Induction of diploid gynogenesis in southern flounder (Paralichthys lethostigma) with homologous and heterologous sperm

Effective methods for induction of diploid gynogenesis in North American flounder of the genus Paralichthys are needed to initiate monosex culture, which will allow growers to take advantage of the more rapid growth and larger size attained by females. To test methods for inducing diploid gynogenesis in southern flounder (Paralichthys lethostigma) using homologous sperm, 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) for 3-4 min in seawater, and then subjecting the eggs to cold shock in 0-2°C seawater for 45-50 min. Cold shock was used to prevent extrusion of the second polar body. Control treatments omitted one or more of these steps to separately assess the effectiveness of UV irradiation and cold shock. Larvae were observed for physical abnormalities and then histologically processed for ploidy determination. Haploid larvae exhibited abnormal external morphology while diploid, gynogenetic diploid, and triploid larvae showed normal morphologies. Cross-sectional areas of erythrocyte nuclei were measured for larvae in each treatment group and significant differences were found. Nuclear areas for treatment groups corresponded to predicted ploidy (triploid>diploid>haploid) and did not differ between normal diploid controls and gynogenetic diploids. These results suggest that the procedures of sperm irradiation and egg cold shock successfully generated gynogenetic diploids. Due to the low volumes of semen produced by male flounder, and to eliminate any potential genetic contribution by homologous sperm, activation of flounder eggs with heterologous sperm was also investigated. Induction of diploid gynogenesis was successful when flounder eggs were fertilized with irradiated (50 J/cm2) sperm from striped mullet (Mugil cephalus), and then cold shocked. This work provides procedures for induction of diploid gynogenesis in southern flounder using homologous and heterologous sperm, and validates a method for verification of ploidy in larval fish.