真鲷虹彩病毒自然感染杂交石斑鱼“褐龙斑”的组织病理分析

褐龙斑是雌性褐石斑鱼(Epinephelus bruneus)和雄性鞍带石斑鱼(E. lanceolatus)杂交产生的子代。作为杂交石斑鱼的新品种,国内外尚没有褐龙斑疾病的报道。2017年7月,某养殖场褐龙斑出现急性死亡,10 d内累积死亡率高达80%。现场调查发现,病鱼外观无明显异常,但反应迟钝,伏底死亡。临床检查和剖检可见脾和肾严重肿大、易碎。组织病理切片观察发现,各组织中存在数量不等的嗜碱性、细胞质均一、直径为10~15 µm的肿大细胞。超薄组织切片中发现,肿大细胞胞质内存在大量直径为130~150 nm的虹彩病毒样颗粒。使用特异性的PCR引物,从病鱼脾、头肾等组织中均检测到真鲷虹彩病毒(Red seabream iridovirus, RSIV)的高强度感染。测定了该病毒主要衣壳蛋白(Major capsid protein, MCP)基因1362 bp的全长编码区,构建了19种(株)虹彩病毒系统发育树,结果显示,该病毒属于虹彩病毒科肿大细胞病毒属RSIV类群。本研究首次描述了褐龙斑虹彩病毒病的组织病理特征,揭示了褐龙斑是RSIV新的敏感宿主,为杂交石斑鱼病毒病的诊断与防治提供了重要的参考依据。     

云纹石斑鱼、鞍带石斑鱼及其杂交F1的DNA甲基化分析

为探讨石斑鱼杂种优势形成过程中基因组DNA甲基化水平的变化,本研究采用甲基化敏感扩增多态性(Methylation-sensitive amplification polymorphism, MSAP)技术检测云纹石斑鱼(Epinephelus moara)、鞍带石斑鱼(Epinephelus lanceolatus)及云纹石斑鱼(♀)×鞍带石斑鱼(♂)杂交F1 3个群体的基因组DNA甲基化水平,分析杂交F1与亲本基因组DNA甲基化水平的差异。结果显示,云纹石斑鱼和鞍带石斑鱼的基因组DNA属于甲基化程度较高的类群;云纹石斑鱼、鞍带石斑鱼及其杂交F1的DNA总甲基化率分别为60.62%、59.38%和55.78%,DNA全甲基化率分别为31.37%、30.67%和29.27%,DNA半甲基化率分别为29.25%、28.71%和26.51%;杂交F1的DNA总甲基化率、全甲基化率和半甲基化率均低于双亲,并存在极显著差异(P<0.01),3个群体的全甲基化率均大于半甲基化率。研究表明,云纹石斑鱼(♀)×鞍带石斑鱼(♂)杂交F1 DNA甲基化水平与杂种优势呈负相关,杂交F1 DNA甲基化水平的降低可能是形成快速生长等杂种优势的原因之一。     

鞍带石斑鱼肌肉营养成分及氨基酸含量分析

采用常规营养物质测定方法对鞍带石斑鱼Epinephelus lanceol肌肉营养成分及氨基酸含量进行了定量分析,并开展了相关营养分析。结果表明,鞍带石斑鱼新鲜肌肉中粗蛋白含量19．5％、粗脂肪含量7．69％、水分含量70．5％、粗灰分含量1．01％,氨基酸种类有18种,其中8种必需氨基酸含量为43．43％。鞍带石斑鱼作为一种高蛋白、氨基酸含量丰富的养殖鱼类,具有较高的食用和营养价值。     

赤点石斑鱼与鞍带石斑鱼杂交子一代肌肉营养成分分析与评价

为分析赤点石斑鱼与鞍带石斑鱼杂交子一代的营养组成,参照国家标准,测定了体质量(182.84±29.35) g杂交石斑鱼肌肉的常规营养成分、氨基酸和脂肪酸组成,并对肌肉营养价值进行了评定。试验结果显示,杂交石斑鱼肌肉水分、粗蛋白、粗脂肪和灰分的含量分别为(74.07±0.71)%、(21.52±0.78)%、(4.03±0.15)%和(1.29±0.07)%。肌肉鲜样中测定了17种氨基酸,总量为(19.88±0.15)%;必需氨基酸和鲜味氨基酸总量分别为(8.64±0.13)%和(7.64±0.16)%,必需氨基酸指数为85.19,必需氨基酸组成符合联合国粮农组织/世界卫生组织标准。肌肉鲜样中含有17种脂肪酸,饱和脂肪酸、单不饱和脂肪酸和多不饱和脂肪酸分别占肌肉脂肪酸总量的(27.63±1.15)%、(22.75±1.22)%和(32.59±1.90)%,其中二十二碳六烯酸和二十碳五烯酸占肌肉脂肪酸总量的(19.27±1.27)%。研究表明,赤点石斑鱼与鞍带石斑鱼杂交子一代具有较高营养价值,可作为新品种进行开发。     

循环水养殖条件下鞍带石斑鱼生长特点研究

通过对鞍带石斑鱼(Epinephelus lanceolatus)为期5个月的养殖监测试验,研究循环水养殖条件下鞍带石斑鱼的生长特点。采用线性拟合、指数拟合、乘幂拟合的方法,分析了鞍带石斑鱼全长、体重生长与养殖时间,以及全长与体重的最佳拟合曲线。结果表明:鞍带石斑鱼全长生长与养殖时间以线性回归为佳(y=2.791x-15.716,R2=0.951 2);体重生长与养殖时间以指数回归最佳(y=2.432 3e0.32x,R2=0.996 5);全长与体重以乘幂回归为最佳(y=0.007 3x3.262 8,R2=0.945 3)。对鞍带石斑鱼全长与体重的幂函数关系分析,表明鞍带石斑鱼属正异速生长型(b=3.262 8),说明本研究条件下的循环水养殖模式适用于鞍带石斑鱼的养殖。     

斜带石斑鱼甘露糖受体基因的克隆表达及其功能

为了研究斜带石斑鱼甘露糖受体(Epinephelus coioides mannose receptor,EcMR)在抗赤点石斑鱼神经坏死病毒(red-spotted grouper nervous necrosis virus,RGNNV)感染中的免疫功能,实验成功克隆与表达了EcMR.结果显示,EcMR cDNA全...     

斜带石斑鱼生长激素cDNA克隆及其在大肠杆菌中的融合表达

通过构建斜带石斑鱼垂体cDNA文库,克隆了其生长激素(GH)全长cDNA。斜带石斑鱼GH全长cDNA为955bp,编码的多肽为204aa。应用PCR方法把编码GH成熟肽的cDNA片段克隆到表达载体pET-15b,在大肠杆菌BL21(DE3)表达N端含6个组氨酸的融合多肽。SDS-PAGE结果表明,0.4mmol·L-1IPTG诱导表达的蛋白约为24kDa,主要为不溶性的包含体。细菌裂解液沉淀溶于6mol·L-1盐酸胍后,用Ni2+-NTA树脂进行亲和分离纯化,纯化产物在SDS-PAGE上表现为一条24kDa的蛋白带。在黑鲷GH放射免疫分析系统中,纯化产物能与黑鲷GH竞争结合GH抗体,表明大肠杆菌表达的斜带石斑鱼GH融合多肽具有GH免疫活性。     

鞍带石斑鱼繁育群体遗传多样性分析

鞍带石斑鱼作为最大型的石斑鱼,生长速度快,有明显的生长优势,在石斑鱼的产业发展中起到举足轻重的作用。为了解人工养殖和选育活动对鞍带石斑鱼遗传多样性的影响,本文采用微卫星分子标记技术,对广东、海南和福建三个省份共五个代表性采集点的鞍带石斑鱼繁育群体的遗传变异信息进行了研究。群体内遗传多样性分析显示,5个群体等位基因(Na)的平均数目为7.326(6.375-8.380),观测杂合度(Ho)平均值为0.711(0.625-0.775),期望杂合度(He)平均值为0.705(0.684-0.734),多态信息含量(PIC)平均值为0.659(0.633-0.693)。其中,来自福建厦门翔安区的鞍带石斑鱼繁育群体遗传多样性最高。分子方差分析(AMOVA)结果显示,5.36%的遗传变异来自群体间,95.45%来自所有个体间。群体间遗传分化指数(Fst)及遗传距离结果显示,GC和CP群体聚为一支,再与AT群体聚为一支,再与XA群体距为一支,HL群体为独立一支。通过系统进化树分析显示,鞍带石斑鱼繁育群体交叉在一起,没有形成明显的地理格局分布。总而言之,这三省五地的鞍带石斑鱼繁育群体遗传多样性较高,没有明显的驯化迹象。整体研究表明,鞍带石斑鱼繁育群体仍具有较高的遗传多样性,品种受亲本近交影响而出现衰退的可能性不高,人工繁育技术的不完善及养殖管理不规范可能是导致品种病害频发及养殖成活率低的原因。本研究为鞍带石斑鱼种质评价和人工选育提供理论依据。     

鞍带石斑鱼Epinephelus lanceolatus (Bloch)繁殖生物学的初步研究

本文对鞍带石斑鱼的繁殖生物学特性进行了初步研究，该鱼在台湾已可进行商业化育苗生产，但在广东沿海还有待摸索。     

鞍带石斑鱼Epinephelus lanceolatus(Bloch)中间培育技术的初步研究

将体长2.4～3.00锄,平均体长2.64 cm;体重2.37～3.16 g,平均体重2.68 g的鞍带石斑鱼鱼苗在水温17.扣24～6℃、海水盐度31.2条件下进行室外水泥池培育.采取逐渐添加淡水降低盐度至18～19;以鱼体重4%～6%日投喂量投喂用鱼浆或鲜虾浆与幼鳗粉料混合制成的湿性饲料的方式.经过52 d的培育,鱼苗体长增至4.7～6.8 cm,平均体长增长3.12 era/尾,增长率121.44%;体重增至5.73～8.64g,平均体重增重4.84 g/尾,增重率180.6%.培育存活率97.4%.     

Isolation and characterization of collagens from the skin of giant grouper (Epinephelus lanceolatus)

ABSTRACT

Acid-solubilized collagen (ASC) and pepsin-solubilized collagen (PSC) were extracted from the skin of giant groupers (Epinephelus lanceolatus) with yields of 39.51 and 19.12%, respectively. ASC and PSC consisted of two different α chains (α1 and α2) and were characterized to be type I collagen with no disulfide bond. The imino acid contents of the ASC and PSC from giant grouper skin were 189 and 181 per 1,000 residues, respectively. The maximum endothermic temperatures (T_max) of ASC and PSC measured by differential scanning calorimetry (DSC) were 31.71 and 31.33°C, respectively. The denaturation temperatures of ASC and PSC measured by viscometry were 29.84 and 29.05°C, respectively. The maximum solubility in 0.5 M acetic acid was observed at pH 5 and pH 6 for ASC and PSC, respectively. A sharp decrease in solubility was observed for both ASC and PSC in the presence of NaCl above 3% (w/v).     

龙胆石斑鱼引种及人工育苗技术的初步研究

从台湾引进龙胆石斑鱼受精卵 2 5 0 g ,35 .71× 1 0 4粒 ,孵出仔鱼 2 5× 1 0 4尾 ,经 6 1d培育 ,成功地培育出 33mm幼鱼 1 .0 381× 1 0 4尾 ,成活率 4 %。并针对初孵仔鱼、开口仔鱼、 30d稚鱼、 6 0d幼鱼、90d幼鱼三个生长发育阶段的特征进行了实验和观察     

Experimental evaluation of inorganic fertilization in larval giant grouper (Epinephelus lanceolatus Bloch) production

A major constraint in successful larviculture of groupers has been the small gape of the larvae and hence the requirement for small prey at first feeding. In this study, we examined how maintaining a phosphate concentration of 100 μg P L^?1 and an inorganic nitrogen (N) level of 700 μg N L^?1 via weekly fertilization with inorganic fertilizers affected phytoplankton, zooplankton and giant grouper larval survival in relation to a control group that was provided with rotifers immediately after larvae hatched. Unicellular algae, zooplankton within the size ranges of 10–50 μm and 50–100 μm and survival of giant grouper larvae were all significantly higher in the fertilized treatment compared with the control. Stomach analysis revealed that ciliates and flagellates were actively consumed by larval fish in the fertilized group, whereas few rotifers were consumed in the control. We conclude that the inorganic fertilization method provides high densities of suitable‐sized prey for larval groupers at the onset of exogenous feeding before they are able to consume larger, commercially available rotifers and copepods.     

龙胆石斑鱼源美人鱼发光杆菌的生物学特性与系统发育学分析

对从1尾病死的观赏用龙胆石斑鱼Epinephelus lanceolatus L.中分离到的细菌,进行了形态特征、理化特性和对抗菌类药物的敏感性等较系统的表观生物学性状鉴定。同时,测定了16S rRNA基因序列、分析了相关细菌相应序列的同源性、构建了系统发生树。结果表明,供试两株纯培养菌（编号：HQ061227-1、HQ061227-2）为发光杆菌属Photobacterium（Beijerinck 1889）的美人鱼发光杆菌美人鱼亚种P.damselae subsp.damselae（Love et al.1982;Smith et al.1991）,用HQ061227-1株作为代表菌株的16S rRNA基因序列长度（不包括引物结合区）为1469bp（GenBank登录号：EF635307）,与GenBank数据库中美人鱼发光杆菌美人鱼亚种的同源性在99%。药敏试验结果显示,对供试37种抗菌药物中的青霉素G等4种耐药,对头孢唑啉等32种敏感,对氨苄青霉素低敏。     

Natural outbreak of Streptococcus agalactiae (GBS) infection in wild giant Queensland grouper, Epinephelus lanceolatus (Bloch), and other wild fish in northern Queensland, Australia

Ninety‐three giant Queensland grouper, Epinephelus lanceolatus (Bloch), were found dead in Queensland, Australia, from 2007 to 2011. Most dead fish occurred in northern Queensland, with a peak of mortalities in Cairns in June 2008. In 2009, sick wild fish including giant sea catfish, Arius thalassinus (Rüppell), and javelin grunter, Pomadasys kaakan (Cuvier), also occurred in Cairns. In 2009 and 2010, two disease epizootics involving wild stingrays occurred at Sea World marine aquarium. Necropsy, histopathology, bacteriology and PCR determined that the cause of deaths of 12 giant Queensland grouper, three wild fish, six estuary rays, Dasyatis fluviorum (Ogilby), one mangrove whipray, Himantura granulata (Macleay), and one eastern shovelnose ray, Aptychotrema rostrata (Shaw), was Streptococcus agalactiae septicaemia. Biochemical testing of 34 S. agalactiae isolates from giant Queensland grouper, wild fish and stingrays showed all had identical biochemical profiles. The 16S rRNA gene sequences of isolates confirmed all isolates were S. agalactiae; genotyping of selected S. agalactiae isolates showed the isolates from giant Queensland grouper were serotype Ib, whereas isolates from wild fish and stingrays closely resembled serotype II. This is the first report of S. agalactiae from wild giant Queensland grouper and other wild tropical fish and stingray species in Queensland, Australia.     

斑石鲷irf7基因克隆及其在病毒感染下的表达

为探究斑石鲷(Oplegnathus punctatus)干扰素调节因子irf7在虹彩病毒(Iridovirus)感染过程中的作用机制,本研究通过PCR扩增获得了斑石鲷irf7基因CDS区序列,对其序列特征进行分析,并在组织水平和细胞水平研究该基因在病毒感染中的表达模式。结果显示,Opirf7基因CDS区全长为1332 bp,编码443个氨基酸的多肽,具有干扰素调节因子家族的保守结构域。荧光定量PCR (qRT-PCR)结果显示,Opirf7基因在健康个体的不同组织中均有表达,在肝脏中表达量最高,在皮肤和肠等免疫组织中的表达量也较高。对斑石鲷腹腔注射虹彩病毒诱导抗病毒免疫反应,在组织水平检测Opirf7对病毒感染的响应模式。与对照组(0 h)相比,Opirf7在免疫组织中的表达水平有不同程度的升高。在细胞水平,建立poly I: C感染的斑石鲷脑细胞系模型,利用qRT-PCR检测Opirf7的表达变化。结果显示,poly I: C刺激后,脑细胞系Opirf7的表达量显著升高。结果表明,Opirf7在斑石鲷抗病毒病的免疫应答过程中发挥重要作用。     

Establishment of a new cell line from the heart of giant grouper,Epinephelus lanceolatus (Bloch), and its application in toxicology and virus susceptibility

A new marine fish cell line, derived from the heart of giant grouper, Epinephelus lanceolatus (Bloch), was established and characterized. The cell line was designated as ELGH and subcultured with more than 60 passages. The ELGH cells were mainly composed of fibroblast-like cells and multiplied well in Leibovitz's L-15 medium supplemented with 10% foetal bovine serum (FBS) at 28 °C. Chromosome analysis indicated that the modal chromosome number was 48. The fluorescent signals were detected in ELGH when transfected with green fluorescent protein reporter plasmids. The 50% cytotoxic concentration (CC₅₀) of the extracellular products (ECPs) from Streptococcus iniae and Vibrio alginolyticus E333 on ELGH cells was 60.02 and 12.49 μg mL⁻¹, respectively. Moreover, the ELGH cells showed susceptibility to Singapore grouper iridovirus (SGIV), but not to soft-shelled turtle iridovirus (STIV), red-spotted grouper nervous necrosis virus (RGNNV) and spring viremia of carp virus (SVCV), which was demonstrated by the presence of a severe cytopathic effect (CPE) and increased viral titres. In addition, electron microscopy observation showed that abundant virus particles were present in the infected cells. Taken together, our data above provided the potential utility of ELGH cells for transgenic and genetic manipulation, as well as cytotoxicity testing and virus pathogenesis.