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Simple Summary  The purpose of this paper was to determine which genes in  Aquatica leii , an endemic Chinese firefly, are suitable for use as reference genes in future RT-qPCR studies. This is the first example of an effective knockdown in fireflies and an important methodological advance for continuing research in  A. leii . The results of this study will help improve accuracy and reliability to normalize RT-qPCR data in  A. leii  for further molecular analysis. Since luciferase is very abundant in the adult light organ of fireflies generally, this is an encouraging result and of interest not just for  A. leii  researchers but other researchers who plan to perform RNAi or RT-qPCR in fireflies, or other nonmodel insects, as well.    Abstract   Aquatica leii  Fu and Ballantyne is a species of rare aquatic firefly and endemic in China. It is considered good material to study the molecular mechanism of sexual flash communication systems. To improve conservation and behavioral research strategies, large-scale genetic studies involving gene-expression analysis are required and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the most commonly used method. However, there have been very few reports on appropriate reference genes in any species of firefly. Here, we evaluated eight widely utilized reference genes including  18S ,  Actin ,  Reep5 ,  Odc1 ,  Tub ,  Gapdh ,  Ef1a  and  S27Ae  for their expression stabilities in  A. leii  under three different conditions, i.e., life stage, tissue and dsRNA injection. Based on the gene stability ranking calculated by RefFinder, which integrates four algorithms (geNorm, delta Ct method, NormFinder, and BestKeeper), we recommend  S27Ae  and  Reep5  as the most appropriate reference genes for molecular studies in different life stages;  Ef1a  and  Odc1  for different tissues;  Tub  and  Odc1  for RNAi studies. The most appropriate reference genes in all treatments are  S27Ae  and  Tub  .  The results of this study will help improve accuracy and reliability to normalize RT-qPCR data in  A. leii  for further molecular analysis.

Selection and Validation of Suitable Reference Genes for RT-qPCR Analysis in the Rare Aquatic Firefly Aquatica leii (Coleoptera: Lampyridae)