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ACTA

UNIVERSITATIS

UPSALIENSIS

UPPSALA

2008

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Science and Technology 453

Functional Studies of SomeImmune Relevant Genesin a Crustacean

HAIPENG LIU

ISSN 1651-6214ISBN 978-91-554-7252-8urn:nbn:se:uu:diva-9194

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TO MY PARENTS

List of Papers

This thesis is based on the following papers, which will be referred to in the text by their Roman numerals: I Liu H. and Söderhäll I. (2007). Histone H2A as a transfection

agent in crayfish hematopoietic tissue cells. Dev Comp Immunol, 31:340-6.

II Liu H., Jiravanichpaisal P., Söderhäll I., Cerenius L. and Söderhäll K.

(2006). Antilipopolysaccharide factor interferes with white spot syndrome virus replication in vitro and in vivo in the crayfish Paci-fastacus leniusculus. J Virol, 80:10365-71.

III Liu H., Jiravanichpaisal P., Cerenius L., Lee BL., Söderhäll I.

and Söderhäll K. (2007). Phenoloxidase is an important compo-nent of the defense against Aeromonas hydrophila infection in a crustacean, Pacifastacus leniusculus. J Biol Chem, 282: 33593-8.

IV Wu C., Söderhäll I., Kim Y., Liu H. and Söderhäll K. (2008).

Hemocyte lineage marker proteins in a crustacean, the freshwater crayfish, Pacifastacus leniusculus. Proteomics (In press).

Reprints were made with permission from the publishers. ©Elsevier Ltd. 2007 ©American Society for Microbiology, 2006 ©American Society for Biochemistry and Molecular Biology, 2007 ©WILEY-VCH Verlag GmbH & Co. KGaA, 2008

Contents

Introduction.....................................................................................................9 Pattern recognition proteins (PRPs) in arthropods ...................................10 Melanization and prophenoloxidase activating system (proPO-system)..14 Antimicrobial peptides (AMPs) ...............................................................16 Coagulation ..............................................................................................19 Cellular immune response........................................................................20 Hematopoiesis ..........................................................................................22 Antiviral immune response ......................................................................23

Objectives .....................................................................................................28

Results and discussion ..................................................................................29 Histone H2A as a transfection agent in crayfish hematopoietic tissue cells (Paper I) ...........................................................................................29 Antilipopolysaccharide factor interferes with white spot syndrome virus replication in vitro and in vivo in the crayfish Pacifastacus leniusculus (Paper II) ...............................................................................30 Phenoloxidase is an important component of the defense against Aeromonas hydrophila infection in a crustacean, Pacifastacus leniusculus (Paper III) ..............................................................................31 Hemocyte lineage marker proteins in a crustacean, the freshwater crayfish, Pacifastacus leniusculus (Paper IV)..........................................32

Concluding remarks ......................................................................................34

Svensk sammanfattning (Summary in Swedish) ..........................................37

Acknowledgements.......................................................................................39

References.....................................................................................................41

Abbreviations

2-DE Two dimensional gel electrophoresis �GBP �-1, 3-glucan binding protein AMPs Antimicrobial peptides ALF Antilipopolysaccharide factor Dscam Down syndrome cell adhesion molecule dsRNA Double stranded RNA ECPs Extracellular products EGF Epidermal growth factor EST Expressed sequence tag GCs Granular cells GFP Green fluorescent protein HLS Hemocyte lysate supernatant Hpt Hematopoietic tissue IgSF Immunoglobulin super family KPI Kazal type proteinase inhibitor LGBP Lipopolysaccharide and �-1, 3-glucan binding protein LPS Lipopolysaccharide MS Mass spectra NLSs Nuclear localization signals PCNA Proliferating cell nuclear antigen PGN Peptidoglycan PGRPs Peptidoglycan recognition proteins PO Phenoloxidase ppA Propheonoloxidase activating enzyme PRPs Pattern recognition proteins proPO Prophenoloxidase RNAi RNA interference RT-PCR Reverse transcriptase polymerase chain reaction SGCs Semigranular cells siRNA Small interference RNA SOD Superoxide dismutase SSH Suppression subtractive hybridization TEP Thioester-containing protein TGase Transglutaminase WSSV White spot syndrome virus YHV Yellow head virus

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Introduction

Unlike vertebrate immunity which is composed of innate and adaptive re-sponses, invertebrates rely solely on multiple innate defense reactions to combat infections. These reactions include the protection by physical barri-ers together with local (eg. epithelial immunity) and systemic immune re-sponses. Two main components, the humoral and cellular systems, are in-volved in the systemic innate immune response in invertebrates, both of which are activated upon immune challenge. A hallmark of the humoral host-defense is the challenge-induced synthesis and secretion of immune factors, eg. antimicrobial peptides (AMPs), that accumulate in the hemo-lymph where they act on invading microbes. The cellular response by hemo-cytes involves phagocytosis, nodule formation, and encapsulation of patho-gens. Further, two other reactions, coagulation and melanization are acti-vated immediately upon injury/infection and play critical roles. Melanization is the response whereby melanin is formed at invading parasite surfaces and wound sites, resulting in the release of toxic reactive oxygen species and immobilization of intruders. The defense strategies are diverse for different pathogens, but many of them are evolutionarily conserved such as produc-tion of AMPs, activation of phagocytic cells and generation of toxic metabo-lites. Important progress has been made into key aspects of the crustacean immune system, including the role of melanization, AMPs, coagulation, phagocytosis and reactive oxygen metabolism. Some of these well-conserved immune response pathways (eg. melanization and production of AMPs) are, to some extent, well understood at the biochemical level (Bachère et al. 2004; Cerenius and Söderhäll 2004). However, knowledge of the immune defense systems is still too limited in crustaceans. Research is hampered from a lack of knowledge of genomic data and genome organiza-tion of these animals. So far, no real established cell lines are available for crustaceans. Therefore, the hematopoietic tissue (Hpt) cell cultures in fresh-water crayfish, Pacifastacus leniusculus, provide a useful tool for gene func-tional studies in crustaceans (Söderhäll et al. 2005). The freshwater crayfish mounts an immune response against pathogens through the use of both hu-moral and cellular responses to activate multiple cellular and humoral re-sponses including melanization, blood coagulation, the production of AMPs, and the phagocytosis/encapsulation of invading microorganisms by hemo-cytes. A schematic view of innate immune responses of crayfish is shown in Figure 1. The main components involved in innate immunity of crustaceans and some other invertebrate models are described in summary. Several fac-

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tors have been identified from crayfish P. leniusculus (Table 1) (Iwanaga and Lee 2005).

Figure 1. Schematic view of innate immune responses in crayfish P. leniusculus.

Pattern recognition proteins (PRPs) in arthropods Innate immune activation is based on recognition of pathogen molecules not present in the host. A number of soluble molecules binding to and lysing microbes have been found in invertebrates. For instance, lectin-like proteins can bind to carbohydrates present on microbial cell walls followed by activa-tion of immune response to kill/remove the invading microbes. These recog-nition molecules for non-self materials have been named as pattern recogni-tion proteins (Medzhitov and Janeway 1997). Pathogen-associated molecules not found in other multicellular organisms, eg. lipopolysaccharide (LPS)/peptidoglycan (PGN) from bacterial cell walls, �-1, 3-glucan from fungal cell walls, and double stranded RNA (dsRNA) of viruses, can be rec-ognized by these recognition proteins based on the particular structures of the invading microbes. Current studies have emphasized the important roles of non-self recognition molecules both in the vertebrate and the invertebrate

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Table 1. Immune factors found in hemocytes, plasma and tissues of the fresh-water crayfish (P. leniusculus) modified from Iwanaga and Lee, 2005.

Name Mass (kDa) Activity

Prophenoloxidase system Prophenoloxidase (proPO) 76 Precursor of phenoloxidase ProPO activating enzyme (ppA) 48 Serine protease cleaves proPO

LPS and �-1,3-glucan binding proteins (LGBP) �-1, 3-glucan binding protein 100 Recognition protein in proPO activation LGBP 40 ProPO activation, opsonin and adhesion

Mannose-binding lectin 28 Secreted from hemocytes upon challenge Cell adhesion proteins Masquerade-like protein I 129/134 Non-catalytic cell adhesion protein Peroxinectin 76 Cell adhesion molecule

Proteinase inhibitor Kazal-type inhibitors 10-30 Kazal type proteinase inhibitor �2-Macroglobulin 190 x 2 Complement-like activity Pacifastin 155 Serine protease inhibitor with a unique

transferrin chain Subtilisin inhibitor 28 Proteinase inhibitor Antibacterial proteins Astacidine 1 and 2 1.6-1.8 Antimicrobial peptide Anti-LPS factor 13.5 Antiviral activity Carcinine-like peptides 14-20 Antimicrobial peptide Crustin-like peptides 10.2-15.2 Antimicrobial peptide Hemagglutinin 420 Hemagglutinating activity Lysozyme 14 Cell lysis

Others Astakine 8.7 Cytokine-like activity in hematopoiesis Cytosolic ferritin 440 A storage protein for ferric ion Hemocyanin 360 O2 transporter, phenoloxidase-like activ-

ity, and produces antimicrobial peptides Masquerade-like protein II 40 Hemocyte secreted protein upon challenge Masquerade-like protein III 46 Binding activity to partially digested

insoluble Lys-type PGN Transglutaminase (TGase) 87 Cross-linking Vitellogenin-related protein 210 x 2 A plasma clotting protein, lipoprotein- like and TGase substrate

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immune systems. Recognition of microbial carbohydrates, that are distinct from those of host cells is the first step to trigger the innate immunity. Many recognition molecules, such as LPS or/and �-1, 3-glucan binding proteins (LBP, �GBP or LGBP), peptidoglycan recognition proteins (PGRPs), and lectins have been identified in various invertebrates and different biological functions have been suggested for these molecules following their binding to the microbes (Lee and Söderhäll 2002).

Lectins/agglutinins are sugar-binding proteins usually without catalytic activity. Various biological activities such as the cellular and tissue transport of carbohydrates and glycoproteins (Vasta 1992), cell adhesion (Vasta et al. 1999), opsonization (Cerenius et al. 1994) and nodule formation (Koizumi et al. 1999), involve the interaction between lectins and carbohydrates. Some lectins such as �GBP and LGBP have been suggested to enhance activation of the prophenoloxidase (proPO)-system in invertebrates. In crustaceans, �GBP was firstly cloned and identified for its properties involved in the acti-vation of the proPO-system (Cerenius et al. 1994). Crayfish �GBP is present in plasma and recognizes �-1, 3-glucan to promote the activation of the proPO-system and also to act as an opsonin to increase the phagocytic activ-ity. The �GBP and glucan complex may bind to crayfish granular hemocytes surface via its RGD (Arg-Gly-Asp) motif, suggesting binding to an integrin-like protein and then spreading and degranulation of crayfish hemocytes is induced. An integrin �-subunit has been characterized as a candidate recep-tor for the �GBP and glucan complex (Cerenius et al. 1994; Duvic and Söderhäll 1990; Duvic and Söderhäll 1992; Thörnqvist et al. 1994). �GBP also interacts with an extracellular superoxide dismutase (SOD) involved in binding with peroxinectin (a cell adhesive and opsonic peroxidase) to the cell surface. Binding of peroxinectin to the cell surface may signal into the cell via an integrin for further cellular responses such as cell adhesion and phagocytosis (Johansson et al. 1999b). Moreover, LGBP has been identified from crayfish hemocytes and it shows binding activity to both LPS and �-1, 3-glucans but not peptidoglycans (Lee et al. 2000). Other LGBPs and �GBPs were also shown to mediate the activation of the proPO-system (Ma and Kanost 2000) as well as induction of AMPs (Kim et al. 2000). These mole-cules do not exhibit any glucanase activity although they have similar pri-mary structure to bacterial glucanases (Beschin et al. 1998; Cerenius et al. 1994; Ochiai and Ashida 2000). Two other inducible immunolectins C-type lectin (Yu et al. 1999) and an LPS specific lectin (Yu and Kanost 2000) were identified from tobacco hornworm, Manduca sexta, and were both found to activate the proPO-system. C-type lectins and calcium dependent lectins were suggested to be involved in immune recognition in invertebrates (Vasta et al. 1999; Weis et al. 1998). Constitutively expressed LPS-binding lectins were indentified from silkworm, Bombyx mori (Koizumi et al. 1999) and the American cockroach, Periplaneta americana (Jomori and Natori 1992). These LPS-binding molecules showed a bacterial clearance activity and an opsonic effect, respectively. In horseshoe crab, many different lectins

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(tachylectins) were shown to be involved in critical immune responses such as hemagglutinating activity and antibacterial activity (Kawabata and Iwanaga 1999).

The crayfish masquerade-like protein (PlMasI) was identified as a pattern recognition molecule in hemocytes. It has binding activity to LPS, �-1, 3-glucans, Gram-negative bacteria, and yeast. Besides, PlMasI has an opsonic and cell adhesive activity (Huang et al. 2000; Lee and Söderhäll 2001) and is a multifunctional protein. It exists usually in an intact form and is processed by an unknown proteolytic enzyme when binding to microbes. Its amino acid sequence shows homology to serine proteinases except for the substitu-tion of one amino acid of the catalytic triad resulting in loss of enzyme activ-ity. Two other masquerade-like proteins PlMasII and PlMasIII were also isolated from crayfish. PlMasII was released from exocytosis of crayfish granular hemocytes triggered by a calcium ionophore, LPS-PGN or peroxi-nectin (Sricharoen et al. 2005). PlMasIII was recently separated from cray-fish hemocyte lysate supernatant (HLS) by its binding property to a partially digested insoluble Lys-type PGN (unpublished data). The functions of these two proteins in crayfish still need further studies. A shrimp clip domain ser-ine proteinase homolog (c-SPH) has been shown to act as a cell adhesion molecule (Lin et al. 2006). To date, several serine proteinase homologues have been identified from various invertebrate animals and they have impor-tant biological roles such as cell adhesion activity (Huang et al. 2000), an-timicrobial activity (Lee and Söderhäll 2001), pattern recognition, opsoniza-tion, and as a component of the proPO-system (Kwon et al. 2000; Lee et al. 2002; Piao et al. 2005). Therefore, this demonstrates that serine proteinase homologous proteins play critical roles in invertebrates even though these molecules do not show any proteinase activity.

Besides, Toll/Toll-like receptors are an evolutionary ancient family of pat-tern recognition receptors playing important roles in innate immune re-sponses. Most of these studies were carried out in insects (Leulier and Le-maitre 2008). A novel Toll receptor gene was identified from Penaeus monodon but no change of gene expression of this gene after white spot syn-drome virus (WSSV) infection was observed (Arts et al. 2007). Another Toll homolog was also isolated from Chinese shrimp F. chinensis, which was distinctly modulated by bacterial or viral stimulation (Yang et al. 2008). However, the mechanism for how this Toll homolog is regulated by micro-bial challenge is still unknown.

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Melanization and prophenoloxidase activating system (proPO-system) The melanization reaction is a rapid immune response in invertebrates ob-served at the site of cuticular injury or on the surface of invading parasites. This melanotic reaction results from the synthesis and deposition of melanin mediated by proPO activation (Cerenius et al. 2008). The proPO cascade is an efficient nonself-recognition system in invertebrates, which includes sev-eral proteins involved in the immune defense resulting in melanin produc-tion, cell adhesion, encapsulation, and phagocytosis triggered by recognition of LPS/PGNs from bacteria and �-1, 3-glucans from fungi. Several PRPs, such as LGBP and �GBP, involved in the proteinase cascade have been well studied in crayfish (Cerenius and Söderhäll 2004). Crayfish proPO is synthe-sized in the hemocytes, and released into plasma by exocytosis triggered by PRPs. After binding with microbial components, PRP molecules induce activation of proteinases in the proPO-system. Finally, proPO is proteolyti-cally converted into active phenoloxidase (PO) by the prophenoloxidase activating enzyme (ppA) (Aspán et al. 1995), an endogenous trypsin-like serine proteinase. A recombinant defensin-like peptide comprising the clip domain of ppA from crayfish shows antibacterial activity in vitro against Gram-positive bacteria indicating a multiple function of crayfish ppA and possible for other ppAs too (Wang et al. 2001a). Many proPO sequences have been studied from different invertebrates, such as insects, ascidians, mollusks, echinoderms, millipedes, bivalves, and brachiopods (Cerenius and Söderhäll 2004), since the first primary structure of proPO was identified in crayfish (Aspán et al. 1995). Several ppAs and cofactors have been identi-fied from insects: a beetle, Holotrichia diomphalia, a silkworm, Bombyx mori, and a tobacco hornworm, Manduca sexta (Cerenius and Söderhäll 2004). These proteins all exist as zymogens of typical serine proteinases based on their primary structure and are similar to Drosophila serine pro-teinases involved in the regulation of the developing embryo. Other proPO activating factors (masquerade-like serine proteinase homologues) have been characterized from the coleopteran insects H. diomphalia and Tenebrio mo-litor, which have no proteinase activity but an essential role in activation of the proPO-system in these insects (Kwon et al. 2000; Lee et al. 2002; Piao et al. 2005). The primary structures of these proteins show a serine proteinase domain lacking the catalytic triad residues necessary for serine proteinase activity similar to the crayfish masquerade-like protein PlMasI (Lee and Söderhäll 2001), a Drosophila masquerade protein (Murugasu-Oei et al. 1995), horseshoe crab factor D (Kawabata et al. 1996) and mosquito infec-tion-responsive serine proteinase-like protein (ispl5) (Dimopoulos et al. 1997).

Importantly, the activation of the proPO-system can also trigger cellular responses including phagocytosis, nodule formation, and encapsulation (Pa-per III) (Johansson and Söderhäll 1995). Functional studies of crayfish PO

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suggest that PO was required in crayfish defense against an infection by a highly virulent and pathogenic bacterium, Aeromonas hydrophila, to cray-fish. Silencing of crayfish proPO led to increased bacterial growth, lower phagocytosis, lower PO activity, lower nodule formation, and higher mortal-ity when infected with this bacterium. In contrast, if there was a higher PO activity by silencing of pacifastin (an inhibitor of the crayfish proPO activa-tion cascade), it resulted in lower bacterial growth, increased phagocytosis, increased nodule formation, and delayed mortality (Paper III). Recent details have been collected on the activation of proPO-system and more specifically on the regulation of the proPO activating proteinase cascade by zymogenic serine proteinases, serine proteinase homologues and PRPs (Gorman et al. 2007; Park et al. 2006; Park et al. 2007; Piao et al. 2007; Piao et al. 2005). Evidence strongly implies that the melanization cascade (the proPO activat-ing system) provides, or is closely associated with factors stimulating cellu-lar defense by aiding phagocytosis (Cerenius et al. 2008). Other molecules such as peroxinectin, a cell adhesion protein from crayfish and shrimp (Johansson 1999a; Lin et al. 2007; Sritunyalucksana et al. 2001), can gain their function under the activation of proPO-system. There are reports that proPO binds to the outer membrane of some hemocytes by which a strict spatial localization of the melanization response can be enhanced (Ling and Yu 2005; Mavrouli et al. 2005). Studies from Anopheles indicate that PO may be involved in blood clotting process (Agianian et al. 2007), promoting cellular defense reactions and increasing the efficiency of plasma coagula-tion.

Given the toxic intermediates produced by PO, the initiation of proPO-system must be tightly controlled to avoid the production of deleterious ef-fectors. In crayfish, an efficient inhibitor against ppA is pacifastin, which contains a light chain with nine proteinase inhibitor subunits and a heavy chain with three transferrin lobes (Liang et al. 1997). This molecule has been grouped into a new class of proteinase inhibitors named as pacifastin-like serine proteinase inhibitors which inhibit the proPO-system in different in-sects (Simonet et al. 2002; Vanden Broeck et al. 1998). Silencing of paci-fastin always resulted in 100% mortality of crayfish challenged with A. hy-drophila, indicating that endogenous proteinase inhibitors could, perhaps, be employed to protect certain tissues or cells by inhibiting unnecessary pro-duction of highly toxic and reactive compounds (Paper III). Some proteinase inhibitors preventing overreaction of ppA (Hergenhahn et al. 1987) and a PO inhibitor directly inhibiting the activity of PO (Daquinag et al. 1995; Daqui-nag et al. 1999; Levashina et al. 1999; Sugumaran and Nellaiappan 2000) have been studied from different arthropods. A well studied serpin encoded by spn27A was shown to inhibit the proPO activating proteinase in Droso-phila. Mutations in spn27A led to uncontrolled melanization and increased lethality (De Gregorio et al. 2002). Impaired melanotic encapsulation of the parasitoid wasp Leptolinina boulardi and increased parasitoid survival rate have been found in the fly hosts by injections of serpin27A into Drosophila

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larvae (Nappi et al. 2005). Genetic studies revealed several serpin genes affecting the melanization response toward different pathogens in both Dro-sophila (Ligoxygakis et al. 2002) and Anopheles mosquitoes (Michel et al. 2005). RNA interference (RNAi) of SRPN6, another member of the family, the expression of which was strongly up-regulated by parasite infection, did not change the number of developing parasites in A. gambiae but rather in-fluenced the balance between lysis and melanization during clearance of killed parasites (Abraham et al. 2005). Recent studies indicate that some specific pathogens have developed strategies to block proPO activation by generating serpin type inhibitors or other factors specifically interfering with the proteolytic activation of proPO cascade. Endogenous effectors, micro-bial-derived factors and non-proteinaceous factors have been identified for their capacities to interfere with active PO (Daquinag et al. 1995; Eleftheri-anos et al. 2007; Lu and Jiang 2007; Shi et al. 2006; Zhao et al. 2005). Sev-eral clip-domain serine proteases have also been shown to be involved in limiting parasite numbers and/or affecting the regulation of melanization (Volz et al. 2006; Volz et al. 2005). Additionally, hemocyanin, the respira-tory protein produced by the hepatopancreas and localized in the plasma from many arthropods and mollusks exhibits PO activity under certain con-ditions which may also play critical roles in immune defense against micro-bial invasions (Baird et al. 2007; Decker and Rimke 1998; Jaenicke and Decker 2008; Jiang et al. 2007; Lee et al. 2004; Nagai et al. 2001).

Antimicrobial peptides (AMPs) AMPs have been shown to be rapidly elicited after microbe presentation and be active against many bacteria and/or fungi (Boman 2000). More than 910 AMPs have been discovered in plants, invertebrates, and vertebrates. These effectors show a great diversity in structural features, biological properties and functions, as well as in their tissue distribution and expression. They have a wide range of antimicrobial activity against Gram-positive and Gram-negative bacteria, filamentous fungi, viruses and protozoa. In D. melanogaster, different AMPs are primarily produced in the fat body and secreted into the blood in response to a microbial infection. Genetic analyses have revealed that the Toll and Imd pathways regulate AMP gene expres-sion. The Toll pathway plays a key role in the defense against Gram-positive bacterial and fungal infections, whereas the Imd pathway controls most re-sponses to Gram-negative bacterial infection (Ferrandon et al. 2007; Leulier and Lemaitre 2008). The expression of these peptides is also locally regu-lated in the surface epithelia (Tzou et al. 2000). In crustaceans, most of the AMPs reported have been isolated from hemocytes.

It is well known that AMPs play critical roles in innate immunity in crus-taceans. The first crustacean AMP was identified as a proline-rich 6.5 kDa peptide from the shore crab Carcinus maenas (Schnapp et al. 1996). In crus-

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tacean AMPs, the penaeidin and crustin families have been well studied. The penaeidin family, identified from both Litopenaeus vannami and L. setiferus, is a broad-spectrum AMP family containing a proline-rich N-terminal do-main and a C-terminal domain with six cystein residues (Destoumieux et al. 1997; Gross et al. 2001). Recently, an increasing number of penaeidin/penaeidin-like AMPs have been studied in various species of shrimps (Barracco et al. 2005; Chiou et al. 2007; Chiou et al. 2005; Kang et al. 2007), in which some penaeidins show strong bactericidal activities. Ge-nomic structure and transcriptional regulation of the penaeidin gene family were also reported from L. vannamei, showing that each penaeidin class is encoded by a unique gene and isoform diversity is generated by polymor-phism within each penaeidin gene locus (O'Leary and Gross 2006).

The crustin family, eg. crustin peptide (CM1) initially identified in the hemoyctes of the crab C. maenas is cystein-rich, hydrophobic and shows specific activity against Gram-positive marine bacteria (Relf et al. 1999). This family also contains crustins from L. vannamei and L. setiferus which have an amino terminal glycine rich repeat region of 40-50 amino acid resi-dues in addition to the cystein-rich C-terminal, but no antibacterial activity has been tested for these crustins (Bartlett et al. 2002). Over 50 crustin se-quences have been reported from a variety of decapods, including crab, lob-ster, shrimp and crayfish. These crustins purified as native proteins or ex-pressed recombinant are all killing Gram-positive bacteria (Smith et al. 2008). Some crustins are usually found to be constitutively expressed in hemocytes and the expression might be enhanced 2-3 times upon bacterial challenge in shrimp and lobster (Hauton et al. 2006; Hauton et al. 2007). Other crustin-like putative antibacterial proteins were investigated in Ameri-can lobster, Chinese shrimp, Brazilian penaeid shrimp, and black tiger shrimp. Some of these recombinant crustin-like proteins also showed strong antibacterial activities against Gram-positive bacteria (Amparyup et al. 2008; Christie et al. 2007; Rosa et al. 2007; Zhang et al. 2007a; Zhang et al. 2007b). However, gene expression studies have shown that some crustins were con-stitutively expressed often at high levels, but no consistent patterns of change were observed in expression following challenge with bacteria. This variable response to infection is enigmatic but indicates that these proteins could perform additional functions, perhaps as immune regulators in recov-ery from wounding, trauma or physiological stress (Smith et al. 2008).

Several AMPs, such as astacidin1, astacidin2 and crustins1-3 have been identified in freshwater crayfish. Astacidin1 inhibits growth of both Gram-positive and Gram-negative bacteria. A synthetic astacidin1 showed similar activity as the authentic astacidin1 against Gram-positive bacteria but rather less or no activity against Gram-negative bacteria. The astacidin1 can be produced by a proteolytic cleavage from hemocyanin under acidic conditions. This process and release of astacidin1 from hemocyanin was enhanced when crayfish was injected with LPS or glucan (Lee et al. 2003). Astacidin2 has a broad range of antibacterial activity against both Gram-positive and Gram-

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negative bacteria. The transcript of Plcrustin1 was increased in both hemo-cytes and hematopoietic tissue as a response to challenge with Gram-negative non-pathogenic bacteria Acinetobacter ssp. or E. coli as well as by a highly pathogenic Gram-negative bacterium A. hydrophila (Jiravanichpaisal et al. 2007).

Recently, another well known cationic protein, antilipopolysaccharide fac-tor (ALF) originally isolated from the horseshoe crab Limulus polyphemus (Aketagawa et al. 1986; Muta et al. 1987) has also been widely studied in crustaceans. L. polyphemus ALF binds to LPS and shows a strong antibacte-rial activity, especially on the growth of Gram-negative bacteria. A shrimp ALF was shown to be effective in protecting animals from Gram-negative bacterial infection (Pan et al. 2007). Interestingly, the crayfish ALF was up-regulated by a WSSV challenge and was shown to be involved in antiviral function against WSSV. Silencing of ALF specially resulted in higher rates of WSSV propagation both in the animals and in a cell culture of Hpt from crayfish. In contrast, overexpression of ALF in the animals by the admini-stration of UV-treated WSSV led to lower viral replication and a partial pro-tection against a subsequent challenge with the active virus (Paper II). ALF from shrimp was also up-regulated in L. vannamei upon WSSV challenge (Robalino et al. 2007a). Silencing of LvALF1 resulted in a significant in-crease of mortality in L. vannamei by V. penaeicida and F. oxysporum indi-cating that LvALF1 has a role in protecting shrimp from both bacterial and fungal infections but not WSSV infection even if it was up-regulated by WSSV infection (de la Vega et al. 2008). This is different from crayfish ALF which showed antiviral property in both in vivo and in vitro in crayfish (Pa-per II). The mechanism for antiviral activity of crayfish ALF is still un-known. Further studies showed that recombinant ALFPm3, but not ALFPm2, from shrimp P. monodon affected on the viral infection by inhibiting the attachment of virus to the crayfish Hpt cells suggesting that ALF may act on the entry of virus to host cells (unpublished data). Several studies have re-ported the molecular cloning, sequencing and expression analysis of ALF in various crustaceans (Gross et al. 2001; Imjongjirak et al. 2007; Liu et al. 2005; Nagoshi et al. 2006; Somboonwiwat et al. 2006; Supungul et al. 2004). Recombinant ALF or a synthetic peptide containing part of the ALF se-quence exhibited antimicrobial activity against fungi, Gram-positive and Gram-negative bacteria (de la Vega et al. 2008; Imjongjirak et al. 2007; Li et al. 2007a; Somboonwiwat et al. 2005). ALFPm3 was recently shown to have binding activity to Gram-negative and Gram-positive bacteria as well as their major cell wall components LPS and lipoteichoic acid, respectively, suggesting that ALFPm3 executes its antibacterial activity via binding to components of the bacterial cell wall (Somboonwiwat et al. 2008). These data together suggest that ALFs play critical roles against bacteria, fungi, and virus in crustaceans. The functional characterization of ALF in anti-WSSV response will bring interesting insights into the antiviral defense in crustaceans. Analysis of ALF genomic organization has also been performed

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in different species such as shrimp and crab which may be helpful for our understanding of the regulation of gene expression under microbial chal-lenges (Imjongjirak et al. 2007; Li et al. 2007a; Tharntada et al. 2008).

Besides, trypanocidal and leishmanicidal activities of different AMPs iso-lated from aquatic animals have also been investigated, in which tachyplesin from Tachypleus tridentatus showed the most potent peptide for killing completely L. braziliensis and trypomastigote T. cruzi in lower concentra-tions (Lofgren et al. 2008). Further, a hepatopancreas-specific C-type lectin from F. chinesis exhibits antimicrobial activity (Sun et al. 2008) and its ex-pression could be up-regulated following challenge of shrimp with bacteria or virus. A newly isolated shrimp ovarian peritrophin from F. merguiensis (Loongyai et al. 2007) and a male specific anionic AMP scygonadin from Scylla serrata (Wang et al. 2007b) both have antimicrobial activities.

Coagulation Blood coagulation is a first line of defense composing a part of the whole invertebrate immune system. Clotting is important in reducing hemolymph loss and for initiating wound healing in crustaceans. It is also a critical im-mune defense to form a secondary barrier to infection, immobilized bacte-ria/fungi and hence promote their killing. Mechanisms of coagulation have been established based on crayfish (Hall et al. 1995; Hall et al. 1999; Kopácek et al. 1993; Wang et al. 2001b) and horseshoe crab (Iwanaga et al. 1992) as models. The clot formation is linked with the release of antimicro-bial effectors in the horseshoe crab (Iwanaga 2002; Iwanaga and Lee 2005). A new study showed that a protein stablin promotes the formation of the clotting mesh and the immobilization of invading microbes at injury sites (Matsuda et al. 2007).

In crustaceans, clotting occurs through the polymerization of the clotting protein catalyzed by a Ca2+ ion dependent transglutaminase (TGase), which is released from hemocytes upon the stimulation by foreign organisms or tissue damage (Hall et al. 1999; Wang et al. 2001b). The clotting protein of crayfish was the first cloned from a crustacean and it is a high density lipo-glycoprotein (HDL) composed of two identical 210 kDa subunits held to-gether via disulfide bonds. Each of the 210 kDa subunits contains both lysine and glutamine in the side chains which are recognized by TGase to become covalently linked to each other (Kopácek et al. 1993). The primary structure of crayfish clotting protein suggests that it belongs to the vitellogenin super-family (Hall et al. 1999). Whereas, the shrimp clotting protein shows 36% identities to the crayfish clotting protein and lower similarities to insect vitel-logenin, apolipoprotein B and mammalian von Willebrand factor (Yeh et al. 1999). TGase and clottable protein have been suggested to be involved in the blood coagulation system of many crustaceans, such as shrimp, lobster, freshwater giant prawn, and sand crayfish (Fuller and Doolittle 1971; Koma-

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tsu and Ando 1998; Yeh et al. 1998). These clotting proteins have similar amino acid composition and N-terminal sequences but no homology to horseshoe crab coagulogen. The mortality rate of shrimps after infection of V. penaecida and WSSV was significantly higher in TGase and clotting protein depleted animals indicating the essential roles of these two molecules in shrimp immune protection against microbial challenges (Maningas et al. 2008).

Cellular immune response The cellular immune responses mainly involve phagocytosis and encapsula-tion by hemocytes. Hemocytes can internalize various particles including bacteria, yeast, Sephadex beads, and dsRNA. Phagocytosis is one of the earlier immune responses in invertebrates. It is an evolutionary conserved, complex process based on recognition, engulfment, and intracellular destruc-tion of invading microorganisms. Phagocytosis is initiated by the attachment of the phagocyte to the target particle, and followed by cytoskeleton modifi-cation, internalization, and destruction of the engulfed target within phagosomes. The importance of the blood cells in combating infection is visible when phagocytosis is blocked or in mutants lacking blood cells (Braun et al. 1998; Elrod-Erickson et al. 2000).

Crayfish has an open circulating system which is filled with hemolymph containing hemocytes. These hemocytes originate from the hematopoietic tissue and mature hemocytes can be divided into the following three cell types based on their structural and functional features: hyaline cells (HCs), semigranular cells (SGCs), and granular cells (GCs) (Söderhäll and Smith 1983). The hyaline cells are small, spherical and have no or few granules. These cells constitute � 5% of all mature hemocytes and function in the phagocytic removal of microbial pathogens and dead cells. The SGCs con-tain small granules and participate in encapsulation as well as phagocytosis. Both GCs and SGCs store the components of the proPO activating system and participate in cytotoxic reactions in addition to generate and store AMPs and other immune effectors (Söderhäll et al. 1985).

Hemocytes also play a role in surveillance of healthy and damaged base-ment membranes and to encapsulate and destroy aberrant tissue. It has been shown in Lepidoptera that hemocytes are able to recognize virally infected cells (Trudeau et al. 2001). Phagocytosis is critical for host defense against invading pathogens and for the removing of apoptotic cells produced during development (Aderem and Underhill 1999). Several types of receptor pro-teins have been well-documented to be involved in phagocytosis (Stuart and Ezekowitz 2008). These include members of the scavenger receptor family, the epidermal growth factor (EGF)-domain protein Eater, complement-like opsoins, and the immunoglobulin super family (IgSF)-domain protein down syndrome cell adhesion molecule (Dscam) (Kocks et al. 2005; Pearson et al.

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1995; Ramet et al. 2001; Watson et al. 2005). For example, inhibition of phagocytosis by depletion of the receptor Eater in D. melanogaster dramati-cally decreased survival of flies after gastrointestinal bacterial challenges by feeding with the pathogen Serratia marcescens (Kocks et al. 2005). Phago-cytosis was also the primary mechanism of parasite elimination in the Aedes Aegypti/Plasmodium gallinaceum model (Hillyer et al. 2003). In another recent study the authors suggest that phagocytosis is involved in the antiviral response against WSSV, which is regulated by a novel Rab-dependent com-plex in shrimp (Wu et al. 2008).

Some other proteins including proteins related to CD36, PGRP family members, and an LPS recognition protein with six EGF repeats, were also shown to favor phagocytosis or play a role in this process (Franc et al. 1996; Garver et al. 2006; Ju et al. 2006; Ramet et al. 2002). In A. gambiae, the thioester dependent binding of thioester-containing protein (TEP1) to the surface of bacteria promoted their phagocytosis. TEP1 and integrin BINT2 have been well documented to be involved in phagocytosis (Levashina et al. 2001; Moita et al. 2006).

Dscam, a hypervariable immunoglobulin domain containing protein in in-sects, was recently described to bind to bacteria and affect phagocytosis in mosquitoes (Dong et al. 2006). Dscam was first characterized in D. melanogaster as an axonal pathfinder (Schmucker et al. 2000) and as a puta-tive phagocytic receptor (Watson et al. 2005). The Dscam gene comprises a cluster of variable exons flanked by constant exons, which could theoreti-cally generate as many as 38,016 and 32,000 isoforms in Drosophila and Anopheles, respectively, by alternative splicing. Secreted isoforms of Dscam could be detected in the hemolymph, and silencing of Dscam by RNAi in cultured mosquito cells was shown to decrease phagocytosis of E. coli and S. aureus. Further, it was proposed that challenges of a mosquito cell line with different elicitors induce the production of distinct isoforms through regula-tion of the alternative splicing of Dscam. Moreover, in vivo depletion of a given microbe-induced isoform in adult mosquitoes apparently increased their susceptibility to the corresponding microbe but not to unrelated bacteria (Dong et al. 2006). These data raise the possibility that the different Dscams may be important in recognizing different pathogens. Hypervariable Dscam has been found in other insects such as Apis mellifera, Tribolium castaneum, and Bombyx mori. Diversified Dscam was newly reported from crustacean Daphnia magna and Daphnia pulex. The Dscam of Daphnia generates up to 13,000 different transcripts by the alternative splicing of variable exons based on the genomic analysis (Brites et al. 2008). Dscam transcripts were also found in crayfish (P. leniusculus) nerve cords, hemocytes and Hpt cells (unpublished data) indicating that Dscam might play important roles in the nervous and immune system of crustaceans. The molecular diversity of Dscam transcripts is maintained across major insect orders indicating a con-served role of this gene.

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Encapsulation is a dramatic defense response against invading parasites too large to be phagocytosed by individual hemocytes. Meanwhile, nodula-tion is multicelluar hemocytic aggregates entrapping a large number of bac-teria in an extracellular material (Gillespie et al. 1997). In crayfish, peroxi-nectin and PlMasI were shown to be involved in these processes (Johansson et al. 1995; Lee and Söderhäll 2001). Peroxinectin was initially purified as a hemocyte adhesive molecule functioning as opsonin to promote phagocyto-sis during the immune response (Johansson and Söderhäll 1988). It was the first reported molecule showing both cell adhesion and peroxidase activities. PlMasI is also involved in clearance of bacteria in crayfish. Intact PlMasI was isolated from hemocytes which functions as a heterodimer containing two subunits with molecular masses of 134 kDa and 129 kDa. This molecule has binding activity to LPS, �GBP, Gram-negative bacteria and yeast, and is processed by a proteolytic enzyme after binding to bacteria or yeast cell walls. The processed 33 kDa protein from the heterodimer showed cell adhe-sion activity. E. coli coated with PlMasI was shown to be removed more rapidly than E. coli alone in crayfish suggesting that the PlMasI has both cell adhesive and opsonic activities (Lee and Söderhäll 2001).

Hematopoiesis Hematopoiesis is the process where hemocytes form and mature and subse-quently enter the blood circulation. This process involves proliferation, commitment and differentiation from undifferentiated hematopoietic cells (Barreda and Belosevic 2001; Medvinsky and Dzierzak 1999). In crusta-ceans, the number of free hemocytes varies in response to environmental stress, endocrine activity during the moulting cycle, and infection (Jiravanichpaisal et al. 2006a; Johansson et al. 2000). Thus, new hemocytes are necessary to be continuously produced from a separate organ named as hematopoietic tissue. However, the mechanisms for the release of blood cells into circulation in crustaceans are still unknown. In the crayfish P. leniuscu-lus, mature hemocytes arise from the Hpt which is situated on and covers the dorsal and dorsolateral sides of the stomach and is surrounded by connective tissue (Chaga et al. 1995). The Hpt contains different cell types based on their morphology. In P. leniusculus Hpt at least five different types of cells which might correspond to different developmental stages of GCs and SGCs can be identified (Chaga et al. 1995). Morphological studies of hematopoi-etic tissue have been carried out in the lobster Homarus americanus (Martin et al. 1993), blue crab Carcinus sapidus (Johnson 1987), shrimp Sicyonia ingentis (Hose et al. 1992), and the black tiger shrimp P. monodon (van de Braak et al. 2002b). But no details of hemocyte maturation and release have been shown in these crustaceans.

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Hematopoiesis is tightly regulated by transcription factors that promote cell diversification (Orkin 1998; Orkin 2000; Sieweke and Graf 1998). Many hematopoietic factors have been characterized and they are conserved across taxonomic groups including both protostomian and deuterostomian organ-isms, ranging from flies to humans (Fossett et al. 2001b). In Drosophila, five genes: Serpent (Srp), Lozenge (Lz), U-shaped (Ush), glial cell missing (Gcm), and friend of GATA (FOG) have been identified to be involved in the hematopoietic lineage commitment (Fossett and Schulz 2001a; Lebestky et al. 2000; Rehorn et al. 1996). The D. melanogaster embryo contains two primary blood cell lineages, the plasmatocytes and the crystal cells, and both of them originate from a common hemocyte progenitor expressing the GATA protein Srp (Lebestky et al. 2000). The Lz-homologue PlRunt has been shown to have a role in crayfish hematopoiesis (Söderhäll et al. 2003). The Hpt of crayfish was shown to be actively proliferating. Injection of a �-1, 3-glucan results in severe loss of hemocytes that will cause an accelerated maturation of hemocyte precursors in the Hpt followed by release into the circulation of new cells, which may develop into functional SGCs and GCs expressing the proPO transcript (Söderhäll et al. 2003). Astakine, an en-dogenous cytokine like-factor containing a prokineticin domain from cray-fish P. leniusculus (Söderhäll et al. 2005), has been shown to play a critical role in the differentiation and growth of hematopoietic stem cells in vitro. In addition, two specific protein markers, a two domain Kazal type proteinase inhibitor (KPI) and an extracellular superoxide dismutase (SOD), were found in SGCs and GCs, respectively. The proliferating cell nuclear antigen (PCNA), a unique marker for Hpt cells, was detected by searching the ex-pressed sequence tags (EST)-library of Hpt cells. The newly described hemocyte lineage marker protein genes from crayfish P. leniusculus provide new clues for the differentiation of different stages of the crayfish blood cells (Paper IV). These data together may be helpful for future studies to understand the connection between SGCs, GCs, and precursor cells in Hpt and also the role of the growth factors (eg. astakine) as regulators of hemo-cyte maturation and development in crustaceans.

Antiviral immune response The molecular mechanisms that underlie the majority of crustacean antiviral immune responses are still unknown and only starting to be addressed. Re-cently, many studies using different techniques have been carried out on host-WSSV interactions in crustaceans (Paper II) (Lan et al. 2006; Wang et al. 2006a; Wang et al. 2007a; Zhao et al. 2007). WSSV is an enveloped, large circular double stranded DNA virus containing about 300 Kbp (van Hulten et al. 2001; Yang et al. 2001). Viruses are the most serious pathogens for shrimp. WSSV is one of these viruses that causes up to 100% mortality

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within 7-10 days in commercial shrimp farms. This virus has a wide host range including salt and brackish water penaeid shrimps, crab, spiny lobster, and freshwater shrimp and crayfish. It has been reported that virus-inhibiting proteins could be produced and some genes were up-regulated upon viral infection in crustaceans (Dhar et al. 2003; He et al. 2005; Pan et al. 2005; Pan et al. 2000; Rojtinnakorn et al. 2002; Roux et al. 2002). Genes induced by viral infections and genes whose expression is associated with the ability of shrimp to survive from viral infections have been widely reported (Table 2). Crayfish ALF was shown to inhibit WSSV replication both in vivo and in vitro (Paper II). The mechanism of this inhibition still needs further investi-gations. A LGBP gene was up-regulated in WSSV infected shrimp suggest-ing that shrimp LGBP is an inducible acute-phase protein that may play a critical role in shrimp-WSSV interaction (Roux et al. 2002). PmAV was found in virus resistant shrimp which has a C-type lectin-like domain (CTLD). Recombinant PmAV protein displayed a strong antiviral activity in

Table 2. Proteins/genes involved in anti-WSSV responses or interacting with WSSV in crustaceans

Proteins/genes involved in anti-WSSV or interacting with WSSV Species

Actin Litopenaeus vannamei ALF Pacifastacus leniusculus Beta-integrin Marsupenaeus japonicus Calreticulin Fenneropenaeus chinensis Caspase-3 like gene Penaeus monodon C-type lectin Litopenaeus vannamei FcLectin Fenneropenaeus chinensis Hemocyanin Penaeus japonicus LGBP Penaeus stylirostris Manganese superoxide dismutase Fenneropenaeus chinensis PmAV Penaeus monodon PmCBP Penaeus monodon PmRab7 Penaeus monodon Rab GTPase Penaeus japonicus Ran protein Penaeus japonicus Syntenin Penaeus monodon Syntenin-like protein gene Penaeus monodon

inhibiting virus-induced cytopathic effect in fish cells in vitro. Further ex-periments showed that PmAV did not bind to the WSSV implying that the antiviral mechanism of this protein was not due to inhibition of the attach-ment of virus to target host cell (Luo et al. 2003). A beta-integrin was found

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to interact with a WSSV envelope protein VP187 containing the RGD motif. Soluble integrin, integrin-specific antibody and an RGD containing peptide could block the WSSV infection in vivo and in vitro. Silencing of beta-integrin efficiently inhibited the virus infection. These data suggest that this beta-integrin may function as a cellular receptor for WSSV infection (Li et al. 2007b). A syntenin and its protein partner alpha-2-macroglobulin co-precipitated with each other and both of them were up-regulated in the acute-phase of a WSSV infection (Tonganunt et al. 2005). A chitin-binding protein (PmCBP) interacted with a WSSV067C protein and showed up-regulation at the late stage of WSSV infection (Chen et al. 2007). Actin microfilaments were shown to interact with VP26 in shrimp (Xie and Yang 2005). A VP28 of WSSV was suggested to bind to shrimp cells as an attachment protein and could help the virus to enter into the cytoplasm (Yi et al. 2004). Moreover, hemocyanin, the respiratory protein of arthropods and mollusks, was found to delay the infection of WSSV in vivo in P. japonicus (Lei et al. 2008). Another C-type-lectin (LvLT) was decreased initially in the first 2 h and then increased to a much higher level after 4 h challenge of WSSV in shrimp (Ma et al. 2007). Some other genes were only found to be up-regulated as a re-sponse to WSSV infection in the animals but no mechanistic studies were performed. These genes include a Ras-related nuclear protein (Ran protein) gene (Han and Zhang 2007), a caspase-3 like gene (Wongprasert et al. 2007), calreticulin (Luana et al. 2007), a Rab GTPase gene (Wu and Zhang 2007), manganese superoxide dismutase (Zhang et al. 2007c), Fclectin (Liu et al. 2007), and a syntenin-like protein gene (Bangrak et al. 2002). These genes might also play certain roles in antiviral responses and more investigations are needed to elucidate the details.

Viral entry into host cells needs endocytosis machineries of the host. Im-portantly, PmRab7, a shrimp small GTPase protein binding directly to VP28 of WSSV, was suggested to be involved in WSSV infection (Sritunyalucksana et al. 2006). Silencing of PmRab7 dramatically inhibited WSSV-VP28 RNA and protein expression. Future work is still necessary to elucidate if this PmRab7 localizes on the surface of the host cells. Further, the silencing of PmRab7 also inhibited yellow head virus (YHV) replication in the YHV-infected shrimp. These data suggest that PmRab7 is a common cellular factor required for WSSV or YHV replication in shrimp (Ongvarrasopone et al. 2008). However, in this study, some shrimp injected with dsRNA did not show any antiviral protection and some of the RT-PCR results of host gene/viral gene were not in agreement with each other in the quantitation of their transcripts. Thus, the data presented in this study may need more detailed investigations for fully understanding of the mechanism by which PmRab7 interacts with WSSV. Besides, another RabGTPase from P. japonicus was reported to regulate shrimp hemocytic phagocytosis through a proposed protein complex consisting of the PjRab, beta-actin, tropomyosin, and an envelope protein VP466 of WSSV, indicating a

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possible role of phagocytosis involved in viral invasion in shrimp (Wu et al. 2008).

Apoptosis has been observed in viral target organs of WSSV-infected shrimp. Caspases are central effectors in apoptosis. When a shrimp Marsu-penaeus japonicus PjCaspase gene was silenced, the WSSV-induced apop-tosis was significantly inhibited which resulted in an increase of viral copies, indicating that apoptosis played a key role in antiviral processes of shrimp (Wang et al. 2008). Whereas, knocking down caspase-3 by RNAi reduces mortality in Pacific white shrimp challenged with a low dose of WSSV but not a high-dose of WSSV, suggesting that apoptosis may increase rather than decrease mortality in WSSV-challenged shrimp (Rijiravanich et al. 2008). The percentage of apoptotic hemocytes in WSSV-infected crayfish was very low, but it was significantly higher than that in the sham-injected crayfish on day 3 or 5 post-infection (Jiravanichpaisal et al. 2006c). Apoptosis was con-sidered as a defence responsible for eliminating the virus (Anggraeni and Owens 2000), whereas another study suggested that progressive occurrence of apoptosis may be the cause of death in P. monodon infected with YHV (Khanobdee et al. 2002). One study clearly showed that apoptosis occurs following WSSV infection in P. monodon, but the importance of this process for the mortality still needs further investigations (Wongprasert et al. 2003). Controversy remains for the role of apoptosis in antiviral responses in crus-taceans so far. This may be due to different species used for the assays, the big variations of animals, and different experimental manipulations etc. Therefore, further studies are still encouraged of the role of apoptosis in the antiviral response in crustaceans.

RNAi has been proven to be a natural antiviral mechanism in plants (Lecellier and Voinnet 2004), fruit flies (Galiana-Arnoux et al. 2006; Wang et al. 2006b; Zambon et al. 2006), mosquitoes (Keene et al. 2004) and nema-todes (Lu et al. 2005; Wilkins et al. 2005). RNAi technique now is explored as an alternative and more specific approach to counteract virus infections in shrimps. Injection of dsRNA/small interference RNA (siRNA) specific to viral genes can block viral disease progression. This effect has been con-firmed with different unrelated viruses. Viral replication was efficiently sup-pressed with injection of WSSV-specific dsRNA/siRNA in penaeid shrimp (Kim et al. 2007; Robalino et al. 2005; Xu et al. 2007). Lower YHV replica-tion was observed in shrimp primary cell cultures by transfecting the cells with dsRNA targeted to the viral nonstructural genes (Tirasophon et al. 2005). Inhibition of YHV replication by cognate dsRNA significantly re-sulted in lower mortality in the black tiger shrimp (Tirasophon et al. 2007; Yodmuang et al. 2006). Meanwhile, recent studies revealed the existence of both innate (non-sequence specific) and RNAi related (sequence specific) antiviral phenomena in a crustacean model (Robalino et al. 2005; Robalino et al. 2007b; Robalino et al. 2004; Westenberg et al. 2005). However, the protection induced by dsRNA could be overwhelmed by a higher dose (8-

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fold) of infectious virus. The mechanism of this difference between higher and lower amount of infectious virus remains unknown. The protective effi-ciency for WSSV infection by specific dsRNAs varied between different viral genes targeted and no reason has been addressed for these differences in this study. It remains unclear whether the antiviral protection by virus-specific long dsRNA is the result of RNAi mechanism alone or the combina-tion of innate immune activation and RNAi. Further, the readout from these studies was mainly based on the cumulative mortalities and no mechanism so far is available for these antiviral phenomena. Importantly, these studies suggest a possible evolutionary link (recognition of dsRNA) between innate antiviral immunity in invertebrates and vertebrates. It would be interesting to elucidate the possible role, potency and application of dsRNA in crustacean antiviral immunity in future.

Some researches suggested other approaches to protect the animals from infection of WSSV. For instance, a number of WSSV envelope proteins, such as VP28, have been proposed to be involved in viral infectivity based on the ability of specific antibodies to attenuate WSSV-induced mortality in vivo. When injected intramuscularly or administered orally with VP28, the shrimps obtained a higher and prolonged survival rates after WSSV chal-lenge (Witteveldt et al. 2004a; Witteveldt et al. 2004b). Different pro-tein/DNA vaccinations against WSSV infection were reported for the protec-tion of shrimp/crayfish (Jha et al. 2006; Kumar et al. 2008; Li et al. 2006; Rout et al. 2007; Vaseeharan et al. 2006; Witteveldt et al. 2006). Other anti-bodies against WSSV envelope proteins, such as VP68, VP281 and VP466 were also shown to reduce and delay the mortality of shrimp challenged with WSSV (Wu et al. 2005). However, strong inactivation of WSSV by some normal rabbit sera was observed in a manner independent of anti-VP28 anti-bodies questioning the potential of anti-VP28 preimmune antibodies to spe-cifically neutralize WSSV (Robalino et al. 2006). Thus, further investiga-tions are necessary for the availability of these antibodies against different viral proteins. Some other immune stimulants have also been described such as: glucans derived from yeast (Chang et al. 2003; Huang and Song 1999), LPS from bacteria (Takahashi et al. 2000), inactivated viruses (Bright Singh et al. 2005; Melena et al. 2006), and dsRNA (Robalino et al. 2005; Robalino et al. 2004; Sarathi et al. 2008; Yodmuang et al. 2006). Among these im-mune stimulants, viral proteins and dsRNA might be of particular interest for further investigations, because as virus-associated molecules they are likely to be the targets of immune recognition in the context of natural viral infec-tions (Robalino et al. 2007b).

Taken together, all these studies may help us for our understanding of the virus-host interactions, which may provide further promising therapeutic treatment and prevention of serious aquaculture viral diseases.

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Objectives

This thesis covers the study of a novel method for dsRNA transfection in crayfish Hpt cell cultures, functional studies of immune relevant genes in-volved in host-pathogens interactions (including WSSV and A. hydrophila, respectively) and isolation of hemocyte lineage marker proteins from cray-fish P. leniusculus.

Crayfish Hpt cell cultures are useful tools for gene functional study in crustaceans. To avoid the toxicity of commercial chemicals such as Effec-tene or liposome-based transfection reagents for delivering the dsRNA in crayfish Hpt cell cultures, a novel method using histone H2A as a transfec-tion agent for dsRNA transfection was established (Paper I).

To select immune-related genes against viral infection, differentially ex-pressed genes against a challenge of WSSV were isolated from crayfish hemocytes by suppression subtractive hybridization (SSH). Some of these up-regulated genes were chosen for further studies. The antiviral study of one of these up-regulated genes, anti-LPS factor, was performed using RNAi both in vivo and in vitro in crayfish P. leniusculus to interpret the role of this factor in antiviral responses (Paper II).

To elucidate the immune protection of PO against a highly pathogenic bacterium, A. hydrophila, isolated from crayfish, PO activity was investi-gated after a bacterial infection using RNAi in crayfish (Paper III).

Two dimensional gel electrophoresis (2-DE) followed by mass spectra (MS) analysis was performed to identify proteins specific for different hemocyte lineages in crayfish. Different marker proteins for Hpt cells, SGCs and GCs were isolated. Further functional study on one of these marker genes was also carried out by RNAi in crayfish Hpt cell cultures (Paper IV).

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Results and discussion

Histone H2A as a transfection agent in crayfish hematopoietic tissue cells (Paper I) RNAi is a widely used technique to investigate gene functions (Friedman and Perrimon 2004; Vanhecke and Janitz 2005). RNAi studies are also commonly performed nowadays with large dsRNA molecules in inverte-brates. Functional studies on immunity in crustacean are limited by the lack of permanent cell lines. We have succeeded in developing crayfish Hpt cell cultures that provide us with a useful tool for interesting gene functional studies. The crustacean cells are usually sensitive to the classical commercial transfection agents such as Effectene or liposome-based transfection system. The present study provides a novel protocol for dsRNA interference medi-ated by histone H2A from calf thymus (TypeII-A) in crayfish Hpt cell cul-tures.

Histones are nuclear proteins with features of DNA-condensing capacity and nuclear localization signals (NLSs), which allow them to act as vectors for delivering nucleic acids into cells. Histone H2A, one of the histone oc-tamers that package genomic DNA in eukaryotes has been widely used in transfection studies (Balicki and Beutler 1997; Balicki et al. 2000; Singh and Rigby 1996). The mechanism of DNA-delivering activity of histone H2A has been suggested as follows: (i) electrostatically driven DNA binding and condensation by histone and (ii) then nuclear import of these histone H2A·DNA polyplexes via NLSs in the histone H2A (Balicki et al. 2002). Based on these theories, we first tested whether histone H2A worked as a dsRNA transfection reagent in crayfish Hpt cell cultures. The results showed that the dsRNA transfection into Hpt cells mediated by histone H2A resulted in the reduction of crayfish ALF transcript at 2 days post-transfection and almost no transcript could be detected by RT-PCR at the 5th and 9th day post dsRNA transfection.

Since naked dsRNA could also be taken up by cells reported in other ani-mals without any transfection reagent (Feinberg and Hunter 2003; May and Plasterk 2005), we then determined the silencing efficiency between naked dsRNA alone and dsRNA transfection mediated by histone H2A. The results suggested that the silencing efficiency was much higher in histone H2A me-diated dsRNA transfection, indicating that a transfection agent is required for reaching a complete RNA silencing effect.

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To optimize the minimum amount of dsRNA for an efficient RNA silenc-ing, a series of ALF dsRNA was used to transfect Hpt cells mixed with his-tone H2A as a transfection reagent. The result showed that the crayfish ALF gene expression could be silenced completely with as little as 0.56 μg/ml of ALF dsRNA in combination with histone H2A.

Additionally, histone H2A-mediated transfection was also compared with Effectene or liposome based transfection methods, in which histone H2A mediated transfection showed higher silencing efficiency for both of ALF and serine proteinase inhibitor (PAPI I) genes constitutively expressed in crayfish Hpt cell cultures. To evaluate toxicity of different transfection re-agents, the viabilities of crayfish Hpt cells were determined at 12 h after inoculation with different transfection reagents. The 96.9%, 79.4%, 92.1%, 90.2% and 92.7% of cell viabilities were observed for histone H2A, Effec-tene® transfection reagent, LipofectaminTM2000, Lipofectin® reagent and calcium phosphate transfection kit, respectively. Thus this novel method showed higher cell viability with non-toxic effect to the cell cultures.

Finally, these data together indicate that calf thymus histone H2A is an ef-fective mediator of transfection without any additional agents, which is con-sistent with a previous study showing similar transfection results in mam-malian cells (Balicki et al. 2002). This method is cheaper and highly suitable for primary cells or sensitive cell cultures of crustacean animals such as crayfish and shrimp.

Antilipopolysaccharide factor interferes with white spot syndrome virus replication in vitro and in vivo in the crayfish Pacifastacus leniusculus (Paper II) To identify immune genes against WSSV infection in P. leniusculus, we generated SSH libraries from hemocytes isolated from a single crayfish pre- and post-viral challenge, and 1305 cDNAs were screened from which 231 up-regulated clones were sequenced and analyzed. Several functional classes were characterized in crayfish, including immune-related genes such as AMPs and molecules involved in pathogen recognition/proteinase cascades.

To understand how these induced molecules contribute to the control of WSSV infection, several of the up-regulated immune genes were chosen for further investigations. Among the transcripts enriched after the viral infec-tion were several clones with sequence homology to the Limulus ALF. A full-length cDNA clone encoding a 120-amino-acid protein was identified, and its sequence showed 28-32% sequence identities to ALFs from other crustaceans. Besides, the crayfish ALF sequence contains conserved features, such as the positions of two conserved cysteine residues and the putative LPS binding site located between these two cysteines.

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Since crayfish ALF was up-regulated upon WSSV infection, we per-formed further studies to see if the ALF expression could be increased by injection of UV-inactivated virus and further to determine whether these evaluated ALF levels could influence the outcome of the viral infection. Pretreating animals with UV-inactivated virus resulted in higher ALF levels, slower viral replication, and a slower increase in mortality values upon injec-tion of the active virus than those in animals receiving active virus only.

To assess further the role of ALF in protecting against WSSV, RNAi both in vivo and in vitro was employed. Injection or inoculation of ALF dsRNA resulted in a partial reduction of ALF transcription levels in the whole ani-mal and crayfish Hpt cell cultures, respectively, which was accompanied with an increase of VP28 mRNA levels. No difference of the ALF transcrip-tion was observed in control treatment with green fluorescent protein (GFP) dsRNA. Further, silencing of ALF in the animals with subsequent WSSV infection resulted in a faster disease progress than in animals that had received control dsRNA. These data suggest that ALF in some way either directly or indirectly affects the replication of WSSV and thus has an antivi-ral effect toward this virus.

Phenoloxidase is an important component of the defense against Aeromonas hydrophila infection in a crustacean, Pacifastacus leniusculus (Paper III) Phenoloxidase mediated melanization has been suggested to play a key role in recognition of and defense against microbial infections in invertebrates (Cerenius and Söderhäll 2004).

Preliminary results showed that the proPO and pacifastin (a proteinase in-hibitor specific against the PO-activating enzyme of crayfish) mRNA tran-scripts were up-regulated 3 h post challenge with A. hydrophila in crayfish. To determine the role of PO in the host defense against this highly patho-genic bacterium, we performed gene silencing of the proPO or the pacifastin gene separately using dsRNA interference in live animals. The efficiency of RNAi silencing was determined by quantitative RT-PCR which showed that the proPO mRNA transcript was reduced to 57%. This reduction was also accompanied with lower PO activity. The level of PO activity decreased by 33% in proPO-silenced animals. When these animals were challenged with A. hydrophila, the time needed to reach 80% cumulative mortality decreased 4.8-fold from 48h to 10 h, suggesting that reduction of PO activity could result in a faster and higher mortality following a bacterial challenge than that in control animals. Meanwhile, the growth of A. hydrophila in proPO silenced animals increased 2.3-fold compared to control animals indicating that proPO may act in a bacterium-killing pathway.

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Further study showed that after silencing the inhibitory domain of the pacifastin gene (44% reduction in mRNA transcription level), the PO activ-ity was enhanced 1.4-fold above levels of the control animals. When the pacifastin silenced animals were challenged with A. hydrophila, there was a significant increase in the mean survival time (p<0.01, Student’s t test) com-pared to control treatment. The accumulated mortality reached 80% within 48 h in the control group, whereas the same mortality was reached 64 h after infection in the animals with an enhanced PO activity. Further, the higher PO activity was accompanied by a strong decrease of bacterial CFUs (40% reduction), indicating that enhanced PO activity was correlated with both increased survival time and enhanced bacterial clearance.

Experiments were also performed to reveal the correlation between PO ac-tivity and phagocytosis by determining the change of the phagocytic rate if the PO activity was decreased or increased by RNAi of proPO or pacifastin, respectively. Compared to GFP dsRNA-treated animals (2.1%), decreased PO activity in the hemolymph gave a significant 57% decrease of the phago-cytic activity (0.9% of phagocytic bacteria, p<0.01, Student’s t test), whereas an increase in phagocytosis was observed in the pacifastin gene silenced animals (3.7%, p<0.05, Student’s t test). Moreover, nodule formation was quite often seen in PO activity-enhanced, but to a lesser extent in proPO-silenced, animals during our experiments. Therefore, it is likely that the phagocytic and nodule-forming activities in crayfish are associated with PO activity. Taken together, we assume that PO participates not only in the melanization of the parasites but also somehow enhances other cellular ac-tivities such as phagocytosis and/or nodule formation. Taken together, these data therefore suggest that PO is required in crayfish defense against an in-fection by A. hydrophila, a highly virulent and pathogenic bacterium to cray-fish.

Hemocyte lineage marker proteins in a crustacean, the freshwater crayfish, Pacifastacus leniusculus (Paper IV) Hemocytes, the immune competent cells, are important sources for produc-ing original immune effectors in crustaceans. Therefore, hematopoiesis is a crucial element of antimicrobial immune response for crustaceans.

To reveal the hematopoietic process at the molecular level, we performed the 2-DE based proteomic assays to isolate some proteins that are specially present in the different hemocyte types such as Hpt cells, semigranular and granular hemocytes in P. leniusculus. Different spots were selected for MS analysis followed by cDNA cloning using degenerate primers designed from MS results. Two specific protein markers, KPI and SOD, were detected in SGCs and GCs respectively. A hypothetical protein (22.4 kDa) was found from Hpt, but the transcript of this protein could be detected in SGCs by RT-

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PCR. We then tried to search the EST-library of crayfish Hpt cells and got the PCNA as a unique marker for Hpt cells.

To determine the specific expression of the above protein markers, we analyzed the transcription of the corresponding genes in different cell types by RT-PCR. The results showed that the PCNA, KPI and SOD genes are only expressed in the Hpt, SGCs and GCs, respectively. Since crayfish Hpt PCNA expression varied between animals we next decided to analyze ex-pression of this gene after a challenge with microbial elicitor such as lami-narin or crude LPS to mimic a microbial challenge and to reveal whether the cell cycles (and thereby PCNA expression) are stimulated by the presence of microbial polysaccharides. A high expression of PCNA was always ob-served and most animals also showed induction of the SOD transcript in the Hpt as a response to this challenge, indicating that differentiation as well as proliferation was stimulated in the crayfish Hpt. To confirm that SOD could be used as a marker protein for GCs, a Western blot using cell lysate from separated Hpt, SGCs, or GCs was performed. The result showed that the SOD protein could only be detected in GCs. The KPI transcript was never present in Hpt regardless of any challenge indicating that this putative pro-teinase inhibitor could be used as a marker for SGCs late differentiation.

To determine whether silencing of PCNA in cultured Hpt cells could af-fect differentiation measured as an increased transcription of SOD or KPI, we performed RNAi of PCNA. The RT-PCR results showed that the PCNA gene expression was substantially silenced (60% silencing) but no appear-ance of SOD or KPI could be detected. Interestingly, when comparing the morphology of the silenced cells a significant decrease in cell attachment and spreading was observed (15 ± 6% spread cells compare to 40 ± 3% in the GFP dsRNA treated cells) suggesting that PCNA may play a role in es-tablishing cell adhesion and spreading, and differentiation of the Hpt cells.

In conclusion, we have identified three different proteins/transcripts that can be used as indicators/markers for hematopoietic cell proliferation and specific differentiation into SGCs or GCs. These protein markers PCNA, KPI and SOD will be useful for further detailed studies to reveal the connec-tion between SGCs, GCs and precursor cells in Hpt and also the role of astakine (Söderhäll et al. 2005) as regulator of this process in P. leniusculus, as well as in other crustaceans.

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Concluding remarks

Crustaceans (eg. crayfish and shrimp) mount strong innate immune re-sponses against microbes such as bacteria and viruses. Studies on the mechanism of these responses have been hampered by absence of genome, tools for genetic manipulation and mutants, and stable long-term cell lines for in vitro studies. We have succeeded in developing a Hpt cell culture from crayfish which can be a useful tool for gene functional studies in crustaceans (Söderhäll et al. 2003). This Hpt cultures can also be used to replicate WSSV and to study host-virus interactions (Jiravanichpaisal et al. 2006b). In this thesis, a novel dsRNA mediated RNAi technology was developed for crusta-cean cells (Paper I). Further, we used RNAi to elucidate the function of im-mune-relevant genes (ALF and proPO) in the immune responses against different pathogens in crayfish (Paper II and Paper III). RNAi of a marker protein gene was also carried out in crayfish Hpt cells (Paper IV).

RNAi is a powerful tool for gene functional studies (Fire et al. 1998). Meanwhile, RNAi is also a widely studied mechanism for antiviral responses (Galiana-Arnoux et al. 2006; Keene et al. 2004; Lecellier and Voinnet 2004; Lu et al. 2005; Wang et al. 2006b; Wilkins et al. 2005; Zambon et al. 2006). Compared to other pharmacological interventions, RNAi is an attractive antiviral therapeutic because it allows interference with the target gene in a highly sequence-specific manner, and therefore essential viral genes can be targeted by design with little or no risk of unexpected off-target effects. Re-cent studies have demonstrated that the viral disease progression can be blocked by injecting shrimp with dsRNA/siRNA specific to viral genes. This strategy is effective against three unrelated viruses: WSSV, Taura syndrome virus (TSV) and YHV (Robalino et al. 2004; Yodmuang et al. 2006). The mechanism for this phenomenon is still not clear. In these studies, positive strand RNA virus (eg. TSV and YHV) and DNA virus (eg. WSSV) often induce the formation of dsRNAs during their infectious cycles such as ge-nomic replicative intermediates, intramolecular interactions within viral transcripts, and bi-directional transcription (Weber et al. 2006). And these dsRNAs might engage the shrimp RNAi pathway, resulting in effective anti-viral responses. Thus it will be of interest to investigate whether viral dsRNA accumulates in crustacean cells infected with WSSV, TSV, or YHV. Two components of RNA silencing, Argonaute and Dicer-1, have been char-acterized from P. monodon (Dechklar et al. 2008; Su et al. 2008; Unajak et al. 2006). Based on the studies in crustaceans above, plants (Lecellier and Voinnet 2004), fruit flies (Galiana-Arnoux et al. 2006; Wang et al. 2006b;

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Zambon et al. 2006), mosquitoes (Keene et al. 2004), and nematodes (Lu et al. 2005; Wilkins et al. 2005), it is likely that RNAi indeed exists as a natural antiviral mechanism in shrimp and other crustaceans. These studies and fur-ther investigations of genes involved in RNAi in crustaceans should provide the possibility by reverse-genetic approaches to test this hypothesis directly.

In crustaceans, the number of freely circulating hemocytes varies in re-sponse to environmental stress, endocrine activity during the moulting cycle, and infection. Exposure to non-self molecules can cause a dramatic drop in total hemocyte count and the animals may die of an infection that they oth-erwise usually resist (Jiravanichpaisal et al. 2006a; Johansson et al. 2000). The pathogens (WSSV and A. hydrophila) used in this thesis also affect the hemocytes in crayfish P. leniusculus. For instance, infection of WSSV has a significant effect on the proportion of different hemocyte types in the ani-mals. The number of GCs was significantly higher in WSSV-injected cray-fish compared to control animals (Jiravanichpaisal et al. 2001). Studies from crayfish and shrimp suggest that SGCs are more susceptible to WSSV and the virus replicates more rapidly in SGCs than GCs resulting in the gradually decreased SGCs from blood circulation (Jiravanichpaisal et al. 2006c; Wang et al. 2002). Other studies showed that the total numbers of circulating hemocytes in shrimp were dramatically decreased after infection by WSSV (Guan et al. 2003; van de Braak et al. 2002a; Wongprasert et al. 2003). Se-creted extracellular toxins and enzymes have been described which were suggested to be associated with the virulence of A. hydrophila (Allan and Stevenson 1981; Ljungh and Wadstrom 1981). The dramatic mortality of crayfish infected with A. hydrophila was probably caused by the secretion of toxin (eg. hemolysin) from this highly pathogenic bacterium to crayfish (Jiravanichpaisal unpublished). In this study, crude extracellular products (ECPs) obtained from culture supernatants of bacteria were used. The results showed that ECPs induced cytotoxicity of crayfish SGCs and GCs in a dose- and time-dependent manner. Besides, the toxins were found to affect the hematopoietic cells in vitro. All cells were lysed within 20 h after treatment with ECPs of A. hydrophila. It is likely that infection of A. hydrophila results in loss of hemocytes/hemocyte progenitors in crayfish. Previous study showed that injection of a �-1, 3-glucan rapidly reduced the numbers of crayfish hemocytes followed by a recovery to the same level after 4-6 h (Smith and Söderhäll 1983). After a decrease in the number of hemocytes by an injection of �-1, 3-glucan, the parasite Aphanomyces astaci which nor-mally is present only in the cuticle and does not cause mortality of the cray-fish, can grow rapidly within the body cavity and kills the crayfish a few days later (Persson et al. 1987). This probably indicates the importance of normal hemocyte numbers for crayfish defense against pathogens. On the other hand, immune-relevant molecules from crustacean hemocytes are es-sential in defense against invasive pathogens (Cerenius et al. 2008; Johans-son et al. 2000). The data from Paper II and III also demonstrate that silenc-ing of immune-relevant genes (ALF or proPO) from crayfish hemocytes led

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to rapid infection of WSSV and A. hydrophila, respectively. Therefore, im-mediate regeneration of hemocytes is critical for the survival of the animals against pathogenic intruders.

Previous study showed that Hpt of crayfish is the organ in which prolif-eration of hemocytes occurs. Injection of a �-1, 3-glucan caused a severe loss of hemocytes followed by a rapid recovery due to release from the Hpt organ (Söderhäll et al. 2003). This study also indicated that the hemocytes were synthesized and partly differentiated in the Hpt, but the final differen-tiation into functional hemocytes was not completed until they were released into the circulation. Hematopoiesis is tightly regulated by the lineage re-stricted activity of various factors that promote cell diversification (Orkin 1998; Orkin 2000; Sieweke and Graf 1998). The hematopoietic tissue has been studied in several crustaceans, but the mechanisms for the release of blood cells into circulation in crustaceans are still unclear. In Paper IV, the identification of three different proteins/transcripts can be used as mark-ers/indicators for hematopoietic cell proliferation and specific differentiation into SGCs or GCs. Thus, the study of of hemocyte lineage marker proteins would be useful for future investigations of hematopoiesis under normal or microbial challenge conditions in crustaceans.

In conclusion, all these data together give more insights into the functions of immune-relevant genes through RNAi techniques as well as some clues into a model for the classification of hemocyte lineages in crustaceans.

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Svensk sammanfattning (Summary in Swedish)

Kräftdjur, liksom övriga ryggradslösa djur, äger trots avsaknaden av ett för-värvat immunförsvar, effektiva mekanismer för att bekämpa patogener som virus och bakterier. Deras blodkroppar, hemocyter, aktiveras av olika sub-stanser från mikroorganismer och är sedan kapabla att bekämpa dessa genom exempelvis fagocytos, produktion av antimikrobiella ämnen, melanininkaps-ling med flera immunmekanismer. I denna avhandling har några av dessa mekanismer verksamma i sötvattenskräftan Pacifastacus leniusculus stude-rats. Behovet av att förstå kräftdjurens immunförsvar i detalj är stort efter-som odling av olika kräftdjur, t . ex. räkor, har blivit en global storindustri.

För detta arbete krävs arbetsredskap, i synnerhet möjligheter att hantera olika hemocyttyper in vitro samt att på ett systematiskt sätt påverka olika genuttryck i dessa. En viktig del i avhandlingen behandlar därför metoder att introducera dubbelsträngat RNA in i blodkroppar och i celler från det blod-kroppsbildande organet för att specifikt slå ut olika genuttryck. Eftersom dessa celler är mycket känsliga och lätt reagerar för främmande ämnen visa-de sig många protokoll och kommersiella preparationer som används för att transfektera andra djurceller fungera dåligt med kräftceller. Därför utprova-des en metod baserad på användningen av histonet H2A för att introducera det dubbelsträngande RNA:t. Den visade sig effektivt kunna slå ut önskade genuttryck. Fördjupade studier av blodkroppar och deras bildning ur det hematopoietiska organet kräver vidare markörproteiner/genaktiviteter speci-fika för respektive blodkroppstyp. Här har vi, med proteinkemiska metoder kompletterade med studier av genuttryck, identifierat markörer specifika för cellerna i det hematopoietiska organet samt för de två talrikaste mogna cell-typerna ute i hemolymfan. PCNA, ett protein inblandat i DNA-replikation, produceras av omogna blodkroppar i det hematopoietiska organet men sak-nas i de mogna fritt cirkulerande hemocyterna. Mogna blodkroppar av den semigranulära typen utmärks av att de innehåller en speciell proteinasinhibi-tor tillhörande den s.k. Kazalklassen. De granulära hemocyterna producerar inte denna inhibitor men bildar, i motsats till semigranulära celler, enzymet superoxiddismutas. Med hjälp av dessa markörer blir det nu möjligt att de-taljstudera både blodkroppsdifferentiering och -produktion vid till exempel ett sjukdomsangrepp.

RNA interferens användes sedan för att följa hur kräftan bekämpande mi-krobiella infektioner. Två för djuret dödliga patogener användes, dels vit-

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fläcksvirus (WSSV), dels bakterien Aeromonas hydrophila. I synnerhet WSSV är ett stort hot mot all odling av kräftdjur och orsakar enorma eko-nomiska skador årligen. Vid dessa försök hittades för första gången någonsin i något kräftdjur ett endogent protein med kapacitet att fördröja WSSV-virusets replikation. Proteinet, känt från andra sammanhang som anti-lipopolysackaridfaktor (ALF), visade sig kunna kraftigt reducera WSSV-replikation i såväl cellkulturer som i levande djur. I det senare fallet fördröj-des även själva sjukdomsförloppet. Om däremot produktionen av ALF hind-rades med RNA interferens av ALF-uttrycket replikerade viruset sig snabbt och djuret dog tidigt.

Melaninbildning är ett verksamt vapen mot många patogener och större parasiter blir ofta helt inkapslade i melanin. Här visade sig att de ämnen som bildas när aktivt fenoloxidas reagerar även är verksamma mot en bakteriein-fektion. Om fenoloxidasmängden reducerades med RNA interferens blev resultatet att den för kräftan dödliga A. hydrophila-infektionen fick ett snab-bare förlopp. Däremot, om fenoloxidashalten ökades genom att förhindra genuttrycket för en specifik inhibitor av fenoloxidasaktiveringen, försvåra-des bakteriens tillväxt och infektionen fick då ett snabbt förlopp. Resultatet kan åtminstone till en del förklaras av att någon eller några ämnen vilka bil-das av det aktiva fenoloxidassystemet stimulerar blodkropparnas fagocytos av bakterien.

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Acknowledgements

This work was carried out at the Department of Comparative Physiology and supported by grants from Swedish Science Research Council and Formas (K.S.), the Carl Trygger Foundation (I.S.), and Sweden Asia Research Link (to P.J. and K.S.). I hope to thank all those who have helped me throughout this study. In par-ticular, I would like to express my appreciations to: Prof. Kenneth Söderhäll, my supervisor, for helping me to develop my “scientific brain” from “a single neuron”. He taught me how to focus on a problem, approach it carefully and present it precisely. Without his support, this work would never have been possible. In addition to being an exemplary mentor, he has been a great friend to travel together to Charleston (USA), Åland (Finland) and Obergurgal (to Austria by car from Sweden). I also thank him for wonderful discussions about life during the last five years. Thank you so much for your encouragements, suggestions and always great recommendations. I will try to make a tough life to be an easy one like what you said. I will always be honored to be called as his student. Co-supervisor Dr. Irene Söderhäll, for always kindness, practical useful tips and suggestions, discussions, chemical supplying and special dishes for Swedish Christmas. Co-supervisor Dr. Lage Cerenius, for generous discussions, proposals, manuscripts preparations and helpful suggestions in Lab-teaching. Engineer Ragnar Ajaxon, super Lab-police officer, for always technical assistant and on-time repairing of equipments. I also appreciated ever for your picking me up at Arlanda airport and taking care of me when I first arrived in Sweden five years ago. Dr. Maria Lind Karlberg, for interesting discussions and wonderful or-ganizations of our international parties. Prof. Olof Tottmar, for often late lunch discussions, help for Lab-teaching and preparations.

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Dr(s). Pikul Jaravanichpaisal, for the second Lab-police lady, Lab-fighting, my co-author, helpful tips in cell cultures, tasty Thai-dishes and always complaining about me. Anbar Kodabandeh, for sequencing and always encouragement. Marianne Andersson and Rose-Marie Löfberg for practical and secretarial helps. Present Lab-friends, Xionghui Lin, Christoph Mentzendorf, Wenlin Wu, Chenglin Wu, Tobias Backström, Daniel Brelin, and Yanjiao Zhang for great time together. All former Lab-friends from Department of Comparative Physiology, Chenghua Li, Karin Johansson, Tove Andrén, Young-A Kim, Pelle, Siripavee Sricharoen, Amornrat Tangprasittipap and Sirinit Tharntada. We did have fun together. All my former and present friends in Sweden, Geng Tian, Jia Mi, Wei Lin, Sa Li, Yupeng Lai, Li Yang, Jan Andersson, Tong Sun, Zhihu Zhao family, Wei Shi, Yang Yu, Zhuo Bao, Lunmei Huang, Mei Hong, Jie Chen, and……, too many friends to be listed here for space limitation. I am thank-ful to all of you for friendship and joyful time in Sweden. Prof. Bok Luel Lee, for project discussions and corrections of manuscript. Prof. Shun-ichiro Kawabata, for great help and recommendation in Fu-kuoka, Japan. Thanks to Yasuyuki Matsuda, Manabu Iijima and all other lab guys for helps and nice lunches/dinners together. I did have a good time with you guys. Prof. Xun Xu, for bringing me into this research field in innate immunity and always supporting for my study and life. I am so appreciated with your generous and kindness. Prof. Kejian Wang and Prof. Weiquan Liu, for always helpful suggestions and encouragements. I will never forget your suggestions that totally changed my life. All my friends in China, Linli Xu, Jigui Wang, Benxu Wang, Liwei Qiu, Yu Liu, Xingang Mou, Kejia Huang, Xiangyuan Fu, Xiaobao Liu, and other friends for generous helps and encouragements during these years. All my relatives, specially, Hongyan Lü and Sifu Lü for more than twenty years support. I will never forget what you did for me in the last two decades. All Pacifastacus leniusculus, whose sacrifice made this work possible. Last, my parents, for being the best, words are not enough to express my appreciations, for your love and support. I could not finish without all of you. Thank you all so much!

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