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.     

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.     

Tenacibaculosis induction in the Senegalese sole (Solea senegalensis) and studies of Tenacibaculum maritimum survival against host mucus and plasma

Tenacibaculum maritimum, the aetiological agent for marine tenacibaculosis, is one of the most significant pathogens that threaten Senegalese sole, Solea senegalensis (Kaup), aquaculture. Because no immersion challenge with T. maritimum has been reported previously for this flatfish species, this study aimed to optimize bacterial yields as well as to establish a challenge model for tenacibaculosis induction. Several approaches were performed to optimize bacterial culture conditions, including treatment with non‐ionic surfactants, detergents, cellulase hydrolysis and strong shaking. A prolonged bath challenge was performed for 24 h under two different temperatures, 16 and 23 °C. Moreover, mucus and plasma bactericidal activities against T. maritimum were also assessed. Culturing bacteria with strong shaking and continuous shaking provided suitable culture conditions to obtain higher bacterial yields without aggregation and fluctuation, contrary to most other treatments that showed a huge amount of bacterial aggregates. A prolonged bath method for 24 h, without skin or gill scarification, was considered suitable for disease induction with high mortality rates. Moreover, data regarding mucus and plasma bactericidal activities suggested that there is a lack of host innate immune response against T. maritimum or that this particular pathogen presents evading strategies against Senegalese sole.     

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.     

Morphopathological features of a severe ulcerative disease outbreak associated with Tenacibaculum maritimum in cultivated sole, Solea senegalensis (L.)

This study describes morphopathologic changes in naturally infected farmed Senegalese sole affected by tenacibaculosis caused by Tenacibaculum maritimum. Macroscopic observation, in addition to light microscopy and scanning electron microscopy, was used to study the lesions. Main lesions were characterized by complete loss of epidermis and dermis, as well as extensive necrosis of muscle layers. Mild-to-moderate inflammatory response with the presence of macrophages was noted around hyaline degenerated muscle cells. Gram-negative filamentous bacteria could be detected only at the dermis. Under scanning electron microscopy, filamentous bacteria located over the scales without epithelium could be observed. These findings together with the isolation and PCR detection of the bacteria in kidney and skin tissues suggest that once the bacteria reach the dermis, probably through eroded epidermis, they are able to proliferate and produce enzymes that are responsible for the damage in the underlying tissues.     

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.     

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.     

Effective replacement of protein‐bound amino acids by crystalline amino acids in Senegalese sole (Solea senegalensis) juveniles

The objective of this study was to evaluate the potential of free amino acids to replace protein‐bound amino acids in diets for Solea senegalensis juveniles. For this purpose, fish with 4.26 g mean initial weight were fed during 55 days with diets containing 500 g kg^?1 protein and 100 g kg^?1 lipid, based on fish meal and fish oil as main protein and lipid sources (control diet), or diets where 20%, 35% and 50% of fish meal were replaced by crystalline amino acids, using L‐lysine HCl as lysine source. To access the efficiency of the crystalline lysine source, another diet was formulated with 35% fish meal replacement but using L‐lysine sulphate instead of L‐lysine HCl as lysine source. Overall, growth performance, feed utilization, nutrient retention, whole‐body and amino acid composition did not show significant differences among groups. In conclusion, up to 50% protein‐bound amino acids can be replaced by crystalline amino acids in diets for Senegalese sole juveniles without negative effects in fish performance. Additionally, it was shown that L‐lysine sulphate and L‐lysine‐HCl may be used interchangeably by Senegalese sole juveniles, with the potential economic benefits involved.     

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.     

Effectiveness of a divalent vaccine for sole, Solea senegalensis (Kaup), against Vibrio harveyi and Photobacterium damselae subsp. piscicida

The protection of cultured sole, Solea senegalensis, against Vibrio harveyi and Photobacterium damselae subsp. piscicida was evaluated following the use of a divalent vaccine prepared with formalized whole cells and extracellular products of virulent strains of both pathogenic microorganisms and administered by the immersion route. Two prolonged immersions of 5-10 g fish in the divalent bacterin at a 1-month interval gave high levels of protection similar to those obtained when the respective monovalent vaccines were administered by the intraperitoneal route [relative percentage of survival (RPS) values >70%], which indicates that the former procedure can be a useful strategy with small fish. The high protection afforded by the divalent vaccine in sole lasted for 4 months after which the RPS values against both pathogens decreased significantly.     

Enhancement of non‐specific immune responses and disease resistance on oral administration of Nyctanthes arbortristis seed extract in Oreochromis mossambicus (Peters)

The immunomodulatory effect of the seed extract of an Indian medicinal plant Nyctanthes arbortristis L. (NAT) on non‐specific immune responses and disease resistance of tilapia, Oreochromis mossambicus was investigated. Chloroform extract was administered orally as a feed supplement at doses of 0.01%, 0.1% or 1% for 3 weeks. Non‐specific immune parameters such as serum lysozyme and alternate complement haemolytic (ACH₅₀) activities, and cellular reactive oxygen species (ROS), reactive nitrogen intermediate (RNI) and myeloperoxidase production and disease resistance against live virulent Aeromonas hydrophila were investigated after 1, 2 or 3 weeks of feeding with chloroform extract‐supplemented diet. The results of this study indicated that feeding tilapia for 2 weeks with selected doses of chloroform extract of NAT seeds significantly enhanced serum lysozyme, alternate complement activities and cellular ROS, RNI and MPO production. It was evident from the disease resistance test that feed supplemented with NAT seed extract at 0.1% or 1% level significantly reduced the mortality of O. mossambicus and a 3‐week feeding with 0.1% extract‐supplemented diet appears to be the optimal regimen for maximal disease resistance. Thus, the study indicates the scope of using the NAT extract as an immunoprophylactic in finfish aquaculture.     

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.     

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.     

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.     

Iodine‐enriched rotifers and Artemia prevent goitre in Senegalese sole (Solea senegalensis) larvae reared in a recirculation system

To study the effect of dietary supplementation of iodine in Solea senegalensis, larvae were randomly distributed in six tanks. Larvae in three tanks were given rotifers and Artemia enriched with iodine in addition to Rich Advance or Super Selco from 2 days after hatch (DAH) until 31 DAH. Larvae in a second set of three tanks were fed control rotifers and Artemia, enriched only with Rich Advance or Super Selco. Samples were collected at 2, 5, 10, 15 and 31 DAH to determine dry weight, total length, myotome height and thyroid status. Larvae fed the iodine‐enriched diet had significantly higher weight at 31 DAH and higher levels of whole body iodine concentration, compared to control larvae. At 31 DAH, larvae from the control treatment showed typical goitrous thyroid follicles. Thyroid cells of larvae from this treatment appeared columnar or afollicular, with the colloid partly or completely depleted, representative of hyperplasia (goitre). The lower growth rate in fish larvae from the control treatment was possibly a consequence of the hyperplasia, and the iodine enrichment prevented Senegalese sole larvae from developing goitre. This study demonstrates the importance of iodine enrichment of live feed for fish reared in a recirculation system.     

Endolimax piscium sp. nov. (Amoebozoa), causative agent of systemic granulomatous disease of cultured sole,Solea senegalensis Kaup

A new amoeba species pathogenic for Senegalese sole is described based on ultrastructural analysis and SSU rDNA phylogenetic inference. The parasite presents round to ovoid trophozoites (<5 μm) with a high degree of intracellular simplification. No mitochondria were observed, but mitosome‐like organelles were present. No cysts could be detected. Phylogenetic analysis confirmed the Senegalese sole parasite as an amitochondriate Archamoeba related to Endolimax nana and Iodamoeba spp., and we tentatively describe it as a new species in the genus Endolimax, Endolimax piscium. However, the genetic distance with E. nana is quite large, with only 60% pairwise identity between both SSU rDNA genotypes. Although the overall topology of the Archamoebae cladograms containing E. piscium was consistent, the support for the branching of Endolimax spp. relative to its closest neighbours was variable, being higher with distance or parsimony‐based inference methods than with ML or Bayesian trees. The use of stringent alignment sampling masks also caused instability and reduced support for some branches, including the monophyly of Endolimax spp. in the most conservative data sets. The characterization of other Archamoebae parasitizing fish could help to clarify the status of E. piscium and to interpret the large genetic distance observed between Endolimax species.     

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.