biochemical mechanism of chlorantraniliprole resistance in the diamondback moth, plutella xylostella...

Journal of Integrative Agriculture2014, 13(11): 2452-2459 November 2014RESEARCH ARTICLE

© 2014, CAAS. All rights reserved. Published by Elsevier Ltd.doi: 10.1016/S2095-3119(14)60748-6

Biochemical Mechanism of Chlorantraniliprole Resistance in the Diamondback Moth, Plutella xylostella Linnaeus

HU Zhen-di1, 2*, FENG Xia1, 2*, LIN Qing-sheng1, 2, CHEN Huan-yu1, 2, LI Zhen-yu1, 2, YIN Fei1, 2, LIANG Pei3 and GAO Xi-wu3

1 Institute of Plant Protection, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, P.R.China2 Guangdong Provincial Key Laboratory of High Technology for Plant Protection, Guangzhou 510640, P.R.China3 Department of Entomology, China Agricultural University, Beijing 100193, P.R.China

Abstract

The insecticide chlorantraniliprole exhibits good efficacy and plays an important role in controlling the diamondback moth, Plutella xylostella Linnaeus. However, resistance to chlorantraniliprole has been observed recently in some field populations. At present study, diamondback moths with resistance to chlorantraniliprole (resistant ratio (RR) was 82.18) for biochemical assays were selected. The assays were performed to determine potential resistance mechanisms. The results showed that the selected resistant moths (GDLZ-R) and susceptible moth could be synergized by known metabolic inhibitors such as piperonyl butoxide (PBO), triphenyl phosphate (TPP) and diethyl-maleate (DEM) at different levels (1.68-5.50-fold and 2.20-2.89-fold, respectively), and DEM showed the maximum synergism in both strains. In enzymes assays, a high level of glutathione-S-transferase (GST) was observed in the resistant moth, in contrast, moths that are susceptible to the insecticide had only 1/3 the GST activity of the resistant moths. The analysis of short-term exposure of chlorantraniliprole on biochemical response in the resistant strain also showed that GST activity was significantly elevated after exposure to a sub-lethal concentration of chlorantraniliprole (about 1/3 LC50, 12 mg L-1) 12 and 24 h, respectively. The results show that there is a strong correlation between the enzyme activity and resistance, and GST is likely the main detoxification mechanism responsible for resistance to chlorantraniliprole in P. xylostella L., cytochrome P450 monooxygenase (P450) and carboxy-lesterase (CarE) are involved in to some extent.

Key words: Plutella xylostella, chlorantraniliprole, resistance, biochemical mechanism

INTRODUCTION

The diamondback moth, Plutella xylostella Linnaeus, is considered to be the most important pest of cruciferous vegetables throughout the world, costing upwards of US$ 4-5 billion annually (Furlong et al. 2013). Due to its abil-ity to rapidly form resistance to insecticides (Athanas-sios and Eunice 1999; Zhao et al. 2006), there are few

common insecticides that are still effective in controlling this pest (Huang and Wu 2003). Chlorantraniliprole is a reduced-risk insecticide which was recently registered for use in many agricultural settings as an alternative to traditional insecticides (Lahm et al. 2005, 2009; Loriatti et al. 2009). In insects, anthranilic diamides selectively bind to ryanodine receptors (RyR) in muscle and nervous tissue, resulting in an uncontrolled release of calcium from internal stores in the sarcoplasmic reticulum. The cacium release within the cells leads to feeding cessation,

Received 2 August, 2013 Accepted 3 January, 2014HU Zhen-di, Tel: +86-20-87597577, E-mail: [email protected]; Correspondence GAO Xi-wu, Tel: +86-10-62732974, E-mail: [email protected]; FENG Xia, Tel: +86-20-87597577, E-mail: [email protected]* These authors contributed equally to this work.

Biochemical Mechanism of Chlorantraniliprole Resistance in the Diamondback Moth, Plutella xylostella Linnaeus 2453

© 2014, CAAS. All rights reserved. Published by Elsevier Ltd.

lethargy, muscle paralysis and ultimately death of target organisms (Cordova et al. 2006; Sattella et al. 2008).

Susceptible baseline studies or toxicity tests of field populations were conducted on the diamondback moth in China during 2010-2011, revealed that the majority of populations were susceptible to chlorantraniliprole (Chen et al. 2010; Hu et al. 2010; Wang et al. 2010). Similar results were also obtained for populations from Brazil (Silva et al. 2012). However, the risk of resistance is notably high because growers are making indiscriminate use of chlorantraniliprole. High levels of resistance in some field populations of P. xylostella L. in Guangdong Province, China, have been monitored in 2012 (Hu et al. 2012; Wang and Wu 2012).

Studies with P. xylostella L. suggested that multiple resistance mechanisms enable it to overcome the ef-fect of insecticides. Elevated levels of detoxification enzymes, mutation of the insecticide target-site and reduced penetration of insecticide have all been cited as resistance mechanisms (Noppum et al. 1989; Baxter et al. 2010; Sonoda 2010). For a study of chlorantra-niliprole resistance in P. xylostella L., the full-length cDNA of a ryanodine receptor gene (PxRyR) was cloned and characterized recently (Wang et al. 2012). Further studies at the molecular level indicated that high level of diamide cross-resistance in P. xylostella L. was associated with a target-site mutation in the C-termi-nal membrane-spanning domain of the RyR (Troczka et al. 2012). In other species, tribufos (DEF) has been reported to synergize the toxicity of chlorantraniliprole to C. rosaceana, indicating possible involvement of esterase in resistance to chlorantraniliprole (Huang and Sun 1989; Sial and Burnner 2012). Jiang et al. (2012) reported that piperonyl butoxide (PBO) had synergistic effects (synergism ratio (SR) was 9.3 and 8.3, respec-tively) with chlorantraniliprole against two populations of Leptinotarsa decemlineata. In contrast, Lai and Su (2011) considered that PBO, diethyl-maleate (DEM), DEF and triphenyl phosphate (TPP) did not significant-ly enhance the toxicity of chlorantraniliprole to three Spodoptera exigua strains. These differences might be due to species difference because these studies have characterized chlorantraniliprole resistance in other species. In P. xylostella L., a study using certain inhib-itors of metabolic enzymes has suggested that enhanced enzymatic detoxification may play a role to some extent (Wang et al. 2012). However, little is known about the

possible biochemical mechanisms underlying resistance to chlorantraniliprole in P. xylostella L.

In this work, we investigate the effect of selection of P. xylostella L. strain for resistance to chlorantraniliprole, the effect of metabolic synergists on toxicity, and the detoxification enzymes assays of larvae. Furthermore, we also test the effect of short-term exposure to chlo-rantraniliprole on biochemical response to check for the involvement of detoxifying enzymes in insecticide bio-chemical resistance to the insecticide. The goals of this study are to further understand biochemical resistance to chlorantraniliprole, to examine the role that detoxifying enzymes have on resistance to chlorantraniliprole and to offer valuable information for chlorantraniliprole resistance management of P. xylostella L.

RESULTS

Toxicity of chlorantraniliprole against P. xylostella L. in different strains

The leaf dip bioassays showed that chlorantraniliprole was toxic to the unselected strain with LC50 value of 7.97 mg L-1. After ten generations of selection for resistance, susceptibility of resistant strain (GDLZ-R) significantly decreased in leaf dip bioassay with LC50 value of 41.91 mg L-1. The leaf dip bioassay showed a 5.26-fold increase in the LC50 value of the GDLZ-R strain after ten consecutive generations of selection as compared to the unselected strain (a field population which was collected from Lianzhou City, Guangdong Province, China and used to develop the resistant strain, GDLZ-R). As compared to the susceptible strain (LC50=0.51 mg L-1), the high level of resistance to chlorantranilipro-le was evolved in the GDLZ-R strain (resistant ratio (RR)=82.18) (Table 1).

Synergism of several enzyme inhibitors

PBO, TPP and DEM were normally considered as the inhibitor of P450, GST and CarE, respectively. The effects of three inhibitors on chlorantraniliprole toxicity in the GDLZ-R strain and susceptible strain are shown in Table 2. In GDLZ-R strain, the synergistic ratios of PBO, TPP and DEM on chlorantraniliprole were 1.68-,

2454 HU Zhen-di et al.


2.50- and 5.50-fold, respectively. DEM showed the maximum synergism. The 95% fiducial limits of LC50 values between chlorantraniliprole and chlorantra-niliprole+PBO/TPP overlapped. In susceptible moth, synergistic ratios (2.20-2.89-fold) of PBO, TPP and DEM on chlorantraniliprole were observed. DEM also showed the maximum synergism, but the 95% fiducial limits of LC50 values between chlorantraniliprole and chlorantraniliprole+PBO/TPP/DEM overlapped. This suggests that P450, CarE and GST could be all involved in the metabolic detoxification of chlorantraniliprole to some extent in both strains. However, PBO and TPP have the similar synergism in the two strains (1.1-1.3 times). Only DEM has higher synergism in

the GDLZ-R strain (1.9 times), which may imply that GST could contribute more to the chlorantraniliprole resistance in this pest.

Activities of detoxification enzymes

In order to determine the potential role of detoxification enzymes in resistance to chlorantraniliprole in GDLZ-R strain. The resistant and susceptible strains were ana-lyzed for their activities of P450, GST and CarE. As the results showed in Table 3, the activity of GST was significantly higher in the third-instar larvae from the GDLZ-R strain compared to the susceptible strain (3.34-fold) when determined using CDNB as substrate. But for P450 and CarE, no significant difference was found between the two strains (1.08- and 1.14-fold). This in-dicates a possible role of GST in conferring resistance to chlorantraniliprole in the GDLZ-R strain. And the findings support the results of the synergism test.

Biochemical response of P. xylostella L. to chlorantraniliprole-induced stress

To further check for the involvement of detoxifying

Table 2 Toxicity of chlorantraniliprole in combination with synergists to different strains of P. xylostella L. Strain Treatment1) LC50 mg L-1 (95% FL) Slope (±SE) Chi-square SR2)

GDLZ-R Chlorantraniliprole 45.40 (30.69-75.15) 2.25 (±0.59) 0.15 -Chlorantraniliprole+PBO 27.08 (16.52-38.09) 2.57 (±0.49) 1.61 1.68Chlorantraniliprole+TPP 18.16 (8.39-32.82) 1.44 (±0.42) 1.51 2.50Chlorantraniliprole+DEM 8.26 (4.35-12.79) 1.61(±0.27) 4.19 5.50

S Chlorantraniliprole 0.55 (0.42-0.71) 2.38 (±0.27) 0.21 -Chlorantraniliprole+PBO 0.24 (0.17-0.34) 1.87 (±0.30) 3.24 2.20Chlorantraniliprole+TPP 0.20 (0.12-0.27) 1.91 (±0.30) 2.66 2.75Chlorantraniliprole+DEM 0.19 (0.10-0.22) 1.77 (±0.30) 1.87 2.89

1) PBO, piperonyl butoxide; TPP, triphenyl phosphate; DEM, diethyl-maleate.2) SR, synergism ratio.

Table 3 Activities of detoxification enzymes in different strains of P. xylostella L. Enzyme/substrate Strain Enzyme activity1) RateP450/p-NA S 33.12±4.48 a 1.00

GDLZ-R 35.94±1.77 a 1.08CarE/α-NA S 37.52±2.16 a 1.00

GDLZ-R 42.74±10.06 a 1.14GST/CDNB S 10.20±0.39 a 1.00

GDLZ-R 34.12±9.69 b 3.341) Enzyme activity was showed as means±SE (mOD min-1 mg-1 protein for CarE

and GST, nmol mg-1 protein 30 min-1 for P450). Means in the column followed by the same letter were not significantly different (α=0.05, t-test).

enzymes in chlorantraniliprole resistance, variations in detoxification enzymes activities for both the susceptible and resistant strains were studied after exposure of the third-instars larvae to sub-lethal concentration of chlo-rantraniliprole for 6, 12 and 24 h. For each exposure time, activities of P450 and CarE were not significantly changed in GDLZ-R strain (Figs. 1-B and 2-B). How-ever, GST activities were significantly enhanced after 12 and 24 h of exposure as compared to the control

Table 1 Toxicity of chlorantraniliprole against different strains of Plutella xylostella L.Strain1) LC50 mg L-1 (95% FL)2) Slope (±SE) Chi-square RR3)

Unselected 7.97 (5.46-12.25) 1.85 (±0.25) 0.47 15.63GDLZ-R 41.91 (31.52-56.53) 3.14 (±0.45) 0.41 82.18S 0.51 (0.21-4.17) 0.72 (±0.22) 2.151) Unselected, the strain was collected from Lianzhou City, Guangdong Province,

China, which was not treated with chlorantraniliprole; GDLZ-R, the strain was developed from the unselected strain with chlorantraniliprole; S, susceptible strain.

2) 95% fiducial limits (95% FL) estimated using Polo plus program (LeOra Software).

3) RR, resistant ratio. The same as below.



(Fig. 3-B). In the susceptible strain, activities of P450 were increased after 6 and 12 h exposed (Fig. 1-A). An increase in CarE and GST activity was also observed after 6 h of chlorantraniliprole exposure (Fig. 2-A and Fig. 3-A). The findings suggest that all these three enzymes are involved in the metabolic detoxification of chlorantraniliprole in the susceptible strain, just as synergist tests suggested. But in the GDLZ-R strain, the insensitive response of the enzymes to chlorantra-niliprole induction are observed, that may be due to the constitution of higher enzyme activity.

DISCUSSION

Chlorantraniliprole is one of a new class of insecticides that is highly effective against P. xylostella L. (Chen et al. 2010). However, resistance to this insecticide in field populations in China presents a major risk to the effective life of this insecticide (Hu et al. 2010; Wang

et al. 2012). The effective life of new insecticides can be prolonged by the implementation of a resistance manage-ment program (Croft et al. 1987). The success of such program depends on better understanding of resistance characteristics, such as the relative risk of resistance evolution and resistance mechanisms.

To further understand chlorantraniliprole resistance, we selected P. xylostella L. larvae for investigation. As significant level of resistance (RR=82.18) to chlo-rantraniliprole in P. xylostella L. was observed after selection was continued through 10 generations. The results obtained in this study were not surprising at all as P. xylostella L. is well known for its ability to rapidly develop resistance to almost all classes of insecticides (Zhao et al. 2006; Sparks et al. 2012; Furlong et al. 2013). We also found that a high level of resistance against chlorantraniliprole could occur in a relatively shorter time in the field, especially in South China, where selection pressures are likely to be much higher than those imposed in laboratory conditions and where pop-ulations are likely to be more heterogeneous. However,

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Fig. 1 P450 activity of control and chlorantraniliprole-exposed P. xylostella L. larvae after 6, 12 and 24 h exposure. Data represent the means±SE of three replicates. * indicates significant difference from control and chlorantraniliprole. The same as below.

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Fig. 2 CarE activity of control and chlorantraniliprole-exposed P. xylostella L. larvae after 6, 12 and 24 h exposure.



due to low stability of resistance to chlorantraniliprole observed by Sial et al. (2011) in Choristoneura rosacea-na and Wang et al. (2012) in P. xylostella L., the strategy of the chemical rotations from different modes of action classes can be applied operationally to reduce resistance selection pressure in the field (Shen and Wu 1995).

P450, esterase (including CarE) and GST have been considered to be important in the metabolism of pyre-throids, organophosphorus pesticides (Ops), carbamates and novel insecticides in several insects including P. xylostella L. (Doichuanngam and Thornhill 1989; Huang and Sun 1989; Harold and Sun 2000). Since chlorantraniliprole was a new class of insecticide, very little was known about its biochemical mechanisms.

In the present work, we demonstrated biochemi-cal resistance characteristics of chlorantraniliprole in P. xylostella L. The synergism of the toxicity of chlo-rantraniliprole with certain inhibitors both in the two strains, suggests that chlorantraniliprole biochemical resistance was mediated by P450, CarE and GST to some

extent, but GST might contribute more in this pest. In detoxifying enzymes assays, a high level of GST activ-ity was observed in the GDLZ-R strain, as opposed to the susceptible strain, which supports the results of the inhibition assays.

There are some differences in the biochemical re-sponse to short-term exposure to chlorantraniliprole between the different strains of P. xylostella L. In the susceptible strain, all of the three detoxification enzymes could be induced significantly in short treatment time (6 h), but in the GDLZ-R strain, only GST was induced significantly in a relative long treatment time (12 and 24 h). From the above results, we deduced that at the initial point of chlorantraniliprole resistance development, many detoxification enzymes, such as P450, CarE and GST were all involved in the metabolic detoxification of chlorantraniliprole. But when resistance was developed to some extent, the insensitive responses of the enzymes were observed, that may be due to the constitution of higher enzymes activity. It was also possible that the mechanisms responsible for resistance to the same chemical may vary from one population to another (Smirle et al. 1998), so it was reasonable that detoxifi-cation mechanism observed in the GDLZ-R strain may not be the same as those present in the susceptible strain. Nevertheless, the results of biochemical response of P. xylostella L. to chlorantraniliprole-induced stress also indicate that all of the P450, CarE and GST are involved in chlorantraniliprole resistance to some extent, but GST might be contribute more in this pest.

CONCLUSION

In summary, all findings of the present study suggested that there is a strong correlation between the enzyme activity and chlorantraniliprole resistance in P. xylostella L., and GST is likely the main detoxification mechanism responsible for resistance, although P450 and CarE can-not be ruled out. However, the individual GST enzyme involved in chlorantraniliprole resistance has not been identified. Therefore, further study in more details, such as resistance might be attributed to increases of one or more GST enzymes, gene amplification or more commonly through increases in transcriptional rate, and so on, are needed.

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Fig. 3 GST activity of control and chlorantraniliprole exposed P. xylostella L. larvae after 6, 12 and 24 h exposure.



MATERIALS AND METHODS

Insects

The susceptible strain (S) of P. xylostella L. was kindly provided by the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing, China. This strain was maintained in the laboratory for more than 5 years without exposure to any insecticides.

The resistant strain (GDLZ-R) was developed from a field population which was collected from Lianzhou City, Guangdong Province, China. In the first selection, larvae were exposed to LC50 of the unselected strain, and then the concentration of insecticide used to select subsequent generation was ≈LC50 based on the results of bioassays from the previous generation. The number of larvae used for each generation varied 1 000-2 000 depending on availability. After 10 generations of continuous selection, the strain exhibiting high resistance was used for further biochemical assays.

Both strains were maintained in the laboratory under controlled conditions of (25±2)°C, (65±5)% relative humidity, and 16 h L: 8 h D photoperiod. Adults were fed on a 10% honey solution and allowed to lay eggs on leaf mustard (Brassica juncea) seedlings.

Insecticide and other chemicals

The formulated insecticide used for our study was chlorantraniliprole (200 g L-1 suspension concentrate, SC), obtained from DuPont Agricultural Chemical Ltd., USA. Other chemicals used were reduced form of nicotinamide-adenine dinucleotide phosphate (NADPH), reduced glutathione (GSH), phenyl thiourea (PTU), phenylmethyl sulfonylfluoride (PMSF), 1,4-dithiothreitol (DTT), 1-chloro-2,4-dinitrobenzene (CDNB), and p-nitroanisole (p-NA) were purchased from Sigma Chemical Co., USA. Ethylene diamide tetraacetic acid (EDTA) was acquired from Sinapharm Chemical Reagent Co. Ltd., Shanghai, China. Bovine serum albumin (BSA), alpha-naphthyl acetate (α-NA), fast blue RR salt and Triton X-100 were obtained from Beijing Solarbio Science and Technology Co. Ltd., Beijing, China. TPP, PBO, DEM and the other chemicals were of analytical grade and purchased from Guangzhou Jiayan Biotechnolongy Co. Ltd., Guangzhou, China.

Bioassay

For bioassay studies, we adopted the cabbage leaf dip method of Tabashnik et al. (1987). Insecticide solutions were prepared as serial dilutions with distilled water. Cabbage leaf discs (8 cm in diameter) were cut with a metal punch and dipped

in treatment solutions for about 10 s, then airdried at room temperature for about 2 h. One treated disc and ten 1-d-old third-instars of P. xylostella L. larvae were transferred to each plastic Petri dish (9 cm in diameter) and kept at (25±2)°C, (65±5)% RH with a photoperiod of 16 h L:8 h D. Leaves treated with distilled water alone were used as control. The mortality was assessed in 48 h after treatment and the larvae that did not respond when prodded were considered to be dead.

For analysis of the synergistic effect of synergists on chlorantraniliprole, 200 mg L-1 of each PBO, DEM and TPP was prepared in the first treatment solution, and then a series of solution made up with distilled water as described above. All of the tests were replicated three times. Bioassays that showed mortality rates >10% in the control were discarded, and the whole bioassay was repeated.

Preparation of enzymes

Fifteen larvae of third-instars were sampled for three replications in each enzyme assay. In each independent replication, 5 larvae were homogenized with 1 mL homogenization buffer (0.1 mol L-1 sodium phosphate buffer, pH 7.5, containing 1 mmol L-1 EDTA, 0.1 mmol L-1 DTT, 1 mmol L-1 PTU, and 1 mmol L-1 PMSF for P450 assay; 0.2 mol L-1 sodium phosphate buffer, pH 6.0 for CarE and GST assay). The homogenate was centrifuged at 12 000 r min-1 for 15 min at 4°C. The resulting clear supernatant was collected and used as an enzyme source for the analysis of the activity of P450, CarE and GST.

Detoxification enzyme assay

P450 activity was tested using a similar method by Qian et al. (2008). The substrate used for P450 activity assay was p-NA. The reaction solution contained 56.25 μL crude homogenate, 56.25 μL 0.1 mol L-1 sodium phosphate buffer (pH 7.5, containing 1 mmol L-1 EDTA, 0.1 mmol L-1 DTT, 1 mmol L-1 PTU, and 1 mmol L-1 PMSF), 125 μL 0.2 μmol L-1 p-NA, and 12.5 μL 9.6 mmol L-1 NADPH. Incubation was carried out at 34°C for 30 min in a water-bath and the optical density was recorded at 405 nm using a micro-plate spectra-photometer reader (Themo Scientific, Multiskan Spectrum, USA). Enzyme activity was calculated from a p-NA standard curve.

CarE activity was determined using a slight modification to the method described by Han et al. (1998). 25 μL of 0.2 mol L-1 sodium phosphate buffer (pH 6.0) and 200 μL mixture solution containing 1 mmol L-1 α-NA acetate and 0.5 g L-1 fast blue RR salt was added into each micro-plate well. The reaction was initiated by addition of 25 μL enzyme stock solution. Optical density at 450 nm was recorded at intervals of 12 s for 10 min in 27°C using the same micro-plate reader as above.

The similar method described by Zhu et al. (2000) was adopted for testing GST activity using CDNB as the substrate. The reaction solution contained 50 μL of enzyme stock



solution, 50 μL of 0.2 mol L-1 sodium phosphate buffer (pH 7.5), 100 μL of CDNB and 100 μL GSH. Optical density at 340 nm was recorded at intervals of 10 s for 5 min in 27°C using the same micro-plate reader as above.

Exposure to chlorantraniliprole

The short-term biochemical response of P. xylostella L. to chlorantraniliprole was studied by examining the activities of detoxifying enzymes. The method described by Ali et al. (2011) was adapted for these measurements. 150 third-instars of P. xylostella L. larvae from susceptible and resistant populations were exposed to sub-lethal doses of chlorantraniliprole (about 1/3 LC50 of each strain) by the leaf dipped method along with their controls. Three replicates were used in each experiment. The larvae were kept in a transparent plastic container with treated cabbage leaf (L×W×H=20 cm×14 cm×9 cm, covered with mesh cloth) for 24 h at (25±2)°C, (65±5)% relative humidity. Larvae which died due to natural causes during this period were discarded and only alive larvae were collected at 6, 12 and 24 h respectively, immediately frozen in liquid nitrogen and then stored at -80°C for the next short-term biochemical response analysis. And the enzymes activities were assayed by the methods described above.

Protein assay

Total protein content of the enzyme solution was determined by the Bradford method using bovine serum albumin as the standard (Bradford 1976).

Statistical analysis

The LC50, fiducial limit, slope and chi-square of toxicity bioassays were estimated by the Polo plus program (LeOra software 2002). The resistance ratio (RR) was estimated as RR=LC50 of testing strain/LC50 of susceptible strain. Synergism ratio (SR) was calculated as SR=LC50 value of insecticide alone/LC50 of value of insecticide with a synergist.

The data obtained from enzyme assays were analyzed by Microsoft Office Excel 2007 and presented as mOD min-1 mg-1 protein for CarE and GST, nmol mg-1 protein 30 min-1 for P450. Mean enzyme activities recorded in larvae from GDLZ-R strain were compared with those from larvae in the S strain using t-test. Significance was accepted at α=0.05 in all statistical tests used in this study.

AcknowledgementsThis project was sponsored by the Special Fund for Agro-Scientific Research in the Public Interest of China (201103021), the President Foundation of Guangdong Academy of

Agricultural Sciences, China (201206) and the Guangdong Natural Science Foundation, China (S2013010012529).

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(Managing editor SUN Lu-juan)

biochemical mechanism of chlorantraniliprole resistance in the diamondback moth, plutella xylostella...

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