谷氨酰胺对斜带石斑鱼GF-1细胞中C-Myc蛋白的表达与神经坏死病毒复制的影响

为探讨C-Myc表达、谷氨酰胺代谢和神经坏死病毒复制三者之间的关系,本研究首先克隆了斜带石斑鱼鳍条细胞(GF-1)中的C-Myc基因(GF-1-C-Myc),结果显示GF-1-CMyc基因cDNA全长814 bp,开放阅读框(ORF)为285 bp,编码95个氨基酸(aa),有亮氨酸拉链结构域与螺旋-环-螺旋(HLH)结构域。实验表达和纯化了GF-1-C-Myc蛋白,并制备其多克隆抗体。采用实时定量PCR技术(qRT-PCR)与免疫印迹法(WB)检测了GF-1-C-Myc基因的表达和神经坏死病毒的复制。结果显示,缺乏谷氨酰胺会同时抑制GF-1-C-Myc基因的表达和神经坏死病毒(NNV)的复制,添加谷氨酰胺可同时促进GF-1-C-Myc的表达和NNV的复制;此外,NNV感染可上调GF-1-C-Myc基因的表达,并显著消耗GF-1细胞培养液中的谷氨酰胺。研究表明,GF-1-C-Myc基因可调控宿主谷氨酰胺代谢,从而有利于神经坏死病毒的复制。本结果为防控NNV的感染提供了参考。     

生物饵料对赤点石斑鱼不同养殖阶段的影响

通过对赤点石斑鱼(Epinephelus akaara)亲本强化、苗种繁育阶段的饵料进行研究,结果表明:采用牡蛎作为赤点石斑鱼亲本营养强化饵料使得亲本成活率达89.33%,所产的卵子平均受精率达78.9%、孵化率达80.1%,均显著高于使用南美白对虾和蛏子组(P<0.05);苗种培育至摄氏卤虫阶段,采用鱼油(4 g/L)与经小球藻强化轮虫和虾青素(5 g/L)混合代替,成活率29.53%,全长增长率175.00%,效果最佳;苗种培育至摄氏桡足类阶段,采用鱼油(4 g/L)与桡足类和虾青素(5 g/L)培育,成活率和全长增长率均显著高于其他组(P<0.05);而后期采用软颗粒料(鳗粉:鳀鱼=3:2,添加虾青素5 g/kg)可极显著提高赤点石斑鱼的养成成活率和全长增长率(P<0.01)。     

Effects of dietary protein and lipid levels with different protein‐to‐energy ratios on growth performance,feed utilization and body composition of juvenile red‐spotted grouper,Epinephelus akaara

An 8‐week feeding trial was conducted to assess dietary protein and lipid levels on growth performance, feed utilization and body composition of juvenile red‐spotted grouper (7.85 ± 0.03 g fish^?1). Nine semi‐purified diets were formulated containing varying protein levels (440–520 g kg^?1, dry matter) and lipid levels (60–120 g kg^?1, dry matter). The weight gain of juvenile Epinephelus akaara was affected by dietary protein (p = .005) and its interaction with dietary lipid (p = .020). Viscerosomatic index, intraperitoneal fat ratio and whole‐body lipid level increased with increasing dietary lipid level (p < .001). Nitrogen retention was not affected by dietary protein and lipid, while lipid retention decreased with increasing dietary lipid level (p < .001). The plasma blood urea nitrogen increased with increasing dietary protein level (p = .003). This study showed that diet with 520 g kg^?1 protein and 60 g kg^?1 lipid with 30.58 mg kJ^?1 P:E provided a maximal growth for this species. Moreover, an increase in dietary lipid levels (from 60 to 90 g kg^?1) could reduce the protein requirement (from 520 to 480 g kg^?1) without affecting the growth performance, while higher fat deposition was observed in fish fed high‐lipid diets.     

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。     

Vibrio harveyi (VirB11) recombinant vaccine development against vibriosis in orange‐spotted grouper (Epinephelus coioides)

Despite significant improvements in aquaculture to compensate wild catch, disease organisms have thrived in limiting its national and global potential. Using antibiotics, in a bid to remedy the havoc, has given rise to complications, attracting attention to disease prevention by immune enhancement against diseases. Grouper production has been inhibited for the threats of bacterial infection, particularly of Vibrio origin. Considering the rise in vibriosis cases, improved vaccines are necessary; moreover, recombinant vaccines, the choice for trial in the present experiment have been effective and more specific in improving immunity. The current work deals with grouper immune system enhancement with a recombinant vaccine developed from VirB11 gene in Vibrio harveyi. VirB11 was cloned in V. harveyi for recombinant vaccine development against vibriosis in orange‐spotted grouper (Epinephelus coioides). As indicated by the results, recombinant VirB11 protein showed effectiveness in conferring protection against vibriosis with observable specific antibody response in enzyme‐linked immunosorbent assay (ELISA) analysis; a significant increase (p < 0.05) in antibody levels was observed after a week and after 8 weeks post‐vaccination. From the weeks post‐vaccination, log2 (antibody titres) in the sera of vaccinated groups reached a peak of 14.2 at week 5 in the vaccinated group in comparison with a peak of approximately 5 and 2 in adjuvant and PBS controls. As indicated by the challenge results, 90% relative survival was observed in vaccinated group and 13% relative survival in control group I (adjuvant control). The cumulative performance of protein concludes VirB11 commendable for recombinant vaccine development.     

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。     

荧光定量PCR法检测点带石斑鱼神经坏死病毒研究

根据己公布的神经坏死病毒全基因序列,应用引物设计软件依据Gene Bank所收录的各种石斑鱼神经坏死病毒RNA2基因组全序列选择保守区域,根据Taqman探针引物设计原则,设计Taq Man探针及其引物。采用Taq Man探针技术,并且将假病毒技术构建的含有神经坏死病毒RNA2基因组的MS2噬菌体假病毒作为阳性质控品,建立Taq Man探针检测点带石斑鱼神经坏死病毒的荧光定量PCR方法,拟合标准曲线,能够在病毒检测中进行绝对定量,能检测到10个病毒拷贝模板数,在临床检测中得到很好运用。本研究建立实时荧光定量RT-PCR,有助于日常检验工作,为鱼类神经坏死病毒的筛选和监测提供基础,有助于控制神经坏死病的疫情传播。     

Rapid detection of SNPs in candidate genes regulating the growth of orange‐spotted grouper,Epinephelus coioides (Hamilton, 1822), using semiconductor sequencing

Orange‐spotted grouper (Epinephelus coioides) is one of the most important marine food species in Southeast Asia and China and has been cultured for decades. In this study, we fully utilized the limited capacity of semiconductor sequencing, the high efficiency of long‐range PCR for target enrichment and a non‐indexed pooling strategy to screen single‐nucleotide polymorphisms (SNPs) in a breeding population of orange‐spotted grouper. Forty‐one genomic DNA fragments, with a total length of approximately 180 kb, including 22 candidate genes that control growth, and from a DNA pool of 20 heaviest and 22 lightest individuals of the sampled population were successfully sequenced using an Ion Torrent Personal Genome Machine. 3 503 466 clear reads were produced with a length of 192 ± 56 bp, 86.8% of which were mapped to the reference with an average coverage depth of 2567‐fold and physical coverage of 98.8%. Finally, 1623 high‐quality SNPs were adopted. Compared with Sanger sequencing of three random common regions, the sensitivity and specificity of our approach were 39.4% and 100.0% respectively. A mutation located at the third position of the previously labelled start codon of growth hormone receptor type 1 invalidated the start codon. Furthermore, comparison of the frequencies of genotypes and alleles of this site between the two extreme groups, prediction of signal peptide and identification of conservative mRNA sequences suggested that the functional start codon is likely located at the position of another downstream in‐frame ATG in the mutant. These detected SNP markers will provide important tools for the selective breeding of orange‐spotted grouper.     

温度变化对七带石斑鱼早期发育及开口摄食的影响

观察比较了9个温度梯度(13、15、17、19、21、23、25、27、29℃)对七带石斑鱼(Epinephelus septemfasciatus)受精卵孵化时间、孵化率和畸形率的影响。用在(21±0.5)℃条件下孵出的健康仔鱼进行耐饥饿和摄食实验,实验温度处理设两种方式:一、处理温度始终保持不变;二、将温度在处理48 h后统一调节至21℃。实验期间,每天统计仔鱼死亡数,测定不投饵条件下的存活指数(SAI),并观察仔鱼投饵后的开口摄食情况及其形态发育状况。结果表明:1)受精卵孵化的最适温度范围为17~23℃,当温度低于13℃或高于27℃时,受精卵不能孵化。21℃时,受精卵孵化率最高,为(93.67±1.52)%;而畸形率最低,为(1.06±1.06)%。当温度低于或高于21℃时其孵化率降低,而畸形率升高;2)在13~28℃范围内,初孵仔鱼的SAI值随着温度的上升先升高后逐渐降低,21℃时的SAI值最高,为(20.26±0.44)%。各组温度保持不变时,13℃和27℃的SAI值分别为(2.18±0.01)%、(8.47±0.28)%;调整一致后分别为(6.90±0.44)%、(13.30±0.31)%。温度调节组与不调节组相比,其SAI值明显提高。温度调节后,每个梯度组的畸形率也出现了不同程度的降低;3)不同温度处理的仔鱼开口摄食存在差异。21℃时,仔鱼的初始摄食率和饱食率分别可达80.0%和40.0%。随着温度偏离21℃,摄食率和饱食率均逐渐降低。温度调节恢复组的仔鱼摄食情况改善明显,其中17℃不变和恢复组对比变化最为明显:当保持17℃不变时仔鱼的摄食率和饱食率分别为20.0%、13.3%,而处理48 h后恢复到21℃后,两者分别提高到66.7%和33.3%,分别提高了2.3和1.5倍。     

Antiparasitic activity of the medicinal plant Dillenia suffruticosa against the marine leech Zeylanicobdella arugamensis (Hirudinea) and its phytochemical composition

Zeylanicobdella arugamensis (Annelida: Hirudinea), a marine parasitic leech, is currently affecting different species of cultured groupers, hybrid groupers, snappers and sea bass in Malaysia. Dillenia suffruticosa (Dilleniaceae), a medicinal plant found in Sabah, has been selected in our experiment to kill the leeches as a natural control method. The leech‐infested hybrid groupers were collected from aquaculture facilities, and the isolated leeches were challenged against methanol extract of D. suffruticosa leaves. The experiment was carried out using various concentrations of the extracts such as 25, 50 and 100 mg/ml. The methanol extract showed significant antiparasitic activity against Z. arugamensis with 100% mortality at a concentration of 100 mg/ml in 14.39 ± 3.75 min., followed by 50 and 25 mg/ml in 32.97 ± 9.29 and 41.77 ± 5.40 min., respectively. The phytochemical composition of the extract was determined using GC‐MS analysis to understand the nature of the principal compounds responsible for its antiparasitic properties. The leaves of D. suffruticosa demonstrated the presence of different bioactive compounds of various natures with varying percentages. Thus, it could be revealed that the methanol extract of D. suffruticosa mainly contains vital phytochemical compounds and showed an effective antiparasitic activity against the harmful leeches of hybrid groupers.