黄颡鱼与长须黄颡鱼种间的RAPD标记

黄颡鱼属(Pelteobagrus Bleeker, 1865-属鲇形目( Siluri formes), 科(Bagridae-。       

Mitochondrial DNA sequence of yellow catfish (Pelteobagrus fulvidraco)

Yellow catfish (Pelteobagrus fulvidraco) belongs to the family Bagridae, which is one of the most important economic freshwater aquaculture species in China. In this study, we reported the complete sequence of the mitochondrial genome of P. fulvidraco. The complete mitochondrial genome is 16,527 bp in length, including the typical structure of 22 transfer RNA genes, 13 protein-coding genes, two ribosomal RNA genes, and the non-coding control region. Both the termination-associated sequence and critical central conserved sequences (CSB-D, CSB-E, and CSB-F) was also detected.     

Effects of ammonia on growth,digestion and antioxidant capacity in juvenile yellow catfish Pelteobagrus fulvidraco (Richardson, 1846)

Effects of ammonia stress on food ingestion, growth, digestion and antioxidant capacity were investigated in juvenile yellow catfish Pelteobagrus fulvidraco (Richardson) with initial body weights of 20.24 ± 0.18 g. The fish were reared in triplicate in 15 experimental tanks at a rate of 30 fish per tank for 56 days. Water was maintained at a dissolved oxygen (DO) level of over 6.2 mg L^?1, pH 7.2–7.6, and temperature of 29.0 ± 1.5°C under a natural 12L: 12D photoperiod. Survival, food ingestion (FI), specific growth rate (SGR), food conversion efficiency (FCE), apparent digestibility coefficient (ADC), total antioxidant capacity (T‐AOC), levels of glutathione (GSH) and malonaldehyde (MDA), and activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH‐PX) of the juveniles were determined in total ammonia nitrogen (NH₃‐N+NH₄‐N) levels of 0 (control group), 3.36, 6.72, 13.44 and 26.88 mg L^?1. The results show that the survival, FI, SGR, FCE, and ADC decreased significantly with an increase in total ammonia nitrogen (p < .05), and a significantly negative correlation between SGR and total ammonia nitrogen levels (p < .05). T‐AOC, SOD, CAT, GSH‐PX and GSH in the blood, liver and gills were found to decline significantly with an increase in the total ammonia nitrogen level (p < .05), while the MDA in the blood, liver and gills was elevated significantly with the increase in total ammonia nitrogen (p < .05). The results indicate a threshold in the induction of the T‐AOC and activities of antioxidant enzymes in yellow catfish tissues with a total ammonia nitrogen increase. In the present study the total ammonia nitrogen threshold thus changed from 6.72 mg L^?1 in the juvenile yellow catfish.     

黄颡鱼免疫球蛋白M重链基因原核表达研究

在前期研究克隆得到黄颡鱼(Pelteobagrus fulvidraco)全长cDNA的基础上, 为进一步研究黄颡鱼免疫球蛋白M(IgM)重链的生物学功能, 设计特异性引物PCR扩增获得了编码成熟免疫球蛋白M重链基因的编码序列。将该基因编码片段连接到原核表达载体pQE30中, 构建黄颡鱼IgM重链的重组表达质粒IgM-pQE30。该重组质粒经酶切和测序鉴定后, 转入表达宿主大肠杆菌M15中诱导表达, 在30℃下, 经0.2 mmol/L IPTG诱导表达8 h, 可获得大量以包涵体形式存在的黄颡鱼IgM重链蛋白, 经SDS-PAGE电泳和Western blotting 分析表明, 新增的62 kDa蛋白条带与预期值相符, 且能与鼠源抗6×His的单克隆抗体特异性结合, 证明黄颡鱼IgM重链基因获得了高效原核表达。为进一步纯化该蛋白制备特异抗体, 研究其生物学功能奠定了基础。     

Isolation and characterization of twelve novel microsatellites in yellow catfish, Pelteobagrus fulvidraco

The yellow catfish Pelteobagrus fulvidraco is a freshwater fish species. Due to overfishing and pollution of freshwater ecosystems, the wild stocks of this fish reduced substantially. We isolated and characterized 12 polymorphic microsatellites of this species. The number of alleles at the 12 microsatellite loci ranged from four to eight, with an average of 6.6/locus. The average observed heterozygosity was 0.72, whereas the expected heterozygosity ranged from 0.60 to 0.86 (average: 0.80). All 12 microsatellites conformed to Hardy–Weinberg Equilibrium and were in linkage equilibrium. These 12 novel microsatellites could facilitate studies of genetic diversity and population structure of the yellow catfish to supply necessary information of conservation of the yellow catfish.     

Mitochondrial genome of the yellow catfish Pelteobagrus fulvidraco and insights into Bagridae phylogenetics

The mitochondrial genome (mitogenome) can provide important information for understanding phylogenetic analysis and molecular evolution. Herein, we amplified the complete mitogenome sequence of Pelteobagrus fulvidraco. The mitogenome was 16,526 bp in length and included 13 protein-coding genes (PCGs), 22 transfer RNA genes, two ribosomal RNA genes and a non-coding control region (D-loop). Both the organization and location of genes in the mitogenome were consistent with those from Siluriformes fishes previously published in GenBank. The phylogenetic relationships based on Bayesian inference (BI) and Maximum likelihood (ML) methods showed that P. fulvidraco has close relationships with Pelteobagrus eupogon and Tachysurus intermedius, suggesting that P. fulvidraco belongs to Tachysurus. This study provides evidence that Tachysurus, Pseudobagrus and Leiocassis do not form monophyly, but that these three genera form a monophyletic group. Our results provide reference for further phylogenetic research of the Bagridae species.     

Heritable targeted inactivation of myostatin gene in yellow catfish (Pelteobagrus fulvidraco) using engineered zinc finger nucleases

Yellow catfish (Pelteobagrus fulvidraco) is one of the most important freshwater aquaculture species in China. However, its small size and lower meat yield limit its edible value. Myostatin (MSTN) is a negative regulator of mammalian muscle growth. But, the function of Mstn in fish remains elusive. To explore roles of mstn gene in fish growth and create a strain of yellow catfish with high amount of muscle mass, we performed targeted disruption of mstn in yellow catfish using engineered zinc-finger nucleases (ZFNs). Employing zebrafish embryos as a screening system to identify ZFN activity, we obtained one pair of ZFNs that can edit mstn in yellow catfish genome. Using the ZFNs, we successfully obtained two founders (Founder July29-7 and Founder July29-8) carrying mutated mstn gene in their germ cells. The mutated mstn allele inherited from Founder July29-7 was a null allele (mstn(nju6)) containing a 4 bp insertion, predicted to encode function null Mstn. The mutated mstn inherited from Founder July29-8 was a complex type of mutation (mstn(nju7)), predicted to encode a protein lacking two amino acids in the N-terminal secretory signal of Mstn. Totally, we obtained 6 mstn(nju6/+) and 14 mstn(nju7/+) yellow catfish. To our best knowledge, this is the first endogenous gene knockout in aquaculture fish. Our result will help in understanding the roles of mstn gene in fish.     

Characterization and expression analysis of seven selenoprotein genes in yellow catfish Pelteobagrus fulvidraco to dietary selenium levels

BackgroundSelenium (Se) appears in the selenoproteins in the form of selenocysteine (Sec) and is important for the growth and development of vertebrates. The present study characterized seven selenoproteins, consisting of the GPX1, GPX3, GPX4, SELENOW, SELENOP, TXNRD2 and TXNRD3 cDNAs in various tissues of yellow catfish, explored their regulation to dietary Se addition.Methods3′ and 5′ RACE PCR were used to clone full-length cDNA sequences of seven selenoprotein genes (GPX1, GPX3, GPX4, SELENOW, SELENOP, TXNRD2 and TXNRD3). Their molecular characterizations were analyzed, including conservative motifs and the SECIS elements. The phylogenetic trees were generated through neighbor-joining (NJ) method with MEGA 6.0 with 1000 bootstrap replications. Quantitative real-time PCR was used to explore their mRNA tissue distribution in the heart, anterior intestine, dorsal muscle, head kidney, gill, liver, brain, spleen and mesenteric fat. Yellow catfish (mixed sex) were fed diets with dietary Se contents at 0.03 (low Se), 0.25 (adequate Se) and 6.39 (high Se) mg Se/kg, respectively, for 12 weeks, and their spleen, kidney, testis and brain were used for the determination of the mRNA levels of the seven selenoproteins.ResultsThe seven selenoproteins had similar domains to their corresponding members of other vertebrates. They were widely expressed in nine tissues, including heart, liver, brain, spleen, head kidney, dorsal muscle, mesenteric fat, anterior intestine and gill, but showed tissue-dependent expression patterns. Dietary Se addition affected the expression of the seven genes in spleen, kidney, testis and brain tissues of yellow catfish.ConclusionTaken together, our study demonstrated the characterization, expression and regulation of seven selenoproteins, which increased our understanding of the biological functions of Se and selenoproteins in fish.     

黄颡鱼卵致病性绵霉的分离鉴定与药敏特性

【目的】对黄颡鱼水霉病病原进行分离鉴定,并对其药敏特性进行研究。【方法】参照传统方法对患水霉病的黄颡鱼卵上的丝状真菌进行分离,通过人工感染实验证实分离菌株的致病性,然后根据形态学特征和ITS rDNA序列分析对致病菌株进行鉴定,并进一步采用倍比稀释法研究其药敏特性。【结果】从患水霉病的黄颡鱼卵上分离到8株丝状真菌,经人工感染试验证实菌株YC对黄颡鱼卵具有致病性,并进一步研究了其形态与药敏特性,开展了ITS rDNA序列分析。结果表明,菌株YC为透明管状结构,中间无横隔;游动孢子囊多呈圆筒形、棍棒形或穗状,游动孢子发育成熟后不断从孢子囊顶端释放出来,成团聚集在游动孢子囊口,并经过一个时期的静休后,成团脱落或直接分散在水中游动;次生孢子囊具有典型的侧生现象;藏卵器呈球形或梨形,大多与雄器异枝,少数与雄器同枝,含1?15个卵孢子;成熟卵孢子中生或亚中生,偏生一个大油球。其ITS rDNA序列与GenBank基因库中绵霉属菌株自然聚类,同源性高达99%,与异丝绵霉(Achlya klebsiana)菌株CBS101.49(GenBank登录号AF119579)的亲缘关系最近。结合形态特征与ITS序列鉴定的结果,判定菌株YC为异丝绵霉(Achlya klebsiana)。此外,在实验选用的中草药和消毒剂中,黄连和异噻唑啉酮分别对菌株YC的抑菌效果最好,其对菌株YC的最小抑菌浓度分别为256 mg/L和2 mg/L。【结论】首次分离了黄颡鱼卵致病性异丝绵霉菌株YC,并确定了其药敏特性,可以作为该病防治用药的依据。     

黄颡鱼暴发性出血病病原菌的分离鉴定及其生物膜形成特性

【背景】安徽省当涂县某池塘养殖黄颡鱼发生暴发性出血病,而当前对该病的病原存在争议。【目的】确定引起黄颡鱼暴发性出血病的病原菌,并明确分离菌株的生物膜形成特性,为从抗生物膜形成角度防治病原菌感染提供参考。【方法】取濒死期黄颡鱼病变脏器分别接种EPC细胞与培养基(TSB琼脂平板和血琼脂平板)分离病原,并通过人工感染回归试验确定其致病性;采用表型鉴定与16S rRNA基因序列分析相结合的方法鉴定分离菌株,并对其生物膜形成最佳条件、成膜能力及携带的生物膜形成相关基因进行研究。【结果】从病变脏器中分离纯化到一株优势菌株(HSY-2),对黄颡鱼的半数致死量为1.05×106 CFU/mL。经形态学、生化特性和细菌16S rRNA基因测序等分析确定分离株HSY-2为简达气单胞菌。其形成生物膜的最佳条件是将细菌接种TSB培养基于30 °C培养静置96 h,可形成中等强度的生物膜。同时,分离菌株携带气单胞菌甘油-3-磷酸脱氢酶D编码基因glpD、S-核糖同型半胱氨酸裂解酶基因luxS和LuxI家族蛋白同系物编码基因ahyI三种生物膜形成相关基因,但未检测到甘露糖敏感型血凝素菌毛合成蛋白Q编码基因。【结论】本实验为进一步研究简达气单胞菌生物膜形成的调控机制打下基础,并且从抗生物膜形成角度防治简达气单胞菌感染提供了参考。     

黄颡鱼仔稚鱼胃肠发育的显微和超微结构研究

利用光学显微技术和透射电镜技术,观察和研究了出膜后1-35日龄黄颡鱼(Pelteobagrus fulvidraco)仔稚鱼的胃肠发育.水温为23-25℃时,2日龄仔稚鱼的消化道分化出口咽腔、食道、胃、肠;3日龄肠道分化为前肠、中肠、后肠.3日龄黄颡鱼开口摄食时其胃贲门部黏膜层下出现胃腺,为已有鱼类研究报道中胃腺最早出现的日龄.超微结构显示3日龄胃腺细胞中可见胃蛋白酶原颗粒和丰富的管泡系统,为典型的泌酸胃酶细胞;随日龄增加,胃蛋白酶原颗粒越来越丰富而管泡系统越来越不明显.3日龄时前肠吸收细胞胞质中可见脂肪泡,后肠吸收细胞胞质中可见蛋白质胞饮体.直到25日龄后肠吸收细胞胞质中尚可见蛋白质胞饮体.以七结果表明黄颡鱼在3日龄开口摄食时消化道具备细胞外消化功能,但此功能不完善,期间继续通过胞饮作用等细胞内消化来弥补胞外消化的不足,直到25-30日龄后细胞外消化功能发育完善.采用符合其生理机能发育过程的投喂管理策略可以有效提高大规格苗种培育的成活率.     

Molecular characterization of ten zinc (Zn) transporter genes and their regulation to Zn metabolism in freshwater teleost yellow catfish Pelteobagrus fulvidraco

BackgroundZn is an essential trace element for vertebrates, and Zn uptake and transport is related with the ZIP family of Zn transporters. Meantime, Zn also influenced the expression of ZIP family members.MethodsWe cloned and characterized the full-length cDNA sequences of ten Zn transport-relevant genes (ZIP1, ZIP3, ZIP6, ZIP7, ZIP8, ZIP9, ZIP10, ZIP11, ZIP13 and ZIP14) from yellow catfish Pelteobagrus fulvidraco, investigated their mRNA tissue expression. These ZIP mRNA expression was also assessed in the primary hepatocytes and intestinal epithelial cells of yellow catfish in response to three Zn levels (0, 30 μM and 60 μM, respectively).ResultsAll these genes shared the similar domains with the corresponding members in mammals. The mRNA expression of the ten ZIP genes was detected in nine-tested tissues, but variable among these tissues. Flow cytometry analysis and confocal microscopy observation indicated that intracellular free Zn²⁺ concentration in hepatocytes and intestinal epithelial cells increased with increasing Zn incubation concentration at both 24 h and 48 h. Zn incubation differentially influenced mRNA levels of ZIP transporters in the hepatocytes and intestinal epithelial cells, in a time- and cells-dependent manners. In the hepatocytes, at 24 h, compared to the control, Zn addition down-regulated mRNA levels of ZIP1, ZIP3, ZIP6, ZIP7, ZIP8, ZIP9, ZIP11 and ZIP14; however, ZIP10 mRNA levels were lower in 60 μM Zn group than those in the control and 30 μM Zn group. At 48 h, mRNA levels of ZIP1, ZIP6, ZIP7, ZIP9, ZIP10 and ZIP14 declined with increasing Zn incubation concentrations; ZIP3 mRNA levels were the lowest in 60 μM Zn group and showed no significant differences between the control and 30 μM Zn group. In the intestinal epithelial cells, at 24 h, Zn addition down-regulated mRNA levels of ZIP1, ZIP6, ZIP7, ZIP8, ZIP9, ZIP10, ZIP11, ZIP13 and ZIP14; ZIP3 mRNA levels were lower in 60 μM Zn group than those in the control and 30 μM Zn group. At 48 h, Zn addition up-regulated mRNA levels of ZIP6 and ZIP9, but down-regulated mRNA levels of ZIP8, ZIP10 and ZIP13. ZIP7, ZIP11 and ZIP14 mRNA abundances were the lowest in 60 μM Zn group and showed no significant differences between the control and 30 μM Zn group.ConclusionFor the first time, our study characterized ten ZIP family members in yellow catfish, explored their mRNA tissue expression. Their regulation to Zn addition were also investigated in the hepatocytes and intestinal epithelial cells of yellow catfish. Our study revealed the mechanism of cells exposed to Zn addition and provided novel insights for the regulatory mechanism of Zn homeostasis.     

Isolation and identification of pelteobagrin, a novel antimicrobial peptide from the skin mucus of yellow catfish (Pelteobagrus fulvidraco)

Fish skin mucus has recently been recognized to be a potential source of antimicrobial peptides, which provides the first line of defense against invading pathogens. This study reports the purification and characterization of a novel linear antimicrobial peptide, pelteobagrin, from the skin mucus of yellow catfish (Pelteobagrus fulvidraco, Richardson). Pelteobagrin is 20 amino acids in length (GKLNLFLSRLEILKLFVGAL) and shows no clear homology with any known bioactive peptides. MALDI-TOF MS indicated the molecular mass of the purified peptide was 2244.4 Da, which is in good agreement with pelteobagrin's predicted molecular weight of 2244.8 Da. Pelteobagrin exhibited antibacterial activity against Gram-positive and Gram-negative bacteria as well as fungi, and the activity was relatively salt-insensitive as it was not affected by NaCl concentrations of up to 137 mM. Moreover, pelteobagrin displayed no hemolytic activity to rabbit red blood cells. Transmission electron microscopy suggested that pelteobagrin might kill bacteria via acting on both the cell wall and the cytoplasmic membrane of bacteria. These results suggest that pelteobagrin might be involved in the innate defense system in yellow catfish.     

黄颡鱼卵水霉病

黄颡鱼是我国重要的淡水名优鱼类之一,近年其养殖发展迅速,已经形成重要的产业[1]。由于黄颡鱼的苗种繁育阶段其受精卵极易患水霉病,严重制约黄颡鱼的苗种规模化生产。开展黄颡鱼受精卵水霉病的病原学、病原生物学特性以及控制方法研究具有较为重要的理论意义与实际应用价值。     

Molecular characterization,tissue distribution and kinetic analysis of carnitine palmitoyltransferase I in juvenile yellow catfish Pelteobagrus fulvidraco

Up to date, only limited information is available on genetically and functionally different isoforms of CPT I enzyme in fish. In the study, molecular characterization and their tissue expression profile of three CPT Iα isoforms (CPT Iα1a, CPT Iα1b and CPT Iα2a) and a CPT Iβ isoform from yellow catfish Pelteobagrus fulvidraco is determined. The activities and kinetic features of CPT I from several tissues have also been analyzed. The four CPT I isoforms in yellow catfish present distinct differences in amino acid sequences and structure. They are widely expressed in liver, heart, white muscle, spleen, intestine and mesenteric adipose tissue of yellow catfish at the mRNA level, but with the varying levels. CPT I activity and kinetics show tissue-specific differences stemming from co-expression of different isoforms, indicating more complex pathways of lipid utilization in fish than in mammals, allowing for precise control of lipid oxidation in individual tissue.     

Effects of water temperature and initial weight on growth,digestion and energy budget of yellow catfish Pelteobagrus fulvidraco (Richardson, 1846)

The effects of water temperature and body weight on feeding, growth, and energy budget were inevitable in the yellow catfish Pelteobagrus fulvidraco (Richardson, 1846), an important fish cultivated in China. This study explores the interaction of water temperature and body weight on both energy utilization strategy and energy conversion efficiency to promote further healthy culture of yellow catfish. Fish with body weights of 6 g (Group S), 16 g (Group M) and 35 g (Group B) were reared in 15 circular glass steel cylinders 80 cm in diameter × 70 cm in height (180 L) at water temperatures of 21, 24, 27, 30 and 33°C (3 replicates for each temperature) for 42 days to investigate effects of water temperature and body weight on the feeding, growth, digestion and energy budget in yellow catfish. Results showed that the levels of dry matter, protein and energy in the body were significantly affected by water temperature (p < .05). Feeding, growth, feed conversion efficiency, digestion and energy allocation parameters were significantly related to both water temperature and body weight (p < .05). Yellow catfish had higher maximal food consumption (C_max), food intake rate, specific growth rate, food conversion efficiency, appear digestibility coefficient, and growth energy allocation (G) at 24–30°C, and optimal growth at a water temperature of 27°C. Two‐factor analysis of variance revealed that there was reciprocation of both water temperature and body weight on the above parameters. At the optimal temperature of 27°C, the value of energy for growth (G) was the highest, and the value of energy for feces (F) produced was the lowest. Yellow catfish with various body weights had energy budget equations of 100 A = 63.70 R + 36.30 G in Group S, 100 A = 62.54 R + 37.46 G in Group M, and 100 A = 67.47 R + 32.53 G in Group B if the equations were described as percentage of the proportion of the assimilation energy. Therefore, the optimal temperature was 27°C according to its feeding, growth and digestion.     

Effects of dietary whole and defatted Arthrospira platensis (Cyanobacterium) on growth,body composition and pigmentation of the yellow catfish Pelteobagrus fulvidraco

The cyanobacterium Arthrospira platensis is regarded as a potential future source of food for humans due to its superior nutritional value compared with many conventional crops. An 8-week feeding trial was conducted to evaluate nutritional and pigmentation effects of whole A. platensis, defatted A. platensis and its lipid extracts in diets for the yellow catfish Pelteobagrus fulvidraco. Four percent whole A. platensis (AP4), 0.4% A. platensis lipid extracts (AL0.4), and 4% defatted A. platensis (AR4) were used as experimental diets, and a diet without any A. platensis was as the control diet (AP0). The AP4 and AL0.4 groups had no effects on the growth performance and feed utilization in yellow catfish; however, the AR4 group was significantly lower than the AP0 group in terms of specific growth rate and feed efficiency (P < 0.05). AP4 and AL0.4 groups significantly increased the body protein of fish (P < 0.05). The skin color was significantly improved in the AP4 and AL0.4 groups, as well as the fish skin and plasma lutein contents (P < 0.05). Correlations between skin color and the lutein contents in liver and muscles were weak, whereas the correlations between skin color and the lutein contents in skin and plasma were significant and strong. The results suggest that whole A. platensis and its lipid extracts can be functional additives to improve whole body protein and skin color of fish, while defatted A. platensis showed no advantage in yellow catfish diets.

     

Sex differences in the expression of GH/IGF axis genes underlie sexual size dimorphism in the yellow catfish (Pelteobagrus fulvidraco)

正Dear Editors,Sexual dimorphism is the systematic difference in size,shape,color,physiology,and behavior,between male and female individuals of the same species(Mei and Gui,2015).Some studies have indicated that the traits of sexual dimorphism in vertebrates are the consequences of sex-biased gene expression and are controlled by multiple critical genes during growth and development(Williams and Carroll,2009).However,the exact molecular mechanism underlying sexual dimorphism remains unclear.Sexual dimorphism with respect to size has been observed in many