Organophosphate insecticides that are heavily used in agriculture for pest control have caused growing environmental problems and public health concerns worldwide. Ironically, insecticide resistance develops quickly in major lepidopteran pests, partially via their microbial symbionts. To investigate the possible mechanisms by which the microbiota confers insecticide resistance to Lepidoptera, the model organism silkworm Bombyx mori (Lepidoptera: Bombycidae) was fed different antibiotics to induce gut dysbiosis (microbiota imbalance). Larvae treated with polymyxin showed a significantly lower survival rate when exposed to chlorpyrifos. Through high-throughput sequencing, we found that the abundances of Stenotrophomonas and Enterococcus spp. changed substantially after treatment. To assess the roles played by these two groups of bacteria in chlorpyrifos resistance, a germ-free (GF) silkworm rearing protocol was established to avoid the influence of natural microbiota and antibiotics. Monoassociation of GF silkworms with Stenotrophomonas enhanced host resistance to chlorpyrifos, but not in Enterococcus-fed larvae, consistent with larval detoxification activity. GC-μECD detection of chlorpyrifos residues in feces indicated that neither Stenotrophomonas nor Enterococcus degraded chlorpyrifos directly in the gut. However, gut metabolomics analysis revealed a highly species-specific pattern, with higher levels of essential amino acid produced in the gut of silkworm larvae monoassociated with Stenotrophomonas. This critical nutrient provisioning significantly increased host fitness and thereby allowed larvae to circumvent the deleterious effects of these toxic chemicals more efficiently. Altogether, our study not only suggests a new mechanism for insecticide resistance in notorious lepidopteran pests but also provides a useful template for investigating the interplay between host and gut bacteria in complex environmental systems.

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.