Characterization and Expression Analysis of a Complement Component Gene in Sea Cucumber (Apostichopus japonicus)

CHEN Zhong, ZHOU Zunchun, YANG Aifu, DONG Ying, GUAN Xiaoyan,JIANG Bei, and WANG Bai



Characterization and Expression Analysis of a Complement Component Gene in Sea Cucumber ()

CHEN Zhong, ZHOU Zunchun*, YANG Aifu, DONG Ying, GUAN Xiaoyan,JIANG Bei, and WANG Bai

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The complement system plays a crucial role in the innate immune system of animals. It can be activated by distinct yet overlapping classical, alternative and lectin pathways.In the alternative pathway, complement factor B (Bf) serves as the catalytic subunit of complement component 3 (C3) convertase, which plays the central role among three activation pathways. In this study, the Bf gene in sea cucumber (), termed, was obtained by rapid amplification of cDNA ends (RACE). The full-length cDNA ofwas 3231bp in length barring the poly (A) tail. It contained an open reading frame (ORF) of 2742bp encoding 913 amino acids, a 105bp 5’-UTR (5’-terminal untranslated region) and a 384bp 3’-UTR. AjBf was a mosaic protein with six CCP (complement control protein) domains, a VWA (von Willebrand factor A) domain, and a serine protease domain. The deduced molecular weight of AjBf protein was 101kDa. Quantitative real time PCR (qRT-PCR) analysis indicated that the expression level ofinwas obviously higher at larval stage than that at embryonic stage. Expression detection in different tissues showed thatexpressed higher in coelomocytes than in other four tissues. In addation,expression in different tissues was induced significantly after LPS or PolyI:C challenge. These results indicated that AjBf plays an important role in immune responses to pathogen infection.

sea cucumber;; complement factor B; LPS challenge; PolyI:C challenge

1 Introduction

The complement system is a highly sophisticated biological reaction system, which plays a crucial role in body defense as a part of both the innate and the adaptive immune systems (Nonaka and Yoshizaki, 2004). This system can be activated by a series of limited proteolytic cascades through three parallel pathways. The classical pathway (CP) is activated by antibody-antigen (Ab-Ag) complexes and is a major effector of the acquired immune system that arose in the jawed vertebrate lineage. The other two, including the alternative pathway (AP) and the lectin pathway (LP), function in innate immune defense, and are considered to be important in invertebrates defending against microbial infections (Nonaka, 2014; Sekine., 2001; Fujita., 2004). These three pathways merge at the proteolytic activation step of the complement component 3 (C3), and the activation fragments of C3 induce several effecter mechanisms against the pathogens and facilitate the immune response (.). In mammalians, complement factor B (Bf) serves as the catalytic subunit of C3 convertase in the alternative pathway (Pangburn, 1983) while in the classical pathway, this function is subjected to C2 (Ishii., 1993; Wei., 2009). In addition, mammalian Bf and C2 have identical modular structures, including three complement control protein (CCP) domains at the N- terminus, one von Willebrand factor A (VWA) domain and one serine protease (SP) domain at the C-terminus (Horiuchi., 1993; Tuckwell., 1997). The VWA and CCP structural modules contain binding sites for C3b and C4b, respectively (Tuckwell., 1997; Xu and Volanakis, 1997).

The most ancestral pathway to activate C3 is mediated by factor B (Prado-Alvarez., 2009). This mechanism, presenting in sea anemone(.), carpet-shell clam(Prado-Alvarez., 2009), horseshoe craband(Zhu., 2005; Tagawa., 2012), sea urchin(Gross., 1999), ascidian(Yoshizaki., 2005; Nicola., 2014), lamprey(Nonaka, 1994) and amphioxus(He., 2008) resembles the alternative pathway from mammals and constitutes an ‘archeo-complement system’. These Bf proteins often have an atypical structure compared with mammalian Bf or C2. For example, sea urchin (Smith., 1998), horseshoe crab (Zhu., 2005) and sea anemone Bf proteins (.) all have five CCP domains, while tunicate Bf possesses four CCP domains with two additional low-density lipoprotein receptor (LDLR) domains. Some marine invertebrates contain more than one isoform of Bf gene. For example, two homologs of Bf gene were found in sea anemone(Kimura., 2009) and horseshoe crab(Tagawa., 2012), while three were found in sea urchin(Hibino., 2006), ascidian(Yoshizaki., 2005) and amphioxus(Huang., 2008), respectively. The presence of multiple Bf genes in marine species may be resulted from gene duplication and exon shuffling (Yoshizaki., 2005). However, the difference in functions among Bf isomers is still unclear. Despite the distinctive characteristics, the described invertebrate Bf proteins have been considered orthologues of the mammalian Bf and C2 (Nonaka and Kimura, 2006).

Sea cucumber () belongs to echinoderms, which locates between invertebrates and chordates systematically, and occupies an important evolutionary position. It is a reference point for inferring the evolution of deuterostome immune systems. Sea cucumber is also an economically important aquaculture species. In previous studies, a Bf gene of, named-2 (JN- 634069.1) has been cloned and studied on its transcriptional expression after LPS challenge (Zhong., 2012). AjBf-2 shows similarity to the Bf genes of other invertebrates in molecular structure, and is expressed in all determined tissues and has been proved to be involved in the immune response against Gramnegative bacteria. Here, complement Bfgene in, named, was cloned and characterized. The expression patterns ofwere investigated in different tissues at embryonic and larval developmental stages. Furthermore, the gene expression profiles after LPS and PolyI:C challenge were also analyzed in the different tissues of. We also compared the structure and expression patterns ofwith those ofwhich was cloned by Zhong. (2012). Our study aimed to enrich the information of echinoderms complement components that are complementary to comparative and evolutionary aspects of immunity.

2 Materials and Methods

2.1 Full-Length cDNA Cloning of

The partial sequences ofwere obtained from the ESTs ofby cDNA libraries sequencing (Yang., 2009; Zhou., 2014). To obtain the full-length cDNA of, rapid amplication of cDNA ends (RACE) PCR was carried out using a SMART RACE cDNA Amplification Kit (Clontech, Japan). The primer-F and-R were designed according to the known EST sequence of AjBfgene (Table 1). The primer pairs of-F,-R and UPM primer (supplied by Clontech) were used for 3’ RACE and 5’RACE, respectively. The PCR products were purified, cloned into pMD19-T vector (Takara, Japan) and subsequently sequenced. Finally, the full-length cDNA ofwas obtained by assembling the 3’ RACE and 5’RACE fragments.

Table 1 Primers for RACE and qRT-PCR used in this study

2.2 Characterization and Phylogenetic Analysis of AjBf

For sequence analysis, the Bf amino acid sequences were either identified by simple key word searches, or with BLAST searches using sea urchin Bf amino acid sequences at NCBI (http://www.ncbi.nlm.nih.gov/BLAST). Protein sequences retrieved from public database were used for open reading frame (ORF) and domain searches, alignment, and phylogenetic reconstruction. ORF was predicted using Open Reading Frame Finder (http:// www.ncbi.nlm.nih.gov/gorf/gorf.html). The protein domains were predicted by the Simple Modular Architecture Reach Tool (SMART, http://smart.embl-heidelberg.de/). Translation and protein analysis were performed using ExPaSy tools (http://us.expasy.org/tools). Multiple alignment of the Bf was performed using the program ClustalX version 2.0 (Larkin., 2007). Phylogenetic tree was constructed using the neighbor-joining method based on the deduced full-length amino acid sequences within the Molecular Evolutionary Genetics Analysis (MEGA 4.0) package (Tamura., 2007). Data were analyzed using Poisson correction, and gaps were removed by complete deletion. The topological stability of the tree was evaluated by 1000 bootstrap replications.

2.3 Sampling and Immune Challenge

Mature sea cucumbers were collected from Guanglu Island (Dalian, China) in early July. Animals were artificially spawned by flowing sea water (20–21℃) stimulation. During the development from fertilized eggs to 1mm-long juvenile, sea cucumbers were cultured in filtered sea water (temperature: 20–21℃, salinity: 32, pH: 7.8). To investigate the expression of AjBfgene at different developmental stages, samples from every developmental stages including unfertilized eggs, fertilized eggs, cellulous stages, blastula prior to hatching, gastrula, early auricularia, auricularia, late auricularia, doliolaria, pentactula and 1-mm long juvenile, were collected by sieving using 60µm filter, pelleted by centrifugation (Labnet Spectrafuge, USA) and then stored in 1.5mL microcentrifuge tubes. All samples were frozen immediately with liquid nitrogen and then stored at −80℃ prior to RNA isolation.

To investigate the AjBfgene expression in different tissues, the intestine, respiratory tree, coelomocytes, body wall and tube feet tissues were separated from 15 healthy individuals (3 pools with 5 individuals each pool) (average body weight 10.2g). These tissues from different individuals were mixed respectively. Coelomocytes were collected according to the methods of Santiago-Cardona (Santiago-Cardona,2003). Intestine, respiratory tree, body wall and tube feet were excised, briefly rinsed in filtered seawater, and frozen immediately with liquid nitrogen and then stored at −80℃.

To investigate the expression patterns of AjBf gene after immune challenge, Two different immune challenges were conducted by coelomic injection for each sea cucumber with 500µL PolyI:C (100mgmL−1) or LPS (1mgmL−1). PolyI:C (Sigma, P9582) and LPS (Sigma, L2880) were diluted with phosphate buffered saline (PBS, pH= 7.4). The sea cucumbers injected with 500µL PBS were treated as the control group (Ramírez-Gómez, 2009). The sea cucumbers without any injection were treated as the blank group (0h). The different tissues were collected for quantitative real-time PCR (qRT-PCR) analysis after the sea cucumber were injected for 4h, 12h, 24h, 48h and 72h, respectively.At each sampling time, the tissues taken from 15 different individuals (3 pools with 5 individuals each pool) were mixed in three 1.5mL microcentrifuge tubes, and thrown immediately into liquid nitrogen and then stored at −80℃.

2.4 Quantitative Real-Time PCR

Total RNA was isolated using the RNAprep pure Tissue Kit (Tiangen, Beijing, China) according to the manufacturer’s instructions. The quality and quantity of extracted total RNA were measured using the NanoPhotometer (Implen GmbH, Munich, Germany) and agarose gel electrophoresis. First strand cDNA synthesis was performed in a volume of 20µL with 900ng total RNA, 25pmol Oligo dT Primer, 50pmol random 6mers, 4µL 5×PrimeScriptTM buffer, 1µL PrimeScriptTM RT enzyme Mix I (PrimeScriptTM RT reagent Kit, TaKaRa, China). Reactions were conducted by incubating at 37℃ for 15min, and then at 85℃ for 5s to deactivate the enzyme.

To know the AjBf gene expression profiles ofat different developmental stages, in different tissues and after LPS or PolyI:C challenge, equal amounts of cDNA template were used in qRT-PCR. The cytochrome b (Cytb) gene was used as the reference gene (Yang., 2009), and the relative expression of the AjBf gene was assessed using qRT-PCR in the Mx3000pTMdetection system (Applied Stratagene, USA). The reactions were performed in a total volume of 20µL containing 10µL of 2×SYBR Green Master mix (SYBR PrimeScript_RT-PCR Kit II, TaKaRa), 0.4µL of ROX Reference Dye II, 2µL of cDNA template, and 0.4µmol L−1of each primer (Table 1). The qRT-PCR program was 95℃ for 30s, followed by 40 cycles of 95℃ for 10s, 56℃ for 25s and 72℃ for 25s. Melting curve analysis of amplification products was performed at the end of each PCR reaction to confirm that only one PCR product was amplified and detected. In addition, the amplicons were checked by agarose gel with a 100bp ladder to confirm the correct amplicon sizes.

2.5 Statistical Analysis

All the statistical analyses about the expression level ofmRNA were normalized by Cytb gene. We used the Relative Expression Software Tool 384 v.2 (REST) (Pfaffl., 2002) to study themRNA expression profiles. The crossing-point (CP) values were converted to fold differences by relative quantification method. The mathematical model used is based on the PCR efficiencies and the mean crossing point deviation between the control and treatment groups by pairwise fixed reallocation randomization test. The statistically significant difference was defined as<0.05.

3 Results

3.1 AjBf Gene Isolation and Sequencing

Based on our cDNA libraries sequencing of(Yang., 2009), an EST, 1624bp in length, was obtained, which was similar to the partial sequence of sea urchinin Genbank. The full-length cDNA sequence of AjBf gene was obtained by RACE PCR and sequencing. The assembled sequence showed that the full-length cDNA sequence of AjBf gene was 3231bp barring the poly (A) tail. It contained a 105bp 5’-UTR (terminal untranslated region), an open reading frame (ORF) of 2739bp encoding 913 amino acids and a 384bp 3’-UTR with a polyadenylation signal (AATAAA motif) (Fig.1).

AjBf had a deduced molecular weight of 101kDa. The deduced amino acid sequences of AjBf contained a possible signal peptide, suggesting that it was a secreted protein. Like the other Bf or C2 family members, AjBf was a mosaic protein including CCPs, a VWA domain, and a serine protease domain (Fig.2). It was interesting that AjBf had six CCPs, while both AjBf-2 (Zhong., 2012) and SpBf (sea urchin) had five, and most of vertebrate Bf or C2 proteins had three. In addition, one Arg- Phe (RF) bond, the putative cleavage site for C1s and/or factor D to activate Bf, was located at residues 448–449 before the VWA domain of AjBf (Fig.3). In the Serine Protease domain of AjBf, the critical catalytic triad was His 698, Asp742 and Ser851. There were eight consensus recognition sequences for N-linked glycosylation through- out the AjBf sequence (Fig.1). Four were located in the CCPs, one was found in the region between the CCPs and the VWA domain, one was in the VWA domain, and the other two were in the serine protease domain. A CCP domain was typically about 60 amino acids long with a number of conserved residues. These amino acids in- cluded four cysteines, three glycines, two prolines, two tyrosines (or phenylalanine) and one tryptophan, all of which were located in specific positions within the domain (Reid and Day, 1989). The aligned results of six CCP domains from AjBf were shown in Fig.4. Two disulfide bonds were formed between the four cysteines and maintain the topology of the domain. Although some of the consensus amino acids were missing from AjBf, all four cysteines were present in each CCP, suggesting that these domains fold as expected.

Fig.1 The deduced protein sequence of AjBf. The full-length cDNA has been deposited in GenBank with accession number HQ993063. The signal peotide is underlined. The nucleotides of initiation codon and the polyadenylation signal are underlined too. The N-linked glycosylation sites are shaded.

Fig.2 Structural feature of AjBf. Schematic representations of AjBf were predicted by SMART program. Signal peptide determined by the SignalP program is shown in red. Abbreviations indicate as follows: CCP, complement control protein; VWA, von Willebrand factor A; Tryp_SPc, serine protease.

Fig.3 Alignment of the VWA and serine protease domains from AjBf and the other Bf/C2 family proteins. To simplify the analysis, the CCP domains were deleted, and the remaining domains were aligned with CLUSTAL W program. Domains and amino acids with functional significance are labeled. Accession numbers for sequences used in this alignment can be found in the legend for Fig.5. Aj: A. japonicus; Sp: Strongylocentrotus purpuratus; Hr: Halocynthia roretzi; Hs: Homo sapiens; Mm: Mus musculus; Ol: Oryzias latipes; Xl: Xenopus laevis; Nv: Nematostella vectensis; Lc: Lethenteron camtschaticum; Br: Branchiostoma belcheri tsingtauense.

3.2 Phylogenetic Analysis

Pairwise alignments were used to calculate percentage of amino acid similarities and identities between AjBf and all the other Bf porteins without the CCP domains. The AjBf amino acids showed 86.4% similarity with AjBf-2, and 19.2%–31.2% similarities with Bfs in some other species. Consequently, to assess the relationships among the Bf protein sequences, alignments of full length of the AjBf and other Bf/C2 proteins were used for phylogenetic analysis (Fig.5). The evolutionary tree analysis showed that the Bf genes were evolved from a common ancestor in the evolutionary development of two directions, one cluster was the invertebrates and cephalochordates, and another was the Urochordates and vertebrates. Sea cucumber had the closest relationship with sea urchin, and they were involved in one cluster with sea anemone and Branchiostomas.

Fig.4 Alignment of the AjBf CCPs. The six CCPs from AjBf are aligned to each other with the consensus amino acids shown in bold and at the bottom. Two disulfide bonds maintain the CCP domain structure and are formed between the first and third cysteine, and the second and fourth cysteine. The alignment was done with CLUSTAL W.

Fig.5 Phylogenetic tree. The tree was constructed using ClustalW and MEGA (3.1). The numbers of branches are bootstrap values for 1000 replicates. The GenBank accession numbers for the sequences are as follows: AjBf-2 (Apostichopus japonicus), AEP68015; SpBf (Strongylocentrotus purpuratus), NP_999700; HrBf (Halocynthia roretzi), AAK00631; HsBf (Homo sapiens), NP_001701; MmBf (Mus musculus), AAC05283; OlBf (Oryzias latipes), NP_001098275; XlBf (Xenopus laevis), BAA06179; NvBf (Nematostella vectensis), BAH22728; LcBf (Lethen- teron camtschaticum), BAA02763; BrBf (Branchiostoma belcheri tsingtauense), ABY28382; HsC2 (Homo sapiens), AAB97607; MmC2 (Mus musculus), AAA63294; BtC2 (Bos taurus), AAI03358; XlC2 (Xenopus laevis), ABB- 85337; OmC2 (Oncorhynchus mykiss), NP001117673.

3.3 AjBf Gene Expression Analysis

The qRT-PCR analysis was employed to determine specific expression patterns of AjBf gene at eleven developmental stages of sea cucumber. The expression level of AjBf gene in unfertilized eggs was higher than that in the multi-cell and blastula stages, and AjBfgene expression increased significantly from the gastrulas to the juveniles. The highest expression level was detected in the late auricularias, about 10-fold higher than that in unfertilized eggs (<0.05). The AjBf gene expression levels were obviously higher at larval stages than those at embryonic stages (Fig.6).

Fig.6 The relative transcript abundance of AjBf gene at different developmental stages of A. japonicus determined through qRT-PCR analysis. 1, Unfertilized egg; 2, Fertilized egg; 3, Cellulous stage; 4, Blastula; 5, Gastrula; 6, Early auricularia; 7, Auricularia; 8, Late auricularia; 9, Doliolaria; 10, Pentactula; 11, Juvenile. Each symbol and vertical bar represents the mean±S.D (n=3). Asterisks represent a significant difference at the level of P<0.05 relative to unfertilized egg.

The AjBf gene expression in different tissues including intestine, respiratory tree, coelomocytes, tube feet and body wall, were also detected. The results indicated thattranscripts were detected in all tested tissues, the lowest expression level was found in the intestine and thehighest level was found mainly in the coelomocytes, which was about 30-fold higher than that in intestine (Fig.7).

Fig.7 The relative mRNA levels of AjBf gene in different tissues. I, intestine; BW, body wall; C, coelomocytes; F, tube feet; RT, respiratory tree. Each vertical bar represents the mean±S.D (n=3). Expression levels in all tissues are presented relatively to that in the intestine tissue (1×).

AjBf gene expression was significantly induced by LPS and PolyI:C in the tested tissues. After LPS challenge, AjBf gene expression in coelomocytes increased at 12h, and peaked at 48h (about 9 fold), then decreased at 72h. It is noted that AjBf gene expression levels were significantly up-regulated at 4h and 72h in body wall. In respiratory tree, AjBf gene expression increased significantly at three time points except of 12h and 48h. While, significant up-regulations of AjBf gene were just observed at 4h and 24h in tube feet. In intestine, the expression levels of AjBf gene were relatively lower at 12h and 72h, and higher at 4h (Fig.8A).

After PolyI:C challenge, AjBf gene expressed strongly in body wall, 9-fold higher than that of the control group at 4h. In coelomocytes, the expression level increased at 4h after challenge, and reached a peak at 24h (about 3.5 fold), then decreased gradually to the similar level as in the LPS group. AjBf gene expression was significantly up-regulated in intestine at 72h. In tube feet and respiratory tree, it was similar to the LPS challenge group (Fig.8B).

Fig.8 Fold induction of AjBf gene challenged by LPS (A) and PolyI:C (B) in five tissues. Relative expression of AjBf gene was expressed as fold changes over control samples taken at the same time interval as normalized to change in expression in the Cytb control. Each vertical bar represents the mean±S.D (n=3). Asterisks represent a significant difference at the level of P<0.05 relative to appropriate control.

4 Discussion

In the present study, we isolated and characterized the full-length cDNA of the complement factor B-like gene in sea cucumber. Its expression profiles in normal tissues and after LPS and PolyI:C challenges were also analyzed. Complement component C3, a key specific protease in the alternative pathway, had been proved to be present in the sea cucumber (Zhou., 2011). The isolation of Bf and C3 provided a strong evidence for the presence of an alternative activation pathway in sea cucumber.

Like other members of the Bf/C2 family, AjBf had a mosaic structure including CCPs, a von Willebrand factor A (VWA) domain, and a serine protease domain. The alignment between AjBf and vertebrate Bf/C2 proteins revealed amino acids in conserved positions for serine protease activity, a conserved factor D cleavage site, and Mg2+binding sites.

We used to believe that five CCP domains is the ancestral structure of Bf/C2 protein family. The recently identified members of Bf/C2 from horseshoe crab and sea urchin have five CCP domains (Zhu., 2005; Smith., 1998), and sea anemone has two Bf homologue with three and five CCP domains, respectively (.), while the vertebrate Bf/C2 members all have three CCP domains, suggesting that vertebrate Bf/C2 may have lost two (Horiuchi., 1993; Hourcade., 1995). In this study, six CCPs were found in AjBf, which was more than AjBf-2 and Bf in other species. Compared with AjBf-2, AjBf has an extra CCP domain and a signal peptide. Six CCPs rather than five may more likely be the ancestral type. Considering the important functions of CCPs in Bf for binding to C3b in vertebrates, the three CCPs close to the VWA domain of AjBf might be more important for binding functions than the three N-terminal CCPs.

Phylogenetic analysis showed that the Bf gene of sea cucumber has the closest relationship with that of sea urchin, and they are in one cluster with those of sea anemone and Branchiostomas. Based on the sequences alignment and phylogenetic trees, the complement Bf-like homology of sea cucumber is close to Bf rather than C2. In vertebrates, the Bf/C2 families continue to differentiate to Bf and C2. AjBf may be one of the most primitive members of the Bf/C2 protein family.

The existing of AjBf and AjC3 in sea cucumber indicated that its simple complement pathway is similar to the complement system in amphioxus, which is also composed of C3 and Bf (He., 2008). This simple sea cucumber complement pathway might function like the alternative pathway in vertebrates (Tuckwell., 1997). Thetranscripts were persistent during the embryogenesis. The expression level oftranscripts in unfertilized eggs was significantly higher than those in the multi-cell and blastula stages. It was significantly increased from the gastrulas to the juveniles, and the highest level was detected in the late auricularias (Fig.6). This suggested that complement-mediated defense system may function in unfertilized eggs and early embryos. The increase ofexpression level in the gastrulas period probably meant that the embryos are protected by the fertilization envelope, and do not require a functional defense system. From the gastrulas, because of the organ formation and the change of life habits, thetranscriptional levels increased significantly.

The gene encoding AjBf expressed specifically in coelomocytes than in other tissues. Coelomocytes have long been considered to be the essential immune effector cells of the immune response in echinoderm, so it is not surprising that AjBf gene showed intensive expression in the coelomocytes (Al-Sharif., 1998). For-2, high level expression was found in both coelomocytes and body wall, while the expression was only in a detectable level in other tissues (Zhong., 2012). The expressions ofand-2 were both up-regulated in coelomocytes after LPS challenge, and followed by a decrease. However, the time to peak value was different.expression reached the peak at 48h, while-2 reached the peak at 12h. In body wall, the expressions ofand-2 both were significantly up-regulated at 4h after LPS challenge. It was inferred that both AjBf and AjBf-2 are involved in the immune response of sea cucumber to LPS challenge, while their functions need to be further studied.

LPS is a major component of cell walls of gram- negative bacteria and an extremely useful immune-activating substance. Many immune-related genes have been documented to respond to LPS challenge. PolyI:C, the synthetic analog of dsRNA, can be treated as a potent immunoregulatory agent for viral infection. Welsh found that human serum can deplete RNA virus through either the classical complement pathway component C4 or the alternative complement pathway component factor B (Welsh, 1977). Furthermore, it was reported that LPS and dsRNA could effectively increase phagocytes and phagocytic activities in coelomocytes of the sea cucumber(Ramírez-Gómez., 2010). In this study, we found that the AjBf gene expression increased significantly after LPS challenge, which was consistent with gene C3 as we reported early (Zhou., 2011). Thus both Bf and C3 may be involved in the immune response against the bacteria pathogen by alternative pathway. In PolyI:C group,AjBf expression was significantly induced in different tissues, implying that the sea cucumber responds to RNA virus by activating the alternative complement pathway.

In sea cucumber, coelomocytes are the essential immune effector cells, and body wall is the first barrier against pathogen invasion. Except these two tissues, immune responses of other three tissues, including respiratory tree, intestine and tube feet, were simultaneously detected in our study. Positive results showed that they were all involved in immune responses in sea cucumber.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (Nos. 31272687 and 30972 272), Science and Technology Project of Liaoning Province (2014203006), Ocean and Fisheries Project of Liao- ning Province (201301), and Science and Technology Project of Dalian City (2013J21DW025).

Al-Sharif, W. Z., Sunyer, J. O., Lambris, J. D., and Smith, L. C., 1998. Sea urchin coelomocytes specifically express a homologue of the complement component C3., 160: 2983-2997.

Franchi, N., and Ballarin, L., 2014. Preliminary characterization of complement in a colonial tunicate: C3, Bf and inhibition of C3 opsonic activity by compstatin., 46 (2): 430-438.

Fujita, T., Matsushita, M., and Endo, Y., 2004. The lectin-com- plement pathway: Its role in innate immunity and evolution., 198: 185-202.

Gross, P. S., Al-Sharif, W. Z., Clow, L. A., and Smith, L. C., 1999. Echinoderm immunity and the evolution of the complement system., 23: 429-442.

He, Y., Tang, B., Zhang, S., Liu, Z., Zhao, B., and Chen, L., 2008. Molecular and immunochemical demonstration of a novel member of Bf/C2 homolog in amphioxus: Implications for involvement of hepatic cecum in acute phase response., 24: 768-778.

Hibino, T., Loza-Coll, M., Messier, C., Majeske, A. J., Cohen, A. H., Terwilliger, D. P., Buckley, K. M., Brockton, V., Nair, S. V., Berney, K., Fugmann, S. D., Anderson, M. K., Pancer, Z., Cameron, R. A., Smith, L. C., and Rast, J. P., 2006. The immune gene repertoire encoded in the purple sea urchin genome., 300 (1): 349-365.

Horiuchi, T., Kim, S., Matsumoto, M., Watanabe, I., Fujita, S., and Volanakis, J. E., 1993. Human complement factor B: cDNA cloning, nucleotide sequencing, phenotypic conversion by site-directed mutagenesis and expression., 30: 1587-1592.

Hourcade, D. E., Wagner, L. M., and Oglesby, T. J., 1995. Analysis of the short consensus repeats of human complement factor B by site-directed mutagenesis.,270: 19716-19722.

Huang, S., Yuan, S., Guo, L., Yu, Y., Li, J., Wu, T., Liu, T., Yang, M., Wu, K., Liu, H., Ge, J., Yu, Y., Huang, H., Dong, M., Yu, C., Chen S., and Xu, A., 2008. Genomic analysis of the immune gene repertoire of amphioxus reveals extraordinary innate complexity and diversity.,18 (7): 1112- 1126.

Ishii, Y., Zhu, Z. B., Macon, K. J., and Volanakis, J. E., 1993. Structure of the human C2 gene., 151 (1): 170-174.

andMulti-com- ponent complement system of Cnidaria: C3, Bf, and MASP genes expressed in the endodermal tissues of a sea anemone, . , 214: 165-

Larkin, M. A., Blackshields, G., Brown, N. P., Chenna, R., Mc- Gettigan, P. A., McWilliam, H., Valentin, F., Wallace, I. M., Wilm, A., Lopez, R., Thompson, J. D., Gibson, T. J., and Higgins, D. G., 2007. Clustal W and Clustal X version 2.0., 23: 2947-2948.

Nonaka, M., 1994. Molecular analysis of the lamprey complement system., 4: 437-446.

Nonaka, M., 2014. Evolution of the complement system., 80: 31-43.

Nonaka, M., and Kimura, A., 2006. Genomic view of the evolution of the complement system., 58: 701-713.

Nonaka, M., and Yoshizaki, F., 2004. Primitive complement system of invertebrates.,198: 203-215.

Pangburn, M. K., 1983. Activation of complement via the alternative pathway.,42: 139-143.

Pfaffl, M. W., Horgan, G. W., and Dempfle, L., 2002. Relative expression software tool (REST(c)) for group-wise comparison and statistical analysis of relative expression results in real-time PCR., 30 (9): e36.

Prado-Alvarez, M., Rotllant, J., Gestal, C., Novoa, B., and Figueras, A., 2009. Characterization of a C3 and a factor B-like in the carpet-shell clam,., 26: 305-315.

Ramírez-Gómez, F., Aponte-Rivera, F., Méndez-Castaner, L., and García-Arrarás J. E., 2010. Changes inpopulations following immune stimulation with different molecular patterns., 29: 175-185.

Ramírez-Gómez, F., Ortíz-Pineda, P. A., Rivera-Cardona, G., andGarcía-Arrarás, J. E., 2009. LPS-induced genes in intestinal tissue of the sea cucumber., 4: 61-78.

Reid, K. B., and Day, A. J., 1989. Structure-function relationships of the complement components., 10: 177-180.

Santiago-Cardona, P. G., Berríos, C. A., Ramírez, F., andGar- cía-Arrarás, J. E., 2003. Lipopolysaccharides induce intestinal serum amyloid A expression in the sea cucumber.,27: 105-110.

Sekine, H., Kenjo, A., Azumi, K., Ohi, G., Takahashi, M., Kasukawa, R., Ichikawa, N., Nakata, M., Mizuochi, T., Matsushita, M., Endo, Y., and Fujita, T., 2001. An ancient lectin-dependent complement system in an ascidian: Novel lectin isolated from the plasma of the solitary ascidian, Halocynthia roretzi.,167: 4504-4510.

Smith, L. C., Shih, C. S., and Dachenhausen, S. G., 1998. Coe- lomocytes express SpBf, a homologue of factor B, the second component in the sea urchin complement system., 161: 6784-6793.

Tagawa, K., Yoshihara, T., Shibata, T., Kitazaki, K., Endo, Y., Fujita, T., Koshiba, T., and Kawabata, S., 2012. Microbe- specific C3b deposition in the horseshoe crab complement system in a C2/factor B-dependent or -independent manner., 7 (5): e36783.

Tamura, K., Dudley, J., Nei, M., and Kumar, S., 2007. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0., 24: 1596-1599.

Tuckwell, D. S., Xu, Y., Newham, P., Humphries, M. J., and Volanakis, J. E., 1997. Surface loops adjacent to the cation- binding site of the complement factor B von Willebrand factor type A module determine C3b binding specificity., 36: 6605-6613.

Wei, W., Wu, H., Xu, H., Xu, T., Zhang, X., Chang, K., and Zhang, Y., 2009. Cloning and molecular characterization of two complement Bf/C2 genes in large yellow croaker ()., 27: 285- 295.

Welsh, R. M., 1977. Host cell modification of lymphocytic choriomeningitis virus and Newcastle disease virus altering viral inactivation by human complement.,118: 348-354.

Xu, Y., and Volanakis, J. E., 1997. Contribution of the complement control protein modules of C2 in C4b binding assessed by analysis of C2/factor B chimeras., 158: 5958-5965.

Yang, A. F., Zhou, Z. C., He, C. B., Hu, J. J., Chen, Z., Gao, X. G., Dong, Y., Jiang, B., Liu. W. D., Guan, X. Y., and Wang, X. Y., 2009. Analysis of expressed sequence tags from body wall, intestine and respiratory tree of sea cucumber ()., 296: 193-199.

Yoshizaki, F. Y., Ikawa, S., Satake, M., Satoh, N., and Nonaka, M., 2005. Structure and the evolutionary implication of the triplicated complement factor B genes of a urochordate ascidian,., 56: 930-942.

Zhong, L., Zhang, F., and Chang, Y., 2012. Gene cloning and function analysis of complement B factor-2 of., 33: 504-513.

Zhou, Z. C., Dong, Y., Sun, H. J., Yang, A. F., Chen, Z., Gao, S., Jiang, J. W., Guan, X. Y., Jiang, B., and Wang, B., 2014. Transcriptome sequencing of sea cucumber (Apostichopus japonicus) and the identification of gene-associated markers., 14 (1): 127-138.

Zhou, Z. C., Sun, D. P., Yang, A. F., Dong, Y., Chen, Z., Wang, X. Y., Guan, X. Y., Jiang, B., and Wang, B., 2011. Molecular characterization and expression analysis of a complement component 3 in the sea cucumber ()., 31 (4): 540-547.

Zhu, Y., Thangamani, S., Ho, B., and Ding, J. L., 2005. The ancient origin of the complement system.,24: 382-394.

(Edited by Qiu Yantao)

DOI 10.1007/s11802-015-2696-8

ISSN 1672-5182, 2015 14 (6): 1096-1104

© Ocean University of China, Science Press and Spring-Verlag Berlin Heidelberg 2015

(July 11, 2014; revised November 5, 2014; accepted September 1, 2015)

* Corresponding author. Tel: 0086-411-84678047 E-mail: zunchunz@hotmail.com