Seasonal variation of phosphatidylcholine hydroperoxides in blood of sweet smelt Plecoglossus altivelis

Sweet smelt was reared at two fishery experimental stations for 5 months from June to October. Every 2 weeks blood was collected from the caudal vessels and, subsequently, the phosphatidylcholine hydroperoxide contents and the fatty acid compositions in the blood were determined by high-performance liquid chromatography and gas chromatography, respectively. The seasonal variation of the contents of accumulated hydroperoxides and fatty acids in the sweet smelt blood were observed in both experimental stations. Sweet smelt started performance of cucumber-like or watermelon-like aroma in the middle of July and the aroma was enhanced in August. The content of phosphatidylcholine hydroperoxides and the amount of total fatty acid in the fish blood, in terms of possible precursors of volatile compounds, were also extremely high in the same period. According to lipid peroxidation mechanisms, the strong characteristic aroma of sweet smelt during July to August might be due to the high contents of accumulated lipid hydroperoxides and polyunsaturated fatty acids in their tissues.     

Molecular cloning of cone opsin genes and their expression in the retina of a smelt, Ayu (Plecoglossus altivelis, Teleostei)

Five cone opsin genes of landlocked ayu fish (Plecoglossus altivelis) were cloned, and the expression patterns of these genes were investigated. AYU-LWS, -RH2-1, -RH2-2, -SWS1-1, and -SWS1-2 were isolated and had high (more than 75%) identity with red, green, green, UV, and UV-sensitive opsin, respectively, genes of other fish reported previously. The results of Southern blotting experiments showed that each gene is present as a single copy. Gene expression was measured by RT-PCR using four populations collected from rivers and a lake in spring and summer. The results of the RT-PCR experiment showed that AYU-SWS1-2 was highly expressed, whereas AYU-SWS1-1 was scarce. Two RH2 opsins were expressed simultaneously in the same individual, and the expression ratio between these opsins changed among populations. In situ hybridization revealed that AYU-LWS and -RH2-1 were expressed in the double cones and that AYU-RH2-2 and -SWS1-2 were expressed in the long and short single cones (LSC and SSC), respectively. It was shown that an individual ayu expresses two RH2 opsins simultaneously in different types of cone cells.     

香鱼消化道及肝脏的形态结构特征

采用解剖及石蜡切片显微技术观察了香鱼消化道及肝脏的组织学结构。香鱼消化道由口咽腔、食道、胃及肠构成。口咽腔大且狭长,其底壁前部有一对粘膜褶,两颌边缘着生宽扁梳状齿,腭骨及舌骨具齿,犁骨无齿;舌由基舌骨突出部分覆盖粘膜构成,舌粘膜上皮为复层扁平上皮,含有较多的杯状细胞和味蕾。食道、胃及肠均由粘膜层、粘膜下层、肌层及外膜构成。食道粘膜层上皮为复层扁平上皮,杯状细胞发达。胃呈V形,由贲门部、胃体部及幽门部组成,胃壁粘膜上皮为单层柱状上皮,贲门部与胃体部的固有层中有胃腺。肠较短,由前、中、后肠构成,肠壁粘膜上皮为单层柱状上皮,其游离面具微绒毛;上皮细胞间有杯状细胞。幽门盲囊有350～400条,其组织学结构与肠相同。肝脏单叶,外被浆膜;肝细胞形态不规则,肝小叶界限不明显。     

长江下游发现香鱼的稚鱼

长江靖江江段位于长江最下游,接近河口段。在对这一江段沿岸的鱼类进行调查的过程中,发现在2004年5月11日的渔获物中,有1尾体长70.8mm、体重3．34g的香鱼（Plecoglossus altivelis）稚鱼。这是香鱼在长江流域的首次分布报道。     

Indirect effects of the algivorous fish Plecoglossus altivelis altivelis on the growth of two insectivorous benthic fish

1. Animals exploiting different resources may nevertheless interact if one species indirectly alters the abundance and distribution of the food of the other. To analyse this indirect effect, we conducted experiments in artificial pools and in the field to investigate the influence of the algivorous fish Plecoglossus altivelis altivelis (known as the ayu) on two species of insectivorous benthic fish, Pseudogobio esocinus esocinus and the goby Gymnogobius petschiliensis .
2. In the pool experiments, algal biomass was not correlated with the number of ayu, but the percentage of blue-green bacteria rose as the number increased. The number of aquatic macroinvertebrates on the upper surface of ceramic tiles placed in the pool bed decreased as the number of ayu increased.
3. Although ayu and the benthic species did not interact directly, the reduction in invertebrate abundance on the upper surface of tiles in the pool reduced the growth rate of the benthic insectivores.
4. In field experiments, the introduction of ayu into habitats with P. esocinus esocinus or G. petschiliensis reduced the growth rate of these benthic fish. In the field experiment that was carried out over 5 years in the G. petschiliensis habitat, the population density of the goby decreased when ayu were stocked.
5. The ayu is a strong interactor or bioengineer in streams, affecting not only benthic algae but also aquatic invertebrates and fishes. We conclude that to predict the outcome of interspecific interactions amongst fishes in streams with high algal production, possible indirect effects must be considered alongside better known direct effects.     

Bacteriophage control of Pseudomonas plecoglossicida infection in ayu Plecoglossus altivelis

Two previously isolated phages were used to examine the therapeutic effects against Pseudomonas plecoglossicida infection in ayu Plecoglossus altivelis. Phage PPp-W4 (Podoviridae) inhibited the in vitro growth of P. plecoglossicida more effectively than Phage PPpW-3 (Myoviridae), and a mixture (PPpW-3/W-4) of the 2 phages exhibited the highest inhibitory activity. In phage therapy experiments, ayu were fed P. plecoglossicida-impregnated feed (10(7) CFU fish(-1)) and then fed phage-impregnated feed (10(7) PFU fish(-1)). Mortalities of fish receiving PPpW-3, PPpW-4, PPpW-3/W-4, and a control fish receiving no phages were 53.3, 40.0, 20.0 and 93.3%, respectively. Phage (PPpW-3/W-4)-receiving fish also showed high protection against water-borne infection with P. plecoglossicida. In a field trial, when phage (PPpW-3/W-4)-impregnated feed was administered to ayu in a pond where the disease occurred naturally, daily mortality of fish decreased at a constant level (5% d(-1)) to one-third after a 2 wk period. The causal relationship of phages in this phenomenon was verified by the long-lasting appearance of administered phages in the kidneys of the fish, and a disappearance of P. plecoglossicida from apparently healthy fish. Neither phage-resistant organisms nor phage-neutralizing antibodies were detected in diseased fish or apparently healthy fish, respectively. These results indicate the potential for phage control of the disease.     

Isolation of different types of birnavirus from ayu Plecoglossus altivelis and amago salmon Oncorhynchus rhodurus cultured in the same geographic area

A birnavirus was recently isolated from cultured ayu Plecoglossus altivelis on Shikoku island, Japan. The diseased fish displayed vertebral or vertical curvature and mild haemorrhage around the brain. Cytopathic effects (CPE) of the virus, including cell roundness, filamentous change and cell lysis, were observed in CHSE-214, RTG-2 and RSBK-2 cells. The virus isolated from ayu, designated the AY-98 strain, was found to be antigenically related to the marine birnavirus (MABV) Y-6 strain that originated from yellowtail Seriola quinqueradiata. AY-98 had a bi-segmented RNA genome and the same nucleotide sequence in the 310 bp VP2/NS junction as MABV Y-6. At the same time that the ayu epizootics occurred, another birnavirus (AM-98) was isolated from amago salmon Oncorhynchus rhodurus which were cultured 66 km away from the ayu farm. AM-98 showed a similar CPE and had the same host cell ranges as AY-98. However, AM-98 was serologically similar to the VR-299 strain of infectious pancreatic necrosis virus (IPNV) and their nucleotide sequences in the VP2/NS junction region showed 98% homology without changes at the amino acid level. In this study, the ayu strain AY-98 was grouped into MABV, whereas the amago salmon strain AM-98 was grouped into IPNV. This indicates that the 2 birnaviruses originated from different sources in spite of the fact that the places where they were isolated are close to one another. The results in this paper show a new aspect of the traditional consensus that the same serogroup of birnavirus distribute in close geographic areas.     

Identification of albumin-bound fatty acids as the major factor in serum-induced lipid accumulation by cultured cells

Factors responsible for the high lipogenic activity of rabbit serum were investigated using an assay procedure based on the gravimetric determination of the 24 hr increase in cell lipid. Cellular synthesis of fatty acids was inhibited by the presence of serum in the assay medium. Approximately 90% of the increase in cell lipid produced by serum fractions was due to triglyceride accumulation. Fractionation of rabbit serum by precipitation with ammonium sulfate or by ultracentrifugation in high density medium, both indicated that three-quarters of its lipogenic activity was associated with albumin. The lipoproteins prepared by ultracentrifugation also exhibited about one-half the activity of whole serum. The lipogenic activity of albumin was confirmed by the high potency of the albumin isolated in a nearly pure form from proteins of d>1.21 by precipitation with trichloroacetic acid and extraction with ethanol. As judged from chemical and isotopic analysis, neither the lipid content nor the lipid composition of the albumin was appreciably altered during its isolation. Of the albumin-bound lipids, only the free fatty acids, as determined by DEAE column chromatography, were present in an amount sufficient to account for the observed increase in cell triglycerides. In control experiments with horse serum of low lipogenic activity, the proteins of d>1.21 also possessed low activity in conjunction with a low content of free fatty acid. However, the albumin isolated from the latter preparation exhibited the high lipogenic activity of rabbit serum albumin. Chemical and isotopic analysis of the recovered horse serum albumin revealed that its free fatty acid content was the same as that of rabbit serum albumin. These results indicated that the isolation of horse serum albumin was attended by a substantial increase in its free fatty acid content. When the rabbit serum and horse serum content of media were adjusted to provide equivalent concentrations of albumin-bound fatty acids, the rabbit liver cells grown on the former media accumulated more lipid than cells grown on the latter media. This difference was shown to be due to the higher concentration of albumin per micro mole of fatty acid in horse serum as compared with rabbit serum. Consequently, the albumin to fatty acid ratio also controls the lipogenic activity of a serum. A linear relationship is presented which relates the cell lipid content to the molar ratio of albumin to free fatty acids and to the absolute concentration of free fatty acids in the medium.     

Isolation and characterization of 49 polymorphic microsatellite loci in the ayu,Plecoglossus altivelis

A total of 49 novel polymorphic microsatellite markers in the ayu fish, Plecoglossus altivelis, were successfully isolated and characterized. The number of alleles per locus, observed heterozygosity, and expected heterozygosity ranged from two to 25 (11.3 on average), from 0.125 to 1.000 (0.648 on average), and from 0.123 to 0.957 (0.751 on average), respectively. These microsatellite markers will be useful for both studying population structure and identifying quantitative trait loci that control evolutionarily and economically important traits of ayu.     

Adherence of Flavobacterium psychrophilum on the body surface of the ayu Plecoglossus altivelis

Bacterial cold water disease in the ayu Plecoglossus altivelis caused by Flavobacterium psychrophilum is a serious problem in the Japanese freshwater culture industry. The distribution and activity of this bacterium on the body surface of the ayu in the infection process was investigated. The survival of F. psychrophilum in tap water showed that this bacterium might sustain its infectivity for 24 h. In an experimental infection, juvenile ayu were immersed in water containing 10(8.9) CFU/ml F. psychrophilum, and the progressing infection was followed by scanning electron microscopy during a 24-h period. This bacterium was observed in the ayu for 24 h adhering to the lower jaw and caudal peduncle, where the epidermis tissue was collapsed. This study showed that bacterial suspension in water sustains the activity of this bacterium. F. psychrophilum attaches especially to the jaw and caudal peduncle, growing at these sites, collapsing the dermal structure and invading the tissues.     

Effects of chemotherapeutics on bacterial ecology in the water of ponds and the intestinal tracts of cultured fish, ayu (Plecoglossus altivelis).

Drug-resistant gram-negative bacilli conferred with R factors were isolated with high frequencies from the intestinal tracts of ayu (Plecoglossus altivelis) cultured in ponds, in which chemotherapeutics had often been used, and with relatively low frequencies from ayu which received no administration of chemotherapeutics. Drug-resistant bacteria were also isolated at low frequencies from the intestinal tracts of wild ayu in rivers, as well as from the water of ayu-culturing ponds and some of them carried R factors. The drug-resistant bacteria carrying R factors were Aeromonas liquefaciens, Citrobacter, Enterobacter cloacae, Escherichia coli, Hafnia and unidentified strains. All the R factors were classified as the Fi-(F) type, except the two R factors detected in an E. coli strain and in an unidentified strain.     

Efficacy of oral vaccine against bacterial coldwater disease in ayu Plecoglossus altivelis

The development of a practical vaccination method against bacterial coldwater disease (BCWD) in ayu Plecoglossus altivelis and the efficacy of oral administration of formalin-killed cells (FKCs) of Flavobacterium psychrophilum was investigated. The FKC was administrated at a dose of 0.1-0.2 g kg(-1) body weight to juvenile ayu (0.5 g body weight) every day for 2 wk or on 5 days over 2 wk. Experimental immersion challenge at 3 and 7 wk after vaccination showed significantly higher survival rates than the controls. The results show the effectiveness of oral vaccination against BCWD in ayu.     

宁海地区香鱼弧菌病病原菌鉴定

摘要：【目的】香鱼弧菌病对中国沿海地区的香鱼养殖业造成了巨大的危害,然而,病原不明导致了防治上的许多问题。本文鉴定了引起宁海地区香鱼爆发性弧菌病的病原。【方法】采用TCBS平板分离优势菌;采用回归感染试验确认病原菌,采用改进的寇氏法计算LD50;采用形态学观察、生理生化特征测定、细菌特异性引物PCR扩增检测及细菌16S rRNA和金属蛋白酶（MP）基因序列分析鉴定细菌;采用药敏实验测定它对部分抗生素的敏感性。【结果】分离并鉴定优势菌株ayu-H080701为宁海地区香鱼弧菌病的病原菌,它对香鱼的半致死量为1.2×104 CFU。形态学观察和生理生化特征测定表明,ayu-H080701与鳗利斯顿氏菌最为接近。PCR扩增检测表明,细菌16S rRNA 基因通用引物和鳗利斯顿氏菌MP基因特异引物均能扩增到预期大小的特异性条带。ayu-H080701与鳗利斯顿氏菌16S rRNA基因核苷酸序列同源性最高,为99.4%～99.5%,与同属的海弧菌和美人鱼发光杆菌分别为94.3%和91.9%;ayu-H080701与鳗利斯顿氏菌MP氨基酸序列同源性高达97.6%～98.8 %,与其它弧菌科成员则低于75.6 %,系统进化树分析也揭示ayu-H080701与鳗利斯顿氏菌进化相关性最高。【结论】引起宁海地区香鱼弧菌病的菌株ayu-H080701被鉴定为鳗利斯顿氏菌。     

Development of immersion vaccine for bacterial cold-water disease in ayu Plecoglossus altivelis

Flavobacterium psychrophilum (F. psychrophilum) is the causative agent of bacterial cold-water disease (BCWD) that occurs in ayu Plecoglossus altivelis. Formalin-killed cell of F. psychrophilum has long been studied as an immersion vaccine for BCWD. In this study, we explored the possibility of F. psychrophilum collagenase (fpcol) for use as the immersion vaccine. BCWD convalescent ayu sera contained specific IgM antibodies against somatic F. psychrophilum and fpcol, meaning that fpcol is a promising antigen for the vaccine development. The recombinant fpcol was successfully expressed in Escherichia coli and Brevibacillus chosinensis (B. chosinensis). The culture supernatant of the B. chosinensis was used as an immersion vaccine solution. The vaccinated ayu were then challenged by soaking into F. psychrophilum culture. In two experimental groups, the relative percentages of survivals were 63 and 38%, respectively, suggesting that fpcol is promising as the immersion vaccine for ayu-BCWD.     

香鱼甘油-3-磷酸脱氢酶基因的克隆与表达

GPDH(glycerol-3-phosphate dehydrogenase)是合成脂肪代谢中间产物甘油-3-磷酸的关键酶。通过设计简并引物从香鱼肝cDNA文库中克隆GPDH基因,该基因cDNA序列全长577个核苷酸,单一大的开放阅读框编码一个由351个氨基酸组成的分子量为37.9kD的蛋白。蛋白序列分析表明,香鱼GPDH(aGPDH)与亚洲胡瓜鱼的GPDH序列同源性最高。系统进化树分析表明,GPDH的物种进化关系与目前接受的物种分类关系基本一致。实时荧光定量PCR结果显示,aGPDH基因在香鱼肝、脾、肾、脑、心和肌肉组织均有表达。香鱼咸淡水适应以后,肝、脾、脑、心和肌肉的aGPDH的mRNA表达水平下调。成功构建重组表达质粒pET-32a-GPDH。SDS-PAGE试验表明,目的蛋白可以在大肠杆菌中大量表达;并制备了抗血清,能与目的蛋白起强的特异性反应,但不与细菌自身蛋白起反应。本研究有助于进一步理解鱼类盐度适应过程中的脂肪代谢调控机制。