The oyster immunity

Developmental and Comparative Immunology 80 (2018) 99e118

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

The oyster immunity Lingling Wang 1, Xiaorui Song 1, Linsheng Song* Liaoning Key Laboratory of Marine Animal Immunology and Disease Control, DalianOcean University, Dalian 116023, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 23 March 2017 Received in revised form 21 May 2017 Accepted 21 May 2017 Available online 3 June 2017

Oysters, the common name for a number of different bivalve molluscs, are the worldwide aquaculture species and also play vital roles in the function of ecosystem. As invertebrate, oysters have evolved an integrated, highly complex innate immune system to recognize and eliminate various invaders via an array of orchestrated immune reactions, such as immune recognition, signal transduction, synthesis of antimicrobial peptides, as well as encapsulation and phagocytosis of the circulating haemocytes. The hematopoietic tissue, hematopoiesis, and the circulating haemocytes have been preliminary characterized, and the detailed annotation of the Pacific oyster Crassostrea gigas genome has revealed massive expansion and functional divergence of innate immune genes in this animal. Moreover, immune priming and maternal immune transfer are reported in oysters, suggesting the adaptability of invertebrate immunity. Apoptosis and autophagy are proved to be important immune mechanisms in oysters. This review will summarize the research progresses of immune system and the immunomodulation mechanisms of the primitive catecholaminergic, cholinergic, neuropeptides, GABAergic and nitric oxidase system, which possibly make oysters ideal model for studying the origin and evolution of immune system and the neuroendocrine-immune regulatory network in lower invertebrates. © 2017 Elsevier Ltd. All rights reserved.

Keywords: Oyster Innate immune system Hematopoiesis Immune priming Apoptosis and autophagy Neuroendocrine-immune regulation

1. Introduction Oysters are worldwide aquaculture animals, which belong to the second largest animal phylum Mollusca. Oysters not only play vital roles in terrestrial and marine ecosystems, but also contribute to the coastal economy of many countries (Guo, 2009; Ponder and Lindberg, 2008). As sessile and filter-feeding marine animals living in estuarine and intertidal regions, oysters have to cope with harsh and dynamically environmental changes including a wide range of biotic (bacterial and viral) and abiotic (dynamic variation in temperature, salinity and prolonged desiccation) stresses. The accumulating evidences indicate that oysters have formed a highly complex immune system with remarkable discriminatory properties and it can respond to different pathogens as well as environmental stress during the long time evolution and adaption (Corporeau et al., 2012; Zhang et al., 2012a, 2015b). It is generally recognized that oysters lack the lymphocytemediated adaptive immunity, and they only possess innate immunity which is a common character of both vertebrates and

* Corresponding author. E-mail address: [email protected] (L. Song). 1 These authors contribute equally to this article. http://dx.doi.org/10.1016/j.dci.2017.05.025 0145-305X/© 2017 Elsevier Ltd. All rights reserved.

invertebrates. The innate immunity consists of a cellular and a humoral arm. The cellular immune reaction including phagocytosis and encapsulation is performed by circulating haemocytes with subsequent pathogen destruction via enzyme activity and release of oxygen metabolite. Humoral immunity refers to the immune response that happens in body fluid. This process begins from the immune recognition of the conserved pathogen-associated molecular patterns (PAMPs) presenting in microbes by the germline encoded pathogen-associated pattern recognition receptors (PRRs). Upon PAMP recognition, PRRs activate intracellular signaling pathways, and then trigger the synthesis of antimicrobial effectors (Akira et al., 2006). The hematopoietic tissue, hematopoiesis, and the circulating haemocytes have been preliminary characterized, and the maternal immune transfer and immune priming are also suggested to exist in oyster (Jema a et al., 2014; Li et al., 2017; Zhang et al., 2014b). Besides, apoptosis and autophagy are proved to be functional in the host defense system of oyster (Hughes et al., 2010; Moreau, 2014; Pierrick et al., 2015). Recently, a complex neuroendocrine-immune (NEI) regulatory network was reported in oyster which could modulate the immune responses by releasing various neurotransmitters and hormones (Chang et al., 2015; Liu et al., 2016d). On this point, we summarize the research progress of oyster immunity with the emphasis on (1) the molecular constitution and the defense mechanism of innate immune system,

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(2) maternal immune transfer, (3) immune priming, (4) hematopoiesis, (5) apoptosis, (6) autophagy as well as (7) the NEI regulatory work. 2. The molecular constitution of innate immune system As invertebrate, oysters rely exclusively on the sophisticated cascade of innate immune reactions to eliminate invaders. To mount effective and flexible defense responses against diverse pathogens, oysters have developed diversified repertoires of receptors, regulators, and/or effectors including PRRs, and antimicrobial proteins (AMPs), as well as many other molecules involved in the key processes of agglutination, phagocytosis, and encapsulation. The following is the summary about the molecular constitution of oyster immune system, mainly focusing on immune recognition, signaling pathway and effector synthesis involved in cellular and humoral immunity. 2.1. Immune recognition Oysters have developed a sophisticated repertoire of PRRs to recognize diversiform microorganisms. Most of the PRRs serve as multi-functional proteins, not only in immune recognition, but also in the elimination of invading microbes. So far, various PPRs, such as peptidoglycan recognition proteins (PGRPs), lectins (including Ctype lectins, galectins and sialic acid-binding Ig-like lectins), tolllike receptors (TLRs), Gram-negative binding proteins (GNBPs), scavenger receptors (SRs) and fibrinogen-related proteins (FREPs) have been identified in oysters (Table 1). 2.1.1. Peptidoglycan recognition proteins PGRPs are kind of highly conserved PRRs to recognize the peptidoglycans (PGN) in the cell wall of invading bacteria (Royet et al., 2011). Nine PGRP genes are predicated in the oyster genome (Zhang et al., 2015b), and six of them (CgPGRP-S1S, -S1L, -S2, -S3, -S4 and CgPGRP-L) have been identified and characterized to be short PGRP type with a conserved PGRP/amidase domain in their C-terminus (Iizuka et al., 2014; Itoh and Takahashi, 2008, 2009; Yang et al., 2016). However, the recombinant PGRP-S1S (rPGRP-S1S) do not have any bactericidal activity. It can agglutinate Escherichia coli and induce secretion of granular contents by haemocyte degranulation (Iizuka et al., 2014). Although the molecular weight of CgPGRP-L is close to that of long PGRP groups, it is homologous to short PGRPs and possesses an additional goose-type (g-type) lysozyme domain, suggesting that CgPGRP-L may have both binding and lytic functions against the bacterial cell wall (Itoh and Takahashi, 2009). Recently, several genes encoding PGRP/amidase domains were identified by searching the Pacific oyster EST database and phylogenetic analysis suggested a positive Darwinian selection in the CgPGRP family (Swaminathan et al., 2006; Zhang and Yu, 2013). Although all the oyster PGRP genes contain conserved PGRP/ amidase domain, the critical residues involved in specific PGN recognition show a certain degree of mutation, which may create a more flexible response to different microbial challenges. 2.1.2. Toll-like receptors TLRs are evolutionarily conserved from the cnidarians to mammals with essential role in host defense and numerous TLRs have been identified in different organisms. Although the innate immune responses mediated by TLRs share a common ancient ancestry both in vertebrates and invertebrates, the domain organization, activation mode and functions are different (Imler and Zheng, 2004; Kanzok et al., 2004). An expanded set of 83 TLR genes is predicted in the oyster genome, which can be divided into five groups based on the

patterns of sequence relatedness: V (vertebrate-type), P (protostome-like with LRRCT [leucine-rich repeat C-terminal]-LRRNT [leucine-rich repeat N-terminal] ectodomains), sP (short protostome-like without LRRCT-LRRNT ectodomains), sPP (short protostome-like with LRRCT-LRRNT ectodomains) and Ls (LRRCTspecific ectodomains) (Zhang et al., 2015b). Especially, sP-type TLRs are most greatly expanded in oyster, similar with equally extensive expanded P-type TLRs in Drosophila (Rock et al., 1998) and V-type TLRs in the sea urchin (Rast et al., 2006), suggesting that speciesspecific TLR gene expansion is a frequent, independent occurrence in metazoan phylogeny. Meanwhile, the large number of Lsand P-type TLRs in oyster suggests that these genes are derived from oyster-specific expansions and may play a major role in host defense. As the important molecules in innate immune recognition, the expressions of TLRs induced by single or different combinations of bacteria/ostreid herpesvirus type 1 (OsHV-1) stimulation are different, underscoring the high degree of divergence of immune function within the tandemly duplicated, tightly linked TLR genes in oyster (Zhang et al., 2015b). So far, at least six oyster TLRs have been identified to participate in immune response. CgToll-1 is highly expressed in hemolymph of C. gigas, and its expression increases dramatically after the challenge of Vibrio anguillarum (Zhang et al., 2011c). Another four Vtype CgTLRs share comparable subcellular localization patterns and they distribute on late endosomes and plasma membranes. CgTLR1-4 can activate NF-kB reporter in a dose-dependent manner, suggesting an ancient and conserved link between TLRs and NF-kB signaling in oysters. However, they all lack the activities of specific PAMPs recognition (Zhang et al., 2013). Recently, another TLR (CgTLR-6) was identified from C. gigas, which exhibited not only broad recognition spectrum for bacteria and fungi, but also affinity to lipopolysaccharide (LPS) and PGN. The antibody blockage of CgTLR6 did not significantly inhibit the phagocytic rates of haemocytes. These results collectively imply that CgTLR6 is a newly described non-phagocytic oyster receptor to mediate the humoral immune response by recognizing PAMPs on the invaders (Wang et al., 2016). TLR genes in oyster are hypervariable and most of them are expressed differentially after bacterial and viral challenges, which underscore the high degree of functional divergence within the tandemly duplicated, tightly linked TLR genes. 2.1.3. Lectins Lectins are carbohydrate-binding proteins to bind glycans of glycoproteins, glycolipids, or polysaccharides with high affinity. They are not only involved in the recognition of potential pathogens, but also participate in downstream reaction cascades, such as agglutination, immobilization, and complement-mediated opsonization and killing (Liener, 2012). Presently, at least seven groups of lectins have been identified in oyster including C-type, P-type, Ftype, I-type, galectins, ficolins and chitinase-like lectins (Badariotti et al., 2007; Xiang et al., 2014; Yamaura et al., 2008). Three extracellular lectins of them including C-type lectins (CLecs), galectins and siglecs are discussed in this circumstance with the emphases on their functions, such as cell adhesion, cell signaling, glycoprotein clearance and pathogen recognition in oyster. CLecs, a large family of proteins found almost in all metazoans, can bind carbohydrates in a calcium-dependent manner (Robinson et al., 2006; Weis et al., 1998). Generally, CLecs contain at least one carbohydrate-recognition domain (CRD) with about 120 residues. Presently, a more general term “C-lectin domain containing proteins” (CTLDCs) has been introduced to define the proteins with one or more CTLDs, regardless of carbohydrate or calcium binding ability (Zelensky and Gready, 2005). CLecs are abundant in oyster, and widely distributed in various tissues. For Pacific oyster C. gigas, there is an expanded set of 266 CTLDCs annotated in its genome,


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Table 1 The PRRs reported in oysters. Gene name

Accession No.

Peptidoglycan recognition proteins CgPGRP-S1S AB425335.1 CgPGRP-S1L AB425336.1 CgPGRP-S2 AB425337 CgPGRP-S3 AB425338.1 CgPGRPS4 EKC26200 CgPGRP-L AB473552 Lectins CgClec-1 AB308130 CgClec-2 JH818449 CgClec-3 e CgClec-4 XP_011415552 CgClec-5 EKC39564 CgClec-6 e CgClec-7 e CvML e poLEC2 HQ827789 poLEC1 FJ812172 C-type lectin-1 KP689431.1 PmF-lectin HQ199600 CgGal Ab308370 poGal1 FJ812171 poGal2 HQ014601 PfGal FJ267519 CvGal DQ779197 Toll-like receptors CgToll-1 HQ174216 CgTLR1 KC700617 CgTLR2 KC700618 CgTLR3 KC700619 CgTLR4 KC700620 TLR6 EKC38225 Gram-negative binding proteins Cg-bGBP-1 NM_001305364 Cg-bGBP-2 AB377115 poLGBP FJ775601 Scavenger receptorslike PmSR-B KX889917.1 Fibrinogen-related proteins CgFREP-1 CgFREP-2 CgFREP-3 CgFREP-4 CgFREP-5

Species

Domain

Reference

C. C. C. C. C. C.

PGRP/amidase domain, defensin-like domain PGRP/amidase domain, defensin-like domain PGRP/amidase domain e PGRP/amidase domain PGRP/amidase domain, goose-type lysozyme domain

(Iizuka et al., 2014; Itoh and Takahashi, 2008; Yang et al., 2016)

One CRD One CRD

(Yamaura et al., 2008) (Li et al., 2015a,b) Unpublished (Jia et al., 2016) (Jia et al., 2016) Unpublished Unpublished (Jing et al., 2011) Unpublished Unpublished Unpublished (Chen et al., 2011b) (Yamaura et al., 2008) (Zhang et al., 2011b) (Zhang et al., 2011a) (Wang et al., 2011) (Tasumi and Vasta, 2007)

gigas gigas gigas gigas gigas gigas

(Yang et al., 2016) (Itoh and Takahashi, 2009)

C. gigas C. gigas C. gigas C. gigas C. gigas C. gigas C. gigas C. virginica P. fucata P. fucata P. martensii P. fucata C. gigas P. fucata P. fucata P. fucata C.virginica

One CRD One CRD One CRD One CRD One CRD e e e e One CRD four CRDs Two tandem GRDs Two tandem GRDs four CRDs

C. C. C. C. C. C.

LRRNT, 19 LRR TIR domain, LRRCT, 7 LRR TIR domain, 6 LRR TIR domain, LRRCT/NT, 18 LRR, TIR domain, LRRCT/NT, 7 LRR TIR domain, LRRCT, 5 LRR

(Zhang et al., 2011c) (Zhang et al., 2013)

C. gigas C. gigas P. fucata

e e e

(Itoh et al., 2010a) (Itoh et al., 2010a) (Zhang et al., 2010)

P. martensii

e

Direct Submission

C. C. C. C. C.

fibrinogen-like fibrinogen-like fibrinogen-like fibrinogen-like fibrinogen-like

gigas gigas gigas gigas gigas gigas

gigas gigas gigas gigas gigas

and seven CgCLecs have been identified. CgCLec-1, CgCLec-2 and CgCLec-4 are secreted lectins, and CgCLec-3, CgCLec-5 and CgCLec6 locate at intracellular, while CgCLec-7 is a transmembrane lectin (unpublished data in our laboratory). CgCLec-1 is expressed only in specialized basophilic cells in the digestive gland (Yamaura et al., 2008), while other CgCLecs and mucosal C-type lectin (CvML) from C. virginica can be detected in various tissues (Jia et al., 2016; Jing et al., 2011; Li et al., 2015b). Generally, there are four Ca2þbinding sites in the CRD domain, among which, site 2 contains two remarkable amino acid motifs, EPN (Glu-Pro-Asn)/QPD (Gln-ProAsp) and WND (Trp-Asn-Asp). In Ca2þ-binding site 2 of CgCLec-2, the motif is EPN. However, it is QPE (Glu-Pro-Asn) in CgCLec-4, QYE (Glu-Tyr-Asn, a non-a typical motif) in CgCLec-5, YPD in CvML, and WHD in CgCLec-6, showing considerable variety in oyster (Jia et al., 2016; Jing et al., 2011; Li et al., 2015b). The mRNA expressions of almost all the oyster CLecs are up-regulated after bacteria stimulations. Moreover, all the recombinant proteins of rCgCLec-2 to rCgCLec-7 can bind various PAMPs and display strong binding abilities to different microorganisms. rCgCLec-2 and rCgCLec-4 exhibit antimicrobial activity against Gram-positive bacteria and fungi. Furthermore, rCgCLec-2 and rCgCLec-6 can function as opsonins to enhance the phagocytic activity of oyster

domains domains domains domains domains

(Wang et al., 2016)

(Zhang et al., 2012b)

haemocytes towards V. splendidus (Jia et al., 2016; Li et al., 2015b). Especially, rCgCLec-2 can interact with ascidian MBL-associated serine proteases (MASPs), which is similar as that of vertebrate Clecs (Li et al., 2015b). The expansion of oyster CLecs is associated with their functional diversities, which contributes to the carbohydrate binding activity and specificity. Galectin family is defined by a conserved CRD with a canonical amino acid sequence and affinity primary for b-galactoside (Sato et al., 2009). Previous studies demonstrate that bivalve galectins can target glycans on the surfaces of bacteria and parasites, and play a crucial role in innate immune responses (Morga et al., 2011; Rabinovich and Gruppi, 2005; Sato et al., 2009). CgGal from C. gigas is a prototype galectin with a single galactose-binding domain, but its expression in haemocytes does not change significantly after injection of V. tubiashii (Yamaura et al., 2008). CvGal from C. virginica with four unique CRD organization domains can recognize exogenous carbohydrate ligands, which is clearly different from most mammalian galectins. CvGal can particularly facilitate the recognition of a variety of potential microbial pathogens, unicellular algae and preferentially Perkinsus trophozoites. The strong interaction between CvGal and P. trophozoites suggests that CvGal can function as a haemocyte surface receptor for the parasite,

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and mediates the phagocytosis (Tasumi and Vasta, 2007). PoGal1 and PoGal2 are two tandem-repeat type galectins characterized from P. fucata with four and two CRDs respectively. Their mRNA expressions are significantly up-regulated after V. alginolyticus stimulation, suggesting their involvements in the immune response against bacterial infection (Wang et al., 2011; Zhang et al., 2011a, 2011b). The phylogenetic analysis suggests that the oyster galectin CRDs share a common ancient and the multiple individual CRDs of each galectin are originated by repeated duplication of a single galectin gene (Tasumi and Vasta, 2007; Zhang et al., 2011b). Siglec, a subcollection of immunoglobulin superfamily (IgSF) with a single N-terminal V-set immunoglobulin-like domain as sialic acid-binding CRD, is involved in the host-pathogen recognition, cell-cell interactions and subsequent signaling pathways in the immune and nervous systems (Crocker et al., 2007). So far, oyster siglecs have only been identified in C. hongkongensis (Chsalectin) and C. gigas (CgSiglec-1). Chsalectin protein, as counterpart of siglec in the invertebrate, contains a conserved complement component C1q domain (He et al., 2011a). CgSiglec-1 is composed of two I-set immunoglobulin (Ig) domains, one transmembrane (TM) domain and two ITIM motifs, and shares a higher sequence similarity with vertebrate CD22 homologs (Liu et al., 2016a). Both of Chsalectin and CgSiglec-1 can bind sialic acid containing proteins and their mRNA transcripts are significantly up-regulated after Vibrio infection. Additionally, rChsalectin displays strong antibacterial activity, and CgSiglec-1 can bind LPS and PGN. The blockade of CgSiglec-1 by specific polyclonal antibodies can enhance the LPSinduced cell apoptosis, phagocytosis towards V. splendidus and the release of cytokines, such as CgTNF-1, CgIFNLP and CgIL-17 (He et al., 2011a; Liu et al., 2016a). These results highlight the essential roles of siglecs in immune recognition and host defense against bacterial infection in oyster. F-type lectins (fucolectins) are fucose-binding proteins, which have been reported in P. martensii (PmF-lectin) and C. gigas. PmFlectin mRNA transcripts are abundant in haemocytes and gill and they are dramatically up-regulated after a challenge with V. alginolyticus, suggesting that PmF-lectin is involved in the innate immune response (Chen et al., 2011b). Two chitinase-like proteins (CgClp1 and CgClp2) have been identified from C. gigas, and they are involved in oyster immunity by increasing mRNA expressions in haemocytes after immune challenges (Badariotti et al., 2006, 2007). A novel ficolin-like protein (ChFCN) identified from C. hongkongensis contains a typical signal peptide and a fibrinogenrelated domain but does not contain the additional ficolin collagenlike domain. The mRNA expression of ChFCN is increased after microbial challenges and its recombinant protein can bind and agglutinate microorganisms, and enhance the phagocytosis (Xiang et al., 2014). 2.1.4. Gram-Negative Binding Protein (GNBP) The GNBP family, including three members GNBP, the LPS and b1, 3-glucan binding protein (LGBP), and b-1, 3-Glucan binding proteins (bGBP), can bind Gram-negative bacteria, LPS and GLU (Christophides et al., 2002). So far, there are only two cDNAs coding bGBPs (CgbGBP-1 and CgbGBP-2) reported in oyster. The domain structures of CgbGBPs are similar to other invertebrate bGBPs. Integrin recognition sites are identified in CgbGBP-1, but not in CgbGBP-2. The recombinant CgbGBP-2 enhances the phenoloxidase (PO) activity of haemocyte under the presence of laminarin, but rCgbGBP-1 does not show this enhancement. It is suggested that CgbGBPs in the Pacific oyster have evolved with different immunological functions, CgbGBP-1 for haemocyte-related functions through integrin and CgbGBP-2 for PO activation (Itoh et al., 2010a). The LGBP isolated from P. fucata (poLGBP), possesses a potential polysaccharide-binding motif, a glucanase motif, a LPS-binding site

and a b-1, 3-linkage of polysaccharide. Its mRNA transcripts are specifically expressed in digestive gland and significantly upregulated after bacteria or LPS stimulation, suggesting that poLGBP is an inducible acute-phase protein and plays an important function in digestion as digestive enzyme and pattern recognition receptor (Zhang et al., 2010). 2.1.5. Scavenger receptors (SRs) SRs represent a large family of endocytic receptors with multifunction to bind and internalize a variety of microbial pathogens, as well as modified or endogenous molecules derived from the host, and contribute to a range of physiological or pathological processes (Mukhopadhyay and Gordon, 2004). According to their multidomain structure, SRs are classified into six different classes (classes A, B, C, D, E and F) (Areschoug and Gordon, 2009). However, the information about oyster SRs is extremely limited, and only one SR-B has been identified in the P. martensii. The increased genomic information indicates that there is an expansion of SRs and about 71 members have been annotated in oyster, and more especially, they have experienced high levels of diversification (McDowell et al., 2016; Zhang et al., 2015b). The mRNA transcripts of multiple SRs are highly up-regulated in response to summer mortality syndrome (Fleury et al., 2010; Huvet et al., 2004), hypoxia (David et al., 2005) and bacterial challenge (McDowell et al., 2014), suggesting their significant roles in adaption to complex environment. 2.1.6. Fibrinogen-related proteins (FREPs) FREPs, a kind of fibrinogen-like (FBG) domain containing proteins with high levels of sequence diversity, play an important role as immune PRRs (Middha and Wang, 2008). Oyster FREPs exhibit expansion and diversification, and there are about 190 FREPs with more than 200 FBG domains predicted in the genome of C. gigas. These FREPs are highly expressed in gill, suggesting their major roles in oyster immune system (Huang et al., 2015). Phylogenetic analysis reveals that CgFREPs are derived from oyster-specific expansion with individual hypermutation (Zhang et al., 2015b). For instance, five types of FREP (CgFREP-1 to CgFREP-5) can be amplified with a single pair of primers, confirming their high diversity (Zhang et al., 2012b). The compatibility polymorphism hypothesis proposes that FREP mutation increases the range of germline-encoded immune recognition in oyster to counter antigenically-varied parasites (Adema, 2015; Hanington and Zhang, 2010). 2.2. The signaling pathway in immune response The transmission of extracellular signals into their intracellular targets is mediated by a network of interacting proteins that regulate a large number of cellular processes. Cumulative efforts over the past decades have allowed the elucidation of several signaling mechanism in oyster, such as the TLR signaling pathway, NF-kB signaling pathway, mitogen-activated protein kinase (MAPK) signaling cascade, Prophenol/phenol oxidase cascade and complement pathway in oyster. 2.2.1. The toll-like receptors signaling pathway TLRs have been established to play an essential role in the activation of innate immunity by recognizing specific patterns of microbial components. The characterization of TLR and its signaling pathway is one of milestone events and immune routines in immunology during the last decades (Kanzok et al., 2004; Takeda and Akira, 2004). The accumulating evidences have demonstrated that a series components of TLR signaling pathway exist in oyster, such as TLR, MyD88, IRAK, TRAF, ECSIT, IKK, IkB and NF-kB. MyD88 serves as a critical cytosolic adaptor modulating TLR


signaling pathway, and it consists of an N-terminal death domain as well as a C-terminal TIR domain (Horng and Medzhitov, 2001; Hultmark, 1994). The oyster genome encodes an expanded set of 10 MyD88 genes, indicating that MyD88 genes have undergone a considerable expansion coupled to TLRs (Zhang et al., 2015b). So far, eight MyD88s (CgMyD88-A to CgMyD88-D, CgMyD88-1, CgMyD882, truncated CgMyD88-T1 and CgMyD88-T2) have been identified in Pacific oyster C. gigas. CgMyD88-A to CgMyD88-D, CgMyD88-1 and CgMyD88-2 are typical MyD88s, containing both death domain and TIR domain (Du et al., 2014; Xin et al., 2016), whereas two truncated MyD88s only have TIR domain (Xu et al., 2015b). The expressions of CgMyD88-B, CgMyD88-C, CgMyD88-T1 and CgMyD88-T2 can be induced after bacterial challenge, while that of CgMyD88-A and CgMyD88-D are suppressed. Moreover, CgMyD88B and CgMyD88-C can promote the activation of NF-kB signaling pathway, while the other four CgMyD88s fail or even suppress the activities of CgMyD88-B and CgMyD88-C on the activation of NF-kB signaling (Xin et al., 2016; Xu et al., 2015b). Although the expressions MyD88 genes were up-regulated during OsHV-1 infection before mortality occurred (Du et al., 2014; Renault et al., 2011), there was no significant association between MyD88 expression level and surviving spat after virus infection, suggesting that MyD88 could be interpreted as a marker of infectious processes (Segarra et al., 2014a, 2014b). Tumor necrosis factor (TNF) receptor-associated factors (TRAFs) are a family of adaptor proteins involved in signaling by both TNF receptors and TIR receptors (Inoue et al., 2000). To date, seven members of TRAF family (TRAF1 through to TRAF7) have been identified in mammals, among which TRAF1, TRAF2, TRAF3 and TRAF7 are confirmed to exist in oyster. CgTRAF2 and CgTRAF3 from C. gigas and PfTRAF3 from pearl oyster P. fucata can respond to the challenges with V. alginolyticus or OsHV-1, and they are supposed to be involved in a MAVS-mediated immune signaling pathway (Huang et al., 2014, 2016, 2012b). However, the mRNA expression of TRAF7 homolog (ChTRAF7), isolated from C. hongkongensis, is suppressed in the haemocytes after bacterial infection, indicating that ChTRAF7 may play a unique role in signal transduction during the immune response of oysters (Fu et al., 2011b). Moreover, TRAF1 is overexpressed in the parasite infected oyster C. virginica, while not in C. gigas, reflecting the greater cellular disorder caused by parasite in the C. virginica (Tanguy et al., 2004). Evolutionarily conserved signaling intermediate in Toll pathways (ECSIT) is specific for the Toll/IL-1 pathways and bridges TRAF6 to MEKK-1 (Barton and Medzhitov, 2003). The homolog of ECSIT (ChECSIT) has been identified in oyster C. hongkongensis and it shares similar structural characteristics with other known ECSIT proteins. ChECSIT is located primarily in the cytoplasm, and its overexpression can stimulate the transcriptional activity of an NFkB reporter gene in HEK293T cells, suggesting its involvement in immune responses (Qu et al., 2015a). The other related genes in TLR signaling pathway, such as SARM, ARM-TIR, IG-TIR, TIR-TPR, EGF-TIR and Orphan-TIR are also annotated in oyster genome, which suggests the existence of the canonical and complete TLR signaling pathway in oyster. The information is helpful to understand the origin, evolution and crucial roles in innate immunity of TLR signaling pathway. 2.2.2. The canonical NF-kB signaling pathway NF-kB signaling pathway is considered as canonical intracellular signaling cascades with the central role of transcription factor Rel/ NF-kB family. The three key molecules, Rel/NF-kB (Huang et al., 2012a; Montagnani et al., 2004; Wu et al., 2007), IkB (Montagnani et al., 2008; Xu et al., 2015a; Zhang et al., 2009; Zhang et al., 2011f) and IKK (Escoubas et al., 1999; Xiong et al., 2008), are all identified in the Pacific oyster C. gigas and pearl oyster P. fucata.

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LPS and V. alginolyticus stimulations apparently up-regulate the expression level of PfRel, and transiently stimulate poIkB degradation, but do not influence the expression level of PfiKK (Wu et al., 2007; Xiong et al., 2008; Zhang et al., 2009). There are three CgIkBs identified in C. gigas, which inhibit Rel-dependent NF-kB activation in HEK293 cells in a dose-dependent manner and respond to bacterial challenge (Xu et al., 2015a; Zhang et al., 2011f). Unlike CgIkB1 and CgIkB2, CgIkB3 lacks PEST (regions rich in proline, glutamic acid, serine, and threonine) domain and C-terminal casein kinase II phosphorylation site, and only contains a conserved degradation motif with five ankyrin repeats. It is consistent with the multiple forms of vertebrate IkB to regulate NF-kB by distinct mechanisms (Moynagh, 2005). There is no significant change of IkB2 expression after experimental infection in adult oysters, whereas it is significantly increased in spat C. gigas after OsHV-1 infection. Furthermore, there is a positive correlation between IkB2 expression level and viral DNA amounts, which suggests the crucial role of IkB2 in virus recognition and cell activation (Segarra et al., 2014a, 2014b). 2.2.3. Mitogen-Activated Protein Kinases (MAPK) pathway MAPKs compose a family of protein kinases with conserved function and regulation. Several ESTs homologous of the MAPK pathway components, CgMAPKK1, CgMAPKK2, Cgc-jun, Cgphosphatase, Cgfocal and CgFAK have been screened from the cDNA library of oyster C. gigas, and a novel p38 MAPK (Chp38) and JNK (ChJNK) are identified from C. hongkongensis. Chp38 and ChJNK are constitutively expressed in various oyster tissues and developmental stages. Their expression levels in haemocytes are significantly up-regulated after bacteria and PAMPs stimulations. Moreover, the overexpression of Chp38 and ChJNK can significantly enhance the transcriptional activities of AP-1-Luc in HEK293T cells (Qu et al., 2016, 2017). These results suggest the involvement of MAPK in the innate immunity of oyster. Further investigations in molecular structure and functions are required for the precise defining of all molecules involved in MAPK pathway of oysters. 2.2.4. Complement pathway Complement system, consisting of more than 40 plasma proteins that function either as enzymes or as binding proteins, is organized in three activation pathways in the vertebrates, named classical, alternative and lectin pathways. All the three pathways share the common step of the activating the central component C3, but they differ according to the nature of recognition (Carroll, 2004; Sarma and Ward, 2011). Several complement molecule homologs have been found in oysters, such as C3, MBL, C1q, ficolin and MASP. Large numbers of fibrinogen domain-containing proteins, Clectin domain containing proteins and FREPs, have been identified in oyster (Huang et al., 2015; Zhang et al., 2015b). CgCLec-2, CgCLec3 and CgCLec-4 all contain the typical CRDs with ligand binding activities similar as MBL in vertebrates. They can recognize and bind a variety of PAMPs and microbes, exhibit microbial agglutination activities, and significantly facilitate the phagocytosis of haemocytes towards V. splendidus (Jia et al., 2016; Li et al., 2015b). Ficolins, belonging to the FREP family, are oligomeric proteins characterized by an N-terminal collagen-like domain and a C-terminal FBG domain. A novel ficolin-like protein (designated ChFCN) has been identified from C. hongkongensis, which is ubiquitously expressed in several tissues with the highest expression level in gills. Recombinant ChFCN proteins (rChFCN) bind Saccharomyces cerevisiae, S. haemolyticus and E. coli K-12, but not V. alginolyticus. Furthermore, the rChFCN protein agglutinates Gram-negative bacteria E. coli K-12 and enhances the phagocytosis of C. hongkongensis haemocytes in vitro (Xiang et al., 2014). MASP is a key enzyme which associates with MBL or ficolins and contributes to the activation of lectin complement pathway (Takahashi et al.,

104


2008). MASP-like molecule (CgMASPL-1) is identified from C. gigas, and it interacts with rCgCLec-2 to form a collectin/MASP complex, which is similar to that in higher vertebrates to be involved in the activation of lectin pathway (Li et al., 2015b). C1q is the first subcomponent of the C1 complex of the classical pathway and the major connecting link between innate and adaptive immunity. As a PRR molecule, C1q can engage a broad range of ligands via its globular (gC1q) domain and modulate immune cells, probably via its collagen region (Bohlson et al., 2007; Kishore et al., 2004). An expanded set of 321 C1q domaincontaining proteins (C1qDC) is found in oyster genome, but only one C1q-like protein has been identified with a typical vertebratetype collagen domain (Gerdol et al., 2015; Zhang et al., 2015b). The genes encoding C3, factor B-like molecules, CD109, alpha-2 macroglobulins and thioester-containing proteins, as well as other related genes in the alternative complement pathway have also been reported in oyster (Zhang et al., 2015b), although their physiological functions are not yet well understood. Collectively, a rudimentary complement system with a set of expanded and diversified genes is suggested to exist in oyster. 2.2.5. Prophenol/phenol oxidase cascade The prophenoloxidase system is the origin of melanin production and it is considered to be an innate defense mechanism in invertebrate. ProPO cascade is intimately associated with the appearance of factors stimulating cellular defense by aiding phagocytosis and encapsulation reactions (Cerenius et al., 2008; € derha €ll and Cerenius, 1998; Sugumaran, 2002). In oysters, PO is So found to exist as an inactive form of proPO both in plasma component of hemolymph and hemocytes, and can be activated by exogenous protease (trypsin and chymotrypsin) as well as other modulators/activator (LPS, zymosan or laminarin). Meanwhile, many factors can inhibit PO-like activities through repressing the spontaneous PO activity (Aladaileh et al., 2007a; Hellio et al., 2007; Luna-Acosta et al., 2011; Thomas-Guyon et al., 2009). The proPO activating system in Sydney rock oysters is a proteinase cascade in which Ca2þ-dependent serine proteinases proteolytically convert proPO into active PO. The activated PO is a tyrosinase-like enzyme responsible for both monophenolase and diphenolase activity (Aladaileh et al., 2007a). POs have been demonstrated to involve in haemolymphatic immune defense in oysters. PO activity is significantly suppressed in Sydney rock oysters suffered from QX disease (Butt and Raftos, 2008; Peters and Raftos, 2003). Similarly, PO activity is also inhibited by the presence of P. marinus in C. virginica (Jordan and Deaton, 2005). Laccase-type enzyme of POs in Pacific oyster C. gigas haemocytes inhibits the bacteria growth in the presence of a specific chemical inhibitor of PO or proPO activation. The antibacterial activities of haemocyte lysate supernatant involve POs, and more particularly laccase catalysed reactions. The oyster ProPO system is not well understood, and it is in urgent need to investigate the molecular composition and more specifically on the regulation of the proPO-activating proteinase cascade. 2.3. The generation of immune effectors Immune effectors are usually induced by PRR recognition and produced by epithelial cells from various organs. They are active against a large range of pathogens or sensitive for the environmental stress. These effectors are essential molecules in the oyster immunity system and they are utilized as executors for the incapacitation and elimination of invaders. The following is a general overview of the recent knowledge about immune effectors (Table 2), such as AMPs, lysozymes, cytokines, antioxidant enzymes and acute phase proteins, and draw their repertoire in oyster immune response.

2.3.1. Antimicrobial peptides and proteins (AMPs) AMPs comprise a chemically and structurally heterogeneous family of molecules, possessing some distinguishable characteristics, such as small molecules, cationic and amphipathic structures (Hof et al., 2001; Zasloff, 2002). So far, there are over 30 AMPs identified from oyster, and four of them are defensins which share high similarities with the arthropod defensin family. Oyster defensins are constitutively expressed in specific tissues such as mantle (CgDefm), gill (CvAOD) or haemocytes (CgDefhs). CgDefh1 and CgDefh2 identified from C. gigas haemocytes share around 80% identity with mantle CgDefm. Recombinant CgDefm is active in vitro against Gram-positive bacteria but displays no or a limited activity against Gram-negative bacteria and fungi (Gonzalez et al., 2007a; Gueguen et al., 2006; Seo et al., 2005). Big defensin (BigDefs) is a kind of antimicrobial polypeptide (8e11 kDa) which is only reported in marine invertebrates (Arthropoda, Mollusca and Cephalochordata) (Rosa et al., 2011). Oyster big defensin (CgBigDefs) is a diverse family of AMPs composed of three representative members, named CgBigDef1, CgBigDef2 and CgBigDef3, which are specifically expressed in the haemocytes. All CgBigDefs contain a hydrophobic N-terminal domain and a cationic C-terminal domain, which resembles vertebrate b-defensins. The mRNA transcripts of CgBigDef1 and CgBigDef2 are up-regulated after bacteria challenge, while CgBigDef3 is not induced (Rosa et al., 2011). Two membrane antimicrobial proteins, bactericidal/permeability-increasing protein (CgBPI) and macrophage expressed gene 1-like protein (CgMpeg1), have been identified to have antimicrobial properties in oyster (Gonzalez et al., 2007b; He et al., 2011b). CgBPI, the first identified invertebrate BPI, is expressed in haemocytes after an immune challenge and constitutively expressed in various tissue epithelia of both challenged and unchallenged oysters (Gonzalez et al., 2007b). A novel cysteine-rich antimicrobial peptide, CgPep33, isolated from the enzymatic hydrolysates by digestion of oyster with alcalase and bromelin, exhibits strong activity against bacteria and fungi (Liu et al., 2008b). CgPrp with an acidic region and a cationic proline-rich region is expressed in haemocytes and up-regulated after bacterial challenge. Intriguingly, CgPrp exists in some haemocytes and exhibits strong synergistic antimicrobial activity with CgDefs (Gueguen et al., 2009). Recently, two other novel AMPs Molluscidin and b-thymosin homologous peptide were purified from the gill and mantle of the Pacific oyster, C. gigas respectively, which possessed potential antimicrobial activities against Gram-positive and Gram-negative bacteria (Nam et al., 2015; Seo et al., 2013). Besides, histones and histone-derived peptides (histone H4, histone-H2A-derived antimicrobial peptides Molluskin, CgH1/H5, CvH2B-1, CvH2B-2, CvH2B-3 and CvH2B-4) have been confirmed to exhibit antimicrobial activity and play a potential immune defense role in oysters (Dorrington et al., 2011; Sathyan et al., 2012; Seo et al., 2010, 2011). Seven antioxidant peptides (SCAP 1-7) containing unique amino acid composition from oyster S. cucullata play important roles in antioxidant activity (Umayaparvathi et al., 2014). A great sequence diversity and extraordinary expression profile polymorphism of AMP gene among individuals are reported to be re et al., 2015; Schmitt the characteristics of oyster AMPs (Bache et al., 2010). The multigenic families of CgDefs, CgPrps and CgBigDefsdisplay a variety of variations in gene structure and copy re et al., 2015). The copy number of Cgdef gene is of number (Bache highly variable (14-53 copies) and the copy number of Cgprp also varies from 4 to 18 among oyster individuals (Schmitt et al., 2010, 2013). CgDefs and CgPrps are clustered into distinct groups in phylogenetic analyses, indicating that they are produced by different evolutionary paths (Schmitt et al., 2010). In addition to their antimicrobial activities, oyster AMPs possess numerous unexplored functions such as antiviral, chemotactic


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Table 2 The immune effectors reported in oysters. Gene name Antimicrobial peptides AOD Cg-Defm Cg-Defh1 Cg-Defh2 Cg-BPI CgPep33 Cg-Prp cvH2B-1 Cg-BigDef1 Cg-BigDef2 Cg-BigDef3 cvH2B-2 cvH2B-3 cvH2B-4 histone H4 Cg-Mpeg1 Molluskin Molluskin cgMolluscidin Cg-Ubiquitin SCAP 1-7 Cg-H1/H5 b-thymosin Lysozymes cv-lysozyme 1 cv-lysozyme 2 cv-lysozyme 3 CGL-1 CGL-2 CGL-3 Lysozyme I-type Lysozyme Cytokines CgIL-17-1 CgIL-17-2 CgIL-17-3 CgIL-17-4 CgIL-17-5 CgIL-17-6 PfiL-17 CgIFNLP CgTNF-1 Antioxidant enzymes ChSOD Cu/Zn SOD Cg-EcSOD MnSOD PoMnSOD PfSOD Pm-SOD Oe-EcSOD Oe-SOD PoCAT ChCat-1 ChCat-2 CAT GPX GPX GST s-GST p-GST m-GST u-GST m-GST u-GST s-GST MGST3 GST Acute phase proteins PmHSP20 PmHSP40 PmHSP90

Species

Tissue

Accession no.

Reference

C. virginica C. gigas C. gigas C. gigas C. gigas C. gigas C. gigas C. virginica C. gigas C. gigas C. gigas C. virginica C. virginica C. virginica C. virginica C. gigas C. madrasensis S. cucullata C. gigas C. gigas S. cucullata C. gigas C. gigas

gill mantle haemocytes haemocytes haemocytes e haemocytes gill haemocytes haemocytes haemocytes gill gill gill e all tissues all tissues all tissues gill gills e gills mantle

e AJ565499 DQ400101 DQ400102 BQ427321 e BQ426670 e FP004575 CU989616 AM862886 e e e HM130521 e HQ720145 HQ720147 e e e e e

(Seo et al., 2005) (Gueguen et al., 2006) (Gonzalez et al., 2007a) (Gonzalez et al., 2007a) (Gonzalez et al., 2007b) (Liu et al., 2008b) (Gueguen et al., 2009) (Seo et al., 2010) (Rosa et al., 2011) (Rosa et al., 2011) (Rosa et al., 2011) (Seo et al., 2011) (Seo et al., 2011) (Seo et al., 2011) (Dorrington et al., 2011) (He et al., 2011b) (Sathyan et al., 2012) (Sathyan et al., 2012) (Seo et al., 2013) (Seo et al., 2013) (Umayaparvathi et al., 2014) (Poirier et al., 2014) (Nam et al., 2015)

C. virginica C. virginica C. virginica C. gigas C. gigas C. gigas Ostrea edulis C. Hongkongensis

hemolymph digestive tubules digestive gland mantle mantle mantle digestive gland

BAE47520 BAE93114 BAG41979 BAD19059 BAF48044 AB307634 AB179776 JF320829

(Itoh et al., 2007; Xue et al., 2004) (Xue et al., 2007) (Xue et al., 2010) (Itoh et al., 2010b; Matsumoto et al., 2006) (Itoh and Takahashi, 2007) (Itoh et al., 2010b) (Matsumoto et al., 2006) (Xu et al., 2016)

C. gigas C. gigas C. gigas C. gigas C. gigas C. gigas P. fucata C. gigas C. gigas

e e e e e e e e e

EF190193 KJ531893 KJ531894 KJ531895 KJ531896 KJ531897 AGC24392 JH816585.1 JH818057

(Roberts et al., 2008) (Li et al., 2014) (Li et al., 2014) (Li et al., 2014) (Li et al., 2014; Xin et al., 2015) (Li et al., 2014) (Wu et al., 2013) (Zhang et al., 2015b) (Sun et al., 2014)

C. hongkongensis P. fucata C. gigas C. gigas P. fucata P. fucata P. martensi Ostrea edulis Ostrea edulis P. fucata C.hongkongensis C.hongkongensis C. gigas C. gigas P. fucata C. gigas C. gigas C. gigas C. gigas C. gigas C. ariakensis C. ariakensis C. ariakensis P. martensi P. fucata

e haemocytes, gill haemocyte e e e e e e e haemocytes haemocytes e e e e e e e e e e e e e

KJ741847 JX013537 DQ010420 EU420128 KF017277 JX013537 KP689432.1 GU320696 GU320695 HQ703465 HM147934 HM147935 EF687775 EF692639 GU362541 AJ557140 AJ577235 AJ557140 AJ558252 AJ557141 EU908274 EU908273 EU908270 HQ284164 GU362542

Unpublished (Anju et al., 2013) (Gonzalez et al., 2005) (Park et al., 2009) (Zhang et al., 2013) (Anju et al., 2013) Unpublished (Morga et al., 2011) (Morga et al., 2011) (Guo et al., 2011) (Zhang et al., 2011d) (Zhang et al., 2011d) (Jo et al., 2008) (Jo et al., 2008) Unpublished Unpublished (Boutet et al., 2004) (Boutet et al., 2004) (Boutet et al., 2004) (Boutet et al., 2004) Unpublished Unpublished Unpublished (Chen et al., 2011a) Unpublished

P. martensi P. martensi P. martensi

e e e

KM977565 JF929907 KJ010545

Unpublished (Li et al., 2016a) (Liang et al., 2015) (continued on next page)

106


Table 2 (continued ) Gene name

Species

Tissue

Accession no.

Reference

PfHSP70 CgHSP20 CgHSP68 CgHSP70 CgHSP70 CgHSP70 CgHSP70B CgHSC70 CgHSP90 CvHSP70 CaHSP70 CcHSP70 OeHSC70 OeHSP70 OeHSP70 OeHSP70 CvMT-I A CvMT-I B CvMT-II A CvMT-II B CvMT-II C CvMT-II D CvMT-II E CvMT-II F CvMT-II G CvMT-II H CvMT-III CvMT-IV CgMT CgMT2 CgMT-IV CrMT ChSAA

P. fucata C. gigas C. gigas C. gigas C. gigas C. gigas C. gigas C. gigas C. gigas C. viginica C. ariakensis C. columbiensis Ostrea edulis. Ostrea edulis. Ostrea edulis. Ostrea edulis. C. viginica C. viginica C. viginica C. viginica C. viginica C. viginica C. viginica C. viginica C. viginica C. viginica C. viginica C. viginica C. gigas C. gigas C. gigas C. rivularis C. hongkongensis

e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e

ABJ97378 EKC40046 AB122062 CAC83009 AB122063 BAD15286 AB549340 CAC83683 EF687776 AJ271444 AAO41703 ABC02062 CAC83684 AAM46635 AAM46634 CAC83010 AY331695 AY331699 AY331700 AY331701 AY331702 AY331703 AY331704 AY331705 AY331706 AY331707 DQ354066 DQ117912 AJ242657 AJ297818 AM265551 JN225502 KF156832

Unpublished Unpublished Unpublished (Boutet et al., 2003b) Unpublished Unpublished Unpublished (Boutet et al., 2003b) (Choi et al., 2008) (Rathinam et al., 2000) Unpublished Unpublished (Boutet et al., 2003a) (Piano et al., 2004) (Piano et al., 2004) (Boutet et al., 2003a) (Jenny et al., 2004)

activities (Segarra et al., 2014b; Thomas et al., 2002) and opsonization (Iovine et al., 1997). It indicates that oysters have developed sophisticated immune strategies by diversification of immune effectors to survive from infection. 2.3.2. Lysozymes Lysozyme is a family of glucoside hydrolases to play major biological roles in biodefense as antibacterial and immunemodulating agents. In addition, lysozymes can function as important digestive enzymes in some animals (Callewaert and Michiels, 2010; Shailesh and Sahoo, 2008; Xue et al., 2010). According to the structure and sources, lysozymes are classified as phage-type, bacteria type, plant type, invertebrate type (i-type), chicken type (c-type) and goose type (g-type) (Callewaert and Michiels, 2010). The oyster lysozyme activity was firstly reported in the hemolymph and skin mucus from the C. virginica in 1967 (McDade and Tripp, 1967). Subsequently, the lysozyme and lysozyme-like activities are detected in various tissues of oyster, and multiple i-type lysozymes have been identified and characterized in C. virginica (CVL-1, CVL-2 and CVL-3) and C. gigas (CGL-1, CGL-2 and CGL-3), respectively. CVL-1 is distributed in the outer surface tissues (mantle and gills) as well as hemolymph with strong antimicrobial activity (Itoh et al., 2007; Xue et al., 2004), and CGL-1 exhibits lysozyme activity and antibacterial activity which is main detected in mantle tissue (Itoh et al., 2010b; Matsumoto et al., 2006), indicating the main roles of CVL-1 and CGL-1 in host defense. CVL-2 and CGL-2 are phylogenetically homologous proteins, and both of them are digestive lysozymes expressed in the digestive gland (Itoh and Takahashi, 2007; Xue et al., 2007). Phylogenetically, CVL-3 and CGL-3 are homologous to digestive lysozymes of CGL-2 and CVL-2. However, the important characters (e.g. distinctive N-terminal amino acid sequence) and functions (e.g. antibacterial activity) are more close to CGL-1, suggesting that CVL-3 and CGL-3 represent a

(Jenny et al., 2004)

(Jenny et al., 2006) (Tanguy and Moraga, 2001) Unpublished Unpublished (Qu et al., 2014a)

transitional lysozyme form with potential functions both in immunity and digestion (Itoh et al., 2010b; Xue et al., 2010). These results collectively suggest that i-type lysozymes in oyster exert functions in an adaptive switching mechanism between host defense and digestion as some vertebrate c-type lysozymes. 2.3.3. Cytokines Cytokines comprise a large number of regulatory molecules, such as IL, interferons (IFN), TNF and chemokines, and most of them function in vertebrate immune defense (Dinarello, 2000; O'Shea et al., 2002). The first invertebrate IL-17 homolog was identified in oyster, and its transcript abundance in haemocytes increased rapidly after bacteria challenge (Roberts et al., 2008). Six IL17s have been identified from oyster C. gigas (designated as CgIL17-1 to CgIL17-6), and all of them contain the classical cysteine knot of fourconserved cysteines as human IL17s. Their mRNA expression levels in haemocytes are up-regulated after PAMPs stimulation (Li et al., 2014). CgIL17-5 can activate the transcription factors NF-kB, CREB and ATF-1, and involve in the corresponding signal pathways in HEK293T cells. It also augments the IL6 synthesis in HuVEC cells, exhibiting similar activity as human IL17 in inflammatory response. Additionally, CgIL17-5 protein displays broad bacterial binding spectrum, and significantly inhibits the growth of Micrococcus luteus and E. coli (Xin et al., 2015). A IFN-like protein (CgIFNLP) with typical structure homolog of vertebrate IFN and its receptor CgIFNR-3 are identified in oyster. CgIFNR-3 interacts with CgIFNLP in vitro, and the expressions of CgIFNLP and CgIFNR-3 in haemocytes are both significantly up-regulated after poly (I:C) stimulation. Moreover, the CgIFNLP protein induces apoptosis and phagocytosis of oyster haemocytes, and inhibits the proliferation of A549 cell in vitro. As a receptor of IFNLP, CgIFNR-3 can activate JAK/ STAT pathway in oyster (Zhang et al., 2015c, 2016). A TNF superfamily member (CgTNF-1) with typical TNF domain is also


identified in oyster, and rCgTNF-1 not only regulates apoptosis and phagocytosis of haemocytes, but also modulates PO, lysozyme and anti-bacterial activities (Sun et al., 2014). Moreover, 23 TNF homologs are annotated in the genome of C. gigas, and 15 of them are C. gigas-specific. These TNF homologs are formed by tandem and/or segmental duplication events, and most of the duplicated pairs are evolved under purifying selection with consistent tissue expression patterns (Gao et al., 2015). 2.3.4. The antioxidant enzymes The application of oxygen by aerobic organisms during normal metabolism always produces reactive oxygen species (ROS). Some ROS are highly toxic and deleterious to cells and tissues, and they are highly induced especially when the organism is attacked by invaders or contaminant exposures (Aguirre et al., 2005; Winterbourn, 2008). To protect cells from the damage caused by abundant ROS, organisms have evolved functional antioxidant defense system to rapidly and efficiently remove ROS from the intracellular environment. Antioxidant enzymes, such as superoxide dismutase, catalase and glutathione peroxidase (GPx), directly scavenge free radicals and related reactants, and form the first defense line in organisms (Rodriguez et al., 2004; Tom aszapico and Cotomontes, 2005). Detoxification enzymes, e.g. glutathione-Stransferase, also play an important role in clearance of superoxide anion to protect against extracellular oxidative damage (Masella et al., 2005; Strange et al., 2001). SOD is a kind of ubiquitous metalloenzyme which converts superoxide anion into hydrogen peroxide and water. There are many reports about the importance of SODs in immune response and their roles in protecting cells against various challenges (Petrov et al., 2016; Zelko et al., 2002). Based on the metal content, SODs are categorized into six types, and two of them, Cu/ZnSOD and MnSOD, have been isolated and characterized in oysters. The extracellular SOD (CgEcSOD), a major plasma protein, has been thoroughly investigated in Pacific oyster C. gigas. In addition to antioxidant activity, CgEcSOD functions as PRR through a RGD motif. The rCgEcSOD binds LPS, PGN and poly (I:C), as well as various microorganisms (Gonzalez et al., 2005; Liu et al., 2016c). It is also reported that CgEcSOD plays a crucial role in the immune response against V. splendidus by promoting the internalization as a bridge molecule between virulence factor OmpU and the specific ligands of CgIntegrin (Duperthuy et al., 2011). In addition, a paradoxical role of CgEcSOD in the immune response is also revealed after bacterial stimulation. The mRNA expression of CgEcSOD in haemocytes is significantly up-regulated at the initial phase, but decreased sharply at 48 h post V. splendidus stimulation. Whereas the mRNA and protein levels of CgEcSOD are both down-regulated significantly upon the secondary challenge of live V. splendidus (Liu et al., 2016c). The paradoxical role of SOD is also reported in other oyster species. For example, the expression level of pfSOD in haemocytes increases after LPS stimulation, reaches the highest level at 8 h, and then drops to basal levels at 36 h in pearl oyster (Anju et al., 2013). The mRNA expressions of MnSOD and CuZnSOD in C. hongkongensis are both up-regulated firstly and then return to normal level after infection by V. alginolyticus (Yu et al., 2011). The mechanism underlying this confliction is still obscure and it is speculated as a particular strategy to benefit the host of surviving from the invasion. CAT is one of the key antioxidant enzymes involved in protection against immune infection and oxidative stress by decomposing hydrogen peroxide (Alfonso Prieto et al., 2009; Chelikani et al., 2004). Although CAT is ubiquitous and present in all aerobic organisms from bacteria to mammals, the information on CAT in oyster is quite limited. CAT genes have been identified in C. gigas (Jo et al., 2008), C. hongkongensis (Zhang et al., 2011e) and P. fucata

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(Guo et al., 2011), and all of them possess the characteristic features of catalase family. Higher-levels of CAT mRNA expression are detected in haemocytes of C. hongkongensis and intestine of P. fucata after bacterial challenge, indicating that CAT is necessary in the immune responses against bacterial infection (Guo et al., 2011; Liu et al., 2008b; Zhang et al., 2011e). Moreover, two CAT homologs are identified in C. hongkongensis, and this is the first report of the presence of two catalase genes in a single marine bivalve. Recombinant ChCAT-1 and ChCAT-2 proteins both display strong CAT activities, and the S2 cells carrying ChCAT-1 or ChCAT-2 show a higher degree of resistance to H2O2. It highlights the involvements of both ChCAT-1 and ChCAT-2 in host protection against pathogen infection and oxidative stress in C. hongkongensis (Zhang et al., 2011e). GPx is another important peroxidase enzyme to catalyze the reduction of lipid hydroperoxides and free hydrogen peroxide to and Maiorino, 2013; Comhair water and oxygen (Brigelius-Flohe and Erzurum, 2005). The activity of GPx has been detected in several oysters, while GPx gene is only cloned from Pacific oyster C. gigas. A higher expression level of CgGPx is detected in gill after Cadmium (Cd) treatment, indicating its role in the physiological changes related to metabolism and cell protection (Jo et al., 2008). GSTs represent a major group of detoxification enzymes including multiple cytosolic and membrane-bound GST isoenzymes (Masella et al., 2005; Strange et al., 2001). Several cytosolic GSTs have been characterized in oysters with tissue-specific, time- and treatment-dependent expression patterns. The expression level of GST is a crucial factor in determining the sensitivity of cells to a broad spectrum of toxic chemicals. For example, the expressions of u- and m-GST as well as p- and s-GSTs in the digestive gland of C. gigas can be used as a possible marker of hydrocarbon and pesticide exposure in monitoring programs, respectively (Boutet et al., 2004). In C. ariakensis, the expression profiles of eight GSTs after exposure to Prorocentrum lima are distinctly different, and the enzyme activity of total GST increase in gill, indicating that the GSTs isoenzymes play divergent physiological roles in the detoxification (Zou et al., 2015). A novel microsomal GST, named as PmMGST3, is identified from the pearl oyster P. martensii. Cd treatment significantly increases PmMGST3 mRNA levels in gill and hepatopancreas, while bacterial challenge initially depresses its expression and then increases its level in haemocytes, gill and hepatopancreas in a time-dependent manner. Moreover, PmMGST3 is found to possess glutathione-dependent peroxidase activity with cumene hydroperoxide as a substrate, suggesting that PmMGST3 plays an important role in cellular defense against oxidative stress (Chen et al., 2011a). 2.3.5. Acute phase proteins Acute phase proteins, a family of plasma proteins, are synthesized and secreted during the acute phase response (Cray et al., 2009; Eckersall and Bell, 2010). Several acute phase proteins have been well studied in oysters, especially heat shock proteins (HSPs), metallothioneins (MTs) and Serum amyloid A (SAA). HSPs are a family of ubiquitous and highly conserved stress proteins that help organisms to modulate stress response and protect them from environmentally induced cellular damage. HSPs also function as potent activators of the innate immune system (Robert, 2003; Srivastava, 2002). HSPs with molecular mass from 15 to 110 kDa are classified into several families according to their apparent molecular mass. HSP20 (Lei et al., 2016), HSP40 (Li et al., 2016a), HSP68 (Unpublished data), HSP70 (Boutet et al., 2003a, 2003b; Wang et al., 2009; Zhang and Zhang, 2012) and HSP90 (Fu et al., 2011a) have been identified from oysters. They are highly induced by thermal stress (Li et al., 2016a; Zhang and Zhang, 2012), salinity (Li et al., 2016a), osmotic stress (Fu et al., 2011a), bacterial infection (Fu et al., 2011a; Li et al., 2016a; Wang et al., 2009), a great

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variety of heavy metals (Choi et al., 2008; Piano et al., 2004; Zhang and Zhang, 2012) as well as chemical stressors, such as antifouling biocides (Park et al., 2016), polynuclear aromatic hydrocarbons (Cruz-Rodrıguez and Chu, 2002), and malachite green (Zhang and Zhang, 2012). Remarkably, an expand set of 88 HSP70 genes are annotated in oyster genome, and phylogenetic analysis reveals a cluster of 71 oyster HSP70 genes, which suggest that the expansion is specific to the oyster. After heat stress, the average expression levels of all HSP70 genes increase 13.9-fold, and a 2000-fold increase in expression is induced for five highly inducible HSP70 genes, indicating that HSPs are central to the oyster defense against many stresses (Zhang et al., 2012a, 2015a, 2015b). The genomic expansion and massive up-regulation of HSP genes greatly benefit the euryhaline and eurythermal characteristics of oyster. MTs are typically low relative molecular mass, sulfhydryl-rich metal-binding proteins. Their expression are highly induced by a variety of metals, thus they play fundamental roles in the homeostasis of essential metals and detoxification of trace metals (Coyle et al., 2002; Dabrio et al., 2002). It is notable that oyster MTs comprise three or more metal-binding domains which are evolved from a series of duplication events to produce the greatest structural diversity (Jenny et al., 2004; Tanguy and Moraga, 2001). At least three distinct MT gene families are identified in C. virginica, and CvMT-I is proposed to be a prototypical member ofab-domain MT subfamily. It can be converted into bb-domain CvMT-III, abdomain CvMT-IV and CvMT-II with solely one to four a-domains upon a duplication event (Jenny et al., 2004, 2006). These data extend our knowledge of the evolutionary diversification of MTs, and indicate the differences in metal-binding preferences between isoforms in the same family. SAA is a major positive acute-phase protein involved in the modulation of numerous immunological responses against infection, trauma or stress (Cocco et al., 2010; Malle et al., 2009). The first oyster SAA (referred to as ChSAA) is identified and characterized from the C. hongkongensis, which shares a high identity with other known acute-phase SAA proteins. ChSAA is constitutively expressed in various tissues and its expression is acutely and significantly up-regulated in haemocytes after challenged by numerous pathogens. The over-expression of ChSAA leads to significant increase of NF-kB activity in HEK293T cells (Qu et al., 2014a). These results suggest that ChSAA is likely to be a member of the A-SAA family involved in antipathogen responses in C. hongkongensis.

enzymatic activities are detected during the ontogenesis of the Pacific oyster C. gigas. Two antioxidant enzymes, SOD and CAT, and 19 hydrolytic enzymes, including phosphatases, esterases, proteases, and glycosidases are all present in oocytes and early larvae stages (Luna-Gonz alez et al., 2004; Song et al., 2016b). PO activity is also detected in oocytes of oyster (Thomas-Guyon et al., 2009). Moreover, the transcripts of at least 30 candidate immune genes, such as PRRs (e. g. CgCLEC3, CgTLR4, CgLBP/BPI, Cgintegrin b-1 and CgEcSOD), hematopoiesis-related genes (CgDrac3, Cgtal and CgGATA3), signal pathway molecules (CgMyD88, CgECSIT, CgTRAF3, CgRel and CgIKK) and immune effectors (CgIL17-5, Cgdefh2, CgSOD and CgCAT), are expressed during early stages (oocytes and 2-4 cell embryos), but they decrease or cannot be et al., 2007). It detected in later stages (Song et al., 2016b; Tirape suggests that various immune molecules (mRNA or proteins), can be transferred from female parent and provide sufficient immune protection for the embryo development. 3.2. Role of maternally derived immune factors The fertilization of oyster occurs in vitro and oyster larvae will experience embryonic period for approximate nine hours after newly fertilization. The environmental factors such as pathogens, temperature and pollutants always affect the development of embryo. Numerous immune related molecules are demonstrated to be maternal origin and respond to bacterial challenge during the early embryonic period. The expressions of IL17-5 and defh2 in the haemocytes of C. gigas are significantly up-regulated at 12 h post bacterial challenge. Moreover, IL17-5 plays critical role in immune defense against bacterial challenge, which appears in the digestive gland of bacterial challenged blastula larvae (Song et al., 2016b). LPS challenge can trigger pro-PO activating system in early embryo stage of C. gigas. In contrast, the PO activity is totally suppressed by PO-specific inhibitors such as b-2-mercaptoethanol, sodium diethyldithiocarbonate and tropolone in the early embryo stage of oyster (Thomas-Guyon et al., 2009). Vitellogenin proteins, the main nutrient source for the developing embryos and larvae, are also reported to function in immune defense response via its pleiotropic activities of recognizing PAMPs and hemagglutinating against microbes (Li et al., 2008). Although the maternal transfer of vitellogenin is also found in oyster C. gigas, the immune function of vitellogenin is still not well recognized (Corporeau et al., 2012). 3.3. The Trans-Generational Immune Priming (TGIP)

3. The maternal immune transfer Maternal transfer of immunity is defined that offspring obtains the immunological capacity via eggs from parental generation, which plays a crucial role in protecting the vulnerable offspring at early stages of life. The transmission of maternal immunity has been well described in vertebrates as well as some invertebrates, such as insects, crustaceans and molluscs (Poorten and Kuhn, 2009; Swain and Nayak, 2009; Yue et al., 2013). 3.1. Transfer of maternally derived immune factors Most molluscs, especially oysters, are broadcast spawners. They release gametes into water for in vitro fertilization. Recently, with the wide application of high throughput proteomic techniques, many maternally derived immune factors are identified in oyster eggs or embryos. Immune effectors are generally considered as the main maternally derived immune factors because of their direct scavenge against invaders. Oyster eggs or larvae possess remarkable antibacterial and agglutination activities against pathogens. Ontogenetic variations of antioxidant enzymatic and hydrolytic

TGIP refers the enhanced immunity of offsprings to the pathogen that has been encountered by their parents, which is also considered as another form of immune priming across generations. To date, solid evidences of TGIP have been reported in several invertebrates, including oysters (Barbosa-Solomieu et al., 2005; Roth et al., 2010; Yue et al., 2013). The treatment with poly (I:C) can significantly enhance the immune protection of oyster against OsHV-1. The survival rate of offspring from poly (I:C)-treated parents is two times higher than that of control when they are exposed to OsHV-1, which provides an effective management tool to control OsHV-1 (Green et al., 2016). Although the mechanism of TGIP is still not clear, the epigenetic regulation is believed to play a crucial role in the TGIP. Maternal experience of pathogen or other immune stimulation not only affects the maternal transfer of immunity from female parent to eggs, but also has a profound influence on offspring's phenotype (Ottaviani et al., 2013). It is hypothesized that the TGIP should be related with epigenetic modifications, and increasing reports have suggested that maternal environment exhibits the approved epigenetic effect on development of offspring and their


behavior (Curley et al., 2011). 4. The occurrence, maturation and differentiation of haemocytes Oyster possesses an opening circulatory system and all of its tissues are infiltrated in hemolymph. Thus circulating haemocytes is of vital importance in participating in many biological processes especially in immunological homeostasis (Cochennec-Laureau et al., 2003; Lemaitre and Hoffmann, 2007). Oyster haemocytes are considered to be the counterpart of vertebrate leukocytes and involved in both humoral and cellular immune responses, such as synthesis of antimicrobial peptides, encapsulation, and phagocytosis (Cochennec-Laureau et al., 2003; Ottaviani, 2011; Song et al., 2010). Here we summarize the research progress of hematopoiesis and hematopoietic tissue, and haemocyte classification in oyster.

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mammal macrophages. Large cell diameter, rough cell membrane and extended pseudopodia of phagocytes in C. gigas are revealed by the scanning electron microscopy (Jiang et al., 2016b, 2016c; Lin et al., 2014). In addition, cytochemical staining analysis indicates that oyster phagocytes possess distinct cytochemical pattern compared with that of phagocytic cells in mammals, such as abundant acid hydrolase NAE, oxidase MPX, as well as low abundance of polysaccharides and heparin/histamine granules (Jiang et al., 2016b; Wang et al., 2017; Xue and Renault, 2000). The haemocytes of invertebrates are less differentiated than vertebrate leucocytes. Recent evidences from other invertebrates suggest that haemocytes also have stem cell-like behavior and the circulating haemocytes of bivalves are just certain types at different development stages (de Freitas Rebelo et al., 2013; Ottaviani, 2011). The origin of hematopoietic tissue and haemocytes are also needed to be investigated by using hematopoietic and haemocyte specific molecules. What's more, except for morphological and cytochemical criteria, new classification standards are in urgent need.

4.1. The occurrence, maturation and hematopoietic tissue 5. The immune priming in oyster Studies in both vertebrate and invertebrate model animals reveal that the hematopoietic development is conserved across €derha €ll, 2011; different species (Galloway and Zon, 2003; Lin and So Orkin and Zon, 2008). Recently, gill was reported to be the potential hematopoiesis site of adult oyster. The adult somatic precursor cells with active DNA synthesis and exuberant hemocytes production are identified from the unreported irregularly folded structure (IFS) et al., in oyster gill, which can differentiate into haemocytes (Jemaa 2014; Li et al., 2017). Stem Cell Leukemia (SCL) is essential for development of all hematopoietic lineages as well as vascular cuyer and Hoang, 2004; Qian et al., 2007; system in vertebrates (Le Solek et al., 2013). The homolog of SCL is also identified in oyster C. gigas. As a promising hematopoietic marker, CgSCL is highly expressed in the haemocytes, and the knock down of CgSCL significantly down-regulates the haemocyte-specific genes (e.g. Integrin, ECSOD, GATA3). The expression profile of CgSCL during the ontogeny demonstrates that the hematopoietic system of the Pacific oyster occurs at the blastula stage, and migrates to the dorsal region in trochophore and vesicle tissues around adductor in Dveliger larvae (Song et al., 2016a). Besides, CgAstakine, a homolog of hematopoietic growth factor prokineticin, is also identified in oyster, and the recombiant CgAstakine protein significantly promotes the proliferation of haemocytes both in vitro and in vivo (Li et al., 2016d). These results offer us a new clue to study the origin of the hematopoietic tissue and haemocytes. 4.2. Haemocytes classification The classification of C. gigas haemocytes was reported early in re et al., 1988; Chagot et al., 1992). Most of the the 1980's (Bache studies are focused on morphological description, and the haemocytes are classified into granulocytes and hyalinocytes (agranular) based on the presence of the cytoplasmic granules (Aladaileh et al., 2007b; Ashton-Alcox and Ford, 1998; de Freitas Rebelo et al., 2013). They are further identified and separated into agranulocytes, semi-granulocytes and granulocytes morphologically by flow cytometry and Percoll® density gradient centrifugation (Wang et al., 2017). The granulocytes are characterized functionally as the main phagocytic and encapsulating population. Meanwhile, the lysosome activity and the productions of ROS and NO are all mainly concentrated in granulocytes under both normal and immuneactivated situations, suggesting that the granulocytes are the main immunocompetent haemocytes in oyster C. gigas (Wang et al., 2017). Furthermore, phagocytes are also separated by fluorescence activated cell sorting in C. gigas, which share similar features with

It has long been thought that invertebrates lack the capacity for immunological memory that can be exploited in vaccination programs to prevent disease outbreaks (Hauton and Smith, 2007). However, the increasing evidences prove that invertebrates can mount sophisticated immune responses with unique forms of immune-specificity and immune-memory (Little et al., 2003; Roth and Kurtz, 2009; Rowley and Powell, 2007). Most of the evidences from insects and other arthropods confirm that the exposure to a non-lethal dose of a pathogen or its products can provide protection later in life against a lethal dose of the same pathogen ~ o et al., 2016). These studies have led to the hy(Contreras Gardun pothesis that invertebrate immunity can be influenced by previous encounters with pathogens or their products, which provide protection against reinfection via phenomena termed “innate im~ o et al., 2016; Netea et al., 2011, mune-priming” (Contreras Gardun 2016). Though the knowledge of immune priming at molecular and cellular level is still not available for most observations in invertebrate, plenty of evidences for innate immune memory or priming in oyster have been found, and a number of candidate mechanisms have arisen from such searches. 5.1. Hematopoiesis Haemocytes interact with numerous foreign particles and lead the subsequent activation of phagocytosis, apoptosis, encapsulation as well as other cell responses. The invasion of malaria parasite in mosquito triggers a long-lived response characterized by increasing abundance of granulocytes and enhanced immunity to bacteria that indirectly reduces survival of parasites (Rodrigues et al., 2010). In oyster, total haemocyte counts (THC), number of regenerated haemocytes and expression levels of hematopoiesis related genes (e.g. CgRunx1 and CgBMP7) are all significantly increased after the secondary challenge with live V. splendidus (Zhang et al., 2014b), suggesting the involvement of hematopoiesis during the immune priming. Serum transfer experiments demonstrate that serum immune factors might enhance immune reaction by regulating THC, apoptosis, proliferation and phagocytosis (Zhang et al., 2014b). 5.2. Phagocytosis Phagocytosis is major mechanism of cellular immune reaction. In oyster, a specifically enhanced phagocytic activity of haemocytes, including both phagocytic rate and phagocytic index, is found after the secondary challenge with live V. splendidus. Besides, the

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expressions of six putative genes involved in the process of phagocytosis, CgIntegrin, CgPI3K, CgRho J, CgMAPKK, CgRab 32, and CgNADPH oxidase, are also significantly up-regulated after secondary challenge (Zhang et al., 2014b). The results indicate that phagocytosis may play critical role during the immune priming. 5.3. Diversification of immune receptors Although invertebrates lack adaptive immune system homologous to that of vertebrates, some fascinating receptor systems are found to have the potential for somatic diversification processes (Du Pasquier, 2006). The most important examples are the Down Syndrome Cell Adhesion Molecule (Dscam) in arthropods (Watson et al., 2005), FREPs in molluscs (Huang et al., 2015), and the Sp185/ 333 proteins in echinoderms (Buckley and Smith, 2007). These somatic diversification processes produce a receptor repertoire in the absence of antigen, while the specific repertoires are induced with a greater association affinity for the eliciting pathogen. A sophisticated repertoire of PRRs have been found in oyster, such as FREPs, PGRPs, Lectins, TLRs, C1qDC proteins, which have been introduced above. Remarkably, a total of 268 IgSF members containing 1-36 Ig domains are identified from the oyster genome (Zhang et al., 2012a). The IgSF members play important roles in the immune response of oyster. After challenges with PAMPs and microorganisms, the expression levels of 116 and 166 IgSF members are significantly up-regulated, respectively. Plenty members display highly specify for different challenge, suggesting that a functional divergence occur during the evolution of oyster IgSF. Several members undergo alternative splicing and single nucleotide mutation to generate tens of thousands of protein isoforms after the challenge of V. splendidus (Zhang et al., 2012a, 2015b) suggesting a potential mechanism of the immune priming. Several members of IgSF (CgJAM-A-L and CgSiglec-1) are further characterized in oyster. CgJAM-A-L and CgSiglec-1 both contain several typical I-set Ig domains and possess broad recognition spectrum. Their mRNA transcripts are significantly increased after microbe stimulation (Liu et al., 2016a, 2016b). Furthermore, the blockade of CgSiglec-1 by specific polyclonal antibodies enhance the LPS-induced cell apoptosis, phagocytosis towards V. splendidus and the release of cytokines, such as CgTNF-1, CgIFNLP and CgIL-17, suggesting that CgSiglec-1 inhibit the activity of apoptosis, phagocytosis and cytokine release in oyster haemocytes (Liu et al., 2016a). It indicate that oysters possess an abundant and diverse collection of IgSF members which play crucial roles in the immune response. However, the diversified forms of IgSF and their roles in immune priming need to be further investigated. 6. Apoptosis Apoptosis is a multifunctional process, which is mainly performed by a family of cysteine protease known as caspase (cysteinyl aspartate-specific proteinase) (Elmore, 2007; Johnstone et al., 2002; Riedl and Shi, 2004). Numerous findings indicate that most biotic and abiotic stressors, such as bacteria, viruses, parasite, heat, salinity, air exposure and heavy metal, could induce apoptosis of hemocytes in oyster. The typical features of apoptosis, such as cell shrinkage and blebbing, chromatin condensation, DNA fragmentation and translocation of a phospholipid phosphatydilserine into the outer leaflet of the cell membrane, have been described in oysters (Gervais et al., 2016; Sokolova et al., 2004; Sunila and Labanca, 2003; Terahara et al., 2003). The mechanisms and signaling pathways underlying apoptosis as well as its roles in hostpathogen interactions have been preliminary studied in oyster (Kiss, 2010; Sokolova, 2009).

Currently, it is clear that there are two major apoptotic pathways in oysters, the intrinsic apoptotic signaling and extrinsic or death receptor pathway (Kiss, 2010; Sokolova, 2009; Zhang et al., 2011d). The intrinsic apoptotic signaling pathway is triggered by the initiator caspase-9, which is activated by cytochrome c released from mitochondria during apoptosis (Elmore, 2007; Johnstone et al., 2002; Riedl and Shi, 2004). The initiator caspase-2 (Li et al., 2015a; Xiang et al., 2013; Zhang et al., 2011d, 2014a), antiapoptosis member Bcl-2 (Renault et al., 2011), the inhibitor of apoptosis proteins (IAP) (Qu et al., 2015b), and pro-apoptotic genes BAX/BAK (Xiang et al., 2015) have been described in oysters (Li et al., 2015a; Xiang et al., 2013; Zhang et al., 2011d, 2014a). The transcripts of Cgcaspase-2, two Bax/Bak homologous genes in C. hongkongensis, and one CgIAP2 gene, all increased after treatment with bacteria or toxins, suggesting their roles in apoptosis as well as bacterial defense (Li et al., 2015a; Medhioub et al., 2013; Xiang et al., 2013; Zhang et al., 2011d, 2014a). Notably, Bcl-2 and IAP, the kernel element of intrinsic apoptosis, are significantly upregulated after OsHV-1 infection. It suggests that over-expression of IAP could be a reaction to OsHV-1 infection, which may induce the apoptotic process (Segarra et al., 2014a). In addition, CgIAP2 interacts with Cgcaspase-2 through BIR2 domain in oyster C. gigas, providing direct evidence that CgIAP2 participates in apoptosis inhibition (Qu et al., 2015b). The apoptosis-related genes are found to be highly enriched in the genome of the Pacific oyster C. gigas. Specially, there are 48 genes coding inhibitor of apoptosis proteins (IAPs) in its genome, indicating that a powerful anti-apoptosis system exists in this organism (Zhang et al., 2012a). The extrinsic pathway primarily involves initiator caspase-8, which is activated by binding of several death receptors such as tumor necrosis factor receptor (TNFR), Fas, DR4, and DR5, via Fasassociated protein with death domain (FADD) (Elmore, 2007; Johnstone et al., 2002; Riedl and Shi, 2004). Several components of this pathway were identified recently from oysters. The initiator caspase-8s have been described in C. gigas and C. virginica, with a low identity with other invertebrate and vertebrate caspase-8s (Li et al., 2015a; Xiang et al., 2013; Zhang et al., 2011d, 2014a). Remarkably, the role of Chcaspase-8 in anti-bacterial response has been demonstrated in C. hongkongensis (Xiang et al., 2013). However, the transcript of CgCaspase8-2 is only up-regulated post poly(I:C) challenge, but no change is observed after bacterial challenge, suggesting that CgCaspase8-2 is specifically involved in immune response against viruses (Li et al., 2015a). At least 13 genes encoding TNFR-like proteins are annotated in the C. gigas genome, and four of them possess a death domain (Zhang et al., 2012a). Moreover, only one FADD-like protein has been identified from C. gigas, and its expression is up-regulated by bacterial stimulation (Zhang et al., 2011d, 2015b), suggesting that these apoptosisrelated genes participate in innate immunity. Executioner caspases are the central components of the intrinsic and extrinsic apoptotic pathway. Executioner caspase-3 and caspase-1 (caspase-7-like protein) both possess executioner caspase activity and they are capable of inducing cell death in vitro (Qu et al., 2014b). Specially, the over-expression of CgCaspase-3 leads to the phosphatidylserine exposure on the external plasma membrane and the cleavage of poly (ADP-ribose) polymerase, which reduce cell viability of haemocytes, and finally result in cell apoptosis (Xu et al., 2016). Furthermore, enzyme-linked immunosorbent assay and surface plasmon resonance analysis reveal that CgCaspase-3 possess high binding specificity and moderate binding affinity to LPS. Meanwhile, the cell apoptosis mediated by CgCaspase-3 in vivo is significantly inhibited by the treatment of LPS (Xu et al., 2016), suggesting that CgCaspase-3 serve as an intracellular LPS receptor, and the interaction of LPS with CgCaspase-3 specifically inhibits the cell apoptosis induced by


CgCaspase-3. The existence of caspase-independent apoptotic pathway has been proposed in oyster. Cd2þ is known to induce apoptosis in oyster haemocytes. However, there is no decrease in the mitochondrial membrane potential or activation of caspases in response to Cd2þ in the apoptotic range (Sokolova et al., 2004). Besides, the treatment of a pan-caspase inhibitor fails to prevent apoptosis of C. virginica hemocytes following infection with the intracellular parasite P. marinus (Hughes et al., 2010). These results suggest the existence and involvement of caspase-independent pathways in oyster. Apoptosis of immune cells play important roles in protection against parasites and pathogens by the innate immune system. In oysters, the induction of apoptosis upon contact with pathogens or parasites, and the parasite-induced inhibition of apoptosis have been described. The treatment with oligopeptides containing integrin-binding Arg-Gly-Asp (RGD) motif inhibits haemocyte adhesion and spreading, suggesting that integrin may function for regulation of apoptosis in oyster haemocytes (Terahara et al., 2003, 2005). During phagocytosis of live or heat-killed marine bacteria Planococcus citraeus, apoptosis is induced in the haemocytes of the Pacific oyster C. gigas. The increased level of apoptosis is associated with the oxidative burst of haemocytes, suggesting that apoptosis may be induced by oxidative damage to haemocytes during bacterial killing (Terahara and Takahashi, 2008). It is interesting that in vivo infection with P. marinus induced apoptosis faster in C. gigas hemocytes (3 days' postinfection) than that in C. virginica hemocytes (7 days’ postinfection). The faster induction of apoptosis may be an effective defense mechanisms against P. marinus infection (Terahara and Takahashi, 2008). Many downstream elements of apoptotic signaling cascades, such as b-adrenergic signaling pathway components, MAPK, Rho, Protein kinase A and cAMP, appear to be involved in oyster haemocytes apoptosis (Lacoste et al., 2002). A highly evolutionary conserved apoptosis pathway is confirmed to exists in oyster and it plays a key role in homeostasis and functioning of the oyster immune system, especially in parasite virulence and establishment of infections. However, the composition and induction mechanism of apoptotic pathways in oyster are apparently different from those defined in invertebrate models. Further studies are urgently needed to determine the molecular mechanisms underlying pathogen-induced modulation of apoptosis in oyster to provide significant insights into immune avoidance and the evolution of the host-parasite arms race. 7. Autophagy Autophagy is a cellular homeostatic process and it plays housekeeping roles in the organisms. Accelerated evidences demonstrate that autophagy is also an innate immunity mechanism against intracellular bacteria and viruses. Its role as an immune defense mechanism has been extensively studied in higher eukaryotes (Deretic, 2006; Deretic and Levine, 2009; Levine and Deretic, 2007). Autophagy has been previously described blue mussels and abalone (Bai et al., 2012; Moore, 2008; Moore et al., 2007). Until recently, the autophagy pathway is first reported in oysters (Pierrick et al., 2015). Autophagy-related ATG genes are identified in the genome of Pacific oyster and they appear to be closer to human autophagy genes. Besides, the membrane-bound form of LC3 which is associated with autophagosomes and the activation of autophagy is found in oysters, suggesting that there is an autophagy flux in oysters (Pierrick et al., 2015). The role of autophagy during experimental infections has tested, and the infections of V. aestuarianus and OsHV-1 both trigger autophagy in oyster. When autophagy is induced by V. aestuarianus and OsHV-1,

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bacterial and viral DNA are both clearly decreased during the infection. However, bacterial DNA increases when autophagy is inhibited, while the viral DNA does not, suggesting the clearance of pathogens by autophagy is different (Pierrick et al., 2015). More work is required to understand the mechanism of autophagy in the clearance of different pathogens. 8. The neuroendocrine immunomodulation in oyster The NEI regulatory network is proposed on the existence of afferent-efferent pathways between immune and neuroendocrine structures, which refers to a unified feedback network consisted of nervous system, endocrine system and immune system. During immune response, the endocrine system optimizes immune activities to eliminate the invading pathogens and restore the wellbeings of vertebrates (Song et al., 2015; Steinman, 2004; Sternberg, 2006). Recently, a simple but complete NEI system is revealed in oysters, which can modulate immune response via a “nervous-haemocyte”-mediated neuroendocrine immunomodulatory axis (NIA)-like pathway (Liu et al., 2016d, 2017). Various hormones, neurotransmitters, key enzymes and receptors have been identified in oyster. In particular, molluscs have been proved to be the most primitive animals with a complete NEI system. This section intends to summarize the knowledge of the neuroendocrine system and its immunomodulation function in oyster. 8.1. The catecholaminergic immunomodulation The catecholaminergic neuroendocrine system is mainly composed of catecholamines (CA), CA metabolic enzymes and CA receptors (Kvetnansky et al., 2009; Lucasmeunier et al., 2003). CA, a family consisting of dopamine (DA), norepinephrine (NE) and epinephrine (EN), is one of the very first appeared neurotransmitters during the ontogenesis of oyster larvae. It acts as signal at the earliest developmental stages to regulate cell proliferation, differentiation, and neurogenesis (Zakharova, 2010; Zhou et al., 2012). In adult oysters, the synthesis and release of CAs have been reported in hemolymph, mantle and gills (Lacoste et al., 2001d, 2002). Three critical catecholamine metabolic enzymes, dopa decarboxylase (DDC), phenylalanine hydroxylase (PAH) and monoamine oxidase (MAO), as well as homologs of both a1-and a2 -adrenergic receptors are all identified in oysters (Aladaileh et al., 2008; Boutet et al., 2004; Liu et al., 2016d; McDowell et al., 2014; Yang et al., 2012). These results suggest that oyster possesses a complete catecholaminergic neuroendocrine system which is structurally similar to those in higher organisms but not C. elegans with only dopaminergic neuroendocrine system. The immune regulation is one of the most important activities modulated by CAs under biotic and abiotic stress during oyster larval stages (Liu et al., 2015b). NE exhibits negative effects on haemocyte apoptosis and phagocytosis through the receptors on oyster haemocytes (Lacoste et al., 2001d; Liu et al., 2016d; Liu et al., 2017). For example, the treatment with NE decreases the total protein contents and the activities of PAH and acid phosphatase in Sydney rock oyster S. glomerata (Aladaileh et al., 2008). NE is associated with increased susceptibility to bacterial infection in adult oyster C. gigas (Adamo, 2008). CAs respond to stress via badrenoceptor-cAMP signaling pathway (Kvetnansky et al., 2009; Lacoste et al., 2001a, 2001b), while NE modulates the reactive oxygen species production in haemocyte via b-adrenergic receptors in C. gigas (Lacoste et al., 2001c, 2002). Furthermore, a comprehensive immune-related network comprising of PRRs, intracellular receptors, signaling transducers and immune effectors is modulated by NE-responsive NeurimmiRs (microRNAs interacting with NEI system), which is indispensable for oyster haemocytes to respond

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against stress and infection (Chen et al., 2015). 8.2. The cholinergic immunomodulation Acetylcholine (ACh) is a significant neurotransmitter synthesized by choline acetyltransferase (ChAT) for cholinergic nervous system. It is hydrolyzed by acetylcholinesterase (AChE) with the products of choline and acetic acid ready for re-uptake and resynthesis (Deiana et al., 2010). Recently, the cholinergic nervous system is identified in nematodes, molluscs and arthropods (Shi et al., 2014, 2012, 2015). It provides a basis for the understanding of its modulation on the invertebrate immune system. The enzyme activities of AChE are observed in haemocyte lysis of C. gigas and tissues including gill, mantle, gut and muscle of C. hongkongensis (Zha et al., 2013). In addition, a homolog of muscarinic ACh receptor (CgmAChR) is identified from C. gigas, and its mRNA is widely distributed in various tissues (Liu et al., 2016d). These evidences prove the existence of the cholinergic anti-inflammatory pathway in oyster. The cholinergic neuroendocrine system can be activated by immune stimulation, and tends to negatively regulate the immune response at a long-time scale (Liu et al., 2016d). In C. gigas, ACh influences the mRNA expressions of TNF, transcription factors AP-1 and NF-kB, as well as the apoptosis and phagocytosis of haemocytes via the mediation of intracellular second messenger Ca2þ (Liu et al., 2016e). The regulation of ACh is accomplished with the involvement of its relevant receptors. For example, the activation of muscarinic cholinergic receptors is coupled to inhibitory cardiac modulation (Kim et al., 2004). CgmAChR, receptor of Ach in oyster, functions in cholinergic neuroendocrine-immune system and contributes to the regulation of TNF expression and apoptosis process (Liu et al., 2016f). Interestingly, ACh conducts synergistic regulation together with neurotransmitters such as enkephalin (ENK) during the immune response. CgmAChR rapidly binds ENK at early stage to eliminate the invading pathogens, but then bind ACh instead of ENK during late stages to avoid hyperimmunity (Liu et al., 2017). Besides, an invertebrate-specific miRNA cgi-miR-2d in C. gigas is proved to repress the synthesis/release of Ach and uptake of choline by targeting oyster choline transporter-like proteins (CgCTL1) in haemocytes during the early stage of Vibrio infection (Chen et al., 2016). 8.3. The immune regulation of nitric oxidase system Nitric oxide (NO) is an important gaseous signaling molecule involved in a broad range of physiological processes, such as vascular regulation (Figueroa et al., 2002; Jiang et al., 2013a), neural transmission (Straub et al., 2007) and especially immune defense (Bogdan, 2001; Bogdan et al., 2000). In oysters, protozoan parasite P. marinus could activate the NO response both in vitro and in vivo. The treatment with NO synthase (NOS) inhibitor and NO donor cause the decrease of NO levels, thereby leading to a significant inhibition in the proliferation of P. marinus. The results indicate that NO plays a role in the defenses of C. virginica to experimental infection with the protozoan parasite P. marinus (Villamil et al., 2007). NOS is the most crucial member in NO system for its exclusive role in de novo synthesis of NO (Jiang et al., 2013b). According to the structure and activity features, vertebrate NOS are divided into three isoforms, neuronal (n) NOS, inducible (i) NOS and endothelial (e) NOS (Bruckdorfer, 2005). However, only one NOS isoform (CgNOS) is found in oyster haemocytes, which is structurally similar to deuterostomen NOS, but biochemically similar to both nNOS and iNOS (Jiang et al., 2014). The newly synthesized CgNOS is targeted to the cell membrane through CgPSD-95, where it is re-activated to modulate the immune defense. The CgNOS

protein shares a similar structure and translocation process with nNOS, exhibiting similar transcriptional activation and immunomodulatory effects as iNOS. The co-stimulation of LPS and TNF-a induce the translocation of CgNOS from the membrane to the cytoplasm within the oyster haemocytes (Jiang et al., 2016a). Furthermore, the induction and activation of CgNOS are initiated by the activation of CgNF-kB and CgSTAT via the PI3K-Akt pathway. These results suggest that CgNOS is an ancient NOS which exerts all of the functions allocated to diverse isoforms of mammalian NOSs (Jiang et al., 2016a). 8.4. The immune regulation of neuropeptides Neuropeptides encompass a diverse class of cell signaling molecules that are produced and released from neurons through a kely, 2013; Stewart et al., 2014). regulated secretory pathway (Je Seventy four putative neuropeptide genes have been identified from oyster genome (Zhang et al., 2012a). There are more than 300 predicted bioactive peptide precursors, including three newly identified neuropeptide precursors PFGx8amide, RxIamide and Wx3Yamide, the gonadotropin-releasing hormone (GnRH) and two egg-laying hormones (ELH) (Stewart et al., 2014; Zhang et al., 2012a). Two GnRH-related peptides (CgGnRH-A and CgGnRH-G) are characterized by mass spectrometry from the extracts of visceral ganglia of C. gigas (Bigot et al., 2012). Both CgsNPFR (short neuropeptide F receptor)-like receptor and LFRFamide are highly expressed in the oyster central nervous system (Bigot et al., 2014). Moreover, Leucine- and Met- ENK are detected in haemocytes and hemolymph of C. gigas (Liu et al., 2008a). Met-ENK is firstly observed on the marginal of the dorsal half of D-hinged larvae of C. gigas (Liu et al., 2015b), while its specific transmembrane receptor (CgDOR) is also characterized with abundant mRNA transcripts in various tissues (Liu et al., 2015a). Similar to that in vertebrates, neuropeptides identified in oyster also play important roles in the immune regulation. Oyster LeuENK regulates CAT activity and H2O2 content via binding with opioid neuropeptide receptors on oyster immunocytes (Liu et al., 2008a). With the increased concentration of Leu-ENK, the activities of CAT are up-regulated while the H2O2 contents are decreased (Martin et al., 2008). During the ontogenesis of oyster, the activated enkephalinergic nervous system regulates the expressions of immune factors, the antibacterial and PO activities, as well as the cytokine concentration through the neurotransmitter Met-ENK (Liu et al., 2015b). CgDOR specific for Met-ENK can modulate the haemocyte phagocytic and antibacterial functions through the second messengers Ca2þ and cAMP, which are requisite for pathogen elimination and homeostasis maintenance in oyster (Liu et al., 2015a). The synergistic regulation of ENK and ACh/NE is revealed by transcriptomics and proteomic analysis. Cytokines and transcription factors such as TNF, AP-1 and NF-kB play important roles in the immunomodulation of ENK/ACh/NE (Liu et al., 2016d). It is hypothesized that the haemocytes, as immune cells, constitute the “haemocyte-nervous” neuroendocrine-immune axis (NIA) with the nervous system to mediate the neural immunomodulation in oyster (Liu et al., 2017). 8.5. The GABAergic immune regulation The GABAergic system is a ubiquitous inhibitory neurotransmitter system which cooperates with excitatory glutamatergic system to exert a profound effect on the nervous system as well as immune cells. In vertebrates, GABAergic system is composed of four primary components: (i) GABA synthase, glutamic acid decarboxylase; (ii) GABA catabolism enzyme, GABA transaminase (GABA-T); (iii) GABA transporters; and (iv) GABA receptors (Dionisio et al.,


2011; Murphy et al., 2005; Shen et al., 2014). Recently, a GAD homolog (CgGAD) and GABA have been identified from oyster C. gigas. CgGAD is mostly located in granulocytes, while GABA mainly exists in hemolymph. Both of them dynamically respond to LPS stimulation. CgGAD is capable of promoting the production of GABA after transfected into HEK293 cell line, and GABA could remarkably inhibit the immune response stimulated by LPS, such as NOS concentration, the phagocytosis and apoptosis rates of immunocytes (Li et al., 2016b, 2016c). The present results firstly demonstrate that the amino acid neurotransmitters exist in oysters and they are involved in the immune regulation. Oysters have evolved a primitive-in-structure but complicatedin-function NEI system which is similar to that in vertebrates. This NEI system participates in many physiological processes, especially in the immune regulation of oyster. However, the information about the NEI system is still limited and the lack of appropriate techniques (e.g. techniques of neurobiology in invertebrates) also hinders the research progress. Moreover, the neuroendocrine system is an extremely complicated network, and any slight change can result in totally different consequences. More comprehensive and ecological investigations will be critical for a better understanding of the regulation patterns of the NEI system. 9. Conclusion remarks In the course of long-term evolution, oysters have developed an array of effective strategies to recognize and eliminate various invaders by employing a set of molecules and cells. In the past years, there is a growing volume of information on the gene organization and adaptive characteristics of immune system, molecular and cellular mode of immune response, evolutionary importance of neuroendocrine system as well as the modulation pattern in NEI. Further investigations will be focused on the mechanisms in mounting protective immune responses against infection and their crosstalk with respect to the adaptation of oyster to the hostile environment. The controversy about the immunological memory in invertebrates also urges our efforts on the immune priming in oysters to assist in understanding the nature and evolution of immunity and the connections between immune defense in invertebrates and vertebrates. The rapid development of multi-omics and the utilization of modern biotechnology will be powerful tool to deepen our understanding about the immune system of mollusc and evolution of this largest and most diverse group in the multitude of invertebrate taxa. Acknowledgement The authors thank Dr. Sheng Ma for critical reading of the manuscript. This work was supported by NSFC (No. 31530069), earmarked fund (CARS-48) from Modern Agro-industry Technology Research System, and the Dalian high level talent innovation support program (2015R020), the Research Foundation for Talented Scholars in Dalian Ocean University (to L. S.). References Adamo, S., 2008. Norepinephrine and octopamine: linking stress and immune function across phyla. Invertebr. Surviv. J. 5, 12e19. Adema, C.M., 2015. Fibrinogen-related Proteins (FREPs) in Mollusks, Pathogen-host Interactions: Antigenic Variation v. Somatic Adaptations. Springer International Publishing, pp. 111e129. Aguirre, J., Rios-Momberg, M., Hewitt, D., Hansberg, W., 2005. Reactive oxygen species and development in microbial eukaryotes. Trends Microbiol. 13, 111e118. Akira, S., Uematsu, S., Takeuchi, O., 2006. Pathogen recognition and innate immunity. Cell 124, 783e801. Aladaileh, S., Rodney, P., Nair, S.V., Raftos, D.A., 2007a. Characterization of

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