Teleost fish are the most diverse group of vertebrates and provide opportunities to study the evolution of sex determination (SD) systems. Using genomic and functional analyses, we identified a male-specific duplication of anti-Müllerian hormone ( amh ) gene as the male master sex-determining (MSD) gene in Sebastes schlegelii . By resequencing 10 males and 10 females, we characterized a 5 kb-long fragment in HiC_Scaffold_12 as a male-specific region, which contained an amh gene (named amhy ). We then demonstrated that amhy is a duplication of autosomal amh that was later translocated to the ancestral Y chromosome. amha and amhy shared high-nucleotide identity with the most significant difference being two insertions in intron 4 of amhy . Furthermore, amhy overexpression triggered female-to-male sex reversal in S. schlegelii , displaying its fundamental role in driving testis differentiation. We developed a PCR assay which successfully identified sexes in two species of northwest Pacific rockfish related to S. schlegelii . However, the PCR assay failed to distinguish the sexes in a separate clade of northeast Pacific rockfish. Our study provides new examples of amh as the MSD in fish and sheds light on the convergent evolution of amh duplication as the driving force of sex determination in different fish taxa.

Black rockfish is an economically important fish in East Asia. Little mention has been paid to the sperm cryopreservation in black rockfish. In this study, the optimal cryodiluent was selected from 48 combinations by detecting various sperm parameters. Transcriptome and methylome analysis were further performed to explore the molecular mechanism of inevitable cryoinjuries. The results showed that cryopreservation had negative effects on the viability, DNA integrity, mitochondrial activity, total ATPase and LDH of sperm even with optimal cryodiluent (FBS + 15% Gly). Transcriptome and methylome analysis revealed that the expression of 179 genes and methylation of 1266 genes were affected by cryopreservation. These genes were enriched in GO terms of death, G-protein coupled receptor signaling pathway, response to external stimulus and KEGG pathways of phospholipase D signaling pathway and xenobiotic and carbohydrate metabolism pathways. The role of PIK3CA and CCNA2 were highlighted in the protein-protein interaction network, and the sperm quality-related imprinted gene mest was identified among the 7 overlapping genes between transcriptome and methylome. Overall, the cryodiluent for black rockfish sperm was optimized, providing a feasible method for cryopreservation. The transcriptome and methylome data further demonstrated the underlying molecular mechanisms of cryoinjuries, proving clues for improvement of cryopreservation method of black rockfish.

The cell wall protein 66 gene ( cwp66 ) encodes a cell wall protein, which is the second major cell surface antigen of C. difficile . Although immunological approaches, such as antibodies and purified recombinant proteins, have been implemented to study the role of Cwp66 in cell adhesion, no deletion mutant of the cwp66 gene has yet been characterized.