Molecular Dissection On The Role Of A Cytoplasmic Peptidoglycan Recognition Protein RfPGRP-L2 And IMD-like Pathway In Maintaining The Homeostasis Of Gut Microbiota In Rhynchophorus Freeugineus Olivier

Red palm weevil(RPW),Rynchophorus ferrugineus Olivier,is an invasive pest which seriously infests the palm trees.It has been proved that there are abundant microorganisms in the intestinal tract of this pest,and changes of gut microflora can significanly affect physiological fitness,such as growth and development,food digestion,immunity and resistance to pathogens.Peptidoglycan recognition proteins(PGRPs),as a kind of pattern recognition receptor,play multiple important roles in animals.Immune overreaction or immune disorder can both impair the beneficial gut microbiota and also cause losses to the host physiological traits.Thus,there is a sophisticated system for modulating the immune intensity to tolerate symbiotic bacteria while removing the pathogens.Conservative domain prediction found that RfPGRP-L2 has no transmembrane domain and the signal peptide,suggesting that RfPGRP-L2 is a cyoplasmic peptidoglycan recognition protein.RT-qPCR results showed that RfPGRP-L2 highly expressed in the immunity-related tissues such as hemolymph,gut and fat body.The expression level of RfPGRP-L2 in fat body and gut were significantly up-regulated after the challenge of Escherichia coli DH5? and Staphylococcus aureus.Futhermore,in vitro assays revealed rRfPGRP-L2 results in the obvious agglutination of E.coli and S.aureus,indicating that RfPGRP-L2 can serve as the recognition receptor to recognize pathogentic bacteria.When RfPGRP-L2 was silenced,the ability of individuals to clear the invaded eGFP-tagged E.coli in gut and hemolymph was significantly depressed as compared to the controls.Additionally,gut bacterial load in RfPGRP-L2-silenced larvae was drastically increased and the bacterial compositions were also alered significantly.Interestingly,we found that the expression level of RfCecropin in gut was dramaticly reduced by RfPGRP-L2 silencing.When RfRelish was silenced,the ability of individuals to clear the pathogenic bacteria in hemolymph and gut was significantly compromised.Furthermore,the silenced larvae exhibited increased gut bacterial load,and the relative abundance of some gut bacteria was changed as compared to controls.Collectively,RfPGRP-L2 acts as a recognition receptor to discriminate pathogens to activate the production of AMPs via the RfRelish-mediated IMD-like pathway to maintain the homeostasis of gut microbiota.