Cardenolide toxin diversity impacts monarch butterfly growth and sequestration

In coevolutionary interactions, host plants accrue novel chemical defenses that specialist herbivores counter by detoxification and sometimes sequestration. We recently found unusual nitrogen- and sulfur-containing (N,S-) cardenolides in some milkweeds-highly toxic compounds that monarch butterflies (Danaus plexippus) detoxify during sequestration. We hypothesized that the N,S-cardenolides in Asclepias curassavica (uscharin and voruscharin) would reduce caterpillar performance and sequestration more than other abundant related cardenolides (15-hydroxy-calotropin, frugoside, calactin). Cardenolides generally increased feeding relative to controls, but voruscharin was not stimulatory and substantially reduced growth efficiency. Exposure to either N,S-cardenolide produced the lowest sequestration and reduced sequestration efficiency, consistent with detoxification limiting toxin retention. We next tested whether toxin mixtures impose additional costs relative to individual compounds. We prepared two mixtures, one with equal concentrations of five cardenolides and a 'realistic mixture' reflecting natural proportions. Relative to the average of single compounds, mixtures reduced feeding, growth, sequestration, and sequestration efficiency, indicating phytochemical diversity effects exceeded expectations from an additive model. The two mixtures similarly reduced growth, but feeding on the realistic mixture yielded the lowest sequestration. We conclude that coevolution can produce highly specialized defense metabolites such as N,S-cardenolides that thwart even sequestering herbivores, and that phytochemical mixtures strengthen plant defense.