The honey bee acetylcholinesterase 1 (AmAChE1) has been suggested to be related to stress response as judged from its elevated expression level under brood rearing-suppressed conditions. To further investigate the involvement of AmAChE1 expression in the stress response and its physiological functions, we analyzed altered expression profiles of AmAChE1 induced by diverse stress factors. In addition, transcription profiles of several heat shock protein (Hsp) genes ( hsps ) and the vitellogenin (Vg) gene ( vg ) known as general stress markers were investigated as positive references. Among the tested stress conditions, AmAChE1 expression was induced under the brood rearing-suppressed, crowding and heat shock conditions. The hsps , particularly hsp70 and hsp90 , responded to seven of nine stress conditions tested, confirming that hsp expression profiles can serve as a general stress marker. Taken together, AmAChE1 expression is not suitable for using as a stress marker due to its limited response. Nevertheless, AmAChE1 expression appears to be connected, at least in part, to heat shock response and other pathways. Considering that AmAChE1 likely regulates the ACh titer particularly in non-neuronal tissues, thereby modulating the signal cascades mediated by mAChR, the AmAChE1 expression profile under different conditions likely provides important information on its physiological roles in honey bees.

To investigate the acaricide toxicity and resistance mechanisms in the Varroa mite, it is essential to understand the genetic responses of Varroa mites to acaricides, which are usually evaluated by transcriptional profiling based on quantitative real‐time polymerase chain reaction (qPCR). In this study, to select reference genes showing consistent expression patterns regardless of the acaricide treatment or the type of tissue, Varroa mites treated with each of the three representative acaricides (coumaphos, fluvalinate, and amitraz) were processed for transcriptomic analysis, from which eight genes (NADH dehydrogenase [NADHD], glyceraldehyde‐3‐phosphate dehydrogenase [GAPDH], eukaryotic translation elongation factor 1 α 1 [eEF1A1], eukaryotic translation elongation factor 2 [eEF2], ribosomal protein L5 [RpL5], Actin, tubulin α‐1D chain [α‐tubulin], and Rab1) were selected as candidates. The transcription profiles of these genes, depending on the treatment of the three acaricides or across different tissues (cuticle, legs, gut/fat bodies, and synganglion), were analyzed using qPCR with four validation programs, BestKeeper, geNorm, NormFinder, and RefFinder. Following acaricide treatment, eEF1A1 and NADHD showed the least variation in their expression levels, whereas the expression levels of α‐tubulin and RpL5 were the most stable across different tissue groups. Rab1/GAPDH and Actin/eEF2 showed the least stable expression patterns following acaricide treatments and across different tissues, respectively, requiring precautions for use. When vitellogenin gene expression was analyzed by different reference genes, its expression profiles varied significantly depending on the reference genes, highlighting the importance of proper reference gene use. Thus, it is recommended using eEF1A1 and NADHD as reference genes for the comparison of the effects of acaricide on the whole body, whereas α‐tubulin and RpL5 are recommended for investigating the tissue‐specific expression profiles of target genes.