Integrated Time-Series Biochemical, Transcriptomic and Metabolomic Analyses Reveal Key Pathways and...

Chironomids are abundant aquatic macroinvertebrates with known pollutant tolerance, but the molecular effects of chlorantraniliprole (CAP) remain unclear. In this study, we exposed Propsilocerus akamusi larvae to LC₁₀ and LC₅₀ of CAP, sampled at 48 h and 144 h, and integrated biochemical assays, metabolomics, and transcriptomics to profile stress responses. Our results showed that CAP elevated antioxidant enzymes and protein carbonyls, indicating severe oxidation, especially at LC₅₀. At 48 h, LC₁₀ activated JNK-mediated antioxidative and drug metabolism pathways, whereas LC₅₀ altered glycolysis, cofactor biosynthesis, and fatty acid metabolism. At 144 h, LC₁₀ enriched proteasome and oxidative phosphorylation pathways; chaperones PaHsp70 and PaHsp90 were upregulated by 4.14- and 2.08-fold, respectively, and were negatively correlated with lipid and carbohydrate metabolites, indicating a metabolic shift toward protein repair. In contrast, LC₅₀ CAP triggered protein processing in the endoplasmic reticulum (ER) and TCA cycle dysregulation, with upregulation of PaCalreticulin linked to energy crisis markers, reflecting severe energy collapse. After knockdown of PaHsp70, the survival rates of chironomid larvae exposed to LC₁₀ and LC₅₀ CAP significantly decreased from 80.8% to 65.8% and from 43.3% to 28.3%, respectively. Collectively, our results revealed that low CAP concentrations triggered early detoxification followed by proteasome- and Hsp-mediated strategies to sustain protein homeostasis whereas high concentrations directly disrupted core metabolism and ultimately caused energy collapse and lethal ER stress in chironomid larvae. These findings provide mechanistic insights into the chronic effects of CAP on aquatic insects.