Abstract
The crustacean Daphnia magna is an important model in multi-disciplinary scientific fields such as genetics, evolutionary developmental biology, toxicology, and ecology. Recently, the draft genome sequence and transcriptome data became publicly available for this species. Genetic transformation has also been achieved via the introduction of plasmid DNA into the genome. The identification of a screenable marker gene and generation of mutant strains are essential to further advance D. magna functional genomics. Because crustaceans are closely related to insects, we hypothesized that, similar to Drosophila genetic studies, eye color-related genes can function as marker genes in Daphnia. 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 the multiplicity of white orthologs, we analyzed the 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 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. magna and the DapmaSt mutants would be an important resource for genetic transformation of this species in the future.
Highlights
The branchiopod crustacean, Daphnia magna, commonly referred to as the water flea, is a model organism for various scientific fields such as genetics, evolutionary developmental biology, toxicology, and ecology
The phylogenetic analysis showed that one transporter was grouped with the scarlet gene and the others with the white gene (Fig 3), whereas no brown ortholog was reported in the D. magna genome
These results suggest that D. magna has a single scarlet ortholog and six white orthologs
Summary
The branchiopod crustacean, Daphnia magna, commonly referred to as the water flea, is a model organism for various scientific fields such as genetics, evolutionary developmental biology, toxicology, and ecology. Together with transcriptome data, is publicly available [1] Genetic manipulation tools, such as RNA interference [2] and genome editing [3], have been developed. To evaluate chemicals with hormone-like activities towards Daphnia at the level of gene expression, generation of the biosensor daphniid harboring a reporter gene that responds to ecdysteroids or juvenile hormones has been attempted [9,10]. In these transformation experiments, fluorescent protein genes were used as visible markers
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