Comprehensive metabolomics reveals defense‐driven metabolic reprogramming via withanoside biosynthesis under insect herbivory in Withania somnifera

Plants exhibit dynamic physiological and biochemical responses to herbivory that often involve extensive metabolic reprogramming. Withania somnifera (Ashwagandha), a globally important medicinal plant, is susceptible to herbivory by the pest Henosepilachna vigintioctopunctata, resulting in significant yield losses. Understanding herbivory-induced metabolic responses is essential for elucidating plant-insect interactions and identifying potential defense-associated metabolites. In this study, we investigated the metabolic responses of W. somnifera leaves to H. vigintioctopunctata infestation using a comprehensive metabolomics approach combining liquid chromatography-high-resolution accurate mass spectrometry and gas chromatography-mass spectrometry. Our findings revealed that the herbivore triggers a significant metabolic shift by upregulating secondary metabolites, withanosides and phenylpropanoids. Furthermore, primary metabolites from carbohydrate metabolism were significantly affected. In addition, elemental analysis using ICP-OES revealed the accumulation of micronutrients due to pest infestation in W. somnifera. Notably, we also found plant defense metabolites, specifically withanolides and withanosides, within the herbivore pests themselves. The accumulation of withanosides in response to pest infestation was further confirmed through gene expression analysis. Additionally, feeding on Withania leaf extract containing withanolides adversely affected beetle physiology and survival. Collectively, our findings demonstrate that herbivory induces substantial metabolic plasticity in W. somnifera, particularly involving withanolide glycosylation and phenylpropanoid metabolism. This study provides a valuable framework for understanding herbivory-associated metabolic responses and identifies candidate pathways for future functional and comparative studies.