A role for transferrin in triggering apoptosis in Helicoverpa armigera

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Food Safety and Toxicology

A role for transferrin in triggering apoptosis in Helicoverpa armigera cells treated with 2-tridecanone Lei Zhang, Junping Gao, and Xiwu Gao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02505 • Publication Date (Web): 28 Sep 2018 Downloaded from http://pubs.acs.org on September 29, 2018

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Journal of Agricultural and Food Chemistry

A role for transferrin in triggering apoptosis in

1 2

Helicoverpa armigera cells treated with 2-tridecanone

3

Lei Zhang1, Junping Gao2, Xiwu Gao1*

4

1

Department of Entomology, China Agricultural University, Beijing, 100193, PR China.

5

2

Beijing Key Laboratory of Development and Quality Control of Ornamental Crops, China Agricultural University,

6

Beijing, 100193, PR China.

7 8

ABSTRACT:

9

(Lycopersicon hirsutum f. glabratum), can induce the expression of Helicoverpa armigera

10

transferrin (HaTrf), which is necessary for insect growth and development. To gain further insight

11

into the mechanism of HaTrf in the response to 2-tridecanone, we measured the iron and H2O2

12

levels in the hemolymph during exposure to 2-tridecanone, and then explored the effect of

13

transferrin downregulation in a H. armigera fat body cell line exposed to 2-tridecanone. We found

14

that the reduction of HaTrf levels via RNA interference (RNAi) caused rapid apoptotic cell death

15

during exposure to 2-tridecanone. There have been no reports about transferrin genes related to

16

apoptosis induced by plant allelochemical. Our results indicate that HaTrf mediates the inhibition

17

of apoptotic cell death during exposure to 2-tridecanone and provides insight into the importance

18

of transferrin in the interaction between plants and insects.

19

KEYWORDS: Transferrin; Apoptosis; Helicoverpa armigera; 2-tridecanone; RNA

20

interference

2-tridecanone, a plant allelochemical present in a large range of tomato species

21 22

*Corresponding Author: Professor Xi-Wu Gao

ACS Paragon Plus Environment


23

Department of Entomology

24

China Agricultural University

25

Beijing, 100193, PR China

26

Tel/fax: +86 1 62732974

27

Email: [email protected]

28

INTRODUCTION

29

Insects have evolved various strategies to increase their performance and fitness, and plants

30

have also evolved efficient strategies to defend themselves against pest insects. Plant

31

allelochemicals may be potential alternatives for controlling pests1. However, the mechanisms by

32

which insects increase their ability to adapt to host plants are still largely unknown. Despite many

33

studies of insect transferrins, their specific functions are still poorly understood2. In mammals and

34

plants, transferrin genes were involved in iron transport, while limited evidence indicate that

35

transferrin may be involved in iron transport in insects3, 4, many studies suggest that insect

36

transferrin has some type of immune function5. Consistent with these functions, transferrin is

37

present in hemolymph and has been detected in other extracellular fluids that contain immune

38

proteins2, 5-9. Transferrins contribute to adaptation of insects to various stresses10-13. In Apriona

39

germari, transferrin mRNA and protein levels are up-regulated in response to cold shock (4 °C),

40

sterile wounding, and paraquat (10 mM) exposure10. Some groups have reported that the mRNA

41

expression of transferrin is up-regulated by the xenobiotic cypermethrin in cypermethrin-resistant

42

Culex pipiens pallens strains, and that transferrins might confer insecticide resistance14, 15. Insect transferrin is an antioxidant protein and may function in antioxidative defenses10, 11,

43 44

16-21

. Many researchers have also suggested that insect transferrin is involved in oxidative


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stress-induced apoptosis, just like heat shock, H2O2, or fungal challenge, but little is known about

46

the pathway and molecular mechanism of this anti-apoptotic effect10, 11. Transferrin controls the

47

level of free iron and rapidly binds free iron in the hemolymph, which protects tissues from

48

iron-induced oxidative stress18, 22, 23. Some researchers have hypothesized that insect transferrin

49

may play an important role in the defense against the generation of hydroxyl radicals, which are

50

produced by an Fe2+ catalyzed reaction from H2O2 and are highly reactive, damaging cells by

51

oxidizing DNA, membrane lipids and proteins24. Transferrin reduces the levels of these hydroxyl

52

radicals by sequestering iron, thereby reducing damage10. Consistent with these hypotheses, RNAi

53

interference (RNAi) mediated reduction of transferrin transcript accumulation caused rapid

54

apoptotic cell death in fat bodies during exposure to stress in A. germari and Protaetia

55

brevitarsis11, and then iron and H2O2 levels are increased in the hemolymph of P. brevitarsis11.

56

In a previous study we found that 2-tridecanone, a plant allelochemical present in a large

57

range of tomato species (Lycopersicon hirsutum f. glabratum), induced the expression of

58

Helicoverpa

59

(dsRNA)-mediated depletion of HaTrf in larvae decreased their tolerance to 2-tridecanone, and

60

larval growth was significantly inhibited following feeding with HaTrf dsRNA; thus, HaTrf is

61

necessary for insect growth and development9. To gain further insight into the functional role of

62

HaTrf in the response to 2-tridecanone, we measured the iron and H2O2 levels in the hemolymph

63

during exposure to 2-tridecanone challenge, and then explored the effects of transferrin

64

downregulation on the response of a H. armigera fat body cell line to 2-tridecanone. Our results

65

may provide insight into how HaTrf mediates the inhibition of apoptotic cell death during

66

exposure to 2-tridecanone and reveal a mechanism for plant and insect interaction.

armigera

transferrin

(HaTrf)9

.

Furthermore,


double

stranded

RNA


67 68

MATERIALS AND METHODS

69

Insect strain and cell line

70

The H. armigera population used in this study was collected from Handan in Hebei Province, China

71

in 1998, and reared on an artificial diet in an air conditioned room maintained at 26 ±1 °C, 70-80 %

72

relative humidity, and with a photoperiod of 16:8 (L:D). The artificial diet was supplied as described

73

in our previous study37. Adults were held under the same conditions and supplied with a 10% sugar

74

solution. The homologous H. armigera fat body cell line was generously provided by Dr. Huan

75

Zhang (Zoology, CAS, China). The cell line was routinely maintained with sf900-II insect

76

serum-free medium (Thermo Fisher Scientific, USA) supplemented with 10% heat-inactivated fetal

77

bovine serum (Gibco, USA) at 27 °C.

78 79

Collection of hemolymph

80

Hemolymph was collected in cold test tubes by cutting off the head of 6th H. armigera larvae. The

81

ten 6th instar larvae were used to collect the hemolymph from each treatment. The collected

82

hemolymph was centrifuged at 10,000 g for 10 min to remove hemocytes and cell debris, and the

83

supernatant was recovered for experimentation.

84 85

Measurement of total iron in hemolymph

86

To determine total iron concentration in H. armigera larvae, we collected hemolymph from the 6th

87

instar larvae at 24 h after exposed to artificial diet containing 2-tridecanone (10 mg/g, w/w) and

88

HaTrf dsRNA (35 µg/g, w/w). Iron level in hemolymph was measured using the QuantiChrom™


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Iron Assay Kit (BioAssay Systems, Hayward, CA, USA). This method utilizes a chromogen that

90

forms a blue-colored complex specifically with Fe2+. Fe3+ in the sample is reduced to Fe2+, thus

91

allowing for the determination of total iron concentration. The intensity of the color, measured at

92

590 nm, is directly proportional to the iron concentration in the sample. The experiment

93

performed for six experimental replicates. 10 µL hemolymph was used for each experiment. Iron

94

concentration was expressed as µg mL-1 hemolymph.

were

95 96

Measurement of hydrogen peroxide

97

H2O2 concentration was measured using the Hydrogen Peroxide Assay Kit (Beyotime, China).

98

This assay is based on the oxidation of Fe2+ to Fe3+ by H2O2 under acidic conditions. The Fe3+

99

binds to the xylenol orange indicator dye to form a stable colored complex, which can be

100

measured at 560 nm. Assays were performed for six experimental replicates. 10 µL hemolymph

101

was used for each experimentation. The H2O2 concentration was expressed as µM mL

102

hemolymph.

-1

103 104

The viability of 2-tridecanone-treated H. armigera fat body cells

105

H. armigera fat body cells were seeded into wells of 96-well plates (5 × 103 cells/well) containing

106

0.1 mL sf900-II insect serum-free medium supplemented with 10% heat-inactivated fetal bovine

107

serum. Ten biological replicates were performed for each treatment. After 12 hours, the

108

supplemented medium was discarded and 0.1 mL new supplemented medium (containing 10%

109

fetal bovine serum) plus/minus 2-tridecanone (at a final concentration of 125 µM, based on LC20

110

=125 µM determined at 48 h) was added into the wells and incubated for 0, 12, 24, 48 and 72 h at



111

27 °C. Following incubation, 10 µL of Cell Counting Kit-8 reagent (Dojindo Laboratories, Japan)

112

was added to each well, and cells were incubated for 4 h according to the manufacturer's

113

instructions. The number of active cells was estimated by measuring the absorbance at 450 nm

114

using a spectrophotometer (Bio-Rad). The fat body cells were not treated with 2-tridecanone as the

115

control at each time point. The cell viability was calculated according to the following formula:

116

Cell viability(%)=[Abs(S)−Abs(B)]/[Abs(C)−Abs(B)]×100.

117

Where Abs (S), Abs (B) and Abs (C) are the absorbance of the sample, blank and control,

118

respectively.

119 120

The expression levels of HaTrf after treatment with 2-tridecanone

121

H. armigera fat body cells were seeded in wells of 6-well plates (9×105cells/well) containing 2

122

mL supplemented medium. After 12 h, the supplemented medium was discarded, and 2 mL new

123

supplemented medium (containing 10% fetal bovine serum) mixed with 125 µM 2-tridecanone

124

was added to each well. Cells were then incubated for 0, 12, 24 and 48 h. Three biological

125

replicates were performed for each treatment. Total RNA was isolated from H. armigera fat body

126

cells using Trizol reagent (Invitrogen, USA) according to the manufacturer’s instructions. An

127

additional DNaseI digestion was performed using RNase-Free DNaseI (TaKaRa, Japan). Two

128

micrograms of each RNA sample were used as the template for synthesis of the first-strand cDNA,

129

using oligo-dT18 primer and M-MLV Reverse Transcriptase (TaKaRa, Japan). One microliter of

130

cDNA (1000 ng/µL) was used as the template to amplify HaTrf and EF-1a. The levels of HaTrf

131

transcripts were determined by qPCR by using primers qHaTrf-F and qHaTrf-R (Table 1), and

132

levels of EF-1a transcripts were determined using primers qEF-F and qEF-R (Table 1). Each


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sample was analyzed in triplicate and normalized to the internal control, EF-1α37. A standard curve

134

was generated for each set of primers, and the efficiency for all of the primer pairs was 100%.

135

Statistical analyses were performed using GraphPad Prism 5.0 software, and a P value less than 0.05

136

was considered statistically significant.

137 138

The effect of RNAi-induced silencing of HaTrf on H. armigera fat body cells

139

Synthesis of HaTfr dsRNA was the same as our previous report. Based on the HaTrf sequence and

140

predicted

141

(http://www.dkfz.de/signaling/e-rnai3/), we designed specific primers using DNAMAN 6.0

142

software. A 667-bp fragment of HaTrf (position 204–870) was amplified and cloned into

143

pMD-18simple-T (TaKaRa Dalian, China), using the dsRNAi-Tf1 and dsRNAi-Tf2 primers

144

(Table 1) containing the additional T7 promoter sequences. GFP dsRNA, which was used as the

145

control, was synthesized using the same procedures with primers dsGFP-F and dsGFP-R (Table 1).

146

Purified plasmids served as templates for synthesis of GFP and HaTrf dsRNA using the

147

MEGAscript T7 transcription kit (Ambion, Austin, TX, USA) with an extended transcription time

148

of 6 h at 37 °C. The resulting dsRNA was digested by DNase I and RNase to remove DNA and

149

any single-stranded RNA, and finally dissolved in DEPC water (1 mL diethylprocarbonate, DEPC

150

diluted with 1 L dd H2O2)9.

possible

interference

sites

obtained

using

online

prediction

software

151

H. armigera fat body cells seeded in wells of 6-well plates (9×105 cells/well) containing 2

152

mL supplemented medium were transfected with 5 µg HaTrf dsRNA or GFP dsRNA for 6 h. The

153

supplemented medium was then discarded, and 2 mL new supplemented medium (containing 10%

154

fetal bovine serum) was added to each well. Cells were incubated for 12, 24 and 36 h. Three



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biological replicates were performed for each treatment. The dsRNA-mediated depletion of HaTrf

156

transcripts was determined by qPCR by using primers qHaTrf-F and qHaTrf-R (Table1). Levels of

157

EF-1a transcripts were determined using the qEF-F and qEF-R primers as previously described

158

(Table 1). Each sample was analyzed in triplicate and normalized to the internal control, EF-1α37.

159

Statistical analyses were performed using GraphPad Prism 5.0 software, and a P value less than 0.05

160

was considered statistically significant.

161 162

Table 1 Primers used in this study Primer name

Sequence

Application

dsRNAi-Tf1

5’-taatacgactcactatagggCAAGCAGACATCGTGCCAGT-3’

HaTrf dsRNA synthesis

dsRNAi-Tf2

5’-taatacgactcactatagggGTCTAGCTGCCCAGGAACAG-3’

HaTrf dsRNA synthesis

dsGFP-F

5’-taatacgactcactatagggagaCAGTGCTTCAGCCGCTAC-3’

GFP dsRNA synthesis

dsGFP-R

5’-taatacgactcactatagggagaGTTCACCTTGATGCCGTTC-3’

GFP dsRNA synthesis

qHaTrf-F

5’-GGTCCAAAGTGCCGATCAAATCCAAA-3’

Real-Time PCR

qHaTrf-R

5’-GCGAGCTAATTCATTCAACTTCTCTCTCA-3’

Real-Time PCR

qEF-F

5’- AGGAGTTGCGTCGTGGTTA-3’

Real-Time PCR

qEF-R

5’-GACTTGATGGACTTAGGGTTGT-3’

Real-Time PCR

163 164

The effects of HaTrf RNAi on the tolerance of H. armigera fat body cells to 2-tridecanone

165

H. armigera fat body cells seeded in wells of 96-well plates (5 × 103cells/well) containing 0.1 mL

166

supplemented medium were transfected with 0.25 µg HaTrf dsRNA or GFP dsRNA for 6 h. Ten

167

biological replicates were performed for each treatment. The supplemented medium was then


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discarded, and 0.1 mL new supplemented medium (containing 10% fetal bovine serum)

169

plus/minus 2-tridecanone (125 µM) was added into the wells, and cells were incubated for 12 h or

170

24 h at 37 °C. Following incubation, the Cell Counting Kit-8 reagent (Dojindo Laboratories, Japan)

171

was used to measure cell viability. Briefly, 10 µL of Cell Counting Kit-8 reagent was added to

172

each well, and cells were incubated for 4 h according to the manufacturer's instructions.

173 174

Double-immunofluorescence staining

175

H. armigera fat body cells seeded in wells of 6-well plates (9×105cells/well) containing 2 mL

176

supplemented medium were transiently transfected with 5 µg HaTrf dsRNA or GFP dsRNA for 6 h.

177

Then the supplemented medium was discarded, and 2 mL new supplemented medium (containing

178

10% fetal bovine serum) plus/minus 2-tridecanone (125 µM) was added into the wells, and cells

179

were incubated for 12 h at 37 °C. After incubation, H. armigera fat body cells were fixed in 4%

180

neutral buffered paraformaldehyde (NBP) for 15 min and then treated with 0.5% Triton X-100 at

181

room temperature for 10 min. The cells were stained with DAPI for 10 min. Then, the fat body

182

cells were double-labeled according to the in situ cell death detection kit (Roche Applied Science)

183

as follows. Polyclonal antibodies against HaTrf were raised in male New Zealand rabbits, and

184

prepared and purified polyclonal anti-HaTrf antibody was stored at -70°C. The cells were washed

185

three times in PBS and then pre-incubated in PBS containing 10% goat serum for blocking at

186

37 °C for 1 h. After incubation at 37°C for 1 h with 1:2000 (v/v) diluted antiserum against HaTrf

187

in PBS containing 1% BSA, the cells were washed in PBS with three times. They were then

188

incubated in 1:10000 (v/v) diluted goat anti-rabbit IgG conjugated to tetramethyl rhodamine

189

isothiocyanate (Santa Cruz Biotech., Inc.) in PBS containing 1% BSA for 30 min. Following three



190

successive washes in PBS for 1h, the cells were incubated in a TUNEL reaction mixture

191

containing terminal deoxynucleotidyl transferase (TdT) and fluorescein-conjugated dUTP at 37°C

192

for 1 h. After additional washing with PBS and wet mounting, HaTrf and apoptosis in the fat body

193

cells were visualized by laser scanning confocal microscopy Fv1000 (Olympus, Japan), and

194

images were processed with Photoshop CS, version 8 (Adobe, CA).

195 196

RESULTS

197

H2O2 and iron concentration in the hemolymph

198

We previously found that the level of HaTrf mRNA in larvae was dramatically upregulated

199

by 2-tridecanone9. In order to investigate the relationship between HaTrf and tolerance to

200

2-tridecanone, we first tested whether HaTrf expression level is correlated with iron and H2O2

201

levels in the hemolymph of wildtype H. armigera larvae and larvae expressing HaTrf dsRNA or

202

GFP dsRNA exposed to 2-tridecanone. We found that iron and H2O2 levels increased in the

203

hemolymph after exposure to 2-tridecanone for 24 h (Fig. 1A and B) and were significantly

204

elevated in HaTrf dsRNA-treated larvae compared to GFP dsRNA-treated larvae (Fig. 1A and B).

205

Combined exposure to 2-tridecanone and HaTrf dsRNA significantly increased iron and H2O2

206

levels compared with other treatments (Fig. 1A and B). These findings indicate that during

207

exposure to 2-tridecanone, HaTrf expression level is correlated with iron and H2O2 levels in the

208

hemolymph.

209

[Figure 1] position

210 211

The effect of 2-tridecanone on H. armigera fat body cell viability and HaTrf expression


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We used the Cell Counting Kit-8 to determine the effects of 2-tridecanone on the viability of

213

H. armigera fat body cells. Cell viability decreased dramatically after 12 h to 72 h of treatment

214

with 125 µM 2-tridecanone (Fig. 2A). The relative expression level of HaTrf compared with the

215

untreated group was higher at each time point after treatment with 125 µM 2-tridecanone (12, 24

216

and 48 h) (Fig. 2B). The expression level of HaTrf increased dramatically at 24 h after treatment

217

with 2-tridecanone, and then decreased significantly at 48 h after treatment (Fig. 2B).

218 219

The efficiency of HaTrf dsRNA-mediated RNAi in H. armigera fat body cells

220

The efficacy of RNAi was evaluated in fat body cells treated with HaTrf dsRNA for 12, 24,

221

and 36 h. The HaTrf dsRNA-treated cells showed a significant reduction of HaTrf expression

222

compared to cells treated with only GFP dsRNA (Figs 2C, 2D, 2E). The expression level of HaTrf

223

in cells transfected with HaTrf dsRNA decreased by 79% and 63% at 12 and 24 h, respectively,

224

compared with the control (Fig. 2C, 2D), but no significant inhibition of transcription was

225

observed after 36 h (Fig. 2E).

226

[Figure 2] position

227 228

The effects of HaTrf RNAi in H. armigera fat body cells

229

The Cell Counting Kit-8 was used to investigate the effect of HaTrf RNAi on H. armigera fat

230

body cells. We found that the survival rate of fat body cells treated with HaTrf dsRNA for 12 and

231

24 h decreased compared with the GFP dsRNA controls (Fig. 3). After 12 h, 74% of fat body cells

232

treated with HaTrf dsRNA were viable compared with 88% of cells treated with GFP dsRNA (Fig.

233

3A). At 24 h, the 67% of fat body cells treated with HaTrf dsRNA were viable compared with 84%



234

for the GFP dsRNA control (Fig. 3B). The cell viability (63%) of the group treated with a mixture

235

of HaTrf dsRNA and 125 µM 2-tridecanone was significantly lower compared to the other

236

treatments (73% for GFP dsRNA and 2-tridecanone, 74% for HaTrf dsRNA, and 88% for GFP

237

dsRNA) at 12 h (Fig. 3A). At 24 h, the cell viability of the group treated with a mixture of HaTrf

238

dsRNA and 125 µM 2-tridecanone (56%) was significantly lower compared to other treatments

239

(71% for GFP dsRNA and 2-tridecanone, 67% for HaTrf dsRNA treatment, and 84% for GFP

240

dsRNA treatment) (Fig. 3B). These results suggest that dsRNA-mediated depletion of HaTrf

241

significantly increases the susceptibility of fat body cells to 2-tridecanone.

242

[Figure 3] position

243 244

Double-immunofluorescence staining

245

We next determined whether the level of HaTrf is correlated with apoptotic cell death. Fig. 4

246

shows the effect of HaTrf RNAi on 2-tridecanone-induced apoptotic cell death. Fluorescence

247

microscopy revealed that induction of apoptotic cell death in the HaTrf dsRNA-treated fat body

248

cells was higher than in the GFP dsRNA-treated control (Fig. 4). Compared with cells treated only

249

with GFP dsRNA, more apoptotic cell death was observed in cells treated with GFP dsRNA

250

combined with 2-tridecanone (Fig. 4). Treatment of fat body cells with HaTrf dsRNA combined

251

with 2-tridecanone further accelerated apoptotic cell death (Fig. 4). These results suggest that

252

dsRNA-mediated depletion of HaTrf increases the susceptibility of fat body cells to

253

2-tridecanone-induced apoptotic cell death.

254

[Figure 4] position

255


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DISCUSSION

257

Although insect transferrin is involved in the inhibition of stress-induced apoptosis, little is

258

known as to why insect transferrin is upregulated during exposure to plant allelochemical and how

259

transferrin acts as a defense against stress in insects. To gain further insight into the function of

260

HaTrf in response to 2-tridecanone, we explored the effect on transferrin upregulation on the

261

response of H. armigera to 2-tridecanone, a plant allelochemical that induces the expression of

262

HaTrf in larvae9. We previously found that dsRNA-mediated depletion of HaTrf in larvae

263

decreased their tolerance to 2-tridecanone and significantly inhibited larval growth9. Here, we

264

studied the effect of reduction of HaTrf expression during exposure to 2-tridecanone, and found

265

that iron and H2O2 levels were significantly increased in the hemolymph of HaTrf RNAi-treated

266

larvae. Iron is an essential nutrient for living organisms, is required for a wide variety of metabolic

267

processes, including electron transfer, oxygen transport, nitrogen fixation, gene regulation, DNA

268

biosynthesis, and immunity25-27. Additionally, iron is involved in cuticle formation, tanning,

269

melanization, and wound healing in insects28. Iron is released from macromolecular complexes

270

under a number of conditions in which cells are exposed to oxidative stress, and this plays a

271

central role in generating harmful oxygen species29, 30. H2O2 as a byproduct of reactions catalyzed

272

by oxidases, is typically produced in the mitochondria and peroxisomes31,

273

exposure, the increased cell oxygen consumption leads to increased O2 and H2O2 production33, 34.

274

In addition, H2O2 is introduced into the cytoplasm via leakage from a range of organelles and

275

readily diffuses through the cell membrane. It is generally believed that H2O2 is poorly reactive to

276

cells alone, but rather reacts with intracellular transition metals like iron to form much more

277

damaging species, such as the hydroxyl radical11, 12, 31.


32

. During stress


278

Consistent with our previous findings in larvae, we observed that the viability of fat body

279

cells decreased dramatically after 12 to 72 h of treatment with 125 µM 2-tridecanone (Fig. 2A),

280

and HaTrf mRNA levels in the fat body cells were upregulated during exposure to 2-tridecanone

281

(Fig. 2B). The survival rate of fat body cells treated with HaTrf dsRNA for 12 and 24 h decreased

282

compared with the control, and cell viability was significantly lower in fat body cells treated with

283

a mixture of HaTrf dsRNA and 125 µM 2-tridecanone compared to other treatments at 24 h (Fig.

284

3B). There was a decrease in expression level of HaTrf in transfected HaTrf dsRNA cells observed

285

at 12 and 24 h after treatment but not after 36 h (Figure 2C, 2D, 2E), this may be because the fat

286

body cell has presumably contains numerous RNases, and different fat body cells environments at

287

each time point may require different concentrations of dsRNA to trigger gene silencing.

288

Furthermore, dsRNA-mediated depletion of HaTrf increased the susceptibility of fat body cells to

289

2-tridecanone-induced apoptotic cell death (Fig. 4). These results suggest that dsRNA-mediated

290

depletion of HaTrf significantly increases the susceptibility of fat body cells to 2-tridecanone.

291

Decreased levels of HaTrf led to increased iron and H2O2 levels and resulted in rapid

292

induction of apoptotic cell death. These results indicate that HaTrf may inhibit

293

2-tridecanone-induced apoptotic cell death, potentially by regulating iron levels. In fact, the

294

reduction of transferrin levels was previously shown to result in increased oxidative stress, a key

295

mediator of stress-induced apoptosis18, suggesting that transferrin has an antioxidant function16-18.

296

Under stress conditions, the increase in iron may further potentiate the effects of oxidative stress

297

due to the formation of hydroxyl radicals by the Fenton reaction, which yields the highly reactive

298

and toxic hydroxyl radical from the reaction of Fe2+ with H2O211, 12, 29, 35, 36. Therefore, reduction in

299

HaTrf during exposure to 2-tridecanone may induce apoptotic cell death via the production of


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300

hydroxyl radicals from the Fenton reaction.

301

Our results indicate that RNAi-mediated reduction of HaTrf in H. armigera rapidly induced

302

apoptotic cell death in response to 2-tridecanone, suggesting the involvement of insect transferrin

303

in the protection against cell death was induced by this plant allelochemical. Our findings may

304

explain how HaTrf mediates the inhibition of apoptotic cell death and provides insight into the

305

importance of transferrins in the interaction between plants and insects. Plant allechemicall may

306

be a potential alternative agent for controlling pest. These researches provide a theoretical basis to

307

develop more environmentally friendly insecticides and strategies for the safety of the food and

308

pest control for future.

309 310

Abbreviations Used

311

RNAi, RNA interference; HaTrf, Helicoverpa armigera transferrin; NBP, Neutral buffered

312

paraformaldehyde; DAPI, 4', 6-diamidino-2-phenylindole.

313 314

Acknowledgments

315

This research was supported by the National Natural Science Foundation of China (31601655) and

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China Postdoctoral Science Foundation (2016T90153, 2015M581208).

317 318

AUTHOR INFORMATION

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Corresponding Author

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* Telephone: +86-010-62732974. Fax: +86-010-62732974. Email: [email protected]

321

ORCID



322

Xiwu Gao: 0000-0003-3854-2449

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Funding

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This research was supported by the National Natural Science Foundation of China (31601655) and

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China Postdoctoral Science Foundation (2016T90153, 2015M581208)

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Notes

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The authors declare no competing financial interest.

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Figure caption

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Figure 1. H2O2 and iron concentration in the hemolymph of HaTrf dsRNA-treated larvae and controls. (A)

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Iron concentration in the hemolymph of HaTrf dsRNA- and 2-tridecanone-treated H. armigera larvae and the GFP


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dsRNA-treated controls. (B) H2O2 concentration in the hemolymph of HaTrf dsRNA- and 2-tridecanone-treated H.

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armigera larvae and GFP dsRNA-treated controls. Bars represent mean ± SD of replicated experiments (n=6).

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Bars sharing the different letter are significantly different at P < 0.05 (Tukey’s HSD tests).

435 436

Figure 2. The effect of HaTrf dsRNA-mediated RNAi silencing on H. armigera fat body cells. (A) The viability

437

of 2-tridecanone-treated H. armigera fat body cells (n=6); (B) The relative expression levels of HaTrf in fat body

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cells after treatment with 2-tridecanone; (C) The levels of HaTrf in HaTrf dsRNA-treated H. armigera fat body

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cells at 12 h; (D) The levels of HaTrf in HaTrf dsRNA-treated H. armigera fat body cells at 24 h; (E) The levels of

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HaTrf in HaTrf dsRNA-treated H. armigera fat body cells at 36 h. Bars represent mean ± SD of replicated

441

experiments (n=3). Bars sharing the different letter are significantly different at P < 0.05 (Tukey’s HSD tests).

442 443

Figure 3. The effects of 2-tridecanone and HaTrf RNAi on H. armigera fat body cells. (A) The viability of

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2-tridecanone-treated H. armigera fat body cells at 12 h (n=6); (B) The viability of 2-tridecanone-treated H.

445

armigera fat body cells at 12 h (n=6). Cell: untreated fat body cells; Cell+2-13: 2-tridecanone-treated fat body

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cells; dsGFP: fat body cells transfected with dsGFP; dsGFP+2-13: fat body cells transfected with dsGFP, then

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treated with 125 µM 2-tridecanone; dsTF: fat body cells transfected with dsHaTrf; dsTF+2-13: fat body cells

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transfected with dsHaTrf, then treated with 125 µM 2-tridecanone. Bars represent mean ± SD of replicated

449

experiments (n=10). Bars sharing the different letter are significantly different at P < 0.05 (Tukey’s HSD tests).

450 451

Figure 4. Effect of RNAi-mediated silencing of HaTrf on 2-tridecanone-induced cell apoptosis. Apoptosis

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(green) and HaTrf (red) were detected in fat body cells transfected with dsHaTrf or the dsGFP control. Merged

453

confocal images are shown in the fourth column. Scale bar=10 µm. dsHaTrf-2-13: fat body cells transfected with



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dsHaTrf, then treated with 125 µM 2-tridecanone for 12 h; dsHaTrf: fat body cells transfected with dsHaTrf;

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dsGFP-2-13: fat body cells transfected with dsHaGFP, then treated with 125 µM 2-tridecanone for 12 h; dsGFP:

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fat body cells transfected with dsGFP; 1: DAPI staining showing the nucleus; 2: 5-Carboxyfluorescein (FAM)

457

labeling of apoptotic cells; 3: Red signal showing the expression of HaTrf; 4: merged fluorescent signals.


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Figure 1. H2O2 and iron concentration in the hemolymph of HaTrf dsRNA-treated larvae and controls. (A) Iron concentration in the hemolymph of HaTrf dsRNA- and 2-tridecanone-treated H. armigera larvae and the GFP dsRNA-treated controls. (B) H2O2 concentration in the hemolymph of HaTrf dsRNA- and 2-tridecanonetreated H. armigera larvae and GFP dsRNA-treated controls. Bars represent mean ± SD of replicated experiments (n=6). Bars sharing the different letter are significantly different at P < 0.05 (Tukey’s HSD tests). 59x44mm (300 x 300 DPI)



Figure 2. The effect of HaTrf dsRNA-mediated RNAi silencing on H. armigera fat body cells. (A) The viability of 2-tridecanone-treated H. armigera fat body cells (n=6); (B) The relative expression levels of HaTrf in fat body cells after treatment with 2-tridecanone; (C) The levels of HaTrf in HaTrf dsRNA-treated H. armigera fat body cells at 12 h; (D) The levels of HaTrf in HaTrf dsRNA-treated H. armigera fat body cells at 24 h; (E) The levels of HaTrf in HaTrf dsRNA-treated H. armigera fat body cells at 36 h. Bars represent mean ± SD of replicated experiments (n=3). Bars sharing the different letter are significantly different at P < 0.05 (Tukey’s HSD tests). 78x51mm (300 x 300 DPI)


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Figure 3. The effects of 2-tridecanone and HaTrf RNAi on H. armigera fat body cells. (A) The viability of 2tridecanone-treated H. armigera fat body cells at 12 h (n=6); (B) The viability of 2-tridecanone-treated H. armigera fat body cells at 12 h (n=6). Cell: untreated fat body cells; Cell+2-13: 2-tridecanone-treated fat body cells; dsGFP: fat body cells transfected with dsGFP; dsGFP+2-13: fat body cells transfected with dsGFP, then treated with 125 µM 2-tridecanone; dsTF: fat body cells transfected with dsHaTrf; dsTF+2-13: fat body cells transfected with dsHaTrf, then treated with 125 µM 2-tridecanone. Bars represent mean ± SD of replicated experiments (n=10). Bars sharing the different letter are significantly different at P < 0.05 (Tukey’s HSD tests). 42x22mm (300 x 300 DPI)



Figure 4. Effect of RNAi-mediated silencing of HaTrf on 2-tridecanone-induced cell apoptosis. Apoptosis (green) and HaTrf (red) were detected in fat body cells transfected with dsHaTrf or the dsGFP control. Merged confocal images are shown in the fourth column. Scale bar=10 µm. dsHaTrf-2-13: fat body cells transfected with dsHaTrf, then treated with 125 µM 2-tridecanone for 12 h; dsHaTrf: fat body cells transfected with dsHaTrf; dsGFP-2-13: fat body cells transfected with dsHaGFP, then treated with 125 µM 2tridecanone for 12 h; dsGFP: fat body cells transfected with dsGFP; 1: DAPI staining showing the nucleus; 2: 5-Carboxyfluorescein (FAM) labeling of apoptotic cells; 3: Red signal showing the expression of HaTrf; 4: merged fluorescent signals. 125x131mm (300 x 300 DPI)


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47x37mm (300 x 300 DPI)


A role for transferrin in triggering apoptosis in Helicoverpa armigera

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