1 Introduction
E. integriceps, commonly known as Sunn pest, belongs to the order Hemiptera and family Scutelleridae. Sunn pest is a serious pest of cereals causes severe quantitative and qualitative (destruction of gluten protein) damage to crops (sometime up to 100%) by feeding on leaves, stems and grains (Konarev et al., 2011; Amiri and Bandani, 2013; Dolgikh et al., 2014; Yandamuri et al., 2014).

Pesticides as a major approach of insect pest control worldwide are associated with significant hazards to the environment and human health. As a result, there is a need to develop more benign and ecologically safe alternative methods for pest control. Since there is less molecular data about this important pest, Sunn pest molecular gene identification can be targets for developing new tools in integrated pest management.

Between different genes, this study focused on Sunn pest V-ATPase, cytochrome P450, and polygalacturonase gene identification because of their importance.

Vacuolar-type ATPases (V-ATPase) are multisubunit enzymes, which are composed of V0 (membrane bound subunits) and V1 (peripherally associated subunits) complex. The V1 complex that catalyzes the hydrolysis of ATP includes eight different subunits (A, B, C, D, E, F, G and H) and V0 that forms the proton conducting pore contains subunits with the a, c, c',c″, and d isoforms (Jefferies and Michael, 2007; Ma et al., 2011).

V-ATPases are multisubunit and multifunctional enzymes in eukaryotic cells that are ubiquitously found in the membranes of intracellular compartments, such as lysosomes, vacuoles, secretary granules, the trans-Golgi network and coated vesicles or are also found in the plasma membranes of cells.

V-ATPases are involved in the pH homeostasis and membrane secondary active transport processes energization in cell plasma membranes; while they pumps H⁺ (coupled with ATP hydrolysis) from the cytoplasm into the lumen of the organelles likelysosomes, secretory vesicles, and endosomes to acidify them in every eukaryotic cell or even can acidify whole insect’s midgut. Acidification is required for multiple cellular processes and modifying fluid and electrolyte transport (Jefferies et al., 2008; Wieczorek et al., 2009; Yang et al., 2009; Baumann and Walz, 2012; Holliday, 2014; Lu et al., 2014; Sahara et al., 2014).

Cytochrome P450 was another gene selected in this research. Cytochrome P450 monooxygenases are present in almost all organisms such as bacteria, fungi, plants and animals (Feyereisen, 2006). They are superfamily of heme-containing ubiquitous enzymes that play vital roles in herbivorous insect biology and physiology. Cytochrome P450s are important enzymes because their fundamental physiological functions in the hormone, pheromones, fatty acids, and steroids regulation such as juvenile hormone (JH) or 20- hydroxyecdysone (Feyereisen, 2012), biosynthesis or inactivation of endogenous or xenobiotic compounds (pesticides and plant phytochemicals) by catalyzing oxidation reactions (Feyereisen, 2005; Nielsen and Moller, 2005; Scott, 2008; Mizutani and Ohta, 2010; Schuler, 2011), insect growth, development, reproduction and evolutionary adaptation to environments (Hu et al., 2014; Chen et al., 2015; Yu et al., 2015). Insect P450s can induce insecticide resistance in pest population by catalysis of many reactions (Hardwick, 2008; Zhou et al., 2010).

According diverse functions, there are multiple P450 forms. More than 1000 cytochrome P450 genes have been reported in insects and new sequencing technologies facilitate rapidly increasing of this number (Nelson, 2009; Ai et al., 2011).

Plant innate immunity is based on an ancient system of molecules that defend the host against infection. Pectin is a complex carbohydrate and one of major component of plant physiological barrier against pathogens and insects. Polygalacturonases (PGs; EC 3.2.1.15) are produced by bacteria, fungi, nematodes and insects (Jaubert et al., 2002; De Lorenzo et al., 2003; Allen and Mertens, 2008; Prabhu et al., 2012). Insect PGs are hydrolytic enzymes degrade the α(1-4) linkages between the D-galacturonic acid residues of homogalacturonan, the main component of pectin and can play important role in plant-pest interactions (Kalunke et al., 2015). Fungi and bacteria polygalact-uronase have been studied extensively, but there are less PGs reports from several species of insects belonging to different taxa (Garcia-Maceira et al., 2001; Gotesson et al., 2002; D’Ovidio et al., 2004; Shackel et al., 2005; Frati et al., 2006; Allen and Mertens, 2008; Prabhu et al., 2012).

Therefore, the objectives of this research were to: 1) Molecular identify V-ATPase, cytochrome P450, and polygalacturonase genes in the Sunn pest genome for the first time; and 2) to study alteration in expression of salivary glands and gut polygalacturonase in response to different host plants.

2 Results
2.1 Identification and Homologous alignment
By searching in database, the E. integriceps V-ATPase subunit E and P450 protein homologues were found in various insects. The other protein or homologue sequences used in this study were downloaded from GenBank database. Protein alignment of V-ATPase subunit E and P450 demonstrated a high homology to that of Riptortus pedestris (Hemiptera: Alydidae) (97% identity) and Dendroctonus rhizophagus (Coleoptera: Curculionidae) (81% identity) respectively (Figure 1 and 2).

Figure 1 The alignment of amino acid sequences of V-ATPase subunit E from E. integriceps with the sequences of other insect V-ATPase subunit E: Plutella xylostella (Lepidoptera: Plutellidae), Culex quinquefasciatus (Diptera: Culicidae), Bombyx mori (Lepidoptera: Bombycidae), Danaus plexippus (Lepidoptera: Nymphalidae), Acyrthosiphon pisum (Hemiptera: Aphididae), Zootermopsis nevadensis (Isoptera: Termopsidae), Riptortus pedestris (Hemiptera: Alydidae), Cerapachys biroi (Hymenoptera: Formicidae), Papilio xuthus (Lepidoptera: Papilionidae), Leptinotarsa decemlineata (Coleoptera: Chrysomelidae), Maconellicoccus hirsutus (Hemiptera: Coccoidea), Coptotermes formosanus (Isoptera: Rhinotermitidae), Nilaparvata lugens (Hemiptera: Delphacidae), Anopheles darling (Hemiptera: Culicidae)

Figure 2 The alignment of amino acid sequences of cytochrome P450 from E. integriceps with the sequences of other insect cytochrome P450:  Leptinotarsa decemlineata (Coleoptera: Chrysomelidae), Dendroctonus ponderosae (Coleoptera: Curculionidae), Tribolium castaneum (Coleoptera: Tenebrionidae), Aedes aegypti (Diptera: Culicidae)

A 238 bp partial cDNA encoded a protein was denoted as E. integriceps V-ATPase subunit E, based on the BLASTx analysis indicating homology with V-ATPase subunit E Protein from Riptortus pedestris (Hemiptera: Alydidae) (97% identity), Culex quinquefasciatus (Diptera: Culicidae), Zootermopsis nevadensis (Isoptera: Termopsidae) (95%), Bombyx mori (Lepidoptera: Bombycidae) (94%) and Danaus plexippus (Lepidoptera: Nymphalidae) (94%). This cDNA sequence has been deposited in GenBank under accession number KR045860. The next 154 bp partial cDNA encoded a protein that it’s BLASTx analysis revealed homology with cytochrome P450 from Dendroctonus rhizophagus (Coleoptera: Curculionidae) (81% identity), D. valens (Coleoptera: Curculionidae) (79%), Aedes albopictus (Diptera: Culicidae) (43%), and Leptinotarsa decemlineata (Coleoptera: Chrysomelidae) (43%). Therefore, it was referred to the protein as E. integriceps cytochrome P450. This cDNA sequence has been deposited in GenBank under accession number KR045862. High level of conservation of the amino acid sequence among different insect V-ATPase subunit E proteins and also cytochrome P450 proteins indicates that these proteins are highly conserved during the evolution of insects.

Also the last partial cDNA encoded a 251 bp sequence that homology search revealed 100% identity with Lygus lineolaris (Hemiptera: Miridae) polygalacturonase PG1 mRNA, so was denoted as E. integriceps polygal- acturonase (PG). The PG sequence was deposited to GenBank with accession number KP295959.

2.2 Phylogenetic analysis
To understand the relationship between insect V-ATPase subunit E and cytochrome P450 and Sunn pest corresponding genes, phylogenetic analysis were performed using cDNA sequences available in GenBank. Neighbor-joining trees were constructed using amino acid sequences and a poisson-corrected distance with bootstrap test of 1000 replications. Phylogenetic analysis of proteins from insect species in the different orders such as Hemiptera, Diptera, Hymenoptera, Coleoptera, and Lepidoptera showed that V-ATPase subunit E and cytochrome P450 from the Sunn pest E. integriceps was closely related to R. pedestris (Hemiptera: Alydidae) and A. darlingi (Diptera: Culicidae) (Figure 3 and 4). The molecular evolution of V-ATPase genes showed a high level of conservation and a correlation with the taxonomy of the selected species.

Figure 3 Phylogenetic tree of amino acid sequences of V-ATPase subunit E of Sunn pest and other insects. Neighbour- joining method with genetic distance calculated. The numbers above the branches indicate the percentages of times that the species are grouped together in the bootstrap trees. The scale bar indicates the number of substitutions per site for a unit branch length

Figure 4 Phylogenetic tree of amino acid sequences of cytochrome P450 of Sunn pest and other insects. Neighbour- joining method with genetic distance calculated. The numbers above the branches indicate the percentages of times that the species are grouped together in the bootstrap trees. The scale bar indicates the number of substitutions per site for a unit branch length

2.3 Sunn pest response to different host plants
This experiment was done to investigate the effects of different hosts on the transcript level of PG gene. Wheat is the preferred host for the Sunn pest. However, the insect can also survive on barley, rye and triticale. To gain insight on the impact of different host types on the expression of PG gene, the expression patterns of the gene in the Sunn pest larvae feeding on wheat, barley, rye and triticale kernels were examined. To determine tissue and host dependent expression of PG, total RNA samples from the salivary glands and midgut were analyzed by real-time quantitative PCR. The results showed that this gene was differentially expressed in Sunn pest tissues and in response to different host plants kernels. In other words, the expression of PG gene was affected by host genus. This polygalacturonase was expressed only in the gut and there wasn’t any detectable PG transcript in the salivary glands (Figure 5A). For the Sunn pest larvae fed with barley, rye and triticale, gut PG mRNA expression decreased significantly comparing with that of the wheat fed larvae. Results showed that barley, rye and triticale caused 39%, 100% and 100% reduction in PG expression respectively (p<0.05) (Figure 5B). In conclusion, all these results indicated that the application of all-plant-based diet could decrease gene expression when compared with wheat.

Figure 5 Relative expression of polygalacturonase in fifth instar larvae of E. integriceps. A: in salivary glands and gut of wheat fed Sunn pest. B: in gut of insects feeding on either barley (B), rye (R) or triticale (T) in comparison with transcript levels in insects feeding on wheat (W) kernels. Ct values were first normalized to the endogenous control gene 18S ribosomal RNA gene, followed by normalization to the control without treatment using the 2-∆∆Ct method. Each kinetic point was performed in triplicate on 5 pooled larvae. Asterisks indicate significant difference (P<0.05) according to the iteration test (Rest 2009 Software). The values represent averages with vertical bars indicating SE

3 Discussion
E. integriceps causes serious economic losses to cereals especially to wheat and barley in wide areas of the world almost every year. Despite the importance of this key pest, almost no functional genomics data exist for the Sunn pest. In this study, for the first time cDNAs encoding V-ATPase subunit E, cytochrome P450 and polygalacturonase were isolated and identified from the Sunn pest, E. integriceps.

V-ATPases are multifunctional and important genes ofdifferent insect species and have potential for pest management (Baumann and Walz, 2012; Holliday, 2014; Lu et al., 2014; Sahara et al., 2014). Midgut lumen acidification by V-ATPases was reported in different insects such as Bombyx mori (Wieczorek et al., 2000; Lu et al., 2013), Aedes aegypti (Weng et al., 2003) and the American visceral leishmaniasis vector Lutzomyia longipalpis (Ramalho-Ortigão et al., 2007).

Other than acidifying, in insects, V-ATPases function as midgut, salivary glands and labial glands epithelia cation transporter (Wieczorek et al., 2009; Thakur et al., 2014). Also Huang et al. (2006) reported V-ATPases seem to associate with gregarine infection of the mosquito Aedes albopictus. As well as Lu et al. (2013) observed V-ATPases play important role in the defense response against Bombyx mori Nucleopolyhe-drovirus.

In insects, cytochrome P450s enzymes are very important, large and diverse group of enzymes that play vital roles in many different physiological processes (Hu et al., 2014; Chen et al., 2015; Yu et al., 2015). Therefore, identification of Sunn pest P450 could be useful in pest management strategies. To date, however, no P450 gene from E. integriceps has been reported. Our research is the first report of this vital gene in the Sunn pest.

There are many convincing evidence for P450s in several insect species. For example identification of Cytochrome P450 Genes have been reported in Cnaphalocrocis medinalis (Lepidoptera: Pyralidae) (Chen et al., 2015); diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae)(Hu et al., 2014; Yu et al., 2015); the mosquito, Aedes aegypti (Diptera: Culicidae) (Elgarj and Wajidi, 2013); Locusta migratoria (Orthoptera: Acrididae)(Guo et al., 2012), Asian corn borer, Ostrinia furnacalis (Lepidoptera: Crambidae) (Zhang et al., 2011), and the red flour beetle, Tribolium castaneum (Coleoptera: Tenebrionidae) (Xu et al., 2009).

Molecular characterization of polygalacturonases from agricultural pests will be helpful in formulating and selecting pest resistant crops. Phytophagous insect’s salivary glands PGs are considered as a main cause of plant damage (Boyd et al., 2002; Frati et al., 2006).

Following finding Sunn pest polygalacturonase gene (PG), we investigated whether and how different host plants affect the transcription levels of this gene. Host plant quality can significantly influence gene expression and the insect growth (Oppert et al. 2010; Vogelweith et al., 2011; Wang et al., 2013; Spit et al., 2014; Wang et al., 2015). Likewise in this study results clearly indicated that host plants quality can affect gene expression. So transcript level of PG was most abundant in the wheat fed Sunn pest larvae in compare to other hosts. Such results were observed in other insects. Phytophagous insects may exhibit differential expression patterns of various genes in response to plant defense (Bown et al., 1997).

Frati et al. (2006) reported PG activity in the salivary glands and the gut of different species of mirid bugs [Lygus rugulipennis Popp., L. pratensis (L.), Orthops kalmi (L.), Adelphocoris lineolatus (Goeze) and Closterotomus norwegicus (Gmelin)]. They also examined effect of different plant source polygalacturonase- inhibiting proteins on inhibition of mirid PGs. PGs of all the mirid bugs were inhibited by PGIPs from Phaseolus vulgaris.

Polygalacturonase-inhibiting proteins (PGIPs) are plant cell wall leucine-rich repeat glycoproteins that upregulated and counteract insect and patogen polygalacturonases (Frati et al., 2006; Janni et al., 2008; Prabhu et al., 2012; Tamburino et al., 2012; Kalunke et al., 2015). They can bind to the plant cell and play an important role in plant defense with inhibiting the pectin-depolymerizing activity of PGs. Sevaral studies reported plant cell wall PGIPs have been inhibited the PGs of insects and pathogens (Al-Obaidi et al., 2010; Prabhu et al., 2012; Bashi et al., 2013; Ferrari et al., 2013; Kalunke et al., 2015).

No plant species or mutants totally lacking PGIP activity has been characterized so far (Kalunke et al., 2015). Di Giovanni et al., (2008) genome analysis showed PGIPs exist as a single gene in diploid wheat species. However, other plant species have more PGIPs, such as two in Arabidopsis thaliana (Ferrari et al., 2003), and sixteen in Brassica napus (Hegedus et al., 2008). Al-Obaidi et al., (2010) reprted PGIP from barley.

We observed PG expression in the Sunn pest feed on rye and triticale was zero. Allen and Mertens (2008) reported isolation of three unique cDNAs encoding putative polygalacturonase from the tarnished plant bug, Lygus lineolaris (Hemiptera: Miridae). They studied differentially expression of Lygus PG genes in all feeding stages and observed a zero expression reading in at least one life stage of this hemipteran too. Also, Walker and Allen (2010) reported cotton-fed bugs had consistently lower PG transcript levels than those reared on artificial diet.

To conclude, we have for the first time identified three different genes encoding V-ATPase, cytochrome P450, and polygalacturonase from an important agriculture pest E. integriceps and analyzed polygalacturonase expression in different tissues of the Sunn pest reared on various hosts. Variation in host plant, strongly affects the level of polygalacturonase expression.

4 Materials and Methods
4.1 Insect rearing
Sunn pest adults were collected from wheat field during spring 2014. The insects were maintained on soaked wheat kernels and water in the laboratory as described by Allahyari et al. (2010). To determine the effect of different host species on gene expression, 100 eggs for each replicate (three replicates were considered for each treatment) in each treatment were reared separately on various hosts including wheat, barley, rye and triticale kernels. The insects were reared to fifth-instar when used for sample collection. In order to establish the developmental stage and to synchronize the treated fifth instar larvae, the molt was observed every day and the newly emerged larvae were collected every 24 h and placed on fresh soaked kernels and one day after ecdysis, fifth instar larvae were collected for gene expression experiment.

4.2 Tissue sampling
To obtain selected tissue samples (midgut and salivary glands), E. integriceps fifth instar larvae were dissected in sterile water under a stereoscopic microscope. Each tissue (midgut and salivary glands) was collected and pooled from individual insects and were transferred into a microtube containing 0.5 mL TRI reagent (~ 30 mg wet tissue) (Sigma, Aldrich).

4.3 RNA extraction and cDNA cloning
Samples were processed for total RNA extraction by TRI reagent using instruction provided by the manufacturer. To remove potential DNA contamination, total RNA samples were treated with DNase I. First strand cDNA was prepared with RNA (DNA free) and the oligo-dT primer using AccuPower® RocketScript™ Cycle RT PreMix cDNA synthesis kit (Bioneer, Korea). All cDNA concentrations were determined using a Nanodrop 1000 Thermo Scientific, diluted (ten-fold) and used for the next PCR reactions consisting of preheating 95°C for four min, followed by 30 cycles of 95°C for 30 s, 55°C for 30 s and 72°C for 30 s, and a final extension step of 72°C for seven min. Each 25µL PCR reaction contained 1µL of cDNA template, 2.5 µL of 10x PCR buffer, 1.5 µL of MgCl₂ (25 mmol/µL), 2µL of dNTP (2 mmol/µL each), 1.5 µL of forward and 1.5 µL of reverse primers (10 pmol/µL) (E primers for V-ATPase, P450 primers for cytochrome P450, PG primers for polygalacturonase, Table 1), 0.2 µL of Taq polymerase (5 U/µL), and 14.8 µL of ddH₂O. E and P450 primers were designed from two highly conserved regions of other insect genes and PG primers were obtained from the tarnished plant bug,Lygus lineolaris (Walker and Allen, 2010) (Table 1). Six microlitter of the PCR product was analyzed on a 1% TAE-ethidium bromide gel and the target band was isolated from the gel and recovered and purified by using the AccuPrep Gel Extraction Kit (Bioneer, Korea), cloned into the pTG19-T PCR cloning vector (Vivantis) and transformed into Escherichia Coli DH5α competent cells. Colonies with inserts were screened under standard ampicillin conditions. Recombinant plasmids were sequenced from both directions and hereby, gene sequences were confirmed by sequencing. The sequencing reaction was performed commercially.

Table 1 The primer sequences were used in the study

4.4 Sequences and phylogenetic analysis
The sequences of the V-ATPase, cytochrome P450, and polygalacturonase cDNA were confirmed by a homology search of other corresponding gene sequences known within the Blast program available on the GenBank database of National Center for Biotechnology Information (NCBI) website (http:// www.ncbi.nlm.nih.gov/BLAST/). The amino acid sequences of corresponding gene homologs from different organisms were retrieved from GenBank database. Sequences were aligned by using ClustalX, and phylogenetic trees were constructed by the neighbor-joining method with a Poisson correction model (1000 bootstrap replications to check for repeatability of the results) using the MEGA 5.0 software.

4.5 Qualitative gene expression assays
Real time PCR was employed to determine the expression of polygalacturonase in various tissues of E. integriceps in response to different hosts. The reactions were performed with Eva green master mix on a Rotor-Gene® Q system (QIA-Gene). Gene-specific primers (re-PG and 18S, Table 1) were designed using Primer Blast. PCR amplification using forward and reverse primers resulted in an approximately 100 bp fragment within the coding region of each gene. E. integriceps specific 18S ribosomal RNA gene (18S) was used as internal control. The 18S primers were designed according to Sunn pest 18S ribosomal RNA sequence in GenBank with accession number KP890857 by using primer blast online software (authors unpublished data). Prior to PCR, cDNA preparations from developmental stages were quantified. For every sample, three technical replicates were performed. Each reaction was performed with 5 μl of cDNA, 0.5 μM of forward and reverse primer (re-PG and 18S for separate reactions) and 4 μl of Eva green master mix in 20 μl total volumes. The PCR amplifications were performed with the following cycling conditions: one cycle at 95°C (15 min), followed by 40 cycles of denaturation at 95°C (15 seconds), annealing at 62°C for 20 sec and extension at 72°C for 20 sec. At the end of the program a melting curve for each primer (65-94°C read every 1°C) was acquired to ensure that only single products were generated.

The data on gene expression in different tissues of E. integriceps were normalized by subtracting cycle threshold (Ct) values from the corresponding 18S Ct values. The relative expression level of genes in different tissues was determined by comparative Ct method (2^-∆∆Ct) (Pfaffl, 2001).

4.6 Statistical Analysis
The data were given as means ± SE. Statistical significance was established as P<0.05. The significance of differences in the gene expression was determined by t-test using the Rest 2009 Software (Pfaffl et al., 2002). The PG expression levels were normalized relative to those of the control gene (18S).

5 Author contributions
Azam is a student and carried out the experiments and drafted the manuscript. Ali is Azam’s supervisor and participated in the design of the study, helped in statistical analysis and correction of the manuscript. Ali also paid from his grant for the project expenditure. Morteza is Azam’s advisor. All authors read and approved the final manuscript.

6 Acknowledgements
This research was founded by a grant (No. 86025.11) from the Iran National Science Foundation (INSF).

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