Generation of white-eyed Daphnia magna mutants lacking scarlet function

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.

Fig 1. Putative structures of orthologs of eye transporter proteins in Daphnia magna.

The green, light blue, yellow, dark blue, purple and pink boxes indicate the Walker A/P-loop, Q-loop, ABC signature motif, Walker B, D-loop, and Switch/H-loop. Red boxes show transmembrane regions.

Fig 2. Amino acid alignment of nucleotide binding domains of Scarlet (St), White (W) in Daphnia magna (Dma), Daphnia pulex (Dp), Tribolium castaneum (Tc), Bombyx mori (Bm), Aedes aegypti (Aa), and Drosophila melanogaster (Dm).

The accession number of each protein is shown in S1 Table.

Fig 3. Phylogenetic tree of the amino acid sequences of eye pigment transporters.

The percentages of the replicate tree in which the associated taxa clustered together in the bootstrap test (1,000 replicates) are shown next to the branches. The bar indicates branch length and corresponds to the mean number of differences (P < 0.05) per residue along each branch. Evolutionary distances were computed using the p-distance method. The accession number of each protein is shown in S1 Table.

Fig 4. RNA interference of the scarlet ortholog in D. magna.

(A) The typical phenotype of embryos injected with the Scarlet-targeting siRNA. The cephalic regions of control siRNA-injected (siControl) and scarlet siRNA-injected (siScarlet) embryos are magnified. The ce and se refer to the compound eye and single eye (ocellus). (B) Gene expression profile of scarlet in embryos injected with Scarlet siRNAs. Error bars indicate the standard error of the mean (n = 3). *P < 0.05 (Student’s t-test).

Fig 5. Knockout of the DapmaSt in D. magna.

(A) Schematic gene structure of DapmaSt and the partial sequences of a wild-type (WT) and two DapmaSt mutants (MT1 and MT2). The green, light blue, yellow, dark blue, purple, and pink boxes indicate the Walker A/P-loop, Q-loop, ABC signature motif, Walker B, D-loop, and Switch/H-loop. Red boxes show transmembrane regions. The target sites for gRNA are indicated by bold letters and insertions are indicated by red letters. The protospacer adjacent motif (PAM) sequence is colored in grey. (B) The phenotype of DapmaSt mutant with the cephalic regions of a wild type and the DapmaSt mutant (MT2) are magnified. The ce and se refer to the compound eye and single eye (ocellus). (C) Polymerase chain reaction for genotyping of WT, MT1, and MT2. The amplified genomic DNA fragments were resolved by agarose gel electrophoresis. (D) Comparison of fecundity between WT, MT1, and MT2. The cumulative number of offspring was counted at each clutch. Error bars indicate the standard error of the mean (n = 3). n.s. indicates P > 0.05 (Student’s t-test).