Ailanthone, a natural quassinoid from Ailanthus altissima, has been reported to exhibit insecticidal activity, yet its molecular mechanism remains elusive. Here, we show that ailanthone acts as a non-orthosteric inhibitor of 20-hydroxyecdysone (20E) signaling by promoting ubiquitin-proteasome-dependent degradation of the ecdysone receptor (EcR). In both Drosophila melanogaster and Ostrinia furnacalis, ailanthone treatment inhibited insect growth and metamorphosis. Mechanistically, in D. melanogaster, these developmental defects were attributable to disruption of 20E signaling, as evidenced by suppressed expression of 20E early response genes. We further found that ailanthone suppresses 20E signaling not through competitive binding to the EcR ligand-binding pocket. Instead, it promotes ubiquitination and proteasomal degradation of EcR, leading to EcR depletion. This degradation is associated with reduced interaction between EcR and the chaperone Hsp83. These results identify a novel mode of 20E antagonism and highlight ailanthone as a promising lead compound for further development as an insect growth regulator.
Chiral fungicides constitute a substantial proportion of commonly used pesticides; however, the spread of antibiotic resistance genes (ARGs) in agricultural settings under repeated exposure to different enantiomers is still unclear. Here, the prevalence and potential risks of ARGs were investigated in the soil-earthworm ecosystem when exposed to hexaconazole (HEX) enantiomers. Metagenomic analysis indicated that R-HEX and S-HEX facilitated the dissemination of ARGs in soil without earthworms, but this effect diminished with successive exposures. Notably, S-HEX exhibited a greater effect on ARG profiles compared to R-HEX. Furthermore, HEX enantiomers, particularly S-HEX, increased the mobility potential of ARGs and enhanced host pathogenicity, thereby contributing to elevated ecological risks. Specifically, exposure to the HEX enantiomer led to an enrichment of dominant pathogenic ARG hosts, such as Enterobacteriaceae. Importantly, earthworms alleviated the enantiomer-driven dissemination of ARGs and weakened their association with mobile genetic elements (MGEs). Overall, this study offers novel perspectives on the enantiomer-driven propagation of ARGs induced by the chiral pesticide hexaconazole in soil and highlights the role of earthworms in mitigating ARG contamination. These findings contribute significantly to the risk assessment and remediation strategies for chiral pesticides.
Volatility, the propensity to dissipate from solid or liquid state to the air, is an important property of certain pesticides and small molecules mediating chemical communication. Volatility is the function of a compound's vapor pressure, which depends on intra- and intermolecular forces given by the chemical structure. The sesquiterpenoid juvenile hormones (JHs), vital to insect development and reproduction, have been known for vapor-borne activity in vivo. The same applies to some synthetic JH mimics (juvenoids), which serve as biorational insecticides of the insect growth regulator (IGR) class. However, neither volatility of the natural JHs nor the mode of vapor-phase JH or juvenoid action have been experimentally defined. Here, we employ a reporter assay based on the insect JH receptor (JHR) to determine to what extent diverse JHR ligands - five natural JH homologs and six synthetic juvenoids of two types (aliphatic and aromatic) - exert JHR agonist activity over distance. Our microtiter plate volatility assay showed that specific vapor-borne activity and effective concentrations of the agonists agreed with their empirical or calculated vapor pressure values. While most of the natural JHs and the aliphatic juvenoids produced moderate to extensive vapor-borne activity across the assay plate, the aromatic juvenoids did not. These experiments have resolved volatility of juvenile hormones and their mimics at the molecular action level. The data bear significance for proper designing of JH signaling assays and screens for JHR modulating compounds, where any contaminating vapor-borne activity is detrimental. Physiological effect of JH as an airborne endocrine signal is discussed.
The ribosomal protein S6 kinase (S6K) transduces the anabolic signals from nutrients and is involved in insect development and reproduction, however, its role in the regulation of the insecticide detoxification is still unclear. In this study, LsS6K was cloned and characterized from Laodelphax striatellus. RT-qPCR analysis revealed tissue-specific expression of LsS6K with the highest expression level in the fat body, and thiamethoxam exposure significantly down-regulated the expression of LsS6K at both the mRNA and protein levels. Functional analysis using RNA interference (RNAi) showed that knockdown of LsS6K significantly increased the tolerance of L. striatellus nymphs to thiamethoxam with concomitant upregulation of activity of cytochrome P450 monooxygenases (CYPs). Comparative transcriptome analysis identified six up-regulated P450 genes in dsLsS6K-injected nymphs compared with dsEGFP-injected nymphs, among which LsCYP4DE1 and LsCYP4C1 showed significant upregulation upon exposure to thiamethoxam. Further RNAi revealed that knockdown of LsCYP4DE1 and LsCYP4C1 significantly decreased the tolerance of nymphs to thiamethoxam. These results suggest that LsS6K is a negative regulator of thiamethoxam tolerance, and provide valuable insight into the cross-talk between nutrient signaling pathways and the detoxification regulatory network.
The desert locust, Schistocerca gregaria, remains one of the most destructive agricultural pests, posing a serious threat to crop production and food security. Current control strategies rely heavily on synthetic insecticides, raising concerns about environmental contamination and the development of insecticide resistance. Glutathione S-transferases (GSTs) are major phase II detoxification enzymes involved in xenobiotic metabolism and insecticide tolerance; however, their physiological role in reproductive tissues remains poorly understood. This study aimed to purify and characterize ovarian GST from S. gregaria and evaluate the inhibitory effects of selected plant-derived flavonoids as potential GST-targeting compounds. Ovarian GST activity was purified by GSH-affinity chromatography, yielding a predominant protein band with an apparent molecular mass of approximately 24 kDa. The purified enzyme exhibited maximum activity at pH 8.5 and showed the highest catalytic activity toward 1-chloro-2,4-dinitrobenzene (CDNB), followed by 1,2-dichloro-4-nitrobenzene (DCNB) and ethacrynic acid. Among the inhibitors tested, Cibacron Blue showed the greatest inhibitory potency, followed by bromosulfophthalein and ethacrynic acid, with IC₅₀ values of 0.48, 1.17, and 3.0 nM, respectively. The Michaelis-Menten constants (Kₘ) for glutathione (GSH) and CDNB were 0.10 and 0.086 mM, respectively. Kinetic analyses revealed that quercetin and delphinidin chloride competitively inhibited GST activity with respect to CDNB. In contrast, quercetin exhibited non-competitive inhibition toward GSH, whereas delphinidin chloride acted as a competitive inhibitor. These results demonstrate that ovarian GST possesses distinct kinetic characteristics and can be effectively inhibited by naturally occurring flavonoids. Although these findings are based on in vitro biochemical analyses, they suggest that inhibition of ovarian GST may reduce the detoxification capacity of S. gregaria and provide a biochemical basis for future in vivo investigations. Collectively, this study identifies ovarian GST as a potential biochemical target and highlights the potential of flavonoid-based GST inhibitors for further evaluation as synergists in integrated desert locust management.
Pronounced sex-specific differences exist in the toxicological traits of spider mite species. Our previous work showed that female Tetranychus cinnabarinus exhibit significantly higher tolerance to acaricides than males, primarily driven by elevated detoxification enzyme activity. However, the molecular basis underlying this sex-specific difference remains unclear. Here, we used pyridaben and cyflumetofen as representative acaricides to dissect the molecular mechanisms underlying sex-specific differences in detoxification metabolism between female and male mites. After 48 h of cyflumetofen exposure, GST activity increased significantly in female mites. Following pyridaben exposure, the activities of both P450 (24 h and 48 h) and CCE (48 h) increased significantly in female mites. Under the same conditions, only P450 activity increased significantly in male mites after 48 h of pyridaben exposure. Proteomic profiling identified 33 differentially expressed detoxification enzymes, predominantly from the major detoxification families P450, GST, and CCE; among them, 26 were significantly upregulated in females relative to males. Six detoxification enzymes, including CYP392A3, CYP389C5, TcGSTd02, TcGSTd13, TcCCE39, and TcCCE52, were selected for functional characterization. We successfully obtained six active recombinant detoxification enzymes through heterologous expression. IC50 and in vitro metabolism assays showed that these recombinant proteins display both shared and distinct capacities for metabolizing or sequestering cyflumetofen and pyridaben. RNAi and bioassay results demonstrated that silencing CYP389C5, TcGSTd02, and TcCCE52 resulted in more pronounced changes in acaricide susceptibility in female than in male mites. Collectively, this study demonstrates that the sex-biased protein abundance identifies candidate biochemical contributors to differential susceptibility in female and male mites.
