Cytochrome P450-Mediated Î»-Cyhalothrin-Resistance in a Field Strain

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Agricultural and Environmental Chemistry

Cytochrome P450-mediated lambda-cyhalothrin-resistance in a field strain of Helicoverpa armigera from northeast China Ziguo Wang, Shanshan Jiang, David Mota-Sanchez, Wei Wang, Xinru Li, Yulin Gao, Xupeng Lu, and Xue-Qing Yang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b07308 • Publication Date (Web): 18 Mar 2019 Downloaded from http://pubs.acs.org on March 18, 2019

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Journal of Agricultural and Food Chemistry

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For submission to: Journal of Agricultural and Food Chemistry

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Cytochrome P450-mediated lambda-cyhalothrin-resistance in a field

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strain of Helicoverpa armigera from northeast China

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Zi-Guo Wang1, a, Shan-Shan Jiang1, a, David Mota-Sanchez2, Wei Wang1, Xin-Ru Li1,

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Yu-Lin Gao3, Xu-Peng Lu 4, Xue-Qing Yang1, *

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1

College of Plant Protection, Shenyang Agricultural University, Shenyang, 110866,

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Liaoning, China

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2

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48824, USA

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3

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Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China

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4

16

Liaoning, China

Department of Entomology, Michigan State University, East Lansing, Michigan

State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of

Green Agricultural Technology Center of Liaoning Province, Shenyang, 110034,

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a These

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*Corresponding author: Xue-Qing Yang

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ORCID: 0000-0002-3919-8013

authors contributed equally to this work

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Tel./ Fax: (+86)024-88487148, email: [email protected]

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Running title: P450s mediate lambda-cyhalothrin resistance in H. armigera.

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ACS Paragon Plus Environment


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ABSTRACT

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Resistance to pyrethroid and organophosphate insecticides has been a growing

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problem in the management of cotton bollworm Helicoverpa armigera (Hübner)

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populations in the Yangtze River and Yellow River valleys of China, but resistance

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status and mechanisms of H. armigera populations from northeast China is less

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documented. In this study, a field strain collected from Shenyang in northeast China

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(SYR) is up to 16-fold more resistant than a susceptible strain (SS) to

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lambda-cyhalothrin, while the resistance level to phoxim remained low (2.6-fold).

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Synergist tests and enzymatic assays show that increased cytochrome P450

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monooxygenase (P450) activity is the main mechanism for lambda-cyhalothrin

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resistance. Eight out of 10 genes from CYP6 and CYP9 subfamilies were significantly

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overexpressed in the SYR strain, and CYP6AE11 was the mostly overexpressed P450

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(59-fold). These results suggest that overexpression of multiple P450 enzymes

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contributes to lambda-cyhalothrin resistance in the SYR strain of H. armigera from

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northeast China.

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KEYWORDS: Helicoverpa armigera; northeast China; cytochrome P450; pyrethroid

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resistance; lambda-cyhalothrin

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INTRODUCTION The cotton bollworm Helicoverpa armigera (Hübner) is one of the major pests in 1, 2.

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Asia, Africa and Australia

Control of H. armigera has mainly depended on

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chemical insecticides, including pyrethroid (Pys) and organophosphate insecticides

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(OPs) over the past 30 years in China 3. Due to its strong capacity to develop

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resistance to insecticides 4, this polyphagous pest has gained a prominent pest status in

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China, and causes severe economic losses in crops such as cotton, corn, sunflowers

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and vegetable crops5.

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Although genetically modified Bt cotton was introduced and commercialized in

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China in 1997 6, the application of chemical insecticides for H. armigera control has

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continued based on the “refuge” strategy in Bacillus thuringiensis (Bt) crops, and the

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spraying has continued for other non-Bt crops 7, 8. As a result, insecticide resistance of

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H. armigera to chemical insecticides remains problematic in China. In China,

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resistance of H. armigera to chemical insecticides has been documented mainly in

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field populations collected from the Yangtze River and Yellow River valleys

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but there have been few reports from northeast China.

3, 9-11,

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H. armigera cannot overwinter in northeast China. However, it annually extends

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its geographic range through seasonal migration between northern and northeast

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China over the Bohai Sea

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during spring and summer and then return to the south during the fall 12. During this

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process, H. armigera may likely encounter different degrees of selection pressure, e.g.

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the various insecticides used in different areas along these migration routes, inducing

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resistance

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insecticides for H. armigera control in corn fields in most areas of China, including

12.

12.

Moths migrate into northern China to northeast China

Lambda-cyhalothrin and phoxim are the most commonly used



13.

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northeast China

However, the resistant status of H. armigera populations from

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northeast China to lambda-cyhalothrin and phoxim is less documented, and the

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underlying resistance mechanisms have not yet been documented. Mechanisms of pyrethroid resistance have been attributed to increased

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14-16.

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insecticide detoxification and/or target site mutations

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that metabolic resistance to pyrethroids is primarily associated with cytochrome P450

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monooxygenases

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P450-dependent resistance was observed in laboratory-selected resistance strain of H.

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armigera to esfenvalerate

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resistance to esfenvalerate and fenvalerate in populations of H. armigera from China

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11,19,

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of CarEs to pyrethroid resistance is less documented in H. armigera. Up-regulated

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esterases have been found in a fenvalerate-resistant H. armigera strain

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recently, Chen et al. (2017) demonstrated that resistance to lambda-cyhalothrin is

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associated with elevated CarE enzyme activity 24. In addition, target-site mutations of

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the voltage-gated sodium channel (VGSC) gene have been linked to field-evolved

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pyrethroid resistance in H. armigera. Head et al. (1998) observed that two mutations

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(D1561V and E1565G) at domain III-IV of the HaVGSC gene were associated with

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knockdown resistance (kdr) to pyrethroids in Heliothis virescens and H. armigera 25.

Australia

20,21,

(P450s)

,

17

10.

followed

by

Previous studies indicate

carboxylesterases

(CarEs)

18.

P450s have also been associated with field-evolved

and Pakistan 22. Compared with P450s, the extent of contribution

23,

and more

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The aim of this work was to detect the resistance level of a field-collected H.

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armigera population from northeast China to lambda-cyhalothrin, and to uncover the

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underlying resistance mechanisms. It is reported here that moderate resistance to

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lambda-cyhalothrin in H. armigera from northeast China is the result of an

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overexpression of multiple P450 genes.

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MATERIALS AND METHODS


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Insects. The susceptible H. armigera strain (SS) was procured from a colony

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provided by the Key Laboratory for Biology of Plant Diseases and Insect Pests,

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Institute of Plant Protection, Chinese Academy of Agricultural Sciences (Beijing,

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China). This colony has been maintained in the laboratory without exposure to

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insecticides for more than 50 generations at 25±1 ºC, 70±5 % humidity with a

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photoperiod of 14:10 h of light : dark. Larvae were fed an artificial diet based on

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wheat germ and soybean powder 3. Moths were supplied with a 10% honey solution.

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A total of 68 larvae of the field strain of H. armigera (SYR) were collected from

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the flower belt (Tagetes erecta) of a corn field (123.57°E, 41.84°N) in Shenyang city,

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Liaoning province, northeast China on September 30, 2017. This strain was reared in

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the laboratory using the same conditions as the SS strain.

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Chemicals. Piperonyl butoxide (PBO), diethyl maleate (DEM), S,S,S-tributyl

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phosphorothrithioate (DEF), 1-chloro-2,4-dinitrobenzene (CDNB), 7-ethoxycoumarin,

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and 7-hydroxycoumarin were purchased from Sigma Chemical Corporation (St. Louis,

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MO, USA). The α-naphthyl acetate (α-NA), fast blue B salt, NADPH, glutathione

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(GSH), eserine, and technical grade of lambda-cyhalothrin and phoxim with greater

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than 98% purity were obtained from Aladdin Reagent (Shanghai, China). All other

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chemicals and solvents used were reagent grade.

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Bioassays and synergism tests. Toxicity for the SS and F1 generation of SYR of H.

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armigera was determined by topical application 3 with slight modifications. A stock

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solution of lambda-cyhalothrin and phoxim was prepared in acetone and diluted to

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five concentrations of lambda-cyhalothrin (2, 10, 50, 250, 1000 mg/L), and phoxim (2,

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10, 50, 250, 1000 mg/L) with acetone for these bioassays. One µL of each

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concentration of insecticide solution was applied onto the thoracic dorsum of third

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instar larvae (day 1). Three replicates of 24 larvae were used for each concentration.



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For synergism tests, PBO (an inhibitor of P450s), DEM (an indirect inhibitor of

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GSTs), and DEF (an inhibitor of CarEs) were applied 1h before the insecticide

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treatments. Synergists were applied onto the thoracic dorsum of third instar larvae at a

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dosage of 10 μg/larva. Larvae treated with acetone or synergist alone were used as a

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controls. After treatment, larvae were held individually on artificial diet in a 24-well

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tissue culture plate. Mortality was recorded at 24, 48 and 72 hours post exposure (hpe).

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A larva was considered dead when it did not respond to a gentle stimulation by an ink

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brush.

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Enzyme sample extraction. Three replicates of five third-instar larvae of H.

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armigera from SS and the F1 generation of SYR were homogenized on ice in a mortar

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using 2.5mL of different buffers 15. In brief, for crude enzyme preparations for P450,

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CarE, and GST analysis, 2ml of 100 mM sodium phosphate (1mM EDTA, 1mM DTT,

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1mM PMSF, pH 7.8), Tris–HCl/CaCl2 (25 mM/1 mM, pH 7.0), and 50 mM sodium

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phosphate (pH 7.2) were used, respectively. The homogenates were centrifuged at

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14,000 g for 30 min at 4°C, and the supernatants were collected as the enzyme source

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for CarE and GST. The protein concentration of the enzyme source was determined

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using bovine serum albumin as a standard according to the Bradford method

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Resuspended microsomal membranes obtained by centrifugation at 100,000g for 60

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min at 4°C were used as P450 enzymes. P450 concentrations were determined by

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reduced versus oxidized spectroscopy according to the method of Omura and Sato

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(1964) 27.

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Cytochrome P450 dependent 7-ethoxycoumarin-O-dealkylation (ECOD) assay.

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The P450 activity was determined by measuring ECOD activity using

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7-ethoxycoumarin as substrate using the method described by Tang et al. (2011)

149

with slight modifications. The reaction was carried out in a 1.5 mL Eppendorf tube


26.

30

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with 141 μl of sodium phosphate buffer (100 mM, pH 7.8), 50 μl of the enzyme, 5 μl

151

of 7-ethoxycoumarin (2 mM), and 4 μl of NADPH (10 mM). After 10 min of

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incubation at 30°C, the excess NADPH was removed as described by Stumpf &

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Nauen (2001)

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trichloroaceticacid (TCA). The homogenates were centrifuged at 12,000 g for 10 min

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at 4°C, and a volume of 200 μl of the supernatant was used to detect the generated

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7-hydroxycoumarin at an excitation wavelength of 358 nm and an emission

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wavelength of 456 nm on a luminescence spectrometer (Perkin Elmer LS50B).Three

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biological

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7-ethoxy-coumarin O-dealkylated min-1 mg protein-1.

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Carboxylesterase assay. The CarE activity was detected using α-NA as substrate

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according to the method adapted from Yang and Zhang (2013) 30. An 200μl reaction

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volume comprised of 100 μl enzyme preparation, 95 μl of Tris– HCl/CaCl2 (25 mM/1

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mM, pH 7.0), 1.0 mM eserine and 5 μl of α-NA (0.1 mM). After 10 min of incubation

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at 30 °C, the reaction was quenched by the addition of 25 μl of 0.42% sodium dodecyl

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sulfate (SDS) and stained by adding 25 μl of 0.2% fast blue B salt at room

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temperature in the dark for 30 min. The product of the reaction was measured at a

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wavelength of 600 nm on an Infinite M200 PRO multimode Microplate Reader

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(Tecan, Männedorf, Switzerland). Each sample was analyzed in triplicate. A standard

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curve was prepared with α-NA. Each test was conducted in triplicate. The activity was

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expressed as nmol α-naphthol min-1mg protein-1.

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Glutathione S-transferase assay. The conjugation activity of reduced glutathione

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was measured using CDNB as the substrate

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consisted of 88 μl of sodium phosphate buffer (50 mM, pH 7.2), 100 μl of the enzyme

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preparation, 2 μl of GSH (100 mM), and 10μl of CDNB (30mM). The absorbance at

29,

and then the reaction was terminated by 60 μl of 15%

replicates

were

tested.

The

activity

31.

was

expressed

as

nmol

The reaction mixture of 200 µl



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340nm was measured at time zero (t0) and 1 min later (t1) at 30°C, and the absorbance

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change (A340-t1− A340-t0) was used to calculate the GST activity with an extinction

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coefficient of 9.6 mM-1 cm-1. Each test was conducted in triplicate. The activity is

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expressed as nmol 2,4-dinitrophenyl-glutathione min-1 mg protein-1.

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Real-time quantitative PCR of H. armigera P450 genes. Total RNA was extracted

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from three pooled third-instar larvae of H. armigera using RNAiso Plus (Takara,

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Dalian, China) according to the manufacturer's instructions. The quality and

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concentration of extracted RNA was measured on a spectrophotometric Nano

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Drop2000c (Thermo Fisher Scientific, Waltham, MA, USA). The first strand cDNA

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was synthesized from 1 μg total RNA using a PrimeScriptTM RT Regent Kit with

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gDNA Eraser (Takara, Dalian, China) according to the manufacturer's instructions. In

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this study, ten P450 genes belonging to the CYP6 and CYP9 subfamilies that are

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potentially involved in pyrethroid resistance in H. armigera

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this

189

(http://bioinfo.ut.ee/primer3-0.4.0/).

study

(Table

S1).

Primers

were

designed

22 32, 33,

by

were selected in using

Primer3

190

Real-time quantitative PCR (RT-qPCR) was carried out on a CFX-96 (BioRad,

191

USA) thermal cycler. A 20μl reaction volume comprised of 10μl SYBR®Premix Ex

192

TaqTM (TaKaRa, Dalian, China), 1μl cDNA, 0.6 μl primer was amplified under the

193

following conditions: 3 min at 95 °C, followed by 40 cycles of 95 °C for 5 s, and 60

194

°C for 30 s. A melting curve was conducted after amplification to make sure the PCR

195

product was specific. RNase-free water instead of cDNA templates and RNA without

196

transcription were used as negative controls to exclude the potential genomic DNA

197

contamination. Each reaction was carried out in triplicate and each test was repeated

198

with three independent mRNA samples. The expression was normalized to transcript

199

levels of reference genes RPS18 and EF-1α, and relative gene expression was


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calculated using the 2−ΔΔCt method 34.

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Mutation detection. Ten third-instar larvae from SS and SYR strains of H. armigera

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were genotyped for the presence of D1561V and E1565G mutations at the HaVGSC

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gene (domain III-IV). Genomic DNA was extracted using DNA IQ System (Promega).

204

Amplification of the domain III-IV region of the HaVGSC gene was performed as

205

previously described 27. Primer pair HaVGSC-F and HaVGSC-R was used to amplify

206

a ~800bp fragment using PrimeSTAR® HS DNA Polymerase (Takara, Dalian, China).

207

The PCR conditions were 35 cycles of 98 °C for 10 s, 55 °C for 15 s, and 72 °C for 60

208

s, and finally 72 °C for 5 min. The PCR products were visualized by 1.5% agarose gel

209

electrophoresis, purified using Biospin Gel Extraction Kit (Bioer Technology Co.,

210

Ltd., Hangzhou, China), and then sequenced at Shanghai Sangon Biotech Co., Ltd.,

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China. Sequence data were analyzed using BioEdit software v. 7.2.5.

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Data analysis. Dose-mortality data were analyzed by Probit Analysis to estimate the

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slope and intercept of the dose–response line. Mortality was corrected using the

214

Abbott formula (1925). The differences in enzymatic activities and mRNA expression

215

were assessed by one way analysis of variance (ANOVA) using SPSS v.12.0 (IBM

216

Inc., Chicago, IL). The results were plotted using GraphPad Prism 5 (GraphPad

217

Software, CA).

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RESULTS

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Insecticide resistance in SYR strain. The toxicity of lambda-cyhalothrin and

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phoxim was measured by topical application to the susceptible strain (SS) and the

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SYR strain of H. armigera collected from northeast China. The susceptible (SS)

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larvae showed a LD50 value of 0.009 µg/larva and 0.059 µg/larva to

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lambda-cyhalothrin and phoxim. The SYR population showed a LD50 value of 0.144

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µg/larva and 0.151 µg/larva to lambda-cyhalothrin and phoxim, respectively, at 48



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hours post exposure (Table 1). The SYR strain displayed a moderate level of

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resistance to lambda-cyhalothrin, with a resistance ratio (RR) of 16-fold compared to

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the susceptible strain (SS). In contrast, the level of resistance to phoxim in strain SYR

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is low (2.6-fold) and not significantly different from strain SS (Table 1). When

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discriminating dosages of 0.009µg/larva and 0.05µg/larva of lambda-cyhalothrin were

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tested, the levels of mortality ranged between 8.33 and 33.33% for the SYR

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population, which was significantly different from the SS strain (Figure 1A). In the

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case of phoxim, 0.06 µg/larva and 0.10 µg/larva were tested, but mortality in the SYR

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strain was not significantly different from the SS strain (Figure 1B).

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Synergistic effects of PBO, DEM and DEF with lambda-cyhalothrin. The

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synergistic effects of PBO, DEM and DEF with lambda-cyhalothrin against SS and

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SYR strains are shown in Table 2. After pretreatment with PBO and DEF, significant

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synergistic effects were observed in both SS and SYR strains of H. armigera. PBO

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had a synergism ratio (SR) value of 2.25 on lambda-cyhalothrin in the SS strain,

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albeit it was not mathematically significant due to overlapping 95% confidence

240

intervals. In contrast, PBO had a significant synergistic effect on lambda-cyhalothrin

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in the SYR strain with a SR of 4.97. The low synergistic effect of DEF in the SYR

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strain was not significant when compared to the pyrethroid application alone (SR

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value of 2.06). A negative synergistic effect of DEM on lambda-cyhalothrin was

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observed in SS strain, but there were no observed effects on the SYR strain (Table 2).

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Enzymatic assays. Activities of three detoxification enzyme systems were

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determined in both strains of H. armigera. The P450, GST and CarE, activities in

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third instar larvae in strain SS were 0.32±0.13 nmol 7-ethoxy-coumarin

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O-dealkylatedmin-1 mg protein-1, 1.81±0.79 nmol min-1 mg protein-1, and 2.99±0.80

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nmol α-naphthol min-1 mg protein-1

respectively; in the strain SYR these were


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2.21±0.84 nmol 7-ethoxy-coumarin O-dealkylatedmin-1 mg protein-1, 4.20±0.97 nmol

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min-1 mg protein-1, and 6.03±0.36 nmol α-naphthol min-1 mg protein-1, respectively.

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Results show that P450, CarE, and GST activities in the SYR strain were 6.90-, 2.32-,

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and 2.02-fold higher, respectively, than in the SS strain (Figure 2).

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Transcription analysis of P450 genes. The results of RT-qPCR indicate that 8 of 10

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P450 genes, including CYP9A14 (F=93.598, P=0.001), CYP9A17 (F=140.995,

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P

Cytochrome P450-Mediated Î»-Cyhalothrin-Resistance in a Field Strain

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