19, 167- 171 (1983)
Studies on the Acetylcholinesterase of Anopheles Resistant and Susceptible to Organophosphate Carbamate Insecticides’ JANET Division
and Physiology, Department Riverside, California
of Entomology. 92521
Received August 6, 1982; accepted September 30, 1982 Acetylcholinesterase from fourth instar Anopheles albimanus larvae was studied in vitro. The acetylcholinesterase from both the resistant and susceptible strains behaved as a single enzyme “type,” with straight pseudo first-order insecticide inhibition lines which intersected the Y axis at 100%. The enzyme from resistant larvae was more slowly inhibited than the susceptible enzyme; bimolecular rate constants (ki) differed by approximately 1.2- to dfold for a range of organophosphorous compounds and 17- to 1.570-fold for the carbamates. There was a good correlation between the levels of resistance and the acetylcholinesterase inhibition rates. INTRODUCTION
albimanus from El Salvador.
The relationship between the degree of enzyme inhibition and the resistance levels is also investigated.
Organophosphorous and carbamate insecticides act by inhibiting acetylcholinesterase (AChE). Some resistant strains of arthropods, e.g., Tetranychus urticae (l), T. paci$cus (2), T. telarium (3), Boophilus microplus (4, S), Musca domestica (6), Anopheles albimanus (7), and Nephotettix cincticeps (8) have developed a modified
AChE which is insensitive to some of these insecticides. As such a modified enzyme does not confer resistance to the entire spectrum of organophosphorous compounds and carbamates, we need to know which insecticides are affected, and what, if any, interaction there is between this resistance and other resistance mechanisms that may be present in field populations. The present study looks at the inhibitory effect of various insecticides on the altered AChE of An. ’ These studies were supported in part by Special State Funds for Mosquito Control Research appropriated annually by the California Legislature. 4 Present address: Department of Entomology, London School of Hygiene and Tropical Medicine, Keppel St. (Gower St.), London WClE 7HT, England. To whom correspondence should be addressed.
Two strains of mosquitoes were used. The EP-R strain originated from La Libertad, El Salvador (1970), and was subsequently selected at the larval stage with propoxur for eight generations. Larvae of this strain were then selected every generation with ethyl parathion until 1982. This was originally the OP-R strain utilized in the study of Ayad and Georghiou (7). The GORGAS strain is a laboratory strain from the Gorgas Memorial Laboratory in Panama which has never been exposed to insecticides. Resistance levels were determined by bioassaying fourth instar larvae. Groups of thirty larvae were held in waxed paper cups with 100 ml of water, to which 1 ml of insecticide solution in acetone was applied. Mortality was determined after 24 hr. There was no mortality in control cups treated with 1 ml of acetone. The staining intensity of acetylcholinesterase with acetylthiocholine iodide was vi167 00483575/83 $3.00 Copyriabt All &bts
0 1983 by Academic Press, Inc. of npmduction in any form mscrved.
sualized on filter paper by the method of Miyata et al. (9). Fenitroxon was produced from fenitrothion by the method of Falscheer and Cook (10). Structural identity of this and other oxygen analogs of the organophosphorous compounds used in the AChE inhibition study were determined by nuclear magnetic resonance spectrometry and any necessary purification was by thin-layer chromatography. Stock solutions of inhibitor (1 x 10m2M) were prepared in acetone and dilutions were made in glass-distilled water. The concentration of acetone in the final incubation mixture did not exceed 1% and had no effect on enzyme activity. The enzyme solution was prepared by homogenizing 1000 fourth instar larvae in 0.1 M phosphate buffer at pH 7.5 at a grinding ratio of 100 larvae/ml buffer. The homogenate was centrifuged at 10,000 rpm for 30 min and the supernatent was then used as the source of the enzyme. AChE activity was measured at 25°C and pH 7.5 by the technique of Ellman et al. (11) with acetylthiocholine iodide as the substrate. Inhibition studies were carried out by forcing 10 ~1 of inhibitor under the surface of 1 ml of enzyme. At 15set intervals, loo-p.1 samples of this mixture were
added to cuvettes containing 2.9 ml of substrate plus 5,5-dithiobis(2-nitrobenzoic acid). The absorbance at 420 nm was recorded for 5 min. The bimolecular rate constant (ki) was calculated by the method of Aldridge (12). The slopes of the plots were obtained by linear regression analysis. RESULTS
Resistance levels. Table 1 indicates the resistance levels of EP-R to a number of organophosphates and carbamates compared to GORGAS. The highest levels of resistance (> lOOO- and 73-fold) were to the carbamates propoxur and carbaryl. Resistance to the organophosphorous compounds tested ranged from 37.5-fold for fenitrothion to l.Zfold for temephos. Filter paper test. There was insufficient difference in staining intensity between the 200 resistant and susceptible adults or larvae that were tested to allow consistently accurate determination of the presence or absence of the modified AChE using this method. Differentiation of resistant and susceptible individuals was not improved by immersing the filter paper in the substrate/stain mixture rather than spraying it. In vitro enzyme inhibition. Table 2 gives
TABLE 1 LC,, Values for Resistant and Susceptible Anopheles albimanus for a Range of Organophosphate and Carbamate Insecticides
LC,, (mfdl) Temephos Fenthion Methyl chlorpyrifos Ethyl chlorpyrifos Phenthoate Fenitrothion Malathion Parathion Chlorphoxima Carbaryl Propoxur
0.006 0.03 0.02 0.008 0.035 0.04 0.35 0.007 0.012 0.89 0.39
0.007 0.17 0.03 0.22 0.25 1.5 6.2 0.25 0.08 65.3 >390
1.2 5.0 1.3 26.8 7.2 37.5 17.7 36.2 6.8 73 > 1000
OF ACETYLCHOLINESTERASE 2
Rate Constants (k,)” for Inhibition of Acetylcholinesterase Susceptible Anopheles albimanus Measured in the Absence
lo-’ x ki (M-l
from Resistant of Substrate
0.38 0.21 1.8
0.46 0.28 3.5 0.39 327 94.9 6.8 53.6 5.5 36.0 42.4
1.2 1.3 2.0
Fenoxon Chlorpyrifoxon methyl Chlorpyrifoxon Fenitrothion Fenitroxon Malaoxon Methyl paraoxon Paraoxon Chlorphoxon Carbaryl Propoxur
58.1 71 4.3 10.3 4.6 2.1 0.003
5.6 1.3 1.6 5.2 1.2 17.3 1570
a ki values quoted are averages of not less than five replications.
the bimolecular rate constants (ki) for various organophosphorous compounds and carbamates against EP-R and GORGAS. In the case of fenitrothion, ki values were calculated for both the P=O and P=S analogs for the susceptible strain. This demonstrated, as expected, that the P=O compound had higher activity than the P=S analog (approximately [email protected]
). In all cases, the ki values for the resistant strain were greater than those for the susceptible, i.e., the AChE of the resistant strain was less sensitive to inhibition than that of the susceptible. However, the differences between the resistant and susceptible strains were low (1.2- to 5.6-fold) for TABLE Ranking
of Resistance albimanus
The LC,, ratios for the organophosphorous compounds and carbamates reported here differ by &threefold from those obtained in the earlier studies by Georghiou et 3
(RR) and ki Ratios (k, R) for Multiple Compared to a Laboratory Susceptible
Decreasing inhibition differential
RR Propoxur Carbaryl Fenitrothion Parathion Chlorpyrifos Malathion Chlorphoxim Fenthion Chlorpyrifos
all compounds with the exception of propoxur and carbaryl. Table 3 shows that there is a good correlation between the LC,, ratios (RR) and the ki ratios of the resistant and susceptible strains. The greater the level of resistance the greater the difference in ki. Thus, propoxur has the highest RR and the greatest ki ratio of all the compounds tested, whereas chlorpyrifos methyl has a low RR and a low ki ratio.
L, R Propoxur Carbaryl Fenitroxon Paraoxon Chlorpyrifoxon Malaoxon Chlorpyrifoxon Chlorphoxon Fenoxon
al. (13) and Ayad and Georghiou (7). This is accounted for in part by the fact that most of the LC,,, values of the susceptible GORGAS strain used in the present study are somewhat higher than those of the susceptible “Haiti’ ’ strain used in the earlier work. The EP-R strain has also been under parathion selection since 1971, whereas the OP-R strain of Ayad and Georghiou (7) had been under strong selection with propoxur prior to being selected for an additional 17 generations with parathion, which may also account for some of the differences. It is not known whether carbamates select more strongly for the altered AChE mechanism than the organophosphorous compounds. However, it may be significant that in Nephotettix cincticeps, the appearance of the resistant AChE was not observed until after the development of carbamate resistance on top of the existing organophosphorous resistance (8). The lower LC5,, ratios may suggest that the EP-R strain used in these experiments was not homozygous for the altered AChE mechanism. However, the pseudo firstorder carbaryl and propoxur inhibition lines for the EP-R enzyme were straight, whereas an equal mixture of EP-R and GORGAS enzymes produced a curved line. This indicates that the EP-R strain is behaving as a single enzyme “type,” unlike the EP-RGORGAS mixture which contains enzymes with different susceptibility to inhibition. The inhibition lines for the EP-R enzyme with propoxur also intersected the Y axis at lOO%, which indicates that the enzyme did not contain a small amount of susceptible enzyme which may have been very rapidly inhibited by the high concentrations of insecticide required to progressively inhibit the EP-R enzyme. The bimolecular rate constant (,4J for all the organophosphorous compounds and carbamates tested revealed that the AChE of the EP-R strain is less sensitive to inhibition than that of GORGAS. This indicates that the altered AChE mechanism probably confers resistance to a broad spectrum of
organophosphorous compounds and carbamates. But, there is only a large difference in sensitivity with propoxur and carbaryl. The ki values of paraoxon reported here are considerably lower than those reported previously (7). This may be due to the use of a nonrepurified sample of paraoxon in the earlier inhibition study. The differences in resistant and susceptible ki values reported here for the organophosphorous compounds are generally low (1.3- to 5.6fold). Devonshire (6) found differences in ki of a similar order of magnitude in resistant and susceptible house flies, which produced low-level resistance (2- to lo-fold). This resistance mechanism was shown to interact with other resistance mechanisms to produce high-level resistance (14). An insensitive AChE in Nephotettix cincticeps also shows similar ki ratios for malaoxon propoxur and carbaryl to those reported here (8, 15, 16). However, the ki ratio reported for Tetranychus urticae for paraoxon are much larger (17). There is a good correlation between the RR and the ki ratios of the resistant (EP-R) and susceptible GORGAS strains. The greater the levels of resistance, the larger the difference in ki values. The only exception to this is chlorpyrifos methyl, which has the same ki ratio as malaoxon but a lower RR. The correlation between RR and ki ratios may indicate that the altered AChE mechanism is the only resistance mechanism present in this population, and this is in agreement with the monofactorial inheritance of resistance in this population (13). We have, as yet, no simple method to determine the extent of the altered AChE mechanism in field populations. The filter paper technique described for N. cincticeps (9) is too insensitive for use with either adults or larvae of An. albimanus, even though there is a distinct difference in the K, values of the susceptible and resistant strains for acetylthiocholine (7). The EP-R laboratory strain may, therefore, be useful
as a standard to help in the determination of the frequency of the AChE mechanism in the field. The present study indicates that this altered AChE mechanism extends to a wide range of organophosphorous and carbamate compounds, although the resistance levels it confers toward organophosphorous compounds are low.
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