Analysis Of Microbial Diversity Of Mosquitoes And Culicoides On Zhoushan Island, China.

Objective:To analyze the composition and characteristics of microorganisms such as viruses,bacteria carried by blood-sucking mosquitoes and midges on Zhoushan Island,and to find out the similarity and uniqueness of different species of mosquitoes and midges.Search for potential new microorganisms,and provide scientific basis for the prevention and control strategies for mosquito-midge diseases on Zhoushan Island.Methods:From June to September 2021,mosquitoes and midges were collected in five habitats by light trapping.The Cytochrome c oxidase subunit I(COI)gene sequence was amplified and sequenced by Polymerase Chain Reaction(PCR),and the species of mosquitoes and midges were determined by morphological identification.The samples were divided into 28 groups according to the species,habitat and obvious blood-sucking of mosquitoes and midges,including 23 groups of mosquitoes and 5 groups of Culicoides.Ribonucleic Acid(RNA)was extracted from 28 groups of samples,and pathogen screening and mategenome sequencing were performed by PCR.Remove splices,low-quality and low-complexity reads,and use fastp software to generate high-quality genome sequencing data.Used Kraken2,Refseq and Non-Redundant Protein Sequence Database(NR)to identify microorganisms with reads which had removed the host.The Kraken2 software was used to annotate the reads of viruses,bacteria and protozoa in all samples.And the Bracken was used to calculate the relative abundance of bacteria and protozoa in the samples.MEGAHIT software was used to assemble reads to get contigs.Based on National Center for Biotechnology Information’s(NCBI)Nucleotide Sequence Database(NT)and NR,used BLAST software to annotate contigs.The"meta"model in Prodigal was used to predict viral protein,and the sequence with amino acid sequence<90%or nucleotide sequence<85%is set as a new sequence.We used the online tool CD-search to predict the conservative domain,and used R package ggplot2 and MEGA to visualize the results.The deep learning method Deep Vir Finder was used to explore the sequences that may be viruses in Dark matter(the sequences that cannot be compared with known species).Non-measured multidimensional scale analysis,displacement multivariate analysis of variance,nonparametric multivariate analysis of variance,Welch’s t test and other statistical methods were used to compare the differences of microbiota in different groups,and the random forest model was used to find the key bacterial species with differences between different species groups.Results:More than 16600 samples were collected on Zhoushan Island,and experts used morphology to identify Culicoides at the scene.The COI gene sequence was amplified by PCR and 311 mosquitoes were identified by sequencing sampling.A total of 8 species of mosquitoes and 3 species of Culicoides were identified.28 groups of samples were screened,and the PCR results of Flavivirus,Bunyavirus,Nairovirus,Alphavirus,Liaoning virus,Banna virus,Bluetongue virus and Rickettsia were negative.A total of 28 groups of samples were sequenced,and we found that the microbial composition of mosquitoes and midges were different.99%of the viral reads in Culicoides were Polycipiviridae,while the common viridae in the mosquito virus group were Ifluviridae(30.03%),Phasmaviridae(23.09%),Xinmoviridae(21.82%),Laviviridae(13.44%)and Rhabdoviridae(8.40%),and the high abundance of Amoebophilaceae bacteria in midges was 0.PERMANOVA analysis of species showed that the composition of virus groups carried by mosquitoes and midges in Zhoushan Island was related to the species(P=0.001,R~2=0.6129).Using Welch’s t test,we found that there are more unknown sequences annotated as viruses in Culicoides(P=0.046).Through phylogenetic analysis,we found the sequence of the suspected new virus in Culicoides,and named Culicoides Thimiri virus(4186bp),Culicoides Iflavus1(10785bp),and Culicoides narna-like virus(2485bp).The displacement multivariate analysis of variance showed that the bacterial group carried by mosquitoes and midges was mainly related to species(n=9,P=0.001),but not to habitat(n=5,P=0.957).Using the random forest model,it is found that Proteus is the key bacterial species with the greatest difference between different media groups,followed by Mycoplasma and Staphylococcus,with the importance of more than 1.25.The results of mosquito and midge sequencing showed that the reads sequences of Wolbacchia(5522869),Rickettsia(14690)and Plasmodium(352670215)were spliced into contigs.We tried to isolate and culture rickettsia,but the result was negative.In mosquito and midge samples,the sequences of Leishmania and Theileria transmitted by other blood-sucking arthropods,as well as the Encephalitozoon that exist in the environment and are opportunistically pathogens in the host were found.Conclusion:In this study,the composition of mosquitoes’and Culicoides’virus group and bacteria group in Zhoushan Island was mainly statistically relevant to the host species,and different host species have different core virus groups.Compared with mosquitoes,Culicoides may have more unknown sequences annotated as viruses in this study.The sequences of several suspected new viruses were found in Culicoides and Culex tritaeniorhynchus,providing research support for the prevention and control of diseases transmitted by mosquitoes and Culicoides in Zhoushan Island.Sequences of Wolbacchia,Rickettsia,Plasmodium and other arthropods were found in mosquito and midge samples,it is necessary to monitor the microbiome and related pathogens of the blood-sucking arthropods in this area.