Metabolic compensation via gluconeogenesis explains the non-essentiality of glycogen phosphorylase as an insecticidal target in Plutella xylostella

Benzoylphenylurea (BPU) insecticides disrupt insect chitin formation, yet their molecular target is debated. Although resistance-associated mutations map to the Chitin Synthase (CHS) gene, direct inhibition of CHS has not been demonstrated. Given the structural similarity of BPUs to mammalian glycogen phosphorylase (GP) inhibitors, GP has been proposed as a potential target controlling chitin precursor flux. We characterized Plutella xylostella GP (PxGP) and found that the human GP inhibitor GPI potently inhibits both recombinant PxGP (IC₅₀ = 2.96 nM) and native larval GP activity (57.5% reduction), while the BPU diflubenzuron showed no effect. Despite this biochemical inhibition, neither GPI treatment nor RNAi-mediated PxGP knockdown (87.6% suppression) caused mortality or developmental defects. Mechanistic analysis revealed coordinated downregulation of glycogenolysis genes (trehalase 69%, hexokinase 77%) and robust upregulation of gluconeogenolysis genes (PEPCK 3.95-fold, G-6-Pase 3.34-fold). Critically, protein catabolism (29.4% decline at 72 h) provided substrates for gluconeogenolysis by releasing amino acids. Metabolite profiling validated the full substrate-to-product pathway: transient glucose shortfall was followed by massive accumulation of trehalose (7.4-fold) and glucose-6-phosphate (6.5-fold), confirming a quantitatively sufficient metabolic rescue via de novo glucose synthesis. These findings demonstrate that BPUs do not target GP, and that GP inhibition alone is not lethal due to metabolic compensation. This highlights a fundamental metabolic plasticity that must be considered in future target-based insecticide design.