J. Med. Entomol. 42(5): 830Ð837 (2005)

Transcription

1 VECTOR CONTROL, PEST MANAGEMENT, RESISTANCE, REPELLENTS Effectiveness of Methoprene, an Insect Growth Regulator, Against Temephos-Resistant Aedes aegypti Populations from Different Brazilian Localities, Under Laboratory Conditions IMA APARECIDA BRAGA, 1, 2, 3 CÍCERO BRASILEIRO MELLO, 4 ISABELA REIS MONTELLA, 1, 2 JOSÉ BENTO PEREIRA LIMA, 1, 2 ADEMIR DE JESUS MARTINS JÚNIOR, 1, 2 PRISCILA FERNANDES VIANA MEDEIROS, 1, 2 1, 2, 5 AND DENISE VALLE Departamento de Entomologia, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, RJ, CEP , Brazil J. Med. Entomol. 42(5): 830Ð837 (2005) ABSTRACT The susceptibility of Aedes aegypti (L.) larvae from several Brazilian populations to the juvenile hormone analog methoprene and the organophosphate insecticide temephos were investigated. Populations from Natal (northeastern region), Macapá (northern region), and Jardim América, Rio de Janeiro (southeastern region) are temephos-resistant (RR , 13.3, and 15.8, respectively), whereas populations from Presidente Prudente (southeastern region) and Porto Velho (northern region) exhibit only an incipient temephos-altered susceptibility status (RR and 2.6, respectively). Biochemical assays revealed alterations of the enzymes implicated in metabolic resistance, glutathione S-transferase, mixed function oxidases and esterases, among these populations. DoseÐresponse assays showed at most a low resistance to methoprene of all populations tested, irrespective of their temephos resistance level. However, sequential exposure of Macapá and Natal populations to temephos and methoprene indicated a potential cross-resistance when larvae are exposed to both insecticides. Nevertheless, susceptibility of the Brazilian Ae. aegypti populations to methoprene alone suggests this insect growth regulator could substitute for temephos in the control of the dengue vector in the country. KEY WORDS methoprene, Aedes aegypti, temephos, vector control, insect growth regulator 1 Departamento de Entomologia, Fundação Oswaldo Cruz, Av. Brasil 4365, Manguinhos, Rio de Janeiro, RJ , Brazil. 2 Instituto de Biologia do Exército, Rio de Janeiro, Brazil. 3 Secretaria de Vigilância em Saúde, Ministério da Saúde, Brasõ lia, Brazil. 4 Universidade Federal Fluminense, Niterói, Brazil. 5 Corresponding author, dvalle@ioc.þocruz.br. PRESENTLY, DENGUE IS A MAJOR public health problem at Brazil, where almost 70% of the municipalities are infested by the vector Aedes aegypti (L.) (SVS 2004). The organophosphate temephos has been used since 1967 and until 2000, it was the sole larvicide used in Ae. aegypti control programs (DNERu 1968, Funasa 2001). Its widespread use resulted in temephos resistance, detected in several Brazilian municipalities since 1999 (Macoris et al. 1999, 2003; Funasa 2000; Lima et al. 2003; Braga et al. 2004). The biolarvicide Bacillus thuringiensis variety israelensis (Bti) and the juvenile hormone (JH) analog methoprene are larvicides that can be substituted for temephos in localities where Ae. aegypti exhibits resistance to this organophosphate. Both are approved by the World Health Organization (WHO) and by the Brazilian Health Ministry for use in potable water (Chavasse and Yap 1997, Funasa 2001). Bti applications against Þeld populations started during 2001, but tests performed with presently available formulations revealed a low persistence under simulated Þeld conditions (Lima 2003). The second envisaged alternative, methoprene, is a member of the third generation of pesticide chemicals. It has been suggested (Williams 1967) that insects would not be able to develop resistance to juvenile hormone analogs, because these compounds mimic an endogenous hormone. However, resistant strains in different insect orders have been developed after selection pressure under laboratory conditions (Cerf and Georghiou 1972, Dyte 1972, Benskin and Vinson 1973, Wilson and Fabian 1986), including mosquitoes (Brown and Brown 1974). Methoprene-tolerant Þeld populations also have been detected, and some exhibited cross-resistance to conventional chemical insecticides (Kadri 1974, Amin and White 1984, Dame et al. 1998, Cornel et al. 2000). Although a target site for methoprene action has been found and cloned (Wilson and Fabian 1986, Ashok et al. 1998), in several instances methoprene resistance has been associated to activation of the esterases and mixed function oxidases, responsible for the metabolic resistance to conventional chemical in /05/0830Ð0837$04.00/ Entomological Society of America

2 September 2005 BRAGA ET AL.: EFFECT OF METHOPRENE ON TEMEPHOS-RESISTANT Ae. aegypti 831 secticides, including organophosphates (Cerf and Georghiou 1972, 1974; Brown and Brown 1974). Furthermore, both classes of enzymes are known to be involved with the metabolism of endogenous JH (Yu and Terriére 1971, Bergé et al. 1998, Sutherland et al. 1998, Feyereisen 1999, Lu et al. 1999). In the present work, we investigated the susceptibility of Ae. aegypti larvae derived from some Brazilian populations to methoprene in laboratory assays. Some of these Þeld populations are resistant to temephos and exhibit multiple mechanisms of metabolic resistance. Our research was performed to determine the activity of this insect growth regulator as an alternative larvicide for the control of Ae. aegypti. Materials and Methods Mosquitoes. Rockefeller (RCK) larvae were used as the susceptible control strain. Field populations from the following locations were tested: Jardim América district (JDA) at Rio de Janeiro city, Rio de Janeiro (RJ) state; Natal (NAT) at Rio Grande do Norte State (RN); Presidente Prudente (PPR) at São Paulo state (SP) and; Porto Velho (PVE) at Rondônia state (RO). Oviposition traps (ovitraps) (Braga et al. 2000) were used to collect mosquitoes according to procedures recommended by the Brazilian Ae. aegypti resistance monitoring program (Lima et al. 2003, Braga et al. 2004), covering each entire locality. Ovitraps also were used to collect mosquitoes from Macapá (MCP) at Amapá State (AP), but in this locality only sites under high insecticide pressure (organophosphates and pyrethroids) were chosen. Eggs from the different strains were allowed to hatch for 1 h, and larvae were kept at 26 1 C and fed daily with dog food (Dog Criador, Purina, Paulõ nia, SP, Brazil). Under these conditions, after 5 to 6 d, late third or early fourth instars resulted and were used in bioassays as described below. Insecticides. Metoprag 20 EC (the Brazilian name for Biopren) a synthetic pyrethroid, obtained from Bernardo Quõ mica Comércio e Indústria (São Vicente, SP), containing 50% of each S and R isomers was used. A stock solution (1 g/liter) was prepared in ethanol and stored at 4 C up to 6 mo. A 100 mg/liter ethanolic Metoprag solution was freshly prepared for use in bioassays. Larvae were exposed to Metoprag concentrations varying from 0.5 to 50 g/liter. Several lots of technical grade temephos, obtained from Prodelyn Quõ mica (Sorocaba, SP, Brazil) were used. Each of these lots has been or is being used in the Brazilian Ae. aegypti temephos resistance monitoring program. As discussed elsewhere, temephos is previously calibrated with the Rockefeller strain to deþne lethal concentrations (Braga et al. 2004). Dose Response Bioassays. Temephos doseðresponse bioassays were performed by exposing larvae to a range of 10 different insecticide concentrations (four replicates of 20 larvae per concentration), according to WHO protocol (WHO 1981a, b). Resistance ratios (RR 50 and RR 90 ) for temephos for the various Þeld strains were calculated by comparison with lethal concentrations (LC 50 and LC 90 ) obtained for the RCK strain. Four tests were run with mosquitoes from each population. Although different absolute values have been found, lethal concentrations are always compared with those obtained with a parallel test performed with the RCK strain. For methoprene doseðresponse assays, four replicates (containing 10 larvae each) were exposed continuously to serial dilutions of Metoprag (250 ml/ replicate), until death or adult emergence, following procedures described previously by Braga et al. (2005). Absolute ethanol (1 ml/liter water) was used as control. Feeding of larvae and removal of dead specimens and of live adults were each performed every other day. Two or three tests were run with mosquitoes from each population. Sequential Exposure to Temephos and Methoprene. Larvae from two temephos-resistant populations (MCP and NAT) and from the susceptible RCK strain were sequentially exposed to temephos and methoprene. To accomplish that, early L3 were Þrst exposed to a sublethal temephos concentration for 24 h. The temephos dosages used (between LC 25 and LC 50 ) corresponded to , 0.012, and 0.02 g/liter for Rockefeller strain, MCP, and NAT vector populations, respectively. For each population, 44Ð52 replicates were used. Replicates used throughout this assay consisted of 20 larvae. After 24 h of exposure, dead larvae were scored (Tem 24 h), and the remaining larvae were washed with clean water and divided in two groups. One group was transferred to clean water, and further mortality was quantiþed at the end of the assay (Tem Þnal). For each population, four replicates were used for this group. The second group was transferred to a 5 g/ liter Metoprag solution and observed until complete emergence of the untreated control group (see Braga et al for standardization of methoprene bioassay). For this group (Tem met), 16Ð20 replicates were used for each population. Controls consisted of untreated larvae (Control) and of larvae exposed only toa5 g/liter Metoprag (Meth). In both groups, four replicates were used for each population. All groups were followed until complete emergence of untreated controls. Biochemical Assays. One-day-old adult females, reared in the absence of insecticide and frozen at 70 C until used were tested in biochemical assays. The activity of acetylcholinesterase (AChE), glutathione S-transferase (GST), esterases (ESTs), and mixed function oxidases (MFOs) was quantiþed according to slight modiþcations of the protocols recommended by the Centers for Disease Control (Brogdon 1989, CDC 1998) and by Hemingway (1998). Brießy, each mosquito was homogenized in 300 l of Milli-Q water, and four aliquots of 25 l were used for AChE activity quantiþcation. The remainder was centrifuged, and the supernatants were used to quantify enzymes responsible for the metabolic activity and for dosage of total proteins. All assays were performed in 96-well microtiter plates, with individual females,

3 832 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 42, no. 5 analyzed in duplicate. The activity of all enzymes was measured in homogenate of each female in a 3-h interval. For the majority of the populations, 70Ð150 individual mosquitoes were analyzed. Absorbance was determined in a ThermoMax microplate reader (Molecular Devices, Sunnyvale, CA). AChE activity was measured for 60 min in the presence or in the absence of propoxur, an AChE inhibitor, and absorbance was read at 405 nm. Data were expressed as residual AChE activity in the presence of propoxur, and parameters deþned by Hemingway (1998) have been used to discriminate between susceptible and altered populations. Reactions to quantify glutathione S-transferase activity and heme content, an indirect measure of the MFO, were read at 340 and 620 nm, respectively. Esterases were quantiþed with three different substrates. When -or -naphtyl acetate ( NA and NA) were used, reactions proceeded for 15 min, and absorbance was read at 540 nm after addition of Fast Blue (o-tetrazotized dianisidine) and sodium dodecyl sulfate. Reactions with p-nitrophenyl acetate (pnpa) were read at 15-s intervals during 2 min at 405 nm. Total proteins were quantiþed at 620 nm with the Bio-Rad protein assay/dye reagent concentrate ( ). Bovine serum albumin was used to construct a standard curve (Bradford 1976). Analysis of Data. Both temephos and methoprene doseðresponse assays were submitted to log-probit transformation and lethal concentrations (LCs) were calculated through linear regression analysis with a conþdence interval of 95% (Raymond 1985). Resistance ratios were calculated by comparison with results obtained with the RCK strain. The criteria proposed by Mazzari and Georghiou (1995) were used to classify resistance ratios as high ( 10-fold), moderate (between 5 and 10) or low ( 5). Results of enzymatic activities obtained from individual mosquitoes were standardized for protein content and plotted as the frequency of individuals observed in each class of activity. The enzymatic proþles of different populations were compared through nonparametric one-way analysis of variance (ANOVA) (KruskalÐWallis) with DunnÕs multiple comparison posttest. When mosquitoes were sequentially exposed to temephos and methoprene, no mortality has been found in controls. In this case, mortalities of experimental groups were compared through parametric one-way ANOVA followed by BonferroniÕs multiple comparison test. In both cases, ANOVA tests were performed using GraphPad Prism version 3.00 for Windows (GraphPad Software Inc., San Diego, CA). Results Figure 1 shows the doseðmortality relationship for larvae of the susceptible strain and of Þeld populations treated with temephos (Fig. 1A and B) or with methoprene (Fig. 1C). Mortality obtained with temephos varied according to the lot of the insecticide used (Braga et al. 2004). Nevertheless, tests carried out with Þeld populations were conducted in parallel assays Fig. 1. Log-dose probit mortality lines for larvae of Ae. aegypti populations from different localities exposed to temephos (A and B) and to methoprene (C). The RCK strain was used as a susceptibility control. NAT, Natal, RN; PVE, Porto Velho, RO; PPR, Presidente Prudente, SP; JDA, Jardim América, Rio de Janeiro, RJ. Temephos bioassays for JDA were performed with different lots of insecticide, relative to the other vector populations, and are shown in a different panel. Parallel tests with RCK strain were run for each batch of insecticide. with the RCK strain. Accordingly, temephos dose response of the JDA population was done with a temephos batch distinct from that used in the other populations, and the results are shown in distinct panels (Fig. 1A and B). All populations presented an altered response to temephos compared with RCK. Among the Þeld populations tested, two (PPR and PVE) exhibited only an incipient altered temephos-

4 September 2005 BRAGA ET AL.: EFFECT OF METHOPRENE ON TEMEPHOS-RESISTANT Ae. aegypti 833 Table 1. Resistance ratios to temephos and to methoprene of Ae. aegypti larvae from different localities at Brazil, relative to the Rockefeller strain Temephos Methoprene Slope Slope a b c RR 50 RR 90 LC 50 LC 90 RR 50 RR 90 RCK Region* State** Locality*** F d SE SP PPR F N RO PVE F N AP MCP F nd e nd nd nd nd SE RJ JDA F NE RN NAT F * Brazilian regions: SE, southeastern; N, northern; NE, northeastern. ** States: SP, São Paulo; RO, Rondônia; AP, Amapá; RJ, Rio de Janeiro; RN, Rio Grande do Norte. *** Localities: PPR, Presidente Prudente; PVE, Porto Velho; MCP, Macapá; JDA, Jardim América; NAT, Natal. a Slopes, with 95% conþdence intervals, are depicted in probit units. b Resistance ratio (RR) corresponds to the ratio of a given lethal concentration (LC) between the population under test and the control Rockefeller strain. c Lethal concentrations of methoprene are liven in micrograms per liter. d F indicates the generation used in the tests. e nd, not determined. susceptible status, whereas the other three (JDA, NAT, and MCP) were resistant (Table 1). Low temephos resistance ratios were found for PPR and PVE. However, RR values between 8.3 and 24.4, indicative of moderate and high temephos resistance, respectively, were found for JDA, NAT, and MCP. Low RR values were obtained in methoprene doseð response assays, performed for the same populations, irrespective of their temephos resistance status (Fig. 1C; Table 1). In no case was a methoprene RR higher than 5 obtained. In this respect, it is noteworthy that mosquitoes from NAT, highly temephos resistant, exhibited methoprene RR values around 0.8, suggesting a slightly higher susceptibility (although not signiþcant) to methoprene than the RCK strain. Additionally, slope values obtained in methoprene doseðresponse assays were lower than those obtained in temephos bioassays (Table 1). This Þnding suggests higher heterogeneity of methoprene response, compared with the organophosphate (Ferrari 1996). Biochemical assays were performed for all populations and for the insecticide-susceptible RCK strain. Acetylcholinesterase, the target site for organophosphates, exhibited an unaltered proþle in all the populations examined (data not shown). However, alterations of the enzymes responsible for the metabolic resistance were detected in all the other cases. Figure 2 shows the enzymatic proþles obtained for GST, MFO, and EST (using pnpa as substrate). In all cases, the enzymatic proþles of experimental populations differed signiþcantly from those observed for the RCK strain (P 0.05 or P 0.001). This Þnding is in agreement with the higher temephos resistance ratio observed for those populations in relation to the control strain (Table 1). Equivalent GST proþles were found only for PVE and JDA strains (P 0.05). Differences between the control and PVE strains were signiþcant (P 0.05), whereas differences between the other three strains (JDA, MCP, and NAT) and the RCK strain were highly signiþcant (P 0.001). It is interesting to note that, among the above-mentioned populations, only PVE showed a low temephos resistance ratio, the other three being classiþed as highly resistant (RR 90, Table 1). The proþles of MFO did not differ signiþcantly between PVE and NAT (P 0.05). All other comparisons revealed highly signiþcant differences (P 0.001), both between each population and the RCK strain and among the different experimental populations. Again, the only exception was PVE that differed signiþcantly from RCK (P 0.05). When esterases were considered, differences in relation to the Rockefeller strain were highly signiþcant for all populations (P 0.001). Esterase proþles were equivalent only between JDA and NAT (P 0.05). These are populations exhibiting the highest temephos resistance ratios (Table 1). A high temephos resistance ratio also was observed for MCP (Table 1). Figure 2 shows the esterase proþle of this population also is highly altered. Accordingly, comparison of esterase proþle between MCP and JDA or between MCP and NAT revealed signiþcant differences (P 0.05), whereas all other comparisons among populations resulted in highly signiþcant differences (P 0.001). Sequential exposure of susceptible RCK larvae to temephos and methoprene induced a higher mortality compared with larvae exposed only to methoprene (P 0.05; Fig. 3A). However, when the temephosresistant MCP population was submitted to the same procedure (Fig. 3B), mortality of both groups of larvae (temephos and methoprene or methoprene alone) was equivalent (P 0.05). On the other hand, mortality of NAT larvae exposed to both chemicals (Fig. 3C) was slightly lower than mortality after exposure to methoprene alone (P 0.05). Discussion This work presents the Þrst evaluation of methoprene against Brazilian temephos-resistant Ae. aegypti.

5 834 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 42, no. 5 Fig. 2. Rate of activity of enzymes supporting metabolic resistance among different Ae. aegypti Brazilian populations and the RCK strain. Individual mosquitoes were assayed and results are expressed as the percentage of the population exhibiting different classes of activity. The number of mosquitoes used (n) is indicated below each graphic. Enzymes shown are GST, MFO, and EST, with pnpa as substrate. Populations evaluated are Porto Velho, RO (PVE); Jardim América, Rio de Janeiro, RJ (JDA), Macapá, AP (MCP); and Natal, RN (NAT). A varied level of temephos resistance was found among the vector populations tested. Nevertheless, only a slight methoprene altered susceptibility status was found in all cases, suggesting mechanisms that have been selected with the organophosphate do not confer cross-resistance to this juvenile hormone analog. Higher activity of ESTs, MFOs, and GSTs, all known to be involved with insecticide metabolic resistance,

6 September 2005 BRAGA ET AL.: EFFECT OF METHOPRENE ON TEMEPHOS-RESISTANT Ae. aegypti 835 Fig. 3. Effect of the sequential exposure of Ae. aegypti larvae to temephos and methoprene on the total mortality of Rockefeller strain (A), Macapá, AP (B), and Natal, RN (C) populations. Each bar corresponds to the mean and standard deviation of mortality in replicates containing 20 larvae each; the exception is the Tem 24 h bar (C) for which results from the 44 replicates were pooled. The number of replicates of 20 larvae used in each case is indicated below the corresponding bar. Concentration of temephos used for each strain (corresponding to LC 25 Ð LC 50 ) is shown below the bar Tem 24 h. Control, untreated larvae; Tem 24 h, mortality after 24 h of exposure to temephos in the concentrations given below each bar; Tem Þnal, mortality of larvae that survived 24 h of exposure to temephos were washed and held in clean water; Tem meth, mortality of larvae that were treated with temephos for 24 h, washed, and exposed to methoprene; and Meth, mortality of larvae exposed to methoprene only. In all groups, except Tem 24 h, mortality was recorded when all larvae from the control group emerged as adults. were found in many populations examined. Because no alteration in the proþle of the organophosphate target site, AChE, was detected, it is reasonable to assume that, in these cases, metabolic activation is involved with temephos resistance. In Ae. aegypti, elevated EST levels is the more frequent metabolic mechanism associated with organophosphate resistance (Mazzari and Georghiou 1995, Hemingway 2000, Macoris et al. 2003). Accordingly, higher EST levels were found in all populations tested in this work, even PVE, which presents a low temephos resistance ratio. Relation between MFOs and organophosphate resistance is less common in Ae. aegypti. Bioassays with the synergist piperonyl butoxide implicated this class of enzymes with temephos resistance in two Cuban localities (Rodrõ guez et al. 2004). However, the same procedure did not reveal association between MFOs and organophosphate resistance in Santiago (Cuba) neither in Venezuelan municipalities (Rodrõ guez et al. 1999, Bisset et al. 2001). On the other hand, MFO activation has been more frequently related to pyrethroid resistance (Kumar et al. 2002, Brengues et al. 2003). MFO alteration in some temephos-resistant Brazilian populations was found. However, this Þnding could be related to pyrethroid resistance. Cypermethrin has been used in the country since 1999 in the control of adults (Braga et al. 2004). Accordingly, cypermethrin resistance has been recently detected in JDA and NAT populations (Pereira-da-Cunha 2005). The enzymes GST are classically implicated in metabolic resistance to pyrethroids and DDT (Hemingway 2000). Activation of GST enzymes also has been found in organophosphate resistant Ae. aegypti populations from Cuba and Venezuela (Rodrõ guez et al. 2000, Bisset et al. 2001). However, at least one of these populations (Santiago, Cuba) also is pyrethroid-resistant (Rodrõ guez et al. 1999). Activation of GST in the Brazilian populations tested also could be related to resistance to pyrethroids. Bioassays with Brazilian populations revealed low resistance ratios to methoprene. Esterases and MFO have already been associated with resistance to this juvenile hormone analog in other insects (Cerf and Georghiou 1972, 1974; Brown and Brown 1974). Both classes of enzymes were activated in MCP and NAT strains. We then investigated whether previous activation of these enzymes, through exposure to temephos, could induce cross-resistance to methoprene. As expected, sequential exposure of susceptible RCK strain larvae to temephos and methoprene resulted in higher mortality compared with larvae exposed to temephos or methoprene alone. In contrast, mortality of MCP larvae was equivalent after exposure to temephos alone, methoprene alone or temephos plus methoprene. Mortality of NAT strain larvae exposed to temephos and methoprene was lower than mortality with methoprene alone, suggesting that a temephos-resistance mechanism, elicited by the previous exposure to this organophosphate, could protect larvae from methoprene. However, it should be remembered that, when exposed only to methoprene,

7 836 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 42, no. 5 the susceptibility of Ae. aegypti from NAT strain was slightly higher than Rockefeller (RR ). These results seem to indicate a potential crossresistance between organophosphates and the juvenile hormone analog. Bioassays with synergists are envisaged, to elucidate this question in detail. Nevertheless, our data indicate a lack of resistance to methoprene and point to the feasibility of using this insect growth regulator in the control of temephos-resistant Ae. aegypti Brazilian populations. However, the continuous monitoring of the resistance of the dengue vector to insecticides is essential to enable the establishment of sustainable control strategies. Acknowledgments We thank Sabrina C. G. Carvalho, Ta nia M. R. Santos, Henrique M. C. Ramon, and José L. Silva for technical assistance; the Entomology Laboratories from the Secretarias Estaduais de Saúde from Rio Grande do Norte and Rondônia and from Sucen/MariliaÐSão Paulo for providing mosquito eggs; Bernardo Quõ mica Comércio e Indústria for the Metoprag sample; and the reviewers of this article that greatly collaborated to the improvement of the analysis and the text. Use of trademark names does not imply endorsement by the Health Ministry but is intended only to assist in identiþcation of a speciþc product. This work was supported by Fundação de Amparo à Pesquisa (FAPERJ), Conselho Nacional de Desenvolvimento Cientõ Þco e Tecnológico (CNPq), Fundação Oswaldo Cruz, Fundação Nacional de Saúde and Secretaria de Vigila ncia em Saúde. References Cited Amin, A. M., and G. B. White Resistance potential of Culex quinquefasciatus against the insect growth regulators methoprene and dißubenzuron. Entomol. Exp. Appl. 36: 69Ð76. Ashok, M., C. Turner, and T. G. 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