Environmental clines in organismal defensive traits are usually attributed to stronger selection by enemies at lower latitudes or near the host’s range center. Nonetheless, little functional evidence has supported this hypothesis, especially for coevolving plants and herbivores. We quantified cardenolide toxins in seeds of 24 populations of common milkweed ( Asclepias syriaca ) across 13 degrees of latitude, revealing a pattern of increasing cardenolide concentrations toward the host's range center. The unusual nitrogen-containing cardenolide labriformin was an exception and peaked at higher latitudes. Milkweed seeds are eaten by specialist lygaeid bugs that are even more tolerant of cardenolides than the monarch butterfly, concentrating most cardenolides (but not labriformin) from seeds into their bodies. Accordingly, whether cardenolides defend seeds against these specialist bugs is unclear. We demonstrate that Oncopeltus fasciatus (Lygaeidae) metabolized two major compounds (glycosylated aspecioside and labriformin) into distinct products that were sequestered without impairing growth. We next tested several isolated cardenolides in vitro on the physiological target of cardenolides (Na + /K + -ATPase); there was little variation among compounds in inhibition of an unadapted Na + /K + -ATPase, but tremendous variation in impacts on that of monarchs and Oncopeltu s. Labriformin was the most inhibitive compound tested for both insects, but Oncopeltus had the greater advantage over monarchs in tolerating labriformin compared to other compounds. Three metabolized (and stored) cardenolides were less toxic than their parent compounds found in seeds. Our results suggest that a potent plant defense is evolving by natural selection along a geographical cline and targets specialist herbivores, but is met by insect tolerance, detoxification, and sequestration.

Insect artificial diets are not only an important tool for mass rearing, nutritional research, and maintaining laboratory colonies but also for studying insect‐plant interactions. For herbivorous insects able to sequester plant toxins, feeding and sequestration assays based on artificial diet allow for the investigation of physiological, ecological, and evolutionary questions which may be difficult to study using real plants representing complex chemical environments. We developed a simple artificial diet, consisting of sunflower meal pressed into pills, for the milkweed bugs Oncopeltus fasciatus (Dallas) and Spilostethus saxatilis (Scopoli) (Heteroptera: Lygaeidae), which are capable of sequestering cardenolides and colchicum alkaloids, respectively. We assessed insect performance, suitability of the diet for sequestration assays, and its shelf life. Compared to sunflower seeds which are widely used as a laboratory maintenance diet for milkweed bugs, no differences were found in terms of weight development, presence of deformities, speed of development, or mortality. Importantly, after feeding O. fasciatus and S. saxatilis sunflower pills enriched with crystalline ouabain (cardenolide) or colchicine (colchicum alkaloid), respectively, sequestration was observed in both species. Moreover, as a prerequisite to test ecological hypotheses, our method allows for adequate concentration control and homogenous distribution of toxins across the diet. Under relatively warm conditions (27 °C and 60% r.h.), the new diet was stable for up to 10 days when used for feeding assays with adult bugs. Therefore, studies focusing on the role of plant toxins in predator–prey interactions and plant defense, but also insecticide research could benefit from using this approach.

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