Analysis And Functional Verification Of Three Enzymes In The Embryonic Development Of Parthenogenetic Haemaphysalis Longicornis

As obligate blood-sucking ectoparasites,ticks have a wide range of hosts and strong reproductive capacity.Ticks are the world's second most popular vector of diseases after mosquitoes and can cause a variety of zoonoses,threatening livestock products and human health.Haemaphysalis longicornis is widely distributed in China and exhibits two reproductive populations: bisexual population(HLBP)and obligate parthenogenetic population(HLPP).HLPP can complete their life-cycle without fertilization.Vitellin plays an important role in eggs with high content and it continuously degrades under the regulation of various hydrolases to provide various nutrients and energy for embryonic development.This study focused on HLPP,using molecular biology,biochemistry,and immunology to evaluate the dynamic changes of vitellin subunits in eggs and analyzed the content of free amino acids and heme in its degradation products.The results showed that vitellin were degraded during embryonic development and 3 and 1 new protein bands appeared on days 13 and 17,respectively,but the vitellin was not completely degraded.The detection of free amino acids showed that the content of histidine gradually increased at different developmental stages and the content of glutamic also gradually increased after the day 5,while the remaining amino acids revealed a downward trend and participated in various metabolism.The content of heme did not have significant differences during embryonic development because of the combination of heme with vitellin to protect the ticks from oxidative damage.On the other hand,it also combined with specific enzymes to facilitate the recognition of vitellin by hydrolases to regulate degradation.Quantitative real-time PCR and fluorescent substrates detection showed that the expression of cathepsin B was highest on day 15 and its activity was high throughout embryonic development.The expression of cathepsin D reached the highest on day 3,and its activity showed a gradual decline.The mRNA transcript of acid phosphatase was highest on day 9 and the enzyme activity gradually increased during embryonic development.Enzymes and enzymes inhibitors incubation with total proteins showed that the inhibitors of three enzymes inhibited enzymes activity and hindered the degradation of vitellin;after adding three enzymes,the degree of degradation of vitellin was increased,indicating that three enzymes regulate vitellin degradation.When adding a mixture of two enzymes,compared with the addition of a single enzyme,the degradation of vitellin was accelerated,which indicated that there exists a synergy between two enzymes.RNAi was performed on engorged female ticks and the effect of genes silencing was detected by qPCR.The oviposition of the ticks and the hatching rate of the eggs were recorded which revealed that knocking down the expression of enzymes genes did not affect the oviposition,but the eggs hatching rate were significantly decreased.This may because the three enzymes were involved in the degradation of vitellin.On the other hand,the three enzymes played a role in other vital activities such as apoptosis,immune response and activation of growth factors or hormones.Therefore,the three enzymes have potential as vaccine candidates.The number of proteins,common proteins,differential proteins and specific proteins during five periods were analyzed by DIA method.The results showed that proteins participate in many pathways,such as the lysosomal pathway,citrate cycle,sugar metabolism and glutathione metabolism,involving multiple proteins such as the nucleotide exchange factor sil1,glutathione,malate dehydrogenase,and NADP-dependent isocitrate dehydrogenase.Analysis of specific proteins during five stages revealed that Ran-binding protein promoted the development and elongation of appendages in early stages;ribosomal proteins ensured proteins synthesis on day 9 and tissues or organs differentiation;aspartate aminotransferase on day 13 regulated mitochondria polarization and apoptosis which might be involved in the degradation of appendages;AIP1 was identified at the end of embryonic development,which was involved in dynamic reorganization of the actin cytoskeleton and it could enhance the breakdown of actin filaments and played an important role in maintaining cell morphology.This study explored the degradation of vitellin in HLPP and the regulation of vitellin and embryonic development by three enzymes.We also evaluated the changes,functions,and interactions of enzymes and dug important proteins related to embryonic development.The research also wanted to clarify the similarities and differences between HLPP and HLBP to provide a theoretical basis for exploring the special reproductive mechanism of parthenogenesis and to seeking candidates for tick vaccines and providing a scientific theory for tick control.