Tick 14-3-3 Protein Regulates The Absorption And Transport Mechanisms Of Host-derived Molecular In Ticks

Ticks are specialized obligate haematophagous ectoparasites,and are an important vector ectoparasite that pose a serious risk to human health and animal husbandry development.The blood of the host provides the ultimate source of energy and nutrition for the blood-feeding arthropods during their development and reproduction,containing albumin,hemoglobin,lipoproteins,immunoglobulins,cytokines,hormones and inorganic salts.However,the absorption and transport mechanisms of most host blood-derived molecules in ticks remain unclear.Moreover,there are no reports on the absorption and transport mechanisms of host-derived iron and autophagy molecule Beclin-1.To elucidate the complex biological relationship between ticks and hosts and explore new targets for tick prevention and control,this study identified the key molecule 14-3-3 in tick extracellular vesicles（EVs）and its components,and further investigated the mechanism of 14-3-3 regulation of host-derived iron and Beclin-1 absorption and transport in ticks.The findings offer a new perspective on the complex relationship between ticks and hosts at the molecular level,providing new ideas for tick-borne disease prevention and control.The main research findings are as follows:1.Tick hemolymph EVs are closely associated with host proteins transport.Combined with 4D label-free proteomics,transmission electron microscopy,immunoblotting and immuno-electron microscopy,this study revealed that tick hemolymph EVs exhibited typical cup-shaped vesicle morphology,with an average size of approximately 135 nm.Several EV markers,such as RhCD9,RhTSG101,Rh14-3-3,and the iron transport protein Ferritin-2,were identified,together with various host proteins,including histone H2A and alpha-2-macroglobulin.Furthermore,in vitro experiments revealed that PKH26-labeled hemolymph EVs can be internalized by tick salivary gland and ovarian cells,indicating that EVs are a crucial pathway for tick-host molecular transport.2.The tick 14-3-3 protein plays a crucial role in tick feeding,development and reproduction across different developmental stages and tissue organs.Knockdown of the Rh14-3-3ζgene by ds RNA significantly affects tick blood-feeding,development and oviposition.Using proteomics,quantitative PCR,immunoblotting and TUNEL staining,Rh14-3-3ζknockdown disrupts the balance of hemolymph proteases,downregulates Cathepsin B（CTSB）and Cathepsin D（CTSD）protein levels,upregulates Cathepsin L（CTSL）and Cathepsin F（CTSF）protein levels,and promotes hemolymph protease hydrolysis of bovine hemoglobin and albumin.In the midguts,Rh14-3-3ζknockdown upregulates CTSB,CTSD,ATG8 and ATG8-PE protein levels,promoting midgut autophagy.In the salivary glands,Rh14-3-3ζknockdown upregulates Caspase-7 and Bax protein levels,leading to the colocalization of Bax with mitochondria and promoting salivary gland cell apoptosis.3.The tick Rh14-3-3 protein regulates the transport and absorption of host-derived iron via ferritin.Using quantitative PCR,immunoblotting,tissue ion quantification,Native PAGE and Prussian blue staining,it was found that the knockdown of Rh14-3-3ζsignificantly increased Fe²⁺concentration in the midgut and upregulated Ferritin-1 protein levels while downregulating Ferritin-2 protein levels.Furthermore,the knockdown inhibited hemolymph iron transport and decreased hemolymph iron concentration.However,it increased the Fe³⁺content and RhFerritin-1 protein levels in salivary gland and ovary tissues,indicate that absorbing and storage of more iron from hemolymph.4.The transport of ferritin is regulated by ZIP13.RhZIP13 is necessary for iron loading and secretion facilitated by the tick iron transporter protein RhFerritin-2.During blood-feeding,RhZIP13and HRG1 expression are induced,while RhZIP14 expression is inhibited.and knockdown of RhZIP13affects tick blood feeding.Protein analysis via immunoblotting and Native PAGE indicates that RhZIP13 knockdown results in increased levels of midgut RhFerritin-1 and stored iron,reduced secretion of RhFerritin-2 into the hemolymph,and lower levels of RhFerritin-1 and stored iron in salivary glands,fat bodies and ovarian tissues.This ultimately leads to the release of stored iron from Ferritin-1 to maintain life activities.Additionally,RhFerritin-1 expression is induced and RhZIP13expression is downregulated in response to capillary feeding of ferric ammonium citrate（FAC）to inhibit iron transport.RhZIP13 is localized in the Golgi apparatus,indicating its role in regulating both the secretion pathway of Ferritin-2 and iron export in tick midgut.5.The tick Rh14-3-3 protein regulates the uptake and transport of host-derived Beclin-1.To investigate the dynamics of hemolymph Beclin-1 concentration in R.haemaphysaloides and Haemaphysalis longicornis ticks,it was observed that there was an increment in hemolymph Beclin-1concentration in parallel with the increasing blood feeding,but it decreased significantly after engorgement.Through immunoprecipitation and plasmid co-transfection,it was discovered that tick RhAkt phosphorylates the Ser295 site on host Beclin-1 to facilitate its interaction with Rh14-3-3.These findings suggest that Beclin-1 is involved in tick physiological processes through hemolymph transport.In summary,this study successfully isolated and identified tick hemolymph EVs and their associated protein components.The study highlights the critical role of the EV-associated molecule known as 14-3-3 in tick feeding,development,and reproduction.The study focused on the molecular mechanism of 14-3-3 regulating the absorption and transport of host-derived iron through ferritin and ZIP13,as well as its molecular interaction with Beclin-1 to regulate its absorption and transport in tick bodies.This study provides preliminary insights into the complex relationship between ticks and hosts at the molecular level,offering new directions for tick and tick-borne disease prevention and control.