BmATG5, BmATG6 and BmATG8 Are Involved in Autophagy and Apoptosis During Metamorphosis Induced by Cadmium in Bombyx mori

Cadmium (Cd) is a pervasive environmental contaminant with potent cytotoxic effects in a wide range of organisms. Although autophagy and apoptosis are recognized as major cellular responses to heavy metal stress, the molecular basis of Cd-induced cell death in insects remains insufficiently understood. In this study, we used fifth-instar day-4 (5L4D) larvae of Bombyx mori and the silkworm-derived Bm-12 cell line to investigate the involvement of three core autophagy-related proteins, Bombyx mori Autophagy-related protein 5(BmATG5), Bombyx mori Autophagy-related protein 6(BmATG6), and Autophagy-related protein 8(BmATG8), in Cd-induced autophagy and apoptosis. Exposure to CdCl2 markedly induced autophagic and apoptotic responses in both larval midgut tissue and Bm-12 cells, as demonstrated by monodansylcadaverine(MDC) staining, Lyso-Tracker Red staining, DAPI and Hoechst 33258 staining, and DNA fragmentation assays. qPCR and Western blot analyses showed significant upregulation of BmATG5, BmATG6, and BmATG8 following Cd exposure. Notably, the cleaved forms tBmATG5-N (24 kDa) and tBmATG6-C (35 and 37 kDa), as well as the lipidated form BmATG8-PE (12 kDa), accumulated substantially under Cd stress. In parallel, intracellular Ca2+ levels and calpain activity were significantly increased, suggesting activation of a calcium-dependent regulatory pathway. Pharmacological inhibition experiments further indicated that autophagy and apoptosis are functionally interconnected during the Cd response. Collectively, these findings demonstrate that BmATG5, BmATG6, and BmATG8, together with their processed forms, play central roles in coordinating autophagy–apoptosis crosstalk during Cd-induced cytotoxicity in Bombyx mori. This study provides new mechanistic insight into heavy metal toxicity in insects and expands our understanding of stress-induced programmed cell death during silkworm metamorphosis.