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.

Helicoverpa punctigera (native budworm) is an important pest species in crops across Australia. From the third instar onward, this species causes severe damage to crop plants: therefore, caterpillars need to be managed at an early stage of their development. In our experiment, we raised H. punctigera on an artificial diet, which included different concentrations of the natural insecticides Spinetoram and Azadirachtin. The survival of the larvae, growth and body mass gain was recorded over 17 days. Only caterpillars raised on lowest toxin concentrations survived and molted successfully to the fifth instar, but had slower growth and body mass gain compared to the insecticide-free control group. Caterpillars fed on higher toxin concentrations never molted to the next instar or died in the first few days. To test how the toxins influence physiological conditions including metabolic rate and water loss, surviving fifth instar larvae were exposed to thermolimit respirometry: starting at 25°C following a constant increasing temperature ramping rate of 0.25°C –1 , until reaching the critical thermal maxima (CT max ). Caterpillars raised on a non-lethal dose of insecticides had higher metabolic rates and lost more water compared to the control group. Insects that have seem to consume more energy per mg tissue and have a higher water loss at high temperatures. Non-lethal concentrations of insecticides on pest insects physiology may reduce their impact on crops and may enable more targetted insecticide application.

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.

Sabotaging milkweed by monarch caterpillars is a textbook example for disarming plant defence. By severing leaf veins, monarchs are thought to prevent toxic latex flow to their feeding site. Here we show that sabotaging by monarch caterpillars is not an avoidance strategy. Instead, caterpillars actively ingest outflowing latex to increase sequestration of toxic latex cardenolides. Comparisons with caterpillars of the related non-sequestering common crow butterfly revealed three lines of evidence supporting our hypothesis. First, monarchs sabotage inconsistently and therefore the behaviour is not mandatory to feed on milkweed, while sabotaging in crows precedes every feeding event. Second, monarchs eagerly drink latex, while crow caterpillars spit out latex during sabotaging. Third, monarchs raised on detached leaves sequestered more cardenolides when latex was supplemented artificially. Hence, we conclude, that monarchs converted the ″sabotage to avoid″ strategy of their relatives into a ″sabotage to consume″ strategy for acquiring toxins for defence.