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.     

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.     

Morphofunctional ontogeny of the urinary system of the European sea bass Dicentrarchus labrax

European sea bass (Dicentrarchus labrax) are euryhaline fish that tolerate wide salinity fluctuations owing to several morphofunctional adaptations. Among the osmoregulatory sites (tegument, branchial chambers, digestive tract, urinary system), little is known about the kidney and the urinary bladder. The present study describes the ontogeny of the urinary system (kidney and urinary bladder) and focuses on the progressive expression of the Na⁺/K⁺-ATPase in the cells of these ion-transporting epithelia. A structural approach has shown that two pronephric urinary tubules are already present at hatching while the urinary bladder starts to differentiate. The glomus, an ultrafiltration site, occurs at day 5 (D5). The opisthonephros differentiates at D19/25 from the pronephric collecting tubules, then it rapidly grows longer and becomes folded. Na⁺/K⁺-ATPase immunolocalization and transmission electron microscopy show that ionocyte-like cells line the urinary tubules and the dorsal wall of the urinary bladder from D2/D5 on. Tubule ionocytes present a basolateral-localized fluorescence. Ionocytes of the collecting ducts and of the dorsal wall of the bladder present a fluorescence distributed in the whole cytoplasm. Fluorescence becomes stronger in later stages, suggesting a progressively increasing functionality of the urinary system in active ion transports. This observation is closely correlated with the ontogeny of osmoregulatory abilities. In juvenile and preadult fish kept in seawater, osmolality measurements demonstrate that urine is isotonic to blood. At low salinity, urine is hypotonic to blood in both stages. The capacity to produce hypotonic urine increases during ontogeny, a fact that suggests an increasing involvement of the urinary system in osmoregulation. The occurrence and the progressive functionality of the urinary system during the ontogeny, along with those of other osmoregulatory sites, are major adaptations allowing the sea bass to live in habitats of variable salinity such as lagoons and estuaries.     

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.     

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.     

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.     

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.     

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.     

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.     

Functional expression of the oligopeptide transporter PepT1 from the sea bass (Dicentrarchus labrax)

Complementary RNA, derived from the intestine of the sea bass Dicentrarchus labrax and putatively coding for a pH-dependent oligopeptide transporter PepT1 (SLC15 family), was injected in Xenopus oocytes that were subsequently tested with electrophysiological techniques. Transport-associated currents were observed when various di- or tripeptides were applied at concentrations ranging between 0.1 and 10 mM. No currents were generated by histidine nor by other single amino acids. Sea bass PepT1 also exhibited presteady-state currents in the absence of substrates. Acidic pH slowed down the relaxation time constant of these currents and shifted both Q/V and τ/V relationships toward more positive voltages. Michaelis–Menten analysis of the transport currents showed an increase in apparent substrate affinity at acidic pH, which was very similar to that exhibited by the related transporter from zebrafish (Danio rerio), but in contrast, did not demonstrate a significant effect of pH on the maximal transport current.     

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.     

Diplectanidae (Monogenea) infestations of sea bass,Dicentrarchus labrax (L.), from the Spanish Mediterranean area

In the present study,Diplectanum laubieri was redescribed from material obtained from wild and cultured fish. The population dynamics ofD. aequans andD. laubieri under culture conditions was studied. The prevalence and mean intensity (average number of worms per parasitized fish) were 80.64% and 112 forD. aequans and 67.74% and 59.61 forD. laubieri, respectively. Both Diplectanidae exhibited seasonal variations, with maximal infection levels occurring in winter, suggesting continuous parasites recruitment, and infection levels tended to increase with host age. Male hosts were more heavily infested than females. The apparent positive association betweenD. aequans andD. laubieri was not confirmed statistically. The Diplectanidae showed definite and different patterns of gill distribution. Histopathological gill damage mainly consisted of hyperplasia of the epithelium, fusion of branchial lamellae and the presence of hemorrhagic and inflammatory foci with leucocytic infiltration. The results demonstrated the pathological importance of both Diplectanidae for the cultivation of sea bass.     

Development of the digestive tract of sea bass (Dicentrarchus labrax L). Light and electron microscopic studies

The developing gut of sea bass was studied by light and electron microscopy, four phases being established. Phase I, from hatching to the opening of the mouth, was a lecitotrophic period, in which the gut appeared as a straight undifferentiated tube lined by a simple epithelium that became stratified in the most caudal region. The epithelial cells increased in length towards the caudal zone, as did the number and height of the apical microvilli and the magnitude of the lamellar structures in their basal region. Cilia were more numerous in the caudal region than in the rest of the gut. Signs of lipid but not of protein absorption were found in the epithelial cells at this phase. Phase II, from the opening of the mouth to the complete resorption of the yolk sac, was a lecitoexotrophic period in which an esophagus, a gastric region, an intestine and a rectum, the last two separated by a valve, were present. During this phase the differentiation of the gut started at the esophagus and the rectum. In the esophagus, the epithelium became stratified and goblet cells containing acid mucosubstances, including sulphomucins, appeared. In the epithelial cells of the rectum, supranuclear vacuoles and an incipient endocytotic apparatus that seemed to be involved in the absorption and digestion of proteins were found. In both regions the mucosa was folded. Phase III, from the complete resorption of the yolk sac to the appearance of the first gastric glands, initiated the exclusively exotrophic period. During this phase the intestine formed the mucosa folds, while the first pyloric caeca and the epithelial cells acquired the ultrastructural features of mature absorptive cells with many lipid inclusions. Goblet cells containing neutral mucosubstances appeared and increased in number in both the intestine and the rectum. Neutral mucosubstances were also present in the cells lining the gastric region. During phase IV, from the appearance of the first gastric glands onwards, the intestinal absorptive surface increased with the formation of new pyloric caeca and two intestinal loops. The stomach acquired its definitive anatomy and histology with the development of the caecal and pyloric regions alongside differentiated gastric glands. The glandular cells had the ultrastructural features of the cells that secrete both pepsinogen and hydrochloride acid in the adult teleost stomach. Accepted: 7 February 2001     

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.     

Development of the digestive tract of sea bass (Dicentrarchus labrax L). Light and electron microscopic studies

The developing gut of sea bass was studied by light and electron microscopy, four phases being established. Phase I, from hatching to the opening of the mouth, was a lecitotrophic period, in which the gut appeared as a straight undifferentiated tube lined by a simple epithelium that became stratified in the most caudal region. The epithelial cells increased in length towards the caudal zone, as did the number and height of the apical microvilli and the magnitude of the lamellar structures in their basal region. Cilia were more numerous in the caudal region than in the rest of the gut. Signs of lipid but not of protein absorption were found in the epithelial cells at this phase. Phase II, from the opening of the mouth to the complete resorption of the yolk sac, was a lecitoexotrophic period in which an esophagus, a gastric region, an intestine and a rectum, the last two separated by a valve, were present. During this phase the differentiation of the gut started at the esophagus and the rectum. In the esophagus, the epithelium became stratified and goblet cells containing acid mucosubstances, including sulphomucins, appeared. In the epithelial cells of the rectum, supranuclear vacuoles and an incipient endocytotic apparatus that seemed to be involved in the absorption and digestion of proteins were found. In both regions the mucosa was folded. Phase III, from the complete resorption of the yolk sac to the appearance of the first gastric glands, initiated the exclusively exotrophic period. During this phase the intestine formed the mucosa folds, while the first pyloric caeca and the epithelial cells acquired the ultrastructural features of mature absorptive cells with many lipid inclusions. Goblet cells containing neutral mucosubstances appeared and increased in number in both the intestine and the rectum. Neutral mucosubstances were also present in the cells lining the gastric region. During phase IV, from the appearance of the first gastric glands onwards, the intestinal absorptive surface increased with the formation of new pyloric caeca and two intestinal loops. The stomach acquired its definitive anatomy and histology with the development of the caecal and pyloric regions alongside differentiated gastric glands. The glandular cells had the ultrastructural features of the cells that secrete both pepsinogen and hydrochloride acid in the adult teleost stomach.     

Molecular characterization, 3D modelling and expression analysis of sea bass (Dicentrarchus labrax L.) interleukin-10

Interleukin-10 (IL-10) is a pleiotropic cytokine generally known for its relevance in the resolution of inflammation, but that also has immunostimulatory properties. Here is described the isolation and characterization of the sea bass IL-10 (sbIL-10) cDNA and gene. The sbIL-10 gene encodes a 187 amino acid protein and comprises a five exon-four intron structure as other known IL-10 genes. Important structural residues are maintained in the sbIL-10 protein, including the four cysteines responsible for the two intra-chain disulfide bridges reported for human IL-10. The 3D structure of sbIL-10 was predicted. This first homology model of a fish IL-10 reveals a high degree of compatibility between the dimeric quaternary architectures of sbIL-10 and its mammalian counterparts. The phylogenetic analysis clusters sbIL-10 with other IL-10s, apart from IL-10-related molecules. The involvement of IL-10 in sea bass immune responses was demonstrated by investigating the expression profiles of IL-1beta and IL-10 in the head-kidney and spleen following intraperitoneal injection of UV-killed Photobacterium damselae ssp. piscicida. Furthermore, involvement of IL-10 in the resolution of inflammation is for the first time suggested in fish, due to the delayed maximal mRNA levels of sbIL-10 compared to those of the pro-inflammatory IL-1beta.     

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

Several endocrine cell types were ultrastructurally characterized during the differentiation of the intestine and rectum of sea bass (Dicentrarchus labrax L.) larvae. Only one cell type (type I) was found in the posterior region of the undifferentiated gut of 5-day-old larvae (phase I). Types V and VI were found in both the intestine and rectum, types II, III and IV in the intestine, and types VII and VIII in the rectum of 9- and 12-day-old larvae (phase II), the rectum alone showing signs of functional differentiation. In phase III larvae, in which both the intestine and rectum were differentiated, types IX, X, XI, XII, XIII, XIV and XV were found in the intestine, only types X, XI and XII being seen in the rectum. Besides these, a new cell type, XVI, was observed in the intestine of 55- and 60-day-old larvae (phase IV), in which the digestive tract was completely differentiated. The endocrine cells appearing in phases I and II showed very scarce secretory granules and the ultrastructural features of undifferentiated cells. Some endocrine cell types in the earliest developmental stages were related to some of those found later. A maturational process of the endocrine cell types paralleled the differentiation of the intestine and rectum, with an apparent increase in the number of secretory granules accompanying organelle development.