Swift regulation of nicotinic acetylcholine receptors (nAChRs) and glutathione S-transferase (GST) enables the rapid detoxification of thiacloprid in pine sawyer beetles.

Thiacloprid, a neonicotinoid insecticide, has become one of the major control agents for the pine sawyer beetle, Monochamus alternatus Hope, however, the mechanism of detoxification is unknown. We demonstrate that glutathione S-transferases (GSTs) and nicotinic acetylcholine receptors (nAChRs) are involved in the rapid detoxification of thiacloprid in M. alternatus larvae. The activity of detoxification enzyme GSTs was significantly higher, while the activity of acetylcholinesterase (AChE) was inhibited under thiacloprid exposure. The inhibition of AChE activity led to lethal over-stimulation of the cholinergic synapse, which was then released by the rapid downregulation of nAChRs. Meanwhile, GSTs were overexpressed to detoxify thiacloprid accordingly. A total of 3 nAChR and 12 GST genes were identified from M. alternatus, among which ManAChRα2 and MaGSTs1 were predicted to confer thiacloprid tolerance. RNA interference (RNAi) was subsequently conducted to confirm the function of ManAChRα2 and MaGSTs1 genes in thiacloprid detoxification. The successful knock-down of the ManAChRα2 gene led to lower mortality of M. alternatus under LC30 thiacloprid treatment, and the suppression of the MaGSTs1 gene increased the mortality rate of M. alternatus. However, the mortality rate has no significant difference with controls when thiacloprid was fed together with both dsMaGSTs1 and dsManAChRα2. Molecular docking modeled the molecular basis for interaction between MaGSTs1/ManAChR and thiacloprid. This study highlights the important roles that ManAChRα2 and MaGSTs1 genes play in thiacloprid detoxification through transcriptional regulation and enzymatic metabolization, and proposes a new avenue for integrated pest management that combines pesticides and RNAi technology as an efficient strategy for M. alternatus control.

Associated bacteria of a pine sawyer beetle confer resistance to entomopathogenic fungi via fungal growth inhibition.

BACKGROUND: The entomopathogenic Beauveria bassiana is a popular fungus used to control the Japanese pine sawyer, Monochamus alternatus Hope, the key vector of pine wood nematode (Bursaphelenchus xylophilus) that is the causal agent of pine wilt disease, resulting in devastating losses of pines in China and Portugal. However, recent studies have demonstrated that some insect-associated bacteria might decrease fungal toxicity and further undermine its biological control efficacy against M. alternatus. Thus, it is of great significance to uncover whether and how associated bacteria of M. alternatus become involved in the infection process of B. bassiana. RESULTS: Here, we show that axenic M. alternatus larvae died significantly faster than non-axenic larvae infected by four increasing concentrations of B. bassiana spores (Log-rank test, P < 0.001). The infection of B. bassiana significantly changed the richness and structure of the beetle-associated bacterial community both on the cuticle and in the guts of M. alternatus; meanwhile, the abundance of Pseudomonas and Serratia bacteria were significantly enriched as shown by qPCR. Furthermore, these two bacteria genera showed a strong inhibitory activity against B. bassiana (One-way ANOVA, P < 0.001) by reducing the fungal conidial germination and growth rather than regulating host immunity. CONCLUSIONS: This study highlights the role of insect-associated bacteria in the interaction between pest insects and entomopathogenic fungi, which should be taken into consideration when developing microbial-based pest control strategies.

Positive effects of the tea catechin (-)-epigallocatechin-3-gallate on gut bacteria and fitness of Ectropis obliqua Prout (Lepidoptera: Geometridae).

Ectropis obliqua Prout (Lepidoptera: Geometridae) is the most devastating insect pest of tea plants in China and infests thousands of hectares of tea plantations in China annually. (-)-Epigallocatechin-3-gallate (EGCG) is a major phenolic compound in tea leaves and has a strong antibacterial function. Here, we show that EGCG can effectively improve the fitness of E. obliqua larvae and present the reason by which EGCG promotes larval fitness. In this study, we compared the fitness difference among Control, Antibiotic and Treatment of larvae. The fitness of larvae treated with EGCG and antibiotic was similar and better than that of control group. We also demonstrated that EGCG treatment could significantly reduce species richness and abundance of gut bacteria in E. obliqua larvae. Hence that we speculate that EGCG promotes larval fitness and is associated with ECGG antimicrobial activity. In short, our study provides evidence of the E. obliqua larvae have adapted to secondary compounds found in tea leaves, and may even benefit from these compounds. Our study also contributes to a greater understanding of the reason involved in plant-insect interactions.

Comparative analysis of the immune system of an invasive bark beetle, Dendroctonus valens, infected by an entomopathogenic fungus.

Dendroctonus valens LeConte is one of the most economically important forest pest in China. Leptographium procerum, a mutualistic fungus can assist the host beetle in overcoming the pine's chemical defenses, and Beauveria bassiana, an entomopathogenic fungus has shown high beetle killing efficiency. Considering that the D. valens immune system remains unknown at the genomic level, a mutualistic and antagonistic fungus associated with the beetle provides an ideal model for studying immune interactions between the insect and associated fungi. Here, B. bassiana killed most tested larvae more effectively than L. procerum and Tween. The entomopathogenic fungus provoked stronger responses than the symbiotic fungus at the transcriptome level. We identified 185 immunity-related genes, including pattern recognition receptors, signal modulators, members of immune pathways (Toll, IMD, and JAK/STAT), and immune effectors. Quantitative real-time PCR analysis confirmed that several recognition receptors and effector genes were activated at 1 or 2 days post infection, while the effector genes were suppressed at 4 days post infection by B. bassiana, respectively. In contrast, effector genes were upregulated in response to L. procerum. Together, this study provides a comprehensive sequence resource and insight into the D. valens immune system and lays a basis for understanding the molecular aspects of the interaction between the host and associated fungi.

Identification and Expression Patterns of Anoplophora chinensis (Forster) Chemosensory Receptor Genes from the Antennal Transcriptome.

The citrus long-horned beetle (CLB), Anoplophora chinensis (Forster) is a destructive native pest in China. Chemosensory receptors including odorant receptors (ORs), gustatory receptors (GRs), and ionotropic receptors (IRs) function to interface the insect with its chemical environment. In the current study, we assembled the antennal transcriptome of A. chinensis by next-generation sequencing. We assembled 44,938 unigenes from 64,787,784 clean reads and annotated their putative gene functions based on gene ontology (GO) and Clusters of Orthologous Groups of proteins (COG). Overall, 74 putative receptor genes from chemosensory receptor gene families, including 53 ORs, 17 GRs, and 4 IRs were identified. Expression patterns of these receptors on the antennae, maxillary and labial palps, and remaining body segments of both male and female A. chinensis were performed using quantitative real time-PCR (RT-qPCR). The results revealed that 23 ORs, 6 GRs, and 1 IR showed male-biased expression profiles, suggesting that they may play a significant role in sensing female-produced sex pheromones; whereas 8 ORs, 5 GRs, and 1 IR showed female-biased expression profiles, indicating that these receptors may be involved in some female-specific behaviors such as oviposition site seeking. These results lay a solid foundation for deeply understanding CLB olfactory processing mechanisms. Moreover, by comparing our results with those from chemosensory receptor studies in other cerambycid species, several highly probable pheromone receptor candidates were highlighted, which may facilitate the identification of additional pheromone and/or host attractants in CLB.