Abstract:

Abstract: 【Aim】 To determine the function of a cytoplasmic peptidoglycan recognition protein, RfPGRP-L2, in the maintenance and regulation of homeostasis of gut microbiota in the invasive insect pest, Rhynchophorus ferrugineus, so as to provide scientific basis and action targets for the development of new insect pest control strategies targeting to destroy the homeostasis of gut microbiota. 【Methods】 The sequence characteristics of RfPGRP-L2 were analyzed by bioinformatics methods. RT-qPCR was used to analyze the expression levels of RfPGRP-L2 in the different tissues (head, fat body, epidermis, foregut, mid-/hindgut and hemolymph) of the healthy 4th instar larvae of R. ferrugineus and in the gut and fat body of the 4th instar larvae of R. ferrugineus challenged with Escherichia coli DH5α and Staphylococcus aureus by injection with 1 μL bacterial suspension with the OD₆₀₀ value of 1.6 and oral feeding with sugarcane slices smeared with 1 mL bacterial suspension with the OD₆₀₀ value of 1.6, respectively. Prokaryotic expression of RfPGRP-L2 was carried out, and in vitro assays were applied to determine the agglutination and antibacterial activity of the recombinant RfPGRP-L2 to E. coli DH5α and S. aureus. After RNAi of RfPGRP-L2, the number of gut bacterial colonies of E. coli in the hemolymph and gut of the 4th instar larvae of R. ferrugineus was determined. The expression levels of antimicrobial peptide genes in the fat body and gut of the 4th instar larvae of R. ferrugineus after RNAi of RfPGRP-L2 were detected by RT-qPCR. By bacterial 16S rRNA-based high-throughput sequencing, the effect of RNAi of RfPGRP-L2 on the gut microbiota composition of the healthy 4th instar larvae of R. ferrugineus was analyzed.【Results】 The SMART analysis revealed that RfPGRP-L2 has no transmembrane domain and signal peptide, suggesting that RfPGRP-L2 is a cyoplasmic peptidoglycan recognition protein. The RT-qPCR results showed that RfPGRP-L2 was highly expressed in the immunity-related tissues, such as the hemolymph, gut and fat body of the healthy 4th instar larvae of R. ferrugineus. The expression level of RfPGRP-L2 in the fat body of the 4th instar larvae of R. ferrugineus was significantly up-regulated upon the challenge with injected E. coli and S. aureus for 6 h and 12 h, respectively. After oral feeding of E. coli for 6 h, the expression level of RfPGRP-L2 in the gut of the 4th instar larvae of R. ferrugineus was also increased significantly. Furthermore, in vitro assays revealed that the recombinant RfPGRP-L2 could result in the obvious agglutination of E. coli and S. aureus, suggesting that RfPGRP-L2 could recognize the two pathogenic bacteria. When RfPGRP-L2 was silenced in the 4th instar larvae of R. ferrugineus, the ability to clear the invaded EGFP-tagged E. coli in the gut and hemolymph was significantly impaired as compared to the control group, the expression level of antimicrobial peptide gene RfCecropin in the gut was significantly reduced, the number of gut bacterial colonies in the healthy 4th instar larvae was significantly increased compared to the control group, and the gut bacterial composition was also altered significantly. 【Conclusion】 The cyoplasmic peptidoglycan recognition protein RfPGRP-L2, acting as a pattern recognition receptor to discriminate pathogens and to activate the immune signaling pathways in intestinal epithelial cells, can promote the expression of antimicrobial peptide gene to modulate the homeostasis of gut microbiota in R. ferrugineus.

Key words: Rhynchophorus ferrugineus, peptidoglycan recognition protein, expression profile, gut microbiota, immune response, immune homeostasis