Selection of valid reference genes for quantitative real-time PCR in Cotesia chilonis (Hymenoptera: Braconidae) exposed to different temperatures.

In quantitative real-time PCR (qRT-PCR), data are normalized using reference genes, which helps to control for internal differences and reduce error among samples. In this study, the expression profiles of eight candidate housekeeping genes, 18S ribosomal (18S rRNA), elongation factor (EF1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein L10 (RPL10), ribosomal protein L17 (RPL17), histone 3 (H3), arginine kinase (AK), amd β-Actin (ACTB), were evaluated in the parasitic wasp Cotesia chilonis in response to different temperatures. Specifically, the performance and stabilities of these genes were compared in adult wasps maintained in a growth condition at 27°C (normal storage conditions) and in adults obtained from pupae refrigerated at 4°C for five days (cold storage conditions). Data were analyzed using the ΔCt method, BestKeeper, NormFinder, and geNorm. The optimal numbers and stabilities of reference genes varied between the two temperature treatments (27°C and 4°C). In samples stored at normal developmental temperature (27°C), the requirement for normalization in response to low temperature exposures was three genes (18S, H3, AK), whereas normalization in response to high temperature exposures required only two reference genes (GAPDH, ACTB). In samples stored at cold temperature (4°C), for low temperature exposures two reference genes (RPL17, RPL10) were required for standardization, while following high temperature exposures three reference genes (18S, H3, ACTB) were needed. This study strengthens understanding of the selection of reference genes before qRT-PCR analysis in C. chilonis. The reference genes identified here will facilitate further investigations of the biological characteristics of this important parasitoid.