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The water flea Daphnia magna is a small freshwater planktonic animal in the Cladocera. In this study, we assembled the genome of the D. magna NIES strain, which is widely used for gene targeting but has no reported genome. We used the long-read sequenced data of the Oxford nanopore sequencing tool for assembly. Using 3,231 genetic markers, the draft genome of the D. magna NIES strain was built into ten linkage groups (LGs) with 483 unanchored contigs, comprising a genome size of 173.47 Mb. The N50 value of the genome was 12.54 Mb and the benchmarking universal single-copy ortholog value was 98.8%. Repeat elements in the D. magna NIES genome were 40.8%, which was larger than other Daphnia spp. In the D. magna NIES genome, 15,684 genes were functionally annotated. To assess the genome of the D. magna NIES strain for CRISPR/Cas9 gene targeting, we selected glutathione S-transferase omega 2 (GST-O2), which is an important gene for the biotransformation of arsenic in aquatic organisms, and targeted it with an efficient make-up (25.0%) of mutant lines. In addition, we measured reactive oxygen species and antioxidant enzymatic activity between wild type and a mutant of the GST-O2 targeted D. magna NIES strain in response to arsenic. In this study, we present the genome of the D. magna NIES strain using GST-O2 as an example of gene targeting, which will contribute to the construction of deletion mutants by CRISPR/Cas9 technology.

Although many efforts have been made to understand the toxic effects of metals in aquatic invertebrates, there are limited data regarding metal toxicity in natural ecosystems, as most previous studies were conducted under controlled laboratory conditions. To address this data gap, we analyzed toxic effects and molecular responses in the marine rotifer Brachionus koreanus and the marine copepod Tigriopus japonicus following in vivo exposure to a seawater sample collected from a polluted region in South Korea. Inductively coupled plasma-mass spectrometry (ICP-MS) analysis of the field seawater sample found a variety of metals. Exposure to several dilutions of the field seawater sample impacted several endpoints in both species, including mortality and reproduction. Interestingly, the rotifer and copepod test species exhibited different patterns of effects on reactive oxygen species (ROS) and antioxidant enzymatic activities, suggesting that different regulatory mechanisms may be activated in the two species in response to exposure to toxic chemicals. Our study helps to better understand the defense mechanisms activated in aquatic invertebrates in response to metal-induced oxidative stress induced by contaminated seawater.

Sorption of organic pollutants on microplastics can be an alternative uptake route for organic pollutants in aquatic organisms. To assess the combined effects of microplastics and organic pollutants, we employed phenotypic and transcriptomic analyses to the responses of the marine rotifer Brachionus koreanus to environmentally relevant concentrations of nano-sized microplastic (0.05 µm), water-accommodated fractions of crude oil, and binary mixtures thereof. Our multigenerational in vivo experiments revealed more than additive effects on population growth of B. koreanus in response to combined exposure, while a single exposure to nano-sized microplastic did not induce observable adverse effects. Synergistic transcriptome deregulation was consistently associated with dramatically higher numbers of differentially expressed genes, and increased gene expression was associated with combined exposure. The majority of synergistic transcriptional alteration was related to metabolism and transcription, with impaired reproduction resulting from energetic reallocation toward adaptation. As further supported by chemistry analysis for polycyclic aromatic hydrocarbons sorption on microplastic, our findings imply that nano-sized microplastics can synergistically mediate the effects of organic pollutants in aquatic organisms.

Besides the adverse biological effects induced by microplastics (MPs), the effects associated with sorption of ambient pollutants on MPs are considered as an emerging environmental problem as MPs act as a mediator of pollutants. The present study examines the combined effects of nano(micro)plastics (NMPs) and arsenic (As) by exposing the marine rotifer Brachionus plicatilis to MP particles at the micro-scale (6 μm) and nano-scale (nanoplastics, NPs) (50 nm) along with As. In vivo toxicity, bioaccumulation, and biochemical reactions were used to examine the effects of combined exposure. The results of in vivo experiments showed that As toxicity increased with NP exposure, whereas toxicity was alleviated by MPs, indicating a different mode of action between NPs and MPs in combination with As. The highest level of As bioaccumulation was detected in NP + As groups, and followed by MP + As and As-only exposure groups, whereas no significant difference between groups was shown for As metabolites. In addition, the activity of several ATP-binding cassette proteins that confer multixenobiotic resistance, which is responsible for efflux of As, was activated by As but significantly inhibited by NP exposure, supporting the findings of in vivo experiments. Our results show that the effects of combining exposure to As with NP and MPs differ depending on particle size and provide an in-depth understanding of both environmental pollutants.

We measured the generation of intracellular reactive oxygen species (ROS) and the enzymatic activity and expression of glutathione-S transferase (GST) in response to temperature changes in the temperate copepod Tigriopus japonicus and the Antarctic copepod Tigriopus kingsejongensis. The levels of ROS and GST enzymatic activity were slightly elevated (P

iTRAQ proteomic profiling was conducted to examine the proteomic responses of the Antarctic copepod Tigriopus kingsejongensis under ultraviolet B (UVB) exposure. Of the 5507 proteins identified, 3479 proteins were annotated and classified into 25 groups using clusters of orthologous genes analysis. After exposing the T. kingsejongensis to 12 kJ/m2 UVB radiation, 77 biological processes were modulated over different time periods (0, 6, 12, 24, and 48 h) compared with the control. A Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed that UVB exposure in T. kingsejongensis downregulated ribosome and glyoxylate and dicarboxylate metabolism at all time points. Furthermore, antioxidant and chaperone proteins were highly downregulated in response to UVB exposure, causing protein damage and activating apoptotic processes in the 48 h UVB exposure group. These proteomic changes show the mechanisms that underlie the detrimental effects of UVB on the cellular defense systems of the Antarctic copepod T. kingsejongensis.

The toxicity of arsenic (As) has been reported to be different depending on their chemical forms. However, its toxicity mechanisms largely remain unknown. In this study, to investigate toxicity mechanism of As in marine zooplanktons, namely, the rotifer Brachionus plicatilis and the copepod Paracyclopina nana, metabolites of As were analyzed by using a high-performance liquid chromatography coupled with inductively coupled plasma mass spectrometry with in vivo toxicity and antioxidant responses in response to inorganic As, including arsenate (AsV) and arsenite (AsIII). While AsIII was more toxic than AsV in both organisms, the rotifer B. plicatilis exhibited stronger tolerance, compared to the copepod P. nana. The As speciation analysis revealed differences in biotransformation processes in two species with B. plicatilis having a more simplified process than P. nana, contributing to a better tolerance against As in the rotifer B. plicatilis compared to P. nana. Moreover, the levels of GSH content and the regulation of omega class glutathione S-transferases were different in response to oxidative stress between B. plicatilis and P. nana. These results suggest that the rotifer B. plicatilis has a unique survival strategy with more efficient biotransformation and antioxidant responses, compared to P. nana, conferring higher tolerance to As.

To understand effects of two widely used antidepressant on the antioxidant defense mechanism in the marine rotifer Brachionus koreanus, we assessed acute toxicity and measured population growth, reactive oxygen species (ROS) levels, glutathione (GSH) levels, and antioxidant enzymatic activities (GST, GR, and SOD) in response to fluoxetine hydrochloride (FLX) and sertraline hydrochloride (SER). The no observed effect concentration-24 h of fluoxetine and sertraline were 1000 μg/L and 450 μg/L, respectively, whereas the median lethal concentration (LC50)-24 h of fluoxetine and sertraline were 1560 μg/L and 507 μg/L, respectively. Both fluoxetine and sertraline caused significant reduction (P0.05) in the population growth rate indicating that both antidepressants have a potential adverse effect on life cycle parameters of B. koreanus. The intracellular ROS level and GSH level were significantly modulated (P0.05) in response to fluoxetine and sertraline. In addition, antioxidant enzymatic activities have shown significant modulation (P0.05) in response to FLX and SER in B. koreanus. Furthermore, transcriptional profiles of antioxidant genes (GSTs, SODs, and GR) have shown modulation in response to FLX compared to SER-exposed B. koreanus. Our results indicate that fluoxetine and sertraline induce oxidative stress, leading to reduction in the population density and modulation of antioxidant defense mechanism in the marine rotifer B. koreanus.

Brachionus spp. rotifers, which are widely distributed in aquatic environments, have been proposed as model organisms for ecotoxicological studies. Although the genomes of several rotifers belonging to the genus Brachionus have been assembled, the genome for the freshwater rotifer Brachionus angularis remains unknown. In this study, we analyzed the whole-genome sequence of B. angularis, which revealed a total length of 56.5 Mb and 21 contigs. The N50 and the GC content were 5.42 Mb and 23.66%, respectively. A total of 13,952 genes were predicted. Of them, we identified the main detoxification-related gene families, including those for cytochrome P450, glutathione S-transferase (GST), and the ATP-binding cassette transporter. In comparison with other Brachionus rotifers, massive species-specific expansion in GST sigma genes was found in B. angularis. This whole-genome analysis of B. angularis provides a basis for molecular ecotoxicological studies and provides useful biological tools for comparative studies of the evolution of detoxification mechanisms in Brachionus spp.

To investigate the adverse effect of two widely used pharmaceuticals, paracetamol (acetaminophen [APAP]) and oxytetracycline (OTC) on the marine rotifer Brachionus rotundiformis (B. rotundiformis), the animals were exposed to various environmentally-relevant concentrations. Up to date, acetaminophen and oxytetracycline have been considered as toxic, if used above threshold concentration, i.e. overdosed. However, this study demonstrated these two pharmaceuticals even at low concentration (i.e., μg/L scale) elicited oxidative stress through the generation of reactive oxygen species (ROS) along with the increased glutathione S-transferase activity, despite no-observed effect in in-vivo population growth. To validate the adverse effects of the two pharmaceuticals at relatively low concentrations, mRNA expression analysis was performed of the entire set of genes encoding 26 cytochrome P450s (CYPs) of phase I and 19 glutathione S-transferases (GSTs) of phase II of the rotifer B. rotundiformis. The mRNA expression analysis suggested specific genes CYP3045A2 and GSTσ1, GSTσ4, and GSTω1 take part in detoxification of APAP and OTC, resulting in no significant changes in the population growth and undetermined no observed effect concentration (NOEC) in the marine rotifer B. rotundiformis.