| Turbot, Scophthalmus maximus, is a main species of the marine fish cultured in Europe, South America and Asia. Turbot culture is an important pillar industry of China’s marine aquaculture in recent years, with the annual output of nearly 60 thousand tons and value of more than 5 billion yuan, accounting for 80% of the total annual output of the world. Thus, genetic improvement of turbot seed culture characters will maximize profit potential for the culture of this species. The application of chromosome set manipulation techniques (e.g., the induction of triploidy, tetraploidy and gynogenesis) have been studied as a means to improve the production of many cultured fish species, which also offers the ideal material for the studies of genetic linkage mapping, QTL mapping and genomic research. Induced triploidy resulting in genetic sterility and gynogenesis can be used to obtain all-female stocks. Therefore, in order to establish the techniques for industrial production of all female and triploidy turbot juveniles, the present study objectives are:I) to determine the optimal conditions to induce meiotic and mitotic gynogenesis and tetraploidy in turbot,2) to study early development and viability of gynogentic and tetraploid progenies,3) to determination the strict period for gonadal differentiation and Genetic sex determination system.Over more than 3 years implementation, the following aspects of achievements were obtained:(1) Meiotic gynogenesis induction in turbotHertwig effect and orthogonal experiment design were employed to determine the optimal protocol for the induction of meiogynogenetic diploids of turbot with cryopreserved sperm of red sea bream (Pagrus major). With or without cold shock treatment neither of the turbot embryos fertilized with untreated sperm of red sea bream could survive beyond hatching. All of the turbot larvae hatched from eggs activated by UV irradiated sperm of red sea bream at the dosage of 64.8-72.0 mJ·cm-2 were typical haploids. Under the fertilization and hatchery temperature of 14.5±0.5℃, the maximal diploidization of the meiogynogenetic haploids of turbot could be achieved in -2℃ cold water shock treatment for 45 min to the eggs 6.5 min after fertilization with the UV-irradiated sperm of red sea bream. Two batches of meiogynogenetic diploids of turbot were produced by the above UV and cold shock treatments, and a comparison of the survival and growth between one batch of the meiogynogenetic diploids and their half-sib normal diploids had been lasted for 17 months. Up to the 60 days after hatching, the survival rate of the meiogynogenetic dipoloids was only 0.61%, which was less than a tenth of that of their half-sib’s. Thereafter, survival rates of both groups were similar and well above 90%. The growth rate of meiogynogenetic diploids had been lower than that of their half-sib’s upto the 14th months after the hatching, and became equal to that of their half-sib’s by the end of 17th months after the hatching, which was possible the result of growth advantage of more females in the meiogynogenetic dipoids.(2) Comparative study in embryonic developmental morphology and rates of different ploidy of turbotThe developmental morphology and rates of turbot embryos, which includes hybrid diploid, haploid and gynogenetic diploid induced by cryopreserved sperm of red sea bream, and normal diploid and triploid induced by turbot sperm, were observed and compared. As a result, under the hatchery condition of water temperature of 14.5±0.5 ℃, pH of 7.8-8.2, and salinity of 29.7, the developing rates of both gynogenetic diploid and triploid were slower than normal diploid from fertilization to hatching stage, while that of triploid was faster than gynogenetic diploid. Before blastula stage, the developing rate decreases in the sequence of haploid, hybrid diploid, normal diploid, triploid and gynogenetic diploid. After that, the development of haploid slowed down and was detained for a long time in the gastrula stage. From blastula stage to tail bud stage, the developing rate decreased in the sequence of hybrid diploid, normal diploid, triploid, gynogenetic diploid and haploid. The hybrid diploid embryos sank down to the bottom of hatching net from now on, and all died before the end of heart beating stage. The time from fertilization to hatching for normal diploid, triploid, gynogenetic diploid and haploid was 109h15m,109h30m,112h15m and 124h40m, respectively, while the hatching rate of them was 83.4%,44.6%,42.4% and 4.5%, respectively. The morphological abnormalities such as body slightness, obscureness, and organ differentiation, were observed in the hybrid diploid that died before the end of heart beating stage. Typical haploid syndrome (i.e., morphological abnormality) was also observed in the haploid embryo. No significant morphological difference among gynogenetic diploid, triploid and normal diploid embryos was observed in this study. However, compared to the normal diploid, both gynogenetic diploid and triploid had a lower hatching rate and higher rate of abnormality.(3) Mitotic gynogenesis induction in turbotA protocol for artificial induction of mitotic gynogenesis was developed in turbot involving a combination of UV irradiation of cryopreserved red sea bream sperm, followed by the application of a hydrostatic pressure shock to the activated eggs. Under the strict control of steady water temperature (14.5±0.5 ℃) pre-shock, the optimal initiation time for pressure shock of blocking the first mitotic was determined to be 85-90 min after fertilization, while the optimal intensity of pressure and duration time were determined to be 75 MPa and 6 min, respectively. The lowest total hatching rate, averaged 2.26 ±1.91%, was observed in mitogynogenetic diploids, only account about 10% of that in meiogynogenetic diploids. In addition, above 40% proportions of mitogynogenetic larvae exhibited severe deformities on the head, vertebral column or tail, whereas the rest developed a morphologic normal appearance with similar to normal control groups (hybrid diploid treated by pressure shock) and meiogynogenetic diploids. All of the embryos in sham control groups died before hatching. One batch of mitogynogenetic diploids of turbot was produced using eggs from four different female parents. The diploid status was verified by cytometre flow, while the homozygosity was primarily confirmed by unilocus analysis of seven microsatellites markers. Survival rate of mitogynogenetic diploids was significantly lower in the larval and juvenile period (3.25% in the period 1-40 DAH and 51.08% in the period 40-60 DAH), while the similar high survival rate was observed in the young fish period (in the period 60 DAH-5 months) compared to their "half-sib" diploids. At 5 months, the mitotic gynogenetic diploid had grown to 16.18±11.30 g in body weight and 9.18±2.03 cm in total length in a manner significant lower than that of the "half-sib" diploids (33.02±6.23 g and 12.24±0.77 cm, respectively).(4) Autotetraploid induction in turobtA protocol for artificial induction of autotetraploid was developed in turbot by the application of a hydrostatic pressure shock to the normal activated eggs. Under the strict control of steady water temperature (14.5±0.5 ℃) pre-shock and duration time of 6 min, the optimal initiation time for pressure shock of blocking the first mitotic was determined to be 75-80 min after fertilization, while the optimal intensity of pressure was determined to be 75 MPa, respectively. Autotetraploid was induced in two large-scales followed the optimum conditions. The hatching rates of induced tetraploid were 16.54% and 11.40%, respectively, significant lower than the normal diploid group (71.62%). Survival rates of tetraploid were significantly lower in the larval and juvenile period (0.43% and 0.16% in the period 1-60 DAH), while the similar high survival rate was observed in the young fish period (in the period 60 DAH-12 months) compared to normal diploid. Tetraploid percentage rates of the induced individuals were checked at the newly hatched larvae stage by NORs-Ag stained method and 5 months stage by chromosome analysis, accouting for 46.67% and 5% respectively, showing a remarkable declined rate. The remaining 465 induced individuals were selected based on erythrocytes measurement, while 23 individuals showed a proximately twice time of erythrocytes volume compared with that of the normal diploid.(5) Effects of temperature on sex differentiation and genetic sex determination system in turbotA detailed understanding of sexual development and its timing is critical to control sex and optimize culture. Structural and cellular sex-distinguishing markers were identified histologically, and then used to describe ovarian development in female and testicular development in male turbot. In presumptive ovaries of turbot, development of an ovarian cavity first occurs in fish of 37.43 mm total length (TL,45 dph). In presumptive testes, the formation of seminiferous tubules first occurs in fish of 42.83 mm TL (55 dph). The effect of temperature on sex determination in turbot was addressed in a separate experiment. Juvenile southern flounder were rared at 18℃ from an initial mean size of 12.30±1.45 mm TL (20 dph),25 ℃ from an initial mean size of 12.30±1.45 mm (20 dph),20.05±1.24 mm (28 dph),30.11±2.15 mm (40 dph),43.00±3.64 mm (54 dph),51.53±2.34 mm TL (65 dph) respectively, to a final mean size above 70 mm TL (90 dph). The sex of each fish was then determined by macroscopic and histological examination of the gonads. Sex ratios were not significantly different from 1:1 in 18℃ group. High induced phenotypic sex reversal in juvenile turbot of an initial mean size both 12.30±1.45 mm and 20.05±1.24 mm TL, producing a higher proportion of males (73.43±3.88% and 74.40±6.36% males, respectively, P<0.05). Raising initial mean size at 25 ℃ temperature held sex ratios close to 1:1. These findings indicate that sex differentiation in turbot is sensitive to high temperature from an initial mean size of 20.05±1.24 mm to 30.11±2.15 mm and sex determination can be affected by high temperature. Sex ratios of 4 batches of meiogynogenetic diploid were 82.86%、85.00%、76.67%、75.81% female, significantly biased from 1:1, consistent with the theoretical value (82.2% F:17.8% M) deduced based on genetic recombination in meiogynogenesis from a female heterozygous hypothesis (ZW/ZZ). Sex ratios of mitogynogenetic diploid were 1:1. The progenies in hybrid of meiogynogenetic males and normal female exhibited proximate 1:1 sex ratios in two separate batches. These findings indicate a ZW/ZZ model of genetic sex determination in turbot. |
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