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14 pages, 7 figures, 2 tables, supplementary material https://doi.org/10.1016/j.ygeno.2021.04.007.-- Availability of data and materials: All dataset generated during this study are included (in this article and as supplementary information files) or are available in public repositories. Turbot Genome Sequencing data are at NCBI databases (Bioproject: PRJNA631898) (https://www.ncbi.nlm.nih.gov/genome/?term=turbot) and BioProject es PRJNA649485 (https://www.ncbi.nlm.nih.gov/bioproject/649485; Accessión numbers: SRX8843737, SRX8843739, SRX8843738), Background: Understanding sex determination (SD) across taxa is a major challenge for evolutionary biology. The new genomic tools are paving the way to identify genomic features underlying SD in fish, a group frequently showing limited sex chromosome differentiation and high SD evolutionary turnover. Turbot (Scophthalmus maximus) is a commercially important flatfish with an undifferentiated ZW/ZZ SD system and remarkable sexual dimorphism. Here we describe a new long-read turbot genome assembly used to disentangle the genetic architecture of turbot SD by combining genomics and classical genetics approaches. Results: The new turbot genome assembly consists of 145 contigs (N50 = 22.9 Mb), 27 of them representing >95% of its estimated genome size. A genome wide association study (GWAS) identified a ~ 6.8 Mb region on chromosome 12 associated with sex in 69.4% of the 36 families analyzed. The highest associated markers flanked sox2, the only gene in the region showing differential expression between sexes before gonad differentiation. A single SNP showed consistent differences between Z and W chromosomes. The analysis of a broad sample of families suggested the presence of additional genetic and/or environmental factors on turbot SD. Conclusions: The new chromosome-level turbot genome assembly, one of the most contiguous fish assemblies to date, facilitated the identification of sox2 as a consistent candidate gene putatively driving SD in this species. This chromosome SD system barely showed any signs of differentiation, and other factors beyond the main QTL seem to control SD in a certain proportion of families, This work was supported by the Spanish Ministry of Economy and Competitiveness, Grant: AGL2014-57065-R, by the European Union's Horizon 2020 Research and Innovation Programme under grant agreement No 81792 (AQUA-FAANG) and by Consellería de Educación, Universidade e Formación Profesional. Xunta de Galicia, Grant number: ED431C 2018/28, With funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S)

The knowledge of oxygen availability and consumption is one of the prerequisites for the development of aquatic systems (Brown et al., (1984), since low levels of available oxygen impose limitations on aquatic poikilotherms (Fernandez and Rantin, 1989). The effect of a prolonged (21 days) decrease in dissolved oxygen was compared on growth of juvenile turbot (Petssa maxima). Turbot (Psetta maxima) is a flatfish that has been cultured for many years in commercial aquaculture. Since this species is usually cultured at high densities, up to 40 Kg m-2 (Martinez-Tapia and Fernandez Pato, 1991), the effects of lower dissolved oxygen concentrations are an important factor for the optimization of commercial scale aquaculture. Initial weight of turbot was of 10,6±1,3 g; at the end of the 21 days experiment, juvenile turbot exposed to a lower dissolved oxygen concentration grew less (15,9±0,5 g) compared to those cultured at normal concentrations (17,8±0,6 g) (p

SPARC/osteonectin is a multifunctional matricellular glycoprotein, which is expressed in embryonic and adult tissues that undergo active proliferation and dynamic morphogenesis. Recent studies indicate that Sparc expression appears early in development, although its function and regulation during development are largely unknown. In this report, we describe the isolation, characterization, post-embryonic developmental expression and environmental thermal regulation of sparc in turbot. The full-length turbot sparc cDNA contains 930. bp and encodes a protein of 310 amino acids, which shares 77, 75 and 80% identity with human, frog and zebrafish, respectively. Results of whole-mount in situ hybridization reveal a dynamic expression profile during post-embryonic turbot development. Sparc is expressed differentially in the cranioencephalic region; mainly in jaws, branchial arches, fin folds and rays of caudal, dorsal and anal fins. Furthermore, ontogenetic studies demonstrated that Sparc gene expression is dynamically regulated during post-embryonic turbot development, with high expression during stage-specific post-embryonic remodeling. Additionally, the effect of thermal environmental conditions on turbot development and on ontogenetic sparc expression was evaluated. © 2015 Elsevier B.V.

Polybrominated diphenyl ethers (PBDEs) are a class of global flame retardants whose residues have markedly increased in fish and human tissues during the last decade. They belong to persistent and toxic contaminants which needmore attention and toxicological study as their degradation in the environment is not well understood. This study characterizes 2,2′,4,4′- tetrabromodiphenyl ether (BDE-47) bioconcentration, elimination and biotransformation in juvenile turbot Psetta maxima, in order to evaluate the risk of its bioaccumulation in the marine environment. During this study, juvenile turbot were exposed to environmentally relevant BDE-47 concentrations from 0.001 to 1 μg/L for 16 days via aqueous exposure. This study found that juvenile turbot bioconcentrated and biotransformed BDE-47. There was no difference in standard length or weight between control and exposed fish. Uptake and elimination of BDE-47 by the turbot during exposure were examined in controlled laboratory experiments. Bioconcentration of BDE-47 was similar in fish from all treatment groups. Bioconcentration was rapid and increased with exposure time, since the bioconcentration factor (BCF) was higher. However, elimination was slower in comparison and low elimination was detected after 10 days in clean water. BDE-47 concentration in water influences the BCF. Half-life ranged between 37 and 108 days and theoretical times t90 could range from 120 to 358 days. The present study demonstrates a stepwise debromination of BDE-47 to BDE-28. MAE-PCI Versión del editor

5 páginas, 2 figuras, 2 tablas, Plastic responses to environmental conditions are crucial among ectothermic organisms, and any traits induced early in ontogeny may have long-term effects. Here, we studied how turbot larvae altered their morphology in three different thermal environments and assessed whether different thermal regimes affected the survival, growth, development and morphology of juvenile turbot at the end of metamorphosis. Therefore, newly hatched Turbot (Scopthalmus maximux L.) larvae were divided into three groups and reared at 14, 18 and 22°C until 80 days after hatching. Environmental temperature was seen to affect several turbot traits. Low temperature induced low dorsal and anal fin ray counts, but had no effect on gross body morphology. There were differences in growth and skeletal development rates, but no differences in survival. There were also no differences in the skeletal malformations occurring in response to environmental temperature treatment. Collectively, these results supported the idea of the existence of a thermal plastic response, as found, in other fish species, This work was funded by the Spanish Science and Innovation Ministry projects AGL2008-00392/ACU and ALG2011-23581 to J. Rotllant; E. Torres-Nuñez was supported by the PhD fellowship FPI BES-2009-016797

Background, aim and scope The environmental presence of polybrominated diphenyl ethers (PBDEs), among which BDE-47 and BDE-99 are particularly abundant, makes toxicity data necessary to assess the hazard risk posed by PBDE to aquatic organisms. This study examines the effects of BDE-47 and BDE-99 on embryo-larval stages of the marine flatfish turbot. Materials and methods The turbot embryos were exposed at nominal concentrations of BDE-47 and BDE-99 for 6 days. Selected dose levels were relevant for investigating sublethal and lethal effects. Results Both tested compounds caused lethal toxicity as well as non-lethal malformations during embryo development. We found a high toxic potency of BDE-47 compared to BDE-99 (LC50 values for embryos and larvae, respectively, BDE-47: 27.35 and 14.13 μg L−1; BDE-99: 38.28 and 29.64 μg L−1). Discussion The present study shows high sensitivity of fish early life stages (ELS) to PBDE compounds. Based on environmental concentrations of dissolved PBDEs from various aquatic ecosystems, waterborne BDE-47 and BDE- 99 pose little risk of acute toxicity to marine fish at relevant environmental concentrations. Conclusions Turbot fish ELS proved to be an excellent model for the study of ecotoxicity of contaminants in seawater. The results demonstrate harmful effects of PBDE on turbot ELS at concentrations in the range of parts per billion units. Recommendations and perspectives In the perspective of risk assessment, ELS endpoints provide rapid, costeffective and ecologically relevant information, and links should be sought between these short-term tests and effects of long-term exposures in more realistic scenarios. MAE-PCI CTM2009-10908 Versión del editor 2,6510

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

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

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

11 pages, 3 figures, 2 tables, supporting information http://www.genetics.org/cgi/content/full/177/4/2457/DC1, A consensus microsatellite-based linkage map of the turbot (Scophthalmus maximus) was constructed from two unrelated families. The mapping panel was derived from a gynogenetic family of 96 haploid embryos and a biparental diploid family of 85 full-sib progeny with known linkage phase. A total of 242 microsatellites were mapped in 26 linkage groups, six markers remaining unlinked. The consensus map length was 1343.2 cM, with an average distance between markers of 6.5 ± 0.5 cM. Similar length of female and male maps was evidenced. However, the mean recombination at common intervals throughout the genome revealed significant differences between sexes, ~1.6 times higher in the female than in the male. The comparison of turbot microsatellite flanking sequences against the Tetraodon nigroviridis genome revealed 55 significant matches, with a mean length of 102 bp and high sequence similarity (81–100%). The comparative mapping revealed significant syntenic regions among fish species. This study represents the first linkage map in the turbot, one of the most important flatfish in European aquaculture. This map will be suitable for QTL identification of productive traits in this species and for further evolutionary studies in fish and vertebrate species, This study was supported by a project from the Xunta de Galicia local government (PGIDIT04RMA261014PR)

This study was carried out to compare key haematological features of diploid (2n) and triploid (3n) turbot as a first step towards the assessment of the ability of 3n turbot to withstand sub-optimal culture conditions. Morphometric indices of erythrocytes were determined on blood smears by light microscopy. Triploidy significantly (P < 0.001) increased all morphometric indices measured in the erythrocytes, including size, surface, and volume, except for the size of minor nuclear axis. The increase in cell size was larger for the major (31.0%) than for the minor (8.3%) axis, thus rendering erythrocytes of 3n turbot more ellipsoidal. The increase in erythrocyte volume (45.9%) was close to the theoretical expected 50% increase as a result of one extra chromosome set. Haematological indices were measured automatically by a haematological Coulter Counter. Triploid turbot had lower numbers of red blood cells (RBC: 1.84 cells pL 1 in 2n vs. 1.27 cells pL 1 in 3n; P < 0.001) but they were of a larger size (Mean corpuscular volume [MCV]: 145.51 fL in 2n vs. 181.78 fL in 3n; P < 0.001). However, the decrease in RBC was not compensated by the increase in MCV, and thus, triploidy decreased the haematocrit (Hct: 26.80% in 2n vs. 23.11% in 3n; P < 0.001) and total blood haemoglobin concentration (Hb: 73.74 g l 1 in 2n vs. 67.54 g l 1 in 3n; P < 0.05). In contrast, mean corpuscular hemoglobin (MCH: 40.27 pg in 2n vs. 53.28 pg in 3n; P < 0.001) was higher for 3n turbot as a result of their larger erythrocytes although MCH concentration (MCHC: 0.28 pg fL 1 in 2n vs. 0.29 pg fL 1 in 3n did not significantly differ. RBC, Hct and MCV were also determined manually using light microscopy. In general, discrepancies between the two methods were small (overall ¨7%) but the Coulter Counter tended to overestimate RBC and Hct (and thus to underestimate MCV). Nevertheless, relative differences between ploidies were very similar, thus verifying triploidy-associated changes in hematological features. These changes, as determined in the present study, are essential when evaluating the feasibility of triploid turbot for intensive aquaculture systems in which unfavorable situations may occur., 0,0000

17 figures, 8 pages, 1 table, Fish in which gynogenesis has been induced have all their chromosomes inherited from the mother and, if females are the homogametic sex, they usually are all females. Because turbot females grow faster than males, the production of all-female populations is highly desirable. The sperm of turbot is of poor quality and its larvae are small and fragile. These circumstances represent a challenge for the induction of gynogenesis in the turbot. As a first step towards this goal, effective conditions for the induction of gynogenesis through UV irradiation of sperm followed by a cold shock were established. When diluted 1:10 with Ringer-200 saline solution and placed in a thin layer (~0.3 mm), a dose-dependent effect of UV light on sperm motility was found. The dose at which both the amount of motile sperm and the duration of sperm motility was reduced to 50% of the original value (ID-50) was ~28,000 erg mm−2. A typical Hertwig effect was elicited with a dose of 30,000 erg mm−2. The resulting embryos exhibited the typical “haploid syndrome” and died shortly after hatching. Application of a cold shock (−1 to 0°C for 25 min starting at 6.5 min after fertilization) to activated eggs with UV-irradiated (30,000 erg mm−2) and diluted (1:10) sperms restored diploidy and resulted in the production of gynogenetic diploids (2n=44 chromosomes). These conditions were used in a pilot-scale experiment and found effective in inducing gynogenesis in ~47,000 eggs. The rate of gynogenesis induction was 100% as verified by an analysis with microsatellite DNA markers. Survival of the gynogenetics was approximately 10% of diploids at 6 months of age, although growth was similar during this period. If this species turns out to have female homogamety, as is the case in most pleuronectiformes examined so far, the method presented here is the first necessary step for the production of all-female populations of this economically important species, Research funded by Spanish Government CICYT grant MAR95-1855 to P.M.

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., 1,5280

The purpose of this work was to determine the efficacy of marking turbot, Scophthalmus maximus (L.), eggs and larvae with alizarin complexone (ALC) for use in enhancement programmes and in studies of the ecology and behaviour of the species. The main aim was to determine the optimum dosage and treatment time for large batches of larvae destined for release into the sea.Eggs and larvae were immersed in a range of ALC concentrations (0–60 mg L–1) for various times (3–24 h). On day 8 after hatching, eggs marked with the different ALC dosages had 99% marking success and had mainly good-quality or very-good-quality marks, whereas larvae achieved 100% success and had mainly very-good-quality marks. The best results were obtained after marking larvae with ALC at 60 mg L–1 for 6–24 h.During the first months after marking, the fluorescent rings formed in the otoliths were easily detected when directly examined with a UV microscope, no prior preparation of the otoliths was necessary, but after 7 months the marks become faint as the otoliths get thicker, so it is necessary to cut or polish the otoliths before examination., 1

Female turbot in different stages of gonadal maturation (initial, average or advanced) were injected i.m. with 5¡dkg PV of LHRHa. A limited response was detected in the initial gonadal maturation group, whereas induced spawning was experienced by 100% of average and advanced females, with a high level of synchronization. The number of ovulatory cycles was low, eggs quality variable and egg production high. New works are neccesary to improve the performance of this technique., Hembras de rodaballo en distintos estados de maduración gonadal (inicial, medio o avanzado) recibieron una inyección i.m. de 51L1Kg PV de LHRHa. La respuesta ha sido escasa o nula en hembras en desarrollo gonadal inicial, mientras que se ha inducido la puesta con un alto nivel de sincronización en el 100% de las hembras en estado medio o avanzado. El número de ciclos ovulatorios ha sido bajo, la calidad de las puestas variable y el volumen de huevos elevado. Se consideran necesarios nuevos trabajos al objeto de optimizar el rendimiento de esta técnica de control de reproducción.

n August 1983, 305 wild O-group turbot (Scophthalmus maximus L.) were captured at selected beaches of the Ría de Vigo, north-west Spain, for use in fattening trials. The fish were maintained in a flow-through system (0.015 m3 kg-1h-1) at ambient seawater temperatures (13° to 18°C) and salinities (32 to 35‰) for a period of 17 mo, and were fed a moist paste prepared in the laboratory. They were divided into two groups, the larger group having a mean individual weight at the start of experiments of 9.7 g, the smaller group having a mean individual weight of 5.7 g. Over the 17 mo period, the two groups grew to final mean wet weights of 1 905 and 1 123 g, respectively. Monthly food conversion indices, on a wet weight basis, varied between 2.1 and 5.9%. Survival during the 17 mo period varied between 90.0 and 93.1%. In a second experiment from November 1983 to February 1985, we tested the relationship between monthly growth rate and stocking density for two different groups of fish at 4 to 25 kg m-3 and at 6 to 75 kg m-3. Growth was essentially the same for both groups., 2