The fruit fly, Drosophila melanogaster, is a good model organism in various areas of biological science. Since D. melanogaster has been thought to be adapted to the chemical stress environment caused by the overripen, decay and fermented fruits, identification of the genes involved in chemical tolerance and investigation of their expression patterns are essential for better understanding of the physiological evolution in D. melanogaster. For investigation of the gene expression level, quantitative real-time PCR (qRT-PCR) can be applied to quantify gene expression level and selection of reliable reference gene(s) for normalization is an accurate step. In the present study, therefore, we validated the expression stabilities of ten candidate reference genes using three softwares (geNorm, NormFinder and BestKeeper) in D. melanogaster exposed to different concentrations of acetic acid, ethanol and 2-phenylethanol. Although three programs resulted in slightly different gene stability ranks, but overall tbp encoding TATA box binding protein was most stable gene in acetic acid and ethanol exposed fly, while nd encoding NADH dehydrogenase was the most suitable reference gene in the case of 2-phenylethanol treatment. In the comparison of three chemical treatment condition, nd was also suggested to be most optimal reference gene. In addition, optimal number of reference gene for accurate normalization was calculated by geNorm pairwise analysis, and selection of multiple reference genes was suggested to be better for target gene normalization method than use of a single reference gene.

Drosophila melanogaster and Drosophila suzukii are the species of the family Drosophilidae. Although these two fruit flies are taxonomically close species, D. suzukii is thought to be evolutionally adapted to the flesh or maturing fruits, whereas D. melanogaster is adapted to more fermented environments. According to the previous studies, several environmental toxins, such as acetic acid, ethanol, methanol and phenylacetate, ect., have been identified from rotten fruit and fermentation procedures. Considering the differences of distinct habitat between two flies, D. melanogaster is hypothesized to exhibit higher tolerance to the chemical toxins than D. suzukii. Therefore, in this study, we compared the tolerance and susceptibility of two fruit flies to three chemicals (acetic acid, ethanol, 2-phenlyethano).