六带石斑鱼染色体核型和银染研究

以六带石斑鱼为材料,利用腹腔注射法体内注射植物血球凝集素和秋水仙碱获得大量处于分裂中期的头肾细胞,头肾细胞经低渗处理、卡诺氏液固定后,常规自然晾干法制作成染色体标本后,进行染色体核型分析和银染研究。结果显示,六带石斑鱼的二倍体染色体数目为2n=48,核型为2n=2st+46t,NF=48;Ag-NORs仅有一对位于第24对染色体即亚端部着丝点染色体的短臂上。     

布氏石斑鱼的染色体核型、银染和C-带

为了补充布氏石斑鱼细胞遗传学数据, 并为石斑鱼染色体进化研究以及石斑鱼遗传育种等工作奠定基础, 实验利用Giemsa染色、C-带和Ag-NORs方法对布氏石斑鱼的染色体核型以及异染色质和具有转录活性的核仁组织区在染色体上的分布位置和数量进行了研究。结果表明,布氏石斑鱼二倍体染色体数目为48, 其染色体组由48条端部着丝粒染色体组成, 绝大多数染色体的着丝粒位置呈异染色质带, 仅在第24对染色体靠近着丝粒的位置检测到银染带, 以上结果均表明, 布氏石斑鱼属于较为原始的类群。     

日本东京湾云纹石斑鱼染色体核型研究

采用PHA体内直接注射法制备了云纹石斑鱼(Epinephelus moara TemminckSchlegel1842)东京湾群体头肾组织染色体标本并分析其核型。结果显示,其东京湾群体二倍体染色体数为48,其中,亚中部着丝粒3对;其余21对染色体为端部着丝粒染色体,其染色体臂数NF为54,核型公式为:2n=48,6sm+42t,NF=54;第24对染色体的短臂的形态具有多态性。该鱼染色体臂数高于石斑鱼类的原始核型类群,属于石斑鱼属的特化类群。云纹石斑鱼日本东京湾群体与厦门群体染色体核型存在显著差异,东京湾群体比厦门群体多1对亚中部着丝粒染色体,可能是长期隔离导致染色体重组进化形成的。结果表明,云纹石斑鱼不同地理群体间存在丰富的遗传差异,可利用不同地理群体间的种内杂交优势改良云纹石斑鱼种质。     

石斑鱼染色体研究进展

石斑鱼(Epinephelus)属鲈形目、鮨科石斑鱼亚科、石斑鱼属,约100种,具有分布广、生长快、个体大和肉质鲜美等特点.石斑鱼是分布于热带和亚热带海域的大型鱼类,少数种类在温带水域也可遇见.     

云纹石斑鱼(♀)×鞍带石斑鱼(♂)杂交后代染色体核型分析

本研究对云纹石斑鱼(Epinephelus moara♀)×鞍带石斑鱼(Epinephelus lanceolatus♂)杂交后代利用植物血球凝集素(PHA)及秋水仙素通过活体注射法制作染色体标本。选用头肾和鳃组织制作细胞悬浊液,并对冷滴片和热滴片法进行比较,结合空气干燥法制作染色体分裂相玻片,经吉姆萨染色后,在显微镜下观察筛选清晰完整的分裂相。杂交子代(俗称云龙斑)选取75个分裂相,统计染色体数目并进行核型分析。结果显示,云龙斑二倍体染色体数为48,其中,19对染色体为端部着丝粒染色体,2对为亚中部着丝粒染色体,3对为亚端部着丝粒染色体,其染色体臂数(NF)为58,核型公式为2n=48,4sm+6st+38t。杂交后代云龙斑与父母本染色体数目相同,都为2n=48,但云龙斑染色体与其父母本组型不同,在遗传过程中发生了复杂的染色体重新配对和变异。本研究为杂交后代"云龙斑"的种质鉴定、亲缘关系分析、杂交育种等提供了重要的科学根据。     

棕点石斑鱼(Epinephelus fuscoguttatus♀)×鞍带石斑鱼(Epinephelus lanceolatus♂)F1染色体制备方法及核型分析

探索了在鱼类大小规格不同情况下制备鱼类染色体的最佳策略和方法,并对棕点石斑鱼(Epinephelus fuscoguttatus♀)×鞍带石斑鱼(Epinephelus lanceolatus♂)杂交子代(俗称珍珠龙胆)的染色体核型进行研究.本研究根据预实验的结果,对所选取的珍珠龙胆幼鱼,采用小鱼游泳法进行前处理,取其鳍条并以冷、热两种不同的滴片方法制备染色体,观察染色体形态并对其染色体核型进行分析.结果显示,选用的染色体制备方法能获得图像清晰、形态良好的细胞分裂相;不同的滴片处理方法细胞分裂指数存在一定差异,即热滴片法的有丝分裂指数为2.58％,明显高于冷滴片法的有丝分裂指数(0.83％),此外,热滴片法所获得的染色体的质量也明显优于冷滴片法.珍珠龙胆石斑鱼二倍体染色体数为48,22对染色体为端部着丝粒染色体,2对染色体为亚端部着丝粒染色体,其染色体臂数(NF)为52,核型公式为2n=48,4st+44t.珍珠龙胆与父本鞍带石斑鱼的染色体数目、核型基本一致,而与母本棕点石斑鱼的核型存在很大差异.研究表明,珍珠龙胆的2n数与其亲本相同,其核型特点符合典型的高位类群鱼类核型特征.在对珍珠龙胆与其亲本的核型进行比较的同时,对已报道的25种石斑鱼进行了核型的比较和综合分析,为石斑鱼的种质鉴定、遗传资源保护利用、育种提供基础资料和理论依据.     

巨石斑鱼染色体核型分析

以巨石斑鱼为材料,采用PHA、秋水仙素体外培养肾细胞直接制片法,分析了巨石斑鱼染色体核型.结果表明,巨石斑鱼核型为2n=48=48t,臂数NF=48.     

巨石斑鱼染色体核型分析

以巨石斑鱼为材料,采用PHA、秋水仙素体外培养肾细胞直接制片法,分析了巨石斑鱼染色体核型.结果表明,巨石斑鱼核型为2n=48=48t,臂数NF=48.     

七带石斑鱼的形态和生物学性状

2010年在广东省饶平县柘林湾进行了七带石斑鱼（Epinephelus septemfasciatus）海水网箱人工养殖研究,观察和研究了人工养成的七带石斑鱼的形态特征和生物学性状。通过观察、测量、解剖,对114尾七带石斑鱼的外部形态、可量比性状和内部结构进行了研究。结果表明,七带石斑鱼体呈椭圆形,侧扁,体侧具7条褐色横带,第1条横带仅伸达主鳃盖骨上方边缘,第6条横带分成2条,体侧横带具有不规则的白色斑纹,各鳍边缘白色,背鳍鳍棘部褐色和白色横带相间,鳍条部具有由白色横带延伸而成的3个白斑。胃呈卜型,幽门盲囊发达;肠道长度为体长的0.7～1.4倍,与七带石斑鱼为肉食性鱼类的特征相一致。     

七带石斑鱼仔鱼摄食习性的观察

在人工育苗条件下,采用轮虫与牡蛎受精卵两种开口饵料,3种不同的投喂方式：开口单喂轮虫(A)、开口单喂牡蛎受精卵(B)、开口将牡蛎受精卵和轮虫混合投喂(C),分析对比不同饵料对仔鱼开口效果。通过观察5日龄仔鱼日摄食与4~20日龄仔鱼消化道饱满度的变化,探讨仔鱼摄食习性及光照与摄食的关系。结果发现,七带石斑鱼孵化开口后,投喂3d牡蛎受精卵者比直接投喂轮虫对仔鱼生长有明显的效果;10日龄前,B组在全长、肛前距、口裂宽方面都大于A、C两组,B组全长的特定生长率为4.969%,分别是A、C两组的2.5与2.4倍,生长速度最快;5日龄仔鱼在09∶00~10∶00与13∶00~14∶00时间段,有两个明显的摄食高峰期,光照强度分别为720~1 018 lx与865~923 lx;4日龄仔鱼其饱食率较低,只有40%达到了3级摄食量,4~10日龄的仔鱼饱食率波动幅度较大,10日龄后开始逐渐上升,13日龄后基本可以达到100%。     

Protection conferred against viral nervous necrosis by simultaneous inoculation of aquabirnavirus and inactivated betanodavirus in the sevenband grouper, Epinephelus septemfasciatus (Thunberg)

An aquabirnavirus (ABV) and a formalin-inactivated betanodavirus [redspotted grouper nervous necrosis virus (RGNNV)] were investigated for their potential to prevent RGNNV-induced viral nervous necrosis (VNN) in the sevenband grouper, Epinephelus septemfasciatus (Thunberg). Three groups of fish were injected intramuscularly with ABV, intraperitoneally with inactivated RGNNV (iRGNNV) or with both ABV and iRGNNV. At 3, 7, 14, 21 and 28 days post-injection (p.i.), fish were challenged by intramuscular injection of RGNNV. Control fish, which received neither ABV nor iRGNNV, showed high mortalities in all RGNNV challenges. Fish that received only ABV exhibited relative percent survival (RPS) of >60 against RGNNV challenges at 3, 7, 14 and 21 days p.i., but not at 28 days p.i., while fish that received only iRGNNV showed significantly higher protection against RGNNV challenges only at 21 and 28 days p.i. In contrast, fish that received both ABV and iRGNNV showed 60 or higher RPS against all RGNNV challenges. Fish inoculated with iRGNNV with or without ABV exhibited similar high titres of neutralizing antibodies to RGNNV at 14, 21 and 28 days p.i. These results indicate that combined inoculation with iRGNNV and ABV conferred both rapid non-specific and delayed specific protection against VNN.     

Dry shipper cryopreservation of seven‐band grouper (Epinephelus septemfasciatus Thunberg) spermatozoa

This study examined the usage of a dry shipper for cryopreservation of Epinephelus septemfasciatus (Thunberg) spermatozoa. Milt was diluted 1:49 with 5% dimethyl sulfoxide plus 95% foetal bovine serum for cryopreservation. Computer‐assisted sperm analysis was used to analyse sperm motility, while fertilization and hatching trials were conducted to gauge the applicability of the cryopreservation method for aquaculture. We showed that cooling rates of the dry shipper were stable for 14 days and could be manipulated by the use of different sized freezing straws and use of a simple polystyrene foam container (5 × 5 × 12 cm and 1 cm thickness on all sides with the upper layer exposed). Dry shipper cryopreserved spermatozoa had significantly lower post‐thaw per cent motility and velocity than fresh sperm, but linearity of movement was unchanged. Fertilization and hatching rates were not significantly different at all tested sperm to egg ratios (3000:1–243000:1). The results indicated that 0.33 mL of milt when cryopreserved was sufficient to fertilize up to 450 g of oocytes. Application of this technology will help improve seed production in aquaculture and further develop breeding and genetics studies.     

Neutralizing antibody levels for protection against betanodavirus infection in sevenband grouper, Epinephelus septemfasciatus (Thunberg), immunized with an inactivated virus vaccine

An inactivated betanodavirus, red‐spotted grouper nervous necrosis virus (RGNNV), is a vaccine candidate for viral nervous necrosis (VNN). The present study was conducted to examine inoculation doses of the vaccine and neutralizing antibody titre levels to protect fish against VNN. Young sevenband grouper, Epinephelus septemfasciatus, averaging 25.4 g, were immunized at 25 °C water temperature by a single intraperitoneal injection of formalin‐inactivated RGNNV. Fish immunized at vaccine doses of 10^8.5, 10^8.0, 10^7.5, 10^7.0 and 10^6.5 TCID₅₀ per fish produced antibodies at mean titres of 1:907, 1:511, 1:259, 1:197 and 1:96, respectively, at 20 days post‐immunization (p.i.). Neutralizing antibodies were not detected in any control fish (titre <1:80). When fish were challenged with RGNNV (10^5.0 and 10^4.0 TCID₅₀/fish) at 20 days p.i., cumulative mortalities of the fish groups immunized with 10^8.5, 10^8.0, 10^7.5 and 10^7.0 TCID₅₀ per fish were significantly lower than those of the control group, and the relative percent survival values were higher than 60% in fish groups immunized with 10^7.5 TCID₅₀ per fish or higher doses. However, no significant differences were found in mortality between the group immunized with 10^6.5 TCID₅₀ per fish and the control group. From these results, it was deduced that the minimum effective inoculation dose of the vaccine is 10^7.0 TCID₅₀ per fish and the minimum mean neutralizing antibody titre giving significant protection is approximately 1:200. This antibody titre level is a possible measure of vaccine efficacy against VNN in sevenband grouper, instead of a virus challenge test.     

Protective immunity of sevenband grouper, Epinephelus septemfasciatus Thunberg, against experimental viral nervous necrosis

This paper describes the protective immune responses of sevenband grouper, Epinephelus septemfasciatus Thunberg, immunized with live piscine nodavirus, the causative agent of viral nervous necrosis (VNN), or the Escherichia coli – expressed recombinant coat protein. Nodavirus-neutralizing antibodies were detected at titres ranging from 1:158 to 1:1257 in serum of sevenband grouper which survived intramuscular injection with the virus, by a cell culture assay system. The virus-neutralizing ability of immune serum was also confirmed by injecting virus previously treated with serum into fish. This indicates establishment of acquired immunity in survivors and thus explains why survivors from natural infection are resistant to recurrence of the disease. Young sevenband grouper were immunized twice by intramuscular injections with the recombinant coat protein. Immunized fish produced neutralizing antibodies at high titres for at least 110 days and showed significantly lower mortalities in virus challenge tests. These results suggest the potential for vaccination against VNN in sevenband grouper, which is susceptible to piscine nodavirus at all life-stages.     

Molecular cloning of gonadotropin cDNA in sevenband grouper, Epinephelus septemfasciatus

The cDNA for glycoprotein hormone (GPH) α-subunit, β-subunits of the FSH (GtH I) and LH (GtH II) were isolated, cloned, and sequenced from sevenband grouper pituitary using RACE PCR. These results show that sevenband grouper has two different types of gonadotropins FSH (GtH I) and LH (GtH II), as other teleosts.     

七带石斑鱼和云纹石斑鱼幼鱼在封闭循环水条件下的生长特性

观测研究了七带石斑鱼(Epinephelus septemfasciatus)幼鱼和云纹石斑鱼(Epinephelus moara)幼鱼在封闭式循环养殖系统中的生长特性,用SPSS 17.0软件中的Curve Estimation对相关数据进行模型分析与参数估计。结果表明,经过122 d的养殖,七带石斑鱼幼鱼平均体质量由(114.836±25.343)g增加到(213.861±38.604)g,相对增长率为0.707%,全长(TL)与养殖时间(t)的函数关系式为TL=-0.006t3+1.622t+13.954,体质量(W)与体长(BL)的关系式为W=0.436BL2.055;云纹石斑鱼幼鱼平均体质量由(79.620±13.007)g增加到(238.086±46.307)g,相对增长率为1.631%,全长(TL)与养殖时间(t)的函数关系式为TL=-0.013t2+2.008t+11.540,体质量(W)与体长(BL)的关系式为W=0.018BL3.083。两种幼鱼丰满度生长差异不显著(P0.05),都保持在2.2-3.4之间。     

Histopathological studies on viral nervous necrosis of sevenband grouper, Epinephelus septemfasciatus Thunberg, at the grow-out stage

Abstract Viral nervous necrosis caused by sevenband grouper nervous necrosis virus (SGNNV) has occurred in grow-out stages (0-3 years old) of sevenband grouper, Epinephelus septemfasciatus, since the 1980s. In the present study, based on histopathological features of the central nervous system (CNS) in naturally diseased fish, pernasal infection experiments using grow-out fish were performed and pernasal infection was established as a putative invasion route of SGNNV. The definite SGNNV-targeted cells were determined by histopathological studies including indirect fluorescent antibody test and electron microscopy. Nerve cells in the olfactory lobe were most extensively necrotized with vacuolation followed by infiltration of microglia and macrophages. Purkinje cells and Golgi cells were extensively infected in the cerebellum. Megalocells and small nerve cell nuclei were also infected in the preoptic area, thalamus, medulla oblongata and spinal cord. Only a few small nerve cells were infected in the olfactory bulb and optic tectum. The retina of some diseased fish displayed vacuolated bipolar cells of the inner nuclear layer and in the ganglion cell layer. These SGNNV-infected nerve cells displayed viroplasmic inclusions containing virions, vacuoles and myelin-like structures. Based on observed histopathological changes, the lesion of the CNS was characterized by encephalitis but not encephalopathy.     

Field tests of Poly(I:C) immunization with nervous necrosis virus (NNV) in sevenband grouper, Epinephelus septemfasciatus (Thunberg)

It was recently reported that Poly(I:C) immunization with live nervous necrosis virus (NNV) confers protection in sevenband grouper, Epinephelus septemfasciatus (Thunberg), from NNV infection. In the present study, we conducted field tests with sevenband grouper for the evaluation of Poly(I:C) immunization efficacy. In the first experiment, sevenband grouper were immunized with NNV followed by Poly(I:C) administration 7 weeks before natural occurrence of viral nervous necrosis (VNN). Survival rate of the naïve fish was 71.0%, whereas that of the immunized fish was 99.8%. In the second experiment, sevenband grouper were immunized 10 months before VNN occurrence and survival rate of the non‐treated and vaccinated fish was 79.5% and 97.5%, respectively. In the third experiment, we administered Poly(I:C) to sevenband grouper at 20 days after natural occurrence of VNN. The survival rate of the non‐treated fish was 9.8%, whereas that of fish administered Poly(I:C) was 93.7%. Based on these results, it was concluded that Poly(I:C) immunization conferred protection in fish against NNV infection in field tests and the protection lasted more than 10 months. Furthermore, even after occurrence of VNN, fish mortality could be reduced by Poly(I:C) administration and there was an unexpected curative effect on VNN‐affected fish.     

七带石斑鱼繁殖群体“突眼”症病原菌的分离与鉴定

2007~2008年间,对暴发"突眼"症的七带石斑鱼繁育群体,从细菌学、寄生虫学和病毒学等方面进行了全面的筛查和实验研究。从具有明显"突眼"症的七带石斑鱼病灶组织中分离出1株致病力极强的优势菌CB1008,经人工感染试验证实为此次七带石斑鱼"突眼"症的致病菌。该菌革兰氏染色呈阴性,电镜下观察菌体呈短杆状,极生单鞭毛。通过API-32E鉴定系统和菌体常规形态特征、培养特性和生理生化反应指标测定,以及16S rDNA测序分析等综合鉴定,菌株CB1008为弧菌属哈维氏弧菌Vibrio harveyi。药敏试验结果表明,该菌株对氯霉素、萘啶酸、四环素、氟哌酸和氟罗沙星等5种药物较为敏感。