Chelonus formosanus is belonging to Hymenoptera, Braconidae, and Chelonus Panzer(Zhang 2008). Chelonus formosanus is the natural enemy of eggs and larvae of Noctuidae, such as Spodoptera exigua, Spodoptera litura and Spodoptera frugiperda(He et al. 2002). It has great biological control value. Previous studies have found that the development period of Chelonus formosanus is divided into four stages, and the duration of each stage decreases with an increase in temperature (Yu 2023). Chelonus formosanus reached a mating rate of 97.5% under specific conditions: temperature of 28℃, diet of 10% honey mixed with 10% sugar water, male/female ratio of 1:7, pairing time of 2 days, and space size of 12×4×8 cm. This led to an increase in the sexual reproduction ratio and population expansion(Wang et al. 2023a). To screen the suitable storage conditions for Chelonus formosanus, Wang Luchao (2023b)suggested that 7-day-old pupae of Chelonus formosanus should be stored at 13℃ for no more than 15 days. Biological analysis has been conducted in the early stages, and it is essential to investigate the process of host searching or conduct further research to get biological information from more exact and accurate gene expression data for further study.
Gene expression analysis can provide biological insights such functional gene signals and metabolic pathways in the reproductive, developmental, and cellular processes of organisms, which researchers utilize for further research(Pfaffl 2001). Real-time fluorescent quantitative PCR (RT-PCR) is widely used in gene expression analysis because of its high-cost performance and accuracy(Imai et al. 2014, Wang et al. 2019,Wei et al. 2023,). The RT-qPCR is divided into relative fluorescence quantification and absolute fluorescence quantification. The experimental results may be inaccurate due to variations across and within the samples when using relative fluorescence quantification technology. Therefore, the expression of stable internal reference genes is needed for standardized correction( Bustin 2002, Guénin et al. 2009, Wang et al. 2018). The internal reference genes are generally traditional housekeeping genes, such as β-actin gene (β-actin), glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH), α-extension factor gene (EF1-α), ribosomal protein gene (RPS, RPL), ubiquitin-binding enzyme (UBC), translation extension factor gene (TEF) and so on(Sękalska et al. 2006, Nolan et al. 2006, Shi and Zhang 2016)However, there is no universal reference gene, and the correction of unscreened reference genes may make the result inaccurate or even wrong(Zhu et al. 2019).
In this study, the reference genes, including GAPDH, ACT, TU, TF, HSP, and RP, were screened based on transcriptome sequencing of Chelonus formosanus. The internal reference stability was analyzed using BestKeeper, GeNorm, NormFinder, Delta, and RefFinder methods. Internal reference genes that exhibited stable expression in tissues subjected to high and low temperature stress as well as insecticide treatment were selected to provide an ideal reference gene for studying gene expression in Chelonus formosanus.