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

Although being famous for sequestering milkweed cardenolides, the mechanism of sequestration and where cardenolides are localized in caterpillars of the monarch butterfly ( Danaus plexippus ) is still unknown. While monarchs tolerate cardenolides by a resistant Na+/K+-ATPase, it is unclear how closely related species such as the non-sequestering common crow ( Euploea core ) 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 defenses 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. ### Competing Interest Statement B.S. is a consultant, and D.D. is a part-time employee of TransMIT GmbH, Giessen, Germany. All other authors declare no conflicts of interest.