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.

The effects of 17α-methyltestosterone on gonadal histology and gene expression along hypothalamic-pituitary-gonadal axis, germ cells, sex determination, and hypothalamus-pituitary-thyroid axis in zebrafish (Danio rerio).

As a synthetic androgen, 17α-methyltestosterone (MT) is widely used in aquaculture to induce sex reversal and may pose a potential risk to aquatic organisms. This ecological risk has attracted the attention of many scholars, but it is not comprehensive enough. Thus, the adverse effects of MT on zebrafish (Danio rerio) were comprehensively evaluated from gonadal histology, as well as the mRNA expression levels of 47 genes related to hypothalamic-pituitary-gonadal (HPG) axis, germ cell differentiation, sex determination, and hypothalamus-pituitary-thyroid (HPT) axis. Adult zebrafish with a female/male ratio of 5:7 were exposed to a solvent control (0.001% dimethyl sulfoxide) and three measured concentrations of MT (5, 51 and 583 ng/L) for 50 days. The results showed that MT had no significant histological effects on the ovaries of females, but the frequency of late-mature oocytes (LMO) showed a downward trend, indicating that MT could induce ovarian suppression to a certain extent. The transcriptional expression of activating transcription factor 4b1 (atf4b1), activating transcription factor 4b2 (atf4b2), calcium/calmodulin-dependent protein kinase II delta 1 (camk2d1), calcium/calmodulin-dependent protein kinase II delta 2 (camk2d2) and calcium/calmodulin-dependent protein kinase II inhibitor 2 (camk2n2) genes in the brain of females increased significantly at all treatment groups of MT, and the mRNA expression of forkhead box L2a (foxl2) and ovarian cytochrome P450 aromatase (cyp19a1a) genes in the ovaries were down-regulated by 5 and 583 ng/L group, which would translate into inhibition of oocyte development. As compared to females, MT had relatively little effects on the reproductive system of males, and only the transcriptional alterations of synaptonemal complex protein 3 (sycp3) and 17-alpha-hydroxylase/17,20-lyase (cyp17) genes were observed in the testes, not enough to affect testicular histology. In addition, MT at all treatments strongly increased corticotropin-releasing hormone (crh) transcript in the brain of females, as well as deiodinase 2 (dio2) transcript in the brain of males. The paired box protein 8 (pax8) gene was significantly decreased at 51 or 583 ng/L of MT in both female and male brains. The above results suggest that MT can pose potential adverse effects on the reproductive and thyroid endocrine system of fish.

Physiological and transcriptional effects in the male western mosquitofish (Gambusia affinis) following exposure to dexamethasone.

Dexamethasone (DEX) is a synthetic glucocorticoid widely found in a variety of aquatic environments and has potential adverse effects on aquatic organisms. This study was to assess the toxic effects of exposure to different concentrations (0, 5 and 50 µg/L) of DEX for 60 days on adult male mosquitofish (Gambusia affinis). Morphological analyses of skeleton and anal fin, histological effects of testes and livers, and transcriptional expression levels of genes related to reproductive and immune system were determined. The results showed that exposure to DEX significantly increased 14L and 14D values of hemal spines, which suggested DEX could affect skeleton development and result in more masculine characteristics in male fish. In addition, the damage to testis and liver tissue was observed after DEX treatment. It also enhanced mRNA expression of Erß gene in the brain and Hsd11b1 gene in the testis. The findings of this study reveal physiological and transcriptional effects of DEX on male mosquitofish.

Toxic effects of bisphenol AF on the embryonic development of marine medaka (Oryzias melastigma).

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.

Transcriptome Analysis Revealed the Advantages of Room Temperature Preservation of Concentrated Oocystis borgei Cultures for Use in Aquaculture.

Oocystis borgei, a microalgae species employed for regulating the quality of aquaculture water, demonstrates the capacity to adsorb noxious substances, curtail the growth of detrimental bacteria, and outcompete blooming cyanobacteria. It can be concentrated by natural sedimentation and stored at room temperature, making it costless and simple to transport and use. To study the mechanism of adaptation to room temperature preservation, O. borgei was concentrated (1.19 × 107-1.21 × 107 cell/mL) and stored for 50 days at low (5 °C, LT), normal (25 °C, NT), and high (35 °C, HT) temperatures, respectively. Polysaccharide content, lipid content, cell survival, and resuscitation were evaluated. RNA-Seq was also used to examine how concentrated O. borgei responded to temperature. During storage, there was an increase in polysaccharide content and a decrease in lipid content, with both being significantly upregulated in the LT and HT groups. Survival and cell density were highest in the NT group. The RNA-Seq analysis revealed extensive differences in transcript levels. ATP synthesis was inhibited in the LT group due to the reduced expression of PsaD, PsaE, PsaF, PsaK, and PsaL. Under HT, the formation of reactive oxygen species (ROS) was facilitated by low levels of redox-related genes (nirA) and high levels of oxidative genes (gdhA, glna, and glts). The findings suggest that storing concentrated O. borgei at room temperature is optimal for microalgae preservation, enhancing theoretical research in this field. Our study provides further theoretical and practical support for the development of O. borgei as a live ecological preparation for aquaculture microalgae ecology management.

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.

Histology, physiology, and glucose and lipid metabolism of Lateolabrax maculatus under low temperature stress.

Spotted sea bass (Lateolabrax maculatus) is a popular and important commercial fish throughout the world, but it is unknown whether introducing domesticated fish to locations that experience cold weather might alter physiological performance. In this study, we evaluated the behavior, fatty acid content, histological analysis of liver and gills, liver enzymatic activity in carbohydrate and lipid metabolism, and gene expression in liver related to carbohydrate and lipid metabolism of spotted sea bass acclimatized at 22 °C (control), 16 °C, 10 °C, 8 °C, and 4 °C for 24 h, and 8 °C for 4 days. When L. maculatus was exposed to acute cold stress for 24 h, the gill showed curling, lamellar disorganization, lamellar epithelium hyperplasia, and formed aneurysms inside of the secondary lamellae. Long term stress over four days resulted in severe lamellar epithelium hyperplasia and curling. Continued extreme cold exposure (4 °C) in L. maculatus caused liver HK, PK levels and LDH activities to achieve a peak value at 0 h, and decreased over time. These indicated that glucose metabolism might play critical roles in the initial time of stress. Results of carbohydrate and lipid metabolism showed that lipids appear to play roles in prolonged cold stress. The constitutive transcriptional levels of six genes related to glucose (G6Pase) and lipid metabolism (PPAR-α, PPAR-γ) and mTOR signal pathway (eif4ebp1, eif4ebp2, mlst8) genes increased significantly in most groups during cold stress.

Histological and transcriptional effects of androstenedione in adult zebrafish (Danio rerio).

As a natural androgen, androstenedione (AED) may pose potential risks to aquatic organisms due to its ubiquitousness in aquatic environments. Here we assessed the adverse effects of AED on histology of gonads, as well as mRNA expression levels of 34 genes concerned with hypothalamic-pituitary-gonadal (HPG) axis, germ-cell differentiation and sex differentiation in zebrafish (Danio rerio). Adult zebrafish were exposed to solvent control and three measured concentrations of 0.2, 2.3 and 23.7 µg/L AED for 60 days. The results showed that AED did not induce any obvious histological effects in the ovaries and testes. Of the investigated genes, transcriptional expression levels of amh and cyp11c1 genes in the ovaries of females were significantly increased by AED at 2.3 or 23.7 µg/L. However, different exposure concentrations of AED significantly inhibited mRNA expression of gnrh3, atf4b1 and cyp19a1b in the brain of males. In the testes of males, AED at 2.3 µg/L led to a significant induction of sox9b gene, but it at 23.7 µg/L down-regulated nr5a1b gene. These observed transcriptional changes indicated that AED could pose potential androgenic effects in zebrafish.

Molecular characterization of Pod1 during sex development in Chinese tongue sole (Cynoglossus semilaevis).

Pod1 encodes a Class II bHLH transcription factor involved in the development of a number of tissues such as gonad, spleen, lungs and heart. However, to date, little is known about its function in teleosts. In this study, we cloned and characterized Pod1 gene from Cynoglossus semilaevis. This gene contains three exons and two introns, with the full-length cDNA of 918 nucleotides that encodes a 183 amino acid protein with a conserved bHLH domain. Realtime quantitative PCR revealed that Pod1 was predominantly expressed in the testes of C. semilaevis. In different stages of testes development, Pod1 expression was undetectable up to 120 days after hatching (dah), and then increased at 210 dah and 1 year after hatching (yah). Furthermore, in situ hybridization (ISH) analysis revealed that Pod1 was mainly localized in the germ cells of testes, but was not detected in ovarian cells; which suggested its possible functions in spermatogenesis of C. semilaevis. The methylation profile analysis of Pod1 genomic sequence in the gonads showed that the differences in their putative promoter regions of Pod1 among ovary, male and pseudo-male testes were not obvious. Thus, further research might be needed to evaluate whether Pod1 expression is regulated by epigenetic level.

Characterization and expression pattern of r-spondin1 in Cynoglossus semilaevis.

r-spondin1 (rspo1) encodes a secreted protein that is involved in the determination and differentiation of the mammalian ovary. However, little information is yet available for teleosts. Here, we identified a homologue of rspo1 in Cynoglossus semilaevis. The full-length cDNA of rspo1 had a length of 2,703 bp with an open reading frame of 834 bp, encoding a protein with a length of 277 amino-acids. rspo1 expression was detected via qRT-PCR in various tissues, and significant sexually dimorphic expression was observed in the gonads. Furthermore, ISH located rspo1 in germ cells such as spermatogonia, spermatocytes, spermatids, spermatozoa, and oocytes, as well as in somatic cells of the gonads. Following knockdown of rspo1 in an ovarian cell line, the expressions of wnt4a, ß-catenin, foxl2, and StAR were highly affected; wnt4a and ß-catenin were significantly downregulated, whereas foxl2 and StAR were significantly upregulated. In summary, these data suggest that rspo1 may be involved in the regulation of ovarian development and differentiation through a conserved pathway, while the function of the gene in the testis remains elusive.

A first generation BAC-based physical map of the half-smooth tongue sole (Cynoglossus semilaevis) genome.

BACKGROUND: Half-smooth tongue sole (Cynoglossus semilaevis Günther) has been exploited as a commercially important cultured marine flatfish, and female grows 2-3 times faster than male. Genetic studies, especially on the chromosomal sex-determining system of this species, have been carried out in the last decade. Although the genome of half-smooth tongue sole was relatively small (626.9 Mb), there are still some difficulties in the high-quality assembly of the next generation genome sequencing reads without the assistance of a physical map, especially for the W chromosome of this fish due to abundance of repetitive sequences. The objective of this study is to construct a bacterial artificial chromosome (BAC)-based physical map for half-smooth tongue sole with the method of high information content fingerprinting (HICF). RESULTS: A physical map of half-smooth tongue sole was constructed with 30, 294 valid fingerprints (7.5 × genome coverage) with a tolerance of 4 and an initial cutoff of 1e-60. A total of 29,709 clones were assembled into 1,485 contigs with an average length of 539 kb and a N50 length of 664 kb. There were 394 contigs longer than the N50 length, and these contigs will be a useful resource for future integration with linkage map and whole genome sequence assembly. The estimated physical length of the assembled contigs was 797 Mb, representing approximately 1.27 coverage of the half-smooth tongue sole genome. The largest contig contained 410 BAC clones with a physical length of 3.48 Mb. Almost all of the 676 BAC clones (99.9%) in the 21 randomly selected contigs were positively validated by PCR assays, thereby confirming the reliability of the assembly. CONCLUSIONS: A first generation BAC-based physical map of half-smooth tongue sole was constructed with high reliability. The map will promote genetic improvement programs of this fish, especially integration of physical and genetic maps, fine-mappings of important gene and/or QTL, comparative and evolutionary genomics studies, as well as whole genome sequence assembly.

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.

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.

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.

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.

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.

The impact of co-exposure to polystyrene microplastics and norethindrone on gill histology, antioxidant capacity, reproductive system, and gut microbiota in zebrafish (Danio rerio).

In recent years, studies have focused on the combined ecological risks posed by microplastics and other organic pollutants. Although both microplastics and progestin residues are frequently detected in the aquatic environments, their ecological implications remain unknown. Adult zebrafish were exposed to polystyrene microplastics (PS, 200 nm, 200 µg/L), norethindrone (NET, 69.6 ng/L), and their mixture (200 µg/L PS + 63.1 ng/L NET) for 30 days. The results demonstrated that exposure to PS and NET resulted in gill damage. Notably, the PS and PS+NET exhibited a significant decrease in glutathione (GSH) and oxidized glutathione (GSSG) content, as well as reduced antioxidase activity in the gills. The oxidative stress in PS+NET primarily originated from PS. The PS, NET, or their mixture resulted in a decrease in testosterone (T) and estradiol (E2) levels in female. Furthermore, compared to NET, the PS+NET showed a significant reduction in E2 levels, thereby augmenting the inhibitory effect on reproductive ability mediated by NET. However, males showed an increase in 11-ketodihydrotestosterone (11-KT) content, accompanied by a significant decrease in spermatogonia (Sg) and increase in spermatocytes (Sc). Consequently, it can be inferred that PS enhances the androgenic effect of NET. In female fish brain, NET alone resulted in transcriptional down-regulation of partial hormone receptors; however, co-administration of PS effectively mitigated the interference effects. Furthermore, transcriptional downregulation of 17-alpha-hydroxylase (cyp17), hydroxysteroid 3-beta dehydrogenase (hsd3b), estrogen receptor 1 (esr1), and estrogen receptor 2a (esr2b) genes in the ovary was found to be associated with the androgenic activity induced by NET. Moreover, in comparison to PS or NET alone, PS+NET resulted in a notable decrease in Cetobacterium abundance and an increase in Aeromonas population, suggesting that the co-exposure of PS+NET may exacerbate intestinal burden. The findings highlight the importance of studying the combined toxicity of PS and NET.

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.