Browsing by Subject "Milkweed bugs"

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Physiological and ecological implications of sequestered cardenolides in the milkweed bugs (Heteroptera: Lygaeinae)
(2022) Pokharel, Prayan; Petschenka, Georg
Milkweed bugs (Heteroptera: Lygaeinae) have a predilection for toxic plants, and possess a distinctive black and red coloration. Although many milkweed bugs are generalist seed predators, they commonly feed on plants in the family Apocynaceae (milkweed) which often contain toxic cardenolides. Cardenolides inhibit the ubiquitous Na+/K+-ATPase, an essential animal enzyme mediating essential physiological functions. Milkweed bugs possess pronounced insensitivity towards cardenolides due to a few amino acid substitutions in the Na+/K+-ATPase (i.e., target site insensitivity) and sequester cardenolides for protection against their predators. The overarching question remains whether chemical defenses, in aposematic individuals sequestering toxins, incur physiological costs, such as effects on growth or other fitness parameters like longevity and fecundity, production of color pigments, and handling oxidative stresses, and/or ecological costs, such as universality of toxin defense. Using an artificial diet, I raised larvae of four milkweed bug species (Oncopeltus fasciatus, Caenocoris nerii, Spilostethus pandurus and Arocatus longiceps) and a closely related pyrrhocorid bug species (Pyrrhocoris apterus) on three increasing dietary doses of cardenolides, and assessed the increase in growth by recording the mass until adult. Additionally, I investigated the life-history parameters only in O. fasciatus. To understand if milkweed bugs exhibit honest signaling, using same artificial diet treatment, the color intensity of O. fasciatus was measured by taking photographs in each larval stage until adulthood. To understand if toxin sequestration in milkweed bugs imposes oxidative stress, biomarkers of oxidative stress was measured through biochemical assays for lipid peroxidation (malondialdehyde, or MDA), superoxide dismutase (SOD), and total glutathione content (GSH). To understand why protection against certain predators is not observed in all bug species although they feed on seeds containing cardenolides, I tested if the outcome of predator- prey interaction was mediated by the structural variation within the same class of compound or by the insect species. For this purpose, I raised two milkweed bug species (Lygaeus equestris and Horvathiolus superbus) on the seeds of two phylogenetically unrelated host plants (Ranunculaceae: Adonis vernalis and Plantaginaceae: Digitalis purpurea) from which the bugs sequestered cardenolides, and carried out predation assays with lacewing larvae. The amount of toxins sequestered by the milkweed bugs was estimated using high performance liquid chromatography. My research revealed that dietary plant toxins increased growth in the sequestering specialists (O. fasciatus and C. nerii) but not in the sequestering generalist, S. pandurus, despite all possessing toxin-resistant Na+/K+-ATPases. Under exposure to the dietary toxins, the growth of A. longiceps nymphs (resistant and non-sequestering) was unaffected, while that of P. apterus (non-resistant and non-sequestering) was impaired. In addition, O. fasciatus nymphs developed to adults faster and lived longer as adults under toxin exposure when compared to individuals raised on the control diet, but produced significantly fewer offspring unless being transferred to a toxin-free diet after reaching adulthood. Furthermore, I showed that O. fasciatus raised on the high and medium levels of dietary cardenolides had significantly lower levels of GSH. Bugs with more GSH levels had brighter warning signals but these signals were not related to sequestration. Besides physiological aspects, the chance of milkweed bugs surviving a predator attack strongly depended on the structural differences of sequestered toxins. Overall, I found that cardenolide consumption exerts a positive effect on overall fitness in milkweed bugs, a conclusion in disagreement with current theory predicting costs of sequestration. Oxidative state may be a fundamental aspect where costs lie in aposematic individuals sequestering toxins, and the effect of plant-toxin sequestration on predators is affected by the structural variation of defensive compounds and therefore depends on the ecological context, i.e., host-plant use. My dissertation provides insight into the implications of physiology and ecology on sequestering aposematic insects, giving us a better understanding of plant-insect-predator interactions.
Sequestration of plant toxins in milkweed bugs (Heteroptera: Lygaeinae) : physiological implications and mechanisms
(2023) Espinosa del Alba, Laura; Petschenka, Georg
Insect herbivores and plants together are a crucial component of terrestrial macro-biodiversity. Within the realm of plant-insect interactions, phytophagy by insects triggered an “arms-race” dynamic resulting in escalatory adaptation and counter-adaptation over time. This coevolution led to complex phenomena such as sequestration of plant toxins by specialized insects, with the main aim to deter predators. Although sequestration is an extensively reported phenomenon, many physiological aspects and underlying mechanisms remain largely unexplored. Milkweed bugs (Heteroptera: Lygaeinae) constitute a versatile model ideally suited for studying both areas due to their particular evolutionary history. They are primarily associated with plant species in the Apocynaceae which commonly produce cardenolides, but remarkably some milkweed bug species secondarily evolved novel associations with phylogenetically disparate plant families supplying new sources of chemically related or unrelated toxins. Using as model milkweed bugs the cardenolide specialist Oncopeltus fasciatus and Spilostethus saxatilis, a species that shifted to sequestration of the chemically unrelated colchicoids, the present thesis first aimed to develop a new artificial diet that allowed the incorporation of the desired types and amounts of toxins without impairing insect performance. Taking a simplified approach, an artificial diet presented in a pill form and made of 100% organic sunflower meal was established. Despite the fact that the new diet has remarkable energy and nutrient differences with sunflower seeds (the laboratory diet), no differences in terms of insect performance were found between the two diets in O. fasciatus and S. saxatilis. Moreover, the new diet presented an acceptable concentration accuracy and shelf-life for short-term toxin feeding assays. Once established, the new diet was used to investigate the effects of cardenolides and colchicoids on several life-history traits of S. saxatilis, and to compare them with the effect of cardenolides in O. fasciatus. Although both classes of toxins have different molecular targets (cardenolides: Na+/K+-ATPase; colchicoids: tubulin), S. saxatilis was able to sequester them at a cost-free level. In fact, an increased performance was observed in O. fasciatus and an according trend was found in S. saxatilis after dietary exposure to cardenolides and colchicoids, respectively. Among cardenolides, labriformin is especially toxic for milkweed-specialist Na+/K+-ATPases in vitro. Nevertheless, it was shown to have no costs in terms of growth and fertility at the whole organism level for O. fasciatus. This finding might be an example of reciprocal evolution between milkweed plants and its herbivores, where highly toxic cardenolides specifically targeted to insect specialists are counteracted by tolerance, detoxification, and sequestration strategies. To assess the role of sequestration beyond normal physiological conditions, O. fasciatus and S. saxatilis were orally infected with the bacterium Pseudomonas entomophila. Neither cardenolides nor colchicoids provided a higher resistance or tolerance. Regarding mechanisms of sequestration, the other overarching research topic of the present thesis, both in vivo (whole animal) and in vitro (isolated digestive tracts) approaches showed no reciprocal competition for the same transport mechanism between chemically related and unrelated toxins. Furthermore, the digestive tract of milkweed bugs did not seem to be a critical mediator as it is for other non-sequestering and sequestering species. The time course of sequestration for the model species was resolved from three days to one hour, and the higher levels of colchicoids detected in S. saxatilis compared to the level of cardenolides in O. fasciatus might indicate an early acquisition of defenses with the shift from cardenolide to colchicoid-containing plants. Finally, a hint to preadaptation mechanisms to resist novel toxins was documented in Spilostethus pandurus, a species that belongs to the same genus as S. saxatilis, thereby providing a basis for future investigations.
A simple artificial diet for feeding and sequestration assays for the milkweed bugs Oncopeltus fasciatus and Spilostethus saxatilis
(2023) Espinosa del Alba, Laura; Petschenka, Georg
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.