DNA methylation has frequently been implicated in sex determination and differentiation in teleost species. In order to detect the DNA methylation patterns established during sexual differentiation in tiger pufferfish T. rubripes, we performed comprehensive whole genome methylation sequencing and analyses of the gonads of male, female, and pseudo male. We obtained a total of 33.12, 32.44, and 31.60 Gb clean data for male, female, and pseudo male, with a sequencing depth of 66.44×, 60.47× and 54.86×, respectively. The methylation level of cytosine (C) residues in the genomic DNA from gonads was 11.016%, 10.428%, and 11.083% in male, female, and pseudo male, respectively. More than 65% of C methylation was at CpG sites, and less than 1% was at CHG and CHH sites. In each regulatory element, there were low methylation levels on both sides of the transcription start site, and higher methylation levels in exons, introns, and downstream of genes. The highest mCpG was on chromosome 8 and the lowest mCpG was on chromosome 5. Comparisons of whole-genome DNA methylation between pairs of samples revealed that there were 3,173 differentially methylated regions (DMRs) between female and male, and 3,037 DMRs between male and pseudo male, corresponding to 0.232% and 0.223% of the length of the genome, respectively. There were only 1,635 DMRs between female and pseudo male, representing 0.127% of the length of the genome. A number of differentially methylated genes (DMGs) related to sex determination and differentiation were selected, such as amhr2 and pfcyp19a. After Bisulfite Sequencing PCR (BSP) verification, amhr2 was exhibited low methylation level in normal males and pseudo male, and high methylation level in normal females but pfcyp19a showed low methylation level in normal females and high methylation level in normal males and pseudo males. These results provide information about the molecular epigenetic mechanisms of DNA methylation during low-temperature induced masculinization of tiger pufferfish, and increase our understanding of the mechanisms of sex determination and differentiation in this important aquaculture fish species.

In this study, we selected natural polyploidy loach (diploid, triploid and tetraploid) and hybrid F₁ generation obverse cross (4 × 2) and inverse cross (2 × 4) by diploids and tetraploids as the research model. The MSAP (methylation-sensitive amplified polymorphism) reaction system was established by our laboratory to explore methylation levels and pattern diversification features at the whole genome level of the polyploidy loach. The results showed that the total methylation and full methylation rates decreased on increased ploidy individuals; moreover, the hemimethylation rate showed no consistent pattern. Compared with diploid loach, the methylation patterns of tetraploid sites changed 68.17%, and the methylation patterns of triploid sites changed 73.05%. The proportion of hypermethylation genes is significantly higher than the proportion of demethylation genes. The methylation level of reciprocal cross F₁ generation is lower than the male diploid and higher than the female tetraploid. The hemimethylation and total methylation rate of the cross hybrid F₁ generation is significantly higher than the orthogonal F₁ generation (p < 0.01). After readjusting, the methylation pattern of genome DNA of reciprocal hybrids changed 69.59% and 72.83%, respectively.

Natural tetraploid loach (Misgurnus anguillicaudatus) living in the Changjiang River basin and adjacent area in China has been considered a true genetic tetraploidy (4n = 100) with four sets of homologous chromosomes. Here, we reported its further supporting evidence provided by the cold‐shock androgenesis. Viable androgenetic progeny appeared when eggs were cold‐shocked at 3°C for 60 min, starting 5 min after fertilization with sperm of natural tetraploid males, although the survival rate was significantly lower than control group. The majority (87%) of androgenetic embryos were diploid (2n = 50) and all‐paternal inheritance was verified in larval stage by microsatellite genotypes. Microscopic observations confirmed the elimination of both egg nucleus and second polar body from a fertilized egg, followed by the cleavage exclusively with sperm nucleus. Thus, the appearance of normal diploid androgenetic progeny from sperm of natural tetraploid loach revealed the presence of four chromosome sets in tetraploid males.

To explore the induction of low temperature the Tiger Puffer (Takifugu rubripes) In this study, the influence of temperature on the pituitary gland during masculinization was investigated through chronic hypothermia stress experiments. Metabolomics was used to analyze the metabolic regulatory network of the pituitary gland under hypothermia stress. ELISA technology was employed to determine the activity content of oxidative stress-related enzymes in the pituitary gland. Further, TUNEL fluorescence labeling and qPCR were used to detect the apoptosis level of pituitary cells. Finally, to assess the impact of low-temperature stress on muscle tissue, HE staining and qPCR techniques were employed. The results showed that after 45 days of low-temperature stress, the differential metabolites of the pituitary gland were mainly enriched in the amino acid metabolic signaling pathway, and the contents of amino acids such as GSH and its synthetic precursors in the pituitary tissue changed significantly. The contents of oxidative stress indicators such as ROS and MDA all showed a trend of first increasing and then decreasing. The qPCR results of TUNEL fluorescence labeling and apoptosis-related genes were consistent, indicating that the apoptotic level of pituitary cells first increased and then decreased with the stress process. Histological analysis revealed that low temperature led to muscle cell atrophy and increased interstitial space in muscle tissue. The expression changes in genes related to muscle development further confirmed that low temperature significantly inhibited muscle growth and development. Therefore, this study speculates that after being subjected to chronic low-temperature stress, the pituitary gland of the red-finned Oriental pufferfish can alleviate the oxidative stress response of the body by strengthening the amino acid metabolic pathway, and the fish body has shown a physiological trend of gradually adapting to low-temperature stress, but the growth and development of muscles are still significantly inhibited. The results of this study can provide theoretical support for understanding the physiological adaptation mechanism of the red-finned Oriental pufferfish to low-temperature stress and lay a foundation for subsequent in-depth exploration of the pituitary response mechanism to low temperatures.