Background: The ubiquitous, freshwater microcrustacean Daphnia pulex provides a model system for both human health research and monitoring ecosystem integrity. It is the first crustacean to have a well annotated, reference genome assembly that revealed an unusually high gene count highlighted by a large gene orphanage,-i.e., previously uncharacterized genes. Daphnia are capable of either clonal or sexual reproduction, making them ideally suited for genetic manipulation, but the establishment of gene manipulation techniques is needed to accurately define gene functions. Although previous investigations developed an RNA interference (RNAi) system for one congener D. magna, these methods are not appropriate for D. pulex because of the smaller size of their early embryos. In these studies, we develop RNAi techniques for D. pulex by first determining the optimum culture conditions of their isolated embryos and then applying these conditions to the development of microinjection techniques and proof-of-principle RNAi experiments.Results: We found that isolated embryos were best cultured on a 2% agar plate bathed in 60 mM sucrose dissolved in M4 media, providing optimal conditions for microinjections. Then, we injected double-stranded (ds)RNA specific to the Distal-less gene (Dll), which is a homeobox transcription factor essential for limb development in invertebrates and vertebrates. Injected embryos presented with defects in the second antenna and appendage development, and dsRNA induced the degradation of Dll mRNAs, indicating that this technique successfully inhibited transcription of the target gene.Conclusions: We developed a microinjection system for RNAi studies in D. pulex. These techniques add to the growing genomic toolbox and enhance the genetic tractability of this important model for environmental, evolutionary, and developmental genomics.
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