Male-biased gonadal differentiation in hatchery-reared blackthroat seaperch Doederleinia berycoides and development of molecular markers to elucidate sex differentiation mechanisms

In the seed production of commercially valuable blackthroat seaperch Doederleinia berycoides , the sex ratio is often skewed toward males. To address this imbalance, several environmental and dietary approaches have been examined; however, the issue remains unresolved. Assessing the effectiveness of such treatments requires reliable methods for early sex identification before stocking hatchery-reared juveniles into the wild. However, the processes of sex differentiation and the potential for protandry remain unexamined in this species. In this study, observations of wild populations showed no significant sex-ratio bias, and no histological evidence of gonads indicated protandrous sex change. This suggests that the male-biased sex ratio observed in hatchery populations is unlikely to result from protandry but is instead more likely driven by environmental factors associated with artificial rearing conditions. Histological analysis of early developmental stages of hatchery-reared juveniles (80–125 days post hatching, dph) and 1-year-old fish revealed that the presence of ovarian cavities in both undifferentiated gonads, testes, and ovaries, indicating that ovarian cavity formation is not a reliable marker for sex identification. Moreover, because oocytes were still not observed even at 120 dph, histological sex identification prior to stocking hatchery-reared juveniles was considered impossible. Consequently, RNA-seq and differentially expressed gene analyses were employed for the first time in this species to identify genetic markers of sex differentiation. qPCR analysis showed predominant expression of cyp11b1 , dmrt1 , and amh in the testis of the 2-year-old male, and foxl2a and wnt-4a in the ovary of the 2-year-old female, respectively. Notably, foxl2a expression was also detected in female fin tissue, suggesting the potential for non-lethal female sex identification. These findings provide insights into the molecular mechanisms of sex differentiation, support discrimination between the sexes, and clarify the occurrence of phenotypically normal females in hatchery-reared blackthroat seaperch.