). This strain was characterized by the absence of two loci coding for effector delivery systems, a MARTX complex and a T6SS that we show to be linked to an even higher survival of injected oysters when invalidated in a moderately virulent strain, but had no effect on cytotoxicity. We have not gone further in the frame of article 1 into the characterization of the cytoxicity determinants of V. splendidus. However, it

GENERAL CONCLUSION

The objective of the present thesis was to characterize the mechanisms underpinning the association of the oyster Crassostrea gigas with vibrio populations in health and disease.

Oysters possess a remarkable ability to maintain an immune homeostasis in presence of an abundant and diverse microbiota, which paradoxically is in close contact with immunocompetent tissues (Schmitt et al. 2012). In this state, preferential and positive associations of vibrio populations can be sustained within oysters without the development of disease. However, abiotic and biotic stressors acting on the oyster holobiont (the oyster and its associated microbiota) can trigger a dysbiosis in which the proliferation of opportunistic vibrio species leads to oyster death ( de Lorgeril, et al. 2018). In this thesis we have been interested in characterizing these two states, which question both the tolerance of the oyster immune system and its manipulation by vibrios.

The study of the homeostatic state led us to characterize a V. splendidus population associated with healthy oysters (article 1). We identified this population as moderately virulent, and found that the O-antigen, a major membrane determinant of Gram-negative bacteria, is subjected to important selection pressures in V. splendidus, which are exerted by both the host immune system and environmental predators (grazers). We indeed found that O-antigen structures that favor resistance to environmental predators result in an increased activation of the oyster immune system and a reduced virulence in that host. Our results suggest 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. We found that not only Vibrio species are able to modulate their O-antigen structure to reduce immunogenicity but some species are also rather resistant to the host immune effectors.

However, by comparing Vibrio species positively and negatively associated with oyster tissues we found that resistance to host antimicrobial defenses is not sufficient to colonize a healthy oyster.

In parallel, we investigated the association of vibrios when homeostasis between the immunity and the microbiota of the oyster is disrupted. We focused on several vibrio populations associated with the Pacific Oyster Mortality Syndrome. We first developed a comparative study of the mechanisms of virulence in two species V. crassostreae and V.