The physiological functions of ABC transporters in Tribolium castaneum and their role in protecting insects from pesticides and their metabolites

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The ATP-binding cassette (ABC) transporters belong to a large superfamily of proteins that have important physiological functions in all living organisms. Most are integral membrane proteins that transport a broad spectrum of substrates across cellular membranes. While ABC transporters from vertebrates have been extensively studied, there is only little insight into their physiological functions in insects. Therefore, we conducted a comprehensive RNAi study and silenced the expression of all 73 ABC genes identified in the genome of the red flour beetle, Tribolium castaneum. We monitored development to screen for evident phenotypes that could link the respective gene with a specific function. Using this approach, we identified ten genes that lead to abnormal phenotypes during development when their expression was knocked-down. Two of these genes are associated with eye pigmentation (TcABCG-9A/B), two are related to protein biosynthesis (TcABCE-3A and TcABCF-2A), two appear to have functions in lipid transport affecting waterproofing of the epicuticle (TcABCG-4C and TcABCH-9C), one seems to be involved in an ecdysterone-related processes (TcABCG-8A), and the other three may have other developmental functions (TcABCA-9A/B and TcABCB-5A). In the proposed research, we will pursue several aspects related to the functions of selected ABC transporters in T. castaneum. 1) We aim to clarify the physiological function of TcABCG-8A and address the question how this ABC transporter modulates ecdysterone responses. This will involve the analysis of transport properties using vesicles from baculovirus-infected insect cells that express different mutated versions of TcABCG-8A. 2) We will examine the roles of TcABCG-4C and TcABCH-9C in the transport of cuticular lipids. Next to GC/MS determination of the lipid composition in the cuticle of control and dsRNA-injected larvae, we will identify the transport substrates using vesicle preparations of insect cells expressing these TcABC genes. 3) We will further analyze the sulfonylurea receptor homolog TcABCC-9A, which has been suggested to mediate the insecticidal effects of benzoylurea compounds known to inhibit chitin synthesis. Utilizing a newly developed membrane preparation from T. castaneum pupal elytrae, we will perform in vitro analysis to verify or falsify this hypothesis. 4) As T. castaneum is also an excellent model for investigating insecticidal effects, we will perform an RNAi screen to identify TcABC genes that are involved in the elimination of insecticides and/or their metabolites. This study will be complemented by fluid secretion assays using large Malpighian tubules isolated from the superworm, the larvae of the tenebrionid beetle, Zophobas artratus, which is related to T. castaneum and equally amenable to RNAi. 4) Finally, we will systematically address the question whether inhibitors of ABC transporters are suited to increase susceptibility to insecticides of tolerant Tribolium strains.