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proteomic analysis of salivary gland and secreted saliva in three planthopper species
Journal of Proteomics xxx (xxxx) xxx–xxx
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Combined transcriptomic/proteomic analysis of salivary gland and secreted saliva in three planthopper species Hai-Jian Huanga,b, Jia-Bao Lub, Qiao Lib, Yan-Yuan Baob, Chuan-Xi Zhangb, a b
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Department of Entomology, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China Institute of Insect Science, Zhejiang University, Hangzhou 310058, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Planthopper Transcriptome Proteome Salivary glands Saliva Comparison
The planthoppers are piercing-sucking pests that continuously inject saliva into host plants using specialized stylets. However, knowledge on the constituent and function of planthopper saliva proteins was still limited. In this study, the transcriptomic and proteomic approach were adopted to characterize the composition of salivary glands and their secreted saliva in three planthoppers, respectively. Gene repertoires of salivary glands in brown planthopper (Nilaparvata lugens, BPH), white-backed planthopper (Sogatella furcifera, WBPH) and small brown planthopper (Laodelphax striatellus, SBPH) were very similar, which actively involved in protein synthesis and energy metabolism. Comparative analysis of saliva proteome was performed among three planthoppers and other reported insect species. The saliva composition in three planthoppers was diverse, with 55 saliva proteins commonly identified in more than two species. A few proteins, including serine protease, carboxylesterase, aminopeptidase N, lipophorin, elongation factor, carbonic anhydrase, and calcium binding protein were ubiquitous distributed in different insects, indicating conserved function of saliva. While, the majority of saliva proteins were specifically identified in planthoppers, which might be the evolutional adaptation of insects to different hosts. Our work gained insight into the interaction between insect and host plant through salivary approach, and provided a good resource for functional characterization of effectors. Biological significance: Secreted saliva from insects is attracting immense research interest on the global level due to the crucial roles in determining the compatibility between the insects and their hosts. The three planthoppers: brown planthopper (Nilaparvata lugens, BPH), small brown planthopper (Laodelphax striatellus, SBPH), and whitebacked planthopper (Sogatella furcifera, WBPH) caused serious damage to rice plants throughout Asia. However, knowledge on the composition and function of their secreted saliva proteins was limited. Our study characterizes the global gene expression of salivary glands and their secreted saliva by Illumina sequencing technology and LC–MS/MS analysis, respectively. By comparative analysis, the ubiquitous and specific saliva compounds in different insects were unveiled.
1. Introduction Saliva, an oral secretion predominantly produced from salivary glands, mediate the intimate interaction between insect and host plant during all feeding stages, especially the probing and ingestion process [1]. It was the mixture of bioactive compounds that played multiplied roles in lubrication, digestion, penetration, and handling plant defenses [2–5]. Recently, with the development of sequencing technology, the composition of salivary glands and secreted saliva in several herbivorous insects have been unveiled [6–12]. Saliva proteins such as SHP, C002, and NlShp were indispensable for insect feeding [5,13,14]. However, current knowledge on saliva was still limited, especially the saliva composition that ubiquitously or specifically existed among
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insect species. The brown planthopper (Nilaparvata lugens, BPH), small brown planthopper (Laodelphax striatellus, SBPH), and white-backed planthopper (Sogatella furcifera, WBPH) are most destructive insect pests that belong to Delphacidae [15]. Although all three species use rice as their main food source, their host range was a bit different. The BPHs were extremely monophagous that restricted to rice plant, while the WBPHs and SBPHs were oligophagous that can survived on several Poaceae plants including rice, wheat and maize [16,17]. As an important tissue for insect feeding, the salivary glands played critical roles in host selection. It was of great interest to unveil the different gene repertoires of this secretory tissue. Moreover, saliva directly mediate the plant and insect interaction. The larger complement of saliva
Corresponding author. E-mail address: [email protected] (C.-X. Zhang).
https://doi.org/10.1016/j.jprot.2017.11.003 Received 30 August 2017; Received in revised form 30 September 2017; Accepted 1 November 2017 1874-3919/ © 2017 Elsevier B.V. All rights reserved.
Please cite this article as: Huang, H.-J., Journal of Proteomics (2017), http://dx.doi.org/10.1016/j.jprot.2017.11.003
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proteins was essential for insect to combat a greater diversity of plant defenses [18]. Only a few effector proteins were reported in aphid [19–21]. The potential effector molecules that associated with different host adaption in planthoppers were still unknown. In this study, we characterize the global gene expression of salivary glands and their secreted saliva by Illumina sequencing technology and Liquid Chromatography–Mass Spectrometry/Mass Spectrometry (LC–MS/MS) analysis, respectively. By parallel comparison, we found that the gene repertoires of three species were very similar, but their saliva secretion varied. In addition, we compared the saliva proteins of planthoppers with other insect species. The ubiquitous and specific saliva compounds were unveiled. Our result provided a deeper understanding on plant-insect interaction.
by the reference score and normalized from 0 to 1. BPH, WBPH, and SBPH were used as reference library, respectively. Four quadrants were derived from a threshold value of 0.4, which empirically represented a commonly used threshold for peptide similarity as follows: approximately 30% amino acid identity over approximately 30% of the peptide length. BSR of X-axis < 0.4 and Y-axis < 0.4 represents no BLAST match in reference; X-axis < 0.4 and Y-axis > 0.4 represents conserved peptides in reference and query species peptides on the Y-axis; Xaxis > 0.4 and Y-axis > 0.4 represents conserved peptides in all species; X-axis > 0.4 and Y-axis < 0.4 represents conserved peptides in reference and query peptides on the X-axis.
2. Material and methods
The planthoppers were removed from rice plants by gentle shaking, and the fifth instar nymphs were transferred to sterile diets with 2.5% sucrose. The diet was prepared under aseptic conditions and filtered through a 0.22 μm syringe filter (Millipore, MA, USA). Appropriately 100 planthoppers were trapped in each glass tube with 1 mL diet provided between two layers of Parafilm (Neenah, WI, USA). After 24 h feeding, the diets were collected from the space between the two layers of Parafilm with a pipet. Ultrafiltration was conducted using a 3-kDa molecular-weight cutoff Amicon Ultra-4 Centrifugal Filter Device (Millipore) at 5000g at 4 °C for 30 min. The concentrated samples were precipitated using a trichloro-acetic acid protein precipitation kit (Sangon, Shanghai, China). The pellets were solubilized in 200 μL of SDT buffer (4% sodium dodecyl sulfate (SDS; Sigma, St. Louis, MO, USA), 100 mM dithiothreitol (DTT; Sigma), 150 mM Tris-HCl pH 8.0) and incubated in hot water for 15 min, and then centrifuged at 14,000g for 45 min.
2.6. Collection of SBPH and WBPH saliva
2.1. Insect strains The BPH, SBPH, and WBPH population were originally collected from a rice field in Huajiachi Campus of Zhejiang University, Hangzhou, China. The insects were reared at 26 ± 0.5 °C on rice seeding (Oryza sativa strain Xiushui 134) with 50 ± 5% humidity under a 16:8 h light:dark photoperiod. 2.2. Collection of salivary glands and cDNA library preparation The fifth-instar insect of BPHs, SBPHs, and WBPHs were anesthetized on ice and their salivary glands were dissected as described previously (Fig. S1) [22]. Total RNA were extracted from 200 salivary glands using RNAiso plus (TaKaRa, Dalian, China). After determination of RNA integrity and quantity, the poly (A)+ RNA was purified from 20 μg pooled total RNA using oligo (dT) magnetic beads. Fragmentation was conducted in the presence of divalent cations at 94 °C for 5 min and the cleaved RNA was transcribed according to manufacturer's instructions. After end-repairing and adaptor ligation, the products were PCRamplified and purified using a QIAquick PCR purification kit (Qiagen, Hilden, Germany) to create a cDNA library.
2.7. In-solution digestion Protein digestion was conducted according to Yu et al. recommendation [25]. Briefly, the soluble samples were concentrated with a 3-kDa filtration unit and alkylated with 200 mM IAA for 1 h. To remove DTT and other low-molecular-weight components, the samples were washed by UA buffer (8 M Urea, 150 mM Tris-HCl pH 8.0) twice, and 25 mM NH4HCO3 (Sigma) twice. Digestion was performed using the trypsin in NH4HCO3 buffer overnight at 37 °C. After centrifugation, the digested peptides were desalted using C18 pipette tip (Thermo Scientific, Rockford, IL61101, USA) according to manufacturer's protocol. Then, the samples were concentrated by vacuum drying and dissolve in 30 μL of 0.1% (v/v) formic acid.
2.3. Illumina sequencing and transcriptome assembly The cDNA library was performed using Illumina sequencing platform and the raw data from the images were generated using Solexa GA pipeline 1.6. After removal of low quality reads, processed reads were assembled using Short Oligonucleotide Analysis Package (SOAP) de novo software and clustered with TIGR Gene Indices (TGI) Clustering tools.
2.8. LC–MS/MS analysis
2.4. Unigene annotation and peptide prediction
LC–MS/MS analysis was performed as follow: The digested peptides (20 μL) were loaded onto the trap column at a flow rate of 10 μL/min by Thermo Scientific Easy nanoLC 1000 (Thermo Fisher Scientific, MA, USA). After trap equilibration, the samples were eluted with a linear gradient of buffer A (0.1% formic acid) and buffer B (84% acetonitrile and 0.1% formic acid) at a flow rate of 250 nL/min. The chromatographic system includes a trapping column (75 μm × 2 cm, nanoviper, C18, 3 μM, 100 Å) and an analytical column (50 μm × 15 cm, nanoviper, C18, 2 μM, 100 Å). Separated MS data were analyzed using Thermo LTQ-Orbitrap Velos Pro (Thermo Fisher Scientific) equipped Nanospray Flex ionization source and FTMS (Fourier transform ion cyclotron resonance mass analyzer) combined with Thermo LTQOrbitrap Elite equipped Ion Trap analyzer. The top 20 ions were chosen from a full mass scan (300–2000 m/z) by collision induced decomposition (1.0 m/z isolation width, 35% collision energy, 0.25 activation Q, 10 ms activation time). Dynamic exclusion duration was 60 s. Survey scans for MS1 were acquired at a resolution of 30,000 at m/z 400. The MS/MS spectra were searched against the three peptide databases of salivary glands as we generated above using SEQUEST HT
The assembled unigenes were analyzed by searching the GenBank databases using BLASTX algorithm (http://www.ncbi.nlm.nih.gov/). Gene Orthology (GO) and KEGG Orthology (KO) annotations of the unigenes were determined using Blast2go (http://www.blast2go.org/) and InterProScan software (http://www.ebi.ac.uk/Tools/pfa/iprscan/). In addition, the unigenes were subjected to the Clusters of Orthologous Groups (COG) database (http://www.ncbi.nlm.nih.gov/COG/). The coding sequence (CDS) of each unigene was analyzed using blastx and Estscan software 3.03 [23]. The generated peptide database was used to support the proteomic analysis. 2.5. BLAST score ratio (BSR) test BSR tests [24] were used to global visualize the degree of proteome similarity between BPH, WBPH, and SBPH. BLAST software (http:// blast.ncbi.nlm.nih.gov/Blast.cgi) was used for the reference comparison. The BSR index was calculated by dividing the BLAST query score 2
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Table 1 Statics of three transcriptomes. Species
Raw reads
Clean reads
Unigene
N50 (bp)
Unigene mean length (bp)
Predicted protein (Total transcriptome)
Predicted protein (Top 1000 genes)
Signal peptide (Top 1000 genes)
BPH SBPH WBPH
45,781,625 44,298,976 47,687,228
44,582,696 40,212,148 43,244,348
43,070 44,113 42,210
1869 1399 2063
1066 864 1140
22,176, 51.5% 24,340, 55.1% 24,383, 57.8%
903 936 917
171, 18.9% 158, 16.9% 152, 16.6%
grouped together.
search engine configured with a Proteome Discoverer 1.4 workflow (Thermo Fischer Scientific, Bremen, Germany), respectively. The search parameters were set as follow: 10 ppm mass tolerances for MS; 0.8 Da tolerances for MS/MS; trypsin as proteolytic enzyme; 2 missed cleavages allowed; oxidation and deamidated as dynamic modifications; carbamidomethyl as a static modification. In addition, high peptide confidence was extracted and an automatic decoy database search was performed with false discovery rate (FDR) ≤ 0.01. The identified proteins with unipep ≥2 or with a signal peptide were selected as the saliva proteins.
3. Results 3.1. Transcriptome assembly, gene annotation, and protein prediction The three transcriptome libraries of salivary glands (TLSG) from BPH, SBPH, and WBPH were constructed using the Illumina sequencing platform GAII (Tables 1, S2). After removing the low quality data, a total of 43,070 unigenes in BPH were generated with a mean length of 1066 bp and N50 of 1869 bp. For SBPH, sequence data contained 44,113 unigenes with a mean length of 864 bp and N50 of 1399 bp. The TLSG of WBPH contained 42,210 unigenes with a mean length of 1140 bp and N50 of 2063 bp. The coding sequences were firstly predicted by searching against the NCBI NR, SwissProt, COG, and KEGG databases. A total of 19,566 unigenes (45.6% of all sequences in BPH), 21,525 unigenes (48.9% of all sequences in SBPH), 21,643 unigenes (51.4% of all sequences in WBPH) returned a significant BLASTX hit (Table 1). The remnant unigenes were further predicated by ESTScan software and additional 2610 (BPH), 2815 (SBPH), and 2740 (WBPH) putative proteins were found (Table 1). These predicted proteins provided query databases for the raw data of the proteome.
2.9. Bioinformatic analysis The SignalP 4.1 Server (http://www.cbs.dtu.dk/services/SignalP/) was used to predict potential signal peptides. The EditSeq 5.01 program (DNASTAR, Madison, WI, USA) was used to predict the Mw and isoelectric point (pI). According to GO annotation, the identified saliva proteins were classified into three main categories (biological process, cellular component and molecular function) using BGI WEGO (http:// wego.genomics.org.cn/cgi-bin/wego/index.pl). 2.10. Tissue-specific expressions analysis The insect tissue from salivary glands, guts, fat body, ovary, testis, and carcass were dissected under a Leica S8AP0 stereomicroscope, and the whole insect body was used as a reference. Total RNA was extracted as was described above. First-strand cDNA was synthesized using ReverTraAce qPCR RT Master Mix with a gDNA Remover Kit (ToYoBo, Osaka, Japan) to remove any contaminating genomic DNA. RNA with no reverse transcriptase was used as the no-template control (NTC). Quantitative real-time PCR was run on a CFX Connect Real-Time System (Bio-Rad, Hercules, CA, USA) using iQ SYBR Green Supermix Kit (Bio-Rad). The first-strand cDNA and a no-reverse-transcription control were used as templates for three biological replicates under the following reaction program: an initial denaturation step at 95 °C for 3 min followed by 40 cycles at 95 °C for 10 s and 60 °C for 30 s. The gene-specific primers were designed using the Primer Premier 5.0 program. The housekeeping gene for β-actin and GAPDH was used as an internal control (Table S1). The ΔΔCt method was used to evaluate the quantitative variation in the transcript levels as described previously [26].
3.2. Function analysis of high expression genes in salivary gland To overview the function of salivary gland, the 1000 most highly expressed genes (Top 1000 genes) were selected for further analysis. It was interestingly to find that the ribosomal proteins (99 genes in BPH, 84 genes in SBPH, and 83 genes in WBPH) played the dominant in this secretory tissue (Table S2), indicating the evaluated protein synthetic process. More than 16% of predicted protein contained a signal peptide (Table 1), which might be delivered into saliva through the eukaryotic endoplasmic reticulum–Golgi pathway [1]. Genes which were previously identified as saliva proteins of BPH (such as carboxylesterase, annexin-like protein, cathepsin, mucin-like protein), were also abundantly expressed in the salivary gland of three species (Table S2). We subsequently compared the COG annotation data between the total transcriptome and the top 1000 genes in three species, respectively (Fig. 1). The function sets of total transcriptome in salivary glands were very similar among three species. Increased percentage of function sets associated with energy production, translation, and posttranslational modification were detected in top 1000 genes, indicating an increased energy production and protein synthetic process in salivary glands.
2.11. Comparative analysis of insect saliva proteins The saliva proteins from Halyomorpha halys [12], Nephotettix cincticeps [10], Acyrthosiphon pisum [9,18], Macrosiphum euphorbiae [27], Diuraphis noxia [11], and Myzus persicae [18,28] were extracted according to published sequence information. Then, comparison of saliva proteins from BPH, SBPH, and WBPH was performed. Only the saliva proteins identified in at least two planthoppers' saliva were underwent further analysis. The ubiquitous and specific saliva proteins in planthoppers and other insect species were identified using BLAST alignment (ftp://ftp.ncbi.nlm.nih.gov/blast) with a significance cutoff of Evalue < 10− 5. The proteins showed high sequence similarity were further verified by BLAST search against NCBI database. Only the proteins exhibited high sequence similarity and similar annotation were
3.3. BSR analysis of three transcriptomes The BSR analysis was used to global visualize of the degree of proteome similarity between all three transcriptomes. The results showed that more than half of salivary gland genes were conserved among three species, indicating the close relationship of planthoppers (Fig. 2). Using the predicted proteins of SBPH as a query, we found that 21.2% of SBPH genes showed high similarity with WBPH (but not with BPH), while only 6.2% of SBPH genes showed high similarity with BPH (not with WBPH). Similar results were observed when using proteins of 3
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Fig. 1. Percentages of COG function set annotation between top 1000 genes and total transcriptome in BPH (A), SBPH (B), and WBPH (C). The capital letter in abscissa represents as follow: A, RNA processing and modification; B, chromatin structure and dynamics; C, energy production and conversion; D, cell cycle control, cell division, chromosome partitioning; E, amino acid transport and metabolism; F, nucleotide transport and metabolism; G, carbohydrate transport and metabolism; H, coenzyme transport and metabolism; I, lipid transport and metabolism; J, translation, ribosomal structure and biogenesis; K, transcription; L, replication, recombination and repair; M, cell wall/membrane/envelope biogenesis; N, cell motility; O, posttranslational modification, protein turnover, chaperones; P, inorganic ion transport and metabolism; Q, secondary metabolites biosynthesis, transport and catabolism; R, general function prediction only; S, function unknown; T, signal transduction mechanisms; U, intracellular trafficking, secretion, and vesicular transport; V, defense mechanisms; W, extracellular structures; Y, nuclear structure; Z, cytoskeleton.
phosphate dehydrogenase (GAPDH) were correlated with oxidoreductase. We identified a total of 236 proteins from secreted saliva in SBPH by shotgun LC–MS/MS analysis (Table S4). Among them, 50 proteins contained a signal peptide. Genes such as annexin-like protein, salivap3, plancitoxin-1, stubble-2, carboxylesterase, carboxypeptidase, carbonic anhydrase, and mucin-like protein were also identified in BPH saliva (Fig. 4). GO analysis showed that the gene set associated with cell (28 proteins), cellular process (32 proteins), metabolic process (21 proteins), binding (29 proteins), and catalytic activity (28 proteins) played the dominant in SBPH saliva, which was very similar with that of BPH (Fig. 3B). For ion binding genes, EF-hand domain-containing protein, spectrin, and annexin-like protein participated in calcium binding; titin and E3 ubiquitin-protein ligase participated in zinc binding; adenylosuccinate synthetase participated in magnesium binding. There were five genes exhibited peptidase activity, including stubble-2, ATP-dependent Clp protease, prophenoloxidase activating factor, proteasome, and carboxypeptidase. Other genes including L-2hydroxyglutarate dehydrogenase, 26S proteasome non-ATPase regulatory, and peroxidase participated in oxidoreductase activity. A total of 177 proteins were identified in secreted saliva of WBPH, with 43 proteins containing a signal peptide (Table S5). There were 47 proteins showed high similarity with saliva proteins in BPH or SBPH (Fig. 4), including carboxylesterase, plancitoxin-1, annexin like protein, salivap-3, lipophorin, and venom dipeptidyl peptidase. Similar GO
WBPH as a query, indicating the closer relationship between SBPH and WBPH. 3.4. Saliva proteins of three planthoppers In our previous work, a total of 58 saliva proteins of BPH was identified using the databases generated by Xue et al. [29]. As the TLSG of BPH was available now, we search the MS/MS spectra against this new database. Moreover, the saliva proteins identified by Liu et al. [30] were also adopted and combined with our results. A total of 149 BPH saliva proteins were screened out and listed in Table S3. Among them, 6 saliva proteins (three unknown proteins, EF-hand motif protein isoform 1, serpin, and maltase A1) were uniquely identified using TLSG of BPH as reference database. There were 29 saliva proteins commonly existed in our saliva sample and Liu's [30], which included 19 secretory proteins (i.e., saliva-2, saliva-3, saliva-5, saliva-7, carboxylesterase precursor, and aminopeptidase N). As much as 78 proteins were specifically identified in Liu's saliva samples, and 36 proteins were specifically identified in our saliva samples. Gene Ontology (GO) analysis showed that the majority of saliva proteins participated in metabolic process (27 proteins) and cellular process (21 proteins) (Fig. 3A). While 27 and 24 proteins related with catalytic activity and binding, respectively. Genes such as annexin-like protein, EF-hand motif protein, and regucalcin-like participated in calcium binding. Other genes including glycolate oxidase, peroxiredoxin 1-like, and glyceraldehyde-34
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Fig. 2. BSR analysis of three planthoppers. (A) BSR obtained from comparing BPH to WBPH and SBPH. (C) BSR obtained from comparing SBPH to BPH and WBPH. (D) BSR obtained from comparing WBPH to SBPH and BPH. Each point in the figure represents a single peptide in reference library.
species, we group the proteins according to the gene annotation and sequence similarity. Proteins that commonly identified in planthopper were listed in Table 2. There were 12 proteins existed in all three planthoppers (Fig. 4). Among them, the serine protease, carboxylesterase, leucinerich repeat-containing protein were reported in H. halys saliva; the elongation factor were extensively distributed in aphid; other saliva proteins including plancitoxin-1, annexin-like protein, saliva-3, vitellogenin, and four unknown proteins were exclusively distributed in planthoppers. There were 43 saliva proteins commonly existed in two planthopper (Fig. 4), of which the aminopeptidase N, lipophorin, GAPDH, actin, Nesprin-1, EF-hand domain containing protein, carbonic anhydrase, protein lava lamp, and TBC1 domain containing protein were also found in other insect saliva. The majority of saliva proteins were exclusively identified in planthoppers, indicating the speciesspecificity of saliva secretion. The saliva proteins commonly identified in aphid were also compared with the other insect species (Table 2). Eight proteins, which were predicted to be the major constituent of aphid saliva, were selected. To our expectation, the majority of proteins (glucose dehydrogenase, structural sheath protein, trehalase, effector C002, effector Mp1 Pinto1, effector Me10/Mp58, and cytochrome oxidase) were exclusively identified in aphid saliva (Table 2). Only the ribosomal protein, which expected to be abundant and involved in cellular core functions was also found in the saliva of N. lugens, H. halys, and N. cincticeps.
distribution of WBPH saliva was observed when compared with BPH or SBPH (Fig. 3C). Genes such as lysosomal alpha-mannosidase, ablim, protein prickle-like, and aminopeptidase N were predicted to binding zinc. The glutaredoxin and GAPDH were correlated with oxidoreductase activity. 3.5. Identification of salivary gland-highly expression genes Based on the information of gene annotation and signal peptide, 20 saliva genes in SBPH and WBPH were selected for spatial expression analysis, respectively. For SBPH, a total of 15 genes were verified to be highly expressed in salivary glands, especially the placental protein, annexin-like protein, stubble-2, and six unknown proteins, which exclusively expressed in this secretory tissue (Fig. 5). For WBPH, we found 12 genes expressed highly in salivary glands. Among them, the multiple inositol polyphosphate phosphatase 1-like, venom dipeptidyl peptidase, PI-PLC X domain-containing protein 1like, salivap-5, stubble-2, and six unknown proteins were exclusively expressed in salivary glands (Fig. 5). 3.6. Comparative analysis of insect saliva proteins To date, saliva proteins collected from bug (H. halys), leafhopper (N. cincticeps), and several aphid species (i.e. A. pisum, M. euphorbiae, D. noxia, M. persicae) have been identified by MS analysis. To unveil the ubiquitous and specific saliva proteins in planthoppers and other insect 5
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Fig. 3. Gene ontology classification of the saliva components in BPH (A), SBPH (B), and WBPH (C).
4. Discussion
large-portion of high expressed genes in salivary gland could be secreted, and participated in metabolism process. The insect continually secreted saliva during feeding, which needs a metabolically active factory to support protein synthesis. An enrichment of genes associated with protein synthesis, transport, and energy metabolism were also reported in salivary gland of Anopheles gambiae [31], Ixodes pacificus [32], Bemisia tabaci [33], and A. pisum [34], indicating the conserved biological function of this secretary tissue. The transcriptome of salivary gland from three planthoppers showed high similarity, but SBPH and WBPH were closer related when compared with BPH (Fig. 2). This might be explained by host
Saliva, which was crucial in mediating the interaction between herbivorous insect and host plant, played multiple roles in digestion, detoxification, and protection against host defense response. In the present study, we gain insight into the salivary gland and its secreted saliva of three planthopper species. By transcriptomic and proteomic approach, the saliva proteins that ubiquitously and specifically existed among insect species were unveiled. The salivary gland was an important secretary tissue that associates with insect feeding and viral transmission. Our study revealed that a 6
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response [47,48]. The function of carbonic anhydrase in insect saliva needs further investigation. Calcium binding proteins, including regucalcin [9,10,46], calreticulin [11,46], calmodulin [46], cadherin [6], annexin-like protein [46], and other EF-hand domain containing protein [10,12] were reported in most insect saliva. In response to stylet penetration, the calcium-triggered occlusion was induced, preventing sieve-tube sap from continuous leakage [49]. It was demonstrated that the presence of saliva induced dispersed forisomes to revert back to the contracted state, which functioned like EDTA [50]. The calcium-binding activity of EFhand domain containing protein (a saliva protein in N. cincticeps) was also verified, indicating the important role of saliva in calcium regulation [51]. However, current study found that different kinds of calcium binding proteins were distributed in insect saliva. This could be explained in two aspects: 1) the concentration of calcium binding proteins in some insect saliva was too low to be detected [11,52]; 2) the defense mechanism varied among host plants, and the different calcium binding protein was the result of evolutionary adaptation. Saliva proteins associated with oxidoreductase were ubiquitous identified in planthoppers and other insect species. When attacked by sucking insects, the soluble antioxidants in host plant enhance the effectiveness of the defensive system, and the oxidoreductase enzymes in saliva served to counter them [53]. The presence of glucose dehydrogenase, peroxiredoxin, and peroxidase in aphid saliva were presumed to involve in detoxification and suppression of plant defense responses [27]. In planthoppers, the glycolate oxidase, peroxiredoxin, GAPDH, peroxidase, and glutaredoxin were ubiquitous secreted, which might play a similar role with that of aphid. The glutaredoxin, for example, are essential for maintaining the cellular redox homeostasis and coping with toxicity [54]. It is likely that the oxidoreductase in saliva detoxified the secondary metabolic products and maintained the level of reactive oxygen species (ROS) in plant tissues. There were 42 saliva proteins uniquely identified in three planthoppers (but not in other insect speices), indicating a similar feeding habit of three species. Among them, annexin-like protein, plancitoxin-1, vitellogenin, saliva-3 and four unknown proteins were identified in all three planthoppers (Table 1). Annexin-like protein was presumed to participate in calcium binding. Different from insect annexins, these proteins were the uniquely identified in planthopper, which contained only 1–3 annexin repeats and have a putative signal peptide [46]. The ubiquitous presentation of annexin-like protein indicated that the planthoppers evolved new strategy to handle calcium regulation. The plancitoxin-1, also known as deoxyribonuclease, was a lethal factor responsible for the hepatotoxicity in mammal [55,56]. By degrading nuclear DNA, plancitoxin-1 caused caspase-independent apoptosis of cell [57]. It was intriguing to unveil such toxic proteins in saliva of planthoppers. The salivary toxins were supposed to be caused by virus [58] and saliva-induced apoptosis in host was reported in mosquito, which facilitated viral transmission by inhibiting the host immune cells [59,60]. Planthoppers harbor multiply symbiotic virus that can be horizontal transmitted [61,62]. The relationship between salivary plancitoxin-1 and viral transmission needs further investigation. Five proteins showing no homology with other species were commonly existed in three planthoppers. Among them, only the function of saliva-3 was unveiled as an indispensable factor for plant-associated feeding. Saliva directly mediate the interaction between insect and host plant. The secreted salivary gland proteins, which were under selection pressure for functional adaptation, showed a rapid diversification [63]. The specificity of saliva proteins were also reported in bugs, leafhoppers, and aphids [10,12,18]. Approximately 40% of the secreted proteins in leafhopper were not similar to known proteins [10]. The salivary genes of C002, ACYPI009881, ACYPI008224, and ACYPI006346 were also unique to aphid [7,13]. It was intriguing to uncover the function of these planthopper-specific genes in Poaceae-associated feeding.
Fig. 4. The Venn diagram of identified saliva proteins in three planthoppers.
specificity. The SBPH and WBPH were oligophagous, while BPH was monpohagous. Gene modulation occurred when insect transformed to different hosts [35–37], resulting in contraction or expansion of gene families associated with detoxification, digestion, and chemoreception [35,38]. As a matter of fact, the closer relationship between SBPH and WBPH was verified by viral transmission. South rice black-streaked dwarf virus, a member of Fijivirus that caused serious damage to wheat and rice plant, could be acquired by SBPH and WBPH, but not BPH [39]. The component of BPH saliva has been investigated by shotgun LC–MS/MS for several biological replicates (Table S2). The results showed that the saliva protein identified in different laboratories or different biological repeats in the same laboratory were diversified. Of 149 BPH saliva proteins reported so far, only a few proteins, such as carboxylesterase, salivap-3, salivap-5, and mucin like protein, were commonly identified in all experiments. Similar results were also found in A. pisum [9,18] and M. persicae [18,28], with different saliva proteins reported in the same insect species. In this study, we found that the frequency of saliva proteins identified in different replicates was closely related with their abundance. This might be caused by the concentration of collected saliva samples, or the saliva secretion was diversified in different conditions (i.e., BPH strain and rice variety). The different BPH saliva proteins identified in different replicates indicates variable, but also conservative of saliva secretion. Comparison of saliva proteins among insect species, we found serine protease, carboxylesterase, aminopeptidase N, lipophorin, elongation factor, GAPDH, carbonic anhydrase, and calcium binding protein were ubiquitous distributed in insect saliva, which played basic roles in insect feeding (Table 2). Among them, the serine protease, carboxylesterase, and aminopeptidase N were proteolytic enzymes [26,40,41]. We presumed that the presence of these enzymes might be correlated with digestive function of saliva. Extra-oral digestion was ubiquitous reported in predatory insect species [42]. Through injection of hydrolytic enzymes, the liquefied contents are withdrawn via the stylets, which greatly increase the efficiency of nutrient extraction [43]. The commonly distribution of digestive enzymes in herbivorous saliva indicated that the extra-oral digestion was also important for phloem feeding insects. In addition, the carboxylesterase can metabolize the insecticides or xenobiotics [44], while aminopeptidase N degraded the conjugated glutathione [45]. The commonly distribution of these proteins indicated a detoxifying role of insect saliva as well. Carbonic anhydrases, known for their role in reversible catalyzing the conversion of carbon dioxide to bicarbonate, were identified in N. lugens, L. striatellus, N. cincticeps, and M. euphorbiae. In our previous study, knocking down the expression of carbonic anhydrase resulted in premature death of BPH, but this gene did not influence the pH value of saliva or salivary glands [46]. The carbonic anhydrase in tobacco was reported to bind salicylic acid (SA), which modulated the plant defense 7
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Fig. 5. Tissue-specific expression (in fold) of genes encoding saliva proteins in SBPH and WBPH. SG, salivary gland; Fb, fat body; Ov, ovary; Te, testis; Ca, the remaining carcass; Wh, whole insect body.
planthopper feeding on Poaceae other than rice plant.
Although the saliva of three planthoppers demonstrated similar function categories, variation in saliva protein were found. Salivary action is responsible for host selection [64]. Traits such as substrate specificity of detoxification enzymes, avoidance or suppression of induced defenses were potential mechanisms underlying host breadth [65,66]. Different constituents of saliva have been reported in aphid species, with more proteins identified in polyphagous M. persicae [18]. High level of saliva proteins could help polyphagous species fit variable environments [67]. In this study, more saliva proteins were found in WBPH and SBPH, and a portion of them were common in two species (Fig. 4). Considering the different feeding habit, the close relationship of salivary gland proteins and secreted saliva between WBPH and SBPH could be explained by host adaptation. Some saliva proteins or salivary gland proteins in WBPH or SBPH might be indispensable for
5. Conclusion In this study, comparative analysis of salivary glands and secreted saliva in three planthoppers were performed at a transcriptomic and proteomic level. The salivary glands of three planthoppers were metabolic active, and the oligophagous SBPH and WBPH were closer related when compared with monpohagous BPH. The planthoppers secreted a large number and diverse repertoire of saliva proteins, which contained insect-ubiquitous saliva proteins and planthopper-specific proteins. This work gained insight into the evolutional adaptation of insects to host plant by salivary approach, and would be useful in pest management. 8
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Table 2 Comparative analysis of planthopper saliva proteins with other insect species. Annotation
Genome ID
N.l
L.s
S.f
Elongation factor Aminopeptidase N Lipophorin GAPDH Serine protease Carboxylesterase Leucine-rich repeat-containing protein Carbonic anhydrase Protein lava lamp Plancitoxin-1 Annexin_like protein Salivap_3 Uncharacterized Uncharacterized Uncharacterized Uncharacterized Vitellogenin Actin Nesprin-1 EF-hand domain containing protein TBC1 domain family member Structural maintenance of chromosomes protein Lysosomal alpha-mannosidase Venom dipeptidyl peptidase 4 PI-PLC X domain-containing protein 1-like Carboxylase Salivap-5 La-related protein CG11505 isoform X3 Transforming growth factor-beta-induced protein ig-h3 Uncharacterized Peritrophin_like_protein Mucin like protein Protein_disulfide_isomerase Leucine–tRNA ligase, cytoplasmic-like Uncharacterized Thyroid receptor-interacting protein Myosin Presequence protease Exosome component Laminin AT-rich interactive domain-containing protein Kinectin-like isoform THO complex subunit Centrosomal protein Intraflagellar transport protein Nucleoprotein Centrosomin Kinesin-associated protein Guanine nucleotide-binding protein Uncharacterized protein Glycine receptor Arrestin Spectrin Tetratricopeptide repeat protein DnaJ-like protein Ribosomal Glucose dehydrogenase Uncharacterized, putative sheath Trehalase Effector C002 Effector (Mp1 Pinto1) Effector (Me10/Mp58) Cytochrome oxidase
NLU023845 NLU025909 NLU010815 NLU026274 Several NLU028181 NLU025113 NLU007482 NLU001907 NLU020028 several NLU027618 NLU023223 NLU026221 NLU012896 NLU005924 NLU019208 NLU010270 NLU002722 NLU024064 NLU003550 NLU023991 NLU010922 NLU004783 NLU028828 NLU002883 NLU022620 NLU008087 NLU004268 NLU007429 NLU003617 NLU004518 NLU001990 NLU005007 NLU025004 NLU005236 NLU010186 NLU021532 NLU005952 NLU012725 NLU013185 NLU005357 NLU010210 NLU002233 NLU010377 NLU028559 NLU022979 NLU020280 NLU020395 NLU016453 NLU023442 NLU006361 NLU007265 NLU012904 NLU005039 Several Several ACYPI009881 Several ACYPI008617 ACYPI006346 ACYPI008224 ACYPI007006
Y Y Y Y Y Y Y Y
Y
Y Y Y Y Y Y Y
Y Y Y Y Y Y Y Y Y Y Y Y Y
Y Y Y Y Y Y Y Y Y Y Y
Y Y
Y Y Y Y Y Y Y Y Y Y Y Y Y Y
Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y
Y
Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y
H.h
N.c
M.e
A.p
M.p
D.n
Y Y Y Y
Y Y Y Y
Y Y
Y
Y Y Y Y
Y
Y
Y
Y Y Y Y
Y
Y Y Y Y
Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y
Y
Y Y Y Y Y Y Y Y
Y Y Y
Y Y
Y Y Y
Y Y Y
Y Y
Y
Comparison of saliva proteins from N.l, N. lugens (BPH); L.s, L. striatellus (SBPH); S.f, S. furcifera (WBPH) with other insect species of H.h, H. halys [12]; N.c, N. cincticeps [10], A.p, A. pisum [9,18], M.e, M. euphorbiae [27], D.n, D. noxia [11], and M.p, M. persicae [18,28]. Only the proteins exhibited high sequence similarity and similar annotation were grouped together.
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Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jprot.2017.11.003.
The http://dx.doi.org/10.1016/j.jprot.2017.11.003 associated with this article can be found, in the online version.
Conflict of interests All authors have no conflicts of interest to declare. 9
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Acknowledgements
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11
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Journal of Proteomics journal homepage: www.elsevier.com/locate/jprot
Combined transcriptomic/proteomic analysis of salivary gland and secreted saliva in three planthopper species Hai-Jian Huanga,b, Jia-Bao Lub, Qiao Lib, Yan-Yuan Baob, Chuan-Xi Zhangb, a b
⁎
Department of Entomology, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China Institute of Insect Science, Zhejiang University, Hangzhou 310058, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Planthopper Transcriptome Proteome Salivary glands Saliva Comparison
The planthoppers are piercing-sucking pests that continuously inject saliva into host plants using specialized stylets. However, knowledge on the constituent and function of planthopper saliva proteins was still limited. In this study, the transcriptomic and proteomic approach were adopted to characterize the composition of salivary glands and their secreted saliva in three planthoppers, respectively. Gene repertoires of salivary glands in brown planthopper (Nilaparvata lugens, BPH), white-backed planthopper (Sogatella furcifera, WBPH) and small brown planthopper (Laodelphax striatellus, SBPH) were very similar, which actively involved in protein synthesis and energy metabolism. Comparative analysis of saliva proteome was performed among three planthoppers and other reported insect species. The saliva composition in three planthoppers was diverse, with 55 saliva proteins commonly identified in more than two species. A few proteins, including serine protease, carboxylesterase, aminopeptidase N, lipophorin, elongation factor, carbonic anhydrase, and calcium binding protein were ubiquitous distributed in different insects, indicating conserved function of saliva. While, the majority of saliva proteins were specifically identified in planthoppers, which might be the evolutional adaptation of insects to different hosts. Our work gained insight into the interaction between insect and host plant through salivary approach, and provided a good resource for functional characterization of effectors. Biological significance: Secreted saliva from insects is attracting immense research interest on the global level due to the crucial roles in determining the compatibility between the insects and their hosts. The three planthoppers: brown planthopper (Nilaparvata lugens, BPH), small brown planthopper (Laodelphax striatellus, SBPH), and whitebacked planthopper (Sogatella furcifera, WBPH) caused serious damage to rice plants throughout Asia. However, knowledge on the composition and function of their secreted saliva proteins was limited. Our study characterizes the global gene expression of salivary glands and their secreted saliva by Illumina sequencing technology and LC–MS/MS analysis, respectively. By comparative analysis, the ubiquitous and specific saliva compounds in different insects were unveiled.
1. Introduction Saliva, an oral secretion predominantly produced from salivary glands, mediate the intimate interaction between insect and host plant during all feeding stages, especially the probing and ingestion process [1]. It was the mixture of bioactive compounds that played multiplied roles in lubrication, digestion, penetration, and handling plant defenses [2–5]. Recently, with the development of sequencing technology, the composition of salivary glands and secreted saliva in several herbivorous insects have been unveiled [6–12]. Saliva proteins such as SHP, C002, and NlShp were indispensable for insect feeding [5,13,14]. However, current knowledge on saliva was still limited, especially the saliva composition that ubiquitously or specifically existed among
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insect species. The brown planthopper (Nilaparvata lugens, BPH), small brown planthopper (Laodelphax striatellus, SBPH), and white-backed planthopper (Sogatella furcifera, WBPH) are most destructive insect pests that belong to Delphacidae [15]. Although all three species use rice as their main food source, their host range was a bit different. The BPHs were extremely monophagous that restricted to rice plant, while the WBPHs and SBPHs were oligophagous that can survived on several Poaceae plants including rice, wheat and maize [16,17]. As an important tissue for insect feeding, the salivary glands played critical roles in host selection. It was of great interest to unveil the different gene repertoires of this secretory tissue. Moreover, saliva directly mediate the plant and insect interaction. The larger complement of saliva
Corresponding author. E-mail address: [email protected] (C.-X. Zhang).
https://doi.org/10.1016/j.jprot.2017.11.003 Received 30 August 2017; Received in revised form 30 September 2017; Accepted 1 November 2017 1874-3919/ © 2017 Elsevier B.V. All rights reserved.
Please cite this article as: Huang, H.-J., Journal of Proteomics (2017), http://dx.doi.org/10.1016/j.jprot.2017.11.003
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proteins was essential for insect to combat a greater diversity of plant defenses [18]. Only a few effector proteins were reported in aphid [19–21]. The potential effector molecules that associated with different host adaption in planthoppers were still unknown. In this study, we characterize the global gene expression of salivary glands and their secreted saliva by Illumina sequencing technology and Liquid Chromatography–Mass Spectrometry/Mass Spectrometry (LC–MS/MS) analysis, respectively. By parallel comparison, we found that the gene repertoires of three species were very similar, but their saliva secretion varied. In addition, we compared the saliva proteins of planthoppers with other insect species. The ubiquitous and specific saliva compounds were unveiled. Our result provided a deeper understanding on plant-insect interaction.
by the reference score and normalized from 0 to 1. BPH, WBPH, and SBPH were used as reference library, respectively. Four quadrants were derived from a threshold value of 0.4, which empirically represented a commonly used threshold for peptide similarity as follows: approximately 30% amino acid identity over approximately 30% of the peptide length. BSR of X-axis < 0.4 and Y-axis < 0.4 represents no BLAST match in reference; X-axis < 0.4 and Y-axis > 0.4 represents conserved peptides in reference and query species peptides on the Y-axis; Xaxis > 0.4 and Y-axis > 0.4 represents conserved peptides in all species; X-axis > 0.4 and Y-axis < 0.4 represents conserved peptides in reference and query peptides on the X-axis.
2. Material and methods
The planthoppers were removed from rice plants by gentle shaking, and the fifth instar nymphs were transferred to sterile diets with 2.5% sucrose. The diet was prepared under aseptic conditions and filtered through a 0.22 μm syringe filter (Millipore, MA, USA). Appropriately 100 planthoppers were trapped in each glass tube with 1 mL diet provided between two layers of Parafilm (Neenah, WI, USA). After 24 h feeding, the diets were collected from the space between the two layers of Parafilm with a pipet. Ultrafiltration was conducted using a 3-kDa molecular-weight cutoff Amicon Ultra-4 Centrifugal Filter Device (Millipore) at 5000g at 4 °C for 30 min. The concentrated samples were precipitated using a trichloro-acetic acid protein precipitation kit (Sangon, Shanghai, China). The pellets were solubilized in 200 μL of SDT buffer (4% sodium dodecyl sulfate (SDS; Sigma, St. Louis, MO, USA), 100 mM dithiothreitol (DTT; Sigma), 150 mM Tris-HCl pH 8.0) and incubated in hot water for 15 min, and then centrifuged at 14,000g for 45 min.
2.6. Collection of SBPH and WBPH saliva
2.1. Insect strains The BPH, SBPH, and WBPH population were originally collected from a rice field in Huajiachi Campus of Zhejiang University, Hangzhou, China. The insects were reared at 26 ± 0.5 °C on rice seeding (Oryza sativa strain Xiushui 134) with 50 ± 5% humidity under a 16:8 h light:dark photoperiod. 2.2. Collection of salivary glands and cDNA library preparation The fifth-instar insect of BPHs, SBPHs, and WBPHs were anesthetized on ice and their salivary glands were dissected as described previously (Fig. S1) [22]. Total RNA were extracted from 200 salivary glands using RNAiso plus (TaKaRa, Dalian, China). After determination of RNA integrity and quantity, the poly (A)+ RNA was purified from 20 μg pooled total RNA using oligo (dT) magnetic beads. Fragmentation was conducted in the presence of divalent cations at 94 °C for 5 min and the cleaved RNA was transcribed according to manufacturer's instructions. After end-repairing and adaptor ligation, the products were PCRamplified and purified using a QIAquick PCR purification kit (Qiagen, Hilden, Germany) to create a cDNA library.
2.7. In-solution digestion Protein digestion was conducted according to Yu et al. recommendation [25]. Briefly, the soluble samples were concentrated with a 3-kDa filtration unit and alkylated with 200 mM IAA for 1 h. To remove DTT and other low-molecular-weight components, the samples were washed by UA buffer (8 M Urea, 150 mM Tris-HCl pH 8.0) twice, and 25 mM NH4HCO3 (Sigma) twice. Digestion was performed using the trypsin in NH4HCO3 buffer overnight at 37 °C. After centrifugation, the digested peptides were desalted using C18 pipette tip (Thermo Scientific, Rockford, IL61101, USA) according to manufacturer's protocol. Then, the samples were concentrated by vacuum drying and dissolve in 30 μL of 0.1% (v/v) formic acid.
2.3. Illumina sequencing and transcriptome assembly The cDNA library was performed using Illumina sequencing platform and the raw data from the images were generated using Solexa GA pipeline 1.6. After removal of low quality reads, processed reads were assembled using Short Oligonucleotide Analysis Package (SOAP) de novo software and clustered with TIGR Gene Indices (TGI) Clustering tools.
2.8. LC–MS/MS analysis
2.4. Unigene annotation and peptide prediction
LC–MS/MS analysis was performed as follow: The digested peptides (20 μL) were loaded onto the trap column at a flow rate of 10 μL/min by Thermo Scientific Easy nanoLC 1000 (Thermo Fisher Scientific, MA, USA). After trap equilibration, the samples were eluted with a linear gradient of buffer A (0.1% formic acid) and buffer B (84% acetonitrile and 0.1% formic acid) at a flow rate of 250 nL/min. The chromatographic system includes a trapping column (75 μm × 2 cm, nanoviper, C18, 3 μM, 100 Å) and an analytical column (50 μm × 15 cm, nanoviper, C18, 2 μM, 100 Å). Separated MS data were analyzed using Thermo LTQ-Orbitrap Velos Pro (Thermo Fisher Scientific) equipped Nanospray Flex ionization source and FTMS (Fourier transform ion cyclotron resonance mass analyzer) combined with Thermo LTQOrbitrap Elite equipped Ion Trap analyzer. The top 20 ions were chosen from a full mass scan (300–2000 m/z) by collision induced decomposition (1.0 m/z isolation width, 35% collision energy, 0.25 activation Q, 10 ms activation time). Dynamic exclusion duration was 60 s. Survey scans for MS1 were acquired at a resolution of 30,000 at m/z 400. The MS/MS spectra were searched against the three peptide databases of salivary glands as we generated above using SEQUEST HT
The assembled unigenes were analyzed by searching the GenBank databases using BLASTX algorithm (http://www.ncbi.nlm.nih.gov/). Gene Orthology (GO) and KEGG Orthology (KO) annotations of the unigenes were determined using Blast2go (http://www.blast2go.org/) and InterProScan software (http://www.ebi.ac.uk/Tools/pfa/iprscan/). In addition, the unigenes were subjected to the Clusters of Orthologous Groups (COG) database (http://www.ncbi.nlm.nih.gov/COG/). The coding sequence (CDS) of each unigene was analyzed using blastx and Estscan software 3.03 [23]. The generated peptide database was used to support the proteomic analysis. 2.5. BLAST score ratio (BSR) test BSR tests [24] were used to global visualize the degree of proteome similarity between BPH, WBPH, and SBPH. BLAST software (http:// blast.ncbi.nlm.nih.gov/Blast.cgi) was used for the reference comparison. The BSR index was calculated by dividing the BLAST query score 2
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Table 1 Statics of three transcriptomes. Species
Raw reads
Clean reads
Unigene
N50 (bp)
Unigene mean length (bp)
Predicted protein (Total transcriptome)
Predicted protein (Top 1000 genes)
Signal peptide (Top 1000 genes)
BPH SBPH WBPH
45,781,625 44,298,976 47,687,228
44,582,696 40,212,148 43,244,348
43,070 44,113 42,210
1869 1399 2063
1066 864 1140
22,176, 51.5% 24,340, 55.1% 24,383, 57.8%
903 936 917
171, 18.9% 158, 16.9% 152, 16.6%
grouped together.
search engine configured with a Proteome Discoverer 1.4 workflow (Thermo Fischer Scientific, Bremen, Germany), respectively. The search parameters were set as follow: 10 ppm mass tolerances for MS; 0.8 Da tolerances for MS/MS; trypsin as proteolytic enzyme; 2 missed cleavages allowed; oxidation and deamidated as dynamic modifications; carbamidomethyl as a static modification. In addition, high peptide confidence was extracted and an automatic decoy database search was performed with false discovery rate (FDR) ≤ 0.01. The identified proteins with unipep ≥2 or with a signal peptide were selected as the saliva proteins.
3. Results 3.1. Transcriptome assembly, gene annotation, and protein prediction The three transcriptome libraries of salivary glands (TLSG) from BPH, SBPH, and WBPH were constructed using the Illumina sequencing platform GAII (Tables 1, S2). After removing the low quality data, a total of 43,070 unigenes in BPH were generated with a mean length of 1066 bp and N50 of 1869 bp. For SBPH, sequence data contained 44,113 unigenes with a mean length of 864 bp and N50 of 1399 bp. The TLSG of WBPH contained 42,210 unigenes with a mean length of 1140 bp and N50 of 2063 bp. The coding sequences were firstly predicted by searching against the NCBI NR, SwissProt, COG, and KEGG databases. A total of 19,566 unigenes (45.6% of all sequences in BPH), 21,525 unigenes (48.9% of all sequences in SBPH), 21,643 unigenes (51.4% of all sequences in WBPH) returned a significant BLASTX hit (Table 1). The remnant unigenes were further predicated by ESTScan software and additional 2610 (BPH), 2815 (SBPH), and 2740 (WBPH) putative proteins were found (Table 1). These predicted proteins provided query databases for the raw data of the proteome.
2.9. Bioinformatic analysis The SignalP 4.1 Server (http://www.cbs.dtu.dk/services/SignalP/) was used to predict potential signal peptides. The EditSeq 5.01 program (DNASTAR, Madison, WI, USA) was used to predict the Mw and isoelectric point (pI). According to GO annotation, the identified saliva proteins were classified into three main categories (biological process, cellular component and molecular function) using BGI WEGO (http:// wego.genomics.org.cn/cgi-bin/wego/index.pl). 2.10. Tissue-specific expressions analysis The insect tissue from salivary glands, guts, fat body, ovary, testis, and carcass were dissected under a Leica S8AP0 stereomicroscope, and the whole insect body was used as a reference. Total RNA was extracted as was described above. First-strand cDNA was synthesized using ReverTraAce qPCR RT Master Mix with a gDNA Remover Kit (ToYoBo, Osaka, Japan) to remove any contaminating genomic DNA. RNA with no reverse transcriptase was used as the no-template control (NTC). Quantitative real-time PCR was run on a CFX Connect Real-Time System (Bio-Rad, Hercules, CA, USA) using iQ SYBR Green Supermix Kit (Bio-Rad). The first-strand cDNA and a no-reverse-transcription control were used as templates for three biological replicates under the following reaction program: an initial denaturation step at 95 °C for 3 min followed by 40 cycles at 95 °C for 10 s and 60 °C for 30 s. The gene-specific primers were designed using the Primer Premier 5.0 program. The housekeeping gene for β-actin and GAPDH was used as an internal control (Table S1). The ΔΔCt method was used to evaluate the quantitative variation in the transcript levels as described previously [26].
3.2. Function analysis of high expression genes in salivary gland To overview the function of salivary gland, the 1000 most highly expressed genes (Top 1000 genes) were selected for further analysis. It was interestingly to find that the ribosomal proteins (99 genes in BPH, 84 genes in SBPH, and 83 genes in WBPH) played the dominant in this secretory tissue (Table S2), indicating the evaluated protein synthetic process. More than 16% of predicted protein contained a signal peptide (Table 1), which might be delivered into saliva through the eukaryotic endoplasmic reticulum–Golgi pathway [1]. Genes which were previously identified as saliva proteins of BPH (such as carboxylesterase, annexin-like protein, cathepsin, mucin-like protein), were also abundantly expressed in the salivary gland of three species (Table S2). We subsequently compared the COG annotation data between the total transcriptome and the top 1000 genes in three species, respectively (Fig. 1). The function sets of total transcriptome in salivary glands were very similar among three species. Increased percentage of function sets associated with energy production, translation, and posttranslational modification were detected in top 1000 genes, indicating an increased energy production and protein synthetic process in salivary glands.
2.11. Comparative analysis of insect saliva proteins The saliva proteins from Halyomorpha halys [12], Nephotettix cincticeps [10], Acyrthosiphon pisum [9,18], Macrosiphum euphorbiae [27], Diuraphis noxia [11], and Myzus persicae [18,28] were extracted according to published sequence information. Then, comparison of saliva proteins from BPH, SBPH, and WBPH was performed. Only the saliva proteins identified in at least two planthoppers' saliva were underwent further analysis. The ubiquitous and specific saliva proteins in planthoppers and other insect species were identified using BLAST alignment (ftp://ftp.ncbi.nlm.nih.gov/blast) with a significance cutoff of Evalue < 10− 5. The proteins showed high sequence similarity were further verified by BLAST search against NCBI database. Only the proteins exhibited high sequence similarity and similar annotation were
3.3. BSR analysis of three transcriptomes The BSR analysis was used to global visualize of the degree of proteome similarity between all three transcriptomes. The results showed that more than half of salivary gland genes were conserved among three species, indicating the close relationship of planthoppers (Fig. 2). Using the predicted proteins of SBPH as a query, we found that 21.2% of SBPH genes showed high similarity with WBPH (but not with BPH), while only 6.2% of SBPH genes showed high similarity with BPH (not with WBPH). Similar results were observed when using proteins of 3
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Fig. 1. Percentages of COG function set annotation between top 1000 genes and total transcriptome in BPH (A), SBPH (B), and WBPH (C). The capital letter in abscissa represents as follow: A, RNA processing and modification; B, chromatin structure and dynamics; C, energy production and conversion; D, cell cycle control, cell division, chromosome partitioning; E, amino acid transport and metabolism; F, nucleotide transport and metabolism; G, carbohydrate transport and metabolism; H, coenzyme transport and metabolism; I, lipid transport and metabolism; J, translation, ribosomal structure and biogenesis; K, transcription; L, replication, recombination and repair; M, cell wall/membrane/envelope biogenesis; N, cell motility; O, posttranslational modification, protein turnover, chaperones; P, inorganic ion transport and metabolism; Q, secondary metabolites biosynthesis, transport and catabolism; R, general function prediction only; S, function unknown; T, signal transduction mechanisms; U, intracellular trafficking, secretion, and vesicular transport; V, defense mechanisms; W, extracellular structures; Y, nuclear structure; Z, cytoskeleton.
phosphate dehydrogenase (GAPDH) were correlated with oxidoreductase. We identified a total of 236 proteins from secreted saliva in SBPH by shotgun LC–MS/MS analysis (Table S4). Among them, 50 proteins contained a signal peptide. Genes such as annexin-like protein, salivap3, plancitoxin-1, stubble-2, carboxylesterase, carboxypeptidase, carbonic anhydrase, and mucin-like protein were also identified in BPH saliva (Fig. 4). GO analysis showed that the gene set associated with cell (28 proteins), cellular process (32 proteins), metabolic process (21 proteins), binding (29 proteins), and catalytic activity (28 proteins) played the dominant in SBPH saliva, which was very similar with that of BPH (Fig. 3B). For ion binding genes, EF-hand domain-containing protein, spectrin, and annexin-like protein participated in calcium binding; titin and E3 ubiquitin-protein ligase participated in zinc binding; adenylosuccinate synthetase participated in magnesium binding. There were five genes exhibited peptidase activity, including stubble-2, ATP-dependent Clp protease, prophenoloxidase activating factor, proteasome, and carboxypeptidase. Other genes including L-2hydroxyglutarate dehydrogenase, 26S proteasome non-ATPase regulatory, and peroxidase participated in oxidoreductase activity. A total of 177 proteins were identified in secreted saliva of WBPH, with 43 proteins containing a signal peptide (Table S5). There were 47 proteins showed high similarity with saliva proteins in BPH or SBPH (Fig. 4), including carboxylesterase, plancitoxin-1, annexin like protein, salivap-3, lipophorin, and venom dipeptidyl peptidase. Similar GO
WBPH as a query, indicating the closer relationship between SBPH and WBPH. 3.4. Saliva proteins of three planthoppers In our previous work, a total of 58 saliva proteins of BPH was identified using the databases generated by Xue et al. [29]. As the TLSG of BPH was available now, we search the MS/MS spectra against this new database. Moreover, the saliva proteins identified by Liu et al. [30] were also adopted and combined with our results. A total of 149 BPH saliva proteins were screened out and listed in Table S3. Among them, 6 saliva proteins (three unknown proteins, EF-hand motif protein isoform 1, serpin, and maltase A1) were uniquely identified using TLSG of BPH as reference database. There were 29 saliva proteins commonly existed in our saliva sample and Liu's [30], which included 19 secretory proteins (i.e., saliva-2, saliva-3, saliva-5, saliva-7, carboxylesterase precursor, and aminopeptidase N). As much as 78 proteins were specifically identified in Liu's saliva samples, and 36 proteins were specifically identified in our saliva samples. Gene Ontology (GO) analysis showed that the majority of saliva proteins participated in metabolic process (27 proteins) and cellular process (21 proteins) (Fig. 3A). While 27 and 24 proteins related with catalytic activity and binding, respectively. Genes such as annexin-like protein, EF-hand motif protein, and regucalcin-like participated in calcium binding. Other genes including glycolate oxidase, peroxiredoxin 1-like, and glyceraldehyde-34
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Fig. 2. BSR analysis of three planthoppers. (A) BSR obtained from comparing BPH to WBPH and SBPH. (C) BSR obtained from comparing SBPH to BPH and WBPH. (D) BSR obtained from comparing WBPH to SBPH and BPH. Each point in the figure represents a single peptide in reference library.
species, we group the proteins according to the gene annotation and sequence similarity. Proteins that commonly identified in planthopper were listed in Table 2. There were 12 proteins existed in all three planthoppers (Fig. 4). Among them, the serine protease, carboxylesterase, leucinerich repeat-containing protein were reported in H. halys saliva; the elongation factor were extensively distributed in aphid; other saliva proteins including plancitoxin-1, annexin-like protein, saliva-3, vitellogenin, and four unknown proteins were exclusively distributed in planthoppers. There were 43 saliva proteins commonly existed in two planthopper (Fig. 4), of which the aminopeptidase N, lipophorin, GAPDH, actin, Nesprin-1, EF-hand domain containing protein, carbonic anhydrase, protein lava lamp, and TBC1 domain containing protein were also found in other insect saliva. The majority of saliva proteins were exclusively identified in planthoppers, indicating the speciesspecificity of saliva secretion. The saliva proteins commonly identified in aphid were also compared with the other insect species (Table 2). Eight proteins, which were predicted to be the major constituent of aphid saliva, were selected. To our expectation, the majority of proteins (glucose dehydrogenase, structural sheath protein, trehalase, effector C002, effector Mp1 Pinto1, effector Me10/Mp58, and cytochrome oxidase) were exclusively identified in aphid saliva (Table 2). Only the ribosomal protein, which expected to be abundant and involved in cellular core functions was also found in the saliva of N. lugens, H. halys, and N. cincticeps.
distribution of WBPH saliva was observed when compared with BPH or SBPH (Fig. 3C). Genes such as lysosomal alpha-mannosidase, ablim, protein prickle-like, and aminopeptidase N were predicted to binding zinc. The glutaredoxin and GAPDH were correlated with oxidoreductase activity. 3.5. Identification of salivary gland-highly expression genes Based on the information of gene annotation and signal peptide, 20 saliva genes in SBPH and WBPH were selected for spatial expression analysis, respectively. For SBPH, a total of 15 genes were verified to be highly expressed in salivary glands, especially the placental protein, annexin-like protein, stubble-2, and six unknown proteins, which exclusively expressed in this secretory tissue (Fig. 5). For WBPH, we found 12 genes expressed highly in salivary glands. Among them, the multiple inositol polyphosphate phosphatase 1-like, venom dipeptidyl peptidase, PI-PLC X domain-containing protein 1like, salivap-5, stubble-2, and six unknown proteins were exclusively expressed in salivary glands (Fig. 5). 3.6. Comparative analysis of insect saliva proteins To date, saliva proteins collected from bug (H. halys), leafhopper (N. cincticeps), and several aphid species (i.e. A. pisum, M. euphorbiae, D. noxia, M. persicae) have been identified by MS analysis. To unveil the ubiquitous and specific saliva proteins in planthoppers and other insect 5
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Fig. 3. Gene ontology classification of the saliva components in BPH (A), SBPH (B), and WBPH (C).
4. Discussion
large-portion of high expressed genes in salivary gland could be secreted, and participated in metabolism process. The insect continually secreted saliva during feeding, which needs a metabolically active factory to support protein synthesis. An enrichment of genes associated with protein synthesis, transport, and energy metabolism were also reported in salivary gland of Anopheles gambiae [31], Ixodes pacificus [32], Bemisia tabaci [33], and A. pisum [34], indicating the conserved biological function of this secretary tissue. The transcriptome of salivary gland from three planthoppers showed high similarity, but SBPH and WBPH were closer related when compared with BPH (Fig. 2). This might be explained by host
Saliva, which was crucial in mediating the interaction between herbivorous insect and host plant, played multiple roles in digestion, detoxification, and protection against host defense response. In the present study, we gain insight into the salivary gland and its secreted saliva of three planthopper species. By transcriptomic and proteomic approach, the saliva proteins that ubiquitously and specifically existed among insect species were unveiled. The salivary gland was an important secretary tissue that associates with insect feeding and viral transmission. Our study revealed that a 6
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response [47,48]. The function of carbonic anhydrase in insect saliva needs further investigation. Calcium binding proteins, including regucalcin [9,10,46], calreticulin [11,46], calmodulin [46], cadherin [6], annexin-like protein [46], and other EF-hand domain containing protein [10,12] were reported in most insect saliva. In response to stylet penetration, the calcium-triggered occlusion was induced, preventing sieve-tube sap from continuous leakage [49]. It was demonstrated that the presence of saliva induced dispersed forisomes to revert back to the contracted state, which functioned like EDTA [50]. The calcium-binding activity of EFhand domain containing protein (a saliva protein in N. cincticeps) was also verified, indicating the important role of saliva in calcium regulation [51]. However, current study found that different kinds of calcium binding proteins were distributed in insect saliva. This could be explained in two aspects: 1) the concentration of calcium binding proteins in some insect saliva was too low to be detected [11,52]; 2) the defense mechanism varied among host plants, and the different calcium binding protein was the result of evolutionary adaptation. Saliva proteins associated with oxidoreductase were ubiquitous identified in planthoppers and other insect species. When attacked by sucking insects, the soluble antioxidants in host plant enhance the effectiveness of the defensive system, and the oxidoreductase enzymes in saliva served to counter them [53]. The presence of glucose dehydrogenase, peroxiredoxin, and peroxidase in aphid saliva were presumed to involve in detoxification and suppression of plant defense responses [27]. In planthoppers, the glycolate oxidase, peroxiredoxin, GAPDH, peroxidase, and glutaredoxin were ubiquitous secreted, which might play a similar role with that of aphid. The glutaredoxin, for example, are essential for maintaining the cellular redox homeostasis and coping with toxicity [54]. It is likely that the oxidoreductase in saliva detoxified the secondary metabolic products and maintained the level of reactive oxygen species (ROS) in plant tissues. There were 42 saliva proteins uniquely identified in three planthoppers (but not in other insect speices), indicating a similar feeding habit of three species. Among them, annexin-like protein, plancitoxin-1, vitellogenin, saliva-3 and four unknown proteins were identified in all three planthoppers (Table 1). Annexin-like protein was presumed to participate in calcium binding. Different from insect annexins, these proteins were the uniquely identified in planthopper, which contained only 1–3 annexin repeats and have a putative signal peptide [46]. The ubiquitous presentation of annexin-like protein indicated that the planthoppers evolved new strategy to handle calcium regulation. The plancitoxin-1, also known as deoxyribonuclease, was a lethal factor responsible for the hepatotoxicity in mammal [55,56]. By degrading nuclear DNA, plancitoxin-1 caused caspase-independent apoptosis of cell [57]. It was intriguing to unveil such toxic proteins in saliva of planthoppers. The salivary toxins were supposed to be caused by virus [58] and saliva-induced apoptosis in host was reported in mosquito, which facilitated viral transmission by inhibiting the host immune cells [59,60]. Planthoppers harbor multiply symbiotic virus that can be horizontal transmitted [61,62]. The relationship between salivary plancitoxin-1 and viral transmission needs further investigation. Five proteins showing no homology with other species were commonly existed in three planthoppers. Among them, only the function of saliva-3 was unveiled as an indispensable factor for plant-associated feeding. Saliva directly mediate the interaction between insect and host plant. The secreted salivary gland proteins, which were under selection pressure for functional adaptation, showed a rapid diversification [63]. The specificity of saliva proteins were also reported in bugs, leafhoppers, and aphids [10,12,18]. Approximately 40% of the secreted proteins in leafhopper were not similar to known proteins [10]. The salivary genes of C002, ACYPI009881, ACYPI008224, and ACYPI006346 were also unique to aphid [7,13]. It was intriguing to uncover the function of these planthopper-specific genes in Poaceae-associated feeding.
Fig. 4. The Venn diagram of identified saliva proteins in three planthoppers.
specificity. The SBPH and WBPH were oligophagous, while BPH was monpohagous. Gene modulation occurred when insect transformed to different hosts [35–37], resulting in contraction or expansion of gene families associated with detoxification, digestion, and chemoreception [35,38]. As a matter of fact, the closer relationship between SBPH and WBPH was verified by viral transmission. South rice black-streaked dwarf virus, a member of Fijivirus that caused serious damage to wheat and rice plant, could be acquired by SBPH and WBPH, but not BPH [39]. The component of BPH saliva has been investigated by shotgun LC–MS/MS for several biological replicates (Table S2). The results showed that the saliva protein identified in different laboratories or different biological repeats in the same laboratory were diversified. Of 149 BPH saliva proteins reported so far, only a few proteins, such as carboxylesterase, salivap-3, salivap-5, and mucin like protein, were commonly identified in all experiments. Similar results were also found in A. pisum [9,18] and M. persicae [18,28], with different saliva proteins reported in the same insect species. In this study, we found that the frequency of saliva proteins identified in different replicates was closely related with their abundance. This might be caused by the concentration of collected saliva samples, or the saliva secretion was diversified in different conditions (i.e., BPH strain and rice variety). The different BPH saliva proteins identified in different replicates indicates variable, but also conservative of saliva secretion. Comparison of saliva proteins among insect species, we found serine protease, carboxylesterase, aminopeptidase N, lipophorin, elongation factor, GAPDH, carbonic anhydrase, and calcium binding protein were ubiquitous distributed in insect saliva, which played basic roles in insect feeding (Table 2). Among them, the serine protease, carboxylesterase, and aminopeptidase N were proteolytic enzymes [26,40,41]. We presumed that the presence of these enzymes might be correlated with digestive function of saliva. Extra-oral digestion was ubiquitous reported in predatory insect species [42]. Through injection of hydrolytic enzymes, the liquefied contents are withdrawn via the stylets, which greatly increase the efficiency of nutrient extraction [43]. The commonly distribution of digestive enzymes in herbivorous saliva indicated that the extra-oral digestion was also important for phloem feeding insects. In addition, the carboxylesterase can metabolize the insecticides or xenobiotics [44], while aminopeptidase N degraded the conjugated glutathione [45]. The commonly distribution of these proteins indicated a detoxifying role of insect saliva as well. Carbonic anhydrases, known for their role in reversible catalyzing the conversion of carbon dioxide to bicarbonate, were identified in N. lugens, L. striatellus, N. cincticeps, and M. euphorbiae. In our previous study, knocking down the expression of carbonic anhydrase resulted in premature death of BPH, but this gene did not influence the pH value of saliva or salivary glands [46]. The carbonic anhydrase in tobacco was reported to bind salicylic acid (SA), which modulated the plant defense 7
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Fig. 5. Tissue-specific expression (in fold) of genes encoding saliva proteins in SBPH and WBPH. SG, salivary gland; Fb, fat body; Ov, ovary; Te, testis; Ca, the remaining carcass; Wh, whole insect body.
planthopper feeding on Poaceae other than rice plant.
Although the saliva of three planthoppers demonstrated similar function categories, variation in saliva protein were found. Salivary action is responsible for host selection [64]. Traits such as substrate specificity of detoxification enzymes, avoidance or suppression of induced defenses were potential mechanisms underlying host breadth [65,66]. Different constituents of saliva have been reported in aphid species, with more proteins identified in polyphagous M. persicae [18]. High level of saliva proteins could help polyphagous species fit variable environments [67]. In this study, more saliva proteins were found in WBPH and SBPH, and a portion of them were common in two species (Fig. 4). Considering the different feeding habit, the close relationship of salivary gland proteins and secreted saliva between WBPH and SBPH could be explained by host adaptation. Some saliva proteins or salivary gland proteins in WBPH or SBPH might be indispensable for
5. Conclusion In this study, comparative analysis of salivary glands and secreted saliva in three planthoppers were performed at a transcriptomic and proteomic level. The salivary glands of three planthoppers were metabolic active, and the oligophagous SBPH and WBPH were closer related when compared with monpohagous BPH. The planthoppers secreted a large number and diverse repertoire of saliva proteins, which contained insect-ubiquitous saliva proteins and planthopper-specific proteins. This work gained insight into the evolutional adaptation of insects to host plant by salivary approach, and would be useful in pest management. 8
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Table 2 Comparative analysis of planthopper saliva proteins with other insect species. Annotation
Genome ID
N.l
L.s
S.f
Elongation factor Aminopeptidase N Lipophorin GAPDH Serine protease Carboxylesterase Leucine-rich repeat-containing protein Carbonic anhydrase Protein lava lamp Plancitoxin-1 Annexin_like protein Salivap_3 Uncharacterized Uncharacterized Uncharacterized Uncharacterized Vitellogenin Actin Nesprin-1 EF-hand domain containing protein TBC1 domain family member Structural maintenance of chromosomes protein Lysosomal alpha-mannosidase Venom dipeptidyl peptidase 4 PI-PLC X domain-containing protein 1-like Carboxylase Salivap-5 La-related protein CG11505 isoform X3 Transforming growth factor-beta-induced protein ig-h3 Uncharacterized Peritrophin_like_protein Mucin like protein Protein_disulfide_isomerase Leucine–tRNA ligase, cytoplasmic-like Uncharacterized Thyroid receptor-interacting protein Myosin Presequence protease Exosome component Laminin AT-rich interactive domain-containing protein Kinectin-like isoform THO complex subunit Centrosomal protein Intraflagellar transport protein Nucleoprotein Centrosomin Kinesin-associated protein Guanine nucleotide-binding protein Uncharacterized protein Glycine receptor Arrestin Spectrin Tetratricopeptide repeat protein DnaJ-like protein Ribosomal Glucose dehydrogenase Uncharacterized, putative sheath Trehalase Effector C002 Effector (Mp1 Pinto1) Effector (Me10/Mp58) Cytochrome oxidase
NLU023845 NLU025909 NLU010815 NLU026274 Several NLU028181 NLU025113 NLU007482 NLU001907 NLU020028 several NLU027618 NLU023223 NLU026221 NLU012896 NLU005924 NLU019208 NLU010270 NLU002722 NLU024064 NLU003550 NLU023991 NLU010922 NLU004783 NLU028828 NLU002883 NLU022620 NLU008087 NLU004268 NLU007429 NLU003617 NLU004518 NLU001990 NLU005007 NLU025004 NLU005236 NLU010186 NLU021532 NLU005952 NLU012725 NLU013185 NLU005357 NLU010210 NLU002233 NLU010377 NLU028559 NLU022979 NLU020280 NLU020395 NLU016453 NLU023442 NLU006361 NLU007265 NLU012904 NLU005039 Several Several ACYPI009881 Several ACYPI008617 ACYPI006346 ACYPI008224 ACYPI007006
Y Y Y Y Y Y Y Y
Y
Y Y Y Y Y Y Y
Y Y Y Y Y Y Y Y Y Y Y Y Y
Y Y Y Y Y Y Y Y Y Y Y
Y Y
Y Y Y Y Y Y Y Y Y Y Y Y Y Y
Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y
Y
Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y
H.h
N.c
M.e
A.p
M.p
D.n
Y Y Y Y
Y Y Y Y
Y Y
Y
Y Y Y Y
Y
Y
Y
Y Y Y Y
Y
Y Y Y Y
Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y
Y
Y Y Y Y Y Y Y Y
Y Y Y
Y Y
Y Y Y
Y Y Y
Y Y
Y
Comparison of saliva proteins from N.l, N. lugens (BPH); L.s, L. striatellus (SBPH); S.f, S. furcifera (WBPH) with other insect species of H.h, H. halys [12]; N.c, N. cincticeps [10], A.p, A. pisum [9,18], M.e, M. euphorbiae [27], D.n, D. noxia [11], and M.p, M. persicae [18,28]. Only the proteins exhibited high sequence similarity and similar annotation were grouped together.
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Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jprot.2017.11.003.
The http://dx.doi.org/10.1016/j.jprot.2017.11.003 associated with this article can be found, in the online version.
Conflict of interests All authors have no conflicts of interest to declare. 9
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Acknowledgements
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