Reference Gene Selection for Quantitative Gene Expression Analysis in Argynnis hyperbius

<i>Argynnis hyperbius</i> (Lepidoptera: Nymphalidae)<i>,</i> 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 (<i>ACT</i>, <i>α-TUB</i>, <i>AK</i>, <i>GAPDH</i>, <i>EF1α</i>, <i>BTF3</i>, <i>RPS3</i>, <i>RPL10</i>, <i>RPL32</i>, and <i>RPL27</i>) were selected, and their expression stability under different developmental stages, genders, and temperature treatments was evaluated using the <i>RefFinder</i> website. The results indicated that selecting a pair of reference genes could achieve the most reliable normalization analysis under all experimental conditions. Specifically, <i>AK</i> and <i>EF1α</i> were the most stably expressed reference genes across different developmental stages; <i>ACT</i> and <i>RPL32</i> showed the most stable expression in different adult sexes; and <i>EF1α</i> and <i>RPL27</i> exhibited stable expression under different temperature treatments. Additionally, this study used <i>EF1α</i> and <i>RPL32</i>, the most stable reference genes from all results, to normalize and analyze the relative transcription levels of <i>HSP90</i> 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 <i>A. hyperbius.</i>