BAX, a novel cell pro-apoptotic protein, involved in hemocytes early antiviral immune response in fresh water crayfish, Procambarus clarkii

BAX, a novel cell pro-apoptotic protein, involved in hemocytes early antiviral immune response in fresh water crayfish, Procambarus clarkii

Fish & Shellfish Immunology 55 (2016) 384e392 Contents lists available at ScienceDirect Fish & Shellfish Immunology journal homepage: www.elsevier.com...

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Fish & Shellfish Immunology 55 (2016) 384e392

Contents lists available at ScienceDirect

Fish & Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi

Full length article

BAX, a novel cell pro-apoptotic protein, involved in hemocytes early antiviral immune response in fresh water crayfish, Procambarus clarkii Zhi-Qiang du School of Life Science and Technology, Inner Mongolia University of Science and Technology, Baotou, Inner Mongolia Autonomous Region 014010, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 1 March 2016 Received in revised form 4 June 2016 Accepted 8 June 2016 Available online 10 June 2016

Apoptosis plays an important role in various biological processes and acts as a host defending mechanism by which infected cells are eliminated to restrict the virus propagation scale. Bax is a crucial proapoptotic protein, which mediates the release of cytochrome c from mitochondrion to cytosol in mammalian. However, its role in invertebrate is still obscure. Here, a novel pro-apoptotic protein gene was identified from hemocytes of red swamp crayfish. There was a Bcl-2 domain in the C-terminus of PcBax, which possessed 497 amino acids residues. And an important transmembrane region existed in the C-terminus of Pc-Bax, which implied that Pc-Bax located in mitochondrial membrane. Besides, Pc-Bax was expressed at a relative high level in hemocytes, and a relative low expression levels in hepatopancreas, gills, and intestine. In hemocytes, Pc-Bax transcript was rapidly up-regulated from 12 h to 36 h after WSSV infection. And there was the same trend for Pc-Bax protein expression level in hemocytes after WSSV infection. Results of qRT-PCR testing for VP28 gene showed WSSV replication was obviously enhanced after Pc-Bax knockdown. Meantime, hemocytes apoptosis was suppressed in Pc-Bax knockdown crayfish after WSSV injection, compared with the dsGFP injection group and normal group. Taken together, these results revealed that crayfish hemocytes apoptosis scale was enhanced to suppress WSSV replication by up-regulating Bax protein expression level after WSSV infection. © 2016 Elsevier Ltd. All rights reserved.

Keywords: Procambarus clarkii BAX Pro-apoptotic protein Antiviral innate immune

1. Introduction To keep a regular development pattern, multicellular organisms evolve to form a dynamic balance between cell proliferation and apoptosis [1]. Although most of cells must be protected to serve in the following stages of development by cell survival factors, damaged or superfluous cells must be removed by apoptosis [2]. Cells which are undergoing apoptosis have obvious morphological changes including chromatin condensation, apoptotic body formation, cell shrinking, cell member blebbing, and fragmentation [3]. At the same time, there are some important molecular events in cell apoptosis process. For example, changes of relative expression level between anti- and pro-apoptotic proteins. Apoptosis plays an important role in various processes including cell differentiation, cell development, and cell proliferation, and so on [4]. Furthermore, apoptosis acts as a host defending mechanism by which infected cells are eliminated to restrict the propagation scale of virus [5]. When organisms encounter pathogens invasion or

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other environment stress stimulus, apoptosis can be triggered to adjust cell metabolism to adapt to environment changing [6]. This cell suicidal death process was studied and well documented in animals from the genetic, biochemical, and morphological levels. For example, Bax inhibitor 1 protein was involved in innate immunity of Procambarus clarkii. RNA interference result showed that WSSV replication was evidently suppressed and the apoptosis was enhanced under Pc-BI-1 knock down [7]. Another anti-apoptotic protein was inhibitors of apoptosis (IAPs) identified in Litopenaeus vannamei. Research results showed that Lv-IAP1 and Lv-IAP3 might participate in host defense against WSSV infection, and Lv-IAP2 might be involved in the regulation of antimicrobial peptides [8]. Accumulating evidences in animals have revealed that apoptosis was triggered and controlled by the conserved B-cell lymphoma-2 (Bcl-2) family proteins [9]. The Bcl-2 family proteins were composed of pro-apoptotic molecules (Bax, Bak, etc.) and antiapoptotic molecules (Bcl-2, Bcl-xL, etc.) [10]. The roles of these two kinds of molecules were preliminarily confirmed in mammalian systems. For example, apoptosis could be inhibited in the Bcl-2 transgenic mice which expressed high levels of exogenous Bcl-2 protein [11]. It was also inhibited in the mice which were

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deficient in pro-apoptotic factor Bax or Bak protein. And the extent of cell apoptosis was elevated when both of these two factors were lost [12]. Besides, Bcl-2 family proteins regulated apoptosis through regulating or changing mitochondria structure and function, which were crucial in deciding the fate of a cell [13]. Lots of evidences in mammalian systems illustrated that anti-apoptotic Bcl-2 family proteins prevented mitochondrial release of cytochrome c, which was required for Apaf-1 apoptosome formation and consequent caspase activation [14]. Meanwhile, pro-apoptotic Bcl-2 proteins assisted cytochrome c release from mitochondria by changing the mitochondrial membrane permeability [15]. To date, some Bcl-2 family proteins have been identified in mammal and insects. However, few pro- and anti-apoptotic molecules were identified in crustaceans [8]. There were two antiapoptotic proteins respectively found in Procambarus clarkia and L. vannamei. Until now, pro-apoptotic proteins have not been identified in crustaceans. In present study, a novel full-length cDNA of the Bax protein has been identified from red swamp crayfish, P. clarkii (named as Pc-Bax). Like other Bax proteins of higher vertebrate animals, Pc-Bax possessed an important transmembrane region in the C-terminus [9,16]. The expression profile of Pc-Bax has been examined in hemocytes in response to the infection of WSSV (white spot syndrome virus). Obvious up-regulation expression levels in mRNA and protein suggested Pc-Bax was involved in defending reaction against virus. Besides, results of RNAi assay implied that Pc-Bax was a pro-apoptotic protein. And replication of WSSV was enhanced through suppressing cell apoptosis after PcBax was knocked down in crayfish [17,18]. Taken together, these results revealed that Pc-Bax participated in the crayfish defending reaction against WSSV infection. Hemocytes apoptosis scale could be enhanced to suppress WSSV replication, by increasing endogenous Bax protein expression level after WSSV infection. 2. Materials and methods 2.1. Immunity challenge and tissues collection P. clarkii (approximately 15e20 g each) were bought from an aquatic market in Baotou, Inner Mongolia autonomous region, China. They were temporarily cultured in laboratory tanks filled with fresh water. For WSSV challenge assay, we injected WSSV (3.2  107 copies per crayfish) into the abdominal segment of crayfish [19]. There were totally sixty crayfishes. And ten crayfishes were arranged for every time point (0, 12, 24, 36, 48, and 72 h). Hemolymph was taken from the ventral sinus at different time points after WSSV injection, using a 5 ml sterile syringe preloaded with 500 ml anticoagulant (10% sodium citrate, pH 7). Then it was centrifuged immediately at 800  g for 5 min (4  C) to isolate hemocytes [20]. Hemolymph and hemocytes from unchallenged crayfish were also collected using the same method. Other normal tissues from unchallenged crayfish such as hepatopancreas, gills, and intestine were collected for total RNA extraction. 2.2. RNA extraction and cDNA synthesis Total RNA of four tissues (hemocytes, hepatopancreas, gills, and intestine) from unchallenged crayfish and hemocytes from WSSVchallenged crayfish at different time points were extracted using BIOZOL reagents (Hangzhou, China) according to the manufacturer’s protocol. Then they were dissolved in DEPC treated water. Electrophoresis on 1% agarose gel free of RNase was carried out to test RNA quality. The first strand cDNA synthesis was carried out in 25 ml reaction volume containing 5 mg RNA, 1 ml M-MLV reverse transcriptase (Promega USA), 1 mM dNTP mixture using SMART F (50 -tac ggc tgc gag aag acg aca gaa ggg-30 ), and oligo anchor R (50 -

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gac cac gcg tat cga tgt cga ct16v-30 ) at 42  C for 2 h [21]. 2.3. Gene cloning The specific forward and reverse primers (F1:50 -gta tac aaa tta cca ccg cga g-30 ; R1:50 -agc tct tga cca agt agg gtt a-30 ) were designed based on the nucleotide sequences of an Expressed Sequence Tag (EST) we obtained from transcriptome sequencing. The sample used to perform transcriptome sequencing was lymph organ tissue of adult crayfish. And transcriptome sequencing was finished in the Beijing Genomics Institute-Shenzhen (BGI, Shenzhen, China). The F1 and 30 anchor R primer (50 -gac cac gcg tat cga tgt cga c-30 ) were used to amplify the 30 end of the cDNA. The 50 PCR primer (50 -tac ggc tgc gag aag acg aca gaa-30 ) and the R1 were used to amplify the 50 end of the cDNA. Polymerase chain reaction (PCR) amplification was carried out as follows: a cycle of 94  C for 3 min and 35 cycles of 94  C for 30 s, 53  C for 45 s, and 72  C for 90 s, followed by an additional extension at 72  C for 10 min. PCR products were in-gel purified using the gel purification kit (Sangon, Shanghai, China) as described in the manufacturer’s protocol, followed by the ligation into the pMD-18T vector (TaKaRa) and transformed into the competent DH5a cells [22]. The positive recombinants were identified through blue-white color selection in ampicillin-containing LB plates and the PCR screening using the two specific primers (F1 and R1), respectively. Positive clones were sequenced by Sangon companies (Shanghai, China). 2.4. Sequence alignment and phylogenetic analysis After obtaining Pc-Bax genes sequences, BLASTx online analysis was carried out on the website (http://www.ncbi.nlm.nih.gov/). Translation of the amino acid sequences and prediction of the deduced protein were performed with ExPASy (http://www. expasy.org) [20]. Signal peptides sequence and structural domain were predicted using SMART (Simple Modular Architecture Research Tool) (http://www.smart.embl-heidelberg.de/) [21]. At last, amino acids sequences alignment with the homogenous sequences from other species, which were selected by the BLASTx analysis results, was performed using MEGA 6.0 software. Phylogenetic analysis was carried out using Neighbor Joining (NJ) methods of MEGA 6.0 based on the amino acid sequences. To estimate the reliability, 1000 bootstraps were selected for the NJ tree. 2.5. Quantitative real-time PCR analysis for expression pattern of Pc-Bax Total RNA was extracted from different tissues of normal crayfish and hemocytes from WSSV-challenged crayfish (at 12, 24, 36, 48 and 72 h time points). And then, RNA (5 mg) from each tissue was used to reverse transcribe the first strand of cDNA, which was used as a template in PCR reactions. For quantitative real-time PCR (qRTPCR) analysis, cDNA templates were diluted 50-fold in nucleasefree water, and were used as templates. A pair of primers (Pc-BaxRT-F: 50 -tat agt tgg ctc att agc ag-30 ; Pc-Bax-RT-R: 50 -ata cta agt gaa gat gac tg-30 ) were used in qRT-PCR. The specific primers, 18S RNART-F(50 -tct tct tag agg gat tag cgg-30 ) and 18S RNA-RT-R(50 -aag ggg att gaa cgg gtt a-30 ), were used to amplify crayfish 18S RNA gene as inner control. At the same time, total DNA was extracted from hemocytes of normal crayfish and hemocytes from WSSV-challenged crayfish (at 12, 24, 36, 48 and 72 h time points). Then they were used for detection of WSSV VP28 gene expression. The qRT-PCR was performed following the manufacturer’s instruction of SYBR Premix Ex Taq (Takara, Japan) using a real-time thermal cycler (Bio-Rad, USA) in a total volume of 20 ml containing 10 ml of 2  Premix Ex Taq, 2 ml of the 1:50 diluted cDNA, and

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4 ml (1 mM) each of the forward and reverse primer [23]. The amplification procedure consisted of an initial denaturation step at 95  C for 3 min and then 40 cycles of 95  C for 15 s, 58  C for 30 s, followed by melting from 65  C to 95  C. To confirm that only one fragment was amplified, the melting curve was also analyzed for amplification products, and was performed at the end of each PCR reaction. Three parallel experiments were carried out to increase the integrity (three different batches of crayfish were selected to carry out immune challenge, and then extracted RNA, synthesized cDNA, performed qRT-PCR). Furthermore, expression level of PcBax was shown as relative expression values, which were calculated according to 2DDCT [24]. The data obtained were subjected to statistical analysis followed by an unpaired sample t-test. Significant difference was accepted at P < 0.05. Extremely significant difference was accepted at P < 0.01. 2.6. Antibody preparation and western blot analysis for Pc-Bax protein A polypeptide containing sixty amino acids residues from 367th site to 426th site in the open reading frame (ORF) of Pc-Bax was synthesized by Sangon company (Shanghai, China). When synthesized polypeptide was used as protein antigen to produce polyclonal rabbit antiserum, a method described in a previous study was referenced [25]. Western blot was carried out to investigate endogenous Pc-Bax protein’s expression profiles at protein level in crayfish hemocytes after WSSV infection. Total proteins were extracted from hemocytes of unchallenged crayfish using buffer A (40 mmol Tris-HCl, 1 mmol phenylmethylsulfonyl fluoride, and 3 mmol ethylenediaminetetraacetic acid) [26]. In the same method, proteins were respectively extracted from hemocytes of WSSV-challenged crayfish at 12, 24, 36, 48, and 72 h time points after WSSV injection. The proteins were then electrically transferred onto the nitrocellulose membrane, and bands were detected via immunoblotting assay [27]. The crayfish b-actin was used as inner reference. The antibody against b-actin was prepared in the same method like antibody against Pc-Bax. Besides, the nitrocellulose membrane was scanned, and density of immonoreactive bands was measured using “Image J” software. 2.7. RNAi assay of Pc-Bax gene Pc-Bax dsRNA was synthesized using the method described previously in another article [7]. Crayfish was randomly divided into three groups (40 for each group), including Pc-Bax dsRNA (dsPc-Bax) injection group, GFP dsRNA (dsGFP) injection group, and normal group. Pc-Bax and GFP DNA fragments were amplified using Pc-Bax-Fi (50 -gcg taa tac gac tca cta tag gtt acc tag att acc taa tca ta30 ) and Pc-Bax-Ri (50 -gcg taa tac gac tca cta tag gcc aac cac ctg cct ctt gta cc-30 ), GFP-Fi (50 -gcg taa tac gac tca cta tag gtg gtc cca att ctc gtg gaa c-30 ) and GFP-Ri (50 -gcg taa tac gac tca cta tag gct tga agt tga cct tga tgc c-30 ), respectively. The sequence of T7 promotor was underlined in the primers above [28]. The DNA fragments obtained by PCR will be used as templates for dsRNA synthesis. The dsRNA synthesis system (with a 50 ml total volume) were devised according to this following methods: 8 mg DNA templates, 20 ml 5  transcription buffer, 2.4 ml A/U/C/GTP each (10 mM Fermentas, USA), 60 U RNasin (TaKaRa, Japan), 40 U T7 RNA polymerase (Fermentas, USA), mixed with RNase-Free water to total volume 50 ml. After incubated at 37  C for 5 h, the solution was added to final volume to 100 ml, with 20 ml 10  DNase I buffer, 6 ml DNase I (Fermentas, USA) and 14 ml RNase-Freewater. And then, the mixed solution was incubated again at 37  C for 1 h in order to remove the DNA templates [29]. After this step, dsRNA was extracted with

phenol/chloroform and precipitated with ethanol, then resuspended in 40 ml RNase-Free water. The prepared dsRNA (50 mg) was injected into the abdominal segment of each crayfish, and the second injection was given 24 h later to enhance the RNAi efficiency. And then, the total RNA was isolated from hemocytes of three groups’ crayfish at 12 h after the second injection. In order to evaluate the knockdown efficiency of Pc-Bax, qRT-PCR was carried out, using with primers Pc-Bax-RT-F (50 -tat agt tgg ctc att agc ag-30 ) and Pc-Bax-RT-R (50 -ata cta agt gaa gat gac tg-30 ). 2.8. WSSV replication and hemocytes apoptosis rate detection after WSSV infection in dsPc-Bax injected crayfish To identify the antiviral role of Pc-Bax in innate immune system of crayfish, WSSV was immediately injected into crayfish after knockdown of Pc-Bax was proved to be successful. At 36 h after WSSV injection, hemocytes were isolated from hemolymph taken from each group crayfish (5 crayfish) ventral sinus. And then, genome was extracted from hemocytes. VP28 (an envelope protein gene of WSSV) was detected to check WSSV proliferation in three groups, using qRT-PCR. The special primers were VP28-F (50 -ctc cgc aat gga aag tct ga-30 ) and VP28-R (50 -ggg tga agg agg agg tgt t-30 ) [30]. At the same time, apoptosis rate of hemocytes in three groups was detected using TUNEL method at 36 h after WSSV injection. The detailed test methods could be found in our previous article [7]. Briefly, the hemocytes were dropped on glass slides coated with 2% gelatin and fixed with 4% paraformaldehyde (dissolved in PBS, pH 7.4), after washed once with 5 ml anticoagulant solution, and twice with 5 mL PBS, Then hemocyes on the slide were treated with 1% triton X-100, washed twice with 1 ml PBS. The hemocytes apoptosis was detected with the In Situ Cell Death Detection Kit, Fluorescein (Roche, Germany) following the manufacture’s instruction, and the hemocytes were directly analyzed under a fluorescence microscope. A total of 500 cells were counted to determine the apoptosis rate (the number of apoptosis cells/the number of total cells). 3. Results 3.1. Cloning of Pc-Bax cDNA The cDNA sample of P. clarkii hemocytes was used as template for PCR amplification. A 1840 bp intact cDNA sequence of target gene was cloned. After BLAST online analysis, this target gene was nominated as P. clarkii Bax gene (Pc-Bax). The open reading frame of Pc-Bax encoded 497 amino acids residues, which possessed a 99amino acids residues B-Cell lymphoma (BCL) domain in C-terminus (Fig. S1). And a transmembrane region was prospected in the Cterminus of Pc-Bax, which included 23 amino acids residues (from 469th to 491st residue). 3.2. Multiple alignments for amino acids sequences and phylogenetic analysis To analyze Pc-Bax amino acids sequence difference with other Bax proteins, some Bax proteins were chosen to perform sequence multiple alignments on the base of online BLASTx alignment analysis results. These fourteen Bax proteins included Homo sapiens Bax (GenBank number: AAH14175), Macaca mulatta Bax (GenBank number: NP_001247945), Bos taurus Bax (GenBank number: NP_776319), Capra hircus Bax (GenBank number: AII22864), Felis catus Bax (GenBank number: NP_001009282), Myotis davidii Bax (GenBank number: XP_006770030), Rattus norvegicus Bax (GenBank number: NP_058755), Cricetulus griseus Bax (GenBank number: NP_001230949), Xenopus tropicalis Bax (GenBank number:

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NP_989185), Danio rerio Bax (GenBank number: AAF66960), Salmo salar Bax (GenBank number: ACI67445), Sparus aurata Bax (GenBank number: AGU38820), Cyprinus carpio Bax (GenBank number: AHV90609), and Ictalurus punctatus Bax (GenBank number: NP_001187866). The sequence alignment was done between PcBax and these fourteen Bax proteins. And the results showed that similarity was not very high among these Bax proteins (Fig. 1). At the same time, there were some obvious sequence characteristics. Among all these Bax proteins, there was a B-Cell lymphoma (BCL) domain and a transmembrane region in the C-terminus of Bax proteins. Besides, phylogenetic analysis by MEGA 6.0 software revealed that these Bax proteins could be divided into two branches. Pc-Bax located in the same branch with Danio rerio Bax, Salmo salar Bax, Sparus aurata Bax, and Cyprinus carpio Bax (Fig. S2). 3.3. Expression profiles analysis of Pc-Bax in mRNA and protein levels The qRT-PCR was carried out to detect the expression level of PcBax in crayfish different tissues. The results showed that Pc-Bax was expressed in hemocytes at a relative high level, compared with the expression levels in hepatopancreas, gills, and intestine (Fig. 2). The time course expression profile of Pc-Bax in hemocytes was also analyzed. In the hemocytes of WSSV-injected crayfish, Pc-Bax expression level was up-regulated from 12 h to 36 h post-viral challenge, and obviously decreased to normal level from 48 h to 72 h (Fig. 3A). And the Pc-Bax expression level up-regulated to the maximum level at 24 h after WSSV infection. At the same time, WSSV replication was showed in Fig. 3B, by testing the relative expression level of VP28 gene. The western blot result showed that the expression level of endogenous Pc-Bax protein in crayfish hemocytes was upregulated from 12 h to 72 h after WSSV infection (Fig. 4A). And the Pc-Bax protein expression level increased to maximum level at 24 h after WSSV infection (Fig. 4B). The up-regulation trends in both mRNA and protein level suggested that Pc-Bax participated in crayfish antiviral immune response, especially in the early period. 3.4. WSSV replication was enhanced after knocking down of Pc-Bax The RNAi assay was carried out to detect Pc-Bax role in crayfish antiviral innate immunity. After the second dsRNA injection, total RNA was isolated from hemocytes of three groups’ crayfish to detect target gene’s expression level. The results showed that PcBax was successfully knocked down compared with other two groups (Fig. 5). Subsequently, WSSV was injected into three groups’ crayfish abdominal segment to study WSSV replication after Pc-Bax knockdown. At 36 h after WSSV injection, genomic DNA was extracted from hemocytes of three groups’ crayfish. And VP28 was checked to study WSSV replication. The qRT-PCR results showed that WSSV replication was obviously enhanced after Pc-Bax knockdown, compared with the dsGFP injection group and normal group (Fig. 6). 3.5. Hemocytes apoptosis was suppressed after knocking down of Pc-Bax After Pc-Bax was successfully knocked down, VP28 relative expression level was tested to study WSSV replication. Meantime, hemocytes apoptosis rate in three groups was also detected using TUNEL method at 36 h after WSSV injection. The results showed that hemocytes apoptosis was suppressed in Pc-Bax knockdown crayfish after WSSV injection, compared with the dsGFP injection group and normal group (Fig. 7). And the percent of hemocytes

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apoptosis in dsPc-Bax injection group was a little higher than normal crayfish which was not injected WSSV. 4. Discussion Apoptosis is the prevalent name of programmed cell death (PCD), which is characterized by nuclear chromatin condensation, DNA fragments forming, decrease of plasma membrane phospholipids asymmetry, and appearance of apoptosome, and so on [31]. Regulation of apoptosis is very important for normal growth and development, especially for homeostasis maintenance. Occurrence of apoptosis disorders can induce severe outcomes. At the same time, apoptosis is a special kind of cell death with evolutionally conserved mechanism. In mammalian cells, apoptosis is controlled and regulated by various cellular signals from intracellular or extracellular. However, it was mainly induced through two pathways (intrinsic pathway and extrinsic pathway) [32]. The intrinsic pathway is also called as Bcl-2 regulated pathway, and the extrinsic pathway is called as death receptor pathway. In both intrinsic and extrinsic pathways, induction of apoptosis is correlated with the activation of caspases proteases [33]. The extrinsic apoptosis pathway can be triggered by binding between ligands with death receptors. The death receptors are members of tumor necrosis factor receptor (TNFR) family localizing on cell surface, with an intracellular death domain [34]. Meanwhile, the intrinsic apoptosis pathway can be triggered by developmental cues, growth factor, nutrient deprivation, and wide-ranging cytotoxic stimuli, and so on [35]. The intrinsic apoptosis pathway is regulated by the Bcl-2 proteins family, which control cytochrome c release from mitochondria. The induction of intrinsic apoptosis mainly relies on the ratio of pro-apoptotic proteins to antiapoptotic proteins [34]. To date, approximately 25 different types of Bcl-2 family proteins have been identified in mammals. Conversely, there is only one or two Bcl-2 family proteins prospected to exist in insects’ genome [36]. And some aspects of the Bcl-2 family proteins’ molecular mechanisms, which were involved in the mitochondrial outer membrane permeabilization process in apoptosis, still remained controversial. However, some features of Bax proteins action have been revealed in the last couple of decades. For example, Bax proteins are inactive as a monomer and locate in the cytosol in normal conditions. And Bax proteins need to be activated and translocated to the mitochondrial outer membrane, when cells encounter challenges which could induce apoptosis [37]. In present study, a novel pro-apoptotic protein gene was identified from the hemocytes of red swamp crayfish (P. clarkii), nominated as Pc-Bax. There was a Bcl-2 domain in the C-terminus of Pc-Bax, which possessed 497 amino acids residues. Like other vertebrate animals’ Bax proteins, there was a transmembrane region in the C-terminus of Pc-Bax, which included 23 amino acids residues (from 469th to 491st residue). This feature might imply that Pc-Bax locate in the mitochondrial membrane. Besides, the tissue distribution and time course expression profile of Pc-Bax was studied by qRT-PCR. The results showed that Pc-Bax was expressed at a relative high level in hemocytes, and a relative low expression levels in hepatopancreas, gills, and intestine (Fig. 2). In crayfish hemocytes, the Pc-Bax was rapidly up-regulated from 12 h to 36 h after WSSV infection. Then the expression level recovered to normal from 48 h to 72 h (Fig. 3). In protein level, there was the same trend for Pc-Bax expression level after WSSV infection (Fig. 4). These rapidly up-regulated expression trends in both mRNA and protein level implied that Pc-Bax participated in the crayfish early period antiviral immune response. In order to analyze the biological functions of Pc-Bax, RNAi assay was performed. The results of qRT-PCR testing for VP28 gene

Fig. 1. Multiple alignment of Pc-Bax with other Bax proteins, including Homo sapiens Bax (Hs-Bax, AAH14175), Macaca mulatta Bax (Mm-Bax, NP_001247945), Bos taurus Bax (BtBax, NP_776319), Capra hircus Bax (Ch-Bax, AII22864), Felis catus Bax (Fc-Bax, NP_001009282), Myotis davidii Bax (Md-Bax, XP_006770030), Rattus norvegicus Bax (Rn-Bax, NP_058755), Cricetulus griseus Bax (Cg-Bax, NP_001230949), Xenopus tropicalis Bax (Xt-Bax, NP_989185), Danio rerio Bax (Dr-Bax, AAF66960), Salmo salar Bax (Ss-Bax, ACI67445), Sparus aurata Bax (Sa-Bax, AGU38820), Cyprinus carpio Bax (Cc-Bax, AHV90609), and Ictalurus punctatus Bax (Ip-Bax, NP_001187866). The numbers on the right indicate amino acid position of different sequences. Different colors represent different amino acid conservations.

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Fig. 1. (continued).

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Fig. 2. Tissue distribution of Pc-Bax in normal red swamp crayfish. Ten crayfishes were arranged for RNA extraction of normal group. 18 S RNA was used as inner reference.

Fig. 4. Western blot result for the time course expression profiles of Pc-Bax protein in hemocytes of crayfish after WSSV infection. (A) Western blot result for Pc-Bax protein. (B) Semi-quantification analysis for western blot result. Actin was used as the control.

Fig. 3. Time course expression profiles of Pc-Bax (A) and VP28 (B) in hemocytes of crayfish after WSSV infection. There were totally sixty crayfish for this assay. And ten crayfish were arranged for every time point (0, 12, 24, 36, 48, and 72 h). 18S RNA was used as the inner reference. The asterisks indicate significant differences (*: P < 0.05, **: P < 0.01) from the control.

Fig. 5. Result of RNAi assay for Pc-Bax. dsGFP injection group and normal group were used as the control. The data are expressed as the ratio of Pc-Bax to 18S RNA mRNA. The asterisks indicate significant differences (**: P < 0.01) from the control.

showed that WSSV replication was obviously enhanced after PcBax knockdown (Fig. 6). At the same time, hemocytes apoptosis was suppressed in Pc-Bax knockdown crayfish after WSSV injection, compared with the dsGFP injection group and normal group

(Fig. 7). Taken together, these results revealed that crayfish could improve hemocytes apoptosis scale to suppress WSSV replication, by up-regulating Bax protein expression level after WSSV infection.

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References

Fig. 6. Pc-Bax knockdown increased the replication of WSSV in crayfish. “NormalþWSSV” stands for the normal crayfish are injected with WSSV; “dsGFPþWSSV” stands for the crayfish group which is injected with GFP dsRNA and then challenged with WSSV; “dsPc-BaxþWSSV” stands for the crayfish group which is injected with PcBax dsRNA and then challenged with WSSV. Five crayfish were arranged for every group. The data are expressed as the ratio of VP28 to 18S RNA mRNA. The asterisks indicate significant differences (**: P < 0.01) from the control.

Fig. 7. Pc-Bax knockdown decreased the hemocytes apoptosis ratio of crayfish infected with WSSV. “Normal” stands for the normal crayfish without WSSV injection; “NormalþWSSV” stands for the normal crayfish are injected with WSSV; “dsGFPþWSSV” stands for the crayfish group which is injected with GFP dsRNA and then challenged with WSSV; “dsPc-BaxþWSSV” stands for the crayfish group which is injected with PcBax dsRNA and then challenged with WSSV. Five crayfish were arranged for every group. The asterisks indicate significant differences (**: P < 0.01) from the control.

Acknowledgments This work was supported by the National Natural Science Foundation of China (Grant No. 31460698).

Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.fsi.2016.06.015.

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