Cardenolide Intake, Sequestration, and Excretion by the Monarch Butterfly along Gradients of Plant Toxicity and Larval Ontogeny

Abstract

Monarch butterflies, Danaus plexippus, migrate long distances over which they encounter host plants that vary broadly in toxic cardenolides. Remarkably little is understood about the mechanisms of sequestration in Lepidoptera that lay eggs on host plants ranging in such toxins. Using closely-related milkweed host plants that differ more than ten-fold in cardenolide concentrations, we mechanistically address the intake, sequestration, and excretion of cardenolides by monarchs. We show that on high cardenolide plant species, adult butterflies saturate in cardenolides, resulting in lower concentrations than in leaves, while on low cardenolide plants, butterflies concentrate toxins. Butterflies appear to focus their sequestration on particular compounds, as the diversity of cardenolides is highest in plant leaves, lower in frass, and least in adult butterflies. Among the variety of cardenolides produced by the plant, sequestered compounds may be less toxic to the butterflies themselves, as they are more polar on average than those in leaves. In accordance with this, results from an in vitro assay based on inhibition of Na + /K + ATPase (the physiological target of cardenolides) showed that on two milkweed species, including the high cardenolide A. perennis, extracts from butterflies have lower inhibitory effects than leaves when standardized by cardenolide concentration, indicating selective sequestration of less toxic compounds from these host plants. To understand how ontogeny shapes sequestration, we examined cardenolide concentrations in caterpillar body tissues and hemolymph over the course of development. Caterpillars sequestered higher concentrations of cardenolides as early instars than as late instars, but within the fifth instar, concentration increased with body mass. Although it appears that large amounts of sequestration occurs in early instars, a host switching experiment revealed that caterpillars can compensate for feeding on low cardenolide host plants with substantial sequestration in the fifth instar. We highlight commonalities and striking differences in the mechanisms of sequestration depending on host plant chemistry and developmental stage, which have important implications for monarch defense.

Transcriptomics of monarch butterflies (Danaus plexippus) reveals that toxic host plants alter expression of detoxification genes and down‐regulate a small number of immune genes

“…Furthermore, our chemical analyses comparing foliage and frass cardenolide composition identified specific cardenolides in frass that are not present in foliage, including several with high polarity. This result, consistent with a recent study (Jones et al, ), suggests that some of the cardenolides excreted via frass are likely modified forms, created through detoxification processes. Thus, CYP450 genes may play a role in this modulation, but future studies are needed to directly examine their function.…”

“…Therefore, the up‐regulation of ABC transporters when feeding on low‐cardenolide milkweed compared to feeding on high‐cardenolide milkweed might be related to an increased rate of cardenolide sequestration. Although larvae may exhibit a relatively lower sequestration rate when feeding on A. curassavica than A incarnata , given the drastic difference in foliage cardenolide concentration (95‐fold), the total amount of cardenolide sequestered in larval tissue likely is much higher when fed A. curassavica , which has been demonstrated in previous studies (Decker, Soule, de Roode, & Hunter, ; Jones et al, ).…”

“…Previous studies have demonstrated that monarch larvae can regulate the level of cardenolide sequestration, as indicated by the fact that cardenolide concentration in larval hemolymph and milkweed leaves do not show a linear relationship (Rosenthal & Berenbaum, ). Interestingly, monarchs concentrate cardenolides when feeding on low‐cardenolide plants and sequester at a lower rate when feeding on plants with a very high concentration of cardenolides (Jones et al, ; Malcolm, ). Notably, our results show that all the differentially expressed ABC transporters were up‐regulated in larvae fed Asclepias incarnata , a milkweed species with very low‐cardenolide concentrations.…”

“…We could not use first instars due to size limitations. Also, second‐instar larvae sequester the highest amounts of cardenolides relative to their body mass (Jones, Peschenka, Flacht, & Agrawal, ). Upon hatching, we reared larvae individually in Petri dishes on cuttings from different plants of either A. incarnata or A. curassavica .…”

Variation of chemical compounds in wild Heliconiini reveals ecological factors involved in the evolution of chemical defenses in mimetic butterflies

“…Nevertheless, other local ecological factors may influence the evolution of chemical defenses in mimetic species. In butterflies for instance, deterrent compounds, as well as precursors for their synthesis, can be acquired by caterpillars during feeding on specific host plants (Jones, Petschenka, Flacht, & Agrawal, 2019;Nishida, 2002). Chemical defenses may thus vary among species depending on their diet (Engler & Gilbert, 2007).…”