Abstract

The tea geometrid Ectropis grisescens is a destructive defoliator in tea plantations, and its management relies heavily on chemical insecticides, raising concerns about resistance development and environmental safety. Botanical insecticides offer a potential alternative, yet little is known about the insecticidal mode of action of the plant-derived compound methyl benzoate (MB) against insect pests. Here, we systematically investigated the bioactivity, transgenerational effects, and mode of action of MB against E. grisescens under laboratory conditions by integrating bioactivity assays, age-stage, two-sex life table analysis, transcriptomic profiling, biochemical analyses, and RNA interference (RNAi). MB exhibited strong contact and fumigation toxicity against E. grisescens , significantly inhibited oviposition and egg hatchability, and also caused significant suppression of population growth in the F 1 generation. Time-course transcriptomics revealed an early induction of cuticle- and membrane-related responses followed by marked repression of mitochondrial oxidative phosphorylation pathways. Consistent with these patterns, MB significantly inhibited the activities of mitochondrial Complex I and Complex V, reduced mitochondrial membrane potential and ATP content, and induced excessive reactive oxygen species accumulation and oxidative stress. Furthermore, silencing of mitochondrial genes encoding Complex I ( EgNDUFB2 and EgNDUFS4 ) and Complex V ( EgATP5J ) subunits markedly increased larval susceptibility to MB and exacerbated mitochondrial dysfunction. Collectively, these results demonstrate that MB disrupts mitochondrial energy metabolism by inhibiting Complexes I and V, thereby inducing energy depletion and oxidative damage. These findings provide mechanistic insight into MB toxicity against E. grisescens and contribute to a broader understanding of the complex mechanisms underlying botanical insecticides.