Breeding Biotechnology, Sex Determination, and Growth in Southern Flounder, Paralichthys lethostigma

Abstract

Southern flounder (Paralichthys lethostigma) support valuable, but declining US fisheries. This species is therefore a strong candidate for aquaculture to mitigate fishing impacts and stabilize seafood supply. Because female flounder reach substantially larger sizes than males, all-female culture is desirable for commercial aquaculture. Hence, a thorough understanding of sexual development, its timing and regulation by temperature is essential for optimization of flounder aquaculture.

To better understand ovarian and testicular development in southern flounder, structural and cellular sex-distinguishing markers were studied using histological methods. We found that histologically discernible sex differentiation occurs in southern flounder at ~75-120 mm TL and that early differentiation is considerably delayed relative to its Japanese congener, P. olivaceus. High (28°C) and low (18°C) water temperatures, produced a higher proportion of males (96% and 78% males, respectively). The sex ratio at a mid-range (23°C) temperature was not different from 1:1. This suggests that southern flounder possess a temperature sensitive mechanism of sex determination. Growth was also affected by temperature with the temperature that maximized females inducing better growth.

Aromatase cytochrome P450 (P450arom) is responsible for estrogen biosynthesis and plays a critical role in ovarian differentiation. We cloned ovarian P450arom and developed a qRT-PCR for assessment of early sex differentiation. The deduced amino acid sequence for southern flounder P450arom is very similar to P450arom in other teleosts. Comparison of P450arom intron sequences of fish within and between different populations revealed substantial inter-individual variation that may affect sex determination responses. Ovary and spleen exhibited high levels of P450arom mRNA, while P450arom was weakly detected in testis, brain, and gill. Gonads sampled from wild flounder spanning the period of sex differentiation initially exhibited a low level of P450arom gene (fish 40-55 mm TL) followed by bifurcation into two clearly segregated groups beginning at ~65 mm TL. Through histology we show high and low P450arom mRNA relates to ovarian and testicular differentiation, respectively. This work imparts a powerful tool for better understanding mechanisms of sex determination and rapidly defining conditions that influence sex.

Effective methods for induction of diploid gynogenesis in southern flounder are needed to initiate all-female culture. To test methods for inducing gynogenesis in this species, four treatments, named for their expected outcome, were employed: haploid, diploid, triploid, and gynogenetic diploid. Diploid gynogenesis was induced by activating egg development with UV-irradiated flounder sperm (70 J/cm²) and then 'cold shocking' the eggs to prevent second polar body extrusion. Control treatments omitted one or more of these steps to separately assess the effectiveness of UV irradiation and cold shock. Haploid larvae exhibited abnormal external morphology while diploid, gynogenetic diploid, and triploid larvae appeared normal. Erythrocyte nuclear sizes for treatment groups corresponded to predicted ploidy (triploid>diploid>haploid) and did not differ between normal diploids and gynogenetic diploids, suggesting that our procedures were successful. To eliminate any possible paternal genetic contribution during gynogenesis, activation of flounder eggs with striped mullet (Mugil cephalus) sperm was also tested. Induction of diploid gynogenesis was successful when flounder eggs were fertilized with irradiated mullet sperm and cold shocked. This work provides procedures for induction of diploid gynogenesis in southern flounder and validates a method for verification of ploidy in larval fishes.

These studies establish the schedule of southern flounder sex differentiation based on results of gonadal histology and patterns of P450arom gene expression. We also demonstrate that sex determination in this species is temperature sensitive. This knowledge, along with methods for induction of gynogenesis, can be utilized for creation of all-female southern flounder populations for aquaculture. These findings may also benefit fisheries management by providing methods for sex assessment and guiding stock enhancement.