High Resistance to Pyrethroid Insecticides ... - Semantic Scholar

VECTOR CONTROL, PEST MANAGEMENT, RESISTANCE, REPELLENTS

High Resistance to Pyrethroid Insecticides Associated with Ineffective Field Treatments in Triatoma infestans (Hemiptera: Reduviidae) from Northern Argentina MARIÁ INE´S PICOLLO,1, 2 CLAUDIA VASSENA,1, 3 PABLO SANTO ORIHUELA,1 SILVIA BARRIOS,1 MARIO ZAIDEMBERG,4 AND EDUARDO ZERBA1, 3

J. Med. Entomol. 42(4): 637Ð642 (2005)

ABSTRACT Field populations of Triatoma infestans Klug were collected during 2002 from four villages in northern Argentina (El Chorro, La Toma, El Sauzal, and Salvador Mazza), after application of deltamethrin and other pyrethroids was ineffective. High levels of resistance to the pyrethroid insecticides deltamethrin, ␤-cypermethrin, ␤-cyßuthrin, and lambda-cyhalothrin were detected in all of the evaluated populations. The resistance ratio to pyrethroids determined by topical application ranged from 50.5 (deltamethrin, El Sauzal) to 667.6 (␤-cyßuthrin, Salvador Mazza). None of the pyrethroid-resistant insects was resistant to the organophosphorus insecticide fenitrothion. Topical application of piperonyl butoxide to the most deltamethrin-resistant population (Salvador Mazza) led to slight reduction in levels of resistance. Activity of P450 monooxygenase, measured in individual insects through ethoxycoumarine-O-deethylase, showed a slight but noticeable difference in the distribution of activities between susceptible and resistant populations. The total percentage of insects below 0.48 pmol of 7-OH coumarine/min/insect was 36.4 for Salvador Mazza population and 64.3 pmol of 7-OH coumarine/min/insect for CIPEIN strain. Whereas a low level of resistance to deltamethrin was previously related to monooxygenase activity in T. infestans, the high levels of resistance shown by these populations seem to involve monooxygenase in combination with other resistance mechanisms, for example, insensitivity of nervous membrane. Research on T. infestans resistance is in progress to improve Chagas vector control programs in Latin America and to implement resistance management strategies. KEY WORDS Triatoma infestans, insecticide resistance, resistance ratios, monooxygenases

Triatoma infestans Klug (Hemiptera: Reduviidae) is the major vector of American trypanosomiasis or Chagas disease in Argentina. Pyrethroid insecticides have been used to control this pest during the past 20 yr (Zerba 1999). Possible development of resistance in Þeld populations exposed to vector control programs with insecticides has been evaluated in our laboratory since 1997. Low resistance levels (resistance ratios, RRs) to deltamethrin were initially found (resistance ratio ranged from 2.0 for San Luis colony to 7.9 for Salta colony) that were not correlated with failures of chemical control in the Þeld (Picollo et al. 2000). Laboratory studies on the population with highest deltamethrin resistance (Salta) in comparison with the susceptible strain (CIPEIN), suggested enhanced

1 Centro de Investigaciones de Plagas e Insecticidas (CITEFACONICET), Juan B. De La Salle 4397 (B1603ALO), Villa Martelli, Provincia de Buenos Aires, Argentina. 2 E-mail: [email protected]. 3 Universidad Nacional de General San Martõń, Escuela de Postgrado, Provincia de Buenos Aires, Argentina. 4 Coordinacio ´ n Nacional de Control de Vectores. Ministerio de Salud de la Nacio´ n, Buenos Aires, Argentina.

degradative activity by cytochrome P450 monooxygenases (Gonza´lez Audino et al. 2004). In 2002, deltamethrin-resistant Þeld populations were detected in several localities close to where the Salta colony was collected. The new region with detected resistance is located in northern Argentina (San Martõń Department, Salta Province), in the area bordering Bolivia. At the same time, the Health Authorities for Vector Control reported ineffectiveness of the application of deltamethrin and other pyrethroid insecticides in infested houses of this endemic area. To evaluate the resistance proÞle and the resistance mechanisms in these Þeld populations from four villages in this region (El Chorro, La Toma, El Sauzal, and Salvador Mazza), we bioassayed the insecticides recommended for the chemical control of this vector against susceptible and collected resistant populations. The monooxygenase synergist piperonyl butoxide (PBO) and the esterase synergist triphenyl phosphate (TPP) were used to evaluate the relative role of these enzymes in the resistance. The enhanced activity of cytochrome P450 microsomal monooxygenases was further characterized using biochemical measurements.

0022-2585/05/0637Ð0642$04.00/0 䉷 2005 Entomological Society of America

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Fig. 1. Map of the northern Argentina (Salta Province) showing study sites where T. infestans were collected.

Materials and Methods Study Sites. Field T. infestans were collected in November 2002 from infested houses of an endemic area in northern Salta Province. The vector control campaign intervention based on pyrethroid insecticides was considered ineffective in this area due presence of insects assessed by household and ofÞcial reports a week after the intervention. In this area, insecticides have been intensively used for T. infestans and mosquito chemical control. Four sites located in the San Martõń Department (22⬚ 5⬘ S, 63⬚ 7⬘ E) were chosen. Sites were El Chorro, La Toma, and El Sauzal, which are rural areas, and Salvador Mazza, an urban area. They are small villages located 50 km from Tartagal city (Fig. 1). A high percentage of infested houses was detected in this area since 2002. Data on the evolution of dwelling infestation and the insecticides used are summarized in Table 1. Table 1.

Insects. T. infestans nymphs and adults were individually collected with entomological forceps from the infested houses of the four localities. We found an average of 30 insects per house. Further generations of the Þeld insects were bred in the laboratory. CIPEIN is an insecticide-susceptible strain maintained in our laboratory since 1975 without any exposure to insecticides (Picollo et al. 1976). Insects were reared at 28 ⫾ 1⬚C, 50% RH, and a photoperiod of 12:12 (L:D) h and were fed on pigeons weekly. From all colonies, 3-d-old Þrst instars, starved since eclosion (mean weight 1.2 ⫾ 0.2 mg), were selected for toxicity tests according to the World Health Organization protocol (1994) and for the measurement of the enzymatic activity (Gonza´lez Audino et al. 2004). Chemicals. Technical grade insecticides used for bioassay were deltamethrin (97%, Bayer, Buenos Aires, Argentina), ␤-cypermethrin (99.4%, Chemotec-

Percentage of houses infested with T. infestans after vector control intervention in the north of Salta, Argentina % infested houses

Date

Insecticides used

El Chorro n ⫽ 125

June 1998 Dec. 2001 June 2002 Nov. 2002 Feb. 2003

Deltamethrin, cypermethrin Deltamethrin, ␤-cypermethrin ␤-Cypermethrin, cypermethrin Cypermethrin, ␤-cypermethrin ␤-Cypermethrin

16.7 55.6 87.9 76.3 88.6

Empty cells represent no data available. n is number of houses per locality.

La Toma n ⫽ 26

80.9

El Sauzal n ⫽ 95

S. Mazza n ⫽ 1400

32.1

10.4 41.4 45.4 90

75 88.0

July 2005 Table 2.

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639

Bioassay statistics and resistant ratios for deltamethrin in field populations of T. infestans from Salta, Argentina Population

n

Slope ⫾ SE

LD50 ng/insect (95% CL)

RR (95% CL)

CIPEINa El Chorro La Toma El Sauzal S. Mazza

125 220 100 90 90

2.10 ⫾ 0.66 1.90 ⫾ 0.14 1.90 ⫾ 0.14 1.47 ⫾ 0.11 1.37 ⫾ 0.11

0.13 (0.12Ð0.15) 12.8 (11.0Ð14.9) 11.3 (6.1Ð23.3) 6.5 (2.3Ð19.4) 31.1 (12.9Ð84.7)

99.0 (78.2Ð125.3) 86.9 (68.1Ð111.0) 50.5 (30.7Ð83.1) 133.1 (105.6Ð167.7)

Topical application of 0.2 ␮l of acetone solution to dorsal abdomen of 3-d-old Þrst instars. LD50 values are in nanograms per insect. RR ⫾ 95% CL is calculated according to Robertson and Preisler (1992). a Susceptible strain.

nica, Buenos Aires, Argentina), ␤-cyßuthrin (98.7%, Bayer, Yorkshire, United Kingdom), lambda-cyhalothrin (99.7%, Syngenta, Basel, Switzerland), and fenitrothion (97%, Sumitomo, Osaka, Japan). The synergists tested were PBO (92.3%, Chemotecnica) and triphenyl phosphate (TTP) (99%, Janssen Chimica, Belgium). 5,5⬘-Dithiobis-2-nitrobenzoic (DTNB), 7-ethoxycoumarin (7-EC), 7-OH-coumarin (umbelliferone), eserine, and Fast Blue B were from Sigma (St. Louis, MO). All solvents were analytical grade purchased from Merck (Buenos Aires, Argentina). Topical Application Bioassays. Insecticides or synergists were delivered in 0.2 ␮l of acetone to the dorsal abdomen of Þrst instars (WHO 1994). PBO or TPP were delivered 1 h before the insecticide application at the maximum sublethal dose (based on the susceptible strain): 1 ␮g per insect (Vassena et al. 2000). For synergism studies, we used Salvador Mazza population because of the high resistance level and the amount of biological material. At least 10 nymphs per dose and per replicate were treated for each dose. Control groups received acetone, PBO, or TPP. A minimum of three doses, giving ⬎0 and ⬍100% mortality at 24 h after insecticide treatment, were used for each treatment. Each experiment was replicated at least three times. The treated insects were kept in an environmental chamber (Lab-Line Instruments, Melrose Park, IL) at 28 ⫾ 1⬚C, 50 Ð 60% RH, and a photoperiod of 12:12 (L:D) h. Mortality was assessed at 24 h after insecticide application. Insects were considered dead if they were unable to walk from the center to the border of a 7-cm Þlter paper disc, alone or when they were mechanically stimulated (WHO 1994). Cytochrome P450 Monooxygenase. Activity was measured using 7-ethoxycoumarin-O-deethylation (ECOD) on intact tissue on microplate (Bouvier et al. 1998). Fluorescence was determined using microplate ßuorescence reader (Packard Fluorocount), with 400-nm excitation and 440-nm emission Þlters. First instar abdomens were placed individually into wells of a 96-well microplate containing 100 ␮l of 0.05 M phosphate buffer, and 4 mM 7-EC. The reaction was stopped after incubation time by adding 100 ␮l of glycine buffer (10⫺4 M), pH 10.4. To precipitate the abdomens in the wells, microplates were centrifuged at 2,000 ⫻ g for 30 s in a refrigerated centrifuge for microplates (4237 R, ALC International SRL, Cologna Monzese, Italy) before and after the incubation of the enzymatic reaction at 30⬚C. For each population, sim-

ilar wells receiving glycine buffer previous to incubation were used for blanks. Statistical Analysis. Mortality data were corrected using AbbottÕs formula (Abbott 1925). Bioassay data from each T. infestans population were pooled and analyzed based on probit analysis (LitchÞeld and Wilcoxon 1949). Doses giving 50% lethality (LD50 values) obtained in probit were expressed as nanograms of insecticide per insect. To compare lethal dose and estimate whether the LD50 of resistant populations differed signiÞcantly from the LD50 of the susceptible strain, the ratio of LD50 and the 95% CL for the ratio were calculated. If the 95% conÞdence interval includes 1, then the LD is not signiÞcantly different (Robertson and Preisler 1992). The biochemical data were plotted as the percentage of individuals responding within a particular range of values of enzyme activity (Sokal and Rohlf 1980). A threshold between susceptible and resistant colonies was established, and the total percentage of insects below the threshold was calculated for both populations.

Results Resistance ratios to deltamethrin were assessed in T. infestans Þrst instars whose parents were collected in houses that has been unsuccessfully treated with pyrethroid insecticides. The toxicity data estimated for resistant (El Chorro, La Toma, El Sauzal, and Salvador Mazza) and susceptible (CIPEIN) insects are shown in Table 2. All Þeld populations exhibited high degrees of resistance compared with the insecticide-susceptible strain. The resistance ratios ranged from 50.5 to 133.1. These high levels of resistance were not previously observed in T. infestans or any other vector of Chagas disease. There was no difference between the resistance ratios to deltamethrin of the four resistant populations (the conÞdence limits overlapped). The resistance status of each population used in this study toward ␤-cypermethrin, ␤-cyßuthrin, lambdacyhalothrin, and fenitrotion is shown in Table 3. All the deltamethrin-resistant populations also were resistant to ␤-cypermethrin, ␤-cyßuthrin, and lambdacyhalothrin, three pyrethroids that had been used for chemical control on T. infestans in the Þeld. The results showed high degrees of resistance, similar to those

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Table 3.

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Toxicity of insecticides against susceptible (CIPEIN) and deltamethrin-resistant T. infestans from Salta, Argentina

Insecticide

␤-Cypermethrin

␤-Cyßuthrin

lambda-Cyhalothrin

Fenitrothion

Population

n

Slope ⫾ SE

LD50 ng/ i (95% CL)

CIPEIN El Chorro La Toma El Sauzal S. Mazza CIPEIN El Chorro La Toma El Sauzal S. Mazza CIPEIN El Chorro La Toma El Sauzal S. Mazza CIPEIN El Chorro La Toma El Sauzal S. Mazza

150 180 180 180 120 150 120 120 124 157 120 120 120 150 120 150 120 120 90 120

1.54 ⫾ 0.11 1.22 ⫾ 0.09 0.94 ⫾ 0.07 1.12 ⫾ 0.08 2.40 ⫾ 0.25 1.91 ⫾ 0.13 1.27 ⫾ 0.13 2.11 ⫾ 0.17 2.20 ⫾ 0.17 1.07 ⫾ 0.09 1.63 ⫾ 0.13 0.91 ⫾ 0.08 1.06 ⫾ 0.08 0.75 ⫾ 0.06 0.98 ⫾ 0.09 1.42 ⫾ 0.11 1.66 ⫾ 0.15 1.53 ⫾ 0.14 2.39 ⫾ 0.25 1.41 ⫾ 0.14

0.24 (0.11Ð0.55) 106.5 (73.6Ð164.9) 63.7 (46.9Ð88.5) 62.2 (35.2Ð118.2) 72.8 (62.2Ð87.1) 0.46 (0.09Ð1.26) 27.1 (7.6Ð70.7) 23.4 (11.9Ð43.5) 9.5 (5.4Ð16.4) 105.1 (43.0Ð352.2) 0.11 (0.09Ð0.56) 53.3 (25.7Ð123.9) 26.2 (9.3Ð75.7) 11.9 (2.9Ð55.6) 19.6 (6.6Ð58.8) 21.6 (5.3Ð67.9) 27.0 (1.8Ð69.3) 25.7 (21.9Ð30.2) 36.1 (31.8Ð41.1) 23.4 (19.8Ð27.8)

RR (95% CL) 451.2 (328.0Ð620.6) 270.1 (190.2Ð383.6) 263.5 (116.6Ð595.3) 308.4 (229.6Ð414.4) 171.7 (137.1Ð215.0) 148.4 (119.9Ð183.7) 60.3 (54.3Ð67.0) 667.6 (540.4Ð824.8) 288.7 (178.3Ð467.4) 141.7 (90.9Ð221.1) 64.5 (39.2Ð106.1) 106.2 (69.5Ð162.2) 1.29 (0.97Ð1.70) 1.54 (1.16Ð2.05) 1.78 (1.36Ð2.34) 1.39 (1.03Ð1.88)

LD50 values are in nanograms per insect. RR ⫾ 95% CL is calculated according to Robertson and Preisler (1992).

found for deltamethrin. Topical application of ␤-cypermethrin gave the highest resistance ratios for all samples, ranging from 270.1 to 451.2. None of the pyrethroid-resistant populations was resistant to fenitrothion, an organophosphorus insecticide that had not been used in Argentina for controlling T. infestans for the past 20 yr. The resistance ratios ranged from 1.29 for El Chorro to 1.78 for El Sauzal. Topical application of cytochrome P450 inhibitor PBO to the most resistant population (Salvador Mazza) before deltamethrin led to slight reduction in the levels of resistance. At LD50, PBO reduced the resistance ratios from 133.1 to 101.1 (Table 4). The topical application of the esterase inhibitor TPP did not increase the toxicity of the insecticide, suggesting that these enzymes were not involved in deltamethrin resistance of Salvador Mazza population (Table 4). Activity of P450 monooxygenases showed a slight but noticeable difference in the distribution of 7-OHcoumarine activities between susceptible and resistant populations (Fig. 2). Considering 0.48 pmol of 7-OH coumarine per minute as a reasonable threshold between the populations, the total percentage of insects below the threshold was 64.3 for CIPEIN and 36.4 for the resistant population.

Discussion This study reported the Þrst case of pyrethroid resistance at a high enough level to cause control failures in Þeld populations of T. infestans from northern Argentina. The laboratory toxicity tests indicated that resistance to topically applied deltamethrin existed at a high level in the Þeld populations collected in four villages of the same area (El Chorro, La Toma, El Sauzal, and Salvador Mazza). The resistance ratios to deltamethrin estimated here (99.0, 86.9, 50.5, and 133.1 respectively) were consistently higher than those previously estimated for a Þeld population of the same area collected in September 1999 (RR ⫽ 7.9 to deltamethrin), when chemical control was still successful. Based on these data, it can be assumed that RRs ⱖ 50 measured by topical application are a signal of potential control failures in the Þeld, whereas RRs ⱕ 8 indicate that resistant populations are being successfully controlled. Although this information is not sufÞciently complete to correlate laboratory test and Þeld chemical control, it is valuable information in the development of practical resistance management strategies. Moreover, it will be useful for the study of variations in pyrethroid susceptibility of Þeld populations of Rhodnius prolixus Stahl, another vector of

Table 4. Effect of the pretreatment with PBO and TPP on the toxicity of deltamethrin against susceptible (CIPEIN) and pyrethroidresistant T. infestans Deltamethrin Alone ⫹ PBO ⫹ TPP

CIPEIN

Salvador Mazza

n

Slope ⫾ SE

LD50 (CL)

n

Slope ⫾ SE

LD50 (CL)

RR (CL)

125 90 90

2.10 ⫾ 0.66 2.32 ⫾ 0.24 2.18 ⫾ 0.25

0.13 (0.12Ð0.15) 0.14 (0.12Ð0.15) 0.13 (0.12Ð0.14)

120 180 120

1.37 ⫾ 0.11 1.07 ⫾ 0.79 1.45 ⫾ 0.13

31.1 (12.9Ð84.7) 19.5 (11.4Ð35.1) 33.8 (11.5Ð81.4)

133.1 (105.6Ð167.7) 101.1 (72.1Ð141.9) 133.9 (86.7Ð206.6)

Pretreatment by topical application of PBO 1 ␮g/insect or TPP 1 ␮g/insect, 1 h before the deltamethrin application. LD50 values are in nanograms per insect. RR ⫾ 95% CL is calculated according to Robertson and Preisler (1992).

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Fig. 2. Histogram indicating the range of monooxygenase activities exhibited by susceptible and resistant individuals in microplate assays.

Chagas disease without detected resistance to insecticides (Molina de Fernańdez et al. 2004). Based on the records of the Vector Control National Programs, the resistant Þeld insects have been successfully controlled with other pyrethroids (␤-cypermethrin, ␤-cyßuthrin, and lambda-cyhalothrin) for the past 20 yr. Thus, resistance to these pyrethroid may be due to cross-resistance to deltamethrin, selection with each insecticide, or both. Our toxicity studies have indicated that the pyrethroid-resistant insects have not developed cross-resistance to fenitrothion and that the resistance was not completely reduced by pretreatment with PBO or TPP. The biochemical results found a slight but noticeable difference in the distribution of 7-OH-coumarine activities between susceptible and resistant populations. This result is not conclusive evidence but suggests monooxygenase involvement as a collaborative resistance mechanism. Lack of PBO synergism also has been found in the presence of strong synergism by propynyl ethers in permethrin-resistant Heliothis virescens (Boddie) (Rose et al. 1995), suggesting that monooxygenase involvement in pyrethroid resistance should not be

discarded when PBO fails to synergies toxicity. The authors suggested that monooxygenases present in a population might mean that a synergist capable of interacting with one isozyme may not be capable of interacting with another. Resistance to pyrethroids in insects has been correlated with enhanced metabolism by enzymes (Oppenoorth 1985), in particular, a higher cytochrome P450s activity was measured in pyrethroid resistance in house ßy, Musca domestica L. (Lee and Scott 1989). Hung and Sun (1989), using ethoxycoumarine as a substrate, found that larval homogenates of fenvalerate-resistant diamondback moth, Plutella xylostella (L.), possessed 100-fold higher activity than susceptible insects. Bouvier et al. (1998) reported higher ethoxycoumarin deethylase activity measured at individual level in microtitration plates, in deltamethrinresistant codling moth, Cydia pomonella (L.). The role of monooxygenases was previously linked to deltamethrin resistance in T. infestans and R. prolixus, the two major vectors of Chagas disease in America. Vassena et al. (2000) demonstrated that the pyrethroid resistance in a Brazilian T. infestans (RR ⫽

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7.0) and a Venezuelan R. prolixus (RR ⫽ 11.4) was decreased by piperonyl butoxide, suggesting oxidative metabolism as cause of resistance. Moreover, Gonza´lez Audino et al. (2004), using biochemical assays, established the role of enhanced detoxication in a deltamethrin-resistant colony of T. infestans (RR ⫽ 7.9) from Argentina. They found ECOD activities to be 1.8-fold higher in the resistant colony, with values 108.0 pg of 7-OH/min/insect for resistant colony and 61.3 pg of 7-OH/min/insect for the susceptible strain. These values correspond to 0.67 and 0.38 pmol of 7-OH/min/insect, respectively. However, all deltamethrin-resistant populations previously studied showed moderate resistance levels (RR ⱕ 11.4). The high resistance to pyrethroid in the Þeld populations studied here, suggests that a further mechanism, such as reduced nerve sensibility (i.e., kdr), may be involved in the resistance. The likely contribution of sodium channel mutations as an important resistance mechanism has been investigated in other hemipterans (Soderlund and Knipple 2003). Biochemical, electrophysiological, and molecular investigations are in advance to demonstrate this hypothesis. Meanwhile, fenitrothion could be used for chemical control of pyrethroid resistant populations, according to the low resistance ratios found in this study. Acknowledgments We thank to Drs. Cynthia Spillmann and Sonia Blanco and technicians of the Coordinacio´ n Nacional de Control de Vectores del Ministerio de Salud de Argentina for coordination of Þeld sampling. This investigation received Þnancial support from the United Nations Development Programme/ World Bank/World Health Organization Special Programme for Research and Training in Tropical Diseases, and the Consejo Nacional de Investigaciones Cientõ´Þcas y Te´ cnicas of Argentina.

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(Hemiptera, Reduvidae) from Argentina. Mem. Inst. Oswaldo Cruz, Rõó de Janeiro 99: 335Ð339. Hung, C., and H. Sun. 1989. Microsomal monooxygenase in diamond-back moth larvae resistant to fenvalerate and piperonyl butoxide. Pestic. Biochem. Physiol. 33: 168 Ð 175. Lee, S., and J. Scott. 1989. Microsomal cytochrome P450 monooxygenases in the houseßy. Biochemical changes associated with pyrethroid resistance and Phenobarbital induction. Pestic. Biochem. Physiol. 35: 1Ð10. Litchfield, J. T., and F. Wilcoxon. 1949. A simpliÞed method of evaluating dose-effect experiments. J. Exp. Ther. 96: 99 Ð110. Molina de Ferna´ ndez, D., A. Soto Vivas, and H. Barazarte. 2004. Susceptibilidad a insecticidas piretroides en cepas de campo de Rhodnius prolixus Stal (Hemiptera: Reduviidae) de Venezuela. Entomotropica, Caracas, Venezuela 19: 1Ð5. Oppenoorth, F. J. 1985. Biochemistry and genetics of insecticide resistance, pp. 731Ð773. In G. A. Kerkut, C. I. Gilbert [eds.], Comprehensive insect physiology, biochemistry and pharmacology, vol. 12. Pergamon, Oxford, United Kingdom. Picollo, M. I., E. Wood, E. N. Zerba, S. A. Licastro, and M. A. Ru´ veda. 1976. Laboratory test for measuring toxicity of insecticides in Triatoma infestans, Klug. Acta Bioquim. Latinoam., Buenos Aires, Argentina X: 67Ð70. Picollo, M. I., C. Vassena, and E. Zerba. 2000. Detection and monitoring of insecticide resistance in Triatoma infestans and Rhodnius prolixus. In Proceedings of XXI International Congress of Entomology, Foz do Iguazu´ , Brazil. Robertson, J. L., and H. K. Preisler. 1992. Pesticide bioassays with arthropods. CRC, Boca Raton, FL. Rose, R. L., L. Barbhaiya, R. Michael Roe, G. Rock, and E. Hodson. 1995. Cytochrome P450-associated insecticide resistance and the development of biochemical diagnostic assays in Heliothis virescens. Pestic. Biochem. Physiol. 51: 178 Ð191. Soderlund, D. M., and D. C. Knipple. 2003. The molecular biology of knockdown resistance to pyrethroid insecticides. Insect Biochem. Mol. Biol. 33: 563Ð577. Sokal, R., and F. Rohlf. 1980. Introduccio´ n a la Bioestadõ´stica, Editorial Reverte´ S.A., Barcelona, Spain. Vassena, C. V., M. I. Picollo, and E. N. Zerba. 2000. Insecticide resistance in Brazilian Triatoma infestans and Venezuelan Rhodnius prolixus. Med. Vet. Entomol. 14: 51Ð55. [WHO] World Health Organization. 1994. Protocolo de evaluacio´ n de efecto insecticida sobre Triatominos. Acta Toxicol. Arg. 2: 29 Ð32. Zerba, E. 1999. Susceptibility and resistance to insecticides of Chagas disease vectors. Medicina 59. 2: 41Ð 46. Received 25 August 2004; accepted 9 March 2005.