Development of resistance to carbamates and organophosphorus compounds in Anopheles albimanus in nature.

Brief communications/Notes Development of resistance to carbamates and organophosphorus compounds in Anopheles albimanus in nature G. P. GEORGHIOU, 1 V. ARIARATNAM, 1 & S. G. BREELAND 2 Carbamate and organophosphorus insecticides are currently being studied in the global malaria eradication effort as alternative chemicals of choice for use against DDT- and dieldrin-resistant Ano- pheles mosquitos. Although they are more expensive and less persistent than DDT as residual sprays for the walls of buildings, many of these chemicals have outstanding toxicity for adult and larval mosquitos. Such toxicity remains largely unaffected by existing DDT or dieldrin resistance, and until recently it appeared that selection pressure on Anopheles species did not result in resistance to these chemicals. This paper reports the appearance of high levels of resistance to several carbamate and organophos- phorus insecticides in a natural population of Anopheles albimanus in coastal El Salvador. The observed resistance was found in an area that has been under intensive treatment with organophos- phorus insecticides for several years for cotton insect control and may be attributed to indirect selection pressure by these treatments. The first signs of impending resistance to organo- phosphorus compounds in field populations of Anopheles were reported by the World Health Orga- nization in 1965 and consisted of suspected slight increases in tolerance to malathion in A. albimanus in Guatemala and Nicaragua. 3 Subsequently, Breeland et al. (1970) demonstrated that aerial appli- cations of this chemical in the area of La Libertad, El Salvador, in March 1969 were significantly less toxic to caged wild-caught adult A. albimanus than to similarly exposed adults of a laboratory colony. Assays with the standard WHO testing kit verified these conclusions. Breeland and his co-workers pointed out that the decrease in susceptibility to malathion in El Salvador occurred only in areas of intensive cotton cultivation. These areas are being 1 Department of Entomology. University of California, Riverside, Calif. 92502, USA. ' Central America Malaria Research Station, San Sal- vador, El Salvador. 'WHO (1965) Information circular on insecticide resistance, insect behaviour and vector genetics, No. 56, p. 24. subjected during six months of the year, at almost weekly intervals, to heavy applications of several organophosphorus insecticides, including trichlorfon, parathion, parathion-methyl, and malathion (Bree- land et al., 1970). Organophosphorus insecticides have been em- ployed extensively on cotton in El Salvador for more than 10 years. Since A. albimanus breeds also in the vicinity of cotton fields (Breeland et al., 1970), it may be assumed that it is subjected to indirect selection pressure by these chemicals. The reported field resistance to malathion in Anopheles is of signifi- cance in the global malaria eradication programme since this compound, the organophosphorus com- pound fenitrothion, and the carbamate propoxur are the only compounds among more than 1 400 candi- date materials to have been tested successfully through most stages of the WHO international col- laborative programme for evaluating and testing new insecticides (Wright et al. 1969). For almost a decade, studies have been carried out in our laboratory at the University of California, Riverside, on the potentialities of promising new insecticides and on mechanisms of resistance to them in mosquitos. Intensive selection pressure in the laboratory on A. albimanus from Panama and Haiti using m-cumenyl methylcarbamate, propoxur, and fenitrothion has resulted in only minimal resis- tance to these insecticides, not exceeding 3 times the LC50 for larvae, despite the presence of DDT- and dieldrin-resistant genes in the parental populations (Georghiou, 1963, 1969; Georghiou & Calman, 1969; Georghiu & Metcalf, 1963). Such results have been considered to be encouraging indications that high resistance to carbamates or organophosphorus compounds in Anopheles would, if it ever developed, require extremely prolonged selection pressure. Following publication of the WHO reports of impending resistance of Anopheles to organophos- phorus compounds, we obtained a collection of this 1 Also: Ariaratnam, V. & Georghiou, G. P. (1971) unpub- lished document WHO/VBC/71.277. 2831A - 551 552 BRIEF COMMUNICATIONS/NOTES Table 1. Insecticide resistance levels in Anopheles albimanus, El Salvador, in June 1970 and February 1971 Haiti/Panama strain El Salvador strains Insecticide (susceptible) June 1970 February 1971 LC5o (ppm) slope LCso (ppm) slope RRa LC5o (ppm) slope RRa propoxur 0.39 7.7 0.53 7.1 1.4 64.1 0.43 164.4 carbaryl 0.89 6.6 1.02 5.9 1.1 15.7 0.8 16.9 parathion-methyl 0.0065 3.0 0.022 3.5 3.4 0.17 2.1 26.2 parathion 0.0031 6.8 0.0098 3.1 3.2 0.057 1.9 18.4 malathion 0.085 2.9 0.25 2.7 2.9 1.1 1.8 12.9 fenitrothion 0.025 3.8 0.045 5.3 1.8 0.23 2.9 9.2 dichlorvos 0.11 7.1 0.11 7.0 1.0 0.29 5.9 2.6 Dursban 0.0063 4.7 - - - 0.015 3.6 2.4 fenthion 0.023 6.0 0.026 5.8 1.1 0.028 4.1 1.2 Abate 0.005 2.6 0.0049 2.6 1.0 0.006 3.0 1.2 a Resistance ratio = (LC5o El Salvador strain)/(LC5o) susceptible strain. species from the area of Hacienda Melara, La Libertad, El Salvador, in June 1970, and colonized the insects in our laboratory. Our studies provided definitive evidence of the appearance of tolerance to organophosphorus compounds in larvae, amounting to the following increases in LC50 values: 3.2x for parathion, 3.4 x for parathion-methyl, and 2.9 x for malathion (Ariaratnam & Georghiou, 1971). 1 More- over, when this larval population was subjected to rigorous selection pressure by propoxur, resistance rose within three generations to more than 100 x for propoxur, 74.8 x for carbaryl, 35.5 x for para- thion, 36.9 x for parathion-methyl, 20 x for mala- thion, and 9.6 x for fenitrothion (Ariaratnam & Georghiou, op. cit.). Thus it was apparent that A. albimanus was indeed capable of developing resis- tance to carbamates and organophosphorus com- pounds and that the population of this species in the La Libertad area had attained a tolerance level that would readily lead to high resistance upon rigorous selection pressure by propoxur. We have continued to study the situation in El Salvador and can now report that additional samples collected from the same locality 8 months later in February 1971 show considerably increased resis- tance to carbamates and organophosphorus com- pounds. The samples consisted of approximately 500 gravid females obtained from the field and per- mitted to oviposit at the CAMRS laboratory. 1 Some 3 000 eggs from these females were then shipped to our Riverside laboratory. Larvae of the F. and F2 generations resulting from the shipment were tested for susceptibility to a variety of insecticides. Parallel tests were performed on larvae of a strain of Haiti/Panama origin, which were susceptible to carbamates and organophos- phorus compounds but resistant to dieldrin and moderately resistant to DDT. The colonies were maintained in 30 x 30 x 30 cm screened cages at 29°C and 60-70% R.H. and provided with white mice for blood meals. The larvae were kept in enamelled pans at 29°C and fed on a 50:50 mixture of brewers' yeast and Purina Laboratory Chow. 2 Larvae were tested in the early fourth instar in groups of 20 in 250-ml waxed paper cups containing 100 ml of water. The insecticides used were technical or analytical grade samples in standard w/v acetone solutions of the desired concentrations, 1 ml of which was added to 100 ml of water. The results were observed 24 hours later. Tests were repeated on at least 5 different days and the data subjected to probit analysis (Finney, 1952) on an IBM 360/50 computer. The results (Table 1) clearly indicate that the 1 Central America Malaria Research Station, San Salva- dor, El Salvador. ' Ralston Purina Co., St Louis, Mo., USA. CARBAMATE AND OP RESISTANCE IN A. ALBIMANUS 553 population of A. albimanus at Melara acquired con- siderable resistance to a variety of insecticides be- tween June 1970 and February 1971. Resistance was highest to the carbamates propoxur and carbaryl and the organophosphorus compounds parathion, parathion-methyl, malathion, and fenitrothion, and only slight to dichlorvos and Dursban,' whereas there was no resistance to fenthion and Abate.2 This is in agreement with the characteristic subgroup specificity of resistance to organophosphorus com- pounds observed in other insects (March, 1960; Georghiou & Hawley, 1971). A high degree of resistance is evident not only in increased LC50 values and the resultant resistance ratios, but also in characteristically low slope values (b) of the log- dose-probit-mortality regression lines. This is especially apparent with the carbamates, where b is 0.8 for carbaryl and 0.43 for propoxur. Similar results were obtained when resistance to propoxur and parathion was assayed in glass beakers instead of waxed paper cups: there was a small decrease in the LC50 for both the susceptible and the resistant strains but there was no significant change in the level of resistance obtained. High resistance-especially in the aquatic stage, which is bioassayed by continuous exposure of the organisms for 24 hours-would suggest that a com- bination of increased metabolism and decreased penetration of the insecticide may be responsible for resistance. The role of reduced penetration in producing considerably magnified levels of resistance has been amply demonstrated in the housefly (Plapp & Hoyer, 1968; Georghiou, 1971). Until appropriate tests on metabolism are carried out in the present strain, it is not possible to offer a biochemical inter- pretation of the broad spectrum of resistance we have observed, especially in regard to the involvement of carbamates in such resistance. Studies with various synergists on the propoxur-selected strain mentioned earlier, whose resistance spectrum closely resembles that observed in the present study, suggest that mixed-function oxidases and carboxylesterases are involved in metabolism (Ariaratnam & Georghiou, op. cit.). Mixed-function oxidases and a reduced rate of penetration have been shown to be responsible for propoxur resistance in larvae of Culex pipiens fatigans (Shrivastava et al., 1969). Although the precise mechanisms of resistance in 1 0,0-diethyl 0-(3,5,6-trichloro-2-pyridyl phosphoro- thioate. ' 0,0,0',O'-tetramethyl 0,0'-thiodi-p-phenylene phos- phorothioate. the present case are unknown, it is evident that the observed pattern of resistance to organophosphorus compounds corresponds closely to the extensive use of parathion, parathion-methyl, and malathion on cotton in El Salvador during the past several years. Fenitrothion has not been employed in this area, however, and the 9.2-fold resistance to this insecti- cide may be true cross resistance 1 in view of the close similarity of its molecule to that of parathion-methyl. A similar situation exists in California, where Aedes nigromaculis resistant to organophosphorus com- pounds showed a 75-fold cross resistance to feni- trothion although this compound has not been used commercially in the State (Schaefer & Wilder, 1970). In the housefly, the coexistence of resistance to fenitrothion and to parathion-methyl has been shown to be due to enhanced degradation of the activation products-the oxygen analogues of these pesticides- by phosphatase action (Hollingworth et al., 1967). The involvement of carbamates in such resistance is less well understood, however, in view of the rela- tively limited use that has been made of these insec- ticides in the study area. It is possible that resistance to these materials has resulted from selection by carbaryl, which is applied to rice and occasionally to cotton and maize (corn), and may have been en- hanced by the broad-spectrum resistance to organo- phosphorus compounds present in the population. The coexistence of propoxur, malathion, and fenitrothion resistance in A. albimanusin El Salvador, and its possible effect on malaria eradication in Central America, require careful assessment, since these three chemicals have been considered to be the outstanding alternative insecticides for use against DDT- and dieldrin-resistant anophelines. ACKNOWLEDGEMENTS This investigation was supported in part by Public Health Service research grant CC 00301 from the Center for Disease Control, Atlanta, Ga., USA. REFERENCES Ariaratnam, V. & Georghiou, G. P. (1971) Nature (Lond.), 232, 642 Breeland, S. G. et al. (1970) Bull. Wld. Hlth Org., 43, 627 Finney, D. J. (1952) Probit analysis, London, Cambridge University Press 1 " Cross resistance" refers to those cases in which one mechanism confers protection against a variety of toxicants. 9 554 BRIEF COMMUNICATIONS/NOTES Georghiou, G. P. (1963) Effects of carbamate insecti- cide selection pressure on Anopheles albimanus Wied. In: Proceedings and papers of the thirty-first annual conference of the California Mosquito Control Associa- tion, Visalia, Calif., p. 80 Georghiou, G. P. (1969) Wld Rev. Pest Control, 8, 86 Georghiou, G. P. (1971) Proceedings of the second Inter- national Congress of Pesticide Chemistry, Tel Aviv, London, Gordon & Breach, Vol. 2, p. 77 Georghiou, G. P. & Calman, J. R. (1969) Bull. Wld Hlth Org., 40, 97 Georghiou, G. P. & Hawley, M. K. (1971) Bull. Wld Hlth Org., 45, 43 Georghiu, G. P. & Metcalf, R. L. (1963) Science, 140, 301 Hollingworth, R. M. et al. (1967) J. agric. Food Chern., 15, 250 March, R. B. (1960) Misc. Publ. ent. Soc. Amer., 2, 139 Plapp, F. W. Jr., & Hoyer, R. F. (1968) J. econi. Enit., 61, 1298 Schaefer, C. H. & Wilder, W. H. (1970) J. econ. Ent., 63, 1224 Shrivastava, S. P. et al. (1969) Bull. Wld Hith Org., 42 931 Wright, J. W. et al. (1969) Bull. Wid Hlth Org., 40, 67 Suppression of a field population of Aedes aegypti by malathion thermal fogs and Abate larvicide Y. H. BANG,1 N. GRATZ,2 & C. P. PANT 3 In previous field studies, a field population of Aedes aegypti was suppressed by larviciding water containers with 1 % Abate 4 sand granules.5 How- ever, the decline in the population following initial treatment was gradual, a factor that might limit the use of a larvicidal treatment by itself during an epidemic of dengue haemorrhagic fever. Immediate control of adult populations can be achieved by the application of insecticidal thermal logs, although the application must be repeated several times, as it has been found in previous field trials at the ARU (unpublished data) that the mosquito density returns to the pretreatment level within a week. A combined treatment by the two different methods i.e., adulticid- ing and larviciding, appears to be desirable in com- bining an emergency operation and a long-term con- trol programme. The exact rate of the reduction in adult mosquito populations and their recovery in an 1 Entomologist, WHO Aedes Research Unit, Bangkok, Thailand. Present address: WHO East Africa Aedes Research Unit, Dar es Salaam, Tanzania. 2 Entomologist, Vector Biology and Control, World Health Organization, Geneva, Switzerland. 3 Project Leader, WHO Aedes Research Unit, Bangkok, Thailand. 4 O,O,O',O'-tetramethyl O,O'-thiodi-p-phenylene phos- phorothioate. 'Bang, Y. H. et al. (1969) Pilot studies of Abate (OMS 786) as a larvicide for controlling Aedes aegypti in Bangkok (Unpublished document WHO/VBC/69.164). area where all larval habitats are treated with Abate is not known. The investigation reported in this paper was car- ried out in Bangkok by the World Health Organiza- tion Aedes Research Unit (ARU) to determine the efficacy of malathion thermal fogs, applied soon after larviciding by 10% Abate sand granules, on the sup- pression of a field population of Ae. aegypti and its recovery. This was one of a series of field studies undertaken by the ARU to devise emergency control measures that can be recommended when an epi- demic occurs. Description of the area The study was carried out in a housing area of Bang Sue, in the northern part of Bangkok, which had an area of approximately 7 ha, was 830 m in length and 85 m in width, and was divided into two unequal parts by a playground (0.5 ha); the two parts were used for evaluation of the two different treatments. There are 52 apartment buildings, and 304 houses in the treated area: 172 in site A and 132 in site B. The structure of the houses at the two sites were alike and similar to those described by Bang et al.5 To determine the rate of reduction of the adult mosquito population in an area where there was no mosquito infiltration from untreated neighbour- 2831B