Selection and evaluation of RT-qPCR reference genes for expression analysis in the tiny egg parasitoid wasp, Trichogramma dendrolimi matsumura (Hymenoptera: Trichogrammatidae)

The egg parasitoid, Trichogramma spp., is an important biological control agent used against a broad range of Lepidopteran pests in agriculture and forestry. The biology of Trichogramma has been studied in details. Further studies should focus on the molecular mechanisms of Trichogramma by qualifying the expression of related genes It is critical to select appropriate reference genes for normalizing RT-qPCR results and establishing a robust method for quantifying target gene expression. This study aimed to identify and validate appropriate reference genes for use in RT-qPCR analysis of Trichogramma dendrolimi. Ten candidate housekeeping genes, namely beta-actin (ACTIN), forkhead box O (FOXO), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), heat shock protein 90 (HSP90), ribosomal protein L10a (RPL10a), L18 (RPL18), L28 (RPL28), S13 (RPS13), S15 (RPS15), and superoxide dismutase (SOD), were tested for their suitability as reference genes for developmental stage (3rd, 4th, 5th, 6th, 7th, 8th, 9th, and 10th day after parasitization), tissue (head, thorax, and abdomen of adults), sex of adults (male and female), and temperature (17 degrees C, 25 degrees C, and 32 degrees C). According to the GeNorm analysis, a robust analysis should involve using an appropriate combination of reference genes, namely, at least three genes for different development stages, two genes for different tissues, two genes for different sex, and two genes for different temperatures, respectively. According to the RelFinder method by the integrated results of GeNorm, NormFinder, BestKeeper, and the Delta Ct method, we identified the developmental stage-specific reference genes SOD, GAPDH, and ACTIN; tissue-specific reference genes RPL18 and RPS15; sex-specific reference genes RPL18 and SOD; and temperature-specific reference genes RPL18 and RPL10a. This study provides a standardized procedure for the quantification of gene expression in T. dendrolimi and will be helpful for future biological control programs using Trichogramma wasps.