Taxonomic Study On Blood-sucking Culicoides Latreille(Diptera: Ceratopogonidae) From The Southwestern China

Objective: To investigate the species diversity of blood-sucking Culicoides in the Southwestern China and lay the foundation for rapid,accurate identification of blood-sucking midges.Methods: 1.The Culicoides species were collected using insect net capture,Malaise traps and light trap,slided by neutral balsam,identified by morphological characteristics.With consulting reference,sorting the list of Culicoides from the Southwestern China.2.Genomic DNA was extracted from the Culicoides.Mitochondrial cytochrome oxidase I(COⅠ)gene and Cytochrome b(Cytb)gene was amplified by consensus primer polymerase chain reaction(PCR)and sequenced.The phylogenetic relationships among Culicoides were reconstructed based on sequences of the COⅠ and Cytb genes using neighbor-joining method(NJ),maximum likelihood method(ML),maximum parsimony method(MP).Results: 1.There are 129 Culicoides species,belonging to 10 subgenera in the Southwestern China,and 63 species in Sichuan,22 species in Chongqing,41 species in Guizhou,89 species in Yunnan,17 new record species in these areas.2.The component analysis based on COⅠ and Cytb gene sequences show that the average contents of A+T to COⅠ and Cytb gene in Culicoides is 66.7% and 79.7%,higher than contents of C+G.Sequence alignment of COⅠ and Cytb gene results consistent with the results of morphological identification.The COⅠ sequences were submitted to the database of Genbank,similarity was very high with matching species.Estimate genetic distances of COⅠ and Cytb gene,and statistical analysis showed that intraspecific genetic distances of the COⅠ sequence was different from that of interspecies,subgenral and intergeneric genetic distances,respectively;Intraspecific,interspecific,subgenral,intergeneric genetic distances of Cytb sequence differed from each other.NJ,ML and MP methods are used to reconstruct the phylogenetic relationships separately based on the COI,NJ and MP methods used based on the Cytb.The phylogenetic relationship within Culicoides from such trees in this study indicates that the topologies of all the phylogenetic trees were quite similar;Distinguished between different species of Culicoides clearly;The different subgenus and species formed monophyletic groups respectively;The same species gathered closely with each other,no cross between different species;Further clarified the phylogenetic relationship of Culicoides combined with morphological features.Conclusion: 1.Enrich the species diversity and improve the geographical zoning of Culicoides in the southwest of China,provide the basic data for the control of the blood-sucking Culicoides and the diseases they carry in these areas.2.In this study,we verified the effectiveness of Mitochondrial DNA COⅠ and Cytb genes in species identification,distincyion and phylogenetic analysis of Culicoides(especially similar species),and laid the foundation for further development of more effective molecular markers.