Innate Immune Proteins in a Crustacean Pacifastacus Leniusculus

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List of Papers

This thesis is based on the following papers, which will be referred to in the text by their Roman numerals:

I. Wu, Chenglin, Söderhäll, I., Kim, Y. A., Liu, H., Söderhäll, K. (2008). Hemocyte lineage marker proteins in a crustacean, the freshwater crayfish, Pacifastacus leniusculus. Proteomics 8: 4226-4235.

II. Söderhäll, I., Wu, Chenglin, Novotny, M., Lee B. L., Söderhäll, K. (2009). A novel protein acts as a negative regulator of proPO activation and melanization in the freshwater crayfish Pacifastacus leniusculus. Journal of Biological Chemistry 284: 6301-6310.

III. Liu, H.*, Wu, Chenglin*, Matsuda, Y., Shun-ichiro Kawabata, Lee, B. L., Söderhäll, K. and Söderhäll, I. (2011). Peptidoglycan activation of the proPO-system without a peptidoglycan receptor protein (PGRP)? Developmental Comparative Immunology 35(1): 51-61. (* Co-first au- thor)

IV. Wu, Chenglin, Söderhäll, K., Söderhäll, I. Two novel ficolin-like proteins (FLPs) act as pattern recognition molecules for invading pathogens in the freshwater crayfish, Pacifastacus leniusculus. Proteomics (In press)

Reprints were made with permission from the publishers.

Contents

Introduction ...... 9 Hematopoiesis ...... 11 Hematopoietic tissue (Hpt) in crustacean ...... 13 Transcription factors and marker proteins ...... 13 Cytokines are important for the hematopoiesis in crustacean ...... 15 The proPO-system and melanization ...... 17 ProPO activation ...... 17 ProPO-system is involved in cellular responses ...... 19 Importance of proPO-system in arthopods ...... 19 Control of proPO activation and PO activity ...... 20 Pattern recognition proteins (PRPs) ...... 21 SPHs ...... 22 PGRPs ...... 23 GNBPs ...... 24 LGBP and GBP/-1,3-glucan recongition protein (/GRP) ...... 25 Lectins ...... 26 Fibrinogen-related domain (FReD) containing proteins ...... 27 Dscams ...... 28 Toll-like receptors (TLRs) ...... 30 Thioester-containing proteins (TEPs) ...... 31 Objectives ...... 36 Results and Discussion ...... 37 Hemocyte lineage marker proteins in P. leniusculus (Paper I) ...... 37 A novel protein acts as a negative regulator of proPO activation and melanization in P. leniusculus (Paper II) ...... 38 Peptidoglycan activation of the proPO-system without a PGRP (Paper III) ...... 40 Two FLPs act as pattern recognition molecules for invading pathogens in P. leniusculus (Paper IV) ...... 42 Concluding remarks ...... 44 Svensk sammanfattning ...... 46 Acknowledgements ...... 48 References ...... 51 Abbreviations

2-DE Two dimensional gel electrophoresis GBP -1, 3-glucan binding protein A2M Alpha-2-macroglobulin AMPs Antimicrobial peptides ALF Antilipopolysaccharide factor Dscam Down syndrome cell adhesion molecule dsRNA Double stranded RNA EST Expressed sequence tag FLP Ficolin-like protein 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 spectrometry PCNA Proliferating cell nuclear antigen PGN Peptidoglycan PGRPs Peptidoglycan recognition proteins ppA Propheonoloxidase activating enzyme PRPs Pattern recognition proteins proPO Prophenoloxidase RNAi RNA interference RT-PCR Reverse transcriptase polymerase chain reaction SGCs Semigranular cells SPH Serine proteinase homologue SOD Superoxide dismutase SSH Suppression subtractive hybridization TEP Thioester-containing protein TGase Transglutaminase WSSV White spot syndrome virus Introduction

Production from crustacean aquaculture is an important income for many developing/low-income countries (Rosenberry, 1998). However, the increase in aquaculture also has many negative effects, for instance, it causes environmental destruction and the farming often faces severe disease problems. To optimize aquaculture conditions and avoid disease outbreaks, the development of tools for rapid recognition and control of pathogens are urgently needed (Bachère, 2000). Crayfish are immunologically related to other more economically important crustaceans, so insight into crayfish immunity and their defence system is valuable for aquaculture development and good for optimizing farming conditions. Vertebrate immunity is composed of innate and adaptive response, and it is widely accepted that adaptive immunity is restricted to jawed vertebrates. Invertebrates including arthropods, lack lymphocytes and antibody-based adaptive immune system and, therefore, only rely on innate immune system to defend themselves against different pathogens. The innate immune system seems to be enough to protect them against infections or intruders, and it includes cellular and humoral mechanisms, both of which are activated upon immune challenge. The cellular response mediated by hemocytes (blood cells) in hemolymph involves nodule formation, phagocytosis, encapsulation of pathogens and coagulation (Johansson and Söderhäll, 1989; Ratcliff et al., 1985; Theopold et al., 2002). Moreover, hemocytes are also involved in an- other response, melanization, which is activated immediately upon injury/infection and normally is localized to the place of injury or at the surfaces of invading microorganisms, so that toxic phenol intermediates or melanin are produced in the melanization, and the intruders are immobilized and killed (Lee et al., 2002b; Söderhäll and Cerenius, 1998). Humoral defense is characterized by synthesis and secretion of immune components after challenge, for example: antimicrobial peptides (AMPs) can accumulate in hemolymph to defend against invading microorganisms (Lemaitre and Hoffmann, 2007). But it is important to keep in mind that cellular and humoral responses are connected to each other, and can not be separated completely in the immune response. There are various strategies for the invertebrates to combat different invading pathogens, but most of these are evolutionarily conserved such as: activation of phagocytic cells, production of AMPs and generation of toxic or reactive oxygen species (ROS).

9 The last decade, progress has been made in different aspects of crustacean immunity, especially the understanding at molecular and biochemical level of some highly conserved immune response pathways (eg. melanization, production of AMPs and clotting) (Bachère et al., 2004; Cerenius et al., 2008 and 2010). Many factors involved in innate immunity have been identified in freshwater crayfish Pacifastacus leniusculus (Table 1). However, the research of crustaceans is hampered by a lack of access to genomic data and of well-developed cell lines. Therefore, the hematopoietic tissue (Hpt) cell cultures technique developed in the crayfish P. len i us cul u s, provides a useful tool for gene functional studies in crustaceans (Söderhäll et al., 2005), since an efficient method for RNA silencing is at hand for these cells (Liu and Söderhäll, 2007).

Table 1. Immune factors found in freshwater crayfish (P. leniusculus). Proteins and peptides Accession Mass Activity and Reference number (kDa) Prophenoloxidase (proPO) activating system proPO CAA58471 76 Precursor of phenoloxidase (Aspán et al., 1991 and 1995) ProPO activating enzyme (ppA) CAB63112 48 Serine protease that cleaves proPO (Wang et al., 2001a)

Serine protease homologue 1 (SPH1) AAX55746 40 Hemocyte secreted protein upon challenge (Sricharoen et al., 2005) SPH2a/ SPH2b ACB41379/ 46 Binding activity to partially digested insoluble Lys-type PGN (Paper ACB41380 III)

Pattern recognition proteins

LGBP CAB65353 40 ProPO activation, opsonin and adhesion (Lee et al., 2000) βGBP CAA56703 100 ProPO activation (Cerenius et al., 1994; Duvic et al., 1990) Mannose-binding lectin AAX55747 28 Secreted from hemocytes upon challenge (Sricharoen et al., 2005) Ficolin-like protein 1 (FLP1) ADM89628 55 Bacterial binding and Phagocytosis (Paper IV) FLP2 ADM89629 53 Bacterial binding and Phagocytosis (Paper IV) Down syndrome cell adhesion molecule HQ596367 200 Pattern recognition protein (Watthanasurorot et al., 2011a) (Dscam)

Cell adhesion proteins

Masquerade-like protein I CAA72032 129/134 Non-catalytic cell adhesion protein (Huang et al., 2000; Lee and Söderhäll, 2001) Peroxinectin CAA62752 76 Cell adhesion molecule (Johansson et al., 1995)

Proteinase inhibitor

KPI1 X79512 23 Four Kazal domain, inhibited chymotrypsin or subtilisin (Johansson et al., 1994) KPI2 EU433325 21 Two Kazal domain, specifically expressedin semigranular cell, inhibit strongly subtilisin and weakly to trypsin (Paper I; Donpudsa et al., 2010b) KPI8 CF542313 13 Two Kazal domain, inhibit trypsin (Donpudsa et al., 2010b) Other Kazal-type inhibitors 10-30 Kazal type proteinase inhibitors (Cerenius et al., 2010c) 2-Macroglobulin 1 (A2M1) HQ596363 190 Universal proteinase inhibitor (unpublished data). A2M2-isoform 1 HQ596364 190 × 2 Universal proteinase inhibitor; Substrate of transglutaminase (Hergen- A2M2-isoform 2 HQ596365 hahn et al., 1988; Hall et al., 1989 and 1994; unpublished data) A2M2-isoform 3 HQ596366 Pacifastin AAC64660/ 155 Serine protease inhibitor with a unique transferrin chain (Liang et al., AAC64661 1997) Subtilisin inhibitor 28 Proteinase inhibitor (Aspán et al., 1990) Serpin-type proteinase inhibitor CAA57964 47 Serine proteinase inhibitor (Liang et al., 1995)

Antibacterial proteins

Astacidine 1 AAO47336.1 1.9 Antimicrobial peptide cleaved from hemocyanin (Lee et al., 2004) Astacidine 2 ABH05920 1.8 Antimicrobial peptide (Jiravanichpaisal et al., 2007) Pl-crustin 1 ABP88042 13.4 Antimicrobial peptide (Inhibit Micrococcus luteus M1 11) (Donpudsa et al., 2010a; Jiravanichpaisal et al., 2007; Sricharoen et al., 2005) Pl-crustin 2 ABP88043 12.3 Antimicrobial peptide (Inhibit Micrococcus luteus M1 11) (Donpudsa et al., 2010a; Jiravanichpaisal et al., 2007; Sricharoen et al., 2005)

10 Pl-crustin 3 ABP88044 16.5 Antimicrobial peptide (Jiravanichpaisal et al., 2007) Hemagglutinin 420 Hemagglutinating activity (Kopácek et al., 1993a)

Anti-virual proteins

Anti-LPS factor ABQ12866 13.5 Antiviral activity (Liu et al., 2006) gC1qR HQ596361 27 Prevents White spot syndrome virus replication (Watthanasurorot et al., 2010)

Hematopoietic factor

Astakine 1 AAX14635 8.7 Hematopoiesis proliferation and differentiation of hemocytes (Söder- häll et al., 2005) Astakine 2 ABQ23255 9 Differentiation of granular cells (Lin et al., 2010) Crustacean hematopoietic factor (CHF) GQ497446 9 Downstream of astakine 1; Inhibitor the apoptosis of hemocyte (Lin et al., 2011) Hemocyte homeostasis-associated pro- ADN43413 13 Putative role in hemocyte homeostasis of crayfish (Prapavorarat et al., tein (HHAP) 2010)

Others

Ferritin 440 A storage protein for ferric ion (Huang et al., 1996) Hemocyanin AAM81357 360 O2 transporter, phenoloxidase-like activity, and produces antimicrobial peptides (Lee et al., 2004) Transglutaminase (TGase) AAK69205 87 Cross-linking/Coagulation; Keep Hpt cells at undifferentiated stage (Lin et al., 2008; Wang et al., 2001b) Clotting protein AAD16454 210 × 2 A plasma clotting protein, lipoprotein-like and TGase substrate (Sricharoen et al., 2005) Thioester-containing protein (TEP)-iso- HQ596368 164 Opsonic activity (In manuscript) form 1 TEP isoform 2 HQ596369 Vitelline membrane outer layer protein I AAX54597 20 Granular proteins of hemocyte (Sricharoen et al., 2005) (VMO-I). Calreticulin HQ596062 Apoptosis regulation (Watthanasurorot et al., in manuscript) Fatty acid binding protein ABE77153 15 Retinoic acid-dependent signaling pathway (Söderhäll et al., 2006) Superoxide dismutase AAD25400 25 Binding protein for peroxinectin (Johansson et al., 1999a)

Hematopoiesis

Hematopoiesis is the formation and development of new hemocytes. This is the process whereby undifferentiated hematopoietic stem cells develop into mature cells and are involved in proliferation, commitment and differentiation. Over the years, the fruit fly Drosophila melanogaster has become a major invertebrate model to study hematopoiesis (Minakhina and Steward, 2010). Drosophila hematopoiesis gives rise to three types of circulating hemocytes, and it occurs in two waves (embryogenesis and larval stages) during development (Crozatier et al., 2007; Krzemien et al., 2007). In the first wave, hemocytes develop from the early embryo head mesoderm and supply the pool of circulating blood cells (Wood and Jacinto, 2007). The

11 lymph gland produces the second wave that composes the adult hemocytes, and different cell-specific markers have been used for investigating the differentiation of hemocytes (Sinenko et al., 2009). There is no hemocyte synthesis in Drosophila adult, but as is the case in crayfish. In crustaceans, normally three different classes of hemocytes, hyaline cells (HCs), semigranular cells (SGCs), and granular cells (GCs) are observed within the hemolymph according to the morphological characteristics (cell shape, cell size, degree of cytoplasmic granule), and all types of hemocytes are important in immob- ilizing or destroying invasive pathogens (Cerenius et al., 2008; Gargioni and Barracco, 1998). In crayfish, the HCs are small and spherical cells containing no or few granules, and they are involved in phagocytosis (Söderhäll et al., 1986). The SGCs with different amount of secretory granules are responsible for encapsulation or phagocytosis; the GCs have large amount of secretory granules, and they are the major cells that store and release the prophenoloxidase activating system (proPO-system) and other molecules like AMPs (Sricharoen et al., 2005). SGCs and GCs degranulate and secret their granular content as an immune response to non-self microbial cell wall components, and they can be cytotoxic and lyse foreign eukaryotic cells (Söderhäll et al., 1985). Different hemocytes also participate in the clotting process. All crustaceans have an open circulatory system, and therefore the hemolymph needs to be rapidly clotted to prevent the invasion of microorganisms when the animals are injured (Hall et al., 1999; Maningas et al., 2008). In crustaceans, the number of free hemocytes can vary a lot in different individuals and also vary in response to environmental stress, hypoxia, and endocrine activity during the moulting cycle (Jiravanichpaisal et al., 2006a; Le Moullac et al., 1998; Söderhäll et al., 2003). In addition, the hemocyte number drops dramatically when the animal is injected with laminarin or infected with virus such as white spot syndrome virus (WSSV), a major viral pathogen of cultured shrimp (Guan et al., 2003; Persson et al., 1987; Söderhäll et al., 2003; van de Braak et al., 2002a). In crayfish and shrimp, the SGCs contain higher virus loads and exhibit faster infection rates, and therefore, they are suggested to be more susceptible to WSSV infection (Jiravanichpaisal et al., 2006b; Wang et al., 2002). Recently, a viral responsive protein, named hemocyte homeostasis-associated protein (HHAP) was found up-regulated upon WSSV infection in shrimp Penaeus monodon. Gene silencing of shrimp PmHHAP with dsRNA results in significant decrease in circulating hemocyte number and a high shrimp mortality. Severe damage of hemocytes was also observed in vivo and in vitro, which indicates that PmHHAP is essential for shrimp survival (Prapavorarat et al., 2010). A homologious protein was also found in crayfish P. leniusculus with much lower expression level compared with that of shrimp, but crayfish does not die if this gene is silenced (Prapavorarat et al., 2010).

12 Hematopoietic tissue (Hpt) in crustacean Generally, hemocytes do not divide in the circulatory system of crustaceans (Jiravanichpaisal et al., 2006a; Söderhäll and Cerenius, 1992). New hemocytes are continuously and proportionally produced from a separate organ called Hpt. However, there are not many published studies elucidating the mechanism by which blood cells are released into the circulation in crustaceans. In the lobster Homarus americanus, shore crab Carcunis maenas or crayfish P. l en i us cu lu s, mature hemocytes come from a thin sheet-like Hpt which is surrounded by a connective tissue and situated on dorsal side of the stomach (Chaga et al., 1995; Gary et al., 1993). In healthy animals, the release of hemocytes from Hpt in crayfish is under circadian regulation, which is a direct result of rhythmic expression of astakine (a hematopoietic cytokine) (Watthanasurorot et al., 2011b).The connection between the circulating hemocytes and the hemocyte precursors in the Hpt is still unclear, and the proposed hemocyte lineages have mainly been based on morphological characters. For instance, morphological studies of the Hpt was carried out in blue crab C. sapidus (Johnson, 1987), shrimp Sicyonia ingentis (Hose et al., 1992), lobster H. americanus (Martin et al., 1993), and black tiger shrimp P. monodon (van de Braak et al., 2002b), but these studies did not reveal any molecular details about maturation and release of the hemocytes. These studies did not show that the new synthesized hemocytes were released directly from the Hpt or similar tissues, or stored somewhere and released upon activation when they were needed.

Transcription factors and marker proteins Hematopoiesis is the lifelong production of blood cells and is tightly regulated by various transcription factors that promote or limit cell diversification (Orkin, 2000). Several hematopoietic transcription factors have been characterized and those are conserved across taxonomic groups including both protostomian and deuterostomian animals, ranging from flies to humans (Fossett et al., 2001a, b). The crystal cells and the plasmatocytes are two main hemocyte lineages in D. melanogaster embryo, and they develop from a common hemocyte precursor expressing the GATA protein Serpent (Srp) (Lebestky et al., 2000). Several other genes encoding transcription factors are: glial cell missing (Gcm), Gcm2, U-shaped (Ush), lozenge (Lz), and friend of GATA (FOG), and they are involved in the hematopoietic lineage commitment in D. melanogaster. Gcm and Gcm2 promote plasmatocyte development; Gcm and Gcm2 inhibit Lz activation to control the size of the crystal cell population; Ush also limits crystal cell development, while Lz promotes crystal cell development; Srp acts upstream of the other factors and

13 is required for late plasmatocyte differentiation (Bataillé et al., 2005; Fossett and Schulz, 2001a). During larval development of D. melanogaster, the lymph gland is a hematopoietic organ in which pluripotent hemocyte progenitors proliferate and differentiate into mature hemocytes. The largest lobe of the larval lymph gland is sub-divided into the posterior signaling center (PSC), the medullary zone (MZ) and the cortical zone (CZ). The MZ contains pluripotent prohemocytes (PH), niche cells that regulate the choice between progenitor quiescence and hemocyte differentiation (Sinenko et al., 2009). These cells migrate into the CZ as they differentiate into three kinds of hemocytes (Jung et al., 2005). Cells of the PSC express the Hedgehog (Hh) signaling molecule, which instructs cells within the neighboring MZ to maintain a hematopoietic precursor state while preventing hemocyte differentiation (Krzemien et al., 2007; Mandal et al., 2007). In the differentiation of niche cells of Drosophila larval, Srp is essential for Hh activation in niche cells, whereas the Ush and suppressor of Hairless prevent Hh expression in hemocyte progenitors and differentiated hemocytes (Tokusumi et al., 2010), which demonstrates the requirement of them in the control of niche cell differentiation and blood cell precursor maintenance. Hematopoietic stem cells (HSCs) are in the lymph gland of embryo or young larvae, and they give rise to a hematopoietic lineage. In addition, a zinc finger protein (Zfrp8), also called programmed cell death 2 (PDCD2) is highly conserved from flies to humans and is important for the maintenance of the HSCs (Minakhina and Steward, 2010). In P. l en i us cu lu s, the Lz-homologue PlRunt was shown to be important in crayfish hematopoiesis (Söderhäll et al., 2003), and all mature hemocytes are expressing proPO while less than 3 % of the Hpt cells express this transcript, so we suggested that the presence of the proPO transcript can be used as a marker for final hemocyte maturation (Söderhäll et al., 2003). In P. leniusculus, the Hpt was studied by light and electron microscopy and the cells were subdivided into five morphologically different cell types that might correspond to different developmental stages of SGCs and GCs (Chaga et al., 1995). In the five different cell types, proPO mRNA is restricted to type 4 cells, and expression of PlRunt in Hpt in this stage is low in all cell types. A short period prior to the release of the cells into circulation, the PlRunt transcript was induced significantly whereas proPO expression was delayed until the hemocytes matured and reached the circulation (Söderhäll et al., 2003). This showed that the final development into functional SGCs or GCs happens after their release from the Hpt. Absence of proPO transcript in a majority of Hpt cells indicates that this protein is not required until the cells are released into the circulation, which is of considerable interest since this protein is a very important protein in innate immune reactions as well as during sclerotization of the cuticle (Söderhäll et al., 2003; Söderhäll and Cerenius, 1998).

14 Both proPO and PlRunt mentioned above are not specifically expressed in different type of crayfish hemocytes. In order to study the differentiation of different hemocyte lineages, specific marker proteins for different hemocyte types are needed. Söderhäll and Smith (1983) developed a technique (percoll gradient centrifugation) to separate different crayfish hemocyte types. This technique has helped to elucidate the hemocyte specific functions (Jiravanichpaisal et al., 2006a), and markers for the hemocyte types are now at hand in P. len i us cul u s (Paper I).

Cytokines are important for the hematopoiesis in crustacean Cytokines are small cell-signaling proteins that are secreted by numerous cells of the immune system and by glial cells of the nervous system, and are involved in intercellular interactions or communications (Smithgall, 1998; Wang et al., 2009). Hematopoietic cytokines/hematopoietic growth factors are required for the proliferation and differentiation of hemocyte precursors into mature hemocytes (Baker et al., 2007; Smithgall, 1998). PVF2, a PDGF/ VEGF like growth factor, from fruit fly D. melanogaster and astakine 1 (ast1) from crayfish are the only two hematopoietic growth factors that have been shown to promote proliferation of hemocyte precursors in invertebrates. So far, no homologue of PVF2 has been identified in crustaceans. Ast1, as a homologue to vertebrate prokineticins, was first characterized in crayfish P. leniusculus. Ast1 contains a signal peptide and a prokineticin domain (10 cysteines with conserved spacing pattern) and is found to be mainly expressed in SGCs. In vivo and in vitro experiments proved its abil- ity to stimulate the migration and proliferation of Hpt cells (Söderhäll et al., 2005). Prokineticin (PROK) domain-containing proteins belong to a family of small secreted proteins of about 80 amino acids, for instance, the mamba in- testinal toxin 1 originally isolated from venom of black memba Dendroaspis polylepis (Joubert and Strydom, 1980), Bv8 isolated from skin secretions of frog Bombina variegate (Mollay et al., 1999) and mammalian PROK1 and PROK2 (Li et al., 2001; Wechselberger et al., 1999; Melchiorri et al., 2001). All of them contain an AVIT sequence in the N-terminal and a PROK domain in the C-terminal, and induce gastrointestinal smooth muscle contrac- tion (Joubert and Strydom, 1980; Li et al., 2001; Mollay et al., 1999). Moreover, both PROK1 and PROK2/Bv8 were shown to stimulate the differentiation of murine and human bone marrow cells into the macrophage lineage (Dorsch et al., 2005; LeCouter et al., 2004). The N-terminal AVIT is crucial for the interaction with prokineticin receptors (PROKRs) in all vertebrate PROKs. The PROK-like proteins (astakines) from invertebrates are different, and all the astakines found so far lack

15 the AVIT region in the N-terminal (Lin et al., 2010; Söderhäll et al., 2005), which suggests that astakines have different receptors. By using covalent cross-linking of ast1 to intact Hpt cell surface, ATP synthase beta-subunit, as a receptor for ast1, was detected on Hpt cells and not on mature crayfish hemocytes (Lin et al., 2009). With the finding of a second astakine, astakine 2 (ast2), invertebrate astakines are classified into two groups, ast1 and ast2. So far, ast1 is found in crayfish and spider, whereas ast2 is widely distributed in several invertebrates (Lin et al., 2010). Up to now, astakine-like sequences have been found in around twenty different invertebrate species. Ast1 and ast2 have different roles in hemocyte lineage differentiation. In P. leniusculus, ast1 induces the proliferation and differentiation to SGCs in the primary Hpt cell culture, whereas ast2 may function in the differentiation to GCs in vivo (Lin et al., 2010). Many factors are known to be necessary for the crayfish hematopoiesis. For instance, a novel crustacean hematopoietic factor (CHF) was found as a downstream gene of ast1 in P. leniusculus. Like ast1, CHF is also a small cysteine rich protein, which has high similarity to the N-terminal region of vertebrate cysteine-rich motor neuron 1 (CRIM1). CHF can be induced by ast1 in vivo and in vitro in crayfish. Silencing of CHF resulted in the apoptosis of Hpt cells and severe loss of blood cells in the animal, which suggests that CHF is critical for survival of hemocytes and Hpt cells by prevent the apoptosis (Lin et al., 2011). In crustaceans, transglutaminase (TGase) is mainly known for its role in the plasma clotting reaction (Chen et al., 2005; Kopácek et al., 1993b; Maningas et al., 2008; Osaki et al., 2002; Wang et al., 2001b). Recently, TGase mRNA expression as well as enzyme activity was found to be very high in the Hpt, lesser in SGCs and very low in GCs in P. leniusculus. TGase is important for keeping the Hpt cells in an undifferentiated stage inside the Hpt, and the Hpt cells start to migrate when the mRNA expression of TGase is blocked with dsRNA (Lin et al., 2008).

16 The proPO-system and melanization

Melanization is a process that phenoloxidase (PO) catalyzes the hydroxyla- tion of monophenols to o-diphenols (monophenolase activity) and the oxidation of o-diphenols to o-quinones (diphenolase activity), followed by several intermediate steps which result in the formation of melanin, a dark brown pigment. Some of the steps are catalyzed by PO, whereas other steps occur spontaneously. In vertebrates, melanin provides pigmentation and protection and is important for the development of the central nervous system and the eyes, but the pigment is also associated with some diseases like melanomas. In most invertebrates, PO is often produced by hemocytes and released to the plasma upon immuno-stimulation. By contrast, in vertebrates the corresponding enzyme, tyrosinase, is a membrane-bound protein and located in a specialised organelle, the melanosome. Tyrosinase is also a redox enzyme and activity is similar to that of phenoloxidase (Cerenius et al., 2010b). The melanization reaction is an important immune reaction in arthropods (Cerenius et al., 2008). It is a rapid immune response important in killing of microbial pathogens, and melanin depositions are often observed on the surface of invading pathogens in the hemocoel or at the site of cuticular injury. The first proPO cDNA was reported and characterized in P. leniusculus (As- pán et al., 1995). To date, more than forty proPO sequences have been cloned and characterized from different invertebrates (ascidians, bivalves, brachiopods, echinoderms, insects, millipedes, and mollusks), and some species have more than one PO, for instance, there are three POs in fruit fly D. melanogaster, nine POs in mosquito Anopheles gambiae and three in beetle Tribolium castaneum (Cerenius et al., 2008; Gerardo et al., 2010). Arthropod POs are copper-containing proteins, and they are similar to arthropod hemo- cyanins (Burmester, 2001; Terwilliger and Ryan, 2006). Hemocyanin, as res- piratory protein, exhibits PO activity under certain conditions, which indicates its role in immune defence (Cerenius et al., 2008). For instance, in horseshoe crab, some clotting factor components convert hemocyanin into PO by forming a complex without proteolytic cleavage (Nagai and Kawabata, 2000). In crayfish, the hemocyanin subunit 2 exhibited relatively low phenoloxidase activity if it was cleaved at the N-terminal part with trypsin (Lee et al., 2004).

ProPO activation In invertebrates, the proPO-system is a non-self recognition system, which is very efficient and can be triggered by tiny amount of microbial components, eg. -1, 3-glucans from fungi and lipopolysaccharides (LPSs) / pep-

17 tidoglycans (PGNs) from bacteria. Pattern recognition proteins (PRPs) are responsible for detecting and responding to the microbial components. The proPO-system includes several PRPs, such as -1, 3-glucans binding protein (GBP), lipopolysaccharide and -1, 3-glucans binding protein (LGBP). Several serine protease zymogens involved in the proteinase cascade have been well studied (Cerenius et al., 2008). In P. l en i us cu lu s , the proPO-system is located in secretory granules of SGCs and GCs, and is released into the hemocoel by exocytosis triggered by PRPs upon bacterial infection, like the clotting system of horseshoe crab (Theopold et al., 2004). After the recognition and binding of the PRPs to cell wall components of pathogens, a serine proteinase cascade is activated in the proPO-system, and the final step in this process is the conversion of proPO into PO by the prophenoloxidase activating enzyme (ppA) (Aspán et al., 1995). The N-terminal part of crayfish ppA is similar to those of the horseshoe crab big defensin and mammalian beta-defensins, which contain clip-domain with a similar disulfide-bonding pattern (Saito et al., 1995; Wang et al., 2001a). The recombinant clip domain of the endogenous trypsin-like serine proteinase ppA from crayfish accordingly shows antibacterial activity in vitro against Gram-positive bacteria suggesting a multiple function of crayfish ppA, and this is possible for other ppAs (Wang et al., 2001a). Several ppAs and cofactors have been identified from insects: M. sexta, Holotrichia diomphalia, and Bombyx mori, (Cerenius and Söderhäll, 2004). Crayfish and B. mori ppAs alone can produce an active PO (Satoh et al., 1999; Wang et al., 2001a), but M. sexta or H. diomphalia ppA need a cofactor to generate active PO (Jiang et al., 1998 and 2003). The primary structures of these proteins show that they all exist as zymogens of typical serine proteinases (Jiang and Kanost, 2000). In addition, masquerade-like proteins (Mas) and serine proteinase homologues (SPHs) are involved in activation of the proPO-system in invertebrates. Mas and SPHs have no proteinase activity since they lack the catalytic triad residues which are necessary for serine proteinase activity. These proteins have been characterized from many species, such as insects (D. melanogaster, H. diomphalia and T. molitor) (Kwon et al., 2000; Lee et al., 2002a; Murugasu-Oei et al., 1995), P. leniusculus and Limulus spp. (Kawabata et al., 1996; Lee and Söderhäll, 2001). Moreover, Tenebrio Spätzle -processing enzyme (SPE) was proved to process proPO and pro-Spätzle, and the injection of Lys-type PGN into T. molit- or larvae induced both AMP production and melanin formation, which shows that Toll and proPO cascades overlap and share common serine proteases (Cerenius et al., 2010b; Kan et al., 2008; Roh et al., 2009).

18 ProPO-system is involved in cellular responses Except for the melanization, activation of the proPO-system can also trigger cellular responses including cell adhesion, degranulation, phagocytosis, nodule formation, and encapsulation (Johansson and Söderhäll, 1995; Liu et al., 2007a). In T. molitor, PO participates in cell adhesion and/or clumping of hemocytes after binding to the hemocytes membrane (Lee et al., 1999). There are reports showing that proPO binds to the surface of some hemocytes resulting in a strict spatial localization of the melanization response in M. sexta (Ling and Yu, 2005; Mavrouli et al., 2005). However these observa- tions may be incorrect and due to the hydrophobic properties of PO which can result in non-specific binding. Studies from A. gambiae indicate that PO activity is required for coagulation by causing lipophorin particles to co- alesce into the sheet structures (Agianian et al., 2007), which increase the efficiency of hemolymph coagulation and cellular defense reactions. Some studies imply that the proPO-system provides factors to stimulate cellular defense by increasing phagocytosis (Cerenius et al., 2008). Moreover, host immune molecules can also gain their functions when they are generated concomitant with the activation of the proPO-system. For instance, peroxinectin, a cell adhesion protein in crayfish and shrimp, is stored in the secretory granules of hemocytes, and it is released during exocytosis and proPO- system activation to mediate cell attachment and spreading (Johansson, 1999b; Johansson and Söderhäll, 1988; Lin et al., 2007; Sritunyalucksana et al., 2001). In P. l en i us cu lu s, high rate of phagocytosis and nodule formation were observed when pacifastin (an efficient inhibitor of the proPO activation cascade) was silenced and the crayfish was infected with the bacterium, Aeromonas hydrophila, whereas silencing of proPO resulted in the opposite result (Liu et al., 2007).

Importance of proPO-system in arthopods A recent study showed that an active PO has different antibacterial effect to bacteria in vitro, and reduction of bacterial growth was stronger when dopamine was used as substrate as compared to L-dopa (Cerenius et al., 2010a). The importance of the proPO-system to the immune response is widely accepted in invertebrates, but it is controversial in some insects such as fruit fly D. melanogaster and mosquito A. gambiae (Leclerc et al., 2006; Schnitger et al., 2007). Most studies suggest an important role for the proPO-system. In D. melanogaster and A. gambiae, the PO activity was reported to be redundant, but these insects possess several proPO genes, and a functional study needs to be carefully designed, in order to clarify the role of proPO. A recent study showed that melanization was important to combat

19 bacterial infections in the flies with defective Toll and Imd pathways (Tang et al., 2006).

Control of proPO activation and PO activity Although melanin formation is essential for host defence in crustaceans and insects, the initiation of proPO-system needs to be tightly regulated due to the danger to the animal of unwanted production of quinone intermediates and melanization in places where it is not appropriate (Söderhäll and Cereni- us, 1998). Pacifastin, as an efficient inhibitor of crayfish ppA, forms a new family of proteinase inhibitors (Liang et al., 1997). This family was named as pacifastin-like serine proteinase inhibitor, and it can inhibit the proPO- system in many insects (Simonet et al., 2002; Vanden Broeck, et al., 1998). Pacifastin is a high molecular weight and heterodimeric inhibitor, and it is composed of one light chain containing nine protease inhibitor subunits and a heavy chain that contains three transferrin lobes (Liang et al., 1997). Chal- lenging crayfish with A. hydrophila results in 100% mortality when pacifastin was silenced, indicating that endogenous pacifastin may participate in inhibiting the production of inappropriate toxic compounds (Liu et al., 2007a). Several genes expressing serine proteinase inhibitors (Serpins) were shown to be involved in the melanization in A. gambiae (Michel et al., 2005) and D. melanogaster (Ligoxygakis et al., 2002). For example, Drosophila Serpin 27A can inhibit the proPO activating enzyme, PPAE and prevent melanin synthesis. Mutations in Serpin 27A led to severe melanization and increased lethality (De Gregorio et al., 2002; Ligoxygakis et al., 2002). The survival rate of parasitoid wasp Leptolinina boulardi increased when Ser- pin27A was injected into Drosophila larvaes (Nappi et al., 2005). Serpin 28D was strongly induced upon injury, and it also regulated hemolymph PO activity. It is suggested that Spn27A confines the melanin produced to the wound site, while Spn28D controls its initial release (Scherfer et al., 2008). Expression of SRPN6 was induced by Escherichia coli and malaria parasites, and depletion of SRPN6 delayed the lysis of parasite without changing the number of developing parasites in A. gambiae, indicating that SRPN6 acts on parasite clearance by inhibiting melanization and promoting parasite lysis (Abraham et al., 2005). Microplitis demolitor bracovirus carried by the wasp M. demolitor encodes a protein Egf1.0, which disabled melanization reaction in M. sexta by inhibiting both the activity of PAP3 and also preven- ted processing of SPH1, and SPH2 (Beck and Strand, 2007; Lu et al., 2008). In addition to inhibitors of the proPO-system activation, some PO molecules that inhibit the activity of PO have been studied in different arthropods. For instance, phenoloxidase inhibitors (POIs), have been found in Musca domestica, A. gambiae and M. sexta (Daquinag et al., 1999; Lu and

20 Jiang, 2007; Shi et al., 2006). Several clip-domain serine proteases were involved in limiting parasite numbers and/or affected the regulation of melanization (Volz et al., 2005 and 2006). Recently, a 43-kDa protein was described as a melanization-inhibiting protein (MIP) in T. molitor. Recombin- ant MIP could inhibit melanin synthesis in vitro, and melanin synthesis was markedly induced when MIP was silenced in Tenebrio larvae (Zhao et al., 2005). A protein with similar function has been found in crayfish and shrimp (Paper II and Angthong et al., 2010). For comparison, the factors that interfere with PO activity or proPO activation process are summarized in Table 2.

Table 2. Factors interfering with PO activity or proPO activation (modified from Cerenius et al., 2008) Name Type of compound Source Activity Negative regulators of proPO activation

Egf1.0 26-kDa protein MdB virus Prevents host proPO activation by interfering with PAP-3 (Beck and (polydnavirus) Strand, 2007) Vn50 50-kDa protein Cotesia rubecola An SPH that down-regulates proPO activation in host (Zhang et al., parasitoid venom 2004a) Pacifastin Proteinase inhibitor Crustaceans Inhibitor of ppA (Liang et al., 1997) Serpins-1J,-3,-6 Manduca sexta Inhibitors of PAPs (Jiang et al., 1997) Serpins-4,-5 M. sexta Inhibitors of upstream proteinases (Tong et al., 2005) Spn27A 48-kDa protein Drosophila melanogaster Inhibition of proPO activation (De Gregorio et al., 2002; Ligoxygakis et al., 2002) Spn28D 59-kDa protein D. melanogaster Limits PO availability by controlling its initial release (Scherfer et al., 2008) SRPN1,-2 Peptidoglycan fragment Anopheles gambiae Inhibition of M. sexta PAP (Michel et al., 2006) T-4P2 Synthetic Competitive inhibitor of melanization cascade (Park et al., 2006)

Negative regulators of PO activity or melanin synthesis

ST A hydroxystilbene Photorhabdus luminiscens Used by insect pathogen to inhibit host PO (Eleftherianos et al., 2007) Tm MIP 43-kDa protein Tenebrio molitor Interferes with melanin production from phenolics (Zhao et al., 2005) Pl MIP 43-kDa protein crayfish Melanization inhibiting protein (Paper II) Pm MIP 36-kDa protein shrimp Melanization inhibiting protein (Angthong et al., 2010) POI Proteins with cysteine Many insects (Shi et al., Interferes with melanization by binding reaction intermediates(?) knot motifs 2006; Lu and Jiang, 2007)

Pattern recognition proteins (PRPs)

Innate immunity is not just a non-specific immune response characterized by phagocytosis, but it can also discriminate between non-self and self. A set of conserved host proteins known as PRPs are important for non-self recogni-

21 tion to sense specific and conserved molecules called pathogen associated molecular patterns (PAMPs) such as LPS of Gram-negative bacteria, PGN of Gram-positive bacteria, and -1, 3-glucan of fungi (Janeway et al., 2002; Medzhitov et al., 2002). Recognition of PAMPs initiates various innate immune responses, such as the activation of the opsonic and lytic complement pathways in vertebrates, the proPO activating cascades in arthropods, the activation of phagocytic cells, and production of AMPs (Bachère et al., 2004; Cerenius and Söderhäll 2004; Fujita et al., 2004a, b). Factor C in horseshoe crab is the first PRP detected in any animal and found to bind LPS. Shortly thereafter a LPS-binding protein (LBP) was found in rabbit (Nakamura et al., 1986; Tobias et al., 1986), and a glucan- binding protein was characterized in cockroach Blaberus craniifer (Söder- häll et al., 1988). Significant advances have been achieved in elucidating the functions of PRRs in vertebrates and invertebrates (Akira et al., 2006; Kurata et al., 2006; Royet et al., 2004; Yu et al., 2002). Many kind of recognition molecules have been found, such as SPHs, LPS and -1,3-glucan binding protein (LGBP), -1,3-glucan binding protein (GBP), PGN recognition proteins (PGRPs) and lectin, insect thioester-containing proteins (TEPs), Down syndrome cell adhesion molecules (Dscams), and Toll/TLRs (Cerenius et al., 2010b; Iwanaga and Lee, 2005; Pal and Wu, 2009).

SPHs Serine proteases belong to the trypsin protease family, which is characterized by an active site that contains H, D, and S amino acids named as the catalytic triad (Stroud et al., 1974). In vertebrates, the functions of serine proteases are diverse, and they play important roles in blood clotting, food digestion, and the complement system (Jiravanichpaisal et al., 2006a; Wu et al., 2005). In arthropods, the activation of proPO-system is mediated by a serine proteinase cascade (Cerenius et al., 2008). In addition to serine proteinases, non-catalytic clip domain SPHs, lacking protein hydrolytic activity by non-synonymous amino acid substitutions in the catalytic residues, have been identified from both vertebrates and invertebrates. A SPH, named masquerade (mas), was first found in D. melanogaster, and it is required for stabilizing muscle attachment in the embryo (Murugasu-Oei, et al., 1995). SPHs exhibit different biological functions such as antimicrobial activity (Almeida et al., 1991; Kawabata et al., 1996), adhesion activity (Barthalay et al., 1990; Huang et al., 2000), growth factor activity (Nakamura et al., 1989) or as pattern recognition proteins (Lee and Söderhäll, 2001), and they have also been identified as critical factors for the activation/regulation of the proPO-system in insects (Cerenius et al., 2008). All these SPHs contain one or more clip domains. The clip domain is suggested to be involved in protein-protein interactions, regulation of protease

22 activity, and antimicrobial activity in arthropods (Jiang et al., 2000). In crustaceans, several SPHs have been identified with different biological properties such as cell adhesion activity (Huang et al., 2000; Lee and Söderhäll, 2001; Lin et al., 2006), binding to virus (Sriphaijit et al., 2007), or acting as immune molecules induced by challenge (Amparyup et al., 2007; Rattana- chai et al., 2005; Sricharoen et al., 2005). Previously, Pl-MasI and Pl-SPH1, have been isolated from P. l en i us cu lu s. Pl-MasI was identified as a PRP in hemocytes. It has binding activity to LPS, -1, 3-glucan, and Gram-negative bacteria (Lee and Söderhäll, 2001). This protein also has opsonic and cell adhesive activity (Huang et al., 2000; Lee and Söderhäll, 2001). Pl-SPH1 is shown to be released by exocytosis of crayfish granular hemocytes triggered by a calcium ionophore, LPS-PGN or peroxinectin (a crayfish cell adhesion and degranulation factor) (Sricharoen et al., 2005). The expression of Pl- MasI and Pl-SPH1 is high in hemocytes, suggesting an important role of this protein in crayfish immune responses (Sricharoen et al., 2005). Pl-SPH2, Pl- SPH1 and LGBP were found in several different PGN-binding assays. RNA interference of Pl-SPH2, Pl-SPH1 or LGBP in the crayfish Hpt cell culture resulted in lower PO activity following activation of the proPO-system by soluble Lys-type PGN, which may suggest that these crayfish SPHs function as PRPs (Paper III).

PGRPs A wide range of bacterial patterns is recognized by the immune systems in vertebrates and many invertebrates, while some insects like D. melanogaster seem to mainly recognize PGNs. PGN is a polymer containing glycan strands of alternating N-acetylglucosamine and N-acetylmuramic acid that are cross-linked to each other by short peptide bridges. PGN from Gram- positive bacteria is different from Gram-negative bacteria and Bacillus species by the replacement of meso-diaminopimelic acid (DAP) with Lysine (Lys) at the third amino acid in the peptide chain. In insects, the Lys-type PGN activates the Toll signaling pathway, whereas the DAP-type PGN stimulates Imd signaling pathway, both of which result in AMPs expression, and activation of the proPO cascade (Lemaitre and Hoffmann, 2007). PGRPs have been studied from mammals to insects especially in Drosophila. The first member of the PGRP family was discovered in the silkworm B. mori as a protein that initiated the activation of proPO-system in the presence of PGN (Yoshida et al., 1996). Insects have several PGRPs, classified into short (S) and long (L) forms. The short forms are present in the hemolymph, cuticle, and fat-body cells, and sometimes in epidermal cells in the gut and hemocytes, whereas the long forms are mainly expressed in hemocytes.

23 Drosophila PGRPs are similar to bacteriophage T7 lysozyme (Kim et al., 2003; Royet et al., 2005; Steiner, 2004). Thirteen Drosophila PGRPs can be divided into two subgroups with either pattern recognition or enzymatic properties. Like bacteriphage T7 lysozyme, many PGRPs (PGRP-SC1, LB, SB1) have amidase activity that can reduce or eliminate biological activity of PGN by cleaving peptides from glycan chains (Mellroth et al., 2003; Zaid- man-Remy et al., 2006). Other subgroup of PGRPs (PGRP-SA, SD, LA, LC, LD, LE, LF) that act as PRPs have a serine substitution in the position corresponding to the Cys zinc ligand (Mellroth et al., 2003). PGRPs have been reported in insects, molluscs, echinoderms, and vertebrates, but in a genomic analysis of an insect, pea aphid (Acyrthosiphon pisum) no PGRP gene was detected, which suggests a diversity of immune responses among insect species (Gerardo et al., 2010). Up to now, no PGRP with pattern recognition properties has been described in any crustacean. Re- cently, the complete genome data of the crustacean Daphnia pulex was achieved, but only PGRP genes with putative enzymatic properties were detected (McTaggart et al., 2009). Therefore, the mechanism for how PGN induces activation of the proPO-system in crustacean is still unknown.

GNBPs Gram-negative binding proteins were divided into two distinct groups, characterized by the absence or presence of a cysteine rich (CR) domain. The CR domain of GNBP helps to bind pathogenic cell wall compounds. All GNBP genes of D. melanogaster, Apis mellifera and B. mori have a CR domain, whereas only two out of seven GNBP genes of A. gambiae have this domain (McTaggart et al., 2009). B. mori GNBPs contain a glucanase-like (GLU) domain in their C-terminal and have high affinity to the surface of Gram- negative bacteria (Lee et al., 1996). However, the crucial amino acids required for the glucanase activity in B. mori GNBPs are not conserved in Drosophila. A. gambiae and crustacean D. pulex have GNBPs with and without a putative catalytic site in GLU, respectively. In D. pulex, eleven GNBP genes were revealed by complete genome sequencing, which is higher in number in comparison to D. melanogaster (three members) or A. gambiae (six members). Phylogenetic analysis showed that the D. pulex GNBPs fall into four clades. D. melanogaster and A. gambiae GNBPs were mainly clustered in clade I and IV, respectively. Most of GNBP clade II, III, IV proteins have both CR domain and active GLU domain, while GNBP clade I proteins have a CR domain and an inactive GLU domain. (McTaggart et al., 2009). In Drosophila, three GNBPs have been isolated, and they show strong affinity for LPS and -1,3 glucan in vitro (Medzhitov et al., 2000). GNBP1 and PGRP-SA form a complex in the hemolymph to sense Lys-type PGN

24 and induce AMP synthesis (Gobert et al., 2003; Wang et al., 2006). In Tenebrio, GNBP1 and PGRP-SA recognize Lys-type PGN and this results in both proPO activation and Toll pathway activation (Kan et al., 2008). Inter- estingly, Drosophila GNBP1 has glycosyl hydrolase activity and degraded Lys-type PGN into small fragments that are susceptible for PGRP-SA recognition in D. melanogaster (Filipe et al., 2005; Wang et al. 2006). The Droso- phila GNBP3 is important for the detection of fungal infection since GNBP3 mutants were susceptible to fungal infection and failed to activate the Toll pathway (Gottar et al., 2006). GNBP3 protein shares high similarity with that of silkworm B. mori lepidopteran glucan recognition proteins known to bind fungal -1,3-glucan (Ochiai et al., 1988).

LGBP and GBP/-1,3-glucan recongition protein (/GRP) In 1988, a GBP was identified in B. craniifer (Söderhäll et al., 1988) followed by a GRP in B. mori (Ochiai and Ashida, 1988). This is a group of proteins similar to GNBPs, and they contain a region with sequence similarity to several glucanases such as -1,3-glucanase A1 of Bacillus circulans WL-12 (Yahata et al., 1990), -1,3-glucanase of the sea urchin Strongylo- centrotus purpuratus (Bachman et al., 1996), and the subunit of clotting factor G from horseshoe crab Tachypleus tridentatus (Muta et al., 1995), but it lacks glucanase activity that hydrolyze -1,3-glucans. It is suggested that these invertebrate proteins have evolved from a -1,3-glucanase gene, which has lost its glucanase activity during evolution, but the glucan binding activity has been retained (Cerenius et al., 1994). In P. leniusculus, LGBP was identified from hemocytes, it contains a true -1,3-glucan recognition protein domain, and it does not have any carbohydrate hydrolyzing activity. This protein was shown to bind to intact E. coli, or commercial LPS and to -1,3-glucans such as curdlan and laminarin (Lee et al., 2000), and is involved in the activation of the proPO-system after it binds to -1,3-glucan. Crayfish GBP was found in plasma, and this protein may bind to an integrin-like protein through a RGD (Arg-Gly-Asp) motif, which activates the spreading and degranulation of crayfish hemocytes (Barracco, et al., 1991). GBP interacted with an extracellular superoxide dismutase (SOD), which is involved in binding with peroxinectin (a cell adhesive and opsonic peroxidase) to the cell surface. The binding of the cell surface SOD to peroxinectin may mediate, or regulate, cell adhesion and phagocytosis (Johansson et al. 1999a). Other GBPs and LGBPs such as the GRP1 and GRP2 from hornworm M. sexta, GRP from moth, Plodia interpunctella, or LGBP from earthworm Eisenia foetida, were also shown to

25 mediate the activation of the proPO-system (Beschin et al., 1998; Fabrick et al., 2003; Jiang et al., 2004; Ma and Kanost, 2000).

Lectins Lectins, as sugar-binding proteins, are often complex, multi-domain containing proteins, and are capable of agglutinating cells. Their sugar-binding activity can usually be ascribed to a domain named carbohydrate-recognition domain (CRD). The specificity of lectins is usually defined in terms of the mono-saccharide(s) or simple oligosaccharides that inhibit lectin-induced ag- glutination. Although lectins were first discovered in plants, they are present in all phyla, including bacteria and animals (Malagoli et al., 2010). Lectins play an important role in cell-to-cell or cell-to-matrix interaction, glycopro- tein trafficking, protein folding, signal transduction, fertilization, development and self/non-self discrimination (Vasta et al., 2004). Lectins may be classified by structural or functional criteria. With regards to the structural composition, at least eight families have been identified in animals on the basis of the CRD domain: C-type lectin, P-type lectin, I-type lectin, Calnex- in/calreticulin, S-type lectin (Galectin), L-type lectin, R-type lectin and F- type lectin (fucolectin) (Dodd et al., 2001; Malagoli et al., 2010). According to the final location, the lectins fall broadly into two categories (intracellular and extracellular). Lectins of three structural groups (Calnexin, L-type, P- type) are located mostly intracellularly, in luminal compartments. Lectins in the remaining structural groups (Galectins, C-type, I-type, R-type, F-type lectin) are lectins that function largely outside the cell, and are secreted or localized to the plasma membrane. But some lectins such as S-type lectins, a major group of animal lectins, are found both intracellularly and extracellu- larly (Drickamer 1988; Dodd et al., 2001). In C-type lectins, CRD contains conserved carbohydrate-binding sites, Ca2+-binding sites and also a triplet motif, either Glu-Pro-Asn (EPN) or Gln-Pro-Asp (QPD). The triplet motifs are important for ligand-binding specificity of the proteins to mannose/GlcNAc or galactose/GalNAc, respectively (Drickamer, 1993). C- type lectins share a moderate similarity in primary amino acid sequence, but higher similarity in structure, which indicates that the existing C-type lectins might have evolved from a same ancestor molecule as a result of functional differentiation. So far, C-type lectins have been characterized in both vertebrates and invertebrates. C-type lectins are Ca2+ dependent proteins, and they are the most diverse family of animal lectins. According to the domain architecture, C-type lectins are separated into seven groups, which is consistent with the result of phylogenetic analysis of C-type lectin domain sequences and also functional similarity. As the number of determined sequences grew, seventeen groups

26 have been suggested to classify all the C-type lectins (Zelensky and Gready, 2005). It is clear now that not all proteins containing C-type CRDs can actu- ally bind carbohydrates, and the domains in these proteins were named as C- type lectin-like domains (Zelensky and Gready, 2005). C-type lectins were suggested be involved in immune recognition in invertebrates (Weis et al., 1998). A number of C-type lectins have been reported in different invertebrate species. In insect, 22 genes encoding C-type lectin domains have been discovered in A.gambiae (Osta et al., 2004), while there are 32 genes in D. melanogaster (Adams et al., 2000), 279 genes in Caenorhabditis elegans (Schulenburg et al., 2008), and 4 C-type lectins (immulectins) in M. sexta (Yu et al., 2006a). C-type lectins have also been identified in echinoderm (Kouzuma et al., 2003), cnidaria (Wood-Charlson et al., 2009), porifera (Gundacker et al., 2001) and mollusk (Takagi et al., 1994). The cDNA sequences that encode C-type lectins have been extensively reported in crustaceans like shrimp, crab and crayfish (Song et al., 2010; Wang et al., 2010). Many different C-type lectins are isolated from invertebrates, and the functions of C-type lectins are likewise diverse. Invertebrate C-type lectins are involved in diverse innate immune responses, which include activation of the proPO-system (Yu and Kanost, 2000), phagocytosis (Luo et al., 2006), cell adhesion and hemocyte mediated nodule formation or encapsulation (Ling and Yu, 2006). They also contribute to antifungal immune response (Willment and Brown, 2008), antibacterial activity (Schroder et al., 2003; Sun et al., 2008), and also play roles in viral infection (Zhao et al., 2009). In crustacean, C-type lectins display different structures (one or two CRDs), expression profiles and functions. These crustacean C-type lectins have ligand binding specificities for carbohydrates such as LPS and GlcNAc, and most of them possess bacterial agglutinating activity and exhibit antimicrobial activity against some bacteria, fungi or even virus. A number of papers have presented results that indicate a role for C-type lectins in host response to WSSV infection, but so far no conclusive evidences of the mechanism is at hand (Luo et al., 2003; Song et al., 2010; Zhao et al., 2009).

Fibrinogen-related domain (FReD) containing proteins FReD containing proteins are carbohydrate binding proteins, and they play important roles in recognizing broad classes of pathogens. Ficolin and mannose binding lectin (MBL) are C-type lectins, and they are sorted into collectins composed of a lectin domain and a collagenous region. However, these two collagenous lectins differ in their lectin domains: ficolin has a FReD and MBL has CRD (Holmskov et al., 2003; Zelensky et al.,

27 2005). Both are involved in the complement system, which represents one of the major humoral systems of the host innate response. In ficolins, the FReD consists of about 200-250 amino acid residues and is characterized by the presence of forty highly conserved residues, mostly hydrophobic, amino acid residues including four cysteines (Runza et al., 2008). Three types of ficolins have been identified in human: L-ficolin, M-ficolin and H-ficolin, and two types in mouse: ficolin A and ficolin B (Endo et al., 2007). Because of the conserved FReD, all ficolins have a common binding specificity for GlcNAc (Fujita et al., 2004b). In P. len i us cul u s, a lectin MBL- like protein has been found to be released by exocytosis of granular hemocytes triggered by LPS-PGN or a calcium ionophore (Sricharoen et al., 2005). Many FReD containing proteins have been described in various animal species. This is a family of glycoproteins that are similar to fibrinogen ( and chains), tenascins, ficolins or angiopoietins (Adema et al., 1997; Lu et al., 1998; Zhang et al., 2001). In invertebrates, many FReD containing proteins have been reported. A melanization inhibition protein MIP, acts as a regulat- or of phenoloxidase-induced melanization in crayfish, P. leniusculus and in shrimp, P. monodon (Paper II and Angthong et al., 2010). A FReD containing protein was indentified as an inhibitor of differentiation of R8 photoreceptor cells in the developing eye of D. melanogaster (Baker et al., 1990). In amphioxus Branchiostoma belcheri, BbFREP was able to bind to both the Gram-negative bacterium E. coli and the Gram-positive bacterium Staphylo- coccus aureus (Fan et al., 2008). Aslectin, a mosquito (Armigeres subalbatus) FREP, was up-regulated by bacterial challenge and could bind to the bacteria Micrococcus luteus and E. coli (Wang et al., 2004b). Tachylectin 5A and Tachylectin 5B from horseshoe crab, T. tridentatus, recognize N-acetyl group containing molecules and agglutinate all types of human erythrocytes and different bacteria (Gokudan et al., 1999). The freshwater snail, Biom- phalaria glabrata produces a number of fibrinogen-related proteins (FREPs), and the diversification of FREPs occurs at genomic and transcriptional level through gene conversion and alternative splicing (Zhang et al., 2004b). Diverse FREP proteins are present in the hemolymph and show a pattern of bands of 50-150 kDa, the larger forms of FREP (95-125 kDa) bind to a variety of microbes (Gram-positive and Gram-negative bacteria, yeast), while the smaller forms of FREP bind to the trematode Echinostoma paraensei sporocysts or soluble parasite-derived secretory/excretory products (Zhang et al., 2004b; Zhang et al., 2008).

Dscams Down syndrome cell adhesion molecules belong to the immunoglobulin superfamily (IgSF) (Schmucker et al., 2009), and they function in cell surface

28 recognition, cell adhesion, cell connection, and cell signaling (Garver et al., 2008). DSCAM was first characterized from human chromosome band 21q22 2-22.3, a region critical for Down syndrome (Yamakawa et al., 1998). Normally, it contains ten Ig domains, six fibronectin type III domains, one transmembrane domain and a cytoplasmic domain. Human DSCAM or Drosophila Dscam were originally studied for their role in axon guidance in the embryonic central nervous system. In Droso- phila, Dscam, Dock (an SH3/SH2 adaptor protein) and Pak (serine/threonine signaling kinase) function together to guide axons of nerves to an intermediate aim in the embryo (Ang et al., 2003; Schmucker et al., 2000). Alternative splicing leads to huge diversity of Dscam that helps in connectivity and development of neurons (Schmucker et al., 2000). The Drosophila Dscam exhibited a striking molecular diversity, which had never been found in vertebrate Dscam (Schmucker et al., 2000). A similar diversity produced by the alternative splicing was also shown in mosquito A. gambiae (Dong et al., 2006), wax moth B. mori, and beetle T. castaneum (Watson et al., 2005). Drosophila Dscam can potentially generate more than 38,000 isoforms through alternative splicing, and these isoforms are both spatially and temporally regulated (Schmucker et al., 2000; Watson et al., 2005). Tremendous diversity of Drosophila Dscams indicates the intriguing possibility for them to recognize a variety of pathogens. In mosquito A. gambiae, AgDscam produced pathogen challenge-specific splice form repertoires as a response to different infection agents. Transient silencing of AgDscam compromises the mosquito's resistance to infections with bacteria (even for the opportunistic microbes) and the malaria parasite Plasmodium (Dong et al., 2006). Interestingly, certain spliced forms of Drosophila Dscam were found to bind to E. coli while others did not. Moreover, once some specific isoforms of Dscam was knocked down by RNAi, the phagocytosis rate to E. coli was reduced by 30%. (Watson et al., 2005). This data arise the hypothesis that different spliced isoforms of Dscam can detect specific pathogenic epitopes and lead to phagocytosis by the hemocytes. However, no evidence to date has shown Dscams bind pathogens in vivo and that the hemocytes in Droso- phila selectively expressed specific Dscam isoforms in response to different pathogens. In the crustacean, Daphnia Dscam can generate up to 13,000 different transcripts by alternative splicing of variable exons. According to genealogy of the Dscam gene family from vertebrates and invertebrates, it is confirmed that Dscam evolved from a non-diversified form before the evolutional split of insects and crustaceans (Brites et al., 2008). A first shrimp LvDscam from L. vannamei was recently isolated and characterized. The LvDscam protein had an extracellular domain but lacked the expected transmembrane domain and cytoplasmic tail, which is different from all other known Dscams.

29 However, no functional studies were performed (Chou et al., 2009). In P. leniusculus, PlDscams have been indentified. Certain alternative spliced isoforms of PlDscam were specifically up-regulated by different bacterial species. They bound to different bacteria such as S. aureus and Pseudomonas aeruginosa. It was shown that PlDscams were directly involved in immune defence (Watthanasurorot et al., 2011a).

Toll-like receptors (TLRs) Toll receptors were first identified in a pathway required for the dorso-vent- ral axis of the Drosophila embryo, and later in its importance for AMPs synthesis (Lemaitre and Hoffmann, 2007). Due to homology between the intracellular regions of the mammalian interleukin-1 receptor (IL-1R) and the Drosophila protein Toll, mammalian domain is named as Toll-IL1-Receptor (TIR) domain and the proteins as TLRs. TLRs are membrane-spanning proteins with extracellular leucine-rich repeat (LRR) motifs and an intracellular TIR domain. There are eleven TLRs identified in mammals, and they sense different PAMPs with or without the help of other cofactors/ligands (Barton et al., 2002; Pal and Wu, 2009). For instance, TLR3 appear to act alone as homodimers, which bind to double-stranded RNA and activate of NF-B (Alexopoulou et al., 2001). But TLR2 needs to form heterodimers with several (but not all) other TLRs, including TLR1 and TLR6. The TLR 1/2 het- erodimer is specific for triacylated lipopeptides, whereas the TLR 2/6 com- bination binds diacylated lipopeptides (Ozinsky et al., 2000). Recent genomic data from diverse organisms suggest that putative Toll receptors/TLRs exist in animal phyla, and are present in most eumetazoans, with the exception of platyhelminthes (Leulier et al., 2008). In arthropods, the number of putative Toll genes is around 10, but in the sea urchin S. purpuratus the count is a remarkable 222 (Cerenius et al., 2010b; Sodergren et al., 2006), which indicates the requirement for further detailed studies of Toll receptors from other invertebrates than insects. So far, functional studies of Toll are carried out in a small number of insects, especially in Drosophila (Leulier and Lemaitre, 2008). Unlike vertebrate TLRs, insect Toll does not function directly as a pattern recognition receptor, but plays a central role in mediating the immune defense to different types of infections. Toll receptor activation in Drosophila, M. sexta and T. molitor is mediated by the binding of the ligand/cytokine Spätzle to the receptor Toll (Lemaitre and Hoffmann, 2007). Ten TLRs have been characterized in D. melanogaster, but only one has been shown to play a role in defense against Gram-positive bacteria, fungi and virus (Zambon et al., 2005). In the case of Gram-positive bacteria and fungi, the recognition of PGRP-SA and GNBP1 to Lys-type PGNs or GNBP3 to fungal -1,3-glucans, triggers a serine proteinase cascade, which cleaves the Drosophila pro-Spätzle before it binds to Toll and results in AMP

30 production (Lemaitre et al., 2003). Most Drosophila Tolls are not up-regulated upon infection, but a recent study showed that the Toll pathway is required for efficient inhibition of Drosophila X virus (DXV) replication in the fruit fly (Zambon et al., 2005). Moveover, some Drosophila TLRs like the Toll-2 and Toll-8 are involved in developmental and neural functions (Eldon et al., 1994; Seppo et al., 2003). As an evolutionary ancient family of PRPs, TLRs may also be important for innate immune responses in crustaeans. Up to now, four TLRs have been identified in shrimp P. monodon, L. vannamei, F. chinensis, and M. japonicus (Arts et al. 2007; Mekata et al., 2008; Yang et al., 2007b; Yang et al., 2008). For example, expression of MjToll in M. japonicus was significantly increased by PGN stimulation but not by other microbial components (Mekata et al., 2008). LvToll was not responsive to dsRNA in L. vannamei. Silencing of LvToll did not increase the shrimp susceptibility to viral infection. In contrast, LvToll dsRNA injection activated an antiviral response, suggesting that LvToll is not a dsRNA receptor and plays no role in dsRNA- induced antiviral immunity (Labreuche et al., 2009). A recent result showed that Fc-Spz (Spätzle homologue-Toll receptor ligand) was up-regulated in shrimp F. chinensis injected with V. anguillarum or WSSV. Up-regulation of crustin 2 was found when recombinant Fc-Spz was injected into crayfish (Procambarus clarkii) (Shi et al., 2009). In conclusion, function of crustacean TLRs is relatively unknown and comes only from expression analysis. Therefore, the mechanism for how these TLRs are regulated in microbial challenge is still unknown, and the detailed information about how Toll functions in the crustacean immune system still needs further rigorous tests since more TLRs may exist in a single crustacean.

Thioester-containing proteins (TEPs) Thioester-containing proteins belong to a family of proteins that are characterized by a unique intrachain -cysteinyl--glutamyl thioester bond (Chu and Pizzo, 1994). This protein family includes the universal protease inhibit- or alpha-2-macroglobulin (A2M), complement components C3, C4, C5, gly- coprotein CD109, pregnancy zone protein (PZP), C3 and PZP-like A2M domain-containing 8 (CPAMD8), and a set of TEPs found only in some invertebrates (Blandin and Levashina, 2004; Fujito et al., 2010). The thioester bond is present in the inside of TEP molecules, and only small nucleophiles such as methylamine, hydrazine and ammonia can access and react with it (Fujito et al., 2010). It is necessary to expose the thioester bond to activate the TEP molecules, which is achieved through a proteolytic cleavage either by attacking proteases for A2Ms or by a specific complement protease com-

31 plex for C3 (Blandin and Levashina, 2004). The thioester bond mediates the covalent and non-covalent cross-linking to attacking proteases (A2Ms), or the attachment of TEPs to activating non-self and self surfaces (complement factors) (Blandin and Levashina, 2004). Upon cleavage of A2Ms, a large conformational change occurs, and this results in the entrapment of proteinase, which is accompanied by cleavage of thioester, and exposure to the carboxy terminal receptor region. In the case of complement factors, the proteolytic activation generates two products, a small anaphylatoxin fragment and a larger fragment that can combine covalently with the surface of a pathogen through the hydrolysis of the thioester. The small fragments recruit macrophages to the site of infection, and the large one lead to the phagocytosis or lysis via membrane attack complex, respectively. Taken together, the invading pathogens and non-self attacking proteases are the main targets for the covalent binding of the C3 and A2M. Members of the A2M subfamily are abundant components of plasma in arthropods and mammals (Armstrong et al., 1996; Sottrup-Jensen et al., 1989b). The A2M genes have been cloned and characterized in different vertebrates and invertebrates, including humans, rat, mouse, pig, chicken, frog, carp and scallop (Ma et al., 2010). Recently, some A2Ms have been characterized in arthropods, for instance, horseshoe crab Limulus polyphemus (Enghlid et al., 1990), mud crab Scylla serrata (Vaseeharan et al., 2007), and different shrimps like Chinese shrimp Fenneropen aeuschinesis (Ma et al., 2010). Immuno-stimulation assay showed that the PGN, LPS, or bacteria including A. hydrophila, Vibrio alginolyticus, Lactococcus garviae, or even virus such as WSSV infections could induce A2M mRNA expression (Ho et al., 2009; Ma et al., 2010; Qin et al., 2010). The A2M contains three functional domains, a thioester domain, a bait region and a receptor binding domain (RBD). The RBD of A2M is located at the C-terminal and is exposed after the proteolytic activation of A2M (violet color domain in Fig. 1). Low density lipoprotein receptor-related protein (LRP), a receptor distributed in hepatocytes, macrophages and fibroblasts, binds to the exposed RBD, which subsequently leads to the clearance of the complex through circulation in vertebrates (Samonte et al., 2002; Sottrup- Jensen et al., 1989a), or through granular amebocytes in arthropods (Melchi- or et al., 1995). Alternative splicing exists in TEPs, and may serve to extend the repertoire of inhibited proteases, and this may be a novel mechanism for recognition of non-self structural patterns in the absence of the large repertoire of receptors of the adaptive immune response in vertebrates (Blandin and Levashina, 2004). In A2M, a bait region, a short stretch of about forty amino acid residues and located upstream of the thioester motif, showed alternative splicing. This region exposed on the surface of A2M molecule contains many cleavage sites for various proteinases, and therefore is apt to be hydrolytically attacked (Ma et al., 2010). D. melanogaster TEP2 has five spliced isoforms because of the alternative splicing exons similar to anaphyl-

32 atoxin (ANATO) in complement C3 or bait region in A2M (Lagueux et al., 2000). C3 proteins are mainly found in vertebrates, but C3 genes have been identified in some protostomes, the ecdysozoan horseshoe crab (Zhu et al., 2005), the lophotrochozoan squid (Castillo et al., 2009) and the mollusk clam (Prado-Alvarez et al., 2009). C3 is also found in some deuterostome invertebrates such as sea urchin (Al-Sharif et al., 1998), ascidian (Nonaka et al., 1999), and sea anemone (Kimura et al., 2009). On the contrary, no complement component genes but a set of TEPs were found in insects D. melanogaster and A. gamiae. There are 6 and 19 TEP homologues in D. melanogaster and A. gamiae, respectively, and some of them were demon- strated to function in the immune defense as pattern recognition proteins (PRPs) (Christophides et al., 2002; Lagueux et al., 2000; Levashina et al., 2001). In mosquito A. gambiae, TEP1 is one of the best-characterized thioester- containing proteins, and it bears structural and functional similarities to the mammalian complement factor C3. TEP1 lacks two domains: the ANATO domain and the C-terminal C345C domain, suggesting different mechanisms exist in TEP1 activation (Fig. 1) (Baxter et al., 2007). TEP1 was secreted by hemocytes as a monomer, and played a crucial role in phagocytosis of bacteria (Levashina et al., 2001). After the TEP1 was cleaved by an unidentified protease, the C-terminal part bound to the surface of Gram-negative or Gram-positive bacteria with its intact thoester site (Levashina et al., 2001). Mosquito TEP1 also bound to and mediated killing of midgut stages of the rodent malaria parasite Plasmodium berghei. Silencing of TEP1 completely abolished refractoriness in refractory strain of adult mosquitoes, and increased the number of developing parasites in susceptible strain, suggesting that TEP1 is a determinant of malaria vectorial capacity in mosquito, and affects the mosquito’s capacity to transmit malaria (Blandin et al., 2004). In addition, two leucine-rich repeat proteins (LRRs) form a complex that is required for maintaining mature TEP1 in circulation, and this complex inhibits TEP1 activation in the absence of an infectious agent (Fraiture et al., 2009). In D. melanogaster, expression of Tep1, 2 and 4 were up-regulated in fat body upon bacterial challenge. The expression of Teps is JAK/STAT dependent, since gain of function JAK/STAT components lead to concomitant Teps expression (Lagueux et al., 2000). Drosophila S2 cell was used as a model system to study the phagocytosis of Candida albicans, the major fungal pathogen of humans, by screening an RNAi library. Macroglobulin complement related, also known as TEP6 (Blandin and Levashina, 2004), is shown to be secreted by Drosophila S2 cells. TEP6 binds specifically to the surface of C. albicans, and promotes the subsequent phagocytosis of S2 cells of this fungus (Stroschein-Stevenson et al., 2006). This study also shown that TEP2 is required for efficient phagocytosis of E. coli (but not for C. albicans or S.

33 aureus), while TEP3 is required for the efficient phagocytosis of S. aureus (but not C. albicans or E. coli). Taken together, all data suggests that this family of fly proteins distinguishes different pathogens for subsequent phagocytosis (Stroschein-Stevenson et al., 2006). However, Bou Aoun et al., (2010) showed that Drosophila TEPs (TEP 2, 3, 4) are not strictly required in the body cavity to fight several bacterial and fungal infections, since the flies with single, double or triple mutants of Tep 2, 3, 4 did not have higher death rate in comparison with control group. Therefore, these controversial results encourage further studies to clarify the role of TEPs in D. melanogaster, A. gambiae and other invertebrates. Three thioester-containing proteins have been cloned and characterized in P. leniusculus. Sequence comparison by using these three crayfish TEPs and various TEPs from other organisms indicated that crayfish TEPs could be classified into two subfamilies: two belong to A2Ms (Pl-A2M1, Pl-A2M2) and one belongs to insect TEP-like gene (Pl-TEP). Schematic drawing of crayfish TEPs and representative TEPs are shown in Fig. 1. A typical ANATO domain and C345C domain in C3 are not observed in A2Ms and insect TEPs. The thioester motif (GCGEQ) is located at similar corresponding regions of all aligned TEPs (Fig. 1). In many TEPs, a histidine (His) residue about 100 amino acids downstream of the thioester motif affects binding specificity of the TEPs. This histidine is found in Pl-A2M1, Pl-A2M2, and this histidine is replaced by an asparagine (Asn) in Pl-TEP. In many vertebrate A2Ms, the conserved histidine site is replaced by an Asn. TEPs are glycoproteins with variant N-linked glycosylation sites. Like vertebrare A2Ms and complement factors, two crayfish A2Ms are cysteine abundant proteins and the cysteines are located dispersedly. Crayfish Pl-TEP only contains eight cysteines and a cluster of six cysteine residues is located at the C-terminus of Pl-TEP, which form a unique structure shared with mosquito TEP1 (Fig. 1).

34 Figure 1. Multiple alignments of crayfish Pl-A2M1, Pl-A2M2 and Pl-TEP with other representative thioester-containing proteins by schematic drawing. P. leniusculus Pl-A2M1 (HQ596363), Pl-A2M2 (HQ596364) and Pl-TEP (HQ596368), mosquito A. gambiae TEP1 (AAG00600), Homo sapiens A2M (P01023) and C3 (NP_000055) are used for schematic drawing. The different fillings in the rectangles correspond to different domains or regions. Red stars point to the thioester sites. Black stars are putative N-glycosylation sites. Black thin vertical bars in the figure stand for cysteines. Black thick vertical bars are the conserved residues about 100 amino acids downstream of thioester sites. Blue thick vertical bars indicate the signal peptides.

35 Objectives

The work presented in this thesis was aimed to find new proteins in order to further understand the mechanisms of hematopoiesis, regulation of proPO -system and pattern recognition in the freshwater crayfish P. leniusculus.

Astakine, a hematopoietic cytokine/hematopoietic growth factor, has been found to stimulate proliferation of Hpt cells. To obtain tools for more detailed investigations about the connection between SGCs, GCs and precursor cells in Hpt of P. len i us cul u s, 2-DE followed by MS analysis was performed to find proteins specific for different hemocyte lineages. Different marker proteins for Hpt cells, SGCs and GCs were identified (Paper I).

The toxic quinone substances and intermediates formed during melanization can kill invading pathogens, but are also harmful to the host. Therefore, this reaction has to be tightly regulated. Recently, a new negative regulator of melanization was identified from a beetle T. molitor. To isolate similar proteins from crayfish, western blot and 2-DE coupled with MS analysis were chosen. The function of this crayfish melanization inhibiting protein was analyzed (Paper II).

To reveal the mechanism by which PGN induces activation of the proPO- system in P. len i us cul u s, different forms of Lys-type PGN were used to pull down putative PGRPs from plasma or a hemocyte lysate supernatant (HLS). The PGN-binding proteins were separated and analyzed with MS, and the roles of these proteins were investigated further (Paper III).

To isolate PAMP-binding molecules from the plasma or HLS of crayfish, different bacteria including S. aureus were used as an affinity matrix to pull down bacterial binding proteins, followed by the analysis with 2-DE and MS. Further studies were performed to explore the function of these pattern recognition proteins (Paper IV).

36 Results and Discussion

Hemocyte lineage marker proteins in P. l e n i usc u lu s (Paper I) In crustaceans and other invertebrates, the circluating hemocytes play an important role in the protection of the animal against invading microorganisms. Therefore, hematopoiesis, the life-long production of hemocytes (blood cells), is essential for antimicrobial immune response of crustaceans. The number of free hemocytes can vary a lot between different individuals and decrease dramatically during an infection (Lorenzo et al., 1999; Persson et al., 1987; Söderhäll et al., 2001). Thus new hemocytes need to be produced from Hpt. Three morphologically different classes of hemocytes, the HCs, SGCs and GCs are observed in the hemolymph of crustaceans. We have carried out 2-DE followed by MS analysis with Hpt cells, SGCs and GCs to identify proteins associated with development of different hemocyte types in P. leniusculus. After cDNA cloning using degenerate primers designed from the MS result, two specific proteins, one two domain Kazal type proteinase inhibitor (KPI) and one extracellular superoxide dismutase (SOD), were found in SGCs and GCs, respectively. A protein spot from Hpt cells was also sequenced and cloned, but the transcription of this hypothetic- al protein was found in SGC too. However, by searching an expressed sequence tags (EST)-library of Hpt cells, the proliferating cell nuclear antigen (PCNA) was detected as a unique marker for Hpt cells. We determined the transcription of the corresponding genes, and found that PCNA, KPI and SOD were specifically expressed in Hpt, SGC and GC, respectively. Since transcription of PCNA in Hpt varied between animals, we decided to analyze expression of this gene after challenge with microbial polysaccharides (laminarin or LPS) to confirm whether the cell cycle (mediated by PCNA expression) is stimulated by these elicitors. Although the results of the injections are variable, the transcript levels of PCNA and SOD always increased in the Hpt cells, whereas the KPI transcript never was present in Hpt regardless of any challenge. Moreover, four hour after injection with crude LPS, the KPI protein increased in plasma, but this increase was not the result of increased transcription since KPI expression was not significantly changed after LPS injection. We also performed a western blot experiment using cell lysates from separated Hpt, SGC or GC, and found that the SOD protein could only be detected in GC, which confirmed that SOD could be used as a marker protein for GC. To determine if silencing of PCNA in cultured Hpt cells could affect differentiation measured by increased transcription, we performed RNAi experiments. The results showed that there is no appearance of SOD or KPI, al-

37 though PCNA was significantly silenced (at least 60%) in the Hpt cells, indicating that differentiation was not induced by PCNA silencing. But when comparing the morphology of the silenced Hpt cells with the controls, a sub- stantial decrease in cell attachment and spreading was seen (15 ± 6 % spread cells compare to 40 ± 3% in the GFP dsRNA treated), which suggests the importance of PCNA in establishing cell adhesion and spreading. These marker proteins (PCNA, KPI and SOD) will help to perform more detailed studies in future to understand the connection between SGCs, GCs and precursor cells in Hpt, and also the role of astakine (ast) as regulator of this process (Söderhäll et al., 2005). Accordingly, Lin et al., (2010) showed that ast1 stimulated differentiation of Hpt cells along the SGC lineage, since KPI expression is induced. Moreover, the presence of ast1 is needed to prevent apoptosis of Hpt cells (Lin et al., 2011), while ast2 seems to play a role in maturation of GCs (Lin et al., 2010).

A novel protein acts as a negative regulator of proPO activation and melanization in P. leniusculus (Paper II) The melanization reaction is an important innate immune response of invertebrates. In arthropods and most other invertebrates melanin synthesis is achieved by the proPO-system, a proteolytic cascade similar to vertebrate complement (Söderhäll and Cerenius, 1998; Cerenius et al., 2008). Active PO oxidizes o-diphenols into quinones that are toxic to microorganisms. This process is followed by several intermediate steps which result in formation of melanin to restrict the spreading of microorganisms within the host (Cerenius et al., 2008; Söderhäll and Cerenius, 1998). Since toxic quinone and intermediates are harmful to the host, it is necessary to tightly control the melanization reaction. Recently, Zhao et al. (2005) discovered a novel 43 kDa protein from the hemolymph of the beetle T. molitor (named Tm-MIP) which acts as a negative regulator of melanin synthesis. The target of this protein is presently unknown. Tm-MIP was consumed during melanization, and no similarity was found between Tm-MIP and any other known proteins (Zhao et al., 2005). Interestingly, the Tm-MIP-antibody recognized a protein in crayfish plasma, and this crayfish 43 kDa protein also was involved in regulating the proPO- system and melanization. Therefore, this protein was named as Pl-MIP. A sequence analysis shows that Pl-MIP is totally different from the Tm-MIP, and contains a C-terminal domain similar to vertebrate fibrinogens. The fibrinogen-related domain (FReD) is the substrate-recognition domain of vertebrate L-ficolins known as activators of vertebrate complement. However, Pl-MIP did not show any hemagglutinating activity as is common for vertebrate

38 ficolins. The structural similarities of Pl-MIP with ficolins indicate interesting parallels in the regulation between proteolytic cascades involved in defence in vertebrates and invertebrates. The similar antigenicity of Tm-MIP and Pl-MIP is most likely due to the Asp-rich regions that are common to both proteins. Tm-MIP contains a region in its central part with 11 Asp residues, whereas Pl-MIP has Asp-rich region containing 5 Asp residues with four Asp residues in one row, which is probably recognized by the Tm- MIP antibody. Pl-MIP was expressed at fairly low levels in hepatopancreas and eyestalk, whereas high expression occurred in nerve, heart and intestine, while the expression was absent in hemocytes and Hpt. Pl-MIP was expressed as a recombinant protein in a baculovirus vector expression system and was purified to homogeneity. When recombinant Pl- MIP (Pl-rMIP) was added to HLS containing an inactive proPO-system, Pl- rMIP could inhibit LPS-PGN or laminarin induced PO-activity in a dose dependent manner, whereas no such inhibition was achieved when the proPO was activated prior to incubation with the Pl-rMIP. To examine whether Pl- MIP disappeared when the proPO-system was activated during melanization of the hemolymph, we induced activation of proPO by LPS-PGN in the presence or absence of the PO inhibitor PTU after preincubation with Pl-rMIP. As we expected, it did not result in oxidation of L-Dopa and Pl-rMIP was unaffected in the presence of PTU. On the contrary, when PTU was absent from the reaction mixture, oxidation of L-Dopa proceeded and Pl-rMIP disappeared completely. Our results suggest that Pl-MIP functions by two different mechanisms: One is to inhibit proPO activation partially and the other is to block or delay melanin formation, once proPO is activated. An Asp-rich region is located close at the putative Ca2+-binding sites in the FReD. To investigate whether the antigenic Asp-rich region is important for the function of Pl-MIP, site-directed mutagenesis was perfomed by deleting the four-Asp amino acids in the recombinant protein. As anticipated the mutant form of MIP lacking four-Asp amino acids lost its original function, which implic- ates that it is involved in regulating the proPO activating cascade. The sequence similarity between Pl-MIP and the FReD of human L-ficolin was used to build a homology model of Pl-MIP 3-D structure. The model suggests that Pl-MIP molecule is stabilized by two cysteine bridges. The binding site for Ca2+ ions as described in the L-ficolin structure and similar to Ta- chylectin 5A is well conserved in Pl-MIP. In conclusion, a FReD containing protein was isolated and shown to be a negative regulator of proPO-system activation and melanin synthesis.

39 Peptidoglycan activation of the proPO-system without a PGRP (Paper III) Recognition of microbial polysaccharides by PRPs triggers the proPO-system, and results in melanin synthesis and deposition on the surface of invading pathogens. As PAMPs, PGN and its derivatives induces AMPs through the Toll signaling or the Imd signaling pathways, and it can also trigger activation of the melanization cascade in insects (Kan et al., 2008; Roh et al., 2009). PGRPs have been reported in insects, molluscs, echinoderms, and vertebrates. So far, no PGRP with pattern recognition properties has been described in any crustacean. Therefore, it is of interest to reveal the mechanism by which PGN induces activation of the proPO-system in crustacean. High PO activity was induced by PGN as well as by curdlan, suggesting that soluble Lys-type PGN is an inducer of the proPO-system in crayfish and hereby as efficient as the -1,3-glucan, curdlan, at the same concentration. These results also indicate that a putative PGRP is likely to be present in crayfish. In order to reveal the mechanism by which PGN induces activation of the proPO-system in P. leniusculus, different forms of PGN (partially lysozyme digested or TCA-treated insoluble Lys-type PGN and soluble Lys- type PGN) were used to pull down proteins from plasma or HLS of crayfish. To achieve the amino acid sequences of the binding proteins, we performed RACE-PCR with degenerate primers designed from the MS sequence. However, no PGRP-like protein was detected by “pull-down” assays in this study. Instead, a novel serine proteinase homologue, Pl-SPH2, was found and isolated as a 30 kDa protein from hemocytes, by its binding property to different forms of Lys-type PGN, and two isoforms of Pl-SPH2 were isolated based on the sequencing results. Two other proteins, Pl-SPH1 and LGBP were also found in several different PGN-binding assays. The clip domain containing non-catalytic SPHs are important for the activation/regulation of the proPO-system in insects (Cerenius et al., 2008). Crayfish Pl- SPH2 is similar to Pl-MasI, Pl-SPH1, shrimp SPHs and insect SPHs, and structurally, Pl-SPH2 exhibits features of the clip domain family of SPHs with an amino-terminal disulfide knotted clip domain and a SP-like domain in the carboxyl terminus. The putative catalytic domain, from Asn146 to Ser- 424, is characteristic of trypsin-like serine proteinases. The serine proteinase domain contains the conserved His205 and Asp255 except for Ser356 residue which is replaced by Glycine, indicating that this protein is a non-catalytic serine proteinase. A 30 kDa band was produced from the intact recombinant Pl-SPH2 after trypsin cleavage, suggesting that a putative serine proteinase recognition site exists. In crustaceans, several SPHs have been identified with different biological properties such as cell adhesion activity (Huang et al., 2000; Lee and Söderhäll, 2001; Lin et al., 2006 ), binding to virus (Sriphaijit et al., 2007), and some with yet unknown function (Sricharoen et al., 2005; Rattanachai et al., 2005; Amparyup et al., 2007).

40 PGRPs can be receptors and scavengers to PGN. Recent studies show that PGRP-LB can cleave DAP-PGN and PGRP-SC is active as amidase using both DAP-type PGN and Lys-type PGN as substrates (Mellroth et al., 2003; Zaidman-Remy et al., 2006). PGRP-SC1B as amidase is similar to T7 lysozyme, and a sequence comparison with T7 lysozyme shows that five amino acid residues which are required for the enzymatic activity are conserved in PGRP-SC1B, although Lys-128 in T7 lysozyme is replaced by threonine, which does not significantly block amidase activity (Mellroth et al., 2003). PGRP-SA and PGRP-LCx, act as PGN receptors, both have a serine substitution in the position corresponding to the Cys-130 zinc ligand in T7 lysozyme. This replacement modifies one of the three potential zinc ligands and makes these proteins as inactive enzymes. Thus it is suggested that this serine substitution is a prerequisite for making a PGRP active as a pattern recognition receptor (Lemaitre and Hoffmann, 2007). By blasting available crustacean EST-databases with Drosophila PGRP-SA protein sequence, we found more than ten crustacean candidate PGRP proteins, but all of these proteins contain cysteine in the position corresponding to the Cys-130 in T7 lysozyme. This indicates that these PGRP-like proteins in crustaceans most likely function as amidases and possibly to down-regulate the immune response induced by PGN or as digestive enzyme (Bischoff et al., 2006; Zaid- man-Remy et al., 2006). Recently, the complete genome sequence of the crustacean D. pulex was reported, but only PGRPs with enzymatic properties have so far been detected (McTaggart et al., 2009). Moreover, in a recent genomic analysis of an insect, pea aphid (A. pisum) no PGRP gene was detected, and this result points to a diversity of immune responses among insect species (Gerardo et al., 2010). Therefore, these results indicate that PGRPs acting as receptors for PGN may be absent in crustaceans. Pl-SPH1, Pl-SPH2 and LGBP were found in the “pull-down" assays. In order to elucidate the roles of these three proteins in the Lys-type PGN dependent proPO activation in crayfish, we designed dsRNA specific to Pl- SPH1, Pl-SPH2 or LGBP in order to carry out RNA interference in the Hpt cell culture, and the silencing efficiency was more than 80%. RNAi was followed by determining the degree of proPO activation by adding soluble Lys- type PGN to the Hpt cell culture. Interestingly, the results showed that the PO activity induced by soluble Lys-type PGN was significantly lower in Pl- SPH1, Pl-SPH2 or LGBP silenced cells when they were compared to the control cells treated with GFP dsRNA or without any dsRNA treatment. In summary, the results show that Lys-type PGN is a trigger of proPO-system activation in a crustacean, and that two Pl-SPHs are involved in this activation possibly by forming a complex with LGBP and possibly without a PGRP.

41 Two FLPs act as pattern recognition molecules for invading pathogens in P. leniusculus (Paper IV) In order to isolate PAMP-binding molecules, different bacteria including S. aureus were used as an affinity matrix to find binding proteins from crayfish plasma or HLS according to a method previously described by Lee and Söderhäll (2001). Two 57 kDa proteins were found to bind to S. aureus. These proteins generated two strings of protein spots in 2-DE, maybe because of modifications after translation. The MS result further confirms that these two strings of spots only constitute two proteins. RACE-PCR and degenerate primers designed from the MS sequence were used to determine the amino acid sequence of these 57 kDa proteins. The sequences show that they are FReD containing proteins with some similarity to ficolins. Therefore, we named these proteins as ficolin-like protein 1 (FLP1) and FLP2. The FLPs have a FReD in their C-terminal and a repeat region in their N-terminal region with structural similarities to the collagen-like domain of vertebrate ficolins and MBLs. A phylogenetic analysis revealed that FLP1 and FLP2 were clustered separately from the other FReD containing proteins in this tree. An analysis of tissue distribution further showed that the FLPs were mainly expressed in Hpt and in the hepatopancreas. Recombinant FLPs (rFLPs) bound to A. hydrophila, E. coli and S. aureus in a bacterial adsorption assay. The rFLPs agglutinated Gram-negative bacteria E. coli and A. hydrophila in the presence of Ca2+. To investigate whether FLP1 and FLP2 function during a bacterial infection, rFLPs were used to coat S. aureus and A. hydrophila and then the coated bacteria were separately injected into crayfish. Then hemolymph was bled from injected crayfish and the number of bacteria was determined. A. hydrophila is a highly pathogenic bacterium to crayfish and kills crayfish within 6-24 hours. Usually, A. hydrophila is not cleared but instead multiply rapidly before killing the animal. Our results showed that the bacterial number of BSA coated A. hydrophila increased nearly three folds between 1 and 4 h, while the bacterial number of rFLP1 coated A. hydrophila group increased less than one fold. This indicates that FLP1 may be important for the clearance and removal of A. hydrophila from hemolymph. The bacterial number is still increasing, but at a significantly lower rate. The clearance assays were performed with FLP1 and FLP2 with similar results. S. aureus is not a pathogenic bacterium for P. leniusculus, and it can be cleared rapidly within hours. When S. aureus was coated with FLP1, the number of bacteria remaining in the crayfish hemocoel after 1 h was higher than in the group of animals injected with BSA coated S. aureus. However after 1 h, the rate of clearance was similar in both groups. To reveal whether this effect was due to a general inhibition of growth, the rFLPs were tested for their effect on bacterial growth in vitro. However, none of the bacterial species used in this study were affected by the addition of rFLPs. That the initial clearance rate is slower after FLPs coating suggests that S. aureus might be protected by the proteins against the

42 immune system of crayfish, whilst FLPs may help crayfish to clear the viru- lent bacterium A. hydrophila. When crayfish Hpt cells are treated with bacteria, the cells lyse as a response to Gram-negative bacteria, while Gram-positive bacteria have no or a very small effect on these cells. When BSA coated E. coli was added to Hpt cells, more than 70% of the cells lysed within ten hours, while the death rate decreased to below 50 % if the Hpt cells were incubated with the same bacteria coated with FLPs. The death rate of the Hpt cells that are affected with BSA or FLP coated S.aureus are low (around 6 %), which is similar to that of the control Hpt cells without any treatment. Similar results were obtained when other Gram-positive and Gram-negative bacteria were used. This further corroborated previous results that FLP helped to remove Gram-negative bacteria from crayfish hemolymph. Taken together, these results suggest that FLPs may function as PRPs in the immune response of crayfish.

43 Concluding remarks

There are various strategies for invertebrates to combat different invading pathogens. In the past decades, progress has been made in the knowledge of different aspects of crustacean immunity, especially some highly conserved immune response pathways have been discovered (eg. proPO-system and melanization, AMP defence and coagulation system). A technique for Hpt cell cultures and RNAi were developed in P. leniusculus, which facilitates studies about hematopoiesis in crustaceans (Liu and Söderhäll, 2007; Söder- häll et al., 2003 and 2005). Due to lack of genomic data, protein identifica- tions in crustaceans rely on MS analysis and RACE-PCR. In this thesis, these methods were used to identify three hemocyte lineage marker proteis, and these have later been used to increase our understanding of the mechanism of crayfish hematopoiesis (Paper I). A new negative regulator of melanization induced by proPO-system was identified by western blot using a het- erologous antibody together with 2-DE and MS analysis. (Paper II). Further some proteins involved in the recognition of Lys-type PGN or Gram-positive and Gram-negative bacteria were also found in P. l en i us cu lu s (Paper III and Paper IV). During pathogenic infection, crayfish hemocytes are consumed, and have to be replaced by new hemocytes released from Hpt, a separate tissue where synthesis of new hemocytes occurs (Jiravanichpaisal et al., 2006a; Söderhäll et al., 2003). The proposed hemocyte lineages have earlier mainly been based on morphological characters (Chaga et al., 1995; Martin et al., 1993; van de Braak et al., 2002b). We have now identified three marker proteins (PCNA, KPI, SOD) for Hpt cells, SGCs and GCs of P. leniusculus, respectively (Paper I), which also suggested that SGCs and GCs are differentiated as separated lineages. So far, these crayfish lineage marker proteins have been used to determine the function of a new group of cytokines, the astakines, and the combined study showed that ast1 and ast2 play different roles in proliferation and differentiation of crayfish pro-hemocyte (Lin et al., 2010). Also a crustacean hematopoiesis factor (CHF), a downstream gene of ast1, was studied in vivo and in vitro in the Hpt cells of crayfish (Lin et al., 2011). Once formed, the hemocytes are released into the circulation and function in different innate immune reactions. Recognition of microbial polysaccharide by PRPs triggers the proPO-system and results in melanin synthesis. The melanin/intermediate deposits on the surface of invading pathogens helps to kill them, but this process needs

44 to be tightly controlled (Söderhäll et al., 1982). A previous study showed that a proteinase inhibitor, pacifastin, regulated the activation of the proPO-system by efficient inhibiting the crayfish ppA (Liang et al., 1997). In this study, we identified a novel protein Pl-MIP that acts as a negative regulator of proPO activation and melanization (Paper II). Crayfish MIP and Tenebrio MIP do not affect phenoloxidase activity in itself, but instead they interfere with the melanization reaction from quinone compounds to melanin (Zhao et al., 2005). Results suggest that the Pl-MIP may keep the proPO-system in a non-active form until certain inducers such as PAMPs or pathogens are present. Moreover, a similar shrimp protein (PmMIP) has been detected in P. monodon (Angthong et al., 2010). Thus we have shown that the activation and acitivity of proPO and PO, respectively, is tightly controlled in arthropods. Pl-MIP contains a FReD domain similar to ficolins, but did not show any hemagglutinating activity as is common for the vertebrate ficolins. Two new bacteria binding ficolin-like proteins (FLPs) were also identified in crayfish plasma (Paper IV). These FLPs both have a FReD and a collagen-like domain similar to vertebrate ficolins, which are involved in the clearance of non-self pathogens through the lectin pathway of complement system. Re- combinant FLPs need Ca2+ to bind and agglutinate bacteria, and N-acetyl-D- mannosamine and N-acetyl-D-glucosamine are efficient inhibitors for the bacterial binding of FLPs. Moreover, the FLPs play a role in clearing Gram- negative bacteria, but not Gram-positive bacteria injected into the hemolymph, and they also help to protect Hpt cells when the Gram-negative bacteria were coated with FLPs. All these results suggest that FLPs may function as PRPs in crayfish. Up to now, many PRPs have been characterized in P. len i us cul u s: LGBP, GBP, Pl-MBL, Pl-FLPs, Pl-SPH1, Pl-MasI, and they are all involved in the proPO activation. In D. melanogaster and T. molitor, GNBP1 and PGRP-SA are needed to induce AMP synthesis, and Tenebrio GNBP1 is part of a complex responsible for binding to Lys-type PGN together with Tenebrio PGRP- SA resulting in proPO activation and AMP synthesis (Gobert et al., 2003; Kan et al., 2008). A similar activation pathway may exist in crustaceans. By using different forms of Lys-type PGN to “pull-down” putative PGN binding proteins from crayfish HLS and plasma (Paper III), a recognition complex containing Pl-SPH1, Pl-SPH2 and LGBP, but no PGRP homologue was identified by the binding property to PGN. No putative PGRP as receptor was detected after searching available crustacean sequence databases. The results in this thesis present marker proteins for hemocyte lineages and some innate immune proteins in a crustacean P. leniusculus, which further our understanding of the hematopoiesis, the activation of proPO-system and related immune responses.

45 Svensk sammanfattning

Ryggradslösa djur som t.ex. insekter och kräftdjur saknar antikroppar och har inget adaptivt immunförsvar med minnesfunktion utan måste klara sig med nedärvda system. De senaste decenniernas forskning kring dessa system har klarlagt hur immunsystemet har utvecklats från enklare former till vårt eget komplexa immunsystem. Många ryggradslösa djur lever i miljöer där de är omgivna av mängder av bakterier och svampar. Därför behöver djuren snabbt känna igen främmande molekyler som härrör från potentiella parasiter, och då snabbt starta effektiva försvarssystem. Det viktigaste hos förs- varssystemen hos alla ryggradslösa djur är därför förmågan att snabbt kunna reagera på främmande molekyler från exempelvis bakterier. Ett sådant system är det så kallade profenoloxidas-aktiverande systemet, som till sin funktion liknar däggdjurens komplementsystem, och aktiveras av utomordentligt små mängder mikrobiella sockermolekyler. I aktiv form medverkar detta till att försvara djuren mot farliga parasiter. Syftet med arbetet i denna avhand- ling har varit att med hjälp av proteinanalyser upptäcka flera nya proteiner som är delaktiga i kräftans immunförsvar, och att hitta verktyg för att kunna studera de olika blodkroppstypernas funktion. Blodkropparna (hemocyterna) hos kräftor som cirkulerar i hemolymfan spelar en nyckelroll i att förhindra mikrober att skada djuret. Det finns två huvudtyper av blodkroppar i den sötvattenskräfta som studerats, dels de semigranulära cellerna och dels de granulära cellerna. Båda blodkroppstyperna innehåller det profenoloxidas- aktiverande systemet. Hos kräftdjur bildas blodkroppar i en speciell blodkroppsbildande vävnad som kan plockas ut ur djuret och vi kan studera stamcellerna in vitro. En teknik för att odla dessa blodkroppsbildande celler har gjort det möjligt att studera funktionen hos flera olika immun-relaterade proteiner, bl.a genom RNA-interferensteknik. Tekniken har också möjliggjort identifieringen av en ny grupp cytokiner som kan inducera differentiering av stamcellerna och därmed kan regleringen av differentiering till mogna blodkroppar studeras. I ett första arbete identifierades tre olika proteiner, som specifikt förekom- mer i stamceller, semigranulära respektive granulära celler och som därför kan användas som markörer för differentiering av de båda blodkroppstyperna. En proteas-inhibitor som tillhör gruppen Kazal-inhibitorer men med mycket speciell struktur är unik för de semigranulära cellerna, medan ett super- oxid-dismutas (SOD) utgör en markör för de granulära blodkropparna. I sen-

46 are studier har dessa proteiner använts för att klarlägga funktionen hos en ny grupp cytokiner, de s.k. astakinerna. Det profenoloxidas-aktiverande systemet leder till melanin-bildning, och flera av de intermediärer som bildas under denna process är toxiska för såväl parasiter som för värddjuret. Därför är det mycket viktigt att detta system är noga reglerat, så att det inte aktiveras vid fel tid eller på fel plats. Vi har identifierat ett protein i plasma som innehåller en fibrinogen-liknande domän, och som fungerar som en negativ regulator för det profenoloxidas- aktiverande systemet. Proteinet, kallat Pl-MIP, kan både fördröja aktiveringen av prophenoloxidas och hindra att melanin bildas där det inte behövs. Genom att framställa rekombinant Pl-MIP och även införa förändringar i det rekombinanta proteinet, kunde vi visa att närvaro av fyra asparaginsyra molekyler i rad i fibrinogendomänen är nödvändiga för att Pl-MIP skall vara aktivt. Om Pl-MIP är frånvarande i plasma sker melanisering mycket snabbt. Det profenoloxidas-aktiverande systemet aktiveras när sockerfragment från mikroorganismer binder till igenkänningsmolekyler i plasma eller på cellytor. I insekter aktiveras systemet främst av beta-glukaner från svamp och peptidoglykaner från bakterier genom att dessa binder till ett pep- tidoglykanbindande protein, ett PGRP. PGRP med bunden peptidoglykan bil- dar sedan ett aktiverande komplex med andra proteiner, bl.a. flera serinpro- teashomologer (SPH). I kräfta aktiveras systemet av glukaner, men också av lipopolysackarider från bakterier. Vi har nu vidare visat att peptidoglykaner från Gram-poistiva bakterier kan aktivera prophenoloxidas, men att det troligen inte sker via ett PGRP. Vi kunde dock genom att använda lösligt peptidoglykan isolera ett nytt SPH som behövs för denna aktivering. Genom att söka i de genom från andra krustaceér som finns publicerade kunde vi inte i någon art finna tecken på att det finns PGRP i dessa djur. Detta visar att det finns en diversitet i den exakta mekanismen för hur aktiveringen av prophenoloxidas går till. Genom att använda bakterier som adsorberande partiklar isolerades ytterligare två tidigare okända proteiner från kräftplasma. Dessa proteiner in- nehåller liksom Pl-MIP en fibrinogen-lik domän, men dessutom en collagen- lik domän vilket gör att proteinerna till sin struktutr påminner om ryg- gradsdjurens ficolliner., och de benämndes därför “ficolin-like proteins” (FLP). Precis som ficoliner har FLP förmåga att agglutinera bakterier, och våra försök visar att de troligen har betydelse för kräftans försvar mot Gram- negativa bakterier. Dessa studier har bidragit till ytterligare förståelse av ryg- gradslösa djurs immunförsvar.

47 Acknowledgements

The work of this thesis was carried out at Evolutionary Biology Center, De- partment of Comparative Physiology at Uppsala University and was founded by Swedish Science Research Council and The Swedish Research Council Formas.

I would like to express my sincerest gratitude to everyone who has helped and supported me in various ways through the years. I especially want to thank:

My supervisor Docent Irene Söderhäll, your help and support over the past several years have been very much appreciated! Thank you for advice, en- couragement and patience of correcting my writing, for project discussions that make the work so much easier to keep going, also for the special dishes of Swedish Christmas parties.

My co-supervisor Prof. Kenneth Söderhäll, thank you for teaching me scientific thinking and writing, for the patience of correcting my writing, for giving me the opportunity to work independently, for all the constructive cri- ticism, for interesting discussions at project report and various journal clubs, also for showing me how to be a professional scientist.

Dr. Lage Cerenius, I enjoyed our lunch conversations, thanks for helpful suggestion in Lab-teaching, for reviewing and commenting my paper.

Engineer Ragnar Ajaxon, our “lab police”, for your kind help and efficient technical assistance, for the Christmas present and interesting talk about Sweden, also for taking care of the crayfish for us many years.

Prof. Olof Tottmar, thanks for your help in lab-teaching cell biology lab and preparation in animal physiology lab.

Dr. Maria Lind Karlberg, thanks for often interesting discussion, for your kind help and helpful suggestion in the lab.

Prof. Monika Schmitz, for often late lunch talks, for the snacks and cakes, and for great help in animal physiology lab.

48 Dr. Pikul Jaravanichpaisal, our regular visiting professor and good friend, for your always kind help and all the scientific and non-scientific discussions, for the tasty Thai-dishes and different cakes.

Rose-Marie Löfberg and Marianne Andersson, for efficiently taking care of all documents and financial details.

All the wonderful present and former members of the Department of Com- parative Physiology: Haipeng Liu, as a good example to follow and a good party organizer, thank you for being an excellent senior and kind help and all the suggestions. Xionghui Lin, my senior fellow from Xiamen University, Third Institute of Oceanography (State Oceanic Administration) to Uppsala University, thanks for sharing your lab experience and enriching scientific discussion, also for your always friendly help. Christoph Mentzendorf, for the nice lunch conversations and your training stories. Yanjiao Zhang, I am really impressed by your enthusiasm in life, thanks for the kind help, for the Dongbei dishes, and for the nice travel plans. Wenlin Wu, for the interesting discussion about China and Sweden. Margherita Pergolizzi, for your excellent Tilamisu cake and your stories. Susanne Trombley, for the nice conver- sation, and for being a good coagent in animal physiology lab. Chadanat Noonin, for the helps in the lab, and for the collaboration. Tobias Back- ström, for the nice talking and information about postdoc application. Apiruck Watthanasurorot, for the interesting discussion and the special Tai-style songs in the lab. Walaiporn Charoensapsri, for working together during research and writing. Puttharat Baopraserkul, for the kind helps in the lab. Seiko Johansson, for the things about Fukuoka and Japan. And also other members: Chenghua Li, Yingbo Lin, Yasuyuki Matsuda, Shibata Toshio, Sirinit Tharntada, Tipachai Vatanavicharn, Sushao Donpudsa, Adisak Prapavorarat, Markus, thanks for your kind help and interesting discussion in the lab, it is very nice to work with all of you.

My other fellow lab-teachers: John Pettersson and Sofia Hemmilä (It would be fun again to lubricate the separating funnels, rinse the small syr- inges and sucking the KCl for the electrodes).

All other friends in Uppsala, Wei Lin, Yang Yu, Jian Liu, Rongqin Ke, Geng Tian, Zhenghua Xu, Jijuan Gu, Kai Gao, Tigar, Sa Wang, Guihong Cai, Hao Guan, Zai Lian, Afu, Chengxi Shi, Jizhi Hu, Goren, David, Richard, Tobias, Robertino, Haolin Sheng, Chongyan Sun, Jing- han Zhang, Xuan Jiang, Man Chen etc. for your company and help in different ways, which make my life in Sweden more joyful.

49 Prof. Xun Xu, for your recommendation and your always supporting for my study and life. Prof. Feng Yang, my former supervisor (M.Sc.), for guidance and leading me into the field of molecular biology. Researcher Limei Xu, for helpful suggestions and encouragements.

Xuna Wu, Zaizheng Shen, Yueling Shen and Chenxi Shen, my older sister and her family; Jie Xiong and Yintao Li family, thanks for your love and support.

Last, my parents (Mashe Wu and Meixia Wang) and parents in-laws (Shuangjiu Xiong and Baoxian Yuan), for endless love and being support- ive all the time, and for trusting me with my decisions. My wife Jing Xiong, for sharing life with me, for being an understanding and encouraging com- panion. My little daughter Banglei Wu, for all the happy you bring to me.

Chenglin Wu 2011-02-18 Uppsala University, Sweden

50 References

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