Identification and validation of reference genes for RT-qPCR analysis in Sclerodermus guani (Hymenoptera: Bethylidae)

Gene expression studies in organisms are often conducted using reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR), and the accuracy of RT-qPCR results relies on the stability of reference genes. We examined ten candidate reference genes in Sclerodermus guani, a parasitoid wasp that is a natural enemy of long-horned beetle pests in forestry, including ACT, EF1 alpha, Hsc70, Hsp70, SRSF7, alpha-tubulin, RPL7A, 18S, 28S, and SOD1, regarding variable biotic and abiotic factors such as body part, life stage, hormone, diet, and temperature. Data were analysed using four dedicated algorithms (Delta Ct, BestKeeper, NormFinder, and geNorm) and one comparative tool (RefFinder). Our results showed that the most stable reference genes were RPL7A and EF1 alpha regarding the body part, SRSF7 and Hsc70 regarding the diet, RPL7A and alpha-tubulin regarding the hormone, SRSF7 and RPL7A regarding the life stage, and SRSF7 and alpha-tubulin regarding temperature. To ascertain the applicability of specific reference genes, the expression level of the target gene (ACPase) was estimated regarding the body part using the most stable reference genes, RPL7A and EF1 alpha, and the least stable one, SOD1. The highest expression level of ACPase was observed in the abdomen, and the validity of RPL7A and EF1 alpha was confirmed. This study provides, for the first time, an extensive list of reliable reference genes for molecular biology studies in S. guani.