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Background: Blotched snakehead (Channa maculata) displays significant sexual dimorphism, with males exhibiting faster growth rates and larger body sizes compared to females. The cultivation of the all-male population of snakeheads holds substantial economic and ecological value. Nonetheless, the intricate processes governing the development of bipotential gonads into either testis or ovary in C. maculata remain inadequately elucidated. Therefore, it is necessary to determine the critical time window of sex differentiation in C. maculata, providing a theoretical basis for sex control in production practices. Methods: The body length and weight of male and female C. maculata were measured at different developmental stages to reveal when sexual dimorphism in growth initially appears. Histological observations and spatiotemporal comparative transcriptome analyses were performed on ovaries and testes across various developmental stages to determine the crucial time windows for sex differentiation in each sex and the sex-related genes. Additionally, qPCR and MG2C were utilized to validate and locate sex-related genes, and levels of E2 and T were quantified to understand sex steroid synthesis. Results: Sexual dimorphism in growth became evident starting from 90 dpf. Histological observations revealed that morphological sex differentiation in females and males occurred between 20 and 25 dpf or earlier and 30–35 dpf or earlier, respectively, corresponding to the appearance of the ovarian cavity or efferent duct anlage. Transcriptome analyses revealed divergent gene expression patterns in testes and ovaries after 30 dpf. The periods of 40–60 dpf and 60–90 dpf marked the initiation of molecular sex differentiation in females and males, respectively. Male-biased genes (Sox11a, Dmrt1, Amh, Amhr2, Gsdf, Ar, Cyp17a2) likely play crucial roles in male sex differentiation and spermatogenesis, while female-biased genes (Foxl2, Cyp19a1a, Bmp15, Figla, Er) could be pivotal in ovarian differentiation and development. Numerous biological pathways linked to sex differentiation and gametogenesis were also identified. Additionally, E2 and T exhibited sexual dimorphism during sex differentiation and gonadal development. Based on these results, it is hypothesized that in C. maculata, the potential male sex differentiation pathway, Sox11a–Dmrt1–Sox9b, activates downstream sex-related genes (Amh, Amhr2, Gsdf, Ar, Cyp17a2) for testicular development, while the antagonistic pathway, Foxl2/Cyp19a1a, activates downstream sex-related genes (Bmp15, Figla, Er) for ovarian development. Conclusions: This study provides a comprehensive overview of gonadal dynamic changes during sex differentiation and gametogenesis in C. maculata, establishing a scientific foundation for sex control in this species. Plain language summary: Blotched snakehead (Channa maculata) exhibits significant sexual dimorphism, as males display faster growth rates and larger body sizes compared to females. The cultivation of the all-male population of snakeheads holds substantial economic and ecological value. However, the mechanisms underlying sex determination and differentiation in C. maculata remain insufficiently elucidated. In this study, sexual dimorphism in growth became evident starting from 90 dpf through the measurement of body length and weight of male and female C. maculata at different developmental stages. Histological observations indicated that morphological sex differentiation in females and males occurred at 20–25 dpf or earlier and 30–35 dpf or earlier, respectively, corresponding to the appearance of the ovarian cavity or efferent duct anlage. Transcriptome analyses revealed divergent gene expression patterns in male and female gonads after 30 dpf, suggesting that the period preceding 30 dpf might be the critical time window for sex control in C. maculata. The periods of 40–60 dpf and 60–90 dpf marked the initiation of molecular sex differentiation in females and males, respectively. Male-biased genes (Sox11a, Dmrt1, Amh, Amhr2, Gsdf, Ar, Cyp17a2) likely play crucial roles in testicular differentiation and spermatogenesis, while female-biased genes (Foxl2, Cyp19a1a, Bmp15, Figla, Er) could be pivotal in ovarian differentiation and oogenesis. Additionally, numerous biological pathways linked to sex differentiation and gametogenesis were identified. Moreover, sexual dimorphism was observed in the levels of E2 and T during gonadal differentiation and development. Based on these findings, it is hypothesized that in C. maculata, the potential male sex differentiation pathway, Sox11a–Dmrt1–Sox9b, activates downstream sex-related genes (Amh, Amhr2, Gsdf, Ar, Cyp17a2) for testicular development, while the antagonistic pathway, Foxl2/Cyp19a1a, activates downstream sex-related genes (Bmp15, Figla, Er) for ovarian development. This study provides a comprehensive overview of gonadal dynamic changes during sex differentiation and gametogenesis in C. maculata, thereby establishing a scientific foundation for sex control in this species. Highlights: Sexual dimorphism in growth became apparent starting from 90 dpf. The morphological differentiation of ovary and testis occurred at 20–25 dpf or earlier and 30–35 dpf or earlier, respectively, and the period preceding 30 dpf may be the critical time for sex control in C. maculata. Male-biased genes (Sox11a, Dmrt1, Amh, Amhr2, Gsdf, Ar, Cyp17a2) likely play crucial roles in testicular differentiation and spermatogenesis, whereas female-biased genes (Foxl2, Cyp19a1a, Bmp15, Figla, Er) could be pivotal in ovarian differentiation and oogenesis. The fate of undifferentiated primordial gonads in C. maculata may be determined by the antagonistic action of the Sox11a–Dmrt1–Sox9b and Foxl2/Cyp19a1a pathways. [ABSTRACT FROM AUTHOR]

Simple Summary: There is a significant sexual dimorphism in the blotched snakehead (Channa maculata). However, studies on sex differentiation and gonadal development of C. maculata are limited. Factor in the germ line alpha (Figla) is an oocyte-specific basic helix–loop–helix (bHLH) transcription factor, involved in ovarian development. In the present research, the expression patterns and cellular location of Figla, as well as the CpG methylation of its promoter, demonstrated that Figla participated in ovarian differentiation and development in C. maculata. Meanwhile, the gene disruption of Figla was realized by the CRISPR/Cas9 system for further functional research. Figla is one of the earliest expressed genes in the oocyte during ovarian development. In this study, Figla was characterized in C. maculata, one of the main aquaculture species in China, and designated as CmFigla. The length of CmFigla cDNA was 1303 bp, encoding 197 amino acids that contained a conserved bHLH domain. CmFigla revealed a female-biased expression patterns in the gonads of adult fish, and CmFigla expression was far higher in ovaries than that in testes at all gonadal development stages, especially at 60~180 days post-fertilization (dpf). Furthermore, a noteworthy inverse relationship was observed between CmFigla expression and the methylation of its promoter in the adult gonads. Gonads at 90 dpf were used for in situ hybridization (ISH), and CmFigla transcripts were mainly concentrated in oogonia and the primary oocytes in ovaries, but undetectable in the testes. These results indicated that Figla would play vital roles in the ovarian development in C. maculata. Additionally, the frame-shift mutations of CmFigla were successfully constructed through the CRISPR/Cas9 system, which established a positive foundation for further investigation on the role of Figla in the ovarian development of C. maculata. Our study provides valuable clues for exploring the regulatory mechanism of Figla in the fish ovarian development and maintenance, which would be useful for the sex control and reproduction of fish in aquaculture. [ABSTRACT FROM AUTHOR]

The cyp19a1 gene codes aromatase that converts androgen to estrogen, which plays a central role in early female differentiation and ovarian development in teleosts. For the blotched snakehead (Channa maculata), an important aquaculture fish that is susceptible to hormone-induced sex reversal, two aromatase genes were characterized in the present study, cyp19a1a and cyp19a1b. We analyzed gene expression and the epigenetic state of cyp19a1a and cyp19a1b in different adult tissues: the gonad and brain from normal XX females (XX-F), normal XY males (XY-M), sex-reversal females (XY-F) induced by estrogen, and YY super-males (YY-M), and gonads at different development stages. Cyp19a1a exhibited strong female-biased expression patterns in the ovary, and cyp19a1b dominantly expressed in the brain with no sex bias. Cyp19a1a's expression pattern in the XY-F ovary was similar to that in the XX-F ovary, with a relatively high expression level, which was far higher than that in XY-M and YY-M testis. Meanwhile, CpG methylation levels of cyp19a1a promoter were lower in XX-F and XY-F ovaries compared with XY-M and YY-M testis. A significantly negative correlation between the CpG methylation levels and cyp19a1a expression was elucidated in XX-F, XY-M, XY-F, and YY-M gonads. Furthermore, the strong female-biased cyp19a1a expression was closely related to ovarian differentiation and maturation, and the overall methylation levels of cyp19a1a promoter were inversely correlated with cyp19a1a expression. There were no detectable sexually dimorphic differences in cyp19a1b expression and CpG methylation levels of cyp19a1b promoter in the brain and gonad between sexes in C. maculata, thus the function of cyp19a1b in C. maculata needs further research. Our research illustrates that cyp19a1a is closely related to estrogen production, ovary differentiation/maintenance, and sex reversal, and epigenetic modification plays a crucial part in maintaining the sexual dimorphic expression of cyp19a1a, ovarian differentiation and oogenesis in C. maculata. [ABSTRACT FROM AUTHOR]