Effects of incubation temperatures on embryonic development in the Asian yellow pond turtle

The Asian yellow pond turtle, Mauremys mutica (Cantor), is a potential aquaculture target in China owing to the higher values for food and remedy than other species of turtle. In this study, color and morphological changes of fertilized eggs were observed during embryogenesis, and the effects of incubation temperature on embryonic development were analyzed. Both calcium layer and membrane layer are thicker in the middle portion of egg-shell than that in the terminal portion, and become thinner after embryo hatching than before embryonic development. Significant change in the white spot and subsequent white ring on the eggshell occurs during embryonic development. Of five different incubation temperatures used to investigate the effects of incubation temperatures on embryonic development, 29.0 +/- 0.5 degrees C was optimal for embryo survival and development. Moreover, the incubation temperature of 33.0 +/- 0.5 degrees C was harmful effect to embryonic development. The data provide important and useful information for husbandry and management of the Asian yellow pond turtle. (c) 2006 Elsevier B.V. All rights reserved.

EST-based identification of genes expressed in the hypothalamus of male orange-spotted grouper (Epinephelus coioides)

The orange-spotted grouper, Epinephelus coioides, is an important marine aquaculture fish, but its large-scale aquaculture has been hindered by the rarity of natural males because it is a protogynous hermaphroditic fish. Hypothalamus-pituitary-gonad is an important endocrine axis in regulating reproduction and sex differentiation. To reveal the molecular mechanism of hypothalamic physiological functions, we performed he studies on identification of genes expressed in the hypothalamus of male orange-spotted grouper using EST and RT-PCR strategy. A total of 1006 ESTs were sequenced, and 402 (39.96%) clones were identified as known genes and 604 (60.04%) as unknown genes. The 402 clones of known gene products represent transcripts of 18 1 genes. Moreover, the expression patterns of 26 unknown genes were analyzed in various tissues, such as liver, kidney, spleen, fat, heart, muscle, pituitary, hypothalamus, telencephalon, cerebellum, midbrain, medulla oblongata, ovary and testes. Five different categories of expression patterns were observed from them. Several unknown ESTs, such as DN551996, DN551998, DN552082, and DN552070, were detected to be hypothalamus-specific, brains-specific, or hypothalamus and gonad-specific genes. Interestingly, DN551996, not only exhibiting expression differences between ovary and testis, but also showing sex-dependent differences in hypothalamus of grouper, might play significant role in grouper reproduction or sex inversion. Further functional studies on these genes will provide more information on molecule regulation mechanism of sex inversion in groupers. (c) 2006 Elsevier B.V All fights reserved.

Differential expression of three Paralichthys olivaceus Hsp40 genes in responses to virus infection and heat shock

Heat shock proteins (Hsps) are a family of highly conserved cellular proteins present in all organisms, mediating a range of essential housekeeping and cytoprotective functions as well-known molecular chaperons and recently as regulators of the immune response. By subtractive suppression hybridization, three Hsp40 homologues have been identified in the flounder (Paralichthys olivaceus) embryonic cells (FEC) after treatment with UV-inactivated turbot (Scophthalmus maximus L.) rhabdovirus (SMRV), termed PoHsp40A4, PoHsp40B6 and PoHsp40B11, whose encoded proteins all possess the conserved DnaJ domain, a signature motif of the Hsp40 family. Based on different protein structure and phylogenetic analysis, they can be categorized into two subfamilies, PoHsp40A4 for Type I Hsp40, PoHsp40B6 and PoHsp40B11 for Type 11 Hsp40. Further expression analysis revealed two very different types of kinetics in response either to heat shock or to virus infection, with a marked induction for PoHsp4OA4 and a weak one for both PoHsp40B6 and PoHsp40B11. A very distinct tissue distribution of mRNA was also revealed among the three genes, even between PoHsp40B6 and PoHsp40B11. This is the first report on the transcriptional induction of Hsp40 in virally stimulated fish cells, and the differential expressions might reflect their different roles in unstressed and stressed cells. (c) 2005 Elsevier Ltd. All rights reserved.

Seasonal variation of virioplankton in a eutrophic shallow lake

Lake Donghu is a typical eutrophic freshwater lake in which high abundance of planktonic viruses was recently revealed. In this study, seasonal variation of planktonic viruses were observed at three different trophic sites, hypertrophic, eutrophic, and mesotrophic regions, and the correlation between their abundances and other aquatic environmental components, such as bacterioplankton, chlorophyll a, burst size, pH, dissolved oxygen, and temperature, was analyzed for the period of an year. Virioplankton abundance detected by transmission electron microscope (TEM) ranged from 5.48 x 10(8) to 2.04 x 10(9) ml(-1) in all the sites throughout the study, and the high abundances and seasonal variations of planktonic viruses were related to the trophic status at the sampled sites in Lake Donghu. Their annual mean abundances were, the highest at the hypertrophic site (1.23x10(9) ml(-1)), medium at the eutrophic site (1.19x10(9) ml(-1)), and the lowest at the mesotrophic site (1.02x10(9) ml(-1)). The VBR (virus-to-bacteria ratio) values were high, ranging from 49 to 56 on average at the three sampled sites. The data suggested that the high viral abundance and high VBR values might be associated with high density of phytoplankton including algae and cyanobacteria in this eutrophic shallow lake, and that planktonic viruses are important members of freshwater ecosystems.

Identification of a novel C2 domain factor in ovaries of orange-spotted grouper (Epinephelus coioides)

Follicle consists of an oocyte and a lot of surrounding follicular cells, and significant interactions exist between the oocyte and the somatic cells. In this study, a novel cDNA has been screened from a subtractive cDNA library between tail bud embryos and blastula embryos in the protogynous hermaphrodite orange-spotted grouper (Epinephelus coioides). Its full-length cDNA is 821 bp, and has an ORF of 414 by for encoding a peptide of 137 aa, which shows 38%, 37%, 33%, and 33% homology with 4 putative proteins screened from zebrafish (Danio rerio). Conserved domain search in NCBI reveals a single C2 domain existing in the C2 domain superfamily proteins, and has only 7 beta strands in comparison with 8 beta strands of C2 domains in other C2 domain superfamily proteins. Artificial sex reversal, RT-PCR analysis and Western blot detection demonstrated ovary-specific expression of the C2 domain factor, and therefore the novel gene was designated as E. coioides ovary-specific C2 domain factor, EcOC2 factor. Moreover, predominant expression of EcOC2 factor was further revealed in grouper mature ovary, and its strong immunofluorescence signals were located between granulosa cells and oocyte zona radiata in grouper mature follicles. The data indicate that the novel EcOC2 factor might be a main component that associates between granulosa cells and the oocyte during oocyte maturation, and might play significant roles in regulating oocyte maturation and ovulation. Further studies on its developmental behaviour and physiological functions will elucidate the interactions between oocyte and the surrounding somatic cells and the underlying molecular mechanisms. (C) 2005 Elsevier Inc. All rights reserved.

Molecular characterization of three Rana grylio virus (RGV) isolates and Paralichthys olivaceus lymphocystis disease virus (LCDV-C) in iridoviruses

Three Rana grylio virus (RGV) isolates and lymphocystis disease virus (LCDV-C) were molecularly characterized by antigenicity comparison, Western blot detection of viral polypeptides, restriction fragment length polymorphism analysis of viral genomes, and MCP sequence analysis. Significant antigenicity differences existed among the three RGV isolates and LCDV-C. Western blot detection indicated that the viral polypeptides of three RGV isolates could be recognized by the anti-RGV9807 serum, whereas no bands were observed in the LCDV-C, and significant differences exist among the band patterns of three RGV isolates. Restriction fragment length polymorphism (RFLP) analysis was performed by digesting genomic DNA of the four iridovirus isolates with restriction endonucleases HindIII, KpnI, XbaI and BamHI. On the whole, obvious discrepancies existed between LCDV-C and RGV isolates, and some significant band pattern differences were also revealed between RGV9808 and RGV9506 (or RGV9807) in the profiles of restriction endonucleases Xbal, Kpn I and BamHI. PCR amplification and sequence analysis of MCP gene sequence further revealed their phylogenetic relationship among the three RGV isolates, LCDV-C and other iridoviruses. RGV9506, RGV9807 and RGV9808 are clustered together with other ranaviruses, such as FV3, BIV, TFV and ENHV, although the RGV9808 is more close to EHNV than to other ranaviruses. Additionally, LCDV-C is clustered with LCDV-1, the type species of genus Lymphocystisvirus. The current study provides clear evidence that significant genetic difference exists among the three RGV isolates. Therefore, further work on comparative genomic studies will contribute significantly to understanding of their taxonomic position and pathological mechanism. (C) 2005 Elsevier B.V. All rights reserved.

Molecular cloning and expression pattern of 14 kDa apolipoprotein in orange-spotted grouper, Epinephelus coioides

A novel fish-specific apolipoprotein (apo-14 kDa) has been recently cloned from eel and pufferfish. However, its expression pattern has not been elucidated. in this study, EcApo-14 has been screened from hypothalamic cDNA library of male orange-spotted grouper, which shows 62.9%, 51%, 46.9%, 43.2%, and 31.9% identities to Apo-14 of European flounder, pufferfish, Japanese eel, gibel carp, and grass carp, respectively. RT-PCR analysis reveals that this gene is first transcribed in neurula embryos and maintains a relatively stable expression level during the following embryogenesis. EcApo-14 transcripts are at a very high level during embryonic and early larval development in the yolk syncytial layer (YSL), and decrease in YSL and form intense staining in liver at 3 days after hatching. In adult tissues, EcApo-14 is predominantly expressed in liver and brain. The data suggested that EcApo-14 might play an important role in liver and brain morphogenesis and growth. (c) 2005 Elsevier Inc. All rights reserved.

Molecular identification and expression analysis of natural resistance associated macrophage protein (Nramp) cDNA from Japanese flounder (Paralichthys olivaceus)

Natural resistance associated macrophage protein (Nramp) controls partially innate resistance to intracellular parasites. Its function is to enhance the ability of macrophages to kill pathogens. However, little is known about the structure and function of Nramp in lower vertebrates such as teleosts. We have recently isolated a cDNA encoding Nramp from Japanese flounder (Paratichthys olivaceus). The full-length cDNA of the Nramp is 3066 bp in length, including 224 bp 5' terminal UTR, 1662 bp encoding region and 1180 bp 3' terminal UTR. The 1662-nt open reading frame was found to code for a protein with 554 amino acid residues. Comparison of amino acid sequence indicated that Japanese flounder Nramp consists of 12 transmembrane (TM) domains. A consensus transport motif (CTM) containing 20 residues was observed between transmembrane domains 8 and 9. The deduced amino acid sequence of Japanese flounder had 77.30%, 82.71%, 82.67%, 79.64%, 80.72%, 90.97%, 91.16%, 60.14%, 71.48%, 61.69%, 72.37% identity with that of rainbow trout Nramp alpha and beta, channel catfish Nramp, fathead minnow Nramp, common carp Nramp, striped sea bass Nramp, red sea bream Nramp, mouse Nramp 1 and 2, human Nramp 1 and 2, respectively. RT-PCR indicated that Nramp transcripts were highly abundant in spleen, head kidney, abundant in intestine, liver and gill, and less abundant in heart. The level of Nramp mRNA in embryos gradually increases during embryogenesis from 4 h (8 cell stage) to 80 h (hatched stage) after fertilization. (c) 2005 Elsevier Ltd. All rights reserved.

A population of red-transparent, triploid Carassius auratus

A red-transparent population, distributed in Pingxiang of Jiangxi Province, was identified to be triploid Carassius auratus by DNA content measurement and chromosome analysis. Artificial propagation experiments indicated that the red-transparent triploid Carassius auratus could reproduce by gynogenesis. (c) 2005 The Fisheries Society of the British Isles.

Identification of one intron loss and phylogenetic evolution of Dfak gene in the Drosophila melanogaster species group

Intron loss and its evolutionary significance have been noted in Drosophila. The current study provides another example of intron loss within a single-copy Dfak gene in Drosophila. By using polymerase chain reaction (PCR), we amplified about 1.3 kb fragment spanning intron 5-10, located in the position of Tyr kinase (TyK) domain of Dfak gene from Drosophila melanogaster species group, and observed size difference among the amplified DNA fragments from different species. Further sequencing analysis revealed that D. melanogaster and D. simulans deleted an about 60 bp of DNA fragment relative to other 7 Drosophila species, such as D. elegans, D. ficusphila, D. biarmipes, D. takahashii, D. jambulina, D. prostipennis and D. pseudoobscura, and the deleted fragment located precisely in the position of one intron. The data suggested that intron loss might have occurred in the Dfak gene evolutionary process of D. melanogaster and D. simulans of Drosophila melanogaster species group. In addition, the constructed phylogenetic tree based on the Dfak TyK domains clearly revealed the evolutionary relationships between subgroups of Drosophila melanogaster species group, and the intron loss identified from D. melanogaster and D. simulans provides a unique diagnostic tool for taxonomic classification of the melanogaster subgroup from other group of genus Drosophila.

Expression pattern and developmental behaviour of cellular nucleic acid-binding protein (CNBP) during folliculogenesis and oogenesis in fish

In vertebrates, folliculogeneis establishes an intricate system for somatic cell-oocyte interaction, and ultimately leads to the acquisition of their respective competences. Although the formation process and corresponding interactions are strikingly similar in diverse organisms, knowledge of genes and signaling pathways involved in follicle formation is very incomplete and the underlying molecular mechanisms remain enigmatic. CNBP has been identified for more than ten years, and the highest level of CNBP transcripts has been observed in adult zebrafish ovary, but little is known about its functional significance during folliculogeneis and oogenesis. In this study, we clone CNBP cDNA from gibel carp (Carassius auratus gibelio), and demonstrate its predominant expression in gibel carp ovary and testis not only by RTPCR but also by Western blot. Its full-length cDNA is 1402 bp, and has an ORF of 489 nt for encoding a peptide of 163 aa. And its complete amino acid sequence shared 68.5%-96.8% identity with CNBPs from other vertebrates. Based on the expression characterization, we further analyze its expression pattern and developmental behaviour during folliculogeneis and oogenesis. Following these studies, we reveal an unexpected discovery that the CagCNBP is associated with follicular cells and oocytes, and significant distribution changes have occurred in degenerating and regenerating follicles. More interestingly, the CagCNBP is more highly expressed in some clusters of interconnected cells within ovarian cysts, no matter whether the cell clusters are formed from the original primordial germ cells or from the newly formed cells from follicular cells that invaded into the atretic oocytes. It is the first time to reveal CNBP relevance to folliculogeneis and oogenesis. Moreover, a similar stage-specific and cell-specific expression pattern has also been observed in the gibel carp testis. Therefore, further studies on CNBP expression pattern and developmental behaviour will be of significance for understanding functional roles of CNBP during gametogenests. (c) 2005 Elsevier B.V. All rights reserved.

Inductive expression and characterization analysis of Paralichthys olivaceus pigment epithelium-derived factor in a virally infected cell line

Pigment epithelium-derived factor (PEDF) is acknowledged to be a non-inhibitory member of the serine protease inhibitor (serpin) superfamily, with antiangiogenesis, and neuroprotective and immumoregulatory function, mainly in the tissues of nervous system. Here, A PEDF gene homolog, Paralichthys olivaceus PEDF (PoPEDF), was isolated from flounder embryonic cells (FEC) treated with UV-inactivated Grass carp hemorrhage virus (GCHV) and subsequently identified as a differentially expressed gene. The full length of PoPEDF cDNA is 1803 bp with an open reading frame of 1212 bp encoding a 403-amino-acid protein. This deduced protein contains an N-terminal signal peptide, a glycosylation site, a consensus serpin motif, and a 34-mer and a 44-mer fragment, all of which are very conserved in the PEDF family. PoPEDF gene exhibits a conserved exon-intron arrangement with 8 exons and 7 introns. This conserved evolutionary relationship was further confirmed by a phylogenetic analysis, where fish PEDFs and mammalian members formed a well-supported clade. Constitutive expression of PoPEDF was widely detected in many tissues. In response to UV-inactivated GCHV or poly(I:C), PEDF mRNA was upregulated in FEC cells with time. This is the first report on the transcriptional induction of PEDF in virally infected cells. (C) 2005 Elsevier Inc. All rights reserved.

Identification of a Spindlin homolog in gibel carp (Carassius auratus gibelio)

Spindlin has been suggested to play an important role during the transition from oocyte maturation to embryo development in mouse, but its homolog similar to the mouse Spindlin in molecular and expression characterization has not been identified up to now in other vertebrates. In this study, a full length of cDNA sequence is cloned and sequenced from the gibel carp (Carassius auratus gibelio). It contains 1240 nucleotides with an open reading frame of 771 nt encoding 257 amino acids. Based on its amino acid sequence alignment and comparison analysis with the known Spin family proteins, the newly cloned Spin is named Carassius auratus gibelio Spindlin (CagSpin). Its product could be detected from mature eggs to blastula embryos, but its content decreased from the two-cell stage, and could not be detected after the gastrula stage. It suggests that the CagSpin should be a maternal protein that is expressed during oocyte maturation, and plays a crucial role in early cleavage of embryogenesis. CagSpin is the first homolog similar to mouse spindlin identified in fish, and also in other vertebrates. GST pull-down assay reveals the first biochemical evidence for the association of CagSpin and p-tubulin, the microtubule component. Therefore, CagSpin may play important functions by interacting with beta-tubulin and other spindle proteins during oocyte maturation and egg fertilization. (c) 2005 Elsevier Inc. All rights reserved.