The joint action of malathion and IBP against malathion-resistant and -susceptible strains of Anopheles stephensi

Bulletin ofthe WorldHealth Organization, 62 (3): 445-449(1984) © World Health Organization 1984 The joint action of malathion and IBP against malathion-resistant and -susceptible strains of Anopheles stephensi JANET HEMINGWAY 1 Malathion resistance in an Anopheles stephensi strain from Pakistan is dependent on a single gene, which results in increased degradation of malathion to the monocarboxylic acid. Mixtures of malathion and the fungicide IBP (O,O-bis(J-methylethyl) S- phenylmethyl phosphorothioate) were tested against this resistant strain and a laboratory susceptible strain. The mixtures were more toxic to both the resistant andsusceptible insects than either IBP or malathion alone. The high degree ofsynergism with the mixtures against the susceptible strain would not be expected ifIBP were simply competing with malathion for a common carboxylesterase detoxification enzyme. The reason for the high degree of synergism in the susceptible strain is unknown. As insecticide resistance becomes more common and fewer new insecticides are produced, there is greater need for alternative measures to combat resistance. These measures may include the use of economically and toxicologically acceptable insecti- ticide mixtures. Potentiation (synergism) and antag- onism of insecticidal action have been shown for some mixtures of chemicals in certain insects. For example, DEF (S,S,S,-tributyl phosphorotrithioate) and temephos showed a high degree of synergism against a temephos-resistant strain of Culex quinque- fasciatus Say (1), and IBP (O,O-bis(I-methylethyl) S-phenylmethyl phosphorothioate)a and malathion had a high joint toxic effect against malathion-resis- tant Nephotettix cincticeps Uhler (2). Anopheles stephensi Liston, a major vector of malaria, is malathion-resistant over much of its range (3, 4). Malathion resistance in A. stephensi in Lahore, Pakistan, was inherited monofactorially, and resistant homozygotes were 23 times more resist- ant than susceptible homozygotes (5). Metabolism and synergist studies indicate that this resistance was due to increased degradation at the carboxylester linkage of malathion (6). This paper investigates the effect of mixtures of IBP and malathion against this malathion-resistant strain and a laboratory suscept- ible strain of A. stephensi. ' Research Fellow, Department of Entomology, London School of Hygiene and Tropical Medicine, Keppel Street/Gower Street, London WC1E 7HT, England. a IBP is the common name of a fungicide in Japan, approved by the Japanese Ministry of Agriculture. MATERIALS AND METHODS Two strains of A. stephensi were used: (1) ST, a susceptible strain originating from an area near Delhi, India, and reared in the laboratory since the late 1940s; and (2) ST MAL, derived from ST LA (a strain from Lahore, Pakistan, colonized since 1978) and selected at the adult stage for 19 generations with 507o malathion for up to 8 h. All adult tests were carried out by the tarsal contact method using the standard WHO test kit. Insecticide papers were prepared by impreg- nating rectangles of Whatman's No. 1 filter-paper (12 x 15 cm) with 0.7 ml of the required concen- tration of insecticide or fungicide (at least 99% pure) in acetone. A further 1.2 ml of acetone was used to spread the solution evenly on the paper. Control papers were treated with 1.9 ml of acetone. Earlier analysis had shown that ST MAL males were significantly more resistant to malathion than the females. As control operations are primarily aimed at females, only the females were used for further experiments. There was no difference between the response of males and females of the ST stock to malathion. Log dosage-probit mortality lines (LD-P) were produced for each insecticide and for the various mixtures of insecticides tested. The lines were fitted using maximum likelihood analysis. The LD50 values for each of the pure compounds were plotted on isobolograms. A line was drawn between the two 4417 -445- J. HEMINGWAY LD5o values and three equidistant points were taken along this line. These represented the proportions of the two active ingredients in the mixtures to be tested. Plots of the actual LD50 values for these mixtures on the isobolograms then demonstrated whether poten- tiation or antagonism had occurred. (For a full explanation of this technique, see Hewlett & Plackett (7).) RESULTS The percentage mortalities obtained after exposure to malathion or IBP alone are given in Table 1 for ST and ST MAL. Mortalities were recorded separately for males and females. The values for both sexes of the ST colony were pooled as there was no difference in their response. The males of ST MAL were Table 1. Percentage mortalities of ST and ST MAL after exposure to varying concentrations of malathion or IBP (figures in parentheses are the numbers tested) Malathion IBP Dosage (yg/cm2) ST ST MAL ST ST MAL (9 1166.4 100 (80) 777.6 99.2 (130) 680.4 80 (100) 583.2 54.2 (120) 486 29.2 (120) 388.8 21.6 (120) 220.2 0 (80) 95 (100) 194.4 74.3 (140) 152.2 56 (100) 110 18.6 (140) 97.2 100 (136) 10 (100) 81 2 (100) 64.8 72 (200) 0 (37) 59.9 54 (200) 55.1 28 (200) 48.6 8.5 (142) 32.4 0 (98) 18.2 100 (60) 15.2 96 (100) 12.2 87 (100) 10.5 85 (60) 9.1 36.7 (120) 8 28 (100) 6.1 5 (80) 3.9 0 (80) consistently less tolerant to IBP than the females, which is in direct contrast to malathion alone, where the males were more resistant than the females. All subsequent experiments with the ST MAL stock utilized only females. The results for both males and females show that IBP is less toxic to ST MAL than to ST, but the resistance ratio (RR) is only 2.5 compared with 55 for malathion. The LD5o values and their fiducial limits from the malathion and IBP lines were as follows: LD50 (jg/cm2) RR Malathion: ST ST MAL IBP: ST ST MAL 9.6± 0.3 528.9 ± 19.8 59.5 ± 0.8 149.9 ± 5.4 55 2.5 These LD5o values were used to produce the isobolograms in Fig. 1 and 2 for ST and ST MAL respectively. The predicted LD50 values of the mix- tures for each of the three equidistant points (A-C) along these isobol lines are given in the inset tables in Fig. 1 and 2. A number of dilutions of these mixtures were prepared. In addition, a fourth set of mixtures of 45 Ag/cm2 and 108 Ag/cm2 of IBP and varying con- centrations of malathion were tested against ST and ST MAL respectively. These correspond to the con- centration of IBP at point C on each of the iso- bolograms. LDsc5 0 30 60 yg /cm2 LDSIBP Fig. 1. Isobologram for malathion and IBP showing the predicted and actual LD5o values for the four mixtures tested against ST. 446 ACTION OF MALATHION AND IBP ON ANOPHELES STEPHENSSI MIXTURES MAL / IeP estimates of the LD5o values for each of the mixtures A B C was made and plotted on Fig. 2. PREDICTED 435 350 258 Four control replicates were run for each experi- ACTUAL ment using filter-papers that had been treated with . D ~~~316 .6+17 108-4*6R 6e8 4 !3LD50 acetone alone. No control mortalities occurred in any 1 2 3 test. The plots of the actual LD50 values for the mixtures in Fig. 1 and 2 indicate whether there is potentiation, additive action, or antagonism of malathion by IBP. A #~; As all the points lie below the isobol line for these . mixtures, potentiation or synergism of action must C have occurred. (Points which lie above the line would 2 3, have indicated antagonism, while those on the line 0 40 250O 120 LD%Sl8P imply joint additive action.) pg cm2 Fig. 2. Isobologram for malathion and IBP showing the predicted and actual LD50 values for the four mixtures tested against ST MAL. The results of exposure of ST to mixtures 1-4 are given in Table 2. These results were used to produce LD-P lines. The calculated proportions of malathion and IBP in each of the mixtures at the LD5o value were then plotted on Fig. 1 (points 1-4). Table 3 gives the percentage mortalities for ST MAL after exposure to mixtures 1-4. Maximum likelihood fitting of LD-P lines indicated that the response of ST MAL to mixtures 1-3 was non-linear. Although the points for the mixtures deviate from a straight line, the best DISCUSSION Exposure of malathion-resistant and -susceptible colonies of A. stephensi to mixtures of malathion and IBP indicated that the mixtures were more toxic to both colonies than either compound on its own. A similar potentiation of the action of malathion by IBP has been shown in the green rice leafhopper by Miyata & Saito (8) and Miyata et al. (9) who suggested that, since both compounds contain a carboxylester bond, the potentiation of action was due to competition of the two compounds for a common detoxication enzyme. This was supported in their study by the Table 2. Percentage mortalities of ST after exposure to four mixtures of malathion and IBP (figures in parentheses are the numbers tested) Mixtures Dosage of mixture (Ug/cm2) 1 2 3 4a 47.5 93.2 (44) 34.8 98.6 (71) 100 (72) at mal. conc. of ".5" 23.8 90 (100) 100 (80) at mal. conc. o' 2 23.2 97 (100) 98.4 (123) at mal. conc. of 1 17.4 89 (100) 66.2 (142) at mal. conc. of 0.5 15.8 82 (100) 61.1 (144) at mal. conc. of0.4 15 100 (123) 49.6 (131) at mal. conc. of 0.3 14.5 80.9 (110) 21.3 (122) at mal. conc. of 0.2 11.9 72 (100) 11.6 67.5 (120) 5.6 96.2 (59) 22 (100) 40 (100) 4.1 85.6 (95) 2 70.8 (72) 2 (50) 3 (100) 1.35 49.5 (190) 0.675 11.4 (35) ' Mixture 4 was used in a dose of 45 'ag/cm2 of IBP plus 2.5, 2, 1, 0.5, 0.4, 0.3 and 0.2 Ag/cm2 of malathion. b mal. conc. = malathion concentration in fg/cm2. 1000 60C LD50 MALATHION 40C ,ug1I1112 447 J. HEMINGWAY Table 3. Percentage mortalities of ST MAL after exposure to four mixtures of malathion and IBP (figures in paren- theses are the numbers tested) Mixtures Dosage of mixture (Lg/cm2) 1 2 3 4a 435 70.8 (144) 350 58 (100) 92 (100) 100 (64) at mal. conc. oflOOb 290 60.3 (63) 100 (45) at mal. conc. of 80 258 100 (62) 89.9 (99) at mal. conc. of 75 253 42 (100) 84.9 (132) at mal. conc. of 70 233.3 81 (100) 54.8 (115) at mal. conc. of 65 217.5 8.9 (112) 48.9 (92) at mal. conc. of 60 175 70 (100) 49.1 (106) at mal. conc. of 50 172 99.2 (125) 37.5 (32) at mal. conc. of 40 145.8 61 (100) 0 (42) at mal. conc. of 30 129 84.4 (122) 116.7 70 (100) 87.5 44.3 (79) 86 80.8 (130) 64.5 16 (100) 56.2 (146) 60 53.3 (90) 53.5 17.1 (123) 40.2 0 (140) Mixture 4 was used in a dose of 108 yg/cm2 of IBP plus 100, 80, 75, 70, 65, 60, 50, 40 and 30 Ag/cm2 of malathion. mal. conc. = malathion concentration in Ag/cm2. synergistic action of IBP and malathion observed in the resistant but not in the susceptible strain, and the fact that IBP appeared to have an inhibitory effect on '4C-malathion degradation in vitro. The present study on A. stephensi indicates that the interaction of the two compounds in this species may be more com- plex, because the effect of the mixtures is as great on the susceptible as on the resistant strain. It has already been shown that malathion resistance in this popu- lation of A. stephensi is due to an increased degra- dation of malathion to the monocarboxylic acid in the resistant as compared to the susceptible strain (6). Therefore, if simple competition for a common carboxylesterase detoxification enzyme was the sole source of the mixture's potentiation in this species, a much higher degree of synergism would have been expected in the. resistant strain compared to the susceptible. As this was not the case, further investi- gations of the joint action of IBP and malathion are required to elucidate the full interaction of these mixtures at the biochemical level. ACKNOWLEDGEMENTS The author wishes to thank Dr T. Miyata for supplying a sample of IBP, and G. Davidson, C. F. Curtis and P. Rawlings for their constructive criticisms of this manuscript. RESUME ACTION DE L'ASSOCIATION MALATHION-IBP SUR DES SOUCHES D'ANOPHELES STEPHENSI SENSIBLES OU RESISTANTES AU MALATHION La resistance au malathion chez une souche d'Anopheles stephensi provenant du Pakistan depend d'un gene unique dont l'existence entraine une degradation accrue du mala- thion en acide monocarboxylique. Des melanges de mala- 448 ACTION OF MALATHION AND IBP ON ANOPHELES STEPHENSI 449 thion et d'IBP (un thiophosphate de di-O, (methyl-1-ethyle) et d'S-phenyImethyle dote d'un pouvoir fongicide) ont e experimentes sur la souche resistante ci-dessus et sur une souche sensible du laboratoire. Les melanges se sont montres plus toxiques que l'IBP ou le malathion seuls, A la fois pour les insectes resistants et les insectes sensibles. Si I'IBP et le malathion entraient simplement en competition vis-a-vis d'une meme carboxylesterase assurant la detoxi- cation, on n'aurait pas pu obtenir l'effet de potentialisation intense observe avec ces melanges sur la souche sensible. On ignore pourquoi l'effet de synergie est aussi eleve chez la souche sensible. REFERENCES 1. RANASINGHE, L. E. & GEORGHIOU, G. P. Comparative modification of insecticide-resistance spectrum of Culex pipiens fatigans Wied by selection with temephos and temephos synergist combinations. Pesticide science, 10: 502 (1979). 2. YOSHIOKA, K. ET AL. Synergism of IBP and insecticides against resistant green rice leafhopper, Nephotettix cinc- ticeps Uhler. Proceedings of the Association of Plant Protection, Sikoku, 10: 49 (1975). 3. HERATH, P. R. J. & DAVIDSON, G. Studies on the nature of malathion resistance in a population of Anopheles stephensi from southern Iran. Mosquito news, 41: 531 (1981). 4. RATHOR, M. R. & TOQIR, A. 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