Fish represent the most species-rich group within the phylum Chordata, possessing exceptional nutritional and ornamental value. Global aquaculture, particularly finfish farming, is experiencing rapid expansion worldwide, and fish serve as crucial model organisms for vertebrate developmental biology and functional genomics research. However, traditional breeding methods are plagued by limitations such as low precision and lengthy breeding cycles. Currently, gene editing technologies represented by the CRISPR/Cas system, base editing, and prime editing have provided revolutionary tools for dissecting gene function, modeling human diseases, targeted trait improvement, and ecological adaptation studies. This review describes the evolutionary history of gene editing technology, compares gene delivery strategies in fish embryos, and highlights landmark applications in key areas, including gene function research, aquaculture breeding, ornamental fish coloration regulation, and human disease model construction. Finally, we propose that innovation should be pursued while ensuring biosafety and regulatory compliance, to promote the transformation of fish gene editing toward large-scale and safe application.

The Tomato hind (Cephalopholis sonnerati) is an economically important marine fish species. As the skin serves as the primary barrier against pathogens, establishing a skin cell line is crucial for in-depth studies of fish immunology. In this study, a continuous and stable cell line, designated TGSK, was successfully established from the skin tissue of tomato hind for the first time. The cells exhibited robust growth and a fibroblast-like morphology in Leibovitz’s L-15 medium supplemented with 20% fetal bovine serum at 28°C, and have been stably subcultured for over 63 passages. Karyotype analysis revealed a diploid chromosome number of 2n = 48. The species origin was verified by sequencing the mitochondrial COI gene.The cells maintained good viability after cryopreservation in liquid nitrogen and were successfully transfected with exogenous plasmids to express fluorescent proteins. To investigate its immunological utility, a dynamic transcriptomic analysis was performed on TGSK cells following infection with Vibrio harveyi. The results showed that the number of differentially expressed genes increased over time post-infection and were significantly enriched in key innate immune and inflammatory pathways, including the Toll-like receptor, NOD-like receptor, C-type lectin receptor, NF-κB, and necroptosis signaling pathways. In conclusion, the TGSK cell line established here exhibits stable growth and well-defined characteristics. It not only provides a valuable tool for germplasm conservation and immunology research of tomato hind but also, through the infection model, elucidates the dynamic regulatory network of early host immune responses, thereby laying a solid foundation for future research on disease resistance mechanisms in fish.

Sex-specific markers are crucial molecular tools for identifying the genetic sex and enhancing the economic value of fish, and server as an important foundation for understanding sex determination mechanisms. Hemibagrus guttatus is a rare freshwater fish known for its delicate flavor and high nutritional value. Due to overfishing, wild populations have been severely impacted, and artificial breeding techniques are still underdeveloped. Cultivating more egg-producing females is beneficial for expanding aquaculture scale and promoting species conservation. However, the long-term lack of effective sex identification methods has hindered both the efficiency of artificial breeding and research on the sex determination of H. guttatus. In this study, whole-genome resequencing was performed on 20 males and 20 females, generating a total of 224.77 Gb of male data and 259.82 Gb of female data. Through comparative genomic analysis between male and female, 40 candidate male-specific sequences were identified. After further PCR validation, a male-specific marker was successfully developed and named HG46M2. Subsequently, the marker was applied to identify the genetic sex in three additional H. guttatus populations. Moreover, the genetic sex of the population feed with Limnodilus spp. was identified by HG46M2 marker and determined the sex reversal from male to female in breeding. This study provides a rapid and effective method for determining the genetic sex of H. guttatus. The sex-specific marker developed here will contribute to improving the efficiency of artificial breeding, facilitate sex-controlled breeding programs, and holds great significance for research on sex determination and conservation efforts for H. guttatus.

This study establishes a CRISPR/Cas9 gene-editing protocol for the tiger grouper (Epinephelus fuscoguttatus) for the first time. We immobilized zygotes in agarose molds and used custom-pulled microinjection needles to deliver 2-3 nL of ribonucleoprotein (RNP) targeting the cyp19a1a gene and foxl2 gene into each egg at the I-VIIIcell stage, respectively. For cultivating gene-edited fry, we firstly established a protocol for hatching and meticulous rearing of Epinephelus eggs in room. The embryonic survival was detected at 72 h post-fertilization, we found the embryonic survival was at 16% in the experimental group versus 90% in the control group. Additionally, the mutation of the injected larvae was detected at 4 days post-fertilization (dpf). Two type of mutation in both cyp19a1a gene and foxl2 gene were observed with 15% mutation efficiency, respectively. Moreover, we also detected the mutation of the gene-edited fry at 60 dpf in the fin-clip. We found that 5 out of 53 individuals was detected mutation displaying three different mutation types in cyp19a1a gene. Until 120d, the experimental group are still 53 individuals surviving, corresponding to a larval survival rate of 10%, approximately 24% in the control group. This protocol provides a powerful tool for dissecting sex-determination mechanisms and for developing optimized breeding strategies in tiger grouper.

The rapid expansion of aquaculture breeding has increased the need to identify wild genetic resources for germplasm management and sustainable breeding. However, distinguishing wild from domesticated populations remains challenging in species with recent domestication histories and low genetic differentiation. In this study, we used the large yellow croaker (Larimichthys crocea) as a case study species to develop specific insertion/deletion (InDels)-based identification methods for wild and domesticated populations based on whole-genome resequencing data. Four large-fragment InDels with significant allelic frequency differences between populations were selected as one marker panel. In addition, four coding-region InDels predicted to alter amino acid sequences were selected as another marker panel based on allele-frequency differences, coding-region annotation, and supporting SNP-based Fst or nucleotide-diversity signals. Principal component analysis (PCA) based on these selected InDel loci showed partial separation with some overlap between wild and domesticated samples. Therefore, multi-locus scoring models were established by assigning scores to wild-predominant alleles. Receiver operating characteristic (ROC) analysis showed strong discriminatory performance for both panels, with area under the ROC curve (AUC) values of 0.9608 and 0.9771 and classification agreement rates with known sample status of 90.0% and 91.7%, respectively. These results indicate that targeted InDel markers can provide a practical PCR-based approach for distinguishing wild and domesticated large yellow croaker populations with limited genetic differentiation. This workflow may provide a reference for marker development in other recently domesticated species, although further validation in independent populations and target species is needed.

Cephalopholis sonnerati represents a commercially important marine teleost. As the skin serves as the primary barrier against pathogens, the establishment of a skin-derived cell line is considered essential for advancing immunological research in this species. In this study, a novel continuous cell line was successfully developed from tomato hind skin tissue and has been designated TGSK. The cells were observed to exhibit robust proliferation and a fibroblast-like morphology when maintained in Leibovitz’s L-15 medium supplemented with 20% fetal bovine serum at 28°C, and stability was maintained for over 63 passages. The karyotype was determined to be diploid with a chromosome number of 2n = 48. The species origin was verified by sequencing the mitochondrial COI gene. The cells maintained good viability after cryopreservation in liquid nitrogen and were successfully transfected with exogenous plasmids to express fluorescent proteins. To investigate its immunological utility, dynamic transcriptomic profiling was conducted following challenge with Vibrio harveyi. It was observed that the repertoire of differentially expressed genes expanded progressively post-infection and was significantly enriched in innate immune and inflammatory pathways, including the Toll-like receptor, NOD-like receptor, C-type lectin receptor, NF-κB, and necroptosis signaling pathways. In conclusion, the TGSK cell line established here exhibits stable growth and well-defined characteristics. It not only provides a valuable tool for germplasm conservation and immunology research of tomato hind but also, through the infection model, elucidates the dynamic regulatory network of early host immune responses, thereby laying a solid foundation for future research on disease resistance mechanisms in fish.

The tomato grouper (Cephalopholis sonnerati) is a commercially valuable species in mariculture, however, infections caused by Vibrio, particularly Vibrio harveyi (V. harveyi), present significant challenges to sustainable aquaculture. To elucidate the immune response to V. harveyi, a novel intestinal cell line of tomato grouper was established, designated TGGI (tomato grouper gastrointestinal), which was cultured in L-15 containing 20 % FBS at 28 °C for over one year and underwent 65 passages. The TGGI cell line shew typical epithelial-like morphology. The origin was confirmed by sequence of COI gene, and karyotype analysis revealed a diploid chromosome number of 2n = 48. TGGI was transfected with pEGFP-N3 plasmid, then emitted strong green fluorescent signals, suggesting potential utility as a tool of exogenous gene functional researches. Furthermore, transcriptomic analysis was performed on TGGI cell lines after infection with V. harveyi at different time points, the results revealed that the majority of differentially expressed genes (DEGs) were enriched in immune-related pathways, including the IL-17, NF-ĸB, Toll-like receptor signaling pathway. Indicating that TGGI cells can recognize pathogen-associated antigens and activate immune responses. In summary, a novel intestinal cell line of grouper, designated TGGI, was successfully established and was suitable for investigating bacterial immune responses. TGGI is expected to play a significant role in multiple fields, particularly by providing a powerful ex vivo tool for elucidating the immune response mechanisms underlying bacterial infection of the intestine.

Monopterus albus is a protogynous hermaphroditic fish that changes from female to male, but the underlying sex change mechanism remains as-yet unknown. In this study, we firstly cloned and characterized the sequence and protein structure of unc-13d of M. albus. We found that the genomic structure of unc-13d was different from other species. Expression was detected in the developing gonad by applying qRT-PCR and in situ hybridization. We found that the expression of unc-13d in the ovotestis was higher than in the ovary and testes. A strong signal of unc-13d was detected in oocytes and granulosa cells in the ovary and spermatogonia and primary spermatocytes in the testes. We found that the promoter methylation of unc-13d was negatively correlated with gene expression in developing gonads, especially at site 114. A dual-luciferase assay was designed and revealed that dmrt1 regulates promoter activity opposite to foxl2. In summary, during sex reversal, DNA methylation affects the binding of the transcription factor dmrt1 and foxl2 in the promoter region through methylation and demethylation interactions to regulate the expression of unc-13d during gonadal development.

Nr5a2 (nuclear receptor subfamily 5, group a, member 2) is involved in gonad development and sex hormone synthesis. In this study, the full length of Nr5a2f and Nr5a2m were obtained by Nr5a2 variable splicing from Andrias davidianus, and the tissue distribution was detected. We identified Nr5a2f of 2455 bp and Nr5a2m of 2150 bp length, encoding 479 and 325 amino, respectively. We first characterized Nr5a2f and Nr5a2m gene expression in developing gonads. Results showed that Nr5a2f had significantly high expression in the ovary and little expression in other tissues, during the sex differentiation and sex reversal, Nr5a2f expression was gradually decreased in the ovary and the expression in the testis was significantly lower than in the ovary from 1 year to 6 year old. Significantly high expression was observed in the ovary and reversal ovary, while low expression was in the testis and reversal testis. While Nr5a2m expression exhibited the opposite profile, high expression was observed in the brain and testis. During sex differentiation and sex reversal, high expression was shown in the testis and low expression in the ovary from one year to six years old and significantly higher expression emerged in testis and reversal testis than in ovary and reversal ovary. In situ hybridization, results showed that Nr5a2f began to express in female undifferentiated gonads and the expression level increased from 48 dpf to 91, while Nr5a2m was expressed in male undifferentiated gonads. Three RNA interference sites were designed and we detected that site 293 exhibited a significant inhibitory effect in ovary cells. After Nr5a2f expression was inhibited by site 293, we observed that female-based gene Nr5a2f, foxl2 and cyp19 expression were decreased, while the male-based gene dmrt1 and cyp17 expression was increased. These results suggested that Nr5a2f and Nr5a2m exhibited different expression patterns in the process of sex differentiation, which provided a foundation for further functional characterizations.

As a fundamental taxonomic group within vertebrates, fish represent an invaluable resource for investigating the mechanisms underlying sex determination and differentiation owing to their extensive geographical distribution and rich biodiversity. Within this biological cohort, the processes of sex determination and differentiation are intricately governed by both genetic factors and the complex interplay of environmental cues. While variations in external environmental factors, particularly temperature, can exert a modulatory influence on sex differentiation in fish to a limited degree, genetic factors remain the primary determinants of sexual traits. Hermaphroditic fish display three distinct types of sexual transitions: protandry (male to female), protogyny (female-to-male), bidirectional sex change (both directions serially). These fish, characterized by their unique reproductive strategies and sexual plasticity, serve as exemplary natural models for elucidating the mechanisms of sex differentiation and sexual transitions in fish. The present review delves into the histological dynamics during gonadal development across three types of sequential hermaphroditic fish, meticulously delineating the pivotal characteristics at each stage, from the inception of primordial gonads to sexual specialization. Furthermore, it examines the regulatory genes and associated signaling pathways that orchestrate sex determination and differentiation. By systematically synthesizing these research advancements, this paper endeavors to offer a comprehensive and profound insight into the intricate mechanisms governing sex differentiation in sequential hermaphroditic fish.

Hemibagrus guttatus is a commercially valuable freshwater fish in the Pearl River Basin, renowned as the "King of Freshwater Fish." Due to habitat degradation and overfishing, its wild population has declined sharply, leading to its listing as a National Key Protected Wild Animal of Class II in China. Artificial breeding is therefore crucial for conservation, yet progress is hindered by the lack of clear sexual dimorphism and poor understanding of its sex differentiation mechanism. In this study, we performed high-throughput RNA sequencing (RNA-seq) to compare gonadal transcriptomes of male and female H. guttatus. A total of 3245 differentially expressed genes (DEGs) were identified, including 3122 male-biased and 123 female-biased DEGs, which clustered into three distinct expression patterns. Enrichment analysis revealed that genes associated with the TGF-β (Transforming Growth Factor-beta) and GnRH (Gonadotropin-Releasing Hormone) signaling pathways were significantly enriched in the female gonads, suggesting their potential roles in gonadal differentiation. From the DEG set, we further highlighted five genes with pronounced sex-biased expression: rbm46 (RNA Binding Motif Protein 46) exhibited gonad-specific expression, whereas myc (v-myc avian myelocytomatosis viral oncogene homolog), angptl4 (Angiopoietin-Like 4), sox9 (SRY-Related HMG-Box Gene 9), and fzd2 (Frizzled Class Receptor 2) showed marked expression differences between male and female gonads. These findings provide insights into the molecular mechanisms underlying sex differentiation in H. guttatus, offer potential molecular markers for sex identification, and establish a scientific basis for germplasm conservation and the optimization of breeding techniques.

为解析黄鳝(Monopterus albus)性逆转机制,实验以雌、雄、间性发育黄鳝为研究对象,分析不同组织、不同发育时期性腺、甲基睾丸酮处理性腺及Zebularine处理性腺原代细胞后dynlt3 基因表达模式的变化及其在性腺中的表达定位.性腺转录组测序结果显示,基因全长1082 bp,开放阅读框354 bp,编码117 个氨基酸.生物信息学分析显示,dynlt3 基因编码蛋白质的二级结构包含37.96％的α-螺旋,29.20％的β-折叠,32.85％的无规则卷曲.系统进化分析结果显示,黄鳝DYNLT3 氨基酸序列与硬骨鱼纲中底鳉(Fundulus heteroclitus)同源性最高.实时荧光定量PCR结果表明,dynlt3 基因在黄鳝肌肉和脑具有较高表达,心脏次之,在其它各组织表达量较低.在性腺的不同发育时期,在间性后期和雄性中表达量显著性高于雌性与间性早期.甲基睾酮处理后黄鳝卵巢组织结构发生明显退化,卵母细胞退化且数量减少,结缔组织间出现空泡结构;dynlt3 基因在卵巢中表达量显著性下调.原位杂交分析dynlt3 基因在性腺组织中的表达定位结果显示,在不同性腺发育时期均检测到dynlt3 阳性信号,间性性腺组织中dynlt3 基因主要在精原细胞和初级精母细胞中表达,精巢组织中dynlt3 基因可在精原细胞与初级精母细胞中,卵巢组织阳性信号较弱,主要在Ⅱ时相卵母细胞细胞质中表达.Zebularine处理性腺原代细胞后,dynlt3 基因表达无显著性差异.以上研究表明,dynlt3 参与黄鳝性腺发育过程,并在黄鳝性逆转及性腺细胞发育过程中发挥重要作用,但是甲基化可能不参与其基因表达调控.

Monopterus albus is a hermaphroditic fish with sex reversal from ovaries to testes via the ovotestes in the process of gonadal development, but the molecular mechanism of the sex reversal was unknown. We produced transcriptomes containing mRNAs and lncRNAs in the crucial stages of the gonad, including the ovary, ovotestis and testis. The expression of the crucial lncRNAs and their target genes was detected using qRT‒PCR and in situ hybridization. The methylation level and activity of the lncRNA promoter were analysed by applying bisulfite sequencing PCR and dual-luciferase reporter assays, respectively. This effort revealed that gonadal development was a dynamic expression change. Regulatory networks of lncRNAs and their target genes were constructed through integrated analysis of lncRNA and mRNA data. The expression and DNA methylation of the lncRNAs MSTRG.38036 and MSTRG.12998 and their target genes Psmβ8 and Ptk2β were detected in developing gonads and sex reversal gonads. The results showed that lncRNAs and their target genes exhibited consistent expression profiles and that the DNA methylation levels were negatively regulated lncRNA expression. Furthermore, we found that Ptk2β probably regulates cyp19a1 expression via the Ptk2β/EGFR/STAT3 pathway to reprogram sex differentiation. This study provides novel insight from lncRNA to explore the potential molecular mechanism by which DNA methylation regulates lncRNA expression to facilitate target gene transcription to reprogram sex differentiation in M. albus, which will also enrich the sex differentiation mechanism of teleosts. Monopterus albus is a hermaphroditic fish that undergoes sex reversal from female to male via intersex during the process of the gonadal differentiation which was an ideal model for epigenetic modification research. After laying eggs, the female M.albus reversal to the intersex. So that the female have a shorter stage and smaller body size which cause low egg production. In the present study, we produced the transcriptomes which contain mRNA and lncRNA in the crucial stage of the gonad including ovary, ovotestis and testis. This effort reveals that gonadal development was a dynamic expression changes. Regulatory networks of lncRNAs and its target genes were constructed though integrated analysis of lncRNA and mRNA data. We found DNA methylation was negatively associated with lncRNA (MSTRG.38036 and MSTRG.12998) expression in developing gonads. Additionally, 17α-methyltestosterone inhibit the expression of lncRNA and increase methylation. Furthermore, we found that Ptk2β probably regulates cyp19a1 expression via the Ptk2β/EGFR/STAT3 pathway to reprogram sex differentiation. The present study on the gonadal differentiation of M. albus provides novel insights from lncRNA to explore potential molecular mechanism. In the future, function of the lncRNA will be further studied and the gene editing technology will be applied to cultivate the female with high fecundity to improve the yield of fish fry.

In order to solve the problems in paternity test and genealogical management for improved variety breeding of Monopterus albus, we obtained 16 microsatellite markers with high polymorphism through whole genome searching of and following confirmation, and established two multiplex PCR sets by using 16 high polymorphic microsatellite markers and successfully applied to the parentage assignment for 11 full-sib M. albus families. The average allele number(Na) of 16 microsatellite was 5.562; the average of abserved heterozygosite(Ho) was 0.627; the average of expected heterozygosite(He) was 0.619; the average of polymorphic information content (PIC) was 0.564. Parentage analysis reveals that the combined exclusion probability of first parent given only the genotype of the offspring (CE-1P) was 0.999 999 99, the combined exclusion probability of a second candidate parent given the genotype of the offspring and one known parent (CE-2P) was 0.999 999 91, and combined exclusion probability of a parent pair given only the genotype of the offspring (CE-PP) was 0.999 964 76. The simulated identification rate of the 11 M. albus families was 99.96%, and the actual identification rate was 95%. Furthermore, the simulation analysis shows that the identification rate could reach over 95% in the case of 200 candidate parental individuals with known sexes and 150 candidate parental individuals with unknown sexes. The cluster analysis shows that 108 offspring could be clustered correctly except two, and the accuracy rate was 98.18%. The paternity method of M. albus established in this study provides important technical support for the future breeding program and management of germplasm resources of M. albus.

Farmed chinese giant salamander (Andrias davidianus) was an important distinctive economically amphibian that exhibited male-biased sexual size dimorphism. Fgf9 and rspo1 genes antagonize each other in Wnt4 signal pathway to regulate mammalian gonadal differentiation has been demonstrated. However, their expression profile and function in A. davidianus are unclear. In this study, we firstly characterized fgf9 and rspo1 genes expression in developing gonad. Results showed that fgf9 expression level was higher in testes than in ovaries and increased from 1 to 6 years while rspo1 expression was higher in ovaries than in testes. In situ hybridization assay showed that both fgf9 and rspo1 genes expressed at 62 dpf in undifferentiated gonad, and fgf9 gene was mainly expressed in spermatogonia and sertoli cells in testis while strong positive signal of rspo1 was detected in granular cell in ovary. During sex-reversal, fgf9 expression was significantly higher in reversed testes and normal testes than in ovaries, and opposite expression pattern was detected for rspo1. When FH535 was used to inhibit Wnt/β-catenin pathway, expression of rspo1, wnt4 and β-catenin was down-regulated. Conversely, expression of fgf9, dmrt1, ftz-f1 and cyp17 were up-regulated. Furthermore, when rspo1 and fgf9 were knocked down using RNAi technology, respectively. We observed that female biased genes were down regulated in ovary primordial cells after rspo1 was knocked down, while the opposite expression profile was observed in testis primordial cells after fgf9 was knocked down. These results suggested that fgf9 and rspo1 played an antagonistic role to regulate sex differentiation in the process of the gonadal development and provided a foundation for further functional characterizations. The data also provided basic information for genome editing breeding to improve the Chinese giant salamander farming industry.

The Asian swamp eel ( Monopterus albus ) is an excellent model species for studying sex change and chromosome evolution. M. albus is also widely reared in East Asia and South-East Asia because of its great nutritional value. The low fecundity of this species (about 300 eggs per fish) greatly hinders fries production and breeding programs. Interestingly, about 3–5% of the eels could remain as females for 3 years and lay more than 3,000 eggs per fish, which are referred to as non-sex-reversal (NSR) females. Here, we presented a new chromosome-level genome assembly of such NSR females using Illumina, HiFi, and Hi-C sequencing technologies. The new assembly (Mal.V2_NSR) is 838.39 Mb in length, and the N50 of the contigs is 49.8 Mb. Compared with the previous assembly obtained using the continuous long-read sequencing technology (Mal.V1_CLR), we found a remarkable increase of continuity in the new assembly Mal.V2_NSR with a 20-times longer contig N50. Chromosomes 2 and 12 were assembled into a single contig, respectively. Meanwhile, two highly contiguous haplotype assemblies were also obtained, with contig N50 being 14.54 and 12.13 Mb, respectively. BUSCO and Merqury analyses indicate completeness and high accuracy of these three assemblies. A comparative genomic analysis revealed substantial structural variations (SVs) between Mal.V2_NSR and Mal.V1_CLR and two phased haplotype assemblies, as well as whole chromosome fusion events when compared with the zig-zag eel. Additionally, our newly obtained assembly provides a genomic view of sex-related genes and a complete landscape of the MHC genes. Therefore, these high-quality genome assemblies would provide great help for future breeding works of the swamp eel, and it is a valuable new reference for genetic and genomic studies of this species.

Previous studies have shown that high-temperature treatment during thermosensitive periods (TSPs) induces >70% Nile tilapia to become pseudomales (XX phenotypic males) in all-XX families mainly by downregulating the 178-estradiol (E2) levels. Commercially, methyltestosterone (MT) is used to induce the sex reversal of XX individuals in Nile tilapia and produce all-male larvae. In order to reduce the dosage of methyltestosterone (MT), it is very necessary to investigate the possibility of obtaining all-male Nile tilapia larvae using combination treatment with high-temperature and low dose of MT. In this study, three families were developed by crossing XX females with XY males and were used for subsequent analysis. The results demonstrated that the male ratio in T + 0 MT (36 degrees C high-temperature treatment and 0 mu g/g MT in diet) was higher than in the C + 0 MT (28 degrees C culturing water temperature and 0 mu g/g MT in diet) or C + 2 MT (28 degrees C culturing water temperature and 2 mu g/g MT in diet) group and lower than in C + 10 MT. The lowest MT concentration achieving a 100% male ratio was 10 mu g/g in the high-temperature-treated group and 20 mu g/g in the control group. High-temperature and MT combination treatment further downregulated E2 levels compared to the control and various MT-treated groups. High-temperature treatment significantly increased 11-KT levels in XX Nile tilapia trunks, and 11-KT levels in T + 0 MT were significantly higher than in C + 0 MT or C + 2 MT and were not significantly different from that in XY. Interestingly, 11-KT levels in the XX Nile tilapia trunk in the C + 10 MT, C + 20 MT, T + 10 MT and T + 20 MT groups were significantly higher than in the XY group. High-temperature treatment significantly downregulated mRNA and protein expression levels of Cyp19a1a in 21 dpf (days post fertilization) XX Nile tilapia gonads (T + 0 MT). High-temperature and MT combination treatment further downregulated mRNA and protein expression levels of Cyp19a1a in 21 dpf XX Nile tilapia gonads (T + 10 MT) to levels similar to those in the XY group. In contrast to Cyp19a1a, high-temperature and MT combined treatment significantly upregulated expression levels of Dmrt1 in 21 dpf XX Nile tilapia gonads (T + 10 MT), and expression levels of Dmrt1 in T + 10 MT were higher than in XY. Furthermore, we found that high-temperature and low dose of MT combined treatments synergistically promote the growth of Nile tilapia. These results illustrated the potential applicability of high-temperature and low dose of MT combined treatment for all-male production of Nile tilapia larvae used in aquaculture industry.

Background: Monopterus albus is a hermaphroditic and economically farmed fish that undergoes sex reversal from ovary to testis via ovotestis during gonadal development. The epigenetic changes that are associated with gonadal development in this species remain unclear. Methods: We produced DNA methylome, transcriptome, and chromatin accessibility maps of the key stages of gonad development: ovary, ovotestis, and testis. The expression of the key candidate genes was detected using qRT-PCR and in situ hybridization and the methylation levels were analysed using bisulphite sequencing PCR. Promoter activity and regulation were assessed using dual-luciferase reporter assays. Results: Gonadal development exhibits highly dynamic transcriptomic, DNA methylation, and chromatin accessibility changes. We found that DNA methylation status, especially of the transcription start site, was significantly negatively correlated with gene expression while chromatin accessibility exhibited no correlation with gene expression during gonadal development. The epigenetic signatures revealed many novel regulatory elements and genes involved in sex reversal, which were validated. DNA methylation detection and site mutation of plastin-2 promoter, as a candidate gene, revealed that DNA methylation could impact the binding of transcription factor dmrt1 and foxl2 through methylation and demethylation to regulate plastin-2 expression during gonadal development. Conclusions: These data provide novel insights into epigenetic modification and help elucidate the potential molecular mechanism by which dynamic modification of DNA methylation plays a crucial role in gonadal development.

Background The Chinese giant salamander Andrias davidianus is an important amphibian species in China because of its increasing economic value, protection status and special evolutionary position from aquatic to terrestrial animal. Its large genome presents challenges to genetic research. Genetic linkage mapping is an important tool for genome assembly and determination of phenotype-related loci. Results In this study, we constructed a high-density genetic linkage map using ddRAD sequencing technology to obtain SNP genotyping data of members from an full-sib family which sex had been determined. A total of 10,896 markers were grouped and oriented into 30 linkage groups, representing 30 chromosomes of A. davidianus . The genetic length of LGs ranged from 17.61 cM (LG30) to 280.81 cM (LG1), with a mean inter-locus distance ranging from 0.11(LG3) to 0.48 cM (LG26). The total genetic map length was 2643.10 cM with an average inter-locus distance of 0.24 cM. Three sex-related loci and four sex-related markers were found on LG6 and LG23, respectively. Conclusion We constructed the first High-density genetic linkage map and identified three sex-related loci in the Chinese giant salamander. Current results are expected to be a useful tool for future genomic studies aiming at the marker-assisted breeding of the species.

Y The swamp eel (Monopterus albus) is one economically important fish in China and South-Eastern Asia and a good model species to study sex inversion. There are different genetic lineages and multiple local strains of swamp eel in China, and one local strain of M. albus with deep yellow and big spots has been selected for consecutive selective breeding due to superiority in growth rate and fecundity. A high-quality reference genome of the swamp eel would be a very useful resource for future selective breeding program. In the present study, we applied PacBio single-molecule sequencing technique (SMRT) and the high-throughput chromosome conformation capture (Hi-C) technologies to assemble the M. albus genome. A 799Mb genome was obtained with the contig N50 length of 2.4 Mb and scaffold N50 length of 67.24 Mb, indicating 110-fold and similar to 31.87-fold improvement compared to the earlier released assembly (similar to 22.24 Kb and 2.11 Mb, respectively). Aided with Hi-C data, a total of 750 contigs were reliably assembled into 12 chromosomes. Using 22,373 protein-coding genes annotated here, the phylogenetic relationships of the swamp eel with other teleosts showed that swamp eel separated from the common ancestor of Zig-zag eel similar to 49.9 million years ago, and 769 gene families were found expanded, which are mainly enriched in the immune system, sensory system, and transport and catabolism. This highly accurate, chromosome-level reference genome of M. albus obtained in this work will be used for the development of genome-scale selective breeding.