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The viral 2A peptides have recently been used for bicistronic expression in various organisms. In this system, a single mRNA that codes for two proteins flanking the 2A peptide can be translated simultaneously into each protein by ribosomal skipping at this peptide sequence. Here, we tested the function of the Thosea asigna insect virus 2A (T2A) peptide in the branchiopod crustacean Daphnia magna-an emerging model of evolutionary developmental biology. First, we used transgenic Daphnia that expresses a potential bicistronic RNA containing mCherry and histone H2B- green fluorescent protein (GFP) open reading frames upstream and downstream of the T2A sequence, respectively. Microscopic observation revealed difference of localization of the two proteins in the cell, homogenous distribution of mCherry and nuclear localization of H2B-GFP. Second, we changed localization of mCherry from cytoplasm to plasma membrane by attachment of a consensus myristoylation motif in the bicistronic reporter. RNA that codes for this new bicistronic reporter was injected into eggs. At gastrulation stage, we found spectrally distinct fluorescence with enough intensity and resolution to detect membrane localized mCherry and nuclear GFP. These results indicate that the T2A peptide functions in D. magna and T2A-mediated bicistronic expression would be a promising tool for evo-devo studies of this species., (© 2017 Wiley Periodicals, Inc.)

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system (Cas) is widely used for mediating the knock-in of foreign DNA into the genomes of various organisms. Here, we report a process of CRISPR/Cas-mediated knock-in via non-homologous end joining by the direct injection of Cas9/gRNA ribonucleoproteins (RNPs) in the crustacean Daphnia magna, which is a model organism for studies on toxicology, ecology, and evolution. First, we confirmed the cleavage activity of Cas9 RNPs comprising purified Cas9 proteins and gRNAs in D. magna. We used a gRNA that targets exon 10 of the eyeless gene. Cas9 proteins were incubated with the gRNAs and the resulting Cas9 RNPs were injected into D. magna eggs, which led to a typical phenotype of the eyeless mutant, i.e., eye deformity. The somatic and heritable mutagenesis efficiencies were up to 96% and 40%, respectively. Second, we tested the CRISPR/Cas-mediated knock-in of a plasmid by the injection of Cas9 RNPs. The donor DNA plasmid harboring the fluorescent reporter gene was designed to contain the gRNA recognition site. The co-injection of Cas9 RNPs together with the donor DNAs resulted in generation of one founder animal that produced fluorescent progenies. This transgenic Daphnia had donor DNA at the targeted genomic site, which suggested the concurrent cleavage of the injected plasmid DNA and genomic DNA. Owing to its simplicity and ease of experimental design, we suggest that the CRISPR/Cas-mediated knock-in method represents a promising tool for studying functional genomics in D. magna.

We examined Daphnia pulex in Japan to clarify if they were representative of indigenous populations or colonized recently. Phylogenetic analysis of mtDNA suggests that Japanese lineages of D. pulex are immigrants from North America, and are hybrids formed with Daphnia pulicaria prior to this immigration. Based on the mtDNA sequences, the D. pulex individuals aggregated into four distinct genetic groups (JPN 1-4) comprising a total of 21 haplotypes. Surprisingly, microsatellite analysis with 12 loci revealed only a single multilocus genotype per genetic group, suggesting that D. pulex populations in Japan are comprised of asexual individuals, derived from only four clones. According to the reported mutation rate of mtDNA, JPN 1 and 2, now widely distributed across Japan, were estimated to have invaded between 680 yr and 3400 yr ago, while JPN 3 and 4 colonized much more recently. Results also indicated that the invasion of some clones could not be attributed to recent human activities and most likely occurred by rare natural events. Since the evolutional longevity of asexual clones is thought to be limited, genetic diversity of D. pulex in Japan has the possibility of decreasing in the near future without addition of novel gene flow. [ABSTRACT FROM AUTHOR]