The Study Of DNA Barcoding Technology Of Mosquitoes Of The Genus Culex Based On COI Gene

Mosquitoes of the genus Culex (Diptera:Culicidea) are distributed worldwide. Up to now,82 species from China have been reported, which are classified into 9 subgenus. Many species in the genus are important disease vectors. For mastering and control mosquito-born diseases,the identification and classification is very important. The method of morphology is common but there are some limitations for morphological classification. In this case, we need urgently a fast, precise, automated and global tool for classification and identification.Mitochondrial cytochrome C oxidase I subunits gene (mtDNA COI) owns the most data information in gene database,whose structure and evolutionary dynamics have been clearly understood. The structure is relatively conservative and interspecific variation. In the research of phylogeny of closely related species, the structure can provide lots of phylogenetic information. DNA barcoding based on the mtDNA COI gene is simple and efficient.Twenty-two mosquitoes species of the genus Culex were collected which have been identified with the method of morphology. Then mitochondrial DNA was extracted from 73 Culex mosquitoes and used for COI amplification, cloning and sequencing.73 COI sequences of Culex were obtained by BLAST analysis. Sequence characteristics were further analyzed. Twenty-two of seventy-three sequences combined with 16 Culex COI sequences retrieved from GenBank were used for phylogenic analysis. A Neighbor-Joining (NJ) tree was constructed by MEGA5.1 using Chironomus nepeanensis as the outgroup. Then, genetic relationship and taxonomic status of the genus Culex in China were inferred based on the phylogenetic tree. Moreover, it is discussed whether the method of DNA barcoding is feasible in identification of mosquitoes species of Culex.The results of the research as follows:(1) The nucleotide composition,codon frequences and amino acids compositon of mtDNA COI gene, there are some bias.(2) 100% bintraspecific distance of COI gene of mosquito is less than 2%, and interspecific distance is more than 2% except some mosquitoe species.(3) We found maybe there are cryptic species in Cx.modestus. Culex Subgenus Barraudius belong to Culex Subgenus Culex with the COI molecular markers.Culex mimulus and Culex murrelli are probably the different names of Culex orientalis.(4) Morphological taxonomy results were further confirmed by mtDNA COI sequencing and analysis. Sequence and phylogenic analysis were in accordance with morphological and molecular taxonomy.(5) The phylogenetic tree based on 38 species of mtDNA COI showed that:eight subgenus were better distinguish; the Pipiens group and the Sitiens group are claded to a branch. The subgenus except Subgenus Barraudius and Subgenus Neoculex can gather closely which have good monophyly. Culex okinawae group should be promoted to the Subgenus Eumelanomyia. The result also revealed that Culex territens is a single species. The reason may be due to the members of this subgenus in this study are less,so this subgenus evolutionary needs further study.