Sequence analysis of mtDNA COI barcode region revealed three haplotypes within Culex pipiens assemblage

Members of the Culex (Culex) pipiens assemblage are known vectors of deadly encephalitides, periodic filariasis, and West Nile virus throughout the world. However, members of this assemblage are morphologically indistinguishable or hard to distinguish and play distinct roles in transmission of the diseases. The current study aimed to provide further evidence on utility of the two most popular nuclear (ITS2-rDNA) and mitochondrial (COI barcode region) genetic markers to identify members of the assemblage. Culex pipiens assemblage specimens from different climate zones of Iran were collected and identified to species level based on morphological characteristics. Nucleotide sequences of the loci for the specimens plus available data in the GenBank were analyzed to find species specific genetic structures useful for diagnosis purposes. ITS2 region was highly divergent within species or populations suggesting lack of consistency as a reliable molecular marker. In contrast, sequence analysis of 710 bp of COI gene revealed three fixed haplotypes named here "C, T, H" within the assemblage which can be distinguished by HaeIII and AluI enzymes. There were a correlation between the haplotypes and the world climate regions, where the haplotypes H/T and C are present mainly in temperate and tropical regions of the world, respectively. In the New world, Australia, and Japan only haplotype H is found. In conjunction between tropical and temperate regions such Iran, China, and Turkey, a mix of C/H or C/H/T are present. Although, the haplotypes are not strictly species-specific, however, Cx. quinquefasciatus was mainly of haplotype C. Due to the lack of mating barrier and questionable taxonomic situation of the complex members, the mentioned haplotypes in combination with other morphological and molecular characters might be used to address the genetic structure of the studied populations.