Cardenolides, toxicity, and the costs of sequestration in the coevolutionary interaction between monarchs and milkweeds

Abstract

For highly specialized insect herbivores, plant chemical defenses are often co-opted as cues for oviposition and sequestration. In such interactions, can plants evolve novel defenses, pushing herbivores to trade off benefits of specialization with costs of coping with toxins? We tested how variation in milkweed toxins (cardenolides) impacted monarch butterfly (Danaus plexippus) growth, sequestration, and oviposition when consuming tropical milkweed (Asclepias curassavica), one of two critical host plants worldwide. The most abundant leaf toxin, highly apolar and thiazolidine ring-containing voruscharin, accounted for 40% of leaf cardenolides, negatively predicted caterpillar growth, and was not sequestered. Using whole plants and purified voruscharin, we show that monarch caterpillars convert voruscharin to calotropin and calactin in vivo, imposing a burden on growth. As shown by in vitro experiments, this conversion is facilitated by temperature and alkaline pH. We next employed toxin-target site experiments with isolated cardenolides and the monarch's neural Na⁺/K⁺-ATPase, revealing that voruscharin is highly inhibitory compared with several standards and sequestered cardenolides. The monarch's typical >50-fold enhanced resistance to cardenolides compared with sensitive animals was absent for voruscharin, suggesting highly specific plant defense. Finally, oviposition was greatest on intermediate cardenolide plants, supporting the notion of a trade-off between benefits and costs of sequestration for this highly specialized herbivore. There is apparently ample opportunity for continued coevolution between monarchs and milkweeds, although the diffuse nature of the interaction, due to migration and interaction with multiple milkweeds, may limit the ability of monarchs to counteradapt.

Keywords: chemical ecology; coevolution; milkweeds Asclepias; monarch butterfly (Danaus plexippus); plant–insect interactions.

Fig. 1.

Chemical conversion of milkweed cardenolides by monarch caterpillars. (A) A visualization of metabolomic data showing the differences in the chemical composition across sample groups (n = 4 per group; significance tested by PERMANOVA). After data curation, over 7,000 chemical features (m/z) were generated with MS data collected in positive ionization mode and visualized with a Bray–Curtis distance matrix. Ellipses represent the region of 95% confidence. (B) Voruscharin was converted to calactin and calotropin when fed to monarch caterpillars. Shown are means ± SE concentrations as determined by UV-HPLC (n = 3 to 9). Data bars very close to zero had no detectable cardenolides. Note that the caterpillars fed A. curassavica were reared on this diet from hatching and had an order of magnitude higher cardenolides than other treatments which were dosed only during the fourth instar. (C) The basic skeleton of cardenolides and the chemical structures of calactin, calotropin, and voruscharin.

Fig. 2.

Connections between plant cardenolides, monarch sequestration, and monarch growth. (A) Sequestration was predicted by the concentration of the dominant plant cardenolide, voruscharin, although this compound was itself not sequestered (it was converted to calactin and calotropin). (B) In multiple regression, sequestered cardenolides were predictive of monarch growth (whereas plant cardenolides were not), indicating a cost of converting or storing cardenolides. The weak relationships of growth predicted by total leaf cardenolides and leaf voruscharin are shown in SI Appendix, Fig. S6. Monarch image credit: Frances Fawcett.

Fig. 3.

The difference in inhibitory impacts of isolated cardenolides on the highly sensitive porcine Na⁺/K⁺-ATPase versus the milkweed-adapted monarch butterfly Na⁺/K⁺-ATPase. Data are presented as the molar concentration of plant toxin necessary to cause 50% inhibition of the animal enzyme, or IC₅₀, on the y-axis. Thus, higher values on the log scale y-axis indicate that the enzyme is more resistant to the cardenolide. The monarch Na⁺/K⁺-ATPase is substantially more resistant (it typically takes a higher concentration of the plant compounds to inhibit the enzyme). Sequestered+ indicates that these compounds are sequestered intact from consumed leaves as well as converted products from consumed voruscharin and uscharin. Each bar is a mean of three to nine replicates (each based on a six-concentration inhibition curve) ± SE (where not visible, SEs are too small). The arrow representing the polarity of cardenolides is based on elution through a C18 HPLC column (apolar stationary phase).

Fig. 4.

Monarch oviposition was a quadratic function of total leaf cardenolides, with the highest number of eggs laid on intermediate cardenolide plants. Ten butterflies were released to freely oviposit in a large greenhouse common garden (n = 212 plants). Although raw data are shown, plant height was included and was significant in the statistical model. Zero egg values have been jittered. Monarch image credit: Frances Fawcett.