WORLD HEALTH ORGANIZATION WHO/VBC/79.736 ORGANISATION MONDIAL£ DE LA SANTE ENGLISH ONLY IND£X£;., INSECTICIDE SUSCEPTIBILITY OF VECTORS OF CHAGAS' DISEASE IN VENEZUElA by 1 2 M. J. Nelson, and P. Colmenares ABSTRACT The level of dieldrin susceptibility was determined for the Chagas' disease vector Rhodnius prolixus in 11 Venezuelan states and for Triatoma maculata in 8 states. The only high level of resistance found was in R. prolixus in the state of Trujillo, where resistance was found in 9 out of the 10 villages sampled. That the resistance was genetically determined was shown by the close similiarity of resistance level of the field-collected insects from each village and their F offspring. There was a low level 1 of tolerance to fenthion and propoxur but no tolerance to fenitrothion. INTRODUCTION Chagas' disease in Venezuela is controlled mostly by application of residual insecticides to houses and annexes (animal shelters, sheds, etc.) to kill the main triatomine vector, Rhodnius prolixus, and the less important vector, Triatoma maculata. For many years dieldrin has been used in houses because of its long residual life, and gamma BHC has been used in animal shelters because it is less toxic to poultry and livestock. In 1969, resistance of R. prolixus to dieldrin was found in Trujillo, and varying degrees of tolerance were found in the states of Yaracuy, Tachira, Cojedes and Portuguesa (Busvine, 1970; Gonzalez-Valdivieso et al., 1971; Cockburn, 1972; Nocerino, 1976). Cross-resistance to gamma BHC was found in Trujillo, and tolerance to fenthion and propoxur was observed. Also, tolerance of T. maculata to dieldrin was found in Trujillo. In the present paper, the susceptibility mapping of the two species to dieldrin is extended to several new states, and the susceptibility of several dieldrin-resistance strains to three organophosphorus compounds is determined. MATERIALS AND METHODS During 1976 to 1978 numerous collections of R. prolixus and T. maculata were made from houses and animal shelters throughout Venezuela for studies of taxonomy, distribution and epidemiology. Laboratory colonies were established from each state in 4 1, wide-mouth glass jars with netting tops and folded paper inside, and fed every two weeks on chickens. Some of the original field collections were small, and insufficient insects were available for susceptibility testing until the F generation or later. 1 1 PAHO Research and Reference Center on Vector Biology and Control, Apartado 2171, Las Delicias, Maracay, Venezuela. 2 Ministerio de Sanidad y Asistencia Social, Direccion de Malariologia, Maracay, Venezuela. The issue of this document does not constitute Ce document ne constitue pas une publication. formal publication. lt should not be reviewed, 11 ne doit faire l'objet d'aucun compte rendu ou abstracted or quoted without the agreement of resume ni d'aucune citation sans l'autorisation de the World Health Organization. Authors alone !'Organisation Mondiale de la Sante. Les opinions are responsible for views expressed in signed exprimees dans les articles signes n·engagent articles. que leurs auteurs. WHO/VBC/79. 736 page 2 Two of the strains, the Cojedes dieldrin-susceptible and the Santa Domingo dieldrin­ resistant were chosen as standard reference strains for all insecticide testing, and the susceptibility was monitored each generation. The Cojedes strain was started from collections from various municipalities in the state of Cojedes during 1976. The Santa Domingo strain was first colonized in 1969 by the Pesticide Evaluation Service of Venezuela and had been selected for resistance to dieldrin papers by a process described by Nocerino (1976). This strain was in the tenth generation when the Service provided approximately 50 insects in mid- 1976. Each generation was selected for dieldrin resistance by seven days exposure of fifth instar nymphs to 4% dieldrin papers. Methods for susceptibility testing were adapted from unpublished document WHO/VBc/75.587. Insecticide-impregnated papers, procured from WHO, were cut into strips 3 x 15 cm and inserted into glass test-tubes 20 x 2.5 cm diameter. Fifth instar nymphs were distributed 10 to a tube five days after feeding. Between observations the racks of tubes were held in a dark cabinet in a room without temperature or humidity control. Temperature extremes varied from 24.5 to 33.5°C and humidity from 36 to 55%. Three different kinds of observation regimes were employed. 1. Fixed time, varying concentration: R. prolixus and T. maculata from all of the states except Trujillo were exposed to various concentrations of dieldrin papers for 48 hours, knockdown was recorded, and the insects were transferred to clean test-tubes with untreated paper for another 48 hours before final mortality was recorded. 2. Fixed concentration, varying times: As only one concentration of each of the organophosphorus and carbamate compounds was available, exposure time was more varied. Knock­ down was noted after the appropriate exposure time in each tube, and the insects were transferred to a clean tube for 48 hours holding before final mortality was recorded. 3. Continuous exposure. For the 10 villages from Trujillo, insufficient numbers of insects were available from each village to use either of the above two methods. Also, most Trujillo strains were resistant to dieldrin, and it was of interest to observe the response for periods longer than 48 hours. Therefore, all Trujillo strains were exposed continuously to all of the insecticides and several observations of knockdown were made on each tube. For dieldrin, observations were made daily for seven days. For fenthion, fenitrothion and propoxur, observations were made hourly for eight hours, then every two hours for the next four hours, then every four hours until all insects were dead. All data were graphically plotted by log of dosage against probits of mortality (or knock­ down), and a straight line was fitted to the points by eye. When variable concentration at fixed time was the dosage, the concentrations for 50% and 95% mortality (Lc50 and Lc95 ) and knockdown (KC and Kc ) were read from the graph. When variable time at fixed concentration 5 95 was the dosage? the times for 50% and 95% mortality (LT5o and LT95) and knockdown (KT50 and KT95) were determined. RESULTS Laboratory strains Table 1 shows the results of susceptibility monitoring during five generations of the Cojedes dieldrin-susceptible strain. There was an increase in susceptibility from generation to generation. By the F5 generation the LC5o was less than half that of the F1 generation. The slope (S) of the dosage-mortality curve remained unchanged. WHo/vsc/79.736 page 3 As seen in Table 2, the Santo Domingo strain was highly resistant to dieldrin, and the resistance increased from generation to generation. F numbers refer to number of generations after the sub-colony was brought to the laboratory. Field strains No strains of T. maculata were resistant to dieldrin and only the Trujillo strains of R. prolixus. As seen in Table 3, the diagnostic d0sage of 4% accounted for lOO% mortality for every strain of T. maculata, and 1.6% resulted in 80-100% mortality. For R. prolixus (Table 4), 4% dieldrin caused lOO% mortality in all strains except Cojedes and Guarico, where occasional survivors were observed. The dosage of 1.6% resulted in at least 80% mortality in all strains except Yaracuy (42%). In Table 5 the tests of 4% dieldrin against the 10 strains from the state of Trujillo are shown by village. After the standard two-day exposure, which knocks dowtl all insects of the susceptible Cojedes strain, knockdown was 0-60% in all Trujillo strains except La Cortadora, which had only three insects available for testing. Even after seven days exposure, knock­ down was 4-79% in eight strains and lOO% in only two strains. Geographically, the villages sampled in Trujillo formed roughly three clusters (Fig. 1). For the strains from Rio Morosmoy, El Tablon and Betico, knockdown after two days exposure to dieldrin was 1-5%. In Paramito, Palo Negro and Cuba, approximately 20 km away, knockdown was 0-10%. Between these two groups lay Santo Domingo, La Vera, Montana Peraza and La Cortadora, where knockdowns were much higher, at 33-100%. The percentage knockdown for each strain of field-collected (P) insects was remarkably similar to that of the F1 generation progeny of each strain of field collected insects that had not been exposed to insecticide in the laboratory. Knockdown times of the Fl Trujillo strains against fenthion, fenitrothion and propoxur are shown in Table 6. The Cojedes colonized strain (F4) was tested at the same time, in parallel with the Trujillo strains, and forms the baseline susceptibility for comparison. Results of previous tests with Cojedes strain are also given, and are similar to the F4 tests. The colonized Santo Domingo strain was also run in parallel. The knockdown times for SD were only slightly longer than COJ. The resistance ratio (R) was only 1.1 for each of the three insecticides. For Trujillo, six of the eight strains were less tolerant to fenitrothion than was Cojedes. All were somewhat more tolerant to fenthion (R= from 1.1 to 1.9) and propoxur (R= from 1.2 to 2.5). The propoxur data are less reliable because fewer insects were tested and the impregnated papers were eight months old, three months older than the official WHO expiry date. However, it is notable that all insecticides caused lOO% knockdown, and that there was no appreciable flattening of the dosage-mortality curves. DISCUSSION The occurrence of occasional survivors of R. prolixus to 4% dieldrin after two days exposure for the Cojedes and Guarico strains is not cause for alarm, but susceptibility in these two states should be monitored yearly to detect any increase in tolerance.