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)。     

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.     

The effects of the tank colour on growth performance and physiological responses in fingerling grouper,Epinephelus coioides

In this study, the effects of different background colours on growth performance and physiological parameters of Epinephelus coioides were investigated. One hundred eighty E. coioides (average weight 18.16 ± 0.07 g) were distributed in black, white and blue 300‐L tanks and fed to satiation for 60 days. The tank colour had no significant effect on the weight gain, condition factor, hepatosomatic index and specific growth rate (p > .05). The lowest amount of viscerosomatic index and the highest carcass protein were recorded in the white tank (p < .05). Daily food intake, feed conversion ratio and plasma glucose were significantly higher in the blue tank (p < .05). The highest levels of haematocrit and plasma triglyceride were observed in the white tank, while the total protein and albumin were higher in the black tank compared to the other tanks. The tank colour had no significant effects on the plasma cortisol level (p > .05). Our findings showed that colours used in this study did not evoke stress and thus had no effect on growth, but some metabolic adaptation occurred in each colour to achieve optimum growth.     

clpV is a key virulence gene during in vivo Pseudomonas plecoglossicida infection

Interaction between bacterial pathogen and aquatic animal host is exceedingly complex, which involves large dynamic changes in gene expression during different stages of the disease. However, research on identifying key virulence genes based on the dynamics of gene expression changes of a one‐sided bacterial pathogen in tissue has not been reported so far across different stages of infectious disease. The clpV for the T6SS of Pseudomonas plecoglossicida was identified for a candidate for key virulence gene based on dynamic changes of gene expression. For the Epinephelus coioides infected using clpV‐RNAi strain, no deaths were observed up to 20 dpi. The spleens, kidneys and livers of all the E. coioides that received clpV‐RNAi strain failed to develop visible nodules at 5–8 dpi, with the swelling gradually disappearing. The burdens of clpV‐RNAi strain in the spleen and blood were greatly reduced at most of the time points after injection, and the burdens of clpV‐RNAi strain in the head kidneys and trunk kidneys also had a sharp reduction from 72 to 120 hpi. This paper provides a new insight into the discovery of key virulence genes of pathogens in infected tissue systems.     

广西工厂化循环水养殖石斑鱼水质处理效果

通过集成海水养鱼、生化处理、设施工程、环境工程、电器自控和水质监控等技术,建立一套适合广西地区应用的工厂化循环水石斑鱼养殖系统。该养殖系统处理流程为:鱼池→沉淀池→蛋白质分离器→生物滤池→海马齿浮床→紫外杀菌,生物滤池包括A、B、C三个通道,A、B通道均由珊瑚石作为一、二级生物过滤和生化球作为三级过滤,C通道由珊瑚石作为一级过滤和椰子壳碎作为二、三级过滤。结果显示:沉淀池处理可以显著降低鱼池的COD,氨氮、亚硝酸盐氮、硝酸盐氮和磷酸盐的质量浓度(P<0.05);经海马齿浮床系统处理后,硝酸盐氮质量浓度显著低于沉淀池、蛋白质分离器处理池、生物滤池A、B通道(P<0.05);整体上,C通道水质指标略低于A、B通道,但差异不明显(P>0.05)。该研究结果可为广西沿海发展循环水养殖技术以及养殖生产方式转变提供可靠的科学数据和技术参考。     

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。     

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.     

硒源及硒水平对斜带石斑鱼幼鱼生长性能、免疫酶活性和全鱼及脊椎骨硒含量的影响

本试验在不同硒源及硒水平下,研究其对斜带石斑鱼(Epinephelus coioide)幼鱼的生长性能、免疫酶活性和全鱼及脊椎骨硒含量的影响,确定斜带石斑鱼幼鱼对饲料中硒的最适需要量以及相对生物学利用率。选取810尾初重为(8.99±0.03)g的斜带石斑鱼幼鱼,随机分为9组,每组3个重复。试验分别以亚硒酸钠(Se-S)和蛋氨酸硒(Se-Met)为硒源,在基础饲料中分别添加0(对照)、0.3、0.6、0.9和1.2mg/kg的硒。基础饲料硒水平为0.48mg/kg,Se-S添加组饲料硒水平分别为0.82、1.15、1.32和1.79mg/kg;Se-Met添加组饲料硒水平分别为0.81、1.18、1.51和1.94mg/kg。养殖周期为10周。结果表明:1)饲料中不同硒水平对斜带石斑鱼幼鱼增重率(WGR)和特定生长率(SGR)均有显著影响(P<0.05),对斜带石斑鱼幼鱼的成活率(SR)和饲料系数(FCR)无显著影响(P>0.05)。以Se-S为硒源,当饲料硒水平为1.15mg/kg时,WGR和SGR最高;以Se-Met为硒源,当饲料硒水平为1.18mg/kg时,WGR和SGR最高。2)斜带石斑鱼幼鱼血清碱性磷酸酶(AKP)及肝脏总超氧化物歧化酶(T-SOD)、谷胱甘肽过氧化物酶(GSH-Px)、谷胱甘肽还原酶(GR)活性和丙二醛(MDA)含量均受到饲料硒水平的显著影响(P<0.05)。3)鱼体和脊椎骨硒含量均随着饲料中硒水平的增加而呈线性升高,且脊椎骨硒含量低于鱼体。综上所述,分别以Se-S和Se-Met为硒源,SGR为评价指标,斜带石斑鱼幼鱼对饲料中硒的最适需要量分别为0.98和1.01mg/kg。在本试验条件下,斜带石斑鱼幼鱼Se-Met的生物利用率比Se-S高,相当于Se-S的112.5%。     

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, Ec MR)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全长4 793 bp,共编码1 446个氨基酸。Ec MR的蛋白结构域包括1个信号肽(signal peptide)、1个富含蓖麻类β型三叶草结构域(RICIN)、1个Ⅱ型纤维连接蛋白结构域(FNⅡ)、8个串联的C-型凝集素样结构域(CLECTs)以及1个跨膜结构域(transmembrane region)。实时定量PCR(qRTPCR)和细胞免疫荧光(IF)分析结果显示,Ec MR在斜带石斑鱼的8个组织中均有表达,其mRNA的相对表达量顺序为鳃头肾脑脾脏肝脏外周血心脏肌肉。在研究Ec MR是否参与RGNNV入侵过程中时发现,RGNNV可以在GF-1细胞系中快速增殖,同时显著激活Ec MR的表达。为进一步探究RGNNV对GF-1细胞系的影响,本实验通过双染色法检测了RGNNV感染GF-1细胞系后的细胞凋亡情况,研究表明,RGNNV感染可以促进GF-1细胞系的凋亡,并且细胞凋亡率随着RGNNV感染时间延长而增加。同时,qRT-PCR和酶活性检测结果显示,RGNNV的感染可以显著促进凋亡相关基因的转录水平以及Caspase-3、Caspase-8、Caspase-9的酶活性水平。综上所述,本研究成功克隆了EcMR,并揭示了其在RGNNV入侵过程中一定的相关性。本研究结果将为石斑鱼病毒性疾病的防控提供参考。