Enhancing the accuracy of qPCR normalization in Bactrocera zonata: A systematic study for the selection of condition-optimized reference genes for gene expression studies

Bactrocera zonata (Diptera: Tephritidae) is a highly destructive polyphagous pest that infests a wide range of economically important fruit crops. Understanding its growth, development, reproduction, and responses to environmental stresses is essential for developing effective management strategies. Gene expression analysis is an important approach for elucidating the molecular mechanisms underlying these biological processes. Quantitative PCR (qPCR) is widely used for gene expression analysis but requires validated reference genes for accurate normalization. This study evaluated the expression stability of eight candidate reference genes ( GAPDH , G6PDH , TUBA1A , TUBB , RPL19 , EEF1A2 , SOD , and ATP6V0A1 ) across six biological and experimental conditions: sex, tissues, developmental stages, gamma irradiation, temperature stress, and dsRNA treatment. Stability was assessed using the comparative delta-Ct and standard deviation method, geNorm, NormFinder, BestKeeper, and RefFinder. GAPDH showed the highest stability under gamma irradiation and dsRNA treatment, TUBA1A across developmental stages and temperature stress, RPL19 between adult males and females, and TUBB across tissues. These findings demonstrate condition-dependent reference-gene stability and provide validated reference genes for reliable qPCR normalization in B. zonata .