| Ticks are specialized hematophagous ectoparasites,which not only elicit inflammation,anemia,and paralysis through the toxic effects of their blood-sucking and toxin-release mechanisms at the host’s bite site,but more significantly,they serve as vectors for numerous pathogens(including viruses,bacteria,rickettsiae,and protozoa).This aspect holds immense significance in the realms of veterinary medicine and public health.In Heilongjiang Province,the principal tick species encompass Ixodes persulcatus,Dermacentor silvarum,and Haemaphysalis conicinna.Among these,I.persulcatus reigns as the dominant tick species and primary vector for tick-borne diseases within the province.In recent years,the advent of high-throughput sequencing technology has underscored the pivotal role of vector arthropod microbiota.Distinctions and alterations in the microbiota can exert multifarious effects on vector organisms,encompassing modifications in vector competence triggered by immune system impacts and adaptations in reproduction driven by incubation-related influences.Concurrently,intratick bacteria may also represent potential pathogens for vertebrates.In order to comprehensively comprehend the composition and variances in microbiota across distinct tick species,genders,and feeding stages,and to acquire pertinent bacterial strains,as well as to discern immune-related alterations induced by microbiota changes,the ensuing experiments were conducted within this study.The objective was to obtain tick-borne bacteria via culturomics cultureomics,thereby furnishing a scientific foundation and material and technical reservoir for the detection,diagnosis,treatment,prevention,and control of associated diseases.Initially,free ticks and bloodsucking ticks were gathered from diverse regions within Heilongjiang Province.Following morphological and molecular biological identification,microbiome analysis was conducted via high-throughput sequencing on three distinct tick types,namely male and female I.persulcatus,D.silvarum,and H.conicinna,across various feeding stages.The objective was to elucidate the structure,compositional attributes,and alterations within the bacterial flora under divergent factors.Subsequently,employing a cultureomics approach,bacteria residing within the digestive tracts of ticks were isolated,cultured,and subjected to sequencing for taxonomic classification and pathogen annotation.Finally,through comparative genome analysis,insights into tick immune-related genes were collected,enabling an investigation into the level of immune-related expression and the phenotypic disparities arising from microbiome changes.The primary research findings are as follows:1.A total of 1953 free ticks and 254 blood-sucking ticks were collected from distinct regions within Heilongjiang Province.These ticks were subjected to both morphological and molecular identification,revealing their classification as I.persulcatus,D.silvarum and H.conicinna.Microbiome sequencing outcomes that encompass different tick species,genders and feeding stages unveiled notable variations in bacterial abundance and flora structure among the three tick species.Following blood satiation,the bacterial abundance(Chao 1 index)of all tick species exhibited a significant decrease.All operational taxonomic units(OTUs)were classified into 26 phyla,67 orders,159 families,279 genera,and 627 species.At the phylum level,Proteobacteria and Firmicutes emerged as the predominant phyla across all tick species.Furthermore,the primary commensal bacteria of the ticks were identified as Bacteroidetes,Actinobacteria and Acidobacteria.The relative abundance of the Proteobacteria phylum exhibited an increasing trend after tick satiation,while Firmicutes phylum displayed a decreasing trend.Among all tick samples,Pseudomonas were identified as characteristic genera of I.persulcatus,Rickettsia and Arsenophonus of D.silvarum,and Bacteroides of H.conicinna.A pairwise comparison of subordinate levels within different subgroups revealed significant alterations in Rickettsia members in both I.persulcatus and D.silvarum subsequent to blood saturation.2.By employing cultureomics techniques,a total of 426 bacterial strains were successfully isolated and cultured from the digestive tracts of ticks.These strains were distributed across 4phyla,4 orders,11 families,17 genera,and 74 species.At the phylum level,Bacilli and Gammaproteobacteria emerged as the dominant groups,constituting 46.01% and 52.35% of the isolated strains,respectively.At the family level,Staphylococcaceae,Enterococcaceae,and Morganellaceae exhibited prominence,accounting for 36.62%,15.49%,and 13.85% respectively.At the species level,the 426 isolates were classified into 74 distinct species,with Staphylococcus roterodami being the most prevalent,comprising 8.69% of the isolates.Evolutionary analysis,subsequent to clustering the 426 isolated bacteria,revealed the formation of four major branches among the different phyla.Notably,the Pseudomonas phylum exhibited the greatest evolutionary distance from the Bacteroidetes phylum,while demonstrating a closer relationship with the Firmicutes phylum.The Pseudomonas phylum,which accounted for the highest number of isolates,further exhibited the formation of six branches within its entirety,encompassing a total of eight families.Four of these families were classified under a single branch,while the remaining four families formed separate branches.To detect known animal and zoonotic pathogenic bacteria,the16 S r RNA gene sequencing technique was employed for the analysis of the 426 bacterial isolates,utilizing a multiple bacterial pathogen detection(MBPD)approach.The results unveiled a total of25 genera and 65 species that exhibited a similarity of over 99% to published documented or potentially pathogenic bacteria.A majority of these strains were identified as opportunistic pathogens.3.By employing an antibiotic cocktail administered via injection and oral ingestion on host animals,followed by artificial bite experiments,the findings demonstrated that the utilization of antibiotics on host animals exerted a profound influence on the gastrointestinal microbial composition of I.persulcatus.Specifically,it elicited a discernible decrease in both the diversity and abundance of bacterial species.Moreover,subsequent midgut PAS staining of distinct subgroups of samples exhibited a notable reduction in the thickness of the peritrophic matrix(PM)within the antibiotic-treated cohort,as compared to the control ticks.Furthermore,the expression levels of four proteins responsible for preserving the integrity of the peritrophic mechanism were significantly diminished.A comparative analysis between the complete genome of I.persulcatus and proteins associated with the innate immunity-related signaling pathway in Drosophila identified 26 pertinent proteins that plausibly participate in signaling processes within I.persulcatus,specifically via the Toll,IMD,and JAK/STAT pathways.Transcriptome sequencing of samples derived from both the antibiotic-treated and control groups provided compelling evidence that the differentially expressed genes were substantially enriched for activities encompassing the activation of NF-κB-induced kinase,regulation of the JNK signaling pathway,and oxidoreductase activity during biological processes,suggesting the involvement of enriched genes in the innate immune response to microbial changes in I.persulcatus.Concurrently,q PCR validation subsequent to screening of proteins involved in the three pivotal signaling pathways of innate immunity corroborated the under-expression of key effectors within these pathways under antibiotic treatment.In this research,we conducted a comprehensive examination of the microbiomes inhabiting three prominent tick species belonging to the Ixodesmidae family within Heilongjiang Province.Employing the cultureomics approach,we isolated intra-tick bacteria and subsequently conducted an investigation into the alterations occurring in midgut flora homeostasis,intestinal physical barriers,and innate immunity-related proteins at the transcriptional level of ticks following the administration of antibiotics.Profound insights were gained into the distinctive characteristics of tick flora under diverse influencing factors,the tick innate immunity changes amidst the disruption of midgut flora equilibrium.Isolation of bacteria serves to furnish a solid scientific foundation and invaluable material and technical resources for the detection,diagnosis,treatment,prevention,and control of associated diseases. |
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