Journal of Invertebrate Pathology 127 (2015) 115–121

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Peroxiredoxin 1 protects the pea aphid Acyrthosiphon pisum from oxidative stress induced by Micrococcus luteus infection Yongdong Zhang a, Zhiqiang Lu a,b,⇑ a b

Department of Entomology, College of Plant Protection, Northwest A&F University, Yangling, Shaanxi 712100, China Key Laboratory of Plant Protection Resources and Pest Management, Ministry of Education, Northwest A&F University, Yangling, Shaanxi 712100, China

a r t i c l e

i n f o

Article history: Received 13 December 2014 Revised 9 March 2015 Accepted 19 March 2015 Available online 26 March 2015 Keywords: Acyrthosiphon pisum Reactive oxygen species Peroxiredoxin Oxidative stress Infection

a b s t r a c t Reactive oxygen species (ROSs) are generated in organisms in response to infections caused by invading microbes. However, excessive ROSs will inflict oxidative damage on the host. Peroxiredoxins (Prxs) are antioxidative enzymes that may eliminate ROSs efficiently. In this study, ApPrx1 from the pea aphid Acyrthosiphon pisum was cloned, and its function was investigated in vitro and in vivo. In the presence of DTT, recombinant ApPrx1 protein from Escherichia coli showed antioxidative activity by eliminating H2O2 effectively. The H2O2 levels were significantly higher in Micrococcus luteus-infected aphids than in uninfected aphids, and ApPrx1 expression was remarkably up-regulated when the aphids were infected with M. luteus or injected with H2O2. When ApPrx1 expression was reduced by dsRNA injection, the survival of the aphids decreased significantly after M. luteus infection. Knockdown of ApPrx1 decreased M. luteus loads inside the aphids 48 h post-infection. While under infection conditions, the H2O2 levels were much higher in ApPrx1 knockdown aphids than in dsGFP-injected aphids, indicating that the decreased survival of the aphids was caused by increased oxidative stress. Taken together, our results reveal that ApPrx1 plays a protective role in oxidative stress caused by bacterial infection. Ó 2015 Published by Elsevier Inc.

1. Introduction Reactive oxygen species (ROSs), such as superoxide anion, hydrogen peroxide, and hydroxyl radical, are generated by living organisms to fight against foreign microbe invasion (Apel and Hirt, 2004). However, the persistence of high levels of reactive oxygen molecules will exert oxidative stress on the host cells and cause oxidative damage to DNA, proteins, and lipids (Wiseman and Halliwell, 1996; Dandona et al., 2001). To prevent ROS toxicity, organisms have evolved antioxidant enzymes, including superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), and

Abbreviations: AMPs, antimicrobial peptides; BCA, bicinchoninic acid ; BSA, bovine serum albumin; CFU, colony formation unit; DTT, dithiothreitol; HEPES, 4(2-hydroxyethyl)-1- piperazineethanesulfonic acid; HRP, horse radish peroxidase; IPTG, isopropyl b-D-1-thiogalactopyranoside; LB, Luria-Bertani; OD, optical density; ORF, open reading frame; PBS, phosphate buffered saline; PGRG, peptidoglycan receptor protein; PVDF, polyvinylidene difluoride; ROSs, reactive oxygen species; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TBS, trisbuffered saline; TBST, tris-buffered saline with Tween-20. ⇑ Corresponding author at: Department of Entomology, College of Plant Protection, Northwest A&F University, Yangling, Shaanxi 712100, China. Tel.: +86 29 8709 1997. E-mail address: [email protected] (Z. Lu). http://dx.doi.org/10.1016/j.jip.2015.03.011 0022-2011/Ó 2015 Published by Elsevier Inc.

peroxiredoxin (Prx), also known as thioredoxin peroxidase (TPx) (Corona and Robinson, 2006). Since the first cytosolic Prx1 was identified in Saccharomyces cerevisiae (Kim et al., 1988), the Prx family subsequently has been discovered in all organisms. All Prxs are cysteine-based peroxidases classified as 1-Cys or 2-Cys based on the number of conserved cysteine residues. The 2-Cys members are further subdivided into typical and atypical 2-Cys according to their structures and reaction mechanisms (Rhee et al., 2001). Due to their antioxidant properties, Prxs have been reported to participate in immune responses following pathogen infection in many species of invertebrates (Arockiaraj et al., 2012; Zhang et al., 2014) and vertebrates (Ishii et al., 2012; Mu et al., 2013). The participation of Prxs in immunity has also been observed in insects, including Bombyx mori (Lee et al., 2005; Zhang and Lu, 2015), Anopheles stephensi (Peterson and Luckhart, 2006), Drosophila melanogaster (Radyuk et al., 2010), Locusta migratoria (Wang et al., 2013), Spodoptera litura (Chen et al., 2014), and Ostrinia furnacalis (Liu et al., 2014). The pea aphid Acyrthosiphon pisum was hypothesized to have limited immunity due to the absence of genes essential to insect immune systems (Gerardo et al., 2010; Laughton et al., 2011). Several studies have indicated that ROS may contribute to A. pisum

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immune responses against pathogens (Altincicek et al., 2011; Schmitz et al., 2012). In the present study, we performed the first evaluation of ROS in A. pisum and characterized the function of ApPrx1 in the pea aphid’s defense against Micrococcus luteus.

using three biological replicates. The primers used for qPCR are listed in Table 1.

2. Materials and methods

The entire open reading frame (ORF) of ApPrx1 was amplified using two specific primers containing Nde I and Xho I restriction sites (Table 1) and then cloned into the pMD 19-T vector (Takara, Dalian, China). The ApPrx1 gene was excised with Nde I and Xho I, subcloned into the His-tagged expression vector pET28, and transformed into Rosetta (DE3) cells (Novagen, Darmstadt, Germany). A single transformant was cultured in 5 ml Luria–Bertani (LB) broth supplemented with 30 lg/ml kanamycin at 37 °C overnight. Subsequently, 3 ml of the culture was used to inoculate 300 ml LB broth, and when the optical density at 600 nm reached 0.6– 0.8, 0.5 mM isopropyl b-D-1-thiogalactopyranoside (IPTG) was added and the culture incubated for an additional 5 h at 25 °C. The cells were harvested by centrifugation at 8000g at 4 °C for 10 min, and the pellet was resuspended in 5 ml PBS with 100 ll protease inhibitor cocktail (Roche). The suspension was then sonicated for 2 min on ice and centrifuged at 12,000g at 4 °C for 15 min. The supernatant was separated using Ni2+-NTA resin (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The purified protein was quantified using a bicinchoninic acid (BCA) protein assay and visualized on a 15% SDS– PAGE gel. Western blot analysis was used to further detect ApPrx1. The separated gel was transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Darmstadt, Germany), which was then blocked for 4 h with blocking buffer (Tris-buffered saline (TBS), 5% skim milk powder, pH 8.3). The membrane was incubated with an anti-His-tag monoclonal antibody (Santa Cruz Biotechnology Inc., Dallas, TX, USA) (1:10,000) for 2 h, and washed five times with TBST (TBS, 0.1% Tween-20, pH 8.3). The membrane was then probed with a horseradish peroxidase (HRP)-conjugated secondary antibody (Santa Cruz Biotechnology Inc.) (1:20,000) for 1 h at room temperature. The protein bands were visualized using a Western blot detection kit (Advansta , Menlo Park, CA, USA) on a chemiluminescence imaging system (Clinx, Shanghai, China).

2.1. Aphids and treatments The pea aphids used in this study were captured in Yunnan, China and maintained on Vicia faba (broad bean) seedlings in a growth incubator at 21 ± 1 °C and 70 ± 5% relative humidity, on a 16-h light (L):8-h dark (D) photoperiod. The newly emerged female adults were placed on broad bean seedlings and allowed to produce progeny for 2 days. The adults were then removed, and the nymphs were reared on the plants until they became wingless adults. These generally concordant adult aphids were used for the treatments. For bacterial infection, M. luteus was cultured to log phase with an optical density (OD) of 1 at 600 nm and harvested by centrifugation. The pellet was resuspended in sterilized 0.85% NaCl solution to bring the final M. luteus cell suspension to 1010 colony forming units (CFU)/ml. The adult aphids were iceanaesthetized and injected using a capillary dipped into either the bacterial suspension or the sterilized 0.85% NaCl solution, according to Altincicek et al. (2011). For H2O2 treatments, aphids were injected using a micro-injector apparatus Nanoliter 2000 (World Precision Instruments, Sarasota, FL, USA) with 46 nl H2O2 at a 10-lM final concentration by diluting a 30% stock with sterilized phosphate buffered saline (PBS), while PBS only was used in the control. 2.2. RNA extraction, cDNA synthesis, and genomic DNA preparation Total RNA from each sample of 10 aphids was extracted using Trizol reagent (Roche, Basel, Switzerland) and purified using the Direct-zol RNA Miniprep kit (Zymo research, Irvine, CA, USA) according to the manufacturer’s instructions. The cDNA was synthesized using a reverse transcription system (Roche). The genomic DNA was isolated using the TIANamp Stool DNA Kit (Tiangen, Beijing, China) in accordance with the manufacturer’s instructions.

2.5. Production of recombinant ApPrx1

2.6. Peroxidase activity assay 2.3. Bioinformatic analysis and phylogenetic tree construction NCBI bioinformatics tools (http://blast.ncbi.nlm.nih.gov/Blast. cgi) were used to determine the conserved domains in ApPrx1. The theoretical isoelectric point and molecular weight were predicted using the ExPASy tool (http://web.expasy.org/compute_pi/ ). Homologous Prx1 sequences from other species were retrieved from the NCBI server and aligned using ClustalX2. The phylogenetic analysis was performed using MEGA (version 4.0, Tamura et al., 2007) with the neighbor-joining method. 2.4. Quantitative real-time PCR Quantitative real-time PCR (qPCR) was used to measure the expression levels of ApPrx1 in A. pisum after treatment. qPCR was performed using FS Essential DNA Green Master mix (Roche) in a 20-ll total volume on the CFX96 Real-Time System (Bio-Rad, Hercules, FL, USA). The cycling conditions were 94 °C for 1 min, followed by 40 cycles of 94 °C for 10 s, 60 °C for 10 s, and 72 °C for 30 s. After cycling completion, melt curve analysis was performed at 65 °C. The pea aphid ribosomal protein L7 (RpL7) gene (Nakabachi et al., 2005) was used as the endogenous reference. Relative expression was calculated using the 2 DDCt method (Schmittgen and Livak, 2008). Measurements were performed

The peroxidase activity of ApPrx1 was measured as described by Yu et al. (2011). Increasing concentrations of ApPrx1 (0, 12.5, 25, 50, 75, and 100 lg/ml) were added to 100 mM 4-(2-Hydroxyethyl)-1piperazineethanesulfonic acid (HEPES) buffer (pH 7.0) with or

Table 1 Primers used in this study. Primer

Sequence

Quantitative real-time PCR qPrx1F qPrx1R qRpl7F qRpl7R Recombinant protein expression ePrx1F

GCATATGGTTTTAGTACTTGAACAAC

ePrx1R

ACTCGAGTTATTCAACTGTTTTGA

dsRNA synthesis dsPrx1F dsPrx1R dsGFPF dsGFPR

AAAATTCAAGGGCACTGCTG CTGAAGTGGCTATCGCATGA TTGAAGAGCGTAAGGGAACTG TATTGGTGATTGGAATGCGTTG

a

GGCTAATGAATTTGCGGCTA TGGAATGCTTGAACAAGACG GTGTTCAATGCTTTTCCCGT a CAATGTTGTGGCGAATTTTG a a

Underline showed the Nde I and Xho I restriction enzyme sites. a Only gene-specific parts of the primers are listed. These are preceded by the T7 adaptor TAATACGACTCACTATAGGG for dsRNA synthesis.

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without 10 mM dithiothreitol (DTT) in a final volume of 1 ml for each reaction. After a 10-min incubation at 37 °C, H2O2 at a final concentration of 200 lM was added to the reactions, which were then incubated again at 37 °C for 10 min. The reactions were stopped by adding 100 ll 100% (w/v) trichloroacetic acid. The amount of residual H2O2 was determined by measuring the absorbance of the red-colored ferrithiocyanate complex at 490 nm after adding 200 ll Fe(NH4)2(SO4)2 and 100 ll 2.5 M KSCN to the reaction mixture. 2.7. DNA cleavage assay by the metal-catalyzed oxidation (MCO) system The capacity of ApPrx1 to protect DNA from oxidative nicking was measured as previously described (Wang et al., 2008). The reaction mixtures, containing 3.3 mM DTT, 16.5 lM FeCl3 and various concentrations (24, 48, 72, 96, and 120 lg/ml) of ApPrx1 in PBS, were incubated in 50-ll total volumes at 37 °C for 2 h. Then, 800 ng supercoiled pUC19 plasmid DNA was added to the reaction mixtures, which were incubated at 37 °C for another 2.5 h. After incubation, 10 ll each mixture was subjected to 1% agarose gel electrophoresis. Bovine serum albumin (BSA; 120 lg/ml) was used as a control. 2.8. H2O2 measurement Whole body H2O2 levels in aphids were determined as described previously (Pan et al., 2012). M. luteus-infected and uninfected aphids were collected in 50 mM sodium phosphate buffer (pH 7.4) containing 2 mg/ml catalase inhibitor 3-amino-1,2,4trizole. After homogenization, samples were filtered through a 10 K molecular weight cutoff spin filter (Millipore). The elution from each experimental group was collected and tested using the Hydrogen Peroxide Assay Kit (Invitrogen, Grand Island, NY, USA). The fluorescence intensity was detected at an excitation/emission = 550 nm/590 nm using a fluorescence microplate reader

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(Tecan, Männedorf, Switzerland). The values were normalized to the total amount of protein present in the samples. Ten aphids from each group were used for the assays. 2.9. Double-stranded RNA synthesis The pea aphid cDNA was used as template to amplify a 206-bp fragment of the coding sequence of ApPrx1 using the primers listed in Table 1. Gel-purified (Omega, Norcross, GA, USA) PCR products were used to synthesize dsRNA using the T7 RiboMAX™ Express RNAi System (Promega, Madison, WI, USA) according to the manufacturer’s instructions. The purified dsRNA was quantified spectrophotometrically at 260 nm, subjected to agarose gel electrophoresis to determine purity and integrity, and then stored at 80 °C until use. dsGFP was also synthesized and served as a control. 2.10. RNA interference and A. pisum survival assay The aphids were synchronized on ice and injected with 46 nl 6.2 lg/ll dsRNA and then placed on broad bean seedlings for 3 days before RNA extraction or for 2 days prior to inoculation with M. luteus. Aphid survival was recorded at 12-h intervals. The H2O2 levels and CFUs of M. luteus were determined after infection. For CFU measurements, the aphids were surface-sterilized by washing briefly with 75% ethanol and then washing with sterilized 0.85% NaCl solution twice to remove the ethanol. The cuticle of each aphid sample was then broken in 100 ll sterilized saline solution. The sample mixtures were diluted and spread onto LB agar plates. The bacterial colonies were counted after a 40-h incubation at 37 °C. 2.11. Statistical analyses The Student’s t test was used to compare H2O2 levels and CFUs between the controls and treated samples at each time point. The

Fig. 1. Characterization of ApPrx1 gene. (A) Comparison of the deduced amino acid sequences of ApPrx1 from Acyrthosiphon pisum, AaPrx (XP_001648521.1) from Aedes aegypti, TcPrx (XP_970797.1) from Tribolium castaneum, AcPrx (ADT65134.1) from Apis cerana cerana, BmPrx (NP_001037083.1) from Bombyx mori, DmPrx (NP_477510.1) from Drosophila melanogaster. Identical amino acid residues are shared in black. The amino acids conserved in catalytic regions are marked by asterisk. Conserved regions are boxed, and two sequences motifs (Region I and II) essential for dimer formation in 2-Cys Prx are underlined. (B) A phylogenetic tree generated by MEGA vesion 4.0 with Prxs from various insect species, including Acyrthosiphon pisum, Helicoverpa armigera (ABW96360.1), Spodoptera litura (AHF48620.1), Bombyx mori (NP_001037083.1), Danaus plexippus (EHJ66348.1), Drosophila melanogaster (NP_477510.1), Tribolium castaneum (XP_970797.1), Aedes aegypti (XP_001648452.1), Anopheles gambiae (XP_308081.2), Nasonia vitripennis (XP_001601016.1), Camponotus floridanus (EFN65636.1), Apis cerana cerana (ADT65134.1), Apis mellifera (XP_003249289.1), Toxoptera citricida (AAU84947.1), Maconellicoccus hirsutus (ABM55645.1) and Riptortus pedestris (BAN20543.1). (C) Structure of the ApPrx1 gene. The exons are indicated with white boxes, and the introns with black boxes. The translational start codon is marked by ‘‘D’’.

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respectively (Fig. 1A). ApPrx1 has two highly conserved cysteinecontaining motifs (FFYPLDFTFVCPTEI and HGEVCPA) (Fig. 1A). The two functional cysteine regions flanked by valine and proline residues (VCP) are conserved in the catalytic motif of Prx. Phylogenetic relationship analysis showed that ApPrx1 had closer orthologous relationships with three other hemiptera Prxs: TciPrx, MhPrx, and RpPrx. (Fig. 1B). Comparison of the amplicon sequences from genomic DNA and cDNA revealed that the ApPrx1 gene spans 3786 bp and contains three introns and four exons (Fig. 1C). 3.2. Expression and purification of recombinant ApPrx1 To investigate the function of ApPrx1, recombinant ApPrx1 was expressed and purified from Escherichia coli. The recombinant ApPrx1 protein was expressed as approximately 22 kDa in size, in both soluble and inclusion body forms (Fig. 2A). The soluble ApPrx1 was further purified using a Ni2+-NTA spin column, and the purified protein was detected by Western blotting using an anti-His antibody (Fig. 2B). 3.3. Characterization of recombinant ApPrx1

Fig. 2. Expression and purification of recombinant ApPrx1 protein. Protein samples were analyzed by SDS–PAGE (A) and Western blot (B). Lane 1, protein marker; lane 2 and 3, uninduced and induced over-expression of pET-28-ApPrx1 in BL21(DE3) cells; lane 4 and lane 5, supernatant and pellet of E. coli cell lysate, respectively; lane 6, purified recombinant ApPrx1 protein.

fold change was analyzed using one-way analysis of variance to determine the significance of gene expression. The Gehan– Breslow–Wilcoxon test was used to analyze the survival curves. 3. Results 3.1. Cloning and sequence analysis of ApPrx1 By searching AphidBase (http://www.aphidbase.com/), we identified the ApPrx1 gene (XM_001946102.2), which showed high similarity to previously reported Prx1 genes. The ApPrx1 gene contains a 582-bp ORF encoding a 193-amino acid protein with a predicted molecular mass of 21.51 kDa and an isoelectric point of 5.64. Multiple sequence alignment of the deduced protein sequence of ApPrx1 indicated that the ApPrx1 sequence had 75%, 70%, 70%, 70%, and 69% similarity to Prxs from Apis cerana, Tribolium castaneum, B. mori, D. melanogaster, and Aedes aegypti,

To examine the antioxidant activity of the recombinant protein, the ability of the ApPrx1 protein to remove H2O2 was measured in vitro in the presence or absence of DTT. As expected, when DTT was present, the rate of H2O2 degradation gradually increased with the concentration of ApPrx1, while BSA did not affect H2O2 degradation (Fig. 3A). In the absence of DTT, no apparent H2O2 degradation was observed (Fig. 3B). Additionally, the ApPrx1 protein could also protect the pUC19 plasmid DNA in a dosagedependent manner from cleavage caused by oxidative damage with DTT as an electron donor (Fig. 4). 3.4. Exogenous H2O2 and bacterial infection up-regulated ApPrx1 expression in A. pisum To confirm the involvement of ApPrx1 in oxidative stress, we examined its expression levels when aphids were given excess H2O2. ApPrx1 expression was up-regulated at 3 h, after which it declined to normal levels (Fig. 5A). To determine whether bacterial infection induced oxidative stress in pea aphids, we compared the levels of H2O2 in M. luteus-infected and uninfected aphids. The H2O2 levels were significantly higher in the infected aphids at 6, 12, and 24 h post-infection. At 48 h, the H2O2 concentration in infected aphids decreased to a lower level compared with the controls (Fig. 5B). The expression of ApPrx1 was also measured after bacterial infection. The results showed that ApPrx1 expression

Fig. 3. Activity assay of recombinant ApPrx1 protein. (A) H2O2 elimination in the presence of different concentration of ApPrx1 or BSA with DTT. The incubation time of the reaction mixture was 10 min. (B) H2O2 elimination in the presence or absence of DTT with 100 lg/ml ApPrx1 for different incubation times. Removal of H2O2 by ApPrx1 was monitored by measurement of the decrease in absorbance at 490 nm. Each value is given as the mean ± SE of three replicates.

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Fig. 4. ApPrx1 protected DNA from oxidative damage in a mixed-function oxidation system. Lane 1, pUC19 plasmid only; lane 2, pUC19 plasmid + FeCl3; lane 3, pUC19 plasmid + DTT; lane 4–9, pUC19 plasmid + FeCl3 + DTT + purified ApPrx1 (24, 48, 72, 96, 120 lg/ml, respectively); lane 10, pUC19 plasmid + FeCl3 + DTT + 120 lg/ml BSA. SF, supercoiled form; NF, nicked form.

Fig. 5. Detection of ApPrx1 expression and H2O2 levels in A. pisum. Expression analysis of ApPrx1 in M. luteus (A) and H2O2 (C)-challenged A. pisum. Sterilized saline injected samples were used as controls. Values shown are the mean (±SE) of three biological replicates. (B) Detection of H2O2 levels in M. luteus infected A. pisum. M. luteus treated aphids were collected in 50 mM PBS buffer (pH 7.4) containing 2 mg/ml of catalase inhibitor 3-amino-1,2,4-trizole. After homogenization, samples were filtrated through a 10 K molecular weight cutoff spin filter. The elution from each experimental group was collected and the fluorescence intensity was tested. The values were normalized by the total amount of proteins in the sample. Saline treated aphids were used as control. 10 aphids for each sample were used for measurement. ⁄, ⁄⁄ and ⁄⁄⁄ indicate statistical significance expression at p < 0.05, p < 0.01 and p < 0.001, respectively.

was highly induced at 6 and 12 h post-infection with M. luteus (Fig. 5C). 3.5. RNA interference verified the protective role of ApPrx1 in A. pisum The effect of RNA interference (RNAi) on ApPrx1 was determined by qPCR. Compared with dsGFP injection, ApPrx1 expression was reduced by 35% when the aphids were injected with dsApPrx1 for 3 days (Fig. 6A). The function of ApPrx1 against oxidative stress, which was induced by M. luteus infection, was investigated by measuring the survival of aphids after RNAi-mediated knockdown of ApPrx1. Knockdown of ApPrx1 reduced aphid survival after M. luteus infection (Fig. 6B). The CFU data showed that knockdown of ApPrx1 significantly decreased the concentration of M. luteus inside the aphids after 48 h of infection (Fig. 6C). Under the conditions of infection, H2O2 levels were much higher in ApPrx1 knockdown aphids than in dsGFP-injected aphids (Fig. 6D). These results indicate that the reduced survival of aphids was caused by increased oxidative stress. 4. Discussion The pea aphid harbors both obligate and facultative symbiotic bacteria. This makes it a valuable model for studying the molecular interactions between hosts and either beneficial or harmful microbes (International Aphid Genomics Consortium, 2010). Genomic analyses and other studies have indicated that the pea aphid has an inferior immune system compared with the fruit fly

and other insects. The most striking differences in microbial recognition and defense genes between the pea aphid and other studied insects are the pea aphid’s lack of peptidoglycan receptor proteins and antimicrobial peptides (Gerardo et al., 2010; Laughton et al., 2011). Altincicek et al. (2011) showed that the E. coli K-12 XL1Blue strain, which is more sensitive to oxidative stress than other E. coli K-12 strains, exhibited a significant lag phase before multiplying and killing aphids. Studies of cellular immunity also showed that ROS were present in pea aphid hemocytes (Schmitz et al., 2012). All of these studies have provided clues suggesting that ROS play a role in the pea aphid’s defense against pathogens. To obtain direct evidence that ROS play a role in pea aphid immunity, H2O2 levels were measured after M. luteus infection in our study. H2O2 levels in the aphids increased persistently within 24 h after M. luteus infection and then decreased to lower levels at 48 h (Fig. 5B). In D. melanogaster, rapid synthesis of ROS is triggered by natural gut infection (Ha et al., 2005). The dynamic cycle of ROS generation and elimination is vital in D. melanogaster, because the flies lack the capacity to remove ROS and therefore had increased mortality (Ha et al., 2005). When Lutzomyia longipalpis sand flies were infected by Serratia marcescens, their H2O2 concentrations exhibited a significant increase after blood feeding (Diaz-Albiter et al., 2012). In Anopheles gambiae, hemolymph H2O2 levels increased significantly 24 h after the ingestion of a Plasmodium berghei-infected blood meal, and induction of ROS detoxification enzymes prevented a further increase in hemolymph H2O2 levels (Molina-Cruz et al., 2008). Notably, infection with Wolbachia, a maternally transmitted symbiotic bacterium,

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Fig. 6. Effect of dsRNA-mediated knockdown in ApPrx1 expression, A. pisum survival, bacterial persistence and H2O2 levels after M. luteus infection. (A) Effect of ApPrx1 dsRNA interference. 0.3 lg dsApPrx1 was injected into each newly emerged aphid. Three days after injection, total RNA was extracted from three aphids for transcript analysis with qPCR. The control sample was from the aphids injected with dsGFP. Values shown are the mean (±SE) of three experiments. (B) Effect of dsRNA-mediated knockdown on the survival of A. pisum after M. luteus infection. n = 25. (C) Bacterial persistence in dsRNA injected aphids after M. luteus infection. Black bars indicate mean values, n = 8. (D) H2O2 levels in dsRNA injected aphids after M. luteus infection. ⁄ and ⁄⁄⁄ indicate statistical significance expression at p < 0.05 and p < 0.001, respectively.

led to induction of oxidative stress and an increased level of H2O2 in its mosquito host A. aegypti (Pan et al., 2012). However, consistent ROS exposure will lead to serious oxidative damage to the host’s tissues and cells. The antioxidant function of Prxs, which eliminate peroxides in cooperation with thioredoxin, has been demonstrated in several insect species. In the present study, we cloned and characterized the H2O2 detoxification enzyme ApPrx1, which can eliminate H2O2 efficiently in vitro in the presence of DTT (Fig. 3A and B). Based on the fact that excess H2O2 in A. pisum was removed 48 h after M. luteus infection (Fig. 5B), we speculated that ApPrx1 may take part in H2O2 removal. We observed that transcript levels of ApPrx1 were remarkably up-regulated in A. pisum after M. luteus infection (Fig. 5C). In B. mori, the expression levels of BmTPx were much higher in the fat body of larvae after exposure to baculovirus infection (Lee et al., 2005). The up-regulation of Prx expression was also found in A. stephensi after infection by the malaria parasite (Peterson and Luckhart, 2006). In particular, the role of Prx in prophylactic immunity has been suggested due to the high expression of Prx in gregarious locusts (Wang et al., 2013). To further validate the role of ApPrx1 in pea aphid defense against M. luteus infection, RNAi based on dsRNA injection was performed. The survival of aphids decreased significantly after M. luteus infection when ApPrx1 was knocked down (Fig. 6B). Compared to the control group, the dsApPrx1 injected group had lower bacterial load (Fig. 6C) and higher H2O2 level (Fig. 6D), suggesting that the reduction of bacterial load might be caused by increased H2O2 level, and the decreased aphid survival rate was the result of oxidative stress. In Schistosoma mansoni, it was demonstrated that when sporocysts were cultured with dsRNA targeting GST26, GST28, Prx1/2, and GPx, but not SOD, their susceptibility to H2O2-induced oxidative stress increased significantly (Mourão et al., 2009). In vitro destruction of S. mansoni sporocysts

by hemocytes of the snail Biomphalaria glabrata increased in larvae treated with Prx1/2 dsRNA (Mourão et al., 2009). In S. litura, the knockdown of SlTpx by dsRNA resulted in accelerated insect death during Nomuraea rileyi infection (Chen et al., 2014). Altogether, our present study provides evidence that bacterial infection results in the generation of H2O2 in pea aphids. Consequently, elevated H2O2 levels lead to oxidative stress and death in aphids. ApPrx1 is involved in the clearance of H2O2 and protects aphids from oxidative stress induced by bacterial infection. Acknowledgment This work was financially supported by the National Natural Science Foundation of China (No. 31272497). References Altincicek, B., Braak, B., Laughton, A.M., Udekwu, K.I., Gerardo, N.M., 2011. Escherichia coli K-12 pathogenicity in the pea aphid, Acyrthosiphon pisum, reveals reduced antibacterial defense in aphids. Dev. Comp. Immunol. 35, 1089–1095. Apel, K., Hirt, H., 2004. Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu. Rev. Plant Biol. 55, 373–399. Arockiaraj, J., Easwvaran, S., Vanaraja, P., Singh, A., Othman, R.Y., Bhassu, S., 2012. Immunological role of the thiol-dependent peroxiredoxin gene in Macrobrachium rosenbergii. Fish Shellfish Immunol. 33, 121–129. Chen, H., Yin, Y.P., Feng, E.Y., Li, Y., Xie, X., Wang, Z.K., 2014. Thioredoxin peroxidase gene is involved in Spodoptera litura: gene cloning, expression, localization and function. Dev. Comp. Immunol. 44, 76–85. Corona, M., Robinson, G.E., 2006. Genes of antioxidant system of the honey bee: annotation and phylogeny. Insect Mol. Biol. 15, 687–701. Dandona, P., Mohanty, P., Ghanim, H., Aljada, A., Browne, R., Hamouda, W., Prabhala, A., Afzal, A., Garg, R., 2001. The suppressive effect of dietary restriction and weight loss in the obese on the generation of reactive oxygen species by leukocytes, lipid peroxidation, and protein carbonylation. J. Clin. Endocrinol. Metab. 86, 355–362.

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Peroxiredoxin 1 protects the pea aphid Acyrthosiphon pisum from oxidative stress induced by Micrococcus luteus infection.

Reactive oxygen species (ROSs) are generated in organisms in response to infections caused by invading microbes. However, excessive ROSs will inflict ...
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