Generation of white-eyed Daphnia magna mutants lacking scarlet function

The crustacean Daphnia magna is an important model in multi-disciplinary scientific fields such as genetics, evolutionary developmental biology, toxicology, and ecology. Recently, draft genome sequence and transcriptome data became publicly available for this species. Genetic transformation by introduction of plasmid DNA into a genome has been achieved. To further advance D. magna functional genomics, identification of a screenable marker gene and generation of its mutant are indispensable. Because Daphnia is more closely related to insects among crustaceans, we hypothesized that eye color-related genes can function as a marker gene as used in Drosophila genetics. We searched orthologs of Drosophila eye pigment transporters White, Scarlet, and Brown in the genome of D. magna. Amino acid sequence alignment and phylogenetic analysis suggested that D. magna has six white and one scarlet orthologs, but lacks the brown ortholog. Due to a multiplicity of white orthologs, we analyzed function of the scarlet ortholog, DapmaSt, using RNA interference. DapmaSt RNAi embryos showed disappearance of black pigments both in the compound eye and in the ocellus, suggesting that DapmaSt is necessary for black pigmentation in Daphnia eyes. To disrupt DapmaSt by using the Crispr/Cas9 system, we co-injected DapmaSt-targeting gRNAs with Cas9 mRNAs into eggs and established white-eyed DapmaSt mutant lines that lack eye pigments throughout their lifespan. Our results suggest that DapmaSt can be used as a transformation marker in D. magng+a and the DapmaSt mutants would be an important resource for genetic transformation of this species in the future.