Development of microsatellite markers for blunt snout bream Megalobrama amblycephala using 5'-anchored PCR

A rapid method for isolating microsatellite loci in blunt snout bream, based on the 5'-anchored polymerase chain reaction technique, revealed 522 microsatellite loci (consisting of 442 dinucleotide, 4 trinucleotide and 76 tetranucleotide repeats). Of the 25 loci characterized, 10 turned out to be highly polymorphic. The number of alleles per locus ranged from 3 to 17 while the expected heterozygosity ranged from 0.4899 to 0.9355 in population of selected strain F(7 ) and from 0.5786 to 0.9556 in wild population from Lake Liangzi. These markers are useful as tools for the detection of genetic variation levels in selected strains and wild populations of blunt snout bream for germplasm conservation.     

Molecular cloning and function analysis of insulin-like growth factorbinding protein 1a in blunt snout bream(Megalobrama amblycephala)

Insulin-like growth factor-binding protein 1(IGFBP-1), a hypoxia-induced protein, is a member of the IGFBP family that regulates vertebrate growth and development. In this study, full-length IGFBP-1a cDNA was cloned from a hypoxia-sensitive Cyprinidae fish species, the blunt snout bream(Megalobrama amblycephala). IGFBP-1a was expressed in various organs of adult blunt snout bream, including strongly in the liver and weakly in the gonads. Under hypoxia, IGFBP-1a mRNA levels increased sharply in the skin, liver, kidney, spleen, intestine and heart tissues of juvenile blunt snout bream, but recovered to normal levels after 24-hour exposure to normal dissolved oxygen. In blunt snout bream embryos, IGFBP-1a mRNA was expressed at very low levels at both four and eight hours post-fertilization, and strongly at later stages. Embryonic growth and development rates decreased significantly in embryos injected with IGFBP-1a mRNA. The average body length of IGFBP-1a-overexpressed embryos was 82.4% of that of the control group, and somite numbers decreased to 85.2%. These findings suggest that hypoxia-induced IGFBP-1a may inhibit growth in this species under hypoxic conditions.     

团头鲂人工同源四倍体、自繁后代、倍间交配后代的染色体组型及形态遗传特征

以2n团头鲂为对照,对诱导产生的同源四倍体、自繁后代(4n-F1)和倍间交配后代(正交3n和反交3n)的染色体组型及形态遗传特征进行比较分析。结果发现:(1)团头鲂四倍体(包括4n和4n-F1)和正反倍间交配三倍体的染色体众数分别为96和72,是2n团头鲂的2倍和1·5倍;在四倍体团头鲂组型排列中,sm1的四条较大的染色体明显可见,可视为标记染色体;(2)9个比例性状的测量结果显示,多倍体的体长/体高、体长/头长比例值显著小于2n团头鲂(P<0·05);而对于背棘长/体长比例值,多倍体则显著大于2n团头鲂(P<0·05);(3)29个参数的主成分分析结果表明,团头鲂同源4n、4n-F1、倍间交配3n及2n团头鲂等5个不同倍性群体的传统形态差别很大部分是由躯干部的形态差异,主要是体长/体高引起的,可作为团头鲂多倍体与二倍体群体鉴别的形态依据;(4)聚类结果显示,正交3n和反交3n相聚类,亲缘关系最近,然后,它们与4n-F1聚类后,再与4n奠基群体聚类,与二倍体群体的聚类关系较远[动物学报51(3):455-461,2005]。     

The fates of zebrafish Hox gene duplicates

In his 1970 book, Susumu Ohno stressed the importance of gene duplication in the evolution of the vertebrate genome and body plan. He elaborated the idea that duplication events provide novel genetic material on which evolution may act. Data are accumulating to show that extensive duplication events, perhaps incorporating the duplication of entire genomes, occurred in the lineage leading to teleost fishes. These duplications may have been pivotal in the explosive radiation of this highly successful vertebrate group. Thus, the teleosts provide us with an ideal opportunity to investigate the fates and functions of duplicated genes. A convenient system for these studies is the zebrafish, Danio rerio, which has become a popular genetic and embryological model.     

Isolation and characterization of polymorphic microsatellite loci in Wuchang bream (Megalobrama amblycephala)

Wuchang bream (Megalobrama amblycephala) is an economically important fish in China. From a (GT)₁₃‐enriched genomic library, 20 microsatellites were developed. Nine of these 20 loci were polymorphic in a test population with allele numbers ranging from two to four, and the observed and expected heterozygosities ranging from 0.2609 to 0.7826 and from 0.3739 to 0.7546, respectively. In the cross‐species amplifications, six of these nine loci were also polymorphic in white amur bream (Parabramis pekinensis). These polymorphic microsatellite loci are potentially useful for population genetics of Wuchang bream and its closely related species.     

Dietary valine requirement of juvenile blunt snout bream (Megalobrama amblycephala Yih, 1955)

An 8‐week feeding trial was carried out to test the hypothesis that adequate dietary valine might improve growth, feed utilization and protein content in blunt snout bream, whereas a valine deficiency might have adverse effects on these parameters. Six isonitrogenous (34% crude protein) and isoenergetic (14.2 MJ kg^?1 digestible energy) experimental diets were formulated to contain graded valine levels (0.66, 0.95, 1.26, 1.55, 1.87 and 2.16% of dry weight) at about 0.30% increments replaced by equal proportions of glycine. At the end of the experiment the survival rate was not significantly affected by the dietary valine level. Final weight, weight gain (WG), specific growth rate (SGR), feed efficiency ratio (FER) and protein efficiency ratio (PER) increased with an increasing dietary valine level up to the 1.26% diet, and thereafter remained relatively constant. Dietary valine levels significantly affected the viscerosomatic index, but not the hepatosomatic index or condition factor. The dietary valine levels significantly affected the protein contents of whole body and plasma. Dietary valine supplementation significantly increased the plasma valine concentration, but not the levels of other branched‐chain amino acids (isoleucine and leucine). Based on SGR and FER, the optimal dietary valine requirements of juvenile blunt snout bream were determined to be 1.32% of the diet (3.88% of dietary protein) and 1.26% of the diet (3.71% of dietary protein), respectively, using broken‐line regression analysis.     

An AFLP-based approach for the identification of sex-linked markers in blunt snout bream, Megalobrama amblycephala (Cyprinidae)

Sex-specific DNA markers are useful for studying sex-determination mechanisms and establishment of monosex populations. Three widely spaced geographical populations (Liangzi, Poyang and Yuni Lakes in China) of blunt snout bream (Megalobrama amblycephala) were screened with AFLPs to search for sex-linked markers. Female and male pools (10 individuals in each pool) from each population were screened using 64 different primer combinations. A total of 4789 genomic fragments were produced, with a mean frequency of 75 bands per primer pair. Three different primer combinations produced putative sex-associated amplifications and were selected for individual screening in the three populations. However, none showed sex specificity when we converted these three markers into sequence characterized amplified region markers and evaluated all the individuals from the three populations.     

Microsatellite analysis of variation among wild,domesticated,and genetically improved populations of blunt snout bream(Megalobrama amblycephala)

In the present study,the genetic diversity of one selected strain(Pujiang No.1),two domesticated populations(GA and HX)and four wild populations(LZ,YN,SS and JL)of blunt snout bream(Megalobrama amblycephala)was analyzed using 17microsatellite markers.The results showed that an average of 4.88-7.65 number of alleles(A);an average of 3.20-5.33 effective alleles(Ne);average observed heterozygosity(Ho)of 0.6985-0.9044;average expected heterozygosity(He)of 0.6501-0.7805;and the average polymorphism information content(PIC)at 0.5706-0.7226.Pairwise FST value between populations ranged from0.0307-0.1451,and Nei’s standard genetic distance between populations was 0.0938-0.4524.The expected heterozygosities in the domesticated populations(GA and HX)were significantly lower than those found in three wild populations(LZ,SS and JL),but no difference was detected when compared with the wild YN population.Likewise,no difference was found between the four wild populations or two domesticated populations.The expected heterozygosity in Pujiang No.1 was higher than the two domesticated populations and lower than the four wild populations.Regarding pairwise FST value between populations,permutation test P-values were significant between the GA,HX and PJ populations,but not between the four wild populations.These results showed that the expected heterozygosity in the selected strain of blunt snout bream,after seven generations of selective breeding,was lower than that of wild populations,but this strain retains higher levels of genetic diversity than domesticated populations.The genetic differences and differentiation amongst wild populations,domesticated populations and the genetically improved strain of blunt snout bream will provide important conservation criteria and guide the utilization of germplasm resources.     

Adequate dietary protein level enhances stress resistance and immune status of blunt snout bream (Megalobrama amblycephala Yih, 1955)

This study hypothesized that an optimum dietary protein level might play an important role in improving stress tolerance, enhancing an immune function, and ultimately minimizing temperature stress. For this purpose, the present study conducted a 10‐week feeding trial followed by a 7‐day stress experiment to evaluate the effects of dietary protein levels (28%–36%) on the physiological performances and hepatic Hsp70 gene expression in blunt snout bream Megalobrama amblycephala fry under a high temperature challenge. Fish fry (initial weight, 16.08 ± 0.03 g, n = 25) were fed with their respective diets to apparent satiation, and samples taken once before stress and four times during high temperature stress days (0.125, 0.5, 2 and 7 days). Serum total protein and cholesterol contents before stress were affected by dietary protein levels; during stress these parameters showed no significant changes up until day 2, showing some changes thereafter. Regardless of 0.125, 0.5, 2 or 7 days during stress, cortisol and aspartate aminotransferase levels in the group fed 32% dietary protein were significantly lower than in the other groups. At 0.125, 0.5 or 2 days during stress, the complement component 3 (C3) and 4 (C4) levels increased significantly up to certain levels, declining thereafter. The expression level of Hsp70 mRNA before stress was not affected by dietary protein levels, but increased significantly from 0.125 to 0.5 days during stress, and was reduced thereafter. Overall, low cortisol and high C3, C4 and Hsp70 mRNA levels were found in the group fed 32% protein throughout the stress experiment, which indicated that an adequate protein level enhances stress resistance and immunity in fish.     

Dietary carbohydrate/lipid ratios affect stress, oxidative status and non-specific immune responses of fingerling blunt snout bream, Megalobrama amblycephala

This study aimed to evaluate the effects of dietary carbohydrate/lipid (CHO:L) ratios on stress, liver oxidative status and non-specific immune responses of fingerling blunt snout bream. Fish were fed six isonitrogenous and isoenergetic diets containing various CHO:L ratios for 10 weeks. After the feeding trial, fish were challenged by Aeromonas hydrophila and survival rate was recorded for the next 10 days. The lowest plasma cortisol, lactate, aspartate aminotransferase and alanine aminotransferase were all observed in fish fed a CHO:L ratio of 5.64. They were significantly (P < 0.05) lower than those of fish offered the lowest CHO:L ratio, but showed little difference (P > 0.05) with those of fish fed the highest CHO:L ratio. This also held true for liver catalase and glutathione peroxidase activities, whereas the opposite was true for liver reduced glutathione contents, plasma lysozyme and alternative complement (ACH50) activities. Contrary to leucocyte counts, plasma glucose levels, liver malondialdehyde contents, blood haemoglobin contents and erythrocyte numbers all increased significantly (P < 0.05) with decreasing dietary CHO:L ratios. The highest plasma total protein and globulin content both observed in fish fed a CHO:L ratio of 2.45 was significantly (P < 0.01) higher than that of fish offered the lowest CHO:L ratio, but showed no statistical difference (P > 0.05) with that of the other groups. After challenge, fish fed the lowest CHO:L ratio obtained significantly (P < 0.05) low survival rate. However, survival rate showed little difference (P > 0.05) as dietary CHO:L ratios ranged from 3.67 to 24.20. The results of this study indicated that high dietary lipid may cause metabolic stress of fingerling blunt snout bream, as might consequently lead to the elevated liver oxidation rates, impaired liver function, depressed immunity and reduced resistance to A. hydrophila infection of this species, whereas the opposite was true for carbohydrate enriched diets.     

团头鲂黏蛋白基因Muc5b克隆及表达分析

摘要：黏液（mucus）在鱼体防御外界病原侵袭、信息传递、调节渗透压等方面具有重要作用。黏蛋白（mucin）作为黏液的基础骨架组分,与其相关的研究正受到广泛的关注。在本研究中,作者克隆获得团头鲂（Megalobrama amblycephala）Muc5b mRNA 的部分序列3895 bp,并通过qRT-PCR分析了Muc5b在团头鲂不同组织的表达分布及其在捕捞应激后在鳃和表皮中的表达变化。序列分析结果显示,团头鲂Muc5b与鲤等脊椎动物的Muc5b有较高的同源性,其N端含有黏液蛋白特异性结构域：三个VWD区域,三个C8区域,二个TIL区。组织表达分析结果表明,Muc5b在鳃和表皮表达量相对较高,在脑、脾、肾中表达水平较低,在肝、肠道几乎不表达。捕捞应激后1 h时鳃中Muc5b显著降低（P < 0.05）,24 h时恢复初始水平;表皮中4 h时Muc5b显著上升（P < 0.05）,24 h时恢复到初始水平。     

Hox gene expression reveals regionalization along the anteroposterior axis of the zebrafish notochord

 The vertebrate Hox genes have been shown to confer regional identity along the anteroposterior axis of the developing embryo, especially within the central nervous system (CNS) and the paraxial mesoderm. The notochord has been shown to play vital roles in patterning adjacent tissues along both the dorsoventral and mediolateral axes. However, the notochord’s role in imparting anteroposterior information to adjacent structures is less well understood, especially as the notochord shows no morphological distinctions along the anteroposterior axis and is not generally described as a segmental or compartmentalized structure. Here we report that four zebrafish hox genes: hoxb1, hoxb5, hoxc6 and hoxc8 are regionally expressed along the anteroposterior extent of the developing notochord. Notochord expression for each gene is transient, but maintains a definite, gene-specific anterior limit throughout its duration. The hox gene expression in the zebrafish notochord is spatially colinear with those genes lying most 3’ in the hox clusters having the most anterior limits. The expression patterns of these hox cluster genes in the zebrafish are the most direct molecular evidence for a system of anteroposterior regionalization of the notochord in any vertebrate studied to date. Received: 30 March 1998 / Accepted: 16 June 1998     

团头鲂Toll样受体3基因的克隆及特征研究

由草鱼呼肠孤病毒(Grass carp reovlrus,GCRV)引起的草鱼出血病,是对草鱼危害最大的传染性疾病之一.GCRV是双链RNA病毒,属呼肠孤病毒科(Reoviridae),水生呼肠孤病毒属(Aquareovirus),G亚型[1].GCRV主要引起草鱼、青鱼等在鱼种阶段发生出血病.