Validation of quantitative real-time PCR reference genes and spatial expression profiles of detoxication-related genes under pesticide induction in honey bee, Apis mellifera

Abstract

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.

Validation of reference genes for quantitative real‐time polymerase chain reaction assay of honeybee under various pesticide treatment conditions

“…Therefore, in this study, we investigated the expression stabilities of five candidate reference genes in honeybees under various pesticide treatment conditions based on C q distribution analysis and using four statistical algorithms (NormFinder, BestKeeper, geNorm, and RefFinder). Consistent with previous reports (Jeon et al 2020;Kim et al 2020Kim et al , 2022Köhsler et al 2020;Moon et al 2018b;Wang et al 2019), the four algorithms resulted in different gene stability ranks. NormFinder visualizes the log-transformed C q values and relative transcription levels (Andersen et al 2004), whereas BestKeeper determines the geometric mean of the C q values and SD values based on the raw C q data of the candidates (Pfaffl et al 2004).…”

“…1). Although GAPDH has previously been suggested as an optimal reference gene for qRT-PCR assays in honeybee samples (Jeon et al 2020;Moon et al 2018b), recent studies have ranked GAPDH as the least stable gene in honeybees treated with pesticides (Kim et al 2022;Kim, Cho, & Lee 2021). This implies that GAPDH expression in honeybees may be significantly altered by pesticides, and therefore, it may not be an appropriate reference gene for evaluating gene expression in honeybees subjected to various pesticide treatments.…”

“…6). A single reference gene might be appropriate if the target gene expression levels normalized to it are not significantly different from those normalized against multiple reference genes (Jeon et al 2020;Kim et al 2020Kim et al , 2022. In addition, selection of one reference gene with consistent expression stability across various conditions is easier than selection of multiple reference genes with similar expression stabilities.…”

“…2020; Kim et al . 2022; Kim, Kim, & Kim 2021), NormFinder (version 0.953) (Andersen et al . 2004), BestKeeper (version 1) (Pfaffl et al .…”

“…To evaluate the expression stability of the five candidate reference genes in pesticide-treated honeybees under different conditions, including exposure durations and different doses, five analysis methods were employed: C q distribution (Jeon et al 2020;Kim et al 2022;, NormFinder (version 0.953) (Andersen et al 2004), BestKeeper (version 1) (Pfaffl et al 2004), geNorm (version 3.2) (Vandesompele et al 2002), andRefFinder (Xie et al 2012). The C q distribution of candidate reference genes was estimated using SigmaPlot 14.0, and the arithmetic means (AM), standard deviation (SD), and coefficient of variation (CV) values were calculated (CV = SD/AM) (Fig.…”