Cadmium (Cd) is an important heavy metal pollutant in the Bohai Sea. Mitochondria are recognized as the key target for Cd toxicity. However, mitochondrial responses to Cd have not been fully investigated in marine fishes. In this study, the mitochondrial responses were characterized in gills of juvenile flounder Paralichthys olivaceus treated with two environmentally relevant concentrations (5 and 50 μg/L) of Cd for 14 days by determination of mitochondrial membrane potential (MMP), observation of mitochondrial morphology and quantitative proteomic analysis. Both Cd treatments significantly decreased MMPs of mitochondria from flounder gills. Mitochondrial morphologies were altered in Cd-treated flounder samples, indicated by more and smaller mitochondria. iTRAQ-based proteomic analysis indicated that a total of 128 proteins were differentially expressed in both Cd treatments. These proteins were basically involved in various biological processes in gill mitochondria, including mitochondrial morphology and import, tricarboxylic acid (TCA) cycle, oxidative phosphorylation (OXPHOS), primary bile acid biosynthesis, stress resistance and apoptosis. These results indicated that dynamic regulations of energy homeostasis, cholesterol metabolism, stress resistance, apoptosis, and mitochondrial morphology in gill mitochondria might play significant roles in response to Cd toxicity. Overall, this study provided a global view on mitochondrial toxicity of Cd in flounder gills using iTRAQ-based proteomics.

Nickel and cobalt are essential elements that become toxic at high concentrations. Little is known about nickel and cobalt toxicity in aquatic animals. This study aimed to investigate acute and chronic toxicity of nickel and cobalt in Japanese flounder (Paralichthys olivaceous), with emphasis on oxidative stress reactions, histopathological changes, and differences in gene expression. The lethal concentration for 50% mortality (LC₅₀) in 3 and 8 cm Japanese flounder exposed to nickel for 96 h was found to be 86.2 ± 0.018 and 151.3 ± 0.039 mg/L; for cobalt exposure, LC₅₀ was 47.5 ± 0.015 and 180.4 ± 0.034 mg/L, respectively. Chronic nickel and cobalt exposure caused different degrees of oxidative enzyme activity changes in gill, liver, and muscle tissues. Erythrocyte deformations were detected after acute or chronic exposure to nickel and cobalt. the nickel and cobalt exposure also caused pathological changes such as spherical swelling over other gill patches, rod-like proliferations in the gill patch epithelial cell layer, and disorder in hepatocyte arrangement, cell swelling, and cytoplasm loosening. RNA-Seq indicated that there were 184 upregulated and 185 downregulated genes in the liver of Japanese flounder exposed to 15 mg/L nickel for 28 d. For cobalt, 920 upregulated and 457 downregulated genes were detected. Among these differentially expressed genes, 162 were shared by both nickel and cobalt exposure. In both nickel and cobalt, pathways including fatty acid elongation, steroid biosynthesis, unsaturated fatty acid biosynthesis, fatty acid metabolism, PPAR signaling, and ferroptosis were significantly enriched. Taken together, these results aided our understanding of the toxicity of nickel and cobalt in aquatic animals.

Green tide outbreaks caused by overgrowth of Ulva prolifera in the Yellow Sea of China can cause serious ecological stress with concomitant economic hardships, especially to marine fisheries. In this study, short-term effects (14 days) were evaluated using fresh algae U. prolifera (FU), and a 7-day assessment of the effects of decomposing U. prolifera (DU) algal effluent was conducted to determine the effects on the environmental and intestinal microbiota, intestinal transcriptome and mortality of the commercial marine benthic fish, Japanese flounder (Paralichthys olivaceus). The results revealed that algal degradation altered the microbial community structure of fish farm water and fish intestines and increased the relative abundance of the pathogens Flavobacteriaceae in water and Vibrio in fish intestines. Fish intestinal tissue structure appeared to be damaged, as indicated in pathological sections, and transcriptome analysis showed intestinal inflammation after exposure, which may have caused an increase in fish mortality. The degradation of U. prolifera led to a bloom of potential pathogenic bacteria and the inflammation of fish intestines, which resulted in disease in the flounder population that reduced fish harvests and might pose a potential health threat.

Mercury isotope ratios in fish tissues have been used to infer sources and biogeochemical processes of mercury in aquatic ecosystems. More experimental studies are however needed to understand the internal dynamics of mercury isotopes and to further assess the feasibility of using fish mercury isotope ratios as a monitoring tool. We exposed Olive flounder (Paralichthys olivaceus) to food pellets spiked with varying concentrations (400, 1600 ng/g) of methylmercury (MeHg) and inorganic mercury (IHg) for 10 weeks. Total mercury (THg), MeHg concentrations, and mercury isotope ratios (δ²⁰²Hg, Δ¹⁹⁹Hg, Δ²⁰⁰Hg) were measured in the muscle, liver, kidney, and intestine of fish. Fish fed mercury unamended food pellets and MeHg amended food pellets showed absence of internal δ²⁰²Hg and Δ¹⁹⁹Hg fractionation in all tissue type. For fish fed IHg food pellets, the δ²⁰²Hg and Δ¹⁹⁹Hg values of intestine equilibrated to those of the IHg food pellets. Kidney, muscle, and liver exhibited varying degrees of isotopic mixing toward the IHg food pellets, consistent with the degree of IHg bioaccumulation. Liver showed additional positive δ²⁰²Hg shifts (∼0.63‰) from the binary mixing line between the unamended food pellets and IHg food pellets, which we attribute to redistribution or biliary excretion of liver IHg with a lower δ²⁰²Hg to other tissues. Significant δ²⁰²Hg fractionation in the liver and incomplete isotopic equilibration in the muscle indicate that these tissues may not be suitable for source monitoring at sites heavily polluted by IHg. Instead, fish intestine appears to be a more suitable proxy for identifying IHg sources. The results from our study are essential for determining the appropriate fish tissues for monitoring environmental sources of IHg and MeHg.

Microplastics and polychlorinated biphenyls are ubiquitous in the marine environments. To illuminate their combined biological impacts, juvenile Japanese flounder (Paralichthys olivaceus) were exposed to 500 ng/L PCBs alone or 500 ng/L PCBs plus 2, 20, and 200 μg/L 10-μm porous MPs for 21 days. Compared to PCBs alone, co-exposure to PCBs and 20, 200 μg/L MPs reduced fish body length and body weight, and the concurrence of MPs aggravated PCBs-induced thyroid-disrupting effects, including significantly decreased L-thyroxine and L-triiodothyronine levels, more severe damage to the thyroid tissue and gill morphology, and disturbance on the expression of hypothalamus-pituitary-thyroid axis genes. The PCBs concentrations in the seawater were decreased dramatically with the increase of MPs concentrations, confirming that MPs absorbed PCBs from the seawater. Our results demonstrated that MPs enhanced the thyroid disruption of PCBs, suggesting that the risk of MPs and thyroid-disrupting chemicals on marine organisms should be paid more attention.

The potential ecological impacts of elevated suspended sediments (SS) in coastal areas due to human activities remain unclear. In particular, physiological response of benthic fish to SS exposure in polluted environment has not been documented. We determined sub-lethal toxicity of polluted and non-polluted SS to olive flounder. Test organism was exposed to varying concentrations of SS (0–4000 mg L⁻¹) and real-time oxygen consumption rate (OCR) was measured for 12 h. The early-juvenile was sensitive to SS, particularly at >500 mg L⁻¹, but late-juvenile was tolerant up to 4000 mg SS L⁻¹. Metal polluted SS (HQₘₑₜₐₗ > 1) increased OCR in general, particularly at >1000 SS mg L⁻¹. Combined effect of copper and SS exposure on fish was either synergistic or antagonistic. Overall, potential adverse effect of polluted SS on fish greatly varied at different life stage and/or by metal pollution gradients.

This study investigated the anti-estrogenic effects of chronic exposure to a new marine pollutant, semicarbazide (SMC; 1, 10, and 100μg/L), in female Paralichthys olivaceus, as well as the associated mechanism. After 130days of exposure, plasma 17β-estradiol and testosterone concentrations, and hepatic estrogen receptors, vitellogenin, and choriogenin mRNA levels decreased significantly in SMC-exposed groups. Moreover, down-regulation of genes in the hypothalamic-pituitary-gonadal axis, including gonadotropin-releasing hormone, gonadotropic hormones and their receptors, the steroidogenic acute regulatory protein, 17α-hydroxylase, 17β-hydroxysteroid dehydrogenase, and cytochrome P450 19A, was observed after SMC exposure. Furthermore, the kisspeptin/g protein-coupled receptor 54 (kiss/gpr54) system and gamma-aminobutyric acid-ergic (GABAergic) system were also affected by SMC: SMC significantly down-regulated mRNA expression of kiss2, gpr54, and the GABA synthesis enzyme gad67. Our results demonstrated for the first time that environmentally relevant concentrations of SMC exerted anti-estrogenicity in female flounders, providing theoretical support for ecological risk assessments of SMC in marine environments.

This study examined the influence of environmental concentrations of Aroclor 1254 (10, 100, and 1000ng/L) on metamorphosis of Paralichthys olivaceus, and analyzed the mechanisms in relation to thyroid disruption. Results showed that 100 and 1000ng/L Aroclor 1254 delayed metamorphosis and that 1000ng/L Aroclor 1254 caused abnormal morphology. Thyroxine and triiodothyronine levels in the control group were significantly elevated at metamorphic climax, but treatment with 100 and 1000ng/L delayed the increase in thyroid hormones (THs) and retarded metamorphic processes. In larvae exposed to 1000ng/L Aroclor 1254, TH levels at metamorphic climax were significantly lower than those of the control group at the same metamorphic stage. We suggest that the effects of Aroclor 1254 on larval metamorphosis can be explained by disruption of thyroid homeostasis. These findings provide a new perspective and biological model for thyroid-disrupting chemicals (TDCs) screening and investigating interference of thyroid function by TDCs.

We studied the morphological and biochemical changes of mitochondria-rich cells (MRCs) of a demersal teleost, Paralichthys olivaceus, during exposure to 0.98, 2.97 and 4.95kPa pCO2. The apical opening area of MRCs increased 2.2 and 4.1 times by 24h exposure to 2.97 and 4.95kPa pCO2, respectively, while the cross-sectional area or density of MRCs did not change. Gill Na+/K+-ATPase activity more than doubled at 72h and then returned to the pre-exposure level at 168h in 0.98kPa pCO2, while it increased 1.7 times at 24h at 4.95kPa. These results indicate that the apical opening area of MRCs and the gill Na+/K+-ATPase activity may be used as an indicator of acute (up to 72h), but not chronic, impacts of high (>1kPa) seawater CO2 conditions in P. olivacues. Limitations of those parameters as indices of CO2 impacts are discussed.

In this study, individual Japanese flounder were intraperitoneally injected with 2μg tributyltin-d27 (TBT-d27) fish⁻¹. Blood samples were collected on day 7 after injection. TBT-binding protein types 1 and 2 (TBT-bp1, -bp2) in the blood serum were quantified by western blotting analysis. As a result, the concentration of TBT-bp2 in TBT-d27 treated group increased to 220% of that in the solvent control, whereas the TBT-bp1 concentration decreased to 65% of that in the solvent control. Additionally, a positive relationship between the concentrations of TBT-bp2 and TBT was observed in blood sera of wild and cultured flounder. We suggest that TBT-bp2 is produced in response to TBT exposure and may play an important role in fish physiology.