302 ISJ 15: 302-315, 2018 ISSN 1824-307X RESEARCH REPORT The mRNA expression profiles demonstrating versatile roles of glutathione S-transferase genes in the mollusk Chlamys farreri M Wang1, L Wang3,4,5, D Ni1, Q Yi3,4,5, X Wang6, Z Jia1, L Song2,3,4,5* 1CAS Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China 2Laboratory for Marine Fisheries Science and Food Production Processes, National Laboratory for Marine Science and Technology, Qingdao 266237, China 3Liaoning Key Laboratory of Marine Animal Immunology, Dalian Ocean University, Dalian 116023, China 4Liaoning Key Laboratory of Marine Animal Immunology and Disease Control, Dalian Ocean University, Dalian 116023, China 5Dalian Key Laboratory of Disease Prevention and Control for Aquaculture Animals, Dalian Ocean University, Dalian 116023, China 6College of Marine Science and Biological Engineering, Qingdao University of Science & Technology, Qingdao 266042, China Accepted August 27, 2018 Abstract Glutathione S-transferase (GST) is a superfamily of multifunction enzymes with varying catalytic roles in cellular detoxification to protect hosts against oxidative damage. In the present study, six GST genes were identified from Chlamys farreri, including CfGSTω, CfGSTσ-1, CfGSTσ-2, CfGSTρ, CfGSTζ and CfmGST. CfGSTs shared high similarities with their counterparts from other species, and were clustered with their homologues into the corresponding clades in the phylogenetic tree, respectively. We investigated the distribution of their mRNA transcripts in different tissues and their temporal expression profiles in hemocytes after microbe stimulations by quantitative real-time PCR. The six CfGST genes were detectable in all the tested tissues, including hemocytes, muscle, mantle, gill, hepatopancreas, and gonad. Stimulations with various microbes drastically induced the mRNA transcripts of all the CfGSTs with different expression profiles. For examples, CfGSTω could be induced by three kinds of microbes, including Vibrio anguillarum, Micrococcus luteus and Pichia pastoris, whereas CfmGST could be only induced by V. anguillarum. These results indicated a powerful detoxification system of GSTs in scallop. Moreover, the distinct mRNA expression profiles of CfGSTs indicated their versatile and immune-challenge specific roles in the mollusk C. farreri. Key Words: Chlamys farreri; Glutathione S-transferase; innate immunity Introduction The innate immunity acts as the first defense line for all multicellular animals and almost the only mechanism for invertebrates to protect themselves against microbial invaders (Hoffmann et al., 1999). Many innate immune responses, especially hemocytes-mediated phagocytosis, were accompanied with respiratory burst and followed by mass production of reactive oxygen species (ROS) (Liu et al., 2009; Jia et al., 2018). The production of ROS is an effective way to eliminate invading microbes; however, it has been already proved to be ___________________________________________________________________________ Corresponding author: Linsheng Song Dalian Ocean University Dalian 116023, China E-mail: lshsong@dlou.edu.cn; lshsong@qdio.ac.cn a double-edged sword (Benedetti et al., 2015). Low concentration of ROS is beneficial for activating signaling pathways mediating various responses to kill or eliminate foreign invaders (He and Klionsky, 2009). While extremely high levels of ROS may be detrimental to biological macromolecules, and lead to cellular dysfunctions, increase cell damage and finally threaten hosts’ survival (Martindale and Holbrook, 2002). Therefore, almost all the aerobic organisms have developed an antioxidant system to remove excessive ROS and maintain the redox balance (Halliwell, 2006). The antioxidant system is constituted by a series of antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), peroxiredoxin (PRX), thioredoxin peroxidase (TPX), thioredoxin reductase (TRX), glutathione peroxidase (GPX), glutathione reductase (GRX), 303 glutathione-S-transferase (GST) and many other non-enzymatic antioxidant molecules (Harris, 1992). Among all these antioxidant enzymes, GST (EC: 2.5.1.18) is a superfamily of multifunction enzymes, which play varying catalytic roles in cellular detoxification and protect hosts from oxidative damage (Strange et al., 2001). By now, at least 15 different classes of GSTs have been identified and characterized in numerous organisms according to their structural, catalytic and immune features, including alpha (α), beta (β), delta (δ), epsilon (ε), kappa (κ), lambda (λ), mu (μ), omega (ω), phi (φ), pi (π), sigma (σ), tau (τ), theta (θ), zeta (ζ) and rho (ρ) (Hayes et al., 2005). The microsomal GSTs, members of the membrane associated protein in eicosanoid and glutathione metabolism (MAPEG) protein family, also play pivotal roles in antioxidant reaction (Morgenstern et al., 1982). Although no criteria were developed to classify GSTs in marine organisms, the expression profiles and enzyme activities of GSTs have been investigated in some aquatic species, such as abalone Haliotis diversicolor (Ren et al., 2009), bay scallop Argopecten irradians (Wang et al., 2017a), disk abalone Haliotis discus discus (Wan et al., 2008; Sandamalika et al., 2018), green-lipped mussels Perna viridis (Li et al., 2013), intertidal copepod Tigriopus japonicas (Lee et al., 2007), manila clam Venerupis philippinarum (Xu et al., 2010; Li et al., 2012; Zhang et al., 2012a,b; Li et al., 2015), marine mussels Mytilus galloprovincialis (Wang et al., 2013; Li et al., 2015), pearl oyster Pinctada martensii (Chen et al., 2011), razor clam Solen grandis (Yang et al., 2012), ridge-tail white prawn Exopalaemon carinicauda (Duan et al., 2013), and sea cucumber Apostichopus japonicas (Shao et al., 2017; Zhang et al., 2017a,b). Some of these GSTs from aquatic species were involved in innate immunity and could respond to invading microbes, for examples, the sigma class GST from H. diversicolor was significantly induced post bacteria challenged (Ren et al., 2009), while the mRNA expression level of a GST gene in S. grandis was significantly up-regulated in hemocytes after being stimulated by β-1, 3-glucan (Yang et al., 2012). The Zhikong scallop Chlamys farreri is one of the most important commercial species which is widely cultivated in the northern coastal provinces of China (Li et al., 2015b; Song et al., 2015). With the rapid expansion of intensive culture and environmental deterioration, scallops have frequently suffered from various diseases. The knowledge about the antioxidant system and its function in response to invading microbes may provide a better understanding of innate immune mechanisms in scallop and potential development of disease control strategies in scallop farming. In previous reports, several antioxidant enzyme genes have been identified and investigated in C. farreri, such as SOD (Ni et al., 2007; Wang et al., 2018), CAT (Li et al., 2008), PRX (Cong et al., 2009), and GPX (Mu et al., 2010). Moreover, the cDNA sequence of a pi (π) class GST and its expression profiles in response to Benzo[α]pyrene exposure was also reported in C. farreri (Miao et al., 2011). However, compared with other antioxidant enzymes in scallop, the information of GSTs is rather rare and fragmentary and more investigation is needed to illustrate their exact roles in the innate immunity. In the present study, six novel GST genes were identified in C. farreri based on the analysis of expression sequence tag (EST) sequences (Wang et al., 2009) with the main objectives (1) to characterize the molecular features of CfGST genes (2) to detect the tissue distribution and temporal mRNA expression profiles of their mRNA transcripts, and (3) to compare these features to lead a better understanding of their versatile roles in C. farreri. Materials and methods Scallops, immune stimulation and sample collection Adult scallops with an average 55 mm in shell length were collected from a local farm in Qingdao, China, and maintained in aerated seawater at about 15 °C. Approximately 120 scallops were employed for microbe stimulation assay. After acclimated for two weeks, 30 scallops were kept in tanks containing live Vibrio anguillarum strain M3 (kindly provided by Prof. Zhaolan Mo) at a final concentration of 1.0 × 108 colony forming units (CFU) mL-1, and defined as Gram-negative bacteria stimulation group. Another 30 scallops were transferred to the tanks containing live Micrococcus luteus (28001, Microbial Culture Collection Center, China) at a final concentration of 1.0 × 108 CFU mL-1, and defined as Gram-positive bacteria stimulation group. The third 30 scallops were transferred to the fungi-containing tanks with live Pichia pastoris strain GS115 (PA17237, Thermo Fisher Scientific, USA) at a final concentration of 1.0 × 108 CFU mL-1, and defined as fungi stimulation group. And the last 30 scallops were employed as the control group. Five individuals from each group were randomly sampled at 0, 3, 6, 12, 24 and 48 hours post stimulation (hps), respectively. The hemolymphs were collected from the adductor muscle using syringes and centrifuged at 800 g, 4 °C for 10 min to harvest the hemocytes for RNA preparation. Hemocytes, muscle, mantle, gill, hepatopancreas and gonad from five untreated scallops were collected to determine the mRNA transcripts distribution of CfGST genes. RNA isolation and cDNA synthesis Raw RNA was isolated from the hemocytes and other tissues of scallops using RNAiso plus reagent (9108, Takara, Japan). The first-strand synthesis was performed with M-MLV (M1705, Promega, USA) using the DNase I (RQ1, M6101, Promega, USA) treated raw RNA as template and adaptor primer-oligo(dT) as primer (Table 1). The reaction were carried out at 42 °C for 1 h, terminated by heating at 95°C for 5 min. A homopolymeric tail was added to the 5` end of the cDNA using terminal deoxynucleotidyl transferase (TdT, 2230, Takara, Japan) and dCTP (U1221, Promega, USA) and the obtained product were subsequently stored at -80 °C till use. cDNA cloning of the full-length CfGST genes The full-length cDNA sequences of CfGST genes were obtained by rapid-amplification of cDNA ends (RACE) technique based on the analysis of EST sequences (Wang et al., 2009). All the primers 304 used in this assay were listed in Table 1. All PCR amplification was performed in a TP-600 PCR Thermal Cycler (Takara, Japan). The PCR products were gel-purified and then cloned into the pMD19-T simple vector (3271, Takara, Japan), and then transformed into the competent cells Escherichia coli strain Top10 (CB104, Tiangen, China). The positive recombinants were identified through anti-Ampicillin selection and verified via PCR screening with sequencing primers M13-47 and RV-M (Table 1). Five positive clones were sequenced with a 3730XL automated sequencer (Thermo Fisher Scientific, USA). Bioinformatics analysis of sequences The search for protein sequence similarity was conducted with blast+ 2.2.18. The deduced amino acid sequences were analyzed with DNAStar Lasergene suite 7.1.0.44 using the EditSeq module. SignalP 3.0 was employed to predict the presence and location of signal peptide. The protein domain and motif features were predicted by Simple Modular Architecture Research Tool (SMART) 5.1. A phylogenic NJ tree was constructed with MEGA 5.05. To derive confidence value for the phylogeny analysis, bootstrap trials were replicated 1000 times. Real-time PCR analysis of relative mRNA expression levels The mRNA expression profiles of CfGST genes were detected via quantitative real-time PCR (qRT-PCR). All qRT-PCR reactions were performed with the SYBR premix Ex Taq (Tli RNaseH Plus, RR420, Takara, Japan) in a 7500 Real-Time Detection System (Thermo Fisher Scientific, USA). All the primers used in qRT-PCR assay were listed in Table 1. The mRNA expression leveld of CfGST genes were normalized to that of elongation factor 1 α (EF-1α) gene for each sample, according to our previous reports (Wang et al., 2016b, 2017b). The comparative CT method (2-ΔΔCt method) was used to analyze the relative mRNA expression level of GST genes (Schmittgen and Livak, 2008). All data were given as means ± S.D. (n = 5). The data were subjected to one-way analysis of variance (one-way ANOVA) followed by a multiple comparison via IBM SPSS Statistics 19.0.0.0, and the p values less than 0.05 were considered statistically significant. Table 1 Primers used in the present research Primer Sequence (5`-3`) Brief information CfGSTω-Race-F1 GGTAATGAAGTCGCTGCCTGCTGT Gene specific primer for 3` RACE CfGSTω-Race-F2 CTTTTATAAAAGTTACGCAGCAGG Gene specific primer for 3` RACE CfGSTω-Race-R1 AAAGGACAGAACCTCATGCTATACAGC Gene specific primer for 5` RACE CfGSTω-Race-R2 GAATCTTTAGAGTGTGATTTGAGA Gene specific primer for 5` RACE CfGSTσ-1-Race-F1 GCTGACCGAGTTCTTTAAGTA Gene specific primer for 3` RACE CfGSTσ-1-Race-F2 TAAGAAGAAAACTTTCGATTCAGT Gene specific primer for 3` RACE CfGSTσ-2-Race-F1 ACTTCGAAAGTGACGAGACTAAGAAGG Gene specific primer for 3` RACE CfGSTσ-2-Race-F2 CTATTCCTAAGTTTGCCAAAATCTTCACAA Gene specific primer for 3` RACE CfGSTσ-2-Race-R1 CAAGTACCGGCAGCTGACCAGTGGGCATCTTTT Gene specific primer for 5` RACE CfGSTσ-2-Race-R2 TAATGGTATCTTCTTCGAATGTTTGCCCGG Gene specific primer for 5` RACE CfGSTρ-Race-F1 CAGTTTGCTTATGGGGATAAGTTCACT Gene specific primer for 3` RACE CfGSTρ-Race-F2 GCCACTGTGGTACGATTTGGCTGCGACATA Gene specific primer for 3` RACE CfGSTζ-Race-F1 GGCTGATGCGTGTCTGGTTCCTCAGGT Gene specific primer for 3` RACE CfGSTζ-Race-F2 GAAACAGTTCCCTACCATTGCTCGTCTAAA Gene specific primer for 3` RACE CfGSTζ-Race-R1 ACCTGAGGAACCAGACACGCATCAGCCATTGTC Gene specific primer for 5` RACE CfGSTζ-Race-R2 CCATTCCATTTTACACCTCGTCCC Gene specific primer for 5` RACE CfmGST-Race-F1 GGAATGTAAACCAACGTTATCGGACCC Gene specific primer for 3` RACE CfmGST-Race-F2 GGATCCGGCAACAGCCCTGATGTACTT Gene specific primer for 3` RACE CfmGST-Race-R1 GGTCCGATAACGTTGGTTTACATTCCT Gene specific primer for 5` RACE CfmGST-Race-R2 GGTTAGCGTACACCGACTTTCGAA Gene specific primer for 5` RACE CfGSTω-qRT-F TCGTTAGAGTAACCACCAGGA Gene specific primer for real-time PCR CfGSTω-qRT-R ATGCTATACAGCCTTAGTTTCCC Gene specific primer for real-time PCR CfGSTσ-1-qRT-F AGTTTGGTTTGGCGGGAG Gene specific primer for real-time PCR CfGSTσ-1-qRT-R TGCGTACTTAAAGAACTCGGTC Gene specific primer for real-time PCR CfGSTσ-2-qRT-F CACCACCATCTATCTAAGGACAC Gene specific primer for real-time PCR CfGSTσ-2-qRT-R GTATCTTCTTCGAATGTTTGCCC Gene specific primer for real-time PCR CfGSTρ-qRT-F TACCAAGACTCCAAGCCTACTACGA Gene specific primer for real-time PCR CfGSTρ-qRT-R GTCCTTCAATTCTCCTTCCAGCCA Gene specific primer for real-time PCR CfGSTζ-qRT-F GAGATAAGGTGACAATGGCGG Gene specific primer for real-time PCR CfGSTζ-qRT-R TTTAGACGAGCAATGGTAGGGA Gene specific primer for real-time PCR CfmGST-qRT-F TAACCCGGAGGACTGTGCCA Gene specific primer for real-time PCR CfmGST-qRT-R ATGACACCTTCTGATGCGTTCCAC Gene specific primer for real-time PCR CfEF-1α-qRT-F ATCCTTCCTCCATCTCGTCCT Internal control for real-time PCR CfEF-1α-qRT-R GGCACAGTTCCAATACCTCCA Internal control for real-time PCR adaptor primer-oligo (dT) GGCCACGCGTCGACTAGTACT17VN Olido (dT) primer for cDNA synthetize adaptor primer GGCCACGCGTCGACTAGTAC Anchor primer for 3` RACE adaptor primer-oligo (dG) GGCCACGCGTCGACTAGTACG10HN Anchor primer for 5` RACE M13-47 CGCCAGGGTTTTCCCAGTCACGAC Vector primer for sequencing RV-M GAGCGGATAACAATTTCACACAGG Vector primer for sequencing 305 Fig. 1 Nucleotide and deduced amino acid sequences of six CfGSTs (A: CfGSTω, B: CfGSTσ-1, C: CfGSTσ-2, D: CfGSTρ, E: CfGSTζ, F: CfmGST). The nucleotides and amino acids are numbered along the left margin. Capital letters indicated coding sequence, small letters indicated UTRs. The GST_N/GST_C/MAPEG domains are in shade. The single typical polyadenylation signal was underlined. The asterisk and bold font indicated the stop codon Results Identification and classification of CfGSTs genes Six different CfGST genes were identified from the EST database and the full-length cDNA sequences were obtained via RACE technique. Based on the deduced protein sequences identities and phylogenetic analysis with other GSTs, the CfGSTs were classified into five classes, including two in sigma (CfGSTσ-1 and CfGSTσ-2) and one each in omega (CfGSTω), rho (CfGSTρ), zeta (CfGSTζ) and the microsomal GST isoenzyme (CfmGST), respectively. The main sequence features of these GST genes were illustrated in Figure 1 and Table 2. The cDNA sequences of these six CfGST genes were deposited to GenBank 306 Table 2 Sequence features of the six GSTs in scallop Feature CfGSTω CfGSTσ-1 CfGSTσ-2 CfGSTρ CfGSTζ CfmGST Accession Number GQ240291 EU183306 GQ240292 EU183305 GU361617 GQ403696 EST cl23ct28cn28 cl124ct131cn139 cl327ct342cn359 cl51ct57cn59 rscag0_004919 rscag0_001764 cDNA length (bp) 945 1089 776 954 696 647 5` UTR length (bp) 85 46 68 48 21 112 3` UTR length (bp) 140 425 90 231 39 79 ORF length (bp) 720 618 618 675 636 456 Polyadenylation signal sites 1 1 0 1 0 1 Deduced polypeptide length (aa) 239 205 205 224 211 151 Domain information GST_N+ GST_C GST_N+ GST_C GST_N+ GST_C GST_N+ GST_C GST_N+ GST_C MAPEG Calculated molecular mass (kDa) 27.65 23.22 23.02 25.76 24.20 16.86 Theoretical isoelectric point 7.261 8.849 5.339 6.201 6.417 8.386 Best hits by blastX (protein, taxa, E_value, Score, Identity) GSTω-2, [Haliotis discus discus], 1e-90, 279, 57% GSTσ, [Argopecten irradians], 2e-59, 199, 52% GSTσ, [Argopecten irradians], 6e-114, 334, 78% GSTρ, [Solea senegalensis], 4e-54, 185, 45% GSTζ, [Cyprinus carpio], 1e-84, 259, 59% mGST-1, [Xenopus tropicalis], 4e-45, 156. 52% database under the following accession numbers: GQ240291 (CfGSTω), EU183306 (CfGSTσ-1), GQ240292 (CfGSTσ-2), EU183305 (CfGSTρ), GU361617 (CfGSTζ) and GQ403696 (CfmGST). CfmGST consisted of an open reading frame (ORF) of 456 bp encoding a polypeptide of 151 amino acid residues with the calculated molecular mass of 16.86 kDa, while CfGSTω, CfGSTσ-1, CfGSTσ-2, CfGSTρ and CfGSTζ consisted of 239, 205, 205, 224 and 211 amino acid residues, respectively. Among these five cytosolic CfGSTs, CfGSTω had the highest calculated molecular mass (27.65 kDa) and CfGSTSσ-2 had the lowest one (23.02 kDa), which were consistent with most identified mammalian GSTs with the calculated molecular mass ranging from 23 kDa to 28 kDa as heterodimers or homodimers. The theoretical isoelectric points of these six putative CfGSTs proteins were calculated from 5.339 to 8.849. These six CfGSTs were annotated using blastx algorithm and each of them showed high identities (from 45% to 78%) with those from other vertebrate or invertebrate species. The assignment of six CfGSTs to the omega, sigma, rho, zeta and microsomal GST isoenzymes was clearly supported by the phylogenetic analysis of all these six CfGSTs along with those previous identified ones from other vertebrate and invertebrate species. These six CfGSTs were separated into five groups in the phylogenetic tree and each GST class formed their own clades (Fig. 2). Tissue distribution of CfGSTs mRNA The tissue-specific expression patterns of these six CfGSTs mRNA transcripts have been investigated in the present study. These six CfGST genes were detectable in all the examined tissues, including hemocytes, muscle, mantle, gill, hepatopancreas and gonad, although there were noticeable variations in the mRNA expression levels among different tissues. The highest mRNA expression levels of CfGSTω, CfGSTσ-1 and CfGSTζ were found in hemocytes (Fig. 3A,B,E), CfGSTρ and CfmGST were found to be most abundantly expressed in hepatopancreas (Fig. 3D,F), while the CfGSTσ-2 mRNA transcripts highest expressed in gill (Fig. 3C). Moreover, the mRNA abundance of different CfGSTs was also variable within one single tissue, CfGSTσ-1 was the most abundant GST in hemocytes, while CfGSTρ was the most scarce one (Fig. 4). Expression profiles of the CfGSTs genes after V. anguillarum stimulation The mRNA transcripts of CfGSTs exhibited differential expression profiles post V. anguillarum stimulation (Fig. 5). The relative mRNA expression levels of CfGSTω, CfGSTσ-1, CfGSTσ-2, CfGSTζ and CfmGST were all significant up-regulated within 3 or 6 hps and reached to the peak at 12 hps, which was 26.18-fold, 13.19-fold, 23.08-fold, 18.28-fold and 15.81-fold of the origin levels (p < 0.05), respectively (Figure 5A, B, C, E and F), while no significant change was observed in the mRNA expression profiles of CfGSTρ during V. anguillarum stimulation (Fig. 5D). Additionally, within the two sigma class CfGSTs, the immune responses of CfGSTσ-2 were more rapidly and intensely than those of CfGSTσ-1 (Fig. 5B,C). Expression profiles of the CfGSTs genes after M. luteus stimulation The M. luteus stimulation affected the mRNA expression profiles of these six CfGSTs differentially 307 Fig. 2 Consensus phylogenetic analysis based on the amino acid sequences of GSTs from different organisms. The evolutionary history was inferred using the Neighbor-Joining method. The bootstrap consensus tree inferred from 1000 replicates was taken to represent the evolutionary history of the taxa analyzed. All positions containing gaps and missing data were eliminated. The numbers at the forks indicated the bootstrap values. The dark circles stood for sequences from C. farreri. The sequences and their accession numbers are as follows, omega class: Chlamys farreri (ADF32018), Crassostrea gigas (XP_011429380), Danio rerio (NP_001002621), Haliotis discus discus (ABO26600), Haliotis madaka (ALU63761), Perna viridis (AGN03944); sigma class: Argopecten irradians (ANG56313), C. farreri (ACF25904), C. farreri (ADF32019); Hyriopsis cumingii (AGU68336), Pinctada fucata (JAS04242), Ruditapes philippinarum (AEW46325); rho class: C. farreri (ACF25903); Cyprinus carpio (BAS29983); Ruditapes philippinarum (AEW46331); Sebastes schlegelii (ANW83217); Siniperca chuatsi (ACI32418); Solea senegalensis (BAG12568); zeta class: Chlamys farreri (ADD82544); Cyprinus carpio (BAS29981); Oplegnathus fasciatus (ADY80028); Xenopus laevis (XP_018084636); microsomal: C. farreri (ADF45336), Gallus gallus (NP_001129022), Microtus ochrogaster (XP_005364596), Osmerus mordax (ACO10098), Sinonovacula constricta (ALC77324), Xenopus tropicalis (NP_001011245) 308 Fig. 3 Tissue distribution of six CfGSTs mRNA transcripts detected by qRT-PCR (A: CfGSTω, B: CfGSTσ-1, C: CfGSTσ-2, D: CfGSTρ, E: CfGSTζ, F: CfmGST). The mRNA expression level of CfGSTs in hemocytes, mantle, gill, hepatopancreas and gonad were normalized to that of muscle. Vertical bars represented mean ± S.D. (n = 5), and bars with different characters indicated significantly different (p < 0.05) (Fig. 6). The relative mRNA expression levels of CfGSTω and CfGSTρ were all significant up-regulated within 3 hps and reached to the peak at 6 hps, which was 27.03-fold and 28.73-fold of the origin levels (p < 0.05), respectively (Fig. 6A,D), and those of CfGSTζ were significant up-regulated at 6 hps and reached the peak at 12 hps (15.18-fold, p < 0.05, Fig. 6E). While no significant difference in CfGSTσ-1, CfGSTσ-2 and CfmGST mRNA expression was observed (Fig. 6B,C,F). 309 Fig. 4 Quantification of abundance of different CfGST isoforms in hemocytes of untreated scallops. The abundance were calculated relative to EF-1α gene and shown as (CtGSTs-CtEF-1α) -1. Vertical bars represented mean ± S.D. (n = 5), and bars with different characters indicated significant difference (p < 0.05) Expression profiles of the CfGSTs genes after P. pastoris stimulation Only two CfGSTs, CfGSTρ and CfGSTζ, were drastically induced during P. pastoris stimulation (Fig. 7). The mRNA expression level of CfGSTρ was significantly up-regulated firstly at 3 hps (4.94-fold, p < 0.05) and then reached to the peak expression level at 6 hps, which was 18.36-fold of the origin levels (p < 0.05, Fig. 7D). While the CfGSTζ were significantly induced at 6 hps (6.53-fold, p < 0.05) and reached its highest expression level at 12 hps (18.78-fold, p < 0.05, Fig. 7E). Although these six CfGSTs expressions in the normal group were slightly fluctuant throughout the experiment, no significant difference was observed (Figs 5,6,7). Discussion Glutathione S-transferases are a well characterized protein family of multifunctional isoenzymes ubiquitously identified in many aerobic organisms from bacteria to animals, and play pivotal roles in the oxidative stress responses and detoxification pathways (Hayes et al., 2005). In the present study, the full-length cDNA sequences of six different GST genes, including CfGSTω, CfGSTσ-1, CfGSTσ-2, CfGSTρ, CfGSTζ and CfmGST, were identified from C. farreri. Their sequence features, high similarities with other previous identified GTSs and the phylogenetic relationship collectively suggested that they are novel invertebrate GSTs and may have similar function with GSTs from other invertebrates. In the GST family, at least 15 different classes of GSTs have been identified and characterized in numerous aerobic organisms according to their different primary structures, enzyme properties, physiological functions and immune activities (Strange et al., 2001). According to their functional differences, GST isoforms would express differentially in various tissues. Accumulating research achievements on tissue-specific expression profiles of GSTs in aquatic organisms have revealed that GSTs are generally abundantly expressed in the mantle, gills, hepatopancreas and gonad (Li et al., 2008; Ren et al., 2009; Mu et al., 2010; Xu et al., 2010; Chen et al., 2011; Li et al., 2012; Yang et al., 2012; Zhang et al., 2012a; Duan et al., 2013; Li et al., 2013; Wang et al., 2013; Shao et al., 2017), indicating that different tissue-specific expression pattern of GSTs were associated with their differential susceptibility to antioxidant damage. In the present study, the mRNA transcripts of the six CfGST genes could be detected in all tested tissues, including hemocytes, muscle, mantle, gill, hepatopancreas and gonad, suggesting that they would be involved in many crucial physiologic or immune processes of scallop. And, there were noticeable variations in the tissue-specific expression pattern of CfGSTs. Hemocytes have been demonstrated to play irreplaceable roles in the innate immune response of invertebrates mainly through phagocytosis, which was usually companied with oxidative stress, and tubules of gill filaments were confirmed to be the hematopoietic position in Mollusks (Li et al., 2017a). In the present study, almost all the six CfGSTs were high expressed in hemocytes, and CfGSTσ-1, CfGSTσ-2, CfGSTρ and 310 Fig. 5 Temporal mRNA expression profiles of six CfGSTs detected by qRT-PCR in scallop hemocytes post V. anguillarum stimulation (A: CfGSTω, B: CfGSTσ-1, C: CfGSTσ-2, D: CfGSTρ, E: CfGSTζ, F: CfmGST). Each values was shown as mean ± S.D. (n = 5), and bars with different characters indicated significant difference (p < 0.05) CfmGST were found to be most abundantly expressed in gills, indicating that these GSTs would act as efficient immune effectors in scallop. While CfGSTω, CfGSTσ-1, CfGSTσ-2, CfGSTρ and CfmGST were highly expressed in hepatopancreas, which was consistent with the opinion that hepatopancreas was the major organ for detoxification of xenobiotics in marine invertebrates (Doi et al., 2004). Similar phenome has been observed in M. galloprovincialis, in which tissue distribution study revealed that MgGSTa, MgGSTS2, MgGSTS3 transcripts were highly expressed in hemocytes, while MgGSTS1 mRNA was most abundantly expressed in hepatopancreas. Additionally, previous reports have demonstrated that some low constitutively expressed GSTs might 311 Fig. 6 Temporal mRNA expression profiles of six CfGSTs detected by qRT-PCR in scallop hemocytes post M. luteus stimulation (A: CfGSTω, B: CfGSTσ-1, C: CfGSTσ-2, D: CfGSTρ, E: CfGSTζ, F: CfmGST). Each values was shown as mean ± S.D. (n = 5), and bars with different characters indicated significant difference (p < 0.05) performed a crucial role in the detoxification process, while high constitutively expressed GSTs might involve in protecting the cell against endogenous oxidative stress (Zhang et al., 2012a). It could be speculated that CfGSTσ-1 perhaps played a pivotal role in the detoxification process. So, we hypothesized based on these results that each of the GST classes with different tissues distributions might be involved in some specific physiological functions in the basal metabolism of scallop. Mollusks highly rely on innate immunity, and hemocytes-mediated phagocytosis is considered as a main arm of innate immune defense strategies (Song et al., 2015; Wang et al., 2016a). Infection of microbes could induce hemocytes-mediated phagocytosis accompanied with respiratory burst and 312 Fig. 7 Temporal mRNA expression profiles of six CfGSTs detected by qRT-PCR in scallop hemocytes post P. pastoris stimulation (A: CfGSTω, B: CfGSTσ-1, C: CfGSTσ-2, D: CfGSTρ, E: CfGSTζ, F: CfmGST). Each values was shown as mean ± S.D. (n = 5), and bars with different characters indicated significant difference (p < 0.05) followed by mass production of ROS in various organisms ranging from invertebrate to vertebrate (Halliwell, 2006; Benedetti et al., 2015). Compared with vertebrate GSTs, rare information about the mRNA expression profiles of different classes of GSTs is available in mollusks, considering their indispensable roles in antioxidant system (Song et al., 2015). In the present study, almost all the identified CfGSTs were high expressed in hemocytes. So this tissue was selected as candidate for investigating the temporal mRNA expression profiles of CfGSTs post various microbe stimulations. Among of the previous identified GSTs from aquatic species, some could be induced by foreign stimulus or invading microbes and be involved in innate immunity (Ren et al., 2009; Mu et al., 2010; Chen et al., 2011; Li et al., 2012; Yang et al., 2012; Duan et al., 2013; Wang et al., 2013; Shao et al., 2017). In 313 the present study, it was observed that the mRNA transcripts of these six CfGST genes all drastically increased after one or two kinds of microbe stimulation. For examples, CfGSTσ-1, CfGSTσ-2 and CfmGST could only respond to the stimulation of V. anguillarum, while both CfGSTω and CfGSTρ could be significantly induced by two kinds of microbe stimulation, which indicated that they could be involved in the innate immune response of scallop against different invading pathogens. Interestingly, CfGSTζ could respond to all the three kinds of microbe stimulation with similar expression profiles, indicating CfGSTζ was involved in the innate immune responses to more microbes and its modulation to different invading microbes might share the similar mechanism. Similar phenome has been observed in V. philippinarum, in which all the VpGSTs showed differential response profiles depending on the concentrations of various toxicants and exposure times. Additionally, CfGSTσ-2 with low basal mRNA expression level responded to invading V. anguillarum more rapidly and intensely than CfGSTσ-1, similarly, the basal mRNA expression level of EscytMnSOD in hemocytes was higher than that of EsmtMnSOD by approximately two times, which indicated that EscytMnSOD might play a more routine role in the physiological activity of crabs (Wang et al., 2015). These differences in their mRNA expression profile indicated that CfGSTσ-1 might play a routine role in the detoxification process, while CfGSTσ-2 would mainly be involved in the response to invading pathogens. In summary, the full-length cDNA sequences of six GST genes, including CfGSTω, CfGSTσ-1, CfGSTσ-2, CfGSTρ, CfGSTζ and CfmGST, were obtained from C. farreri. All the CfGSTs were constitutively expressed in all the tested tissues and they were drastically but differentially induced post different microbe stimulation. Based on these obtained results, it could be hypothesized that CfGSTs were involved in the defense responses of C. farreri against bacterial infection. Additionally, the difference in their temporal mRNA expression patterns against various microbe stimulation indicated that CfGSTs would play pivotal but different roles in the innate immune responses of scallop. Acknowledgement This research was supported by the National Natural Science Foundation of China (31530069). 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