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.     

Epidermal response of the Japanese eel to environmental stress

Nonspecific responses of Japanese eels to environmental stress were monitored by assaying various lytic activities in eel epidermal extract. In fish maintained at 10 and 30 °C for up to 10 days, epidermal proteolytic activities due to serine protease and aminopeptidase and hemolytic activity varied within a 2-fold value range. Other proteolytic activities, due to cathepsins B and L, in the fish at 30 °C increased for up to 8 days and were 3.4 and 2.9-fold over those in fish maintained at 10 °C, respectively. This was accompanied by a 3.0-fold increase in bacteriolytic activity. Other forms of stress were exerted on the fishes by immersing them in a suspension of Flavobacterium columnare or giving them intraperitoneal injections of Edwardsiella tarda over 72 h. Although serine protease and aminopeptidase activities and hemolytic activity in the fishes exposed to F. columnare changed marginally, and were similar to those in the control fish, cathepsins B and L activities in the infected fishes increased more than 1.5-fold over their initial values over a 48 h period, along with a 4.5-fold increase in bacteriolytic activity. No marked change was detected in any of the lytic activities of the fishes exposed to E. tarda. These findings indicate that epidermal cathepsins B and L probably participate in bacteriolysis associated with Japanese eel skin and that their activities are elicited by environmental stimuli and may be an important nonspecific response of eels. Abbreviations: Cbz – carbobenzoxy; MCA – 4-methylcoumaryl-7-amide.     

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.     

Evaluation and characterization of a novel probiotic Lactobacillus pentosus PL11 isolated from Japanese eel (Anguilla japonica) for its use in aquaculture

In the present study, a potential Lactobacilli probiotics were isolated from Japanese eels (Anguilla japonica) and characterized and evaluated for their possible use in eel farming. Sixteen Lactobacilli were isolated from intestines of Japanese eels, using selective media. The lactobacilli strains (represented as PL1 to PL16) were screened by their ability to produce digestive enzyme. Among these, three strains (PL11, PL13 and PL16) producing four digestive enzymes (amylase, cellulase, protease and phytase) simultaneously were characterized further using API ZYM kit. From these, PL11 (Lactobacillu (L.) pentosus) was identified as potential probiotics candidate producing 15 enzymes among 20 tested. Further examination of biological activities of PL11 revealed tolerance against pH, artificial bile juice and antibacterial activity against several fish pathogenic bacteria. The in vitro competitive exclusion assay also revealed 88.4% reduction in adhesion of fish pathogen (Edwardsiella tarda) by PL11 to host intestinal mucus. In vitro incubation of Japanese eel foregut with Baclight‐labelled PL11 showed colonization of the enterocyte surface by confocal and scanning electron microscopy. In summary, PL11 isolated from eels could serve as a potential probiotics with acid and bile tolerance, production of digestive enzymes, antibacterial activity and inhibition of fish pathogen adhesion to intestinal mucus.     

Two forms of cytochrome P450 cDNA from 3-methylcholanthrene-treated European eel Anguilla anguilla

ABSTRACT:     The cytochrome P450 (CYP) represents a large group of microsomal monooxygenases that catalyze drugs as well as a host of lethal environmental contaminants such as dioxins, leading to either detoxification and excretion from the animal or generation of carcinogenic intermediates. In the present study two forms of cDNA were cloned (Eu MC1 and Eu MC2) for European eel CYP1A genes by polymerase chain reaction (PCR) techniques. The cDNA of Eu MC1 was 3368 bp long coding 521 amino acid residues, and that of Eu MC2 was 2464 bp long coding 517 amino acid residues. Identities of deduced amino acid sequences between Eu MC1 and Japanese eel CYP1A1 and that between Eu MC2 and the second form of Japanese eel CYP1A were 98% and 97%, respectively, showing decisively that Eu MC1 and Eu MC2 are orthologous to Japanese eel CYP1A1 and the second form of CYP1A, respectively. A striking difference between the two eel species was that the Eu MC1 peptide was two amino acid residues longer than that of the Japanese eel CYP1A1. Existence of two loci of CYP1A in Japanese and European eels may suggest that the two forms of CYP1A exist widely among the eel species, because the divergence between the two eel species has been shown to be close to the basal divergence among eels. The identities in CYP1A may help to estimate genetic distance between European and Japanese eels.     

The eel immune system: present knowledge and the need for research

The European eel, Anguilla anguilla, is one of the most important warm water fish species cultured in southern Europe and the Mediterranean as well as in northern countries including Germany, the Netherlands and Denmark. The Japanese eel, A. japonica, is an important cultured fish in several Asiatic countries including Japan, China and Taiwan. During recent decades, research has been performed to elucidate the immune response of these species against different pathogens (viruses, bacteria or parasites). Nevertheless, there is very limited information in terms of both cellular and humoral immune responses. This review summarizes the present knowledge relating to the eel immune system and includes new data.     

A review of extracellular virulence product of Vibrio species important in diseases of cultivated shrimp

Shrimp aquaculture is an important industry that experiences significant losses from Vibrio species, especially at the larval and juvenile stages. Proteinaceous virulence factors, including alkaline proteases, metalloproteases, cysteine proteases and alkaline serine proteases, have been identified as important elements in Vibrio pathogenesis. This review summarizes current knowledge regarding the principal pathogenic Vibrio species in shrimp, with emphasis on relevant exotoxins and their modes of action, principal characteristics and molecular database. This pathogenic factors and their relation with other molecules produced by microorganisms may be help to understand the virulence mechanisms present in Vibrio strain.     

The pathogenic helminth parasites of eels

Although 63 and 55 species of helminths have been reported from each species of Atlantic eel and from 29 to 19 for each species of Pacific eel only the monogeneans Pseudodactylogyrus bini and P. anguillae and the nematode Anguillicola crassus, originally specific to species of Pacific eels, can be considered serious pathogens. None of the three are normally pathogenic to their preferred natural eel host species in the wild. Pseudodactylogyrus spp. only cause serious local gill damage when present on a host in large numbers under optimal conditions that facilitate transmission. This is the case in eel aquaculture, where infections can be controlled by drugs. Anguillicola crassus is only pathogenic to Anguilla anguilla and A. rostrata when Atlantic eels are introduced to the far east or when the parasites have been introduced to Europe. Here the parasite life cycle differs in that A. crassus can infect a wide range of intermediate hosts, employ paratenic hosts and survive as larvae for months in the swimbladder wall. This makes it an excellent colonizer. Its major pathogenic effects on eels result from haemorrhaging in, and thickening of, the swimbladder wall. It reduces the oxygen concentration in the swimbladder, reducing its ability to function as a hydrostatic organ, and increases the stress response of eels. In shallow lakes at warm temperatures this can result in mass mortalities. It is also feared that the parasite affects the ability of eels to migrate to the Sargasso Sea and so contributes to the decline in eel populations. Control by drug treatment is possible in culture, but not in the wild.     

美国金鳟和日本鳗鲡精浆的化学组成和渗透压

本研究采用原子吸收分光光度法(AAS)和气相渗透压仪测定了人工养殖美国金鳟和日本鳗鲡精浆的化学组成和渗透压。研究结果表明,Na+、K+是精浆中的主要离子,可能在维持精浆渗透压和精子活动能力中有重要的作用。人工养殖美国金鳟与日本鳗鲡相比,精浆离子含量各项指标除K含量较高之外(P<0.01),其它各项指标均无显著性差异。本实验的两种鱼与其它种类海淡水鱼类比较,各种离子含量均较高;美国金鳟精浆渗透压值与其它鱼类相似,均保持在300 mOsm/kg的水平。     

The impact of Anguillicola crassus on European eels

The impact of the parasitic swimbladder nematode, Anguillicola crassus Kuwahara, Niimi & Itagaki, on European eel populations is assessed with reference to published research on its origin and rapid dissemination, life cycle and transmission dynamics, and its pathogenic effect. The parasite was originally endemic to East Asia, but has transferred from its native host, the Japanese eel, Anguilla japonica Temminck & Schlegel, to the European eel, Anguilla anguilla (L.) and American eel, Anguilla rostrata (Le Seur). Anguillicola crassus is a very successful colonizer and is now known to occur in four continents (Asia, Europe, Africa and America). The nematode can severely impair swimbladder function and has caused mortalities in both farmed and wild populations in the presence of other stressors. Anguillicola crassus may impair the capacity of European eels to complete the spawning migration, although direct evidence is not available to support this hypothesis. Areas for future research are recommended.     

In vivo gonadotropic effects of recombinant Japanese eel follicle-stimulating hormone

Recombinant Japanese eel follicle-stimulating hormone (rjeFSH) produced by methylotrophic yeast was subjected to in vivo bioassay to assess its gonadotropic activities and availability to artificially induce maturation of this species. Intramuscular injections of rjeFSH into male eels at doses of 0.1 and 1.0 U/g body weight were repeated 3 times during 12 days. The rjeFSH significantly increased plasma 11-ketotestosterone levels, and induced both testicular growth and spermatogenesis in a dose-dependent manner. These results demonstrate that rjeFSH is effective in promoting male eel gamatogenesis in vivo. Instead of scarce native eel FSH, abundant recombinant eel FSH is now ready for future application to maturation induction of Japanese eel.     

酶法提取烤鳗下脚料水解动物蛋白的研究

以烤鳗下脚料为原料，经蛋白酶水解后，提取水解动物蛋白，使废充物得到充分利用。通过正交实验，确定了木瓜蛋白酶、枯草杆菌蛋白酶、胃蛋白酶的最佳水解条件，选择了木瓜蛋白酶为最佳水解酶。     

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.     

日本鳗鲡人工催产后亲鱼恢复培养与再催产效果

日本鳗鲡(Anguillajaponica)产后亲鱼的恢复培养全过程包括:产后亲鱼的海水淡化、诱导开口以及亲鱼培育。对2种不同淡化方式的结果比较发现,日本鳗鲡亲鱼的海水淡化方式以缓慢连续的方式较好,即每天换水1次,每次淡化量为总盐度的3%～4%,总淡化时间为1个月。淡化后的亲鱼存活率分别为:自然产卵的产后亲鱼达到100%,人工授精的产后亲鱼为86.3%,而难产的产后亲鱼则为81.5%。在各种开口驯养方式中,以水蚯蚓为开口饵料的驯养效果较好。诱导亲鱼开始摄食所需时间最短,为18d;摄食量也最大,达到平均体重的2.5%。从水蚯蚓逐步转为全人工饵料后,进行日常亲鱼培育。经18个月的恢复培养后,使产后亲鱼的平均体重雌鳗330g、雄鳗150g分别恢复到765g和470g。但2次繁殖的各项指标中,催熟率、成熟系数和性腺发育情况远远低于野生鳗,而催产率和幼苗存活率则略高于野生鳗,其原因尚有待进一步研究。     

鲤脑垂体匀浆液和人绒毛膜促性腺激_省略_性腺激素及性类固醇激素含量的影响

雄鳗注射５－６次、雌鳗注射９－１０次鲤脑体匀浆液（ＣＰＥ）＋人绒毛膜促性腺激素（ＨＣＧ）能分别诱导精巢和卵巢发育成熟。在雌雄鳗鲡，注射ＣＰＥ＋ＨＣＧ可显著增加端脑、间脑、中脑和下丘脑ｍＧｎＲＨ的含量，而对后脑和延髓ｍＧｎＲＨ的影响较小；注射ＣＰＥ＋ＨＣＧ增加雄鳗后脑和延髓ｃＧｎＲＨ－Ⅱ含量，对雌鳗脑区ｃＧｎＲＨ－Ⅱ则无显著影响。雌雄鳗鲡每次注射ＣＰＥ＋ＨＣＧ后１天，血清促性腺激素（ＧｔＨ）急剧上升     

Discovering the dominance of the non-native European eel in the upper reaches of the Tone River system,Japan

To investigate the presence of non-native anguillid eels in Japanese waters, 141 eels were collected from seven sampling sites throughout the Tone River system. Genetic species identification showed an extraordinary dominance of the non-native European eel Anguilla anguilla in the uppermost site of the study area. Estimation of age from otoliths suggested that the European eels were introduced into the river in the 1990s, corresponding to previous reports from other Japanese water systems. Comparison of the von Bertalanffy growth curve parameters indicated that the European eels caught in the Tone River system appeared to have a similar or even higher growth rate than the same species in the original habitats in Europe. The long-term inhabitation and the normal development of European eels in Japanese waters suggest that regulations prohibiting the release of non-native eels and safeguards against accidental escape from culture ponds must be strictly maintained in order to ensure the conservation of the native Japanese eel.     

Assay of Biochemical Compositions of Two Indian Fresh Water Eel with Special Emphasis on Accumulation of Toxic Heavy Metals

This study investigated lipid and protein composition of two river eel available in India, Mastacembelus armatus and Anguilla bengalensis bengalensis, to ascertain their nutritional value. The carnivorous nature of eel makes it susceptible for bioaccumulation and biomagnification of toxic metal in their body; therefore, the measurement of two toxic heavy metals, mercury and arsenic, in the muscle tissues of these two eel species was also determined. The results showed that though the lipid content of both the species was quite low in comparison to European or Japanese eel, the protein content was appreciably high, and the value was quite similar in both species (～24%). The fatty acid profile and amino acid profile of the two species were also determined to quantify the amount of essential fatty acid as well as essential amino acid in their muscle tissue. The amount of total mercury, organic mercury, and arsenic was found to be non-remarkable which indicates the beneficial and safe consumption of these species.     

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。