Reference Gene Selection for Quantitative Gene Expression Analysis in Argynnis hyperbius

Argynnis hyperbius (Lepidoptera: Nymphalidae), as an important environmental indicator species, has shown a gradual decline in its species richness amid intensifying climate change and increasing environmental pressures. Screening for optimal reference genes is fundamental to studying their physiological and adaptive changes using RT-qPCR technology. In this study, 10 commonly used reference genes (ACT, α-TUB, AK, GAPDH, EF1α, BTF3, RPS3, RPL10, RPL32, and RPL27) were selected, and their expression stability under different developmental stages, genders, and temperature treatments was evaluated using the RefFinder website. The results indicated that selecting a pair of reference genes could achieve the most reliable normalization analysis under all experimental conditions. Specifically, AK and EF1α were the most stably expressed reference genes across different developmental stages; ACT and RPL32 showed the most stable expression in different adult sexes; and EF1α and RPL27 exhibited stable expression under different temperature treatments. Additionally, this study used EF1α and RPL32, the most stable reference genes from all results, to normalize and analyze the relative transcription levels of HSP90 under different temperatures, revealing significant differences between the 4 °C group and both the 26 °C and 37 °C groups. The findings of this study will significantly improve the reliability of RT-qPCR detection and lay a foundation for in-depth research on the gene expression, physiology, and biochemical mechanisms of A. hyperbius.