Single-cell transcriptome landscape of zebrafish liver reveals hepatocytes and immune cell interactions in understanding nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is becoming the most common chronic liver disease in the world. Immunity is the major contributing factor in NAFLD; however, the interaction of immune cells and hepatocytes in disease progression has not been fully elucidated. As a popular species for studying NAFLD, zebrafish, whose liver is a complex immune system mediated by immune cells and non-immune cells in maintaining immune tolerance and homeostasis. Understanding the cellular composition and immune environment of zebrafish liver is of great significance for its application in NAFLD. Here, we established a liver atlas that consists of 10 cell types using single-cell RNA sequencing (scRNA-seq). By examining the heterogeneity of hepatocytes and analyzing the expression of NAFLD-associated genes in the specific cluster, we provide a potential target cell model to study NAFLD. Additionally, our analysis identified two subtypes of distinct resident macrophages with inflammatory and non-inflammatory functions and characterized the successive stepwise development of T cell subclusters in the liver. Importantly, we uncovered the possible regulation of macrophages and T cells on target cells of fatty liver by analyzing the cellular interaction between hepatocytes and immune cells. Our data provide valuable information for an in-depth study of immune cells targeting hepatocytes to regulate the immune balance in NAFLD.

Norethindrone alters growth, sex differentiation and gene expression in marine medaka (Oryzias melastigma).

Norethindrone (NET) is a widely used synthetic progestin, which appears in water environments and threatens aquatic organisms. In this study, marine medaka (Oryzias melastigma) larvae were exposed to 7.6 and 80.1 ng/L NET for 190 days. The effects of NET on growth, sex differentiation, gonad histology and transcriptional expression profiles of hypothalamic-pituitary-gonadal (HPG) axis-related genes were determined. The results showed that exposure to 80.1 ng/L NET caused an all-male marine medaka population and significantly decreased the growth of males. Exposure to 7.6 ng/L NET increased the ratio of males/females in the marine medaka population, decreased the growth of males and delayed the ovary maturation in females. However, the sperm maturation was accelerated by 7.6 or 80.1 ng/L NET. In females, the transcription levels of cytochrome P450 aromatase (cyp19a1a) and progesterone receptor (pgr) in ovaries, glucocorticoid receptor (gr) and vitellogenin (vtg) in livers were suppressed after exposure to 7.6 ng/L NET, which may cause delayed ovary maturation. In males, NET significantly decreased the transcription levels of follicle stimulating hormone ß (fshß) and Luteinizing hormone ß (lhß)in the brain, Estrogen receptor ß (erß)，gr and pgr in the liver, and vitellogenin receptor (vtgr) in the testes, while NET of 80.1 ng/L led to a significant up-regulation of steroidogenic acute regulatory protein (star) in the testes of males. These results showed that NET could influence growth, sex differentiation and gonadal maturation and significantly alter the transcriptional expression levels of HPG axis-related genes.

Disease Association and Druggability of WD40 Repeat Proteins.

WD40 repeat (WDR) domains are protein interaction scaffolds that represent one of the largest protein families in human, and a first WDR inhibitor-an allosteric antagonist of polycomb repressive complex 2-just entered the clinic. A systematic analysis of the CORUM database of protein complexes shows that WDR is the most represented domain in transcriptional regulation and one of the most prevalent in the ubiquitin proteasome system, two pathways of high relevance to drug discovery. Parsing the literature and the vulnerability of cancer cell lines to CRISPR knockout indicates that WDR proteins are targets of interest in oncology and other disease areas. A quantitative analysis of WDR structures reveals that druggable binding pockets can be found on multiple surfaces of these multifaceted protein interaction platforms. These data support the development of chemical probes to further interrogate WDR proteins as an emerging therapeutic target class.

The substrate binding domains of human SIAH E3 ubiquitin ligases are now crystal clear.

BACKGROUND: Seven in absentia homologs (SIAHs) comprise a family of highly conserved E3 ubiquitin ligases that play an important role in regulating signalling pathways in tumorigenesis, including the DNA damage repair and hypoxia response pathways. SIAH1 and SIAH2 have been found to function as a tumour repressor and a proto-oncogene, respectively, despite the high sequence identity of their substrate binding domains (SBDs). Ubiquitin-specific protease USP19 is a deubiquitinase that forms a complex with SIAHs and counteracts the ligase function. Much effort has been made to find selective inhibitors of the SIAHs E3 ligases. Menadione was reported to inhibit SIAH2 specifically. METHODS: We used X-ray crystallography, peptide array, bioinformatic analysis, and biophysical techniques to characterize the structure and interaction of SIAHs with deubiquitinases and literature reported compounds. RESULTS: We solved the crystal structures of SIAH1 in complex with a USP19 peptide and of the apo form SIAH2. Phylogenetic analysis revealed the SIAH/USP19 complex is conserved in evolution. We demonstrated that menadione destabilizes both SIAH1 and SIAH2 non-specifically through covalent modification. CONCLUSIONS: The SBDs of SIAH E3 ligases are structurally similar with a subtle stability difference. USP19 is the only deubiquitinase that directly binds to SIAHs through the substrate binding pocket. Menadione is not a specific inhibitor for SIAH2. GENERAL SIGNIFICANCE: The crystallographic models provide structural insights into the substrate binding of the SIAH family E3 ubiquitin ligases that are critically involved in regulating cancer-related pathways. Our results suggest caution should be taken when using menadione as a specific SIAH2 inhibitor.

Morphological Characters and Transcriptome Profiles Associated with Black Skin and Red Skin in Crimson Snapper (Lutjanus erythropterus).

In this study, morphology observation and illumina sequencing were performed on two different coloration skins of crimson snapper (Lutjanus erythropterus), the black zone and the red zone. Three types of chromatophores, melanophores, iridophores and xanthophores, were organized in the skins. The main differences between the two colorations were in the amount and distribution of the three chromatophores. After comparing the two transcriptomes, 9200 unigenes with significantly different expressions (ratio change ≥ 2 and q-value ≤ 0.05) were found, of which 5972 were up-regulated in black skin and 3228 were up-regulated in red skin. Through the function annotation, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the differentially transcribed genes, we excavated a number of uncharacterized candidate pigment genes as well as found the conserved genes affecting pigmentation in crimson snapper. The patterns of expression of 14 pigment genes were confirmed by the Quantitative real-time PCR analysis between the two color skins. Overall, this study shows a global survey of the morphological characters and transcriptome analysis of the different coloration skins in crimson snapper, and provides valuable cellular and genetic information to uncover the mechanism of the formation of pigment patterns in snappers.

Genome sequencing, comparative analysis, and gene expression responses of cytochrome P450 genes in Oryzias curvinotus provide insights into environmental adaptation.

The mangrove fish (Oryzias curvinotus) serves as a model for researching environmental adaptation and sexual development. To further such research, we sequenced and assembled a high-quality 842 Mb reference genome for O. curvinotus. Comparative genomic analysis revealed 891 expanded gene families, including significantly expanded cytochrome P450 (CYP) detoxification genes known to be involved in xenobiotic defense. We identified 69 O. curvinotus CYPs (OcuCYPs) across 18 families and 10 clans using multiple methods. Extensive RNA-seq and qPCR analysis demonstrated diverse spatiotemporal expression patterns of OcuCYPs by developmental stage, tissue type, sex, and pollutant exposure (17ß-estradiol (E2) and testosterone (MT)). Many OcuCYPs exhibited sexual dimorphism in gonads, suggesting reproductive roles in steroidogenesis, while their responsiveness to model toxicants indicates their importance in environmental adaptation through enhanced detoxification. Pathway analysis highlighted expanded CYP genes in arachidonic acid metabolism, drug metabolism, and steroid hormone biosynthesis. This chromosome-level genomic resource provides crucial biological insights to elucidate the functional roles of expanded CYPs in environmental adaptation, sexual development, early life history, and conservation in the anthropogenically impacted mangrove habitats of O. curvinotus. It also enables future ecotoxicology research leveraging O. curvinotus as a pollution sentinel species.

Toxic Effects of Bisphenol AF Exposure on the Reproduction and Liver of Female Marine Medaka (Oryzias melastigma).

In recent years, bisphenol AF (BPAF) in aquatic environments has drawn attention to its ecological risks. This study aims to investigate the toxic effects of BPAF (188.33 µg/L) exposure for 30 days on female marine medaka (Oryzias melastigma). On the 10th and 30th day of exposure, the toxicity was evaluated using histological analysis of the liver and ovaries and the transcription levels of genes related to the antioxidant system, immune system, and hypothalamic-pituitary-gonadal (HPG) axis. Findings revealed that (1) BPAF exposure caused vacuolation, karyopyknosis and karyolysis in the liver of marine medaka, and the toxic impact augmented with duration; (2) exposure to BPAF for 10 days facilitated the growth and maturation of primary ova, and this exposure had a comparatively inhibitory effect after 30 days; (3) exposure to BPAF resulted in a biphasic regulation of the transcriptional abundance of genes involved in antioxidant and inflammatory response (e.g., il-8, cat), with an initial up-regulation followed by down-regulation. Additionally, it disrupted the transcriptional pattern of HPG axis-related genes (e.g., 3ßhsd, arα). In conclusion, 188.33 µg/L BPAF can alter the expression levels of functionally related genes, impair the structural integrity of marine organisms, and pose a threat to their overall health.

A high-quality chromosome-level genome assembly of the Chinese medaka Oryzias sinensis.

Oryzias sinensis, also known as Chinese medaka or Chinese ricefish, is a commonly used animal model for aquatic environmental assessment in the wild as well as gene function validation or toxicology research in the lab. Here, a high-quality chromosome-level genome assembly of O. sinensis was generated using single-tube long fragment read (stLFR) reads, Nanopore long-reads, and Hi-C sequencing data. The genome is 796.58 Mb, and a total of 712.17 Mb of the assembled sequences were anchored to 23 pseudo-chromosomes. A final set of 22,461 genes were annotated, with 98.67% being functionally annotated. The Benchmarking Universal Single-Copy Orthologs (BUSCO) benchmark of genome assembly and gene annotation reached 95.1% (93.3% single-copy) and 94.6% (91.7% single-copy), respectively. Furthermore, we also use ATAC-seq to uncover chromosome transposase-accessibility as well as related genome area function enrichment for Oryzias sinensis. This study offers a new improved foundation for future genomics research in Chinese medaka.

Reproductive toxic effects of chronic exposure to bisphenol A and its analogues in marine medaka (Oryzias melastigma).

As awareness of BPA's health risks has increased, many countries and regions have implemented strict controls on its use. Consequently, bisphenol analogues like BPF and BPAF are being increasingly used as substitutes. However, these compounds are also becoming increasingly prevalent in the environment due to production, use and disposal processes. The oceans act as a repository for various pollutants, and recent studies have revealed the extensive presence of bisphenols (BPs, including BPA, BPF, BPAF, etc.) in the marine environment, posing numerous health hazards to marine wildlife. Nevertheless, the reproductive toxicity of these chemicals on marine fish is not comprehensively comprehended yet. Thus, the histological features of the gonads and the gene expression profiles of HPG (Hypothalamic-Pituitary-Gonadal) axis-related genes in marine medaka (Oryzias melastigma) were studied after exposure to single and combined BPs for 70 days. The effects of each exposure group on spawning, embryo fertilization, and hatching in marine medaka were also assessed. Furthermore, the impacts of each exposure group on the genes related to methylation in the F2 and F3 generations were consistently investigated. BPs exposure was found to cause follicular atresia, irregular oocytes, and empty follicles in the ovary; but no significant lesions in the testis were observed. The expression of several HPG axis genes, including cyp19b, 17ßhsd, 3ßhsd, and fshr, resulted in significant changes compared to the control group. The quantity of eggs laid and fertilization rate decreased in all groups treated with BPs, with the BPAF-treated group showing a notable reduction in the number of eggs laid. Additionally, the hatching rate showed a more significant decline in the BPF-treated group. The analysis of methylated genes in the offspring of bisphenol-treated groups revealed significant changes in the expression of genes including amh, dnmt1, dnmt3ab, mbd2, and mecp2, indicating a potential transgenerational impact of bisphenols on phenotype through epigenetic modifications. Overall, the potential detrimental impact of bisphenol on the reproduction of marine medaka emphasizes the need for caution in considering the use of BPAF and BPF as substitutes.

Effects of salinity on behavior and reproductive toxicity of BPA in adult marine medaka.

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.

Systematic selection of suitable reference genes for quantitative real-time PCR normalization studies of gene expression in Lutjanus erythropterus.

Quantitative real-time PCR (qRT-PCR) has been widely employed for the study of gene expression in fish, and accurate normalization is crucial. In this study, we aimed to identify the most stably expressed genes in various tissues, different developmental stages, and within astaxanthin treatment groups in Lutjanus erythropterus. Twelve candidate genes (EEF1A, CYB5R3, DLD, IDH3A, MRPL17, MRPL43, NDUFS7, PABPC1, PAGR1, PFDN2, PSMC3, and RAB10) were examined via qRT-PCR. We employed geNorm and NormFinder to assess their stability. The results revealed that RAB10 and PFDN2 exhibited relatively stable expression patterns across different tissue and astaxanthin treatment groups, while NDUFS7 and MRPL17 proved to be the most reliable reference gene combinations across various developmental stages. The stability of these selected genes was further validated by assessing the expression of two target genes, CRADD and CAPNS1, across developmental stages, reinforcing the reliability of NDUFS7 as it closely aligned with transcriptome-wide expression patterns at these stages. The present results will help researchers to obtain more accurate results in future qRT-PCR analysis in L. erythropterus.

Toxic effects of environmental concentration Bisphenol AF exposure on the survival, growth and reproduction of adult male Oryzias curvinotus.

Bisphenol AF (BPAF) is a novel environmental endocrine disruptor, and is widely detected in the aquatic environment, which is a potential threat to the health of fish. In this study, male Oryzias curvinotus were exposed to environmental concentrations (0.93 and 9.33 µg/L) of BPAF for 21 days. The effects of BPAF on survival, growth, reproduction, liver and testis histology, and gene transcriptional profiles of O. curvinotus were investigated. The results showed that the survival rate of male O. curvinotus slight decrease with increasing BPAF concentration, and there was no significant effect on body length, body weight, and K-factor. BPAF (9.33 µg/L) caused significant changes in testicular structure and reduced spermatid count in O. curvinotus. Changes in transcript levels of some antioxidant-related genes in gills and liver following BPAF exposure, imply an effect of BPAF on the immune system. After BPAF exposure, chgs and vtgs were up-regulated, validating the estrogenic effect of BPAF. In the hypothalamic - pituitary - gonadal axis (HPG) results, erα, erγ and cyp19a1b were all up-regulated in the brain, and the 0.93 µg/L BPAF group was more up-regulated than the 9.33 µg/L BPAF group. In testis, BPAF significantly up-regulated the mRNA expression level of cyp17a1 and cyp11b, while significantly down-regulated mRNA expression level of cyp11a, and cyp19a1 was significantly down-regulated only in the 0.93 µg/L BPAF group. In conclusion, environmental levels of BPAF have adverse effects on the survival and reproduction of O. curvinotus, and the potential toxic effects of environmental levels of BPAF cannot be ignored.

Short-term exposure to norethisterone affected swimming behavior and antioxidant enzyme activity of medaka larvae, and led to masculinization in the adult population.

Norethisterone (NET), one of the synthetic progestins, is detected with increasing frequency in the water environment and distributed in the ocean, with a potential toxicity risk to marine organisms. However, current studies on the adverse effects of progestins (including NET) in aquatic environments have focused on freshwater organisms, mainly fish. In the present, marine medaka (Oryzias melastigma) larvae were exposed to 91.31 ng/L NET for 10 days, and then the swimming behavior, oxidation-antioxidant-related enzyme activities, sex and thyroid hormone levels, and the gene transcription patterns of the larvae were measured. After NET treatment, medaka larvae were raised in artificial seawater until 5 months of age, and the sex ratio was counted. Ten-day exposure to 91.31 ng/L NET inhibited swimming behavior, of marine medaka larvae, which showed that the time in the resting state was significantly prolonged, while the time in the large motor state was significantly reduced; disrupted oxidative-antioxidant system, significantly up-regulated the enzymatic activities of reactive oxygen species (ROS), malondialdehyde (MDA), and glutathione peroxidase (GSH-Px); affected the hormone levels of larvae, lowered 11- keto testosterone (11-KT) and triiodothyronine (T3) concentrations. RNA-seq results showed that 91.31 ng/L NET exposure for 10 days changed the transcript levels of 275 genes, of which 28 were up-regulated and 247 were down-regulated. Differentially expressed genes (DEGs) were mainly significantly enriched in piwi interacting RNA (piRNA), gonadal development, gametogenesis, and steroidogenesis biological processes, etc. After removing NET exposure and returning to breeding for 140 days, a significant increase in male proportions (69.67%) was observed in sexually mature medaka populations in the NET-treated group. These results show that exposure to 91.31 ng/L NET for 10 days can lead to various adverse effects on marine medaka larvae. These findings shed light on the potential ecological risks of synthetic progestins to marine organisms.

The first high-quality chromosome-level genome of Eretmochelys imbricata using HiFi and Hi-C data.

Eretmochelys imbricata, a critically endangered sea turtle inhabiting tropical oceans and protected across the world, had an unknown genome sequence until now. In this study, we used HiFi reads and Hi-C technology to assemble a high-quality, chromosome-level genome of E. imbricata. The genome size was 2,138.26 Mb, with contig N50 length of 123.49 Mb and scaffold N50 of 137.21 Mb. Approximately 97.52% of the genome sequence was anchored to 28 chromosomes. A total of 20,206 protein-coding genes were predicted. We also analyzed the evolutionary relationships, gene family expansions, and positive selection of E. imbricata. Our results revealed that E. imbricata diverged from Chelonia mydas 38 million years ago and had enriched olfactory receptors and aging-related genes. Our genome will be useful for studying E. imbricata and its conservation.

Effects of bisphenols on lipid metabolism and neuro-cardiovascular toxicity in marine medaka larvae.

Bisphenols are environmental endocrine disruptors that have detrimental effects on aquatic organisms. Using marine medaka larvae, this study explored the effects of bisphenol compounds [bisphenol A (BPA), bisphenol S (BPS), bisphenol F (BPF), and bisphenol AF (BPAF)] on the early growth and development of aquatic organisms. Marine medaka larvae were exposed to bisphenol compounds at concentrations of 0.05, 0.5, and 5 µM for 72 h, and changes in heartbeat rate, behavior, hormone levels, and gene expression were determined. Bisphenols were shown to have a toxic effect on the cardiovascular system of larvae and can cause neurotoxicity and endocrine disruption, such as changes to thyroid-related hormones. Functional enrichment showed that bisphenols mainly affect lipid metabolism and cardiac muscle contraction of larvae, which implied that the main toxic effects of bisphenols on marine medaka larvae targeted the liver and heart. This study provides a theoretical foundation for evaluating the toxicological effects of bisphenols on the early development of aquatic organisms.

The First High-Quality Chromosome-Level Genome of the Lutjanus erythropterus (Bloch, 1790) Using Single-Tube Long Fragment Reads and Hi-C Technologies.

Lutjanus erythropterus (Bloch, 1790), a Perciformes from the Lutjanidae family, is a commercially important species because of its taste and abundance. Despite the increase in genome resources in recent years, few genome assemblies are available within this fish family for comparative and functional studies. In this study, we determined the chromosomal genome of Crimson snapper using high-throughput Single-Tube Long Fragment Reads sequencing technology and Hi-C data. The final assembly size was 973.04 Mb with contig and scaffold N50 values of 1.51 and 40.65 Mb, respectively. We successfully scaffolded 95.84% of the genome sequence onto 24 chromosomes ranging in length from 19.37 to 49.48 Mb. A total of 22,663 genes and 13,877 gene families were identified in the genome, with 29 gene families being L. erythropterus-specific. A phylogenetic analysis using single-copy gene families showed that L. erythropterus and Larimichthys crocea had the closest genetic relationship with a divergence time of ∼47.7 Ma. This new genomic resource will facilitate comparative genomic studies as well as genetic breeding programs for L. erythropterus.

Analysis of opsin gene family of Crimson snapper (Lutjanus erythropterus).

Opsin is a fellow of the G protein-coupled receptors (GPCRs) superfamily. It can be divided into visual and non-visual opsin according to whether it is directly involved in visual imaging. Opsin plays an important role in visual image formation and the regulation of non-image forming functions such as circadian entrainment in the growth, development and evolution of fish. Crimson snapper belongs to Perciforme mainly found in the Indo-West Pacific and the South China Sea. It is one of the most influential economic fishes in the South China Sea. In order to study the existence and expression of opsin gene in Crimson snapper, we sequenced the genome and tissue sample transcriptome of Crimson snapper. In this study, 32 opsin genes were identified from the genome of Crimson snapper. The length of these genes ranged from 1061 bp to 86203 bp and were distributed on 15 different chromosomes. The analysis of opsin gene family of Crimson snapper showed that the sws2 had two extra copies as compared with that of Zebrafish. Domain and motif analysis revealed that all the 32 opsin genes have seven-(pass)-transmembrane domain receptors (7TM receptors) each, and the opsin family contained 10 common motifs. The expression level of opsin gene, confirmed by RT-qPCR, was analyzed by using nine tissues transcriptome databases of Crimson snapper. The results showed that almost all opsin genes were highly expressed in the retina and brain, except opn7a and opn7b which were expressed in intestine and red skin, and almost no expression in other tissues. Our results provide a comprehensive basic knowledge for the opsin gene family of Crimson snapper, which has significance for the study of the function of opsin in Lutjanidaes.

Comparative Analysis of Central Corneal Thickness in Four Fish Models.

To understand the left-right asymmetry of vertebrate eyes, this study measured the central corneal thickness (CCT) of Oryzias curvinotus, Oryzias melastigma, Oryzias latipes, and zebrafish with optical coherence tomography. The results showed that the CCTs were significant differences among different species and groups, even between the right and left eyes of each fish. The values of the CCTs (mean ± SD, µm) for the four species were 104.71 ± 14.49, 61.88 ± 8.63, 64.76 ± 10.36 and 56.96 ± 10.48, respectively. Moreover, comparing the two wild groups of O. curvinotus from Sanya on N18° and Gaoqiao on N21°, the CCT value for the low-latitude group was 104.71 ± 14.49 µm, greater than the high latitude group 76.13 ± 5.70 µm significantly (t-test, p = 0.0001). Lastly, the paired Student's t-test revealed that significant CCT differences existed between the left and right eye for all four species and groups, of which zebrafish and O. melastigma were belonging to the left thicker type in contrast to the others. This study laid a foundation for understanding the causes of the difference in CCT, and also provided possible fish models for human researches on keratomileusis, glaucoma, and other corneal diseases.

Effects of bisphenol AF on growth, behavior, histology and gene expression in marine medaka (Oryzias melastigma).

Bisphenol AF (BPAF) is one of the substitutes for bisphenol A (BPA), which has endocrine-disrupting, reproductive and neurological toxicity. BPAF has frequently been detected in the aquatic environment, which has been a long-term threat to the health of aquatic organisms. In this study, female marine medaka (Oryzias melastigma) were exposed to 6.7 µg/L, 73.4 µg/L, and 367.0 µg/L BPAF for 120 d. The effects of BPAF on behavior, growth, liver and ovarian histology, gene transcriptional profiles, and reproduction of marine medaka were determined. The results showed that with the increase of BPAF concentration, the swimming speed of female marine medaka showed an increasing trend and then decreasing trend. BPAF (367.0 µg/L) significantly increased body weight and condition factors in females. BPAF (73.4 µg/L and 367.0 µg/L) significantly delayed oocyte maturation. Exposure to 367.0 µg/L BPAF showed an increasing trend in the transcript levels of lipid synthesis and transport-related genes such as fatty acid synthase (fasn), sterol regulatory element binding protein (srebf), diacylglycerol acyltransferase (dgat), solute carrier family 27 member 4 (slc27a4), fatty acid-binding protein (fabp), and peroxisome proliferator-activated receptor gamma (pparγ) in the liver. In addition, 6.7 µg/L BPAF significantly down-regulated the expression levels of antioxidant-related genes [superoxide dismutase (sod), glutathione peroxidase (gpx), and catalase (cat)], and complement system-related genes [complement component 5 (c5), complement component 7a (c7a), mannan-binding lectin serine peptidase 1 (masp1), and tumor necrosis factor (tnf)] were significantly up-regulated in the 73.4 and 367.0 µg/L groups, which implies the effect of BPAF on the immune system in the liver. In the hypothalamic-pituitary-ovarian axis (HPG) results, the transcription levels of estrogen receptor α (erα), estrogen receptor ß (erß), androgen receptor (arα), gonadotropin-releasing hormone 2 (gnrh2), cytochrome P450 19b (cyp19b), aromatase (cyp19a), and luteinizing hormone receptor (lhr) in the brain and ovary, and vitellogenin (vtg) and choriogenin (chg) in the liver of 367.0 µg/L BPAF group showed a downward trend. In addition, exposure to 367.0 µg/L BPAF for 120 d inhibited the spawning behavior of marine medaka. Our results showed that long-term BPAF treatment influenced growth (body weight and condition factors), lipid metabolism, and ovarian maturation, and significantly altered the immune response and the transcriptional expression levels of HPG axis-related genes.

Long-term exposure to bisphenol A and its analogues alters the behavior of marine medaka (Oryzias melastigma) and causes hepatic injury.

Bisphenols (BPA, BPF, and BPAF) are widely present in the aquatic environment and have various adverse effects on aquatic organisms. However, their hepatic toxicity in marine fish is not fully understood. Hence, we examined the growth parameters, histological features, antioxidant defense mechanisms, and gene expression profiles in the livers of marine medaka after exposure to single and combined bisphenols for 70 days. The final body weight and final body length of males exposed to BPAF were significantly higher than those in the control group, and the survival rate was significantly lower. Bisphenol exposure led to vacuolization and local lesions in the liver, especially in the BPAF group, and altered antioxidant enzyme activity in the liver, leading to oxidative stress. In addition, after bisphenol exposure, marine medaka showed anxiolytic effects and a significant reduction in swimming distance compared with that in the control group. As determined by RNA-seq, bisphenol exposure altered multiple biological pathways in the liver, such as fatty acid biosynthesis, fatty acid metabolism, and ribosome biogenesis pathways, with significant changes in gene expression levels. In particular, chgs and vtgs were significantly up-regulated after BPAF exposure, suggesting an estrogenic effect. Overall, bisphenols can adversely affect the growth and metabolism of marine medaka. BPF and BPAF may not be ideal substitutes for BPA.