Browsing by Person "Espinosa del Alba, Laura"

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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.