Species Diversity And DNA Barcodes Of Culicoides In Hainan Island

Culicoides belongs to the family Ceratopogonidae of Diptera,is one genus with great medical meanings.All species of Culicoides can bite human and animals,and some species are vectors of many diseases.However,several factors such as the small body size,high species abundance,difficulty for slides specimen preparation and the existence of species complex,make identification of Culicoides very difficult.The technique which can identify Culicoides quickly and accurately will promote the progress of taxonomy and their control.Hainan island,locating in the tropical area of China,has high species diversity of Culicoides,and serious damages to animals and people's health were brought by Culicoides.Based on the field investigation,our research studied the morphology,species composition of Culicoides from different habitats.Besides,DNA barcodes were also sequenced from the species we collected.The main contents and results are as follows:1.Different methods including nets,Malaise traps and light traps were used to collect the specimens from 8 cities of the island.Species were identified based on morphological characters after slide specimens were prepared.Forty species from 7 subgenera and 1 addatinal species could not be arrange to any subgenera were totally identified,including one new record species of China,and four new records species of Hainan Island.A key to sub-genus were given,and images of the wing of 36 species were illustrated.2.Species composition of the Culicoides from three different habitats including a chicken farm Wenchang City,a pig farm from Qionghai City and mangroves forest of Wenchang Bamenwan,were studied by light trap.Ten and thirteen Culicoides species were separated collected from chicken and pig farm,and C.arakawai is the dominant species in both habitats.The total number of individuals and the number of dominant species were consistent with the trend of temperature.C.flavipunctatus is the dominant species of the mangroves in Wenchang,and still abundant in January.3.The cytochrome oxidase I gene(COI)was selected as marker for DNA barcode.COI from a total of 172 individuals of 41 species of Culicoides were sequenced.All sequences were aligned with GenBank and barcoding of life database(BOLD).Seventy-one sequences of 22 species can be identified with similarity over 98%,consist with the identification results based on morphological characters.In addition,the DNA barcodes of 19 species were first sequenced in this study and the DNA barcode database of common species of Culicoides in Hainan Island was constructed.4.Base constitution of the COI fragment we sequenced were analyzed from 45 individuals of 15 species of 6 subgenera.The content of bases A+T(65.2%)was much higher than the content bases of C+G(34.8%),showing significant AT bias which was consistent with mitochondrial DNA of insect.The interspecific genetic distance is 37 times of the intraspecific genetic distance(0.301/0.008),much larger than the rules for separating species which is 10 times in animals..This results confirmed COI can be used as DNA barcodes to identify species of the genus Culicoides.5.A phylogenetic tree was constructed by Neighbor-Joining method for 15 species of 6 subgenera.Individuals of the same species were well clustered into 1 clade,reflect DNA bacodes could be used for species identification of Culicoides.The results of this study increased the species number of Culicoides of Hainan Island,enriched BOLD of Culicoides DNA barcode,and initially analyzed the species composition of Culicoides of different habitats of Hainan Island.We provided a tool kit for identifying Culicoides much more quickly and accurately based on our images of wing patterns and DNA barcodes we provided.Our results is helpful to discover the diversity of Culicoides,and their control which is significant for ensuring animal husbandry production and human health.