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Most pathogens start the process of infection at the mucosal surfaces and therefore the mucosal immune response plays an essential role in the course of the infection. Due to the Senegalese sole (Solea senegalensis Kaup) condition of flatfish, the present comparative study aimed to analyse several immune-related enzymes as well as the bactericidal activity in the skin mucus from ocular and blind sides. For this purpose, Senegalese sole juveniles were bath challenged with a sub-lethal dose of Tenacibaculum maritimum for 24 h and sampled at 1, 2 and 3 weeks. The haematological profile and immune-related parameters were also measured in plasma in order to evaluate the systemic immune response after T. maritimum challenge. Results from this study showed that most parameters tested increased in skin mucus of bath challenged fish compared to unchallenged ones. In contrast, the sub-lethal dose tested did not influence the haematological profile including peripheral numbers the different leucocyte types. No variations were observed in plasma lysozyme, peroxidase, protease and haemolytic complement activities between unchallenged and bath challenged fish. This study suggests that the studied innate immune-related molecules are constitutively present in both skin mucus sides but at different levels. Interestingly, the levels of most parameters measured were higher on the ocular side than on the blind side, possibly due to the higher exposure to invasion by waterborne microorganisms on this side. Therefore, the present study brings some insights regarding local immune responses after bacterial challenge in skin mucus from the ocular and blind sides in one of the most valuable flatfish species in southern Europe. Image 1 • Systemic and local immune responses of Senegalese sole bath-challenged with T. maritimum were evaluated. • A comparative study was carried out in skin mucus of Senegalese sole from ocular and blind sides. • Immune parameters increased in skin mucus of challenged fish compared to unchallenged ones. • At systemic level no variations were found between unchallenged and challenged groups. • The most parameters measured were higher on the ocular side than on the blind side. [ABSTRACT FROM AUTHOR]

Macroalgae represent valuable sources of functional ingredients for fish diets, and the influence of supplemented aquafeeds on growth performance has been studied for some fish and seaweed species. In the present work, the potential immunomodulation exerted by U. ohnoi (5%) as dietary ingredient was investigated in Senegalese sole. After feeding with the experimental diets for 90 d, fish immune response before and after challenge with Photobacterium damselae subsp. piscicida (Phdp) was assessed. In absence of infection, systemic immune response was not modified by 5% U. ohnoi dietary inclusion for 90 d. Thus, no differences in liver and head kidney immune gene transcription or serum lysozyme, peroxidase, antiprotease and complement activities were observed based on the diet received by Senegalese sole specimens. Regarding mucosal immune parameters, no changes in gene transcription were detected in the skin and gills, whilst only tnf , cd4 and cd8 were significantly up-regulated in the intestine of fish fed with U. ohnoi , compared to the values obtained with control diet. On the contrary, when S. senegalensis specimens were challenged with Phdp , modulation of the immune response consisting in increased transcription of genes encoding complement (c1q4, c3, c9), lysozyme g (lysg), tumor necrosis factor alpha (tnfα) as well as those involved in the antioxidant response (gpx, sodmn) and iron metabolism (ferrm, hamp- 1) was observed in the liver of fish fed with U. ohnoi. In parallel, decreased inflammatory cytokine and complement encoding gene transcription was displayed by the spleen of fish receiving the algal diet. Though mortality rates due to Phdp challenge were not affected by the diet received, lower pathogen loads were detected in the liver of soles receiving U. ohnoi diet. Further research to investigate the effects of higher inclusion levels of this seaweed in fish diets, feeding during short periods as wells as to assess the response against other pathogens needs to be carried out. • Solea senegalensis was fed low inclusion level Ulva ohnoi (5%) for 90 days. • Dietary U. ohnoi modulates S. senegalensis immune response during Phdp infection. • Lower presence of Phdp is detected in the liver of S. senegalensis fed U. ohnoi. [ABSTRACT FROM AUTHOR]

Sulfated polysaccharides derived from green seaweeds exhibit many beneficial biological activities and have great potential to be used as nutraceutical in aquaculture. In this work, we evaluated the effects of the sulfated polysaccharide ulvan from Ulva ohnoi on Senegalese sole (Solea senegalensis) juveniles at the transcriptomic level. Cytotoxicity assay performed in liver primary cell cultures from sole determined that the different ulvan concentrations assayed did not impair cell viability. Juveniles were intraperitoneally (IP) injected with ulvan (0.5 mg/fish) followed by a challenge with Photobacterium damselae subsp. piscicida (Phdp) at 7 days. RNASeq analyses at 2 days post injection (dpi) revealed that 402 transcripts were differentially expressed in liver between ulvan IP injected and control groups before the challenge. Genes related to bacterial and antiviral defence, complement system, chemokines, proteasomes and antigen presentation were upregulated in ulvan treated groups. A detailed expression analysis of sixteen genes related to innate and adaptive immune system was performed in two systemic tissues: liver and spleen. Ulvan injection provoked the upregulation of tlr22 and a transient inflammatory response was initiated in both liver and spleen at 2 dpi. As consequence, expression of acute phase proteins, antimicrobial peptides and complement genes was induced. Moreover, expression of mhcI , mhcII , psmb10 and bcl6 was also induced 2 dpi. At 2 dpi with Phdp , inflammatory cytokines and genes related to bacterial and antiviral defense, iron metabolism, complement system and antigen presentation were differentially modulated in survival juveniles previously IP injected with ulvan. Moreover, mortality was retarded in ulvan treated juveniles. These results provide new evidence about the role of ulvan as a bioactive compound with immunomodulatory activity in Senegalese sole as well as its possible use as vaccine adjuvant against Phdp. This is the first published study that evaluates the transcriptomic response of Senegalese sole IP injected with ulvan. • The sulfated polysaccharide ulvan (Ulva ohnoi) is non cytotoxic in S. senegalensis. • Ulvan activates the acute phase response of the immune system after IP injection. • Ulvan modulates the immune response after challenge with Phdp. • Ulvan is proposed as potential candidate as nutraceutical and/or vaccine adjuvant. [ABSTRACT FROM AUTHOR]

Nervous necrosis virus (NNV) reassortant strains RGNNV/SJNNV have emerged as a potent threat to the Mediterranean marine aquaculture industry, causing viral encephalopathy and retinopathy (VER) in Senegalese sole (Solea senegalensis). In this study, a cheap and practical vaccine strategy using bacterial inclusion bodies made of the coat protein of a virulent reassortant strain of this betanodavirus was devised. The nanostructured recombinant protein nanoparticles, VNNV-CNP, were administered without adjuvant to two groups of juvenile sole, one by intraperitoneal injection and the other by oral intubation. Specific antibodies were raised in vivo against the NNV coat protein via both routes, with a substantial specific antibody expansion in the injected group 30 days post homologous prime boost. Expression levels of five adaptive immune-related genes, cd8a, cd4, igm, igt and arg2, were also quantified in intestine, spleen and head kidney. Results showed cd4 and igm were upregulated in the head kidney of injected fish, indicating activation of an adaptive systemic response, while intubated fish exhibited a mucosal response in the intestine. Neither route showed significant differential expression of cd8a. The specific antibody response elicited in vivo and the lack of any signs of toxicity over the 6-week study period in young fish (n = 100), evidences the potential of the nanoparticle as a vaccine candidate. • We test a nanoparticle vaccine made of nodavirus RGNNV/SJNNV coat protein in sole. • Oral and i.p. delivery routes were compared via antibody and gene expression data. • Injection without adjuvant triggered a strong systemic specific antibody response. • Oral intubation evoked mainly a mucosal immune response. • Nanoparticle VNNV-CNP is immunogenic in S. senegalensis juveniles. [ABSTRACT FROM AUTHOR]

Mercury (Hg) is globally recognized as a persistent chemical contaminant that accumulates in marine biota, thus constituting an ecological hazard, as well as a health risk to seafood consumers. Climate change-related stressors may influence the bioaccumulation, detoxification, and toxicity of chemical contaminants, such as Hg. Yet, the potential interactions between environmental stressors and contaminants, as well as their impacts on marine organisms and seafood safety, are still unclear. Hence, the aim of this work was to assess the bioaccumulation of Hg and neuro-oxidative responses on the commercial flat fish species Solea senegalensis (muscle, liver, and brain) co-exposed to dietary Hg in its most toxic form (i.e., MeHg), seawater warming (ΔT°C = +4 °C), and acidification (pCO2 = +1000 µatm, equivalent to ΔpH = −0.4 units). In general, fish liver exhibited the highest Hg concentration, followed by brain and muscle. Warming enhanced Hg bioaccumulation, whereas acidification decreased this element's levels. Neuro-oxidative responses to stressors were affected by both climate change-related stressors and Hg dietary exposure. Hazard quotient (HQ) estimations evidenced that human exposure to Hg through the consumption of fish species may be aggravated in tomorrow's ocean, thus raising concerns from the seafood safety perspective. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Most teleosts undergo a thyroid hormone (TH) regulated larval to juvenile transition known as metamorphosis. In Pleuronectiformes (flatfish), metamorphosis is most dramatic, and one eye of the symmetric pelagic larvae migrates to the opposite side of the head, giving rise to an asymmetric benthic juvenile with both eyes on the same side of the body. Asymmetric development occurs mostly in the head. To understand the genetic mechanisms underlying this developmental change we have generated a Solea senegalensis metamorphosing flatfish head transcriptome. Our results reveal that THs acting as integrative factors direct a stepwise genetic program that initiates a specific organismal level response followed by cell specific responses that lead to the long-term changes that characterise the post-metamorphic identity and physiology of the head. Flatfish head asymmetric development during metamorphosis and its TH dependency is conserved thus we consider the findings in sole most likely representative of all flatfish species. • 440 genes regulated by THs during sole head metamorphosis bring about complex post-natal development of the head • Differentially expressed genes during sole head metamorphosis present six possible different patterns • Sole head metamorphic transcriptome reveals a dynamic hierarchical gene regulatory network [ABSTRACT FROM AUTHOR]

Immunonutrition is a promising and viable strategy for the development of prophylactic measures in aquaculture. Ulvan, a sulphated marine polysaccharide from green seaweeds, has many biological activities including the immunomodulatory ones. The aim of this study was to assess the short and long-term effects of an ulvan-rich extract obtained from U. ohnoi as immunonutrient in Senegalese sole juveniles. In this work, an ulvan-rich extract from Ulva ohnoi has been obtained by the hot water method and isolated by ethanol precipitation. The FTIR analysis revealed that the ulvan-rich extact had very similar characteristics to previously published ulvan spectra. The total sulfate and protein content was 24.85 ± 3.98 and 0.91 ± 0.04 %, respectively. In vitro assays performed in Senegalese sole (Solea senegalensis) macrophages showed that the ulvan obtained in this study did not compromise the cell viability at concentrations up to 1 mg ml−1 and expression levels of lyg , irf1 , il6 , il10 , c7 , tf and txn were significantly upregulated in a concentration dependent-manner. Finally, S. senegalensis juveniles were fed basal diets and diets supplemented with the ulvan-rich extract at ratios 1 and 2 % for 30 days and then, challenged with Photobacterium damselae subsp. piscicida (Phdp). Thereafter, ulvan was withdrawn from the diet and all juveniles were fed the basal diet for 30 days. At 30 days post withdrawal (dpw), juveniles were challenged with Phdp. The expression profiles of a set of genes related to the immune system in spleen were evaluated as well as the lysozyme, peroxidase and bactericidal activity in plasma. Dietary effects of 1 % ulvan resulted in a boost of the immune response and increased disease resistance at short-term whereas juveniles fed diets supplemented with 2 % ulvan showed a significant decrease in the bactericidal activity and lack of protection against Phdp. At long-term (30 days after the withdrawal of ulvan), an improved response was observed in juveniles previously fed 1 % ulvan. • The ulvan-rich extract obtained shows suitable features to be use as immunonutrient. • The ulvan-rich extract shows immunomodulatory effects in a concentration dependent-manner. • Dietary administration of 1 % ulvan enhance immune response and disease resistance in Solea senegalensis juveniles. • Dietary administration of 2 % ulvan resulted in lack of protection against Phdp. [ABSTRACT FROM AUTHOR]

Understanding vertebral bone development is essential to prevent skeletal malformations in farmed fish related to genetic and environmental factors. This is an important issue in Solea senegalensis , with special impact of spinal anomalies in postlarval and juvenile stages. Vertebral bone transcriptomics in farmed fish mainly comes from coding genes, and barely on miRNA expression. Here, we used RNA-seq of spinal samples to obtain the first comprehensive coding and miRNA transcriptomic repertoire for postlarval and juvenile vertebral bone, covering different vertebral phenotypes and egg-incubation temperatures related to skeleton health in S. senegalensis. Coding genes, miRNA and pathways regulating bone development and growth were identified. Differential transcriptomic profiles and suggestive mRNA-miRNA interactions were found between postlarvae and juveniles. Bone-related genes and functions were associated with the extracellular matrix, development and regulatory processes, calcium binding, retinol and lipid metabolism or response to stimulus, including those revealed by the miRNA targets related to signaling, cellular and metabolic processes, growth, cell proliferation and biological adhesion. Pathway enrichment associated with fish skeleton were identified when comparing postlarvae and juveniles: growth and bone development functions in postlarvae, while actin cytoskeleton, focal adhesion and proteasome related to bone remodeling in juveniles. The transcriptome data disclosed candidate coding and miRNA gene markers related to bone cell processes, references for functional studies of the anosteocytic bone of S. senegalensis. This study establishes a broad transcriptomic foundation to study healthy and anomalous spines under early thermal conditions across life-stages in S. senegalensis , and for comparative analysis of skeleton homeostasis and pathology in fish and vertebrates. • Novel mRNA- and miRNA-sequencing was obtained from Senegalese sole vertebrae. • Postlarval and juvenile transcriptomes under early thermal conditions were profiled. • A transcriptome baseline was set up for studying spinal anomalies in Senegalese sole. • New skeleton transcriptome data useful for comparative studies in fish and vertebrates. [ABSTRACT FROM AUTHOR]

Viral encephalopathy and retinopathy (VER), caused by nervous necrosis virus (NNV), is one of the most threatening diseases affecting marine farmed fish, especially in early developmental stages. In addition, subclinical infections are commonly detected in both farmed and wild fish. In recent years numerous efforts have been made to achieve effective vaccines for the protection of different fish species against NNV infection. Vaccination experiments are always conducted on pathogen-free animals, but subclinically infected individuals may respond differently to immunization. In this study, we have observed a different response between experimentally-induced-NNV carrier Senegalese sole and NNV-free animals when immunized with a BEI-inactivated vaccine, whereas mock-vaccinated carrier fish behaved like the NNV-free group. Analysis of the immune response after vaccination showed specific antibody production as well as up-regulation of the genes coding for the proinflammatory cytokines IL-6 and IL-8, the antiviral protein HERC4 and the cytotoxic T lymphocyte (CTL) and T helper markers CD8 and CD4 in vaccinated NNV-free and mock-vaccinated carrier individuals. However, no significant antibody production was detected and only cd4 up-regulation was recorded in the vaccinated carrier fish. After challenge, viral replication was similar in the vaccinated groups up to 15 days post-challenge (dpc), although thereafter the viral load in the vaccinated carrier fish reached values similar to the control fish, whereas in the vaccinated NNV-free sole remained 1.5–2 log lower. At 2 dpc 10% mortality was recorded in the vaccinated carrier group that did not correlate with a higher viral titre, but with an overexpression of inflammation-related genes. These results suggest that an acute inflammatory reaction could have been the cause of the fish death. Excepting the values recorded in this episode, poor modulation of the immune response was observed at the early days post-challenge. However, at 30 dpc the IgM levels in the vaccinated carrier fish were significantly higher than those of the NNV-free sole. Longer post-vaccination studies would be necessary to clarify if this antibody increase may be relevant for fish protection. • NNV inactivated vaccine performs differently in NNV-free and NNV-carrier sole. • Subclinical infection confers limited protection to sole against NNV challenge. • Acute phase reaction may have caused post-challenge mortality in vaccinated carriers. [ABSTRACT FROM AUTHOR]

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.

Fish hatcheries must adapt larval feeding protocols to feeding behavior and metabolism patterns to obtain more efficient feed utilization. Fish larvae exhibit daily ingesting rhythms rather than ingesting food continuously throughout the day. The aim of this study was to determine the daily patterns of feed intake, protein digestibility, protein retention and catabolism in Senegalese sole post-larvae (Solea senegalensis; 33 days post-hatching) using 14C-labeled Artemia protein and incubation in metabolic chambers. Sole post-larvae were fed at 09:00, 15:00, 21:00, 03:00 and 09:00+1 day; and those fed at 09:00, 21:00, 03:00 and 09:00+1 day showed significantly higher feed intake than post-larvae fed at 15:00 h (P=0.000). Digestibility and evacuation rate of ingested protein did not change during the whole cycle (P=0.114); however, post-larvae fed at 21:00 and 03:00 h showed the significantly highest protein retention efficiency and lowest catabolism (P=0.002). Therefore, results confirm the existence of daily rhythmicity in feeding activity and in the utilization of the ingested nutrients in Senegalese sole post-larvae.

Global aquaculture production continues to increase rapidly. One of the most important species of marine fish currently cultivated in Southern Europe is Solea senegalensis, reaching more than 300 Tn in 2017. In the present work, 14 Bacterial Artificial Chromosome (BAC) clones containing candidate genes involved in the immune system (b2m, il10, tlr3, tap1, tnfα, tlr8, trim25, lysg, irf5, hmgb2, calr, trim16, and mx), were examined and compared with other species using multicolor Fluorescence in situ Hybridization (mFISH), massive sequencing and bioinformatic analysis to determine the genomic surroundings and syntenic chromosomal conservation of the genomic region contained in each BAC clone. The mFISH showed that the groups of genes hmgb2-trim25-irf5-b2m; tlr3-lysg; tnfα-tap1, and il10-mx-trim16 were co-localized on the same chromosomes. Synteny results suggested that the studied BACs are placed in a smaller number of chromosomes in S. senegalensis that in other species. Phylogenetic analyses suggested that the evolutionary rate of immune system genes studied is similar among the taxa studied, given that the clustering obtained was in accordance with the accepted phylogenetic relationships among these species. This study contributes to a better understanding of the structure and function of the immune system of the Senegalese sole, which is essential for the development of new technologies and products to improve fish health and productivity.

Gastrointestinal (GI) microbiota has a relevant role in animal nutrition, modulation of the immune system and protection against pathogen invasion. Interest in algae as source of nutrients and functional ingredients for aquafeeds is increasing in order to substitute conventional feedstuffs by more sustainable resources. The diet is an important factor in the modulation of the microbiota composition, and functional ingredients have been proposed to shape the microbiota and contribute benefits to the host. However, fish microbiome research is still limited compared to other hosts. Solea senegalensis is a flat fish with high potential for aquaculture in South Europe. In this study, a characterization of the microbiome of S. senegalensis (GI) tract and the effects of feeding Ulva ohnoi supplemented diet has been carried out. Differences in the composition of the microbiota of anterior and posterior sections of S. senegalensis GI tract have been observed, Pseudomonas being more abundant in the anterior sections and Mycoplasmataceae the dominant taxa in the posterior GI tract sections. In addition, modulation of the GI microbiota of juvenile Senegalese sole fed for 45 days a diet containing low percentage of U. ohnoi has been observed in the present study. Microbiota of the anterior regions of the intestinal tract was mainly modulated, with higher abundance of Vibrio spp. in the GI tract of fish fed dietary U. ohnoi.

Concerns about safety, applicability and functionality associated with live probiotic cells have led to consideration of the use of non-viable microorganisms, known as paraprobiotics. The present study evaluated the effects of dietary administration of heat-inactivated cells of the probiotic strain Shewanella putrefaciens Pdp11 on the intestinal microbiota and immune gene transcription in Solea senegalensis. Results obtained were evaluated and compared to those described after feeding with viable Pdp11 cells. S. senegalensis specimens were fed with basal (control) diet or supplemented with live or heat inactivated (60 °C, 1 h) probiotics diets for 45 days. Growth improvement was observed in the group receiving live probiotics compared to the control group, but not after feeding with a probiotic heat-inactivated diet. Regarding immune gene transcription, no changes were observed for tnfα, il-6, lys-c1, c7, hsp70, and hsp90aa in the intestinal samples based on the diet. On the contrary, hsp90ab, gp96, cd4, cd8, il-1β, and c3 transcription were modulated after probiotic supplementation, though no differences between viable and heat-inactivated probiotic supplemented diets were observed. Modulation of intestinal microbiota showed remarkable differences based on the viability of the probiotics. Thus, higher diversity in fish fed with live probiotic cells, jointly with increased Mycoplasmataceae and Spirochaetaceae to the detriment of Brevinemataceae, was detected. However, microbiota of fish receiving heat-inactivated probiotic cells showed decreased Mycoplasmataceae and increased Brevinemataceae and Vibrio genus abundance. In short, the results obtained indicate that the viable state of Pdp11 probiotic cells affects growth performance and modulation of S. senegalensis intestinal microbiota. On the contrary, minor changes were detected in the intestinal immune response, being similar for fish receiving both, viable and inactivated probiotic cell supplemented diets, when compared to the control diet.

Fish vary in their ability to biosynthesise long-chain polyunsaturated fatty acids (LC-PUFA) depending upon the complement and function of key enzymes commonly known as fatty acyl desaturases and elongases. It has been reported in Solea senegalensis the existence of a Δ4 desaturase, enabling the biosynthesis of docosahexaenoic acid (DHA) from eicosapentaenoic acid (EPA), which can be modulated by the diet. The present study aims to evaluate the combined effects of the partial replacement of fish oil (FO) with vegetable oils and reduced environmental salinity in the fatty acid composition of relevant body compartments (muscle, hepatocytes and enterocytes), the enzymatic activity over α-linolenic acid (ALA) to form n-3 LC-PUFA through the incubation of isolated hepatocytes and enterocytes with [1-14C] 18:3 n-3, and the regulation of the S. senegalensis fads2 and elovl5 in the liver and intestine. The presence of radiolabelled products, including 18:4n-3, 20:4n-3 and EPA, provided compelling evidence that a complete pathway enabling the biosynthesis of EPA from ALA, establishing S. senegalensis, has at least one Fads2 with ∆6 activity. Dietary composition prevailed over salinity in regulating the expression of fads2, while salinity did so over dietary composition for elovl5. FO replacement enhanced the proportion of DHA in S. senegalensis muscle and the combination with 20 ppt salinity increased the amount of n-3 LC-PUFA in hepatocytes.

The hatching enzymes or choriolysins are key proteases in fish life cycle controlling the release of larvae to surrounding environment that have been suggested as target for novel biotechnological uses. Due to the large amounts of eggs released by the flatfish Solea senegalensis, during the spawning season, the hatching liquid properties and choriolysin-encoding genes were investigated in this species. A genomic analysis identified four putative genes referred to as SseHCEa, SseHCEb, SseLCE and SseHE. The phylogenetic analysis classified these paralogs into two clades, the clade I containing SseHCE paralogs and the clade II containing two well-supported subclades named as HE and LCE. The two SseHCE paralogs were intron-less and both genes were tandemly arrayed very close in the genome. The synteny and gene rearrangement identified in the flatfish lineage indicated that the duplication of these two paralogs occurred recently and they are under divergent evolution. The genes SseHE and SseLCE were structured in 8 exons and 7 introns and the synteny was conserved in teleosts. Expression studies confirmed that the four genes were expressed in the hatching gland cells and they migrate co-ordinately from the head to around the yolk sac close to the hatch with specific temporal and intensity expression profiles. Although the mRNA levels of the four genes peaked in the hours previous to larval hatching, the SseHCE and SseLCE paralogs kept a longer expression than SseHE after hatching. These expression patterns were consistent even when larvae were incubated at different temperatures that modified hatching times. The analysis of hatching-liquid using SDS-PAGE and zymography analyses of hatching liquid identified a major band of expected choriolysin size. The optimal pH for protease activity was 8.5 and inhibition assays using EDTA demonstrated that most of the activity in the hatching liquid was due to metalloproteases with Ca2+ ions acting as the most effective metal to restore the activity. All these data provide new clues about the choriolysin evolution and function in flatfish with impact in the aquaculture and the blue cosmetic industry. [ABSTRACT FROM AUTHOR]

Harmful effects of triclosan (TCS) have been reported on several organisms; however, effects on early life stages of marine vertebrates are limited. Therefore, the objective of this work was to assess the effects of TCS during early development of the flatfish Solea senegalensis after initial characterization of cholinesterases (ChEs) and determination of selected biochemical markers baseline levels. Characterization of ChEs and determination of biochemical markers baseline levels of cholinergic activity, energy metabolism and oxidative stress were analysed in sole at 3 days after hatching (dah) and at the onset and end of metamorphosis. To assess TCS effects, fish were exposed during 96h to 30-500 μg L−1 TCS until 3 dah. Fish at 13 dah were exposed during 48h to 200-1,500 μg L−1 TCS and maintained until complete metamorphosis. Effects on survival, malformations, length, metamorphosis progression and biochemical markers were evaluated. The main ChE active form present in sole early life stages is acetylcholinesterase and baseline levels of oxidative stress and energy metabolism biomarkers changed according to fish developmental stage. Triclosan induced malformations (EC 50 = 180 μg L−1 at 3 dah), decreased growth (95 μg L−1 at 3 dah; 548 μg L−1 at 24 dah) and affected metamorphosis progression (391 μg L−1 at 17 dah). Impairment of antioxidant system was observed, with TCS affecting catalase at the end of metamorphosis test, however, no oxidative damage on lipids was detected. Glutathione S-transferase was the most sensitive endpoint during early larval test (LOEC = 30 μg L−1). Exposure to TCS affected S. senegalensis at individual and sub-individual levels, both at early larval stage and during the critical period of metamorphosis. Image 1 • Triclosan induced malformations and decreased growth on sole early life stages. • Triclosan accelerated sole metamorphosis at transient stages. • Biochemical enzymatic markers were the most sensitive endpoints to triclosan exposure. • Triclosan induced alterations on antioxidant system without causing oxidative damage. [ABSTRACT FROM AUTHOR]

Photobacteriosis caused by Photobacterium damselae subsp. piscicida (Pdp) remains one of the main infectious diseases affecting cultured fish in Mediterranean countries. Diverse vaccine formulations based in the use of inactivated bacterial cells have been used with unsatisfactory results, especially in newly cultured species like sole (Solea senegalensis). In this work, we describe the use of the outer membrane receptor (FrpA) of the siderophore piscibactin produced by Pdp as a novel subunit vaccine against photobacteriosis. FrpA has been cloned and expressed in Escherichia coli under an arabinose-inducible promoter. A recombinant protein (rFrpA) containing the pelB localization signal and a His tag was constructed to obtain a pure native form of the protein from E. coli outer membranes. The immunogenicity of rFrpA, and its protective effect against photobacteriosis, was tested by i.p. injection of 30 μg of the protein, mixed with Freund's adjuvant, in sole fingerlings with two immunizations separated by 30 days. Results showed that using either pure rFrpA or whole cells as immobilized antigens in ELISA assays, rFrpA induces the production of specific antibodies in sole. An experimental infection using fish vaccinated with rFrpA or formalin-killed whole cells of Pdp showed that both groups were protected against Pdp infection at similar levels, with no significant differences, reaching RPS values of 73% and 79%, respectively. Thus, FrpA constitutes a promising antigen candidate for the development of novel more effective vaccines against fish photobacteriosis. • The siderophore piscibactin of P. damselae subsp piscicida is transported into the cell by the outer membrane protein FrpA. • FrpA was cloned and expressed in E. coli under an arabinose-inducible promoter. • Native FrpA was obtained from the outer membranes of E. coli by addition of a pelB sequence and a His tag to frpA sequence. • FrpA was immunogenic in fish since it induced specific antibodies when it was injected in sole fingerlings. • Fish immunized with FrpA achieved a 73% (RPS) protection against experimental infections with P. damselae subsp piscicida. [ABSTRACT FROM AUTHOR]

Coastal marine areas are highly vulnerable to the exposure to various types of stressors and impact of chemical pollution resulting from increasing anthropogenic activities, namely pollution by metals and polycyclic aromatic hydrocarbons (PAHs). To assess ecosystem quality and functions, biomarkers can provide information about the presence and adverse effects of pollutants. Accordingly, the present study was conducted to evaluate the chronic (28 days) biologic effects of putatively contaminated sediments from the Zarzis area, located in the south of the Gulf of Gabes on the Southern Tunisian coast, on the marine flatfish Solea senegalensis. Sediments were collected at three sampling sites, impacted by wastewater discharges, aquaculture activities, and industrial contamination, and then surveyed for metals (Cd, Cu, Cr, Hg, Zn, and Pb) and organic contaminants (polycyclic aromatic hydrocarbons). The quantified biomarkers involved the determination of oxidative stress, phase II metabolism, and the extent of lipid peroxidation (catalase, CAT; glutathione peroxidase activity: total and selenium-dependent, T-GPx and Se-GPx; activities of glutathione-S-transferases, GSTs; levels of lipid peroxidation, by means of the thiobarbituric acid reactive substances assay, TBARS) and neurotoxicity (activity of acetylcholinesterase, AChE). S. senegalensis exposed to potentially contaminated sediments, collected near the aquaculture facility, presented the highest values for the generality of biomarkers tested, and a significant inhibition of AChE activity. A few lesions have been also recorded in the gills and liver tissues of S. senegalensis following chronic exposure. However, the observed lesions in gills (e.g., epithelial lifting, lamellar fusion, gills hyperplasia and hypertrophy, and leukocyte infiltration) and liver (cytoplasmic vacuolation, enlargement of sinusoids, foci of necrosis, and eosinophilic bodies) were of minimal pathological importance and/or low prevalence that did not significantly affect the weighted histopathological indices. Finally, the biological responses evidenced by this flatfish can be potentially caused by metal and PAH pollution occurring in specific areas in the southeast of Tunisia. The type and extent of the observed biochemical alterations strongly suggest that the contaminated sediments from the surveyed areas could cause early adverse biological effects on exposed biota. [ABSTRACT FROM AUTHOR]

One bottleneck to sustainability of fish aquaculture is the control of infectious diseases. Current trends include the preventive application of immunostimulants and prebiotics such as polysaccharides. The present study investigated how yeast β-glucan (Y), microalgal polysaccharide-enriched extracts (MAe) and whole Phaeodactylum tricornutum cells (MA) modulated the gut microbiome and stimulated the immune system in Senegalese sole (Solea senegalensis) when administered by oral intubation. Blood, intestine and spleen samples were taken at 3 h, 24 h, 48 h and 7 days after treatment. The short-term response (within 48 h after treatment) consisted of up-regulation of il1b and irf7 expression in the gut of the Y treated group. In contrast, administration of MAe decreased expression of tnfa and the chemokine cxc10 in the gut and spleen. Both treatments down-regulated the expression of irf3 with respect to the control group. Lysozyme activity in plasma decreased at 48 h only in the MAe-treated soles. Medium-term response consisted of the up-regulation of clec and irf7 expression in the gut of the Y, MAe and MA groups and of il1b mRNAs in the spleen of the MA group compared to the control group. Microbiome analysis using 16S rDNA gene sequencing indicated that the intestine microbiome was dominated by bacteria of the Vibrio genus (>95%). All the treatments decreased the relative proportion of Vibrio in the microbiome and Y and MAe decreased and MA increased diversity. Quantitative PCR confirmed the load of bacteria of the Vibrio genus was significantly decreased and this was most pronounced in Y treated fish. These data indicate that orally administrated insoluble yeast β-glucans acted locally in the gut modulating the immune response and controlling the Vibrio abundance. In contrast, the MAe slightly reduced the Vibrio load in the intestine and caused a transient systemic anti-inflammatory response. The results indicate that these polysaccharides are a promising source of prebiotics for the sole aquaculture industry. • Insoluble yeast β-glucans increased il1b mRNAsand reduced Vibrio loads in the gut indicating a major local action. • Microalgal extracts triggered a systemic response with a decrease of innate immune defenses and reduction of Vibrio loads. • Whole-cell microalgae increased the diversity of gut microbiota. [ABSTRACT FROM AUTHOR]

Global aquaculture production continues to increase rapidly. One of the most important species of marine fish currently cultivated in Southern Europe is Solea senegalensis , reaching more than 300 Tn in 2017. In the present work, 14 Bacterial Artificial Chromosome (BAC) clones containing candidate genes involved in the immune system (b2m , il10 , tlr3 , tap1 , tnf α, tlr8 , trim25 , lysg , irf5 , hmgb2 , calr , trim16 , and mx), were examined and compared with other species using multicolor Fluorescence in situ Hybridization (mFISH), massive sequencing and bioinformatic analysis to determine the genomic surroundings and syntenic chromosomal conservation of the genomic region contained in each BAC clone. The mFISH showed that the groups of genes hmgb2-trim25-irf5-b2m ; tlr3-lysg ; tnfα-tap1 , and il10-mx-trim16 were co-localized on the same chromosomes. Synteny results suggested that the studied BACs are placed in a smaller number of chromosomes in S. senegalensis that in other species. Phylogenetic analyses suggested that the evolutionary rate of immune system genes studied is similar among the taxa studied, given that the clustering obtained was in accordance with the accepted phylogenetic relationships among these species. This study contributes to a better understanding of the structure and function of the immune system of the Senegalese sole, which is essential for the development of new technologies and products to improve fish health and productivity. [ABSTRACT FROM AUTHOR]

Senegalese sole (Solea senegalensis) is a species with a high commercial value that exhibits a reproductive dysfunction in males born and raised in captivity (F1) that hinders their sustainable culture. The present study evaluates the sperm quality and dopaminergic pathway of males born in the wild environment and of F1 males. Traditional sperm analyses were performed, finding only significant differences in curvilinear velocity (VCL) and no significant differences in viability and total motility. No differences in global sperm methylation were observed either in spermatozoa or brain between the two groups (F1 and wild-born males). However, our results point to a different sperm molecular signature between wild fish and fish born in captivity, specifically the differential expression in miR-let7-d and miR-200a-5p between these two groups. miR-let7-d has been correlated with spermatogenesis and sex preferences, whereas the miR-200 family is implied in target innervation of dopaminergic neurons in zebrafish. When we analysed the dopaminergic pathway, no differences were found in terms of different mRNA expression of dopaminergic markers. However, some differences were detected in terms of tyrosine hydroxylase protein expression by western blot analysis, thus suggesting an altered post-transcriptional regulation in F1 males. The results of this study suggest that an altered sperm miRNA signature in F1 males could be one possible mode of transmission of reproductive dysfunction to the progeny. Solea senegalensis males born in captivity exhibit a reproductive dysfunction that hinders their sustainable culture. The present study detected lower tyrosine hydroxylase protein expression in the brain and an altered sperm miRNA signature in these males. This molecular alteration in germ cells may play a role in the transmission of reproductive dysfunction to the progeny. [ABSTRACT FROM AUTHOR]

Abstract The progressive growth of aquaculture implicates a dependence on large water amounts, which are submitted to disinfection processes, namely ozonation. Considering the importance of genomic integrity, it is critical to improve the knowledge on ozone-related genotoxic hazard to organisms reared in recirculating aquaculture systems (RAS) applying ozonation. Therefore, genetic damage induced by ozone exposure in the Senegalese sole (Solea senegalensis) was assessed, combining the comet and the erythrocytic nuclear abnormalities (ENA) assays, reflecting different damage levels, i.e. DNA and chromosomal damage, respectively. Fish were subjected to a daily 6-h ozone (0.15 mg L−1) exposure, repeated for 3 consecutive days, simulating a short-term event of overozonation. To assess the temporal impact of the previous event, the progression of damage was evaluated 7 days later, following transference to ozone-free water or to 0.07 mg L−1 ozone, a routinely adopted level in RAS. Both endpoints pointed to the ozone genotoxic potential, displaying DNA oxidation as a possible mechanism of damage. Overall, the present findings pointed out the genotoxic hazard of ozone to fish, highlighting the importance of these types of studies and contributing to improve aquaculture practices, namely in RAS systems. These early genotoxic signals may be a prelude to negative repercussions on fish health, which may affect the aquaculture productivity. The present findings recommend precautions in relation to accidental or intentional overozonation in fish-farming, even when short-term events are considered. The strategies to mitigate the impact of ozonation in S. senegalensis may include a dietary extra supplementation of antioxidants (regularly, or punctually in cases of overozonation). Graphical abstract Unlabelled Image Highlights • Knowledge gap concerning the ozone-related genotoxicity to organisms reared in recirculating aquaculture systems (RAS); • Early genotoxic signals may have negative effects on fish health, which may affect the aquaculture productivity; • Ozone-induced genotoxicity in Solea senegalensis, assessed by using the comet and erythrocytic nuclear abnormalities assays; • Both endpoints point the ozone genotoxic potential, displaying DNA oxidation as a possible mechanism of damage; • Present findings may contribute to improve aquaculture practices, namely in RAS systems. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract Tenacibaculosis, caused by Tenacibaculum maritimum , continues to inflict substantial losses among cultured marine species, particularly in the Senegalese sole. However, the immune mechanisms in fish involved in fighting against this disease are still poorly understood. Thus, the present study aimed to investigate the skin mucus's terminal carbohydrate composition, several immune-related enzymes (i.e. lysozyme, peroxidase, proteases and antiproteases), the haemolytic activity of complement and the bactericidal activity in the skin mucus and plasma of the Senegalese sole in a time-course study following a bath challenge with T. maritimum. The haematological profile and the kinetics of cell migration post-infection were also considered. The bath challenge induced slight variations in the terminal carbohydrate composition of Senegalese sole skin mucus. In general, results from this study showed a delay in the mucosal immune response compared to that found at the systemic level (i.e. blood and plasma). For instance, a significant increase in the skin mucus's lysozyme, complement, protease and antiprotease activities were observed at the end of the experiment (14 d post-challenge). Interestingly, the higher activity of these enzymes could be related to the skin mucus's bactericidal capacity and haemolytic complement activity, suggesting that these enzymes play an important role in the defence against Gram-negative bacteria. The haematological profile revealed a significant increase in circulating neutrophils in challenged fish after 48 and 72 h, which was positively correlated to the increments observed in peroxidase and lysozyme activities, respectively, in the plasma of challenged fish at the same time. Although the route of entry and the survival strategy of T. maritimum are still not fully elucidated, results from the present study will contribute to this endeavour through the study of the mucosal immune responses of fish against this particular pathogen. Graphical abstract Image 1 Highlights • Fish immune mechanisms involved in tenacibaculosis are still poorly understood. • T. maritimum bath challenge not revealed great variations in the carbohydrates profile in fish skin mucus. • A delay in the mucosal immune response was observed compared to that found at systemic level (plasma). • Skin mucus had a stronger bactericidal activity than that from plasma against bacterial species tested. • A general increase was observed in all immune parameters evaluated in skin mucus at the end of the trial. [ABSTRACT FROM AUTHOR]

Abstract The pathogenic bacteria Photobacterium damselae subsp. piscicida affects the development of Solea senegalensis culture. Vaccines made with inactivated cells have produced a relative protection against the sickness, however the administration of subcellular and purified antigens as vaccine could increase the effectiveness of the immune response. Thus, the aim of this work was the determination of antigens of P. damselae subsp. piscicida involved in the specific immune response of S. senegalensis. Fish were immunized by intraperitoneal injection (i.p.) with inactivated extracellular polymeric substances (ECP) and whole cells of P. damselae subsp. piscicida , and Freund's incomplete adjuvant. Two months later fish were boosted with the same antigens. Serum from fish was collected to determine by ELISA the title of antibodies against subcellular fractions of bacteria (ECP, capsule, outer membrane proteins, O antigen and formalized whole cells). Significant differences were found between control and immunized fish, but differences between first immunization and booster were only found for O antigen and capsule. Western blots derived from 2D-PAGE of ECP and Outer Membrane Proteins (OMP), using sole immunized serum, detected two high reactive antigens from ECP. Proteins were identified, by mass spectrometry, as ATP-dependent metalloprotease and Telurite resistance proteins. In the case of OMP, three antigenic proteins were detected and identified as Nrfa Y218f, Anti-oxidant AhpC/TSA, and a protein domain DNA binding heat shock related. Highlights • Sole specimens were immunized with inactivated cells of P. damselae subsp. piscicida and ECP to obtain antiserum. • Specific immune response was detected by ELISA using P. damselae subsp. piscicida antigens. • 2D Western blots of ECP and OMP, using sole immunized serum, identified five immunogenic proteins. • Two reactive antigens were identified from ECP: ATP-dependent metalloprotease and Telurite resistance proteins. • In OMP were detected three proteins: Nrfa Y218f, Anti-oxidant AhpC/TSA, and a protein domain DNA binding heat shock related. [ABSTRACT FROM AUTHOR]

Skeletal anomalies affect animal welfare and cause important economic problems in aquaculture. Despite the high frequency of skeletal problems in reared Solea senegalensis, there is lack of information regarding the histological features of normal and deformed vertebrae in this flatfish. The aim of this study was to describe the histopathological and radiographical appearance of vertebral body anomalies. Sixty-seven juvenile fish were radiographically examined 104 or 105 days after hatching. Through radiographic images, vertebral segments were selected and processed for histopathological examination from 7 normal and 7 affected fish. Alterations in bone shape and vertebral fusion were the most significant anomalies in the vertebral bodies. These alterations occurred most frequently between the last 3 abdominal vertebrae and the first 10 caudal centra. Radiographically, deformed vertebrae showed flattening of the endplates and narrowing of the intervertebral spaces. The radiographic findings concurred with the histological lesions where affected vertebrae exhibited irregular endplates and changes in trabecular bone. Radiolucent cartilaginous tissue was evident in the endplates of the deformed vertebra and, in some cases, the cartilaginous material extended from the growth zone into the intervertebral space. These changes were likely the primary alterations that led to vertebral fusion. Fused vertebrae were often reshaped and showed a reorganization of the trabeculae. The formation of metaplastic cartilage is frequent in a variety of anomalies affecting teleost species. [ABSTRACT FROM AUTHOR]

Abstract Subcellular cadmium (Cd) partitioning was investigated in the liver of two marine fish species, the European sea bass Dicentrarchus labrax and the Senegalese sole Solea senegalensis , dietary exposed to an environmentally realistic Cd dose for two months followed by a two-month depuration. The two species displayed different handling strategies during the depuration period. Cd was largely bound to detoxifying fractions such as heat stable proteins (HSP) including metallothioneins (MT) in sea bass, while Cd was more linked to sensitive fractions such as organelles in sole. Whole liver concentrations and subcellular partitioning were also determined for essential elements. The greatest impairment of essential metal homeostasis due to Cd exposure was found in sole. These elements followed the Cd partitioning pattern, suggesting that they are involved in antioxidant responses against Cd toxicity. Cd consumption diminished sole growth in terms of body weight, probably due to lipid storage impairment. The contrasting partitioning patterns showed by the two species might imply different pathways for Cd elimination from the liver. In sea bass, MT-bound Cd would be excreted through bile or released into blood, crossing the cell membrane via a protein transporter. In sole, MRG-bound Cd would be sequestered by organelles before being released into the blood via vesicular exocytosis. These distinct strategies in cellular Cd handling in the liver might account for differential sensitivity to Cd toxicity and differential Cd excretion pathways between the two marine fish species. Graphical abstract Image 1 Highlights • Sea bass and sole displayed different Cd subcellular partitioning in the liver. • Cd was largely bound to metallothionein-like proteins in the sea bass liver. • Cd was mainly linked to organelles in the sole liver. • Essential metals followed the Cd partitioning pattern in hepatic cells. • Handling strategies might account for sensitivity and Cd excretion. [ABSTRACT FROM AUTHOR]

Gastrointestinal (GI) microbiota has a relevant role in animal nutrition, modulation of the immune system and protection against pathogen invasion. Interest in algae as source of nutrients and functional ingredients for aquafeeds is increasing in order to substitute conventional feedstuffs by more sustainable resources. The diet is an important factor in the modulation of the microbiota composition, and functional ingredients have been proposed to shape the microbiota and contribute benefits to the host. However, fish microbiome research is still limited compared to other hosts. Solea senegalensis is a flat fish with high potential for aquaculture in South Europe. In this study, a characterization of the microbiome of S. senegalensis (GI) tract and the effects of feeding Ulva ohnoi supplemented diet has been carried out. Differences in the composition of the microbiota of anterior and posterior sections of S. senegalensis GI tract have been observed, Pseudomonas being more abundant in the anterior sections and Mycoplasmataceae the dominant taxa in the posterior GI tract sections. In addition, modulation of the GI microbiota of juvenile Senegalese sole fed for 45 days a diet containing low percentage of U. ohnoi has been observed in the present study. Microbiota of the anterior regions of the intestinal tract was mainly modulated, with higher abundance of Vibrio spp. in the GI tract of fish fed dietary U. ohnoi. [ABSTRACT FROM AUTHOR]

There is a growing consensus that future technological developments of aquaculture systems should account for the structure and function of microbial communities in the whole system and not only in fish guts. In this study, we aimed to investigate the composition of bacterioplankton communities of a hatchery recirculating aquaculture system (RAS) used for the production of Senegalese sole (Solea senegalensis) juveniles. To this end, we used a 16S rRNA gene based denaturing gradient gel electrophoresis (DGGE) and pyrosequencing analyses to characterize the bacterioplankton communities of the RAS and its water supply. Overall, the most abundant orders were Alteromonadales, Rhodobacterales, Oceanospirillales, Vibrionales, Flavobacteriales, Lactobacillales, Thiotrichales, Burkholderiales and Bdellovibrionales. Although we found a clear distinction between the RAS and the water supply bacterioplankton communities, most of the abundant OTUs (≥50 sequences) in the hatchery RAS were also present in the water supply. These included OTUs related to Pseudoalteromonas genus and the Roseobacter clade, which are known to comprise bacterial members with activity against Vibrio fish pathogens. Overall, in contrast to previous findings for sole grow-out RAS, our results suggest that the water supply may influence the bacterioplankton community structure of sole hatchery RAS. Further studies are needed to investigate the effect of aquaculture practices on RAS bacterioplankton communities and identification of the key drivers of their structure and diversity. [ABSTRACT FROM AUTHOR]

Abstract In the present work, recent studies that contributed for improving dietary protein in practical microdiets for Senegalese sole larvae are revised, in an attempt to overcome some of the current problems in this species larviculture, such as the difficult early adaptation to inert diets and highly variable growth rates. Different diet formulation strategies were employed in an attempt to improve Senegalese sole larvae capacity to utilize and deposit protein throughout metamorphosis, to further maximize growth potential: (1) increasing indispensable amino acids (IAA) content; (2) meeting the ideal IAA profile by adjusting the dietary AA profile to the larval body AA profile; and (3) decreasing the complexity (molecular weight) of dietary protein to increase its digestibility. Either manipulating the quality or the complexity of dietary protein had impact on the larvae capacity to utilize protein and direct it for growth. Sole larvae seem to be able to adapt their digestive functions and metabolic capacity to dietary protein. Moreover, increasing the dietary IAA/DAA ratio and changing the degree of hydrolysis of dietary protein affected the expression pattern of muscle growth related genes, with consequences on muscle cellularity and potential for growth. The expression of DNA methyltransferases was altered in response to changes in dietary protein. The novelty of such information in fish may trigger further studies on the effect of dietary protein on the epigenetic regulation of growth. In conclusion, optimal protein quality for Senegalese sole seems to change during larval development. Whereas the inclusion of a moderately hydrolysed protein comes up as a promising way to improve growth in early larval stages, larger peptides and intact protein seem to be more suitable to sole post-larvae and young juveniles. Therefore, these results suggest that dietary protein fraction formulation of microdiets for Senegalese sole should be adapted to each developmental stage, what has important consequences for practical larval microdiets formulation and feeding protocols. Highlights • Manipulating dietary protein quality or complexity had different effects on sole larvae capacity to utilize and retain protein. • Changing dietary IAA/DAA ratio or protein complexity affected myogenesis, which reflected on sole somatic growth potential. • More than dietary AA profile, changing dietary protein complexity shows as a promising way to improve growth in sole early larvae. • Dietary protein formulation of microdiets for Senegalese sole should be adapted to each developmental stage. [ABSTRACT FROM AUTHOR]

In the present study, selected temperature drops were examined to investigate the effects of live chilling on stress and welfare in turbot and sole. This study demonstrated that rapid temperature drops from 11°C-12°C and 18°C-18.7°C to 4°C-0°C in turbot resulted in higher blood sodium and potassium at 0°C (164 mmol l−1, 4.4 mmol −1) compared to 4°C (153 mmol l−1, 3.1 mmol −1), indicating osmoregulatory disturbance. A rapid temperature drop from 18°C to 0°C in Senegal sole also resulted in higher blood sodium and potassium at 0°C (164 mmol l−1, 4.8 mmol −1) compared to the control group at 18°C (157 mmol l−1, 3.2 mmol −1). Based on present findings, we conclude that immersion in ice water will have a negative effect on the animal osmoregulatory capacity, and we recommend that turbot and sole are stunned before slaughter. [ABSTRACT FROM AUTHOR]

Abstract This study evaluates the in vitro protein hydrolysis of four microalgae (Tisochrysis lutea , Nannochloropsis gaditana , Tetraselmis suecica and Scenedesmus almeriensis) by intestinal proteases of gilthead sea bream (Sparus aurata) and Senegalese sole (Solea senegalensis). The hydrolysis of protein was monitored at different sampling times by electrophoretic techniques, and the quantification of the free amino acids released by proteases. Overall, S. aurata or S. senegalensis proteases hydrolysed microalgae protein in a similar way. The highest hydrolysis values (coefficient of protein degradation, CPD > 70%) were obtained for Tisochrysis and Nannochloropsis biomasses, which showed a progressive and almost complete proteolysis at the end of the in vitro assay. Tetraselmis and Scenedesmus protein was also hydrolysed, but SDS-PAGE revealed that two protein fractions remained virtually intact at the end of the in vitro assay. The final amount of free amino acids released in vitro by the fish digestive enzymes ranged from 9 to 25 g 100 g protein−1. A linear relationship between CPD and the amount of free amino acids released was found, a fact that suggests that microalgae protein is hydrolysed efficiently by the digestive proteases of both fish species. The present study provides information about the protein availability from selected microalgae, which will aid in the initial evaluation of the microalgae as potential protein sources in feeds of two important farmed fish species. Graphical abstract Unlabelled Image Highlights • The in vitro proteolysis of four microalgae by intestinal enzymes of two farmed fish is assessed. • Gilthead sea bream and Senegalese sole digestive proteases are able to hydrolyse the protein of microalgae. • Tisochrysis lutea and Nannochloropsis gaditana show the higher proteolysis values. • These microalgae biomasses can be used as potential ingredient for aquafeeds. [ABSTRACT FROM AUTHOR]

Mercury (Hg) is globally recognized as a persistent chemical contaminant that accumulates in marine biota, thus constituting an ecological hazard, as well as a health risk to seafood consumers. Climate change-related stressors may influence the bioaccumulation, detoxification, and toxicity of chemical contaminants, such as Hg. Yet, the potential interactions between environmental stressors and contaminants, as well as their impacts on marine organisms and seafood safety, are still unclear. Hence, the aim of this work was to assess the bioaccumulation of Hg and neuro-oxidative responses on the commercial flat fish species Solea senegalensis (muscle, liver, and brain) co-exposed to dietary Hg in its most toxic form (i.e., MeHg), seawater warming (ΔT°C = +4 °C), and acidification (pCO2 = +1000 µatm, equivalent to ΔpH = −0.4 units). In general, fish liver exhibited the highest Hg concentration, followed by brain and muscle. Warming enhanced Hg bioaccumulation, whereas acidification decreased this element’s levels. Neuro-oxidative responses to stressors were affected by both climate change-related stressors and Hg dietary exposure. Hazard quotient (HQ) estimations evidenced that human exposure to Hg through the consumption of fish species may be aggravated in tomorrow’s ocean, thus raising concerns from the seafood safety perspective.

Betanodaviruses [nervous necrosis virus (NNV)] are the causative agent of the viral encephalopathy and retinopathy, a disease that affects cultured Senegalese sole (Solea senegalensis). NNV reassortants, combining genomic segments from redspotted grouper nervous necrosis virus (RGNNV) and striped jack nervous necrosis virus (SJNNV) genotypes, have been previously isolated from several fish species. The wild-type reassortant wSs160.03, isolated from Senegalese sole, has been proven to be more virulent to sole than the parental genotypes (RGNNV and SJNNV), causing 100% mortality. Mutations at amino acids 247 (serine to alanine) and 270 (serine to asparagine) in the wSs160.03 capsid protein have allowed us to obtain a mutant reassortant (rSs160.03247+270), which provokes a 40% mortality decrease. In this study, the RNA-Seq technology has been used to comparatively analyze Senegalese sole transcriptomes in two organs (head kidney and eye/brain) after infection with wild-type and mutant strains. A total of 633 genes were differentially expressed (DEGs) in animals infected with the wild-type isolate (with higher virulence), whereas 393 genes were differentially expressed in animals infected with the mutant strain (37.9% decrease in the number of DEGs). To study the biological functions of detected DEGs involved in NNV infection, a gene ontology (GO) enrichment analysis was performed. Different GO profiles were obtained in the following subclasses: (i) biological process; (ii) cellular component; and (iii) molecular function, for each viral strain tested. Immune response and proteolysis have been the predominant biological process after the infection with the wild-type isolate, whereas the infection with the mutant strain induces proteolysis in head kidney and inhibition of vasculogenesis in nervous tissue. Regarding the immune response, genes coding for proteins acting as mediators of type I IFN expression (DHX58, IRF3, IRF7) and IFN-stimulated genes (ISG15, Mx, PKR, Gig1, ISG12, IFI44, IFIT-1, to name a few) were upregulated in animals infected with the wild-type isolate, whereas no-differential expression of these genes was observed in samples inoculated with the mutant strain. The different transcriptomic profiles obtained could help to better understand the NNV pathogenesis in Senegalese sole, setting up the importance as virulence determinants of amino acids at positions 247 and 270 within the RNA2 segment.

Abstract This study aimed to evaluate the protective effects of four autochthonous bacteria isolated from juvenile sole (Solea senegalensis) intestine as dietary probiotic supplement against bacterial pathogen infection and handling/transport stressors. Growth performance and immune responses were evaluated after 85 days of feeding trial. Sole (IBW = 16.07 ± 0.11 g) were fed six experimental diets, a control diet (CTRL, without the dietary probiotic supplementation), and five diets supplemented with probiotic bacteria: PB1 (Shewanella hafniensis), PB2 (Enterococcus raffinosus), PB3 (Shewanella hafniensis + Arthrobacter soli), PB4 (Pseudomonas protegens + Arthrobacter soli) and PB5 (Shewanella hafniensis + Arthrobacter soli + Enterococcus raffinosus). All bacteria were selected based on their in vitro antimicrobial activity. After the growth trial, fish were submitted to a stress factor (transport) and then each dietary group was divided in two additional groups: non-infected (placebo) and infected with Photobacterium damselae subsp. piscicida. Immune and antioxidant responses were evaluated at day 10 post-infection. In infection trial A, fish were infected on the same day of transport, whereas in trial B fish were infected after a 7-day recovery from the transport stress. At the end of the feeding trial, fish fed with PB2 and PB4 showed lower final body weight when compared with the other dietary groups. Respiratory burst activity and nitric oxide production were not affected by probiotic supplementation. Fish fed with PB5 presented lower peroxidase activity compared to CTRL. Lysozyme and alternative complement pathway activity (ACH50) showed no significant differences between treatments. The innate immune responses were significantly affected after handling stress and bacterial infection. In trial A, the ACH50 levels of infected fish were significantly lower than the placebo groups. On the other hand, in trial B fish infected with Pdp demonstrated higher ACH50 levels when compared to placebos. Peroxidase levels were strongly modulated by bacterial infection and handling stress. In trials A and B, infection had a clear downgrade effect in peroxidase levels. Lipid peroxidation, catalase, glutathione S-transferase and glutathione reductase were altered by both bacterial infection and transport. Overall, dietary probiotic supplementation did not influence growth performance of sole. The immune and oxidative defenses of sole responded differently to infection depending on the probiotic and the synergy between pathogen infection and transport. Highlights • Intestine autochthonous bacteria were used as probiotic in Senegalese sole. • Diets PB2 and PB4 resulted in lower final body weight compared with control. • Respiratory burst and nitric oxide were not affected by probiotic supplementation. • Oxidative stress and innate immunity were altered by handling and infection. [ABSTRACT FROM AUTHOR]

Background: Solea senegalensis (Kaup, 1858) is a commercially important flatfish species, belonging to the Pleuronectiformes order. The taxonomy of this group has long been controversial, and the karyotype of the order presents a high degree of variability in diploid number, derived from chromosomal rearrangements such as Robertsonian fusions. Previously it has been proposed that the large metacentric chromosome of S. senegalensis arises from this kind of chromosome rearrangement and that this is a proto-sex chromosome. Results: In this work, the Robertsonian origin of the large metacentric chromosome of S. senegalensis has been tested by the Zoo-FISH technique applied to two species of the Soleidae family (Dicologlossa cuneata and Dagetichthys lusitanica), and by comparative genome analysis with Cynoglossus semilaevis. From the karyotypic analysis we were able to determine a chromosome complement comprising 2n = 50 (FN = 54) in D. cuneata and 2n = 42 (FN = 50) in D. lusitanica. The large metacentric painting probe gave consistent signals in four acrocentric chromosomes of the two Soleidae species; and the genome analysis proved a common origin with four chromosome pairs of C. semilaevis. As a result of the genomic analysis, up to 61 genes were annotated within the thirteen Bacterial Artificial Chromosome clones analysed. Conclusions: These results confirm that the large metacentric chromosome of S. senegalensis originated from a Robertsonian fusion and provide new data about the chromosome evolution of S. senegalensis in particular, and of Pleuronectiformes in general. [ABSTRACT FROM AUTHOR]

Consecutive treatments with recombinant follicle-stimulating and luteinizing hormones (rFsh and rLh, respectively) stimulate spermatogenesis and potentiate sperm production in pubescent specimens of the oligospermic Senegalese sole ( Solea senegalensis ). However, sperm production in response to the hormones is highly variable, and the steroidogenic potential of the testis may be diminished due to sustained hormone supply. Here, we compared the effectiveness of low (9 μg/kg) and high (18 μg/kg) doses of rFsh and rLh to improve sperm production in adult sole during late winter-early spring (onset of the natural spawning period), and in autumn under a controlled temperature of 12 °C (period of testicular recrudescence). Treatment with rFsh over six weeks during spring, followed by a single rLh injection, did not enhance sperm production, possibly because of an advanced stage of sexual maturation of the males, as reflected by high Lh plasma levels (~17 ng/ml) before rFsh treatment. In contrast, in autumn, when the Lh circulating levels were much lower (~3 ng/ml), the low doses of rFsh and rLh generated a four-times increase in sperm production, whereas the high doses of the hormones were ineffective. However, treatment with rLh, regardless of the effect of rFsh, improved the motility of spermatozoa during both spring and autumn. These data confirm that consecutive rFsh and rLh treatments increase sperm production and quality in adult sole males, although they seem to be highly sensitive to the rFsh dose. The efficiency of recombinant gonadotropins also appears to be season-dependent despite the asynchronous nature of the sole testis. [ABSTRACT FROM AUTHOR]

Farmed Senegalese sole ( Solea senegalensis ) still show a high incidence of vertebral anomalies that limit its intensive production and hamper its economic profitability. A great effort towards understanding how fish species develops and grows in captivity has been undertaken in the last decade, with particular emphasis to how different biotic and abiotic factors affect its skeletal development. Although some work has been performed on its skeletal development and expression patterns of key genes in developmental signaling pathways, a detailed description of the above-mentioned processes is still lacking. Here, the progression of skeletal development of cranial, appendicular and axial skeleton has been described through the implementation of an acid free double staining protocol; while the gene expression pattern of vitamin A (VA) and thyroid hormones (THs) signaling pathways were assessed through quantitative PCR (qPCR) during larval fish development under a standard larval rearing protocol and in Senegalese sole larvae fed with increased dietary VA levels (8-fold increase) during the Artemia feeding phase (from 6 to 27 dph). The ontogenetic study allowed us to identify the onset and follow-up the development of most skeletal structures in this fish species, and to gather some insights on the potential onset of deformities originated at the larval to juvenile transition affecting Senegalese sole rearing. The pro-metamorphosis stage is when the caudal fin vertebrae are formed, where the most common skeletal deformities are found in Senegalese sole. A highly coordinated expression of VA- and TH-related genes has been revealed during larval to juvenile transition, while a comparative gene expression analysis in larvae fed control and high dietary VA content identified the specific timing of VA and THs signaling disruption through which VA and THs directly or indirectly might increase the incidence of skeletal deformities in this species. The present research work represents an important step forward towards the proper identification of the onset of some skeletal deformities and urge the investigation of nutritional and rearing conditions during the switch of larval behavior - from pelagic to benthonic - in order to overcome them in this important species for southwestern European aquaculture. [ABSTRACT FROM AUTHOR]

In this study, the short- and long-term effects of embryo incubation temperatures (16, 18 and 20 °C) on development and growth of the flatfish Senegalese sole ( Solea senegalensis ) was determined by investigating the expression patterns of the epigenetic regulators DNA methyltransferases (dnmt) and histone 3 (H3) and genes belonging to the retinoic acid (RA), insulin-like growth factors (IGFs) and hypothalamic-pituitary-thyroid (HPT) axes. Results indicated that egg incubation temperature affected embryo development, but not survival, and incubation at 16 °C significantly delayed development. Coincident with these effects, levels of muscle-specific dnmt3aa transcripts and histone H3 protein levels were significantly different between the 16 and 20 °C groups at hatch. The larvae from eggs incubated at 20 °C relative to the 16 °C group had significantly higher transcript levels of four genes belonging to the HPT axis ( trhr1a , tshr , thrb and dio2 ), four genes of the RA axis (aldh1a2 , cyp26a1 , rara2 , rarg ), igfbp1 and the glycolytic enzyme gapdh2 . Taken together the data suggest that higher egg incubation temperatures enhance energy production, which accelerates cell proliferation and larval development and that hatching is a key moment for the regulation of epigenetic mechanisms. Long-term effects of egg and larval incubation temperatures were revealed by a higher mRNA abundance of the thyroid-related genes tgb and tpo and the RA degrading enzyme cyp26a1 in pre- and metamorphic larvae when they were incubated at 20 °C as embryos and may be related to the earlier initiation of metamorphosis in the pelagic larval stages. Evaluation of growth in pelagic larvae and juveniles after weaning (one trial from 42 to 119 and another from 164 to 247 days post-hatch using a longitudinal approach) revealed that juveniles from embryos incubated at 20 °C had a higher growth rate. All these data demonstrate that the thermal regime during embryogenesis modulated mechanisms that regulate larval plasticity and caused imprinting evident in juvenile sole as persistent changes in key endocrinological pathways and growth performance. [ABSTRACT FROM AUTHOR]

Abstract: Senegalese sole has been shown to be highly susceptible to betanodavirus infection, although virulence differences were observed between strains. To study the mechanisms involved in these differences, we have analysed the replication in brain tissue of three strains with different genotypes during 15 days after bath infection. In addition, possible portals of entry for betanodavirus into sole were investigated. The reassortant RGNNV/SJNNV and the SJNNV strain reached the brain after 1 and 2 days postinfection, respectively. Although no RGNNV replication was detected until day 3–4 postinfection, at the end of the experiment this strain yielded the highest viral load; this is in accordance with previous studies in which sole infected with the reassortant showed more acute signs and earlier mortality than the RGNNV and SJNNV strains. Differences between strains were also observed in the possible portals of entry. Thus, whereas the reassortant strain could infect sole mainly through the skin or the oral route, and, to a minor extent, through the gills, the SJNNV strain seems to enter fish only through the gills and the RGNNV strain could use all tissues indistinctly. Taken together, all these results support the hypothesis that reassortment has improved betanodavirus infectivity for sole. [ABSTRACT FROM AUTHOR]

Feeding is a complex process including food uptake, digestion and processing of the absorbed nutrients, therefore it is required to cover the postprandial period to obtain a global and integrated view of the digestive function. As in the case of fish larvae where the animals may eat ad libitum , it is more adequate to study the feeding and digestion patterns on a daily basis. The aim of the present work was to present an overview on the digestive process throughout the daily cycle in Senegalese sole larvae and post-larvae. The work has included the assessment of feeding and digestion patterns, as well as the regulation of the digestive processes, with the global objective to improve the knowledge of the physiological basis underlying the digestive function and, as consequence, to improve feeding efficiency. [ABSTRACT FROM AUTHOR]

Betanodaviruses [nervous necrosis virus (NNV)] are the causative agent of the viral encephalopathy and retinopathy, a disease that affects cultured Senegalese sole (Solea senegalensis). NNV reassortants, combining genomic segments from redspotted grouper nervous necrosis virus (RGNNV) and striped jack nervous necrosis virus (SJNNV) genotypes, have been previously isolated from several fish species. The wild-type reassortant wSs160.03, isolated from Senegalese sole, has been proven to be more virulent to sole than the parental genotypes (RGNNV and SJNNV), causing 100% mortality. Mutations at amino acids 247 (serine to alanine) and 270 (serine to asparagine) in the wSs160.03 capsid protein have allowed us to obtain a mutant reassortant (rSs160.03247+270), which provokes a 40% mortality decrease. In this study, the RNA-Seq technology has been used to comparatively analyze Senegalese sole transcriptomes in two organs (head kidney and eye/brain) after infection with wild-type and mutant strains. A total of 633 genes were differentially expressed (DEGs) in animals infected with the wild-type isolate (with higher virulence), whereas 393 genes were differentially expressed in animals infected with the mutant strain (37.9% decrease in the number of DEGs). To study the biological functions of detected DEGs involved in NNV infection, a gene ontology (GO) enrichment analysis was performed. Different GO profiles were obtained in the following subclasses: (i) biological process; (ii) cellular component; and (iii) molecular function, for each viral strain tested. Immune response and proteolysis have been the predominant biological process after the infection with the wild-type isolate, whereas the infection with the mutant strain induces proteolysis in head kidney and inhibition of vasculogenesis in nervous tissue. Regarding the immune response, genes coding for proteins acting as mediators of type I IFN expression (DHX58, IRF3, IRF7) and IFN-stimulated genes (ISG15, Mx, PKR, Gig1, ISG12, IFI44, IFIT-1, to name a few) were upregulated in animals infected with the wild-type isolate, whereas no-differential expression of these genes was observed in samples inoculated with the mutant strain. The different transcriptomic profiles obtained could help to better understand the NNV pathogenesis in Senegalese sole, setting up the importance as virulence determinants of amino acids at positions 247 and 270 within the RNA2 segment. [ABSTRACT FROM AUTHOR]

Numerous metabolic and physiological processes are rhythmically regulated in order to accomplish the animal energetic daily demands. The precise knowledge of how metabolic fluxes are modulated is essential to adjust the dietary formulation to satisfy fish energetic demands. In the present study, nutrient (lipid and protein) metabolism and swimming activity were assessed, during a complete daily cycle in Senegalese sole post-larvae (63 days post-hatching), using tube-feeding technique and video-recording approaches. Lipid absorption and retention efficiency were higher in post-larvae fed during the night than those fed during the day. Protein metabolism was not affected by daily cycle. Regarding the swimming activity, a period of higher swimming activity was recorded at 04:00 and at 12:00 h followed by a basal activity of approximately 20% during the rest of the day. Results suggest that post-larvae present higher lipid absorption and retention capacities during the night. The higher swimming activity observed from the mid of the night period until early hours was most likely sustained by a higher feed intake. In conclusion, these results provide useful insight in how fish modulates metabolism to accommodate physiological needs in relation to energy reserves. Sole post-larvae presents a low capacity to digest dietary lipid during daytime. In the future these findings may be used to improve dietary formulations and increase sole growth performance. [ABSTRACT FROM AUTHOR]

The inclusion of organic UV filters in personal care products (PCPs) has increased in recent years. 4-Methylbenzylidene camphor (4MBC) is one of the most used UV filters, and thus it is commonly found in aquatic ecosystems, with proved negative effects on aquatic organisms. Effects on early life stages of marine vertebrates are largely unknown. Therefore, the main goal of this work was to evaluate 4MBC effects on Senegalese sole ( Solea Senegalensis Kaup, 1858) larvae at different levels of biological organization. S. senegalensis were exposed to increasing concentrations of 4MBC from egg stage until 96 h. Mortality, growth, malformations, behaviour and biochemical responses, including enzymatic biomarkers were studied. The exposure to 4MBC until 96 h post-fertilization (hpf) induced mortality and malformations in a dose-response manner. Besides, reduced growth with increasing concentrations was observed. The exposure to 4MBC also caused alterations on behaviour, including overall lower swimming time during light and dark periods. Biomarker alterations caused by 4MBC included imbalance of neurotransmission related endpoints (increased acetylcholinesterase activity) and decreased activity of enzymes related to anaerobic metabolism (lower cellular lactate dehydrogenase activity) at the lower concentrations tested. Furthermore, our results suggest that 4MBC do not induce oxidative stress in S. senegalensis larvae, since catalase and lipid peroxidation levels were not significantly altered by 4MBC. S. senegalensis revealed to be a good model species for vertebrate animal testing in the marine environment. Sub-lethal concentrations of 4MBC induced toxic effects at all organizational levels. Swimming behaviour was a sensitive endpoint and showed that exposure to 4MBC causes impairment on response to light stimulus which is possibly linked with the observed imbalances on cholinesterase activity in larvae. Conservation concerns along distribution range of S. senegalensis should consider that increasing levels of UV filters in marine environment might have impact on the ecology of the species. [ABSTRACT FROM AUTHOR]

Senegalese sole ( Solea senegalensis ) has been proposed as a high-potential species for aquaculture diversification in Southern Europe. It has been demonstrated that a proper feeding regimen during the first life stages influences larval growth and survival, as well as fry and juvenile quality. The bacterial strain Shewanella putrefaciens Pdp11 (SpPdp11) has shown very good probiotic properties in Senegalese sole, but information is scarce about its effect in the earliest stages of sole development. Thus, the aim of this study was to investigate the effect of SpPdp11, bioencapsulated in live diet, administered during metamorphosis (10–21 dph) or from the first exogenous feeding of Senegalese sole (2–21 dph). To evaluate the persistence of the probiotic effect, we sampled sole specimens from metamorphosis until the end of weaning (from 23 to 73 dph). This study demonstrated that probiotic administration from the first exogenous feeding produced beneficial effects on Senegalese sole larval development, given that specimens fed this diet exhibited higher and less dispersed weight, as well as increases in both total protein concentration and alkaline phosphatase activity, and in non-specific immune response. Moreover, real-time PCR documented changes in the expression of a set of genes involved in central metabolic functions including genes related to growth, genes coding for proteases (including several digestive enzymes), and genes implicated in the response to stress and in immunity. Overall, these results support the application of SpPdp11 in the first life stages of S. senegalensis as an effective tool with the clear potential to benefit sole aquaculture. [ABSTRACT FROM AUTHOR]