Metabolomic insights into western corn rootworm tolerance to dsRNA (Snf7 or Ssj1) ingestion

The western corn rootworm (WCR, Diabrotica virgifera virgifera) is a devastating pest of maize in the United States, causing approximately $2 billion in annual economic losses. RNA interference (RNAi) is a highly specific gene-silencing mechanism that enables targeted suppression of essential insect genes through ingestion of double-stranded RNA (dsRNA). However, WCR can evolve resistance to RNAi under selection pressure. Here, we investigated the metabolic responses associated with WCR larval tolerance to dsRNA targeting DvSnf7 or DvSsj1. Surviving, well-developed larvae from both field-derived and lab-reared colonies were exposed for 14 days to LC50 concentrations of either dsRNA via insect diet assays and analyzed using UPLC-Orbitrap-MS/MS. These larvae exhibited significant target gene knockdown and showed reduced survival, weight gain, and molting rates compared to control larvae. Global metabolomic profiling identified 724 metabolites, with 82% confirmed using authentic standards and 72% matched with the Human Metabolome Database. Partial least squares-discriminant analysis revealed highly similar metabolic profiles in WCR larvae exposed to the two dsRNA treatments, both distinct from controls, and no significant variation between the two WCR colonies regardless of RNAi exposure. Of the identified metabolites, 308 were significantly altered compared to controls (P < 0.05). Pathway enrichment analysis implicated glutathione metabolism, pyrimidine metabolism, and arginine–proline metabolism as potential contributors to dsRNA tolerance. These findings suggest that WCR activates specific metabolic pathways to counteract RNAi-induced stress. This study provides mechanistic insights into altered metabolic networks associated with WCR tolerance to dsRNA, with implications for the sustainability of RNAi-based integrated pest management strategies.