WORLD HEALTH ORGANIZATION ORGANISATION MONDIAL£ DE LA SANTE
WHO/VBC/79.736 ENGLISH ONLY IND£X£;.,
INSECTICIDE SUSCEPTIBILITY OF VECTORS OF CHAGAS' DISEASE IN VENEZUElA by M. J. Nelson,
1
and P. Colmenares
2
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 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. 1
The issue of this document does not constitute formal publication. lt should not be reviewed, abstracted or quoted without the agreement of the World Health Organization. Authors alone are responsible for views expressed in signed articles.
Ce document ne constitue pas une publication. 11 ne doit faire l'objet d'aucun compte rendu ou resume ni d'aucune citation sans l'autorisation de !'Organisation Mondiale de la Sante. Les opinions exprimees dans les articles signes n·engagent que leurs auteurs.
WHO/VBC/79. 736 page 2 Two of the strains, the Cojedes dieldrin-susceptible and the Santa Domingo dieldrinresistant 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 mid1976. 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. 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.
Knock-
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 knockdown), 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 (Lc 50 and Lc 95 ) 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 (KT 50 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 F 1 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, knockdown 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. The close similarity between the knockdowns of the field-collected and F1 generation of each strain demonstrates that the resistance level in the populations was genetically determined, and not a result of differences in alimentation or habitat between field and colonized insects, or weakening of insects during transport from the field to the laboratory. GonzalezValdivieso et al. (1971) found that F 1 progeny of survivors of dieldrin exposure were sometimes less susceptible to dieldrin exposure than their parents and sometimes more susceptible.
WHo/vBc/79.736 page 4 High The present study covered a zone in central Trujillo state about 25 km long. levels of resistance were found throughout the zone, with no indication of diminished tolerance towards the borders. Further studies are necessary to define the geographical distribution of resistant and tolerant populations throughout the state. Populations at the borders of the resistant area should be tested annually to determine if resistance is spreading. There was evidently no tolerance in Trujillo to fenitrothion and only a very low degree of tolerance to fenthion and propoxur. Every one of the villages had been sprayed frequently for the past 30 years, at first with DDT, then with dieldrin. In the early 1970s, after the discovery of dieldrin resistance in Trujillo, many different insecticides were tried, including diazinon, malathion, fenthion, jodfenphos and propoxur. Dieldrin and gamma BHC were used very little. During 1976 and 1977 dieldrin and gamma BHC were again used and fenthion was tried. As fenitrothion had not been used previous to these tests, this may account for the complete susceptibility to this compound. Field trials with fenitrothion are now in progress in Trujillo. Fenthion and propoxur should still be quite effective for control for some time. CONCLUSIONS (1) The only state in Venezuela found with a high level of dieldrin resistance by R. prolixus was Trujillo. No resistant strains of T. maculata were found. (2) The level of resistance in the Trujillo villages was genetically determined.
(3) A low level of tolerance was found against fenthion and propoxur, but not fenitrothion. (4) The complete distribution of dieldrin resistance in Trujillo should be determined. ACKNOWLEDGEMENTS The authors gratefully acknowledge the advice and guidance of Dr R. J. Tonn, PAHO/DPC, Washington, formerly Project Leader of the Center; Dr N. G. Gratz and Dr R. Pal, WHO/VBC, Geneva; and Dr R. Busvine, formerly of the London School of Tropical Medicine and Hygiene, now retired. Dr F. Nocerino kindly provided the insects to begin the Santo Domingo substrain. Jose Guerra, Antonio Guerra and Armando Cardozo assisted with the laboratory tests, Freddy Rodriguez maintained the colonies from the various states, and field teams made the collections. REFERENCES Busvine, J. R. (1970) Chagas' disease control and the possibilities of resistance in triatomids. (Unpublished document WHO/VBC/70.195)
Gonzalez-Valdivieso, F. E., Sanchez Diaz, B. & Nocerino, F. (1971) ~S~u~s~c~e~t~i~b~~~·l~~~·~t~~~~~r~o~l~~~·x~u~s to chlorinated hydrocarbon insecticides in Venezuela (Unpublished Nocerino, F. (1976) Susceptibilidad de R. prolixus y T. maculata a los insecticidas en Venezuela. Boletin de la Direccion de Malariolo ia Saneamiento Ambiental Venezuela, 16 (3), 276-283 (WHO VBC 75.565, English translation)
WHO/VBC/79.736 page 5
TABLE 1. SUSCEPTIBILITY MONITORING OF THE COLONIZED COJEDES DIELDRIN-SUSCEPTIBLE STRAIN TO DIELDRIN IMPREGNATED PAPERS, SHOWING PER CENT. MORTALITIES (NUMBERS TESTED IN PARENTHESES) , CONCENTRATIONS NECESSARY TO KILL 50% AND 95% OF THE INSECTS (LC , Lc ), SLOPE, AND RESISTANCE LEVEL 50 95 Concentration of dieldrin (7o) Generation 0.4 Fl F2 F3 F4 F5 2 (40) 0 (40) 15 (20) 30 (40) 15 (20) 0.8 9 (57) 7 (60) 55 (19) 78 (40) 70 (20) 1.6 61(67) 73(67) 95 (20) lOO (40) lOO (20) 4.0 99 (68) lOO (60) 100 (20) lOO (40) 95 (20) 1.4 1.3 3.2 2.3 1.7 1.1 1.3 2.3 1.7 2.3 1.9 2.0
LC50
LC95
s
R
0.93 0.54 0.41 0.46
o. 75 0.57 0.64
s = Slope of dosage -mortality curve= Lc 95 :LC 50 R = Resistance level compared to F 1 generation = LC -
50
of generation F :LC of F • 1 i 50
RESISTANCE LEVELS OF THE COLONIZED SANTO DOMINGO DIELDRIN-RESISTANT TABLE 2. STRAIN, SELECTED EACH GENERATION BY EXPOSURE FOR SEVEN DAYS TO 4% DIELDRIN-IMPREGNATED PAPERS Per cent, mortality 15 13 10 7 3 3
Generation
Number tested 808 2 187 4 523
F2 F3 F4 F5 F6 F7
3 650 4 647 2 285
WHO/VBC/79. 73 6 page 6
TABLE 3.
SUSCEPTIBILITY TESTING OF T. MACULATA FROM EIGHT STATES IN VENEZUELA AGAINST DIELDRIN-IMPREGNATED PAPERS
State
Generation
Per cent. concentration 0.2 0.4 0.8 1.6 4.0 0.2 0.4 0.8 1.6 4.0 0.4 0.8 1.6 4.0 1.6 4.0 0.2 0.4 0.8 1.6 4.0 0.4 0.8 1.6 4.0 0.4 0.8 1.6 4.0 0.2 0.4 0.8 1.6 4.0
Per cent. mortality 8 6 30 80 lOO 0
Number tested 25 50 50 50 50 30 59 59 70 70 20 60 80 70 20 30 20 61 82 80 70 31 30 30 30 20 30 40 30 20 20 20 20 30
Anzoategui
Fl-2
Aragua
P-F
2
2 32 86 lOO 0 47 93 lOO 85 lOO 0 5 33 88 lOO 13
Bolivar
F2-3
Carabobo
F3' F5 Fl, F4
Cojedes
Falcon
F2
60 lOO lOO 5 60 98 lOO 0 15 65 lOO lOO
Lara
Fl-4
Portuguesa
p
WHO/VBC/79.736 page 7
TABLE 4.
SUSCEPTIBILITY TESTING OF R.PROLIXUS FROM 10 STATES IN VENEZUELA AGAINST DIELDRIN-IMPREGNATED PAPERS
State
Generation
Per cent. concentration 0.8 1.6 4.0 4.0 0.4 0.8 1.6 4.0 4.0 4.0 0.4 0.8 1.6 4.0 0.8 1.6 4.0 0.4 0.8 1.6 4.0 0.8 1.6 4.0 0.4 0.8 1.6 4.0
Per cent. mortality 0 89 lOO lOO 0 3 83 lOO 97 93 40 52 96 lOO 0 90 lOO 5 36 86 lOO 0 80 lOO 3 4 42 lOO
Number tested 5 9 18 105 59 60 60 60 85 30 40 60 98 108 20 20 157 20 50 50 50 10
Anzoategui
FJ-4
Barinas Carabobo
p
Fl
Cojedes Gml'rico Miranda
p
Fl F2-4
Portuguesa
P-F
1
Sucre
F3
Tachira
Fl, F7
10 53 39 50 60 80
Yaracuy
Fl-2
WHO/VBC/79. 736 page 8
TABLE 5. SUSCEPTIBILITY TESTS OF R. PROLIXUS FROM 10 VILLAGES IN THE STATE OF TRUJILLO BY CONTINUOUS EXPOSURE TO PAPERS IMPREGNATED WITH 4% DIELDRIN. PER CENT. KNOCKDOWN AFTER TWO AND SEVEN DAYS EXPOSURE
Generation p
F1 Number tested Exposure 2 days 46 73 27 75 17 10 12 42 58 3 0 0 0 0 13
Strain Exposure 2 days 1. 2. 3. 4. El Tab1o'n Rio Morosmoy Par ami to Betico Pa1o Negro Cuba San to Domingo La Vera Montana Peraza 0 1 0 5 6 10 33 45 60 lOO 7 days 4 7 19 19 29 30 75 79 lOO lOO Number tested
7 days
3 0 0 0 26 38 87 78 lOO
31 34 21 64 87 8 38 68 38
s. 6. 7.
0 63 31 34
B. 9.
10. La Cortadora
-
-
-
WIIO/VBc/79. 73 6
pagee 9
TABLE 6. SUSCEPTIBILITY OF R. PROLIXUS TO FENTHION, FENITROTHION AND PROPOXUR BY CONTINUOUS EXPOSURE TO IMPREGNATED PAPERS. TIMES FOR SO% AND 95% KNOCKDOWN (KT AND KT ) GIVEN IN HOURS 50 95
FENTHION Strain (2.5%) KT 50 KT95 A. Trujillo F 1 18 16 22 18 16 28 18 32 26 31 31 32 65 32 1.8 1.2 30 1.6 1.1 30 1.4 1.5 10 1.7 1.2 30 2.0 1.1 20 2 .3 1.9 10 1.8 1.2 40 19
FENITROTHION (1.0%) R N KT 50 KT95
PROPOXUR (1.0%)
s
s
R
N KT 50 KT95
s
R
N
l. El Tablon
30 29 27 35 31 I
1.6 0.8 50 1.5 0.8 30 1.7
24 21
30 25
1.2 1.5 20 1.2 1.3 10
2. Rio Morosmoy 3. Par ami to 4. Betico 5. Palo Negro 6. Santa Domingo 7. La Vera
19 16 20 19 20 26 19
o. 7 24
19 33 24 40
22 43 42 46 -
-
-
-
1.8 0.8 80 1.6 0.8 30 1.2 0.8 20 1.5 1.1 60 2.0 1.6 30
1.2 1.2 10 1.3 2.1 10 1.8 1.5 10 1.2 2.5 20
25 40 38
B. Montana Peraza B. Cojedes colony a 1. F4 parallelb 2. F previous3
15 18
21 28
-
-
-
-
1.6
-
-
1.4
- 30
23 25
39 37
1.7
-
60
16 12
25 16
- 40
1.5 1.2 70
1.5 1.1 80
1.3 0.8 50
c.
Santa Domingo colony a 1. F5 parallelb 2. F4 previous17 26 1.5 1.1 40 25 20 37 28 1.5 1.1 60 1.4 0.9 20 17 22 40 33 2.4 1.1 20 1.5 1.4 40
-
-
-
-
-
~ Tests run continuously in parallel to Trujillo. b
-Tests done previously.
KT determined by separate replicates for each exposure time.
R N
Resistance level
=
KT
50
of each village:
KT
50
Cojedes F 4
Number observed for each time period.
WHO/VBC/79.736 page 10
FIG. 1. RELATIVE LOCATION OF 10 VILLAGES IN THE STATE OF TRUJILLO, INDICATING PER CENT. KNOCKDOWN OF RHODNIUS PROLIXUS FROM EACH VILLAGE AFTER EXPOSURE TO 4% DIELDRIN IMPREGNATED PAPERS FOR 48 HOURS
c .... Betico
El Tabl6n
_o___
x. . . N
Rio Morosmoy _l __ )
5
La Vera
45
~ntaDa ( 33
c )
La Cortadora
100
)
Peraza
Santo Domingo
Paramito
C) 6
Palo Negro 10 Km