军曹鱼淋巴囊肿病毒主衣壳蛋白基因全序列分析

军曹鱼(Rachycentron canadum)亦称海鲡,是我国南方沿海一带的重要海水网箱养殖对象。2005年8月,广东省海水网箱养殖的军曹鱼首次暴发类似的淋巴囊肿病,病鱼的口唇、鳃、鳍、尾及体表等处,可看到大小不一的单个或成群的肿瘤,个别网箱的感染率在80%以上,死亡率近30%。病鱼形象丑陋,严重影响其市场价值,造成了较大的经济损失。淋巴囊肿病(Lymphocystis disease)发现于1874年,1965年正式确认该病病原为淋巴囊肿病毒[1],现已知可感染9目34科140种以上鱼类。我国在20世纪90年代陆续在养殖石斑鱼、鲈鱼、牙鲆中发现淋巴囊肿病[2-5],随后对其病原…     

淡水瓦氏雅罗鱼基因文库的构建和抗冻蛋白基因同源序列的筛选

极地海域中的鱼类非常耐寒。它们的血液里常含有两种能降低其体内冰点的蛋白质：抗冻糖蛋白（AFGP）和不含糖的抗冻蛋白或抗冻多肽（AFP）。这些蛋白的编码基因已经被克隆,并将黄盖鲽AFP导人虹鳟鱼和大西洋鲑鱼,而改变了它们的抗冻基因进行了分离研究。所用实验材料鱼为中国内蒙古北部生活的瓦氏雅罗鱼（Leuciscus waleckii),属鲤形目（Lypciniformes),鲤科（Lyprinidae),雅罗鱼亚科（Leucisinae),基本按标准方法构建瓦氏雅罗鱼的基因库。只在包装反应至30分钟时,再加一份BHB2690制备物,并延长30分钟反应时间。所得库的成斑率大于1×10^5pfu。已知AFP基因常常多至40个拷贝,故已够用。采用通用的原位分子杂交和Southern分析进行瓦氏雅罗鱼AFP基因同源序列的分离及其亚克隆分析。以北美黄盖鲽AFP基因片段为探针,从瓦氏雅罗鱼DNA库中筛选了三个阳性克隆。图一所示为其中一个的双份膜及其复筛结果。图二为这个阳性克隆DNSA经过三种限制酶切后,同时与北美黄盖鲽AFP基因片段和该基因0．4KbcDNA两种探针的southern分析结果。其中Sac酶切在两个杂交中的均能产生一条2．7Kb的杂交带。图三对两个阳性克隆的southern分析证明：对这个杂交片段的亚克隆是成功的。这是首次在中国一种耐寒淡水鱼基因组内发现并得到AFP基因同源序列,将用于转基因鱼的研究。     

在现代鱼和化石鱼性的同种异形

性的同种异形,就是雌性与雄性之间第二性徵的不同,在鱼类此种现象是相当广泛而普通的。这种现象常带有暂时性,表现在产卵期,在雄鱼作为交配的装饰(在鲑鱼和一些其他的鱼)。性的同种异形,大半是表现在鱼的成年阶段。雌雄性的不同或在于他们身体的大小不一,比例上的差别,或在于腹鳍及臀鳍的结构不同,雄鱼的腹鳍或臀鳍时常充任交配的器官。鱼为了保护自己的幼鱼,雄者常较雌者大(大多数鲶及其他鱼类)。但普通雌鱼较雄鱼大——鲤、(鱼八)、鳗、鲽、鲻、撲(?)、黄瓜鱼、鲷等,而且某些鱼的雄性仅只一寸     

淡水瓦氏雅罗鱼基因文库的构建和抗冻蛋白基因同源序列的筛选

极地海域中的鱼类非常耐寒。它们的血液里常含有两种能降低其体内冰点的蛋白质：抗冻糖蛋白（ＡＦＧＰ）和不含糖的抗冻蛋白或抗冻多肽（ＡＦＰ）。这些蛋白的编码基因已经被克隆,并将黄盖鲽ＡＦＰ导入虹鳟鱼和大西洋鲑鱼,而改变了它们的抗冻性能。为开展中国的抗冻转基因鱼研究,本项目对中国北方的耐寒淡水鱼的抗冻基因进行了分离研究。所用实验材料鱼为中国内蒙古北部生活的瓦氏雅罗鱼（Ｌｅｕｃｉｓｃｕｓｗａｌｅｃｋｉｉ）,属鲤形目（Ｌｙｐｃｉｎｉｆｏｒｍｅｓ）,鲤科（Ｌｙｐｒｉｎｉｄａｅ）,雅罗鱼亚科（Ｌｅｕｃｉｓｉｎａｅ）。基本按标准方法构建瓦氏雅罗鱼的基因文库,只在包装反应至３０分钟时,再加一份ＢＨＢ２６９０制备物,并延长３０分钟反应时间。所得文库的成斑率大于１×１０５ｐｆｕ。已知ＡＦＰ基因常常多至４０个拷贝,故已够用。采用通用的原位分子杂交和Ｓｏｕｔｈｅｒｎ分析进行瓦氏雅罗鱼ＡＦＰ基因同源序列的分离及其亚克隆分析。以北美黄盖鲽ＡＦＰ基因片段为探针,从瓦氏雅罗鱼ＤＮＡ文库中筛选了三个阳性克隆。图一所示为其中一个的双份膜及其复筛结果。图二为这个阳性克隆ＤＮＡ经过三种限制酶切后,同时与北美黄盖鲽ＡＦＰ基因片段和该基因０．４ＫｂｃＤＮ     

海淡水养殖中华鲟免疫特性的比较

实验以淡水养殖的4龄中华鲟(Acipenser sinensis)为研究对象, 经海水驯化后实施全海水养殖, 探究中华鲟在海水养殖条件下血液理化指标和免疫组织结构变化。结果显示: 与淡水养殖个体相比, 海水养殖后中华鲟的血红蛋白(HB)、红细胞比容(HCT)和白细胞数(WBC)均显著升高(P<0.05), 红细胞数(RBC)与各类白细胞占比无显著变化(P>0.05); 超氧化物歧化酶(SOD)、丙二醛(MDA)、溶菌酶(LZM)、免疫球蛋白M(IgM)、碱性磷酸酶(AKP)和酸性磷酸酶(ACP)等6种免疫生化指标与淡水个体无显著差异(P>0.05)。对免疫组织的观察发现, 海水养殖中华鲟头肾组织中细胞聚集, 形态分布更整齐, 黑色素巨噬细胞中心增多; 脾脏组织的淋巴细胞和红细胞分布更加密集。研究结果表明, 海水养殖会在一定程度上增强中华鲟的免疫和造血功能, 保持较好的生理状态。     

关于“松江鲈鱼”

“松江鲈鱼”是我国四大名鱼之一(其他是黄河的鲤鱼,松花江的鲑鱼和兴凯湖的白鱼我国自古以来,一直在养殖着的,也有四大名鱼,叫“青鱼、鲩、鲢、鳙”),亦名“四鳃鲈”(因鳃盖上有四条鲜红的颜色).三国演义里,左慈对曹操道:“……天下鲈鱼只两鳃,惟松江鲈鱼有四鳃…….”南宋有一位诗     

鲑鱼隐鞭虫病及其防治策略

鲑鱼隐鞭虫病的病原为Cryptobiasalmositica。据报道,北美西海岸的所有大西洋鲑(Oncorhynchusspp.)均有该虫的寄生。该病除了通常情况下经河流中的水蛭传播外,在特定的条件下可直接经鱼类传播。鲑隐鞭虫病的临床症状包括贫血、食欲减退、脾脏肿大、全身性水肿、腹水性腹胀。被感染的鱼类需要付出巨大的能量代价,表现出明显的新陈代谢及游泳功能的衰退。鱼体对缺氧相当敏感,免疫系统也受到抑制。鱼类对隐鞭虫病的感染严重程度和死亡率可因鱼种而存在差异。预防感染的有效策略包括选育抗隐鞭虫的新品种,“抗隐鞭虫感染”在遗传上是孟德尔显性遗传。在抗隐鞭虫的鱼体内,虫体可被宿主免疫系统的“补体旁路途径”所裂解。在“隐鞭虫耐受性鱼”(处于感染状态但未发病)的血液中存在一种天然的抗蛋白酶———α2巨球蛋白,可以中和分泌的金属蛋白酶(毒力因子)。因此,繁殖转基因“隐鞭虫耐受性”鲑鱼是一种很好的选择,而无需通过疫苗和药物来治疗隐鞭虫病。康复后的鱼体具有对该病的免疫保护力,因而,利用这一特点采取疫苗免疫也是防治隐鞭虫病的另一种策略。我们所研制的致弱疫苗单一剂量免疫鱼体可获得至少两年的免疫保护。免疫保护机制包括产生“抗体结合补体”、强化吞噬作用及细胞介导的细胞毒作用。氯化氮氨菲啶是防治隐鞭虫病的有效药物,它通过溶解虫体而杀灭寄生虫,也可在线粒体中富集,显著影响有氧呼吸的顺利进行并造成线粒体的损伤。通过药物与抗体连接可以显著增强药效,这种利用“免疫化学疗法”控制鲑隐鞭虫病的策略,其优点在于减小了药物剂量,因而大大降低了化学治疗所引起的副作用。对于食用性鱼类而言,减少药物在鱼体的富集符合食品安全的需要     

军曹鱼营养需求与饲料研究进展

目前, 养殖鱼类已成为满足人类对水产品需求的主要来源。军曹鱼(Rachycentron canadum)具有生长速度快、肉质细嫩鲜美、营养价值高等特点, 已成为我国南方重要的海水网箱养殖鱼类。然而, 目前饲养军曹鱼仍主要依靠冰鲜杂鱼, 特别是大规格鱼的养殖, 这严重制约其养殖业的健康发展, 研发适合不同生长阶段需要的配合饲料非常必要。文章重点总结了军曹鱼对蛋白质、脂肪、氨基酸、脂肪酸、微量营养素等的需求, 饲料中鱼粉和鱼油替代研究, 饲料添加剂应用及其饲料产业发展现状, 以期为军曹鱼营养需求和配合饲料开发的深入研究提供参考。     

红树林与水产养殖系统初步研究

为研究红树林对养殖水体的净化效应,选择3种红树植物海桑、秋茄和桐花树,每种分别按45％、30％和15％的面积比例种植于滩涂海水养殖塘,塘中养殖美国红鱼和星洲红鱼1年后收获,养殖期间通过对水质和鱼的生长情况进行监测,结果显示,种植红树植物的养殖塘水质均有不同程度的改善,可以减少鱼类病害。其中以种植比例为45％的桐花树养殖塘水质最好,鱼类生长速度最快。     

饥饿和再投喂对日本黄姑鱼生长及其体生化组成的影响

日本黄姑鱼(Nibea japonica)俗称黑毛,属鲈形目,石首鱼科,黄姑鱼属,分布于中国东海及日本南部海域[1,2].其营养丰富、生长快、病害少、易于养殖,是东海区海水养殖的理想品种.     

Neoparamoebic gill infections: host response and physiology in salmonids

Amoebic gill diseases (AGD) caused primarily by the amphizoic Neoparamoeba spp. have been identified as significant to fish health in intensive aquaculture. These diseases have consequently received significant attention with regard to disease pathophysiology. Neoparamoeba perurans has been putatively identified as the aetiological agent in salmonids, with other species such as turbot Psetta maxima and sea bass Dicentrarchus labrax also affected. Similarly, Neoparamoeba spp. have also been identified in co‐infections with other gill diseases in salmonids. While infection of the gills results in an acute multifocal hyperplastic host response, reduced gill surface area and increased mucous cell densities, ion regulation and respiration in terms of blood gasses are only marginally affected. This may be partially attributed to reserve respiratory capacity and a reduction in mucous viscosity allowing for a greater flushing of the gill, so reducing the gill mucus boundary layer. Clinical and acute infections result in significant cardiovascular compromise with increases in aortic blood pressure, and systemic vascular resistance in Atlantic salmon, Salmo salar, which are not seen in rainbow Oncorhynchus mykiss and brown trout Salmo trutta. Increases in vascular resistance appear to be due to vascular constriction potentially reducing blood flow to the heart in compromised fishes, the overall effect being to lead to a compensatory tissue remodelling and change in cardiac shape in chronically infected fishes. The combined effect of reduced gill surface area and cardiovascular compromise leads to a significant reduction in swimming performance and increases in the routine metabolic rate that lead to an increase in the overall metabolic cost of disease.     

Comparative ionic flux and gill mucous cell histochemistry: effects of salinity and disease status in Atlantic salmon (Salmo salar L.)

Two experiments were conducted to assess the physiological effects of freshwater exposure and amoebic gill disease (AGD) in marine Atlantic salmon (Salmo salar L.). The first experiment monitored marine salmon during a 3 h freshwater exposure, the standard treatment for AGD in Tasmania. The second experiment described the gill mucous cell histochemistry for freshwater adapted and seawater acclimated fish (AGD affected and unaffected) for possible correlations to ionoregulation. When exposed to freshwater, marine Atlantic salmon experienced a minor ionoregulatory dysfunction represented by a net efflux of Cl(-) ions at 3 h. AGD affected fish experienced the net efflux of Cl(-) ions 1 h sooner, and had a significantly greater net efflux of total ammonia. Changes to gill mucous cell populations corresponded to differing salinity and the presence of AGD. In AGD affected fish, these populations significantly differed between lesion and non-lesion associated areas of the gill filament. Our results have shown changes in the ionoregulatory capacity of Atlantic salmon due to freshwater exposure and AGD. Gill mucous cell histochemistry indicates the potential importance of the mucous layer in ionoregulation and disease. In comparison to previous studies on rainbow trout, these results suggest that Atlantic salmon have a greater short-term ionoregulatory capacity.     

18S ribosomal DNA-based PCR identification of Neoparamoeba pemaquidensis, the agent of amoebic gill disease in sea-farmed salmonids

Neoparamoeba pemaquidensis is a parasomal amoeboid protozoan identified as the agent of amoebic gill disease (AGD) in Atlantic salmon Salmo salar reared in sea-pens in Tasmania, Australia, and coho salmon Oncorhynchus kisutch farmed on the west coast of the USA. Outbreaks of AGD caused by immunologically cross-reactive paramoebae have also been reported in sea-farmed salmonids in several other countries. Complete 18S rDNA sequences were determined for respective paramoebae isolated from infected gills of salmon from Tasmania and Ireland, and N. pemaquidensis isolates from the USA and UK, including representative free-living isolates. Alignments over 2110 bp revealed 98.1 to 99.0% sequence similarities among isolates, confirming that paramoebae implicated in AGD in geographically distant countries were homologous and belonged to the same species, N. pemaquidensis. The results supported previous findings that N. pemaquidensis exists as a widely distributed, amphizoic marine protozoan. Partial 18S rDNA sequences were obtained for the ultrastructurally similar species, N. aestuarina, and for the morphologically similar but non-parasomal amoeba Pseudoparamoeba pagei. N. aestuarina had 95.3 to 95.7% sequence similarities with N. pemaquidensis strains, which distinguished 2 closely related but separate species. Neoparamoeba spp. were not analogous to P. pagei or to other marine Gymnamoebia. We designed 4 oligonucleotide primers based on elucidated 18S rDNA sequences and applied them to single-step and nested 2-step PCR protocols developed to identify N. pemaquidensis to the exclusion of apparently closely related and non-related protistan taxa. Nested PCR was able to detect the AGD parasite from non-purified, culture-enriched net microfouling samples from Atlantic salmon sea-pens in Tasmania, and confirmed that N. pemaquidensis was also responsible for AGD in chinook salmon O. tshawytscha in New Zealand. Our sequence and PCR analyses have now shown that AGD affecting 3 different salmonid species farmed in 4 countries are associated with N. pemaquidensis. A species-specific diagnostic PCR provides for the first time, a highly specific detection and identification assay for N. pemaquidensis that will facilitate future ecological and epidemiological studies of AGD.     

Influence of salmonid gill bacteria on development and severity of amoebic gill disease

The relationship between salmonid gill bacteria and Neoparamoeba sp., the aetiological agent of amoebic gill disease (AGD) was determined in vivo. Fish were divided into 4 groups and were subjected to following experimental infections: Group 1, amoebae only; Group 2, Staphylococcus sp. and amoebae; Group 3, Winogradskyella sp. and amoebae; Group 4, no treatment (control). Fish (Groups 1, 2 and 3) were exposed to potassium permanganate to remove the natural gill microflora prior to either bacterial or amoebae exposure. AGD severity was quantified by histological analysis of gill sections to determine the percentage of lesioned filaments and the number of affected lamellae within each lesion. All amoebae infected groups developed AGD, with fish in Group 3 showing significantly more filaments with lesions than other groups. Typically lesion size averaged between 2 to 4 interlamellar units in all AGD infected groups. The results suggest that the ability of Neoparamoeba sp. to infect filaments and cause lesions might be enhanced in the presence of Winogradskyella sp. The possibility is proposed that the prevalence of more severe AGD is due to the occurrence of Winogradskyella sp. at high concentrations on the gills.     

Virulence factors of Aeromonas salmonicida subsp. salmonicida strains associated with infections in turbot Psetta maxima

Virulence factors for Aeromonas salmonicida subsp. salmonicida (ASS) strains isolated from cultured turbot Psetta maxima L. are unknown with regard to this host. The presence of virulence genes associated with different stages of ASS infection in salmonids (vapA, tapA, fla, ascV, ascC, aexT, satA and aspA) was analysed using a polymerase chain reaction (PCR) technique in ASS strains isolated from turbot. Other ASS strains isolated from salmonids and environmental A. salmonicida (AS) strains were included for comparison. The presence of the genes was evaluated with respect to ASS virulence in turbot based on intraperitoneal and bath challenges. The genetic profile, including all of the genes studied, that was linked to virulent behaviour after intraperitoneal challenge was significantly more frequent in strains isolated from turbot than in those from salmonids or the environment. The data prove that it is not possible to predict the virulence of ASS in turbot based only on the presence of all genes tested. Moreover, the combined PCR results of vapA, aexT, ascV and ascC were useful for separating most of the ASS from environmental A. salmonicida strains. An association between virulence or genetic profile and the geographical or facility origin of the strains was not found.     

Acid–base and respiratory effects of confinement in Atlantic salmon affected with amoebic gill disease

Amoebic gill disease (AGD) in cultured salmonids causes severe multifocal hyperplastic lesions in the gills with the potential to influence respiratory and acid–base physiology. Atlantic salmon Salmo salar affected with AGD were surgically implanted with dorsal aortic catheters and, following recovery, were confined for 5 min ( n  = 16) or left undisturbed ( n  = 8). Confinement caused an acute extracellular acidosis that was corrected in 6 h amongst surviving fish. There was a gradual increase in plasma lactate concentrations that peaked at 1 h post-confinement then declined by 9 h recovery. In a second experiment, AGD-affected fish were confined then recovered either in a tank of static water ( n  = 9) or while being forced to swim at 1·5 body lengths s⁻¹ ( n  = 6). There was no significant difference between fish recovered by swimming and those in static water in terms of recovery of the acute extracellular acidosis and lactate accumulations coincident with exhaustive exercise. Confinement severely compromised the survival of AGD-affected Atlantic salmon, although survivors appeared to recover similarly to other studies. Forced swimming of AGD-affected Atlantic salmon following confinement did not facilitate recovery and is unlikely to be a useful strategy for mitigating the effects of stressful episodes such as crowding and fish movement and commercial handling.     

Major histocompatibility polymorphism associated with resistance towards amoebic gill disease in Atlantic salmon (Salmo salar L.)

The association between major histocompatibility (MH) polymorphism and the severity of infection by amoebic gill disease (AGD) was investigated across 30 full sibling families of Atlantic salmon. Individuals were challenged with AGD for 19days and then their severity of infection scored by histopathological examination of the gills. Fish were then genotyped for the MH class I (Sasa-UBA) and MH class II alpha (Sasa-DAA) genes using polymorphic repeats embedded within the 3' untranslated regions of the Sasa-UBA and Sasa-DAA genes. High variation in the severity of infection was observed across the sample material, ranging from 0% to 85% gill filaments infected. In total, seven Sasa-DAA-3UTR and ten Sasa-UBA-3UTR marker alleles were identified across the 30 families. A significant association between the marker allele Sasa-DAA-3UTR 239 and a reduction in AGD severity was detected. There was also a significant association found between AGD severity and the presence of two Sasa-DAA-3UTR genotypes. While the associations between MH allele/genotypes and AGD severity reported herein may be statistically significant, the small sample sizes observed for some alleles and genotypes means these associations should be considered as suggestive and future research is required to verify their biological significance.     

Cultured gill-derived Neoparamoeba pemaquidensis fails to elicit amoebic gill disease (AGD) in Atlantic salmon Salmo salar

Amoebic gill disease (AGD) affects the culture of Atlantic salmon Salmo salar in the southeast of Tasmania. The disease is characterised by the presence of epizoic Neoparamoeba spp. in association with hyperplastic gill tissue. Gill-associated amoebae trophozoites were positively selected by plastic adherence for culture in seawater, where they proliferated using heat-killed E. coli as a nutrient source. One isolate of gill-harvested amoebae designated NP251002 was morphologically consistent to N. pemaquidensis under light, fluorescence and transmission electron microscopy. Rabbit anti-N. pemaquidensis antiserum bound to NP251002, and N. pemaquidensis small subunit (SSU) ribosomal DNA (18S rDNA) was detected in NP251002 genomic DNA preparations using PCR. A high degree of similarity in the alignment of the NP251002 18S rDNA PCR amplicon sequence with reference isolates of N. pemaquidensis suggested conspecificity. While short-term culture (72 h) of gill-harvested amoebae does not affect the capacity of amoebae to induce AGD, Atlantic salmon challenged with NP251002 after the trophozoites had been 34 and 98 d in culture exhibited neither gross nor histological evidence of AGD. It is not known if NP251002 were avirulent at the time of isolation, had down-regulated putative virulence factors or virulence was inhibited by the culture conditions. Therefore, the time in culture could be a limiting factor in maintaining virulence using the culture technique described here.