Molecular Mechanisms Of HaMMR1-like-or HaIBP-mediated Cellular Phagocytosis Promoted By 20E In Helicoverpa Armigera

Innate immunity of insects includes cellular immunity and humoral immunity,and cellular immunity is composed of phagocytosis,encapsulation and nodulation.Phagocytosis consists of two key steps,namely,the recognition of pathogens by pattern recognition proteins followed by the recruitment of immune cells,and immune cells phagocytose pathogens through cytoskeletal reorganization to form phagosome.20-Hydroxyecdysone（20E）is an active form of ecdysone,which plays an important role in the regulation of innate immune responses（e.g.,phagocytosis）in insects,but the regulatory mechanism remains unclear.In this study,to eliminate the interference of complex components of insect hemolymph,we used Helicoverpa armigera epidermal cell line（HaEpi）as a model to study the mechanism of 20E regulation on phagocytosis.Macrophage mannose receptor 1-like protein（GenBank ID:XP₀21189909,HaMMRl-like）,a pattern recognition protein,and integrin binding protein（GenBank ID:XP₀21195104,HaIBP）,a protein that may be involved in the cytoskeletal reorganization,were selected as the targets to study their functions in phagocytosis and verify whether they responded to 20E signaling.The results showed as follows:1.20E promoted the phagocytosis of HaEpi cells to bacteria,and 20E nuclear receptor EcR/USP knockdown weakened the phagocytosis of HaEpi cells to bacteria.2.20E promoted the extension of HaEpi cells and knockdown of EcR/USP inhibited the extension of HaEpi cells.3.HaMMRl-like was highly expressed in the fat body of H.armigera during metamorphosis and was induced by 20E.HaMMRl-like bound to the surface of bacteria and HaEpi cells,as well as promoted phagocytosis of hemocytes and HaEpi cells to bacteria;4.20E induced the expression of HaIBP in HaEpi through EcR/USP.Knockdown of HaIBP inhibited the phagocytosis of HaEpi cells and the extension of HaEpi cells.Overexpression of HaIBP significantly promoted the extension of HaEpi cells,but inhibited the phagocytic ability of HaEpi cells which may be due to the extreme extension of HaEpi cells.In conclusion,20E promotes phagocytosis,and HaMMR1-like and HaIBP mediate 20E signaling to regulate the two key steps of phagocytosis（cellular recruitment and extension）.Our study proves that HaEpi can be used as a model to study the molecular mechanism of 20E regulating phagocytosis in vitro.This study enriches the research on the molecular mechanism of endocrine regulation of insect innate immunity and provides an important molecular target for biological control of pests.