Selection and Validation of Stable Reference Genes for RT-qPCR in Diaphorencyrtus aligarhensis-A Predominant Parasitoid of Diaphorina citri.

Diaphorencyrtus aligarhensis parasitizes the Asian citrus psyllid (ACP), Diaphorina citri, the primary insect vector responsible for transmitting Huanglongbing (HLB), a severe citrus disease. Screening of appropriate reference genes is a critical prerequisite for reliable RT-qPCR analysis, which is essential for investigating the functions of target genes in D. aligarhensis across diverse experimental conditions. However, to date, no validated reference genes have been reported for this species. This study assessed seven housekeeping genes in D. aligarhensis under six conditions (developmental stage, body tissue, population, temperature, diet, and starvation) using five stability algorithms (geNorm, BestKeeper, NormFinder, RefFinder, and ∆Ct). The results identified the most suitable reference genes for specific experimental conditions: EIF5A and RPL32 for the developmental stage; RPL13 and H3 for population comparisons; RPS6 and GAPDH for different feeding diets; RPL32 and RPS6 for starvation; RPL7A and RPS6 for different body tissues (head, thorax, abdomen) and temperature gradients (5 °C, 15 °C, 25 °C, 35 °C). Furthermore, the expression profiles of HSP70 were markedly different when normalized to the most versus the least stable reference genes across body tissues, diets, starvation durations, and temperatures. Our findings establish the first set of RT-qPCR reference genes for D. aligarhensis, providing a useful foundation for functional genomics research on this biological control agent.