Microinjection-Based RNA Interference Method in the Water Flea, Daphnia pulex and Daphnia magna

It is well known that most daphnid species have several attractive life history characteris‐ tics such as cyclical parthenogenesis, environmental sex determination, and predator-in‐ duced defense formation. Recent advances in high-throughput omics technologies make it easy to obtain a huge number of potential candidate factors involved in environmental stimuli-triggered phenotypic alterations. Furthermore, our group has developed a micro‐ injection system to introduce foreign materials such as nucleotides and chemicals into the early-stage (one-cell stage) egg of Daphnia pulex and Daphnia magna. Consequently, we es‐ tablished a microinjection-based RNAi system that allows arbitrary gene functions to be investigated. However, this microinjection system does not seem to have pervaded in the daphnid research community due to its low throughput and high level of skills required. In this chapter, we review the microinjection method and its RNAi system in water fleas, D. pulex and D. magna, providing some technical tips and making challenging proposals for the development of novel high-throughput RNAi methods. Finally, we provide an overview of recently developed gene functional analysis methods such as overexpression and genome-editing systems.