Microsatellite polymorphism and population subdivision in natural populations of European sea bass Dicentrarchus labrax (Linnaeus, 1758)

Polymorphism of microsatellite markers was used to study the genetic variability and structure in natural populations of European sea bass Dicentrarchus labrax. The data consisted of six microsatellite loci analysed for 172 individuals from three samples collected in the Golfe-du-Lion (France) and one sample collected in the Golfo-de-Valencia (Spain). Our goals were (i) to assess the level of genetic variability as revealed by these markers, (ii) to estimate the genetic differentiation among natural populations within a restricted area, and (iii) to evaluate how microsatellite loci fit the predictions of the two most widely used mutation models (the infinite allele model and the stepwise mutation model). As expected, our results indicate that the genetic polymorphism is very high when compared with previously used genetic markers, the mean expected heterozygosity per locus ranging between 0.69 and 0.93. We also found that all loci but one fitted the infinite allele model better. Using this model as a lower limit, we could extrapolate from the observed diversity effective population sizes on the order of 35 000 individuals. Our results also suggest that there may be a slight genetic differentiation between the two gulfs (F_ST= 0.007, P < 0.05), indicating that the corresponding populations are likely to be dynamically independent. This finding for a species with high dispersal abilities, if confirmed, has important beatings on fish-stock assessment.     

A combined AFLP and microsatellite linkage map and pilot comparative genomic analysis of European sea bass Dicentrarchus labrax L

European sea bass (Dicentrarchus labrax L., Moronidae, Teleostei) sustains a regional fishery and is commonly farmed in the Mediterranean basin, but has not undergone much long-term genetic improvement. An updated genetic linkage map of the European sea bass was constructed using 190 microsatellites, 176 amplified fragment length polymorphisms and two single nucleotide polymorphisms. From the 45 new microsatellite markers (including 31 type I markers) reported in this study, 28 were mapped. A total of 368 markers were assembled into 35 linkage groups. Among these markers, 28 represented type I (coding) markers, including those located within the peptide Y, SOX10, PXN1, ERA and TCRB genes (linkage groups 1, 7, 16, 17 and 27 respectively). The sex-averaged map spanned 1373.1 centimorgans (cM) of the genome. The female map measured 1380.0 cM, whereas the male map measured 1046.9 cM, leading to a female-to-male (F:M) recombination rate ratio of 1.32:1. The intermarker spacing of the second-generation linkage map of the European sea bass was 3.67 cM, which is smaller than that of the first-generation linkage map (5.03 cM). Comparative mapping of microsatellite flanking regions was performed with five model teleosts and this revealed a high percentage (33.6%) of evolutionarily conserved regions with the three-spined stickleback.     

Gonadal sexual differentiation of European sea bass (Dicentrarchus labrax,L. 1758) of fingerlings in different size classes

This report describes a study of the gonad differentiation in fingerlings of European sea bass (Dicentrarchus labrax) belonging to different size groups. Fishes were divided into four groups according to their weight: Group 1 (4.5 ± 0.7 g), Group 2 (9.2 ± 0.8 g), Group 3 (14.8 ± 1.8 g), and Group 4 (21.8 ± 2.3 g). In all groups, low percentages of undifferentiated or early sexually differentiated fish were found. Higher percentages of fully differentiated ovaries were found in Groups 1 and 3. Fully differentiated testes occurred in 90% of Group 4. Ninety percent of fish in Group 2 had gonads with intratesticular oocytes. This highlights the occurrence of intersexual cases and suggests that at this size, the fish is susceptible to phenotypic plasticity. The results of the present study show that sexual differentiation of the gonads is found in fish at 4.5 g weight. The ambiguous relationship between the observed gonad morpho-functional characteristics and fish size suggests a subject-dependent sexual differentiation.     

QTL for body weight,morphometric traits and stress response in European sea bass Dicentrarchus labrax

Natural mating and mass spawning in the European sea bass (Dicentrarchus labrax L., Moronidae, Teleostei) complicate genetic studies and the implementation of selective breeding schemes. We utilized a two‐step experimental design for detecting QTL in mass‐spawning species: 2122 offspring from natural mating between 57 parents (22 males, 34 females and one missing) phenotyped for body weight, eight morphometric traits and cortisol levels, had been previously assigned to parents based on genotypes of 31 DNA microsatellite markers. Five large full‐sib families (five sires and two dams) were selected from the offspring (570 animals), which were genotyped with 67 additional markers. A new genetic map was compiled, specific to our population, but based on the previously published map. QTL mapping was performed with two methods: half‐sib regression analysis (paternal and maternal) and variance component analysis accounting for all family relationships. Two significant QTL were found for body weight on linkage group 4 and 6, six significant QTL for morphometric traits on linkage groups 1B, 4, 6, 7, 15 and 23 and three suggestive QTL for stress response on linkage groups 3, 14 and 23. The QTL explained between 8% and 38% of phenotypic variance. The results are the first step towards identifying genes involved in economically important traits like body weight and stress response in European sea bass.     

Embryonic ionocytes in the European sea bass (Dicentrarchus labrax): Structure and functionality

Early ionocytes have been studied in the European sea bass (Dicentrarchus labrax) embryos. Structural and functional aspects were analyzed and compared with those observed in the same conditions (38 ppt) in post hatching stages. Immunolocalization of Na⁺/K⁺‐ATPase (NKA) in embryos revealed the presence of ionocytes on the yolk sac membrane from a stage 12 pair of somites (S), and an original cluster around the first gill slits from stage 14S. Histological investigations suggested that from these cells, close to the future gill chambers, originate the ionocytes observed on gill arches and gill filaments after hatching. Triple immunocytochemical staining, including NKA, various Na⁺/K⁺/2Cl^? cotransporters (NKCCs) and the chloride channel “cystic fibrosis transmembrane regulator” (CFTR), point to the occurrence of immature and mature ionocytes in early and late embryonic stages at different sites. These observations were completed with transmission electronic microscopy. The degree of functionality of ionocytes is discussed according to these results. Yolk sac membrane ionocytes and enteric ionocytes seem to have an early role in embryonic osmoregulation, whereas gill slits tegumentary ionocytes are presumed to be fully efficient after hatching.     

Combined effects of physicochemical variables (pH and salinity) on sperm motility: characterization of sperm motility in European sea bass Dicentrarchus labrax

Gamete activation in fish is an important step in terms of artificial fertilization of oocytes, cryopreservation studies and other experimental manipulations. Salinity and pH differences in activation media affect to sperm motility and fertilizing ability. These experiments were therefore designed to investigate the combined effects of pH (range 5.0–9.0) and salinity (20, 30, 37, and 45‰) of activation media on sperm motility of European sea bass Dicentrarchus labrax. The best results were obtained at salinity 37‰ and a pH of 9.0. Our results also demonstrated that non-progressive motility at salinity 45‰ was observed in the range of 5.0–9.0 pH. In conclusion, spermatozoa can be motile at a wide range of pH and salinity values although the percent of motile spermatozoa and motility duration are negatively affected by low pH values.     

Sexual differentiation and juvenile intersexuality in the European sea bass (Dicentrarchus labrax)

Sexual differentiation was studied at the histological level using a mixture of 30 families of sea bass Dicentrarchus labrax . Most of the fish (93%) differentiated into males as usually observed in farmed populations. All testes were differentiated when the males reached 12 cm and no more undifferentiated fish were found from 419 days post-fertilization (p.f.). In 28% of the males, among the biggest, sexual differentiation had already begun at 168 days p.f. (8.3–9.5 cm) and these fish started spermatogenesis in their first year of life. The other males differentiated later and remained immature at the end of their first year of life. Ovaries could be identified at the histological level from the age of 168 days p.f. (7.9–9.0 cm) and the females became significantly longer than the males from the age of 191 days p.f., i.e. during the process of ovarian differentiation. In the studied group, 62% of the males developed intratesticular oocytes. Such intersexuality had no consequence on growth rate. Intratesticular oocytes were also recorded in testes of wild males originating from Atlantic (Britain and Gulf of Gascogne) and West Mediterranean showing that juvenile intersexuality is not restricted to farmed populations but is a widespread phenomenon in sea bass.     

The effect of environmental salinity on the proteome of the sea bass (Dicentrarchus labrax L.)

The European sea bass, Dicentrarchus labrax L., tolerates a range of salinities from freshwater to hyper-saline. To study differences in protein expression, fish were reared in both freshwater and seawater. After 3-month acclimation, gill and intestine epithelia were collected and the soluble protein extracted. In all, 362 spots were differentially expressed in the gills and intestines of fishes reared in seawater compared to those from freshwater. Fifty differential protein spots were excised from a colloidal Coomassie-stained gel. Nine separate protein spots were identified unambiguously by mass spectrometry and database searching. Among the six proteins over-expressed in gill cells in seawater, five were cytoskeleton proteins and one was the aromatase cytochrome P450. In gill cells under freshwater conditions, the two over-expressed proteins identified were the prolactin receptor and the major histocompatibility complex class II β -antigen. In intestinal cells under freshwater conditions, the Iroquois homeobox protein Ziro5 was upregulated over ninefold. The expression of these proteins, their possible direct or indirect roles in the adaptation of D. labrax to salinity, and their correspondences with a previous study are discussed.     

A microsatellite linkage map of the European sea bass Dicentrarchus labrax L

A genetic linkage map of the European sea bass (Dicentrarchus labrax) was constructed from 174 microsatellite markers, including 145 new markers reported in this study. The mapping panel was derived from farmed sea bass from the North Adriatic Sea and consisted of a single family including both parents and 50 full-sib progeny (biparental diploids). A total of 162 microsatellites were mapped in 25 linkage groups. Eleven loci represent type I (coding) markers; 2 loci are located within the peptide Y (linkage group 1) and cytochrome P450 aromatase (linkage group 6) genes. The sex-averaged map spans 814.5 cM of the sea bass genome. The female map covers 905.9 cM, whereas the male map covers only 567.4 cM. The constructed map represents the first linkage map of European sea bass, one of the most important aquaculture species in Europe.     

Molecular cloning and characterization of sea bass (Dicentrarchus labrax, L.) Tapasin

Mammalian tapasin (TPN) is a key member of the major histocompatibility complex (MHC) class I antigen presentation pathway, being part of the multi-protein complex called the peptide loading complex (PLC). Several studies describe its important roles in stabilizing empty MHC class I complexes, facilitating peptide loading and editing the repertoire of bound peptides, with impact on CD8⁺ T cell immune responses. In this work, the gene and cDNA of the sea bass (Dicentrarchus labrax) glycoprotein TPN have been isolated and characterized. The coding sequence has a 1329 bp ORF encoding a 442-residue precursor protein with a predicted 24-amino acid leader peptide, generating a 418-amino acid mature form that retains a conserved N-glycosylation site, three conserved mammalian tapasin motifs, two Ig superfamily domains, a transmembrane domain and an ER-retention di-lysine motif at the C-terminus, suggestive of a function similar to mammalian tapasins. Similar to the human counterpart, the sea bass TPN gene comprises 8 exons, some of which correspond to separate functional domains of the protein. A three-dimensional homology model of sea bass tapasin was calculated and is consistent with the structural features described for the human molecule. Together, these results support the concept that the basic structure of TPN has been maintained through evolution. Moreover, the present data provides information that will allow further studies on cell-mediated immunity and class I antigen presentation pathway in particular, in this important fish species.     

Induction of Tetraploid Gynogenesis in the European Sea Bass (Dicentrarchus labrax L.)

A preliminary study on tetraploid gynogenetic induction in the European sea bass was performed by pressure-blocking the second polar body release and the first cleavage in eggs fertilized with ultraviolet-irradiated sperm. Fertilization of eggs with genetically inactivated sperm produced only haploid development that terminated around hatching. Pressure treatments (8.500 psi for 2 min) applied at 6 and 65 min after fertilization (a.f.) produced variable levels (7–95%) of tetraploid larvae at hatching. A small proportion of mosaics (3.8n/4.2n) was also recorded.     

Aspects of the reproductive biology of the bass, Dicentrarchus labrax L. II. Fecundity and pattern of oocyte development

The European bass, Dicentrarchus labrax L., in common with most temperate marine teleosts, exhibits a distinct seasonal reproductive cycle. Its reproductive strategy is characterized by a high absolute fecundity (2-25 × 10⁵), both absolute and relative fecundity increasing with size and age. The cyclic pattern of oocyte development has been determined by histology and from oocyte size frequency analysis. Results show that the bass exhibits group-synchronous oocyte development and is a 'fractional spawner', i.e. it spawns a number (3–4) of discrete clutches in quick succession, successive clutches containing fewer oocytes. All oocytes which are recruited into the secondary growth phase (> 110 μrn) are either spawned that season or become atretic, i.e. no secondary oocytes are held over for the subsequent reproductive season.     

The effects of capture, net retention and preservation upon lengths of larval and juvenile bass, Dicentrarchus labrax (L.)

Relationships between live and preserved lengths of larval and juvenile bass, Dicentrarchus labrax , subjected to simulated capture, net retention and preservation were investigated. Bass larvae and juveniles shrank during the period of net retention following death. Preserved lengths of larvae, but not of juveniles, decreased significantly when increased periods of net retention preceded preservation. The time which larvae or juveniles remained in 4% formaldehyde in distilled water buffered with 3gl⁻¹ sodium acetate trihydrate or in 70% ethanol in distilled water neutralized with 1gl⁻¹ calcium carbonate powder, had no significant effect upon preserved length. Appropriate data were pooled to calculate transformation factors for estimating live from preserved length. Shrinkage phenomena, and their effects, are discussed. It is suggested that larval bass captured and preserved during standard ichthyoplankton surveys are unsuitable for determination of live length.     

Ceratomyxa spp. (Protozoa: Myxosporea) infections in wild and cultured sea bass, Dicentrarchus labrax, from the Spanish Mediterranean area

Ceratomyxa labracis and Ceratomyxa diplodae , parasitic in the gall bladder of wild and cultured Mediterranean sea bass ( Dicentrarchus labrax L.) were studied. Prevalences were clearly higher in cultured fish, especially for C. labracis . Starvation seems to favour the appearance of ceratomyxosis. Infection varied seasonally, with maxima in winter and minima in summer, a pattern almost contrary to temperature. Prevalence of C. labracis decreased in older fish. No clear influence of host sex was observed, and the apparent negative association between the two Ceratomyxa spp. was not confirmed statistically. Different degrees of histopathological damage of the gall bladder were observed by light and electron microscopy, mainly consisting of vacuolation, deformation or even necrosis of epithelial cells, and thickening and inflammation of the subepithelial connective tissue. Trophozoites appeared frequently lining the epithelium, closely attached to the cell surface or even forming invaginations in it. The importance of ceratomyxosis for sea bass and the influence of culture and stress conditions are discussed.     

Production and Characterization of a Continuous Embryonic Cell Line from Sea Bass (Dicentrarchus labrax L.)

Continuous cell lines represent an important tool both for biological studies and for their applications in marine biotechnology. In this article we describe the production and characterization of a continuous adherent cell line, named DLEC, derived from early embryos of the European sea bass Dicentrarchus labrax L. (Actinopterygii, Moronidae). Cells were obtained by disrupting 2- to 12-hour-old embryos and culturing resulting cells at 18°C in RPMI medium containing 5% fetal calf serum (FCS) and 10% supernatant fraction of the embryo homogenate. After 8 weeks culture medium was replaced with Liebovitz's L15 medium containing 10% FCS and DLEC cells started proliferation. Subsequently, they were continuously cultured until the 50th passage without evident changes in their morphology. DLEC cells show a fibroblast-like shape and a modal chromosome number of 48, as do the wild-type cells; conversely the constant presence of six to nine meta-submetacentric elements in the karyotype (vs. zero to two in the wild-type) indicates the occurrence of chromosomal rearrangements during stabilization. DLEC cells are sensitive to substances known to induce differentiation of mammalian cells such as retinoic acid and phorbol esters. They have been transfected using liposomes with a commercial plasmid vector containing a reporter gene, thus suggesting a possible importance as an alternative expression system of recombinant vertebrate proteins in teleost cells.     

Coupling of heart rate and locomotor activity in sole, Solea solea (L.), and bass, Dicentrarchus labrax (L.), in their natural environment by using ultrasonic telemetry

Field measurements of distance moved and heart rate in sole, Solea solea (L.) and bass, Dicentrarchus labrax (L.) using ultrasonic telemetry revealed two different strategies. Heart rate in the sole increases during activity, accurately reflecting fluctuation in metabolic rate and so can be used as a measure of metabolic rate in the field. In contrast, the relatively stable value of the heart rate in bass during the whole tracking period whatever the activity level suggests that in this species heart rate in the field cannot be associated with metabolic rate determination.     

A model for acute iron overload in sea bass (Dicentrarchus labrax L.)

Evaluation of several parameters involved in iron metabolism was carried out after intraperitoneal (i.p.) injection with iron dextran (IDx) in sea bass (Dicentrarchus labrax L.). After treatment, a rapid mobilization of IDx from the peritoneal cavity to other organs was observed. This was followed by a modification of normal peripheral blood iron parameters. Total iron (TI) and transferrin saturation (TS) rose rapidly, to 4.14 microg/ml and 83.7%, respectively, on day 3. In contrast, unsaturated iron binding capacity (UIBC) dropped from 3.19 microg/ml (at day 0) to 0.90 microg/ml on day 3. Tissue iron content was determined by atomic absorption spectometry (AAS). Three days post-IDx injection, values of iron concentration in liver, spleen and head kidney were significantly higher than control values (15, 6 and 9-fold increase, respectively). Samples of liver, spleen and head kidney were processed for routine histology, and the Perl's method was used for iron staining. Histological sections of the IDx-treated animals showed iron deposition in all tissues studied. In the liver, the iron was evenly distributed over the whole organ, being present in the hepatocytes. In the head kidney and spleen, the iron deposition was mainly observed in the melanomacrophage centres (MMCs). The present study characterizes several parameters involved in iron metabolism, and develops a fish model, of iron overload, which can be used in further studies of iron toxicity and iron-induced susceptibility to bacterial infections.     

Molecular cloning and characterization of sea bass (Dicentrarchus labrax,L.) calreticulin

Mammalian calreticulin (CRT) is a key molecular chaperone and regulator of Ca²⁺ homeostasis in endoplasmic reticulum (ER), also being implicated in a variety of physiological/pathological processes outside the ER. Importantly, it is involved in assembly of MHC class I molecules. In this work, sea bass (Dicentrarchus labrax) CRT (Dila-CRT) gene and cDNA have been isolated and characterized. The mature protein retains two conserved motifs, three structural/functional domains (N, P and C), three type 1 and 2 motifs repeated in tandem, a conserved pair of cysteines and ER-retention motif. It is a single-copy gene composed of 9 exons. Dila-CRT three-dimensional homology models are consistent with the structural features described for mammalian molecules. Together, these results are supportive of a highly conserved structure of CRT through evolution. Moreover, the present data provides information that will allow further studies on sea bass CRT involvement in immunity and in particular class I antigen presentation.     

Vitamin E in early stages of sea bass (Dicentrarchus labrax) development

This study reports titration of vitamin E levels in the sea bass (Dicentrarchus labrax) using high-pressure liquid chromatography. The first part of the work is devoted to vitamin E detection in: (1) plasma of maturing females and males characterized by different body sizes; (2) seminal fluid and eggs; and (3) developing embryos of sea bass fed with vitamin E. In the second part of the study, variations of vitamin E levels during larval development are analyzed. The results show a direct correlation between plasma vitamin E content and body size for both adult male and female sea bass. High vitamin E levels were found in seminal fluid, in eggs before and after fertilization, and in embryos during development and at hatching, whereas vitamin E level was low in dead embryos and in embryos with limited survival. During larval development, the vitamin E content decreased slowly but steadily during the first four days of larval growth; subsequently, it progressively increased from day 9 to day 40. In teratogenic larvae, vitamin E content was significantly higher than in normal larvae. This study provides evidence on how vitamin E exerts an antioxidant defense in sea bass reproduction.