title: Accurate identification of Culicidae at aquatic developmental stages by MALDI-TOF MS profiling.
abstract: BACKGROUND
  The identification of mosquito vectors is generally based on morphological criteria, but for aquatic stages, morphological characteristics may be missing, leading to incomplete or incorrect identification. The high cost of molecular biology techniques requires the development of an alternative strategy. In the last decade, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) profiling has proved to be efficient for arthropod identification at the species level.
  
  
  METHODS
  To investigate the usefulness of MALDI-TOF MS for the identification of mosquitoes at aquatic stages, optimizations of sample preparation, diet, body parts and storage conditions were tested. Protein extracts of whole specimens from second larval stage to pupae were selected for the creation of a reference spectra database. The database included a total of 95 laboratory-reared specimens of 6 mosquito species, including Anopheles gambiae (S form), Anopheles coluzzi (M form), Culex pipiens pipiens, Culex pipiens molestus, Aedes aegypti and 2 colonies of Aedes albopictus.
  
  
  RESULTS
  The present study revealed that whole specimens at aquatic stages produced reproducible and singular spectra according to the mosquito species. Moreover, MS protein profiles appeared weakly affected by the diet provided. Despite the low diversity of some MS profiles, notably for cryptic species, clustering analyses correctly classified all specimens tested at the species level followed by the clustering of early vs. late aquatic developmental stages. Discriminant mass peaks were recorded for the 6 mosquito species analyzed at larval stage 3 and the pupal stage. Querying against the reference spectra database of 149 new specimens at different aquatic stages from the 6 mosquito species revealed that 147 specimens were correctly identified at the species level and that early and late developmental stages were also distinguished.
  
  
  CONCLUSIONS
  The present work highlights that MALDI-TOF MS profiling may be useful for the rapid and reliable identification of mosquito species at aquatic stages. With this proteomic tool, it becomes now conceivable to survey mosquito breeding sites prior to the mosquitoes' emergence and to adapt anti-vectorial measures according to the mosquito fauna detected.
authors.affiliation: Aix Marseille Université, Unité de Recherche en Maladies Infectieuses et Tropicales Emergentes (URMITE), UM63, CNRS 7278, IRD 198 (Dakar, Sénégal), Inserm 1095, WHO collaborative center for rickettsioses and other arthropod borne bacterial diseases, Faculté de Médecine, 27 bd Jean Moulin, 13385, Marseille cedex 5, France. caiusdieme@yahoo.fr.|Laboratoire d'Ecologie Vectorielle et Parasitaire, Université Cheikh Anta Diop de Dakar, Dakar, Senegal. caiusdieme@yahoo.fr.
authors.forename: Constentin
authors.initials: C
authors.lastname: Dieme
authors.affiliation: Aix Marseille Université, Unité de Recherche en Maladies Infectieuses et Tropicales Emergentes (URMITE), UM63, CNRS 7278, IRD 198 (Dakar, Sénégal), Inserm 1095, WHO collaborative center for rickettsioses and other arthropod borne bacterial diseases, Faculté de Médecine, 27 bd Jean Moulin, 13385, Marseille cedex 5, France. yssoufamina@yahoo.fr.
authors.forename: Amina
authors.initials: A
authors.lastname: Yssouf
authors.affiliation: Department of Virology, Institut Pasteur, Arboviruses and Insect Vectors, Paris, France. avega@pasteur.fr.
authors.forename: Anubis
authors.initials: A
authors.lastname: Vega-Rúa
authors.affiliation: Aix Marseille Université, Unité de Recherche en Maladies Infectieuses et Tropicales Emergentes (URMITE), UM63, CNRS 7278, IRD 198 (Dakar, Sénégal), Inserm 1095, WHO collaborative center for rickettsioses and other arthropod borne bacterial diseases, Faculté de Médecine, 27 bd Jean Moulin, 13385, Marseille cedex 5, France. jmberenger@free.fr.
authors.forename: Jean-Michel
authors.initials: JM
authors.lastname: Berenger
authors.affiliation: Department of Virology, Institut Pasteur, Arboviruses and Insect Vectors, Paris, France. anna-bella.failloux@pasteur.fr.
authors.forename: Anna-Bella
authors.initials: AB
authors.lastname: Failloux
authors.affiliation: Aix Marseille Université, Unité de Recherche en Maladies Infectieuses et Tropicales Emergentes (URMITE), UM63, CNRS 7278, IRD 198 (Dakar, Sénégal), Inserm 1095, WHO collaborative center for rickettsioses and other arthropod borne bacterial diseases, Faculté de Médecine, 27 bd Jean Moulin, 13385, Marseille cedex 5, France. didier.raoult@gmail.com.
authors.forename: Didier
authors.initials: D
authors.lastname: Raoult
authors.affiliation: Aix Marseille Université, Unité de Recherche en Maladies Infectieuses et Tropicales Emergentes (URMITE), UM63, CNRS 7278, IRD 198 (Dakar, Sénégal), Inserm 1095, WHO collaborative center for rickettsioses and other arthropod borne bacterial diseases, Faculté de Médecine, 27 bd Jean Moulin, 13385, Marseille cedex 5, France. philippe.parola@univ-amu.fr.
authors.forename: Philippe
authors.initials: P
authors.lastname: Parola
authors.affiliation: Aix Marseille Université, Unité de Recherche en Maladies Infectieuses et Tropicales Emergentes (URMITE), UM63, CNRS 7278, IRD 198 (Dakar, Sénégal), Inserm 1095, WHO collaborative center for rickettsioses and other arthropod borne bacterial diseases, Faculté de Médecine, 27 bd Jean Moulin, 13385, Marseille cedex 5, France. almeras.lionel@gmail.com.
authors.forename: Lionel
authors.initials: L
authors.lastname: Almeras
mesh_terms: D000818:Animals; D015415:Biomarkers; D009033:Culicidae / Q000737:chemistry* / Q000145:classification*; D007814:Larva / Q000737:chemistry / Q000145:classification; D011679:Pupa / Q000737:chemistry / Q000145:classification; D013045:Species Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D015415:Biomarkers