ABCB transporters as key adaptations of herbivorous insects to toxic cardiac glycosides

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Although plants have evolved a plethora of noxious secondary compounds to ward off their herbivore enemies, these defenses are not insurmountable and even the most toxic plants have some insects that manage to feed on them. One such highly toxic class of compounds are cardiac glycosides (cardenolides and bufadienolides). Adaptation to these compounds has become a text book example for convergent evolution of resistance that circumvents noxious effects by target-site insensitivity of the Na,K-ATPase. However, it is obvious that in addition to target-site insensitivity transport mechanisms play a decisive role in the adaptation to these toxins. Such transport mechanisms may prevent uptake from the gut, regulate excretion, protect sensitive tissues or guarantee the transfer into specialized insensitive compartments of the body. Our current data strongly support a role of ATP-binding cassette subfamily B (ABCB) transporters as decisive players in the transport network mediating adaptation to cardiac glycosides. The aim of this study is to elucidate the importance of ABCB proteins for cardenolide transport in the large milkweed bug, Oncopeltus fasciatus. O. fasciatus is adapted to the cardenolides in its food plants and is insensitive to their inhibiting effect on the Na,K-ATPase. The bugs are aposematically colored and sequester the toxins in their defense fluids which are released upon attack by predators. This necessitates a transport from the gut into the hemolymph and further on against a concentration gradient into the storage compartments of the defense fluids. At the same time the nervous tissue needs to be protected by active efflux carriers from an accumulation of cardenolides that might impair nerve excitability. This project starts by identifying ABCB full and half transporters from transcriptomes of O. fasciatus followed by a determination of their tissue distributions and a functional characterization by in vivo and in vitro assays. RNAi mediated knock-down experiments combined with functional tests of excretion and resorption by the Malpighian tubules will be used to analyze the importance of specific gene transcripts. Effects of transcript knock-down combined with injection or oral administration of cardenolides will in addition be investigated by phenotypic screening and HPLC analyses of hemolymph and defense fluids. The study will thereby elucidate the transport network for cardenolides and further our understanding of the relevance of ABCB transporters in the adaptations of insect herbivores to toxic plant compounds. This contributes valuable knowledge to the still widely under-investigated processes of xenobiotic transport across insect epithelia.