Enantiospecific Uptake and Depuration Kinetics of Chiral Metoprolol and Venlafaxine in Marine Medaka (Oryzias melastigma): Tissue Distribution and Metabolite Formation.

The increasing use of chiral pharmaceuticals has led to their widespread presence in the environment. However, their toxicokinetics have rarely been reported. Therefore, the tissue-specific uptake and depuration kinetics of two pairs of pharmaceutical enantiomers, S-(-)-metoprolol versus R-(+)-metoprolol and S-(+)-venlafaxine versus R-(-)-venlafaxine, were studied in marine medaka (Oryzias melastigma) during a 28-day exposure and 14-day clearance period. The toxicokinetics of the studied pharmaceuticals, including uptake and depuration rate constants, depuration half-life (t1/2), and bioconcentration factor (BCF), were reported for the first time. The whole-fish results demonstrated a higher S- than R-venlafaxine bioaccumulation potential, whereas no significant difference was observed between S- and R-metoprolol. O-desmethyl-metoprolol (ODM) and α-hydroxy-metoprolol (AHM) were the main metoprolol metabolites identified by suspect screening, and the ratios of ODM to AHM were 3.08 and 1.35 for S- and R-metoprolol, respectively. N,O-Didesmethyl-venlafaxine (NODDV) and N-desmethyl-venlafaxine (NDV) were the main venlafaxine metabolites, and the ratios of NODDV to NDV were 1.55 and 0.73 for S- and R-venlafaxine, respectively. The highest tissue-specific BCFs of the four enantiomers were all found in the eyes, meriting in-depth investigation.

Assembling the perfect bacterial genome using Oxford Nanopore and Illumina sequencing.

A perfect bacterial genome assembly is one where the assembled sequence is an exact match for the organism's genome-each replicon sequence is complete and contains no errors. While this has been difficult to achieve in the past, improvements in long-read sequencing, assemblers, and polishers have brought perfect assemblies within reach. Here, we describe our recommended approach for assembling a bacterial genome to perfection using a combination of Oxford Nanopore Technologies long reads and Illumina short reads: Trycycler long-read assembly, Medaka long-read polishing, Polypolish short-read polishing, followed by other short-read polishing tools and manual curation. We also discuss potential pitfalls one might encounter when assembling challenging genomes, and we provide an online tutorial with sample data (github.com/rrwick/perfect-bacterial-genome-tutorial).

Evolutionary differentiation of androgen receptor is responsible for sexual characteristic development in a teleost fish.

Teleost fishes exhibit complex sexual characteristics in response to androgens, such as fin enlargement and courtship display. However, the molecular mechanisms underlying their evolutionary acquisition remain largely unknown. To address this question, we analyse medaka (Oryzias latipes) mutants deficient in teleost-specific androgen receptor ohnologs (ara and arb). We discovered that neither ar ohnolog was required for spermatogenesis, whilst they appear to be functionally redundant for the courtship display in males. However, both were required for reproductive success: ara for tooth enlargement and the reproductive behaviour eliciting female receptivity, arb for male-specific fin morphogenesis and sexual motivation. We further showed that differences between the two ar ohnologs in their transcription, cellular localisation of their encoded proteins, and their downstream genetic programmes could be responsible for the phenotypic diversity between the ara and arb mutants. These findings suggest that the ar ohnologs have diverged in two ways: first, through the loss of their roles in spermatogenesis and second, through gene duplication followed by functional differentiation that has likely resolved the pleiotropic roles derived from their ancestral gene. Thus, our results provide insights into how genome duplication impacts the massive diversification of sexual characteristics in the teleost lineage.

Functionalized calcium phosphate nanoparticles to direct osteoprotegerin to bone lesion sites in a medaka (Oryzias latipes) osteoporosis model.

Calcium phosphate (CaP) is the inorganic part of hard tissues, such as bone, teeth and tendons, and has a high biocompatibility and good biodegradability. Therefore, CaP nanoparticles functionalized with DNA encoding bone anabolic factors are promising carrier-systems for future therapeutic development. Here, we analysed CaP nanoparticles in a genetically modified medaka fish model, where osteoporosis-like lesions can be induced by transgenic expression of receptor activator of nuclear factor kappa-B ligand (Rankl). Rankl-transgenic medaka were used to visualize and understand effects of microinjected functionalized CaP nanoparticles during modulation of osteoclast activity in vivo. For this, we synthetized multi-shell CaP nanoparticles by rapid precipitation of calcium lactate and ammonium hydrogen phosphate followed by the addition of plasmid DNA encoding the osteoclastogenesis inhibitory factor osteoprotegerin-b (Opgb). An additional layer of poly(ethyleneimine) was added to enhance cellular uptake. Integrity of the synthesized nanoparticles was confirmed by dynamic light scattering, scanning electron microscopy and energy dispersive X-ray spectroscopy. Fluorescently labelled CaP nanoparticles were microinjected into the heart, trunk muscle or caudal fins of Rankl-transgenic medaka embryos that expressed fluorescent reporters in various bone cell types. Confocal time-lapse imaging revealed a uniform distribution of CaP nanoparticles in injected tissues and showed that nanoparticles were efficiently taken up by macrophages that subsequently differentiated into bone-resorbing osteoclasts. After Rankl induction, fish injected with Opg-functionalized nanoparticles showed delayed or absent degradation of mineralized matrix, i.e. a lower incidence of osteoporosis-like phenotypes. This is proof of principle that CaP nanoparticles can be used as carriers to efficiently deliver modulatory compounds to osteoclasts and block their activity.

Forced expression of α2,3-sialyltransferase IV rescues impaired heart development in α2,6-sialyltransferase I-deficient medaka.

Sialic acids (Sias) are often linked to galactose (Gal) residues by α2,6- and α2,3-linkages in glycans of glycoproteins. Sias are indispensable for vertebrate development, because organisms deficient in some enzymes in the Sia synthetic pathway are lethal during the development. However, it remains unknown if the difference of Siaα2,6Gal or α2,3Gal linkage has a critical meaning. To find a clue to understand significance of the linkage difference at the organism level, medaka was used as a vertebrate model. In embryos, Siaα2,6Gal epitopes recognized by Sambucus nigra lectin (SNA) and Siaα2,3Gal epitopes recognized by Maackia amurensis lectin (MAA) were enriched in the blastodisc and the yolk sphere, respectively. When these lectins were injected in the perivitelline space, SNA, but not MAA, impaired embryo body formation at 1 day post-fertilization (dpf). Most Siaα2,6Gal epitopes occurred on N-glycans owing to their sensitivity to peptide:N-glycanase. Of knockout-medaka (KO) for either of two ß-galactoside:α2,6-sialyltransferase genes, ST6Gal I and ST6Gal II, only ST6Gal I-KO showed severe cardiac abnormalities at 7-16 dpf, leading to lethality at 14-18 dpf. Interestingly, however, these cardiac abnormalities of ST6Gal I-KO were rescued not only by forced expression of ST6Gal I, but also by that of ST6Gal II and the ß-galactoside:α2,3-sialyltransferase IV gene (ST3Gal IV). Taken together, the Siaα2,6Gal linkage synthesized by ST6Gal I are critical in heart development; however, it can be replaced by the linkages synthesized by ST6Gal II and ST3Gal IV. These data suggest that sialylation itself is more important than its particular linkage for the heart development.

Sexually Dimorphic Regulation of Gonadotrope Cell Hyperplasia in Medaka Pituitary via Mitosis and Transdifferentiation.

The 2 pituitary gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), regulate the reproductive function in all vertebrates. While many studies have investigated the regulation of gonadotropin production and release by sex steroid feedback, its role on the regulation of gonadotrope cell number remains unclear. Using medaka as a model and an optimized protocol to restore physiological sex steroids levels following gonadectomy, we show that gonadal sex steroids not only decrease fshb transcript levels, but also Fsh cell number in both sexes. We then investigated the origin of Fsh cell hyperplasia induced by gonadectomy. In both sexes, bromodeoxyuridine incubation shows that this is achieved via Fsh cell mitosis. In situ hybridization reveals that new Fsh cells also originate from transdifferentiating Tsh cells in females, but not in males. Both phenomena are inhibited by sex steroid supplementation via feeding. In males (but not females), gonadectomy (without recovery with sex steroid supplementation) also reduces sox2 transcript levels and Sox2-immunopositive population size, suggesting that Sox2 progenitors may be recruited to produce new Fsh cells. Opposite to Fsh cells, gonadectomy decreases lhb levels in both sexes, and levels are not restored by sex steroid supplementation. In addition, the regulation of Lh cell number also seems to be sex dependent. Removal of gonadal sex steroids stimulates Lh cell mitosis in male (like Fsh cells) but not in females. To conclude, our study provides the first evidence on sexually dimorphic mechanisms used in the fish pituitary to remodel gonadotrope populations in response to sex steroids.

Toxicity of perfluorobutanesulfonate on gill functions of marine medaka (Oryzias melastigma): A time course and hypoxia co-exposure study.

Perfluorobutanesulfonate (PFBS) is found in hypoxia regions. Results of previous studies have shown that hypoxia was capable of altering the inherent toxicity of PFBS. However, regarding gill functions, hypoxic influences and time course progression of toxic effects of PFBS remain unclear. In this study, with the aim to reveal the interaction behavior between PFBS and hypoxia, adult marine medaka Oryzias melastigma were exposed for 7 days to 0 or 10 µg PFBS/L under normoxic or hypoxic conditions. Subsequently, to explore the time-course transition in gill toxicity, medaka were exposed to PFBS for 21 days. The results showed that hypoxia dramatically increased the respiratory rate of medaka gill, which was further enhanced by exposure to PFBS; although exposure to PFBS under normoxic conditions for 7 days did not alter respiration, exposure to PFBS for 21 days significantly accelerated the respiration rate of female medaka. Concurrently, both hypoxia and PFBS were potent to interrupt the gene transcriptions and Na+, K+-ATPase enzymatic activity that play pivotal roles in the osmoregulation in gills of marine medaka, consequently disrupting homeostasis of major ions in blood, such as Na+, Cl-, and Ca2+. In addition, composition and diversity of the microbiome residing on surfaces of the gill were profiled by using amplicon sequencing. Acute exposure to hypoxia for only 7 days caused a significant decrease in diversity of the bacterial community of gill whatever the presence of PFBS, while PFBS exposure for 21 days increased the diversity of gill microbial community. Principal component analysis revealed that, compared with PFBS, hypoxia was the predominant driver of gill microbiome dysbiosis. Depending on duration of exposure, a divergence was caused in the microbial community of gill. Overall, the current findings underline the interaction between hypoxia and PFBS on gill function and demonstrate the temporal variation in PFBS toxicity.

Evaluation of pancreatic δ- cells as a potential target site of graphene oxide toxicity in Japanese medaka (Oryzias latipes) fish.

In continuation to our previous investigations on graphene oxide (GO) as an endocrine disrupting chemical (EDC), in the present experiment, we have investigated endocrine pancreas of Japanese medaka adults focusing on δ-cells in the islet organs as an endpoint. Breeding pairs of adult male and female fish were exposed to 0 mg/L (control) or 20 mg/L GO by continuous immersion (IMR) for 96 h, or to 0 µg/g or 100 µg/g GO by a single intraperitoneal (IP) administration and depurated 21 days in a GO-free environment. Histological investigations indicated that the endocrine cells are concentrated in one large principal islet, and several small secondary islets scattered within the mesentery near the liver and intestine. The cells of the islet organ are in various shapes with basophilic nuclei and eosinophilic cytoplasm. Immunohistochemical evaluation using rabbit polyclonal antisomatostatin antibody indicated that immunoreactivity is localized either at the periphery or at the central region in principal islets, and throughout the secondary islets, and found to be enhanced in fish exposed to GO than controls. The soma of δ-cells exhibits neuron-like morphology and have filopodia like processes. Cell sorting as non-communicating δ-cells (NCDC), communicating cells (CC), and non- δ-cells (NDC) indicated that within an islet organ, the population of NDCC is found to be the least and NDC is the highest. Our data further indicated that GO-induced impairments in the islet organs of medaka pancreas are inconsistent and could be affected by the exposure roots as well as the sex of the fish.

Genome Editing of Medaka.

Medaka (Oryzias latipes), along with zebrafish (Danio rerio), is a useful experimental model fish. Here, we describe a simple method for generating medaka gene knockout strains using an automated microchip electrophoresis system. We also describe a method for targeted gene knockin using a plasmid carrying a sequence that does not cause off-target effects in medaka. Additionally, knockin method without plasmid cloning is described.

Low particle concentrations of nanoplastics impair the gut health of medaka.

The environmental occurrence of nanoplastics (NPs) is now evident but their long-term impacts on organisms are unclear, limiting ecological and health risk assessment. We hypothesized that chronic exposure to low particle concentrations of NPs can result in gut-associated toxicity, and subsequently affect survival of fish. Japanese medaka Oryzias latipes were exposed to polystyrene NPs (diameter 100 nm; 0, 10, 104, and 106 items/L) for 3 months, and histopathology, digestive and antioxidant enzymes, immunity, intestinal permeability, gut microbiota, and mortality were assessed. NP exposures caused intestinal lesions, and increased intestinal permeability of the gut. The trypsin, lipase, and chymotrypsin activities were increased, but the amylase activity was decreased. Oxidative damage was reflected by the decreased superoxide dismutase and alkaline phosphatase and increased malondialdehyde, catalase, and lysozyme. The integrated biomarkers response index values of all NP-exposed medaka were significantly increased compared to the control group. Moreover, NP exposures resulted in a decrease of diversity and changed the intestinal microbiota composition. Our results provide new evidence that long-term NPs exposure impaired the health of fish at extremely low particle concentrations, suggesting the need for long-term toxicological studies resembling environmental particle concentrations when assessing the risk of NPs.

Dysfunction of liver-gut axis in marine medaka exposed to hypoxia and perfluorobutanesulfonate.

With objectives to explore the interactive mode on the function of liver-gut axis, adult marine medaka were exposed for 7 days to environmentally realistic concentrations of perfluorobutanesulfonate (PFBS) (0 and 10 µg/L) under normoxia or hypoxia condition. Furthermore, PFBS exposure was extended to 21 days to reveal the temporal progression in toxicity. The results showed that hypoxia exposure significantly disturbed lipid metabolism, caused oxidative damage, and induced inflammation in the livers of medaka. The composition of gut microbiota was also drastically shifted by hypoxia acute exposure. In contrast, the effect of PFBS was much milder. Hypoxia was thus the determinant of the combined toxicity. Depending on the exposure duration, a time-course recovery from PFBS innate toxicity was generally noted. Overall, the present study underlines the hypoxic and temporal variation in the dysregulation of liver-gut axis by PFBS, which is expected to support a comprehensive ecological risk assessment.

Toxicity and Estrogenicity of Bisphenol TMC in Oryzias melastigma via In Vivo and In Silico Studies.

Bisphenol 4-[1-(4-hydroxyphenyl)-3,3,5-trimethylcyclohexyl] phenol (BPTMC), as a substitute for bisphenol A, has been detected in environments. However, the ecotoxicological data of BPTMC are extremely scarce. Here, the lethality, developmental toxicity, locomotor behavior, and estrogenic activity of BPTMC at different concentrations (0.25-2000 µg/L) in marine medaka (Oryzias melastigma) embryos were examined. In addition, the in silico binding potentials of O. melastigma estrogen receptors (omEsrs) with BPTMC were assessed by docking study. Low-concentration BPTMC exposure (including an environmentally relevant concentration, 0.25 µg/L) resulted in stimulating effects, including hatching rate, heart rate, malformation rate, and swimming velocity. However, elevated concentrations of BPTMC led to an inflammatory response, changed heart rate and swimming velocity in the embryos and larvae. In the meantime, BPTMC (including 0.25 µg/L) altered the concentrations of estrogen receptor, vitellogenin, and endogenous 17 ß-estradiol as well as the transcriptional levels of estrogen-responsive genes in the embryos or/and larvae. Furthermore, elaborate tertiary structures of omEsrs were built by ab initio modeling, and BPTMC exerted potent binding potential with three omEsrs with -47.23, -49.23, and -50.30 kJ/mol for Esr1, Esr2a, and Esr2b, respectively. This work suggests that BPTMC has potent toxicity and estrogenic effects in O. melastigma.

An RNA-seq time series of the medaka pituitary gland during sexual maturation.

Directing both organismal homeostasis and physiological adaptation, the pituitary is a key endocrine gland in all vertebrates. One of its major tasks is to coordinate sexual maturation through the production and release of hormones stimulating gonad development. In order to study its developmental dynamics in the model fish medaka (Oryzias latipes), we sampled both the pituitary and the ovaries of 68 female fish. Of these, 55 spanned the entire course of sexual maturation from prepubertal juveniles to spawning adults. An additional 13 showed either considerably faster or slower growth and development than the majority of fish. We used histological examination of the ovaries to determine a histological maturation stage, and analyzed the pituitary glands using RNA-seq optimized for low input. Taken together, these data reveal the timing of hormone production priorities, and form a comprehensive resource for the study of their regulation.

Charge-dependent negative effects of polystyrene nanoplastics on Oryzias melastigma under ocean acidification conditions.

Marine nanoplastics (NPs) have attracted increasing global attentions because of their detrimental effects on marine environments. A co-existing major environmental concern is ocean acidification (OA). However, the effects of differentially charged NPs on marine organisms under OA conditions are poorly understood. We therefore investigated the effects of OA on the embryotoxicity of both positively and negatively charged polystyrene (PS) NPs to marine medaka (Oryzias melastigma). Positively charged PS-NH2 exhibited slighter aggregation under normal conditions and more aggregation under OA conditions than negatively charged PS-COOH. According to the integrated biomarker approach, OA reversed the toxicity of positively and negatively charged NPs towards embryos. Importantly, at environmental relevant concentrations, both types of PS-NPs could enter the embryos through chorionic pores and then transfer to the larvae. OA reversed the internalization of PS-NH2 and PS-COOH in O. melastigma. Overall, the reversed toxicity of PS-NH2 and PS-COOH associated with OA could be caused by the reversed bioavailability of NPs to O. melastigma, which was attributed to altered aggregation of the NPs in acidified seawater. This finding demonstrates the charge-dependent toxicity of NPs to marine fish and provides new insights into the potential hazard of NPs to marine environments under OA conditions that could be encountered in the near future.

A comparative study on endocrine disrupting effects of leachates from virgin and aged plastics under simulated media in marine medaka larvae (Oryzias melastigma).

Marine plastic pollution has garnered substantial attention, but the potential endocrine disrupting effects of plastic leachates in marine organisms remain unclear. In this study, the larvae of marine medaka (Oryzias melastigma) were exposed to the leachates from virgin and aged plastics soaked in simulated seawater and fish digest for 3 days. The concentrations of vitellogenin (VTG), estradiol (E2), and 11-ketotestosterone (11-KT), as well as the transcripts of endocrine-related genes were measured in the larvae. The results revealed that endogenous E2 was more sensitive to plastic leachates than VTG and 11-KT, which was significantly affected by 26.7 % of all plastic leachates. Among all genes, estrogen receptor α was impacted mostly, being up-regulated by 53.3 % of leachates from aged plastics. The comparative results demonstrated that the leachates from plastics with different statuses caused a greater difference than those from plastics in different simulated media, and the leachates from aged plastics resulted in higher endocrine disrupting effects than those from virgin plastics. In addition, seven leached additives (plasticizers and flame retardants) could explain 25.6 % of the hormonal effects using redundancy analysis, indicating that other additives in the plastic leachates can also play important roles in regulating the endocrine system of O. melastigma larvae.

Critical Role of the Cortical Alveolus Protease Alveolin in Chorion Hardening In Vivo at Medaka Fertilization.

Alveolin is a cortical alveolus proteinase that is secreted in the perivitelline space (PVS) at fertilization to act on the chorion. Purified alveolin is known to induce chorion hardening in vitro by processing zona pellucida B (ZPB), a major chorion component. However, in vivo function of alveolin remains unclear; thus, in this study, the effects of alveolin efficiency (Alv-/-) at the organism level were investigated using the medaka, Oryzias latipes. The Alv-/- fertilized eggs were mechanically fragile; however, they developed normally and left offspring as long as they were carefully handled before hatching. A mechanical press test showed that the Alv-/- fertilized eggs were six times more fragile than the wild-type eggs. They were 35% larger owing to the enlarged PVS, 34% thinner, and permeable to even 10 kDa FITC-dextran. These results are consistent with the transmission electron microscopy observation that the periphery of the inner layers was highly porous in the Alv-/- chorion. In chorion hardening, the alveolin-mediated processing of ZPB and the transglutaminase (TGase)-mediated crosslinking of chorion components are the key steps. This study was the first to show that alveolin also processed TGase concomitantly with ZPB, which greatly facilitated the crosslinking. Thus, alveolin was concluded to be the primary trigger for chorion hardening in vivo. Furthermore, fertilization in a balanced salt solution could partially improve the impaired chorion hardening of the Alv-/- eggs fertilized in water, probably through an alveolin-independent mechanism.

Chronic nanoplastic exposure induced oxidative and immune stress in medaka gonad.

Nanoplastic (NP) pollution is a global issue because of its widespread occurrence and potential toxicity. Many studies have investigated the impacts of the short-term toxicity of NPs on organisms. Until now, only a few studies have assessed the toxicological effects of prolonged exposure to NPs at low concentrations in fish. In this study, the effects of NPs (nano-polystyrene microspheres, diameter: 100 nm) on immune and oxidative stress response, histopathology, and survival in medaka were evaluated. The effects of different concentrations (0, 10, 104, and 106 particles/L) of nanoplastics were studied in medaka Oryzias latipes after 3 months of exposure. Lysozyme enzyme activity, oxidative stress-related biomarkers (i.e., superoxide dismutase, catalase, and glutathione peroxidase), and malondialdehyde levels were decreased under NP exposure. The gonadal histology showed that high NP exposure (106 particles/L) inhibited the process of spermatogenesis and oogenesis processes, implying delayed maturation of the gonad. Furthermore, the IBR and PCA analysis revealed the potential biotoxicity of NPs and the total survival rate of medaka was significantly reduced due to the long-term exposure to NPs. Overall, prolonged exposure to low concentrations of NPs is harmful to the health of medaka gonads. In the long run, this may threaten the fish reproduction and population, suggesting the need for long-term toxicological studies to predict the aquatic animal health in nature.

Individual identification of inbred medaka based on characteristic melanophore spot patterns on the head.

With disease progression, individual differences appear, even in an animal disease model with genetic homogeneity. Therefore, non-invasive long term observation and individual identification is desirable for late-onset diseases. To this end, the natural markings used in ecological studies are preferable to the external invasive markings used in animal husbandry and fisheries management. Here, we propose using the distribution pattern of melanophore spots on the head of an inbred strain of medaka, a small fish model organism with monotonous pigmentation, as biometric identifier. Long term and variation analyses show different patterns whose characteristics can be attributed to individual animals. These findings were also valid in a non-inbred medaka strain and will help individual follow-up of late-onset disease medaka models for the elucidation of the pathogenesis and drug discovery.

Zebrafish and medaka T1R (taste receptor type 1) proteins mediate highly sensitive recognition of l-proline.

In vertebrates, nutritional tastants, such as amino acids and sugars, are recognized by G-protein-coupled receptors of the taste receptor type 1 (T1R) family. Previous studies have shown that fish T1Rs are functionally distinct from mammalian T1Rs in certain regards. Here, we report the existence of oral receptors with high sensitivity to amino acids in zebrafish and medaka fish. We describe the construction of multiple cell lines stably expressing functional T1Rs (from medaka fish or zebrafish) with a chimeric G-protein (G16gust44) using the Flp-In system. Through functional assays with these cell lines, medaka fish and zebrafish were confirmed to possess particular T1Rs highly sensitive to l-proline, possibly reflecting the physiological importance of l-proline in teleosts, in line with previous studies.

Developmental toxicity and transcriptome analysis of equine estrogens in developing medaka (Oryzias latipes) using nanosecond pulsed electric field incorporation.

Equine estrogens (EQs) are steroidal hormones isolated from the urine of pregnant mares and are used in the formulation of human medications. This study initially investigated the embryonic developmental toxicity of equilin (Eq) and equilenin (Eqn) in medaka (Oryzias latipes). Malformations were observed in embryos exposed to nominal concentrations of 1 and 10 mg/L of Eq and Eqn. Delayed hatching was observed at 1 mg/L of Eq. To further investigate the molecular mechanism of developmental toxicity caused by Eq and Eqn, transcriptome and bioinformatics analyses were performed. Among 2016 and 3855 total differentially expressed genes (DEGs), 1117 DEGs overlapped between Eq. (55.4 % of total DEGs) and Eq. (29.0 % of total DEGs). Gene ontology indicated effects in terms related to blood circulation and cell junctions. Pathway analyses using DEGs revealed that both Eq and Eqn treatments at 10 mg/L affected various KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways, such as neuroactive ligand-receptor interaction, mitogen-activated protein kinase signaling, retinol metabolism, and cytokine-cytokine receptor interaction. These results suggest that the disruption of these KEGG pathways is involved in the developmental toxicity of EQs in medaka embryos.