Epidermal proteases of the Japanese eel

A fluorescent-sensitive assay was used to demonstrate the protease activity in the dorsal skin of Japanese eel (Anguilla japonica). Two distinct extracts were separately prepared from skin mucus and epidermal cell layers, with no mutual contamination. The epidermal extract was sensitive to various substrates, whereas there was no, or only marginal, susceptibility to the same substrates for the mucous extract. Optimum hydrolysis pHs of the epidermal extract was variable and below pH 7.0, and the optimum hydrolysis temperatures were between 40 and 50 °C. In addition, Tos-Phe-Ch₂Cl, chymostatin, CdCl₂, CuCl₂, HgCl₂ and ZnCl₂ inhibited protease activities to different extents. Several other reagents specifically affected the protease activities, and their induced effects were useful for the identification of epidermal proteases. The findings indicate that a proteolytic factor, exhibiting various enzymological specificities, is retained within epidermal cell layers of Japanese eel. This factor is composed of 4 distinct proteases, such as cathepsins L and B-like proteases, a serine protease and an aminopeptidase.     

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.     

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.     

中草药添加剂对日本鳗鲡生长和非特异性免疫效应的研究

在基础饲料中分别添加0.5 g/kg、1.0 g/kg和1.5 g/kg的自制中草药免疫添加剂,配制成3种试验饲料,以基础饲料为对照组饲料,每处理设3个平行样,对体重为30~40 g的日本鳗鲡(Anguilla japonica)进行为期60 d的饲养试验,每20 d取样一次,以相对增重率、成活率和血清中的溶菌酶(LZM)、一氧化氮合酶(NOS)、碱性磷酸酶(AKP)和超氧化物歧化酶(SOD)活性为指标,探讨了中草药免疫添加剂对日本鳗鲡生长和非特异性免疫效应的影响。结果显示:饲料中添加中草药免疫添加剂可以显著提高日本鳗鲡的增重率,降低饲料系数(P<0.05)。试验组日本鳗鲡血清中的LZM、NOS、AKP和SOD等活性均高于对照组。因此,中草药免疫添加剂具有显著促生长和增强机体免疫能力的作用,建议在实际生产中的添加量为1.0 g/kg较为合适。     

人工条件下日本鳗鲡胚胎及早期仔鱼发育的生物学特征

在多年对日本鳗鲡人工育苗技术研究的基础上,通过采用对胚胎和早期仔鱼的活体观察、计算机图文分析以及组织切片等方法,系统研究了鳗鲡胚胎及孵化后19d前仔鱼的生物学特性。研究结果表明,在水温(23±0.5)℃条件下,日本鳗鲡从受精卵到仔鱼出膜需约38h30min,总积温为885.50℃·h。其特征与一般硬骨鱼类胚胎发育基本相同,鳗鲡胚胎发育可分为14期,为典型的盘状卵裂,原肠作用通过细胞的下包和内卷完成。但日本鳗鲡早期仔鱼与一般硬骨鱼类的仔鱼存在较大差异,表现为无色透明的血细胞和S形心管;开放型的口腔,发达且不能闭合的上下颌和3对颌齿;孵化后3～9d在身体两侧出现由神经细胞和支持细胞构成发达的感觉丘,其数目为6～9对不等;以及形态不断变化的尾垂体等生物学特性。分析认为这些生物学特性决定了日本鳗鲡仔鱼的孵化环境可能相对较稳定,以及仔鱼摄食方式可能为触碰后咬食的方式。     

鳗鲡繁殖生物学研究进展

鳗鲡是优质养殖鱼类,但鳗鲡苗种完全依靠天然捕捞。近年来鳗鲡苗种的短缺限制了鳗鲡养殖业的进一步发展,开展鳗鲡人工繁殖技术研究势在必行。文章从鳗鲡的催熟与催产、产后鳗鲡的某些繁殖生物学以及鳗鲡胚胎和仔鱼发育等方面简要回顾了鳗鲡繁殖生物学的研究概况,并着重介绍了日本的最新研究进展,日本已将人工苗培育至20多厘米的成鱼,实现了实验室内鳗鲡由卵到成鱼的全人工养殖。文章最后指出人工育苗中存在的问题,并提出今后应从内因和外因两方面研究,才能真正实现鳗鲡苗种规模化生产。     

Population structure of the Japanese eel Anguilla japonica as examined by mitochondrial DNA sequencing

SUMMARY: To examine the population structure of the Japanese eel Anguilla japonica , mitochondrial DNA sequence analysis was made for various samples of glass eels that might reflect genetic characteristics of spawning aggregates. The mitochondrial DNA from a total of 51 glass eels collected at Tanegashima Island, Kanagawa and Ibaraki in different periods within an inshore migrating season was sequenced for a 615 base-pair fragment from the tRNA^Thr gene to the central part of the control region. The DNA region was so variable that no individual was found to possess the same sequence, although average sequence differences within samples (1.07–1.63) were not large. Average sequence differences between samples (1.22–1.57) were comparable to those within samples, suggesting no genetic heterogeneity among samples. Tree analysis of the sequences showed neither a geographical nor temporal structure of population. Furthermore, although all DNA sequences from the present study were different from one another, two sequences were found to be the same with those reported from individuals collected in different localities in different years. Altogether, the present mitochondrial DNA sequence analysis of glass eels did not provide any evidence for genetic subdivision of the Japanese eel population.     

日本鳗鲡腐皮病病原菌的分离及鉴定

从患腐皮病的日本鳗鲡(Anguilla japonica)体表溃烂处分离到1株病原菌(322A),对其进行了人工感染实验和生理生化分析,测定了该菌株的16S rRNA基因序列和促旋酶(gyrase)B亚单位gyrB基因序列,并分别构建系统发育树。结果显示:感染实验证实菌株322A具有致病性;生理生化分析鉴定该菌株属于气单胞菌属(Aero-monassp.);16S rRNA基因分析显示,该菌株与气单胞菌属细菌的同源性均在99%~100%,构建的系统树显示,菌株322A与嗜水气单胞菌(A.hydrophila(FJ462702))亲缘关系最近;gyrB基因分析表明,该菌株与A.hydrophila种内序列的相似性为96%~98%,种间序列的相似性为94%~95%,构建的系统树结果显示,该菌株与A.hydrophila(FJ608553、FJ608552、AF208259)聚为一个分支。综合上述实验结果,菌株322A可鉴定为嗜水气单胞菌(A.hydrophila)。     

长江口环境条件对日本鳗鲡(Anguilla japonica)鳗苗溯河洄游的影响

为掌握长江口日本鳗鲡的苗汛特征与捕捞生产现状,于2017—2020年在长江口开展了定点监测和走访调查,分析了长江口鳗苗的汛期规律、捕捞努力量及捕捞产量和产值。研究发现,长江口鳗苗旺发期为2－4月,主要捕捞区域分布在东旺沙、佘山岛和南汇嘴附近水域。长江口鳗苗捕捞努力量呈现下降趋势,有效网口面积从2017年的78.72万m²,下降到2020年的50.40万m²,下降了36%。长江口鳗苗汛期的单位捕捞努力量渔获量 (CPUE)和总捕捞产量呈现波动变化趋势,2017和2020年较高,分别为(4 474±256) 尾/100 m²和(5 220±1 063)尾/100 m²,2018和2019年较低,分别为(1 917±335)和(1 365±257) 尾/100 m²。研究表明,长江口鳗苗生产值受到捕捞总产量影响,近4年来逐渐下降。建议进一步加强长江口鳗苗的资源监测和科学评估,指导规范鳗苗捕捞生产。     

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.     

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,...     

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。     

Genetic variability of the Japanese eel Anguilla japonica (Temminck & Schlegel) related to latitude

Abstract— Isozyme genotypes of 400 glass eels recruiting to 4 localities along the east Asian coast, stretching from Taiwan to the Yalu River of northeastern China, were studied using starch gel electrophoresis. Geographic cline was found to exist in two loci: NADP-isocitrate dehygenase-1 and 6-phosphogluconate dehydrogenase. Frequencies of the most common allele of these two loci increased from south to north. In the latitudinal range of 25°N to 40°N, the magnitude of difference of IDH100 and PGD100 was 13% and 9% respectively. However, deviations from Hardy-Weinberg equilibrium were found to be insignificant in both loci at three of the four localities. The cline was, therefore, unlikely to have resulted from selection. Migration time-lag from different parts of the continent to the spawning ground in the western Pacific was suggested to be a possible reason for the formation of the cline.     

Environmental DNA analysis reveals the spatial distribution,abundance, and biomass of Japanese eels at the river‐basin scale

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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。