The use of Western blot and electroimmunotransfer blot assays to monitor bacterial kidney disease in experimentally challenged Atlantic salmon, Salmo salar L.

Abstract. Western blot (WB) and electroimmunotransfer blot (EITB) methods were used to monitor bacterial soluble antigens and scroconversion profiles, respectively, in Atlantic salmon, Salmo salar L., experimentally challenged with Renibacterium salmoninarum. The two challenged populations exhibited differences in susceptibility to R. salmoninarum permitting an evaluation of the methods in fish populations harbouring different levels of the pathogen, WB detection of R. salmoninarum soluble antigens in serum provided a sensitive method for detecting pathogen presence during the active infection stages. For comparison, results for drop-plate culture and direct fluorescent antibody techniques were obtained. During the recovery phase, surviving fish were negative for the pathogen by these methods. However, >90% of challenged fish sampled from early infection stages to the end of the experimental period (141 days post-infection) were scropositive by the EITB assay for Atlantic salmon immunoglobulin reactive with R. salmoninarum soluble antigens. The results suggest that these methods for detecting R. salmoninarum or its soluble antigens may be useful during active infections. However, asymptomatic fish populations exposed to the pathogen, perhaps having recovered from an active infection and representing potential disease carrier sources, may be more readily identified by antigen-specific antibodies using methods such as the EITB assay.     

Interspecific germ cell transplantation: a new light in the conservation of valuable Balkan trout genetic resources?

Interspecific transplantation of germ cells from the brown trout Salmo trutta m. fario and the European grayling Thymallus thymallus into rainbow trout Oncorhynchus mykiss recipients was carried out in order to improve current practices in conservation of genetic resources of endangered salmonid species in the Balkan Peninsula. Current conservation methods mainly include in situ efforts such as the maintenance of purebred individuals in isolated streams and restocking with purebred fingerlings; however, additional ex situ strategies such as surrogate production are needed. Steps required for transplantation such as isolation of high number of viable germ cells and fluorescent labeling of germ cells which are to be transplanted have been optimized. Isolated and labeled brown trout and grayling germ cells were intraperitoneally transplanted into 3 to 5 days post hatch rainbow trout larvae. Survival of the injected larvae was comparable to the controls. Sixty days after transplantation, fluorescently labeled donor cells were detected within the recipient gonads indicating successful incorporation of germ cells (brown trout spermatogonia and oogonia—27%; grayling spermatogonia—28%; grayling oogonia—23%). PCR amplification of donor mtDNA CR fragments within the recipient gonads additionally corroborated the success of incorporation. Overall, the transplantation method demonstrated in this study presents the first step and a possible onset of the application of the germ cell transplantation technology in conservation and revitalization of genetic resources of endangered and endemic species or populations of salmonid fish and thus give rise to new or improved management strategies for such species.     

Development of monoclonal antibodies to PK'X', the causative agent of proliferative kidney disease

Abstract. Two monoclonal antibody probes were produced against PK'X' cells. The parasites were partially purified by filtration and centrifugation of kidney tissue from rainbow trout with proliferative kidney disease and used as antigen for immunization of mice. The resulting monoclonal antibodies reacted with PK'X' cell antigens in enzyme-linked immunosorbent assay and immunohistochemistry tests. One antibody (Mab 12) appeared to be specific for PK'X'in kidney tissue, while the other (Mab 18) cross-reacted with host cell antigens in the kidney tubules. These probes are invaluable tools for the investigation of parasite surface antigens and life cycle studies.     

Serological techniques for detection of lymphocystis virus in fish

Three serological techniques (indirect immunofluorescence test, flow cytometry, and indirect dot–blot immunoenzymatic assay) have been evaluated for the detection of lymphocystis viral antigens using a gilt-head seabream cell line, SAF-1, and fish leukocytes. Six lymphocystis disease virus (LCDV) isolates from gilt-head seabream, and one reference strain (ATCC VR 342), were tested. Detection of viral LCDV antigens in SAF-1 cells and fish leukocytes by indirect immunofluorescence test occurs at similar periods (5–7 d post-inoculation), and viral antigens were detected as cytoplasmic inclusions located at the periphery of inoculated cells. The percentages of cells with LCDV antigens obtained by flow cytometry were very low, ranging between 0.9% at 5 d post-inoculation and 19.7% at 10 d post-inoculation. The optimal concentration of viral stocks detected by indirect dot–blot immunoenzymatic assay was 0.5 μg ml^–1, when purified viral stocks were used as antigens. Inoculated and uninoculated SAF-1 cells could not be distinguished using LCDV antiserum binding. On the basis of these results, indirect immunofluorescence and flow cytometry tests appear to be the best serological methods to detect LCDV antigens in both SAF-1 cells and fish leukocytes.     

鲤疱疹病毒3型ORF136基因编码蛋白多克隆抗体的制备与鉴定

为了对鲤疱疹病毒3型(Cyprinid herpesvirus 3,CyHV-3)ORF136基因编码蛋白进行功能研究和血清学诊断,本实验通过对ORF136基因推导的第31~157位氨基酸序列进行PCR扩增,并与原核载体pET-32a(+)连接,转化至大肠杆菌Rosetta(DE3)感受态后进行IPTG诱导表达,将纯化后的重组蛋白免疫新西兰白兔(Oryctolagus cuniculus)以制备ORF136多克隆抗体,运用Western blot和间接免疫荧光技术对抗体进行鉴定。结果表明,重组融合表达蛋白大小与预期一致,约为35 kD,且主要分布在包涵体中。Western blot分析显示,免疫兔后获得的纯化ORF136多克隆抗体能特异性识别纯化的CyHV-3和感染CyHV-3的KS细胞;间接免疫荧光分析进一步表明ORF136多抗能识别感染CyHV-3的KS细胞。ORF136多克隆抗体的制备为ORF136蛋白功能研究和CyHV-3血清学诊断方法的建立提供了重要基础。     

大银鱼生物学与渔业生态学研究进展

大银鱼是东亚特有的小型经济鱼类,相关研究工作主要由我国科技工作者开展。本文对大银鱼分类、生长、繁殖、孵化与胚胎发育、移植与产量波动以及对其他鱼类的影响等研究进展进行了归纳和总结。更正后的大银鱼拉丁学名存在尚未被广泛使用的问题。多年的移植增殖使大银鱼产生了大量新的遗传变异,形态上也有所改变,丰富了大银鱼的种质资源。不同纬度地区大银鱼快速生长的月份不同,虽然北方生长季节短,但大银鱼个体却不小于南方。大银鱼能否稳定地转变为食鱼性决定了其生长速率和最终个体大小,其种群内出现个体大小分化的饵料资源条件尚不清楚。种群内是否出现个体大小分化决定了大银鱼的性选择模式,而相关的研究尚未开展。大银鱼为一次性产卵鱼类,其自然受精率高于人工受精率,因此,在增养殖中若能保留合适的繁殖群体量则无需人工投放受精卵。大银鱼的胚胎发育及其影响因素已经比较清楚,足以指导受精卵的生产。大银鱼是耐盐碱鱼类,可用于发展盐碱水域渔业。饵料生物资源的过度消耗被认为是大银鱼产量跌入低谷的原因,但相关的定量生态学研究有待开展,以指导大银鱼的可持续稳产。本研究还针对大银鱼种群爆发对土著鱼类影响的问题,提出了应对策略。     

Worldwide status of burbot and conservation measures

Although burbot ( Lota lota Gadidae) are widespread and abundant throughout much of their natural range, there are many populations that have been extirpated, endangered or are in serious decline. Due in part to the species' lack of popularity as a game and commercial fish, few regions consider burbot in management plans. We review the worldwide population status of burbot and synthesize reasons why some burbot populations are endangered or declining, some burbot populations have recovered and some burbot populations do not recover despite management measures. Burbot have been extirpated in much of Western Europe and the United Kingdom and are threatened or endangered in much of North America and Eurasia. Pollution and habitat change, particularly the effects of dams, appear to be the main causes for declines in riverine burbot populations. Pollution and the adverse effects of invasive species appear to be the main reasons for declines in lacustrine populations. Warmer water temperatures, due either to discharge from dams or climate change, have been noted in declining burbot populations at the southern extent of their range. Currently, fishing pressure does not appear to be limiting burbot populations world-wide. We suggest mitigation measures for burbot population recovery, particularly those impacted by dams and invasive species.     

广西沿海及其邻近海区拟穴青蟹群体遗传多样性的RAPD分析

采用随机扩增多态DNA（random amplified polymorphic DNA,RAPD）技术检测广西沿海及其邻近海区拟穴青蟹（Scylla paramamosain）6个地理群体的遗传变异和遗传结构,8条10 bp寡核苷酸随机引物扩增99个个体,分析其中的44个位点,31个位点表现出多态性,在种水平的多态位点百分率为70.45%。POPGENE分析结果显示,6个群体的多态位点百分率为29.55%～54.55%,平均为36.97%;群体的遗传多样性自高至低排列为钦州湾群体〉党江群体〉珍珠湾群体〉闸口群体〉清化群体〉流沙湾群体;群体内的遗传变异大于群体间的遗传变异,群体间的遗传分化程度较大。AMOVA分析显示,群体内遗传变异占87.03%,群体间遗传变异占12.97%,群体间发生中等程度遗传分化。Mantel检测结果表明,拟穴青蟹6个群体间的遗传距离与地理距离之间的相关性不显著。聚类分析表明,群体间聚类无明显的地域性分布格局。     

Oral immunization of rainbow trout, Salmo gairdneri Richardson, against vibriosis with vaccines protected against digestive degradation

Abstract. In order to reduce digestive degradation of vaccines against vibriosis when administered orally to fish, two methods of protecting the antigens were tested. Lyophilized vaccine was either incorporated into a slow-release pellet or coated by an acid-resistant film. These vaccines were compared to both oral vaccination by an unprotected vaccine as well as to standard vaccination methods of immersion and injection. However, mortalities after an artificial challenge showed that the efficacy of the protected vaccines was lower than that of the unprotected vaccine given orally and the standard vaccination procedures. The reason for this is probably that the important antigens of vibriosis vaccines are lipopolysaccharides which are little affected by gastric digestion, and the coating of the vaccine or incorporation into slow-release pellets resulted only in reduced absorption of the antigens.     

翘嘴鳜选育群体的生长和遗传特征分析

以湖南和江苏的野生翘嘴鳜(Siniperca chuatsi)为基础群体选育了6个群体,包括4个家系选育群体和F_1、F_2两个群体选育群体。对4个家系选育群体的生长特性进行了分析,并利用微卫星标记技术对6个翘嘴鳜选育群体的遗传多样性进行了检测。分析结果表明,翘嘴鳜纯种家系的生长速度显著快于不同地理种群的杂交系,杂交系未表现出生长的杂种优势,家系群体的杂合性与生长性能不对应。4个家系选育群体的特有等位基因数量比两个群体选育群体高出约29.33%,表明不同地理种群翘嘴鳜家系的建立可以丰富翘嘴鳜选育群体的遗传多样性水平。翘嘴鳜选育群体间遗传分化显著(F_(st)=0.4388),两个群体选育群体与4个家系选育群体间的遗传距离较远,表明将群体选育的个体与家系选育的个体进行杂交育种有可能获得杂种优势。筛选到G14437特有等位基因可作为江苏翘嘴鳜家系(JCJC)的群体特异性分子标记;G5_(530)等位基因可作为湖南和江苏翘嘴鳜杂交系与翘嘴鳜基础群体和其他选育群体区分的特异性分子标记。     

基于线粒体控制区的许氏平鲉养殖群体与野生群体比较研究

为研究许氏平鲉养殖群体与野生群体遗传结构及遗传多样性状况,采用PCR扩增获得许氏平鲉线粒体DNA控制区高变区片段,并对其进行比对分析。结果显示,在长度为451 bp的线粒体控制区片段中,养殖群体单倍型多样度(0.540±0.067~0.815±0.021)明显低于野生群体(0.883±0.053~0.944±0.028),而核苷酸多样度(0.001±0.001~0.007±0.004)与野生群体(0.004±0.003~0.007±0.004)相差不大,遗传多样性水平均较低。在52个单倍型中,养殖群体仅占12个,且有6个单倍型与野生群体共享。群体间遗传分化指数和AMOVA分析结果显示,养殖群体和野生群体之间以及养殖群体之间的遗传分化较大,而野生群体间遗传变异较小,组群间的遗传分化较小且不显著(ΦCT=-0.013;P0.05)。单倍型最小跨度树和NJ系统发育树均未检测到明显的谱系结构。     

Humoral immune response in Atlantic salmon, Salmo salar L., to cellular and extracellular antigens of Aeromonas salmonicida

Abstract. The putative virulence factors of Aeromonas salmonicida , the aetiological agent of furunculosis in salmonids, are candidates for protective antigens in effective vaeeines against furunculosis. In this report, the authors have compared the immunogenieily of eell-associated and extracellular antigens of A. salmonicida in Atlantic salmon, Salmo salar L., to that in rabbit. The animals were immunized with formalin-killed whole cells and formalin-inactivated extracellular products (ECP), either separately or in combination. The ability of the antigens to induce antibody production was studied by elisa and Western blotting techniques. These results confirm previous reports that far more structures are immunogenie in rabbit compared to the antibody responses elicited in salmon. However, in both species, some antigens were dominant, including a caseinolytic protease in addition to the A-protein and high and low MW LPS.     

Patterns of phenotypic and genetic variability show hidden diversity in Scottish Arctic charr

Abstract –  This study examined the degree and pattern of variability in trophic morphology in Arctic charr ( Salvelinus alpinus L. ) at three spatial scales: across 22 populations from Scotland and between and within two adjacent catchments (Laxford and Shin) in northern Scotland. In addition, the variability at six microsatellite loci between and within the Laxford and Shin systems was determined. Habitat use by charr differed significantly between populations. The pattern of variability in trophic morphology, known to influence foraging ability in charr, showed a very high degree of between-population variation with at least 52% of population pairs showing significant differences in head shape. Trophic morphology and genetic variation was also high over small geographical scales; variation being as high between charr from lakes within the same catchment, as between adjacent catchments. The pattern of both phenotypic and genotypic variation suggests a mosaic of variation across populations with geographically close populations often as distinct from each other as populations with much greater separation. Very low levels of effective migrants between populations, even within the same catchment, suggest that this variation is being maintained by very low straying rates between phenotypically and genetically distinct populations, even when there is no apparent barrier to movement. We conclude that the genetic and phenotypic integrity of charr populations across Scotland is high and that this adaptive radiation constitutes a 'hidden' element of diversity in northern freshwater systems. Two consequences of this are that the population (rather than the species) makes a more rational unit for the consideration of conservation strategies and that the habitat requirements and therefore management needs may differ significantly between populations.     

鱼类生殖细胞移植的研究进展及应用前景

生殖细胞移植是指将供体的生殖细胞移植到同种或异种受体体内,供体生殖细胞嵌合到受体性腺,经过增殖、分化并最终发育为功能性配子的过程。作为辅助生殖技术,它不仅为珍稀濒危动物的繁育和保护提供了新途径,同时也为生殖干细胞的功能研究提供了有效手段。鱼类生殖细胞移植研究首先在模式鱼类斑马鱼中开展,经过十多年的发展,取得了一系列突破性的进展:主要包括先后建立了以胚胎、仔鱼和成鱼为受体的生殖细胞移植体系,精原和卵原干细胞的发现拓宽了供体生殖细胞的选择,受体的选择与制备方法的完善。该技术在缩短鱼类性成熟周期、性控育种、珍稀濒危鱼类保护等方面具有巨大的应用前景,已成功在多种淡水和海水鱼类中开展了研究和应用。本文结合作者的研究实践和经验,系统地梳理和总结了鱼类生殖细胞移植的研究进展,指出了该技术实践应用的关键问题,并探讨了其应用前景。     

Survival and growth of local and transplanted blue mussels (Mytilus trossulus, Lamark)

Consumer demand for fish and shellfish has led to an increase in aquatic species movement for aquaculture purposes. One potential drawback to the successful transplantation of animals for aquaculture is unpredictable performance due to local adaptation effects. This study used a common environment experiment to examine the potential for local adaptation in Mytilus trossulus (Lamark) on the east coast of Vancouver Island, British Columbia (BC). Newly settled mussels were collected from two sites, Chemainus and Quadra Island (approximately 150 km apart), reared in cages at the Quadra collection site, and mortality and shell length were monitored. Mussels were cage‐reared for 384 days with an overall survival of 59% (Quadra) and 39% (Chemainus). The Quadra mussels were initially smaller (5.07 mm) than the Chemainus mussels (5.90 mm), but the two were similar at the end of the experiment (23.5 and 23.6 mm). Transplanted mussels had a significantly higher mortality over the course of the experiment, primarily due to a severe episode in the early summer, and had significantly lower relative growth rates at three of 11 measurement dates. Overall, the local mussels performed better than the transplanted mussels. This study demonstrates the potential for local adaptation effects between populations of mussels separated by only a relatively small geographic distance.     

Evaluation of genetic characteristics of wild and cultured populations of the Japanese pearl oyster <Emphasis Type="Italic">Pinctada fucata martensii</Emphasis> by using AFLP markers

Genetic characteristics of four wild (Mie, Fukui, Shimane and Nagasaki) and five cultured populations (selectively bred for 12 years with an origin of Ehime population) of the Japanese pearl oyster, Pinctada fucata martensii, were evaluated by using AFLP markers. Six primer pairs generated 1019 loci in total, among which 45.2–55.1 % was polymorphic among populations. Although there was no significant difference in gene diversity between wild (0.170–0.174) and cultured (0.158–0.173) populations, genetic relatedness in cultured populations (0.316–0.450) was about three times higher than that in wild populations (0.110–0.165). In addition, genetic differentiation was about twenty times larger in cultured populations (Nei’s distance: 0.0111) than in wild populations (Nei’s distance: 0.0005). These results mean that selective breeding can cause marked inbreeding as well as large genetic differentiation among cultured populations in a short period. On the other hand, it was suggested that genetic homogenization in the wild, probably due to a large-scale transport of cultured oysters, had progressed in the sea around Japan. In conclusion, it is necessary to prevent inbreeding by the reconsideration of the style of selective breeding in cultured populations, while the escape of gametes or spats of cultured strains in the wild should be avoided for the preservation of genetic characteristics in native populations of P. f. martensii.     

马氏珠母贝不同地理种群内自繁和种群间杂交子一代主要性状的比较

为了培育马氏珠母贝新品种，开展了不同地理种群内自繁和种群间杂交的工作，获得了3个自繁群体和3个杂交群体，BB1、DD1和SS1分别来自北海野生种群(BW)、大亚湾野生种群(DW)和三亚野生种群(SW)自繁一代；BDl、BS1和DSl分别来自BW与DW、BW与SW以及DW与SW杂交一代。运用方差分析对6个子一代群体的5个主要性状(壳长、壳宽、壳厚、总体重和壳重)进行了比较，3个杂交群体未表现出杂种优势。但获得的6个群体将成为进一步选育的基本群体。子一代的变异性增加为进一步选育奠定了基础。     

斑鳢基因组中微卫星分布特征及野生种群遗传结构分析

斑鳢(Channa maculata)是华南地区的本土经济鱼类,也是杂交鳢的亲本之一。养殖个体逃逸可能会对野生种群产生影响,存在种质混杂的风险,亟须开展野生资源的遗传背景分析。该研究分析了斑鳢基因组中微卫星标记的分布特征,筛选获得20个多态性位点构建多重PCR体系,对广州、化州、江华、南宁、阳江和邵武6个野生群体的遗传多样性和遗传结构进行了分析。结果显示,6个野生群体各位点的等位基因(Na)为3~28、有效等位基因(Ne)为1.28~14.88、观测杂合度(Ho)为0.10~1.00、期望杂合度(He)为0.14~0.95以及多态信息含量(PIC)为0.13~0.95。UPGAM系统进化树结果显示,化州和福建种群遗传关系最近,化州和江华种群遗传关系最远。该研究结果将为斑鳢的遗传监测和亲缘关系鉴定提供技术支持,为斑鳢种质资源养护及管理提供参考。     

Significance of straying in salmonids and implications for ranching

Salmonids are famous for their homing precision and straying is usually regarded as a failure of individuals to achieve the population norm. However, without straying there would be no salmonid populations throughout much of their present range, as much of that area has been colonized by modern salmonids over the past 8000–15000 years. Straying is a normal population strategy entirely appropriate to a colonizing phase of a species and hatchery populations are comparable to colonizing wild populations. The real challenge for ranching is to ensure that the learned components of the homing process are so well ingrained that any genetic tendency to stray can be over-ridden.     

Humoral immune response of turbot, Scophthalmus maximus (L.), to antigens from Tetramicra brevifilum Matthews & Matthews, 1980 (Microspora)

Abstract. The humoral immune response of turbot, Scophthalmus maximus (L.), to antigens from the microsporean parasite Tetramicra brevifilum Matthews & Matthews, 1980, was studied. Thirty days after intraperitoneal immunization with whole T. brevifilum spores in Freund's complete adjuvant, double indirect ELISA indicated that initial production of antibodies to parasite surface antigens was considerably higher than production of antibodies to the antigens contained in a crude extract (CE) of spores. Following re-immunization without adjuvant on day 30, levels of antibodies to surface antigens gradually declined, whilst levels of antibodies to CE antigens increased. The antibody response of intraperitoneally immunized fish was characterized by Western blotting of total soluble antigens obtained by heating and reduction of T. brevifilum spores at 95–100°C in Tris-HCl buffer containing SDS and dithiothreitol: a series of bands with molecular weights between 20 and 53 kDa was recognized by immunized turbot sera. Four additional bands (with molecular weights between 15 and 18kdA) were recognized by serum from re-immunized fish. ELISA studies of sera from naturally infected fish revealed a surprisingly low incidence of strong T. brevifilum seropositivity (61% individuals); antibodies to surface antigens predominated in seropositive individuals. The low background response levels and high sensitivity of the ELISA used in this study indicate that the assay is of value for the monitoring of serum antibody levels in turbot. However, given the relatively low seropositivities observed in naturally infected turbot, particularly to CE antigens, the use of anti- T. brevifilum serum antibody levels for the diagnosis of infection by this parasite may lead to false negative results.