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      Identification of French Guiana anopheline mosquitoes by MALDI-TOF MS profiling using protein signatures from two body parts

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          Abstract

          In French Guiana, the malaria, a parasitic infection transmitted by Anopheline mosquitoes, remains a disease of public health importance. To prevent malaria transmission, the main effective way remains Anopheles control. For an effective control, accurate Anopheles species identification is indispensable to distinguish malaria vectors from non-vectors. Although, morphological and molecular methods are largely used, an innovative tool, based on protein pattern comparisons, the Matrix Assisted Laser Desorption / Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) profiling, emerged this last decade for arthropod identification. However, the limited mosquito fauna diversity of reference MS spectra remains one of the main drawback for its large usage. The aim of the present study was then to create and to share reference MS spectra for the identification of French Guiana Anopheline species. A total of eight distinct Anopheles species, among which four are malaria vectors, were collected in 6 areas. To improve Anopheles identification, two body parts, legs and thoraxes, were independently submitted to MS for the creation of respective reference MS spectra database (DB). This study underlined that double checking by MS enhanced the Anopheles identification confidence and rate of reliable classification. The sharing of this reference MS spectra DB should make easier Anopheles species monitoring in endemic malaria area to help malaria vector control or elimination programs.

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          Emerging tools for identification of arthropod vectors.

          The rapid and reliable identification of arthropod vector species is an essential component of the fight against vector-borne diseases. However, owing to the lack of entomological expertise required for the morphological identification method, development of alternative and complementary tools is needed. This review describes the main methods used for arthropod identification, focusing on the emergence of protein profiling using MALDI-TOF MS technology. Sample preparation, analysis of reproducibility, database creation and blind tests for controlling accuracy of this tool for arthropod identification are described. The advantages and limitations of the MALDI-TOF MS method are illustrated by emphasizing different hematophagous arthropods, including mosquitoes and ticks, the top two main vectors of infectious diseases.
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            Matrix-Assisted Laser Desorption Ionization - Time of Flight Mass Spectrometry: An Emerging Tool for the Rapid Identification of Mosquito Vectors

            Background The identification of mosquito vectors is typically based on morphological characteristics using morphological keys of determination, which requires entomological expertise and training. The use of protein profiling by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), which is increasingly being used for the routine identification of bacteria, has recently emerged for arthropod identification. Methods To investigate the usefulness of MALDI-TOF-MS as a mosquito identification tool, we tested protein extracts made from mosquito legs to create a database of reference spectra. The database included a total of 129 laboratory-reared and field-caught mosquito specimens consisting of 20 species, including 4 Aedes spp., 9 Anopheles spp., 4 Culex spp., Lutzia tigripes, Orthopodomyia reunionensis and Mansonia uniformis. For the validation study, blind tests were performed with 76 specimens consisting of 1 to 4 individuals per species. A cluster analysis was carried out using the MALDI-Biotyper and some spectra from all mosquito species tested. Results Biomarker mass sets containing 22 and 43 masses have been detected from 100 specimens of the Anopheles, Aedes and Culex species. By carrying out 3 blind tests, we achieved the identification of mosquito vectors at the species level, including the differentiation of An. gambiae complex, which is possible using MALDI-TOF-MS with 1.8 as the cut-off identification score. A cluster analysis performed with all available mosquito species showed that MALDI-Biotyper can distinguish between specimens at the subspecies level, as demonstrated for An gambiae M and S, but this method cannot yet be considered a reliable tool for the phylogenetic study of mosquito species. Conclusions We confirmed that even without any specific expertise, MALDI-TOF-MS profiling of mosquito leg protein extracts can be used for the rapid identification of mosquito vectors. Therefore, MALDI-TOF-MS is an alternative, efficient and inexpensive tool that can accurately identify mosquitoes collected in the field during entomological surveys.
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              Identification of Belgian mosquito species (Diptera: Culicidae) by DNA barcoding.

              Since its introduction in 2003, DNA barcoding has proven to be a promising method for the identification of many taxa, including mosquitoes (Diptera: Culicidae). Many mosquito species are potential vectors of pathogens, and correct identification in all life stages is essential for effective mosquito monitoring and control. To use DNA barcoding for species identification, a reliable and comprehensive reference database of verified DNA sequences is required. Hence, DNA sequence diversity of mosquitoes in Belgium was assessed using a 658 bp fragment of the mitochondrial cytochrome oxidase I (COI) gene, and a reference data set was established. Most species appeared as well-supported clusters. Intraspecific Kimura 2-parameter (K2P) distances averaged 0.7%, and the maximum observed K2P distance was 6.2% for Aedes koreicus. A small overlap between intra- and interspecific K2P distances for congeneric sequences was observed. Overall, the identification success using best match and the best close match criteria were high, that is above 98%. No clear genetic division was found between the closely related species Aedes annulipes and Aedes cantans, which can be confused using morphological identification only. The members of the Anopheles maculipennis complex, that is Anopheles maculipennis s.s. and An. messeae, were weakly supported as monophyletic taxa. This study showed that DNA barcoding offers a reliable framework for mosquito species identification in Belgium except for some closely related species.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: MethodologyRole: Project administrationRole: Resources
                Role: Data curationRole: Formal analysisRole: MethodologyRole: Project administrationRole: Writing – review & editing
                Role: InvestigationRole: MethodologyRole: Resources
                Role: Formal analysisRole: MethodologyRole: Project administrationRole: Resources
                Role: InvestigationRole: MethodologyRole: ResourcesRole: Visualization
                Role: InvestigationRole: MethodologyRole: Project administrationRole: Resources
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: MethodologyRole: SupervisionRole: ValidationRole: Writing – original draft
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                20 August 2020
                2020
                : 15
                : 8
                : e0234098
                Affiliations
                [1 ] Unité de Parasitologie et Entomologie, Département de Microbiologie et Maladies Infectieuses, Institut de Recherche Biomédicale des Armées, Marseille, France
                [2 ] Aix Marseille Université, IRD, AP-HM, SSA, UMR Vecteurs–Infections Tropicales et Méditerranéennes (VITROME), IHU—Méditerranée Infection, Marseille, France
                [3 ] Unite d’Entomologie Médicale, Institut Pasteur de la Guyane, Cayenne, French Guiana
                [4 ] Centre d’Epidémiologie et de Santé Publique des Armées, Marseille, France
                [5 ] Medical Entomology Unit, Institut Pasteur de Madagascar, Antananarivo, Madagascar
                Fisheries and Oceans Canada, CANADA
                Author notes

                Competing Interests: The authors declare that they have no competing interests.

                Author information
                http://orcid.org/0000-0003-0490-5774
                Article
                PONE-D-20-14339
                10.1371/journal.pone.0234098
                7444543
                32817616
                f436018f-e2aa-425c-9339-1b5e878be885
                © 2020 Briolant et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 25 May 2020
                : 23 July 2020
                Page count
                Figures: 4, Tables: 1, Pages: 16
                Funding
                Funded by: DGA
                Award ID: PDH-2-NRBC-2-B-2113
                This work has been supported by the Délégation Générale pour l’Armement (DGA, MoSIS project, Grant no PDH-2-NRBC-2-B-2113).
                Categories
                Research Article
                Medicine and Health Sciences
                Medical Conditions
                Infectious Diseases
                Disease Vectors
                Insect Vectors
                Mosquitoes
                Biology and Life Sciences
                Species Interactions
                Disease Vectors
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                Biology and Life Sciences
                Zoology
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                Physical Sciences
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                Mass Spectrometry
                Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry
                Research and Analysis Methods
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                Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry
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