Identification Of The RhBcl-2 And RhBax Homologs From The Tick Apoptosis Moleculars And Study Of Their Application Potential

Ticks are obligatory blood-feeding arthropods and vectors of many pathogens.Ticks usually stay attached to their hosts and feed for several days or even weeks.The salivary glands of ticks mediate diverse functions that ensure tick survival.Tick-borne pathogens(TBP)are transmitted to the host by the saliva used during tick feeding.The salivary glands of female ticks degenerate rapidly within four days after engorgement.Salivary gland degeneration begins at the early stage of rapid feeding and may be caused by apoptosis and autophagy related to the increase of ecdysone in the hemolymph.During degeneration,the granular acini undergo DNA fragmentation and caspase enzyme activity increases.According to the comprehensive analysis of the global protein changes,some proteins associated with apoptosis and autophagy changed and some proteins linked to the degradation of DNA and proteins were consistently upregulated.However,the mechanism of these effects is unknown.Bcl-2(B-cell lymphoma-2)family,the best characterized molecules are mainly involved in regulating the signaling pathway of cell apoptosis,via the interaction between anti-apoptotic and pro-apoptotic molecules.Currently,the regulatory mechanism of Bcl-2 molecules during apoptosis has been attracting a wide research attention.Recent studies that are conducted in mammals had revealed that Bcl-2 family molecules are beneficial to find new drugs for cancer and to monitor the developmental stages of tumor.Moreover,arthropod based study findings indicated that Bcl-2 family molecules are used to biologically control pests.Therefore,the review article has compiled recent research findings regarding the role of Bcl-2 family molecules during apoptosis,in different species.The reports about Bcl-2 family molecules of ticks are still lacking,and their function and application potential need to be studied.To study the mechanism and the application potential of Bcl-2 family molecules in the process of tick salivary gland apoptosis,we identified the Rh Bcl-2 and Rh Bax molecules and evaluated the possibility of application in prevention and treatment by RNA interference technique.The ORF regions of Bcl-2 and Bax genes were cloned from the c DNA of fully engorged R.haemaphysaloides salivary glands and named Rh Bcl-2 and Rh Bax.Rh Bcl-2 ORF has 576 bp and encodes a protein of 192 amino acid residues.Rh Bax ORF has 648 bp and encodes a protein with 216 amino acid residues.Rh Bcl-2 had the conserved BH1,BH2,BH3,and BH4 domains,but Rh Bax had the conserved BH1 and BH3 domains.After mouse immunizations,specific polyclonal antibodies(Pc Ab)were created in response to the recombinant proteins.Reverse transcription quantitative PCR(RT-q PCR)and western blot were used to detect the prensence of Rh Bcl-2 and Rh Bax in ticks.TUNEL assays were used to determine the apoptosis level in the salivary glands of female ticks at different feeding times after gene silencing.The interactions between Rh Bcl-2 and Rh Bax were identified by co-transfection and GST pull-down assays.Finally,The RT-q PCR assay demonstrated that the transcription of Rh Bax genes increased significantly during the engorged periods of the tick developmental stages(engorged larvae,nymphs,and adult females).Transcriptional levels of Rh Bcl-2 and Rh Bax in the female salivary glands increased more significantly than in other tissues post engorgement.Co-transfection and GST pull-down assays showed that Rh Bcl-2 and Rh Bax could combine with each other,but failed to combine without the BH3 domain.To identify the function of Rh Bcl-2 and Rh Bax,the genes of Rh Bcl-2 and Rh Bax were in vivo targets of RNA interference.Rh Bcl-2 treatment significantly restrained tick feeding.In contrast,Rh Bax interference had no effect on tick feeding.TUNEL assays showed that salivary gland interference by Rh Bcl-2 ds RNA increased apoptosis levels,but Rh Bax was similar to the control.The results showed the potential of Rh Bcl-2 molecule as target molecules for tick biologic control,and provided a basis for further elucidating the mechanism of Bcl-2 family molecules in the process of tick salivary gland apoptosis.