Selection of reference genes for RT-qPCR analysis in Trichogramma chilonis (Hymenoptera: Trichogrammatidae)

Abstract

• Suitable reference genes were determined in Trichogramma chilonis . • RPS23 and EF2 are for developmental stages in T. chilonis. • ZFP268 and EF2 are for feeding with different diets in T. chilonis. • ZFP268 and RPL13 are for undergoing different temperatures in T. chilonis. • EF2 and RPL44 are for T. chilonis on insecticides treatments. Trichogramma chilonis is an important natural enemy for control of various Lepidoperan crop pests. The biology of T. chilonis is well-studied, but the molecular mechanisms of this biology require further study. Screening suitable reference genes is a vital step for use of RT-qPCR to understand underlying molecular physiology. In the present study, nine candidate reference genes including elongation factor 2 ( EF2 ), ribosomal proteins ( RPS23 , RPL13 , and RPL44 ), malate dehydrogenase ( MDH ), eukaryotic translation initiation factor 3 subunit F ( EIF3F ), zinc finger protein 268 ( ZFP268 ), muscle specific protein 20 ( MP20 ), and ATP synthase subunit alpha ( ATP5F1A ) were evaluated at different conditions including development stage, diet, temperature, and insecticide treatments. Four common algorithms (the Delta Ct method, geNorm , BestKeeper , and NormFinder ) and RefFinder were used to analyze gene expression stability. Our results indicated that two reference genes used for normalization were sufficient, and the optimal combinations were: RPS23 and EF2 for developmental stages, ZFP268 and EF2 for feeding with different diets, ZFP268 and RPL13 for temperature treatments, and EF2 and RPL44 for insecticide treatments. The results provide preliminary determination of suitable reference gene for standard RT-qPCR analyses in T. chilonis , which might establish the foundation for further molecular biology research.