Selection of stable reference genes for quantitative real‐time PCR in the Varroa mite, Varroa destructor

• Published in: Archives of insect biochemistry and physiology Vol. 110; no. 3; pp. e21905 - n/a
• Main Authors: Lee, Joonhee, Kim, Young Ho, Kim, Kyungmun, Kim, Dongwon, Lee, Si Hyeock, Kim, Sanghyeon
• Format: Journal Article
• Language: English
• Published: United States Wiley Subscription Services, Inc 01.07.2022
• Subjects: acaricide; Acaricides; Acaricides - pharmacology; Actin; Actins - genetics; Animals; Coumaphos; Dehydrogenase; Dehydrogenases; Elongation; Fluvalinate; Gene expression; Gene Expression Profiling; Genes; Glyceraldehyde; Glyceraldehyde-3-phosphate dehydrogenase; Insecticides; Mites; NADH; NADH dehydrogenase; Nicotinamide adenine dinucleotide; Polymerase chain reaction; qPCR; Real-Time Polymerase Chain Reaction; reference gene; tissue; Tissues; Toxicity; Transcriptomics; Translation; Translation elongation; Translation elongation factor 2; Tubulin; Tubulin - genetics; Varroa destructor; Varroa mite; Varroidae - genetics; Vitellogenin; acaricide; reference gene; qPCR; tissue; Varroa mite
• ISSN: 0739-4462; 1520-6327
• DOI: 10.1002/arch.21905

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. Highlights The stabilities of eight reference genes were investigated in acaricide‐treated whole bodies or across different tissues of Varroa mites. It is recommended to use eEF1A1/NADHD for acaricide treatment and α‐tubulin/RpL5 for cross‐tissue comparison. The stabilities of eight reference genes (NADHD, GAPDH, eEF1A1, eEF2, RpL5, Actin, α‐tubulin, and Rab1) were validated in each acaricide (coumaphos, fluvalinate, or amitraz)‐treated whole bodies or across different tissues (cuticle, legs, gut/fat bodies, and synganglion) of untreated Varroa mites. As a result, we recommended to use eEF1A1/NADHD for acaricide treatment and α‐tubulin/RpL5 for cross‐tissue comparison.