Development of an efficient RNA interference method by feeding for the microcrustacean Daphnia

Charles A. Schumpert, Jeffry L. Dudycha, Rekha C. Patel

RNA interference (RNAi) is an important molecular tool for analysis of gene function in vivo. Daphnia, a freshwater microcrustacean, is an emerging model organism for studying cellular and molecular processes involved in aging, development, and ecotoxicology especially in the context of environmental variation. However, in spite of the availability of a fully sequenced genome of Daphnia pulex, meaningful mechanistic studies have been hampered by a lack of molecular techniques to alter gene expression. A microinjection method for gene knockdown by RNAi has been described but the need for highly specialized equipment as well as technical expertise limits the wider application of this technique. In addition to being expensive and technically challenging, microinjections can only target genes expressed during embryonic stages, thus making it difficult to achieve effective RNAi in adult organisms. In our present study we present a bacterial feeding method for RNAi in Daphnia. We used a melanic Daphnia species (Daphnia melanica) that exhibits dark pigmentation to target phenoloxidase, a key enzyme in the biosynthesis of melanin. We demonstrate that our RNAi method results in a striking phenotype and that the phenoloxidase mRNA expression and melanin content, as well as survival following UV insults, are diminished as a result of RNAi. Overall, our results establish a new method for RNAi in Daphnia that significantly advances further use of Daphnia as a model organism for functional genomics studies. The method we describe is relatively simple and widely applicable for knockdown of a variety of genes in adult organisms.