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.     

Isolation of a novel polyomavirus,related to Japanese eel endothelial cell‐infecting virus,from marbled eels,Anguilla marmorata (Quoy & Gaimard)

Marbled eels, Anguilla marmorata (Quoy & Gaimard), cultured in Taiwan exhibited haemorrhage and mortality in January 2012. The severely diseased eels bled from the gills and showed congestion of the central venous sinus of the gill filaments and haemorrhage throughout the body similar to viral endothelial cell necrosis of eel. In this study, a novel polyomavirus (AmPyV) was isolated from the diseased eels using the AMPF cell line established from the pectoral fin of healthy marbled eels. AmPyV was found to encode a long T‐antigen orthologous gene. Phylogenetic analysis showed that AmPyV was closely related to Japanese eel endothelial cell‐infecting virus. PCR assays revealed AmPyV infection throughout the systemic organs. AmPyV proliferated in the AMPF, EK‐1 and EO‐2 cells at temperatures 25–30 °C, and the progeny virus yields were 10^7.0, 10^7.4 and 10^7.7 TCID₅₀ mL^?1, respectively. The purified virions were icosahedral particles, 70–80 nm in diameter. No clinical signs or mortality was observed among the eels injected with the virus; however, the virus was reisolated from the brain, eyes, kidneys, fins and gills of infected eels 2 month after injection. Our results suggest that AmPyV exhibits a latent infection. Pathogen of the disease needs to study further.     

Hen egg yolk and skinned krill as possible foods for rearing leptocephalus larvae of Anguilla japonica Temminck & Schlegel

Usual diets for rearing leptocephalus larvae of Japanese eel Anguilla japonica include eggs of the endangered spiny dogfish Squalus acanthias (SE). We investigated the effects of alternative food materials, hen egg yolk (HEY) and exoskeleton‐free (skinned) Antarctic krill (SAK), on the growth and survival of eel larvae. We found that feed comprising whole krill including exoskeleton (WAK) containing higher levels of fluoride (37.89 mg kg^?1) was acutely toxic to eel larvae exposed to this alone. In contrast, extract from SAK containing lower concentrations of fluoride (4.25 mg kg^?1) showed no apparent adverse effects. Growth of larvae fed a mixture of SE and SAK in a feed trial of 58 days [mean body weight (BW), 6.0 mg] was about twofold higher than that of larvae fed a mixture of SE and WAK (3.2 mg) (P < 0.01). A mixture of HEY and SAK also had some dietary benefits for eel larvae, enabling them to survive for up to 58 days and to grow significantly (mean BW, 2.4 mg), compared with their initial weight (mean BW, 0.2 mg) (P < 0.001). Although additional nutritional improvements are needed, the present results suggest that combination diet HEY and SAK may be a good alternative to SE as an effective diet for eel larvae.     

Natural dietary additive yellow loess as potential antibiotic replacer in Japanese eel,Anguilla japonica: Effects on growth,immune responses,serological characteristics and disease resistance against Edwardsiella tarda

The present experiment was conducted to evaluate the efficacy of an additive derived from the nature as an alternative of dietary antibiotic in Japanese eel, Anguilla japonica. Six experimental diets were formulated to contain no antibiotics or additive (yellow loess/YL) (control/CON), three graded levels of yellow loess at 5 (YL₅), 10 (YL₁₀) and 20 g/kg (YL₂₀), oxytetracycline at 5 (OTC) and amoxicillin at 10 g/kg amoxicillin (AMX) of diet. Weight gain (WG) and specific growth rate (SGR) from fish fed CON or YL₅ diets were significantly lower than those of fish fed YL₂₀ or OTC diets. Among non‐specific enzyme, lysozyme activity of fish fed YL₂₀, OTC or AMX was detected to be significantly higher than that from fish fed CON or YL₅ diets, whereas superoxide dismutase (SOD) activity of the fish fed CON was significantly lower than that for fish fed other experimental diets. Challenge test with bacteria, Edwardsiella tarda, showed improved disease resistance among the fish fed different levels of natural additive without any statistical difference from those fed antibiotics (OTC and/or AMX) supplemented diets. Therefore, these results demonstrated the potential of natural feed additive, yellow loess to replace oxytetracycline and/or amoxicillin in Japanese eel, A. japonica.     

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。     

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.     

Isolation and characterization of bacteria causing mortality in early stage larvae of captive‐bred Japanese eels (Anguilla japonica Temminck & Schlegel)

Bacteria with lethal effect on eel larvae were isolated from moribund captive‐bred eel larvae and their 16S rRNA gene sequences were analysed. Nucleotide sequence of 10 isolates showed highest similarity with Lacinutrix algicola, Crocinitomix catalasitica and Pseudoalteromonas rubra. Age‐dependent changes in the susceptibility of eel larvae were observed in response to challenge by a highly lethal isolate. Compared with 10 and 11 days after hatching (DAH), larval susceptibility to the highly lethal isolate was lower at 18 DAH. We found that the bacterial isolates have lethal effect on the captive‐bred eel larvae, especially at the early developmental stage. These results will be useful to establish appropriate culture practices for eel larvae that will improve the success of mass production of glass eels for aquaculture.     

Detection of Herpesvirus anguillae (AngHV‐1) in European eel Anguilla anguilla (L.) originating from northern Poland—assessment of suitability of selected diagnostic methods

The Community Action Plan requests EU member states to implement measures that ensure the recovery of the severely depleted European eel stocks. One of the main threats is posed by Anguillid herpesvirus 1 (AngHV‐1) leading to increased mortality in both wild and farmed eels. Following recommendations of the OIE to minimize the risk of obtaining false‐negative results, the main aim of the study was to optimize diagnostic methods for AngHV‐1 detection using conventional PCR, nested PCR and in situ hybridization assay. While 53.3% of the individual organ samples were tested positive for AngHV‐1 by PCR, the additional virus analysis via nested PCR revealed that the actual prevalence was 93.3%. In the cell cultivation passages, a cytopathic effect was hardly found in the first two rounds. In the third passage onto cell cultures, a lytic CPE was detected. The identification and confirmation of the viruses obtained from cell cultures as well as directly from the organ tissues were proceeded by PCR, nested PCR and sequencing of the PCR products. While no positive signal was detectable in the first round by PCR using samples from the third cell culture passages, the nested PCR provided weak but visible positive signals.     

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)。     

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。     

A preliminary experiment regarding the natural induction of gonadal development in female Japanese eels without hormone treatment

To test whether gonadal development of female eels could be promoted without any exogenous hormone treatments, we observed the effect of water temperature manipulation. After 3–5 months of water temperature treatments, three silver eels showed higher gonadosomatic indices (GSI). In particular, one eel in the 5–15°C fluctuating temperature treatment group (5°C daytime and 15°C night‐time) had the highest GSI of 8.5 with secondary yolk globule stage oocytes of large diameter (OD; 412 μm) after 3 months, which indicated definite gonadal development compared with those in the initial states (mean GSI, 2.4; OD, 226.7 μm). The 5–15°C fluctuating temperature and constant 5°C groups had low oocyte breakdown (atretic) rates. Because daily temperature fluctuations and cold daytime water are experienced by vertically migrating silver eels in the ocean, these temperature conditions may be key maturation process components that could be useful for hormone‐free artificial maturation.     

Site fidelity,diel and seasonal activities of yellow‐phase Japanese eels (Anguilla japonica) in a freshwater habitat as inferred from acoustic telemetry

Japanese eels (Anguilla japonica) perform large‐scale oceanic migrations between their spawning ground and growth habitats in continental waters during life history. However, between these migrations, they spend most of time in growth habitats such as rivers. To investigate the diel and seasonal activities, homing behaviour and home range of yellow‐phase Japanese eels in the lower reach of the Tone River, we tracked them throughout a year using a fine‐scale positioning system (VPS) based on acoustic telemetry. The tracked eels were generally nocturnal, but not exclusively. They were mainly mobile from spring to autumn, with little or no activity observed during winter. A transport‐release experiment showed that most eels returned to their original capture area within 13 days after release. The eels had very small home ranges (mean ± SD = 0.085 ± 0.068 km²), core areas (0.014 ± 0.014 km²) and linear home ranges (744 ± 268 m). They also tended to be distributed on one particular side of the river (right or left bank) and in one particular shore type (revetment or vegetation), rarely moving from one to the other. This study provides evidence for nocturnal, dormancy, homing behaviours, limited habitat use and small home range size in Japanese eels. The eels clearly showed strong fidelity to a “familiar” site, which contrasts with the long distances travelled during upstream and downstream migration phases in the river, and during spawning migrations in the ocean.     

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.     

Effects of feeding stimulants on the feed consumption,growth and survival at glass eel and elver stages in the European eel (Anguilla anguilla)

Elvers and glass eels of Anguilla anguilla were fed diets containing two types of feeding stimulants (FS) that were based on processed marine (MBFS) and yeast proteins (YBFS). Elvers (1.5 ± 0.3 g) were fed seven diets (MBFS and YBFS diets at 20 g kg^?1, 40 g kg^?1 and 60 g kg^?1 plus control) for 60 days. Glass eels (250 ± 100 mg) were weaned to 60 g kg^?1 MBFS, 60 g kg^?1 YBFS and control diets for 30 days. Diets containing 60 g kg^?1 FS had a beneficial effect in terms of growth, homogenous size distribution and feed intake in elvers. Elvers fed 60 g kg^?1 MBFS and YBFS diets grew 11.9% and 5.6% faster than the control group. No differences in growth and size distribution were detected in glass eels fed 60 g kg^?1 MBFS and YBFS diets. However, FS affected the digestive system maturation; fish fed the 60 g kg^?1 MBFS and YBFS diets showed higher and intermediate values in the specific enzyme activities in comparison with the control group. This study revealed that the incorporation of FS into a pelleted diet was beneficial on the overall performance of European glass eels and elvers. However, the observed results were different depending on the eel’s stage of development, as well as the type and inclusion level of the FS.     

Demonstration of infectious pancreatic necrosis virus strain VR-299 in Japanese eel, Anguilla japonica Temminck & Schlegel

Abstract. Six isolates of infectious pancreatic necrosis virus (IPNV) were obtained from trout or eel farms in Taiwan in 1987. By using the electrophoretic analysis of ³⁵S-methionine-labelled early viral polypeptide patterns and silver-stained ds RNA gel patterns, a comparison of these isolates with the three archetypal IPNV serotypes (AB, SP and VR-299) was made. They were all identical with VR-299, both in the RNA and early viral polypeptide patterns. From the results obtained, it was apparent that the VR-299 serotype of IPNV was just as widespread in eel as in trout in Taiwan. This is the first case of VR-299 isolation from eel.     

Co‐Infection with Pseudomonas anguilliseptica and Delftia acidovorans in the European eel,Anguilla anguilla (L.): a case history of an illegally trafficked protected species

Inspections by customs agents at Barcelona airport discovered 420 kg of contraband glass eels prepared for shipment to Hong Kong. After confiscation of these animals by police, they were transported to holding facilities to be maintained until after a judicial hearing. Upon arrival, they were separated into two groups and held under ambient flow‐through conditions in fresh water. During their captivity period, several peaks in mortality occurred and multiple bacterial strains were isolated from moribund animals. Sequencing of 16S rDNA was used to determine specific identity of the isolates. An initial isolation of Pseudomonas anguilliseptica was treated with oxytetracycline. A subsequent isolation of Delftia acidovorans proved resistant to oxytetracycline and was treated with gentamicin in combination with sulphadiazine–trimethoprim. Once the health condition of the animals was stabilized, they were partitioned into groups and subsequently released as part of a restocking effort for the species following the guidelines of Regulation (EC) 1100/2007 (Anon 2007). This represents the first record for both bacterial species in the host Anguilla anguilla in the Spanish Mediterranean.