Insecticide Resistance Mechanisms of Brazilian Aedes aegypti ...

Am. J. Trop. Med. Hyg., 77(3), 2007, pp. 467–477 Copyright © 2007 by The American Society of Tropical Medicine and Hygiene

Insecticide Resistance Mechanisms of Brazilian Aedes aegypti Populations from 2001 to 2004 Isabela Reis Montella, Ademir Jesus Martins, Priscila Fernandes Viana-Medeiros, José Bento Pereira Lima, Ima Aparecida Braga, and Denise Valle* Laboratório de Fisiologia e Controle de Artrópodes Vetores, Instituto Oswaldo Cruz, Rio de Janeiro, Brazil; Instituto de Biologia do Exército, Rio de Janeiro, Brazil; Secretaria de Vigilância em Saúde, Ministério da Saúde, Brasília, DF, Brazil

Abstract. In Brazil, Aedes aegypti resistance to temephos, used since 1967, was detected in several municipalities in 2000. Organophosphates were substituted by pyrethroids against adults and, in some localities, by Bti against larvae. However, high temephos resistance ratios were still detected between 2001 and 2004. Field-simulated assays confirmed a low temephos residual effect. Acethylcholinesterase and Mixed Function Oxidase profiles were not altered. In contrast, higher Esterase activity, studied with three substrates, was found in all examined populations collected in 2001. From 2001 to 2004, a slight reduction in ␣-Esterase (EST) and ␤-EST activity together with a gradual increase of p-nitrophenyl acetate (PNPA)-EST was noted. Gluthathione-S-transferase alteration was encountered only in the northeast region in 2001, spreading the entire country thereafter. In general, except for ␣-EST and ␤-EST, only one enzyme class was altered in each mosquito specimen. Data are discussed in the context of historic application of insecticides in Brazil. on alterations of common target sites or on detoxification mechanisms, can impair the use of a given product. Biochemical mechanisms of resistance are mainly associated with either a change in insecticide target site sensitivity in the central nervous system (sodium channels, GABA receptors, and acetylcholinesterase) or an increased rate of insecticide detoxification. This latter mechanism, known as metabolic resistance, involves three major groups of enzymes: Esterases (ESTs), Glutathione-S-transferases (GSTs), and Mixed Function Oxidases (MFOs).10 We describe the quantification of temephos resistance and the evaluation of potentially associated mechanisms in several Ae. aegypti Brazilian populations in two and three monitoring rounds, respectively. These evaluations were performed in accordance with the MoReNAa network, which supports the Brazilian Dengue Control Program (PNCD), which is, to our knowledge one of the largest and most complex strategies of dengue control among those coordinated at a national level. The biochemical assays were performed according to a protocol11 slightly modified from the Centers for Disease Control (CDC) and the World Health Organization (WHO) recommendations.12,13 A functional classification of vector populations with respect to enzymatic activity is presented. Resistance mechanisms detected in different populations during the course of these three rounds of monitoring are discussed with regard to the recent insecticide choices made by the Brazilian Dengue Control Program.

INTRODUCTION Dengue is currently the most important vector-borne human disease causing arbovirus in the world. The primary vector of dengue is Aedes aegypti, and in Brazil, an extensive country of 8.5 million km2, high infestation levels prevail in all regions except in the two southernmost states. Three dengue virus sorotypes (DEN 1, 2, and 3) circulate simultaneously in Brazil, now considered endemic to this disease.1,2 Chemical insecticides are still a major tool in the control of Ae. aegypti. In Brazil, since 1967, organophosphates (OP) have been the principal weapon against this vector, subjecting mosquito populations to an intense selection pressure. After the dengue outbreak of 1986,3,4 OP use was intensified against larvae and adults. As a consequence, OP-resistant vector populations disseminated throughout the country, the first cases being detected in 1999. In that year the Brazilian Health Ministry started the coordination of an integrated program (Rede Nacional de Monitoramento da Resistência de Aedes aegypti a Inseticidas, MoReNAa) designed to monitor Ae. aegypti insecticide resistance.5 Initially based solely on the detection and quantification of temephos resistance, through bioassays, results of this network were an important aid for the Health Ministry in the definition of novel control strategies, leading to temephos substitution in 2001 by the biolarvicide Bacillus thuringiensis var. israelensis (Bti) in those localities with temephos-resistant vector populations. Simultaneously, OPs were replaced by pyrethroids (PY) in the control of adults throughout the country. These decisions were based exclusively on bioassays, because at that time, the biochemical mechanisms responsible for insecticide resistance in Brazilian dengue vector populations were not known.5–7 In the second monitoring round (2001), we confirmed temephos resistance of Ae. aegypti populations from several municipalities of the northeast and southeast regions.8 Alterations in the susceptibility status to pyrethroids were also detected in some localities in 2001 and in 2002–2003.9 Cross-resistance among different insecticide classes, based

MATERIALS AND METHODS Mosquitoes. Mosquito eggs from several Brazilian regions (Figure 1) were collected in three different periods (2001, 2002–2003, and 2004). According to the monitoring program, the same municipality was not necessarily evaluated in all periods, enabling surveillance in a greater number of localities. Ovitraps were adopted to collect mosquitoes complying with MoReNAa network recommendations.7,8,14 In almost all cases, F1 (preferably) or F2 specimens were used. Thirdinstar larvae and 1-day-old adult females were subjected to temephos bioassays and biochemical tests, respectively. The Rockefeller (RCK) strain, reared continuously in the laboratory for many years, was used as the susceptible reference lineage in all situations. Larval bioassays. Temephos bioassays performed with

* Address correspondence to Denise Valle, Laboratório de Fisiologia e Controle de Artrópodes Vetores, Instituto Oswaldo Cruz, Fiocruz, Av. Brasil 4365, 21040-900 Rio de Janeiro, RJ, Brazil. E-mail: dvalle@ioc.fiocruz.br

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FIGURE 1. Localization of Brazilian municipalities and districts (in the case of Rio de Janeiro municipality) where Ae. aegypti populations were evaluated. In Brazil map, thick lines indicate limits among regions. Only evaluated states are delimited, in light gray.

populations collected in 2001 have been described elsewhere.8 Temephos samples available to the Brazilian Dengue Control Program in 2002–2003 (Prodelyn 94%) and 2004 (Fersol 90%) were calibrated before use in the bioassays. This was carried out according to parameters discussed in Braga and others8 using the RCK strain as the susceptibility reference lineage. The temephos diagnostic dose used in bioassays with samples collected in 2002–2003 was 0.0084 mg/L. Temephos dose-response assays were conducted essentially as Braga and others,8 in accordance with WHO protocol.15 Resistance ratios (RR95) were calculated by comparison with the lethal

concentration obtained for the RCK strain. At least four assays were run with mosquitoes from each population. In each diagnostic dose and dose-response assay, 240 and 800 larvae were used, totaling 960 and 3,200 specimens, respectively. Field-simulated assays. According to the parameters established by MoReNAa,16,17 mosquitoes from vector populations exhibiting temephos RR95 > 10 were exposed, in fieldsimulated conditions, to the temephos dose adopted to control Ae. aegypti in the field. Four mosquito populations (Table 1, bold) were submitted to field-simulated temephos bioassays: Belém (PA), Natal (RN), Maceió (AL), and Jardim América district (municipality of Rio de Janeiro, RJ). These

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INSECTICIDE RESISTANCE IN BRAZILIAN AE. AEGYPTI

TABLE 1 Temephos resistance status of Ae. aegypti populations from different Brazilian localities in three rounds of monitoring Locality Region†

State‡

Municipality

Rio de janeiro

SE

RJ

2002/2003

2004*

District

Collection of eggs

Percent mort DD§

Bangu Barra da Tijuca Botafogo Ilha do Governador Jacarepaguá/Taquara Jardim América** Penha Pilares Rocha Miranda São Cristóvão Tijuca

Dec/2002 Jan/2003 Jan/2003 Dec/2002–Jan/2003 Dec/2002 Dec/2002 Jan/2003 Dec/2002 Dec/2002 Dec/2002 Jan/2003

26.3 16.3 50.8 22.5 75.9 31.1 16.9 11.3 23.4 35.9 34.3

May/2003 May/2003 Mar/2003 Jul/2003 Feb/2003 Nov/2002 Dec/2002 Apr/2003

94.0 90.0 50.5 33.3 29.6 96.1 26.3 88.8

Cabo Frio Campos dos Goytacazes Itaperuna Niterói N. Iguaçu São José v. Rio Preto Três Rios Volta Redonda

RR95¶

9.0

11.8 7.7

11.1

Jan Jan Jan Jan Jan

12.6 10.1 18.4 12.3 15.3

5.4

Jan

12.1

Aug Aug Aug Aug

18.6 12.6 12.6 12.5

Oct Oct

23.5 25.8

Nov/2003 Aug Aug

21.4 11.2 26.2

8.2 7.4 4.0 9.3 7.2

5.5 4.2 16.8 11.7 12.8 3.0 8.2 5.2

Natal** Mossoró Parnamirim Caicó

AL

Maceió** Arapiraca

May/2003 Apr/2003

38.4 53.4

10.8 8.7

Aracajú Barra dos Coqueiros Itabaiana

Apr/2003

28.9

6.6

SE

N

PA

Dom Eliseu Belém** Belém

CW

GO

Goiânia

S

RS

Ijuí Santa Rosa

Marambaia Jul/2003

96.4

RR95¶

Jan Jan

RN

NE

Collection of eggs

3.3 Abr Jan

1.41 1.7

* In 2004, only temephos dose-response bioassays were performed. † Regions: CW, Central-West; N, North; NE, Northeast; S, South; SE, Southeast. ‡ States: AL, Alagoas; GO, Goiás; PA, Pará; RN, Rio Grande do Norte; RJ, Rio de Janeiro; RS, Rio Grande do Sul; SE, Sergipe. § Rate of mortality after exposure of larvae to the temephos diagnostic dose. ¶ RR, resistance ratio. ** Results shown in bold correspond to populations that were also submitted to bioassays against temephos in simulated field conditions (Fig. 2).

bioassays, designed to determine the persistence of temephos effect, were conducted both indoors and outdoors. In each milieu, three randomly dispersed 50-L containers, filled with tap water, were used for each population under test and for RCK. On the first day, each recipient received 5 g of the same granulated temephos formulation (1%) adopted for in the field by PNCD. This resulted in a concentration of 1 ppm, exactly the same applied by field personnel. Three additional recipients (RCK) remained without temephos treatment. Three times a week, 10 L from each recipient was replaced by fresh water. Weekly, 50 L3 larvae of the corresponding populations were added to each container, and mortality was quantified both 24 and 48 hours later, when remaining larvae were discarded. Biochemical assays. One-day-old adult females, reared in the absence of insecticide and frozen at −70°C until use, were submitted to biochemical assays. The activity of Acetylcholinesterase, GSTs, ESTs, and MFOs was quantified according to slight modifications of the protocols recommended by CDC12,18 and WHO.13 This modified protocol is partially de-

scribed by Braga and others,19 details of which protocol are shown in Valle and others.11 EST activity was evaluated with three different substrates: ␣-naphthyl (referred to as ␣-EST), ␤-naphthyl (␤-EST), and p-nitrophenyl (PNPA-EST) acetate. As an internal control, at each monitoring round, RCK was submitted to novel biochemical tests. Analysis and interpretation of bioassays. Bioassays carried out with the diagnostic dose (DD) were evaluated according to criteria defined by Davidson and Zahar.20 Mortality > 98% and < 80% is indicative of susceptibility and resistance, respectively, and intermediate mortality levels suggest an incipient altered susceptible status. Results from different temephos dose-response assays of the same mosquito population were compared by analysis of variance (ANOVA) tests, exactly as described before.8 If significant differences were found (P < 0.05), the discrepant assay was discarded, and the others were pooled. When no significant differences among the assays were detected (P > 0.05), data were pooled and submitted to probit analysis, using the computer program GW-Basic 2.01 (1984) to define

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lethal concentrations (LCs). RRs were calculated through comparison with results obtained for the reference strain, RCK. The criteria proposed by Mazzarri and Georghiou21 was used to classify RRs in high (> 10-fold), medium (between 5- and 10-fold), and low (< 5-fold). Analysis of biochemical assays. Results of enzymatic activities obtained from individual mosquitoes were corrected according to total proteins. Different graphic representations have been used and will be presented. In all cases, the enzymatic profiles were compared with those obtained for the RCK strain. For each enzyme, the RCK 99th percentile was calculated. Afterward, for each population and for each enzyme under

FIGURE 2. Temephos persistence evaluation, in field-simulated conditions, of some populations exhibiting high RR95 (> 10.0) in laboratory bioassays.

test, the percent of specimens with enzymatic activity above the RCK 99th percentile was obtained. Activities were classified as “unaltered,” “incipiently altered,” or “altered” if the rate was < 15%, between 15% and 50%, or > 50%, respectively. This criterion is discussed below. Populations with altered activity of two or more enzymes were submitted to Spearman correlation to study whether the same individuals exhibited higher activity of different enzymes. RESULTS Bioassays. Only temephos resistance of larvae was monitored in the scope of this work. However, previous data on adult resistance to the PY cypermethrin9 are available. Results of temephos bioassays performed in 2001 are shown elsewhere.8 In Rio de Janeiro State, temephos applications were officially interrupted during 2001.9,22 Nevertheless, quantification of temephos resistance that year still indicated high resistance levels,8 and since then, in general only a slight reduction of resistance levels (or no reduction at all) has been observed. Some vector populations, such as those from the districts of Penha, Jacarepaguá, and Tijuca (Rio de Janeiro city, RJ), exhibited an increase in RR in 2004 compared with 2002–2003. This is probably because of cross-resistance with PY insecticides, introduced in the state to control mosquito adults since 2001. Alternatively, increase in the resistance status of these populations might be attributed to the uncontrolled use of different classes of insecticides by the local population. Regarding other RJ municipalities, apart from Rio de Janeiro city, temephos resistance monitoring was not performed in 2004. This was a strategic decision of the MoReNAa/ PNCD to enable the evaluation of other localities, mainly the northeastern (NE), northern (N), and southern (S) regions. Accordingly, four municipalities in RN state (NE region) were monitored in 2004. All of them displayed elevated temephos resistance levels despite the official substitution of temephos by Bti in this state in 2001. Very high levels of temephos resistance (RR > 20.0) were witnessed in Barra dos Coqueiros and Itabaiana, two Sergipe (SE) municipalities evaluated in 2004. In the former city, this corresponds to twice the RR quantified in 2001.8 In the NE region, generally higher RR levels were noted in 2004 compared with previous evaluations.8 This may reflect either a non-effective interruption of temephos application or potential cross-resistance with insecticides later uncontrollably introduced. High RR levels were also present in the N region, first evaluated by our group in 2004. Only the Brazilian CW (2002–2003) and S (2004) regions did not exhibit significant alterations of temephos susceptibility. Regarding the latter region, this may reflect the absence of dengue epidemics in RS, the southernmost state and, as a consequence, the lack of an intense insecticide selection pressure. Tests with the temephos diagnostic dose were in accordance with the corresponding dose-response assay. No population was classified as susceptible with this qualitative temephos assay. With few exceptions, bioassays with the DD indicated resistance to temephos. Only five mosquito populations (evaluated in 2002–2003) exhibited mortality rates indicative of an incipient alteration of susceptibility: Goiânia, in GO state and Cabo Frio, Campos dos Goytacazes, São José do


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FIGURE 3. Different graphic representations of biochemical assays. In all cases, PNPA-EST activity profiles are shown. A and B, Histograms with the activity of Rockefeller strain and mosquitoes from Bangu district, Rio de Janeiro, RJ, respectively. C, Box plot and D, Scatter graphic of vectors from different districts of Rio de Janeiro municipality. bng, Bangu; cgn, Campo Grande; jcp, Jacarepaguá; jam, Jardim América; pen, Penha; rel, Realengo; rmi, Rocha Miranda; scr, São Cristóvão. Note that in C and D, Rockefeller (RCK) profile is included for comparison.

Vale do Rio Preto, and Volta Redonda, in RJ state. Accordingly, in all cases, the RR of these populations was compatible with low or moderate resistance. It should be noted that cypermethrin bioassays with Ae. aegypti adults already pointed to a decrease in the susceptibility to this class of insecticides between 2001 and 2002–2003, despite its recent use.9 Field-simulated tests. Figure 2 shows the results obtained in a field-simulated assay with four mosquito populations previously classified as highly resistant (RR > 10) in the laboratory. The original localities of each one of the populations, the periods of egg collection and the corresponding temephos RR are marked in bold in Table 1. In the containers placed indoors, mortality rates up to 70% were attained until the 10th to 11th week for all the populations evaluated, except for larvae from Jardim América district that presented low mortality levels already on the 3rd week (Figure 2, middle). However, in outdoor conditions, mortality values > 70% were not observed after the fourth week for any population. Again in this case, larvae from Jardim América district exhibited the lowest residual temephos level (Figure 2, bottom). The abrupt decrease in mortality observed outdoors between the third and fifth weeks reflects a diminished insecticide effect, probably a consequence of the resistant status of these vector populations as-

sociated with the elevated climate temperatures verified from the second to the fourth week (Figure 2, top). It is relevant to note that a decreased temephos activity was still effective against RCK larvae, the insecticide susceptible control lineage: high mortality levels were obtained for this strain up to the end of the field-simulated assays in both indoor and outdoor conditions, respectively, 7 and 12 weeks. Results shown in Figure 2 were obtained after exposing larvae for 24 hours. Records attained after 48 hours of exposure were essentially equivalent, as already noted in other simulated assays.23 Because mortality (1.5–12%) in the untreated controls has been observed after a 48-hour exposure (but not 24 hours), these data were not taken into account. Graphic representation of biochemical assays. Quantification of enzymatic activities related to insecticide resistance is generally represented as depicted in the example of Figure 3A and B (PNPA-EST profiles of, respectively, Rockefeller strain and Bangu district, Rio de Janeiro, RJ, collected in 2001). This type of graphic displays the enzymatic profile of a given population discriminated in different classes of activity. In this way, a different graphic is necessary for each enzyme and for each population. Data from the monitored vector populations are compared with the profile of a standard susceptible lineage. Evaluations are usually made by visual com-

472 parison of the histograms or by statistical analysis of parametric measures, such as mean and SD.24–26 This graphic representation is not convenient when a high number of populations are simultaneously evaluated, as is the case of the MoReNAa network. Besides, in many circumstances where field populations are considered, parametric measures do not provide the most informative parameters: in the context of insecticide resistance monitoring, special attention should be given precisely to populations that exhibit an incipient altered status. What is generally verified in these cases is a great majority of susceptible specimens together with a few altered individuals presented at a low frequency (Figure 3B). Detection of the mechanisms implicated in these situations will enable an intervention in the field before resistance attains a threshold that hampers vector control. We propose two different graphic representations. In both cases, the profiles of different populations are shown in one graphic and are directly compared with the susceptible reference lineage (here represented by RCK). One of them (Figure 3C, box plot) deals with the median instead of the mean. The box limits (upper and lower quartiles) enclose the distribution of the central half of the population (25–75%), and the vertical lines indicate the higher and lower measures obtained. In the other representation (Figure 3D, scatter graphic), the activity of each individual is signaled. This kind of graphic allows discrimination as to whether the enzymatic activity of a given population exhibits an unaltered and homogeneous distribution (Figure 3D, RCK and scr) or whether it is completely altered and heterogeneous (Figure 3D, rmi). However, it is ideal to detect altered individuals in a population whose distribution is otherwise normal, as in Figure 3D, for instance of Bangu (bng) or Penha (pen) vectors, two examples of relevant populations in the context of insecticide resistance monitoring. Biochemical assays. The quantification of several enzymes in mosquito populations from different Brazilian regions was performed in the course of three insecticide monitoring rounds. Table 2 shows the number of specimens evaluated in each assay together with their corresponding median values. Initial analysis was undertaken through comparison of RCK medians with those for the populations under test. A classification of the activity is included according to criteria outlined in the Materials and Methods section and presented elsewhere (see also Discussion).11 The values were used to classify the activity as unaltered (light gray), incipiently altered (dark gray), or altered (black) if it was < 15, between 15 and 50, or > 50%, respectively. According to this classification, an unaltered profile of Acetylcholinesterase (ACE), target site of organophosphates (OP), was found in the three rounds of monitoring in all populations examined, regardless of their Brazilian region. The same was true, in practically all cases, for MFO, enzymes associated with metabolic resistance against virtually all classes of insecticides. A slight alteration of MFO was only noted in two districts of Rio de Janeiro city (RJ) in 2001: Realengo, which was not further monitored, and Jardim América, which exhibited subsequent unaltered activity. In contrast, there was alteration in EST activity in all populations examined in 2001 when at least one of three substrates was used. In general, a slight reduction in ␣-EST and ␤-EST activity was noted between 2001 and 2004. PNPA-EST activ-

MONTELLA AND OTHERS

ity, in opposition, showed a gradual increase during the course of the three periods of evaluation. The most surprising results were with reference to GST, a class of enzymes generally involved in Phase 2 detoxification mechanisms and associated with metabolic resistance to organochlorines.27 From the material collected during the 2001 monitoring, increased GST activity was noted in the populations from the NE but not from the SE region. Mosquitoes from all the NE localities evaluated that year exhibited some degree of GST alteration. In 2002–2003, all the samples evaluated (in three Brazilian regions) presented increased GST activity at varying levels. In 2004, there was an unequivocally altered activity in the 16 populations examined. It is outstanding that this year, in 9 of 16 vector populations, 90% or more of the individuals presented GST activity above the RCK 99th percentile. These data indicate a strong selection pressure, probably a consequence of the national decision to introduce a different class of insecticides in the control of adults (see Discussion). We studied whether the same individuals displayed simultaneous alteration for more than one enzyme in each population. Spearman correlation analysis revealed that this was not so, with the exception of ␣-EST and ␤-EST, which are strongly correlated, even in the susceptible RCK strain (data not shown).

DISCUSSION The Brazilian Ae. aegypti Insecticide Resistance Monitoring network started in 1999 to study potential resistance to the OP temephos, the sole larvicidae against the country’s dengue vector since 1967. Although previous experience of the laboratories was very heterogeneous, this program has acquired the capacity to evaluate the resistance status of many localities simultaneously. Initially projected to perform qualitative evaluations to detect temephos resistance,7 quantitative assays were later introduced into the activities of the program, leading to the determination of RRs. The criterion defined by Mazzarri and Georghiou21 to classify temephos resistance was adopted. According to this criterion, populations with a resistance ratio (RR95) > 10.0 are highly resistant, i.e., potential lack of effective control if the insecticide continues to be administered in the field. As far as we know, there is no report in the literature that this was really true with Ae. aegypti populations. For this reason, it was decided to perform field-simulated assays with these populations, as an additional control, before suggesting temephos application interruption in a given locality. In these assays, mosquito populations with RRs > 10.0 were submitted to the temephos dose effectively used in the field, and its residual effect was evaluated. We confirmed a decrease in the temephos residual effect in these cases up to 4 weeks in outdoor conditions. Considering this larvicide is routinely applied in Brazil four to six times a year, these results point to the inadequacy of temephos in the control of larvae from populations with high RR. However, retrospectively, it is presently clear that when temephos use is only interrupted in localities where Ae. aegypti RR is > 10.0, resistance decrease is gradual, impairing temephos reuse for several years. In this sense, it was proposed, in a recent meeting of the MoReNAa/PNCD, that RRs

TABLE 2 Quantification of the enzymatic activity in Brazilian Aedes aegypti populations in three insecticide resistance monitoring rounds.


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> 3.0 should dictate temephos substitution by another larvicidae.22 Considering that Brazil has ∼5,600 municipalities, this implies a huge modification in the operational aspects of vector control. It is also relevant to note that organophosphates were the only class of insecticides used in the control of both larvae and adults of Ae. aegypti for > 30 years before the first suspicions of temephos resistance were communicated by field personnel in 1998. After resistance confirmation, through bioassays, organophosphates were substituted by cypermethrin for adult control. Neither organochlorines nor carbamates (CA) were considered, the first class being precluded from Brazil since 1994 and CA share the target site with OP—thereby setting the stage for a potential cross-resistance scenario. One must keep in mind that this decision was based only on bioassay data, because at that time, there was no biochemical protocol available to the monitoring network that could unravel resistance mechanisms. Cypermethrin use against Ae. aegypti adults, effectively initiated in 2001, was immediately followed by the decision to monitor resistance to pyrethroids (PY). In this way, it would be possible to anticipate potential vector control problems derived from resistance. However, it was verified that, in contrast to the slow acquisition of resistance to OP, alteration in the susceptible status of Ae. aegypti populations to PY increased very fast.9 It became evident that study of the biochemical mechanisms was a necessary step to elucidate the dynamics of resistance in different regions of the country. Both CDC12,18 and WHO13 protocols were tested and resulted in a modified procedure10 that was applied to several mosquito populations in three rounds of monitoring. Modifications in the biochemical assays included some differences in the presentation of the results, as well as a proposal for the classification of enzyme activity. As stated in the Results section, the representation in the graphic of the enzymatic activity of each individual from a given population (Figure 3D) is an accurate way to evaluate the resulting profile. Several populations are plotted in the same graphic, together with the susceptible strain, a procedure that enables direct comparison with the “unaltered” profile. Finally, with this format, populations presenting few individuals with altered activity are quickly detected. It should be noted that, in the context of resistance monitoring, these are the most important, because identification of incipiently altered populations allows interference before resistance spreads throughout the population. Several attempts were made to define a classification of enzyme activity in vector populations. We decided to start evaluations with ACE, because the functional criterion adopted by WHO for this enzyme is generally accepted: ACE activity is said to be altered if the CA propoxur inhibition is < 70%.13 According to this criterion, all the populations analyzed in the scope of this work have ACE unaltered activity. However, all non-parametric tests used showed significant differences in the ACE activity of field populations in relation to the reference strain, Rockefeller (data not shown). We opted to define a classification of enzyme activity that was representative of the WHO functional criterion adopted for ACE, regardless of its statistical significance. It was also considered that enzymatic profiles do not necessarily have a normal distribution, mainly when a resistance character has been

recently introduced in a given population. For this reason non-parametric measures were considered, e.g., the median and the 99th percentile. Comparisons were made with RCK. The cut-off point established to discriminate between “unaltered” and “incipiently altered” activities, 15% of individuals above the RCK 99th percentile, was arbitrarily chosen as the value that best correlates with the WHO evaluation criterion of ACE activity with the populations quantified in the scope of this work. The cut-off points defined for ACE were applied to the other enzymes as well. In several populations, the activity of more than one enzyme was altered. Because all the enzymes were quantified in each mosquito homogenate, it was possible to compare the activity of different enzymes in the same individual. Absence of correlation indicated that in the evaluated populations different specimens generally presented an alteration of each enzyme, the exceptions being ␣-EST and ␤-EST, which exhibited a strong correlation, even in the susceptible RCK strain. Although the possibility of co-selection of specific alleles for these enzymes can not be discarded, as has been found for Culex,28,29 this result might also indicate a lack of specificity of the substrates used. Accordingly, LimaCastelani and others30 verified that Ae. aegypti ␣-EST can hydrolyze ␤-naphthyl acetate, applied as the ␤-EST substrate. As already pointed out by Hemingway and Karunaratne,31 often in insecticide resistance mechanism–related literature, the terminology for EST enzymes is not homogeneous, making it difficult to compare results among different groups. Based on the definitions of Aldridge,32 Okuda,33 and the International Union for Biochemistry and Molecular Biology (IUBMB),34 we opted to group the ESTs evaluated here into two classes. 1) A-EST (EC 3.1.1.2) are not inhibited by OP and are able to hydrolyze these same compounds. Because these enzymes act on aromatic esthers, like phenyl-acetate, they are also referred to as arylesterases. 2) B-EST (EC 3.1.1.1) are stequiometrically inhibited by OP and do not hydrolyze these compounds. Because of their wide range of substrates, ␤-ESTs are also known as non-specific ESTs, aliesterases, or carboxylesterases. Both ␣− and ␤-naphthyl acetates are general substrates used in the identification of ESTs actively expressed against insecticides. To distinguish the enzymes acting on these substrates from the aforementioned IUBMB classification, we designated as ␣-EST and ␤-EST the hydrolytic activities measured against ␣− and ␤-naphthyl acetate, respectively. This terminology does not mean we are dealing with individual molecular entities because, as already remarked, B-ESTs are non-specific and there is evidence that ␣− and ␤-naphthyl acetates can be used by the same molecular species.30 It is also important to stress that the ␣-EST and ␤-EST activities here presented are distinct from the ␣-EST and ␤-EST genes already detected in C. pipiens.28,31 Convention dictates that only B-ESTs are present in insects.31,35,36 However, PNPA, adopted as an EST substrate in insecticide resistance mechanism evaluations, is classified as an A-EST substrate.34 PNPA-EST activity dynamics, as observed in several populations in three rounds of resistance monitoring, was distinct from ␣-EST/␤-EST profiles, confirming the simultaneous existence of different EST enzymes in Brazilian Ae. aegypti populations and suggesting the presence of hydrolytic ESTs (A-ESTs) in these vectors. More detailed studies, based on the effect of inhibitors on purified enzymes,


are needed to elucidate this question. Accordingly, as described by Wheelock and others,37 “one of the major obstacles in the biochemical identification and characterization of Carboxylesterases is the choice of substrate to measure activity in the presence of multiple enzymes.” An elevated ␣-EST and ␤-EST activity is associated with OP resistance.21,38,39 The slight decrease in activity of these enzymes observed in the majority of populations between 2001 and 2004 could be related to the official interruption of temephos use, initiated in 2001. However, as highlighted above, the decline in ␣-EST and ␤-EST activity was slower than expected in this period. This might reflect a continued selection pressure caused by either a delayed effective temephos interruption or to a potential cross-reaction with other insecticides uncontrollably administered. Contrary to ␣-EST and ␤-EST, an increase in PNPA-EST activity was observed in the same period. This was concomitant with a pronounced GST activity increase and with the substitution of OP by PY in the control of adults, initiated in 2001. There was no correlation between PNPA-EST and GST activities, suggesting the selection of independent resistance mechanisms in these vector populations in different individuals. GST plays an important role in insecticide resistance. This enzyme was implicated in the detoxification of OP40,41 and in the protection against PY toxicity.42 In Ae. aegypti, GST is also associated with metabolic-based DDT resistance.27 This insecticide was widely used in the 1940 decade in Brazil, mainly in the NE region, in the campaign against urban yellow fever.43 Accordingly, in the first evaluation of resistance mechanisms performed in the scope of MoReNAa (2001), vector populations with altered GST activity were detected only in the NE region, suggesting maintenance of resistant alleles since then (although Brazil was considered free of Ae. aegypti at that time).44 Later dissemination of GST alteration to populations from other Brazilian regions could reflect either a migration of individuals among populations or a selection through PY or both. It is interesting to note that several authors have recently verified, by means of different genetic markers, that Brazilian Ae. aegypti populations are genetically differentiated. In particular, vectors from the NE region exhibit distinct characteristics compared with the others (Bracco JE, unpublished data).1,45,46 Alteration in the sodium channel, the PY target site, is another potential resistance mechanism. Although individuals from several Brazilian Ae. aegypti populations have been analyzed, the classic Kdr mutation, normally associated with PY resistance,10,47 was not detected. Nevertheless, other mutations have been detected that could account for the resistance phenotype (Martins AJ, unpublished data).48 Relation of these mutations and PY resistance and their frequency distribution are being studied. Although bioassays are the gold standard to detect insecticide resistance, biochemical assays can provide essential information in the rational insecticide choice. In this context, bioassays and the study of resistance mechanisms have been useful in the definition of the strategies adopted by the Brazilian dengue control program. Retrospectively, the dynamics of enzyme activity observed in the three monitoring rounds herein presented reflects the recent changes in insecticide choices for dengue vector control in Brazil. In this sense, the slight decrease in ␣-EST and ␤-EST seems to be a consequence of temephos interruption, whereas GST and PNPA-

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EST activity increase could account for PY insecticides introduction in the control of adults. Notwithstanding, precise correlations between biologic and biochemical assays could not be established for each evaluated population. Lack of correlation might partially be explained by differences in enzyme activity throughout development, already detected in different mosquitoes for EST and GST.30,49–51 In our case, one must be aware that temephos bioassays are carried out with larvae, whereas biochemical tests are carried out on adults. We are presently studying the biochemical resistance mechanisms in both larvae and adults from the monitored vector populations. We also intend to identify the subsets of GST whose activity is increased in PY resistant field populations. Although many questions still remain unanswered, the MoReNAa network has advanced considerably in the understanding of the resistance status and mechanisms in Brazilian Ae. aegypti populations. It is outstanding that the knowledge acquainted with all these assays is being taken into account by the Dengue Control Program to define rational control strategies adapted to local situations in a productive interaction between basic research and Public Health. In this sense, the effects and persistence of alternative insecticides, like the Insect Growth Regulators, are being evaluated in both laboratory and field conditions to determine their application with regard to the climatic and operational reality of Brazil. Received September 27, 2006. Accepted for publication March 18, 2007. Acknowledgments: The authors thank the Secretaria de Vigilância em Saúde and the Programa Nacional de Controle da Dengue for technical assistance and the Secretarias Estaduais and Municipais de Saúde from the different localities evaluated, for mosquito egg collection. We also thank Dr. W. G. Brogdon, for evaluation and validation of our biochemical protocol, Tânia Maria Rodrigues dos Santos and Nathalia Giglio Fontoura for technical assistance with, respectively, laboratory and field simulated bioassays, and Heloisa Diniz for preparing the map. The manuscript was reviewed and revised by Mitchell Raymond Lishon, native of Chicago, IL. Financial support: This work was supported by Secretaria de Vigilância em Saúde/Ministério da Saúde, Programa de Desenvolvimento e Inovação Tecnológica em Saúde Pública/Fundação Oswaldo Cruz, Vice-Presidência de Serviços de Referência e Ambiente/Fundação Oswaldo Cruz, Conselho Nacional de Desenvolvimento Científico e Tecnológico, and Financiadora de Estudos e Projetos do Rio de Janeiro. Authors’ addresses: Isabela Reis Montella, Ademir Jesus Martins, Priscila Fernandes Viana-Medeiros, Jose´ Bento Pereira Lima, and Denise Valle, Laboratório de Fisiologia e Controle de Artrópodes Vetores, Instituto Oswaldo Cruz, Fiocruz, Av. Brasil 4365, 21040-900 Rio de Janeiro, RJ, Brazil/Instituto de Biologia do Exército, Rua Francisco Manuel 102, 20911-270 Rio de Janeiro, RJ, Brazil, Telephone: 55-21-2580-6598, E-mail: dvalle@ioc.fiocruz.br. Ima Aparecida Braga, Secretaria de Vigilância em Saúde, Ministério da Saúde, Esplanada dos Ministérios, Bloco G, Ed. Sede do Ministério da Saúde, 1° andar, 70058-900 Brasília, DF, Brazil.

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