Artificial induction of mito-gynogenetic diploids in large yellow croaker (Pseudosciaena crocea) by hydrostatic pressure

The present study investigated conditions for inducing mito-gynogenetic (endomitosis) diploids by hydrostatic pressure in the large yellow croaker Pseudosciaena crocea. In haploid control groups, the development of eggs was activated with ultraviolet radiated semen. All fry presented typical haploid syndrome in the haploid control groups, and were verified as haploids using cytometry. After hydrostatic pressure treatment, morphologically normal fry reappeared at different frequencies according to the intensity and time of pressure shock. Fry with normal appearance in the pressure treated groups were verified as gynogenetic double haploids (GDHs), containing only one allele from the female parent at all four diagnostic microsatellite loci. For a fixed duration of 3 min, the optimal intensity of blocking the first mitosis was determined to be 40 Mpa, which was similar to that of blocking the second meiosis. There was a “window” of starting time, from 36.1 min to 38.1 min post-insemination at 25.0±1.0°C, within which the production of GDHs was not significantly different. Maximum production of morphologically normal fries, 9.36%±2.97% of developed eggs, was found when the eggs were shocked with hydrostatic pressure at 40 Mpa for 3 min, starting from 38.1 min post insemination at 25.0±1.0°C.     

Artificial induction of mito-gynogenetic diploids in large yellow croaker (Pseudosciaena crocea) by hydrostatic pressure

The present study investigated conditions for inducing mito-gynogenetic(endomitosis) diploids by hydrostatic pressure in the large yellow croaker Pseudosciaena crocea.In haploid control groups,the development of eggs was activated with ultraviolet radiated semen.All fry presented typical haploid syndrome in the haploid control groups,and were verified as haploids using cytometry.After hydrostatic pressure treatment,morphologically normal fry reappeared at different frequencies according to the intensity and...     

STUDY ON EMBRYONIC DEVELOPMENT AND EARLY GROWTH OF TRIPLOID AND GYNOGENETIC DIPLOID LEFI—EYED FLOUNDER, PARALICHTHYS OLIVACEUS （T. et S. ）

The early effects of chromosomal manipulation of eggs and sperm on the yields of trip-loid and gynogenefic diploid larvae of Paralichthys olivaceus were investigated. Triploidy was achieved by cold shocking fen.ilized eggs at 0 - 2℃ for 45 minutes duration 5 minutes after fen.ilization, and the in-duced triploidy rates were 31.2% - 50% and the relative hatching rates were 53.3% - 99%. Gynnge-aetic diploids were obtained when eggs were inseminated with irradiated sperm and cold shocked at 0 -2℃ for 45 minutes duration 5 minutes after fertihzation. The induced gynogenetic diploid rates and the relative hatching rates were 94 % - 96 % and 48.5 % - 68.5 % respectively. The embryonic development of the triploid experimental group and of the gynogenetic diploid experimental group was delayed at first compared with the control group. But from the gastrula stage, it was not delayed anymore. There were no significant differences in the growth of the triploid experimental group larvae and the control group larvae, and in the growth of the gynogenetic diploid experimental group larvae and the control group larvae ac-cording to Student‘s t-test (α = 0.05). The relationship between the early growth of the triploid experi-mental group larvae and that of gynogenetic diploid experimental group larvae was also studied.     

Microsatellite-Centromere Mapping in Japanese Scallop(Patinopecten yessoensis) Through Half-Tetrad Analysis in Gynogenetic Diploid Families

Gene-centromere mapping is an essential prerequisite for understanding the composition and structure of genomes. Half-tetrad analysis is a powerful tool for mapping genes and understanding chromosomal behavior during meiosis. The Japanese scallop(Patinopecten yessoensis), a cold-tolerant species inhabiting the northwestern Pacific coast, is a commercially important marine bivalve in Asian countries. In this study, inheritance of 32 informative microsatellite loci was examined in 70-h D-shaped larvae of three induced meiogynogenetic diploid families of P. yessoensis for centromere mapping using half-tetrad analysis. The ratio of gynogenetic diploids was proven to be 100%, 100% and 96% in the three families, respectively. Inheritance analysis in the control crosses showed that 51 of the 53 genotypic ratios observed were in accordance with Mendelian expectations at the 5% level after Bonferroni correction. Seven of the 32 microsatellite loci showed the existence of null alleles in control crosses. The second division segregation frequency(y) of the microsatellite loci ranged from 0.07 to 0.85 with a mean of 0.38, suggesting the existence of positive interference after a single chiasma formation in some chromosomes in the scallop. Microsatellite-centromere distances ranged from 4 c M to 42 c M under the assumption of complete interference. Information on the positions of centromeres in relation to the microsatellite loci will represent a contribution towards the assembly of genetic maps in the commercially important scallop species.     

Seasonal variation of the glycogen enzyme activity in diploid and triploid Pacific oyster gonad during sexual maturation

The glycogen content and the activities of two key enzymes in glycogen metabolism, glycogen phosphorylase and gly- cogen synthetase, in the gonad of diploid and triploid Pacific oysters (Crassostrea gigas) were compared during maturation. The glycogen content in the gonad of diploids decreased with gametogenesis (by 85.7%), but the glycogen content in the gonad of trip- loids did not vary significantly. Activity of glycogen phosphorylase (GP) in the gonad of diploids decreased with gametogenesis (by 55.5%), while GP activity of triploids did not vary significantly during maturation. Activity of glycogen synthetase (GS) in the gonad of diploids increased slightly with gametogenesis, reaching a peak in June. Activity of GS declined sharply from June to July, which might be due to gonad spawning. GS activity of triploid oysters in spawning time (July and August) was significantly higher than that in other months, which might be explained with a ‘compensating’ mechanism for the higher glycogen content in triploids.     

Establishment and characterization of a testicular Sertoli cell line from olive flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis>

The culture of Sertoli cells has become an indispensable resource in studying spermatogenesis. A new Sertoli cell line (POSC) that consisted predominantly of fibroblast-like cells was derived from the testis of the olive flounder Paralichthys olivaceus and sub-cultured for 48 passages. Analysis of the mtDNA COI gene partial sequence confirmed that the cell line was from P. olivaceus. Cells were optimally maintained at 25°C in DMEM/F12 medium supplemented with fetal bovine serum, basic fibroblast growth factor, and epidermal growth factor. The growth curve of POSC showed a typical “S” shape. Chromosome analysis revealed that the cell line possessed the normal P. olivaceus diploid karyotype of 2n=48t. POSC expressed dmrt1 but not vasa, which was detected using RT-PCR and sequencing. Immunocytochemistry revealed that the cells exhibited the testicular Sertoli cell marker FasL. Therefore, POSC appeared to consist of testicular Sertoli cells. Bright fluorescent signals were observed after the cells were transfected with pEGFP-N3 plasmid, with the transfection efficiency reaching 10%. This research not only offers an ideal model for further gene expression and regulation studies on P. olivaceus, but also serves as valuable material in studying fish spermatogenesis, Sertoli cell-germ cell interactions, and the mechanism of growth and development of testis.     

Molecular identification,expression pattern,and in-vitro bioactivity analysis of insulin-like growth factor 2 in olive flounder Paralichthys olivaceus

Insulin-like growth factors (IGFs) are key regulators of development and growth. Here, we characterized the igf2 gene from olive flounder (Paralichthys olivaceus) and determined its temporal and spatial expression. We set up an in-vitro protein expression system in eukaryotic human embryonic kidney (HEK293T) cells and explored its effects on cell proliferation. The flounder igf2 cDNA contained a 648-bp open reading frame (ORF) encoding a protein of 215 amino acids (aa), which spanned the complete signal peptide (47 aa), mature peptide (70 aa), and E domain (98 aa). In adult flounder, igf2 mRNA was detected in all selected tissues. In early development, igf2 mRNA was detected throughout development from unfertilized eggs to hatching-stage embryos. In-situ hybridization analysis indicated that igf2 mRNA was specially expressed in the brain region, floor plate, hypochord, otic vesicle, and pectoral fin during embryogenesis. Western blotting analysis indicated that the soluble recombinant flounder IGF2 protein was successfully produced through eukaryotic expression in HEK293T cells. In addition, the recombinant IGF2 protein significantly promoted the proliferation of human cervical carcinoma (HeLa) and HEK293T cells. These results provide new information about the structural and functional conservation, expression patterns, and biological activity of the igf2 in teleosts.

     

Screening of eye-position related genes with DD-RT-PCR and RDA in the hybrids between Japanese flounder Paralichthys olivaceus and stone flounder Kareius bicoloratus

Flatfish or flounder moves one eye to change body proportion into vertebral asymmetry during metamorphosis, during which some become sinistral while others dextral. However, the mechanism behinds the eye-position has not been well understood. In this research, hybrids between Japanese flounder(♀) and stone flounder (♂) show mixed eye-location in both dextral type and sinistral type, and thus become good samples for studying the eye-migration. mRNAs from pro-metamorphosis sinistral and dextral hybrids larvae...     

Zoospore-derived monoecious gametophytes in Undaria pinnatifida (Phaeophyceae)

The annual life cycle of the brown seaweed Undaria pinnatifi da(Harvey)Suringer comprises a macroscopic diploid sporophyte stage and a microscopic haploid gametophyte stage.In 2011,an unusual zoospore-derived monoecious gametophyte isolate(designated as line 10-5-3)of U.pinnatifi da was observed.To understand this phenomenon,a comprehensive screening of eighty-two previously identifi ed male gametophyte cultures,isolated from three randomly selected cultivars(lines 10,7,and 5)was performed.Thirty-six of the isolates developed both antheridia and oogonia on the same fi lamentous fragment in a standard gametogenesis test(SGT: 18°C,60 μmol photons/(m 2·s)).Selfi ng of the monoecious gametophyte or crossing it with a normal male gametophyte both gave rise to morphologically normal sporophytic offspring.However,crossing resulted in a much higher fertilization rate(89.7%).The hybrid and selfed sporophytic offspring were grown to maturity in fl ow tanks at an ambient temperature of 10–18°C over a period of 69 days.Active zoospores were released from both types of mature sporophylls.The majority of these developed into male gametophytes,while 15%–20% developed into the observed monoecious structures on the same fi lament.Using PCR amplifi cation it was found that all the monoecious gametophyte isolates and the sporophytic offspring resulting from the selfi ng and crossing lacked the femalelinked microsatellite sequence(a part of the locus Up-AC-2A8,GenBank accession No.AY738602.1),indicating their male nature.U.pinnatifi da is an invasive species in some regions and the implications of the above fi ndings for this species in nature are briefl y discussed.     

Development of inter-family nuclear transplant embryos by transplanting the nuclei from the loach blastulae into the non-enucleated zebrafish eggs

The developmental fate of the pronuclei in recombined embryos obtained by transplanting the donor nuclei into the non-enucleated eggs remains controversial in the case of fish.In the present study,the nuclei from the loach blastulae were transplanted into non-enucleated zebrafish eggs,the resulting 9 inter-family nuclear transplant embryos developed to larval stages,Although the development timing of the nuclear transplants resembled that of zebrafish,chromosome examination revealed that most of the recombined embryos were diploids with karyotype characteristic of loach,which was also proved by RAPD analysis.Moreover,3 out of the 9 larval fish formed barb rudiments specific to loach.It was therefore concluded that the nuclear transplant larval fish were inter-family nucleo-cytoplasmic hybrids;and that only the donor nuclei were involved in the development of the nuclear transplant embryos,while the pronuclei in the non-enucleated eggs were likely automatically eliminated during the development.     

Mutations in exons of the CYP17-II gene affect sex steroid concentration in male Japanese flounder (Paralichthys olivaceus)

As a specific gene of fish,cytochrome P450c17-Ⅱ(CYP17-Ⅱ) gene plays a key role in the growth,development and reproduction level of fish.In this study,the single-stranded conformational polymorphism(SSCP) technique was used to characterize polymorphisms within the coding region of CYP17-Ⅱ gene in a population of 75 male Japanese flounder(Paralichthys olivaceus).Three single nucleotide polymorphisms(SNPs) were identified in CYP17-Ⅱ gene of Japanese flounder.They were c.G594A(p.G188R),c.G939A and c.G1502A(p.G490D).SNP1(c.G594A),located in exon 4 of CYP17-Ⅱ gene,was significantly associated with gonadosomatic index(GSI).Individuals with genotype GG of SNP1 had significantly lower GSI(P < 0.05) than those with geno-type AA or AG.SNP2(c.G939A) located at the CpG island of CYP17-Ⅱ gene.The mutation changed the methylation of exon 6.Indi-viduals with genotype AA of SNP2 had significantly lower serum testosterone(T) level and hepatosomatic index(HSI) compared to those with genotype GG.The results suggested that SNP2 could influence the reproductive endocrine of male Japanese flounder.How-ever,the SNP3(c.G1502A) located in exon 9 did not affect the four measured reproductive traits.This study showed that CYP17-Ⅱ gene could be a potentially useful candidate gene for the research of genetic breeding and physiological aspects of Japanese flounder.     

Quantitative comparison of the expression of myogenic regulatory factors in flounder (Paralichthys olivaceus)embryos and adult tissues

MyoD,Myf5,and myogenin are myogenic regulatory factors that play important roles during myogenesis.It is thought that MyoD and Myf5 are required for myogenic determination,while myogenin is important for terminal differentiation and lineage maintenance.To better understand the function of myogenic regulatory factors in muscle development of flounder,an important economic fish in Asia,real-time quantitative RT-PCR was used to characterize the expression patterns of MyoD,Myf5, and myogenin at early stages of embryo development,and in different tissues of the adult flounder.The results show that,Myf5 is the first gene to be expressed during the early stages of flounder development, followed by MyoD and myogenin.The expressions of Myf5,MyoD,and myogenin at the early stages have a common characteristic:expression gradually increased to a peak level,and then gradually decreased to an extremely low level.In the adult flounder,the expression of the three genes in muscle is much higher than that in other tissues,indicating that they are important for muscle growth and maintenance of grown fish.During embryonic stages,the expression level of MyoD might serve an important role in the balance between muscle cell differentiation and proliferation.When the MyoD expression is over 30%of its highest level,the muscle cells enter the differentiation stage.     

Proteomic aspects of infection by lymphocystis disease virus in Japanese flounder (Paralichthys olivaceus)

To reveal the key factor in self-healing from LCDV (lymphocystis disease virus)-infec Japanese flounder (Paralichthys olivaceus), serum proteins from self-healing and sick Japanese floun were separated by two-dimensional electrophoresis to screen differentially expressed proteins. Prot spots demonstrating changes greater than two-fold in the expression level were digested and furt identified in capillary liquid chromatography tandem mass spectrometry (LC-MS/MS). Two immuni relevant proteins were thus identified as transferrin and the complement component C3 of Japan flounder. These findings suggest that the two proteins may play important roles in the self-healing lymphocystis in Japanese flounder. This is an important theoretical foundation to promote self-healing LCDV-infected Japanese flounder by improving their innate immunity.     

Screening of eye-position related genes with DD-RT-PCR and RDA in the hybrids between Japanese flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis> and stone flounder <Emphasis Type="Italic">Kareius bicoloratus</Emphasis>

Flatfish or flounder moves one eye to change body proportion into vertebral asymmetry during metamorphosis, during which some become sinistral while others dextral. However, the mechanism behinds the eye-position has not been well understood. In this research, hybrids between Japanese flounder(♀) and stone flounder (♂) show mixed eye-location in both dextral type and sinistral type, and thus become good samples for studying the eye-migration. mRNAs from pro-metamorphosis sinistral and dextral hybrids larvae were screened with classical differential display RT-PCR (DD-RT-PCR) and representational difference analysis of cDNA (cDNA-RDA); 30 and 47 putative fragments were isolated, respectively. The cDNA fragments of creatine kinase and trypsinogen 2 precursor genes isolated by cDNA-RDA exhibited eye-position expression patterns during metamorphosis. However, none of the fragments was proved to be related to flatfishes’ eye-position specifically. Therefore, further studies and more sensitive gene isolated methods are needed to solve the problems. Supported by National Natural Science Foundation of China (No. 30600455) and the National Basic Research Program of China (973 Program, No.2004CB117402)     

Application of quantitative PCR method in detection of Lymphocystis disease virus China （LCDV-cn） in Japanese flounder （Paralichthys olivaceus）

Lymphocystis disease causes serious economic losses in the fish farming industry. The causative agent of the disease is Lymphocystis disease virus China (LCDV-cn), which has a wide range of hosts. Based on competitive quantitative PCR technology, we established a method to quantify the LCDV-cn in tissue. Results demonstrate that the average amount of LCDV-cn in the peripheral blood of infected flounder with evident tumors is about 106virions/ml while the average amount in those flounder with no evident tumor but cultured with the flounder with evident tumor is about 104virions/ml. No virus was found in the negative samples of flounder.     

Genome-Wide Identification of heat shock protein 10/60 Genes in Japanese Flounder(Paralichthys olivaceus) and Their Regulated Expression After Bacterial Infection

Heat shock proteins 10/60(hsp10/60) are a family of conserved ubiquitously expressed heat shock proteins which are produced by cells in response to exposure to stressful conditions. Besides the chaperone and housekeeping functions, they are also known to be involved in immune response during bacterial infection. In this study, we identified and annotated 10 hsp10/60 genes through bioinformatic analysis in Japanese flounder(Paralichthys olivaceus). Among them one member of hsp10(hspe) family and nine members of hsp60(hspd) family were identified. Phylogenetic and selection pressure analysis showed that the hsp10/60 genes were evolutionarily constrained and their function was conserved. Besides, hsp10/60 genes were involved in different embryonic and larval stages and acted as the sentinel role in an unchallenged organism. In addition, we also observed the expression patterns of hsp10/60 genes after Edwardsiella tarda infection, for the first time in Japanese flounder. Eight out of 10 genes were differentially expressed after bacterial challenges, the significantly regulated expressions of flounder hsp10/60 genes after bacterial infections suggested their involvement in immune response in flounder. Our results provide valuable information for clarifying the evolutionary relationship, and early insights of the immune functions of hsp10/60 genes in Japanese flounder.