多重RT-PCR体系检测4种虾病毒的方法

根据多重RT-PCR的技术原理,利用对虾传染性表皮与造血组织坏死症病毒、白斑综合征病毒、黄头病毒和桃拉综合征病毒的基因序列分别设计了4对特异引物,建立多重RT-PCR体系用于虾4种病毒的检测。多重RT-PCR体系能特异地扩增出IHHNV、WSSV、YHV和TSV的目的片段:TSV特异性扩增片段508 bp,WSSV 特异性扩增片段435 bp,IHHNV 特异性扩增片段301 bp 和YHV。特异性扩增片段614 bp。结果表明,多重PCR虾病毒检测系统具有较高的特异性和敏感性,并对其它对虾病原呈阴性。IHHNV、TSV、WSSV和YHV模板在多重PCR虾病毒检测体系中的检测下限分别为0.1,1,0.02和0.2 pg。病毒感染病料检测试验中,该检测体系的检测结果与单纯PCR的检测结果呈现出较好的吻合度。     

Edwardsiellosis in farmed rainbow trout (Oncorhynchus mykiss)

Edwardsiella tarda was isolated from naturally infected rainbow trout (Oncorhynchus mykiss) in raceway culture on a commercial fish farm where the fish were kept at a density of 110 kg m^?3 and at a water temperature of 14°C and with a photoperiod of 13 h light:11 h dark. The clinical signs of diseased fish (150 ± 20 mm standard length) were anorexia and lethargy. The most striking lesions in the fish were in the liver. There were hyperaemia and haemorrhages; on histopathological examination, the liver displayed inflammatory infiltrate in portal area, focal necrosis, dilatation of blood sinus and activation of sinusoidal cells. Infection experiments, performed 2 years after isolation of the original culture of E. tarda, were carried out under laboratory conditions at water temperatures of 15, 18 or 24°C. All experimental fish (common carp, Prussian carp, tench), intraperitoneally injected with 8 × 10⁶ cells, demonstrated a total resistance to E. tarda.     

Stocked rainbow trout (Oncorhynchus mykiss) affect space use by Warpaint Shiners (Luxilus coccogenis)

Due to widespread stocking, Rainbow Trout (Oncorhynchus mykiss Walbaum) are perhaps the most widely distributed invasive species in the world. Nonetheless, little is known about the effects of stocked Rainbow Trout on native non‐game species. We conducted experiments in an artificial stream to assess the effects of hatchery Rainbow Trout on home range and behaviour of Warpaint Shiners (Luxilus coccogenis Cope), a common minnow frequently found in stocked Southern Appalachian streams. We used the LoCoH algorithm to generate polygons describing the home ranges used by Warpaint Shiners. When a stocked trout was present Warpaint Shiners: (a) increased home range size by 57%, (b) were displaced into higher velocity mesohabitats, and (c) reduced mean overlap between the home ranges of individual warpaint shiners. Rainbow Trout did not significantly affect the edge/area ratio of Warpaint Shiner home ranges. Warpaint Shiner density (two and five fish treatments) did not significantly affect any response variable. Displacement from preferred microhabitats and increases in home range size likely result in increased energy expenditure and exposure to potential predators (i.e., decreased individual fitness) of Warpaint Shiners when stocked trout are present.     

Metabolism of exogenous histamine in rainbow trout (Oncorhynchus mykiss)

Information on the metabolism of exogenous histamine in fish is of much concern regarding the effect of dietary histamine on fish. Histamine catabolic enzymes, diamine oxidase and histamine N-methyl transferase were measured in the tissues of rainbow trout. Diamine oxidase was detected in the stomach, pylorus caeca and intestine. Histamine N-methyl transferase was detected only in the liver.A change in the contents of histamine and its metabolites was observed in the tissues of rainbow trout after oral administration of histamine. A large amount of imidazole acetic acid was observed in the serum, kidney, liver and muscle. On the other hand, 1-methyl histamine was detected only in the liver. Histamine and its metabolites, imidazole acetic acid and 1-methyl histamine were metabolized and diminished within 48 hr in all tissues.These results showed that histamine was metabolized by two metabolic routes in rainbow trout. One is the main pathway producing imidazole acetic acid by intestinal diamine oxidase and the other is the complementary pathway producing 1-methyl histamine by liver N-methyl-transferase.     

Identification of a novel gill-specific calpain from rainbow trout (Oncorhynchus mykiss)

Calpains are calcium-dependent neutral proteases responsible for many cellular functions. The two forms of calpain ubiquitously expressed in mammalian tissues are known as μ-calpain and m-calpain. We report here the identification of a novel calpain that is similar to but distinct from the μ- and m-calpains in rainbow trout. The cDNA of the novel gene is 2623 bp in length with a single open reading frame. The predicted protein (676 amino acids) contains the conserved calpain characteristic domains that include: domain I (pro peptide), II (cysteine catalytic site), III (electrostatic switch), and IV (calmodulin-like) with five Ca²⁺-binding EF hands. Northern blot and RT-PCR analyses demonstrated that the novel calpain gene is predominantly expressed in rainbow trout gills. Comparison of the novel protein with the ubiquitously expressed calpains and several mammalian tissue-specific calpains revealed that the novel calpain is an orthologue of the mammalian digestive tract specific calpain (calpain 9).     

Androgen metabolism in the skin of the rainbow trout (Oncorhynchus mykiss)

Pieces of skin of male and female rainbow trout (Oncorhynchus mykiss) were incubated with testosterone and 11-ketotestosterone as substrates. In immature fish the conversion rate was low. In non-spawning adult males 11-ketotestosterone was reduced to 5α-11KDHT (up to 5.2%). In the fish in spawning condition the 5α-reduction rate was only about 1 to 2%. In the same specimens incubated with testosterone a high 11β-hydroxylase activity (23.8-25% in the male and 13% in the female skin) was found. Similar sex specific differences were observed for the occurence of 5α-reduced metabolites (about 20% in the male and 13% in the female tissue).

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Résumé Des fragments de peau de truites arc en ciel (Oncorhynchus mykiss, males ou femelles, ont été incubés avec de la testostérone ou de la 11-cétotestostérone, utilisées comme substrats. Chez les poissons immatures, les taux de conversion restent faibles. Chez les males adultes ne donnant pas de sperme, la 11-cétotestostérone est réduite en 5α-androstane-17β-ol-3,11-dione (jusqu'à 5.2%). Chezles poissons en conditions de fraie, le taux de 5α-réduction est seulement de l'ordre de 1 à 2%. Pour ce derniers individus, les incubations de peau en présence de testostérone montrent l'existence d'une forte activité 11β-hydroxylase (23.8-25% pour le male, et 13% pour la femelle). Des différences liées au sexe sont observées de la même manière dans la production de métabolites 5α-réduits (environ 20% avec le tissu male et 13% avect le tissu femelle).

     

虹鳟生长性状的随机回归分析

生长和抗逆是水产动物遗传育种工作中最重要的农艺性状,虹鳟的生长性状关乎虹鳟规模化养殖的生产经济效益,为了从遗传上精细解析虹鳟的生长性状,我们从渤海、丹麦、挪威、唐纳森和加利福尼亚5个虹鳟(Oncorhynchus mykiss)种系间的双列杂交开始,进行了连续4代的继代选育。本研究测量了第4代总共4368个实验个体在516日龄、608日龄、668日龄、883日龄和1036日龄5个时间点的生长数据。采用随机回归测定日模型,对虹鳟生长性状进行了动态遗传分析。根据贝叶斯信息准则,确定3阶勒让德多项式为拟合体重和体长的加性遗传效应和永久环境效应变化的最优子模型。利用双变量随机回归模型同时分析体重和体长两个生长性状。它们的遗传力在400～1000日龄之间呈现递减趋势,分别从0.288下降到0.164和从0.469下降到0.186,并且在该生长区间内体长的遗传力始终高于体重的遗传力。无论体重还是体长性状,在不同日龄之间的遗传相关都随着生长间隔的增大而降低,但是两个性状在生长初期和后期之间的遗传相关较高(遗传相关系数0.75以上),尤其是体重(遗传相关系数0.85以上),该研究结果为虹鳟早期的遗传选育提供了理论支撑。两个性状之间在相同日龄之间的遗传相关均在0.75以上,在不同日龄之间的遗传相关随着生长间隔的增大由0.83下降到0.63。以上的研究结论为虹鳟生长性状(主要是体长和体重)的遗传选育提供了理论基础,同时也为虹鳟的体长和体重两个性状的联合选育提供了精确的遗传分析结果,由于两性状在前期有较高的遗传相关,因此建议在虹鳟生长前期(400日龄)进行联合选择。     

Embryonic expression of UCP2 in rainbow trout (Oncorhynchus mykiss)

Uncoupling proteins are mitochondrial anions transporters that dissociate respiration from ATP synthesis through proton leaks. Uncoupling protein 2 reportedly plays a role in several physiological processes such as energy partitioning, nutrition, and fatty acid metabolism. The mRNA expression of rainbow trout UCP2 genes (UCP2A and UCP2B) was monitored during embryogenesis and early larval development. Both genes were recruited early and displayed similar steadily decreasing patterns from fertilization until hatching. The expression of UCP2A and UCP2B appeared significantly differentiated after hatching and during the yolk sac absorption, with UCP2A displaying higher expression. We suggest that UCP2 expression profiles in the rainbow trout embryo could be associated with the utilization of lipids as a source of energy during development.     

Computer-controlled freezing of rainbow trout Oncorhynchus mykiss (Walbaum) spermatozoa for routine programmes

The effects of extender composition and freezing rate on cryopreservation efficiency of refrigerated spermatozoa of rainbow trout Oncorhynchus mykiss (Walbaum) were evaluated in order to test the suitability of a computer-controlled ultrafreezer to cryopreserve milt samples obtained in field conditions and stored for several hours. A very highly significant first-order interaction between freezing rate and the type of extender was found. Six of the eight experimental variants did not differ significantly, resulting, after fertilization of eggs with cryopreserved sperm, in a range of 62.3–74.8% of eyed embryos. This procedure was effective for samples stored at 1 °C for 2 days.     

Indirect estimation of stomach volume of rainbow trout Oncorhynchus mykiss (Walbaum)

The main hypothesis of this study was that if stomach volume is correlated with food intake it can be estimated without laborious and destructive direct measurement. Rainbow trout, Oncorhynchus mykiss (Walbaum), ca. 500–1300 g, were starved for 1, 4, 8 or 16 days at 15°C after which they were fed in excess with dry pellets containing known amounts of X‐ray‐dense markers. Immediately after feeding the fish were killed, X rayed and weighed. Then the stomach was dissected, its contents removed and weighed, and stomach volume was measured. X‐ray plates were developed and feed intake was estimated based on the amount of marker. All measured variables correlated positively with stomach volume. The best fit for linear regression models was obtained for fish starved for 4 days, where stomach content (dry mass) explained 94%, food intake (based on X‐ray measurement) 77% and fish mass 62% of the variation in stomach volume. However, as stomach content measurement can be a lethal, or at least very stressful, event for the fish, the accuracy of food intake measurement (X‐ray) could be increased using multiple regression. In multiple linear regressions, R²‐values varied between 0.79 (16‐day starvation) and 0.91 (1‐day starvation) with food intake and fish mass as explanatory variables for stomach volume. These results indicate that the stomach volume in rainbow trout can be estimated satisfactorily using indirect methods, which are not detrimental to the fish, although feeding history may affect the accuracy of the estimates.     

A test of a feed budget model for rainbow trout, Oncorhynchus mykiss (Walbaum)

A feed budget model, which enables farmers to adapt a general model to local conditions, was tested on rainbow trout, Oncorhynchus mykiss (Walbaum). The model has two major components; component 1 is used to calculate the energy required for one unit of biomass gain (g or kg) and component 2 estimates the daily growth increment (g or kg). Both components can be adjusted in relation to prevailing environmental conditions and, together, they express the daily energetic requirement of the specific species or strain. Fish fed using the model as a guide had a high rate of growth and good feed conversion. The model accurately predicted growth over the experiment, and is presented as a means of calculating the daily feed budget in fish farming. Growth of fish fed using self-feeders is presented for comparison with the results obtained using the model.     

Seasonal variation of muscle metabolic organization in rainbow trout (Oncorhynchus mykiss)

This study examined how muscle metabolic organization varied during an annual cycle in which rainbow trout (Oncorhynchus mykiss) were held in outdoor holding ponds in which they were exposed to natural changes in temperature (range 0.2 to 15.6°C) and photoperiod. We examined the activities of glycolytic and mitochondrial enzymes in red and white muscle to evaluate whether trout enhance their capacity for lipid and carbohydrate oxidation during cold-acclimization. When assayed at habitat temperature, the enzyme activities generally increased in spring to reach a maximum in summer followed by a decrease in the fall. This led to significantly higher activities at warm than cold periods for all enzymes measured in red muscle and all but one in white muscle. The activities at 10°C provided little evidence for compensatory adjustments of aerobic capacity. Particularly in red muscle, enzyme levels at 10°C were generally lower during cold than warm periods. The variation of enzyme activities throughout the cycle was not due to changes in protein concentration, as the same responses were observed when activities were expressed per g wet mass or per mg protein. Although the aerobic capacity did not increase with cold-acclimatization, the relative capacity for lipid oxidation was higher in winter than in summer trout. In contrast, the relative capacity for aerobic glycolysis was higher in summer than in winter trout. Thus, the metabolic capacities of trout muscle undergo seasonal reorganization.     

Isolation and partial characterization of rainbow trout (Oncorhynchus mykiss) gill mucin

Gill mucin from rainbow trout was isolated utilizing two rounds of cesium chloride density ultracentrifugation followed by gel filtration on Sepharose CL-2B. Neither density ultracentrifugation nor gel filtration alone was sufficient for purification of the mucin. Isolated gill mucin had a density of 1.5 g/ml and eluted at the void volume of the Sepharose CL-2B column. Silver-stained reducing 6% polyacrylamide gel electrophoresis of gill mucin produced a band at the origin with a smear entering the separating gel. There was no evidence of a link protein in gill mucin on reducing 12% polyacrylamide gel electrophoresis. Gill mucin had an amino acid profile similar to that of mucins in other species. Specifically, 35.1% of the total amino acids were represented by threonine and serine, while another 27.5% were alanine and proline. Gill mucin contained galactose (26.7 ± 3.2%), galactosamine (22.5 ± 4.4%), glucose (16.6 ± 8.7%), fucose (16.1 ± 1.5%), glucosamine (12.0 ± 1.9%) and mannose (5.1 ± 4.4%). Uronic acid levels from purified mucin were very low (0.7 ± 0.1%). Sialic acid was also present (0.06 g/g of mucin protein). The periodic acid-Schiff assay routinely utilized for detection of mucins was relatively insensitive for detection of gill mucin (6 × less sensitive than for pig gastric mucin) so a rabbit antiserum was raised. The antiserum produced profiles similar to the periodic acid-Schiff assay of fractions following gel filtration. Immunofluorescence of formalin-fixed rainbow trout gill tissue sections showed that the antiserum detected mucin within branchial goblet cells.     

Virulence of Flavobacterium psychrophilum isolates in rainbow trout Oncorhynchus mykiss (Walbaum)

Flavobacterium psychrophilum, the causative agent of bacterial cold‐water disease (BCWD) in freshwater‐reared salmonids, is also a common commensal organism of healthy fish. The virulence potential of F. psychrophilum isolates obtained from BCWD cases in Ontario between 1994 and 2009 was evaluated. In preliminary infection trials of rainbow trout juveniles, significant differences (0% to 63% mortality) in the virulence of the 22 isolates tested were noted following intraperitoneal injection with 10⁸ cfu/fish. A highly virulent strain, FPG 101, was selected for further study. When fish were injected intraperitoneally with a 10⁶, 10⁷ or 10⁸ cfu/fish of F. psychrophilum FPG 101, the 10⁸ cfu/fish dose produced significantly greater mortality (p < 0.05). The bacterial load in spleen samples collected from fish every 3 days after infection was determined using rpoC quantitative polymerase chain reaction amplification and by plate counting. Bacterial culture and rpoC qPCR were highly correlated (R² = 0.92); however, culture was more sensitive than the qPCR assay for the detection of F. psychrophilum in spleen tissue. Ninety‐seven per cent of the asymptomatic and the morbid fish had splenic bacterial loads of <2.8 log₁₀ gene/copies and >3.0 log₁₀ gene copies/reaction, respectively, following infection with 10⁸ cfu/fish.     

Immunogenicity of amoebic antigens in rainbow trout, Oncorhynchus mykiss (Walbaum)

Abstract. Rainbow trout developed a humoral immune response against numerous antigens of sonicated amoebae which were emulsified with Freund's complete adjuvant and injected into the peritoneum. The amoebae were cultured from the gills of Atlantic salmon, Salmo salar L., affected by amoebic gill disease. Antibodies in fish sera were detected by both enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Non-spedfie reactivity in fish serum against Escherichia coli, the bacterium used in co-cultivation of amoebae in vitro, was removed by immunoadsorption. Results obtained using ELISA and immunoblotting were comparable and indicated no significant difference in response to immunization with 10, 50 or 250 μg of sonieatcd amoebic protein. Amoebae contained immunogenie components of > 100, 100, 89, 49, 37 and 34kDa.     

Oxygen-induced gas bubble disease in rainbow trout, Oncorhynchus mykiss (Walbaum)

Abstract. An Aquatector oxygen injection unit was used to supersaturate a hatchery water supply to 200% oxygen saturation (18–20mg/l) and increase the total gas pressure to 120% of saturation. Nitrogen saturation was reduced to near 100%. Rainbow trout, Oncorhynchus mykiss (Walbaum), held in the treated water developed signs of gas bubble disease in 4 days, and 50% died within 20 days. We demonstrated that supersaturated total gas pressure due to excessive oxygen saturation causes gas bubble disease in the absence of supersaturated nitrogen gas. It is recommended that users of oxygen injection systems closely adjust the amount of oxygen added to the water to keep the total gas pressure near saturation.