Immune responses and gut morphology of Senegalese sole (Solea senegalensis,Kaup 1858) fed monospecies and multispecies probiotics

The current study aimed to determine the effects of dietary probiotic supplementation on growth, gut morphology and non‐specific immune parameters in Senegalese sole (Solea senegalensis) juveniles during a 1‐month trial. Fish were fed for 1‐month two diets with 1.0 or 4.6 × 10⁶ CFU kg^?1) of probiotic A (Bacillus sp., Pediococcus sp., Enterococcus sp. and Lactobacillus sp.) and two diets with 3.5 or 8.6 × 10⁵ CFU kg^?1 of probiotic B (Pediococcus acidilactici) and tested against an unsupplemented diet (control). Growth performance, as well as respiratory burst activity, nitric oxide (NO), alternative complement pathway (ACH50), lysozyme and peroxidase activities, was not affected by the dietary treatments. Probiotic supplementation tended to increased growth homogeneity between tanks having diet A₁ the best possible alternative to decrease costs associated to size grading. Villous length and number of goblet cells of the anterior intestine did not vary among treatments. Muscle duodenal layer was significantly thicker in fish fed probiotic A compared to probiotic B, when included at the lowest level (A₂ versus B₂). The current study indicate that the use of the multispecies probiotic at 1.0 × 10⁶ CFU kg^?1 might enhance protection against pathogen outbreak and increase nutrient absorption, whereas at the highest concentration could reduced size dispersion among tanks.     

Comparing skeletal development of wild and hatchery-reared Senegalese sole (Solea senegalensis, Kaup 1858): evaluation in larval and postlarval stages

The Senegalese sole is a marine pleuronectiform that naturally occurs in Southern Europe and Mediterranean region where it is being produced in aquaculture, in particular in Portugal and Spain. The aim of this study was to assess the quality of hatchery-reared larvae in comparison with those reared in the wild, and determine to which extension wild growing larvae are also affected by skeletal deformities. The main structures affected included those forming the axial skeleton, the caudal fin complex and both anal and dorsal fins, with the most prevalent anomalies affecting caudal vertebrae and arches. Hatchery-reared fish presented a higher incidence of deformities (79%) compared with the 19% observed in wild specimens. In wild postlarvae collected in Autumn no deformities were observed. This work clearly shows that wild Senegalese sole present less skeletal deformities than those hatchery-reared during larval stages, indicating a selective mortality of wild deformed fish and/or an effect of aquaculture-related rearing conditions in the development of skeletal deformities in sole.     

High stocking density induces crowding stress and affects amino acid metabolism in Senegalese sole Solea senegalensis (Kaup 1858) juveniles

Fish held at high stocking densities are generally exposed to chronic stress situations that impose severe energy demands and may predispose the fish to infection. Senegalese sole Solea senegalensis (Kaup) juveniles (78.8±18.9 g body weight) were maintained at low (LSD; 4 kg m^?2 at the end of the experiment), medium (MSD; 9 kg m^?2) and high (HSD; 14 kg m^?2) stocking densities during a period of 63 days. Although disease outbreaks were observed in fish reared at HSD, growth and food consumption did not vary among different treatments. Results from plasma cortisol and free amino acids (FAAs) showed significant differences among different rearing densities pointing to HSD as stressful rearing condition. However, higher plasma glucose and osmolality levels indicated that fish held at MSD may also be under stress. The higher usage of FAAs from the HSD group may be due to the higher demand for energy production in order to cope with stressful rearing conditions, higher rate of protein synthesis or due to synthesis of other important metabolites related to stress response. Therefore, results from the present study point out to HSD as a stressful rearing condition and suggest that crowding stress may affect amino acid requirements.     

New insight on vertebral anomalies in cultured Senegalese sole (Solea senegalensis,Kaup) at early stages of development

Senegalese sole (Solea senegalensis, Kaup) is a promising flatfish species in aquaculture. However, skeletal anomalies are still a great concern in sole farming. Investigation of this issue is crucial to improving larval quality and optimizing production. The aim of this study was to thoroughly assess anomalies in the rachis of reared sole at early developmental stages. Sole (n = 507) were sampled at 31 or 32 days after hatching (dah). The specimens were stained with alcian blue and alizarin red and evaluated for the detection of vertebral deformities. Most fish presented 9:34:3 vertebrae in abdominal, caudal and caudal complex regions, respectively. Remarkably, all specimens showed at least one spinal anomaly. Alterations of neural/haemal elements, as well as deformities of hypurals, parhypural and epural, were recurrent. Vertebral body anomalies and/or vertebral column deviations were identified in 52% of the individuals. Vertebral deformations and fusions were common, especially in caudal complex. ‘Minor’ anomalies were predominant, and some of the detected disorders might be a result of non‐/low‐pathological processes. These results contribute a new insight into the main skeletal anomalies affecting cultured sole larvae. Further research is required to determine their impact on fish welfare and external appearances at commercial stages.     

Optimal dietary protein levels in juvenile Senegalese sole (Solea senegalensis)

A study was undertaken to determine the dietary protein level for optimal growth performance and body composition of juvenile Senegalese sole. Five experimental extruded diets were formulated to contain increasing levels of protein [430, 480, 530, 570 and 600 g kg^?1 dry matter (DM)] and a constant lipid level, ranging from 100 to 130 g kg^?1 DM. Triplicate groups of 35 sole (initial body weight: 11.9 ± 0.5 g) were grown over 84 days in 60‐L tanks supplied with recirculated seawater. Fish were fed by means of automatic feeders in eight meals per day. At the start and end of the trial, whole‐body samples were withdrawn for proximate composition analysis. At the end of 84 days of experimental feeding, daily weight gain and specific growth rate in fish fed diets P43 and P48 were significantly lower than those found in fish fed higher protein level diets (P53, P57 and P59). Similarly, feed efficiency was also significantly lower in fish fed diet P43 than in fish fed all other dietary treatments. Sole juveniles fed lower protein level diets (P43 and P48) showed a significantly lower protein content than fish fed the higher dietary protein level treatments (P53, P57 and P60). Changes within the tested dietary protein levels did not affect significantly protein productive value or total nitrogen (N) losses in fish. However, daily N gain was significantly higher (P < 0.05) in fish fed diets P53 and P60 than in fish fed the lowest protein level diet (P43). Data from the present study indicate that diets for juvenile Senegalese sole should include at least 53% crude protein to maintain a good overall growth performance. Based on a second‐order polynomial regression model, the daily crude protein requirement for maximum whole‐body N gain as estimated here for Senegalese sole juveniles was 6.43 g kg^?1 body weight day^?1 which corresponds to a value of 1.03 g N intake kg^?1 body weight day^?1. If the present data are expressed on a dietary crude protein concentration basis, the allowance for maximum protein accretion (N gain) would be met by a diet containing a crude protein level of 600 g kg^?1.     

Stocking density affects Senegalese sole (Solea senegalensis,Kaup) growth independently of size dispersion,evaluated using an individual photo‐identification technique

To assess how initial size dispersion affects Senegalese sole (Solea senegalensis, Kaup) growth, 128 fish were stocked either under homogeneous (HOM) or heterogeneous (HET) initial size dispersion (standard length, CV 11.8% and 29.7% respectively), and also under low (LD) and high (HD) stocking density (60% and 180% of bottom occupation). After 105 days, growth parameters, evaluated at an individual level using linear mixed‐effects models, were not significantly affected by initial size dispersion, LD fish growing significantly faster than HD fish. Variance of individual growth rate distributions of weight and standard length were lowest for LD‐HOM fish, indicating the most regular growth, and highest for LD‐HET. The diminution of the coefficient of variation along time in HET groups (between 13% and 15.4% for weight, and between 4.3% and 5.8% for standard length), and its slight decrease in the HOM groups (between 1% and 3% for both parameters), is consistent with the absence of strong competition between individuals.     

Immunohistochemical diagnosis of tenacibaculosis in paraffin‐embedded tissues of Senegalese sole Solea senegalensis Kaup, 1858

A sensitive and specific immunohistochemical technique was developed to improve the diagnosis of tenacibaculosis and to better understand its pathogenesis. Senegalese sole Solea senegalensis Kaup, 1858 were inoculated subcutaneously with a bacterial suspension of Tenacibaculum maritimum, and samples were taken at different hours post‐inoculation. Sections from different organs were used as positive controls. In addition, a total of 128 field samples from different organs collected from tenacibaculosis outbreaks were used. Tenacibaculum maritimum antigens were detected in several organs of experimentally infected Senegalese sole and in at least one of the tissues from fish suffering from natural tenacibaculosis previously confirmed by culture and PCR‐based methods. In fish collected during outbreaks, a strong positive reaction was detected in ulcerative skin areas. Moreover, bacterial antigen was identified inside scale pockets and in sites of the skin with mild lesion. In kidney and spleen, evident immunostaining of bacterial antigen was detected in both naturally and experimentally infected fish. Besides, the presence of T. maritimum in the intestinal tract without associated histological changes suggests that this organ may act as a reservoir for T. maritimum. The results of this study confirm the usefulness of IHC for the diagnosis of tenacibaculosis in paraffin‐embedded tissues.     

Effect of dietary administration of probiotics on growth and intestine functionality of juvenile Senegalese sole (Solea senegalensis, Kaup 1858)

The effects of the dietary administration of two bacterial probiotic strains (Ppd11 and Pdp13) from the Alteromonadaceae family for 60 days, were assessed by measuring growth and feed efficiency, activities of leucine aminopeptidase and alkaline phosphatase and structural changes in the intestine of juvenile Senegalese sole. In addition, the profile of intestinal microbiota was studied by Denaturing Gradient Gel Electrophoresis. Growth and nutrient utilization were significantly higher in fish receiving probiotics than in those fed the control diet. No differences were observed in proximal composition between treatments, though higher lipid muscle content was measured in fish receiving Pdp13. Those fish also exhibited higher activities of AP when compared to Ppd11 and control groups. The profile of intestinal microbiota clearly separated those fish receiving probiotics from those of the control group. Microscopical examination revealed accumulation of lipid droplets in the enterocytes of fish receiving the control diet, but not in those fed on probiotics. Interactions between those structural changes and growth performance are discussed.     

Effect of dietary carbohydrate-to-lipid ratios on growth, lipid deposition and metabolic hepatic enzymes in juvenile Senegalese sole (Solea senegalensis, Kaup)

A study was undertaken to determine the effect of various dietary carbohydrate‐to‐lipid ratios on growth performance, whole‐body composition and tissue lipid content in Senegalese sole (Solea senegalensis) juveniles. Data on the dietary regulation of key hepatic enzymes of the lipogenic and glycolytic pathways (glucose‐6‐phosphate dehydrogenase, G6PD; malic enzyme, ME; fatty acid synthetase, FAS; pyruvate kinase, PK and glucokinase, GK) were also generated. Four isonitrogenous (crude protein: 52% dry matter (DM)) diets were formulated to contain one of two lipid levels (11% and 21% DM). Within each dietary lipid level, the nature of the carbohydrate fraction (raw or extruded peas) was varied. Triplicate groups of 54 sole (initial body weight: 23.6±1.2 g) were grown in recirculated seawater over 67 days. Fish were fed using automated feeders. At the end of the study, whole‐body, liver, viscera and muscle samples were withdrawn for analyses. During the experimental period, the mean fish weight about doubled in all treatments. No significant differences were found in growth performance (ranging from 1.1% to 1.4% body weight day^?1) among dietary treatments. High‐fat diets increased whole‐body fat content. Similarly, daily fat gain ranged from 0.54 to 0.78 g kg^?1 day^?1 and highest values were found in fish fed high‐lipid diets. Dietary treatments also affected tissue lipid content (liver, viscera and muscle), with highest values in fish fed high‐fat diets. The nature of dietary carbohydrates had little influence on performance criteria, but affected tissue lipid deposition. The activities of G6PD, ME and FAS were depressed by elevated levels of dietary lipid, confirming the inhibitory effect of dietary fats on lipid biosynthesis. At both dietary lipid levels, ME and FAS activities were little affected by dietary carbohydrate. Activities of PK and GK were not affected by the starch level of the diets. In Senegalese sole juveniles, the lipogenic pathway is more susceptible to modulation by dietary means (particularly through lipid intake) than the glycolytic pathway.     

塞内加尔鳎规模化人工繁育技术研究

2005~2006年连续两年对引进苗种养成的塞内加尔鳎Solea senegalensis全人工养殖亲鱼进行了繁育技术研究。两年间分别在威海和海阳两个实验基地共对33尾成熟雌鱼进行了LHRH-a肌肉注射催产和人工采卵授精,正常获产31尾,共获得受精卵7·3×106粒,平均受精率为64·3%,受精卵的平均孵化率为80·5%。两个实验基地分别培育出全长21~33mm苗种249万尾和全长22~36mm苗种112万尾,苗种培育成活率分别达到62·7%和58·2%。本研究成功地通过人工催产的方法,稳定获得塞内加尔鳎批量受精卵,并达到苗种生产量连续超过100万尾的规模化育苗水平。     

Growth, stress response and free amino acid levels in Senegalese sole (Solea senegalensis Kaup 1858) chronically exposed to exogenous ammonia

Stressful husbandry conditions are likely to affect growth and amino acid metabolism in fish. In this study, chronic ammonia exposure was used to test the effects of a stressor on growth and amino acid metabolism of Senegalese sole juveniles. The fish were exposed for 52 days to 11.6 mg L⁻¹ [low‐TAN (L‐TAN)] or 23.2 mg L⁻¹ [high‐TAN (H‐TAN)] of total ammonia nitrogen (TAN), or to 0 mg L⁻¹ (Control). Growth in L‐TAN groups was slightly but significantly different from the Control groups [relative growth rate (RGR=0.35±0.13 and 0.52±0.23% day⁻¹ respectively)]. In H‐TAN groups, growth was severely affected (RGR=0.01±0.13% day⁻¹). Stress parameters (plasma cortisol and glucose) showed slight or no significant differences between treatments. Plasma free amino acid (FAA) concentrations were affected after H‐TAN treatment. Increases in glutamine and aspartate concentrations in H‐TAN fish suggest alterations in amino acid metabolism related to nitrogen excretion processes. Some of the changes in FAA concentrations also suggest mobilization to energy supply and synthesis of metabolites related to stress response. Therefore, Senegalese sole seem to adapt to the L‐TAN concentration tested, but the H‐TAN concentration reduced growth and affected amino acid metabolism.     

Feed deprivation in Senegalese sole (Solea senegalensis Kaup, 1858) juveniles: effects on blood plasma metabolites and free amino acid levels

The effects of prolonged feed deprivation were assessed on blood plasma metabolites and free amino acid levels in Solea senegalensis. Juvenile specimens were maintained at two experimental conditions (24 h fasted and 21 days feed-deprived). In feed-deprived fish, relative growth rate and hepatosomatic index as well as plasma triglycerides and proteins levels were significantly lower. However, plasma cortisol levels were higher in feed-deprived fish, while plasma glucose and lactate values were not significantly different between treatments. Furthermore, feed-deprived fish showed higher levels of total plasma free amino acids than 24 h fasted fish. In 21 days feed-deprived sole, an increase in plasma cortisol levels may suggest a functional role in mobilizing energy due to the catabolic action of this hormone in teleosts. Higher levels of glutamine, arginine and ornithine in 21 days feed-deprived fish may be indicative of a dual role for these amino acids: ammonia detoxification and carbon source for gluconeogenesis. The increased plasma glucogenic and branched-chain amino acid levels, together with the maintenance of plasma glucose in 21 days feed-deprived sole, suggest active liver gluconeogenic processes supported by tissue proteolysis.     

Feed intake and growth performance of Senegalese sole (Solea senegalensis Kaup, 1858) fed diets with partial replacement of fish meal with plant proteins

To be able to study nutrient requirement and utilization in any species, a diet supporting normal feed intake and growth equally well as a traditional fish meal‐based diet is needed. Additionally the formulation of the diet should allow low levels of the nutrient under study. When studying the amino acid metabolism and requirements, one cannot rely on the fish meal‐based diets as fish meal are nicely balanced according to requirements. Therefore the current study aimed to develop a plant protein‐based diet (with low fish meal inclusion) to be used in the nutritional studies of Senegalese sole juveniles supporting feed intake and growth close to that obtained in a fish meal‐based control feed. Two experiments were conducted to evaluate whether Senegalese sole juveniles would accept and utilize diets containing high plant protein inclusion. For testing the acceptance of high plant protein inclusion, two diets were formulated: a reference diet that contained fish meal as the main protein source (450 g kg^?1 dry matter) whereas in the test diet, fish meal was substituted by a mixture of plant ingredients (soybean meal, corn and wheat gluten) with l ‐lysine supplementation. In order to improve the palatability, 50 g kg^?1 squid meal was added to both diets. The indispensable amino acids (IAA) profile of the test diet was made similar to the control diet by adding crystalline amino acids. Further, automatic feeders were used to improve the feed intake. Fish (24 g initial body weight) were fed the diets for a period of 4 weeks. As fish accepted both diets equally well, a second study was undertaken to test the growth performance. Fish (6 g initial BW) were fed the diets for a period of 12 weeks. The use of automatic feeders to deliver the feed and the addition of both squid and balancing the indispensable amino acids resulted in growth performance and accretion not differing from the fish meal fed control. It can be concluded that juvenile Senegalese sole are able to grow and utilize high plant‐protein diets when both diet composition and feeding regime are adequate for this species.     

Effect of feeding time on dietary protein utilization and growth of juvenile Senegalese sole (Solea senegalensis)

The present study aimed to determine the effect of feeding time on growth and nitrogen excretion in juvenile sole. An 84‐day growth trial was conducted, in which food was supplied to three triplicate groups of juvenile Senegalese sole (3 g wet weight) at different schedules – diurnal, nocturnal and mixed. At the end of the growth trial, ammonia and urea excretion was assessed during a 24 h cycle. Improved growth (1.3% vs. 0.9% day^?1, specific growth rate), higher nitrogen retention (0.35 vs. 0.27 g N kg^?1 day^?1), lower ammonia excretion (209 vs. 272 mg N‐NH₄ kg^?1 day^?1) and lower total nitrogen excretion (278 vs. 352 mg N kg^?1 day^?1) were found in daytime‐fed fish compared with night‐fed fish. Fish in the mixed feeding regime showed intermediate values of ammonia and total nitrogen excretion, but did not differ from day‐fed fish regarding the other parameters. Results indicate that juvenile sole at a period of their life cycle appear to use more efficiently dietary protein for somatic growth under a diurnal than under a nocturnal feeding regime. This suggest that at least during a time‐window in the juvenile rearing a diurnal feeding regime might be more effective in the production of this species.     

In vitro effect of the red alga Hydropuntia cornea (J. Agardh) on the respiratory burst activity of sole (Solea senegalensis, Kaup 1858) phagocytes

The stimulatory effect of different extracts from the red alga Hydropuntia cornea (formerly Gracilaria cornea) on the respiratory burst activity of sole phagocytes was evaluated in vitro. Aqueous and ethanolic extracts and extracellular polysaccharidic fractions from algal cultures were assayed. Phagocytes, isolated from sole kidney, were incubated in vitro with different algal fractions at concentrations of 10, 5, 2 and 1 mg mL⁻¹. As a positive control, commercial β‐glucan from the alga Euglena gracilis was used. The results obtained indicate that an ethanolic extract from H. cornea, at 10 mg mL⁻¹, is capable of enhancing superoxide anion production in sole phagocytes when the cells are incubated for 30 min with the bacterium P. damselae ssp. piscicida and the extract. On the other hand, the commercial β‐glucan leads to a significant increase at highest concentration, 10 mg mL⁻¹, after 30 min of incubation.     

Isolation of lymphocystis disease virus from sole, Solea senegalensis Kaup, and blackspot sea bream, Pagellus bogaraveo (Brunnich)

Two viruses were isolated from cultured sole, Solea senegalensis, and wild blackspot sea bream, Pagellus bogaraveo, and preliminarily characterized as lymphocystis disease viruses (LCDVs). Viral isolates were characterized by morphological, biochemical and biophysical properties. In addition, the susceptibility of four fish cell lines was also tested. LCDV isolates developed cytopathic effects on the SAF-1 cell line at 5 and 6 days post-infection and reached titres of 10(6) TCID50 mL(-1). The antigenic and structural protein analysis of the two new LCDV isolates showed identical profiles to that obtained for LCDV strain Leetown NFH (ATCC VR-342), used as a reference viral strain, and for an LCDV isolate collected from gilt-head sea bream, Sparus aurata, cultured in southern Spain. Molecular confirmation was performed by polymerase chain reaction. Specific primers for LCDV produced a 270-bp DNA fragment, the expected size for LCDV.     

Effect of dietary administration of Porphyridium cruentum on the respiratory burst activity of sole, Solea senegalensis (Kaup), phagocytes

The stimulatory effect of the red microalga Porphyridium cruentum on respiratory burst activity of sole phagocytes was evaluated in vivo . Oral administration of a diet supplemented with lyophilized P. cruentum cells (10 g kg⁻¹) stimulated respiratory burst activity after 4 weeks feeding in sole vaccinated with Photobacterium damselae subsp. piscicida bacterin.     

Use of the probiotic Shewanella putrefaciens Pdp11 on the culture of Senegalese sole (Solea senegalensis, Kaup 1858) and gilthead seabream (Sparus aurata L.)

The application of probiotics on aquatic animals is increasing for a better fish welfare status as well as an environment-friendly activity which are actual demands of modern aquaculture industry. A bacterium from skin mucus of healthy gilthead seabream (Sparus aurata L.) has been isolated and identified as Shewanella putrefaciens Pdp11. Different studies have been done to know its application as probiotic in the Senegalese sole and gilthead seabream farming. This article reviews the studies carried out with this probiotic microorganism focusing on the current knowledge of its in vitro and in vivo mechanisms of action. The results suggested that the probiotic S. putrefaciens Pdp11, due to its beneficial effects, could be used in the aquaculture activity of both species.     

Cellular and humoral immune responses of Senegalese sole,Solea senegalensis (Kaup), following challenge with two Photobacterium damselae subsp. piscicida strains from different geographical origins

The present study aimed to investigate leucocyte responses to inflammation as well as some innate immune parameters of Senegalese sole, Solea senegalensis, following challenge with two strains of Photobacterium damselae subsp. piscicida belonging to the European and Japanese clones described for this bacterium. Pathogenicity assays were performed to assess the virulence of each Photobacterium damselae subsp. piscicida strain for sole. Subsequently, fish were intraperitoneally injected with phosphate‐buffered saline (control) or two concentrations (2 × 10² and 2 × 10⁶ CFU mL^?1) of each bacterial strain and sampled after 6 and 24 h. Results showed that the European isolate induces a higher degree of response than the Japanese strain. While blood neutrophilia and monocytosis correlated well with the increase in neutrophil and macrophage numbers in the peritoneal cavity, fish infected with the European isolate presented higher peritoneal cell numbers than fish challenged with the Japanese strain. In addition, alternative complement pathway activity and respiratory burst of head kidney leucocytes increased significantly in fish infected with the European isolate. The enhanced innate immune response displayed by Senegalese sole challenged with the European isolate is probably due to the higher degree of virulence presented by this Photobacterium damselae subsp. piscicida strain.