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Fipronil resistance mechanisms in the rice stem borer, Chilo suppressalis Walker
PESTICIDE Biochemistry & Physiology
Pesticide Biochemistry and Physiology 89 (2007) 169–174 www.elsevier.com/locate/ypest
Fipronil resistance mechanisms in the rice stem borer, Chilo suppressalis Walker Xiaotao Li a, Qingchun Huang b
a,*
, Jianzhong Yuan b, Zhenhua Tang
a,b,*
a Shanghai Key Lab of Chemical Biology, School of Pharmacy, East China University of Science and Technology, Shanghai, China Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China
Received 26 March 2007; accepted 11 June 2007 Available online 15 June 2007
Abstract Fipronil resistance mechanisms were studied between the laboratory susceptible strain and the selective field population of rice stem borer, Chilo suppressalis Walker in the laboratory. The borer population was collected from Wenzhou county, Zhejiang province. After five generations of selection, fipronil resistance ratio was 45.3-fold compared to the susceptible strain. Synergism experiments showed that the synergistic ratios of PBO, TPP and DEF on fipronil in susceptible and resistant strains of C. suppressalis were 7.55-, 1.93and 2.91-fold, respectively, and DEM showed no obvious synergistic action on fipronil. Activities of carboxylesterase and microsomal-O-demethylase in the resistant strain were 1.89- and 1.36-fold higher that in susceptible strain, and no significant difference of glutathione-S-transferase activity was found between the resistant and susceptible strains. The Km and Vmax experiments also demonstrated that fipronil resistance of C. suppressalis was closely relative to the enhanced activities of carboxylesterase and microsomalO-demethylase. Moreover, cross-resistance between fipronil and other conventional insecticides and the multiple resistant properties of the original Wenzhou’s population were also discussed. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Chilo suppressalis Walker; Fipronil; Synergism; Resistance mechanism
1. Introduction The rice stem borer, Chilo suppressalis Walker is one of the most important insect pests of many subtropical and tropic paddyfields in Asia, North Africa and Southern Europe. In China, it is mainly found in the Yangtze drainage areas and hilly areas of southern provinces beyond Yangtze River. This insect feeds on rice causing significant economic losses to farmers. Besides, it also causes great damage to other crops including water bamboo, sugarcane, sorghum, corn, wheat, millet, horsebean and rape [1]. Since 1970s, there have been several outbreaks of pest attack in China because of the *
Corresponding authors. Address: Shanghai Key Lab of Chemical Biology, School of Pharmacy, East China University of Science and Technology, Shanghai, China. Fax: +86 21 6425 2603. E-mail addresses: [email protected] (Q. Huang), tangzh@sh163. net (Z. Tang). 0048-3575/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.pestbp.2007.06.002
changes in the rice cultivation system and the use of hybrid varieties [2,3]. The major control measure for the rice stem borer is the application of chemicals, which are known to cause resistance and control problems. Before 1980s, rice stem borer was mainly controlled by BHC, trichlorphon, parathion, parathionmethyl and chlordimeforn, however, these insecticides were banned for the bioaccumulation in food chain, chronic toxicity and/or high resistance at the end of 1980s. From the beginning of 1980s to the middle of 1990s, some organophosphate and nereistoxin insecticides such as methamidophos, triazophos, monosultap and dimehypo were mainly used to control the pest. However, because of long-term use of insecticides, some studies reported that rice stem borer is resistant to lots of conventional insecticides [4–11]. At the end of 1990s, fipronil and abamectin were used for control and showed excellent effects. Recently, there are few reports about rice stem borer with low or middle level of resistance to fipronil [12,13].
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Fipronil [(±)-5-amino-1-(2,6-dichloro-a,a,a-trifluorop-tolyl)-4-trifluoromethylsulfinylpyr-azole-3-carbonitrile] is the first member of the phenylpyrazole insecticide. Its mode of action involves disruption of chloride ion flow by interacting at the GABA-gated chloride ionophore of the central nervous system [14,15], or fipronil potently blocks glutamate-activated chloride channel and strongly inhibits glutamate-induced chloride ion flow [16]. Moreover, its oxidative sulfone is reported as the most effective inhibitor of glutamate-gated chloride channel than other metabolites [17]. Since 1997 the first use in Wentai region of Zhejiang province, fipronil has been used extensively in the whole Yangtze middle and downriver and Southeast coastal areas, and the dosage was increased year after year. Herein, the purpose of this study is to investigate the characterization and mechanisms of fipronil resistance in C. suppressalis, in order to offer some guidance for fipronil resistance management and reasonable use. 2. Materials and methods 2.1. Insects The strain of rice stem borer was obtained from Nanjing Agricultural University, China. This strain was originally collected from a mountain area of Taihu county, Anhui province, and raised in laboratory without any contact with insecticides from 2000. For obtaining more susceptible strain to fipronil, the strain was selected from single egg mass that was produced by one-pair mating adult under conditions free of insecticides from 2005. The most sensitive larvae to fipronil were used as the susceptible strain in the experiments. A resistant field population of C. suppressalis was collected from Wenzhou, Zhejiang province in 2005, where some conventional insecticides were normally used to control this pest each year and fipronil had been mainly used in recent years. The laboratory resistant strain was obtained by five-generation selection on the field population. The resistance ratio was increased from initial 4.1 to 45.3 against fipronil. All insects were reared in laboratory according to the seeding method reported by Shang et al. [18]. The rearing conditions were 28 ± 1°C, photoperiod 16L:8D and the relative humidity >80%. 2.2. Insecticides and chemicals Fipronil (87% a.i.) was obtained from Rhone-Poulenc Company in France. Abamectin (B1a, 98%), fenitrothion (93.2% a.i.), chlorpyrifos (99.4 a.i.), monosultap (94% a.i.) and alphamethrin (96% a.i.) were supplied by Shanghai Pesticide Research Institute in China. Piperonyl butoxide (PBO) was from Fluka A G, packed in Switzerland. Triphenyl phosphate (TPP) was from Sinopharm Group Chemical Reagent, China (Shanghai, China). Diethyl maleate (DEM) was supplied by Beijing Xudong Reagent
Factory (Beijing, China). S,S,S-tributylphosphorothioate (DEF), l-glutathione reduced (GSH), phenylthiourea (PTU) and NADPH were from Sigma Chemical (St. Louis, USA). a-Naphthyl acetate (a-NA), 1-chloro-2,4dinitrobenzene (CDNB), editic acid (EDTA) and p-nitroanisole (p-NA) were produced by Shanghai Reagent Factory (Shanghai, China). 1,4-Dithiothreitol (DTT) was from Shanghai Institute of Biochemistry, Chinese Academy of Sciences. Phenylmethyl sulfonyl fluoride (PMSF) was from E. Merck, Darmstsdt. 5,5 0 -Dithiobis-(2-nitrobenzoic acid) (DTNB) was from Sino-American Biotechnology Company. Fast blue RR salt, actylcholine (ATCHI), bovine serum albumin (BSA) and Coomassie brilliant blue G-250 were purchased from Fluka Chemie GmbH (Buchs, Italy). 2.3. Bioassay and synergism Technical grade insecticide and synergist were dissolved in acetone and five serially diluted concentrations were prepared so that each was one-half of the previous concentration. Ten fourth-instar larvae (9–11 mg) were treated with 1 ll of the dilution on the middle-abdomen notum by a hand microapplicator (Burkard, UK). Each test was replicated three times and the acetone treatment was used as control. In the synergism experiments, 1 ll of synergists with maximal non-lethal dosage was topically applied on each larvae about 1 h before insecticide treatment, the corresponding concentrations of PBO, TPP, DEF and DEM were set as 6.0, 10.0, 5.0 and 5.5 g/L, respectively. The test larvae were then reared individually at 28 ± 1 °C with adequate food. Mortality was assessed at 48-h posttreatment and the data were analyzed by probit analysis using the POLO program. 2.4. Enzyme assays Carboxylesterase (CarE) activity was measured according to the method described by Han et al. [19] with minor modification. Twenty larvae were homogenized in 2 ml phosphate buffer (0.02 mol/L, pH 7.0) at 0 °C. The homogenate was centrifuged at 5000g for 15 min at 4 °C. The supernatant was collected as enzyme solution. An aliquot of 0.1 ml enzyme solution was put into 0.9 ml mixed solution of a-NA and Fast blue RR salt (10 mg a-NA and 20 mg Fast blue RR salt dissolved in 2 ml acetone, and fixed cubage to 25 ml by adding phosphate buffer (0.02 mol/L, pH 7.0), then the mixed solution was filtrated), and this reacting mixture contained 105 mol/L eserin. The change rate of optical density at 450 nm was recorded with Beckman DU-650 spectrophotometer (Beckman Company, USA). Glutathione-S-transferase (GST) activity was measured with the method described by Kao et al. [20] with minor modification. Twenty larvae were homogenized in 2 ml Tris–HCl buffer (0.1 mol/L, pH 8.0) at 0 °C. The homoge-
171 Note: NT denoted no treatment. SD represented the sensitive degree of the test larvae to the toxicity of fipronil. The resistance ratio (RR) indicated that LD50 value of resistant strain was divided by LD50 value of susceptible strain. The value followed by the same letter was not significantly different (LSD test, P < 0.05).
— 0.35 0.25 0.15 0.13 0.09 (0.0032–0.0283)a (0.0281–0.0613)ab (0.0413–0.0949)b (0.0674–0.1796)bc (0.0883–0.1859)c (0.1207–0.2905)d 0.0156 0.0443 0.0633 0.1053 0.1240 0.1721 1.568 ± 0.466a 1.883 ± 0.411b 2.683 ± 0.642d 1.688 ± 0.405a 2.354 ± 0.489c 2.255 ± 0.514bc — 1.38 1.74 1.92 1.92 1.92 (0.0060–0.0089)c (0.0043–0.0064)b (0.0026–0.0057)ab (0.0019–0.0056)a (0.0019–0.0056)a (0.0019–0.0056)a 0.0073 0.0053 0.0042 0.0038 0.0038 0.0038 3.018(±0.353)c 2.557(±0.319)b 2.237(±0.258)ab 2.032(±0.478)a 2.032(±0.478)a 2.032(±0.478)a 12.45 10.04 16.82 15.57 14.75 NT
LD50 (95% FL) (lg/larva)
0.02 0.04 0.08 0.10 0.15 NT
The responses of the fourth-instar larvae of C. suppressalis to fipronil in different generations of the susceptible and resistant strains after selection are presented in Table 1. For the susceptible strain taming, LD50 value of fipronil against the fourth-instar larvae of parental generation was 0.0073 lg/larva and it decreased to 0.0038 lg/larva after three generations of selection, the susceptibility of population to fipronil increased 1.92-fold. For the resistant strain, LD50 value of parental generation was 0.0156 lg/larva and it increased to 0.1721 lg/larva after five generations of selection, their susceptibility to fipronil was decreased 11.03-fold. Thus, the resistance level of the fourth-instar larvae to fipronil increased 45.3-fold in comparison with the susceptible strain. Moreover, the slope value of regression curve suggests that the individuals of population after selection have greater heterogeneity compared with the population before selection.
0 1 2 3 5 6
3.1. Susceptibility taming and resistance selection
Survival percent (%)
3. Results and analysis
Number of larvae
All data were subjected to analysis of variance (ANOVA) and means separated using least significant difference (LSD) test. The level of significance (P) was set at 5% (SAS Institute, 1988).
Concentration (g/L)
2.6. Data analysis
Generation
Protein content was determined by the Bradford method using bovine serum albumin as the standard [22].
Table 1 Responses of the fourth-instar larvae to fipronil in the susceptible and resistant strains of C. suppressalis
2.5. Protein assay
4000 4000 1500 1000 2500 NT
Slope ± SE Slope ± SE
SD
Resistant strain selection Susceptible strain taming
LD50 (95% FL) (lg/larva)
SD
RR
nate was centrifuged at 10,000g for 15 min at 4 °C. The supernatant was collected as enzyme solution. The reaction mixture contained 0.1 ml enzyme solution, 1.4 ml Tris–HCl buffer (0.1 mol/L, pH 8.0) and 0.05 ml GSH (40 mmol/L), After preincubation for 5 min 25 °C, 0.06 ml CDNB (30 mmol/L) was added. The change rate of optical density at 340 nm was recorded with Beckman spectrophotometer. Microsomal-O-demethylase activity was measured by referring the method of Qu et al. [9] and Yang et al. [21] with minor modification. Twenty larvae were homogenized in 2 ml phosphate buffer (0.1 mol/L, pH 7.8, containing 1 mmol/L EDTA, 1 mmol/L DTT, 1 mmol/L PTU, 1 mmol/L PMSF) at 0 °C. The homogenate was centrifuged at 12,000g for 15 min at 0 °C. The supernatant was collected as enzyme solution. The reaction mixture contained 0.1 ml enzyme solution, 0.4 ml phosphate buffer (0.1 mol/L, pH 7.8) and 0.25 ml p-NA (6.0 mmol/L), After preincubation for 5 min at 34 °C, 0.25 ml NADPH (2.0 mmol/L) was added to start the reaction, and the mixture was shaken during incubation at 34 °C for 30 min. The change value of optical density at 405 nm was recorded with Beckman spectrophotometer.
2.1 8.2 15.1 27.7 32.6 45.3
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3.2. Resistance to fipronil and other insecticides The topical toxicities of fenitrothion, chlorpyrifos, monosultap, abamectin and alphamethrin to the fourthinstar larvae of the susceptible and resistant strains in C. suppressalis population are shown in Table 2. The strains of rice stem borer revealed different responses to the actions of two organophosphates, fenitrothion and chlorpyrifos, and seemed to possess higher cross-resistance to fenitrothion with 23.1-fold of RR value whilst almost no cross-resistance to chlorpyrifos (LSD test, P < 0.05). Furthermore, the strains showed lower cross-resistance to monosultap with 3.2-fold of RR value and median crossresistance to abamectin and alphamethrin with 8.9- and 7.0-fold of RR value, respectively. The results seemed to suggest that the development of resistance of the laboratory selective strain to fipronil had potentially evolved the decreased susceptibility to organophosphate and nereistoxin, even pyrethroid insecticides or abamectin, and/or the field population had developed multiple resistant properties before the laboratory selection.
3.3. Synergism of PBO, TPP, DEF and DEM The synergistic ratios (SR) of PBO, TPP, DEF and DEM on fipronil in susceptible and resistant strains of C. suppressalis are listed in Table 3. For the susceptible strain, TPP and DEF all showed no obvious synergistic action on fipronil, whereas PBO seemed to play antagonistic effect on fipronil with 0.4-fold of SR value. For the resistant strain, PBO and TPP as well as DEF exhibited stronger synergism (SR
3.02, 2.10 and 2.76), which was 7.55-, 1.93- and 2.91-fold higher than that in susceptible strain. However, no synergism of DEM on fipronil was found in both strains. These implied that microsomal-O-demethylase and esterase might be involved in fipronil resistance in the C. suppressalis population and that glutathione-S-transferase might be less important for the strains in conferring the fipronil resistance. 3.4. Activity of enzymes The activity of CarE, microsomal-O-demethylase and GST from the susceptible and resistant strains were tested in vitro, and the results are presented in Table 4. The activities of CarE and microsomal-O-demethylase of the resistant strain were 1.89- and 1.36-fold higher than that of Table 4 Activity of enzymes in susceptible and resistant strains of C. suppressalis Enzymes
Strains
Activity
R/S ratio
Carboxylesterase
S R
16.957 ± 0.516 32.032 ± 0.465
1.89bB
Microsomal-O-demethylase
S R
0.534 ± 0.062 0.726 ± 0.083
1.36abAB
Glutathione-S-transferase
S R
3.853 ± 0.350 3.872±0.053
1.00aA
Note: Units of CarE, GST and microsomal-O-demethylase were DOD450Æmin1 (mg pro)1, DOD340Æmin1 (mg pro)1, and nmolÆmin1 (mg pro)1, respectively. R/S ratio represented the enzyme activity in the resistant strain was divided by the enzyme activity in the susceptible strain. The value followed by the same letter was not significantly different (LSD test, P < 0.05 and P < 0.01).
Table 2 Responses of the fourth-instar larvae to different insecticides in the susceptible and resistant strains of C. suppressails Insecticides
Fipronil Fenitrothion Chlorpyrifos Monosultap Abamectin Alphamethrin
Susceptible strain
Resistant strain 1
RR 1
Slope ± SE
LD50 (95% FL) (lg larva )
Slope ± SE
LD50 (95% FL) (lg larva )
2.032 ± 0.478 2.529 ± 0.374 0.983 ± 0.278 2.229 ± 0.269 1.676 ± 0.197 2.222 ± 0.460
0.0038 (0.0019–0.0056) 0.8066 (0.4382–1.2925) 1.2278 (0.5512–8.6369) 3.9616 (3.1231–5.0526) 0.00036 (0.00020–0.00054) 0.0251 (0.0154–0.0389)
2.255 ± 0.514 3.832 ± 0.923 2.827 ± 0.558 1.572 ± 0.360 2.427 ± 0.483 3.206 ± 0.751
0.1721 (0.1207–0.2905) 18.594 (13.4357–24.5737) 2.0488 (1.3800–3.0432) 12.8147 (7.6215–27.1288) 0.0032 (0.0021–0.0047) 0.1751 (0.1188–0.2619)
45.3 23.1 1.7 3.2 8.9 7.0
Note: RR denoted LD50 value of resistant strain was divided by LD50 value of susceptible strain.
Table 3 Synergism of PBO, TPP, DEF and DEM on fipronil in susceptible and resistance strains of C. suppressalis Treatments
Fipronil Fipronil + PBO Fipronil + TPP Fipronil + DEF Fipronil + DEM
Susceptible strain
Resistance strain
SR2/SR1
Slope ± SE
LD50 (95%FL) (lg/larva)
SR1
Slope ± SE
LD50 (95%FL) (lg/larva)
SR2
2.032 ± 0.478 1.825 ± 0.299 5.884 ± 1.051 2.051 ± 0.479 4.045 ± 0.591
0.0038 0.0095 0.0035 0.0040 0.0038
— 0.40 1.09 0.95 1.00
2.255 ± 0.514 2.907 ± 0.568 2.533 ± 0.321 2.954 ± 0.339 2.162 ± 0.277
0.1721 0.0569 0.0819 0.0623 0.1501
— 3.02 2.10 2.76 1.15
(0.0019–0.0056)a (0.0068–0.0152)b (0.0030–0.0041)a (0.0022–0.0063)a (0.0032–0.0045)a
(0.1207–0.2905)c (0.0423–0.0791)a (0.0678–0.1023)b (0.0528–0.0745)ab (0.1217–0.1923)c
7.55c 1.93b 2.91bc 1.15a
Note: The synergistic ratio (SR) indicated that LD50 value without synergist was divided by LD50 value with synergist. The value followed by the same letter was not significantly different (LSD test, P < 0.05).
X. Li et al. / Pesticide Biochemistry and Physiology 89 (2007) 169–174 Table 5 Km and Vmax values for carboxylesterase and microsomal-O-demethylase in susceptible and resistant strains of C. suppressalis Enzymes
Strains
Km
Ratio
Vmax
Ratio
Carboxylesterase
S R
69.942 15.267
0.22
35.971 29.534
0.82
Microsomal-O-demethylase
S R
17.291 25.438
1.47
3.188 2.679
0.84
Note: Units of Km and Vmax for CarE was mmol/L and DOD450Æmin1 (mg pro)1; Units of Km and Vmax for microsomal-Odemethylase was nmol/L and nmolÆmin1 (mg pro)1. Ratio denoted that Km or Vmax of the resistant strain was divided by Km or Vmax of the susceptible strain.
the susceptible strain, respectively, whereas the activities of glutathione-S-transferase from the two strains showed no significant difference (LSD test, P < 0.05). The results substantiated that microsomal-O-demethylase and CarE played important effects in enhancing the resistance of C. suppressalis to fipronil, and the increased activity of CarE seemed to be the major inducer relative to the larval resistance. 3.5. Km and Vmax values The results of the kinetic study for CarE and microsomal-O-demethylase are shown in Table 5. The Km values of the CarE and microsomal-O-demethylase in the resistant strain were 0.22- and 1.47- fold higher than that in the susceptible strain, respectively. Data meant that the affinity of CarE increased and the affinity of microsomal-O-demethylase declined significantly in the resistant strain, indicating that the enhancing activity of CarE might be the key factor for improving the resistance. Moreover, the Vmax values of the CarE and microsomal-O-demethylase in the resistant strain were 0.82- and 0.84-fold less than that in the susceptible strain, respectively. The results suggested that the resistance to fipronil might be the modifications of CarE and microsomal-O-demethylase protein of C. suppressalis larvae and the multiple resistance background of the original Wenzhou’s population already existed to other insecticides. 4. Discussion Fipronil is high toxic to both piercing–sucking and chewing insects and has shown excellent activity against a broad spectrum of insect orders [15], and has shown no obvious cross-resistance to other action mechanism insecticides [23]. Many researches indicated that fipronil was transformed to the more toxic sulfone metabolites by cytochrome P450-mediated microsomal monooxygenase in insect [17,24,25]. However, due to increased frequency and years of use, fipronil resistance had appeared in insects. Its resistance mechanism was demonstrated as the low permeability of the cuticle, monooxygenase metabolism, and/ or the increased activity of detoxification enzymes in
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Musca domestica and Plutella oxylostella [26–28]. Recently, Goff et al. found that two site mutations in RDL subunit of GABA-receptor caused the high level of resistance to fipronil in Drosophila simula [29]. Based on these reports, we speculated that the low level of resistance was associated with the increased enzymatic detoxification, and GABAreceptor protein mutations caused the high resistance to fipronil in insects. Preliminary study on fipronil resistance in field strain C. suppressalis had been done by Jiang et al. [13]. Their synergism experiments indicated that esterase and glutathione-Stransferase played important role in the fipronil resistance of C. suppressalis. Our current study with laboratory taming strain C. suppressalis show that the role of glutathioneS-transferase may not be important because DEM is not synergistic to fipronil and the activity of glutathione-Stransferase is not significantly different between the resistant and susceptible strains. However, carboxylesterase and microsomal-O-demethylase might play important roles in fipronil resistance of C. suppressalis because TPP, DEF and PBO had significant synergistic activity to fipronil in the resistant strain. Moreover, enzyme activity assays and the kinetic experiments such as Km and Vmax also demonstrated that fipronil resistance of C. suppressalis was closely relative to the enhanced activities of carboxylesterase and microsomal-O-demethylase. As far as we know, cross-resistance is a very serious problem in insecticide resistance management. Our current study seemed to show that cross-resistance existed, more or less, among fipronil and fenitrothion and abamectin. Fortunately, the resistance mechanisms for conventional organophosphate insecticides in C. suppressalis have been extensively studied. Konno et al. had demonstrated that esterase played a critical role with different isoenzymes for resistance of C. suppressalis to organophosphate insecticides [4–6]. Han et al. and Peng et al. successively indicated that resistant mechanisms in C. suppressalis to feniteothion and methamidophos were the enhanced activity of carboxylesterase, microsomal-O-demethylase and N-demethylase as well as the insensitivity of AChE [7,8]. The same resistant mechanism was further found in C. suppressalis to triazophos and monosultap [9–11,30]. In paper, we also found that AChE activity of the resistant strain was 2.38-fold higher than that of the susceptible strain, and fenitrothion I50 ratio of R/S was 1.54 (undelivered data). So we assume that insensitive AChE is implicated in multiple resistance of the resistant strain because the original Wenzhou’s population was ever controlled by organophosphate insecticides. This might be the reason that the increased carboxylesterase activity was tangly involved in resistance mechanism. The reason that chlorpyrifos, however, in the susceptible strain is nearly as effective as in the resistant strain was the increased monooxygenase activity. Chlorpyrifos would convert into oxon derivative advantageously and its effective toxicity would become stronger through cytochrome P450-mediated monooxygenase reactions. Furthermore, although fipronil and
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abamectin have different acting sites on GABA-receptor, fipronil resistance changes the metabolism of glutamate and the function of GABA-gated chloride channels, thus might induce cross-resistance between fipronil and abamectin. From the above-mentioned, we could easily eliminate the superficial disorder that the laboratory taming strain already possessed the multiple resistance to organophosphate and fipronil, and conclude that the enhanced activity of microsomal-O-demethylase was the main inducer for fipronil resistance in C. suppressalis, glutathione-S-transferase seemed not participate in fipronil resistance. Of course, the well-defined elucidations of fipronil resistance still need the laboratory mono-resistant strain of C. suppressalis. Acknowledgments We thank Dr. V. Shyam kumar, P.G. Department of Sericulture Karnatak University, Dharwad, India for critical review and helpful discussion of the manuscript. We also thank the National Key Project for Basic Research (2003CB114402), the National Natural Science Foundation of China (30400295), the Shanghai Foundation of Science and Technology, and the Shanghai Education Commission for financial support. References [1] X.T. Li, Q.C. Huang, Z.H. Tang, An review on the development of resistant mechanism in Chilo suppressalis, World Pesticides 28 (2006) 17–20, 44. (in Chinese). [2] J.C. Fang, Z.W. Du, X.N. Cheng, Increasing harm tendency of rice stem borers and their control strategy in China, Entomol. Knowl. 35 (1998) 193–197 (in Chinese). [3] C.Y. Diao, M.T. Wang, Y.Q. Zhu, The reasons of the increase of rice stem borer in paddy field and the approaches of controlling strategies in south Jiangsu Province, Plant Prot. Tech. Ext. 21 (2001) 7–9 (in Chinese). [4] Y. Konno, T. Shishido, Resistance mechanism of the rice stem borer to organophosphorus insecticides, J. Pesticide Sci. 10 (1985) 285–287. [5] Y. Konno, N.N. Gakkaishi, Studies on resistance mechanism and synergism in the OP-resistance rice stem borer, Chilo suppressalis Walker, Agrochemical Bioregulators 14 (1989) 373–381. [6] Y. Konno, Carboxylesterase of the rice stem borer Chilo suppressalis Walker (Lepidoptera: Pyralidae). responsible for feniteothion resistance as sequesting protein, J. Pestic. Sci. 21 (1996) 425–429. [7] Q.F. Han, P.J. Zhuang, Z.H. Tang, The mechanism of resistance to fenitrothion in the rice stem borer Chilo suppressalis Walker, Acta Entomol. Sin. 38 (1995) 266–271 (in Chinese). [8] Y. Peng, C.K. Chen, Z.J. Han, Y.C. Wang, Resistance measurement of Chilo suppressalis from Jiangsu province and its resistance mechanism to methamidophos, Acta Phytophylacica Sin. 28 (2001) 173–177 (in Chinese). [9] M.J. Qu, Z.J. Han, X.J. Xu, L.N. Yue, Triazophos resistance mechanisms in rice stem borer (Chilo suppressalis Walker), Pesticide Biochem. Physiol. 77 (2003) 99–105. [10] X.F. Li, Z.J. Han, C.K. Chen, G.Q. Li, Y.C. Wang, Monitoring for resistance of rice stem borer (Chilo suppressalis Walker) to 4 conventional insecticides, J. Nanjing Agric. Univ. 24 (2001) 43–46 (in Chinese).
[11] T. Deguchi, T. Narahashi, H.G. Hass, Mode of action of nereistoxin on neuromuscular transmission in the flog, Pestic. Biochem. Physiol. 1 (1971) 196–201. [12] M.Z. Cao, J.L. Shen, J.Z. Zhang, M. Lu, X.Y. Liu, W.J. Zhou, Monitoring of insecticide resistance and inheritance analysis of triazophos resistance in the striped stem borer (Lepidoptera: Pyralidae), Chinese J. Rice Sci. 18 (2004) 73–79. [13] W.H. Jiang, Z.J. Han, M.L. Hao, Preliminary study on risistance of fipronil in rice stem borer (Chilo suppressalis Walker), Chinese J. Rice Sci. 19 (2005) 577–579. [14] L.M. Cole, R.A. Nicholson, J.E. Casida, Action of phenylpyrazole insecticides at the GABA gated chloride channel, Pesticide Biochem. Physiol. 46 (1993) 47–54. [15] D.B. Grant, A.E. Chalmers, M.A. Wolff, H.B. Hoffman, D.F. Bushey, Fipronil: action at the GABA receptor, Rev. Toxicol. 2 (1998) 147–156. [16] T. Ikeda, X. Zhao, Y. Kono, J.Z. Yeh, T. Narahashi, Fipronil modulation of glutamate induced chloride currents in cockroach thoracic ganglion neurons, Neurotoxicology 24 (2003) 807–815. [17] X. Zhao, J.Z. Yeh, V.L. Salgado, T. Narahashi, Sulfone metabolite of fipronil blocks c-amino-butyric acid and glutamate-activated chloride channels in mammalian and insect neurons, J. Pharm. Exp. Ther. 314 (2005) 363–373. [18] Z.Z. Shang, Y.S. Wang, Y.H. Zou, Study on rearing method of rice stem borer Chilo suppressalis Walker, Acta Entomological Sinica 22 (1979) 164–167 (in Chinese). [19] Z.J. Han, G.D. Moores, D. Jan, L.D. Alan, Association between biochemical marks and insecticide resistance in the cotton aphid, Aphisgossypii Glover, Pesticide Biochem. Physiol. 62 (1998) 164–171. [20] C.H. Kao, C. Fu, C.N. Sum, Parathion and methyl parathion resistance in diamondback moth (Lepidoptera: Plutellidae) larvae, J. Econ. Entomol. 82 (1989) 1299–1304. [21] Y. Yang, Y. Wu, S. Chen, G.J. Devine, I. Denholm, P. Jewess, G.D. Moores, The involvement of microsomal oxidases in pyrethroid resistance in Helicoverpa armigera from Asia, Insect Biochem. Mol. Biol. 34 (2004) 763–773. [22] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248–254. [23] F. Colliot, K.A. Kukorowski, D.W. Hakins, D.A. Roberts, Fipronil: A new soil and foliar broad spectrum insecticide, Proc. Brighton Crop Protect. Conf-Pests Diseases 29 (1992) 34. [24] E.W. Durham, M.E. Scharl, B.D. Siegfried, Toxicity and neurophysiological effects of fipronil and its oxidative sulfone metabolite on European corn borer larvae, Pesticide Biochem. Physiol. 71 (2001) 97–106. [25] J.E. Mulrooney, D. Goli, Efficacy and degradation of fipronil applied to cotton for control of Anthonomus grandis (Coleoptera: Curculionidae), J. Econ. Entomol. 92 (1999) 1364–1368. [26] Z.M. Wen, J.G. Scott, Genetic and biochemical mechanisms limiting fipronil toxicity in the LPR strain of house fly, Musca domestica, Pesticide Sci. 55 (1999) 988–992. [27] M. Kristensen, J.B. Jespersen, M. Knorr, Cross-resistance potential of fipronil in Musca domestica, Pest Manag. Sci. 60 (2004) 894–900. [28] M. Mohan, G.T. Gujar, Local variation in susceptibility of the diamonback moth Plutella oxylostella Linnaeus to insecticides and role of detoxification enzymes, Crop Protection 22 (2003) 495– 504. [29] G.L. Goff, A. Hamon, J.B. Berge, M. Amichot, Resistance to fipronil in Drosophila simulans: influence of two point mutations in the RDL GABA receptor subunit, J. Neurochem. 92 (2005) 1295–1305. [30] Z.J. Han, Z.J. Han, Y.C. Wang, C.K. Chen, Biochemical features of a resistant population of the rice stem borer, Chilo suppressalis Walker, Acta Entomol. Sin. 46 (2003) 161–170.
Pesticide Biochemistry and Physiology 89 (2007) 169–174 www.elsevier.com/locate/ypest
Fipronil resistance mechanisms in the rice stem borer, Chilo suppressalis Walker Xiaotao Li a, Qingchun Huang b
a,*
, Jianzhong Yuan b, Zhenhua Tang
a,b,*
a Shanghai Key Lab of Chemical Biology, School of Pharmacy, East China University of Science and Technology, Shanghai, China Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China
Received 26 March 2007; accepted 11 June 2007 Available online 15 June 2007
Abstract Fipronil resistance mechanisms were studied between the laboratory susceptible strain and the selective field population of rice stem borer, Chilo suppressalis Walker in the laboratory. The borer population was collected from Wenzhou county, Zhejiang province. After five generations of selection, fipronil resistance ratio was 45.3-fold compared to the susceptible strain. Synergism experiments showed that the synergistic ratios of PBO, TPP and DEF on fipronil in susceptible and resistant strains of C. suppressalis were 7.55-, 1.93and 2.91-fold, respectively, and DEM showed no obvious synergistic action on fipronil. Activities of carboxylesterase and microsomal-O-demethylase in the resistant strain were 1.89- and 1.36-fold higher that in susceptible strain, and no significant difference of glutathione-S-transferase activity was found between the resistant and susceptible strains. The Km and Vmax experiments also demonstrated that fipronil resistance of C. suppressalis was closely relative to the enhanced activities of carboxylesterase and microsomalO-demethylase. Moreover, cross-resistance between fipronil and other conventional insecticides and the multiple resistant properties of the original Wenzhou’s population were also discussed. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Chilo suppressalis Walker; Fipronil; Synergism; Resistance mechanism
1. Introduction The rice stem borer, Chilo suppressalis Walker is one of the most important insect pests of many subtropical and tropic paddyfields in Asia, North Africa and Southern Europe. In China, it is mainly found in the Yangtze drainage areas and hilly areas of southern provinces beyond Yangtze River. This insect feeds on rice causing significant economic losses to farmers. Besides, it also causes great damage to other crops including water bamboo, sugarcane, sorghum, corn, wheat, millet, horsebean and rape [1]. Since 1970s, there have been several outbreaks of pest attack in China because of the *
Corresponding authors. Address: Shanghai Key Lab of Chemical Biology, School of Pharmacy, East China University of Science and Technology, Shanghai, China. Fax: +86 21 6425 2603. E-mail addresses: [email protected] (Q. Huang), tangzh@sh163. net (Z. Tang). 0048-3575/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.pestbp.2007.06.002
changes in the rice cultivation system and the use of hybrid varieties [2,3]. The major control measure for the rice stem borer is the application of chemicals, which are known to cause resistance and control problems. Before 1980s, rice stem borer was mainly controlled by BHC, trichlorphon, parathion, parathionmethyl and chlordimeforn, however, these insecticides were banned for the bioaccumulation in food chain, chronic toxicity and/or high resistance at the end of 1980s. From the beginning of 1980s to the middle of 1990s, some organophosphate and nereistoxin insecticides such as methamidophos, triazophos, monosultap and dimehypo were mainly used to control the pest. However, because of long-term use of insecticides, some studies reported that rice stem borer is resistant to lots of conventional insecticides [4–11]. At the end of 1990s, fipronil and abamectin were used for control and showed excellent effects. Recently, there are few reports about rice stem borer with low or middle level of resistance to fipronil [12,13].
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Fipronil [(±)-5-amino-1-(2,6-dichloro-a,a,a-trifluorop-tolyl)-4-trifluoromethylsulfinylpyr-azole-3-carbonitrile] is the first member of the phenylpyrazole insecticide. Its mode of action involves disruption of chloride ion flow by interacting at the GABA-gated chloride ionophore of the central nervous system [14,15], or fipronil potently blocks glutamate-activated chloride channel and strongly inhibits glutamate-induced chloride ion flow [16]. Moreover, its oxidative sulfone is reported as the most effective inhibitor of glutamate-gated chloride channel than other metabolites [17]. Since 1997 the first use in Wentai region of Zhejiang province, fipronil has been used extensively in the whole Yangtze middle and downriver and Southeast coastal areas, and the dosage was increased year after year. Herein, the purpose of this study is to investigate the characterization and mechanisms of fipronil resistance in C. suppressalis, in order to offer some guidance for fipronil resistance management and reasonable use. 2. Materials and methods 2.1. Insects The strain of rice stem borer was obtained from Nanjing Agricultural University, China. This strain was originally collected from a mountain area of Taihu county, Anhui province, and raised in laboratory without any contact with insecticides from 2000. For obtaining more susceptible strain to fipronil, the strain was selected from single egg mass that was produced by one-pair mating adult under conditions free of insecticides from 2005. The most sensitive larvae to fipronil were used as the susceptible strain in the experiments. A resistant field population of C. suppressalis was collected from Wenzhou, Zhejiang province in 2005, where some conventional insecticides were normally used to control this pest each year and fipronil had been mainly used in recent years. The laboratory resistant strain was obtained by five-generation selection on the field population. The resistance ratio was increased from initial 4.1 to 45.3 against fipronil. All insects were reared in laboratory according to the seeding method reported by Shang et al. [18]. The rearing conditions were 28 ± 1°C, photoperiod 16L:8D and the relative humidity >80%. 2.2. Insecticides and chemicals Fipronil (87% a.i.) was obtained from Rhone-Poulenc Company in France. Abamectin (B1a, 98%), fenitrothion (93.2% a.i.), chlorpyrifos (99.4 a.i.), monosultap (94% a.i.) and alphamethrin (96% a.i.) were supplied by Shanghai Pesticide Research Institute in China. Piperonyl butoxide (PBO) was from Fluka A G, packed in Switzerland. Triphenyl phosphate (TPP) was from Sinopharm Group Chemical Reagent, China (Shanghai, China). Diethyl maleate (DEM) was supplied by Beijing Xudong Reagent
Factory (Beijing, China). S,S,S-tributylphosphorothioate (DEF), l-glutathione reduced (GSH), phenylthiourea (PTU) and NADPH were from Sigma Chemical (St. Louis, USA). a-Naphthyl acetate (a-NA), 1-chloro-2,4dinitrobenzene (CDNB), editic acid (EDTA) and p-nitroanisole (p-NA) were produced by Shanghai Reagent Factory (Shanghai, China). 1,4-Dithiothreitol (DTT) was from Shanghai Institute of Biochemistry, Chinese Academy of Sciences. Phenylmethyl sulfonyl fluoride (PMSF) was from E. Merck, Darmstsdt. 5,5 0 -Dithiobis-(2-nitrobenzoic acid) (DTNB) was from Sino-American Biotechnology Company. Fast blue RR salt, actylcholine (ATCHI), bovine serum albumin (BSA) and Coomassie brilliant blue G-250 were purchased from Fluka Chemie GmbH (Buchs, Italy). 2.3. Bioassay and synergism Technical grade insecticide and synergist were dissolved in acetone and five serially diluted concentrations were prepared so that each was one-half of the previous concentration. Ten fourth-instar larvae (9–11 mg) were treated with 1 ll of the dilution on the middle-abdomen notum by a hand microapplicator (Burkard, UK). Each test was replicated three times and the acetone treatment was used as control. In the synergism experiments, 1 ll of synergists with maximal non-lethal dosage was topically applied on each larvae about 1 h before insecticide treatment, the corresponding concentrations of PBO, TPP, DEF and DEM were set as 6.0, 10.0, 5.0 and 5.5 g/L, respectively. The test larvae were then reared individually at 28 ± 1 °C with adequate food. Mortality was assessed at 48-h posttreatment and the data were analyzed by probit analysis using the POLO program. 2.4. Enzyme assays Carboxylesterase (CarE) activity was measured according to the method described by Han et al. [19] with minor modification. Twenty larvae were homogenized in 2 ml phosphate buffer (0.02 mol/L, pH 7.0) at 0 °C. The homogenate was centrifuged at 5000g for 15 min at 4 °C. The supernatant was collected as enzyme solution. An aliquot of 0.1 ml enzyme solution was put into 0.9 ml mixed solution of a-NA and Fast blue RR salt (10 mg a-NA and 20 mg Fast blue RR salt dissolved in 2 ml acetone, and fixed cubage to 25 ml by adding phosphate buffer (0.02 mol/L, pH 7.0), then the mixed solution was filtrated), and this reacting mixture contained 105 mol/L eserin. The change rate of optical density at 450 nm was recorded with Beckman DU-650 spectrophotometer (Beckman Company, USA). Glutathione-S-transferase (GST) activity was measured with the method described by Kao et al. [20] with minor modification. Twenty larvae were homogenized in 2 ml Tris–HCl buffer (0.1 mol/L, pH 8.0) at 0 °C. The homoge-
171 Note: NT denoted no treatment. SD represented the sensitive degree of the test larvae to the toxicity of fipronil. The resistance ratio (RR) indicated that LD50 value of resistant strain was divided by LD50 value of susceptible strain. The value followed by the same letter was not significantly different (LSD test, P < 0.05).
— 0.35 0.25 0.15 0.13 0.09 (0.0032–0.0283)a (0.0281–0.0613)ab (0.0413–0.0949)b (0.0674–0.1796)bc (0.0883–0.1859)c (0.1207–0.2905)d 0.0156 0.0443 0.0633 0.1053 0.1240 0.1721 1.568 ± 0.466a 1.883 ± 0.411b 2.683 ± 0.642d 1.688 ± 0.405a 2.354 ± 0.489c 2.255 ± 0.514bc — 1.38 1.74 1.92 1.92 1.92 (0.0060–0.0089)c (0.0043–0.0064)b (0.0026–0.0057)ab (0.0019–0.0056)a (0.0019–0.0056)a (0.0019–0.0056)a 0.0073 0.0053 0.0042 0.0038 0.0038 0.0038 3.018(±0.353)c 2.557(±0.319)b 2.237(±0.258)ab 2.032(±0.478)a 2.032(±0.478)a 2.032(±0.478)a 12.45 10.04 16.82 15.57 14.75 NT
LD50 (95% FL) (lg/larva)
0.02 0.04 0.08 0.10 0.15 NT
The responses of the fourth-instar larvae of C. suppressalis to fipronil in different generations of the susceptible and resistant strains after selection are presented in Table 1. For the susceptible strain taming, LD50 value of fipronil against the fourth-instar larvae of parental generation was 0.0073 lg/larva and it decreased to 0.0038 lg/larva after three generations of selection, the susceptibility of population to fipronil increased 1.92-fold. For the resistant strain, LD50 value of parental generation was 0.0156 lg/larva and it increased to 0.1721 lg/larva after five generations of selection, their susceptibility to fipronil was decreased 11.03-fold. Thus, the resistance level of the fourth-instar larvae to fipronil increased 45.3-fold in comparison with the susceptible strain. Moreover, the slope value of regression curve suggests that the individuals of population after selection have greater heterogeneity compared with the population before selection.
0 1 2 3 5 6
3.1. Susceptibility taming and resistance selection
Survival percent (%)
3. Results and analysis
Number of larvae
All data were subjected to analysis of variance (ANOVA) and means separated using least significant difference (LSD) test. The level of significance (P) was set at 5% (SAS Institute, 1988).
Concentration (g/L)
2.6. Data analysis
Generation
Protein content was determined by the Bradford method using bovine serum albumin as the standard [22].
Table 1 Responses of the fourth-instar larvae to fipronil in the susceptible and resistant strains of C. suppressalis
2.5. Protein assay
4000 4000 1500 1000 2500 NT
Slope ± SE Slope ± SE
SD
Resistant strain selection Susceptible strain taming
LD50 (95% FL) (lg/larva)
SD
RR
nate was centrifuged at 10,000g for 15 min at 4 °C. The supernatant was collected as enzyme solution. The reaction mixture contained 0.1 ml enzyme solution, 1.4 ml Tris–HCl buffer (0.1 mol/L, pH 8.0) and 0.05 ml GSH (40 mmol/L), After preincubation for 5 min 25 °C, 0.06 ml CDNB (30 mmol/L) was added. The change rate of optical density at 340 nm was recorded with Beckman spectrophotometer. Microsomal-O-demethylase activity was measured by referring the method of Qu et al. [9] and Yang et al. [21] with minor modification. Twenty larvae were homogenized in 2 ml phosphate buffer (0.1 mol/L, pH 7.8, containing 1 mmol/L EDTA, 1 mmol/L DTT, 1 mmol/L PTU, 1 mmol/L PMSF) at 0 °C. The homogenate was centrifuged at 12,000g for 15 min at 0 °C. The supernatant was collected as enzyme solution. The reaction mixture contained 0.1 ml enzyme solution, 0.4 ml phosphate buffer (0.1 mol/L, pH 7.8) and 0.25 ml p-NA (6.0 mmol/L), After preincubation for 5 min at 34 °C, 0.25 ml NADPH (2.0 mmol/L) was added to start the reaction, and the mixture was shaken during incubation at 34 °C for 30 min. The change value of optical density at 405 nm was recorded with Beckman spectrophotometer.
2.1 8.2 15.1 27.7 32.6 45.3
X. Li et al. / Pesticide Biochemistry and Physiology 89 (2007) 169–174
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3.2. Resistance to fipronil and other insecticides The topical toxicities of fenitrothion, chlorpyrifos, monosultap, abamectin and alphamethrin to the fourthinstar larvae of the susceptible and resistant strains in C. suppressalis population are shown in Table 2. The strains of rice stem borer revealed different responses to the actions of two organophosphates, fenitrothion and chlorpyrifos, and seemed to possess higher cross-resistance to fenitrothion with 23.1-fold of RR value whilst almost no cross-resistance to chlorpyrifos (LSD test, P < 0.05). Furthermore, the strains showed lower cross-resistance to monosultap with 3.2-fold of RR value and median crossresistance to abamectin and alphamethrin with 8.9- and 7.0-fold of RR value, respectively. The results seemed to suggest that the development of resistance of the laboratory selective strain to fipronil had potentially evolved the decreased susceptibility to organophosphate and nereistoxin, even pyrethroid insecticides or abamectin, and/or the field population had developed multiple resistant properties before the laboratory selection.
3.3. Synergism of PBO, TPP, DEF and DEM The synergistic ratios (SR) of PBO, TPP, DEF and DEM on fipronil in susceptible and resistant strains of C. suppressalis are listed in Table 3. For the susceptible strain, TPP and DEF all showed no obvious synergistic action on fipronil, whereas PBO seemed to play antagonistic effect on fipronil with 0.4-fold of SR value. For the resistant strain, PBO and TPP as well as DEF exhibited stronger synergism (SR
3.02, 2.10 and 2.76), which was 7.55-, 1.93- and 2.91-fold higher than that in susceptible strain. However, no synergism of DEM on fipronil was found in both strains. These implied that microsomal-O-demethylase and esterase might be involved in fipronil resistance in the C. suppressalis population and that glutathione-S-transferase might be less important for the strains in conferring the fipronil resistance. 3.4. Activity of enzymes The activity of CarE, microsomal-O-demethylase and GST from the susceptible and resistant strains were tested in vitro, and the results are presented in Table 4. The activities of CarE and microsomal-O-demethylase of the resistant strain were 1.89- and 1.36-fold higher than that of Table 4 Activity of enzymes in susceptible and resistant strains of C. suppressalis Enzymes
Strains
Activity
R/S ratio
Carboxylesterase
S R
16.957 ± 0.516 32.032 ± 0.465
1.89bB
Microsomal-O-demethylase
S R
0.534 ± 0.062 0.726 ± 0.083
1.36abAB
Glutathione-S-transferase
S R
3.853 ± 0.350 3.872±0.053
1.00aA
Note: Units of CarE, GST and microsomal-O-demethylase were DOD450Æmin1 (mg pro)1, DOD340Æmin1 (mg pro)1, and nmolÆmin1 (mg pro)1, respectively. R/S ratio represented the enzyme activity in the resistant strain was divided by the enzyme activity in the susceptible strain. The value followed by the same letter was not significantly different (LSD test, P < 0.05 and P < 0.01).
Table 2 Responses of the fourth-instar larvae to different insecticides in the susceptible and resistant strains of C. suppressails Insecticides
Fipronil Fenitrothion Chlorpyrifos Monosultap Abamectin Alphamethrin
Susceptible strain
Resistant strain 1
RR 1
Slope ± SE
LD50 (95% FL) (lg larva )
Slope ± SE
LD50 (95% FL) (lg larva )
2.032 ± 0.478 2.529 ± 0.374 0.983 ± 0.278 2.229 ± 0.269 1.676 ± 0.197 2.222 ± 0.460
0.0038 (0.0019–0.0056) 0.8066 (0.4382–1.2925) 1.2278 (0.5512–8.6369) 3.9616 (3.1231–5.0526) 0.00036 (0.00020–0.00054) 0.0251 (0.0154–0.0389)
2.255 ± 0.514 3.832 ± 0.923 2.827 ± 0.558 1.572 ± 0.360 2.427 ± 0.483 3.206 ± 0.751
0.1721 (0.1207–0.2905) 18.594 (13.4357–24.5737) 2.0488 (1.3800–3.0432) 12.8147 (7.6215–27.1288) 0.0032 (0.0021–0.0047) 0.1751 (0.1188–0.2619)
45.3 23.1 1.7 3.2 8.9 7.0
Note: RR denoted LD50 value of resistant strain was divided by LD50 value of susceptible strain.
Table 3 Synergism of PBO, TPP, DEF and DEM on fipronil in susceptible and resistance strains of C. suppressalis Treatments
Fipronil Fipronil + PBO Fipronil + TPP Fipronil + DEF Fipronil + DEM
Susceptible strain
Resistance strain
SR2/SR1
Slope ± SE
LD50 (95%FL) (lg/larva)
SR1
Slope ± SE
LD50 (95%FL) (lg/larva)
SR2
2.032 ± 0.478 1.825 ± 0.299 5.884 ± 1.051 2.051 ± 0.479 4.045 ± 0.591
0.0038 0.0095 0.0035 0.0040 0.0038
— 0.40 1.09 0.95 1.00
2.255 ± 0.514 2.907 ± 0.568 2.533 ± 0.321 2.954 ± 0.339 2.162 ± 0.277
0.1721 0.0569 0.0819 0.0623 0.1501
— 3.02 2.10 2.76 1.15
(0.0019–0.0056)a (0.0068–0.0152)b (0.0030–0.0041)a (0.0022–0.0063)a (0.0032–0.0045)a
(0.1207–0.2905)c (0.0423–0.0791)a (0.0678–0.1023)b (0.0528–0.0745)ab (0.1217–0.1923)c
7.55c 1.93b 2.91bc 1.15a
Note: The synergistic ratio (SR) indicated that LD50 value without synergist was divided by LD50 value with synergist. The value followed by the same letter was not significantly different (LSD test, P < 0.05).
X. Li et al. / Pesticide Biochemistry and Physiology 89 (2007) 169–174 Table 5 Km and Vmax values for carboxylesterase and microsomal-O-demethylase in susceptible and resistant strains of C. suppressalis Enzymes
Strains
Km
Ratio
Vmax
Ratio
Carboxylesterase
S R
69.942 15.267
0.22
35.971 29.534
0.82
Microsomal-O-demethylase
S R
17.291 25.438
1.47
3.188 2.679
0.84
Note: Units of Km and Vmax for CarE was mmol/L and DOD450Æmin1 (mg pro)1; Units of Km and Vmax for microsomal-Odemethylase was nmol/L and nmolÆmin1 (mg pro)1. Ratio denoted that Km or Vmax of the resistant strain was divided by Km or Vmax of the susceptible strain.
the susceptible strain, respectively, whereas the activities of glutathione-S-transferase from the two strains showed no significant difference (LSD test, P < 0.05). The results substantiated that microsomal-O-demethylase and CarE played important effects in enhancing the resistance of C. suppressalis to fipronil, and the increased activity of CarE seemed to be the major inducer relative to the larval resistance. 3.5. Km and Vmax values The results of the kinetic study for CarE and microsomal-O-demethylase are shown in Table 5. The Km values of the CarE and microsomal-O-demethylase in the resistant strain were 0.22- and 1.47- fold higher than that in the susceptible strain, respectively. Data meant that the affinity of CarE increased and the affinity of microsomal-O-demethylase declined significantly in the resistant strain, indicating that the enhancing activity of CarE might be the key factor for improving the resistance. Moreover, the Vmax values of the CarE and microsomal-O-demethylase in the resistant strain were 0.82- and 0.84-fold less than that in the susceptible strain, respectively. The results suggested that the resistance to fipronil might be the modifications of CarE and microsomal-O-demethylase protein of C. suppressalis larvae and the multiple resistance background of the original Wenzhou’s population already existed to other insecticides. 4. Discussion Fipronil is high toxic to both piercing–sucking and chewing insects and has shown excellent activity against a broad spectrum of insect orders [15], and has shown no obvious cross-resistance to other action mechanism insecticides [23]. Many researches indicated that fipronil was transformed to the more toxic sulfone metabolites by cytochrome P450-mediated microsomal monooxygenase in insect [17,24,25]. However, due to increased frequency and years of use, fipronil resistance had appeared in insects. Its resistance mechanism was demonstrated as the low permeability of the cuticle, monooxygenase metabolism, and/ or the increased activity of detoxification enzymes in
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Musca domestica and Plutella oxylostella [26–28]. Recently, Goff et al. found that two site mutations in RDL subunit of GABA-receptor caused the high level of resistance to fipronil in Drosophila simula [29]. Based on these reports, we speculated that the low level of resistance was associated with the increased enzymatic detoxification, and GABAreceptor protein mutations caused the high resistance to fipronil in insects. Preliminary study on fipronil resistance in field strain C. suppressalis had been done by Jiang et al. [13]. Their synergism experiments indicated that esterase and glutathione-Stransferase played important role in the fipronil resistance of C. suppressalis. Our current study with laboratory taming strain C. suppressalis show that the role of glutathioneS-transferase may not be important because DEM is not synergistic to fipronil and the activity of glutathione-Stransferase is not significantly different between the resistant and susceptible strains. However, carboxylesterase and microsomal-O-demethylase might play important roles in fipronil resistance of C. suppressalis because TPP, DEF and PBO had significant synergistic activity to fipronil in the resistant strain. Moreover, enzyme activity assays and the kinetic experiments such as Km and Vmax also demonstrated that fipronil resistance of C. suppressalis was closely relative to the enhanced activities of carboxylesterase and microsomal-O-demethylase. As far as we know, cross-resistance is a very serious problem in insecticide resistance management. Our current study seemed to show that cross-resistance existed, more or less, among fipronil and fenitrothion and abamectin. Fortunately, the resistance mechanisms for conventional organophosphate insecticides in C. suppressalis have been extensively studied. Konno et al. had demonstrated that esterase played a critical role with different isoenzymes for resistance of C. suppressalis to organophosphate insecticides [4–6]. Han et al. and Peng et al. successively indicated that resistant mechanisms in C. suppressalis to feniteothion and methamidophos were the enhanced activity of carboxylesterase, microsomal-O-demethylase and N-demethylase as well as the insensitivity of AChE [7,8]. The same resistant mechanism was further found in C. suppressalis to triazophos and monosultap [9–11,30]. In paper, we also found that AChE activity of the resistant strain was 2.38-fold higher than that of the susceptible strain, and fenitrothion I50 ratio of R/S was 1.54 (undelivered data). So we assume that insensitive AChE is implicated in multiple resistance of the resistant strain because the original Wenzhou’s population was ever controlled by organophosphate insecticides. This might be the reason that the increased carboxylesterase activity was tangly involved in resistance mechanism. The reason that chlorpyrifos, however, in the susceptible strain is nearly as effective as in the resistant strain was the increased monooxygenase activity. Chlorpyrifos would convert into oxon derivative advantageously and its effective toxicity would become stronger through cytochrome P450-mediated monooxygenase reactions. Furthermore, although fipronil and
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abamectin have different acting sites on GABA-receptor, fipronil resistance changes the metabolism of glutamate and the function of GABA-gated chloride channels, thus might induce cross-resistance between fipronil and abamectin. From the above-mentioned, we could easily eliminate the superficial disorder that the laboratory taming strain already possessed the multiple resistance to organophosphate and fipronil, and conclude that the enhanced activity of microsomal-O-demethylase was the main inducer for fipronil resistance in C. suppressalis, glutathione-S-transferase seemed not participate in fipronil resistance. Of course, the well-defined elucidations of fipronil resistance still need the laboratory mono-resistant strain of C. suppressalis. Acknowledgments We thank Dr. V. Shyam kumar, P.G. Department of Sericulture Karnatak University, Dharwad, India for critical review and helpful discussion of the manuscript. We also thank the National Key Project for Basic Research (2003CB114402), the National Natural Science Foundation of China (30400295), the Shanghai Foundation of Science and Technology, and the Shanghai Education Commission for financial support. References [1] X.T. Li, Q.C. Huang, Z.H. Tang, An review on the development of resistant mechanism in Chilo suppressalis, World Pesticides 28 (2006) 17–20, 44. (in Chinese). [2] J.C. Fang, Z.W. Du, X.N. Cheng, Increasing harm tendency of rice stem borers and their control strategy in China, Entomol. Knowl. 35 (1998) 193–197 (in Chinese). [3] C.Y. Diao, M.T. Wang, Y.Q. Zhu, The reasons of the increase of rice stem borer in paddy field and the approaches of controlling strategies in south Jiangsu Province, Plant Prot. Tech. Ext. 21 (2001) 7–9 (in Chinese). [4] Y. Konno, T. Shishido, Resistance mechanism of the rice stem borer to organophosphorus insecticides, J. Pesticide Sci. 10 (1985) 285–287. [5] Y. Konno, N.N. Gakkaishi, Studies on resistance mechanism and synergism in the OP-resistance rice stem borer, Chilo suppressalis Walker, Agrochemical Bioregulators 14 (1989) 373–381. [6] Y. Konno, Carboxylesterase of the rice stem borer Chilo suppressalis Walker (Lepidoptera: Pyralidae). responsible for feniteothion resistance as sequesting protein, J. Pestic. Sci. 21 (1996) 425–429. [7] Q.F. Han, P.J. Zhuang, Z.H. Tang, The mechanism of resistance to fenitrothion in the rice stem borer Chilo suppressalis Walker, Acta Entomol. Sin. 38 (1995) 266–271 (in Chinese). [8] Y. Peng, C.K. Chen, Z.J. Han, Y.C. Wang, Resistance measurement of Chilo suppressalis from Jiangsu province and its resistance mechanism to methamidophos, Acta Phytophylacica Sin. 28 (2001) 173–177 (in Chinese). [9] M.J. Qu, Z.J. Han, X.J. Xu, L.N. Yue, Triazophos resistance mechanisms in rice stem borer (Chilo suppressalis Walker), Pesticide Biochem. Physiol. 77 (2003) 99–105. [10] X.F. Li, Z.J. Han, C.K. Chen, G.Q. Li, Y.C. Wang, Monitoring for resistance of rice stem borer (Chilo suppressalis Walker) to 4 conventional insecticides, J. Nanjing Agric. Univ. 24 (2001) 43–46 (in Chinese).
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