Mechanisms of midgut injury induced by chronic trace concentrations of chlorantraniliprole in silkworm (Bombyx mori).

The diamide insecticide chlorantraniliprole (CAP) is widely applied for pest control, and its environmental residues pose toxic risks to non-target species. However, the mechanisms underlying the developmental toxicity of environmentally relevant chronic exposure to trace concentrations of CAP in non-target lepidopteran insects remain poorly characterized. Here, we used the silkworm (Bombyx mori) as a lepidopteran model to investigate the toxic effects of chronic exposure to trace concentrations of CAP on development and physiology. Chronic exposure to trace concentrations of CAP significantly inhibited larval growth, delayed larval-pupal metamorphosis, reduced cocoon quality, and caused midgut structural damage and defective remodeling. Mechanistically, CAP induced time-dependent physiological disturbances in Bombyx mori. Early exposure activated RyR-mediated Ca2+ release and caused calcium overload, whereas compensatory SERCA activation transiently alleviated Ca2+ accumulation. With prolonged exposure, CAP induced mitochondrial damage, ROS accumulation, and NADPH/ATP depletion, which impaired SERCA activity, triggered secondary calcium overload, and established a Ca2+-ROS-NADPH/ATP-SERCA dysregulation loop. This loop promoted calpain-mediated ATG5 cleavage and NtATG5 generation, thereby driving an autophagy-to-apoptosis transition in midgut cells. Moreover, substantial NADPH depletion impaired enzymes involved in 20E metabolism and, together with disrupted Ca2+ homeostasis, interfered with 20E pulse formation. These disturbances ultimately impaired midgut remodeling, delayed development, and reduced cocoon quality. This study provides mechanistic evidence for the chronic developmental toxicity of CAP and experimental support for the ecological risk assessment of diamide insecticide residues in agroecosystems.