Cynoglossus semilaevis, commonly known as the Chinese tongue sole, is a popular aquaculture flatfish species.[1] They are native to China's northern coast but have experienced overfishing these past three decades.[1][2] Tongue sole farming began in 2003 and they have since become a popular, expensive seafood.[2] However, tongue soles have created issues for farmers due to their pathogen susceptibility and uneven sex ratio.[2]
Cynoglossus semilaevis have a female heterogametic sex-determination system, with females being ZW and males being ZZ.[3] 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.[4] 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.[5] Pseudomales have female ZW chromosomes, but become physiologically male.[6] 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.[3][5] This change occurs due to a combination of genotypic sex determination and temperature-dependent sex determination.[7] 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).[3][8] Furthermore, the offspring of the pseudomales reared at 28°C had a sex-reversal rate of 94% despite being reared at 22°C.[3] 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.[9]
A single SNP, Cyn_Z_6676874, allows certain females to become pseudomales.[10] Females with a thymine at this site can undergo sex reversal, but females with an adenine cannot.[10] There are also three known female-specific molecular markers that can be used to distinguish between the various tongue sole sexes.[5] 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.[5]
Gene expression in the gonads of males and females are different before sex determination, after sex determination, and after exposure to higher temperatures.[11] Prior to sex determination, males and females have differentially expressed genes (DEGs) related to muscle development.[11] 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.[11] 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.[11]
Pseudomale gonads have more similar gene expression patterns to males than females.[7] DEGs between males and pseudomales played a role in spermatogenesis and energy metabolism.[11] Whereas, DEGS between females and pseudomales were related to steroid hormones, helicase activity, sexual differentiation, and development.[11]