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.

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.

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.

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.

Illumina-based RNA-seq was used to determine the short-term transcriptomic responses of Paralichthys olivaceus gill to an environmentally relevant level of water accommodated fraction (WAF) of crude oil. 213,979 transcripts and 128,482 unigenes were obtained. Differential expression analysis revealed that 1641 and 2142 genes were significantly up- and down-regulated. Enrichment analysis identified a set of GO terms and putative pathways involved in the response of P. olivaceus to WAF exposure. Analysis of the transcripts revealed the effective protective mechanisms of P. olivaceus to reduce the toxic effects of WAF. Moreover, WAF exposure induced the metabolism of energy substrates, and downstream pathway genes were modified to provide protection against toxic damage. Transcripts analysis demonstrated that the genes involved in circadian rhythm signaling were regulated in gills of P. olivaceus exposed to WAF. These results provide insights into the mechanisms of WAF-induced toxicity in fishes and into the WAF-sensitive biomarkers in P. olivaceus.

We investigated levels of the pollutant tributyltin (TBT) in blood of pufferfishes (six species), Japanese sea perch, red sea bream, Japanese common goby, Japanese flounder, rockfish, conger eel, and sea mullet collected off the coast of northern Kyushu, Japan. We found considerable levels of TBT (1.4–190ng/mL) accumulated in the blood of these fish. Blood TBT concentrations were 1.3–22.5times liver concentrations and 4.9–78times muscle concentrations, except in conger eel and mullet. We detected TBT (16–111ng/mL-blood) in the plasma of the fine-patterned puffer (Takifugupoecilonotus) year-round, without any apparent seasonal trend. These results suggest that fish inhabiting coastal areas of Kyushu, Japan, continue to be contaminated with TBT.

The relationship between chemical exposure and disease outbreak in fish has not been fully defined due to the limitations of experimental systems (model fish and pathogens). Therefore, we constructed a system using the Japanese flounder, Paralichthys olivaceus, and viral haemorrhagic septicemia virus (VHSV), and evaluated it by heavy oil (HO) exposure. The fish were exposed to HO at 0.3, 0.03, 0.003, and 0g/L following VHSV infection at doses of 10².⁵ or 10³.⁵ tissue culture infectious dose (TCID)₅₀/fish. As a result, groups given the dual stressors showed more than 90% mortality. Although VHSV infection at 10².⁵ and 10³.⁵ TCID₅₀/fish without HO exposure also induced high mortality, at 68.8% and 81.3%, respectively, HO exposure induced faster and higher mortality in the virus carrier fish, indicating that chemical stressors raise the risk of disease outbreak in fish. The experimental system established in this study could be useful for chemical risk assessment.

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.

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.