Immune Transcripts of the Pacic Oyster, Crassostrea gigas Larvae that Change in Expression as a Result of Bacteria Challenge

Background: Marine molluscs living in intertidal and estuarine areas, such as oysters, face numerous pathogen challenges during their development. Infection from bacteria such as Vibrio alginolyticus, represents a major factor affecting larval development and frequently leads to high mortality of the pacic oyster, Crassostrea gigas. The oyster immune response is known to play an important role in protecting the animal during development by mitigating the consequences of infection. Results: In this study, we undertook a comprehensive analysis of the immune response of C. gigas to V. alginolyticus challenge. We sequenced the transcriptome of C. gigas at 0, 6, 12, 24, 48 and 72 hours post infection. After RNA-seq, the raw reads are available through the NCBI Sequence Read Archive under accession number PRJNA623063. After ltering, a total of 58.24 Gb clean reads were produced and assembled using the reference genome of C. gigas. The distribution of quality Q30 was higher than 90.88% for each sample and the GC content ranged from 41.27% to 42.91%. When compared with sequences in the COG, GO, KEGG, Swiss-Prot, and NR databases, there were 1267, 1112, 2187, 682, 1133 differentially expressed genes annotated at 6, 12, 24, 48, 72 hours post infection respectively. Numerous immune-related genes displayed differential expression that varied over time: toll-like receptors, tripartite motif proteins, Lectin-like factors, scavenger receptors, signaling pathway components such as Myeloid differentiation factor 88, and stress proteins such as Heat shock 70 kDa protein were all found to be higher in abundance following V. alginolyticus challenge compared to control. For analysis, these genes were divided into several categories such as pattern recognition receptors, brinogen-like proteins, damage associated molecular patterns, complement factors, etc. These general categories allowed us to generate an immune response prole for C. gigas over the rst 72 hours of infection. These results indicate that bacterial infection induce a complex pattern of immune gene expression in C. gigas larvae. Conclusion: Our study will facilitate targeted investigation into the function of specic immune factors

The Paci c oyster, C. gigas, is an important aquaculture oyster throughout the world, the global production of this species had expanded to 4.38 million tonnes, more than any other species of sh, molluscs or crustacea (FAO, http://www.fao.org/ shery/culturedspecies/Crassostrea_gigas/en). Because of this economic relevance, and the relative ease to culture it in controlled lab conditions, C. gigas has emerged as an important model organism for studying bivalve biology. Despite this, our understanding of the immune mechanisms that protect C.giga is still largely unknown. Based on our current knowledge, there are only a few studies that focus on advancing our understanding about the oyster immune response against bacterial challenge [5][6][7][8][9]. The genome of C. gigas was reported in 2012, highlighting genes involved in apoptosis, environmental stress adaptability and shell formation. The immune system was given speci c attention, but how the immune genes changed in expression after bacterial challenge were still unelucidated [10].
Studies that have attempted to characterize the immune capabilities of C. gigas following challenge with a pathogen or pathogen-associated molecular pattern (PAMP) have primarily focused on assessing speci c factors. For example, Gueguen et al. (2003) investigated transcriptional changes in four immunerelated genes of C.gigas following challenge with V. anguillarum, V. metshnikovii, V. tubiashii and V. S322 [11]. Tirapé et al. (2007) selected to focus on 18 immune-related genes from C.gigas EST library and analyzed their expression pattern after Vibrio tasmaniansis, V. anguillarum and Micrococcus luteus challenge during oyster ontogenesis [7]. Song et al. (2016) reported some immune-related genes expression change after V. splendidus infection at embryonic and larval development stages of the Paci c oyster, such as phagocytosis (Integrin b-1), hematopoiesis (Gata3) ,immune recognition (C-type lectin 3, TLR4 and Caspase-3), immune elimination (Interleukin 17 − 5, defensin, superoxide dismutase, catalase and heat shock protein 70) and signaling transduction (REL and serine kinase I kinase) [5]. While these studies provide valuable insight into the immune repertoire of C. gigas and how it is implemented in the face of infection, it does not provide a comprehensive overview of the organism-wide response.
Pro ling the change of abundance of immune-related transcripts following challenge with pathogens or pathogen-associated molecular patterns (PAMPs) provides insight into how the immune response is directed against pathogen insult. Considering that bacterial infection can have serious consequences for the survival of oysters, especially during the larvae stages [6], and that in the past decades, the knowledge of immune processes in adult molluscs has been studied well, however the information in larval stages is scarce. In this study, we sequenced the transcriptome of larval C. gigas at 0, 6, 12, 24, 48, 72 hours post V. alginolyticus exposure by means of Illumina RNA-Seq technology. The general transcriptome was selectively analyzed to identify transcripts that displayed differential expression that varied between exposed and control animals and that also mapped to a putative immune role based on Gene Ontology (GO) searches. Numerous immune-related genes displayed differential expression that varied over time: toll-like receptors (TLRs), tripartite motif proteins (TRIM), Lectin-like factors, scavenger receptors, signaling pathway components such as Myeloid differentiation factor 88 (MyD88), and stress proteins such as Heat shock 70 kDa protein (Hsp70) were all found to be higher in abundance following V. alginolyticus challenge compared to control. For analysis, these genes were divided into several categories such as pattern recognition receptors (PRRs), brinogen-like proteins (FREPs), damage Page 4/32 associated molecular patterns (DAMPs), complement factors, etc. These general categories allowed us to generate an immune response pro le for C. gigas over the rst 72 hours of infection. To our knowledge, this is the rst study to undertake a comprehensive transcriptome analysis of the immune responses of C.gigas to bacterial challenge. It will facilitate targeted investigation into the function of speci c immune factors that may explain the diversity and evolution of invertebrate immune molecules and lead to the development of effective measures to improve the performance of oyster culture.

Transcriptome sequence assembly
The raw reads are available through the National Center for Biotechnology Information Sequence Read Archive under accession number PRJNA623063. Quality assessment of the RNA-Seq data indicated that the distribution of quality Q30 was higher than 90.88% for each sample and the GC content ranged from 41.27-42.91%. After ltering, 69.13-73.40% clean reads were successfully matched to the C.gigas genome by the TopHat2 software (Table 1). These results demonstrate that the sequencing quality was quite good, suggesting that the later transcriptome analysis results are reliable. were down-regulated. All the DEGs at each time point were mapped to a variety of GO annotations and three main functional classi cations were determined ( Fig. 1 and see Additional le 2): cellular component, molecular function, and biological process. Based on the GO analysis results, most immunerelated genes were enriched in the category of biological process. Pathway enrichment analysis identi ed signi cantly enriched immune pathways in DEGs and showed that speci c immune pathways were signi cantly altered following V. alginolyticus challenge (Fig. 2). KEGG pathway analysis suggested that these DEGs could be assigned to speci c immune-related pathways such as Toll-like receptor signaling pathway, NOD-like receptor signaling pathway, mitogen-activated protein kinases (MAPK) signaling pathway and Apoptosis were enriched in these signaling pathways ( Fig. 3 and see Additional le 3). The signi cantly up-regulated expressed immune-related genes analysis caused by V. alginolyticus According to the analysis of GO terms and KEGG pathways, we selected the up-regulated immune-related genes with a strict threshold criterion (Log 2 Fold Change (FC) ≥ 1.5 and FDR < 0.01) (see Additional le 4). Our analyses indicate that 12 immune-related transcripts were increased in abundance at each time point after V. alginolyticus challenge (Fig. 4). Various well-known immune-related genes such as TLRs, peptidoglycan-recognition protein, heat shock protein, complement C1q protein, interleukin 17-like protein, and myeloid differentiation primary response protein MyD88, were identi ed separately and divided into general categories based on their function during an immune response. These categories included; TLRs, PRRs, DAMPs, Complement, Immune effector, Cytokines, Tripartite motif-containing proteins and Immune signaling pathway (see Additional le 5).

Pattern Recognition Receptors
PRRs can directly recognize and bind conserved PAMPs on the surface of invaders and play an important role in the innate immune defense response. Numerous PRRs were found in higher abundance in C.gigas transcriptome after V. alginolyticus infection compared to controls. At nearly every time point post challenge various PRRs were were up-regulated. A majority of PRRs genes exhibited higher expression levels from 6 hpi to 24 hpi and the expression of them reduced rapidly at 48 h post-stimulation, some of them then were increased at 72 hpi. Scavenger receptor class F member 2, scavenger receptor cysteinerich domain superfamily protein (precursor), C-type receptor 2 and macrophage mannose receptor 1 were down-regulated (see Additional le 5: Table S1).
It is well known that TLRs as pattern recognition receptors play a vital role in initiating innate immune responses against pathogens challenge. A variety of TLRs expression were obviously up-regulated after V. alginolyticus infection (see Additional le 5: Table S2). The abundance of numerous C. gigas TLRs genes increased steadily between 6 hpi − 24 hpi, then dropped gradually at 48 hpi − 72 hpi and some TLRs genes were raised again at 72 hpi.

Damage associated molecular patterns
DAMPs are endogenous danger signals that can activate immune cells and originate from damaged or necrotic tissues. A common group of proteins that are often categorized as DAMPs are the heat shock proteins (HSPs), which have a vital role 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]. In our study, we found that the transcriptome abundance of Hsp70 12A was notably increased by bacterial infection compared to controls at time points 6, 12, 24, 48, and 72 h respectively. The expression level of Hsp70 12A steadily declined at 12 hpi and increased at 24 hpi, then decreased at 48 hpi and raised at 72 hpi (see Additional le 5: Table S3). We also found that abundance of Hsp 68 decreased within 12 h against V. alginolyticus challenge, then the abundance of this transcript increased by bacterial infection compared to controls among 24 hpi − 72 hpi (see Additional le 5: Table  S3).

Complement
The complement system plays a key role in innate immunity and is widely involved in physiological and pathological processes, such as phagocytosis, cytolysis, in ammation, solubilization of immune complexes, clearance of apoptotic cells and promotion of humoral immune responses [13][14][15]. In this study, four transcripts that re ect Complement component receptor 1-like protein, complement C1q tumor necrosis factor-related protein 3, complement C1q-like protein 4 and complement C1q-like protein 3, were identi ed in the C.gigas transcriptome after bacterial infection. Abundance of the complement component receptor 1-like protein (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 le 5: Table S4). The abundance of other three complements increased at 6 hpi and the decreased level appeared at 48 hpi, and then sharp raised up the maximum level at 72 hpi.
Tripartite motif-containing (TRIM) proteins TRIM proteins have been implicated in multiple cellular functions including apoptosis, immune signaling, antiviral activity, cell proliferation, differentiation and oncogenesis. TRIM proteins including 23 TRIM2, 2 TRIM3, TRIM75 and E3 ubiquitin-protein ligase displayed increased expression post bacterial infection in our study (see Additional le 5: Table S5). Five TRIM2 were found to be most abundant at 6 hpi after which, the abundance dropped immediately at the other ve time points, seven TRIM2 and TRIM75 were sharp reached the maximum level at 72 hpi, TRIM3 were raised up the highest level at 12 hpi and then subsequently decreased progressively.

Cytokines
Cytokines are produced and secreted by cells in response to various stimulation and play vital roles in the host defense response to pathogens by performing myriad immune-relevant roles. In this study, we found three IL17 were notable up-regulated at different time points after bacterial infection (see Additional le 5: Table S6). The transcriptome expression of two IL17 steadily declined at 12 hpi and increased at 24 hpi, then dropped immediately at 48 hpi and raised at 72 hpi, the minimum level of IL17 appeared at 48 hpi.

Immune signaling pathway
MyD88 is a key adapter molecule in TLR signaling pathway and played an important role in transmitting upstream information and pathogen defense. Once MyD88 has associated with the receptor TIR domain, IL-1 receptor-associated kinase 4 (IRAK4) is recruited to the receptor complex, subsequently interact with TNF receptor-associated factor 6 (TRAF 6) to activate downstream signals, such as nuclear factor-κB (NF-κB) and MAPK, inducing the production of in ammatory cytokines and/or type I interferon to eliminate invading pathogens.
In this study, we found the expression of TLR, MyD88, TRAF family protein and IRAK4 were signi cantly up-regulated following bacterial challenge (see Additional le 5: Table S7). Five MyD88 genes expression were signi cantly increased at each time point post infection. Among these time points, MyD88 were expressed the highest level at 6 hpi, then dropped immediately to a minimum at 48 hpi and subsequently increased progressively. Two TRAF family protein were notable up-regulated at different time points post bacterial infection and one TRAF family protein was found to be most abundant at 6 hpi after which, the abundance declined gradually at the other ve time points. Abundance of two IRAK4 transcript reached a maximum level at 6 h post-stimulation, and then subsequently decreased progressively.
Validation of genes expression pro les by Quantitative Realtime PCR (qRT-PCR) To validate the transcriptome expression pro les, ten representative up-regulation immune-related DEGs were selected for qRT-PCR analysis at 6 h, 12 h, 24 h, 48 h and 72 h after treatment with the elongation factor 1 α (EF1-α) gene as internal control (Fig. 5). The overall trend of qRT-PCR-based expression patterns of ten selected genes Hsp70, Peptidoglycan-recognition protein SC2 (PRR), TLR6, IL17, Complement C1q-like protein 4 (C1q4), MyD88, Serine protease inhibitor dipetalogastin (SPI), Alternative oxidase, mitochondrial (AOM), TNF receptor-associated factor family protein (TRAFP) and Fibrinogen C domain-containing protein 1(FC1) were similar to those obtained by transcriptome analysis, indicating that the reliability of the expression pro ling determined by RNA-seq was reliable and accurate, although the expression fold changes of most genes measured by qRT-PCR were more than those from transcriptome analysis.

Discussion
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 calci ed 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-speci c transcriptome during ontogenesis.

Pattern recognition receptors
PRRs are an important part of the innate immune response. This general classi cation of immune receptors is de ned 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, brinogen-related proteins, LPS-binding proteins, peptidoglycan recognition proteins and scavenger receptors) have been reported from invertebrates, including C. gigas [19][20][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), brinogen-like proteins (FREPs), calciumdependent (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 le 5: Table S1). Besides TLRs, which are discussed in detail below, other PRRs found in this transcriptome study have also been identi ed 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 upregulated 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.  [26][27][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 signi cantly post bacterial challenge. Consistent with our study, up-regulation of FREPs were also reported in snail B. glabrata [28][29][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 Ca 2+ [21]. In our study, we also nd 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 rst 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, ies, 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 in ammatory cytokines and/or type I interferon to clear invading pathogens. Along with the TLRs identi ed 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, identi ed 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 grampositive bacteria Staphylococci aureus, Micrococcus luteus, and fungi Pichia pastoris in oysters [41]. The Tolls identi ed 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 signi cantly upregulated after LPS, PGN and zymosan (GLU) challenge [43]. Similar to above studies, we have also found some TLRs/Tolls expression upregulation 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 in ammatory 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 signi cantly 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 Paci c 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 identi ed 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][52][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 classi ed 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  [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, in ammation, solubilization of immune complexes, clearance of apoptotic cells and promotion of humoral immune responses [13][14][15].
There are four types of protein in the complement system, including inherent components, regulatory molecules, complement receptors and speci c protein fragments that can be activated [57]. Complement components such as C1q, mannose-binding lectin and colin 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 rst identi cation of a complement homolog found in the sea urchin in 1996 [60]. The recognition molecules and associated serine proteases, colins, 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][62][63][64][65][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][69][70][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 le 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 nger (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 identi ed 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 identi ed 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 Paci c oyster after LPS challenge and function as a regulator of immune response against bacterial challenge [84]. Seo et al.
reported the ubiquitin was puri ed from the gill of C.gigas and identi ed as antimicrobial [85]. Wu et al.
found an Rbx1 which belonged to the RING-nger 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 signi cantly up-regulated in the C. gigas transcriptome after bacterial challenge. Rouvier et al. rst reported that IL-17 as cytolytic T-lymphocyte (CTL)-associated antigen 8, is a T-cell factor with proin ammatory activity [88]. It is well known that IL-17 is an important member of the proin ammatory 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 in ammatory response [87,90]. IL-17 genes have also been identi ed in mollusks such as the pearl oyster P. fucata [91], the triangle-shell pearl mussel, H. cumingii [93], the Paci c oyster C.gigas [87]. suggested that IL17-5 acted as an in ammatory 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.

Conclusions
The transcriptome of C. gigas larvae at 0, 6, 12, 24, 48, 72 hours post V. alginolyticus exposure was sequenced by means of Illumina RNA-Seq technology. This is the rst study to undertake a comprehensive transcriptome analysis of the immune responses of C. gigas to bacterial challenge. Numerous immune-related genes displayed differential expression that varied over time: toll-like receptors, tripartite motif proteins, Lectin-like factors, scavenger receptors, signaling pathway components such as MyD88, and stress proteins such as Hsp70 were all found to be higher in abundance following V. alginolyticus challenge compared to control. These genes were divided into several categories such as PRRs, FREPs, DAMPs, complement factors, etc. These general categories allowed us to generate an immune response pro le for C. gigas over the rst 72 hours of infection. These results will facilitate targeted investigation into the function of speci c immune factors that may explain the diversity and evolution of invertebrate immune molecules and lead to the development of effective measures to improve the performance of oyster culture.

Oyster larvae and bacteria treatment
Adult individuals of oyster C. gigas were collected from a farm in Yantai, Shandong Province, China, and acclimatized in laboratory for over one week before subsequent experiments. Sperms and eggs were scratched from gonads of male and female oysters with toothpicks. The fertilization was obtained by mixing sperms and eggs, and then the fertilized embryos were cultured in aerated seawater at 26 ℃.
D-hinged larvae were obtained using 300 mesh and challenged by the nal concentration of 6 × 10 8 CFU of V. alginolyticus. Larvae without any treatment were designated as the control group. At the beginning of the bacterial challenge experiment, the samples were washed two times using ltered seawater and DEPC respectively at 1, 200 rpm. Then adding 500 µL of TRIzol Reagent (Invitrogen, USA) and stored at -80℃ for later use. After bacterial challenge, the tissues were obtained by above methods at the following

RNA-seq data analysis
For ensuring the accuracy of subsequent analysis, Raw data generated from Illumina sequencing were preprocessed to remove the low-quality sequence and adaptor sequences reads. Raw sequences were transformed into clean reads after data processing. These clean reads were then mapped to the reference genome (C. gigas genome) sequence using Tophat2 tools [96]. Only reads with a perfect match or one mismatch were further analyzed and annotated based on the reference genome. Cu inks was used to assemble the mapped reads and then the reads were compared with the annotation of the reference genome to acquire new transcripts and fragments per kilobase of transcript (FPKM) value was used as an indicator to measure the level of gene expression [97]. The obtained unigenes were used for a BLAST search and functionally annotated against the NR, Swiss-Prot, GO, COG, KOG (Eukaryotic Orthologous Groups), KEGG and Pfam databases.
The differentially expressed transcripts analysis DESeq software [98] applied to experiments with biological repetition was then used to determine the differential gene expression of every 2 groups of samples with a threshold criterion of Log 2 FC ≥ 1 and FDR < 0.01, for up-regulated and down-regulated transcripts. GO analysis was performed to determine the biological implications of the differentially expressed genes. For enrichment analysis, all differentially expressed genes were mapped to terms in GO and KEGG databases, and then we searched for signi cantly (P ≤ 0.05) enriched GO and KEGG terms in DEGs compared with the overall transcriptome.
Validation using quantitative real-time PCR (qRT-PCR) To con rm our Illumina sequencing data, ten representative genes including Hsp70, PRR, TLR6, IL17, C1q4, MyD88, SPI, AOM, TRAFP and FC1 were selected for qRT-PCR. Gene-speci c primers of all the genes above were designed using Primer 3.0 and EF1-α gene was chosen as an internal reference gene The oysters used in this study were marine-cultured animal and were collected from a local farm in Yantai, China. There are no speci c permissions required for the sampling of these oysters. All experiments were conducted with approval from the Experimental Animal Ethics Committee, Beibu Gulf University and Zhejiang University, China.

Consent for publication
Not applicable.

Availability of data and material
The transcriptome datasets supporting the results of this study are available in the NCBI BioProject repository (Accession Number PRJNA623063) and in the SRA database (https://submit.ncbi.nlm.nih.gov/subs/sra/SUB7232205/overview, SUB7232205 ).
48h after bacteria challenge. Sheet 5. The up-regulated immune-related genes with a strict threshold criterion (Log 2 FC≥1.5 and FDR<0.01) at 72h after bacteria challenge.
Additional le 5 (Microsoft Word): Categories based on function during an immune response over the rst 72 hours of infection. The le includes following datasheets: Additional le 6 (xls.): Ten representative genes and the internal reference gene primers.    Venn diagram of up-regulated expressed immune-related genes.