Plasma citrulline and arginine kinetics in juvenile channel catfish, Ictalurus punctatus, given oral gabaculine

Experiments with young mammals have indicated that renal arginine synthesis is a major endogenous source of this amino acid. The kidney readily synthesizes arginine from intestinally derived citrulline, which is produced from glutamate, the so-called intestinal-renal axis. The present experiment was conducted to ascertain whether or not citrulline and arginine are synthesized in channel catfish via this pathway by administering gabaculine, an effective inhibitor of ornithine aminotransferase, and then monitoring circulating levels of arginine, citrulline, glutamate and ornithine. A conditioning diet was fed to apparent satiation to triplicate groups of channel catfish initially averaging 150 g/fish. After 2 weeks of acclimation, fish were lightly anesthetized with MS-222 and were intubated with a gelatin capsule containing gabaculine (3 mg/kg body weight) in one dose. Then, 24 and 48 h after gabaculine treatment, blood plasma was obtained and analyzed for amino acids via HPLC. Oral gabaculine administration significantly (p < 0.05) decreased plasma concentrations of citrulline (–41.8%) and arginine (–8.9%). It also increased (p < 0.05) plasma glutamine (+17.5%) and ornithine (+256.6%). A reasonable explanation for the increase in plasma glutamine and the decrease in citrulline and arginine is offered by the inhibition of glutamine degradation and a decrease in synthesis of citrulline caused by gabaculine. Results of this experiment suggest that endogenous synthesis of citrulline occurs in channel catfish, most likely at the small intestine, and it may serve as a de novo source of arginine. Therefore, the physiological needs of fish tissues and organs for arginine appear to be met via both dietary arginine and the endogenous synthesis of this amino acid.     

Amino Acid Profile of Various Body Tissues and Eggs of Discus Fish,Symphysodon aequifasciata

Abstract

Amino acid profiles of various body tissues from juvenile and adult discus fish, Symphysodon aequifasciata, were compared. Results from whole-body tissue revealed significant difference (P < 0.05) between the levels of lysine, phenylalanine, alanine, aspartic acid, and serine among the two different fish sizes. Levels of arginine, histidine, leucine, methionine, phenylalanine, and threonine were also higher in muscle tissue of adult fishes. Analysis of gill tissues showed significant difference in levels of histidine, isoleucine, lysine, methionine, threonine, tryptophan, aspartic acid, glutamic acid, proline, and serine were significantly different among the different fish stages. This study also showed that among all ten essential amino acids, muscle had the highest levels of histidine, isoleucine, lysine, and tryptophan; gills had the highest level of arginine; while levels of methionine and threonine were higher in the intestinal tissue. Regression analysis between the calculated whole-body A/E ratio of discus juveniles and with quantitative dietary amino acid requirements of several fish species also showed significant correlation, with highest r values (> 0.90) obtained with tilapia, common carp, channel catfish, and Japanese eel. This profile may be useful as a preliminary reference index to evaluate suitability of various ingredients in terms of amino acid profile for development of economical diet for discus farming.     

Enhanced Disease Resistance to Enteric Speticemia in Channel Catfish,Ictalurus punctatus,Administered Lytic Peptide

Lytic peptides have been shown in vitro to be toxic to a wide range of fish bacterial pathogens, including Edwardsiella ictaluri, the causative agent for enteric septicemia. Fingerling channel catfish, Ictalurus punctatus, were challenged with an injection of Edwardsiella ictaluri, and lytic peptide was administered with a single intraperitoneal injection 24 hours later. In a second group, lyptic peptide was also administered by osmotic pump 7 days before the bacterial challenge. A range of peptide concentrations (20-400µg/g fish) was used. Mortalities were recorded for 7 days after injection of bacteria, and E. icraluri was isolated and quantified by standard plate counts from livers. Various tissues were examined for abnormalities due to bacteria and peptide treatment. Fish injected with lytic peptide at 400 µg/g had up to a 95% reduction in bacteria and a 65% reduction in mortality compared to complete mortality in bacteria-injected controls. Fish implanted with osmotic pumps had 99.3% reduction in bacterial counts and had 6% mortality. Tissue samples from infected fish showed changes typical of reaction to immunologic stimuli and infection, while control fish injected with saline or peptide had normal gills, spleens, and kidneys. This study shows the potential for enhancing channel catfish disease resistance to E. ictaluri and other diseases by transferring the gene coding for the lytic peptide into channel catfish.     

Branchiomyces infection in farm-reared channel catfish, Ictalurus punctatus (Rafinesque)

Four cases (representing outbreaks in four different ponds on three farms) of branchial mycosis caused by Branchiomyces spp. were identified in channel catfish fry during the summer of 1996. Mortalities ranged from a few hundred to several thousand fish per pond. Significant gross and histopathological findings from these four cases were limited to the gills. All fry examined had fungal mycelia that were mainly but not entirely confined to the base of the primary lamellae and the gill arches. These fungal hyphae were intravascular and occluded vessels in the gill tissues. The present paper describes the fungal characteristics and pathology of branchial mycosis caused by Branchiomyces spp. in channel catfish.     

Characterization of nonautonomous Tc1-like transposable elements of channel catfish (Ictalurus punctatus)

Putative nonautonomous transposable elements from channel catfish (Ictalurus punctatus) were identified. They were named Tipnon elements for Tc1-like transposable elements from channel catfish that are nonautonomous. These elements were defined by their terminal repeats that share identity to members of the known Tc1/mariner transposon superfamily. They show structural similarities to Tc1-like elements, but share little sequence identity beyond the terminal inverted repeats. They do not harbor any amino acid blocks that show similarities to the Tc1-like or other transposases and thus may represent truly nonautonomous transposons in channel catfish. They are abundant in the channel catfish genome with a copy number of 32000, having 500 base pair per copy, this family of nonautonomous transposon-like elements account for 1.6% of the channel catfish genomic DNA. Their high abundance and transposon-like terminal repeats indicate that they may play important roles in gene evolution and in genomic architecture of catfish. Similarity search for potential coding capacity of the Tipnon elements revealed that they contain sequence blocks that can potentially encode amino acid blocks similar to the para-type sodium channel proteins in cockroaches or house flies, proteins that function in the central nervous system as voltage-gated sodium transporters. Sequences surrounding the terminal inverted repeats are divergent from those used by the reconstructed Sleeping Beauty fish transposase.     

Evaluation of Experimental and Practical Diets for Bluegill Lepomis macrochirus and Hybrid Bluegill L. cyanellus × L. macrochirus

Two experiments were conducted to identify appropriate experimental and practical diets for bluegill Lepomis macrochirus and hybrid bluegill L. cyanellus × L. macrochirus reared in aquaria. In the first study, four experimental diets and five commercial diets were evaluated in juvenile hybrid bluegill initially weighing 4.8 g/fish. The experimental diets contained casein (CAS), casein + gelatin (CAWGEL), casein + L‐arginine‐HCI (CAS/ARG), or casein + gelatin + crystalline amino acids (CAS/AA) as sources of crude protein. The commercial diets included three diets formulated to meet the nutritional requirements of rainbow trout Oncorhynchus mykiss and two diets formulated to meet the nutritional requirements of channel catfish Ictalurus punctatus. Each diet was fed twice daily at a rate of 4% of body weigh/d to triplicate groups of fish. At the end of the 8‐wk feeding trial, weight gain was significantly higher in fish fed the best commercial rainbow trout diets (205–217%) compared to fish fed diets formulated for channel catfish (87–104%). Weight gain and feed efficiency (FE) of fish fed the experimental diets (5346% and 0.19–0.32, respectively) were significantly lower than those of fish fed the commercial rainbow trout diets (143–217% and 0.49–0.64, respectively). In the second experiment, the same dietary treatments were fed for 8 wk to juvenile bluegill initially weighing 3.7 g/fish. Each diet was fed twice daily at a rate of 4% of body weight/d to triplicate groups of fish. Weight gain was significantly higher in fish fed commercial trout diets (291–402%) compared to fish fed diets formulated for channel catfish (164–191%). Weight gain and FE of fish fed CAS/ARG were significantly higher than those of fish fed the commercial catfish diets, but significantly lower than those of fish fed the best commercial trout diets. Results of this study indicate that commercial rainbow trout diets are preferable to commercial channel catfish diets for culture of bluegill and hybrid bluegill. More research is needed to identify appropriate experimental diets for this group of fishes.     

斜带石斑鱼IgM、IgZ和IgD重链基因的克隆

应用RACE方法获得斜带石斑鱼膜结合型免疫球蛋白M(membrane-bound immu-noglobulin M,mIgM),膜结合型免疫球蛋白D(mIgD),分泌型免疫球蛋白Z(secretory immu-noglobulin Z,sIgZ)的重链基因。斜带石斑鱼膜结合型IgM重链恒定区包含3个恒定区结构域(μ1,μ2,μ3)以及两个跨膜外显子(TM1,TM2),TM1外显子与μ3结构域末端相连接。氨基酸序列相似性分析结果显示,斜带石斑鱼mIgM各恒定区与牙鲆mIgM恒定区相似性最高,为53%~78%。mIgD的cDNA全长为3 375 bp,开放阅读框包含3 006 bp,其恒定区由1个μ1外显子,7个δ外显子以及跨膜区组成。斜带石斑鱼IgD恒定区与鳜IgD各恒定区氨基酸序列相似性最高,δ1~δ7的相似性分别为75.5%、75.8%、65.4%、76.6%、88.1%、90.6%、82.8%,TM结构域为82.7%。sIgZ的基因结构与其他硬骨鱼类sIgZ的结构相似,包括4个外显子和3个内含子,内含子长度分别为222、129和458 bp。利用半定量PCR分别检测了这3种基因在斜带石斑鱼各器官/组织中的表达,发现mIgM在头肾、肾脏、脑、脾脏、肠、鳃、心脏和胸腺中均有表达;mIgD的mRNA在头肾、肾脏以及胸腺中有较高的表达,在肠中表达量较低;sIgZ mRNA主要分布于淋巴组织如头肾、肾及脾脏中,而在鳃、心脏和胸腺中的丰度较低。     

Detection of channel catfish virus in channel catfish,Ictalurus punctatus (Rafinesque): use of a nucleic acid probe*

A nucleic acid probe for channel catfish virus (CCV) was constructed using recombinant DNA techniques. This probe consisted of a specific viral DNA fragment generated by digestion of CCV DNA with the restriction enzyme EcoRI. The probe was used to examine DNA isolated from tissues of fish that had been injected with CCV. Viral DNA was detected in some tissues of various injected fish. The sensitivity limit of detection was determined to be one viral DNA per cell.     

The amino acid and fatty acid compositions of selected tissues of the dolphin fish (Coryphaena hippurus) and their nutritional implications

The amino acid and fatty acid compositions of muscle, gill, intestine, liver, ovaries, and whole bodies from captive juvenile and adult, and wild adult dolphin fish (Coryphaena hippurus) were determined. Essential amino acid ratios (A/E ratios) of the whole body and individual tissues were calculated and compared. In addition, the concept of a metabolic A/E ratio calculated from known daily protein turnover rates and from the amino acid profiles is introduced. Fatty acid profiles of phospholipid (PL) and triglyceride (TG) fractions in muscle and liver were also compared.

There were few differences in the amino acid and fatty acid profiles of tissues between the different groups of dolphin fish examined. Linolenic series fatty acids were quantitatively the most important polyunsaturates in tissue PL fractions. C22: 6n−3 was the major polyunsaturate present, accounting for approximately 20% of the total PLs in muscle and liver. There was no significant difference in tissue essential amino acid patterns between adult fish raised in captivity, and adult fish caught in the wild. However, the muscle of juvenile fish was lower in arginine and histidine contents than the muscle of adult fish. Ovaries had the lowest arginine, histidine, and tryptophan contents of all tissues measured.

Calculated A/E ratios of the muscle and whole body, and A/E ratios based on the metabolic rates for protein turnover in muscle, gill, and intestine, were similar. All ratios predicted an ontological increase in dietary arginine, histidine, and tryptophan needs of dolphin fish consistent with the changes noted in tissue amino acid contents.

It is concluded that the calculated A/E ratios and fatty acid profiles of dolphin fish tissues may be useful guides in formulating diets for the culture of this species. The results obtained suggest that the requirements for arginine, histidine, and tryptophan may differ between subadult and adult fish. Dietary needs for linolenic series HUFAs, especially C22:6n−3, are likely high throughout the life of this fish.     

Histologic changes in channel catfish,Ictalurus punctatus Rafinesque,fed diets containing graded levels of gossypol–acetic acid

A study was performed to evaluate the histologic changes among fingerling channel catfish, Ictalurus punctatus Rafinesque, fed purified diets containing gossypol from gossypol–acetic acid. The catfish were maintained on diets with 0, 300, 600, 900, 1200 or 1500 mg gossypol kg^?1 diet for 12 weeks, and histologic samples from the stomach, proximal and distal intestines, pancreas, liver, and spleen were obtained from fish in all groups. Stomach sections exhibited significant gastric gland necrosis in fish fed 600 mg gossypol kg^?1 diet or higher. Non‐existent‐to‐mild enterocyte vacuolization loss, inflammatory cell infiltration, and hyperplastic lamina propria were noted in the intestinal sections from gossypol‐fed fish, but no significant differences in severity scores were noted. The pancreas from fish fed doses of gossypol above 600 mg gossypol kg^?1 diet exhibited significant mild‐to‐severe necrosis, and livers from fish fed 900 and 1500 mg gossypol kg^?1 diet exhibited significantly higher pigment deposition. No other significant histologic differences were observed in the fish fed diets containing gossypol–acetic acid. The data in this study indicates that at least 600–900 mg gossypol kg^?1 purified diet can cause statistically significant histologic changes in fingerling channel catfish, suggesting that gossypol should remain at concentrations below 600 mg gossypol kg^?1 diet.     

Comparative challenge model of Flavobacterium columnare using abraded and unabraded channel catfish, Ictalurus punctatus (Rafinesque)

The early entry of the fish pathogen Flavobacterium columnare and enhancement by abrasion was studied in channel catfish, Ictalurus punctatus (Rafinesque), using the polymerase chain reaction and a species-specific primer set for a bacterial 16S rRNA gene product. Evaluations were conducted following an abrasion bath immersion challenge with F. columnare. Abrasion, a practice which has historically been used prior to bacterial challenge, had significant effects on the early entry of the pathogen and on cumulative percent survival (CPS). The FvpF1-FvpR1 primer set was useful in detecting the early entry of F. columnare in mucus, skin, gill, blood, liver and trunk kidney tissues in both abraded and unabraded fish following immersion challenge at 29 +/- 2 degrees C. Bacteria were detected earlier in all tissues in abraded fish, except in the trunk kidney. These differences were not significant, except in the case of blood. Mucus, skin and gill tissues were positive for F. columnare earliest regardless of treatment (after 5 min in abraded fish and after 15 min in unabraded fish). CPS following challenge with F. columnare was significantly affected by abrasion, which supports the use of abrasion for the F. columnare challenge model for channel catfish.     

Molecular cloning and sequencing of hemoglobin-β gene of channel catfish, Ictalurus punctatus Rafinesque

The hemoglobin-β gene of channel catfish, Ictalurus punctatus, was cloned and sequenced. Total RNA from head kidneys was isolated, reverse transcribed and amplified. The sequence of the channel catfish hemoglobin-β gene consists of 600 nucleotides. Analysis of the nucleotide sequence reveals one open reading frame and 5′- as well as 3′-untranslated regions. The open reading frame of the sequence potentially encodes 148 amino acids with a calculated molecular mass of 16.3 kDa. The pI and charge at pH 7.0 of the deduced hemoglobin-β protein were 7.28 and 0.47, respectively. Overall, 22 amino acid residues were conserved throughout the sequences, including His64 and His93, the sites for heme-binding. Unlike the counterpart of other common cultured fish such as Salmo salar, Oncorhynchus nerka, Oncorhynchus mykiss, Cyprinus carpio and Ctenopharyngodon idella, the hemoglobin-β of channel catfish did not have cysteine. The amino acid sequence of channel catfish hemoglobin-β shows 84% homology with that of Silurus asotus (both are in the order Siluriformes). However, comparison with those of other fish species shows homology ranging from 53 to 68%. Structural analysis by the 3D-PSSM program displays that channel catfish hemoglobin-β has eight α-helices, A–H.     

半滑舌鳎(Cynoglossus semilaevis) Nramp基因克隆与表达分析及SNP筛选

天然抗性相关巨噬细胞蛋白(Natural resistance-associated macrophage protein, Nramp)属于膜整合转运蛋白,具有抑制胞内寄生菌侵染、调节巨噬细胞的抗菌活性等作用。本研究对半滑舌鳎(Cynoglossus semilaevis)Nramp基因进行了克隆和表达分析,并对其与抗鳗弧菌感染相关的单核苷酸多态性(Single Nucleotide Polymorphism, SNP)位点进行了筛选。该基因cDNA序列全长3717 bp,其中开放阅读框(Open reading frame, ORF)1677 bp,所编码蛋白含有558个氨基酸,该蛋白具有Nramp家族的典型特征,包括10个跨膜区(Transmembrane, TM)、1个由20个氨基酸残基组成的胞质内转运蛋白特征结构域(Consensus Transport Motif, CTM)。半滑舌鳎Nramp的ORF末端有1个类似于脊椎动物Nramp2中的铁反应控制蛋白结合位点(Iron-responsive regulatory protein-binding site, IRE)。半滑舌鳎Nramp与其他14个物种的Nramp氨基酸序列同源性在63%?91%之间,系统进化分析表明,半滑舌鳎Nramp和所有鱼类Nramp聚集为一簇,与其他物种Nramp2的亲缘关系较近。实时荧光定量PCR分析显示,Nramp基因在半滑舌鳎脾脏和肾脏中的表达量最高,而在肌肉和性腺中的表达量最低;在哈维氏弧菌感染的半滑舌鳎肾脏、脾脏和肝脏中表达量呈升高趋势,而在鳃中则表现为下调趋势。利用直接测序法检测感染鳗弧菌后同一家系的233个个体(抗病个体165个,感病个体68个),共检测到15个SNP位点,对其中3个SNP 位点即 SNP-g.3113(T→C)、SNP-g.3125(A→G)和 SNP-g.3164(A→T)进行测序分型后发现,SNP- g.3125(A→G)的等位基因(G)频率和基因型(GG)频率与半滑舌鳎抗鳗弧菌疾病呈极显著相关(P<0.01)。研究结果表明,Nramp 基因不同基因型对半滑舌鳎的抗病能力有着极其重要的影响,SNP-g.3125(A→G)可作为潜在的抗性遗传标记位点。本研究将为半滑舌鳎抗性品系培育提供技术支持。     

斑点叉尾鮰病毒囊膜蛋白ORF6在昆虫细胞中的表达

为构建基于杆状病毒表达系统的CCV新型亚单位疫苗,将CCV的囊膜蛋白基因ORF6克隆至杆状病毒转座载体pFastBacTM 1质粒中,并将阳性重组转座质粒转化进含穿梭载体Bacmid的感受态细胞DH10Bac中,获得重组子rBacmid-ORF6。在脂质体介导下将该重组子转染sf9昆虫细胞,获得重组杆状病毒AC-ORF6。AC-ORF6感染的sf9昆虫细胞,经超薄切片电镜观察,可见重组杆状病毒呈多粒包埋,经间接免疫荧光、Western-blotting检测,CCV的ORF6蛋白可以在感染了AC-ORF6的sf9细胞中表达。研究表明,获得了插入ORF6基因的重组杆状病毒,并且该基因可以在重组杆状病毒介导下在昆虫细胞中表达,从而为基于CCV ORF6的杆状病毒亚单位疫苗研究奠定了基础。     

Digestibility of Alternative Protein‐Rich Feedstuffs for Channel Catfish,Ictalurus punctatus

Apparent digestibility coefficients (ADCs) of amino acids, crude protein, gross energy, and dry matter of canola meal, corn gluten feed, fish offal meal, shrimp and fish offal meal, poultry by‐product meal, and hydrolyzed feather meal were determined for channel catfish, Ictalurus punctatus. Experimental diets contained 30% test ingredient, 69.5% casein‐based reference diet, and 0.5% chromic oxide. Groups of 20 fish (102.45 g) were fed the experimental diets twice a day at 3.5% body weight. Fecal samples were collected in triplicate daily at 0000 h and 0600 h after settlement into collection devices. Shrimp and fish offal meal and corn gluten feed presented significantly lower (P < 0.05) ADC for dry matter (59.5 and 39.3%) suggesting their low value as feedstuffs for catfish feeds. Although ADC values for crude protein were above 80% for all the test ingredients, amino acid digestibility varied significantly, except for histidine whose ADC remained constant regardless of the protein source. Lysine was the first limiting amino acid in most of the test ingredients, except in fish offal meal and poultry by‐product meal. Among the protein sources tested, only fish offal meal and poultry by‐product meal met channel catfish amino acid requirements for a 28% digestible protein grow‐out diet.     

Estimating Amino Acid Requirement of Brazilian Freshwater Fish from Muscle Amino Acid Profile

Information on nutritional requirement of some Brazilian farmed fish species, especially essential amino acids (EAA) requirements, is scarce. The estimation of amino acids requirements based on amino acid composition of fish is a fast and reliable alternative. Matrinxa, Brycon amazonicus, and curimbata, Prochilodus lineatus, are two important Brazilian fish with potential for aquaculture. The objective of the present study was to estimate amino acid requirements of these species and analyze similarities among amino acid composition of different fish species by cluster analysis. To estimate amino acid requirement, the following formula was used: amino acid requirement = [(amount of an individual amino acid in fish muscle tissue) × (average totalEAA requirement among channel catfish, Ictalurus punctatus, Nile tilapia, Oreochromis niloticus, and common carp, Cyprinus carpio)]/(average fish muscle totalEAA). Most values found lie within the range of requirements determined for other omnivorous fish species, in exception of leucine requirement estimated for both species, and arginine requirement estimated for matrinxa alone. Rather than writing off the need for regular dose–response assays under the ideal protein concept to determine EAA requirements of curimbata and matrinxa, results set solid base for the study of tropical species dietary amino acids requirements.