| Ticks are hematophagous ectoparasites that are the second largest media pathogensonly to mosquitoes. Ticks can transmit a variety of pathogens which can causehuman,domestic animals and wildlife severe illness,even death. The tick-bornepathogens include fungal, viruses, rickettsiae, spirochaetes, bacterias and protozoa.ticks and tick-borne diseases caused huge econimic loss in livestockindustry,.Hyalomma asiaticum is a widespread tick species in the Palearcticzoogeographic region, norstwest of China and Central Asia, which mainly transmitsCrimean-Congo hemorrhagic feve and Q fever.The traditional method is to usepesticides to control ticks,but drug residues and other issues have become increasinglyprominent,therefore seeking a more healthy and environmentally method is moreimportant.In the process of obtaining a blood meal,tick’s salivary glands secrete largeamounts of active substances,these active substances can hinder the coagulation of thehost,modulate the host immune response and promote the development andreproduction of pathogens,It’s important for obtaining a blood meal and transmissionof pathogens. Therefore, the active substance from the tick’s salivary glands becomethe focus of the study. In the process of blood coagulation,serine proteases areextremely important functional molecules,while the body also haveserine protease’snegative regulatory molecules.Kunitz inhibitors are an important class of serineprotease inhibitors which possess one or more Kunitz domains,Kunitz proteaseinhibitors are ubiquitous,being found in many organisms with anti-coagulation.Recentstudies have shown that Kunitz protease inhibitors are the most abundant proteins inIxodes Scapularis’s salivary glands.In order to promise a long-time blood feeding process,ticks use Kunitz protease inhibitors and other salivary proteins to resist thehost’s coagulation and immune mechanism.However,if the Kunitz protease inhibitorsin Hyalomma asiaticum has not been reported.In this work, we described two new genes with Kunitz domain, B26and P15, fromthe salivary glands of Hyalomma asiaticum. In order to investigate the biologicalmechanism and characteristics.By means of prokaryotic expression,we accessanticoagulant activity substances that encoded by these two genes to study itsactivity.We detected the expression characteristics of the two genes in differentdevelopmental stages and organs. Then verify anticoagulant activity substances thatencoded by these two genes through immunity protection.Finally,The success of tickfeeding was investigated by measuring the feeding periods, the total number of ticksengorged, engorgement weight, survival, and oviposition by the use of RNAi.Theresults show that P15can cause the inhibition of Trypsin.In APTT assays, Comparedwith the GST protein, B26prolonged the coagulation time of citrated rabbit plasma ina dose-dependent manner,P15also prolonged the coagulation time but the differencewas not significant.B26immunity protection shows that Compared with GST-injectedticks there was a significant effect on the engorgement body weight(P<0.001),P15immunity protection shows that Compared with GST-injected ticks there was asignificant effect on the attachments rate of24h and feedingperiod(days)(P<0.001).RT-PCR analysis showed that B26was expressed most richly atlarvae and salivary glands,P15was expressed most richly at eggs andhemolymph.Compared with buffer-injected ticks, there was a significant effect on theattachment rate at24h through silence of B26and P15by RNAi.In summary,B26andP15have certain biological functions in blood-sucking. |
