Comparative analysis of CpG islands in four fish genomes

There has been much interest in CpG islands (CGIs), clusters of CpG dinucleotides in GC-rich regions, because they are considered gene markers and involved in gene regulation. To date, there has been no genome-wide analysis of CGIs in the fish genome. We

Distribution of IgM, IgD and IgZ in mandarin fish, Siniperca chuatsi lymphoid tissues and their transcriptional changes after Flavobacterium columnare stimulation

The gene sequences of three different immunoglobulin (Ig) heavy chains, namely IgM, IgD and IgZ, were cloned from mandarin fish (Siniperca chuatsi) recently. In this study the distribution of these three kinds of Ig-producing cells in lymphoid-related tissues as head kidney, spleen, gill and intestine were investigated by using in situ hybridization, and their transcriptional changes were also analyzed by quantitative real-time PCR during 8 weeks after immunization. IgM-producing cells could be detected obviously and abundantly in all the tissues examined. A few numbers of IgD and IgZ positive cells were both detected in head kidney and spleen. IgZ positive cells could be detected in gill moderately while IgD showed negative results, otherwise no IgD or IgZ positive cells could be detected in intestine. After stimulated with bacterial pathogen Flavobacterium columnare G(4), the transcripts of these three Ig genes exhibited quite different kinetics. Significantly increased transcription of IgM gene was observed in almost all the tissues examined especially in boosted group. In contrast with IgM, seldom strong increase was examined for IgD and IgZ genes. For IgD, it seemed that the first injection could stimulate the immune response easier, since in almost all the tissues significant increase was detected at 1 or 2 weeks after injection. For IgZ, boosted injection could not enlarge the up-regulation of gene expression of first injection. This is the first case to report the transcriptional kinetics of three Ig genes in teleost after bacterin immunization. (C) 2008 Elsevier B.V. All rights reserved.

Characterization of C-C chemokine receptor subfamily in teleost fish

Chemokines and their receptors play important roles in nervous and immune systems. Little information, however, exists concerning this gene family in teleost fish. In the present Study, 17 C-C chemokine receptors genes were identified from Danio rerio, 9 from Gasterosteus aculeatus, 10 from Oryzius latipes, 8 from Takifugu rubripes and 5 from Tetraodon nigroviridis. Phylogenetic analysis showed that the orthologs to mammalian CCR6, 7, 8, 9 and CCRL1 receptors were evident in zebrafish, but the clear orthologs to mammalian CCR1, 2, 3, 4, 5 and 10 were not found in zebrafish. The gene structure of zebrafish CCR (zfCCR) was further analyzed. The open reading frame of zfCCR3-1, zfCCR3-3, zfCCR6-1, zfCCR6-2, zfCCR8-2 contain one exon, and two exons were identified for zfCCR2-1, zfCCR2-2, zfCCR4 and zfCCRLI-1, three exons for zfCCR3-2, zfCCR5 and zfCCR7, four exons for zfCCR8-1 and zfCCR9-1. The expression analyses showed that in zebrafish, most C-C chemokine receptor genes Were expressed in fertilized eggs and oocytes, and all the receptor genes were expressed in larval stages. The zfCCR2-2, zfCCR3-1, zfCCR4 and zfCCR6-2 genes were expressed in all normal organs examined, whereas not for zfCCR2-1, zfCCR3-3, zfCCR6-1, zfCCR8-1, zfCCR9-2 and zfCCRL1-2. The expression of zfCCR3-2, zfCCR5, zfCCR7, zfCCR9-1 and zfCCRLI-1 were detected in the majority organs. and zfCCR8-2 and zfCCR8-3 detected only in brain. The differential expression pattern of different paralogues in organs may indicate their difference in function, which requires further investigation. (C) 2008 Elsevier Ltd. All rights reserved.

Rapid growth cost in “all-fish” growth hormone gene transgenic carp: Reduced critical swimming speed

Evidence has accumulated that there is a trade-off between benefits and costs associated with rapid growth. A trade-off between growth rates and critical. swimming speed (U-crit) had been also reported to be common in teleost fish. We hypothesize that growth acceleration in the F-3 generation of "all-fish" growth hormone gene (GH) transgenic common carp (Cyprinus carpio L.) would reduce the swimming abilities. Growth and swimming performance between transgenic fish and non-transgenic controls were) compared. The results showed that transgenic fish had a mean body weight 1.4-1.9-fold heavier, and a mean specific growth rate (SGR) value 6%-10% higher than the controls. Transgenic fish, however, had a mean absolute U-crit (cm/s) value 22% or mean relative Ucrit (BL/s) value 24% lower than the controls. It suggested that fast-growing "all-fish" GH-transgenic carp were inferior swimmers. It is also supported that there was a trade-off between growth rates and swimming performance, i.e. faster-growing individuals had lower critical swimming speed.

Short and long peptidoglycan recognition proteins (PGRPs) in zebrafish, with findings of multiple PGRP homologs in teleost fish

Peptidoglycan recognition protein (PGRP) specifically binds to peptidoglycan and is considered to be one of the pattern recognition proteins in the innate immunity of insect and mammals. Using a database mining approach and RT-PCR, multiple peptidoglycan recognition protein (PGRP) like genes have been discovered in fish including zebrafish Danio rerio, Japanese pufferfish TakiFugu rubripes and spotted green pufferfish Tetraodon nigroviridis. They share the common features of those PGRPs in arthropod and mammals, by containing a conserved PGRP domain. Based on the predicted structures, the identified zebrafish PGRP homologs resemble short and long PGRP members in arthropod and mammals. The identified PGRP genes in T. nigroviridis and TakiFugu rubripes resemble the long PGRPs, and the short PGRP genes have not been found in T. nigroviridis and TakiFugu rubripes databases. Computer modelling of these molecules revealed the presence of three alpha-helices and five or six beta-strands in all fish PGRPs reported in the present study. The long PGRP in teleost fish have multiple alternatively spliced forms, and some of the identified spliced variants, e.g., tnPGRP-L3 and tnPGRP-L4 (in: Tetraodon nigroviridis), exhibited no characters present in the PGRP homologs domain. The coding regions of zfPGRP6 (zf: zebrafish), zfPGRP2-A, zfPGRP2-B and zfPGRP-L contain five exons and four introns; however, the other PGRP-like genes including zfPGRPSC1a, zfPGRPSC2, tnPGRP-L1-, tnPGRP-L2 and frPGRP-L (fr: Takifugu rubripes) contain four exons and three introns. In zebrafish, long and short PGRP genes identified are located in different chromosomes, and an unknown locus containing another long PGRP-like gene has also been found in zebrafish, demonstrating that multiple PGRP loci may be present in fish. In zebrafish, the constitutive expressions of zfPGRP-L, zfPGRP-6 and zfPGRP-SC during ontogeny from unfertilized eggs to larvae, in different organs of adult, and the inductive expression following stimulation by Flavobacterium columnare, were detected by real-time PCR, but the levels and patterns varied for different PGRP genes, implying that different short and long PGRPs may play different roles in innate immune response. (c) 2007 Elsevier Ltd. All rights reserved.

Improvement and observation of immunoelectron microscopic method for the localization of frog Rana grylio virus (RGV) in infected fish cells

In this paper, to understand the roles of amorphous structures which were observed within the viromatrix of Rana grylio virus (RGV), an improved immunoelectron microscopy (IEM) method was developed to detect the localization of RGV in carp Epithelipma papulosum cyprinid (EPC) cells. Infected EPC cells were fixed with 4% paraformaldehyde-0.25% glutaraldehyde mixture, dehydrated completely, and embedded in LR White resin. This method allowed good ultrastructural preservation and specific labeling with anti-RGV antibodies. The results of IEM showed that colloidal gold mainly bound to the capsids of viral particles at the stage of viral assembly, while during the viral maturation colloidal gold bound to the envelop of virions. In addition, within the viromatrix, the amorphous structures, including dense floccules, membranous materials and tubules, also had strong colloidal gold signals, revealing that those amorphous structures were participated in RGV assembly. In contrast, no significant gold labeling signals were obtained in negative controls. The present study not only provided further evidence that amorphous structures within the viromatrix were involved in the process of RGV assembly, but also developed an improved IEM method for studying the interaction between iridovirus and host cells. (C) 2006 Elsevier Ltd. All rights reserved.

Isolation and characterization of a rhabdovirus from co-infection of two viruses in mandarin fish

Co-infection of two viruses has been observed in mandarin fish (Siniperca chuatsi), but the two viruses have not been characterized. In this study, a rhabdovirus has been isolated from the co-infected two viruses extracted from the diseased mandarin fish, and its morphological structure and partial biochemical and biophysical characteristics have been observed and analyzed. The isolated rhabdovirus has a typical bullet shape, and is therefore called S. chttatsi rhabdovirus (SCRV). And, the isolated rhabdovirus produced a higher titer (10(8.5) TCID50 ml(-1)) than did the co-infecting viruses (10(6.5) TCID50 ml(-1)). Subsequently, the viral genome RNA was extracted, and used as template to clone the complete nucleoprotein (N) gene by RT-PCR amplification. Cloning and sequencing of the SCRV N protein revealed 42%-31% amino acid identities to that of trout rhabdovirus 903/87 and the rhabdoviruses in genus Vesiculovirus. SDS-PAGE separation of the isolated SCRV and other two rhabdoviruses also revealed obvious polypeptide profile difference. Moreover, the anti-SCRV N protein antibody was prepared, and the anti-SCRV N protein antibody only could recognize the SCRV N protein, whereas no antigenicity was detected in other two rhabdoviruses. The data suggested that the SCRV should be a rhabdovirus member related to the genus Vesiculovirus in the Rhabdoviridae. (c) 2006 Elsevier B.V. All rights reserved.

Electron microscopic examination of the viromatrix of Rana grylio virus in a fish cell line

Rana grylio virus (RGV), a Ranavirus belonging to the family Iridoviridae, assembles in the viromatrix which is a factory for viral genome replication and particle assembly. Ultrastructural studies of the viromatrix will clarify the pathway of assembly. The viromatrix and quantitative changes in RGV infected epithelipma papulosum cyprini (EPC) cells, one of fish cell lines, were studied by electron microscopy. It was shown that viromatrices were adjacent to the nucleus, and the electron density was lower than that of the surrounding cytoplasm. The viromatrix contained virus particles with different forms, electron-dense materials and amorphous structures which included tubules and membranous materials. Tubules were often observed in direct continuity with empty capsids. Several bundles of intermediate filaments were seen alongside the viromatrix and crystalline aggregates. Large clusters of mitochondria occurred in proximity to viromatrix. A total of 990 cells profiles were examined. The results showed that 394 cells contained viromatrix: 89.3% contained one, and 10.7% contained two to four viromatrices. The number of viromatrices increased gradually and reached a peak at 16 h p.i. The viromatrix area at 24 h p.i. increased up to 7.4 +/- 0.69 mu m(2) which was three-times lower than that at 6 h p.i. The number of empty capsids within viromatrix was generally more than that of "full" particles at different time points, and there was a strong positive correlation between them. (c) 2005 Elsevier B.V. All rights reserved.

Developments in transgenic fish in the people's Republic of China

In the People's Republic of China, genetically modified (GM) fish are being developed primarily to produce desirable alterations to growth rates or feed-conversion efficiency. Up to the present, no transgenic fish have been commercially approved for human consumption. This review introduces advances in the People's Republic of China in transgenic fish studies, biosafety studies of fast-growth GM fish, and the regulation of GM fish.

Factors affecting the efficiency of somatic cell nuclear transplantation in the fish embryo

Procedures to improve somatic cell nuclear transplantation in fish were evaluated. We reported effects of nonirradiated recipient eggs, inactivated recipient eggs, different combinations between recipient eggs and donor cells, duration of serum starvation, generation number, and passage number of donor cells on developmental rates of nuclear transplant (NT) embryos. Exposure to 25,000 R of gamma-rays inactivated recipient eggs. Single nucleus of cultured, synchronized somatic cell from gynogenetic bighead carp (Aristichthys nobilis) was transplanted into nonirradiated or genetically inactivated unfertilized egg of gibel carp (Carassius auratus gibelio). There was no significant difference in developmental rate between nonirradiated and inactivated recipient eggs (27.27% vs. 25.71%, respectively). Chromosome count showed that 70.59% of NT embryos contained 48 chromosomes. It showed that most NT embryos came from donor nuclei of bighead carp, which was supported by microsatellite analysis of NT embryos. But 23.53% of NT embryos contained more than 48 chromosomes. It was presumed that those superfluous chromosomes came from nonirradiated recipient eggs. Besides, 5.88% of NT embryos were chimeras. Eggs of blunt-snout bream (Megalobrama amblycephala) and gibel carp were better recipient eggs than those of loach (Misgurnus anguillicaudatus) (25% and 18.03% vs. 8.43%). Among different duration of serum starvation, developmental rate of NT embryos from somatic nuclei of three-day serum starvation was the highest, reaching 25.71% compared to 14.14% (control), 20% (five-day), and 21.95% (seven-day). Cultured donor cells of less passage facilitated reprogramming of NT embryos than those of more passage. Recloning might improve the developmental rate of NT embryos from the differentiated donor nuclei. Developmental rate of fourth generation was the highest (54.83%) and the lowest for first generation (14.14%) compared to second generation (38.96%) and third generation (53.01%). (C) 2002 Wiley-Liss, Inc.

Molecular variation of Bothriocephalus acheilognathi Yamaguti, 1934 (Cestoda : Pseudophyllidea) in different fish host species based on ITS rDNA sequences

The molecular variation in Bothriocephalus acheilognathi Yamaguti, 1934 from 11 species of freshwater fish collected in Australia, China, the Czech Republic, England and Hawaii was investigated by determining the nucleotide sequences of the internal transcribed spacer region. The length of the first and second internal transcribed spacer sequences of multiple individuals ranged from 553 to 571 bp and 553 to 615 bp, and the G + C content from 53.1 to 53.5%. The percentage sequence divergence varied between 0 and 0.9% in the ITS1 and 0 and 6.6% in the ITS2, respectively, indicating the occurrence of intraspecific variation. It is demonstrated that the fragment length variation resulted primarily from microsatellite polymorphisms present in the ITS region, especially in the ITS2 region. Phylogenetic analyses revealed that B. acheilognathi examined in this study consisted of three closely related genotypes with certain degrees of host-specificity, and the genotype representing isolates from Cyprinus carpio L. was the most common and diverse form within the species B. acheilognathi.

Resting metabolism and heat increment of feeding in mandarin fish (Siniperca chuatsi) and Chinese snakehead (Channa argus)

Resting metabolism was measured in immature mandarin fish Siniperca chuatsi weighing 42.1-510.2 g and Chinese snakehead Channa argus weighing 41.5-510.3 g at 10, 15, 20, 25, 30 and 35 degreesC. Heat increment of feeding was measured in mandarin fish weighing 202.0 (+/-14.0) g and snakehead weighing 200.8 (+/-19.3) g fed swamp leach Misgurnus anguillicaudatus at 1% body weight per day at 28 degreesC. In both species, weight exponent in the power relationship between resting metabolism and body weight was not affected by temperature. The relationship between resting metabolism and temperature could be described by a power function. The temperature exponent was 1.39 in mandarin fish and 2.10 in snakehead (P < 0.05), indicating that resting metabolism in snakehead increased with temperature at a faster rate than in mandarin fish. Multiple regression models were used to describe the effects of body weight (W, g) and temperature (T, C) on the resting metabolism (R-s, mg O-2/h): In R-s = - 5.343 + 0.772 In W + 1.387 In T for the mandarin fish and In R-s = -7.863 + 0.801 ln W + 2.104 In T for the Chinese snakehead. The proportion of food energy channelled to heat increment was 8.7% in mandarin fish and 6.8% in snakehead. (C) 2000 Elsevier Science Inc. All rights reserved.

Occurrence of Bothriocephalus acheilognathi in cyprinid fish from three lakes in the flood plain of the Yangtze River, China

Cyprinid fish, Hemiculter leucisculus, Cultrichthys erythropterus and Culter dabryi, were sampled from Liangzi, Honghu and Tangxun lakes in the flood plain of the Yangtze River. The cestode Bothriocephalus acheilognathi Yamaguti, 1934 was found in the 3 lakes, but C. erythropterus sampled from Liangzi lake was found uninfected due probably to the small sample size. Findings of the cestode in the 3 lakes represent the first record of the parasite in the flood plain of the Yangtze River, indicating that B. acheilognathi may be distributed much wider in China than previously recognized.

Time course of foreign gene integration and expression in transgenic fish embryos

Using a nuclear transplantation approach, the integration and expression of the green fluorescent protein (GFP) gene in the embryogenesis of transgenic leach (Misgurnus anguillicaudatus Cantor) have been studied. The GFP gene expression is first observed at the gastrula stage, which is consistent with the initiation of cell differentiation of fish embryos. The time course of the foreign gene expression is correlated with the regulatory sequences. The expression efficiency also depends on the gene configuration: the expression of pre-integrating circular plasmid at early embryos is higher than that of the linear plasmid. The integration of the GFP gene is first detected at the blastula stage and lasts for quite a long period. When two types of different plasmids are co-injected into fertilized eggs, the behavior of their integration and expression is not identical.

Studies on the biology of Chaoborus flavicans (Meigen) (Diptera : Chaoboridae) in a fish-free eutrophic pond, Japan

Population dynamics of Chaoborus flavicans larvae of various instars was studied from November 1986 to December 1987 in a eutrophic, fish-free pond, Japan. First and 2nd instar larvae were observed from late April to late October, indicating a reproductive period of about half a year. C. flavicans overwintered in the 4th instar larvae. In water column samples, total density of all instars was 680-23 680 m(-2), and pupal density 0-2 600 m(-2); larvae of the Ist, 2nd, and 3rd instars showed 5-6 density peaks in 1987, suggesting that 5-6 generations occur during a year (peaks of the 4th instar larvae were not clear, probably due to their longer development than those of younger instars). In sediment samples, no Ist and 2nd instar larvae were found, 3rd instar larvae were found occasionally but density of the 4th instar larvae was 280-18 600 m(-2), and pupal density varied between 0-502 m(-2). Fouth instar larvae accumulated in sediment in the cold season and in the water column in the warm season; high temperature and low oxygen concentration were the most important factors limiting the distribution of larvae in the sediment in summer in the NIES pond. The dry weight of total C. flavicans larvae was 0.08-4.2 g m(-2) in sediment samples and 24-599 mu g l(-1) (0.10-2.40 g m(-2)) in water column samples. Comparisons of maximum densities in the NIES pond in different years and in waters of different trophic status show that density is generally higher in eutrophic than in oligotrophic habitats.