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

虽然植物已经进化出了大量有毒的次级化合物来抵御它们的食草性敌人，但这些防御并不是不可逾越的，即使是最有毒的植物也有一些昆虫设法以它们为食。强心苷（cardenolides和bufadienolides）是一类毒性很高的化合物。对这些化合物的适应已经成为耐药性趋同进化的教科书例子，这种耐药性通过Na，K-ATP酶的靶位点不敏感性来规避有害作用。然而，很明显，除了靶点不敏感性外，转运机制在适应这些毒素方面起着决定性作用。这种转运机制可以防止从肠道摄取，调节排泄，保护敏感组织或保证转移到身体的专门不敏感区室。我们目前的数据有力地支持ATP结合盒亚家族B（ABCB）转运蛋白在介导强心苷适应的转运网络中起决定性作用。本研究的目的是阐明的重要性，ABCB蛋白的大马利筋错误，Oncopeltus fasciatus的Cardenoprotein运输。O. fasciatus适应于其食用植物中的Cardenolides，并且对它们对Na，K-ATPase的抑制作用不敏感。这些虫子是警戒色的，在被捕食者攻击时释放的毒素会被隔离在它们的防御液中。这需要从肠道运输到血淋巴中，并进一步逆着浓度梯度进入防御液的储存室。同时，神经组织需要主动外排载体的保护，以防止可能损害神经兴奋性的强心苷的积累。本项目首先从O. fasciatus，然后通过体内和体外测定确定它们的组织分布和功能表征。RNAi介导的敲低实验结合分泌和吸收的马氏管的功能测试将被用来分析特定基因转录的重要性。此外，还将通过表型筛选和血淋巴和防御液的HPLC分析研究转录物敲低与注射或口服给予强心内酯组合的作用。该研究将阐明Cardenolides的运输网络，并进一步了解ABCB转运蛋白在昆虫植食性动物对有毒植物化合物的适应中的相关性。这有助于有价值的知识，仍然广泛调查的过程中异生物质运输昆虫上皮细胞。

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.