Researches On The Diversity And Function Of The Bacterial Community In Haemaphysalis Longicornis

Ticks are important pathogenic vectors,which can transmit various pathogens and cause diseases.They are extremely harmful to human health,animal husbandry,and wildlife.In addition to the pathogen,many non-pathogenic bacteria in ticks,such as symbiotic bacteria and opportunistic bacteria,participate in the host nutrition metabolism,immune defense,reproductive development,and resistance to harsh conditions,relating to the colonization and transmission of pathogens.At present,the research on the interaction between bacteria and ticks is mainly concentrated on a few endosymbiotic bacteria.The structure and function of the bacterial community and the mechanism of its action in the process of host reproduction and development are still unclear.Haemaphysalis longicornis is widely distributed in China and can transmit a variety of pathogens,such as New Bunia virus,Huaiyang Mountain virus,Spirochaeta,Taylonia,Babesia,Erikettsia and Rickettsia,with serious threats to human and animal health.This study took H.longicornis as the object and applied molecular biological techniques such as quantitative PCR and high-throughput sequencing,combined with co-occurrence network analysis and community construction null model,to systematically analyze the bacterial community structure and function,bacterial interaction,and community construction process in H.longicornis.Through the joint analysis of multiple omics,the effects of bacterial community changes on the reproductive development of female ticks were clarified at the level of transcription and translation,and the dynamic changes and mechanisms of the bacterial community in the reproductive development of H.longicornis were revealed.The main findings are as follows:Proteobacteria is the dominant bacteria in adult ticks of H.longicornis,and its relative abundance increases significantly after female ticks are engorged.At the genus level,Brevibacterium,Coxiella,and Pseudomonas accounted for a large proportion,and the relative abundance of Coxiella increased significantly after female ticks engorged.There is no significant difference in α-diversity of female ticks between before and after blood-sucking.The α-diversity of unfed male ticks was significantly higher than that of unfed or engorged female ticks.There were major B vitamins and essential amino acid synthesis pathways in the bacteria genomes.The VB2,VB3,VB5,VB6,VB9,coenzyme FAD(FMN),NAD+/NADP+,and lysine,tryptophan,phenylalanine,tyrosine,threonine,isoleucine,leucine,and valine synthesis pathways are complete.The relative abundance of Pseudomonas is the largest in unfed ticks and contains the largest number of genes for these pathways.Brevibacterium contains several gene species,followed by Rhodococcus and Stenotrophomon.The VB3 synthesis pathway is complete in the Coxiella genome,and it participates in the synthesis of multiple VB and essential amino acids.These pathways were not significantly different between males and females,but the number of genes in female ticks was greater than that of male ticks.Tetracycline treatment significantly impacted the bacterial composition and community construction process in ticks and their tissues.The relative abundance of Proteobacteria increased significantly after tetracycline injection,whereas Firmicutes decreased significantly.The relative abundance of Coxiella increased significantly,while Pseudomonas decreased significantly.The α-diversity has no significant difference,while β-diversity significantly changed.A small number of bacteria with relatively low abundance in the midgut changed significantly after injection.In contrast,the dominant bacteria Ralstonia did not change significantly.Proteobacteria was always the dominant phylum in the bacterial composition of the ovary,and Coxiella was the dominant genera,with no significant difference before and after injection.The composition and change trend of bacteria in the malpighian tubule and salivary gland were similar.The control group takes Actinobacteriota as the dominant bacteria,while the experimental group takes Proteobacteria as the dominant bacteria.After injection,the relative abundance of Rhodococcus and Variovorax decreased significantly.The Shannon index of the salivary gland increased significantly,and other diversity indexes did not change significantly after injection.The relationship between bacteria in ticks is more complex,and the synergy is enhanced.It significantly negatively affected the digestive,reproductive,and excretory systems within 48 hours after injection.The structural complexity of the flora in the ovary,salivary gland,and malpighian tubule increased,and the bacterial synergetic effect in the salivary gland and malpighian tubule increased significantly,while the antagonistic effect in the ovarian was significantly enhanced.The structure of midgut bacteria tended to be simple,and the antagonistic effect increased significantly.The process of bacterial community construction in ticks is always stochastic,with the drift process significantly enhanced after tetracycline injection.The process of bacterial community construction in the ovary and midgut has changed greatly;the random process of midgut bacteria has increased significantly,while the deterministic process of the ovary has increased significantly.Tetracycline injection stimulated the proliferation and density of Coxiella,and total bacteria in the ovary increased significantly but not significantly in ticks.The change of bacterial community structure could inhibit the development and reproductive process of the engorged female ticks,including its body color,turned black,their behavior was not active,the development of the ovary and midgut being blocked,the development of the oocyte was delayed,the normal apoptosis process of the salivary gland was inhibited,the reproductive efficiency was significantly reduced,the egg appeared abnormal such as depression,drying,color darkening,and the larva hatching rate was significantly reduced.The change in bacterial community significantly affected the gene expression level of female ticks,and the vitellogenin(Vg)and vitellogenin receptor(Vg R)genes directly relating to vitellogenesis were significantly up-or down-regulated.The transcription of differentially expressed genes and KEGG significantly enriched pathways in the midgut,fat body,and ovary,which were mainly down-regulated,and the negative effect of the bacterial community on ticks gradually increased over time.The significantly upregulated genes are mainly involved in the cell process,genetic information processing,and metabolism.The up-regulated genes relating to apoptosis and phagosomes revealed a kind of cytotoxicity.Significantly down-regulated genes are mainly involved in metabolism and cell process,including N-glycan biosynthesis,amino acid metabolism,lysosome,ECM receptor interaction,hormone synthesis,focal adhesion kinase,and so on,indicating that the change of bacterial community affects the development of H.longicornis through amino acid metabolism,lipid synthesis,and protein decomposition.The change in the bacterial community significantly affects the metabolic process of H.longicornis.After treatment,the types of differential metabolites increased continuously from the 2nd day to the 4th day.The KEGG enrichment analysis showed that on the second day,125 differential metabolite enrichment pathways were mainly down-regulated and mainly involved in metabolism,including three significant enrichment pathways: nicotinate and nicotinamide metabolism,ether lipid metabolism,and glycerophospholipid metabolism.On the 4th day,the enrichment pathway was mainly up-regulated and involved in metabolism,among which the glycerophospholipid metabolism pathway enrichment difference was extremely significant.The down-regulation pathway was mainly involved in metabolism,including three significantly enriched pathways: nicotinate and nicotinamide metabolism,galactose metabolism,and ether lipid metabolism.Combined multiple omics showed that high-dose tetracycline(150 μg/tick)affects the relative abundance and metabolic level of the key bacteria in female ticks.The change in the differential enrichment pathway is mainly due to the down-regulation of amino acid metabolism,which affects multiple signal pathways such as lipid metabolism,glucose metabolism,transcription,and translation.It was found that the reproductive status was improved to some extent through T50 VB supplementation.The effect of tetracycline on the reproductive development of ticks may come from the coregulation of bacterial communities and tick hosts.The results are helpful in understanding the structure and function of the bacterial community and its role in the reproductive process in H.longicornis for further clarifying the molecular regulatory mechanism of the interaction between bacterial community and tick reproduction and development.It can provide a theoretical basis for finding new targets for tick control,which has important scientific significance and potential application value.