Genome-wide identification of 769 G protein-coupled receptor (GPCR) genes from the marine medaka Oryzias melastigma.

The marine medaka Oryzias melastigma is a useful fish model for marine and estuarine ecotoxicology studies and can be applied to field-based population genomics because of its distribution in Asian estuaries and other coastal areas. We identified 769 full-length G protein-coupled receptor genes in the O. melastigma genome and classified them into five distinct classes. A phylogenetic comparison of GPCR genes in O. melastigma to humans and two other small fish species revealed a high-level orthological relationship. Purinergic and chemokine receptors were highly differentiated in humans whereas significant differentiation of chemosensory receptors was evident in fish species. Our results suggest that the GPCR gene families among the species used in this study exhibit evidence of sporadic evolutionary processes. These results may help improve our understanding of the advanced repertoires of GPCR and expand our knowledge of physiological mechanisms of fish in response to various environmental stimuli.

Toxicity and speciation of inorganic arsenics and their adverse effects on in vivo endpoints and oxidative stress in the marine medaka Oryzias melastigma.

Here, we investigate the effects of acute and chronic exposure to arsenate (AsV) and arsenite (AsIII) in the marine medaka Oryzias melastigma. In vivo effects, biotransformation, and oxidative stress were studied in marine medaka exposed to the two inorganic arsenics for 4 or 28 days. An investigation of embryonic development revealed no effect on in vivo parameters, but the hatching rate increased in the group exposed to AsIII. Exposure to AsIII also caused the greatest accumulation of arsenic in medaka. For acute exposure, the ratio of AsV to AsIII was higher than that of chronic exposure, indicating that bioaccumulation of inorganic arsenic can induce oxidative stress. The largest increase in oxidative stress was observed following acute exposure to AsIII, but no significant degree of oxidative stress was induced by chronic exposure. During acute exposure to AsV, the increase in the enzymatic activity of glutathione-S-transferase (GST) was twice as high compared with exposure to AsIII, suggesting that GST plays an important role in the initial detoxification process. In addition, an RNA-seq-based ingenuity pathway analysis revealed that acute exposure to AsIII may be related to cell-cycle progression. A network analysis using differentially expressed genes also revealed a potential link between the generation of inflammatory cytokines and oxidative stress due to arsenic exposure.

Non-negligible Toxicity to Fish in the Early Life Stages Triggered by Aqueous Leachate of Takeaway Plastic Containers.

Ordering takeout is a growing social phenomenon and may raise public health concerns. However, the associated health risk of compounds leaching from plastic packaging is unknown due to the lack of chemical and toxicity data. In this study, 20 chemical candidates were tentatively identified in the environmentally relevant leachate from plastic containers through the nontargeted chemical analysis. Three main components with high responses and/or predicted toxicity were further verified and quantified, namely, 3,5-di-tert-butyl-4-hydroxycinnamic acid (BHC), 2,4-di-tert-butylphenol (2,4-DTBP), and 9-octadecenamide (oleamide). The toxicity to zebrafish larvae of BHC, a degradation product of a widely used antioxidant Irganox 1010, was quite similar to that of the whole plastic leachate. In the same manner, RNA-seq-based ingenuity analysis showed that the affected canonical pathways of zebrafish larvae were quite comparable between BHC and the whole plastic leachate, i.e., highly relevant to neurological disease, metabolic disease, and even behavioral disorder. Longer-term exposure (35 days) did not cause any effect on adult zebrafish but led to decreased hatching rate and obvious neurotoxicity in zebrafish offspring. Collectively, this study strongly suggests that plastic containers can leach out a suite of compounds causing non-negligible impacts on the early stages of fish via direct or parental exposure.

Changes in Induced-Antipredation Defense Traits and Transcriptome Regulations of Daphnia magna in Response to 5-HT1A Receptor Antagonist.

The serotonin signaling system plays a crucial role in regulating the ontogeny of crustaceans. Here, we describe the effects of different concentrations of the 5-hydroxytryptamine 1A receptor antagonist (WAY-100635) on the induced antipredation (Rhodeus ocellatus as the predator), morphological, behavioral, and life-history defenses of Daphnia magna and use transcriptomics to analyze the underlying molecular mechanisms. Our results indicate that exposure to WAY-100635 leads to changes in the expression of different defensive traits in D. magna when faced with fish predation risks. Specifically, as the length of exposure to WAY-100635 increases, high concentrations of WAY-100635 inhibit defensive responses associated with morphological and reproductive activities but promote the immediate negative phototactic behavioral defense of D. magna. This change is related to the underlying mechanism through which WAY-100635 interferes with gene expression of G-protein-coupled GABA receptors by affecting GABBR1 but promotes serotonin receptor signaling and ecdysteroid signaling pathways. In addition, we also find for the first time that fish kairomone can significantly activate the HIF-1α signaling pathway, which may lead to an increase in the rate of immediate movement. These results can help assess the potential impacts of serotonin-disrupting psychotropic drugs on zooplankton in aquatic ecosystems.

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.

Synergistic adverse effects of microfibers and freshwater acidification on host-microbiota interactions in the water flea Daphnia magna.

Microfibers are the most common type of microplastics in freshwater environments. Anthropogenic climate stressors, such as freshwater acidification (FA), can interact with plastic pollution to disrupt freshwater ecosystems. However, the underlying mechanisms responsible for the interactive effects of microfibers and FA on aquatic organisms remain poorly understood. In this study, we investigated individual Daphnia magna-microbiota interactions affected by interactions between microfibers and FA (MFA). We found that the accumulated amount of microfibers in pH-treatment groups was significantly higher than in the control groups, resulting in negative consequences on reproduction, growth, and sex ratio. We also observed that MFA interactions induced immunity- and reproduction-related biological processes. In particular, the abundance of pathogenic bacteria increased only in MFA groups, indicating that MFA interactions can cause intestinal damage. Our integrated analysis of microbiomes and host transcriptomes revealed that synergistic adverse effects of MFAs are closely related to changes in microbial communities, suggesting that D. magna fitness and the microbial community are causally linked. These finding may help elucidate the toxicity mechanisms governing the responses of D. magna to microfibers and acidification interactions, and to host-microbiome-environment interactions.

Nanoplastics induce epigenetic signatures of transgenerational impairments associated with reproduction in copepods under ocean acidification.

Ocean acidification (OA) is one of many major global climate changes that pose a variety of risks to marine ecosystems in different ways. Meanwhile, there is growing concern about how nanoplastics (NPs) affect marine ecosystems. Combined exposure of marine organisms to OA and NPs is inevitable, but their interactive effects remain poorly understood. In this study, we investigated the multi- and transgenerational toxicity of NPs on copepods under OA conditions for ten generations. The findings revealed that OA and NPs have a synergistic negative effect on copepod reproduction across generations. In particular, the transgenerational groups showed reproductive impairments in the F1 and F2 generations (F1T and F2T), even though they were never exposed to NPs. Moreover, our epigenetic examinations demonstrated that the observed intergenerational reproductive impairments are associated with differential methylation patterns of specific genes, suggesting that the interaction of OA and NPs can pose a significant threat to the sustainability of copepod populations through epigenetic modifications. Overall, our findings provide valuable insight into the intergenerational toxicity and underlying molecular mechanisms of responses to NPs under OA conditions.

Oxidative stress-mediated synergistic deleterious effects of nano- and microplastics in the hypoxia-conditioned marine rotifer Brachionus plicatilis.

While pollution due to nano- and micro-sized plastics (NMPs) and hypoxic conditions both occur in coastal areas, the deleterious potential of co-exposure to hypoxia and NMPs (hypoxia and micro-sized plastics, HMPs; hypoxia and nano-sized plastics, HNPs) is largely unclear. Here, we provide evidence for multigenerational effects of HMP and HNP in the marine rotifer Brachionus plicatilis by investigating changes in its life traits, antioxidant system, and hypoxia-inducible factor (HIF) pathway using an orthogonal experimental design, with nanoscale and microscale particles measuring 0.05 µm and 6.0 µm in diameter, respectively, and hypoxic conditions of 0.5 mg/L for six generations. Combined exposure to NMPs and hypoxia caused a significant decrease in fecundity and overproduction of reactive oxygen species (ROS). The HIF pathway and circadian clock genes were also significantly upregulated in response to HMP and HNP exposure. In particular, synergistic deleterious effects of HNP were evident, suggesting that size-dependent toxicity can be a major driver of the effects of hypoxia and NMP co-exposure. After several generations of exposure, ROS levels returned to basal levels and transcriptomic resilience was observed, although rotifer reproduction remained suppressed. These findings help eluciating the underlying molecular mechanisms involved in responses to plastic pollution in hypoxic conditions.

Comparative genome analysis of the monogonont marine rotifer Brachionus manjavacas Australian strain: Potential application for ecotoxicology and environmental genomics.

This is the first study to analyze the whole-genome sequence of B. manjavacas Australian (Aus.) strain through combination of Oxford Nanopore long-read seq, resulting in a total length of 108.1 Mb and 75 contigs. Genome-wide detoxification related gene families in B. manjavacas Aus. strain were comparatively analyzed with those previously identified in other Brachionus spp., including B. manjavacas German (Ger.) strain. Most of the subfamilies in detoxification related families (CYPs, GSTs, and ABCs) were highly conserved and confirmed orthologous relationship with Brachionus spp., and with accumulation of genome data, clear differences between genomic repertoires were demonstrated the marine and the freshwater species. Furthermore, strain-specific genetic variations were present between the Aus. and Ger. strains of B. manjavacas. This whole-genome analysis provides in-depth review on the genomic structural differences for detoxification-related gene families and further provides useful information for comparative ecotoxicological studies and evolution of detoxification mechanisms in Brachionus spp.

The genome of the freshwater monogonont rotifer Brachionus rubens: Identification of phase I, II, and III detoxification genes.

Monogonont rotifers are common species in aquatic environments and make model species for ecotoxicology studies. Whole genomes of several species of the genus Brachionus have been assembled, but no information on the freshwater rotifer Brachionus rubens has been reported. In this study, the whole-genome sequence of B. rubens was successfully assembled using NextDenovo. The total length of the genome was 132.7 Mb (N50 = 2.51 Mb), including 122 contigs. The GC contents accounted for 29.96% of the genome. Aquatic organisms are always exposed to various external stresses, and a comprehensive genomic analysis is needed to better understand the adverse effects on organisms. This paper focuses on the ecotoxicological aspect and conducted genome analysis of representative gene families involved in detoxification mechanisms against environmental stressors. Specifically, we identified cytochrome P450 genes (CYPs) of phase I, glutathione S-transferase genes (GSTs) of phase II, and ATP-binding cassette transporter genes (ABCs) of phase III in the genome of B. rubens. Gene duplications were found in CYP, GST, and ABC genes, as is the case for other Brachionus rotifers. Our results suggest that these detoxification-related gene families have evolved in a species-specific and/or lineage-specific manner. This paper improves our understanding of how the freshwater Brachionus rotifers respond to environmental stressors in a molecular ecotoxicology context.

The Genome of the Marine Rotifer Brachionus manjavacas: Genome-Wide Identification of 310 G Protein-Coupled Receptor (GPCR) Genes.

The marine rotifer Brachionus manjavacas is widely used in ecological, ecotoxicological, and ecophysiological studies. The reference genome of B. manjavacas is a good starting point to uncover the potential molecular mechanisms of responses to various environmental stressors. In this study, we assembled the whole-genome sequence (114.1 Mb total, N50 = 6.36 Mb) of B. manjavacas, consisting of 61 contigs with 18,527 annotated genes. To elucidate the potential ligand-receptor signaling pathways in marine Brachionus rotifers in response to environmental signals, we identified 310 G protein-coupled receptor (GPCR) genes in the B. manjavacas genome after comparing them with three other species, including the minute rotifer Proales similis, Drosophila melanogaster, and humans (Homo sapiens). The 310 full-length GPCR genes were categorized into five distinct classes: A (262), B (26), C (7), F (2), and other (13). Most GPCR gene families showed sporadic evolutionary processes, but some classes were highly conserved between species as shown in the minute rotifer P. similis. Overall, these results provide potential clues for in silico analysis of GPCR-based signaling pathways in the marine rotifer B. manjavacas and will expand our knowledge of ligand-receptor signaling pathways in response to various environmental signals in rotifers.

Local lymph node assay: 5-bromo-d-deoxyuridine-ELISA method for comparative study in assessing chemical potencies and skin sensitization in BALB/c and CBA/J strains.

Local lymph node assay (LLNA) is a predictive in vivo method to provide estimates of relative potency and to contribute to risk assessment/risk management regarding skin sensitizing potency of chemicals and formulations as a stand-alone alternative test. In addition, LLNA is relatively rapid and cost-effective compared to the Buehler method (Guinea pig test), and confers important animal welfare benefits. CBA/J and BALB/c strains are widely commercially available and have been evaluated by formal LLNA validation studies. However, the LLNA method using BrdU with ELISA, unlike other LLNA methods (OECD TG 429, 442 A, 442B), has not been previously validated. Therefore, in this study a validation method was performed to evaluate if the LLNA:BrdU-ELISA method could also be used to identify sensitizers among chemicals listed in OECD TG 429 using CBA/J and BALB/c strains. Here, we newly found that the LLNA:BrdU-ELISA validation method correctly identified 12 of 13 sensitizers in the BALB/c, 11 of 13 sensitizers in the CBA/J, and 3 of 5 non-sensitizers were identified in the two strains. Collectively, we found that the results of LLNA:BrdU-ELISA method provide a similar level of performance for accuracy and sensitivity in two mouse strains BALB/c and CBA/J.

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.

iTRAQ-based proteomic profiling, pathway analyses, and apoptotic mechanism in the Antarctic copepod Tigriopus kingsejongensis in response to ultraviolet B radiation.

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.

Design and Testing of Augmented Reality-Based Fluorescence Imaging Goggle for Intraoperative Imaging-Guided Surgery.

The different pathways between the position of a near-infrared camera and the user's eye limit the use of existing near-infrared fluorescence imaging systems for tumor margin assessments. By utilizing an optical system that precisely matches the near-infrared fluorescence image and the optical path of visible light, we developed an augmented reality (AR)-based fluorescence imaging system that provides users with a fluorescence image that matches the real-field, without requiring any additional algorithms. Commercial smart glasses, dichroic beam splitters, mirrors, and custom near-infrared cameras were employed to develop the proposed system, and each mount was designed and utilized. After its performance was assessed in the laboratory, preclinical experiments involving tumor detection and lung lobectomy in mice and rabbits by using indocyanine green (ICG) were conducted. The results showed that the proposed system provided a stable image of fluorescence that matched the actual site. In addition, preclinical experiments confirmed that the proposed system could be used to detect tumors using ICG and evaluate lung lobectomies. The AR-based intraoperative smart goggle system could detect fluorescence images for tumor margin assessments in animal models, without disrupting the surgical workflow in an operating room. Additionally, it was confirmed that, even when the system itself was distorted when worn, the fluorescence image consistently matched the actual site.

Complete mitochondrial genomes of two marine monogonont rotifer Brachionus manjavacas strains.

The complete mitochondrial genomes of Brachionus manjavacas German strain were 10,721 bp (mitochondrial DNA I) and 12,274 bp (mitochondrial DNA II) in size, while the complete mitochondrial genomes of B. manjavacas Australian strain were 10,889 bp (mitochondrial DNA I) and 12,443 bp (mitochondrial DNA II) in size. Of 12 protein-coding genes (PCGs), 99.6% of amino acid sequences were identical between the two strains. Of 12 PCGs of both B. manjavacas strains, three genes (ND1, ATP6, and ND5) had incomplete stop codon T. Furthermore, ATA was the start codon for ND4, ND5, and CO3 genes, whereas that for other PCGs was ATG in both strains. The base compositions of 12 PCGs in the B. manjavacas strains were similar, indicating that the mitochondrial genome of the two strains was structurally conserved over evolution. The gene structure and its orientation of 12 PGCs of B. manjavacas strains were identical, as shown in other marine Brachionus rotifers and the freshwater Brachionus rotifers, while the freshwater rotifer B. calyciflorus had an additional cytochrome b gene in the mitochondrial DNA I.

The genome of the minute marine rotifer Proales similis: Genome-wide identification of 401 G protein-coupled receptor (GPCR) genes.

The minute marine rotifer Proales similis is a potential model species for ecotoxicological and ecophysiological studies. Therefore, the provision of whole-genome data for P. similis is an easy way to deepen understanding of the molecular mechanisms involved in response to various environmental stressors. In this research, we assembled the whole-genome sequence (32.7 Mb total, N50 = 2.42 Mb) of P. similis, consisting of 15 contigs with 10,785 annotated genes. To understand the ligand-receptor signaling pathway in rotifers in response to environmental cues, we identified 401 G protein-coupled receptor (GPCR) genes in the P. similis genome and compared them with those from other species. The 401 full-length GPCR genes were classified into five distinct classes: A (363), B (18), C (7), F (2), and other (11). Most GPCR gene families have undergone sporadic evolutionary processes. However, some classes were highly conserved between species. Overall, this result provides new information about GPCR-based signaling pathways and the evolution of GPCRs in the minute rotifer P. similis, and it expands our knowledge of ligand-receptor signaling pathways in response to various environmental cues.

The genome of the euryhaline rotifer Brachionus paranguensis: Potential use in molecular ecotoxicology.

Brachionus spp. rotifers have been proposed as model organisms for ecotoxicological studies. We analyzed the whole-genome sequence of B. paranguensis through NextDenovo, resulting in a total length of 106.2 Mb and 71 contigs. The N50 and the GC content were 4.13 Mb and 28%, respectively. A total of 18,501 genes were predicted within the genome of B. paranguensis. Prominent detoxification-related gene families of phase I and II detoxifications have been investigated. In parallel with other Brachionus rotifers, high gene expansion was observed in CYP clan 3 and GST sigma class in B. paranguensis. Moreover, species-specific expansion of sulfotransferase (SULTs) and gain of UDP-glucuronosyltransferases (UGTs) through horizontal gene transfer has been specifically found within B. plicatilis complex. This whole-genome analysis of B. paranguensis provides a basis for molecular ecotoxicological studies and provides useful information for comparative studies of the evolution of detoxification mechanisms in Brachionus spp.