日本鳗鲡幼体的耳石微化学分析及其环境指示元素筛选

在长江口日本鳗鲡鳗苗捕捞汛期,于长江靖江段采集日本鳗鲡幼体,采用同步辐射X射线荧光(XRF)定量分析方法测定了幼体矢耳石中14种元素的含量,分析了在海洋与淡水早期生活史阶段的微化学特征。结果显示,耳石中Ca和Sr为常量元素,Ba、Fe、Mn、Zn的含量也在1-10μg/g之间,Cr、Ni、Se、Co、Cu、Au则在1 mg/g以下。Ni、Cu、Mn、Cr、Co、Zn和Sr含量的稳定性较好,变异系数在30%以下;Se、Au的含量低且极不稳定,变异系数达40.8-75.0%;Fe在不同个体间存在显著的含量差异(p=0.007<0.05),但其他元素不存在个体差异。在海洋生活史阶段,Mn、Se、Co、Ba为强富集元素(BCFa-e >1000),Sr、Fe、Zn、Ni、Cu为中等富集元素(BCFa-e在100~1000)。除Sr、Ba和Se外,卵黄囊期和柳叶鳗期的耳石富集系数并无显著差异(p>0.05)。在淡水生活史阶段,Sr和Co为强富集元素(BCFa-e >1000),Se、Zn、Cu、Ba、Mn为中等富集元素(BCFa-e在100~1000),Ni、Fe为低富集元素(BCFa-e在10~100)。与海水阶段相比,耳石在淡水阶段的Fe、Ba、Mn、Se、Co、Ni富集系数均大幅减小,而对Sr、Zn、Cu的富集能力有所增大。分析表明,耳石内的Sr、Ba、Ni、Co为环境强响应元素,Fe和Mn为环境弱响应元素,Zn、Cu和Se为环境负响应元素,幼鳗自海洋至淡水的迁徙过程中,前者存在明显的时滞效应,但后两者的日间含量波动较大,缺乏响应的规律性。本文认为,用作鱼类迁移行为或栖息地环境变化的指示元素,需要具备耳石富集效应强、时滞效应小、不同环境间含量差异大、稳定性好、且为非必需元素等特点。从本研究结果看,只有Sr和Ba两种元素符合这些条件 。     

日本鳗鲡胶原蛋白和小清蛋白的过敏原性

以日本鳗鲡皮与肌肉组织为研究对象,采用碱溶、酸溶、盐析、冻干等方法纯化得到胶原蛋白,采用加热、饱和硫酸铵分级盐析、DEAE-Sepharose阴离子交换层析等方法纯化得到两种亚型的小清蛋白(PV-Ⅰ和PV-Ⅱ),纯化的目标蛋白经动物特异性抗体的免疫印迹实验确证。酶联免疫吸附测定和免疫印迹分析结果显示,纯化的胶原蛋白和小清蛋白分别与鱼类过敏患者阳性血清发生特异性反应,且二者之间无免疫交叉反应。体外模拟胃液消化实验和SDS-PAGE分析结果显示,胶原蛋白和小清蛋白均具有较高的消化稳定性。结果提示,日本鳗胶原蛋白和小清蛋白具有较高的消化稳定性和免疫原性,二者可引发不同患者的IgE介导特异性超敏反应。     

Growth and survival of eel leptocephali (Anguilla japonica) in low-salinity water

We investigated the effects of low-salinity water on the growth, survival, and activity of artificially reared Japanese eel larvae (Anguilla japonica), proceeding from the assumption that such water quality saves energy due to lower cost for osmoregulation. We reared 5-day-old larvae in 0%, 10%, 30%, 50%, 70%, and 100% seawater (SW) with or without diet for 13 days. All larvae reared in 0% and 10% SW died within 6 days, while larvae in 70% and 100% SW survived until 9 days. Larvae in 30% and 50% SW further survived until 13 days without diet. Significant growth in body depth was observed in 30% and 50% SW after 7 days rearing with diet (0.65 ± 0.02 and 0.62 ± 0.02 mm, respectively) as compared with the initial size (0.49 ± 0.03 mm), while no significant growth was observed under the other salinity conditions examined. Larvae swam actively in the light (about 2000 lx) in 50%, 70%, and 100% SW, while they were apparently inactive in 0%, 10%, and 30% SW. The long-term rearing trial showed a 2.2-fold higher 2-month cumulative survival rate in 50% SW (18.2%) than in 100% SW (8.2%). The body depth of larvae in 50% SW (1.58 ± 0.47 mm) was also significantly larger than in 100% SW (1.32 ± 0.35 mm). These findings indicate that the intermediate salinity can result in better growth and survival performance in Japanese eel larvae.     

鳗鲡疱疹病毒的分离与鉴定

为获知鳗鲡"脱粘败血病"与病毒的关系,实验用蔗糖密度梯度离心的方法从发病的欧洲鳗鲡内脏器官组织匀浆液中纯化了病毒粒子,负染后利用电镜观察;进一步用EO细胞对病毒进行了分离、培养,并对感染病毒的细胞进行超薄切片,电镜观察;然后,提取病毒DNA,利用鳗鲡疱疹病毒的PCR检测方法对其进行了鉴定。结果显示,接种匀浆上清液的EO细胞出现细胞融合的病变效应;分离病毒的病毒粒子具囊膜,大小约为200 nm;从感染病毒的细胞上清液DNA中扩增出特异性条带,序列测定与比对分析表明,该序列与鳗鲡疱疹病毒欧洲株(AngHV-1)的序列完全一致。研究表明,利用EO细胞分离了一株鳗鲡病毒,经形态观察和DNA分析,确认该病毒为鳗鲡疱疹病毒,命名为AngHV-FJ。该研究为深入开展鳗鲡疱疹病毒的致病机制及鳗鲡"脱粘败血病"的防控研究奠定了重要基础。     

Fluctuation in the distribution of low-salinity water in the North Equatorial Current and its effect on the larval transport of the Japanese eel

Surface water in the North Equatorial Current (NEC) is composed of southern low‐salinity water diluted by precipitation to less than 34.2 psu and northern, high‐salinity tropical water greater than 34.8 psu. Analyses of 27‐year historical data, observed in winter and summer along the longitude 137°E by the Japan Meteorological Agency, shows that an obvious salinity front (34.5 psu) generated by the two water masses was usually located around 15°N. However, the salinity front has been moving northward during the past three decades. El Niño/Southern Oscillation (ENSO) affected salinity in the surface layer, while temperature changed in the middle layer. The salinity front sometimes moved southward, mainly south of 5°N, and the movement was well correlated with the southern oscillation index (SOI). Because precipitation at Yap (9.5°N, 138.1°E) fluctuated with SOI, this spike‐like southward movement of the salinity front was probably affected by reduction of low‐salinity water during El Niño in the north‐western Pacific Ocean. However, ENSO only induced such large southward movements of the salinity front when the time lag between the low precipitation and low SOI was short (within four months). This salinity front is quite important for long‐distance migrating fish such as the Japanese eel because the eels spawn just south of the salinity front in the NEC. This behaviour suggests that the movement of the salinity front associated with ENSO may control the success of larval transport from the spawning ground in the NEC to the nursery ground in East Asia. In fact, catch of the Japanese eel larvae in Japan was well correlated with fluctuation of SOI and the location of the salinity front, and lower catch occurred during El Niño. The salinity front has moved from 13°N to 17°N during the past three decades. Considering that conditions of larval transport are worse north of 15°N, we suggest that decadal‐scale linear decrease of glass eel catch during the past three decades also can be explained by the displacement of the salinity front.     

Purification and characterization of two novel cysteine protease inhibitors, Eel-CPI-2 and Eel-CPI-3, in the skin mucus of the Japanese eel Anguilla japonica

We have discovered multiple acidic cysteine protease inhibitors, in addition to the known Eel-CPI-1, in the skin mucus extract of the Japanese eel Anguilla japonica by using the two-dimensional gel system of gelatin reverse zymography. Two of the acidic inhibitors, which we have named Eel-CPI-2 and Eel-CPI-3, were purified to homogeneity by anion exchange chromatography on a column of DEAE-Sepharose CL-6B, followed by fast protein liquid chromatography on Superdex 75 10/300 GL and HiTrap Q HP columns. The amino acid compositions of Eel-CPI-2 and Eel-CPI-3 were found to be almost identical and closely similar to that of the eel galectin AJL1. The molecular masses of Eel-CPI-2 and Eel-CPI-3 were elucidated to be 16,089.080 and 16,089.137 Da, respectively, by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The apparent dissociation constant of Eel-CPI-2 and Eel-CPI-3 for cysteine protease papain was determined to be 1.79 × 10⁻⁷ and 1.05 × 10⁻⁷ M, respectively, by a quartz crystal microbalance technique.     

Fine structure and differentiation of the alimentary canal in captive-bred Japanese eel Anguilla japonica preleptocephali

ABSTRACT:   The fine structure of the alimentary canal in preleptocephali produced by artificially matured Japanese eel was examined. At 1 day posthatch (dph), the alimentary canal was found only above the dorsal side of the yolk mass, and the epithelium was composed of a single layer of epithelial cells. By 5 dph, the alimentary canal was divided into three segments based on the structure of the epithelial cells: foregut, midgut and hindgut, corresponding to the future esophagus, intestine and rectum, respectively. After 7 dph, the epithelium in the foregut was surrounded by a circular muscle layer, suggesting a role in the transportation of food materials. The epithelial cells of the midgut exhibited well-developed membranous structures, which are deduced to be invaginations of the cytoplasmic membrane. Pinocytotic invaginations and vacuoles were observed in the epithelial cells of the hindgut; this observation suggests that this region is involved in the uptake of food. Significant changes in morphological features of the epithelial cells in each segment were observed until 7 dph; however, these were not evident between 7 dph and 13 dph. Consequently, the differentiation of the alimentary canal was completed by 7 dph, and preleptocephalus had developed the ability to absorb food by 7 dph.     

养鳗池塘生态系统中矿物元素及其影响因素

对比分析了6口处于不同养殖阶段的日本鳗鲡(Anguill japonica)池塘水体、水源、底泥及饲料中的矿物元素,探讨了池塘生态系统中矿物元素的主要影响因素和生产管理对策,结果表明:1)养鳗池塘水体矿物元素与所处养殖阶段投入饲料的品种相关,底泥中矿物元素部分在养殖过程中积累而来;2)主成分分析结果表明钙、镁、铝元素是...     

Distinct regional profiles of trace element content in muscle of Japanese eel Anguilla japonica from Japan, Taiwan, and China

Trace element distribution patterns were examined in Japanese eel from five Asian regions for the purpose of identifying the elements and patterns that could be used to determine provenance. Distinctive patterns were found in eels from the Japanese prefectures of Miyazaki, Kagawa, and Shizuoka, and from Taiwan and China. The levels of six trace elements, selenium, mercury, copper, manganese, zinc, and arsenic, in muscle tissue varied among eels of different origins, and factorial analysis of the elemental patterns showed that Factor 1 was attributable to selenium and mercury levels and Factor 2 to zinc and copper levels. Multivariate analysis showed that the patterns of elemental composition in fish muscle were mainly differentiated by these two factors.     

Changes in serum vitellogenin levels and immunohistochemical localization of vitellogenin in hepatic cells during ovarian development in the Japanese eel

ABSTRACT: The present study was designed to quantify changes in serum vitellogenin (VTG) levels and to localize VTG in hepatic cells immunohistochemically during artificial maturation of the Japanese eel Anguilla japonica . Hormone treatment resulted in a gradual rise in serum VTG levels, in which the highest values were attained at the migratory nucleus stage. Hepatocytes responded to hormone treatment by increased immunoreactivity; both staining area and intensity were enhanced with ongoing gonadal development. Moreover, cytoplasmic vacuoles increased in number during artificial maturation, occupying large areas inside hepatocytes. The unusual appearance of hepatocytes may reflect the effect of artificial maturation.     

Variability of the Pacific North Equatorial Current and its implications on Japanese eel (Anguilla japonica) larval migration

The zonal velocity produced by a Regional Ocean Modeling System (ROMS)‐based Pacific Ocean circulation model was validated against in situ measurements along the 137°E longitude. The Pacific model successfully reproduced the position and the shape of the North Equatorial Current (NEC) as well as the latitude of maximum surface velocity in the NEC region (8–17°N). The flow field produced by the Pacific model was then used to conduct numerical Lagrangian experiments, in which passive particles were released along a transect (142.5°E, 12.5–17°N) that traverses the known Japanese eel spawning area, and the effects of NEC strength and bifurcation latitude on the particle advection in the northwest Pacific were studied. Our results suggest that, in the 20‐yr period (1993–2012), the variability of the currents alone can cause interannual variability of one order of magnitude in the Kuroshio Entrance (KE), the percentage of particles entering the Kuroshio, the range of which varies from 43% in 1997 to 6% in 2012. The yearly‐averaged KE is not sensitive to the NEC bifurcation latitude. Instead, it is controlled by the average zonal velocity of a fixed domain (125–143°E, 13.5–17°N) and related to a recently‐developed climate index, the Philippines–Taiwan Oscillation (PTO). During the positive phase of the PTO, the zonal velocity in the domain, hence the yearly‐averaged KE, increases, and the opposite is true in the negative phase of the PTO. Considering only the trajectories, diel vertical migrations (DVM) in the top 400 m do not significantly affect Japanese eel larval transport, as incorporating DVM schemes does not increase the KE.     

中国近海日本鳗鲡玻璃鳗体组织营养成分及氨基酸、脂肪酸组成的比较

为了解日本鳗鲡玻璃鳗的早期营养需求,分别对中国近海6个不同地点玻璃鳗体组织一般营养成分、氨基酸和脂肪酸含量进行了分析。结果显示,野生玻璃鳗体组织中水分含量为78.62%±2.03%,干重基础下粗蛋白质含量为69.08%±0.64%、粗脂肪含量为11.96%±1.33%、粗灰分含量为11.45%±1.30%。在台州、舟山、南通和东台4个采样点的样品中,水分含量无显著差异,而粗蛋白含量以舟山点最高、粗脂肪含量最低,并且舟山点样品肠道内发现可见内容物。玻璃鳗样品中氨基酸含量以谷氨酸、天门冬氨酸、赖氨酸、亮氨酸最高,半胱氨酸最低;除舟山点外,各采样点氨基酸含量无明显差异。不同洄游地点玻璃鳗组织中脂肪酸含量均以C16:0、 C18:1n9、 C20:5n3(EPA)、 C22:5n3、 C22:6n3(DHA)为主要成分,其中EPA和DHA共占脂肪酸总量的31.59%±1.00%,不同洄游地点样品必需脂肪酸含量差异明显。研究表明,必需氨基酸、必需脂肪酸对于玻璃鳗的正常生长存活具有重要意义。此外,对比不同地点营养物质组成及稚鱼饵料理论需求量分析,推测舟山点海洋环境中包含更适合玻璃鳗稚鱼摄食的食物,...     

Lysis of pathogenic bacteria by epidermal cathepsins L and B in the Japanese eel

Cysteine proteases, cathepsins L and B, in Japanese eel epidermis are suspected bacteriolysins against aquatic pathogens. This work examines the lysis of three species of gram-negative pathogenic bacteria by the eel epidermal extract according to the assay method for cysteine proteases. The optimum pH for the lysis of the three species of bacteria were commonly determined to be 5.0, where both of eel epidermal cathepsins L and B caused active proteolysis. Four kinds of specific inhibitors for cysteine proteases strongly inhibited these catheptic proteolyses and bacteriolyses. The activities for the lysis of three types of bacteria were at a similar level, but the effects on the bacterium which infects predominantly eel skin largely varied with individual fish. These results suggest that epidermal cathepsins L and B are responsible for lysis of any pathogenic bacteria in the nonspecific defense of Japanese eel.     

Development of an emulsion prepared with lipophilized gelatin and its application for hormone administration in the Japanese eel Anguilla japonica

A new water-in-oil-in-water (W/O/W) type emulsion using lipophilized gelatin (LG) and cotton-seed oil was developed for a novel method of hormone administration in fishes. LG was prepared by attaching palmitic anhydride to gelatin. The best conditions for preparing stable emulsions were determined where the final concentrations of LG in the emulsion and the volume ratio of LG solution to cotton-seed oil were 2% and 1:2, respectively. Hormone releasing properties of LG emulsion were compared with a water-in-oil (W/O) type emulsion prepared with Freund's incomplete adjuvant (FIA) and saline solution by monitoring plasma profiles of salmon gonadotropin (sGtH) II in the Japanese eel. sGtH II is gradually released from the LG emulsion. LG emulsion had hormone-releasing properties different from those of the saline solution and the FIA emulsion. Immature female Japanese eel (BW, 566 to 1017 g) received a weekly intramuscular injections (total of 10 injections) of the LG emulsion, the FIA emulsion or the saline solution, each of which contained sGtH fraction. The LG emulsion was found to be more effective in inducing ovarian maturation. In mature eel, final maturation and ovulation could be induced by simultaneous administration of sGtH fraction and 17-hydroxyprogesterone using LG W/O/W emulsion.     

Fluorescence localization of epidermal cathepsins L and B in the Japanese eel

Histochemical localization of proteolytic activities in the dorsal epidermis of Japanese eel was demonstrated by fluorescent microscopy utilizing 4-methoxy-2-naphthylamide (4M$\beta$NA) derivatives as substrates and 5-nitrosalicylaldehyde as a trapping agent. Carbobenzoxy-L-phenylalanyl-L-arginyl-4M$\beta$NA (Cbz-Phe-Arg-4M$\beta$NA) and Cbz-Arg-Arg-4M$\beta$NA were used for direct detection of cathepsins L and B activities, respectively, in fresh frozen sections and unfixed cells of the eel epidermis. The fluorescing areas, where Cbz-Phe-Arg-4M$\beta$NA was hydrolyzed by cathepsin L, were shown in mucus secretory cells and club cells and broadly around skin surface. The fluorescing areas due to Cbz-Arg-Arg-4M$\beta$NA hydrolysis by cathepsin B were localized similarly in these tissues. The fluorescing intensity for both catheptic activities in mucus secretory cells was higher than that in club cells, where small fluorescing granules were distributed. These results indicate that eel cathepsins L and B are stored in epidermal secretory cells at different levels and probably serve as defense factors before or after secretion by these cells. Abbreviations: Cbz – carbobenzoxy; 4M$\beta$NA – 4-methoxy-2-naphthylamide; NSA – 5-nitrosalicylaldehyde.     

The first success of glass eel production in the world: basic biology on fish reproduction advances new applied technology in aquaculture

The eel has long been esteemed as an important food fish in the world, especially in Japan, and has been used as an experimental fish for many fields of fish physiology. However, the decreases in eel resources have been a serious concern in recent years. The catches of glass eels as seedlings for aquaculture have shown a long-term decrease in both Europe and East Asia. To increase eel resources, the development of techniques for artificial induction of maturation and spawning and rearing their larvae have been eagerly desired. Recent progress of reproductive physiology of fish, especially mechanisms of oocyte maturation and ovulation in female and of spermatozoa maturation in male, facilitate to establish techniques for hormonal induction of maturation and spawning in sexually immature eels. With persistent effort to development of rearing techniques of larvae, we have first succeeded to produce glass eel. These applied techniques are may contribute to understand the basic reproductive physiology of the eel.     

发酵豆粕部分替代鱼粉对日本鳗鲡生长性能和体内矿物元素的影响

在日本鳗鲡（Anguill japonica）饲料中分别添加0、50 g·kg-1、100 g·kg-1、150 g·kg-1和200 g·kg-1的发酵豆粕,相应替代饲料中鱼粉使用量的0、5%、10%、15%和20%,饲养体质量为（37.62±0.16）g日本鳗鲡50 d,研究发酵豆粕对鳗鱼生长性能和体内矿物元素的影响。结果显示：1）随着发酵豆粕替代鱼粉的比例升高,鳗鱼增重率和特定生长率呈先上升后下降的趋势,其中15%替代组增重率和特定生长率高于其他组,但差异不显著（P﹥0.05）,5%替代组饲料系数显著低于其他组（P〈0.05）。从生长性能上看,15%是发酵豆粕替代鳗鱼饲料中鱼粉的最适比例;2）发酵豆粕替代鱼粉后鳗鱼饲料中的钾（K）、铁（Fe）、镁（Mg）等元素质量分数升高,而铝（Al）元素质量分数降低,按变化幅度大小排列Al〉K〉铜（Cu）〉硼（B）〉钙（Ca）〉钠（Na）〉磷（P）〉Fe,但替代组与对照组鳗鱼肌肉中矿物元素K、Ca和P差异不显著（P〉0.05）,而无论是肌肉还是皮肤中矿物元素Cu质分数量均降低（P〈0.05）,因此,发酵豆粕替代鱼粉后应对饲料中矿物元素添加量适当进行调整。