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Turbot (Scophthalmus maximusL., 1758) is a valuable commercial fish species classified as endangered. The conservation and sustainability of the turbot populations require knowledge of the population’s genetic structure and constant monitoring of its biodiversity. The present study was performed to evaluate the population structure of turbot along the Bulgarian Black Sea coast using seven pairs of microsatellites, two mitochondrial DNA (COIII and CR) and 23 morphological (15 morphometric and 8 meristic) markers. A total of 72 specimens at three locations were genotyped and 59 alleles were identified. The observed number of alleles of microsatellites was more than the effective number of alleles. The overall mean values of observed (Ho) and expected heterogeneity (He) were 0.638 and 0.685. A high rate of migration between turbot populations (overall mean of Nm = 17.484), with the maximum value (19.498) between Shabla and Nesebar locations, was observed. This result corresponded to the low level of genetic differentiation amongst these populations (overall mean Fst = 0.014), but there was no correlation between genetic and geographical distance. A high level of genetic diversity in the populations was also observed. The average Garza-Williamson M index value for all populations was low (0.359), suggesting a reduction in genetic variation due to a founder effect or a genetic bottleneck. Concerning mitochondrial DNA, a total number of 17 haplotypes for COIII and 41 haplotypes for CR were identified. The mitochondrial DNA control region showed patterns with high haplotype diversity and very low nucleotide diversity, indicating a significant number of closely-related haplotypes and suggesting that this population may have undergone a recent expansion. Tajima’s D test and Fu’s FS test suggested recent population growth. Pairwise Fst values were very low. The admixture and lack of genetic structuring found pointed to the populations analysed probably belonging to the same genetic unit. Therefore, a proper understanding and a sound knowledge of the level and distribution of genetic diversity in turbot is an important prerequisite for successful sustainable development and conservation strategies to preserve their evolutionary potential.

8 Pág. Departamento de ​Mejora Genética Animal (INIA), This work was supported by the European Union’s Seventh Framework Programme (KBBE.2013.1.2-659 10 under Grant Agreement No. 613611 FISHBOOST project), the European Commission Horizon 2020 (H2020) Framework Programme through grant agreement no 727315 MedAID project (Mediterranean Aquaculture Integrated Development), by Ministerio de Ciencia e Innovación (CGL2016-75904-C2), MCIN/AEI/https://doi.org/10.13039/501100011033 (PID2020-114426GB-C22 and PID2020-114426GB-C2), Xunta de Galicia (GRC, ED431C 2020-05) and Centro singular de investigación de Galicia accreditation 2019–2022, and the European Union (European Regional Development Fund—ERDF), Fondos Feder “Unha maneira de facer Europa”. MK and MP were also supported by Ministry of Education, Youth and Sports of the Czech Republic—project Biodiverzity (CZ.02.1.01/0.0/0.0/16_025/0007370). The Roslin Institute was partly funded by Biotechnology and Biological Sciences Research Council Institute Strategic Programme grants (BBS/E/D/20241866, BBS/E/D/20002172 and BBS/E/D/20002174).

Pathogenic bacterium discovery using 16S rRNA high throughput sequencing has become more commonplace. It offers advantages of being high-throughput screening, cost-effective (less than $15 per sample), and rapid (less than one week). Fish pathogens causing aquatic bacterial diseases outbreak represent a major threat to aquaculture industry. A haemorrhagic disease in turbot (Scophthalmus maximus) outbreaks with unknown etiology in Haiyang, China. Initial analyses suggested it was a bacterial disease based on gross pathology of diseased fish containing ascitic fluid from the distended abdomen, a swollen intestine, an inflamed and red mouth. To fully understand the accurate etiology, distribution of pathogenic bacterium and comprehensive information on the diseased turbots is required. Tested tissues (ascites, heart, eye, mouth, intestine, spleen, and liver) with typical symptoms of diseased turbots were collected and 16 s rRNA sequencing was performed using the Illumina Mi-Seq platform. Sequencing results revealed generally low microbial diversity (< 50 %) in different infected tissues, but the microbiota did exhibit a little higher diversity in the intestine than in the other tissues. A key finding was that Edwardsiella was the most abundant genus detected in all the fish tissues. It was indicated that Edwardsiella spp. was the main causative pathogen in the diseased turbot. The evidence also suggested that Edwardsiella spp. as a severely pathogenic bacteria may cause disruption of the healthy balance in the host. Our study was a new attempt to investigate unknown pathogens in clinical marine fish specimens through the high-throughput next-generation sequencing technology.

In the Turkish Black Sea coasts fish is particularly consumed because of high protein supplies, essential amino acids, vitamin, and mineral content. Fish are exposed to contaminants such as heavy metals in polluted waters. The aims of the study are to determine heavy metals in Pleuronectiformes species from Sinop coasts of the Black Sea and determine the health risks due to the consumption of contaminated fish. Pleuronectiformes species Scophthalmus maximus (Linnaeus, 1758) belonging to Scophthalmidae family, Arnoglossus laterna (Walbaum, 1792) belonging to Bothidae family and Pegusa lascaris (Risso, 1810) belonging to Soleidae family were collected from Sinop coasts of the Black Sea in fishing season of 2016. Heavy metals (Hg, Cd, Pb, Cu and Zn) in edible tissues were determined by ICP-MS (Agilent 7700x). The limit values given by the international and national organizations did not exceed in the muscle tissues of turbot, Mediterranean scald fish and sand sole. Overall Zn was detected in higher concentrations in all species followed by Cu, Pb, Hg and Cd. The results obtained from the analyses of the maximum levels of the metals except Hg were found in turbot. The highest Hg (0.021±0.007 µg g -1 wet wt.) was found in sand sole. However, the high amounts of Cd, Pb, Cu and Zn in turbot were 0.011±0.004, 0.07±0.005,1.32±0.28 and 14±3 µg g -1 wet wt., respectively. The EDIs and EWIs of the metals were estimated taking into account the mean of metal in all fish samples and the mean consumption of fish per day/week for adults. These results are normally significantly lower than the recommended values of FAO/WHO. Estimated HIs of all the considered metals were below the value of 1, therefore the metals in fish samples do not toxic any apparent threat to the population and these fishes are healthy for consumption. Keywords: Scophthalmus maximus , Arnoglossus laterna , Pegusa lascaris , heavy metals, Black Sea

Turbot (Scophthalmus maximus) is an important marine fish both in Europe and North China. Although there are plenty of studies on the reproduction of turbot, the complete cytological process of spermatogenesis remains unclear. In this study, we investigated the submicroscopic structure of total 23 types of male germ cells throughout the breeding season, with a relatively complete process of the primary spermatocytes. We found that the spermatid tail formed early at Spermatid II, and there were at least 16 spherical mitochondria in the spermatozoa. The hepatosomatic index (HSI) and gonadosomatic index (GSI) both peaked during the breeding season. Preliminary analysis showed that the vitality of mature sperm was negatively correlated with the proportion of sperm deformity. The serum 3,5,3′-triiodothyronine (T3), 5-hydroxytryptamine (5-HT), testosterone (T), 17α,20β-Dihydroxy-4-pregnen-3-one (17α,20β-DHP), and 17β-estradiol (E2) all increased during the maturity period, with the change of T content most noticeable. Whereas in the testis, an overall high level of 11-ketotestosterone (11-KT) was more remarkable. The expression and localization of androgen receptor (AR) mRNA showed that the AR was highly expressed at the stages of II (15 – 70 g), with a slight rebound at the mature stages [IV(2200 g) to V(2500 g)], whose change was ahead to the changes of T and 11-KT. Fluorescence in situ hybridization (FISH) analysis showed that the AR mainly distributed in but not limited to Sertoli cells. This study represents the most complete overview of the reproductive cycle and spermatogenesis of turbot, which provides an important reference for the reproduction research and the guidance of flatfish breeding.

Proteomics has been recently introduced in aquaculture research, and more methodological studies are needed to improve the quality of proteomics studies. Therefore, this work aims to compare three sample preparation methods for shotgun LC–MS/MS proteomics using tissues of two aquaculture species: liver of turbot Scophthalmus maximus and hepatopancreas of Mediterranean mussel Mytilus galloprovincialis. We compared the three most common sample preparation workflows for shotgun analysis: filter-aided sample preparation (FASP), suspension-trapping (S-Trap), and solid-phase-enhanced sample preparations (SP3). FASP showed the highest number of protein identifications for turbot samples, and S-Trap outperformed other methods for mussel samples. Subsequent functional analysis revealed a large number of Gene Ontology (GO) terms in turbot liver proteins (nearly 300 GO terms), while fewer GOs were found in mussel proteins (nearly 150 GO terms for FASP and S-Trap and 107 for SP3). This result may reflect the poor annotation of the genomic information in this specific group of animals. FASP was confirmed as the most consistent method for shotgun proteomic studies, however, the use of the other two methods might be important in specific experimental conditions (e.g., when samples have a very low amount of protein).

International audience; Opportunistic green macroalgae blooms increasingly affect coastal areas worldwide. Understanding their impacts on organisms that use this zone, such as juvenile flatfish, is critical. By combining stable isotope data, digestive tract contents and community analyses of flatfish and their potential prey (benthic macroinvertebrates) from two North-East Atlantic sandy beaches (one impacted by blooms and one not), we detected similar and species-specific trophic changes among three co-occurring species (sand sole, plaice and turbot). Across flatfish species, juveniles displayed more opportunistic foraging behavior at the impacted site. Differently, plaice and sand sole relied more on the additional basal resource (Ulva spp.) than turbot. Finally, sand sole and turbot presented a stronger diet shift at the impacted site than plaice. We hypothesize that the species-specific response to the blooms are mostly driven by how the flatfish detect their prey (using visual and/or chemical cues) and when they forage (diurnal or nocturnal foraging).

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

Enteromyxosis, caused by Enteromyxum scophthalmi, is one of the most devastating diseases stemming from myxozoan parasites in turbot (Scophthalmus maximus L.), being a limiting factor for its production. The disease develops as a cachectic syndrome, associated to catarrhal enteritis and leukocytic depletion, with morbidity and mortality rates usually reaching 100%. To date, no effective treatment exists and there are different unknown issues concerning its pathogenesis. The gross and microscopic lesions associated to enteromyxosis have been thoroughly described, and several morphopathological studies have been carried out to elucidate the mechanisms of this host-parasite interaction. More recently, efforts have been focused on a multidisciplinary approach, combining histopathology and transcriptome analysis, which has provided significant advances in the understanding of the pathogenesis of this parasitosis. RNASeq technology was applied at early and advanced stages of the disease on fishes histologically evaluated and classified based on their lesional degree. In the same way, the transcriptomic data were analyzed in relation to the morphopathological picture and the course of the disease. In this paper, a comprehensive review of turbot enteromyxosis is presented, starting from the disease description up to the most novel information extracted by an integrated approach on the infection mechanisms and host response. Further, we discuss ongoing strategies toward a full understanding of host-pathogen interaction and the identification of suitable biomarkers for early diagnosis and disease management strategies. This work was supported by the European Regional Development Fund (ERDF) together with the Spanish Ministry of Economy, Industry and Competitiveness through the projects AGL2015–67039–C3–1–R and AGL2015–67039–C3–3–R. DR was supported by a Newton International Fellowship from The Royal Society (NF160037) SI

WOS: 000499795100001, Scientific & Technological Research Council of TurkeyTurkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [TUBITAK-112O920]; Bulgarian Academy of Sciences (BAS), This research was supported by the Scientific & Technological Research Council of Turkey (TUBITAK-112O920), Bulgarian Academy of Sciences (BAS).

Highly dense linkage maps enable positioning thousands of landmarks useful for anchoring the whole genome and for analysing genome properties. Turbot is the most important cultured flatfish worldwide and breeding programs in the fifth generation of selection are targeted to improve growth rate, obtain disease resistant broodstock and understand sex determination to control sex ratio. Using a Restriction-site Associated DNA approach, we genotyped 18,214 single nucleotide polymorphism in 1,268 turbot individuals from 31 full-sibling families. Individual linkage maps were combined to obtain a male, female and species consensus maps. The turbot consensus map contained 11,845 markers distributed across 22 linkage groups representing a total normalised length of 3,753.9 cM. The turbot genome was anchored to this map, and scaffolds representing 96% of the assembly were ordered and oriented to obtain the expected 22 megascaffolds according to its karyotype. Recombination rate was lower in males, especially around centromeres, and pairwise comparison of 44 individual maps suggested chromosome polymorphism at specific genomic regions. Genome comparison across flatfish provided new evidence on karyotype reorganisations occurring across the evolution of this fish group This study has been supported by the FISHBOOST project (ref. 613611) from the European Community's Seventh Framework Programme (FP7/2007-2013) and by Consellería de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia local government (ref. GRC2014/010) SI

The impact of escapees from aquaculture is of general concern for the sustainability of natural resources. Turbot Scophthalmus maximus is a marine flatfish of great commercial value whose land-based aquaculture started approx. 40 yr ago; hence, a low impact of escapees is expected on wild populations. However, enhancement of wild stocks using farmed turbot has been carried out along the Northeast Atlantic coasts in the last decades. Recently, a broad panel of single nucleotide polymorphism (SNP) markers (755 SNPs; 1 SNP Mb−1) has been used to evaluate the genetic structure of turbot throughout its distribution range, constituting the baseline to evaluate the impact of farmed fish in the wild. Two distinct origins were identified for farmed turbot (F_ORI1 and F_ORI2; FST = 0.049), which differentiated from wild populations after 5 generations of selection (average FST = 0.059), and consistent evidence of adaptation to domestication was de - tected. A notable proportion of fish of farmed ancestry was detected in the wild (15.5%), mainly in the North Sea, where restocking activities have taken place, determining genetic introgression in wild populations. Conversely, effects of land-based aquaculture appear negligible. A simulation exercise supported panels of 40 and 80 SNPs to identify fishes of F_ORI1 and F_ORI2 ancestry in the wild, respectively. Application to empirical data showed an assignment success (wild/farmed ancestry) of approx. 95% in comparison with the full SNP dataset. The SNP tools will be useful to monitor turbot of farmed ancestry in the wild, which might represent a risk, considering the lower fitness of farmed individuals The project was funded by the 7th Framework Programme for research (FP7) under ‘Knowledge-Based Bio-Economy — KBBE’, Theme 2: ‘Food, Agriculture and fisheries, and Biotechnologies’ Project identifier: FP7-KBBE-2012-6-singlestage Grant agreement no.: 311920 ‘The development of tools for tracing and evaluating the genetic impact of fish from aquaculture: AquaTrace’ and the Spanish Regional Government Xunta de Galicia GRC2014/010. Ciência sem Fronteiras/CAPES − Brazil supported the fellowship for the stay of F.D.P. at USC SI

International audience; Turbot (Scophthalmus maximus) is a valuable flatfish with current selection programs to increase growth rate in the fifth generation. Disentangling the genetic architecture of growth traits has biological and productive relevance. Growth-related quantitative trait loci (QTL) and associated markers have been previously reported in turbot. Here, we tested the performance of 39 markers associated with three growth-related traits (body weight (BW), body length (BL) and condition factor (FK)) in 18 families for their potential application in marker assisted selection (MAS) in this species. A cost-effective selective genotyping approach was developed by checking the statistical power of small samples at the extreme of the phenotypic distributions. Associations between growth traits and markers were performed using two statistical approaches (contingency chi-square tests and stepwise regression analysis) to identify the most robust set of markers. Twenty markers were detected by both methods, mostly linked to previously reported growth-related QTL and encompassing 11 linkage groups (LGs). The stepwise regression analysis revealed that a subset of seven markers explained nearly 40% and 60% of the phenotypic variation at genotypic and allelic level, respectively, for BW and BL. Seven markers for BW and BL and other two for FK showed association at population level, when all family data were pooled. Validated markers might be useful to go a step forward to improve growth performance through more refined MAS, and as physical anchors for identifying causal genes underpinning growth traits in turbot.

15 páginas, 4 figuras, 4 tablas.-- The original publication is available at www.springerlink.com, Viral haemorrhagic septicaemia virus (VHSV) is one of the major threats to the development of the aquaculture industry worldwide. The present study was aimed to identify genes differentially expressed in several turbot (Scophthalmus maximus) families showing different mortality rates after VHSV. The expression analysis was conducted through genome-wide expression profiling with an oligo-microarray in the head kidney. A significant proportion of the variation in the gene expression profiles seemed to be explained by the genetic background, indicating that the mechanisms by which particular species and/or populations can resist a pathogen(s) are complex and multifactorial. Before the experimental infections, fish from resistant families (low mortality rates after VHSV infection) showed high expression of different antimicrobial peptides, suggesting that their pre-immune state may be stronger than fish of susceptible families (high mortality rates after VHSV infection). After infection, fish from both high- and low-mortality families showed an up-modulation of the interferon-induced Mx2 gene, the IL-8 gene and the VHSV-induced protein 5 gene compared with control groups. Low levels of several molecules secreted in the mucus were observed in high-mortality families, but different genes involved in viral entrance into target cells were down-regulated in low-mortality families. Moreover, these families also showed a strong down-modulation of marker genes related to VHSV target organs, including biochemical markers of renal dysfunction and myocardial injury. In general, the expression of different genes involved in the metabolism of sugars, lipids and proteins were decreased in both low- and high-mortality families after infection. The present study serves as an initial screen for genes of interest and provides an extensive overview of the genetic basis underlying the differences between families that are resistant or susceptible to VHSV infection., We wish to thank the Spanish Ministerio de Ciencia e Innovación for funding through project CSD2007-00002 “Aquagenomics” of the Consolider-Ingenio 2010 program

38 p., 7 figuras, 1 tabla y bibliografía, The kinetics of the antibody production and the protection at challenge were studied in turbot inoculated with various scuticociliate antigen preparations: live ciliates putatively attenuated through long-term in vitro culture (Trial 1) and formalin-killed ciliates without or with GERBU adjuvant in Trials 2, 3, and 4. Antigen used in killed preparations was a mixture of 3 different ciliate isolates (V3) in the case of Trials 2 and 3, whereas in Trial 4, monovalent (V1), trivalent (V3) or pentavalent (V5) antigens were used. A booster injection was administered 28 to 29 d post-priming in all trials. Fish were challenged with virulent live ciliates after the immunization protocol, testing 2 challenge times in Trial 2 (t1 and t2). No protection was obtained in Trial 1 with live ciliates, which in turn were not completely attenuated. Using killed-ciliate formulations, protection was high only in Trial 3 when a low dose (50 000 ciliates fish-1) was used for challenge. In Trial 1, heat-inactivated sera of antigen-inoculated fish agglutinated the homologous ciliate, although no specific antibodies were detectable by ELISA. In contrast, high specific antibody levels were detected in antigen-inoculated fish in Trials 2 and 4, and their amount increased progressively, usually peaking after challenge. No advantage was obtained from the use of V5 antigens compared to V1 or V3. No good correlation was observed in most cases between serum antibody levels and protection. Although the use of GERBU adjuvant generally increased the specific immune response, some undesired side effects indicate a need to adjust dosage and/or improve the formulation., This study was partly funded by project PTR1995-0604-OP from the Spanish Ministerio de Ciencia y Tecnología

Many estimators and algorithms have been developed to infer the genealogical relationships from molecular marker data when there is no pedigree information. Most pairwise methods provide estimates in a continuous range that needs to be converted into genealogical relationships (namely full-sibs, half-sibs and unrelated) if there is a previous knowledge of the population structure. Transformations are usually based on arbitrary thresholds, but the possibility of correcting the coancestry estimates via explicit pedigree reconstruction methods has been suggested. Using molecular data for ten highly polymorphic microsatellite loci on a population of turbot (Scophthalmus maximus) with a known genealogy, the behaviour of four pairwise marker-based coancestry estimators and the molecular coancestry has been evaluated. The population consisted on 138 families with 4 full-sib individuals each and one family with 8 full-sib individuals (up to 15 half-sib families were also present due to the sharing of parents between some of the full-sibs families). Our results suggested that transforming the pairwise estimators and the molecular coancestry to family relationships through the establishment of thresholds performs slightly better than the explicit pedigree reconstruction method, when accuracy is measured in a pairwise basis. However, if joint relationships between more than two individuals were tested at a time, the threshold methods led to a high percentage of incongruous triads of full-sib individuals, with Mendelian incompatibilities appearing in congruous full-sib families (more than 70% and 60% in our study, respectively)., The explicit pedigree reconstruction approach, due to its nature, is free from such problems. Therefore, the pedigree reconstruction approach seems to be a valuable tool to provide a congruent and compatible pedigree when the pairwise marker-based coancestry matrices or the molecular coancestry need to be transformed. (C) 2007 Elsevier B.V. All rights reserved.

The environmental impacts of a water re-circulating system for fish farming were studied through the case study of an inland turbot farm located in Brittany (France). Life Cycle Assessment methodology was used to evaluate the potential environmental impact through the following indicators: Eutrophication Potential, Acidification Potential, Global Warming Potential, Net Primary Production Use and Non Renewable Energy Use. Two methods were used to assess the farm's nitrogen, phosphorus and solids emissions: nutrient measurement accounting and nutrient balance modelling. The two methods gave similar results for solids and phosphorus emissions, while for nitrogen the measurement-based approach resulted in half the emissions predicted by the model. The uncertainty regarding the potential gaseous nitrogen emissions led us to assess impacts according to three scenarios, differing with respect to emissions of N2, N2O and NH3. This approach illustrates that the uncertainty concerning nitrogenous emissions to the atmosphere leads to uncertainty with respect to the production system's Eutrophication Potential and its Global Warming Potential. The comparison of our results with similar results for large rainbow trout production in a flow-through system points out the impacts associated with the high level of energy consumption in the studied re-circulating system (i.e. Non Renewable Energy Use, Global Warming potential, Acidification Potential). The nitrogenous gas emissions of re-circulating systems require further studies, in order to precisely identify the substances involved and the technological solutions allowing reduced impacts.

The survival, growth, gonadal development and sex ratios of gynogenetic diploid turbot Scophthalmus maximus between 9 and 36 months of age were investigated. Gynogenesis was induced by activation of freshly collected eggs with diluted (1 : 10 with Ringer) and UVirradiated (30000 erg mm 2) sperm, followed by a cold shock at 1 to 0 C for 25 min, starting at 6 5 min after fertilization. The exclusive maternal inheritance of the resulting gynogenetic diploids was confirmed in all individuals used in the experiment by microsatellite markers. Mean total length and mass throughout the experiment was higher in the controls than in the gynogenetics. The gonadal development in gynogenetic males proceeded normally, and in gynogenetic females it was delayed during the first sexual maturation but was normal during the second one. The sex ratio was 1 male (M) : 1 female (F) in the controls, as expected, but 1 M : 3 F in the gynogenetics. In a second, independent experiment, carried out with fish originating from different broodstock, however, the sex ratio was 1 M : 1 F in the controls and 0 M : 1 F (i.e. 100% females) in the gynogenetics. Together, these results suggested that the main sex-determining genetic component in turbot fits well to female homogamety, in accordance with data from other flatfishes. These results indicate that it will be possible to obtain near or all-female turbot stocks through induced gynogenesis or by crosses involving hormonally sex-reversed fish., 2

11 pages, 4 figures, 1 table, Triploidy was induced in the turbot (Scophthalmus maximus, L.) by applying cold shocks shortly after fertilization. The combined effects of the timing of cold shock commencement after fertilization, cold shock duration and cold shock temperature were investigated. Ploidy was assessed by counting the number of nucleoli per nucleus (NOR) in larvae and also by measuring erythrocyte size in juveniles. A clear peak in triploidy induction was obtained when shocks were started between 6 and 7 min after fertilization at a pre-shock temperature of 13–14°C. With this timing, shocks of 20-min duration at 0°C gave >90% triploidy, with survival about 80% of the untreated controls. In order to ensure both high triploidy rates and high survival, it was necessary to carefully maintain the water temperature just below 0°C. Experiments with small and large volumes of eggs were performed in order to determine how changes in the relative volumes of eggs and chilled water could affect survival and triploidy induction. The best combination to induce triploidy in the turbot was as follows: shock commencement 6.5 min after fertilization, shock duration 25 min, and shock temperature between 0 and −1°C. With this combination, 100% triploidy could consistently be induced with survival 60% of the untreated control. This was successfully applied to a large volume of eggs (~300 ml; 1 ml ~800 eggs) in order to mass-produce triploid turbot. Triploids had lower survival rate than diploids at hatching but similar thereafter, with the ability to complete the different stages of larval rearing, indicating the viability to produce triploid turbot under farming conditions, Research was funded by CICYT grant MAR95-1855 from the Spanish Government to P.M.

[Background] Gene expression analysis by reverse transcription quantitative PCR (qPCR) is the most widely used method for analyzing the expression of a moderate number of genes and also for the validation of microarray results. Several issues are crucial for a successful qPCR study, particularly the selection of internal reference genes for normalization and efficiency determination. There is no agreement on which method is the best to detect the most stable genes neither on how to perform efficiency determination. In this study we offer a comprehensive evaluation of the characteristics of reference gene selection methods and how to decide which one is more reliable when they show discordant outcomes. Also, we analyze the current efficiency calculation controversy. Our dataset is composed by gonad samples of turbot at different development times reared at different temperatures. Turbot (Scophthalmus maximus) is a relevant marine aquaculture European species with increasing production in the incoming years. Since females largely outgrow males, identification of genes related to sex determination, gonad development and reproductive behavior, and analysis of their expression profiles are of primary importance for turbot industry., [Results] We analyzed gene stability of six reference genes: RPS4, RPL17, GAPDH, ACTB, UBQ and B2M using the comparative delta-CT method, Bestkeeper, NormFinder and GeNorm approaches in gonad samples of turbot. Supported by descriptive statistics, we found NormFinder to be the best method, while on the other side, GeNorm results proved to be unreliable. According to our analysis, UBQ and RPS4 were the most stable genes, while B2M was the least stable gene. We also analyzed the efficiency calculation softwares LinRegPCR, LREanalyzer, DART and PCR-Miner and we recommend LinRegPCR for research purposes since it does not systematically overestimate efficiency., [Conclusion] Our results indicate that NormFinder and LinRegPCR are the best approaches for reference gene selection and efficiency determination, respectively. We also recommend the use of UBQ and RPS4 for normalization of gonad development samples in turbot., This study was supported by a project from the Spanish Ministerio de Ciencia e Innovación (AGL2010-22326-C02-01). Diego Robledo was supported by a FPU fellowship from the Ministerio de Educación Cultura y Deporte of Spanish Governement.

Linkage maps based on markers derived from genes are essential evolutionary tools for commercial marine fish to help identify genomic regions associated with complex traits and subject to selective forces at play during exploitation or selective breeding. Additionally, they allow the use of genomic information from other related species for which more detailed information is available. Sole (solea solea L.) is a commercially important flatfish species in the North Sea, subject to overexploitation and showing evidence of fisheries-induced evolutionary changes in growth- and maturation-related traits. Sole would definitely benefit from a linkage map to better understand how evolution has shaped its genome structure. This study presents a linkage map of sole based on 423 single nucleotide polymorphisms derived from expressed sequence tags and 8 neutral microsatellite markers. The total map length is 1233.8 cM and consists of 38 linkage groups with a size varying between 0 to 92.1 cM. Being derived from expressed sequence tags allowed us to align the map with the genome of four model fish species, namely medaka (Oryzias latipes), Nile tilapia (Oreochromis niloticus), three-spined stickleback (Gasterosteus aculeatus) and green spotted pufferfish (Tetraodon nigroviridis). This comparison revealed multiple conserved syntenic regions with all four species, and suggested that the linkage groups represent 21 putative sole chromosomes. The map was also compared to the linkage map of turbot (Scophthalmus maximus), another commercially important flatfish species and closely related to sole. For all putative sole chromosomes (except one) a turbot homolog was detected, confirming the even higher degree of synteny between these two flatfish species. ispartof: PLoS One vol:9 issue:12 ispartof: location:United States status: published

Prey items of 0- and 1-group plaice Pleuronectes platessa, sole Solea solea, brill Scophthalmus rhombus, turbot S. maximus and dab Limanda limanda of the surf zone of a Belgian sandy beach, included hyperbenthic (e.g; mysids), endobenthic (e.g. Polychaetes) and epibenthic (e.g. shrimps) species. Little dietary overlap was observed. If diet overlap did occur, it mainly involved prey species that are dominant in the surf zone of Belgian beaches, such as shrimps and mysids. These results suggest an opportunistic utilization by flatfish of the available food resources in surf zone ecosystems. Also, two strategically different feeding habits could be distinguished between the five flatfish species. Turbot and brill mainly fed on large, highly mobile prey (e.g. fish, mysids) and had a rather narrow prey spectrum, whereas plaice, dab and sole ate more benthic prey (e.g. Polychaetes) and had a broader prey spectrum.

The present study describes an explanatory model that simulates growth and body composition of fish larvae. Its objective is to improve the understanding of the growth process in larval fish, and by doing so, to enable the optimisation of the feeding strategies in larviculture. The model is driven by nutrient intake, with the absorbed dietary nutrients being used for energy production or for biosynthetic processes, and it is based on the stoichiometry of intermediary metabolism. The model was parameterised using literature data and validated for the African catfish and turbot. According to the model, high unavoidable losses of amino acids due to imbalances between the dietary and the larval amino acid profiles lead to an increase in lipid deposition in periods of high food intake. In catfish, this occurs with the contribution of gluconeogenesis. Model simulations indicate that an increase in the dietary protein level of currently used diets would stimulate growth and lead to a reduction in lipid deposition, providing the dietary AA profile is well balanced. An increase of 25% in the dietary protein level may stimulate growth (dry matter) by 15% for the African catfish, and 140% for turbot. Model simulations also show that a 25% increase in dietary lipid level may lead to a small protein-sparing effect, and to an increase of 46 and 36% in larval lipid content for African catfish and turbot, respectively. Due to its mechanistic nature, the present model can be used for other fish species, and in particular in the development of tentative feeding strategies when new species are brought into cultivation.

Ichthyophonus sp. is reported for the first time in Mugil capito (thinlip grey mullet) and Li a saliens (leaping grey mullet). The fungus was also found in L. aurata (golden grey mullet), Dicentrarchus labrax (sea bass), Sparus aurata (gilthead sea bream) and Scophthalmus maximus (turbot), whereas Mugil cephalus (grey mullet) was not parasitized. In fish sampled periodically, the highest prevalences were observed in sea bass and the lowest in turbot. Among the fish sampled occasionally, the fungus was found associated to an epizootic in thinlip grey mullet. Ichthyophonus was never found in fish weighing

Ionic (Na+, K+, Cl−, PO43−, pH), total CO2, total calcium and protein concentrations in the plasma and endolymph of the inner ear were compared in trout Oncorhynchus mykiss and turbot Scophthalmus maximus. In both species, saccular endolymph was characterized by high levels of K+ and total CO2 and in trout by an alkaline pH. The kinetic characteristics of proton secretion across the saccular epithelium of trout were investigated using a titration technique in which isolated saccules were mounted as closed sacs. The rate of proton secretion depends strongly on the pH of the Ringer’s solution and secretion stops at a pH below 7.2. Proton secretion is driven by an energy-dependent mechanism involving basolateral ouabain-sensitive Na+/K+ exchangers. Proton secretion was partially inhibited by acetazolamide and completely inhibited in Na+-free Ringer or in the presence of 1 mmol l−1 amiloride. A cellular model stressing the importance of proton exchange through the saccular epithelium is proposed to explain the regulation of endolymph pH, a crucial factor for the deposition of otolith calcium.

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The turbot (Scophthalmus maximus) is a commercially valuable flatfish and one of the most promising aquaculture species in Europe. Two transcriptome 454-pyrosequencing runs were used in order to detect Single Nucleotide Polymorphisms (SNPs) in genes related to immune response and gonad differentiation. A total of 866 true SNPs were detected in 140 different contigs representing 262,093 bp as a whole. Only one true SNP was analyzed in each contig. One hundred and thirteen SNPs out of the 140 analyzed were feasible (genotyped), while Ш were polymorphic in a wild population. Transition/transversion ratio (1.354) was similar to that observed in other fish studies. Unbiased gene diversity (He) estimates ranged from 0.060 to 0.510 (mean = 0.351), minimum allele frequency (MAF) from 0.030 to 0.500 (mean = 0.259) and all loci were in Hardy-Weinberg equilibrium after Bonferroni correction. A large number of SNPs (49) were located in the coding region, 33 representing synonymous and 16 non-synonymous changes. Most SNP-containing genes were related to immune response and gonad differentiation processes, and could be candidates for functional changes leading to phenotypic changes. These markers will be useful for population screening to look for adaptive variation in wild and domestic turbot. This study was supported by the Consolider Ingenio Aquagenomics (CSD200700002), the Science and Education Spanish Ministry (AGL2009-11782) and the Xunta de Galicia (09MMA011261PR) projects SI

12 páginas, 11 figuras, 3 tablas, Dietary long chain polyunsaturated fatty acids (FA) have been recognized of crucial importance in early development of vertebrates, contributing to the impressive morphological and physiological changes both as building blocks and to energy production. The importance of lipids along development depends on ontogenetic, phylogenetic and environmental parameters. The expression patterns of FA metabolism genes have not been characterized in developing fish embryos nor compared to lipid class profiles. Full lipid metabolism only occurred after hatching, as revealed by alterations in lipid profiles and FA gene expression. Nonetheless, transcriptional changes of some FA genes were already present in embryos at notochord formation. Many genes displayed an expression profile opposed to the decrease of lipids along the development, while others responded solely to starvation. Transcription of most genes involved in FA metabolism had a strong correlation to PPARs' mRNA levels (α1, α2, β, γ). The comparison of mRNA expression of the genes with the lipid profiles produced new insights into the FA metabolism and regulation during the development of turbot larvae, providing the basis for future studies including comparative approaches with other vertebrate species., This work was developed under the research project PTDC/MAR/68885/2006, funded by the Portuguese Foundation for Science and Technology (FCT) and by the “Programa Operacional Ciência e Inovação 2010” (POCI 2010), co-financed by the FEDER European Community fund.

The adjuvant effect of Quillaja saponaria saponin (QSS) on protection of turbot fry was investigated with immersion vaccination of formalin-killed Vibrio anguillarum O1 and various concentrations of QSS (5, 25, 45 and 65 mg/L). Fish were challenged at days 7, 14 and 28 post-vaccination. Significantly high relative percent of survival (RPS) ((59.1 ± 13.6)%, (81.7 ± 8.2)%, (77.8 ± 9.6)%) were recorded in the fish that received bacterins immersion with QSS at 45 mg/L, which is comparable to the positive control group vaccinated by intraperitoneal injection (IP). Moreover, a remarkably higher serum antibody titer was also demonstrated after 28 days in the vaccinated fish with QSS (45 mg/L) than those vaccinated fish without QSS (p < 0.05), but lower than the IP immunized fish (p < 0.05). Significant upregulation of IgM gene expression has also been identified in the tissues of skin, gill, spleen and kidney from the immunized fish in comparison to the control fish. Taken together, the present study indicated that QSS was able to dramatically evoke systemic and mucosal immune responses in immunized fish. Therefore, QSS might be a promising adjuvant candidate for fish vaccination via an immersion administering route.

Growth traits represent a main goal in aquaculture breeding programs and may be related to adaptive variation in wild fisheries. Integrating quantitative trait loci (QTL) mapping and next generation sequencing can greatly help to identify variation in candidate genes, which can result in marker-assisted selection and better genetic structure information. Turbot is a commercially important flatfish in Europe and China, with available genomic information on QTLs and genome mapping. Muscle and liver RNA-seq from 18 individuals was carried out to obtain gene sequences and markers functionally related to growth, resulting in a total of 20,447 genes and 85,344 single nucleotide polymorphisms (SNPs). Many growth-related genes and SNPs were identified and placed in the turbot genome and genetic map to explore their co-localization with growth-QTL markers. Forty-five SNPs on growth-related genes were selected based on QTL co-localization and relevant function for growth traits. Forty-three SNPs were technically feasible and validated in a wild Atlantic population, where 91% were polymorphic. The integration of functional and structural genomic resources in turbot provides a practical approach for QTL mining in this species. Validated SNPs represent a useful set of growth-related gene markers for future association, functional and population studies in this flatfish species. Fil: Robledo, Diego. Universidad de Santiago de Compostela; España Fil: Fernández, Carlos. Universidad de Santiago de Compostela; España Fil: Hermida, Miguel. Universidad de Santiago de Compostela; España Fil: Sciara, Andres Angel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Álvarez Dios, José Antonio. Universidad de Santiago de Compostela; España Fil: Cabaleiro, Santiago. Centro Tecnológico del Cluster de la Acuicultura de Galicia; España Fil: Caamaño, Rubén. Centro Tecnológico del Cluster de la Acuicultura de Galicia; España Fil: Martínez, Paulino. Universidad de Santiago de Compostela; España Fil: Bouza, Carmen. Universidad de Santiago de Compostela; España

4 páginas, 2 tablas, 4 figuras, Triploid turbot (Scophthalmus maximus) produced by cold shock showed no differences in their gross body morphology and skeleton characteristics compared with their diploid full-sibs., This research was carried out with the financial support of the Spanish Ministry of Science and Innovation (AGL2008-00392/ACU) and FPI (BES-2009-016797) and FPI-IEO grants to ET and JHU respectively.

The objective of this study was to determine the relative essential fatty acid (EFA) growth-promoting activities of pure arachidonic (AA, 20:4n−6) and docosahexaenoic (DHA, 22:6n−3) acids and various concentrations of these two acids in the diet of juvenile turbot (Scophthalmus maximus). Casein-based, semi-purified diets containing 15% fish oil or 14% hydrogenated coconut oil/oleic acid (1:1) supplemented with 1% 20:4n−6, 1% 22:6n−3 or 1% of various combinations of these two acids were fed to duplicate groups of 26 juvenile turbot for 11 weeks. In this trial, feeding the diet containing 20:4n−6 as the only highly unsaturated fatty acid (HUFA) resulted in higher growth and survival than any of the mixtures of the two fatty acids or 22:6n−3 alone. The diet containing 22:6n−3 as the sole HUFA resulted in the lowest growth and survival of all dietary treatments. The control diet with 15% fish oil resulted in a greater growth rate than any of the pure HUFA-supplemented diets. There was a significant effect of dietary lipid on the somatic index of the brain but not heart, kidney or liver. The percentage of lipid in the liver, but not of heart, brain, eyes, gills or kidney, was influenced by dietary lipid, with the highest percentage in fish supplemented with DHA alone. After 11 weeks, the 20:4n−6 and 22:6n−3 levels in whole-body total lipids were strongly influenced by the content of these fatty acids in the diets. The relative effect of dietary levels of these two fatty acids on their content in fish lipids varied considerably among the various organs and tissues of the fish that were analyzed. Brain and eye lipids were generally highest in 22:6n−3 while gill and kidney lipids were consistently higher in 20:4n−6 than the other organs analyzed. The effect of dietary 20:4n−6 on the content of that HUFA in organ lipid was greatest in gill and liver. The greatest impact of dietary 22:6n−3 level on content of that acid in organ lipid was seen in gill and kidney. There were also significant effects of dietary HUFA content on organ lipid levels of saturated, mono-unsaturated fatty acids and other members of the n−3 and n−6 PUFA, and HUFA series. The present study suggests that the EFA growth-promoting activity of arachidonic acid provides strong support for the contention that dietary 20:4n−6 is essential for juvenile turbot.

5 pages, 13 figures., Histological examination of moribund specimens of turbot grow-out facilities in northwest Galicia (Spain) revealed lesions severe enough to be responsible for the diseased condition and death of many fish. The lesions were caused by 2 agents, the microsporidian Tetrarnicra brevifjlum, already described from the same host farmed in the same region and a histophagous ciliate. Both agents were found In mlxed infections; the dominant role in pathogenesls was played by the histophagous ciliate which was tentatively assigned to the subclass Hymenostomata, order Scuticociliatida Small, 1967.

8 páginas, 4 tablas, 7 figuras, The isocitrate dehydrogenase activity specific for NADP + has been studied in early turbot larvae and compared with that of their adult counterparts. The activity showed a broad optimum pH between 7.9 and 9.0, and an optimum temperature of 400C (with an E, of 23.3 k J/tool) and was inhibited by high ionic strength and heavy metals. The apparent K, values for the substrates were 16.7 pM for isocitrate, 3.5 for NADP +, 29.6 pM for Mk~ + and 3.3/JM for the Mg-isocitrate complex. The effect of the absence of Mk~ + on the enzyme affinity for its substrates is also reported. The enzyme stability was decreased by iodoacetic acid and this effect reverted by isocitrate. The enzyme activity was inhibited by metabolites such as citrate, oxoglutarate and adenine dinucleotides. It was also fully inhibited by the concerted action of glyoxylate and oxaloacetate., This work has been supported by a grant from the Xunta de Galicia No. XUGA40201B90.

Background The turbot (Scophthalmus maximus) is a highly appreciated European aquaculture species. Growth related traits constitute the main goal of the ongoing genetic breeding programs of this species. The recent construction of a consensus linkage map in this species has allowed the selection of a panel of 100 homogeneously distributed markers covering the 26 linkage groups (LG) suitable for QTL search. In this study we addressed the detection of QTL with effect on body weight, length and Fulton's condition factor. Results Eight families from two genetic breeding programs comprising 814 individuals were used to search for growth related QTL using the panel of microsatellites available for QTL screening. Two different approaches, maximum likelihood and regression interval mapping, were used in order to search for QTL. Up to eleven significant QTL were detected with both methods in at least one family: four for weight on LGs 5, 14, 15 and 16; five for length on LGs 5, 6, 12, 14 and 15; and two for Fulton's condition factor on LGs 3 and 16. In these LGs an association analysis was performed to ascertain the microsatellite marker with the highest apparent effect on the trait, in order to test the possibility of using them for marker assisted selection. Conclusions The use of regression interval mapping and maximum likelihood methods for QTL detection provided consistent results in many cases, although the high variation observed for traits mean among families made it difficult to evaluate QTL effects. Finer mapping of detected QTL, looking for tightly linked markers to the causative mutation, and comparative genomics are suggested to deepen in the analysis of QTL in turbot so they can be applied in marker assisted selection programs.

Production of triploid individuals is a relevant goal for the aquaculture industry due to the benefits associated with their sterility and growth. Thus, methods for assessing triploidy have been developed based on genome, chromosome or gene triploid-associated properties. In this study, we developed a new cheap, technically simple and accurate method to validate triploidy in turbot (Scophthalmus maximus) based on microsatellite markers. Five crosses were performed to produce diploid and triploid progenies that were used to validate this molecular tool. Flow cytometry, one of the most widely used and accurate techniques for ploidy determination, was used as reference to contrast results. A set of four highly polymorphic and largely distant to centromere microsatellites was selected for this purpose. Ploidy was easily evaluated according to the maximum number of alleles at the microsatellite loci tested, diploids showing two and triploids three. These microsatellites were combined in a single multiplex and were able to identify triploids with 100% accuracy in all analyzed crosses., 2

We studied the variation at 30 anonymous and 30 expressed sequence tag (EST)-associated microsatellites in 4 natural populations of turbot Scophthalmus maximus living in habitats with different salinity and temperature conditions. We identified putative divergent selection effects on 3 genes: the fibroblast growth factor receptor, the β microglobuline, and the trap alpha gene for translocon associate protein. The markers closely linked to these genes showed significant deviations from the neutral expectations using 2 different statistical methods in several pairwise population comparisons involving samples from salty and brackish environments. Our results confirmed the weak genetic structure among populations from the northeast Atlantic and the low but significant genetic differentiation of turbot from the Baltic Sea. These results suggest that populations from the Baltic–Atlantic transition area could be accumulating adaptive polymorphisms in the face of high gene flow This work was funded by the grant PGIDIT06PXIB261178PR from Xunta de Galicia, a Consolider Ingenio Aquagenomics project (CSD2007-00002) from Spanish Ministry of Education and Science, and an Isidro Parga Pondal Fellowship to R.V. SI

8 pages, 4 figures, 1 table, In order to further investigate the reproductive physiology of triploid turbot, Scophthalmus maximus, the gonadal development and the incidence of apoptosis were studied in diploid and triploid fish at up to 28. months of age. Within the period studied, diploid males completed the reproductive cycle and produced spermatozoa, while the most advanced cell type observed in triploid males was spermatids. In diploid females, preovulatory oocytes were observed, whereas in triploid females a nearly total meiosis blockade was evident with only a small number of oogonia achieving the perinucleolar oocyte stage. Testosterone levels in males were similar regardless of ploidy level with peaks observed immediately before the completion of spermatogenesis; however, peaks were advanced in triploids. Estradiol levels remained very low in triploid females. In diploid males, apoptosis was observed only at the start of spermatogenesis and during testicular regression. In contrast, triploid males had an overall higher rate of apoptotic cells which was constant throughout the reproductive cycle. In diploid females, few or no apoptotic cells were observed in contrast to triploid females where these numbers were markedly increased followed by an overall downward trend as oogenesis progressed. These data suggest that apoptosis plays a role in the control of germ cell number and fate and this regulatory mechanism is magnified in females bearing three sets of chromosome. Thus, the sex-related effects of triploidy on gonadal development in turbot, and likely in fish, include an increase in apoptosis, particularly in females, Research at the FP lab is partially funded by project CDS2007-00002 “Aquagenomics” from the Spanish Ministry of Science

When evaluating the environmental impacts of finfish production systems, both regional impacts (e.g., eutrophication) and global impacts (e.g., climate change) should be taken into account. The life cycle assessment (LCA) method is well suited for this purpose. Three fish farms that represent contrasting intensive production systems were investigated using LCA: rainbow trout (Oncorhynchus mykiss) in freshwater raceways in France, sea-bass (Dicentrarchus labrax) in sea cages in Greece, and turbot (Scophtalmus maximus) in an inland re-circulating system close to the seashore in France. Two main characteristics differentiated the three farm systems: feed use and energy use. Emission of nitrogen and phosphorus accounted for more than 90% of each farm's potential eutrophication impact. In the trout and sea-bass systems, feed production was the major contributor to potential climate change and acidification impacts and net primary production use (NPPU). In these systems, the main source of variation for environmental impacts was the feed conversion ratio. Results from this study indicate that the sea-bass cage system was less efficient than the trout raceway system, with a higher level of potential eutrophication (65% greater) and NPPU (15% greater). The turbot re-circulating system was a high energy-consumer compared to the trout raceway system (four times higher) and the sea-bass cage system (five times higher). Potential climate change and acidification impacts were largely influenced by energy consumption in the turbot re-circulating system. In the turbot re-circulating system 86% of energy use was due to on-site consumption, while in the sea-bass cage farming system 72% of energy use was due to feed production. These results are discussed in relation to regional contexts of production and focus attention on the sensitivity of each aquatic environment and the use of energy carriers.

International audience; The relative roles of genetic (GSD) and temperature sex determination (TSD) were investigated in turbot, Scophthalmus maximus, with the objective of producing all-female stocks for aquaculture. Androgen treated males (ATM) and oestrogen treated females (OTF) were produced after testing various 17 alpha-methyltestosterone or 17 beta-oestradiol treatments delivered orally (3 or 5 mg steroids/kg food) from 35 days postfertilization (dpf) during either 500 or 700 degree-days respectively. GSD was analyzed by checking the sex ratio of families (n = 33) obtained by crossing control parents with other control parents, either ATM or OTF. TSD was investigated on 8 families reared at three temperatures (15 degrees C, 17 degrees C or 24 degrees C) from weaning to 100 dpf. All the steroid treatments were efficient in producing either 100% mate or 100% female populations demonstrating the production of androgen sex-inversed males and oestrogen sex-inversed females. The results of the GSD analysis suggested that most of the data fit to a ZZ/ZW model of sex determination. However, some crosses deviated from this model and other minor factors may be involved. interestingly, minor effects of temperature and a limited but significant interaction between family and temperature have been observed. In conclusion, the present data suggest that a major ZZ/ZW genetic model is involved in turbot sex determination. Unexpected sex ratios deviating from this model are discussed, including a possible minor effect of environmental factors, such as temperature. (C) 2009 Elsevier B.V. All rights reserved.

In a radiotracer study designed to evaluate the fate of metals from fish prey to predator fish, we measured the trophic transfer of 7 trace elements (Am, Cd, Co, Cr, Cs, Mn, Zn) from juvenile Mediterranean sea bream Sparus auratus to 3 predator species, the teleosts Psetta maxima and Sparus auratus, and the elasmobranch Scyliorhinus canicula. Prey S. auratus were labeled by exposure to metal radioisotopes in solution for a period of 3 wk and were then fed to predators, after which the metal retention in the predator was assessed. Mean assimilation efficiencies (AEs) of ingested metals in the predator fishes ranged from 6 to 15% for Am, to 63 to 74% for Cs; significant differences in AEs were noted between species for Co and Mn only. Efflux rate constants (ke) of assimilated metal did not differ consistently among predators for any metals. The mean ke ranged from 0.003 d-1 for Mn in S. canicula to 0.02 d-1 for Cd and Cs in all 3 species and for Co in S. canicula. Trophic transfer factors (TTF) for each metal were determined in predator fishes to assess the potential for metal biomagnification. TTF, calculated by dividing the product of metal AE and ingestion rate (IR) by ke, exceeded 1 only for Mn, Zn and Cs. For most likely scenarios of IR and AE, TTF was

Apoptosis was monitored in male and female diploid and triploid turbot encompassing a full gametogenic cycle. Results indicate that apoptosis is involved in the normal regulation of spermatogenesis progression and male germ cell fate, and provide the first evidence that the differential effects of triploidy on gonadal development include the induction of apoptosis in females., 2,0410

Genetic (GSD) and temperature sex determination (TSD) have been investigated in the turbot, Scophthalmus maximus using sex inversed males (androgen treated males=ATM) and females (oestradiol treated female-s= OTF). ATM and OTF have been produced through feeding treatments using 17[alpha]-methyltestosterone and 17[beta]-oestradiol at a dosage of 3 or 5 mg/kg food starting from 35 days post fertilization (dpf) during periods of 500 or 700° days. 16 ATM and 6 OTF were mated with normal females or males. The 22 resulting progenies and 11 control families were reared at 17°C. For TSD evaluation, parts of 8 of these 33 families (2 from ATM and 6 from controls) have been reared at 15°C, 17°C or 24°C from 35 dpf to 100 dpf. Then all progenies (GSD and TSD) have been reared at 17°C until 11 months old (100 g body weight) for the sex ratio assessment. For 27 progenies reared at 17°C, sex ratios perfectly fit in with the hypothesis of a male homogamety ZZ/ZW system (X-square test; P

Biomarkers of exposure & effect:: validationBiomarker: MNExposure/effect represented: genetic damageStudy type (in vitro, animals, humans): fishMode of exposure (if in vivo) (acute, chronic, root of exposure):chronicTissue/biological material/sample size:4*103 erythrocytes/animalMethod of analysis: MN testSensitivity (LOD): low baseline frequencyIntra-individual variation: large range of variabilityInter-individual variation:0-2 MN frequencyDose-response: 2.95 MN/1000 cells compared to 1MN/1000 cells in the control, p

10 pages, 5 figures, 1 table.-- Available online Jul 1, 2005., This study determined the effect of triploidy on the survival, growth and gonadal development of turbot from 6 to 48 months of age. From 6 to 24 months of age (first sexual maturity), survival was similar in both ploidies (P > 0.05). From 24 to 48 months of age, after the first sexual maturity, survival was 91.9% in diploids and 100% in triploids, which did not exhibit the post-spawning-associated mortality. Growth was similar for both ploidies during the first year of life. After that, triploids grew significantly (P < 0.05) more that diploids, with more marked differences after each spawning season. From 24 to 48 months, the average weight difference between both ploidies was 11.4 ± 1.9%, ranging from 4.3 to 23.0%. At 47 months of age, the biomass of triploids was 10.3% greater in total weight and 14.3% greater in eviscerated weight. Gonads of triploid males were similar to that of diploids, whereas in triploid females, they were significantly smaller and rudimentary. A histological analysis carried out at 47 months of age showed complete sterility of triploids in both sexes. Sex ratio was 1 male (M):0.6 female (F), for diploids, significantly (P < 0.05) different from 1:1, and 1 M:3.3 F for triploids, significantly (P < 0.05) different from 1:1 and from the diploids. Since females grow more than males, culture of triploids benefited from the high female ratio, which helped to reduce size dispersion. In addition, their sterility allowed better performance by avoiding the reduction in growth that takes place during the spawning periods. Together, these observations indicate that triploidy induction can be an interesting option for turbot aquaculture, especially for the production of large-size fish of more than 2 years of age.

12 páginas, 4 figuras, 4 tablas, Vibriosis is one of the main causes of mortality in all stages of fish. In larvae, further improvements on the control and knowledge of bacterial diseases require the development of models for experimental infections. A reproducible model for in vivo infections of first feeding turbot (Scophthalmus maximus) larvae with the pathogen Vibrio (Listonella) anguillarum is described. Experimental challenges were carried out under hatchery conditions considering different ways of delivery of V. (Listonella) anguillarum strain 90-11-287 serotype O1 to turbot larvae: via rotifers (orally) and via water of the larval rearing tanks (bath). The effect of the supernatant of the bacteria culture was also considered and discussed. The model proposed is based on the incorporation of the bacteria into previously enriched rotifers and the delivery to the larvae during the rotifer feeding period. The effect of infections was already evident 24–72 h after the first challenged day. Final mortalities (days 8–14) in infected larvae were 56–72% with respect to unchallenged larvae. Mortalities achieved by applying the protocol of infection are reliable, reproducible and adequate for experimental purposes. The model of infection via rotifers proposed in this study is a useful tool for experimental purposes in the larval rearing of marine fish., This work was supported by funds of the European Commission (Project PROBE, Contract No. Q5RS-2000-31457). Dr. J.A. Va´zquez had a postdoctoral contract (CSIC-I3P-PC 2003) financed by the European Social Fund.

Scophthalmus maximus is an important commercially aquaculture fish species. We tackle the search for new microsatellites using two different approaches: an enriched partial genomic library and a screening of all turbot DNA sequences deposited in GenBank. Out of 15 genomic library derived loci, five gave working primer pairs, with expected heterozygosities (HE) ranging from 0.13 to 0.91. Out of seven loci derived from database sequences, two amplified successfully with an HE ranging from 0.56 to 0.63. The average allele number of these microsatellites was 5.7 per locus with a range between two and 15.

11 páginas, 7 figuras, 2 tablas, A bacterial strain, characterized as Vibrio pelagius (Hq 222), was isolated from a turbot, Scophthalmus maximus (L.), larvae mass mortality in a commercial fish farm in Spain. Turbot larvae, post-larvae (0.2 g) and juveniles (5 and 15 g) were experimentally infected. The bacterium appeared to be very virulent for larvae and post-larvae, LD50 being, This work was funded by Project 1FD97-044-C03- 03 from FEDER funds.

9 páginas, 5 figuras, The effect of a pathogenic Vibrio pelagius, isolated during a mass mortality of turbot larvae, on the non-specific immune response of turbot, Scophthalmus maximus (L.), macrophages was studied both in vitro and in vivo. The in vitro treatment of head kidney (HK) macrophages with viable V. pelagius caused a significant inhibition of the chemiluminescence (CL) response in comparison with untreated macrophages, while incubation with heat-killed bacteria did not affect this response. In vivo, the intraperitoneal injection of V. pelagius resulted in a significant inhibition of the CL response in infected fish at days 1 and 4 post-infection compared with the control fish response. The HK macrophage nitric oxide (NO) production was enhanced by in vitro incubation with intermediate doses of viable V. pelagius (5 × 103 and 5 × 104 bacteria mL−1) and higher doses of the heat-killed bacteria (5 × 104–5 × 106 bacteria mL−1). In both cases, the NO inhibitorN-ω -nitro-L-arginine was capable of down-regulating the specific NO induction caused by incubation with the bacterial treatments. In contrast, incubation with ECPs at higher doses caused a reduction in NO production. In vivo, a significant enhancement in NO production was also observed in macrophage supernatants at day 10 post-infection. Lysozyme concentration in the serum was also significantly increased in the experimentally infected fish at days 4 and 10 post-injection. In addition, viable V. pelagius and its ECPs significantly reduced HK macrophage viability in vitro, whereas no significant differences in viability were observed during the incubation with heat-killed bacteria. As NO production was enhanced in the experimentally infected fish, the inhibitory effect of the NO donor, S-nitroso-acetyl-penicillamine (SNAP), was tested in vitro in a cell-free assay. The results showed that growth of V. pelagius was significantly inhibited using SNAP at a high concentration (1 mm)., This work was partially supported by project 1FD97-0044-C03-03 from FEDER, and a grant from Caixa Galicia (Spain).