(AChE) in insecticide resistance based on the differential inhibition by ..... We thank Cheminova Agro (Wayne, New Jersey, USA and. Lemvig, Denmark) for ...
JAE 122 (1998) J. Appl. E11t. 122.269-273 (1998) < 1998, Blackwell Wisseiischafts-Verlag, Berlin ISSN 093 1-2048
Altered acetylcholinesterase associated with organophosphate resistance in Rhyzoperfha dominica (F.) (Col., Bostrichidae) populations from Brazil and the United States R. N. C. Guedes, K. Y. Zhu and S. Kambhampati Department of Entomology, Kansas State University, Manhattan, KS 66506, USA
Abstract: Microplate assay format was used to examine the susceptibility of acetylcholinesterase (AChE) to inhibition by malaoxon and chlorpyrifos-methyl oxon in individual adult insects of an organophosphate susceptible and two resistant populations (Marshall and Uberlindia) of Rhysopertha dorninica (F.).AChE from these resistant populations was significantly less sensitive to inhibition by malaoxon, but not by chlorpyrifos-methyl oxon, suggesting that other resistance mechanisms might also contribute to the overall resistance, at least to chlorpyrifos-methyl. The remaining AChE activities in the presence of 1 0 - 4 malaoxon ~ were 22.8 and 20.3% in the Marshall and Uberlfindia populations, respectively, whereas the remaining activity in the susceptible population was 18.9%. Both resistant populations showed about 27% of remaining activity in the presence of lo-' M chlorpyrifos-methyl oxon, whereas the remaining activity in the susceptible population was 26.1 Yo.One cycle of selection of the resistant populations with chlorpyrifos-methyl decreased the sensitivity to chlorpyrifos-methyl oxon (z3%), but increased their sensitivity to malaoxon (3-7%), suggesting that selection with chlorpyrifos-methyl might result in deselection for resistance to malaoxon in these populations. Such effects on the sensitivity of AChE by different organophosphate selections suggest that different modifications in AChE structure might have occurred in these resistant populations of R. dominica.
1 Introduction
of the involvement of altered acetylcholinesterase (AChE) in insecticide resistance based on the differential Chemical control is one of the most frequently used inhibition by malaoxon (GUEDESet al., 1997a). The methods for stored-product insect management in storobjectives of the present study were to examine the age facilities. Malathion has been used for postharvest frequency of the altered-AChE phenotypes, and to insect control in the United States since the late 1950s investigate the relationship of malaoxon and chlorwhereas pirimiphos-methyl and chlorpyrifos-methyl pyrifos-methyl oxon resistance with respect to altered have been used as grain protectants since mid-1980s R . dominica. AChE from (GUEDESet al., 1996). Frequent use of insecticides in storage facilities resulted in the development of resistance to these chemicals in stored-product insects and control failures due to resistance have been reported in various countries (CHAMPand DYTE, 1976; BADMIN, 2 Material and methods 1990; GUEDES, 1990a; GUEDES, 1990b; SUB- 2.1 Insects RAMANYAM and HAGSTRUM, 1996). A standard laboratory colony of R. dominica has been mainIt appears, however, that the rate of development of tained in the Laboratory of Stored-Product Insects, Departresistance in field populations of stored-product insects ment of Entomology, Kansas State University, Manhattan is relatively slow. Malathion resistance in the lesser (Kansas, USA), and was used as source of susceptible insects grain borer, Rhyzopertha dominica (F.) (Col., Bos- for comparisons. Insecticide resistant populations of R. domtrichidae), in Brazil and the United States usually does inica were collected from Uberlfindia County (Minas Gerais, not exceed the 10-fold level, whereas resistance to pir- Brazil) and Marshall County (Kansas, USA) and maintained imiphos-methyl and chlorpyrifos-methyl were less than in laboratory without insecticide selection. These two popu10-fold and 100-fold, respectively (GUEDESet al., 1996). lations have previously been shown to be resistant to malaSuch a slow rate of resistance development in stored- thion, pirimiphos-methyl, and chlorpyrifos-methyl (GUEDES product insects probably attributes to the characteristics et al., 1996) and both of them showed some degree of insensitivity of AChE to inhibition by malaoxon (GUEDES et al., of stored-product ecosystems (e.g. restricted insecticide 1997a) and increased phosphotriesterase activity (GUEDES et applications and the periodic turnovers of commodities al., 199713). Two more R. dominica populations used in this in a facility) (SUBRAMANYAM and HAGSTRUM, 1996). study, Uberlgndia- 1 and Marshall- 1, were established from In a recent survey of biochemical mechanisms of the survivors of UberlZndia and Marshall, respectively, after resistance in field-collected populations of R . dominica one cycle of selection with chlorpyrifos-methyl at LC9, from Brazil and the United States, we provided evidence (GUEDES et al., 1996). The individual insect populations were u. S. Copyright Clearance Center Code Statement:
093 1-2048/98/22054269 $ 14.00/0
R. N . C. Guedes et al.
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reared on whole wheat under constant conditions of 25 and 65 i 5% RH.
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2.2 Chemicals Technical grade oxon analogs of malathion, pirimiphosmethyl, and chlorpyrifos-methyl were supplied by Cheminova Agro (Lemvig, Denmark), DowElanco (Indianapolis, USA), and Zeneca Agrochemicals (Bracknell, UK), respectively. Triton X-100, 5,5'-dithiobis (2-nitrobenzoate) (DTNB), and acetylthiocholine iodide (ATC) were purchased from Sigma Chemical Co. (St. Louis, Missouri, USA).
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a 2.3 Measurement of AChE activity AChE activity was measured based on the method of ELLMAN et al. (1961) using ATC as substrate. Enzyme inhibition by organophosphate compounds was determined in the presence of substrate (ZHU et al., 1996). To minimize the possible degradation of the insecticide inhibitors in the reactions, relatively high concentrations of inhibitors and short reaction time were used. Measurements were made at ambient temperature in a V,, kinetic microplate reader (Molecular Devices Corp., Menlo Park, CA) accomodated with SOFT,,, software. Random samples of adult insects of each population were collected and immediately frozen at - 70°C. For the susceptible, UberlBndia, and Marshall populations of R . dominica, 12 nonsexed adults were homogenized in 7.2ml of ice-cold 0.1 M phosphate buffer (pH 7.5) containing 0.3% (v/v) Triton X-100. The insect homogenate was centrifuged at 10000 g,, for 15min at 4°C. About 3.0ml of the supernatant was collected and transferred to new vials as enzyme source. Diluted insect homogenate (50 pl) and acetone solution with or without insecticide (10 pl) were equilibrated in a flat-bottom microtitre plate at room temperature, with eight replicates for each insect homogenate. Assays were started by adding 90 pl of 0.1 M phosphate buffer (pH 7.5) containing DTNB and ATC. The final concentration was 0.4mM for DTNB and 0.5 mM for ATC. Assays were run for a maximum of 15 min at room temperature ( z 25"C), but only data at 5 min was used in the analysis since they provided better fit in the linear regressions provided by SOFT,,, program. For single-insect assays, individual R. dominica adults were homogenized in 0.6 ml of ice-cold 0.1 M phosphate buffer (pH7.5) containing 0.3% (v/v) Triton X-100, and three inhibition assays (i.e. without inhibitor, with lop4M malaoxon, and with 10-6M chlorpyrifos-methyl oxon) were run simultaneously. The mean remaining (i.e. uninhibited) activity in each inhibition assay was expressed as percentage remaining activity. Data was analyzed by using the procedures PROC REG and PROC COR in SAS (SAS INSTITUTE, 1987).
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3 Results As expected, the remaining AChE activity of R. dom0.01 0.1 1 10 100 inica populations declined with increase of insecticide Concentration of chlorpyrifos-methyl oxon (pM) concentration (fig. 1). The greatest difference between susceptible and resistant populations of R. dominica Fig. 1. Inhibition of AChEfrom R. dominicapopulations were obtained with l O P 4of~ malaoxon and 10-6M of by malaoxon, pirimiphos-methyl oxon, and chlorpyrifoschlorpyrifos-methyl oxon. Therefore, these con- methyl oxon. The degree of inhibition is expressed as a centrations were used to discriminate between insec- percentage of the remaining activity of AChE ( f SE) ticide-sensitive and insensitive AChE variants in individual insects. Similar to the reports of BONNINGand HEMINGWAY (1991) on the low efficiency of inhibition to AChE by pirimiphos-methyl oxon in mosquito oxon did not provide strong inhibition to AChE from [Culex pipiens (L.)] populations, pirimiphos-methyl the lesser grain borer. Therefore, the establishment of
27 1
Insensitive acetylcholinesaterase in R. c/on~Ozic.o Populations
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Fig. 2. Acetylcholinesterase (AChE) activity (mean S E ) of R. dominica populations without the presence of inhibitors ( A ) and in the presence of lop4M malaoxon (B) and 10- M chlorpyrifos-methyl oxon ( C ) . AChE activity in absence of inhibitors is expressed as the slope of a linear regression of absorbance readings on time. Remaining AChE activity (%) in the presence of inhibitors was calculated as mean inhibited activity x 100/activity. Means followed by the same letter within a graphic do not differ significantly at 5% probability by Duncan’s multiple range test
a reliable discriminating concentration of pirimiphosmethyl oxon for resistant phenotype identification was not possible. AChE activities and remaining activities (%), estimated using both inhibitors for each R. dominica population, are presented in fig. 2. The field-collected resistant populations (i.e. Marshall and Uberlilndia) had lower AChE activities as compared with the susceptible population, but showed an increase in AChE activity after one cycle of selection with chlorpyrifos-methyl (fig. 2A). In contrast, the susceptible population showed lower remaining activity than the field-collected populations when only malaoxon was used to inhibit the eazyme activity (fig. 2B,C). The resistant populations after selection with chlorpyrifos-methyl showed an increase in remaining activity with chlorpyrifos-methyl oxon and a decrease in remaining activity with malaoxon (fig. 2). Discrimination of phenotypes in all five populations of R. dominica was carried out using malaoxon (fig. 3a) and chlorpyrifos-methyl oxon (fig. 3b) as AChE inhibitors. The field-collected resistant populations had more individuals with less sensitive AChE towards malaoxon than the susceptible population, but did not differ from the susceptible sample in AChE sensitivity to chlorpyrifos-methyl oxon, which agreed with fig. 2. Bivariate plots of percentage remaining AChE activity in the presence of malaoxon or chlorpyrifos-methyl oxon suggested a possible cross-insensitivity of AChE to these organophosphate compounds (fig. 4), because they show a positive correlation between the percentage remaing activity of AChE in the presence of malaoxon or chlorpyrifos-methyl oxon. The results of the Marshall and Marshall-l populations were similar to those from the Uberliindia and UberlAndia-I populations,
respectively; therefore only the results from latter populations were shown.
4 Discussion The field-collected populations of R. dominica showed low levels of resistance to malathion (6.9- and 9.1-fold for Marshall and Uberliindia, respectively) and moderate levels to chlorpyrifos-methyl (22.6- and 70.1-fold for Marshall and Uberliindia, respectively) (GUEDESet al., 1996). Results from this study indicated that these resistant populations possessed AChE less sensitive to inhibition by malaoxon. The percentage remaining activity of AChE in the presence of 1 0 - 4 malaoxon ~ was significantly higher in the resistant populations than in the susceptible population. However, there was no significant difference in percentage remaining activity between the susceptible and resistant populations with respect to the inhibition of AChE by chlorpyrifosmethyl oxon. Therefore, chlorpyrifos-methyl resistance was probably due to other mechanism(s). The levels of insensitivity of AChE to malaoxon in the resistant populations were relatively low. However, the effect of this target site insensitivity can be important since it can be potentiated by other resistance mechanisms, especially metabolic ones (ZHU and BRINDLEY, 1992). Our previous study indicated that increased phosphotriesterase activity appeared to play an important role conferring organophosphate resistance in these resistant populations (GUEDES et al., 1997b). The combination of these resistance mechanisms is likely to be copotentiation, resulting in a relatively high resistance. A low degree of AChE insensitivity was also reported in Drosophila melanogaster Merger (Dipt., Dro-
R . N . C. Guedes et al.
212 60
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Fig. 3. Frequency distributions of remaining AChE activity (%) in R. dominica populations subjected to in vitro inhibition with ( A ) l O U 4 malaoxon ~ and (B) 1 0 - 6 chlorpyrijos-methyl ~ oxon. Distributions were split into 5% intervals
sophilidae), where different alterations in AChE responsible for low degrees of insensitivity to insecticides could lead to high levels of insecticide resistance (FOURNIER et al., 1992; PRAVALORIO and FOURNIER,1992). Correlations between insensitivity to malaoxon and insensitivity to chlorpyrifos-methyl oxon suggested a weak cross-insensitivity of AChE to both insecticides that tends to be weakened even more with further selection with chlorpyrifos-methyl. As expected, further selection of the field populations with chlorpyrifosmethyl led to an increase of the frequency of individuals that were more resistant to chlorpyrifos-methyl oxon as compared with the susceptible and the original field populations of R. dominica. This was associated with a decrease of the mean AChE sensitivity to inhibition by chlorpyrifos-methyl oxon and an increase of AChE sensitivity to inhibition to malaoxon. These results imply that further selection of these populations with chlorpyrifos-methyl might have resulted in deselection of different AChE alleles which encode AChE with
different responses to inhibition by different organophosphates. These findings suggest that different modifications in AChE structure might have developed independently in the resistant populations of R. dominica in response to different insecticides. If this is the case, rotation of different organophosphates may be a helpful tactic in resistance management programs for this insect species.
Acknowledgements We thank Cheminova Agro (Wayne, New Jersey, USA and Lemvig, Denmark) for supplying malaoxon, DowElanco (Indianapolis, Indiana, USA) for supplying chlorpyrifosmethyl oxon, and Zeneca Agrochemicals (Bracknell, UK) for supplying pirimiphos-methyl oxon. Financial support was provided by Coordenaqgno de AperfeiGoamento de Pessoal de Nivel Superior-CAPES/Brazilian Ministry of Education and by seed grants from the K.S.U. Department of Ento-
273
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mology. This manuscript is contribution no. 97-22-5 from the Kansas Agricultural Experiment Station.
References BADMIN,J. S., 1990: IRAC survey of resistance of stored grain pests: results and progress. In: 5th international
working conference on stored-product protection. Ed. by F.; DUCOM.P. Bordeaux: Institut FLEURRAT-LESSARD, National de la Recherche Agronomique, 973-981. BONNING,B. C.; HEMINGWAY, J., 1991: The efficacy of acetylcholinesterase in organophosphorus and carbamate resistance in Culexpipiens L. from Italy. Pestic. Biochem.. Physiol. 40, 143-148. CHAMP,B. R.; DYTE,C. E., 1976: F A 0 global survey of pesticide susceptibility of stored grain pests. Rome: FAO/UN. ELLMAN, G. L.; COURTNEY, K. D.; ANDRES,V. JR; FEATHERSTONE, R. M., 1961: A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7,88-95. FOURNIER, D.; BRIDE,J.-M.; HOFFMAN,F.; KARCH,F., 1992: Acetylcholinesterase: two types of modifications confer resistance to insecticides. J. Biol. Chem. 267, 14270-1 4274. GUEDES,R. N. C., 1990a: Resisttncia a inseticidas: desafio para o controle de pragas de grlos armazenados: Seiva 50, 24-29. -, 1990b: Manejo integrado para a proteglo de grlos armazenados contra insetos. Rev. Bras. Armaz. 15, 3 4 8 . -; DOVER,B. A.; KAMBHAMPATI, S., 1996: Resistance to chlorpyrifos-methyl, pirimiphos-methyl, and malathion in Brazilian and U.S. populations of Rhyzopertha dominica (Coleoptera: Bostrichidae). J. Econ. Entomol. 89, 27-32. -; KAMBHAMPATI, S.; DOVER,B. A.; ZHU, K. Y., 1997a: Biochemical mechanisms of organophosphate resistance in Rhyzopertha dominica (Coleoptera: Bostrichidae) from the United States and Brazil. Bull. Entomol. Res. 87,581586. -; ZHU, K. Y.; DOVER,B. A.; KAMBHAMPATI, S., 1997b: Partial characterization of phosphotriesterase from organophosphate-susceptible and resistant Rhyzopertha dominica (Coleoptera: Bostrichidae). Pestic. Biochem. Physiol. 57, 156164. M.; FOURNIER,D., 1992: Drosophila acePRALAVORIO, tylcholinesterase: characterization of different mutants resistant to insecticides. Biochem. Genet. 30, 77-83. SAS INSTITUTE,1987: SAS user’s guide: statistics, version 5.15. Cary: SAS Institute. SUBRAMANYAM, BH.; HAGSTRUM, D. W., 1996: Resistance measurement and management. In: Integrated management of insects in stored products. Ed. by SUBRAMANYAM, BH.; HAGSTRUM, D. W. New York: Marcel Dekker, 331-397. ZHU, K. Y.; BRINDLEY,W. A,, 1992: Significance of carboxylesterase and insensitive acetylcholinesterase in con~ ferring organophosphate resistance in Lygus hesperus populations. Pestic. Biochem. Physiol. 43, 223-23 1. -; LEE, S. H.; CLARK,J. M., 1996: A point mutation of acetylcholinesterase associated with azinphosmethyl resistance and reduced fitness in Colorado potato beetle. Pestic. Biochem. Physiol. 55, 100-108. Authors’ addresses: Dr R. N. C. GUEDES,Departamento de Biologia Animal, Universidade Federal de Vigosa, Vigosa, Minas Gerais 36571.000, Brasil. Dr K.Y. ZHU(corresponding author), Dr S. KAMBHAMPATI, Department of Entomology, 123 Waters Hall, Kansas State University, Manhattan, Kansas 66506, U S A .
