Acquisition and Analysis Methods for Hi-C Data from Medaka Early Embryos.

The three-dimensional (3D) structure of the genome undergoes dynamic changes during the developmental process. While Hi-C is a technique that enables the acquisition of genome 3D structure data across various species and cell types, existing Hi-C analysis programs may face challenges in detecting and comparing structures effectively depending on the characteristics of the genome or cell type. Here, we describe a method for acquiring Hi-C data from medaka early embryos and quantifying the structural changes during the developmental process.

Water-accommodated fractions of heavy and light oils impact DNA integrity, embryonic development, and immune system of Japanese medaka at early life stages.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous contaminants generally found in complex mixtures. PAHs are known to cause pleiotropic effects on living organisms, including developmental defects, mutagenicity, carcinogenicity and immunotoxicity, and endocrine disruptions. The main goal of this study is to evaluate the toxicity of water-accommodated fractions (WAFs) of oils in two life stages of the Japanese medaka, larvae and juveniles. The deleterious effects of an acute exposure of 48 h to two WAFs from Arabian light crude oil (LO) and refined oil from Erika (HO) were analyzed in both stages. Relevant endpoints, including ethoxy resorufin-O-deethylase (EROD) activity, DNA damage (Comet assay), photomotor response, and sensitivity to nervous necrosis virus (NNV) infection, were investigated. Larvae exposed to both oil WAFs displayed a significant induction of EROD activity, DNA damage, and developmental anomalies, but no behavioral changes. Deleterious effects were significantly increased following exposure to 1 and 10 µg/L of LO WAFs and 10 µg/L of HO WAFs. Larval infection to NNV induced fish mortality and sharply reduced reaction to light stimulation. Co-exposure to WAFs and NNV increased the mortality rate, suggesting an impact of WAFs on fish defense capacities. WAF toxicity on juveniles was only observed following the NNV challenge, with a higher sensitivity to HO WAFs than to LO WAFs. This study highlighted that environmentally realistic exposure to oil WAFs containing different compositions and concentrations of oil generated high adverse effects, especially in the larval stage. This kind of multi-marker approach is particularly relevant to characterize the toxicity fingerprint of environmental mixtures of hydrocarbons and PAHs.

Modular control of vertebrate axis segmentation in time and space.

How the timing of development is linked to organismal size is a longstanding question. Although numerous studies have reported a correlation of temporal and spatial traits, the developmental or selective constraints underlying this link remain largely unexplored. We address this question by studying the periodic process of embryonic axis segmentation in-vivo in Oryzias fish. Interspecies comparisons reveal that the timing of segmentation correlates to segment, tissue and organismal size. Segment size in turn scales according to tissue and organism size. To probe for underlying causes, we genetically hybridised two closely related species. Quantitative analysis in ~600 phenotypically diverse F2 embryos reveals a decoupling of timing from size control, while spatial scaling is preserved. Using developmental quantitative trait loci (devQTL) mapping we identify distinct genetic loci linked to either the control of segmentation timing or tissue size. This study demonstrates that a developmental constraint mechanism underlies spatial scaling of axis segmentation, while its spatial and temporal control are dissociable modules.

An evolutionarily distinct Hmgn2 variant influences shape recognition in Medaka Fish.

Protein sequence diversification significantly impacts physiological traits. In this study, using medaka fish (Oryzias latipes), we identify a novel protein variant affecting shape preference behavior. Re-analysis of sequencing data reveals that LOC101156433 encodes a unique Hmgn2 variant with unusual subnuclear localization, clustered separately from the Hmgn2 clades of other species. Medaka mutants with this variant showed reduce telencephalic regions and altered shape preference, suggesting a link between protein sequence variation and behavioral changes. Additionally, this Hmgn2 variant is common in Acanthopterygii fishes, which are adapted to a variety of environments, indicating its potential evolutionary significance. Our findings highlight the relationship between amino acid sequence variation and the development of new molecular and behavioral adaptations, providing insights into the visual shape perception system in fish.

Evolution of cardiac tissue and flow mechanics in developing Japanese Medaka.

The effects of pressure drop across cardiac valve cushion regions and endocardial wall strain in the early developmental stages of a teleost species heart are poorly understood. In the presented work, we utilize microscale particle image velocimetry (µPIV) flow measurements of developing medaka hearts from 3 to 14 dpf (n = 5 at each dpf) to quantify the pressure field and endocardial wall strain. Peak pressure drop at the atrioventricular canal (ΔPAVC) and outflow tract (ΔPOFT) show a steady increase with fish age progression. Pressure drops when non-dimensionalized with blood viscosity and heart rate at each dpf are comparable with measurements in zebrafish hearts. Retrograde flows captured at these regions display a negative pressure drop. A novel metric, Endocardial Work (EW), is introduced by analyzing the ΔPAVC-strain curves, which is a non-invasive measure of work required for ventricle filling. EW is a metric that can differentiate between the linear heart stage (< 100 Pa-%), cardiac looped chamber stage (< 300 Pa-%), and the fully formed chamber stage (> 300 Pa-%).

Negligible additive effect of environmental concentrations of fragmented polyethylene terephthalate microplastics on the growth and reproductive performance of Java medaka exposed to 17ß-estradiol and bisphenol A.

To investigate whether environmental concentrations of fragmented polyethylene terephthalate (PET) microplastics (MPs) have additional or combined effects on endocrine-disrupting activity, Java medaka (Oryzias javanicus) were exposed to 17ß-estradiol (E2; 5, 10, 50, and 100 ng L-1), bisphenol A (BPA; 5, 10, 50, and 100 µg L-1), and E2 and BPA combined with PET MPs (1 and 100 particles L-1) for 200 days. The growth parameters, such as body length and weight, were significantly decreased by the highest concentrations of E2 and BPA. A significant reduction in egg production was observed in female fish exposed to BPA, with an additive toxic effect of PET MPs. A female-biased sex ratio was observed in fish exposed to both chemicals. Exposure to E2 significantly increased the hepatosomatic index (HSI) in both sexes, while no significant effect was observed in the gonadosomatic index (GSI). Exposure to BPA significantly increased the HSI in female fish and decreased the GSI in both sexes of fish. An additive effect of PET MPs was observed on the GSI value of female exposed to BPA. Significant elevations in vitellogenin (VTG) levels were observed in both sexes due to exposure to E2 and BPA. Additive effects of PET MPs were observed on VTG levels in males exposed to E2 and BPA. Taken together, even long-term treatment with PET MPs induced only a negligible additive effect on the endocrine-disrupting activity in Java medaka at environmentally relevant concentrations.

Microglial activation and pyroptosis induced by nano-TiO2 in marine medaka brain.

Recently, nano-titanium dioxide (nano-TiO2) has been widely distributed over surface water. However, there are few reports on its effects on the central nervous system of fish. In this study, we investigated whether nano-TiO2 enters the medaka brain after exposure and its effect on the brain. Marine medaka brains were examined after exposure to 0.01 g/L nano-TiO2 for 3, 10, and 20 d. Nano-TiO2-like particles were found in the telencephalon of treated fish. There was no obvious brain histopathological injury. The number of irregular mitochondria with absent cristae increased. Gene expression of the apoptosis-related genes, casp8, bcl2b, and bax, decreased significantly in the nano-TiO2 group at 3 d. In contrast, the pyroptosis-related genes, gsdmeb and casp1, and inflammation-related factor, il18, increased significantly. As an activated microglia marker, mRNA expression of cd68 increased significantly in the nano-TiO2 treated group. Moreover, CD68 protein expression also increased significantly at 10 d. Altogether, we show that nano-TiO2 can alter mitochondrial morphology in the telencephalon of medaka, leading to microglial activation and pyroptosis.

Contrasting the distribution kinetics of microplastics and nanoplastics in medaka following exposure and depuration.

Direct ingestion of micro/nanoplastics (MNPs) results in significant accumulation in gastrointestinal (GI) tract of fish. The breathing process of fish makes MNPs easily retained in their gills. However, the uptake of MNPs in other fish organs remains largely unknown, let alone their kinetic processes. Herein, microplastics (MPs) and nanoplastics (NPs) in vivo imaging and precise quantification in various tissues (GI tract, gill, liver, brain, eye, and skin) of seawater (SW)- and freshwater (FW)- acclimated medaka Oryzias melastigma were achieved at an environmentally relevant concentration. Subsequently, the distribution kinetics of MNPs was investigated over a 96-h uptake and 48-h depuration period. MNPs were quickly and mostly captured in GI tract and gill of O. melastigma, and then transferred to liver and brain likely via blood circulation. Such transport was more efficient for NPs as compared to MPs, as evidenced by the consistently higher bioconcentration factors in both SW and FW conditions. The detection of MNPs in eye and skin of O. melastigma was more of an adsorption process, although the specific mechanisms of adsorption and absorption process can hardly be clearly differentiated. This study presented distribution kinetics of MNPs in O. melastigma and highlighted their possible transportation among tissues.

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.

In Vivo Injection of Reversible Optically Controlled Short Interfering RNA into Japanese Medaka Embryos (Oryzias latipes) to Regulate Gene Silencing.

Photoswitchable ortho-functionalized tetrafluorinated azobenzene-modified siRNAs (F-azo-siRNAs) were synthesized using solid-phase phosphoramidite chemistry. The activity of an F-azo-siRNA targeting enhanced green fluorescence protein (eGFP) in transgenic (Tg) Japanese Medaka (Oryzias latipes) was reversibly photocontrolled with blue (470 nm) and green (530 nm) light, to activate and inactivate the siRNA, respectively. This study highlights the first reversible in vivo study with photoswitchable siRNA. Controlling siRNA function reversibly in vivo could open new opportunities for biotech research to better understand gene function and cellular mechanisms.

Leptin A deficiency affecting the mitochondrial dynamics of aged oocytes in medaka (Oryzias latipes).

Mitochondrial dysfunction and metabolic disorder have been associated to age-related subfertility, however, the precise molecular mechanism controlling the development of fertile oocytes in aging females remains elusive. Leptin plays an important role in the maintenance of energy homeostasis, as both excessive or insufficient levels can affect the body weight and fertility of mice. Here, we report that leptin A deficiency affects growth and shortens reproductive lifespan by reducing fertility in medaka (Oryzias latipes). Targeted disruption of lepa (lepa-/-) females reduced their egg laying and fertility compared to normal 3-month-old females (lepa+/+ sexual maturity), with symptoms worsening progressively at the age of 6 months and beyond. Transcriptomic analysis showed that differentially expressed genes involved in metabolic and mitochondrial pathways were significantly altered in lepa-/- ovaries compared with the normal ovaries at over 6 months old. The expression levels of the autophagy-promoting genes ulk1a, atg7 and atg12 were significantly differentiated between normal and lepa-/- ovaries, which were further confirmed by quantitative polymerase chain reaction analysis, indicating abnormal autophagy activation and mitochondrial dysfunction in oocyte development lacking lepa. Transmission electron microscopy observations further confirmed these mitochondrial disorders in lepa-deficient oocytes. In summary, these research findings provide novel insights into how leptin influences female fertility through mitochondrial-mediated oocyte development.

Conversion of Retinoids along the Marine Food Chain Contributes to Adverse Impacts on the Spine, Liver, and Intestinal Health of the Marine Medaka (Oryzias melastigma).

Marine microalgae serve as an aquaculture bait. To enhance algal cell growth and breeding profits, high-intensity light conditions are standard for cultivating bait microalgae, potentially altering microalgal metabolite production. This research revealed that Thalassiosira pseudonana, when subjected to high-intensity light conditions, accumulated significant quantities of retinal (RAL) that transferred through the food chain and transformed into all-trans retinoic acid (atRA) in marine medaka. The study further explored the toxic effects on individual fish and specific tissues, as well as the mechanisms behind this toxicity. The accumulation of atRA in the liver, intestine, and spinal column resulted in structural damage and tissue inflammation, as well as oxidative stress. It also down-regulated the gene transcription levels of key pathways involved in immune function and growth. Furthermore, it disrupted the homeostasis of the intestinal microbial communities. The implications for wildlife and human health, which are influenced by the regulation of microalgal metabolite accumulation and their transfer via the food chain, require further investigation and could hold broader significance.

Rapid body colouration changes in Oryzias celebensis as a social signal influenced by environmental background.

Rapid body colouration changes in some animals, such as chameleons and octopuses, serve dual functions: camouflage and intraspecific communication. It has been hypothesized that these colouration changes originally evolved to provide camouflage and subsequently were co-opted as social signals; however, experimental model systems that are suitable for studying such evolutionary processes are limited. Here, we investigated the relationship between rapid colouration changes of the blackened markings and aggressive behaviours in male Oryzias celebensis, an Indonesian medaka fish, under triadic relationships (two males and one female) or three males conditions with two different environmental backgrounds. In an algae-covered tank, mimicking the common laboratory rearing conditions, males with blackened markings exhibited more frequent attacks towards different conspecific individuals compared with non-blackened males and females. The blackened males were seldom attacked by non-blackened males and females. By contrast, neither aggressive behaviours nor black colouration changes were observed in the transparent background condition with a brighter environment. These indicated that the blackened markings in O. celebensis serve as a social signal depending on the environmental backgrounds. Considering that such colouration changes for camouflage are widely conserved among teleost fishes, the traits are likely to be co-opted for displaying social signals in O. celebensis.

Stereoselective Bioconcentration and Neurotoxicity of Perfluoroethylcyclohexane Sulfonate in Marine Medaka.

Perfluoroethylcyclohexane sulfonate (PFECHS) is an emerging per- and polyfluoroalkyl substance used to replace perfluorooctane sulfonate (PFOS), mainly in aircraft hydraulic fluids. However, previous research indicates the potential neurotoxicity of this replacement chemical. In this study, marine medaka (Oryzias melastigma) was exposed to environmentally relevant concentrations of PFECHS (concentrations: 0, 0.08, 0.26, and 0.91 µg/L) from the embryonic stage for 90 days. After exposure, the brain and eyes of the medaka were collected to investigate the bioconcentration potential of PFECHS stereoisomers and their effects on the nervous systems. The determined bioconcentration factors (BCFs) of PFECHS ranged from 324 ± 97 to 435 ± 89 L/kg and from 454 ± 60 to 576 ± 86 L/kg in the brain and eyes of medaka, respectively. The BCFs of trans-PFECHS were higher than those of cis-PFECHS. PFECHS exposure significantly altered γ-aminobutyric acid (GABA) levels in the medaka brain and disrupted the GABAergic system, as revealed by proteomics, implying that PFECHS can disturb neural signal transduction like PFOS. PFECHS exposure resulted in significant alterations in multiple proteins associated with eye function in medaka. Abnormal locomotion was observed in PFECHS-exposed medaka larvae, which was rescued by adding exogenous GABA, suggesting the involvement of disrupted GABA signaling pathways in PFECHS neurotoxicity.

Assessment of environmental exposure to betamethasone on the reproductive function of female Japanese medaka (Oryzias latipes).

Betamethasone has been extensively used in medicine in recent years and poses potential hazards to aquatic organisms. This study investigated the reproductive toxic effects of betamethasone exposure in fish, employing female Japanese medaka (Oryzias latipes) as a model. Betamethasone exposure at environmentally relevant concentrations (0, 20, 200, and 2000 ng/L) for a period of 15 weeks resulted in its high accumulation in the ovary, leading to abnormal oogenesis in female Japanese medaka. The production of gonadotropins (LH and FSH) in the pituitary gland was inhibited, and sex steroid biosynthesis in the ovary was significantly influenced at the transcriptional level. The imbalance of androgens and estrogens resulted in a decrease in the E2/T ratio and hepatic VTG synthesis, and the suppression of estrogen receptor signaling was also induced. Furthermore, betamethasone exposure delayed spawning and reduced fertility in the F0 generation, and had detrimental effects on the fertilization rate and hatchability of the F1 generation. Our results showed that environmental betamethasone had the potential to adversely affect female fertility and steroid hormone dynamics in fish.

Neuropeptide Y receptor Y8b (npy8br) regulates feeding and digestion in Japanese medaka (Oryzias latipes) larvae: evidence from gene knockout. / 神经肽受体Y8b（npy8br）调节日本青鳉（Oryzias latipes）仔鱼的摄食和消化： æ¥è‡ªåŸºå› æ•²é™¤çš„è¯æ®.

Neuropeptide Y receptor Y8 (NPY8R) is a fish-specific receptor with two subtypes, NPY8AR and NPY8BR. Changes in expression levels during physiological processes or in vivo regulation after ventricular injection suggest that NPY8BR plays an important role in feeding regulation; this has been found in only a few fish, at present. In order to better understand the physiological function of npy8br, especially in digestion, we used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology to generate npy8br-/- Japanese medaka (Oryzias latipes). We found that the deletion of npy8br in medaka larvae affected their feeding and digestion ability, ultimately affecting their growth. Specifically, npy8br deficiency in medaka larvae resulted in decreased feed intake and decreased expression levels of orexigenic genes (npy and agrp). npy8br-/- medaka larvae fed for 10 d (10th day of feeding) still had incompletely digested brine shrimp (Artemia nauplii) in the digestive tract 8 h after feeding, the messenger RNA (mRNA) expression levels of digestion-related genes (amy, lpl, ctra, and ctrb) were significantly decreased, and the activity of amylase, trypsin, and lipase also significantly decreased. The deletion of npy8br in medaka larvae inhibited the growth and significantly decreased the expression of growth-related genes (gh and igf1). Hematoxylin and eosin (H&E) sections of intestinal tissue showed that npy8br-/- medaka larvae had damaged intestine, thinned intestinal wall, and shortened intestinal villi. So far, this is the first npy8br gene knockout model established in fish and the first demonstration that npy8br plays an important role in digestion.

Metabolome analysis revealed the critical role of betaine for arsenobetaine biosynthesis in the marine medaka (Oryzias melastigma).

Arsenobetaine (AsB), a non-toxic arsenic (As) compound found in marine fish, structurally resembles betaine (GB), a common methyl donor in organisms. This study investigates the potential role of GB in AsB synthesis in marine medaka (Oryzias melastigma) using metabolomic analysis. Dietary exposure to arsenate (As(V)) and varying GB concentrations (0.05% and 0.1% in diets) increased total As and AsB bioaccumulation, particularly in marine medaka muscle. Metabolomic analysis revealed that GB played a crucial role in promoting up-regulation in methylthioadenosine (MTA) by modulating the methionine cycle and down-regulation in glutathione (GSH) by modulating the glutathione cycle. Methionine metabolism and GSH, potentially binding again to exogenous GB, could synchronously produce more non-toxic AsB. Combining verification experiments of differential metabolites of Escherichia coli in vitro, GB, GSH, S-adenosylmethionine (SAM), and arsenocholine (AsC) entered methionine and glutathione metabolism pathways to generate more AsB. These findings underscore the GB's crucial regulatory role in modulating the synthesis of AsB. This study provides vital insights into the interplay between the structural analogs GB and AsB, offering specific strategies to enhance the detoxification mechanisms of marine fish in As-contaminated environments.

A second hit by PFOS exposure exacerbated developmental defects in medaka embryos with a history of ancestral BPA exposure.

Bisphenol-A (BPA), a known endocrine-disrupting chemical (EDC) in plastics and resins, has been found to induce heritable health effects in fish and mammals, affecting directly exposed individuals and indirectly their progenies in subsequent generations. It is not clearly understood if subsequent generations of the BPA-exposed ancestors have increased sensitivity to the second hit by the chemicals of emerging concern. To understand this, the present study examined the effects of developmental exposure to perfluorooctanesulfonic acid (PFOS), which has been a global contaminant recently, in embryos whose ancestors were exposed to BPA. Two lineages of medaka (Oryzias latipes) were established: 1) the BPA lineage in which the F0 generation was exposed to 10 µg/L BPA during early development and 2) the control lineage with no BPA exposure in the F0 generation. These lineages were raised up to the F4 generation without further exposure. The embryos of the F4 generation were exposed to PFOS at 0, 0.002, 0.02, 0.2, 2, and 20 mg/L concentrations. Early developmental defects resulting in mortality, delayed hatching, teratogenic phenotypes, and altered gene expression were examined in both lineages. The expression level of genes encoding DNA methyltransferases and genes responsible for oxidative stress defense were determined. Following environmentally relevant PFOS exposure, organisms with a history of BPA exposure displayed significant changes in all categories of developmental defects mentioned above, including increased expression of genes related to oxidative stress, compared to individuals without BPA exposure. The present study provides initial evidence that a history of ancestral BPA exposure can alter sensitivity to developmental disorders following the second hit by PFOS exposure. The variable of ancestral BPA exposure could be considered in mechanistic, medical, and regulatory toxicology, and can also be applied to holistic environmental equity research.

Effects of whole life-cycle exposure to carbaryl on reproduction of female marine medaka (Oryzias melastigma) and their offspring.

Carbaryl is widely used as a highly effective insecticide which harms the marine environment. This study aimed to assess the reproductive toxicity of chronic carbaryl exposure on female marine medaka and their female offspring. After a 180-day exposure from embryonic period to adulthood, females exhibited reduced attraction to males, decreased ovulation, increased gonadosomatic index and a higher proportion of mature and atretic follicles. These reproductive toxic effects of carbaryl may stem from changes in hormone levels and transcription levels of key genes along the HPG axis. Furthermore, maternal carbaryl exposure had detrimental effects on the offspring. F1 females showed the reproductive disorders similar to those observed in F0 females. The significant changes in the transcription levels of DNA methyltransferase and demethylase genes in the F0 and F1 generations of ovaries indicate changes in their DNA methylation levels. The changes in DNA methylation levels in F1 female marine medaka may lead to changes in the expression of certain reproductive key genes, such as an increase in the transcription level of cyp19a, which may be the reason for F1 reproductive toxicity. These findings indicate that maternal exposure may induce severe generational toxicity through alterations in DNA methylation levels. This study assesses the negative impacts of whole life-cycle carbaryl exposure on the reproductive and developmental processes of female marine medaka and its female offspring, while offering data to support the evaluation of the ecological risk posed by carbaryl in marine ecosystems.

Chronic exposure to environmental concentrations of benzo[a]pyrene causes multifaceted toxic effects of developmental compromise, redox imbalance, and modulated transcriptional profiles in the early life stages of marine medaka (Oryzias melastigma).

Polycyclic aromatic hydrocarbons (PAHs) accumulate and integrate into aquatic environments, raising concerns about the well-being and safety of aquatic ecosystems. Benzo[a]pyrene (BaP), a persistent PAH commonly detected in the environment, has been extensively studied. However, the broader multifaceted toxicity potential of BaP on the early life stages of marine fish during chronic exposure to environmentally relevant concentrations needs further exploration. To fill these knowledge gaps, this study assessed the in vivo biotoxicity of BaP (1, 4, and 8 µg/L) in marine medaka (Oryzias melastigma) during early development over a 30-day exposure period. The investigation included morphological, biochemical, and molecular-level analyses to capture the broader potential of BaP toxicity. Morphological analyses showed that exposure to BaP resulted in skeletal curvatures, heart anomalies, growth retardation, elevated mortality, delayed and reduced hatching rates. Biochemical analyses revealed that BaP exposure not only created oxidative stress but also disrupted the activities of antioxidant enzymes. This disturbance in redox balance was further explored by molecular level investigation. The transcriptional profiles revealed impaired oxidative phosphorylation (OXPHOS) and tricarboxylic acid (TCA) cycle pathways, which potentially inhibited the oxidative respiratory chain in fish following exposure to BaP, and reduced the production of adenosine triphosphate (ATP) and succinate dehydrogenase (SDH). Furthermore, this investigation indicated a potential connection to apoptosis, as demonstrated by fluorescence microscopy and histological analyses, and supported by an increase in the expression levels of related genes via real-time quantitative PCR. This study enhances our understanding of the molecular-level impacts of BaP's multifaceted toxicity in the early life stages of marine medaka, and the associated risks.