Research progress on the mollusc immunity in China

Developmental and Comparative Immunology 39 (2013) 2–10

Contents lists available at SciVerse ScienceDirect

Developmental and Comparative Immunology journal homepage: www.elsevier.com/locate/dci

Review

Research progress on the mollusc immunity in China Lingling Wang a, Limei Qiu a, Zhi Zhou a,b, Linsheng Song a,⇑ a b

Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China Graduate University of Chinese Academy of Sciences, Beijing 100049, China

a r t i c l e

i n f o

Article history: Available online 31 July 2012 Keywords: Immune recognition Signal transduction Immune effector Catecholaminergic system Mollusc China

a b s t r a c t The economical and phylogenic importance of mollusc has led an increasing number of investigations giving emphasis to immune defense mechanism. This review discusses the advances in immunological study of mollusc in China, with special reference to dominant aquaculture species over the past decades. As an invertebrate group, molluscs lack adaptive immunity and consequently they have evolved sophisticated strategies of innate immunity for defense against pathogens. This review aims to present the various immunologically significant pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs), lectins, lipopolysaccharide and b-1, 3-glucan binding protein (LGBP), scavenger receptors (SRs) employed by mollucans. This work also highlights immune proteolytic cascade, TLR signaling pathway and an extensive repertoire of immune effectors including antimicrobial peptide, lysozyme, antioxidant enzyme and heat shock protein. Further, the review presents the preliminary progress made on the catecholaminergic neuroendocrine system in scallop and its immunomodulation function to throw light into neuroendocrine-immune regulatory network in lower invertebrates. Ó 2012 Elsevier Ltd. All rights reserved.

Contents 1. 2.

3. 4.

5. 6.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Immune recognition. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Toll-like receptors (TLRs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Lectins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Lipopolysaccharide and b-1,3-glucan binding protein (LGBP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Scavenger receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Other PRRs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Signal transduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Immune effectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Antimicrobial peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Lysozyme . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Antioxidant enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Heat shock protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The immunomodulation of neuroendocrine system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2 3 3 3 4 4 4 5 5 5 6 6 7 8 8 8 8

1. Introduction

⇑ Corresponding author. Address: Institute of Oceanology, Chinese Academy of Sciences, 7 Nanhai Rd., Qingdao 266071, China. Tel.: +86 532 82898552; fax: +86 532 82880645. E-mail address: [email protected] (L. Song). 0145-305X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.dci.2012.06.014

Mollusca is one among the major phyla of invertebrates and it shows great diversity inasmuch as approximately 200,000 living species are distributed in various ecosystem including terrestrial, freshwater and marine environments (Ponder and Lindberg, 2008). Significantly, majority of the mollusc are marine, constituting about 23% of all the known marine organisms. In China, 2557

L. Wang et al. / Developmental and Comparative Immunology 39 (2013) 2–10

species of Mollusca have been recorded, almost 1/8 of the whole marine creatures (Cui et al., 2006). Many marine mollusc are of commercial importance, and there have been more than 30 major species cultured in China; in 2009, the culture of mollusc including scallops, oysters, mussels, nacres, clams, abalones and snails, achieve 77.67% (10.77 million tons) of the total China’s mariculture outputs (Wang and Mu, 2010). However, in recent years, mollusc aquaculture has been facing a set back due to challenges emanating from diseases caused by pathogenic infections. This situation in turn paved the way for increased number of investigations world over to have thorough understanding on the immune mechanisms displayed by this group. The study of mollusc immune mechanism is also found to be significant on account of its unique immune evolution status. Despite the immunity in the most popular invertebrate model organisms, Drosophila and Caenorhabditis elegans, have been well characterized, our knowledge on mollusc immunity is quite meager. As an invertebrate group, molluscs lack the complexity of adaptive immunity system and they rely on innate immunity mediated by both cellular and humoral components to defense against pathogens (Loker et al., 2004). They share the conserved immune components and pathways with drosophila and nematode, such as the immune effectors and immune signal transduction cascades, etc. Significantly, recent studies has revealed the existence of specific immune priming in marine mollusc to some important pathogens; certainly this finding would pave the way for further investigations emphasizing the evolution of adaptive immunity in invertebrates (Cong et al., 2008, 2009; Hanington et al., 2010). The present review attempts to summarize the research progress so far accomplished in the field of mollusc immunity in China, mainly focusing on immune recognition, signal transduction and effector synthesis involved in cellular and humoral immunity. 2. Immune recognition The discrimination of non-self from self appears to be the initial step of immune response, generally known as immune recognition. Though molluscs lack the antigen recognition receptor, they could recognize potential dangerous substances mainly via pattern recognition receptors (PRRs). These PRRs can sense pathogen-associated molecular patterns (PAMPs) which are invariant molecular signatures in potential pathogens, as well as endogenous sources. For the past decades, various families of conserved PRRs have been identified and verified to attest their important roles in immune recognition in mollusc. This section briefly represents the mollusc PRRs such as Toll-like receptors (TLRs), lectins, LPS and b-1,3-glucan binding protein (LGBP), and scavenger receptors (SRs) so far studied in China. 2.1. Toll-like receptors (TLRs) TLRs play key roles in sensing various non-self substances and then activating immune system to build up the front-line against invading pathogens. Although TLRs are widely distributed in nearly all animal phyla (Song et al., 2011a), the reports on TLRs in mollusc are still quite meager, till date only two TLR genes have been identified from Chlamys farreri (CfToll-1) and Crassostrea gigas (CgToll1), and one TLR EST fragment in Argopecten irradians (Song et al., 2006). The mRNA expression of CfToll-1 was up-regulated by LPS stimulation in a dose-dependent manner (Qiu et al., 2007b), showing highest expression in haemocytes; it’s expression level in haemocytes increased dramatically after the challenge of bacteria Vibrio anguillarum, suggesting the ability of this receptor (CfToll1) to sense bacterial invasion and subsequent involvement in the immune response of mollusc. The further knowledge of this

3

Fig. 1. The CRD domain structure and the motif of the second Ca2+-binding site (above the domain) in the scallop C-type lectins.

receptor, by identifying novel components and elucidating diverse pattern recognition mechanism in mollusc, would enhance our comprehension of the innate immune response. 2.2. Lectins Significant investigations have been made approving the involvement of lectin in non-self recognition, agglutination, opsonization and phagocytosis in invertebrates (Matsubara and Ogawa, 2006). The following highlights current knowledge on mollusc lectins giving emphasis to the structure and function of lectins derived from the scallop in China. Scallop C-type lectins differ significantly in the organization and amino acid sequences of carbohydrate-recognition domain (CRD). To our knowledge, most of known mollusc lectins have single CRD, while multi-CRD lectins have been identified from scallop recently. For example, there are three and four CRDs in CfLec-3 and CfLec-4 from C. farreri, respectively, which are similar to the lectins found in arthropods (Fig. 1) (Liu et al., 2007; Watanabe et al., 2006). Each CRD contains four Ca2+-binding sites, and the site 2 is considered to be involved in carbohydrate-binding specificity (Zelensky and Gready, 2005). Four type motifs of Ca2+-binding site 2, EPN, EPD, QPD and YPT, have been characterized in scallop. EPN motif, which determined the mannose or similar sugar binding specificity (Weis et al., 1998), was identified in CfLec-3, CfLec-4 and CfLec-5. EPD motif, with similar function to EPN, was conserved in most scallop lectins, such as CfLec-1, CfLec-2, CfLec-3, CfLec-4 from C. farreri, as well as AiCTL-2, AiCTL-6, AiCTL-7 from A. irradians. The motif QPD, dictating the binding specificity to galactose or similar sugar (Weis et al., 1998), was only found in AiCTL1. YPT, a special motif identified in CfLec-3, has never been found in any other known C-type lectins. The contribution of motif YPT to the Ca2+-binding capacity or carbohydrate specificity still requires further investigation. Based on the preliminary picture in sequence diversity, much more attention has been paid to the greatly diverse functions of C-type lectin identified from mollusc. Lectins with similar carbohydrate-binding specificity may distinguish different invading microbes in humoral immune system. For example, CfLec-1, CfLec-2, Cflec-3 and CfLec-5 from C. farreri, agglutinated Escherichia coli, Staphylococcus haemolyticus, Pseudomonas stutzeri and Pichia pastoris, respectively, though they all possessed mannose-binding specificity. It is noteworthy that those scallop C-type lectins with

4

L. Wang et al. / Developmental and Comparative Immunology 39 (2013) 2–10

the same motif also had different PAMPs binding spectrums. CfLec1 containing the motif EPD could bind LPS, PGN and mannan in vitro, while CfLec-2 with the same motif could also bind zymosan additionally. The potential mechanism of different binding and agglutinating capability for those lectins remains concern. Interestingly, some scallop lectins with similar carbohydrate-binding specificity also synergistically mediated cellular adhesion. For example, both CfLec-1 and CfLec-2 could bind to the surface of scallop haemocytes and recruit haemocytes to enhance their encapsulation in vitro. The methodical coordination of C-type lectins in different conditions endowed the scallop with greatly enhanced capability of immune defense. Galectins are probably the most conserved and ubiquitous family found in multicellular organisms among the various lectin types (Christophides et al., 2002; Janeway and Medzhitov, 2002; Medzhitov and Janeway, 2002). Interestingly, there were quadruple-CRDs galectins which were only found in mollusc, including AiGal1 (Song et al., 2010) and AiGal2 (Song et al., 2011b) from bay scallop, CvGal from Crassostrea virginica (Tasumi and Vasta, 2007) and galectin from Pearl Oyster Pinctada fucata (Wang et al., 2011a; Zhang et al., 2011a). From the phylogenetic point of view, AiGal1 and AiGal2 clustered with galectins from oyster to form a single clade distinct from other invertebrate galectins, suggesting that the bivalve galectin CRDs might share a common ancient. The joining of more different CRDs in the same galectin may provide the possibility to specifically crosslink more distinct types of ligands, as opposed to homo-functional crosslinking by multimeric prototype galectins (Cooper, 2002). For instance, the recombinant AiGal2 displayed high binding and agglutinating activities toward V. anguillarum, Micrococcus luteus, E. coli, Vibrio fluvialis and Edwardsiella tarda, and the agglutinating activity could be inhibited totally by D-galactose and lactose. It also recruited haemocytes and promoted haemocytes encapsulation, and similar phenomenon was observed in some other lectins from scallop (Song et al., 2011b) and insect (Yu and Kanost, 2004). Since there is no antibody-mediated immunity in invertebrates, abundant lectins with diverse expression profiles and bioactivities might function as nonclonal effectors in the immune system. In the past years, the studies on mollusc lectins in China have provided us with a growing volume of information about their molecular structure, gene organization, evolutionary relationships as well as biological roles. Future progress will elucidate the contribution of those lectins in mounting protective immune responses against infection and their crosstalk with other PRRs with respect to pathogen recognition. 2.3. Lipopolysaccharide and b-1,3-glucan binding protein (LGBP) As a member of PRR, LGBP can recognize and bind LPS and b-1,3glucan, and involved in various biological functions, including the activation of the prophenoloxidase (pro-PO) system, cytolysis, bacterial aggregation and opsonic reaction. This acts as a significant molecule responsible for recognition of invader and induction of downstream innate immune response in arthropods. One LGBP (PoLGBP) was identified from the pearl oyster, P. fucata, the most important bivalve for seawater pearl production in China. The remarkable up-regulation of mRNA transcripts in digestive gland signifying that PoLGBP could be effectively induced by LPS or bacteria challenge, as the LGBPs derived from other marine invertebrates (Nikapitiya et al., 2008). The LGBP (CfLGBP) gene cloned from C. farreri also showed upregulation after V. anguillarum challenge indicating its sensitivity to bacterial infection. Apart from LPS and b-glucan, the recombinant CfLGBP could recognize and bind also PGN, and exhibited obvious agglutination activity towards Gram-negative bacteria E. coli, Gram-positive bacteria Bacillus subtilis and fungi P. pastoris in vitro (Yang et al., 2010c). The preliminary information of mollusc LGBP

would likely pave the way for the further understanding on the mechanism of innate immune system in invertebrates. 2.4. Scavenger receptor Scavenger receptors (SR), the main endocytic receptors exhibit distinctive ligand-binding properties to recognize a wide range of ligands including microbial surface constituents and intact microbes (Mukhopadhyay and Gordon, 2004). At present, the knowledge of the molecular feature and function of SR in invertebrates is quite meager, and there are only two invertebrate SRs so far identified, one from Drosophila (dSR-CI) and the other from C. farreri (CfSR). The dSR-CI can recognize both gram-negative and gram-positive bacteria, but not yeast. CfSR appears to be a novel type SR showing structural difference from others characterized so far. Besides the conserved six scavenger receptor cysteine-rich (SRCR) domains, it contains one UPAR-like and one ShK toxin-like domain, both have not been found in any other SRs. CfSR shared a similar attachment site with anchor protein Sgp-2 and it was mainly detected on the outer surface of haemocytes, indicating that CfSR was anchored on the outer-membrane of cells. The recombinant CfSR displayed a broader ligand binding ability not only to acetylated low density lipoprotein, dextran sulfate but various PAMPs, including LPS, PGN, zymosan particle and mannan. CfSR appears to be one of the most primitive SR found so far in invertebrates, likely symbolizing the ancient forms of immune recognition. Interestingly, recent evidences favored that several pathogens have evolved mechanisms to evade recognition by SR (Areschoug et al., 2008; Pinheiro da Silva et al., 2007; Serghides et al., 2006), and other pathogenic microorganisms have exploited SR for their own benefit, mainly as a portal of entry into host cells (Dubuisson et al., 2008; Rodrigues et al., 2008; Yalaoui et al., 2008). The importance of SR in the immune defense will be further underlined, as the cognition of SRs may offer an entrance to potential mechanisms by which pathogens can subvert pattern recognition and establish infection interaction (Areschoug and Gordon, 2009). 2.5. Other PRRs Some other PRRs such as peptidoglycan recognition proteins (PGRPs), globular C1q-domain-containing (C1qDC) proteins and thioester-containing Proteins (TEPs), have also been characterized from mollusc. PGRP is a PRR specifically binding to peptidoglycan, and two mollusc PGRPs have been identified, including AiPGRP from A. irradians (Ni et al., 2007b), CfPGRP-S1 from C. farreri (Su et al., 2007). These scallop PGRPs were of short type with a conserved amidase_2/PGRP domain in their C-terminus. The recombinant CfPGRP-S1 exhibited high affinity to PGN and moderate affinity to LPS, and agglutinating activities to bacteria M. luteus, B. subtilis as well as E. coli. Moreover, the recombinant CfPGRP-S1 functioned as a bactericidal amidase to degrade PGN and strongly inhibit the growth of E. coli and Staphyloccocus aureus in the presence of Zn2+ (Yang et al., 2010b). The C1q-domain-containing (C1qDC) proteins, a family of versatile PRRs are able recognize various ligands including several PAMPs via their globular C1q (gC1q) domain (Bohlson et al., 2007). A C1qDC protein with LPS binding activity (CfC1qDC) (Zhang et al., 2008) and another one with remarkably aggregating activity to P. pastoris (AiC1qDC-1) have been characterized from C. farreri and A. irradians, respectively (Kong et al., 2010). Since large number of C1qDC proteins have been identified in invertebrate, such as 168 C1qDC proteins in Mediterranean mussel Mytilus galloprovincialis (Gerdol et al., 2011), invertebrate C1qDC proteins would function powerfully in the recognition strategy and provide clues for the knowledge of evolution of the complement system. TEPs are a family of proteins characterized by the unique intrachain b-cysteinyl-c-glutamyl thioester bond and a propensity for

L. Wang et al. / Developmental and Comparative Immunology 39 (2013) 2–10

multiple conformationally sensitive binding interactions (Blandin and Levashina, 2004). Previous studies have demonstrated that insect TEPs are involved in the innate immune defense as PRRs (Christophides et al., 2002). Recently, a scallop TEP was identified from C. farreri, and it possessed canonical thioester motif GCGEQ, proteolytic cleavage sites and catalytic histidine residues similar to C3 molecules, as well as some additional features different from C3 molecules (Zhang et al., 2007). However, there were overall stringent similarities between the deduced amino acid sequence of CfTEP and those of previously known TEP family members. The similar up-regulation of TEP transcripts in Anopheles gambiae and Drosophila was also observed in scallop after bacteria challenge. It is of great interest that seven different CfTEP transcripts were produced by alternative splicing and displayed different expression patterns in gonads in response to different bacterial challenges, signifying the important role of diverse CfTEP transcripts in the immune recognition of scallops (Zhang et al., 2009c). The investigation of CfTEP, as well as the knowledge of AiFREP as PRR (Zhang et al., 2009b), would help for further understanding on the structure and function of the complement-like system in marine mollusc. Those PRRs from various mollusc species seem to be versatile for recognizing and binding PAMPs and thus indispensable components in innate immunity of marine invertebrate. The immune system has developed in a stepwise way by progressive improvement of basic and ancestral functions that help molluscs to survive in their hostile environment. Although it is not quite clear yet the mechanisms for awareness of microbes in mollusc, the recent discovery suggests that many aspects of immune recognition in innate immunity are more sophisticated and complex. 3. Signal transduction After recognition of the PAMPs on the invasive pathogens, a part of PRRs would repress the growth of pathogens or kill them directly, whereas majority of PRRs would activate corresponding signal pathways to eliminate the pathogens. The process of signal transduction is essential to the downstream cascades of immune reactions, and two main signal pathways, the immune proteolytic cascades and TLR signaling pathway are discussed here. The immune proteolytic cascades can be activated after the recognition of PAMPs by PRR to trigger complement and phenoloxidase activities to eliminate the invasive pathogens. Serine protease cascade is one of important immune proteolytic cascades, which consists of serine proteases and serine protease inhibitors. The first mollusc serine protease and serine protease inhibitor were characterized in C. farreri and A. irradians, respectively (Zhu et al., 2006, 2007), followed by other two serine proteases identified in A. irradians and P. fucata (Zhang et al., 2009a; Zhu et al., 2008). The three serine proteases contain an amino-terminal clip domain and a carboxyl-terminal trypsin-like domain. The scallop serine protease inhibitor consists of six Kazal-type domains, whose crucial P1 positions were Val, Arg, Ser, Lys, Leu and Leu, respectively, implying the serine protease inhibitor might be able to inhibit many kinds of proteases. Further, the mRNA expression levels of those serine proteases and serine protease inhibitors show obvious up-regulation upon bacteria challenge, indicating their involvement in the immune signal transduction of scallops. TLR signaling pathway is one of the crucial intracellular immune signaling pathways. A series of components in the TLR signaling pathway have been found in mollusc including TLR from C. farreri and C. gigas (Qiu et al., 2007a; Zhang et al., 2011b), MyD88 and TRAF6 from C. farreri (Qiu et al., 2007b, 2009), IjB from A. irradians, clam Meretrix meretrix and C. gigas (Mu et al., 2010b; Yang et al., 2011b; Zhang et al., 2011c), Rel/NF-jB homologue from P. fucata and abalone Haliotis diversicolor supertexta (Jiang and Wu, 2007; Wu et al., 2007). These components have been verified to

5

response against the immune stimulation with different pattern. For example, the expression level of scallop TLR mRNA in primary cultured haemocytes was down-regulated after LPS stimulation at lower concentration, while up-regulated at higher concentration. The expression level of MyD88 in haemocytes of oyster Crassostrea ariakensis was induced by ompR, an outer membrane protein of pathogenic organism (Zhu and Wu, 2008). The results of CfTLR study have suggested the existence of a primitive TLR signaling pathway in scallop C. farreri involving the immune signaling to activate the diverse downstream reaction including antioxidant, anti-bacteria and apoptosis (Wang et al., 2011c). The expression levels of scallop TLR, MyD88, TRAF6, IjB and NF-jB mRNA in haemocytes were all up-regulated after LPS stimulation. After scallop TLR was knockdown by RNAi technique, the mRNA transcripts of MyD88, TRAF6, IjB, NF-jB and G-type lysozyme in haemocytes all decreased, while those of superoxide dismutase (SOD) and catalase (CAT) increased. Furthermore, the cumulative mortality of the TLR-suppressed scallops significantly arose after Listonella anguillara challenge. As the scallop TLR signaling pathway appears as the most primitive one so far observed, it would be an intriguing drive to further understanding not only the immune defense mechanism but also the evolution of TLR signaling pathway.

4. Immune effectors Innate immunity encompasses a complex array of defense reactions, in which immune effectors are synthesized as executor for the incapacitation and elimination of invaders. The information on mollusc immune effectors is crucial to better understand the immune defense mechanisms, and provides the potentially feasible solutions for disease control in practical aquaculture. As described above, some mollusc PRRs not only recognize or bind various PAMPs, but also function as the immune effectors involved in eliminating the invaders, such as lectin and PGRP in scallop (Yang et al., 2010a,b, 2011a). Apart from the identification of some classic mollusc immune effectors such as antimicrobial peptide (AMP), lysozyme, antioxidant enzyme and heat shock protein (HSP), several of their effect mechanisms have also been characterized. This section briefly documents the research progress on the classic immune effectors.

4.1. Antimicrobial peptides In mollusc, the investigation of AMPs mainly focuses on mussels Mytilus edulis and M. galloprovincialis, and those mollusc AMPs are usually named as the classification of defensin, mytilin, myticin and mytimycin, which is originally nominated in mussels. More than twenty AMPs belonging to the former three types, along with some other potential AMPs have been identified from different mollusc including scallop, clam, abalone, oyster and mussel in China (details in Table 1). Defensins, a group of small cysteine-rich cationic proteins possess remarkable microbicidal activity. Four defensins have been reported in clam, scallop and abalone (Wei et al., 2003; Zhao et al., 2007a, 2010a); among these, AiBD from A. irradians was detected mainly in haemocytes and slightly in haemocytes-rich gills, and could respond drastically to Vibrio stimulation, while the abalone defensin expressed only in the hepatopancreas responding to both gram-positive and negative bacteria stimulation. Moreover, RpBD from Ruditapes philippinesis, VpBD from Venerupis philippinarum and AiBD displayed a broad-spectrum antibacterial activity to both gram-positive and negative bacteria, and AiBD even had a strong fungicidal activity. The results indicated that mollusc defensins were involved in the host immune response of specific tissues

6

L. Wang et al. / Developmental and Comparative Immunology 39 (2013) 2–10

Table 1 AMPs identified by Chinese scientists. Species

AMP Name

Antibacterial spectrum

Reference

Ruditapes philippinesis Chlamys farreri

RPD-1 N-terminus of H2Aa VpBD

Gram-positive and negative Gram-positive and negative

(Wei et al., 2003) (Li et al., 2007)

Gram-positive and negative

(Zhao et al., 2010a)

Hd-DEF

Has not yet elucidated or tested

(Hong et al., 2008)

AiBD Hc theromacin CgPep33

Gram-positive and negative bacteria, fungi Has not yet elucidated or tested Gram-positive and negative, fungi

Mytilus coruscus

Myticin A

Mytilus coruscus Mytilus coruscus

Mytilin 1–3 Mytilin 1–8b Myticin 1–9b

Gram-positive bacteria, specially to the Bacillus megaterium, tumor cell lines including SW l990, L929 and LoVo Has not yet elucidated or tested Has not yet elucidated or tested Has not yet elucidated or tested

(Zhao et al., 2007b) (Xu et al., 2010) (Liu et al., 2007, 2008) (Zhong et al., 2005)

Venerupis philippinarum Haliotis discus hannai Ino Argopecten irradians Hyriopsis cumingii Crassostrea gigas

a b

(Wang et al., 2010) (Liao et al., 2010) (Liao et al., 2010)

Functional-like AMPs. Only cDNA sequences obtained.

against bacterial infection and contribute greatly to the clearance of invading bacteria. Mytilin and myticin, possessing eight cysteines and similar molecular weight, are most plentiful AMPs in mussels. In China, 11 mytilin and 10 myticin have been reported in the same organism Mytilus coruscus, with high polymorphism level in their cDNA sequences (Wang et al., 2010). To be more specific, 70 and 88 variation sites were found in eight different mytilin and nine myticin cDNAs, whose total length was around 300 bp (Liao et al., 2010). Such a diversity of mytilin and myticin is particularly noticeable, and might endue them with broader antibacterial activity spectrum. (Mitta et al., 2000). For example, a myticin (Myticin A) from M. coruscus exhibited strong inhibitory effects specifically on the growth of gram-positive bacteria and even tumor cells (Zhong et al., 2005). These studies lay the foundation for further research about the molecular diversity and its mechanism of antibacterial peptides from mollusc. There are also other potential AMPs reported in mollusc, which may be the production of enzymatic hydrolysis. CgPep33 from C. gigas is a cysteine-rich antimicrobial peptide. Studies revealed that it has wide inhibitory spectrum on gram-positive/negative bacteria and fungi (Liu et al., 2008). The other known potential AMP is derived from histone H2A of C. farreri (Li et al., 2007). The N-terminal 39 amino acid of histone H2A exhibited significant similarity with novel vertebrate AMPs such as parasin I, buforin I and hipposin, and predicted to be an antimicrobial peptide to interact with membranes by Antimicrobial Peptide Predictor (http://www.aps.unmc.edu/AP/ prediction/prediction_main.php). The recombinant product of the N-terminus in P. pastoris has proven its antibacterial activity against both gram-positive and gram-negative bacteria, indicating the existence of complex mechanism to produce various potential AMPs in mollusc.

type lysozymes has been under dispute for many years. The discovery of c, i and g-type lysozyme in mollusc has provided important clues to clarify the relationship of the three types of lysozymes. For instance, Ding et al. (2011) observed that the three type lysozymes diverged from a common precursor, and i and c-type lysozyme were closer to the ancestor than g-type lysozymes, and c-type appears as the basal lysozyme conforming the previously suggestion. Moreover, there was high content of cysteine residues in mollusc lysozymes; for example, i-type lysozyme in M. meretrix (MmeLys) contains as many as 15 cysteine residues, and c-type lysozyme of H. discus hannai (HdLysC) has eight cysteine residues. It was presumed that the more cysteine residues in mollusc lysozyme may enable them stable in high-osmolarity seawater with a tightly structure (Zhao et al., 2007b). The lysozyme expression patterns in different tissues apparently reveal their different biological functions. The mRNA transcripts of g-type lysozyme in scallop (CfLysG) and i-type lysozyme in M. meretrix (MmeLys) were most abundantly expressed in gills and hepatopancreas, while the higher expression level of HdLysC and i-type lysozyme in V. philippinarum (VpLYZ) were detected in mantle and haemocyte, respectively. Moreover, CfLysG displayed a wider range of lytic activities against the gram-positive bacteria than the gram-negative bacteria, supporting the report on the g-type lysozymes from Japanese flounder and orange-spotted grouper (Zhao et al., 2007b); in contrary to this, MmeLys exhibited greater activity against gram-negative bacteria. The results are consistent with the assumption that in addition to digestive capability, mollusc lysozymes possess bactericidal effect and different bacteriolytic activity inclinations depending on their respective structure characteristics.

4.2. Lysozyme

Reactive oxygen species (ROS) are free radicals such as superox ide anion ðO 2 Þ, hydrogen peroxide (H2O2), hydroxyl radical (OH ), which constantly generated when the organism is attacked by invaders or contaminant exposures (Aguirre et al., 2005), and excessive production of ROS would lead to oxidative stress, loss of cell function, and ultimately apoptosis or necrosis (Kappus and Sies, 1981; Bao et al., 2009; Manduzio et al., 2003; Winston, 1991). However, in order to protect the tissues from the oxidative stress, there exist an interacting network of antioxidant enzymes such as SOD, CAT, glutathione peroxidase (GPx), glutathione-Stransferase (GST) and glutathione reductase (GR). In mollusc, a large number of antioxidant enzymes have been identified over

There are six distinct types of lysosomes such as chicken (c), goose (g), invertebrate (i), phage, bacterial and plant-type, naming was based on the organisms in which lysozymes were identified, So far there are three i-type (Xie et al., 2011; Yue et al., 2011; Zhao et al., 2010b), two g-type (Zhao et al., 2007b; Zou et al., 2005) and one c-type mollusc lysozymes (Ding et al., 2011) reported in China. Among the three types of animal lysozymes, c and i-type lysozymes are often reported in invertebrate, while g-type from vertebrate, although two g-type lysozymes have been recently identified in mollusc. The evolutionary relationship of different

4.3. Antioxidant enzymes

7

L. Wang et al. / Developmental and Comparative Immunology 39 (2013) 2–10

the last decades, and their response against microbe challenge and environmental stress have been studied. This part of the review is devoted to summarize briefly the work on SOD, CAT, GPx and GST done in China. The SODs are a class of antioxidant enzymes catalyzying the dismutation of superoxide into oxygen and hydrogen peroxide. Six intracellular and one extracellular Cu/ZnSOD have been identified in different mollusc animals (Li et al., 2010a,b; Xu et al., 2010b; Zhu et al., 2010; Ni et al., 2007a; Bao et al., 2009). Apart from this, much attention has also been paid to MnSOD as considering its role as a major scavenger of damaging ROS metabolites in the mitochondrial matrix. There are three MnSODs characterized in mollusc, including MnSOD from A. irradians (Bao et al., 2008), TgmMnSOD from Tegillarca granosa (Li et al., 2011), and ChMnSOD from Crassostrea hongkongensis (Yu et al., 2011). According to their primary sequence analysis, it is confirmed that the three mollusc MnSODs contain putative signal peptide, which may help MnSODs transport to mitochondria after translation. The fusion proteins of ChMnSOD exhibited a punctate GFP fluorescence pattern that largely coincided with the staining pattern of the mitochondria-specific dye MitoTracker (Yu et al., 2011). It is therefore concluded that most mollusc MnSOD are located in mitochondria just as their homolog. Most of mollusc SODs are constitutive proteins and their transcripts exhibit a rapid elevation after microbe infection (Bao et al., 2008; Li et al., 2010a; Ni et al., 2007a). Hence, their expression and enzyme activity level could be used as a significant criterion to evaluate the health condition of mollusc animals. However, the underlying mechanisms still invite further exploration. CAT is one of the central enzymes involved in scavenging the high level of ROS by catalyzing the decomposition of hydrogen peroxide to gaseous oxygen and water molecules (Chelikani et al., 2004). This enzyme appears to be ubiquitous by its presence in archaea, prokaryotes and eukaryotes, while only one mollusc CAT gene (CfCAT from C. farreri) was reported. The deduced amino acid sequence of CfCAT is significantly homologous to CATs from other animals, plants and bacteria. Several highly conserved motifs were identified including the proximal heme-ligand signature sequence RLFSYNDTH, the proximal active site signature FNRERIPERVVHAKGGGA, and the three catalytic amino acid residues of HisAsn-Tyr. CfCAT appears to be a constitutive and inducible protein responding to bacterial infection, and is involved in eliminating excess ROS (Li et al., 2008). GPx are a family of enzymes catalyzing the reduction of various organic hydroperoxide and hydrogen peroxidase using glutathione as hydrogen donor (Mills, 1957). The selenium-dependent GPx

(Se-GPx) and non-selenium glutathione peroxidase (non-Se-GPx) are considered the two main types of GPx (Arthur, 2000). And some Se-GPx have been identified from mollusc in China (Mu et al., 2010a; Wang et al., 2011b; Wu et al., 2010; Jiang et al., 2010). The transcripts of these GPxs were universally detected, but the expression patterns were quite variable. For example, the transcripts of CfGPx3 from C. farreri were significantly high in haemocytes, while that of MmeGPx from M. meretrix was mainly expressed in hepatopancreas. Significantly, most of GPx are sensitive to bacterial infection. For instance, the expression of MmeGPx in hepatopancreas was early up-regulated in the Vibrio-resistant resistant population after the immersion challenge of bacteria (Wang et al., 2011b). The GSTs catalyze the nucleophilic attack of the sulfur atom of glutathione (GSH) to an electrophilic group on metabolic products or xenobiotic compounds (Blanchette et al., 2007; Hayes and Pulford, 1995), and three pi- and one sigma-type GSTs have been identified in China. All three pi-type GSTs share a high similarity in sequence, and they are all highly susceptive to benzo[a]pyrene exposure, suggesting their antioxidant role and their expression could be a useful candidate biomarker for monitoring environmental contaminants such as PAHs (Miao et al., 2011; Xu et al., 2010a). The sigma-type abGST has shown significant expression in the haemocytes, gill, mantle and digestive gland of bacteria-challenged abalone, and the total GSTs enzyme in the four tissues is also induced by bacteria challenge (Ren et al., 2009). 4.4. Heat shock protein The HSPs are a family of ubiquitous proteins that help organisms to modulate stress response and protect them from environmentally induced cellular damage. According to apparent molecular mass, they are classified into several families, including HSP90 (83–90 kDa), HSP70 (66–78 kDa), HSP60, HSP47, and small heat shock protein (sHSP; 15–43 kDa). A great number of HSPs, including HSP22, HSP40, HSP70 and HSP90 have been identified from mollusc (Table 2). So far the progress of mollusc HSPs mainly focuses on their sequence analysis and expression profile under different stimulation, such as temperature, salinity, heavy mental, pollutant chemicals, as well as bacteria invasion (Table 2). Considering that mollusc animals are constantly exposed to the changing environment, HSPs would probably buffer all kinds of environmental stress and play important roles in the homeostasis maintenance of mollusc. The future exploration on the activities of mollusc HSP to minimize the biochemical, physiological and histological

Table 2 HSPs identified from most commonly cultured bivalve mollusks in China. Species

Name

Classification

Stimulations

Tegillarca granos Meretrix meretrix

Tg-sHSP MmeHsc71

Small HSPs HSP70

V. parahaemolyticus, LPS (Yue et al., 2011) V. parahaemolyticus (Yue et al., 2011)

Venerupis philippinarum

VpHSP40 VpsHSP-1 VpsHSP-2

HSP40 Small HSPs Small HSPs

V. anguillarum, Cu2+, Cd2+ (Li et al., 2011) V. anguillarum, Cd2+ (Li et al., 2010b) V. anguillarum, Cd2+ (Li et al., 2010b)

Haliotis discus hannai Ino

HSP70 HdhHSP90

HSP70 HSP90

V. anguillarum, heat (Cheng et al., 2007) Selenium (Yang et al., 2011a)

Argopecten irradians

AiHSP90 AiHSP70 AiHSP22

HSP90 HSP70 HSP22

V. anguillarum, M. luteus (Gao et al., 2008) Naphthalin, V. anguillarum (Song et al., 2006) Cd2+, Pb2+ and Cu2+ (Zhang et al., 2010a)

Chlamys farreri

CfHSP90 CfHSP70 CfHSP22

HSP90 HSP70 Small HSP

Cd2+, Pb2+ and Cu2+ (Gao et al., 2007) (Wu et al., 2003) V. anguillarum (Zhang et al., 2010b)

Crassostrea hongkongensis Crassostrea ariakensis Pinctada fucata

ChHSP90 HSP70 HSP70

HSP90 HSP70 HSP70

High salinity, V. alginolyticus (Fu et al., 2011) Heat shock (Zhang et al., 2003) Heat shock, high salinity (Huang et al., 2007)

8

L. Wang et al. / Developmental and Comparative Immunology 39 (2013) 2–10

alteration of host will contribute to a better understanding of mollusc defensive mechanisms against environmental stress. 5. The immunomodulation of neuroendocrine system The crosstalk between neuroendocrine and immune system constitutes a complex neuroendocrine-immune regulatory network to maintain the host homeostasis in all vertebrates. In this network, the neuroendocrine system can optimize the immune response to eliminate the invasive pathogens and restore the host homeostasis. The immunomodulation of the neuroendocrine system is indispensable to most fine-tuned innate immune response, such as inflammatory reaction, but it is still far from well understood in invertebrate. The preliminary progress on the catecholaminergic neuroendocrine system and its immunomodulation function in scallop C. farreri is summarized to throw light into mollusc neuroendocrine-immune regulatory network. Catecholaminergic neuroendocrine system is mainly composed of catecholamines, catecholamine metabolic enzymes and catecholamine receptors. Recently, most components of this system have been characterized in scallop. The catecholamines (dopamine, norepinephrine and epinephrine) are all detected in the haemolymph of scallop (Zhou et al., 2011b). Four crucial catecholamine metabolic enzymes have also been identified from scallop, including phenylalanine hydroxylase (Zhou et al., 2011c), dopa decarboxylase (Zhou et al., 2011e), dopamine beta hydroxylase (Zhou et al., 2011d) and monoamine oxidase (Fig. 2) (Zhou et al., 2011a). Moreover, an adrenoceptor cDNA fragment was found in scallop EST library (Wang et al., 2009). These studies could demonstrate the existence of a catecholaminergic system in scallop similar to that of vertebrates. Scallop catecholaminergic neuroendocrine system could be activated after the immune stimulation, and then modulate immune response against bacteria challenge. The mRNA expression of four crucial catecholamine metabolic enzymes was induced after infection, and catecholamines concentration in the haemolymph of scallops also increased significantly. High concentration of norepinephrine and epinephrine repressed the increase of SOD, CAT and lysozyme activities in haemolymph after bacteria challenge, and the antagonist of adrenoceptors also modulated negatively the activities of CAT and lysozyme (Zhou et al.,

2011b). The catecholaminergic neuroendocrine system also modulates immune reaction through the adjustment of catecholamine metabolic enzyme activities. For example, dopa decarboxylase could prompt the encapsulation of scallop haemocytes, and the ROS level in haemocytes shows significant decrease when dopa decarboxylase activity was inhibited (Zhou et al., 2011e). Since the immunomodulation of neuroendocrine system in mollusc is more complex and important than what we have learned, it is worth to further investigate the interaction network and modulation manner. 6. Conclusion remarks In the long course of evolution, molluscs have developed an array of effective strategies to recognize and eliminate various invaders by employing a set of molecules. It must be emphasized that the knowledge presented in this review is based on only a few mollusc species, mainly bivalves in China. From this restricted sampling, we are unable to catch sight of unitive laws of immune system applying to all the mollusc animals, however, it is evident that basic immune reactions such as immune recognition, signal transduction and effector synthesis involved in mollusc immunity can be accomplished in a variety of ways. Many immune molecules such as cytokines, and system such as neuroendocrine-immune regulatory network and complement system have not been investigated in detail, and future studies could reveal their molecular constitution and regulation network, even greater complexity in the mollusc immunity. Additionally, the potential advancement of our cognition to the immune system in mollusc, through the incorporation of newly exploit and novel molecular techniques as well as the development of comparative immunology, is expected. The development of genomics, transcriptomics, proteomics and metabolomics, and potential utilization of related methods, also would deepen the knowledge of mollusc immunity on the whole. Acknowledgments The authors would like to thank Dr. Kappalli Sudha for revision of English language in the manuscript. Work cited in this review was supported by grants (Nos. 30925028 and 30730070 to L.S., 31072192 to L.W., 31001129 to L.Q.) from NSFC, 973 program (No. 2010CB126404 to L.W.), Shandong Provincial Natural Science Foundation (No. JQ201110 to L.W.), the Knowledge Innovation Program of the Chinese Academy of Sciences (No. KZCX2-YW-QN206 to L.W.), and the earmarked fund for Modern Agro-industry Technology Research System. References

Fig. 2. The metabolism of catecholamines in the mollusc. The phenylalanine hydroxylase (PAH), dopa decarboxylase (DDC) and dopamine beta hydroxylase (DBH) are responsible of the anabolism of catecholamines, while the monoamine oxidase (MAO) is involved in the catabolism of catecholamines.

Aguirre, J., Rios-Momberg, M., Hewitt, D., Hansberg, W., 2005. Reactive oxygen species and development in microbial eukaryotes. Trends Microbiol. 13 (3), 111–118. Areschoug, T., Gordon, S., 2009. Scavenger receptors: role in innate immunity and microbial pathogenesis. Cell. Microbiol. 11 (8), 1160–1169. Areschoug, T., Waldemarsson, J., Gordon, S., 2008. Evasion of macrophage scavenger receptor A-mediated recognition by pathogenic streptococci. Eur. J. Immunol. 38 (11), 3068–3079. Arthur, J., 2000. The glutathione peroxidases. Cell. Mol. Life Sci. 57 (13), 1825–1835. Bao, Y., Li, L., Zhang, G., 2008. The manganese superoxide dismutase gene in bay scallop Argopecten irradians: cloning, 3D modelling and mRNA expression. Fish Shellfish Immunol. 25 (4), 425–432. Bao, Y., Li, L., Xu, F., Zhang, G., 2009. Intracellular copper/zinc superoxide dismutase from bay scallop Argopecten irradians: its gene structure, mRNA expression and recombinant protein. Fish Shellfish Immunol. 27 (2), 210–220. Bao, Y., Wang, Q., Liu, H., Lin, Z., 2011. A small HSP gene of bloody clam (Tegillarca granosa) involved in the immune response against Vibrio parahaemolyticus and lipopolysaccharide. Fish Shellfish Immunol. 30 (2), 729–733. Blanchette, B., Feng, X., Singh, B.R., 2007. Marine glutathione S-transferases. Mar. Biotechnol. 9 (5), 513–542. Blandin, S., Levashina, E.A., 2004. Thioester-containing proteins and insect immunity. Mol. Immunol. 40 (12), 903–908.

L. Wang et al. / Developmental and Comparative Immunology 39 (2013) 2–10 Bohlson, S.S., Fraser, D.A., Tenner, A.J., 2007. Complement proteins C1q and MBL are pattern recognition molecules that signal immediate and long-term protective immune functions. Mol. Immunol. 44 (1–3), 33–43. Chelikani, P., Fita, I., Loewen, P.C., 2004. Diversity of structures and properties among catalases. Cell. Mol. Life Sci. 61 (2), 192–208. Cheng, P., Liu, X., Zhang, G., He, J., 2007. Cloning and expression analysis of a HSP70 gene from Pacific abalone (Haliotis discus hannai). Fish Shellfish Immun. 22 (1– 2), 77–87. Christophides, G.K., Zdobnov, E., Barillas-Mury, C., Birney, E., Blandin, S., Blass, C., et al., 2002. Immunity-related genes and gene families in Anopheles gambiae. Science 298 (5591), 159–165. Cong, M., Song, L., Wang, L., Zhao, J., Qiu, L., Li, L., et al., 2008. The enhanced immune protection of Zhikong scallop Chlamys farreri on the secondary encounter with Listonella anguillarum. Comp. Biochem. Physiol. B: Biochem. Mol. Biol. 151 (2), 191–196. Cong, M., Song, L., Qiu, L., Li, C., Wang, B., Zhang, H., et al., 2009. The expression of peptidoglycan recognition protein-S1 gene in the scallop Chlamys farreri was enhanced after a second challenge by Listonella anguillarum. J. Invertebr. Pathol. 100 (2), 120–122. Cooper, D.N., 2002. Galectinomics: finding themes in complexity. Biochim. Biophys. Acta 1572 (2–3), 209–231. Cui, M., Sun, Q., Xu, Z., 2006. Prospects of the development and application of the molluscs. Aquaculture Breeding 818. Ding, J., Li, J., Bao, Y., Li, L., Wu, F., Zhang, G., 2011. Molecular characterization of a mollusk chicken-type lysozyme gene from Haliotis discus hannai Ino, and the antimicrobial activity of its recombinant protein. Fish Shellfish Immunol. 30 (1), 163–172. Dubuisson, J., Helle, F., Cocquerel, L., 2008. Early steps of the hepatitis C virus life cycle. Cell. Microbiol. 10 (4), 821–827. Fu, D., Chen, J., Zhang, Y., Yu, Z., 2011. Cloning and expression of a heat shock protein (HSP) 90 gene in the haemocytes of Crassostrea hongkongensis under osmotic stress and bacterial challenge. Fish Shellfish Immun. 31 (1), 118–125. Gao, Q., Song, L., Ni, D., Wu, L., Zhang, H., Chang, Y., 2007. cDNA cloning and mRNA expression of heat shock protein 90 gene in the haemocytes of Zhikong scallop Chlamys farreri. Comp. Biochem. Physiol. B: Biochem. Mol. Biol. 147 (4), 704– 715. Gao, Q., Zhao, J., Song, L., Qiu, L., Yu, Y., Zhang, H., et al., 2008. Molecular cloning, characterization and expression of heat shock protein 90 gene in the haemocytes of bay scallop Argopecten irradians. Fish Shellfish Immun. 24 (4), 379–385. Gerdol, M., Manfrin, C., De Moro, G., Figueras, A., Novoa, B., Venier, P., et al., 2011. The C1q domain containing proteins of the Mediterranean mussel Mytilus galloprovincialis: a widespread and diverse family of immune-related molecules. Dev. Comp. Immunol. 35 (6), 635–643. Hanington, P.C., Forys, M.A., Dragoo, J.W., Zhang, S.M., Adema, C.M., Loker, E.S., 2010. Role for a somatically diversified lectin in resistance of an invertebrate to parasite infection. Proc. Natl. Acad. Sci. USA 107 (49), 21087–21092. Hayes, J.D., Pulford, D.J., 1995. The glutathione S-transferase supergene family: regulation of GST and the contribution of the isoenzymes to cancer chemoprotection and drug resistance. Crit. Rev. Biochem. Mol. Biol. 30 (6), 445–600. Hong, X., Sun, X., Zheng, M., Qu, L., Zan, J., Zhang, J., 2008. Characterization of defensin gene from abalone Haliotis discus hannai and its deduced protein. Chin J Oceanol Limnol. 26 (4), 375–379. Huang, G., Qu, N., Yu, D., Li, L., 2007. Cloning and expression analysis of heat shock protein 70 gene in pearl oyster Pinctada fucata. China J Fisheries Sci. 14 (5), 726–732. Janeway Jr., C.A., Medzhitov, R., 2002. Innate immune recognition. Annu. Rev. Immunol. 20 (1), 197–216. Jiang, Y., Wu, X., 2007. Characterization of a RelYNF-kappaB homologue in a gastropod abalone, Haliotis diversicolor supertexta. Dev. Comp. Immunol. 31 (2), 121–131. Jiang, J., Zhang, D., Ma, J., Su, T., Jiang, S., 2010. Molecular characterization and expression analysis of a selenium dependent glutathione peroxidase on pearl oyster Pinctada fucata. J. Fujian Agric. Forest. Univ. Nat. Sci. Ed. 39 (6), 614–621. Kappus, H., Sies, H., 1981. Toxic drug effects associated with oxygen metabolism: redox cycling and lipid peroxidation. Experientia 37 (12), 1233–1241. Kong, P., Zhang, H., Wang, L., Zhou, Z., Yang, J., Zhang, Y., et al., 2010. AiC1qDC-1, a novel gC1q-domain-containing protein from bay scallop Argopecten irradians with fungi agglutinating activity. Dev. Comp. Immunol. 34 (8), 837–846. Li, C., Song, L., Zhao, J., Zhu, L., Zou, H., Zhang, H., et al., 2007. Preliminary study on a potential antibacterial peptide derived from histone H2A in hemocytes of scallop Chlamys farreri. Fish Shellfish Immunol. 22 (6), 663–672. Li, C., Ni, D., Song, L., Zhao, J., Zhang, H., Li, L., 2008. Molecular cloning and characterization of a catalase gene from Zhikong scallop Chlamys farreri. Fish Shellfish Immunol. 24 (1), 26–34. Li, C., Sun, H., Chen, A., Ning, X., Wu, H., Qin, S., et al., 2010a. Identification and characterization of an intracellular Cu, Zn-superoxide dismutase (icCu/Zn-SOD) gene from clam Venerupis philippinarum. Fish Shellfish Immunol. 28 (3), 499–503. Li, H., Sun, X., Cai, Z., Cai, G., Xing, K., 2010b. Identification and analysis of a Cu/Zn superoxide dismutase from Haliotis diversicolor supertexta with abalone juvenile detached syndrome. J. Invertebr. Pathol. 103 (2), 116–123. Li, C., He, J., Su, X., Li, T., 2011. A manganese superoxide dismutase in blood clam Tegillarca granosa: molecular cloning, tissue distribution and expression analysis. Comp. Biochem. Physiol. B: Biochem. Mol. Biol. 159 (1), 64–70.

9

Liao, Z., Liu, M., Wang, R., Wu, M., Yang, L., Lu, T., et al., 2010. CDNA clone and sequence analysis of mytilin and myticin from Mytilus coruscus. J. Fish. China 34 (7), 1025–1033. Liu, Y.C., Li, F.H., Dong, B., Wang, B., Luan, W., Zhang, X.J., et al., 2007. Molecular cloning, characterization and expression analysis of a putative C-type lectin (Fclectin) gene in Chinese shrimp Fenneropenaeus chinensis. Mol. Immunol. 44 (4), 598–607. Liu, Z., Dong, S., Xu, J., Zeng, M., Song, H., Zhao, Y., 2008. Production of cysteine-rich antimicrobial peptide by digestion of oyster (Crassostrea gigas) with alcalase and bromelin. Food Control 19 (3), 231–235. Loker, E.S., Adema, C.M., Zhang, S.-M., Kepler, T.B., 2004. Invertebrate immune systems—not homogeneous, not simple, not well understood. Immunol. Rev. 198, 10–24. Manduzio, H., Monsinjon, T., Rocher, B., Leboulenger, F., Galap, C., 2003. Characterization of an inducible isoform of the Cu/Zn superoxide dismutase in the blue mussel Mytilus edulis. Aquat. Toxicol. 64 (1), 73–83. Matsubara, H., Ogawa, T., 2006. K. MURAMOTO. Structures and functions of C-type lectins in marine invertebrates. Tohoku J. Agric. Res. 57 (1/2), 71–86. Medzhitov, R., Janeway Jr., C.A., 2002. Decoding the patterns of self and nonself by the innate immune system. Science 296 (5566), 298–300. Miao, J.J., Pan, L.Q., Liu, N., Xu, C.Q., Zhang, L., 2011. Molecular cloning of CYP4 and GSTpi homologues in the scallop Chlamys farreri and its expression in response to Benzo[a]pyrene exposure. Mar. Genomics 4 (2), 99–108. Mills, G.C., 1957. Hemoglobin catabolism. I. Glutathione peroxidase, an erythrocyte enzyme which protects hemoglobin from oxidative breakdown. J. Biol. Chem. 229 (1), 189. Mitta, G., Vandenbulcke, F., Roch, P., 2000. Original involvement of antimicrobial peptides in mussel innate immunity. FEBS Lett. 486 (3), 185–190. Mu, C., Ni, D., Zhao, J., Wang, L., Song, L., Li, L., et al., 2010a. CDNA cloning and mRNA expression of a selenium-dependent glutathione peroxidase from Zhikong scallop Chlamys farreri. Comp. Biochem. Physiol. B: Biochem. Mol. Biol. 157 (2), 182–188. Mu, C., Yu, Y., Zhao, J., Wang, L., Song, X., Zhang, H., et al., 2010b. An inhibitor kappaB homologue from bay scallop Argopecten irradians. Fish Shellfish Immunol. 28 (4), 687–694. Mukhopadhyay, S., Gordon, S., 2004. The role of scavenger receptors in pathogen recognition and innate immunity. Immunobiology 209 (1–2), 39–49. Ni, D., Song, L., Gao, Q., Wu, L., Yu, Y., Zhao, J., et al., 2007a. The cDNA cloning and mRNA expression of cytoplasmic Cu, Zn superoxide dismutase (SOD) gene in scallop Chlamys farreri. Fish Shellfish Immunol. 23 (5), 1032–1042. Ni, D., Song, L., Wu, L., Chang, Y., Yu, Y., Qiu, L., et al., 2007b. Molecular cloning and mRNA expression of peptidoglycan recognition protein (PGRP) gene in bay scallop (Argopecten irradians, Lamarck 1819). Dev. Comp. Immunol. 31 (6), 548– 558. Nikapitiya, C., De Zoysa, M., Lee, J., 2008. Molecular characterization and gene expression analysis of a pattern recognition protein from disk abalone, Haliotis discus discus. Fish Shellfish Immunol. 25 (5), 638–647. Pinheiro da Silva, F., Aloulou, M., Skurnik, D., Benhamou, M., Andremont, A., Velasco, I.T., et al., 2007. CD16 promotes Escherichia coli sepsis through an FcR gamma inhibitory pathway that prevents phagocytosis and facilitates inflammation. Nat. Med. 13 (11), 1368–1374. Ponder, W., Lindberg, D., 2008. Phylogeny and Evolution of the Mollusca. Qiu, L., Song, L., Xu, W., Ni, D., Yu, Y., 2007a. Molecular cloning and expression of a Toll receptor gene homologue from Zhikong Scallop, Chlamys farreri. Fish Shellfish Immunol. 22 (5), 451–466. Qiu, L., Song, L., Yu, Y., Xu, W., Ni, D., Zhang, Q., 2007b. Identification and characterization of a myeloid differentiation factor 88 (MyD88) cDNA from Zhikong scallop Chlamys farreri. Fish Shellfish Immunol. 23 (3), 614–623. Qiu, L., Song, L., Yu, Y., Zhao, J., Wang, L., Zhang, Q., 2009. Identification and expression of TRAF6 (TNF receptor-associated factor 6) gene in Zhikong scallop Chlamys farreri. Fish Shellfish Immunol. 26 (3), 359–367. Ren, H.L., Xu, D.D., Gopalakrishnan, S., Qiao, K., Huang, W.B., Wang, K.J., 2009. Gene cloning of a sigma class glutathione S-transferase from abalone (Haliotis diversicolor) and expression analysis upon bacterial challenge. Dev. Comp. Immunol. 33 (9), 980–990. Rodrigues, C.D., Hannus, M., Prudencio, M., Martin, C., Goncalves, L.A., Portugal, S., et al., 2008. Host scavenger receptor SR-BI plays a dual role in the establishment of malaria parasite liver infection. Cell Host Microbe 4 (3), 271–282. Serghides, L., Patel, S.N., Ayi, K., Kain, K.C., 2006. Placental chondroitin sulfate Abinding malarial isolates evade innate phagocytic clearance. J. Infect. Dis. 194 (1), 133. Song, L., Xu, W., Li, C., Li, H., Wu, L., Xiang, J., et al., 2006. Development of expressed sequence tags from the bay scallop, Argopecten irradians irradians. Mar. Biotechnol. (NY) 8 (2), 161–169. Song, X., Zhang, H., Zhao, J., Wang, L., Qiu, L., Mu, C., et al., 2010. An immune responsive multidomain galectin from bay scallop Argopectens irradians. Fish Shellfish Immunol. 28 (2), 326–332. Song, L., Wang, L., Qiu, L., Zhang, H., 2011a. Bivalve immunity. Adv. Exp. Med. Biol., 70844–70865. Song, X., Zhang, H., Wang, L., Zhao, J., Mu, C., Song, L., et al., 2011b. A galectin with quadruple-domain from bay scallop Argopecten irradians is involved in innate immune response. Dev. Comp. Immunol. 35 (5), 592–602. Su, J., Ni, D., Song, L., Zhao, J., Qiu, L., 2007. Molecular cloning and characterization of a short type peptidoglycan recognition protein (CfPGRP-S1) cDNA from Zhikong scallop Chlamys farreri. Fish Shellfish Immunol. 23 (3), 646–656.

10

L. Wang et al. / Developmental and Comparative Immunology 39 (2013) 2–10

Tasumi, S., Vasta, G.R., 2007. A galectin of unique domain organization from hemocytes of the eastern oyster (Crassostrea virginica) is a receptor for the protistan parasite Perkinsus marinus. J. Immunol. 179 (5), 3086–3098. Wang, J.T., Mu, Y.T., 2010. Management situation and suggestions of shellfish’s culture in China. China’s Fish. Econ. 28 (3), 43–47. Wang, L., Song, L., Ni, D., Zhang, H., Liu, W., 2009. Alteration of metallothionein mRNA in bay scallop Argopecten irradians under cadmium exposure and bacteria challenge. Comp. Biochem. Physiol.: Toxicol. Pharmacol. 149 (1), 50– 57. Wang, R., Liu, M., Liao, Z., Lu, T., Wu, M., He, G., 2010. Purification and identification of Mytilins from Mytilus coruscus. J. Fish. China 34 (1), 153–159. Wang, A., Wang, Y., Gu, Z., Li, S., Shi, Y., Guo, X., 2011a. Development of expressed sequence tags from the pearl oyster Pinctada martensii Dunker. Mar. Biotechnol. (NY) 13 (2), 275–283. Wang, C., Huan, P., Yue, X., Yan, M., Liu, B., 2011b. Molecular characterization of a glutathione peroxidase gene and its expression in the selected Vibrio-resistant population of the clam Meretrix meretrix. Fish Shellfish Immunol. 30 (6), 1294– 1302. Wang, M., Yang, J., Zhou, Z., Qiu, L., Wang, L., Zhang, H., et al., 2011c. A primitive Toll-like receptor signaling pathway in mollusk Zhikong scallop Chlamys farreri. Dev. Comp. Immunol. 35 (4), 511–520. Watanabe, A., Miyazawa, S., Kitami, M., Tabunoki, H., Ueda, K., Sato, R., 2006. Characterization of a novel C-type lectin, Bombyx mori multibinding protein, from the B. mori hemolymph: mechanism of wide-range microorganism recognition and role in immunity. J. Immunol. 177 (7), 4594–4604. Wei, Y.X., Guo, D.S., Li, R.G., Chen, H.W., Chen, P.X., 2003. Purification of a big defensin from Ruditapes philippinesis and its antibacterial activity. Sheng wu hua xue yu sheng wu wu li xue bao Acta Biochimica et Biophysica Sinica 35 (12), 1145–1148. Weis, W.I., Taylor, M.E., Drickamer, K., 1998. The C-type lectin superfamily in the immune system. Immunol. Rev. 163 (1), 19–34. Winston, G.W., 1991. Oxidants and antioxidants in aquatic animals. Comp. Biochem. Physiol. C 100 (1–2), 173–176. Wu, L., Song, L., Xu, W., Qiu, L., Li, H., Su, J., et al., 2003. Identification and Cloning of Heat Shock Protein 70 Gene from Scallop Chlamys farreri. High Technol. Lett. 13 (11), 75–79. Wu, X., Xiong, X., Xie, L., Zhang, R., 2007. Pf-Rel, a Rel/nuclear factor-kappaB homolog identified from the pearl oyster, Pinctada fucata. Acta Biochim. Biophys. Sin. (Shanghai) 39 (7), 533–539. Wu, C., Mai, K., Zhang, W., Ai, Q., Xu, W., Wang, X., et al., 2010. Molecular cloning, characterization and mRNA expression of selenium-dependent glutathione peroxidase from abalone Haliotis discus hannai Ino in response to dietary selenium, zinc and iron. Comp. Biochem. Physiol.: Toxicol. Pharmacol. 152 (2), 121–132. Xie, S., Cong, L., Zhang, H., Wang, D., 2011. Molecular cloning and recombinant expression of i-type lysozyme from oyster Crassostrea gigas. Biotechnol. Bull. 4, 127–132. Xu, C., Pan, L., Liu, N., Wang, L., Miao, J., 2010a. Cloning, characterization and tissue distribution of a pi-class glutathione S-transferase from clam (Venerupis philippinarum): response to benzo[alpha]pyrene exposure. Comp. Biochem. Physiol.: Toxicol. Pharmacol. 152 (2), 160–166. Xu, H.H., Ma, H., Hu, B.Q., Lowrie, D.B., Fan, X.Y., Wen, C.G., 2010b. Molecular cloning, identification and functional characterization of a novel intracellular Cu-Zn superoxide dismutase from the freshwater mussel Cristaria plicata. Fish Shellfish Immunol. 29 (4), 615–622. Xu, Q., Wang, G., Yuan, H., Chai, Y., Xiao, Z., 2010c. cDNA sequence and expression analysis of an antimicrobial peptide, theromacin, in the triangle-shell pearl mussel Hyriopsis cumingii. Comp. Biochem. Physiol. B: Biochem. Mol. Biol. 157 (1), 119–126. Yalaoui, S., Huby, T., Franetich, J.F., Gego, A., Rametti, A., Moreau, M., et al., 2008. Scavenger receptor BI boosts hepatocyte permissiveness to Plasmodium infection. Cell Host Microbe 4 (3), 283–292. Yang, J., Qiu, L., Wei, X., Wang, L., Zhou, Z., Zhang, H., et al., 2010a. An ancient C-type lectin in Chlamys farreri (CfLec-2) that mediate pathogen recognition and cellular adhesion. Dev. Comp. Immunol. 34 (12), 1274–1282. Yang, J., Wang, W., Wei, X., Qiu, L., Wang, L., Zhang, H., et al., 2010b. Peptidoglycan recognition protein of Chlamys farreri (CfPGRP-S1) mediates immune defenses against bacterial infection. Dev. Comp. Immunol. 34 (12), 1300–1307. Yang, J.L., Qiu, L.M., Wang, L.L., Wei, X.M., Zhang, H.A., Zhang, Y., et al., 2010c. CfLGBP, a pattern recognition receptor in Chlamys farreri involved in the immune response against various bacteria. Fish Shellfish Immunol. 29 (5), 825–831. Yang, J., Wang, L., Zhang, H., Qiu, L., Wang, H., Song, L., 2011a. C-type lectin in Chlamys farreri (CfLec-1) mediating immune recognition and opsonization. PLoS ONE 6 (2), e17089. Yang, Q., Yang, Z., Li, H., 2011b. Molecular characterization and expression analysis of an inhibitor of NF-kappaB (IkappaB) from Asiatic hard clam Meretrix meretrix. Fish Shellfish Immunol. 31 (3), 485–490. Yu, X.Q., Kanost, M.R., 2004. Immulectin-2, a pattern recognition receptor that stimulates hemocyte encapsulation and melanization in the tobacco hornworm, Manduca sexta. Dev. Comp. Immunol. 28 (9), 891–900. Yu, Z., He, X., Fu, D., Zhang, Y., 2011. Two superoxide dismutase (SOD) with different subcellular localizations involved in innate immunity in Crassostrea hongkongensis. Fish Shellfish Immunol. 31 (4), 533–539. Yue, X., Liu, B., Xue, Q., 2011. An i-type lysozyme from the Asiatic hard clam Meretrix meretrix potentially functioning in host immunity. Fish Shellfish Immunol. 30 (2), 550–558.

Zelensky, A.N., Gready, J.E., 2005. The C-type lectin-like domain superfamily. FEBS J. 272 (24), 6179–6217. Zhang, Q., Wu, X., Gao, J., Pan, J., 2003. Cloning, Southern blotting and RT-PCR analysis of a cDNA fragment encoding of 70 kDa heat shock cognate protein (Hsc70) from the oyster (Crassostrea ariakensis). Acta Zool Sinica. 49 (5), 708– 712. Zhang, H., Song, L., Li, C., Zhao, J., Wang, H., Gao, Q., et al., 2007. Molecular cloning and characterization of a thioester-containing protein from Zhikong scallop Chlamys farreri. Mol. Immunol. 44 (14), 3492–3500. Zhang, H., Song, L., Li, C., Zhao, J., Wang, H., Qiu, L., et al., 2008. A novel C1q-domaincontaining protein from Zhikong scallop Chlamys farreri with lipopolysaccharide binding activity. Fish Shellfish Immunol. 25 (3), 281–289. Zhang, D., Jiang, S., Ma, J., Jiang, J., Pan, D., Xu, X., 2009a. Molecular cloning, characterization and expression analysis of a clip-domain serine protease from pearl oyster Pinctada fucata. Fish Shellfish Immunol. 26 (4), 662–668. Zhang, H., Wang, L., Song, L., Song, X., Wang, B., Mu, C., et al., 2009b. A fibrinogenrelated protein from bay scallop Argopecten irradians involved in innate immunity as pattern recognition receptor. Fish Shellfish Immunol. 26 (1), 56– 64. Zhang, H., Wang, L., Song, L., Zhao, J., Qiu, L., Gao, Y., et al., 2009c. The genomic structure, alternative splicing and immune response of Chlamys farreri thioester-containing protein. Dev. Comp. Immunol. 33 (10), 1070–1076. Zhang, L., Wang, L., Song, L., Zhao, J., Qiu, L., Dong, C., et al., 2010a. The involvement of HSP22 from bay scallop Argopecten irradians in response to heavy metal stress. Mol. Biol. Rep. 37 (4), 1763–1771. Zhang, L., Wang, L., Zhao, J., Qiu, L., Song, L., Dong, C., et al., 2010b. The responsive expression of heat shock protein 22 gene in zhikong scallop Chlamys farreri against a bacterial challenge. Aquac Res. 41 (2), 257–266. Zhang, D., Jiang, S., Hu, Y., Cui, S., Guo, H., Wu, K., et al., 2011a. A multidomain galectin involved in innate immune response of pearl oyster Pinctada fucata. Dev. Comp. Immunol. 35 (1), 1–6. Zhang, L., Li, L., Zhang, G., 2011b. A Crassostrea gigas Toll-like receptor and comparative analysis of TLR pathway in invertebrates. Fish Shellfish Immunol. 30 (2), 653–660. Zhang, Y., He, X., Yu, Z., 2011c. Two homologues of inhibitor of NF-kappa B (IkappaB) are involved in the immune defense of the Pacific oyster, Crassostrea gigas. Fish Shellfish Immunol. 30 (6), 1354–1361. Zhao, J., Song, L., Li, C., Ni, D., Wu, L., Zhu, L., et al., 2007a. Molecular cloning, expression of a big defensin gene from bay scallop Argopecten irradians and the antimicrobial activity of its recombinant protein. Mol. Immunol. 44 (4), 360– 368. Zhao, J., Song, L., Li, C., Zou, H., Ni, D., Wang, W., et al., 2007b. Molecular cloning of an invertebrate goose-type lysozyme gene from Chlamys farreri, and lytic activity of the recombinant protein. Mol. Immunol. 44 (6), 1198–1208. Zhao, J., Li, C., Chen, A., Li, L., Su, X., Li, T., 2010a. Molecular characterization of a novel big defensin from clam Venerupis philippinarum. PLoS ONE 5 (10), e13480. Zhao, J., Qiu, L., Ning, X., Chen, A., Wu, H., Li, C., 2010b. Cloning and characterization of an invertebrate type lysozyme from Venerupis philippinarum. Comp. Biochem. Physiol. B: Biochem. Mol. Biol. 156 (1), 56–60. Zhong, Y., Liu, J., Zhang, J., Feng, W., Jiao, B., 2005. Cloning, expression and biological characterization of Myticin A, an antimicrobial peptide from mussel (Mytilus). Acad. J. Second Military Med. Univ. 26 (1), 65–68. Zhou, Z., Wang, L., Gao, Y., Wang, M., Zhang, H., Qiu, L., et al., 2011a. A monoamine oxidase from scallop Chlamys farreri serving as an immunomodulator in response against bacterial challenge. Dev. Comp. Immunol. 35 (7), 799–807. Zhou, Z., Wang, L., Shi, X., Zhang, H., Gao, Y., Wang, M., et al., 2011b. The modulation of catecholamines to the immune response against bacteria Vibrio anguillarum challenge in scallop Chlamys farreri. Fish Shellfish Immunol. 31 (6), 1065–1071. Zhou, Z., Wang, L., Wang, M., Zhang, H., Wu, T., Qiu, L., et al., 2011c. Scallop phenylalanine hydroxylase implicates in immune response and can be induced by human TNF-a. Fish Shellfish Immunol. 31 (6), 856–863. Zhou, Z., Wang, L., Yang, J., Zhang, H., Kong, P., Wang, M., et al., 2011d. A dopamine beta hydroxylase from Chlamys farreri and its induced mRNA expression in the haemocytes after LPS stimulation. Fish Shellfish Immunol. 30 (1), 154–162. Zhou, Z., Yang, J., Wang, L., Zhang, H., Gao, Y., Shi, X., et al., 2011e. A dopa decarboxylase modulating the immune response of scallop Chlamys farreri. PLoS ONE 6 (4), e18596. Zhu, B., Wu, X., 2008. Identification of outer membrane protein ompR from rickettsia-like organism and induction of immune response in Crassostrea ariakensis. Mol. Immunol. 45 (11), 3198–3204. Zhu, L., Song, L., Chang, Y., Xu, W., Wu, L., 2006. Molecular cloning, characterization and expression of a novel serine proteinase inhibitor gene in bay scallops (Argopecten irradians, Lamarck 1819). Fish Shellfish Immunol. 20 (3), 320–331. Zhu, L., Song, L., Zhao, J., Xu, W., Chang, Y., 2007. Molecular cloning, characterization and expression of a serine protease with clip-domain homologue from scallop Chlamys farreri. Fish Shellfish Immunol. 22 (5), 556–566. Zhu, L., Song, L., Mao, Y., Zhao, J., Li, C., Xu, W., 2008. A novel serine protease with clip domain from scallop Chlamys farreri. Mol. Biol. Rep. 35 (2), 257–264. Zhu, D., Li, H., Gao, X., Su, H., He, C., 2010. Molecular cloning and sequence analysis of an intracellular Cu/Zn-superoxide dismutase gene from hard clam (Meretrix meretrix). Biotechnol. Bull. 11, 123. Zou, H., Song, L., Xu, W., Yang, G., 2005. Molecular cloning and characterization analysis of cDNA encoding g-type lysozyme from scallop (Argopecten irradians). High Technol. Lett. 15 (7), 101–106.