Figures and data
Structural comparison of benzoylphenylurea (BPU) insecticides and a mammalian glycogen phosphorylase inhibitor (GPI) reveals a shared acylurea scaffold.
Chemical structures of four BPU insecticides (Diflubenzuron, Teflubenzuron, Novaluron, and Flufenoxuron) and a human glycogen phosphorylase inhibitor (GPI) are shown. All share a central N,N’-diphenylurea (acylurea) core (dashed boxes). BPUs are characterized by halogenated (F, Cl) aromatic substituents and in some cases by additional ether or alkyl side chains. The GPI contains a methoxyphenyl group bearing an additional methylurea side chain.
Recombinant PxGP is inhibited by a mammalian GP inhibitor (GPI) but not by the benzoylphenylure Diflubenzuron.
(A) Purification of 6×His-tagged PxGP from Sf9 cells. Left panel: Coomassie-stained SDS-PAGE.. Right: Western blot with anti-His antibody. The expected ∼100 kDa band is indicated (red box). M, marker; S, soluble fraction; P, pellet; F, flow-through; lanes 1–4, imidazole elution fractions. (B) Dose-response inhibition by GPI. Activated PxGP-a activity was measured after pre-incubation with GPI (0.16–500 nM). Data (mean ± SEM, n=3) were fit to a four-parameter logistic curve. The calculated IC50 is 2.96 nM. Inset: GPI structure with its characteristic asymmetric side chains (orange). (C) Inhibition assay with DFB (0.54–1700 nM). DFB did not yield a complete sigmoidal inhibition curve, causing ≤62% inhibition at the highest concentration. Inset: DFB structure with its characteristic halogenated benzoyl groups (orange). This rules out PxGP as a direct target of DFB.
The GP inhibitor (GPI) is non-toxic to Plutella xylostella, unlike the benzoylphenylurea Diflubenzuron (DFB).
(A) Developmental stage distribution over time for control, GPI (250, 500 mg/L), and DFB (125, 250 mg/L) treated larvae. DFB causes developmental arrest. (B) Quantitative effects on mortality (top), pupation (middle), and adult emergence (bottom). GPI groups did not differ from control (P > 0.05), whereas DFB caused significant, dose-dependent mortality and severely impaired development (*P < 0.05). Data are mean ± SEM (n=3). Different letters denote statistical significance. (C) Larval phenotypes at 96 h. Control and GPI-treated larvae are normal. DFB-treated larvae show catastrophic molting failure and “double cuticle” (red arrows). Scale bar, 1 mm. (D) Pupal and adult phenotypes. GPI-treated individuals develop normally. DFB treatment results in deformed pupae and trapped pharate adults (red arrows). Scale bar, 1 mm.
Ingestion of GP inhibitor (GPI) induces compensatory upregulation of PxGP transcription.
Relative PxGP mRNA levels in larvae after continuous exposure to 500 mg/L GPI or control (CK). Expression was normalized to PxRPS13 (control set to 1.0 at each time point) using the 2-ΔΔCt method. Data are mean ± SEM (n=3). GPI triggered a rapid and sustained transcriptional increase, peaking at 48 h (3.48-fold). Significance vs. control: *P < 0.05, **P < 0.01, ***P < 0.001 (two-way ANOVA with Sidak’s test).
PxGP sequence conservation and domain architecture.
(A) Schematic of PxGP functional domains: pyridoxal phosphate (PLP) binding site (blue boxes), AMP allosteric site (yellow boxes), and glycogen-binding/active site pocket (blue shading). (B) Multiple sequence alignment of GP from Plutella xylostella (PxGP), other lepidopterans (Helicoverpa armigera HaGP, Spodoptera exigua SeGP, Ostrinia furnacalis OfGP), human (HsGP), and rabbit (OcGP). Identical residues are shaded red. The gray box indicates the 606 bp region (aa 22–224) targeted for RNAi, which overlaps the conserved AMP-binding domain. PxGP shares 82.29% identity with human GP.
Developmental expression profile of PxGP.
Relative PxGP mRNA levels across life stages (Egg, larval instars L1-L4, Pupa, Adult), normalized to PxRPS13 (L1 set to 1.0). Data are mean ± SEM (n=3). Expression peaks in adults. Different letters indicate significant differences (P < 0.05, one-way ANOVA with Tukey’s test).
RNAi-mediated knockdown of PxGP is dose- and time-dependent.
(A) Experimental workflow. Third-instar larvae were injected with dsRNA targeting PxGP (dsGP) at three concentrations or a control dsRNA (dsGFP), then sampled for analysis. (B) Time-course of PxGP mRNA levels post-injection, relative to dsGFP control (set to 1.0). The highest dsGP dose (14,000 ng/µL) caused maximal suppression (87.6%) at 48 h. Data are mean ± SEM (n=3). Significance vs. dsGFP at each time point: **P < 0.01, ***P < 0.001 (one-way ANOVA with Tukey’s test).
PxGP knockdown does not impair development or survival.
(A) Developmental stage distribution post-injection shows similar progression across all groups. (B) Cumulative mortality (top), pupation (middle), and adult emergence (bottom) rates were not significantly different between dsGP and dsGFP control groups (P > 0.05). Data are mean ± SEM (n=3). (C) Representative larval phenotypes at 48 h and 96 h post-injection are normal in both dsGFP and high-dose dsGP groups. Scale bar, 1 mm. (D) Pupae and adults from both groups are morphologically normal. Scale bar, 1 mm.
PxGP knockdown triggers biphasic metabolic reprogramming, culminating in gluconeogenic compensation.
(A, B) Concurrent suppression of downstream genes Trehalase (PxTre) and Hexokinase (PxHex) post-knockdown. (C) Upregulation of gluconeogenic genes PEPCK and G6Pase at 96 h. Data in A-C are mean ± SEM (n=3); *P < 0.05, **P < 0.01, ***P < 0.001 vs. dsGFP control. (D-H) Metabolite levels post-knockdown. (D) Total protein decreased at 72 h (P < 0.01). (E) Glycogen remained stable. (F) Trehalose surged 7.4-fold at 96 h (P < 0.001). (G) Glucose-6-phosphate (G6P) increased 6.5-fold at 96 h (P < 0.001). (H) Free glucose showed a transient dip at 72 h (P < 0.05) before recovery. Data are mean ± SEM (n=3); *P < 0.05, ***P < 0.001 vs. control at each time point.
Effects of PxGP knockdown on GP enzyme activity and the expression of glycogen metabolism-related genes.
(A) GP enzyme activity normalized to sample protein concentration. A significant decrease in activity was detected only at 48 h (P < 0.05). (B) GP enzyme activity per insect. Significant decreases were detected at 24 and 48 h (P < 0.05, **P < 0.01). (C) Expression levels of the GBE gene at different time points after RNAi treatment. (D) Expression levels of the α-Amylase gene at different time points after RNAi treatment. All data are presented as mean ± SEM (n = 3); *P < 0.05, **P < 0.01, ***P < 0.001, dsGFP vs. dsGP treatment groups. An independent samples t-test was used in (A) and (B), and two-way ANOVA with Sidak’s test was used in (C) and (D).
Assessment of fitness cost of PxGP gene silencing by RNAi in Plutella xylostella.
(A) Schematic diagram of fitness cost evaluation at different developmental stages of P. xylostella after dsRNA injection. (B) Food consumption per 10 larvae at different time points. (C) Average body weight per larva at different time points. (D) Pupal weight on day 3 of pupation (n = 21). (E) Adult wing area (n = 20–21). (F) Number of eggs laid by females within 3 days (n = 10–11). All data are presented as mean ± SEM (n = 3); *P < 0.05, dsGP vs. dsGFP treatment group (independent samples t-test).
Binding mode analysis of GPI and DFB with PxGP.
(A) 3D binding model of PxGP with GPI. (B, C) The key intermolecular interactions for GPI (B) and DFB (C). 2D interaction profile analysis of GPI (D) and DFB (E) with PxGP.
MM/GBSA binding free energy analysis and interaction profile of GPI and DFB with PxGP.
Schematic of glycogen metabolism and compensatory glucose homeostasis.
Red arrows: blocked flux from glycogen to G6P; Green arrows: activated gluconeogenesis. Solid arrows represent single enzymatic steps, while dashed arrows indicate multi-step processes.
Enzymatic activity of purified PxGP and inhibition assays using crude larval lysate.
(A) Reaction kinetics of purified, activated PxGP-a (red line) compared to a no-enzyme control (CK, blue line). The specific activity was calculated to be 294.25 ± 3.45 U/mg Prot (mean ± SEM). (B) Inhibition of native GP activity in a crude larval lysate by GPI. Activity was measured at 5, 15, 25, and 45 min post-incubation. (C) Effect of DFB on native GP activity in a crude larval lysate. DFB showed no significant inhibition at any concentration or time point. Data in (B) and (C) are presented as mean ± SEM (n = 3).
Heatmap depicting pairwise sequence similarity of glycogen phosphorylase among six species.
The heatmap displays the percentage of global sequence similarity calculated from pairwise alignments using TBtools. The six species (Plutella xylostella, Homo sapiens, Oryctolagus cuniculus, Helicoverpa armigera, Ostrinia furnacalis and Spodoptera exigua) are arranged in the same order along both axes. The color intensity in each cell corresponds to the similarity percentage, as shown in the color key (from blue (low) to red (high)). Numerical values are shown within the cells.
Expression levels of the PxGP gene after double-stranded RNA injection.
Data are presented as mean ± SEM (n = 3); ***P < 0.001, dsGP vs. dsGFP treatment group (independent samples t-test).
Total protein concentration in test insects subjected to glycogen phosphorylase activity determination, analysis of alternative glycogen-metabolizing enzymes, and fitness cost evaluation.
Data are mean ± SEM (n=3). Significance vs. control: **P < 0.01 (two-way ANOVA with Sidak’s test).
Wing phenotype of adult insects emerged from the dsGFP-treated group.
Wing phenotype of adult insects emerged from the dsGP-treated group.
Primer sequences used in this study.
