Proteomic Analysis of Insecticide Triazophos ... - ACS Publications

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Proteomic Analysis of Insecticide Triazophos-Induced Mating-Responsive Proteins of Nilaparvata lugens Stal (Hemiptera: Delphacidae) Lin-Quan Ge,† Yao Cheng,† Jin-Cai Wu,*,† and Gary C. Jahn‡ † ‡

School of Plant Protection, Yangzhou University, Yangzhou 225009, P.R. China Imaging Science and Information Systems (ISIS) Center, Georgetown University, Suite 603, 2115 Wisconsin Avenue, NW, Washington, DC 20007, United States ABSTRACT: The brown planthopper, Nilaparvata lugens (Stal) (Hemiptera: Delphacidae), is a classic example of a resurgent pest induced by insecticides. It has been demonstrated that triazophos treatment causes an increase in the content of male accessory gland proteins (Acps) that can be transferred to females via mating, influencing female reproduction. However, the mechanism of this type of insecticide-induced Acps in males and the subsequent stimulation of reproduction in females are not well understood. To identify changes in the types of Acps and reproductive proteins in mated females, we conducted a comparative proteomic analysis. Six samples were categorized into four different groups: (1) untreated unmated males compared to treated unmated males (UUM vs TUM); (2) treated unmated males compared to treated mated males (TUM vs TMM); (3) untreated unmated females compared to treated unmated females (UUF vs TUF); (4) treated unmated females compared to treated mated females (TUF vs TMF). Protein expression changes among the four different groups were examined by twodimensional gel electrophoresis (2-DE) and liquid chromatography tandem mass spectrometry (LC-MS/MS). Of the 500600 reproducibly detected protein spots on each gel, 107 protein spots were differentially expressed between the four different groups. Of the 63 proteins identified by LC-MS/MS analysis, 38 were up-regulated and 25 were down-regulated in the four different groups. Some novel proteins related to fecundity were observed including spermatogenesis-associated protein 5, testis development protein NYD-SP6, arginine kinase, actin-5C, vitellogenin, and ovarian serine protease nudel. The elevated expression of novel fecundity proteins in six samples of N. lugens females and males due to exposure to triazophos was confirmed by quantitative real-time PCR (qRT-PCR). The results suggest that these proteins may participate in the reproductive process of N. lugens adult females and males. Our findings fill a gap in understanding the relationship between insecticide-treated males and the stimulated reproduction of N. lugens females.

KEYWORDS: Nilaparvata lugens, triazophos, proteomics, 2-DE, LC-MS/MS, mating, reproductive response

1. INTRODUCTION The brown planthopper (BPH) Nilaparvata lugens (Stal) (Hemiptera: Delphacidae) is a serious pest of rice crops in the temperate and tropical regions of Asia and Australia.1 It is also a classic example of an insecticide-induced resurgent pest.2,3 Previous studies on insecticide-induced stimulation of fecundity in pests concentrated on the reproduction of adult females, but the effect of insecticide on reproduction in adult males has been neglected. Recently, some studies have demonstrated that insecticide treatments enhanced the content of accessory gland proteins (Acps) in BPH males.4,5 Interestingly, insecticidetreated males significantly increased the number of eggs laid by adult females relative to untreated males.4 The main objective of the present study attempts to answer several questions. What types of Acps changed after insecticide treatments? What are the molecular mechanisms of treated males that stimulate fecundity via mating in adult females? r 2011 American Chemical Society

Sperm storage, sperm maintenance, and egg activation in adult females are likely controlled by interactions between eggs, sperm, the female reproductive tract, and molecules contributed by both sexes.68 Males of many insect species transfer seminal fluid proteins (primarily Acps) that influence female reproductive physiology. In Drosophila melanogaster, males produce reproductive molecules that influence a wide range of processes including oogenesis, egg-laying, female receptivity, sperm transfer, and sperm storage.79 Acps transferred during mating are known to affect several behaviors in insects; reduced female receptivity and increased oviposition are the most common behavioral effects.10 Lay et al. (2004) found that male Locusta migratoria (L.) proteins play a significant role in reproduction: they discovered peptides Received: May 8, 2011 Published: August 01, 2011 4597

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Journal of Proteome Research from white secretions of the male accessory glands (MAGs) in developing eggs.11 Although gene discovery has been greatly accelerated by progress in structural and functional genomics, the functions of proteins that depend on post-translational and proteinprotein interactions cannot be inferred through genomic analysis. Proteomics has emerged as a powerful method to gain insight into physiological changes at the cellular level.1218 Two-dimensional gel electrophoresis (2-DE) combined with mass spectrometry (MS) has been frequently used in insect proteomics research.19,20 However, for large-scale characterizations of proteome profiles, an LC-MS/MS shotgun strategy is increasingly utilized. This method has been widely used in human disease research.18,21 Li et al. (2009) also identified thousands of proteins by using this method (LC-MS/MS) in insect studies.22,23 To date, a proteomic analysis of Acps in insecticide-treated males and correspondingly mated females has not been investigated. Using a comparative proteomic analysis of treated males and females, we will fill a gap in the understanding of the molecular mechanisms of the effects of insecticides on male reproduction and material transference during mating. In addition, the present results may provide new insights into the pest resurgence theory.

2. MATERIALS AND METHODS 2.1. Rice Variety and Culture

Rice (Oryza sativa L.) variety Ninjing 4 (japonica rice) was used in the trials. This variety of rice was selected because it is commonly planted in the Jiangsu province of China. Seeds were sown outdoors in cement tanks (height 60 cm, width 100 cm, and length 200 cm) containing standard rice-growing soil. When seedlings reached the six-leaf stage, they were transplanted into 16 cm diameter plastic pots that contained four hills per pot and three plants per hill. All rice plants used in the experiments reached the tillering stage. 2.2. Insect Culture and Insecticide

A laboratory strain of N. lugens that was originally obtained from the China National Rice Research Institute (CNRRI; Hangzhou, China) was reared in a greenhouse at an ecology laboratory at Yangzhou University. N. lugens was kept in the ecology laboratory at 26 ( 1 °C, with 7080% humidity and a 16 h light/8 h dark photoperiod (16L:8D). Technical triazophos (87%) was purchased from the Shenli Pesticide Co. Ltd., in Ningguo, Anhui, China. 2.3. Experiments

Triazophos was dissolved in acetone. Ten percent of an emulsifier was added and diluted to a concentration of 40 ppm on the basis of previous results from a sublethal test.24 A total of 400 third instars per pot were released onto the potted rice plants at the tillering stage. Twenty-four hours after insect release, rice plants at the tillering stage were sprayed with 40 ppm triazophos using a Jacto sprayer (Maquinas Agricolas Jacto S.A., Brazil) equipped with a cone nozzle (1 mm diameter orifice, pressure 45 psi, flow rate 300 mL/min). Control plants at the same stage were sprayed with the same amount of acetone and emulsifier. Each treatment and control was replicated three times. The treated and control plants were covered with cages (screen size: 80-mesh). When the nymphs reached fifth (final) instar, they were collected from the treated and control plants, and nymphs from treated and control plants were individually introduced into a glass jar (diameter 10 cm, height 12 cm) with untreated rice plants in a bioculture box under

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26 ( 1 °C and 16L:8D until adult emergence. After the adults emerged, a brachypterous male and a brachypterous female were put together for mating (a mated female is close to the male on rice plants, while an unmated female is far from the male, according to our observations). Adult males and females were collected 2 days after emergence (2 DAE) as either unmated or mated samples. It has been reported that both brachypterous adult females and males could mate 1 day after emergence and mate repeatedly, that the mating course of the experimental population lasted 70.38 ( 41.77 s, and that the percentage of successful mating was relatively higher.25 Two hundred unmated and 200 mated males or females were used for each replication in each treatment and control. To understand the molecular mechanisms of triazophos on the reproduction of males and females and mating transference, six samples were used for protein extract and 2-DE: (1) untreated unmated males (UUM); (2) treated unmated males (TUM); (3) treated mated males (TMM); (4) untreated unmated females (UUF); (5) treated unmated females (TUF); and (6) treated mated females (TMF). 2.4. Protein Extract and Two-Dimensional Gel Electrophoresis Analysis

For each sample group, 100 mg of adult N. lugens (males or females) was ground into a powder in liquid nitrogen, homogenized on ice in 1 mL of lysis buffer (7 M urea, 2 M thiourea, 4% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS), 30 mM Tris-HCl, and protease inhibitor cocktail), and sonicated (10 10 s pulses) on ice. The homogenate was subjected to centrifugation (12 000 rpm) for 1 h at 4 °C. The protein was precipitated with cold acetone at 20 °C for 2 h and redissolved in rehydration buffer (8 M urea, 2 M thiourea, 4% CHAPS, 100 mM dithiothreitol (DTT), and 2% ampholyte). Protein concentrations were determined by the Bradford method (Bio-Rad, Hercules, CA). For 2-DE, 1000 μg of protein was loaded onto analytical and preparative gels. The Ettan IPGphor isoelectric focusing system (IEF) (Amersham, Piscataway, NJ) was used with pH 310 immobilized pH gradient (IPG) strips that were rehydrated for 12 h in 400 μL of rehydration buffer containing 5 μL of 28% (w/v) DTT. IEF was performed in three steps: 500 V for 1 h, 1000 V for 1 h, and 800 V for 5 h. The gel strips were equilibrated for 15 min in equilibration buffer (50 mM Tris-HCl (pH 8.8), 6 M urea, 30% glycerol, 2% sodium dodecyl sulfate (SDS), 0.01% bromophenol blue, and 130 mM DTT). The gel strip was repeated using the same buffer containing 200 mM iodoacetamide instead of 130 mM DTT for another 15 min. The strips were then subjected to a second dimension of electrophoresis after being transferred onto 12% SDS-polyacrylamide gels (200 mm 200 mm 1 mm) using an Ettan Dalt 2D-gel system (Amersham Biosciences). Electrophoresis was performed using the Hofer SE 600 system (Amersham) at 30 mA per gel for 40 min, followed by 60 mA until the bromophenol blue reached the end of the gel. At least three replicates were performed for each sample. 2.5. Gel Staining and Image Analysis

The gels were fixed in 10% (v/v) acetic acid and 30% ethanol (fixing solution) for 30 min, stained for 90 min in a Coomassie Brilliant Blue (CBB) staining solution (5% (v/v) methanol, 42.5% (v/v) ethanol, 10% (v/v) acetic acid, 30% (v/v) ethanol, 0.2% (w/v) CBB G250, and 0.05% (w/v) CBB R250), and destained in fixing solution twice for 60 min. The gels were further destained in 7% (v/v) acetic acid until the backgrounds were clear. The protein spots were detected, quantified, and matched using PD-Quest 2D-analysis software (Bio-Rad, Hercules, CA). 4598

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Journal of Proteome Research 2.6. In-Gel Tryptic Digestion and Protein Identification by LC-MS/MS

Protein spots were excised from the preparative gels and destained with 100 mM NH4HCO3 and 30% acetonitrile (ACN). After the destain buffer was removed, the gel pieces were lyophilized and rehydrated in 30 μL of 50 mM NH4HCO3 containing 50 ng of trypsin (sequencing grade; Promega, Madison, WI). After digestion overnight at 37 °C, the peptides were extracted three times with 0.1% trifluoroacetic acid (TFA) in 60% ACN. Extracts were pooled and lyophilized. The resulting lyophilized tryptic peptides were dissolved in 5 mg/mL R-cyano4-hydroxycinnamic acid (CHCA) containing 0.1% TFA and 50% ACN. A protein-free gel piece was treated as stated above and used as a control to identify autoproteolyic products derived from trypsin. The samples extracted from N. lugens adults (males or females) were digested with trypsin and analyzed using LC-MS/MS. An automated nano-HPLC (Eksigent Technologies, USA) coupled with a Thermo Finnigan LCQDeca ion trap mass spectrometer (Thermo Electron Corporation, USA) was used. After adding 200 μL of 60% acetonitrile and 0.1% TFA, the samples were kept at room temperature for 60 min. This step was repeated after samples had been transferred into prewashed tubes, and then the mixtures were dried in a speed vac. The dried samples were dissolved in 10 μL of 5% acetonitrile and 0.05% TFA. Two microliters of this solution were separated chromatographically on an RP-18 capillary column (50 μm i.d., Dionex, USA) at a flow rate of 200 nL min1. The gradient profile consisted of a linear gradient from 98% A (acetonitrile/H2O/formic acid, 5/95/0.1) to 50% B (acetonitrile/H2O/formic acid, 80/20/0.1) over 40 min, followed by a linear gradient to 98% B over 5 min. The eluted settings were 1.3 kV, 14 V, and 165 °C for spray voltage, heated capillary voltage, and temperature, respectively. Collision energy was set to 35%. Upon a full scan, a zoom scan was recorded to determine the charge state of the peptide. This was followed by the isolation of the particular mass and an MS/MS scan. The instrument executed a MS/MS measurement for the three most intense peaks from the MS scan. The generated peptide sequence tags were analyzed by the MASCOT software (version 1.9). The data were sent to the National Center for Biotechnology nonredundant (NCBInr) protein database. The search parameters included the potential residue mass modification for carbamidomethylation (C) and oxidation (Met), one missed trypsin cleavage, peptide tolerance of 100 ppm, fragment mass tolerance of 0.5 Da, and a peptide charge of 1+, 2+, and 3+. A confident identification had a statistically significant (p < 0.05) protein score (based on combined LC and mass/mass spectra) and best ion score (based on mass/mass spectra). Redundant proteins that appeared in the database under different names and accession numbers were eliminated. If more than one protein was identified in single spot, the protein with the highest protein score was used. 2.7. RNA Extraction and cDNA Synthesis

Total RNA was isolated from six samples (UUM, TUM, TMM, UUF, TUF and TMF) (20 adult males or 8 adult females from each of the six samples) using an SV Total Isolation System kit (Promega Corporation, Madison, WI, USA). The synthesis of first-strand cDNAs was carried out according to the PrimeScript RT reagent kit (TaKaRa Biotechnology (Dalian) Co., Ltd.). The first-strand cDNA synthesis was performed in a 10 μL total reaction volume containing 0.5 μg of total RNA, 0.5 μL of PrimeScript RT

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enzyme mix I, 0.5 μL of Oligo dT primer (50 μM), 2 μL of random hexamers (100 μM), 2 μL of 5 PrimeScript buffer (for Real Time), X μL of total RNA, and the addition of RNase-free deionized H2O up to 10 μL. The cDNA reverse transcription was performed at 37 °C for 15 min, 85 °C for 5 s, and 4 °C for 5 min. 2.8. Quantitative Real-Time PCR (qRT-PCR) Analysis

The primers used for real-time PCR are listed in Table 5. Aliquots (0.5 μL) of the synthesized first-strand cDNA were amplified by PCR in 10 μL reaction mixtures using a 7500 realtime PCR system (American) with the following cycling regime: 94 °C for 2 min, followed by 3545 cycles of 94 °C for 5 s, 55 °C for 10 s, and 72 °C for 15 s. The actin-1 gene was used as an internal standard. After the amplifications, a melting curve analysis was performed in triplicate and the results were averaged. The values were calculated using three independent biological samples, and the well-known 2 Ct method was employed for the analysis of relative gene expression.26 2.9. Statistical Analysis

Statistical calculations were performed using SPSS statistical software (Version 11.0; SPSS, Inc., USA). All data are expressed as the mean ( standard deviation (SD). Comparisons among multiple groups were performed using one-way analysis of variance (ANOVA) and least significant difference (LSD)-t tests.

3. RESULTS 3.1. 2-DE Analysis of Differentially Expressed Proteins among Six Samples

To investigate the differentially expressed proteins between the six samples, we performed a two-dimensional gel electrophoresis (2-DE) analysis (Figure 1). Each sample was subjected to triplicate runs, and the results were highly reproducible. Coomassie blue staining of 2-DE gels from UUM, TUM, TMM, UUF, TUF, and TMF at day 2 after adult emergence showed 506 ( 36, 474 ( 34, 490 ( 39, 648 ( 52, 647 ( 38, and 615 ( 46 protein spots, respectively, in a pH range from 3 to 10. Statistical analysis of the 2-DE gels revealed that 33, 29, 21, and 24 protein spots from the UUM vs TUM group, TUM vs TMM group, UUF vs TUF group, and TUF vs TMF group, respectively, were differentially expressed (p < 0.05). In the UUM vs TUM group, 14 protein spots were upregulated and 19 protein spots were down-regulated. In the TUM vs TMM group, 17 protein spots were up-regulated and 12 protein spots were down-regulated. In the UUF vs TUF group, 11 protein spots were up-regulated and 10 protein spots were downregulated. In the TUF vs TMF group, 16 protein spots were upregulated and 8 protein spots were down-regulated. In adult males, only four protein spots showed significantly different expression levels related to reproduction in both TUM and TMM relative to UUM (Figure 2). In adult females, only two protein spots showed significantly different expression levels related to reproduction in both TUF and TMF relative to UUF (Figure 2). 3.2. Identification of the Differentially Expressed Proteins by LC-MS/MS

Differentially expressed protein spots among the UUM vs TUM groups, TUM vs TMM groups, UUF vs TUF groups, and TUF vs TMF groups at day 2 after adult emergence were subsequently subjected to LC-MS/MS. Sixty-three differentially expressed proteins were identified from 107 protein spots in four different groups. Of the 63 proteins identified, 38 proteins were up-regulated and 25 proteins were down-regulated (Tables 14). 4599


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Figure 1. Proteomic analysis of six different samples using 2-DE gels. Comparison of protein profiles by 2-DE gels among I (UUM), II (TUM), III (TMM), IV (UUF), V (TUF), and VI (TMF) samples at 2 days after adult emergence. There are six of eight representative spots with similar expression patterns in the UUM (I) vs TUM (II) groups, and four of eight representative spots with similar expression patterns in the TUM (II) vs TMM (III) groups. There are three of six representative spots with similar expression patterns between the TUF and TMF groups relative to UUF.

Only eight proteins in both TUM and TMM showed significantly different expression levels relative to UUM (Figure 3A). Only six proteins in both TUF and TMF showed significantly different expression levels relative to UUF (Figure 3B). As summarized in Tables 14, LC-MS/MS analysis of spot 3 (spermatogenesisassociated protein 5) between TUM and TMM relative to UUM and spot 1 (vitellogenin) between TUF and TMF relative to UUF were shown in Figure 4 as being representative and have a typical spectrum for a peptide mapping at this spot. 3.3. Triazophos-Induced Differential Protein Expression in the TUM vs UUM Group

We compared expression levels between UUM and TUM males at 2 days after adult emergence. We visualized 506 spots from UUM and 474 spots from TUM males (Figure 1-I,II). Sixteen differentially expressed protein spots were randomly excised from 33 spots in the UUM vs TUM group. Ten of 16 were up-regulated and six were down-regulated in TUM relative to UUM. After they were digested in-gel by trypsin, the proteins were identified by LC-MS/MS (Table 1). Their expression levels are shown in Figure 5A. Two glycolytic enzymes (glyceraldehyde-3-phosphate dehydrogenase, Q4U3L0, and mitochondrial 2-oxoglutarate dehydrogenase, Q60HE2) were down-regulated in the TUM relative to UUM. One protein, Act5C (P10987), which plays a role in flight, muscle isoforms, sperm individualization, and mushroom body development, was significantly up-regulated by 18.7-fold in TUM relative to UUM, suggesting that the insecticide treatment significantly increased the flight capacity for males. Two proteins (spermatogenesisassociated protein 5, Q8NB90, and testis development protein

NYD-SP6, Q9BWX1) that are associated with spermatogenesis were also significantly up-regulated by 3.1- and 5.5-fold in TUM relative to UUM, respectively. Arginine kinase (P48610) was down-regulated by 3.3-fold in TUM relative to UUM. An annotated protein, U4/U6 small nuclear ribonucleo protein Prp31 (a pre-mRNA-processing factor 31, Q6NVP6), was down-regulated by 3.5-fold in TUM relative to UUM. Carboxylesterase (Q9GQ01), which participates in mechanisms related to insecticide resistance, was up-regulated by 2.1-fold in TUM relative to UUM. Keratin, type I cytoskeletal 10 (P13645), and keratin, type I cytoskeletal 9 (P35527), which are associated with the structural constituents of the epidermis and cytoskeleton, were up-regulated by 5.9- and 5.1-fold in TUM relative to UUM, respectively. Hsp70 (P29843), which responds to stress, was up-regulated by 4.9-fold in TUM relative to UUM. Other proteins involved in protein biosynthesis (bifunctional protein foID, A3PVV6), ATP binding (myosin heavy chain, muscle, P05661), motor activity (myosin light chain alkali, Q24756), and glucose energy metabolism (enolase, P15007) were also observed, showing different expression levels in TUM relative to UUM. 3.4. Mating-Induced Differential Protein Expression in the TMM vs TUM Group

We compared expression levels among TUM vs TMM males 2 days after mating. We visualized 474 spots from TUM and 490 spots from TMM males (Figure 1-II,III). Twenty differentially expressed protein spots were randomly excised from 29 spots in the TUM vs TMM group. Twelve of 20 were upregulated and eight were down-regulated in TMM relative to 4600



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Figure 2. Cropped 2-DE gel images of six spots, (A) actin-5C, (B) spermatogenesis-associated protein 5, (C) testis development NYD-SP6, (D) arginine kinase, (E) vitellogenin, and (F) ovarian serine protease nudel, with similar expression patterns between groups I (UUM), II (UMM), III (TUM), IV (UUF), V (UMF), and VI (TMF). There were a significant differences between groups UUM vs TUM, TUM vs TMM, UUF vs TUF, and TUF vs TMF, as indicated by one way ANOVA statistical analysis (p < 0.05). Each treatment and control was replicated three times.

TUM. After they were digested in-gel by trypsin, the proteins were identified by LC-MS/MS (Table 2). Their expression levels are shown in Figure 5B. Two proteins that are associated with spermatogenesis (spermatogenesis-associated protein 5, Q8NB90, and testis development protein NYD-SP6, Q9BWX1) were upregulated by 2.4- and 2.8-fold in TMM relative to TUM, respectively. One protein (actin-5C, P10987), associated with flight, muscle isoforms, mushroom body development, and sperm individualization, was also up-regulated by 3.5-fold in TMM relative to TUM. One protein that is associated with high quality sperm (arginine kinase, P4860) was down-regulated by 2.7-fold in TMM relative to TUM. Three of four glycolytic enzymes (phosphoglycerate mutase 2, P15259, fructose-

bisphosphate aldolase, P07764, and transaldolase, Q9EQS0) were up-regulated by 2.3-, 2.1-, and 2.3-fold in TMM relative to TUM, respectively. However, one glycolytic protein (2-oxoglutarate dehydrogenase, mitochondrial, Q60HE2) was down-regulated by 2.0-fold in TMM relative to TUM. Two proteins (myosin heavy chain, muscle, P05661, and myosin light chain alkali, Q24756) that are associated with the structural components of muscle were up-regulated in TMM relative to TUM. One protein (ATP synthase subunit beta, mitochondrial, Q9PTY0) that is associated with hydrogen ion-transporting ATP synthase activity was significantly up-regulated by 2.8-fold in TMM relative to TUM. Hsp70 (heat shock 70 kDa protein 5, P29845), which responds to stress, was up-regulated by 4601


4602

Q24756

16

myosin light chain alkali

mitochondrial

2-oxoglutarate dehydrogenase,

bifunctional protein foID

myosin heavy chain, muscle enolase

carboxylesterase

keratin, type I cytoskeletal 9

dehydrogenase

glyceraldehyde-3-phosphate

14-3-3 protein Zeta

testis development protein YD-SP6 arginine kinase

ribonucleoprotein Prp31

U4/U6 small nuclear


protein 5

spermatogenesis-associated

28057/4.80

Drosophila melanogaster

Homo sapiens (human)

44455/6.46

Macaca fascicularis

OGDH Mlc1

35454/8.30

32985/6.97

Mycobacterium sp.

foID

Drosophila virilis

24674/5.68 46630/5.92

Mhc Eno

Drosophila melanogaster Drosophila melanogaster

59950/5.51

62027/5.14

Glossina moritans morsitans 35446/6.54

64686/6.86 60686/8.56

Xenopus tropicalis Homo sapiens (human) Drosophila melanogaster

59492/5.17 65176/6.52


Nl-EST1 Nilaparvata lugens

KRT9

Gapdh

Zeta

14-3-3-

PHF7 Argk

prpf31

KTR10

Act5C

5

9

8

2 4

5

3

5

3

1 4

1

3

5

13

4

9

20

21

14 17

17

4

18

6

1 8

1

6

7

34

9

peptidesc peptidesd

42

18

41

5 15

19

8

23

19

2 8

2

10

12

42

11

(%)

102

94

76

75 94

82

72

79

74

63 62

67

85

103

94

78

scoree

127

177

129

98 137

101

80

108

90

63 112

67

95

165

263

110

scoref

changes

up-regulated

up-regulated up-regulated

up-regulated

up-regulated

down-regulated

down-regulated

up-regulated down-regulated

down-regulated

up-regulated

up-regulated

up-regulated

up-regulated

expression levels

K.AENGLMVGAELSHILLSLGER down-regulated

R.LAVVANPSHLEAVDPIVQGK.T down-regulated

R.QFTKPSVALAGLSAVDMF

K.LAGADIETYLLEK.A K.VNQIGSVTESIEAHLLAK.K

R.KVEGSEFLIHNEK.F

R.SGGGGGGGLGSGGSIR.S

R.VPVPNVSVVDLTVR.L

R.YLAEVATGDTR.S

K.AGANIGCCKPGCR.K R.GEHTEAEGGVYDISNK.R

K.MPSCNILLLGSQKK.T

R.ALEESNYELEGK.I

R.EIIELPLKQPELFKSYG.I

K.SYELPDGQVITIGNER.F

K.DAGAIAGLNVLR.I

peptides identifiedg

Accession Number was derived from Swiss-Prot database. b Theoretical molecular weight (kDa) and pI from the Swiss-Prot database. c The number of peaks that match the trypsin peptides. d The number of peaks that do not match the trypsin peptides. e Best ion score (based on mass/mass spectra) was from LC-MS/MS identification. Best ion scores were considered successfully identified. f Protein score (based on combined LC and mass/mass spectra) was from LC-MS/MS identification. The proteins had statistically significant protein scores. g Each spot corresponding to a certain protein had at least one of the shown peptides identified.

a

A3PVV6

Q60HE2

P05661 P15007

12 13

15

Q9GQ01

14

P35527

11

C4NFK6

8

10

Q9BWX1 P48610

6 7

Q4U3L0

Q6NVP6

5

9

P13645

4

97904/7.92

SPATA5 Homo sapiens (human)

Q8NB90

41822/5.37

3

actin-5C

heat shock 70 kDa protein cognate 1 Hsc70-1

70643/5.34

P10987

2

Mr/pIb

theoretical matched unmatched coverage best ion protein


P29843

1

organism Drosophila melanogaster

gene name

no.a

no.

protein description

accession

spot

Table 1. Differential Protein Expression of N. lugens Adult Males Identified by LC-MS/MS in TUM Relative to UUM

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4603

Q60HE2

P29845

Q7SY23

P83967

17

18

19

20

KRT10

actin, indirect flight muscle

dehydrogenase

delta-1-pyrroline-5-carboxylate

cognate 5

heat shock 70 kDa protein

dehydrogenase, mitochondrial

2-oxoglutarate

myosin light chain alkali

subunit alpha

electron transfer flavoprotein

troponin C, isoform 2

phosphoglycerate mutase 2

fructose-bisphosphate aldolase

myosin heavy chain, muscle

testis development protein NYD SP6

tropomyosin-1, isoforms 33/34

actin-5C


arginine kinase

mitochondrial

13 11 1

41822/5.37 54526/4.39

Drosophila melanogaster Drosophila melanogaster Homo sapiens (human) 64686/6.86

Homo sapiens (human) 28832/9.01

Art88F

alfh4a1

Hsc70-5

OGDH

Mlcl

ETFA


Danio rerio (zebrafish)

41653/5.37

63195/8.63

73168/5.49

44455/6.46

Macaca fascicularis Drosophila virilis

12822/4.58

Drosophila virilis


1

2

15

7

9

2

4

2

38937/6.92

Drosophila melanogaster 17872/3.91

6

24730/5.78

Drosophila melanogaster 6

4 3

60686/8.56



6 3

Rattus norvegicus 68950/8.65


3 6

8

8

6

20

21

23

6

7

3

7

9

1

21

34

9

8

4

1

12

16

18

peptidesc peptidesd

35624/5.45

68315/5.16

Mr/pIb

19

3

13

16

55

11

18

4

12

5

2

22

42

5

8

5

5

16

13

20

(%)

theoretical matched unmatched coverage


Rattus norvegicus

Cyprinus carpio

TpnC47D Drosophila melanogaster

PGAM2

Ald

Mhc

PHF7

Tm1

Act5C

KRT14

Argk

specific acyl-CoA dehydrogenase, Acadvl

protein 5 keratin, type I cytoskeletal 10

Taldo 1 SPATA5

spermatogenesis-associated

transaldolase

mitochondrial

ATP synthase subunit beta,

organism

76

77

99

95

82

70

76

84

74

85

63

94

94

85

62

75

85

81

81

71

scoree

best ion

76

89

240

140

121

76

121

149

115

115

63

1022

263

100

112

75

92

108

88

97

scoref

protein


K.GYFIQPTIVETK.D

R.QAVTNSANTFYATK.R

DPIVQGK.T

R.LAVVANPSHLEAV-

K.AENGLMVGAELSHILLSLGER

R.TIYAGNAILTLK.S

K.FIVEEDDEAMQK.E

R.KAMEAVAAQGK.A

K.NTPEEIALATVTALSR.T

K.LTQEAVADLER.N

K.AGANIGCCKPGCR.K

K.NKALQNAESEVAALNR.R


R.SGGGGGGGLGSGGSIR.S

R.GEHTEAEGGVYDISNK.R

R.FGMAAALSGTMR.S

R.ALEESNYELEGK.I

R.EIIELPLKQPELFKSYG.I

R.ALAGCDLLTISPK.L

K.NMIAFYDMSR.H


changes

down-regulated

down-regulated

down-regulated

down-regulated

up-regulated

up-regulated

up-regulated

up-regulated

up-regulated

up-regulated

up-regulated

down-regulated

up-regulated

down-regulated

down-regulated

down-regulated

up-regulated

up-regulated

up-regulated

up-regulated

expression levels


a

Q24756

16

P1 3804

Q9BWX1

10

P47948

P49455

9

15

P10987

8

14

P02533

7

P15259

P48610

6

13

P45953

5

P05661

P13645

4

P07764

Q8NB90

3

12

Q9EQS0

2

11

Q9PTY0

1

atp5b

gene name

no.a

no.

protein description

accession

spot

Table 2. Differential Protein Expression of N. lugens Adult Males Identified by LC-MS/MS in TMM Relative to TUM




nudel KRT6C foID Ald Vitellogenin Eno Act5C RpLP2 KRT10 14-3-3 zeta P98159 P48668 A3PVV6 P07764 A7BK94 P15007 P10987 P05839 P13645 Q2F637 4 5 6 7 8 9 10 11 12 13

vitellogenin keratin, type II cytoskeletal 1 glyceraldehyde-3-phosphate dehydrogenase ovarian serine protease nudel keratin, type II cytoskeletal 6C bifunctional protein foID fructose-bisphosphate aldolase vitellogenin enolase actin-5C 60S acidic ribosomal protein P2 keratin, type I cytoskeletal 10 14-3-3 protein zeta A7BK94 P04264 Q4U3L0 1 2 3

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a

up-regulated down-regulated down-regulated down-regulated up-regulated down-regulated up-regulated up-regulated down-regulated down-regulated R.LCDGYEDCPMGEDEL.G R.AIGGGLSSVGGGSSTIK.Y K.NITSAGTDNDQTR.V K.NTPEEIALATVTALSR.T K.HATANAGTGPALEAIK.N R.AAVPSGASTGIYEALELR.D K.SYELPDGQVITIGNER.F R.YVAAYLLAVLGGK.E K.QSLEASLAETEGR.Y R.YLAEVATGDTR.N 292492/4.97 89430/5.57 32985/6.97 38937/6.92 229528/8.68 46663/5.92 41822/5.37 11662/4.59 62063/5.17 28087/4.88

9 4 5 1 22 1 13 2 2 4

13 7 8 2 34 6 34 3 7 9

20 9 36 6 21 17 42 37 14 37

98 87 89 88 114 93 94 115 93 101

222 108 135 88 649 97 263 133 90 215

up-regulated up-regulated up-regulated R.MLFFASMNPANSDSK.A R.SLDLDSIIAEVK.A R.VPVPNVSVVDLTVR.L 429 117 142 103 94 88 15 22 12 17 10 3 14 5 3

Nilaparvata lugens Homo sapiens (human) Glossina morsitans morsitans Drosophila melanogaster Homo sapiens (human) Mycobacterium sp. Drosophila melanogaster Nilaparvata lugens Drosophila melanogaster Drosophila melanogaster Homo sapiens (human) Drosophila melanogaster Bombyx mori Vitellogenin KRT1 Gapdh

229528/8.68 65978/8.16 35454/8.30

coverage best ion (%) scoree unmatched peptidesd matched peptidesc theoretical Mr/pIb organism gene name protein description spot no. accession no.a

Table 3. Differential Protein Expression of N. lugens Adult Females Identified by LC-MS/MS in TUF Relative to UUF

protein scoref


changes expression levels


2.2-fold in TMM relative to TUM. Some proteins involved in functions of ATP binding (heat shock 70 kDa protein 5, P29845, and actin, indirect flight muscle, P83967), FAD or FADH2 binding (specific acyl-CoA dehydrogenase, mitochondrial, P45953), protein binding with the structural constituents of the epidermis and cytoskeleton (keratin, type I cytoskeletal 14, P02533, and keratin, type I cytoskeletal 10, P13645) and actin binding (tropomyosin-1, isoforms 33/34, P49455), and 1-pyrroline-5-carboxylate dehydrogenase activity (delta-1-pyrroline-5-carboxylate dehydrogenase, mitochondrial, Q7SY23) were also observed showing different expression levels in TMM relative to TUM. 3.5. Triazophos-Induced Differential Protein Expression in the TUF vs UUF Group

We compared the expression levels between UUF and TUF females at 2 days after adult emergence. We visualized 648 spots from UUF and 647 spots from TUF females (Figure 1-IV,V). Thirteen differentially expressed protein spots were randomly excised from 21 spots in a preparative gel for the UUF vs TUF group. Seven of 13 were up-regulated and six were downregulated in TUF relative to UUF. Following in-gel digestion by trypsin, the proteins were identified by LC-MS/MS (Table 3) and their expression levels are shown in Figure 6A. Vitellogenin in TUF was up-regulated by 27.4-fold relative to UUF. Ovarian serine protease nudel, which is associated with ovary development and egg activation, was up-regulated by 2.4-fold in TUF relative to UUF. Actin-5C (P10987) in TUF, which is associated with flight, muscle isoforms, sperm individualization, and mushroom body development, was up-regulated by 22.3-fold relative to UUF. Two of three glycolytic enzymes (fructose-bisphosphate aldolase, P07764, and enolase, P15007) in TUF were downregulated by 2.9- and 6.7-fold relative to UUF, respectively. However, another protein (glyceraldehyde-3-phosphate dehydrogenase, Q4U3L0) in TUF, which is associated with glucose energy metabolism, was up-regulated by 2.3-fold relative to UUF. Other proteins with different expression levels in TUF relative to UUF observed included those involved in protein binding with the structural components of the cytoskeleton and epidermis (keratin, type II cytoskeletal 1, P04264, and keratin, type I cytoskeletal 10, P13645), structural of molecule activity (keratin, type II cytoskeletal 6C, P48668), protein domain specific binding (14-3-3 protein zeta, Q2F637), and protein binding (60S acidic ribosomal protein P2, P05389). 3.6. Mating-Induced Differential Protein Expression in the TMF vs TUF Group

We compared expression levels among TUF vs TMF males at 2 days after mating. We visualized 647 spots from TUF and 615 spots from TMF females (Figure 1-V,VI). Fourteen differentially expressed protein spots were randomly excised from 24 spots in a preparative gel in the TMF vs TUF group. Nine were upregulated and five were down-regulated in TMF relative to TUF. After they were digested in-gel by trypsin, the proteins were identified by LC-MS/MS (Table 4), and their expression levels are shown in Figure 6B. Two proteins associated with fecundity of adult females (vitellogenin, AB353856; ovarian serine protease nudel, P98159) in TMF were up-regulated by 2.4- and 5.1-fold relative to TUF, respectively. One protein (actin-5C, P10987) in TMF that is associated with flight, muscle isoforms, sperm individualization, and mushroom body development was down-regulated by 3.5-fold in relative to TUF. One proteolysis protein (cysterine proteinase, O02469) in TMF 4604


4605

P07764

P02533

P05661 02469

5

6

7 8

Q9U943

P13645

13

14

B0WC46

P29843

11

12

14 6

Drosophila melanogaster Nilaparvata lugens

KRT10


Apolipophorins Locusta migratoria


cognate 1 apolipophorins 65980/7.62

371384/8.78

65944/7.62 70609/5.33

Culex quinquefasciatus

4

3

1

4

3


13

41822/5.37

Drosophila pseudoobscura 35214/7.68


4

Homo sapiens (human) 107386/4.97 224385/5.78 60895/5.65

1

38937/6.92

6

7

65980/7.62

54954/5.26


9

12


292492/4.97

matched peptidesc


229528/8.68


Mr/pIb

theoretical

Nilaparvata lugens

organism

Hsc70-1

Tret1

Gapdh2

Act5C

proteinase

Mhc cysteine

KRT14

Ald

KTR1

ATPsyn-beta

nudel

Vitellogenin

gene name

heat shock 70 kDa protein

facilitated trehalose transporter

dehydrogenase 2

glyceraldehyde-3-phosphate

actin-5C

myosin heavy chain, muscle cysteine proteinase


fructose-bisphosphate aldolase

keratin, type II cytoskeletal 1

beta, mitochondrial

ATP synthase subunit

ovarian serine protease nudel

vitellogenin

protein description

10

5

3

8

3

34

30 16

6

1

9

10

13

30

peptidesd

19

1

6

13

13

42

13 26

7

4

16

26

20

19

(%)

89

76

67

78

88

94

116 88

85

63

93

108

98

106

scoree

160

96

67

124

127

263

314 230

118

63

176

174

222

329

scoref

unmatched coverage best ion protein

changes

up-regulated

expression levels

R.SLDLDSIIAEVK.A

R.VAALEAFQSDASK.S

K.VEIIANDQGNR.T

R.FLEQQNQVLQTK.W

R.VPVPNVSVVDLTVR.L


R.ALDSMQASLEAEAK.G R.NVIIEQSWGSPK.I

R.FSSSGGGGGGGR.F

K.NTPEEIALATVTALSR.T

R.FLEQQNQVLQTK.W

R.FTQAGSEVSALLGR.I

down-regulated

down-regulated

up-regulated

up-regulated

up-regulated

down-regulated

up-regulated up-regulated

down-regulated

down-regulated

up-regulated

up-regulated

R.LCDGYEDCPMGEDEL.G up-regulated

K.NFLSSFVIDNIDK.S


Accession Number was derived from Swiss-Prot database. b Theoretical molecular weight (kDa) and pI from the Swiss-Prot database. c The number of peaks which match the trypsin peptides. d The number of peaks which do not match the trypsin peptides. e Best ion score (based on mass/mass spectra) was from LC-MS/MS identification. Best ion scores were considered successfully identified. f Protein score (based on combined LC and mass/mass spectra) was from LC-MS/MS identification. The proteins had statistically significant protein score. g Each spot corresponding to a certain protein had at least one of the shown peptides identify.

a

P04264

4

O44104

Q05825

3

10

P98159

2

P10987

A7BK94

1

9

noa

no.

spot accession

Table 4. Differential Protein Expression of N. lugens Adult Females Identified by LC-MS/MS in TMF Relative to TUF




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Figure 3. Distribution of differentially expressed protein spots. There are 63 differentially expressed proteins revealed by 2-DE gel analysis. Comparisons between I (TUM vs UUM) and II (TMM vs TUM) samples (A) and III (TUF vs UUF) and IV (TMF vs TUF) samples (B) are shown. Eight proteins were differentially expressed in I sample but not in II sample. Twelve proteins were differentially expressed in II sample but not in I sample. Eight proteins were differentially expressed in both I and II samples. Seven proteins were differentially expressed in III sample but not in IV sample. Eight proteins were differentially expressed in IV sample but not in III sample. Six proteins were differentially expressed in both III and IV samples.

that is associated with a nucleophilic cysterine thiol in a catalytic dyad was up-regulated by 2.7-fold relative to TUF. One trehalose transport protein (facilitated trehalose transporter, B0WC46) in TMF was also up-regulated by 3.1-fold relative to TUF. One protein (glyceraldehyde-3-phosphate dehydrogenase 2, O44104) in TMF that is associated with glucose energy metabolism was up-regulated by 4.7-fold relative to TUF. One Hsp70 protein (heat shock 70 kDa protein 1, P29843) in TMF, which is associated with the stress response, was significantly up-regulated by 2.2-fold relative to TUF. Other proteins with different expression levels in TMF relative to TUF observed include those involved in hydrogen ion transport in ATP binding (ATP synthase subunit beta, mitochondrial, Q05825,), protein binding (keratin, type II cytoskeletal 1, P04264, keratin, type I cytoskeletal 14, P02533, and keratin, type I cytoskeletal 10, P13645), lipid binding (apolipophorins, Q9U943), motor activity (myosin heavy chain, muscle, P05661), and fructose-bisphoate aldolase activity (fructose-bisphosphate aldolase, P07764). 3.7. Gene Transcription Profile Analysis by Quantitative Real-Time PCR (qRT-PCR)

To confirm the LC-MS/MS results, we used qRT-PCR to examine gene transcription. Six different genes from N. lugens at 2 days after adult emergence were selected for the analysis. These primer designs were based on expressed sequence tags (ESTs) of the genes of N. lugens except actin-1 and vitellogenin.27,28 The gene-specific primers are listed in Table 5, and actin-1 was chosen as an internal control. As shown in Figure 2, six genes showed consistent mRNA and protein expression patterns (Figure 7).

4. DISCUSSION Previous findings demonstrated that triazophos treatment resulted in an increase in the content of male accessory gland proteins (Acps) that can be transferred to females during mating, influencing female reproduction.4,5 However, the mechanisms of insecticide-induced Acps increase in males and of reproductive stimulation in females are not well understood. To identify changes in Acps and reproductive proteins in mated females, we conducted a comparative proteomic analysis (Figure 1). Sharma et al. (2004) observed modulation in 22 proteins at the expression level in the brown planthopper N. lugens treated with low levels (LC10) of a methyl carbamate insecticide using two-dimensional polyacrylamide gel electrophoresis.29 The

Figure 4. Identification of spot 3 from the TUM sample (A) and spot 1 from the TUF sample (B) by LC-MS/MS. Two proteins were identified as spermatogenesis-associated protein 5 (NCBI GI: 221130788) and vitellogenin (NCBI GI: 154799937) by a Swiss-Prot database search.

different expression of these proteins reflected the overall changes in cellular structure and metabolism after insecticide 4606



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Figure 5. Expression profile of 16 and 20 proteins in TUM relative to UUM (A) and TMM relative to TUM (B), respectively, at 2 days after adult emergence. UUM is untreated unmated males. TUM is treated unmated males. TMM is treated mated males.

treatment.29 However, the present findings showed that there are the same proteins in both UUM and TUM relative to TMM, but only eight proteins exhibited significantly different expression levels in TUM relative to UUM (Figure 3A). Four of eight proteins associated with reproduction in N. lugens adult males had significantly different expression levels (Figure 2). Three of four proteins (actin-5C, spermatogenesis-associated protein 5, and testis development protein NYD-SP6) were significantly upregulated and one protein (arginine kinase) was significantly down-regulated in both TMM and TUM relative to UUM. Actin-5C is associated with flight, muscle isoforms, mushroom body development, and sperm individualization and was significantly up-regulated. Two proteins associated with spermatogenesis (spermatogenesis-associated protein 5 and testis development protein NYD-SP6) were significantly up-regulated. Zhao et al.

(2011) indicated that a 40 ppm triazophos treatment enhanced the flight capacity of N. lugens adult males and females.30 Shen and Cheng (1998) indicated that long distance flight could significantly stimulate N. lugens reproductivity.31 In adult males of Ephesia cautella, large size or heavy weight has been used as an indicator of “good quality” and having better genes and more sperm supply.32,33 Spermatozoa numbers in virgin Aedes aegypti males were associated with the number of days after emergence, body weight, and size.34 Spermatogenesis is a highly regulated, complex process involving both intrinsic and extrinsic regulators and interactions between germ and sertoli cells.35 In D. melanogaster, sperm and Acps, a major component of seminal fluid transferred by males during mating, trigger many physiological and behavioral changes in females.10,3640 Males produce reproductive molecules that influence a wide range of processes including 4607



ARTICLE

Figure 6. Expression profile of 13 and 14 proteins in TUF relative to UUF (A) and TMF relative to TUF (B), respectively, at 2 days after adult emergence. UUF is untreated unmated females; TUF is treated unmated females; TMF is treated mated females.

Table 5. Primers for Quantitative Real-Time PCR Measurements of Expression Levels of Selected Genes accession no.

gene name

gi|156759

Act5C

gi|221130788

SPATA 5

gi|195394017

PHF7

gi|195012053

Argk

gi|154799937

Vg

gi|193683762

nudel

gi|163883730

actin-1

primer (50 30 )

product length

sense: CGATTTGACCGACTACCTG antisense: GTCGAGGGCAACATAGCA sense: ATTCAAAAGAGCAAGACAAC antisense: CCTGGAGGACCATAAAGTAA sense: AAGCCATCTACCACCGCAAGT antisense: TTCCCATTCTCAGCATTTCTTG sense: TGTCCGGTCTGGAAGGTGAG antisense: GGTCTTGGCATCGTTGTGGT sense: GTGGCTCGTTCAAGGTTATGG antisense: GCAATCTCTGGGTGCTGTTG sense: TGACGGAAGGGAAGTGGTAG antisense: GTTGATTTCGGTTGTGCTGA sense: TGGACTTCGAGCAGGAAATGG antisense: ACGTCGCACTTCAGATCGAG

118 bp

4608

172 bp 120 bp 185 bp 93 bp 181 bp 200 bp



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Figure 7. Quantitative real-time PCR data for the mRNA expression levels of Act5C, SPATA5, PHF7, Argk, Vg, and nudel genes in six samples. Values are reported as mean ( standard error, and bars with different letters indicate that means are significantly different at the p < 0.05 level. Values are normalized relative to actin-1 transcript levels. Each treatment and control was replicated three times.

oogenesis, egg-laying, female receptivity, and sperm transfer and storage.79 Xiao et al. (2002) indicated that the putative protein NYD-SP6 may play an important role in stimulating transcription involved in testicular development and spermatogenesis.41 The fecundity of adult female Spodoptera litura F. increases when females mate with male moths with high quality sperm.42 However, a key enzyme (arginine kinase) of L-arginine degradation, which is associated with spermatogenesis and high quality sperm, was down-regulated in both between TMM and TUM relative to UUM in the present investigation. L-Arginine is a necessary amino acid for sperm and is associated with

spermatogenesis. L-Arginine is a precursor in the synthesis of nitric oxide (NO). NO promotes spermatogenesis, enhanced sperm vigor, and sperm insemination ability.4345 The present findings demonstrated that only six proteins exhibited significantly different expression levels in both TMF and TUF relative to UUF (Figure 3B). Two (vitellogenin and ovarian serine protease nudel) of six proteins associated with reproduction in N. lugens adult females were significantly upregulated (Figure 2). During the reproductive phase, vitellogenin (Vg) is synthesized in large amounts in the female fat body, secreted into the hemolymph, and then taken up by the 4609


Journal of Proteome Research developing oocytes by receptor-mediated endocytosis.4650 The expression profile revealed that the Nlvg gene starts to be expressed earlier (on day 3) in brachypterous compared to macropterous females, where the mRNA transcript was observed on day 4.51 Triazophos induced Vg mRNA expression in the fifth instar nymphs, while vitellogenin synthesis is regulated at the mRNA level by translation.52,53 Our previous results have reported that vitellogenin was significantly increased in fat bodies and ovaries of N. lugens adult females after treatment with triazophos and mating.5 Heavy females often have more and larger eggs available for laying and are able to regenerate eggs faster when required.54,55 Males that mate with heavy or average Cnephasia jactatana females may sire more offspring, thus having higher probabilities of successful reproduction.56 Ovarian serine protease nudel plays a role in the Toll signaling pathway, egg activation, eggshell assembly, and maternal specification of dorsal and oocyte development. Experiments with genetically mosaic females indicated that embryonic dorsoventral polarity required nudel gene activity in the somatic tissue.57,58 Presumably, the follicle cells secrete eggshell components around the oocyte during oogenesis. Nudel gene production functions to generate a ventrally localized component of the vitelline envelope that directs the local activation of the Toll ligand.57,58 Hong and Hashimoto (1995) showed that the first protease in this pathway (Toll signaling pathway) is encoded by nudel.59 Lemosy et al. (1998) showed that the serine protease activity of the nudel protein is essential for embryonic dorsoventral polarity and that nudel protease activation serves to trigger the activation of the Toll signaling pathway.60 Hsp70 protein, which is associated with response to stress, was significantly up-regulated in both TMM and TUM relative to UUM. This indicates that triazophos treatment is beneficial to N. lugens for survival, repair, and improved resistance. Under stressful conditions, Hsps migrate into the cell nucleus where they act to repair or protect the nuclear proteins and minimize protein aggregation, thereby preventing genetic damage.61,62 Enolase and caboxylesterase were up-regulated in the UUM vs TUM group. Enolase is a key enzyme in the glycolytic pathway and is ubiquitous in cells from bacteria to higher animals and plants.63 Carboxylesterase was significantly up-regulated after triazophos treatment, indicating that it participates in the mechanisms related to insecticide resistance. Resistance of N. lugens to organophosphorous insecticides is associated with an elevation of carboxylesterase activity.64 Bao et al. (2010) suggest that the Nlcare gene expression level significantly increased in the fourth instar nymphs and on day 3 in the fifth instar nymphs exposed to triazophos as third instars.53 Trehalose transporter protein, which is associated with trehalose transport and sugar transport, was upregulated in the TUF vs TMF group. Trehalose is the major hemolymph sugar in most insects. It is predominantly synthesized in the fat body and released into the hemolymph.65 The trehalose transport gene conferred trehalose permeability to cells, including those of vertebrates as well as insects.66 Cuber et al. (1997) indicated that the trehalose transporter is essential during the germination process in order to translocate trehalose from the cytosol to the external environment.67 Two glycolic enzymes were up-regulated in the TUM vs TMM group. Soluble sugar content, crude body content, and the flight capacity of N. lugens adults after triazophos treatment significantly increased relative to the untreated control.68 An induction of ATP synthase was observed in response to aluminum (Al) in the Al-resistant cultivar of Triticum aestivum.69 A study of the Trypanosoma brucei ATP synthase is

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particularly important since mitochondrial structure and function is developmentally regulated in trypanosomes.70 As female reproductive molecules are identified and their functions are better understood, it will be important to understand the dynamics of their interactions with male reproductive molecules after insecticide treatment and mating. When evolutionary changes confer fitness benefits to one sex at a cost to the other, male and female processes that contribute to reproduction may coevolve antagonistically.71,72 A greater understanding of how females respond to mating genetically and physiologically will help us better understand the molecular aspects of sperm competition,73 sexual conflict,72 and cryptic female choice.74 The present results have significance for the further understanding of pesticide-induced resurgence and pest management. If we clearly understand the changes of proteome levels of N. lugens treated with pesticides, especially proteins related to reproduction, we may develop a new control technique on the basis of reproductive molecular biology from another view, for example, the suppression techniques of reproduction proteins. In addition, proteome analysis of treated males and its transference effect via mating have valuable information for the theory of pest resurgence, because the previous studies of pest resurgence focus on the effect of pesticides on female reproduction.

’ AUTHOR INFORMATION Corresponding Author

*Tel: 86-0514-87979246. Fax: 86-0514-87349917. E-mail: jc.wu@ public.yz.js.cn.

’ ACKNOWLEDGMENT This research was funded in part by National Basic Research Program of China (973 Program) (No. 2010CB126200) and by the National Natural Science Fund of China (No. 30870393). ’ REFERENCES (1) Heinrichs, E. A. Impact of insecticides on the resistance and resurgence of rice planthoppers. In Planthoppers: Their Ecology and Management; Denno, R. F., Perfect, T. J., Eds.; Chapman and Hall Press: New York, 1994; pp 571614. (2) Gao, C. X.; Gu, X. H.; Bei, Y. W.; Wang, R. M. Approach of causes on brown planthopper resurgence. Acta Ecol. Sin. 1988, 8, 155–163. (3) Chelliah, S.; Heinrichs, E. A. Factors affecting insecticide-induced resurgence of the brown planthopper, Nilaparvata lugens on rice. Environ. Entomol. 1980, 9, 773–777. (4) Wang, L. P.; Shen, J.; Ge, L. Q.; Wu, J. C.; Yang, G. Q.; Jahn, G. C. Insecticide-induced increase in the protein content of male accessory glands and its effect on the fecundity of females in the brown planthopper Nilaparvata lugens Stal (Hemiptera: Delphacidae). Crop Prot. 2010, 29, 1280–1285. (5) Ge, L. Q.; Wang, L. P.; Zhao, K. F.; Wu, J. C.; Huang, L. J. Mating pair combinations of insecticide-treated male and female Nilaparvata lugens (Stal) (Hemiptera: Delphacidae) planthoppers influence protein content in the male accessory glands (MAGs) and vitellin content in both fat bodies and ovaries of adult females. Pestic. Biochem. Physiol. 2010, 98, 279–288. (6) Chen, P. S. The functional morphology and biochemistry of insect male accessory glands and their secretions. Annu. Rev. Entomol. 1984, 2, 233–255. (7) Kalb, J. M.; Dibenedetto, A. J.; Wolfner, M. F. Probing the function of Drosophila melanogaster accessory glands by directed cell ablation. Proc. Natl. Acad. Sci. U. S. A. 1993, 90, 8093–8097. 4610


Journal of Proteome Research (8) Aigaki, T.; Fleischmann, I.; Chen, P. S.; Kubli, E. Ectopic expression of sex peptide alters reproductive behavior of female D. melanogaster. Neuron 1991, 7, 557–563. (9) Chen, P. S.; Stumm-Zollinger, E.; Aigaki, T.; Balmer, J.; Bienz, M.; Bohler, P. A male accessory gland peptide that regulates reproductive behavior of female D. melanogaster. Cell 1988, 54, 291–298. (10) Gillott, C. Male accessory gland secretions: modulators of female reproductive physiology and behavior. Annu. Rev. Entomol. 2003, 48, 163–184. (11) Lay, M.; Loher, W.; Hartmann, R. Pathways and destination of some male gland secretions in female Locusta migratoria migratorioides (R&F) after insemination. Arch. Insect Biochem. Physiol. 2004, 55, 1–25. (12) Komatsu, S.; Rakwal, R.; Li, Z. Separation and characterization of proteins in rice (Oryza sativa) suspension cultured cells. Plant Cell, Tissue Organ Cult. 1999, 55, 183–192. (13) Antelman, H.; Scharf, C.; Hecker, M. Phosphate starvation inducible proteins in Bacilus subtilis: proteomics and transcriptional analysis. J. Bacteriol. 2000, 182, 4478–4490. (14) Konishi, H.; Ishiguro, K.; Komatsu, S. A proteomics approach towards understanding blast fungus infection in rice grown under different levels of nitrogen fertilization. Proteomics 2001, 1, 1162–1171. (15) Chen, G.; Gharib, T. G.; Huang, C. C.; Thomas, D. G.; Sheddan, K. A.; Taylor, J. M. G.; Kardia, S. L. R.; Misek, D. E.; Giordano, T. J. Proteomic analysis of lung adenocarcinoma: identification of a highly expressed set of proteins in tumors. Clin. Cancer Res. 2002, 8, 2298–2305. (16) Natera, S. H.; Guerreiro, N.; Djordjevic, M. A. Proteome analysis of differentially displayed proteins as a tool for investigation of symbiosis. Mol. Plant-Microbe Interact. 2000, 13, 995–1009. (17) Ishimura, R.; Ohsako, S.; Kawakami, T.; Sakaue, M.; Aoki, Y.; Tohyama, C. Altered protein profile and possible hypoxia in the placenta of 2,3,7,8-tetrachlorodibenzo-p-dioxin-exposed rats. Toxicol. Appl. Pharmacol. 2002, 185, 197–206. (18) Adachi, J.; Kunmar-Kunmar, C.; Zhang, Y.; Olsen, J. V.; Mann, M. The human urinary proteome contains more than 1500 proteins, including a large proportion of membrane proteins. Genome Biol. 2006, 7, R80, DOI: . (19) Zhou, Z. H.; Yang, H. J.; Chen, M.; Lou, C. F.; Zhang, Y. Z.; Chen, K. P.; Wang, Y.; Yu, M. L.; Yu, F.; Li, J. Y.; Zhong, B. X. Comparative proteomic analysis between the domesticated silkworm (Bombyx mori) reared on fresh mulberry leaves and on artificial diet. J. Proteome Res. 2008, 7, 5103–5011. (20) Hossein Mohaddam, S.; Du, X.; Li, J.; Cao, J. R.; Zhong, B. X.; Chen, Y. Y. Proteome analysis on differentially expressed proteins of the fat body of two silkworm breeds, Bombyx mori, exposed to heat shock exposure. Biotechnol. Bioprocess Eng. 2008, 13, 624–631. (21) de Souza, G. A.; Godoy, L. M.; Mann, M. Identification of 491 proteins in the tear fluid proteome reveals a large number of proteases and protease inhibitors. Genome Biol. 2006, 7, R72. (22) Li, J. Y.; Chen, X.; Fan, W.; Hosseini Moghaddam, S. H.; Chen, M.; Zhou, Z. H.; Yang, H. J.; Chen, J. E.; Zhong, B. X. Proteomic and bioinformatic analysis on endocrine organs of domesticated silkworm, Bombyx mori for a comprehensive understanding of their roles and relateions. J. Proteome Res. 2009, 8, 2620–2632. (23) Li, J. Y.; Chen, X.; Hosseini Moghaddam, S. H.; Chen, M.; Wei, H.; Zhong, B. X. Shortgun proteomics approach to characterizing the embryonic proteome of silkworm, Bombyx mori, at labrum appearance stage. Insect Mol. Biol. 2009, 18, 649–660. (24) Azzam, S.; Wang, F.; Wu, J. C.; Shen, J.; Wang, L. P.; Yang, G. Q.; Guo, H. R. Comparisons of stimulatory effects of a series of concentrations of four insecticides on reproduction in the rice brown planthopper Nilaparvata lugens (Stal) (Hemiptera: Delphacidae). Int. J. Pest Manage. 2009, 55, 347–358. (25) Dai, H. G.; Wu, X. Y.; Feng, C. J.; Yang, Y. H. The relationship between mating behaviour and titer of juvenile hormone in brown planthopper, Nilaparvata lugens (Stal). Acta Entomol. Sin. 1997, 40, 153–158. (26) Livak, K. J.; Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2ΔΔCT method. Methods 2001, 25, 402–408.

ARTICLE

(27) Noda, H.; Kawai, S.; Koizumi, Y.; Matsui, K.; Zhang, Q.; Furukawa, S.; Shimomura, M.; Mita, K. Annotated ESTs from various tissues of the brown planthopper Nilaparvata lugens: A genomic resource for studying agricultural pests. BMC Genomics 2008, 9, 117. (28) Xue, J.; Bao, Y. Y.; Li, B. L.; Cheng, Y. B.; Peng, Z. Y.; Liu, H.; Xu, H. J.; Zhu, Z. R.; Lou, Y. G.; Cheng, J. A.; Zhang, C. X. Transcriptome analysis of the brown planthopper Nilaparvata lugens. PLoS One 2010, 5 (12), e14233, DOI: 10.137/journal.pone.001423. (29) Sharma, R.; Komatsu, S.; Noda, H. Proteomic analysis of brown planthopper: application to the study of carbamate toxicity. Insect Biochem. Mol. Biol. 2004, 34, 425–432. (30) Zhao, K. F.; Shi, Z. P.; Wu, J. C. Insecticide-induced enhance ment of flight capacity of the brown planthopper Nilaparvata lugens Stal (Hemiptera: Delphacidae). Crop Prot. 2011, 30, 475–482. (31) Shen , L.; Cheng , X. N. The effect of migration on reproduction of Nilaparvata lugens (Stal). J. Nanjing Agric. Univ. 1998, 21, 3135. (32) Phelan, P. L.; Barker, J. C. Male size related to courtship success and intersexual selection on tobacco moth Ephesia cautella. Experientia 1986, 42, 1291–1293. (33) Bissoondath, C. J.; Wiklund, C. Effect of male mating history and body size on ejaculate size and quantity in two polyandrous butterflies, Pieris napi and Pieris rapae (Lepidoptera: Pieridae). Funct. Ecol. 1996, 10, 457–464. (34) Ponlawat, A.; Harrington, L. C. Age and body size influence male sperm capacity of the dengue vector Aedes aegypti (Diptera: Culicidae). J. Med. Entomol. 2007, 44, 422–426. (35) Russel, L. D. Morphological and functional evidence for Sertoli germ cell relationship. In The Sertoli Cell; Russel, L. D., Griswold, M. D., Eds.; Cache River Press: Clearwater, FL, 1993; pp 65390. (36) Kuubli, E. Sex-peptides: seminal peptides of the Drosophila male. Cell. Mol. Life Sci. 2003, 60, 1689–1704. (37) Wolfner, M. F. The gifts that keep on giving: physiological functions and evolutionary dynamics of male seminal proteins in Drosophila. Heredity 2002, 88, 85–93. (38) Swanson, W. J.; Vacquier, V. D. The rapid evolution of reproductive proteins. Nat. Rev. Genet. 2002, 3, 137–144. (39) Chapman, T. Seminal fluid-mediated fitness traits in Drosophila. Heredity 2001, 87, 511–521. (40) McGraw, L. A.; Gibson, G.; Clark, A. G.; Wolfner, M. F. Genes regulated by mating, sperm or seminal proteins in mated female Drosophila melanogaster. Curr. Biol. 2004, 14, 1509–1514. (41) Xiao, J. H.; Xu, M.; Li, J. M.; Chan, H. C.; Lin, M.; Zhu, H.; Zhang, W.; Zhou, M.; Zhao, B.; Sha, J. H. NYD-SP6, a novel gene potentially involved in regulating testicular development/spermatogenesis. Biochem. Biophys. Res. Commun. 2002, 291, 101–110. (42) Seth, R. K.; Kaur, J. J.; Reynolds, S. E. Sperm transfer during mating, movement of sperm in the female reproductive tract, and sperm precedence in the common cutworm Spodoptera litura. Physiol. Entomol. 2002, 27, 1–14. (43) Fujisawa, M.; Yamanaka, K.; Tanaka, H.; Okada, H.; Arakawa, S.; Kamidono, S. Expression of endothelial nitric oxide synthase in the sertoli cells of men with infertility of various causes. BJU Int. 2001, 87, 85–88. (44) Zhang, H.; Zheng, R. L. Possible role of nitric oxide on fertile and asthenozoospermic infertile human sperm functions. Free Radical Res. 1996, 25, 347–354. (45) Yamamoto, I.; Tomizawa, M.; Saito, T.; Miyamoto, T.; Walcott, E. C.; Sumikawa, K. Structural factors contributing to insecticidal selective actions of neonicotinoids. Arch. Insect Biochem. Physiol. 1998, 37, 24–32. (46) Byrne, B. M.; Gruber, M.; Ab, G. The evolution of egg yolk proteins. Prog. Biophys. Mol. Biol. 1989, 53, 33–69. (47) Raikhel, A. S.; Dhadialla, T. S. Accumulation of yolk proteins in insect oocytes. Annu. Rev. Entomol. 1992, 37, 217–251. (48) Hagedorn, H. H.; Maddison, D. R.; Tu, Z. The evolution of vitellogenins, cyclorrhaphan yolk proteins and related molecules. Adv. Insect Physiol. 1998, 27, 335–384. (49) Snigirevskaya, E. S.; Raikhel, A. S. Receptor-mediated endocytosis of yolk proteins in insect oocytes. In Progress in Vitellogenesis; 4611


Journal of Proteome Research Raikhel, A. S., Sappington, T. W., Eds.; Reproductive Biology of Vertebrates; Adiyodi, K. G., Adiyodi, R. G., Eds.; Science Publishers, Inc.: Enfield, NH, Plymouth, U.K., 2005; Vol XII, Part B, pp 199228. (50) Tufail, M.; Takeda, M. Insect vitellogenin/lipophorin receptors: molecular structures, role in oogenesis, and regulatiory mechanisms. J. Insect Physiol. 2009, 55, 87–103. (51) Tufail, M.; Naeemullah, M.; Elmogy, M.; Sharma, P. N.; Takeda, M.; Nakamura, C. Molecular cloning, transcriptional regulation, and differential expression profiling of vitellogenin in two wing-morphs of the brown planthopper, Nilaparvata lugens Stal (Hemiptera: Delphacidae). Insect Mol. Biol. 2010, 19, 787–798. (52) Ge, L. Q.; Wu, J. C.; Zhao, K. F.; Chen, Y.; Yang, G. Q. Induction of Nlvg and suppression of Nljhe gene expression in Nilaparvata lugens (Stal) (Hemiptera: Delphacidae) adult females and males exposed to two insecticides. Pestic. Biochem. Physiol. 2010, 98, 269–278. (53) Bao, Y. Y.; Li, L.; Liu, Z. B.; Xue, J.; Zhu, Z. R.; Cheng, J. A.; Zhang, C. X. Triazophos up-regulated expression in the female brown planthooper, Nilaparvata lugens. J. Insect Physiol. 2010, 56, 1087–1094. (54) Cloutier, C.; Duperron, J.; Tertuliano, M.; McNeil, J. N. Host instar, body size and fitness in the koinobiotic parastoid Aphidius negripes. Entomol. Exp. Appl. 2000, 97, 29–40. (55) García-Barros, E. Body size, egg size, and their interspecific relationships with ecological and life history traits in butterflies (Lepidoptera: Papilionoidea, Hesperioidea). Biol. J. Linn. Soc. 2000, 70, 251–284. (56) Iyengar, V.; Eisner, T. Parental body mass as a determinant of egg size and egg output in an arctiid moth (Utetheisa ornatrix). J. Insect Behav. 2002, 15, 309–318. (57) Sch€upbach, T.; Clifford, R. J.; Manseau, L. J.; Price, J. V. Dorsoventral signaling processes in Drosophila oogenesis. In CellCell Interactions in Early Development; Gerhart, J., Ed.; Wiley-Liss: New York, 1991; pp 163174. (58) Stein, D.; Roth, S.; Vogelsang, E.; N€usslein-Volhard, C. The polarity of the dorsoventral axis in the Drosophila embryo is defined by an extracellular signal. Cell 1991, 65, 429–440. (59) Hong, C. C.; Hashimoto, C. An unusual mosaic protein with a protease domain, encoded by the nudel gene, is involoved in defining embryonic dorsoventral polarity in Drosophila. Cell 1995, 82, 785–794. (60) Lemosy, E. K.; Kemler, D.; Hashimoto, C. Role of nudel protease activation in triggering dorsoventral polarization of the Drosophila embryo. Development 1998, 125, 4045–4053. (61) Bensaude, O.; Pinto, M.; Dubois, M. F.; Vanrung, N.; Morange, M. Protein denaturation during heat shock and related stress. In Stress Proteins: Induction and Function; Schlesinger, M. J., Santoro, M. G., Garaci, E., Eds.; Springer-Verlag: Berlin, Germany, 1990; pp 8999. (62) Ohtsuka, K.; Hata, M. Molecular chaperone function of mammalian Hsp 70 and Hsp 40, a review. Int. J. Hyperthermia 2000, 16, 231–245. (63) Canback, B.; Andwesson, S. G. E.; Kurland, C. G. The global phylogeny of glycolytic enzymes. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 6097–6012. (64) Small, G. J.; Hemingway, J. Molecular characterization of the amplified carboxylesterase gene associated with organophosphorus insecticide resistance in the brown planthopper, Nilaparvata lugens. Insect Mol. Biol. 2000, 9, 647–653. (65) Wyatt, G. R. The biochemistry of sugars and polysaccharides in insects. Adv. Insect Physiol. 1967, 4, 287–360. (66) Kikawada, T.; Saito, A.; Kanamori, Y.; Nakahara, Y.; Lwata, K.; Tanaka, D.; Watanabe, M.; Okuda, T. Trehalose transporter 1, a facilitated and high-capacity trehalose transporter, allows exogenous trehalose uptake into cells. Proc. Natl. Acad. Sci. U. S. A. 2007, 104, 11585–11590. (67) Cuber, R.; Eleutherio, E. C. A.; Pereira, M. D.; Panek, A. D. The role of the trehalose transporter during germination. Biochim. Biophys. Acta 1997, 1330, 165–171. (68) Yin, J. L.; Xu, H. W.; Wu, J. C.; Hu, J. H.; Yang, G. Q. Cultivar and insecticide applications affect the physiological development of the brown planthopper, Nilaparvata lugens (Stal) (Hemiptera: Delphacidae). Environ. Entomol. 2008, 37, 206–212.

ARTICLE

(69) Hamilton, C. A.; Good, A. G.; Taylor, G. J. Induction of vacuolar ATPase and mitochondrial ATP synthase by aluminium in aluminiumresistant cultivar of wheat. Plant Phyiol. 2001, 125, 2068–2077. (70) Silvia, V.; Brown, B.; Stanislawski, A.; Quinesha, L.; Williams, P. N. Cloning and characterization of the subunits comprising the catalytic core of Trypanosaoma brucei mitochondrial ATP synthase. Mol. Biochem. Parasitol. 2011, 113, 289–301. (71) Parker, G. A. Sexual selection and sexual conflict. In Sexual Selection and Reproductive Competition in Insects; Blum, M. S., Blum, N. A., Eds.; Academic: New York, 1979; pp 123166. (72) Rice, W. R. Sexually antagonistic male adaptation triggered by experimental arrest of female evolution. Nature 1996, 361, 232–234. (73) Parker, G. A. Sperm competition and its evolutionary consequences in the insects. Biol. Rev. 1970, 45, 525–567. (74) Eberhard, W. G. Female Control: Sexual Selection by Cryptic Female Choice; Princeton University Press: Princeton, NJ, 1996.

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