Vol. 42, no. 2 Journal of Vector Ecology 349 Scientifc Note First report of the endobacteria Wolbachia in natural populations of Culex pipiens in Morocco F.Z. Tmimi 1,2 , M. Bkhache 1 , K. Mounaji 2 , A.B. Failloux 3 , and M. Sarih 1* 1 Institut Pasteur du Maroc, Service de Parasitologie et des Maladies Vectorielles, Place Louis Pasteur, Casablanca 20360, Morocco, mhammed.sarih@pasteur.ma 2 Faculté des Sciences Aïn Chock, Laboratoire de Physiopathologie, Biologie Moléculaire et Biotechnologie, Université Hassan II de Casablanca, Morocco 3 Institut Pasteur, Department of Virology, Arboviruses and Insect Vectors, 25-28 rue du Docteur Roux, Paris 75724, France Widely spread throughout North Africa, members of the Culex pipiens complex are competent vectors of pathogens such as West Nile Virus (WNV) (Krida et al. 2011, Amraoui et al. 2012) and Rif Valley Fever Virus (RVFV) (Moutailler et al. 2008) that afect humans and/or other animals. Culex pipiens includes two forms, pipiens and molestus, which are morphologically identical but genetically diferent. Culex pipiens mosquitoes are naturally infected by Wolbachia, which is an endosymbiotic bacterium able to generate multiple efects in its host, such as cytoplasmic incompatibility (Duron et al. 2006, Yen and Barr 1971). Recently, Wolbachia has attracted much attention as a promising tool to control diseases transmitted by mosquitoes (Atyame et al. 2011, Joubert and O’Neill 2017, Joubert et al. 2016). In Cx. pipiens, this endosymbiotic bacterium is subdivided into fve subclades referred as wPip-I to wPip-V groups (Atyame et al. 2011, Dumas et al. 2013). wPip-I, the most dominant, was found in Sub-Saharan Africa, South America, and Southeast Asia; wPip-II was found in western Europe; and wPip-III was found in North America and Europe (Dumas et al. 2013). wPip-IV was found in Europe, Asia, and north Africa and wPip-V was found in Asia (Atyame et al. 2011, Duron et al. 2006). It has been reported recently that Cx. pipiens populations from Algeria were infected with wPip 31 (wPip-IV group). A Cx. pipiens population from Tunisia was infected with wPip 11 (wPip-I group) in the east and south, whereas regions in the north and west were infected by wPip 31 (wPip-IV group) (Atyame et al. 2011, Duron et al. 2006). In Morocco, no data were available about these bacteria. Here we studied, for the frst time, the diversity of wPip in natural populations of two forms of Cx. pipiens (pipiens and molestus forms) using the two ANK Wolbachia markers ank2 and pk1 in the northern, central, and southern parts of Morocco. Cx. pipiens were collected as larvae using dipping nets from three bioclimatic regions of Morocco. Tangier located in northern Morocco (humid), Casablanca in the west (semi-arid), and Marrakech in the south (arid), at various sites, urban and rural (Figure 1). Te larvae were morphologically identifed using sofware (Brunhes et al. 2000). All specimens were frozen at −20° C until tested. DNA of individual mosquitoes was extracted using DNAzol solution under conditions recommended by the manufacturer. Te mosquitoes were identifed using the multiplex PCR described by Bahnck and Fonseca (2006). For each specimen identifed, the wPip type was determined using PCR-RFLP (Restriction Fragment Length Polymorphism) assays based on two ANK Wolbachia markers ank2 and pk1; all PCR amplifcation conditions were described by Dumas et al. 2013. Digestion of PCR products was performed afer incubation at 37° C according to manufacturer’s instructions. Te DNA fragments were separated by agarose gel (2%) electrophoresis. Te ank2 PCR products digested by the restriction enzyme HinfI allowed the diferentiation of three alleles: type-I (one RFLP fragment: 313 bp), type-IV (293 and 217 bp), and type-V (217 and 195 bp). While pk1 products were digested by two diferent enzymes, TaqI also resulting in the same alleles: I and V with the same fragment size (991, 251, and 107 bp) and III (497, 251, and 107 bp) when the digestion with PstI gives: I (903, 303, 141bp) and V (903, 430bp), while type IV is not digested by PstI. We tested the diversity of Wolbachia in 459 specimens from natural populations of Cx. pipiens in three cities. In Tangier, 155 individuals were tested, 203 in Casablanca, and 101 in Marrakech. Figure 1. Location of the collection sites in Morocco.