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.

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The majority of insects rely on a highly complex and precise olfactory system to detect various volatile organic compounds released by host and non-host plants in environments. The odorant receptors (ORs) are considered to play an important role in odor recognition and the molecular basis of ORs, particularly in coleopterans they are relatively poorly understood. The red palm weevil (RPW), Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae), is one of the most destructive pests of the global palm industry. Although feeding and egg oviposition behaviors of RPW can be repelled by some non-palm plant volatiles, such as α-pinene, geraniol, or 1-octen-3-ol, there is limited understanding of how RPW recognizes the non-host plant volatiles. In this study, three candidate RferOrs were identified from the Rfer-specific clade, and the tissue expression analysis used was mainly expressed in the antennae of both sexes. Functional characterization of RferOr6, RferOr40, and RferOr87 was analyzed by using the Xenopus oocyte expression system, and the results indicated that RferOr6/RferOrco was narrowly tuned to α-pinene. The behavioral experiment showed that α-pinene at the concentrations of 10 and 100 μg/μl can cause a significantly repelled behavioral response of RPW. In conclusion, this study reveals that RferOr6 is an antenna-biased expressed OR used by RPW to detect the volatile compound α-pinene in non-palm plants, and our results provide a foundation for further in vivo functional studies of Or6 in RPW, including in vivo knockout/knockdown and feeding/ovipositing behavioral studies of RPW and further pest control.