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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.

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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.

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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.

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The DMY gene is deleted in all males of the Sanya population (SY-medaka) of the Hainan medaka, Oryzias curvinotus, as recently reported by us. However, due to limited knowledge regarding their population genetic background, it is difficult to explore the possible evolutionary pathway. Herein, we resequenced the whole genome of four populations, including SY-medaka. A total of 56 mitogenomes and 32,826,105 SNPs were identified. We found that the genetic differentiation is highest between SY-medaka and the other populations. The results of the population history of the O. curvinotus suggest that the SY-medaka has been in a bottleneck period recently. Further analysis shows that SY-medaka are the most strongly affected by environmental selection. Moreover, we screened some potential genomic regions, and the genes contained in these regions may explain the potential mechanism of the selection process of the SY-medaka. In conclusion, our study can provide new clues for the adaptation process of medaka in the new environment of Sanya.

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Hainan medaka (Oryzias curvinotus) is distributed in the coastal waters of the South China Sea and is able to adapt to a wide range of salinities. In this study, we characterized O. curvinotus in Sanya River (SY-medaka), which lacks dmy (a male sex-determining gene in O. latipes and O. curvinotus). In a comparison of SY-medaka and Gaoqiao medaka (GQ-medaka), the morphological difference between the two populations does not reach the subspecies level and they can be considered two geographic populations of O. curvinotus. A mitochondrial cytochrome oxidase subunit I (CoI) sequence alignment showed that the sequence identities between SY-medaka and other geographic populations of O. curvinotus are as high as 95%. A phylogenetic analysis of the mitochondrial genome also indicated that SY-medaka belongs to O. curvinotus. Molecular marker-based genetic sex assays and whole genome re-sequencing showed that SY-medaka does not contain dmy. Further, in RNA-Seq analyses of the testis and ovaries of sexually mature SY-medaka, dmy expression was not detected. We speculate that high temperatures resulted in the loss of dmy in SY-medaka during evolution, or the lineage has another sex-determining gene. This study provides a valuable dataset for elucidating the mechanism underlying sex determination in Oryzias genus and advances research on functional genomics or reproduction biology in O. curvinotus.

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The effect of estrogens on Oryzias curvinotus juveniles were investigated by sequencing the transcriptome of O. curvinotus juveniles exposed to 17 ß - estradiol for 24 h. A total of 69,071,524 and 71,210,528 raw reads were obtained for the control group (NC) and 17 ß - estradiol exposure group (E2), respectively. After de novo assembly, total 133,210 unigenes were identified, and 85,837 unigenes (64.44% of 133,210) were annotated. Analysis of the transcriptome showed that exposure to 2 µg/L 17 ß - estradiol led to the up-regulation of 19 genes and down-regulation of 18 genes. The eef1b and rps4x was most suitable as controls for quantitative real-time PCR (qPCR) using Reffinder. Different expression genes enrichment analysis found that exposed to 2 µg/L 17 ß - estradiol affected various physiological processes, including spliceosome, phototransduction, amino sugar and nuclear sugar metabolism, hypotaurine metabolism, and renin-angiotensin system, etc. Exposing O. curvinotus juveniles to increasing concentrations of 17 ß - estradiol (2 ng/L, 20 ng/L, 200 ng/L and 2 µg/L) led to significant up-regulation of vitellogenins (vtgs) and choriogenins (chgs) mRNA expression. The present study is the first high-throughput transcriptome sequencing of O. curvinotus juveniles, which will be useful for future functional analysis of genes related to environmental estrogen exposed, and development of biomarkers.

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In this study, combining the liver transcripts from both sexes by RNA-Seq with DNA sequences by conventional PCRs, we have determined the complete mitogenome of Hainan medaka Oryzias curvinotus collected from the mangrove seawater of the Leizhou Peninsula in tropical South China (Accession no.: KY364884). The mitochondrial genome is 16,676 bp, and its content and structure are highly homologous to those of other teleostean fishes, including 13 protein-coding genes (PCGs), 2 rRNAs, 22 tRNA and 1 control region. Among the PCGs, ATG is used as the initiation codon, except for GTG in COI gene. There are 7 overlapping genes with overlap lengths ranging from 1 to 10 nucleotides (nt), while ten intergenic regions with a total of 66 nt and a maximum interval of 37 nt between tRNAAsn and tRNACys. Moreover, the data from RNA-Seq shows that the significant differences exist in the expression patterns of mitogenomes between male and female.