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Characterization and evolutionary analysis of duplicated C7 in miiuy croaker
Fish & Shellfish Immunology 45 (2015) 672e679
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Short communication
Characterization and evolutionary analysis of duplicated C7 in miiuy croaker Shanchen Wang a, 1, Yunhang Gao b, 1, Chang Shu a, Tianjun Xu a, * a b
Laboratory of Fish Biogenetics & Immune Evolution, College of Marine Science, Zhejiang Ocean University, Zhoushan, 316022, China College of Animal Science and Veterinary Medicine, Jilin Agriculture University, Changchun, 130118, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 April 2015 Received in revised form 24 May 2015 Accepted 28 May 2015 Available online 30 May 2015
The complement system, as one of the most sophisticated innate immune system, plays an important role in defense against invading microorganisms. The complement component C7 participates in the cytolytic phase of complement activation through a series of polymerization reactions with other terminal complement components. In this study, we derived two C7 genes from the whole genome of miiuy croaker which were the consequence of the fish-specific genome duplication. Our data showed that miiuy croaker C7-1 and C7-2 genes shared same structure domains. The analysis of gene synteny showed that high degree conserved of synteny was retained between miiuy croaker and other teleosts, and miiuy croaker had a relatively closer relationship with fugu. The expression of C7-1 and C7-2 in miiuy croaker healthy tissues revealed that they were ubiquitously expressed in all ten tested tissues. Besides, the immune response of C7-1 and C7-2 were different in spleen with Vibrio anguillarum, Staphylococcus aureus, poly I:C and LPS at 24 h post-injection, respectively. Furthermore, the expression patterns of C7-1 and C7-2 were different in liver, spleen and kidney after infected with V. anguillarum at different timepoint. Evolutionary analysis showed that all the ancestral lineages underwent positive selection except for the ancestral lineages of fish C7-2, indicated that the ancestral lineages of fish C7-1 genes undertook more pressures than C7-2 in defense against the invading microorganisms. Meanwhile, a series of maximum likelihood methods were used to explore the evolutionary patterns on extant vertebrates' C7 genes. Three and one positive selection sites were found in extant mammalian C7 genes and fish C7-2 genes, but no positive selection site was found in extant fishes C7-1 genes. The result showed that extant fish C7-2 genes undertook more pressures compared with C7-1. In conclusion, fish C7-1 and C7-2 gene underwent different evolutionary patterns. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Complement C7 Gene duplication Expression patterns Molecular evolution
1. Introduction Immune responses are mediated by two general systems: adaptive immunity and innate immunity. Adaptive immunity can date back to the early period of vertebrates evolution, between the divergence of cyclostomes and cartilaginous fish [1]. Innate immunity system is a very old defense mechanism, and it provides the first line of defense before the adaptive immune system comes into play [2]. Fish, the first of true vertebrates, have evolved adaptive immune responses similar to those in higher vertebrates, but the adaptive immunity in fish is inefficient, therefore they should make
* Corresponding author. E-mail address: [email protected] (T. Xu). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.fsi.2015.05.042 1050-4648/© 2015 Elsevier Ltd. All rights reserved.
full use of the well-developed innate immune system to compensate for their rudimentary antibody response [3]. The complement system, one of the most sophisticated innate immune system [4,5], plays a major role during inflammation and infection, as well as a link between innate and adaptive immunity [6]. The complement activated by the classical, lectin or alternative pathway leads to the formation of the membrane attack complex (MAC) on the surface of complement-opsonized cells [7]. The assembly of MAC involves the aggregation of the lytic complement components C5b, C6, C7, C8 and C9 [8]. C5b connects with C6 via a metastable binding site to form a soluble C5b-C6 complex in the vicinity of the activating cell. Subsequent C7 interacts with the C5b-C6 to produce C5b-7, a trimolecular complex that allows insertion of this complex into the target cell membrane [9,10]. Among MAC components, the terminal complement components (TCC) C6, C7, C8a, C8b and C9 belong to the same gene
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family as perforins and they may have emerged through a series of duplications of an ancestral gene [11]. Consequently, they share several common structural motifs such as thrombospondin (TSP), low-density lipoprotein receptors class A (LDLa), epidermal growth factor precursor (EGF) and the MAC/perforin segment (MACPF). These domains are conserved and also present in teleost counterparts [12]. In addition, mammalian C7 possesses complement control protein (CCP) motifs and factor I MAC (FIMAC) modules in the C-terminal domain except for these conserved motifs [13]. As one member of TCC, C7 fulfills a crucial role in the hydrophiliceamphiphilic transition of MAC, so it plays an essential role in the elimination of invading pathogens. In previous studies, the full set of TCC genes had been identified in teleost, amphibian reptilian/aves and mammals except for the avian C9 gene which was not found in the draft genome sequence of chicken [14]. Besides, two C7 genes were identified in rainbow trout [15], although gene duplication was a common evolutionary event, it was the first time to find gene duplication in the C6eC9 gene family. Miiuy croaker (Miichthys miiuy), as one member of family Sciaenidae, mainly distributes from the Western Japan Sea to the East China Sea. Currently, the molecular immunology, genetics and molecular evolutionary of immune genes were studied more indepth in this species [16e19]. Besides, we have been studying complement system. In our previous studies, the complement component Bf/C2, C3, C4 and C9 genes had been identified and analyzed in miiuy croaker [20e22]. Now we are interested in complement component C7 gene which fulfills a crucial role in the hydrophiliceamphiphilic transition of MAC. In this study, two complement component C7 genes (C7-1 and C7-2) were identified from the whole genome database of miiuy croaker which were in consequence of the fish-specific genome duplication (FSGD or 3R). And we were the first time to analyze the gene synteny of C7-1 and C7-2 in teleost which could provide information for the investigation of the evolutionary relationships and evolutionary mechanism of C7 genes. Furthermore, the expression patterns of C7-1 and C7-2 genes were also analyzed to illuminate the possible role of C7-1 and C7-2 in response to different pathogens infection. Considering the unique status of complement component in evolutionary process of innate and adaptive immunity, we also analyzed the molecular evolution of C7 gene in fishes and mammals. 2. Material and methods 2.1. Sequence analysis and phylogenetic tree construction In order to identify the C7-1 and C7-2 genes, we used available fish genes as queries to search for the transcriptome [23] and whole genome database of miiuy croaker (unpublished data) by local Blast software. The retrieved reconstructed transcripts were translated using ORF Finder (http://www.ncbi.nlm.nih.gov) and GENSCAN [24]. The predicted open reading frames (ORFs) were verified by BLASTP against NCBI non-redundant protein sequence database. The potential protein domains of amino acid sequences were calculated by SMART program [25]. All sequences from other organisms used in this study were derived from GenBank and Ensemble database (Supplementary table S1). All the gene sequences were aligned under codon model by MUSCLE software for its high accuracy and speed [26]. Phylogenetic tree was constructed with the Bayesian approach by MrBayes v3.2 [27] which was running 5,000,000 generation with 25% of trees burned. For Bayesian inference, the GTR þ I þ G model was regarded as the best-fit model by Bayesian information criterion (BIC) using jModeltest software [28].
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2.2. Molecular evolutionary analysis The nonsynonymous and synonymous rate ratio u (dN/dS) stands for the change of selective pressure. The u ¼ 1, <1, and >1 are indicative of neutral evolution, purifying selection and positive selection, respectively. To investigate the evolutionary process of C7-1 and C7-2 genes, the selective pressure was analyzed by CODEML of PAML software [29] and the Hyphy package of Data Monkey Web Server [30]. All the models of PAML and Hyphy package used in this study were carried out as described by Zhu et al. [31]. 2.3. Fish sampling, challenge experiments and RNA extraction Miiuy croakers were obtained from Zhoushan Fisheries Research Institute (Zhejiang, China). All fishes were held in aerated water tanks to allow for acclimatization and evaluation of overall fish health before using in experiments. Only fishes with similar size and body weight were used. One week later, the challenge experiments of miiuy croaker were intraperitoneally infected with Vibrio anguillarum, Staphylococcus aureus, poly I:C and LPS. Fish samples were randomly divided into two groups, injection and control groups. In injection group, fishes were injected with 1 ml of V. anguillarum (1.5 108 CFU/ml), poly I:C (2.5 mg/ml), LPS (1.0 mg/ ml) and S. aureus (1.5 108 CFU/ml), respectively. Meanwhile, the control fishes were injected with 1 ml of physiological water. For injection group with V. anguillarum, fish samples were killed at 6 h, 12 h, 24 h, 36 h, 48 h and 72 h post-injection, respectively. While injection group with poly I:C, LPS and S. aureus, fish samples were killed at 24 h post-injection, respectively. Three tissues (liver, kidney and spleen) of infection were removed and stored at 80 C, at the same time ten tissues (liver, kidney, spleen, heart, eye, fin, brain, gill, muscle and intestines) of uninfected were also removed and keep at 80 C. Total RNA were extracted from the various tissues of adult individuals by Trizol reagent (Qiagen) following the manufacturer's instructions. cDNA was synthesized using QuantScript RT Kit (TIANGEN) according to the manufacturer's protocol. 2.4. miRNA and mRNA transcriptomes and the prediction of target gene Total RNA of spleen tissue which infected with V. anguillarum, poly I:C, LPS and S. aureus after 24 h post-injection and the control group were used to prepare the miRNA and mRNA transcriptomes in our Lab (unpublished). These libraries were used for the deep sequencing by an Illumina platform according to the manufacturer's protocol. In order to predict the miRNA target gene of miiuy croaker C7-1 and C7-2, the miRNA target gene database miRGen 3.0 was used to select the corresponding miRNA that exhibited complementarily with mRNA sequencing. We found that miR-27a3p and miR-1306 were perfect complementary with miiuy croaker C7-1 and C7-2, then we analyzed the expression patterns of these two miRNA using the deep sequencing data. 2.5. Expression analysis of C7-1 and C7-2 genes Three pairs of primers were used to study the expression patterns of C7-1 and C7-2 genes (Supplementary table S2). Healthy tissues and infected tissues were determined using qRT-PCR. The qRT-PCR was run on a 7300 real-time PCR system (Applied Biosystems, USA) using a RealMaster Mix kit (TIANGEN). The reaction carried out without the template was used as blank control. Cycling conditions were as follows: 15 min at 95 C, followed by 45 cycles consisting of 15 s at 95 C and 60 s at 60 C, dissociation curve analysis was performed after each assay to determine target
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specificity. Expression of b-actin was used as internal control for miiuy croaker C7-1 and C7-2 genes expression analysis. We used the Duncan test to detect the significant differences between groups and p < 0.05 represented significant differences. 3. Results and discussion 3.1. Identification of miiuy croaker C7-1 and C7-2 genes The complete coding sequence of miiuy croaker C7-1 and C7-2 were obtained (GenBank accession No. KP689602 and KP689603). The C7-1 cDNA sequence encodes a protein of 830 aa with ORF 2493 bp and the C7-2 consists 2466 bp with its ORF encoding 821 aa. The genomic DNA of miiuy croaker C7-1 contains 17 exons and 16 introns, while miiuy croaker C7-2 includes 15 exons and 14 introns (Fig. 1A). The sizes of C7-1 and C7-2 exons appear to be similar among fish genes, respectively. Comparative genomic structure reflects the evolutionary conservation exists among fishes. The comparison of deduced amino acid sequences shows that miiuy croaker C7-1 shares a 62.0%e80.6% identity with other fishes and the identity of C7-2 among fish ranges from 53.0% to 79.6% (Table 1). Sequences alignment of C7-1 and C7-2 genes from several fishes shows that C7-1 genes share highly conservation with C7-2 genes (Fig. 1B). The miiuy croaker C7-1 and C7-2 genes share the same structure domains which contains two TSP1 domains, one LDLa domain, one MACPF domain, one EGF domain, two CCP domains and two FIMAC domains (Fig. 1C). All the structure domains that are characteristic of TCCs (TSP1, LDLa, MACPF and EGF) are present in miiuy croaker C7-1 and C7-2. 3.2. Gene synteny analyses of C7-1 and C7-2 among fishes In order to explore the evolutionary relationship of C7-1 and C72 genes among fishes, synteny analysis was carried out using the Genomicus database v73.01. Gene synteny around the miiuy croaker C7-1 and C7-2 genes was compared with other fishes. The miiuy croaker C7-1 gene was found on scaffold 625 and it was flanked by c6 and ghrb genes different from zebrafish and cave fish (Fig. 2A). Zebrafish C7-1 gene located between two c6 genes, while cave fish C7-1 gene was flanked by c6 and oxct1b genes. Compared
with other fishes, miiuy croaker C7-1 gene was also surrounded by rnf180, rgs7bpb, c6, ghrb, cdc152, nim1k and gadd45g genes indicated that synteny around the C7-1 gene was conserved among these fishes, but miiuy croaker missed one gadd45g gene. Besides, no oxct1b gene was found in other fishes except zebrafish and cave fish, and two c6 genes were found in zebrafish which was different from other fishes. For the synteny of C7-2 gene, a strong conserved synteny was also remained among these fishes. The C7-2 gene was located on the miiuy croaker scaffold 27 and it was flanked by plcxd3 and rgs7bpa genes different from cod neighbored oxct1b and rgs7bpa, and the plcxd3 was missing in cod (Fig. 2B). Meanwhile, some genes existed in miiuy croaker were not found in some species fishes. For example, the rnf180 gene was not found in medaka and tilapia, and the htr1aa gene was missing in fugu. Besides, the slr gene did not exist in other fishes except for medaka. Although some surrounding genes appeared to miss in some fishes, the high degree conserved of synteny was retained among these fishes. We drew a conclusion that miiuy croaker had a relatively closer relationship with fugu on the views of gene synteny and phylogenetic relationship. 3.3. Gene duplication and evolutionary analysis of C7 gene Despite the fact that duplication of complement genes is a common observation in teleost fish, there is little report of duplicated genes in MAC. In our study, two C7 genes (C7-1 and C7-2) were found in miiuy croaker and they shared highly conservation with other fishes (Fig. 1B). We also find two C7 genes existing in other teleost fishes which experience the fish-specific genome duplication (FSGD or 3R). While one C7 gene exists in spotted gar, human, mouse, pig and cow which undergo the second-round whole-genome duplication (2R). But, no C7 gene is found in lamprey which undergoes the first-round whole-genome duplication (1R) (Fig. 3A). As we all know, the complement system is a very old defense mechanism that emerges at least 600e700 million years ago [3]. Although the complement activity (alternative pathway) and complement components (C3 and factor B) had been characterized in the most ancient vertebrates, the jawless fish, these fish lacked the classical pathway and the MAC [32]. Cartilaginous fish appear to evolve molecular functions in three complement
Fig. 1. (A) Comparisons of genomic structures of miiuy croaker C7-1 and C7-2 genes with other fishes. The lines stand for introns and exons are shown as boxes. (B) Sequences alignment of C7-1 and C7-2 genes in several fishes. (C) The prediction protein domains characteristic of C7 gene.
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Table 1 Scores of identities of C7-1 and C7-2 sequences between miiuy croaker and other fish analyzed by MegAlign. The percents of identities are showed in upper triangle and the percents of divergence are showed in lower triangle. Species
1
2
3
4
5
6
7
8
9
10
11
12
1.Fugu-2 2.Fugu-1 3.Medaka-2 4.Medaka-1 5.Zebrafish-2 6.Zebrafish-1 7.Platyfish-2 8.Platyfish-1 9.Tilapia-2 10.Tilapia-1 11.Miiuy croaker-2 12.Miiuy croaker-1
* 74.5 43.1 72.8 75.9 69.9 39.1 70.4 34.8 72.4 32.2 70.2
55.3 * 79.0 40.0 71.4 56.4 79.2 41.2 76.7 33.3 74.3 29.4
68.5 52.9 * 75.6 73.4 73.3 37.4 75.1 36.0 73.6 38.7 72.5
55.9 70.7 54.1 * 77.2 56.6 77.2 35.0 71.9 31.6 75.7 30.8
52.7 54.2 53.6 52.2 * 64.8 70.2 78.5 71.9 74.8 74.9 72.3
56.6 62.0 55.0 62.1 56.8 * 72.1 58.6 72.0 54.4 69.8 53.5
70.4 52.3 71.4 53.0 54.7 54.8 * 77.3 31.8 74.4 33.0 74.2
55.5 69.1 53.8 72.7 51.8 60.4 52.7 * 74.5 31.1 72.3 30.9
73.4 53.6 72.5 55.3 54.2 55.5 74.8 53.9 * 70.9 24.1 71.0
53.0 72.3 52.6 73.3 52.5 60.7 52.4 73.8 53.6 * 71.1 21.1
74.2 53.0 70.1 52.7 53.0 54.8 73.5 53.9 79.6 54.3 * 71.9
56.5 76.8 55.0 75.9 54.0 63.3 53.8 75.3 55.5 80.6 54.0 *
activation pathways including the MAC [33]. All other vertebrates (including teleost fishes, amphibians, reptiles, birds and mammals) have a well-developed complement system that involves all pathways of complement activation and the MAC. Because the lamprey belongs to cyclostomata so no C7 gene is found in lamprey. We can conclude that the duplication of C7 gene occurs after the divergence of mammals and teleosts and it is in consequence of FSGD. In order to detect whether C7-1 and C7-2 genes underwent different selective pressures in fishes, we estimated the ratio of nonsynonymous to synonymous substitution rates by likelihood method. The Bayesian tree reconstructed with C7 genes (Fig. 3B) was used to explore the evolutionary analysis. Firstly, the one-ratio model assumes one unique u for all branches and the value of the u is estimated to be 0.20 which is smaller than 1 (Table 2). The result shows that a strong purifying selection exists in C7 genes. Secondly, the free-ratio model which allows different u values for each lineage among the tree fits data significantly better than the oneratio model (Table 2). Lastly, the branch-site models are used to test positive selection in ancestral lineages which we are interested. We found one (336) and five (140, 202, 244, 245, 319) positive selection sites in ancestral lineages of mammals and fish C7 genes (Table 2). Meanwhile, three (23, 59, 225) and none positive selection sites were detected in ancestral lineage of fish C7-1 and C7-2
genes (Table 2). For ancestors of mammals, they left the water for land and the environment had changed greatly which the pathogen significantly differed from the aquatic environment. The C7 genes of mammals underwent significantly selection pressure so we detected positive selection sites in ancestors of mammals. Meanwhile, the positive selection happened on the early period of fish evolutionary history. Fishes lived in aquatic environment which was filled with countless types and numerous amounts of pathogens, so they further evolved to adapt to the aquatic environment. As the years went by, some species fishes experienced an additional genome duplication which was called fish-specific genome duplication (FSGD or 3R), so two duplications of C7 gene (C7-1 and C7-2) were found in teleost fishes and it may lead that teleost fishes further adapted to the aquatic environment. Molecular evolutionary analysis showed that the ancestors of C7-1 and C7-2 genes underwent different selection pressures. C7-1 genes experienced positive selection and the C7-2 genes underwent purifying selection in teleost fishes. Compared with C7-2 genes, C7-1 genes undertook more pressures in defense against the invading microorganisms for ancestors of teleost fishes. Furthermore, the multiple ML methods were used to detect the possible positive selection sites in extant fishes and mammals. For extant mammalian C7 genes, seven positive selection sites (219, 220, 238, 325, 346,
Fig. 2. Genomic synteny of C7-1 and C7-2 among several fishes. The same color represented the same genes and the arrows indicated the transcriptional direction. A is the Genomic synteny of C7-1 genes and B is C7-2 genes. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 3. (A) Schematic model proposed for the evolution of C7 members in 2R and 3R. The arrows represented genome duplication. (B) Phylogenetic tree of C7 genes was constructed using MrBayes with Bayesian method. The GTR þ I þ G model was selected for the Bayesian analysis and the consensus tree was built after burning 125,000 trees from 5,000,000 generations. Bayesian posterior probabilities values were indicated.
369, 396) were detected in M8 model and other ML methods also detected positive selection sites (Table 3) in this group. To identify candidates of sites under positive selection, we considered that the positive selection sites should be detected in at least three of the ML methods. So we found three positive selection sites (173, 222, 377) in mammalian C7 genes (Table 3), indicated that mammalian C7 genes faced more selective pressures. To mammals, when their ancestors left water for land, they had adapted to the terrestrial environment. However, the terrestrial environment had changed greatly and the pathogen differed from before with time elapsing. So mammals must further evolve to adapt to the new terrestrial environment. For extant fishes, none and one positive selection site (783) were found in C7-1 and C7-2 genes (Table 3), respectively. Compared with extant mammals, although two duplicated C7 genes existed in teleost fishes, they underwent different selection pressures in defense against the invading microorganisms: C7-1 genes experienced purifying selection and C7-2 genes experienced positive selection. In conclusion, no matter ancestral lineage or extant lineage of mammals, C7 genes underwent positive selection. While, the ancestors of teleost fishes C7-1 genes underwent positive selection and the extant teleost fishes C7-2 genes
underwent positive selection. In order to explore the distribution of positive selection sites of C7 gene in mammals and fish, the known protein structure of human and miiuy croaker C7 genes were used to analysis our data. For mammals, we detected 3 positive selection sites and one located in the MACPF domain (249e450 amino acids) which may create a pore in the target cell membrane leading to cell lysis and death. The variation of site might directly affect the capability of MAC to kill the target cell. For fishes, one positive selection site was detected in C7-2 gene and it located in the second FIMAC domain (772e840 amino acids) which might have an ability to specifically interact with C5b-6 complex [10]. The mutation site might increase the ability of C7-2 protein to interact with C5b-6 complex. Our findings could be used to further explore the functions of C7 gene and the selective mechanisms involved. 3.4. Expression analysis of miiuy croaker C7-1 and C7-2 genes The expression profile of miiuy croaker C7-1 and C7-2 in ten normal tissues (liver, spleen, kidney, intestine, eye, brain, muscle, heart, gill and fin) was determined by qRT-PCR. The expression analysis results showed that they were ubiquitously expressed in
Table 2 Likelihood ratio tests of branch-models and branch-site models on C7 gene. Model Branch-model A: One-ratio B: Omega ¼ 1 C: Free-ratio Branch-site model 1: Null-fishes 2: fishes 3: Null-C7-1 4: C7-1 5: Null-C7-2 6: C7-2 7: Null-mammals 8: mammals a b c
Npa
Ln likelihood
Parameter estimates
72 71 141
24929.17 26282.19 24811.87
u ¼ 0.20 u¼1
74 75 74 75 74 75 74 75
24499.68 24486.93 24502.30 24495.79 24503.96 24502.15 24506.49 24502.25
Numbers of parameters. Only the sites with BPP >0.95 were shown. Twice the difference of ln [likelihood] between the two models compared.
Model compare
Positive selection sitesb
2△lnLc (p-value)
None B vs A C vs A
n/a
2706.05(p ¼ 0.00) 234.61(p ¼ 0.00)
1 vs 2
140,202,244,245,319
25.50(p < 0.01)
3 vs 4
23,59,225
13.03(p < 0.01)
5 vs 6
None
3.62(p < 0.01)
7 vs 8
336
8.48(p < 0.01)
None 1 None 345,723,783,799
d
c
a
b
Fishes Fishes C7-1 C7-2
Codons identified by more than one ML method are underlined. Codons with P values <0.1. Codons with Bayes factor >50. Condons with posterior probability >0.9.
None 565 None 17,118,223,345,532,565,683,688,723,748,770,783,779 211 783
3 173,222,330,377 173,222,225,330,351,352,371,377,393,415 173,207,222,233,236,241,324,326,330,336,361,364,371,377,390,416,734,849 361,377 Mammals C7
219,220, 238,325, 346,369, 396 None None
FELb SLACb PAML M8
Sites under positive selection identified by different methodsa Species Gene
Table 3 Different methods test positive selection on C7 gene in mammals and fishes.
RELc
FUBARd
Total number Sites
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all ten tested tissues (Fig. 4A). Higher expression levels of C7-1 were detected in liver, spleen and brain, while lower expression levels were detected in gill, muscle, eye, heart and kidney. For miiuy croaker C7-2 gene, higher expressions were detected in liver, spleen and kidney, while lower expressions were detected in eye, gill, brain, muscle, heart and intestine. In addition, we explored the expression of C7-1 and C7-2 in spleen which was infected with V. anguillarum, poly I:C, LPS and S. aureus at 24 h post-injection, respectively. For C7-1, expression level was lower than control group after infection with poly I:C and S. aureus, but the expression level was higher than control group after infection with V. anguillarum and LPS. While expression level of C7-2 gene was lower than control group after infection with V. anguillarum and LPS, and the expression level was higher than control group after infection with poly I:C and S. aureus (Fig. 4B). Besides, the deep sequencing results of miR-27a-3p and miR-1306 were contrary to the expression of their target genes (C7-1 and C7-2) indicating that miRNA could restrain the expression of their target genes (Fig. 4C). We will study the miR-27a-3p and miR-1306 and their targets modulating mechanism according this information. Except for above that, we found that expression of C7-1 in spleen with V. anguillarum was significantly higher than other injection groups and it was significantly different from C7-2. In order to explore the response of C7-1 and C7-2 to V. anguillarum, three main immune organs (liver, spleen and kidney) were injected with V. anguillarum. In liver, the expression of C7-1 increased sharply at 6 h, and decreased sharply at 12 h, then gradually increased from 36 h to 72 h. The expression of C7-2 fluctuated up and down from 6 h to 24 h and increased from 36 h to 48 h, then reached its peak at 48 h (Fig. 5A). In spleen, the expression of C7-1 was volatile from 6 h to 36 h and increased from 48 h to 72 h, and the highest expression level occurred at 24 h. Meanwhile, the expression of C72 fluctuated up and down from 6 h to 36 h and increased from 48 h to 72 h (Fig. 5B). In kidney, the expression of C7-1 and C7-2 had a sharply up-regulated and fluctuated up and down from 12 h to 72 h, and the highest expression level occurred at 6 h (Fig. 5C). In our study, ten healthy tissues and infected tissues were used to explore the similarities and differences of expression patterns of C7-1 and C7-2 genes. In ten normal tissues, miiuy croaker C7-1 and C7-2 expressed ubiquitously in all ten tested tissues, although the expression patterns were obvious differences. For example, the highest expression levels of C7-1 and C7-2 were detected in liver, but the expression levels of C7-1 and C7-2 in brain and kidney were different, respectively. In previous study, Zarkadis et al. [34] observed that trout C7 expressed in the brain, heart, intestine and liver, but did not observe any C7 expression in kidney and spleen. While the trout C7-2 expressed in brain, heart, intestine, kidney, liver and spleen [15]. All the results showed that the expression of C7-1 and C7-2 were not exactly the same, implied that C7 gene implemented functions in various tissues. In injection group, the immune response of C7-1 and C7-2 were different in spleen with V. anguillarum, poly I:C, LPS and S. aureus at 24 h post-injection, respectively. Furthermore, the immune responses of C7-1 and C72 were different in three main immune organs after infected with V. anguillarum. These results indicated that immune response and preference of C7-1 and C7-2 were different in same tissues. This was the first time to analyze the expression differences of C7-1 and C7-2, and all the result showed that they had different expression patterns in healthy tissues and infected tissues. In conclusion, it was the first time to derive duplication of C7 gene (C7-1 and C7-2) from miiuy croaker genome which was the consequence of the fish-specific genome duplication, and they shared same structure domains with each other. The evolutionary analysis results showed that fish C7-1 and C7-2 experienced different evolutionary patterns, indicated that they may undertake
678 Fig. 5. Expression analysis in liver (A), spleen (B) and kidney (C) during 6, 12, 24, 36, 48 and 72 h after infection with V. anguillarum. Deviation bars represented the standard errors. Data indicated with asterisk symbol (*) are significantly different (p < 0.05) between challenged group and control group.
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Fig. 4. (A) Expression of C7-1 and C7-2 genes in ten healthy tissues of miiuy croaker. (B) Expression of C7-1 and C7-2 in spleen during 24 h after infection with V. anguillarum, poly I:C, LPS and S. aureus, respectively. (C) The deep sequencing results of miR-27a-3p and miR-1306. In ten healthy tissues, the total RNA from tissues of liver (L), spleen (S), kidney (K), intestine (I), eye (E), brain (B), muscle (M), heart (H), gill (G) and fin (F). Deviation bars represented the standard errors. Values with the same superscript are not significantly different (p > 0.05). Data indicated with asterisk symbol (*) are significantly different (p < 0.05) between challenged group and control group.
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different pressures in defense against the invading microorganisms. Besides, the expression results indicated that the expression patterns of C7-1 and C7-2 were different in healthy tissues and infected tissues, and they may play an important role in defense against pathogen infection. Most important of all, this was also the first time to analyze the gene synteny of fish C7 genes, which provided more significant information to clarify the evolutionary relationship of fish C7 genes. Acknowledgments This study was supported by National Natural Science Foundation of China (31370049) and Zhejiang Province Natural Science Foundation of Distinguished Young Scientists (LR14C040001). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.fsi.2015.05.042. References [1] T. Fujita, M. Matsushita, Y. Endo, The lectin-complement pathway-its role in innate immunity and evolution, Immunol. Rev. 198 (2004) 185e202. [2] J.A. Hoffmann, F.C. Kafatos, C.A. Janeway, R.A. Ezekowitz, Phylogenetic perspectives in innate immunity, Science 284 (1999) 1313e1318. [3] J.O. Sunyer, I.K. Zarkadis, J.D. Lambris, Complement diversity: a mechanism for generating immune diversity? Immunol. Today 19 (1998) 519e523. [4] M.J. Walport, Complement. First of two parts, N. Engl. Med. 344 (2001a) 1058e1066. [5] M.J. Walport, Complement. Second of two parts, N. Engl. Med. 344 (2001b) 1140e1144. [6] W.C. Song, M.R. Sarrias, J.D. Lambris, Complement and innate immunity, Immunopharmacol 49 (2000) 187e198. [7] M.P. Chondrou, D. Mastellos, I.K. Zarkadis, cDNA cloning and phylogenetic analysis of the sixth complement component in rainbow trout, Mol. Immunol. 43 (2006) 1080e1087. [8] H.J. Muller-Eberhard, The membrane attack complex of complement, Annu Rev. Immunol. 4 (1986) 503e528. [9] R. Wurzner, Modulation of complement membrane attack by local C7 synthesis, Clin. Exp. Immunol. 121 (2000) 8e10. [10] R.G. DiScipio, Formation and structure of the C5b-7 complex of the lytic pathway of complement, Biol. Chem. 267 (1992) 17087e17094. [11] A. Mikrou, I.K. Zarkadis, Cloning of the sixth complement component and, spatial and temporal expression profile of MAC structural and regulatory genes in chicken, Dev. Comp. Immunol. 34 (2010) 485e490. [12] T. Katagiri, I. Hirono, T. Aoki, Molecular analysis of complement component C8 beta and C9 cDNAs of Japanese flounder, Paralichthys olivaceus, Immunogenetics 50 (1999) 43e48. [13] M.J. Hobart, B.A. Fernie, R.G. DiScipio, Structure of the human C7 gene and comparison with the C6, C8a, C8b and C9 genes, Immunol 154 (1995) 5188e5194.
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[14] M. Nonaka, A. Kimura, Genomic view of the evolution of the complement system, Immunogenetics 58 (2006) 701e713. [15] A.D. Papanastasious, I.K. Zarkadis, Gene duplication of the seventh component of complement in rainbow trout, Immunogenetics 57 (2005) 703e708. [16] Y.Z. Cheng, T.J. Xu, G. Shi, R.X. Wang, Complete mitochondrial genome of the miiuy croaker Miichthys miiuy (Perciformes, Sciaenidae) with phylogenetic consideration, Mar. Genomics 3 (2010) 201e209. [17] T.J. Xu, Y.Y. Sun, G. Shi, Y.Z. Cheng, R.X. Wang, Characterization of the major histocompatibility complex class II genes in miiuy croaker, PLoS One 6 (2011) e23832. [18] T.J. Xu, Z.H. Zhu, Y.N. Sun, L.P. Ren, R.X. Wang, Characterization and expression of the CXCR1 and CXCR4 in miiuy croaker and evolutionary analysis shows the strong positive selection pressures imposed in mammal CXCR1, Dev. Comp. Immunol. 44 (2014) 133e144. [19] T.J. Xu, F.X. Meng, Y.N. Sun, G. Shi, R.X. Wang, Identification of immune genes of the miiuy croaker (Miichthys miiuy) by sequencing and bioinformatic analysis of ESTs, Fish Shellfish Immunol. 29 (2010) 1099e1105. [20] F.X. Meng, Y.N. Sun, X.Z. Liu, J.X. Wang, T.J. Xu, R.X. Wang, Analysis of C3 suggests three periods of positive selection events and different evolutionary patterns between fish and mammals, PloS One 7 (2012) e37489. [21] F.X. Meng, R.X. Wang, T.J. Xu, Y.N. Sun, Y.Z. Cheng, G. Shi, An unexpected loss of domains in the conservative evolution ninth complement component in a higher teleost, Miichthys miiuy, Fish Shellfish Immunol. 32 (2012) 1171e1178. [22] S.C. Wang, R.X. Wang, T.J. Xu, Genomic characterization and expression pattern of Bf/C2 and C4 in miiuy croaker and molecular evolution analysis on mammals and fishes, Fish Shellfish Immunol. 39 (2014) 423e431. [23] R.B. Che, Y.Y. Sun, D.Q. Sun, T.J. Xu, Characterization of the miiuy croaker (miichthys miiuy) transcriptome and development of immune-relevant genes and molecular markers, PLoS One 9 (2014) e94046. [24] C. Burge, S. Karlin, Prediction of complete gene structures in human genomic DNA, Mol. Biol. 268 (1997) 78e94. [25] I. Letunic, R.R. Copley, B. Pils, S. Pinkert, J. Schultz, P. Bork, SMART5: domains in the context of genomes and networks, Nucleic Acids Res. 34 (2006) 257e260. [26] R.C. Edgar, MUSCLE: multiple sequence alignment with high accuracy and high throughput, Nucleic Acids Res. 32 (2004) 1792e1797. [27] F. Ronquist, J.P. Huelsenbeck, MrBayes 3: Bayesian phylogenetic inference under mixed models, Bioinformatics 19 (2003) 1572e1574. [28] D. Posada, Selection of models of DNA evolution with jModelTest, Methods Mol. Biol. 537 (2009) 93e112. [29] Z. Yang, PAML 4: phylogenetic analysis by maximum likelihood, Mol. Biol. Evol. 24 (2007) 1586e1591. [30] S.L. Pond, S.D. Frost, Datamonkey: rapid detection of selective pressure on individual sites of codon alignments, Bioinformatics 21 (2005) 2531e2533. [31] Z.H. Zhu, R.X. Wang, L.P. Ren, T.J. Xu, Characterization of the CCR3 and CCR9 genes in miiuy croaker and different selection pressures imposed on different domains between mammals and teleosts, Dev. Comp. Immunol. 41 (2013) 631e643. [32] M. Nonaka, M. Takahashi, M. Sasaki, Molecular cloning of a lamprey homologue of the mammalian MHC class and Ⅲ gene, complement factor B, Immunol 152 (1994) 2263e2269. [33] J.A. Jensen, E. Festa, D.S. Smith, M. Cayer, The complement system of the nurse shark: hemolytic and comparative characteristics, Science 214 (1981) 566e569. [34] I.K. Zarkadis, S. Duraj, M. Chondrou, Molecular cloning of the seventh component of complement in rainbow trout, Dev. Comp. Immunol. 29 (2005) 95e102.
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Short communication
Characterization and evolutionary analysis of duplicated C7 in miiuy croaker Shanchen Wang a, 1, Yunhang Gao b, 1, Chang Shu a, Tianjun Xu a, * a b
Laboratory of Fish Biogenetics & Immune Evolution, College of Marine Science, Zhejiang Ocean University, Zhoushan, 316022, China College of Animal Science and Veterinary Medicine, Jilin Agriculture University, Changchun, 130118, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 April 2015 Received in revised form 24 May 2015 Accepted 28 May 2015 Available online 30 May 2015
The complement system, as one of the most sophisticated innate immune system, plays an important role in defense against invading microorganisms. The complement component C7 participates in the cytolytic phase of complement activation through a series of polymerization reactions with other terminal complement components. In this study, we derived two C7 genes from the whole genome of miiuy croaker which were the consequence of the fish-specific genome duplication. Our data showed that miiuy croaker C7-1 and C7-2 genes shared same structure domains. The analysis of gene synteny showed that high degree conserved of synteny was retained between miiuy croaker and other teleosts, and miiuy croaker had a relatively closer relationship with fugu. The expression of C7-1 and C7-2 in miiuy croaker healthy tissues revealed that they were ubiquitously expressed in all ten tested tissues. Besides, the immune response of C7-1 and C7-2 were different in spleen with Vibrio anguillarum, Staphylococcus aureus, poly I:C and LPS at 24 h post-injection, respectively. Furthermore, the expression patterns of C7-1 and C7-2 were different in liver, spleen and kidney after infected with V. anguillarum at different timepoint. Evolutionary analysis showed that all the ancestral lineages underwent positive selection except for the ancestral lineages of fish C7-2, indicated that the ancestral lineages of fish C7-1 genes undertook more pressures than C7-2 in defense against the invading microorganisms. Meanwhile, a series of maximum likelihood methods were used to explore the evolutionary patterns on extant vertebrates' C7 genes. Three and one positive selection sites were found in extant mammalian C7 genes and fish C7-2 genes, but no positive selection site was found in extant fishes C7-1 genes. The result showed that extant fish C7-2 genes undertook more pressures compared with C7-1. In conclusion, fish C7-1 and C7-2 gene underwent different evolutionary patterns. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Complement C7 Gene duplication Expression patterns Molecular evolution
1. Introduction Immune responses are mediated by two general systems: adaptive immunity and innate immunity. Adaptive immunity can date back to the early period of vertebrates evolution, between the divergence of cyclostomes and cartilaginous fish [1]. Innate immunity system is a very old defense mechanism, and it provides the first line of defense before the adaptive immune system comes into play [2]. Fish, the first of true vertebrates, have evolved adaptive immune responses similar to those in higher vertebrates, but the adaptive immunity in fish is inefficient, therefore they should make
* Corresponding author. E-mail address: [email protected] (T. Xu). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.fsi.2015.05.042 1050-4648/© 2015 Elsevier Ltd. All rights reserved.
full use of the well-developed innate immune system to compensate for their rudimentary antibody response [3]. The complement system, one of the most sophisticated innate immune system [4,5], plays a major role during inflammation and infection, as well as a link between innate and adaptive immunity [6]. The complement activated by the classical, lectin or alternative pathway leads to the formation of the membrane attack complex (MAC) on the surface of complement-opsonized cells [7]. The assembly of MAC involves the aggregation of the lytic complement components C5b, C6, C7, C8 and C9 [8]. C5b connects with C6 via a metastable binding site to form a soluble C5b-C6 complex in the vicinity of the activating cell. Subsequent C7 interacts with the C5b-C6 to produce C5b-7, a trimolecular complex that allows insertion of this complex into the target cell membrane [9,10]. Among MAC components, the terminal complement components (TCC) C6, C7, C8a, C8b and C9 belong to the same gene
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family as perforins and they may have emerged through a series of duplications of an ancestral gene [11]. Consequently, they share several common structural motifs such as thrombospondin (TSP), low-density lipoprotein receptors class A (LDLa), epidermal growth factor precursor (EGF) and the MAC/perforin segment (MACPF). These domains are conserved and also present in teleost counterparts [12]. In addition, mammalian C7 possesses complement control protein (CCP) motifs and factor I MAC (FIMAC) modules in the C-terminal domain except for these conserved motifs [13]. As one member of TCC, C7 fulfills a crucial role in the hydrophiliceamphiphilic transition of MAC, so it plays an essential role in the elimination of invading pathogens. In previous studies, the full set of TCC genes had been identified in teleost, amphibian reptilian/aves and mammals except for the avian C9 gene which was not found in the draft genome sequence of chicken [14]. Besides, two C7 genes were identified in rainbow trout [15], although gene duplication was a common evolutionary event, it was the first time to find gene duplication in the C6eC9 gene family. Miiuy croaker (Miichthys miiuy), as one member of family Sciaenidae, mainly distributes from the Western Japan Sea to the East China Sea. Currently, the molecular immunology, genetics and molecular evolutionary of immune genes were studied more indepth in this species [16e19]. Besides, we have been studying complement system. In our previous studies, the complement component Bf/C2, C3, C4 and C9 genes had been identified and analyzed in miiuy croaker [20e22]. Now we are interested in complement component C7 gene which fulfills a crucial role in the hydrophiliceamphiphilic transition of MAC. In this study, two complement component C7 genes (C7-1 and C7-2) were identified from the whole genome database of miiuy croaker which were in consequence of the fish-specific genome duplication (FSGD or 3R). And we were the first time to analyze the gene synteny of C7-1 and C7-2 in teleost which could provide information for the investigation of the evolutionary relationships and evolutionary mechanism of C7 genes. Furthermore, the expression patterns of C7-1 and C7-2 genes were also analyzed to illuminate the possible role of C7-1 and C7-2 in response to different pathogens infection. Considering the unique status of complement component in evolutionary process of innate and adaptive immunity, we also analyzed the molecular evolution of C7 gene in fishes and mammals. 2. Material and methods 2.1. Sequence analysis and phylogenetic tree construction In order to identify the C7-1 and C7-2 genes, we used available fish genes as queries to search for the transcriptome [23] and whole genome database of miiuy croaker (unpublished data) by local Blast software. The retrieved reconstructed transcripts were translated using ORF Finder (http://www.ncbi.nlm.nih.gov) and GENSCAN [24]. The predicted open reading frames (ORFs) were verified by BLASTP against NCBI non-redundant protein sequence database. The potential protein domains of amino acid sequences were calculated by SMART program [25]. All sequences from other organisms used in this study were derived from GenBank and Ensemble database (Supplementary table S1). All the gene sequences were aligned under codon model by MUSCLE software for its high accuracy and speed [26]. Phylogenetic tree was constructed with the Bayesian approach by MrBayes v3.2 [27] which was running 5,000,000 generation with 25% of trees burned. For Bayesian inference, the GTR þ I þ G model was regarded as the best-fit model by Bayesian information criterion (BIC) using jModeltest software [28].
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2.2. Molecular evolutionary analysis The nonsynonymous and synonymous rate ratio u (dN/dS) stands for the change of selective pressure. The u ¼ 1, <1, and >1 are indicative of neutral evolution, purifying selection and positive selection, respectively. To investigate the evolutionary process of C7-1 and C7-2 genes, the selective pressure was analyzed by CODEML of PAML software [29] and the Hyphy package of Data Monkey Web Server [30]. All the models of PAML and Hyphy package used in this study were carried out as described by Zhu et al. [31]. 2.3. Fish sampling, challenge experiments and RNA extraction Miiuy croakers were obtained from Zhoushan Fisheries Research Institute (Zhejiang, China). All fishes were held in aerated water tanks to allow for acclimatization and evaluation of overall fish health before using in experiments. Only fishes with similar size and body weight were used. One week later, the challenge experiments of miiuy croaker were intraperitoneally infected with Vibrio anguillarum, Staphylococcus aureus, poly I:C and LPS. Fish samples were randomly divided into two groups, injection and control groups. In injection group, fishes were injected with 1 ml of V. anguillarum (1.5 108 CFU/ml), poly I:C (2.5 mg/ml), LPS (1.0 mg/ ml) and S. aureus (1.5 108 CFU/ml), respectively. Meanwhile, the control fishes were injected with 1 ml of physiological water. For injection group with V. anguillarum, fish samples were killed at 6 h, 12 h, 24 h, 36 h, 48 h and 72 h post-injection, respectively. While injection group with poly I:C, LPS and S. aureus, fish samples were killed at 24 h post-injection, respectively. Three tissues (liver, kidney and spleen) of infection were removed and stored at 80 C, at the same time ten tissues (liver, kidney, spleen, heart, eye, fin, brain, gill, muscle and intestines) of uninfected were also removed and keep at 80 C. Total RNA were extracted from the various tissues of adult individuals by Trizol reagent (Qiagen) following the manufacturer's instructions. cDNA was synthesized using QuantScript RT Kit (TIANGEN) according to the manufacturer's protocol. 2.4. miRNA and mRNA transcriptomes and the prediction of target gene Total RNA of spleen tissue which infected with V. anguillarum, poly I:C, LPS and S. aureus after 24 h post-injection and the control group were used to prepare the miRNA and mRNA transcriptomes in our Lab (unpublished). These libraries were used for the deep sequencing by an Illumina platform according to the manufacturer's protocol. In order to predict the miRNA target gene of miiuy croaker C7-1 and C7-2, the miRNA target gene database miRGen 3.0 was used to select the corresponding miRNA that exhibited complementarily with mRNA sequencing. We found that miR-27a3p and miR-1306 were perfect complementary with miiuy croaker C7-1 and C7-2, then we analyzed the expression patterns of these two miRNA using the deep sequencing data. 2.5. Expression analysis of C7-1 and C7-2 genes Three pairs of primers were used to study the expression patterns of C7-1 and C7-2 genes (Supplementary table S2). Healthy tissues and infected tissues were determined using qRT-PCR. The qRT-PCR was run on a 7300 real-time PCR system (Applied Biosystems, USA) using a RealMaster Mix kit (TIANGEN). The reaction carried out without the template was used as blank control. Cycling conditions were as follows: 15 min at 95 C, followed by 45 cycles consisting of 15 s at 95 C and 60 s at 60 C, dissociation curve analysis was performed after each assay to determine target
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specificity. Expression of b-actin was used as internal control for miiuy croaker C7-1 and C7-2 genes expression analysis. We used the Duncan test to detect the significant differences between groups and p < 0.05 represented significant differences. 3. Results and discussion 3.1. Identification of miiuy croaker C7-1 and C7-2 genes The complete coding sequence of miiuy croaker C7-1 and C7-2 were obtained (GenBank accession No. KP689602 and KP689603). The C7-1 cDNA sequence encodes a protein of 830 aa with ORF 2493 bp and the C7-2 consists 2466 bp with its ORF encoding 821 aa. The genomic DNA of miiuy croaker C7-1 contains 17 exons and 16 introns, while miiuy croaker C7-2 includes 15 exons and 14 introns (Fig. 1A). The sizes of C7-1 and C7-2 exons appear to be similar among fish genes, respectively. Comparative genomic structure reflects the evolutionary conservation exists among fishes. The comparison of deduced amino acid sequences shows that miiuy croaker C7-1 shares a 62.0%e80.6% identity with other fishes and the identity of C7-2 among fish ranges from 53.0% to 79.6% (Table 1). Sequences alignment of C7-1 and C7-2 genes from several fishes shows that C7-1 genes share highly conservation with C7-2 genes (Fig. 1B). The miiuy croaker C7-1 and C7-2 genes share the same structure domains which contains two TSP1 domains, one LDLa domain, one MACPF domain, one EGF domain, two CCP domains and two FIMAC domains (Fig. 1C). All the structure domains that are characteristic of TCCs (TSP1, LDLa, MACPF and EGF) are present in miiuy croaker C7-1 and C7-2. 3.2. Gene synteny analyses of C7-1 and C7-2 among fishes In order to explore the evolutionary relationship of C7-1 and C72 genes among fishes, synteny analysis was carried out using the Genomicus database v73.01. Gene synteny around the miiuy croaker C7-1 and C7-2 genes was compared with other fishes. The miiuy croaker C7-1 gene was found on scaffold 625 and it was flanked by c6 and ghrb genes different from zebrafish and cave fish (Fig. 2A). Zebrafish C7-1 gene located between two c6 genes, while cave fish C7-1 gene was flanked by c6 and oxct1b genes. Compared
with other fishes, miiuy croaker C7-1 gene was also surrounded by rnf180, rgs7bpb, c6, ghrb, cdc152, nim1k and gadd45g genes indicated that synteny around the C7-1 gene was conserved among these fishes, but miiuy croaker missed one gadd45g gene. Besides, no oxct1b gene was found in other fishes except zebrafish and cave fish, and two c6 genes were found in zebrafish which was different from other fishes. For the synteny of C7-2 gene, a strong conserved synteny was also remained among these fishes. The C7-2 gene was located on the miiuy croaker scaffold 27 and it was flanked by plcxd3 and rgs7bpa genes different from cod neighbored oxct1b and rgs7bpa, and the plcxd3 was missing in cod (Fig. 2B). Meanwhile, some genes existed in miiuy croaker were not found in some species fishes. For example, the rnf180 gene was not found in medaka and tilapia, and the htr1aa gene was missing in fugu. Besides, the slr gene did not exist in other fishes except for medaka. Although some surrounding genes appeared to miss in some fishes, the high degree conserved of synteny was retained among these fishes. We drew a conclusion that miiuy croaker had a relatively closer relationship with fugu on the views of gene synteny and phylogenetic relationship. 3.3. Gene duplication and evolutionary analysis of C7 gene Despite the fact that duplication of complement genes is a common observation in teleost fish, there is little report of duplicated genes in MAC. In our study, two C7 genes (C7-1 and C7-2) were found in miiuy croaker and they shared highly conservation with other fishes (Fig. 1B). We also find two C7 genes existing in other teleost fishes which experience the fish-specific genome duplication (FSGD or 3R). While one C7 gene exists in spotted gar, human, mouse, pig and cow which undergo the second-round whole-genome duplication (2R). But, no C7 gene is found in lamprey which undergoes the first-round whole-genome duplication (1R) (Fig. 3A). As we all know, the complement system is a very old defense mechanism that emerges at least 600e700 million years ago [3]. Although the complement activity (alternative pathway) and complement components (C3 and factor B) had been characterized in the most ancient vertebrates, the jawless fish, these fish lacked the classical pathway and the MAC [32]. Cartilaginous fish appear to evolve molecular functions in three complement
Fig. 1. (A) Comparisons of genomic structures of miiuy croaker C7-1 and C7-2 genes with other fishes. The lines stand for introns and exons are shown as boxes. (B) Sequences alignment of C7-1 and C7-2 genes in several fishes. (C) The prediction protein domains characteristic of C7 gene.
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Table 1 Scores of identities of C7-1 and C7-2 sequences between miiuy croaker and other fish analyzed by MegAlign. The percents of identities are showed in upper triangle and the percents of divergence are showed in lower triangle. Species
1
2
3
4
5
6
7
8
9
10
11
12
1.Fugu-2 2.Fugu-1 3.Medaka-2 4.Medaka-1 5.Zebrafish-2 6.Zebrafish-1 7.Platyfish-2 8.Platyfish-1 9.Tilapia-2 10.Tilapia-1 11.Miiuy croaker-2 12.Miiuy croaker-1
* 74.5 43.1 72.8 75.9 69.9 39.1 70.4 34.8 72.4 32.2 70.2
55.3 * 79.0 40.0 71.4 56.4 79.2 41.2 76.7 33.3 74.3 29.4
68.5 52.9 * 75.6 73.4 73.3 37.4 75.1 36.0 73.6 38.7 72.5
55.9 70.7 54.1 * 77.2 56.6 77.2 35.0 71.9 31.6 75.7 30.8
52.7 54.2 53.6 52.2 * 64.8 70.2 78.5 71.9 74.8 74.9 72.3
56.6 62.0 55.0 62.1 56.8 * 72.1 58.6 72.0 54.4 69.8 53.5
70.4 52.3 71.4 53.0 54.7 54.8 * 77.3 31.8 74.4 33.0 74.2
55.5 69.1 53.8 72.7 51.8 60.4 52.7 * 74.5 31.1 72.3 30.9
73.4 53.6 72.5 55.3 54.2 55.5 74.8 53.9 * 70.9 24.1 71.0
53.0 72.3 52.6 73.3 52.5 60.7 52.4 73.8 53.6 * 71.1 21.1
74.2 53.0 70.1 52.7 53.0 54.8 73.5 53.9 79.6 54.3 * 71.9
56.5 76.8 55.0 75.9 54.0 63.3 53.8 75.3 55.5 80.6 54.0 *
activation pathways including the MAC [33]. All other vertebrates (including teleost fishes, amphibians, reptiles, birds and mammals) have a well-developed complement system that involves all pathways of complement activation and the MAC. Because the lamprey belongs to cyclostomata so no C7 gene is found in lamprey. We can conclude that the duplication of C7 gene occurs after the divergence of mammals and teleosts and it is in consequence of FSGD. In order to detect whether C7-1 and C7-2 genes underwent different selective pressures in fishes, we estimated the ratio of nonsynonymous to synonymous substitution rates by likelihood method. The Bayesian tree reconstructed with C7 genes (Fig. 3B) was used to explore the evolutionary analysis. Firstly, the one-ratio model assumes one unique u for all branches and the value of the u is estimated to be 0.20 which is smaller than 1 (Table 2). The result shows that a strong purifying selection exists in C7 genes. Secondly, the free-ratio model which allows different u values for each lineage among the tree fits data significantly better than the oneratio model (Table 2). Lastly, the branch-site models are used to test positive selection in ancestral lineages which we are interested. We found one (336) and five (140, 202, 244, 245, 319) positive selection sites in ancestral lineages of mammals and fish C7 genes (Table 2). Meanwhile, three (23, 59, 225) and none positive selection sites were detected in ancestral lineage of fish C7-1 and C7-2
genes (Table 2). For ancestors of mammals, they left the water for land and the environment had changed greatly which the pathogen significantly differed from the aquatic environment. The C7 genes of mammals underwent significantly selection pressure so we detected positive selection sites in ancestors of mammals. Meanwhile, the positive selection happened on the early period of fish evolutionary history. Fishes lived in aquatic environment which was filled with countless types and numerous amounts of pathogens, so they further evolved to adapt to the aquatic environment. As the years went by, some species fishes experienced an additional genome duplication which was called fish-specific genome duplication (FSGD or 3R), so two duplications of C7 gene (C7-1 and C7-2) were found in teleost fishes and it may lead that teleost fishes further adapted to the aquatic environment. Molecular evolutionary analysis showed that the ancestors of C7-1 and C7-2 genes underwent different selection pressures. C7-1 genes experienced positive selection and the C7-2 genes underwent purifying selection in teleost fishes. Compared with C7-2 genes, C7-1 genes undertook more pressures in defense against the invading microorganisms for ancestors of teleost fishes. Furthermore, the multiple ML methods were used to detect the possible positive selection sites in extant fishes and mammals. For extant mammalian C7 genes, seven positive selection sites (219, 220, 238, 325, 346,
Fig. 2. Genomic synteny of C7-1 and C7-2 among several fishes. The same color represented the same genes and the arrows indicated the transcriptional direction. A is the Genomic synteny of C7-1 genes and B is C7-2 genes. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 3. (A) Schematic model proposed for the evolution of C7 members in 2R and 3R. The arrows represented genome duplication. (B) Phylogenetic tree of C7 genes was constructed using MrBayes with Bayesian method. The GTR þ I þ G model was selected for the Bayesian analysis and the consensus tree was built after burning 125,000 trees from 5,000,000 generations. Bayesian posterior probabilities values were indicated.
369, 396) were detected in M8 model and other ML methods also detected positive selection sites (Table 3) in this group. To identify candidates of sites under positive selection, we considered that the positive selection sites should be detected in at least three of the ML methods. So we found three positive selection sites (173, 222, 377) in mammalian C7 genes (Table 3), indicated that mammalian C7 genes faced more selective pressures. To mammals, when their ancestors left water for land, they had adapted to the terrestrial environment. However, the terrestrial environment had changed greatly and the pathogen differed from before with time elapsing. So mammals must further evolve to adapt to the new terrestrial environment. For extant fishes, none and one positive selection site (783) were found in C7-1 and C7-2 genes (Table 3), respectively. Compared with extant mammals, although two duplicated C7 genes existed in teleost fishes, they underwent different selection pressures in defense against the invading microorganisms: C7-1 genes experienced purifying selection and C7-2 genes experienced positive selection. In conclusion, no matter ancestral lineage or extant lineage of mammals, C7 genes underwent positive selection. While, the ancestors of teleost fishes C7-1 genes underwent positive selection and the extant teleost fishes C7-2 genes
underwent positive selection. In order to explore the distribution of positive selection sites of C7 gene in mammals and fish, the known protein structure of human and miiuy croaker C7 genes were used to analysis our data. For mammals, we detected 3 positive selection sites and one located in the MACPF domain (249e450 amino acids) which may create a pore in the target cell membrane leading to cell lysis and death. The variation of site might directly affect the capability of MAC to kill the target cell. For fishes, one positive selection site was detected in C7-2 gene and it located in the second FIMAC domain (772e840 amino acids) which might have an ability to specifically interact with C5b-6 complex [10]. The mutation site might increase the ability of C7-2 protein to interact with C5b-6 complex. Our findings could be used to further explore the functions of C7 gene and the selective mechanisms involved. 3.4. Expression analysis of miiuy croaker C7-1 and C7-2 genes The expression profile of miiuy croaker C7-1 and C7-2 in ten normal tissues (liver, spleen, kidney, intestine, eye, brain, muscle, heart, gill and fin) was determined by qRT-PCR. The expression analysis results showed that they were ubiquitously expressed in
Table 2 Likelihood ratio tests of branch-models and branch-site models on C7 gene. Model Branch-model A: One-ratio B: Omega ¼ 1 C: Free-ratio Branch-site model 1: Null-fishes 2: fishes 3: Null-C7-1 4: C7-1 5: Null-C7-2 6: C7-2 7: Null-mammals 8: mammals a b c
Npa
Ln likelihood
Parameter estimates
72 71 141
24929.17 26282.19 24811.87
u ¼ 0.20 u¼1
74 75 74 75 74 75 74 75
24499.68 24486.93 24502.30 24495.79 24503.96 24502.15 24506.49 24502.25
Numbers of parameters. Only the sites with BPP >0.95 were shown. Twice the difference of ln [likelihood] between the two models compared.
Model compare
Positive selection sitesb
2△lnLc (p-value)
None B vs A C vs A
n/a
2706.05(p ¼ 0.00) 234.61(p ¼ 0.00)
1 vs 2
140,202,244,245,319
25.50(p < 0.01)
3 vs 4
23,59,225
13.03(p < 0.01)
5 vs 6
None
3.62(p < 0.01)
7 vs 8
336
8.48(p < 0.01)
None 1 None 345,723,783,799
d
c
a
b
Fishes Fishes C7-1 C7-2
Codons identified by more than one ML method are underlined. Codons with P values <0.1. Codons with Bayes factor >50. Condons with posterior probability >0.9.
None 565 None 17,118,223,345,532,565,683,688,723,748,770,783,779 211 783
3 173,222,330,377 173,222,225,330,351,352,371,377,393,415 173,207,222,233,236,241,324,326,330,336,361,364,371,377,390,416,734,849 361,377 Mammals C7
219,220, 238,325, 346,369, 396 None None
FELb SLACb PAML M8
Sites under positive selection identified by different methodsa Species Gene
Table 3 Different methods test positive selection on C7 gene in mammals and fishes.
RELc
FUBARd
Total number Sites
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all ten tested tissues (Fig. 4A). Higher expression levels of C7-1 were detected in liver, spleen and brain, while lower expression levels were detected in gill, muscle, eye, heart and kidney. For miiuy croaker C7-2 gene, higher expressions were detected in liver, spleen and kidney, while lower expressions were detected in eye, gill, brain, muscle, heart and intestine. In addition, we explored the expression of C7-1 and C7-2 in spleen which was infected with V. anguillarum, poly I:C, LPS and S. aureus at 24 h post-injection, respectively. For C7-1, expression level was lower than control group after infection with poly I:C and S. aureus, but the expression level was higher than control group after infection with V. anguillarum and LPS. While expression level of C7-2 gene was lower than control group after infection with V. anguillarum and LPS, and the expression level was higher than control group after infection with poly I:C and S. aureus (Fig. 4B). Besides, the deep sequencing results of miR-27a-3p and miR-1306 were contrary to the expression of their target genes (C7-1 and C7-2) indicating that miRNA could restrain the expression of their target genes (Fig. 4C). We will study the miR-27a-3p and miR-1306 and their targets modulating mechanism according this information. Except for above that, we found that expression of C7-1 in spleen with V. anguillarum was significantly higher than other injection groups and it was significantly different from C7-2. In order to explore the response of C7-1 and C7-2 to V. anguillarum, three main immune organs (liver, spleen and kidney) were injected with V. anguillarum. In liver, the expression of C7-1 increased sharply at 6 h, and decreased sharply at 12 h, then gradually increased from 36 h to 72 h. The expression of C7-2 fluctuated up and down from 6 h to 24 h and increased from 36 h to 48 h, then reached its peak at 48 h (Fig. 5A). In spleen, the expression of C7-1 was volatile from 6 h to 36 h and increased from 48 h to 72 h, and the highest expression level occurred at 24 h. Meanwhile, the expression of C72 fluctuated up and down from 6 h to 36 h and increased from 48 h to 72 h (Fig. 5B). In kidney, the expression of C7-1 and C7-2 had a sharply up-regulated and fluctuated up and down from 12 h to 72 h, and the highest expression level occurred at 6 h (Fig. 5C). In our study, ten healthy tissues and infected tissues were used to explore the similarities and differences of expression patterns of C7-1 and C7-2 genes. In ten normal tissues, miiuy croaker C7-1 and C7-2 expressed ubiquitously in all ten tested tissues, although the expression patterns were obvious differences. For example, the highest expression levels of C7-1 and C7-2 were detected in liver, but the expression levels of C7-1 and C7-2 in brain and kidney were different, respectively. In previous study, Zarkadis et al. [34] observed that trout C7 expressed in the brain, heart, intestine and liver, but did not observe any C7 expression in kidney and spleen. While the trout C7-2 expressed in brain, heart, intestine, kidney, liver and spleen [15]. All the results showed that the expression of C7-1 and C7-2 were not exactly the same, implied that C7 gene implemented functions in various tissues. In injection group, the immune response of C7-1 and C7-2 were different in spleen with V. anguillarum, poly I:C, LPS and S. aureus at 24 h post-injection, respectively. Furthermore, the immune responses of C7-1 and C72 were different in three main immune organs after infected with V. anguillarum. These results indicated that immune response and preference of C7-1 and C7-2 were different in same tissues. This was the first time to analyze the expression differences of C7-1 and C7-2, and all the result showed that they had different expression patterns in healthy tissues and infected tissues. In conclusion, it was the first time to derive duplication of C7 gene (C7-1 and C7-2) from miiuy croaker genome which was the consequence of the fish-specific genome duplication, and they shared same structure domains with each other. The evolutionary analysis results showed that fish C7-1 and C7-2 experienced different evolutionary patterns, indicated that they may undertake
678 Fig. 5. Expression analysis in liver (A), spleen (B) and kidney (C) during 6, 12, 24, 36, 48 and 72 h after infection with V. anguillarum. Deviation bars represented the standard errors. Data indicated with asterisk symbol (*) are significantly different (p < 0.05) between challenged group and control group.
S. Wang et al. / Fish & Shellfish Immunology 45 (2015) 672e679
Fig. 4. (A) Expression of C7-1 and C7-2 genes in ten healthy tissues of miiuy croaker. (B) Expression of C7-1 and C7-2 in spleen during 24 h after infection with V. anguillarum, poly I:C, LPS and S. aureus, respectively. (C) The deep sequencing results of miR-27a-3p and miR-1306. In ten healthy tissues, the total RNA from tissues of liver (L), spleen (S), kidney (K), intestine (I), eye (E), brain (B), muscle (M), heart (H), gill (G) and fin (F). Deviation bars represented the standard errors. Values with the same superscript are not significantly different (p > 0.05). Data indicated with asterisk symbol (*) are significantly different (p < 0.05) between challenged group and control group.
S. Wang et al. / Fish & Shellfish Immunology 45 (2015) 672e679
different pressures in defense against the invading microorganisms. Besides, the expression results indicated that the expression patterns of C7-1 and C7-2 were different in healthy tissues and infected tissues, and they may play an important role in defense against pathogen infection. Most important of all, this was also the first time to analyze the gene synteny of fish C7 genes, which provided more significant information to clarify the evolutionary relationship of fish C7 genes. Acknowledgments This study was supported by National Natural Science Foundation of China (31370049) and Zhejiang Province Natural Science Foundation of Distinguished Young Scientists (LR14C040001). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.fsi.2015.05.042. References [1] T. Fujita, M. Matsushita, Y. Endo, The lectin-complement pathway-its role in innate immunity and evolution, Immunol. Rev. 198 (2004) 185e202. [2] J.A. Hoffmann, F.C. Kafatos, C.A. Janeway, R.A. Ezekowitz, Phylogenetic perspectives in innate immunity, Science 284 (1999) 1313e1318. [3] J.O. Sunyer, I.K. Zarkadis, J.D. Lambris, Complement diversity: a mechanism for generating immune diversity? Immunol. Today 19 (1998) 519e523. [4] M.J. Walport, Complement. First of two parts, N. Engl. Med. 344 (2001a) 1058e1066. [5] M.J. Walport, Complement. Second of two parts, N. Engl. Med. 344 (2001b) 1140e1144. [6] W.C. Song, M.R. Sarrias, J.D. Lambris, Complement and innate immunity, Immunopharmacol 49 (2000) 187e198. [7] M.P. Chondrou, D. Mastellos, I.K. Zarkadis, cDNA cloning and phylogenetic analysis of the sixth complement component in rainbow trout, Mol. Immunol. 43 (2006) 1080e1087. [8] H.J. Muller-Eberhard, The membrane attack complex of complement, Annu Rev. Immunol. 4 (1986) 503e528. [9] R. Wurzner, Modulation of complement membrane attack by local C7 synthesis, Clin. Exp. Immunol. 121 (2000) 8e10. [10] R.G. DiScipio, Formation and structure of the C5b-7 complex of the lytic pathway of complement, Biol. Chem. 267 (1992) 17087e17094. [11] A. Mikrou, I.K. Zarkadis, Cloning of the sixth complement component and, spatial and temporal expression profile of MAC structural and regulatory genes in chicken, Dev. Comp. Immunol. 34 (2010) 485e490. [12] T. Katagiri, I. Hirono, T. Aoki, Molecular analysis of complement component C8 beta and C9 cDNAs of Japanese flounder, Paralichthys olivaceus, Immunogenetics 50 (1999) 43e48. [13] M.J. Hobart, B.A. Fernie, R.G. DiScipio, Structure of the human C7 gene and comparison with the C6, C8a, C8b and C9 genes, Immunol 154 (1995) 5188e5194.
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