Cynoglossus semilaevis

Cynoglossus semilaevis, commonly known as the Chinese tongue sole, is a popular aquaculture flatfish species. They are native to China's northern coast but have experienced overfishing these past three decades. Tongue sole farming began in 2003 and they have since become a popular, expensive seafood. However, tongue soles have created issues for farmers due to their pathogen susceptibility and uneven sex ratio.

Sex & sex reversal
Cynoglossus semilaevis have a female heterogametic sex-determination system, with females being ZW and males being ZZ. Female tongue soles grow up to 2-4 times larger and faster than males. As such, female tongue soles are preferred by farmers and attempts have been made to breed all-female stocks via artificial gynogenesis. In both the wild and in aquaculture, the sex ratio of Cynoglossus semilaevis is male-skewed due to a number of the females becoming pseudomales. Pseudomales have female ZW chromosomes, but become physiologically male. The offspring of pseudomales have a significantly lower growth rate than the offspring of males, and are more likely to become pseudomales themselves, further skewing the sex ratio. This change occurs due to a combination of genotypic sex determination and temperature-dependent sex determination. Exposure to higher temperatures during the gonadal sex differentiation stage (~56–62 days post-hatching) makes the sex change more likely, with one study finding that the percentage of females who became pseudomales was increased to 73% when reared at a higher temperature (28°C) as opposed to a 14% sex-reversal rate when reared at an ambient temperature (22°C). Furthermore, the offspring of the pseudomales reared at 28°C had a sex-reversal rate of 94% despite being reared at 22°C. This high rate of females becoming pseudomales in the F2 generation was attributed to the offspring inheriting their Z chromosome from their pseudomale father and retaining paternal methylation patterns.

Sex-specific molecular markers
A single SNP, Cyn_Z_6676874, allows certain females to become pseudomales. Females with a thymine at this site can undergo sex reversal, but females with an adenine cannot. There are also three known female-specific molecular markers that can be used to distinguish between the various tongue sole sexes. Two SNPs, SNP_chr_8935925_C_T and SNP_chr_8936186_C_G, and one indel were found to enable accurate differentiation between males, females, and pseudomales.

Gene expression in the gonads
Gene expression in the gonads of males and females are different before sex determination, after sex determination, and after exposure to higher temperatures. Prior to sex determination, males and females have differentially expressed genes (DEGs) related to muscle development. Following sex determination, females have upregulated female-specific genes, figla and foxl2, and downregulation of the male-specific genes, dmrt1 and amh, under both ambient and high-temperature conditions. When exposed to higher temperatures, males, females, and pseudomales have hundreds of DEGs in common that are mainly involved in biological processes and molecular functions.

Pseudomale gonads have more similar gene expression patterns to males than females. DEGs between males and pseudomales played a role in spermatogenesis and energy metabolism. Whereas, DEGS between females and pseudomales were related to steroid hormones, helicase activity, sexual differentiation, and development.