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Defence on Surface of Rhodophyta Halymenia Floresii THESE DE DOCTORAT DE UNIVERSITE BRETAGNE SUD ECOLE DOCTORALE N° 598 Sciences de la Mer et du littoral Spécialité : Biotechnologie Marine Par Shareen A ABDUL MALIK Defence on surface of Rhodophyta Halymenia floresii: metabolomic fingerprint and interactions with the surface-associated bacteria Thèse présentée et soutenue à « Vannes », le « 7 July 2020 » Unité de recherche : Laboratoire de Biotechnologie et Chimie Marines Thèse N°: Rapporteurs avant soutenance : Composition du Jury : Prof. Gérald Culioli Associate Professor Président : Université de Toulon (La Garde) Prof. Claire Gachon Professor Dr. Leila Tirichine Research Director (CNRS) Museum National d’Histoire Naturelle, Paris Université de Nantes Examinateur(s) : Prof. Gwenaëlle Le Blay Professor Université Bretagne Occidentale (UBO), Brest Dir. de thèse : Prof. Nathalie Bourgougnon Professor Université Bretagne Sud (UBS), Vannes Co-dir. de thèse : Dr. Daniel Robledo Director CINVESTAV, Mexico i Invité(s) Dr. Gilles Bedoux Maître de conferences Université Bretagne Sud (UBS), Vannes Title: Systèmes de défence de surface de la Rhodophycée Halymenia floresii : Analyse metabolomique et interactions avec les bactéries épiphytes Mots clés: Halymenia floresii, antibiofilm, antifouling, métabolomique, bactéries associées à la surface, quorum sensing, molecules de défense Abstract : Halymenia floresii, une Rhodophycée présente Vibrio owensii, ainsi que son signal C4-HSL QS, a été une surface remarquablement exempte d'épiphytes dans les identifié comme pathogène opportuniste induisant un conditions de l'Aquaculture MultiTrophique Intégrée (AMTI). blanchiment. Les métabolites extraits de la surface et Ce phénomène la présence en surface de composés actifs de cellules entières de H. floresii ont été analysés par allélopathiques. L'objectif de ce travail a été d'explorer les LC-MS. Une base de données a été constituée à partir mécanismes de défense développés par H. floresii contre d’une analyse métabolomique non ciblée. Quarante et l'épibiose, de détecter et d'identifier les métabolites un putatives actifs ont été identifiés, parmi lesquels les secondaires produits à la surface de l’algue et d'étudier les composés halogénés, des furanones et divers relations avec les bactéries épiphytes. Nous avons ainsi pu inhibiteurs étaient surreprésentés. Fait intéressant, isoler la communauté épibactérienne de H. floresii cultivée les deux premières classes sont connues comme de dans des conditions contrôlées (AMTI) et non contrôlées puissants composés interférant avec le QS. La (échantillons collectés in situ). Les épibactéries isolées ont présence relativement élevée de putatives été criblées in vitro pour analyser les signaux de détection allélopathiques à la surface de H. floresii soutient de Quorum Sensing (QS). Lesextraits produits en surface fortement l'hypothèse selon laquelle ils doivent être ont été analysés pour détecter toute interférence avec le impliqués dans la protection de l'hôte. Des recherches Quorum Sensing. Les épibactéries pathogènes et non- supplémentaires seront nécessaires pour explorer pathogènes ont été différenciées par leur capacité à induire l’ensemble des métabolites secondaires produits par une maladie algale, le blanchiment. H. floresii et leurs rôles chez l’algue. Title: Defence on surface of Rhodophyta Halymenia floresii: metabolomic fingerprint and interactions with the surface-associated bacteria Keywords: Halymenia floresii, antibiofilm, antifouling, metabolomics, surface-associated bacteria, quorum sensing, defence molecules Abstract: The surface of Halymenia floresii, a Mexican Vibrio owensii was identified as an opportunistic Rhodophyta, was observed to be remarkably free of pathogen inducing bleaching in H. floresii which was epiphytes under Integrated MultiTrophic Aquaculture also associated to the presence of its C4-HSL QS (IMTA) conditions. This suggests the presence of signal. The surface and whole cell metabolites allelopathic active compounds released by this extracts from H. floresii specimens cultivated under macroalgae. The aim of this work was to explore the controlled conditions were analysed by means of defence mechanisms developed by H. floresii against LC/MS. An untargeted metabolomic analysis of H. surface epibiosis, to detect and identify the secondary floresii was performed to provide a global metabolic metabolites produced at the surface of the alga, and to fingerprint as a first baseline. We identified ‘41’ active study its relation with surface associated bacteria. For the putatives in H. floresii, among which halogenated first time, we isolated the epibacterial community of H. compounds, furanones and various inhibitors were floresii cultivated under controlled conditions (IMTA) and overrepresented. Interestingly, the first two classes uncontrolled ones (beach-cast material collected in the are well known potent QS interfering compounds. area). The isolated epibacteria were screened in vitro to The relatively higher occurrences of allelopathic analyse Quorum Sensing (QS) signals, and others H. putatives at the surface of H. floresii strongly floresii surface extracts were assayed for any QS supports the hypothesis that they must be involved in interference with them. We differentiated the epibacteria the host protection. Further investigations are significant pathogens from the non-pathogens ones by their needed to explore the secondary metabolites of H. ability to induce bleaching, a well-known algal disease. floresii and their role in the seaweed. ii Defence on surface of Rhodophyta Halymenia floresii: metabolomic profile and interactions with its surface-associated bacteria Shareen Arockiasamy 2020 iii Defence on surface of Rhodophyta Halymenia floresii: metabolomic profile and interactions with its surface-associated bacteria Shareen Arockiasamy 2020 Empty your mind, be formless, shapeless — like water. Now you put water in a cup, it becomes the cup; You put water into a bottle it becomes the bottle; You put it in a teapot it becomes the teapot. Now water can flow or it can crash. Be water, my friend. - Bruce Lee Be careful what you water your dreams with. Water them with worry and fear and you will produce weeds that choke the life from your dream. Water them with optimism and solutions and you will cultivate success. Always be on the lookout for ways to turn a problem into an opportunity for success. Always be on the lookout for ways to nurture your dream - Lao Tzu iv Defence on surface of Rhodophyta Halymenia floresii: metabolomic profile and interactions with its surface-associated bacteria Shareen Arockiasamy 2020 Acknowledgements Wow... Expressing gratitude is such a greatest human emotion and it’s very exciting to start acknowledging the BEings who have been and/or being with me throughout my PhD. This period of ‘3’ years in my life gave me a lot of people and lots and lots of wonderful experiences. And I am very grateful for it. Firstly, I sincerely thank Prof. Gérald Culioli, Associate Professor of the Laboratory of Matériaux Polymères Interfaces Environnement Marin (MAPIEM), Université de Toulon (La Garde) and Dr. Leila Tirichine, Research Director of the Epigénomique des microalgues et interactions avec l’environment, Université de Nantes, for being rapporteurs and examining the work. I also thank other members of jury Prof. Claire Gachon, Professor, Museum National d’Histoire Naturelle and Prof. Gwenaëlle Le Blay, Professor, Université Bretagne Occidentale (UBO) for equally participating in examining my work. I profoundly thank my doctoral school, Ecole Doctorale Sciences de la Mer et du Littoral (EDSML) for constantly supporting throughout my thesis and granting me scholarship for my international mobility. I also thank ECOS-Nord CONACYT for their financial support towards my research stay in Mexico. I sincerely thank GlobalSeaweedSTAR for their financial help towards attending the conference of ISS, 2019, Korea. Well, my Directors of the thesis... Words are not enough to express my deep gratitude to Prof. Nathalie Bourgougnon, firstly for the offering me this wonderful opportunity to pursue my PhD under her wonderful supervision. Her immense knowledge always influenced me to think critically and develop my ideas. I always admired her way of valuing others TIME equally as she values her own. I could not have imagined having a better supervisor for my PhD study. I equally thank my Co-director Dr. Daniel Robledo for sharing his knowledge on his all-time favourite, macroalgae. I really appreciate his guidance in writing the publications for his insightful comments which incented me to widen my perspectives of the subject. Nothing would have been possible without their continuous support to my PhD. My sincere thanks also go to Dr. Gilles Bedoux for his valuable comments and guidance in the chemistry part of the research and his advice on chromatographic analyses is highly valuable. I greatly appreciate his deep knowledge on the subject which really ignited my interest in the field of chemistry. I extend my gratitude to Dr. José Q. Garcia Maldonado for his guidance and comments in the molecular biology part of the research. His direction really helped me a lot and earned me a confidence in the field of molecular biology. I also gratefully acknowledge Prof. Alain Dufour and Dr. Alexis Bazire for their guidance and views in the quorum sensing related work of the research. I also sincerely thank Dr. Bazire for offering me v Defence on surface of Rhodophyta Halymenia floresii: metabolomic profile and interactions with
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