Study On The Changes Of Protein Expression And Phosphorylation Modification During The Process Of Degeneration Of Salivary Gland In Haemaphysalis Longicornis Using DIA Technology

Ticks belong to Arthropoda,Arachnida,Acari,Parasitiformes,Ixodina,Ixodoidea.It is divided into three families: Ixodidae,Argasidae and Nuttalliellidae.Ticks parasitize vertebrates and transmit many pathogens.They are very harmful vectors.Ticks cause local skin hemorrhage,edema and infection when they suck blood.Together with a large number of pathogenic microorganisms,ticks become the second most harmful blood-sucking arthropod after mosquitoes.A variety of tick-borne pathogens can cause zoonosis,greatly affecting the development of animal husbandry,but also threatening human health.Haemaphysalis longicornis,which belongs to Ixodidae,is widely distributed in China.It can spread many dangerous pathogens and seriously endanger human health.Therefore,it is of great significance to carry out relevant research on H.longicornis.The salivary glands of ticks,as an indispensable auxiliary blood-sucking organ,play an important role in their whole life activities.With the female ticks begin to suck blood,their salivary glands develop rapidly and their functions are gradually improved.But when the female tick is engorge and falls off from the host body surface,the salivary gland loses its function and rapidly degenerates.During this period,salivary gland acinar autolysis occurred,and a large number of phagocytic acinar appeared.When the female tick was engorge for 4 days,its salivary gland degenerated until it disappeared.The study of salivary gland degeneration is of great significance for exploring the physiological mechanism of the regulation of tick salivary gland development.Finding proteins that promote rapid salivary gland degeneration is of great help to control tick development and reproduction.Therefore,it plays a key role in controlling the transmission of tick-borne pathogens on another level.The dynamic changes of all proteins in the salivary glands of H.longicornis were studied by iTRAQ quantitative proteomics method in the early stage of this laboratory.However,due to the large demand for samples by iTRAQ technology,it is difficult to achieve phosphorylation enrichment and phosphorylation modified proteomics for small individual samples.In this study,the dynamic regulation and phosphorylation modification of proteins during salivary gland degeneration of H.longicornis were studied by using DIA quantitative proteomics technique,which can be operated with micro-samples and has high reproducibility and stability.A total of 2226 proteins were identified,including 524 differentially expressed proteins.A total of 635 phosphorylated proteins were identified,involving 1957 phosphorylated sites.There were 576 proteins involved in phosphorylation modification,and 1431 proteins involved in phosphorylation modification.Bioinformatics analysis was used to systematically analyze differentially expressed proteins and differentially phosphorylated modification sites.Through a large number of RNA interference work,we found that the salivary gland acinar of female ticks during blood suction showed obvious atrophy compared with normal female ticks after the interference of AMPK,a protein that provides energy for the development of salivary gland,and the body weight of engorgement and the amount of oviposition decreased significantly.Phosphorylation of CDC37,a protein regulating cell cycle function,was down-regulated in the process of salivary gland degeneration.RNAi was performed on CDC37 in unfed female ticks.It was found that the development of salivary glands was also blocked during blood sucking in female ticks.Hippo can promote cell apoptosis,and the phosphorylation modification of Hippo protein in female ticks during salivary gland degeneration is significantly up-regulated.When the gene was interfered with in female ticks with engorgement,the rate of salivary gland degeneration of female ticks slowed down obviously,which indicated that the gene had obvious function of promoting salivary gland degeneration.This study is devoted to exploring the molecular regulatory mechanism of salivary gland degeneration in H.longicornis.The results clearly showed the dynamic changes of protein expression and phosphorylation modification during the process,and found some key proteins which are important for regulating the degeneration of salivary gland,laying a foundation for further effective control of tick development and reproduction,and providing a theoretical basis for integrated control of ticks.