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Ontogenèse Du Microbiote Chez Le Poisson Vivipare Brachyistius Frenatus

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Ontogenèse Du Microbiote Chez Le Poisson Vivipare Brachyistius Frenatus Ontogenèse du microbiote chez le poisson vivipare Brachyistius frenatus. Transmission verticale de symbiotes microbiens pionniers? Mémoire Aurélie Boilard Maîtrise en biologie - avec mémoire Maître ès sciences (M. Sc.) Québec, Canada © Aurélie Boilard, 2021 Ontogenèse du microbiote chez le poisson vivipare Brachyistius frenatus. Transmission verticale de symbiotes microbiens pionniers? Mémoire Aurélie Boilard Sous la direction de : Nicolas Derome, directeur de recherche Résumé Chez les Mammifères, le recrutement du microbiote débute in utero, ce qui restait à démontrer chez d'autres classes de Vertébrés. L’objectif général du projet était de tester si un tel recrutement se produit chez un Vertébré non Mammifère. Nous avons testé, chez le Poisson vivipare Brachyistius frenatus, l'hypothèse selon laquelle la poche utérine est colonisée par un microbiote transmissible aux alevins, conférant à leur propre microbiote une ontogenèse semblable à celle des Mammifères. Le projet visait l’atteinte des objectifs suivant : i) caractériser le mode de transmission du microbiote, ii) établir la composition, la diversité et les relations des communautés bactériennes du microbiote des femelles, des juvéniles et de leur environnement et iii) déterminer l’ontogenèse du microbiote chez B. frenatus. Ce projet a permis de caractériser le mode de transmission du microbiote, sa séquence de recrutement, ainsi que la contribution respective de différentes communautés sources en caractérisant la diversité bactérienne du microbiote des femelles, des juvéniles et de leur environnement avec une approche métagénomique de type code barre. La région V4 du gène de l'ARNr 16S a été ciblée comme marqueur taxonomique bactérien pour identifier les taxons des différents échantillons. Cette étude nous a permis d’identifier le premier cas d’une transmission verticale du microbiote in utero chez un vivipare non Mammifère et les résultats sous- entendent que B. frenatus est peut-être un tout nouveau modèle d’ontogenèse du microbiote. Cette étude a permis l’acquisition des connaissances sur la transmission du microbiote et, dans le contexte de convergence évolutive de la viviparité, elle ouvre à de nouvelles perspectives quant aux avantages évolutifs d'une telle transmission de symbiotes microbiens. ii Abstract In Mammals microbial recruitment starts in utero, something that had not been shown in any other Vertebrate class. The main goal of this project was to test whether this type of recruitment happens in a non-mammalian Vertebrate. We tested in the viviparous fish Brachyistius frenatus the hypothesis under which the uterine pouch is colonized by a microbiome transmissible to the juveniles, conferring them an ontogeny similar to Mammals. This project also aimed to i) characterize the mode of transmission of the microbiota, ii) establish the composition, diversity and relationships between the microbial communities of pregnant females, juveniles and their environment and iii) determine the ontogeny of the microbiota in B. frenatus. We characterized the mode of transmission of the microbiome, explored its recruitment and the contribution of different source communities with a metagenomic approach (bar coding). We targeted the hyper variable region V4 of the small subunit (16S) rRNA gene to determine the presence of a vertical transmission of the microbiome In this study, we confirmed the presence of a vertically transmissible microbiome in the viviparous fish B. frenatus. We documented for the first time an in utero transmission of the microbiota in a non-mammalian viviparous species. Our results also hint that B. frenatus might be a new model of microbiota ontogeny. This study contributes to the acquisition of knowledge on microbiome transmission and, in the context of evolutionary convergence of viviparity, allows the formulation of hypotheses concerning the evolutionary advantages of in utero microbiome transmission. iii Table des matières Résumé ................................................................................................................................................ ii Abstract ............................................................................................................................................... iii Table des matières .............................................................................................................................. iv Glossaire .............................................................................................................................................. v Remerciements .................................................................................................................................. vii Avant-propos .................................................................................................................................... viii Introduction ......................................................................................................................................... 1 1.1 Stratégies de reproductions chez les Vertébrés ......................................................................... 1 1.2 L’holobionte .............................................................................................................................. 2 1.2.1 L’importance du microbiote pour la santé de l’hôte ......................................................... 2 1.2.2 L’acquisition du microbiote chez les Mammifères ............................................................ 5 1.2.3 L’ontogenèse et stratégies d’acquisition initiale du microbiote ........................................ 6 1.3 Approche expérimentale ............................................................................................................ 8 1.4 Brachyistius frenatus, espèce modèle pour notre étude ............................................................ 9 1. 5 Objectifs et hypothèses ........................................................................................................... 11 Chapitre 1 Viviparity : cutting edge strategy for microbiota ontogeny ............................................. 12 1.1 Résumé .................................................................................................................................... 12 1.2 Abstract .................................................................................................................................... 12 Introduction ................................................................................................................................... 13 Methodology .................................................................................................................................. 16 Sampling and dissections .......................................................................................................... 16 Bacterial DNA extraction and amplification ............................................................................ 16 Bioinformatic analyses ............................................................................................................. 17 Statistical analyses .................................................................................................................... 17 Results ........................................................................................................................................... 19 Discussion ...................................................................................................................................... 21 Conclusion ..................................................................................................................................... 27 Acknowledgements ....................................................................................................................... 28 Conflicts of Interests ...................................................................................................................... 28 References ..................................................................................................................................... 29 List of tables and figures ............................................................................................................... 33 Conclusion ......................................................................................................................................... 45 Bibliographie ..................................................................................................................................... 48 Annexe A : Supporting informations ................................................................................................ 58 iv Glossaire ARNr 16S Sous unité 16S du gène de l’ARN ribosomique mitochondrial (ARNr 16S). Fréquemment utilisé comme marqueur taxonomique bactérien puisque ces régions hyperconservées sont entourées de régions hypervariables, qui permettent l’identification des taxons bactériens jusqu’au genre ou à l’espèce. Acquisition horizontale du microbiote Acquisition de symbiotes microbiens par contact direct avec l’environnement. Acquisition verticale du microbiote Transmission de symbiotes microbiens d’un parent à sa progéniture. Dysbiose État de déséquilibre entre le microbiote et l’hôte, peut être causé par différents facteurs intrinsèques et extrinsèques, mène potentiellement à des maladies voir à la mort. Eubiose État d’équilibre au sein de l’holobionte. Gonopodium Modification de la portion antérieure de la nageoire anale chez
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