Molecular characterization and targeting of the hatching enzyme EsHE in Exorista sorbillans identifies abametapir as a potent ovicidal agent.

Exorista sorbillans, a parasitic fly that infests the silkworm Bombyx mori, causes severe economic losses in sericulture. Current control methods are limited by pesticide resistance, underscoring the need for novel strategies targeting essential physiological processes. Here, we identified and characterized the hatching enzyme gene (EsHE) from E. sorbillans, which encodes a zinc-binding metalloprotease highly expressed during egg hatching process and third-instar larval development. Heterologous expression of EsHE revealed that its catalytic domain (EsHEb) exhibits robust proteolytic activity against both native egg chorions and host cocoons, confirming its role in facilitating larval emergence. Screening of metalloprotease inhibitors identified 5,5'-dimethyl-2,2'-bipyridyl (abametapir) as a potent inhibitor of EsHE, achieving 100 % ovicidal efficacy at 2 mM without adversely affecting silkworm viability or key economic traits. Structural and computational analyses demonstrated that EsHE belongs to the ZnMc superfamily, with a conserved active-site cavity stabilized by Zn2+ coordination. Molecular dynamics simulations and MM/PBSA calculations revealed strong binding affinity between EsHE and its substrate tripeptide (Leu-Leu-Glu, LLE), driven primarily by electrostatic interactions, and identified four critical residues essential for substrate recognition. Abametapir disrupts enzyme function by chelating Zn2+, thereby inhibiting egg hatching. Our findings establish EsHE as a promising molecular target for controlling E. sorbillans and position abametapir as an eco-friendly, targeted agent for managing silkworm myiasis, offering a sustainable alternative to conventional pesticides.