The Mechanism Of RH36 Molecule Involved In Tick Reproduction And Oviposition

Ticks are a kind of blood-sucking vector arthropod,which are of great value in prevention and control of human and animal diseases.During blood sucking,there are a large number of active molecules secreted from tick salivary glands.RH36 is an immunomodulatory protein isolated and identified from salivary glands of Rhipicephalus haemaphysaloides.RNAi and vaccination tests of RH36 showed to inhibit tick blood feeding and unexpectedly induced a significant decrease in tick oviposition.Tick reproductive process plays a central role in the prevention and control of ticks,therefore,this paper will study the influence of RH36 gene silencing on tick reproduction,explore the molecular mechanism of this function,to provide the foundation for finding key targets in prevention and control of ticks.1.The relationship between the expression patterns of RH36 and the temporal sequence of tick reproduction.The expression characteristic of RH36 was studied during tick blood feeding by RT-q PCR and Western blot,and found(1)RH36 was expressed in the salivary glands of blood-feeding ticks,not in the hemolymph,fat body and ovary,which indicated the characteristics of the organ specific expression;(2)With the vampire time prolonged,the amount of RH36 expressed in the salivary gland was more and more,and reached the peak in the engorged ticks.These results indicated RH36 was expressed in the late blood-sucking salivary gland,which was highly consistent with the time of tick ovarian development.2.The effect of RH36 gene silencing on the ovarian development porphology and vitellogenesis.Compared to the luciferase gene silencing group,tick ovary development was immature and there were fewer eggs in the oviduct in RH36 gene silencing group,which significantly decreased tick oviposition rate.RH36 gene knockdown inhibited significantly the expression level of yolk protein in the tick ovary at the 3 day after tick engorgement by RT-q PCR and Western Blot;There were fewer yolk protein in the oocyte of RH36 RNAi group than that of luciferase RNAi group.These results indicated the effect of RH36 gene silencing on tick reproduction and oviposition was closely related to ovary development and vitellogenesis.3.The proteomics analysis of tick ovaries after RH36 and Luciferase RNAi.The differential proteins were studied using proteomic analysis and found that there were 25 up-regulated proteins and 19 down-regulated proteins in 5 days fed tick ovaries;while 74 and 134 proteins were up-regulated and down-regulated in 10 days engorged tick ovaries,respectively.It was also found that HSP70,endocytosis and focal adhension were affected notably with RH36 gene silencing after GO functional annotation and KEGG analysis of differentially-expressed proteins,suggesting HSP70,endocytosis and focal adhension were possible pathways influenced by RH36 gene silencing.4.The effect of RH36 gene silencing on tick blood feeding and reproduction via HSP70.After gene cloning of HSP70 family,sequence alignment and evolutionary tree construction were performed and found there were high degrees of similarity between R.haemaphysaloides and other ixodids or homo sapiens,and the evolution of HSP70 family was conservative.HSP70-5 was highly expressed in the fat body,hemolymph,ovary and salivary gland from partially fed and engorged ticks,suggesting HSP70-5 was widely distributed in ticks.HSP70-5 was significantly down-regulated by RH36 gene knockdown in the fed and engorged ticks.Gene silencing of HSP70-5 not only inhibited tick blood sucking,but also down-regulated vitellogenin and RH36 protein.These results showed RH36 and HSP70 interacted with each other at the molecular level,and RH36 can affect vitellogenin via HSP70-5 and finally influence tick oviposition.5.The effect of RH36 gene silencing on tick reproduction and oviposition via focal adhension.In non-muscle cells,actinin,paxillin and parvin were interacted to regulate the cell adhesion and cell proliferation.The transcriptional level of parvin,paxillin and actinin was significantly down-regulated by RH36 gene knockdown in the ovary of engorged ticks.Gene silencing of parvin or actinin not only inhibited tick engorgement rate,oviposition rate and engorgement body weight,but also inhibited ovary development maturation in the late period of engorged tick.At the transcriptional level,actinin gene silencing down regulated RH36,Vg4 and parvin,while parvin gene knockdown redecreased RH36,actinin and paxillin.These results indicated RH36 can affect vitellogenin and ovary development via focal adhension-related parvin and actinin,and actinin affected oocyte maturation by regulating the expression of vitellogenin.6.The effect of RH36 gene silencing on tick reproduction and oviposition via endocytosis.Vg depends on Vg R-mediated clathrin-dependent endocytosis pathway to enter into oocytes.RH36 gene silencing significantly decreased VPS28 and VPS37 genes in the ovary.Vg R gene RNAi analysis showed oviposition rate was notably decreased and ovary development was inhibited significantly;while VPS37 gene knockdown decreased significantly tick engorgement body weight and dramatically suppressed ovary maturation.These results indicated there was some relationship between RH36 and VgR-mediated endocytosis,and RH36 may affect ovary development via VgR and VPS37.In conclusion,RH36 expression level in the late blood-sucking ticks was highly consistent with the time of tick ovarian development.RH36 gene silencing caused tick ovary maturation inhibition and reduction of vitellin expression.By proteomics analysis of tick ovaries,HSP70,endocytosis and focal adhension were affected notably with RH36 gene silencing.RH36 can reduce the expression of vitellogenin and inhibit oviposition by regulating the expression of HSP70-5,focal adhension-related parvin and actinin,or endocytosis-related VPS24 and VPS37.The analysis of this mechanism will be helpful to the exploitation and application in the anti-ticks vaccines and tick-borne diseases prevention and treatment.