Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Studies on Insecticidal Activities and Action Mechanism of Novel Benzoylphenylurea Candidate NK-17

  • Yongqiang Li,

    Affiliations Key Laboratory of Pesticide Chemistry and Application, MOA, Department of Applied Chemistry, College of Science, China Agricultural University, Beijing, People’s Republic of China, State Key Laboratory of Elemento-Organic Chemistry, Institute of Elemento-Organic Chemistry, Nankai University, Tianjin, People’s Republic of China

  • Yaoguo Qin,

    Affiliation Key Laboratory of Pesticide Chemistry and Application, MOA, Department of Applied Chemistry, College of Science, China Agricultural University, Beijing, People’s Republic of China

  • Na Yang,

    Affiliation State Key Laboratory of Elemento-Organic Chemistry, Institute of Elemento-Organic Chemistry, Nankai University, Tianjin, People’s Republic of China

  • Yufeng Sun,

    Affiliation Key Laboratory of Pesticide Chemistry and Application, MOA, Department of Applied Chemistry, College of Science, China Agricultural University, Beijing, People’s Republic of China

  • Xinling Yang,

    Affiliation Key Laboratory of Pesticide Chemistry and Application, MOA, Department of Applied Chemistry, College of Science, China Agricultural University, Beijing, People’s Republic of China

  • Ranfeng Sun,

    Affiliation State Key Laboratory of Elemento-Organic Chemistry, Institute of Elemento-Organic Chemistry, Nankai University, Tianjin, People’s Republic of China

  • Qingmin Wang ,

    wangqm@nankai.edu.cn (QW); lyun@cau.edu.cn (YL)

    Affiliation State Key Laboratory of Elemento-Organic Chemistry, Institute of Elemento-Organic Chemistry, Nankai University, Tianjin, People’s Republic of China

  • Yun Ling

    wangqm@nankai.edu.cn (QW); lyun@cau.edu.cn (YL)

    Affiliation Key Laboratory of Pesticide Chemistry and Application, MOA, Department of Applied Chemistry, College of Science, China Agricultural University, Beijing, People’s Republic of China

Studies on Insecticidal Activities and Action Mechanism of Novel Benzoylphenylurea Candidate NK-17

  • Yongqiang Li, 
  • Yaoguo Qin, 
  • Na Yang, 
  • Yufeng Sun, 
  • Xinling Yang, 
  • Ranfeng Sun, 
  • Qingmin Wang, 
  • Yun Ling
PLOS
x

Abstract

Insecticidal activity of NK-17 was evaluated both in laboratory and in field. It was found that the toxicity of NK-17 against S. exigua was 1.93 times and 2.69 times those of hexaflumuron and chlorfluazuron respectively, and the toxicity of NK-17 against P. xylostella was 1.36 times and 1.90 times those of hexaflumuron and chlorfluazuron respectively, and the toxicity of NK-17 against M. separate was 18.24 times those of hexaflumuron in laboratory, and 5% NK-17 EC at 60 g a.i ha−1 can control S. exigua and P. xylostella with the best control efficiency of about 89% and over 88% respectively in Changsha and Tianjin in field. The insecticidal mechanism of NK-17 was explored for the first time by utilizing the fluorescence polarization method. NK-17 could bind to sulfonylurea receptor (SUR) of B. germanica with stronger affinity comparing to diflubenzuron and glibenclamide, which suggested that NK-17 may also act on the site of SUR to inhibit the chitin synthesis in insect body and the result can well explain that NK-17 exhibited stronger toxicity against B. germanica than diflubenzuron and glibenclamide in vivo.

Introduction

The abundance of insects belonging to the order of Lepidoptera, such as Mythimna separata, Plutella xylostella, Spodoptera exigua and Hyphantria cunea etc., are one type of the most damaging pests for crops and forests. Because they may be able to cause significant damage in the process of agricultural production, a variety of insecticides were utilized to control the Lepidoptera pests since 1950s. The insecticides brought numerous benefits, meanwhile, they have negative effects such as environmental pollution, toxicity to nontarget organisms including mammals, and the insecticide resistance increased year by year. Therefore, researchers were pushed to develop novel, highly efficient, low toxicity, friendly environmental insecticides. Benzoylphenylureas (BPUs), acting on the larval stages of the Lepidoptera insects by inhibiting chitin synthesis as an important type of insect growth regulators (IGRs), have been rapidly developed since the first benzoylphenylurea (diflubenzuron, DFB, Figure 1) was introduced to the market in 1972 [1]. Besides diflubenzuron, hexaflumuron and chlorfluazuron (Figure 1) were some of other widely used insecticides. The 2000 “Presidential Green Chemistry Challenge” was awarded to Dow AgroSciences LLC for their innovation of Sentricon Termite Colony Elimination System, a new paradigm for termite control, which contained hexaflumuron as a major active ingredient [2]. Benzoylphenylureas have a unique mode of action coupled with a high degree of activity on target pests and low toxicity to nontarget organisms [3][4], thus have become a new tool for integrated pest management. Because of the above advantages, benzoylphenylureas have attracted considerable attention for decades [5][15]. In particular, we have designed and synthesized novel benzoylphenylureas containing oxime ether group, and found that these benzoylphenylureas exhibited excellent larvicidal activities against oriental armyworm and mosquito. For example, compound NK-17 (Figure 1) exhibited excellent larvicidal activity against mosquito, which had 90% mortality even at 0.001 mg L−1 [16][17]. According to the comprehensive analysis of bioactivity, physical properties and synthetic procedure etc., we chose compound NK-17 for further development as a novel and potent insecticide. It is very important that evaluation of insecticidal activities is a critical step to a new insecticide candidate with independent intellectual property right before it is introduced to the market. Hence, in this paper we will study insecticidal activities of NK-17 in laboratory and in field to evaluate its application prospects.

thumbnail
Figure 1. Chemical structures of benzoylphenylureas, NK-17 and glibenclamide.

http://dx.doi.org/10.1371/journal.pone.0066251.g001

The initial study results showed that chitin synthetase catalyzing the incorporation of UDP-N-acetylglucosamine (UDP-NAGA) to chitin was clearly inhibited by BPUs in vivo or in situ (isolated integument incubated in a tissue culture medium). However, all of the subsequent studies trying to prove some action of BPUs on any part of the chitin synthesis pathway in insects in cell free systems (rather than in vivo and in situ) failed [18][22]. In addition, BPUs showed no inhibitory actions on fungal chitin synthetase in vivo as well as in cell free systems, which have roughly equivalent chitin synthesis pathways as insects [18]. Therefore, the action mechanism of BPUs remained unresolved. In 2004, Matsumura and co-workers reported that the benzoylurea insecticide diflubenzuron acted on the same target site on the sulfonylurea receptor (SUR) protein as a typical sulfonylurea drug, glibenclamide, in Drosophila melanogaster and Blattella germanica. Furthermore, such action by these chemicals is the cause of their inhibitory effect on chitin synthesis [22][23]. It is a great discovery on the action mechanism research of benzoylurea insecticides.

In recent years, more evidences have been provided that the sulfonylurea receptor (SUR) protein, the action site of DFB, attributing to the ATP-binding cassette (ABC) transporter superfamily proteins [24]. The ABC proteins execute a number of important physiological functions in the biosystems such as chemical exchange, ion channels, receptors, and signal transduction etc.. SUR associates with four pore-forming (Kir6.x) potassium channel subunits (4 SUR subunits and 4 Kir6.x subunits) to form ATP-sensitive potassium channels(KATP). Some researches suggested that the Kir6.x subunits, which constitute the pore of KATP, are located centrally, whereas the SUR subunits are located peripherally [25][27]. The KATP has a unique function among the ABC superfamily of proteins, because the Kir6.x subunits manage transport of potassium, however, the SUR subunits play are regulatory role modulating channel activity. ATP can bind to the SUR subunits to inhibit KATP channel activity and regulate a series of biochemical systems, similarly, sulfonylureas, which are widely used in the treatment of type 2 diabetes mellitus as a ligand of SUR, bind to the SUR subunit in multiple locations so as to inhibit KATP channel activity too.

However, above research about SUR proteins involve in utilizing the isotope labeling technology. Due to the restriction by the level of laboratory condition and safety to the researchers, the isotope labeling technology cannot be quickly and easily used in usual laboratory.

The fluorescence polarization (FP) assay is based on the high affinity binding of the fluorescence probe N-Phenyl-1-naphthylamine (1-NPN) to a specific site of SUR. After absorbing polarized light, free 1-NPN of relatively small molecular weight emits light in all directions due to the fast tumbling rate, resulting in low polarization. When binding to the target protein SUR, the 1-NPN molecules rotate slower due to the larger combined molecular size of the complex. Consequently, they emit radiation in the same direction as that of the incident light, and exhibit higher polarization. When some compounds can displace the 1-NPN molecules from SUR, the disruption of the binding between the 1-NPN molecules and the protein can be identified by decreased polarization. Thus, the FP assay can be used for determination of small molecule chemical ligand binding affinity to SUR in vitro. Hence, in this paper we will adopt FP method to study NK-17 binding affinity to SUR comparing with glibenclamide and DFB and then obtain the effect-dose curves by dose-dependent validation experiments with these compounds, so ascertain the binding mechanism of diflubenzuron and glibenclamide to the SUR, and study the action mechanism of NK-17 for the fisrt time.

Materials and Methods

Insects

Beet armyworm (Spodoptera exigua, S. exigua ) has been reared in the bioassay platform of State Key Laboratory of Elemento-Organic Chemistry, Nankai University since 2008 in the standard laboratory conditions of 27±1°C, 50%∼75% RH and under an Light : Dark (L:D) 14∶10 h photoperiod. Third-instar larvae were raised on the artificial diets and used in bioassay experiments.

Oriental armyworm (Mythimna separate, M. separata) has been introduced into the bioassay platform of State Key Laboratory of Elemento-Organic Chemistry, Nankai University in 1995 under a climatic chamber (25±1 °C, 60%∼70% RH and under an L:D 13∶11 h photoperiod). Fourth-instar larvae were raised on the artificial diets and used in bioassay experiments.

Diamondback moth (Plutella xylostella, P. xylostella) was raised on wild cabbage (Brassica oleracea L) in the greenhouse of the bioassay platform of State Key Laboratory of Elemento-Organic Chemistry, Nankai University plants under standard laboratory conditions (23±1°C, 50%∼60% RH and natural lighting). Third-instar larvae were used in bioassay experiments.

German cockroach (Blattella germanica, B. germanica) was fed dog food and water and maintained in cheesecloth-covered plastic, the rim of which was coated with Vaseline. The cockroach colony was maintained at 27±2°C, 60±5% relative humidity and 12∶12 h (L:D).

Reagents and compounds

Chlorfluazuron (purity 98%) was purchased from Shijiazhuang Jitai Sanmu Pesticide Chemical Industry Co., Ltd. 5% chlorfluazuron EC and hexaflumuron (purity 95%) were purchased from Jiangsu Yangnong Chemical Group Co., Ltd. 5% hexaflumuron EC was purchased from Tianjin Shipule Pesticide Technical DEP. Co., Ltd. Tween-20 and dimethyl sulfoxide (DMSO) were purchased from Alfa Aesar China (Tianjin) Co., Ltd. NK-17 (>99%) was synthesized according to our previously reported method [16]. 5% NK-17 EC was prepared in our research group. All other biochemical reagents including glibenclamide and diflubenzuron were of the highest purity grade from Sigma-Aldrich Chemical Co., Ltd.

Bioassay against S. exigua and P. xylostella in laboratory

The bioassay of NK-17 and contrast compounds chlorfluazuron and hexaflumuron against the beet armyworm (S. exigua) and the diamondback moth (P. xylostella) were tested by the leaf-dip method. For each test sample, a stock solution at a concentration of 200 mg L−1 in DMSO was prepared and then diluted to the required series concentrations with water containing Tween-20. Leaf disks (5 cm×1 cm) from fresh cabbage leaves were dipped into the test solution for 10 s. After air-drying on a filter paper, the leaf disks were individually placed into Petri dishes (7 cm diameter). Third-instar larvae were individually transferred into the Petri dishes. Infested leaves treated with water and DMSO were provided as blank controls. Six replicates (10 larvae per replicate) were performed, and the results were expressed as mean value of replicates. Percentage mortalities were evaluated 72–96 hours after treatment in the culture conditions and corrected with Abbott’s formula [28][30]. When the percentage mortality of the control is less than 5%, the result is directly used. However, when the percentage mortality was less than 20%, the result was corrected by V  =  ((X–Y)/X)*100 (V  =  value of corrected mortality, X  =  livability of the control, Y  =  livability of the treat). The LC50 value (median lethal concentration) was obtained using probit analysis of the concentration-dependent mortality data by software DPS v7.05 for Window XP [31]. The biological data against S. exigua and P. xylostella in laboratory are listed in Table 1 and Table 2.

Bioassay against M. separate in laboratory

The insecticidal activities of NK-17 and the contrast hexaflumuron against M. separata were evaluated by foliar application according to the reported procedure [28][29]. For the foliar armyworm tests, individual corn leaves were placed on moistened pieces of filter paper in Petri dishes. The leaves were then sprayed with the test solution and allowed to dry. The dishes were infested with 10 fourth-instar oriental armyworm larvae. Percentage mortalities were evaluated 96 h after treatment. Each treatment was performed three times. The biological data in Table 3 was the average value of the three tested values.

Evaluation of insecticidal activities against S. exigua and P. xylostella in field

Field trials were obtain permits by Nankai University, and carried out between June and September during the 2011 and 2012 cropping seasons, in the Zhangjiawo vegetable production area, Xiqing district, Tianjin City, China (soil organic matter content of 2%, 42% sand, 30% silt and 28% clay, areas are public) and in Daqiao village, Yuhua district, Changsha City, Hunan Province, China (the soil: red earth, soil organic matter content of 4%, PH: 6.5, areas are public). The selected field sites are two of the most primary crop and vegetable production area in china and represent domain properties of north and south respectively. Because of consecutive cultivation and less effective rotation, the occurrence of the S. exigua and P. xylostella damages was very severe in these regions during the trials. Based on crop nutritional requirements, the field received a broadcast application of 200 kg ha−1 of 15N-10P-25K as starter fertilizer.

The treat plots of the beets were designed in the random block array. Moreover, each treat had four replicates in S. exigua field trials. According to the results of bioassay in the laboratory, 5% hexaflumuron EC at a dosage of 60 g active ingredient (a.i.) ha−1 (IV) was applied as a reference treatment; 5% NK-17 EC was applied at a series of dosage of 15 g a.i. ha−1 (I), 30 g a.i. ha−1 (II) and 60 g a.i. ha−1 (III) in the Tianjin and Changsha trial fields. A nontreated control was also included and had four replicates. The P. xylostella field trials scheme was approximately same as that of S. exigua except for the contrast agent and the crops replaced by 5% chlorfluazuron EC (V) on the cabbages respectively. We investigate and counted the insect numbers before spraying pesticides, 1 day, 3 days, 7 days and 14 days after applying pesticides in the all treat plots.

The data expressed as percentages were arcsine transformed to homogenize variances before analysis, and then the effects of different spraying treatments were examined using analysis of variance (ANOVA) and when the F test was significant at P<0.05, treatment means were compared using the Duncan's new multiple range (DPS, v7.05 for Windows) [32].

Action mechanism study of NK-17

Bioassay against B. germanica in vivo.

Insecticidal activity of NK-17 against cockroach (B. germanica) fifth-instar nymphs was assayed and compared with glibenclamide and diflubenzuron on the basis of the reported method [22] with some minor modifications. The aim of the assay was to determine the activity order of the three compounds, which would be used to compare with the order of bioactivity (SUR binding affinity) in vitro. A series of dilutions of each test compound were prepared in dimethylsulfoxide (DMSO): ethanol (1:1) according to wt/vol and expressed as µg/µL. The test was carried out by delivering a drop of test compound (about 1.0 µL) to the foregoing three abdominal sternites of nymphs of cockroach using a 5 µL Hamilton gas-tight syringe (Sigma-Aldrich). Seven concentrations were chosen to give the mortality percentage between 0% and 100%. Three replicates (10 nymphs per replicate) were performed for each concentration. Control cockroaches received only solvent mixture. Mortalities were recorded after 72 h. Cockroaches that could not respond to touch or always remained ventral side up when turning were considered dead. The molting symptoms was also observed and recorded in each assay.

Preparation of SUR.

The integuments of cockroach were cut into small pieces and homogenized using a precisely fitted glass–glass tissue homogenizer in isotonic MES–sucrose buffer (10 mM MES containing 250 mM sucrose and 2.5 mM MgSO4, adjusted to pH 6.6 with NaOH). The homogenate was centrifuged for 15 min at 1000 g, and then the supernatant was further centrifuged at 10,000 g for 20 min, and the precipitate was obtained and suspended in the same buffer as the preparation of SUR [28].

NK-17 binding to SUR.

To determine the superior concentration for the fluorescence probe, some total volume of 3000 µL reaction solutions were prepared by adding 100 µL SUR preparation prepared above to the proper quantity of MES–sucrose buffer containing different concentrations of N-phenyl-1-naphthylamine (1-NPN). Subsequently, ligands such as glibenclamide, diflubenzuron and NK-17 in different concentrations was respectively added to the mixture containing SUR, buffer and 1-NPN, and then the mixture was incubated for 1 h at room temperature. The binding affinity was assayed by FP.

The FP assay.

The value of FP was measured using Cary Eclipse Fluorescence Spectrofluorimeter (Agilent). The emission wavelength was emitted at 410 nm (with a slit width of 10 nm) and the excitation wavelength was emitted at 337 nm (with a slit width of 10 nm). Polarization is expressed as:

The I and I represent the vertically and horizontally polarized emission intensities, respectively, while polarized excitation light is vertical. The G factor is the instrumental effects and can be expressed as:

The I∥⊥ and I∥∥ represent the vertically and horizontally polarized emission intensities, respectively, while polarized excitation light is horizontal. The polarization of each sample was obtained from an average of 6 measurements each of I and I, and five values of such a set of averages were collected.

Results and Discussion

Effects of insecticides against S. exigua, P. xylostella and M. separate in laboratory

The concentration-effect curves of the bioassay results of NK-17 and contrast compounds against S. exigua are presented in Figure 2. The LC50 values and the slope± SEM of NK-17 were calculated according to the curve and are shown in Table 1. The LC50 (95% confidence limits) value of NK-17 against S. exigua was 3.38 mg L−1 (2.68-4.25 mg L−1), whereas the LC50 (95% confidence limits) values of hexaflumuron and chlorfluazuron were 6.54 mg L−1 (5.45-7.84 mg L−1) and 9.09 mg L−1 (8.20-10.07 mg L−1), respectively. Hence, the toxicity of NK-17 was 1.93 times and 2.69 times respectively those of hexaflumuron and chlorfluazuron.

thumbnail
Figure 2. The concentration-effect curve of NK-17, Hexaflumuron and Chlorfluazuron.

The Y-axis is the probit of mortality of Beet Armyworm, S. exigua, The X-axis is the logarithm value of the concentration of the insecticides, at last, the nymphs were assayed by using of leaves-dip method.

http://dx.doi.org/10.1371/journal.pone.0066251.g002

Similarly, in Table 2 and 3, the LC50 (95% confidence limits) value of NK-17 against P. xylostella was 26.73 mg L−1 (22.73-31.44 mg L−1), and the toxicity of NK-17 was 1.36 times and 1.90 times of hexaflumuron and chlorfluazuron (Figure 3). The LC50 values and the toxicity regression equations of NK-17 were calculated according to the curve and are shown in Table 3. The LC50 (95% confidence limits) value of NK-17 against M. separate was 0.017 mg L−1 (0.011-0.025 mg L−1), and the toxicity of NK-17 was 18.24 times of hexaflumuron.

thumbnail
Figure 3. The concentration-effect curve of NK-17, Hexaflumuron and Chlorfluazuron.

The Y-axis is the probit of mortality of Diamondback Moth, P. xylostella, accordingly, The X-axis is the logarithm value of the concentration of the insecticides, at last, the nymphs were assayed by using of leaves-dip method.

http://dx.doi.org/10.1371/journal.pone.0066251.g003

As NK-17 had definitely stronger toxicity than hexaflumuron and chlorfluazuron, faster action of NK-17 than those compounds could be observed from the mortality of S. exigua, P. xylostella and M. separate at 48 h. More importantly, the investigation of toxic symptoms showed that the action mode of NK-17 is similar, if not identical to hexaflumuron and chlorfluazuron. The treated larvae displayed several obvious characters of moulting defects, which were a double head capsule caused by the disability to shed the old cutile, and incomplete ecdysis caused by loss of haemolymph at the joint of the new head capsule and the thoracic segments, in that the typical molting problems were observed, and some dying insects still attached to the old cuticles.

Evaluation of insecticidal activities against S. exigua in field

Field trials of insecticidal activities against S. exigua were carried out in Tianjin and Changsha during the 2011 and 2012 cropping seasons. The two year results of the field trials on S. exigua in Tianjin were given in Figure 4. It showed that 5% NK-17 EC at the dose of 60 g a.i. ha−1 had better control effect than the comparison 5% hexaflumuron EC at the same dosage. The significance of difference was analyzed using Duncan's new multiple range. The effect data of the trial of Tianjin were indicated in 2011 (Table 4), 1 day after, the control efficiency of 5% NK-17 EC at 60 g a.i. ha−1 was 54.50% comparing to 48.75% of 5% hexaflumuron EC at the same conditions. Therefore, The speed of action of 5% NK-17 EC was faster than 5% hexaflumuron EC. 14 days after, the control efficiency of 5% NK-17 EC at 60 g a.i. ha−1 was 90.19% comparing to 84.71% of 5% hexaflumuron EC. The data of control efficiency in 2012 (Table 5) were similar, if not identical, to that in 2011, for example, the control efficiency of 5% NK-17 EC at 60 g a.i. ha−1 achieved to 90.16% in 14 days comparing to 85.97% of 5% hexaflumuron EC.

thumbnail
Figure 4. Evaluation of 5% NK-17 EC against S. exigua.

The trials was carried out in the cabbages in the Zhangjiawo vegetable production area, Xiqing district, Tianjin City, China, in 2011 and 2012. The 5%NK-17 EC had the better performance on controlling S. exigua in the cabbage field than the commercial insecticides 5% Hexaflumuron.

http://dx.doi.org/10.1371/journal.pone.0066251.g004

The control efficiency of the trial in Changsha was exhibited in Figure 5, and the result was almost same as Tianjin, so did the data of the trial in Changsha (Table 6). 1 day after, the control efficiency of 5% NK-17 EC at 60 g a.i. ha−1 was 50.31% comparing to 43.28% of 5% hexaflumuron EC. Therefore, the speed of action of 5% NK-17 EC was faster than 5% hexaflumuron EC. 14 days after, the control efficiency of 5% NK-17 EC at 60 g a.i. ha−1 was 89.06% comparing to 79.26% of 5% hexaflumuron EC. The data of control efficiency in 2012 (Table 7) were similar, if not identical, to that in 2011, for example, the control efficiency of 5% NK-17 EC at 60 g a.i. ha−1 achieved to 89.97% in 14 days comparing to 85.14% of 5% Hexaflumuron EC.

thumbnail
Figure 5. Evaluation of 5% NK-17 EC against S. exigua.

The trials was carried out in the cabbages in Daqiao village Yuhua district, Changsha City, Hunan Province, China, in 2011 and 2012. The 5% NK-17 EC had the better performance on controlling S. exigua in the cabbage field than the commercial insecticides 5% Hexaflumuron.

http://dx.doi.org/10.1371/journal.pone.0066251.g005

thumbnail
Table 6. Effect of insecticidal progam on S. exigua Changsha City, Hunan Province in 2011.

http://dx.doi.org/10.1371/journal.pone.0066251.t006

thumbnail
Table 7. Effect of insecticidal progam on S. exigua Changsha City, Hunan Province in 2012.

http://dx.doi.org/10.1371/journal.pone.0066251.t007

Hence, it was suggested that 5% NK-17 EC was a promising insecticide as alternative to the high toxic pesticides against S. exigua according to the two years and two sites trials.

Evaluation of insecticidal activities against P. xylostella in field

Firstly, the results of the trials in Tianjin including 2011 and 2012 controlling P. xylostella were given at 1 day, 3 days, 7 days, and 14 days respectively in Figure 6. It showed obviously that 5% NK-17 EC had better control effect than the comparison 5% Chlorfluazuron EC at the all same conditions. The significance of difference was analyzed using Duncan's new multiple range. The effect data of the trial of Tianjin were indicated in 2011 (Table 8), 1 day after, the control efficiency of 5% NK-17 EC at maximum dosage was 52.07% comparing to 44.31% of 5% Chlorfluazuron EC at the same conditions. Therefore, the speed of action of 5% NK-17 EC was faster than 5% Chlorfluazuron EC. 14 days after, the control efficiency of 5% NK-17 EC at maximum dosage was 88.64% comparing to 81.69% of 5% Chlorfluazuron EC. The data of control efficiency in 2012 (Table 9) were about identical to that in 2011, one of which indicated the control efficiency of 5% NK-17 EC at the same maximum dosage achieving to 88.91% in 14 days comparing to 83.03% of 5% Chlorfluazuron EC.

thumbnail
Figure 6. Evaluation of 5% NK-17 EC against P. xylostella.

The trials was carried out in the Zhangjiawo vegetable production area, Xiqing district, Tianjin City, China, in 2011 and 2012. The 5% NK-17 EC had the better performance on controlling P. xylostella in the cabbage field than the commercial insecticides 5% chlorfluazuron.

http://dx.doi.org/10.1371/journal.pone.0066251.g006

thumbnail
Table 8. Effect of insecticidal progam on P. xylostella in Tianjin City in 2011.

http://dx.doi.org/10.1371/journal.pone.0066251.t008

thumbnail
Table 9. Effect of insecticidal progam on P. xylostella in Tianjin City in 2012.

http://dx.doi.org/10.1371/journal.pone.0066251.t009

The results of the trial in Changsha were showed in Figure 7, and the evaluation of the efficiency was almost same as Tianjin, so did the data of the trial in Changsha in 2011 (Table 10). 1 day after, the control efficiency of 5% NK-17 EC at the same maximum dosage was 51.35% comparing to 43.04% of 5% Chlorfluazuron EC. Therefore, the fast action of 5% NK-17 EC was suprior than 5% Chlorfluazuron EC. 14 days after, the control efficiency of 5% NK-17 EC at 60 g a.i. ha−1 was 87.85% comparing to 82.88% of 5% Chlorfluazuron EC. The data of control efficiency in 2012 (Table 11) were similar, if not identical, to that in 2011, one of which showed the control efficiency of 5% NK-17 EC at the maximum dosage achieving to 88.22% in 14 days comparing to 84.37% of 5% Chlorfluazuron EC on the same conditions.

thumbnail
Figure 7. Evaluation of 5% NK-17 EC against P. xylostella.

The trials was carried out in the cabbages in Daqiao village Yuhua district, Changsha City, Hunan Province, China, in 2011 and 2012. The 5% NK-17 EC had the better performance on controlling P. xylostella in the cabbage field than the commercial insecticides 5% chlorfluazuron.

http://dx.doi.org/10.1371/journal.pone.0066251.g007

thumbnail
Table 10. Effect of insecticidal progam on P. xylostella Changsha City, Hunan Province in 2011.

http://dx.doi.org/10.1371/journal.pone.0066251.t010

thumbnail
Table 11. Effect of insecticidal progam on P. xylostella Changsha City, Hunan Province in 2012.

http://dx.doi.org/10.1371/journal.pone.0066251.t011

Hence, it was suggested that 5% NK-17 EC was a promising insecticide as alternative to the high toxic pesticides against P. xylostella depending on the results of the two years and two sites trials.

Action mechanism study of NK-17

Effects of NK-17 against B. germanica in laboratory.

Insecticidal activity of NK-17, diflubenzuron and glibenclamide against B. germanica in vivo was firstly assayed. The aim of the assay was to determine the activity order of the three compounds, which would be used to compare with the order of bioactivity in vitro (SUR binding affinity). According to the concentration-effect curves of the bioassay results presented in Figure 8, the order of toxicity to B. germanica was NK-17 > diflubenzuron > glibenclamide according to the LD50 of 0.12 mg L−1, 0.23 mg L−1, 0.56 mg L−1, respectively. Moreover, the molting symptom of NK-17 was identical to diflubenzuron and glibenclamide, which suggested NK-17 may have the same action mechanism as diflubenzuron and glibenclamide.

thumbnail
Figure 8. The concentration-effect curve of NK-17, Diflubenzuron and Glibenclamide.

The Y-axis is the probit of mortality of cockroach, B. germanica, The X-axis is the logarithm value of the concentration of the insecticides, at last, the nymphs were assayed by using topical application technique. By passing of Probit analysis for NK-17: Slope±SEM = 0.67±0.03, the LD50 (95% confidence limits) value of NK-17 was 0.12 mg·L−1 (0.10–0.13 mg·L−1), whereas the LD50 (95% confidence limits) values of diflubenzuron and glibenclamide were 0.23 mg·L−1 (0.21–0.25 mg·L−1) and 0.56 mg·L−1 (0.42–0.75 mg·L−1), respectively.

http://dx.doi.org/10.1371/journal.pone.0066251.g008

Affinity of NK-17 binding to SUR.

As previously mentioned, by utilizing the isotope labeling technology, diflubenzuron was testified to act on the same target site of SUR proteins as glibenclamide [22]. In this paper, we would like to adopt fluorescence polarization (FP) method to ascertain if NK-17 could also bind in the same site of SUR.

The FP assay is based on the high affinity binding of the fluorescence probe N-phenyl -1-naphthylamine (1-NPN) to a specific site of SUR. After absorbing polarized light, free 1-NPN of relatively small molecular weight emits light in all directions due to the fast tumbling rate, resulting in low polarization. Binding to the target protein SUR, the 1-NPN molecules rotate slower due to the larger combined molecular size of the complex. Consequently, they emit radiation in the same direction as that of the incident light, and exhibit higher polarization. When some compounds can displace the 1-NPN molecules from SUR, the disruption of the binding between the 1-NPN molecules and the protein can be identified by decreased polarization.

First of our work was to test and verify the feasibility of this FP assay by the competitive displacement of 1-NPN with glibenclamide and diflubenzuron from 1-NPN-SUR. The SUR proteins of B. germanica were prepared by the reported method with some minor modifications. However, the concentration of the SUR cannot be accurately measured without its crystal structure, because its gene has never decoded so far. Furthermore, the vesicles containing the SUR were not stable enough for the receptor to bind to the fluorescence probe after a long time. Therefore, the binding affinity of NK-17, diflubenzuron and glibenclamide with SUR were compared within the same set of experiments with the same vesicle preparation, and over 5 replicates must be carried out to further minimize the error.

Because of previous discussion, we had to measure the affinity of the probe binding to SUR to elucidate the dose of the probe very time (Figure 9). At beginning, the FP was quickly raised by increasing the dosage of the probe to indicate the all probe binding to the SUR in order to find the most proper concentration of the probe [33].

thumbnail
Figure 9. Binding of 1-NPN ligand to the sSUR.

Binding of 1-NPN to the SUR protein in the MES-sucrose. As the concentration of the fluorescence probe 1-NPN was gradually elevated, the value of fluorescence polarization was according improved in the origin. When the concentration was increase to 3 µM, the value of FP began to keep a balance. That indicates that the binding of 1-NPN to SUR was saturated, therefore, 3 µM was used as the standand concentration in investing the comparative affinity to other ligands in the experiment.

http://dx.doi.org/10.1371/journal.pone.0066251.g009

With the most proper concentration of the 1-NPN-SUR, NK-17, diflubenzuron and glibenclamide as competitive ligands binding to the SUR were assayed by FP (Figure 10). According to the affinity result of the three compounds, the order was NK-17 > diflubenzuron > glibenclamide, which was consistent with the bioactivity against B. germanica in vivo.

thumbnail
Figure 10. Binding of NK-17, Glibenclamide and diflubenzuron to SURs.

Binding reactions were incubated for 1 h at room temperature. All measurements were done in tetraplicate; data are the average of the all repeat experiments. The affinity was assayed using fluorescence polarization. The lower FP indicates the more intensive receptor-ligand binding affinity.

http://dx.doi.org/10.1371/journal.pone.0066251.g010

Conclusions

Our research on insecticidal activities of NK-17 in laboratory and in field (two years, two trial sites) showed that the toxicity of NK-17 against S. exigua was 1.93 times and 2.69 times respectively those of hexaflumuron and chlorfluazuron, and the toxicity of NK-17 against P. xylostella was 1.36 times and 1.90 times respectively those of hexaflumuron and chlorfluazuron, and the toxicity of NK-17 against M. separate was 18.24 times those of hexaflumuron in laboratory. 5% NK-17 EC at 60 g a.i. ha−1 can control S. exigua with the best control efficiency of about 89% in Changsha and Tianjin in field, which suggested that 5% NK-17 EC a promising insecticide as alternative to the high toxic insecticide against S. exigua. 5% NK-17 EC at 60 g a.i. ha−1 can control P. xylostella with the best control efficiency of over 88% in Changsha and Tianjin, which suggested that 5% NK-17 EC a promising insecticide as alternative to the high toxic pesticides against P. xylostella. Therefore, we conclude that NK-17 is a very comprising insecticide candidate for controlling pests in crops, vegetables and fruits.

We explored the insecticidal mechanism of NK-17 by utilizing fluorescence polarization method for the first time. NK-17 could bind to SUR with stronger affinity comparing to DFB and glibenclamide, and the result can well explained that NK-17 exhibited stronger toxicity against B. germanica than DFB and glibenclamide in vivo. It is suggested that NK-17 may act on the site of SUR to inhibit the chitin synthesis in insect body.

Acknowledgments

We thank Prof. Xiaoming Ou (National engineering research center for Agrochemicals, hunan research institute of chemical industry, Changsha) and Prof. Xishu Gu (Tianjin institute of Plant protection, Tianjin) for helps in field trials. We thank Prof. Shangzhong Liu and Jianjun Zhang (Key Laboratory of Pesticide Chemistry and Application, MOA, Department of Applied Chemistry, College of Science, China Agricultural University) for helps in Cary Eclipse Fluorescence Spectrofluorimeter.

Author Contributions

Conceived and designed the experiments: YL XY QW YL. Performed the experiments: YL YQ NY QW YL. Analyzed the data: YL XY QW YL. Contributed reagents/materials/analysis tools: YL YQ YS RS. Wrote the paper: YL QW YL.

References

  1. 1. Van Daalen JJ, Meltzer J, Mulder R (1972) Selective insecticide with a novel mode of action. Naturwissenschaften 59: 312–313. doi: 10.1007/bf00593360
  2. 2. Green Chemistry website. Available: http://www.epa.gov/greenchemistry/pubs/pgcc/winners/dgca00.html. Accessed 2000.
  3. 3. Oberlander H, Silhacek DL (1998) Mode of action of insect growth regulators in Lepidopteran tissue culture. Pestic Sci 54: 300–302. doi: 10.1002/(sici)1096-9063(1998110)54:3<300::aid-ps830>3.0.co;2-8
  4. 4. Post LC, Vincent WR (1973) A new insecticide inhibits chitin synthesis. Naturwissenschaften 60: 431–432. doi: 10.1007/bf00623561
  5. 5. Yang XL, Wang DQ, Chen FH, Zhang ZN (1998) The synthesis and larvicidal activity of N-aroyl-N′-(5-aryl-2-furoyl)ureas. Pestic Sci 52: 282–286. doi: 10.1002/(sici)1096-9063(199803)52:3<282::aid-ps723>3.0.co;2-j
  6. 6. Xu XY, Qian XH, Li Z, Huang QC, Chen G (2003) Synthesis and insecticidal activity of new substituted N-aryl-N′-benzoylth-iourea compounds. J Fluorine Chem 121: 51–54. doi: 10.1016/s0022-1139(02)00330-5
  7. 7. Qian XH (1999) Quantitative studies on structure activity relationship of sulfonylurea and benzoylphenylurea type pesticides and their substituents bioisosterism using synthons activity contribution. J Agric Food Chem 47: 4415–4418. doi: 10.1021/jf981174r
  8. 8. Kubato S, Shono Y, Matsunaga T, Tsunoda K (2006) Laboratory evaluation of bistrifluron, a benzoylphenylurea compound, as a bait toxicant against Coptotermes formosanus (Isoptera: Rhinotermitidae). J Econ Entomol 99: 1363–1368. doi: 10.1603/0022-0493-99.4.1363
  9. 9. Chen L, Ou XM, Mao CH, Shang J, Huang RQ, et al.. (2007) Synthesis and bioassay evaluation of 1-(4-substitutedideneaminooxymethyl)phenyl-3-(2,6-difluorobenzoyl)ureas. Bioorg Med Chem 3678–3683.
  10. 10. Yoon C, Yang JO, Kang SH, Kim GH (2008) Insecticidal properties of bistrifluron against sycamore lace bug, Corythuchaciliata (Hemiptera: Tingidae). J Pestic Sci 33: 44–50. doi: 10.1584/jpestics.g07-09
  11. 11. Chen L, Wang QM, Huang RQ, Mao CH, Shang J, et al. (2005) Synthesis and insecticidal evaluation of propestcides of benzoylphenylureas. J Agric Food Chem 53: 38–41. doi: 10.1021/jf048561n
  12. 12. Sun RF, Zhang YL, Chen L, Li YQ, Li QS, et al. (2009) Design, synthesis, and insecticidal activities of new N-benzoyl-N’-phenyl-N’-sulfenylureas. J Agric Food Chem 57: 3661–3668. doi: 10.1021/jf900324a
  13. 13. Sun RF, Zhang YL, Bi FC, Wang QM (2009) Design, synthesis, and bioactivity study of novel benzoylpyridazylureas. J Agric Food Chem 57: 6356–6361. doi: 10.1021/jf900882c
  14. 14. Sun RF, Liu YX, Zhang YL, Xiong LX, Wang QM (2011) Design and synthesis of benzoylphenylureas with fluorinated substituents on the aniline ring as insect growth regulators. J Agric Food Chem 59: 2471–2477. doi: 10.1021/jf104578j
  15. 15. Sun RF, Li YQ, Xiong LX, Liu YX, Wang QM (2011) Design, synthesis, and insecticidal evaluation of new benzoylureas containing isoxazoline and isoxazole group. J Agric Food Chem 59: 4851–4859. doi: 10.1021/jf200395g
  16. 16. Sun RF, Lü MY, Chen L, Li QS, Song HB, et al. (2008) Design, synthesis, bioactivity, and structure activity relationship (SAR) studies of novel benzoylphenylureas containing oxime ether group. J Agric Food Chem 56: 11376–11391. doi: 10.1021/jf801901h
  17. 17. Qian XH, Lee PW, Cao S (2010) China: Forward to the Green Pesticides via a Basic Research Program. J Agric Food Chem 58: 2613–2623. doi: 10.1021/jf904098w
  18. 18. Mayer RT, Chen AC (1981) Chitin synthesis inhibiting insect growth regulators do not inhibit chitin synthase. Experientia 37: 337–338. doi: 10.1007/bf01959848
  19. 19. Cohen E, Casida JE (1980) Inhibition of Tribolium gut chitin synthetase. . Pestic. Biochem. Physiol. 13: 129–136. doi: 10.1016/0048-3575(80)90064-4
  20. 20. Meola SM, Mayer RT (1980) Inhibition of cellular proliferation of imaginal epidermal cells by diflubenzuron in pupae of the stable fly. Science 207: 985–987. doi: 10.1126/science.207.4434.985
  21. 21. DeLoach JR, Meola SM, Mayer RT, Thompson JM (1981) Inhibition of synthesis by diflubenzuron in pupae of the stable fly Stomoxys calcitrans. . Pestic. Biochem. Physiol. 15: 172–180. doi: 10.1016/0048-3575(81)90083-3
  22. 22. Abo-Elghar GE, Fujiyoshi P, Matsumura F (2004) Significance of the sulfonylurea receptor (SUR) as the target of diflubenzuron in chitin synthesis inhibition in Drosophila melanogaster and Blattella germanica. Insect Biochem. Mol Biol 34: 743–752. doi: 10.1016/j.ibmb.2004.03.009
  23. 23. Matsumura F (2010) Studies on the action mechanism of benzoylurea insecticides to inhibit the process of chitin synthesis in insects: A review on the status of research activities in the past, the present and the future prospects. Pestic Biochem Physiol 97: 133–139. doi: 10.1016/j.pestbp.2009.10.001
  24. 24. Burke MA, Mutharasan RK, Ardehali H (2008) The Sulfonylurea Receptor, an Atypical ATP-Binding Cassette Protein, and Its Regulation of the KATP Channel. Circ Res 102: 164–176. doi: 10.1161/circresaha.107.165324
  25. 25. Higgins CF, Linton KJ (2004) The ATP switch model for ABC transporters. Nat Struct Mol Biol 11: 918–926. doi: 10.1038/nsmb836
  26. 26. Miki T, Liss B, Minami K, Shiuchi T, Saraya A, et al. (2001) ATP-sensitive K+ channels in the hypothalamus are essential for the maintenance of glucose homeostasis. Nat Neurosci 4: 507–12.
  27. 27. Harris A, Cox S, Burns D, Norey C (2003) Miniaturization of fluorescence polarization receptor-binding assays using CyDye-labeled ligands, .J Biomol Screen8410–420.
  28. 28. Chen L, Huang ZQ, Wang QM, Shang J, Huang RQ, et al. (2007) Insecticidal benzoyl phenylurea-S-carbamate: a new propesticide with two effects of both benzoylphenylureas and carbamates. J Agric Food Chem 55: 2659–2663. doi: 10.1021/jf063564g
  29. 29. Shang J, Sun RF, Li YQ, Huang RQ, Bi FC, et al. (2010) Synthesis and insecticidal evaluation of N-tert-butyl-N′-thio[1-(6-chloro-3-pyridylmethyl)-2-nitroiminoimidazolidine]-N,N′-diacylhydrazines. J Agric Food Chem 58: 1834–1837. doi: 10.1021/jf903642s
  30. 30. Abbott WS (1925) A method of computing the effectiveness of an insecticide. J Econ Entomol 18: 265–267.
  31. 31. Siegel S, Castellan NJ Jr. (1988) Nonparametric statistics for the behavioral sciences. Singapore: McGraw-Hill Book Co.
  32. 32. FAO (1979) Recommended methods for the detection and measurement of resistance of agricultural pests to pesticides: method for adult aphids; FAO method 17. FAO Plant Protection Bulletin 18 , 6.
  33. 33. Sun YF, Biasio FD, Qiao HL, Iovinella I, Yang SX, et al. (2012) Two odorant-binding proteins mediate the behavioural response of aphids to the alarm pheromone (E)-ß-arnesene and structural analogues. PLoS ONE 7(3): e32759. doi: 10.1371/journal.pone.0032759