Ricinine toxicity in susceptible lepidoptera involves perturbations in β-nicotinamide adenine dinucleotide metabolism.

Ricinine, a plant-derived alkaloid, exhibits potent insecticidal activity; however, the molecular mechanisms underlying its differential toxicity among insect species remain largely elusive. This study employed integrated metabolomics and transcriptomics to investigate the adaptation strategies of three lepidopteran insects with distinct host plant ranges-Bombyx mori (B. mori), Spodoptera litura (S. litura), and Samia cynthia ricini (S. cynthia ricini) to ricinine. The three species exhibited sharply divergent toxicological responses: ricinine caused acute lethality in B. mori, severely inhibited the growth of S. litura, but induced no significant adverse effects in S. cynthia ricini. Untargeted metabolomics and transcriptomics revealed that ricinine treatment provoked the most severe metabolic disruption in B. mori, followed by S. litura, while the impact on S. cynthia ricini was minimal. Moreover, the species showed largely species-specific pathway-enrichment patterns, indicating fundamentally different adaptive mechanisms. Integrated analysis identified decreased normalized relative abundances of key metabolites (e.g., adenine, flavin mononucleotide) and significant downregulation of genes associated with NAD/NADH-linked energy metabolism (e.g., nicotinate phosphoribosyltransferase (NPH), malate dehydrogenase (MDH), acetyl-CoA synthetase (ACS)) in susceptible species. These molecular changes were associated with β-nicotinamide adenine dinucleotide (β-NAD) metabolism and oxidative phosphorylation-related pathways. Exogenous β-NAD supplementation alleviated ricinine toxicity in S. litura, supporting the involvement of β-NAD-related metabolism. In contrast, S. cynthia ricini, despite accumulating the highest levels of ricinine, displayed only marginal metabolic and transcriptional perturbations, signifying the evolution of robust innate tolerance mechanisms. Finally, the combined treatment with ricinine and nucleopolyhedrovirus (NPV) produced greater growth inhibition and mortality in S. litura than either treatment alone, which was associated with the marked inhibition of cytochrome P450 detoxification enzyme activity. These findings elucidate evolutionary strategies insects employ to cope with plant alkaloids and provide a molecular basis for developing novel, selective pest control agents.