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The widespread occurrence of perfluorooctane sulfonate (PFOS) and the mass production and application of graphene oxide (GO) lead to their inevitable release and interaction in the environment, which may enhance associated toxic impacts on aquatic organisms. This study elucidates the induction of apoptosis by 60-day chronic single and mixture exposures to environmentally relevant levels of PFOS (0.5 µg/L and 5 µg/L) and GO (1 mg/L) in adult marine medaka Oryzias melastigma. Results showed a significant increase (p < 0.05) in reactive oxygen species (ROS) levels, the apoptotic positive rate in livers, and activities of caspases 3, 8, and 9 in all treated groups compared to the control. PFOS individual and PFOS-GO combined exposures significantly impacted fish growth, upregulated expressions of six apoptosis-related genes including p53, apaf1, il1b, tnfa, bcl2l1, bax, as well as enriched cell cycle and p53 signaling pathways (transcriptomic analysis) related to apoptosis compared to control group. Besides higher ROS production, GO also had a higher binding affinity to proteins than PFOS, especially to caspase 8 as revealed by molecular docking. Overall, PFOS induced ROS-p53-caspase apoptosis pathway through multi-gene regulation during single or mixture exposure, while GO single exposure induced apoptosis through tissue damage and ROS-caspase pathway activation and direct docking with caspase 8 to activate the caspase cascade. Under co-exposure, the PFOS-induced apoptotic pathway overshadowed the GO-induced pathway, due to competition for limited active sites on caspases. These findings will contribute to a better understanding of the apoptosis mechanism and ecological risks of nanomaterials and per- and polyfluoroalkyl substances in marine ecosystems.

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Microplastics and heavy metal pollution frequently co-occur in the marine environment, raising concerns about their potentially harmful impacts on marine fish. This study undertook a comprehensive evaluation of the individual and combined stress effects of polylactide microplastics (PLA-MPs) and chromium (Cr) on marine medaka larvae. Following a 14-day exposure to PLA-MPs (100 µg/L) and Cr (50 µg/L), both individually and in combination, significant increases in heart rate and body length were observed. Notably, the combined exposure to PLA-MPs and Cr caused marked histopathological alterations, including shedding, atrophy, and lysis of the intestinal tissues. Furthermore, both individual and combined exposure induced oxidative stress in fish larvae, leading to changes in various enzyme activity indices. Individual exposure to either PLA-MPs or Cr led to anxious behavior in the larvae, whereas combined exposure not only caused anxious behavior but also altered swimming patterns. These findings suggest that combined exposure to PLA-MPs and Cr can exacerbate the toxic effects on marine medaka larvae.

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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.

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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.

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Naphthenic acids (NAs) are important pollutants in marine crude oils and have obvious toxic effects on marine organisms. However, the effects of NAs on the intestine are largely unknown. Thus, we evaluated the effects of NAs exposure in the intestines of marine medaka. Fish were experimentally exposed to NAs (0.5 mg/L, 5 mg/L, and 10 mg/L) for 96 h and monitored for changes in intestinal histology, markers of oxidative stress, and intestinal microbiome responses. Significant mucosal damage, inflammation, and oxidative stress were observed in the intestines of marine medaka after exposure to NAs. In addition, significant changes in the gut microbiota were observed. Specifically, the relative abundance of Proteobacteria decreased, while that of Verrucomicrobiota increased in the high-concentration exposure group. In addition, nutrient synthesis and metabolism in the gut were affected. The results of this study contribute to a better understanding of the ecological risk of different concentrations of NAs to marine organisms. CAPSULE ABSTRACT: Changes in the gut microbial community of marine medaka (Oryzias melastigma) caused by naphthenic acids in the marine environment were investigated through the assessment of gut inflammatory factors and comprehensive analysis using 16S rDNA high-throughput sequencing. The results indicated the induction of intestinal inflammation and changes in the structural composition of the intestinal flora.

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Salinity is an important environmental factor influencing the toxicity of chemicals. Bisphenol A (BPA) is an environmental endocrine disruptor with adverse effects on aquatic organisms, such as fish. However, the influence of salinity on the biotoxicity of BPA and the underlying mechanism are unclear. In this study, we exposed marine medaka (Oryzias melastigma) to BPA at different salinities (0 psµ, 15 psµ, and 30 psµ) for 70days to investigate the toxic effects. At 0 psµ salinity, BPA had an inhibitory effect on the swimming behavior of female medaka. At 15 psµ salinity, exposure to BPA resulted in necrotic cells in the ovaries but not on the spermatozoa. In addition, BPA exposure changed the transcript levels of genes related to the nervous system (gap43, elavl3, gfap, mbpa, and α-tubulin) and the hypothalamic-pituitary-gonadal (HPG) axis (fshr, lhr, star, arα, cyp11a, cyp17a1, cyp19a, and erα); the expression changes differed among salinity levels. These results suggest that salinity influences the adverse effects of BPA on the nervous system and reproductive system of medaka. These results emphasize the importance of considering the impact of environmental factors when carrying out ecological risk assessment of pollutants.

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Microplastics (MPs) and nanoplastics (NPs) are emerging pollutants in the ocean, but their transfer and toxicity along the food chains are unclear. In this study, a marine rotifer (Brachionus plicatilis)-marine medaka (Oryzias melastigma) food chain was constructed to evaluate the transfer of polystyrene MPs and NPs (70 nm, 500 nm, and 2 µm, 2000 µg/L) and toxicity of 70 nm PS-NPs (0, 20, 200, and 2000 µg/L) on marine medaka after long-term food chain exposure. The results showed that the amount of 70 nm NPs accumulated in marine medaka was 1.24 µg/mg, which was significantly higher than that of 500 nm NPs (0.87 µg/mg) and 2 µm MP (0.69 µg/mg). Long-term food chain exposure to NPs caused microflora dysbiosis, resulting in activation of toll-like receptor 4 (TLR4) pathway, which induced liver inflammation. Moreover, NPs food chain exposure increased liver and muscle tissue triglyceride and lactate content, but decreased the protein, sugar, and glycogen content. NPs food chain exposure impaired reproductive function and inhibited offspring early development, which might pose a threat to the sustainability of marine medaka population. Overall, the study revealed the transfer of MPs and NPs and the effects of NPs on marine medaka along the food chain.

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The marine medaka is emerging as a potential behavioral model organism for ocean studies, namely on marine ecotoxicology. However, not much is known on the behavior of the species and behavioral assays lack standardization. This study assesses the marine medaka as a potential model for chemical communication. We investigated how short exposure to visual and chemical cues mediated the stress response to social isolation with the light/dark preference test (LDPT) and the open field test (OFT). After a 5-day isolation period, and 1 h before testing, isolated fish were randomly assigned to one of four groups: (1) placed in visual contact with conspecifics; (2) exposed to a flow of holding water from a group of conspecifics; (3) exposed to both visual and chemical cues from conspecifics; or (4) not exposed to any stimuli (controls). During the LDPT, the distance traveled and transitions between zones were more pronounced in animals exposed to the conspecific's chemical stimuli. The time spent in each area did not differ between the groups, but a clear preference for the bright area in all animals indicates robust phototaxis. During the OFT, animals exposed only to chemical cues initially traveled more than those exposed to visual or both stimuli, and displayed lower thigmotaxis. Taken together, results show that chemical cues play a significant role in exploratory behavior in this species and confirm the LDPT and OFT as suitable tests for investigating chemical communication in this species.

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Microplastics (MPs) have become a popular research topic due to their potential ramifications on aquatic organisms. To evaluate the ecotoxicological impacts of chronic exposure to different microplastics on marine medaka larvae, we exposed medaka larvae to 200 µg/L of polyethylene (PE-200) and polylactic acid (PLA-200) microplastics for 60 days, respectively. The results indicated that both exposures had no significant effect on fish length/weight and did not result in fish mortality. Notably, the structure of intestinal microbiota was not disrupted either. However, microscopy observations of intestinal tissue suggested that exposure to MPs resulted in inflammation of the intestinal tract of fish and significant atrophy and shedding of small intestinal villus. Linear discriminant analysis Effect Size (LEfSe) showed that intestinal enrichment of Streptomyces occurred in marine medaka larvae in both MPs treatments, while the PE-200 treatment exhibited a significant enrichment. In addition, the PICRUSt2 prediction indicated significant upregulation of the Novobiocin biosynthesis function in gut microbiota in the PE-200 treatment. Overall, multi-level assessment is necessary to determine the risk of exposure of aquatic organisms to MPs.

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The simultaneous presence of micro(nano)plastics (MNPs) and pollutants represents a prevalent environmental challenge that necessitates understanding their combined impact on toxicity. This study examined the distribution of 5 µm (PS-MP5) and 50 nm (PS-NP50) polystyrene plastic particles during the early developmental stages of marine medaka (Oryzias melastigma) and assessed their combined toxicity with triphenyltin (TPT). Results showed that 2 mg/L PS-MP5 and PS-NP50 could adhere to the embryo surface. PS-NP50 can passively enter the larvae and accumulate predominantly in the intestine and head, while PS-MP5 cannot. Nonetheless, both types can be actively ingested by the larvae and distributed in the intestine. 2 mg/L PS-MNPs enhance the acute toxicity of TPT. Interestingly, high concentrations of PS-NP50 (20 mg/L) diminish the acute toxicity of TPT due to their sedimentation properties and interactions with TPT. 200 µg/L PS-MNPs and 200 ng/L TPT affect complement and coagulation cascade pathways and cardiac development of medaka larvae. PS-MNPs exacerbate TPT-induced cardiotoxicity, with PS-NP50 exhibiting stronger effects than PS-MP5, which may be related to the higher adsorption capacity of NPs to TPT and their ability to enter the embryos before hatching. This study elucidates the distribution of MNPs during the early developmental stages of marine medaka and their effects on TPT toxicity, offering a theoretical foundation for the ecological risk assessment of MNPs.

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In order to improve the sensitivity of lateral flow immunoassays (LFIAs) for the detection of piscine vitellogenin (Vtg), a well-established biomarker for environmental estrogens, Au coated Ag nanoflowers (Ag@Au NFs) were used as labeling probes to develop a LFIA for marine medaka Vtg. The synthesized Ag@Au NFs with good monodispersity had an average diameter of 44.1 nm and absorbance peak of 524 nm. When the concentration of goat anti-mouse IgG and anti-Vtg polyclonal antibody (anti-Vtg PAbs) were 1.3 and 0.4 mg/mL, respectively, the detection range of the LFIA was 0.19-25 ng/mL, and the visual detection limit was 0.1 ng/mL, which was approximately 80 times lower than that of LFIAs based on other nanoparticles (Au NPs, Ag NPs, Au NFs, and FM). After evaluation of its specificity and robustness, the usefulness of Ag@Au NFs labeled LFIA was validated by measuring Vtg induction in the plasma of marine medaka exposed to bisphenol A, a weak estrogenic chemical. This highly sensitive lateral flow immunoassay could detect Vtg biomarker within 15 min without the need of expensive and complicated instruments, and thus offered an ultrasensitive and robust on-site detection method for estrogenic activity in field environment.

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Enrofloxacin (ENR) is a widely used fluoroquinolone antibiotic that is frequently detected in the environment. Our study assessed the impact of short-term ENR exposure on the intestinal and liver health of marine medaka (Oryzias melastigma) using gut metagenomic shotgun sequencing and liver metabolomics. We found that ENR exposure resulted in imbalances of Vibrio and Flavobacteria and enrichments of multiple antibiotic resistance genes. Additionally, we found a potential link between the host's response to ENR exposure and the intestinal microbiota disorder. Liver metabolites, including phosphatidylcholine, lysophosphatidylcholine, taurocholic acid, and cholic acid, in addition to several metabolic pathways in the liver that are closely linked to the imbalance of intestinal flora were severely maladjusted. These findings suggest that ENR exposure has the potential to negatively affect the gut-liver axis as the primary toxicological mechanism. Our findings provide evidence regarding the negative physiological impacts of antibiotics on marine fish.

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Diflubenzuron, an insecticide commonly used in marine fish farming, has been detected in various marine environments. However, its potential impact on marine fish remains largely unknown. This study investigated the reproductive toxicity of chronic diflubenzuron exposure in female marine medaka (Oryzias melastigma). Marine medaka were exposed continuously to environmentally relevant concentrations of diflubenzuron (0.1, 1, and 10 µg/L) or a solvent control from the fertilized egg to adulthood. In exposed female marine medaka, the gonadosomatic index (GSI) and the number of laid eggs were significantly reduced. Moreover, diflubenzuron-exposed female marine medaka showed altered ovarian histopathology, with an increased relative proportion of immature oocytes and atretic follicles and a decreased relative proportion of mature oocytes. Maternal exposure to diflubenzuron also inhibited the development of the F1 generation, significantly reducing the hatching rate of F1 embryos and significantly increasing the malformation rate of F1 larvae. Furthermore, changes in hormone levels and expression of genes along the hypothalamus-pituitary-gonad-liver (HPGL) axis were observed, which may be the fundamental reason for all the reproductive toxic effects mentioned above. These results provide new insights into the impact of diflubenzuron on the female marine medaka reproductive system and underscore the importance of investigating the potential environmental risks of diflubenzuron in the marine environment.

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Bisphenol AF (BPAF), an emerging environmental endocrine disruptor, has been detected in surface waters worldwide and has adverse effects on aquatic organisms. The accumulation of BPAF in oceans and its potential toxic effect on marine organisms are important concerns. In this study, the effects of BPAF (10, 100, 1, and 5 mg/L) on marine medaka (Oryzias melastigma) were evaluated, including effects on the survival rate, heart rate, hatchability, morphology, and gene expression in embryos. The survival rate of marine medaka embryos was significantly lower after treatment with 5 mg/L BPAF than in the solvent control group. Exposure to 1 mg/L and 5 mg/L BPAF significantly reduced hatchability. Low-dose BPAF (10 µg/L) significantly accelerated the heart rate of embryos, while high-dose BPAF (5 mg/L) significantly decreased the heart rate. BPAF exposure also resulted in notochord curvature, pericardial edema, yolk sac cysts, cardiovascular bleeding, and caudal curvature in marine medaka. At the molecular level, BPAF exposure affected the transcript levels of genes involved in the thyroid system (dio1, dio3a, trhr2, tg, and thra), cardiovascular system (gata4, atp2a1, and cacna1da), nervous system (elavl3 and gap43), and antioxidant and inflammatory systems (sod, pparß, and il-8) in embryos. These results indicate that BPAF exposure can alter the expression of functional genes, induce abnormal development, and reduce the hatching and survival rates in marine medaka embryos. Overall, BPAF can adversely affect the survival and development of marine medaka embryos, and BPAF may not be an ideal substitute for BPA.

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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.

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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.

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Microplastics could accumulate and enrich antibiotics in the aquatic environment. Despite this, the joint effects of microplastics and antibiotics on aquatic organisms are not clear. Here, we investigated the changes of microbial interactions in both gill and gut of marine medaka exposed to polystyrene microbeads (PS) and/or tetracycline for 30 days by using co-occurrence network analysis based on 16S rRNA gene amplicon sequences. We found that the single and combined effects of PS and tetracycline were more profound on the gut than on the gill microbiome. SourceTracker analysis showed that the relative contributions from the gill microbiome to the gut microbiome increased under combined exposure. Moreover, the combined exposure reduced the complexity and stability of the gut microbial network more than those induced by any single exposure, suggesting the synergistic effects of PS and tetracycline on the gut microbiome. The PS and tetracycline combined exposure also caused a shift in the keystone taxa of the gut microbial network. However, no similar pattern was found for gill microbial networks. Furthermore, single and combined exposure to PS and/or tetracycline altered the associations between the gut network taxa and indicator liver metabolites. Altogether, these findings enhanced our understanding of the hazards of the co-occurring environmental microplastics and antibiotics to the fish commensal microbiome.

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Triphenyltin (TPT) is an endocrine contaminant that is often detected in the environment. However, the mechanism of the effects of TPT on biological systems is not fully understood. Here we exposed marine medaka (Oryzias melastigma) to TPT for 21 days. Brain transcriptome, intestinal content metabolism group, and behavior analysis were carried out. Through the comprehensive analysis of multiomics for the in-depth understanding of the ways related to health improvement, we determined that the glycine-serine-threonine metabolic axis was most perturbed by TPT. Through behavioral analysis, it was found that there was behavioral hyperactivity in the exposed group; behavioral hyperactivity may be caused by the interference of TPT with the neuroendocrine system. In order to gain a full understanding of the impacts of TPT on human health, transcriptomic screening of differential genes and an impartial attitude based on bioinformatics were used. Gene-disease interaction analysis using the Comparative Toxicogenomics Database (CTD) revealed the possible effects of TPT on human health. Finally, based on these findings, the relevant adverse outcome pathway (AOP), which is the "epigenetic modification of PPARG leading to adipogenesis," was identified from AOP Wiki. Further research is required to validate the potential AOP of TPT.

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Here we propose a carbon release model that divides fish-released carbon into two sources (ingested food and the fish body), and three forms (dissolved organic carbon (DOC), CO2, and particulate carbon (PC)). We quantified the daily carbon budget of a marine fish Oryzias melastigma by feeding the fish radiocarbon-labeled living rotifer. We found that 91%-92%, 25%-47%, 28%-50%, 20%-31%, and 8%-9% of the ingested food carbon was absorbed, assimilated, and released as DOC, CO2, and PC, respectively. Fish body carbon dissimilated/catabolized and released as 0.053-0.12 d-1 at two daily food rations. DOC, CO2, and PC accounted for 39%-42%, 39%-45%, and 16%-19% of the released fish body carbon, respectively. Our study shows that the fish transformed substantial fractions of their daily ingested food and dissimilated body carbon into DOC, and fish may be an important source of DOC in the ocean.

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Marine ecosystems are currently subjected to dual stresses of chemical pollution and climate change. Through a series of laboratory experiments, this study investigated the impact of exposure to chemical contaminant such as DDT or copper (Cu), in combination with cold or warm temperature extremes on the marine medaka fish Oryzias melastigma. The results showed that extreme seawater temperatures (i.e., 15 and 32 °C in sub-tropical Hong Kong) exacerbated adverse chemical impacts on the growth performance of O. melastigma, in particular at the high thermal extreme. This was likely associated with an interruption of oxygen consumption and aerobic scope. Most importantly, the results of acclimation experiments, as reflected by thermal tolerance polygons, showed that chemical exposure substantially narrowed the thermal tolerance of the medaka, making them more vulnerable to temperature changes and extreme thermal events. Under dual stresses of thermal extremes and chemical exposure, the medaka switched their metabolic pathway to anaerobic respiration that might deplete their energy reserve for chemical detoxification. Although stress proteins such as heat shock proteins (HSP90) were up-regulated for cellular protection in the fish, such a defensive mechanism was repressed with intensifying dual stresses at high temperature and high chemical concentration. Bioconcentration of DDT or Cu generally increased with increasing temperature and its exposure concentration. Overall, these complex chemical-temperature interactions concomitantly exerted a concerted adverse impact to O. melastigma. The temperature-dependent toxicity of DDT or Cu shown in this study clearly demonstrated the potential challenge brought by the risk of chemical pollution under the impact of global climate change.

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