The complete mitochondrial DNA sequence of Triplophysa venusta was determined using a PCR-based method in our present study. The genome is totally 16 574 bp in length and contains 13 protein-coding genes, 2 ribosomal RNA, 22 transfer RNA genes, and 1 non-coding control region. The overall base composition of T. venusta is A 27.8%, T 26.9%, C 26.9%, and G 18.4%, with an A + T bias of 54.7%. This complete mitogenome of T. venusta provides a basic data for studies on species identification, molecular systematics, and conservation genetics. The phylogenetic trees consistently showed that the Family Cobitidae was divided into three major clades: the subfamily Botiidae, the subfamily Nemaeheilidae, and the subfamily Cobitinae. Cobitinae and Nomaeheilidae formed a main Loach clade, with Botiidae clustered at the basal.

Chinese tongue sole (Cynoglossus semilaevis) has a ZZ/ZW sex determination system, but the genotypic female (ZW) can be sex-reversed into phenotypic males, namely, pseudomales. Pseudomale fish can produce only Z-type sperm but not W sperm. However, the molecular mechanism is unclear. To screen the key genes involved in pseudomale sperm abnormalities, we analysed the transcriptomic profiles of pseudomale and male sperm. In comparison to male sperm, 592 differentially expressed genes (DEGs) were identified in pseudomale sperm, including 499 upregulated and 93 downregulated genes. KEGG analysis indicated that the FoxO signalling pathway, especially the foxo3a and foxo6-like genes, may play an important role in spermatogenesis. The DEGs were mainly distributed on sex chromosomes, with 158 downregulated genes on the Z chromosome and 41 upregulated genes on the W chromosome. A specific area (14–15 M) on the Z chromosome was identified, which enriched eight DEGs inside the ~1 M region. In addition, there were five gene alleles on the sex chromosomes, which showed the opposite transcription pattern (upregulated for the W allele, downregulated for the Z allele). This study has provided valuable data for screening candidate genes involved in the pseudomale sperm abnormality.

Phosphorylation is a post-translational modification that contributes to versatile protein functions in spermatogenesis, and the variations they generate usually results in abnormal spermatogenesis or sperm dysfunction. The sex-reversal phenomenon exists in Chinese tongue sole under certain conditions such that individuals with a ZW genotype can acquire a male phenotype and are thus called pseudomales. Pseudomale tongue sole can reach sexual maturity but produce only Z-type sperm, and the Z sperm carries paternal epigenetic information. Whether phosphorylation plays a role in the sperm abnormality of pseudomales is unknown. In this study, a phosphoproteomic analysis was performed to compare protein phosphorylation profiles between pseudomale and male testes. Altogether, we identified 14,253 phosphopeptides matching with 4843 proteins, with 1329 differentially phosphorylated peptides corresponding to 1045 differentially phosphorylated proteins (DPPs). Phosphorylation at 781 sites was upregulated and at 548 sites was downregulated. Four motifs were identified among differentially phosphorylated peptides, which were “SP”, “SD”, “RxxS”, and “TP”. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses suggested that the cell cycle and DNA/RNA processing were significantly enriched with the genes encoding DPPs. To analyze DPP function in depth, a protein-protein interaction network was constructed, and Ran-binding protein 2 was found to play a central role in spermatogenesis by regulating several processes such as the cell cycle, eukaryotic translation, ubiquitination, and minichromosome maintenance. In kinase-associated network analyses, two “mitogen-activated protein kinase (Mapk)-centered” clusters were identified that may account for abnormal spermatogenesis in pseudomales. One cluster was centered on Mapk6, which predominantly regulated the cell cycle by interacting with several cyclin-dependent kinases, and the other was centered on the “testis-expressed kinase 1-like (Tesk1l)/Pim1l-Mapk4l- testis-expressed 14 (Tex14)” kinase cascade, which might contribute to spermatogenesis by regulating β-catenin. Taken together, these data suggested the new candidates involved in pseudomale sperm abnormalities and provided clues to discover the phosphorylated regulatory mechanism underlying tongue sole spermatogenesis.