Screening Of Disease Agents And Host Species Of Black Flies In Guizhou Province

Objective: To identify blackfly species in Guizhou Province with combined data of DNA barcodes, 5′ region of mitochondrial cytochrome c oxidase I gene(COI), and morphological characteristics. To screen disease agents with specific regions of cytochrome b gene(Cytb) and hepatitis B virus DNA as genetic markers,and to determine host species through sequences of Cytb gene. Methods: Samples of black flies were collected with a vehicle-mounted trap, from July to September, 2014,collecting sites as Leishan, Rongjiang, Sansui, Jiangkou, Dejiang, Guanling, Kaiyang counties in Guizhou Province, with two collection sites each county. Collected female black flies were fixed with 95% alcohole and then identified under a stereo-microscope, genitalia were dissected off separately and mounted on slides as vouchers. Genomic DNA was extracted, followed by amplification and sequencing for COI gene. Black flies species were identified by comprehensive data of Barcode of Life Data Systems(BOLD), phylogenetic analysis and morphology. Disease agent species were identified by GenBank blasting with sequences of Cytb and HBV DNA.Host vertebrate and avian species were determined by blasting Cytb sequences,obtained by sequencing the PCR products for amplifying Cytb, in GenBank. Results:The total counts of specimens collected were 49,622, all belonged to order Insecta except 1 individual of order Entotrophi. Insecta specimens fall into 8 orders and 145 families, including 38 families of Coleoptera, 37 of Diptera, 30 of Lepidoptera, 29 of Hymenoptera, 7 of Hemiptera, 2 of Odonata, 1 of Plecoptera, and 1 of Neuroptera.Among 702 black flies collected, 531 individuals were female and 171 male. Five hundred and twenty one of 531 female were identified as 23 species of Simulium,including S. asakoae(153 individuals), S. quinquestriatum(156), S. bidentatum(97),S. chamlongi(9), S. ornatum(2), S. erythrocephala(1), and 17 undetermined species(designated as S. 1- S. 17, 103 individuals total). The value of 4% ofKimura-2-parameter distance was employed as a threshold for species diagnosis.Protozoan pathogens were not detected. HBV DNA sequences were detected in only one female black fly. Host Cytb DNA sequences were found in 4 female black flies,with one belonging to Homo sapiens(in a S. asakoae), another to Capra hircus(in a S.bidentatum), and others to an unknown avian species(in a S. bidentatum and an unknown Simulium species). Conclusion: Twenty three blackflies species were identified, including S. asakoae, a new species record of Guizhou Province. Protozoan pathogens were not founded in all 531 black fly individuals detected. HBV DNA sequences were detected in one S. quinquestriatum. A proof of H. sapiens, C. hircus and an avian species as host of Simuliidae in Guizhou was given. And S. asakoae, S.bidentatum, and an unknown species of Simulium were documented as blood feeding species.