Honey bee is not only considered an important pollinator in agriculture, but is also widely used as a model insect in biological sciences, thanks to its highly evolved sociality, specialization of labor division, and flexibility of colony management. For an intensive investigation of the seasonal and labor-dependent expression patterns of its genes, accurate quantification of the target gene transcription level is a fundamental step. To date, quantitative real-time PCR (qRT-PCR) has been widely used for rapid quantification of gene transcripts, with reliable reference gene(s) for normalization. To this end, in an attempt to search for reliable reference genes, the amplification efficiencies of six candidate reference genes (rp49, rpL32, rpS18, tbp, tub, and gapdh) were determined. Subsequently, four genes (rpL32, rpS18, tbp, and gapdh) with PCR efficiencies of 90% to 110% were evaluated for their expression stabilities with three programs (geNorm, NormFinder, and BestKeeper) and used for normalization of seasonal expression patterns of target genes in the forager and nurse heads. Although the three programs revealed slightly different results, two genes, rpS18 and gapdh, were suggested to be the optimal reference genes for qRT-PCR-based determination of seasonal and labor-specific gene expression profiles. Furthermore, the combined use of these two genes yielded a more accurate normalization, compared with the use of a single gene in the head of honey bee. The validated reference genes can be widely used for quantification of target gene expression in honey bee head although it is still remained to be elucidated the expression levels of the selected reference genes in specific tissues in head.

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.

Drosophila suzukii Matsumura (Drosophilidae) is a devasting invasive pest affecting berry crops and cherry production throughout North America, South America, and Europe. This species has two different morph phenotypes, winter and summer morphs, which are temperature dependent. Chemical control is the most widely used management approach for managing the spotted wing drosophila, D. suzukii. Little is known regarding the differential response of the two seasonal morphs to insecticides. In this study, we identified a high number of differentially expressed genes likely involved in phase I, II, and III of detoxification pathways and other cuticular proteins in winter morphs as compared to summer morphs. Specifically, several detoxification genes (phase I: Cyp4e3, Cyp4s3, Cyp6d5, Cyp49a1, Cyp318a1; phase II: GstD10, Ugt35Bb, Ugt37b1, Ugt58Fa; phase III: Mdr65) were overexpressed more than two‐fold in winter morph. Additionally, we determined the median lethal concentration, LC50, values of Malathion, Cyantraniliprole, Imidan, Zeta‐cypermethrin, and Spinetoram insecticides to compare the insecticide susceptibility against two seasonal morphs of D. suzukii. For most of the pesticides tested, there were no differences between the LC50 values, between summer and winter morphs, however, we found that winter morph exhibited an LC50 value of Spinetoram 3.7‐fold, significantly higher than that of the summer morph. Overall, we demonstrated that seasonally induced different morphological phenotypes may result in different transcriptional response of phases I, II, and III of the detoxification pathways and other cuticular proteins. However, we found different responses to at least one insecticide, Spinetoram. It remains to be determined what are underlying physiological differences that lead to these changes in response to Spinetoram.

Two taxonomically similar Drosophila species, Drosophila melanogaster and Drosophila suzukii, are known to have distinct habitats: D. melanogaster is mostly found near overripe and fermented fruits, whereas D. suzukii is attracted to fresh fruits. Since chemical concentrations are typically higher in overripe and fermented fruits than in fresh fruits, D. melanogaster is hypothesized to be attracted to higher concentrations of volatiles than D. suzukii. Therefore, the chemical preferences of the two flies were compared via Y-tube olfactometer assays and electroantennogram (EAG) experiments using various concentrations of 2-phenylethanol, ethanol, and acetic acid. D. melanogaster exhibited a higher preference for high concentrations of all the chemicals than that of D. suzukii. In particular, since acetic acid is mostly produced at the late stage of fruit fermentation, the EAG signal distance to acetic acid between the two flies was higher than those to 2-phenylethanol and ethanol. This supports the hypothesis that D. melanogaster prefers fermented fruits compared to D. suzukii. When comparing virgin and mated female D. melanogaster, mated females showed a higher preference for high concentrations of chemicals than that of virgin females. In conclusion, high concentrations of volatiles are important attraction factors for mated females seeking appropriate sites for oviposition.

In quantitative real‐time polymerase chain reaction (qRT‐PCR), target gene expression levels are normalized to internal reference gene(s) that are stably expressed across different conditions to determine whether they are up‐ or down‐regulated. Therefore, it is essential to select appropriate reference gene(s) for the accurate comparison of target gene expression across different experimental conditions. Honeybee colonies can be damaged due to pesticide exposure, resulting in a decline of their population. Determination of gene expression levels is important for understanding the physiological response of honeybees to pesticide exposure. Therefore, in this study, we used qRT‐PCR to analyze the expression stability of five candidate reference genes (RPS5, RPS18, GAPDH, ARF1, and RAB1a) in honeybees subjected to treatment with different dosages and exposure durations of seven pesticides (acetamiprid, imidacloprid, flupyradifurone, fenitrothion, carbaryl, amitraz, and bifenthrin) using four programs (geNorm, NormFinder, BestKeeper, and RefFinder). Subsequently, the expression levels of the target genes (PER, FOR, and EGR1) were calculated using different normalization methods and compared. Based on our collective results, we propose RPS5 as the most appropriate reference gene for the normalization of target gene expression levels in qRT‐PCR assays for honeybees under various conditions of pesticide exposure, including pesticide type, exposure duration, and concentration.