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Keywords:

  • Triatoma infestans;
  • insecticide;
  • resistance;
  • Bolivia
  • Triatoma infestans;
  • insecticide;
  • résistance;
  • Bolivie
  • Triatoma infestans;
  • insecticida;
  • resistencia;
  • Bolivia

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Objective  To define the insecticide resistance status of Triatoma infestans to deltamethrin (pyrethroid), malathion (organophosphate) and bendiocarb (carbamate) in Bolivia.

Methods  Fifty populations of T. infestans were sampled in Bolivian human dwellings. Quantal response data were obtained by topical applications of 0.2 μl of insecticide–acetone solutions on nymphs N1 of the F1 generations. For most populations, dose–mortality relationships and resistance ratios (RR) were analysed. Discriminating concentrations were established for each insecticide with a susceptible reference strain and used on the other field populations. A tarsal-contact diagnostic test using insecticide impregnated papers was designed to rapidly identify deltamethrin-resistant populations in the field.

Results  Discriminating concentrations for topical applications were 5, 70 and 120 ng active ingredient per insect for deltamethrin, bendiocarb and malathion, respectively. The diagnostic concentration for deltamethrin was 0.30% for the 1-h exposure by tarsal contact. All populations sampled in human dwellings exhibited significant levels of resistance to deltamethrin, from 6 to 491 and varied among regions. Resistant populations did not recover complete susceptibility to deltamethrin when the synergist piperonyl butoxide (PBO) was used. None of the sampled populations exhibited significant resistance to bendiocarb (all RR50 < 1.8) or malathion (all RR50 < 2.2).

Conclusion  In Bolivia, most ‘domestic’T. infestans populations are resistant to deltamethrin. Because insecticide vector control is the only selection pressure, resistance likely originates from it. Switching from pyrethroids to organophosphates or carbamates could be a short-term solution to control this vector, but other alternative integrated control strategies should also be considered in the long term.

La résistance aux insecticides de Triatoma infestans (Hemiptera, Reduviidae), vecteur de la maladie de Chagas en Bolivie

Objectif:  Définir la situation de la résistance de Triatoma infestans au deltaméthrine (pyréthroïdes), au malathion (composé organophosphoré) et au bendiocarbe (carbamate) en Bolivie.

Méthodes:  Cinquante populations de T. infestans ont étééchantillonnées dans des habitations humaines en Bolivie. Des données de réponses quantiques ont été obtenues par l’application topique de 0,2 μl de solutions acétonique d’insecticide sur les nymphes N1 des générations F1. Pour la plupart des populations, les relations dose-mortalité et les rapports de résistance (RR) ont été analysés. Les concentrations discriminantes ont étéétablies pour chaque insecticide avec une souche sensible de référence et utilisées sur les autres populations de terrain. Un test de diagnostic de contact tarsal utilisant du papier imprégné d’insecticide a été conçu pour identifier rapidement les populations résistantes au deltaméthrine sur le terrain.

Résultats:  Les concentrations discriminantes pour les applications topiques étaient de 5, 70 et 120 ng de substance active par insecte pour le deltaméthrine, le bendiocarbe et le malathion, respectivement. La concentration de diagnostic pour le deltaméthrine était de 0,30% pour une heure d’exposition par contact tarsal. Toutes les populations échantillonnées dans les habitations humaines présentaient des niveaux importants de résistance au deltaméthrine de 6 à 491 et variant selon les régions. Les populations résistantes n’ont pas retrouvé une sensibilité complète au deltaméthrine lorsque le synergiste pipéronyle-butoxite (BPO) a été utilisé. Aucune des populations échantillonnées ne présentaient une résistance importante au bendiocarbe (tous les RR50 < 1,8) ou au malathion (tous les RR50< 2,2).

Conclusion:  En Bolivie, la plupart des populations «domestiques» de T. infestans sont résistantes au deltaméthrine. Parce que la lutte antivectorielle par insecticide est la seule pression de sélection, la résistance viendrait probablement de là. Passer des pyréthroïdes aux organophosphorés ou aux carbamates pourrait être une solution à court terme pour contrôler ce vecteur, mais d’autres stratégies alternatives de lutte intégrée devraient également être envisagées à long terme.

Resistencia a insecticidas del Triatoma infestans (Hemiptera, Reduviidae), vector de la enfermedad de Chagas en Bolivia

Objetivo:  Definir el estatus de resistencia del Triatoma infestans frente a deltametrin (piretroide), malatión (organofosfato) y bendiocarb (carbamato) en Bolivia.

Métodos:  Se muestrearon cincuenta poblaciones de T. infestans en hogares Bolivianos. Se obtuvieron datos de respuesta cuantal mediante aplicaciones tópicas de 0.2 μl de solución de insecticida-acetona en nínfas N1 de generaciones F1. Para la mayoría de las poblaciones se analizó la relación entre la dosis y la mortalidad, y las tasas de resistencia. Se establecieron las concentraciones discriminatorias para cada insecticida utilizando una cepa de referencia susceptible, y se utilizaron en otras poblaciones de campo. Se establecieron las concentraciones discriminatorias para cada insecticida con una cepa de referencia susceptible y utilizada en otras población de campo. Se diseñó una prueba de contacto tarsal utilizando papeles impreganados de insecticida, para identificar rápidamente sobre el terreno las poblaciones deltametrin resistentes .

Resultados:  Las concentraciones discriminatorias para aplicaciones tópicas fueron 5, 70 y 120 ng de ingrediente activo por insecto para deltametrin, bendiocarb y malation respectivamete. La concentración diagnóstica para deltametrin era 0.30% para una hora de exposición por contacto tarsal. Todas las poblaciones muestreadas en lugares habitados por humanos, tenían niveles significativos de resistencia al deltametrin, de 6 a 491 con variación entre regiones. Las poblaciones resistente no recuperaron completamente la susceptibilidad al deltametrin cuando se utilizó el butóxido de piperonilo (BOP). Ninguna de las poblaciones muestreadas tenía una resistencia significativa frente al bendiocarb (todos RR50 <1.8) o malation (todos RR50 < 2.2).

Conclusión: En Bolivia, la mayoría de las poblaciones “domésticas” de T. infestans son resistentes a deltametrin. Pues que el control vectorial con insecticida es la única presión de selección, probablemente es el orígen de la resistencia. El cambiar de piretroides a organofosforados o carbamatos sería una solución a corto plazo para este vector, pero otras estrategias de control integrado también deberían considerarse como alternativas a largo plazo.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

With more than 10 million human cases, Chagas disease is one of the most important parasitic diseases in Latin America. It is caused by the protozoan Trypanosoma cruzi (Kinetoplastida, Trypanosomatidae) and is the major cause of cardiopathy in the world (Yacoub et al. 2008). The parasite is transmitted mainly by blood-sucking insects of the Triatominae family (Heteroptera, Reduviidae), which are responsible of more than 80% of human cases (Schofield 1994). In Bolivia, 55% of the territory is considered endemic for Chagas disease and involves around four million people at-risk (approximately 50% of the total population). As well as in all the countries of the ‘southern cone’ (Southern Peru, Bolivia, Argentina, Paraguay, Brazil), the kissing bug Triatoma infestans is the main vector (World Health Organization 2002). It entirely completes its life cycle in human dwellings (in intra- and peridomicile environments) and present vector control strategies are therefore based on indoor and outdoor sprayings of persistent insecticides (pyrethroids). In Bolivia, this spraying is the responsibility of the Ministry of Health, which manages a National Program for the Control of Chagas Disease (NPCCD) (Rojas Cortez 2007).

Despite some good results obtained with insecticide sprayings in the southern cone countries since the 1950s (Zerba 1999), endemic vector-borne transmission still occurs in large areas of Southern Peru, Bolivia and Argentina. Several factors may explain the maintenance of Chagas transmission in these regions (Gürtler et al. 2007), among which insecticide resistance has recently been pointed out as one of the most significant (Vassena et al. 2007). Indeed, since 1997, resistance to pyrethroids has been detected in certain areas of Argentina (Vassena & Picollo 2003; Gonzalez Audino et al. 2004) and in 2002, failures in field insecticide treatments have been associated with deltamethrin resistance (Picollo et al. 2005). Recently, high levels of deltamethrin resistance have been observed in Bolivia, in the vicinity of Yacuiba, Sucre and Mataral (Santo Orihuela et al. 2008; Toloza et al. 2008), confirming preliminary data obtained in 2003–2005 (Vassena et al. 2007). In Bolivia, however, the geographical extent and the magnitude of insecticide resistance remains unknown despite some recent alarming reports of treatment failures by technicians of the NPCCD.

The aim of this study is therefore to assess the resistance status of various Bolivian field populations of T. infestans to deltamethrin, the pyrethroid insecticide used by the Bolivian NPCCD. An organophosphate insecticide (malathion) and a carbamate (bendiocarb) have been proposed as alternatives to deltamethrin in Bolivia (Vassena et al. 2007) and therefore are also tested here. A diagnostic concentration for each of the three tested insecticide is set for topical applications. A tarsal-contact diagnostic test is also proposed to rapidly assess the resistance status of field populations to deltamethrin. This is the first large-scale study in Bolivia.

Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Triatoma infestans populations

Fifty field populations of T. infestans were collected between 2006 and 2009 by means of active search in infested human dwellings (Pinchin et al. 1981) by the personnel of the NPCCD before routine insecticide treatments. Localities were chosen without any previous knowledge of the T. infestans population resistance status. Three localities were in the Cochabamba Department 20 in the Chuquisaca Department 20 in the Tarija Department and seven in the La Paz Department, covering most of the geographical distribution of the species in this country (only populations from the Santa-Cruz Department are lacking) (Figure 1).

image

Figure 1.  Distribution of Triatoma infestans in Bolivia (shaded area) and sample locations for insecticide resistance studies. Names of Departments are in italics.

Download figure to PowerPoint

In Chivisivi (La Paz Department), a T. infestans population (normal phenotype) was also captured with sticky traps in a sylvatic rocky environment 500 m away from any house or human activities. Sylvatic populations are considered as independent entities without any evident interchanges with ‘human dwelling’ populations (Noireau 2009). Field populations were reared in the insectary following the method of Núñez and Segura (1987) until the F1 generation from which first-instar nymphs (N1) were used for the bioassays. The laboratory strain CIPEIN (Picollo et al. 1976) was used as a susceptible reference strain for bioassays.

Chemicals

Technical grade insecticides were deltamethrin (100% purity, AgrEvo, Berkham, UK), malathion (98.4% purity, Supelco, Bellefonte PA, USA) and bendiocarb (96% purity, Aventis CropScience (Bayer), Cambridge, UK). Piperonyl butoxide (PBO) (Sigma-Aldrich, France) was used as a synergist for deltamethrin.

Topical application bioassays

Serial dilutions of the insecticides in acetone were prepared, and 0.2 μl of solution was topically applied on the dorsal surface of the abdomen of each N1 (WHO 1994, CIPEIN CITEFA 2001), with a 10-μl Hamilton Microliter 701 micro-syringe (Hamilton Co, Reno, NE, USA) mounted on a repeating dispenser (Hamilton PB600). The mean weight of N1 was 1.69 mg (± 0.31). At least 15 (and up to 100) insects per dose per replicate were used. At least three replicates were carried out for each insecticide/population experiment. For each experiment, at least four doses flanking the LD50 (insecticide dose that killed 50% of the population) and causing >0 or <100% mortality were used. For each experiment, a control group received only acetone. Treated and control insects were maintained in a climatic chamber (Sanyo MLR 351-H, Japan) under controlled conditions of temperature (27 ± 1 °C), relative humidity (60 ± 5% RH) and photoperiod 12:12 h (light:dark). Mortality was recorded at 24 h. The criterion for mortality was the inability of the nymphs to walk out of a filter paper disc of 7 cm diameter (Vassena et al. 2000; Picollo et al. 2005).

Probit analysis (Finney 1971) was performed on mortality data using probit ver.2 software (Raymond et al. 1993). When results exhibited a large Chi-squared for the Log-probit lines (which was the case for only the El Chaco population for deltamethrin and San Francisco del Inti population for bendiocarb) and because there was no sign of systematic deviation from linear regression, the heterogeneity factor H (Finney 1971) was computed to continue the computations. Resistance ratios (RR) were computed relative to the susceptible reference strain CIPEIN as inline image. When lines were parallel, RRs were computed at LD50 (i.e. RR50) (Finney 1971) and in addition, at LD90 (i.e. RR90) when probit lines were not parallel (Robertson et al. 2007).

Bioassays with piperonyl butoxide (PBO)

A dose of 1000 ng active ingredient (a.i.) per insect PBO (Vassena et al. 2000) in 0.2 μl acetone solution was first topically applied on the dorsal surface of the abdomen of N1. Insects were then left 1 h before being processed for topical application of insecticide as explained above. Then, RRs were computed relative to the reference strain CIPEIN, and the percentage of effective reduction in RR50 (with and without PBO) was computed as:

  • image

If the only mechanism of resistance is based on detoxifying enzymes such as oxidases (cytochrome P450), Preduc% would be expected to be 100% (i.e. a complete recovery of susceptibility to deltamethrin) as RR50 with PBO would be 1.

If Preduc% is significantly different from 0% (i.e. it exists some synergistic effect with PBO) but also significantly different from 100% (i.e. the population does not recover total susceptibility to insecticide with PBO), then in addition to detoxifying oxidases, others types of resistance mechanisms are likely to be involved (Scott 1990). The significance in reduction of RRs was estimated by computing the RR of the population ‘with’ and ‘without’ application of PBO (RRPBO = DL50 without PBO/DL50 with PBO). Therefore, Preduc% was considered as not significantly different from 0 if the value one was inside the 95% confidence interval of RRPBO.

Determination of diagnostic doses (DD) for topical application assays

For a given insecticide, the diagnostic concentration is twice the minimum concentration that causes 100% mortality of individuals of the susceptible strain (WHO 2006). At a diagnostic dose, the usual World Health Organization (WHO) rough threshold for detecting resistance is 80% mortality (Brown & Pal 1973) and therefore, with a single dose assay, it is possible to rapidly detect the presence of resistant individuals in a field population. Diagnostic concentrations for each of the tested insecticides were determined with the CIPEIN strain. A Log-probit base line was first computed and then, a trial-and-error process was used with concentrations lower and higher than the computed LD99. For each concentration, at least 25 (and up to 50) N1 were used. This process was repeated several times to determine the observed LD100 and then, the diagnostic concentration was set to 2 × LD100.

Design of a tarsal-contact diagnostic test for field use

Graduated series of filter papers (Whatman no. 1) 12 × 15 cm were impregnated with 2 ml of insecticide solution (insecticide diluted in acetone with silicone oil [Dow Corning 556 cosmetic grade 3.6 mg/cm2]) following WHO (1996). Depending on their developmental stage, groups of 5–10 T. infestans of the CIPEIN strain were allowed to walk on an impregnated filter paper during 1 h, below WHO plastic cones which maintained the insects on the paper. Then, as for topical application bioassays, insects were returned to plastic glass with folded paper in climatic chambers (27 ± 1 °C, 60 ± 5% RH), for the 24-h dosage/mortality relationship. For each concentration, at least 20 insects were used. At least three replicates were carried out for each bioassay. For each developmental stage, Log-probit lines were estimated and lethal concentrations 99 (LC99) were computed. Then, ‘trial and error’ assays were carried out with impregnated papers flanking the LC99 to determine the observed LC100. The concentration for diagnostic paper was set to 2 × LC100.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Resistance to deltamethrin

The minimum dose of deltamethrin that caused 100% mortality in the CIPEIN strain of T. infestans when topically applied was 2.5 ng a.i./insect in agreement with the computed LD99 of 1.72 ng a.i./insect (95% confidence interval: 1.29–2.53). Therefore, the discriminating dose was set to 5 ng a.i./insect. At this dose, few populations exhibited 100% mortality and for the majority of them, mortality was <80% indicating that most of the tested populations were resistant (Table 1). In resistant populations, mortality rates were generally far below 80% and often close or equal to zero, indicating high levels of resistance, as also evidenced by large resistance ratios (RR50) which ranged from 6 to 491. However, regional differences existed: Most of the susceptible populations were from La Paz Department, a few from Chuquisaca and none from the Tarija Department. RRs were higher in Tarija Department (mean RR = 183, SD = 138, n = 10) than in Chuquisaca Department (mean RR = 68, SD = 114, n = 14) and La Paz Department (mean RR = 6.6, SD = 4, n = 2). Similarly, the mean mortality rate M at the diagnostic dose was lower in Tarija (= 18.3%, SD = 16.2, n = 12) than in Chuquisaca (= 51%, SD = 33.1, n = 21) and La Paz (= 75.2%, SD = 35.8, n = 7). The sylvatic population was susceptible (100% mortality at the diagnostic dose). Generally, Log-probit lines were parallel to that of the reference strain CIPEIN, except for some populations for which slopes values were significantly lower (Yuchan, San Francisco del Inti, Estación Caiza, Saladito, San Antonio, Tentami, Villa El Carmen, Sausal, Barrial, Barrio Itaty).

Table 1.   Deltamethrin. Log-probit lines, lethal doses (LD50), resistance ratios (RRs) at LD50 and LD90 for various field populations of Triatoma infestans (CH for Chuquisaca, CO for Cochabamba, LP for La Paz and TJ for Tarija Departments, respectively). LD50 are in ng a.i./insect. RR50 and RR90 are computed relative to the susceptible reference strain CIPEIN. For LD50, RR50 and RR90 the 95% confidence interval is in parenthesis. For the percentage of mortality at the diagnostic dose (5 ng a.i./insect for deltamethrin), the number of N1 tested is in parenthesis
T. infestans populationProbit lineLD50 (95% CI)RR50 (95% CI)RR90 (95% CI)% Mortality with diagnostic assay (n tested insects)
n tested insectsSlope (standard error)χ2 (df) P
  1. *Population from peridomestic environments only.

CIPEIN (sensible reference strain)7392.73 (0.21)1.29 (4) P = 0.860.24 (0.22–0.26)100
Barrio Itaty (CH)2021.44 (0.20)1.11 (4) P = 0.8967.33 (49.02–96.83)277 (234–328)735 (505–1070)6 (50)
Hicupe (CH)44 (45)
Huacareta (CH)66 (101)
Huacaya* (CH)67 (95)
Icla (CH)1742.79 (0.46)1.85 (3) P = 0.601.69 (1.35–2.00)6.9 (5.5–8.9)88 (49)
Jatun Cka Cka* (CH)3092.06 (0.37)7.68 (5) P = 0.171.29 (0.98–1.55)5.2 (4.2–6.5)98 (45)
La Abra (CH)40 (40)
La Mendoza (CH)40 (63)*
Corso (CH)3223.81 (0.47)3.84 (3) P = 0.281.44 (1.30–1.57)5.9 (4.9–7.1)100 (40)
El Chaco (CH)2982.34 (0.59)43.3 (4) P = 02.04 (1.73–2.42)8.5 (5.2–10.9)72 (229)
Machareti (CH)772.13 (0.15)1.38 (3) P = 0.718.07 (4.27–11.17)33 (24–47)17 (77)
Rodeo Porvenir* (CH)3192.59 (0.33)0.56 (3) = 0.902.65 (2.31–3.03)11 (9–13)82 (83)
Pajcha (CH)1083.40 (0.79)0.52 (2) P = 0.775.08 (4.25–6.37)21 (16–28)48 (56)
Sotomayor* (CH)743.47 (1.15)0.14 (3) = 0.983.32 (2.09–4.05)14 (9–19)58 (40)*
Sucre (CH)48 (180)
Tambo Ackachilla (CH)2542.97 (0.45)0.62 (3) = 0.891.26 (1.02–1.47)5 (4–6)100 (57)
Tambo Atajo (CH)2422.64 (0.42)1.56 (4) = 0.811.30 (1.01–1.54)5 (4–7)91 (58)
Tentami (CH)3221.38 (0.23)2.19 (4) = 0.7078.44 (61.76–103.83)323 (283–367)936 (630–1390)0 (35) 5 (57)*
Tiguipa Estación (CH) Intradomicile1622.47 (0.76)0.94 (2) P = 0.6255.98 (34.78–90.43)230 (150–353)0 (52)
Tiguipa Estación (CH) Peridomicile10071.59 (0.18)3.55 (3) P = 0.3134.47 (27.13–43.43)142 (121–166)307 (225–419)3 (62)
Tihuacana* (CH)583.72 (1.26)0.68 (3) P = 0.873.27 (2.14–4.07)13 (9–19)77 (40)
Anamo (CO)73 (22)
Mataral (CO)3243.11 (0.35)5.94 (3) P = 0.113.35 (2.88–3.78)14 (11–17)67 (148)
Tunal Kassa (CO)92 (53)
Candial (LP)100 (17)
Chivsivi (LP)677.09 (2.73)5 10−6 (2) = 0.991.41 (1.00–1.86)*5.8 (3.3–10.1)100 (11) 100 (39) sylvatic
Colopampa (LP)705.04 (1.02)1.83 (3) P = 0.612.31 (1.84–2.73)9.5 (6.5–13.8)95 (22)
El Palomar* (LP)100 (25)
Los Sotos (LP)0 (54)
Malavi* (LP)76 (45)
Parani (LP)2713.09 (0.38)9.90 (5) = 0.080.91 (0.80–1.04)3.7 (3.0–4.6)92 (36)
Sapini (LP)63 (8)
Barrial (TJ)4401.46 (0.14)5.92 (3) P = 0.1240.11 (32.21–49.23)165 (146–187)424 (328–548)31 (70)
Estación Caiza (TJ)6971.14 (0.14)2.28 (3) P = 0.5149.46 (39.50–60.06)204 (184–227)924 (692–1233)1 (96)
La Grampa (TJ)6 (65)
Quinchao (TJ)1531.36 (0.21)2.69 (3) = 0.4431.70 (21.19–54.45)131 (106–161)530 (312–900)15 (92)
Rio Negro (TJ)8 (84) 33 (33)*
Saladito (TJ)2121.39 (0.18)4.04 (3) = 0.2523.34 (16.72–35.51)96 (80–115)274 (187–401)17 (139)
San Antonio (TJ)6661.24 (0.12)8.16 (5) P = 0.1576.37 (62.45–96.95)315 (281–353)1157 (866–1546)9 (128)
San Francisco del Inti (TJ)2731.01 (0.16)4.71 (5) = 0.457.41 (4.61–10.69)30 (26–35)189 (137–260)43 (92)
Sausal (TJ)1711.51 (0.34)1.29 (2) P = 0.52119.12 (87.00–189.04)491 (409–589)1170 (684–2002)5 (79)
Tierras Nuevas (TJ)4731.76 (0.18)0.671 (4) P = 0.9535.82 (30.34–42.09)148 (125–174)38 (288)
Villa El Carmen (TJ)4381.36 (0.20)1.33 (3) P = 0.7252.00 (41.35–64.00)214 (190–241)3 (94)
Yuchan (TJ)24280.82 (0.04)8.74 (5) P = 0.128.10 (6.73–9.57)33 (30–36)405 (333–493)44 (468)

Evaluation of PBO synergist effect

The percentage of effective reduction in RR50 for deltamethrin with PBO was variable and ranged 0–74% (Table 2). PBO significantly reduced the RR50 values in some of the tested populations: Estación Caiza, San Antonio, Yuchan and Tiguipa (RRPBO range [1.1–1.6], [1.7–3.0], [1.6–2.2] and [2.6–5.4)], respectively) indicating that oxidative mechanisms are likely to play a significant but variable role in deltamethrin resistance. However, other mechanisms may be involved because the reduction was significant, but never reached 100%.

Table 2.   Deltamethrin. Probit lines with prior piperonyl butoxide (PBO) application for some field populations of T. infestans, resistance ratios (RRs) at LD50 and percentage of effective reduction (Preduc) in RR50 with PBO (see formula in text). A 100% reduction would indicate a complete recovery of susceptibility to deltamethrin with PBO
T. infestans populationProbit lineLD50 (95% CI)RR50with PBO (95% CI)Preduc% in RR50
n tested insectsSlope (standard error)χ2 (df) P
  1. *Indicates that Preduc% is significant (i.e. RRPBO > 1). In parenthesis, the 95% confidence intervals of the computed parameters LD50 and RR50.

El Chaco5013.16 (0.32) 1.52 (3) P = 0.67 3.01 (2.73–3.39)12.4 (10.4–14.8) 0
Estación Caiza3441.60 (0.22) 1.04 (3) P = 0.7937.52 (28.36–46.38)153 (125–186)25*
San Antonio1251.37 (0.32) 1.35 (3) P = 0.7133.79 (22.72–64.61)144 (106–195)54*
Tiguipaperidomicile4131.46 (0.18) 0.94 (3) P = 0.81 9.07 (5.72–12.42) 38 (31–45)74*
Tierras Nuevas1191.72 (0.42) 0.21 (2) P = 0.9033.82 (24.47–52.39)139 (106–184) 6
Villa El Carmen 411.12 (0.78) 0.44 (2) P = 0.8039.17147 (91–238)31
Yuchan4701.09 (0.12)11.22 (6) P = 0.08 4.32 (2.97–5.81) 18 (15–20)47*

Susceptibility to bendiocarb

The minimum dose of bendiocarb that caused 100% mortality in the CIPEIN strain of T. infestans when topically applied was 35 ng a.i./insect in agreement with the computed LD99 of 32.63 ng a.i./insect (95% confidence interval: 26.26–41.01) (Table 3). Therefore, the discriminating dose was set to 70 ng a.i./insect. At this discriminating dose, the percentage of mortality observed in field ‘domestic’ populations were all usually >95–97% indicating the absence of resistance to bendiocarb. Some kind of tolerance could be evoked because most RR50 were significantly >1 (their 95% confidence intervals did not flank this value), but only slightly above (range 1.4–1.8). On the contrary, for some populations such as Tambo Ackachilla and Tambo Atajo, mortality at the diagnostic dose was close to the 80% threshold and therefore, resistance might be in the process of appearance. These populations should be monitored.

Table 3.   Bendiocarb. Log–probit lines, lethal doses (LD50 and LD90), resistance ratios (RRs) at LD50 and LD90 for various field populations of Triatoma infestans (CH for Chuquisaca, CO for Cochabamba and TJ for Tarija Departments, respectively). LD50 are in ng a.i./insect. RR50 and RR90 are computed relative to the susceptible reference strain CIPEIN. For LD50, RR50 and RR90 the 95% confidence interval is in parenthesis. For the percentage of mortality at the diagnostic dose (70 ng a.i./insect for bendiocarb), the number of N1 tested is in parenthesis
T. infestans populationProbit lineLD50 (95% CI)RR50 (95% CI)RR90 (95% CI)% Mortality diagnostic assay (n tested insects)
n tested insectsSlope (standard error)χ2 (df) P
  1. *Population from peridomestic environment only.

CIPEIN (sensible reference strain)8814.99 (0.5556)10.9 (6) P = 0.0911.15 (10.41–11.93)100
Barrio Itaty (CH) 100 (15)
Huacaya (CH)*100 (23)
Icla (CH)96 (28)
Sucre (CH)     100 (16)
Tambo Ackachilla (CH)88 (25)
Tambo Atajo (CH)81 (11)
Tentami (CH)100 (30)
Tiguipa Estación intradomicile (CH)100 (14)
Mataral (CO)93 (120)
Tunal Kassa (CO)100 (24)
Los Sotos (LP)3693.45 (0.56)3.9 (4) P = 0.4217.67 (15.86–19.70)1.6 (1.4–1.8)100 (88)
Barrial (TJ)11244.36 (0.28)10.5 (5) P = 0.0619.56 (18.56–20.66)1.8 (1.4–2.1)98 (102)
Berrety Chaco (TJ)1216.09 (1.85)0.78 (2) P = 0.7015.18 (12.56–17.00)1.4 (1.1–1.6)100 (22)
Estación Caiza (TJ)14153.32 (0.31)9.90 (5) P = 0.0817.73 (16.54–18.83)1.6 (1.4–1.8)95 (79)
La Grampa (TJ)91 (99)
Laime (TJ)100 (21)
Lajitas (TJ)95 (58))
Quinchao (TJ)100 (65)
Saladito (TJ)97 (100)
San Antonio (TJ)94 (93)
San Francisco del Inti (TJ)3282.48 (2.22)23.8 (1) P = 018.80 (8.54–41.32)1.7 (0.8–3.5)3.1 (0.5–19)100 (74)
Sausal (TJ)100 (108)
Supitin (TJ)100 (29)
Tierras Nuevas (TJ)12113.33 (0.27)15.0 (8) P = 0.0620.82 (18.48–23.46)1.7 (1.5–2.2)2.5 (1.8–3.5)94 (266)
Villa El Carmen (TJ)98 (115)
Villa Primavera (TJ)3755.28 (0.59)1.90 (2) P = 0.3820.70 (19.28–22.08)1.8 (1.4–2.4)100 (51)
Yuchan (TJ)5183.16 (0.61)11.8 (4) P = 0.0216.52 (14.30–19.09)1.5 (1.2–1.8)2.1 (1.3–3.3)100 (40)

Susceptibility to malathion

The minimum dose of malathion that caused 100% mortality in the CIPEIN strain of T. infestans when topically applied was 60 ng a.i./insect, and therefore, the discriminating dose was set to 120 ng a.i./insect (Table 4). At this discriminating dose, the percentage of mortality observed in field ‘domestic’ populations were all >91% indicating the absence of resistance to malathion. For the three populations for which Log-probit lines were computed, RR50 were significantly >1, but only slightly above this value (range: 1.5–2.2) indicating that no marked resistance existed. Slopes of the Log-probit lines were generally high, as a consequence of homogeneity of response of the T. infestans populations to this insecticide.

Table 4.   Malathion. Log-probit lines, lethal doses (LD50 and LD90), resistance ratios (RRs) at LD50 and LD90 for various field populations of Triatoma infestans (CO for Cochabamba, LP for La Paz and TJ for Tarija Departments, respectively). LD50 are in ng a.i./insect. RR50 and RR90 and are computed relative to the susceptible reference strain CIPEIN. For LD50, RR50 and RR90 the 95% confidence interval is in parenthesis. For the percentage of mortality at the diagnostic dose (120 ng a.i./insect for malathion), the number of N1 tested is in parenthesis
T. infestans populationProbit lineLD50 (95% CI)RR50 (95% CI)RR90 (95% CI)% Mortality with diagnostic assay (n tested insects)
n tested insectsSlope (standard error)χ2 (df) P
CIPEIN (sensible reference strain)7229.63 (0.74)6.53 (4) P = 0.1617.73 (17.27–18.16)100
Mataral (CO) 91 (77)
Los Sotos (LP)100 (63)
Barrial (TJ) 99 (74)
Cortaderal (TJ)100 (33)
Estación Caiza (TJ)100 (80)
Ibopeiti (TJ)100 (41)
La Grampa (TJ)1703.89 (0.12)2.25 (3) P = 0.5238.81 (33.62–45.85)2.2 (1.8–2.7)3.4 (2.4–5.0) 96 (50)
Laime (TJ)1416.42 (1.04)7.06 (3) P = 0.0731.14 (28.47–35.13)1.8 (1.3–2.4)100 (78)
Lajitas (TJ) 95 (58)
Quinchao (TJ)100 (60)
Saladito (TJ) 99 (71)
San Antonio (TJ)100 (60)
San Francisco del Inti (TJ)100 (46)
Sausal (TJ)100 (85)
Tierras Nuevas (TJ)100 (64)
Villa El Carmen (TJ)100 (72)
Yuchan (TJ)35714.49 (2.02)4.62 (3) P = 0.2026.67 (26.09–27.29)1.5 (1.2–1.8)100 (59)

Contact-tarsal test with deltamethrin

Table 5 summarizes the results for the Log-probit lines and the determination of the observed LC100 obtained for each larval developmental stage. For all nymphal stages, the LC100 were close to 0.15% except for N1, for which it was 0.09%. Because the diagnostic test should be used in field conditions (i.e. where all T. infestans developmental stages are captured) during routine surveillance, a single consensual diagnostic concentration that could be used for all developmental stages would be easier to handle. Results indicate that except for N1, a consensual LC100 would be 0.15% and therefore that the diagnostic concentration would be set to 0.30%. So, the protocol for the diagnostic test would be as follows: (i) expose field captured triatomines (all developmental stages, including N1) to 0.30% deltamethrin impregnated filter papers for 1 h in groups of 5–10 individuals; (ii) transfer the insects in resting cups with folded paper at room temperature (if possible at approximately 27 °C); and (iii) compute the percentage of mortality after 24 h and use the WHO criterion (Brown & Pal 1973) to conclude. However, special attention should be paid in the analysis of N1 mortality (see Discussion).

Table 5.   Log-probit analysis, computation of the lethal concentrations 50 and 99 (LC50 and LC99) and determination of observed LC100 in tarsal-contact bioassays with deltamethrin impregnated papers for each nymphal stage of Triatoma infestans from the CIPEIN strain. LC50, LC99 and LC100 are in % according to the World Health Organization (1996) definition
T. infestans developmental stageProbit lineLC50 (95% CI)LC99 (95% CI)Observed LC100
n tested insectsSlope (standard error)χ2 (df) P
N12605.15 (0.85)0.88 (3) P = 0.830.037 (0.032–0.041)0.10 (0.083–0.162)0.09
N25703.81 (0.35)6.32 (7) P = 0.500.045 (0.042–0.049)0.18 (0.151–0.248)0.12
N36364.69 (0.38)5.50 (7) P = 0.600.052 (0.048–0.056)0.162 (0.141–0.196)0.15
N44578.64 (1.03)6.23 (4) P = 0.180.074 (0.069–0.078)0.138 (0.125–0.161)0.15
N52004.57 (1.86)0.80 (3) P = 0.850.050 (0.004–0.067)0.161 (0.124–1.413)0.15

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Insecticide pressure on Bolivian ‘domestic’ populations of T. infestans comes from vector control measures of the NPCCD. Present results indicate that resistance to deltamethrin is widespread and high. In some places in the centre and south of Bolivia, where the NPCCD reported insecticide field treatments failures, RRs for deltamethrin where >50, which is the rough threshold computed by Picollo et al. (2005) above which field treatment failures were also reported in Argentina. Few populations are still susceptible, of which the sylvatic is one. The field diagnostic test for deltamethrin designed in this study is simple to use and could adequately be carried out by the technicians of the NPCCD to complete the geographical distribution of deltamethrin resistance in Bolivia. The diagnostic concentration of 0.30% deltamethrin on impregnated papers would probably overestimate the diagnostic concentration for N1. If only N1 are tested and 100% mortality is found, the test would conclude that the population is susceptible even though it is resistant. However, in field conditions, the capture of N1 only is very unlikely and generally, a mixture of all development stages is sampled which would make the test effective. Testing N1 could still be informative if they survive the discriminating dose because their survival would indicate that the resistance level in the population is high.

Villages in Tarija Department (South Bolivia) exhibited the higher levels of resistance (generally RR > 150) and correlates to the lower percentages of mortality at the diagnostic dose (generally <10%). These are in agreement with results from T. infestans populations from southern Bolivia (Vassena et al. 2007; Santo Orihuela et al. 2008) and Brazil (Vieira Sonoda et al. 2009). The significant decrease in RR with PBO in some populations, such as Yuchan, San Antonio, Tiguipa and Estación Caiza, indicated that oxidases played a significant role in deltamethrin resistance (Ffrench-Constant & Roush 1990). However, because the populations did not recover complete susceptibility with PBO (and even, for some populations, there was no decrease in RR) other mechanisms would be involved such as esterases or kdr (Santo Orihuela et al. 2008). In T. infestans, glutathione S-transferases are known to participate in pyrethroid and organophosphate resistance (Sivori et al. 1997) and therefore may also participate in the observed deltamethrin resistance. The heterogeneity in the resistance levels to deltamethrin and in the mechanisms involved (variations in PBO response) are likely to originate from non-homogenous insecticide pressure (frequencies of interventions vary among villages and insecticide doses may vary among ‘domestic’ and ‘peridomestic’ environments, the later being more difficult to treat (Gürtler et al. 2004)). In addition T. infestans populations are generally highly structured genetically (although not isolated) (Pérez de Rosas et al. 2007, 2008; Abrahan et al. 2008; Pizarro et al. 2008; Segura et al. 2009) and this also can partly account for the observed differences in resistance patterns.

Because there is no marked resistance to bendiocarb and malathion (as measured by diagnostic dose assays), alteration of insecticide biding-sites for acetylcholine esterase is unlikely to occur in T. infestans (McCord & Yu 1987). Therefore, because these enzymes are strongly suspected for deltamethrin-resistant T. infestans populations in Bolivia, they might also be involved for bendiocarb, giving various RR50 > 1 (although only slightly above one). However, to better characterize the metabolic resistance, biochemical assays would be needed (Hemingway 1998).

In addition to metabolic resistance, the knockdown resistance (kdr) mechanism is common in many insect species resistant to pyrethroids. Its mode of action consists of pointed mutations in the sodium channel genes leading to nerve insensitivity and cross-resistance with DDT (Soderlund & Knipple 2003). Because the tested populations of T. infestans did not recover total susceptibility to deltamethrin with PBO (and even in some cases, PBO did not have any effect), and because RR are high, the most probable complementary mechanism is the kdr mechanism which has already been suggested by Santo Orihuela et al. (2008) for T. infestans populations of Argentina.

In combination with insecticide resistance reports in Argentina, our results also raise the question of the appearance of insecticide resistance in the Andean populations of T. infestans. On the other hand, non-Andean populations of T. infestans of Paraguay and from some regions of Argentina seem to be susceptible to pyrethroids despite the same insecticide pressure. The classification of T. infestans populations into two distinct groups – Andean and non-Andean – has long been highlighted (Panzera et al. 2007), and differences in ecological responses of the two groups have been mentioned (Cataláet al. 2006). Andean populations of T. infestans from Bolivia seem to have the potential to develop insecticide resistance faster than non-Andean populations. Correlating genetic and insecticide resistance data will bring new insights into resistance management programmes.

Resistance profiles of T. infestans might be more complex and diversified than expected, reflecting the various insecticide pressures and genetic structures of the populations. A better management of chemicals can be a short-term solution to insecticide resistance (Tabashnick 1990). Because our results indicated that there was no resistance to bendiocarb, this molecule could be suggested as a short-term alternative where resistance to deltamethrin is high. In that case, a careful monitoring should be carried out in areas where Anopheles pseudopunctipennis, a malaria vector, also occurs in sympatry (Lardeux et al. 2008) and is simultaneously controlled by the Bolivian Ministry of Health with insecticide indoor sprayings.

New control strategies including integrated approaches are needed to definitively overcome the resistance problem of T. infestans. Housing improvements and ecosystem management using an Eco-Bio-Social approach (WHO 2002, Gürtler et al. 2007) are strongly encouraged. The use of insecticidal paints using organophosphates (Amelotti et al. 2009) is promising but poses different challenges at the operational level. Insecticide rotations or mixtures of organophosphates or carbamates with pyrethroids and the search for insecticide synergy would also be worth considering. Restoration of pyrethroid susceptibility using low rates of other insecticides (Bielza et al. 2007) could also be studied. Finally, high-tech strategies using genetics (such as paratransgenesis) are promising (Durvasula et al. 2008) but more time is needed to render them fully operational.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We thank R. Lopez, E. Sinani, J. Vidaurre, M. Gallardo, R. Rodriguez, P. Vidaurre, the personnel of the SEDES of La Paz, Cochabamba, Chuquisaca and Tarija Departments as well as the National Program for Control of Chagas Disease of Bolivia for their help in collecting and rearing the T. infestans populations. We are grateful to F. Chandre (IRD-LIN) for assessing the manuscript and improving it. This investigation received financial support from the UNICEF/UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR); project ID A70384. The authors declare that they have no conflict of interest.

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  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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