Identification and validation of reference genes for gene expression analysis in Aphidius gifuensis (Hymenoptera: Aphidiidae)

Reference genes have been utilized in estimating gene expression levels using quantitative reverse transcriptase-quantitative polymerase chain reaction (qRT-PCR) analysis. Aphidius gifuensis Ashmaed is one of the most widely used biological control agents for aphids. The biological properties of this species have been studied in detail, and current investigations are focused on elucidating the regulatory mechanisms in its host However, the appropriate reference genes for target gene expression studies have not been identified. In this study, the expression profiles of 12 candidate reference genes were evaluated under different experimental conditions(development stage, sex, tissue type, diet) by using dedicated algorithms, including geNorm, Normfinder, BestKeeper, and Delta Ct. In addition, RefFinder was used to rank the overall stability of the candidate genes. Finally, we recommend three optimal reference genes for the normalization of qRT-PCR data in the presence of specific variables, which include ACTB, RPL13, and PPI for different developmental stages; RPS18, ACTB, and RPL13 for sexes; RPL13, PRII3, and RPS18 in different tissue types; and RPL13, RPL27, and ACTB in diverse diets. The present study has identified optimal reference genes that could be used in estimating the expression levels of specific genes under these conditions following the Minimum Information for publication of Quantitative real-time PCR Experiments (MIQE) guidelines, which would facilitate in advancements in functional genomics research on A. gifuensis.