Validation of Quantitative Real-time PCR Reference Genes and Spatial Expression Profiles of Detoxication-related Genes Under Pesticide Induction in Honey Bee, Apis Mellifera

Recently, pesticides have been suggested to be one of the factors responsible for the large-scale decline in honey bee populations, including colony collapse disorder. The identification of the genes that respond to pesticide exposure based on their expression is essential for understanding the xenobiotic detoxification metabolism in honey bees. For the accurate determination of target gene expression by quantitative real-time PCR, the expression stability of reference genes should be validated in honey bees exposed to various pesticides. Therefore, in this study, to select the optimal reference genes, we analyzed the amplification efficiencies of five candidate reference genes (RPS5, RPS18, GAPDH, ARF1, and RAD1a) and their expression stability values using four programs (geNorm, NormFinder, BestKeeper, and RefFinder) across samples of five body parts (head, thorax, gut, fat body, and carcass) from honey bees exposed to seven pesticides (acetamiprid, imidacloprid, flupyradifurone, fenitrothion, carbaryl, amitraz, and bifenthrin). Among these five candidate genes, a combination of RAD1a and RPS18 was suggested for target gene normalization. Subsequently, expression levels of six genes (AChE1, CYP9Q1, CYP9Q2, CYP9Q3, CAT, and SOD1) were normalized with a combination of RAD1a and RPS18 in the different body parts from honey bees exposed to pesticides. Among the six genes in the five body parts, the expression of SOD1 in the head, fat body, and carcass was significantly induced by six pesticides. In addition, among seven pesticides, flupyradifurone statistically induced expression levels of five genes in the fat body.