For invasive insects, the potential roles of gut microbiota in exploiting new food resources and spreading remain elusive. Red palm weevil (RPW), Rhynchophorus ferrugineus Olivier, is an invasive destructive pest which feeds on nutrient-poor tender tissues and has caused extensive mortality of palm trees. The microbes associated with insects can improve their nutrition assimilation. However, experimental evidence on the interactions between RPW and its gut microbiota is still absent. The aim of this study is to determine the dynamics changes and the bacterial entomotype in the RPW gut and its potential physiological roles. Here, we confirmed RPW harbors a complex gut microbiota mainly constituted by bacteria in the families Enterobacteriaceae, Lactobacillaceae, Entomoplasmataceae, and Streptococcaceae. RPW gut microbiota exhibited a highly stable microbial community with low variance in abundance across different life stages and host plants. Furthermore, the abundance of Enterobacteriaceae was markedly increased but that of Acetobacteraceae was reduced significantly after administration of antibiotics. Although no significant effects were found on the body weight gain of RPW larvae, these alterations dramatically decreased the concentration of hemolymph protein and glucose while that of hemolymph triglyceride increased. In the gut of wild-caught RPW larvae, seven bacterial species in the genera Klebsiella, Serratia, Enterobacter, and Citrobacter were shown to have an ability to degrade cellulose. Together, RPW accommodate a stable gut microbiota which can degrade plant polysaccharides and confer their host optimal adaptation to its environment by modulating its metabolism.

The immune system of animals, including insects, is the vital factor to maintain the symbiotic interactions between animals and their associated microbes. However, the effects of gut microbiota on insect immunity remain mostly elusive. Red palm weevil (RPW), Rhynchophorus ferrugineus Olivier, is a destructive pest of palm trees worldwide, which has forged alliances with its gut microbiota. Here, we found that the aposymbiotic insects succumbed at a significantly faster rate than conventionally reared (CR) ones upon bacterial infection. Physiological assays confirmed that CR insects had stronger antimicrobial activity and higher phenoloxidase activity in contrast to germfree (GF) ones, indicating that the systemic immune responses of GF individuals were compromised markedly. Interestingly, under the bacterial challenge conditions, the reassociation of gut microbiota with GF insects could enhance their survival rate by rescuing their immunocompetence. Furthermore, comparative transcriptome analysis uncovered that 35 immune-related genes, including pathogen recognition receptors, effectors and immune signaling pathway, were significantly downregulated in GF insects as compared to CR ones. Collectively, our findings corrobate that intestinal commensal bacteria have profound immunostimulatory effects on RPW larvae. Therefore, knowledge on the effects of gut microbiota on RPW immune defenses may contribute to of set up efficient control strategies of this pest.

Red palm weevil (RPW), Rhynchophorus ferrugineus Olivier, is a destructive pest for palm trees worldwide. Recent studies have shown that RPW gut is colonized by microbes and alterations in gut microbiota can significantly modify its hemolymph nutrition content. However, the exact effects of gut microbiota on RPW phenotype and the underlying mechanisms remain elusive. Here germ-free (GF) RPW larvae were generated from dechorionated eggs which were reared on sterilized artificial food under axenic conditions. Compared with controls, the larval development of GF RPW individuals was markedly depressed and their body mass was reduced as well. Furthermore, the content of hemolymph protein, glucose and triglyceride were dropped significantly in GF RPW larvae. Interestingly, introducing gut microbiota into GF individuals could significantly increase the levels of the three nutrition indices. Additionally, it has also been demonstrated that RPW larvae monoassociated with Lactococcus lactis exhibited the same level of protein content with the CR (conventionally reared) insects while feeding Enterobacter cloacae to GF larvae increased their hemolymph triglyceride and glucose content markedly. Consequently, our findings suggest that gut microbiota profoundly affect the development of this pest by regulating its nutrition metabolism and different gut bacterial species show distinct impact on host physiology. Taken together, the establishment of GF and gnotobiotic RPW larvae will advance the elucidation of molecular mechanisms behind the interactions between RPW and its gut microbiota.

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 Rhynchophorus ferrugineus 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 RfSpätzle 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, RfColeoptericin and RfCecropin, were downregulated significantly by RfSpätzle knockdown, indicating that their secretion is under the regulation of the RfSpätzle-mediated signaling pathway. After being challenged by pathogenic microbes, the cumulative mortality rate of RfSpätzle-silenced individuals was drastically increased as compared to that of the controls. Further analysis indicated that these larvae possessed the diminished antibacterial activity. Moreover, RfSpätzle knockdown altered the relative abundance of gut bacteria at the phylum and family levels. Taken together, these findings suggest that RfSpätzle is involved in RPW immunity to confer protection and maintain the homeostasis of gut microbiota by mediating the production of antimicrobial peptides.

The exposure to plant defense chemicals has negative effects on insect feeding activity and modifies insect gut microbial community composition. Dendroctonus valens is a very destructive forest pest in China, and harbors a large diversity and abundance of gut microorganisms. Host pine defensive chemicals can protect the pines from attack by the holobiont. In this study, boring length of D. valens feeding on 0 mg/g α-pinene and 9 mg/g α-pinene concentration in phloem media for 6 and 48 h were recorded, and their gut bacterial communities were analyzed in parallel. Nine milligram per gram α-pinene concentration significantly inhibited boring length of D. valens and altered its gut microbial community structure after 6 h. The inhibition of boring length from 9 mg/g α-pinene in diets ceased after 48 h. No significant differences of the bacterial communities were observed between the beetles in 0 and 9 mg/g α-pinene concentration in phloem media after 48 h. Our results showed that the inhibition of the feeding behavior of D. valens and the disturbance to its gut bacterial communities in 9 mg/g α-pinene concentration in phloem media after 6 h were eliminated after 48 h. The resilience of gut bacterial community of D. valens may help the beetle catabolize pine defense chemical.