Transcriptional plasticity of different ABC transporter genes from Tribolium castaneum contributes to diflubenzuron resistance

The development of insecticide resistance challenges the sustainability of pest control and several studies have shown that ABC transporters contribute to this process. ABC transporters are known to transport a large range of chemically diverse molecules across cellular membranes, and therefore the identification of ABC transporters involved in insecticide resistance is difficult. Here, we describe a comprehensive strategy for the identification of whole sets of ABC transporters involved in insecticide resistance using the pest beetle, Tribolium castaneum (Tc) as a model. We analyzed the expression of ABCA to ABCC genes in different tissues and developmental stages using larvae that were sensitive or resistant to diflubenzuron (DFB). The mRNA levels of several ABC genes expressed in excretory or metabolic tissues such as midgut, Malpighian tubules or fat body were markedly upregulated in response to DFB. Next, we monitored mortality in the presence of the ABC inhibitor verapamil, and found that it causes sensitization to DFB. We furthermore established a competitive assay for the elimination of DFB, based on Texas Red (TR) fluorescence. We monitored TR elimination in larvae that were treated with DFB or different ABC inhibitors, and combinations of them. TR elimination was decreased significantly in the presence of DFB, verapamil and the ABCC inhibitor MK-571. The effect was synergized when DFB and verapamil were both present suggesting that the transport of TR and DFB involves overlapping sets of ABC transporters. Finally, we silenced the expression of DFB-responding ABC genes by RNA interference and then followed the survival rates after DFB exposure. Mortality increased particularly when specific ABCA and ABCC genes were silenced. Taken together, we were able to show that different ABC transporters expressed in metabolic and excretory tissues contribute to the elimination of DFB. Up- or down-regulation of gene expression occurs within a few days already at very low DFB concentrations. These results suggests that transcriptional plasticity of several ABC genes allows adaptation of the efflux capacity in different tissues to eliminate insecticides and/or their metabolites.