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

Abstract

The egg parasitoid Trichogramma spp. is an important biological control agent used against multiple species of Lepidopteran pest in forestry and agriculture. Due to the importance of Trichogramma spp. in biocontrol programs, its biological characteristics have been studied in detail, and current investigations should focus on the molecular biology of these tiny parasitoids. Real-time quantitative PCR (qPCR) is considered as the standard method for quantifying the gene expression of organisms. Surprisingly, the appropriate reference genes to ensure robust qPCR have not been documented at all for the Trichogramma genus. This study aimed to identify suitable reference genes for use in qPCR procedure of Trichogramma dendrolimi. Nine candidate housekeeping genes, namely glyceraldehyde-3-phosphate dehydrogenase (GAPDH), forkhead box O (FOXO), superoxide dismutase (SOD), beta-actin (ACTIN), ribosomal protein L10a (RPL10a), L18 (RPL18), L28 (RPL28), S13 (RPS13), and S15 (RPS15), 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, 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 temperature, respectively. According to the RelFinder method and by assessing the integrated values from using the {Delta}Ct method, GeNorm, NormFinder, and BestKeeper, we identified the developmental stage-specific reference genes SOD, GAPDH, and ACTIN; tissue-specific reference genes RPL18 and RPS15; sex-specific reference genes SOD and RPL18; and temperature-specific reference genes RPL18 and RPL10. When testing the use of stable vs. unstable reference genes, the substantial differences were observed in the estimation expression of a hypothetical target gene, HSP90, in response to temperature. The present study provides a robust method for the measurement of gene expression in T. dendrolimi and will be helpful for future biological control programs using Trichogramma wasps.