Selection of stable reference genes for real-time quantitative PCR in honey bee pesticide toxicity studies

Honey bees are exposed to various pesticides via both acute and chronic routes. Transcriptional profiling based on quantitative real-time PCR could be used as a representative method to assess the effects of pesticides on honey bee physiology. In this method, the identification of reference genes left unaffected by pesticide treatment is required for accurate analysis; however, there have been no reports regarding such reference genes. In this study, the expression level fluctuations of two putative reference genes (arf1 and rab1), along with three commonly used reference genes (rpS18, actin, and gapdh), were investigated in different stages and tissues following treatment with fluvalinate, coumaphos, imidacloprid, and flupyradifurone, and their performances were compared using geNorm, BestKeeper, NormFinder and RefFinder. The expression stability of rab1a was the highest, followed by that of arf1, rpS18, actin, and gapdh, suggesting that rab1 and arf1 can be employed as reliable reference genes in pesticide studies.