Reference genes for expression studies in different developmental stages of Oryctes rhinoceros, the coconut rhinoceros beetle

Oryctes rhinoceros L. (Coleoptera: Scarabaeidae), the coconut rhinoceros beetle (CRB), is a primary pest of coconut in South and Southeast Asia and the Pacific Islands. The beetle has the potential to severely affect the economies of local communities, many of whom are marginal and small farmers who rely on coconut as the main source of livelihood. Reverse transcription-quantitative real-time PCR (qRT-PCR) based targeted gene expression analysis has emerged as a powerful tool due to its sensitivity and reproducibility. However, calculating the relative expression of target genes requires normalization with reference genes across specific experimental conditions. To identify suitable reference gene(s) possessing stability, we selected six prospective genes (viz., NADH, ACTIN, EF1A, RPL3, SDHA, and ARF6) and evaluated them for their potential use as reference gene(s) across different developmental stages of O. rhinoceros. A comprehensive approach based on five statistical models viz., GeNorm, BestKeeper, NormFinder, RefFinder and the Delta Ct value, was utilized, and based on the obtained stability values of candidate genes, a consensus ranking was generated. The expression levels of NADH, EF1A and RPL3 were observed to be the most stable across the developmental stages with significant statistical reliability. Further, this study identified NADH/EF1A as the most reliable reference gene combination which could provide robust normalization of RT-qPCR data in gene expression studies in O. rhinoceros. This is the first report identifying the suitable reference genes for normalizing gene expression in O. rhinoceros across different developmental stages, facilitating future elucidation of gene expressions in this species.