Comprehensive evaluation of candidate reference genes for gene expression studies in Lysiphlebia japonica (Hymenoptera: Aphidiidae) using RT-qPCR

Abstract Lysiphlebia japonica (Ashmead) is a predominant parasitoid of cotton-melon aphids in the fields of northern China with a proven ability to effectively control cotton aphid populations in early summer. For accurate normalization of gene expression in L . japonica using quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR), reference genes with stable gene expression patterns are essential. However, no appropriate reference genes is L . japonica have been investigated to date. In the present study, 12 selected housekeeping genes from L . japonica were cloned. We evaluated the stability of these genes under various experimental treatments by RT-qPCR using four independent (geNorm, NormFinder, BestKeeper and Delta Ct) and one comparative (RefFinder) algorithm. We identified genes showing the most stable levels of expression: DIMT , 18S rRNA , and RPL13 during different stages; AK , RPL13 , and TBP among sexes; EF1A , PPI , and RPL27 in different tissues, and EF1A , RPL13 , and PPI in adults fed on different diets. Moreover, the expression profile of a target gene ( odorant receptor 1 , OR1 ) studied during the developmental stages confirms the reliability of the chosen selected reference genes. This study provides for the first time a comprehensive list of suitable reference genes for gene expression studies in L . japonica and will benefit subsequent genomics and functional genomics research on this natural enemy.