饥饿对条石鲷仔稚鱼生长发育的影响

采用实验生态学方法研究了饥饿对条石鲷仔稚幼鱼的影响.结果表明,水温18、22和26 ℃时,初孵仔鱼的PNR点分别出现在6～7 d、6～7 d和4～4.5 d.仔鱼开口摄食时间随温度降低而滞后,22 ℃时初次摄食率最高,26 ℃时初次摄食率降至50%以下.温度对仔稚幼鱼耐饥饿的能力影响明显,18和22 ℃时,前期仔鱼半数死亡时间为4～5 d,而13日龄仔鱼半数死亡时间为2.5 d,24日龄稚鱼的为1.5～2 d,实验鱼饥饿3 d后,大部分已经不能恢复摄食.在26 ℃时,前期仔鱼和13日龄仔鱼的半数死亡时间为1.5 d,24日龄稚鱼为2～2.5 d,而45日龄幼鱼在22和26 ℃时半数死亡时间都为6 d,表明随着生长发育,苗种对饥饿耐受能力明显增强.饥饿对条石鲷幼鱼的生长影响显著(P＜0.05),饥饿条件下的仔鱼全长和体重增加远远低于对照组,甚至出现体重的负增长,30日龄幼鱼在饥饿条件下的体重损失率达到50%以上,而45日龄幼鱼饥饿后的体重损失率约为10%,13日龄仔鱼阶段是条石鲷早期生长发育阶段中较为敏感的阶段.     

Effects of water temperature on mortality in Megalocytivirus‐infected rock bream Oplegnathus fasciatus (Temminck et Schlegel) and development of protective immunity

Rock bream iridovirus (RBIV) causes huge losses, especially in rock bream Oplegnathus fasciatus. Rock bream injected with RBIV and held at 29, 26, 23 or 20 °C had 100% mortality. Conversely, all infected fish held at 17 °C survived even after the temperature was progressively increased to 26 °C at 100 dpi. Rock bream exposed to virus and held for 2, 4 and 7 days at 23/26 °C before the temperature was reduced to 17 °C had mortality rates of 26.6/73.2%, 66.6/100% and 93.4/100%, respectively, through 100 dpi. When surviving fish had the water temperature increased from 17 to 26 °C at 100 dpi, they did not exhibit signs of disease and had low virus copy numbers (below 10³). To investigate the development of a protective immune, rock bream were infected with RBIV and held at 23 °C before shifting the water temperature to 17 °C at 4 dpi. All injected fish survived until 120 dpi. While 100% of the previously unexposed fish died, 80.2% of the previously infected fish survived. When the survivors were rechallenged again at 160 dpi, no further mortality occurred. The high survival rate of fish following rechallenge with RBIV indicates that protective immunity was established in the surviving rock bream.     

Dietary eicosapentaenoic acid requirement of juvenile rock bream,Oplegnathus fasciatus

A feeding trial was conducted to evaluate the optimum dietary level of eicosapentaenoic acid (EPA, 20:5n‐3) based on growth and non‐specific immune responses in juvenile rock bream. A basal diet without EPA supplementation was used as a control, and six other diets were prepared by supplementing with 4, 8, 12, 16, 20 or 40 g of EPA per kg diet. The actual EPA concentrations of the diets were 0.5, 4.3, 8.5, 13.0, 16.8, 21.0 and 41.2 g of EPA per kg diet, and the diets were abbreviated as EPA_0.5, EPA_4.3, EPA_8.5, EPA_13.0, EPA_16.8, EPA_21.0 and EPA_41.2, respectively. Triplicate groups of fish averaging 1.06 ± 0.01 g (mean ± SD) were fed one of the seven experimental diets at the apparent satiation for 8 weeks. At the end of the feeding trial, weight gain, specific growth rate and feed efficiency of fish fed EPA_16.8, EPA_21.0 and EPA_41.2 diets were significantly higher than those of fish fed EPA_0.5, EPA_4.3, EPA_8.5 and EPA_13.0 diets (p < .05). Superoxide dismutase activity of fish fed EPA_16.8, EPA_21.0 and EPA_41.2 diets were significantly higher than those of fish fed EPA_0.5, EPA_4.3 and EPA_8.5 diets. Fish fed EPA_21.0 and EPA_41.2 diets showed significantly higher lysozyme activity than did fish fed EPA_0.5, EPA_4.3, EPA_8.5 and EPA_13.0 diets. The broken‐line analysis of weight gain indicated that the optimum dietary EPA level was 16.7 g/kg diet. These results suggested that the optimum dietary EPA level in juvenile rock bream could be greater than 16.7 g/kg diet but less than or equal to 16.8 g/kg diet based on the broken‐line analysis and the ANOVA test of weight gain.     

Expression and serological application of a capsid protein of an iridovirus isolated from rock bream, Oplegnathus fasciatus (Temminck & Schlegel)

Iridoviruses infect a wide variety of wild and cultured fish. Those iridoviruses belonging to the genus Ranavirus, in the Iridoviridae family, cause systemic disease in infected animals with a high morbidity and mortality. This paper reports the cloning, sequencing, and expression of the rock bream iridovirus (RBIV) major capsid protein (MCP) in an Escherichia coli expression system for subsequent immunological studies. The completeness of the expressed protein was confirmed by peptide mass fingerprinting (PMF) analysis using MALDI-TOF MS. The recombinant MCP (rMCP)-specific mouse polyclonal antibody reacted with the viral 52 kDa protein, indicating that this rMCP induces an immunological response. Fish antibodies induced against iridovirus infection were also detected using ELISA when rMCP was used as an antigen. As a result, it was found that many cultured rock bream (92.5%) were naturally infected with iridovirus and that the rMCP might be useful for serological tests.     

南海区驯养条石鲷亲鱼的初次性成熟和产卵

2008年4月初在广东省饶平县对2006年5月人工孵化和培育的2龄条石鲷（ Oplegnathus fasciatus）成熟亲鱼进行激素诱导,研究和观察条石鲷亲鱼初次性成熟的繁殖生物学。结果显示,在南海区全人工养殖的条石鲷亲鱼初次性成熟年龄为2龄,成熟亲鱼的最小型为全长245 mm、体质量610 g,最大个体为全长300 mm、体质量1450 g;产卵季节为4月10日~7月15日,产卵盛期为4月中旬至6月下旬;为升温产卵型鱼类,产卵温度为20.0~28.8℃,适宜的产卵温度为20.7~27.6℃;雌、雄亲鱼发育同步,个体大小和成熟年龄差别不大;雌鱼属于一年一次分批产卵类型,产卵期超过3个月;24尾初次性成熟雌性亲鱼的总产卵量为3180.0×104粒,日最高产卵量为341.5×104粒;其产卵量和受精卵质量与水温的变化关系密切,受精卵的浮卵率随温度的变化而变化,在水温相对衡定时,浮卵率相对稳定并维持在80%~95%的较高水平;受精卵平均卵径为（0.860±0.023）mm,油球径为（0.191±0.009）mm。     

The optimum dietary docosahexaenoic acid level based on growth and non‐specific immune responses in juvenile rock bream,Oplegnathus fasciatus

A feeding trial was conducted to determine the optimum level and effect of incremental dietary levels of docosahexaenoic acid (DHA, 22:6n‐3) on growth and non‐specific immune responses in juvenile rock bream, Oplegnathus fasciatus. A basal diet without DHA supplementation was used as a control, and six other diets were prepared by supplementing with 4, 8, 12, 16, 20 or 40 g kg^?1 DHA. These diets included no eicosapentaenoic acid and/or arachidonic acid contents. The actual DHA concentrations of the diets were 1, 4.8, 8.9, 13.1, 17.6, 21.2 and 41.4 g kg^?1 diet (DHA_1.0, DHA_4.8, DHA_8.9, DHA_13.1, DHA_17.6, DHA_21.2 and DHA_41.4 respectively). At the end of feeding trial, final body weight, weight gain, specific growth rate and feed efficiency of fish fed the DHA_13.1, DHA_17.6, DHA_21.2 and DHA_41.4 diets were significantly higher than those fed the other diets (P < 0.05). The broken‐line analysis of weight gain indicates that the optimum dietary DHA level is 11.9 g kg^?1. Fish fed DHA_1.0 had the highest hepatosomatic index, an increase in plasma cholesterol, triglyceride, low‐density lipoprotein and aspartate aminotransferase levels, as well as a decrease in high‐density lipoprotein. Superoxide dismutase activity of fish fed DHA_13.1 and DHA_17.6 diets was significantly higher than that of fish fed DHA_1.0, DHA_4.8 and DHA_8.9 diets. Fish fed the DHA_17.6, DHA_21.2 and DHA_41.4 diets showed significantly higher lysozyme activity than those fish fed DHA_1.0, DHA_4.8 and DHA_8.9 diets. Therefore, the optimum dietary DHA level could be greater than 11.9 g kg^?1 but less than 13.1 g kg^?1 in diet.     

Studies on epizootic iridovirus infection among red sea bream,Pagrus major (Temminck & Schlegel), cultured in Taiwan

Since 1993, an epizootic viral disease has occurred in net-cage cultured red sea bream, Pagrus major (Temminck & Schlegel), in Peng-hu Island located on the south-western coast of Taiwan. The diseased fish exhibited abnormal swimming and were lethargic, but few visible external signs were observed. The cumulative mortality because of the disease sometimes reached 50-90% over 2 months. Histopathogical studies of the affected fish showed enlarged basophilic cells in the gill, kidney, heart, liver and spleen. These necrotic cells were Feulgen-positive and stained blue using Giemsa. Transmission electron microscopy revealed icosahedral virions in the cytoplasm of the necrotic cells. The viral particles consisted of a central nucleocapsid (75-80 nm) and envelope, and were 120-150 nm in diameter. These results suggest that the virus belongs to the Iridoviridae. Using polymerase chain reaction (PCR), approximately 570 bp fragments were produced from the viral DNA using as a template 1-F and 1-R primers derived from red seabream iridovirus (RSIV) from red sea bream in Japan. Similar results were also obtained using nested-PCR with different primer sets (1-F, 2-R and 2-F, 1-R). Although the size and some features of epizootics of this virus differed from RSIV in Japan, it shows close genetic affinities with the latter and it is suggested that RSIV has been introduced to Taiwan.     

我国养殖大菱鲆病毒性红体病及其流行情况调查

大菱鲆病毒性红体病(Viral reddish body syndrome,VRBS)是一种新发现的感染我国养殖大菱鲆的流行性疾病。本文描述了病鱼的外观症状和解剖病理特征,报道了该病的病原及疾病流行情况调查结果。外观检查发现,病鱼的体表无明显损伤,但腹面沿脊椎骨附近皮下淤血、发红,鳍及鳍基部充血、发红;病鱼贫血,血液凝固性差;肾脏肿大,呈灰白色。组织病理学研究显示病鱼脾组织中存在大量肥大细胞,电镜切片中可见大量平均直径约125nm的二十面体病毒粒子,即大菱鲆红体病虹彩病毒(Turbot reddish body iridovirus,TRBIV)。将过滤除菌的含TRBIV的病鱼脾组织匀浆液,经腹腔注射进行人工感染,感染鱼在3周内的累积死亡率达85．7％,死亡大菱鲆表现出腹面和鳍边发红等外观症状,并在感染鱼脾组织切片中观察到大量同样的病毒粒子,由此证实TRBIV是我国养殖大菱鲆病毒性红体病的病原。疾病流行情况调查显示,该病多在养成期大菱鲆中流行,高发季节为每年的8～12月。     

松江鲈白介素15(TfIL-15)的结构特征与重组表达

为研究白介素15(Interleukin-15, IL-15)在松江鲈(Trachidermus fasciatus Heckel)先天免疫中的功能,本研究利用RACE技术克隆得到松江鲈IL-15基因(命名为TfIL-15)的全长cDNA序列,其长度为1140 bp,包括5¢-非编码区(5¢-UTR) 165 bp、开放阅读框(ORF) 522 bp和3¢UTR 453 bp。在5¢UTR区域,存在4个读码框外的AUG翻译起始位点。基因ORF编码173个氨基酸(aa),其中,前59 aa为信号肽序列。成熟肽全长为114 aa,预测分子量为12.975 kDa,理论等电点为5.15。同源比对发现,鱼类IL-15变异程度较高,TfIL-15与其他鱼类IL-15同源性在23%~61%之间。多序列比对和三维结构构建结果显示,TfIL-15具有典型4个α螺旋二级结构,形成二硫键的4个半胱氨酸高度保守。qRT-PCR分析表明,TfIL-15广泛表达于松江鲈各组织中。腹腔注射脂多糖(Lipopolysaccharides, LPS)后,TfIL-15 mRNA在血液、皮肤、肝脏和脾脏中均上调表达。在皮肤和血液中,刺激后2 h表达量迅速上调至最高峰,分别为对照组的74倍和41倍。脾脏和肝脏在刺激后12 h分别达到对照组的3倍和18倍。肝脏中,刺激后96 h,表达量再次上调至对照组的86倍。上述结果表明,TfIL-15可能参与了松江鲈抵抗外界刺激的先天免疫过程。另外,通过构建TfIL-15成熟肽的原核表达载体,成功获得重组蛋白,为进一步研究TfIL-15蛋白的功能奠定了基础。     

中国养殖斑石鲷(Oplegnathus puncatus)上皮囊肿病的发现及显微观察

2015年,山东省某养殖场工厂化养殖的斑石鲷(Oplegnathus puncatus)幼鱼(全长为15 cm左右)因病陆续死亡,15d内累积死亡率达40％以上.现场调查发现,发病池水温为21℃,盐度为30.患病鱼群散开、不聚集.病鱼身体侧偏,活力差,常贴底或者贴壁,严重者随着水流漂流.病鱼呼吸困难,口部持续张开,鳃盖开合频繁,对投喂的食物无反应.但病鱼反应灵敏,难以捕捉.临床检查和剖检可见病鱼鳃表面覆盖着大量黏液,鳃丝有损伤,肠道无食物.取病鱼鳃丝制成水浸片,在光学显微镜下观察,鳃丝上可见到许多直径约为30-70 μm的囊肿物,外观圆形或卵圆形,呈浅黄棕色.在苏木精-伊红染色的石蜡切片中,病鱼次级鳃丝末端粘连,许多上皮细胞膨大呈囊肿状.囊肿嗜碱性,内部均质化.在扫描电子显微镜下观察,病鱼鳃丝呈棍棒化,鳃小片被大量黏液覆盖,表面光滑的囊肿细胞镶嵌其间.通过上述疾病现场调查、病鱼的临床检查、鳃组织的光学显微镜和扫描电子显微镜病理观察,可以初步确定该病为斑石鲷上皮囊肿病.这是上皮囊肿病在中国养殖斑石鲷中首次被发现和记载.     

Establishment of cell lines from a tropical grouper,Epinephelus awoara (Temminck & Schlegel), and their susceptibility to grouper irido- and nodaviruses

Four tropical marine fish cell lines have been established from the eye, fin, heart and swim bladder of grouper, Epinephelus awoara (Temminck & Schlegel). Optimum media and temperature conditions for maximum growth were standardized. The eye and swim bladder cells were mostly epithelial, but the fin and heart cells were mostly fibroblastic. The viability of cells was 95% after 1 year of storage in liquid nitrogen (-196 degrees C). Besides these four cell lines, previously established grouper brain, kidney and liver cell lines were also used for a viral susceptibility study which showed that all the cell lines were sensitive to grouper iridovirus, whereas only brain, fin and liver cell lines were susceptible to the yellow grouper nervous necrosis virus (a nodavirus). Electron microscopy studies of the grouper irido- and nodaviruses in ultrathin sections of infected cells showed an abundance of viral particles in the cytoplasm of the virus-infected cells indicating the effective replication of these two viruses. It is suggested that these cell lines can be used for the isolation of putative fish specific viruses and provide a valuable tool to study the mechanisms of host-pathogen interactions. Furthermore, these cell lines upon transfection, using pEGFP-C1 and pEGFP-aMT2.5 (ayu metallothionein promoter), produced significant fluorescent signals indicating their utility for exogenous studies.     

Establishment and characterization of a fibroblast cell line derived from the dorsal fin of red sea bream,Pagrus major (Temminck & Schlegel)

The establishment and partial characterization of a continuous cell line from the dorsal fin of red sea bream, Pagrus major, are described. The cell line, designated RSBF‐2, has been subcultured for more than 100 passages since its initiation in November 2000. It was optimally maintained at 28 °C in Leibovitz L‐15 medium with 10% foetal bovine serum. Propagation of RSBF‐2 cells was serum dependent and exhibited low plating efficiency (<1.7%). Aside from long‐term cryopreservation, the cells could also be kept at 4 °C for 72 days. The distribution of the chromosome number was 38–98 with a mode of 48. The RSBF‐2 cell line was susceptible to red sea bream iridovirus but only produced a few rounded and refractory cells. Virus‐inoculated RSBF‐2 cells were then subcultured to generate a persistently infected cell line. RSBF‐2 was also very sensitive to the extracellular products of Photobacterium damselae ssp. piscicida and produced significant fluorescent signals after transfection with pEGFP‐C3. Analysis of mitochondrial cytochrome b gene sequences revealed 99% identity between the cell line and Pagrus major.     

Establishment and characterization of a new cell line derived from kidney of grouper, Epinephelus akaara (Temminck & Schlegel), susceptible to Singapore grouper iridovirus (SGIV)

A marine fish cell line derived from the kidney of red-spotted grouper, Epinephelus akaara, designated as EAGK was established and characterized. The EAGK cells multiplied well in Leibovitz's L-15 medium containing 10% foetal bovine serum at 25 °C and have been subcultured for more than 90 passages. Karyotyping, chromosomal typing and ribosomal RNA (rRNA) genotyping analysis revealed that EAGK had a modal diploid chromosome number of 82 and was a fibroblast cell line originated from grouper. A severe cytopathic effect was observed in EAGK cells incubated with Singapore grouper iridovirus (SGIV), but not with soft-shelled turtle iridovirus, viral nervous necrosis virus or spring viraemia of carp virus. SGIV replication was further confirmed by immunofluorescence, electron microscopy and virus titre determination. Bright fluorescence was observed after transfection with fluorescent protein reporter plasmids, indicating that EAGK cells can be used to identify gene functions in vitro. In addition, the cell organelles including mitochondria and endoplasm reticulum changed and aggregated around virus factories after SGIV infection, suggested that the EAGK cell line could be an important tool for investigation of iridovirus-host interactions.     

Effect of dietary lipid oxidation with vitamin C and E supplementation on fillet quality of red sea bream,Pagrus major (Temminck & Schlegel) during storage

An experiment was conducted to determine the effects of different levels of dietary vitamin C (VC) and E (VE) supplementation on fillet quality of red sea bream fed oxidized fish oil (OFO). Fish with an average body weight of 205.0 g were fed four test diets for 9 weeks. Control diet contained fresh fish oil (FFO) with 100 mg kg^?1 of VE and 500 mg kg^?1 of VC (FFO100E/500C). The other three diets contained OFO with varying levels of VE (mg kg^?1) and VC (mg kg^?1) (OFO100E/500C, OFO200E/500C and OFO200E/1000C). After feeding trial, two fillets from each fish by hand filleting were stored in a refrigerator at 4°C for 96 h during analyses. Results showed that fish fed OFO increased fillet thiobarbituric acid reactive substances (TBARS) and K‐value, and decreased fillet VC and VE concentrations during storage time. Supplementation of VC did not have any detectable effect on fillet quality. Increasing dietary VE supplementation increased fillet VE concentrations, reduced fillet TBARS and K‐value values of red sea bream. Therefore, we suggest that dietary supplementation of 200 mg kg^?1 of vitamin E could improve fillet oxidative stability of red sea bream fed OFO.     

Edwardsiellosis in farmed turbot,Scophthalmus maximus (L.), associated with an unusual variant of Edwardsiella tarda: a clinical,aetiological and histopathological study

During 2005 and 2010, a survey of edwardsiellosis on eight turbot, Scophthalmus maximus (L.), farms was conducted in China. This report presents the detailed results of the study on this disease. Diseased turbot displayed two distinct types of gross signs: black discoloration of the dorsal skin on the posterior portion of the body; and red cutaneous foci on the ventral side. Internally, the most pronounced clinical signs in all fish examined were enlarged kidneys. The causal agent of the disease was finally proved to be one species of bacterium that was identified as Edwardsiella tarda by physiological and biochemical tests, API 32E and 16S ribosomal RNA sequence analysis. It is noteworthy that unlike the commonly described E. tarda strains, the isolates in this study were non‐motile strains without flagella. A histopathological study revealed that E. tarda infection was systemic in turbot and that kidney showed the most significant pathological changes, including acute focal necrosis, an influx of macrophages and formation of granuloma. The most common histopathological characteristics of this disease are the proliferation of macrophage in various organs and formation of granuloma. In addition, this article also gave background information on the disease and presented the results of virulence tests with the E. tarda strain identified in this study.