Selection and evaluation of reference genes for quantitative real-time polymerase chain reaction normalization in <i>Pieris melete</i> (Lepidoptera, Pieridae)

Accurate normalization of gene expression data in real-time quantitative polymerase chain reaction (RT-qPCR) relies on the identification of stably expressed reference genes, which remains uncharacterized in Pieris melete Ménétriés, a cruciferous crop pest with agricultural significance. This study systematically evaluated eight candidate reference genes (GAPDH, α-tub, β-actin, 18S, β-tub, EF1α, RPL27, RPS15) across four experimental conditions: developmental stages, tissues, temperature stresses, and diapause stages. Stability rankings were determined using four algorithms (geNorm, NormFinder, BestKeeper, ΔCt method) integrated via RefFinder. Results revealed that at different condition-specific stability patterns, RPL27 and EF1α were optimal for developmental stages, RPL27 paired with 18S were suitable for tissue analyses; EF1α and α-tub were stable at different temperature stresses, and RPL27 combined with RPS15 were stably expressed during diapause. Pairwise variation analysis confirmed that dual reference genes sufficiently enhanced normalization accuracy. This work provides the validated reference genes panel for P. melete, addressing a critical gap in molecular studies of this pest and ensuring robust gene expression analyses for future research on diapause regulation and pest control strategies.