DNA Barcoding For Sandfly Identification In China And Transcriptomic Analysis Of Phlebotomus Chinensis(Diptera: Psychodidae)

Sandfly is a small blood-sucking insects,belonging to the Family Psychodidae,widely distributed in China.More than 40 species of sandflies were recorded based on the morphological characteristics.Part of them can transmit sandfly fever,leishmaniasis and Bartonella disease,which has important medical significance.Therefore,it is necessary to establish objective distinguishing method,understand the seasonal fluctuation and bloodthirsty habits.The strategy of this project was following: Firstly,sandflies were collected from Henan,Sichuan and Hainan provinces in China and investigate the ecological habits of local sand flies.And the blood-sucking habits of the sandflies were analyzed by molecular asssay.The seasonal fluctuation was recorded by monitoring sandflies number of Hainan.Secondly,sandflies were identified according to the key of morphological characteristics,and mtDNACytb,COI and rDNA 18 S gene fragments corresponding to the samples were amplified analyzed.The intraspecifc and interspecific divergence was estimated,and compared with species by morphology.The mtDNA-Cytb,COI and rDNA-18 S for sandflies species identification as DNA barcoding were determined.Thirdly,the total mRNA of head and thoracic abdomen,and the female and male Phlebotomus chinensis were sequenced by highthroughput sequencing techniques,making annotation and differential expression level analysis.The results were as follows:1.There were differences between ecological habits of sandfly in Sichuan and Henan provinces,blood animals containing pigs,rabbits,chickens,dogs,cattle and human beings.Breeding sites of sandfly collected from the Sichuan was the cave and soil cave,near the habitat,from Henan was the cave and home.The main blood source of sandfly from Sichuan was pig,while Henan was chicken.Human and dog blood were detected in sample from both Sichuan and Henan,which higher proportion of human blood was 41.67% in Henan province.2.A total of 377 specimens were captured by light trap in Haikou City from Aug.2015 to Sept.2016.The double peaks were in June and November.3.The seven sandfly species were identified by morphological characteristics,including Ph.chinensis Newstead,Ph.stantoni Newstead,Sergentomyia bailyi Sinton,Se.iyengari Sinton,Sergentomyia koloshanensis Yao & Wu,Se.squamirostris Newstead,Se.squamipleuris.The mtDNA-COI,Cytb and r DNA-18S(excluding Se.koloshanensis)sequences were obtained.The results of showed that mtDNA-COI,Cytb and rDNA-18 S could be as DNA barcodes for identification of these sandfly species.4.There were some sibling species in Sergentomyia bailyi complex.The mtDNA-COI sequence of Se.bailyi in China is closely related to it in Thailand,which was different from species A and B.It was renamed as Se.bailyi C by molecular markers.5.A total of 133,835,342 reads were obtained by the high-throughput sequencing group.Further assembly of 32,628 unigene was obtained,which were known to be biologically annotated.Respectively,annotated to the Nr,Swiss-Prot,KEGG,COG and GO databases.A total of 4374 genes were screened and GO-rich analysis was performed.The expression of 10 genes was confirmed by RT-PCR and were consistent with the results of sequencing.The differential expression genes of the head and body,or male and female of Ph.chinensis were closely related to their function,and the difference genes between sexes,head and thoratic abdomen were also related to their sensation,feeding and reproductive function.