In vivo fish models for visualizing Aeromonas hydrophila invasion pathway using GFP as a biomarker

The invasion pathway of Aeromonas hydrophila in vivo was studied in Crucian carp (Carassius auratus gibelio) with a virulent strain A. hydrophila J-1 transformed with a plasmid encoding green fluorescent protein (pGFPuv) (A. h J-1^GFP), which had similar virulence characteristics (haemolysin, extracellular proteases production, toxicity for EPC cells, survival in fish serum and LD₅₀ value) as the parent strain. Fish were divided into four experimental groups: (1) normal fish; (2) bacteria bath challenged, unwounded fish; (3) skin artificially wounded by scalpel, bacterial bath challenged fish; and (4) skin mucus layer partially removed by paper towel, bacterial bath challenged fish. The number of bacteria from blood, gills, kidney, muscle, liver and intestine were detected at 2, 4, 8, 12, and 24 h post-challenge. High bacterial numbers were observed in the muscle of the artificial wound group, in the kidney of the mucus removed group and in the gills of all groups. In conclusion, the gills and damaged skin are likely to be the main routes of entry for A. hydrophila, and GFP can be used as a real-time biomarker to study intimate host–pathogen interaction in fish.     

A novel fluorescent protein purified from eel muscle

We discovered that some isolated eel skeletal muscle cells exhibited green fluorescence under a fluorescence stereomicroscope, and we successfully isolated a novel fluorescent protein from the eel muscle homogenate. The protein was a monomer with a molecular mass of 16.5–17 kDa and showed minor and major peaks at 280 and 493 nm, respectively, in the absorption spectrum. The molar extinction coefficient at 493 nm was 41,300 M⁻¹ cm⁻¹ and A280/A493 was 0.083. Excitation and emission spectra of the protein showed maxima at 493 and 527 nm, respectively. Heat treatment at 95°C for 10 min or 5% trichloroacetic acid treatment of the protein caused aggregation of the protein but did not release any fluorescent components such as FAD into the supernatant after centrifugation. Fluorescence of the protein remained after native PAGE, but not after SDS-PAGE. These results indicate that the purified fluorescent protein is not a flavoprotein, and that its fluorescent chromophore is a covalently bound one, such as green fluorescent protein (GFP) from jellyfish Aequoria victoria, but that its fluorescence requires its native conformation within the protein. Based on these results, we can conclude that the fluorescent protein obtained from eel skeletal muscle is a novel GFP-like protein.     

Three‐dimensional visualization of green fluorescence protein‐labelled Edwardsiella tarda in whole Medaka larvae

The invasive fish pathogen Edwardsiella tarda is common in aquatic environments and causes the environmentally and economically destructive emphysematous putrefactive disease called edwardsiellosis. In order to understand the organism's infection pathway, medaka larvae (Oryzias latipes) were immersion‐infected with E. tarda labelled with green fluorescence protein (GFP) and then visualized in three dimensions under confocal laser microscopy and light‐sheet fluorescence microscopy. Confocal microscopy revealed GFP‐labelled E. tarda in the mouth, head, gill bridges, gill cover, skin, membrane fin, gastrointestinal tract and air bladder, and in the caudal vein, somite veins, caudal artery and caudal capillaries. Light‐sheet microscopy additionally showed GFP‐labelled E. tarda in the pharyngeal cavity, muscle of the pectoral fin and cardiac atrium and ventricle. These findings suggest that during its infection of fish, E. tarda initially adheres to, and invades, the epithelial cells of the skin, gills and gastrointestinal tract (through the pharyngeal cavity); E. tarda then enters the blood vessels to access organs, including the air bladder and heart.     

红色荧光蛋白基因在转基因唐鱼中遗传和表达的稳定性分析

为了评估转红色荧光蛋白(red fluorescent protein,RFP)基因唐鱼新品系的稳定性,研究了RFP基因在不同世代转基因唐鱼中的遗传和表达情况。荧光显微镜下观察显示,RFP基因在F6和F10代所检测的组织器官中均有表达,并且两个世代间相同组织部位的表达水平相似。F6和F10代个体分别配对繁殖实验表明,RFP基因在转基因唐鱼后代中的遗传仍然符合孟德尔分离规律,而且培育出的转基因个体表型特征无显著差异。利用PCR技术在F2、F6和F10代转基因唐鱼基因组中扩增外源性肌球蛋白轻链2启动子、RFP基因编码区和整合位点上下游侧翼区域(片段总长度为4 883 bp),测序结果显示,3个世代间的外源基因序列完全相同,没有发生碱基缺失或突变等现象。研究表明,红色荧光蛋白基因在转基因唐鱼传代培育过程中保持了稳定的遗传和表达。     

Rhabdomyosarcoma in the Japanese medaka, Oryzia latipes (Temminck & Schlegel) and guppy, Poecilia reticulata Peters

Abstract. Three cases of skeletal muscle neoplasms occurring in two small fish species used in carcinogen tests are reported. The cases illustrated a wide range of histologic patterns, and consisted of a well-differentiated juvenile type, a well-differentiated pleomorphic type and a poorly differentiated pleomorphic type. A rhabdomyosarcoma with juvenile type features developed in the Japanese medaka, Oryzias latipes , exposed to 0·5 ml/1 of the solvent dimethylformamide. A pleomorphic type rhabdomyosarcoma occurred in one medaka and in one guppy, Poecilia reticulata , exposed to the carcinogen methylazoxymethanol acetate. These cases indicate the potential for skeletal muscle cells in medaka and guppy to become neoplastic but, because the tumours occurred at a low frequency, there may not necessarily be a chemical aetiology.     

Effects of dietary protein and lipid levels on growth,body composition and flesh quality of juvenile topmouth culter,Culter alburnus Basilewsky

Nine experimental diets at three protein (35%, 40% and 45% crude protein) and lipid (5%, 8% and 11% crude lipid) levels with variable digestible protein to digestible energy (DP/DE) ratios ranged from 21.9 to 27.8 g protein MJ^?1 were fed to topmouth culter (Culter alburnus Basilewsky) fingerlings (initial weight 6.5 ± 0.9 g) in triplicated groups (30 fish per replicated) for a period of 10 week to assess the optimum dietary DP/DE ratio and the protein sparing effect by utilizing dietary lipid. 27 cages of 1.5 m³ capacity placed in a lake located in Wuhan were used for rearing the fish. At the end of the experiment, maximum weight gain and thermal‐unit growth coefficient was found in fish fed diet D4 with 45% protein, 8% lipid and P/E ratio of 26.2 g protein MJ^?1, but without a significant difference compared to fish fed diet D5 with 40% protein, 8% lipid and DP/DE ratio of 25.3 g protein MJ^?1. The best flesh quality evaluated by muscle collagen content was found in fish fed D5. High fat accumulation with increasing dietary lipid levels was observed in whole body but not in muscle tissue. Hence, it may be concluded that the optimum formulation for maximum growth and quality of topmouth culter is a diet containing 40% protein and 8% lipid with a resultant DP/DE ratio of 25.3 g protein MJ^?1. In addition, the protein sparing effect by inclusion lipid was observed but limited.     

Study on the glass transition for several processed fish muscles and its protein fractions using differential scanning calorimetry

ABSTRACT:   The glass transition behavior of processed fish muscles (bonito, tuna, mackerel, sea bream, cod) and its muscle protein fractions (sarcoplasmic and myofibrillar proteins) were studied using differential scanning calorimetry. Each dried processed fish muscle and the extracted protein fractions showed clear glass transition phenomenon. The T _g values of muscles and myofibrillar proteins from red muscle fishes tended to be lower than those from white muscle fishes though there was no difference on T _g of sarcoplasmic proteins. The T _g value of whole muscle was considerably lower than that of extracted protein fractions because of the plasticizing effects of low molecular weight materials contained in the muscle.     

Gene transfer for Japanese flounder fertilized eggs by particle gun bombardment

ABSTRACT:   Fish transgenesis has progressed considerably. However, the technique of gene transfer for most marine aquaculture species has not been established. A method to introduce foreign genes into fertilized eggs of Japanese flounder Paralichthys olivaceus by particle gun bombardment was developed by the authors. A recombinant plasmid which contains the Japanese flounder keratin gene promoter linked to the green fluorescence protein (GFP) gene was introduced into fertilized eggs by particle gun bombardments twice at 250 psi. In each experimental group, 25 000–35 000 eggs were treated. However, the survival rate was 12.8% which is lower than that of the control groups (56.8%). Of 2606–5205 the embryos survived for 24 h, 43–61 GFP positive embryos were obtained in one experiment, giving a final gene transfer efficiency of 1.4%. All GFP-positive embryos developed and hatched normally. GFP expression was observed in epithelial tissue throughout the developmental stages. At 3 months after gene transfer, foreign DNA was detected by genome polymerase chain reaction analysis in 37 of 69 fry (53.6%). These results suggest that the particle gun method is an effective method to use with fertilized Japanese flounder eggs.     

Characterization of grouper nervous necrosis virus (GNNV)

Grouper nervous necrosis virus (GNNV) was isolated from moribund grouper larvae, Epinephelus sp., using a fish cell line GF-1. The present study describes the biochemical and biophysical properties of GNNV and the expression of GNNV in diseased grouper larvae. Viral protein was detectable in most of the GNNV-infected GF-1 cells by the fluorescent antibody technique (FAT) after 12 h post-infection (p.i.), although no cytopathic effect (CPE) appeared at that time. Clear CPE developed on the third day, and complete disintegration of the monolayer occurred over the subsequent two days. The infectivity of GNNV can be blocked following treatment at 60 °C for 1 h. GNNV was sensitive to pH 3 and pH 10–12 with a 4 log₁₀ drop in infectivity. Purified GNNV was analysed by SDS–PAGE, and then stained with periodic acid silver. The positive staining indicated that its two capsid proteins were glycoproteins. Genomic RNAs of GNNV were extracted from purified virions and analysed. The molecular weights of genomic RNAs were 1.02 × 10⁶ and 0.50 × 10⁶ Da. The T2 region of the coat protein gene of GNNV was amplified by polymerase chain reaction (PCR), and the multiple alignment of the T2 sequence of two GNNV isolates with four genotypes of fish nodaviruses revealed that these two isolates (GNNV9410 and GNNV9508) belong to the red-spotted grouper nervous necrosis virus (RGNNV) genotype. The tissue distribution of GNNV in naturally infected grouper larvae was investigated by in situ hybridization using a dig-labelled probe, which showed that GNNV was not only detected in the brain and retina, but also in the gill, skeletal muscle, liver, pyloric gland, intestine and blood cells in the heart.     

Qualitative and quantitative changes of F<Subscript>o</Subscript>F<Subscript>1</Subscript>-ATPase in Japanese flounder and red sea bream associated with rearing temperatures

ABSTRACT:   Fast skeletal muscles of Japanese flounder Paralichthys olivaceus and red sea bream Pagrus major were examined for quantitative and qualitative changes of mitochondrial ATP synthase (F_oF₁-ATPase) in association with rearing temperatures. The specific activities of F_oF₁-ATPase from Japanese flounder reared at 10°C, 15°C and 25°C for 4 weeks were determined to be 81 ± 11, 74 ± 13 and 83 ± 11 nmol/min·mg mitochondrial protein, respectively. The corresponding activity from red sea bream reared at 8°C for 5 weeks was determined to be 65 ± 9 nmol/min·mg mitochondrial protein, which was higher than 33 ± 9 nmol/min·mg mitochondrial protein in fish reared at 23°C. The contents of α- and β-F₁-ATPase in total mitochondrial proteins were not significantly different between fish reared at different temperatures for the two fish species. However, the contents of β-F₁-ATPase in the total fast skeletal muscle extracts, prepared from Japanese flounder reared at 10°C, were 2.1- and 2.9-fold higher than those for fish reared at 15°C and 25°C, respectively. The corresponding content from red seabream reared at 8°C was 2.2-fold higher than that for fish reared at 23°C. Therefore, the changes in F_oF₁-ATPase depending on rearing temperatures were species-specific.     

Effects of dietary protein and lipid levels on growth,feed utilization and body composition in red‐tailed catfish juveniles (Hemibagrus wyckioides,Chaux & Fang 1949)

A feeding trial was conducted in a recycling water system during 10 weeks to determine the optimal protein to lipid ratio in Asian red‐tailed catfish (Hemibagrus wyckioides). Six diets of two protein levels (390 and 440 g kg^?1) with three lipid levels (60, 90 and 120 g kg^?1) were formulated. Fish (1.96 g) were fed six diets with four replicates to apparent satiation at a stocking density of 50 fish per tank (500 L). Faeces were collected in cultured tanks at the end of the feeding trial for digestibility measurement. Significantly, improved growth performances (P < 0.01) and higher feed utilization (P < 0.001) were observed in fish fed with higher lipid diets. However, higher protein diets did not significantly improve fish growth but they reduced FCR (P < 0.001) and protein efficiency ratio (P < 0.01). Higher lipid diets also resulted in significantly increased adipose‐somatic index, carcass fat and reduced moisture of the fish. The study revealed the protein sparing effect of dietary lipid in the catfish and highest growth performance was found by fish fed 390 g kg^?1 protein and 120 g kg^?1 lipid diet with P/E ratio of 20.48 mg protein kJ^?1. DP/DE ratio for maximal growth rate in diets was 21.48 mg protein kJ^?1.     

The dietary protein requirement of a bagrid catfish, Mystus nemurus (Cuvier & Valenciennes), determined using semipurified diets of varying protein level

Triplicate groups of Mystus nemurus (Cuvier & Valenciennes) were fed isoenergetic semipurified diets containing seven dietary protein levels from 200 to 500 g kg^–1 diet for 10 weeks. Dietary protein was supplied by graded amounts of a protein mixture (tuna muscle meal:casein:gelatine) at a fixed ratio of 50:37.5:12.5. Mystus nemurus fingerlings of initial weight 7.6 ± 0.2 g were fed close to apparent satiation at 2.5% of their body weight per day in two equal feedings. Growth performance and feed utilization efficiency increased linearly with dietary protein level from 202 to 410 g kg^–1 diet and declined with protein levels of 471 g kg^–1 diet or above. Protein efficiency ratio and apparent net protein utilization started to decline when the fish were fed with dietary protein levels exceeding 471 g kg^–1 diet. Fish fed with lower protein diets (202–295 g kg^–1 diet) had significantly ( P  < 0.05) higher carcass lipid content compared with fish fed with higher protein diets. Carcass lipid contents were inversely related to moisture content. Dietary protein did not significantly affect fish carcass protein and ash content. Using two-slope broken-line analysis, the dietary protein requirement for M. nemurus based on percentage weight gain was estimated to be 440 g kg^–1 diet with a protein to energy ratio of 20 mg protein kJ^–1 gross energy. This level of protein in the diet is recommended for maximum growth of M. nemurus fingerlings weighing between 7 and 18 g under the experimental conditions used in this study.     

C-protein (MyBP-C) isoforms from carp ordinary and dark muscles and muscle type-specific binding to myosin

ABSTRACT:   C-protein is a myosin-associated protein of vertebrate striated muscle, and its function and properties have been extensively examined. However, there has been no report of C-protein of fish skeletal muscle so far. C-protein was identified in carp skeletal muscle by immunoassay using antibody against chicken C-protein, and the muscle-type specific C-protein was purified from carp ordinary and dark muscles for the first time. Although C-protein could be prepared from crude myosin by the reported procedure, C-protein degraded appreciably during the purification steps. Accordingly, C-protein was selectively extracted from the muscle with 0.15 M K-phosphate buffer (pH 5.8), and purified by ammonium sulfate fractionation, followed by AF-blue chromatography. Myosin free from the accessory proteins was obtained by diethylaminoethyl (DEAE) chromatography and used to assay the binding of C-protein with myosin. Ordinary muscle C-protein bound to ordinary muscle myosin in a saturable manner, but its maximum amount of binding was approximately twice that of dark muscle myosin. Similarly, dark muscle C-protein bound to dark muscle myosin much more than to ordinary muscle myosin. These results suggest that C-protein isoforms specifically bound with myosin isoforms originated from the same type of muscle.     

拟态弧菌的绿色荧光蛋白标记及其在感染草鱼体内的动态分布

为了示踪研究拟态弧菌感染草鱼的动态过程,将增强型绿色荧光蛋白编码基因EGFP克隆至质粒pBAD24,并转化到拟态弧菌04-14菌株构建荧光标记重组菌.重组菌经阿拉伯糖诱导后,能高效表达EGFP蛋白;荧光显微镜观察和流式细胞仪检测均发现重组菌能够发出明显的绿色荧光信号,且传至30代后质粒稳定率仍为100％;生物学特性检测结果显示,与野生株相比,重组菌的形态、生长特性和细胞黏附性均未发生明显改变.用标记重组菌浸泡感染草鱼,定点采集鳃、肠道、肌肉、头肾、脾脏和肝脏,借助荧光信号检测4d内细菌在不同组织脏器中的动态分布.结果发现感染4h后即可在肠道和鳃中检测到绿色荧光信号,标记菌检出量分别为3.60×108和2.36×106 CFU/g,直至10 h,其含量无明显变化,12 h后含菌量逐渐下降,但持续存在直至鱼死亡.标记菌在肌肉、头肾、脾脏和肝脏中呈现相似的动力学,感染24 h后才检测到荧光信号,24～ 85 h时间段含菌量呈现先增加后下降的变化,48 h达到峰值,检出量分别为9.58×104(肌肉)、8.75×104(头肾)、1.50×104(脾脏)和4.50×104 CFU/g(肝脏),但均低于肠道中的检出量,结果表明肠道是拟态弧菌黏附定植与繁殖的主要靶器官.     

绿色荧光蛋白基因重组子的构建及其在鱼类受精卵中的表达

通过体外重组，将绿色荧光蛋白基因编码序列克隆到鲤鱼肌动蛋白基因启动子下游，构建成能在鱼体内表达绿色荧光蛋白的重组分子。用限制性内切酶PvuI酶切线性化后，通过显微注射法导入金鱼受精卵内，在转化后48h，可观察到绿色荧光，经PCR初步筛选后，对显阳性个体进行Southern杂交检测，结果表明，转化个体表现出较强的杂交信号。这说明绿色荧光蛋白基因能在金鱼体内整合、表达。     

The use of green fluorescent protein as a marker for monitoring a probiotic Bacillus S11 in the black tiger shrimp Penaeus monodon

To monitor the probiotic Bacillus S11 (BS11) in vivo , wild-type cells were transformed with the green fluorescent protein (GFP)-expressing plasmid, pAD44-12, carrying the gfp mut3a gene under the constitutive Bacillus cereus UW85 promoter, and its non-GFP control plasmid pAD. Transformants with pAD44-12 (BS11-GFP), not pAD (BS11-pAD), expressed detectable but not too high levels of green fluorescence. Chloramphenicol resistance (BS11-GFP, BS11-pAD) and GFP fluorescence (BS11-GFP) as markers suggested that plasmid retention was 78–79% for both BS11-GFP and BS11-pAD cells after approximately 50 generations of growth without antibiotic selection. When mixed into shrimp feed at a final concentration ∼10⁵ CFU g⁻¹, the inclusion of viable transformed bacteria in fed shrimps was observed. After feeding shrimp three times daily in 400-L cement tanks for 9 weeks, no significant differences in the average shrimp weight, the number of BS11 in either the culture water or in the shrimp's gut were seen between shrimp fed BS11-GFP and BS11-pAD or BS11, suggesting that expression of the gfp mut3a gene has no detectable effect on BS11 properties and shrimp growth. Histological examination of sections of shrimp's intestines following feeding with BS11-GFP demonstrated that BS11-GFP in shrimp feed survived and adhered onto the shrimp intestines' surface. BS11-GFP thus has good potential as a non-invasive marker tag for short-term experiments.     

1H NMR‐based metabolomics studies on the effect of size‐fractionated fish protein hydrolysate,fish meal and plant protein in diet for juvenile turbot (Scophthalmus maximus L.)

This study was designed to evaluate changes in the metabolic profile of liver and muscle of turbot (Scophthalmus maximus L.) fed fishmeal‐based diet, diets containing size‐fractionated fish protein hydrolysate and plant protein‐based diet using ¹H NMR‐based metabolomics approach combined with the growth. Fish protein hydrolysate (FPH) was obtained by enzymatic treatment, permeate fraction was obtained as UF by ultrafiltered step, and retentate fraction was retained as RF. FM diet contained fish meal used as a single protein source. Four other diets (PP, UF, FPH and RF) contained 180 g kg^?1 diet fish meal. 54, 55 and 55 g kg^?1 dry diet UF, FPH and RF were supplemented to UF, FPH and RF diets. All diets were formulated to be isolipidic and isonitrogenous fed to five triplicate groups of turbot (16.05 ± 0.03 g) for 68 days. O‐PLS‐DA in FM versus UF, FM versus FPH, FM versus RF and FM versus PP resulted in a reliable model for muscle and liver tissue, while O‐PLS‐DA in UF versus FPH and UF versus RF only showed metabolites changes in liver tissue. Results indicated that metabolite changes among the different treatments were consistent with the growth tendency.     

Protein synthesis in tissues of fed and starved carp,acclimated to different temperatures

The temperature dependence of the rates of protein synthesis in the red and white skeletal muscle of the carp (Cyprinus carpio) was measured using a method which involved a single injection of tritiated phenylalanine. Plasma and muscle-free phenylalanine quickly reached a plateau level at all temperatures. During the plateau phase the incorporation of label into protein was liner. Muscle from fish previously acclimated to either a low temperature (8°C) or a high temperature (28°C), showed marked differences in the rates of protein synthesis. The results show that cold acclimation is associated with significantly higher rates of protein synthesis (p<0.001) in both red and white muscle. Arrhenius activation energies, derived from the rates of protein synthesis at the different experimental temperatures, were similar for both red and white muscle in fish acclimated to warm or cold temperatures. Measurements for both acclimated groups over the temperature range 8–34°C showed that the activation energy for the process of protein synthesis was 86.7 kJ/mol and 78.7 kJ/mol for the red and white muscle respectively.