Single and combined effects of increased temperature and methylmercury on different stages of the marine rotifer Brachionus plicatilis.

Rapid, anthropogenic activity-induced global warming is a severe problem that not only raises water temperatures but also shifts aquatic environments by increasing the bioavailability of heavy metals (HMs), with potentially complicated effects on aquatic organisms, including small aquatic invertebrates. For this paper, we investigated the combined effects of temperature (23 and 28 °C) and methylmercury (MeHg) by measuring physiological changes, bioaccumulation, oxidative stress, antioxidants, and the mitogen-activated protein kinase signaling pathway in the marine rotifer Brachionus plicatilis. High temperature and MeHg adversely affected the survival rate, lifespan, and population of rotifers, and bioaccumulation, oxidative stress, and biochemical reactions depended on the developmental stage, with neonates showing higher susceptibility than adults. These findings demonstrate that increased temperature enhances potentially toxic effects from MeHg, and susceptibility differs with the developmental stage. This study provides a comprehensive understanding of the combined effects of elevated temperature and MeHg on rotifers. ENVIRONMENTAL IMPLICATION: Methylmercury (MeHg) is a widespread and harmful heavy metal that can induce lethal effects on aquatic organisms in even trace amounts. The toxicity of metals can vary depending on various environmental conditions. In particular, rising temperatures are considered a major factor affecting bioavailability and toxicity by changing the sensitivity of organisms. However, there are few studies on the combinational effects of high temperatures and MeHg on aquatic animals, especially invertebrates. Our research would contribute to understanding the actual responses of aquatic organisms to complex aquatic environments.

Multigenerational effects of elevated temperature on host-microbiota interactions in the marine water flea Diaphanosoma celebensis exposed to micro- and nanoplastics.

Rising ocean temperatures are driving unprecedented changes in global marine ecosystems. Meanwhile, there is growing concern about microplastic and nanoplastic (MNP) contamination, which can endanger marine organisms. Increasing ocean warming (OW) and plastic pollution inevitably cause marine organisms to interact with MNPs, but relevant studies remain sparse. Here, we investigated the interplay between ocean warming and MNP in the marine water flea Diaphanosoma celebensis. We found that combined exposure to MNPs and OW induced reproductive failure in the F2 generation. In particular, the combined effects of OW and MNPs on the F2 generation were associated with key genes related to reproduction and stress response. Moreover, populations of predatory bacteria were significantly larger under OW and MNP conditions during F2 generations, suggesting a potential link between altered microbiota and host fitness. These results were supported by a host transcriptome and microbiota interaction analysis. This research sheds light on the complex interplay between environmental stressors, their multigenerational effects on marine organisms, and the function of the microbiome.

Combined effects of nanoplastics and elevated temperature in the freshwater water flea Daphnia magna.

Global warming and nanoplastics (NPs) are critical global issues. Among NPs, one of the most hazardous types of plastics, polystyrene (PS), poses ecotoxicological threats to several freshwater organisms. The degree of toxicity of PS-NPs is strongly influenced by various environmental factors. This study illustrates the combined effects of temperature and PS-NPs on the water flea Daphnia magna. The sensitivity of D. magna to PS-NPs was tested under control (23 °C) and elevated temperatures (28 °C). As a result, increased temperatures influenced the uptake and accumulation of PS-NPs. Co-exposure to both higher temperatures and PS-NPs resulted in a drastic decrease in reproductive performance. The level of oxidative stress was found to have increased in a temperature-dependent manner. Oxidative stress was stimulated by both stressors, leading to increased levels of reactive oxygen species and antioxidant enzyme activity supported by upregulation of antioxidant enzyme-related genes under combined PS-NPs exposure and elevated temperature. In the imbalanced status of intracellular redox, activation of the p38 mitogen-activated protein kinase signaling pathway was induced by exposure to PS-NPs at high temperatures, which supported the decline of the reproductive capacity of D. magna. Therefore, our results suggest that PS-NPs exposure along with an increase in temperature significantly affects physiological processes triggered by damage from oxidative stress, leading to severely inhibited reproduction of D. magna.

Physiological effects and molecular response in the marine rotifer Brachionus plicatilis after combined exposure to nanoplastics and copper.

Because nanoplastics (NPs) can transport pollutants, the absorption of surrounding pollutants into NPs and their effects are important environmental issues. This study shows a combined effect of high concentrations of NPs and copper (Cu) in the marine rotifer Brachionus plicatilis. Co-exposure decreased the growth rate, reproduction, and lifespan. The highest level of NP ingestion was detected in the co-treated group, but the Cu concentration was higher in the Cu single-exposure group. ERK activation played a key role in the downstream cell signaling pathway activated by the interaction of NPs and Cu. The increased sensitivity of B. plicatilis to Cu could be due to the impairment of MXR function caused by a high concentration of NPs, which supports our in vivo experiment results. Our results show that exposure to NPs could induce the dysfunction of several critical molecular responses, weakening resistance to Cu and thereby increasing its physiological toxicity in B. plicatilis.

Heavy metals and metalloid in aquatic invertebrates: A review of single/mixed forms, combination with other pollutants, and environmental factors.

Heavy metals (HMs) and metalloid occur naturally and are found throughout the Earth's crust but they are discharged into aquatic environments at high concentrations by human activities, increasing heavy metal pollution. HMs can bioaccumulate in higher organisms through the food web and consequently affect humans. In an aquatic environment, various HMs mixtures can be present. Furthermore, HMs adsorb on other environmental pollutants, such as microplastics and persistent organic pollutants, causing a synergistic or antagonistic effect on aquatic organisms. Therefore, to understand the biological and physiological effects of HMs on aquatic organisms, it is important to evaluate the effects of exposure to combinations of complex HM mixtures and/or pollutants and other environmental factors. Aquatic invertebrates occupy an important niche in the aquatic food chain as the main energy link between higher and lower organisms. The distribution of heavy metals and the resulting toxic effects in aquatic invertebrates have been extensively studied, but few reports have dealt with the relationship between HMs, pollutants, and environmental factors in biological systems with regard to biological availability and toxicity. This review describes the overall properties of individual HM and their effects on aquatic invertebrates and comprehensively reviews physiological and biochemical endpoints in aquatic invertebrates depending on interactions among HMs, other pollutants, and environmental factors.

Differential susceptibility to arsenic in glutathione S-transferase omega 2 (GST-O2)-targeted freshwater water flea Daphnia magna mutants.

To examine the role of glutathione S-transferase omega class (GST-O2) genes in the biotransformation and detoxification in Daphnia magna, various responses such as in vivo endpoints, arsenic speciation, enzymatic activities, and gene expression pathways related to arsenic metabolism were investigated in wild-type (WT) and GST-O2-mutant-type (MT) fleas produced by CRISPR/Cas9. Sensitivity to arsenic in MT fleas was higher than in WT fleas. Also, the reduction rate of arsenate (AsV) to arsenite (AsIII) in the MT group was significantly lower and led to accumulation of higher arsenic concentrations, resulting in decreased protection against arsenic toxicity. Relative mRNA expression of other GST genes in the GST-O2-targeted MT group generally increased but the enzymatic activity of GST decreased compared with the WT group. Oxidative stress on arsenic exposure was more strongly induced in the MT group compared with the WT group, resulting in a decrease in the ability to defend against toxicity in GST-O2-targeted mutant D. magna. Our results suggest that GST-O2 plays an important role in arsenic biotransformation and detoxification functions in D. magna.

Short- and long-term single and combined effects of microplastics and chromium on the freshwater water flea Daphnia magna.

In this study, we investigated the individual and combined effects of microplastics (MPs) and chromium (Cr) on the freshwater water flea Daphnia magna by measuring mortality, bioaccumulation, antioxidative response, multixenobiotic resistance activity, and sestrin-related mitochondrial biogenesis in short-term assays and in vivo endpoints including reproduction and adult survival rate in long-term assays. Exposure to MPs, Cr, and their combination caused significant deleterious effects and acute toxicity in D. magna. Alterations in oxidative stress occurred in the groups treated with MPs and Cr alone and together. However, upon co-exposure to MPs, the Cr concentration, measured by inductively coupled plasma optical emission spectroscopy, decreased, suggesting that MPs and Cr interact with each other. Based on enzymatic activities, we noted a decrease in MP egestion via inhibition of P-glycoprotein activity in the MP-exposed groups, and multidrug resistance-associated protein activity increased in some of the MP-exposed animals depending on Cr concentration. On the other hand, MP exposure seemed to lead to mitochondrial transcription dysfunction induced by Cr via sestrin-related mitochondrial biogenesis. Overall, these results indicate that co-exposure to MPs and Cr causes acute toxicity in D. magna but lacks the chronic toxicity (21 days) and mitochondrial dysfunction caused by Cr exposure alone.

Molecular evidence for suppression of swimming behavior and reproduction in the estuarine rotifer Brachionus koreanus in response to COVID-19 disinfectants.

The increased use of disinfectants due to the spread of the novel coronavirus infection (e.g. COVID-19) has caused burden in the environment but knowledge on its ecotoxicological impact on the estuary environment is limited. Here we report in vivo and molecular endpoints that we used to assess the effects of chloroxylenol (PCMX) and benzalkonium chloride (BAC), which are ingredients in liquid handwash, dish soap products, and sanitizers used by consumers and healthcare workers on the estuarine rotifer Brachionus koreanus. PCMX and BAC significantly affected the life table parameters of B. koreanus. These chemicals modulated the activities of antioxidant enzymes such as superoxide dismutase and catalase and increased reactive oxygen species even at low concentrations. Also, PCMX and BAC caused alterations in the swimming speed and rotation rate of B. koreanus. Furthermore, an RNA-seq-based ingenuity pathway analysis showed that PCMX affected several signaling pathways, allowing us to predict that a low concentration of PCMX will have deleterious effects on B. koreanus. The neurotoxic and mitochondrial dysfunction event scenario induced by PCMX reflects the underlying molecular mechanisms by which PCMX produces outcomes deleterious to aquatic organisms.

Identification and characterization of homeobox gene clusters in harpacticoid and calanoid copepods.

In this study, we have identified the entire complement of typical homeobox (Hox) genes (Lab, Pb, Dfd, Scr, Antp, Ubx, Abd-A, and Abd-B) in harpacticoid and calanoid copepods and compared them with the cyclopoid copepod Paracyclopina nana. The harpacticoid copepods Tigriopus japonicus and Tigriopus kingsejongensis have seven Hox genes (Lab, Dfd, Scr, Antp, Ubx, Abd-A, and Abd-B) and the Pb and Ftz genes are also present in the cyclopoid copepod P. nana. In the Hox gene cluster of the calanoid copepod Eurytemora affinis, all the Hox genes were present linearly in the genome but the Antp gene was duplicated. Of the three representative copepods, the P. nana Hox gene cluster was the most compact due to its small genome size. The Hox gene expression profile patterns in the three representative copepods were stage-specific. The Lab, Dfd, Scr, Pb, Ftz, and Hox3 genes showed a high expression in early developmental stages but Antp, Ubx, Abd-A, and Abd-B genes were mostly expressed in later developmental stages, implying that these Hox genes may be closely associated with the development of segment identity during early development.

The freshwater water flea Daphnia magna NIES strain genome as a resource for CRISPR/Cas9 gene targeting: The glutathione S-transferase omega 2 gene.

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.

Arsenic exposure combined with nano- or microplastic induces different effects in the marine rotifer Brachionus plicatilis.

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.

Different effects of nano- and microplastics on oxidative status and gut microbiota in the marine medaka Oryzias melastigma.

Plastic is regarded as a major environmental concern. In particular, nanoplastics and microplastics (NMPs) are attracting global attention due to their potential impact on aquatic organisms. Here, we examined the effects of NMPs (50 nm polystyrene microbead nanoplastics [NPs] and 45 µm microplastics [MPs]) on oxidative status and gut microbiota in the marine medaka Oryzias melastigma. The NP-exposed group exhibited stronger oxidative stress with higher activation levels of antioxidants compared to the MP-exposed group. However, the MP-exposed group demonstrated induction of intestinal damage (e.g., increased mucus ratio) with further alterations of gut microbiota, compared to the NP-exposed group. In particular, MPs caused more significant alterations of microbiota composition at both phylum and genus levels. Thus, in this study we show distinct toxicity pathways of NPs and MPs, an oxidative stress-mediated pathway (e.g., antioxidants) induced by NP exposure and dysbiosis of gut microbiota in association with immune dysfunction induced by MP exposure. Our results are helpful for expanding our knowledge about the impacts of NMPs as potentially harmful substances in the aquatic environment.

Genome-wide identification and transcriptional modulation of histone variants and modification related genes in the low pH-exposed marine rotifer Brachionus koreanus.

Histone modification is considered to be a major epigenetic control mechanism. These modifications (e.g. acetylation, phosphorylation, and methylation) may affect the interaction of histones with DNA and/or regulate DNA-based processes (e.g., recombination, repair, replication, and transcription) and chromatin remodeling complexes. Despite their significance in metazoan life and evolution, few studies have been conducted to identify genes undergoing epigenetic control modification in aquatic invertebrates. In this study, we identified whole core histones (70 total genes) and post-translational modification (PTM) histone genes (63 total genes) in the marine rotifer Brachionus koreanus through whole-genome analysis, and annotated them according to the human nomenclature. Notably, upon comparative analysis of cis-regulatory motif sequences, we found that B. koreanus core histone protein structures were similar to those of mammals. Furthermore, to examine the effect of parental low pH stress on the offspring's epigenetic regulation, we investigated the expression of PTM genes in two generations of B. koreanus exposed to low pH conditions. Given that the B. koreanus genome does not possess DNA methyltransferase 1 and 3 genes, we concluded that histone genes could be involved as an important epigenetic mechanism in B. koreanus. Therefore, the histone-associated genes identified in this study could be useful for ecotoxicological studies and facilitate the application of chromatin immunoprecipitation sequencing using high-throughput DNA sequencing based on the genome-wide identification of transcription factor binding sites in rotifers.