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

Abstract

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.

Autochthonous Transmission of West Nile Virus by a New Vector in Iran, Vector-Host Interaction Modeling and Virulence Gene Determinants

“…biotypes for individual (blood-fed) Cx. pipiens specimens based on protocols described previously [ 36 ].…”

Morphological and molecular identification of Culicidae mosquitoes (Diptera: Culicidae) in Lorestan province, Western Iran

“…Multiplex real-time PCR was performed in a 20 mL reaction volume using HotStarTaq Master Mix Kit (Qiagen). Real-time PCR was performed with an initial denaturation step 95 °C (15 min), followed by 50 cycles denaturation at 95 °C (15 s), annealing at 60 °C (20 s), extension at 72 °C (30 s), and a final elongation at 40 °C (30 s) ( Shahhosseini et al., 2018b ).…”

“…Infection of Culicidae mosquitoes to arboviruses, especially west Nile virus, in other studies have been investigated in Western Iran (Shahhosseini et al, 2016b(Shahhosseini et al, , 2017a(Shahhosseini et al, , 2017b(Shahhosseini et al, , 2018bShahhosseini and Chinikar, 2016a;Chinikar et al, 2013aChinikar et al, , 2013bMeshkat et al, 2015;Shah-Hosseini et al, 2014). There are few studies related to fauna, ecology, biology, resting and blood feeding behaviors of Culicidae mosquitoes in Lorestan province, Western Iran.…”

From qualitative to quantitative insect metabarcoding: an in tandem multilocus mosquito identification methodology

“…DNA barcoding is a taxonomic molecular method, usually implicating the cytochrome oxidase subunit I (COI) gene for animal species discrimination 4 . In cases where COI is not sufficient, other taxonomically important genomic loci are used as 12S mtDNA, 16S mtDNA, Cytochrome B ITS, 18S and 28S rDNA, EF-1a and NADH [5][6][7][8][9][10] . DNA metabarcoding is a recently developed molecular approach that engages next-generation sequencing (NGS) into a high-throughput identification of species in a mixed population 11 .…”