Identification and Characterisation of Putative Glutathione S-Transferase Genes from Daktulosphaira vitifoliae (Hemiptera: Phylloxeridae)

Identification and Characterisation of Putative Glutathione S-Transferase Genes from... Abstract Glutathione S-transferases (GSTs) in insects are widely known for their role in the detoxification of both endogenous and xenobiotic compounds. Grape phylloxera, Daktulosphaira vitifoliae (Fitch) (Hemiptera: Phylloxeridae) is a serious grape pest, which causes great economic damage in vineyards, and has currently spread throughout the world. In this study, eight putative GST genes were identified by analyzing the transcriptomes of grape phylloxera. Phylogenetic analyses showed that there are seven cytosolic DviGSTs and one microsomal DviGST. These cytosolic DviGSTs are clustered into four different classes including two delta genes, one omega gene, one theta gene, and three sigma genes. Among candidate cytosolic DviGSTs, a conserved N-terminal domain and a less conserved C-terminal domain were identified. For the candidate microsomal DviGST, three transmembrane regions were predicted. Multiple sequence alignment analysis of the candidate microsomal DviGST was conducted with other insect microsomal GSTs and the result showed that there is a conserved sequence pattern. Semiquantitative polymerase chain reaction was used to examine the tissue expression of these transcripts, and the results revealed that DviGSTs were ubiquitously expressed in the head and the body, but DviGSTd1, DviGSTd2, DviGSTs2, and DviGSTs3 were abundantly expressed in the head and body. This is the first study of the molecular characteristics of GST genes in grape phylloxera. Our results will provide a molecular basis for future studies of the detoxification mechanisms in grape phylloxera. Glutathione S-transferases (GSTs) are multifunctional enzymes widely distributed in both prokaryotic and eukaryotic cells (Qin et al. 2013). They play important roles not only in the detoxification of endogenous and xenobiotic compounds but also in intracellular transport, biosynthesis of hormones, and protection against oxidative stress (Enayati et al. 2005). In insects, the essential role of GSTs is considered to be the detoxification of a diversity of harmful compounds including plant allelochemicals, insecticides, and heavy metals (Francis et al. 2005, Vlahović et al. 2016). As detoxification enzymes, the GSTs family can catalyze the conjugation of the tripeptide glutathione (GSH) (at the N-terminal end) to electrophilic centers of xenobiotic compounds, resulting in water soluble conjugates, which can be easily excreted (Salinas and Wong 1999, Hayes et al. 2005). Three major GSTs families have been described, including cytosolic GSTs, mitochondrial GSTs, and microsomal GSTs (Hayes et al. 2005). Insect GSTs are separated into cytosolic GSTs and microsomal GSTs. The membrane-bound microsomal GSTs are structurally and evolutionarily distinct from the cytosolic GSTs, and their numbers are much lower than that of cytosolic GSTs (Enayati et al. 2005). The cytosolic GSTs are members of six major classes (delta, epsilon, omega, theta, sigma, and zeta) along with several unclassified genes. Among these groups, the delta and epsilon classes are insect specific, and the omega, theta, sigma, and zeta classes have a wider taxonomic distribution (Chelvanayagam et al. 2001, Ketterman et al. 2011). The secondary structure of cytosolic GSTs includes two domains: a conserved N-terminal domain, responsible for glutathione GSH binding (G-site), and a less conserved C-terminal domain, which contains a hydrophobic substrate binding site (H-site) (Ketterman et al. 2011). For microsomal GSTs, there is an unambiguous sequence pattern D-P-x-V-E-R-V-R-R-A-H-x-N-D-x-E-N-I-L-P (where x is any amino acid) (Bresell et al. 2005). Grape phylloxera, Daktulosphaira vitifoliae (Fitch) (Hemiptera: Phylloxeridae), is an aphid-like pest that specially infests grapes (Vitis spp.). It is native to the Northeastern United States and can induce the formation of leaf and root galls on American Vitis species (Wapshere and Helm 1987, Nabity et al. 2013). Galls on grape roots are called radicicoles, and galls on grape leaves like a pocket are known as gallicoles (Granett et al. 2001). After accidently being imported into Europe in the 1800s, this pest caused serious economic damage to European vineyards, then was spread across the continent, and finally throughout the world (Granett et al. 2001). In China, grape phylloxera is a quarantine pest and has invaded vineyards in the Baqiao Shaanxi province and the Huaihua, Hunan province (Du et al. 2014, Wang et al. 2015). Radicicoles have been found in vineyards of Baqiao and Huaihua, but gallicoles have not been found on leaves. Radicicoles cause progressively more severe root damage, yield loss, and eventual vine death because they provide entry for soil-borne fungi and bacteria disrupting the functionality of the roots and are also more difficult to control than galls on the leaf (Forneck and Huber 2009, Powell et al. 2013). At present, resistant rootstocks derived from native American Vitis are the primary control tool for phylloxera management, because they can produce different secondary components which have an adverse effect on the growth of phylloxera groups (Granett et al. 2001, Du et al. 2009). In insects, GSTs are enzymes involved in detoxification of a diversity of harmful compounds including secondary host metabolites. DviGSTs may play an important role in resistance of phylloxera to secondary host metabolites, insecticides, and heavy metals. Currently, a great number of genes encoding GSTs have been identified in insects, but the studies in Aphididae are limited (Strode et al. 2008, Zhou et al. 2012). In total, 20 GSTs have been identified in Acyrthosiphon pisum (Harris) (Hemiptera: Aphididae), 21 GSTs in Myzus persicae (Sulzer) (Hemiptera: Aphididae), 16 GSTs in Rhopalosiphum padi (L.) (Hemiptera: Aphididae), 21 GSTs in Sitobion avenae (F.) (Hemiptera: Aphididae), and 2 GSTs in Aphis gossypii (Glover) (Hemiptera: Aphididae) (Ramsey et al. 2010, Gawande et al. 2014, Zhang et al. 2017). GSTs have not been previously studied in grape phylloxera. In this study, we report (a) the identification and sequence analyses of seven cytosolic GST genes and one microsomal GST gene in grape phylloxera; (b) construction of phylogenetic trees of these genes; and (c) investigation of the tissue distribution of these genes by semiquantitative polymerase chain reaction (PCR). The research presented here provides a basis for the future functional characterization of these GSTs from grape phylloxera. Materials and Methods Insect Grape phylloxera used in this study were collected from Kyoho grape vine roots in Baqiao of Xi’an (Shaanxi province, China) (109.07°E, 34.27°N). These roots with phylloxera were maintained at 24°C and 45–55% relative humidity under a 24-h dark photoperiod in a climate chamber (Griesser et al. 2015). Tissues from parthenogenetic female adults including the head and body were separated under a microscope using a sharp blade. Heads consisted of antenna, mouthparts, and part of the head (including the eyes and brain), and the other sections consisted of only body parts. This study included three independent replicates using 1,000 adults in each replication. All dissected samples were immediately frozen in liquid nitrogen and then stored at −80°C until use. Homology Searches Previously, five transcriptome datasets of grape phylloxera were released in GenBank (Accession numbers: SRX1202871, SRX1202869, SRX1202868, SRX1202867, and SRX1202866). In this study, the annotated GST gene sequences from Hemiptera, such as A. gossypii, A. pisum, and Laodelphax striatellus (Fallen) (Hemiptera: Delphacidae), were applied to identify putative GST genes by blasting the transcriptomes of grape phylloxera. Sequence Analyses The identified gene fragments were assembled by DNAMAN6.0 software (Lynnon Biosoft, CA). The open reading frames (ORFs) were predicted using ORF Finder software (http://www.ncbi.nlm.nih.gov/gorf/gorf.html, date last accessed 2 December 2017). The NCBI Conserved Domains and Protein Classification (https://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml, date last accessed 2 December 2017) were searched to predict the GSTs conserved domains. The TMHMM Server v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM/, date last accessed 2 December 2017) was used to predict transmembrane domains. The ExPASy Proteomics Server (http://www.expasy.org/tools/, date last accessed 2 December 2017) was used to predict molecular weight (Mw) and the theoretical isoelectric point (pI). Multiple sequences were aligned using Clustal muscle (http://www.ebi.ac.uk/Tools/msa/muscle/, date last accessed 2 December 2017). RNA Extraction and cDNA Synthesis The total RNA of all samples was extracted, respectively, using an RNAiso Plus Kit (Takara, Japan) following the manufacturer’s protocol. Each RNA sample was treated with DNase I (MBI Fermentas, Amherst, NY) to efficiently eliminate the genomic DNA. Total RNA was dissolved in RNase-free water and the integrity of total RNA was confirmed by 1% agarose gel electrophoresis. RNA concentration was determined using Infinite 200 Pro NanoQuant (Tecan, Männedorf, Switzerland). For semiquantitative PCR, the first strand of cDNA was synthesized from 1 μg of total RNA using the Revert Aid First Strand cDNA Synthesis Kit (Fermentas) and then stored at −20°C before use. Sequence Verification and Semiquantitative PCR To confirm the full-length cDNA sequences of identified DviGSTs, specific primers were designed using Primer Premier 5.0 (PREMIER Biosoft International, Palo Alto, CA) and synthesized (Sangon Biotech, Shanghai, China; Supp Table 1 [online only]) to amplify the ORFs. The PCR amplification procedures were as follows: 3 min at 95°C, followed by 33 cycles of 30 s at 95°C, 60 s at 56–62°C (depending on gene-specific primers), and 60 s at 72°C, with a final elongation for 8 min at 72°C. The reaction system consisted of 25 of 12.5-μl Taq Master Mix (Novoprotein, Shanghai, China), 8.5 μl of ddH2O, 1 μl of each primer (10 μM), and a 2-μl sample of cDNA. PCR products were analyzed by electrophoresis in a 1% agarose gel and photographs were taken by GelDoc XR System (Bio-Rad, Hercules, CA). Target bands were gel purified with an agarose gel DNA recovery Kit (Bioteke, Beijing, China) and then cloned into a pMD19-T vector (Takara). The products were transformed into Escherichia coli competent cells and then sequenced by Sangon Biotech. Semiquantitative PCR primers (Supp Table 2 [online only]) were designed based on the ORFs of nucleotide sequences using Primer Premier 5.0. The grape phylloxera actin gene (accession number: KX890128) was used as an internal control. Amplification conditions were 95°C for 3 min; 33 cycles of 95°C for 30 s, 52–62°C for 30 s, 72°C for 60 s; then a final extension at 72°C for 5 min. The reaction volume was 25 μl with 12.5-μl Taq Master Mix, 8.5 μl of ddH2O, 1 μl of each primer (10 μM), and a 2-μl sample cDNA (Zhao et al. 2017). Photographs of 1% agarose gels were taken by the GelDoc XR System (Bio-Rad) and then cropped by Photoshop 7.0.1 (Adobe, San Jose, CA). Image J software (https://imagej.nih.gov/ij/, date last accessed 2 December 2017) was used to quantify the density of the bands. The photograph was opened in image J software and then analyzed by using functions of ‘Edit, Image and Analyze’ to measure the density with five replicate operations (Chen et al. 2014, Yang et al. 2017). Statistical analyses were performed using SPSS 20.0 for windows. Differences in densities of DviGSTs between head and body were analyzed with an independent sample t- test, as all data were normally distributed. Phylogenetic Analyses MEGA5.0 software was used to construct phylogenetic trees by using the neighbor-joining method. Bootstraps of the supporting tree branches were constructed with 1,000 replications. In total, 64 GST amino acid sequences were used for the phylogenetic analyses. The accession numbers of these amino acid sequences are shown in Figs. 3 and 4. In total, 52 cytosolic GSTs from five classes (delta, epsilon, omega, theta, and sigma) from 23 insect species were used to construct a cytosolic GSTs phylogenetic tree and 12 microsomal GSTs from 10 insect species were used to construct a microsomal GSTs phylogenetic tree. The phylogenetic tree was visualized by using the Evolview web server (www.evolgenius.info/evolview, date last accessed 2 December 2017). Results Identification and Characterization of DviGSTs Eight GST transcripts, including seven cytosolic GSTs and one microsomal GST, were identified in the grape phylloxera. The ORFs were deposited in the GenBank database and the accession numbers are listed in Table 1. The cytosolic GSTs (DviGSTd1, DviGSTd2, DviGSTt1, DviGSTs1, DviGSTs2, DviGSTs3, and DviGSTo1) contain 201 to 238 amino acid residues. The predicted molecular weight (Mw) ranged from 58734.46 Da to 70215.89 Da and the theoretical isoelectric points (pI) varied from 5.15 to 5.23. Multiple sequence alignment analyses showed that the cytosolic GSTs contained a G-site in the N-terminal domain and an H-site in the C-terminal domain (Fig. 1). DviGSTd1 and DviGSTd2 contain a conserved serine residue at the N-terminal domain, which functions as a catalytic active residue (Fig. 1A). The microsomal GST, DviGSTm, contains 158 amino acid residues. Its Mw is 46384.17 Da and the pI is 5.09. DviGSTm contains a conserved motif consisting of 16 amino acids and is a member of the membrane-associated proteins in eicosanoid and glutathione metabolism (MAPEG). Three transmembrane domains were found in DviGSTm (Fig. 2). Table 1. List of GSTs in D. vitifoliae GST class  Gene name  ORFs (aa)/length(bp)  Molecular weight (Da)  Isoelectric point  GenBank ID  Delta  DviGSTd1  238/717  67329.84  5.19  MF197746  DviGSTd2  217/654  62660.00  5.15  MF197747  Theta  DviGSTt1  217/654  67431.13  5.21  MF197748  Omega  DviGSTo1  238/717  70215.89  5.19  MF197749  Sigma  DviGSTs1  203/612  58734.46  5.23  MF197750  DviGSTs2  205/618  59041.10  5.20  MF197751  DviGSTs3  201/606  63005.26  5.20  MF197752  Microsomal  DviGSTm  158/477  46384.17  5.09  MF197753  GST class  Gene name  ORFs (aa)/length(bp)  Molecular weight (Da)  Isoelectric point  GenBank ID  Delta  DviGSTd1  238/717  67329.84  5.19  MF197746  DviGSTd2  217/654  62660.00  5.15  MF197747  Theta  DviGSTt1  217/654  67431.13  5.21  MF197748  Omega  DviGSTo1  238/717  70215.89  5.19  MF197749  Sigma  DviGSTs1  203/612  58734.46  5.23  MF197750  DviGSTs2  205/618  59041.10  5.20  MF197751  DviGSTs3  201/606  63005.26  5.20  MF197752  Microsomal  DviGSTm  158/477  46384.17  5.09  MF197753  View Large Fig. 1. View largeDownload slide Sequence alignment of cytosolic GSTs from D. vitifoliae and other insects. A: Delta class; B: theta class; C: omega class; and D: sigma class. Red: GSH binding sites; green: substrate binding sites. The key serine residue for the catalytic is boxed. Dvi: D. vitifoliae, the accession numbers of DviGSTs are listed in Table 1. Slit: Spodoptera litura (F.) (Lepidoptera: Noctuidae) (SlitGSTd4: AIH07597.1), Bmor: Bombyx mori (L.) (Lepidoptera: Bombycidae) (BmorGSTd2: NP_001036974.1), Agos: A. gossypii (AgosGSTd1: AML23851.1; AgosGSTs1-2: AFM78642.1- AFM78643.1), Sfur: Sogatella furcifera (Horváth) (Hemiptera: Delphacidae) (SfurGSTd2: AFJ75818.1), Cflo: Camponotus floridanus (Buckley) (Hymenoptera: Formicidae) (CfloGSTo1: EFN62827.1), Acol: Atta colombica (Guerin) (Hymenoptera: Formicidae) (AcolGSTo1: KYM78756.1), Nvit: Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae) (NvitGSTo1: NP_001165912.1; NvitGSTt2: NP_001165925.1), Nlug: Nilaparvata lugens (Stal) (Hemiptera: Delphacidae) (NlugGSTs1: AFJ75803.1), Lstr: L. striatellus (LstrGSTs3: AEY80032.1), Lnig: Lasius niger (L.) (Hymenoptera: Formicidae) (LnigGSTt1: KMQ97840.1), Aech: Acromyrmex echinatior (Forel) (Hymenoptera: Formicidae) (AechGSTt1: EGI60287.1) Fig. 1. View largeDownload slide Sequence alignment of cytosolic GSTs from D. vitifoliae and other insects. A: Delta class; B: theta class; C: omega class; and D: sigma class. Red: GSH binding sites; green: substrate binding sites. The key serine residue for the catalytic is boxed. Dvi: D. vitifoliae, the accession numbers of DviGSTs are listed in Table 1. Slit: Spodoptera litura (F.) (Lepidoptera: Noctuidae) (SlitGSTd4: AIH07597.1), Bmor: Bombyx mori (L.) (Lepidoptera: Bombycidae) (BmorGSTd2: NP_001036974.1), Agos: A. gossypii (AgosGSTd1: AML23851.1; AgosGSTs1-2: AFM78642.1- AFM78643.1), Sfur: Sogatella furcifera (Horváth) (Hemiptera: Delphacidae) (SfurGSTd2: AFJ75818.1), Cflo: Camponotus floridanus (Buckley) (Hymenoptera: Formicidae) (CfloGSTo1: EFN62827.1), Acol: Atta colombica (Guerin) (Hymenoptera: Formicidae) (AcolGSTo1: KYM78756.1), Nvit: Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae) (NvitGSTo1: NP_001165912.1; NvitGSTt2: NP_001165925.1), Nlug: Nilaparvata lugens (Stal) (Hemiptera: Delphacidae) (NlugGSTs1: AFJ75803.1), Lstr: L. striatellus (LstrGSTs3: AEY80032.1), Lnig: Lasius niger (L.) (Hymenoptera: Formicidae) (LnigGSTt1: KMQ97840.1), Aech: Acromyrmex echinatior (Forel) (Hymenoptera: Formicidae) (AechGSTt1: EGI60287.1) Fig. 2. View largeDownload slide Sequence alignment of microsomal GSTs from D. vitifoliae and other insects. The green box: three transmembrane domains, red: conserved motif. Dvi: D. vitifoliae, Cqui: Culex quinquefasciatus (Say) (Diptera: Culicidae) (CquiGSTm: XP_001868657.1), Aaeq: Aedes aegypti (L.) (Diptera: Culicidae) (AaeqGSTm: XP_001658060), Tcas: Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) (TcasGSTm: XP_968617), Hvir: Heliothis virescens (F.) (Lepidoptera: Noctuidae) (HvirGSTm: ADH16761.1), Slit: S. litura (SlitGSTm: AIH07604.1). Fig. 2. View largeDownload slide Sequence alignment of microsomal GSTs from D. vitifoliae and other insects. The green box: three transmembrane domains, red: conserved motif. Dvi: D. vitifoliae, Cqui: Culex quinquefasciatus (Say) (Diptera: Culicidae) (CquiGSTm: XP_001868657.1), Aaeq: Aedes aegypti (L.) (Diptera: Culicidae) (AaeqGSTm: XP_001658060), Tcas: Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) (TcasGSTm: XP_968617), Hvir: Heliothis virescens (F.) (Lepidoptera: Noctuidae) (HvirGSTm: ADH16761.1), Slit: S. litura (SlitGSTm: AIH07604.1). Phylogenetic Analyses In the phylogenetic tree of cytosolic GSTs, the DviGSTs were assigned to four clades representing different GST classes (Fig. 3). DviGSTd1 and DviGSTd2 fell into the group of the delta class. DviGSTt1 is on the branch of theta class. DviGSTo1 is a member of the omega class. DviGSTs1, DviGSTs2, and DviGSTs3 are in the sigma class group and are closely related to AgosGSTs1 and AgosGSTs2 in A. gossypii. In the phylogenetic tree of microsomal GSTs, DviGSTm is closely associated with RpedGSTm of Riptortus pedestris (F.) (Hemiptera: Alydidae) with 61% bootstrap support (Fig. 4). Fig. 3. View largeDownload slide Phylogenetic analysis of cytosolic GSTs. The tree was constructed by neighbor-joining method using the MEGA software. The GenBank accession numbers are included after the GST names. The cytosolic GSTs of D. vitifoliae are marked with black circles. The clade in lightblue indicates the Delta class; the clade in pink indicates the Epsilon class; the clade in lightgreen indicates the Theta class; the clade in grey indicates the Sigma class; the clade in yellow indicates the Omega class. Dvi: D. vitifoliae, Bmor: B. mori, Csup: Chilo suppressalis (Walker) (Lepidoptera: Crambidae), Pxut: Papilio Xuthus (L.) (Hymenoptera: Pteromalidae), Slit: S. litura, Agos: A. gossypii, Amel: Apis mellifera (L.) (Hymenoptera: Apidae), Bger: Blattella germanica (L.) (Dictyoptera: Blattellidae), Cpun: Cryptocercus punctulatus (Scudder) (Blattodea: Cryptocercidae), Tmol: Tenebrio molitor (L.) (Coleoptera: Tenebrionidae), Sfur: S. furcifera, Znev: Zootermopsis nevadensis (Hagen) (Isoptera: Archotermopsidae), Nlug: N. lugens, Lstr: L. striatella, Dmag: Daphnia magna (Straus) (Crustacea: Cladocer), Mqua: Melipona quadrifasciata (Lep.) (Hymenoptera: Apidae), Nvit: N. vitripennis, Lnig: L. niger, Aech: A. echinatior, Cmed: Cnaphalocrocis medinalis (Guenée) (Lepidoptera: Pyralidae), Asin: Anopheles sinensis (Wiedemann) (Diptera: Culicidae), Dant: Delia antiqua (Meigen) (Diptera: Anthomyiidae), Cflo: C. floridanus. Fig. 3. View largeDownload slide Phylogenetic analysis of cytosolic GSTs. The tree was constructed by neighbor-joining method using the MEGA software. The GenBank accession numbers are included after the GST names. The cytosolic GSTs of D. vitifoliae are marked with black circles. The clade in lightblue indicates the Delta class; the clade in pink indicates the Epsilon class; the clade in lightgreen indicates the Theta class; the clade in grey indicates the Sigma class; the clade in yellow indicates the Omega class. Dvi: D. vitifoliae, Bmor: B. mori, Csup: Chilo suppressalis (Walker) (Lepidoptera: Crambidae), Pxut: Papilio Xuthus (L.) (Hymenoptera: Pteromalidae), Slit: S. litura, Agos: A. gossypii, Amel: Apis mellifera (L.) (Hymenoptera: Apidae), Bger: Blattella germanica (L.) (Dictyoptera: Blattellidae), Cpun: Cryptocercus punctulatus (Scudder) (Blattodea: Cryptocercidae), Tmol: Tenebrio molitor (L.) (Coleoptera: Tenebrionidae), Sfur: S. furcifera, Znev: Zootermopsis nevadensis (Hagen) (Isoptera: Archotermopsidae), Nlug: N. lugens, Lstr: L. striatella, Dmag: Daphnia magna (Straus) (Crustacea: Cladocer), Mqua: Melipona quadrifasciata (Lep.) (Hymenoptera: Apidae), Nvit: N. vitripennis, Lnig: L. niger, Aech: A. echinatior, Cmed: Cnaphalocrocis medinalis (Guenée) (Lepidoptera: Pyralidae), Asin: Anopheles sinensis (Wiedemann) (Diptera: Culicidae), Dant: Delia antiqua (Meigen) (Diptera: Anthomyiidae), Cflo: C. floridanus. Fig. 4. View largeDownload slide Phylogenetic analysis of microsomal GSTs. The tree was constructed by neighbor-joining method using the MEGA software. The GenBank accession numbers are included after the GST names. The microsomal GST of D. vitifoliae is marked with a black circle. Dvi : D. vitifoliae, Hvir: H. virescens, Slit: S. litura, Dmag: D. magna, Cqui: C. quinquefasciatu, Apis: A. pisum, Sfur: S. furcifera, Nlug: N. lugens, Lstr: L. striatella, Rped: R. pedestris. Fig. 4. View largeDownload slide Phylogenetic analysis of microsomal GSTs. The tree was constructed by neighbor-joining method using the MEGA software. The GenBank accession numbers are included after the GST names. The microsomal GST of D. vitifoliae is marked with a black circle. Dvi : D. vitifoliae, Hvir: H. virescens, Slit: S. litura, Dmag: D. magna, Cqui: C. quinquefasciatu, Apis: A. pisum, Sfur: S. furcifera, Nlug: N. lugens, Lstr: L. striatella, Rped: R. pedestris. Fig. 5. View largeDownload slide Expression of DviGSTs in different tissues determined by semi quantitative PCR. The Actin gene was used as an internal control. H: head; B: body; NTC: no template control. Fig. 5. View largeDownload slide Expression of DviGSTs in different tissues determined by semi quantitative PCR. The Actin gene was used as an internal control. H: head; B: body; NTC: no template control. Tissue Distribution Semiquantitative PCR was conducted to confirm the expression of the identified DviGSTs (Fig. 5) and Image J was used to quantify the density of each band. There were significant differences between the expression of DviGSTt1, DviGSTo1, DviGSTs1, and DviGSTm in the head and body (P < 0.05), and the expression levels of them were significantly higher in the body than in the head (Table 2). DviGSTd1, DviGSTd2, DviGSTs2, and DviGSTs3 were highly expressed almost equally in the head and body (P > 0.05) (Table 2). In the head, the expressions of DviGSTt1, DviGSTo1, DviGSTs1, and DviGSTm were lower than that of other DviGSTs. In the body, DviGSTo1 and DviGSTm were substantially lower expressed than other DviGSTs. Table 2. Density quantification of DviGSTs expression levels Gene name  Tissues  t  P-value  Head  Body  DviGSTd1  256.08 ± 0.53  256.46 ± 0.71  −0.424  0.683  DviGSTd2  265.27 ± 0.35  266.47 ± 0.49  −1.964  0.085  DviGSTt1  183.63 ± 0.48  230.57 ± 0.73  −53.501  <0.001  DviGSTo1  66.04 ± 0.45  140.36 ± 0.46  −114.846  <0.001  DviGSTs1  152.19 ± 0.42  205.35 ± 0.84  −56.452  <0.001  DviGSTs2  239.59 ± 0.49  240.56 ± 0.60  −1.253  0.246  DviGSTs3  252.12 ± 0.30  252.70 ± 0.98  −0.566  0.587  DviGSTm  85.33 ± 0.45  139.13 ± 0.83  −57.045  <0.001  Actin  80.36 ± 0.41  79.74 ± 0.54  0.906  0.391  Gene name  Tissues  t  P-value  Head  Body  DviGSTd1  256.08 ± 0.53  256.46 ± 0.71  −0.424  0.683  DviGSTd2  265.27 ± 0.35  266.47 ± 0.49  −1.964  0.085  DviGSTt1  183.63 ± 0.48  230.57 ± 0.73  −53.501  <0.001  DviGSTo1  66.04 ± 0.45  140.36 ± 0.46  −114.846  <0.001  DviGSTs1  152.19 ± 0.42  205.35 ± 0.84  −56.452  <0.001  DviGSTs2  239.59 ± 0.49  240.56 ± 0.60  −1.253  0.246  DviGSTs3  252.12 ± 0.30  252.70 ± 0.98  −0.566  0.587  DviGSTm  85.33 ± 0.45  139.13 ± 0.83  −57.045  <0.001  Actin  80.36 ± 0.41  79.74 ± 0.54  0.906  0.391  Independent sample t-test was performed to access the differences in densities of DviGSTs between head and body. Values are means ± standard errors (df = 8; significant difference, P < 0.05). View Large Discussion In the plant-insect ecosystem, an arms race exists between the plant and insect in defending against the negative influence of each other (Gawande et al. 2014). Plants have biosynthesized a variety of secondary metabolites that function as insect toxins (Rodriguez 1983). The ability to metabolize toxins may affect insects’ choice of host plants (Zangerl and Berenbaum 1993). Several secondary metabolites associated with resistance to insects have been found in grapes. Du et al. (2009) used gas chromatography—mass spectrometry to analyze the chemical components of Kyoho grape roots and rootstocks of 5BB grape and found that 5BB contained large amounts of eucalyptol and limonene and Kyoho roots were rich in limonene. Eucalyptol extracted from Hemizonia fitchii (Asterales: Asteraceae) is moderately effective as a feeding repellent and highly effective as an ovipositional repellent against adults of A. aegypti (yellow fever mosquito; Klocke et al. 1987). Limonene is responsible for the resistance of the Chinese pine to Dendroctonus valens LeConte (Coleoptera: Curculionidae) (Jia et al. 2008). However, Kyoho are susceptible to grape phylloxera. Even in the resistant rootstocks (Beta, 5BB, SO4, and 140Ru), the survival rates of grape phylloxera on feeding sites on roots can reach 47.5, 47.00, 41.00, and 37.50%, respectively (Du et al. 2008). This implies that grape phylloxera have a fairly effective detoxification system for overcoming the secondary metabolites in these grape varieties. Insect cytosolic GSTs are major phase II detoxification enzymes and play a pivotal role in detoxifying the secondary metabolites of various hosts (Sheehan et al. 2001). The effect of cereal plant allelochemicals on the activity of aphid glutathione S-transferases was studied by Leszczynski et al. (1994). This study revealed that glutathione S-transferase activity was significantly higher in the aphids (Metopolophium dirhodum (Wlk.) (Hemiptera: Aphididae), S. avenae and R. padi) that fed on the moderately resistant wheat variety (Grana) than those that fed on the susceptible variety (Emika). Induction of GST activity in M. persicae in response to secondary metabolites from Brassica plants was determined using different host plant species and the results showed that GST activity from aphids reared on Sinapis alba (L.) was prominent higher than that of aphids reared on the other two plant species (Vicia faba (L.) (Rosales: Leguminosae) and Brassica napus (L.) (Capparales: Cruciferae)) (Francis et al. 2005). Therefore GSTs in aphids can be highly associated with their resistance to secondary metabolites. In this study, we identified eight DviGST transcripts of grape phylloxera and all of them were expressed in the head and body. Considering the survival rates of grape phylloxera on feeding sites on rootstocks, we speculated that these DviGSTs may be involved in protecting grape phylloxera against secondary host metabolites. In addition to detoxifying plant secondary compounds, insect GSTs also play an important role in protecting organisms against external harmful factors, including bacterial infections, temperature challenges, heavy metals, and insecticides. In S. litura, SlGSTs1 is expressed in the larval midgut and higher expression levels are associated with increased levels of bacterial infections (Huang et al. 2011). The expression pattern of AccGSTZ1 in Apis cerana cerana (Fabricius) (Hymenoptera: Apidae) under oxidative stress revealed that its transcription was significantly upregulated by temperature challenges and H2O2 treatment (Yan et al. 2012). In L. striatellus, six genes (LsGSTd1, LsGSTm, LsGSTs1, LsGSTs2, LsGSTs3, and LsGSTt1) were noticeably more highly expressed in the midgut and/or the malpighian tubules after the insects were exposed to sublethal concentrations of each of six insecticides (Zhou et al. 2012). AccGSTS1 has been identified in A. cerana cerana, and the mRNA level of AccGSTS1 was higher in the fat body and in the midgut in response to abiotic stresses (cold, heat, UV, H2O2, HgCl2, and insecticides) (Yan et al. 2013). GSTs may be very important in insects such as grape phylloxera that live in a complex underground environment that may expose them to various external harmful factors including bacterial infections and heavy metals. The DviGSTs may help in protecting phylloxera against these harmful underground environmental factors. Delta class GSTs in insects are involved in metabolizing insecticides (Enayati et al. 2005, Li et al. 2007, Liu et al. 2014). A conserved serine residue at position 10 of the N-terminal domain plays an important role in catalytic activity (Sheehan et al. 2001). The N-terminal catalytic serine residue has also been found in DviGSTd1 and DviGSTd2. In the cytosolic GSTs phylogenetic tree, DviGSTd1 and DviGSTd2 are clustered in the Delta group. In this group, SlitGSTd4 is involved in the resistance of S.litura to chlorpyrifos, and SfurGSTd2 is associated with the resistance of S.furcifera to beta-cypermethrin (Zhou et al. 2013, Zhang et al. 2016). These results suggest that DviGSTd1 and DviGSTd2 may have a role in detoxification of insecticides. Sigma GSTs are abundant and conserved in many insect species and are considered to have various detoxification roles among different insect orders. These GSTs can act as antioxidants in conjugating lipid peroxidation and are involved in detoxification of insecticides (Gawande et al. 2014, Liu et al. 2015). Molecular screening of several insecticides and known plant derived natural compounds with AgosGSTs1 and AgosGSTs2 protein models of A. gossypii have been conducted and the results revealed that AgosGSTs1 can bind the insecticide, piperonyl butoxide, and AgosGSTs2 can bind the plant-derived natural compounds tannin (Gawande et al. 2014). In the cytosolic GSTs phylogenetic tree, DviGSTs1 and DviGSTs3 are closely related to AgosGSTs1, AgosGSTs2, and DviGSTs1 and DviGSTs3 were abundantly expressed in the body. These important structural characteristics suggest that DviGSTs may be involved in detoxification of xenobiotics. In conclusion, eight GST genes have been identified in grape phylloxera. Seven of them were placed into four different classes of cytosolic GSTs and the remaining one was classified as a microsomal GST. The phylogenetic analysis and tissue distribution of these genes have been presented. 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R.. 2013. Genomic insights into the glutathione S-transferase gene family of two rice planthoppers, Nilaparvata lugens (Stål) and Sogatella furcifera (Horváth)(Hemiptera: Delphacidae). PLoS ONE  8: e56604. Google Scholar CrossRef Search ADS PubMed  © The Author(s) 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Environmental Entomology Oxford University Press

Identification and Characterisation of Putative Glutathione S-Transferase Genes from Daktulosphaira vitifoliae (Hemiptera: Phylloxeridae)

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Abstract

Abstract Glutathione S-transferases (GSTs) in insects are widely known for their role in the detoxification of both endogenous and xenobiotic compounds. Grape phylloxera, Daktulosphaira vitifoliae (Fitch) (Hemiptera: Phylloxeridae) is a serious grape pest, which causes great economic damage in vineyards, and has currently spread throughout the world. In this study, eight putative GST genes were identified by analyzing the transcriptomes of grape phylloxera. Phylogenetic analyses showed that there are seven cytosolic DviGSTs and one microsomal DviGST. These cytosolic DviGSTs are clustered into four different classes including two delta genes, one omega gene, one theta gene, and three sigma genes. Among candidate cytosolic DviGSTs, a conserved N-terminal domain and a less conserved C-terminal domain were identified. For the candidate microsomal DviGST, three transmembrane regions were predicted. Multiple sequence alignment analysis of the candidate microsomal DviGST was conducted with other insect microsomal GSTs and the result showed that there is a conserved sequence pattern. Semiquantitative polymerase chain reaction was used to examine the tissue expression of these transcripts, and the results revealed that DviGSTs were ubiquitously expressed in the head and the body, but DviGSTd1, DviGSTd2, DviGSTs2, and DviGSTs3 were abundantly expressed in the head and body. This is the first study of the molecular characteristics of GST genes in grape phylloxera. Our results will provide a molecular basis for future studies of the detoxification mechanisms in grape phylloxera. Glutathione S-transferases (GSTs) are multifunctional enzymes widely distributed in both prokaryotic and eukaryotic cells (Qin et al. 2013). They play important roles not only in the detoxification of endogenous and xenobiotic compounds but also in intracellular transport, biosynthesis of hormones, and protection against oxidative stress (Enayati et al. 2005). In insects, the essential role of GSTs is considered to be the detoxification of a diversity of harmful compounds including plant allelochemicals, insecticides, and heavy metals (Francis et al. 2005, Vlahović et al. 2016). As detoxification enzymes, the GSTs family can catalyze the conjugation of the tripeptide glutathione (GSH) (at the N-terminal end) to electrophilic centers of xenobiotic compounds, resulting in water soluble conjugates, which can be easily excreted (Salinas and Wong 1999, Hayes et al. 2005). Three major GSTs families have been described, including cytosolic GSTs, mitochondrial GSTs, and microsomal GSTs (Hayes et al. 2005). Insect GSTs are separated into cytosolic GSTs and microsomal GSTs. The membrane-bound microsomal GSTs are structurally and evolutionarily distinct from the cytosolic GSTs, and their numbers are much lower than that of cytosolic GSTs (Enayati et al. 2005). The cytosolic GSTs are members of six major classes (delta, epsilon, omega, theta, sigma, and zeta) along with several unclassified genes. Among these groups, the delta and epsilon classes are insect specific, and the omega, theta, sigma, and zeta classes have a wider taxonomic distribution (Chelvanayagam et al. 2001, Ketterman et al. 2011). The secondary structure of cytosolic GSTs includes two domains: a conserved N-terminal domain, responsible for glutathione GSH binding (G-site), and a less conserved C-terminal domain, which contains a hydrophobic substrate binding site (H-site) (Ketterman et al. 2011). For microsomal GSTs, there is an unambiguous sequence pattern D-P-x-V-E-R-V-R-R-A-H-x-N-D-x-E-N-I-L-P (where x is any amino acid) (Bresell et al. 2005). Grape phylloxera, Daktulosphaira vitifoliae (Fitch) (Hemiptera: Phylloxeridae), is an aphid-like pest that specially infests grapes (Vitis spp.). It is native to the Northeastern United States and can induce the formation of leaf and root galls on American Vitis species (Wapshere and Helm 1987, Nabity et al. 2013). Galls on grape roots are called radicicoles, and galls on grape leaves like a pocket are known as gallicoles (Granett et al. 2001). After accidently being imported into Europe in the 1800s, this pest caused serious economic damage to European vineyards, then was spread across the continent, and finally throughout the world (Granett et al. 2001). In China, grape phylloxera is a quarantine pest and has invaded vineyards in the Baqiao Shaanxi province and the Huaihua, Hunan province (Du et al. 2014, Wang et al. 2015). Radicicoles have been found in vineyards of Baqiao and Huaihua, but gallicoles have not been found on leaves. Radicicoles cause progressively more severe root damage, yield loss, and eventual vine death because they provide entry for soil-borne fungi and bacteria disrupting the functionality of the roots and are also more difficult to control than galls on the leaf (Forneck and Huber 2009, Powell et al. 2013). At present, resistant rootstocks derived from native American Vitis are the primary control tool for phylloxera management, because they can produce different secondary components which have an adverse effect on the growth of phylloxera groups (Granett et al. 2001, Du et al. 2009). In insects, GSTs are enzymes involved in detoxification of a diversity of harmful compounds including secondary host metabolites. DviGSTs may play an important role in resistance of phylloxera to secondary host metabolites, insecticides, and heavy metals. Currently, a great number of genes encoding GSTs have been identified in insects, but the studies in Aphididae are limited (Strode et al. 2008, Zhou et al. 2012). In total, 20 GSTs have been identified in Acyrthosiphon pisum (Harris) (Hemiptera: Aphididae), 21 GSTs in Myzus persicae (Sulzer) (Hemiptera: Aphididae), 16 GSTs in Rhopalosiphum padi (L.) (Hemiptera: Aphididae), 21 GSTs in Sitobion avenae (F.) (Hemiptera: Aphididae), and 2 GSTs in Aphis gossypii (Glover) (Hemiptera: Aphididae) (Ramsey et al. 2010, Gawande et al. 2014, Zhang et al. 2017). GSTs have not been previously studied in grape phylloxera. In this study, we report (a) the identification and sequence analyses of seven cytosolic GST genes and one microsomal GST gene in grape phylloxera; (b) construction of phylogenetic trees of these genes; and (c) investigation of the tissue distribution of these genes by semiquantitative polymerase chain reaction (PCR). The research presented here provides a basis for the future functional characterization of these GSTs from grape phylloxera. Materials and Methods Insect Grape phylloxera used in this study were collected from Kyoho grape vine roots in Baqiao of Xi’an (Shaanxi province, China) (109.07°E, 34.27°N). These roots with phylloxera were maintained at 24°C and 45–55% relative humidity under a 24-h dark photoperiod in a climate chamber (Griesser et al. 2015). Tissues from parthenogenetic female adults including the head and body were separated under a microscope using a sharp blade. Heads consisted of antenna, mouthparts, and part of the head (including the eyes and brain), and the other sections consisted of only body parts. This study included three independent replicates using 1,000 adults in each replication. All dissected samples were immediately frozen in liquid nitrogen and then stored at −80°C until use. Homology Searches Previously, five transcriptome datasets of grape phylloxera were released in GenBank (Accession numbers: SRX1202871, SRX1202869, SRX1202868, SRX1202867, and SRX1202866). In this study, the annotated GST gene sequences from Hemiptera, such as A. gossypii, A. pisum, and Laodelphax striatellus (Fallen) (Hemiptera: Delphacidae), were applied to identify putative GST genes by blasting the transcriptomes of grape phylloxera. Sequence Analyses The identified gene fragments were assembled by DNAMAN6.0 software (Lynnon Biosoft, CA). The open reading frames (ORFs) were predicted using ORF Finder software (http://www.ncbi.nlm.nih.gov/gorf/gorf.html, date last accessed 2 December 2017). The NCBI Conserved Domains and Protein Classification (https://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml, date last accessed 2 December 2017) were searched to predict the GSTs conserved domains. The TMHMM Server v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM/, date last accessed 2 December 2017) was used to predict transmembrane domains. The ExPASy Proteomics Server (http://www.expasy.org/tools/, date last accessed 2 December 2017) was used to predict molecular weight (Mw) and the theoretical isoelectric point (pI). Multiple sequences were aligned using Clustal muscle (http://www.ebi.ac.uk/Tools/msa/muscle/, date last accessed 2 December 2017). RNA Extraction and cDNA Synthesis The total RNA of all samples was extracted, respectively, using an RNAiso Plus Kit (Takara, Japan) following the manufacturer’s protocol. Each RNA sample was treated with DNase I (MBI Fermentas, Amherst, NY) to efficiently eliminate the genomic DNA. Total RNA was dissolved in RNase-free water and the integrity of total RNA was confirmed by 1% agarose gel electrophoresis. RNA concentration was determined using Infinite 200 Pro NanoQuant (Tecan, Männedorf, Switzerland). For semiquantitative PCR, the first strand of cDNA was synthesized from 1 μg of total RNA using the Revert Aid First Strand cDNA Synthesis Kit (Fermentas) and then stored at −20°C before use. Sequence Verification and Semiquantitative PCR To confirm the full-length cDNA sequences of identified DviGSTs, specific primers were designed using Primer Premier 5.0 (PREMIER Biosoft International, Palo Alto, CA) and synthesized (Sangon Biotech, Shanghai, China; Supp Table 1 [online only]) to amplify the ORFs. The PCR amplification procedures were as follows: 3 min at 95°C, followed by 33 cycles of 30 s at 95°C, 60 s at 56–62°C (depending on gene-specific primers), and 60 s at 72°C, with a final elongation for 8 min at 72°C. The reaction system consisted of 25 of 12.5-μl Taq Master Mix (Novoprotein, Shanghai, China), 8.5 μl of ddH2O, 1 μl of each primer (10 μM), and a 2-μl sample of cDNA. PCR products were analyzed by electrophoresis in a 1% agarose gel and photographs were taken by GelDoc XR System (Bio-Rad, Hercules, CA). Target bands were gel purified with an agarose gel DNA recovery Kit (Bioteke, Beijing, China) and then cloned into a pMD19-T vector (Takara). The products were transformed into Escherichia coli competent cells and then sequenced by Sangon Biotech. Semiquantitative PCR primers (Supp Table 2 [online only]) were designed based on the ORFs of nucleotide sequences using Primer Premier 5.0. The grape phylloxera actin gene (accession number: KX890128) was used as an internal control. Amplification conditions were 95°C for 3 min; 33 cycles of 95°C for 30 s, 52–62°C for 30 s, 72°C for 60 s; then a final extension at 72°C for 5 min. The reaction volume was 25 μl with 12.5-μl Taq Master Mix, 8.5 μl of ddH2O, 1 μl of each primer (10 μM), and a 2-μl sample cDNA (Zhao et al. 2017). Photographs of 1% agarose gels were taken by the GelDoc XR System (Bio-Rad) and then cropped by Photoshop 7.0.1 (Adobe, San Jose, CA). Image J software (https://imagej.nih.gov/ij/, date last accessed 2 December 2017) was used to quantify the density of the bands. The photograph was opened in image J software and then analyzed by using functions of ‘Edit, Image and Analyze’ to measure the density with five replicate operations (Chen et al. 2014, Yang et al. 2017). Statistical analyses were performed using SPSS 20.0 for windows. Differences in densities of DviGSTs between head and body were analyzed with an independent sample t- test, as all data were normally distributed. Phylogenetic Analyses MEGA5.0 software was used to construct phylogenetic trees by using the neighbor-joining method. Bootstraps of the supporting tree branches were constructed with 1,000 replications. In total, 64 GST amino acid sequences were used for the phylogenetic analyses. The accession numbers of these amino acid sequences are shown in Figs. 3 and 4. In total, 52 cytosolic GSTs from five classes (delta, epsilon, omega, theta, and sigma) from 23 insect species were used to construct a cytosolic GSTs phylogenetic tree and 12 microsomal GSTs from 10 insect species were used to construct a microsomal GSTs phylogenetic tree. The phylogenetic tree was visualized by using the Evolview web server (www.evolgenius.info/evolview, date last accessed 2 December 2017). Results Identification and Characterization of DviGSTs Eight GST transcripts, including seven cytosolic GSTs and one microsomal GST, were identified in the grape phylloxera. The ORFs were deposited in the GenBank database and the accession numbers are listed in Table 1. The cytosolic GSTs (DviGSTd1, DviGSTd2, DviGSTt1, DviGSTs1, DviGSTs2, DviGSTs3, and DviGSTo1) contain 201 to 238 amino acid residues. The predicted molecular weight (Mw) ranged from 58734.46 Da to 70215.89 Da and the theoretical isoelectric points (pI) varied from 5.15 to 5.23. Multiple sequence alignment analyses showed that the cytosolic GSTs contained a G-site in the N-terminal domain and an H-site in the C-terminal domain (Fig. 1). DviGSTd1 and DviGSTd2 contain a conserved serine residue at the N-terminal domain, which functions as a catalytic active residue (Fig. 1A). The microsomal GST, DviGSTm, contains 158 amino acid residues. Its Mw is 46384.17 Da and the pI is 5.09. DviGSTm contains a conserved motif consisting of 16 amino acids and is a member of the membrane-associated proteins in eicosanoid and glutathione metabolism (MAPEG). Three transmembrane domains were found in DviGSTm (Fig. 2). Table 1. List of GSTs in D. vitifoliae GST class  Gene name  ORFs (aa)/length(bp)  Molecular weight (Da)  Isoelectric point  GenBank ID  Delta  DviGSTd1  238/717  67329.84  5.19  MF197746  DviGSTd2  217/654  62660.00  5.15  MF197747  Theta  DviGSTt1  217/654  67431.13  5.21  MF197748  Omega  DviGSTo1  238/717  70215.89  5.19  MF197749  Sigma  DviGSTs1  203/612  58734.46  5.23  MF197750  DviGSTs2  205/618  59041.10  5.20  MF197751  DviGSTs3  201/606  63005.26  5.20  MF197752  Microsomal  DviGSTm  158/477  46384.17  5.09  MF197753  GST class  Gene name  ORFs (aa)/length(bp)  Molecular weight (Da)  Isoelectric point  GenBank ID  Delta  DviGSTd1  238/717  67329.84  5.19  MF197746  DviGSTd2  217/654  62660.00  5.15  MF197747  Theta  DviGSTt1  217/654  67431.13  5.21  MF197748  Omega  DviGSTo1  238/717  70215.89  5.19  MF197749  Sigma  DviGSTs1  203/612  58734.46  5.23  MF197750  DviGSTs2  205/618  59041.10  5.20  MF197751  DviGSTs3  201/606  63005.26  5.20  MF197752  Microsomal  DviGSTm  158/477  46384.17  5.09  MF197753  View Large Fig. 1. View largeDownload slide Sequence alignment of cytosolic GSTs from D. vitifoliae and other insects. A: Delta class; B: theta class; C: omega class; and D: sigma class. Red: GSH binding sites; green: substrate binding sites. The key serine residue for the catalytic is boxed. Dvi: D. vitifoliae, the accession numbers of DviGSTs are listed in Table 1. Slit: Spodoptera litura (F.) (Lepidoptera: Noctuidae) (SlitGSTd4: AIH07597.1), Bmor: Bombyx mori (L.) (Lepidoptera: Bombycidae) (BmorGSTd2: NP_001036974.1), Agos: A. gossypii (AgosGSTd1: AML23851.1; AgosGSTs1-2: AFM78642.1- AFM78643.1), Sfur: Sogatella furcifera (Horváth) (Hemiptera: Delphacidae) (SfurGSTd2: AFJ75818.1), Cflo: Camponotus floridanus (Buckley) (Hymenoptera: Formicidae) (CfloGSTo1: EFN62827.1), Acol: Atta colombica (Guerin) (Hymenoptera: Formicidae) (AcolGSTo1: KYM78756.1), Nvit: Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae) (NvitGSTo1: NP_001165912.1; NvitGSTt2: NP_001165925.1), Nlug: Nilaparvata lugens (Stal) (Hemiptera: Delphacidae) (NlugGSTs1: AFJ75803.1), Lstr: L. striatellus (LstrGSTs3: AEY80032.1), Lnig: Lasius niger (L.) (Hymenoptera: Formicidae) (LnigGSTt1: KMQ97840.1), Aech: Acromyrmex echinatior (Forel) (Hymenoptera: Formicidae) (AechGSTt1: EGI60287.1) Fig. 1. View largeDownload slide Sequence alignment of cytosolic GSTs from D. vitifoliae and other insects. A: Delta class; B: theta class; C: omega class; and D: sigma class. Red: GSH binding sites; green: substrate binding sites. The key serine residue for the catalytic is boxed. Dvi: D. vitifoliae, the accession numbers of DviGSTs are listed in Table 1. Slit: Spodoptera litura (F.) (Lepidoptera: Noctuidae) (SlitGSTd4: AIH07597.1), Bmor: Bombyx mori (L.) (Lepidoptera: Bombycidae) (BmorGSTd2: NP_001036974.1), Agos: A. gossypii (AgosGSTd1: AML23851.1; AgosGSTs1-2: AFM78642.1- AFM78643.1), Sfur: Sogatella furcifera (Horváth) (Hemiptera: Delphacidae) (SfurGSTd2: AFJ75818.1), Cflo: Camponotus floridanus (Buckley) (Hymenoptera: Formicidae) (CfloGSTo1: EFN62827.1), Acol: Atta colombica (Guerin) (Hymenoptera: Formicidae) (AcolGSTo1: KYM78756.1), Nvit: Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae) (NvitGSTo1: NP_001165912.1; NvitGSTt2: NP_001165925.1), Nlug: Nilaparvata lugens (Stal) (Hemiptera: Delphacidae) (NlugGSTs1: AFJ75803.1), Lstr: L. striatellus (LstrGSTs3: AEY80032.1), Lnig: Lasius niger (L.) (Hymenoptera: Formicidae) (LnigGSTt1: KMQ97840.1), Aech: Acromyrmex echinatior (Forel) (Hymenoptera: Formicidae) (AechGSTt1: EGI60287.1) Fig. 2. View largeDownload slide Sequence alignment of microsomal GSTs from D. vitifoliae and other insects. The green box: three transmembrane domains, red: conserved motif. Dvi: D. vitifoliae, Cqui: Culex quinquefasciatus (Say) (Diptera: Culicidae) (CquiGSTm: XP_001868657.1), Aaeq: Aedes aegypti (L.) (Diptera: Culicidae) (AaeqGSTm: XP_001658060), Tcas: Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) (TcasGSTm: XP_968617), Hvir: Heliothis virescens (F.) (Lepidoptera: Noctuidae) (HvirGSTm: ADH16761.1), Slit: S. litura (SlitGSTm: AIH07604.1). Fig. 2. View largeDownload slide Sequence alignment of microsomal GSTs from D. vitifoliae and other insects. The green box: three transmembrane domains, red: conserved motif. Dvi: D. vitifoliae, Cqui: Culex quinquefasciatus (Say) (Diptera: Culicidae) (CquiGSTm: XP_001868657.1), Aaeq: Aedes aegypti (L.) (Diptera: Culicidae) (AaeqGSTm: XP_001658060), Tcas: Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) (TcasGSTm: XP_968617), Hvir: Heliothis virescens (F.) (Lepidoptera: Noctuidae) (HvirGSTm: ADH16761.1), Slit: S. litura (SlitGSTm: AIH07604.1). Phylogenetic Analyses In the phylogenetic tree of cytosolic GSTs, the DviGSTs were assigned to four clades representing different GST classes (Fig. 3). DviGSTd1 and DviGSTd2 fell into the group of the delta class. DviGSTt1 is on the branch of theta class. DviGSTo1 is a member of the omega class. DviGSTs1, DviGSTs2, and DviGSTs3 are in the sigma class group and are closely related to AgosGSTs1 and AgosGSTs2 in A. gossypii. In the phylogenetic tree of microsomal GSTs, DviGSTm is closely associated with RpedGSTm of Riptortus pedestris (F.) (Hemiptera: Alydidae) with 61% bootstrap support (Fig. 4). Fig. 3. View largeDownload slide Phylogenetic analysis of cytosolic GSTs. The tree was constructed by neighbor-joining method using the MEGA software. The GenBank accession numbers are included after the GST names. The cytosolic GSTs of D. vitifoliae are marked with black circles. The clade in lightblue indicates the Delta class; the clade in pink indicates the Epsilon class; the clade in lightgreen indicates the Theta class; the clade in grey indicates the Sigma class; the clade in yellow indicates the Omega class. Dvi: D. vitifoliae, Bmor: B. mori, Csup: Chilo suppressalis (Walker) (Lepidoptera: Crambidae), Pxut: Papilio Xuthus (L.) (Hymenoptera: Pteromalidae), Slit: S. litura, Agos: A. gossypii, Amel: Apis mellifera (L.) (Hymenoptera: Apidae), Bger: Blattella germanica (L.) (Dictyoptera: Blattellidae), Cpun: Cryptocercus punctulatus (Scudder) (Blattodea: Cryptocercidae), Tmol: Tenebrio molitor (L.) (Coleoptera: Tenebrionidae), Sfur: S. furcifera, Znev: Zootermopsis nevadensis (Hagen) (Isoptera: Archotermopsidae), Nlug: N. lugens, Lstr: L. striatella, Dmag: Daphnia magna (Straus) (Crustacea: Cladocer), Mqua: Melipona quadrifasciata (Lep.) (Hymenoptera: Apidae), Nvit: N. vitripennis, Lnig: L. niger, Aech: A. echinatior, Cmed: Cnaphalocrocis medinalis (Guenée) (Lepidoptera: Pyralidae), Asin: Anopheles sinensis (Wiedemann) (Diptera: Culicidae), Dant: Delia antiqua (Meigen) (Diptera: Anthomyiidae), Cflo: C. floridanus. Fig. 3. View largeDownload slide Phylogenetic analysis of cytosolic GSTs. The tree was constructed by neighbor-joining method using the MEGA software. The GenBank accession numbers are included after the GST names. The cytosolic GSTs of D. vitifoliae are marked with black circles. The clade in lightblue indicates the Delta class; the clade in pink indicates the Epsilon class; the clade in lightgreen indicates the Theta class; the clade in grey indicates the Sigma class; the clade in yellow indicates the Omega class. Dvi: D. vitifoliae, Bmor: B. mori, Csup: Chilo suppressalis (Walker) (Lepidoptera: Crambidae), Pxut: Papilio Xuthus (L.) (Hymenoptera: Pteromalidae), Slit: S. litura, Agos: A. gossypii, Amel: Apis mellifera (L.) (Hymenoptera: Apidae), Bger: Blattella germanica (L.) (Dictyoptera: Blattellidae), Cpun: Cryptocercus punctulatus (Scudder) (Blattodea: Cryptocercidae), Tmol: Tenebrio molitor (L.) (Coleoptera: Tenebrionidae), Sfur: S. furcifera, Znev: Zootermopsis nevadensis (Hagen) (Isoptera: Archotermopsidae), Nlug: N. lugens, Lstr: L. striatella, Dmag: Daphnia magna (Straus) (Crustacea: Cladocer), Mqua: Melipona quadrifasciata (Lep.) (Hymenoptera: Apidae), Nvit: N. vitripennis, Lnig: L. niger, Aech: A. echinatior, Cmed: Cnaphalocrocis medinalis (Guenée) (Lepidoptera: Pyralidae), Asin: Anopheles sinensis (Wiedemann) (Diptera: Culicidae), Dant: Delia antiqua (Meigen) (Diptera: Anthomyiidae), Cflo: C. floridanus. Fig. 4. View largeDownload slide Phylogenetic analysis of microsomal GSTs. The tree was constructed by neighbor-joining method using the MEGA software. The GenBank accession numbers are included after the GST names. The microsomal GST of D. vitifoliae is marked with a black circle. Dvi : D. vitifoliae, Hvir: H. virescens, Slit: S. litura, Dmag: D. magna, Cqui: C. quinquefasciatu, Apis: A. pisum, Sfur: S. furcifera, Nlug: N. lugens, Lstr: L. striatella, Rped: R. pedestris. Fig. 4. View largeDownload slide Phylogenetic analysis of microsomal GSTs. The tree was constructed by neighbor-joining method using the MEGA software. The GenBank accession numbers are included after the GST names. The microsomal GST of D. vitifoliae is marked with a black circle. Dvi : D. vitifoliae, Hvir: H. virescens, Slit: S. litura, Dmag: D. magna, Cqui: C. quinquefasciatu, Apis: A. pisum, Sfur: S. furcifera, Nlug: N. lugens, Lstr: L. striatella, Rped: R. pedestris. Fig. 5. View largeDownload slide Expression of DviGSTs in different tissues determined by semi quantitative PCR. The Actin gene was used as an internal control. H: head; B: body; NTC: no template control. Fig. 5. View largeDownload slide Expression of DviGSTs in different tissues determined by semi quantitative PCR. The Actin gene was used as an internal control. H: head; B: body; NTC: no template control. Tissue Distribution Semiquantitative PCR was conducted to confirm the expression of the identified DviGSTs (Fig. 5) and Image J was used to quantify the density of each band. There were significant differences between the expression of DviGSTt1, DviGSTo1, DviGSTs1, and DviGSTm in the head and body (P < 0.05), and the expression levels of them were significantly higher in the body than in the head (Table 2). DviGSTd1, DviGSTd2, DviGSTs2, and DviGSTs3 were highly expressed almost equally in the head and body (P > 0.05) (Table 2). In the head, the expressions of DviGSTt1, DviGSTo1, DviGSTs1, and DviGSTm were lower than that of other DviGSTs. In the body, DviGSTo1 and DviGSTm were substantially lower expressed than other DviGSTs. Table 2. Density quantification of DviGSTs expression levels Gene name  Tissues  t  P-value  Head  Body  DviGSTd1  256.08 ± 0.53  256.46 ± 0.71  −0.424  0.683  DviGSTd2  265.27 ± 0.35  266.47 ± 0.49  −1.964  0.085  DviGSTt1  183.63 ± 0.48  230.57 ± 0.73  −53.501  <0.001  DviGSTo1  66.04 ± 0.45  140.36 ± 0.46  −114.846  <0.001  DviGSTs1  152.19 ± 0.42  205.35 ± 0.84  −56.452  <0.001  DviGSTs2  239.59 ± 0.49  240.56 ± 0.60  −1.253  0.246  DviGSTs3  252.12 ± 0.30  252.70 ± 0.98  −0.566  0.587  DviGSTm  85.33 ± 0.45  139.13 ± 0.83  −57.045  <0.001  Actin  80.36 ± 0.41  79.74 ± 0.54  0.906  0.391  Gene name  Tissues  t  P-value  Head  Body  DviGSTd1  256.08 ± 0.53  256.46 ± 0.71  −0.424  0.683  DviGSTd2  265.27 ± 0.35  266.47 ± 0.49  −1.964  0.085  DviGSTt1  183.63 ± 0.48  230.57 ± 0.73  −53.501  <0.001  DviGSTo1  66.04 ± 0.45  140.36 ± 0.46  −114.846  <0.001  DviGSTs1  152.19 ± 0.42  205.35 ± 0.84  −56.452  <0.001  DviGSTs2  239.59 ± 0.49  240.56 ± 0.60  −1.253  0.246  DviGSTs3  252.12 ± 0.30  252.70 ± 0.98  −0.566  0.587  DviGSTm  85.33 ± 0.45  139.13 ± 0.83  −57.045  <0.001  Actin  80.36 ± 0.41  79.74 ± 0.54  0.906  0.391  Independent sample t-test was performed to access the differences in densities of DviGSTs between head and body. Values are means ± standard errors (df = 8; significant difference, P < 0.05). View Large Discussion In the plant-insect ecosystem, an arms race exists between the plant and insect in defending against the negative influence of each other (Gawande et al. 2014). Plants have biosynthesized a variety of secondary metabolites that function as insect toxins (Rodriguez 1983). The ability to metabolize toxins may affect insects’ choice of host plants (Zangerl and Berenbaum 1993). Several secondary metabolites associated with resistance to insects have been found in grapes. Du et al. (2009) used gas chromatography—mass spectrometry to analyze the chemical components of Kyoho grape roots and rootstocks of 5BB grape and found that 5BB contained large amounts of eucalyptol and limonene and Kyoho roots were rich in limonene. Eucalyptol extracted from Hemizonia fitchii (Asterales: Asteraceae) is moderately effective as a feeding repellent and highly effective as an ovipositional repellent against adults of A. aegypti (yellow fever mosquito; Klocke et al. 1987). Limonene is responsible for the resistance of the Chinese pine to Dendroctonus valens LeConte (Coleoptera: Curculionidae) (Jia et al. 2008). However, Kyoho are susceptible to grape phylloxera. Even in the resistant rootstocks (Beta, 5BB, SO4, and 140Ru), the survival rates of grape phylloxera on feeding sites on roots can reach 47.5, 47.00, 41.00, and 37.50%, respectively (Du et al. 2008). This implies that grape phylloxera have a fairly effective detoxification system for overcoming the secondary metabolites in these grape varieties. Insect cytosolic GSTs are major phase II detoxification enzymes and play a pivotal role in detoxifying the secondary metabolites of various hosts (Sheehan et al. 2001). The effect of cereal plant allelochemicals on the activity of aphid glutathione S-transferases was studied by Leszczynski et al. (1994). This study revealed that glutathione S-transferase activity was significantly higher in the aphids (Metopolophium dirhodum (Wlk.) (Hemiptera: Aphididae), S. avenae and R. padi) that fed on the moderately resistant wheat variety (Grana) than those that fed on the susceptible variety (Emika). Induction of GST activity in M. persicae in response to secondary metabolites from Brassica plants was determined using different host plant species and the results showed that GST activity from aphids reared on Sinapis alba (L.) was prominent higher than that of aphids reared on the other two plant species (Vicia faba (L.) (Rosales: Leguminosae) and Brassica napus (L.) (Capparales: Cruciferae)) (Francis et al. 2005). Therefore GSTs in aphids can be highly associated with their resistance to secondary metabolites. In this study, we identified eight DviGST transcripts of grape phylloxera and all of them were expressed in the head and body. Considering the survival rates of grape phylloxera on feeding sites on rootstocks, we speculated that these DviGSTs may be involved in protecting grape phylloxera against secondary host metabolites. In addition to detoxifying plant secondary compounds, insect GSTs also play an important role in protecting organisms against external harmful factors, including bacterial infections, temperature challenges, heavy metals, and insecticides. In S. litura, SlGSTs1 is expressed in the larval midgut and higher expression levels are associated with increased levels of bacterial infections (Huang et al. 2011). The expression pattern of AccGSTZ1 in Apis cerana cerana (Fabricius) (Hymenoptera: Apidae) under oxidative stress revealed that its transcription was significantly upregulated by temperature challenges and H2O2 treatment (Yan et al. 2012). In L. striatellus, six genes (LsGSTd1, LsGSTm, LsGSTs1, LsGSTs2, LsGSTs3, and LsGSTt1) were noticeably more highly expressed in the midgut and/or the malpighian tubules after the insects were exposed to sublethal concentrations of each of six insecticides (Zhou et al. 2012). AccGSTS1 has been identified in A. cerana cerana, and the mRNA level of AccGSTS1 was higher in the fat body and in the midgut in response to abiotic stresses (cold, heat, UV, H2O2, HgCl2, and insecticides) (Yan et al. 2013). GSTs may be very important in insects such as grape phylloxera that live in a complex underground environment that may expose them to various external harmful factors including bacterial infections and heavy metals. The DviGSTs may help in protecting phylloxera against these harmful underground environmental factors. Delta class GSTs in insects are involved in metabolizing insecticides (Enayati et al. 2005, Li et al. 2007, Liu et al. 2014). A conserved serine residue at position 10 of the N-terminal domain plays an important role in catalytic activity (Sheehan et al. 2001). The N-terminal catalytic serine residue has also been found in DviGSTd1 and DviGSTd2. In the cytosolic GSTs phylogenetic tree, DviGSTd1 and DviGSTd2 are clustered in the Delta group. In this group, SlitGSTd4 is involved in the resistance of S.litura to chlorpyrifos, and SfurGSTd2 is associated with the resistance of S.furcifera to beta-cypermethrin (Zhou et al. 2013, Zhang et al. 2016). These results suggest that DviGSTd1 and DviGSTd2 may have a role in detoxification of insecticides. Sigma GSTs are abundant and conserved in many insect species and are considered to have various detoxification roles among different insect orders. These GSTs can act as antioxidants in conjugating lipid peroxidation and are involved in detoxification of insecticides (Gawande et al. 2014, Liu et al. 2015). Molecular screening of several insecticides and known plant derived natural compounds with AgosGSTs1 and AgosGSTs2 protein models of A. gossypii have been conducted and the results revealed that AgosGSTs1 can bind the insecticide, piperonyl butoxide, and AgosGSTs2 can bind the plant-derived natural compounds tannin (Gawande et al. 2014). In the cytosolic GSTs phylogenetic tree, DviGSTs1 and DviGSTs3 are closely related to AgosGSTs1, AgosGSTs2, and DviGSTs1 and DviGSTs3 were abundantly expressed in the body. These important structural characteristics suggest that DviGSTs may be involved in detoxification of xenobiotics. In conclusion, eight GST genes have been identified in grape phylloxera. Seven of them were placed into four different classes of cytosolic GSTs and the remaining one was classified as a microsomal GST. The phylogenetic analysis and tissue distribution of these genes have been presented. This research can serve as a basis for additional functional research of DviGSTs that is needed to demonstrate the exact roles of these GSTs in the grape phylloxera. Supplementary Data Supplementary data are available at Environmental Entomology online. Acknowledgments We thank X. L. Huang from Northwest A&F University (Shaanxi, China) and S. Y. Ning from Shaanxi Institute of Zoology (Shaanxi, China) for substantial help in discussions and editing the manuscript; Dr. William Harvey Reissig from Cornell University (New York) for proofreading this manuscript. This work was supported by Science and Technology Department of Shaanxi Province: Prevention and Control of Crop Disease (K332021401). References Bresell, A., Weinander R., Lundqvist G., Raza H., Shimoji M., Sun T. H., Balk L., Wiklund R., Eriksson J., and Jansson C.. 2005. Bioinformatic and enzymatic characterization of the MAPEG superfamily. FEBS J . 272: 1688– 1703. 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Environmental EntomologyOxford University Press

Published: Feb 1, 2018

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