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      Phagocytosis and Respiratory Burst Activity in Lumpsucker ( Cyclopterus lumpus L.) Leucocytes Analysed by Flow Cytometry

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          Abstract

          In the present study, we have isolated leucocytes from peripheral blood, head kidney and spleen from lumpsucker ( Cyclopterus lumpus L.), and performed functional studies like phagocytosis and respiratory burst, as well as morphological and cytochemical analyses. Different leucocytes were identified, such as lymphocytes, monocytes/macrophages and polymorphonuclear cells with bean shaped or bilobed nuclei. In addition, cells with similar morphology as described for dendritic cells in trout were abundant among the isolated leucocytes. Flow cytometry was successfully used for measuring phagocytosis and respiratory burst activity. The phagocytic capacity and ability were very high, and cells with different morphology in all three leucocyte preparations phagocytised beads rapidly. Due to lack of available cell markers, the identity of the phagocytic cells could not be determined. The potent non-specific phagocytosis was in accordance with a high number of cells positive for myeloperoxidase, an enzyme involved in oxygen-dependent killing mechanism present in phagocytic cells. Further, high respiratory burst activity was present in the leucocytes samples, verifying a potent oxygen- dependent degradation. At present, the specific antibody immune response could not be measured, as immunoglobulin or B-cells have not yet been isolated. Therefore, analyses of the specific immune response in this fish species await further clarification. The present study presents the first analyses of lumpsucker immunity and also the first within the order Scopaeniformes.

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          Most cited references29

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          Innate immunity of fish (overview).

          The innate immune system is the only defence weapon of invertebrates and a fundamental defence mechanism of fish. The innate system also plays an instructive role in the acquired immune response and homeostasis and is therefore equally important in higher vertebrates. The innate system's recognition of non-self and danger signals is served by a limited number of germ-line encoded pattern recognition receptors/proteins, which recognise pathogen associated molecular patterns like bacterial and fungal glycoproteins and lipopolysaccharides and intracellular components released through injury or infection. The innate immune system is divided into physical barriers, cellular and humoral components. Humoral parameters include growth inhibitors, various lytic enzymes and components of the complement pathways, agglutinins and precipitins (opsonins, primarily lectins), natural antibodies, cytokines, chemokines and antibacterial peptides. Several external and internal factors can influence the activity of innate immune parameters. Temperature changes, handling and crowding stress can have suppressive effects on innate parameters, whereas several food additives and immunostimulants can enhance different innate factors. There is limited data available about the ontogenic development of the innate immunological system in fish. Active phagocytes, complement components and enzyme activity, like lysozyme and cathepsins, are present early in the development, before or soon after hatching.
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            The genome sequence of Atlantic cod reveals a unique immune system.

            Atlantic cod (Gadus morhua) is a large, cold-adapted teleost that sustains long-standing commercial fisheries and incipient aquaculture. Here we present the genome sequence of Atlantic cod, showing evidence for complex thermal adaptations in its haemoglobin gene cluster and an unusual immune architecture compared to other sequenced vertebrates. The genome assembly was obtained exclusively by 454 sequencing of shotgun and paired-end libraries, and automated annotation identified 22,154 genes. The major histocompatibility complex (MHC) II is a conserved feature of the adaptive immune system of jawed vertebrates, but we show that Atlantic cod has lost the genes for MHC II, CD4 and invariant chain (Ii) that are essential for the function of this pathway. Nevertheless, Atlantic cod is not exceptionally susceptible to disease under natural conditions. We find a highly expanded number of MHC I genes and a unique composition of its Toll-like receptor (TLR) families. This indicates how the Atlantic cod immune system has evolved compensatory mechanisms in both adaptive and innate immunity in the absence of MHC II. These observations affect fundamental assumptions about the evolution of the adaptive immune system and its components in vertebrates.
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              Morphologic and functional characterization of granulocytes and macrophages in embryonic and adult zebrafish.

              The zebrafish is a useful model organism for developmental and genetic studies. The morphology and function of zebrafish myeloid cells were characterized. Adult zebrafish contain 2 distinct granulocytes, a heterophil and a rarer eosinophil, both of which circulate and are generated in the kidney, the adult hematopoietic organ. Heterophils show strong histochemical myeloperoxidasic activity, although weaker peroxidase activity was observed under some conditions in eosinophils and erythrocytes. Embryonic zebrafish have circulating immature heterophils by 48 hours after fertilization (hpf). A zebrafish myeloperoxidase homologue (myeloid-specific peroxidase; mpx) was isolated. Phylogenetic analysis suggested it represented a gene ancestral to the mammalian myeloperoxidase gene family. It was expressed in adult granulocytes and in embryos from 18 hpf, first diffusely in the axial intermediate cell mass and then discretely in a dispersed cell population. Comparison of hemoglobinized cell distribution, mpx gene expression, and myeloperoxidase histochemistry in wild-type and mutant embryos confirmed that the latter reliably identified a population of myeloid cells. Studies in embryos after tail transection demonstrated that mpx- and peroxidase-expressing cells were mobile and localized to a site of inflammation, indicating functional capability of these embryonic granulocytes. Embryonic macrophages removed carbon particles from the circulation by phagocytosis. Collectively, these observations have demonstrated the early onset of zebrafish granulopoiesis, have proved that granulocytes circulate by 48 hpf, and have demonstrated the functional activity of embryonic granulocytes and macrophages. These observations will facilitate the application of this genetically tractable organism to the study of myelopoiesis.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2012
                24 October 2012
                : 7
                : 10
                : e47909
                Affiliations
                [1 ]Department of Biology, University of Bergen, Bergen, Norway
                [2 ]Aqua Kompetanse AS, Flatanger, Norway
                University Medical Center Freiburg, Germany
                Author notes

                Competing Interests: NV is employed by Aqua Kompetanse AS. This company offers services for environmental and general fish health management to regional farmers and regulatory authorities. They are not involved in lumpsucker production, but perform general supervision of salmon farms using lumpsucker as cleaner fish as part of their general activities. Recently, NV graduated as master in aquamedicine in the group of Professor HIW at UiB. His employment is not related to any companies’ consultancy, patents and products in development or marketed products, and as such, this does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials.

                Conceived and designed the experiments: GTH HIW. Performed the experiments: GTH NV RAJ KU LS HIW. Analyzed the data: GTH. Contributed reagents/materials/analysis tools: HIW. Wrote the paper: GTH HIW.

                Article
                PONE-D-12-22351
                10.1371/journal.pone.0047909
                3480447
                23112870
                f270a25c-f9df-4ca0-89bc-6f8d2b925aea
                Copyright @ 2012

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 12 July 2012
                : 17 September 2012
                Page count
                Pages: 11
                Funding
                This project is funded by University of Bergen, Norway [ http://www.uib.no]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology
                Immunology
                Immune System
                Lymphoid Organs
                Immunity
                Immune Defense
                Immunity to Infections
                Immunologic Techniques
                Immunoassays
                Immune Cells
                Immune Response
                Marine Biology
                Fisheries Science

                Uncategorized
                Uncategorized

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