循环水高密度养殖珍珠龙胆石斑鱼效果研究

为研究循环水高密度养殖珍珠龙胆石斑鱼[Epinephelus lanceolatu(♂)×Epinephelus fuscoguttatus(♀)]的养殖效果,在自行研制的循环水养殖系统中进行了试验。试验中对珍珠龙胆石斑鱼生长指标及养殖系统主要水质指标进行分析测定。结果显示,养殖期间的水质指标:水温26~29℃,盐度25~30,溶氧(DO)≥8 mg/L,氨氮浓度0.20~1.16 mg/L,亚硝酸盐氮0.05~0.40 mg/L。试验共持续250 d,分3个生长阶段:第1阶段87 d,密度由13.82 kg/m3增加到28.89 kg/m3,存活率95.28%,平均体重由(150±18)g增加到(329±42)g,特定生长率(SGR)为(0.90±0.06)%;第2阶段106 d,密度由28.89 kg/m3增加到53.36 kg/m3,存活率90.44%,平均体重由(329±42)g增加到(672±66)g,SGR为(0.67±0.02)%;第3阶段57 d,密度由46.98 kg/m3增加到69.50 kg/m3,存活率98.6%,平均体重由(676±52)g增加到(1 014±75)g,SGR为(0.71±0.02)%。养殖期间的平均SGR为(0.76±0.02)%,总存活率84.9%,饲料系数1.04,投入产出比为1∶2.02。本研究成果可为高密度养殖珍珠龙胆石斑鱼提供参考。     

云纹石斑鱼♀与赤点石斑鱼♂杂交的初步研究

为石斑鱼种间杂交经济型利用提供基础资料,以云纹石斑鱼为母本,赤点石斑鱼为父本进行种间远源杂交,对杂交子一代的生长及越冬作了系统观察.结果表明,用人工授精的方法可以取得杂交受精卵,受精卵的受精率、孵化率均在90％左右,杂交子一代比对照组仔鱼畸形率高;杂交子一代的生长趋势接近母本云纹石斑鱼,体色接近父本赤点石斑鱼,在室内可以正常越冬.云纹石斑鱼♀与赤点石斑鱼♂进行经济型杂交是可行的.     

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.     

鱼源芽孢杆菌在石斑鱼稚鱼育苗中的应用研究

利用芽孢杆菌SE5和DE5单独及联合强化桡足类,研究SE5和DE5对斜带石斑鱼稚鱼生长、存活率、水质及水体和稚鱼体内细菌总数和弧菌数量的影响。试验结果表明,饲养14d时,SE5组和DE5组体长显著高于对照组,混合组体长和体质量显著高于对照组。饲养28d时,SE5组和DE5组体长和体质量均高于对照组,混合组体长和体质量显著高于对照组。饲养28d时,SE5组和DE5组存活率显著高于对照组。14d时SE5组和混合组水体NH3-N显著低于对照组,28d时各组NH3-N均低于对照组。14d时,混合组水体NO2-N显著高于对照组,28d时各试验组NO2-N均显著高于对照组。试验结束时(28d),各试验组水体细菌总数均高于对照组,SE5组和DE5组水体弧菌数量高于对照组;DE5组和混合组活饵细菌总数高于对照组,各试验组活饵弧菌数量高于对照组,其中混合组显著高于对照组;各试验组稚鱼体内细菌总数高于对照组,其中DE5组显著高于对照组。DE5组稚鱼体内弧菌数量显著高于对照组,SE5组高于对照组。     

斜带石斑鱼神经坏死病毒CP基因 shRNA干扰载体的构建及效果评价

利用Invitrogen公司的在线生物学软件分析斜带石斑鱼神经坏死病毒CP基因,设计针对CP基因不同位置的小发卡RNA(short hairpin RNA,shRNA)干扰序列[其结构特征为正链(19 nt)-环(4 nt)-负链(19 nt)]。化学合成这些序列,并退火连接为双链干扰片段,将双链干扰片段定向克隆到干扰载体pENTRTM/U6中,构建shRNA干扰载体pshRNA-124、pshRNA-896和pshRNA-NNV。然后,用脂质体转染法分别将3种shRNA干扰载体和pEGFP-CP基因共转染导入黑头呆鱼(FHM)肌肉细胞,荧光显微镜观察细胞荧光强度,分析荧光抑制效率,Real-time RT-PCR检测CP基因mRNA的表达水平变化。结果表明,在pEGFP-CP与shRNA干扰载体共转染组,pshRNA-124、pshRNA-896、pshRNA-NNV的荧光抑制效率分别为47%、68%、51%。3种shRNA干扰载体都有干扰效果,均能干扰绿色荧光蛋白的表达,其中pshRNA-896干扰效率最好。Real-time RT-PCR检测表明,干扰质粒pshRNA-124、pshRNA-896、pshRNA-NNV对pEGFP-CP基因的沉默效率分别约为60%、96%和55%,与对照组相比差异显著(P<0.05)。研究表明,靶向斜带石斑鱼神经坏死病毒CP基因的shRNA干扰载体构建成功,为进一步运用RNA干扰技术进行CP基因的功能研究奠定了基础。     

Infection and pathology in Queensland grouper,Epinephelus lanceolatus, (Bloch), caused by exposure to Streptococcus agalactiae via different routes

Since 2007, 96 wild Queensland groupers, Epinephelus lanceolatus, (Bloch), have been found dead in NE Australia. In some cases, Streptococcus agalactiae (Group B Streptococcus, GBS) was isolated. At present, a GBS isolate from a wild grouper case was employed in experimental challenge trials in hatchery‐reared Queensland grouper by different routes of exposure. Injection resulted in rapid development of clinical signs including bilateral exophthalmia, hyperaemic skin or fins and abnormal swimming. Death occurred in, and GBS was re‐isolated from, 98% fish injected and was detected by PCR in brain, head kidney and spleen from all fish, regardless of challenge dose. Challenge by immersion resulted in lower morbidity with a clear dose response. Whilst infection was established via oral challenge by admixture with feed, no mortality occurred. Histology showed pathology consistent with GBS infection in organs examined from all injected fish, from fish challenged with medium and high doses by immersion, and from high‐dose oral challenge. These experimental challenges demonstrated that GBS isolated from wild Queensland grouper reproduced disease in experimentally challenged fish and resulted in pathology that was consistent with that seen in wild Queensland grouper infected with S. agalactiae.     

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.     

Microbial communities associated with early stages of intensively reared orange‐spotted grouper (Epinephelus coioides)

The gut microbiota plays key roles in the health and general welfare of fish larvae, the present study characterized the bacterial communities associated with grouper Epinephelus coioides larvae during a period of 22 days post hatch (DPH) in an intensive hatchery using both cultivation‐based and cultivation‐independent approaches. Both approaches confirmed that bacteria were present in the gut of larvae before and after the onset of exogenous feeding, and the number of cultiviable bacteria increased gradually from 2 DPH to 22 DPH. A more complex bacterial profile was present in larvae fed fertilizer oyster eggs for 4 days (8 DPH), probably as a result of the onset of exogenous feeding. Interestingly, similar internal microbiota were observed in larvae fed fertilized oyster eggs for 4 days (8 DPH) and rotifers for 2 weeks (22 DPH), although different microbial communities were present in the two feeds. This might suggest that the gut environment of E. coioides larvae selects for a common microbiota, which is more closely related with the rearing water than the two feeds. Therefore, bacterial community of the rearing water may play a critical role in the establishment of gut microbiota of fish larvae and more attention should be paid to its practical modulation by using probiotics. In addition, some potentially beneficial bacteria, such as Lactococcus spp., were the major components of the microbiota associated with fertilized oyster eggs, while these bacteria were not detected in larvae samples.