Abstract
S. pupariae is a newly discovered species of parasitoid wasps. Research into its development, behavioral genetics, and molecular mechanisms provides valuable insights for improving integrated pest management strategies. Quantitative real-time PCR (qRT-PCR) is the most commonly used method for analyzing gene expression. This method requires the identification of stable reference genes to accurately evaluate transcriptional level variations. In this study, eight candidate reference genes (TUB, TBP, RPS18, GAPDH, 18S rRNA, RPL32, Actin, and EF1-α) were identified and evaluated for their suitability as reference genes. Gene expression levels across different developmental stages were analyzed using three software tools, GeNorm, NormFinder, and BestKeeper, and the online tool RefFinder. The overall ranking of reference gene stability was as follows: RPS18 > 18S rRNA > RPL32 > GAPDH > Actin > TUB > TPB > EF1-α. Ultimately, RPS18 was determined to be the most stable reference gene.