Metabolic Adaptations in Phosphine‐Resistant Tribolium castaneum Driven by Mitochondrial Enzyme Variability and Gene Expression

ABSTRACT Phosphine fumigation is essential for controlling storage pests like Tribolium castaneum, but its frequent application has resulted in resistance, primarily due to mutations in the Dihydrolipoamide dehydrogenase (DLD) gene associated with the rph2 allele. This study demonstrates that the Patiala population exhibits homozygous resistance variations across populations, contrasting with the susceptibility observed in laboratory cultures. Our assessment of mitochondrial DLD and Cytochrome c oxidase (COX) activities showed significantly elevated levels in the Patiala population, with increases of approximately sevenfold for DLD and 6.92‐fold for COX, indicating mitochondrial adaptations for enhanced energy production. Kinetic analyses of DLD in the resistant Patiala population showed a higher Vmax (0.005 mmol/min) and a significantly increased Km (16.66 mM), indicating variations in maximal enzyme activity and substrate affinity. Furthermore, resistant T. castaneum populations displayed substantial upregulation of DLD and COX gene expression, with DLD expression increasing by 10.53‐fold and COX expression peaking at 102.57‐fold in Patiala. Pearson correlation analysis indicated strong positive correlations (r > 0.8) between enzymatic activity and gene expression for both DLD and COX, suggesting a coordinated role in resistance mechanisms. The PCA biplot illustrated distribution patterns of enzymatic activity and gene expression among field‐resistant populations, highlighting the association between increased resistance and elevated enzymatic activity and gene expression levels. Therefore, the upregulation of DLD and COX activities in resistant populations underscores their critical roles in counteracting phosphine, reflecting metabolic reprogramming for improved energy production under stress. The study of phosphine‐resistant Tribolium castaneum populations. It involves collecting field populations, performing genotypic assessments through CAPS marker analysis, isolating mitochondria for enzyme assays, and conducting gene expression assessments. Key findings include elevated DLD and COX enzyme activity and gene expression variations in the resistant populations. Research Highlights Significant variations in phosphine resistance in Tribolium castaneum populations, with high resistance in Karnal, Hapur, Moga, and Patiala; moderate in Delhi and Sonipat; weak in Shillong; and susceptibility in lab cultures. Increased mitochondrial DLD and COX activities in