Molecular Mechanisms For 20E Signal Regulation Of Cellular Immunity Mediated By HaCTL3 And HaGBPB In Helicoverpa Armigera

20-hydroxyecdysone(20E)is an important hormone that regulates molting and metamorphosis.It is also involved in regulating various immune responses of insects against pathogen invasion,such as phagocytosis and encapsulation.Phagocytosis is the removal of smaller pathogens(such as bacteria,fungi,etc.),while the encapsulation is responsible for clearing larger pathogens(such as eggs of parasitoids,nematodes,etc.).The first and most important step during the phagocytosis and encapsulation reaction is the identification of invaders and the recruitment of hemocytes,typically by pattern recognition proteins(PRPs),such as C-type lectins(CTL).In addition to recruiting hemocytes to the surface of the invader,the encapsulation reaction also requires that the subsequent hemocytes are transformed into an adherent state,layer by layer wrapping around the invader,forming a multicellular cyst sheath.Growth-blocking peptide(GBP)can promote the hemocytes from a non-adherent(spherical)state to an adherent(stretched)state.However,the molecular mechanism underlying the regulation of innate immunity by 20E,especially cellular immunity(such as phagocytosis,encapsulation,etc.)remains unclear.In this study,we investigated the function of a PPR HaCTL3,and two GBP-binding proteins(HaGBPBl and HaGBPB2)involved in cellular immunity of Helicoverpa armigera.By using western blot,immunocytochemistry,in vitro protein activity analysis,RNAi and isothermal titration calorimetry(ITC)methods,we obtained the following results:1.HaCTL3 was up-regulated in fat body,hemocytes and plasma during metamorphosis,and 20E induction and bead challenge increased HaCTL3 expression.The up-regulated HaCTL3 promoted the phagocytosis and encapsulation.Further,Ha?-integrin promoted HaCTL3-mediated phagocytosis.2.HaGBPB1 and HaGBPB2 was up-regulated in plasma during metamorphosis,and both 20E injection and bead challenge promoted the release of HaGBPB1 and HaGBPB2 at least partially from oenocytoid cells into plasma.The N-terminal rather than C-terminal domain of HaGBPB1 is responsible for binding HaGBP subsequently and inhibiting hemocytic encapsulation.In summary,we found that HaCTL3 mediated 20E signaling to recruit blood cells,thereby promoting phagocytosis and encapsulation.HaGBPB bound HaGBP and eliminated redundant HaGBP in the hemolymph,thereby mediating 20E signaling to suppress hemocytic extension and encapsulation.Hence,20E exhibits a regulatory effect on both steps of encapsulation,not only initiating the encapsulation by regulating the expression of PRP,but also terminating encapsulation by regulating the release of GBPB to avoid excessive stimulation of oenocytoids which may cause damage.This study deepens our understanding of the molecular mechanism of 20E regulation of cellular immunity and provides a noval potential targets for pest control.