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Acetylcholinesterase and its reduced sensitivity to inhibition by paraoxon in organophosphate-resistant Lygus hesperus knight (Hemiptera: Miridae)
PESTICIDE
BIOCHEMISTRY
AND
PHYSIOLOGY
36, 22-28 (l!?N)
Acetylcholinesterase and Its Reduced Sensitivity to Inhibition by Paraoxon in Organophosphate-Resistant Lygus hesperus Knight (Hemiptera: Miridae) K.Y. ZHUAND W.A. BRINDLEY Department of Biology, Utah State Universiry Agricultural Experiment Station, Utah State University, Logan, Utah 84322 Received June 16, 1989; accepted August 16, 1989 Acetylchohnesterase (AChE) from Lygus hesperus Knight was partially characterized and examined to determine if insensitive AChE contributed to insecticide resistance in the field. The optimal pH and temperature were 8.5 and 35-40°C, respectively. The specific activity in head homogenates was higher than in thoracic or abdominal homogenates. AChE was likely to be a membrane-associated enzyme. The kinetic parameters, K,,, and V,,, determined with acetylthiocholine iodide at 38”C, were 1.2 x 10e4 M and 43.4 nmol/min/mg protein, respectively, for the susceptible (S) population and 1.1 x lop4 M and 45.3 nmoVmin/mg protein, respectively, for the resistant (R) population. Neither parameter differed signiticantly between the two populations (P > 0.05). The bimolecular rate constant, kj, in the S and R populations was 2.8 X 16 and 8.3 x lo3 M-‘mm-‘, respectively, for paraoxon at 25”C, which indicates that AChE from the R population was about 34-fold less sensitive to paraoxon inhibition than AChE from the S population. AChE from the S population was homogenous to paraoxon inhibition, while AChE from the R population appeared to be heterogenous. o IWOAcademic PRSS, IIIC.
INTRbDUCTION
cholinesterase (AChE; EC 3.1.1.7) to inhibition by organophosphorus and carbamate insecticides is a mechanism that has been found in many arthropods, such as spider mites, cattle ticks, house flies, mosquitoes, green rice leafhoppers, and Egyptian cotton worms (2). Such a mechanism may be solely responsible for resistance (3) or it may reinforce resistance caused by other mechanisms (4). In L. hesperus, nothing is known about AChE and its sensitivity to inhibition by insecticides. The purposes of this study were to characterize AChE activity and to examine whether insensitive AChE contributed to resistance in L. hes-
The development of insecticide resistance in Lygus hesperus Knight is a serious problem for the alfalfa seed industry. Field populations of L. hesperus resistant to trichlorfon were shown to have four- to sixfold higher esterase activity to 1-naphthyl acetate, and enhanced carboxylesterase (CarE)’ activity was considered to be an important factor causing the resistance (1). However, extremely high resistance levels associated with incomplete synergism of DEF to trichlorfon in bioassays (W. A. Brindley et al. unpublished) and large differences in LCsO values with relatively small difference in CarE activity between perus. the susceptible and some resistant populaMATERIALS AND METHODS tions suggestedthat other mechanisms contributed to the resistance. Insects. L. hesperus collected from an alA reduction in the sensitivity of acetyl- falfa seed field near Nampa, Idaho, and from alfalfa hay fields near Logan, Utah, were used as resistant (R) and susceptible ’ Abbreviations used: CarE, carboxylesterase; (S) insects, respectively. AChE, acetylcholinesterase; R, resistant; S, susceptiChemicals. The following chemicals ble; ASCh, acetylthiocholine iodide; DTNB, 5,5’-dithiobis-2-nitrobenzoic acid. were purchased from Sigma Chemical Co. 22 0048-3575190 $3.00 Copyright 0 1990 by Academic Press. Inc. All riglIt.3 of re.pmdwtion in any form reserved.
ACETYLCHOLINESTERASE
(St. Louis, MO): acetylthiocholine iodide (ASCh), bovine serum albumin, 5,5’-dithiobis-2nitrobenzoic acid (DTNB), eserine sulfate. Paraoxon (diethyl 4nitrophenyl phosphate), 95% pure, was obtained from Chem Service (West Chester, PA). Enzyme preparations. Groups of 20-50 adult L. hesperus were homogenized in 510 ml of ice-cold 0.05 M phosphate buffer, pH 8.5 (pH 7.5 for study of paraoxon inhibition), using a Potter-Elvehjem homogenizer in an ice bath with a motor-driven Teflon pestle. The homogenate was centrifuged at 10,000g for 20 min at 4°C in a Beckman LS-65B ultracentrifuge. The supernatant was kept on ice and used as the enzyme source for AChE assays. The separation of subcellular fractions for AChE was designed according to Crankshaw et al. (5) and Aldunate et al. (6). Forty bugs were homogenized in 10 ml of 0.05 M phosphate buffer, pH 8.5. The homogenate was filtered with two layers of cheesecloth to remove the gross debris. The filtered homogenate was brought up to a volume of 10ml with the same buffer, and then divided into halves, one of which was used as crude homogenate. The other half was centrifuged at 470g for 10 min to obtain a pellet which contained nuclei and cell debris. The supernatant was further centrifuged at 10,000g for 20 min to obtain a mitochondrial pellet. The resultant supernatam was again centrifuged at 105,OOOgfor 90 min to obtain the microsomal pellet, and the final supernatant corresponding to the cytosol was used as the soluble fraction. AChE assays. AChE activity was measured according to the method of Ellman et al. (7) with some modifications. A 2.9-ml aliquot of 0.05 M phosphate buffer, pH 8.5, was pipetted into four test tubes. Three of them received a lOO-~1aliquot of the homogenate and the one without homogenate was run as the control. One hundred microliters of 10 mM DTNB reagent and 20 pJ of 75 mM ASCh were pipetted into these four test tubes. The mixture was prepared in an
IN L.
23
hesperus
ice bath and ASCh was added just before incubation. After incubation for 15 min at 38°C in a shaking water bath (the enzyme activity remained linear for at least 24 min), the reaction was stopped by adding 100 ~1 of 0.322 mit4 eserine sulfate; the final concentration of eserine in the mixture (10 ~.LM) completely inhibited enzyme activity (1). One hundred microliters of homogenate was added to the control to correct for interference by the homogenate. Absorbance at 412 nm was determined against the control with a spectrophotometer. Inhibition of AChE by paraoxon was studied based on the method of Oppenoorth (8). The homogenate was preincubated with an equal volume of 2 or 4 x lo-’ M paraoxon (dissolved in acetone and diluted with 0.05 M phosphate buffer, pH 7.5) for various periods of time between 5 and 60 min at 25°C in a shaking water bath. After ASCh and DTNB were added, the mixture was further incubated for 20 min under the same conditions. The reaction was stopped by the addition of eserine, and the rest of the procedure was as described above. At the same time, a similar experiment, but preincubated with the buffer containing an appropriate amount of acetone instead of paraoxon, was carried out to correct for denaturation of AChE. Protein concentrations of each enzyme preparation were determined according to the method of Bradford (9) using bovine serum albumin as a standard. Data analysis. The kinetic parameters, Km and Vmax,were determined by Lineweaver-Burk transformations (10). Bimolecular rate constants (kj) were calculated as described by Main and Iverson (11). The data were tested by t tests or analyses of variance. Duncan’s multiple range test (12) was used in the multiple comparison; P 6 0.05 was considered significant. RESULTS
Figure 1 shows the AChE activity toward ASCh at pH values between 5.5 and 9.0 at 35°C. Although the peak activity occurred
ZHU AND BRINDLEY
24
201
5.0
.
’
6.0
.
’
7.0
’
’
6.0
.
’
-4.5
’
9.0
-4.0
Concentration
PH FIG. 1. EfJect of pH on the hydrolysis of ASCh by AChE from L. hesperus. Temperature of reaction was 35°C. Each point represents the mean of three determinations (each with triplicate incubations). Vertical bars indicate standard deviations of the mean.
at pH 8.5, the optimal pH for the AChE appeared to be fairly broad. The activities at pH 7.5 and 9.0 were, respectively, only 10.6 and 0.5% less than at pH 8.5. Temperature effects on AChE activity were studied from 15 to 55°C (Fig. 2). Activity increased gradually to 35°C but decreased rapidly when temperatures were higher than 45°C. The optimal temperature was 35dO’C. Figure 3 shows the AChE activity from
20
30 Temperature
40
50
-2.5
of Substrate
(Log M)
(%)
.
.O
3.
x lop5 to 8 x 10e3 M ASCh. The activity increased from the concentration of 6.25 x lo-’ to 2 x 10m3M where the peak activity was obtained. The distribution of AChE activities in the head, thoracic, and abdominal homogenates is shown in Table 1. Most of the total activity was located in the thorax and abdomen. However, the specific activity of AChE was much higher in the head homogenate than in the thoracic or abdominal homogenates. The total activity of the AChE was almost evenly distributed in four subcellular fractions (Table 2). However, only about 85% of the crude homogenate activity was recovered in these four fractions. The highest specific activity was found in the mitochondrial fraction and then in the microsomal fraction. The specific activities were relatively low in the nuclei and cell 6.25
TABLE 1 of AChE Activity in Different Body Parts of Adult L. hesperusa
60
2. Effect of temperature on the hydrolysis of ASCh by AChE from L. hesperus. Each point represents the mean of three determinations (each with triplicate incubations). Vertical bars indicate standard deviations of the mean. FIG.
-3.0
Effect of substrate concentration on the hydrolysis of ASCh by AChE from L. hesperus. Each point represents the mean of three determinations (each with triplicate incubations). Vertical bars indicate standard deviations of the mean. FIG.
Distribution 10
-3.5
BUY PM
Head ThOraX
Abdomen
% Total activity 14.2 + 0.6 50.5 f 3.5 35.2 f 4.1
specific activity (nmoVmin/mg protein) 86.1 f 7.5 28.7 k 2.6 19.9 * 1.7
a Results are the mean 2 SD of three determinations with triplicate incubations).
(each
ACETYLCHOLINESTERASE
20 1
TABLE2 Subcellular Distribution of AChE Activity in Adult L. hesperus”
Fraction Crude homogenate Nuclei and cell debris Mitochondria Microsome Soluble traction % Recovery
% Total activity
15
Specific activity (nmoUmin/mg
25
IN L. hcsperus
0 0
S population R population / .
>
protein)
5’
2
100 11.2 29.9 20.8 23.0 84.9
r + k k
2.3 2.4 2.6 2.4
37.0 19.0 82.3 68.3 21.8
2 * 2 f -+
1.9 4.0 3.8 4.2 2.9
;; 0 0
10
0,: . /
--/
a Fractions obtained by centrifugation as described under Materials and Methods. Results are the mean 2 SD of three determinations (each with triplicate incubations).
debris fraction, as well as in the soluble fraction. Table 3 shows the protein content, the total activity, and the specific activity of AChE for both sexes of the S and R populations. The total activities between the two populations and between the two sexes were the same (P > 0.05), but the specific activities were different in both cases (P < 0.05). Insects in the R population contained more protein leading to these differences. A difference in the protein content between the S and R populations was simply due to their body size. The insects collected from alfalfa seed fields were usually larger than those from alfalfa hay fields. Figure 4 and Table 4 show a LineweaverBurk (double reciprocal) plot and the kinetic parameters (K, and V,,) for AChE hydrolyzing ASCh in the S and R populations at 38°C. There were no significant dif-
-10
1’
0
I
1
,
10
20
30
I
40
Sex
Protein ww9
S
Female Male Female Male F df P
531.7 2 26.3 434.5 2 41.1 898.8 ” 37.0 629.6 -+ 24.1 110.90 3, 8 co.001
R
I
60
ml
ferences between two populations in either parameter. There was a significant difference in the inhibition of AChE by paraoxon between the S and R populations in the two series of five paired experiments (Fig. 5). When the homogenates prepared from the resistant insects were preincubated with paraoxon at 1 x 10e7 M, the activities were blocked only about 7% in the first 10 min and remained the same up to 1 hr of preincubation. Although 23.5% of the AChE activity was inhibited by 2 x 10m7M paraoxon in the first 5 min, the inhibition increased rapidly up to 10 min and then increased only
Total activity (nmoL!min/bug) a b c d
I
50
FIG. 4. Lineweaver-Burk (double reciprocal) plot of 100 x (I/v) vs ll[sl for AChEfrom S or R populations of L. hesperus. Each point is based on the mean of three determinations (each with triplicate incubations). Regression coeficient (r) > 0.99 (P < 0.001).
TABLE 3 Quantity of Protein and Total and Specific Activities of AChE in S and R Populations Population
/
-
18.6 21.3 18.6 20.3
2 2.0 a 2 2.4 a +- 2.0 a 2 1.9 a 1.26 3, 8 NSb
of L. hesperus”
Specific activity (nmoYmin/mg protein) -34.9 2 2.1 a 49.0 e 0.7 b 20.7 k 2.9 c 32.4 + 3.9 a 56.00 3, 8
B Results are the mean k SD of three determinations (each with triplicate incubations). Means within columns followed by the same letter are not significantly different (P > 0.05; Duncan’s (1955) multiple range test). b Not sign&ant at P > 0.05.
ZHU AND BRINDLEY
26
TABLE 4 Kinetic Parameters of AChE Hydrolyzing Acetylthiocholine Population
4, (PM
s R
in S and R Populations of L. hesperus”
VIn* (nmoGnin/mg protein)
Regression coefficient (r)
43.4 f 2.6 45.3 + 5.4
>0.99 BO.99
119.4 + 4.1 106.2 2 17.6
sign&kc
(P)
a Results are the mean 2 SD of three determinations (each with triplicate incubations). There is no significant difference in K,,, or V,,,, values between the two populations (P > 0.05; t test).
about 5% up to 1 hr of preincubation. In contrast, AChE activities in the susceptible insects were much more sensitive to inhibition by paraoxon at either concentration, and both time-inhibition curves were linear and intercepted the origin. The ki (bimolecular rate constant) was (2.8 -+ 0.1) X ld M- ‘min- ’ (mean f SE) for the S population (calculated from the two paraoxon concentrations) and (8.3 + 1.7) X lo3 M- ‘min- ’ for the R population (calculated from the second part of the curve for 2 X 10m7M paraoxon). The rate of inhibition of the R population was about 34-fold slower than that of the S population. DISCUSSION
The optimal pH for AChE from L. hesperus (8.5) resembles those of other insects:8-9 for Musca domestica (13), 8.0 for
Acheta domesticus (14), 7.5 for Aphis cifricola (15)) 7-9 for Chironomus riparius (16), and 8.5 for Munduca sexta (17).
AChE activity was pH dependent only at pH <8 and seemed to be tolerant to high pH. However, whether this tolerance is due to substrate specificity (18) or species specificity is unknown. As to be expected of an enzyme, the activity of the AChE from L. hesperus with respect to temperature provided a bellshaped curve (Fig. 2). The optimal temperature appeared to be 35-40°C, which was slightly higher than 30-3X for M. domestica (13), 30°C for A. citricolu (15) and C. riparius (16), and 37°C for M. senta (17). Inhibition by excess substrate is one feature which distinguishes AChE from butrylcholinesterase (18). L. hesperus AChE was moderately inhibited at high concentrations of ASCh (Fig. 3) and shows a broad optimum of L. hesperus AChE for substrate concentrations. An explanation for the high total activity in the abdomen with little nervous tissue is not readily available. Since the concentration of ASCh used in this study was reasonably low (0.48 n&f), it is unlikely that the high total activity in abdominal homogenates was due to nonspecific enzymes which hydrolyze ASCh at high concentrations (19). It seems likely that the AChE activity in L. hesperus is membrane bound. Only about 23% total activity remained in the supematant after centrifugation at 105,OtlOg for 90 min. In Boophilus microplus, only 10% of AChE activity remained in the 100,OOOgsupernatant (20). In C. riparius larvae, about 85% of total activity remained
2z‘-------o-.-.-w-ori v)
&k
1
50
0
s. 40 = s Y
5 2
0
2x10-’
n
1x10-7
n
0
1
20
0
0
1
n
30
s
z
1x10-’
-*-o-e-
l
I I0 0
.
“\
S population R population
I 10
.
I. 20
2x10-7
n
0
I 30
.
.\. 40
I 50
.
I 60
1
l%m (min) FIG. 5. Inhibition ofAChEfrom S or R populations of L. hesperus by 1 or 2 x 10m7 M paraoxon with various periods of time. Each point is based on the mean of three duplicate incubations.
ACETYLCHOLINESTERASE
in the 10,OOOgsupernatant and 76% in the 100,OOOgsupernatant (16). The kinetic rate constant, K,, for AChE from L. hesperus is within similar ranges of 1.67 x 10e4 M for A. domesticus (14), 6.7 x 10m5M for A. citricolu (15), 2.54 x 10V5 M for C. riparius (16), 2.7 x 10m4M for M. sexta larvae (17) and 1.5 x low4 Mfor adult (21), and 3.3 x 10e5 M for Triutoma infestans (22). Neither kinetic parameter, K, nor V-, in the R population differed from the S population (Table 4), which indicated that the afhnity of AChE to the substrate (ASCh) and catalytic efficiency of AChE in the two populations were essentially identical. Although it seems possible to determine the kj value for the AChE from the R population more accurately with higher paraoxon concentrations, it is obvious that the sensitivity of the AChE to the inhibition by paraoxon was significantly different from that of the S population (Fig. 5). AChE from the R population was about 34-fold less sensitive than that from the S population. This seemsto be the first time that this kind of resistance mechanism has been found in hemipteran insects, although a similar mechanism had been found in other arthropods (2). The inhibition pattern of AChE by 2 x 10e7 M paraoxon in the R population was very similar to that in the S population during the first few minutes, but it slowed dramatically in about 10 min. This suggests that AChE from the R population was heterogeneous and contained a small amount of susceptible enzyme that could be inhibited very rapidly. Although the sensitivity of AChE to inhibition by paraoxon was reduced in the R population, the K, and V,, values were the same as those in the S population, indicating that there was no change in catalytic activity toward ASCh. If this reflects AChE catalytic ability to the natural substrate, the insects might have an advantage for their survival, in comparison with some other arthropods in which insensitivity to inhibitors
IN L. hesperus
27
was associated with decreased affinity (higher K,,J to the substrate (23) or reduced catalytic efficiency (lower V-> (20,24) because AChE can normally catalyze the hydrolysis of acetylcholine regardless of its insensitivity to the inhibitor. Animals may have a considerable excess of AChE relative to what they strictly need (25), but this would not necessarily obviate the advantage of L. hesperus’ insensitive AChE. Pest managers concerned with L. hesperus should be aware of this possible dimension to the insect’s resistance to insecticides. ACKNOWLEDGMENTS This research was supported by grants from the Western Region Pesticide Impact Assessment Program and Western Region Integrated Pest Management Special Grants Program, and Projects 505 and 577 of the Utah Agricultural Experiment Station, Utah State University, Logan, Utah. Approved as journal paper No. 3855. REFERENCES 1. K. Y. Zhu, “Properties of Esterases from Lygus hesperus Knight (Hemipera: Miridae) and the Roles of the Esterases in Insecticide Resistance,” MS thesis, p. 116, Utah State University, Logan, 1989. 2. F. J. Oppenoorth, Biochemistry and genetics of insecticide resistance, in “Comprehensive Insect Physiology, Biochemistry and Pharmacology” (G. A. Kerkut and L. I. Gilbert, Eds.), Vol. 12, pp. 731-773, Pergamon, New York, 1985. 3. J. Hemingway and G. P. Georghiou, Studies on the acetylcholinesterase of Anopheles albimanus resistant and susceptible to organophosphate and carbamate insecticides, Pestic. Biothem. Physiol. 19, 167 (1983). 4. R. K. Tripathi and R. D. O’Brien, Insensitivity of acetylcholinesterase as a factor in resistance of house flies to the organophosphate Rabon, Pestic. Biochem. Physiol. 3, 495 (1973). 5. D. L. Crankshaw, H. K. Hetnarski, and C. F. Wilkinson, Microsomal NADPH-cytochrome c reductase from the midgut of the southern armyworm (Spodoptera eridania), Insect Biothem. 9, 43 (1979). 6. J. Aldunate, Y. Repetto, M. E. Letelier, and A. Morello, The carboxylesterases of Trypanosoma cruzi epimastigotes, Comp. Biochem. Physiol. B 86, 67 (1987). 7. G. L. Ellman, D. K. Courtney, V. Andres, and R. M. Featherstone, A new and rapid colori-
28
ZHU
AND
metric determination of acetylcholinesterase activity, Biochem. Pharmacol. 7, 88 (1961). 8. F. J. Oppenoorth, Two different paraoxonresistant acetylcholinesterase mutants in the house fly, Pestic. Biochen. Physiol. 18, 26 (1982).
9. 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, 248 (1976). 10. J. E. Bell and E. T. Bell, “Proteins and Enzymes,” p. 499, Prentice-Hall, Englewood Cliffs, NJ, 1988. 11. A. R. Main and F. Iverson, Measurement of the affinity and phosphorylation constants goveming irreversible inhibition of cholinesterases by di-isopropyl phosphorofluoridate, Biochem. J. 100, 525 (1%6). 12. D. B. Duncan, Multiple range and multiple F tests, Biomefrics 11, 1 (1955). 13. A. Silver, “The Biology of Cholinesterases,” p. 596, Amer. Elsevier, New York, 1974. 14. A. H. Lee, R. L. Metcalf, and C. W. Keams, Purification and some properties of house cricket (Acheta domesticus) acetylcholinesterase, J. Insect Physiol. 4, 267 (1974). IS. S. Manulis, I. Ishaaya, and A. S. Perry, Acetylcholinesterase of Aphis citricola: Properties and significance in determining toxicity of systemic organophosphorus and carbamate compounds, Pestic. Biochem. Physiol. 15, 267 (1981). 16. R. L. Detra and W. J. Collins, Characterization of cholinesterase activity in larval Chironomus riparius Meigen (=Thummi kiefer), Insect Biothem. 16,733 (1986). 17. D. S. Lester and L. I. Gilbert, Characterization of acetylcholinesterase activity in the larval brain
BRINDLEY
of Manduca sexta, Insect Biochem. 17, 99 (1987). 18. K. Bui and R. F. Ochillo, Characterization of cholinesterase of muscularis muscle of Bufo marinus, Comp. Biochem. Physiol. C 87, 107 (1987).
19. H. R. Smissaert, S. Voerman, L. Oostenbrugge, and N. Renooy, Acetylcholinesterases of organophosphate-susceptible and resistant spider mites, J. Agric. Food Chem. 18, 66 (1970). 20. J. Nolan and H. J. Schnitzerling, Characterization of acetylcholinesterase of acaricide-resistant and susceptible strains of the cattle tick Boophilus microplus (Can.), Pestic. Biochem. Physiol. 5, 178 (1975). 21. D. J. Prescott, J. G. Hildebrand, J. R. Sanes, anil S. J. Jewett, Biochemical and developmental studies of acetylcholine metabolism in the central nervous system of the moth Manduca sexta, Comp. Biochem. Physiol. C 56, 77 (1977). 22. E. Wood, E. Zerba, M. Picollo, and S. de Lacastro, Partial purification and characterization of Triatoma infestans head acetylcholinesterase, Insect Biochem. 9, 595 (1979). 23. A. L. Devonshire and G. D. Moores, Different forms of insensitive acetylcholinesterase in insecticide-resistant house flies (Musca domestica), Pestic. Biochem. Physiol. 21, 336 (1984). 24. A. L. Devonshire, Studies of the acetylcholinesterase from houseflies (Musca domestica L.) resistant and susceptible to organophosphorus insecticides, Biochem. J. 149, 463 (1975). 25. H. R. Smissaert, F. M. Abd El Hamid, and W. P. J. Overmeer, The minimum acetylcholinesterase (AChE) fraction compatible with life derived by aid of a simple model explaining the degree of dominance of resistance to inhibitors in AChE “mutants,” Biochem. Pharmacol. 24, 1043 (1975).
BIOCHEMISTRY
AND
PHYSIOLOGY
36, 22-28 (l!?N)
Acetylcholinesterase and Its Reduced Sensitivity to Inhibition by Paraoxon in Organophosphate-Resistant Lygus hesperus Knight (Hemiptera: Miridae) K.Y. ZHUAND W.A. BRINDLEY Department of Biology, Utah State Universiry Agricultural Experiment Station, Utah State University, Logan, Utah 84322 Received June 16, 1989; accepted August 16, 1989 Acetylchohnesterase (AChE) from Lygus hesperus Knight was partially characterized and examined to determine if insensitive AChE contributed to insecticide resistance in the field. The optimal pH and temperature were 8.5 and 35-40°C, respectively. The specific activity in head homogenates was higher than in thoracic or abdominal homogenates. AChE was likely to be a membrane-associated enzyme. The kinetic parameters, K,,, and V,,, determined with acetylthiocholine iodide at 38”C, were 1.2 x 10e4 M and 43.4 nmol/min/mg protein, respectively, for the susceptible (S) population and 1.1 x lop4 M and 45.3 nmoVmin/mg protein, respectively, for the resistant (R) population. Neither parameter differed signiticantly between the two populations (P > 0.05). The bimolecular rate constant, kj, in the S and R populations was 2.8 X 16 and 8.3 x lo3 M-‘mm-‘, respectively, for paraoxon at 25”C, which indicates that AChE from the R population was about 34-fold less sensitive to paraoxon inhibition than AChE from the S population. AChE from the S population was homogenous to paraoxon inhibition, while AChE from the R population appeared to be heterogenous. o IWOAcademic PRSS, IIIC.
INTRbDUCTION
cholinesterase (AChE; EC 3.1.1.7) to inhibition by organophosphorus and carbamate insecticides is a mechanism that has been found in many arthropods, such as spider mites, cattle ticks, house flies, mosquitoes, green rice leafhoppers, and Egyptian cotton worms (2). Such a mechanism may be solely responsible for resistance (3) or it may reinforce resistance caused by other mechanisms (4). In L. hesperus, nothing is known about AChE and its sensitivity to inhibition by insecticides. The purposes of this study were to characterize AChE activity and to examine whether insensitive AChE contributed to resistance in L. hes-
The development of insecticide resistance in Lygus hesperus Knight is a serious problem for the alfalfa seed industry. Field populations of L. hesperus resistant to trichlorfon were shown to have four- to sixfold higher esterase activity to 1-naphthyl acetate, and enhanced carboxylesterase (CarE)’ activity was considered to be an important factor causing the resistance (1). However, extremely high resistance levels associated with incomplete synergism of DEF to trichlorfon in bioassays (W. A. Brindley et al. unpublished) and large differences in LCsO values with relatively small difference in CarE activity between perus. the susceptible and some resistant populaMATERIALS AND METHODS tions suggestedthat other mechanisms contributed to the resistance. Insects. L. hesperus collected from an alA reduction in the sensitivity of acetyl- falfa seed field near Nampa, Idaho, and from alfalfa hay fields near Logan, Utah, were used as resistant (R) and susceptible ’ Abbreviations used: CarE, carboxylesterase; (S) insects, respectively. AChE, acetylcholinesterase; R, resistant; S, susceptiChemicals. The following chemicals ble; ASCh, acetylthiocholine iodide; DTNB, 5,5’-dithiobis-2-nitrobenzoic acid. were purchased from Sigma Chemical Co. 22 0048-3575190 $3.00 Copyright 0 1990 by Academic Press. Inc. All riglIt.3 of re.pmdwtion in any form reserved.
ACETYLCHOLINESTERASE
(St. Louis, MO): acetylthiocholine iodide (ASCh), bovine serum albumin, 5,5’-dithiobis-2nitrobenzoic acid (DTNB), eserine sulfate. Paraoxon (diethyl 4nitrophenyl phosphate), 95% pure, was obtained from Chem Service (West Chester, PA). Enzyme preparations. Groups of 20-50 adult L. hesperus were homogenized in 510 ml of ice-cold 0.05 M phosphate buffer, pH 8.5 (pH 7.5 for study of paraoxon inhibition), using a Potter-Elvehjem homogenizer in an ice bath with a motor-driven Teflon pestle. The homogenate was centrifuged at 10,000g for 20 min at 4°C in a Beckman LS-65B ultracentrifuge. The supernatant was kept on ice and used as the enzyme source for AChE assays. The separation of subcellular fractions for AChE was designed according to Crankshaw et al. (5) and Aldunate et al. (6). Forty bugs were homogenized in 10 ml of 0.05 M phosphate buffer, pH 8.5. The homogenate was filtered with two layers of cheesecloth to remove the gross debris. The filtered homogenate was brought up to a volume of 10ml with the same buffer, and then divided into halves, one of which was used as crude homogenate. The other half was centrifuged at 470g for 10 min to obtain a pellet which contained nuclei and cell debris. The supernatant was further centrifuged at 10,000g for 20 min to obtain a mitochondrial pellet. The resultant supernatam was again centrifuged at 105,OOOgfor 90 min to obtain the microsomal pellet, and the final supernatant corresponding to the cytosol was used as the soluble fraction. AChE assays. AChE activity was measured according to the method of Ellman et al. (7) with some modifications. A 2.9-ml aliquot of 0.05 M phosphate buffer, pH 8.5, was pipetted into four test tubes. Three of them received a lOO-~1aliquot of the homogenate and the one without homogenate was run as the control. One hundred microliters of 10 mM DTNB reagent and 20 pJ of 75 mM ASCh were pipetted into these four test tubes. The mixture was prepared in an
IN L.
23
hesperus
ice bath and ASCh was added just before incubation. After incubation for 15 min at 38°C in a shaking water bath (the enzyme activity remained linear for at least 24 min), the reaction was stopped by adding 100 ~1 of 0.322 mit4 eserine sulfate; the final concentration of eserine in the mixture (10 ~.LM) completely inhibited enzyme activity (1). One hundred microliters of homogenate was added to the control to correct for interference by the homogenate. Absorbance at 412 nm was determined against the control with a spectrophotometer. Inhibition of AChE by paraoxon was studied based on the method of Oppenoorth (8). The homogenate was preincubated with an equal volume of 2 or 4 x lo-’ M paraoxon (dissolved in acetone and diluted with 0.05 M phosphate buffer, pH 7.5) for various periods of time between 5 and 60 min at 25°C in a shaking water bath. After ASCh and DTNB were added, the mixture was further incubated for 20 min under the same conditions. The reaction was stopped by the addition of eserine, and the rest of the procedure was as described above. At the same time, a similar experiment, but preincubated with the buffer containing an appropriate amount of acetone instead of paraoxon, was carried out to correct for denaturation of AChE. Protein concentrations of each enzyme preparation were determined according to the method of Bradford (9) using bovine serum albumin as a standard. Data analysis. The kinetic parameters, Km and Vmax,were determined by Lineweaver-Burk transformations (10). Bimolecular rate constants (kj) were calculated as described by Main and Iverson (11). The data were tested by t tests or analyses of variance. Duncan’s multiple range test (12) was used in the multiple comparison; P 6 0.05 was considered significant. RESULTS
Figure 1 shows the AChE activity toward ASCh at pH values between 5.5 and 9.0 at 35°C. Although the peak activity occurred
ZHU AND BRINDLEY
24
201
5.0
.
’
6.0
.
’
7.0
’
’
6.0
.
’
-4.5
’
9.0
-4.0
Concentration
PH FIG. 1. EfJect of pH on the hydrolysis of ASCh by AChE from L. hesperus. Temperature of reaction was 35°C. Each point represents the mean of three determinations (each with triplicate incubations). Vertical bars indicate standard deviations of the mean.
at pH 8.5, the optimal pH for the AChE appeared to be fairly broad. The activities at pH 7.5 and 9.0 were, respectively, only 10.6 and 0.5% less than at pH 8.5. Temperature effects on AChE activity were studied from 15 to 55°C (Fig. 2). Activity increased gradually to 35°C but decreased rapidly when temperatures were higher than 45°C. The optimal temperature was 35dO’C. Figure 3 shows the AChE activity from
20
30 Temperature
40
50
-2.5
of Substrate
(Log M)
(%)
.
.O
3.
x lop5 to 8 x 10e3 M ASCh. The activity increased from the concentration of 6.25 x lo-’ to 2 x 10m3M where the peak activity was obtained. The distribution of AChE activities in the head, thoracic, and abdominal homogenates is shown in Table 1. Most of the total activity was located in the thorax and abdomen. However, the specific activity of AChE was much higher in the head homogenate than in the thoracic or abdominal homogenates. The total activity of the AChE was almost evenly distributed in four subcellular fractions (Table 2). However, only about 85% of the crude homogenate activity was recovered in these four fractions. The highest specific activity was found in the mitochondrial fraction and then in the microsomal fraction. The specific activities were relatively low in the nuclei and cell 6.25
TABLE 1 of AChE Activity in Different Body Parts of Adult L. hesperusa
60
2. Effect of temperature on the hydrolysis of ASCh by AChE from L. hesperus. Each point represents the mean of three determinations (each with triplicate incubations). Vertical bars indicate standard deviations of the mean. FIG.
-3.0
Effect of substrate concentration on the hydrolysis of ASCh by AChE from L. hesperus. Each point represents the mean of three determinations (each with triplicate incubations). Vertical bars indicate standard deviations of the mean. FIG.
Distribution 10
-3.5
BUY PM
Head ThOraX
Abdomen
% Total activity 14.2 + 0.6 50.5 f 3.5 35.2 f 4.1
specific activity (nmoVmin/mg protein) 86.1 f 7.5 28.7 k 2.6 19.9 * 1.7
a Results are the mean 2 SD of three determinations with triplicate incubations).
(each
ACETYLCHOLINESTERASE
20 1
TABLE2 Subcellular Distribution of AChE Activity in Adult L. hesperus”
Fraction Crude homogenate Nuclei and cell debris Mitochondria Microsome Soluble traction % Recovery
% Total activity
15
Specific activity (nmoUmin/mg
25
IN L. hcsperus
0 0
S population R population / .
>
protein)
5’
2
100 11.2 29.9 20.8 23.0 84.9
r + k k
2.3 2.4 2.6 2.4
37.0 19.0 82.3 68.3 21.8
2 * 2 f -+
1.9 4.0 3.8 4.2 2.9
;; 0 0
10
0,: . /
--/
a Fractions obtained by centrifugation as described under Materials and Methods. Results are the mean 2 SD of three determinations (each with triplicate incubations).
debris fraction, as well as in the soluble fraction. Table 3 shows the protein content, the total activity, and the specific activity of AChE for both sexes of the S and R populations. The total activities between the two populations and between the two sexes were the same (P > 0.05), but the specific activities were different in both cases (P < 0.05). Insects in the R population contained more protein leading to these differences. A difference in the protein content between the S and R populations was simply due to their body size. The insects collected from alfalfa seed fields were usually larger than those from alfalfa hay fields. Figure 4 and Table 4 show a LineweaverBurk (double reciprocal) plot and the kinetic parameters (K, and V,,) for AChE hydrolyzing ASCh in the S and R populations at 38°C. There were no significant dif-
-10
1’
0
I
1
,
10
20
30
I
40
Sex
Protein ww9
S
Female Male Female Male F df P
531.7 2 26.3 434.5 2 41.1 898.8 ” 37.0 629.6 -+ 24.1 110.90 3, 8 co.001
R
I
60
ml
ferences between two populations in either parameter. There was a significant difference in the inhibition of AChE by paraoxon between the S and R populations in the two series of five paired experiments (Fig. 5). When the homogenates prepared from the resistant insects were preincubated with paraoxon at 1 x 10e7 M, the activities were blocked only about 7% in the first 10 min and remained the same up to 1 hr of preincubation. Although 23.5% of the AChE activity was inhibited by 2 x 10m7M paraoxon in the first 5 min, the inhibition increased rapidly up to 10 min and then increased only
Total activity (nmoL!min/bug) a b c d
I
50
FIG. 4. Lineweaver-Burk (double reciprocal) plot of 100 x (I/v) vs ll[sl for AChEfrom S or R populations of L. hesperus. Each point is based on the mean of three determinations (each with triplicate incubations). Regression coeficient (r) > 0.99 (P < 0.001).
TABLE 3 Quantity of Protein and Total and Specific Activities of AChE in S and R Populations Population
/
-
18.6 21.3 18.6 20.3
2 2.0 a 2 2.4 a +- 2.0 a 2 1.9 a 1.26 3, 8 NSb
of L. hesperus”
Specific activity (nmoYmin/mg protein) -34.9 2 2.1 a 49.0 e 0.7 b 20.7 k 2.9 c 32.4 + 3.9 a 56.00 3, 8
B Results are the mean k SD of three determinations (each with triplicate incubations). Means within columns followed by the same letter are not significantly different (P > 0.05; Duncan’s (1955) multiple range test). b Not sign&ant at P > 0.05.
ZHU AND BRINDLEY
26
TABLE 4 Kinetic Parameters of AChE Hydrolyzing Acetylthiocholine Population
4, (PM
s R
in S and R Populations of L. hesperus”
VIn* (nmoGnin/mg protein)
Regression coefficient (r)
43.4 f 2.6 45.3 + 5.4
>0.99 BO.99
119.4 + 4.1 106.2 2 17.6
sign&kc
(P)
a Results are the mean 2 SD of three determinations (each with triplicate incubations). There is no significant difference in K,,, or V,,,, values between the two populations (P > 0.05; t test).
about 5% up to 1 hr of preincubation. In contrast, AChE activities in the susceptible insects were much more sensitive to inhibition by paraoxon at either concentration, and both time-inhibition curves were linear and intercepted the origin. The ki (bimolecular rate constant) was (2.8 -+ 0.1) X ld M- ‘min- ’ (mean f SE) for the S population (calculated from the two paraoxon concentrations) and (8.3 + 1.7) X lo3 M- ‘min- ’ for the R population (calculated from the second part of the curve for 2 X 10m7M paraoxon). The rate of inhibition of the R population was about 34-fold slower than that of the S population. DISCUSSION
The optimal pH for AChE from L. hesperus (8.5) resembles those of other insects:8-9 for Musca domestica (13), 8.0 for
Acheta domesticus (14), 7.5 for Aphis cifricola (15)) 7-9 for Chironomus riparius (16), and 8.5 for Munduca sexta (17).
AChE activity was pH dependent only at pH <8 and seemed to be tolerant to high pH. However, whether this tolerance is due to substrate specificity (18) or species specificity is unknown. As to be expected of an enzyme, the activity of the AChE from L. hesperus with respect to temperature provided a bellshaped curve (Fig. 2). The optimal temperature appeared to be 35-40°C, which was slightly higher than 30-3X for M. domestica (13), 30°C for A. citricolu (15) and C. riparius (16), and 37°C for M. senta (17). Inhibition by excess substrate is one feature which distinguishes AChE from butrylcholinesterase (18). L. hesperus AChE was moderately inhibited at high concentrations of ASCh (Fig. 3) and shows a broad optimum of L. hesperus AChE for substrate concentrations. An explanation for the high total activity in the abdomen with little nervous tissue is not readily available. Since the concentration of ASCh used in this study was reasonably low (0.48 n&f), it is unlikely that the high total activity in abdominal homogenates was due to nonspecific enzymes which hydrolyze ASCh at high concentrations (19). It seems likely that the AChE activity in L. hesperus is membrane bound. Only about 23% total activity remained in the supematant after centrifugation at 105,OtlOg for 90 min. In Boophilus microplus, only 10% of AChE activity remained in the 100,OOOgsupernatant (20). In C. riparius larvae, about 85% of total activity remained
2z‘-------o-.-.-w-ori v)
&k
1
50
0
s. 40 = s Y
5 2
0
2x10-’
n
1x10-7
n
0
1
20
0
0
1
n
30
s
z
1x10-’
-*-o-e-
l
I I0 0
.
“\
S population R population
I 10
.
I. 20
2x10-7
n
0
I 30
.
.\. 40
I 50
.
I 60
1
l%m (min) FIG. 5. Inhibition ofAChEfrom S or R populations of L. hesperus by 1 or 2 x 10m7 M paraoxon with various periods of time. Each point is based on the mean of three duplicate incubations.
ACETYLCHOLINESTERASE
in the 10,OOOgsupernatant and 76% in the 100,OOOgsupernatant (16). The kinetic rate constant, K,, for AChE from L. hesperus is within similar ranges of 1.67 x 10e4 M for A. domesticus (14), 6.7 x 10m5M for A. citricolu (15), 2.54 x 10V5 M for C. riparius (16), 2.7 x 10m4M for M. sexta larvae (17) and 1.5 x low4 Mfor adult (21), and 3.3 x 10e5 M for Triutoma infestans (22). Neither kinetic parameter, K, nor V-, in the R population differed from the S population (Table 4), which indicated that the afhnity of AChE to the substrate (ASCh) and catalytic efficiency of AChE in the two populations were essentially identical. Although it seems possible to determine the kj value for the AChE from the R population more accurately with higher paraoxon concentrations, it is obvious that the sensitivity of the AChE to the inhibition by paraoxon was significantly different from that of the S population (Fig. 5). AChE from the R population was about 34-fold less sensitive than that from the S population. This seemsto be the first time that this kind of resistance mechanism has been found in hemipteran insects, although a similar mechanism had been found in other arthropods (2). The inhibition pattern of AChE by 2 x 10e7 M paraoxon in the R population was very similar to that in the S population during the first few minutes, but it slowed dramatically in about 10 min. This suggests that AChE from the R population was heterogeneous and contained a small amount of susceptible enzyme that could be inhibited very rapidly. Although the sensitivity of AChE to inhibition by paraoxon was reduced in the R population, the K, and V,, values were the same as those in the S population, indicating that there was no change in catalytic activity toward ASCh. If this reflects AChE catalytic ability to the natural substrate, the insects might have an advantage for their survival, in comparison with some other arthropods in which insensitivity to inhibitors
IN L. hesperus
27
was associated with decreased affinity (higher K,,J to the substrate (23) or reduced catalytic efficiency (lower V-> (20,24) because AChE can normally catalyze the hydrolysis of acetylcholine regardless of its insensitivity to the inhibitor. Animals may have a considerable excess of AChE relative to what they strictly need (25), but this would not necessarily obviate the advantage of L. hesperus’ insensitive AChE. Pest managers concerned with L. hesperus should be aware of this possible dimension to the insect’s resistance to insecticides. ACKNOWLEDGMENTS This research was supported by grants from the Western Region Pesticide Impact Assessment Program and Western Region Integrated Pest Management Special Grants Program, and Projects 505 and 577 of the Utah Agricultural Experiment Station, Utah State University, Logan, Utah. Approved as journal paper No. 3855. REFERENCES 1. K. Y. Zhu, “Properties of Esterases from Lygus hesperus Knight (Hemipera: Miridae) and the Roles of the Esterases in Insecticide Resistance,” MS thesis, p. 116, Utah State University, Logan, 1989. 2. F. J. Oppenoorth, Biochemistry and genetics of insecticide resistance, in “Comprehensive Insect Physiology, Biochemistry and Pharmacology” (G. A. Kerkut and L. I. Gilbert, Eds.), Vol. 12, pp. 731-773, Pergamon, New York, 1985. 3. J. Hemingway and G. P. Georghiou, Studies on the acetylcholinesterase of Anopheles albimanus resistant and susceptible to organophosphate and carbamate insecticides, Pestic. Biothem. Physiol. 19, 167 (1983). 4. R. K. Tripathi and R. D. O’Brien, Insensitivity of acetylcholinesterase as a factor in resistance of house flies to the organophosphate Rabon, Pestic. Biochem. Physiol. 3, 495 (1973). 5. D. L. Crankshaw, H. K. Hetnarski, and C. F. Wilkinson, Microsomal NADPH-cytochrome c reductase from the midgut of the southern armyworm (Spodoptera eridania), Insect Biothem. 9, 43 (1979). 6. J. Aldunate, Y. Repetto, M. E. Letelier, and A. Morello, The carboxylesterases of Trypanosoma cruzi epimastigotes, Comp. Biochem. Physiol. B 86, 67 (1987). 7. G. L. Ellman, D. K. Courtney, V. Andres, and R. M. Featherstone, A new and rapid colori-
28
ZHU
AND
metric determination of acetylcholinesterase activity, Biochem. Pharmacol. 7, 88 (1961). 8. F. J. Oppenoorth, Two different paraoxonresistant acetylcholinesterase mutants in the house fly, Pestic. Biochen. Physiol. 18, 26 (1982).
9. 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, 248 (1976). 10. J. E. Bell and E. T. Bell, “Proteins and Enzymes,” p. 499, Prentice-Hall, Englewood Cliffs, NJ, 1988. 11. A. R. Main and F. Iverson, Measurement of the affinity and phosphorylation constants goveming irreversible inhibition of cholinesterases by di-isopropyl phosphorofluoridate, Biochem. J. 100, 525 (1%6). 12. D. B. Duncan, Multiple range and multiple F tests, Biomefrics 11, 1 (1955). 13. A. Silver, “The Biology of Cholinesterases,” p. 596, Amer. Elsevier, New York, 1974. 14. A. H. Lee, R. L. Metcalf, and C. W. Keams, Purification and some properties of house cricket (Acheta domesticus) acetylcholinesterase, J. Insect Physiol. 4, 267 (1974). IS. S. Manulis, I. Ishaaya, and A. S. Perry, Acetylcholinesterase of Aphis citricola: Properties and significance in determining toxicity of systemic organophosphorus and carbamate compounds, Pestic. Biochem. Physiol. 15, 267 (1981). 16. R. L. Detra and W. J. Collins, Characterization of cholinesterase activity in larval Chironomus riparius Meigen (=Thummi kiefer), Insect Biothem. 16,733 (1986). 17. D. S. Lester and L. I. Gilbert, Characterization of acetylcholinesterase activity in the larval brain
BRINDLEY
of Manduca sexta, Insect Biochem. 17, 99 (1987). 18. K. Bui and R. F. Ochillo, Characterization of cholinesterase of muscularis muscle of Bufo marinus, Comp. Biochem. Physiol. C 87, 107 (1987).
19. H. R. Smissaert, S. Voerman, L. Oostenbrugge, and N. Renooy, Acetylcholinesterases of organophosphate-susceptible and resistant spider mites, J. Agric. Food Chem. 18, 66 (1970). 20. J. Nolan and H. J. Schnitzerling, Characterization of acetylcholinesterase of acaricide-resistant and susceptible strains of the cattle tick Boophilus microplus (Can.), Pestic. Biochem. Physiol. 5, 178 (1975). 21. D. J. Prescott, J. G. Hildebrand, J. R. Sanes, anil S. J. Jewett, Biochemical and developmental studies of acetylcholine metabolism in the central nervous system of the moth Manduca sexta, Comp. Biochem. Physiol. C 56, 77 (1977). 22. E. Wood, E. Zerba, M. Picollo, and S. de Lacastro, Partial purification and characterization of Triatoma infestans head acetylcholinesterase, Insect Biochem. 9, 595 (1979). 23. A. L. Devonshire and G. D. Moores, Different forms of insensitive acetylcholinesterase in insecticide-resistant house flies (Musca domestica), Pestic. Biochem. Physiol. 21, 336 (1984). 24. A. L. Devonshire, Studies of the acetylcholinesterase from houseflies (Musca domestica L.) resistant and susceptible to organophosphorus insecticides, Biochem. J. 149, 463 (1975). 25. H. R. Smissaert, F. M. Abd El Hamid, and W. P. J. Overmeer, The minimum acetylcholinesterase (AChE) fraction compatible with life derived by aid of a simple model explaining the degree of dominance of resistance to inhibitors in AChE “mutants,” Biochem. Pharmacol. 24, 1043 (1975).