It is well established that marine molluscs possess an effective and robust immune response that protects them against infection [16]. To date, however, most of the studies that have characterized the immune responses of these animals have focused on adult individuals, while the immune response in juveniles/larvae has received less attention. For oysters and other bivalves, the developmental stages from trochophore to larvae are indispensable as calcified shells will be formed and the immune system becomes mature during this developmental stage [17]. During this developmental period, there can be lethal consequences should any infectious agent impact the developing organism. Before any functional differences in the immunological capabilities between larval and adult organisms can be assessed, it is important to establish a baseline of those immune factors that are likely relevant during the larval stage. In this study, the expression of immune-related genes in the larvae of C. gigas after challenge by the bacterial pathogen V. alginolyticus was studied to demonstrate the immune-specific transcriptome during ontogenesis.
Pattern recognition receptors
PRRs are an important part of the innate immune response. This general classification of immune receptors is defined by their ability to directly recognize different PAMPs such as lipopolyssacharide (LPS) and peptidoglycan (PGN) from bacteria, β-1,3-glucan from fungi, or double-stranded RNA from viruses [18]. To date, several PPRs (e.g TLRs, fibrinogen-related proteins, LPS-binding proteins, peptidoglycan recognition proteins and scavenger receptors) have been reported from invertebrates, including C. gigas [19–21]. As reported here, numerous PPRs have been detected as presenting increased abundance in the C. gigas transcriptome following bacterial challenge. C. gigas TLRs, peptidoglycan recognition proteins (PGRPs), scavenger receptors (SRs), fibrinogen-like proteins (FREPs), calcium-dependent (C-type) mannose receptor, macrophage mannose receptor (MMR), lectins, C-type receptor, protein toll, collectin-12 and hemolymph lipopolysaccharide-binding protein (LPS-BP) were found in higher abundance in challenged groups compared to controls (see Additional file 5: Table S1). Besides TLRs, which are discussed in detail below, other PRRs found in this transcriptome study have also been identified in C. gigas [18], Eriocheir sinensis [22], the Sydney Rock Oyster Saccostrea glomerata [23], Limulus polyphemus [24] from PGRPs, SRs, FREPs, C-type mannose receptor, MMR, lectins, and protein toll to collectin-12 and LPS-BP. PGRPs which can recognize peptidoglycan and peptidoglycan-containing bacteria play an indispensable role in innate immunity for invertebrates and vertebrates, due to its outstanding ability in detecting and eliminating invasive bacteria. Yang et al. [25] reported the up-regulated expression of PGRPs in Zhikong scallop C. farreri after bacterial challenge and the recombinant protein exhibited strong agglutination activity to the Gram-positive bacteria Micrococcus luteus and Bacillus subtilis. Similar to this result, we also found the transcription expression of PGRPs were obviously up-regulated after V. alginolyticus infection and a clearly time-dependent expression pattern of PGRPs was observed. It is conceivable that the C.gigas PGRPs found in this study could serve not only recognize bacterial invasion, but also play a role in eliminating the pathogen.
Adema et al. and Li et al. found that a role for invertebrate FREPs in recognition of parasite-derived molecules and FREPs are proteins with at least a fibrinogen-like (FBG) domain, they are widespread in Mollusca [26–28], and play curial roles in the innate immune response as PRRs. In this study, we found the gene expression of FREPs were up-regulated significantly post bacterial challenge. Consistent with our study, up-regulation of FREPs were also reported in snail B. glabrata [28–30], amphioxus Branchiostoma belcheri [31], mosquito Armigeres subalbatus [32], C. gigas [20], and Anopheles gambiae [33, 34]. It is inferred that the FREPs found in this study can play important roles as PRRs in the innate immune response and inhibit infection from pathogens.
SRs are a group of heterogeneous molecules on the surface of phagocytes and play an important role in innate immunity, which can recognize and mediate engulfment of a variety of pathogenic substances to eliminate the invading pathogens. An up-regulation SR induced by LPS, PGN and β-glucan was observed in the scallop C. farreri and SR recombinant protein can interact with LPS, PGN and the fungal particles mannan and zymosan in the presence of Ca2+ [21]. In our study, we also find a related SR up-regulated under similar circumstances. These results show that PRRs playing as detecting PAMPs in invertebrates as they are in mammalian. C-type lectin superfamily have been reported to play a potential role in the activation of complement system in C.gigas [35] and mediate pathogen recognition and cellular adhesion in C. farreri [36]. C-type lectins, collectins and macrophage mannose receptor formed this family and it was investigated that they were also associated with the cell membrane and phagocytosis [37]. For oysters with only innate immune system to defend various pathogen infection, a series of PRRs might give C.gigas an ability against invading pathogens.
Toll-like receptors
Tolls or TLRs play an indispensable role in initiating innate immune responses against pathogens challenge. Since the first description of Toll and its role in the immune response against fungal infection in Drosophila melanogaster [38], a large number of TLRs have been reported and functionally characterized in various species. As an ancient family of evolutionary conserved PRRs, TLRs, which are found in many species including mammals, flies, crustaceans and molluscs, are playing as crucial roles in immune system [23]. They play a key role in early host defense against invading pathogens by recognizing conserved PAMPs and activating downstream signaling pathways which can induce the production of inflammatory cytokines and/or type I interferon to clear invading pathogens. Along with the TLRs identified in this study, TLRs have also been reported in C.gigas genome [10] and other molluscs such as the pearl oyster Pinctada fucata martensii [39], Hyriopsis cumingii [40]. Wang et al. reported that TLR6, identified in C.gigas, could function as an important pattern recognition receptor in the early detection and response against invading gram-negative bacteria V. anguillarum, V. splendidus, and gram-positive bacteria Staphylococci aureus, Micrococcus luteus, and fungi Pichia pastoris in oysters [41]. The Tolls identified from a marine crab, Portunus trituberculatus have been reported to participate in the host defense against V. alginolyticus, Candida lusitaniae and white spot syndrome virus [42]. A study indicated that the expression of a Toll from E. sinensis was significantly upregulated after LPS, PGN and zymosan (GLU) challenge [43]. Similar to above studies, we have also found some TLRs/Tolls expression up-regulation from our analysis of the C.gigas transcriptome, including transcripts that represent protein tolls, TLR6, TLR13, TLR1 TLR2 and TLR4. Given the consistent increase in expression, it could be inferred that TLRs likely play an essential role in bacteria recognition and subsequent activation of the innate immunity.
It is well known that TLRs play an indispensable role in recognition of invaders and their stimulation by PAMP ligands such as LPS and DAMP ligands such as Hsp70 induces a TLR signaling pathway which plays a vital role in the immune defense against pathogen infection by activating the diverse downstream reaction including anti-oxidant, anti-bacteria and apoptosis [44, 45]. MyD88 is a widespread and important adaptor for TLR/IL-1R family, and primarily recruited to activate TLR after the recognition of PAMPs/DAMPs by TLRs. Once MyD88 has associated with the receptor TIR domain, IRAK4 is recruited to the receptor complex, subsequently interact with TRAF6 to activate downstream signals, such as NF-κB and MAPK, inducing the production of inflammatory cytokines and/or type I interferon to eliminate invading pathogens.
In the present study, we also found the expression of TLR, MyD88, TRAF family protein and IRAK4 were significantly up-regulated following bacterial challenge, but NF-κB has not been found in the C. gigas transcriptome. Similar to our results, Jiao et al. showed an up-regulated expression of MyD88 post stimulation by LPS in P. fucata martensii [46], and these results were echoed by Wang et al. that reported both MyD88 and TRAF6 were up-regulated after LPS stimulation [47]. Tang et al. found IRAK4 could inhibit MyD88-induced NF-κB activation in Pacific oyster with Oyster herpesvirus-1 microvariant (OsHV-1 mvar), V. alginolyticus, and poly I:C challenge [48]. Considering the above results and that our C.gigas transcriptome analysis contains multiple TLR and MyD88 transcripts that demonstrate an increase in abundance following bacterial challenge, it was inferred that a MyD88-dependent TLR signaling pathway also existed in C.gigas larvae and TLRs and the adaptor protein MyD88 respond to a range of pathogens, indicating they play an important role in the innate immunity of oyster.
Damage associated molecular patterns
DAMPs are endogenous danger signals that can activate immune cells and originate from damaged or necrotic tissues. To date, various DAMPs such as HSPs and high-mobility group box 1(HMGB1) have been identified and characterized in invertebrates and vertebrates [49, 50]. A common group of proteins that are often categorized as DAMPs are the HSPs. As stress proteins, HSPs are ubiquitous and evolutionarily conserved, known to exist in all living organisms [51–53]. They play indispensable roles in protecting cells against environmental stresses such as heat shock, heavy metal exposure, bacterial infection or almost any sudden changes inducing protein damage in the cellular environment [12]. According to their molecular weight, these proteins have been classified into several families, such as HSP90 (85–90 kDa), HSP70 (68–73 kDa), HSP60, HSP47, and low molecular mass HSPs (16–24 kDa) [54]. Among these proteins, HSP70s have been studied extensively and are most responsible for intracellular chaperone and extracellular immunoregulatory functions as DAMPs [50]. Recently, the role of HSP70s in activating innate immunity and participating in host response to bacteria infection have been reported in mollusks, such as the up-regulation of HSP70 expression in Pinctada martensii against V. alginolyticus, the increased expression of HSP70 in Mytilus galloprovincialis by V. anguillarum stimuli, the significant expression of HSP70 in Mytilus coruscus affected by V. alginolyticus and V. harveyi. [50, 53, 55]. Song et al. showed that HSP70 would provide cytoprotection in the C. gigas larvae after V. splendidus challenge [5]. In this study, we found HSP-70 kDa and HSP-68 kDa expression were increased in C.gigas after V. alginolyticus challenge and a clearly time-dependent expression pattern of HSP70 was observed. The expression of the HSP70 gene increased at 6 hpi and the maximum level appeared at 24 hpi, and then dropped gradually. Consistent with our study, HSP70 expression up-regulated after the bacteria challenge were also reported in other mollusks, such as Sepiella maindroni [12], P. martensii [50], Laternula elliptica [54], Mytilus coruscus [55], P. fucata [56]. Our results suggested that HSP70 might be involved in oyster response to pathogenic infection and the up-regulated mRNA expression of HSP70 following infection response indicated that the HSP70 might play an important role in oyster immune response.
Complement
The complement system plays a key role in innate immunity against infection and is widely involved in physiological and pathological processes. The functions of the complement system include phagocytosis, cytolysis, inflammation, solubilization of immune complexes, clearance of apoptotic cells and promotion of humoral immune responses [13–15].
There are four types of protein in the complement system, including inherent components, regulatory molecules, complement receptors and specific protein fragments that can be activated [57]. Complement components such as C1q, mannose-binding lectin and ficolin are PRRs that function as recognizing potential pathogens during immune responses and activating different complement pathways in vertebrates [58]. The key component of the classical complement pathway C1q involved in widespread immunological processes such as apoptotic cells elimination, bacteria and retrovirus recognition, cell adhesion and cell growth modulation provides a major connection between innate and acquired immunity [59].
Although the complement system has been well studied in mammals, little is known about complement components in invertebrates. With the first identification of a complement homolog found in the sea urchin in 1996 [60]. The recognition molecules and associated serine proteases, ficolins, MBL-associated serine proteases (MASPs), C1q1, CaC1q2, C3 and C2/Complement factor B (Bf) and C1q-domain containing protein have been recently reported in invertebrates [61–66]. In innate immunity, C1q-domain containing proteins can be considered as specialized PRPs because they have the ability to bind pathogens directly through PAMPs and to trigger phagocytosis [67]. A series of the complement related recognition molecules may be induced when foreign microorganisms invade or pathogen-related carbohydrate detection occurs in invertebrate animals. In present study, we used bacteria V. alginolyticus to infect the oyster larvae, and then analysed the transcriptome data at different time points. The result showed that the complement component expression was up-regulated after pathogen challenge. Similar to our results, the C1q domain-containing transcripts up-regulation expression has been reported in mussel, abalone and clam hemocytes against Gram-positive, Gram-negative bacteria and cell wall components (LPS, glucan and peptidoglycan) [68–71]. Interestingly, our study found that abundance of CR1L transcript increased gradually and reached a maximum level at 48 h post-stimulation, then declined progressively and down regulated straightly at 72 hpi (see Additional file 5: Table S4). It was inferred that the complement system of the oyster larvae could be activated by V. alginolyticus to defense bacteria within 48 h post-stimulation, then the complement system was inhibited, which would suppress the oyster immune response to pathogen challenge. Based on the result above, it could be speculated that the complement played an important role in oyster immune response.
Tripartite motif-containing proteins
TRIM family consist of more than 70 members of proteins, which are characterized by the presence of three different types of domains: RING finger (R) domain, B-box (B) and a coiled coil (CC) domain. The R domain of many TRIM proteins has been reported to function as E3 ubiquitin ligases by binding to both ubiquitin E2-conjugating enzymes and target proteins to facilitate the selective ubiquitination of the target, whereas the B and CC domains may be involved in protein interactions and homo/heterodimerization [72]. E3 ubiquitin ligases play as indispensable roles during ubiquitation process and are involved in the regulation of both innate and adaptive immune response [73, 74]. Nabika et al. found that the tumor necrosis factor-α (TNFα) was increased by ubiquitin and lipopolysaccharide synergistically in the murine macrophage cell line RAW 264, related to the modulatory mechanisms of the immune response [75].
To date, many TRIM proteins have been identified and studied well in mammals [76, 77]. TRIM proteins have been implicated in multiple cellular functions including apoptosis, immune signaling, antiviral activity, cell proliferation, differentiation and oncogenesis. Recent studies have shown that many members of the TRIM superfamily are expressed in response to interferons (IFNs) and are involved in a broad range of biological processes that are associated with innate immunity [78, 79]. Members of TRIM family of E3 ligases are reported as important regulators of innate immunity, such as TLR signaling pathway were regulated by TRIM proteins [80, 81].
However, the knowledge about TRIM proteins in mollusks is still limited. Rosani et al. reported that TRIM2 was expressed after ostreid herpesvirus 1-positive challenge in C.gigas [82]. Song et al. found that TRIM3 had the ability to suppress cell proliferation by inactivating p38 signaling pathway and play as a role of tumor inhibitor in cervical cancer [83]. In this study, we identified TRIM proteins (e.g TRIM2, TRIM3 and TRIM75) and E3 ubiquitin-protein ligase as being up-regulated expression after bacterial challenge. Similar to our results, E3 ubiquitin-protein ligase was up-regulated expression in the Pacific oyster after LPS challenge and function as a regulator of immune response against bacterial challenge [84]. Seo et al. reported the ubiquitin was purified from the gill of C.gigas and identified as antimicrobial [85]. Wu et al. found an Rbx1 which belonged to the RING-finger family of Ubiquitin ligase E3 involved in the immune response of abalone Haliotis diversicolor supertexta [86]. According to these above results, we can infer that TRIM protein obtained in the present study might be involved in oyster response to pathogenic infection.
Cytokines
Cytokines are small proteins including interleukins, IFNs, colony-stimulating factors (CSFs), and TNFs, which are produced and secreted by cells in response to various stimuli. They play vital roles in the host defense response to pathogens by performing myriad immune-relevant roles. [87].
Few of the hallmark cytokines known from mammalian immune studies possess direct ortholog in invertebrates, however, conserved protein domains allow for invertebrate studies to target appealing candidates for functional evaluations. One cytokine that has been shown to be consistent between vertebrates and invertebrates is IL-17, which was significantly up-regulated in the C. gigas transcriptome after bacterial challenge. Rouvier et al. first reported that IL-17 as cytolytic T-lymphocyte (CTL)-associated antigen 8, is a T-cell factor with proinflammatory activity [88]. It is well known that IL-17 is an important member of the proinflammatory cytokine family and plays an indispensable role in the eliminating of extracellular bacteria [89]. IL17 also can activate NF-κB and MAPK signal pathways, then induce other cytokines secretion and immune cells migration, further trigger the inflammatory response [87, 90]. IL-17 genes have also been identified in mollusks such as the pearl oyster P. fucata [91], the triangle-shell pearl mussel, H. cumingii [93], the Pacific oyster C.gigas [87].
Similar to our results, Li et al. showed that five IL-17 genes identified from the Pacific oyster genome were significantly up-regulated in hemocytes challenged with PAMPs including LPS, heat-killed V. alginolyticus and PGN [87]. Wu et al. and Roberts et al. reported the IL17 gene expression in hemocytes of oysters was increased after challenges of bacteria and could activate transcription factors such as NF-κB, which suggested that IL17-5 acted as an inflammatory cytokine transmitting signals, and play crucial roles in immune recognition and bacteria elimination [93, 94]. An IL-17 in pearl oyster P. fucata has been reported that it was involved in the immune response to LPS and poly(I:C) stimulation, participated in and activated NF-κB signal pathway in mammalian cells [95]. Based on these above results, it is inferred that the IL-17 found in this study can play important roles in the innate immune response and inhibit infection from pathogens.