Abstract: 【Aim】 This study aims to determine the role of a C-type lectin, RfCTL-S1, in immune defense in larvae of the red palm weevil, Rhynchophorus ferrugineus. 【Methods】 The sequence characteristics of RfCTL-S1 of R. ferrugineus were analyzed by bioinformatics methods. The expression levels of RfCTL-S1 across the different tissues (head, integument, foregut, mid-/hindgut, hemolymph and fat body) of the 4th instar larvae of R. ferrugineus were assayed by RT-qPCR. The abundance of RfCTL-S1 transcripts in the fat body and gut 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 was also detected by RT-qPCR. Moreover, the expression levels of the antimicrobial peptide genes, including RfAttacin, RfCecropin, RfColeopericin and RfDefensin, in the fat body and gut of the 4th instar larvae of R. ferrugineus after RNAi of RfCTL-S1 were also quantified by RT-qPCR. After RfCTL-S1 was silenced by RNAi, the ability of R. ferrugineus individuals to clear the invaded EGFP-tagged E. coli in the hemolymph and the number of cultivable bacterial colonies in the gut of the 4th instar larvae of R. ferrugineus were evaluated. 【Results】 The bioinformatics analysis revealed that RfCTL-S1 contains a signal peptide and the carbohydrate recognition domain (CRD) with EPN motif, but without transmembrane domain, suggesting that RfCTL-S1 is a secretory protein with mannose-binding activity. RT-qPCR results showed that RfCTL-S1 was highly expressed in the hemolymph of the 4th instar larvae of R. ferrugineus. In the fat body of R. ferrugineus larvae at 24 h after injection of E. coli, the expression level of RfCTL-S1 was significantly down-regulated as compared with that of the control injected with PBS. The expression level of RfCTL-S1 in the fat body of R. ferrugineus larvae at 6 h after injection of S. aureus was significantly higher than that at 24 h after injection of S. aureus. In the gut of R. ferrugineus larvae, the expression level of RfCTL-S1 at 12 h after challenge with S. aureus was significantly higher than those at 6 and 24 h after challenge with S. aureus. After RfCTL-S1 was silenced by RNAi, the expression levels of three antimicrobial peptide genes RfCecropin, RfColeoptericin and RfDefensin in the fat body of R. ferrugineus larvae were significantly reduced, and the ability of R. ferrugineus larvae to clear the invaded EGFP-tagged E. coli in the hemolymph was significantly impaired as compared to the control. However, no significant difference was determined in the number of cultivable bacterial colonies in the larval gut between the RNAi group and the control group. 【Conclusion】 Secretory RfCTL-S1 protein can mount the expression of antimicrobial peptide genes by recognizing bacteria and activating the corresponding immune signaling pathways in the fat body of R. ferrugineus larvae, thus to eliminate invaded pathogens.

Key words: Rhynchophorus ferrugineus, C-type lectins, innate immunity, antimicrobial peptide, pathogen, RNA interference