Cortisol and liver metabolism of immature American eels,Anguilla rostrata (LeSueur)

Plasma and liver composition, liver enzyme activities, and metabolite flux in isolated hepatocytes have been studied in immature American eels,Anguilla rostrata, injected daily IP with saline or cortisol (0.35 mg/kg eel). Plasma cortisol values were significantly increased above saline controls in those eels receiving cortisol at 3h and 6h following the final (tenth) injection. On day 6 and 10 of injection plasma cortisol levels were significantly below saline controls 24h following cortisol injection. Plasma glucose values were significantly depressed in the cortisol-injected eels at both 6 and 24h following the final (tenth) injection. At the 24h sampling time, plasma protein had significantly increased, but there was no change in either plasma amino acid or fatty acid levels. An increased hepatosomatic index was attributed to a major increase in total lipids, as both protein and glycogen contents were decreased. Of the liver enzymes assayed, significant activity changes occurred only for lactate dehydrogenase (decreased), mitochondrial citrate synthase (increased) and phosphoenolpyruvate carboxykinase (increased) 24h following the final (tenth) cortisol injection. Although enzyme activity changes implied increased liver gluconeogenesis, the absolute rate of lactate, alanine, and aspartate incorporation into glucose declined in viable hepatocytes isolated from cortisol-injected eels compared to the saline controls. Relative changes in metabolite flux did support a preferential increase in gluconeogenesis from amino acids. These results are consistent with the increase in amino acid gluconeogenesis as a result of cortisol administration implied in previous studies, but failed to show a definitive cortisol effect on this pathway in the eel liver. It is suggested that other hormones (e.g. thyroxine, catecholamines, glucagon) may interact in a complex way with cortisolin vivo to bring about the biochemical changes observed in this study. The rapid clearance of exogenously injected cortisol noted in this study makes causal relationships between the injected hormone and any observed metabolic effect in the intact animal difficult.     

Changes in plasma glucagon and insulin associated with fasting in sea bass (Dicentrarchus labrax)

Juvenile sea bass (Dicentrarchus labrax) were fasted for 22 days and changes in plasma insulin, glucagon and glucose levels, as well as glycogen and protein content in liver and muscle were analyzed. Glucagon increased initially on the 4th day of fasting. The glucagon/insulin ratio (G/I) increased from a value of 0.11±0.02 (24h of fasting) to 0.21±0.05 (4th day of fasting). Thereafter, both glucagon and insulin levels decreased and remained at low concentrations until the 22nd day of fasting. Plasma glucose levels fell at the beginning of fasting, stabilized between the 4th and 8th day, and gradually declined during the rest of the experiment. There was a body weight loss of 15% and a significant decrease in both the hepatosomatic index and mesenteric fat. The decrease in the percentage of muscle proteins was not significant, while liver glycogen content showed a sharp decline.     

Movements of the European eel (Anguilla anguilla) in a chalk stream

Abstract – The movements of wild European eels (Anguilla anguilla L.) were monitored continuously over a 2‐year period in a tributary of the River Itchen, Hampshire, UK, using a passive integrated transponder (PIT) antenna system. The time of these movements was then related to a number of monitored and calculated environmental parameters. No eel movements were recorded in the winter months when mean stream temperature was below 10 °C. The numbers of movements peaked in May, when mean water temperatures and day length were increasing. The movement of eels was significantly (P < 0.001, R² = 0.45, N = 72) correlated with the time of sunset, with the majority (72%) recorded during the hours of darkness. The results of this investigation have implications for the design/timing of surveys of yellow eel populations and their habitat associations and suggest that successfully determining the microhabitat preferences of active resident eels will require remote monitoring throughout the 24‐h period.     

欧洲鳗短钩拟指环虫病及其鳃组织病理

报道了患短钩拟指环虫病欧洲鳗的症状、流行状况和鳃显微组织病理.游动和呼吸频率异常、鳃丝浮肿和鳃上粘液增多为该病的主要症状.该病尽管在冬季也有发生,但水温在26℃以上的春末、夏季和秋初是最易发病的流行季节,流行出现明显的季节性变化.患病欧洲鳗鳃的组织病理变化主要表现出5种类型,其一是鳃小片上皮细胞增生,使邻近的鳃小片相连;其二是鳃小片粘液细胞增生,同样使鳃小片连成一片,增生的粘液细胞经阿利新蓝(Alclan blue)和过碘酸雪夫氏(Periodic acid schiff)二者联合染色后呈蓝紫色的染色反应,属于Ⅳ型的粘液细胞;其三是鳃小片肿大,但单层扁平上皮细胞无肿大现象,上皮细胞层与毛细血管相分离,鳃小片上皮细胞坏死,上皮细胞层破裂,血细胞外溢,鳃小片上皮细胞坏死脱落后,失去鳃小片原有的结构;其四是鳃小片肿大同时伴随单层扁平上皮细胞肿大,进一步发展,鳃小片上皮细胞坏死脱落后,鳃小片同样失去原有的结构;其五是鳃小片毛细血管严重充血扩张,比原毛细血管扩张几倍到十几倍,形成充满红细胞的棒状到球状的动脉瘤.结果表明鳃上皮细胞增生、粘液细胞增生、鳃小片肿大、上皮细胞坏死脱落和严重充血成动脉瘤等病理变化都可导致患病欧洲鳗呼吸困难,轻者影响其生长,重者最终因呼吸衰竭而死亡.     

Migratory history of the giant mottled eel (Anguilla marmorata) in the Bonin Islands of Japan

Chino N, Arai T. Migratory history of the giant mottled eel ( Anguilla marmorata ) in the Bonin Islands of Japan.
Ecology of Freshwater Fish 2010: 19: 19–25. © 2009 John Wiley & Sons A/S
Abstract –  In order to elucidate the migratory history and habitat use of the giant mottled eel Anguilla marmorata, in the Bonin Islands of Japan, strontium (Sr) and calcium (Ca) concentrations in otoliths were examined by X-ray electron microprobe analysis. The X-ray intensity map and line analyses showed three fluctuation patterns: (i) freshwater residence, (ii) continuous residence in brackish water, and (iii) residence in freshwater after recruitment, while returning to brackish water. Based on these results and previous studies, the migratory behaviours of A. marmorata appear to differ in each habitat in response to inter- and intra-specific competition. The results suggest that A. marmorata has a flexible pattern of migration, with an ability to adapt to various habitats and salinities.     

A new species of freshwater eel <Emphasis Type="Italic">Anguilla luzonensis</Emphasis> (Teleostei: Anguillidae) from Luzon Island of the Philippines

Anguilla luzonensis, a new species of freshwater eel, family Anguillidae, is described on the basis of 29 specimens collected from the Pinacanauan River system, which is a tributary of the Cagayan River on northern Luzon Island of the Philippines. The new species and A. celebesensis, which is distributed in the same region, both have variegated color marking and broad maxillary bands of teeth, but show statistically significant differences in the following characters of the new species: predorsal-fin length 28.6–33.1% of total length (TL); preanal length 39.6–44.8% of TL; trunk length 26.4–31.3% of TL; distance between the verticals through the anus and origin of the dorsal fin 9.3–13.9% of TL; length of gape 35.5–46.9% of head length; the total number of vertebrae 103–107; and number of abdominal vertebrae 40–42. These morphological differences, together with the present knowledge about anguillid eel taxonomy, genetics, and ecology, strongly support the presence of the new species A. luzonensis in the area of northern Luzon Island of the Philippines.     

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.     

外源性促性腺激素诱导日本鳗鲡精子发生和成熟的作用机制

用兔抗血清对抗促黄体素生成素受体（LHR）或称绒毛膜促性腺激素受体（CGR）和雄激素受体（AR）进行LHR和AR免疫组织化学定位,以揭示外源性促性腺激素（鲤脑垂体激素和hCG）诱发日本鳗鲡精子发生及其内分泌机制。结果表明,经过注射激素处理后的实验组与注射前的对照组相比较,其精巢发育和精子发生出现十分显著的变化。组织学切片观察显示,激素处理前鳗鲡精巢处于精原细胞增殖期,而两种激素混合注射后第10天,实验组可见精小叶中精原细胞的有丝分裂和初级与次级精母细胞的数量显著的增加。注射后第35天,靠近生殖上皮除有少量精原细胞外,精小叶中有大量初级精母细胞和次级精母细胞和少数精子细胞以及管腔中存在少量精子。在注射后第83天,日本鳗鲡完成了精子发生和精巢发育成熟以及释精。免疫组织化学染色结果进一步揭示,激素处理前,LH受体免疫活性分布在生殖上皮,显示强的免疫阳性反应;激素处理后,LH受体定位在Sertoli细胞和间质细胞以及精原细胞和初级与次级精母细胞的胞膜上,均显示强的免疫阳性反应。激素处理前,雄激素受体定位在生殖上皮和早期生精细胞的胞膜上;激素处理后,AR则定位在这些生精细胞的核或胞质,而精子细胞和精子显示免疫阴性反应。这些结果首次证明了这两种激素诱导鳗鲡精子发生和成熟的作用机制是通过LH受体和雄激素受体的介导。     

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)。     

A simple method to distinguish two commercially valuable eel species in Japan <Emphasis Type="Italic">Anguilla japonica</Emphasis> and <Emphasis Type="Italic">A. anguilla</Emphasis> using polymerase chain reaction strategy with a species-specific primer

In order to distinguish the two eel species Anguilla japonica and A. anguilla irrespective of unprocessed and processed samples, a quick, convenient method using polymerase chain reaction (PCR) with a species-specific primer was established. The comparison of partial sequences of the mitochondrial 16S ribosomal RNA gene between A. japonica and A. anguilla facilitated designing of a primer pair common to both A. japonica and A. anguilla and a primer specific to A. japonica. PCR was carried out with the three primers for total 110 specimens of A. japonica and A. anguilla. PCR products with the species-specific primer showed two bands for A. japonica and one band for A. anguilla in an agarose gel electrophoresis. This analytical procedure required only a short period and is more convenient than PCR-restriction fragment length polymorphism, one of the standard methods employed for fish species identification.     

日本鳗鲡TRAF3基因的克隆及功能研究

为探究鱼类TRAF3在鱼类抗病毒免疫应答中的功能及作用机制,实验利用逆转录PCR克隆获得了日本鳗鲡TRAF3转录本 (AjTRAF3),利用生物信息学软件分析了AjTRAF3的结构特征,利用实时荧光定量 PCR(qPCR)、双荧光素酶报告系统以及免疫共沉淀等方法对其表达规律、功能及作用机理进行了初步分析。AjTRAF3的开放阅读框长度为1 707 bp,编码568个氨基酸。序列结构分析结果显示,AjTRAF3由N端的环结构域2个锌指结构域以及1个螺旋结构域和C端高度保守的TRAF-C (MATH)结构域组成。qPCR结果显示,AjTRAF3在日本鳗鲡各组织中均有表达,脑组织中表达量最高,其次为头肾,心脏中的表达量最低。Poly I:C刺激6 h后,日本鳗鲡脾脏组织中AjTRAF3上调倍数最高,为对照组的15.83倍。迟缓爱德华氏菌感染24 h后,日本鳗鲡脾脏组织中AjTRAF3上调倍数最高,为对照组的31.47倍。此外,本研究构建了AjTRAF3真核表达质粒,发现过表达AjTRAF3能显著上调炎症及抗病毒相关基因的表达,可显著增强AjIFN2、AjIFN4和NF-κB启动子荧光素酶活性。并能显著上调由AjRIG-IN、AjMAVS、AjIRF3诱导的AjIFN2、AjIFN4和NF-κB启动子活性。免疫荧光结果显示,AjTRAF3主要定位于细胞质中,且与AjMAVS存在共定位。免疫共沉淀结果显示,AjTRAF3通过MATH结构域与AjMAVS相互结合,缺失该结构域后,其与AjMAVS的相互作用消失,推测AjTRAF3可通过介导RIG-I/MAVS信号转导途径调控鱼类的抗病毒免疫应答。本研究结果为进一步揭示鱼类TRAF3的生物学功能奠定了基础。     

美洲鳗鲡及其养殖水体分离耐药菌的多样性和耐药性分析

为了更有针对性地防控水产动物细菌性病害的发生和流行,本实验对美洲鳗鲡及其养殖水体耐药细菌的种属特征及耐药情况开展了相关研究。首先采集美洲鳗鲡不同部位(表皮、鳃、肠道)及其养殖水体的样品,经5种抗菌药物平板筛选耐药菌株,然后采用K-B纸片扩散法检测细菌对抗菌药物的耐药性,同时测定耐药菌株的16S rDNA序列,进而分析耐药菌的种属分布和多重耐药性。结果显示,经耐药平板筛选分离纯化得到108株细菌,分别属于气单胞菌属、柠檬酸杆菌属、不动杆菌属等20个属;其中,93.5%的菌株对3种(含)以上的抗菌药物具有耐药性,86.1%的菌株对3类(含)以上的药物具有抗性。对阿莫西林的耐药率高达90.7%,对四环素、利福平以及磺胺类和酰胺醇类药物类的耐药率为60%~80%,对头孢噻肟、新霉素以及喹诺酮类的耐药性弱(低于20%)。美洲鳗鲡肠道(0.40)、表皮(0.41)、鳃部(0.42)及水样(0.47)菌群的多重耐药指数显示各生态样品耐药程度较为严重,尤以水样为最。各菌属中,柠檬酸杆菌属(0.58)和克雷伯菌属(0.61)的多重耐药指数最高,而不动杆菌属(0.21)则相对较低。美洲鳗鲡及养殖水体普遍存在多重耐药菌株,对此应引起足够的重视;水产动物及养殖环境耐药细菌对某些水产用药如诺氟沙星、新霉素等耐药率低,可将其做为水产动物细菌性疾病治疗的首选药物。     

Effects of cortisol on hepatic carbohydrate metabolism and responsiveness to hormones in the sea raven,Hemitripterus americanus

The sea raven, Hemitripterus americanus, is a sit-and-wait, low metabolic rate, marine teleost. The objective of this study was to determine i) whether cortisol implantation (50 mg. kg^-1) for 7 days altered hepatocyte metabolism, and hepatocyte responsiveness to epinephrine, glucagon and insulin, and ii) whether 8 weeks of food-deprivation modified the above response. Cortisol implantation significantly increased hepatocyte total glucose production and oxidation from alanine compared to the sham group. There was no cortisol effect on glycogen breakdown, suggesting that the activation of other pathways, including gluconeogenesis, are required to account for the increased glucose production. Epinephrine-mediated (10^-5M) glycogen breakdown and insulin-mediated (10^-8M) total glucose production were enhanced in hepatocytes of cortisol implanted sea ravens, but there were no change in any glucagon (10^-7M) effects. The enhanced glycogen breakdown in the absence of similar increases in total glucose production with epinephrine indicates mobilization of carbohydrate reserves for endogenous use by the liver. Food-deprivation for 8 weeks significantly decreased condition factor, plasma cortisol concentration and liver glycogen content in the sea raven, but had no effect on plasma glucose concentration. Hepatocyte total glucose production and flux rates from alanine increased significantly with food-deprivation. Moreover, food-deprivation increased responsiveness of total hepatocyte glucose production to the actions of glucagon and insulin, but not to epinephrine; none of these effects were modified by cortisol implantation. Our results indicate that cortisol in the sea raven exerts both a direct and an indirect or permissive effect on hepatocyte metabolism by modifying hepatocyte responsiveness to epinephrine and insulin stimulation. Cortisol implantation did not modify the effects of glucagon or food-deprivation in this species.     

Biochemical composition of eggsin relation to egg quality in the Japanese eel, Anguilla japonica

This paper presents the relationship between egg quality and egg biochemical composition of cultured and wild Japanese eel, Anguilla japonica. Eggs were obtained by artificialinduction of maturation. Fertilization and hatching rates were used as characteristics of egg quality. Egg quality characteristics showed large variation; fertilization rate, 0–96; hatching rate, 0–84%. The biochemical composition also showed a large variation. There was no marked relationship between egg quality and fatty acid contents of eggs, except for n-6 highly unsaturated fatty acids (HUFA). Both the fertilization and hatching ratesincreased proportionally withincreases of the -tocopherol g(-Toc) contentin eggs. A more significant correlation was found between the amount of -Toc relative to the amount of HUFA and egg quality. The results of this study show that the egg quality of Japanese eel is affected by the –Toc level, andin particular, the ratio of -Toc to HUFAin the eggs. Abbreviations: BHT – butylhydroxytoluene; EFA – essential fatty acids; FAME – fatty acid methyl esters; HPLC – high performance liquid chromatography; HUFA – highly unsaturated fatty acids; NADH – nicotinamide adenine dinucleotide; NADPH - nicotinamide adenine dinucleotide phosphate; ROS – reactive oxygen species; -Toc –-tocopherol.     

Protective immune responses of recombinant outer membrane proteins OmpF and OmpK of Aeromonas hydrophila in European eel (Anguilla anguilla)

Outer membrane proteins (Omps) of Gram‐negative bacteria have been proven to be efficient subunit vaccines against bacteriosis. In this study, OmpF and OmpK of Aeromonas hydrophila were expressed, and their immune protective effects in European eel (Anguilla anguilla) were evaluated. The genomic DNA of A. hydrophila 322A was used as a template, and two kinds of prokaryotic expression plasmids, pET‐32a‐OmpF and pET‐32a‐OmpK, were constructed. Recombinant OmpF protein (r‐OmpF) and r‐OmpK were purified and were proven to have antigenicity by Western‐blot analysis. r‐OmpF and r‐OmpK were used as immunogens to immunize European eel by intraperitoneal injection. The mRNA expression of 6 immune‐related genes (IgM, IL‐10, IRF3, IRF7, LysG4 and HexB) in the liver tissues of eels at 1 hr, 3 hr, 6 hr, 12 hr, 24 hr, 72 hr and 10 days postimmunization was analysed by real‐time PCR. At 30 dpi, the serum antibody response was measured by ELISA. Fish were attacked at 15 dpi by live 322A to assess the protective immunity of r‐OmpF and r‐OmpK. All the six tested genes responded to r‐OmpF or r‐OmpK vaccination at varying degrees. The serum antibody titre of r‐OmpF‐ and r‐OmpK‐immunized groups was 1:1,600 and 1:3,200 respectively. In addition, r‐OmpF gave 35.5% of the relative immune protection rate to European eels, while r‐OmpK gave 70.0%. By analysing the protective immunity and the regulatory role in the immune‐related gene expression of the two recombinant proteins that were studied, it was found that r‐OmpK was a potential vaccine candidate against A. hydrophila.     

抗冻剂对日本鳗鲡精子活力及运动时间的影响

利用精子稀释液(0.5%氯化钠+0.25%氯化钾+0.25%葡萄糖)分别配制4%、8%、12%、16%、20%、24%的二甲亚砜(DMSO)、甘油(Glycerol)、1,2-丙二醇(1,2-Propanediol)、乙二醇(Ethyleneglycol)四种抗冻剂,对日本鳗鲡精子进行激活处理,观察记录精子在不同浓度抗冻剂中的活力和5种运动(激烈运动、快速运动、慢速运动、摆动、死亡)时间。结果表明:4%~24%的四种抗冻剂都可以激活日本鳗鲡精子,其中二甲亚砜浓度为12%时,精子活力最高,平均可达到78.3%;甘油浓度为8%时,精子活力最高,平均可达到72.8%;12%的1,2-丙二醇和乙二醇激活精子后,精子活力都可以达到最高,分别是47.6%和57.8%。日本鳗鲡精子在8%的二甲亚砜、8%的甘油、8%的1,2-丙二醇和8%的乙二醇中,激烈运动时间均为最长,分别为11.8 s、11.1 s、6.0 s和2.9 s;用8%的甘油、16%的二甲亚砜、16%的1,2-丙二醇和12%的乙二醇激活日本鳗鲡精子后,精子存活时间均最长,分别达到154.6 s、45 s、20 s和34 s。与其它几种抗冻剂相比,8%的甘油能显著延长日本鳗鲡精子的寿命。四种抗冻剂对日本鳗鲡精子都有一定的毒性作用,处理后精子的活力和寿命均降低。     

含嗜水气单胞菌气溶素的免疫刺激复合物的制备及其对欧洲鳗的免疫效果

将嗜水气单胞菌气溶素(AerA)基因定向连接到pET-32a(+)表达载体,转化大肠杆菌BL21（DE3）,SDS-PAGE分析表明,硫氧还蛋白-气溶素融合蛋白（Trx-AerA）表达量占重组菌总蛋白量的65.5%。将上述融合蛋白与商品化的QuilA混合,分别添加Mega-10、卵磷脂、胆固醇获得Trx-AerA ISCO Ms。其中同时添加Mega-10、卵磷脂、胆固醇组蛋白质回收率最高为10.46%,仅添加QuilA组蛋白回收率为1.82%,两者差异显著（P＜0.05）。分别采用Trx-AerA、Trx-AerA ISCOMs、Trx ISCOMs经腹腔注射免疫欧洲鳗,免疫30d后,每个实验组取5尾实验鱼采集血清,按1：200稀释通过ELSIA法检测抗体水平,同时采用菌数为2.0×106CFU的嗜水气单胞菌ZN1株进行攻击,测定免疫效力。结果显示：Trx-AerA免疫组相对保护率为0%（3/15）,Trx-AerA ISCOMs免疫组为83.3%（13/15）,Trx ISCOMs免疫组为50%（9/15）; 3个免疫组针对气溶素的特异性抗体水平OD450值分别为0.19,0.52,0.36,Trx-AerA ISCOMs免疫组、Trx-AerA免疫组显著高于Trx ISCOMs免疫组（P＜0.05）。结果表明影响嗜水气单胞菌免疫效力的除了气溶素的抗体水平外,ISCOMs等也能够增强非特异性免疫保护应答。