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

Molecular Networking and On-Tissue Chemical Derivatization for Enhanced Identification and Visualization of Steroid Glycosides by MALDI Mass Spectrometry Imaging

“…To demonstrate the potential of OTCD MALDI MSI for investigating the spatial organization of metabolic networks with sampling areas below 5 μm 2 39 it was not possible to visualize cardiac glycoside distributions in the fat body of the larvae. However, we observed the accumulation of calotropin/ calactin, calotoxin/hydroxycalotropin/hydroxycalactin, and the precursor calotropagenin in the fat body with an increased accumulation in the outer layer of the fat tissue (Figure 3e and Figure S16a).…”

Metabolization and sequestration of plant specialized metabolites in insect herbivores: Current and emerging approaches

“…Testing for sequestration in intact insect specimens (i.e., without dissecting) requires avoiding contamination by gut contents which otherwise could result in false positive results. Starvation of insects prior to analyses should reduce the amount of plant chemicals in the insect gut but may be an insufficient approach given that insects can accumulate and retain plant toxins in the gut [as recently described for cardenolides in monarch butterfly caterpillars (Dreisbach et al, 2022)]. Alternatively, insects may be fed a PSM-free diet to more actively purge their gut from remaining PSMs.…”

“…This method has been used successfully for dananine caterpillars ( Petschenka and Agrawal, 2015 ) but might be useful for a range of insects. To study the fate of plant compounds during the insect gut passage in caterpillars (and probably other insect larvae or adults), dissecting freeze-dried insect specimens should be replaced by a modified approach based on freeze drying guts dissected from fresh caterpillars ( Dreisbach et al, 2022 ). During this procedure, live caterpillars are dissected under ice-cold PBS to expose the gut.…”

“…The authors were able to visualize the absorption of glucosinolates in the front part of the gut to avoid the activation of these secondary metabolites in the gut. By combining high resolution in mass and space, MALDI MSI was used to reveal differences and provide spatially resolved molecular insight for plant toxin sequestration in the monarch butterfly ( D. plexippus ) and the common crow ( Euploea core ) at the low-micrometer scale ( Dreisbach et al, 2022 ). Therefore, both studies demonstrate the potential of MSI to investigate the biochemistry of insect-plant interactions in the spatial context of insect tissues and cells.…”

Metabolization and Sequestration of Plant Specialized Metabolites in Insect Herbivores: Current and Emerging Approaches

“…This method has been used successfully for dananine caterpillars (Petschenka and Agrawal, 2015) but might be useful for a range of insects. To study the fate of plant compounds during the insect gut passage in caterpillars (and probably other insect larvae or adults), dissecting freezedried insect specimens should be replaced by a modified approach based on freeze drying guts dissected from fresh caterpillars (Dreisbach et al, 2022). During this procedure, live caterpillars are dissected under ice-cold PBS (phosphate buffered saline) to expose the gut.…”