Despite sequestration of toxins being a common coevolutionary response to plant defence in phytophagous insects, the macroevolution of the traits involved is largely unaddressed. Using a phylogenetic approach comprising species from four continents, we analysed the ability to sequester toxic cardenolides in the hemipteran subfamily Lygaeinae, which is widely associated with cardenolide-producing Apocynaceae. In addition, we analysed cardenolide resistance of their Na þ /K þ -ATPases, the molecular target of cardenolides. Our data indicate that cardenolide sequestration and cardenolide-resistant Na þ /K þ -ATPase are basal adaptations in the Lygaeinae. In two species that shifted to non-apocynaceous hosts, the ability to sequester was secondarily reduced, yet NaATPase resistance was maintained. We suggest that both traits evolved together and represent major coevolutionary adaptations responsible for the evolutionary success of lygaeine bugs. Moreover, specialization on cardenolides was not an evolutionary dead end, but enabled this insect lineage to host shift to cardenolide-producing plants from distantly related families.

Species of the heteropteran subfamily Lygaeinae possess special subcuticular compartments to store cardiac glycosides, plant‐derived defensive compounds, which they release upon predator attack. In adults of the large milkweed bug, Oncopeltus fasciatus, these storage compartments have previously been described as a modified integument, forming a fluid‐filled dorsolateral space. Here we use three‐dimensional imaging of serial histological sections and synchrotron radiation‐based micro‐computed tomography data to reveal the morphology of these storage compartments and the mechanisms used for the release of a cardiac glycoside‐rich fluid upon attack. Our comparative analysis revealed that the morphology and release mechanism vary among the species investigated. By reconstructing these traits on a recent molecular phylogeny of the Lygaeinae, we demonstrate that the adaptations for the storage and release of cardiac glycosides have evolved in a stepwise manner.

No abstract

Antipredator defense of herbivorous insects often relies on the potential toxicity of defensive chemicals sequestered from their host plants. The colorful Lygaeinae (Heteroptera: Lygaeidae) store a concentrated mixture of toxic cardenolides (cardiac glycosides) in specialized storage compartments of the bugs' integument, from which they are released upon attack. Larvae and adults of the large milkweed bug Oncopeltus fasciatus (Heteroptera: Lygaeinae) are specialized to feed on cardenolide‐containing milkweeds in the plant genus Asclepias and display a conspicuous red and black colorations. To investigate whether O. fasciatus gained improved protection by feeding on a toxic host plant (Asclepias syriaca), compared to a nontoxic alternative (sunflower seeds), we fed nymphs and adults of O. fasciatus to the golden orb‐weaver Nephila senegalensis. While visually oriented vertebrates, such as avian predators, have been intensively investigated for their reaction to defensive compounds and aposematic coloration, less attention has been paid to invertebrate predators. Their different perceptual abilities can provide important opportunities for testing hypotheses on warning coloration and chemical defenses. The predation trials showed that the bugs fed on Asclepias were significantly less likely to be killed than the bugs reared on a cardenolide‐free diet. This suggests that sequestered cardenolides in O. fasciatus nymphs and adults represent a significant fitness advantage on an individual level against this invertebrate predator. Yet, when testing for avoidance learning in the spiders, negative experience did not change the way how similar prey was attacked at the next encounter. In this case, visual or chemical aposematism thus does not seem to matter for predator learning.