Senegalese Sole Research Articles

Taurine Supplementation to Plant-Based Diets Improves Lipid Metabolism in Senegalese Sole.

Taurine is a sulphur-containing amino acid with important physiological roles and a key compound for the synthesis of bile salts, which are essential for the emulsion and absorption of dietary lipids. This study aimed to evaluate the effects of taurine supplementation to low-fishmeal diets on the metabolism of taurine, bile acids, and lipids of Senegalese sole. A fishmeal (FM) and a plant-protein-based (PP0) diet were formulated, and the latter was supplemented with taurine at 0.5 and 1.5% (diets PP0.5 and PP1.5). Diets were assigned to triplicate tanks containing 35 fish (initial weight ~14 g) for 6 weeks. Fish from the PP0 treatment presented lower taurine and bile-acid concentrations compared with the FM treatment, and a downregulation of cyp7a1 and abcb11 was observed. Triolein catabolism decreased in PP0-fed fish, resulting in increased hepatic fat content and plasma triglycerides, while no effects on plasma cholesterol were observed. Taurine supplementation to plant-based diets resulted in a higher taurine accumulation in fish tissues, increased bile-acid concentration, and upregulation of cyp7a1 and abcb11. Hepatic fat content and plasma triglycerides decreased with increasing dietary taurine supplementation. Taurine supplementation mitigated part of the negative effects of plant-based diets, leading to better lipid utilisation.

Immune Response of Senegalese Sole against Betanodavirus Mutants with Modified Virulence.

Nervous necrosis virus (NNV), genus Betanodavirus, the etiological agent of the viral encephalopathy and retinopathy (VER), presents a genome with two positive-sense single-stranded RNA segments. Striped jack nervous necrosis virus (SJNNV) and red-spotted grouper nervous necrosis virus (RGNNV), together with reassortants RGNNV/SJNNV, are the betanodaviruses predominantly isolated in Southern Europe. An RGNNV/SJNNV reassortant isolated from Senegalese sole (wt160) causes high mortalities in this fish species. This virus presents differences in the sequence of the 3’ non-coding region (NCR) of both segments compared to RGNNV and SJNNV reference strains. Previously, it has been reported that the reversion of two of these differences (nucleotides 1408 and 1412) in the RNA2 3’NCR to the SJNNV-type (recombinant r1408-1412) resulted in a decrease in sole mortality. In the present study, we have applied an OpenArray® to analyse the involvement of sole immune response in the virulence of several recombinants: the r1408-1412 and two recombinants, developed in the present study, harbouring mutations at positions 3073 and 3093 of RNA1 3’NCR to revert them to RGNNV-type. According to the correlation values and to the number of expressed genes, the infection with the RNA2-mutant provoked the most different immune response compared to the immune response triggered after the infection with the rest of the viruses, and the exclusive and high upregulation of genes related to the complement system. The infection with the RNA1-mutants also provoked a decrease in mortality and their replication was delayed at least 24 h compared to the wt160 replication, which could provoke the lag observed in the immune response. Furthermore, the infection with the RNA1-mutants provoked the exclusive expression of pkr and the downregulation of il17rc.

Dietary Natural Plant Extracts Can Promote Growth and Modulate Oxidative Status of Senegalese Sole Postlarvae under Standard/Challenge Conditions.

Simple SummaryOxidative stress has a direct impact on the welfare of fish, affecting growth performance and health status. Natural plant extracts present a high antioxidant capacity, due to a diversity and abundant content of polyphenols. Thus, the aim of this work was to identify if plant extracts, such as curcumin, green tea, and grape seeds, can promote oxidative status, and ultimately, enhance the growth and physiological stress response of postlarvae. Our results showed that plant extracts can improve the growth and oxidative status of the fish. Moreover, they may help fish to cope under stressful conditions. Dietary formulations with natural supplements may be a viable strategy to improve fish robustness during early life stages, and can therefore contribute to the development of aquafeeds and promote the sustainability of aquaculture production.Plant extracts are known for their high content and diversity of polyphenols, which can improve fish oxidative status. A growth trial with Senegalese sole postlarvae (45 days after hatching) fed with one of four experimental diets—control (CTRL), and supplemented with curcumin (CC), green tea (GT), and grape seed (GS) extracts—was performed to assess if supplementation could improve growth performance and oxidative status. At the end of the growth trial, postlarvae were submitted to a thermal stress to assess their robustness. Sole growth was improved by CC and GS diets when compared to those fed the CTRL. CC and CTRL postlarvae presented the lowest oxidative damage (lipid peroxidation and protein carbonylation values). Stress-related biomarkers (heat shock protein 70 and glutathione-S-transferase) decreased in CC fish compared to those fed the CTRL diet, which might be due to a direct antioxidant capacity. In contrast, oxidative damage increased in GT and GS sole reared in standard conditions. However, after a thermal stress, GT and GS diets prevented the increase of protein carbonylation content and the decrease of antioxidant glutathione, depending on exposure time. Overall, dietary supplementation with natural extracts modulated oxidative status and stress response after a short/long-term exposure to temperature.

BEI Inactivated Vaccine Induces Innate and Adaptive Responses and Elicits Partial Protection upon Reassortant Betanodavirus Infection in Senegalese Sole.

Nervous necrosis virus (NNV), the causative agent of viral encephalopathy and retinopathy (VER), is one of the most threatening viruses affecting marine and freshwater fish species worldwide. Senegalese sole is a promising fish species in Mediterranean aquaculture but also highly susceptible to NNV and VER outbreaks, that puts its farming at risk. The development of vaccines for aquaculture is one of best tools to prevent viral spread and sudden outbreaks, and virus inactivation is the simplest and most cost-effective method available. In this work, we have designed two inactivated vaccines based on the use of formalin or binary ethylenimine (BEI) to inactivate a reassortant NNV strain. After vaccination, the BEI-inactivated vaccine triggered the production of specific IgM-NNV antibodies and stimulated innate and adaptive immune responses at transcriptional level (rtp3, mx, mhcii and tcrb coding genes). Moreover, it partially improved survival after an NNV in vivo challenge, reducing the mid-term viral load and avoiding the down-regulation of immune response post-challenge. On the other hand, the formalin-inactivated vaccine improved the survival of fish upon infection without inducing the production of IgM-NNV antibodies and only stimulating the expression of herc4 and mhcii genes (in head-kidney and brain, respectively) during the vaccination period; this suggests that other immune-related pathways may be involved in the partial protection provoked. Although these vaccines against NNV showed encouraging results, further studies are needed to improve sole protection and to fully understand the underlying immune mechanism.

Genetic Estimates for Growth and Shape-Related Traits in the Flatfish Senegalese Sole.

Simple SummaryTo increase competitiveness, the aquaculture flatfish industry demands animals with optimal growth rates and a high shape quality. Genetic breeding is an essential tool to achieve these goals but it requires the estimation of the genetic components of these traits under industrial conditions. The current study provides phenotypic data and genetic parameters of eight traits related to growth and shape quality. The high heritabilities and correlations obtained support that genetic breeding programs can be successfully implemented in Senegalese sole to optimize production.Shape quality is very important in flatfish aquaculture due to the impact on commercialization. The Senegalese sole (Solea senegalensis) is a valuable flatfish with a highly elliptic body that slightly changes with age and size, and it is prone to accumulating malformations during the production cycle. The present study aims to investigate the genetic parameters of two growth traits (weight and standard length) and six shape quality predictors (ellipticity, three body heights (body height at the pectoral fin base [BHP], body maximum height [BMH] and caudal peduncle height [CPH]) and two ratios (BMH/BHP and BMH/CPH)). These traits were measured before the on-growing stage (age ~400 days (d)) and at harvest (~800 d). Phenotypic data, heritabilities and genetic and phenotypic correlations between the traits are presented and discussed. High or very high heritabilities (0.433–0.774) were found for growth traits, body heights and ellipticity and they were higher at 400 than 800 d. In contrast, the ratios of BMH/BHP and BMH/CPH were less heritable (0.144–0.306). Positive and very high (>0.95) correlations between growth traits and the three heights were found and decreased with age. In contrast, ellipticity had negative and medium-high genetic correlations with growth traits and heights, indicating fish selected for bigger size would also become rounder. The ratio of BMH/CPH showed low genetic correlations with all traits and provided complementary information to ellipticity for a better fitting to the expected lanceolate body morphology of sole. The genetic correlations for all traits at both ages were very high, indicating that selection before entering the growth-out stage in recirculation aquaculture systems is recommended to accelerate genetic gains.

Cadmium Accumulation and Kinetics in Solea senegalensis Tissues under Dietary and Water Exposure and the Link to Human Health

Bioaccumulation of cadmium was assessed in different tissues of the benthic fish Solea senegalensis. Juvenile Senegalese soles were simultaneously exposed to cadmium-contaminated diet (Hediste diversicolor) and water during 14 days and allowed to depurate for another 14 days. Cadmium content was measured in muscle, gills, liver and intestine, with recorded values increasing in these tissues in this same order. Muscle showed a considerably lower cadmium accumulation after 14 days of uptake. Cadmium kinetics in juvenile Senegalese soles revealed that the highest uptake flux of this metal occurred in the intestine. Cadmium depuration from the liver was not detected, which suggests the existence of a storage compartment for this metal in Solea senegalensis during uptake and depuration. Comparisons between maximum acceptable values for cadmium in the muscle, the Target Hazard Quotient and the Estimated Weekly Intake, indicated that acceptable limits were not exceeded, and the muscle of juvenile Senegalese soles could be considered safe for human consumption.

Skeletal Anomalies in Senegalese Sole (Solea senegalensis, Kaup) Fed with Different Commercial Enriched Artemia: A Study in Postlarvae and Juveniles.

Simple SummaryEnrichment products for Artemia spp. metanauplii are commonly used to enhance the nutritional quality of this live prey offered to fish during conventional larval feeding. However, there are few reports on the influence of such enrichments on the development of skeletal anomalies in Senegalese sole, a major problem for this flatfish aquaculture. This study evaluated the frequency of vertebral anomalies in postlarvae and juvenile Senegalese sole fed with Artemia spp. metanauplii enriched with four commercial products (EA, EB, EC, and ED) in a fish farm. The results show a high percentage of individuals with skeletal anomalies in every dietary group. Some types of anomalies were very frequent in all diet-age groups, indicating the presence of a common trend or mainstay of vertebral deformities. Despite some variations in the frequency of anomalies among diets, it was not possible to establish a clear effect of the enrichment products on the development of vertebral deformities at both rearing stages, probably for the “masking effect” of other rearing conditions. The multivariate statistical technique, as the correspondence analysis, indicated a different anomaly pattern among ages, where bone adaptative responses may be implied.The high incidence of skeletal anomalies in Senegalese sole (Solea senegalensis) still constitutes a bottleneck constraining its production. There are diverse commercially available products for the enrichment of live preys, but few reports of their influence on skeletogenesis in Senegalese sole. This study evaluated the presence of vertebral anomalies in postlarvae and juvenile Senegalese sole fed with Artemia spp. metanauplii enriched with four commercial products (EA, EB, EC, and ED) in a fish farm. The most frequent alterations consisted of deformations of the neural/haemal arches and spines and fusions and deformations of hypurals, epural, or parhypural. The correspondence analysis ordered fish from each age in separated semiaxis, indicating the presence of different anomaly patterns for the two sampled stages. The results showed only very light changes in the frequency of vertebral abnormalities among tested enrichment products, i.e., individuals from EC and EA lots displayed less vertebral body anomalies and/or vertebral column deviations at 31 and 105 days after hatching, respectively. The existence of a large shared malformation pattern in all the experimental groups leads to impute to the rearing conditions as the main driving factor of the onset of such group of anomalies, probably masking some dietary effect.

Kisspeptin Influences the Reproductive Axis and Circulating Levels of microRNAs in Senegalese Sole.

Kisspeptin regulates puberty and reproduction onset, acting upstream of the brain–pituitary–gonad (HPG) axis. This study aimed to test a kisspeptin-based hormonal therapy on cultured Senegalese sole (G1) breeders, known to have reproductive dysfunctions. A single intramuscular injection of KISS2-10 decapeptide (250 µg/kg) was tested in females and males during the reproductive season, and gonad maturation, sperm motility, plasma levels of gonadotropins (Fsh and Lh) and sex steroids (11-ketotestosterone, testosterone and estradiol), as well as changes in small non-coding RNAs (sncRNAs) in plasma, were investigated. Fsh, Lh, and testosterone levels increased after kisspeptin injection in both sexes, while sperm analysis did not show differences between groups. Let7e, miR-199a-3p and miR-100-5p were differentially expressed in females, while miR-1-3p miRNA was up-regulated in kisspeptin-treated males. In silico prediction of mRNAs targeted by miRNAs revealed that kisspeptin treatment might affect paracellular transporters, regulate structural and functional polarity of cells, neural networks and intracellular trafficking in Senegalese sole females; also, DNA methylation and sphingolipid metabolism might be altered in kisspeptin-treated males. Results demonstrated that kisspeptin stimulated gonadotropin and testosterone secretion in both sexes and induced an unanticipated alteration of plasma miRNAs, opening new research venues to understand how this neuropeptide impacts in fish HPG axis.

Dietary Antioxidant Supplementation Promotes Growth in Senegalese Sole Postlarvae.

Somatic growth is a balance between protein synthesis and degradation, and it is largely influenced by nutritional clues. Antioxidants levels play a key role in protein turnover by reducing the oxidative damage in the skeletal muscle, and hence promoting growth performance in the long-term. In the present study, Senegalese sole postlarvae (45 days after hatching, DAH) were fed with three experimental diets, a control (CTRL) and two supplemented with natural antioxidants: curcumin (CC) and grape seed (GS). Trial spanned for 25 days and growth performance, muscle cellularity and the expression of muscle growth related genes were assessed at the end of the experiment (70 DAH). The diets CC and GS significantly improved growth performance of fish compared to the CTRL diet. This enhanced growth was associated with larger muscle cross sectional area, with fish fed CC being significantly different from those fed the CTRL. Sole fed the CC diet had the highest number of muscle fibers, indicating that this diet promoted muscle hyperplastic growth. Although the mean fiber diameter did not differ significantly amongst treatments, the proportion of large-sized fibers (>25 μm) was also higher in fish fed the CC diet suggesting increased hypertrophic growth. Such differences in the phenotype were associated with a significant up-regulation of the myogenic differentiation 2 (myod2) and the myomaker (mymk) transcripts involved in myocyte differentiation and fusion, respectively, during larval development. The inclusion of grape seed extract (GS diet) resulted in a significant increase in the expression of myostatin1. These results demonstrate that both diets (CC and GS) can positively modulate muscle development and promote growth in sole postlarvae. This effect is more prominent in CC fed fish, where increased hyperplastic and hypertrophic growth of the muscle was associated with an upregulation of myod2 and mymk genes.

Assessment of Growth, Lipid Metabolism and Gene Expression Responses in Senegalese Sole Larvae Fed With Low Dietary Phospholipid Levels.

Phospholipids (PL) are essential molecules for larval growth and development. In this study, growth, lipid metabolism and gene expression responses associated with different dietary PL levels in pelagic sole larvae were evaluated. In a first trial, the long-term effects on growth and survival of two experimental microdiets (MD) containing high (High-PL) or low (Low-PL) PL levels were tested and compared to a diet based on live prey (rotifers). The MD were supplied from 3 to 10 days post-hatch (dph) and Artemia from day 8 to 29 dph. High-PL fed larvae had higher dry mass (1.2-fold) than Low-PL fed larvae at 8 dph and both MD were smaller (2.9-fold) than larvae fed live preys. However, a compensatory growth (33% between 8 and 20 dph) occurred when MD were substituted by Artemia and by the end of the trial no significant differences in mass or survival occurred between the dietary treatments. In a second trial, growth, lipid metabolism and gene expression profiles of larvae fed with MD up to 8 dph were analyzed. Growth data confirmed that mass of larvae fed with High-PL was higher (1.3-fold) than the those fed Low-PL and they had lower levels of triacylglycerol (2.8-fold), cholesterol (1.2-fold) and cetoleic acid (1.7-fold). Histological analysis indicated an excess of lipid vacuoles in larvae fed with Low-PL and the expression analysis revealed a coordinated response to enhance lipid mobilization since the expression of genes involved in PL intermediate synthesis, PL remodeling as well as eight apolipoprotein was up-regulated. The down-regulation of apolipoprotein apob2 in larvae fed with Low-PL indicated a specific regulation by PL levels. The present work provides insight into the responses associated with dietary PL in early fish larvae, which will be of use for future studies aimed as designing effective larval sole diets.

Effect of Dietary Manganese and Zinc Levels on Growth and Bone Status of Senegalese Sole (Solea senegalensis) Post-Larvae.

Essential dietary trace elements, such as zinc (Zn) and manganese (Mn), critically influence a wide range of physiological, metabolic, and hormonal processes in fish larvae and post-larvae. Despite their importance for normal fish growth and skeletal development, trace mineral nutrition has not been extensively studied in the early stages of development of fish. Post-larvae of an emergent aquaculture species, Senegalese sole (Solea senegalensis), were the subject of this study in order to better understand the effects of diet supplementation of trace minerals upon fish larval development and performance. Sole post-larvae were fed a combination of organic Mn (45 and 90mgkg-1 feed) and organic Zn (100 and 130mgkg-1 feed) and survival, growth, mineral deposition rates, and vertebral bone status were assessed. Our results showed that although no significant effect was found on the growth performance of Senegalese sole post-larvae, Mn and Zn supplementation to a commercial microdiet for marine fish larvae at higher dietary levels (Mn at 90mgkg-1 and Zn at 130mgkg-1) improved larval survival, decreased the severity of vertebral malformations, and increased the deposition of Mn in bone.

Effects of Water Acidification on Senegalese Sole Solea senegalensis Health Status and Metabolic Rate: Implications for Immune Responses and Energy Use

Increasing water CO2, aquatic hypercapnia, leads to higher physiological pCO2 levels in fish, resulting in an acidosis and compensatory acid-base regulatory response. Senegalese sole is currently farmed in super-intensive recirculating water systems where significant accumulation of CO2 in the water may occur. Moreover, anthropogenic releases of CO2 into the atmosphere are linked to ocean acidification. The present study was designed to assess the effects of acute (4 and 24 h) and prolonged exposure (4 weeks) to CO2 driven acidification (i.e., pH 7.9, 7.6, and 7.3) from normocapnic seawater (pH 8.1) on the innate immune status, gill acid-base ion transporter expression and metabolic rate of juvenile Senegalese sole. The acute exposure to severe hypercapnia clearly affected gill physiology as observed by an increase of NHE3b positive ionocytes and a decrease of cell shape factor. Nonetheless only small physiological adjustments were observed at the systemic level with (1) a modulation of both plasma and skin humoral parameters and (2) an increased expression of HIF-1 expression pointing to an adjustment to the acidic environment even after a short period (i.e., hours). On the other hand, upon prolonged exposure, the expression of several pro-inflammatory and stress related genes was amplified and gill cell shape factor was aggravated with the continued increase of NHE3b positive ionocytes, ultimately impacting fish growth. While these findings indicate limited effects on energy use, deteriorating immune system conditions suggest that Senegalese sole is vulnerable to changes in CO2 and may be affected in aquaculture where a pH drop is more prominent. Further studies are required to investigate how larval and adult Senegalese sole are affected by changes in CO2.

Phenotypic and Molecular Characterization of Lacinutrix venerupis Isolated from Atlantic Horse Mackerel Trachurus trachurus.

The aim of the present study was to characterize two gram-negative bacterial strains that were isolated from diseased Atlantic Horse Mackerel Trachurus trachurus in 2017. Based on the results obtained from the biochemical and chemotaxonomic characterization, the isolates were identified as Lacinutrix spp. The highest similarity of the 16S rRNA gene sequences was obtained with the strain L.venerupis CECT 8573T (99.1%), while other species showed similarities of 98% (L.jangbogonensis) and 97% (L.algicola and L.mariniflava). Molecular characterization by repetitive element (REP)-PCR and enterobacterial repetitive intergenic consensus (ERIC)-PCR, as well as proteomic characterization by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS), demonstrated heterogeneity between the strains from the Atlantic Horse Mackerel and the type strain, CECT 8573T . The virulence of one of the isolates for Turbot Scophthalmus maximus, European Sea Bass Dicentrarchus labrax, Senegalese Sole Solea senegalensis, and Rainbow Trout Oncorhynchus mykiss was assessed under experimental conditions. No mortalities were recorded after intraperitoneal injections with high doses of bacteria (1×109 CFU/mL). Thus, further studies are necessary to elucidate the impact of this bacterial species as a fish pathogen.

Metabolic and Stress Responses in Senegalese Soles (Solea senegalensis Kaup) Fed Tryptophan Supplements: Effects of Concentration and Feeding Period.

Simple SummaryIn order to improve fish welfare in fish farms, feed additives (tryptophan, Trp) were assayed in a cultured species, the Senegalese sole (Solea senegalensis). It has been observed in previous studies that fish fed Trp supplements for several days prior to a stress challenge improved their physiological stress response. Therefore, this feeding strategy could be applied in commercial fish farms before submitting fish to stressful zootechnical procedures, such as grading, sampling, slaughter, and, consequently, fish welfare would be improved.The objective of this study was to assess the impact of different dietary Trp concentrations on the stress and metabolism response of juvenile Senegalese soles (Solea senegalensis). Fish (38.1 ± 1.9 g) were fed different Trp-enriched feeds (0%, 1% and 2% Trp added) for two and eight days, and later exposed to air stress for three min. Samples were taken pre- and 1 h post-stress (condition). Plasma cortisol, lactate, glucose and proteins were significantly affected by the sampling time, showing higher values at 1 h post-stress. Trp concentration in food also had significant effects on lactate and glucose levels. However, the feeding period did not affect these parameters. Post-stress values were higher than in the pre-stress condition for every plasma parameter, except for lactate in two days and 1% Trp treatment. Nevertheless, cortisol, glucose and lactate did not vary significantly between pre- and post-stress samplings in fish fed the 1% Trp-enriched diet for two days. The lack of variability in cortisol response was also due to the high pre-stress value, significantly superior to pre-stress control. The exposure time to Trp feeding did not significantly affect any enzyme activity; however, Trp added and condition influenced protein-related enzyme activities. In spite of decreasing stress markers, Trp-enriched diets altered the protein metabolism.

Dietary Tryptophan Induces Opposite Health-Related Responses in the Senegalese Sole (Solea senegalensis) Reared at Low or High Stocking Densities With Implications in Disease Resistance.

High rearing densities are typical conditions of both inland and onshore intensive aquaculture units. Despite obvious drawbacks, this strategy is nonetheless used to increase production profits. Such conditions inflict stress on fish, reducing their ability to cope with disease, bringing producers to adopt therapeutic strategies. In an attempt to overcome deleterious effects of chronic stress, Senegalese sole, Solea senegalensis, held at low (LD) or high density (HD) were fed tryptophan-supplemented diets with final tryptophan content at two (TRP2) or four times (TRP4) the requirement level, as well as a control and non-supplemented diet (CTRL) for 38 days. Fish were sampled at the end of the feeding trial for evaluation of their immune status, and mortalities were recorded following intra-peritoneal infection with Photobacterium damselae subsp. piscicida. Blood was collected for analysis of the hematological profile and innate immune parameters in plasma. Pituitary and hypothalamus were sampled for the assessment of neuro-endocrine-related gene expression. During the feeding trial, fish fed TRP4 and held at LD conditions presented higher mortalities, whereas fish kept at HD seemed to benefit from this dietary treatment, as disease resistance increased over that of CTRL-fed fish. In accordance, cortisol level tended to be higher in fish fed both supplemented diets at LD compared to fish fed CTRL, but was lower in fish fed TRP4 than in those fed TRP2 under HD condition. Together with lower mRNA levels of proopiomelanocortin observed with both supplementation levels, these results suggest that higher levels of tryptophan might counteract stress-induced cortisol production, thereby rendering fish better prepared to cope with disease. Data regarding sole immune status showed no clear effects of tryptophan on leucocyte numbers, but TRP4-fed fish displayed inhibited alternative complement activity (ACH50) when held at LD, as opposed to their HD counterparts whose ACH50 was higher than that of CTRL-fed fish. In conclusion, while dietary tryptophan supplementation might have harmful effects in control fish, it might prove to be a promising strategy to overcome chronic stress-induced disease susceptibility in farmed Senegalese sole.

In Silico Detection and FISH Analysis to Determine Location of miRNAs in Solea Senegalensis Chromosomes Using BACs

MicroRNAs (miRNAs) are small non-coding RNAs that play a very important role in gene expression through regulation of mRNA cleavage and translation. The Senegalese sole, Solea senegalensis (Kaup 1858), is a flatfish species that shows great potential for marine aquaculture. Nevertheless, the existence of sexual dysfunction of males reared in captivity, high larval mortality and diseases have hampered its production. The integration of sequence information with data on chromosomal physical location is useful for understanding genetic processes and is essential for comparative analysis, QTL mapping and positional cloning of genes. In this study we employed an in silico approach to identify potential miRNAs in a S. senegalensis BAC library. Mature sequences were obtained from miRBase and other sources and a BLAST analysis was carried out against sequences from a S. senegalensis BAC library. Sequences showing similarity were extracted and subjected to an analysis of their secondary structure. This information was combined with BAC-FISH data to assign miRNAs to chromosomes. In addition, a phylogenetic analysis of some miRNAs families was carried out and target genes were predicted against a reference transcriptome. After secondary structure analysis, 45 groups of sequences showed similarity to mature miRNAs. Of these, 19 were assigned to Senegalese Sole chromosomes using BAC-FISH data and thirty-four unique mature sequences targeted 12387 sequences from a S. senegalensis reference transcriptome. Phylogenetic trees showed different levels of support for clustering among types of miRNAs and S. senegalensis sequences usually were closed to other Pleuronectiformes. These results will be helpful to understand how miRNAs affect gene expression in this species and biological processes of interest for its aquaculture. Nevertheless, experimental validation through, for example, RT-PCR, northern blotting or microarrays is required.

Dietary Fatty Acid Metabolism is Affected More by Lipid Level than Source in Senegalese Sole Juveniles: Interactions for Optimal Dietary Formulation.

This study analyses the effects of dietary lipid level and source on lipid absorption and metabolism in Senegalese sole (Solea senegalensis). Juvenile fish were fed 4 experimental diets containing either 100 % fish oil (FO) or 25 % FO and 75 % vegetable oil (VO; rapeseed, linseed and soybean oils) at two lipid levels (~8 or ~18 %). Effects were assessed on fish performance, body proximate composition and lipid accumulation, activity of hepatic lipogenic and fatty acid oxidative enzymes and, finally, on the expression of genes related to lipid metabolism in liver and intestine, and to intestinal absorption, both pre- and postprandially. Increased dietary lipid level had no major effects on growth and feeding performance (FCR), although fish fed FO had marginally better growth. Nevertheless, diets induced significant changes in lipid accumulation and metabolism. Hepatic lipid deposits were higher in fish fed VO, associated to increased hepatic ATP citrate lyase activity and up-regulated carnitine palmitoyltransferase 1 (cpt1) mRNA levels post-prandially. However, lipid level had a larger effect on gene expression of metabolic (lipogenesis and β-oxidation) genes than lipid source, mostly at fasting. High dietary lipid level down-regulated fatty acid synthase expression in liver and intestine, and increased cpt1 mRNA in liver. Large lipid accumulations were observed in the enterocytes of fish fed high lipid diets. This was possibly a result of a poor capacity to adapt to high dietary lipid level, as most genes involved in intestinal absorption were not regulated in response to the diet.

High dietary lipid level is associated with persistent hyperglycaemia and downregulation of muscle Akt-mTOR pathway in Senegalese sole (Solea senegalensis).

High levels of dietary lipids are incorporated in feeds for most teleost fish to promote growth and reduce nitrogen waste. However, in Senegalese sole (Solea senegalensis) previous studies revealed that increasing the level of dietary lipids above 8% negatively affect growth and nutrient utilization regardless of dietary protein content. It has been shown that glucose regulation and metabolism can be impaired by high dietary fat intake in mammals, but information in teleost fish is scarce. The aim of this study was to assess the possible effect of dietary lipids on glucose metabolism in Senegalese sole with special emphasis on the regulation of proteins involved in the muscle insulin-signalling pathway. Senegalese sole juveniles (29 g) were fed two isonitrogenous diets (53% dry matter) for 88 days. These two diets were one with a high lipid level (∼17%, HL) and a moderate starch content (∼14%, LC), and the other being devoid of fish oil (4% lipid, LL) and with high starch content (∼23%, HC). Surprisingly, feeding Senegalese sole the HL/LC diet resulted in prolonged hyperglycaemia, while fish fed on LL/HC diet restored basal glycaemia 2 h after feeding. The hyperglycaemic phenotype was associated with greater glucose-6-phosphatase activity (a key enzyme of hepatic glucose production) and lower citrate synthase activity in the liver, with significantly higher liver glycogen content. Sole fed on HL/LC diet also had significantly lower hexokinase activity in muscle, although hexokinase activity was low with both dietary treatments. The HL/LC diet was associated with significant reductions in muscle AKT, p70 ribosomal S6-K1 Kinase (S6K-1) and ribosomal protein S6 (S6) 2 h after feeding, suggesting down regulation of the AKT-mTOR nutrient signalling pathway in these fish. The results of this study show for the first time that high level of dietary lipids strongly affects glucose metabolism in Senegalese sole.

Dietary supplementation with vitamin k affects transcriptome and proteome of Senegalese sole, improving larval performance and quality.

Nutritional factors strongly influence fish larval development and skeletogenesis, and may induce skeletal deformities. Vitamin K (VK) has been largely disregarded in aquaculture nutrition, despite its important roles in bone metabolism, in γ-carboxylation of Gla proteins, and in regulating gene expression through the pregnane X receptor (Pxr). Since the mechanisms mediating VK effects over skeletal development are poorly known, we investigated the effects of VK-supplementation on skeletal development in Senegalese sole larvae, aiming to identify molecular pathways involved. Larvae were fed live preys enriched with graded levels of phylloquinone (PK) (0, 50, and 250 mg kg(-1)) and survival rate, growth, VK contents, calcium content and incidence of skeletal deformities were determined, revealing an improvement of larval performance and decreasing the incidence of deformities in VK-supplemented groups. Comparative proteome analysis revealed a number of differentially expressed proteins between Control and Diet 250 associated with key biological processes including skin, muscle, and bone development. Expression analysis showed that genes encoding proteins related to the VK cycle (ggcx, vkor), VK nuclear receptor (pxr), and VK-dependent proteins (VKDPs; oc1 and grp), were differentially expressed. This study highlights the potential benefits of increasing dietary VK levels in larval diets, and brings new insights on the mechanisms mediating the positive effects observed on larval performance and skeletal development.

Use of probiotics intercropped with plant protein diets and their influence on the growth performance and immunological status of Senegalese sole (Solea senegalensis)

Use of probiotics intercropped with plant protein diets and their influence on the growth performance and immunological status of Senegalese sole (Solea senegalensis)