LAUSR

Simple Summary Reference genes are the basis for exploring the function of target genes in insects. Recently, we identified a large number of chemoreceptor genes by transcriptome sequencing in different tissues of Cimex hemipterus (F.) (Hemiptera: Cimicidae). Additionally, we plan to study the expression patterns of these genes and clarify their functions. However, there is no report on the screening and application of the reference genes in C. hemipterus. In the present study, the expression-level stability of 10 candidate reference genes in C. hemipterus under various experimental conditions was evaluated through qRT-PCR combined with RefFinder (which integrates four computational programs: geNorm, NormFinder, BestKeeper, and ∆Ct). Results show that the optimal combination of reference genes for each experimental condition was as follows: RPL8 and EF1α for the developmental stage, RPL8 and RPS16 for adult sex, RPL8 and RPL11 for adult tissue, RPL8 and β-tubulin for gas stimulation, and RPL8 and NADH for temperature. The results will lay a foundation for the follow-up study on the expression and function of the target genes of tropical bed bugs. Abstract 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.