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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.

SUBMITTER: Ismail NIB

PROVIDER: S-EPMC6235260 | biostudies-literature |

REPOSITORIES: biostudies-literature

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The first-generation Daphnia magna linkage map.

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Two Doublesex1 mutants revealed a tunable gene network underlying intersexuality in Daphnia magna.

Project description:In recent years, the binary definition of sex is being challenged by repetitive reports about individuals with ambiguous sexual identity from various animal groups. This has created an urge to decode the molecular mechanism underlying sexual development. However, sexual ambiguities are extremely uncommon in nature, limiting their experimental value. Here, we report the establishment of a genetically modified clone of Daphnia magna from which intersex daphniids can be readily generated. By mutating the conserved central sex determining factor Doublesex1, body-wide feminization of male daphniid could be achieved. Comparative transcriptomic analysis also revealed a genetic network correlated with Doublesex1 activity which may account for the establishment of sexual identity in D. magna. We found that Dsx1 repressed genes related to growth and promoted genes related to signaling. We infer that different intersex phenotypes are the results of fluctuation in activity of these Dsx1 downstream factors. Our results demonstrated that the D. magna genome is capable of expressing sex in a continuous array, supporting the idea that sex is actually a spectrum.

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Intragenic tandem repeats in Daphnia magna: structure, function and distribution.

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Annotation of the Daphnia magna nuclear receptors: comparison to Daphnia pulex.

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Titanium dioxide nanoparticles increase sensitivity in the next generation of the water flea Daphnia magna.

Project description:The nanoparticle industry is expected to become a trillion dollar business in the near future. Therefore, the unintentional introduction of nanoparticles into the environment is increasingly likely. However, currently applied risk-assessment practices require further adaptation to accommodate the intrinsic nature of engineered nanoparticles. Combining a chronic flow-through exposure system with subsequent acute toxicity tests for the standard test organism Daphnia magna, we found that juvenile offspring of adults that were previously exposed to titanium dioxide nanoparticles exhibit a significantly increased sensitivity to titanium dioxide nanoparticles compared with the offspring of unexposed adults, as displayed by lower 96 h-EC(50) values. This observation is particularly remarkable because adults exhibited no differences among treatments in terms of typically assessed endpoints, such as sensitivity, number of offspring, or energy reserves. Hence, the present study suggests that ecotoxicological research requires further development to include the assessment of the environmental risks of nanoparticles for the next and hence not directly exposed generation, which is currently not included in standard test protocols.

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Tryptophan hydroxylase (TRH) loss of function mutations induce growth and behavioral defects in Daphnia magna.

Project description:Tryptophan hydroxylase (TRH) is the rate limiting enzyme in the serotonin synthesis. CRISPR-Cas9 technology was used to generate seven indel TRH mutants in Daphnia magna. Mono-allelic indel TRH-/+ clones showed normal levels of serotonin, measured by both immunohistochemistry and mass spectrometry (LC-MS/MS), whereas bi-allelic indel TRH-/- clones showed no detectable levels of serotonin. Life history and behavioral responses of TRH-/- clones showed the anti-phenotype of those exposed to selective serotonin reuptake inhibitors (SSRI). Mutants lacking serotonin grew less and hence reproduced latter, produced smaller clutches of smaller offspring and responded to a greater extent to light than wild type individuals. Mono-allelic indel TRH-/+ individuals showed the intermediate phenotype. The SSRI fluoxetine enhanced offspring production in all clones and decreased the response to light only in those clones having serotonin, thus indication that behavioral effects of this drug in D. magna are associated to serotonin. Results obtained with the TRH mutants are in line with reported ones in TRH knockouts of Caenorhabditis elegans, Drosophila and mice, indicating that there is one gene encoding TRH, which is the serotonin limiting enzyme in both the central and the periphery nervous system in Daphnia and that deprivation of serotonin increases anxiety-like behavior.

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TALEN-mediated homologous recombination in Daphnia magna.

Project description:Transcription Activator-Like Effector Nucleases (TALENs) offer versatile tools to engineer endogenous genomic loci in various organisms. We established a homologous recombination (HR)-based knock-in using TALEN in the crustacean Daphnia magna, a model for ecological and toxicological genomics. We constructed TALENs and designed the 67?bp donor insert targeting a point deletion in the eyeless mutant that shows eye deformities. Co-injection of the TALEN mRNA with donor DNA into eggs led to the precise integration of the donor insert in the germ line, which recovered eye deformities in offspring. The frequency of HR events in the germ line was 2% by using both plasmid and single strand oligo DNA with 1.5?kb and 80?nt homology to the target. Deficiency of ligase 4 involved in non-homologous end joining repair did not increase the HR efficiency. Our data represent efficient HR-based knock-in by TALENs in D. magna, which is a promising tool to understand Daphnia gene functions.

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Testing hypotheses for maternal effects in Daphnia magna.

Project description:Maternal effects are widely observed, but their adaptive nature remains difficult to describe and interpret. We investigated adaptive maternal effects in a clone of the crustacean Daphnia magna, experimentally varying both maternal age and maternal food and subsequently varying food available to offspring. We had two main predictions: that offspring in a food environment matched to their mothers should fare better than offspring in unmatched environments, and that offspring of older mothers would fare better in low food environments. We detected numerous maternal effects, for example offspring of poorly fed mothers were large, whereas offspring of older mothers were both large and showed an earlier age at first reproduction. However, these maternal effects did not clearly translate into the predicted differences in reproduction. Thus, our predictions about adaptive maternal effects in response to food variation were not met in this genotype of Daphnia magna.

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