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Phagocytosis is a fundamental innate immune process primarily executed by vertebrate leucocytes. Fish erythrocytes are involved in immune functions, although their role in phagocytosis remains poorly investigated. Consequently, this study aimed to examine the potential phagocytic mechanisms of fish erythrocytes. To this end, systemic blood erythrocytes were isolated from the teleost gilthead seabream (Sparus aurata) and European sea bass (Dicentrarchus labrax) and incubated for various times (20, 40, 60, 80, 100, and 120 min) with formalin-inactivated bacteria or heat-killed yeasts labelled with fluorescein isothiocyanate. Their putative phagocytic properties and respiratory burst activity were investigated. Our results did not indicate variations in the phagocytic ability or phagocytic capacity of erythrocytes of seabream or sea bass incubated with the bacteria, whereas no activity was detected in the case of incubation with yeast. Additionally, no respiratory burst activity was detected in the erythrocytes of either fish species under any of the experimental conditions tested. Using fluorescence microscopy, it was observed that erythrocytes could bind bacteria to their surface membranes. However, this attachment process was rarely seen with yeast cells. In contrast, examination of the fine structure of erythrocytes using transmission electron microscopy showed distinctive inward and outward folding (pseudopodia formation) and some cytoplasmic vesicles in both species. The results of our study indicate that erythrocytes from gilthead seabream and sea bass did not exhibit phagocytic capabilities when exposed to these specific target particles. Additional studies are necessary to gain more insights into how they contribute to the immune system of fish., (Copyright © 2025 Elsevier Ltd. All rights reserved.)

Inorganic arsenic (As) is one of the most toxic pollutants in the water. We have studied their effects on the marine teleost European sea bass (Dicentrarchus labrax) at 2 and 10 days of 5 μM of As 2 O 3 (sub-lethal doses) waterborne exposure. Arsenic accumulates in liver and gill tissues. The expression profile of five genes (bax, blc2, casp3, casp8 and casp9) involved in apoptosis cell death confirmed apoptotic effects in liver, slight changes in gill and no effects in skin according with the histopathology findings. Total IgM level and peroxidase activities were increased at 2 and 10 days, respectively. The bactericidal activity was decreased at 2 days after As exposure. A general decrease of cellular immune activities with significant differences in the case of respiratory burst activity was observed after 2 and 10 days of exposure. This work describes for the first time the effects of As exposure on European sea bass., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Antimicrobial peptides (AMPs) are increasingly recognized as a critical first line of defence against many pathogens. The genes encoding these peptides are expressed in numerous tissue and cell types from a wide variety of different species including mammals, amphibians, fish, and insects. In this study, we report that the AMPs called piscidins were primarily present in the mast cells (MCs) of fish and were only identified in fish belonging to the Order Perciformes. It is striking that histamine was seen to have a similar evolutionary history, since the only piscine MCs endowed with this molecule are in the Perciformes. We also show that both MCs and professional phagocytic granulocytes were armed with different piscidin molecules. In contrast, macrophages were devoid of these AMPs. More importantly, we found by immunoelectron microscopy that piscidins were delivered to the bacteria-containing phagosome of granulocytes upon phagocytosis, suggesting a role for these AMPs in the killing of both extracellular and intracellular pathogenic bacteria.

Although neuroendocrine-immune system interaction has been shown in teleost fish, no study has evaluated the role of melatonin (Mel) on fish immune response even considering that it is affected by the photoperiod. Gilthead seabream (Sparus aurata L.) and sea bass (Dicentrarchus labrax L.) head-kidney leucocytes were incubated with Mel (0-control-, 20 pM-400 microM) and leucocyte viability and main innate cellular immune parameters were evaluated. Overall, seabream and sea bass head-kidney leucocytes incubated with low (similar to physiological) doses of Mel unchanged the innate immune response, whereas very high (pharmacological) dosages did. Phagocytosis was not affected by any Mel treatment while the peroxidase activity was significantly inhibited with the highest Mel concentration. In contrast, the sea bass respiratory burst activity was increased in a dose-dependent manner with 400 nM Mel or higher. Further studies are needed to clarify whether there are interactions between the fish pineal gland, and its hormone Mel, and the fish immune system., ((c) 2007 Wiley-Liss, Inc.)

Although it is believed that fish fry depend fundamentally on their innate defence mechanisms, the ontogeny of fish innate immune cells is poorly understood. In the present study, we have used a specific monoclonal antibody against acidophilic granulocytes (AGs), the main professional phagocytic cell type of the bony fish gilthead seabream, to study their localization during the development of the main lymphomyeloid organs, namely the head kidney, spleen and thymus, and of the two major portals for pathogen entry, namely the gills and intestine. AGs were observed in the posterior intestine and in the blood earlier than in the haematopoietic kidney (21 vs. 27 days post-hatching, dph). AGs were observed scattered between other cells of the haematopoietic lineage in the head kidney of larvae, but were grouped around the blood vessels of this organ in juveniles and adults, where they were also much more numerous. In the spleen and in the thymus, AGs were observed much later (62 dph) and appeared scattered. AGs were also observed in the gill lamella and the posterior intestine near the anus throughout development.

The cellular innate immune response of gilthead seabream (Sparus aurata L.) against the myxozoan Enteromyxum leei was studied. Enteromyxosis was transmitted by maintaining uninfected fish (recipients) together with infected animals. A group of fish not exposed to the infection served as controls. After 10, 22, 38, 52 and 108 days, control and recipient fish were sampled and leucocyte subpopulations and cellular immune responses (leucocyte peroxidases, phagocytosis, respiratory burst and cytotoxicity) of the head-kidney leucocytes were determined. The percentage of acidophilic granulocytes was significantly lower in non-parasitized and parasitized recipient fish than in control fish after 22 days but no significant differences were seen between non-parasitized and parasitized recipient animals. The leucocyte peroxidase content, phagocytosis and respiratory burst activity were seen to have decreased significantly at different sampling times in both non-parasitized and parasitized recipient fish with respect to the controls, whereas cytotoxic activity was up to 2.3 times higher than in control fish. Within the recipient group, little difference was observed in the studied parameters between non-parasitized and parasitized fish. These data demonstrate that cytotoxic activity may have an important role in the defence of gilthead seabream against the myxosporean E. leei. Immunological implications of E. leei infections are discussed.

The early innate immune response of the teleost gilthead seabream (Sparus aurata L.) against xenogeneic cells was studied. Fish received a single intraperitoneal injection of xenogeneic cells (tumour cell line), following which leucocyte mobilization, degranulation, peroxidase content, respiratory burst and phagocytic and cytotoxic activities were determined in both peritoneal exudate leucocytes (PELs) and head-kidney leucocytes (HKLs). The total number of PELs increased from 4 h post-injection until the end of the experiment (3 days). Interestingly, flow cytometric analysis of PEL and HKL suspensions revealed variations in the proportion of cell types. The percentage of HK acidophilic granulocytes significantly increased after 72 h, whereas PE acidophils increased after 4 h. Moreover, numbers of PE lymphocytes and monocyte-macrophages significantly increased during the experiment. The peroxidase content of the leucocytes was unaffected, although PEL degranulation was largely enhanced. This liberation of peroxidases correlated well with the enhancement of the oxidative respiratory burst activity in PELs, reflecting leucocyte activation. However, phagocytosis only increased in PELs 4 h after intraperitoneal injection, whereas the cytotoxic activity of HKLs increased 1 and 2 days post-injection but, in general, decreased in the PELs. Our data thus demonstrate that the appearance of xenogeneic cells involves leucocyte mobilization and innate immune-response activation at the site of invasion and in the head-kidney. Involvement of the various leucocyte types and potential modes of activation are discussed.

The interaction between chitin particles and gilthead seabream (Sparus aurata L.) head-kidney leucocytes, as well as their effects on the main innate cellular immune responses were studied. Three different chitin particle-sizes were tested: unfiltered, <10 microM and >10 microM. Leucocytes were able to phagocytose only the chitin particles of <10 microM but not the >10 microM ones. Leucocytes were incubated with different concentrations (0 to 1000 microg ml(-1)) of the above chitin particles for 1, 4, 24 or 48 h and their effects on leucocyte viability and the innate cellular immune system were evaluated. Leucocytes incubated with chitin for 48 h maintained their viability as determined by the MTT viability test. Leucocyte phagocytosis of bacteria after chitin incubation for 1 or 4 h was enhanced by the highest chitin concentration tested of each of the chitin fractions studied, while the respiratory burst activity was unaffected. As regards leucocyte natural cytotoxic activity against tumour cells, prior incubation of leucocytes with chitin particles for 1 or 4 h increased while incubation for 24 or 48 h reduced the cytotoxic activity in a dose dependent manner. Statistically significant differences between the different chitin concentrations and between the three chitin particle-size fractions were detected. To conclude, gilthead seabream head-kidney leucocytes were able to phagocytose chitin particles smaller than 10 microM, and the main cellular innate immune activities were enhanced as a consequence of prior incubation with chitin particles.

A flow cytometric method was adapted to evaluate phagocytosis by gilthead seabream leucocytes after incubation with yeast cells (Saccharomyces cerevisiae). Head-kidney leucocytes were incubated in vitro for different times in different proportions with heat-killed fluorescein isothiocyanate (FITC)-labeled yeast cells to study the kinetics of phagocytosis. Attached and internalized yeast cells were differentiated by quenching FITC-labeled S. cerevisiae with trypan blue dye. Only internalized cells kept their FITC fluorescence after quenching. Monocyte-macrophages and acidophilic granulocytes showed phagocytic activity, as demonstrated by transmission electron microscopy (TEM). From the ultrastructural features of the phagocytic process, it was observed that cytoplasmic granule membranes fused with the phagocyte membrane at the point where the yeast cell was attached to the phagocyte surface. This observation led us to adapt a colorimetric method to study peroxidase (myeloperoxidase and eosinophil peroxidase) release, since both are considered to be markers of the degranulation that occurs in seabream head-kidney leucocytes in response to yeast cells. Head-kidney leucocytes were incubated with calcium ionophore (CaI), phorbol myristate acetate (PMA), or yeast cells for different periods of time (0-30 min) to study the kinetics of peroxidase release. The results obtained indicate that CaI and yeast cells, but not PMA, stimulate the degranulation (by about 44.51% and 21.04%, respectively, at 30 min) of seabream head-kidney leucocytes., (Copyright 2003 Wiley-Liss, Inc.)

Thrombocytes and platelets, beyond their primary function in hemostasis, seem to play an active role in inflammation. As regards their phagocytic ability, the results to date are confusing, incomplete, and somewhat contradictory. Whereas the interaction of avian thrombocytes or mammalian platelets with bacteria both in vitro and in vivo has received wide attention, almost no information exists on the topic in "lower" vertebrates. The aim of this work is to review the available information on the phagocytic properties of thrombocytes and platelets. Particular attention is payed to the ontogeny of these cells, the soluble factors involved in the inflammatory process derived from them, and their interaction with particulate material, mainly with bacteria., (Copyright 2002 Wiley-Liss, Inc.)

The various cell types involved in fish phagocytic defence have not been properly established because of the morphological heterogeneity of leucocytes and the lack of appropriate cell-surface markers. In this study, we report the production and characterisation of a monoclonal antibody, G7, which specifically recognises gilthead seabream acidophilic granulocytes, as assayed by immunofluorescence and immunoelectron microscopy. The antibody reacted with 40%-50% of head-kidney and peritoneal exudate leucocytes and 10%-20% of spleen and peripheral blood leucocytes. More importantly, G7(+) acidophils constituted 85% of the head-kidney leucocytes showing phagocytic activity towards the fish pathogenic bacterium Vibrio anguillarum. The results are discussed in relation to the role played by this cell type in fish immune responses.

In this paper we optimize a flow cytometric method for evaluating the phagocytic activity of leucocytes in gilthead seabream (Sparus aurata L.) and characterize the phagocytic cells observed. Optimal conditions were established for the fluorescein-labelling and analysis of the bacterium Vibrio anguillarum by flow cytometry. Head-kidney leucocytes were incubated with the heat-killed fluorescein isothiocyanate (FITC)-labelled bacteria for different periods, during which the kinetics of phagocytosis was studied. Attached and interiorized bacteria were distinguished. Although phagocytic ability reached a maximum after 60 min, phagocytic capacity reached its maximum at 20 min. The amount of ingested bacteria per phagocyte was estimated from the mean fluorescence of the leucocytes. Cytochalasin B or colchicine was used to inhibit phagocytosis. Monocyte-macrophages and acidophilic granulocytes showed phagocytic activity as demonstrated by transmission electron microscopy. In conclusion, the technique presented allows the screening of thousands of cells, and individual cell evaluation, by quantifying interiorized particles in fish phagocytes. Our ultrastructural results demonstrate that V. anguillarum is actively phagocytized by seabream macrophages and acidophilic granulocytes.

Background: Although phagocytosis is considered the main nonspecific defense mechanism in fish, the significance of the influence of bacterial properties and assay conditions on this mechanism has not been established. We report the influence of leukocyte source, bacterial species, presence or absence of a bacterial wall, bacterial status (live or dead), and bacterial opsonization on in vitro phagocytosis by sea bass (Dicentrarchus labrax L.) macrophages., Methods: Leukocytes were challenged with bacteria under different conditions and ultrastructurally studied. The number of phagocytic macrophages and the number of bacteria ingested per macrophage were quantified by microscopical counting, and the data obtained were statistically analyzed following general linear models., Results: Macrophages from sea bass peritoneal exudate showed a greater capacity to engulf bacteria than did those isolated from blood, which, in turn, had greater engulfment properties than did those isolated from head-kidney. Greater phagocytic activity was observed in the assays carried out with Escherichia coli than in those carried out with Salmonella typhimurium, especially when the TA1950 S. typhimurium strain (with wall) was used. Macrophages showed greater phagocytic activity against live bacteria. Bacterial opsonization had a variable effect on phagocytosis. Sea bass macrophages did not always ingest the same number of bacteria., Conclusions: Sea bass macrophages have strong bacterial phagocytic activity. Significant differences were found that had been influenced by bacterial properties as well as by assay conditions. Positive correlation between the number of phagocytic macrophages and the number of bacteria ingested per phagocyte was observed.

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, overlapping 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) Intracellular 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.

The gilthead seabream is a protandrous seasonal breeding teleost that is an excellent model for studying the testicular regression process which occurs in both seasonal testicular involution and sex reversion. Little is known about the cell types and the molecular mechanisms involved in such processes, mainly because of the lack of appropriate methods for testis dissociation, and testicular cell isolation, culture and functional characterization. We have previously reported that gilthead seabream acidophilic granulocytes infiltrate the testis at post-spawning stage, settle close to the spermatogonia and accumulate intracellular interleukin-1&bgr;. In this paper, we report several flow cytometry based assays which allow to establish the role played by gilthead seabream testicular acidophilic granulocytes and permits their quantification.

In this paper the possible involvement of the mannose-receptor on the non-specific recognition and phagocytosis of heat killed yeast cells (Saccharomyces cerevisiae) by gilthead seabream (Sparus aurata L.) head-kidney leucocytes was established by studying the ability of different sugars to inhibit the uptake of the yeast cells by leucocytes. Leucocytes were preincubated for 30 min with different concentrations of sugar (α-mannan, d-mannose, d-fucose, l-fucose, d-glucose, d-glucosamine and n-acetyl-glucosamine, all of them described as specific ligands of the vertebrate mannose-receptor) and afterwards incubated with FITC-labelled yeast cells for phagocytosis assays. The phagocytic ability (percentage of cells with one or more ingested yeast cells within the total cell population) and capacity (number of ingested yeast cells per cell) of leucocytes was analysed by flow cytometry. The results demonstrate the potential existence of a specific receptor-sugar or receptor-yeast cell binding process, which was saturable, specific and dose-dependent. More specifically, when leucocytes were preincubated with appropriate doses of d-mannose, d- or l-fucose, d-glucose or n-acetyl-glucosamine the phagocytosis of yeast cells by head-kidney leucocytes was partially blocked. Seabream leucocytes were also preincubated with chloroquine, a lysosomotropic drug which downregulates (in a nonspecific manner) the expression of mannose-receptors in mammals, before phagocytosis assays were performed. The results demonstrated that the phagocytosis of yeast was completely blocked by this substance. The overall results seem to corroborate the presence of the mannose-receptor in seabream phagocytes, which is involved in the non-specific binding and phagocytosis of yeast cells by head-kidney leucocytes. [Copyright &y& Elsevier]

Phenoxyethanol is routinely used in seabream aquaculture to minimise fish stress response despite the secondary negative effects which have been observed. In this study, two different doses (60 and 200 μl/l) of phenoxyethanol, sedative and narcotic, were tested for their ability to reduce the stress caused in gilthead seabream (Sparus aurata L.) by crowding. Blood glucose and serum cortisol concentrations were measured as stress indicators. In order to study the effects of the treatment on the innate immune system of crowded specimens, two parameters of the innate immune response, serum complement activity and phagocytosis, were assessed. The results show that anaesthesia itself produced a stress response in the fish and affected the immune system, although the effects were greater with the narcotic dose. When the effects of anaesthesia on crowded fish were analysed, the results pointed to a slight reduction in stress as a result of the sedative dose of phenoxyethanol (lower increase in cortisol and lower reduction in phagocytosis). However, additive negative effects were seen in crowded fish when the narcotic dose of phenoxyethanol was used. Since the use of phenoxyethanol is a common practice in aquaculture, the significance of the results should be considered. [Copyright &y& Elsevier]