Cardiac glycosides are a large class of secondary metabolites found in plants. In the genus Asclepias, cardenolides in milkweed plants have an established role in plant–herbivore and predator–prey interactions, based on their ability to inhibit the membrane-bound Na+/K+-ATPase enzyme. Milkweed seeds are eaten by specialist lygaeid bugs, which are the most cardenolide-tolerant insects known. These insects likely impose natural selection for the repeated derivatisation of cardenolides. A first step in investigating this hypothesis is to conduct a phytochemical profiling of the cardenolides in the seeds. Here, we report the concentrations of 10 purified cardenolides from the seeds of Asclepias curassavica. We report the structures of new compounds: 3-O-β-allopyranosyl coroglaucigenin (1), 3-[4′-O-β-glucopyranosyl-β-allopyranosyl] coroglaucigenin (2), 3′-O-β-glucopyranosyl-15-β-hydroxycalotropin (3), and 3-O-β-glucopyranosyl-12-β-hydroxyl coroglaucigenin (4), as well as six previously reported cardenolides (5–10). We test the in vitro inhibition of these compounds on the sensitive porcine Na+/K+-ATPase. The least inhibitory compound was also the most abundant in the seeds—4′-O-β-glucopyranosyl frugoside (5). Gofruside (9) was the most inhibitory. We found no direct correlation between the number of glycosides/sugar moieties in a cardenolide and its inhibitory effect. Our results enhance the literature on cardenolide diversity and concentration among tissues eaten by insects and provide an opportunity to uncover potential evolutionary relationships between tissue-specific defense expression and insect adaptations in plant–herbivore interactions.

Milkweed–herbivore systems are characterized by cardenolide chemical defenses and specialized herbivore adaptations such as physiological target site insensitivity. Cardenolide defenses in milkweeds can vary in terms of the total concentration, differences in the polarity of individual cardenolides, and the substitution of the steroidal structures that can contribute to the molecule's reactivity. The variability in cardenolide defenses could represent the plant's response to natural selection and adaptation of resistant herbivores and is a characteristic of phenotype-matching between defensive and offensive traits resulting from coevolution. Here, we test the phenotypic match of the cardenolide composition of seeds of Asclepias curassavica and those sequestered by nymphs and adults of the specialized seed herbivore Oncopeltus fasciatus, combined with tests of the inhibitory capacity of a subset of seed cardenolides against the Na+/K+-ATPase of O. fasciatus and a non-adapted insect (Drosophila melanogaster). We compare this with the inhibitory capacity against the highly sensitive porcine Na+/K+-ATPase. Among the five most abundant cardenolides present in milkweed seeds, glucopyranosyl frugoside, glucopyranosyl gofruside, and glucopyranosyl calotropin were significantly more abundant in the seeds than in the adults and nymphs; the bugs contained higher concentrations of the deglucosylated compounds. The most abundant compound, glucopyranosyl frugoside, was also the most inhibitory for O. fasciatus, but O. fasciatus was significantly more tolerant to all compounds compared to D. melanogaster and the highly sensitive porcine enzyme. Our results add to the evidence that O. fasciatus sequesters specific individual cardenolides from its Asclepias host plants that are not directly linked to the concentration and inhibitory potency.