Changes And Roles Of Cathepsin D In The Embryonic Development Of Haemaphysalis Longicornis

As a kind of obligate blood-sucking parasites,ticks mainly parasitize mammals,birds,reptiles and amphibians.They can spread bacteria,fungi,viruses,spirochetes and rickettsia which cause varieties of diseases,such as bacterial diseases,Lyme disease,hemorrhagic fever,forest encephalitis,Q heat,and spotted fever.Vitellin is a hemoglycolipoprotein.As a nutrient source,it provide amino acids,sugars and lipids for egg development through programmed degradation.This process requires a variety of hydrolases.Studies about the role of enzymes in vitellin degradation and embryonic development mainly focused on insects.In ticks,there is only few researches about Rhipicephalus microplus.Cathepsin D belongs to aspartic protease which is an important acidic hydrolase.The relationship between Cathepsin D and vitellin has not been reported in Haemaphysalis longicornis.In the current study,DNA sequencing,qPCR,biochemistry,molecular biology and statistics were used to investigate the change of Cathepsin D during embryonic development as well as the role of Cathepsin D in vitellin degradation.The results show that the expression level of Cathepsin D was weak from day 1 to day 9,then increased significantly on 11 days and reached the highest expression.The expression level decreased gradually after 11 days.Enzyme activity analysis showed that Cathepsin D was active throughout the embryonic development.It decreased slightly from day 1 to day 5,then increased rapidly from day 7 to day 13.The highest activity was on 13 days and then gradullydecreased until 17 days.After interrupt Cathepsin D in unfed and engorged femal ticks,we found that the oviposition of engorged femal experimental group was 0.13g,which showed no obvious difference compared with control.The hatching rate of the experimental group was 57.22%,and the difference between the two groups was obvious.The oviposition of unfed femal experimental group was 0.09g,which was almost the same with control group.The hatching rate of the experimental group was 65.38%,and the hatching rate of the control group was 84.97%and the difference was obvious.These results indicated that the hatchability of eggs was affected after interfering with the Cathepsin D,but the oviposition was not been affected.The crude extract of 13^th day eggs was incubated with different concentrations of Cathepsin D and Cathepsin D inhibitor Pepstain A.When 1 U of cathepsin D was added,the vitellin subunits did not change significantly.When 3 U of Cathepsin D was added,the subunits had a certain degree of degradation.When 6 U of Cathepsin D was added,the subunits were basically degraded.After incubation with the inhibitor,the degradation of the vitellin subunits were inhibited,indicating that Cathepsin D is involved in the degradation of vitellin.The above results provide foundation for further understanding on the development process of ticks and the regulation of enzyme.