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The biochemical nature of malathion resistance in Anopheles stephensi from Pakistan
PESTICIDE
BIOCHEMISTRY
AND
PHYSIOLOGY
The Biochemical
17, 149-155
(1982)
Nature of Malathion Resistance stephensi from Pakistan
in Anopheles
JANET HEMINGWAY* Department
of Entomology,
London School of Hygiene Street), London WClE
and Tropical 7HT, England
Medicine,
Keppel
Street
(Go+ver
Received September 29, 1981; accepted December 3, 1981 Malathion resistance in Anopheles stephensi from Pakistan was synergized by triphenyl phosphate, primarily a carboxylesterase inhibitor. There was a slight degree of antagonism with piperonyl butoxide. The major metabolite of malathion in larvae of both the resistant and susceptible strains was malathion monocarboxylic acid. Resistant larvae produced about twice as much of this product as the susceptible larvae. This suggests that a qualitative or a quantitative change in a carboxylesterase enzyme may be the basis of malathion resistance in this strain. Analysis of general esterase levels to a- and fi-naphthyl acetate showed that there was no quantitative change in the amount of carboxylesterase enzyme present in the resistant strain as compared to the susceptible. INTRODUCTION
Malathion and phenthoate resistance have been detected in Anopheles stephensi from near Lahore, Pakistan (1, 2). Laboratory selection with malathion for 14 generations increased the level of resistance to phenthoate and vice versa. This suggested that the same mechanism could be responsible for resistance to both compounds. As both malathion and phenthoate contain a carboxylester bond, the resistance may be based on a common detoxication pathway for the two insecticides. Genetic analysis indicated that only one major biochemical mechanism was involved in malathion resistance, as resistance was inherited monofactorally (2). Synergist and metabolic studies of malathion resistance have, therefore, been undertaken to give further information on the detoxication pathway underlying resistance in this population. MATERIALS
AND METHODS
The following
laboratory colonies of A. ST, a laboratory insecticide-susceptible population originating from an area near Dehli, India, and
stephensi were used:
’ Present address: Division of Toxicology and Physiology, Department of Entomology, University of California, Riverside, California 92521.
maintained as a colony since 1947; ST LA, a population from Lahore, Pakistan, colonized in 1974, resistant to malathion and phenthoate; ST MAL, a colony derived from ST LA by adult selection with 5% malathion for up to 8 hr in every generation (adults of the F,, generation were 24x more resistant to malathion than ST); and ST LAMA, a colony derived from ST LA by larval selection for five generations with 0.5 ppm malathion (larvae of the F, generation were 3~ more resistant to malathion than those of ST). Chemicals
The following chemicals were used: piperonyl butoxide (PB) ([3,4-(methylenedioxy)d-propylbenzyl] n-butyl diethyleneglychol ether); triphenyl phosphate (TPP); SV, (O,O-dimethyl-O-phenyl phosphorothioate); malathion (O,O-dimethyl-S-(1,2di-(ethoxycarbonyl)ethyl)phosphorodithioate) (greater than 99% pure); malaoxon (O,O-dimethyl-S-(1,2-di-(ethoxycarbonyl)ethyl)phosphorothioate); malathion monocarboxylic acid (O,O-dimethyl-S-(l-carboxy-2-ethoxycarbonyl)phosphorodithioate); malathion dicarboxylic acid (0, Odimethyl S-(1,2-dicarboxy)phosphorodithioate) (produced by the method of March et al. (3)); DMPDT (O,O-dimethyl phos149 0048-3575/82/020149-07$02.00/O Copyright All rights
0 1982 by Academic Press, Inc. of reproduction in any form reserved
150
JANET
HEMINGWAY
phorodithionic acid); DMPT (O,O-dimethyl (A) After homogenizing larvae in a small phosphorothionic acid); DMP (O,Oamount of anhydrous sodium sulfate twice dimethyl phosphate); C14-labeled malathion in 5 ml hexane, the homogenate was filtered was obtained from the Radiochemical and the residue washed with a further 5 ml Centre, Amersham (sp act, 4.6 mCi/mM) of hexane. The filtrates were combined (labeled in both carbon atoms of the suc- and evaporated to dryness on a rotary cinyl part of the molecule, the labeled evaporator. This procedure was repeated malathion was diluted I:2 with nonlabeled using diethyl ether and acetonitrile. Metabmalathion in absolute ethanol to give a olites were extracted from the larval test working solution of O.l%, which had an ac- medium using the same procedure with tivity of 8,410,005.5 cprn/ml); and DCQ hexane and diethyl ether as solvents. The (2,6-dibromoquinone-4-chlorimide). Pre- remaining water was then evaporated to coated plastic thin-layer chromatography dryness. All extracts were subsequently re(TLC) sheets coated with 0.2 mm silica gel dissolved in 0.1 ml of the relevant solvent. N-HR/uv,, were used. The purity of all (B) At the conclusion of the insecticide chemicals was regularly assessed by TLC. exposure period the larvae were removed TLC plates were sprayed with 0.5% DCQ in and thoroughly washed with 5 ml of discyclohexane and heated to 100°C to visu- tilled water, which was added to the test alize the spots. medium. The pH of the test medium was then adjusted to pH 7 with 0.1 N sodium Synergist Studies hydroxide and hydrochloride acid. MalaAdult mosquitoes were exposed to the thion and malaoxon in the medium were synergist or insecticide by tarsal contact than partitioned into 10 ml of chloroform. using the standard WHO test kits. Test pa- The water was acidified to pH 2.5 with hypers were prepared by impregnating rect- drochloric acid and again partitioned with angles of Whatmans No. 1 filter paper (12 x 10 ml of chloroform. Larvae were homog15 cm) with the required concentration of enized in 3 ml of distilled water and extracsynergist or insecticide spread at 3.6 ~gl tion was carried out in the same way. At least three replicates were carried out cm2. Any survivors of the synergist/ insecticide treatment were hand-mated and with each extraction method. Controls their progeny again selected in the same were run without the larval homogenate to way. determine the level of nonenzymatic hydrolysis of malathion under the experiEsterases mental conditions used. Total nonspecific esterase activity was assayed spectrophotometrically by the Analysis (after Extraction Method A) method of van Asperen (4) and by the filter TLC plates were prerun with chloroform paper spot technique of Pasteur and Geor- for 1% hr prior to adding extracts. Extracts ghiou (5) using adult mosquitoes. Values in were placed on the origin line of the TLC the former were corrected for the weight of plate using a 5-~1 capillary tube. TLC plates the insect and the amount of homogenate were run with a hexane:diethyl ether (1:3) used. solvent system (6). A set of standard metabolites was run on each plate to check the Larval Maiathion Metabolism position of metabolites. Rf values were reBatches of 100 fourth-instar larvae were producible. exposed to 2 ppm malathion in 100 ml of Quantification distilled water for up to 3 hr. Metabolites were_ extracted by either of the following After extraction of metabolites by methods. method A and running on TLC plates, me-
NATURE
OF MALATHION
RESISTANCE
tabolites were reeluted with either hexane, diethyl ether, or acetonitrile. The solutions were placed in counting vials and air-dried, then 100 ~1 of absolute alcohol, 40 ~1 of distilled water, and 4 ml of scintillation fluid (Packard Scintillation cocktail, Scintillator 299) were added to each counting vial. Samples were counted in a scintillation counter for 10 min or up to 10,000 counts. After extraction method B extracts were placed into vials, air-dried, and treated as
Synergist Studies
The results of selection of ST MAL with TPP + malathion for five generations are given in Table 1. The survivors of the second and third selected generation were all males. They were hand-mated to virgin females of ST MAL which had previously been exposed to 5% malathion for 6 hr. The results show that there was a high level of synergism of malathion by TPP in ST MAL. There was no increase in the percentage survival over five generations of selection with TPP + malathion. This suggests that only one mechanism is involved in this resistance. Malathion was also synergized by SV,. PB had a slight antagonistic effect on malathion in ST MAL. Esterase Activity (a) Filter paper spot test. There was no obvious increase in esterase activity in the
of TPP,
SV,,
or PB Pretreatment
151
stephensi
Mean absorbance 5 SD 632.4 + 10.9 621.7 ? 9.9
Population ST ST MAL
This shows that there is no gross quantitative change in the amount of carboxylesterase enzyme in the resistant (ST MALI) and susceptible (ST) lines ofA. stephensi as measured by the activity with (Y- and /3naphthyl acetate. Larval Resistance
Larval tests were carried out to determine the activity, if any, of the malathion resistance gene in the fourth-instar larvae. Results of larval selection for five generations are given in Table 2. The progeny of ST LAMA (5) were allowed to complete their development and were tested against 5% malathion for 6 hr as l-day-old adults. There was 15.2% mortality in these tests (sample size = 256). A sample of ST LA adults tested against 5% malathion at the start of the larval selection program showed 58.4% mortality and a sample tested at the same time as ST LAMA (6) showed 59.3%
TABLE The Effect
Anopheles
R strain compared to the S strain, when either (Y-or fl-naphtyl acetate was used as a substrate. (b) Spectrophotometric assay. Both ST and ST MAL showed a normal distribution of absorbance values after the enzyme assay with cr-naphtyl acetate. The mean value for 100 insects from each strain are therefore given:
above. RESULTS
IN
1
on Malathion
Resistance
in Anopheles
stephensi
(ST LA)”
Synergist treatment Generation 1 2 3 4 5
Malathion (6 hr) 24.1 28.2 26.3 30.1 29
TPP (20%) 0 (46) 0 (42)
0 (18) 0 (41)
TPP + malathion SV, + malathion sv, (10%) (1 w (1 W 95 95.3 98.2 92.5 loo
ww (192) (216) (40) (10)
0 (42) 0 (16) 0 (24
90.7 (86) 95.5 (44 92.9 (14)
a The fgures are percentage mortalities with the number of mosquitoes
tested
PB (1%)
PB + malathion (6 hr) --
0 (18) 0 (21) 0 (14)
25 (188) 9.9 (152) 26.3 (99)
in parentheses.
152
JANET
HEMINGWAY TABLE
2
The Percentage Mortalities of Fourth-Instar ST and ST LA after Exposure to Various Concentrations of Malathion for 24 hr and the Response of ST LA After Five Generations of Larval Selection with Malathion Concentration Colony ST ST ST ST ST ST ST
LA LAMA LAMA LAMA LAMA LAMA
a Figures
0.25
0.5
1.0
71.6 (201)” 59.1 (513) 10.8 (83)
87.7 (98) 77.9 (703) 39.1 (23)
96.7 (388) 92.4 (263)
100 (120) 99.1 (114)
85.4 66.3 34.6 4.9 are numbers
(192) (243) (231) (284)
21.6 (116)
tested.
mortality. It therefore appears that larval selection with malathion also selects the adult resistance gene. The converse is also true. Larvae of the F19 generation of ST MAL showed 4% (sample size = 150) and 30% (sample size = 40) mortality after exposure to 0.5 and 1 ppm malathion, respectively. The same gene, therefore, appears to produce adult and larval malathion resistance. All metabolism experiments were carried out on the larvae as extraction of metabolites was simpler from the larval rather than the adult test medium. Three hours exposure to 2 ppm malathion was used for metabolism studies as this was the maximum time that did not give mortality of the S larvae during the exposure period. Malathion
(ppm)
0.125
(2) (3) (4) (5)
in parentheses
of malathion
Metabolism
The appearance of malathion metabolites after various time exposures to 2 ppm malathion was determined. Controls showed little or no nonenzymatic hydrolysis of malathion. The results in Table 3 show that the malathion-resistant ST MAL strain produced malathion monoacid more rapidly than the susceptible ST strain. DMPDT, DMPT, and DMP were detected at the same time in ST and ST MAL. There was no qualitative difference in the metabolites produced by the susceptible and resistant larvae. A quantitative measure of metabolite production was obtained by exposing two lots of 100 fourth-instar larvae,of each of ST
and ST MAL to 2 ppm of [C14]malathion for 3 hr. The total recoveries of radioactivity using extraction method A were 79 and 82%, respectively, in resistant and susceptible strains. These results are given in Table 4. The monocarboxylic acid is the major breakdown product of malathion in both the resistant and susceptible stocks from both larval and test media extractions. In spite of a much higher excretion of products to the water in R larvae the amount of malathion/malaoxon is approximately equal in the two strains. This indicates that the resistance is not due to reduced penetration. There was more unmetabolized malathion remaining in the ST test medium than in the ST MAL test medium at the conclusion of the larval exposure period. Seventy percent of the total radioactivity recovered from the ST test was unmetabolized malathion, whereas 42% of the malathion remained unmetabolized in the ST MAL test. Using extraction method B, 89 and 91% recovery of radioactivity was achieved in resistant and susceptible strains, respectively. The results in Table 5 show a similar pattern to those in Table 4. Again using this extraction procedure, there is no evidence of reduced penetration in the malathionresistant population. Total malathion/ malaoxon recovery from susceptible and resistant larvae was similar. However, total malathion/malaoxon recovery from test medium and larvae was greater in the ST
NATURE
OF
MALATHION
RESISTANCE
TABLE Presence
(+)
20
Malathion Malaoxon Malathion monoacid Malathion d&id DMPDT
153
srephensi
3
or Absence (-) of Malathion and Its Metabolites in ST MAL and ST Larvae Test Medium (H@) after Various Timed Exposures to Malathion Minutes
Metabolite
IN Anophefes
40
ST __ L H,O
ST MAL __ L Hz0
ST L H,O
+ -
+
+ -
+
+ -
+
-t
-
-
-
-
-
-
-
-
-
-
-
-
exposure
to 2 ppm malathion
60
ST MAL __ L Hz0
(L) and the
120
80
180
ST ~ L Hz0
ST MAL L Hz0
ST __ L Hz0
ST MAL ___ L Hz0
ST __ L H20
ST MAL L Hz0
ST __ L Hz0
ST’ MAL -L Hz0
+
+ -
+
+ -
+
+ -
+
+ +
+
+ +
+
+ +
+
+ +
+
+
+
-
+
-
+
+
+
-
+
+
+
-
+
+
+
-
+
+
+
-
-
-
-
-
-
-
-
-
-
-
+
_
+
-
+
-
+
t
~~~)-------------+-+-+-+++++
test than in the ST MAL test. Proportions of unmetabolized malathion remaining after the conclusion of the test were similar for both extraction procedures; the same applied to proportions of metabolites produced by ST and ST MAL larvae. These results strongly indicate that the primary metabolic pathway for malathion in both ST and ST MAL is the production of malathion monoacid. DISCUSSION
Synergist studies suggest the involvement of a carboxylesterase enzyme in malathion resistance in ST MAL. Malathion metabolism studies showed that the TABLE Amounts
major metabolic products of malathion in both ST and ST MAL were the malathion monocarboxylic acids. Until Welling et al. (7) showed that a strain of resistant house fly could oxidatively degrade malaoxon to form the monoacid, malathion monocarboxylic acid was considered to be strictly a carboxylesterase-produced degradative product. However, the effect of the synergists TPP and PB on ST MAL do not suggest the involvement of oxidative degradation of malathion. TPP, a carboxylesterase inhibitor, gave almost complete synergism of malathion resistance, and TPP + malathion selection for five generations failed 4
of Malathion and Various Metabolites (Expressed as Percentages of the Total Recovery) in Larvae and the Test Medium after Exposing Fourth-lnstar Larvae of ST and ST MAL to 2 ppm Malathion for 3 hr
Susceptible Compound Malathion/axon Malathion monoacid DMPDT DMPT DMP Total metabolites Total recovery of C14-label applied
Resistant
-._ Water
Larvae
Water
Larvae
1.05
69.6
1.1
40.99
0.63 0.33
25.4 1.3
1.5 0.52
52.38 0.71
1.4
0.23
2.55
0.2 29.3
57.9
82.4
79.2
-
154
JANET
Amount of Radioactivity and Test Medium after
in Various Extracts Exposing Fourth-Znstar
HEMINGWAY TABLE (Expressed Larvae
5 as Percentages of the Total Recovery) of .S? and ST MAL to 2 ppm Malathion
ST Extract Chloroform (pH 7) Chloroform (pH 2.5) Water Larval residue Total metabolites Total recovery of P-Label applied
in Larvae for 3 hr
ST MAL
Larvae
Water
Larvae
Water
1.14 1.2 0.22 0.54
74.6 21.4 0.9
1.16 2.87 0.24 0.58
46.8 47.9 0.45
24.26
51.46
91.3
89.1
to select out a second resistance mechanism. SV, + malathion selection for three generations also gave a similar result. SV, is primarily an oxidation blocker (8) which when converted to its PO analog inhibits hydrolysis (7). This synergistic effect of SV, may, therefore, be due to blockage of either oxidative or hydrolytic mechanisms; however, PB, primarily an oxidase inhibitor, did not show any synergistic action against malathion in the resistant strain. The slight antagonism with PB may be due to inhibition of the oxidase-mediated malathion-to-malaoxon conversion. Resistance in ST MAL appears to be due to an elevation in carboxylesterase products. The possibility of a quantitative change in esterase levels was therefore investigated. A total esterase assay showed no difference between the absorption in resistant and susceptible individuals when a-naphthy1 acetate was used as the substrate. Preliminary electrophoretic investigations also indicated that no single esterase band shows enhanced activity with either a- or /3-nephthyl acetates. (This will be reported in more detail later.) Therefore, if malathion resistance in ST MAL is due to increased detoxication of malathion by a carboxylesterase enzyme, there must either be a qualitative change in that enzyme, or if there is a quantitative change of a carboxylesterase enzyme which attacks. malathion, it must have very low activity with either (Yor P-naphthyl acetate. Malathion resistance in ST MAL resem-
bles the situation in the Fresno strain of Culex tarsalis (9). However, it differs from the situation in other arthropod strains where esterase levels are changed with malathion resistance. Reduced B-esterase activity has been demon&rated in several strains of Musca domestica (10) and the CM strain of Chrysomya putoria (11). The Blauvelt strain of. Tetranychus urticae shows no significant differences in A or B esterase activity as measured by a- or /3naphthyl acetate. It has, however, slightly higher esterase activity with P-naphthyl benzoate when compared to the susceptible strain (12). Increased esterase activity to p-naphthyl acetate in organophosphateresistant strains has been shown in Nephotettix cincticeps, Laodelphax striatellus, Myzus persicae, Culex pipiens, and Cufex quinquefusciatus (13- 19). Genetic analysis of ST MAL showed that malathion resistance was inherited monofactorially. There was cross-resistance between malathion and phenthoate, another organophosphorus compound with a carboxylester bond, but no resistance to organophosphorous compounds without a carboxylester bond in ST MAL (2). The present study provides further evidence for carboxylesterase involvement in malathion resistance in this population. As enzyme assays have indicated no quantitative change in the level of esterases in ST MAL, a study of the enzyme kinetics of the malathio&arboxylesterase interaction is required to confirm these findings.
NATURE
OF
MALATHION
RESISTANCE
ACKNOWLEDGMENTS
The author wishes to thank Miss W. A. Matthews for samples of malathion monocarboxylic acid. Thanks also to G. Davidson, S. Miles, and P. Rawlings for their constructive criticism of this paper. This work was supported by a Medical Research Council Studentship. REFERENCES
1. M. R. Rathor and G. Toqir, Malathion resistance in Anopheles stephensi (LISTON) in Lahore, Pakistan, Mosq. News 40, 526 (1980). 2. J. Hemingway, The genetics of malathion resistance in Anopheles stephensi from Pakistan, Ann. Trop. Med. Parasitol., in press. 3. R. B. March, T. R. -Fukuto, R. L. Metcalf, and M. A. Maxon, Fate of p-malathion in the laying hen, white mouse and American cockroach, J. Econ.
Entomol.
13,
303
11.
12.
13. 14.
49, 185 (1956).
4. K. van Asperen, A study of house fly esterases by means of a sensitive calorimetric method, 1. Znsect Physiol. 8, 401 (1962). 5. N. Pasteur and G. P. Georghiou, Filter paper test for rapid determination of phenotypes with high esterase activity in organophosphate resistant mosquitoes, Mosq. News 41, 181 (1981). 6. W. A. Matthews, The metabolism of malathion in vivo by two strains of Rhyzopertha dominica (F), the lesser grain borer, Pestic. Biochem. Physiol.
10.
(1980).
7. W. Welling, A. W. Devries, and S. Voerman, Oxidative cleavage of a carboxylester bond as a mechanism of resistance to malaoxon in the house fly, Pestic. Biochem. Physiol. 4, 31 (1974). 8. F. J. Oppenoorth, Resistance in insects: The role of metabolism and the possible use of synergists, Bull. WHO 44, 195 (1971). 9. F. Matsumura and A. W. A. Brown, Studies on
15. 16.
IN
Anopheles
sfephetlsi
155
carboxylesterase in malathion resistant Culex tarsalis. J. Econ. Entomoi. 56, 381 (1963). K. van Asperen and F. J. Oppenoorth, Organophosphate resistance and esterase activity in house flies, Entomol. Exp. Appl. 2, 48 (1959). M. G. Townsend and J. R. Busvine, The mechanism of malathion resistance in the blowfly Chrysomya putoria, Entomol. Exp. Appl. 12, 243 (1969). F. Matsumura and G. Voss, Mechanism of malathion and parathion resistance in the twospotted spider mite, Tetranychus urticae. J. Econ. Entomol. 57,911 (1964). K. Kojima and S. Kitakata, Mechanisms of resistance to malathion in the green rice leaf-hopper. Botyu Kagaku 28, 17 (1963). K. Ozaki, The resistance to organophosphorus insecticides of the green rice leaf-hopper, Nephotettix cincticeps Uhler and the small brown planthopper Laodelphax striatellus Fallen, Rev. Plant Prot. Res. 2, 1 (1%9). B. F. Stone and A. W. A. Brown, Mechanisms of resistance to fenthion in Culex pipiensfatigam Wied, Bull. WHO 40, 401 (1%9). N. Pasteur and G. Sinegre, Esterase polymorphism and sensitivity to Dursban organophos. phate insecticide in Culex pipiens population. Biochem.
Genef.
13,
789
(1975).
17. G. P. Georghiou, N. Pasteur, and M. K. Hawley, Linkage relationships between organophosphate resistance and a highly active esterase B in Culex quinquefasciatus from California, J. &on. Entomol. 73, 301 (1980). 18. P. M. Needham and R. M. Sawicki, Diagnosis of resistance to organophosphorus insecticides in MYZUS persicar (Sulz), Nature (London) 230, 125 (1971). 19. A. L. Devonshire and R. M. Sawicki, Insecticide resistant Myzus persicae as an example of evolution by gene duplication, Nature (London) 280, 140 (1979).
BIOCHEMISTRY
AND
PHYSIOLOGY
The Biochemical
17, 149-155
(1982)
Nature of Malathion Resistance stephensi from Pakistan
in Anopheles
JANET HEMINGWAY* Department
of Entomology,
London School of Hygiene Street), London WClE
and Tropical 7HT, England
Medicine,
Keppel
Street
(Go+ver
Received September 29, 1981; accepted December 3, 1981 Malathion resistance in Anopheles stephensi from Pakistan was synergized by triphenyl phosphate, primarily a carboxylesterase inhibitor. There was a slight degree of antagonism with piperonyl butoxide. The major metabolite of malathion in larvae of both the resistant and susceptible strains was malathion monocarboxylic acid. Resistant larvae produced about twice as much of this product as the susceptible larvae. This suggests that a qualitative or a quantitative change in a carboxylesterase enzyme may be the basis of malathion resistance in this strain. Analysis of general esterase levels to a- and fi-naphthyl acetate showed that there was no quantitative change in the amount of carboxylesterase enzyme present in the resistant strain as compared to the susceptible. INTRODUCTION
Malathion and phenthoate resistance have been detected in Anopheles stephensi from near Lahore, Pakistan (1, 2). Laboratory selection with malathion for 14 generations increased the level of resistance to phenthoate and vice versa. This suggested that the same mechanism could be responsible for resistance to both compounds. As both malathion and phenthoate contain a carboxylester bond, the resistance may be based on a common detoxication pathway for the two insecticides. Genetic analysis indicated that only one major biochemical mechanism was involved in malathion resistance, as resistance was inherited monofactorally (2). Synergist and metabolic studies of malathion resistance have, therefore, been undertaken to give further information on the detoxication pathway underlying resistance in this population. MATERIALS
AND METHODS
The following
laboratory colonies of A. ST, a laboratory insecticide-susceptible population originating from an area near Dehli, India, and
stephensi were used:
’ Present address: Division of Toxicology and Physiology, Department of Entomology, University of California, Riverside, California 92521.
maintained as a colony since 1947; ST LA, a population from Lahore, Pakistan, colonized in 1974, resistant to malathion and phenthoate; ST MAL, a colony derived from ST LA by adult selection with 5% malathion for up to 8 hr in every generation (adults of the F,, generation were 24x more resistant to malathion than ST); and ST LAMA, a colony derived from ST LA by larval selection for five generations with 0.5 ppm malathion (larvae of the F, generation were 3~ more resistant to malathion than those of ST). Chemicals
The following chemicals were used: piperonyl butoxide (PB) ([3,4-(methylenedioxy)d-propylbenzyl] n-butyl diethyleneglychol ether); triphenyl phosphate (TPP); SV, (O,O-dimethyl-O-phenyl phosphorothioate); malathion (O,O-dimethyl-S-(1,2di-(ethoxycarbonyl)ethyl)phosphorodithioate) (greater than 99% pure); malaoxon (O,O-dimethyl-S-(1,2-di-(ethoxycarbonyl)ethyl)phosphorothioate); malathion monocarboxylic acid (O,O-dimethyl-S-(l-carboxy-2-ethoxycarbonyl)phosphorodithioate); malathion dicarboxylic acid (0, Odimethyl S-(1,2-dicarboxy)phosphorodithioate) (produced by the method of March et al. (3)); DMPDT (O,O-dimethyl phos149 0048-3575/82/020149-07$02.00/O Copyright All rights
0 1982 by Academic Press, Inc. of reproduction in any form reserved
150
JANET
HEMINGWAY
phorodithionic acid); DMPT (O,O-dimethyl (A) After homogenizing larvae in a small phosphorothionic acid); DMP (O,Oamount of anhydrous sodium sulfate twice dimethyl phosphate); C14-labeled malathion in 5 ml hexane, the homogenate was filtered was obtained from the Radiochemical and the residue washed with a further 5 ml Centre, Amersham (sp act, 4.6 mCi/mM) of hexane. The filtrates were combined (labeled in both carbon atoms of the suc- and evaporated to dryness on a rotary cinyl part of the molecule, the labeled evaporator. This procedure was repeated malathion was diluted I:2 with nonlabeled using diethyl ether and acetonitrile. Metabmalathion in absolute ethanol to give a olites were extracted from the larval test working solution of O.l%, which had an ac- medium using the same procedure with tivity of 8,410,005.5 cprn/ml); and DCQ hexane and diethyl ether as solvents. The (2,6-dibromoquinone-4-chlorimide). Pre- remaining water was then evaporated to coated plastic thin-layer chromatography dryness. All extracts were subsequently re(TLC) sheets coated with 0.2 mm silica gel dissolved in 0.1 ml of the relevant solvent. N-HR/uv,, were used. The purity of all (B) At the conclusion of the insecticide chemicals was regularly assessed by TLC. exposure period the larvae were removed TLC plates were sprayed with 0.5% DCQ in and thoroughly washed with 5 ml of discyclohexane and heated to 100°C to visu- tilled water, which was added to the test alize the spots. medium. The pH of the test medium was then adjusted to pH 7 with 0.1 N sodium Synergist Studies hydroxide and hydrochloride acid. MalaAdult mosquitoes were exposed to the thion and malaoxon in the medium were synergist or insecticide by tarsal contact than partitioned into 10 ml of chloroform. using the standard WHO test kits. Test pa- The water was acidified to pH 2.5 with hypers were prepared by impregnating rect- drochloric acid and again partitioned with angles of Whatmans No. 1 filter paper (12 x 10 ml of chloroform. Larvae were homog15 cm) with the required concentration of enized in 3 ml of distilled water and extracsynergist or insecticide spread at 3.6 ~gl tion was carried out in the same way. At least three replicates were carried out cm2. Any survivors of the synergist/ insecticide treatment were hand-mated and with each extraction method. Controls their progeny again selected in the same were run without the larval homogenate to way. determine the level of nonenzymatic hydrolysis of malathion under the experiEsterases mental conditions used. Total nonspecific esterase activity was assayed spectrophotometrically by the Analysis (after Extraction Method A) method of van Asperen (4) and by the filter TLC plates were prerun with chloroform paper spot technique of Pasteur and Geor- for 1% hr prior to adding extracts. Extracts ghiou (5) using adult mosquitoes. Values in were placed on the origin line of the TLC the former were corrected for the weight of plate using a 5-~1 capillary tube. TLC plates the insect and the amount of homogenate were run with a hexane:diethyl ether (1:3) used. solvent system (6). A set of standard metabolites was run on each plate to check the Larval Maiathion Metabolism position of metabolites. Rf values were reBatches of 100 fourth-instar larvae were producible. exposed to 2 ppm malathion in 100 ml of Quantification distilled water for up to 3 hr. Metabolites were_ extracted by either of the following After extraction of metabolites by methods. method A and running on TLC plates, me-
NATURE
OF MALATHION
RESISTANCE
tabolites were reeluted with either hexane, diethyl ether, or acetonitrile. The solutions were placed in counting vials and air-dried, then 100 ~1 of absolute alcohol, 40 ~1 of distilled water, and 4 ml of scintillation fluid (Packard Scintillation cocktail, Scintillator 299) were added to each counting vial. Samples were counted in a scintillation counter for 10 min or up to 10,000 counts. After extraction method B extracts were placed into vials, air-dried, and treated as
Synergist Studies
The results of selection of ST MAL with TPP + malathion for five generations are given in Table 1. The survivors of the second and third selected generation were all males. They were hand-mated to virgin females of ST MAL which had previously been exposed to 5% malathion for 6 hr. The results show that there was a high level of synergism of malathion by TPP in ST MAL. There was no increase in the percentage survival over five generations of selection with TPP + malathion. This suggests that only one mechanism is involved in this resistance. Malathion was also synergized by SV,. PB had a slight antagonistic effect on malathion in ST MAL. Esterase Activity (a) Filter paper spot test. There was no obvious increase in esterase activity in the
of TPP,
SV,,
or PB Pretreatment
151
stephensi
Mean absorbance 5 SD 632.4 + 10.9 621.7 ? 9.9
Population ST ST MAL
This shows that there is no gross quantitative change in the amount of carboxylesterase enzyme in the resistant (ST MALI) and susceptible (ST) lines ofA. stephensi as measured by the activity with (Y- and /3naphthyl acetate. Larval Resistance
Larval tests were carried out to determine the activity, if any, of the malathion resistance gene in the fourth-instar larvae. Results of larval selection for five generations are given in Table 2. The progeny of ST LAMA (5) were allowed to complete their development and were tested against 5% malathion for 6 hr as l-day-old adults. There was 15.2% mortality in these tests (sample size = 256). A sample of ST LA adults tested against 5% malathion at the start of the larval selection program showed 58.4% mortality and a sample tested at the same time as ST LAMA (6) showed 59.3%
TABLE The Effect
Anopheles
R strain compared to the S strain, when either (Y-or fl-naphtyl acetate was used as a substrate. (b) Spectrophotometric assay. Both ST and ST MAL showed a normal distribution of absorbance values after the enzyme assay with cr-naphtyl acetate. The mean value for 100 insects from each strain are therefore given:
above. RESULTS
IN
1
on Malathion
Resistance
in Anopheles
stephensi
(ST LA)”
Synergist treatment Generation 1 2 3 4 5
Malathion (6 hr) 24.1 28.2 26.3 30.1 29
TPP (20%) 0 (46) 0 (42)
0 (18) 0 (41)
TPP + malathion SV, + malathion sv, (10%) (1 w (1 W 95 95.3 98.2 92.5 loo
ww (192) (216) (40) (10)
0 (42) 0 (16) 0 (24
90.7 (86) 95.5 (44 92.9 (14)
a The fgures are percentage mortalities with the number of mosquitoes
tested
PB (1%)
PB + malathion (6 hr) --
0 (18) 0 (21) 0 (14)
25 (188) 9.9 (152) 26.3 (99)
in parentheses.
152
JANET
HEMINGWAY TABLE
2
The Percentage Mortalities of Fourth-Instar ST and ST LA after Exposure to Various Concentrations of Malathion for 24 hr and the Response of ST LA After Five Generations of Larval Selection with Malathion Concentration Colony ST ST ST ST ST ST ST
LA LAMA LAMA LAMA LAMA LAMA
a Figures
0.25
0.5
1.0
71.6 (201)” 59.1 (513) 10.8 (83)
87.7 (98) 77.9 (703) 39.1 (23)
96.7 (388) 92.4 (263)
100 (120) 99.1 (114)
85.4 66.3 34.6 4.9 are numbers
(192) (243) (231) (284)
21.6 (116)
tested.
mortality. It therefore appears that larval selection with malathion also selects the adult resistance gene. The converse is also true. Larvae of the F19 generation of ST MAL showed 4% (sample size = 150) and 30% (sample size = 40) mortality after exposure to 0.5 and 1 ppm malathion, respectively. The same gene, therefore, appears to produce adult and larval malathion resistance. All metabolism experiments were carried out on the larvae as extraction of metabolites was simpler from the larval rather than the adult test medium. Three hours exposure to 2 ppm malathion was used for metabolism studies as this was the maximum time that did not give mortality of the S larvae during the exposure period. Malathion
(ppm)
0.125
(2) (3) (4) (5)
in parentheses
of malathion
Metabolism
The appearance of malathion metabolites after various time exposures to 2 ppm malathion was determined. Controls showed little or no nonenzymatic hydrolysis of malathion. The results in Table 3 show that the malathion-resistant ST MAL strain produced malathion monoacid more rapidly than the susceptible ST strain. DMPDT, DMPT, and DMP were detected at the same time in ST and ST MAL. There was no qualitative difference in the metabolites produced by the susceptible and resistant larvae. A quantitative measure of metabolite production was obtained by exposing two lots of 100 fourth-instar larvae,of each of ST
and ST MAL to 2 ppm of [C14]malathion for 3 hr. The total recoveries of radioactivity using extraction method A were 79 and 82%, respectively, in resistant and susceptible strains. These results are given in Table 4. The monocarboxylic acid is the major breakdown product of malathion in both the resistant and susceptible stocks from both larval and test media extractions. In spite of a much higher excretion of products to the water in R larvae the amount of malathion/malaoxon is approximately equal in the two strains. This indicates that the resistance is not due to reduced penetration. There was more unmetabolized malathion remaining in the ST test medium than in the ST MAL test medium at the conclusion of the larval exposure period. Seventy percent of the total radioactivity recovered from the ST test was unmetabolized malathion, whereas 42% of the malathion remained unmetabolized in the ST MAL test. Using extraction method B, 89 and 91% recovery of radioactivity was achieved in resistant and susceptible strains, respectively. The results in Table 5 show a similar pattern to those in Table 4. Again using this extraction procedure, there is no evidence of reduced penetration in the malathionresistant population. Total malathion/ malaoxon recovery from susceptible and resistant larvae was similar. However, total malathion/malaoxon recovery from test medium and larvae was greater in the ST
NATURE
OF
MALATHION
RESISTANCE
TABLE Presence
(+)
20
Malathion Malaoxon Malathion monoacid Malathion d&id DMPDT
153
srephensi
3
or Absence (-) of Malathion and Its Metabolites in ST MAL and ST Larvae Test Medium (H@) after Various Timed Exposures to Malathion Minutes
Metabolite
IN Anophefes
40
ST __ L H,O
ST MAL __ L Hz0
ST L H,O
+ -
+
+ -
+
+ -
+
-t
-
-
-
-
-
-
-
-
-
-
-
-
exposure
to 2 ppm malathion
60
ST MAL __ L Hz0
(L) and the
120
80
180
ST ~ L Hz0
ST MAL L Hz0
ST __ L Hz0
ST MAL ___ L Hz0
ST __ L H20
ST MAL L Hz0
ST __ L Hz0
ST’ MAL -L Hz0
+
+ -
+
+ -
+
+ -
+
+ +
+
+ +
+
+ +
+
+ +
+
+
+
-
+
-
+
+
+
-
+
+
+
-
+
+
+
-
+
+
+
-
-
-
-
-
-
-
-
-
-
-
+
_
+
-
+
-
+
t
~~~)-------------+-+-+-+++++
test than in the ST MAL test. Proportions of unmetabolized malathion remaining after the conclusion of the test were similar for both extraction procedures; the same applied to proportions of metabolites produced by ST and ST MAL larvae. These results strongly indicate that the primary metabolic pathway for malathion in both ST and ST MAL is the production of malathion monoacid. DISCUSSION
Synergist studies suggest the involvement of a carboxylesterase enzyme in malathion resistance in ST MAL. Malathion metabolism studies showed that the TABLE Amounts
major metabolic products of malathion in both ST and ST MAL were the malathion monocarboxylic acids. Until Welling et al. (7) showed that a strain of resistant house fly could oxidatively degrade malaoxon to form the monoacid, malathion monocarboxylic acid was considered to be strictly a carboxylesterase-produced degradative product. However, the effect of the synergists TPP and PB on ST MAL do not suggest the involvement of oxidative degradation of malathion. TPP, a carboxylesterase inhibitor, gave almost complete synergism of malathion resistance, and TPP + malathion selection for five generations failed 4
of Malathion and Various Metabolites (Expressed as Percentages of the Total Recovery) in Larvae and the Test Medium after Exposing Fourth-lnstar Larvae of ST and ST MAL to 2 ppm Malathion for 3 hr
Susceptible Compound Malathion/axon Malathion monoacid DMPDT DMPT DMP Total metabolites Total recovery of C14-label applied
Resistant
-._ Water
Larvae
Water
Larvae
1.05
69.6
1.1
40.99
0.63 0.33
25.4 1.3
1.5 0.52
52.38 0.71
1.4
0.23
2.55
0.2 29.3
57.9
82.4
79.2
-
154
JANET
Amount of Radioactivity and Test Medium after
in Various Extracts Exposing Fourth-Znstar
HEMINGWAY TABLE (Expressed Larvae
5 as Percentages of the Total Recovery) of .S? and ST MAL to 2 ppm Malathion
ST Extract Chloroform (pH 7) Chloroform (pH 2.5) Water Larval residue Total metabolites Total recovery of P-Label applied
in Larvae for 3 hr
ST MAL
Larvae
Water
Larvae
Water
1.14 1.2 0.22 0.54
74.6 21.4 0.9
1.16 2.87 0.24 0.58
46.8 47.9 0.45
24.26
51.46
91.3
89.1
to select out a second resistance mechanism. SV, + malathion selection for three generations also gave a similar result. SV, is primarily an oxidation blocker (8) which when converted to its PO analog inhibits hydrolysis (7). This synergistic effect of SV, may, therefore, be due to blockage of either oxidative or hydrolytic mechanisms; however, PB, primarily an oxidase inhibitor, did not show any synergistic action against malathion in the resistant strain. The slight antagonism with PB may be due to inhibition of the oxidase-mediated malathion-to-malaoxon conversion. Resistance in ST MAL appears to be due to an elevation in carboxylesterase products. The possibility of a quantitative change in esterase levels was therefore investigated. A total esterase assay showed no difference between the absorption in resistant and susceptible individuals when a-naphthy1 acetate was used as the substrate. Preliminary electrophoretic investigations also indicated that no single esterase band shows enhanced activity with either a- or /3-nephthyl acetates. (This will be reported in more detail later.) Therefore, if malathion resistance in ST MAL is due to increased detoxication of malathion by a carboxylesterase enzyme, there must either be a qualitative change in that enzyme, or if there is a quantitative change of a carboxylesterase enzyme which attacks. malathion, it must have very low activity with either (Yor P-naphthyl acetate. Malathion resistance in ST MAL resem-
bles the situation in the Fresno strain of Culex tarsalis (9). However, it differs from the situation in other arthropod strains where esterase levels are changed with malathion resistance. Reduced B-esterase activity has been demon&rated in several strains of Musca domestica (10) and the CM strain of Chrysomya putoria (11). The Blauvelt strain of. Tetranychus urticae shows no significant differences in A or B esterase activity as measured by a- or /3naphthyl acetate. It has, however, slightly higher esterase activity with P-naphthyl benzoate when compared to the susceptible strain (12). Increased esterase activity to p-naphthyl acetate in organophosphateresistant strains has been shown in Nephotettix cincticeps, Laodelphax striatellus, Myzus persicae, Culex pipiens, and Cufex quinquefusciatus (13- 19). Genetic analysis of ST MAL showed that malathion resistance was inherited monofactorially. There was cross-resistance between malathion and phenthoate, another organophosphorus compound with a carboxylester bond, but no resistance to organophosphorous compounds without a carboxylester bond in ST MAL (2). The present study provides further evidence for carboxylesterase involvement in malathion resistance in this population. As enzyme assays have indicated no quantitative change in the level of esterases in ST MAL, a study of the enzyme kinetics of the malathio&arboxylesterase interaction is required to confirm these findings.
NATURE
OF
MALATHION
RESISTANCE
ACKNOWLEDGMENTS
The author wishes to thank Miss W. A. Matthews for samples of malathion monocarboxylic acid. Thanks also to G. Davidson, S. Miles, and P. Rawlings for their constructive criticism of this paper. This work was supported by a Medical Research Council Studentship. REFERENCES
1. M. R. Rathor and G. Toqir, Malathion resistance in Anopheles stephensi (LISTON) in Lahore, Pakistan, Mosq. News 40, 526 (1980). 2. J. Hemingway, The genetics of malathion resistance in Anopheles stephensi from Pakistan, Ann. Trop. Med. Parasitol., in press. 3. R. B. March, T. R. -Fukuto, R. L. Metcalf, and M. A. Maxon, Fate of p-malathion in the laying hen, white mouse and American cockroach, J. Econ.
Entomol.
13,
303
11.
12.
13. 14.
49, 185 (1956).
4. K. van Asperen, A study of house fly esterases by means of a sensitive calorimetric method, 1. Znsect Physiol. 8, 401 (1962). 5. N. Pasteur and G. P. Georghiou, Filter paper test for rapid determination of phenotypes with high esterase activity in organophosphate resistant mosquitoes, Mosq. News 41, 181 (1981). 6. W. A. Matthews, The metabolism of malathion in vivo by two strains of Rhyzopertha dominica (F), the lesser grain borer, Pestic. Biochem. Physiol.
10.
(1980).
7. W. Welling, A. W. Devries, and S. Voerman, Oxidative cleavage of a carboxylester bond as a mechanism of resistance to malaoxon in the house fly, Pestic. Biochem. Physiol. 4, 31 (1974). 8. F. J. Oppenoorth, Resistance in insects: The role of metabolism and the possible use of synergists, Bull. WHO 44, 195 (1971). 9. F. Matsumura and A. W. A. Brown, Studies on
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IN
Anopheles
sfephetlsi
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17. G. P. Georghiou, N. Pasteur, and M. K. Hawley, Linkage relationships between organophosphate resistance and a highly active esterase B in Culex quinquefasciatus from California, J. &on. Entomol. 73, 301 (1980). 18. P. M. Needham and R. M. Sawicki, Diagnosis of resistance to organophosphorus insecticides in MYZUS persicar (Sulz), Nature (London) 230, 125 (1971). 19. A. L. Devonshire and R. M. Sawicki, Insecticide resistant Myzus persicae as an example of evolution by gene duplication, Nature (London) 280, 140 (1979).