Application of different types of protein hydrolysate in high plant protein diets for juvenile turbot (Scophthalmus maximus)

A 10‐week feeding experiment was conducted to investigate the effects of different types of dietary protein hydrolysate (PH) on growth performance, body composition, trypsin activity and serum transaminase of juvenile turbot. Four high plant protein diets contained different types of PH, fish PH (FPH), yeast PH (YPH), pig blood PH (PBPH) and soy PH (SPH), replacing 10% fishmeal of the basal diet. The basal diet with 30% fishmeal and no PH was used as the control diet (C). Each diet was assigned to triplicate groups of 30 fish. The specific growth rate (SGR) was not significantly different between groups C and FPH, but groups C and FPH showed significantly higher SGR than other groups. The feed efficiency ratio (FER) and protein efficiency ratio (PER) were not significantly different among groups C, FPH and YPH, but groups PBPH and SPH showed significantly lower FER and PER than groups C and FPH. Group PBPH showed significantly higher hepatosomatic index than other groups except YPH. Fish fed YPH showed significantly lower whole‐body protein content, but significantly higher whole‐body lipid content than fish fed diets C, FPH and PBPH. The activities of serum GOT and GPT in group PBPH were higher than those in groups C, FPH and YPH. These results suggested that when used at a low level in high plant protein diets for juvenile turbot, FPH is a good alternative protein source and YPH also has the application potential, but PBPH and SPH can cause negative impacts on fish growth and health.     

Effect of size‐fractionated fish protein hydrolysate on growth and feed utilization of turbot (Scophthalmus maximus L.)

Four experimental diets were fed to turbot to examine the effect of fish hydrolysate and ultra‐filtered fish hydrolysate on growth performance, feed utilization and non‐specific immune response. Fish hydrolysate was produced by enzymatic treatment and size fractionated using ultra‐filtration (UF). The permeate (molecular weight <1000 Da) after UF and the non‐ultra‐filtered fish hydrolysate (NUF) were tested as feed ingredients. Diets UF1, UF2 contained 3.7%, 1.2% ultra‐filtered fish hydrolysate to replace fish meal protein respectively. The diets UF1, NUF were identical in composition except that the molecular weight of fish hydrolysate in the diet. Fish meal was used in the control diet. All diets were made equal in protein, lipid and energy. Each experimental diet was fed to juvenile turbot (27.87 ± 0.04 g) in triplicate for 8 weeks. Results of this study indicate that the best overall growth and feed utilization of turbot juveniles were obtained with a diet containing higher dose of the small molecular weight compounds in fish hydrolysate. Acid phosphatase, alkaline phosphatase, lysozyme and superoxide dismutase activity in serum were not affected by diet. Total antioxidant capacity was improved with increasing level of low molecule weight fish hydrolysate (UF1).     

The plasticity of vision and body development of turbot Scophthalmus maximus larvae Under different light spectra

This study was aimed to find out the effect of five light spectra (white, LDW; blue, LDB; green, LDG; red, LDR and yellow, LDY) on the visual development and growth performance of turbot (Scophthalmus maximus) larvae. The number (per 100 μm) of cone cells, outer nuclear cells, ganglion cells, the ratio of outer nuclear to cone and ganglion cells, lens diameter and the minimum separable angle were determined. Variations in growth performance in total length, body mass and body colour were also examined. The results showed that the LDB group underwent metamorphosis faster than did the other groups and attained the greatest total length, body mass and light sensitivity. The LDB group also completed the body colour variation prior to the other groups. The LDG group exhibited a high mortality rate since 25 dph (days post‐hatching). We arrived at the conclusions that the spectra could affect the vision and body development of turbot larvae specifically and the blue light had the enhancing efficiency during this process, followed by the white and yellow light. The red light imposed an inhibiting effect on these variations. Our study highlighted the significance of light condition and provided the reference for the farming.     

Genetic parameters for resistance against Vibrio anguillarum in turbot Scophthalmus maximus

Genetic parameters for resistance to Vibrio anguillarum in Scophthalmus maximus were estimated using three different statistical models. Data were recorded from an experimental infection performed on 2,400 individuals from 30 full‐sib groups. Cross‐sectional linear model and cross‐sectional threshold probit model were used to analyse the test‐period survival; the cross‐sectional threshold logit models were used to analyse the test‐day survival. The heritability values estimated by cross‐sectional linear model (CSL), cross‐sectional threshold (probit) model (THRp) and cross‐sectional threshold (logit) model (THRl) were 0.202 ± 0.101, 0.296 ± 0.168 and 0.110 ± 0.023, respectively. The correlation coefficients between the full‐sib families’ estimated breeding values (EBVs) based on these three models were over 0.993. An almost identical ranking of families was generated using any of these models. Accuracy of selection using CSL, THRp and THRl models was 0.783, 0.789 and 0.801, respectively. Accuracy of selection based on the THRl model was higher than that based on CSL and THRp models.     

不同溶解氧条件下亚硝酸盐和非离子氨对大菱鲆的急性毒性效应

研究了过饱和氧条件下亚硝酸盐和氨氮对大菱鲆急性毒性效应,同时对比了在正常溶解氧条件下亚硝酸盐和氨氮对大菱鲆急性毒性效应。实验结果表明,过饱和氧条件下亚硝酸盐对大菱鲆的48hLC50值和96hLC50值(95%可信限)分别为467.60mg/L和390.78mg/L,非离子氨对大菱鲆的48hLC50值和96hLC50值(95%可信限)分别为2.40mg/L和1.73mg/L;而正常溶氧条件下,亚硝酸盐对大菱鲆的48hLC50值和96hLC50值(95%可信限)分别为181.07mg/L和130.66mg/L,非离子氨对大菱鲆的48hLC50值和96hLC50值(95%可信限)分别为1.82mg/L和1.14mg/L。亚硝酸盐和非离子氨对大菱鲆均具有一定的毒性,其主要中毒症状表现为,中毒的个体开始急躁不安并沿槽壁狂游,频繁发生相互碰撞或与槽壁摩擦,随后行动减缓并伴有侧游或侧翻动作,呼吸速度减慢,发生昏迷并沉落水底,直至死亡。死亡的大菱鲆鱼体弯曲,体色变淡,鳃盖张开。无论在过饱和氧还是在正常溶氧条件下非离子氨对大菱鲆的毒性都远大于亚硝酸盐的毒性,同时高浓度溶解氧的存在使大菱鲆对这两种毒物的耐受能力得以提高。提出了在大菱鲆循环水养殖过程中可以通过向水体充氧的方式以提高大菱鲆对亚硝酸盐和非离子氨的耐受力。     

Water quality and nitrogen budget in turbot Scophthalmus maximus culture system supplemented with lactic acid bacteria

To understand the nutrient utilization efficiency and distribution in indoor fish culture system under treatment of probiotics, this study examined water quality and nitrogen budget in turbot Scophthalmus maximus culture system supplemented with four strains of lactic acid bacteria (LAB) isolated from fishery products. The results showed that the concentration of ammonia nitrogen (NH?‐N) and nitrite nitrogen (NO?‐N) in LAB treatments were all significantly lower (p < 0.05) than in the control group. The nitrate nitrogen (NO?‐N) in LAB treatments showed no significant differences (p > 0.05) compared with control group. The fish feed and water exchange accounted for 79.07% and 17.02% of total N input respectively. For N output, the drainage and residual diets in LAB treatment accounted for 24.50%–25.80% and 1.33%–1.60% respectively, and they were significantly lower than in the control group (27.60% and 2.20% respectively). Fish growth and lost N in LAB treatments accounted for 27.10%–30.50% and 11.00%–18.50% respectively, and they were both significantly higher (p < 0.05) than in the control group (22.30% and 5.30% respectively). The results indicated that the indigenous LAB strains were capable of improving fish growth, and reducing NH?‐N and NO?‐N level (at concentration of 10⁵ cfu/ml) by directly adding in S. maximus culture water. Moreover, specific strains of LAB may increase nitrogen loss by promoting denitrification process in culture system.     

Evaluating the most suitable nonlinear growth model for turbot (Scophthalmus maximus) in aquaculture 2 (weight application): Multi‐criteria model selection and growth prediction

Seeking the most suitable model to describe the growth of turbot, we analysed growth data of two different turbot (Scophthalmus maximus) strains reared communally in a recirculating aquaculture system. We fitted 10 different nonlinear growth models to individual weight gain data (n = 2,010) during the grow‐out phase. Analyses were carried out for each strain, for sexes within strains and for a pooled data set containing both strains and sexes. To assess the model performance, three different criteria are used. Further, a growth‐simulation was performed to evaluate the shape of the generated curve. This way we could assess the capability of the models to predict future growth. The 3‐parametric Gompertz model achieved the best fit in 42.9% of all cases tested and the lowest Bayesian information criterion in 100% of cases. The model produced realistically shaped curves and asymptotic values matching the biological attributes of the species. In contrast, 5‐parametric functions projected unrealistically shaped curves and predicted improbable mature sizes. Our results show that increasing number of parameters do not lead to increasing goodness of fit, but tend to result in overfitting, and demonstrate the advantages of the 3‐parametric Gompertz model for describing the growth of turbot.     

Effects of silymarin on growth performance,antioxidant capacity and immune response in turbot (Scophthalmus maximus L.)

A 9‐week feeding trial was conducted using triplicate groups of turbot (6.50 ± 0.01 g) to explore the potential effects of silymarin. Three concentrations of silymarin (100, 200, and 400 mg/kg) were added to the plant protein‐based diet. Fish were randomly distributed into fiberglass tanks (30 fish per tank). The results showed that adding 100 mg/kg silymarin significantly improved the growth performance, with no effects on feed utilization. The antioxidant capacity in the liver was significantly improved in the 100 mg/kg and 200 mg/kg silymarin groups by not only inducing the activities of superoxide dismutase (SOD) and catalase but also increasing the messager RNA (mRNA) expression levels of SOD, glutathione peroxidase, and peroxiredoxin 6. Meanwhile, supplying 100 and 200 mg/kg of silymarin enhanced the heights of villi and enterocytes. Silymarin supplementation reduced the mRNA expression of interleukin‐8 and tumor necrosis factor‐α but induced the expression of transforming growth factor‐β (TGF‐β) in the intestine. These results indicated that silymarin was a potential nutraceutical that could enhance the growth performance and health status of turbot fed in a high plant protein diet. Adding 100 mg/kg silymarin to the plant protein diet achieved optimal performance in turbot.     

Study of the distribution of active caspase‐3‐positive cells in turbot,Scophthalmus maximus (L.), enteromyxosis

Enteromyxosis caused by Enteromyxum scophthalmi is one of the parasitizations with a higher economic impact on turbot, Scophthalmus maximus (L.), aquaculture. This myxosporean produces severe catarrhal enteritis with abundant inflammatory infiltrates in the lamina propria‐submucosa (LP), epithelial detachment and leucocyte depletion of the lymphohaematopoietic organs. Some advances made on the pathogenesis pointed to a role of apoptosis in the enteromyxosis. Therefore, the main aim of this work was to employ the TUNEL assay and the anti‐(active caspase‐3) immunohistochemical assay to detect apoptotic cells in both healthy and E. scophthalmi‐infected turbot in order to establish the presence and distribution of apoptotic cells during development of the disease. More apoptotic cells located within the gastrointestinal epithelium were observed in the initial stages of the infection in E. scophthalmi‐infected turbot compared with non‐infected turbot. As the infection progressed, a higher degree of apoptosis occurred in the epithelium of folds heavily parasitized. In the severely infected turbot, apoptosis was also found among the leucocytes of the intestinal inflammatory infiltrates. Moreover, the number of active caspase‐3‐positive cells in the lymphohaematopoietic organs tended to increase with disease severity. In view of the results, increased apoptosis in the epithelium may favour the scaling that occurs during enteromyxosis and cell death of leucocytes in the intestinal LP, contributing to leucocyte depletion in severe cases.     

Effect of 5′‐inosine monophosphate (IMP) and 5′‐guanosine monophosphate (GMP) on the growth,immunity and muscle composition of turbot,Scophthalmus maximus (Linnaeus, 1758)

We evaluated the effect of different concentrations of 5′‐inosine monophosphate (IMP) and 5′‐guanosine monophosphate (GMP) on the growth, immunity and muscle composition of turbot Scophthalmus maximus. Eight diets (containing no IMP or GMP, or 0.5 g/kg IMP, 1.0 g/kg IMP, 2.0 g/kg IMP, 0.5 g/kg GMP, 1.0 g/kg GMP, 2.0 g/kg GMP, or 0.5 g/kg IMP plus 0.5 g/kg GMP) were prepared. A total of 360 fish (average body weight of 105 g) were randomly selected and placed in groups into 24 plastic aquaria (8 treatments × 3 replicates × 15 individuals per plastic aquaria). The tanks were maintained at the temperature of 15 ± 2°C. The experimental diets were fed for 60 days. The specific growth rate (SGR) was significantly higher in S. maximus fed with IMP or GMP compared with fish fed neither IMP nor GMP. The highest SGR was observed in fish fed with 1.0 g/kg IMP. Supplementation with these dietary nucleotides had a positive, but not significant effect on the activity of superoxide dismutase, alkaline phosphatase and acid phosphatase. There was a significant difference in the moisture and crude lipid content of muscle from S. maximus fed the different diets compared with control fish. The highest moisture content was 83.44 for a diet of 0.5 g/kg IMP plus 0.5 g/kg GMP, which was also significantly higher when compared to fish fed alternative diets. The crude lipid content of S. maximus fed diets containing either IMP or GMP was significantly higher than those fed diets without IMP or GMP. Thus, according to these results, the optimal level of dietary IMP is 1.0 g/kg, which correlates with the largest increase in growth performance of S. maximus.     

A production season of turbot larvae Scophthalmus maximus (Linnaeus, 1758) reared on copepods in a Danish (56°N) semi‐intensive outdoor system

Turbot were reared from yolk sack larvae to juvenile in an outdoor semi‐intensive system. Three production cycles were monitored from May to September. A pelagic food chain was established with phytoplankton, copepods and turbot larvae. Abiotic and biotic parameters of lower trophic levels together with turbot larval survival, development, prey electivity and growth were monitored. A decreasing larval survival from 18.4% in May to 13.6% in July and just 7.0% in September was observed. The overall phytoplankton and copepod abundance decreased during the productive season. The turbot larval growth showed significant differences between larvae below (isometric) and above (allometric) 7 mm. Larval fish gut content showed no differences with available prey between production cycles. Therefore, it appears that the available prey concentration is governing their growth in this outdoor system. First‐feeding turbot larvae exhibited active selection for nauplii whereas developed larvae switched to copepodites and adult copepods. Although developing turbot larva exhibited active selection towards copepod size classes, there was no evidence of selective feeding on either of the two dominant copepod species. The turbot larvae's prey ingestion was modelled together with the standing stock of copepod biomass. The model results indicated that the estimated need for daily ingestion exceeded the standing stock of copepods. Hence, the initially established food web was unable to sustain the added turbot larvae with starvation as a consequence. We therefore suggest several solutions to circumvent starvation in the semi‐intensive system.     

The differences in postprandial free amino acid concentrations and the gene expression of PepT1 and amino acid transporters after fishmeal partial replacement by meat and bone meal in juvenile turbot (Scophthalmus maximus L.)

Meat and bone meal (MBM) is a high‐quality alternative protein source used to replace fishmeal (FM). However, the molecular mechanisms of over‐substituted FM by MBM resulted in growth reduction are still not clear. The objective of the study was to evaluate the effect of FM replacement by MBM on the concentration of postprandial free amino acid (FAA) and mRNA abundance of peptide and amino acid transporters in juvenile turbot (Scophthalmus maximus L.). Fish were fed with FM diet (60% FM), MBM diet (33% FM + 34.2% MBM) and MBM + AA diet (MBM diet with essential amino acid (EAA) added to match the AA profile of FM) for 30 days. Results showed that compared with the FM diet, MBM diet led to a reduction in FAA concentration peak values in plasma and muscle. MBM + AA diet significantly elevated the peak values of FAA concentrations to FM diet level in plasma, but not in muscle. Furthermore, compared with FM diet, MBM diet significantly increased gene expression of PepT1 and major amino acid transporters in intestine, whereas MBM diet greatly downregulated gene expression of T‐type amino acid transporter‐1, system ASC amino acid transporter‐2 and cationic amino acid transporter‐2 in muscle. Supplemented EAA did not ameliorate these different effects in intestine and muscle. Overall, this study provided a comprehensive explanation for the relationship between diet, FAA concentrations and AA transportations, which provides a molecular basis for further using MBM to replace FM in aquafeeds.     

Physiological and morphological effects of severe hypoxia,hypoxia and hyperoxia in juvenile turbot (Scophthalmus maximus L.)

We studied the physiological status of juvenile turbot (Scophthalmus maximus L.) under severe hypoxia (1 and 2 mg/L dissolved oxygen, DO), hypoxia (3 and 5 mg/L DO), hyperoxia (11 and 14 mg/L DO) and normoxia (7 mg/L DO, control) conditions. The respiratory rates, haematology parameters, acid–base balance status and gill structure were analysed to find the effects of different DO concentration on turbot. Fish mortality was only observed under severe hypoxia conditions. Severe hypoxia caused an increase in respiratory rates and red blood cell counts, as well as an increase in haemoglobin and haematocrit levels in the fish. In fish exposed to hypoxia conditions, the respiratory rate increased overall as the DO concentration decreased. Lower pCO₂ and HCO₃^? levels led to a high blood pH, while the pO₂ remained stable. In hyperoxia groups, respiratory rate decreased as the DO concentration increased. The levels of pCO₂ and HCO₃^? significantly increased (P < 0.05), while the pO₂ level and blood pH did not change obviously. The gill structure was damaged after prolonged exposure to hyperoxia, but no obvious damage was found in hypoxia groups. The fish that survived the hypoxia or hyperoxia treatment were able to restore the structural integrity of the gills after 14 days' recovery. The results suggest that juvenile turbots can tolerate a wide range of DO concentrations. However, even mild hyperoxia condition (11 mg/L DO), which is widely used in fish culture, has adverse effects on juvenile turbot physiology.     

Effects of feeding frequency on the enzymes and genes involved in oxidative stress in juvenile yellow catfish Pelteobagrus fulvidraco (Richardson) exposed to ammonia

Optimal feeding strategies improve fish growth and health but may be affected by ammonia stress in closed rearing systems such as tanks or ponds. This study aimed to evaluate the effects of feeding frequency and ammonia levels in rearing water on the enzymes and genes involved in oxidative stress of yellow catfish. Experiment (ammonia exposure) and control groups were randomly assigned to one of three feeding frequencies (1, 2 and 4 times daily) for 8 weeks. Weight gain increased as feeding frequency increased from 1 to 4 times daily, but feed conversion ratio values decreased. The highest survival in ammonia group was found when fish was fed 2 times daily. Glutathione peroxidase activity and total antioxidant capacity in liver and brain of fish exposed to ammonia increased as feeding frequency increased from 1 to 4 times daily. Liver malondialdehyde content in control group decreased as feeding frequency increased from 1 to 4 times daily. The lowest liver malondialdehyde content in ammonia group was observed when fish was fed 2 times daily. Liver mRNA expression of superoxide dismutase and catalase in control group increased as feeding frequency increased from 1 to 4 times daily, but the highest superoxide dismutase and catalase expression in ammonia group were observed when fish were fed 2 times daily. This study indicates that higher feeding frequency of yellow catfish exposed to ammonia could result in oxidative stress and poor survival. The optimal feeding frequency of yellow catfish exposed to ammonia is 2 times daily.     

Identification and characterization of pathogens associated with fin erubescence and ulceration of cultured Scophthalmus maximus

Scophthalmas maximus is a precious and main aquacultural species in northern China with an annual output value of more than 1.5 billion yuan. However, with the expansion of breeding range, increased stocking density and variety degradation, a variety of bacterial, parasitic and viral diseases have often occurred and worsened. Among them, bacterial disease is the most serious in farmed S. maximus. Since 2010, an epidemic bacterial disease with primary symptoms of lighter colour of lateral and caudal fins, spotty bleeding, fin base bleeding, abdominal swelling and redness and lesions of internal organs has put broken in S. maximus cultured in Weihai, Longkou and Haiyang, Shandong Province, China. The disease occurred mostly at the seedling and early development stages and propagated very fast, resulting in high mortality. These S. maximus died with obvious surface ulceration in only 3–4 days, causing great economic losses to the aquaculture industry. Beginning in 2010, we conducted epidemiological investigation in farmed S. maximus and isolated two different pathogens from the body of the diseased S. maximus from different farms. In this paper, we observed their morphology, conducted phylogenetic analysis based on their physiological and conventional biochemical characteristics and 16S rRNA gene sequences, preliminarily analysed their pathogenesis and investigated the histopathological changes of the diseased S. maximus. We believed that Vibrio anguillarum and V. parahaemolyticus were the pathogenic bacteria for the epidemic disease in S. maximus.     

Influence of 5′‐inosine monophosphate and 5′‐guanosine monophosphate on growth,feed digestibility and activity of digestive enzymes in turbot Scophthalmus maximus

We evaluated the influence of different proportions of 5′‐inosine monophosphate (IMP) and 5′‐guanosine monophosphate (GMP) on growth, feed digestibility and activity of digestive enzymes of turbot Scophthalmus maximus. Weight gain and daily feed intake were significantly higher in S. maximus fed with IMP or GMP, in comparison with fish fed with neither IMP nor GMP. The growth of 0.05% IMP + 0.05% GMP group was the best, and the intestinal digestive function was improved. The addition of IMP and GMP to fish diets significantly increased the apparent feed digestibility coefficient of dry matter and protein, as well as intestinal protease activity. The highest intestinal protease activity was observed in fish fed with 1 g/kg IMP. However, the lipase activity in hepatopancreas decreased significantly after addition of nucleic acid. According to our results, the optimal level of dietary IMP is 1 g/kg, which is in line with most of the growth performance and feed digestibility.     

Ocular bacterial signatures of exophthalmic disease in farmed turbot (Scophthalmus maximus)

As is the case at other sites in the body (e.g. the gut, skin and mouth), the ocular microbiota plays a crucial role in their host, as disturbances of the composition and function of the ocular microbiota are known to be associated with ocular disorders. Exophthalmic disease (ED) is a significant cause of high mortality in fish species, including farmed turbot (Scophthalmus maximus). However, the relationship between alterations in the ocular microbiota and ED in turbot is unclear. In this work, we collected turbot samples from farmed ponds with ED and healthy samples to understand changes in the ocular microbiota of turbot suffering from ED. We compared the structural and metabolic differences of ocular bacterial communities from farmed turbot with exophthalmic disease and those of healthy controls. Besides less microbial diversity found in turbot with ED regarding the control group, we also found that Aeromonas was the dominant bacteria both in controls and ED samples, but the abundance of Aeromonas was significantly greater in ED individuals. Moreover, the results of correlation test further suggest that Aeromonas overgrowth was correlated with the progress of the disease and shifts in ocular microbiota functional pathways in turbot. These findings emphasize that an increased abundance of Aeromonas serves as an ocular bacterial signature associated with ED in turbot, which provide basic information useful for diagnoses, prevention and treatment of ocular diseases occurring in cultured fish.     

不同加工工艺的饲料对大菱鲆幼鱼生长及养殖水环境的影响

比较了两种加工工艺对饲料(D1、D2)颗粒物理性状的影响,并用其投喂初始体重为16 g的大菱鲆幼鱼64 d,比较其对大菱鲆幼鱼生长、饲料利用和养殖水环境的影响。结果显示,D2组饲料平均颗粒直径、平均百粒重和水中稳定性显著高于D1组(P0.05),但其吸水性、堆积密度和沉降速度显著低于D1组(P0.05)。D2组大菱鲆幼鱼的增重率、特异生长率、摄食率及蛋白质效率显著高于D1组(P0.05)。大菱鲆对D2组饲料中干物质和粗蛋白的表观消化率显著高于D1组(P0.05),但对粗脂肪和总磷的表观消化率无显著影响(P0.05)。投喂18 h后,养殖水体中N、P含量均有了显著升高,D2组每升水中每千克鱼产生的亚硝酸氮含量显著高于D1组(P0.05),但硝酸氮和总氮增加量显著低于D1组(P0.05);D2组活性磷酸盐及总磷酸盐增加量显著低于D1组(P0.05)。研究结果表明,不同的加工工艺显著影响了颗粒饲料的物理性状和饲料利用,并对养殖水环境造成了影响。     

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).     

Construction and expression of DNA vaccine against reddish body iridovirus and evaluation of immune efficacy in turbot (Scophthalmus maximus)

Turbot aquaculture is a very important industry in China. However, it is hampered because of viral reddish body syndrome (VRBS) and high mortality caused by piscine turbot reddish body iridovirus (TRBIV). TRBIV virus is an icosahedron‐like and cytoplasmic DNA virus, belonging to Iridoviridae, Megalocytivirus. In previous studies, we have identified two antigen mimotopes using bioinformatics and constructed prokaryotic expression vectors. In this study, a fragment of major capsid protein (MCP) gene with the two antigenic epitopes was cloned into eukaryotic expression vector pVAX1, to generate a recombinant plasmid pVAX1‐TRBIV‐MCP. The plasmid DNA was transferred into turbot cell line TK using liposome, and transient expression was detected using RT‐PCR. After injection into turbot (Scophthalmus maximus), the expression of the antigen gene was analysed using RT‐PCR and was shown to express in all tested tissues in vaccinated fish 2 and 7 days post‐vaccination. The cumulative mortalities in the vaccinated and unvaccinated control fish were 30% and 88% respectively. Immune responses and upregulation of the expression of chemokine receptor, tumour necrosis factor, interferon and interferon‐induced antiviral molecules were observed in the vaccinated fish 60 h post‐vaccination. These results demonstrate that the vaccinated turbots had higher survival rate and produced specific serum antibodies following the TRBIV challenge. More studies are needed to develop and apply the promising DNA vaccine for virus control in turbot.