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Hemocyte lineage marker proteins in a crustacean, the freshwater crayfish, Pacifastacus leniusculus
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Physiology and Developmental Biology, Comparative Physiology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Physiology and Developmental Biology, Comparative Physiology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Physiology and Developmental Biology, Comparative Physiology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Physiology and Developmental Biology, Comparative Physiology.
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2008 (English)In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 8, no 20, 4226-4235 p.Article in journal (Refereed) Published
Abstract [en]

To identify proteins associated with development of different hemocyte types in the freshwater crayfish Pacifastacus leniusculus, 2-DE followed by MS analysis was carried out with hematopoietic tissue (Hpt) cells, semigranular cells (SGC) and granular cells (GC). Within the hemocyte lineages one two-domain Kazal proteinase inhibitor (KPI) was found to be specific for SGC, while a superoxide dismutase (SOD) was specific for GC at protein as well as at mRNA level. The proliferation cell nuclear antigen (PCNA) was detected at the mRNA level in Hpt cells only. We also provide evidence that SGC and GC most likely differentiate to maturation as separate lineages. We found that after laminarin or lipopolysaccharide (LPS) injection into crayfish, the transcript levels of PCNA and SOD increased in the Hpt cells, whereas the KPI transcript never was present in Hpt regardless of any challenge. RNA interference of PCNA in the Hpt cells led to that most of the cells did not spread or attach to the tissue culture dish. These results suggest that PCNA, KPI and SOD can be used as markers for Hpt cells, SGC and GC, respectively, and in conjunction with these results, a model is proposed how the Hpt responds to a microbial challenge by proliferation and release of Hpt cells.

Place, publisher, year, edition, pages
2008. Vol. 8, no 20, 4226-4235 p.
Keyword [en]
Crustacean hematopoiesis, Hemocyte markers, Kazal, PCNA, SOD
National Category
Immunology
Research subject
Biology with specialization in Comparative Physiology; Immunology
Identifiers
URN: urn:nbn:se:uu:diva-97412DOI: 10.1002/pmic.200800177ISI: 000260717300010PubMedID: 18814328OAI: oai:DiVA.org:uu-97412DiVA: diva2:172353
Available from: 2008-08-18 Created: 2008-08-18 Last updated: 2017-12-14
In thesis
1. Functional Studies of Some Immune Relevant Genes in a Crustacean
Open this publication in new window or tab >>Functional Studies of Some Immune Relevant Genes in a Crustacean
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The freshwater crayfish, Pacifastacus leniusculus, mounts a strong innate immune response against microbes such as viruses and bacteria. In this thesis, a novel RNA interference (RNAi) method mediated with histone H2A was developed and applied in crayfish hematopoietic tissue cell cultures for gene functional studies. Further, the interactions between host (crayfish) and pathogens (white spot syndrome virus and Aeromonas hydrophila, respectively) were studied using RNAi technology in live animals. An antilipopolysaccharide factor isolated from viral challenged crayfish by suppression subtractive hybridization was shown to interfere with the propagation of white spot syndrome virus both in vivo and in vitro in crayfish, suggesting an important role of this factor in antiviral defense. Besides, RNAi of phenoloxidase, a critical immune effector involved in melanization, revealed that phenoloxidase activity is necessary for crayfish immune defense against a highly pathogenic bacterial infection in crayfish. In addition, RNAi was also employed to study a marker protein gene involved in hemocyte maturation in crayfish. Taken together, these studies may provide more insights into the immune responses against pathogen invasion as well as hemocyte ontogenesis in crustaceans.

Place, publisher, year, edition, pages
Uppsala: Universitetsbiblioteket, 2008. 61 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 453
Keyword
anti-LPS factor, crayfish, hemocyte, innate immunity, melanization, phenoloxidase, RNA interference, WSSV
National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-9194 (URN)978-91-554-7252-8 (ISBN)
Public defence
2008-09-12, Lindahlsalen, EBC, Norbyvägen 18A, Uppsala, 10:00 (English)
Opponent
Supervisors
Available from: 2008-08-18 Created: 2008-08-18 Last updated: 2009-04-05Bibliographically approved
2. Innate Immune Proteins in a Crustacean Pacifastacus leniusculus
Open this publication in new window or tab >>Innate Immune Proteins in a Crustacean Pacifastacus leniusculus
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Hemocytes (blood cells) are important in the immune defense against pathogens in invertebrates. In crusteacean, the hemocytes and plasma components mount a strong innate immune response against different pathogens including bacteria and virus. This thesis is aimed to identify marker proteins associated with development of different hemocyte types, and to find a protein involved in the phenoloxidase-induced melanization and other innate immune reactions in freshwater crayfish Pacifastacus leniusculus.

In crustaceans, the hemocytes are produced and partly differentiated in the hematopoietic tissue (Hpt) before they are released into the hemolymph circulation. To investigate the connection between semigranular cells, granular cells and precursor cells in Hpt of P. leniusculus and possibly also in other crustaceans, two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry (MS) analysis was used to identify specific proteins expressed in different hemocytes. The specific expression was analyzed by RT-PCR and western blot. Moreover, RNA interference was used to study the hemocyte differentiation in vivo and in vitro.

Melanin formation is essential for host defence in arthopods, and it needs to be tightly regulated since unwanted production of quinone intermediates or melanization is also dangerous to the animal. By using western blot, 2-DE and MS, a melanization inhibiting protein (MIP) was found to have similar function as mealworm Tenebrio molitor MIP. Both of them interfere with the melanization reaction, but do not affect phenoloxidase activity.

In order to reveal the mechanism by which peptidoglycan (PGN) induces activation of the prophenoloxidase activating system in P. leniusculus, different forms of Lys-type PGN were used to pull down PGN recognition proteins (PGRPs) from plasma or hemocyte lysate supernatant of crayfish. The binding proteins were separated and then analyzed with MS. Results showed that two serine protease homologues are involved in this activation possibly by forming a complex with lipopolysaccharide and β-1,3-glucan binding protein (LGBP) and without a PGRP.

Besides, two ficolin-like proteins (FLPs) have been found from crayfish plasma by using different bacteria including Staphylocuccus aureus as an affinity matrix to pull down bacterial binding proteins, followed by the analysis with 2-DE and MS. Two FLPs can bind to bacteria, and may help crayfish to clear Gram-negative bacteria, but not Gram-positive bacteria injected into the crayfish hemolymph, which suggests that FLPs may function as pattern recognition receptors in the immune response of crayfish.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2011. 72 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 807
Keyword
2-DE, hematopoiesis, marker protein, melaniztion inhibiting protein, proPO-system, pattern recognition protein, PGN, ficolin-like protein
National Category
Immunology
Research subject
Biology with specialization in Comparative Physiology
Identifiers
urn:nbn:se:uu:diva-147600 (URN)978-91-554-8015-8 (ISBN)
Public defence
2011-04-08, Lindahlsalen, Evolutionary Biology Centre, Norbyvägen 18A,, Uppsala, 10:00 (English)
Opponent
Supervisors
Note
Felaktigt tryckt som Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 737Available from: 2011-03-16 Created: 2011-02-26 Last updated: 2011-05-04

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Wu, ChenglinSöderhäll, IreneSöderhäll, Kenneth

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