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Par Madame Fatalmoudou TANDINA AIX-MARSEILLE UNIVERSITE FACULTE DE MEDECINE DE MARSEILLE ECOLE DOCTORALE DES SCIENCES DE LA VIE ET DE LA SANTE THÈSE DE DOCTORAT Présentée publiquement et soutenue devant LA FACULTE DE MEDECINE DE MARSEILLE Le 05 Juillet 2018͒ Par Madame Fatalmoudou TANDINA MISE AU POINT ET APPLICATION DE TECHNOLOGIES INNOVANTES POUR L'ETUDE DES MOUSTIQUES, DE LEUR PREFERENCE TROPHIQUE ET DE LEUR MICROBIOTE Pour obtenir le grade de DOCTEUR d’AIX-MARSEILLE UNIVERSITE Spécialité : Pathologies Humaines - Maladies Infectieuses Membres du Jury de la Thèse : Vincent ROBERT Rapporteur Pascal DELAUNAY Rapporteur Christelle DESNUES Présidente du Jury Ogobara K DOUMBO Examinateur Philippe PAROLA Directeur Laboratoire d’accueil Unité Mixte de Recherche Vecteurs – Infections Tropicales et Méditerranéennes 19-21 boulevard Jean Moulin, 13005 Marseille, IHU Méditerranée Infection 2018 1 AVANT PROPOS Le format de présentation de cette thèse correspond à une recommandation de la spécialité Maladies Infectieuses et Microbiologie, à l’intérieur du Master des Sciences de la Vie et de la Santé qui dépend de l’Ecole Doctorale des Sciences de la Vie de Marseille. Le candidat est amené à respecter des règles qui lui sont imposées et qui comportent un format de thèse utilisé dans le Nord de l’Europe et qui permet un meilleur rangement que les thèses traditionnelles. Par ailleurs, la partie introduction et bibliographie est remplacée par une revue envoyée dans un journal afin de permettre une évaluation extérieure de la qualité de la revue et de permettre à l’étudiant de commencer le plus tôt possible une bibliographie exhaustive sur le domaine de cette thèse. Par ailleurs, la thèse est présentée sur article publié, accepté ou soumis, associé d’un bref commentaire donnant le sens général du travail. Cette forme de présentation a paru plus en adéquation avec les exigences de la compétition internationale et permet de se concentrer sur des travaux qui bénéficieront d’une diffusion internationale. Pr Didier RAOULT 3 REMERCIEMENTS Ce travail de thèse a été réalisé à l’Institut Hospitalo-Universitaire Méditerranée Infection de Marseille au sein de l’unité VITROME. Je tiens à remercier le Professeur Didier RAOULT pour m’avoir financée et accueillie au sein de son laboratoire durant ce travail de thèse. Je tiens à remercier chaleureusement le Professeur Philippe PAROLA, mon directeur de thèse pour m’avoir fait découvrir le domaine de l’entomologie médicale. Son encadrement, sa rigueur scientifique et sa patience m’ont permis de réaliser ce travail. Je voudrais exprimer ma profonde gratitude au Professeur Ogobara DOUMBO, pour m’avoir initié à la recherche. Sa disponibilité, son soutien et ses conseils qui m’ont permis de mener à bien mes travaux de recherche. Je voudrais exprimer ma reconnaissance au Docteur Lionel ALMERAS pour le soutien qu’il m’a apporté au début de ma thèse. Je voudrais remercier également le Professeur Vincent ROBERT pour son aide durant cette dernière année de thèse, qui a été pour moi une belle expérience. Je lui remercie pour sa rigueur scientifique et sa disponibilité constante qui m’ont permis de bien achever ce travail. Je n’oublie pas le Monsieur Jean-Michel BERENGER, pour son aide précieuse au sein de l’insectarium et le partage de sa passion pour l’entomologie avec nous. Mes remerciements s’adressent également aux membres de mon jury : Madame le Docteur Christelle DESNUES qui me fait le très grand honneur de présider le jury de cette thèse. Qu'elle trouve ici l'expression de ma parfaite reconnaissance et mes vifs remerciements. Monsieur le Professeur Vincent ROBERT pour avoir accepté de juger mon travail. 5 Sincères remerciements au Professeur Pascal DELAUNAY qui a accepté d’évaluer mon travail. Veuillez trouver ici l’expression de ma profonde reconnaissance. Je remercie chaleureusement l’ensemble de mes collègues pour leur soutien et leur collaboration dans l’achèvement de ce travail. Je ne saurais terminer mes propos sans adresser un grand merci à tous ceux qui ont contribué, de près ou de loin, à la réussite de ce travail de thèse. 7 DEDICACES Je dédie ce travail à mes parents pour leurs bons conseils et soutien. Mes frères, sœurs, oncles, tantes et cousins pour leurs encouragements indéfectibles. Mon mari pour sa présence et son aide durant ces années de thèse. Feu Professeur Ogobara DOUMBO qui avait co-encadré ce travail. 9 SOMMAIRE Résumé………………………………………………………………………………………. 12/13 Abstract……………………………………………………………………………………… 14/15 I. INTRODUCTION………………………………………………………………... 17 II. Revue de la littérature…………………………………………….……………… 21 Article1 : Tandina F, Doumbo O, Traoré SF, Yaro AS, Parola P, Robert V. (2018), Mosquitoes (Diptera: Culicidae) and mosquito-borne diseases in Mali, West Africa. Parasites & Vectors, IN REVISION ……………………………………………………………………………………….. 25 III. UTILISATION DU MALDI-TOF MS POUR L’IDENTIFICATION DES MOUSTIQUES ET DE LEUR REPAS SANGUIN…………………………………..67 Article 2: Tandina F, Niaré S, Laroche M, Koné AK, Diarra AZ, Ongoiba A, Berenger JM, Doumbo OK, Raoult D, Parola P. (2018), Using MALDI-TOF MS to identify mosquitoes collected in Mali and their blood meals. Parasitology, 7:1-13………………………………….69 Article 3: Tandina F, Laroche M, Davoust B, Doumbo OK, Parola P.(2018), Blood meal identification of cryptic species Anopheles gambiae Giles and Anopheles coluzzii using MALDI-TOF MS. Parasite IN REVISION……………………………………………… 87 ,Article 4: Tandina F֞ , Niare S֞ , Laroche M, Almeras L, Davoust B, Doumbo O, Raoult D Parola P. (2018), Identification of mixed and succevive blood meals of mosquitoes by MALDI-TOF MS protein profiling. IN PREPARATION…………………………………...... 115 IV. UTILISATION DU MALDI-TOF MS ET DE LA CULTUROMIQUE POUR ETUDIER LE MICROBIOTE DIGESTIF DES MOUSTIQUES………………….151 Article 5: Tandina F, Almeras L, Koné AK, Doumbo OK, Raoult D, Parola P. (2016), Use of MALDI-TOF MS and culturomics to identify mosquitoes and their midgut microbiota. Parasites & Vectors, 9 :495………………………………………..………….... 153 V. CONCLUSION GENERALE ET PERSPECTIVES…………………………... 169 10 REFERENCES BIBLIOGRAPHIQUES……………………………………………… 171 ANNEXES……………………………………………………………………………… 175 Article 6: Niaré S, Tandina F, Davoust B, Doumbo O, Raoult D, Parola P, Almeras L. (2017), Accurate identification of Anopheles gambiae Giles trophic preferences by MALDI-TOF MS. Infection, Genetics and Evolution, S1567-1348(17)30315-5…………………………….…… 177 Article 7: Niare S, Almeras L, Tandina F, Yssouf A, Bacar A, Toilibou A, Doumbo O, Raoult D, Parola P. (2017), MALDI-TOF MS identification of Anopheles gambiae Giles blood meal crushed on Whatman filter papers. Plos one, 12, e0183238……………………………… 189 Article 8: Bardet L, Cimmino T, Buffet C, Michelle C, Rathored J, Tandina F, Lagier JC, Khelaifia S, Abrahão J, Raoult D, Rolain JM. (2017), Microbial Culturomics Application for Global Health: Noncontiguous Finished Genome Sequence and Description of Pseudomonas massiliensis Strain CB-1T sp. nov. in Brazil. OMICS, doi: 10.1089/omi.2017.0027……… 207 11 Résumé Les moustiques sont les principaux vecteurs incriminés dans la transmission d’agents pathogènes à l’homme. L’identification précise des espèces de moustiques est importante pour distinguer les espèces vectrices des non vectrices. La détermination de l’origine du repas sanguin des moustiques vecteurs est indispensable dans la compréhension du comportement des espèces vectrices. Ces paramètres sont nécessaires pour la prévention du risque de transmission de maladie infectieuse. Etant originaire et destinée à travailler au Mali, nous avons décidé dans une première partie de ce travail de mettre à jour le répertoire des moustiques du Mali. Dans nos travaux originaux, nous avons travaillé à la mise au point et l’utilisation d’outils innovants comme le MALDI-TOF (Matrix Assisted Laser Desorption Ionisation - Time of Flight) pour l’identification des moustiques, de leur repas sanguin et de leur microbiote digestif. Le premier objectif était donc de faire la mise à jour de la littérature actuelle sur la faune Culicidienne du Mali, notamment les potentiels vecteurs de pathogènes. Nous avons également décrit les avancées dans la lutte contre les moustiques vecteurs et les outils innovants récents pour l'identification des espèces de moustiques utilisés au Mali. Ainsi, nous avons listé 106 espèces de moustiques actuellement enregistrée au Mali dont 28 Anophelinae et 78 Culicinae. Nous avons de plus mis en évidence l’introduction récente de deux espèces de moustiques invasives Aedes albopictus et Culex neavei. Le deuxième objectif était d'évaluer l’efficacité de la spectrométrie de masse MALDI-TOF pour identifier des moustiques collectés sur le terrain au Mali et déterminer leur source de repas sanguin. Ainsi, dans le cadre d’une enquête entomologique au Mali, huit espèces de moustiques ont été identifiées, dont Anopheles gambiae Giles, Anopheles coluzzii, Anopheles arabiensis, Culex quinquefasciatus, Culex neavei, Culex perexiguus, Aedes aegypti et Aedes fowleri. 12 De plus, l’identification de la source de repas de sanguin nous a montré une grande antropophilie de ces moustiques. Nous avons également effectué un travail expérimental pour confirmer la robustesse de la spectrométrie de masse MALDI-TOF pour identifier un grand nombre de sang animaux dans l’abdomen de moustiques gorgés. Au laboratoire, nous avons artificiellement gorgé des femelles de deux espèces (Anopheles gambiae et Anopheles coluzzii) sur différents types de sang d’animaux. Nous avons obtenu 100% d'identification correcte du repas de sang pour les spécimens collectés 1h à 24h après le
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