DNA barcodes corroborating identification of mosquito species and multiplex real-time PCR differentiating Culex pipiens complex and Culex torrentium in Iran

Identifying mosquito species is a fundamental step in risk assessment and implementation of preventative strategies. Moreover, Culex pipiens is the most widespread mosquito vector in several regions of Iran and is the main vector for transmission of West Nile virus (WNV). Mosquitoes were collected at 14 sites in northern regions of Iran in 2015 and 2016. A subset of mosquito specimens was selected for identification confirmation using a DNA-barcoding technique. Construction of a phylogenetic tree showed clustering of mosquito sequences into three main genera: Aedes, Anopheles and Culex with individuals of a single species clustered closely together, regardless of where and when they were collected. Cx. pipiens complex and Cx. torrentium were identified and differentiated using multiplex real-time PCR targeting the gene locus for acetylcholinesterase 2 (ace2) to discriminate between Cx. pipiens pipiens and Cx. torrentium. The CQ11 microsatellite locus was used for discrimination between Cpp. biotypes. The predominant mosquito species in investigated regions were Cx. pipiens pipiens biotype pipiens, but we also detected Culex pipiens pipiens biotype molestus and hybrids of the two pipiens biotypes, as well as Cx. torrentium. The results of this study represent the first certain evidence of the presence of Cx. pipiens pipiens biotype molestus and hybrids between pipiens and molestus forms, and Cx. torrentium in Iran through a molecular identification approach. This report of a potentially important bridge vector for WNV might have key influence in the risk projections for WNV in Iran.