Assessment of housekeeping genes stability for gene transcription regulation analysis of Spodoptera littoralis (Lepidoptera: Noctuidae) under Spodoptera littoralis nucleopolyhedrovirus viral infection

Background Normalization with respect to stable housekeeping genes is important to facilitate gene transcription regulation research and acquire more accurate quantitative polymerase chain reaction (qPCR) data. In the current study, five candidates housekeeping genes of the cotton leafworm, Spodoptera littoralis encoding for Actin (Actin), elongation factor 1-alpha (EF1 alpha), ribosomal protein S3 (RPS3), ribosomal protein 49 (RP49), and Ubiquitin (Ubi), were evaluated as normalization housekeeping genes under Spodoptera littoralis nucleopolyhedrovirus (SpliNPV) viral infection. Methods and Results The qPCR results confirmed the expression of all five housekeeping genes in S. littoralis viral infected larvae. The expression profiles of the housekeeping genes showed that the EF1 alpha, Actin, and RP49 had the minimum average Ct values of 18.41 +/- 0.66, 18.84 +/- 0.90 and 19.01 +/- 0.87 in all infected samples, respectively. While RPS3 and Ubi showed the maximum average Ct of 21.61 +/- 0.51 and 21.11 +/- 0.82, respectively. According to the results of Delta Ct and geNorm analysis, EF1 alpha was ranked as the most stable housekeeping gene during infection time-course. While by using BestKeeper, geNorm and NormFinder, the Ubi, RP49, and RPS3 showed the most genes transcription stability. The obtained results were also validated using the Cytochrome c oxidase (COX) gene transcripts in response to SpliNPV infection. Conclusions The results revealed that EF1 alpha and Ubi were the most stable housekeeping genes to be used for normalizing S. littoralis gene transcription regulation under SpliNPV infection. These findings, provide a significant addition for gene transcription regulation studies of S. littoralis upon infection using SpliNPV as a bio-agent.