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

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

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.

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.

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 gorgement. Ensuite nous avons expérimentalement gorgés trois espèces de moustiques (Anopheles gambiae, Anopheles coluzzii et Aedes albopictus) sur des repas de sang successif et mixte, pour voir si l’espèce de moustique avait un impact sur l’identification du repas de sang par MALDI- TOF MS. Nos résultats révèlent que le MALDI-TOF MS est tout à fait capable d’identifier le repas mixte. Mais en ce qui concerne le repas successif seul le dernier repas de sang est identifié. Cela est peut être du à l’intervalle de jours (trois) qui sépare les deux repas de sangs des moustiques. Donc le premier repas de sang a peut être été digéré par les moustiques. Le troisième objectif de ce travail était d’utiliser la culturomique et le MALDI- TOF pour l’étude du microbiote digestif de moustiques collectés sur le terrain au Mali et à Marseille. Cette approche a révélé une grande diversité du microbiote digestif des moustiques Anopheles gambiae, Aedes albopictus et Culex quinquefasciatus. La majorité de ces bactéries étaient des Grams négatifs et appartiennent au phylum des Protéobacteria. De plus, une nouvelle espèce bactérienne, Pseudomonas massiliensis, a été isolée dans l’estomac des moustiques Anopheles gambiae et Culex quinquefasciatus et leurs eaux de gîte collectées au Mali.

Mots clés : Moustiques, vecteurs, MALDI-TOF MS, Mali

13 Abstract

The first objective of our study was to update the current literature on Malian Culicidan fauna, potential vectors and pathogens transmitted. We also wished to describe the progress made in mosquito vector control and the innovative tools recently used for mosquito identification. Thus, we have listed 106 mosquito species currently recorded in Mali, including 28 Anophelinae and 78 Culicinae. We have also highlighted the recent introduction of two invasive mosquito species, Aedes albopictus and Culex neavei.

The second objective was to evaluate the effectiveness of MALDI-TOF MS for identified mosquitoes collected in Mali and to determine their blood meal source. The results obtained show the ability of MALDI-TOF MS to identify mosquitoes collected in Mali and their source of blood meal. Thus, eight mosquito species has been identified, including Anopheles gambiae Giles, Anopheles coluzzii, Anopheles arabiensis, Culex quinquefasciatus, Culex neavei, Culex perexiguus, Aedes aegypti and Aedes fowleri. In addition, the identification of the blood meal source matched with human blood (n = 619), chicken blood (n = 9), cow's blood (n = 9), donkey blood (n = 6), dog's blood (n = 5) and sheep blood (n = 3).

14 Subsequently, we were able to confirm the robustness of mass spectrometry MALDI-TOF to identify other animal blood samples. We artificially engorged two mosquito species including, Anopheles gambiae and Anopheles coluzzii on eight animal bloods samples. We obtained 100% correct identification of the blood source for samples taken 1 to 24 hours after feeding. Then, we experimentally engorged three mosquito species including Anopheles gambiae, Anopheles coluzzii and Aedes albopictus on successive and mixed blood meals, to see whether the mosquito species had an impact on the blood meal identification using MALDI-TOF MS. The results revealed that MALDI-TOF MS is able to identify mixed blood meals. This may be due to the interval of days (three) between the first and second blood meals of mosquitoes. The first blood meal was indeed digested by the mosquitoes. The third objective of our study was to use MALDI-TOF and culturomics for the microbiota study of the mosquito collected in the field, notably in Marseille and Mali. The culturomics approach revealed a great diversity of the digestive microbiota of the Anopheles gambiae, Aedes albopictus and Culex quinquefasciatus mosquitoes. The majority of bacteria detected in the microbiota of mosquitoes was gram-negative and belong to the Proteobacteria phylum. In addition, a new bacterial species, Pseudomonas massiliensis, was isolated from the Anopheles gambiae and Culex quinquefasciatus microbiota and their breeding waters were collected in Mali.

Key words: Mosquitoes, vectors, MALDI-TOF MS, Mali

I. INTRODUCTION

Les moustiques (Diptera : Culicidae) sont des insectes diptères (Becker N et al. 2010). La distribution des moustiques s’étend à tous les continents, y compris l'Antarctique. La famille des Culicidae comprend actuellement 3 557 espèces valides de moustiques réparties entre les sous-familles Culicinae et Anophelinae (Harbach RE 2013). Les moustiques vecteurs appartiennent principalement aux trois genres Anopheles, Aedes et Culex qui sont responsables de la transmission de nombreux parasites, virus et bactéries pathogènes (Becker N et al. 2010; Dieme et al. 2015; Gould et al. 2017). De nouvelles maladies infectieuses peuvent émerger suite à la colonisation de nouvelles zones écologiques par les vecteurs. L’exemple le plus récent est celui du moustique tigre Aedes (Stegomyia) albopictus (Skuse, 1894) qui était autre fois asiatique mais maintenant se trouve sur la plus part des continents du monde (Muller et al. 2016; Reis et al. 2017). Pour mieux lutter contre les moustiques vecteurs, il est important d'améliorer nos connaissances sur la présence des espèces dans une zone donnée. C’est le travail que nous avons entrepris concernant la faune du Mali (Article1 : Revue de la littérature). Il existe plusieurs méthodes d’identification des moustiques durant les enquêtes de surveillance entomologique sur le terrain. La première méthode est l’identification morphologique basée sur les critères morphologiques en utilisant les clés d’identification. L’utilisation des clés dichotomiques est une stratégie difficile qui nécessite une expertise entomologique considérable. Il est difficile de discriminer les espèces cryptiques, telles que les Anopheles coluzzii et Anopheles gambiae Giles (Gillies MT and Coetzee M 1987). La biologie moléculaire a été utilisée pour la distinction de ces formes cryptiques Anopheles gambiae s.s, et aussi de certaines espèces du complexe Culex pipiens qui sont difficiles a distinguer par la morphologie (Smith and Fonseca 2004). Les limites de la biologie moléculaire comprennent son coût, la nécessité d’un plateau technique adapté et le temps de réalisation.

17 La spectrométrie de masse MALDI-TOF (Matrix Assisted Laser Desorption Ionisation - Time of Flight) est une stratégie protéomique récemment utilisée en routine dans la microbiologie médicale, qui a émergée comme outil dans l’identification des arthropodes (Yssouf et al. 2016). Cet outil a été expérimenté avec des résultats prometteurs pour l’identification des moustiques adultes à partir de l’extrait protéique des pattes des adultes (Yssouf et al. 2013), mais aussi à partir des stades aquatiques, œufs et larves (Dieme et al. 2014; Nebbak et al. 2017; Schaffner et al. 2014). Récemment, elle a été utilisée à partir de spécimen d’arthropodes collectés sur le terrain au cours d’enquêtes de surveillance entomologiques (Sambou et al. 2015; Yssouf et al. 2014). Dans la première partie de mes travaux de thèse, nous avons fait une application de terrain de la technologie MALDI-TOF MS en premier lieu pour identifier les moustiques et ensuite pour déterminer le repas de sang des moustiques (Article2) collectés au Mali. Nous avons aussi confirmé la robustesse de cette technologie à pouvoir identifier le repas de sang des moustiques par l’utilisation d’un large panel de sang d’animaux domestiques et sauvages (Article3). Nous avons par la suite utilisé le MALDI-TOF MS pour identifier le repas de sang successif et mixte de trois espèces de moustiques (Anopheles gambiae Giles, Anopheles coluzzii et Aedes albopictus) que nous avons en élevage dans notre laboratoire. Ensuite, nous avons observés si l’espèce de moustique avait un impact sur l’identification du repas de sang (Article 4). Dans la deuxième partie, nous avons utilisé la technique culturomique et la spectrométrie de masse MALDI-TOF pour étudier le microbiote digestif des moustiques (Article5). La culturomique est une nouvelle technique de culture utilisée pour l’étude du microbiote humain (Lagier et al. 2012). Nous l’avons adaptée à l’étude du microbiote digestif des moustiques collectés sur le terrain notamment dans la ville de Marseille et au Mali.

18 Actuellement le rôle du microbiote des vecteurs (Anopheles, Aedes et Culex) est un nouveau champ de recherche entomologique qui permettra de comprendre les interactions vecteurs/pathogènes et élaborer des stratégies de lutte anti vectorielle (Boissiere et al. 2012; Minard et al. 2013; Tchioffo et al. 2016). Ces dernières années, nous assistons à une augmentation des études axées sur le microbiote de diverses espèces de moustiques et son influence potentielle sur leur compétence vectorielle (Minard et al. 2013; Ricci et al. 2012). Certaines études ont montré l'impact du microbiote digestif des moustiques dans la défense contre les parasites du paludisme, en effet certaines bactéries de la famille des Enterobacteriaceae affectent le développement de Plasmodium falciparum dans l'estomac Anopheles gambiae (Tchioffo et al. 2016). De plus, d'autres études ont suggéré un rôle protecteur des bactéries du microbiote digestif Anopheles gambiae contre les infections plasmodiques (Tchioffo et al. 2016). L'approche culturomique a révélé une grande diversité du microbiote digestif des moustiques Anopheles gambiae (souche sauvage et de laboratoire), Aedes albopictus (souche sauvage et de laboratoire) et Culex quinquefasciatus (souche sauvage). La majorité des bactéries trouvées dans le microbiote des moustiques étaient des Gram-négatifs et appartiennent au phylum des Proteobacteria. La culturomique nous a permis d’isoler 17 nouvelles espèces bactériennes identifiées pour la première fois dans le microbiote digestif de l’espèce Anopheles gambiae. Ensuite nous avons isolé une nouvelle espèce de bactérie dans l’estomac des espèces Anopheles gambiae et Culex quinquefasciatus et dans leurs eaux de gîtes que nous avons nommé Pseudomonas massiliensis. Cette étude montre que l'environnement peut jouer un rôle important dans la diversité bactérienne des moustiques. La technique culturomique et la spectrométrie de masse MALDI-TOF sont des outils prometteurs dans l’étude des moustiques et leur microbiote digestif. Durant mon travail de thèse j’ai participé à d’autres travaux. Ils sont présentés en annexe et ne seront pas commentés.

II. REVUE DE LA LITTERATURE

ARTICLE 1

Mosquitoes (Diptera :Culicidae) and mosquito-borne disease in Mali,

West Africa

Tandina F, Doumbo O, Traoré SF, Yaro AS, Parola P, Robert V. (2018)

Les maladies transmises par les moustiques provoquent un problème de santé publique dans presque toutes les parties du monde. En Afrique de l'Ouest, notamment au Mali, les mesures de lutte anti-vectorielle contribuent à réduire l'impact des maladies transmises par les moustiques, bien que le paludisme reste une menace à la fois pour la morbidité et la mortalité. L'article le plus récent sur le répertoire des moustiques du Mali a été publié en 1961 avec 88 espèces au total. Cette revue se concentre sur les moustiques d'importance médicale parmi lesquels les vecteurs (Anopheles) de Plasmodium et Filaria ainsi que les moustiques vecteurs (Culex et Aedes) des arbovirus. Notre revue fournit une mise à jour concise de la littérature sur les Culicidae au Mali couvrant les zones écologiques dans lesquelles ils se trouvent, les pathogènes transmis et les outils innovants récents pour les enquêtes sur les vecteurs. Elle met en évidence l'introduction récente d'espèces de moustiques envahissantes telles que Aedes albopictus et Culex neavei au Mali. La liste complète des espèces de moustiques actuellement enregistrées au Mali comprend 106 espèces (28 Anophelinae et 78 Culicinae). Il y a probablement un manque de connaissances concernant les moustiques de la sous-famille des Culicinae de la moitié nord du Mali car la plupart des recherches ont été effectuées sur le genre Anopheles et ont eu lieu dans la partie sud du pays. Cette revue pourrait être utile aux décideurs responsables des stratégies de lutte anti-vectorielle et aux chercheurs pour de futures enquêtes sur les moustiques, en particulier les vecteurs des arbovirus émergents.

Parasites & Vectors Mosquitoes (Diptera: Culicidae) and mosquito-borne diseases in Mali, West Africa --Manuscript Draft--

Manuscript Number: PARV-D-18-00181R1

Full Title: Mosquitoes (Diptera: Culicidae) and mosquito-borne diseases in Mali, West Africa Article Type: Review

Section/Category: Dipteran Vectors and Associated Disease

Funding Information: A*MIDEX project Dr. Philippe Parola (ANR-10-IAHU-03)

Abstract: Mosquito-borne diseases cause significant human diseases in almost every part of the world. In West Africa, notably in Mali, vector control measures are contributing towards decreasing the impact of mosquito-borne diseases, although malaria remains a threat to both morbidity and mortality. The most recent overview article on mosquitoes in Mali was published in 1961 with 88 species in total. This review focuses on mosquitoes of medical importance among which the Anopheles vectors of Plasmodium and filaria, as well as the Culex and Aedes mosquito vectors of arboviruses. It aims at providing a concise update of the literature on Culicidae in Mali covering the ecological areas in which it is found, the pathogens transmitted, and recent innovative tools for vector surveys. It highlights the recent introduction of invasive mosquito species such as Aedes albopictus and Culex neavei in Mali. The comprehensive list of mosquito species currently recorded in Mali includes 106 species (28 Anophelinae and 78 Culicinae). There are probable lacks of knowledge concerning mosquitoes of the subfamily Culicinae and northern half of Mali because most researches have been carried out on the Anopheles genus and have taken place in the southern part of the country. It is hoped that this review may be useful to decision makers responsible for vector control strategies and to researchers for future surveys on mosquitoes, particularly the vectors of emerging arboviruses.

Corresponding Author: Philippe Parola IHU Mediterranee Infection Marseille, Marseille FRANCE

Corresponding Author Secondary Information:

Corresponding Author's Institution: IHU Mediterranee Infection Corresponding Author's Secondary Institution:

First Author: Fatalmoudou Tandina First Author Secondary Information:

Order of Authors: Fatalmoudou Tandina Ogobara Doumbo Alpha Seydou Yaro Sékou F Traoré Philippe Parola Vincent Robert

Order of Authors Secondary Information:

Response to Reviewers: The authors' response letter has been included as a supplementary file

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1 Mosquitoes (Diptera: Culicidae) and mosquito-borne diseases in Mali, West Africa 1 2 2 Fatalmoudou Tandina1,2, Ogobara Doumbo2, Alpha Seydou Yaro2, Sékou F. Traoré2, Philippe 3 4 1 3 5 3 Parola * and Vincent Robert 6 7 4 8 9 1 10 5 Aix Marseille Univ, IRD, AP-HM, SSA, VITROME, IHU-Méditerranée Infection, 11 12 6 Marseille, France. 13 14 7 2 Department of Epidemiology of Parasitic Diseases, Malaria Research and Training Center, 15 16 17 8 Faculty of Sciences and Techniques, University of Science, Techniques and Technologies of 18 19 9 Bamako, Bamako, Mali. 20 21 3 22 10 MIVEGEC Unit, IRD-CNRS-Univ. Montpellier, Montpellier, France. 23 24 11 25 26 27 12 * Corresponding author: Prof. Philippe Parola. VITROME, Institut Hospitalo-Universitaire 28 29 30 13 Méditerranée Infection, 19-21 Boulevard Jean Moulin 13385 Marseille Cedex 05, France. 31 32 14 Phone: + 33 (0) 4 13 73 24 01. Fax: + 33 (0) 4 13 73 24 02. 33 34 35 36 15 E-mail addresses: FT: [email protected]; OD: [email protected]; ASY: 37 38 16 [email protected]; SFT: [email protected]; PP: [email protected]; 39 40 41 17 VR: [email protected] 42 43 44 45 18 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 1 63 26 64 65 19 Abstract 1 2 20 Mosquito-borne diseases cause major human diseases in almost every part of the world. In 3 4 5 21 West Africa and notably in Mali, vector control measures help reduce the impact of mosquito- 6 7 22 borne diseases, although malaria remains a threat to both morbidity and mortality. The most 8 9 10 23 recent overview article on mosquitoes in Mali was published in 1961, with a total of 88 11 12 24 species. Our present review focuses on mosquitoes of medical importance among which the 13 14 25 Anopheles vector of Plasmodium and filaria, as well as the Culex and Aedes vectors of 15 16 17 26 arboviruses. It aims to provide a concise update of the Culcidae literature covering the 18 19 27 ecological areas in which it is found but also the transmitted pathogens and recent innovative 20 21 22 28 tools for vector surveys. This review highlights the recent introduction of invasive mosquito 23 24 29 species, including Aedes albopictus and Culex neavei. The comprehensive list of mosquito 25 26 27 30 species currently recorded includes 106 species (28 Anophelinae and 78 Culicinae). There are 28 29 31 probable lacks of knowledge concerning mosquitoes of the subfamily Culicinae and northern 30 31 32 half of Mali because most researches have been carried out on the Anopheles genus and have 32 33 34 33 taken place in the southern part of the country. It is hoped that this review may be useful to 35 36 34 decision makers responsible for vector control strategies and to researchers for future surveys 37 38 39 35 on mosquitoes, particularly the vectors of emerging arboviruses. 40 41 36 42 43 44 37 Keywords: Aedes, Anopheles, Culex, vector 45 46 38 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 2 63 27 64 65 39 Background 1 2 40 Mosquito vectors can transmit several pathogens, including arboviruses, Protozoa and filaria 3 4 5 41 that cause infectious diseases of significant public health concern [1]. To a lesser extent, they 6 7 42 may also transmit bacterial diseases [2]. Mosquitoes of medical importance belong to the 8 9 10 43 Culicidae family and are widely distributed around the world. This large family currently 11 12 44 encompasses 3,556 valid species [3] of mosquitoes distributed between the subfamilies 13 14 45 Culicinae and Anophelinae [4]. The mosquito vectors mainly belong to the three genera of 15 16 17 46 Anopheles, Aedes and Culex. 18 19 47 Anopheles mosquitoes have been continuously studied in Mali since 1906. The first detailed 20 21 22 48 work on mosquitoes in the French Sudan (former name of Mali) has been carried out by Le 23 24 49 Moal (1906) and Bouffard (1908) [5,6]. Since then, several studies have contributed towards 25 26 27 50 our understanding of this subject, but until 1950 there were only twelve known mosquito 28 29 51 species in the country: eight Anopheles spp, one Aedes sp, two Culex spp and one Mansonia 30 31 52 sp (Table 1). These data illustrate the distribution of Anopheles along the Niger River, with 32 33 34 53 some information on their preimaginal development sites and adult resting places. In 1961, 35 36 54 Hamon et al. considerably improved the repertory of the mosquitoes, taking into account 37 38 39 55 previous works and personal observations [7]. Their list contained 88 mosquitoes, including 40 41 56 20 Anophelinae and 68 Culicinae described in Table 1. Nevertheless, many of them only 42 43 44 57 existed in a few places, because the northern half of the country had not yet been surveyed. 45 46 58 Among the Anopheles species, Hamon recognized Anopheles (Cellia) gambiae, An. (Cellia) 47 48 funestus and An. (Cellia) nili as the main malaria vectors [7]. 49 59 50 51 60 Subsequently, Touré et al. carried out studies on the sensitivity of An. gambiae sensu lato 52 53 61 (s.l.) and An. funestus to insecticides and the rates of infection with malaria parasites and 54 55 56 62 filariae in the An. gambiae complex [8–10]. Malaria epidemiological studies have been 57 58 63 conducted by Doumbo et al. in the Malian Sahel to fill the data gap on malaria in that region. 59 60 61 62 3 63 28 64 65 64 The results showed that circulation of the malaria parasites takes place through two main 1 2 65 vectors, the Anopheles gambiae s.s. (chromosomal form Mopti) and Anopheles (Cel.) 3 4 5 66 arabiensis in northern Mali [11]. The Anopheles gambiae sensu stricto (s.s.) was the only 6 7 67 vector found in the far north of the country [12]. Several studies have been conducted on the 8 9 10 68 An. gambiae complex, including An. arabiensis and chromosomal forms of An. gambiae s.s. 11 12 69 targeting the differences in their human blood index (anthropophilic rate) as well as spatial 13 14 70 and seasonal distributions [13–16]. 15 16 17 71 The eco-climatic areas are classified into five facies, i.e., from north to south: the Saharan 18 19 72 zone, the Sahelian zone, the Sudano-Sahelian zone, the Sudanian zone and finally, the 20 21 22 73 Guinean zone (Fig. 1) [17]. In the different eco-climatic areas, the human malaria caused by 23 24 74 Plasmodium spp. continues to be responsible for deaths every year in Mali. This situation is 25 26 27 75 not new as the country has a long history of malaria as the leading cause of morbidity and 28 29 76 mortality, mainly among children under five and pregnant women [18]. 30 31 77 In order to address this public health problem, free mass distribution of long-lasting 32 33 34 78 insecticide-treated mosquito nets (ITNs) has been introduced by the country's public health 35 36 79 services, mainly for these two at-risk populations [18,19]. Despite these control measures, 37 38 39 80 malaria remains endemic with 748 deaths in 2000 and 1,544 in 2015 [20,21]. Anopheles 40 41 81 coluzzii, An. gambiae s.s., An. arabiensis and An. funestus mosquitoes are the dominant 42 43 44 82 vectors species of the Plasmodium parasites, including P. falciparum, P. vivax, P. malariae, 45 46 83 P. ovale wallikeri and P. ovale curtisi [4,20,22]. 47 48 Lymphatic filariasis (LF) is mosquito-borne neglected tropical disease and was considered 49 84 50 51 85 as a public health problem [23]. Lymphatic filariasis, due to the Wuchereria brancrofti 52 53 86 parasite, has the same anopheline vectors as malaria [4,24,25]. It should be noted that since 54 55 56 87 the inception of the Global Program for the Elimination of Lymphatic Filariasis (GPELF), 57 58 59 60 61 62 4 63 29 64 65 88 remarkable progress has been made in this country [23,26]. Indeed, new cases are not reported 1 2 89 indicating an interruption of the transmission [23,27]. 3 4 5 6 90 The Aedes genus contains several vector species of arboviruses, including yellow fever, 7 8 91 dengue, chikungunya, Rift Valley fever and Zika viruses, being responsible for public health 9 10 92 problems around almost the entire world [28 30]. Several arboviruses have been reported as 11 – 12 13 93 being responsible for mortality and morbidity in the country [31,32]. Three outbreaks of 14 15 94 yellow fever have been recently reported, notably in the Sudano-Sahelian area in 1987 (Kati 16 17 18 95 and Kita districts), in 2004 (Kita district), and in 2005 (Bafoulabé district). In these southern 19 20 96 parts of the country, 58 cases and 25 deaths occurred [32,33]. Yellow fever is a hemorrhagic 21 22 23 97 fever transmitted to humans by several species of Aedes mosquitoes including Ae. 24 25 98 (Diceromyia) furcifer and Ae. (Stegomyia) aegypti [32,33]. Rift Valley fever is a viral 26 27 99 hemorrhagic fever affecting both humans and ruminants, and is an emerging disease which is 28 29 30 100 transmitted by several mosquitoes from different genera, including Culex, Aedes and 31 32 101 Anopheles [34–36]. Transmission of Rift Valley fever virus by Aedes may be trans-ovarian. 33 34 35 102 Recently, an outbreak of Rift Valley fever was reported in western Niger, near the border with 36 37 103 Mali [34,37]. 38 39 40 104 The Culex genus contains several potential vectors of pathogens such as microfilaria 41 42 105 Wuchereria bancrofti and flaviviruses [38–40]. A number of mosquito-borne disease control 43 44 45 106 measures have now been developed. The most effective are those directed against the 46 47 107 mosquitoes, including long lasting insecticidal nets and indoor residual spraying [20]. For 48 49 108 mosquito control measures to be effective, it is necessary to deepen our knowledge of the 50 51 52 109 resting behavior of vector species. 53 54 55 110 The purpose of this review is to provide an update of the current literature on the Malian 56 57 58 111 Culicidae fauna, covering the ecological areas where they are distributed and to describe the 59 60 112 potential pathogens transmitted. Special attention is given to mosquito vectors and the main 61 62 5 63 30 64 65 113 bio-ecological characteristics of the mosquito species are detailed for the vector species only. 1 2 114 The table 2 presents an overview of arboviruses detected in Mali. We also mention the 3 4 5 115 progress made in vector mosquito control and recent innovative tools for mosquito species 6 7 116 identification. 8 9 10 117 The complete checklist of mosquitoes currently recorded in Mali includes 106 species (3 of 11 12 13 118 them have 2 subspecies distributed), grouped into 28 Anophelinae and 78 Culicinae (Table 1 14 15 119 and Supplementary file: Table S1). 16 17 18 19 120 20 21 121 The Anophelinae subfamily 22 23 122 The subfamily Anophelinae comprises 488 officially recognized species. Of these, 60 species 24 25 26 123 are important in the epidemiology of malaria, lymphatic filariasis and arboviruses [4]. In 27 28 124 Mali, only Anopheles genus is present, 28 Anopheles species were recorded in various 29 30 31 125 entomological surveys and are listed in the Supplementary file: Table S1 [7,41–43].. 32 33 126 Anopheles vectors 34 35 36 127 Anopheles gambiae s.l., An. funestus and An. nili are the main malaria vectors [22,44]. An. 37 38 128 gambiae populations have shown a considerable heterogeneity in the country [42,45–47]. The 39 40 41 129 An. gambiae s.l. includes 8 valid species of which An. arabiensis, An. coluzzii and An. 42 43 130 gambiae s.s. are distributed in Mali and are important malaria vectors in this country. The 44 45 131 molecular tools used to investigate An. gambiae s.s. have enabled two molecular forms to be 46 47 48 132 differentiated, M and S. The molecular form M refers to the chromosomal form Mopti and 49 50 133 was recently named An. coluzzii by Coetzee et al. [48]. The molecular form S retains its 51 52 53 134 original name, An. gambiae s.s. This molecular form combines two chromosomal forms 54 55 135 known as Savanna and Bamako. These three taxa (An. coluzzii, An. gambiae chromosomal 56 57 58 136 form Savanna, and An. gambiae chromosomal form Bamako) are spread according to the eco- 59 60 137 climatic facies of the country [42,45–47]. 61 62 6 63 31 64 65 138 Anopheles coluzzii (Fig. 2a) is found in the northern Savanna, in the Sahel, and in the irrigated 1 2 139 areas of the inner Niger River delta; it is the most abundant species of the An. gambiae 3 4 5 140 complex in the country. Meanwhile, the Savanna chromosomal form is present in the Sudano- 6 7 141 Sahelian and Sudano-Guinean areas, particularly in the Kayes and Sikasso regions [47]. The 8 9 10 142 chromosomal form of Bamako is limited to the Sudano-Sahelian zone, particularly around 11 12 143 Bamako and in the Sudano-Guinean zone west of Sikasso.. The hybrids and recombinants 13 14 144 between the Bamako and Coluzzii forms are limited to the Kayes region in western Mali.[47]. 15 16 17 145 Anopheles coluzzii and An. gambiae s.s. are considered highly anthropophilic and bite 18 19 146 humans easily, mainly indoors (endophagic) but also outdoors (exophagic). The main biting 20 21 22 147 activity occurs at night, and after blood feeding, females leave (exophilic) or remain 23 24 148 (endophilic) in these homes [49]. These species have a short larval development period and 25 26 27 149 are often found in larval habitats associated with human activity. Immature stages of An. 28 29 150 coluzzii develop in permanent or subpermanent larval settings, such as the central Niger River 30 31 151 delta and irrigated rice fields. Immature stages of An. gambiae s.s. develop in sunny fresh 32 33 34 152 water without raised vegetation [4,50] and develop in temporary water such as puddles and 35 36 153 ponds. Anopheles larval development is optimal six weeks after rice transplantation in the 37 38 39 154 field [42]. Females usually take their first blood meal 24 hours after emergence and a high 40 41 155 proportion feed again the same night after oviposition. The dispersal capacity of the females 42 43 44 156 is low; they are usually found between one and three kilometers from their larval sites [1]. 45 46 157 However, recent studies have demonstrated the potential ability of An. coluzzii to migrate 47 48 over long distances and aestivate [51,52]. Thus, An. gambiae s.s. spreads across several 49 158 50 51 159 biotope types which leads to the species being widely distributed. The majority of mosquitoes 52 53 160 collected in the Sudan-Savanna ecological zone (southern Mali) consist of the sister taxa, An. 54 55 56 161 gambiae s.s. and An. coluzzii [45]. Both species are present in the two Savanna sites, arid 57 58 162 Savanna and humid Savanna. An. coluzzii, however, is predominant in the arid Savanna, and 59 60 61 62 7 63 32 64 65 163 An. gambiae s.s. is predominant in the humid Savanna [45]. Temperature and moisture play 1 2 164 an important role in the density of mosquitoes in the ecological areas [53]. Anopheles 3 4 5 165 gambiae s.s., An. coluzzii and An. arabiensis are the main species represented in all 6 7 166 collections in the various ecological areas [45,54]. 8 9 10 167 Anopheles arabiensis is considered to be a species living in dry savannah-like environments. 11 12 168 This species is described as anthropophilic and zoophilic; when domestic animal hosts are 13 14 169 available, these females prefer to feed on livestock. Furthermore, the An. arabiensis species is 15 16 17 170 more likely to prefer the outside environment for feeding (exophagic) and rest for digestion of 18 19 171 blood meals (exophilic) [1,50]. Moreover, blood feeding usually occurs during the night. The 20 21 22 172 larval habitats are similar to those of An. gambiae s.s., generally temporarily sunny freshwater 23 24 173 and permanent water such as rice paddies.[4,50]. Anopheles gambiae s.l. and An. funestus are 25 26 27 174 collected both from irrigated and non-irrigated zones [55]. In these areas, An. gambiae s.l. is 28 29 175 more abundant than An. funestus [55]. However, the densities of both vectors are dynamic and 30 31 176 are seasonally dependent. For instance, in recent decades An. funestus was more abundant 32 33 34 177 than An. gambiae in non-irrigated areas during the cold dry season; in contrast, in the irrigated 35 36 178 area during the rainy season, An. gambiae s.l. was found to be more abundant than An. 37 38 39 179 funestus [55]. In addition, these mosquito species have also been collected in the rice 40 41 180 cultivation area of Office du Niger, located in the inner delta of the Niger River, in the 42 43 44 181 Sudano-Sahelian area [56]. However, a number of recent studies conducted in non-irrigated 45 46 182 areas have revealed a significant density of An. gambiae complex, compared to An. funestus 47 48 in all seasons [57]. 49 183 50 51 184 The An. funestus subgroup contains six species, including An. aruni, An. confusus, An. 52 53 185 funestus s.s., An. parensis, An. vaneedeni and An. longipalpis, but only An. funestus s.s. is 54 55 56 186 associated with malaria transmission as a vector [3,58,59]. A typical larval habitat for An. 57 58 187 funestus is a permanent and semi-permanent water body with emergent vegetation. These 59 60 61 62 8 63 33 64 65 188 larvae are found in marshes, large sunny ponds, lake shores and rice fields [42,60]. Anopheles 1 2 189 funestus is considered to be highly anthropophilic [60] as the An. gambiae complex [57]. 3 4 5 190 These females usually feed indoors (endophagic) after sunset, with a peak occurring during 6 7 191 the second half of the night. After feeding, they rest indoors (endophilic) on the upper part of 8 9 10 192 the walls. In many areas, An. funestus live inside homes, making them vulnerable to residual 11 12 193 insecticide control measures[1]. 13 14 15 16 194 There is some confusion regarding the taxonomic status of An. (Cellia) sergentii sergentii 17 18 195 and An.(Cellia) sergentii macmahoni, but recently An. macmahoni has been considered as 19 20 21 196 being a subspecies of An. sergentii [61]. Anopheles macmahoni has never been found biting 22 23 197 humans and has no known medical importance [50]. Anopheles sergentii sergentii is known 24 25 198 as the oasis vector or the desert malaria vector due to its distribution within oases across the 26 27 28 199 Saharan belt [50]. This species was collected in Saharan area [41]. The larval habitats are 29 30 200 streams, seepages, canals, irrigation channels, springs, rice fields and most other shallow, 31 32 33 201 unpolluted sites that contain fresh water with a slow current, light emergent vegetation or 34 35 202 algae [50]. An. sergentii sergentii females are described as anthropophilic and zoophilic 36 37 38 203 because they bite both humans and animals. However, this species prefer to feed indoors 39 40 204 (endophagic) and sometimes rest for digestion of blood meal (semi-endophilic) [41]. 41 42 43 44 205 45 46 206 The Culicinae subfamily 47 48 49 207 The Culicinae subfamily includes several tribes, including Aedeomyiini, Aedini, Culicini, 50 51 208 Ficalbiini, Mansoniini, Toxorhynchitini and Uranotaeniini. A total of 78 species of Culicinae 52 53 209 have been recorded in various entomological surveys and are listed in the Supplementary file: 54 55 56 210 Table S1. 57 58 59 60 61 62 9 63 34 64 65 211 In this review, we present in detail only species of the Aedes and Culex genera, because the 1 2 212 potential vectors in Mali belong to these two genera. 3 4 5 213 Aedes as potential vectors 6 7 214 Aedes mosquitoes are dominant vectors of most arboviruses that infect humans and animals 8 9 10 215 worldwide and in West Africa [62]. The distribution of the Aedes mosquitoes and the disease 11 12 216 they transmit depend on the ecological conditions of each area. Hamon et al. reported the 13 14 217 existence of 28 Aedes species (Table 1). Two more Aedes species were then reported in Mali, 15 16 17 218 known as the Ae. opok [63] and Ae. sudanensis [64]. Recently, Muller et al. conducted an 18 19 219 entomological survey and recorded Ae. (Stegomyia) albopictus [65]. According to Hamon et 20 21 22 220 al. and Muller et al., the potential vectors of arboviruses are Ae. aegypti, Ae. albopictus, Ae. 23 24 221 (Stegomyia) africanus, Ae. furcifer, Ae. (Aedimorphus) fowleri, Ae. (Stegomyia) luteocephalus 25 26 27 222 and Ae. (Aedimorphus) ochraceus [7,65]. 28 29 30 223 Aedes aegypti (Fig. 2b) and Ae. albopictus are the major vectors of the dengue virus 31 32 224 (DENV). Of the four viral serotypes of DENV, three (2, 3 and 4) are present in West Africa, 33 34 35 225 particularly at the border between Burkina Faso and Mali [66,67]. In Mali, epidemic 36 37 226 monitoring of DENV is crucial because the Aedes vectors are present and patient serum 38 39 40 227 samples were positive for this viral infection [7,31,65]. The chikungunya virus (CHIKV) 41 42 228 belongs to the Togaviridae family and Alphavirus genus. The CHIKV has the same vectors as 43 44 45 229 DENV and circulates in population at risk of epidemic [7,31,65]. 46 47 48 230 Aedes aegypti is the main vector of the yellow fever virus (YFV) and is the only domestic 49 50 231 vector of this virus in West Africa [62]. In Mali, this species has been reported in towns, 51 52 53 232 villages as well as in natural wooded savannas. Their breeding water is mostly clean or has a 54 55 233 moderate content of organic matter. Females lay their eggs in tree holes and artificial 56 57 58 234 containers such as tires, flower pots, cisterns and abandoned containers, increasing the risk of 59 60 235 urban YFV epidemics in Mali [7]. Aedes aegypti eggs are resistant to desiccation for up to 61 62 10 63 35 64 65 236 one year and are able to maintain vertical transmission, allowing them to be constantly 1 2 237 present during the dry season and to be transported passively [68]. At a favourable 3 4 5 238 temperature, larvae hatch two days after laying, pupation occurs after eight days and adults 6 7 239 emerge between nine and ten days after laying. Females bite during the day in the shade or 8 9 10 240 darkness, with activity peaks in the morning and late afternoon, after feeding, they rest 11 12 241 indoors (endophilic). They appear to prefer human blood to that of domestic animals [1]. 13 14 15 242 Aedes albopictus eggs are resistant to desiccation and can survive for several months [68]. 16 17 18 243 Their passive transport has allowed this species to invade several continents, although it is of 19 20 244 Asian origin. This invasion is linked to their great ecological and physiological plasticity, 21 22 23 245 which allows them to adapt to new biotopes [68]. Their longevity is around 30 days for 24 25 246 females and 18 days for males, under favourable temperature conditions. In 2016, the first 26 27 247 identification of Ae. albopictus species in two areas (Mopti and Bamako along the Niger 28 29 30 248 River) stressed the need to monitor mosquitoes [65]. These females usually bite humans and 31 32 249 other mammalian vertebrates such as rabbits, dogs, cows, squirrels and, occasionally, avian 33 34 35 250 hosts. Their opportunistic trophic preferences make them potentially capable of transferring 36 37 251 infectious agents from animals to humans [68]. This species is exophagic during the day in 38 39 40 252 umbrageous areas and endophagic at sunset and during the night [1]. Egg-laying females are 41 42 253 attracted to containers, buckets, plastic bags, used tires and fishing boats to lay their eggs. 43 44 45 254 Interestingly, all these habitats are created by humans [65]. 46 47 255 Aedes furcifer is a sylvatic vector of YFV and DENV. This species was implicated in the 48 49 256 yellow fever outbreak that occurred in two Sudano-Sahelian areas in the Kati and Kita 50 51 52 257 districts in 1987, in Mali [33]. Their larval sites are primarily tree holes and secondary 53 54 258 puddles on the roadside [7]. 55 56 57 58 59 60 61 62 11 63 36 64 65 259 Finally, at least one species of the Ae. simpsoni complex was recorded in Mali. We ignore if it 1 2 260 is Ae. bromeliae or Ae. lilii or both, although probably not the nominal species Ae. simpsoni 3 4 5 261 s.s. distributed only in Southern Africa [69,70]. 6 7 262 Culex as potential vectors in Mali 8 9 10 263 The Culex genus contains 769 species distributed worldwide [68,71]. In Mali, 28 Culex 11 12 264 species (or subspecies) belonging to four subgenera have been described (Supplementary file: 13 14 265 Table S1) [7,43,71,72]. Among them, Cx. (Oculeomyia) poicilipes, Cx. (Culex) antennatus, 15 16 17 266 Cx. (Culex) quinquefasciatus and Cx. (Culex) neavei are potential vectors of Flavivirus and 18 19 267 lymphatic filariasis [7,35,43,71–75]. Culex females lay their eggs on permanent or temporary 20 21 22 268 water surfaces, their larval habitats are ponds, flooded ground, irrigated crops, river banks and 23 24 269 tree holes [68]. Culex quinquefasciatus (Fig. 2c) is a member of the Cx. (Culex) pipiens 25 26 27 270 complex and is the most abundant species of tropical Africa. Culex quinquefasciatus is widely 28 29 271 distributed in West Africa and is an important vector of Wuchereria bancrofti [24,71]. Culex 30 31 272 quinquefasciatus larvae have been reported in several types of habitat, including clear water, 32 33 34 273 brackish, polluted water with organic matter and human waste, ditches, sewage, latrines and 35 36 274 artificial containers [1]. Females bite humans and domestic animals such as poultry, dogs, cats 37 38 39 275 and pigs. They are endophagic during the night and exophagic from sunset to dawn [1]. They 40 41 276 rest indoors (endophilic) following their blood meals [68]. 42 43 44 277 We recently conducted entomological surveys using classical and innovative tools in order to 45 46 278 identify mosquitoes, such as molecular techniques and MALDI-TOF MS (see below). This 47 48 allowed us to update the list of mosquito vectors in Mali by describing new mosquito species. 49 279 50 51 280 We reported for the first time, Cx. neavei and Cx. perexiguus [71]. Culex neavei species has 52 53 281 been identified in three sites and is considered a potential vector of WNV on the border 54 55 56 282 between Senegal and Mali [43,74]. Other authors have shown that this species is a potential 57 58 283 vector of WNV and USUV [75]. 59 60 61 62 12 63 37 64 65 284 Culex poicilipes is considered a potential vector of Rift Valley Fever Virus (RVFV) in 1 2 285 Barkedji, Senegal [35] and this mosquito species is abundant throughout Mali [7]. Their larval 3 4 5 286 habitats include streams, flooded meadows, swamps, ponds and irrigated farmland along the 6 7 287 Niger River that could increase the risk of transmission of Rift Valley fever. Furthermore, the 8 9 10 288 virus is circulating on the border between Mauritania and Mali, as well as in western Niger 11 12 289 [37,76]. 13 14 290 15 16 17 291 Strategies for mosquito vector control 18 19 292 National malaria control programs, in collaboration with the WHO, have encouraged the use 20 21 22 293 of mosquito nets impregnated with long-lasting insecticide and indoor residual spraying. 23 24 294 These efforts have contributed to a decrease in malaria cases in Mali [20]. There are four 25 26 27 295 classes of insecticides recommended by WHO, namely pyrethroids, organochlorines, 28 29 296 organophosphates and carbamates. Mosquitoes have become resistant to a number of these 30 31 297 insecticides, posing a serious threat to the success of vector control programs[20]. 32 33 34 298 Researchers have reported An. gambiae s.l. resistance mecanisms to several insecticides, 35 36 299 including dichlorodiphenyltrichloroethane (DDT), deltamethrin (PY), lambda-cyhalothrin 37 38 39 300 (PY), bendiocarb (CA) and fenitrothion (OP) [77]. The mutation on two target sites (kdr and 40 41 301 ace-1R) and the metabolic detoxification systems (monooxygenases and esterases) have been 42 43 44 302 identified in An. coluzzii, An. gambiae s.s. and An. arabiensis species [77]. 45 46 303 The attractive toxic sugar bait (ATSB) plant-spraying methods against the An. gambiae 47 48 mosquito have reduced the density and longevity of this vector, suggesting that ATSB's plant 49 304 50 51 305 spraying methods can be used as a new tool to control this species [78]. Recently, Lin Zhu et 52 53 306 al. confirmed the effectiveness of ATSB on malarial vectors in Africa [79]. 54 55 56 57 58 59 60 61 62 13 63 38 64 65 307 Finally, the entomopathogenic fungus Beauveria bassiana treatments significantly reduced 1 2 308 the blood feeding of Cx. quinquefasciatus in the field. These results show that B. bassiana 3 4 5 309 could be a potential new mosquito control alternative [80]. 6 7 310 Larvae control reduces the development of the vector population by using chemical toxins 8 9 10 311 (larvicides), biological toxins, or fish predators as biological controls [20]. Although 11 12 312 larvicides are useful in some contexts, they are only feasible in areas where most larval sites 13 14 313 are easily located, so they are systematically identifiable and can be fully covered by the 15 16 17 314 intervention. This method often has a greater impact on transmission than adulticide methods 18 19 315 that reduce both the density (number) of mosquitoes and their lifespan [20]. 20 21 22 316 Innovative methodologies for mosquito species identification 23 24 25 317 New diseases and new vectors that colonize new territories, where they were previously 26 27 318 absent, are continuously emerging. For example, the tiger mosquito, Ae. albopictus, has been 28 29 30 319 found in almost every continent of the world [65,81]. The mosquito-borne diseases are a 31 32 320 major public health problem worldwide. Formal mosquito identification is essential to 33 34 35 321 effectively control vectors. Morphological identification using dichotomous keys is the most 36 37 322 widely used method for entomological surveys [49]. Currently, entomologists also use 38 39 40 323 identification keys on a CD-ROM [41]. The limits of morphological identification lie in the 41 42 324 distinction between sub-species, in particular the cryptic forms of An. gambiae s.s. [82]. In 43 44 45 325 Mali, molecular methods were used to distinguish An. gambiae s.l. cryptic species and forms 46 47 326 and certain species of the Cx. pipiens complex, which are difficult to distinguish by their 48 49 327 morphology [45,47,72,77]. The limitations of molecular methods are their costs; they also 50 51 52 328 require specific genes and are time-consuming. 53 54 329 Matrix-Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF MS) has 55 56 57 330 revolutionized microbiology and is now routinely used. MALDI-TOF Mass Spectrometry has 58 59 331 been used in medical entomology to identify arthropods [83]. This technique has been used in 60 61 62 14 63 39 64 65 332 the laboratory for the identification of adult mosquitoes from protein extracts from the legs. 1 2 333 Aquatic stages have also been identified, including eggs and larvae [84–86]. MALDI-TOF 3 4 5 334 Mass Spectrometry is now used during entomological surveys. The preliminary study used 6 7 335 field mosquitoes to update the European mosquito repertory [87]. 8 9 10 336 In Mali, MALDI-TOF MS was applied on the mosquitoes and their midgut microbiota 11 12 337 collected in the rural area of Bougoula-hameau in the Sikasso region. This technology was 13 14 338 used to identify Malian mosquitoes from protein extracts from their legs [43]. In addition of 15 16 17 339 the mosquito identification, their blood meal sources were also determined using MALDI- 18 19 340 TOF MS. Specimens collected from three regions in the Sudan-Savanna area (around urban 20 21 22 341 Bamako, the rural area of the Sikasso region and the rural area around Kati) in Mali [72]. In 23 24 342 this country, eight mosquito species have been identified, namely An. gambiae s.s., An. 25 26 27 343 coluzzii, An. arabiensis, Cx. quinquefasciatus, Cx. neavei, Cx. perexiguus, Ae. aegypti and Ae. 28 29 344 fowleri [72]. Indeed, the mosquito blood meal sources were correctly identified and matched 30 31 345 with the blood of human, chicken, cow, donkey, dog and sheep. Thus, this innovative tool 32 33 34 346 successfully identified Malian mosquitoes as well as their blood meal sources and enabled the 35 36 347 first detection of two new mosquito species in Mali known as Cx. neave and, Cx. perexiguus 37 38 39 348 [72]. 40 41 42 349 Conclusions 43 44 45 350 Recent collections of mosquitoes in Mali focus mainly on vector species involved in the 46 47 351 transmission of infectious diseases that cause a public health problem. In this context, the 48 49 352 recent publications only provide information on the ecology, distribution and associated 50 51 52 353 pathogens of Anopheles, Aedes and Culex vectors. We believe that these gaps may be due to 53 54 354 collection techniques and their relevance to public health. Indeed, a large number of vectors 55 56 57 355 belonging to the Culicidae family have been identified, including Ae. aegypti, Ae. albopictus, 58 59 356 An. coluzzii, An. gambiae s.s., An. arabiensis, An. funestus s.s., Cx. poicilipes, Cx. antennatus, 60 61 62 15 63 40 64 65 357 Cx. quinquefasciatus and Cx. neavei species. They are potential vectors for a number of 1 2 358 arboviral, protozoan and filarial pathogens. Our review has contributed to updating the current 3 4 5 359 literature on mosquitoes and mosquito-borne diseases in Mali. This review may be necessary 6 7 360 for a future nationwide entomological field surveys for mosquito vector controls. 8 9 10 361 11 12 13 362 Declarations 14 15 363 Abbreviations 16 17 18 364 Adm.: Aedimorphus; Ae.: Aedes; An.: Anopheles; ATSB: attractive toxic sugar bait; B: 19 20 365 Bacillus; Cx.: Culex (genus); Cux.: Culex (subgenus); Cel: Cellia; CHIKV: chikungunya 21 22 23 366 virus; DENV: dengue virus; Dic.: Diceromyia; GPELF: Global Program for the Elimination 24 25 367 of Lymphatic Filariasis; GNTs: Glue net traps; LF: lymphatic filariasis; MALDI-TOF MS: 26 27 368 Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry; MRTC: 28 29 30 369 Malaria research and training center; Ocu.: Oculeomyia; P.: Plasmodium; RVFV: Rift Valley 31 32 370 fever virus; s.s.: sensu stricto; s.l.: sensu lato; Stg.: Stegomyia; VITROME: Vecteurs 33 34 35 371 Infections Tropicales et Mediterranéennes; WHO: World Health Organization; USUV: Usutu 36 37 372 virus; WNV: West Nile virus; YFV: yellow fever virus 38 39 40 373 41 42 374 Ethics statement 43 44 45 375 Not applicable 46 47 376 48 49 377 Consent for publication 50 51 52 378 Not applicable 53 54 379 55 56 57 380 58 59 381 60 61 62 16 63 41 64 65 382 Availability of data and material 1 2 383 All datasets relating to this study have been included in the main paper and the supplementary 3 4 5 384 files. 6 7 8 385 Competing interests 9 10 386 The authors declare that they have no competing interests. 11 12 13 387 14 15 388 Funding 16 17 18 389 This work has been conducted with the support of the A*MIDEX project (n° ANR-10-IAHU- 19 20 390 03) funded by the Investissements d’Avenir French Government program, managed by the 21 22 23 391 French National Research Agency (ANR). 24 25 392 26 27 393 28 Authors’ contributions 29 30 394 FT wrote the initial draft of the manuscript, and VR, PP, OKD, SFT and ASY added their 31 32 395 contributions and comments. All authors read and approved the final version of the 33 34 35 396 manuscript and consented to its publication. 36 37 38 397 39 40 41 398 Acknowledgment 42 43 44 399 We thank VITROME unit, the IHU Méditerranée-Infection of Marseille, MRTC of Bamako 45 46 400 (Mali) and the IRD of Montpellier for library resources and support during the writing of this 47 48 49 401 review. 50 51 52 402 53 54 403 55 56 57 404 58 59 405 60 61 62 17 63 42 64 65 406 References 1 2 407 3 4 408 1. Becker N, Petric D, Zgomba M, Boase C, Dahl C, Madon M et al. Mosquitoes and 5 6 409 their control. 2nd ed. Springer: Heidelberg, Germany; 2010. 7 8 410 2. Dieme C, Bechah Y, Socolovschi C, Audoly G, Berenger JM, Faye O et al. 9 411 Transmission potential of Rickettsia felis infection by Anopheles gambiae 10 412 mosquitoes. Proc Natl Acad Sci U S A. 2015; 112:8088-8093. 11 12 13 413 3. Harbach RE. Mosquito taxonomic Inventory. 2013. http://mosquito-taxonomic- 14 414 inventory info/. 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Observations sur les Aedes (Aedimorphus) d'Afrique avec 34 656 description de deux nouvelles espèces : Ae. lottei n. sp. et Ae. dialloi n. sp. Bull 35 657 Soc Path exot. 1965;1:101-108. 36 37 658 92. Brès P. Données récentes apportées par les enquêtes sérologiques sur la prévalence 38 39 659 des arbovirus en Afrique, avec référence spéciale à la fièvre jaune. Bull Org 40 660 Mond Santé. 1970;43:223-267. 41 42 661 93. Mayer SV, Tesh RB, Vasilakis N. The emergence of arthropod-borne viral diseases: A 43 662 global prospective on dengue, chikungunya and zika fevers. Acta Trop. 44 663 2017;166:155-163. 45 46 664 47 665 48 49 50 666 51 52 667 53 54 55 668 56 57 669 58 59 670 60 61 62 24 63 49 64 65 671 Tables legends 1 2 672 Table 1. List of Culicidae species reported in Mali since 1908 3 4 5 673 Table 2. Mosquito-borne arboviruses in Mali 6 7 674 Additional file 1: Table S1. Mosquito directory in Mali West Africa 8 9 10 675 11 12 676 Figure legends 13 14 677 Figure 1: Eco-climatic areas and mosquito distribution in Mali. From north to south, there 15 16 17 678 are five zones including the Saharan zone, the Sahelian zone, the Sudano-Sahelian zone, the 18 19 679 Sudanese zone and the Guinean zone. The distribution of some vector mosquitoes is reported, 20 21 22 680 including: Aedes spp., Aedes albopictus, Anopheles spp., Anopheles coluzzii, Anopheles 23 24 681 gambiae and Culex spp. 25 26 27 682 Figure 2: Pictures of three species of mosquitoes that are potential vectors in Mali. a) 28 29 683 Anopheles coluzzii female from laboratory rearing, b) Aedes aegypti female collected from 30 31 684 Gabon, c) Culex quinquefasciatus female collected from Mali. 32 33 34 35 685 36 37 686 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 25 63 50 64 65 15 16 17 18 19 20 21 22 687 Table 1. List of Culicidae species reported in Mali since 1908 Subfamily23 Species References (2000-present) 24 25 26 1908 [5] 1950 [7] 1961 [7] Recent (2000-present) 27 28 Anophelinae29 An. arabiensis An. arabiensis [72,88–90] 30 31 32 An. brohieri 33 34 An. brunnipes [41,89] 35 36 37 An. coluzzii [89,90] 38 39 40 An. coustani An. coustani 51 41 42 An. domicola [41,89] 43 44 45 An. flavicosta 46 47 48 An. funestus An. funestus An. funestus [41,89] 49 50 51 An. gambiae An. gambiae An. gambiae An. gambiae [72,89,90] 52 53 An. hancocki 54 55 56 An. leesoni 57 58 59 An. longipalpis 60 61 62 26 63

64 65 15 16 17 18 19 20 21 22 An. maculipalpis 23 24 An. maliensis [61] 25 26 27 An. nili An. nili 28 29 30 An. obscurus 31 32 An. paludis An. paludis 33 34 35 An. pharoensis An. pharoensis 36 37 38 An. pretoriensis 39 40 52 41 An. rhodesiensis rhodesiensis 42 43 An. rivulorum 44 45 46 An. rufipes An. rufipes rufipes An. rufipes broussesi [89] 47 48 49 An. sergentii sergentii [41] 50 51 An. sergentii macmahoni [61] 52 53 54 An. schwetzi [61] 55 56 57 An. somalicus [61] 58 59 60 61 62 27 63

64 65 15 16 17 18 19 20 21 22 An. squamosus 23 24 An. wellcomei wellcomei 25 26 27 An. ziemanni An. ziemanni An. ziemanni [41] 28 29 Culicinae30 Ad. africana 31 32 Ad. furfurea 33 34 35 Ae. aegypti Ae. aegypti Ae. aegypti Ae. aegypti [6, 72,89] 36 37 38 Ae. africanus Ae. albopictus [65] 39 40 53 41 Ae. argenteopunctatus 42 43 Ae. cumminsii 44 45 46 Ae. circumluteolus 47 48 49 Ae. dalzieli 50 51 Ae. dialloi [91] 52 53 54 55 56 57 Ae. fowleri Ae. fowleri [72] 58 59 60 61 62 28 63

64 65 15 16 17 18 19 20 21 22 Ae. furcifer 23 24 Ae. grahamii 25 26 27 Ae. haworthi 28 29 30 Ae. hirsutus 31 32 Ae. lineatopennis 33 34 35 Ae. luteocephalus 36 37 38 Ae. longipalpis 39 40 54 41 Ae. mattinglyi 42 43 Ae. metallicus 44 45 46 Ae. minutus 47 48 49 Ae. mucidus 50 51 Ae. mixtus 52 53 54 Ae. ochraceus 55 56 57 Ae. opok [63] 58 59 60 61 62 29 63

64 65 15 16 17 18 19 20 21 22 Ae. punctothoracis 23 24 Ae. scatophagoides 25 26 27 Ae. simpsoni s.l. 28 29 30 Ae. stokesi 31 32 Ae. sudanensis [64] 33 34 35 Ae. tarsalis 36 37 38 Ae. taylori 39 40 55 41 Ae. vittatus 42 43 Cq. aurites 44 45 46 Cq. maculipennis 47 48 49 Cq. metallica 50 51 Cx. albiventris 52 53 54 Cx. annulioris 55 56 57 Cx. antennatus 58 59 60 61 62 30 63

64 65 15 16 17 18 19 20 21 22 Cx. argenteopunctatus 23 24 Cx. bitaeniorhynchus Cx. bitaeniorhynchus [61] 25 26 27 Cx. cinereus 28 29 30 Cx. decens 31 32 Cx. duttoni 33 34 35 Cx. grahamii farakoensis 36 37 38 Cx. grahamii grahamii 39 40 56 41 Cx. guiarti Cx. guiarti 42 43 Cx. horridus 44 45 46 Cx. inconspicuosus 47 48 49 Cx. insignis 50 51 Cx. invidiosus 52 53 54 Cx. macfìei 55 56 57 Cx. nebulosus Cx. neavei [72] 58 59 60 61 62 31 63

64 65 15 16 17 18 19 20 21 22 Cx. perfuscus Cx. perexiguus [72] 23 24 Cx. poicilipes 25 26 27 Cx. quasiguiarti 28 29 30 Cx. quinquefasciatus Cx. quinquefasciatus Cx. quinquefasciatus [43,72,88] 31 32 Cx. simpsoni 33 34 35 Cx. trifoliatus 36 37 38 Cx. univittatus 39 40 57 41 Cx. weschei 42 43 Cx. wigglesworthi 44 45 46 Er. dracaenae 47 48 49 Fi. uniformis 50 51 52 Mi. splendens 53 54 Mi. mimomyiaformis 55 56 57 Mi. plumosa 58 59 60 61 62 32 63

64 65 15 16 17 18 19 20 21 22 Mi. mediolineata 23 24 Ma. africana 25 26 27 Ma. uniformis Ma. uniformis 28 29 30 Tr. viridibasis 31 32 33 Tr. brevipalpis conradti 34 35 Ur. balfouri 36 37 38 Ur. chorleyi 39 40 58 41 Ur. ornata 42 43 Ur. mashonanensis 44 45 46 Ur. fusca 47 48 49 688 50 51 689 Abbreviations: Ad.: Aedeomyia; An.: Anopheles; Ae.: Aedes; Cq.: Coquillettidia; Cx.: Culex; Er.: Eretmapodites; Fi.: Ficalbia; Mi.: Mimomyia; 52 53 690 Ma.: Mansonia; Tr.: Toxorhynchites; Ur.: Uranotaenia. 54 691 55 56 692 57 58 59 60 61 62 33 63

64 65 15 16 17 18 19 20 21 22 693 Table 2. Mosquito-borne arboviruses in Mali 23 Virus Source of detection Vertebrate hosts Vector hosts References 24 25 26 Yellow fever Patient serum, mosquitoes Primates Aedes spp. [32,33,92,93] 27 28 29 Dengue Patient serum Primates Aedes spp. [31,93] 30 31 32 Chikungunya Patient serum Primates, birds, cattle and rodents Aedes spp. and Culex spp. [31,92,93] 33 34 Zika Patient serum Primates Aedes spp. [92,93] 35 36 37 Rift Valley fever Patient serum Cows, sheep, camels, goats and Aedes spp., Anopheles spp. [34–37,93] 38 39 other mammals and Culex spp. 40 59 41 42 West Nile Patient serum Birds, horses and other mammals Culex spp. [31,92,93] 43 44 694 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 34 63

64 65 Figure 1 Click here to download Figure Fig1.tiff 60 Figure 2 Click here to download Figure Fig2.tiff 61 Additionnal file S1

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Manuscript: Manuscript ID: PARV-D-18-00181

Full title: Mosquitoes (Diptera: Culicidae) and mosquito-borne diseases in Mali, West Africa

Corresponding author: Philippe PAROLA

Journal: Parasites and Vectors.

Dear Editor,

According to reviewer recommendations, modifications of our manuscript headed

“Mosquitoes (Diptera: Culicidae) and mosquito-borne diseases in Mali, West Africa.”

have been done. We hope that our revisions to the originally submitted version of the

manuscript will correspond to the reviewer expectations and are in compliance with the

Parasites and Vectors guidelines. The changes included in our manuscript are indicated in the

letter “Responses_to_Reviewers_PARV-D-18-00181_R1”. All authors confirm that the

advices to Contributors have been read and that they accepted the conditions set down therein.

All named authors have reviewed the revision and they agreed with the revised submission.

The manuscript has not been submitted elsewhere for possible publication as long as it is

under consideration of Parasites and Vectors.

Sincerely Yours,

Philippe PAROLA

III. UTILISATION DU MALDI-TOF MS POUR L’IDENTIFICATION DES MOUSTIQUES ET DE LEUR REPAS SANGUIN

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.

La spectrométrie de masse à temps de vol avec désorption / ionisation laser assistée par matrice (MALDI-TOF MS) a été récemment décrite comme un outil innovant et efficace pour identifier les arthropodes et le repas de sang des moustiques. Pour tester cette approche dans le contexte d'une enquête entomologique sur le terrain, les moustiques ont été collectés dans cinq zones écologiquement distinctes du Mali. Ainsi, dans le cadre d’un accord interuniversitaire entre l’Université d’Aix-Marseille et l’Université de Bamako nous avons effectué une enquête entomologique de terrain entre le 15 Juillet et le 15 Aout 2016 au Mali, puis analysé le matériel collecté à Marseille Nous avons analysé avec succès les repas de sang de 651 abdomens de moustique écrasés sur le terrain sur Papier filtre Whatman (PFW) en utilisant le MALDI- TOF MS. Les pattes de 826 moustiques ont ensuite été soumises à une analyse MALDI-TOF MS afin d'identifier les différentes espèces de moustiques. Dans notre enquête 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. Les moustiques pour lesquels la spectrométrie de masse MALDI-TOF n'a pas fourni une identification correcte n'étaient en fait pas disponibles dans notre base de données. Ces spécimens ont ensuite été identifiés par la biologie moléculaire, ce qui a permis d’enrichir notre base de données. Les sources de repas de sang des PFWs, dans cette étude, étaient du sang humain (n = 619), du sang de poulet (n = 9), du sang de vache (n = 9), du sang d'âne (n = 6), du sang de chien (n = 5) et du sang de mouton (n = 3). Cette étude renforce le fait que le MALDI-TOF MS est un outil prometteur pour les enquêtes entomologiques.

Parasitology Using MALDI-TOF MS to identify mosquitoes collected in Mali and their blood meals cambridge.org/par

Fatalmoudou Tandina1,2, Sirama Niaré1,2, Maureen Laroche1, Abdoulaye K Koné2, Adama Z Diarra1,2, Abdoulaye Ongoiba2, Research Article Jean Michel Berenger1, Ogobara K Doumbo2, Didier Raoult1 and Philippe Parola1 Cite this article: Tandina F et al. Using MALDI- TOF MS to identify mosquitoes collected in 1Aix Marseille Univ, IRD, AP-HM, IHU-Méditerranée Infection, SSA, VITROME, Marseille, France and 2Department Mali and their blood meals. Parasitology of Epidemiology of Parasitic Diseases, Malaria Research and Training Center, University of Science, Techniques and https://doi.org/10.1017/S0031182018000070 Technologies of Bamako, Mali

Received: 15 July 2017 Revised: 19 December 2017 Abstract Accepted: 21 December 2017 Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF Key words: MS) has been recently described as an innovative and effective tool for identifying arthropods MALDI-TOF MS; mosquito; field; blood meals; and mosquito blood meal sources. To test this approach in the context of an entomological Whatman; Mali survey in the field, mosquitoes were collected from five ecologically distinct areas of Mali. We successfully analysed the blood meals from 651 mosquito abdomens crushed on Author for correspondence: Philippe Parola, Email: philippe.parola@univ- Whatman filter paper (WFPs) in the field using MALDI-TOF MS. The legs of 826 mosquitoes amu.fr were then submitted for MALDI-TOF MS analysis in order to identify the different mosquito species. Eight mosquito species were identified, including Anopheles gambiae Giles, Anopheles coluzzii, Anopheles arabiensis, Culex quinquefasciatus, Culex neavei, Culex perexiguus, Aedes aegypti and Aedes fowleri in Mali. The field mosquitoes for which MALDI-TOF MS did not provide successful identification were not previously available in our database. These specimens were subsequently molecularly identified. The WFP blood meal sources found in this study were matched against human blood (n = 619), chicken blood (n = 9), cow blood (n = 9), donkey blood (n = 6), dog blood (n = 5) and sheep blood (n = 3). This study reinforces the fact that MALDI-TOF MS is a promising tool for entomological surveys.

Introduction Mosquito-borne infectious diseases are a public health concern in tropical countries, and an emerging problem in temperate areas (Becker et al. 2010). The main mosquito vectors, which may transmit pathogens during their blood meals, belong to three main genera, namely Aedes, Culex and Anopheles (Becker et al. 2010). Aedes spp. mosquitoes are vectors for several arboviruses including the Yellow Fever, Dengue, Chikungunya and Zika viruses, which have come to the world’s attention in recent years (Gardner and Ryman, 2010; Vasilakis et al. 2011; Caglioti et al. 2013). Culex mosquitoes are responsible for West Nile Virus (WNV) and Japanese encephalitis virus transmission around the world (Komar, 2003; Anosike et al. 2005; de Wispelaere et al. 2017). Anopheles mosquitoes are the primary vectors of malaria. Female Anopheles mosquitoes are able to transmit six species of Plasmodium to humans: P. falciparum, P. vivax, P. malariae, P. ovale wallikeri, P. ovale curtisi, P. knowlesi and P. simium (WHO, 2016; Brasil et al. 2017). Malarial transmission remains high in Africa, with 117 886 deaths in 2015 (WHO, 2016). In Mali, West Africa, 1544 deaths were recorded as being attrib- utable to malaria in 2015 (WHO, 2016). It is reasonable to assume that the number of malaria-associated deaths remains underestimated. The precise identification of mosquito fauna is essential in entomological surveys, and in order to plan control measures and monitor their impact (Bass et al. 2007). Furthermore, the identification of mosquito blood meal sources is essential to understanding the biting behaviour of mosquito vectors (anthropophilic or zoophilic) (Muturi et al. 2013). Mosquitoes are most frequently identified at the genus and species levels by morphological characteristics and using molecular tools. Morphological identification requires well-trained entomologists using dichotomous identification keys (Gillies MT 1987). Morphological identification continues to be the standard approach for arthropod studies. However, it presents some limits in terms of discriminating cryptic or sibling species. In recent years, molecular tools have emerged and can identify mosquitoes by amplifying different target genes. The target gene, such as the cytochrome c oxidase (COI), internal transcribed spacer 2, IGS regions of rDNA, has been used to satisfactorily identify mosquitoes up to sibling species with great specificity and sensitivity (Folmer et al. 1994). Several approaches have been developed to identify the host vertebrate blood source of © Cambridge University Press 2018 mosquito meals. The main tools include a serological approach which involves precipitin tests and enzyme-linked immunosorbent assays (ELISA) (Fyodorova et al. 2006; Gomes et al. 2013). However, these techniques present several limitations, including the availability of specific antisera against a broad diversity of host species and the cross-reactivity of antibodies for close species. To this end, molecular methods have also been developed to identify 73

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mosquito blood meal sources, such as mammalian blood and 1992). The peak densities and consequentially of anopheline mos- avian blood from Culex pipiens complex (Gomes et al. 2013). quitoes in Mali occur in August (Sogoba et al. 2007). Mosquitoes However, molecular methods also present several constraints, were collected over three consecutive days per week. On each day, such as their cost, the time they take and the need for bulky mosquitoes were aspirated from 10 houses using a mouth aspir- equipment. ator (Model 612, John W Hock, Gainesville, Florida, USA). All Matrix-assisted laser desorption/ionization time-of-flight mass mosquitoes were collected indoors in the morning between 8 spectrometry (MALDI-TOF MS) has recently been used as an am and noon. The mosquito specimens were identified using alternative tool for rapid arthropod identification. The mass spec- morphological criteria (Gillies MT 1987). After being collected, trum from a new sample, generated using MALDI-TOF MS, is mosquito specimens were kept at room temperature (RT) between compared with a library of spectra from a reference database. 2 and 4 h during the female abdomens crushed process and then In our laboratory, the MALDI-TOF MS approach has been rou- were stored at −20 °C. Each mosquito specimen was then indi- tinely used to identify arthropods such as ticks (using their legs) vidually transferred to a 1.5 mL Eppendorf tube labelled with a (Yssouf et al. 2013, 2015; Kumsa et al. 2016), fleas (bodies of reference number, the gender of the specimen, the date and site fleas without the abdomen) (Yssouf et al. 2014b), sand flies of collection. (using their thoraces, wings and legs) (Lafri et al. 2016), adult Mosquito abdomens with visible blood meals were crushed on mosquitoes (using their legs) (Yssouf et al. 2013, 2014a) and WFPs (Whatman International Ltd., Maidstone, England, mosquito larvae (using whole mosquitoes) (Dieme et al. 2014). approved by BSI). Following the entomological stage, all samples Preliminary studies have also reported that MALDI-TOF MS were transported to Aix-Marseille University for mosquito and may be used for mosquito blood meal identification. When the blood meal identification using MALDI-TOF MS in September MS spectra obtained from the abdomen of mosquitoes which and October 2016. had been experimentally engorged on different blood meals source were tested, the MS protein profiles were clearly distinct according to the origin of the mosquito blood meals, up to 24 h post-feeding (Niare et al. 2016). During entomological sur- Preparation of samples for MALDI-TOF MS analysis veys, it may be difficult to preserve samples, and entomologists Mosquito identification frequently use Whatman filter papers (WFPs) to preserve mos- The legs of the specimens were cleaned in 70% (v/v) ethanol for quito blood meals in the field by crushing the engorged abdo- between one to two minutes, then rinsed in high performance mens onto WFPs. liquid chromatography (HPLC) grade water. The legs from each In this study, the goal was to use the proteomic MALDI-TOF mosquito were individually placed in 1.5 mL Eppendorf tubes MS approach to identify mosquitoes collected in Mali and deter- with glass powder (Sigma, Lyon, France), 15 µL of 70% (v/v) for- mine the sources of their blood meals. For this purpose, mosqui- mic acid (Sigma, Lyon, France), and 15 µL of 50% (v/v) aceto- toes were collected in different ecological areas of Mali and tested nitrile (Fluka, Buchs, Switzerland). The samples were crushed by MALDI-TOF MS in Marseille, France. The abdomens of using a TissueLyser device (Qiagen, Hilden, Germany) over engorged female mosquitoes were crushed onto WFP to deter- − three cycles of 30 m s 1 for 60 s (Nebbak et al. 2016). The samples mine the blood meal sources using MALDI-TOF MS. were centrifuged at 200 g for one minute, and 1.5 µL of supernatant of each homogenate was deposited on the MALDI-TOF Materials and methods target plate in quadruplicate (Bruker Daltonics, Wissembourg, France) and covered with 1.5 µL of CHCA matrix solution com- Ethics statement posed of saturated α-cyano-4-hydroxycynnamic acid (Sigma, Consent was obtained from the heads of families where the mos- Lyon, France), 50% acetonitrile (v/v), 2.5% trifluoroacetic acid quitoes were collected. Ethical approval for the collection of mos- (v/v) (Aldrich, Dorset, UK), and HPLC grade water (Yssouf quito was granted by authorities from the National Malaria et al. 2013; Nebbak et al. 2016). The target plate was dried for sev- Control Program (NMCP) and approved by the Faculty of eral minutes at RT and placed in the Microflex LT MALDI-TOF Medicine Ethical Committee, Bamako, Mali (N°2016/113/CE/ Mass Spectrometer (Bruker Daltonics, Wissembourg, France) for FMPOS). The mosquito samples were processed and stored in analysis (Yssouf et al. 2013, 2016; Nebbak et al. 2016). line with the World Health Organization (WHO) Good Laboratory Practices guidance and documents on mosquito sampling handling procedures. Bloody Whatman filter papers (BWFPs) A piece of the WFPs (i.e. about 1 mm2) containing crushed abdo- Collection sites mens from engorged mosquitoes was individually cut using a sterile scalpel and transferred to a new 1.5 mL Eppendorf tube (Niare This study was conducted in three different localities in Mali, et al. 2017). For each piece of WFPs, 20 µL of formic acid (70%, v/v) namely Donéguébougou, Bougoula-hameau and Bamako. In plus 20 µL of acetonitrile (50% v/v) (Fluka, Buchs, Switzerland) Bamako, the collection was performed in the three semi-urban was added and incubated for 10 min at RT. After a fast spin areas of Sotuba, Yirimadio and Missabougou. The geo-positions (i.e. 10 000 rpm for 20 s), 1 µL of the supernatant of each sample of each collection site are as follows: Bougoula-hameau (−5° was loaded onto the MALDI-TOF target plate in quadruplicate 66′13.1′′, 11°30′95.2′′E); Donéguébougou (−7°98′39.8′′N, 12° ′ ′′ and covered with 1 µL of CHCA matrix (Niare et al. 2016). 80 44.9 E) and the semi-urban areas of Bamako, Sotuba (−9° After drying for several minutes at RT, the MALDI-TOF target 18′65.7′N, 8°23′07.4′E), Yirimadio (−9°18′56.5 ′N, 6°23′01.8′′E) ′ ′′ ′ ′′ plate was placed in the Microflex LT MALDI-TOF Mass and Missabougou (−9°18 77.5 N, 8°23 03.9 E). Spectrometer (Bruker Daltonics, Bremen, Germany) for analysis. To control loading on mass spectra steel, matrix quality and MALDI-TOF apparatus performance, the matrix solution was Mosquito collection loaded in duplicate onto each MALDI-TOF plate with or without Mosquitoes were collected from the various sites during the mid- a bacterial test standard (Bruker protein Calibration Standard I) dle of the rainy season between July and August 2016 (WHO, (Niare et al. 2016). 74

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Spectral analysis abdominal proteins of engorged mosquitoes crushed on WFPs were also blindly queried against the database. A sample was con- Protein mass profiles were acquired using a Microflex LT sidered to be correctly and significantly identified at the species MALDI-TOF Mass Spectrometer, with detection in the linear level when the queried spectrum had a log score value (LSV) positive-ion mode at a laser frequency of 50 Hz within a mass ⩾1.8 (Niare et al. 2016). range of 2-20 kDa. The acceleration voltage was 20 kV, and the extraction delay time was 200 ns. Each spectrum corresponded to ions obtained from 240 laser shots performed in six regions of Cluster analysis the same spot and automatically acquired using the AutoXecute of the Flex Control v.2.4 software (Bruker Daltonics, Bremen, Cluster analysis on MSP (MSP, Main Spectrum Profile) spectra Germany). The spectrum profiles obtained from mosquito legs was performed and the comparison of the main spectra given and bloody WFPs were visualized with Flex analysis v.3.3 software by the MALDI-Biotyper software was clustered according to pro- and were exported to ClinProTools version v.2.2 (Bruker Daltonics, tein mass profile (i.e. their mass signals and intensities). We per- Bremen, Germany) and MALDI-Biotyper v.3.0. (Bruker Daltonics, formed hierarchical clustering of the mass spectra of two Bremen, Germany) for data processing (smoothing, baseline sub- specimens per mosquito species using the MSP dendrogram func- traction, peak picking) and evaluated using cluster analysis. tion. The clustering analyses were performed to visualize the Spectra of low quality were excluded from the study. homogeneity level of MS spectra from specimens belonging to the same species level. The resulting MSP dendrogram shows how samples are related to one another. MALDI-TOF identification of mosquitoes We used our in-lab arthropod MALDI-TOF database, which Molecular identification includes spectra obtained from various arthropods listed in Table 1. The database was upgraded with the spectra of three A molecular tool was used to confirm MALDI-TOF MS identifi- Culex quinquefasciatus mosquitoes and one Culex neavei mos- cation in randomly selected mosquitoes. Molecular identification quito collected and molecularly identified during this study. A was also conducted for specimens whose spectra did not match with comparison of the spectrum of each specimen of mosquito legs any mosquito spectrum in our database. When it was demonstrated from Mali was evaluated against the home-made MS reference that a high quality spectrum had been obtained from a mosquito spectra database using the MALDI-Biotyper software v3.0. tool species missing from our database, this new spectrum was added (Bruker Daltonics, Bremen, Germany). The level of significance to the database. DNA extractions from individual mosquito was determined using the log score values (LSVs) provided by heads and thorax samples were performed using the EZ1 DNA the MALDI-Biotyper software v.3.3. corresponding to a matched Tissue Kit (Qiagen, Hilden, Germany) according to the manufac- degree of signal intensities of mass spectra of the query and the turer’s recommendations. A set of primers specifically amplifying reference spectra. LSVs ranged from zero to three. To determine a fragment of 710 bp of the mosquito’s cytochrome c oxidase I the origin of blood meals, MALDI-TOF MS spectra from the gene (mCOI) was used (LCO1490 (forward): 5’-GGTCAAC

Table 1. List of the arthropod species present in our home-made MALDI-TOF MSa database.

Mosquitoes Imago: Aedes albopictus, Ae. excrucians, Ae. vexans, Ae. rusticus, Ae. dufouri, Ae. cinereus, Ae. fowleri, Ae. aegypti, Ae. caspius, Anopheles gambiae Giles, An. coluzzii, An. funestus, An. ziemanni, An. arabiensis, An. wellcomei, An. rufipes, An. pharoensis, An. coustani, An. claviger, An. hyrcanus, An. maculipennis, Culex quinquefasciatus, Cx. pipiens, Cx. modestus, Cx. insignis, Cx. neavei, Mansonia uniformis, Culiseta longiareolata, Orthopodomyia reunionensis, Coquillettidia richiardii and Lutzia tigripes. Larvae: Aedes aegypti, Ae. albopictus, Anopheles gambiae Giles, An. coluzzii, Cx. pipiens, Cx. molestus, Culiseta sp. Sand flies Phlebotomus papatasi, P. (Larrousius) longicuspis, P. (Larrousius) perfiliewi, P. (Larrousius) perniciosus, P. (Paraphlebotomus) sergenti and Sergentomyia minuta Triatomines Triatoma infestans, Rhodnius prolixus, Rh. pictipes, Rh. robustus, Eratyrus mucronatus and Panstrongylus geniculatus Ticks Legs: Amblyomma cohaerens, Am. gemma, Am. variegatum, Dermacentor marginatus, D. reticulatus, Haemaphysalis leachi, Hae. concinna, Hae. spinulosa, Hyalomma marginatum rufipes, H. truncatum, H. detritum, Rhipicephalus decoloratus, Ixodes hexagonus, I. ricinus, Rh. bergeoni, Rh. e. evertsi, Rh. praetextatus, Rh. pulchellus, Rh. sanguineus, Rh. sulcatus, Rh. microplus, Rh. annulatus, Rh. turanicus and Rh. bursa. Hemolymph: Am. variegatum, D. marginatus, H. marginatum rufipes, Rh. bursa and Rh. sanguineus. Mites Leptotrombidium chiangraiensis, L. imphalum and L. deliense Bedbugs Cimex lectularius Lice Pediculus humanus, Damalinia bovis, D. caprae, D. ovis, Haematopinus eurysternus, Linognatus vituli and L. africanus Fleas Ctenocephalides felis, Ct. canis, Archaeopsylla erinacei, Xenopsylla cheopis and Stenoponia tripectinata Abdomen of mosquitoes engorged Anopheles gambiae Giles fed on: Homo sapiens, Equus caballus, Ovis aries, rabbit, Balb/C mouse, Rattus norvegicus, Canis familiaris, Bos taurus, Capra hircus, Gallus gallus, Equus asinus, Tapirus indicus, Tapirus terrestris, Carollia perspicillata, Thraupis episcopus, Erythrocebus patas and Callithrix pygmaea blood Aedes albopictus fed on: Homo sapiens blood Anopheles gambiae Giles blood meals from Ovis aries and Homo sapiens blood Whatman Filter paper

aMALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. 75

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AAATCATAAGATATTGG-3’; HC02198 (reverse): 5’-TAAAC 168 in Donéguébougou, 230 in Sotuba, 125 in Missabougou and TTCAGGGTGACCAAAAAATCA-3’ (Folmer et al. 1994). We 85 in the Yirimadio semi-urban zones of Bamako (Fig. 1). All spe- used gene Acetylcholinesterase-2 to amplify a fragment of cimens collected were morphologically identified to genus level as 610 bp of Culex pipiens and a fragment of 274 bp of Cx. quinque- Anopheles spp. (287/865; 33.18%), Culex spp. (573/865; 66.24%) fasciatus. The primers set were ACEquin (forward): 5′-CCTT and Aedes spp. (5/865; 0.58%). CTTGAATGG CTGTGGCA-3′, ACEpip (forward): 5′-GGAAA For MALDI-TOF analysis, MS spectra of good quality were CAACGACGTATGTACT-3′,B1246s(reverse):5′-TGGAGCC obtained from 272 legs of Anopheles spp. Of these 272 TCCTCTTCACGGC-3′ (Smith and Fonseca, 2004). Anopheles spp. tested against the arthropod MS database, 97% A set of primers specifically amplifying a fragment of 310 bp of (n = 264/272) were identified with a log score value (LSV) ranging the Anopheles gambiae mosquito complex Acomplex_28S_MBF between 1.70 and 2.575. These 264 Anopheles specimens were 5′-AGCKCGTCTTGGTCTGGGG-3′ and Acomplex_28S_MBR identified as Anopheles gambiae Giles (95.80%, n = 253/264), 5′-GCCGACAAGCTCAYTAGTGT-3′ was designed in our labora- Anopheles coluzzii (3.40%, n = 9/264) and Anopheles arabiensis tory based on the work by Fanello et al. and PCR reactions were (0.80%, n = 2/264) (Fig. 2) by MALDI TOF MS. The remaining processed as described (Fanello et al. 2002). Molecular identifica- eight Anopheles spp. were subjected to molecular identification. tion of the blood was carried out on the bloody WFPs from 41 We tested the MS spectra from the legs of 549 Culex spp. specimens randomly selected from the Malian samples, as previ- against our arthropod database. ously described (Niare et al. 2016). Positive PCR products were Of these 549 Culex spp. high-quality spectra, 98% (n = 537/549) then purified and sequenced using the same primers with the were identified as species contained in our database. The 537 Culex BigDye version 1-1 Cycle Sequencing Ready Reaction Mix specimens were identified by MALDI-TOF MS as Cx. quinquefas- (Applied Biosystems, Foster City, CA) and an ABI 3100 automated ciatus (98%, n = 527/537) and Cx. neavei (2%, n = 10/537) from sequencer (Applied Biosystems, Foster City, CA). The sequences Mali (Fig. 3). These 537 Culex obtained LSVs ranging from were assembled and analyzed using the ChromasPro software (ver- 1.713 to 2.611. The remaining twelve Culex spp. were subjected sion 1.34) (Technelysium Pty. Ltd., Tewantin, Australia) and the to molecular identification. NCBI BLAST website (http://blast.ncbi.nlm.nih.gov). The five Aedes specimens were identified by MALDI-TOF MS as Aedes fowleri (n = 4) and Aedes aegypti (n = 1), with log score values ranging between 2.128 and 2.418. Results The MS spectra comparison from different mosquito species with Flex analysis software revealed an intra-species reproducibil- Identification of the mosquitoes by MALDI-TOF MS ity and an inter-species specificity (Fig. 4). Visually, the signals A total of 865 mosquitoes were captured by aspiration in Mali and intensity of mosquito species’ protein profiles (Fig. 4) were from various collection sites, including 257 in Bougoula-hameau, consistent for MALDI-TOF identification and revealed eight

Fig. 1. Ecological patterns and geographic distribution of mosquito collection in Mali. Sikasso: Bougoula-hameau (rural area), Bamako: Sotuba (peri-urban area), Missabougou, Yirimadio (urban areas) and Kati: Doneguebougou (rural area). 76

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Fig. 2. MALDI-TOF MS Identification of 272 Anopheles spp. collected in Mali.

Fig. 3. MALDI-TOF MS Identification of 549 Culex spp. captured in Mali.

different species, namely Anopheles gambiae Giles, An. coluzzii, from two specimens per mosquito species was used to generate An. arabiensis, Cx. quinquefasciatus, Cx. neavei, Culex perexiguus, a dendrogram. Clustering analysis revealed a gathering on distinct Ae. fowleri and Ae. aegypti. Clustering analysis of MSP spectra branches, following the eight species which were loaded 77

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Fig. 4. Comparison of MALDI-TOF MS profiles of eight mosquito species collected in Mali. Spectra analysis was performed using Flex analysis 3.3 software. Abbreviations: a.u., arbitrary units; m/z, mass-to-charge ratio.

(Anopheles gambiae Giles, An. coluzzii, An. arabiensis, Cx. quin- The molecular results were found to be highly consistent with the quefasciatus, Cx. neavei, Cx. perexiguus, Ae. fowleri and Ae. aegypti) MALDI-TOF MS identification. Sequences obtained from Cx. (Fig. 5). The clusters formed were consistent with the intra-species quinquefasciatus and Cx. neavei were shown to share between reproducibility and inter-species specificity visually observed on 98.90 and 100% identity with Genbank (Table 2). protein profiles. Molecular biology was also carried out on the mosquitoes that were not identified by MALDI-TOF MS (low scores), including the eight Anopheles spp. and 12 Culex spp. Sequencing of the Molecular identification of mosquitoes collected in Mali 28S gene was performed to identify the eight Anopheles spp. Molecular biology was performed to confirm the mosquito iden- (3%, n = 8/264). The matching sequences corresponded to tification resulting from the MALDI-TOF MS analyses. For this Anopheles gambiae Giles (n = 4) and An. coluzzii (n = 4), which purpose, we randomly selected 20/253 An. gambiae Giles, 2/9 were shown to share between 98.52 and 100% identity with An. coluzzii, 15/527 Cx. quinquefasciatus, 1/10 Cx. neavei for Genbank (Table 2). sequencing. The 28S gene sequencing of Anopheles corroborated The acetylcholinesterase-2 and COI genes were amplified to the MALDI-TOF MS identification in all cases, with between identify the 12 Culex spp. which were misidentified (2%, n = 12/ 97.51 and 99.27% identity with Genbank sequences (Table 2). 549) by MALDI-TOF MS. The sequences obtained correspond The acetylcholinesterase-2 and COI genes were used to identify to the Cx. quinquefasciatus (n = 11) which were shown to share the Culex species. Sixteen specimens of Cx. quinquefasciatus (n = between 98.90 and 100% identity with Genbank and 100% iden- 15) and Cx. neavei (n = 1) were randomly selected for sequencing. tity with Cx. perexiguus (n =1)(Table 2).

Fig. 5. MSP (Main Spectrum Profile) dendrograms of MALDI-TOF MS spectra of eight mosquito species collected in Mali. Clustering analysis was performed using MALDI Biotyper software. Distance unit corresponds to the relative similarity calculated from the distance matrix. 78

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Table 2. Molecular identification of mosquitoes collected in Mali

%%"$ ((( #!#!# Collection sites Morphological identification MALDI-TOF MS identification Log score value Genes Molecular identification % Identities Genbank Accession number

Sotuba Anopheles spp. Human [1.754] 28S Anopheles coluzzii 100 AF470112.1 Donéguébougou Anopheles spp. Anopheles gambiae Giles [2.470] 28S Anopheles gambiae Giles 99.25 AF470116.1 Donéguébougou Anopheles spp. Human [1.854] 28S Anopheles coluzzii 98.51 AF470113.1 %%"$ !!# Donéguébougou Anopheles spp. Less relevant [1.553] 28S Anopheles coluzzii 98.51 AF470113.1 Donéguébougou Anopheles spp. Less relevant [1.610] 28S Anopheles coluzzii 99.25 KT284724.1

'#$%%## ! % $&% Donéguébougou Anopheles spp. Human [2.008] 28S Anopheles gambiae Giles 98.14 AF470116.1 Donéguébougou Anopheles spp. Anopheles coluzzii [1.864] 28S Anopheles coluzzii 98.51 AF470113.1 Bougoula -hameau Anopheles spp. Anopheles gambiae Giles [2.383] 28S Anopheles gambiae Giles 98.88 AF470116.1 Bougoula-hameau Anopheles spp. Anopheles gambiae Giles [2.389] 28S Anopheles gambiae Giles 99.25 AF470115.1 Bougoula-hameau Anopheles spp. Anopheles gambiae Giles [2.404] 28S Anopheles gambiae Giles 99.25 AF470116.1 Bougoula-hameau Anopheles spp. Less relevant [1.581] 28S Anopheles gambiae Giles 98.51 AF470115.1 Bougoula-hameau Anopheles spp. Human [2.286] 28S Anopheles gambiae Giles 98.88 AF470115.1 Bougoula-hameau Anopheles spp. Human [1.961] 28S Anopheles gambiae Giles 98.88 AF470116.1 Bougoula-hameau Anopheles spp. Anopheles coluzzii [1.964] 28S Anopheles coluzzii 98.51 AF470113.1 79 Bougoula -hameau Anopheles spp. Anopheles gambiae Giles [2.237] 28S Anopheles gambiae Giles 99.27 AF470116.1 Bougoula -hameau Anopheles spp. Anopheles gambiae Giles [2.406] 28S Anopheles gambiae Giles 99.27 AF470116.1 Bougoula -hameau Anopheles spp. Anopheles gambiae Giles [2.390] 28S Anopheles gambiae Giles 98.59 AF470116.1 %!% #!#%# $!&$'% Missabougou Anopheles spp. Anopheles gambiae Giles [2.099] 28S Anopheles gambiae Giles 99.27 AF470116.1 Missabougou Anopheles spp. Anopheles gambiae Giles [2.401] 28S Anopheles gambiae Giles 98.90 AF470116.1 Missabougou Anopheles spp. Anopheles gambiae Giles [2.283] 28S Anopheles gambiae Giles 98.54 AF470116.1 Missabougou Anopheles spp. Anopheles gambiae Giles [2.258] 28S Anopheles gambiae Giles 98.90 AF470116.1 Missabougou Anopheles spp. Anopheles gambiae Giles [2.368] 28S Anopheles gambiae Giles 98.91 AF470116.1 Bougoula-hameau Anopheles spp. Anopheles gambiae Giles [2.293] 28S Anopheles gambiae Giles 98.16 AF470116.1 Bougoula-hameau Anopheles spp. Anopheles gambiae Giles [2.293] 28S Anopheles gambiae Giles 98.13 AF470116.1 Bougoula-hameau Anopheles spp. Anopheles gambiae Giles [2.049] 28S Anopheles gambiae Giles 99.26 AF470116.1 Bougoula-hameau Anopheles spp. Anopheles gambiae Giles [2.311] 28S Anopheles gambiae Giles 97.51 AF470116.1 Bougoula-hameau Anopheles spp. Anopheles gambiae Giles [1.704] 28S Anopheles gambiae Giles 97.84 AF470116.1 Bougoula-hameau Anopheles spp. Anopheles gambiae Giles [2.110] 28S Anopheles gambiae Giles 98.50 AF470116.1 Bougoula-hameau Anopheles spp. Anopheles gambiae Giles [2.233] 28S Anopheles gambiae Giles 97.70 AF470116.1 Donéguébougou Anopheles spp. Anopheles gambiae Giles [2.204] 28S Anopheles gambiae Giles 98.13 AF470116.1 Bougoula-hameau Culex spp. Culex pipiens [2.022] Ace2 Culex quinquefasciatus 99.26 FJ416029.1

(Continued) 7 %%"$ ((( #!#!#%# $ !( !#! 8 %%"$ ((( #!#!# Table 2. (Continued.)

Collection sites Morphological identification MALDI-TOF MS identification Log score value Genes Molecular identification % Identities Genbank Accession number

Bougoula-hameau Culex spp. Culex pipiens [2.229] Ace2 Culex quinquefasciatus 98.90 FJ416029.1 %%"$ !!# Donéguébougou Culex spp. Culex quinquefasciatus [1.791] Ace2 Culex quinquefasciatus 99.26 FJ416029.1 Missabougou Culex spp. Human [2.182] COI Culex quinquefasciatus 99.38 KU920694.1

'#$%%## ! % $&% Missabougou Culex spp. Less relevant [1.610] COI Culex perexiguus 100 KU380476.1 Missabougou Culex spp. Culex pipiens [1.761] Ace2 Culex quinquefasciatus 99.63 FJ416025.1 Missabougou Culex spp. Human [2.042] COI Culex quinquefasciatus 99.08 KU920694.1 Missabougou Culex spp. Culex pipiens [2.139] Ace2 Culex quinquefasciatus 98.90 FJ416025.1 Missabougou Culex spp. Culex pipiens [2.231] Ace2 Culex quinquefasciatus 98.90 FJ416025.1 Missabougou Culex spp. Human [2.514] COI Culex quinquefasciatus 99.84 KU920694.1 Missabougou Culex spp. Culex pipiens [2.088] Ace2 Culex quinquefasciatus 98.90 FJ416029.1 Missabougou Culex spp. Human [2.294] COI Culex quinquefasciatus 99.69 KU920694.1 Yirimadio Culex spp. Culex pipiens [2.032] Ace2 Culex quinquefasciatus 98.90 FJ416029.1

80 Donéguébougou Culex spp. Culex quinquefasciatus [2.000] Ace2 Culex quinquefasciatus 99.26 FJ416029.1 Donéguébougou Culex spp. Culex quinquefasciatus [2.009] Ace2 Culex quinquefasciatus 99.27 FJ416025.1 Donéguébougou Culex spp. Culex quinquefasciatus [2.207] Ace2 Culex quinquefasciatus 98.90 FJ416025.1

%!% #!#%# $!&$'% Bougoula-hameau Culex spp. Culex quinquefasciatus [2.406] Ace2 Culex quinquefasciatus 98.90 FJ416019.1 Bougoula-hameau Culex spp Culex neavei [2.839] COI Culex neavei 99.16 KU380473.1 Bougoula-hameau Culex spp Culex quinquefasciatus [2.037] Ace2 Culex quinquefasciatus 99.11 FJ416029.1 Bougoula-hameau Culex spp Culex quinquefasciatus [2.197] Ace2 Culex quinquefasciatus 99.55 FJ416029.1 Bougoula-hameau Culex spp Culex quinquefasciatus [2.146] Ace2 Culex quinquefasciatus 99.56 FJ416029.1 Bougoula-hameau Culex spp Culex quinquefasciatus [2.383] Ace2 Culex quinquefasciatus 99.56 FJ416029.1 Bougoula-hameau Culex spp Culex quinquefasciatus [2.322] Ace2 Culex quinquefasciatus 99.13 FJ416029.1 Donéguébougou Culex spp. Culex quinquefasciatus [2.445] Ace2 Culex quinquefasciatus 99.57 FJ416029.1 aamuo Tandina Fatalmoudou Donéguébougou Culex spp. Culex quinquefasciatus [2.067] Ace2 Culex quinquefasciatus 98.71 FJ416029.1 Donéguébougou Culex spp. Culex quinquefasciatus [2.151] Ace2 Culex quinquefasciatus 100 FJ416029.1 Missabougou Culex spp. Culex quinquefasciatus [2.244] Ace2 Culex quinquefasciatus 98.68 FJ416029.1 Missabougou Culex spp. Culex quinquefasciatus [2.159] Ace2 Culex quinquefasciatus 99.56 FJ416029.1

MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; Ace 2: acetylcholinesterase-2; COI: the cytochrome c oxidase; %, per cent. tal. et %%"$ ((( #!#!#%# $ !( !#! Parasitology %%"$ ((( #!#!# %%"$ !!#

Table 3. Identification of the blood meals of mosquitoes collected in distinct ecological areas in Mali

'#$%%## ! % $&% Blood meals identified by MALDI-TOF MS

Sites Morphological ID Mosquito identified by MALDI-TOF MS Human Chicken Cow Donkey Dog Sheep Not identified Total

Bougoula-hameau Anopheles Anopheles gambiae Giles 87 3 5 3 98 Anopheles Anopheles coluzzii 3 3 Culex Culex quinquefasciatus 86 5 1 92 Culex Culex neavei 3 3 Donéguébougou Anopheles Anopheles gambiae Giles 46 6 1 2 4 59 Anopheles Anopheles coluzzii 5 5

81 Anopheles Anopheles arabiensis 2 2 Culex Culex quinquefasciatus 93 1 1 5 100 Missabougou Anopheles Anopheles gambiae Giles 13 215

%!% #!#%# $!&$'% Culex Culex quinquefasciatus 69 1 12 82 Sotuba Anopheles Anopheles gambiae Giles 51 3 1 6 61 Culex Culex quinquefasciatus 97 299 Culex Culex neavei 1 1 Yirimadio Aedes Aedes aegypti 1 1 Culex Culex quinquefasciatus 61 2 16 79 Culex Culex neavei 1 1 Total 619 9 9 6 5 3 50 701

ID, Identification; MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. 9 10 Fatalmoudou Tandina et al.

Identification of the bloody WFPs sources by MALDI-TOF MS results of MALDI TOF MS identification (Table 4). The sequences obtained from seventeen bloody WFPs had identities A total 701 abdomens of engorged mosquitoes were crushed in between 98.52 and 100% against Genbank NCBI (Table 4). WFPs in the field in Mali. The 701 bloody BWFPs were submitted for MALDI-TOF MS analysis in Marseille one month after sampling. Of the 701 BWFPs, 651 (93%) high-quality spectra were Discussion obtained. The 651 BWFPs MS high-quality spectra were queried against our blood source MALDI-TOF MS database for identifi- The goal of this work was not to provide precise data on the pres- cation. They matched with spectra from our database, including ence and abundance of various mosquito species in specific areas those of mosquito abdomens engorged with human blood in Mali. Indeed, these data vary according to the type of climate (n = 619), chicken blood (n = 9), cow blood (n = 9), donkey and the seasons. However, we did want to test the usefulness of blood (n = 6), dog blood (n = 5) and sheep blood (n =3) MALDI-TOF MS using mosquitoes collected in the field, as (Table 3). These blood meals were identified using most preliminary studies have used laboratory specimens. MALDI-TOF MS with log score values (LSVs) ranging from The use of MALDI-TOF MS has recently emerged in medical 1.707 to 2.731. The MS spectra comparison of different host entomology, including for the identification of arthropods, their blood revealed an intra-species reproducibility and an inter- blood meals and the detection of potential microorganisms species specificity by Flex analysis (Fig. 6). (Schaffner et al. 2014; Yssouf et al. 2016). The choice of the arthropod body part is critical for specimen identification by MALDI-TOF MS (Yssouf et al. 2016). For example, the legs from adult mosquitoes have been shown to be sufficient for iden- Molecular identification of the bloody mosquito WFPs tification, whereas whole specimens have been used for aquatic A total of 41 bloody WFPs identified by MALDI-TOF MS as stages (larvae) (Nebbak et al. 2017). mosquito abdomens engorged with human blood (n = 21), don- Here, the MS spectra from mosquito legs collected in Mali, key blood (n = 5), chicken blood (n = 4), cow blood (n = 5), dog including 264 Anopheles, 549 Culex and five Aedes, permitted blood (n = 4) and sheep blood (n = 2) were randomly selected MALDI-TOF MS identification. The MS spectrum analyses for sequencing by COI gene amplification. Thirty-three bloody from the mosquito legs revealed an intra-species reproducibility WFPs sequences were obtained which confirmed the accuracy and inter-species specificity consistent with molecular validation of the MS identification. However, for eight bloody WFPs, no (Fig. 5). Accurate identification of mosquitoes queried against quality sequences could be obtained. The results of the PCR the home-made MS database corresponded to 100% concordance based on bloody WFP sequencing highly correlated with the with molecular identification results (Table 2). The consistent

Fig. 6. The MS spectrum alignment from mosquito abdomen engorged on vertebrate host bloods and then crushed on Whatman filters. All bloody WFPs (BWFPs) were obtained from the field mosquitoes collected in Mali and crushed on WFPs. The MS spectrum alignment was performed by Flex analysis 3.3 software. The WFP blood free corresponds to the MS profiles of WFPs where no mosquito blood meal was released. The representative MS spectra from abdominal protein corresponds to Anopheles gambiae Giles abdomens BWFPs feed on human, donkey, cow and sheep blood, and Culex quinquefasciatus abdomens feed on chicken and dog blood. a.u. arbitrary units; m/z mass-to-charge ratio. 82

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Table 4. Molecular identification of the blood from mosquito’s abdomens identification between molecular biology and MALDI-TOF MS crushed on Whatman filter papers was validated by the choice of the 28S gene for Anopheles species Mosquito blood Mosquito blood identification, and the acetylcholinesterase-2 and COI genes for meals sources meals sources Culex species identification. As shown in previous studies, the identification identification by % Accession choice of these genes was highly relevant to discriminate and by MALDI-TOF COI gene Identities number assess the phylogenetic relation between different mosquito spe- MS amplification Genbank Genbank cies (Folmer et al. 1994; Fanello et al. 2002; Smith and Fonseca, Human Homo sapiens 100 KM102136.1 2004). Here, the quality of spectra was a very important element for identification, as more than 98% of the good quality spectra Human Homo sapiens 99.84 KY595668.1 were identified with LSVs >1.8. The MALDI-TOF MS reference Human Homo sapiens 99.64 HM185231.1 database has been updated with other mosquito species. It is Human Homo sapiens 98.52 KM102136.1 necessary to create a reference database, which could subsequently be shared, and open access could be provided for routine arthro- Human Homo sapiens 98.86 KY595669.1 pod identification. In this study, Aedes mosquitoes collected in Human Homo sapiens 98.72 MF058292.1 Mali were correctly identified as Ae. fowleri using a database con- Human Homo sapiens 99.68 KF161694.1 taining reference spectra of this species collected from La Reunion Island only, which is located in the Pacific Ocean. Therefore Human Homo sapiens 99.68 MF058210.1 MALDI-TOF MS appears as an efficient tool for the identification Human Homo sapiens 99.34 MF058210.1 of arthropods collected from distant geographical areas. Human Homo sapiens 100 MF058210.1 For Raharimalala et al. (2017), the usefulness and accuracy of MALDI-TOF MS as a tool to identify vector mosquito species Human Homo sapiens 99.37 MF058210.1 requires the creation of an international database (Raharimalala Human Homo sapiens 98.68 MF058210.1 et al. 2017). In this study, 2.40% of inconsistent MS leg results Human Homo sapiens 99.67 MF058210.1 were attributed to low-quality MS spectra for identification. The MS spectra of some legs (n =9)(Table 2) that matched with ref- Human Homo sapiens 99.18 AY275535.2 erence spectra of mosquito abdomens engorged with human Human Homo sapiens 99.52 MF588867.1 blood were attributed to traces of blood on the legs during the Human Homo sapiens 99.36 MF057217.1 abdomen crushing process onto WFPs. This phenomenon of low- quality spectra, leading to lower identification rates have been Human Homo sapiens 99.18 MF588867.1 reported in arthropod identification such as at the aquatic mos- Human Homo sapiens 99.05 AY922271.1 quito stage (Dieme et al. 2014). According to the reproducibility Human Homo sapiens 99.52 MF588867.1 of MS spectra, the hierarchical clustering showed that all specimens from the same species were grouped in the same branch. Human Homo sapiens 99.35 KM101695.1 These results are similar to previous studies supporting inter- Human Homo sapiens 99.36 KF163046.1 species reproducibility for mosquito identification (Yssouf et al. 2013). Additionally, we stress that MS cannot yet be considered Donkey Equus asinus 99.37 KX683425.1 a reliable tool for the phylogenetic study of mosquito species Chicken Gallus gallus 99.68 KX781318.1 (Yssouf et al. 2013). Chicken Gallus gallus 99.22 KX781318.1 Our results showed that 95% of the collected mosquitoes had fed on human blood. This result is not surprising because all mos- Chicken Gallus gallus 99.21 KX781318.1 quitoes were collected inside homes. The advantage of our Cow Bos taurus 99 KY650678.1 MALDI-TOF approach is its rapidity, effectiveness and reliability Donkey Equus asinus 98.91 KX683425.1 in determining bloody WFPs, since more than 100 bloody WFPs specimens were processed per day. Previously, the authors had Cow Failed –– demonstrated that the profiles of abdominal spectra of mosquito Cow Bos taurus 99 KY650678.1 females engorged on human blood are the same, regardless of Cow Failed –– whether they were crushed or not crushed on WFPs (Niare et al. 2017). Indeed, the home-made database contains filter Cow Failed –– papers with Anopheles gambiae engorged blood such as human Donkey Failed –– blood and sheep blood. These authors tested WFP either with Chicken Gallus gallus 98.52 KX781318.1 the crushed abdomen of a non-engorged mosquito or simply as a control (Niare et al. 2017). These results suggest that MALDI- Dog Failed –– TOF MS is not time-consuming in comparison with molecular Dog Failed –– tools and serological techniques. The eight bloody WFPs which Dog Failed –– failed molecular biology identification may be attributed to blood meal digestion. As previously reported, the time of the Donkey Equus asinus 99.52 KX683425.1 host blood digestion in the mosquito has an impact upon blood Donkey Equus asinus 100 KX683425.1 meal identification by MALDI-TOF MS and molecular biology Sheep Ovis aries 99.84 KP998473.1 (Niare et al. 2016). Moreover, the molecular biology results of the seventeen BWFPs sequences obtained by COI gene amplifica- Sheep Ovis aries 99.21 KR868678.1 tion corroborated the MALDI-TOF MS identification (Table 4). Dog Failed –– Interestingly, as we have recently found that MALDI-TOF may MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass also recognize mixed blood meals (unpublished), we did not find spectrometry; COI, the cytochrome c oxidase; %, per cent. any mixed blood meals either by molecular tools nor by MALDI-TOF. The authors experimentally engorged An. gambiae Giles mosquitoes with a mixture of blood from distinct vertebrate 83

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hosts, such as human, sheep and dogs. Their results demon- Author contributions. PP, TF and NS designed and developed the protocol. strate that mixed mosquito blood meals can be successfully TF and NS performed the protocol. PP, TF, NS and ML analysed the data. identified, depending on the concentration ratio (unpublished). KKA, DZA, OA, BMJ, OD and RD contributed reagents/materials/analysis Recently, some authors have also used the proteomic approach tools. PP, TF and NS wrote the paper. OD and RD contributed to editing to identify the sources of tick mixed blood meals (Onder et al. the paper. All authors agreed to publication. 2013). Financial support. The project has received funding from the Excellence Of the mosquitoes identified by MALDI-TOF MS, A. gambiae Initiative of Aix-Marseille University – A*MIDEX, a French ‘Investissements Giles and Cx. quinquefasciatus were widely distributed across all d’Avenir’ program (No. ANR-11-IDEX-0001-02) and grants from the collection sites. Our work enabled Cx. neavei and Cx. perexiguus Malian Minister of Health, the Foundation Mérieux, UMI3189, and the to be detected for the first time in Mali. Currently, few studies Malian Research and Training Center (MRTC) for the field specimen have been carried out on the Culex species in Mali, particularly collection. on their abundance, ecology and the infectious pathogens transmitted by these vectors. Culex species are widely distributed in West Africa and are found in any type of breeding sites (clear References and polluted water), whereas the Anopheles species colonizes Anosike JC, Nwoke BE, Ajayi EG, Onwuliri CO, Okoro OU, Oku EE, sunny, fresh water (Becker et al. 2010). There is an abundant lit- Asor JE, Amajuoyi OU, Ikpeama CA, Ogbusu FI and Meribe CO erature on these mosquitoes, the well-known distribution of Cx. (2005) Lymphatic filariasis among the Ezza people of Ebonyi state, neavei and Cx. perexiguus in sub-Saharan Africa and their impli- Eastern Nigeria. Annals of Agricultural and Environnemental Medicine cation in the transmission of many arboviruses (Jupp et al. 1986; 12, 181–186. Fyodorova et al. 2006; Nikolay et al. 2012; Fall et al. 2014; Gould Bass C, Williamson MS, Wilding CS, Donnelly MJ and Field LM (2007) et al. 2017). The presence of these potential vectors in Mali might Identification of the main malaria vectors in the Anopheles gambiae species complex using a TaqMan real-time PCR assay. Malaria Journal 6, 155. be of epidemiological importance. Becker N, Petriæ D, Zgomba M, Boase C, Dahl C, Madon M and Kaiser A Our study is the first to use MALDI-TOF MS as a tool for (2010) Mosquitoes and Their Control, 2nd Edn. Heidelberg, Germany: monitoring field mosquitoes in Africa, particularly in Mali, an Springer. endemic malarial area. Moreover, when the MALDI-TOF MS Brasil P, Zalis MG, de Pina-Costa A, Siqueira AM, Junior CB, Silva S, device is bought for clinical microbiology purposes, it can also Areas ALL, Pelajo-Machado M, de Alvarenga DAM, da Silva be used for medical entomology at no additional cost. For Santelli ACF, Albuquerque HG, Cravo P, Santos de Abreu FV, example, at the Dakar hospital in Senegal, the MALDI-TOF MS Peterka CL, Zanini GM, Suarez Mutis MC, Pissinatti A, equipment that was initially bought for clinical microbiology Lourenco-de-Oliveira R, de Brito CFA, de Fatima Ferreira-da-Cruz, has been used for field entomology surveys and has successfully Culleton R and Daniel-Ribeiro CT (2017) Outbreak of human malaria caused by in the Atlantic forest in Rio de Janeiro: a identified Culicoides (Sambou et al. 2015). In Senegal, the acqui- Plasmodium simium molecular epidemiological investigation. Lancet Global Health 5,1038–1046. sition of MALDI-TOF MS equipment has revolutionized bacteri- Caglioti C, Lalle E, Castilletti C, Carletti F, Capobianchi MR and Bordi L ology laboratories and clinical microbiology domains, suggesting (2013) Chikungunya virus infection: an overview. New Microbiology 36, that this technique can be used as a front-line tool in tropical 211–227. countries (Lo et al. 2015). de Wispelaere M, Despres P and Choumet V (2017) European Aedes albopic- Although the time period for blood meal source determin- tus and Culex pipiens Are competent vectors for Japanese encephalitis virus. ation by MALDI-TOF MS was shorter than that of molecular PLoS Neglected Tropical Diseases 11, e0005294. biology or ELISA, the rapidity and low cost of the reagents Dieme C, Yssouf A, Vega-Rua A, Berenger JM, Failloux AB, Raoult D, made this proteomic method a financial and reliable competi- Parola P and Almeras L (2014) Accurate identification of Culicidae at tive strategy. However, the relatively high cost of the machine aquatic developmental stages by MALDI-TOF MS profiling. Parasites &Vectors 7, 544. could be an impediment to implementation of this innovative Fall G, Diallo M, Loucoubar C, Faye O and Sall AA (2014) Vector compe- tool in laboratories. The cost of purchasing the MALDI-TOF tence of Culex neavei and Culex quinquefasciatus (Diptera: Culicidae) from MS equipment in under-developed countries such as Mali Senegal for lineages 1, 2, koutango and a putative new lineage of west Nile (sub-Saharan Africa) could be a limitation to estimating the virus. American Journal of Tropical Medicine and Hygiene 90, 747–754. local vector-borne risk. However, when the device is bought Fanello C, Santolamazza F and della TA, (2002) Simultaneous identification by a microbiology lab it can be used in medical entomology of species and molecular forms of the Anopheles gambiae complex by at no additional cost. PCR-RFLP. Medical and Veterinary Entomology 16, 461–464. Folmer O, Black M, Hoeh W, Lutz R and Vrijenhoek R (1994) DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology Concluding remarks 3, 294–299. Fyodorova MV, Savage HM, Lopatina JV, Bulgakova TA, Ivanitsky AV, The present study successfully identified field mosquitoes and the Platonova OV and Platonov AE (2006) Evaluation of potential west Nile sources of their blood meals using MALDI-TOF MS. The mos- virus vectors in volgograd region, Russia, 2003 (Diptera: Culicidae): species quitoes collected in Mali were correctly identified based on repro- composition, bloodmeal host utilization, and virus infection rates of mos- ducibility and specificity from the protein profiles of leg extracts. quitoes. Journal of Medical Entomology 43, 552–563. The innovative MALDI-TOF MS tool enabled the rapid identifi- Gardner CL and Ryman KD (2010) Yellow fever: a reemerging threat. Clinics cation of eight mosquito species in Mali during entomological in Laboratory Medicine 30, 237–260. surveys. The challenge is to maintain and develop collaboration Gillies MT and Coetzee M (1987) A supplement to the Anophelinae of Africa between north and south to facilitate the acquisition of the south of the Sahara. South African Institute for Medical Research 55, 143p. MALDI-TOF MS equipment. Gomes B, Sousa CA, Vicente JL, Pinho L, Calderon I, Arez E, Almeida AP, Donnelly MJ and Pinto J (2013) Feeding patterns of molestus and pipiens Acknowledgements. We thank all members of the various MRTC sites forms of Culex pipiens (Diptera: Culicidae) in a region of high hybridiza- where mosquitoes were collected. We would also like to acknowledge all the tion. Parasites & Vectors 6, 93. residents of the various concessions where we captured mosquitoes. Gould E, Pettersson J, Higgs S, Charrel R and de Lamballerie X (2017) Emerging arboviruses: why today? One Health 4,1–13. Competing interests. The authors declare that they have no competing Jupp PG, McIntosh BM and Blackburn NK (1986) Experimental assessment interests. of the vector competence of Culex (Culex) neavei theobald with west Nile 84

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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

L’utilisation du MALDI-TOF MS pour identifier le repas sanguin des moustiques a récemment été expérimentée en 2016 dans notre laboratoire (Niare et al. 2016). Dans notre travail, des femelles de deux espèces de moustiques, Anopheles gambiae et Anopheles coluzzii ont été expérimentalement gorgées sur huit différents types de sang d’animaux à savoir Binturong, Springbok, Mouflon, Dromadaire, Ocelot, Jaguar, Babouin et Porc. Nous avons obtenu les spectres protéiques de 240 abdomens de moustiques gorgés sur ces huit sangs différents. Parmi ceux-ci, des spectres de bonne qualité provenant de 72 abdomens de moustiques obtenus entre 1 h à 24 h après le gorgement ont été sélectionnés pour implémenter notre base de données et les 168 autres restants ont été soumis à une analyse de test en aveugle. Nous avons obtenu 100% d'identification correcte du repas de sang pour les spécimens collectés entre 1h et 24h après le gorgement, tandis que pour les spécimens collectés à 36h et à 60h après l'ingestion, le taux d'identification correcte de l'origine du repas du sang a considérablement diminué. Nous confirmons ici que le MALDI-TOF MS peut être utilisée pour identifier l'origine du repas de sang des moustiques fraîchement gorgés. Ce travail ouvre également des perspectives pour de futures études, notamment l’étude du repas de sang interrompu et mixte des moustiques en utilisant le MALDI-TOF MS.

Para site Blood meal identification of cryptic species Anopheles gambiae Giles and Anopheles coluzzii using MALDI-TOF MS --Manuscript Draft--

Manuscript Number: parasite180027R1

Full Title: Blood meal identification of cryptic species Anopheles gambiae Giles and Anopheles coluzzii using MALDI-TOF MS

Article Type: Short Note Order of Authors: Fatalmoudou Tandina

Maureen Laroche

Bernard Davoust

Ogobara K Doumbo

Didier Raoult

Philippe Parola Corresponding Author: Philippe Parola IHU Mediterranee Infection Marseille, Bouches du Rhône FRANCE

Keywords: Blood meal identification; MALDI-TOF MS; Anopheles coluzzii; Anopheles gambiae

Abstract: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI- TOF MS) identification has recently emerged in entomology. Female Anopheles gambiae and Anopheles coluzzii mosquitoes were experimentally engorged on different animal bloods. We obtained the MS spectra from 240 mosquito abdomens engorged on these eight blood meal sources. Among them, high quality MS spectra from 72 mosquito abdomens, obtained between one and 24 hours post- feeding, were selected to upgrade the in-lab MS arthropod database. The remaining 168 specimens were subjected to a blind test analysis against the database. We obtained 100% correct identification of the blood meal source for the specimens collected between one and 24 hours post-feeding, while for the specimens collected between 36 and 60 hours post-feeding, the rate of correct identification dramatically decreased. Hence, MALDI-TOF MS can be used to identify the blood meal origin of freshly engorged mosquitoes. These results confirm its efficacy as an additional tool for entomological surveys.

Author Comments: Dear Editor, According to reviewers' recommendations, modifications of our manuscript "Blood meal identification of cryptic species Anopheles gambiae Giles and Anopheles coluzzii using MALDI-TOF MS." have been done. We have considered all remarks of the reviewers. However, our manuscript had to be shortened to better fit the format of a short note. Therefore, although all comments were answered in the letter of response, not all of them are associated with visible changes in the manuscript. We also did our best to highlight what this paper is adding in terms of results and perspectives. This work confirms previously published studies on the use of MALDI- TOF MS for the identification of mosquito blood meal and opens new questions, particularly regarding the impact of the mosquito species on the identification of the blood meal. Our present results show little influence but as answered to the reviewers, it would be interesting to evaluate this tool on less related mosquitoes. We hope that our revisions to the originally submitted version of the manuscript will correspond to the reviewer expectations and are in compliance with the Parasite guidelines. The changes included in our manuscript are indicated in the letter "Responses_to_Reviewers_ parasite180027_R1". All authors confirm that the advices to Contributors have been read and that they accepted the conditions set down therein. All named authors have reviewed the revision and they agreed with the revised submission. The manuscript has not been submitted elsewhere for possible publication as long as it is under consideration of Parasite.

Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation 91 Sincerely Yours, Prof. Philippe Parola Corresponding author

Response to Reviewers: A letter of response has been enclosed to this revision as a separate file. Thank you.

Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation 92 Manuscript Click here to download Manuscript Tandina et al- parasite180027_R1.docx

1 Blood meal identification of cryptic species Anopheles gambiae Giles and Anopheles coluzzii using MALDI- 2 TOF MS

3 Fatalmoudou Tandina1,2, Maureen Laroche1, Bernard Davoust3, Ogobara K Doumbo2, Philippe Parola1,*

5 1 Aix Marseille Univ, IRD, AP-HM, SSA, VITROME, IHU-Méditerranée Infection, Marseille, France.

6 2Department of Epidemiology of Parasitic Diseases, Malaria Research and Training Center, University of 7 Science, Techniques and Technologies of Bamako, Bamako, Mali.

8 3 Aix Marseille Univ, IRD, APHM, MEPHI, IHU-Méditerranée Infection, Marseille, France.

9 Emails: FT: [email protected]; ML: [email protected]; BD: [email protected]; 10 OD: [email protected]; PP: [email protected].

12 * Corresponding author: Prof. Philippe Parola. VITROME, Institut Hospitalo Universitaire Méditerranée- 13 Infection, 19-21 Boulevard Jean Moulin 13385 Marseille Cedex 05, France. Phone: + 33 (0) 4 13 73 24 01. Fax: 14 + 33 (0) 4 13 73 24 02.

15 Email: [email protected]

93 29 Abstract

30 Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) identification 31 has recently emerged in entomology for the identification of arthropods and their blood meal source. 32 Female Anopheles gambiae were fed on five host blood includingLeopardus pardalis, Arctictis binturong, 33 Antidorcas marsupialus, Panthera onca and Papio hamadryas while Anopheles coluzzii were fed on three hosts 34 such as Camelus dromedarius, Ammotragus lervia and Sus scrofa. We obtained the MS spectra from 240 35 engorged mosquito abdomens and selected high quality one from 72 mosquito abdomens to upgrade the home- 36 made database. We excluded from the analysis the spectra of low quality (n=80) and the remaining 88 specimens 37 were subjected to a blind test analysis against the home-made database. 38 We obtained 100% correct identification of the blood meal source for the specimens collected, 1,12 and 24 hours 39 post-feeding, whereas for the specimens collected 36 hours post-feeding, the correct identification rate decreased 40 dramatically. We confirm here that MALDI-TOF MS can be used to identify the blood meal origin of freshly 41 engorged mosquitoes and opens new perspective for further studies, including the impact of the mosquito species 42 on blood meal identification. 43 44 Keywords: Blood meal identification; MALDI-TOF MS; Anopheles coluzzii; Anopheles gambiae

46 Identification du repas sanguin des espèces cryptiques Anopheles gambiae Giles et Anopheles coluzzii par 47 l’utilisation du MALDI-TOF MS

48 Résumé

49 L’identification par spectrométrie de masse en temps de vol et de désorption / ionisation assistée par matrice 50 (MALDI-TOF MS) a récemment été appliquée à l’entomologie pour l'identification des arthropodes et 51 l'identification de leur source de repas de sang.

52 Des femelles de moustiques Anopheles gambiae et Anopheles coluzzii ont été expérimentalement gorgées sur 53 huit différents sang d’animaux. Nous avons obtenu les spectres protéiques de 240 abdomens de moustiques, 54 parmi lesquels des spectres de bonne qualité provenant de 72 abdomens de moustiques obtenus entre 1 h à 24 h 55 après le gorgement ont été sélectionnés pour implémenter notre base de données .Nous avons exclu de l’analyse 56 les spectres de mauvais qualités (n=80) et les 88 spectres restants ont été soumis à une analyse de test en 57 aveugle. 58 Nous avons obtenu 100% d'identification correcte de la source de sang pour les spécimens collectés 1h à 24h 59 après le gorgement, tandis que pour les spécimens collectés 36h après l'ingestion, le taux d'identification 60 correcte du repas de sang a considérablement diminué. Nous confirmons ici que le MALDI-TOF peut être 61 utilisée pour identifier l'origine du repas de sang des moustiques fraîchement gorgés et ouvrir une nouvelle 62 perspective pour de futures études, tel que l'impact de l’espèce de moustiques sur l'identification du repas 63 sanguin.

94 65 Introduction

66 The analysis and identification of mosquito blood meals is essential to the study of vector bite behavior 67 (anthropophilic or zoophilic). Several methods have been developed to identify the vertebrate host of mosquito 68 blood meals, including serological tools such as precipitin and ELISA tests [3,4]. Although these methods 69 provide valuable information, they present several drawbacks, including the availability of specific antisera 70 against antibodies and their cross-reactivity [3,4]. Molecular biology approach has been adopted as an effective 71 strategy to identify the mosquito blood sources [3,4]. Nevertheless, DNA sequencing can be costly and time 72 consuming [3,4]. 73 The matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) process is 74 based on acidic extraction of proteins from an organism of interest, which are co-crystallized with a matrix. The 75 molecules are ionized and propelled in a flight tube according to their mass-to-charge ratio. The detection of 76 each molecule will generate an individual peak, therefore providing an overall spectrum which will be highly 77 specific of the sample [22]. In the recent years, this process has revolutionized clinical microbiology [21]. 78 Recently, MALDI-TOF MS has emerged as an innovative tool to identify arthropods [8,24,25]. Also, in 79 preliminary reports, MALDI-TOF MS has appeared as a promising tool to identify mosquito blood meals, using 80 mosquitoes experimentally engorged in the lab, as well as using engorged mosquito abdomens crushed on 81 Whatman filter papers (WFPs) collected during entomological field surveys [16-18,23]. In these studies, a home- 82 made MALDI-TOF MS database was established with abdomen MS spectra from mosquitoes freshly engorged 83 on several vertebrate hosts [17,18]. Other recent works have used tandem mass spectrometry approach to 84 identify the ticks and triatomines blood meals [6,7,10,19] by the trypsin digestion of the samples [6,7,10,11,19]. 85 The goal of this study is to confirm the discriminative power of MALDI-TOF MS tool for mosquito blood meal 86 identification using a large panel of host blood vertebrates. For this purpose, Anopheles coluzzii and Anopheles 87 gambiae Giles mosquitoes, two cryptic species among the main vectors of malaria in Africa [1,5], were 88 artificially fed with different host species that were not previously included in the home-made database. 89 Materials and methods 90 Ethical statement

91 The Sus scrofa blood sample was collected by a veterinary specialist, with eth agreement of the owners, in 92 accordance with standards relating to animal welfare. All other animals were living in zoos and were first 93 anesthetized by a veterinarian responsible for monitoring their health. This protocol was approved by the Animal 94 Ethics Committee of Marseille (C2EA-14), and by the French authorities. The blood samples were processed and 95 stored in accordance with the World Health Organization’s Good Clinical Laboratory Practice guidelines and 96 documents on blood sample handling procedures. (Good Clinical Laboratory Practice, 2009). Mosquitoes were 97 raised using the International Conference on Harmonization/Good Laboratory Practices (ICH/GLP) procedures. 98 99 Laboratory rearing of Anopheles gambiae Giles and Anopheles coluzzii

100 Both mosquito species were reared using standard methods at a temperature of 26 ± 1°C, relative humidity of 101 70–90% and over a photoperiod of 12 hours (light/dark). Anopheles coluzzii adult females were artificially fed 102 through a Parafilm-membrane on three blood vertebrates, namely Camelus dromedarius, Ammotragus lervia and 103 Sus scrofa. The An. gambiae Giles females were artificially engorged on five blood vertebrates, namely

95 104 Leopardus pardalis, Arctictis binturong, Antidorcas marsupialus, Panthera onca and Papio hamadryas for 2 105 hours as previously described [17,18]. Engorged females were transferred to a new cage and fed with 10% 106 glucose solution. Five engorged females were harvested between one and 60 hours post-blood feeding, every 12 107 hours (i.e. 1, 12, 24, 36, 48, and 60 hours). The mosquito abdomens were placed in individually labeled vials. A 108 flowchart illustrating the main steps is presented in Figure 1.

109 Spectral analysis 110 The individual mosquito abdomens were manually crushed and prepared as described [17]. MALDI-TOF MS 111 spectra from engorged mosquito specimens were evaluated by analyzing the average spectra obtained from the 112 four spectra of each sample tested using the Flex Analysis 3.3 and ClinPro-Tools 2.2 software. These four 113 spectra correspond to the 4 spots of the same sample on the MALDI-TOF target plate. High quality spectra 114 (n=40) were selected to create a dendrogram. Dendrograms are based on the results of Composite Correlation 115 Index (CCI) matrix. The CCIs are calculated by dividing spectra into intervals and comparing these intervals 116 across a dataset. The composition of correlations of all intervals provides the CCI, which is used as a parameter 117 that defines the distance between spectra [9]. 118 119 Database upgrading and blind tests 120 In order to upgrade the MS arthropod home-made database (Additional file: Table S1), the four high quality 121 spectra from at least three specimens per species that were fed on the same blood and harvested at the same time 122 point, were combined by the automated function of the MALDI-Biotyper software v.3.3. to create 72 reference 123 spectra. Spectra of low quality were excluded from the analysis. Subsequently, a blind test against the updated 124 database was performed with all remaining MS spectra from the abdominals of clogged mosquitoes on eight 125 separate host bloods. The results of the database queries are presented as Log Score Values (LSVs) for each 126 spectrum given corresponding to a matched degree of signal intensities of mass spectra of the query and the 127 reference spectra. LSVs range from 0 to 3 [9]. LSVs greater than 1.8 were considered as the threshold value for 128 relevant identification as previously published [17].

129 Results 130 A total of 240 MS spectra from 150 Anopheles gambiae Giles and 90 Anopheles coluzzii engorged abdomens, 131 collected from 1 to 60 hours post-blood feeding and spotted in quadruplicate, were obtained.

132 Comparison of these MS spectra revealed high reproducibility of protein profiles between the same vertebrate 133 blood samples from mosquito abdomen protein extracts using Flex Analysis software (Figure 2). Among the 240 134 spectra, 72 spectra of the highest quality were used to upgrade the home-made database. This database 135 previously contained reference spectra of several arthropods, including reference spectra derived from the legs of 136 50 mosquito species, as well as reference spectra of Anopheles gambiae abdomens engorged on 17 different 137 hosts (Table S1).

138 We excluded from the analysis the spectra of low quality (n=80) collected from 48 to 60 hours post-blood 139 feeding and the remaining MS spectra from 88 mosquito abdomens collected from 1, 12, 24 and 36 hours , 140 engorged on eight blood vertebrates, were tested against home-made database. The specimens (n=48) collected 141 1, 12 and 24 hours post-feeding, revealed a 100% correct identification of the host blood origin. The log score

96 142 values (LSVs) for these freshly engorged specimens ranged from 1.912 to 2.918 (Table 1). Regarding the 143 specimens collected at 36 hours post-feeding (n=40), the percentage of correct identification (with LSVs ranging 144 from 1.396 to 2.207) dropped to less than 10 %. Among them, 4/40 Anopheles coluzzii abdomens engorged on 145 Sus scrofa blood (n=2) and Ammotragus lervia blood (n=2) were correctly identified with a LSVs ranging from 146 1.800 to 2.207. For the remaining specimens collected at 36 hours (n=36, 90%), an incorrect blood source was 147 identified (Table 1). As for the specimens collected 48 and 60 hours post-feeding (n=80), all MS spectra were 148 low quality.

149 Cluster analysis revealed a grouping on separate mosquito branches according to blood origin (Figure 3). The 150 MSP dendrogram showed that the spectra obtained from the abdomen of An. gambiae Giles, engorged on 151 different felids (Leopardus pardalis and Panthera onca), were close (Figure 3). The MSP dendrogram also 152 revealed that the spectra obtained from the abdomen of Anopheles gambiae Giles, fed on different carnivore 153 blood, were close (Figure 3). 154 155 Discussion and Conclusions 156 157 Our results confirm that MALDI-TOF MS is a promising tool for mosquito blood meals identification. The MS 158 spectra generated from the abdomen of Anopheles gambiae Giles and Anopheles coluzzii revealed specific and 159 reproducible blood up to 24 hours after feeding. In contrast, specimens collected 36 hours post-feeding revealed 160 only 10% of correct identification of the host blood origin, as suggested by preliminary studies [17,18].

161 Cluster analysis revealed that all specimens fed with the same blood were clustered in the same branch (Figure 162 3). Moreover, MALDI-TOF MS generated distinct MS profiles from the abdomens of Anopheles gambiae Giles 163 freshly engorged with bloods of closely related animals, highlighting the specificity of this tool.

164 According to this work and previous reports [17,18], the field application of MALDI-TOF MS blood meal 165 identification requires freshly engorged specimens. However, this is not problematic since field mosquitoes are 166 caught in houses in the morning and traps are collected early, less than 24 hours after the last blood meal [22]. In 167 addition, Sella's score visually determines the time and stage of digestion of mosquito blood meals and may be 168 used to chose the mosquitoes that would give spectra of quality [14].

169 The MALDI-TOF approach can appear limited by the cost of the device and database comprehensiveness. 170 Indeed, the correct identification of blood meals depends strictly on the presence of reference spectra in the 171 database. Specimens without corresponding species reference spectra in the database matched with low log score 172 values (< 1.8). This underlines the need to continue database expansion with MS spectra from new vertebrate 173 hosts [18]. However, reference spectra can be shared, avoiding the difficulties of creating a database. 174 Nevertheless, when the MALDI-TOF device is purchased for clinical microbiology purposes, it can also be used 175 for medical entomology at no additional cost [20].

176 This work open new perspectives and further studies are needed to better study the usefulness of MALDI-TOF 177 MS in medical entomology and in particular for blood meal identification. In this study, the two included 178 mosquito species, Anopheles gambiae Giles and Anopheles coluzzii were fed on different vertebrate bloods.

97 179 However, An. gambiae and An. coluzzi are cryptic species, and it is not known at this stage whether similar 180 results would be obtained by feeding mosquitoes of different genera. Although not noticeable for closely related 181 species, the mosquito proteome might influence the obtained spectra and impact the resulted blood meal 182 identification. If this is the case, the consequence would be the need to build an in-home-made database with a 183 larger number of mosquitoes.

184 MALDI-TOF MS tool has proven its effectiveness in identifying individual blood meals. However, in the field 185 it is important to be able to identify multiple blood meals, as some studies have shown that mixed blood meal 186 can represent up to 10% of mosquitoes collected [13,15]. Multiple host feedings (i.e. single mosquitoes taking 187 blood from different types of hosts) have been observed and describes by other authors in the field of 188 entomological surveys [2,12]. The effectiveness of the MALDI-TOF MS method opens other perspectives such 189 as the identification of interrupted blood meals.

190 Competing interests

191 The authors declare that they have no competing interests.

192 Acknowledgements

193 This work was supported by the French Government under the “Investissements d’avenir” program managed by 194 the French Agence Nationale de la Recherche (ANR), (Méditerranée Infection 10-IAHU-03). 195 Our thanks go to Baptiste Mulot, veterinary surgeon at the Beauval zoological park, who provided animal blood 196 samples. We also thank Sirama Niaré for his help with experimentation.

197 Author contributions

198 PP designed the study. PP and FT designed and developed the protocol. FT performed the experiments. PP, FT, 199 and ML analyzed the data. BD and OD contributed reagents/materials/analysis tools. FT wrote the paper. PP, 200 ML, and OD reviewed and contributed to editing the paper. All authors agreed to publication.

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101 281 Tables

282 Table1. Mosquito engorged abdomens spectra used for the home-made database and identification according to post-feeding period.

Mosquito species Blood meal source Time of Number of Number of LSVs obtained from Mean of Blood meal identified collection specimens used specimens blind tests against DB range by MS (hours) to upgrade the used for blind DB tests Anopheles coluzzii Ammotragus lervia 1-24 9 6 [2.518-2.918] 2.756 Ammotragus lervia Anopheles coluzzii Ammotragus lervia 36 0 5 [1.789 -2.011] 1.873 / Anopheles coluzzii Camelus dromedarius 1-24 9 6 [2.418-2.830] 2.706 Camelus dromedarius Anopheles coluzzii Camelus dromedarius 36 0 5 [1. 597-2.029] 1.764 / Anopheles coluzzii Sus scrofa 1-24 9 6 [2.112-2.711] 2.615 Sus scrofa

102 Anopheles coluzzii Sus scrofa 36 0 5 [1. 397-2.207] 1.905 / Anopheles gambiae Giles Antidorcas marsupialus 1-24 9 6 [2.530-2.807] 2.613 Antidorcas marsupialus Anopheles gambiae Giles Antidorcas marsupialus 36 0 5 [1. 562-1.941] 1.764 / Anopheles gambiae Giles Arctictis binturong 1-24 9 6 [2.585-2.892] 2.754 Arctictis binturong Anopheles gambiae Giles Arctictis binturong 36 0 5 [1.746 -1.892] 1.818 / Anopheles gambiae Giles Leopardus pardalis 1-24 9 6 [2.703-2.860] 2.773 Leopardus pardalis Anopheles gambiae Giles Leopardus pardalis 36 0 5 [1.747 -1.891] 1.818 / Anopheles gambiae Giles Papio hamadryas 1-24 9 6 [2.640-2.853] 2.756 Papio hamadryas Anopheles gambiae Giles Papio hamadryas 36 0 5 [1.652 -2.278] 1.998 / Anopheles gambiae Giles Panthera onca 1-24 9 6 [2.316-2.602] 2.521 Panthera onca Anopheles gambiae Giles Panthera onca 36 0 5 [1. 268-1.395] 1.342 / Total 72 88 283 284 Abbreviation: DB, home-made database.

285 Figure legends 286 Figure 1. Experimental workflow for blood meal identification using MALDI-TOF MS.

287 Figure 2. MALDI-TOF MS spectra of Anopheles gambiae Giles and Anopheles coluzzii abdomen protein 288 extracts engorged on host blood vertebrates. All mosquitoes were collected only times 1and 24 hours post- 289 feeding. Alignment of MS spectra from: H1_An_coluzzii_Camelus dromedarius (A), H24_An_coluzzii_ 290 Camelus dromedarius (B), H1_ An_coluzzii Ammotragus lervia (C), H24_ An_coluzzii Ammotragus lervia(D), 291 H1_ An_coluzzii _Sus scrofa (E), H24_ An_coluzzii _Sus scrofa (F), H1_ An_gambiae _Papio hamadryas (G), 292 H24_ An_gambiae _Papio hamadryas (H), H1_An_gambiae_ Antidorcas marsupialus (I), H24_An_gambiae_ 293 Antidorcas marsupialus (J), H1_An_gambiae_Arctictis binturong (K), H24_An_gambiae_ Arctictis binturong 294 (L), H1_ An_gambiae_ Panthera _ onca (M), H24_An_gambiae_ Panthera _ onca (N), 295 H1_An_gambiae_Leopardus pardalis (O), H24_An_gambiae_ Leopardus pardalis (P). a.u. arbitrary units;m /z 296 mass-to-charge ratio. 297 Figure 3. MSP dendrogram of MALDI-TOF MS spectra from Anopheles gambiae Giles and Anopheles 298 coluzzii abdomens collected 1, 12 and 24 hours post- feeding. MS spectra from two specimens from 1 and 24 299 hours post-feeding and one specimen from 12 hours post-feeding are represented. Blood meal host origins are 300 indicated in the graph. Cluster analysis was performed by MALDI-Biotyper software v.3.3. 301 Additional file 302 Table S1: The list of specimens in the home-made database. The body parts are indicated.

103 Figure 1 Click here to download Figure Figure1-R1-1.tiff 104 Figure 2 Click here to download Figure Figure2-R1-1.tiff

105 Figure 3 Click here to download Figure Figure3-R1-1.tiff 106 Response to reviewers Click here to download Response to reviewers Responses_to_Reviewers_ parasite180027_R1.docx

Reviewer’s comments/requests Author’s responses Reviewer #1: We thank the reviewer his/her comments. This article was maybe Summary a bit dense for a short-note; we’ve tried to shorten it. This paper presents a study that uses MALDI-TOF MS to We could not answer all the questions because some parts were identify blood meals in experimentally fed mosquitoes. It removed from the manuscript. examines a large number of mosquito abdomens to This paper present new data regarding the upgrade the database determine the how long after feeding the blood meal and therefore consolidate the previously original published. This source can be reliably inferred. It is one of a number of work opens new perspectives and further studies are needed to recent papers using mass spectrometry approaches to better study the usefulness of MALDI-TOF MS in medical identify blood meals of arthropods that transmit diseases entomology and in particular for blood meal identification. Here and addresses an interesting topic. apparently the mosquito species does not influence the blood meal identification when closely related mosquito species such as An. gambiae Giles and An. coluzzii are included.

The study has high replication but the experimental design We agree that artificial feeding laboratory conditions will is not particularly powerful for extending the results to probably never mimic the real conditions of mosquitoes’ feeding field collected samples and the conclusion that the results in field nor the reality of variable stages of blood digestion at the confirm the 'efficacy of the approach as an additional tool time of mosquito sampling. However, this approach was for entomological surveys' (line 40) is a bit of a stretch. previously validated on field samples collected in Comoros and Mali (Niare et al 2017, Tandina et al 2018). The blood meals were correctly identified from mosquitoes’ abdomens crushed on Whatman filter papers (WFP) by MALDI-TOF MS (Niare et al 2017). In addition this tool has successfully identified eight mosquito species and their WFP blood meal sources collected in Mali (Tandina et al 2018). We modified lines 40-42: “We confirm here that MALDI-TOF MS can be used to identify the blood meal origin of freshly engorged mosquitoes and opens new perspective for further studies, including the impact of the mosquito species on blood meal identification.”

The authors need to cite more of the other recent work that We have added the references according to the recommendations uses a variety of approaches with mass spectrometry. of the reviewers. These points were added in the introduction section Lines 83-84: “Other recent works have used tandem mass spectrometry approach to identify the ticks and triatomines blood meals [6,7,10,19] by the trypsin digestion of the samples [6,7,10,11,19].” There were two species of host mosquitoes and eight blood We agree with the reviewer that the lack of overlap between the meal sources included in the study. Since the two two experiments does not allow to draw any conclusions on the mosquito species were fed different blood meal sources, impact of the mosquito species on blood meal identification. In there is no power to determine how the method might this short communication, we only to strengthen the previously work for other blood meal sources. published data, illustrating the robustness on the method with a highest diversity of hosts and mosquito species. However, it is true that this information is important for the creation of blood meal databases so it would be an interesting perspective. This point was clarified and added in discussion and conclusions section lines176-183: “This work open new perspectives and further studies are needed to better study the usefulness of MALDI-TOF MS in medical entomology and in particular for blood meal identification. In this study, the two included mosquito species, Anopheles gambiae Giles and Anopheles coluzzii,were fed on differentvertebrate bloods. However, An. gambiae and An. coluzzi are cryptic species, and it is not known at this stage whether similar results would be obtained by feeding mosquitoes of different genera. Although not noticeable for closely related species, the mosquito proteome could influence the obtained spectra and impact the resulted blood meal identification. If this is the case, the consequence would be the need to build an in-home-made

107 database with a larger number of mosquitoes.” The study demonstrates the method is accurate for As answered before, this short note does not allow indeed to draw determining the blood meal source within 24 hours of this conclusion, but this issue should be explored in further feeding, for the experimentally fed specimens. It is not studies. clear how robust the procedure is to variation in the The correct identification of blood meals depends strictly on the mosquito species examined or to blood meal sources not presence of reference spectra in the database. This point was included in the experiment. Since of the eight species for added in the discussion and conclusions section as follow lines blood meal sources, five were test in one mosquito species 171-173: and three in the other (there was no overlap) it is not clear “Specimens without corresponding species reference spectra in if the approach would work across species. the database matched with low log score values (< 1.8). This underlines the need to continue database expansion with MS spectra from new vertebrate hosts [18].” The method works on mosquitoes sampled within 24 hours This point was added in discussion section as follow Lines 166- of taking a blood meal, but how would someone in the 168: field have that information? What criteria would be used to “In addition, Sella's score visually determines the time and stage not identify a blood source or misidentify? of digestion of mosquito blood meals and may be used to chose the mosquitoes that would give spectra of quality [14]. ” Although the method does allow for identification of blood As answered before, this short note does not allow indeed to draw meal sources under a restricted set of conditions. The lack this conclusion, but this issue should be explored in further of testing any of the blood meal sources on both mosquito studies. species is problematic and does not allow them to generalize their results beyond the particular mosquito species/blood meal source combinations examined here. In addition, the method used is the one of the simplest We thank the reviewer for the comments. approaches to MS analysis. Whole abdomens were ground in water and then loaded into the MS. Many other studies extract proteins, run gels and do trypsin digestion (see references above). While the simplicity of the approach is to be admired, because it was not tested in a different experimental design (i.e., factorial combinations of mosquitoes and blood meal sources) and because of the short time frame of detection (0-24 hours post feeding), the study does not strongly support researches taking this approach.

Reviewer #1 More specific comments are provided below. Line 32: The word “engorged” was removed and change by “fed” Abstract according to reviewer recommendation. Line 32: change 'engorged' to 'fed' Experimental design is not clear. Explicitly state This point was clarified lines 32-34: Anopheles gambiae was fed five blood meal sources and “Female Anopheles gambiae were fed on five host blood Anopheles coluzzii was fed three different blood meal including Leopardus pardalis, Arctictis binturong, Antidorcas sources. marsupialus, Panthera onca and Papio hamadryas while Anopheles coluzzii were fed on three hosts such as Camelus dromedarius, Ammotragus lervia and Sus scrofa” Since the method works only for the first 24 hours after We modified lines 40-42 as follow: “We confirm here that feeding, the conclusion that the results confirm the MALDI-TOF MS can be used to identify the blood meal origin of efficacy of the approach as an additional tool for freshly engorged mosquitoes and opens new perspective for entomological surveys is a bit of a stretch. further studies, including the impact of the mosquito species on blood meal identification.” Lines 164-166: “According to this work and previous reports [17,18], the field application of MALDI-TOF MS blood meal identification requires freshly engorged specimens. However, this is not problematic since field mosquitoes are caught in houses in the morning and traps are collected early, less than 24 hours after the last blood meal [22].” Introduction It is true that we referenced a lot of our own papers, mostly The introduction references quite a bit of their own because the number of research teams which use MALDI-TOF previous work (8 of 15 references are self citations), but MS to identify blood meals this way is quite limited. However not that of others. There needs to be reference to other you were right to point out that others use mass spectrometry to papers using mass spec for blood meal analysis and identify arthropods’ blood meals and references were added to the

108 describe how their method differs from other methods: introduction according to your recommendation. These points Honig V, Carolan HE, Vavruskova Z, Massire C, Mosel were added in the introduction section Lines 83-84: MR, Crowder CD, Rounds MA, Ecker DJ, Ruzek D, “Other recent works have used tandem mass spectrometry Grubhoffer L, Luft BJ, Eshoo MW. Broad-range survey of approach to identify the ticks and triatomines blood meals vector-borne pathogens and tick host identification of [6,7,10,19] by the trypsin digestion of the samples Ixodes ricinus from Southern Czech Republic. FEMS [6,7,10,11,19].” Microbiol Ecol. 2017 Nov 1;93(11). doi: 10.1093/femsec/fix129. Keller JI, Ballif BA, St Clair RM, Vincent JJ, Monroy MC, Stevens L. Chagas disease vector blood meal sources identified by protein mass spectrometry. PLoS One. 2017 Dec 12;12(12):e0189647. doi: 0.1371/journal.pone.0189647. Laskay UA, Breci L, Vilcins IM, Dietrich G, Barbour AG, Piesman J, Wysocki VH.Survival of host blood proteins in Ixodes scapularis (Acari: Ixodidae) ticks: atime course study. J Med Entomol. 2013 Nov;50(6):1282-90. Laskay ÜA, Burg J, Kaleta EJ, Vilcins IM, Telford Iii SR, Barbour AG, Wysocki VH. Development of a host blood meal database: de novo sequencing of hemoglobin from nine small mammals using mass spectrometry. Biol Chem. 2012 Mar;393(3):195-201. doi: 10.1515/hsz-2011-0196. Önder Ö, Shao W, Lam H, Brisson D. Tracking the sources of blood meals of parasitic arthropods using shotgun proteomics and unidentified tandem mass spectral libraries. Nat Protoc. 2014 Apr;9(4):842-50. doi: 10.1038/nprot.2014.048.

The single sentence paragraph 2 (lines 72-75) is This paragraph was clarified as following lines 67-72: “Several confusing. methods have been developed to identify the vertebrate host of mosquito blood meals, including serological tools such as precipitin and ELISA tests [3,4]. Although these methods provide valuable information, they present several drawbacks, including the availability of specific antisera against antibodies and their cross-reactivity [3,4]. Molecular biology approach has been adopted as an effective strategy to identify the mosquito blood sources [3,4]. Nevertheless, DNA sequencing can be costly and time consuming [3,4].” Lines 87-88: I did not see the discussion of strengths and This point was added in the discussion and conclusions section. limitations of alternative strategies anywhere in the paper. Lines 169-175: “ The MALDI-TOF approach can appear limited by the cost of the device and database comprehensiveness. Indeed, the correct identification of blood meals depends strictly on the presence of reference spectra in the database. Specimens without corresponding species reference spectra in the database matched with low log score values (< 1.8). This underlines the need to continue database expansion with MS spectra from new vertebrate hosts [18]. However, reference spectra can be shared, avoiding the difficulties of creating a database. Nevertheless, when the MALDI-TOF device is purchased for clinical microbiology purposes, it can also be used for medical entomology at no additional cost [20].” Method Lines 108-109: Give the scientific name as well as more OK, we used the scientific name instead of common name for the descriptive common name (e.g., Dromedary camel instead blood meal sources lines101-105: “Anopheles coluzzii adult of just Dromedary) for the blood meal sources. females were artificially fed through a Parafilm-membrane on three blood vertebrates, namely Camelus dromedarius, Ammotragus lervia and Sus scrofa. The An. gambiae Giles females were artificially engorged on five blood vertebrates, namely Leopardus pardalis, Arctictis binturong, Antidorcas marsupialus, Panthera onca and Papio hamadryas for 2 hours as

109 previously described [17,18].”

Lines 122-123: Parts of the methods need to be described This point was not detailed in the material and methods because in more detail in this paper rather than referring to the this article was maybe a bit dense for a short-note; we’ve tried to authors previous work, in particular, state that whole shorten it. proteins were used in the MS analysis, there was no trypsin digestion or PCR as is done in the other methods. Line 140: The database is referred to as home-made or in- OK, the database in now referred to as “home-made database” lab or reference - choose one term to use throughout. throughout the manuscript. Line 152: Provide more information on the database. Was This point was added in results section lines 134-136: “This it created to ID mosquito species or blood meals? database previously contained reference spectra of several arthropods, including reference spectra derived from the legs of 50 mosquito species, as well as reference spectra of Anopheles gambiae abdomens engorged on 17 different hosts (Table S1).” Are there any spectra of only the blood sources? How The home-made database contains mosquitoes’ legs reference many spectra are included in it? What taxa? What body spectra and the mosquitoes’ engorged abdomen reference spectra. parts? This point was clarified in lines 138-140: “We excluded from the analysis the spectra of low quality (n=80) collected from 48 to 60 hours post-blood feeding and the remaining MS spectra from 88 mosquito abdomens collected from 1, 12, 24 and 36 hours , engorged on eight blood vertebrates, were tested against home- made database.” The body parts used for each arthropod species are indicated on the table S1 (Database). Were the highest quality spectra used to update the The highest quality spectra used to update the database. This database or a random selection? point was clarified in materiel and methods section Lines 120- 123: “In order to upgrade the MS arthropod home-made database (Additional file: Table S1), the four high quality spectra from at least three specimens per species that were fed on the same blood and harvested at the same time point, were combined by the automated function of the MALDI-Biotyper software to create 72 reference spectra.” Figure 1: 36 hour time sampling is not included. We have added 36 hour time in the Figure1. Results The total is 240 MS spectra. This point was clarified as follow: Line 148: Was the total 240 MS? Or 240 x 4 MS? Lines 130-131: “A total of 240 MS spectra from 150 Anopheles gambiae Giles and 90 Anopheles coluzzii engorged abdomens, collected from 1 to 60 hours post-blood feeding and spotted in quadruplicate, were obtained.” Line 132 states four spectra of each sample were tested. This point was clarified in the materials and methods section This needs clarification. Lines 110-113: “MALDI-TOF MS spectra from engorged mosquito specimens were evaluated by analyzing the average spectra obtained from the four spectra of each sample tested using the Flex Analysis 3.3 and ClinPro-Tools 2.2 software. These four spectra correspond to the 4 spots of the same sample on the MALDI-TOF target plate.” Line 156: Explicitly state 1, 12, 24 hr rather than 'between Line 139: We changed “'between one and 24 hours” by “1, 12, 24 one and 24 hours' hours” according to reviewer recommendation.

Line 157: Explain the log score values. What is the LSVs range from 0 to 3. Higher value correspond a stronger possible range? What does a range of 1.9-2.9 mean? Do support. Indeed, a higher value is equal of more peaks in higher or lower values indicate stronger support? common with the reference spectrum. This point was added in material and methods section lines 125-128. “The results of the database queries are presented as Log Score Values (LSVs) for each spectrum given corresponding to a matched degree of signal intensities of mass spectra of the query and the reference spectra. LSVs range from 0 to 3 [9]. LSVs greater than 1.8 corresponding to the origin of blood meals were considered as the threshold value for relevant identification as previously published [17].”

Line 158: Change 'between 36 and 60 hours to ' 36, 48, Line 141: We changed “'between 36 and 60 hours” by “36 hours”

110 60'.

Line 159: This can be stated more clearly: 'revealed an This point was clarified lines142-144: “Regarding the specimens absence of correct identification. collected at 36 hours post-feeding (n=40), the percentage of correct identification (with LSVs ranging from 1.396 to 2.207) dropped to less than 10 %.” Line 160: Provide the percent correctly identified for each The percent for each sampling period were provided, Lines 144- sampling period, does it decay over time or go from 100% 148: “Among them, 4/40 Anopheles coluzzii abdomens engorged to 0%? on Sus scrofa blood (n=2) and Ammotragus lervia blood (n=2) were correctly identified with a LSVs ranging from 1.800 to 2.207. For the remaining specimens collected at 36 hours (n=36, 90%), an incorrect blood source was identified (Table 1). As for the specimens collected 48 and 60 hours post-feeding (n=80), all MS spectra were low quality.” Line 161: The LSV of the single correct identification post This point was clarified lines 147-148: “As for the specimens 24 hours was provided. What was the LSV of the collected 48 and 60 hours post-feeding (n=80), all MS spectra remaining specimens? were low quality.

Line 162: How were the scans chosen for the dendrograms? The best ones? Random? Random spectra among those of highest quality were chosen for the dendrograms. This point was clarified in the material and methods section as follow lines 113-114: “High quality spectra (n=40) were selected to create a dendrogram.” Table 1: It seems like it would be worth using some of the The MS spectra of the samples have low MS spectra quality and 36, 48 and 60 hr samples to update the database? they are not reproducible to be used to update the database

Figure 2: Is there a particular order to the presentation of We changed the order to the presentation of the samples the samples? Perhaps order by mosquito species and then following the reviewer recommendation. by mammal order (carnivora, primate, etc). Revise legend to indicate only times 1 and 24 hr are included. Was there The MALDI-TOF MS technology does not allow to definitively any attempt to validate the peaks? Or compare to MS of correlating the peaks to a particular protein. We do not have blood mosquitoes and blood meal sources separately? profiles alone in home-made database. Figure 3: The lack of pattern in the dendrogram, i.e., for We thank the reviewer comment. samples from the same mosquito species or the same order Anopheles gambiae fed Antidorcas marsupialus groups with the of mammals to cluster together, is a concern. This suggests Anopheles coluzzii fed Camelus dromedarius are on separated that if a blood source or mosquito not in the database use cluster. It was difficult to distinguish because only two specimens for analysis, it might not be identified correctly. In per blood were used to generate a dendrogram. We created a new particular, the An. gambiae_Springbok (Cetartiodactyl) dendrogram with more specimen, it is clearer now. Nevertheless, groups with the An. coluzzii_Dromedary (Artidactyla) the true validation of these approaches comes with the blind test rather than the other An gambiae samples or the other analysis which in this case was able to identify the 4 spots of each Cetartiodactyl samples (pig, mouflon). sample without ambiguity. This point was clarified in the Figure3. Provide statistical support values for the nodes in the In order to visualize MS profiles similarity and distances, dendrogram. hierarchical clustering of the mass spectra of all tested species was performed using the dendrogram function of MALDI Biotyper, version 3.0. This point was added in material and methods section lines 114- 117: “Dendrograms are based on the results of Composite Correlation Index (CCI) matrix. The CCIs are calculated by dividing spectra into intervals and comparing these intervals across a dataset. The composition of correlations of all intervals provides the CCI, which is used as a parameter that defines the distance between spectra [9].” Conclusion/Discussion This point was deleted because we’ve had to shorten this paper. Line 176: This is the first mention of body parts. Were different body parts examined? Line 186: Provide more information on the biology of the In this study, the success of host identification decreases with the system. How helpful is it to be able to detect blood meal advanced stage of mosquito blood meal digestion. A significant sources only up to 24 hours after feeding? How long do success of blood meal identification (100%) was found for

111 the mosquitoes live, etc? mosquitoes with a stage of digestion up to 24 hours, suggesting that mosquito capture on the field must be every 24 hours in the morning during the entomological studies. These points were provided in the discussion section of the manuscript lines 164- 166: “According to this work and previous reports [17,18], the field application of MALDI-TOF MS blood meal identification requires freshly engorged specimens. However, this is not problematic since field mosquitoes are caught in houses in the morning and traps are collected early, less than 24 hours after the last blood meal [22].”

Line 187-188: Since they did not test any of the blood As answered before, this short note does not allow indeed to draw sources in both mosquito species it is not clear that there is this conclusion, but this issue should be explored in further intra-species reproducibility or interspecies specificity. studies.

Line 190: typo? 'inter-the species' Thank you, this point was corrected. Line 195-196: A search in PubMed indicates over 300 For bacterial identification, we agree with the reviewers that articles on the use of MALDI-TOF MS for phylogeny. MALDI-TOF MS tool can be used for the microorganism phylogenic classification. But for the phylogenetic classification of mosquitoes and their blood meals (and even other arthropods such as ticks), MALDI-TOF MS is not yet effective. Are the spectra in a publically available database? All our spectra are available on request as mentioned in this manuscript and others of our research team. The list of specimens in the database is provided as additional data file (Table S1). Reviewer #2: In the manuscript entitled « Blood meal We thank the reviewer for this very interesting comment. This identification of cryptic species Anopheles gambiae Giles present study was submitted at Parasite journal as short-note and Anopheles coluzzii using MALDI-TOF MS », Tandina according the journal instruction. This paper present new data to et al. described the design and testing of a MALDI-TOF upgrading the database and consolidate the previously original spectral database for blood-meal identification in published. We could not answer all the questions because some Anopheles gambiae and A. coluzzii. parts had to be removed from the manuscript to shorten it The authors have previously published the methodology used for constructing such a database from a large panel of vertebrate blood using engorged An. gambiae abdomens (Niaré S. et al. Malaria Journal, 2016 or Niaré S. et al. Infection Genetics and Evolution 2017). Its application with field-collected Anopheles crushed on Whatman paper has also been described by the same authors (Tandina, F et al. Parasitology, 2017 Ref 11). This paper presents an upgrade of the existing database with the blood of new animal species and application to Anopheles coluzzii. From my point of view, the originality of the data presented is not sufficient for an original article, but the present work is appropriate for publication as a short-note.

Reviewer #2 Main comments This point was now added in the discussion and conclusions - The reason for choosing this two particular Anopheles section lines 179-180: “. However, An. gambiae and An. coluzzi species for the study is not clearly explained. are cryptic species, and it is not known at this stage whether similar results would be obtained by feeding mosquitoes of different genera” - The present study is limited to laboratory conditions with We completely agree with the reviewer, for this purpose the new artificially fed females. It would have been really field studies are needed. Here, the present study is short note to interesting to test the upgraded database with spectra confirm the MALDI-TOF MS robustness to identify the large acquired from field samples, such as those already panel of bloods and this tool was efficient to determine the two published in Tandina, F et al. Parasitology, 2017. Anopheles species trophic preferences.

Reviewer #2 Minor comments MALDI-Biotyper software v.3.3. (Bruker Daltonic) was used for - Line135 Was Bruker Daltonic standard method used for MSP generation. This point was now added in the materials and MSP generation? methods sections and Figure 3 legends of the manuscript line122.

112 - Line140 A listing of all species present in the final The list of specimens in the database is provided as additional file upgraded database could be provided as additional data. (Table S1). - Line145: For clarity it would be useful to recall the Cut- This point was clarified and added in material and methods Off value of LSV used (> 1.8?). section lines 125-128:“The results of the database queries are presented as Log Score Values (LSVs) for each spectrum given corresponding to a matched degree of signal intensities of mass spectra of the query and the reference spectra. LSVs range from 0 to 3 [9]. LSVs greater than 1.8 corresponding to the origin of blood meals were considered as the threshold value for relevant identification as previously published [17]” - Line203 The authors cited Onder O et al 2013 (Ref 10), We are completely in accordance with the reviewer, this point but the approach described in this paper was based on LC- was deleted in the manuscript. MS (and no MALDI-TOF MS) to identify mixed blood meals - Line237-240 Misaligned paragraph This point was corrected. - Table 1: the range of LSV (penultimate column) is not an We agree with the reviewer recommendation. This column was informative way to present these values. It would be more added in the Table1. interesting to present the number of LSVs obtained above the Cut-Off value, or at least the LSV average in addition to the range

-Figure 3 legend. Parameters of cluster analysis are not This point was added in material and methods section lines 114- indicated. Please indicate how the distance matrice was 117: “Dendrograms are based on the results of Composite calculated (correlation? Euclidean distances? Spearman Correlation Index (CCI) matrix. The CCIs are calculated by coefficient?) dividing spectra into intervals and comparing these intervals across a dataset. The composition of correlations of all intervals provides the CCI, which is used as a parameter that defines the distance between spectra [9].” Moreover, the authors have developed an extremely All our spectra are available on request as mentioned in this powerful tool for the study of blood meals that will be of manuscript and others of our research team. The list of specimens great interest for the entire community of medical in the database is provided as additional data file (Table S1). entomology. It would be extremely interesting to publish the database on a public data repository such as Zenodo or, as an open-acess collaborative online tool (as proposed in Niare et al. 2017 Plos One).

113 Supplementary Data 1

Click here to access/download Online material Table S1-06_06_18_parasite.pdf

114

ARTICLE 4 Tandina F֞ , Niare S֞ , Laroche M, Almeras L, Davoust B, Doumbo O, Raoult D, Parola P. (2018), Identification of mixed and successive blood meals of mosquitoes by MALDI-TOF MS protein profiling. IN PREPARATION

115

Dans la nature quand un moustique est dérangé dans son repas de sang termine son repas de sang sur un autre hôte différent durant le même cycle gonotrophique ; on parle de repas mixte interrompu. Ce repas de sang peut être double ou triple quand il s’agit de deux ou de trois hôtes différents. Par exemple Anopheles arabiensis qui est décrite comme anthropophile et zoophile; lorsque les hôtes animaux domestiques sont disponibles, ces femelles préfèrent se nourrir sur les animaux (Becker N et al. 2010). Dans cette étude, nous avons développé des protocoles de profil MALDI-TOF MS pour identifier les repas de moustiques successifs et mixtes. Les moustiques ont été expérimentalement gorgés sur un mélange de sang provenant d'hôtes distincts, tels que des humains, des moutons et des chiens. Les résultats révèlent que le MALDI-TOF MS est capable d’identifier des repas de sang mélangé des moustiques avec des scores significatifs. Tous les spectres obtenus à partir des repas de sang successifs des moustiques étaient reproductibles et spécifiques de la dernière source de sang, suggérant que les précédents repas de sang n'influent pas sur l'identification du dernier repas pris. Cela est peut être du à l’intervalle de temps (trois jours) entre le premier et deuxième repas de sang des moustiques. Mais ces résultats éclairent les nouvelles possibilités qu’offre le MALDI-TOF MS au cours d'enquêtes entomologiques.

117

1 Identification of mixed and successive blood meals of mosquitoes by MALDI-TOF MS

2 protein profiling.

Fatalmoudou Tandina1, 2֞ , Sirama Niare1, 2֞ , Maureen Laroche1, Lionel Almeras1,3, Bernard 3

4 Davoust4, Ogobara K Doumbo2, Didier Raoult4, Philippe Parola1*.

6 1Aix Marseille Univ, IRD, AP-HM, SSA, VITROME, IHU-Méditerranée Infection, Marseille,

7 France.

8 2Malaria Research and Training Center, DEAP/FMOS, UMI 3189, University of Science,

9 Techniques and Technology, Bamako, Mali.

10 3Unité de Parasitologie et d’Entomologie, Département des Maladies Infectieuses, Institut de

11 Recherche Biomédicale des Armées, Marseille, France.

12 4Aix Marseille Univ, IRD, AP-HM, MEPHI, IHU-Méditerranée Infection, Marseille, France.

.These authors contributed equally to this work֞ 13

14 E-mail: [email protected]; [email protected]; [email protected];

15 [email protected]; [email protected]; [email protected];

16 [email protected]; [email protected].

18 *Address for correspondence: Philippe Parola. VITROME, IHU-Méditerranée Infection, 19-

19 21 Bd Jean Moulin 13005 Marseille, France. Phone: + 33 (0) 4 13 73 24 01. Fax: + 33 (0) 4

20 13 73 24 02. Email: [email protected].

119 22 Abstract

23 Background: The accurate and rapid identification of mosquito blood meals is critical to

24 study the interactions between vectors and vertebrate hosts and, subsequently, to develop

25 vector control strategies. Recently, MALDI-TOF MS profiling has been shown to be a

26 reliable and effective tool for identifying single blood meals from mosquitoes.

27 Methods: In this study, we developed MALDI-TOF MS profiling protocols to identify

28 successive and mixed blood meals from mosquitoes. The mosquitoes were either successively

29 artificially fed with distinct host bloods or engorged with mixed bloods from distinct

30 vertebrate hosts, such as humans, sheep and dogs.

31 Results: The results revealed a correct identification of mixed blood meals from mosquitoes

32 using MALDI-TOF MS profiling. The MS spectrum from mixed blood meals was identified

33 using LSVs (log score value) greater than 1.8. All MS spectra (n=244) obtained from

34 mosquito successive blood meals were reproducible and specific of the last blood meal,

35 suggesting that the previous blood meals do not impact the identification of the last one.

36 Conclusion: MALDI-TOF MS profiling approach appears as an effective and robust

37 technique to identify the last and mixed blood meals during medical entomologic surveys.

39 Keywords: Aedes, Anopheles, MALDI-TOF MS profiling, mixed, successive, blood meals,

40 entomologic survey.

120 41 1. Introduction

42 Mosquitoes are the major arthropod vectors of human infectious diseases across the world

43 (Becker N et al. 2010). Aedes mosquitoes may transmit arboviral diseases including Yellow

44 Fever, Dengue, Chikungunya and Zika viruses’ infection (Caglioti et al. 2013; Gardner and

45 Ryman 2010; Gould et al. 2017; Vasilakis et al. 2011). Anopheles mosquitoes include the

46 main vectors of malaria in West Africa (Carnevale P et al. 2009). Malaria is the primary cause

47 of morbidity and mortality in Africa mainly amongst the children under five years and

48 pregnant women (WHO 2016). There is no effective vaccine for several mosquito-borne

49 diseases hence and entomological survey and control measures directed against the

50 mosquitoes are essential for the fight against mosquito-borne diseases (Deilgat et al. 2014).

51 Mosquito vectors may feed on various animals and a better understanding of mosquito-borne

52 disease transmission dynamics, including the trophic preferences of mosquitoes, is an

53 important component to evaluate and to improve control measures (Ndenga et al. 2016;

54 Coulibaly et al. 2016; Githeko et al. 1994; Shililu et al. 1998). For example the interaction

55 between Anopheles malaria vectors and host populations is critical to assess the risk of human

56 exposure and determine the Anopheles trophic preference patterns (Fyodorova et al. 2006).

57 Several methods have been used to determine the origin of mosquito blood meals, including

58 precipitin tests, enzyme-linked immunosorbent assays (ELISA) (Fyodorova et al. 2006) and

59 molecular tools (Kent and Norris 2005; Kent 2009; Logue et al. 2016; Ndenga et al. 2016;

60 Prior and Torr 2002). The molecular technique based on host DNA amplification provides

61 greater information on the specific identification of blood meal sources (Kent 2009; Munnoz

62 et al. 2011). However, this molecular method has some limitations, including cost, turnaround

63 time and some vertebrate sequences are not available in Genbank (Egizi et al. 2013; Oshaghi

64 et al. 2006).

121 65 MALDI-TOF MS is a protein profiling-based technique that uses a laser energy absorbing

66 matrix to generate a protein spectrum from an organism of interest (Seng et al. 2010). It has

67 revolutionized clinical microbiology by allowing a quick identification of bacteria at low

68 running costs (Seng et al. 2009; Seng et al. 2013; Schubert and Kostrzewa 2017). In the

69 recent years, the efficiency of matrix-assisted laser desorption/ionization time-of-flight mass

70 spectrometry (MALDI-TOF MS) for the identification of arthropods (Diarra et al. 2017;

71 Halada et al. 2018; Laroche et al. 2017b; Mewara et al. 2018; Tandina et al. 2018; Yssouf et

72 al. 2016) and mosquito blood meals identification (Niare et al. 2017b; Niare et al. 2016) has

73 been reported.

74 Indeed, in preliminary studies, MALDI-TOF MS was able to identify the host blood source

75 from Anopheles gambiae Giles and Aedes albopictus mosquitoes artificially fed on human,

76 horse, sheep, rabbit, mouse, rat and dog blood up to 24 hours post-feeding (Niare et al. 2017b;

77 Niare et al. 2016).

78 The goal of the present study was to determine whether MALDI-TOF MS could be extended

79 to the identification of mixed and successive blood meals. Anopheles gambiae Giles,

80 Anopheles coluzzii and Aedes albopictus mosquitoes were artificially engorged on human

81 blood and subsequently fed on the blood of other vertebrates after complete digestion of the

82 first meal. For the identification of mixed blood meals by MALDI-TOF MS, the mosquitoes

83 were engorged with blood mixed at different concentrations from different vertebrate hosts to

84 mimic interrupted blood meals.

86 2. Materials and Methods

88 2.1.Ethical statement

89 Animal studies were conducted in line with 2010/63/EU directive of the European Parliament

90 and of the Council of September 22, 2010, and in compliance with French Government

122 91 Decree No. 2013-118 of February 1st, 2013. The study was approved by the local Animal

92 Ethics Committee (Comité d’Ethique en Expérimentation Animale, Marseille) and

93 Institutional Animal Care Committee in Marseille, France. Human blood was obtained from

94 the national French blood bank, the “Etablissement Français du Sang” (EFS) accredited by the

95 Institutional Animal Care of IHU Méditerranée Infection.

97 2.2.Mosquito rearing and maintenance

98 Anopheles gambiae Giles, Anopheles coluzzii and Aedes albopictus mosquitoes were reared in

99 our laboratory (Marseille, France) using standard methods at a temperature of 26±1°C,

100 relative humidity of 80±10% and a 12-hour photoperiod in incubators (Panasonic cooled

101 incubator) (Awono-Ambene et al. 2001). Larvae were reared until the pupal stage in trays

102 containing one liter of distilled water supplemented with fish food (TetraMinBaby, Tetra

103 Gmbh, Herrenteich, Germany). Pupae were collected daily and transferred into a mosquito

104 cage (Bug Dorm 1 insect rearing cage, BioQuip products, Taiwan). Adults were fed with a

105 10% glucose solution until the day of the experiment. Three days after emergence, female

106 adult mosquitoes were artificially fed through a Parafilm-membrane (hemotek membrane

107 feeding systems, Discovery Workshops, UK) using fresh heparinized human blood for two

108 hours. Engorged female mosquitoes were transferred to another cage and were fed with a 10%

109 glucose solution on cotton.

110

111 2.3.Successive blood meals

112 To assess the ability of MALDI-TOF MS to identify successive blood meals, 60 An. gambiae

113 Giles, 60 An. coluzzii and 124 Ae. albopictus were firstly engorged on human blood. Twelve

114 hours after feeding, 10 Anopheles and 20 Aedes abdomens were submitted to MALDI-TOF

115 MS analysis while, for each mosquito species, the remaining specimens were dispatched

123 116 equally in six cages. Three days after the first blood meal (after complete digestion), the

117 mosquitoes from each cage were engorged for a second time on six distinct types of vertebrate

118 blood (goat, cow, chicken, dog, sheep and rabbit). The abdomens of all mosquitoes were

119 submitted to MALDI-TOF MS analysis 12 hours after feeding.

120

121 2.4.Mixed blood meals

122 To determine the ability of MALDI-TOF MS to identify interrupted blood meals from

123 mosquito abdomens, the mosquitoes were fed on human blood mixed with dog blood or sheep

124 blood at 75%/25% ratio, 50%/50% and 25%/75% (Table1). To prevent the coagulation of

125 blood mixtures, the various mixtures of blood were previously heated at 56°C in a dry bath

126 for 30 minutes as previously described (Nossel and NIEMETZ 1965). The mosquitoes’

127 abdomens were collected 12 hours after feeding and then submitted to MALDI-TOF-MS

128 analysis.

129 Human, sheep and dog blood was mixed at equal concentrations and given to 25 An. gambiae

130 Giles, 10 An. coluzzii and 23 Ae. albopictus (Table1).

131 A flowchart illustrating the main steps in the experimental workflow for the home-made

132 database creation and blood meal identification by MALDI-TOF MS is presented in Figure 1.

133

134 2.5.Sample preparation for MALDI-TOF analysis

135 Only fully engorged mosquitoes were included in the MS analysis. Engorged females were

136 killed at -20°C prior to dissection. Each mosquito abdomen was separated from the thorax

137 using a sterile scalpel for each sample. The dissected abdomens were manually crushed using

138 sterile pestles (Fischer Scientific, Strasbourg, France) in a 1.5 ml tube containing 50 μL of

139 HPLC-grade water. Ten microliters of the supernatant of the crushed abdomens were

140 homogenized in 20 μL of 70% formic acid and 20 μL of 50% acetonitrile (Fluka, Buchs,

124 141 Switzerland) and centrifuged at 10,000 rpm for 20 seconds. One microliter of supernatant of

142 each sample was deposited on the MALDI-TOF target plate in quadruplicate (Bruker

143 Daltonics, Wissembourg, France) and covered with 1 μL of CHCA matrix solution composed

144 of saturated α-cyano-4-hydroxycynnamic acid (Sigma, Lyon, France), 50% acetonitrile (v/v),

145 2.5% trifluoroacetic acid (v/v) (Aldrich, Dorset, UK) and HPLC-grade water. After drying for

146 several minutes at room temperature, the target was introduced into the Microflex LT

147 MALDI-TOF Mass Spectrometer (Bruker Daltonics, Germany) for analysis. To control

148 loading on mass spectra steel, matrix quality and MALDI-TOF apparatus performance, the

149 matrix solution was loaded in duplicate onto each MALDI-TOF plate with or without a

150 bacterial test standard (Bruker protein Calibration Standard I), as previously described (Niare

151 et al. 2016).

152

153 2.6.MALDI-TOF MS parameters

154 Protein mass profiles were acquired using a Microflex LT MALDI-TOF Mass Spectrometer,

155 with detection in the linear positive-ion mode at a laser frequency of 50 Hz within a mass

156 range of 2–20 kDa. The acceleration voltage was 20 kV, and the extraction delay time was

157 200 ns. Each spectrum corresponded to ions obtained from 240 laser shots performed in six

158 regions of the same spot and automatically acquired using the AutoXecute function of the

159 Flex Control v.2.4 software (Bruker Daltonics, Germany). The spectrum profiles obtained

160 were visualized using Flex analysis v.3.3 software and exported to ClinProTools version v.2.2

161 (Bruker Daltonics, Germany) and MALDI-Biotyper v.3.0. (Bruker Daltonics, Germany) for

162 data processing (smoothing, baseline subtraction, and peak picking) and evaluation with

163 cluster analysis.

164

165 2.7.MALDI–TOF MS spectra analyses

125 166 The reproducibility and specificity of MALDI-TOF MS spectra from the abdomens of

167 mosquitoes which had taken successive or mixed blood meals were evaluated by comparing

168 the average spectra obtained from the four spectra of each sample tested using the Flex

169 Analysis and ClinProTools 2.2 software. As a control, one microliter of host blood was mixed

170 with 20 μL of 70% formic acid and 20 μL of 50% acetonitrile and deposited on the MALDI-

171 TOF target plate in quadruplicate, covered with 1 μL of CHCA matrix solution and compared

172 to the engorged abdomen-derived spectra.

173 The spectra from An. gambiae Giles mixed blood meals of human/dog blood (n=4),

174 human/sheep blood (n=5), and the triple mixture of human/dog/sheep blood (n=5) were added

175 to uploaded into our home-made database (Table1). As for Ae. albopictus the reference

176 spectra of human/dog blood (n=7), human/sheep blood (n=7), and the triple mixture of

177 human/dog/sheep blood (n=3) and An. coluzzii the reference spectra of human/dog blood

178 (n=3), human/sheep blood (n=6), and the triple mixture of human/dog/sheep blood (n=1) were

179 selected to upgrade the database (Table1). This database includes the spectra obtained from

180 various arthropod species and mosquitoes’ abdomens fed on 25 distinct host bloods (Homo

181 sapiens, Equus caballus, Ovis aries, rabbit, Balb/C mouse, Rattus norvegicus, Canis

182 familiaris, Bos taurus, Capra hircus, Gallus gallus, Equus asinus, Tapirus indicus, Tapirus

183 terrestris, Carollia perspicillata, Thraupis episcopus, Erythrocebus patas, Callithrix

184 pygmaea, Leopardus pardalis, Arctictis binturong, Antidorcas marsupialus, Panthera onca ,

185 Papio hamadryas, Camelus dromedarius, Ammotragus lervia and Sus scrofa) (Additional file:

186 TableS1).

187 Main Spectrum Profiles (MSP) were automatically created based on an algorithm using

188 information on peak position, intensity and frequency from the BioTyper MSP Creation

189 Standard Method. The maximum mass error of each single spectrum was 2000 Da, the desired

190 mass error for the MSP was 200 Da.

126 191 2.8.Blind tests

192 2.8.1. Blind test 1

193 The abdomen-derived spectra from Anopheles gambiae Giles which had taken successive

194 blood meals were submitted for a blind test analysis. Similarly, the remaining spectra of the

195 abdomens of the mosquitoes fed with mixed blood meals after excluding those entered in the

196 home-made database were submitted to the same analysis (Table1). The results are presented

197 in the MALDI-Biotyper software v.3.3. as log score values (LSV) that consists in the degree

198 of signal intensity matching between the queried mass spectra and the reference spectra (Niare

199 et al. 2016). LSVs range from 0 to 3. LSVs make it possible to correctly assess the

200 reproducibility between a queried spectrum and a reference spectrum, as it is the result of the

201 thorough comparison of peak positions and intensity between those two spectra. An LSV was

202 obtained for each spectrum of the blindly tested samples (Niare et al. 2016; Laroche et al.

203 2017a).

204 2.8.2. Blind test 2

205 A second blind test against the home-made database upgraded with reference spectra of An.

206 gambiae Giles (database 1) was later performed as described above with MS spectra from

207 engorged abdomens of Ae. albopictus and An. coluzzii after successive and mixed blood meals

208 to evaluate the influence of mosquito species on blood meal identification. Then, Ae.

209 albopictus and An. coluzzii reference spectra (Table1) for successive and mixed blood meals

210 were added to database 1 to create database 2.

211 2.8.3. Blind test3

212 The remaining spectra of the abdomens of the Ae. albopictus and An. coluzzii fed with mixed

213 blood meals were tested against database 2. A LSV was obtained for each of the four spots of

214 each specimen tested.

215

127 216 3. Results

217

218 3.1.Successive blood meals

219 Comparison of the MS spectra from An. gambiae Giles (n=60), Ae. albopictus (n=124) and

220 An. coluzzii (n=60) abdomens after successive blood meals using the Flex Analysis software

221 indicated a high quality and reproducibility of the spectra (Fig. 2). Query of An. gambiae

222 Giles against database 1 (additional file: Table S1) after successive blood meals, showed

223 reliable and accurate identification for 100% of the last blood meal (n= 60) (Table 2). The

224 LSVs for the specimens ranged between 1.826 and 2.764. The spectra from the engorged

225 abdomens of Ae. albopictus and An. coluzzii were then queried against the database upgraded

226 with An. gambiae reference spectra (database 1) and later against the database upgraded Ae.

227 albopictus and An. coluzzii reference spectra (database 2). Before upgrading of database we

228 observed 80% of correct identification with a mean score of 2.242 and after upgrading

229 accurate identification for 100% of the blood meals with a mean score of 2.593 (Table3). The

230 LSVs for the specimens ranged between 1.855 and 2.873.

231

232 3.2.Mixed blood meal identification by MALDI-TOF MS

233 Regarding the human/dog and human/sheep blood mixtures, of , a correct identification was

234 observed for all 52 An. gambiae Giles that fed on mixed blood meals, with LSVs ranging

235 from 2.055 to 2.771 (Table 4).

236 The spectra from engorged abdomens of Ae. albopictus and An. coluzzii were queried against

237 database 1 and database 2. Query against database 1 revealed 51.51% and 51.42% of correct

238 identification of blood mixture from human/dog and human/sheep blood respectively. After

239 upgrading, we obtained accurate identification for 100% of the blood meals against database

240 2, with LSVs ranging from 1.762 to 3.000 (Table5, Table 6).

128 241 Finally, the 20 Anopheles gambiae which had been fed with a triple mixture of blood

242 (human/dog/sheep) were subjected to MS analyses. Their abdomens provided good quality

243 spectra and the blind test led to unambiguous, accurate identification with LSVs ranging from

244 2.258 to 2.846 (Table 4). The following query of Ae. albopictus and An. coluzzii fed with the

245 same triple mixture revealed a percentage 36.36% when blind tested against database 1. After

246 database upgrading with their reference spectra, we observed 100% of the blood meals

247 identification with LSVs ranging from 2.070 to 2.725 (Table7).

248 4. Discussion

249 MALDI-TOF MS consists in three major steps. Firstly, the organism of interest is mixed with

250 a suitable matrix material and applied to a metal plate. Secondly, the co-crystallized sample is

251 irradiated with laser pulses, effecting desorption and ‘soft’ ionization of the organism.

252 Thirdly, molecules are accelerated in an electric field and separated through a flight tube in

253 linear or reflectron mode, according to their mass-to charge ratio. The MS profiles protein

254 obtained are used to discriminate from each specimen tested (Yssouf et al. 2016). MALDI-

255 TOF MS on arthropods has been reported effective on lab models (Diarra et al. 2017; Halada

256 et al. 2018; Laroche et al. 2017b; Mewara et al. 2018; Tandina et al. 2018; Yssouf et al.

257 2016), but also in the field as illustrated by mosquito species MS studies conducted in France

258 and Sweden (Yssouf et al. 2014), or the identification of the invasive mosquito Aedes

259 koreicus in Switzerland and Italy (Suter et al. 2015). Other reports have highlighted the

260 application of MALDI-TOF MS for monitoring sand fly fauna captured in Algeria and Czech

261 Republic (Halada et al. 2018; Lafri et al. 2016). The choice of body parts is crucial for

262 MALDI-TOF MS analyses (Hoppenheit et al. 2013). Each body part provides a fingerprint

263 signature that may be used for identification. For example, we showed that mosquito legs are

264 sufficient to obtain specific and reproducible MS spectra and therefore can be used for

265 mosquito identification (Yssouf et al. 2013). For example Dieme et al. showed that MALDI-

129 266 TOF MS is an effective tool for mosquito aquatic stage identification, using the whole

267 specimen (Dieme et al. 2014).

268 Following the kinetic evolution of the MALDI-TOF spectra according to digestion of the

269 blood meal, this technique was also able to successfully identify mosquito blood meals up to

270 24 hours post feeding (Niare et al. 2017b; Niare et al. 2016).Some studies have shown that

271 mixed or multiple blood meals can amount to up to 10% the mosquitoes in the field (Logue et

272 al. 2016; Moreno et al. 2017), which is considered significant in terms of influencing the

273 transmission of pathogens and maintaining residual infectious diseases. Hence the need to

274 provide tools to monitor such types of blood meals.

275 The reproducibility and specificity of the spectra are critical parameters for accurate use of

276 MALDI-TOF MS, as shown here. The present study shows that the MALDI-TOF MS process

277 is an additional tool for identifying mosquito blood meals, including when mosquitoes have

278 taken successive blood meals on different animals. In our study, the last animal blood meal

279 was correctly and unambiguously identified, although mosquitoes had firstly fed on human

280 blood. This may be due to the interval of days (three) between the first and second blood

281 meals of mosquitoes. The first blood meal was indeed digested by the mosquitoes. In the

282 present study, we obtained accurate identification with high LSVs (100% > 1.8). This

283 admitted threshold for arthropod identification is congruent with previously published studies

284 (Yssouf et al. 2016). Interestingly, for closely mosquito species such as An. gambiae Giles

285 and An. coluzzii there was little influence of the mosquito species on the blood meal

286 identification. This is however not the case for Ae. albopictus. Indeed, for Ae. albopictus and

287 An. coluzzii, we observed 80% of correct identification of the last blood meal against the

288 database 1. In addition, the spectra from Ae. albopictus and An. coluzzii fed with human/dog

289 and human/sheep blood mixtures respectively and tested against database 1 revealed 51.51%

290 and 51.42% of correct identification. These blood mixtures were already in the home-made

130 291 database with Anopheles gambiae Giles species, but we obtained a low score in blind test for

292 half of the identifications. Therefore the mosquito proteins can have an impact on the

293 identification on the blood meal. This probably means that a larger database may be needed to

294 identify blood meals from several mosquito species. However, more and more laboratories

295 (Raharimalala et al. 2017; Sambou et al. 2015; Yssouf et al. 2016) are applying MALDI-TOF

296 to medical entomology and spectra sharing in easy (open access or on request), substantially

297 facilitating database creation.

298 The other limit of our model is, it does not reproduce exactly what is happening in the field.

299 For the identification of mixed blood meals by proteomic approach, Önder et al. have

300 developed an experimental design in the blacklegged ticks (Ixodes scapularis) (Onder et al.

301 2013). Their methodology has successfully identified the mixed blood meal from two

302 vertebrates, according to the concentration ratios. As for the identification of mixed bacterial

303 species, Lin Zhang et al. reported that the major peaks in pluribacterial samples could be

304 assigned to those found in the respective spectra of each bacterium (Zhang et al. 2015). It

305 should be noted that upgrading of the database with mixed meal spectra is essential for

306 accurate identification (Niare et al. 2016). Building a comprehensive database is crucial for

307 accurate arthropod MS identification and, with this in mind, all our reference spectra are

308 available on request. Similarly, Raharimalala et al. have highlighted the need for sharing and

309 have expressed interest in an international database (Raharimalala et al. 2017). The major

310 advantages of using MALDI-TOF MS are its rapidity, effectiveness and reliability in

311 identifying successive blood meals and mixed blood meals. In this study, only a small

312 quantity of host blood was used for MALDI-TOF analysis, and the remaining material can be

313 used for other studies, including the legs to identify mosquitoes at the species level (Yssouf et

314 al. 2013).

131 315 The artificial feeding laboratory conditions will probably never mimic the real conditions of

316 mosquitoes’ feeding in field none the reality of variable stages of blood digestion at the time

317 of mosquito sampling. However, the MALDI-TOF MS approach was previously validated on

318 field samples collected in Comoros and Mali (Niare et al. 2017a; Tandina et al. 2018). The

319 blood meals were correctly identified from mosquitoes’ abdomens crushed on Whatman filter

320 papers (WFP) by MALDI-TOF MS (Niare et al. 2017a; Tandina et al. 2018). In addition this

321 tool has successfully identified eight mosquito species and their WFP blood meal sources

322 collected in Mali.

323 The MALDI-TOF approach can appear limited by the cost of the device and database

324 comprehensiveness. Indeed, the correct blood meal identification is strictly dependent on the

325 reference spectra presence in the database. However, reference spectra can be shared, which

326 circumvent the difficulties of creating a database. When the MALDI-TOF device is bought

327 for clinical microbiology purposes, it can also be used for medical entomology at no

328 additional cost. For example, in Senegal Masse et al. have successfully identified the

329 Culicoides spp. using protein approach MALDI-TOF MS that is economically advantageous

330 compared at molecular tool whose some sequences were not available in Genbank (Sambou et

331 al. 2015).

332 Conclusion

333 We provide here more data to support the fact that MALDI-TOF MS appears as a rapid,

334 reliable and cost-effective method for identifying An. gambiae Giles, An. coluzzii and Ae.

335 albopictus blood meals. Correct identification was observed in all samples tested after the

336 database was upgraded. Although MS profiling has been shown to be effective in this regard,

337 our technique still needs be tested on specimen collected in the field where feeding

338 parameters are less controlled than in laboratory conditions. All spectra of the home-made

339 database are available on request for other research teams.

132 340 Declarations

341

342 Ethics approval and consent to participate

343 Human blood was obtained from the national French blood bank, the “Etablissement Français

344 du Sang” (EFS) accredited by the human and animal ethics committees of the Institutional

345 Animal Care of IHU Méditerranée Infection. Consents for blood donation were collected at

346 EFS. Animal studies were conducted in line with 2010/63/EU directive of the European

347 Parliament and of the Council of September 22, 2010, and in compliance with French

348 Government Decree No. 2013-118 of February 1rst, 2013. The study was approved by the

349 local Ethics Committee (Comité d’Ethique en Expérimentation Animale, Marseille) and

350 Institutional Animal Care Committee in Marseille, France.

351 Consent for publication

352 Not applicable.

353

354 Availability of data and materials

355 All datasets regarding this study are included in the main paper and the additional supporting

356 files.

357 Competing interests

358 The authors declare that they have no competing interests.

359

360 Funding

361 This work has been carried out with the support of the Investissements d’Avenir program of

362 the French government, managed by the French National Research Agency including ANR-

363 11- IDEX-0001-02 (A*MIDEX project) and ANR-10-IAHU-03.

364

133 365 Authors’ contributions

366 AL and PP designed the experiments. FT, SN performed the experiments. FT, SN, ML, LA

367 and PP analyzed the data. PP, OKD, BD and DR provided reagents/materials/analysis tools.

368 FT, ML and NS wrote the paper. PP coordinated the writing of the paper. All authors

369 reviewed the final version.

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138 539 Table and figure legends

540 Table1. Number of mosquito samples submitted to MS analysis

541 Table 2. MALDI-TOF MS identification of An. gambiae successive blood meals

542 (a)Represent the numbers of specimens used to blood meals source identification against the

543 database. All mosquito were firstly engorged on human blood and then secondary fed on goat,

544 dog, cow, sheep and rabbit blood.

545 Table3. MALDI-TOF MS identification of successive blood meals of Aedes albopictus

546 and Anopheles coluzzii.

547 Table4. MALDI-TOF MS identification of Anopheles gambiae mixed blood meals

548 (x)Number of specimens used for blind test analysis.

549 Table5. Influence on the mosquito species on the MALDI-TOF MS identification of

550 Aedes albopictus and Anopheles coluzzii mixed blood meals Human/dog mixed blood

551 meals. (x)Number of specimens used for blind test analysis

552 Table6. Influence on the mosquito species on the MALDI-TOF MS identification of

553 Aedes albopictus and Anopheles coluzzii mixed blood meals. Human/sheep mixed blood

554 meals. (x)Number of specimens used for blind test analysis

555 Table7. Influence on the mosquito species on the MALDI-TOF MS identification of

556 Aedes albopictus and Anopheles coluzzii triple blood meals(x)Number of specimens used for

557 blind test analysis

558 Additional file 1: Table S1. MALDI-TOF MS home-made database

559 Figure legends

560 Figure1. Experimental workflow for mixed and successive blood meal identification

561 using MALDI-TOF MS.

139 562 Figure 2. Representative abdomen-derived MALDI-TOF MS spectra from Anopheles

563 gambiae Giles, Anopheles coluzzii and Aedes albopictus fed on goat (A1,A2,A3), cow

564 (B1,B2,B3), chicken (C1,C2,C3) blood. All mosquitoes were collected 12 hours post-

565 feeding. Au=arbitrary units; m/z=mass-to-charge ratio.

566

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140 582 Table1. Number of mosquito samples submitted to MS analysis Concentration Samples Anopheles gambiae Anopheles coluzzii Aedes albopictus Total Number of samples 14 10 23 47 75%Human/25%Dog Number of specimens for DB / 1 2 3 upgrading Number of samples Mixed blood meals 14 10 23 47 50%Human/50%Dog Number of specimens for DB Human-Dog 4 1 3 8 upgrading Number of samples 14 10 23 47 25%Human/75%Dog Number of specimens for DB / 1 2 3 upgrading Number of samples 4 10 22 36 75%Human/25%Sheep Number of specimens for DB / 2 2 4 upgrading Number of samples 5 14 23 42 Mixed blood meals 141 50%Human/50%Sheep Number of specimens for DB Human-Sheep 5 2 3 10 upgrading Number of samples 4 13 23 40 25%Human/75%Sheep Number of specimens for DB / 2 2 4 upgrading Number of samples Mixed blood meals 20 10 23 53 Equal concentration Number of specimens for DB Human-Dog-sheep 5 1 3 9 upgrading Total / / 89 87 177 353 583

584

585

586

587 588 Table 2. MALDI-TOF MS identification of An. gambiae successive blood meals . All mosquito were firstly engorged on human blood and

589 then secondary fed on goat, dog, cow, sheep and rabbit blood.

Mosquito species First blood meal Second blood Number of Results of MS identification meal specimens used [range LSVs] for blind test An. gambiae Human / 10 Human [1.834-2.320] An. gambiae Human Goat 10 Goat [2.173-2.670] An. gambiae Human Dog 10 Dog [2.139-2.764] An. gambiae Human Cow 10 Cow [1.810-2.396] An. gambiae Human Sheep 10 Sheep [1.802-2.296] An. gambiae Human Rabbit 10 Rabbit [1.826-2.101] Total / / 60 590 142 591

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598

599 600 Table3. MALDI-TOF MS identification of successive blood meals of Aedes albopictus and Anopheles coluzzii.. (x): Number of specimens 601 included in each analysis Mosquito First blood Second Identification of blood LSVs obtained Number of Identification of blood LSVs obtained species meal blood origin before upgrade (DB before upgrade specimen origin after upgrade (DB2) after upgrade meal 1) used for DB upgrade Ae. albopictus Human / Ae. albopictus Human (22) [2.148-2.602] / Ae. albopictus-Human(22) [2.148-2.602] Ae. albopictus Human Cow An. gambiae-Cow (12) [1.889-2.387] 3 Ae. albopictus-Cow(17) [1.941-2.873] An. coluzzii-Mouflon (8) [2.225-2.645] Ae. albopictus Human Sheep An. gambiae-Sheep(2) [1.714-1.745] 3 Ae. albopictus-Sheep(17) [1.972-2.754] An. coluzzii -Mouflon(18) [1.749-2.719] Ae. albopictus Human Goat An. gambiae -Goat(13) [1.762-1.961] 3 Ae. albopictus-Goat(17) [2.206-2.752] An. coluzzii-Mouflon(5) [2.085-2.492] Insuffisant(2) [1.549-1.696]

Ae. albopictus143 Human Chicken An. gambiae -Chicken (20) [1.894-2.294] 2 Ae. albopictus-Chicken(18) [2.117-2.636] Ae. albopictus Human Rabbit An. gambiae -Rabbit(21) [1.429-1.843] 3 Ae. albopictus-Rabbit(19) [2.196-2.535] An. gambiae -Ocelot(1) [1.767] An. coluzzii Human / An. gambiae -Human(10) [1.964-2.421] 1 An. coluzzii-human(9) [2.217-2.421] An. coluzzii Human Cow An. gambiae -Cow (10) [1.648-2.543] 1 An. coluzzii-Cow(9) [2.125-2.543] An. coluzzii Human Sheep An. gambiae -Sheep(10) [1.836-2.584] 1 An. coluzzii-Sheep(9) [1.855-2.584] An. coluzzii Human Goat An. gambiae -Goat (10) [1.637-1.943] 1 An. coluzzii-Goat(9) [2.014-2.239] An. coluzzii Human Chicken An. gambiae -Chicken(4) [1.236-1.38] 1 An. coluzzii-Chicken(9) [1.948-2.819] An. gambiae -Ocelot(4) [1.286-1.373] An. gambiae -Cow(1) [1.467] An. gambiae -Goat(1) [1.341] An. coluzzii Human Rabbit An. gambiae -Rabbit(10) [1.581-1.851] 1 An.coluzzii-Rabbit(9) [2.120-2.643] Total 184 80% 20 164 100% 602 DB: database

603 604 Table4. MALDI-TOF MS identification of Anopheles gambiae mixed blood meals (x)Number of specimens used for blind test analysis.

Mosquito species Concentration of Results of MS LSVs obtained Number Results of MS LSVs obtained from blood meal identification before from blind tests of identification after blind tests against upgrade (a) against specimens upgrade (DB 1) (a) database after upgrade database before used for pgrade DB upgrade Anopheles gambiae 75%Human/25%Dog Human(10) [1.819-2.23] / Human/dog(14) [2.055-2.529] Anopheles gambiae 50%Human/50%Dog Human(5) 4 Human/dog(10) [1.828-2.004] [2.115-2.477] Dog(5) Anopheles gambiae 25%Human/75%Dog Dog(10) [1.615-2.017] / Human/dog(10) [2.161-2.614] Anopheles gambiae 75%Human/25%Sheep Human(3) [1.897-2.047] / Human/Sheep (4) [2.399-2.493] Sheep(1) Anopheles gambiae 50%Human/50%Sheep Sheep(5) [1.146-2.216] 5 / / (4) (4) Anopheles144 gambiae 25%Human/75%Sheep Sheep [2.09-2.372] / Human/Sheep [2.405-2.771] Anopheles gambiae Human/Dog/Sheep Human(6) [1.791-2.38] 5 Human/dog/Sheep(20) [2.258-2.846] Equal concentration Human/dog(4) Human/Sheep(10 Total 14 72 605 DB: database

606

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613 Table5. Influence on the mosquito species on the MALDI-TOF MS identification of Aedes albopictus and Anopheles coluzzii mixed blood 614 meals Human/dog mixed blood meals. (x)Number of specimens used for blind test analysis Mosquito Concentration of Results of MS identification before LSVs obtained Number of Results of MS identification LSVs obtained species blood meal upgrade (DB 1) before upgrade specimen after upgrade (DB 2) after upgrade used for DB upgrade Ae. albopictus 75%Human/25%Dog An. gambiae-Human-Dog (2) [1.739-1.763] 2 Ae. albopictus-Human-Dog(21) [1.762-2.665] Ae. albopictus-Human(20) [1.700-2.140] Ae. albopictus (1) [1.702] Ae. albopictus 50%Human/50%Dog An. gambiae-Hunan-Dog (21) [1.686-2.006] 3 Ae. albopictus-Human-Dog(20) [1.945-2.428] An. gambiae-Human-Dog-Sheep (2) [1.746-1.853] Ae. albopictus 25%Human/75%Dog An. gambiae-Human-Dog (13) [1.736-2.269] 2 Ae. albopictus-Human-Dog(21) [2.067-2.724] An. gambiae-Human-Dog-Sheep (5) [1.899-2.136] 145 Ae. albopictus-Human(5) [2.039-2.156] An. coluzzii 75%Human/25%Dog Ae .albopictus-Human-Dog(4) [1.739-1.929] 1 An. coluzzii-Human-Dog(9) [1.948-2.932] Ae. albopictus-Human(5) [1.927-2.345] An. coluzzii-Human (1) [2.027] An. coluzzii 50%Human/50%Dog Ae. albopictus-Human-Dog(4) [1.810-1.964] 1 An. coluzzii-Human-Dog(9) [1.928-2.722] An. coluzzii-Human(4) [1.876-2.264] An. gambiae-Human(2) [2.036-2.215] An. coluzzii 25%Human/75%Dog Ae. albopictus-Human-Dog(8) [1.722-2.216] 1 An. coluzzii-Human-Dog(9) [2.111-2.835] An. gambiae-Human-Dog-Sheep(2) [1.860-1.904] Total 99 51.51% 10 89 100% 615 DB: database

616

617

618 619 Table6. Influence on the mosquito species on the MALDI-TOF MS identification of Aedes albopictus and Anopheles coluzzii mixed blood 620 meals. Human/sheep mixed blood meals. (x)Number of specimens used for blind test analysis Mosquito Concentration of Results of MS identification LSVs obtained Number of Results of MS identification LSVs obtained species blood meal before upgrade (DB 1) before upgrade specimen used after upgrade (DB 2) after upgrade for DB upgrade Ae. albopictus 75%Human/25%Sheep An.gambiae-Human- Sheep (18) [1.716-1.991] 2 Ae. albopictus-Human- [2.197-2.482] Ae.albopictus -Human (1) [1.68] Sheep(20) An.gambiae-Human-Dog-Sheep(3) [1.837-1.864] Ae. albopictus 50%Human/50%Sheep An.gambiae-Human- Sheep (22) [1.96-2.4] 3 Ae. albopictus-Human- [2.285-2.445] An.gambiae- Sheep (1) [1.922] Sheep(20)

Ae. albopictus 25%Human/75%Sheep An.gambiae-Human- Sheep (1) [1.975] 2 Ae. albopictus-Human- [2.323 -2.567] An.coluzzii- Sheep (20) [1.637-1.958] Sheep(21) Ae.albopictus-cow (1) [2.319] 146 An.coluzzii-mouflon(1) [1.707] An. coluzzii 75%Human/25%Sheep An.coluzzii-Human(1) [2.096] 2 An. coluzzii-Human- Sheep [2.134-3.000] Aedes albopictus- Human(7) [2.020-2.272] (8) Aedes albopictus- Cow(2) [2.168-2.193] An. coluzzii 50%Human/50%Sheep An.gambiae-Human- Sheep(3) [1.855-1.969] 2 An. coluzzii-Human- [2.312-2.608] Ae.albopictus-Human- Sheep(7) [1.805-1.993] Sheep(12) An. coluzzii- Sheep(1) [1.826] Ae. albopictus- Sheep(2) [2.084-2.088] An. gambiae-Human-Dog-Sheep(1) [2.048] An. coluzzii 25%Human/75%Sheep Ae. albopictus-Human- Sheep(3) [1.736-1.748] 2 An. coluzzii-Human- [2.222-2.475] An. coluzzii- Sheep(4) [1.689-1.932] Sheep(11) An. coluzzii-Mouflon(2) [1.714-2.084] Ae. albopictus- Sheep(3) [1.770-2.215] Ae. albopictus-Cow (1) [2.146] Total 105 51.42% 13 92 100% 621 Table7. Influence on the mosquito species on the MALDI-TOF MS identification of Aedes albopictus and Anopheles coluzzii triple blood 622 meals(x)Number of specimens used for blind test analysis Mosquito Concentration of Results of MS identification before LSVs obtained Number of Results of MS LSVs obtained species blood meal upgrade (DB 1) before upgrade specimen used identification after after upgrade for DB upgrade (DB 2) upgrade Ae. albopictus An. gambiae-Human-Dog-Sheep (2) [1.85-2.301] 3 Ae. albopictus -Human- [2.070-2.421] Human/Dog/Sheep An. gambiae-Human- Sheep (21) [1.875-2.49] Dog-Sheep(20) Equal concentration

An. coluzzii An. gambiae-Human-Dog-Sheep (10) [1.743-2.303] 1 An. coluzzii-Human- [2.391-2.725] Human/Dog/Sheep Dog-Sheep (9) Equal concentration

Total 33 36.36% 4 29 100% 623 DB: database 147 624 Table S1. MALDI-TOF MS home-made database

MOSQUITOES Imago : Aedes aegypti, Ae. albopictus, Ae. alternans, Ae. australis, Ae. caspius, Ae. cinereus, Ae. dufouri, Ae. flavifrons, Ae. (Legs for imago and whole mosquitoes for larvae) fowleri, Ae. multiplex, Ae. notoscriptus, Ae. polynesiensis, Ae. procax, Ae. vexans, Ae. vigilax, Ae. vittiger, Anopheles annulipes, An. arabiensis, An. claviger, An. coluzzii, An. coustani, An. funestus, An. gambiae Giles, An. hyrcanus, An. maculipennis, An. pharoensis, An. rufipes, An. wellcomei, An. ziemani, Coquillettidia richiardii, Coquillettidia xanthogaster, Culex annulirostris, Cx. australicus, Cx. insignis, Cx. modestus, Cx. molestus, Cx. neavei, Cx. orbostiensis, Cx. pipiens, Cx. quinquefasciatus, Cx. rima, Cx. sitiens, Cx. watti, Culiseta longiareolata, Lutzia tigripes, Mansonia uniformis, Ochlerothatus rusticus, O. excrucians, Orthopodomyia reunionensis, Verralina funerea Larvae : Ae. albopictus, Ae. aegypti, An. coluzzii, An. gambiae Giles, Cx. molestus, Cx. pipiens. Culiseta sp. LICE Pediculus humanus, Damalinia bovis, D. caprae, D. ovis, Haematopinus eurysternus, Linognatus vituli, L. africanus (Longitudinal cut) FLEAS Archaeopsylla erinacei, Ctenocephalides felis, C. canis Xenopsylla chopis (Whole flea without the abdomen) EtOh : Pulex irritans, Stenoponia tripectinata, Nosopsyllus fasciatus, C. canis TICKS Legs : Amblyomma variegatum, Dermacentor marginatus, D. marginatus infected R. slovaca, D. reticulatus, Haemaphysalis (Legs) concinna, Hae. punctata, Hyalomma. m. rufipes, Ixodes hexagonus, I. ricinus, Rhipicephalus bursa, Rh. sanguineus, Rh. sanguineus infected R. conorii, Rh. sanguineus infected R. massiliae, Rh. sulcatus EtOh : Am. gemma, Am. cohaerens, Am. variegatum, Argas persicus, Hae. leachi, Hae. punctata, Hae. spinulosa, Hy. detritum, Hy. m. rufipes, Hy. truncatum, I. ricinus, O. sonrai, Rh. annulatus, Rh. bergeoni, Rh. bursa, Rh. decoloratus, Rh. e. evertsi, Rh. microplus, Rh. praetextatus, Rh. pulchellus, Rh. sanguineus

148 Hemolymph: Am. variegatum infected R. africae, D. marginatus, Hy. m. rufipes, Rh. bursa, Rh. sanguineus BED BUGS Cimex lectularius, Cimex hemipterus (Heads) TRIATOMINAE Eratyrus mucronatus, Panstrongylus geniculatus, Rhodnius prolixus, Rh. pictipes, Rh. robustus, Triatoma infestans (Legs) SAND FLIES Phlebotomus papatasi, P. longicuspis, P. perfiliewi, P. perniciosus, P. sergenti, Sergentomyia minuta (Thoraces, wings and legs) MITE Leptotrombidium chiangraiensis, L. imphalum, L. deliense (Whole mite) BLATTIDAE Supella longipalpa, Periplaneta americana, Blatta orientalis, Blatella germanica, Blaptica dubia (Legs) FLIES Melophagus ovinus, Hippobosca equina (Legs) ABDOMENS OF ENGORGED MOSQUITOES Anopheles gambiae Giles fed on: Homo sapiens, Equus caballus, Ovis aries, Oryctolagus cuniculus, Mus musculus, Rattus norvegicus, Canis familiaris, Bos taurus, Capra hircus, Gallus gallus, Equus asinus, Tapirus indicus, Tapirus terrestris, Carollia perspicillata, Thraupis episcopus, Erythrocebus patas, Callithrix pygmaea, Leopardus pardalis, Arctictis binturong, Antidorcas marsupialus, Panthera onca , Papio hamadryas blood. Anopheles coluzzii fed on: Camelus dromedarius, Ammotragus lervia and Sus scrofa blood. Aedes albopictus fed on: Homo sapiens blood 625 626 627 Figure1. Experimental workflow for mixed and successive blood meal identification

628 using MALDI-TOF MS.

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149 641 642 Figure 2. Representative abdomen-derived MALDI-TOF MS spectra from Anopheles

643 gambiae Giles, Anopheles coluzzii and Aedes albopictus fed on goat (A1,A2,A3), cow

644 (B1,B2,B3), chicken (C1,C2,C3) blood. All mosquitoes were collected 12 hours post-

645 feeding. Au=arbitrary units; m/z=mass-to-charge ratio.

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IV. UTILISATION DE LA SPECTROMETRIE DE MASSE MALDI-TOF ET DE LA CULTUROMIQUE POUR ETUDIER LE MICROBIOTE DIGESTIF DES MOUSTIQUES

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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.

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Des études sur le rôle du microbiote des insectes ont augmenté ces dernières années. Cela a mené à une intensification des études centrées sur le microbiote de diverses espèces de moustiques, y compris une influence potentielle sur leur compétence vectorielle. Quelques études ont montré l'impact de la flore bactérienne du moustique dans la défense contre les parasites du paludisme, avec Enterobacteriaceae affectant le développement de Plasmodium falciparum au niveau de l’estomac de l’espèce Anopheles gambiae. L'approche culturomique précédemment utilisée dans notre laboratoire pour étudier le microbiote intestinal humain a été appliquée pour évaluer la diversité bactérienne de l'estomac d’Anopheles gambiae (souches sauvages et de laboratoire), Aedes albopictus (souches sauvages et de laboratoire) et Culex quinquefasciatus (souches sauvages). L'influence du statut environnemental sur le microbiote digestif des moustiques a été également étudiée. Les moustiques recueillis sur le terrain ont été identifiés avec précision par l’analyse du MALDI-TOF MS de leurs pattes. Le microbiote de l’estomac des moustiques adultes était composé de quatre phylums : Proteobacteria, Bacteroidetes, Actinobacteria and Firmicutes. La majorité des bactéries trouvées dans le microbiote des moustiques étaient des Gram-négatifs et appartiennent au phylum Proteobacteria. MALDI-TOF MS a permis d’isoler pour la première fois des nouvelles espèces bactériennes dans le microbiote digestif Anopheles gambiae. Dans cette étude, l'approche culturomique s'est révélée être une technique fiable pour explorer la diversité du microbiote des moustiques. La spectrométrie de masse MALDI-TOF a été confirmé comme une technique prometteuse pour identifier les moustiques collectés sur le terrain. La culturomique a permis l'isolement d’une nouvelle espèce bactérienne non associée aux moustiques vecteurs. L'environnement joue un rôle dans la diversité bactérienne du microbiote, ce qui pourrait permettre le développement de nouvelles stratégies de lutte contre les moustiques vecteurs de maladies.

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Tandina et al. Parasites & Vectors (2016) 9:495 DOI 10.1186/s13071-016-1776-y

RESEARCH Open Access Use of MALDI-TOF MS and culturomics to identify mosquitoes and their midgut microbiota Fatalmoudou Tandina1,2, Lionel Almeras1,3, Abdoulaye K. Koné2, Ogobara K. Doumbo2, Didier Raoult1 and Philippe Parola1*

Abstract Background: Mosquitoes transmit a wide range of human parasitic and viral diseases. In recent years, new techniques such as MALDI-TOF MS have been developed to identify mosquitoes at the species level, which is key for entomological surveys. Additionally, there is increasing interest in the mosquito microbiota and its role in vector capacity. Methods: The culturomics approach previously used in our laboratory to study human gut microbiota was applied to evaluate the midgut bacterial diversity of Anopheles gambiae (wild and laboratory strains), Aedes albopictus (wild and laboratory strains) and Culex quinquefasciatus (wild strains) in order to determine the influence of the environmental status on the midgut microbiota of the mosquitoes. Results: Mosquitoes collected in the field were accurately identified by MALDI-TOF MS analysis of their legs. Adult mosquito midgut microbiota was composed of four phyla, including Proteobacteria, Bacteroidetes, Actinobacteria and Firmicutes. The majority of the bacteria detected in the microbiota of mosquitoes were gram-negative and belong to the phylum Proteobacteria. MALDI-TOF MS identified for the first time a new bacterial species from An. gambiae midgut microbiota. Conclusion: In this study, the culturomics approach was found to be a reliable technique for exploring the diversity of the mosquito microbiota. MALDI-TOF MS was confirmed as a promising technique to identify mosquitoes collected in the field. Culturomics allowed the isolation of a new bacterial species not previously associated with mosquito vectors. The environment plays a role in the bacterial diversity of the microbiota, which could enable the development of new control strategies for mosquito-borne disease. Keywords: Anopheles gambiae Giles, Aedes albopictus, Culex quinquefasciatus, Culturomics, MALDI-TOF MS, Microbiota Abbreviations: 16S rRNA, 16S ribosomal ribonucleic acid; B, Bacillus;C,Culex; HPLC, High-performance liquid chromatography; LSV, Log score value; MALDI-TOF MS, Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry; MRTC, Malaria Research and Training Center; URMITE, Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes

* Correspondence: [email protected] 1Unité de Recherche en Maladies Infectieuses et Tropicales Emergentes (URMITE), UM63, CNRS 7278, IRD 198 (Dakar, Sénégal), Inserm 1095, Faculté de Médecine, Aix Marseille Université, 27 bd Jean Moulin, 13385 Marseille cedex 5, France Full list of author information is available at the end of the article

© 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

157 Tandina et al. Parasites & Vectors (2016) 9:495 Page 2 of 11

Background Methods There are over 3500 different species of mosquitoes with Laboratory-reared mosquitoes a worldwide distribution [1]. The most described species An. gambiae Giles laboratory colonies and Ae. albopictus that are able to transmit pathogens to humans and ani- collected in the south of France in June 2013 were main- mals belong to the genera Aedes, Culex and Anopheles tained by breeding at our laboratory in Marseille, France. [1]. Mosquito vectors are not limited to tropical areas, Briefly, Ae. albopictus and An. gambiae from the labora- where malaria, dengue and chikungunya are well-known tory were reared using standard methods at a threats for the local population and travellers [2, 3]. The temperature of 26 ± 1 °C, a relative humidity of 70–90 % tiger mosquito, Aedes albopictus, is an invasive species and a photoperiod of 12 h (light/dark) in standalone that has spread across the world in the last two decades incubators (Panasonic cooled incubator) [15]. After [4]. The global expansion of Ae. albopictus may modify emerging from the pupae, the adults An. gambiae and the worldwide epidemiology of arbovirus and increase Ae. albopictus were fed with a 10 % (w/v) sucrose the risk to humans of mosquito-borne diseases [5]. solution. For egg production, blood meals were given Culex spp. mosquitoes include vectors of human dis- through a parafilm membrane (Hemotek membrane eases such as arboviral diseases and lymphatic filariasis feeding systems, Discovery Workshops, Accrington, [1]. Anopheles gambiae (s.l.) mosquitoes are the major England, UK) using fresh heparinized sheep blood malarial vectors in sub-Saharan Africa [6]. Malaria para- over 1 h [5]. The blood-feeding was performed every sites are transmitted from human to mosquito when a three days, according to the gonotrophic cycle. female Anopheles ingests a gametocyte-infected blood Engorged female An. gambiae and Ae. albopictus were meal [6]. In the mosquito midgut, malaria parasites transferred into another cage and were maintained in undergo a series of complex developmental stages and standard conditions with 10 % (w/v) sucrose solution transmission depends on the success of the different on cotton. Larvae were reared to the nymph stage in transition steps [7]. trays containing distilled water. Pupae were collected Studies on the role of the microbiota within the gut of daily and transferred to mosquito cages (Bug Dorm 1, insects have increased in recent years [8, 9]. This has led BioQuip Products, Gladwick Street, USA). Larvae were to an intensification of studies focused on the microbiota fed with fish food (TetraMin) until the pupal stage [16]. of diverse mosquito species, including a potential influ- To explore the midgut microbiota of the laboratory ence on their vector competence [9, 10]. Some studies mosquito colonies, the midguts of six specimens of An. have shown the impact of the mosquito midgut gambiae and five specimens of Ae. albopictus were microbiota in the defense against malaria parasites, dissected. with Enterobacteriaceae affecting the development of Newly emerged adult An. gambiae specimens from P. falciparum in the An. gambiae mosquito midgut our laboratory colonies were used in the present work. [7]. Moreover, by using antibiotic treatments to clear These anopheline mosquitoes were collected immedi- the midgut microbiota, other studies have suggested a ately after emergence and maintained under standard protective role of An. gambiae midgut bacteria against conditions and fed only with 10 % (w/v) sucrose solution Plasmodium infections [11, 12]. It has also been sug- for three days. Female mosquitoes were engorged on gested that antibiotics in ingested blood enhance the human blood (defibrinated human blood) over one hour. susceptibility of An. gambiae mosquitoes to malarial Anopheles gambiae females (three specimens) engorged infection by disturbing their gut microbiota. In on human blood were sacrificed 24 h after the blood addition, antibiotic exposure increases mosquito sur- meals (at day one) and midgut dissection was immedi- vival and fecundity, which are factors increasing vec- ately performed under sterile conditions. torial capacity [13]. Additionally, one male and one female adult speci- In recent years, a new approach using special cultures, men from the F1 generation, resulting from An. gam- named culturomics, has been developed in our labora- biae female specimens who were fed on blood, were tory for the identification of not only the human gut sacrificed 24 h after their emergence. There were a microbiota, including common bacteria, but also mi- total of 12 An. gambiae specimens, including male nority bacterial populations [14]. Here, we used a cul- (n = 3) and female (n = 3) specimens fed only on turomics approach to study the midgut bacterial sucrose solutions, and females fed on human blood diversity of three mosquito species, including wild (n = 3). In addition, one male and one female adult and laboratory strains. The bacterial patterns of mos- specimen of the F1 generation of these last two quito species reared in the laboratory were compared groups were also tested. In this second experiment to mosquitoes collected in the field within their re- there was a sequential collection (i.e. D1, D3, D8 and spective water sites to assess the effect of the envir- F1) of mosquito midguts. Midguts were collected and onment on bacterial populations. analysed by culturomics.

158 Tandina et al. Parasites & Vectors (2016) 9:495 Page 3 of 11

Mosquitoes in the wild TGG-3'; HC02198 (reverse): 5'-TAA ACT TCA GGG TGA Collection of wild mosquitoes CCA AAA AAT CA-3') [21]. The PCR reaction contained Aedes albopictus was captured in Marseille by human 13 μl of sterile distilled water, 2.5 μl of 10X Phusion HF Buf- landing catches in the garden of the Faculty of Medicine fer (15 mM), 2.5 μl of dNTPs (2 mM), 0.5 μl of each primer in August 2014. The collected Ae. albopictus mosquitoes (10 μM), 0.25 μl of Hot star Taq (5units/μl), 1 μlofMgCl2 were individually conserved in caps prior to transport to (25 mM) and 5 μl of extracted DNA. Reactions were ampli- the laboratory. In Mali, 53 An. gambiae (s.l.) and 204 fied through 35 cycles at the following parameters: 10 min Culex spp. mosquitoes were captured using the CDC at 95 °C, 1 min at 95 °C, 1 min at 40 °C, 1.5 min at light trap from the Sikasso region (south Mali) from 72 °C, followed by a final extension step at 72 °C for April to May, 2014. Specimens were sterilized in 70 % 7min. ethanol (2–10 min) and then rinsed in distilled water. A set of primers specifically amplifying a fragment of Each adult mosquito was transferred individually to a 310 bp of the An. gambiae mosquito complex > Acom- 1.5 ml Eppendorf tube and the specimens were then plex_28S_MBF AGC KCG TCT TGG TCT GGG G kept at -80 °C and sent frozen to the URMITE labora- and > Acomplex_28S_MBR GCC GAC AAG CTC AYT tory (Marseille, France). AGT GT were designed in the URMITE laboratory based on the publication of Fanello et al., and PCR reac- Identification of wild mosquitoes tions were processed as described [22]. Positive PCR The collected specimens were initially identified using products were then purified and sequenced using the morphological criteria [17]. Additionally, each specimen same respective primers with the BigDye version 1–1 was submitted to MALDI-TOF MS for identification as Cycle Sequencing Ready Reaction Mix (Applied previously described [18, 19]. Legs from each mosquito Biosystems, Foster City, CA) and an ABI 3100 were homogenized manually in 20 μl of 70 % (v/v) for- automated sequencer (Applied Biosystems). The se- mic acid and 20 μl of 50 % (v/v) acetonitrile in 1.5 ml quences were assembled and analyzed using the microtubes using pellet pestles (Fischer Scientific, Stras- ChromasPro software (version 1.34) (Technelysium bourg, France). The homogenates were centrifuged at Pty. Ltd., Tewantin, Australia) and BLAST website 10,000 rpm for 20 s, and 1 μl of the supernatant of each (http://blast.ncbi.nlm.nih.gov). sample was deposited on a steel target plate (Bruker Dal- tonicsTM, Wissembourg, France) into four spots for Water from breeding sites each sample [18]. Then, 1 μl of CHCA matrix composed For mosquito colonies reared in the laboratory (i.e. An. of saturated a-cyano-4-hydroxycynnamic acid (SigmaH, gambiae and Ae. albopictus), 200 μl of the laboratory Lyon. France), 50 % (v/v) acetonitrile, 2.5 % (v/v) tri- breeding water was collected with a Pasteur pipette and fluoroacetic acid and HPLC-grade water was directly put into the 1.5 ml Eppendorf sterile tubes for culturo- overlaid on each sample on the target plate, dried for mics analyses. Breeding water was then used for the cul- several minutes at room temperature and introduced turomics experiments to control and compare the into the MALDI-TOF MS instrument for analysis [19]. bacterial diversity between the environmental breeding Protein mass profiles were obtained using Microflex site and the adult mosquito midgut. LT MALDI-TOF Mass Spectrometry (Bruker Daltonics, The breeding water of the wild mosquitoes from Germany) with Flex Control software (Bruker Daltonics, Marseille was recovered with a ladle near the place Germany) as previously described [18, 20]. Measure- where the mosquitoes had been collected on humans. ments were performed in the linear positive-ion mode The water was then transferred to a 15 ml sterile tube within a mass range of 2–20 kDa. Each spectrum corre- and transported to the laboratory. In the Sikasso region, sponds to ions obtained from 240 laser shots performed in Mali, three breeding water sites were selected and in six regions of the same spot. The spectrum profiles collected with ladle sampling, prior to being trans- obtained were visualized with flexAnalysis 3.3 software ferred to a 15 ml sterile tube and transported to the and exported to the MALDI Biotyper v. 3.0 (Bruker laboratory. Then, the samples were stored at -80 °C Daltonics, Germany) [19]. until they were transported frozen to URMITE. Molecular tools were also used to confirm the identifica- Each breeding site was geo-positioned as follows: tion of some mosquitoes. DNA extractions from individual breeding site 1 (-5°66'13.1"N, 11°30'95.2"E); breeding mosquito heads and thorax samples were performed with site 2 (-5°60'80.6"N, 11°30'30.6"E) and breeding site 3 the EZ1 DNA Tissue Kit (Qiagen, Hilden, Germany) accord- (-5°60'73.8"N, 11°30'58.0"E). ing to manufacturer recommendations. A set of primers specifically amplifying a fragment of the mosquito cyto- Mosquito gut dissection chrome c oxidase subunit I gene (mCOI) was used Adult mosquitoes were anesthetized with cold at -20 °C (LCO1490 (forward): 5'-GGTCAACAA ATC ATA AGATAT for 10 min. All midgut mosquitoes from laboratory

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colonies (An. gambiae and Ae. albopictus) and wild mos- covered with 1.5 μl of the matrix solution composed of a quitoes were dissected under sterile conditions. Mosqui- saturated solution of α-cyano-4-hydroxycinnamic acid toes were surface-sterilized in 70 % (v/v) ethanol for 2– (SigmaH, Lyon, France) diluted in 500 μl of acetonitrile 10 min, then rinsed three times in a sterile saline buffer 50 % (v/v), 250 μl of trifluoroacetic acid 10 % (v/v) and 0.9 % (w/v) NaCl (Laboratoires Gilbert, France). The 250 μl of HPLC water and dried for several minutes at midgut was carefully removed under a stereo micro- room temperature. The target plate was then submitted scope (10× magnification) using clean forceps. to MALDI-TOF mass spectrometry for bacterial identifi- Midguts from the An. gambiae laboratory colony, Ae. cation as previously described [25]. To control loading albopictus laboratory colony and Ae. albopictus wild col- on mass spectra steel, matrix quality and MALDI-TOF ony were placed individually and midguts from the An. apparatus performance, the matrix solution was loaded gambiae wild colony and C. quinquefasciatus wild col- in duplicate onto each MALDI-TOF plate with and ony were pooled in sterile Eppendorf tubes containing without a Bacterial Test Standard (Bruker Protein Cali- 200 μl of 0.9 % (w/v) NaCl (Laboratoires Gilbert, France) bration Standard I). A Microflex LT MALDI-TOF mass and homogenized using a single-use pestle and centri- spectrometer (Bruker Daltonics, Germany) was used for fuge and vortex [23, 24]. There were a total of six An. bacterial identification according to the manufacturer’s gambiae from the laboratory in Marseille for the first ex- recommendations. Spectra were recorded in a linear periment and 12 An. gambiae from the laboratory in mode, within a mass range of 2,000 to 20,000 Daltons Marseille for the second experiment; five Ae. albopictus (Da). For each spectrum, data for multiple laser shots from the laboratory in Marseille, four mosquitoes field- were collected, summed and analysed. A maximum of collected in Marseille and twelve mosquitoes field- 100 peaks was used for each spectrum, and these peaks collected in Mali. were compared with the computer database at the Bru- ker base and the lab-specific base at La Timone hospital. Culturomics procedure An isolate was considered to be correctly and signifi- The standard and optimal conditions for the culturomics cantly identified at the species level when the queried approach were used, based on previous work performed spectrum had a log score value (LSV) ≥ 1.9 [26]. Every in our laboratory, notably for research on the human gut unidentified colony was tested successively three times. microbiota [14]. This technique started with pre- When the strain remained unidentified, the 16S rRNA incubation: a special liquid media comprising 15 g/l brain gene was sequenced. Spectra from new bacteria species heart infusion (Becton, Dickinson and Company, Sparks, not yet included in the database and identified by 16S MD 21152 USA; 38800 Le Pont-de-Claix, France), 5 g/l rRNA sequencing were added to the database. Bacto yeast extract (Becton, Dickinson and Company, Sparks, MD 21152 USA; 38800 Le Pont-de-Claix, France), 5 g/l proteose peptone (Oxoid Ltd, Basingstoke, Hamp- 16S rRNA gene sequencing shire, England), 1000 ml of sterile water (Fresenius Kabi Identification with 16S rRNA gene sequencing was per- France, 5 Place de Marivel, 92310 Sèvres, France) and 5 % formed for the bacteria not identified by MALDI-TOF (v/v) sheep blood in aerobic and anaerobic conditions at MS. For this, the bacterial strain was suspended in 28 °C for 1 month. We inoculated them on 5 % (v/v) 200 μl of sterile water and was heated at 100 °C for sheep blood agar (bioMérieux, Marcy l’Etoile, France) 10 min. The 16S rRNA gene was amplified by PCR using after performing ten serial dilutions from 1/10 to 1/10-10, the universal primer pair fd1 and rp2 and an annealing allowing the growth of fastidious bacteria in order to iso- temperature of 52 °C. The PCR products were purified late a maximum of bacterial species. This operation was using a NucleoFast 96 PCR kit (Nanogen, San Diego, carried out every 5 days from day 1 to day 25 (i.e. D1, D5, USA). The sequence reactions were performed with the D10, D15, D20 and D25). Bacterial colonies were then iso- BigDye Terminator v1.1 Cycle Sequencing Kit (Perkin- lated on 5 % (v/v) sheep blood agar after 24 h, and submit- Elmer), with primers fd1, rp2, 536 F, 536R, 800 F, 800R, ted to mass spectrometry (MALDI-TOF MS) for 1050 F and 1050R (Table 1). The products of the se- identification. Bacteria not identified by MALDI-TOF quencing reaction were purified, and the sequences were MS were then submitted to molecular biology for analysed using an ABI PRISM 3130xl Genetic Analyzer taxonomic determination by 16S sequencing. (Applied Biosystems). The obtained sequences were compared with the GenBank database using BLAST soft- Bacterial identification ware. A threshold similarity value of > 98.7 % was Mass spectrometry (MALDI-TOF) chosen for identification at the species level [27]. Below Each bacterial colony obtained from culture was deposed this value, a new species was suspected, and the isolated in duplicate directly onto a MALDI-TOF plate target strain was characterized in detail using phenotypic ana- (Bruker DaltonicsTM, Wissembourg, France) and lyses and electron microscopy and genome sequencing.

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Table 1 List of primers used for 16S rRNA amplification and Mosquitoes reared in the laboratory and their respective sequencing breeding water Primers names Primer sequences (5′–3′) Temperature (°C) In the first experiment, the midgut microbiota of six An. fd1 AGA GTT TGA TCC TGG CTC AG 52 gambiae from the laboratory colony were analysed by rp2 ACG GCT ACC TTG TTA CGA CTT 52 culturomics. A wide variety of bacteria were found at the end of the sequential 1-month culture. A total of ten 536 F CAG CAG CCG CGG TAA TAC 50 distinct bacterial species were identified (Table 2). Bac- 536R GTA TTA CCG CGG CTG CTG 50 terial diversity included three phyla: Proteobacteria 800 F ATT AGA TAC CCT GGT AG 50 (50 %), Firmicutes (30 %) and Bacteroidetes (20 %). Two 800R CTA CCA GGG TAT CTA AT 50 bacterial species, Cedecea lapagei and Serratia marces- 1050 F TGT CGT CAG CTC GTG 50 cens, were isolated in five midguts (83 %) among the six 1050R CAC GAG CTG ACG ACA 50 tested. Enterococcus faecium, Elizabethkingia meningoseptica and Serratia ureilytica were isolated in four midguts (67 %). Elizabethkingia miricola, Enterobacter cloacae, Enterococcus avium, Enterococcus raffinosus and Results Enterobacter kobei were less frequently detected in the Identification of the mosquitoes collected in the field six midguts of the An. gambiae tested (Table 2). Four mosquitoes were collected using the human land- In a second experiment, 12 An. gambiae specimens of ing catches method in the Timone hospital garden the laboratory colony were used. A total of 16 distinct (Marseille, France). They were morphologically identified bacterial species were identified belonging to 12 genera: as Ae. albopictus specimens. The submission of their Enterococcus, Enterobacter, Serratia, Acinetobacter, Eli- legs for MALDI-TOF MS analysis confirmed that the zabethkingia, Microbacterium, Staphylococcus, Strepto- four specimens were Ae. albopictus (LSVs > 1.9). coccus, Cedecea, Pseudomonas, Rhodococcus and In Sikasso, Mali, 257 were mosquitoes captured with Klebsiella in An. gambiae mosquito midguts (Table 3). the CDC light trap, 53 and 204 were identified by This diversity is composed of the bacteria of four phyla: morphologic keys as An. gambiae (s.l.) and 204 Proteobacteria (60 %), Firmicutes (20 %), Bacteroidetes Culex spp. mosquitoes, respectively. Legs of 6 An. (6.67 %) and Actinobacteria (13.33 %) (Table 3). A total gambiae (s.l.)and6Culex spp. specimens were sub- of six bacterial species were common to An. gambiae mitted to MALDI-TOF MS for identification. midgut and breeding water, and seven bacterial species MALDI-TOF MS results confirmed the morpho- were found only in the breeding water (Table 3). logical identification of An. gambiae (s.l.) (LSVs > A wide variety of bacteria in the midgut of Ae. albopic- 1.9) and revealed that Culex spp. specimens were all tus from the laboratory was observed. A total of 11 dis- C. quinquefasciatus (LSVs > 1.9). Among these field- tinct bacterial species were identified in mosquito midguts collected mosquitoes in Mali, six An. gambiae and (Fig. 1). Acinetobacter baylyi were isolated in three mid- six C. quinquefasciatus were selected for analysis of guts (60 %). Acinetobacter guillouiae, Achromobacter xylo- their microbiota. soxidans, Cedecea lapagei, Cedecea neteri, Serratia

Table 2 List of bacteria identified in the midguts of Anopheles gambiae bred under laboratory conditions Bacteria Midgut 1 Midgut 2 Midgut 3 Midgut 4 Midgut 5 Midgut 6 Cedecea lapagei ××××× Enterococcus faecium* ×× ×× Elizabethkingia miricola* ××× Elizabethkingia meningoseptica ×××× Enterobacter cloacae ×× Serratia marcescens ××××× Enterococcus avium* × Enterococcus raffinosus* ××× Serratia ureilytica* ×××× Enterobacter kobei* ×× Bacterial species reported for the first time in Anopheles gambiae are indicated by an asterisk. Presence of bacteria is indicated by ×. Bold corresponds to bacterial species common in the midgut and breeding water

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Table 3 List of bacteria identified in the midguts of laboratory-bred Anopheles gambiae non-engorged or engorged and in breeding water Bacteria Female fed on blood Female sj Male Breeding water Aeromonas hydrophila × Aeromonas jandaei × Klebsiella oxytoca ×× Enterococcus faecium × Enterobacter asburiae* ××× Enterobacter kobei* ×××× Serratia marcescens ×××× Serratia fonticola × Serratia ureilytica* × × Serratia plymuthica × Bacillus cereus × Sphingobacterium multivorum × Oceanobacillus massiliensis × Acinetobacter baylyi* × Elizabethkingia meningoseptica ××× Microbacterium maritypicum* × Staphylococcus epidermidis* × Streptococcus sanguinis* × Streptococcus mitis* × Cedecea lapagei ×× Pseudomonas gessardii* × Rhodococcus erythropolis* × Enterobacter cloacae ××× Bacterial species reported for the first time in Anopheles gambiae are indicated by an asterisk. Presence of bacteria is indicated by ×. Bold corresponds to bacterial species common in the midgut and breeding water Abbreviations: sj, sample from a female fed only on sweet juice, corresponding to the negative control

Fig. 1 Isolation and identification of bacteria in the breeding water and midgut of an Aedes albopictus laboratory colony (Marseille, France). Bacterial species reported for the first time in Aedes albopictus microbiota are indicated by an asterisk (*)

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marcescens, Serratia ureilytica, Staphylococcus epidermi- Raoultella, Robinsonella, Rothia, Shewanella and Serra- dis, Pantoea stewartii and Microbacterium kitamiense tia. Two bacterial species, Bacillus cereus and Entero- were less frequently represented (Fig. 1). The breeding coccus faecium, were common to the An. gambiae wild water of Ae. albopictus was analysed, and five bacterial strains and breeding water (Fig. 3, Table 4). species were identified (Fig. 1). Among them, Serratia marcescens and Microbacterium kitamiense were found Discussion both in the Ae. albopictus mosquito midguts and their This work analysed the midgut microbiota composition breeding water. Three genera, Aeromonas, Carnobacter- of mosquitoes reared in the laboratory and collected in ium and Lactobacillus, were found only in the breeding the wild and compared this midgut microbiota diversity water (Fig. 1). with their respective breeding sites, using an original strategy based on a special culture, the culturomics Mosquitoes collected in the field and their breeding technique. water Furthermore, for the definitive identification of mos- Five distinct bacterial species were isolated from the quitoes collected in the field, a new innovative method midguts of Ae. albopictus field-collected mosquitoes based on the analysis of mosquito leg protein spectra (Marseille), including four genera: Bacillus, Micrococcus, obtained by MALDI-TOF MS was used [19, 28]. Staphylococcus and Serratia (Fig. 2). This approach has been applied here for the first time Serratia marcescens bacteria were common in the Ae. to mosquitoes collected in the field from Africa. This is albopictus mosquito midguts and their breeding water. further evidence that use of MALDI-TOF MS to identify Eleven bacterial species from five genera, Aeromonas, mosquitoes is a rapid, accurate analysis technique, at Clostridium, Enterococcus, Lactococcus and Morga- low cost in terms of consumables [19, 28]. nella, were found only in their breeding water. Several previous studies have already analysed mos- The gut microbiota of An. gambiae in the wild (Mali) quito microbiota and the water of their respective breed- was composed of ten bacterial species from seven gen- ing sites [10]. Most of these studies used molecular era: Enterobacter, Pasteurella, Pseudomonas, Bacillus, approaches, mainly based on analysing sequences of the Enterococcus, Staphylococcus and Kocuria (Fig. 3). 16S ribosomal RNA gene and cultures of mosquito mid- Midgut microbiota of C. quinquefasciatus from Mali gut microbiota [10, 29]. was composed of five bacterial species from five genera: The culturomics approach used in this work revealed Pseudomonas, Escherichia, Propionibacterium, Staphylo- a wide diversity of the midgut microbiota of An. gam- coccus and Lactobacillus (Fig. 3). biae (wild and laboratory strains), Ae. albopictus (wild In breeding water sites, 51 bacterial species were culti- and laboratory strains) and C. quinquefasciatus (wild vated representing 15 genera: Acinetobacter, Aeromonas, strains). Arthrobacter, Bacillus, Clostridium, Delftia, Entero- The majority of the bacteria detected in the microbiota coccus, Lactococcus, Lysinibacillus, Pseudomonas, of mosquitoes were gram-negative and belong to the

Fig. 2 Isolation and identification of bacteria in the breeding water and midgut of Aedes albopictus wild colonies (Marseille, France)

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Fig. 3 Isolation and identification of bacteria in the midgut of Anopheles gambiae and Culex quinquefasciatus wild colonies in Sikasso (Mali). Bacterial species reported for the first time in Anopheles gambiae and Culex quinquefasciatus microbiota are indicated by an asterisk (*) phylum Proteobacteria, similar to other studies [10, 29]. Pseudomonas, Bacillus, Enterococcus, Staphylococcus and However, 17 new bacterial species not previously identi- Kocuria. The gut microbiota of An. gambiae from field col- fied in An gambiae midgut microbiota have been iso- lection (Cameroon) was found to be dominated by Coma- lated here: Serratia ureilytica, Enterobacter kobei, monas, Serratia, Pseudomonas, Burkholderia and Enterococcus faecium, Enterococcus avium, Enterococcus Brevundimonas bacteria by pyrosequencing analysis [30]. raffinosus, Elizabethkingia miricola, Acinetobacter bay- We found that some bacterial species were common lyi, Cedecea neteri, Enterobacter asburiae, Pseudomonas in the midgut of An. gambiae and Ae. albopictus labora- gessardii, Streptococcus sanguinis, Streptococcus mitis, tory strains. Staphylococcus epidermidis, Clostridium perfringens, Comparing the Ae. albopictus laboratory strain with Microbacterium maritypicum, Pseudomonas massiliensis those of the Ae. albopictus wild strain, we observed that and Rhodococcus erythropolis. Serratia marcescens was the only bacterial species found Interestingly, among the bacterial colonies submitted in common. Eight bacterial species were only found in the to MALDI-TOF MS identification, one isolated in breed- midgut of Ae. albopictus in the laboratory. Conversely, four ing water (Mali) was not identified, and corresponded to were specific for the midguts of the Ae. albopictus wild Lactococcus chungangensis according to 16S sequencing. strain. A difference was observed between the midgut This bacterial species was then implemented in the microbiota of Ae. albopictus laboratory and wild strains. In MALDI-TOF MS. addition to the contribution to the knowledge of bacterial Moreover, a bacterial species, Pseudomonas massiliensis, species associated with the microbiota of mosquito vectors, recently isolated in the Timone laboratory, which is under these results suggest that the environment plays a major role description (D. Raoult, personal communication), was iso- in variations of the midgut microbiota diversity of mosqui- lated in the An. gambiae and C. quinquefasciatus midgut toes. All the bacteria isolated from the laboratory and wild and their breeding water collected from Mali. mosquito microbiota and breeding water are ubiquitous in Six bacterial species were commonly found in the midgut the environment and are found in water and soil, as well in of An. gambiae laboratory colonies from Marseille and its association with plants, insects, humans and other animals respective breeding water. Moreover, 12 and seven bacter- [31–33]. These results correlate with other studies; namely ial species were found only in the midgut of An. gambiae that the environmental conditions of the vectors are key de- laboratory colonies from Marseille and its breeding water, terminants in shaping the midgut microbiota [24]. respectively. Three bacterial species (Enterococcus faecium, The main limitation of our growth conditions is that Enterobacter cloacae and Staphylococcus epidermidis)were culturomics does not allow the growth of some strictly commonly found both in the midgut of An. gambiae wild anaerobic bacteria [14]. Strategies are currently under strains and laboratory strains. development by the team culturomics to enable the The gut microbiota of An. gambiae in the wild (Mali) growth of these bacteria considered uncultured by cul- was composed of seven genera: Enterobacter, Pasteurella, turomics [34].

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Table 4 Composition of the microbiota of three breeding Table 4 Composition of the microbiota of three breeding water samples in Sikasso (Mali) water samples in Sikasso (Mali) (Continued) Bacteria Breeding Breeding Breeding Bacteria Breeding Breeding Breeding water 1 water 2 water 3 water 1 water 2 water 3 Acinetobacter lwoffii ××Pseudomonas putida ×× Acinetobacter towneri × Pseudomonas rhodesiae × Aeromonas hydrophila × Pseudomonas tolaasii × Aeromonas veronii × Raoultella ornithinolytica × Arthrobacter gandavensis × Robinsoniella peoriensis × Bacillus cereus ××× Rothia aeria × Bacillus idriensis × Shewanella profunda × Bacillus megaterium × Serratia fonticola ×× Clostridium absonum × Presence of bacteria is indicated by × Clostridium amylolyticum × Bold corresponds to bacterial species common in the midgut and breeding water Clostridium ××× anorexicamassiliense Clostridium aerotolerans × The sample size of laboratory mosquitoes used in this Clostridium butyricum ××study is higher than the number of wild mosquitoes col- Clostridium bifermentans ×××lected; this may explain the increase in the number of bacteria isolated in the laboratory mosquitoes. Clostridium cadaveris × Despite the previous studies of the mosquito midgut Clostridium celerecrescens ×× microbiota, it is still necessary to extend our knowledge Clostridium collagenovorans × in this domain by using new tools for exploration, such Clostridium glycolycum ××as culturomics. This culturomics approach allowed the Clostridium ihumii × isolation of bacterial species not previously associated Clostridium lituseburense ×××with these vectors, and will aid the development of new Clostridium n ××control strategies for mosquito-borne diseases. Clostridium perfringens × Clostridium sardiniense × Conclusions To conclude, diverse bacterial species were found in com- Clostridium sartagoforme × mon in the midguts of adult An. gambiae, Ae. albopictus Clostridium senegalensis × and C. quinquefasciatus and in breeding water. The ma- Clostridium sordellii × jority of the bacterial species belong to the phyla Proteo- Clostridium sphenoides × bacteria and Firmicutes. Culturomics allowed the isolation Clostridium sporogenes × of bacterial species identified for the first time in An. gam- Clostridium tertium ××biae midgut. This study demonstrates a wide diversity of new species of bacteria associated with the mosquito Delftia acidovorans × microbiota, which may be targets for vector control strat- Enterococcus casseliflavus ×××egies. Our study shows that the immediate environment Enterococcus faecium ×××plays an important role in the acquisition of bacteria by Enterococcus hirae ××the mosquito. The innovative culturomics technique and Enterococcus italicus × MALDI-TOF MS application are evidence of the growth Enterococcus mundtii × and correct identification of bacteria, isolated without ambiguity from the mosquito microbiota. Enterococcus termitis

Lactococcus chungangensis ×××Acknowledgments Lysinibacillus sphaericus × We would like to acknowledge Prof. D. Fontenille (IRD, Montpellier, France) for providing Anopheles gambiae laboratory colonies, Dr. Jean-Christophe Pseudomonas chlororaphis × Lagier for sharing his culturomics expertise, Constentin Dieme for help with Pseudomonas corrugata × mosquito dissection, and Sirama Niaré, who introduced me to mosquito identification by MALDI-TOF and sequencing. This work has been carried out Pseudomonas fluorescens × with the support of the A*MIDEX project (No. ANR-11-IDEX-0001-02) funded “ ’ ” Pseudomonas jessenii × by the Investissements d Avenir French Government program, managed by the French National Research Agency (ANR) and the Foundation Mérieux Pseudomonas monteilii × grant to MRTC for the field specimens collection.

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Funding 11. Dong Y, Manfredini F, Dimopoulos G. Implication of the mosquito This manuscript has been reviewed and corrected by American journal midgut microbiota in the defense against malaria parasites. PLoS experts. This work has been carried out thanks to the support of the Pathog. 2009;5:e1000423. A*MIDEX project (n° ANR-11-IDEX-0001-02) funded by the Investissements 12. Beier MS, Pumpuni CB, Beier JC, Davis JR. Effects of para-aminobenzoic acid, d’Avenir French Government programme, managed by the French National insulin, and gentamicin on Plasmodium falciparum development in Research Agency (ANR). anopheline mosquitoes (Diptera: Culicidae). J Med Entomol. 1994;31:561–5. 13. Gendrin M, Rodgers FH, Yerbanga RS, Ouedraogo JB, Basanez MG, Availability of data and material Cohuet A, Christophides GK. Antibiotics in ingested human blood affect Not applicable the mosquito microbiota and capacity to transmit malaria. Nat Commun. 2015;6:5921. 14. Lagier JC, Armougom F, Million M, Hugon P, Pagnier I, Robert C, et al. Authors’ contributions Microbial culturomics: paradigm shift in the human gut microbiome study. Designed and developed the experiments: AL, PP. Performed the experiments: Clin Microbiol Infect. 2012;18:1185–93. TF, KKA. Analyzed the data: AL, TF, PP. Contributed reagents/ materials/ analysis 15. Awono-Ambene HP, Diawara L, Robert V. Comparison of direct and tools: DKO, RD. Wrote the paper: TF, AL, PP. Contributed to the paper redaction: membrane feeding methods to infect Anopheles arabiensis with Plasmodium DKO, RD. All authors read and approved the final version of the manuscript. falciparum. Am J Trop Med Hyg. 2001;64(1–2):32–4. 16. Moutailler S, Bouloy M, Failloux AB. Short report: efficient oral infection of Competing interests Culex pipiens quinquefasciatus by Rift Valley fever virus using a cotton stick The authors declare that they have no competing interests. support. Am J Trop Med Hyg. 2007;76(5):827–9. 17. Hervy J, Le Goff G, Geoffroy B, Herve J and Manga L. Les Anopheles de la Consent for publication région Afro-tropicale, logiciel d’identification et d’ensegnement, Collection Not applicable. didactique. version ORSTOM. 1998. 18. Yssouf A, Flaudrops C, Drali R, Kernif T, Socolovschi C, Berenger JM, Ethics approval and consent to participate et al. Matrix-assisted laser desorption ionization-time of flight mass Consent was obtained from volunteer landing catches collectors. Ethical spectrometry for rapid identification of tick vectors. J Clin Microbiol. approval for the mosquito monitoring was granted by authorities from the 2013;51:522–8. National Malaria Control Program (NMCP) and approved by the faculty of 19. Yssouf A, Socolovschi C, Flaudrops C, Ndiath MO, Sougoufara S, Dehecq JS, medicine ethical committee, Bamako, Mali (N°2011 89 FMPOS). et al. 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166 Tandina et al. Parasites & Vectors (2016) 9:495 Page 11 of 11

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167

V. CONCLUSIONS GENERALES ET PERPECTIVES

A travers cette thèse, nous avons pu mettre à jour le répertoire des moustiques du Mali qui était de 88 espèces en 1961 et maintenant listé à 106 espèces. Nous avons montré l’intérêt de la spectrométrie de masse MALDI- TOF dans le cadre d’une enquête entomologique de terrain, avec l’identification des moustiques collectés au Mali ainsi que la détermination de leur source de repas de sang. Ces résultats sont très prometteurs avec l’acquisition de MALDI TOF par des laboratoires de microbiologie, ou des plateformes technologiques et une utilisation en entomologie sans coût supplémentaire. Ce travail de thèse nous a également permis de mettre en évidence l’introduction récente de deux espèces invasives de moustiques au Mali. Il s’agit des espèces Aedes albopictus et Culex neavei qui sont connus pour être des vecteurs de nombreuses arboviroses. Ceci constitue une alerte et devrait attirer l’attention des autorités sanitaires sur le risque d’émergence ou de réémergence des arboviroses au Mali. Ainsi, des études d’enquêtes entomologiques devront être menées dans le pays surtout dans les zones les moins prospectées. Durant cette thèse, nous avons aussi étudié le microbiote digestif des moustiques de laboratoire et de terrain ainsi que leur eau de gite par la culturomique. Il s’agit des espèces Anopheles gambiae Giles, Aedes albopictus et Culex quinquefasciatus. La culturomique nous a permis d’isoler de nouvelles espèces de bactéries associées pour la première fois à ces moustiques vecteurs. L’étude du microbiote des moustiques et de leur environnement pourrait être développée comme des mesures de lutte contre les moustiques vecteurs de maladies. L’acquisition de la spectrométrie de masse MALDI-TOF au Mali pourrait faciliter nos futures études de terrain.

169

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173

ANNEXES

Durant ma thèse, j’ai collaboré dans les travaux de thèse de deux de mes collègues. Concernant l’article 6, il s’agit de la mise au point de l’identification du repas du moustique vecteur du paludisme Anopheles gambiae Giles par la spectrométrie de masse MALDI-TOF. Ensuite, une mise au point de cet outil a été réalisée sur des spécimens de terrain en écrasant l’abdomen des femelles gorgés sur du papier wathman. Cela a été appliqué sur des moustiques collectés au Comores (Article7). Dans un autre travail (Article8), j’ai participé à la description d’une nouvelle espèce bactérienne nommée Pseudomonas massiliensis Strain CB-1T sp. nov. in Brazil, que nous avons isolé pour la première fois dans l’estomac de Anopheles gambiae Giles et de Culex quinquefasciatus et dans leurs eaux de gîtes collectés au Mali.

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.

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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

RESEARCH ARTICLE MALDI-TOF MS identification of Anopheles gambiae Giles blood meal crushed on Whatman filter papers

Sirama Niare1,2, Lionel Almeras1,3, Fatalmoudou Tandina1,2, Amina Yssouf4, Affane Bacar4, Ali Toilibou4, Ogobara Doumbo2, Didier Raoult1, Philippe Parola1

1 Aix Marseille Universite´, Unite´ de Recherche en Maladies Infectieuses et Tropicales Emergentes (URMITE), UM63, CNRS 7278, IRD 198 (Dakar, Seńe´gal), Inserm 1095, AP-HM, IHU Me´diterraneé Infection, Marseille, France, 2 Malaria Research and Training Center, DEAP/FMOS, UMI 3189, University of Science, Techniques and Technology, Bamako, Mali, 3 Unite´ de Parasitologie et d’Entomologie, De´partement des a1111111111 Maladies Infectieuses, Institut de Recherche Biome´dicale des Armeés, Marseille, France, 4 Malaria Control a1111111111 Program, Moroni, Union of the Comoros a1111111111 * [email protected] a1111111111 a1111111111 Abstract

23(1 $&&(66 Background Citation: Niare S, Almeras L, Tandina F, Yssouf A, Identification of the source of mosquito blood meals is an important component for disease Bacar A, Toilibou A, et al. (2017) MALDI-TOF MS control and surveillance. Recently, matrix-assisted laser desorption/ionization time-of-flight identification of Anopheles gambiae Giles blood mass spectrometry (MALDI-TOF MS) profiling has emerged as an effective tool for mos- meal crushed on Whatman filter papers. PLoS ONE 12(8): e0183238. https://doi.org/10.1371/journal. quito blood meal identification, using the abdomens of freshly engorged mosquitoes. In the pone.0183238 field, mosquito abdomens are crushed on Whatman filter papers to determine the host feed-

Editor: Basil Brooke, National Institute for ing patterns by identifying the origin of their blood meals. The aim of this study was to test Communicable Diseases, SOUTH AFRICA whether crushing engorged mosquito abdomens on Whatman filter papers was compatible

Received: April 7, 2017 with MALDI-TOF MS for mosquito blood meal identification. Both laboratory reared and field collected mosquitoes were tested. Accepted: August 1, 2017 Published: August 17, 2017 Material and methods Copyright: 2017 Niare et al. This is an open Sixty Anopheles gambiae Giles were experimentally engorged on the blood of six distinct access article distributed under the terms of the vertebrate hosts (human, sheep, rabbit, dog, chicken and rat). The engorged mosquito Creative Commons Attribution License, which permits unrestricted use, distribution, and abdomens were crushed on Whatman filter papers for MALDI-TOF MS analysis. 150 What- reproduction in any medium, provided the original man filter papers, with mosquitoes engorged on cow and goat blood, were preserved. A author and source are credited. total of 77 engorged mosquito abdomens collected in the Comoros Islands and crushed on Data Availability Statement: All data of the study Whatman filter papers were tested with MALDI-TOF MS. are within the paper.

Funding: This work has with the support of the Results Ã A MIDEX project (n˚ ANR-11-IDEX-0001-02) The MS profiles generated from mosquito engorged abdomens crushed on Whatman filter funded by the Investissements d’Avenir French Government program, managed by the French papers exhibited high reproducibility according to the original host blood. The blood meal National Research Agency (ANR). host was correctly identified from mosquito abdomens crushed on Whatman filter papers by

Competing interests: The authors have declared MALDI-TOF MS. The MS spectra obtained after storage were stable regardless of the room that no competing interests exist. temperature and whether or not they were frozen. The MS profiles were reproducible for up

PLOS ONE | https://doi.org/10.1371/journal.pone.0183238 August 17, 2017 1 / 16

191 Anopheles blood meal source determination on Whatman filter papers with protein profiling analysis

to three months. For the Comoros samples, 70/77 quality MS spectra were obtained and matched with human blood spectra. This was confirmed by molecular tools.

Conclusion The results demonstrated that MALDI-TOF MS could identify mosquito blood meals from Whatman filter papers collected in the field during entomological surveys. The application of MALDI-TOF MS has proved to be rapid and successful, making it a new and efficient tool for mosquito-borne disease surveillance.

Introduction Malaria is a mosquito-borne infectious disease affecting humans and some animals, which is caused by parasitic protozoans belonging to the Plasmodium genus. Six species are known to infect humans [1,2]. Although malaria mortality rates have decreased in recent years, the disease remains one of the world’s three biggest killers, particularly in sub-Saharan Africa [3]. According to the World Health Organization (WHO), malaria is responsible for more than 400,000 deaths per year, with 90% of deaths occurring in Africa [1]. Female mosquitoes of the Anopheles genus, around 50 species have been described as vectors of malaria transmission. In sub-Saharan Africa, mosquitoes from the Anopheles gambiae complex are the main vectors of malaria transmission [4]. The malaria parasite is transmitted to human during the blood meals of infected female mosquitoes of the Anopheles genus. For a better understanding of the dynamics of malaria transmission, identification of the sources of blood meals is critical to assessing the risk of human exposure and determine the Anopheles trophic preference patterns. For example, evaluation of the Anopheles trophic preference patterns would make it possible to determine anthropophilic behavior in given malaria transmission areas [5]. An understanding of the origin of the mosquito host blood will enable the implementation of better control strategies to reduce the mosquito population and, therefore, transmission of disease. Measuring the proportion of blood meals taken from humans by mosquitoes (the human blood index) is an important component in vectorial capacity estimation. Several techniques are used to identify the source of mosquito blood meals, including serological tools such as precipitin and ELISA tests [6,7]. These methods present several limitations, in terms of antibody specificity and the sensitivity of accurately identifying a large range of host bloods [8–10]. Gradually, molecular biology approaches were adopted as a means of identifying mosquito blood sources. DNA sequence amplification of host blood from mosquitoes has enabled greater sensitivity and specificity in relation to identification [11]. Neverthe- less, the process can be costly and time consuming. Recently, matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) has been used as alternative and rapid tool for mosquito blood meal identification [12]. The MALDI-TOF tool is routinely used to identify microorganisms both from cultures and directly from biological samples. MALDI-TOF MS presents many advantages, including the fact that it is such as reliable and cost-effective technique in comparison to con- ventional phenotypic and molecular methods for identifying microorganisms. Given the increase in emerging diseases such as arbovirus and parasitic diseases transmitted by mosquitoes, the development of new strategies could be essential to vector control. MALDITOF MS was recently used as an innovative, rapid, and reliable tool in entomological studies [13]. How- ever, storage methods and the length of preservation can alter MALDI-TOF spectra for

PLOS ONE | https://doi.org/10.1371/journal.pone.0183238 August 17, 2017 2 / 16

192 Anopheles blood meal source determination on Whatman filter papers with protein profiling analysis

identification purposes [14]. MALDI-TOF MS has also emerged in medical entomology as an effective tool for identifying arthropods. The MS protein extracts from whole specimens or body parts of arthropods have been demonstrated to be an efficient and reproducible method of arthropod identification using MALDI-TOF MS [13]. In a previous study, the proof-of-concept of MALDI-TOF MS was successfully demonstrated through the identification of mosquito blood meals from recently fed mosquitoes. The abdomen proteins extracted from Anopheles gambiae which were either non-engorged or artificially engorged on seven distinct types of vertebrate blood, including human blood, have been suggested at by MALDI-TOF MS analysis. By comparing MALDI-TOF spectra from freshly engorged mosquito abdomens collected between 1 and 24 hours post-feeding, we were able to distinguish blood meal origins [12]. During entomological surveys, Whatman’s filter papers (WFPs) are frequently used in the field to collect and preserve the blood meals of arthropods. For this purpose, we proposed using the Whatman filter paper as a means of preserving samples and adapting to the field conditions. The abdomens of engorged female mosquitoes were crushed on WFPs, which were then transported to the laboratory for analysis and blood identification [15,16]. The advantage of WFPs is that the specimens can be individually crushed and stored at ambient temperature. The WFP collection method appears to be a simple and economical method for storage and preservation [17]. The aim of this study was to test the sensitivity and specificity of MALDI-TOF MS in identifying mosquito blood meal sources from the abdomens of blood-fed females crushed on WFPs. Anopheles gambiae Giles mosquitoes were experimentally fed with various host bloods, before MALDI-TOF analysis. We also attempted to see whether MALDI TOF could be used in the field in areas where malaria is endemic.

Materials and methods Ethical statements and blood sampling This study was conducted in accordance with the World Health Organization’s (WHO) Good Laboratory Practice guidelines and documents on blood sample handling procedures [18]. The mosquitoes were reared in line with the International Conference on Harmonization/ Good Laboratory Practices (ICH/GLP) procedures. Laboratory technicians and students were trained and certified in animal- and insectaria-based experiments. The laboratory procedures adopted in this study were approved by the human and animal ethics committees of the Aix- Marseille University Institutional Animal Care and Use Committee. The animal blood (chicken, rabbit, sheep, rat, and dog) was provided by local animal houses and handled as per French Decree No. 8 87–848 (October 19, 1987, Paris). Human blood was obtained from the Etablissement Franc¸ais du Sang (EFS), under existing agreements between the URMITE laboratory and the EFS [12]. In the field, all procedures adopted in this study were approved by the Comoros ethics committee and carried out with the agreement of the Ministry of Health and leaders of the villages selected for mosquito collection as part of the Programme National de Lutte contre le Paludisme. Informed consent forms were produced and signed by home owners and catchers at each site. The protocol for identifying the host blood was approved by the Scientific Ethics Committee of Marseille (C2EA-14) and by the French authorities.

Mosquito rearing An. gambiae Giles were reared at the insectarium in our laboratory using standard methods [12]. The larvae were kept in water until the nymph stage and fed fish food (TetraMinBaby, Tetra Gmbh, Herrenteich, Germany). Pupae were collected using a dropper and placed into

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plastic receptacles, and then transferred to cages (Bug Dorm 1; Bioquip Products). The emerging adults were fed on 10% glucose until the day of the experiment. The mosquitoes were kept in standard insectary conditions at 26±2˚C, 80% relative humidity and with a 12:12 hour light/ dark cycle. Three days after emerging, the female adults were used in the experiment.

Experiment Bloody Whatman filter paper (BWFPs) process. In the first phase of this study, twenty (n = 20) three-day-old mosquitoes were artificially engorged on human or sheep blood. An. gambiae Giles females were fed through a Parafilm-membrane (hemotek membrane feeding systems, Discovery Workshops, UK) as described [12]. The engorged females were harvested 12 hours after feeding and were anaesthetized at −20˚C for 10 minutes. The abdomens of 10 mosquitoes per host blood source (human or sheep), were first individually crushed on What- man filter papers (Whatman International Ltd. Maidstone England, approved by BSI) and maintained at ambient temperature for a maximum of four hours (drying time). In order to crush the mosquito abdomen on Whatman filter paper, the engorged females were individually deposited on a card. Sterile forceps were used to apply pressure to release the blood meals from the mosquito abdomens. After discharge of the blood meals onto the filter paper, the blood deposits were triturated on the card with a sterile piston. The Whatman paper containing the blood was then individually air dried for four hours before being submitted for MAL- DI-TOF analysis and different storage conditions. They were then used for MALDI-TOF MS analysis. In the second phase, ten mosquitoes were fed on one of four vertebrate host bloods: rabbit, dog, chicken and rat. The abdomens of ten mosquitoes per host blood were crushed on WFPs for MALDI-TOF analysis. Storage of bloody Whatman filter papers. Three storage methods were tested: room temperature, +4˚C, and −20˚C (frozen group). The mosquitoes used for this part of our study were engorged on cow or goat blood. The engorged females were harvested 12 hours after feeding. Each preservation method was applied to 50 An. gambiae Giles bloody WFPs. For each storage method, five specimens per host blood WFP were individually submitted for MALDI-TOF MS analysis after 7, 14, 30, 60 and 90 days.

Mosquito collection in the field The field part of the study was carried out in the Comoros archipelago situated at the northern entrance of the Mozambique Channel, off the eastern coast of Africa, between northeastern Mozambique and northwestern Madagascar. The mosquitoes were captured on three islands (Ngazidja, Moheli and Anjouan) of the Comoros archipelago (11˚ 20’ to 13˚ 4’ S and 42˚ to 45˚ E) [19], between March and August 2015. The mosquitoes were collected by human landing catch. All specimens were captured either inside or outside domestic buildings. Mosqui- toes were morphologically identified using the adult species determination key [20,21]. The engorged female abdomens were crushed on WFPs and preserved at -20˚C, before being used for MALDI-TOF MS analysis in Marseille, France, in April 2016.

Whatman filter paper sample preparation for MALDI-TOF analysis The pieces of bloody WFP containing the crushed abdomen of the engorged mosquito (about 1mm2) were individually cut using a sterile scalpel and transferred to a 1.5 mL tube. Two protein elution methods were used for test preparation. In the first elution method, 20 μL of formic acid (FA) (70% v/v) and 20 μL of acetonitrile (ACN) (50% v/v; Fluka, Buchs, Switzerland) was added and incubated for 10 minutes at room temperature (RT). The benefit of this protocol is the speed of sample preparation for

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MALDI-TOF analysis (protocol 1). In the second method, pieces of bloody WFPs were incubated for 10 minutes in 50 μL of high performance liquid chromatography (HPLC) grade water. Subsequently, 10 μL of the homogenized substance from the crushed bloody WFPs of the incubated mosquito abdomens was added to 40 μL of organic buffers (FA/ACN) (protocol 2). After a fast spin (10,000 rpm for 20 seconds), 1 μL of supernatant of each elution method sample was loaded onto the MALDI-TOF target plate in quadruplicate (Bruker Daltonics, Wissembourg, France) and covered with 1 μL of CHCA matrix solution composed of saturated ċ-cyano-4-hydroxycynnamic acid (Sigma, Lyon, France), 50% acetonitrile (v/v), 2.5% trifluoroacetic acid (v/v) (Aldrich, Dorset, UK) and HPLC-grade water. After drying for several minutes at RT, the MALDI-TOF target plate was introduced into the Microflex LT MALDI-TOF Mass Spectrometer (Bruker Daltonics, Germany) for analysis. As a control for the mass spectra steel loading, matrix quality and MALDI-TOF apparatus performance, a matrix solution was loaded in duplicate onto each MALDI-TOF plate with and without a bacterial test standard (Bruker Bacterial Test Standard, ref: #8255343). In addition, 1 μL of blood and dissected mosquito abdomens from engorged specimens of that host were processed as previously described [12] and loaded in quadruplicate onto the MALDI plate. The MALDI-TOF MS set up parameters were similar to those previously described [12].

Spectra analysis All MS spectra from bloody WFPs with crushed abdomens from mosquitoes fed on human, sheep, rat, rabbit, dog and chicken blood collected at 12 hours after feeding were viewed and analyzed using Flex analysis v.3.3 software. To determine reproducibility and specificity, the bloody WFP MS spectra from the preliminary and secondary phases of this study were aligned with homologous MS spectra of intact abdomens available in the database. The alignment was performed by Flex analysis and using ClinProTools 2.2 (Bruker Daltonics) software. We performed a comparison of MS spectra obtained from our previous work using intact engorged mosquito abdomens with those obtained in this study from abdomens crushed on WFPs to determine the MS spectrum stability of bloody WFPs. The software (Bruker Daltonics) was used to compare the average spectra obtained from the four spectra of each sample.

Blind tests MALDI-TOF MS spectra obtained from engorged mosquito abdomens crushed on WFPs were evaluated against our home-made arthropod database. This database has been upgraded since our proof-of-concept report [12]. The database includes the spectra obtained from (i) the intact abdomens of mosquitoes engorged on 17 host bloods (Homo sapiens, Equus caballus, Ovis aries, rabbit, Balb/C mouse, Rattus norvegicus, Canis familiaris, Bos taurus, Capra hircus, Gallus gallus, Equus asinus, Tapirus indicus, Tapirus terrestris, Carollia perspicillata, Thraupis episcopus, Erythrocebus patas and Callithrix pygmaea), (ii) bloody WFPs obtained from mosquito abdomens crushed after feeding on human or sheep blood, and (iii) various sections from 30 mosquito species (Anopheles gambiae, An. coluzzi, An. funestus, An. ziemanni, An. arabiensis, An. wellcomei, An. rufipes, An. pharoensis, An. coustani, An. claviger, An. hyrcanus, An. maculipennis, Culex quinquefasciatus, Cx. pipiens, Cx. modestus, Cx. insignis, Cx. neavei, Aedes albopictus, Ae. excrucians, Ae. vexans, Ae. rusticus, Ae. dufouri, Ae. cinereus, Ae. fowleri, Ae. aegypti, Ae. caspius, Mansonia uniformis, Orthopodomyia reunionensis, Coquillettidia richiardii and Lutzia tigripes), sandflies (six species: Phlebotomus papatasi, P. (Larrousius) longicuspis, P. (Larrousius) perfiliewi, P. (Larrousius) perniciosus, P. (Paraphlebotomus) sergenti, Sergento- myia minuta), triatomines (six species: Triatoma infestans, Rhodnius prolixus, Rh. pictipes, Rh.

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robustus, Eratyrus mucronatus, Panstrongylus geniculatus), ticks (17 species: Amblyomma cohaerens, Am. gemma, Am. variegatum, Haemaphysalis leachi, Hyalomma marginatum rufipes. H. truncatum, H. detritum, Rhipicephalus decoloratus, Rh. bergeoni, Rh e. evertsi, Rh. praetextatus, Rh. pulchellus, Rh. sanguineus, Rh. microplus, Rh. annulatus, Rh. turanicus, Rh. bursa), mites (three species: Leptotrombidium chiangraiensis, L. imphalum, L. deliense), bedbugs (one species: Cimex lectularius), louse (7 species: Pediculus humanus, Damalinia bovis, D. caprae, D. ovis, Haematopinus eurysternus, Linognatus vituli, L. africanus) and fleas (five species: Ctenoce- phalides felis, Ct. canis, Archaeopsylla erinacei, Xenopsylla cheopis and Stenoponia tripectinata). The level of significance was determined using the log score values (LSVs) given by the MAL- DI-Biotyper software v.3.3, corresponding to matching query and reference mass spectra signal intensities. LSVs ranged from zero to three. A sample was considered to be correctly and significantly identified at the species level when the queried spectrum had a log score value (LSV) 1.8 [12,22,23].

Identification of field mosquito blood meals All bloody WFPs obtained in the field from engorged mosquitoes collected on Comoros were tested by MALDI-TOF MS as described above. To confirm the identification of the mosquito blood meal by MALDI-TOF, molecular identification was attempted on bloody WFPs which were randomly selected (n = 30) from the Comoros samples. DNA extractions from Whatman papers with individual mosquito abdomen samples were performed with the EZ1 DNA Tissue kit (Qiagen, Hilden, Germany) as per the manufacturer’s instructions. DNA extracted from unfed mosquitoes was used as a negative blood meal control. A set of the primers specifically amplifying a fragment of 648 bp for the vertebrate Cytochrome c oxidase I gene (vCOI) was used and the PCR reaction was conducted as described [24]. vCOI positive PCR products were purified using the NucleoFast 96 PCR plate (Machery-Nagel EURL, France), and sequenced using the same primers with the BigDye version 1–1 Cycle Ready Reaction Sequencing mix (Applied Biosystems, Foster City, CA, USA) and an ABI 3100 automated sequencer (Applied Biosystems) to control the amplified products. The sequences were assembled and analyzed using the ChromasPro software (version 1.34) (Technelysium Pty. Ltd., Tewantin, Australia) and BLAST website (Basic Local Alignment Search Tool; http://blast.ncbi.nlm.nih.gov).

Results Bloody WFP MALDI-TOF MS analysis set up The MS spectra from WFPs with and without blood from crushed abdomens were visually analyzed using Flex analysis software (Fig 1). The spectrum of blood-free WFPs had no peaks. The organic buffer and WFPs used as negative controls did not generate any peaks. The comparison of the MS spectra obtained from bloody (human and sheep) WFPs and those obtained from Anopheles engorged abdomens (human and sheep) showed many similar peaks for the same host blood source (Fig 1). The MS spectra of bloody WFPs from specimens fed on human and sheep blood were also compared using the elution method (protocol 1 and protocol 2). The representative spectra of the two bloody WFPs processed with protein elution methods revealed high peak intensities (Fig 1). All MALDI-TOF MS profiles from bloody mosquito WFPs using Flex analysis software clearly showed a reproducible spectrum by blood meal origin (Fig 1). When twenty bloody WFPs were tested against our database, the blind test yielded a 100% correct identification of blood origin. The blind test revealed log score values (LSVs) greater than 1.8 (Table 1). Therefore, the rapid protocol using only organic buffers (protocol 2) was used for all subsequent processing.

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Fig 1. MALDI-TOF MS spectra from An. gambiae Giles (Ang) abdomen protein extracts engorged on vertebrate host bloods and then crushed on Whatman filter papers (WFPs). The MS spectra were generated according to two protocols (P1, P2). Intact Ang match the MS profiles from Anopheles gambiae Giles abdomens crushed on WFPs. The blood-free WFP corresponds to the MS profiles of WFPs where no mosquito blood meal was released. The vertebrate host bloods used for Anopheles gambiae Giles bloody Whatman filter papers (bloody WFPs) were human and sheep. All mosquitoes were collected 12 hours after feeding. a.u. arbitrary units; m/z mass-to-charge ratio. https://doi.org/10.1371/journal.pone.0183238.g001 Assessment of MALDI-TOF MS using bloody WFPs from Anopheles gambiae Giles fed on combination host blood meals After the above set-up was complete, 40 bloody WFPs from engorged Anopheles gambiae Giles fed on rabbit, dog, rat and chicken blood were submitted for MALDI-TOF MS analysis. As the bloody WFP MS spectra were previously reported to be stable, comparison of the 40 bloody WFP MS spectra by Flex analysis software indicated a high reproducibility and specificity by blood meal origin (Fig 2). The bloody WFPs from crushed engorged mosquito abdomens fed on the same host were perfectly superimposable. When we tested the MALDI-TOF MS spectra

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Table 1. Blind tests of the An. gambiae Giles used to set up the protocol optimal for blood meals identification by MALDI TOF from BWFPs. Mosquito Blood feeding High LSVs obtained from blind species source Number of specimens spotted used blind tests tests against database (a) Vertebrate species against database (number per elution mode) identification of blood origin An. gambiae Human 10 (5/5) [1.985–2.930] (10) Human Giles An. gambiae Sheep 10 (5/5) [1.845–2.259] (10) Sheep Giles Total 20

All mosquitoes were collected 12 hours post-feeding to crushing in the whatman filter (a)Into brackets were indicated the number of MS spectrum tested against the database with a LSVs upper 1.8 considered significative https://doi.org/10.1371/journal.pone.0183238.t001

obtained from the 40 bloody WFP abdomens against our MALDI-TOF MS database, the blind test revealed a 100% correct identification of the host blood source in all mosquitoes tested. The log score values (LSVs) ranged from 1.918 to 2.841 (Table 2). A comparison of the MS spectra of crushed abdomen bloody WFPs with those of intact engorged mosquito abdomens loaded in our database indicated great similarity for mosquitoes which were fed on the blood of the same animal. The alignment reveals intra-species reproducibility and inter-species specificity (Fig 2).

Whatman filter storage method for mosquito blood meal identification When five bloody WFPs per storage method (-20˚C, + 4˚C and room temperature) were processed with MALDI TOF MS analysis at a given time point (7, 14, 30, 60 and 90 days), the blind test performed against the database reliably and correctly identified 100% of the bloody WFP (n = 150) mosquitoes tested up to three months. The log score values (LSVs) for these MS bloody WFPs ranged from 1.612 to 2.481 (with 134/150 > 1.8) (Fig 3). Visually, the MS spectra from the stored BWFPs remained stable up to 90 days post-collection (Fig 3). To this end, MS spectra comparison by Flex analysis software between the three storage methods indicated similar profiles quality following these storing modes.

Identification of field mosquito blood meals A total of 216 mosquitoes collected in Comoros were morphologically identified as: 93 An. gambiae s.l. and 123 An. funestus s.l. A total of 77 females were found to be engorged, including 54 An. gambiae s.l. and 23 An. funestus s.l. The engorged female abdomens were crushed on WFPs. Twelve months later, in Marseille, the bloody WFPs were submitted for MALDI-TOF MS analysis to identify the blood meals. The 77 MS spectra obtained from the bloody WFPs were queried against our MALDI-TOF MS database. The blind test reveals that 70/77 MS spectra from Comoros submitted for MALDI TOF analysis matched human blood with high LSV scores (1.818–2.690) (Table 3). The seven remaining MS spectra from Comoros were too low and or of too poor quality to be identified by MALDI-TOF MS. A total of 27 COI sequences were obtained from Comoros bloody WFP samples. The amplified fragment sequences were shown to share between 99.11% and 99.85% similarity with several sequences of human COI (Genbank Accession: KX457614.1, MF278749.1).

Discussion The major advantages of MALDI-TOF MS technology include the rapidity, accuracy and low cost of analyses (under US$1 per sample) [25,26]. This may revolutionize the entomological domain.

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198 Anopheles blood meal source determination on Whatman filter papers with protein profiling analysis

Fig 2. Comparison of MALDI-TOF MS spectra from An. gambiae Giles abdomen protein extracts engorged on vertebrate host bloods and then crushed on Whatman filters. The MS spectrum alignment was performed by Flex analysis 3.3 software. Intact Ang match the MS profiles from Anopheles gambiae Giles abdomens crushed on WFPs. The vertebrate host bloods used for mosquito blood meals were rabbit, dog, chicken and rat. All mosquitoes were collected 12 hours after feeding. a.u. arbitrary units; m/z mass-to-charge ratio. https://doi.org/10.1371/journal.pone.0183238.g002

Limitations of this method of specimen identification include the cost of the device and database comprehensiveness. However, when the MALDI TOF device is bought for clinical microbiology purposes, it can also be used for medical entomology at no additional cost. This is what has hap- pened in Dakar, Senegal, where the Hoˆpital Principal de Dakar laboratory is now equipped with MALDI-TOF devices for clinical microbiology purposes [27], and entomological applications have already been developed, with MALDI-TOF identification of Culicoides [28]. Having previously reported the proof-of-concept for MALDI-TOF’s ability to identify the blood meals of mosquitoes by analyzing their engorged abdomens, here we report another

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Table 2. An. gambiae Giles abdomen crushing in paper filter Whatman to performed the blind tests according blood meal source. Mosquito Blood feeding High LSVs obtained from blind Vertebrate species identification species source Number of specimens spotted used tests against database (a) of blood origin blind tests against database An. gambiae Rabbit 10 [1.918–2.188] (10) Rabbit Giles An. gambiae Dog 10 [2.382–2.728] (10) Dog Giles An. gambiae Rat 10 [1.981–2.481] (10) Rat Giles An. gambiae Chicken 10 [2.662–2.841] (10) Chicken Giles Total 40

promising application in the field of entomology. WFPs are the most common way of collecting and preserving mosquito blood meals in the field. Since a database of several animal blood spectra obtained from engorged mosquitoes has already been created, in this study we used the Whatman filter as a as standard technique for field sample storing and transport during entomological investigations.

Fig 3. Whatman filter storage method for mosquito blood meal identification. Comparison of LSVs obtained following a reference TP database query with MS spectra of An. gambiae Giles fed on cow and goat blood. All specimens were collected 12 hours after feeding and stored up to 90 days (D). The mosquito abdominal proteins crushed on WFPs were stored under three different conditions: -20˚C, + 4˚C and room temperature. The dashed line represents the threshold value for relevant identification (LSVs !1.8). LSV, log score value. https://doi.org/10.1371/journal.pone.0183238.g003

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Table 3. Blind tests of Comoros mosquitoes to identified their blood meals sources by MALDI-TOF MS from whatman paper against TPDB. Sites Samples location Morphological Vertebrate species of Number of High LSVs from blind Date of tested identification of mosquito blood origin correct matching tests database collect Anjouan 16 indoor Anopheles gambiae Human 16 [2.005–2.553] 01/03/ (Chirove) 2015 Anjouan 1 indoor Anopheles funestus Low quality / / 01/03/ (Chirove) 2015 Moheli 1 indoor Anopheles gambiae Human 1 2.124 01/03/ (Hamavouna) 2015 Anjouan(Kowe) 1 indoor Anopheles gambiae Human 1 2.252 01/03/ 2015 Ngazidja 1 indoor Anopheles gambiae Human 1 2.435 01/03/ (Singani) 2015 Ngazidja(Wela) 1 indoor Anopheles gambiae Low quality / / 01/03/ 2015 Ngazidja(Wela) 3 indoor Anopheles gambiae Human 3 [1.869–2.239] 01/03/ 2015 Ngazidja 1 outdoor Anopheles gambiae Human 1 2.141 01/03/ (Chamle) 2015 Anjouan 9 outdoor Anopheles gambiae Human 9 [1.915–2.672] 01/03/ (Chirove) 2015 Moheli 2 outdoor Anopheles gambiae Human 2 [1.818–2.200] 01/03/ (Hamavouna) 2015 Ngazidja 1 outdoor Anopheles gambiae Low quality / / 01/03/ (Singani) 2015 Moheli 1 * Anopheles gambiae Human 1 2.150 01/03/ (Fomboni) 2015 Moheli 1 * Anopheles gambiae Human 1 2.095 01/03/ (Hamavouna) 2015 Ngazidja(Wela) 1 indoor Anopheles gambiae Human 1 2.218 01/03/ 2015 Anjouan(Kowe) 1 outdoor Anopheles gambiae Human 1 2.319 01/03/ 2015 Ngazidja(Wela) 3 outdoor Anopheles gambiae Human 3 [2.152–2.315] 01/03/ 2015 Anjouan 1 * Anopheles gambiae Human 1 2.124 01/03/ (Chirove) 2015 Moheli 1 * Anopheles gambiae Human 1 2.333 01/03/ (Fomboni) 2015 Moheli 1 * Anopheles gambiae Low quality / / 01/03/ (Hamavouna) 2015 Moheli 1 * Anopheles gambiae Low quality / / 01/03/ (Hamavouna) 2015 Moheli 1 * Anopheles gambiae Human 1 2.249 01/03/ (Hamavouna) 2015 Moheli(Wela) 1 * Anopheles gambiae Human 1 2.285 01/03/ 2015 Anjouan 15 indoor Anopheles funestus Human 15 [1.910–2.690] 01/03/ (Chirove) 2015 Anjouan 1 indoor Anopheles funestus Low quality / / 01/08/ (Chirove) 2015 Anjouan 5 outdoor Anopheles funestus Human 5 [1.969–2.639] 01/08/ (Chirove) 2015 Anjouan 1 outdoor Anopheles funestus Low quality / / 01/08/ (Chirove) 2015 (Continued)

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Table 3. (Continued)

Sites Samples location Morphological Vertebrate species of Number of High LSVs from blind Date of tested identification of mosquito blood origin correct matching tests database collect Anjouan 1 outdoor Anopheles funestus Human 1 2.614 01/08/ (Chirove) 2015 Total 77 70 https://doi.org/10.1371/journal.pone.0183238.t003

Our experiment was performed using bloody WFPs from An. gambiae Giles blood meals submitted for MALDI-TOF MS analysis. Interestingly, only a small portion of bloody WFP (about 1mm2) was required to identify the blood meal source. The remaining BWFP can be used for further analysis, such as the molecular detection of plasmodium [29]. The rapidity of the analysis time makes it feasible to identify the origins of the mosquito blood meal using MALDI TOF MS. Only ten minutes per sample was necessary between the crushing process and identification of the blood meals. After our preliminary tests, we chose to process bloody WFPs treated with organic buffers (protocol 2), as it was a simple and effective means of generating MALDI-TOF MS spectra of good quality. It also delivered intra-species reproducibility and inter-species specificity of the origins of mosquito blood meals. One advantage of using water instead of a cocktail of formic acid and acetonitrile is that remains of the host blood can be used for other purposes and techniques, including molecular tools. Our experimental protocol performed on a wide range of animal bloods (rabbit, dog, rat and chicken) confirmed that analyzing WFPs with crushed An. gambiae Giles engorged abdomens yielded spectra with intra-species reproducibility and inter-species specificity. The blind test positively supported the ability of MALDI-TOF MS to identify blood meal sources using WFPs, with high LSVs up to 2.930 (Table 1), when 1.8 had previously been estimated as a reliable threshold for arthropod identification [22,23] and mosquito blood meal identification[12]. During transport from the field to the laboratory, sample storage issues are critical, even when sampling has been adapted to field conditions, such as using WFPs to collect blood or the blood contained in engorged mosquitoes. In our study, the preservation method (- 20˚C, + 4˚C and room temperature) did not affect the identification of mosquito blood meals by MALDI-TOF MS. The stability of the MS profiles from all preservation methods had LSVs over 1.8 for up to three months, which suggests that WFPs are robust and sufficient for preserving field collection samples. This hypothesis was tested on bloody WFPs obtained from mosquitoes collected in Comoros. In Comoros, the bloody WFP samples and all collected mosquitoes were preserved by freezing. The bloody WFPs were transported from Comoros to Marseille at ambient temperature before undergoing MALDI-TOF MS analysis. The time between collecting bloody WFPs from Comoros and performing MALDI TOF MS analysis was about one year. In Comoros, WFPs are commonly used to store blood for malaria studies [30]. Malaria is an endemic disease in Comoros and has long been considered a major public health issue with prevalence over 35% in 2009 in the Union of Comoros. Malaria vectors are An. gambiae s.l. and An. funestus s.l.[20]. An. gambiae is present on the third island while An. funestus s.l.is only present in Anjouan and Moheli [31]. Different strategies have been used by the Malaria Control Program to control both parasites and vectors. The parasitological study carried out during the Malaria Indicator Survey (MIS) in June 2014 showed a significant reduction in prevalence to under 1% in the three islands of the Union of the Comoros, with 1.4% in Grande Comore, 0.5% in Anjouan and 0% in Moheli [32]. However, no entomological studies have been conducted to assess the impact of these operations on malarial transmission in the

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country. We took the opportunity presented by an entomological survey conducted by the PNLP (Programme National de Lutte contre le Paludisme) of Comoros to test MALDI TOF MS’s ability to identify the blood meals of mosquitoes using WFPs. Results were very con- vincing, with 90% of the Comoros engorged mosquito abdomens crushed on WFPs being undoubtedly identified by MALDI TOF MS as human blood sources. The low quality of the seven MS spectra generated by MALDI-TOF MS analysis can be attributed to the MS profile protein alteration [33], as we found in our previous study that MALDI-TOF MS can identify blood meals up to 24 hours after feeding. However, some studies indicate that MS spectra modifications are due to storage methods, such as storing specimens in alcohol [12], or due to extraction models [34]. Apparently, alcohol conservation rapidly modifies the MS spectra of mosquito abdomens fed on different mammals after only two weeks of storage [12]. The results of the blood meal identification of engorged females collected by human land catch in Comoros might be considered surprising. Indeed, the female mosquito is supposed to not have the time to feed on the exposed legs of collectors. However, these mosquitoes may have started to feed on other human hosts before landing on the collectors. Also, malaria has been reported in collectors exposed to human land catch, although the incidence is much lower than in the local population [35]. We used COI sequence analysis, as vertebrate cytochrome c oxidase I gene has a low variability within species [36,37] and might serve as a DNA barcode for the identification of animal species [38].

Conclusions Our results show the correct identification of anopheles vector blood meal sources (field catch and laboratory reared) by MALDI-TOF MS using WFPs. The future use of MALDI TOF in mosquito studies may include to identify the mosquito from legs [13] and the blood meal from the abdomen, and also detect Plasmodium from the cephalothoraxes [39]. Our database could subsequently be shared and open access could be granted for routine arthropod identification either for entomological diagnosis or arthropod monitoring. Other future developments in the field of blood meals include identifying the blood meal mixture, parity status and mosquito age.

Acknowledgments We thank Christophe Flaudrops (URMITE-IRD198, Marseille, France) for his advice on the analysis of MALDI-TOF MS profiles. The authors thank also Nadir Meguini, veterinarian at the El Tarf University, Algeria, who provided animal blood samples.

Author Contributions Conceptualization: Sirama Niare, Lionel Almeras, Ogobara Doumbo, Didier Raoult, Philippe Parola. Funding acquisition: Philippe Parola. Investigation: Sirama Niare. Methodology: Sirama Niare, Lionel Almeras, Fatalmoudou Tandina, Amina Yssouf, Affane Bacar, Ali Toilibou, Didier Raoult. Software: Sirama Niare. Supervision: Sirama Niare, Lionel Almeras, Ogobara Doumbo, Philippe Parola. Validation: Sirama Niare, Lionel Almeras, Ogobara Doumbo, Philippe Parola.

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203 Anopheles blood meal source determination on Whatman filter papers with protein profiling analysis

Visualization: Sirama Niare, Philippe Parola. Writing – original draft: Sirama Niare, Lionel Almeras, Fatalmoudou Tandina, Amina Yssouf, Affane Bacar, Ali Toilibou, Ogobara Doumbo, Didier Raoult, Philippe Parola. Writing – review & editing: Sirama Niare, Lionel Almeras, Fatalmoudou Tandina, Amina Yssouf, Ogobara Doumbo, Didier Raoult, Philippe Parola.

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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.

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OMICS A Journal of Integrative Biology Volume 22, Number 2, 2018 ª Mary Ann Liebert, Inc. DOI: 10.1089/omi.2017.0027

Microbial Culturomics Application for Global Health: Noncontiguous Finished Genome Sequence and Description of Pseudomonas massiliensis Strain CB-1T sp. nov. in Brazil

Lucie Bardet,1 Teresa Cimmino,1 Cle´mence Buffet,1 Caroline Michelle,1 Jaishriram Rathored,1 Fatalmoudou Tandina,1 Jean-Christophe Lagier,1 Saber Khelaiﬁa,1 Joˆnatas Abraha˜o,2 Didier Raoult,1 and Jean-Marc Rolain1

Abstract

Culturomics is a new postgenomics ﬁeld that explores the microbial diversity of the human gut coupled with taxono-genomic strategy. Culturomics, and the microbiome science more generally, are anticipated to transform global health diagnostics and inform the ways in which gut microbial diversity contributes to human health and disease, and by extension, to personalized medicine. Using culturomics, we report in this study the description of strain CB1T ( = CSUR P1334 = DSM 29075), a new species isolated from a stool specimen from a 37-year- old Brazilian woman. This description includes phenotypic characteristics and complete genome sequence and annotation. Strain CB1T is a gram-negative aerobic and motile bacillus, exhibits neither catalase nor oxidase activities, and presents a 98.3% 16S rRNA sequence similarity with Pseudomonas putida. The 4,723,534 bp long genome contains 4239 protein-coding genes and 74 RNA genes, including 15 rRNA genes (5 16S rRNA, 4 23S rRNA, and 6 5S rRNA) and 59 tRNA genes. Strain CB1T was named Pseudomonas massiliensis sp. nov. and classiﬁed into the family Pseudomonadaceae. This study demonstrates the usefulness of microbial culturomics in exploration of human microbiota in diverse geographies and offers new promise for incorporating new omics technologies for innovation in diagnostic medicine and global health.

Keywords: global health, microbial culturomics, Pseudomonas massiliensis, system diagnostics, taxono- genomics

Introduction identity and phylogeny (Stackebrandt and Ebers, 2006; Tindall et al., 2010), genomic G+C content diversity, and DNA-DNA mics techniques represent a new approach to the hybridization (DDH) (Rossello´-Mora, 2006; Wayne et al., Downloaded by Guangxi University for Nationalities from online.liebertpub.com at 02/16/18. For personal use only. Ounderstanding of microbial diversity. Particularly, me- 1987). However, those tools exhibited limitations, such as the tagenomics enabled the identification of a great diversity of variation in the cutoff values between species or genera and the bacterial species, but a consequent number of them did not lack of reproducibility (Welker and Moore, 2010). correspond to an identified organism (Lagier et al., 2012a). The emergence of high-throughput sequencing enabled access Culturomics was introduced to decipher the human gut mi- to the genomic data of many bacterial species, and we recently crobiota, combining high-throughput culture, by the diver- designed a new method, known as taxono-genomics, using these sification of culture conditions, and rapid identification of available sequences, to describe new bacterial species (Rama- the growing colonies by matrix-assisted laser desorption/ samy et al., 2014). Taxono-genomics consists of a systematic ionization time-of-flight mass spectrometry (MALDI-TOF comparison of phenotypic properties, including proteomic in- MS) or 16S rRNA sequencing (Lagier et al., 2015). formation obtained by MALDI-TOF-generated spectrum (Seng Bacterial taxonomy and the identification of a new species et al., 2009), and genome analysis (Fournier et al., 2015). were usually delineated by phenotypic, chemotaxonomic, Using our strategy that combines culturomics and taxono- and genetic parameters such as the 16S rRNA gene sequence genomics on the stool sample of a 37-year-old Brazilian

1Aix-Marseille Universite´, URMITE, UM63, CNRS7278, IRD198, Inserm 1095, IHU-Me´diterrane´e Infection, Marseille, France. 2Laborato´rio de Vı´rus, Departamento de Microbiologia, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil.

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FIG. 1. Reference mass spectrum of Pseudomonas massiliensis strain CB1T. This reference spectrum was generated by comparing 12 individual colonies.

woman, we could isolate and describe strain CB1T, a new formed consent and the agreement of the local ethics com- species that we named Pseudomonas massiliensis. In this mittee of the Institut Fe´de´ratif de Recherche 48 (Marseille, study, we characterize and report the novel isolate P. massi- France) was obtained under Agreement 09–022. The patient liensis sp. nov. strain CB1T ( = CSUR P1334 = DSM 29075), was not taking any antibiotics at the time of sampling and the including the description of its whole genome sequence and fecal sample was frozen at -80°C after collection and was annotation. These characteristics support the circumscription shipped to Marseille, France. of the species P. massiliensis. Strain isolation Material and Methods Culturomics was used, as previously described, to decipher Sample the microbial diversity of the gut microbiota (Lagier et al., A stool sample was consensually collected from an obese 2012a). Brieﬂy, 1 g of the stool sample was preincubated 37-year-old Brazilian patient. The patient gave signed, in- at 37°C in a blood culture bottle and this liquid medium was Downloaded by Guangxi University for Nationalities from online.liebertpub.com at 02/16/18. For personal use only.

FIG. 2. MALDI-TOF MS-based virtual gel view comparing P. massiliensis strain CB1T to other members of the family Pseu- domonadaceae by their generated protein expression proﬁles, or spectra. The x-axis records the m/z value and the y-axis indicates the spectrum number, with corresponding bacterial species indicated on the left. The peak intensity is expressed by a gray scale displayed on the right (arbitrary units). MALDI-TOF, matrix-assisted laser desorption/ionization time-of-ﬂight mass spectrometry.

210 166 BARDET ET AL.

FIG. 3. Phylogenetic tree highlighting the position of P. massiliensis sp. nov. strain CB1T relative to other type strains within the Pseudomonadaceae. Neighbor joining tree based on nearly complete 16S RNA gene sequences of Pseudomonas sp. CB1 sp. nov and related species of genus Pseudomonas sensu stricto. The signiﬁcance of each branch is indicated by a bootstrap value (%) calculated for 1000 subset.

then seeded on agar plates following different culture con- Amplification of the 16S rRNA gene was done by standard ditions and after different time of preincubation. polymerase chain reaction, using the universal primers FD1 and RP2 and HotStarTaq DNA Polymerase (Qiagen, Hilden, Ger- Strain identification many), and sequencing was performed using the Big Dye Ter- minator Sequencing Kit (Thermo Fischer, Vantaa, Finland) and All types of colonies that have grown were identified using the following internal primers: 536F, 536R, 800F, 800R, 1050F, MALDI-TOF-MS as previously described (Seng et al., 1050R, 357F, and 357R, as previously described (Weisburg 2009), using a Microflex spectrometer (Bruker Daltonics, et al., 1991). Strains that exhibited a 16S rRNA sequence simi- Leipzig, Germany). The spectra obtained were compared to larity value of less than 98.7% with their phylogenetically closest the spectra of the continuously incremented Bruker database. species in nomenclature were considered representatives of pu- Colonies exhibiting a spectrum with a MALDI-TOF-MS tative new species (Stackebrandt and Ebers, 2006). score <1.9 were further characterized by the 16S rRNA gene A 16S rRNA phylogenetic tree was realized for the sequence comparison to the GenBank database provided by new species with the closest sequences recovered using a the National Center for Biotechnology Information (NCBI) (Benson et al., 2017). Downloaded by Guangxi University for Nationalities from online.liebertpub.com at 02/16/18. For personal use only.

FIG. 4. Gram staining of P. massiliensis strain CB1T, FIG. 5. Transmission electron microscopy of P. massi- using a DM100 photonic microscope (Leica Microsystems, liensis strain CB1, using Morgani 268D (Philips) at an op- Nanterre, France) at 100 · . erating voltage of 60 kV. The scale bar represents 500 nm.

211 MICROBIAL CULTUROMICS IN GLOBAL HEALTH 167

Table 1. Differential Characteristics of Pseudomonas massiliensis Strain CB1T (Data from This Study), Pseudomonas putida, Pseudomonas ﬂuorescens, and Pseudomonas aeruginosa (Xiao et al., 2009) Properties P. massiliensis P. putida P. ﬂuorescens P. aeruginosa Cell diameter (lm) Oxygen requirement aerobic aerobic aerobic aerobic Gram stain --- - Growth with 5% NaCl +++ + Growth at 4°C -++ - Growth at 42°C +-- + Motility +++ + Endospore formation - Production of Catalase -++ + Oxidase -++ + Urease --- - b-galactosidase --- - b-glucosidase --- - indole --- - Acid produced from D-glucose +++ + Utilization of l-Arabinose -+(-) -- Mannose +-+ - Mannitol +-+ + d-glucose +++ + d-maltose +-- -

nucleotide blast against the 16S rRNA Database in Silva’s the Kimura 2 parameter model and MEGA 6 software (Ta- ‘‘The All-Species Living Tree’’ Project (LTPs119) and mura et al., 2013). the NCBI database (http://blast.ncbi.nlm.gov.gate1.inist.fr/ Blast.cgi). Filtered sequences (over 1450 nt) were aligned Growth conditions using Muscle, and phylogenetic inferences were obtained using the Maximum Likelihood Tree. Phylogenetic infer- The optimal growth conditions of the strain were tested ences were analyzed using the likelihood method based on using aerobic incubation for 2 days on COS medium under Downloaded by Guangxi University for Nationalities from online.liebertpub.com at 02/16/18. For personal use only.

FIG. 6. Graphical circular map of genome. From the outside to center: forward (red) and reverse (blue) strand, contigs (color circle), feature genes indicated in legend, GC plot, GC skew.

212 168 BARDET ET AL.

Table 2. Nucleotide Content and Gene Count Phenotypic characterization Levels of the Genome The colonies were observed on COS medium after an Genome (total) overnight incubation at 37°C. Strain sporulation was tested ° a by applying a 60 C thermo-shock for 20 min to the colonies Attribute Value % of total and its motility was determined by observing a fresh colony between the slides using a DM100 photonic microscope Size (bp) 4,723,534 100 · G+C content (bp) 2,944,883 62.34 (Leica Microsystems, Nanterre, France) at 100 . Gram Coding region (bp) 4,262,037 90.23 staining was performed using the Gram 2-F kit (BioMe´rieux) Total genes 4313 100 and cell morphology was determined using transmission RNA genes 74 1.72 electron microscopy after negative staining. Protein-coding genes 4239 100 Biochemical characteristics were defined using API 20NE, Genes with function prediction 3312 78,13 API 50CH, and API ZYM strips (BioMe´rieux), in line with Genes assigned to COGs 3141 74.10 the manufacturer’s instructions. Catalase and oxidase activ- Genes with peptide signals 874 20.62 ities were determined using commercially available kits: ID Genes with transmembrane helices 517 11.90 Color Catalase (BioMe´rieux) and BD BBL Taxo (Becton- CRISPRs Repeats 0 0 Dickinson, Franklin Lakes, NJ), respectively. aThe total is based on either the size of the genome in base pairs Antibiotic susceptibility testing was performed using the disk or the total number of protein coding genes in the annotated diffusion method on Mueller Hinton Agar (Becton-Dickinson) genome. and was interpreted using the European Committee on Anti- COG, Clusters of Orthologous group. microbial Susceptibility Testing guidelines (www.eucast.org). The 16 antibiotics tested were as follows: ticarcillin, ticarcillin/ different temperatures (25°C, 30°C, 37°C, 45°C, and 55°C). clavulanic acid, piperacillin/tazobactam, ceftazidime, cefepime, imipenem, ertapenem, aztreonam, gentamicin, to- Other atmospheres were tested: aerobic with 5% CO2 and anaerobic and microaerophilic conditions using GENbag bramycin, nalidixic acid, ciprofloxacin, trimethoprim/ anaer and GENbag microaer systems, respectively (Bio- sulfamethoxazole, rifampicin, fosfomycin, and colistin. Me´rieux, Marcy l’E´ toile, France). To determine the optimal pH for growth and NaCl tolerance, ranges of pH (5.4, 7.3, 8.5, Genomic sequencing and assembly and 9.1) and NaCl concentrations (0%, 2%, 5%, and 10% v/w) The DNA of the colonies obtained was extracted using the were tested by inoculation on modified Colombia agar (Sigma- BioRobot EZ1 Advanced XL (Qiagen) after a pre-lysis Aldrich, St. Louis, MO). treatment with lysozyme as previously described (Hadjadj

Table 3. Number of Genes Associated with the 25 General COG Functional Categories Code Value % of totala Description J 180 4.25 Translation A 1 0.02 RNA processing and modification K 299 7.05 Transcription L 148 3.49 Replication, recombination and repair B 2 0.05 Chromatin structure and dynamics D 38 0.90 Cell cycle control, mitosis and meiosis Y 0 0 Nuclear structure V 37 0.87 Defense mechanisms T 218 5.14 Signal transduction mechanisms M 211 4.98 Cell wall/membrane biogenesis Downloaded by Guangxi University for Nationalities from online.liebertpub.com at 02/16/18. For personal use only. N 105 2.48 Cell motility Z 0 0 Cytoskeleton W 0 0 Extracellular structures U 94 2.22 Intracellular trafficking and secretion O 126 2.97 Posttranslational modification, protein turnover, chaperones C 192 4.53 Energy production and conversion G 219 5.17 Carbohydrate transport and metabolism E 431 10.17 Amino acid transport and metabolism F 69 1.63 Nucleotide transport and metabolism H 149 3.51 Coenzyme transport and metabolism I 131 3.09 Lipid transport and metabolism P 256 6.04 Inorganic ion transport and metabolism Q 102 2.41 Secondary metabolites biosynthesis, transport, and catabolism R 502 11.84 General function prediction only S 278 6.56 Function unknown - 451 10.64 Not in COGs

aThe total is based on the total number of protein coding genes in the annotated genome.

213 MICROBIAL CULTUROMICS IN GLOBAL HEALTH 169

Table 4. List of the Genes Associated with Antibiotic Resistance in P. massiliensis Strain CB1T Best blast hit %aa %aa ORF Gene name GC% Size AA Function in GenBank coverage identity 1659 (Bla)Beta-lactamase_ 65.6 364 Zn-dependent Pseudomonas 88 72 class-A hydrolase entomophila L48 3698 (Flq)OqxBgb 62.5 1061 Multidrug efﬂux Pseudomonas 100 90 transporter MexF rhizosphaerae

et al., 2016), obtaining a 50 lL DNA extract. Genomic DNA database, and the predicted proteins were examined using of P. massiliensis was sequenced using MiSeq Technology antiSMASH (Weber et al., 2015). Identification of phage (Illumina, Inc., San Diego, CA) and two applications: paired sequences was performed using PHAge Search Tool (Zhou end and mate pair as previously described (Hadjadj et al., et al., 2011). The identified sequences were compared using 2016). The reads of both applications were trimmed and the BLASTP against the GenBank database. optimal assembly was obtained through SPAdes software Finally, Artemis (Rutherford et al., 2000), DNA Plotter (Bankevich et al., 2012). (Carver et al., 2009), and Mauve alignment tool (Darling et al., 2004) were used for data management, visualization of Sequence analysis and genome annotation genomic features, and multiple genomic sequence alignment, respectively. The genome annotation was performed by Rapid Anno- tation using the Subsystem Technology server (Aziz et al., Comparative genome analysis 2008) and the predicted bacterial protein sequences were compared using BLASTP against the GenBank and the Clus- The new species was compared with the bacteria exhibit- ters of Orthologous Groups (COGs) databases (Evalue 1e-03, ing the closest 16S rRNA sequences and with a complete coverage 0, 7, and identity percent 30%). tRNA and rRNA genome sequence recoverable in the NCBI database. An genes were searched using tRNAScanSE (Lowe and Eddy, annotation of all proteomes was carried out to define the 1997) and RNAmmer (Lagesen et al., 2007), respectively. The distribution of functional classes of predicted genes accord- HHMscan was used to find the PFAM-conserved domains ing to the COGs of proteins (using the same method as for (PFAM-A and PFAM-B domains) (Eddy et al., 2011). The genome annotation). Core genes are defined as genes with an number of transmembrane helices and the lipoprotein signal orthologue in each genome used for comparative analysis. peptides were predicted using Phobius (Ka¨ll et al., 2004). All comparison processes were performed in the Multi-Agent The resistome was analyzed by the Antibiotic Resistance Software System DAGOBAH (Gouret et al., 2011), including Gene-ANNOTation (ARG-ANNOT) database (Gupta et al., Figenix (Gouret et al., 2005) libraries, which provides pipe- 2014). The presence of putative bacteriocins was determined line analysis, and Phylopattern (Gouret et al., 2009) for tree using the bacteriocins of the URMITE (BUR) database (http:// manipulation. drissifatima.wix.com/bacteriocins) (Drissi et al., 2015). The To evaluate the genomic similarity among the studied presence of polyketide synthases and nonribosomal peptide strains, we identified two parameters: dDDH, which exhib- synthetase (NRPS) was also determined using a homemade its a high correlation with DDH (Auch et al., 2010; Meier- Downloaded by Guangxi University for Nationalities from online.liebertpub.com at 02/16/18. For personal use only.

FIG. 7. Phylogenetic tree of the bacteriocin colicin V (WP_040262223.1) in P. massiliensis.

214 170 BARDET ET AL.

FIG. 8. Phylogenetic tree highlighting the position of the bacteriocin peptidase (WP_040263026.1) in P. massiliensis.

Kolthoff et al., 2013), and the average genomic identity of preincubation. The spectrum obtained from MALDI-TOF orthologous gene sequences (AGIOS) (Ramasamy et al., MS for strain CB1T did not obtain a significant score, sug- 2014), which was designed to be independent of DDH. gesting that our isolate could be a new species. The spectrum In this study, we compared the genome sequences was added to our database (Fig. 1). The gel view showed of P. massiliensis CB1T (GenBank accession number spectral differences with other members of the family CCYK01000000) with those of Pseudomonas putida KT2440 Pseudomonadaceae (Fig. 2). (NC_002947.2), Pseudomonas fluorescens Pf0-1 (NC_ Strain CB1T exhibited a 98.2% 16S rRNA sequence identity 007492.2), Pseudomonas protegens Pf-5 (NC_004129.6), with Pseudomonas parafulva strain AJ 2129 (NR_040859.1) Pseudomonas syringae pv. phaseolicola 1448A (CP000058), (Fig. 3), which is under the threshold recommended for de- Pseudomonas brassicacearum subsp. brassicacearum NFM421 lineating a new species (Stackebrandt and Ebers, 2006). (NC_015379.1), Pseudomonas mendocina ymp (NC_009439.1), Consequently, strain CB1T was considered a new strain and and Pseudomonas poae RE*1-1-14 (NC_020209.1). named P. massiliensis sp. nov. and its 16S rRNA sequence was deposited in GenBank under number LK985396. Results Phenotypic characteristics Strain identification and phylogenic analysis Colonies were small, circular, smooth, yellowish, opaque, The P. massiliensis strain CB1T was isolated in June 2014 and a-hemolytic. Cells were gram negative, motile short rod by cultivation on Colombia agar with 5% sheep blood agar (Fig. 4) with a width of 0.5 lm and a length of 2.6 lm (Fig. 5), (COS) (BioMe´rieux) in aerobic conditions after 14 days of and did not exhibit sporulation after thermo-shock. Strain Downloaded by Guangxi University for Nationalities from online.liebertpub.com at 02/16/18. For personal use only.

FIG. 9. Comparison of nonribosomal peptide synthetase (NRPS) of enterobactin of P. massiliensis with enterobactin cluster in other species.

215 MICROBIAL CULTUROMICS IN GLOBAL HEALTH 171

CB1T was mesophilic with a growth between 25°C and 45°C, aerobic, and grew in the presence of 5% CO2 and in a microaerophilic atmosphere, but not anaerobically. Growth did 5172 not require NaCl, with a tolerance at 2%. The colonies could 2391 grow within a pH range from 5.3 to 8.4. Optimal growth was syringae Pseudomonas observed at 37°C in aerobic conditions at a pH of 7.5. Strain CB1T exhibited neither catalase nor oxidase activities. Using API 20 NE, positive reactions were observed for nitrate reduction, esculine, d-mannose, d-mannitol, potas- 4594 sium gluconate, malate, and trisodium citrate. An acid 2503 2915

production assay using an API 50 CH strip gave positive re- mendocina Pseudomonas

actions for glycerol, glucose, fructose, inositol, mannitol, genome (bold). esculine, cellobiose, maltose, lactose, and trehalose. An API ) ZYM strip showed the presence of alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, acid 3246 2721 2936 4796

phosphatase, and naphthol-AS-BI-phosphohydrolase. Nega- poae tive reactions were obtained for other constituents. These

characteristics are compared with other members of Pseu- Pseudomonas Upper Right domonadaceae in Table 1 (Xiao et al., 2009). ( Strain CB1T was resistant to ticarcillin, ticarcillin/clavulanic acid, cefepime, trimethoprim/sulfamethoxazole, rifampicin, and fosfomycin, with an intermediate status for aztreonam, and 3650 3271 2783 3024 was susceptible to the other antibiotics tested. 6108 protegens Pseudomonas Genomic properties The genome (Genbank accession number CCYK01000000) is 4,723,534 bp long with a 62.34% G+C content (Fig. 6). Of the 4313 predicted genes, 4239 are protein-coding genes and 3714 3727 3283 2756 3015 74 are RNAs, including 15 rRNA genes (5 are 16S rRNA, 4 are 6095

23S rRNA, and 6 are 5S rRNA) and 59 tRNA genes. The Pseudomonas properties and statistics of the genome are summarized in brassicacearum Table 2. A total of 3312 genes (78.13%) were assigned to a putative function and 278 (6.56%) were identified as having an unknown function. The distribution of genes into COG func- 3209 3153 390 2857 2551 2670 tional categories is presented in Table 3. 5722 The resistome analysis of P. massiliensis CB1T by ARG- fluorescens ANNOT identified a beta-lactamase class-A gene (1094 bp) Pseudomonas and the multidrug efflux transporter MexF gene (Table 4). Two bacteriocins were detected through the BUR database: colicinV (WP_040262223.1) and peptidase (WP_040263026.1), whose phylogenetic trees are represented in Figures 7 and 8, 2541 2742 2691 2663 2596 2195 2514 5350 respectively. putida

Moreover, 22 toxins and antitoxins that are potentially Pseudomonas attributed to bacterial dormancy and persistence, and could

enhance drug tolerance and biofilm formation were identi- The Numbers of Orthologous Protein Shared Between Genomes Downloaded by Guangxi University for Nationalities from online.liebertpub.com at 02/16/18. For personal use only. fied, as an intact prophage sequence of 47.7 kb (GC%: 60.54). 5. A secondary metabolite biosynthetic gene cluster (NRPS) 4239 75,82 78,19 82,1 81,87 75,38 74,84 77,3 75,19 77,33 82,93 75,07 77,52 81,39 81,19 81,67 74,02 75,67 77,42 77,29 77,55 77,29 74,85 was found and identified as an enterobactin with a size of 74,97 76,34 76,36 76,24 77,27 76,25 massiliensis Table

14.7 kb. The component F of this synthetase showed 68%, Pseudomonas 63%, 62%, and 57% similarity with the respective sequences of Pseudomonas amygdali (WP_010212720.1), Serratia fonticola (WP_024485228.1), Pantoea sp. AS-PWVM4 (WP_ 052469123.1), and Shigella dysenteriae Sd197 (YP_402211.1), respectively (Fig. 9).

Comparison with genomes from other members of the family Pseudomonadaceae Of the compared species, the draft genome of P. massiliensis CB1T (4.72 Mb) is the smallest, with those of P. protegens, P. syringae, P. brassicacearum, P. ﬂuorescens, Average percentage similarity of nucleotides corresponding to orthologous protein shared between genomes (lower left) and numbers of proteins per Species Pseudomonas protegens Pseudomonas massiliensis Pseudomonas putida Pseudomonas ﬂuorescens Pseudomonas brassicacearum Pseudomonas poae Pseudomonas syringae P. putida, P. mendocina, and P. poae being 7.07, 6.11, 6.84, Pseudomonas mendocina

216 172 BARDET ET AL.

FIG. 10. Distribution of genes into COGs functional categories of every compared genome. COGs, Clusters of Ortho- logous groups.

6.44, 6.18, 5.07, and 5.51 Mb, respectively. The G+C content Drancourtella massiliensis (Durand et al., 2016) or Beduini of P. massiliensis CB1T (62.34%) is lower compared with massiliensis (Mourembou et al., 2015). P. protegens (63.30%) and P. mendocina, (64.68%), but In this study, we report the identification and description of higher compared with P. syringae (57.91%), P. brassica- P. massiliensis sp. nov., represented by the strain CB1T, cearum (60.79%), P. fluorescens (60.52%), P. putida isolated from the fecal flora of an obese 37-year-old Brazilian (61.52%), and P. poae (60.85%). woman using this strategy. P. massiliensis (ma.si.li.e¢n.sis. L. The gene content of P. massiliensis CB1T (4239 genes) is gen. masc. n. massiliensis, of Massilia, the Latin name of smaller than those of the compared species, ranging from Marseille where P. massiliensis was first isolated) was de- 4594 to 6108 genes (Table 5) and P. massiliensis CB1T posited in two different collections of microorganisms, the shared a maximum of 2691 orthologous genes with P. Deutsche Sammlung von Mikroorganismen und Zellkulturen brassicacearum (63.5%). The distribution of genes into COG and the Collection de Souches de l’Unite´ des Rickettsies functional categories of every compared genome is presented (CSUR) under numbers DSM 29075 and CSUR P1334, re- in Figure 10. spectively. The 16S rRNA and genome sequences were de- The AGIOS values for P. massiliensis strain CB1T and posited in GenBank under accession numbers LK985396 and studied Pseudomonas species are summarized in Table 5 and CCYK01000000, respectively. P. massiliensis was then iso- ranged from 74.02% to 82.93%, confirming its new species lated in Anopheles gambiae Giles and Culex quinquefascia- status. The dDDH values of those species are represented in tus mosquitoes collected in Mali (Tandina et al., 2016). Table 6 and range from 17.4% – 2.54% to 22.8% – 2.97% The genus Pseudomonas was first proposed by Migula among all the studied species, and from 18.3% – 2.54% 1894, belongs to the family of Pseudomonadaceae, and is one (Pseudomonas caeni) to 21.1% – 2.73% (P. alcaligenes) of the most ubiquitous bacterial genera in the world (Peix between P. massiliensis CB1T and the others. et al., 2009), characterized by gram negative, motile, and aerobic species. Few species of the genus Pseudomonas are Discussion human opportunistic pathogens, mostly represented by Pseudomonas aeruginosa that causes wound infections or The development of high-throughput sequencing gave severe infections in immunocompromised patients, mainly access to a large quantity of information, but it also gen- those with cystic fibrosis, with its biofilm formation (Lee and erates gaps with genomes corresponding to previously Yoon, 2017).

Downloaded by Guangxi University for Nationalities from online.liebertpub.com at 02/16/18. For personal use only. uncultivable bacteria (Marx, 2016). The return to culture- This study attests the usefulness of the culturomics ap- based methods was necessary to isolate such bacterial spe- proach in the characterization of microbial communities. cies and study their properties and behavior (Browne et al., This work intend to extend the knowledge about the reper- 2016). Culturomics was developed to explore the diversity toire of microorganisms that colonize the human gut to better of human microbiota and has the capacity to detect viable understand the composition of its microflora and link it to bacteria and minority populations (Lagier et al., 2015). human health (Hugon et al., 2015, 2017; Lagier et al., 2012b). This high-throughput culture method, combined to taxono- For such purposes, culturomics and metagenomics are com- genomics strategy and applied to human samples from vari- plementary techniques to explore the human microbiota ous regions of the world, enabled the identification of 1057 (Marx, 2016). different bacterial species from the human gut microbiota, including 187 that had never previously been found in human Conclusions and Outlook samples and 197 putative new species (Lagier et al., 2016), of which 47 have officially validated names, such as En- Culturomics, and the microbiome science more generally, terococcus massiliensis (Le Page et al., 2016), Bacillus testis are anticipated to transform global health diagnostics and (Cimmino et al., 2016), Kallipyga gabonensis (Hugon et al., inform the ways in which gut microbial diversity contributes 2013), Enterobacter massiliensis (Lagier et al., 2013) or to human health and disease, and by extension, to personal- Clostridium ihumii (Merhej et al., 2015), and also new genera ized medicine.

217 MICROBIAL CULTUROMICS IN GLOBAL HEALTH 173

Acknowledgments ) The authors thank the Xegen Company (www.xegen.fr/) 2.52% 2.52% 00% 2.52% 2.52% 2.52% 2.52% – – – – – – – for automating the genomic annotation process. butyricum Clostridium

100% Author Disclosure Statement The authors declare that no conﬂicting ﬁnancial interests exist.

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219 MICROBIAL CULTUROMICS IN GLOBAL HEALTH 175

Abbreviations Used DSM ¼ Deutsche Sammlung von AGIOS ¼ average genomic identity of Mikroorganismen orthologous gene sequences MALDI-TOF MS ¼ matrix-assisted laser desorption/ ARG-ANNOT ¼ Antibiotic Resistance ionization time-of-ﬂight mass Gene-ANNOTation spectrometry BUR ¼ Bacteriocins of the URMITE NCBI ¼ National Center for Biotechnology COG ¼ Clusters of Orthologous groups Information CSUR ¼ Collection de Souches de l’Unite´ NRPS ¼ nonribosomal peptide des Rickettsies synthetase DDH ¼ DNA–DNA hybridization PKS ¼ polyketide synthases Downloaded by Guangxi University for Nationalities from online.liebertpub.com at 02/16/18. For personal use only.

220 Communications orales et posters

Communication orale: 1. TANDINA F. “Use of MALDI-TOF MS and culturomics to identify mosquitoes and their midgut microbiota”. Congrès SOPAMYM. Mali, Décembre 2016.

Posters: 1. TANDINA F. “Using MALDI-TOF MS to identify mosquitoes collected in Mali and their blood meals”. Journée scientifique de l’Infectiopôle Sud. Marseille, Juillet 2017. 2. TANDINA F. “Use of MALDI-TOF MS and culturomics to identify mosquitoes and their midgut microbiota”. Journée scientifique de l’Infectiopôle Sud. Marseille, Juillet 2016.

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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. 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 gorgement. Ensuite nous avons expérimentalement gorgés trois espèces de moustiques (Anopheles gambiae, Anopheles coluzzii et Aedes albopictus) sur des repas de sang successif et mixte, pour voir si l’espèce de moustique avait un impact sur l’identification du repas de sang par MALDI-TOF MS. Nos résultats révèlent que le MALDI-TOF MS est tout à fait capable d’identifier le repas mixte. Mais en ce qui concerne le repas successif seul le dernier repas de sang est identifié. Cela est peut être dû à l’intervalle de jours (trois) qui sépare les deux repas de sangs des moustiques. Donc le premier repas de sang a peut-être été digéré par les moustiques. Le troisième objectif de ce travail était d’utiliser la culturomique et le MALDI-TOF pour l’étude du microbiote digestif de moustiques collectés sur le terrain au Mali et à Marseille. Cette approche a révélé une grande diversité du microbiote digestif des moustiques Anopheles gambiae, Aedes albopictus et Culex quinquefasciatus. La majorité de ces bactéries étaient des Grams négatifs et appartiennent au phylum des Protéobacteria. De plus, une nouvelle espèce bactérienne, Pseudomonas massiliensis, a été isolée dans l’estomac des moustiques Anopheles gambiae et Culex quinquefasciatus et leurs eaux de gîte collectées au Mali.

Mots clés : Moustiques, vecteurs, MALDI-TOF MS, Mali

Abstract

Mosquitoes are the main vectors involved in the transmission of pathogens to humans. Accurate identification of mosquito species is crucial to distinguish between vector and non-vector species. The mosquito blood meal determination is fundamental in understanding the behavior of vector species. These parameters are necessary to prevent the risk of transmission of infectious diseases. Originally intended to work in Mali, we decided in the first part of this work, to update the directory of mosquitoes in Mali. In our original work, we have been working on the development and use of an innovative tool, MALDI-TOF (Matrix Assisted Laser Desorption Ionization - Time of Flight), which we used for the mosquito identification, meal blood determination and microbiota study. The first objective of our study was to update the current literature on Malian Culicidan fauna, potential vectors and pathogens transmitted. We also wished to describe the progress made in mosquito vector control and the innovative tools recently used for mosquito identification. Thus, we have listed 106 mosquito species currently recorded in Mali, including 28 Anophelinae and 78 Culicinae. We have also highlighted the recent introduction of two invasive mosquito species, Aedes albopictus and Culex neavei. The second objective was to evaluate the effectiveness of MALDI-TOF MS for identified mosquitoes collected in Mali and to determine their blood meal source. The results obtained show the ability of MALDI-TOF MS to identify mosquitoes collected in Mali and their source of blood meal. Thus, eight mosquito species has been identified, including Anopheles gambiae Giles, Anopheles coluzzii, Anopheles arabiensis, Culex quinquefasciatus, Culex neavei, Culex perexiguus, Aedes aegypti and Aedes fowleri. In addition, the identification of the blood meal source matched with human blood (n = 619), chicken blood (n = 9), cow's blood (n = 9), donkey blood (n = 6), dog's blood (n = 5) and sheep blood (n = 3). Subsequently, we were able to confirm the robustness of mass spectrometry MALDI-TOF to identify other animal blood samples. We artificially engorged two mosquito species including, Anopheles gambiae and Anopheles coluzzii on eight animal bloods samples. We obtained 100% correct identification of the blood source for samples taken 1 to 24 hours after feeding. Then, we experimentally engorged three mosquito species including Anopheles gambiae, Anopheles coluzzii and Aedes albopictus on successive and mixed blood meals, to see whether the mosquito species had an impact on the blood meal identification using MALDI-TOF MS. The results revealed that MALDI-TOF MS is able to identify mixed blood meals. This may be due to the interval of days (three) between the first and second blood meals of mosquitoes. The first blood meal was indeed digested by the mosquitoes. The third objective of our study was to use MALDI-TOF and culturomics for the microbiota study of the mosquito collected in the field, notably in Marseille and Mali. The culturomics approach revealed a great diversity of the digestive microbiota of the Anopheles gambiae, Aedes albopictus and Culex quinquefasciatus mosquitoes. The majority of bacteria detected in the microbiota of mosquitoes was gram-negative and belong to the Proteobacteria phylum. In addition, a new bacterial species, Pseudomonas massiliensis, was isolated from the Anopheles gambiae and Culex quinquefasciatus microbiota and their breeding waters were collected in Mali.

Key words: Mosquitoes, vectors, MALDI-TOF MS, Mali