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Esterase Activity and Resistance to Organophosphorus Insecticides in Larvae of Oryzaephilus surinamensis (L) (Coleoptera: Silvanidae)
J. Asia-Pacific Entomol. 5 (2) : 161-166 (2002) www.entomology.or.kr
Esterase Activity and Resistance to Organophosphorus Insecticides in Larvae of Oryzaephilus surinamensis (L.) (Coleoptera: Silvanidae) 1
2
Eun-Kee Park, Won II Choi and Sung-Eun Lee * Department of Epidemiology and Preventive Medicine, School of Medicine, University of California, Davis, California 95616, USA 'Department of Agricultural Biology, Seoul National University, Suwon 441-744, Korea 2Department of Agricultural Chemistry, Kyungpook National University, Daegu 702-701, Korea
Abstract Acetylcholinesterase and carboxylesterase activities of four strains of Oryzaephilus surinamensis (L.) larvae were examined. The three strains of VOS49, VOSF, and VOSCM were resistant to malathion, fenitrothion, and chlorpyrifos-methyl, respectively, while VOS48 was a susceptible strain. Cross-resistance to all three organophosphate insecticides tested was confirmed in larvae of the three resistant strains. Acetylcholinesterase activity was not correlated to organophosphate resistance in those resistant strains. VOS49, VOSF and VOSCM showed enhanced levels of carboxylesterase activity based on p-nitrophenyl acetate, a.-naphthyl acetate, or p-naphthyl acetate substrates. However, these activities were significantly lower than those of 0. surinamensis adults. PAGE zymograms showed major differences in carboxylesterase band pattern among strains. VOS49 carboxylesterase banding pattern was significantly different from other two resistant strains. Interestingly, these larval esterase band patterns were very similar to those of 0. surinamensis adults. Therefore, carboxylesterase activity of 0. surinamensis plays important role in resistance to organophosphate insecticide from larval stage.
Key words Oryzaephilus surinamensis, resistance, organophosphate insecticide, carboxylesterase
Introduction The use of chemical protectants to control insect pests in stored grain is widespread. This control is largely dependent on the concentration of insecticide to which the stored grain insect pests are exposed. *Corresponding author. E-mail: [email protected] Tel: 82-53-950-7751; Fax: 82-53-953-7233 (Received April 10, 2002; Accepted August 21, 2002)
Two critical factors affect the efficacy of control: Temperature and humidity. Generally, higher temperatures increase the degradation of insecticides. However, this loss is balanced by greater toxicity of a fixed insecticide concentration at higher temperatures (Tyler and Binns, 1982). Variations in humidity are thought to alter both the chemical and biological metabolism of insecticides (Barson, 1983). In Australia, organophosphorus insecticides have been widely used to protect stored grain from insect pests. The site of action of organophosphate insecticides is a key enzyme ofthe insect central nervous system, acetylcholinesterase (AChE) which destroys acetylcholine functioning in the synaptic transmission of nerve impulse. Organophosphate insecticides react with AChE in a similar way to acetylcholine. However, the phosphorylated AChE derivative is much more stable than the acyl derivative and results in clogging of the electric transmission circuit, leading to convulsions, paralysis, and finally to death. Many grain storage insect pests in Australia including Oryzaephilus surinamensis (L.), a major worldwide insect pest of stored grains, have developed resistance initially to malathion, later to fenitrothion and now to chlorpyrifos- methyl (Collins et al., 1993; Herron et al., 1996; Wallbank, 1996). Attia and Frecker (1984) and Subramayam et al. (1989), obtained indications of the possible mechanisms involved in organophosphate insecticide resistance through the use of synergists. Attia and Frecker (1984) showed that piperonyl butoxide (PBO, a cytochrome P450-dependent monooxygenase inhibitor) and S,S,S,-tributyl phosphorothioate (DEF, an carboxylesteraseinhibitor)decreasedresistanceto fenitrothion by up to 36-fold and 14-fold, respectively, in New South Wales strains of organophosphate resistant 0. surinamensis. However, the authors found that those synergists reduced resistance to malathion and dichlorvos by less than 3-fold indicating a more significant role for monooxygenases and esterases in fenitrothion resistance to malathion dichlorvos.
162 J. Asia-Pacific Entomol. Vol. 5 (2002)
Subramanyam et al. (1989) found similar phenomenon that increased mortality of chlorpyrifos-methyl-tolerant 0. surinamensis using PBO and DEF indicated that monoxygenases and esterases could be involved in resistance of this species to chlorpyrifos-methyl, Lee et al. (2000) and Lee and Lees (2001) have found enhanced activities of monoxygenases and carboxylesterases are the principal biochemical mechanisms to confer malathion, fenitrothion and chlorpyrifos-methyl resistance to 0. surinamensis. An interesting study conducted by Milson (1995) have shown that when individual field strains of 0. surinamensis with low resistance to fenitrothion and chlorpyrifos-methyl are selected with these insecticides, the relationship between resistance to chlorpyrifos-methyl and esterase activity is much greater than the relationship between resistance to fenitrothion and esterase activity. These results are very similar to those of Collins et al. (1992). However, these all studies are conducted using only adults of resistant strains. The aim of this study is to examine the possible role of CE and AChE in organophosphate insecticide resistance in larvae of 0. surinamensis.
Materials and Methods Chemicals p-Nitrophenyl acetate (PNPA), a-naphthyl acetate (a -NA) , ~-naphthyl acetate (~-NA), acetylthiocholine iodide (ATChI), 5,5-dithio-bis-(2-nitrobenzenoic acid) (DTNB), a-naphthol, ~-naphthol, p-nitrophenol, Fast Blue B salt (o-diansidine, tetrazotized zinc chloride complex), and tris(hydroxymethyl)-aminomethane (Tris) were all purchased from Sigma (St. Louis, MO). Polytetrafluoroethylene (Fluon) was from ICI Fluoropolymers (Exton, PA). Malathion (91.9% [AI]), fenitrothion (98.6% [AI]) and chlorpyrifos-methyl (94% [AI]) were supplied by Dowelanco (Sydney, Australia). All chemicals were of highest grade commercially available.
Insects Four strains of the saw-toothed grain beetle, 0. surinamensis, were supplied by P. 1. Collins (Department of Primary Industry, Queensland, Australia). VOS48, the organophosphorus insecticide-susceptible strain, has been in continuous laboratory culture since 1973. VOS49 is a malathion-resistant strain collected from the field before introduction of fenitrothion and chlorpyrifos-methyl. It has been maintained under
Eun-Kee Park, Won 11 Choi, Sung-Eun Lee
laboratory culture in .Queensland at 28 °C under malathion selection. VOSF and VOSCM are the composite field strains selected with fenitrothion and chlorpyrifos-methyl, respectively, and maintained under laboratory culture in Queensland at 28 °C under fenitrothion and chlorpyrifos-methyl selection, respectively.
Determination of resistance to organophosphorus insecticides Resistance was assessed by the standard FAO impregnated filter paper assay method (Champ and Dyte, 1976). LD so values of insect strains were determined using malathion, fenitrothion and chlorpyrifos-methyl. Insecticides were dissolved in a mixture of ondina oil: acetone: petroleum ether (1: 1:3, vol:vol). Different concentrations of insecticide solution (0.5 ml) were applied to 7 cm diameter Whatman No.1 filter paper (Whatman, Hillsboro, OR) and solvents were allowed to evaporate. Treated filter papers were then placed in aluminum foil lined PVC trays (80 by 330 by 420 mm) and an aluminum ring (4.3 em i.d. and 2.9 cm high) coated with Fluon was placed on top of each filter paper. Three batches of 40 larvae were exposed to a graded series of four concentrations of malathion, fenitrothion and chlorpyrifos-methyl for 24 h at 28 °C. Exposed larvae were transferred to small vials, containing 1 g rolled oats, for 48 h, and survivors were counted. The LD so and LD 9s values were calculated by Probit analysis (Finney, 1971). Control mortality was accounted for by Abbott's formula (Abbott, 1925). Resistance factors were calculated as the ratio ofLD so and LD 9s values for a resistant strain compared with the susceptible strain, VOS48. Insecticide concentrations and LD so and LD9s values are reported as percent (wt:vol) insecticide in ondina oil.
Preparation of extracts for enzyme assays and determinaton of carboxylesterase and acetylcholinesterase activities Two hundred milligrams of larvae from each of the four strains of insect were homogenized in a pestle and mortar with 3.0 ml of 10 mM Tris-HCl buffer, pH 7.5, containing 0.5% (wt:vol) Triton X-lOO. The homogenateswere filtered through Miracloth (Calbiochem, San Diego, CA) to remove exo-skeletal and tissue debris, and centrifuged at 12,000 g for 20 min using a Sorvall (Kendro Laboratory Products, Newtown, CT) superspeed RC-2B centrifuge. The supernatants were reserved as crude enzyme extracts. Enzyme activities were examined by the method of Lee and Lees (2001).
Esterase Activity and Resistance to OP Insecticides in Oryzaephilus surinamensis
Polyacrylamide gel electrophoresis Native polyacrylamide gel electrophoresis (PAGE) was conducted using a Bio-Rad (Hercules, CA) miniprotein II dual vertical slab cell system and 7.5% acrylamide (wt.vol) gels at pH 8.5 with stacking gels of 3% acrylamide (wt:vol). Samples of carboxylesterase in 20% (wt:vol) sucrose containing bromothymol blue were applied to stacking gels. Electrophoresis was performed at 100 V for 10 min, voltage increased to 200 V and a maximum current 2.5 rnA and continued for about 2 h until the marker dye neared the bottom of the gel. Gels were stained for carboxylesterase as described by Devonshire (1977) with a solution made by adding 30 mM a-NA or 0-NA in acetone (1 ml) to 0.2% (wt:vol) Fast Blue RR salt in 0.2 M phosphate buffer, pH 6.0 (50 ml).
and chlorpyrifos-methyl. PAGE zymograms for larvae ofVOS48, VOS49, VOSF and VOSCM show differences between the strains in band position and in intensity of dye staining. Levels of AChE activity were not significantly different among the four strains (Table 3). However, these levels of AChE in larvae of 0. surinamensis were approximately 4 times lower than those in adults of the insects reported previously (Lee and Lees, 2001). The Michaelis constant (Km) values for ATChI ofthe four strains are not significantly different (Table 3). Interestingly, Km values for ATCW in larvae of 0. surinamensis are almost identical to those in adults of the insects.
Statistical analysis
vas
Strains 48.9F
CM4849
FCM
..- -
Laboratory data on the evaluation of different rates of enzymes were subjected to analysis of variance (ANOVA) followed by Students t-test (a = 0.05) (SAS Institute 1995).
• ••• -...
(I-NA
+
Results LD so values obtained for the three organophosphate insecticides malathion, fenitrothion and chlorpyrifosmethyl for the larvae of the four strains of 0. surinamensis and the resistance factors (RF value) for each resistant strains relative to the larvae of VOS48 are shown in Table 1. The three resistant strains showed cross-resistance to the insecticides tested relative to VOS48. The VOS49 strain had high RF values for LDso or LD9s values to fenitrothion and chlorpyrifos-methyl as well as to malathion. Similar high cross-resistance was found in other two resistant strains based on RF values for both LDso and LD 9s. Levels of CE activity using three substrates are elevated in the three resistant strains of 0. surinamensis relative to VOS48 (Table 2). For all strains the rate of hydrolysis for PNPA was 1.44-2.97 times higher than a-NA or 0-NA. Therefore, the larvae of O. surinamensis may be favorable for hydrolysis of PNP A than other two substrates used in this study. All strains had significantly different CE activity for the three CE substrates. VOSCM had 4.8-7.8 times higher CE activity than VOS48 (Table 2). The results using PAGE (Fig. 1) showed an interesting CE role of 0. surinamensis in the resistance to the three organophosphorus insecticides malathion, fenitrothion
163
~-NA
-. •••
__ <:==A
- ...
Fig. 1. Native polyacrylamide gel electrophoresis (PAGE) of crude extracts of larvae of the four strains of Oryzaephilus surinamensis stained for carboxylesterase activity with a-NA or B-NA and 0.1% Fast Blue RR salt. Standard 15 III aliquots (equivalent to 0.6 mg tissue) were applied to the gels. Zymograms for carboxylesterase activity against a-NA of adults of fours strains of 0. surinamensis were already reported to Lee et al. (2000) and Lee and Lees (200 I). VOS represents Victorian Oryzaephilus surinamensis. VOS48 is an insecticide susceptible strain. VOS49 is a malathionresistant strain collected before the introduction of fenitrothion and chlorpyrifos-methyl. VOSF and VOSCM strains are composite field strains selected with fenitrothion and chlorpyrifos-methyl. 'A' in the figure represents an isozyme of carboxylesterase conferring resistance in the insects against the organophosphorus insecticides and is the enzyme already purified as shown in the previous report Lee et al. (2000).
164 J. Asia-Pacific Entomol. Vol. 5 (2002)
Eun-Kee Park, Won 11 Choi, Sung-Eun Lee
Table 1. LD50 values for larvae of four strains of Oryzaephilus surinamensis in response to malathon, fenitrothion and chlorpyrifos-methyl. Numbers of larvae tested are 480 for all strains Strain
LD 50 (95% FL), gr
1a
LD 95 (95% FL), gr 1a
RF
Malathion VOS48
0.0060 (0.0025-0.0136)
1
0.079 (0.026-5.77)
1
VOS49
0.041
(0.036-0.046)
6.8
0.18
2.3
VOSF
0.026
(0.017-0.046)
4.3
0.096 (0.052-0.62)
1.2
VOSCM
0.087
(0.074-0.10)
14.5
0.64
8.1
(0.14-0.25)
(0.46-1.02)
Fenitrothion VOS48
0.00040 (0.00035-0.00045)
1
0.0013 (0.0010-0.0018)
1
VOS49
0.0019
(0.0010-0.0057)
2.5
om I
(0.0045-2.25)
8.5
VOSF
0.0097
(0.008-0.011)
24.2
0.084
(0.057-0.14)
64.6
VOSCM
0.031
(0.014-0.033)
77.5
0.29
(0.080-1.37)
223
Chlorpyrifos-methyl VOS48
0.00028 (0.00015-0.00050)
1
0.0011 (0.00057-0.0047)
1
VOS49
0.00068 (0.00055-0.00083)
2.4
0.0086 (0.0058-0.015)
7.8
VOSF
0.0017
VOSCM
0.023 (0.019-0.028) gr' of ondina oil.
'LD,. values are in
(0.0013-0.0022)
6.1
0.033
(0.018-0.076)
82.1
0.21
(0.15-0.33)
30.0 191
'Resistance factor (RF) for the two resistant strains have been calculated relative to tbe susceptible strain, VOS48.
Discussions The larvae of the three strains of 0. surinamensis selected for resistance to malathion, fenitrothion and chlorpyrifos-methyl were shown to be resistant to all three organophosphate insecticides relative to YOS48. The degree of cross-resistance was different in the three strains (Table 1). The VOS49 strain is malathionresistant strain collected before fenitrothion and chlorpyrifos-methyl were used in Australia. It shows some resistance to fenitrothion and chlorpyrifosmethyl, althoughit has never been exposedto fenitrothion and chlorpyrifos-methyl. Interestingly, adults of the VOS49 strain had RF values to malathion, fenitrothion and chlorpyrifos-methyl of 16, 24 and 4, respectively (Lee and Lees, 2001). These data have showed that the malathion-resistant strain had different RF values with their growth stages to fenitrothion resistance. However, the other two organophosphates malathion andchlorpyrifos-methyl showedsimilarRF valueswithout significant differences between larvae and adults. Larvae of the fenitrothion-resistant strain (VOSF) of
0. surinamensis had high RF value to fenitrothion. However, it did not show strong cross- resistance to both fenitrothion and chlorpyrifos- methyl. These larval results are identical to the results of Lee and Lees (2001), where adults of VOSF strain had RF values for malathion, fenitrothion, and chlorpyrifosmethyl of 5, 14, and 5, respectively. Obviously, the fenitrothion resistance in 0. surinamensis is not related to malathion and chlorpyrifos-methyl resistance. On the other hand, larvae of VOSCM strain had RF values to malathion, fenitrothion, and chlorpyrifos- methyl of 14.5, 77.5, and 82.1, respectively compared with values of 23, 63, and 30 for three insecticides. These results showed that cross-resistance to chlorpyrifosmethyl and malathion in 0. surinamensis were found, but the relative levels of cross-resistance of chlorpyrifosmethyl and fenitrothion is strong. Milson (1995) has described experiments in which field strains of 0. surinamensis with some resistance to chlorpyrifosmethyl and fenitrothion were selected for several generations with chlorpyrifos-methyl and fenitrothion. There was reasonable correlation (R=0.71) between resistance to fenitrothion and resistance to chlorpyrifos-
Esterase Activity and Resistance to OP Insecticides in Oryzaephilus surinamensis
165
Table 2. Carboxylesterase (CE) activities of larvae of the three strains of Oryzaephilus surinamensis CE activity substrate
Strain PNPA
a-NA
.B -NA
VOS48
5.61 ±0.14(1.0)a
2.43 ±0.15(1.0)a
2.20±0.15(1.0)a
VOS49
8.17±0.47(1.5)b
2.75± 1.27(l.l)b
3.14±0.14(1.4)b
VOSF
10.4 ±0.17(1.9)c
5.72±0.20(2.4)c
5.01 ±0.17(2.3)c
VOSCM
27.2 ±1.02(4.8)d
18.9± 1.28(7.8)d
16.5± 1.09(7.5)d
CE activities are expressed as mmol substrate (p-nitrophenyl acetate, PNPA; a-naphthyl acetate, a-NA; l3-naphthyl acetate, I3-NA) hydrolyzed minlg larvae", Numbers in parentheses indicate the ratio of activity in the resistant strains in comparison with the susceptible strain, VOS48. The data (mean ± SE values) were determined from six replicates. Means in the column followed by the same letter are not significantly different (a=0.05, ANOVA).
methyl. The results presented in Table 2 show the levels of CE activity are elevated in all cases relative to VOS48. Adults of the three resistant strains had 1.5to 2.5-fold higher levels ofCE activity than the larvae. There was no difference in CE activity in adults and larvae of VOS48, the standard susceptible strain. These results indicate that the amount of one or more CE isozymes may increase during growth. Esterases have been shown to act in resistance mechanisms in two ways. The actual increase in hydrolysis of an insecticide to produce a less toxic compound is one mechanism, as suggested by Mackness et al. (1983) and Beeman and Schmidt (1982). In this situation, increased hydrolysis of the carboxylester in malathion by a CE isozyme is consistent with resistance to malathion. Fenitrothion and chlorpyrifos-methyl do not contain a carboxylester group, so detoxification by CE hydrolysis is not likely to have a major role in resistance. In this case, CE may bind, and thus be inhibited by organophosphate insecticides as described by Devonshire (1977). It is possible that these two mechanisms may operate in 0. surinamensis resistance to organophosphates. The role of CE activity in resistance to chlorpyrifos-methyl suggested by Subramanyam et al. (1989) may be in binding chloryrifos-methyl or chlorpyrifos-methyl oxon as this insecticide does not contain a carboxylester group. Lee et al. (2000) purified a major CE isozyme, designated as A, from adults of VOSF strain of 0.
surinamensis conferring resistance to malathion, fenitrothion and chlorpyrifos-methyl. This enzyme had 130 kDa molecular mass and contained two subunits of 65 kDa. With kinetic studies of this purified CE isozyme, Lee et al. (2000) found the activity of purified esterase from VOSF adults was >90% inhibited by 10-1 mM fenitrothon and by 10-4 mM fenitrooxon,. and reactivationrates after inhibition were significantly low, suggesting that fentirothion or fenitrooxon might bind to the enzyme active site irreversibly. This isozyme 'A' also have been found in larvae of VOSF and VOSCM (Fig. 1). Lee and Lees (2001) showed this 'A' band in adults of VOSCM. However, VOS49 does not show the 'A' band in PAGE zymograms. The two very different PAGE patterns for the malathion-resistant strain, VOS49, and the fenitothon- and chlorpyrifos-methylselected strains may support the suggestion that both kinds of CE (hydrolytic and binding) occur in 0. surinamensis. AChE activity from the three resistant strains of 0. surinamensis and the susceptible strain, VOS48, is shown in Table 3. Levels of AChE were similar in the four strains and there was about four times as much AChE (per g tissue) in the adults in comparison with the larvae (Lee and Lees, 2001). These results indicate that the level of AChE in the insects is not related to resistanceagainstmalathion, fenitrothion and chlorpyrifos-methyl in these strains of 0. surinamensis. The Km values for the enzymes from
Table 3. Acetylcholinesterase (AChE) activities and Michaelis constant (Km) values for acetylthiocholine iodide (ATChl) for larvae of the four strains of Oryzaephilus surinamensis Strain VOS48
AChE activity 2.72 ± 0.54 (l.OO)a
Km (mM)
0.037 ± 0.002 (l.OO)a
VOS49
2.55 ± 0.14 (0.94)a
0.041 ± 0.005 (l.ll)a
VOSF
2.46 ± 0.34 (0.90)a
0.036 ± 0.004 (0.97)a
VOSCM
2.74 ± 0.52 (l.OI)a
0.039 ± 0.008 (l.05)a
AChE activities are expressed as mmol ATChI hydrolyzed min'lg larvae". Numbers in parentheses indicate the ratio of activity in the resistant strains in comparison with the susceptible strain, VOS48. The data (mean ± SE values) were determined from six replicates. Means in the column followed by the same letter are . not significantly different (a = 0.05, ANOVA).
166 J. Asia-Pacific Entomol. Vol. 5 (2002)
the four strains were similar, suggesting that they have similar characteristics and are probably not concerned with resistance in these larvae of 0. surinamensis. However, examination of the inhibition of AChE by the organophosphate insecticides themselves and their more toxic' oxon' derivatives would be necessary before a role for AChE in resistance can be ruled out. On the other hand, there are many reports in the literature of involvement of altered AChE in organophosphate insecticide resistance. Tripathi and OBrien (1973) observed a modified AChE from the head ofan organophaophate-resistantstrain ofhouseflies, Musca domestica, which was insensitive to rabon, an organophosphate insecticide. They concluded that one or more amino acid residues close to the active site ofthe enzyme might be altered. Watanabe et al. (1988) reported much stronger inhibition of AChE activity in a susceptible strain (1227-fold for fenitrooxon, 76.S-fold for malaoxon, and 183-fold for dichlorvos) than in an organophosphate-resistant strain ofmosquitoes, Culextriaeniorhynchus. AChE from a methyl parathionresistant strain of the tobacco budworm, Heliothis virescens, was 22-fold less sensitive to methyl paraoxon when compared to a susceptible strain (Brown and Bryson, 1992).Therefore,further assays will be conducted to determine if the organophosphate insecticides or their biochemical products including 'oxon' derivatives differently inhibit larval AChE of 0. surinamensis.
Literature Cited Abbott, W.S. 1925. A method for computing the effectiveness of an insecticide. J. Econ. Entomol. 18: 265-167. Attia, F.I. and T. Frecker. 1984. Cross-resistance spectrum and synergism studies in organophosphorus-resistant strains of Oryzaephilus surinamensis (L.) (Coleoptera: Cucujidae) in Australia. J. Econ. Entomol. 77: 1367-1370. Barson, G. 1983. The effects of temperature and humidity on the toxicity of three organophosphorus insecticides to adult Oryzaephilus surinamensis (L.). Pestic. Sci. 14: 145-152. Beeman, R.W. and BA Schmidt. 1982. Biochemical and genetic aspects of malathion-specific resistance in the Indianmeal moth (Lepidoptera: Pyralidae).J. Econ. Entomol. 75: 945-949. Brown, T.M. and P.K. Bryson. 1992. Selective inhibitors of methyl parathion-resistantacetylcholinesterasefrom Heiothis virescens. Pesctic. Biochem. Physiol. 44: 155-174. Champ, B.R. and C.E. Dyte. 1976. Report of the FAO Global Survey of Pesticide Susceptibility of Stored Grain Pests. FAO Plant Production and Protection Series No.5. FAO, Rome. Italy.
Eun-Kee Park, Won 11 Choi, Sung-Eun Lee
Collins, Pol., H.A. Rose and M. Wegecsanyi, 1992. Enzyme activity in strains of the sawtoothed grain beetle (Coloptera: Cucujidae) differently resistant to fenitrothion, malathion and chlorpyrifos-methyl. J. Econ. Entomol. 85: 1571-1575. Collins, Pol., T.M. Lambkin, B.W. Brideman and C. Pulvirenti. 1993. Resistance to grain-protectant insecticides in coleopterous pests of stored cereals in Queensland, Australia. J. Econ. Entomol. 86: 239-245. Devonshire, A.L. 1977. The properties of a carboxylesterase from the peach-potato aphid Myzus persicae (Sulz.) and its role in conferring insecticide resistance. Biochem. J. 167: 675-683. Finney, OJ. 1971. Probit analysis. 3rd. ed. Cambridge University, London. Herron, G.A., A.D. Clift, G.G. White and H.G. Greening. 1996. Relationships between insecticide use, grain hygiene and insecticide resistance in Oryzaephilus surinamensis (L.) (Coleoptera: Silvanidae) on grain-producing farms. J. Stored Prod. Res. 32: 131-136. Lee, S.E. and E.M. Lees. 2001. Biochemical mechanisms of resistance in strains of Oryzaephilus surinamensis (Coleoptera: Silvanidae) resistant to malathion and chlorpyrifos-methyl. J. Econ. Entomol. 94: 706-713. Lee, S.E., E.M. Lees, and S.c. Campbell. 2000. Purification and characterization of an esterase conferring resistance to fenitrothion in Oryzaephilus surinamensis (L.) (Insecta, Coleoptera, Silvanidae). J. Agric. Food Chern. 48: 49914996. Mackness, M.L, C.H. Walker, D.C. Rowlands and N.R. Price. 1983. Esterase activity in homogenates of three strains of the rust red flour beetle Tribolium castaneum (Herbst). Compo Biochem. Physiol. 74C: 65-68. Milson, J.D. 1995. Esterase activity and resistance to organophosphates in Oryzaephilus surinamensis. MscAgr thesis, The University of Sydney. Sydney. Australia. SAS Institute. 1995. SAS user's guide: statistics. SAS Institute, Cary, NC, USA. Subramanyam, S.H., P.K. Harein and L.K. Cutkomp. 1989. Organophosphate resistance in adults of red flour beetle (Coleoptera: Tenebrionidae) and sawtoothed grain beetle (Coleoptera: Cucujidae) infesting barley on farms in Minnesota. J. Econ. Entomol. 82: 989-995. Tripathi, R.K. and R.D. OBrien. 1973. Insensitivity of acetylcholinesterase as a factor in resistance of houseflies to the organophosphate Rabon. Pestic. Biochem. Physiol. 3: 495-498. Tyler, P.S. and TJ. Binns. 1982. The influence of temperature on the susceptibility to eight organophosphorus insecticides of susceptible and resistant Tribolium castaneum, Oryzaephilus surinamensis and Sitophilus granaries. 1. Stored Prod. Res. 18: 13-19. Wallbank, B.E. 1996. Resistance to organophosphorus grain protectants in Oryzaephilus surinamensis (L.) (Coleoptera: Silvanidae) from off-farm grain storages in New South Wales. Aust. J. Entomol. 35: 193-195. Watanabe, M., S. Takabe, D.H. Kim, R. Arakawa, K. Kamimura and K. Kobashi. 1988. Oxo-type organophosphate-resistant acetylcholinesterase from organophosphate-unsusceptible Culex tritaeniorhynchus. Chern. Pharm. Sull. 36: 312-315.
Esterase Activity and Resistance to Organophosphorus Insecticides in Larvae of Oryzaephilus surinamensis (L.) (Coleoptera: Silvanidae) 1
2
Eun-Kee Park, Won II Choi and Sung-Eun Lee * Department of Epidemiology and Preventive Medicine, School of Medicine, University of California, Davis, California 95616, USA 'Department of Agricultural Biology, Seoul National University, Suwon 441-744, Korea 2Department of Agricultural Chemistry, Kyungpook National University, Daegu 702-701, Korea
Abstract Acetylcholinesterase and carboxylesterase activities of four strains of Oryzaephilus surinamensis (L.) larvae were examined. The three strains of VOS49, VOSF, and VOSCM were resistant to malathion, fenitrothion, and chlorpyrifos-methyl, respectively, while VOS48 was a susceptible strain. Cross-resistance to all three organophosphate insecticides tested was confirmed in larvae of the three resistant strains. Acetylcholinesterase activity was not correlated to organophosphate resistance in those resistant strains. VOS49, VOSF and VOSCM showed enhanced levels of carboxylesterase activity based on p-nitrophenyl acetate, a.-naphthyl acetate, or p-naphthyl acetate substrates. However, these activities were significantly lower than those of 0. surinamensis adults. PAGE zymograms showed major differences in carboxylesterase band pattern among strains. VOS49 carboxylesterase banding pattern was significantly different from other two resistant strains. Interestingly, these larval esterase band patterns were very similar to those of 0. surinamensis adults. Therefore, carboxylesterase activity of 0. surinamensis plays important role in resistance to organophosphate insecticide from larval stage.
Key words Oryzaephilus surinamensis, resistance, organophosphate insecticide, carboxylesterase
Introduction The use of chemical protectants to control insect pests in stored grain is widespread. This control is largely dependent on the concentration of insecticide to which the stored grain insect pests are exposed. *Corresponding author. E-mail: [email protected] Tel: 82-53-950-7751; Fax: 82-53-953-7233 (Received April 10, 2002; Accepted August 21, 2002)
Two critical factors affect the efficacy of control: Temperature and humidity. Generally, higher temperatures increase the degradation of insecticides. However, this loss is balanced by greater toxicity of a fixed insecticide concentration at higher temperatures (Tyler and Binns, 1982). Variations in humidity are thought to alter both the chemical and biological metabolism of insecticides (Barson, 1983). In Australia, organophosphorus insecticides have been widely used to protect stored grain from insect pests. The site of action of organophosphate insecticides is a key enzyme ofthe insect central nervous system, acetylcholinesterase (AChE) which destroys acetylcholine functioning in the synaptic transmission of nerve impulse. Organophosphate insecticides react with AChE in a similar way to acetylcholine. However, the phosphorylated AChE derivative is much more stable than the acyl derivative and results in clogging of the electric transmission circuit, leading to convulsions, paralysis, and finally to death. Many grain storage insect pests in Australia including Oryzaephilus surinamensis (L.), a major worldwide insect pest of stored grains, have developed resistance initially to malathion, later to fenitrothion and now to chlorpyrifos- methyl (Collins et al., 1993; Herron et al., 1996; Wallbank, 1996). Attia and Frecker (1984) and Subramayam et al. (1989), obtained indications of the possible mechanisms involved in organophosphate insecticide resistance through the use of synergists. Attia and Frecker (1984) showed that piperonyl butoxide (PBO, a cytochrome P450-dependent monooxygenase inhibitor) and S,S,S,-tributyl phosphorothioate (DEF, an carboxylesteraseinhibitor)decreasedresistanceto fenitrothion by up to 36-fold and 14-fold, respectively, in New South Wales strains of organophosphate resistant 0. surinamensis. However, the authors found that those synergists reduced resistance to malathion and dichlorvos by less than 3-fold indicating a more significant role for monooxygenases and esterases in fenitrothion resistance to malathion dichlorvos.
162 J. Asia-Pacific Entomol. Vol. 5 (2002)
Subramanyam et al. (1989) found similar phenomenon that increased mortality of chlorpyrifos-methyl-tolerant 0. surinamensis using PBO and DEF indicated that monoxygenases and esterases could be involved in resistance of this species to chlorpyrifos-methyl, Lee et al. (2000) and Lee and Lees (2001) have found enhanced activities of monoxygenases and carboxylesterases are the principal biochemical mechanisms to confer malathion, fenitrothion and chlorpyrifos-methyl resistance to 0. surinamensis. An interesting study conducted by Milson (1995) have shown that when individual field strains of 0. surinamensis with low resistance to fenitrothion and chlorpyrifos-methyl are selected with these insecticides, the relationship between resistance to chlorpyrifos-methyl and esterase activity is much greater than the relationship between resistance to fenitrothion and esterase activity. These results are very similar to those of Collins et al. (1992). However, these all studies are conducted using only adults of resistant strains. The aim of this study is to examine the possible role of CE and AChE in organophosphate insecticide resistance in larvae of 0. surinamensis.
Materials and Methods Chemicals p-Nitrophenyl acetate (PNPA), a-naphthyl acetate (a -NA) , ~-naphthyl acetate (~-NA), acetylthiocholine iodide (ATChI), 5,5-dithio-bis-(2-nitrobenzenoic acid) (DTNB), a-naphthol, ~-naphthol, p-nitrophenol, Fast Blue B salt (o-diansidine, tetrazotized zinc chloride complex), and tris(hydroxymethyl)-aminomethane (Tris) were all purchased from Sigma (St. Louis, MO). Polytetrafluoroethylene (Fluon) was from ICI Fluoropolymers (Exton, PA). Malathion (91.9% [AI]), fenitrothion (98.6% [AI]) and chlorpyrifos-methyl (94% [AI]) were supplied by Dowelanco (Sydney, Australia). All chemicals were of highest grade commercially available.
Insects Four strains of the saw-toothed grain beetle, 0. surinamensis, were supplied by P. 1. Collins (Department of Primary Industry, Queensland, Australia). VOS48, the organophosphorus insecticide-susceptible strain, has been in continuous laboratory culture since 1973. VOS49 is a malathion-resistant strain collected from the field before introduction of fenitrothion and chlorpyrifos-methyl. It has been maintained under
Eun-Kee Park, Won 11 Choi, Sung-Eun Lee
laboratory culture in .Queensland at 28 °C under malathion selection. VOSF and VOSCM are the composite field strains selected with fenitrothion and chlorpyrifos-methyl, respectively, and maintained under laboratory culture in Queensland at 28 °C under fenitrothion and chlorpyrifos-methyl selection, respectively.
Determination of resistance to organophosphorus insecticides Resistance was assessed by the standard FAO impregnated filter paper assay method (Champ and Dyte, 1976). LD so values of insect strains were determined using malathion, fenitrothion and chlorpyrifos-methyl. Insecticides were dissolved in a mixture of ondina oil: acetone: petroleum ether (1: 1:3, vol:vol). Different concentrations of insecticide solution (0.5 ml) were applied to 7 cm diameter Whatman No.1 filter paper (Whatman, Hillsboro, OR) and solvents were allowed to evaporate. Treated filter papers were then placed in aluminum foil lined PVC trays (80 by 330 by 420 mm) and an aluminum ring (4.3 em i.d. and 2.9 cm high) coated with Fluon was placed on top of each filter paper. Three batches of 40 larvae were exposed to a graded series of four concentrations of malathion, fenitrothion and chlorpyrifos-methyl for 24 h at 28 °C. Exposed larvae were transferred to small vials, containing 1 g rolled oats, for 48 h, and survivors were counted. The LD so and LD 9s values were calculated by Probit analysis (Finney, 1971). Control mortality was accounted for by Abbott's formula (Abbott, 1925). Resistance factors were calculated as the ratio ofLD so and LD 9s values for a resistant strain compared with the susceptible strain, VOS48. Insecticide concentrations and LD so and LD9s values are reported as percent (wt:vol) insecticide in ondina oil.
Preparation of extracts for enzyme assays and determinaton of carboxylesterase and acetylcholinesterase activities Two hundred milligrams of larvae from each of the four strains of insect were homogenized in a pestle and mortar with 3.0 ml of 10 mM Tris-HCl buffer, pH 7.5, containing 0.5% (wt:vol) Triton X-lOO. The homogenateswere filtered through Miracloth (Calbiochem, San Diego, CA) to remove exo-skeletal and tissue debris, and centrifuged at 12,000 g for 20 min using a Sorvall (Kendro Laboratory Products, Newtown, CT) superspeed RC-2B centrifuge. The supernatants were reserved as crude enzyme extracts. Enzyme activities were examined by the method of Lee and Lees (2001).
Esterase Activity and Resistance to OP Insecticides in Oryzaephilus surinamensis
Polyacrylamide gel electrophoresis Native polyacrylamide gel electrophoresis (PAGE) was conducted using a Bio-Rad (Hercules, CA) miniprotein II dual vertical slab cell system and 7.5% acrylamide (wt.vol) gels at pH 8.5 with stacking gels of 3% acrylamide (wt:vol). Samples of carboxylesterase in 20% (wt:vol) sucrose containing bromothymol blue were applied to stacking gels. Electrophoresis was performed at 100 V for 10 min, voltage increased to 200 V and a maximum current 2.5 rnA and continued for about 2 h until the marker dye neared the bottom of the gel. Gels were stained for carboxylesterase as described by Devonshire (1977) with a solution made by adding 30 mM a-NA or 0-NA in acetone (1 ml) to 0.2% (wt:vol) Fast Blue RR salt in 0.2 M phosphate buffer, pH 6.0 (50 ml).
and chlorpyrifos-methyl. PAGE zymograms for larvae ofVOS48, VOS49, VOSF and VOSCM show differences between the strains in band position and in intensity of dye staining. Levels of AChE activity were not significantly different among the four strains (Table 3). However, these levels of AChE in larvae of 0. surinamensis were approximately 4 times lower than those in adults of the insects reported previously (Lee and Lees, 2001). The Michaelis constant (Km) values for ATChI ofthe four strains are not significantly different (Table 3). Interestingly, Km values for ATCW in larvae of 0. surinamensis are almost identical to those in adults of the insects.
Statistical analysis
vas
Strains 48.9F
CM4849
FCM
..- -
Laboratory data on the evaluation of different rates of enzymes were subjected to analysis of variance (ANOVA) followed by Students t-test (a = 0.05) (SAS Institute 1995).
• ••• -...
(I-NA
+
Results LD so values obtained for the three organophosphate insecticides malathion, fenitrothion and chlorpyrifosmethyl for the larvae of the four strains of 0. surinamensis and the resistance factors (RF value) for each resistant strains relative to the larvae of VOS48 are shown in Table 1. The three resistant strains showed cross-resistance to the insecticides tested relative to VOS48. The VOS49 strain had high RF values for LDso or LD9s values to fenitrothion and chlorpyrifos-methyl as well as to malathion. Similar high cross-resistance was found in other two resistant strains based on RF values for both LDso and LD 9s. Levels of CE activity using three substrates are elevated in the three resistant strains of 0. surinamensis relative to VOS48 (Table 2). For all strains the rate of hydrolysis for PNPA was 1.44-2.97 times higher than a-NA or 0-NA. Therefore, the larvae of O. surinamensis may be favorable for hydrolysis of PNP A than other two substrates used in this study. All strains had significantly different CE activity for the three CE substrates. VOSCM had 4.8-7.8 times higher CE activity than VOS48 (Table 2). The results using PAGE (Fig. 1) showed an interesting CE role of 0. surinamensis in the resistance to the three organophosphorus insecticides malathion, fenitrothion
163
~-NA
-. •••
__ <:==A
- ...
Fig. 1. Native polyacrylamide gel electrophoresis (PAGE) of crude extracts of larvae of the four strains of Oryzaephilus surinamensis stained for carboxylesterase activity with a-NA or B-NA and 0.1% Fast Blue RR salt. Standard 15 III aliquots (equivalent to 0.6 mg tissue) were applied to the gels. Zymograms for carboxylesterase activity against a-NA of adults of fours strains of 0. surinamensis were already reported to Lee et al. (2000) and Lee and Lees (200 I). VOS represents Victorian Oryzaephilus surinamensis. VOS48 is an insecticide susceptible strain. VOS49 is a malathionresistant strain collected before the introduction of fenitrothion and chlorpyrifos-methyl. VOSF and VOSCM strains are composite field strains selected with fenitrothion and chlorpyrifos-methyl. 'A' in the figure represents an isozyme of carboxylesterase conferring resistance in the insects against the organophosphorus insecticides and is the enzyme already purified as shown in the previous report Lee et al. (2000).
164 J. Asia-Pacific Entomol. Vol. 5 (2002)
Eun-Kee Park, Won 11 Choi, Sung-Eun Lee
Table 1. LD50 values for larvae of four strains of Oryzaephilus surinamensis in response to malathon, fenitrothion and chlorpyrifos-methyl. Numbers of larvae tested are 480 for all strains Strain
LD 50 (95% FL), gr
1a
LD 95 (95% FL), gr 1a
RF
Malathion VOS48
0.0060 (0.0025-0.0136)
1
0.079 (0.026-5.77)
1
VOS49
0.041
(0.036-0.046)
6.8
0.18
2.3
VOSF
0.026
(0.017-0.046)
4.3
0.096 (0.052-0.62)
1.2
VOSCM
0.087
(0.074-0.10)
14.5
0.64
8.1
(0.14-0.25)
(0.46-1.02)
Fenitrothion VOS48
0.00040 (0.00035-0.00045)
1
0.0013 (0.0010-0.0018)
1
VOS49
0.0019
(0.0010-0.0057)
2.5
om I
(0.0045-2.25)
8.5
VOSF
0.0097
(0.008-0.011)
24.2
0.084
(0.057-0.14)
64.6
VOSCM
0.031
(0.014-0.033)
77.5
0.29
(0.080-1.37)
223
Chlorpyrifos-methyl VOS48
0.00028 (0.00015-0.00050)
1
0.0011 (0.00057-0.0047)
1
VOS49
0.00068 (0.00055-0.00083)
2.4
0.0086 (0.0058-0.015)
7.8
VOSF
0.0017
VOSCM
0.023 (0.019-0.028) gr' of ondina oil.
'LD,. values are in
(0.0013-0.0022)
6.1
0.033
(0.018-0.076)
82.1
0.21
(0.15-0.33)
30.0 191
'Resistance factor (RF) for the two resistant strains have been calculated relative to tbe susceptible strain, VOS48.
Discussions The larvae of the three strains of 0. surinamensis selected for resistance to malathion, fenitrothion and chlorpyrifos-methyl were shown to be resistant to all three organophosphate insecticides relative to YOS48. The degree of cross-resistance was different in the three strains (Table 1). The VOS49 strain is malathionresistant strain collected before fenitrothion and chlorpyrifos-methyl were used in Australia. It shows some resistance to fenitrothion and chlorpyrifosmethyl, althoughit has never been exposedto fenitrothion and chlorpyrifos-methyl. Interestingly, adults of the VOS49 strain had RF values to malathion, fenitrothion and chlorpyrifos-methyl of 16, 24 and 4, respectively (Lee and Lees, 2001). These data have showed that the malathion-resistant strain had different RF values with their growth stages to fenitrothion resistance. However, the other two organophosphates malathion andchlorpyrifos-methyl showedsimilarRF valueswithout significant differences between larvae and adults. Larvae of the fenitrothion-resistant strain (VOSF) of
0. surinamensis had high RF value to fenitrothion. However, it did not show strong cross- resistance to both fenitrothion and chlorpyrifos- methyl. These larval results are identical to the results of Lee and Lees (2001), where adults of VOSF strain had RF values for malathion, fenitrothion, and chlorpyrifosmethyl of 5, 14, and 5, respectively. Obviously, the fenitrothion resistance in 0. surinamensis is not related to malathion and chlorpyrifos-methyl resistance. On the other hand, larvae of VOSCM strain had RF values to malathion, fenitrothion, and chlorpyrifos- methyl of 14.5, 77.5, and 82.1, respectively compared with values of 23, 63, and 30 for three insecticides. These results showed that cross-resistance to chlorpyrifosmethyl and malathion in 0. surinamensis were found, but the relative levels of cross-resistance of chlorpyrifosmethyl and fenitrothion is strong. Milson (1995) has described experiments in which field strains of 0. surinamensis with some resistance to chlorpyrifosmethyl and fenitrothion were selected for several generations with chlorpyrifos-methyl and fenitrothion. There was reasonable correlation (R=0.71) between resistance to fenitrothion and resistance to chlorpyrifos-
Esterase Activity and Resistance to OP Insecticides in Oryzaephilus surinamensis
165
Table 2. Carboxylesterase (CE) activities of larvae of the three strains of Oryzaephilus surinamensis CE activity substrate
Strain PNPA
a-NA
.B -NA
VOS48
5.61 ±0.14(1.0)a
2.43 ±0.15(1.0)a
2.20±0.15(1.0)a
VOS49
8.17±0.47(1.5)b
2.75± 1.27(l.l)b
3.14±0.14(1.4)b
VOSF
10.4 ±0.17(1.9)c
5.72±0.20(2.4)c
5.01 ±0.17(2.3)c
VOSCM
27.2 ±1.02(4.8)d
18.9± 1.28(7.8)d
16.5± 1.09(7.5)d
CE activities are expressed as mmol substrate (p-nitrophenyl acetate, PNPA; a-naphthyl acetate, a-NA; l3-naphthyl acetate, I3-NA) hydrolyzed minlg larvae", Numbers in parentheses indicate the ratio of activity in the resistant strains in comparison with the susceptible strain, VOS48. The data (mean ± SE values) were determined from six replicates. Means in the column followed by the same letter are not significantly different (a=0.05, ANOVA).
methyl. The results presented in Table 2 show the levels of CE activity are elevated in all cases relative to VOS48. Adults of the three resistant strains had 1.5to 2.5-fold higher levels ofCE activity than the larvae. There was no difference in CE activity in adults and larvae of VOS48, the standard susceptible strain. These results indicate that the amount of one or more CE isozymes may increase during growth. Esterases have been shown to act in resistance mechanisms in two ways. The actual increase in hydrolysis of an insecticide to produce a less toxic compound is one mechanism, as suggested by Mackness et al. (1983) and Beeman and Schmidt (1982). In this situation, increased hydrolysis of the carboxylester in malathion by a CE isozyme is consistent with resistance to malathion. Fenitrothion and chlorpyrifos-methyl do not contain a carboxylester group, so detoxification by CE hydrolysis is not likely to have a major role in resistance. In this case, CE may bind, and thus be inhibited by organophosphate insecticides as described by Devonshire (1977). It is possible that these two mechanisms may operate in 0. surinamensis resistance to organophosphates. The role of CE activity in resistance to chlorpyrifos-methyl suggested by Subramanyam et al. (1989) may be in binding chloryrifos-methyl or chlorpyrifos-methyl oxon as this insecticide does not contain a carboxylester group. Lee et al. (2000) purified a major CE isozyme, designated as A, from adults of VOSF strain of 0.
surinamensis conferring resistance to malathion, fenitrothion and chlorpyrifos-methyl. This enzyme had 130 kDa molecular mass and contained two subunits of 65 kDa. With kinetic studies of this purified CE isozyme, Lee et al. (2000) found the activity of purified esterase from VOSF adults was >90% inhibited by 10-1 mM fenitrothon and by 10-4 mM fenitrooxon,. and reactivationrates after inhibition were significantly low, suggesting that fentirothion or fenitrooxon might bind to the enzyme active site irreversibly. This isozyme 'A' also have been found in larvae of VOSF and VOSCM (Fig. 1). Lee and Lees (2001) showed this 'A' band in adults of VOSCM. However, VOS49 does not show the 'A' band in PAGE zymograms. The two very different PAGE patterns for the malathion-resistant strain, VOS49, and the fenitothon- and chlorpyrifos-methylselected strains may support the suggestion that both kinds of CE (hydrolytic and binding) occur in 0. surinamensis. AChE activity from the three resistant strains of 0. surinamensis and the susceptible strain, VOS48, is shown in Table 3. Levels of AChE were similar in the four strains and there was about four times as much AChE (per g tissue) in the adults in comparison with the larvae (Lee and Lees, 2001). These results indicate that the level of AChE in the insects is not related to resistanceagainstmalathion, fenitrothion and chlorpyrifos-methyl in these strains of 0. surinamensis. The Km values for the enzymes from
Table 3. Acetylcholinesterase (AChE) activities and Michaelis constant (Km) values for acetylthiocholine iodide (ATChl) for larvae of the four strains of Oryzaephilus surinamensis Strain VOS48
AChE activity 2.72 ± 0.54 (l.OO)a
Km (mM)
0.037 ± 0.002 (l.OO)a
VOS49
2.55 ± 0.14 (0.94)a
0.041 ± 0.005 (l.ll)a
VOSF
2.46 ± 0.34 (0.90)a
0.036 ± 0.004 (0.97)a
VOSCM
2.74 ± 0.52 (l.OI)a
0.039 ± 0.008 (l.05)a
AChE activities are expressed as mmol ATChI hydrolyzed min'lg larvae". Numbers in parentheses indicate the ratio of activity in the resistant strains in comparison with the susceptible strain, VOS48. The data (mean ± SE values) were determined from six replicates. Means in the column followed by the same letter are . not significantly different (a = 0.05, ANOVA).
166 J. Asia-Pacific Entomol. Vol. 5 (2002)
the four strains were similar, suggesting that they have similar characteristics and are probably not concerned with resistance in these larvae of 0. surinamensis. However, examination of the inhibition of AChE by the organophosphate insecticides themselves and their more toxic' oxon' derivatives would be necessary before a role for AChE in resistance can be ruled out. On the other hand, there are many reports in the literature of involvement of altered AChE in organophosphate insecticide resistance. Tripathi and OBrien (1973) observed a modified AChE from the head ofan organophaophate-resistantstrain ofhouseflies, Musca domestica, which was insensitive to rabon, an organophosphate insecticide. They concluded that one or more amino acid residues close to the active site ofthe enzyme might be altered. Watanabe et al. (1988) reported much stronger inhibition of AChE activity in a susceptible strain (1227-fold for fenitrooxon, 76.S-fold for malaoxon, and 183-fold for dichlorvos) than in an organophosphate-resistant strain ofmosquitoes, Culextriaeniorhynchus. AChE from a methyl parathionresistant strain of the tobacco budworm, Heliothis virescens, was 22-fold less sensitive to methyl paraoxon when compared to a susceptible strain (Brown and Bryson, 1992).Therefore,further assays will be conducted to determine if the organophosphate insecticides or their biochemical products including 'oxon' derivatives differently inhibit larval AChE of 0. surinamensis.
Literature Cited Abbott, W.S. 1925. A method for computing the effectiveness of an insecticide. J. Econ. Entomol. 18: 265-167. Attia, F.I. and T. Frecker. 1984. Cross-resistance spectrum and synergism studies in organophosphorus-resistant strains of Oryzaephilus surinamensis (L.) (Coleoptera: Cucujidae) in Australia. J. Econ. Entomol. 77: 1367-1370. Barson, G. 1983. The effects of temperature and humidity on the toxicity of three organophosphorus insecticides to adult Oryzaephilus surinamensis (L.). Pestic. Sci. 14: 145-152. Beeman, R.W. and BA Schmidt. 1982. Biochemical and genetic aspects of malathion-specific resistance in the Indianmeal moth (Lepidoptera: Pyralidae).J. Econ. Entomol. 75: 945-949. Brown, T.M. and P.K. Bryson. 1992. Selective inhibitors of methyl parathion-resistantacetylcholinesterasefrom Heiothis virescens. Pesctic. Biochem. Physiol. 44: 155-174. Champ, B.R. and C.E. Dyte. 1976. Report of the FAO Global Survey of Pesticide Susceptibility of Stored Grain Pests. FAO Plant Production and Protection Series No.5. FAO, Rome. Italy.
Eun-Kee Park, Won 11 Choi, Sung-Eun Lee
Collins, Pol., H.A. Rose and M. Wegecsanyi, 1992. Enzyme activity in strains of the sawtoothed grain beetle (Coloptera: Cucujidae) differently resistant to fenitrothion, malathion and chlorpyrifos-methyl. J. Econ. Entomol. 85: 1571-1575. Collins, Pol., T.M. Lambkin, B.W. Brideman and C. Pulvirenti. 1993. Resistance to grain-protectant insecticides in coleopterous pests of stored cereals in Queensland, Australia. J. Econ. Entomol. 86: 239-245. Devonshire, A.L. 1977. The properties of a carboxylesterase from the peach-potato aphid Myzus persicae (Sulz.) and its role in conferring insecticide resistance. Biochem. J. 167: 675-683. Finney, OJ. 1971. Probit analysis. 3rd. ed. Cambridge University, London. Herron, G.A., A.D. Clift, G.G. White and H.G. Greening. 1996. Relationships between insecticide use, grain hygiene and insecticide resistance in Oryzaephilus surinamensis (L.) (Coleoptera: Silvanidae) on grain-producing farms. J. Stored Prod. Res. 32: 131-136. Lee, S.E. and E.M. Lees. 2001. Biochemical mechanisms of resistance in strains of Oryzaephilus surinamensis (Coleoptera: Silvanidae) resistant to malathion and chlorpyrifos-methyl. J. Econ. Entomol. 94: 706-713. Lee, S.E., E.M. Lees, and S.c. Campbell. 2000. Purification and characterization of an esterase conferring resistance to fenitrothion in Oryzaephilus surinamensis (L.) (Insecta, Coleoptera, Silvanidae). J. Agric. Food Chern. 48: 49914996. Mackness, M.L, C.H. Walker, D.C. Rowlands and N.R. Price. 1983. Esterase activity in homogenates of three strains of the rust red flour beetle Tribolium castaneum (Herbst). Compo Biochem. Physiol. 74C: 65-68. Milson, J.D. 1995. Esterase activity and resistance to organophosphates in Oryzaephilus surinamensis. MscAgr thesis, The University of Sydney. Sydney. Australia. SAS Institute. 1995. SAS user's guide: statistics. SAS Institute, Cary, NC, USA. Subramanyam, S.H., P.K. Harein and L.K. Cutkomp. 1989. Organophosphate resistance in adults of red flour beetle (Coleoptera: Tenebrionidae) and sawtoothed grain beetle (Coleoptera: Cucujidae) infesting barley on farms in Minnesota. J. Econ. Entomol. 82: 989-995. Tripathi, R.K. and R.D. OBrien. 1973. Insensitivity of acetylcholinesterase as a factor in resistance of houseflies to the organophosphate Rabon. Pestic. Biochem. Physiol. 3: 495-498. Tyler, P.S. and TJ. Binns. 1982. The influence of temperature on the susceptibility to eight organophosphorus insecticides of susceptible and resistant Tribolium castaneum, Oryzaephilus surinamensis and Sitophilus granaries. 1. Stored Prod. Res. 18: 13-19. Wallbank, B.E. 1996. Resistance to organophosphorus grain protectants in Oryzaephilus surinamensis (L.) (Coleoptera: Silvanidae) from off-farm grain storages in New South Wales. Aust. J. Entomol. 35: 193-195. Watanabe, M., S. Takabe, D.H. Kim, R. Arakawa, K. Kamimura and K. Kobashi. 1988. Oxo-type organophosphate-resistant acetylcholinesterase from organophosphate-unsusceptible Culex tritaeniorhynchus. Chern. Pharm. Sull. 36: 312-315.