An integrated transcriptome-microbiome host relationship associated with paraben toxicity in the brackish water flea Diaphanosoma celebensis.

Parabens, a group of alkyl esters of p-hydroxybenzoic acid, have been found in aquatic systems in particular, leading to concerns about their potential impact on ecosystems. This study investigated the effects of three commonly used parabens, methylparaben (MeP), ethylparaben (EtP), and propylparaben (PrP), on the brackish water flea Diaphanosoma celebensis. The results showed that PrP had the most adverse impact on survival rates, followed by EtP and MeP, while MeP and EtP induced significant adverse effects on reproductive performance. A transcriptome analysis revealed significant differential gene expression patterns in response to paraben exposure, with MeP associated with the most significant effects. MeP and EtP exposure produced greater disruption in the microbiota of D. celebensis than did PrP compared with control groups, and we identified eight key microbiota, including Ruegeria and Roseovarius. Correlation analysis between transcriptome and microbiome data revealed key interactions between specific microbiota and host gene expression. Certain microbial taxa were associated with specific genes (e.g. cuticle related genes) and toxicological pathways, shedding light on the complex molecular response and in vivo toxicity effects of parabens. These findings contribute to a deeper understanding of the molecular mechanisms underlying paraben toxicity and highlight the importance of considering the ecological impact of chemical contaminants in aquatic ecosystems.

Comparative Microbiome Analysis of Artemia spp. and Potential Role of Microbiota in Cyst Hatching.

Brine shrimp (Artemia spp.) is a significant factor in determining aquaculture production. Since the microbiota of Artemia can colonize the gut in larvae, various microorganisms transmitted from Artemia can affect host larval health. Although the microbiota composition of Artemia would be essential in determining aquaculture productivity, our understanding on microbiome of Artemia is still insufficient. Through our study, we identified the species of Artemia cysts supplied by three different manufacturers (P1, P2, and P3) with investigation of size and hatching efficiency. The species of Artemia from P1 was identified as A. tibetiana, and P2 and P3 was A. franciscana. A. tibetiana hatched from the P1 cysts had the largest body size with the lowest hatching rate. Furthermore, we conducted a comprehensive analysis of the microbiome present in the rearing water and the nauplius whole body from each product. We observed specific microbiota compositions, both beneficial and harmful, depending on the product types and the sample types. Additionally, we found that the microbiota composition in the rearing water was associated with the manufacturing environment, while the compositions in the nauplius whole body were species-specific. Notably, we discovered that an extract containing microbiota from the nauplius sample of P3 increased the hatching rate of A. tibetiana, indicating a positive role in Artemia culture. These findings demonstrate that the microbial communities present in Artemia vary according to the product and/or species, underscoring their significance in aquaculture production.

The combination of osimertinib with Raf inhibitor overcomes osimertinib resistance induced by KRAS amplification in EGFR-mutated lung cancer cells.

Osimertinib is a third-generation epidermal growth factor receptor (EGFR)1 tyrosine kinase inhibitor (TKI) approved for the treatment of EGFR-positive patients exhibiting a T790 M resistance mutation after treatment with an earlier generation of EGFR TKIs. However, resistance to osimertinib inevitably develops despite its efficacy, and the resistance mechanisms are complex and not fully understood. We established cell lines with acquired resistance to osimertinib from gefitinib- or erlotinib-resistant NSCLC cells using a dose-escalation method, and found that they had upregulated levels of phosphorylated ERK1/2. Targeted next-generation sequencing of 143 genes was performed, and interestingly, amplification of KRAS was observed in osimertinib-resistant cells. Transfection of siRNA against the KRAS gene notably reduced the activation of ERK1/2 and AKT and significantly enhanced the induction of apoptosis by osimertinib treatment in osimertinib-resistant cells. LY3009120, a RAF inhibitor, showed a significant synergistic effect with osimertinib on apoptotic cell death in osimertinib-resistant cells. Combined treatment with osimertinib and LY3009120 also demonstrated remarkable synergistic anti-tumor activity in mouse xenografts of these cells. This could be a potential new treatment option for KRAS amplification-induced osimertinib failure.

Emerging nano-scale delivery systems for the treatment of osteoporosis.

Osteoporosis is a pathological condition characterized by an accelerated bone resorption rate, resulting in decreased bone density and increased susceptibility to fractures, particularly among the elderly population. While conventional treatments for osteoporosis have shown efficacy, they are associated with certain limitations, including limited drug bioavailability, non-specific administration, and the occurrence of adverse effects. In recent years, nanoparticle-based drug delivery systems have emerged as a promising approach for managing osteoporosis. Nanoparticles possess unique physicochemical properties, such as a small size, large surface area-to-volume ratio, and tunable surface characteristics, which enable them to overcome the limitations of conventional therapies. These nanoparticles offer several advantages, including enhanced drug stability, controlled release kinetics, targeted bone tissue delivery, and improved drug bioavailability. This comprehensive review aims to provide insights into the recent advancements in nanoparticle-based therapy for osteoporosis. It elucidates the various types of nanoparticles employed in this context, including silica, polymeric, solid lipid, and metallic nanoparticles, along with their specific processing techniques and inherent properties that render them suitable as potential drug carriers for osteoporosis treatment. Furthermore, this review discusses the challenges and future suggestions associated with the development and translation of nanoparticle drug delivery systems for clinical use. These challenges encompass issues such as scalability, safety assessment, and regulatory considerations. However, despite these challenges, the utilization of nanoparticle-based drug delivery systems holds immense promise in revolutionizing the field of osteoporosis management by enabling more effective and targeted therapies, ultimately leading to improved patient outcomes.

Toxic effects of the wastewater produced by underwater hull cleaning equipment on the copepod Tigriopus japonicus.

Unmanaged disposal of wastewater produced by underwater hull cleaning equipment (WHCE) is suspected to induce toxic effects to marine organisms because wastewater contains several anti-fouling compounds. To investigate the effects of WHCE on marine copepod, we examined the toxicity on life parameters (e.g. mortality, development, and fecundity) and gene expression changes of Tigriopus japonicus as model organism. Significant mortality and developmental time changes were observed in response to wastewater. No significant differences in fecundity were observed. Transcriptional profiling with differentially expressed genes from WHCE exposed T. japonicus showed WHCE may induce genotoxicity associated genes and pathways. In addition, potentially neurotoxic effects were evident following exposure to WHCE. The findings suggest that wastewater released during hull cleaning should be managed to reduce physiological and molecular deleterious effects in marine organisms.

Regional analgesia techniques for lumbar spine surgery: a frequentist network meta-analysis.

BACKGROUND: Various regional analgesia techniques are used to reduce postoperative pain in patients undergoing lumbar spine surgery. Traditionally, wound infiltration (WI) with local anesthetics has been widely used by surgeons. Recently, other regional analgesia techniques, such as the erector spinae plane block (ESPB) and thoracolumbar interfascial plane (TLIP) block, are being used for multimodal analgesia. The authors aimed to determine the relative efficacy of these using a network meta-analysis. MATERIALS AND METHODS: The authors searched PubMed, EMBASE, the Cochrane Controlled Library, and Google Scholar databases to identify all randomized controlled trials that compared the analgesic efficacy of the following interventions: ESPB, TLIP block, WI technique, and controls. The primary endpoint was postoperative opioid consumption during the first 24 hours after surgery, while the pain score, estimated postoperatively at three different time periods, was the secondary objective. RESULTS: The authors included 34 randomized controlled trials with data from 2365 patients. TLIP showed the greatest reduction in opioid consumption compared to controls [mean difference (MD) =-15.0 mg; 95% CI: -18.8 to -11.2]. In pain scores, TLIP had the greatest effect during all time periods compared to controls (MD=-1.9 in early, -1.4 in middle, -0.9 in late). The injection level of ESPB was different in each study. When only surgical site injection of ESPB was included in the network meta-analysis, there was no difference compared with TLIP (MD=1.0 mg; 95% CI: -3.6 to 5.6). CONCLUSIONS: TLIP showed the greatest analgesic efficacy after lumbar spine surgery, in terms of postoperative opioid consumption and pain scores, while ESPB and WI are also alternative analgesic options for these surgeries. However, further studies are needed to determine the optimal method of providing regional analgesia after lumbar spine surgery.

Cerebral chronic hypoperfusion in mice causes premature aging of the cerebrovasculature.

Chronic cerebral hypoperfusion (CCH) is the main characteristic of an aged brain showing cerebrovascular alterations. Our previous study that the morphological changes in the pial arteries accompany a decrease in the cerebral blood flow in aged mouse brains, and it raises the question of whether artificial CCH can induce the same changes in brain vessel morphology. Here, we examined the effect of CCH on cerebrovascular morphology. Using a microcoil-induced chronic cerebral hypoperfusion (MCH) model, we induced CCH for 8 and 12 weeks. The cerebrovasculature morphology was evaluated using three-dimensional vessel analysis and compared with that of aging mice. We found the morphological changes in brain vessels of MCH mice, indicating that the CCH can induce cerebrovascular alteration. However, the changes in brain vessel morphology in the MCH mice were different in detail from those in the aging mice. Aging mice showed an increase in vessel tortuosity and thinned string vessels; MCH mice mainly showed thinned string vessels. This suggests that CCH may be a cause of age-related cerebrovascular alterations.

Contrasting toxicity of polystyrene nanoplastics to the rotifer Brachionus koreanus in the presence of zinc oxide nanoparticles and zinc ions.

Emerging contaminants such as nanoplastics and nanoparticles likely experience similar environmental behaviours, fate and effects but our knowledge of their combined toxicity is scanty. This study, therefore, investigated the joint toxicity of polystyrene nanoplastics (PNPs) and zinc oxide nanoparticles (ZnO-NPs) to an ecologically important rotifer Brachionus koreanus, and compared with the joint toxicity of PNPs and Zn ions (Zn-IONs from ZnSO4·7H2O). With increasing concentration, ZnO-NPs formed significant agglomeration with PNPs for up to 1.3 times of the original hydrodynamic size of ZnO-NPs, alongside doubling in their sedimentation and thereby losing 58% of their released Zn ions. In contrast, the availability of Zn-IONs was less affected by the agglomeration and sedimentation of PNPs, with only a loss of 18% of Zn ions at the highest concentration of PNPs. Consequently, as suggested by Concentration Addition and Independent Action models and the Model Deviation Ratios, ZnO-NPs and PNPs exerted an antagonistic interaction whereas Zn-IONs and PNPs exhibited an additive effect. We also advocate the use of the Nonparametric Response Surface method, which is more useful to predict the toxicity of chemical mixtures with interacting effects. Our findings suggested a potential difference between particle-particle and particle-ion interactions, especially at higher test concentrations, which may eventually affect their toxicity. We, therefore, call for a more systematic evaluation of commonly coexisting chemical mixtures which consist of nanoplastics and manufactured nanomaterials.

Transcriptional and toxic responses to saxitoxin exposure in the marine copepod Tigriopus japonicus.

Saxitoxin (STX) is a highly toxic marine neurotoxin produced by phytoplankton and a growing threat to ecosystems worldwide due to the spread of toxic algae. Although STX is an established sodium channel blocker, the overall profile of transcriptional levels in STX-exposed organisms has yet to be described. Here, we describe a toxicity assay and transcriptome analysis of the copepod Tigriopus japonicus exposed to STX. The half-maximal lethal concentration of STX was 12.35 µM, and a rapid mortality slope was evident at concentrations between 12 and 13 µM. STX induced changes in swimming behavior among the copepods after 10 min of exposure. In transcriptome analysis, gene ontology revealed that the genes involved in nervous system and gene expression were highly enriched. In addition, the congenital neurological disorder and nuclear factor erythroid 2-related factor 2-mediated oxidative stress pathways were identified to be the most significant in network analysis and toxicity pathway analysis, respectively. This study provides valuable information about the effects of STX and related transcriptional responses in T. japonicus.

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.

Effects of dinoflagellate Gymnodinium catenatum on swimming behavior and expression of heat shock protein (hsp) genes in the brine shrimp Artemia franciscana.

To understand the effects of the toxic marine dinoflagellate, Gymnodinium catenatum, on the brine shrimp, Artemia franciscana, we examined the acute toxicity and swimming behavior parameters such as swimming speed, swimming distance, and swimming path trajectory with transcriptional regulation of heat shock protein (hsp) genes in response to G. catenatum exposure. Mortality was not observed in response to G. catenatum. In the case of swimming behavior parameters, swimming speed and swimming distance were significantly decreased (P < 0.05) for 5 min at three concentrations (240, 360, and 600 cells/mL) of G. catenatum, whereas no significant change in swimming path trajectory was observed, suggesting that G. catenatum has potential adverse effects on the swimming behavior of A. franciscana. Additionally, the four A. franciscana-hsp genes (hsp26, hsp40, hsp70, and hsp90) were upregulated in response to G. catenatum. In particular, A. franciscana-hsp40 was significantly upregulated in response to 600 cells/mL G. catenatum, suggesting that A. franciscana-hsp genes are highly associated with cellular defense mechanisms and that A. franciscana-hsp40 is a potential biomarker for G. catenatum exposure. Overall, this study improves our understanding of the effects of G. catenatum on the swimming behavior and cellular defense mechanisms of A. franciscana.

Toxicity mechanisms of arsenic compounds in aquatic organisms.

Arsenic is a toxic metalloid that is widely distributed in the environment due to its persistence and accumulative properties. The occurrence, distribution, and biological effects of arsenic in aquatic environments have been extensively studied. Acute and chronic toxicities to arsenic are associated with fatal effects at the individual and molecular levels. The toxicity of arsenic in aquatic organisms depends on its speciation and concentration. In aquatic environments, inorganic arsenic is the dominant form. While trivalent arsenicals have greater toxicity compared with pentavalent arsenicals, inorganic arsenic can assume a variety of forms through biotransformation in aquatic organisms. Biotransformation mechanisms and speciation of arsenic have been studied, but few reports have addressed the relationships among speciation, toxicity, and bioavailability in biological systems. This paper reviews the modes of action of arsenic along with its toxic effects and distribution in an attempt to improve our understanding of the mechanisms of arsenic toxicity in aquatic organisms.

Hydrophobic Surface Coating Can Reduce Toxicity of Zinc Oxide Nanoparticles to the Marine Copepod Tigriopus japonicus.

Coated zinc oxide nanoparticles (ZnO-NPs) are more commonly applied in commercial products but current risk assessments mostly focus on bare ZnO-NPs. To investigate the impacts of surface coatings, this study examined acute and chronic toxicities of six chemicals, including bare ZnO-NPs, ZnO-NPs with three silane coatings of different hydrophobicity, zinc oxide bulk particles (ZnO-BKs), and zinc ions (Zn-IONs), toward a marine copepod, Tigriopus japonicus. In acute tests, bare ZnO-NPs and hydrophobic ZnO-NPs were less toxic than hydrophilic ZnO-NPs. Analyses of the copepod's antioxidant gene expression suggested that such differences were governed by hydrodynamic size and ion dissolution of the particles, which affected zinc bioaccumulation in copepods. Conversely, all test particles, except the least toxic hydrophobic ZnO-NPs, shared similar chronic toxicity as Zn-IONs because they mostly dissolved into zinc ions at low test concentrations. The metadata analysis, together with our test results, further suggested that the toxicity of coated metal-associated nanoparticles could be predicted by the hydrophobicity and density of their surface coatings. This study evidenced the influence of surface coatings on the physicochemical properties, toxicity, and toxic mechanisms of ZnO-NPs and provided insights into the toxicity prediction of coated nanoparticles from their coating properties to improve their future risk assessment and management.

Phenotypic and transcriptomic responses of the rotifer Brachionus koreanus by single and combined exposures to nano-sized microplastics and water-accommodated fractions of crude oil.

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.

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.

The genome of the marine water flea Diaphanosoma celebensis: Identification of phase I, II, and III detoxification genes and potential applications in marine molecular ecotoxicology.

To assemble the genome of the marine water flea Diaphanosoma celebensis, a sentinel model for marine environmental monitoring, we constructed a high-quality genome using PromethION and HiSeq 2500 platforms. The total length of the assembled genome was 100.08 Mb, with N50 = 2.56 Mb (benchmarking universal single-copy orthologs, 96.9%) and consisted of 179 scaffolds. A total of 15,427 genes were annotated, and orthologous gene clusters in D. celebensis were analyzed and compared with those of the cladocerans Daphnia magna and Daphnia pulex. In addition, phase I, II, and III detoxification gene families of cytochrome P450s, glutathione S-transferases, and ATP-binding cassette were fully identified and revealed lineage-specific gene loss and/or expansion, suggesting that the evolution of detoxification gene families likely modulates fitness and susceptibility in response to environmental stressors. The study improves our understanding of the detoxification-related gene system and should contribute to future studies of molecular ecotoxicology in cladoceran species and their responses to emerging 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.

Tyrosine Phosphorylation of the Kv2.1 Channel Contributes to Injury in Brain Ischemia.

In brain ischemia, oxidative stress induces neuronal apoptosis, which is mediated by increased activity of the voltage-gated K+ channel Kv2.1 and results in an efflux of intracellular K+. The molecular mechanisms underlying the regulation of Kv2.1 and its activity during brain ischemia are not yet fully understood. Here this study provides evidence that oxidant-induced apoptosis resulting from brain ischemia promotes rapid tyrosine phosphorylation of Kv2.1. When the tyrosine phosphorylation sites Y124, Y686, and Y810 on the Kv2.1 channel are mutated to non-phosphorylatable residues, PARP-1 cleavage levels decrease, indicating suppression of neuronal cell death. The tyrosine residue Y810 on Kv2.1 was a major phosphorylation site. In fact, cells mutated Y810 were more viable in our study than were wild-type cells, suggesting an important role for this site during ischemic neuronal injury. In an animal model, tyrosine phosphorylation of Kv2.1 increased after ischemic brain injury, with an observable sustained increase for at least 2 h after reperfusion. These results demonstrate that tyrosine phosphorylation of the Kv2.1 channel in the brain may play a critical role in regulating neuronal ischemia and is therefore a potential therapeutic target in patients with brain ischemia.

The genome of the marine monogonont rotifer Brachionus rotundiformis and insight into species-specific detoxification components in Brachionus spp.

The monogonont rotifer Brachionus spp. have been widely used for ecotoxicological studies because of their advantages as one of the most suitable laboratory experimental species. In the present study, we obtained and assembled the whole genome sequence of the rotifer Brachionus rotundiformis, consisting of 13,612 annotated genes with 213 scaffolds and 58 Mb in total length. Focusing on ecotoxicological aspects, we conducted a comparative genome analysis on the gene families involved in detoxification, including four to six sulfotransferase gene families, seven uridine 5'-diphospho-glucuronosyltransferase gene families, and 58, 61, or 70 ATP-binding cassette genes in the genus Brachionus including Brachionus koreanus and Brachionus plicatilis. Our results suggest that these gene families have undergone a species- and/or lineage-specific evolution in response to the surrounding environmental pressure. Our genome resource for B. rotundiformis would be highly useful for future ecotoxicological studies and also provides a better understanding on the view of evolutionary mechanism of detoxification in the genus Brachionus spp.

Multigenerational Mitigating Effects of Ocean Acidification on In Vivo Endpoints, Antioxidant Defense, DNA Damage Response, and Epigenetic Modification in an Asexual Monogonont Rotifer.

Ocean acidification (OA) is caused by changes in ocean carbon chemistry due to increased atmospheric pCO2 and is predicted to have deleterious effects on marine ecosystems. While the potential impacts of OA on many marine species have been studied, the multigenerational effects on asexual organisms remain unknown. We found that low seawater pH induced oxidative stress and DNA damage, decreasing growth rates, fecundity, and lifespans in the parental generation, whereas deleterious effects on in vivo endpoints in F1 and F2 offspring were less evident. The findings suggest that multigenerational adaptive effects play a role in antioxidant abilities and other defense mechanisms. OA-induced DNA damage, including double-strand breaks (DSBs), was fully repaired in F1 offspring of parents exposed to OA for 7 days, indicating that an adaptation mechanism may be the major driving force behind multigenerational adaptive effects. Analysis of epigenetic modification in response to OA involved examination of histone modification of DNA repair genes and a chromatin immunoprecipitation assay, as Bombus koreanus has no methylation pattern for CpG in its genome. We conclude that DSBs, DNA repair, and histone modification play important roles in multigenerational plasticity in response to OA in an asexual monogonont rotifer.