title: Mutual exclusion of Asaia and Wolbachia in the reproductive organs of mosquito vectors.
abstract: BACKGROUND
  Wolbachia is a group of intracellular maternally inherited bacteria infecting a high number of arthropod species. Their presence in different mosquito species has been largely described, but Aedes aegypti, the main vector of Dengue virus, has never been found naturally infected by Wolbachia. Similarly, malaria vectors and other anophelines are normally negative to Wolbachia, with the exception of an African population where these bacteria have recently been detected. Asaia is an acetic acid bacterium stably associated with several mosquito species, found as a dominant microorganism of the mosquito microbiota. Asaia has been described in gut, salivary glands and in reproductive organs of adult mosquitoes in Ae. aegypti and in anophelines. It has recently been shown that Asaia may impede vertical transmission of Wolbachia in Anopheles mosquitoes. Here we present an experimental study, aimed at determining whether there is a negative interference between Asaia and Wolbachia, for the gonad niche in mosquitoes.
  
  
  METHODS
  Different methods (PCR and qPCR, monoclonal antibody staining and FISH) have been used to address the question of the co-localization and the relative presence/abundance of the two symbionts. PCR and qPCR were performed to qualitatively and quantitatively verify the distribution of Asaia and Wolbachia in different mosquito species/organs. Monoclonal antibody staining and FISH were performed to localize the symbionts in different mosquito species.
  
  
  RESULTS
  Here we provide evidence that, in Anopheles and in other mosquitoes, there is a reciprocal negative interference between Asaia and Wolbachia symbionts, in terms of the colonization of the gonads. In particular, we have shown that in some mosquito species the presence of one of the symbionts prevented the establishment of the second, while in other systems the symbionts were co-localized, although at reduced densities.
  
  
  CONCLUSIONS
  A mutual exclusion or a competition between Asaia and Wolbachia may contribute to explain the inability of Wolbachia to colonize the female reproductive organs of anophelines, inhibiting its vertical transmission and explaining the absence of Wolbachia infection in Ae. aegypti and in the majority of natural populations of Anopheles mosquitoes.
authors.affiliation: Scuola di Bioscienze e Medicina Veterinaria, Università di Camerino, 62032, Camerino, MC, Italy. paolo.rossi@unicam.it.
authors.forename: Paolo
authors.initials: P
authors.lastname: Rossi
authors.affiliation: Scuola di Bioscienze e Medicina Veterinaria, Università di Camerino, 62032, Camerino, MC, Italy. irene.ricci@unicam.it.
authors.forename: Irene
authors.initials: I
authors.lastname: Ricci
authors.affiliation: Scuola di Bioscienze e Medicina Veterinaria, Università di Camerino, 62032, Camerino, MC, Italy. alessia.cappelli@unicam.it.
authors.forename: Alessia
authors.initials: A
authors.lastname: Cappelli
authors.affiliation: Scuola di Bioscienze e Medicina Veterinaria, Università di Camerino, 62032, Camerino, MC, Italy. claudia.damiani@unicam.it.
authors.forename: Claudia
authors.initials: C
authors.lastname: Damiani
authors.affiliation: Scuola di Bioscienze e Medicina Veterinaria, Università di Camerino, 62032, Camerino, MC, Italy. ulisse.ulissi@unicam.it.
authors.forename: Ulisse
authors.initials: U
authors.lastname: Ulissi
authors.affiliation: Scuola di Bioscienze e Medicina Veterinaria, Università di Camerino, 62032, Camerino, MC, Italy. mariavittoria.mancini@unicam.it.
authors.forename: Maria Vittoria
authors.initials: MV
authors.lastname: Mancini
authors.affiliation: Scuola di Bioscienze e Medicina Veterinaria, Università di Camerino, 62032, Camerino, MC, Italy. matteo.valzano@unicam.it.
authors.forename: Matteo
authors.initials: M
authors.lastname: Valzano
authors.affiliation: Scuola di Bioscienze e Medicina Veterinaria, Università di Camerino, 62032, Camerino, MC, Italy. aida.capone@unicam.it.
authors.forename: Aida
authors.initials: A
authors.lastname: Capone
authors.affiliation: Dipartimento di Scienze Veterinarie e Sanità Pubblica, DIVET, Università degli Studi di Milano, Milan, Italy. sara.epis@guest.unimi.it.
authors.forename: Sara
authors.initials: S
authors.lastname: Epis
authors.affiliation: Dipartimento di Scienze per gli Alimenti la Nutrizione e l'Ambiente, DeFENS, Università degli Studi di Milano, Milan, Italy. elena.crotti@unimi.it.
authors.forename: Elena
authors.initials: E
authors.lastname: Crotti
authors.affiliation: Dipartimento di Scienze per gli Alimenti la Nutrizione e l'Ambiente, DeFENS, Università degli Studi di Milano, Milan, Italy. bessem.chouaia@unimi.it.
authors.forename: Bessem
authors.initials: B
authors.lastname: Chouaia
authors.affiliation: Scuola di Bioscienze e Medicina Veterinaria, Università di Camerino, 62032, Camerino, MC, Italy. patrizia.scuppa@unicam.it.
authors.forename: Patrizia
authors.initials: P
authors.lastname: Scuppa
authors.affiliation: Department of Microbiology and Molecular Genetic, Michigan State University, Michigan, USA. djoshi@msu.edu.
authors.forename: Deepak
authors.initials: D
authors.lastname: Joshi
authors.affiliation: Department of Microbiology and Molecular Genetic, Michigan State University, Michigan, USA. xizy@msu.edu.
authors.forename: Zhiyong
authors.initials: Z
authors.lastname: Xi
authors.affiliation: Dipartimento di Scienze della Vita, Università di Modena e Reggio Emilia, Modena, Italy. mauro.mandrioli@unimore.it.
authors.forename: Mauro
authors.initials: M
authors.lastname: Mandrioli
authors.affiliation: Dipartimento di Biologia e Biotecnologie "L. Spallanzani", Università di Pavia, Pavia, Italy. luciano.sacchi@unipv.it.
authors.forename: Luciano
authors.initials: L
authors.lastname: Sacchi
authors.affiliation: School of Biological Sciences, Monash University, Clayton, VIC, Australia. scott.oneill@monash.edu.
authors.forename: Scott L
authors.initials: SL
authors.lastname: O'Neill
authors.affiliation: Scuola di Bioscienze e Medicina Veterinaria, Università di Camerino, 62032, Camerino, MC, Italy. guido.favia@unicam.it.
authors.forename: Guido
authors.initials: G
authors.lastname: Favia
mesh_terms: D000330:Aedes / Q000382:microbiology*; D020561:Alphaproteobacteria / Q000302:isolation & purification*; D000818:Animals; D000852:Anopheles / Q000382:microbiology*; D005260:Female; D041981:Gastrointestinal Tract / Q000382:microbiology; D006066:Gonads / Q000382:microbiology*; D008297:Male; D020577:Wolbachia / Q000302:isolation & purification*