Effect of dietary supplementation of vitamins A, D-3, E, and C on yearling rice field eel, Monopterus albus: Serum indices, gonad development, and metabolism of calcium and phosphorus

The objective of this study was to determine the effect of dietary vitamins A, D-3, E, and C on the gonad development, lipid peroxidation, and immune response of yearling rice field eel, Monopterus albus. A 6-wk feeding trial was designed according to an L-16(4(5)) orthogonal design, in which four vitamins, each at four supplementation levels, were arranged. Sixteen diets were mixed with the different vitamin levels and randomly assigned to 16 groups of fish. Increasing dietary vitamin E supplementation level significantly (P <= 0.05) increased the gonadosomatic index and lowered the serum content of malondialdehyde of rice field eel. Increasing dietary vitamin A and C levels also showed similar effect, but the differences were not statistically significant. Serum immunoglobulin M content increased significantly (P <= 0.01) as dietary vitamin C supplementation levels increased. The concentrations of calcium in bones showed significant (P <= 0.05) trend with vitamin D-3 and A supplementation levels, but the bone phosphorus content was not affected by the dietary vitamin levels.

A perspective on fish gonad manipulation for biotechnical applications

The gonad is an essential organ for generating sperm and ova in vertebrates. This review describes several pilot studies on gonad gene manipulation and development in fish. With antisense RNA techniques, we suppressed the gonad development, and thus the fertility, of an antisense gonadotropin-releasing hormone (sGnRH) transgenic common carp. Then, using a tissue-specific exogenous gene excision strategy with sexual compensation, we knocked out the gonad-specific transgene. Under the control of the rainbow trout protamine promoter, the transgenic fish expressed the reporter gene eGFP specifically in the spermary. These results indicate that the fish gonad is a new model organ that can improve contemporary biotechnology experiments. Herein we discuss the potential of fish gonad manipulation for resolving important biosafety problems regarding transgenic fish generation and producing the new transgenic animal bioreactor.

Effect of inclusion of blue-green algae meal on growth and accumulation of microcystins in gibel carp (Carassius auratus gibelio)

Six isonitrogenous (crude protein content: 38%) and isoenergetic (gross energy content: 17 kJ g(-1)) diets were formulated to investigate the effects of inclusion of blue-green algae meal on gibel carp (Carassius auratus gibelio). In each diet, 15% of the protein was supplied by fishmeal; the remainder was supplied by soybean meal and blue-green algae meal. Diet 1 was used as control with no blue-green algae meal whereas the content in diets 2-6 was 15.15, 29.79, 44.69, 59.58 and 74.48%, respectively. Each diet was fed to five groups of gibel carp for 12 weeks in a flow-through system. Final body weight and specific growth rate (SGR) of fish fed diet 5 were significantly lower than the control diet (P < 0.05). Mortality of gibel carp increased with increase in algae meal inclusion (P < 0.05), but there was no significant difference between fish fed diets 3-6 (P > 0.05). Feed conversion efficiency (FCE) decreased with the increase in algae meal inclusion (P < 0.05). Fish-fed diet 6 showed the highest feeding rate (P < 0.05), while there were no significant differences among the other groups (P > 0.05). Apparent digestibility coefficient of dry matter, protein, and energy decreased with increasing algae meal inclusion in the diets (P < 0.05). Aspartate aminotransferase (GOT) activity in the liver was not significantly different among groups (P > 0.05). Liver alanine aminotransferase (GPT) activity of fish-fed diets 4, 5 and 6 was significantly lower than the control diet (diet 1; P < 0.05). Microcystins in the muscle, liver, gallbladder, and spleen increased with increasing algae inclusion (P < 0.05).

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.

Partial compensatory growth in hybrid tilapia Oreochromis mossambicus x O-niloticus following food deprivation

The capacity of hybrid tilapia Oreochromis mossambicus x O. niloticus [23.2 +/- 0.2 g (mean +/- SE)] to show compensatory growth was assessed in an 8-week experiment. Fish were deprived of feed for 1, 2 and 4 weeks, and then fed to satiation for 4 weeks; fish fed to satiation during the experiment served as control. Water temperature gradually declined from 28.1 to 25.5 degrees C throughout the experiment. Specific growth rate (SGR) decreased with progressive food deprivation. At the end of deprivation, body weight was lower in the deprived fish than in the control. Fish deprived for 4 weeks exhibited lower contents of lipids and energy in whole body, and higher moisture content and ratio of protein to energy (P/E) than those of the control; they also consumed feed faster than the control when normal feeding was resumed. All deprived fish showed higher food intake (FI) than that of the control during re-alimentation; however, enhanced SGR was only observed in the fish deprived for 4 weeks. There were no significant differences in digestibility of protein and energy, food efficiency (FE) or energy retention efficiency between the control and deprived fish. At the end of re-alimentation, deprived fish failed to catch up in body weight with the control, while content of moisture, lipids and energy, and P/E in whole body of the deprived fish did not significantly differ from that of the control. The results of the experiment revealed that the hybrid tilapia reared in freshwater showed partial capacity for compensatory growth following food deprivation of 4 weeks, and that growth compensation was due mainly to increased FI, rather than to improved FE.

Identification of immune genes in grass carp Ctenopharyngodon idella in response to infection of the parasitic copepod Sinergasilus major

The parasitic copepod Sinergasilus major is an important pathogen of grass carp Ctenopharyngodon idella. To understand the immune response of grass carp to the copepod infection, suppression subtractive hybridization method was employed to characterize genes up-regulation during the copepod infection in liver and gills of the fish. One hundred and twenty-two dot blot positive clones from infected subtracted library were sequenced. Searching available databases by using these nucleotide sequences revealed that 23 genes are immune-related, including known acute-phase reactants, and four novel genes encoding proteins such as source of immunodominant MHC-associated peptides (SIMP), TNF receptor-associated factor 2 binding protein (T2BP), poliovirus receptor-related protein 1 precursor, glycoprotein A repetitions predominant (GARP). The differential expression of seven immune genes, i.e. GARP, alpha-2-macroglobulin, MHC class I, C3, SIMP, T2BP, transferrin, as a result of infection was further confirmed by RT-PCR, with the up-regulation of alpha-2-macroglobulin, MHC class I, C3, SIMP and T2BP in the liver of infected fish, and down-regulation of SIMP in the gills of infected fish. The present study provides foundation for understanding grass carp immune response and candidate genes for further analysis.

Differential expression of thyroid-stimulating hormone beta subunit in gonads during sex reversal of orange-spotted and red-spotted groupers

We have cloned and characterized the full-length cDNA encoding thyroid-stimulating hormone beta-subunit (TSHbeta) from orange-spotted grouper Epinephelus coioides. It contains 913 nucleotides with an open reading frame encoding 146 amino acids with a 20 amino acid signal peptide. The grouper mature TSHbeta has 75, 70, 61, 59, 41, 42 and 40% identities to that of rainbow trout, Atlantic salmon, zebrafish, European eel, chicken. mouse and human, respectively. RT-PCR analysis indicated that the TSHbeta mRNA was expressed abundantly not only in pituitary but also in gonads. A more interesting finding is to reveal the differential TSHbeta expressions between the ovaries and the transitional gonads or testes in natural individuals of orange-spotted grouper and red-spotted grouper Epinephelus akaara, and in artificial sex reversal individuals of red-spotted grouper induced by MT feeding. In situ hybridization localization provided direct evidence that the TSHbeta was transcribed in the germ cells. In the growing oocytes, the TSHbeta transcripts were concentrated on the ooplasm periphery. In testicular tissues, the intensively expressed TSHbeta cells were found to be spermatogonia and spermatocytes in the spermatogenic cysts. This is the first report of a TSHbeta expressed in the gonads of any vertebrates in addition to the expected expression in the pituitary, and it expresses more transcripts in the gonads during sex reversal or testis than in the ovaries both in E. coioides and E. akaara. Importantly, the TSHbeta identification in germ cells allows us to further investigate the functional roles and the molecular mechanisms in gametogenesis of groupers, especially in sex reversal and in spermatogenesis. (C) 2004 Elsevier Ireland Ltd. All rights reserved.

Differential expression of two Carassius auratus Mx genes in cultured CAB cells induced by grass carp hemorrhage virus and interferon

UV-inactivated GCHV (grass carp hemorrhage virus) is able to induce an antiviral state in cultured CAB cells (crucian carp Carassius auratus blastulae embryonic cells) via the production of interferon (IFN). In the current work, the full-length cDNAs of two Mx genes, termed CaMx1 and CaMx2, have been cloned and sequenced from UV-inactivated GCHV-infected and still IFN-producing CAB cells by suppression subtractive hybridization. Their putative proteins show the characteristically structural features of mammalian IFN-induced Mx proteins, including GTP-binding motif, dynamin family signature and leucine zipper motif. CaMx1 exhibits 85% sequence identity to zebrafish MxA and 72-74% to three Atlantic salmon Mx proteins. CaMx2 is most similar to zebrafish MxE, with 80% identity, and then rainbow trout Mx3, with 52%. Constitutive expression was detected by RT-PCR for CaMx1, but not for CaMx2, in normal CAB cells, but their up-regulations could be induced after treatment with active GCHV, UV-inactivated GCHV and CAB IFN. Distinct kinetics of expression was observed for either CaMx1 or CaMx2 corresponding to the three stimuli, and even between CaMx1 and CaMx2, corresponding to the same stimulus. Upon virus infection, the transcriptional induction was strongly blocked for CaMx2 by cycloheximide (CHX), whereas almost nothing was observed for CaMx1. By contrast, following treatment with CAB IFN, CHX did not inhibit either gene transcription. Collectively, these results suggest that there are very distinct mechanisms for modulating the expression of both CaMx1 and CaMx2 in normal and GCHV-infected CAB cells.

Dynamics of microcystins-LR and -RR in the phytoplanktivorous silver carp in a sub-chronic toxicity experiment

A sub-chronic toxicity experiment was conducted to examine tissue distribution and depuration of two microcystins (microcystin-LR and microcystin -RR) in the phytoplanktivorous filter-feeding silver carp during a course of 80 days. Two large tanks (A, B) were used, and in Tank A, the fish were fed naturally with fresh Microcystis viridis cells (collected from a eutrophic pond) throughout the experiment, while in Tank B, the food of the fish were M. viridis cells for the first 40 days and then changed to artificial carp feed. High Performance Liquid Chromatography (HPLC) was used to measure MC-LR and MC-RR in the M. viridis cells, the seston, and the intestine, blood, liver and muscle tissue of silver carp at an interval of 20 days. MC-RR and MC-LR in the collected Microcystis cells varied between 268-580 and 110-292 mug g(-1) DW, respectively. In Tank A, MC-RR and MC-LR varied between 41.5-99.5 and 6.9-15.8 mug g(-1) DW in the seston, respectively. The maximum MC-RR in the blood, liver and muscle of the fish was 49.7, 17.8 and 1.77 mug g(-1) DW, respectively. No MC-LR was detectable in the muscle and blood samples of the silver carp in spite of the abundant presence of this toxin in the intestines (for the liver, there was only one case when a relatively minor quantity was detected). These findings contrast with previous experimental results on rainbow trout. Perhaps silver carp has a mechanism to degrade MC-LR actively and to inhibit MC-LR transportation across the intestines. The depuration of MC-RR concentrations occurred slowly than uptakes in blood, liver and muscle, and the depuration rate was in the order of blood > liver > muscle. The grazing ability of silver carp on toxic cyanobacteria suggests an applicability of using phytoplanktivorous fish to counteract cyanotoxin contamination in eutrophic waters. (C) 2003 Elsevier Ltd. All rights reserved.

Characterization of cDNA encoding immunoglobulin light chain of the Mandarin fish (Siniperca chuatsi)

Immunoglobulin light chain cDNA sequences of a perciform fish, the mandarin fish Siniperca chuatsi were amplified from head kidney mRNA by reverse transcription (RT)-PCR and RACE methods using degenerated primer and gene specific ones. In cDNA sequences of the VL region, nucleotide exchanges were present mainly within CDRs, although a lesser degree of variability was also found in FRs. Moreover, the length of CDRI and CDR3 in the mandarin fish is shorter than in most other fish species. In the middle of S. chuatsi CL region, a microsatellite sequence (AGC)(6-8) was found, which is also present in another perciform species, the spotted wolffish (Anarhichas minor). The comparison of amino acid sequence of the mandarin fish CL domain with those of other vertebrates showed the highest degree of similarity of 94.5% to the spotted wolffish, while the similarity with rainbow trout (Oncorhynchus mykiss) Ig L1 (62.7%) and channel catfish (Ictalurus punctatus) Ig LG (55.9%) isotypes is also higher. However, there is only 50% identity in the VL regions between the mandarin fish and the wolffish. The sequence similarity of the mandarin fish CL domain with those of higher vertebrate did not readily allow it to be classified as kappa or lambda isotype. The phylogenetic analyses also demonstrated that the CL genes of the mandarin fish and most other teleost fish cluster as a separate branch out of the mammal kappa and lambda branches. (C) 2003 Elsevier B.V. All rights reserved.

Influence of dioxin and metal-contaminated sediment on phase I and II biotransformation enzymes in silver crucian carp

Several biochemical responses were measured in silver crucian carp (Carassius auratus gibelio) after exposure to sediments obtained from contaminated Ya-Er Lake, No, 1 pond, and an unpolluted reference site, Honglian Lake. After 1 week of exposure, a significant induction of the phase I biotransformation enzyme (ethoxylresorufin-o-deethylase, EROD) was found (83-fold of control), whereas the phase II biotransformation enzyme (glutathione S-transferase, GST) exhibited a slight, but significant induction (1,4-fold of control) after 4 weeks of exposure. The level of cellular glutathione in the liver was also slightly elevated after 4 weeks of exposure. The delayed response of GST to the contaminants indicates that the phase I and phase II biotransformation enzymes are regulated differently in fish. The results suggest that EROD is a sensitive bioindicator to assess the toxicity of dioxin-contamined sediment in the laboratory, (C) 1998 Academic Press.

Growth performance of juvenile white sturgeon as affected by feeding regimen

Juvenile (mean +/- SE, 8.6 +/- 0.1 g) white sturgeon Acipenser transmontanus were fed for 8 weeks under one of six feeding regimens: continuously 24 h/d (C24); continuously 12.8 h/d during the day (C12/D), continuously 12.8 h/d at night (C12/N), 6 meals/d (M6), 4 meals/d (M4), and 2 meals/d (M2). Specific growth rate, feed efficiency, and body lipid content were significantly (P < 0.05) affected by the feeding regimen. These variables were highest in the C24 group and lowest in the M2 group; fish in the M6 group showed the second best performance. Specific growth rate and feed efficiency in terms of wet weight in the M6 groups were not significantly different from those in the C24 groups, but specific growth rate in terms of energy and energy retention efficiency were significantly lower. Feeding regimen had no effect on condition factor, hepatosomatic index, coefficient of variation in final body weight, and protein and ash contents. There was no significant difference in these indexes between 12.8-h/d continuous feeding by day or by night. It was concluded that continuous feeding for 24 h/d was the optimum feeding regimen for juvenile white sturgeon.

Gnomon 分布式密码计算环境设计与实现

随着网络技术和信息技术的飞速发展，互联网环境下的安全问题越来越受到 政府、军事和商业部门的重视。密码技术是信息安全的核心技术，密码算法的设 计和实现一直是信息安全学界的重要研究内容。近年来，随着计算技术和网络的 飞速发展，计算工具和模式发生了变化，出现了分布式计算、网格计算和云计算 等新技术。在国内外已经分别采取过大型机、并行计算和分布式计算等方式来提 高密码计算的速度。采用新技术来设计和实现新的密码计算平台，对密码算法的 设计、分析以及应用有重要意义。分布式计算和网格计算在信息安全领域中的应 用是本文的主要研究内容。 首先，通过分析密码计算和网格计算的特点，确定了本文的研究目标，即构 建一个通用的、高效的、可扩展的、可移植的分布式计算环境。其次，分析研究 目标的特点以及遇到的问题，详细讨论系统所采用解决方案的特点及优势。在系 统分析设计的基础上，基于Globus Toolkit 和SWT/JFace 工具包，对Gnomon 分 布式密码计算环境进行了实现，并详细介绍了各个模块的功能。最后，设计和实 现了两个问题实例：大整数因子分解和Rainbow 攻击。针对问题实例，进行了多 组实验，并给出了相应的实验结果和分析。 Gnomon 分布式计算环境为密码的分布式计算提供支持，其易用性和可扩展 性为密码分析和设计人员带来了方便。本文研究成果推动了分布式密码计算的研 究与发展。

Gnomon分布式密码计算环境设计与实现

随着网络技术和信息技术的飞速发展，互联网环境下的安全问题越来越受到政府、军事和商业部门的重视。密码技术是信息安全的核心技术，密码算法的设计和实现一直是信息安全学界的重要研究内容。近年来，随着计算技术和网络的飞速发展，计算工具和模式发生了变化，出现了分布式计算、网格计算和云计算等新技术。在国内外已经分别采取过大型机、并行计算和分布式计算等方式来提高密码计算的速度。采用新技术来设计和实现新的密码计算平台，对密码算法的设计、分析以及应用有重要意义。分布式计算和网格计算在信息安全领域中的应用是本文的主要研究内容。 首先，通过分析密码计算和网格计算的特点，确定了本文的研究目标，即构建一个通用的、高效的、可扩展的、可移植的分布式计算环境。其次，分析研究目标的特点以及遇到的问题，详细讨论系统所采用解决方案的特点及优势。在系统分析设计的基础上，基于Globus Toolkit和SWT/JFace工具包，对Gnomon分布式密码计算环境进行了实现，并详细介绍了各个模块的功能。最后，设计和实现了两个问题实例：大整数因子分解和Rainbow攻击。针对问题实例，进行了多组实验，并给出了相应的实验结果和分析。 Gnomon分布式计算环境为密码的分布式计算提供支持，其易用性和可扩展性为密码分析和设计人员带来了方便。本文研究成果推动了分布式密码计算的研究与发展。