Yang Zhang

Anti-Müllerian hormone (Amh) is a key regulator of sex determination and differentiation in fish. In this study, we identified and characterized a single-copy Amh ortholog, designated CmAmh, in blotched snakehead (Channa maculata), an economically important species in China. The cDNA spans 2340 bp and encodes a 545-amino-acid protein with conserved AMH-N and TGF-β domains. CmAmh exhibited pronounced male-biased gonadal expression, with transcript levels in testes markedly exceeding those in ovaries across all developmental stages (45~365 days post-hatching (dph)). Cellular localization revealed CmAmh transcripts in the Sertoli cells and spermatogonia of the testis and, more weakly, in the oogonia and primary oocytes of the ovary, suggesting a primary role in spermatogenesis and a potential modulatory function in early oogenesis. A strong inverse correlation between promoter CpG methylation and transcriptional activity, hypermethylation in ovaries (82.5~89.7%) versus hypomethylation (15.8~19.1%) in testes, indicated epigenetic regulation, which functionally integrates with hormonal signaling: 17β-estradiol (E₂) suppressed, whereas methyltestosterone (MT) upregulated CmAmh expression. Long-term E₂ exposure induced male-to-female secondary sex reversal (SSR) in 26.7% of XY individuals, whereas MT failed to reverse XX fish, indicating that CmAmh modulation alone is insufficient to override ovarian fate. Furthermore, CRISPR/Cas9-mediated mutagenesis achieved 40~45% editing efficiency, predominantly generating frameshift alleles, providing a platform for future functional studies. Collectively, these findings position CmAmh as a hormone-responsive nodal point within a broader sex-differentiation network. This work provides a theoretical foundation for sex-control strategies in C. maculata aquaculture, while future studies should focus on homozygous mutant generation to definitively establish its loss-of-function phenotype and regulatory networks.

The anti-Müllerian hormone type II receptor (Amhr2) is a critical component of the transforming growth factor-β (TGF-β) signaling pathway and plays essential roles in sex determination and gonadal development in teleosts. However, its function in the blotched snakehead (Channa maculata), an economically important fish in China, remains unexplored. In this study, we cloned and characterized the Amhr2 ortholog from C. maculata, designated CmAmhr2. The gene encodes a 443-amino acid protein containing a conserved STYKc kinase domain. Sequence and phylogenetic analyses revealed that CmAmhr2 is homologous to autosomal Amhr2 in other teleosts. Spatiotemporal expression analyses showed that CmAmhr2 was predominantly expressed in testes, particularly during critical windows of gonadal differentiation. In situ hybridization localized CmAmhr2 transcripts mainly in spermatogonia, with weaker signals in primary spermatocytes, Sertoli cells, and early-stage oocytes (oogonia and primary oocytes). Dietary administration of 30 mg/kg 17β-estradiol (E₂) from 15 to 45 days post-fertilization (dpf) for 30 days induced male-to-female sex reversal, producing neofemales (XY-F) and intersex individuals (XY-I). CmAmhr2 expression levels progressively declined with the degree of gonadal feminization: highest in normal XY male (XY-M) testes, intermediate in XY-I ovotestes, and lowest in fully feminized XY-F and normal XX female (XX-F) ovaries. Furthermore, CRISPR/Cas9-mediated mutagenesis of CmAmhr2 generated frameshift mutations predicted to disrupt the kinase domain. These findings suggest that CmAmhr2 is involved in male sex differentiation and testis development in C. maculata, providing novel molecular insights and a foundation for future sex-control research in aquaculture.

The ecological impact of microplastic pollution in freshwater ecosystems has received growing scientific attention, although research on freshwater species remains limited compared to marine organisms. This study investigates the individual and combined toxicological impacts of polystyrene nanoparticles (PSNPs) and dibutyl phthalate (DBP) on hybrid snakehead (Channa maculata ♀ × Channa argus ♂), a commercially important freshwater fish. PSNPs inhibited growth, induced hepatic and intestinal lesions, and delayed ovarian development, co-exposure with DBP exacerbated these effects. qPCR analysis revealed significant up-regulation of inflammation-related genes in the liver but inhibitory effects in the intestines, indicating that PSNPs and DBP provoke immune modulation and systemic pro-inflammatory responses. Furthermore, PSNPs and DBP induced oxidative damage in the liver and intestines by affecting antioxidant enzyme activity. 16S rRNA sequencing revealed that PSNPs and DBP altered intestinal microbiota composition, particularly reducing Proteobacteria abundance. Correlation analyses indicated negative associations between the abundances of Proteobacteria and Firmicutes and antioxidant parameters (SOD and MDA), suggesting microbiota-mediated impacts on host metabolism and physiological health. These findings highlight the ecological threat of microplastics and phthalates in freshwater environments and underscore the need for targeted conservation strategies.

As a member of the pattern recognition receptor (PRR) class, scavenger receptor class B type 1 (SRB1) plays a key role in innate immunity. Grass carp (Ctenopharyngodon idellus) ranks among the most extensively cultivated freshwater aquaculture species in China. However, little is known about the function of SRB1 in C. idellus. In this research study, a SRB1 gene was identified in C. idellus, named CiSRB1. The full-length cDNA of CiSRB1 is 2486 bp long, with an open reading frame (ORF) of 2486 bp encoding a 497 amino acid (aa) protein containing a conserved CD36 domain. The identified genomic DNA length of CiSRB1 is 20,042 bp, including 12 exons and 11 introns. The predictive analysis of protein interactions revealed that CiSRB1 could interact with the outer capsid proteins of typical GCRV strains. The tissue distribution of CiSRB1 exhibited age-dependent characteristics. CiSRB1 displayed the highest expression in the intestines and moderate levels in muscle, spleen, liver, and brain of one-year-old grass carp while maintaining relatively low levels in three-year-old grass carp. Following grass carp reovirus (GCRV) infection, notable upregulation of CiSRB1 transcripts was observed in major immune tissues (gills, intestines, spleen, and liver). Furthermore, significant differences were found between one-year-old and three-year-old grass carp, with lower CiSRB1 expression levels being detected in the older group. Additionally, a distinct response to GCRV infection was observed in one-year-old and three-year-old grass carp. It was found that one-year-old individuals had a mortality rate of up to 84% 6 days post-infection (dpi), whereas all three-year-old counterparts survived after GCRV infection. The analysis of GCRV copy numbers across tissues revealed substantially higher levels in one-year-old grass carp compared with their older counterparts, confirming the existence of age-dependent susceptibility to GCRV infection in grass carp. Combined with these results, it was speculated that the decline in cell-surface CiSRB1 expression with age may impede reovirus binding to host cells, potentially explaining why older grass carp demonstrated enhanced resistance to GCRV infection. This observation accentuates the importance of CiSRB1 in the context of GCRV infection and provides insights into age-dependent susceptibility to reovirus.

Insulin-like growth factors (IGFs) play central roles in the growth and development of vertebrates. Blotched snakehead (Channa maculata), an economically significant fish, exhibits obvious sexual dimorphism and achieves sexual maturity in one year. However, the role of IGFs in C. maculata remains unknown. Three IGF genes were identified in C. maculata, designated as CmIGF1-1, CmIGF1-2, and CmIGF2. The cDNA sequences of these genes are 1184, 655, and 695 bp, encoding putative proteins of 168, 131, and 215 amino acids, respectively, and all three proteins contain a conserved IGF domain. Quantitative real-time PCR (qPCR) revealed the predominant expression of CmIGFs in the liver of adult fish, with higher expression levels observed in males. Notably, CmIGF1-1, CmIGF1-2, and CmIGF2 displayed analogous expression profiles in the liver across various developmental stages, peaking at 365 days after hatching (dah). Subsequently, 600 individuals at 75 dah, at an early developmental stage, were randomly divided equally into six groups and reared in aerated 2 m × 2 m × 2 m cement ponds at 26.0 ± 1.0 °C. Following a one-week acclimatization period, fish without observed abnormalities were intraperitoneally injected with either 17α-ethynylestradiol (EE₂) or 17α-methyltestosterone (MT) at a dose of 10 μg/g body weight. Three groups underwent short-term hormone treatment, and the remaining three groups underwent long-term hormone treatment, which included five injections at two-week intervals over ten weeks. The analysis of CmIGFs expression levels in the liver under different hormone treatments revealed that EE₂ suppressed the expression of CmIGF1-1 and CmIGF1-2 while promoting CmIGF2 expression. In females, MT up-regulated the expression of CmIGF1-1 and CmIGF2 in a time-dependent manner, but consistently inhibited CmIGF2 expression. In males, MT promoted the expression of CmIGFs in a time-dependent manner, reaching peak levels for CmIGF1-1, CmIGF1-2, and CmIGF2 after 8, 10, and 2 weeks of injection, respectively. Additionally, CmIGF1 and CmIGF2 might exhibit a complementary relationship, with a compensatory increase in CmIGF2 expression in response to low CmIGF1 concentration. These findings highlight the potential key role of IGFs upon growth and their regulation by sex steroid hormones in C. maculata, providing a crucial foundation for future research aimed at elucidating the molecular mechanisms underlying the growth dimorphism between female and male blotched snakeheads.

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.