Abstract
Daphnids are small crustaceans ubiquitous in fresh water; they have been a subject of study in ecology, evolution, and environmental sciences for decades. To understand data accumulated in daphnid biology at the molecular level, expressed sequence tags and a genome sequence have been determined. However, these discoveries lead to the problem of how to understand the functions of newly discovered genes. Double-stranded RNA (dsRNA)-mediated RNA interference (RNAi) is a useful tool to achieve specific gene silencing in nontransformable species. Hence, we established a technique to inject exogenous materials into ovulated eggs and developed a dsRNA-based RNAi method for Daphnia magna. Eggs were collected just after ovulation and injected with dsRNA specific to the Distal-less (Dll) gene, which functions in appendage development in invertebrates and vertebrates. We found that the dsRNA successfully triggered the degradation of Dll mRNAs, which induced the truncation of the second antenna in a dose-dependent manner. This effect was sequence specific in that: (1) an unrelated dsRNA did not induce any morphological abnormalities and (2) two non-overlapping Dll dsRNAs generated the same phenotype. This is the first report of an RNAi technique in D. magna and, together with the emerging genome sequences, will be useful for advancing knowledge of the molecular biology of daphnids.
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Acknowledgments
D. magna and D. pulex genomic sequence data were produced by The Center for Genomics and Bioinformatics at Indiana University and distributed via wFleaBase in collaboration with the Daphnia Genomics Consortium http://daphnia.cgb.indiana.edu. This study was partly supported through a grant from the Ministry of Education, Culture, Sports, Science and Technology (H.W., T.I., Y.S.), the Ministry of the Environment of Japan (H.W., T.I.), and a grant of Long-range Research Initiative by Japan Chemical Industry Association (T.I.). Y.K. is a research fellow of the Japan Society for the Promotion of Science.
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Kato, Y., Shiga, Y., Kobayashi, K. et al. Development of an RNA interference method in the cladoceran crustacean Daphnia magna . Dev Genes Evol 220, 337–345 (2011). https://doi.org/10.1007/s00427-011-0353-9
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DOI: https://doi.org/10.1007/s00427-011-0353-9