Expression of lymphocyte antigenic determinants in developing gut-associated lymphoid tissue of the sea bass Dicentrarchus labrax (L.)

 The monoclonal antibodies DLT15 and DLIg3, which recognize antigenic determinants expressed by T cells and Ig-bearing cells, respectively, allowed the development of gut-associated lymphoid tissue of the teleost fish Dicentrarchus labrax (L.) to be studied. DLT15- immunoreactive cells were first detected in the epithelium of the stomach and intestine at day 30 post-hatching of fish maintained at 16° C. At that age, positive cells were found only in the thymus. Between day 44 and day 81 post-hatching, DLT15-immunoreactive cells became numerous, both in and under the gut epithelium. A gradient in the number of lymphocytes was present, concentrating them towards the anus. Until day 81 post-hatching, DLIg3-immunoreactive cells were not found in the gut, although they were present in the kidney, spleen and thymus earlier. Infrequent Ig-bearing cells were found in the gut mucosa of 1-year-old sea bass. This study showed that the gut-associated lymphoid tissue developed earlier than other lymphoid compartments. It also provided evidence of the predominance of T cells in the gut immune system of the sea bass. Accepted: 30 June 1997     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Effect of periodical water current on the phasing of demand feeding rhythms in sea bass (Dicentrarchus labrax L.)

The demand feeding rhythms of sea bass (Dicentrarchus labrax L.) maintained at rest or subjected to exercise (increase in swimming activity) as a result of an induced water current are compared. Both sea bass kept individually and in group (4 and 8 individuals) shifted their food demands towards the exercise period or to just after the same (the latter especially in individually kept fish). The origin of such a response is discussed in view of innate behaviour, the energy cost of locomotor activity and changes in agonistic behaviour. Induced diurnal exercise favoured diurnal feeding. The periodic exercise regime led to lower rates of daily feeding pattern inversions (diurnal-to-nocturnal or vice-versa). Sea bass were capable of learning to press an exercise sensor and to distinguishing exercise sensors from demand feeding sensors.     

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.     

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.     

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 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.     

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     

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.     

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.     

Ig light chain variability in DNP(494)-KLH immunised sea bass (Dicentrarchus labrax L.): evidence for intra-molecular induced suppression

The coding sequence of the sea bass light chain was obtained by sequential anchored PCR on a head kidney cDNA library of a DNP(494)-KLH immunised sea bass. The cDNA sequence obtained codes for a leader peptide of 21aa and a mature IgL chain of 223aa. Both the amino acid sequence comparisons and neighbour-joining trees showed that the IgL chain of sea bass obtained is of the L1/G type. To study the variability of the light chain, additional PCRs on the cDNA library and cDNA from pooled head kidneys were performed. Multiple alignment of unique sequences (N=17) could be performed without introducing gaps, and showed extremely low variability in CDR1, and no variability in CDR2 or CDR3. A possible explanation for this low variability of the IgL1 chain might be the enhanced expression of monospecific anti-DNP antibodies. The isolation and characterisation of partial genomic and cDNA IgL sequences, which showed normal variability, corroborate this explanation.