Toxicity and protective efficiency of cryoprotectants to flounder (Paralichthys olivaceus) embryos

With the purpose of finding an ideal cryoprotectant or combination of cryoprotectants in a suitable concentration for flounder (Paralichthys olivaceus) embryo cryopreservation, we tested the toxicities, at culture temperature (16 degrees C), of five most commonly used cryoprotectants-dimethyl sulfoxide (Me2SO), glycerol, methanol (MeOH), 1,2-propylene glycol (PG) and ethylene glycol (EG). In addition, cryoprotective efficiency to flounder embryos of individual and combined cryoprotectants were tested at -15 degrees C for 60 min. Five different concentrations of each of the five cryoprotectants and 20 different combinations of these cryoprotectants were tested for their protective efficiency. The results showed that the toxicity to flounder embryos of the five cryoprotectants are in the following sequence: PG < MeOH < Me2SO < glycerol < EG (P < 0.05); whereas the protective efficiency of each cryoprotectant, at -15 degrees C for a period of 60 min, are in the following sequence: PG > Me2SO approximate to MeOH approximate to glycerol > EG (greater symbols mean P < 0.05, and approximate symbols mean P > 0.05). Methanol combined with any one of the other cryoprotectants gave the best protection, while ethylene glycol combined with any one of the other cryoprotectants gave the poorest protection at -15 degrees C. Toxicity effect was concentration dependent with the lowest concentration being the least toxic for all five cryoprotectants at 16 degrees C. For PG, MeOH and glycerol, 20% solutions gave the best protection at -15 degrees C; whereas a 15% solution of Me2SO, and a 10% solution of EG, gave the best protection at -15 degrees C. (c) 2004 Elsevier Inc. All rights reserved.

Cryopreservation of flounder (Paralichthys olivaceus) sperm with a practical methodology

A simple and convenient protocol for the cryopreservation of the flounder (Paralichthys olivaceus) sperm was established for "on the spot" cryopreservation of large quantities of semen. The use of three cryoprotectants, dimethyl sulphoxide (DMSO), glycerol (Gly) and methanol was tested in the method. The percentage of motile sperm present in semen after it had been frozen and thawed in the presence of DMSO, Gly or methanol was 60.5 +/- 3.6, 79.17 +/- 4.5 and 13.25 +/- 4.7%, respectively. The fertilization rates of this sperm were 67.06 +/- 15.1, 76.20 +/- 10.0 and 44.93 +/- 22.6%, while the hatching rates of eggs fertilized with this sperm were 37.40 +/- 8.3, 48.18 +/- 25.7 and 23.35 +/- 10.8%, respectively. It was found that Gly and DMSO were better cryoprotectants than methanol, with Gly giving the best overall results. Under scanning electron microscopy, it could be seen that while the majority of the frozen-thawed sperm remained morphologically normal, some exhibited lost or dilated mitochondria, swollen mid-pieces, broken tails, or damaged cell membrane, which probably caused the decrease in motility and fertility of the frozen-thawed sperm. (C) 2003 Elsevier Science Inc. All rights reserved.

Cloning and expression analysis of myogenin from flounder (Paralichthys olivaceus) and promoter analysis of muscle-specific expression

Myogenin is a bHLH transcription factor of the MyoD family. It plays a crucial role in myoblast differentiation and maturation. We report here the isolation of flounder myogenin gene and the characterization of its expression patterns. Sequence analysis indicated that flounder myogenin shared a similar structure and the conserved bHLH domain with other vertebrate myogenin genes. Flounder myogenin gene contains 3 exons and 2 introns. Sequence alignment and phylogenetic showed that flounder myogenin was more homologous with halibut (Hippoglossus hippoglossus) myogenin and striped bass (Morone saxatilis) myogenin. Whole-mount embryo in situ hybridization revealed that flounder myogenin was first detected in the medial region of somites that give rise to slow muscles, and expanded later to the lateral region of the somite that become fast muscles. The levels of myogenin transcripts dropped significantly in matured somites at the trunk region. Its expression could only be detected in the caudal somites, which was consistent with the timing of somite maturation. Transient expression analysis showed that the 546 bp flounder myogenin promoter was sufficient to direct muscle-specific GFP expression in zebrafish embryos. (c) 2007 Elsevier Inc. All rights reserved.

Induction of diploid gynogenesis in turbot Scophthalmus maximus with left-eyed flounder Paralichthys olivaceus sperm

Turbot Scophthalmus maximus exhibits sexually dimorphic growth, with females growing faster and reaching larger adult sizes than males. Thus, development of techniques for preferentially producing females is necessary to optimize production of these species. In this paper, gynogenetic diploids of turbot were induced by activating egg development with ultraviolet (UV)-irradiated left-eyed flounder Paralichthys olivaceus sperm combined with cold shock to prevent extrusion of the second polar body. The results of UV irradiation experiments showed that survival, motility, and duration of activity of P. olivaceus sperm generally decreased with increase in UV dose. The typical Hertwig's effect was observed after fertilized turbot eggs with UV-irradiated P. olivaceus sperm and the optimal UV dose for gynogenetic haploid production was 36,000 erg mm(-2). At 15 degrees C, appropriate timing of cold shock for retention of the second polar body in turbot eggs was at 6 min after fertilization. Results of different combinations of two shock temperatures (1 or 3 degrees C) and four shock durations (15, 25, 35 or 45 min) at 6 min after fertilization demonstrated that shock of 25 min at 1 degrees C gave the highest production of diploid gynogens (39.58% relative to its diploid control). The results of this study reveal that the use of UV-irradiated P. olivaceus sperm for activation of turbot eggs and cold shock for polar body retention is an effective method to produce gynogenetic offspring.

Effects of hydrostatic pressure on microtubule organization and cell cycle in gynogenetically activated eggs of olive flounder (Paralichthys olivaceus)

Indirect immunofluorescence staining was used to detect cytological changes of isolated blastodisks during mitosis of flounder haploid eggs treated with hydrostatic pressure. Changes in microtubule structure and expected cleavage suppression were observed from blastodisk formation to the third cell cycle, with obvious differences between treated and control eggs. In most eggs, microtubules were disassembled and the nucleation capacity of the centrosome was temporarily inhibited after pressure treatment. Within 15-20 min after treatment, the nucleation capacity of the centrosome began to gradually recover, with slow regeneration of microtubules; approximately 25 min after treatment, the nucleation capacity of the centrosome recovered completely, regenerated distinct bipolar spindles, and the first mitosis ensued. During the second cell cycle, approximately 61% of the embryos were at the two-cell stage, with a monopolar spindle in each blastomere; that treatment was effective was based on second cleavage blockage. Approximately 15% of the eggs still remained at the one-cell stage and had a monopolar spindle (treatment was effective, according to the general model of first cleavage blockage). However, treatment was ineffective in approximately 15% of the embryos (bipolar spindle in each blastomeres) and in another 8% (bipolar spindle in one of the two blastomeres and a monopolar spindle in the other; both mechanisms operating in different parts of the embryo). This is the first report elucidating mitotic gynogenetic diploid induction by hydrostatic pressure in marine fishes and provides a cytological basis for developing an efficient method of inducing mitotic gynogenesis in olive flounder. (C) 2007 Elsevier Inc. All rights reserved.

Identification and analysis of the immune effects of CpG motifs that protect Japanese flounder (Paralichthys olivaceus) against bacterial infection

CpG-containing oligodeoxynucleotides (ODNs) are known to be immunostimulatory in vertebrate systems and can activate both innate and adaptive immune responses. In this report, we described the selection, identification, and analysis of CpG motifs with immunoprotective effects in Japanese flounder. Sixteen CpG ODNs were synthesized and examined for the ability to inhibit bacterial dissemination in Japanese flounder blood. Four ODNs with the strongest inhibitory effects were selected and mixed to form ODNs 4M. In addition, a plasmid, pCN6, was constructed that contains the sequences of the four selected ODNs. When administered into Japanese flounder via intraperitoneal injection, both ODNs 4M and pCN6 could, in dose and time dependent manners, afford short-term protection against the infections of two different bacterial pathogens. Immunological analyses showed that ODNs 4M and, especially, pCN6 activated head kidney macrophages and enhanced serum bactericidal activity via probably the alternative pathway of complement activation. When used as a DNA vaccine to immunize Japanese flounder, pCN6 conferred apparent protections (42.9% and 52.6%, respectively, in terms of relative percent survival) against the challenges of two different fish pathogens at 4-week post-vaccination. Transcriptional analysis showed that vaccination with pCN6 upregulated the expression of the genes encoding NKEF, MHC II alpha, IL-1 beta, Mx, and MHC I alpha. These results demonstrate that ODNs 4M and pCN6 are immunostimulatory in Japanese flounder and can induce short- and long-term nonspecific protections against bacterial infections. (C) 2010 Elsevier Ltd. All rights reserved.

Molecular structure and expression patterns of flounder (Paralichthys olivaceus) Myf-5, a myogenic regulatory factor

Myf-5, a member of the myogenic regulatory factors (MRF), has been shown to be expressed in muscle precursors in early stage zebrafish embryos. The MRFs, including MyoD, Myf-5, Myogenin and MR-F4, belong to the basic Helix-Loop-Helix transcription factors that contain a conserved basic Helix-Loop-Helix (bHLH) domain. To better understand the role of Myf-5 in the development of fish muscles, we have isolated the Myf-5 genomic sequence and cDNA from Flounder (Paralichthys olivaceus), and analyzed its structures and patterns of expression. Promoter analysis identified several putative transcription factor binding sites such as an E-box, NF-Y sites that might confer muscle-specific expression. Myf-5 transcripts were first detected in the paraxial mesoderm that gives rise to slow muscles. During somitogenesis, Myf-5 expression was found in developing somites. Myf-5 expression decreased gradually in somites in the anterior region, but remained strong in the newly formed somites. In the hatching stage, the expression was also detected in other muscle cells such as head muscle and fin muscle. In the growing fish, RT-PCR results showed that Myf-5 was expressed in the skeletal muscle and intestine. (c) 2006 Elsevier Inc. All rights reserved.

Characterization of muscle-regulatory gene, MyoD, from flounder (Paralichthys olivaceus) and analysis of its expression patterns during embryogenesis

Specification and differentiation of skeletal muscle cells are driven by the activity of genes encoding members of the myogenic regulatory factors (MRFs). In vertebrates, the MRF family includes MyoD, Myf5, myogenin, and MRF4. The MRFs are capable of converting a variety of nonmuscle cells into myoblasts and myotubes. To better understand their roles in fish muscle development, we isolated the MyoD gene from flounder (Paralichthys olivaceus) and analyzed its structure and patterns of expression. Sequence analysis showed that flounder MyoD shared a structure similar to that of vertebrate MRFs with three exons and two introns, and its protein contained a highly conserved basic helix-loop-helix domain (bHLH). Comparison of sequences revealed that flounder MyoD was highly conserved with other fish MyoD genes. Sequence alignment and phylogenetic analysis indicated that flounder MyoD, seabream (Sparus aurata) MyoD1, takifugu (Takifugu rubripes) MyoD, and tilapia (Oreochromis aureus) MyoD were more likely to be homologous genes. Flounder MyoD expression was first detected as two rows of presomitic cells in the segmental plate. From somitogenesis, MyoD transcripts were present in the adaxial cells that give rise to slow muscles and the lateral somitic cells that give rise to fast muscles. After 30 somites formed, MyoD expression decreased in the somites except the caudal somites, coincident with somite maturation. In the hatching stage, MyoD was expressed in other muscle cells and caudal somites. It was detected only in muscle in the growing fish.

Effects of cold shock on microtubule organization and cell cycle in gynogenetically activated eggs of olive flounder (Paralichthys olivaceus)

Cytological changes and subsequent mitotic processes were studied in gynogenetically activated eggs of olive flounder subjected to cold-shock treatment using indirect immunofluorescence staining of isolated blastodisks. Obvious differences between controls and treated eggs were detected during early cell division. The developmental process of haploid control was similar to that of the diploid control except several minutes delayed. Spindles disassembled by the cold-shock treatment regenerated soon after treatment, resulting in the occurrence of the first mitosis. The immature daughter centriole was easily depolymerized by cold-shock treatment, leading to the formation of the bipolar spindle in the first cell cycle and the formation of the monopolar spindle in the second cell cycle, resulting in chromosome set doubling. Some two-cell stage eggs had a monopolar spindle in one blastomere and a bipolar spindle in another during the second mitosis. These eggs had a high potency developing into haploid-diploid mosaics. To the best of our knowledge, this study is the first to clarify the mechanism of chromosome set doubling in marine fishes and provides a preliminary cytological basis for developing a reliable and efficient protocol for mitotic gynogenesis induction by cold-shock treatment in olive flounder.

Genetic analysis of meio- and mito-gynogenetic stocks of Paralichthys olivaceus with microsatellite markers

Twenty microsatellite markers (Po1, Po13, Po33, Po35, Po42, Po48, Po56, Po89, Po91, kop6, kop7, kop8, kop12, kop15, kop18, kop21, kop22, kop23, kop26, Po-strl) were used to assess the meiogynogenetic and mitogynogenetic stocks of the left-eyed flounder, Paralichthys olivaceus, which were derived from single pair crossing. Twelve of the 20 loci utilized showed heterozygosity in the female and were mapped in relation to their centromeres in the meiogynogenetic diploid flounder. Microsatellite-centromere map distance, calculated under the assumption of complete interference, ranged from 15.8 cM for kop22 to 50 cM for Po13, Po56 and Po89. Excluding the kop22, the heterozygosities of the rest of the loci were close to 100%, suggesting the occurrence of near complete interference on the chromosome arms that carried these loci. In the mitogynogenetic diploid flounder, each individual showed exact homozygousity and the segregation profiles did not deviate from the Mendelian 1: 1 pattern. The results indicated that there was no lethal gene linked with the loci analyzed. Such high interference accounted for the high recombination rates and large map distances. The Po13 and Po56 loci, Po91 and kop18 loci, kop15 and kop21 loci are tightly linked on the same chromosome arm in pairs.

Effect of ultraviolet irradiation on sperm of the left-eyed flounder, Paralichthys olivaceus

In this study, at proper dosage of ultraviolet (UV) irradiation (180 sec: 36,000 erg/mm(2)), sperm chromosomes of left-eyed flounder, Paralichthys olivaceus, were inactivated, while spermatozoa maintained ability to move and inseminate eggs. Gynogenetic haploids were detected by morphological observation, chromosome counting, and flow cytometer analysis. The ultrastructure of treated sperm was observed under scanning electronic microscope (SEM) and transmission electronic microscope (TEM). The results showed that after being irradiated at lower dosage of irradiation (0-180 sec: 0-36,000 erg/mm(2)), the surface structure of spermatozoa was not affected by UV irradiation, while the inner structures including membrane system and karyoplasm denseness of treated spermatozoa were little changed. However, obvious changes were observed in their membrane system, mitochondria, and nucleus if the dosage of irradiation increased to 240 sec: 48,000 erg/mm(2) or 300 sec: 60,000 erg/mm(2). The sperm survival rates did not change at the lower dosages of the UV irradiation (0-180 sec: 0-36,000 erg/mm(2)) but decreased as the irradiation dosage increased. The motility of treated sperm was lower than that of control group in general but did not change with UV irradiation dosage increasing at the certain range of 0-300 sec: 0-60,000 erg/mm(2).

牙鲆Paralichthys olivaceus（♀）×夏鲆Paralichthys dentaus（♂）分子遗传学及数量遗传学研究

本论文采用扩增片段长度多态性（AFLP）技术对牙鲆Paralichthys olivaceus群体和夏鲆Paralichthys dentaus 群体进行了种群鉴定和遗传结构分析。共用8 对引物对两个群体（每个群体30 个个体）进行了分析。总共产生了379 个条带，条带大小在60－1000bp。牙鲆与夏鲆的遗传多态性分别为53.83%和22.22%。牙鲆群体的多态位点数显著低于夏鲆群体。牙鲆群体和夏鲆群体种的特异条带分别为27.3% 和 29.61%。两群体平均杂和度分别为0.0701 和 0.1556，香农氏多态指数分别为0.1044 和0.2387。群体内平均遗传距离分别为0.0705 （0.0214 到0.1377）和0.1656 （0.0629 到 0.2338），两群体间的平均遗传距离为0.6328。 AFLP 技术是进行种间群体结构分析的一个很好的分子标记方法，对牙鲆和夏鲆亲本群体进行遗传背景析为今后的杂交育种阐明种间杂交的遗传机理奠定基础。 在本论文中我们采用5×8 因子交配设计建立了牙鲆P. olivaceus（♀）× 夏鲆P. dentaus（♂）杂交家系，对生长相关性状的遗传力进行分析。所有家系都混合养殖在同一养殖池中，40 日龄，在养殖池中随机采集600 个个体，测量体长，体宽，体重等数量性状；然后提取杂交子代DNA 进行家系的鉴定。首先从牙鲆的微卫星中筛选了在牙鲆、夏鲆以及杂交子代扩增多态性较好的10 对微卫星引物（Po1, Po13, Po20, Po35, Po42, Po48, Po56, Po58, Po91, Poli 23TUF）。我们采用了三对引物对600 个个体进行了家系鉴定，共有400 多个体鉴定出自己的亲本，成功率达到80％以上。基于以上结果我们认为微卫星标记可以做为一个有效的标记来代替现实的物理标记，并且可以在子代的早期进行亲本的鉴定。 在养殖过程中，我们对自交和杂交家系生长状况进行了跟踪分析，在181刘清华 牙鲆（♀）×夏鲆（♂）分子遗传学及数量遗传学研究 博士学位论文II日龄之前杂交鲆体长和体重均为未表现出杂种优势，杂种优势率值始终为负值，但是绝对值在逐渐减小。从196 日龄之后杂交鲆杂种优势开始表现出来，并且在196 日龄之后杂种优势率显著的增加。256 日龄体长杂种优势率为14.29%，体重杂种优势率为59.78%, 271 日龄体长杂种优势率达到27.36%，体重杂种优势率为 102.32%。 本研究对杂交鲆在40 日龄的体长，体高、尾柄长、尾柄高，以及全长和体重的遗传力进行了估计，5 个性状的体长半同胞遗传力h2 S 为0.00146－0.719，5个性状的全同胞遗传力h2D 为0.00121－0.632，5 个性状的半同胞和全同胞的平均遗传力h2SD 为0.001335－0.6755。其中全长的遗传力最大。实验结果说明对早期幼鱼进行体长、体重等性状实施选育策略可能会显著影响后期杂交鲆的生长。

Size hierarchies affecting the social interactions and growth of juvenile Japanese flounder, Paralichthys olivaceus

Japanese flounder Paralichthys olivaceus (T. & S.)juveniles were size-graded and divided into three groups (small, large, and mixture of small and large flounder), and their social interactions (feeding, aggressive attacking and activity) and growth were investigated. The growth of the small flounder was markedly suppressed by the presence of the large flounder. Large flounder did not significantly suppress the overall food intake of the small flounder but exhibited high aggressive attacking on them and consequently inhibited their activity. Size dominance showed little influence on the aggressive behavior, feeding, activity and growth of the large flounder. The large flounder could not effectively defend the food in excess during the experiments ruling out disproportional food acquisition as the primary mechanism responsible for the size hierarchy effect. Aggressive interaction of the large flounder on the small flounder might be an important cause for the growth retardation of the small flounder. In culture, size grading could markedly improve the growth and survival of the early juvenile flounder. (C) 2004 Elsevier B.V. All rights reserved.

Identification of factors affecting the growth and survival of the settling Japanese flounder larvae, Paralichthys olivaceus

A series of experiments were conducted to identify the factors that affected the growth and survival of the settling flounder larvae Paralichthys olivaceus. Settling larvae 24 days after hatching (DAH) were reared in 10-l experimental tanks up to 40 DAH, and two of the following factors were changed as controlled factors in each experiment: light regime (24L:0D or 12L:12D), prey density (1500, 3000, or 5000 Artemia l(-1)), shelter (sand or no sand) and stocking density (5, 10, or 15 fish l(-1)). Early settling larvae (24-35 DAH) experienced little mortality (less than 10% of the overall mortality) that was not significantly affected by above factors. In contrast, late settling larvae (36-40 DAH) suffered high cannibalistic mortality which was significantly influenced by each of the above factors. Larvae experienced significantly lower mortality at 10 fish l(-1) level than at other densities. Larvae at 15 fish l(-1) level had higher mortality than at 5 fish l(-1) when all other factors were identical. Larvae at 3000 and 5000 Artemia l(-1) treatments survived significantly better than at 1500 Artemia l(-1), but no significant differences in larval mortality were found between the two higher densities. Larvae suffered higher mortality at low prey density or at the absence of sand when they were exposed to longer photoperiod. Low stocking density significantly improved the growth of the settling larvae. The average daily instantaneous growth rate (G) at 5 and 15 fish l(-1) treatments were 0.050 and 0.034, with the coefficient of variation (CV) in final length at 16.4 and 23.5, respectively. Daily instantaneous growth rate increased significantly from 0.033 in the 1500 Artemia l(-1) to 0.041 and 0.045 in the 3000 and 5000 Artennia l(-1), respectively, but no significant difference in larval growth existed between the two higher prey densities. These findings suggested that the optimal prey density for growth and survival of the settling flounder larvae at a stocking density of 5 - 15 fish l(-1) was around 3000 Artemia l(-1) . Larvae that were exposed to 24L showed 20% increase in growth ( G = 0.046, CV = 18.7) than those exposed to 12L ( G = 0.037, CV = 20.5). Longer exposure to light significantly improved larval growth, provided sufficient food was available. Sand substrate did not show significant effects on larval growth, possibly because the larvae spent most of the time swimming or feeding in the water column during this stage. (C) 2003 Elsevier Science B.V. All rights reserved.

Cannibalism in Japanese flounder juveniles, Paralichthys olivaceus, reared under controlled conditions

Laboratory experiments were conducted to determine the effects of sand substratum, light, starvation, fish density and size variation on cannibalism among Japanese flounder juveniles, Paralichthys olivaceus. Cannibalistic behavior (biting and swallowing) increased significantly with starvation except in darkness, where juveniles could not find and attack their prey. Cannibalism occurred more frequently in heterogeneous than in the homogeneous size groups. Cannibalism was more frequent in light than in darkness at each starvation level. With sufficient food present, sand had no significant effect on cannibalism among homogeneous size juveniles, but played an important role in discouraging predation among members of the heterogeneous size groups. With sufficient food present, the rate of cannibalism was generally low and there were no significant differences in cannibalism among fish density levels of the homogeneous juveniles, With starvation, the cannibalism rate was significantly higher in high density groups than in the lower ones. Our results indicate that size variation, starvation and Light are the major factors affecting cannibalism in flounder juveniles, while sand and fish density only have a limited effect on cannibalism, depending on the nature of other factors. (C) 2000 Elsevier Science B.V. All rights reserved.