Biogenetic cantharidin is a promising leading compound to manage insecticide resistance of Mythimna separata (Lepidoptera: Noctuidae).

Cantharidin (CTD) is a natural toxin with effective toxicity to lepidopteran pests. Nevertheless, little information is available on whether pests develop resistance to CTD. After being exposed to CTD (50 mg/L to 90 mg/L) or 10 generations, the resistance ratio of laboratory selected cantharidin-resistant Mythimna separata (Cantharidin-SEL) strain was only elevated 1.95-fold. Meanwhile, the developmental time for M. separata was prolonged (delayed1.65 in males and 1.84 days in females). The reported CTD target, the serine/threonine phosphatases (PSPs), have not been shown significant activity variation during the whole process of CTD-treatment. The activity of detoxification enzymes (cytochrome monooxygenase P450, glutathione-S-transferase (GST) and carboxylesterase) were affected by CTD selection, but this change was not mathematically significant. More importantly, no obvious cross-resistance with other commonly used insecticides was observed in the M. separata population treated with CTD for 10 generations (resistance ratios were all lower 2.5). Overall, M. separata is unlikely to produce target-site insensitivity resistance, metabolic resistance to CTD. Meanwhile, cantharidin-SEL is not prone to develop cross-resistance with other insecticides. These results indicate that CTD is a promising biogenetic lead compound which can be applied in the resistance management on M. separata.

Identifying the Key Role of Plutella xylostella General Odorant Binding Protein 2 in Perceiving a Larval Attractant, (E,E)-2,6-Farnesol.

There is currently a lack of effective olfaction-based techniques to control diamondback moth (DBM) larvae. Identifying behaviorally active odorants for DBM larvae and exploring their recognition mechanisms can provide insights into olfaction-based larval control strategies. Through the two-choice assay, (E,E)-2,6-farnesol (farnesol) was identified as a compound exhibiting significant attractant activity toward DBM larvae, achieving an attraction index of 0.48 ± 0.13. PxylGOBP1 and PxylGOBP2, highly expressed in the antennae of DBM larvae, both showed high affinity toward farnesol. RNAi technology was used to knock down PxylGOBP1 and PxylGOBP2, revealing that the attraction of DBM larvae to farnesol nearly vanished following the knockdown of PxylGOBP2, indicating its critical role in recognizing farnesol. Further investigation into the PxylGOBP2-farnesol interaction revealed the importance of residues like Thr9, Trp37, and Phe118 in PxylGOBP2's binding to farnesol. This research is significant for unveiling the olfactory mechanisms of DBM larvae and developing larval behavior regulation techniques.

Key Contributions of the Overexpressed Plutella xylostella Sigma Glutathione S-Transferase 1 Gene (PxGSTs1) in the Resistance Evolution to Multiple Insecticides.

The overexpression of insect detoxification enzymes is a typical adaptive evolutionary strategy for insects to cope with insecticide pressure. In this study, we identified a glutathione S-transferase (GST) gene, PxGSTs1, that exhibited pronounced expression in the field-resistant population of Plutella xylostella. By using RNAi (RNA interference), the transgenic fly models, and quantitative real-time polymerase chain reaction (RT-qPCR) methods, we confirmed that the augmented expression of PxGSTs1 mediates the resistance of P. xylostella to various types of insecticides, including chlorantraniliprole, novaluron, λ-cyhalothrin, and abamectin. PxGSTs1 was found to bolster insecticide resistance in two ways: direct detoxification and enhancing antioxidative defenses. In addition, our findings demonstrated that pxy-miR-8528a exerts a pivotal influence on forming insecticide resistance in P. xylostella by downregulating PxGSTs1 expression. In summary, we elucidated the multifaceted molecular and biochemical underpinnings of PxGSTs1-driven insecticide resistance in P. xylostella. Our results provide a new perspective for understanding the insecticide resistance mechanism of P. xylostella.

The Mythimna separata general odorant binding protein 2 (MsepGOBP2) is involved in the larval detection of the sex pheromone (Z)-11-hexadecenal.

BACKGROUND: Mythimna separata is a notorious pest causing crop damages at the larval stage. Gaining insight into larval olfaction mechanisms would provide knowledge for olfaction-based management of M. separata larvae. RESULTS: In the present research, (Z)-11-hexadecenal (Z11-16: Ald), a major component of M. separata sex pheromone, was found to attract early-instar larvae of M. separata in a food context. Using a fluorescent binding assay, we found that M. separata general odorant binding protein 2 (MsepGOBP2) exhibited high binding affinity to Z11-16: Ald. Further, silencing of MsepGOBP2 resulted in a sharp reduction of the response to Z11-16: Ald, which could not be mitigated by increasing the concentration of Z11-16: Ald. Additionally, we employed molecular dynamics-based approaches to unravel the interaction details between MsepGOBP2 and Z11-16: Ald, specifically the binding of Z11-16: Ald to MsepGOBP2. CONCLUSION: Z11-16: Ald is attractive to early-instar larvae of M. separata, and MsepGOBP2 is identified to be indispensable in the larval detection of Z11-16: Ald. These results could aid in the development of olfaction-based methods for controlling M. separata in the larval stage. © 2023 Society of Chemical Industry.

Enabling high low-bias performance of Fe2O3 photoanode for photoelectrochemical water splitting.

Fe2O3 is a promising photoanode material used for photoelectrochemical water splitting due to its narrow bandgap and excellent stability in solution. However, because the nanorods shrink and coalesce when annealed under high temperatures, the charge separation and injection efficiencies are suppressed in the conventional nanocoral Fe2O3, resulting in its high bias potential and low photocurrent density. Herein, by improving the radial growth of FeOOH precursor, highly dispersed Fe2O3 nanorods could be prepared. It enabled them to have sufficient light-harvesting and short charge transport distance, high light absorption and charge separation/injection efficiencies, increased photocurrent density and reduced onset potential Von. The optimized Fe2O3 photoanodes obtained a remarkable low-bias photocurrent density of 0.84 mA cm-2 at 1.0 V versus reversible hydrogen electrode (vs. RHE). It was further improved to 1.36 mA cm-2 at 1.0 V vs. RHE with the Von reduced to 0.50 V vs. RHE when post-treated with a solvothermal method and loaded with NiOOH/FeOOH cocatalysts. The applied bias photo-to-current conversion efficiency was maximized to 0.45% at 0.84 V vs. RHE.

The chemosensory protein 1 contributes to indoxacarb resistance in Plutella xylostella (L.).

BACKGROUND: Insecticide resistance continuously poses a threat to agricultural production. Chemosensory protein-mediated resistance is a new mechanism of insecticide resistance discovered in recent years. In-depth research on resistance mediated by chemosensory proteins (CSPs) provides new insight into aid insecticide resistance management. RESULTS: Chemosensory protein 1 in Plutella xylostella (PxCSP1) was overexpressed in the two indoxacarb-resistant field populations and PxCSP1 has a high affinity with indoxacarb. PxCSP1 was upregulated when exposed to indoxacarb and the knockdown of this gene elevated sensitivity to indoxacarb, which demonstrate that PxCSP1 is involved in the indoxacarb resistance. Considering that CSPs may confer resistance in insects via binding or sequestering, we explored the binding mechanism of indoxacarb in PxCSP1-mediated resistance. Using molecular dynamics simulations and site-directed mutation, we found that indoxacarb forms a solid complex with PxCSP1 mainly through van der Waals interactions and electrostatic interactions. Between these, the electrostatic interaction provided by the Lys100 side chain in PxCSP1, and especially the hydrogen bonding between the NZ atom and the O of the carbamoyl carbonyl group of indoxacarb, are the key factors for the high affinity of PxCSP1 to indoxacarb. CONCLUSIONS: The overexpression of PxCPS1 and its high affinity to indoxacarb is partially responsible for indoxacarb resistance in P. xylostella. Modification of indoxacarb's carbamoyl group has the potential to alleviate indoxacarb resistance in P. xylostella. These findings will contribute to solving chemosensory protein-mediated indoxacarb resistance and provide a better understanding of the insecticide resistance mechanism. © 2023 Society of Chemical Industry.

Structural insights into the discrepant synergistic activity of Codlemone and (Z)-8-dodecenol towards Grapholita molesta pheromones.

BACKGROUND: Insect pheromone synergists have been widely used to produce potent pheromone products for environment-friendly pest control. Codlemone (Cod) and (Z)-8-dodecenol (Dod) are two major Grapholita molesta pheromone synergists, with Cod having greater synergism and affinity for G. molesta pheromone binding protein 2 (GmolPBP2). Uncovering structural information key to the different binding affinity of Cod and Dod to GmolPBP2 would gain insights into what causes their synergy activity discrepancy. RESULTS: Binding modes of the two synergists in the binding pocket of GmolPBP2 were analyzed and compared by molecular dynamics-based approaches. Although Cod and Dod were stabilized in a similar hydrophobic pocket, their interaction details with GmolPBP2 were divergent due to the extra double bond (C10═C11) in Cod. The C10═C11 improved the hydrophobic interactions of Cod with around residues. Such hydrophobic interaction improvement was also reflected in the raised importance of Phe11 in the GmolPBP2-Cod interaction. Not only that, the increased hydrophobic forces introduced by the C10═C11 changed the CH2-OH orientation in the GmolPBP2-Cod complex, which improved the H-bond interaction. Electrostatic complementarity analysis further indicated the positive role of C10═C11 in optimizing GmolPBP2-Cod interaction. CONCLUSION: The C10═C11 is thought to contribute greatly to Cod's stronger synergy as a group key to the higher GmolPBP2-affinity, based on which the improvement directions for Cod and Dod were addressed as well. Our findings will aid in the development and optimization of more effective pheromone synergists, resulting in more effective pheromone-based pest management.

Reverse chemical ecology guides the screening for Grapholita molesta pheromone synergists.

BACKGROUND: Pheromone-based management is a leading nonpesticidal strategy among integrated pest management options. Improving the potency of pheromone products by adding synergists would be a practical way to popularize pheromone-based management as well as to reduce pesticide use. RESULTS: Using reverse chemical ecology, synergists for Grapholita molesta sex pheromone were screened. Combined molecular docking and in vitro binding assay led to the determination of four potentially active odorants showing high affinity to G. molesta pheromone binding protein 2 (GmolPBP2). Thereafter, the high affinity between Codlemone and GmolPBP2 was further verified by exploration of GmolPBP2-Codlemone interactions. As the only sex pheromone synergist validated by both laboratory behavioral tests and field trapping, Codlemone was used to optimize commercial sex attractants currently used in G. molesta control. The recommended formulation [(Z)-8-dodecenyl acetate:(E)-8-dodecenyl acetate:Codlemone = 95:4:10] was found to trap about five to six times more G. molesta adults than the commercial sex attractant [(Z)-8-dodecenyl acetate:(E)-8-dodecenyl acetate: (Z)-8-dodecenol = 95:4:1]. CONCLUSION: Codlemone is an excellent pheromone synergist that can be potentially sensed by GmolPBP2, which can remarkably improve the potency of G. molesta sex attractants. It is believed that the introduction of reverse chemical ecology would increase the chance of discovering pheromone synergists, promoting the development of more efficacious pheromone products that can be used in controlling G. molesta and beyond. © 2021 Society of Chemical Industry.

Identification of key residues of carboxylesterase PxEst-6 involved in pyrethroid metabolism in Plutella xylostella (L.).

The long-term and excessive use of insecticides has led to severe environmental problems and the evolution of insecticide resistance in insects. Carboxylesterases (CarEs) are important detoxification enzymes conferring insecticide resistance on insects. Herein, the detoxification process of Plutella xylostella (L.) carboxylesterase 6 (PxEst-6), one representative P. xylostella carboxylesterase, is investigated with cypermethrin, bifenthrin, cyfluthrin and λ-cyhalothrin. RT-qPCR shows that PxEst-6 is highly expressed in the midgut and cuticles of the third instar larvae. Exposure to pyrethroid insecticides resulted in PxEst-6 up-regulation in a short time. Metabolic assays indicate that PxEst-6 has the capacity to metabolize these pyrethroid insecticides. The combination of molecular docking, binding mode analyses and alanine mutations demonstrated that His451, Lys458 and Gln431 were key residues of PxEst-6 for metabolizing pyrethroids and the acetate groups derived from pyrethroids were key sites for being metabolized by PxEst-6. H451- and K458-derived hydrogen bond (H-bond) interactions with the pyrethroid acetate groups and the polar interactions with the pyrethroid acetate group provided by the Q431 sidechain were crucial to the pyrethroids' metabolism by PxEst-6. Our study contributes to revealing the reasons for pyrethroid resistance in P. xylostella, and provides a fundamental basis for the development of novel pyrethroid insecticides.

Structural evidence for pheromone discrimination by the pheromone binding protein 3 from Plutella xylostella.

Insect pheromone binding proteins (PBPs) are believed to have a high degree of pheromone selectivity, acting as the first filter to discriminate specific pheromones from other volatile compounds. Herein, we provide evidence using homology-based model for the pheromone discrimination of Plutella xylostella pheromone binding protein 3 (PxPBP3). Combining molecular dynamics simulations and in vitro binding assays, two dominant sites are determined to be essential for the PxPBP3 to discriminate (Z)-11-hexadecenyl acetate (Hexadecenyl) from (Z)-11-hexadecenal (Hexadecenal). As the first key site for pheromone discrimination, Arg111 is indispensable to the PxPBP3-Hexadecenyl interaction. However, its importance in the binding of Hexadecenal to PxPBP3 is greatly reduced. A second site where pheromone discrimination occurs is a small loop (residues 34-38) in PxPBP3. It is shown that the hydrophobic strength provided by three hydrophobic residues (Phe34, Tyr37, and Trp38) in the small loop is significantly biased in the two complexes formed by PxPBP3 and the two pheromones. The discrimination capacity of PxPBP3 indicates that the P. xylostella pheromones may not share the same peri-receptor pathway, although they both show high affinity to PxPBP3.

Structure dynamics reveal key residues essential for the sense of 1-dodecanol by Cydia pomonella pheromone binding protein 2 (CpomPBP2).

BACKGROUND: Cydia pomonella, a worldwide quarantine fruit pest, causes great damage to fruit production every year. Sex pheromone-mediated control of C. pomonella has been widely used. As an indispensable ingredient of commercial sex attractants, 1-dodecanol (Dod) works to synergize the effect of codlemone in attracting male moths of C. pomonella. The interactions between Dod and its transporter protein, C. pomonella pheromone-binding protein 2 (CpomPBP2), provide inspiration for chemical optimizations to improve the synergistic effects of Dod. RESULTS: In this research, molecular simulations and biological verifications were used in combination to uncover key residues in CpomPBP2 essential for sensing Dod. After performing 150 ns molecular dynamics (MD) simulations, the C1-C12 chain of Dod was found to be locked by the van der Waals energy contributed by the hydrophobic residues Phe12, Leu68, and Ile113, whereas the -OH part of Dod was anchored by the H-bond derived from Glu98 and the salt-bridge derived from Arg109. Because of the importance of these two electrostatic interactions, Glu98 and Arg109 were further verified as key residues in determining the binding affinity between Dod and CpomPBP2. In addition, interactions unfavorable to the binding of Dod were described. CONCLUSION: The research detailed the discovery of key residues involved in CpomPBP2-Dod interactions. Our results provide guidance and caution for the prospective discovery, optimization, and design of novel chemicals with a similar or stronger synergistic effect to codlemone in controlling C. pomonella.

Structural Insights into Two Representative Conformations of the Complex Formed by Grapholita molesta (Busck) Pheromone Binding Protein 2 and Z-8-Dodecenyl Acetate.

Grapholita molesta is a notorious fruit borer globally, causing severe damage to fruit production. To control the pest, one commonly used mean is pheromone-mediated management. As an important sex pheromone, Z-8-dodecenyl acetate (Z8-12: Ac), is often coformulated with other active ingredients to regulate the behavior of G. molesta. To uncover its interactions with G. molesta pheromone binding protein 2 (GmolPBP2) is used to help develop insect attractants. During 200 ns molecular dynamics simulations, two representative conformations of the GmolPBP2-Z8-12: Ac complex are selected. Conformation II at the time of 14-106 ns is dominantly maintained by the hydrophobic interactions and hydrogen bond. In Conformation I, which lasts from 106 to 200 ns, the hydrophobic interactions are enhanced while the hydrogen bond is quite weakened, due to the formation of a more sophisticated hydrophobic binding pocket and the enlargement of hydrogen bond distance. Taking the two conformations as a whole, the affinity between GmolPBP2 and Z8-12: Ac is crucially determined by three hot-spots including Phe11, Trp36, and Ile51. These results would provide a basis for the discovery, optimization, and design of leading compounds potentially active to attract G. molesta.