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Glutathione S-transferase genes in scallops and their diverse expression patterns after exposure to PST-producing dinofagellates

  • Corresponding author: Xiaoli Hu, hxl707@ouc.edu.cn
  • Received Date: 2020-03-15
    Accepted Date: 2020-05-26
    Published online: 2020-06-22
  • Edited by Xin Yu.
  • Jiarun Lou and Jie Cheng have contributed equally to this work.
  • Electronic supplementary material The online version of this article (https://doi.org/10.1007/s42995-020-00050-2) contains supplementary material, which is available to authorized users.
  • The glutathione S-transferases (GSTs) are a superfamily of enzymes that function in cellular protection against toxic substances and oxidative stress. Bivalves could accumulate high concentration of paralytic shellfish toxins (PSTs) from harmful algae. To understand the possible involvement of GSTs in protecting bivalves during PST accumulation and metabolism,the GST genes were systemically analyzed in two cultured scallops,Azumapecten farreri and Mizuhopecten yessoensis,which were reported for PST deposition during harmful algae bloom. A total of 35 and 37 GSTs were identified in A. farreri (AfGSTs) and M. yessoensis (MyGSTs) genome,respectively,and the expansion of the sigma class from the cytosolic subfamily was observed. In both scallop species,sigma class GSTs showed higher expression than other members. The high GSTs expression was detected mainly during/after larvae stages and in the two most toxic organs,hepatopancreas and kidney. After ingesting PST-producing dinoflagellates,all the regulated AfGSTs in the hepatopancreas were from the sigma class,but with opposite regulation pattern between Alexandrium catenella and A. minutum exposure. In scallop kidneys,where PSTs transformed into higher toxicity,more AfGSTs were regulated than in the hepatopancreas,and most of them were from the sigma class,with similar regulation pattern between A. catenella and A. minutum exposure. In M. yessoensis exposed to A. catenella,MyGST-σ2 was the only up-regulated MyGST in both hepatopancreas and kidney. Our results suggested the possible diverse function of scallop GSTs and the importance of sigma class in the defense against PSTs,which would contribute to the adaptive evolution of scallops in marine environments.
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Glutathione S-transferase genes in scallops and their diverse expression patterns after exposure to PST-producing dinofagellates

    Corresponding author: Xiaoli Hu, hxl707@ouc.edu.cn
  • 1. Key Laboratory of Marine Genetics and Breeding (Ocean University of China), Ministry of Education, Qingdao 266003, China
  • 2. Laboratory for Marine Fisheries Science and Food Production Processes, Pilot National Laboratory for Marine Science and Technology (Qingdao), Qingdao 266237, China

Abstract: The glutathione S-transferases (GSTs) are a superfamily of enzymes that function in cellular protection against toxic substances and oxidative stress. Bivalves could accumulate high concentration of paralytic shellfish toxins (PSTs) from harmful algae. To understand the possible involvement of GSTs in protecting bivalves during PST accumulation and metabolism,the GST genes were systemically analyzed in two cultured scallops,Azumapecten farreri and Mizuhopecten yessoensis,which were reported for PST deposition during harmful algae bloom. A total of 35 and 37 GSTs were identified in A. farreri (AfGSTs) and M. yessoensis (MyGSTs) genome,respectively,and the expansion of the sigma class from the cytosolic subfamily was observed. In both scallop species,sigma class GSTs showed higher expression than other members. The high GSTs expression was detected mainly during/after larvae stages and in the two most toxic organs,hepatopancreas and kidney. After ingesting PST-producing dinoflagellates,all the regulated AfGSTs in the hepatopancreas were from the sigma class,but with opposite regulation pattern between Alexandrium catenella and A. minutum exposure. In scallop kidneys,where PSTs transformed into higher toxicity,more AfGSTs were regulated than in the hepatopancreas,and most of them were from the sigma class,with similar regulation pattern between A. catenella and A. minutum exposure. In M. yessoensis exposed to A. catenella,MyGST-σ2 was the only up-regulated MyGST in both hepatopancreas and kidney. Our results suggested the possible diverse function of scallop GSTs and the importance of sigma class in the defense against PSTs,which would contribute to the adaptive evolution of scallops in marine environments.

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Introduction
  • The glutathione S-transferases (GSTs) are a superfamily of enzymes that play a critical role in cellular protection against toxic foreign substances and oxidative stress in almost all life forms (Hayes and Pulford 1995; Mannervik et al. 2005). As the major phase Ⅱ detoxification enzymes, GSTs catalyze the conjugation of reduced glutathione (GSH) with the electrophilic centers of toxic compounds, making them ready for excretion by phase Ⅲ enzymes (Knight et al. 2007; Rodrigues et al. 2019; Sheehan et al. 2001). In addition, GSTs could serve as antioxidants to protect the cells from harmful electrophiles produced during oxidative damage to membranes (Hayes and McLellan 1999). Based on their cellular localization, the GSTs are classified into three subfamilies, namely cytosolic, mitochondrial, and microsomal (also named MAPEG, membrane-associated proteins in eicosanoid and glutathione metabolism) transferases (Hayes and Strange 2000). The most abundant and diverse group of GSTs identified in humans are the cytosolic subfamily enzymes, which are further divided into alpha, mu, pi, theta, sigma, zeta, and omega by their substrate specificity, immunoreactivity properties, and protein sequence homologies (Mannervik et al. 2005). Additional classes are specific to non-mammalian organisms including phi (plants), tau (plants), rho (fish and bivalves), delta (insects), epsilon (insects), and beta (bacteria) GSTs (Sheehan et al. 2001; Soranzo et al. 2004).

    Marine bivalves which feed mainly on microalgae could accumulate toxins during harmful algal blooms (HABs), posing a health hazard to humans and other animals through food chains (Liang et al. 2004). The paralytic shellfish toxins (PSTs) produced by dinoflagellates, especially members of the genus Alexandrum, are the most potent known natural toxins (Cho et al. 2019). Although bivalves could tolerate high concentrations of PSTs due to possessing toxin-resistant amino acids in voltage-gated sodium channel protein, the PST-binding target, ingesting toxic dinoflagellates led to changes of bivalve behavior and physiology with respect to valve closing ability, filtration processes, reproduction, cardiac activity, and immunological status (Gainey 1998; Gainey and Shumway 1988; Lassus et al. 1999; Li et al. 2017; Shumway 1990; Shumway and Gainey 1992; Tran et al. 2010). To counteract these biological effects, detoxification of the harmful substances from the ingested dinoflagellates, and reducing the oxidant stress generated from toxin exposure may be employed in bivalves (Flores et al. 2012; Kim et al. 1999). As the enzyme functions in both detoxification and antioxidation, GST activity was stimulated in oyster, scallop, and clam after PST exposure (Cao et al. 2018; Choi et al. 2006) indicating their involvement in protecting bivalves from the harmful effect of PST accumulation. Meanwhile, single gene expression analysis revealed the transcriptional regulation of GST in oysters after exposure to PST-producing dinoflagellates (Fabioux et al. 2015; Garcia-Lagunas et al. 2013). As a superfamily containing tens of GST genes from different classes, GST activity is largely determined by the expression of all the encoding genes in the cell or certain organs. Therefore, systematically examining the expression regulation of GST genes in bivalves during PSTs or PST-producing algae exposure could improve our understanding of the detoxification and antioxidation mechanism mediated by GSTs of bivalve against PST poisoning. However, research on the genome-wide examination of GST genes in bivalves is still very limited.

    Among bivalve species, scallops exhibited higher PST accumulation capacity and longer retention time than other members (Tan and Ransangan 2015). GST activity variation has been reported in scallops exposed to PSTs (Cao et al. 2018) implying the possible contribution of GST gene regulation to detoxification and PST tolerance of scallop. In the present study, we performed genome-wide identification of GST genes in two scallops, Azumapecten farreri and Mizuhopecten yessoensis, which are both important aquaculture bivalves in China, and have been reported for PST contamination during farming (Chen et al. 2013; Liu et al. 2017). After examining the transcriptomic changes of these genes in the most toxic organs of scallops exposed to PST-producing dinoflagellates, A. catenella and A. minutum, we revealed the variable regulation of scallop GSTs between organs, between scallop species, and between different toxic algae species. Our results suggested the diverse function of scallop GSTs in the defense against the harmful effect of toxic algae, and the involvement of these genes in the adaptive response to the diet contaminated with PST-producing algae.

Results and discussion

    GST genes in scallops A. farreri and M. yessoensis

  • Through genome-wide screening, a total of 35 putative A. farreri GST (AfGST) genes and 37 M. yessoensis GST (MyGST) genes were identified, which could encode proteins containing the GST_N domain (Pfam: PF02798) and GST_C domain (Pfam:PF00043) (Table 1 and Supplementary Fig. S1). Information for these GST genes, including their genome location, encoding protein length and GST domain, SwissProt annotation, and exon number, was summarized in Supplementary Table S1. The ORF (open reading frame) of AfGSTs and MyGSTs varied from 435 to 1851 bp and 312 to 1851 bp in length, encoding 144 to 616 and 103 to 616 amino acids (aa), respectively (Supplementary Table S1). To further investigate the GST structures, conserved domains were annotated in scallop GSTs. All the scallop GSTs composed of the GST domains, and most of them were ranging from 150 to 320 aa in both AfGSTs (33 out of 35) and MyGSTs (33 out of 37) (Supplementary Fig. S1). There was only one longer AfGST-ζ3 and MyGST-ζ3 consisting of 616 aa with two conserved GSTA and GST_C domains, respectively. Also, there was one shorter AfGST and three shorter MyGSTs ranging from 103-138 aa with GST_N and MAPEG domains detected. According to the SwissProt annotation, 29 AfGSTs belonged to the cytosolic subfamily, which were further divided into seven classes, including sigma (14 copies), theta (five copies), omega (four copies), zeta (three copies), pi (one copy), rho (one copy), and mu (one copy), and other one and five AfGSTs were from the mitochondrial and microsomal subfamilies, respectively (Table 1). In M. yessoensis, the 37 MyGSTs were categorized into three subfamilies, including the mitochondrial (1), microsomal (4), and cytosolic (32) GSTs. The 32 cytosolic MyGSTs were further classified as 16 in class sigma, five in theta, three in omega, three in rho, three in zeta, one in pi, and one in mu (Table 1). These classifications were further supported by the phylogenetic analysis (Fig. 1). Compared with vertebrates, mollusks and C. elegans had more cytosolic GST members, mainly due to the abundance of sigma class, which, however, was absent in humans, frog, and zebrafish. Over ten sigma GSTs were found in mollusks, including scallops, oysters, and owl limpet, which was consistent with the previous finding that sigma GSTs were expanded in invertebrate genomes (Flanagan and Smythe 2011). However, the alpha class was only absent in scallops, which was previously regarded as a GST class specific to mammals (Frova 2006), but its presence in oysters (Zou et al. 2015) implied that alpha GST gene might have been lost in the ancestor of scallops.

    GST subfamily Cytosolic Mitochondrial Microsomal
    Alpha Mu Omega Pi Rho Sigma Theta Zeta
    H. sapiens 5 5 2 1 0 0 2 1 1 3
    X. tropicalis 3 1 2 3 0 0 2 1 2 3
    D. rerio 3 3 2 2 1 0 2 1 4 8
    C. elegans 0 0 5 6 0 29 0 1 2 0
    L. gigantea 1 1 4 1 1 11 2 2 1 8
    C. gigas 2 4 5 1 4 10 7 3 1 6
    A. farreri 0 1 4 1 1 14 5 3 1 5
    M. yessoensis 0 1 3 1 3 16 5 3 1 4

    Table 1.  Comparison of GST gene numbers between mollusks and other selected species

    Figure 1.  Phylogenetic analysis of GST amino acid sequences. The ML (maximum-likelihood) tree was constructed with GST sequences from mollusk species and other selected model organisms, with tested LG + G model and bootstrapping of 1000 pseudo-replicates. Values less than 70% were not shown. The AfGSTs and MyGSTs were highlighted in red and blue, respectively (Hs: Homo sapiens; Xt: Xenopus tropicalis; Dr: Danio rerio; Ce: Caenorhabditis elegans; Lg: Lottia gigantean; Cg: Crassostrea gigas; Af: Azumapecten farreri; My: Mizuhopecten yessoensis)

  • Spatiotemporal expression of GSTs in scallops

  • The expression of GSTs in scallop embryos/larvae and adult organs/tissues was analyzed using the transcriptome dataset (Li et al. 2017; Wang et al. 2017). As shown in Fig. 2, during the development of scallops, the sigma class genes, such as AfGST-σ5, -σ7, and -σ10 in A. farreri, and MyGST-σ2, -σ4, -σ5, -σ8, -σ10, and -σ11 in M. yessoensis exhibited relatively higher expression level than other GSTs suggesting the importance of sigma GSTs in scallop development. The expansion of sigma GSTs in scallops might be related to the protection of scallop embryos/larvae from the environmental or internal oxidative stress. In addition to sigma class members, AfGST-µ1, AfGST-ω2, and AfGST-microsomal2 in A. farreri, and MyGST-μ1, MyGST-ω1, -ω2, MyGST-π1, MyGST-ρ3, and MyGST-microsomal1b in M. yessoensis showed moderate expression. Along the entire developmental process, high expression of the GST genes was mostly present at the larval stages, especially after umbo larva, and in the following juvenile stage, implying the involvement of these GSTs in antioxidation or detoxification during scallop metamorphosis and post-larval development.

    Figure 2.  Expression pattern of scallop GSTs during the development of Azumapecten farreri (a) and Mizuhopecten yessoensis (b). The number in each box represents the lg(RPKM) value at the corresponding developmental stage

    In adult scallop tissues, higher expression of GSTs was detected mostly in hepatopancreas and kidney. For example, AfGST-σ5, -σ7 and -σ8 in A. farreri, MyGST-σ2, -σ4, -σ5, -σ8, and -ω2 in M. yessoensis (Fig. 3), most of which were sigma class members, were all highly expressed in hepatopancreas or kidney. Abundant GST transcripts in kidney and liver (or hepatopancreas) were found also in many other organisms, such as rats (Daggett et al. 1998), plaice (Pleuronectes platessa) (Leaver et al. 1993), goldfish (Carassius auratus) (Hao et al. 2008), and bivalves, such as Lamellidens marginalis (Chetty 1995) and Mytilus galloprovincialis (Fitzpatrick et al. 1995). Kidney and liver (or hepatopancreas) are the organs for eliminating toxic substances; for example, liver and kidney function in detoxifying chemicals (Wang et al. 2011) and in removing wastes (Chow et al. 2010), respectively. The scallop GSTs with abundant mRNAs in hepatopancreas and kidney, especially the sigma class members, may well participate in these processes. In addition to hepatopancreas and kidney, MyGST-σ5 was highly expressed in mantle and striated muscle, indicating its possible involvement in the protection of these organs/tissues.

    Figure 3.  Expression pattern of scallop GSTs in adult tissues of Azumapecten farreri (a) and Mizuhopecten yessoensis (b). The number in each box represents the lg(RPKM) value at the corresponding tissue

  • Expression regulation of scallop GSTs after toxic dinoflagellates exposure

  • Dinoflagellates of the genus Alexandrium are the major PST producers in scallop farming, but the toxicities of Alexandrium vary among species and strains due to the different PST analogs which they produce. For example, the A. minutum (AM-1 strain) and A. catenella (ACDH strain) used in this study mainly produce PST analogs of gonyautoxins (GTXs, mainly GTX1-4) and N-sulfocarbamoyl toxins (C1/2), respectively (Anderson 1990; Chang et al. 1997; Hu et al. 2019; Li et al. 2017). For scallops exposed to PST-producing algae, the toxicity varied among organs/tissues. Our previous study showed that scallop hepatopancreas and kidney could accumulate much higher concentration of PSTs than other organs, with hepatopancreas mainly absorbing the incoming PSTs from dinoflagellates directly, whereas kidneys transforming the absorbed toxins into more potent analogs, saxitoxin (STX) and neosaxitoxin (NeoSTX) (Li et al. 2017; Shimizu and Yoshioka 1981). Systematically analyzing the expression profile of GSTs in hepatopancreas and kidney of scallops exposed to different Alexandrium species could deepen our understanding of the defense mechanism against toxic algae in scallops. Therefore, in this study, 102 RNA-Seq libraries (three replicates for each test time point) from scallop hepatopancreas and kidneys of challenged and control groups were constructed, and sequenced to analyze the expression regulation of scallop GSTs after toxic dinoflagellates exposure (Supplementary Table S2).

    In the hepatopancreas of A. farreri, the same five AfGSTs were differentially expressed (DE) after exposure to both A. minutum (5 with P < 0.05 and 3 with FDR < 0.05) and A. catenella (5 with both P < 0.05 and FDR < 0.05). These five genes were AfGST-σ2, -σ4, -σ10, -σ11, and -σ14, all belonging to the sigma class, but with opposite regulation patterns between the two algae exposure, except the up-regulated AfGST-σ14 (Fig. 4a and Supplementary Table S3). After exposure to A. minutum, the four DE AfGSTs were all down-regulated, with AfGST-σ4 presenting only at 15 days after exposure, whereas AfGST-σ2, -σ10, and -σ11 decreased both acutely (1-3 days) and chronically (5-15 days), with AfGST-σ10 being down-regulated at all five sampling time points. In contrast, when being exposed to A. catenella, the four AfGST genes were all significantly up-regulated, and AfGST-σ2, -σ10, and -σ11 were induced both acutely and chronically, while AfGST-σ4 was regulated chronically. Scallop hepatopancreas is the organ absorbing PSTs from the ingested algae (Li et al. 2017). These results indicated that the five AfGST-σ genes might be the key GSTs for the protection of A. farreri hepatopancreas from the harmful effects of PST-producing dinoflagellates. It was reported that sigma class GSTs showed sensitive regulation response against environmental stress inducers (Rhee et al. 2013; Won et al. 2011). The same GST expression in different individuals may be affected by various types of stimulation. In marine invertebrates, the sigma class members were found highly expressed in response to BDE-47 exposure in Venerupis philippinarum and Mytilus galloprovincialis (Li et al. 2015). The GST-mitochondrial gene in Haliotis discus discus was up-regulated after exposure to bacterial lipopolysaccharide, but down-regulated after exposure to Vibrio parahaemolyticus (Sandamalika et al. 2018). Moreover, genes involved in oxidative metabolism such as Catalase and Glutathione peroxidase were also down-regulated after A. minutum exposure in C. gigas (Mat et al. 2013). In this study, according to the PST composition of the two Alexandrium species, mainly producing PST analogs GTX1-4 and C1/2, respectively, therefore, the opposite regulating patterns of the four sigma AfGSTs between exposure to A. minutum and A. catenella may well be related to the different types of PSTs absorbed from the two algae.

    Figure 4.  Temporal expression of scallop GSTs in hepatopancreas and kidney of Azumapecten farreri after Alexandrium minutum and Alexandrium catenella exposure (a), and of Mizuhopecten yessoensis after Alexandrium catenella exposure (b). The heatmap was drawn based on log2(fold change) values (Supplementary Table S3). Exposure time (1, 3, 5, 10, or 15 days) was displayed below the heatmap. Significant regulation with |log2FC| > 2 and P < 0.05 was indicated with "*", and very significant regulation with |log2FC| > 2 and FDR < 0.05 was indicated with "**"

    In A. farreri kidneys, where the ingested PSTs being transformed to higher toxic analogs, more AfGSTs were significantly regulated after exposure to A. minutum (11 with P < 0.05 and 8 with FDR < 0.05) and A. catenella (12 with P < 0.05 and 6 with FDR < 0.05) (Fig. 4a). After exposure to A. minutum, five GSTs were up-regulated, including AfGST-σ5 and -σ14 induced at 1 to 10 days, and AfGST-σ11, -θ3, and -θ5 induced at 15 days post-challenge. Both AfGST-σ11 and -θ5 were also down-regulated at 10 days. The other six AfGSTs were down-regulated, including AfGST-σ7, -σ12, and -θ2 regulated acutely, and AfGST-ρ1, -σ10, -σ12, and -ζ3 regulated at 10 days after exposure. It was reported that the over-expression of GST in Aedes aegypti may increase the capacity of toxin resistance (Helvecio et al. 2019). The majority of regulated AfGSTs in kidney were from the sigma class, suggesting the sigma class members being also the major GSTs involved in detoxification of PSTs in scallop kidney. Meanwhile, the up-regulation of AfGST-θ3 and -θ5 might also contribute to the protection of A. farreri kidney. In the kidney of scallops ingested A. catenella, a similar set of DE AfGSTs was detected as those with A. minutum challenge, except that AfGST-σ4 and -σ8 were regulated only for A. catenella challenge, whereas AfGST-σ11 only for A. minutum challenge (Fig. 4a). Moreover, between the challenges of the two algae, the AfGSTs showing similar up- and down-regulation patterns, whereas their regulated time points were different. For example, after A. minutum exposure, the majority of DE AfGSTs (8) were present at 10 days, and only three genes were regulated at one day. While after ingesting A. catenella, six AfGSTs were regulated at one day and only two genes were regulated at 10 days. In scallop kidney, the ingested PST analogs, GTXs from A. minutum and C1/C2 from A. catenella, could be transformed into more toxic analogs STX and NeoSTX, respectively, whereas both transformations included reductive elimination of o-sulfate groups (Li et al. 2017; Shimizu and Yoshioka 1981). The diverse expression profiles of AfGSTs indicated that, with the challenges of different PST analogs, the AfGSTs functioning in PST detoxification and antioxidation during PSTs transformation were similar in scallop kidneys, but the gene stimulation or inhibition procedure/speed might vary.

    In addition, regarding the highly expressed GSTs in normal scallop hepatopancreas and kidney (Fig. 3), AfGST-σ5, -σ7, and -σ8 were only significantly regulated in kidney but not in hepatopancreas after PST-producing algae exposure, and they were constantly keeping high expression level during the challenge experiments (Supplementary Table S2). The similar pattern has also been reported in genes coding antioxidant enzymes, such as the expression of SuperOxide Dismutase (SOD), CATalase (CAT), and GST-π in mussels remaining stable after short-term exposure to Diarrhetic Shellfish Poisoning (DSP) (He et al. 2019; Prego-Faraldo et al. 2017). Moreover, the expression of SOD1 was high in A. farreri kidney, but its expression was not regulated after A. minutum and A. catenella exposure (Lian et al. 2019). The results here suggested that these highly expressed GSTs probably need to keep playing vital roles in detoxification, such as AfGST-σ5 and -σ7 in hepatopancreas and MyGST-σ5 in both kidney and hepatopancreas. On the other hand, AfGST-σ14 was hardly expressed in normal scallop tissues (Fig. 3), but presented a significant up-regulation in both kidney and hepatopancreas after PST-producing algae exposure. The up-regulation of AfGST-σ14 was mostly from 5 to 10 days, except in kidney after exposed to A. minutum with both acute (one day) and chronical (5-10 day) regulation, which may imply the reaction of AfGST-σ14 to higher toxin stimulation.

    To further understand the different regulation of GSTs between scallop species, we examined MyGSTs transcripts in both hepatopancreas and kidney of M. yessoensis ingesting A. catenella (Fig. 4b). A total of five (2 with FDR < 0.05) and three (2 with FDR < 0.05) regulated sigma GSTs were detected in M. yessoensis hepatopancreas and kidney, respectively, which were less than those identified in A. farreri. For both organs, MyGST-σ2 was the only up-regulated member implying the importance of this sigma class GST in the protection of M. yessoensis from the challenge of PST-producing algae. MyGST-σ1 and -θ5 were both down-regulated in the two organs, whereas MyGST-ω2 and -σ6 were down-regulated only in the hepatopancreas. The species- and organ-specific regulation of GSTs imply the diverse function of scallop GSTs in the defense against the harmful effects of PST accumulation and metabolism, which might contribute to the adaptive evolution of scallops.

Materials and methods

    Genome-wide identification and characterization of GSTs in A. farreri and M. yessoensis

  • Candidate GST genes were identified from the genome (Li et al. 2017; Wang et al. 2017) and transcriptome (Hou et al. 2011; Wang et al. 2013) sequences of A. farreri and M. yessoensis, and then, the coding sequences were translated into amino acid (aa) sequences by ORF finder (https://www.ncbi.nlm.nih.gov/orffinder/). The predicted A. farreri and M. yessoensis GST ORFs were searched with BLASTP setting of e value 1E-05 against public databases including KEGG (https://www.kegg.jp/), UniProt (https://www.uniprot.org/), and NCBI non-redundant (Nr) protein sequence database. The aa sequences with significant blast hit to known GST proteins were obtained as candidate GSTs, and the GST domains were checked by SMART tools (https://smart.embl-heidelberg.de/) and Conserved Domains Database (CDD, https://www.ncbi.nlm.nih.gov/cdd) in these sequences. Only GST protein sequences longer than 100 aa were used for the following analyses.

  • Copy number and phylogenetic analysis of GSTs in A. farreri and M. yessoensis

  • A total of 224 full-length GST protein sequences from A. farreri, M. yessoensis, and other selected organisms, including human (Homo sapiens), frog (Xenopus tropicalis), zebrafish (Danio rerio), nematode (Caenorhabditis elegans), Pacific oyster (Crassostrea gigas), and owl limpet (Lottia gigantean), were employed. The GST protein sequences of the latter six species were obtained from public databases including UniProt (https://www.uniprot.org/), Ensembl (https://asia.ensembl.org/index.html), and NCBI (https://www.ncbi.nlm.nih.gov/) (Supplementary Table S4). Multiple alignment of GST protein sequences was performed using MEGA X and GeneDoc (https://www.softpedia.com/get/Science-CAD/GeneDoc.shtml). The phylogenetic tree was constructed using MEGA X with the maximum-likelihood (ML) method and Le_Gascuel_2008 model (Le and Gascuel 2008). The tree with the highest log likelihood (- 24, 185.01) was shown. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using a JTT model, and then selecting the topology with superior log likelihood value. A discrete Gamma distribution (LG + G) was used to model evolutionary rate differences among sites [7 categories (+ G, parameter = 3.0549)]. All positions with less than 95% site coverage were eliminated, i.e., fewer than 5% alignment gaps, missing data, and ambiguous bases were allowed at any position (partial deletion option). The robustness of the resulting phylogenies was tested by a reanalysis of 1000 bootstrap replicates.

  • Expression of scallop GSTs during developmental stages and in adult organs/tissues

  • Azumapecten farreri and M. yessoensis were collected from Xunshan Group Co., Ltd. (Rongcheng, Shandong Province, China) and Zhangzidao Group Co., Ltd. (Dalian, Liaoning Province, China), respectively. Samples of developmental stages include zygotes, 2-8 cells, blastula, gastrula, trochophore, D-stage larva, early umbo larva, umbo larva, late umbo larva, eyespot larva, and juvenile scallop. The adult organs/tissues include mantle, gill, kidney, hepatopancreas, and striated muscle. The expression profiles of AfGSTs and MyGSTs during developmental stages and in adult organs/tissues were normalized and represented in the form of RPKM (Reads Per Kilobase of exon model per Million mapped reads) using the RNA-Seq data from previous studies (Li et al. 2017; Wang et al. 2017).

  • Expression regulation of scallop GSTs after exposure to PST-producing dinoflagellates

  • Scallops were challenged previously by PST-producing dinoflagellate, with A. farreri being fed with A. minutum (AM-1 strain) and A. catenella (ACDH strain) (Li et al. 2017), respectively. M. yessoensis was fed only A. catenella (ACDH strain). Briefly, A. farreri and M. yessoensis were acclimated in filtered and aerated seawater at 12-13 ℃ for three weeks, and then maintained separately with aeration during the exposure experiments. The two Alexandrium dinoflagellates were cultivated independently (Navarro et al. 2006), and harvested at the late exponential growth phase (Garcia-Lagunas et al. 2013) through centrifugation of 2500 g/10 min. Thereafter, 3 L of dinoflagellate cells were fed to each scallop once a day with a density of 2500 cells/ml.

    Scallops were collected at 0 (control), 1, 3, 5, 10, and 15 days after exposure with three individuals at each time point. For each individual, RNA-Seq libraries were constructed for hepatopancreas and kidney, respectively, and sequenced on the Illumina HiSeq 2500 platform with the strategy of paired-end 250 bp. The expression data for all the GST genes were normalized and represented in the form of RPKM (Supplementary Table S2). Significant differences between the exposed and control groups were determined using the edgeR package with cut-off of |log2FC| > 2 and P value < 0.05, and the very significant difference with cut-off of |log2FC| > 2 and corrected FDR value < 0.05. The heat maps were drawn using pheatmap package with the log2FC values (Supplementary Table S3).

Acknowledgements
  • This work was funded by the National Key R&D Program of China (2019YFC1605704), the National Natural Science Foundation of China (31630081 and 31802292), and Taishan Industry Leading Talent Project.

Author contributions
  • XH and ZB designed the study, JL, XX, and ML carried out the experiments. JL, JC, XX, XZ, and TL analyzed the data. XH, JC, and JL wrote the manuscript. All the authors have read and approved the fnal manuscript.

Data availability
  • The RNA-Seq data used in this study were deposited in NCBI Sequence Read Archive, with the accession numbers as follows: Developmental stages of A. farreri: SRX2444869 (Juveniles), data for other developmental stages are still unpublished; Tissues of A. farreri: SRX2444844, SRX2444846-SRX2444848, SRX2444850, SRX2444856, SRX2444858-SRX2444860, SRX2444863, SRX2444871, SRX2444873-SRX2444876; A. farreri exposed to A. minutum: SRX2445405-SRX2445440; Data for A. farreri exposed to A. catenella are still unpublished; Developmental stages of M. yessoensis: SRX1026991, SRX2238787-SRX2238792; Tissues of M. yessoensis: SRX2238797-SRX2238799, SRX2238801-SRX2238804, SRX2238807, SRX2250257, SRX2250258, SRX2251047, SRX2251049, and SRX2279546; The RNA-seq data of kidney and hepatopancreas after exposed to A. catenella are still unpublished. Some unpublished data are included in our on-going project and will be released soon, therefore, if any readers are interested in these data, they are welcome to contact with the corresponding author for further query.

Compliance with ethical standards

    Conflict of interest

  • The authors declare that they have no confict of interest.

  • Animal and human rights statement

  • This study was conducted in accordance with the Institutional Animal Care and Use Committee of Ocean University of China, and it does not contain any studies with human participants.

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