Molecular Characterization and RNAi-Based Functional Validation of Ache in the Neonicotinoid Stress Response of the Melon Fly, Zeugodacus cucurbitae

The melon fly, Zeugodacus cucurbitae (Coquillett), is a destructive tephritid pest of cucurbit crops, and neonicotinoids remain important tools for its management. Although neonicotinoids primarily target nicotinic acetylcholine receptors (nAChRs), their possible effects on acetylcholinesterase (AChE)-mediated cholinergic homeostasis in Z. cucurbitae remain poorly understood. In this study, we molecularly characterized the Z. cucurbitae Ache gene and evaluated its expression and functional contribution under dinotefuran, acetamiprid, and thiamethoxam stress. Bioinformatics analyses confirmed conserved cholinesterase features, including the FGESAG catalytic motif, catalytic triad, choline-binding site, oxyanion hole, and conserved disulfide-bond residues. Temporal and tissue-specific expression profiling showed high Ache transcript abundance in eggs, third-instar larvae, and adult heads. Larval exposure to LC5, LC20, and LC50 concentrations of the three neonicotinoids caused dose- and time-dependent mortality and significantly induced Ache expression, with dinotefuran producing the strongest transcriptional response. Parental exposure also impaired F1 fecundity, hatchability, larval survival, and adult emergence, with acetamiprid and thiamethoxam causing stronger transgenerational effects than dinotefuran. Oral RNAi significantly increased larval susceptibility upon re-exposure to neonicotinoids after 72 h. These findings indicate that Ache contributes to cholinergic stress response and susceptibility modulation in Z. cucurbitae, supporting its possible potential as an RNAi target for integrated pest management (IPM). However, practical utility at the field level requires further evaluation.