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Predators and parasitoids regulate insect populations and select defense mechanisms such as the sequestration of plant toxins. Sequestration is common among herbivorous insects, yet how the structural variation of plant toxins affects defenses against predators remains largely unknown. The palearctic milkweed bug Lygaeus equestris (Heteroptera: Lygaeinae) was recently shown to sequester cardenolides from Adonis vernalis (Ranunculaceae), while its relative Horvathiolus superbus also obtains cardenolides but from Digitalis purpurea (Plantaginaceae). Remarkably, toxin sequestration protects both species against insectivorous birds, but only H. superbus gains protection against predatory lacewing larvae. Here, we used a full factorial design to test whether this difference was mediated by the differences in plant chemistry or by the insect species. We raised both species of milkweed bugs on seeds from both species of host plants and carried out predation assays using the larvae of the lacewing Chrysoperla carnea. In addition, we analyzed the toxins sequestered by the bugs via liquid chromatography (HPLC). We found that both insect species gained protection by sequestering cardenolides from D. purpurea but not from A. vernalis. Since the total amount of toxins stored was not different between the plant species in H. superbus and even lower in L. equestris from D. purpurea compared to A. vernalis, the effect is most likely mediated by structural differences of the sequestered toxins. Our findings indicate that predator–prey interactions are highly context-specific and that the host plant choice can affect the levels of protection to various predator types based on structural differences within the same class of chemical compounds.

Herbivorous insects encounter diverse plant specialized metabolites (PSMs) in their diet, that have deterrent, anti-nutritional, or toxic properties. Understanding how they cope with PSMs is crucial to understand their biology, population dynamics, and evolution. This review summarizes current and emerging cutting-edge methods that can be used to characterize the metabolic fate of PSMs, from ingestion to excretion or sequestration. It further emphasizes a workflow that enables not only to study PSM metabolism at different scales, but also to tackle and validate the genetic and biochemical mechanisms involved in PSM resistance by herbivores. This review thus aims at facilitating research on PSM-mediated plant-herbivore interactions.

In some aposematic species the conspicuousness of an individual's warning signal and the concentration of its chemical defense are positively correlated. Several mechanisms have been proposed to explain this phenomenon, including resource allocation trade-offs where the same limiting resource is needed to produce both the warning signal and chemical defense. Here, the large milkweed bug (Oncopeltus fasciatus: Heteroptera, Lygaeinae) was used to test whether allocation of antioxidants, that can impart color, trade against their availability to prevent self-damage caused by toxin sequestration. We investigated if (i) the sequestration of cardenolides is associated with costs in the form of changes in oxidative state; and (ii) oxidative state can affect the capacity of individuals to produce warning signals. We reared milkweed bugs on artificial diets with increasing quantities of cardenolides and examined how this affected signal quality (brightness and chroma) across different instars. We then related the expression of warning colors to the quantity of sequestered cardenolides and indicators of oxidative state-oxidative lipid damage (malondialdehyde), and two antioxidants: total superoxide dismutase and total glutathione. Bugs that sequestered more cardenolides had significantly lower levels of the antioxidant glutathione, and bugs with less total glutathione had less luminant orange warning signals and reduced chroma of their black patches compared to bugs with more glutathione. Bugs that sequestered more cardenolides also had reduced red-green chroma of their black patches that was unrelated to oxidative state. Our results give tentative support for a physiological cost of sequestration in milkweed bugs and a mechanistic link between antioxidant availability, sequestration, and warning signals.

1. Sequestration, i.e., the accumulation of plant toxins into body tissues for defence, is primarily observed in specialised insects. Sequestration was frequently predicted to incur a physiological cost mediated by increased exposure to plant toxins and may require resistance traits different from those of non-sequestering insects. Alternatively, sequestering species could experience a cost in the absence of toxins due to selection on physiological homeostasis under permanent exposure of sequestered toxins in body tissues. 2. Milkweed bugs (Heteroptera: Lygaeinae) sequester high amounts of plant-derived cardenolides. Although being potent inhibitors of the ubiquitous animal enzyme Na+/K+-ATPase, milkweed bugs can tolerate cardenolides by means of resistant Na+/K+-ATPases. Both adaptations, resistance and sequestration, are ancestral traits shared by most species of the Lygaeinae. 3. Using four milkweed bug species and the related European firebug (Pyrrhocoris apterus) showing different combinations of the traits ′cardenolide resistance′ and ′cardenolide sequestration′, we set out to test how the two traits affect larval growth upon exposure to dietary cardenolides in an artificial diet system. While cardenolides impaired the growth of P. apterus nymphs neither possessing a resistant Na+/K+-ATPase nor sequestering cardenolides, growth was not affected in the non-sequestering milkweed bug Arocatus longiceps, which possesses a resistant Na+/K+-ATPase. Remarkably, cardenolides increased growth in the sequestering dietary specialists Caenocoris nerii and Oncopeltus fasciatus but not in the sequestering dietary generalist Spilostethus pandurus, which all possess a resistant Na+/K+-ATPase. 4. We then assessed the effect of dietary cardenolides on additional life history parameters, including developmental speed, the longevity of adults, and reproductive success in O. fasciatus. Remarkably, nymphs under cardenolide exposure developed substantially faster and lived longer as adults. However, fecundity of adults was reduced when maintained on cardenolide-containing diet for their entire life-time but not when adults were transferred to non-toxic sunflower seeds. 5. We speculate that the resistant Na+/K+-ATPase of milkweed bugs is selected for working optimally in a ′toxic environment′, i.e. when sequestered cardenolides are stored in the body tissues. Contrary to the assumption that toxins sequestered for defence produce a physiological burden, our data suggest that they can even increase fitness in specialised insects.