Experimental development and field application of innovative tools for arthropods identification

Les arthropodes hématophages tels que les moustiques, les tiques et les puces ont une importance significative en santé publique en raison de leur capacité à transmettre des maladies majeures aux humains et aux animaux. La lutte anti-vectorielle et la surveillance épidémiologique des vecteurs sont essentielles dans la stratégie de lutte contre ces maladies. Cette dernière n'est réussie que grâce à une identification correcte et précise des vecteurs. Ainsi dans ce travail nous avons mis au point les protocoles pour la préparation des échantillons pour l'identification des moustiques adultes et leur stades aquatiques ainsi que des tiques et des puces par MALDI-TOF MS. Cet outil s'est déjà distingué comme étant fiable pour l'identification des arthropodes. La deuxième partie de notre travail a consisté en l'application de ces protocoles sur des larves de moustiques collectées sur terrain durant une enquête entomologique menée dans la ville de Marseille. Lors de cette étude, la pertinence et la fiabilité du MALDI-TOF MS pour l'identification des larves de moustiques collectées sur terrain a été vérifiée. Enfin, nous avons réalisé l'inventaire des communautés virales de trois espèces de moustiques collectées à Marseille par métagénomique, qui a révélé la présence de nombreux nouveaux virus. L'ensemble des résultats présentés dans cette thèse souligne que l'utilisation d'outils innovants tels que le MALDI-TOF MS et la métagénomique pour étudier les vecteurs et les agents qu'ils portent est une stratégie prometteuse qui contribuera dans la connaissance des cycles de transmission zoonotique et des risques potentiels d'émergence des maladies vectorielles en population humaine.Hematophagous arthropods such as mosquitoes, ticks, and fleas are of significant importance in public health because of their ability to transmit major diseases to humans and animals. Vector control and epidemiological vector surveillance are essential in the strategy of combating vector-borne diseases. The latter is successful only by a correct and precise identification of the vectors. Thus in this work, we have developed and improved the protocols of samples preparation for the identification of adult mosquitoes and their aquatic stages, ticks, and fleas by MALDI-TOF MS. This tool has been already distinguished as being reliable for the arthropods identification. The second part of our work consisted in the application of these protocols on mosquito larvae collected in the field during an entomological investigation carried out in the city of Marseille. In this study, the relevance and reliability of MALDI-TOF MS for the identification of mosquito larvae collected in the field were verified. Finally, we carried out the inventory of the viral communities of three mosquito species collected in Marseille by metagenomics, which revealed the presence of numerous new viruses. All the results presented in this thesis emphasize that the use of innovative tools such as MALDI-TOF MS and metagenomics to study vectors and the agents they carry is a promising strategy that will contribute to the knowledge of zoonotic transmission cycles and the potential risks of the emergence of vector-borne diseases in human populations

Identification of Aedes mosquitoes by MALDI-TOF MS biotyping using protein signatures from larval and pupal exuviae

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Mosquito Vectors (Diptera: Culicidae) and Mosquito-Borne Diseases in North Africa

Mosquitoes (Diptera: Culicidae) are of significant public health importance because of their ability to transmit major diseases to humans and animals, and are considered as the world’s most deadly arthropods. In recent decades, climate change and globalization have promoted mosquito-borne diseases’ (MBDs) geographic expansion to new areas, such as North African countries, where some of these MBDs were unusual or even unknown. In this review, we summarize the latest data on mosquito vector species distribution and MBDs affecting both human and animals in North Africa, in order to better understand the risks associated with the introduction of new invasive mosquito species such as Aedes albopictus. Currently, 26 mosquito species confirmed as pathogen vectors occur in North Africa, including Aedes (five species), Culex (eight species), Culiseta (one species) and Anopheles (12 species). These 26 species are involved in the circulation of seven MBDs in North Africa, including two parasitic infections (malaria and filariasis) and five viral infections (WNV, RVF, DENV, SINV and USUV). No bacterial diseases have been reported so far in this area. This review may guide research studies to fill the data gaps, as well as helping with developing effective vector surveillance and controlling strategies by concerned institutions in different involved countries, leading to cooperative and coordinate vector control measures

Mosquito Vectors (Diptera: Culicidae) and Mosquito-Borne Diseases in North Africa

Mosquitoes (Diptera: Culicidae) are of significant public health importance because of their ability to transmit major diseases to humans and animals, and are considered as the world’s most deadly arthropods. In recent decades, climate change and globalization have promoted mosquito-borne diseases’ (MBDs) geographic expansion to new areas, such as North African countries, where some of these MBDs were unusual or even unknown. In this review, we summarize the latest data on mosquito vector species distribution and MBDs affecting both human and animals in North Africa, in order to better understand the risks associated with the introduction of new invasive mosquito species such as Aedes albopictus. Currently, 26 mosquito species confirmed as pathogen vectors occur in North Africa, including Aedes (five species), Culex (eight species), Culiseta (one species) and Anopheles (12 species). These 26 species are involved in the circulation of seven MBDs in North Africa, including two parasitic infections (malaria and filariasis) and five viral infections (WNV, RVF, DENV, SINV and USUV). No bacterial diseases have been reported so far in this area. This review may guide research studies to fill the data gaps, as well as helping with developing effective vector surveillance and controlling strategies by concerned institutions in different involved countries, leading to cooperative and coordinate vector control measures

Virome Diversity among Mosquito Populations in a Sub-Urban Region of Marseille, France

International audienceSome mosquito species have significant public health importance given their ability to transmit major diseases to humans and animals, making them the deadliest animals in the world. Among these, the Aedes (Ae.) genus is a vector of several viruses such as Dengue, Chikungunya, and Zika viruses that can cause serious pathologies in humans. Since 2004, Ae. albopictus has been encountered in the South of France, and autochthonous cases of Dengue, Chikungunya, and Zika diseases have recently been reported, further highlighting the need for a comprehensive survey of the mosquitoes and their associated viruses in this area. Using high throughput sequencing (HTS) techniques, we report an analysis of the DNA and RNA viral communities of three mosquito species Ae. albopictus, Culex (Cx.) pipiens, and Culiseta (Cs.) longiareolata vectors of human infectious diseases in a small sub-urban city in the South of France. Results revealed the presence of a significant diversity of viruses known to infect bacteria, plants, insects, and mammals. Several novel viruses were detected, including novel members of the Rhabdoviridae, Totiviridae, Iflaviviridae, Circoviridae, and Sobemoviridae families. No sequence related to major zoonotic viruses transmitted by mosquitoes was detected. The use of HTS on arthropod vector populations is a promising strategy for monitoring the emergence and circulation of zoonoses and epizooties. This study is a contribution to the knowledge of the mosquito microbiome

Co-infection of bacteria and protozoan parasites in Ixodes ricinus nymphs collected in the Alsace region, France

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Field application of MALDI-TOF MS on mosquito larvae identification

International audienceIn recent years, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has emerged as an efficient tool for arthropod identification. Its application for field monitoring of adult mosquitoes was demonstrated, but identification of larvae has been limited to laboratory-reared specimens. Study aim was to test the success of MALDI-TOF MS in correctly identifying mosquito larvae collected in the field. Collections were performed at 13 breeding sites in urban areas of Marseille, a city in the South of France. A total of 559 larvae were collected. Of these, 73 were accurately morphologically identified, with confirmation either by molecular identification (n = 31) or analysis with MALDI-TOF MS (n = 31) and 11 were tested using both methods. The larvae identified belonged to six species including Culiseta longiareolata, Culex pipiens pipiens, Culex hortensis, Aedes albopictus, Ochlerotatus caspius and Anopheles maculipennis. A high intra-species reproducibility and inter-species specificity of whole larva MS spectra was obtained and was independent of breeding site. More than 92% of the remaining 486 larvae were identified in blind tests against the MS spectra database. Identification rates were lower for early and pupal stages, which is attributed to lower protein abundance and metamorphosis, respectively. The suitability of MALDI-TOF MS for mosquito larvae identification from the field has been confirmed

Assessment of <i>I</i>. <i>ricinus</i> MS spectra reproducibility according to tick body parts and <i>Borrelia</i> infectious status using composite correlation index (CCI).

<p>MS spectra from five specimens per body part and <i>B</i>. <i>afzelii</i> infectious status were analysed using the CCI tool. Body part and infectious status are indicated on the left side of the heat map. Levels of MS spectra reproducibility are indicated in red and blue revealing relatedness and incongruence between spectra, respectively. CCI matrix was calculated using MALDI-Biotyper v3.0. software with default settings (mass range 3.0–12.0 kDa; resolution 4; 8 intervals; auto-correction off). The values correspond to the mean coefficient correlation and respective standard deviations obtained for paired condition comparisons. CCI were expressed as mean ± standard deviation. BI, <i>Borrelia</i>-infected; PF, pathogen-free.</p

Principal component analysis (PCA) from MS spectra of idiosomes and legs of <i>I</i>. <i>ricinus</i> infected or not by <i>Borrelia sp</i>.

<p>PCA dimensional image from MS spectra of <i>I</i>. <i>ricinus</i> idiosomes (A) and legs (B) <i>Borrelia</i>-free (red dots, n = 10), infected by <i>B</i>. <i>afzelii</i> (green dots, n = 6), <i>B</i>. <i>burgdorferi</i> (blue dots, n = 3), <i>B</i>. <i>garinii</i> (yellow dots, n = 2), co-infected by <i>B</i>. <i>garinii</i> and <i>B</i>. <i>burgdorferi</i> (purple dots, n = 1). (C) PCA dimensional image from the same MS spectra of <i>I</i>. <i>ricinus</i> idiosomes (red dots, n = 22) and legs (green dots, n = 22). The contributions of PC1, PC2 and PC3 were 38.4%, 15.5% and 7.2%, respectively. Among the <i>Borrelia</i>-free specimens, five were laboratory reared and the other five came from field collection.</p

MSP dendrogram of MALDI-TOF MS spectra from legs, capitula and half-idiosomes of adult <i>I</i>. <i>ricinus</i> pathogen-free or infected by <i>Borrelia afzelii</i>.

<p>Five specimens per body part and <i>B</i>. <i>afzelii</i> infectious status were used to construct the dendrogram. The dendrogram was created using Biotyper v3.0 software and distance units correspond to the relative similarity of MS spectra. The specimens infected by <i>B</i>. <i>afzelii</i> were indicated by asterisks (*).</p