Gene expression patterns to identify stable reference genes for expression analyses in Carpophilus hemipterus (L.) (Coleoptera: Nitidulidae)

Dried fruit beetle, Carpophilus hemipterus, (Linnaeus, 1758) (Coleoptera: Nitidulidae) is a serious pest of ripened fresh fruit in the orchard and dried fruit in post-processing storage. Despite the economic significance and widespread distribution of C. hemipterus, there is a lack of functional genomics research seeking to elucidate features of developmental biology and molecular physiology for improved pest management. Performing such functional genomics studies requires validated, stably expressed reference genes for accurate normalization of real-time quantitative reverse transcription-PCR (qRT-PCR) data. Here, we identified and validated 12 common insect reference genes (RPL6, RPL4, RPL9, RPS13, RPS18, RPSA, TUB, UBE3A, RPS23, GLU, EF1A, and RBP) for expression stability in C. hemipterus. These genes were assessed across developmental stages and sexes (egg, three larval instars, prepupa, pupa, adult male, and adult female) as well as thermal stress conditions. Stability of candidate genes was estimated using software algorithms geNorm, NormFinder, BestKeeper, comparative delta-Ct, and RefFinder. Two genes UBE3A and RPL9 showed the highest stability across the treatments, and thus were proposed for normalizing the qRT-PCR raw data. Results of this study will strengthen the application of genomics to reveal the fundamental and applied biological aspects in this important beetle species.