Spätzle Homolog-Mediated Toll-Like Pathway Regulates Innate Immune Responses to Maintain the Homeostasis of Gut Microbiota in the Red Palm Weevil, Rhynchophorus ferrugineus Olivier (Coleoptera: Dryophthoridae)

Abstract

Spätzle (Spz) is a dimeric ligand that responds to the Gram-positive bacterial or fungal infection by binding Toll receptors to induce the secretion of antimicrobial peptides. However, whether the Toll-like signaling pathway mediates the innate immunity of <i>Rhynchophorus ferrugineus</i> to modulate the homeostasis of gut microbiota has not been determined. In this study, we found that a Spz homolog, RfSpätzle, is a secretory protein comprising a signal peptide and a conservative Spz domain. RT-qPCR analysis revealed that <i>RfSpätzle</i> was significantly induced to be expressed in the fat body and gut by the systemic and oral infection with pathogenic microbes. The expression levels of two antimicrobial peptide genes, <i>RfColeoptericin</i> and <i>RfCecropin</i>, were downregulated significantly by <i>RfSpätzle</i> knockdown, indicating that their secretion is under the regulation of the <i>RfSpätzle</i>-mediated signaling pathway. After being challenged by pathogenic microbes, the cumulative mortality rate of <i>RfSpätzle</i>-silenced individuals was drastically increased as compared to that of the controls. Further analysis indicated that these larvae possessed the diminished antibacterial activity. Moreover, <i>RfSpätzle</i> knockdown altered the relative abundance of gut bacteria at the phylum and family levels. Taken together, these findings suggest that <i>RfSpätzle</i> is involved in RPW immunity to confer protection and maintain the homeostasis of gut microbiota by mediating the production of antimicrobial peptides.