Reference gene selection for RT-qPCR analysis in two invasive whiteflies after the acquisition of vectored or non-vectored viruses

Custom reference gene selection is essential for reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) in different species of insects and various experiment conditions. In this study, 14 candidate reference genes (HSP40, HSP20, HSP70, HSP90, v-ATPase, RPL29, EF-1, SDHA, Actin, PPM, GAPDH, MyosinL, NADH, and gamma-tubulin) were analyzed using five different programs including Delta Ct method, BestKeeper, geNorm, NormFinder, and ReFinder to validate their use as reference genes in two invasive whiteflies, Bemisia tabaci B and Q, after acquiring the vectored virus, Tomato yellow leaf curl virus (TYLCV), or ingesting the non-vectored virus, Tomato spotted wilt virus (TSWV), respectively. The results showed that HSP40, v-ATPase, and EF-1 were the most stable genes in B. tabaci B (B. tabaci B feeding on the healthy, TYLCV- and TSWV-infected tomato plant), PPLA, SDHA, and RPL29 were the most stable genes in B. tabaci Q (B. tabaci Q feeding on the healthy, TYLCV- and TSWV-infected tomato plant). In addition, EF-1, RPL29, and HSP20 were the most stable reference genes in B. tabaci B and Q. These findings provide the basis for future RT-qPCR-based studies on whitefly-virus interactions. Meanwhile, this report may set a precedent for reference gene selection in insects after the ingestion of non-vectored viruses.