Selection of reference genes for the study of relative gene expression during development of Litopenaeus vannamei larvae

Pacific whiteleg shrimp (Litopenaeus vannamei) production is one of the most economically important aquaculture industries around the world. Gene expression studies during larval development are a key tool to elucidate biological aspects during metamorphosis and the effects of pathological conditions; the adequate quantification of gene expression can provide molecular markers and basic knowledge to optimize culture conditions, including the development of strategies for the prevention and control of diseases. In this context, the selection and validation of reference genes is a critical process to avoid bias due to inaccurate measurements and the consequent misinterpretation of biological processes. In this study, nine candidate reference genes (Clathrin, Cyt-c, SubF0, EF1α, β-Actin, GAPDH, TBP, AK and PK) were selected to test their expression stability during larval development of L. vannamei using geNorm, NormFinder, BestKeeper and the integrated tool RefFinder algorithms. Based on our analysis the most stable gene was SubF0, followed by GAPDH and EF1α. Relative expression of developmental genes (Twist, Mef-2 and Ubx) was quantified using the three most stable reference genes (individually and combined), taking special attention to the expected expression and biological function of these genes. The expression of Mef-2 was the most sensitive to the reference gene. However, the combination of the three most stable reference genes provided consistent results according to the expected expression patterns of developmental genes.