Identification of gene isoforms and their switching events between male and female embryos of the parthenogenetic crustacean Daphnia magna.

The cladoceran crustacean Daphnia exhibits phenotypic plasticity, a phenomenon that leads to diverse phenotypes from one genome. Alternative usage of gene isoforms has been considered a key gene regulation mechanism for controlling different phenotypes. However, to understand the phenotypic plasticity of Daphnia, gene isoforms have not been comprehensively analyzed. Here we identified 25,654 transcripts derived from the 9710 genes expressed during environmental sex determination of Daphnia magna using the long-read RNA-Seq with PacBio Iso-Seq. We found that 14,924 transcripts were previously unidentified and 5713 genes produced two or more isoforms. By a combination of Illumina short-read RNA-Seq, we detected 824 genes that implemented switching of the highest expressed isoform between females and males. Among the 824 genes, we found isoform switching of an ortholog of CREB-regulated transcription coactivator, a major regulator of carbohydrate metabolism in animals, and a correlation of this switching event with the sexually dimorphic expression of carbohydrate metabolic genes. These results suggest that a comprehensive catalog of isoforms may lead to understanding the molecular basis for environmental sex determination of Daphnia. We also infer the applicability of the full-length isoform analyses to the elucidation of phenotypic plasticity in Daphnia.

Mutation of the Cytochrome P450 CYP360A8 Gene Increases Sensitivity to Paraquat in Daphnia magna.

The freshwater crustacean Daphnia magna has traditionally been a model for ecotoxicological studies owing to its sensitivity to many xenobiotics. Because it is used in many toxicity assessments, its detoxification mechanism for xenobiotics is important and requires further study. However, studies related to detoxification genes are limited to transcriptomic profiling, and there are no D. magna mutants for use in the understanding of xenobiotic metabolism in vivo. We report the generation of a D. magna CYP360A8 mutant-the gene is a cytochrome P450 (CYP) clan 3 gene. Based on RNA sequencing of adult D. magna, we found that CYP360A8 has the highest expression level among all CYP genes. At ovarian maturation, its expression level is up-regulated 6-fold compared to the juvenile stages and is maintained thereafter. Using the CRISPR/CRISPR-associated 9 (Cas9) system, we disrupted CYP360A8 by coinjecting CYP360A8-targeting guide RNA and Cas9 proteins into D. magna eggs and established one monoallelic CYP360A8 mutant line. This CYP360A8 mutant had a higher sensitivity to the herbicide paraquat compared to the wild type. We confirmed the up-regulation of CYP360A8 by paraquat. The results demonstrate the role of CYP360A8 in paraquat detoxification. The present study establishes a CYP mutant of D. magna, and this strategy can be a basic platform to document a range of CYP gene-xenobiotic relationships in this species. Environ Toxicol Chem 2021;40:1279-1288. © 2020 SETAC.

Atrazine exposed phytoplankton causes the production of non-viable offspring on Daphnia magna.

This study focuses on the possibility that herbicide-exposed phytoplankton will cause sub-lethal effect on zooplankton. Atrazine, phytoplankton Raphidocelis subcapitata and zooplankton Daphnia magna were chosen as a model chemical and organisms. R. subcapitata was exposed to atrazine at 150 µg/L, harvested and fed to D. magna. While the mothers fed with atrazine-exposed phytoplankton did not show any abnormalities, they produced non-viable offspring. Number of non-viable offspring at the first clutch was high but the number was reduced at later stages and viable offspring was produced. This result indicates that phytoplankton exposed to sub-lethal dose of atrazine affects population dynamics of its predator, D. magna.