In both basic and human health research, there is a pressing need to understand how environmental conditions influence gene functions and, in turn, how individuals and populations cope with changing environments. Invertebrate species have emerged as models for experimental manipulation because of their unique biological attributes, life cycles, large numbers of offspring, easy maintenance, and sophisticated tools for high-throughput biology and open-source informatics . However, the traits observed in laboratories are likely a small subset of the phenotypic variation that is expressed in natural ecosystems. This may partly explain why 15 to 50% or more genes are without experimentally determined functional annotations, even within the best-characterized genomes (e.g., yeast; ).
Daphnia possess several characteristics that make them valuable for environmental, evolutionary, and developmental genomics research–addressing the added complexity of genome-environment interactions. Daphnia are a ubiquitous, and ecologically important member of freshwater lakes and ponds, and have long been used as a sentinel of the integrity of these aquatic ecosystems. More recently with the release of the D. pulex genome , it now serves as a recognized surrogate model for human health research. The D. pulex genome possesses more genes than any previously sequenced animal genome (~31,000), due to a large orphanage of Daphnia genes that likely, allows the organism to respond to its environment . In addition to their short generation time, large brood sizes, and ease of laboratory and field manipulation, Daphnia are capable of either clonal or sexual reproduction, making them ideally suited for genetic studies. At present, however, there are no effective methods for manipulating genes and characterizing gene function, which because of the large gene orphanage limits interspecies extrapolations.
RNA interference (RNAi) is an evolutionarily conserved post-transcriptional gene silencing mechanism, which is triggered by double-stranded (ds)RNA in a sequence specific manner [4, 5]. Since RNAi was first reported in the nematode Caenorhabditis elegans by Fire et al. , it has been used as a powerful tool for the analysis of gene function in many organisms such as zebrafish Danio rerio, planarian Schmidtea meditteranea, cnidarian Hydra magnipapillata, fungus Neurospora crassa, fruit-fly Drosophila melanogaster and mouse Mus musculus. Microinjection is one method of introducing dsRNA into cells, and this method has been successfully developed for the daphnid species, D. magna. In fact, microinjection techniques enabled the application of not only RNAi [13, 14], but also overexpression of foreign genes  and the creation of transgenic individuals . Establishing these techniques in D. pulex will extend the resources for environmental, evolutionary, and developmental genomics research for this species by providing needed tools to characterize gene function.
Distal-less (Dll) and its homologs Dlx genes, which function as homeodomain transcription factors, play one of the major roles in limb development throughout the animal kingdom . Reduction of Dll activity caused defects of distal leg segments in arthropods including insects [18–20], crustaceans (Parhyale hawaiensis, D. magna), the spider Cupiennius salei and the spider mite Tetranychus urticae. Because of its conserved role in limb development, defects in the expression of the Dll gene produce easily recognizable phenotype and ease of evaluation of its phenotype, which is why this endogenous developmental gene was selected as a target in proof-of-principle RNAi in D. pulex.
The goal of this study was to develop a microinjection system for RNAi in D. pulex. Culture requirements for isolated embryos were first determined and then microinjection techniques developed for conducting RNAi experiments using Dll-dsRNA.