Cellular activities during a mixed leucocyte reaction in the teleost sea bass Dicentrarchus labrax

In this investigation a number of "in vitro" activities of sea bass peripheral blood leucocytes (PBL) against allogeneic PBL inactivated by irradiation were studied. Stimulator PBL were cultured with inactivated allogeneic PBL, and direct counting of lymphocytes was done after 2 weeks by immunofluorescence and flow cytometry using mAbs DLT15 and DLIg3 specific for T-cells and B-cells, respectively. In a one-way mixed leucocyte reaction (MLR), results showed an increase of T lymphocytes, whereas B lymphocytes had values similar to those in control PBL. The increase of T-cells in MLR cultures was also confirmed using RT-PCR by analyzing the expression of the T-cell receptor (beta-subunit) mRNA. The addition of 5 microg/ml of cyclosporin A (CsA) to the MLR caused a significant decrease in T-cell proliferation. Leucocytes from MLR cultures displayed an enhanced cytotoxic activity against xenogeneic target cells with respect to control PBL, raising the possibility of the presence of cytotoxic-like T lymphocytes. Cellular activation of PBL was confirmed in 2 weeks MLR by measuring antibody-induced intracellular Ca(++) mobilization with Fura-2 AM. This work represents the first direct quantitative determination of an "in vitro" T-cell activity in a teleost species.     

Neutral amino acid transport by marine fish intestine: role of the side chain

This work was devoted to the study of the structure-affinity relationships in neutral amino acid transport by intestinal brush border of marine fish (Dicentrarchus labrax). The effects of the length of the side chain on kinetics of glycine, alanine, methionine and amino isobutyric acid were investigated. In the presence of K⁺ two components were characterized: one is saturable by increased substrate concentrations, whereas the other can be described by simple diffusion mechanism. Simple diffusion, a passive, non-saturable, Na⁺-independent route, contributes largely to the transport of methionine and to a much lesser extend to alanine, glycine or alphaaminoisobutyric acid uptakes. If a branched chain is present, as in the case of amino isobutyric acid, diffusion is low. A Na⁺-independent, saturable system has been fully characterized for methionine, but not for branched amino acids such as amino isobutyric acid. In the presence of Na⁺ saturable components were shown. Two distinct Na⁺-dependent pathways have been characterized for glycine uptake, with low and high affinities. For alanine and methionine only one Na⁺-dependent high affinity system exists with the same half-saturation concentration and the same maximum uptake at saturable concentrations. Glycine high affinity system has the same half-saturation concentration as methionine or alanine uptake, whereas maximum uptake is lower. The substitution of the hydrogen by a methyl group results in a severe decrease of uptake (aminoisobutyric acid). Mutual inhibition experiments indicate that the same carriers could be responsible for methionine and alanine uptakes and probably glycine Na⁺-dependent uptake. The influence of Na⁺ concentrations (100^-1 mol·l^-1) on amino acid uptake was examined. Glycine, alanine, methionine and amino isobutyric acid transport can be described by a hyperbolic function, with a saturation uptake which is highly increased for methionine. However, the half-saturation concentration does not seem to be strongly affected by the amino acid structure. The effect of Na⁺ concentration (25 and 100 mmol·l^-1) on the kinetics of methionine uptake have been also examined. The maximum uptake of the saturable system clearly shows a typical relationship with concentration.Abbreviations [AA] amino acid concentration - AIB aminoisobutyric acid - [I] Inhibitor amino acid concentration - J _i uptake in the presence of inhibitor - J _o uptake without inhibitor - K _d passive diffusion constant - K _i inhibitor constant - K _t concentration of test amino acid for half-maximal flux - MES 2[N-morpholino]ethanesulphonic acid - V _max maximum uptake at saturable amino acid concentrations - V _tot total amino acid uptake     

The larval development of lateral musculature in gilthead sea bream Sparus aurata and sea bass Dicentrarchus labrax

Fibre-type differentiation of the lateral musculature has been studied in Sparus aurata (L.) and Dicentrarchus labrax (L.) during larval development. Histochemical and ultrastructural techniques show two presumptive muscle layers and two germinative zones of presumptive myoblasts. At hatching, myotomal muscle consists of a monolayer of thin undifferentiated cells near the skin (first germinative zone) overlying another mono-layer of small diameter fibres extending hypaxially and epaxially away from the transverse septum. Below this, there is a much thicker, deep layer of fibres, generally large in diameter and polygonal in shape. The presumptive myoblasts are located between these two layers of fibres in the second germinative zone. Initially, the superficial and deep muscle fibres show high and low myosin ATPase activity, respectively. Both layers grow by generating new fibres from the two mentioned germinative zones. At the end of larval life, the superficial layer changes its histochemical profile from high to low myosin ATPase activity and, at the same time, intermediate or pink muscle fibres can be observed by oxidative activity (the NADH-TR reaction). Morphometric analysis shows a significant increase in mean fibre diameter during successive ages, as shown by the Student's t-test (hypertrophic growth). Skewness and kurtosis values of fibre diameters point to the generation of a new fibre population from the germinative zones (hyperplastic growth).     

cDNA sequence and tissue expression of an antimicrobial peptide, dicentracin; a new component of the moronecidin family isolated from head kidney leukocytes of sea bass, Dicentrarchus labrax

A 483-bp cDNA was isolated from sea bass (Dicentrarchus labrax) head kidney leukocytes, dicentracin, using PCR primers designed from conserved moronecidin domains. Gene bank analysis revealed that dicentracin cDNA belongs to the moronecidin family. As deduced from alignment with Morone chrysops moronecidin, the precursor of 79 aa appeared to be composed of a signal peptide of 22 aa, followed by the mature AMP (antimicrobial peptide) of 22 aa named dicentracin, and a C-terminal extension of 35 aa. Dicentracin precursor displayed 3 aa substitutions with other moronecidin sequence but none in the mature peptide sequence. Using in situ hybridization assay, dicentracin gene expression was observed in 68–71% of peripheral blood leukocytes, kidney leukocytes or peritoneal cavity leukocytes without significant statistical differences. Dicentracin mRNA was observed in most of the granulocytes, as well as in monocytes from both peripheral blood and head kidney, and in macrophages from peritoneal cavity. No expression was observed in thrombocytes or in lymphocytes.     

Detecting the degree of establishment of a non-indigenous species in coastal ecosystems: sea bass <Emphasis Type="Italic">Dicentrarchus labrax</Emphasis> escapes from sea cages in Canary Islands (Northeastern Central Atlantic)

This article gives an insight into the degree of establishment and potential impact of escaped sea bass in shallow coastal habitats of Tenerife. We studied spatial distribution, abundance, size frequency and use of habitat by visual census. Stomach contents and gonadal maturity of escaped sea bass were analysed. Results show that abundance of escaped sea bass is related to distance from to the nearest fish farm and to the habitat complexity. The most frequent size found in the wild corresponds to that of first marketing size. Stomach contents indicate that escaped sea bass were able to exploit natural resources, especially fish. The presence of a female with mature gonads indicates that conditions for successful maturation exist. However, further studies are needed to ensure whether or not escaped sea bass are able to establish self-reproducing populations. Thus, we can conclude that sea bass seem to be already established in shallow coastal habitats of Tenerife, but the population may depend mainly on escapees. As a result of being an opportunistic piscivore, sea bass could be predating on and competing with other native species that are economically important. These results are useful to develop a risk assessment based on quantitative data to predict negative effects. Moreover, management policies such as communication of massive escapes or mitigation measures are needed to prevent future impact on the marine environment. Handling editor: P. Viaroli     

Ontogeny of the endocrine pancreas in sea bass (Dicentrarchus labrax)

Summary The development of the endocrine pancreas of the teleost sea bass (Dicentrarchus labrax, L.) was examined from hatching to 61 days, using the peroxidase-antiperoxidase technique for light microscopy. Mammalian and bonito insulin (mI and bI)-, salmo somatostatin-25 (SST-25)-, somatostatin-14 (SST-14a and b)-, glucagon-, bovine pancreatic polypeptide (PP)-, peptide tyrosine-tyrosine (PYY)- and salmo neuropeptide Y (NPY)-like immunoreactivity was demonstrated. Four ontogenetic stages were established according to the organization and immunostaining of the endocrine cells. One cell strand or primordial cord showing mI/bI- and SST-25/SST-14a-like immunoreactivity was first found at hatching in the dorsal epithelium of the anterior zone of the midgut (stage 1). One primitive islet, comprising outer SST-25/SST-14a- and inner mI/bI- and SST-14a/ SST-14b-immunoreactive cells, was found in 2- to 5-day-old larvae (stage 2). One single islet, in which glucagon-immunoreactive cells appear in the periphery, was found in larvae from 9 to 20 days after hatching (stage 3). One big islet containing, in addition, PP-immunoreactive cells in the outer region and slender cell processes which showed PYY-like immunoreactivity, was found from 25 to 61 days after hatching. During this period, primordial islets, composed of SST-25- and bI-immunoreactive cells, and clustered or isolated pancreatic endocrine cells, close to the pancreatic duct, as well as small and intermediate islets (secondary islets), in which glucagon, PP, PYY and NPY seem to be co-localized, were progressively found (stage 4). The origin of the endocrine pancreas of sea bass, and the ontogenetic and phylogenetic significance, are discussed.     

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.     

Distribution of salmon gonadotrophin releasing-hormone in the brain and pituitary of the sea bass (Dicentrarchus labrax)

Summary The distribution of salmon gonadotrophin-releasing hormone (sGnRH) was studied in the brain and pituitary of two-year-old immature sea bass (Dicentrarchus labrax) by means of an enzymoimmunoassay (EIA) for sGnRH and immunocytochemistry. The EIA for sGnRH is a competitive assay using a tracer made of sGnRH coupled to acetylcholinesterase from an electric eel. The separation of free and bound tracer is achieved by coating the plates with mouse anti-rabbit IgG monoclonal antibodies. Displacement curves generated by sGnRH and extracts from pituitary and different brain regions showed a good parallelism allowing the assay to be used for sGnRH measurements in this species. Although all parts of the brain contained measurable levels of sGnRH, the highest concentrations were found in the pituitary, the olfactory bulbs and the telencephalon. These data were confirmed by immunocytochemistry. Cell bodies were found in the olfactory bulbs, ventral telencephalon, preoptic region and mediobasal hypothalamus. Immunoreactive fibers could be observed in all parts of the brain including the optic tectum, the cerebellum (corpus and valvula), the vagal lobe, the medulla oblongata and the rostral spinal cord. In most cases, these fibers do not form well defined bundles; however, there was clearly a continuum of immunoreactive fibers, extending from the olfactory bulbs to the pituitary, and along which all the cell bodies described above were located. In the ventral telencephalon and the preoptic region, clear pictures of varicose positive fibers contacting immunoreactive perikarya could be observed. These data indicate that sGnRH is most likely an endogenous peptide in the brain of the sea bass, although the presence of other forms of GnRH cannot be excluded at this point. This study also demonstrates that the general organization of the GnRH systems in the sea bass is highly similar to what has been described in most freshwater teleost species, and provides basis for further studies on the neuroendocrine control of gonadotrophin release in this commercially important species.     

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.     

Ontogeny of the endocrine pancreas in sea bass (Dicentrarchus labrax): an ultrastructural study. II. The big and secondary islets

The big and secondary islets of sea bass larvae were characterized ultrastructurally from, 25 to 60 days after hatching. From the 25th day, big islets consisted of inner type II and III, external type I and peripheral type IV cells. From the 55th day, type V cells appeared in limited peripheral areas. Secondary islets, first found in 32-day-old larvae, were made up of inner type II and III, external type I, and peripheral either type IV and V cells (type I islets), or only type V cells (type II islets). Type I cells contained secretory granules with a fine granular, low-medium electron-dense material, whereas the secretory granules of type II cells were smaller and had a high electron-dense core with diffused limits; needle and rod-like crystalloid contents were occasionally found. Type III secretory granules posessed a homogeneous, high or medium electron-dense material with or without a clear halo. Type IV cells had secretory granules with a polygonal dense core embedded in a granular matrix and granules containing a high or medium electron-dense material. Type V cells had secretory granules with a fine granular, high or medium electron-dense content. These cell-types correlated with cells previously identified immuno-cytochemically, as regards to their distribution in the islets, and related to those characterized ultrastructurally in adult specimens. Thus, types I, II, III, IV and V correspond to D₁, B, D₂, A and PP cells, respectively. From the 32nd day onwards, endocrine cells of all the different types were found grouped, type V cells also being observed in isolation close to pancreatic ducts and/or blood vessels. Small groups consisting of type I and II cells were found in 40-day-old larvae. A mitotic centroacinar ductular cell containing some secretory granules similar to those of type I cells, was seen adjacent to a type I cell. As the larvae grew older, the endoplasmic reticulum developed, the number of free ribosomes decreased, and the number and size of the secretory granules increased. Dark type I, II, III, IV and V cells were found in the islets and cell clusters from the 55th day onwards.     

Ontogeny of the endocrine pancreas in sea bass (Dicentrarchus labrax): an ultrastructural study. I. The primordial cord and the primitive,single and primordial islets

The primordial cord and the primitive, single and primordial islets present in the 3 earliest stages of the developing endocrine pancreas of sea bass were studied ultrastructurally. The primordial cord consisted of type I and II cells and was included in the gut. Besides these cell types, X cells were seen in the primitive islet. The single islet was made up of type I, II, III and IV cells. A correlation between these endocrine cell-types and cells previously identified immunocytochemically, was established. Type I, II, III and IV cells, correlated respectively with SST-25-, insulin-, SST-14- and glucagon-immunoreactive cells, and could be related to the D₁, B, D₂ and A cells, respectively, of older larvae and adult sea bass. Each cell type shows characteristic secretory granules from its first appearance. A progressive development of the organelles and an increase in the number and size of the secretory granules, whose ultrastructure also varied, was observed in the endocrine cells of the primordial cord and the succeeding islets. In 25-day-old larvae at the beginning of the fourth developmental stage, the primordial islet, the first ventral islet found, was close to a pancreatic duct and blood vessel, and consisted of type I and II cells whose ultrastructure was similar to that of the type I and II cells in the primordial cord. These data suggest a ductular origin for the pancreatic endocrine cells in the ventral pancreas. It is suggested that although endocrine cells undergo mitosis, their increase in number during the earliest development stages is principally due to the differentiation of surrounding cells.     

Ontogeny of some endocrine cells of the digestive tract in sea bass (Dicentrarchus labrax): An immunocytochemical study

Serotonin- and ten peptide-immunoreactive (IR) cell types were identified in the digestive tract of sea bass (Dicentrarchus labrax L.) larvae of four morphofunctional phases ranging in age from hatching to 61 days. The sequence of appearance and location of endocrine cells during ontogenetic development of the larvae was determined. The differentiation of endocrine cells followed a distal-proximal gradient in the gut which paralleled the morphofunctional differentiation. Serotonin-IR cells were identified in the last portion of the digestive tract from phase I onwards and in the gastric region from phase III, before these regions were morphofunctionally differentiated; met-enkephalin-IR cells were identified from phase II onwards in both the differentiated rectum and the undifferentiated intestine; cholecystokinin (CCK)- and synthetic human gastrin-34-IR cells were located only in the intestine and first found in the undifferentiated intestine of phase II; human gastrin-17-, peptide YY (PYY)- and neuropeptide Y (NPY)-IR cells appeared in the intestine from phase II and in stomach in phase IV, when it showed gastric glands; pancreatic polypeptide (PP)- and glucagon-IR cells were observed in both intestine and stomach, but insulin- and somatostatin-IR cells only in stomach, from phase III, during which the intestine but not the stomach was differentiated. PP- and PYY-, PP- and glucagon-, and PYY- and glucagon-like immunoreactivities coexisted from their first appearance in some cells of the gut.     

Ultrastructure of the peritoneal exudate cells of seawater teleosts,seabream (Sparus aurata) and sea bass (Dicentrarchus labrax)

The peritoneal exudates of seabream and sea bass consist of granulocytes, lymphocytes and macrophages. These cells show conspicuous ultrastructural differences from the same cell-types of blood and head-kidney, which have not been reported previously. Peritoneal exudate granulocytes differ from their corresponding circulating or head-kidney forms in the following way: (a) they are larger in size, and (b) their abundant cytoplasmic granules have some new ultrastructural features, and a new granule population might also be present. Likewise, lymphocytes also show a noticeable difference; they contain a sparse population of small dense cytoplasmic granules. Monocytes, macrophages, and transitional forms between these two cell-types, are also found. The percentage of peritoneal exudate cell-types is different in seabream and sea bass. Macrophages in sea bass represent the most abundant peritoneal exudate cell-type. However, seabream shows lower percentages of macrophages than granulocytes.     

Development and linkage relationships for new microsatellite markers of the sea bass (Dicentrarchus labrax L.)

Twenty-eight polymorphic microsatellites were isolated from the sea bass, Dicentrarchus labrax, using a microsatellite enrichment protocol and selective hybridization with oligonucleotide probes. Analysis for these markers and 11 recently described microsatellites of D. labrax found linkage between 26 loci and revealed eight linkage groups.     

Dynamic expression of immune response genes in the sea bass, Dicentrarchus labrax, experimentally infected with the monogenean Diplectanum aequans

The sea bass, Dicentrarchus labrax, is one of the most extensively farmed marine fishes in the Mediterranean. Under the high-density condition common in aquaculture, the monogenean gill parasite Diplectanum aequans can cause significant economic losses. This study used real-time quantitative PCR to investigate the dynamic expression of immune response genes in sea bass infected with Diplectanum aequans. The target genes, interleukin-1 (IL-1beta, transforming growth factor (TGF-beta and T-cell receptor (TCR-beta), were studied in the gills and spleen of the sea bass from the first day of infection until thirty days post- infection. Our results showed that there was an increase in IL-1beta gene expression in the spleen and gills and in TGF-beta gene expression in the gills of infected fish. These results show that parasitic infection induced a local inflammatory reaction and that reaction was restricted to the site of infection. Finally, the absence of relationship between TCR-beta expression and the parasitic infection suggests that the adaptive immune system is not involved in the response against this parasite.     

Real-time polymerase chain reaction, in situ hybridization and immunohistochemical localization of insulin-like growth factor-I and myostatin during development of Dicentrarchus labrax (Pisces: Osteichthyes)

The distribution of insulin-like growth factor-I (IGF-I) and myostatin (MSTN) was investigated in sea bass (Dicentrarchus labrax) by real-time polymerase chain reaction (PCR), in situ hybridization (ISH) and immunohistochemistry. Real-time PCR indicated that IGF-I mRNA increased from the second day post-hatching and that this trend became significant from day 4. ISH confirmed a strong IGF-I mRNA expression from the first week post-hatching, with the most abundant expression being detected in the liver of larvae and adults. Real-time PCR also showed that the level of MSTN mRNA increased significantly from day 25. The expression of MSTN mRNA was higher in muscle and almost absent in other anatomical regions in both larvae and adults. Interestingly, the lateral muscle showed a quantitative differential expression of IGF-I and MSTN mRNAs in red and white muscle, depending on the developmental stage examined. IGF-I immunoreactivity was detected in developing intestine at hatching and in skeletal muscle, skin and yolk sac. MSTN immunostaining was evident in several tissues and organs in both larvae and adults. Both IGF-I and MSTN proteins were detected in the liver from day 4 post-hatching and, subsequently, in the kidney and heart muscle from day 10. Our results suggest, on the basis of a combined methodological approach, that IGF-I and MSTN are involved in the regulation of somatic growth in the sea bass. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. This research was supported by grants from the Italian Ministero dell’Università e della Ricerca Scientifica e Tecnologica (MIUR) and by the University of Padua (Progetto di Ateneo).     

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.