Experimental infection of turbot, Scophthalmus maximus (L.), by Aeromonas salmonicida subsp. achromogenes and evaluation of cross protection induced by a furunculosis vaccine

Turbot was shown to be sensitive to injection challenges by Aeromonas salmonicida subsp. achromogenes (Asa). A systemic disease was induced and the bacterium was isolated from various internal organs. Histopathological changes involved haemorrhages, necrosis and degeneration in skin and muscle, haemorrhages and necrosis in kidney, degeneration in the heart muscle, and fusion of the secondary gill lamellae. A polyvalent commercial salmon vaccine, containing A. salmonicida subsp. salmonicida as one of five antigens, did not confer protection in turbot against an experimental Asa infection 13 weeks post-vaccination. Vaccination induced a significant antibody response against Asa cells but not against extracellular products of the bacterium. The results of the study indicate that Asa may be a potential threat to turbot farming and that the development of new turbot vaccines is needed.     

A comparative study of susceptibility and induced pathology of cod, Gadus morhua (L.), and halibut, Hippoglossus hippoglossus (L.), following experimental infection with Moritella viscosa

In this study experimental challenges with Moritella viscosa, the causative agent of winter ulcers in salmonids, were performed on juvenile Atlantic cod and Atlantic halibut. The challenges involved both intramuscular (i.m.) and intraperitoneal (i.p.) injections and bath with a strain originally isolated from Atlantic salmon. Cod was found to be significantly more sensitive than halibut to the infection. Both fish species were found to be more sensitive to i.m. than i.p. challenges. Both challenges induced a systemic disease in cod and halibut, but only cod was infected with an experimental bath challenge. Pathognomonic signs were found to be comparable with those described in M. viscosa-infected salmon and turbot. The main distinguishing pathological sign was that the cod showed host response to M. viscosa infection resulting in granuloma formation in infected tissues, which is a known response of cod to a infection with another Gram-negative bacterium, Aeromonas salmonicida. Re-isolation of M. viscosa from kidneys of cod and halibut with clear disease signs was problematic and optimization of isolation measures is needed. The results of this study indicate that M. viscosa infection may be a risk factor in cod farming, but that halibut is more resistant.     

Pathology of Edwardsiella tarda infection in turbot, Scophthalmus maximus (L.)

Macroscopic and histopathological changes in cultured turbot, Scophthalmus maximus (L.), in Spain caused by infection with Edwardsiella tarda are described. Eye tumefaction, inflammation, haemorrhages, ascites and the presence of a purulent fluid were the main macroscopic lesions observed. Histopathological lesions were found in the kidney, spleen and liver. In the kidney and spleen these were characterized by a severe apostematous inflammatory reaction, with a large number of abscesses. The liver was affected to a lesser degree and only some phagocytes loaded with bacteria were observed. Ultrastructural observations indicated that macrophages were the main cell type implicated in the inflammatory response. Most of the bacteria observed within the phagocyte cytoplasm showed no degenerative changes and some were dividing. Degenerative changes observed in macrophages indicate their failure in preventing the infection.     

Dietary protein requirement for young turbot (Scophthalmus maximus L.)

This study was conducted to determine the optimum dietary protein level for young (an initial weight of 89 g) turbot, Scophthalmus maximus L. Duplicate groups of the fish were fed the five isoenergetic diets containing the various protein levels ranging from 290 to 570 g kg^?1 diet for 45 days. Survival was not affected by dietary protein level. Weight gain and feed efficiency were improved with dietary protein level up to 490 g kg^?1 diet. Dietary protein requirement of young turbot using the broken‐line model was estimated to be 494 g kg^?1 diet based on weight gain response. Protein efficiency ratio was not influenced by dietary protein level. The highest protein retention was obtained from the fish fed the 490 g protein kg^?1 diet. Proximate composition of the fish was not significantly affected by dietary protein level. In considering these results, it was concluded that the 494 g protein kg^?1 diet with 100 g lipid kg^?1 diet (15 MJ kg^?1 diet) provided optimal growth of young turbot under these experimental conditions.     

大菱鲆胚胎玻璃化方法研究

用大菱鲆(Scophthalmus maximus)神经期胚对6种抗冻剂的毒性进行检测,发现其毒性排列为1,2-丙二醇(PG)<甲醇(MeOH)<甘油(Gly)<二甲基甲酰胺(DMF)<乙二醇(EG)<二甲亚砜(DMSO).以DMF为主因子配制和筛选出11种玻璃化程度较好的玻璃化液,并用大菱鲆肌节期胚对其中5种玻璃化程度最好的玻璃化液进行检测,结果显示,A4(DMSO 20%+DMF 25%)、A7(DMSO 25%+DMF 20%)较适合于大菱鲆胚胎的平衡处理,利用A4和A7平衡处理神经期胚、肌节期胚、心跳期胚、出膜前期胚,结果显示大菱鲆神经胚和肌节胚对玻璃化液的适应能力较强.实验测试出不同时期胚胎在两种玻璃化液中的成活率,为大菱鲆各期胚胎在玻璃化液中的平衡处理提供了依据.利用大菱鲆肌节胚对不同的平衡步骤进行了筛选,发现五步法的平衡效果较好.     

Cryopreservation of turbot Scophthalmus maximus (L.) sperm: fertilization and hatching rates

The present paper assesses the fertilization and hatching rates of an artificial fertilization series (n=1153) using fresh and cryopreserved sperm from 49 specimens of turbot Scophthalmus maximus (L.), carried out to confirm the results of a previous study, with the ultimate aim of transferring these cryo‐preservation techniques to commercial hatcheries. No significant differences were found between the fertilization rates of the two groups (fresh and cryopreserved sperm) when their respective fertility rates were >69.2%, which was the case in 75% of all fertilizations. Likewise, no significant differences in hatching rates were found. In order to use the cryopreserved sperm more efficiently, a key concern for commercial use of this technique, we also experimented with a lower sperm:diluent ratio (1:1) than used previously at our centre. We also compared the traditional 0.5‐mL straws with 2‐mL cryotubes able to contain a higher volume of sperm, finding no significant differences in the resulting fertilization and hatching rates. In conclusion, the use of cryopreservation for turbot sperm presents major advantages for broodstock management in commercial hatcheries.     

Isolation of a highly pathogenic Vibrio pelagius strain associated with mass mortalities of turbot,Scophthalmus maximus (L.), larvae

A bacterial strain, characterized as Vibrio pelagius (Hq 222), was isolated from a turbot, Scophthalmus maximus (L.), larvae mass mortality in a commercial fish farm in Spain. Turbot larvae, post-larvae (0.2 g) and juveniles (5 and 15 g) were experimentally infected. The bacterium appeared to be very virulent for larvae and post-larvae, LD50 being < 5 bacteria mL(-1) for larvae 1 week post-infection and 3.9 x 10(5) bacteria mL(-1) in post-larvae at day 12 post-infection. The bacterial strain was recovered in pure culture from the internal organs of infected fish. Histological lesions in post-larvae exhibited swelling and necrosis of gill secondary lamellae, sloughing of intestinal mucosa and necrosis of haematopoietic tissue in the kidney. Vibrio pelagius (Hq 222) was able to grow in sterile sea water when incubated at room temperature or at 15 degrees C. Vibrio pelagius (Hq 222) was more adherent to the turbot cell lines TV-1 and TF than Escherichia coli. In both cell lines, the number of adhered bacteria increased with incubation time.     

Characterization of nodavirus and viral encephalopathy and retinopathy in farmed turbot, Scophthalmus maximus (L.)

An outbreak of nodavirus infection in turbot larvae is described with respect to histopathology, immunohistochemistry, cell culture cultivation, RT-PCR amplification and sequence analysis of the capsid protein gene RNA2. Affected turbot developed classical signs of viral encephalopathy and retinopathy (VER) with abnormal swimming behaviour and high mortality levels. In the acute stage of infection, light microscopy revealed vacuolation of the central nervous system (CNS), with positive immunohistochemical staining for nodavirus. Later in the infection, CNS lesions appeared more chronic and contained clusters of cells immunopositive for nodavirus. Bacterial overgrowth in the intestines of the fish may have provoked or influenced the course of the nodavirus infection. We were unable to propagate the virus in cell culture. While RT-PCR using primers designed to detect Atlantic halibut nodavirus gave negative results, further testing with primers complementary to a more conserved region of RNA2 resulted in amplification of a product of the expected size. The entire RNA2 segment was cloned and sequenced. Sequence alignment showed that the turbot nodavirus (TNV) was different from previously described fish nodaviruses. In addition, phylogenetic analysis based on an 823 nt region of the sequence indicated that TNV clustered outside the four established fish nodavirus genotypes, suggesting a fifth genotype within the betanodaviruses.     

Statistical properties and performance of pairwise relatedness estimators using turbot (Scophthalmus maximus L.) family data

The statistical properties and performance of four estimators of pairwise relatedness were evaluated in several scenarios using the microsatellite genotype data from a set of large known full‐sibships of turbot. All estimators showed a significant negative bias for the four kinships commonly used in these studies (unrelated: UR, half‐sibs, full‐sibs and parent–offspring), when allele frequencies of the reference population were estimated from the individuals analysed. When these frequencies were obtained from the base population from which all families proceeded, the bias was mostly corrected. The Wang (W) and Li (L) estimators were the least sensitive to this factor, while the Lynch and Ritland (L&R estimator) was the highest one. The error (mean around 0.130) was very similar in all scenarios for W, L and Queller and Goodnight (QG) estimators, while L&R was the highest error‐prone estimator. Parent–offspring kinship resulted in the lowest error, when using W, L and QG estimators, while UR resulted in the lowest error with the L&R estimator. Globally, W was the best‐performing estimator, although L&R could perform better in specific sampling scenarios. In summary, pairwise estimators represent useful tools for kinship classification in aquaculture broodstock management by applying appropriate thresholds depending on the goals of the analysis.     

Edwardsiellosis in farmed turbot,Scophthalmus maximus (L.), associated with an unusual variant of Edwardsiella tarda: a clinical,aetiological and histopathological study

During 2005 and 2010, a survey of edwardsiellosis on eight turbot, Scophthalmus maximus (L.), farms was conducted in China. This report presents the detailed results of the study on this disease. Diseased turbot displayed two distinct types of gross signs: black discoloration of the dorsal skin on the posterior portion of the body; and red cutaneous foci on the ventral side. Internally, the most pronounced clinical signs in all fish examined were enlarged kidneys. The causal agent of the disease was finally proved to be one species of bacterium that was identified as Edwardsiella tarda by physiological and biochemical tests, API 32E and 16S ribosomal RNA sequence analysis. It is noteworthy that unlike the commonly described E. tarda strains, the isolates in this study were non‐motile strains without flagella. A histopathological study revealed that E. tarda infection was systemic in turbot and that kidney showed the most significant pathological changes, including acute focal necrosis, an influx of macrophages and formation of granuloma. The most common histopathological characteristics of this disease are the proliferation of macrophage in various organs and formation of granuloma. In addition, this article also gave background information on the disease and presented the results of virulence tests with the E. tarda strain identified in this study.     

大菱鲆种质资源研究与开发

综合介绍了大菱鲆种质资源的分类、自然分布和生物学特性。从生化和分子生物学方面阐述了大菱鲆自然群体遗传结构及国内外研究现状。详述了大菱鲆野生资源的利用和养殖开发现状．特别是我国对引进的大菱鲆种质资源的养殖开发。指明了我国目前在引种及养殖方面存在的不足．提出了建立全国性大菱鲆良种引育中心,以保证我国大菱鲆养殖业的持续、稳定、健康发展的建议。     

Non-specific immune response of turbot,Scophthalmus maximus (L.), experimentally infected with a pathogenic Vibrio pelagius

The effect of a pathogenic Vibrio pelagius, isolated during a mass mortality of turbot larvae, on the non-specific immune response of turbot, Scophthalmus maximus (L.), macrophages was studied both in vitro and in vivo. The in vitro treatment of head kidney (HK) macrophages with viable V. pelagius caused a significant inhibition of the chemiluminescence (CL) response in comparison with untreated macrophages, while incubation with heat-killed bacteria did not affect this response. In vivo, the intraperitoneal injection of V. pelagius resulted in a significant inhibition of the CL response in infected fish at days 1 and 4 post-infection compared with the control fish response. The HK macrophage nitric oxide (NO) production was enhanced by in vitro incubation with intermediate doses of viable V. pelagius (5 x 10(3) and 5 x 10(4) bacteria mL(-1)) and higher doses of the heat-killed bacteria (5 x 10(4)-5 x 10(6) bacteria mL(-1)). In both cases, the NO inhibitor N-omega -nitro-L-arginine was capable of down-regulating the specific NO induction caused by incubation with the bacterial treatments. In contrast, incubation with ECPs at higher doses caused a reduction in NO production. In vivo, a significant enhancement in NO production was also observed in macrophage supernatants at day 10 post-infection. Lysozyme concentration in the serum was also significantly increased in the experimentally infected fish at days 4 and 10 post-injection. In addition, viable V. pelagius and its ECPs significantly reduced HK macrophage viability in vitro, whereas no significant differences in viability were observed during the incubation with heat-killed bacteria. As NO production was enhanced in the experimentally infected fish, the inhibitory effect of the NO donor, S-nitroso-acetyl-penicillamine (SNAP), was tested in vitro in a cell-free assay. The results showed that growth of V. pelagius was significantly inhibited using SNAP at a high concentration (1 mM).     

应用时间序列分析模型对大菱鲆选育F1优良家系发育的动态研究

根据Box-Jenkins建模原理,采用ARIMAR(p,d,q)模型,以大菱鲆选育F1优良家系为研究对象,应用时间序列分析方法对在3～27月龄间不同发育阶段体重的生长速度进行动态模拟,建立各家系的预测模型。结果表明,家系E♂1×E♀2和F♂2×E♀4符合ARIMAR(2,0,0)模型(2个家系的模型都不含常数项),家系F♂4×N♀3、E♂2×F♀1和F♂1×F♀4符合ARIMAR(1,0,0)模型(3个家系的模型都不含常数项),且所建模型的残差均为白噪声。由此预测出27～27.5及27.5～28月龄各家系体重生长速度,经与相应实测数据的验证说明,5个家系各自所建模型在一定程度上能够反映大菱鲆体重生长速度的动态变化过程,对各家系体重生长速度的趋势预测有一定的适用性。通过对每一家系后期生长速度的预测,结合前期生长速度的实测值,综合分析体重生长速度的动态变化,为在大菱鲆选择育种过程中确定最佳选择时间提供理论依据。     

Stress responses of larval turbot, Scophthalmus maximus L., exposed to sub-lethal concentrations of petroleum hydrocarbons

Turbot larvae were exposed to dilutions of the water soluble fraction (WSF) of crude oil (1–25%), in a laboratory flow-through system. Whole body immunoreactive cortisol (IRC) content of premetamorphic 345 degree C day and early metamorphosing 450 degree C day larvae (23 and 30 days post hatch, respectively) was elevated when exposed to 25% WSF for 6 h, but the lower WSF concentrations did not induce IRC elevations. Larvae of 450 degree C days exposed to WSF for 6 h and then left in clean sea water for 24 h showed recovery of IRC, whilst the IRC content of 450 degree C day larvae exposed to WSF for 30 h remained elevated. Whole body thyroxine content of 345 degree C day larvae exposed to 25% WSF for 6 h was significantly elevated, which may have implications for larval development/metamorphosis. However, whole body triiodothyronine content of these larvae was not altered by WSF-exposure. Furthermore, larvae of 450 degree C days, WSF-exposed for 6 h or 30 h showed no thyroidal disturbance. Larvae of 345 degree C days exhibited significantly elevated whole body adrenaline and noradrenaline content after exposure for 6 h to 25% WSF; however, more diluted WSF did not influence adrenaline or noradrenaline content. These studies have demonstrated that larvae of 345 and 450 degree C days can successfully mount endocrine stress responses but that environmentally realistic concentrations of petroleum hydrocarbons do not induce these responses.     

Effects of dietary water content on meal size, daily food intake, digestion and growth in turbot, Scophthalmus maximus (L.)

When fed once daily with wet squid, turbot (30–50 g) accustomed to dry pellets require many days to increase intake to meet their feed requirement (≈ 10 mg dry matter g⁻¹ bw meal⁻¹). Adaptation takes 1–2 days if several daily feedings are given. With dried squid, they ingest about 20% of the wet squid bulk because the stomach contents expand when moisturised. In contrast, turbot eat enough wet squid to fill most of the available stomach volume (≈ 7.6 mL 100 g⁻¹ bw). When presented in gelatine capsules, food water content is masked and does not affect the volume ingested. Moistening the contents shortens the delay before gastric emptying starts to one-third (0.6 h) compared with dry food (1.9 h). Daily dry-matter intake increased when dry contents were moistened but only if two or more meals were offered per day. Turbot adapt their digestion to supply water for dry diets but this may add extra metabolic costs. When offered 20 mg dry matter g bw⁻¹ day⁻¹, divided into four equal meals, turbot grew faster and more efficiently with moist than with dry squid. Protein, energy and dry-matter digestibilities were also enhanced. The increased daily protein absorption did not increase ammonia release, indicating that the extra protein was used for somatic growth.     

Alleviation effect of dietary cerium and its complex with chitosan oligosaccharide on cadmium accumulation in juvenile turbot,Scophthalmus maximus L., under cadmium stress

An 8 weeks feeding trial was conducted to investigate the effect of dietary cerium (Ce) and its complex with chitosan oligosaccharide (COS‐Ce) on growth performance and cadmium (Cd) accumulation of turbot, Scophthalmus maximus L. under Cd stress. The basal diet (Diet 0) was formulated without Cd and cerium as the control. Seven other experimental diets (Diets 1–7) were formulated with supplementation of 50 mg Cd²⁺/kg feed, 50 mg Cd²⁺/kg and 50 mg Ce³⁺/kg feed, 50 mg Cd²⁺/kg and 100 mg Ce³⁺/kg feed, 50 mg Cd²⁺/kg and 200 mg Ce³⁺/kg feed, 50 mg Cd²⁺/kg and 50 mg COS‐Ce/kg feed, 50 mg Cd²⁺/kg and 100 mg COS‐Ce/kg feed, and 50 mg Cd²⁺/kg +200 mg COS‐Ce/kg feed. Results of the present study showed that, compared with the control group, the condition factor in fish fed the diet with 50 mg Cd²⁺/kg feed (Diet 1) was significantly lower, whereas the Cd concentration in liver and kidney of fish fed the diet with 50 mg/kg Cd²⁺ (Diet 1) was significantly higher (P < 0.05). The high Cd accumulation of fish fed diets with sole 50 mg/kg Cd (Diet 1) could be significantly decreased by 51.72% after supplementation of 200 mg COS‐Ce/kg in the diet (Diet 7). These results suggested that elevated COS‐Ce could effectively protect against dietary Cd accumulation in turbot.     

一种吻蛭类大鱼蛭在大菱鲆鱼体上的感染

在山东省威海崮山育苗场发现大菱鲆成鱼及幼鱼鱼体感染寄生虫，受感染的鱼由于处于感染初期，并经过及时的处理，所占比例较低。病鱼症状为鱼体体色变黑、瘦弱、离群、不摄食甚至死亡。寄生虫寄生于大菱鲆的体表及鳃部，虫体黑褐色，全长40～50mm。经鉴定，该寄生虫属于环节动物门(Annelida)，蛭纲(Hirudinea)，吻蛭目(Rhynochobdellida Blanchard)，鱼蛭科(Piscicolidae Johnson)，鱼蛭属(Piscicola Blainville)，大鱼蛭(Pisciola magna Yang)。     

Effects of dietary chitosan oligosaccharide complex with rare earth on growth performance and innate immune response of turbot,Scophthalmus maximus L.

An 8‐week‐feeding trial was conducted to investigate the effect of dietary chitosan oligosaccharide complex with rare earth (COS‐REE) on growth performance and innate immune response of turbot, Scophthalmus maximus L. (Initial average weight was (12.1 ± 0.1) g) as well as disease resistance against Edwardsiella tarda. Six practical diets (approximately 53.01% protein and 12.57% lipid) were formulated to contain graded levels (0, 75, 150, 300, 600 and 1200 mg kg^?1) of COS‐REE. Results of the present study showed that, compared to the control group (0 mg kg^?1), the specific growth rate (SGR) was significantly higher in fish fed the diet with 300 mg kg^?1 COS‐REE (P < 0.05), while the feed conversion ratio (FCR) significantly decreased (P < 0.05). The phagocytic index (PI) and the activity of super oxide dismutase (SOD) of serum in fish fed the diet with 300 mg kg^?1 COS‐REE was significantly higher than fish fed the control diet (P < 0.05), but no significant differences were observed in malondialdehyde (MDA) and hepatic metallothionein (MT) concentrations. After 8 weeks, fish were challenged by intraperitoneal injection with E. tarda, and COS‐REE‐treated fish demonstrated increased protection capability. These results suggested that COS‐REE could enhance growth, innate immunity and disease resistance in turbot, and the optimum dose was approximately 300 mg kg^?1.