Genetic and Temperature Manipulation of Southern Flounder (Paralichthys lethostigma) for the Production of Monosex Populations

Abstract

Experiments were conducted to establish methods to produce an all female population of southern flounder (Paralichthys lethostigma) gynogens. The first series of experiments was to optimize the protocol for using UV irradiated black sea bass (Centropristis striata) sperm for the induction of meiogynogenesis. Milt was collected from multiple black sea bass, pooled, and diluted 1:10 using Ringer's solution. The diluted milt was irradiated using an UV Crosslinker with doses ranging from 0-130 J/cm2. The optimal UV dose of 70 J/cm2 was determined by observing a 10 % motility, motility duration of approximately 1 minute, and giving the highest percent hatch (7.73+1.57 %) which was statistically different (P=0.05) than the control. Pressure shock was used to retain the 2nd polar body in southern flounder eggs. Application of 8500 psi for 6 minutes was initiated at varying times 1, 2, and 3 minutes post fertilization with no pressure acting as the negative control. Based upon percentage hatch and survival, the optimal time to apply pressure was either 1 or 2 minutes post fertilization, which was temperature dependent (1 minute at 18oC and 2 minutes at 16oC.) The overall average hatch varied with initiation times and ranged from 1.48 + 0.52% (1 min) to 0.61 + 0.11% (3 min) of the fertilized eggs (average 70.9 + 12.8% fertility.) The development of these techniques can allow aquaculturists to take advantage of the better growth rates of female southern flounder by producing all female offspring.

Three preliminary experiments were also conducted to establish methods to produce cloned, monosex populations of southern flounder through mitotic gynogenesis. The first experiment determined the timing of the first mitotic division of the southern flounder egg. Eggs were fertilized with UV irradiated (70 J/cm2) black sea bass sperm and allowed to normally divide between 17-20oC. There was no correlation between spawning temperature, the timing of the first mitotic division, or the rate of mitosis. The timing of first cleavage varied from 63-88 minutes post fertilization. Once eggs began the first cellular division, 90 % of the eggs had completed this stage within 40 minutes. Using these data, southern flounder eggs were fertilized with UV irradiated (70 J/cm2) black sea bass sperm to create mitogynogens. Percent fertilization was 44.7% and percent hatch was 0%, although some eggs advanced to the blastula stage. Triploidy hatch percentages were 18.0-42.7 % of the normal diploid control percent hatch.

A third series of experiments were designed to test for sex differences among populations from North Carolina and Texas southern flounder raised under the same conditions and shifted to higher temperatures. At the end of the study, there was no significant difference in lengths or weights between the treatments. The flounder that remained in 23oC indicated a trend for larger growth for the Texas flounder while the 28oC tended to grow larger for the North Carolina flounder (not significant.) Final sex ratios were 100% male for all treatments. An additional experiment was conducted to determine the window of temperature dependent sex determination in North Carolina southern flounder. A sample was shifted to 28oC at 60 mm and 90 mm in duplicate to determine when sex differentiation occurred. The North Carolina southern flounder in this study grew better in 23oC until 60 mm and then their growth rate increased in 28oC until approximately 125 mm.