Cardenolide diversity and toxicity in tri trophic interactions Natural selection is expected to drive evolution towards a small number of the most beneficial chemical defences. However in the genus Asclepias there is a high diversity of cardenolides a compound class that target the Na+ K+—ATPase enzyme. The specialised herbivores of Asclepias are hypothesised to impose selection for chemical defences. One major objective of this thesis was to test whether cardenolides in the seeds of Asclepias curassavica defend against the specialist seed bug Oncopeltus fasciatus. For this ten compounds were isolated from A. curassavica seeds three of those new and seven previously reported. Glucopyranosyl frugoside is the most abundant cardenolide in this tissue and there was a significant variation in the compounds’ inhibition of the unadapted Na+ K+—ATPase. To test whether the variability in cardenolides in the seeds could represent the plants response to selection by O. fasciatus; cardenolide analysis of the seeds and O. fasciatus was combined with tests of the inhibitory capacity of a subset of compounds against of O. fasciatus and a non adapted insect target sites. Frugoside and gofruside were more concentrated in the bugs than is available in their diet and had contrasting potency toward the O. fasciatus target site. Thus the accumulation of these compounds could not be explained by their availability in seeds or the biological activity on the bug’s target site. To test an alternative hypothesis that the plant chemodiversity and sequestration patterns of O. fasciatus are explained by natural selection by the third trophic level. The inhibitory properties of cardenolides in O. fasciatus were tested against several predator target sites. Glucopyranosyl frugoside and frugoside were toxic for both resistant and sensitive predators whereas gofruside had varying degrees of inhibitory capacity. This differential effect suggests that predators can contribute to defence diversification in both milkweed plants and milkweed herbivores.