Functional Study Of CD209 And CTL3-mediated Homeostasis Of Enterocuccus Mundtii In Gut And Hemolymph Of Helicoverpa Armigera

Insects lack an adaptive immune system and rely entirely on innate immunity to fight against pathogens.When pathogens invade insects,insects recognize and bind to the highly conserved pathogen-associated molecular patterns on the pathogen's surface through pattern recognition receptors(PRRs),initiating various immune responses,such as phagocytosis and encapsulation and induction of antimicrobial peptides(AMPs).As a PRR,C-type lectins(CTLs)plays an important role in resistance to pathogens.Enterococcus belongs to a group of intestinal symbiotic bacteria,which normally maintain homeostasis in the host gut.However,when it enters into the hemocoel,causing insect diseases,such as sepsis.In the previous study,we found that CTL3 was firstly synthesized in fat body of the cotton bollworm Helicoverpa armigera and then secreted into the hemocoel,thus participating in the hemocyte-mediated phagocytosis and encapsulation.In this study,Enterococcus mundtii as a symbiotic bacteria was isolated from the gut of H.armigera.Then,we investigate the molecular mechanism of two CTLs(CD209 and CTL3)-mediated homeostasis of E.mundtii in the gut and hemolymph.The results are the following.1.CD209 was highly expressed in the midgut during metamorphosis of H.armigera,and the gut microbiome could induce the expression of CD209.E.mundtii inhibited the expression of CD209,but CD209 cannot bind to E.mundtii.Some AMPs(e.g.attacin)were up-regulated after CD209 depletion,and the AMP showed the inhibition activity towards E.mundtii.These suggest that high level of attacin via suppressing the expression of CD209 contributes to maintaining the low level of E.mundtii in the gut.2.Depletion of CTL3 led to increased abundance of E.mundtii in the gut and hemolymph.Meanwhile,depletion of CTL3 decreased the expression of almost all PRRs and AMPs genes in fat body.Further studies showed that E.mundtii could escape from the gut to the hemolymph.CTL3 binds to E.mundtii and promotes hemocytic phagocytosis towards it once E.mundtii entered from gut to the hemocoel.In addition,three AMPs(attacin,lebocin and gloverin)were demonstrated to inhibit the proliferation of E.mundtii.3.Injection of E.mundtii into the hemocoel of H.armigera larvae resulted in delayed growth and development.In summary,we preliminarily draw the following conclusions.1.CD209 may achieve homeostasis of gut E.mundtii by regulating the expression of AMPs.2.CTL3 maintains homeostasis of hemolymph E.mundtii by promoting hemocytic phagocytosis and induction of AMPs.This study enriched our knownledge on the function of insect CTLs,and provided a preliminary reference for the research on the biological control of insect pests by using intestinal symbiotic bacteria.