A C-Type Lectin, RfCTL27, Activates the Immune Defense in the Red Palm Weevil Rhynchophorus ferrugineus (A.G. Olivier, 1791) (Coleoptera: Curculionidae: Dryophthorinae) by the Recognition of Gram-Negative Bacteria.

Red palm weevil, Rhynchophorus ferrugineus (Olivier), is a palm tree insect pest that causes significant damage in the many countries from the Indian sub-continent and southeast Asia into date palm-growing countries of Africa, the Middle East, and the Mediterranean Basin. This study is aimed at determining the role of a C-type lectin, RfCTL27, in the immune defense of RPW larvae. RfCTL27 is a secreted protein that possesses a QPD motif, being integral for the discrimination of Gram-negative bacteria. The abundance of RfCTL27 transcripts in the gut and fat body was significantly higher than that in other tissues. Six hours after injection of Escherichia coli, the expression level of RfCTL27 in the gut of RPW larvae was significantly elevated compared with other groups. At 12 h after injection of E. coli, the expression of RfCTL27 in fat body was dramatically induced in contrast with other treatments. More interestingly, the ability of RPW larvae to clear the pathogenic bacteria in the body cavity and gut was markedly impaired by the silencing of RfCTL27. Additionally, the expression levels of two antimicrobial peptide genes, RfCecropin in the gut and RfDefensin in fat body of RPW larvae, were significantly decreased. Taken together, these data suggested that RfCTL27 can recognize the Gram-negative bacterium and activate the expression of antimicrobial peptides to remove the invaded bacterial pathogens. This study provides a new scientific basis for improving the control efficiency of pathogenic microorganisms against red palm weevils in production practice.

A peptidoglycan recognition protein regulates the immune response of Rhynchophorus ferrugineus Olivier (Coleoptera: Dryophthoridae) during exposure to pathogenic Gram-positive bacteria and fungi.

Red palm weevil (RPW), Rhynchophorus ferrugineus Olivier, is a tremendously destructive insect pest of palm trees worldwide. Although some biological agents have been used to fight against RPW larvae, the control efficiency is still dissatisfactory. This study aimed to determine the role of a peptidoglycan recognition protein (PGRP), RfPGRP-S3, in RPW immunity. RfPGRP-S3 is a secreted protein with a DF (Asp85-Phe86) motif, implying that it can discriminate Gram-positive bacteria. The abundance of RfPGRP-S3 transcripts in the hemolymph was significantly higher than that in other tissues. The expression of RfPGRP-S3 can be markedly induced by challenge with Staphylococcus aureus and Beauveria bassiana. After RfPGRP-S3 was silenced, the ability of individuals to clear the pathogenic bacteria in the body cavity and gut was significantly compromised. Furthermore, silencing RfPGRP-S3 dramatically impaired the survival rate of RPW larvae upon challenge with S. aureus. RT‒qPCR revealed that the expression levels of RfDefensin in the fat body and gut were decreased by RfPGRP-S3 silencing. Taken together, these results demonstrated that RfPGRP-S3 acts as a circulating receptor to promote the expression of the antimicrobial peptide gene upon the discrimination of pathogenic microbes.

Current understanding and perspectives on the potential mechanisms of immune priming in beetles.

Beetles are the most diverse group of insects in Insecta which can be found in almost every habitat and environment on Earth. The possessing of the rapid and effective immune defenses is one of the important factors for their success. It is generally recognized that beetles only rely on the non-specific innate immune defense, without immunological memory, to fight against pathogens. However, there was cumulative evidence for the innate immune memory in invertebrates, including beetles, over the last decades, implying that insect innate immunity is more complex and has more features than previously thought. In beetles, it has been well documented that the specific or nonspecific enhanced immunocompetence can persist throughout development within generations and can even be transferred to the descendents in the next generation. Although insect immune priming might be shaped by epigenetic modifications and transferring effectors, mRNA and microbial signals, the solid experimental evidence to support the causal relationship between any of them and immune priming is still scarce. The combined usage of 'omics' approaches and CRISPR/Cas9 in the appropriate insect models with well-known genetic background, Tribolium castaneum and Tenebrio molitor, will help us to decipher the molecular mechanisms by which immune priming occurs in beetles in depth.

The Effect of Gut Bacteria on the Physiology of Red Palm Weevil, Rhynchophorus ferrugineus Olivier and Their Potential for the Control of This Pest.

Red Palm Weevil (RPW), Rhynchophorus ferrugineus Olivier, is a notorious pest, which infests palm trees and has caused great economic losses worldwide. At present, insecticide applications are still the main way to control this pest. However, pesticide resistance has been detected in the field populations of RPW. Thus, future management strategies based on the novel association biological control need be developed. Recent studies have shown that the intestinal tract of RPW is often colonized by multiple microbial species as mammals and model insects, and gut bacteria have been found to promote the growth, development and immune activity of RPW larvae by modulating nutrient metabolism. Furthermore, two peptidoglycan recognition proteins (PGRPs), PGRP-LB and PGRP-S1, can act as the negative regulators to modulate the intestinal immunity to maintain the homeostasis of gut bacteria in RPW larvae. Here, we summarized the current knowledge on the gut bacterial composition of RPW and their impact on the physiological traits of RPW larvae. In contrast with metazoans, it is much easier to make genetic engineered microbes to produce some active molecules against pests. From this perspective, because of the profound effects of gut bacteria on host phenotypes, it is promising to dissect the molecular mechanisms behind their effect on host physiology and facilitate the development of microbial resource-based management methods for pest control.

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).

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.

Intestinal Microbiota Confer Protection by Priming the Immune System of Red Palm Weevil Rhynchophorus ferrugineus Olivier (Coleoptera: Dryophthoridae).

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.

The Promoting Effect of Gut Microbiota on Growth and Development of Red Palm Weevil, Rhynchophorus ferrugineus (Olivier) (Coleoptera: Dryophthoridae) by Modulating Its Nutritional Metabolism.

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.

An IMD-like pathway mediates the intestinal immunity to modulate the homeostasis of gut microbiota in Rhynchophorus ferrugineus Olivier (Coleoptera: Dryophthoridae).

Most animals have established the mutualistic interactions with their intestinal microbes which provide multiple benefits to their host physiology. However, the mechanisms behind hosts determine the load and composition of gut microbiota are still poorly understood outside dipteran insects. Here, the gene, encoding the NF-κB-like transcription factor Relish, being designated as RfRelish, was identified and analyzed in red palm weevil (RPW), Rhynchophorus ferrugineus Olivier. We revealed that the abundance of RfRelish transcripts in the fat body, hemolymph and gut are significantly higher than that in non-immunity-related tissues, and its expression level can be markedly induced by bacterial challenges. When RfRelish was silenced, the ability of individuals to clear the pathogenic bacteria in body cavity and gut was significantly compromised, suggesting that both the systemic and gut local immunity were impaired dramatically by RfRelish knockdown. Additionally, the silenced insects exhibited increased gut bacterial load, and the relative abundance of some gut bacteria was changed as compared to controls. Collectively, our findings demonstrate that the IMD-like pathway restricts the proliferation of gut bacteria and shapes the commensal community structure in the intestine of R. ferrugineus by mediating the secretion of antimicrobial peptides. We provide a striking example on how an insect pest maintains the homeostasis of gut microbiota via a conserved immune pathway without compromising the advantages of the mutualistic relationships.

Preparation of Red Palm Weevil Rhynchophorus Ferrugineus (Olivier) (Coleoptera: Dryophthoridae) Germ-free Larvae for Host-gut Microbes Interaction Studies.

Red palm weevil (RPW), Rhynchophorus ferrugineus Olivier, is a devastating pest of palm trees worldwide. RPW gut is colonized by diverse bacterial species which profoundly influence host development and nutritional metabolism. However, the molecular mechanisms behind the interactions between RPW and its gut microbiota remain mostly unknown. Antibiotics are usually employed to remove gut bacteria to investigate the impact of gut bacteria on insect fitness. However, administration of antibiotics cannot thoroughly remove gut bacteria for most insect species. Therefore, establishing germfree (GF) organisms is a powerful way to reveal the mutual interactions between gut bacteria and their insect hosts. Here, we describe a protocol to generate and maintain RPW GF larvae, being completely devoid of gut bacteria in laboratory. RPW GF larvae were established from the dechorionated fresh eggs which were reared on the sterilized artificial food under axenic conditions. The establishment of GF larvae set a solid foundation to deeply elucidate the molecular mechanisms behind the interactions between RPW and its gut microbiota.

PGRP-LB homolog acts as a negative modulator of immunity in maintaining the gut-microbe symbiosis of red palm weevil, Rhynchophorus ferrugineus Olivier.

Many notorious insect pests live in the symbiotic associations with gut microbiota. However, the mechanisms underlying how they host their gut microbiota are unknown. Most gut bacteria can release peptidoglycan (PGN) which is an important antigen to activate the immune response. Therefore, how to keep the appropriate gut immune intensity to host commensals while to efficiently remove enteropathogens is vital for insect health. This study is aimed at elucidating the roles of an amidase PGRP, Rf PGRP-LB, in maintaining the gut-microbe symbiosis of Red palm weevil (RPW), Rhynchophorus ferrugineus Olivier. RfPGRP-LB is a secreted protein containing a typical PGRP domain. The existence of five conservative amino acid residues, being required for amidase activity, showed that RfPGRP-LB is a catalytic protein. Expression analysis revealed abundance of RfPGRP-LB transcripts in gut was dramatically higher than those in other tissues. RfPGRP-LB could be significantly induced against the infection of Escherichia coli. In vitro assays revealed that rRfPGRP-LB impaired the growth of E. coli and agglutinated bacteria cells obviously, suggesting RfPGRP-LB is a pathogen recognition receptor and bactericidal molecule. RfPGRP-LB knockdown reduced the persistence of E. coli in gut and load of indigenous gut microbiota significantly. Furthermore, the community structure of indigenous gut microbiota was also intensively altered by RfPGRP-LB silence. Higher levels of the antimicrobial peptide, attacin, were detected in guts of RfPGRP-LB silenced larvae than controls. Collectively, RfPGRP-LB plays multiple roles in modulating the homeostasis of RPW gut microbiota not only by acting as a negative regulator of mucosal immunity through PGN degradation but also as a bactericidal effector to prevent overgrowth of commensals and persistence of noncommensals.

The Gut Entomotype of Red Palm Weevil Rhynchophorus ferrugineus Olivier (Coleoptera: Dryophthoridae) and Their Effect on Host Nutrition Metabolism.

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.

Pine Defensive Monoterpene α-Pinene Influences the Feeding Behavior of Dendroctonus valens and Its Gut Bacterial Community Structure.

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.

Host selection behavior and the fecundity of Plutella xylostella (Lepidoptera: Plutellidae) on multiple host plants.

Insect herbivores often have higher densities on host plants grown in monocultures than those in diverse environments. The underlying mechanisms are thought to be that polyphagous insects have difficulty in selecting food or oviposition sites when multiple host plants exist. However, this hypothesis needs to be extensively investigated. Our field experiments revealed that the population of the diamondback moths, Plutella xylostella (L.) (Lepidoptera: Plutellidae), significantly decreased in a mixed cropping field compared with a monoculture. To determine the reasons for the reduction in population in the mixed cropping field, the takeoff behavior and fecundity of females in no-choice and free-choice laboratory environments were compared by video recordings of host selection by P. xylostella. Adults displayed a significantly higher takeoff frequency in free-choice environments than those in no-choice treatments and preferred landing on Brassica campestris (L.) or Brassica juncea (Coss) plants in contrast with Brassica oleracea (L.). Female adults in the free-choice environment also laid fewer eggs compared with the monoculture. Olfaction experiments demonstrated orientation by P. xylostella to host volatiles when presented with a choice between plant odors and clean air, but females showed no preference when odors from three Brassicaceae species were presented simultaneously. We conclude that mixed cropping alters the host-finding behavior of P. xylostella resulting in reduced oviposition.

Low diversity bacterial community and the trapping activity of metabolites from cultivable bacteria species in the female reproductive system of the Oriental fruit fly, Bactrocera dorsalis Hendel (Diptera: Tephritidae).

Our goal was to identify the bacteria inhabiting the reproductive system of the female oriental fruit fly, Bactrocera dorsalis (Hendel), and evaluate the chemotaxis of B. dorsalis to the metabolites produced by the bacteria. Based on 16S rRNA-based polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), 18 operational taxonomic units (OTUs) were assigned to the five bacterial classes Betaproteobacteria, Alphaproteobacteria, Gammaproteobacteria, Bacilli and Actinobacteria. Nine OTUs were assigned to Gammaproteobacteria, which was the most highly represented class. Enterobacteriaceae constituted the dominant family, and within this family, three genera and five species were identified, including Enterobacter sakazakii, Klebsiella oxytoca, Klebsiella pneumoniae, Raoultella terrigena and Enterobacter amnigenus. In this set, the first two species were the dominant components, and the latter three species were the minor ones. Finally, we found that the metabolites produced by R. terrigena, K. oxytoca and K. pneumoniae were attractive to the B. dorsalis adults, and in field studies, B. dorsalis adults were most attracted to K. oxytoca. Collectively, our results suggest that the female reproductive system plays an important role in the transfer of enterobacteria from the gut to fruit. Our data may prompt the development of a female-targeted population control strategy for this fly.

Immunocompetence of the red turpentine beetle, Dendroctonus valens LeConte (Coleoptera: Curculionidae, Scolytinae): variation between developmental stages and sexes in populations in China.

Immune defense imposes fitness costs as well as benefits, so organisms should optimize, not maximize, their immune function through their life cycle. We investigated this issue in the red turpentine beetle, Dendroctonus valens LeConte (Coleoptera: Curculionidae, Scolytinae), which is a pine-killing invasive beetle in China, though it is usually considered as a secondary pest in its native range of North America. We hypothesized that pathogen pressure may affect these beetles differently throughout their life history. We measured the insect's immunocompetence throughout life, determining encapsulation ability and phenoloxidase activity in larval stages, pupae and adults. Pupae had the highest encapsulation ability, but encapsulation was not different between final instar larvae and adults. Phenoloxidase (PO) activity was highest in final instar larvae and pupae, followed by the second instar larvae and adults. Total phenoloxidase activity increased significantly from the second instar larval stage to pupae, and then decreased in adults. Although the second instar larvae had the lowest phenoloxidase activity, more than 90% of total PO existed in the hemolymph in the form of the active enzyme, as compared with pupae, in which over 60% of PO occurred as a proenzyme. Both active PO and total PO were much higher in females than in males, though no significant differences were detected between the encapsulation ability of male and female adults. This result suggests the existence of a sexual dimorphism of immunocompetence in D. valens adults. Variations in immunocompetence across developmental stages suggest that D. valens adopts diverse investment strategies in immunocompetence during different stages. Potential reasons for variation in immunocompetence among developmental stages and between the sexes of D. valens are discussed.