Organic anion-transporting polypeptides (OATPs) are integral membrane transporters that mediate cellular uptake of a broad range of substrates in humans. The functions of OATPs in insects are less well investigated and only poorly understood. A large number of compounds potentially toxic to insects are organic anions that include secondary plant and insecticide-derived metabolites. Some insect OATP genes are expressed in metabolic, neuroprotective and excreting tissues, and they are co-expressed together with genes known to be involved in detoxification and excretion. Therefore, a role in the elimination of insecticides has been proposed for OATPs, but experimental proof was pending. The aim of this study was to identify OATPs that affect tolerance to insecticides in the red flour beetle, Tribolium castaneum, a genomic model species and stored product pest. We determined expression profiles of TcOATP genes in different tissues and developmental stages and analyzed RNAi phenotypes. We found that some TcOATP genes had particularly high transcript levels in relevant tissues and that knockdown of TcOATP4-C1 led to sever developmental defects during larval–pupal molt. Then, we exposed the larvae to different chemically unrelated insecticides and analyzed transcript levels and mortalities. Some genes were specifically upregulated in response to insecticide treatment, and mortalities observed after administering certain insecticides were significantly increased when specific TcOATPs were silenced. By applying systemic RNAi in T. castaneum, we provide first evidence that OATPs are involved in the elimination of insecticides and hence may contribute to insecticide resistance, which becomes an increasingly serious problem in agriculture and forestry. Graphic abstract

BACKGROUND ATP‐binding cassette transporter (ABC transporter) subfamilies ABCA–C and ABCG–H have been implicated in insecticide detoxification, mostly based on findings of elevated gene expression in response to insecticide treatment. We previously characterized TcABCA–C genes from the model beetle and pest Tribolium castaneum and demonstrated that TcABCA and TcABCC genes are involved in the elimination of diflubenzuron, because RNA interference (RNAi)‐mediated gene silencing increased susceptibility. In this study, we focused on the potential functions of TcABCG and TcABCH genes in insecticide detoxification. RESULTS When we silenced the expression of TcABCG–H genes using RNAi, we noticed a previously unreported developmental RNAi phenotype for TcABCG‐4F, which is characterized by 50% mortality and ecdysial arrest during adult moult. When we knocked down the Drosophila brown orthologue TcABCG‐XC, we did not obtain apparent eye colour phenotypes but did observe a loss of riboflavin uptake by Malpighian tubules. Next, we determined the expression profiles of all TcABCG–H genes in different tissues and developmental stages and analysed transcript levels in response to treatment with four chemically unrelated insecticides. We found that some genes were specifically upregulated after insecticide treatment. However, when we determined insecticide‐induced mortalities in larvae that were treated by double‐stranded RNA injection to silence those TcABCG–H genes that were upregulated, we did not observe a significant increase in susceptibility to insecticides. CONCLUSION Our findings suggest that the observed insecticide‐dependent induction of TcABCG–H gene expression reflects an unspecific stress response, and hence underlines the significance of functional studies on insecticide detoxification. © 2021 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.