Differential Resistance and Cross-Resistance to Three ...

INSECTICIDE RESISTANCE AND RESISTANCE MANAGEMENT

Differential Resistance and Cross-Resistance to Three Phenylpyrazole Insecticides in the Planthopper Nilaparvata lugens (Hemiptera: Delphacidae) XINGHUA ZHAO,1,2 ZUOPING NING,1 YUEPING HE,3 JINLIANG SHEN,1,4 JIANYA SU,1 CONGFEN GAO,1 AND YU CHENG ZHU5

J. Econ. Entomol. 104(4): 1364Ð1368 (2011); DOI: 10.1603/EC11074

ABSTRACT Cross-resistance to two Þpronil analogs, butene-Þpronil and ethiprole, was detected in Þpronil-resistant Þeld populations and a resistant laboratory strain of the planthopper Nilaparvata lugens (Stål) (Hemiptera: Delphacidae), although the two analogs have not been used widely in rice-growing areas in China. The results showed that six Þeld populations with 23.8 Ð 43.3-fold resistance to Þpronil had reached a higher level of cross-resistance to ethiprole (resistance ratio [RR] ⫽ 47.1Ð100.9-fold) and had a minor level of cross-resistance (RR ⫽ 3.4 Ð 8.1-fold) to butene-Þpronil. After 10 generations of selection, the RR to Þpronil increased from 7.3-fold to 41.3-fold. At the same time, the insect increased cross-RR to ethiprole from 16.3-fold to 65.6-fold, whereas it had only minor increase in cross-resistance to butene-Þpronil from 2.8-fold to 4.0-fold. These results conÞrmed that Þpronil-resistant N. lugens could develop a higher level of cross-resistance to ethiprole, although it still maintained a lower level cross-resistance to butene-Þpronil. Our data suggest that ethiprole is not a suitable alternative for controlling N. lugens, once the insect has developed a high level resistance to Þpronil. Further investigation is necessary to understand the cross-resistance mechanisms in N. lugens. KEY WORDS Nilaparvata lugens, Þpronil, ethiprole, butene-Þpronil, cross-resistance

The planthopper Nilaparvata lugens (Stål) (Hemiptera: Delphacidae) is an important rice pest in China and other Asian countries. N. lugens can immigrate with the monsoon. The insect has a high intrinsic growth rate and a strong environmental adaptability. They can easily reach outbreak population levels when environmental conditions are suitable (Wu et al. 1992, Li et al. 1996). In recent years, the outbreaks of N. lugens along the Yangtze River in China seriously threatened rice, Oryza sativa L., production. Because pesticides still play an important role in the control of this pest, cross-resistance in the target insect is a big obstacle for insecticide development. Understanding cross-resistance is an important part of the resistance research, which can not only facilitate the selection of effective alternative insecticides but also can help identify the resistance mechanisms (Shen and Wu 1995). After imidacloprid was phased out due 1 Pesticide Science Department and Plant Protection College of Nanjing Agricultural University, Key Laboratory of Monitoring and Management of Crop Diseases and Insects, Ministry of Agriculture, Nanjing 210095, China. 2 Termite Control Institute of Wujin District, Changzhou, 213159, China. 3 State Key Laboratory Breeding Base for Zhejiang Sustainable Pest and Disease Control, Institute of Plant Protection and Microbiology, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China. 4 Corresponding author, e-mail: [email protected]. 5 Corresponding author: USDAÐARS, Stoneville, MS 38776 (e-mail: [email protected]).

to a rapid resistance increase in N. lugens (Wang et al. 2009), Þpronil, an effective chemical against rice pests, had become one of the main alternatives for N. lugens control. As with imidacloprid, Þpronil soon became less effective against N. lugens due to signiÞcant resistance development (Wang et al. 2009). As the consequence of irrational application in Þelds and its harmful effect on shellÞsh, bees, and the river environment, however, the use of Þpronil had been restricted in China since 2009. Butene-Þpronil, ethiprole and Þpronil are phenylpyrazole analogs. They share same chemical backbone structures and differ in substituent moieties (Fig. 1). The assessment of cross-resistance in this study will provide valuable information for judging whether the two analogs can be used as an alternative to Þpronil and will provide a scientiÞc guideline for correct use of the pesticides in resistance management in N. lugens. Materials and Methods Insects. Six Þeld populations of N. lugens were collected from rice Þelds in 2009 along the Yangtze River in China (Fig. 2): Hexian County in Anhui Province, Fuqing County in Fujian Province, Xiaogan City in Hubei Province, Tongzhou City in Jiangsu Province, Shanggao County in Jiangxi Province, and Tongxiang County in Zhejiang Province. Approximately 800

0022-0493/11/1364Ð1368$04.00/0 䉷 2011 Entomological Society of America

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ZHAO ET AL.: CROSS-RESISTANCE TO PHENYLPYRAZOLE INSECTICIDES

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Fig. 1. International Union of Pure and Applied Chemistry names and chemical structures of Þpronil, ethiprole, and butene Þpronil.

adults or 500 Ð 600 nymphs were collected from each location. These populations were maintained at 26 Ð 30⬚C on rice seedlings (Shanyou 63 at tillering to booting stage) in cages (57 by 57 by 92 cm) placed in the greenhouse. The susceptible strain of N. lugens was collected in 1995 from a rice Þeld near Hangzhou City, Zhejiang Province, and was maintained on rice seedlings (Shanyou 63) at 27 ⫾ 1⬚C without exposure to insecticides.

Fig. 2. Map showing collection sites of six populations of N. lugens tested in this study.

Pesticides. Fipronil (87% technical grade) and ethiprole (96.4% technical grade) were provided by Bayer Crop Sciences Corporation (Hangzhou, China). Butene-Þpronil (90% technical grade) was provided by RAJ Pesticide Corporation (Dalian, China). Pesticide solutions were prepared in acetone and 10% Triton-100 (wt:vol) (emulsiÞer). The concentration was adjusted to a levels similar to the content of active ingredient in commercial emulsiÞable concentrate (EC). Bioassay and Data Analysis. The rice stem dipping method (Zhang and Shen 2000) was adopted for bioassay. Mid-third instars of the F1 or F2 generation, maintained in laboratory conditions without exposure to insecticides, were used in a doseÐresponse bioassay to detect resistance level to the chemicals. The mortality of N. lugens was recorded 96 h after treatment and analyzed by using the doseÐresponse probability value analysis software version 1.5 (U.S. Environmental Protection Agency) for calculating LC/EC values. Standard error of slope (b value) in the equation for toxicity regression, and the LC50 and 95% conÞdence limits (CL) of LC50 values were calculated. LC50 values without overlapping 95% CL were considered to be signiÞcantly different between pesticides. The RR was calculated by dividing the LC50 value of test population by the LC50 value of the susceptible strain. Laboratory Selection and Assessing Resistance and Cross-Resistance Development. The Þpronil-resistant strain was originally collected in 2007 from Xiaogan,

1366 Table 1.

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Resistance to fipronil in N. lugen from six locations of six provinces in 2009 Pop

na

Slope ⫾ SE

LC50 (95% CI) mg (AI)/liter

␹2b

RR

Susceptible strain Hexian, Anhui Province Fuqing, Fujian Province Xiaogan, Hubei Province Shanggao, Jiangxi Province Tongzhou, Jiangsu Province Tongxiang, Zhejiang Province

420 360 360 360 360 360 360

2.25 ⫾ 0.24 1.97 ⫾ 0.30 1.86 ⫾ 0.23 3.16 ⫾ 0.52 4.22 ⫾ 0.74 2.43 ⫾ 0.27 3.75 ⫾ 0.67

0.04 (0.03Ð0.05) 0.95 (0.72Ð1.22) 1.60 (1.28Ð2.00) 1.52 (1.27Ð1.83) 1.73 (1.49Ð2.00) 1.39 (1.14Ð1.67) 1.58 (1.32Ð1.87)

1.888 3.682 3.301 2.484 2.202 7.376 1.083

1.0 23.8 40.0 38.0 43.3 34.8 39.5

a b

Number of insects tested. ␹2 values without an asterisk indicate good Þt of the data to the probit model (P ⬍ 0.05).

Hubei Province, and was reared on the Shanyou 63 rice in greenhouse, with no exposure to any insecticides for eight generations. After that, the colony was screened continuously with Þpronil for 10 generations. The LC50 value (0.29 mg [AI]/liter) of Þpronil in the eighth generation (G8) was used as the starting concentration for the selection. The same rice stemdipping method described above was adopted for resistance selection to Þpronil. Approximately 1,000 third instars of every generation were treated with Þpronil by the rice stem-dipping method and subsequently maintained at 27 ⫾ 1⬚C and a photoperiod of 16:8 (L:D) h for 4 d. Survivors were transferred to another cage containing fresh rice seedlings. By adjusting insecticide concentrations, the mortality for resistance selection was controlled to a range between 40 and 70% to ensure sufÞcient survivors to develop and produce enough progeny for the subsequent insecticide selection. Except for Þpronil, butene-Þpronil and ethiprole have not been widely used for the control of N. lugens in rice-growing areas in China. Therefore, the dose responses to the insecticides could be used to assess whether the Þeld Þpronil-resistant populations developed cross-resistance to the other two analogs. At the same time, the sensitivity of Þpronil-resistant laboratory strain to butene-Þpronil and ethiprole could be assessed by using the same rice seedling-dipping method. Cross-resistance in N. lugens was evaluated by examining a cross-resistance ratio for each compound of interest, which was calculated by dividing the LC50 of the test populations by the LC50 of the susceptible strain, or dividing the LC50 of Þpronil-selected laboratory strain (G18) by the LC50 of initial generation (G8). Table 2.

Results and Analysis Monitoring of Field Populations. By using the rice stem dipping method, the resistance levels to three phenylpyrazole insecticides were monitored in 2009 in six populations of N. lugens from six locations (Fig. 2). Data showed that N. lugens developed 23.8 Ð39.5fold RRs to Þpronil in four populations (Hexian, Tongzhou Xiaogan, and Tongxiang), and 40.0 Ð 43.3-fold RRs in the other two populations (Fuqing and Shanggao) (Table 1). All six N. lugens populations developed a higher level resistance to ethiprole (RR ⫽ 47.1Ð 100.9-fold) (Table 2). RRs to ethiprole were signiÞcantly higher than (e.g., Xiaogan population) or similar to the RR to Þpronil, whereas RRs to butane Þpronil were substantially lower than those to Þpronil and ethiprole. Except for a lower level of resistance to butane Þpronil in Fuqing population (RR ⫽ 8.1-fold), other Þve populations (Xiaogan, Shanggao, Hexian, Tongzhou, and Tongxiang) still maintained a relatively low resistance levels (RR ⫽ 3.4 Ð 4.5-fold) (Table 3). Cross-Resistance to Other Phenylpyrazole Insecticides in Fipronil-Resistant Strain. By using the rice stem-dipping method, the resistance levels to three phenylpyrazole insecticides in the Þpronil-resistant strain collected from Xiaogan, Hubei Province, were determined before and after 10 generations of selection with Þpronil (Table 4). After being screened with Þpronil for 10 generations (from G8 to G18), the LC50 value increased from 0.29 (95% CI ⫽ 0.24 Ð 0.36) mg (AI)/liter (RR ⫽ 7.3-fold) in G8-1.65 (1.43Ð1.85) mg (AI)/liter (RR ⫽ 41.3-fold) in G18, with an increasing rate of 5.6-fold. The initial cross-resistance to ethiprole was 16.3-fold with the LC50 value of 1.63 (1.25Ð2.04)

Resistance to ethiprole in N. lugens from six locations of six provinces in 2009 Pop

na

Slope ⫾ SE


␹2b

RR


420 360 360 360 360 360 360

2.10 ⫾ 0.22 1.94 ⫾ 0.23 1.75 ⫾ 0.22 2.98 ⫾ 0.66 1.93 ⫾ 0.24 1.83 ⫾ 0.22 2.11 ⫾ 0.25

0.10 (0.10Ð0.12) 6.57 (5.11Ð8.13) 7.02 (5.26Ð8.94) 10.09 (3.56Ð17.17) 4.71 (3.49Ð5.95) 7.40 (5.69Ð9.26) 5.18 (4.03Ð6.37)

1.457 0.237 0.790 9.712* 0.255 3.148 0.114

1.0 65.7 70.2 100.9 47.1 74.0 51.8

a b

Number of insects tested. ␹2 values followed by an asterisk indicate not good Þt of the data to the probit model (P ⬍ 0.05).

August 2011 Table 3.

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Resistance to butene-fipronil in N. lugens from six locations of six provinces in 2009 Pop

na

Slope ⫾ SE


␹2b

RR


420 360 360 360 360 360 360

2.29 ⫾ 0.24 2.81 ⫾ 0.32 2.58 ⫾ 0.28 2.18 ⫾ 0.25 2.61 ⫾ 0.31 2.67 ⫾ 0.28 2.09 ⫾ 0.26

0.08 (0.06Ð0.10) 0.36 (0.29Ð0.42) 0.65 (0.54Ð0.76) 0.32 (0.25Ð0.39) 0.31 (0.25Ð0.37) 0.27 (0.22Ð0.32) 0.34 (0.26Ð0.42)

2.374 1.592 1.449 4.035 2.475 4.167 4.098

1.0 4.5 8.1 4.0 3.9 3.4 4.3

a b

Number of insects tested. ␹2 values without an asterisk indicate good Þt of the data to the probit model (P ⬍ 0.05).

mg (AI)/liter in G8, and then increased to 65.6-fold with the LC50 value of 6.56 (5.16 Ð 8.05) mg (AI)/liter in G18 after being selected with Þpronil for 10 generations. Cross-resistance to ethiprole increased by 4.0fold after 10 generations. The initial RR to buteneÞpronil in G8 was 2.8-fold (LC50 ⫽ 0.22 [0.17Ð 0.28] mg [AI]/liter), and it increased to 4.0-fold[LC50 ⫽ 0.32 [0.26 Ð 0.39] mg [AI]/liter]in G18. The cross-resistance to butene-Þpronil increased only 1.5-fold after 10 generations of selection with Þpronil (Table 4). Because the initial (G8) and Þnal (G18) generations had overlapped 95% CI of LC50 values to buteneÞpronil, the increase of cross-resistance to buteneÞpronil was not signiÞcant (P ⬎ 0.05). Discussion After an insect population developed resistance to one insecticide, it may become cross-resistant to one or more other insecticides to which the population has not been exposed. It is a common phenomenon that cross-resistance occurs as a result of exposure to a chemical that has a similar mode of action (MoA) (Shen and Wu 1995). Understanding of cross-resistance level and cross-resistance spectrum may lead to better management of a pest population by using strategies of rotation or mixture of insecticides that have different MoAs (Shen and Wu 1995). Dieldrin, a cyclopentyl diene, has similar action sites as Þpronil. It has been reported that dieldrin-resistant strains or populations of Drosophila, cockroaches, houseßies, and other insects were positively cross-resistant to Þpronil (Cole et al. 1995, Scott and Wen 1997, Kolaczinski and Curtis 2001). Although the insecticides with the same MoA do not necessarily result in the same resistance mechanism, cross-resistance to one or more Table 4.

cross-resistance of fipronil-resistant population to butene-fipronil and ethiprole Susceptible strain

Insecticide

Fipronil Ethiprole Butene-Þpronil a b

insecticides always occurs in a target insect with the same resistance mechanisms to the chemicals (Shen and Wu 1995). The structures of ethiprole, butene-Þpronil, and Þpronil are very similar (Fig. 1), and they all act on ␥-aminobutyric acid (GABA) receptors and interfere with the chloride ion channel to interrupt the normal function of the central nervous system, leading to insect death (Cole et al. 1993, Ikeda et al. 2004, Niu et al. 2007). N. lugens has developed resistance to many insecticides during the past few years. Due to the migratory nature of this planthopper, it is difÞcult to judge whether cross-resistance exists in the insect after these insecticides have been widely used in ricegrowing areas. Providing butene-Þpronil and ethiprole have not been widely used in China, the resistance data from this study are valid in assessing whether the Þpronil-resistant N. lugens developed cross-resistance to the two Þpronil analogs. Liu et al. (2010) reported a low-to-moderate level of Þpronil resistance detected in 2008 in several populations of N. lugens. One year later, the RR increased to a high level (23.8 Ð 43.3-fold), which was signiÞcantly higher than that in 2008. Considering insect may continue to increase resistance to Þpronil, it is urgent to seek alternatives to control Þpronil-resistant planthoppers. Butene-Þpronil and ethiprole are among the options. Therefore, proactive evaluation of cross-resistance to these two insecticides in this study is necessary for guiding the selection of new insecticides for control of Þpronil-resistant populations of N. lugens. In this study, we surveyed Þpronil-resistance and cross-resistance to Þpronil and its two analogs in N. lugens collected in six representative rice-growing areas in China. Our laboratory selection data indicated that these populations would become increasingly re-

Selection original pop (G8)

Selection strain (G18)



RRa


RRa

Increase rate of RRb

0.04 (0.03Ð0.05) 0.10 (0.10Ð0.12) 0.08 (0.06Ð0.10)

0.29 (0.24Ð0.36) 1.63 (1.25Ð2.04) 0.22 (0.17Ð0.28)

7.3 16.3 2.8

1.65 (1.43Ð1.85) 6.56 (5.16Ð8.05) 0.32 (0.26Ð0.39)

41.3 65.6 4.0

5.7 4.0 1.5

RR ⫽ LC50 of selection strain/LC50 of susceptible strain. Increase rate of RR ⫽ LC50 of selection strain (G18)/LC50 of selection original population (G8).

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sistant to Þpronil if they continue to be exposed to Þpronil. To achieve effective control of the pest, alternative chemicals are urgently wanted for relieving selection pressure on the target insect through rotating insecticides. Before this approach is implemented, it is necessary to evaluate cross-resistance in the Þpronil-resistant populations. Our proactive investigation of cross-resistance showed that all of the Þpronilresistant populations are cross-resistant to ethiprole, but fortunately not to butene-Þpronil. Therefore, our data suggest that butene-Þpronil is a potential alternative to Þpronil when insecticide rotation or mixture is required for Þpronil resistance management in N. lugens. Using ethiprole is only recommended in areas where Þpronil has not been extensively applied and the target insect is still susceptible to Þpronil and ethiprole. Considering these insecticides belong to the same class, however, using these alternatives is not recommended as a long-term solution for Þpronil resistance management. Cross-resistance must be constantly monitored. In addition, insecticides from different classes, such as buprofezin, thiamethoxam, nitenpyram, and pymetrozine (Wang et al. 2008), could be considered as potential alternatives against Þpronil-resistant N. lugens. Due to the intensive use of Þpronil for several years, many insects have developed resistance to the insecticide. Reduced sensitivity in resistant insects might be associated with an increase in enzymatic detoxiÞcation, a decrease of GABA receptor sensitivity (Wang et al. 2009), or both. Although three phenylpyrazole analogs have the same MoA on the GABA receptor, the levels of cross-resistance to ethiprobe and buteneÞpronil were signiÞcantly different. Therefore, it is still unclear whether the enzymatic detoxiÞcation or insensitive target site play an important role in the cross-resistance development to ethiprole and butene-Þpronil in Þpronil-resistant strains of N. lugens. Future bioassays with synergists may help elucidate the role of metabolic detoxiÞcation enzymes and develop alternative way to enhance chemical toxicity against target insects. Nevertheless, our data provided valuable information for guiding insecticide selection in Þpronil resistance management and established a foundation for future study on resistance and crossresistance mechanisms to the phenylpyrazole insecticides in N. lugens.

Acknowledgments We thank Jian Chen (USDAÐARS, Stoneville, MS) and Xuming Liu (Kansas State University, Manhattan, KS) for valuable comments and suggestions that improved an early

Vol. 104, no. 4

version of this manuscript. The study was funded by the Test and Demonstrate Program of Replacing High Toxicity Pesticides of the Ministry of Agriculture in China in 2008 (Nongcaifa[2008]No.59).

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