Dietary copper requirement of juvenile grass shrimp,Penaeus monodon,and effects on non-specific immune responses

An 8-week feeding trial was conducted to determine the dietary copper (Cu) requirement and its effect on the non-specific immune responses of juvenile grass shrimp, Penaeus monodon. Purified diets with seven levels (0, 10, 20, 30, 40, 80, 160 mg Cu kg diet(-1) of supplemental Cu were fed to P. monodon (mean initial weight 0.29 +/- 0.004 g). Each diet was fed to three replicate groups of shrimp. The rearing water contained 1.53 microg Cu 1(-1). Shrimp fed diets supplemented with 10 and 20 mg Cu kg diet(-1) had significantly (P < 0.01) greater weight gain, feed efficiency (FE) and protein efficiency ratio (PER) than those fed the unsupplemented control diet and diets supplemented with > or = 40 mg Cu kg diet(-1). Whole body Cu concentration in shrimp generally increased as dietary Cu supplementation increased. Total haemocyte count (THC) was higher in shrimp fed diets supplemented with 10-30 mg Cu kg diet(-1) than shrimp fed the unsupplemented control diet and diets supplemented with > or = 40 mg Cu kg diet(-1). Intracellular superoxide anion (O2-) production ratios were significantly higher in shrimp fed diets supplemented with 10-30 mg Cu kg diet(-1) than shrimp fed the diet supplemented with 160 mg Cu kg diet(-1). Analysis by polynomial regression of weight gain percent, FE and by linear regression of the whole-body Cu retention of shrimp indicated that the adequate dietary Cu concentration in growing P. monodon is about 15-21 mg Cu kg diet (-1). The immune indicators suggest that an adequate dietary Cu concentration for non-specific immune responses in P. monodon is about 10-30 mg Cu kg diet(-1).     

Characterization of oligopeptide transporter (PepT1) in grass carp (Ctenopharyngodon idella)

The oligopeptide transporter (PepT1) is located on the brush-border membrane of the intestinal epithelium, and plays an important role in dipeptide and tripeptide absorptions from protein digestion. In this study, we cloned the PepT1 cDNA from grass carp and characterized its expression profile in response to dietary protein and feed additives (sodium butyrate) treatments. The PepT1 gene encodes a protein of 714 amino acids with high sequence similarity with other vertebrate homologues. Expression analysis revealed highest levels of PepT1 mRNA expression in the foregut of grass carp. In addition, PepT1 mRNA expression exhibited diurnal variation in all three bowel segments of intestine with lower levels of expression in daytime than nighttime. During embryonic development, PepT1 showed a dynamic pattern of expression reaching maximal levels of expression in the gastrula stage and minimal levels in the organ stage. The PepT1 expression showed constant levels from 14 to 34 day post-hatch. To determine whether fish diet of different protein contents may have any effect on PepT1 expression, we extended our research to dietary regulation of PepT1 expression. We found that dietary protein levels had a significant effect on PepT1 gene expression. In addition, PepT1 mRNA levels were higher after feeding with fish meal than with soybean meal. Moreover, in vitro and in vivo sodium butyrate treatments increased PepT1 expression in the intestine of grass carp. The results demonstrate for the first time that PepT1 mRNA expression is regulated in a temporal and spatial pattern during development, and dietary protein and feed additives had a significant effects on PepT1 gene expression in grass carp.     

Morphology of androgenetic and gynogenetic grass carp, Ctenopharyngodon idella (Valenciennes)

Crosses between female carp x male grass carp resulted in androgenetic grass carp and hybrids. Fertilizing grass carp eggs with carp milt that had been irradiated with ultraviolet light for 15 min at 1.0 mW/cm² produced gynogenetic grass carp. We compared the morphology of experimental progeny with that of the parental species to determine if inheritance was strictly matroclinous in gynogenesis and strictly patroclinous in andro-genesis. Dorsal and anal fin rays, lateral line scales, gill rakers, pharyngeal teeth, and the relative length of the dorsal fin all suggested that gynogenetic grass carp were identical with the maternal species and androgenetic grass carp with the paternal species. Hybrids were intermediate between the parents except that the number of anal rays was identical with that in carp. The number of dorsal rays, the body depth, and the pharyngeal teeth in hybrids were more like those in carp than in grass carp, whereas the number of gill rakers was more similar to that in the paternal species.     

Digestion of duckweed (Lemna spp.) by the grass carp (Ctenopharyngodon idella)

A feeding trial was conducted in 55-1 aquaria indoors with the grass carp ( Ctenopharyngodon idella Val.) using fresh duckweed ( Lemna spp.) as food. Faeces dry weight was closely correlated with dry weight of duckweed consumed based upon a linear regression. From this regression, the estimated true digestibility of dry matter was 65%. Samples of duckweed and fish faeces were analysed for crude protein and gross energy. Van Soest's Analysis was conducted on composite samples of duckweed and faeces. Apparent digestibilities were 80% for crude protein, 61% for gross energy, 72% for organic cell contents, and 30% for organic cell wall constituents.     

丙二醛对离体草鱼肠道黏膜细胞的损伤作用

以丙二醛为实验材料, 在草鱼Ctenopharyngodon idella肠道黏膜细胞培养液中加入不同浓度丙二醛, 研究丙二醛不同剂量、不同作用时间下对肠道黏膜细胞生长、细胞形态结构及相关酶活性的变化. 结果显示: 添加(1.23-9.89) mol/L丙二醛在3-9h时显著抑制了离体草鱼肠道黏膜细胞生长及存活率, 以6h时抑制程度较为明显, 导致贴壁细胞减少, 细胞集落面积减小, 其中添加4.94 mol/L丙二醛细胞胞浆内脂肪滴沉积, 空泡变性, 同时线粒体肿胀, 核固缩; 丙二醛对细胞分化成熟有抑制, 且增加细胞器膜的通透性, 导致胞浆酶漏出; 6h时丙二醛处理组培养液中GSH-PX、T-AOC活力显著降低(P0.05). 结果表明: 添加(1.23-9.89) mol/L丙二醛对草鱼肠道黏膜细胞产生了损伤, 表现为抑制细胞生长, 改变细胞形态、结构, 导致膜结构破坏, 且作用程度与添加浓度、作用时间呈正相关关系. 研究认为丙二醛对草鱼肠道黏膜细胞具有显著性的损伤作用.       

Intensive culture of grass carp, Ctenopharyngodon idella, in circular tanks

Fingerling grass carp ( Ctenopharyngodon idella ) were cultured in 380 1 circular fiberglass tanks at stocking densities of 0.53, 1.06, 1.59 and 2.11 fish/1. Fish were fed duckweed ( Lemna minima ) to satiation. Fish grew from 2.7 g to a maximum mean weight of 72.7 g in 88 days. Fish stocked at 0.53 fish/1 grow more rapidly than those stocked at higher densities. Mean daily consumption rates were strongly correlated with oxygen levels and when oxygen levels dropped below 4 mg/1 consumption was reduced approximately 40%. High biomass, growth, and survival rates obtained during this study indicate grass carp are well suited for intensive tank culture.     

A 15 nucleotide deletion mutation in coding region of the RIG-I lowers grass carp (Ctenopharyngodon idella) resistance to grass carp reovirus

RIG-I (Retinoic acid-inducible gene I) is a pivotal receptor that detects numerous RNA and DNA viruses and plays crucial roles in the induction of type I interferons. In the present study, a deletion mutation in CiRIG-I (Ctenopharyngodon idella RIG-I) coding region was detected, its association with resistance/susceptibility to grass carp reovirus (GCRV) was examined, and possible mechanism was analyzed. A 15-bp deletion mutation was found, and the mutation results in a deletion of five amino acids. To investigate the genotypes and alleles, the relevant PCR products were electrophoresed on 2.5% agarose gel. Three genotypes and two alleles were discovered. The general allele was named as A and the deletion mutation allele was named as B. The deletion mutation cancels a predicted phosphorylation site and changes the secondary structure and the probability of peroxisomal targeting signal 1 in CiRIG-I. To explore the correlation between these genotypes and the resistance of grass carp to GCRV, a challenge experiment was carried out. The cumulative mortality in genotype AA (40.70%) and AB (52.73%) was significantly lower than that in genotype BB (71.43%) (P = 0.032). The result demonstrated that genotype AA and AB were resistant to GCRV, while genotype BB was susceptible. The 15-bp deletion mutation lowers the resistance of grass carp to GCRV. This result might provide a potential genetic marker for further investigation of selective breeding of resistant grass carp to GCRV.     

Growth and diet dependant structural adaptations of the digestive tract in juvenile grass carp (Ctenopharyngodon idella, Val.)

The morphology of the intestinal epithelium is described and compared with published data. In the gut of this stomachless fish, three zones can be distinguished based on light and electron microscopic observations of absorptive cells: a rostral zone with numerous fat droplets, a second zone with large P.A.S.-positive supranuclear vacuoles and pinocytotic vesicles, and a third zone without these characteristics. In these zones deep invaginations of the cell membrane is a feature of the basal part of the cells. The presence of pinocytotic vesicles in the second zone is considered evidence of the uptake of undigested protein-like substances.
Juvenile grass carp from 15 to 335 days, fed on either animal or vegetable food, were measured. The growth rates indicate that animal food stimulates rapid growth. The data also show a vegetable diet causes a slight increase in relative gut length, mainly in the length of the first zone.     

How the grass carp (Ctenopharyngodon idella) chooses and chews its food—some clues

Grass carp were allowed to feed on a variety of plants, presented one species at a time. They were filmed and their faeces collected. Canadian pondweed, a grass and a filamentous alga were readily taken; greater duckweed, shining pondweed and the leaves of reedmace were not so acceptable. Limiting factors in feeding seem to vary: with the two pondweeds foraging is limiting, probably because the leaves make it difficult for the fish to extract a single, manageable stem. Transport is limiting with the grass—the fish cannot swallow it quickly enough. Chewing is limiting with reedmace leaves (they are too tough) and the filamentous alga (for no apparent reason). Duckweed is rejected, probably because it has an unpleasant taste. All plants are chewed at the same frequency of jaw movement, independent of the texture of the plant.     

草鱼野生群体遗传变异的微卫星分析

利用12个微卫星标记, 对来自长江水系(邗江、吴江、九江、石首、木洞和万州)、珠江水系(肇庆)和黑龙江水系(嫩江)的8个草鱼野生地理群体进行了遗传多样性及遗传结构等分析。遗传多样性分析显示, 12个微卫星位点均为高度多态位点(PIC=0.755~0.930), 8个草鱼野生群体显示出较高的遗传多样性水平(Ho=0.839~0.893), 其中长江水系的6个群体和肇庆群体的多样性水平高于嫩江群体。瓶颈效应分析显示, 嫩江、肇庆群体及2个长江上游群体(木洞、万州)近期出现了瓶颈效应, 群体数量发生下降。群体间遗传分化指数F^ST及AMOVA分析显示, 群体间出现极显著遗传分化(P<0.01), 整体分化水平较低(F^ST<0.05)。遗传距离分析结果显示, 长江水系的6个群体与肇庆群体遗传距离较近, 与嫩江群体较远; 基于此的UPGMA聚类树显示, 长江水系下游、中游和上游的群体依次聚类, 然后与肇庆群体聚类, 最后与嫩江群体聚类, 遗传距离与地理距离呈现出较强的正相关性。遗传结构分析显示, 所有样本被划分为5个理论群, 肇庆和嫩江群体中个体的遗传结构相对独立, 而长江上游、中游和下游群体中个体的遗传结构则存在一定程度的混杂。综上所述, 中国草鱼野生资源具有较高的遗传多样性, 地理群体间存在遗传分化, 具有进一步遗传改良的潜力; 但部分群体出现的瓶颈效应也需要引起重视。     

Sox genes in grass carp (Ctenopharyngodon idella) with their implications for genome duplication and evolution

The Sox gene family is found in a broad range of animal taxa and encodes important gene regulatory proteins involved in a variety of developmental processes. We have obtained clones representing the HMG boxes of twelve Sox genes from grass carp (Ctenopharyngodon idella), one of the four major domestic carps in China. The cloned Sox genes belong to group B1, B2 and C. Our analyses show that whereas the human genome contains a single copy of Sox4, Sox11 and Sox14, each of these genes has two co-orthologs in grass carp, and the duplication of Sox4 and Sox11 occurred before the divergence of grass carp and zebrafish, which support the "fish-specific whole-genome duplication" theory. An estimation for the origin of grass carp based on the molecular clock using Sox1, Sox3 and Sox11 genes as markers indicates that grass carp (subfamily Leuciscinae) and zebrafish (subfamily Danioninae) diverged approximately 60 million years ago. The potential uses of Sox genes as markers in revealing the evolutionary history of grass carp are discussed.     

The tolerance of grass carp Ctenopharyngodon idella (Val.) to seawatert†

Laboratory tests were conducted to determine the tolerance of 2+ grass carp to an oceanic water. It was found that at a temperature of 20.1°C±0.4°C the maximum survival times of the fish in concentrations of seawater equivalent to 17.5 and 10.5 g/1 sodium chloride were 5 h and approximately 24 days respectively. The importance of these results lies in the consideration of the possibility of grass carp migrating through brackish or seawater from one river system to another. Such a migration would seem to be impossible through seawater but may be possible through brackish water.     

Development of microsatellite DNA markers of grass carp (Ctenopharyngodon idella) and their cross-species application in black carp (Mylopharyngodon piceus)

Thirty-four microsatellite DNA loci were developed for grass carp (Ctenopharyngodon idella) and used to determine the diversity of 42 individuals of grass carp and 16 individuals of black carp collected from Jingzhou fragment (the middle reach) of Yangtze River. All the 34 loci were polymorphic in grass carp. The number of alleles found in grass carp at these loci ranged from 2 to 24. The observed and expected heterozygosities varied from 0.238 to 1.000 and from 0.162 to 0.945, respectively. Thirty of the 34 loci cross-amplified the DNA of black carp (Mylopharyngodon piceus), and 21 of them revealed polymorphism (more than one allele each locus).     

饥饿胁迫对草鱼鱼种非特异免疫力的影响

研究长期饥饿对草鱼(Ctenopharyngodon idellus)鱼种非特异性免疫力的影响。实验选取平均体质量(31.86?1.47g)的草鱼,随机分为2个实验组(对照组和饥饿组),每组三个平行,饥饿处理15、30、45和60d,测定饥饿对头肾和脾脏中自然杀伤(NK)细胞的杀伤活性、血清和肝胰脏中溶菌酶活性、血清中碱性磷酸酶活性的影响。结果表明：受饥饿胁迫的影响,草鱼鱼种自然杀伤性细胞在脾脏和头肾中的杀伤活性显著低于对照组(P<0.05)且不随着饥饿时间的延长发生显著性变化;随着饥饿时间的延长,血清和肝胰脏中溶菌酶呈现先升高后降低的趋势,血清碱性磷酸酶在饥饿15d、45d、60d时显著低于对照组;,饥饿组的碱性磷酸酶活性在饥饿30d以后,维持恒定。由此可见,长时间的饥饿胁迫降低了草鱼鱼种的免疫力。相比较而言,自然杀伤细胞的杀伤活性在评价鱼类的免疫状况时比溶菌酶和碱性磷酸酶更为灵敏。     

Overexpression of Hsp90 from grass carp (Ctenopharyngodon idella) increases thermal protection against heat stress

With homologous DNA probes, we had screened a grass carp heat shock protein 90 gene (CiHsp90). The full sequence of CiHsp90 cDNA was 2793 bp, which could code a 798 amino acids peptide. The phylogenetic analysis demonstrated that CiHsp90 shared the high homology with Zebrafish Grp94. Quantitative RT-PCR analysis showed that CiHsp90 was ubiquitously expressed at lower levels in all detected tissues and up-regulated after heat shock at 34 °C or cold stress at 4 °C. To understand the function of CiHsp90 involving in thermal protection, an expression vector containing coding region cDNA was expressed in E. coli BL21 (DE3) plysS. Upon transfer from 37 °C to 42 °C, these cells that accumulated CiHsp90 peptides displayed greater thermoresistance than the control cells. While incubated at 4°C for different periods, it could also improve the cell viability. After transient transfected recombinant plasmid pcDNA3.1/CiHsp90 into mouse myeloma cell line SP2/0, we found that CiHsp90 could contribute to protecting cells against both thermal and cold extremes. On the contrary, the mutant construct ΔN-CiHsp90 (256-798aa) could abolish the protection activity both in prokaryotic cells and eukaryotic cells. Additionally, both CiHsp90 and ΔN-CiHsp90 peptides could reduce the level of citrate synthase aggregation at the high temperature.