Spatial metabolomics reveal divergent cardenolide processing in the monarch (Danaus plexippus) and the common crow butterfly (Euploea core)

Abstract

Although being famous for sequestering milkweed cardenolides, the mechanism of sequestration and where cardenolides are localized in caterpillars of the monarch butterfly (Danaus plexippus, Lepidoptera: Danaini) is still unknown. While monarchs tolerate cardenolides by a resistant Na+/K+‐ATPase, it is unclear how closely related species such as the nonsequestering common crow butterfly (Euploea core, Lepidoptera: Danaini) cope with these toxins. Using novel atmospheric‐pressure scanning microprobe matrix‐assisted laser/desorption ionization mass spectrometry imaging, we compared the distribution of cardenolides in caterpillars of D. plexippus and E. core. Specifically, we tested at which physiological scale quantitative differences between both species are mediated and how cardenolides distribute across body tissues. Whereas D. plexippus sequestered most cardenolides from milkweed (Asclepias curassavica), no cardenolides were found in the tissues of E. core. Remarkably, quantitative differences already manifest in the gut lumen: while monarchs retain and accumulate cardenolides above plant concentrations, the toxins are degraded in the gut lumen of crows. We visualized cardenolide transport over the monarch midgut epithelium and identified integument cells as the final site of storage where defences might be perceived by predators. Our study provides molecular insight into cardenolide sequestration and highlights the great potential of mass spectrometry imaging for understanding the kinetics of multiple compounds including endogenous metabolites, plant toxins, or insecticides in insects.

Late-instar monarch caterpillars sabotage milkweed to acquire toxins, not to disarm plant defence

“…Whereas monarch caterpillars can tolerate sequestered cardenolides by means of a resistant Na + /K + -ATPase, caterpillars of Euploea naturally consume cardenolide-containing plants but cannot cope with the toxins when injected into their body cavity [ 47 ]. Instead, Euploea caterpillars degrade cardenolides in the gut lumen, presumably to detoxify them [ 48 ]. In the face of antagonistic insect−plant coevolution, monarchs have evolved a number of traits to exploit plant defences (cardenolides) for their own benefit.…”

Testing the selective sequestration hypothesis: Monarch butterflies preferentially sequester plant defences that are less toxic to themselves while maintaining potency to others

“…Further investigation is needed to determine where and when cardenolide modifications occur (e.g. Dreisbach et al., 2023), and whether selective mono‐deglycosylation, potentially catalysed by a β‐glucosidase enzyme or by the basic pH environment in the caterpillar gut, is responsible for the presence of a single carbohydrate motif. β‐Glucosidases are plant enzymes primarily known for their ability to disrupt insect digestion by activating a pro‐toxin through the removal of a carbohydrate motif (Vassão et al., 2018).…”

“…Further investigation is needed to determine where and when cardenolide modifications occur (e.g. Dreisbach et al, 2023), and whether The response variables analysed were μg cardenolide needed for 50% inhibition of the enzyme (i.e. extracts were adjusted by cardenolide concentrations such that the potency of the extracts was tested).…”

“…We define sequestration as the accumulation of plant defence compounds in the insect body, typically following digestion, recognizing that compounds may be modified during the process (Beran & Petschenka, 2022; Dreisbach et al., 2023). Here we address the relative toxicity of plant extracts versus insect‐sequestered compounds to a specialist herbivore to test the prediction that such insects reduce self‐harm by preferentially sequestering compounds that are the least toxic to themselves.…”