The immune system of sea bass, Dicentrarchus labrax, reared in aquaculture.

The sea bass Dicentrarchus labrax is one the most important seawater fish species of south Europe and Mediterranean aquaculture, and studies on its immune system are important for both scientific and applied purposes. In this paper, we summarise the results obtained in studies of the immune system in this species, and present original data on cell-mediated acquired immune response.     

Mammaglobin expression in gynecologic malignancies and malignant effusions detected by nested reverse transcriptase-polymerase chain reaction

The detection of micrometastatic disease remains a challenge for the diagnosis and monitoring of malignant disease. RT-PCR for human mammaglobin (hMAM) was recently shown to provide a sensitive method for assessing circulating breast cancer cells in peripheral blood. This study was aimed at investigating hMAM expression in normal and malignant tissue from the female genital tract and the prostate as well as in malignant effusions derived from gynecologic malignancies. hMAM expression was analyzed with nested RT-PCR in 152 samples of normal (n = 73) and malignant epithelial tissues (n = 79) and in 33 specimens of various normal mesenchymal tissue types. We found hMAM expression was not restricted to the normal mammary gland and breast carcinoma but was also detectable in most specimens of benign and malignant epithelial tissue from the ovary (97% versus 95%), uterus (both 100%), and cervix (91% versus 90%). Notably, hMAM expression was also found in benign prostatic hyperplasia (45%) and in prostate cancer (55%). A much lower expression rate was found in various normal and benign mesenchymal tissues (12%). In keeping with our previous data, hMAM expression was absent in all control samples (n = 124) of peripheral blood and bone marrow from healthy volunteers and patients with hematologic malignancies. In pleural or peritoneal effusions (n = 42) from patients with carcinomas of the breast, endometrium, or ovary, hMAM positivity was noticed in the majority of cases (74%), whereas only 52% of the specimens were cytologically positive for tumor cells. In conclusion, hMAM expression assessed by nested RT-PCR is a sensitive molecular marker for detecting micrometastatic tumor spread into pleural effusions and ascites from patients with breast cancer and various other gynecologic neoplasms.     

Phagocytic defence mechanism in sea bass (Dicentrarchus labrax L.): An ultrastructural study

Background: The ultrastructure of the phagocytic process in fish has not been established in spite of the significant morphofunctional differences detected in the fish immune system with respect to the basic immunological pattern in vertebrates. We report the ultrastructure of the bacterial phagocytic defence mechanism in sea bass (Dicentrarchus labrax L.). Methods: Head-kidney, blood, and peritoneal exudate leukocytes were challenged with Aeromonas salmonicida and Escherichia coli and processed for transmission electron microscopic study. Results: Macrophages challenged with bacteria showed changes in the cell outline, in the chromatin pattern, and in the ultrastructural features of the cytoplasm as a consequence of an activation process. The phagocytic process consists of the following: (1) Bacteria-macrophage contact. One or more spot contacts between the bacterial wall and the phagocyte membrane are observed. (2) Bacteria engulfment. Slight depressions, membrane invaginations, or cytoplasmic processes are formed at the phagocyte surface. Macrophage processes occasionally surround the bacteria, overlaping and roaming parallel, or a single, long pseudopod encircles a bacterium several times. (3) Endocytic vesicle formation. Macrophages show one or more bacteria inside membrane-bound cytoplasmic vesicles. (4) Phagolysosome formation. Some dense granules (lysosomes) fuse with the endocytic vesicle. (5) Intracelular killing/digestion. Bacteria inside the endocytic vesicles are observed both virtually intact or damaged at different digestion stages. Conclusions: Sea bass macrophages possess the mechanisms necessary to both engulf and kill bacteria. Cellular and subcellular events in the morphology of phagocytosis and lysosomal dissolution of bacteria fit the general pattern described for mammals. © 1994 Wiley-Liss, Inc.     

Myelopoiesis in the thymus of the sea bass,Dicentrarchus labrax L. (teleost)

Background: In vertebrates the thymus is primarily regarded as a lymphoid organ whose importance lies in its capacity to produce a large number of lymphocytes that enter the circulation as T cells. In higher vertebrates the organ has also been regarded as a site for my elopoiesis, but this capacity has not been observed in fish. In this study we describe morphologically the presence of intrathymic developing myeloid cells in the sea bass. Methods: The thymus samples were morphologically studied by transmission electron microscopy. Results: We describe the coexistence of cells in different stages of erythropoiesis and granulopoiesis that appear to be developing in situ in some thymus lobes. Degenerated thymocytes and epithelial-reticular cells occur simultaneously in the same areas. Conclusions: The coexistence of different cellular components of erythropoiesis and the heterophilic series of granulopoiesis with areas of necrosis suggests a relationship between both processes that is influenced by the microenvironment. Our observations also suggest that the presence of intrathymic developing myeloid cells may imply a nonimmunological role for the thymus. © 1995 Wiley-Liss, Inc.     

Granulopoiesis in the head-kidney of the sea bass (Dicentrarchus labrax L.): an ultrastructural study

The ultrastructure of granulopoiesis in the head-kidney of the sea bass (Dicentrarchus labrax L.), an euryhaline seawater teleost, was studied. The heterophilic, acidophilic and basophilic series consisting of promyelocyte, myelocyte, metamyelocyte and mature granulocyte stages are described. The granulopoietic pattern was similar to those of higher vertebrates but dissimilar to those of some other fish, suggesting that the vertebrate granulopoietic scheme was probably conserved during the evolution of at least some teleost species. Very scare presumptive blast cells were present. The heterophilic series showed three types of granules that might correspond to nucleated, azurophil and specific granules which have been described in neutrophils from mammals and human bone marrow. Neither a crystalloid nor a dense core was found in the granules of acidophilic granulocytes, where an occasional light zone could be seen. The scarce cells of the basophilic series showed similar characteristics to those described in other fish species.     

Endogenous modification of macronutrient selection pattern in sea bass (Dicentrarchus labrax, L.)

The use of a single diet with a well defined composition to feed fish throughout their life cycle is an oversimplification that probably does not respond to their metabolic requirements. For example, the seasonal reproduction that characterizes most fish species demands changes in nutritional requirements. Bearing this in mind, the macronutrient selection pattern was studied from January to August in twelve individually housed sea bass exposed to a constant photoperiod (12L:12D h) and temperature (23+/-0.5 degrees C). The endogenous "seasonal" effect on food and energy intake regulation and macronutrient selection was determined, using protein (P), carbohydrate (CH), and fat (F) packaged separately into gelatine capsules, a method that prevents the diet chemosensory properties at oropharyngeal level from interfering with macronutrient selection. Energy intake changed monthly, the highest values being recorded in May and June and the lowest values in March and April. The preliminary results illustrated "seasonal" changes in the sea bass macronutrient selection pattern with, which showed a predominantly proteinic selection during April (53% P, 21% CH, 25% F) and lipidic in July (35% P, 19% CH, 42% F); the increase in fat selection from May to July being statistically significant. This is the first evidence supporting the existence of an endogenous rhythm in the "seasonal" energy regulation and macronutrient selection in fish through post-ingestive mechanisms and probably involving chemosensory detection in the gut and/or post-absorptive mechanisms, although the exact mechanisms involved have yet to be clarified.     

Erythropoiesis and thrombopoiesis in the head-kidney of the sea bass (Dicentrarchus labrax L.): an ultrastructural study

The head-kidney of the sea bass is a source of erythropoietic and thrombopoietic cells. No significant lymphopoietic activity is found. Erythropoiesis, thrombopoiesis and granulopoiesis are intermingled, which suggests that only one environmental "niche" exists to modulate the lineage development. No numerous proerythroblasts are present. Erythropoiesis consists of a proerythroblast, basophilic erythroblast, polychromatophilic erythroblast, acidophilic erythroblast, young erythrocyte and old erythrocyte, the latter being nucleated and without nuclear vacuolation. The numbers of the free ribosomes and polyribosomes decrease progressively from proerythroblasts to young erythrocytes, suggesting a high synthetic activity from early on. Immature erythropoietic cells show pits and protrusions correlated to micropinocytotic vesicles, indicating ropheocytosis. The peripheral band of microtubules is the most remarkable cytoplasmic structure in proerythroblasts. Granular cytoplasmic inclusions or lysosomes were not observed in erythropoietic cells. The old erythrocyte reveals an electron-dense homogeneous cytoplasm with occasional mitochondria and a small Golgi apparatus. Thrombopoietic cells comprise both the immature and mature prothrombocyte and adult thrombocyte. Nuclear and cytoplasmic densities increase and the surface connected canalicular system develops during maturation. A marginal band of microtubules is present in the cytoplasm from prothrombocytes to mature thrombocytes. Some pseudopodial processes, dense granules and vesicles, probably indicating passive storage, are also observed.     

Molecular cloning and characterisation of sea bass (Dicentrarchus labrax L.) caspase-3 gene

Caspase-3 is one of the major caspases operating in apoptosis, cleaving and inactivating a number of molecules and largely contributing to the apoptotic phenotype and the dismantling of the apoptoting cell. The opening reading frame of sea bass (Dicentrarchus labrax L.) caspase-3 has 281 amino acids. The complete sequence of caspase-3 shows a very close homology to the correspondent sequence from other vertebrates, in particularly with that of Takifugu rubripes and Oryzias latipes, with 87.7 and 87.9% of similarity, respectively. Furthermore, the sea bass caspase-3 sequence retains the motifs that are functionally important, such as the pentapeptide active-site motif (QACRG) and the putative cleavage sites at the aspartic acids. In the sea bass genome, the caspase-3 gene exists as a single copy gene and is organised in six exons and five introns. A very low expression of caspase-3 was detected by RT-PCR in various organs of non-stimulated sea bass, with slightly higher levels in thymus and heart and was increased in head kidneys of Photobacterium damselae ssp. piscicida infected sea bass. This increased expression was accompanied by the occurrence of high numbers of apoptoting cells with activated caspase-3.     

Role of cholecystokinin and its antagonist proglumide on macronutrient selection in European sea bass Dicentrarchus labrax, L

Teleost fish are able to adjust their energy intake when fed on pure macronutrient sources, although the exact mechanisms regulating macronutrient selection remain unknown. Since cholecystokinin (CCK) has been reported to modify macronutrient selection patterns in mammals, we explored the effect of CCK administered orally to European sea bass on the selection of separately encapsulated macronutrients. CCK doses of 0.05, 0.15 and 0.25 mg/kg BW administered in gelatine capsules for 5 consecutive days produced a significant inhibition of total food intake (21, 28 and 51%, respectively) at highest doses, evenly reducing the quantity of all the macronutrients ingested and, without affecting their relative proportions in the diet. Oral administration of proglumide, a non-specific CCK receptor antagonist, at doses of 5, 15 and 25 mg/kg BW, induced a quantitative total food intake increase of 2, 18 and 44%, respectively, and an increase of 52% in CH and 43% in P quantity ingested at highest dose. Co-administration of proglumide (25 mg/kg BW) and CCK (0.25 mg/kg BW) in a single preload capsule blocked the effects observed with CCK alone. In conclusion, orally administered CCK induced an anorexigenic effect on both total food and single macronutrient intake, an effect that is counteracted by the CCK antagonist proglumide.     

Stromal cells, macrophages and lymphoid cells in the head-kidney of sea bass (Dicentrarchus labrax L.). An ultrastructural study

The ultrastructure of the stromal cells, macrophages and lymphoid cells in the head-kidney of the sea bass (Dicentrarchus labrax L.) was studied. Like mammals, stroma cell types here include endothelial and adventitial cells comprising the sinusoidal wall, fibroblast-like reticular cells related to scarce reticular fibres, and macrophage-type reticulum cells, the last probably corresponding to the resident macrophage population of higher vertebrates. Their possible role in the haemopoietic microenvironment is considered. Monocyte-macrophages, macrophages and melano-macrophages, probably corresponding to ontogenic or functional stages of the same cell type were identified and their functional significances are discussed. Scarce, free lymphoid cells or small clusters of lymphocytes but no lymphopoietic islets were recognizable. Large lymphocytes, small lymphocytes and very scarce developing and mature plasma cells were identified. The lymphoid function and defensive role of the head-kidney were analyzed.     

Blood cells of sea bass (Dicentrarchus labrax L.). Flow cytometric and microscopic studies

Studies of fish blood cells made to date presented numerous problems derived from both the nomenclature and the techniques used. A combination of quantitative and morphological methods is needed if the classification of fish blood cells is to advance from it present provisional state. The aim of the present paper was first to isolate sea bass blood cell populations by flow cytometry and second to characterize then microscopically. Blood cell populations from sea bass (Dicentrarchus labrax L.) were isolated according to their FSC (size) and SSC (granularity) properties by flow cytometry. The isolated populations were then processed for light and transmission and scanning electron microscopic characterization. Sea bass blood leukocytes isolated by flow cytometry consisted of two main cell subpopulations. Subsequent microscopic study of these cells revealed that the first subpopulation was composed of small cells (3-5 microm) of low granularity and consisted of thrombocytes and lymphocytes whereas, the second subpopulation was formed of 6-9 microm sized cells of high granularity consisting of granulocytes and monocyte/macrophages. The combined use of flow cytometry and electron microscopy makes it possible to characterize the different cell types present in sea bass peripheral blood with a high degree of certainty. Although sea bass basically follows the common vertebrate hematological pattern, significant modifications such as the presence of circulating immature erythrocytes, plasma cells and monocyte/macrophages and different forms of thrombocytes can be established with respect to this pattern.     

Ontogeny of the endocrine cells of the stomach of sea bass (Dicentrarchus labrax L.): an ultrastructural study

The endocrine cells present in the developing stomach of sea bass larvae have been characterized ultrastructurally. Only one endocrine cell type (type I) was found in the presumptive stomach of 9- and 12-day-old larvae, one (type II) and five (types III, IV, V, VI and VII) in the aglandular stomach of 32-, and of 39- to 46-day-old larvae, respectively, and five (types III, VIII, IX, X and XI) in the differentiated stomach of 55- and 60-day-old larvae. A maturation process was established for some of these cells. Types I, II and III and types IV and X were thought to be different maturational stages of the same endocrine cell type.     

Location and morphology of chloride cells during the post-embryonic development of the european sea bass,Dicentrarchus labrax

Location and morphology of chloride cells were studied in the sea bass ( Dicentrarchus labrax) from hatching to the juvenile stage to determine the development of the adult osmoregulatory function as seen in adult fish. During the studied developmental sequence changes were observed in the location, number, size and structure of these cells, that were studied by microscopy (light, scanning electron, transmission electron and confocal) and immunocytochemistry. Chloride cells were found on the tegument and on the gills. They were present on the tegument already at hatching, before the development of the gills. Their density as well as their association in multicellular complexes decreased during the postembryonic development. In old larvae and in juveniles, cutaneous chloride cells were associated with the fins, the developing scales and the lateral line. Gills developed gradually during the prelarval stage and the gill arches were present at mouth opening. At that time chloride cells were already numerous on the gill arches. In older larvae, during the progressive development of the gill filaments, chloride cells were numerous on these structures and formed multicellular complexes. Several stages in the differentiation of these cells were studied, including the development of the tubulovesicular system at the end of the prelarval stage, as well as the stratification appearance of the cytoplasm that was concomitant with the considerable development of the tubular system and its association with the endoplasmic reticulum during the larval period. The involvement of different epithelia in the osmoregulatory process during the postembryonic development of this species, as well as the role of chloride cells during successive developmental stages, is discussed.     

Molecular characterisation and structural analysis of an interferon homologue in sea bass (Dicentrarchus labrax L.)

The interferons (IFNs) are a large family of soluble cytokines involved in the immune response against viral pathogens. Three families of IFNs have been identified in mammals (type I, type II and type III) and, recently, homologues of type I and type II genes have been found in various teleost fish species. In this paper we report the cloning of a cDNA encoding an type I IFN molecule from sea bass (Dicentrarchus labrax L.), its expression analysis and gene structure and, finally, its 3D structure obtained by template-based modelling. The sea bass IFN cDNA consists of 1047bp that translates in one reading frame to give the entire molecule containing 185 amino acids. The analysis of the sequence revealed the presence of a putative 22 amino acid signal peptide, two cysteine residues and three potential N-glycosylation sites. The sea bass IFN gene contains four introns as with other type I IFN teleost genes, except medaka that contains three introns. Real time PCR was performed after poly I:C stimulation of DLEC cell line to investigate the expression of sea bass IFN and Mx and an induction was observed for both genes. The predicted 3D structure of sea bass IFN is characterized by an "all-alpha" domain that shows an "up-down bundle" architecture made of six helices (ABB'CDE). The two cysteine residues present in the sequence (i.e. Cys(23) and Cys(126)) are in a position and at a distance that suggest the possible formation of a disulfide bridge that may stabilize the structure. Our results will give the opportunity to investigate more in detail antiviral immune responses in sea bass and add to studies on the evolution of the IFN system in teleosts and vertebrates more generally.