Development and application of innovative technologies for the mosquito study : their preference trophic and their microbiota

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. Nous avons mise à jour la littérature actuelle sur la faune Culicidienne du Mali. Ainsi, nous avons listé 106 espèces de moustiques actuellement enregistrée au Mali dont 28 Anophelinae et 78 Culicinae. Nous avons ensuite évalué l’efficacité du MALDI-TOF MS à identifier des moustiques collectés au Mali et déterminer leur source de repas sanguin. Nous avons confirmé la robustesse du MALDI-TOF MS à identifier un grand nombre de sang d’animaux. Nous avons artificiellement gorgé des femelles de An. gambiae et An. 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 An. gambiae, An. coluzzii et Ae. albopictus sur des repas de sang successif et mixte 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. Enfin nous avons utilisé 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 An. gambiae, Ae. albopictus et Cx. quinquefasciatus.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. Thus, we have listed 106 mosquito species currently recorded in Mali, including 28 Anophelinae and 78 Culicinae. Then, we evaluated 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. Subsequently, we were able to confirm the robustness of MALDI-TOF MS to identify other animal blood samples. We artificially engorged 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 An. gambiae, An. coluzzii and Ae. albopictus on successive and mixed blood meals using MALDI-TOF MS. The results revealed that MALDI-TOF MS is able to identify mixed blood meals. In addition we used 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 An. gambiae, Ae. albopictus and Cx. quinquefasciatus mosquitoes

Blood meal identification in the cryptic species Anopheles gambiae and Anopheles coluzzii using MALDI-TOF MS

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has recently emerged in entomology as a technique to identify arthropods and their blood meal source. In this study, female Anopheles gambiae were fed on five host blood sources: ocelot (Leopardus pardalis), binturong (Arctictis binturong), springbok (Antidorcas marsupialis), jaguar (Panthera onca) and Hamadryas baboon (Papio hamadryas), while Anopheles coluzzii were fed on three hosts: dromedary (Camelus dromedarius), Barbary sheep (Ammotragus lervia) and pig (Sus scrofa). We obtained the MS spectra from 240 engorged mosquito abdomens and selected high quality ones from 72 mosquito abdomens to upgrade our home-made database. We excluded from the analysis any spectra of low quality (n = 80), and the remaining 88 specimens were subjected to a blind test analysis against the home-made database. We obtained 100% correct identification of the blood meal source for the specimens collected, 1, 12 and 24 h post-feeding, whereas for the specimens collected 36 h post-feeding, the correct identification rate decreased dramatically. We confirm here that MALDI-TOF MS can be used to identify the blood meal origin of freshly engorged mosquitoes, which opens new perspectives for further studies, including the impact of the mosquito species on blood meal identification

Blood meal identification in the cryptic species Anopheles gambiae and Anopheles coluzzii using MALDI-TOF MS

International audienceMatrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has recently emerged in entomology as a technique to identify arthropods and their blood meal source. In this study, female Anopheles gambiae were fed on five host blood sources: ocelot (Leopardus pardalis), binturong (Arctictis binturong), springbok (Antidorcas marsupialis), jaguar (Panthera onca) and Hamadryas baboon (Papio hamadryas), while Anopheles coluzzii were fed on three hosts: dromedary (Camelus dromedarius), Barbary sheep (Ammotragus lervia) and pig (Sus scrofa). We obtained the MS spectra from 240 engorged mosquito abdomens and selected high quality ones from 72 mosquito abdomens to upgrade our home-made database. We excluded from the analysis any spectra of low quality (n = 80), and the remaining 88 specimens were subjected to a blind test analysis against the home-made database. We obtained 100% correct identification of the blood meal source for the specimens collected, 1, 12 and 24 h post-feeding, whereas for the specimens collected 36 h post-feeding, the correct identification rate decreased dramatically. We confirm here that MALDI-TOF MS can be used to identify the blood meal origin of freshly engorged mosquitoes, which opens new perspectives for further studies, including the impact of the mosquito species on blood meal identification

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

International audienceMosquito-borne diseases cause major human diseases in almost every part of the world. In West Africa, and notably in Mali, vector control measures help reduce 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 a total of 88 species. Our present review focuses on mosquitoes of medical importance among which the Anopheles vectors of Plasmodium and filaria, as well as the Culex and Aedes vectors of arboviruses. It aims to provide a concise update of the literature on Culicidae, covering the ecological areas in which the species are found but also the transmitted pathogens and recent innovative tools for vector surveys. This review highlights the recent introduction of invasive mosquito species, including Aedes albopictus and Culex neavei. The comprehensive list of mosquito species currently recorded includes 106 species (28 species of the Anophelinae and 78 species of the Culicinae). There are probable gaps in our knowledge concerning mosquitoes of the subfamily Culicinae and northern half of Mali because most studies have been carried out on the genus Anopheles 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

Accurate identification of Anopheles gambiae Giles trophic preferences by MALDI-TOF MS

International audienceThe determination of the trophic preferences of the Anopheles gambiae Giles (Diptera: Culicidae) is a decisive parameter for the monitoring and the prevention of malaria risk transmission. Currently, arthropod blood feeding sources are identified using immunological or molecular biology traditional techniques. Despite the effectiveness of these methods, they present several limitations, and notably, they are time-consuming and costly techniques. A recent study demonstrated that MALDI-TOF MS could be a useful tool for the identification of blood meal origins in freshly engorged mosquitoes. However, the limited number of blood vertebrate species tested to date, did not allow an assessment of the efficiency of MALDI-TOF MS in distinguishing blood MS spectra among close host species, such as humans versus primates. Therefore, in the present study, blood from ten distinct vertebrate host species, including four domestic species, four wild species, and two primates, was selected to control the reliability of MALDI-TOF MS based identification. Host blood species-specific MS profiles, up to 24 h post-feeding in engorged Anopheles abdomens, were confirmed. Blind tests underlined the high specificity of MS spectra for the recognition of each host species, preventing misidentification. Nevertheless, an accurate analysis of the results from MS spectra queried against the MS database revealed that the reliability of identification is directly linked to the comprehensiveness of the MS reference database. Finally, the rapidity, the low-cost reagents, the simplicity of data analysis, and the accuracy of the tool for blood origin determination, make this proteomic strategy a promising complementary method for the elucidation of host/vector interactions

Use of MALDI-TOF MS and culturomics to identify mosquitoes and their midgut microbiota

International audienceBackground: 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

Identification of mixed and successive blood meals of mosquitoes using MALDI-TOF MS protein profiling

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

International audienceBackground: Identification of the source of mosquito blood meals is an important component for disease control and surveillance. Recently, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) profiling has emerged as an effective tool for mosquito blood meal identification, using the abdomens of freshly engorged mosquitoes. In the field, mosquito abdomens are crushed on Whatman filter papers to determine the host feeding patterns by identifying the origin of their blood meals. The aim of this study was to test whether crushing engorged mosquito abdomens on Whatman filter papers was compatible with MALDI-TOF MS for mosquito blood meal identification. Both laboratory reared and field collected mosquitoes were tested.Material and methods: Sixty Anopheles gambiae Giles were experimentally engorged on the blood of six distinct vertebrate hosts (human, sheep, rabbit, dog, chicken and rat). The engorged mosquito abdomens were crushed on Whatman filter papers for MALDI-TOF MS analysis. 150 Whatman filter papers, with mosquitoes engorged on cow and goat blood, were preserved. A total of 77 engorged mosquito abdomens collected in the Comoros Islands and crushed on Whatman filter papers were tested with MALDI-TOF MS. Results The MS profiles generated from mosquito engorged abdomens crushed on Whatman filter papers exhibited high reproducibility according to the original host blood. The blood meal host was correctly identified from mosquito abdomens crushed on Whatman filter papers by MALDI-TOF MS. The MS spectra obtained after storage were stable regardless of the room temperature and whether or not they were frozen. The MS profiles were reproducible for up 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