Microbial Flora Analysis Of Ticks And Fleas Parasitised By Marmota Himalayana

Objective:The purpose of this study was to explore the microbiota community of ticks and fleas collected from Marmota Himalayana in the Qinghai-Tibet Plateau at species-level,aiming to discover tick-borne and flea-borne pathogens or potentially new pathogenic bacterial species and provide etiological information for vector infectious disease prevention and control.Methods:In this study,we analyzed the microbiota community and diversity of each eight pools of ticks and fleas using full-length 16 S r RNA gene amplification sequencing combined with operational phylogenetic units(OPUs)analysis.And we performed deep analysis using metagenomic assembly and binning.Phylogenetic analysis was then performed to further determine the taxonomic position of potential pathogens.Results:(1)The Pac Bio sequencing platform rendered a total of 110,760 and 70,502 highquality reads in respective ticks and fleas,then divided into 1,250 and 689 operational phylogenetic units(OPUs)respectively.The 1250 OPUs in tick samples contained 556 known species and 493 new species,accounting for 48.50% and 41.71% of the total reads,respectively.The 689 OPUs in fleas consisted of 277 known species and 294 new species,accounting for 40.62% and 56.88% of the total sequences,respectively.The Alpha and Beta diversity analyses showed that ticks and fleas had different flora compositions,and ticks harbored a more diverse microbial community than fleas.The core flora of ticks and fleas were Staphylococcus equis and environment-related bacteria belonging to Sphingosinomonas and Staphylococcus.In addition,the known vector-borne pathogen Anaplasma phagocytophilum and the potentially pathogenic Wolbachia,Ehrlichia,Rickettsia,and Bartonella were found in the dominant taxa of ticks and fleas.(2)By phylogenetic analyses based on 16 S r RNA gene sequences,the OPU 421 Anaplasma phagocytophilum identified in ticks formed an independent cluster affiliated with pathogenic Anaplasma phagocytophilum with an average 99.72% of 16 S r RNA gene sequence similarity,possibly representing a known species A.phagocytophilum.The phylogenetic tree of full-length 16 S r RNA gene sequences revealed that the OPU 422 Ehrlichia sp1 identified in ticks was most close to Ehrlichia muris and Ehrlichia muris subsp.eauclairensis with a maximum 16 S r RNA gene sequence similarity of98.4%.Thus OPU 422 could represent a novel species belonging to genus Ehrlichia.The OPU 230 Bartonella sp1 identified in fleas was placed in a clade with Bartonella rochalimae,with an average identity of 98.40±0.01%,indicating OPU 230 could represent a novel species belonging to genus Bartonella.The representative sequences of OPU 427 Rickettsia sp1 in fleas was shown to be clustered with Rickettsia limoniae with an average identity of 97.23±0.025 %,possibly representing a novel species belonging to Rickettsia.(3)A total of 10 metagenomic assembly genomes(MAGs)were obtained from ticks and fleas by metagenomic assembly and binning,including a known species,Providencia heimbachae DFT2,and six novel species belonging to Wolbachia,Bartonella,Anaplasma and Mumia.The Providencia heimbachae is a phytopathogen,with the genome completeness 100%(the contamination was 0%).The phylogenomic analyses based on core genes showed that Anaplasma sp.DTF10 in ticks formed a branch with Anaplasma phagocytophilum,and the two MAGs of Bartonella spp.(DTF8 and DTF9)clustered with B.rochalimae,which was consistent with the phylogenetic analyses of 16 S r RNA gene sequences.Conclusion:The present study has comprehensively described the microbiota community of ticks and fleas collected from Marmota Himalayana in the Qinghai Tibet Plateau at specieslevel for the first time by using metataxonomic analysis based on full-length 16 S r RNA gene amplification sequencing.The ticks and fleas had different flora compositions,and ticks harbored a more diverse microbial community than fleas.The core flora of ticks and fleas were Staphylococcus equis and environment-related bacteria belonging to Sphingosinomonas and Staphylococcus.Notably,the known vector-borne pathogen Anaplasma phagocytophilum and the potentially pathogenic Ehrlichia,Rickettsia,and Bartonella were found in ticks and fleas,which providing etiological information for vector infectious disease prevention and control.