Selective Sequestration of Cardenolides Shapes Chemical Defense in an Aposematic Butterfly

Aposematic insects often sequester plant toxins as chemical defenses, but the extent to which they selectively retain or eliminate specific compounds remains poorly understood. We investigated toxin sequestration in Danaus chrysippus, a chemically defended butterfly that feeds on the cardenolide-rich milkweed Calotropis gigantea. Using untargeted metabolomics, targeted cardenolide quantification, and Na⁺/K⁺-ATPase inhibition assays, we tested whether sequestration is compound-selective and compared the biochemical potency of plant and insect samples. Untargeted profiles showed that caterpillars and adults converge on an insect-specific metabolome, while frass retains a plant-like chemical signature. Targeted analyses showed consistent retention of four cardenolides (calotropagenin, coroglaucigenin, uzarigenin, frugoside) across developmental stages, complete exclusion of uscharin, and progressive loss of calotropin and calotoxin across metamorphosis. Enzyme assays revealed strong differences among sample types: frass showed the strongest inhibition (~ 5-fold weaker than the ouabain standard), adults were intermediate (~ 18-19-fold), and leaves and caterpillars were the weakest (~ 45-58-fold). These potency differences mirrored qualitative composition, frass contained the full set of detected cardenolides, whereas adults retained only a reduced subset. While porcine Na⁺/K⁺-ATPase assays quantify biochemical activity rather than predator deterrence, our results demonstrate clear compound-level selectivity and suggest that physiological filtering shapes the repertoire of plant toxins available for defense in aposematic insects.