Cardenolide toxin diversity impacts monarch butterfly growth and sequestration

In classic coevolutionary interactions, host plants are thought to accrue novel chemical defenses which are later countered by detoxification strategies and sometimes sequestration in specialist herbivores. We recently discovered that unusual nitrogen- and sulfur-containing (N,S-) cardenolides in some milkweed species are highly toxic, and broken down to less toxic forms which are sequestered by monarch butterflies (Danaus plexippus). Here we isolated and purified five dominant cardenolide toxins from the tropical milkweed, Asclepias curassavica, a globally abundant host plant of monarchs, and fed them to caterpillars individually or in mixture. We hypothesized that the two N,S-cardenolides in A. curassavica (uscharin and voruscharin) would reduce caterpillar growth and sequestration more than other abundant related cardenolides (15-Hydroxy calotropin, frugoside, calactin). Overall, cardenolide treatments caused monarchs to feed more and grow more compared to controls; nonetheless, one N,S-cardenolide (voruscharin) was not stimulatory and caused substantial reductions in growth efficiency. Consuming N,S-cardenolides caused caterpillars to sequester the lowest total amounts of cardenolides, and also reduced their efficiency of sequestration. We next tested the phytochemical diversity hypothesis, that toxin mixtures pose a substantial burden for caterpillars compared to individual compounds provided in equimolar concentrations. We prepared two types of mixtures, one containing equal concentrations of the five compounds and another “realistic mixture” where toxin concentrations reflect their natural proportions in leaves. Mixtures had a negative impact on caterpillar feeding, growth, sequestration, and sequestration efficiency compared to the average of single compounds. The equal and realistic mixtures had similar impacts on feeding and growth, but feeding on the realistic mixture resulted in the lowest sequestration. We conclude that as a result of coevolutionary interactions, even sequestering herbivores may be thwarted by highly specialized plant metabolites such as N,S-cardenolides, and that phytochemical mixtures strengthen plant defense. Toxin mixtures likely challenge detoxification and transport of plant defenses, reducing the herbivore’s growth and sequestration.