Selection and Validation of Candidate Reference Genes for qRT-PCR Analysis in Fopius arisanus

Fopius arisanus (Hymenoptera: Braconidae) is an important parasitoid species in tephritid fruit fly biological control and host–parasitoid interaction research. Quantitative real-time PCR (qRT-PCR) is widely used to analyze gene expression, which requires stable reference genes for accurate normalization. However, no systematic evaluation of suitable reference genes for qRT-PCR analysis across F. arisanus tissues has been conducted. We evaluated the expression stability of ten candidate reference genes (Actin, α-tubulin, β-tubulin, RPL37a, EF1-α, EF1-δ, RPL4, RPS18, RPL13, and GAPDH) across five adult tissues from females and males. The stability of these genes was analyzed using the ΔCt method, geNorm, NormFinder, BestKeeper, and RefFinder, and the optimal number of reference genes was determined by geNorm pairwise variation analysis. The results revealed that the optimal reference genes differed between the female and male tissue panels: Actin and RPL13 were the most stable combination for female tissues, whereas RPL13 and RPS18 were recommended for male tissues. Notably, β-tubulin and GAPDH showed the poorest stability in female tissues, whereas α-tubulin and β-tubulin were the least stable in male tissues. The suitability of the stable and unstable reference genes was further validated by normalizing the expression of the target genes vitellogenin (Vg) and dual-specificity phosphatase CDC14A (CDC14A). Our findings represent a systematic evaluation of reference genes for F. arisanus and establish a reliable normalization strategy for future gene expression studies in this parasitoid wasp.