Genetic Identification and Phylogenetic Relationship of Culex pipiens Bioforms and Culext torrentium In Southern Sweden using Mitochondrion and Nuclear DNA Markers

Abstract

Culex (Cx.) pipiens bioforms and Cx. torrentium are vectors of pathogens known to transmit diseases such as West Nile virus in human populations. Traditional classification has remained a challenge in their identification despite observed ecophysiological differences. Being morphologically indistinguishable, molecular methods have proven reliable for their accurate identification. However, not all molecular methods have been explored to determine the most reliable marker for the identification of the bioforms and Cx. torrentium, notwithstanding the evidence of their circulation in Sweden. In this study, mosquito samples were captured in diverse regions (urban, periurban and rural areas) of Southern Sweden in order to genetically identify and determine the geographical distribution of Cx. pipiens bioforms and Cx. torrentium. This was achieved using mitochondrion and nuclear molecular markers including: the mitochondrial cytochrome C oxidase subunit I (CO1) barcode gene region, the CQ11 microsatellite gene and the CPIJ001674 gene. A total of one hundred and eleven (111) mosquitoes were dissected individually and their respective legs removed, homogenized, and subjected to conventional PCR and sequencing. Amplified Fragment Length Polymorphism (AFLP) was used to distinguish Cx. pipiens bioforms and Cx. torrentium based on the CQ11 microsatellite gene. Furthermore, a similar approach targeting the CQ11 microsatellite gene was used in identifying hybrids in one southern site. The COI barcode gene was able to distinguish Cx. pipiens and Cx. torrentium to species level while the CPIJ001674 gene was not a reliable marker for the distinction of the two bioforms and Cx. torrentium in Sweden. Phylogenetic results also revealed Cx. torrentium and Cx. pipiens bioforms clustered separately using the COI barcode and CQ11 genes, but not with the CPIJ001674 gene. The findings further confirmed that the CQ11 microsatellite was a valid diagnostic marker for the distinction of Cx. pipiens bioforms and Cx. torrentium. These findings may provide new insights into the ecology of the Cx. mosquito species since the established molecular markers will serve as proxies for their observed ecophysiological traits. Consequently, aid in virus surveillance against arboviral diseases such as West Nile fever virus, whose key vectors are Cx. mosquitoes.