Identification and Validation of Reference Genes for Expression Analysis Using qRT-PCR in Cimex hemipterus (Hemiptera: Cimicidae)

The tropical bed bug, Cimex hemipterus (F.) (Hemiptera: Cimicidae) is an important public-health pest that feeds on the blood of humans and some other animals. To explore the function of the target genes of C. hemipterus, it is essential to select suitable reference genes for the accurate quantification of gene expression. Here, we selected 10 frequently used reference genes in insects and evaluated their stability in C. hemipterus under various biotic (developmental stage, sex, and tissue) and abiotic (gas stimulation and temperature) conditions through RefFinder (which integrates four computational programs: geNorm, NormFinder, BestKeeper, and ∆Ct). Results indicate that the optimal combination of reference genes for each experimental condition was as follows: RPL8 and EF1α for the developmental stage (eggs, early instar nymphs, late instar nymphs, and adults), RPL8 and RPS16 for adult sex, RPL8 and RPL11 for adult tissue (head, thorax, abdomen, and legs), RPL8 and β-tubulin for gas stimulation (air and carbon dioxide), and RPL8 and NADH for temperature (0, 5, 17, 30, and 37 °C). Finally, the expression pattern of the HSP70 and GR21 genes were analyzed, and the results highlight the importance of appropriate reference-gene selection. Our results provide a comprehensive list of optimal reference genes from C. hemipterus for the first time, which will contribute to accurately analyzing the expression of target genes.