Reference Genes for Expression Analyses by RT-qPCR in Hyblaea puera (Lepidoptera: Hyblaeidae)

Hyblaea puera is a major pest of teak and mangroves. Reliable RT-qPCR normalization requires stable reference genes, none of which have been validated in H. puera. In this study, we assessed the expression stability of ten candidate reference genes under different experimental conditions. Stability was evaluated using the ΔCt method, BestKeeper, NormFinder, and geNorm, and a comprehensive stability ranking was generated using the RefFinder online tool. Our results indicated that amplification efficiencies ranged from 91.67% to 100.82%, with R² values exceeding 0.9901. The optimal reference gene combinations varied by condition: Ribosomal Protein L27 (RPL27) and Ribosomal Protein L10 (RPL10) for temperature treatments; Actin and RPL10 for larval instars; Ribosomal protein S5 (RPS5) and Elongation factor-1α (EF-1a) for adult sexes; RPL10 and EF-1a for tall developmental stages; RPL10 and RPS5 for tissues; as well as EF-1α and Actin for all combined conditions. Finally, the expression profiles of target gene Lethal were evaluated, and the outcomes further confirm the importance of selecting fitting reference genes for normalization of qRT-PCR data. These results provide the evaluated reference gene sets for H. puera, facilitating more accurate RT-qPCR normalization in future molecular studies of host plant adaptation (teak vs. mangroves), temperature tolerance, and larval development in this pest.