Identification Of Mosquito Species And Diversity Analysis Of Their Midgut Microflora In Hainan Province

Objective:Identification of mosquito species collected in 8 cities and counties of Hainan,China.The species and content of midgut flora of each mosquito species were explored,and the composition and characteristics of the intestinal flora were analyzed as well.So as to provide a basic basis for the control of mosquitoes and mosquito-borne infectious diseases in Hainan,China..Methods:Adult mosquitoes were composed by lamp,human and cattle,and the mosquito species were identified by morphology and Cytochrome oxidase subunit 1(COI).Through the Illumina(Hiseq)sequencing platform and the V3-V4hypervariable region of bacterial 16S r RNA were used for high-throughput detection and identification of midgut microflora.Greengene and RDP databases were used for species comparison and annotation.According to the nucleic acid sequence of COI gene,the genetic evolution tree of mosquito species was established by Mega 7.0software,and the molecular origin was determined.The sequencing results were analyzed by Excel 2019 and SPSS 21.0.The Beta and Alpha diversity of microflora were analyzed by R language.Results:1.MK736660(India)and JQ235749(Yunnan)were also close to Ae.Albopictus in Hainan,while MT541282(DF)was a relatively independent population.The genetic relationship of the collected Armigeres subalbatus population was consistent with the physical distance of the sampling sites,and there was also a relatively close genetic relationship between,Culex pipiens pallpens and mosquito species in different parts of Hainan.2.A total of 1016 mosquitoes were collected,including Ae.albopictus,Ar.subalbatus and Cx.pipiens pallpens,of which 1421 OTUs were detected.They belonged to 27phyla,13 classes,25 orders,42 families and 41 genera,five phyla.Proteobacteria,Firmicutes,Bacteroidetes,Actinobacteria and Thermi were the most dominant.Interestingly,60.30%and 23.81%of the bacteria abundance in Ae.albopictus and Ar.subalbatus of Wuzhishan(WZS)are not identified at the genus level respectively,especially 99.14%in Cx.pipiens pallpens.It was also found that the infection rate of,Wolbachia in the midgut of Leong(LD)and WZS very low,accounting only(1.50%)and(0.01%),respectively.There was a notable divergent in the structure of most of the midgut flora among the samples(P<0.05).3.Wolbachia in the midgut of most Ae.albopictus existeds as the main genus in Hainan,and its infection rate in Dingan(DA),Sanya(SY),Lingshui(LS),Haikou(HK)were 84.67%,80.34%,68.77%and 56.89%separately,but the infection rate of LD and WZS were 1.50%and 0.01%separately.There was a statisticaly different in the proportion of Wolbachia abundance in the structure of midgut flora(?~2=322.950,P<0.001).The genetic distance suggested that there had exchange of strains in distinct district,and there were some differences in genes of the same region.Conclusion:Ae.albopictus,,Ar.subalbatus and Cx.pipiens pallpens were closely related to each other in Hainan.The exchange of mosquito species was the main,at the same time,there were also exchanges between mosquito species and other regions.Thermi has not been reported in other regions as the dominant bacteria phylum,it the most of the three major mosquito species of WZS have not been identified at the genus level,and there may be the possibility of a new genus.The composition structure of intestinal flora of mosquito species were similar to that of mosquito species in the same location,and the harmful environment had a great effect on the composition structure of midgut flora of mosquito species.Most of the core bacteria in the midgut of Ae.albopictus exist in Ar.subalbatus and Cx.pipiens pallpens,indicating that there may be an environment covering Ar.subalbatus and Cx.pipiens pallpens in the pathogenic environment of Ae.albopictus.Among the midgut of Ae.albopictus,Wolbachia was the dominant strain in most areas,but the infection rate was very low in LD and WZS.There was significant difference in the composition of Wolbachia in the midgut flora.Genetic distance showed that there was exchange of strains in various regions and genetic diversity of strains in various regions.