VECTOR CONTROL, PEST MANAGEMENT, RESISTANCE, REPELLENTS
Resistance to Malathion and Deltamethrin in Aedes aegypti (Diptera: Culicidae) From Western Venezuela LESLIE C. ALVAREZ,1,2 GUSTAVO PONCE,1 MILAGROS OVIEDO,2 BEATRIZ LOPEZ,1 1,3 AND ADRIANA E. FLORES
KEY WORDS malathion, deltamethrin, resistance, Aedes aegypti, detoxiÞcation enzymes
The control of Aedes aegypti (L.), the principal vector of classic and hemorrhagic dengue in Venezuela and other countries of the world, has been the only option for the prevention and reduction of the transmission of the disease. The reduction of breeding areas and environmental sanitation programs, with the participation of the community, are strategies that have been implemented in recent years, but alone are not sufÞcient for the control of Ae. aegypti. The use of chemical insecticides inside and around homes continues to be the principal tool. Since 1970, in Venezuela and other countries in Latin America, the organophosphates temephos and malathion have been used in control programs against Ae. aegypti (Georghiou et al. 1987, Gratz, 1991), but they have had only temporary success owing to the lack of regularity and sustainability in the majority of cases. Additionally, the massive and extensive use of insecticides in public health as well as agriculture has exerted selective pressure, resulting in the development of resistance to different insecticides in Ae. aegypti and other culicids (WHO 1986). Resistance speciÞcally to malathion was recorded during the 1980s among the principal vector species of the 1 Universidad Autonoma de Nuevo Leon, Facultad de Ciencias Biologicas, Av. Universidad s/d Cd. Universitaria, San Nicolas de los Garza, Nuevo Leo´ n, 66451 Me´ xico. 2 Universidad de los Andes, Nucleo Universitario Rafael Rangel, Villa Universitaria Pampanito, estado Trujllo, 3102, Venezuela. 3 Corresponding author, e-mail: adriana.ß[email protected].
genera Anopheles (Hemingway and Ranson 2000), Culex (Hemingway and Karunaratne 1998), and Aedes (Georghiou et al. 1987, Rawlins 1998). Therefore, in the 1990s, pyrethroids were incorporated, to which species of these three genera rapidly developed resistance (Hemingway and Ranson 2000). Resistance to insecticides can be due mainly to an increase in metabolic activity and/or alterations in the target site. Three families of enzymes are mainly involved in metabolic resistance: carboxylesterases, cytochrome p450 monooxygenases, and glutathione-S-transferases (GST). Gene upregulation or ampliÞcation can be induced or selected in organisms by exposure to insecticides (Hemingway and Ranson 2000). These enzymes catalyze a large range of detoxiÞcation reactions, constituting the Þrst line of enzymatic defense against xenobiotics. They are responsible for removing many metabolic waste products, play essential roles in biosynthetic pathways, and are involved in chemical communication (Scott 1995). In particular, esterases are associated with resistance to organophosphates, carbamates, and to lesser extent pyrethroids. The monooxygenases are involved in the metabolism of pyrethroids and in the activation or detoxiÞcation of organophosphates and to lesser degree methyl-carbamates. Glutathione-S-tranferases play a principal role in the metabolism of DDT to less toxic products and a secondary role in resistance to organophosphates (Hemingway and Ranson 2000).
0022-2585/13/1031Ð1039$04.00/0 䉷 2013 Entomological Society of America
Downloaded from https://academic.oup.com/jme/article-abstract/50/5/1031/904290 by guest on 16 January 2019
J. Med. Entomol. 50(5): 1031Ð1039 (2013); DOI: http://dx.doi.org/10.1603/ME12254
ABSTRACT Resistance to the insecticides deltamethrin and malathion and the enzymes associated with metabolic resistance mechanisms were determined in four Þeld populations of Aedes aegypti (L.) from western Venezuela during 2008 and 2010 using the bottle assay and the microplate biochemical techniques. For deltamethrin, mortality rates after 1 h exposure and after a 24-h recovery period were determined to calculate the 50% knock-downconcentration (KC50) and the lethal concentration (LC50), respectively. For malathion, mortality was recorded at 24 h to determine the LC50. For deltamethrin, resistance ratios of knock-down resistance and postrecovery were determined by calculating the RRKC50 and RRLC50, comparing the KC50 and LC50 values of the Þeld populations and those of the susceptible New Orleans strain. Knock-down resistance to deltamethrin was moderate in the majority of the populations in 2008 (RRKC50 values were between 5- and 10-fold), and only one population showed high resistance in 2010 (RRKC50 ⬎10-fold). Moderate and high postrecovery resistance to deltamethrin was observed in the majority of the populations for 2008 and 2010, respectively. There was signiÞcantly increased expression of glutathione-S-tranferases and mixedfunction oxidases. All populations showed low resistance to malathion in 2008 and 2010 with significantly higher levels of ␣-esterases for 2008 and 2010 and -esterases for 2008.
1032
JOURNAL OF MEDICAL ENTOMOLOGY
Vol. 50, no. 5
There are reports of intensiÞed activity of the esterases and monooxygenases in resistant populations of Anopheles gambiae Giles, Anopheles stephensi Liston, Anopheles subpictus Grassi, Anopheles albimanus Wiedemann, Culex quinquefasciatus Say, and Aedes aegypti (L.) (Brogdon et al. 1999, Vulule et al. 1999, Rodriguez et al. 2001, Flores et al. 2005) caused mainly by gene ampliÞcation, over-regulated transcription, or altered gene expression. GSTs have been reported to be overexpressed in Ae. aegypti and An. gambiae resistant to DDT (Lumjuan et al. 2005). In Venezuela, studies on the monitoring of resistance to insecticides have revealed that Ae. aegypti has developed resistance to different insecticides. In 1958, Quaterman and Schoof (1958) reported resistance to DDT, and later Mouchet (1967) reported resistance to dieldrin/BHC, and Georghiou et al. (1987) documented resistance to organophosphates and carbamates. Mazzarri and Georghiou (1995) and Perez and Molina (2001) detected resistance to organophosphates, carbamates, and pyrethroids in the states of Aragua and Falcon, even though control programs were only recently implemented. Overexpressed levels of esterases determined by biochemical and synergistic assays have been reported in Ae. aegypti of various states in Venezuela, where this enzyme mechanism is responsible for resistance to temephos, chlorpyrifos, and methyl pirimiphos (Mazzarri and Georghiou 1995, Bisset et al. 2001). Additionally, Saavedra et al. (2007) reported the presence of the mutation Ile 1,016 associated with kdr pyrethroid resistance in populations of nine states in Venezuela, revealing the necessity of monitoring other populations in the country where reemergence of dengue has
been observed. The aim of the current study was to evaluate susceptibility to malathion and deltamethrin in populations of Ae. aegypti in western Venezuela in two periods, 2008 and 2010, as well as the role of detoxiÞcation enzymes as a mechanism of resistance. Materials and Methods Mosquitoes. Immature stages of Ae. aegypti were collected during 2008 and 2010 in four localities in western VenezuelaÑPAMPANITO (PTO) at 9⬚ 24⬘42⬙ S, 70⬚ 29⬘39⬙ W, TRES ESQUINAS (TE) situated at 9⬚ 25⬘48⬙ S, 70⬚ 26⬘51⬙ W in the state of Trujillo, LARA in the state of Lara at 10⬚ 03⬘51⬙ S, 69⬚ 29⬘20⬙ W, ˜ A in the state of Tachira at 7⬚ 54⬘57⬙ S, 64⬚ and UREN 24⬘20⬙ W (Fig. 1). The larvae were collected from natural breeding sites and transported to the Insectary Pablo Anduze of the Instituto Experimental J. W. Torrealba, Universidad de los Andes, Venezuela. Colonies were maintained at 25 ⫾ 4⬚C and a photoperiod of 12:12 (L:D) h. The females reared from these larvae were fed on chickens (Gallus gallus domesticus Brisson) for the production of eggs to obtain the parental generations, which were transported to the UANL, Facultad de Ciencias Biolo´ gicas, N.L. Mexico. The eggs were placed in plastic containers with dechlorinated water along with a 50% aqueous solution of powdered liver protein as food source for the subsequent larval stage. Pupae were placed in 250-ml ßasks in cages (30 by 30 cm) until the adults emerged. The male mosquitoes were fed a 10% sugar solution, and the females were fed on rats (Rattus norvegicus (Berkenhout)) for the production of eggs, for which
Downloaded from https://academic.oup.com/jme/article-abstract/50/5/1031/904290 by guest on 16 January 2019
Fig. 1. Collection sites of Ae. aegypti in western Venezuela.
September 2013
ALVAREZ ET AL.: INSECTICIDE RESISTANCE IN A.aegypti FROM VENEZUELA
Each mosquito was homogenized in 100 l of 0.01 M potassium phosphate buffer, pH 7.2, and suspended in up to 2 ml of the same buffer. Aliquots of 100 l were transferred to wells of microtiter plates. Thirty surviving specimens and 30 dead per population were analyzed in triplicate per plate. ␣- and -Esterases, mixed-function oxidases (MFO), GST, and insensitive acetylcholinesterase (iAChE) were quantiÞed according to Brogdon (1989) and CDC (2002) using the NO strain as reference. Absorbance was measured with an UVM-340 Microplate reader (ASYS Hitech GmbH, Eugendorf, Austria) and averaged. Protein concentration was determined according to Brogdon (1984), in case there was some variation in the size of mosquitoes and appropriate dilution of the homogenate was needed. The data for each biochemical assay were evaluated by analysis of variance (ANOVA) and TukeyÕs test was used at a level of signiÞcance of P ⬍ 0.05 to compare means between surviving and dead females in the populations studied with respect to the reference strain. The resistance threshold corresponding to the maximal enzyme activity in the surviving females of the NO strain was compared with the survivors of the four populations studied. We calculated the percentage of individuals exceeding the resistance threshold (CDC 2002) and classiÞed the enzyme mechanisms as nonaltered (NA), incipiently altered (IA), or altered (A), if ⬍15%, 15Ð50%, or ⬎50% of the specimens exceeded the threshold, respectively (Montella et al. 2007). Additionally, the LC50 values and mean enzyme levels of the surviving females exposed to deltamethrin were submitted to linear regression analysis. Correlation (r) was performed to determine the degree of association between the two variables. Finally, two criteria were considered to determine whether an enzyme mechanism was involved in the resistance observed for malathion and deltamethrin: 1) ⬎50% of surviving specimens of the population studied exceeded the resistance threshold; 2) the mean enzyme activity level was signiÞcantly higher in the surviving specimens than in the dead ones and in turn higher than those of the NO strain. A third criterion was considered for deltamethrin: a highly signiÞcant correlation between the LC50 values of deltamethrin and enzyme levels. Results Bioassays. Table 1 shows the results of the resistance to deltamethrin and malathion in Ae. aegypti of Venezuela during the years 2008 and 2010. Knock-down resistance values, KC50, for 2008 were between 0.079 and 0.115 g/bottle and for 2010 between 0.099 and 0.285 g/bottle. There were no changes for 2010 in the populations Pampanito and Tres Esquinas, showing knock-down resistance with RRKC50 ⬍5-fold, while there were differences for the populations Lara and Uren˜ a with increases in KC50 for 2010 with RRKC50 of 7.7- and 10.2-fold, respectively, indicating moderate and high knock-down resistance. With respect to postrecovery resistance, we observed that the LC50
Downloaded from https://academic.oup.com/jme/article-abstract/50/5/1031/904290 by guest on 16 January 2019
ßasks with water, lined inside with Þlter paper, were provided. These eggs corresponded to the F1 and F2 generations, which were used in the majority of the bioassays. All populations of Ae. aegypti were kept at a temperature of 25 ⫾ 2⬚C and relative humidity of 70% ⫾2. Females of the F1 generation, 1Ð3 d after emergence and without blood feeding, were used for the bioassays and biochemical tests. The New Orleans (NO) strain maintained for several years in the laboratory was used as the susceptible reference strain in all situations. Bioassays. The insecticides evaluated were malathion (98.4% purity) and deltamethrin (99.5%) (ChemService, West Chester, PA), which were resuspended in 1 ml of acetone to prepare stock solutions with Þnal concentrations of 500 and 100 g/ml, respectively. Different dilutions were then prepared to obtain concentrations that caused between 2 and 98% mortality. The bioassays were carried out according to the bottle method proposed by the Centers for Disease Control and Prevention (CDC 2002) in Atlanta, GA (Brogdon and McAllister 1998), in which 15Ð20 females were used per bottle. For deltamethrin, the exposure time was 1 h during which observations were made every 10 min to record the number of insects killed and to determine the knock-down effect. Later, the insects were transferred to insecticide-free ßasks, and mortality was recorded at 24 h. For the organophosphate malathion, the insects were exposed for 2 h and then transferred to insecticide-free ßasks; afterwards, the number of recovered mosquitoes was recorded at 1, 2, and 4 h, and the number of dead insects was recorded at 24 h. Each bioassay consisted of bottles containing Þve or six concentrations with four replicates for each insecticide and the control was treated only with acetone. This method was used with all Þeld populations and with the NO reference population. The results obtained were submitted to log-probit regression analysis (Finney 1971) using the Probit program (Raymond 1985) to determine the 50% knock-down dose (KC50) and 50% lethal concentration (LC50) for deltamethrin and LC50 for malathion. The conÞdence intervals were calculated using ␣ ⫽ 0.05, and the signiÞcant difference between the values was determined based on nonoverlapping conÞdence intervals. The mortalities were corrected according to the formula of Abbott (1925) when mortality was observed in the control group. The resistance ratios (RR) were calculated by dividing the LC50 of each population by the LC50 of the NO reference strain, as well as by dividing the KC50 obtained for deltamethrin by the KC50 of the NO strain. Enzymes. Batches of 100 females of each Þeld population and of the reference were exposed to bottles impregnated with the respective LC50 values of deltamethrin for a time period that caused 50% mortality. The dead and survivors were then separated and stored individually at ⫺70⬚C. The same procedure was carried out with malathion using the LC99 determined in the NO strain.
1033
1034
JOURNAL OF MEDICAL ENTOMOLOGY
Vol. 50, no. 5
Table 1. Dose–response values and resistance ratios to deltamethrin and malathion and knock-down doses and resistance ratios to deltamethrin in four strains of Ae. aegypti from Venezuela Insecticide
PTO
TE
2010 Lara
Uren˜ a
NO
PTO
TE
Lara
Uren˜ a 0.294 0.255Ð0.339 1.53 (0.18) 19.6 0.285 0.249Ð0.326 1.63 (0.18) 10.2
0.008 0.053 0.072 0.072 0.031 0.006Ð0.013 0.048Ð0.059 0.059Ð0.088 0.066Ð0.079 0.022Ð0.045 1.27 (0.14) 3.09 (0.39) 1.33 (0.19) 2.9 (0.45) 0.73 (0.13) 6.6 9 9 3.9 0.016 0.079 0.115 0.083 0.110 0.012Ð0.022 0.071Ð0.087 0.105Ð0.127 0.077Ð0.090 0.068Ð0.176 1.15 (0.12) 2.77 (0.38) 3.06 (0.34) 4.33 (0.61) 1.30 (0.35) 4 7.2 5.2 6.9
0.015 0.140 0.203 0.013Ð0.021 0.125Ð0.157 0.176Ð0.233 1.02 (0.13) 2.67 (0.31) 1.79 (0.23) 9.3 13.5 0.028 0.099 0.125 0.021Ð0.034 0.086Ð0.114 0.107Ð0.145 1.42 (0.17) 2.05 (0.25) 1.81 (0.19) 3.5 4.5
0.279 0.245Ð0.317 1.87 (0.24) 18.6 0.216 0.193Ð0.243 2.03 (0.25) 7.7
1.6 2.15 1.21Ð2.12 1.58Ð2.90 0.82 (0.10) 0.77 (0.1) 1.3
1.45 1.34Ð1.57 2.62 (0.26)
2.31 3.07 2.08Ð2.59 2.81Ð3.35 2.19 (0.21) 2.99 (0.36) 1.6 2.1
1.18 3.49 0.90Ð1.54 2.97Ð4.09 0.85 (0.12) 1.53 (0.18) 0.74 2.2
3.05 2.44Ð3.81 1.18 (0.17) 1.9
3.00 2.75 2.79Ð3.24 2.47Ð3.06 3.27 (0.34) 2.85 (0.29) 2.1 1.9
g/bottle. 95% CIs. Slope of regression line Probit-log, standard deviations (⫾SD) are in parentheses. d RRLC50, resistance ratio: LC50 Þeld strain/ LC50 NO strain. e RRKC50, resistance ratio: KC50 Þeld strain/KC50 NO strain. a
b c
values for deltamethrin in the populations evaluated were between 0.031 and 0.072 g/bottle for 2008. Compared with the NO strain, the Pampanito, Tres Esquinas, and Lara populations showed moderate resistance to the pyrethroid with RRLC50 of 6.6-, 9.0-, and 9.0-fold, respectively, while the Uren˜ a population showed low resistance (3.9-fold). For 2010, the LC50 values increased (P ⬍ 0.05) in all populations, showing moderate resistance to deltamethrin for the Pampanito population (9.3-fold) and high resistance (RR LC50 ⬎10-fold) for the Tres Esquinas, Lara, and Uren˜ a populations. The KC50 values for the populations of Pampanito, Tres Esquinas, and Uren˜ a in 2008were higher than the LC50 values, owing to the low recovery percentages after 1 h exposure (0, 0.56, and 0%, respectively), indicating a greater effectiveness of deltamethrin at 24 h. In contrast for 2010, the LC50 values were higher than the KC50 values in the Pampanito, Tres Esquinas, and Lara populations, whose recovery percentages were 9.72, 12.8, and 9.34%, respectively, necessitating a greater amount of deltamethrin to kill the knocked down insects; in the Uren˜ a population, no difference was found between the two values. For the insecticide malathion, the LC50 values varied between 1.18 and 3.49 g/bottle in 2008 and between 2.31 and 3.07 g/bottle in 2010, indicating an increase in the Tres Esquinas population and decrease in Lara for the year 2010. In evaluating the RRLC50 values, we found that all populations studied showed low resistance to the organophosphate in both study periods, with values of RRLC50 less than Þvefold. Enzymes. Of the four enzyme systems evaluated after exposure of the populations to deltamethrin during 2008, we found overexpressed levels of GST in surviving females of the Lara population, with 86.7% of these exceeding the resistance threshold, thus being categorized as an altered enzyme mechanism (A) according to Montella et al. (2007). For 2010, overex-
pression of MFO was recorded in the survivors of the Tres Esquinas population, with 23.3% of the specimens exceeding the resistance threshold (incipiently altered mechanism). In the Pampanito and Tres Esquinas populations, levels of GST were overexpressed, with 100% of survivors exceeding the threshold (altered mechanism) (Tables 2; 3). In examining the association between the LC50 values of deltamethrin and overexpressed enzyme levels for surviving females exposed to LC50 of deltamethirn, there were no signiÞcant correlation values, thereby not fulÞlling in any of the cases the three criteria proposed to associate an enzyme mechanism with resistance to insecticides (Fig. 2). Thus, there was no association of the over-expression of MFO or GST with the resistance observed, suggesting the participation of these enzymes in other functions other than in the metabolism of deltamethrin. Table 2 shows the absorbance means for each enzyme in the populations exposed to malathion during 2008 and 2010. ␣-Esterases were overexpressed during 2008 in the Pampanito, Tres Esquinas, and Lara populations, with 100% of the surviving specimens exceeding the resistance threshold, thus being categorized as an altered mechanism. A similar behavior was observed with -esterases in the Pampanito and Lara populations. MFO appeared to be an incipiently altered mechanism in the Uren˜ a population (33.3%) and GST as an altered mechanism in the Pampanito population (93.3%). For 2010, we found an altered enzyme mechanism for ␣-esterases in the Tres Esquinas, Lara, and Uren˜ a populations with ⬎80% of surviving specimens exceeding the resistance threshold, and the overexpression of MFO in the Uren˜ a population (6.7%) as well (Table 3). Discussion Approximately four decades since their introduction, pyrethroids continue to be one of the principal
Downloaded from https://academic.oup.com/jme/article-abstract/50/5/1031/904290 by guest on 16 January 2019
Deltamethrin LC50a CIb bc (⫾SD) RRLC50d KC50 CI b (⫾SD) RRKC50e Malathion LC50 CI b (⫾SD) RRLC50
2008 NO
1.198 (0.025) 1.196 (0.034)
1.268 (0.091)* 1.172 (0.023)
1.225 (0.022) 1.238 (0.036)
1.246 (0.067)* 1.115 (0.058)
1.314 (0.038)* 0.886 (0.048)
1.178 (0.069) 1.189 (0.064)
0.955 (0.091) 0.941 (0.105)
0.778 (0.098) 0.794 (0.135)
1.195 (0.025)* 1.244 (0.027)
1.029 (0.082) 1.132 (0.119)
0.743 (0.056) 0.821 (0.110)
1.103 (0.061)* 0.969 (0.100)
0.814 (0.101) 0.736 (0.055)
0.806 (0.046) 0.683 (0.057)
0.847 (0.054) 0.737 (0.049)
0.895 (0.092) 0.763 (0.096)
0.661 (0.025) 0.779 (0.035)
0.744 (0.033) 0.780 (0.084)
0.948 (0.090) 0.987 (0.092)
-Esterases
0.766 (0.086) 0.775 (0.054)
␣-Esterases
0.451 (0.09)* 0.367 (0.088)
0.315 (0.038) 0.313 (0.061)
0.230 (0.045) 0.320 (0.076)
0.385 (0.042) 0.379 (0.081)
0.308 (0.069) 0.300 (0.072)
0.325 (0.082) 0.277 (0.091)
0.285 (0.052) 0.273 (0.054)
0.191 (0.045) 0.205 (0.040)
0.242 (0.046) 0.226 (0.047)
0.433 (0.084) 0.334 (0.059)
MFO
2008
0.088 (0.049) 0.101 (0.055)
0.106 (0.010) 0.089 (0.031)
0.071 (0.038) 0.102 (0.031)
0.140 (0.025)* 0.101 (0.017)
0.056 (0.018) 0.052 (0.013)
0.008 (0.021) 0.050 (0.036)
0.098 (0.021)* 0.0005 (0.038)
0.064 (0.014) 0.062 (0.015)
0.095 (0.022) 0.092 (0.021)
0.051 (0.009) 0.020 (0.051)
GST
0.017 (0.010) ⫺0.042 (0.063)
0.012 (0.012) 0.014 (0.009)
0.564 (0.047)* 0.510 (0.047)
0.562 (0.044)* 0.518 (0.036)
0.578 (0.088)* 0.486 (0.029)
0.489 (0.035) 0.596 (0.074)
⫺0.016 (0.038) ⫺0.018 (0.066) 0.003 (0.012) 0.003 (0.026)
0.408 (0.043) 0.348 (0.028)
0.555 (0.045) 0.769 (0.064)
0.636 (0.087) 0.657 (0.055)
0.010 (0.005) ⫺0.013 (0.030)
0.001 (0.006) ⫺0.043 (0.069)
0.012 (0.006) ⫺0.001 (0.006)
0.755 (0.101) 0.917 (0.106)
0.693 (0.114) 0.720 (0.060)
⫺0.104 (0.037) 0.018 (0.016) 0.011 (0.008) 0.004 (0.002)
0.674 (0.502) 0.809 (0.045)
␣-Esterases
⫺0.036 (0.043) 0.004 (0.002)
iAChE
0.766 (0.095) 0.699 (0.076)
0.644 (0.076) 0.605 (0.087)
0.615 (0.097) 0.662 (0.114)
0.807 (0.073) 0.869 (0.092)
0.705 (0.074) 0.713 (0.08)
0.939 (0.051) 0.905 (0.702)
0.925 (0.169) 0.911 (0.066)
0.858 (0.132) 0.924 (0.111)
0.791 (0.139) 0.802 (0.099)
1.100 (0.984) 0.981 (0.118)
-Esterases
2010
0.246 (0.06)* 0.173 (0.041)
0.165 (0.037) 0.143 (0.031)
0.209 (0.062) 0.170 (0.059)
0.204 (0.308) 0.146 (0.022)
0.179 (0.072) 0.122 (0.034)
0.123 (0.025) 0.134 (0.095)
0.175 (0.062) 0.289 (0.088)
0.325 (0.095)* 0.253 (0.104)
0.209 (0.090) 0.189 (0.076)
0.192 (0.053) 0.233 (0.063)
MFO
⫺0.116 (0.078) 0.011 (0.008) 0.009 (0.009) ⫺0.018 (0.044) 0.006 (0.022) ⫺0.030 (0.066) 0.036 (0.054) ⫺0.106 (0.071) 0.010 (0.012) ⫺0.170 (0.057)
0.011 (0.006) 0.006 (0.006) 0.084 (0.002) 0.008 (0.009) 0.0005 (0.020) 0.0009 (0.035) 0.0032 (0.007) 0.0089 (0.010) 0.0009 (0.0087) 0.0270 (0.0071)
0.378 (0.087)* ⫺0.014 (0.036) 0.631 (0.077)* 0.039 (0.014) 0.023 (0.021) 0.019 (0.197) 0.030 (0.007) 0.024 (0.013)
0.048 (0.018) ⫺0.047 (0.067) 0.024 (0.072) 0.112 (0.020) ⫺0.068 (0.097) 0.061 (0.010) 0.084 (0.026) 0.056 (0.013) 0.057 (0.042) 0.058 (0.018)
iAChE
⫺0.039 (0.061) ⫺0.004 (0.038)
GST
Mean absorbance (ⴞSD) in each biochemical test of four strains of Ae. aegypti from Venezuela exposed to LC50 of deltamethrin and LC99 of malathion
ALVAREZ ET AL.: INSECTICIDE RESISTANCE IN A.aegypti FROM VENEZUELA
Downloaded from https://academic.oup.com/jme/article-abstract/50/5/1031/904290 by guest on 16 January 2019
A, alive after exposure to LC50 of deltamethrin or LD99 of malathion; D, dead after exposure to LC50 of deltamethrin or LD99 of malathion. * Mean values higher signiÞcantly (P ⬍ 0.05) in alive specimen compared with dead and with NO strain.
NO Alive Dead PTO Alive Dead TE Alive Dead Lara Alive Dead Uren˜ a Alive Dead
Deltamethrin NO Alive Dead PTO Alive Dead TE Alive Dead Lara Alive Dead Uren˜ a Alive Dead Malathion
Strain
Table 2.
September 2013 1035
1036
JOURNAL OF MEDICAL ENTOMOLOGY
Vol. 50, no. 5
Table 3. Percentage of each Ae. aegypti pop from four sites in western Venezuela exposed to two pesticides with enzyme activity higher than the threshold established by the NO strain Strain/year
-Esterases
MFO
GST
iAchE
2010
2008
2010
2008
2010
2008
2010
2008
2010
0.797a 100 100 100 100 0.987a 0 0 0 0
0.494a 43.3 80 96.7 93.3 0.494a 3.3 0 0 13.3
0.966a 100 100 100 100 1.079a 0 23.3 0 86.7
0.843a 36.7 0 0 20 0.843a 0 0 0 0
0.491a 0 0 0 33.3 0.618a 0 0 0 3.3
0.353a 0 3.3 0 6.7 0.353a 3.3 23.3 0 0
0.118a 93.3 13.3 13.3 26.7 0.075a 86.6 13.3 86.7 86.7
0.125a 0 3.3 3.3 0 0.125a 100 100 0 0
0.023a 13.3 3.3 13.3 10 0.019a 0 20 6.3 3.3
0.026a 6.6 3.3 0 0 0.026a 0 0 50 3.3
a
Maximum absorbance value for NO strain. Cell Shading: unaltered enzyme (no shading), incipiently altered (light gray), and altered (dark gray).
groups of insecticides applied for the control of wide variety of agricultural pests and disease vectors (Davies and Williamson 2009). Resistance to pyrethroids and especially deltamethrin has been reported in different populations of Ae. aegypti in Venezuela (Mazzarri and Georghiou 1995, Perez and Molina 2001, Rodrõ´guez et al. 2007). In the current study, the evolution of resistance to deltamethrin was demonstrated in the populations evaluated during 2008 and 2010, probably owing to the presence of intrinsic genetic characteristics in the species that favor the expression of resistance genes, as well as the selective pressure exerted by the constant use of chemical insecticides for the control of dengue and agricultural pests. The principal economic activity in Uren˜ a is agriculture, and we found that the LC50 of the Ae. aegypti population increased nine times in 2 yr with a RRLC50 of 19.6 in 2010. In addition, there are reports of Ae. aegpyti resistant to pyrethroids owing to cross-resistance to DDT (Hemingway et al. 1989, Brengues et al. 2003). Both compounds share the same target site, the voltage-gated sodium channel, which could be an additional factor present in our populations owing to the selective pressure exerted with the application of DDT for several decades in Venezuela for the control of malaria and other diseases. Rodrõ´guez et al. (2007) associated resistance to deltamethrin in larvae of Ae. aegypti of Venezuela and other Latin American countries with the activity of metabolic enzymes, esterases, and GSTs, as well as cytochrome oxidases (Berge´ et al. 1998). However, we did not Þnd enzyme mechanisms associated with resistance to deltamethrin, suggesting the presence of a nonenzymatic mechanism, such as point mutations in the gene coding for the voltage-gated sodium channel, which have been previously reported in this species (Brengues et al. 2003, Saavedra et al. 2007, Yanola et al. 2011). This may be especially important with the Ile1,016 resistance allele in which Saavedra et al. (2007) observed that 90.6% of Ile1,016/Val1,016 heterozygotes were knocked down after a 1-h exposure to permethrin but that 57.4% of knocked down heterozygotes eventually recovered. Thus, the rate of recovery after knockdown is inherited as an additive genetic trait with
heterozygotes exhibiting an intermediate phenotype. It is important to note that the states of Lara and Tachira (Uren˜ a) are two of the seven Venezuelan states that accounted for the highest number of cases of dengue during 2008 and 2010. Despite efforts by the government agencies to try to control Ae. aegypti in these and other federal territories, the failure of control programs is evident, reßected in the case studies of dengue, which increased from 48,048 in 2008 to 106,725 cases in epidemiological week 43 of 2010 (Ministerio del Poder Popular para Salud 2010). This was possible owing to the resistance of Ae. aegypti to deltamethrin. The Þght against dengue in Venezuela involves the fumigation of critical areas to destroy the adult population of Ae. aegypti and in educational campaigns to make the public aware of the problem and to eliminate foci of contaminated water in which this insect deposits its eggs. Thus, the results obtained in this study contribute important information that must be considered in continuing or halting the use of deltamethrin in control programs. OPS (1997) reported that resistance to malathion in Ae. aegypti has spread throughout the Caribbean, Central America, and South America, but there are reports of populations susceptible to organophosphates in Panama, Cuba (Bisset et al. 2003, 2004), and Vietnam (Houng et al. 2004). In the current study, we found low values of organophosphate resistance (RR ⬍5) in both evaluation periods in agreement with the Þndings of Mazzarri and Georghiou (1997) in populations of Ae. aegypti in the states of Aragua and Falcon, Venezuela, but at odds with Perez and Molina (2009), who found high resistance to malathion in larvae of Ae. aegypti in the municipalities of Girardot, Mario Bricen˜ o Iragorri, and Urdaneta in the state of Aragua, Venezuela (RR50 69.5, 150.6, and 113.5, respectively), with overexpression of esterases and cytochrome P450 oxidases. This suggests that resistance is focal. The populations evaluated during both periods exhibited overexpression of ␣and -esterases and cytochrome oxidases whose participation in the metabolism of malathion in
Downloaded from https://academic.oup.com/jme/article-abstract/50/5/1031/904290 by guest on 16 January 2019
Malathion PTO TE Lara Uren˜ a Deltamethrin PTO TE Lara Uren˜ a
␣-Esterases 2008
September 2013
ALVAREZ ET AL.: INSECTICIDE RESISTANCE IN A.aegypti FROM VENEZUELA
1037
these populations was not determined. This is despite reports associating these enzyme mechanisms with resistance to malathion in Ae. aegypti populations in Venezuela and Latin America (Rodrõ´guez et al. 2007, Perez and Molina 2009). Finally, the current study showed that resistance is dynamic in time and space. There is a need to carry out periodic evaluations that include suscep-
tibility bioassays and to determine the mechanisms associated with resistance to insecticides to obtain complete information about the susceptibility of the target population to the insecticide. There is also a need to design strategies for the management of resistance and to identify the opportune moment to rotate insecticides to maintain the susceptibility of the population in question.
Downloaded from https://academic.oup.com/jme/article-abstract/50/5/1031/904290 by guest on 16 January 2019
Fig. 2. Association of LC50 values of deltamethrin (lines) and four enzyme levels (histograms: mean absorbance values) for surviving Ae. aegypti from four sites in western Venezuela and a susceptible population (NO) in 2008 and 2010. SigniÞcance values for r are in parentheses.
1038
JOURNAL OF MEDICAL ENTOMOLOGY Acknowledgments
Collections of mosquitoes were obtained by the support of the project “Mision Ciencia: Subproyecto Dengue” from FONACYT Venezuela. The study was funded by CONACYT Ciencia Basica and UANL PAICYT, Mexico.
References Cited
tance in Aedes aegypti in the Caribbean area and neighbouring countries. J. Med. Entomol. 24: 290 Ð294. Gratz, N. G. 1991. Emergency control of Aedes aegypti as a disease vector in urban areas. J. Am. Mosq. Control Assoc. 7: 353Ð365. Hemingway, J., R. G. Boddington, and J. Harris. 1989. Mechanisms of insecticide resistance in Aedes aegypti (L.) (Dõ´ptera: Culicidae) from Puerto Rico. Bull. Entomol. Res. 79: 123Ð30. Hemingway, J., and S.H.P.P. Karunaratne. 1998. Mosquito carboxylesterases: a review of the molecular biology and biochemistry of a major insecticide resistance mechanism. Med. Vet. Entomol. 12: 1Ð12. Hemingway, J., and H. Ranson. 2000. Insecticide resistance in insect vectors of human disease. Annu. Rev. Entomol. 45: 371Ð391. Houng, V. D., N.N.T. Bach, D. T. Hien, and L.N.T. Bich. 2004. Susceptibility of Aedes aegypti to insecticides in Vietnam. Dengue Bull. 28: 179 Ð183. Lumjuan, N., L. McCarroll, L. Prapanthadara, J. Hemingway, and H. Ranson. 2005. Elevated activity of an Epsilon class glutathione transferase confers DDT resistance in the dengue vector, Aedes aegypti. Insect. Biochem. Mol. Biol. 35: 861Ð971. Mazzarri, M. B., and G. P. Georghiou. 1995. Characterization of resistance to organophosphate, carbamate, and pyrethroid insecticides in Þeld populations of Aedes aegypti (L) from Venezuela. J. Am. Mosq. Control Assoc. 11: 315Ð322. Ministerio del Poder Popular para la Salud. 2010. Boletõ´n Epidemiolo´ gico No. 43 Gobierno Bolivariano de Venezuela. Ministerio del Poder Popular para la Salud, Venezuela. Montella, I., A. Martins, P. Fernandez, J. Pereira, I. Braga, and D. Valle. 2007. Insecticide resistance mechanisms of Brazilian Aedes aegypti populations from 2001 to 2004. Am. J. Trop. Med. Hyg. 77: 467Ð 477. Mouchet, J. 1967. La resistance aux insecticides chez Aedes aegypti et les espe` ces voisines. Bull. W.H.O. 36: 569 Ð577. (OPS) Organizacion Panamericana de Salud. 1997. Informe sobre el control de Aedes aegypti. Organizacion Panamericana de Salud, Washington, DC. Documento CD. 40. Perez, E. E., and D. Molina. 2001. Resistance of Aedes aegypti to pyretroides in municipalities of Aragua state, Venezuela. J. Am. Mosq. Control Assoc. 17: 166 Ð180. Perez, E. E., and D. Molina. 2009. Resistencia focal a insecticidas organosinteticos en Aedes aegypti (Linneaus, 1762) (Dõ´ptera: Culicidae) de diferentes municipios del estado Aragua, Venezuela. Bol. Mal. Salud Amb. XLIX: 143Ð150. Quaterman, K. D., and H. F. Schoof. 1958. The status of insecticide resistance in arthropods of public health importance in 1956. Am. J. Trop. Med. Hyg. 7: 74 Ð 83. Rawlins, S. C. 1998. Spatial distribution of insecticide resistance in Caribbean populations of Aedes aegypti and its signiÞcance. Rev. Panam. Salud Publica 4: 243Ð51. Raymond, M. 1985. Presentation dÕune programme dÕanalyse logprobit pour Microordinateur cahiers Orstrom. Se´ r Ent Me´ d Parasitol. 23: 117Ð21. Rodrı´guez, M. M., J. A. Bisset, and D. Ferna´ ndez. 2007. Levels of insecticide resistance and resistance mechanisms in Aedes aegypti from some Latin American countries. J. Am. Mosq. Control Assoc. 23: 420 Ð 429. Rodriguez, M. M., J. A. Bisset, D. Molina, C. Diaz, and L. A. Soca. 2001. Adaptacion de los metodos en placas de microtitulacion para la cuantiÞcacio´ n de la actividad de
Downloaded from https://academic.oup.com/jme/article-abstract/50/5/1031/904290 by guest on 16 January 2019
Abbott, W. S. 1925. A method of computing the effectiveness of an insecticide. J. Econ. Entomol. 8: 265Ð267. Berge´, J. B., R. Feyereisen, and M. Amichot. 1998. Cytochrome p450 monooxygenases and insecticide resistance in insects. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 353: 1701Ð1705. Bisset, J. A., M. M. Rodrı´guez, and L. Caceres. 2003. Niveles de resistencia a insecticidas y sus mecanismos en 2 cepas de Aedes aegypti de Panama. Rev. Cubana Med. Trop. 55: 191Ð195. Bisset, J. A., M. M. Rodrı´guez, D. Fernandez, and O. Perez. 2004. Estado de la resistencia a insecticidas y mecanismos de resistencia en larvas del municipio Playa, colectadas durante la etapa intensiva contra el Aedes aegypti en Ciudad de La Habana, 2001Ð2002. Rev. Cubana Med. Trop. 56: 61Ð 66. Bisset, J. A., M. M. Rodrı´guez, D. Molina, C. Dı´az, and L. A. Soca. 2001. Esterasas elevadas como mecanismo de resistencia a insecticidas organofosforados en cepas de Aedes aegpyti. Rev. Cubana Med. Trop. 5: 37Ð 43. Brengues, C., N. J. Hawkes, F. Chandre, L. McCarroll, S. Duchon, P. Guillet, S. Manguin, J. C. Morgan, and J. Hemingway. 2003. Pyrethroid and DDT cross-resistance in Aedes aegypti is correlated with novel mutations in the voltage-gated sodium channel gene. Med. Vet. Entomol. 17: 87Ð94. Brogdon, W. G. 1984. Mosquito protein microassay.I. Protein determinations from small portions of single- mosquito homogenates. Comp. Biochem. Physiol. B. 79: 457Ð 459. Brogdon, W. G. 1989. Biochemical resistance detection: an alternative to bioassay. Parasitol. Today 5: 56 Ð 60. Brogdon, W. G., and J. C. McAllister. 1998. SimpliÞcation of adult mosquito bioassays through use of time-mortality determinations in bottles. J. Am. Mosq. Control Assoc. 14: 159 Ð164. Brogdon, W. G., J. C. McAllister, A. M. Corwin, and C. Cordon-Rosales. 1999. Oxidase-based DDT-pyrethroid cross-resistance in Guatemalan Anopheles albimanus. Pest. Biochem. Physiol. 64: 101Ð111. (CDC) Centers for Disease Control and Prevention. 2002. Evaluating mosquitoes for insecticide resistance: a webbased instruction. (www.cdc.gov/ncidod/wbt/resistance/ assay/microplate/index.htm) Davies, T.G.E., and M. S. Williamson. 2009. Interactions of pyrethroids with the voltage-gated sodium channel. Bayer CropSci. J. 2n: 159 Ð178. Finney, D. J. 1971. Probit analysis, 3rd ed. Cambridge University Press, Cambridge, UK. Flores, A. E., W. Albeldan˜ o–Vazquez, I. Fernandez–Salas, M. H. Badii, H. Loaiza–Becerra, G. Ponce–Garcia, S. Lozano–Fuentes, W. G. Brogdon, W. C. Black, IV, and B. Beaty. 2005. Elevated ␣-esterase levels associated with permethrin tolerance in Aedes aegypti (L.) from Baja California, Mexico. Pest. Biochem. Physiol. 82: 66 Ð78. Georghiou, G. P., M. C. Wirth, H. Tran, F. Saume, and A. B. Knudsen. 1987. Potential for organophosphate resis-
Vol. 50, no. 5
September 2013
ALVAREZ ET AL.: INSECTICIDE RESISTANCE IN A.aegypti FROM VENEZUELA
Kenyan villages using permethrin impregnated nets. Med. Vet. Entomol. 13: 239 Ð244. (WHO) World Health Organization. 1986. Resistance of vectors and reservoirs of disease of pesticides. W.H.O. Tech. Rep. Ser. 737: 1Ð 87. Yanola, J., P. Somboon, C. Walton, W. Nachaiwieng, P. Somwang, and L. Prapanthadara. 2011. High-throughput assays for detection of the F1534C mutation in the voltage-gated sodium channel gene in permethrin-resistant Aedes aegypti and the distribution of this mutation throughout Thailand. Trop. Med. Int. Health 16: 501Ð509. Received 7 November 2012; accepted 6 April 2013.
Downloaded from https://academic.oup.com/jme/article-abstract/50/5/1031/904290 by guest on 16 January 2019
esterasas y glutation-s-tranferasa en Aedes aegypti. Rev. Cubana Med. Trop. 53: 32Ð36. Saavedra, K., L. Urdaneta, S. Rajatileka, M. Moulton, A. E. Flores, I. Fernandez, J. Bisset, M. Rodriguez, P. J. McCall, M. J. Donnelly et al. 2007. Mutations in the voltage gated sodium channel gene associated with pyrethroid resistance in Latin American Aedes aegypti. Insect Mol. Biol. 16: 785Ð798. Scott, J. A. 1995. The molecular genetics of resistance: resistance as a response to stress. Fla. Entomol. 78: 399 Ð 414. Vulule, J. M., R. F. Beach, F. K. Atieli, J. C. McAllister, W. G. Brogdon, J. M. Roberts, R. W. Mwangi, and W. A. Hawley. 1999. Elevated oxidase and esterase levels associated with permethrin tolerance in Anopheles gambiae from
1039
Resistance to Malathion and Deltamethrin in Aedes aegypti (Diptera: Culicidae) From Western Venezuela LESLIE C. ALVAREZ,1,2 GUSTAVO PONCE,1 MILAGROS OVIEDO,2 BEATRIZ LOPEZ,1 1,3 AND ADRIANA E. FLORES
KEY WORDS malathion, deltamethrin, resistance, Aedes aegypti, detoxiÞcation enzymes
The control of Aedes aegypti (L.), the principal vector of classic and hemorrhagic dengue in Venezuela and other countries of the world, has been the only option for the prevention and reduction of the transmission of the disease. The reduction of breeding areas and environmental sanitation programs, with the participation of the community, are strategies that have been implemented in recent years, but alone are not sufÞcient for the control of Ae. aegypti. The use of chemical insecticides inside and around homes continues to be the principal tool. Since 1970, in Venezuela and other countries in Latin America, the organophosphates temephos and malathion have been used in control programs against Ae. aegypti (Georghiou et al. 1987, Gratz, 1991), but they have had only temporary success owing to the lack of regularity and sustainability in the majority of cases. Additionally, the massive and extensive use of insecticides in public health as well as agriculture has exerted selective pressure, resulting in the development of resistance to different insecticides in Ae. aegypti and other culicids (WHO 1986). Resistance speciÞcally to malathion was recorded during the 1980s among the principal vector species of the 1 Universidad Autonoma de Nuevo Leon, Facultad de Ciencias Biologicas, Av. Universidad s/d Cd. Universitaria, San Nicolas de los Garza, Nuevo Leo´ n, 66451 Me´ xico. 2 Universidad de los Andes, Nucleo Universitario Rafael Rangel, Villa Universitaria Pampanito, estado Trujllo, 3102, Venezuela. 3 Corresponding author, e-mail: adriana.ß[email protected].
genera Anopheles (Hemingway and Ranson 2000), Culex (Hemingway and Karunaratne 1998), and Aedes (Georghiou et al. 1987, Rawlins 1998). Therefore, in the 1990s, pyrethroids were incorporated, to which species of these three genera rapidly developed resistance (Hemingway and Ranson 2000). Resistance to insecticides can be due mainly to an increase in metabolic activity and/or alterations in the target site. Three families of enzymes are mainly involved in metabolic resistance: carboxylesterases, cytochrome p450 monooxygenases, and glutathione-S-transferases (GST). Gene upregulation or ampliÞcation can be induced or selected in organisms by exposure to insecticides (Hemingway and Ranson 2000). These enzymes catalyze a large range of detoxiÞcation reactions, constituting the Þrst line of enzymatic defense against xenobiotics. They are responsible for removing many metabolic waste products, play essential roles in biosynthetic pathways, and are involved in chemical communication (Scott 1995). In particular, esterases are associated with resistance to organophosphates, carbamates, and to lesser extent pyrethroids. The monooxygenases are involved in the metabolism of pyrethroids and in the activation or detoxiÞcation of organophosphates and to lesser degree methyl-carbamates. Glutathione-S-tranferases play a principal role in the metabolism of DDT to less toxic products and a secondary role in resistance to organophosphates (Hemingway and Ranson 2000).
0022-2585/13/1031Ð1039$04.00/0 䉷 2013 Entomological Society of America
Downloaded from https://academic.oup.com/jme/article-abstract/50/5/1031/904290 by guest on 16 January 2019
J. Med. Entomol. 50(5): 1031Ð1039 (2013); DOI: http://dx.doi.org/10.1603/ME12254
ABSTRACT Resistance to the insecticides deltamethrin and malathion and the enzymes associated with metabolic resistance mechanisms were determined in four Þeld populations of Aedes aegypti (L.) from western Venezuela during 2008 and 2010 using the bottle assay and the microplate biochemical techniques. For deltamethrin, mortality rates after 1 h exposure and after a 24-h recovery period were determined to calculate the 50% knock-downconcentration (KC50) and the lethal concentration (LC50), respectively. For malathion, mortality was recorded at 24 h to determine the LC50. For deltamethrin, resistance ratios of knock-down resistance and postrecovery were determined by calculating the RRKC50 and RRLC50, comparing the KC50 and LC50 values of the Þeld populations and those of the susceptible New Orleans strain. Knock-down resistance to deltamethrin was moderate in the majority of the populations in 2008 (RRKC50 values were between 5- and 10-fold), and only one population showed high resistance in 2010 (RRKC50 ⬎10-fold). Moderate and high postrecovery resistance to deltamethrin was observed in the majority of the populations for 2008 and 2010, respectively. There was signiÞcantly increased expression of glutathione-S-tranferases and mixedfunction oxidases. All populations showed low resistance to malathion in 2008 and 2010 with significantly higher levels of ␣-esterases for 2008 and 2010 and -esterases for 2008.
1032
JOURNAL OF MEDICAL ENTOMOLOGY
Vol. 50, no. 5
There are reports of intensiÞed activity of the esterases and monooxygenases in resistant populations of Anopheles gambiae Giles, Anopheles stephensi Liston, Anopheles subpictus Grassi, Anopheles albimanus Wiedemann, Culex quinquefasciatus Say, and Aedes aegypti (L.) (Brogdon et al. 1999, Vulule et al. 1999, Rodriguez et al. 2001, Flores et al. 2005) caused mainly by gene ampliÞcation, over-regulated transcription, or altered gene expression. GSTs have been reported to be overexpressed in Ae. aegypti and An. gambiae resistant to DDT (Lumjuan et al. 2005). In Venezuela, studies on the monitoring of resistance to insecticides have revealed that Ae. aegypti has developed resistance to different insecticides. In 1958, Quaterman and Schoof (1958) reported resistance to DDT, and later Mouchet (1967) reported resistance to dieldrin/BHC, and Georghiou et al. (1987) documented resistance to organophosphates and carbamates. Mazzarri and Georghiou (1995) and Perez and Molina (2001) detected resistance to organophosphates, carbamates, and pyrethroids in the states of Aragua and Falcon, even though control programs were only recently implemented. Overexpressed levels of esterases determined by biochemical and synergistic assays have been reported in Ae. aegypti of various states in Venezuela, where this enzyme mechanism is responsible for resistance to temephos, chlorpyrifos, and methyl pirimiphos (Mazzarri and Georghiou 1995, Bisset et al. 2001). Additionally, Saavedra et al. (2007) reported the presence of the mutation Ile 1,016 associated with kdr pyrethroid resistance in populations of nine states in Venezuela, revealing the necessity of monitoring other populations in the country where reemergence of dengue has
been observed. The aim of the current study was to evaluate susceptibility to malathion and deltamethrin in populations of Ae. aegypti in western Venezuela in two periods, 2008 and 2010, as well as the role of detoxiÞcation enzymes as a mechanism of resistance. Materials and Methods Mosquitoes. Immature stages of Ae. aegypti were collected during 2008 and 2010 in four localities in western VenezuelaÑPAMPANITO (PTO) at 9⬚ 24⬘42⬙ S, 70⬚ 29⬘39⬙ W, TRES ESQUINAS (TE) situated at 9⬚ 25⬘48⬙ S, 70⬚ 26⬘51⬙ W in the state of Trujillo, LARA in the state of Lara at 10⬚ 03⬘51⬙ S, 69⬚ 29⬘20⬙ W, ˜ A in the state of Tachira at 7⬚ 54⬘57⬙ S, 64⬚ and UREN 24⬘20⬙ W (Fig. 1). The larvae were collected from natural breeding sites and transported to the Insectary Pablo Anduze of the Instituto Experimental J. W. Torrealba, Universidad de los Andes, Venezuela. Colonies were maintained at 25 ⫾ 4⬚C and a photoperiod of 12:12 (L:D) h. The females reared from these larvae were fed on chickens (Gallus gallus domesticus Brisson) for the production of eggs to obtain the parental generations, which were transported to the UANL, Facultad de Ciencias Biolo´ gicas, N.L. Mexico. The eggs were placed in plastic containers with dechlorinated water along with a 50% aqueous solution of powdered liver protein as food source for the subsequent larval stage. Pupae were placed in 250-ml ßasks in cages (30 by 30 cm) until the adults emerged. The male mosquitoes were fed a 10% sugar solution, and the females were fed on rats (Rattus norvegicus (Berkenhout)) for the production of eggs, for which
Downloaded from https://academic.oup.com/jme/article-abstract/50/5/1031/904290 by guest on 16 January 2019
Fig. 1. Collection sites of Ae. aegypti in western Venezuela.
September 2013
ALVAREZ ET AL.: INSECTICIDE RESISTANCE IN A.aegypti FROM VENEZUELA
Each mosquito was homogenized in 100 l of 0.01 M potassium phosphate buffer, pH 7.2, and suspended in up to 2 ml of the same buffer. Aliquots of 100 l were transferred to wells of microtiter plates. Thirty surviving specimens and 30 dead per population were analyzed in triplicate per plate. ␣- and -Esterases, mixed-function oxidases (MFO), GST, and insensitive acetylcholinesterase (iAChE) were quantiÞed according to Brogdon (1989) and CDC (2002) using the NO strain as reference. Absorbance was measured with an UVM-340 Microplate reader (ASYS Hitech GmbH, Eugendorf, Austria) and averaged. Protein concentration was determined according to Brogdon (1984), in case there was some variation in the size of mosquitoes and appropriate dilution of the homogenate was needed. The data for each biochemical assay were evaluated by analysis of variance (ANOVA) and TukeyÕs test was used at a level of signiÞcance of P ⬍ 0.05 to compare means between surviving and dead females in the populations studied with respect to the reference strain. The resistance threshold corresponding to the maximal enzyme activity in the surviving females of the NO strain was compared with the survivors of the four populations studied. We calculated the percentage of individuals exceeding the resistance threshold (CDC 2002) and classiÞed the enzyme mechanisms as nonaltered (NA), incipiently altered (IA), or altered (A), if ⬍15%, 15Ð50%, or ⬎50% of the specimens exceeded the threshold, respectively (Montella et al. 2007). Additionally, the LC50 values and mean enzyme levels of the surviving females exposed to deltamethrin were submitted to linear regression analysis. Correlation (r) was performed to determine the degree of association between the two variables. Finally, two criteria were considered to determine whether an enzyme mechanism was involved in the resistance observed for malathion and deltamethrin: 1) ⬎50% of surviving specimens of the population studied exceeded the resistance threshold; 2) the mean enzyme activity level was signiÞcantly higher in the surviving specimens than in the dead ones and in turn higher than those of the NO strain. A third criterion was considered for deltamethrin: a highly signiÞcant correlation between the LC50 values of deltamethrin and enzyme levels. Results Bioassays. Table 1 shows the results of the resistance to deltamethrin and malathion in Ae. aegypti of Venezuela during the years 2008 and 2010. Knock-down resistance values, KC50, for 2008 were between 0.079 and 0.115 g/bottle and for 2010 between 0.099 and 0.285 g/bottle. There were no changes for 2010 in the populations Pampanito and Tres Esquinas, showing knock-down resistance with RRKC50 ⬍5-fold, while there were differences for the populations Lara and Uren˜ a with increases in KC50 for 2010 with RRKC50 of 7.7- and 10.2-fold, respectively, indicating moderate and high knock-down resistance. With respect to postrecovery resistance, we observed that the LC50
Downloaded from https://academic.oup.com/jme/article-abstract/50/5/1031/904290 by guest on 16 January 2019
ßasks with water, lined inside with Þlter paper, were provided. These eggs corresponded to the F1 and F2 generations, which were used in the majority of the bioassays. All populations of Ae. aegypti were kept at a temperature of 25 ⫾ 2⬚C and relative humidity of 70% ⫾2. Females of the F1 generation, 1Ð3 d after emergence and without blood feeding, were used for the bioassays and biochemical tests. The New Orleans (NO) strain maintained for several years in the laboratory was used as the susceptible reference strain in all situations. Bioassays. The insecticides evaluated were malathion (98.4% purity) and deltamethrin (99.5%) (ChemService, West Chester, PA), which were resuspended in 1 ml of acetone to prepare stock solutions with Þnal concentrations of 500 and 100 g/ml, respectively. Different dilutions were then prepared to obtain concentrations that caused between 2 and 98% mortality. The bioassays were carried out according to the bottle method proposed by the Centers for Disease Control and Prevention (CDC 2002) in Atlanta, GA (Brogdon and McAllister 1998), in which 15Ð20 females were used per bottle. For deltamethrin, the exposure time was 1 h during which observations were made every 10 min to record the number of insects killed and to determine the knock-down effect. Later, the insects were transferred to insecticide-free ßasks, and mortality was recorded at 24 h. For the organophosphate malathion, the insects were exposed for 2 h and then transferred to insecticide-free ßasks; afterwards, the number of recovered mosquitoes was recorded at 1, 2, and 4 h, and the number of dead insects was recorded at 24 h. Each bioassay consisted of bottles containing Þve or six concentrations with four replicates for each insecticide and the control was treated only with acetone. This method was used with all Þeld populations and with the NO reference population. The results obtained were submitted to log-probit regression analysis (Finney 1971) using the Probit program (Raymond 1985) to determine the 50% knock-down dose (KC50) and 50% lethal concentration (LC50) for deltamethrin and LC50 for malathion. The conÞdence intervals were calculated using ␣ ⫽ 0.05, and the signiÞcant difference between the values was determined based on nonoverlapping conÞdence intervals. The mortalities were corrected according to the formula of Abbott (1925) when mortality was observed in the control group. The resistance ratios (RR) were calculated by dividing the LC50 of each population by the LC50 of the NO reference strain, as well as by dividing the KC50 obtained for deltamethrin by the KC50 of the NO strain. Enzymes. Batches of 100 females of each Þeld population and of the reference were exposed to bottles impregnated with the respective LC50 values of deltamethrin for a time period that caused 50% mortality. The dead and survivors were then separated and stored individually at ⫺70⬚C. The same procedure was carried out with malathion using the LC99 determined in the NO strain.
1033
1034
JOURNAL OF MEDICAL ENTOMOLOGY
Vol. 50, no. 5
Table 1. Dose–response values and resistance ratios to deltamethrin and malathion and knock-down doses and resistance ratios to deltamethrin in four strains of Ae. aegypti from Venezuela Insecticide
PTO
TE
2010 Lara
Uren˜ a
NO
PTO
TE
Lara
Uren˜ a 0.294 0.255Ð0.339 1.53 (0.18) 19.6 0.285 0.249Ð0.326 1.63 (0.18) 10.2
0.008 0.053 0.072 0.072 0.031 0.006Ð0.013 0.048Ð0.059 0.059Ð0.088 0.066Ð0.079 0.022Ð0.045 1.27 (0.14) 3.09 (0.39) 1.33 (0.19) 2.9 (0.45) 0.73 (0.13) 6.6 9 9 3.9 0.016 0.079 0.115 0.083 0.110 0.012Ð0.022 0.071Ð0.087 0.105Ð0.127 0.077Ð0.090 0.068Ð0.176 1.15 (0.12) 2.77 (0.38) 3.06 (0.34) 4.33 (0.61) 1.30 (0.35) 4 7.2 5.2 6.9
0.015 0.140 0.203 0.013Ð0.021 0.125Ð0.157 0.176Ð0.233 1.02 (0.13) 2.67 (0.31) 1.79 (0.23) 9.3 13.5 0.028 0.099 0.125 0.021Ð0.034 0.086Ð0.114 0.107Ð0.145 1.42 (0.17) 2.05 (0.25) 1.81 (0.19) 3.5 4.5
0.279 0.245Ð0.317 1.87 (0.24) 18.6 0.216 0.193Ð0.243 2.03 (0.25) 7.7
1.6 2.15 1.21Ð2.12 1.58Ð2.90 0.82 (0.10) 0.77 (0.1) 1.3
1.45 1.34Ð1.57 2.62 (0.26)
2.31 3.07 2.08Ð2.59 2.81Ð3.35 2.19 (0.21) 2.99 (0.36) 1.6 2.1
1.18 3.49 0.90Ð1.54 2.97Ð4.09 0.85 (0.12) 1.53 (0.18) 0.74 2.2
3.05 2.44Ð3.81 1.18 (0.17) 1.9
3.00 2.75 2.79Ð3.24 2.47Ð3.06 3.27 (0.34) 2.85 (0.29) 2.1 1.9
g/bottle. 95% CIs. Slope of regression line Probit-log, standard deviations (⫾SD) are in parentheses. d RRLC50, resistance ratio: LC50 Þeld strain/ LC50 NO strain. e RRKC50, resistance ratio: KC50 Þeld strain/KC50 NO strain. a
b c
values for deltamethrin in the populations evaluated were between 0.031 and 0.072 g/bottle for 2008. Compared with the NO strain, the Pampanito, Tres Esquinas, and Lara populations showed moderate resistance to the pyrethroid with RRLC50 of 6.6-, 9.0-, and 9.0-fold, respectively, while the Uren˜ a population showed low resistance (3.9-fold). For 2010, the LC50 values increased (P ⬍ 0.05) in all populations, showing moderate resistance to deltamethrin for the Pampanito population (9.3-fold) and high resistance (RR LC50 ⬎10-fold) for the Tres Esquinas, Lara, and Uren˜ a populations. The KC50 values for the populations of Pampanito, Tres Esquinas, and Uren˜ a in 2008were higher than the LC50 values, owing to the low recovery percentages after 1 h exposure (0, 0.56, and 0%, respectively), indicating a greater effectiveness of deltamethrin at 24 h. In contrast for 2010, the LC50 values were higher than the KC50 values in the Pampanito, Tres Esquinas, and Lara populations, whose recovery percentages were 9.72, 12.8, and 9.34%, respectively, necessitating a greater amount of deltamethrin to kill the knocked down insects; in the Uren˜ a population, no difference was found between the two values. For the insecticide malathion, the LC50 values varied between 1.18 and 3.49 g/bottle in 2008 and between 2.31 and 3.07 g/bottle in 2010, indicating an increase in the Tres Esquinas population and decrease in Lara for the year 2010. In evaluating the RRLC50 values, we found that all populations studied showed low resistance to the organophosphate in both study periods, with values of RRLC50 less than Þvefold. Enzymes. Of the four enzyme systems evaluated after exposure of the populations to deltamethrin during 2008, we found overexpressed levels of GST in surviving females of the Lara population, with 86.7% of these exceeding the resistance threshold, thus being categorized as an altered enzyme mechanism (A) according to Montella et al. (2007). For 2010, overex-
pression of MFO was recorded in the survivors of the Tres Esquinas population, with 23.3% of the specimens exceeding the resistance threshold (incipiently altered mechanism). In the Pampanito and Tres Esquinas populations, levels of GST were overexpressed, with 100% of survivors exceeding the threshold (altered mechanism) (Tables 2; 3). In examining the association between the LC50 values of deltamethrin and overexpressed enzyme levels for surviving females exposed to LC50 of deltamethirn, there were no signiÞcant correlation values, thereby not fulÞlling in any of the cases the three criteria proposed to associate an enzyme mechanism with resistance to insecticides (Fig. 2). Thus, there was no association of the over-expression of MFO or GST with the resistance observed, suggesting the participation of these enzymes in other functions other than in the metabolism of deltamethrin. Table 2 shows the absorbance means for each enzyme in the populations exposed to malathion during 2008 and 2010. ␣-Esterases were overexpressed during 2008 in the Pampanito, Tres Esquinas, and Lara populations, with 100% of the surviving specimens exceeding the resistance threshold, thus being categorized as an altered mechanism. A similar behavior was observed with -esterases in the Pampanito and Lara populations. MFO appeared to be an incipiently altered mechanism in the Uren˜ a population (33.3%) and GST as an altered mechanism in the Pampanito population (93.3%). For 2010, we found an altered enzyme mechanism for ␣-esterases in the Tres Esquinas, Lara, and Uren˜ a populations with ⬎80% of surviving specimens exceeding the resistance threshold, and the overexpression of MFO in the Uren˜ a population (6.7%) as well (Table 3). Discussion Approximately four decades since their introduction, pyrethroids continue to be one of the principal
Downloaded from https://academic.oup.com/jme/article-abstract/50/5/1031/904290 by guest on 16 January 2019
Deltamethrin LC50a CIb bc (⫾SD) RRLC50d KC50 CI b (⫾SD) RRKC50e Malathion LC50 CI b (⫾SD) RRLC50
2008 NO
1.198 (0.025) 1.196 (0.034)
1.268 (0.091)* 1.172 (0.023)
1.225 (0.022) 1.238 (0.036)
1.246 (0.067)* 1.115 (0.058)
1.314 (0.038)* 0.886 (0.048)
1.178 (0.069) 1.189 (0.064)
0.955 (0.091) 0.941 (0.105)
0.778 (0.098) 0.794 (0.135)
1.195 (0.025)* 1.244 (0.027)
1.029 (0.082) 1.132 (0.119)
0.743 (0.056) 0.821 (0.110)
1.103 (0.061)* 0.969 (0.100)
0.814 (0.101) 0.736 (0.055)
0.806 (0.046) 0.683 (0.057)
0.847 (0.054) 0.737 (0.049)
0.895 (0.092) 0.763 (0.096)
0.661 (0.025) 0.779 (0.035)
0.744 (0.033) 0.780 (0.084)
0.948 (0.090) 0.987 (0.092)
-Esterases
0.766 (0.086) 0.775 (0.054)
␣-Esterases
0.451 (0.09)* 0.367 (0.088)
0.315 (0.038) 0.313 (0.061)
0.230 (0.045) 0.320 (0.076)
0.385 (0.042) 0.379 (0.081)
0.308 (0.069) 0.300 (0.072)
0.325 (0.082) 0.277 (0.091)
0.285 (0.052) 0.273 (0.054)
0.191 (0.045) 0.205 (0.040)
0.242 (0.046) 0.226 (0.047)
0.433 (0.084) 0.334 (0.059)
MFO
2008
0.088 (0.049) 0.101 (0.055)
0.106 (0.010) 0.089 (0.031)
0.071 (0.038) 0.102 (0.031)
0.140 (0.025)* 0.101 (0.017)
0.056 (0.018) 0.052 (0.013)
0.008 (0.021) 0.050 (0.036)
0.098 (0.021)* 0.0005 (0.038)
0.064 (0.014) 0.062 (0.015)
0.095 (0.022) 0.092 (0.021)
0.051 (0.009) 0.020 (0.051)
GST
0.017 (0.010) ⫺0.042 (0.063)
0.012 (0.012) 0.014 (0.009)
0.564 (0.047)* 0.510 (0.047)
0.562 (0.044)* 0.518 (0.036)
0.578 (0.088)* 0.486 (0.029)
0.489 (0.035) 0.596 (0.074)
⫺0.016 (0.038) ⫺0.018 (0.066) 0.003 (0.012) 0.003 (0.026)
0.408 (0.043) 0.348 (0.028)
0.555 (0.045) 0.769 (0.064)
0.636 (0.087) 0.657 (0.055)
0.010 (0.005) ⫺0.013 (0.030)
0.001 (0.006) ⫺0.043 (0.069)
0.012 (0.006) ⫺0.001 (0.006)
0.755 (0.101) 0.917 (0.106)
0.693 (0.114) 0.720 (0.060)
⫺0.104 (0.037) 0.018 (0.016) 0.011 (0.008) 0.004 (0.002)
0.674 (0.502) 0.809 (0.045)
␣-Esterases
⫺0.036 (0.043) 0.004 (0.002)
iAChE
0.766 (0.095) 0.699 (0.076)
0.644 (0.076) 0.605 (0.087)
0.615 (0.097) 0.662 (0.114)
0.807 (0.073) 0.869 (0.092)
0.705 (0.074) 0.713 (0.08)
0.939 (0.051) 0.905 (0.702)
0.925 (0.169) 0.911 (0.066)
0.858 (0.132) 0.924 (0.111)
0.791 (0.139) 0.802 (0.099)
1.100 (0.984) 0.981 (0.118)
-Esterases
2010
0.246 (0.06)* 0.173 (0.041)
0.165 (0.037) 0.143 (0.031)
0.209 (0.062) 0.170 (0.059)
0.204 (0.308) 0.146 (0.022)
0.179 (0.072) 0.122 (0.034)
0.123 (0.025) 0.134 (0.095)
0.175 (0.062) 0.289 (0.088)
0.325 (0.095)* 0.253 (0.104)
0.209 (0.090) 0.189 (0.076)
0.192 (0.053) 0.233 (0.063)
MFO
⫺0.116 (0.078) 0.011 (0.008) 0.009 (0.009) ⫺0.018 (0.044) 0.006 (0.022) ⫺0.030 (0.066) 0.036 (0.054) ⫺0.106 (0.071) 0.010 (0.012) ⫺0.170 (0.057)
0.011 (0.006) 0.006 (0.006) 0.084 (0.002) 0.008 (0.009) 0.0005 (0.020) 0.0009 (0.035) 0.0032 (0.007) 0.0089 (0.010) 0.0009 (0.0087) 0.0270 (0.0071)
0.378 (0.087)* ⫺0.014 (0.036) 0.631 (0.077)* 0.039 (0.014) 0.023 (0.021) 0.019 (0.197) 0.030 (0.007) 0.024 (0.013)
0.048 (0.018) ⫺0.047 (0.067) 0.024 (0.072) 0.112 (0.020) ⫺0.068 (0.097) 0.061 (0.010) 0.084 (0.026) 0.056 (0.013) 0.057 (0.042) 0.058 (0.018)
iAChE
⫺0.039 (0.061) ⫺0.004 (0.038)
GST
Mean absorbance (ⴞSD) in each biochemical test of four strains of Ae. aegypti from Venezuela exposed to LC50 of deltamethrin and LC99 of malathion
ALVAREZ ET AL.: INSECTICIDE RESISTANCE IN A.aegypti FROM VENEZUELA
Downloaded from https://academic.oup.com/jme/article-abstract/50/5/1031/904290 by guest on 16 January 2019
A, alive after exposure to LC50 of deltamethrin or LD99 of malathion; D, dead after exposure to LC50 of deltamethrin or LD99 of malathion. * Mean values higher signiÞcantly (P ⬍ 0.05) in alive specimen compared with dead and with NO strain.
NO Alive Dead PTO Alive Dead TE Alive Dead Lara Alive Dead Uren˜ a Alive Dead
Deltamethrin NO Alive Dead PTO Alive Dead TE Alive Dead Lara Alive Dead Uren˜ a Alive Dead Malathion
Strain
Table 2.
September 2013 1035
1036
JOURNAL OF MEDICAL ENTOMOLOGY
Vol. 50, no. 5
Table 3. Percentage of each Ae. aegypti pop from four sites in western Venezuela exposed to two pesticides with enzyme activity higher than the threshold established by the NO strain Strain/year
-Esterases
MFO
GST
iAchE
2010
2008
2010
2008
2010
2008
2010
2008
2010
0.797a 100 100 100 100 0.987a 0 0 0 0
0.494a 43.3 80 96.7 93.3 0.494a 3.3 0 0 13.3
0.966a 100 100 100 100 1.079a 0 23.3 0 86.7
0.843a 36.7 0 0 20 0.843a 0 0 0 0
0.491a 0 0 0 33.3 0.618a 0 0 0 3.3
0.353a 0 3.3 0 6.7 0.353a 3.3 23.3 0 0
0.118a 93.3 13.3 13.3 26.7 0.075a 86.6 13.3 86.7 86.7
0.125a 0 3.3 3.3 0 0.125a 100 100 0 0
0.023a 13.3 3.3 13.3 10 0.019a 0 20 6.3 3.3
0.026a 6.6 3.3 0 0 0.026a 0 0 50 3.3
a
Maximum absorbance value for NO strain. Cell Shading: unaltered enzyme (no shading), incipiently altered (light gray), and altered (dark gray).
groups of insecticides applied for the control of wide variety of agricultural pests and disease vectors (Davies and Williamson 2009). Resistance to pyrethroids and especially deltamethrin has been reported in different populations of Ae. aegypti in Venezuela (Mazzarri and Georghiou 1995, Perez and Molina 2001, Rodrõ´guez et al. 2007). In the current study, the evolution of resistance to deltamethrin was demonstrated in the populations evaluated during 2008 and 2010, probably owing to the presence of intrinsic genetic characteristics in the species that favor the expression of resistance genes, as well as the selective pressure exerted by the constant use of chemical insecticides for the control of dengue and agricultural pests. The principal economic activity in Uren˜ a is agriculture, and we found that the LC50 of the Ae. aegypti population increased nine times in 2 yr with a RRLC50 of 19.6 in 2010. In addition, there are reports of Ae. aegpyti resistant to pyrethroids owing to cross-resistance to DDT (Hemingway et al. 1989, Brengues et al. 2003). Both compounds share the same target site, the voltage-gated sodium channel, which could be an additional factor present in our populations owing to the selective pressure exerted with the application of DDT for several decades in Venezuela for the control of malaria and other diseases. Rodrõ´guez et al. (2007) associated resistance to deltamethrin in larvae of Ae. aegypti of Venezuela and other Latin American countries with the activity of metabolic enzymes, esterases, and GSTs, as well as cytochrome oxidases (Berge´ et al. 1998). However, we did not Þnd enzyme mechanisms associated with resistance to deltamethrin, suggesting the presence of a nonenzymatic mechanism, such as point mutations in the gene coding for the voltage-gated sodium channel, which have been previously reported in this species (Brengues et al. 2003, Saavedra et al. 2007, Yanola et al. 2011). This may be especially important with the Ile1,016 resistance allele in which Saavedra et al. (2007) observed that 90.6% of Ile1,016/Val1,016 heterozygotes were knocked down after a 1-h exposure to permethrin but that 57.4% of knocked down heterozygotes eventually recovered. Thus, the rate of recovery after knockdown is inherited as an additive genetic trait with
heterozygotes exhibiting an intermediate phenotype. It is important to note that the states of Lara and Tachira (Uren˜ a) are two of the seven Venezuelan states that accounted for the highest number of cases of dengue during 2008 and 2010. Despite efforts by the government agencies to try to control Ae. aegypti in these and other federal territories, the failure of control programs is evident, reßected in the case studies of dengue, which increased from 48,048 in 2008 to 106,725 cases in epidemiological week 43 of 2010 (Ministerio del Poder Popular para Salud 2010). This was possible owing to the resistance of Ae. aegypti to deltamethrin. The Þght against dengue in Venezuela involves the fumigation of critical areas to destroy the adult population of Ae. aegypti and in educational campaigns to make the public aware of the problem and to eliminate foci of contaminated water in which this insect deposits its eggs. Thus, the results obtained in this study contribute important information that must be considered in continuing or halting the use of deltamethrin in control programs. OPS (1997) reported that resistance to malathion in Ae. aegypti has spread throughout the Caribbean, Central America, and South America, but there are reports of populations susceptible to organophosphates in Panama, Cuba (Bisset et al. 2003, 2004), and Vietnam (Houng et al. 2004). In the current study, we found low values of organophosphate resistance (RR ⬍5) in both evaluation periods in agreement with the Þndings of Mazzarri and Georghiou (1997) in populations of Ae. aegypti in the states of Aragua and Falcon, Venezuela, but at odds with Perez and Molina (2009), who found high resistance to malathion in larvae of Ae. aegypti in the municipalities of Girardot, Mario Bricen˜ o Iragorri, and Urdaneta in the state of Aragua, Venezuela (RR50 69.5, 150.6, and 113.5, respectively), with overexpression of esterases and cytochrome P450 oxidases. This suggests that resistance is focal. The populations evaluated during both periods exhibited overexpression of ␣and -esterases and cytochrome oxidases whose participation in the metabolism of malathion in
Downloaded from https://academic.oup.com/jme/article-abstract/50/5/1031/904290 by guest on 16 January 2019
Malathion PTO TE Lara Uren˜ a Deltamethrin PTO TE Lara Uren˜ a
␣-Esterases 2008
September 2013
ALVAREZ ET AL.: INSECTICIDE RESISTANCE IN A.aegypti FROM VENEZUELA
1037
these populations was not determined. This is despite reports associating these enzyme mechanisms with resistance to malathion in Ae. aegypti populations in Venezuela and Latin America (Rodrõ´guez et al. 2007, Perez and Molina 2009). Finally, the current study showed that resistance is dynamic in time and space. There is a need to carry out periodic evaluations that include suscep-
tibility bioassays and to determine the mechanisms associated with resistance to insecticides to obtain complete information about the susceptibility of the target population to the insecticide. There is also a need to design strategies for the management of resistance and to identify the opportune moment to rotate insecticides to maintain the susceptibility of the population in question.
Downloaded from https://academic.oup.com/jme/article-abstract/50/5/1031/904290 by guest on 16 January 2019
Fig. 2. Association of LC50 values of deltamethrin (lines) and four enzyme levels (histograms: mean absorbance values) for surviving Ae. aegypti from four sites in western Venezuela and a susceptible population (NO) in 2008 and 2010. SigniÞcance values for r are in parentheses.
1038
JOURNAL OF MEDICAL ENTOMOLOGY Acknowledgments
Collections of mosquitoes were obtained by the support of the project “Mision Ciencia: Subproyecto Dengue” from FONACYT Venezuela. The study was funded by CONACYT Ciencia Basica and UANL PAICYT, Mexico.
References Cited
tance in Aedes aegypti in the Caribbean area and neighbouring countries. J. Med. Entomol. 24: 290 Ð294. Gratz, N. G. 1991. Emergency control of Aedes aegypti as a disease vector in urban areas. J. Am. Mosq. Control Assoc. 7: 353Ð365. Hemingway, J., R. G. Boddington, and J. Harris. 1989. Mechanisms of insecticide resistance in Aedes aegypti (L.) (Dõ´ptera: Culicidae) from Puerto Rico. Bull. Entomol. Res. 79: 123Ð30. Hemingway, J., and S.H.P.P. Karunaratne. 1998. Mosquito carboxylesterases: a review of the molecular biology and biochemistry of a major insecticide resistance mechanism. Med. Vet. Entomol. 12: 1Ð12. Hemingway, J., and H. Ranson. 2000. Insecticide resistance in insect vectors of human disease. Annu. Rev. Entomol. 45: 371Ð391. Houng, V. D., N.N.T. Bach, D. T. Hien, and L.N.T. Bich. 2004. Susceptibility of Aedes aegypti to insecticides in Vietnam. Dengue Bull. 28: 179 Ð183. Lumjuan, N., L. McCarroll, L. Prapanthadara, J. Hemingway, and H. Ranson. 2005. Elevated activity of an Epsilon class glutathione transferase confers DDT resistance in the dengue vector, Aedes aegypti. Insect. Biochem. Mol. Biol. 35: 861Ð971. Mazzarri, M. B., and G. P. Georghiou. 1995. Characterization of resistance to organophosphate, carbamate, and pyrethroid insecticides in Þeld populations of Aedes aegypti (L) from Venezuela. J. Am. Mosq. Control Assoc. 11: 315Ð322. Ministerio del Poder Popular para la Salud. 2010. Boletõ´n Epidemiolo´ gico No. 43 Gobierno Bolivariano de Venezuela. Ministerio del Poder Popular para la Salud, Venezuela. Montella, I., A. Martins, P. Fernandez, J. Pereira, I. Braga, and D. Valle. 2007. Insecticide resistance mechanisms of Brazilian Aedes aegypti populations from 2001 to 2004. Am. J. Trop. Med. Hyg. 77: 467Ð 477. Mouchet, J. 1967. La resistance aux insecticides chez Aedes aegypti et les espe` ces voisines. Bull. W.H.O. 36: 569 Ð577. (OPS) Organizacion Panamericana de Salud. 1997. Informe sobre el control de Aedes aegypti. Organizacion Panamericana de Salud, Washington, DC. Documento CD. 40. Perez, E. E., and D. Molina. 2001. Resistance of Aedes aegypti to pyretroides in municipalities of Aragua state, Venezuela. J. Am. Mosq. Control Assoc. 17: 166 Ð180. Perez, E. E., and D. Molina. 2009. Resistencia focal a insecticidas organosinteticos en Aedes aegypti (Linneaus, 1762) (Dõ´ptera: Culicidae) de diferentes municipios del estado Aragua, Venezuela. Bol. Mal. Salud Amb. XLIX: 143Ð150. Quaterman, K. D., and H. F. Schoof. 1958. The status of insecticide resistance in arthropods of public health importance in 1956. Am. J. Trop. Med. Hyg. 7: 74 Ð 83. Rawlins, S. C. 1998. Spatial distribution of insecticide resistance in Caribbean populations of Aedes aegypti and its signiÞcance. Rev. Panam. Salud Publica 4: 243Ð51. Raymond, M. 1985. Presentation dÕune programme dÕanalyse logprobit pour Microordinateur cahiers Orstrom. Se´ r Ent Me´ d Parasitol. 23: 117Ð21. Rodrı´guez, M. M., J. A. Bisset, and D. Ferna´ ndez. 2007. Levels of insecticide resistance and resistance mechanisms in Aedes aegypti from some Latin American countries. J. Am. Mosq. Control Assoc. 23: 420 Ð 429. Rodriguez, M. M., J. A. Bisset, D. Molina, C. Diaz, and L. A. Soca. 2001. Adaptacion de los metodos en placas de microtitulacion para la cuantiÞcacio´ n de la actividad de
Downloaded from https://academic.oup.com/jme/article-abstract/50/5/1031/904290 by guest on 16 January 2019
Abbott, W. S. 1925. A method of computing the effectiveness of an insecticide. J. Econ. Entomol. 8: 265Ð267. Berge´, J. B., R. Feyereisen, and M. Amichot. 1998. Cytochrome p450 monooxygenases and insecticide resistance in insects. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 353: 1701Ð1705. Bisset, J. A., M. M. Rodrı´guez, and L. Caceres. 2003. Niveles de resistencia a insecticidas y sus mecanismos en 2 cepas de Aedes aegypti de Panama. Rev. Cubana Med. Trop. 55: 191Ð195. Bisset, J. A., M. M. Rodrı´guez, D. Fernandez, and O. Perez. 2004. Estado de la resistencia a insecticidas y mecanismos de resistencia en larvas del municipio Playa, colectadas durante la etapa intensiva contra el Aedes aegypti en Ciudad de La Habana, 2001Ð2002. Rev. Cubana Med. Trop. 56: 61Ð 66. Bisset, J. A., M. M. Rodrı´guez, D. Molina, C. Dı´az, and L. A. Soca. 2001. Esterasas elevadas como mecanismo de resistencia a insecticidas organofosforados en cepas de Aedes aegpyti. Rev. Cubana Med. Trop. 5: 37Ð 43. Brengues, C., N. J. Hawkes, F. Chandre, L. McCarroll, S. Duchon, P. Guillet, S. Manguin, J. C. Morgan, and J. Hemingway. 2003. Pyrethroid and DDT cross-resistance in Aedes aegypti is correlated with novel mutations in the voltage-gated sodium channel gene. Med. Vet. Entomol. 17: 87Ð94. Brogdon, W. G. 1984. Mosquito protein microassay.I. Protein determinations from small portions of single- mosquito homogenates. Comp. Biochem. Physiol. B. 79: 457Ð 459. Brogdon, W. G. 1989. Biochemical resistance detection: an alternative to bioassay. Parasitol. Today 5: 56 Ð 60. Brogdon, W. G., and J. C. McAllister. 1998. SimpliÞcation of adult mosquito bioassays through use of time-mortality determinations in bottles. J. Am. Mosq. Control Assoc. 14: 159 Ð164. Brogdon, W. G., J. C. McAllister, A. M. Corwin, and C. Cordon-Rosales. 1999. Oxidase-based DDT-pyrethroid cross-resistance in Guatemalan Anopheles albimanus. Pest. Biochem. Physiol. 64: 101Ð111. (CDC) Centers for Disease Control and Prevention. 2002. Evaluating mosquitoes for insecticide resistance: a webbased instruction. (www.cdc.gov/ncidod/wbt/resistance/ assay/microplate/index.htm) Davies, T.G.E., and M. S. Williamson. 2009. Interactions of pyrethroids with the voltage-gated sodium channel. Bayer CropSci. J. 2n: 159 Ð178. Finney, D. J. 1971. Probit analysis, 3rd ed. Cambridge University Press, Cambridge, UK. Flores, A. E., W. Albeldan˜ o–Vazquez, I. Fernandez–Salas, M. H. Badii, H. Loaiza–Becerra, G. Ponce–Garcia, S. Lozano–Fuentes, W. G. Brogdon, W. C. Black, IV, and B. Beaty. 2005. Elevated ␣-esterase levels associated with permethrin tolerance in Aedes aegypti (L.) from Baja California, Mexico. Pest. Biochem. Physiol. 82: 66 Ð78. Georghiou, G. P., M. C. Wirth, H. Tran, F. Saume, and A. B. Knudsen. 1987. Potential for organophosphate resis-
Vol. 50, no. 5
September 2013
ALVAREZ ET AL.: INSECTICIDE RESISTANCE IN A.aegypti FROM VENEZUELA
Kenyan villages using permethrin impregnated nets. Med. Vet. Entomol. 13: 239 Ð244. (WHO) World Health Organization. 1986. Resistance of vectors and reservoirs of disease of pesticides. W.H.O. Tech. Rep. Ser. 737: 1Ð 87. Yanola, J., P. Somboon, C. Walton, W. Nachaiwieng, P. Somwang, and L. Prapanthadara. 2011. High-throughput assays for detection of the F1534C mutation in the voltage-gated sodium channel gene in permethrin-resistant Aedes aegypti and the distribution of this mutation throughout Thailand. Trop. Med. Int. Health 16: 501Ð509. Received 7 November 2012; accepted 6 April 2013.
Downloaded from https://academic.oup.com/jme/article-abstract/50/5/1031/904290 by guest on 16 January 2019
esterasas y glutation-s-tranferasa en Aedes aegypti. Rev. Cubana Med. Trop. 53: 32Ð36. Saavedra, K., L. Urdaneta, S. Rajatileka, M. Moulton, A. E. Flores, I. Fernandez, J. Bisset, M. Rodriguez, P. J. McCall, M. J. Donnelly et al. 2007. Mutations in the voltage gated sodium channel gene associated with pyrethroid resistance in Latin American Aedes aegypti. Insect Mol. Biol. 16: 785Ð798. Scott, J. A. 1995. The molecular genetics of resistance: resistance as a response to stress. Fla. Entomol. 78: 399 Ð 414. Vulule, J. M., R. F. Beach, F. K. Atieli, J. C. McAllister, W. G. Brogdon, J. M. Roberts, R. W. Mwangi, and W. A. Hawley. 1999. Elevated oxidase and esterase levels associated with permethrin tolerance in Anopheles gambiae from
1039
