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. Author manuscript; available in PMC: 2014 Apr 9.
Published in final edited form as: J Econ Entomol. 2013 Apr;106(2):959–969. doi: 10.1603/ec12284

Wide spread cross resistance to pyrethroids in Aedes aegypti (L.) from Veracruz State Mexico

Adriana E Flores a,*, Gustavo Ponce a, Brenda G Silva a, Selene M Gutierrez a, Cristina Bobadilla a,b, Beatriz Lopez a, Roberto Mercado a, William C Black IV c
PMCID: PMC3980443  NIHMSID: NIHMS568128  PMID: 23786088

Abstract

Seven F1 strains of Aedes aegypti (L.) were evaluated by bottle bioassay for resistance to the pyrethroids d-phenothrin, permethrin, deltamethrin, λ-cyalothrin, bifenthrin, cypermethrin, α-cypermethrin and z-cypermethrin. The New Orleans strain was used as a susceptible control. Mortality rates after a 1h exposure and following a 24h recovery period were determined. The resistance ratio between the 50% knockdown values (RRKC50) of the F1 and New Orleans strains indicated high levels of knockdown resistance (kdr). The RRKC50 with α-cypermethrin varied from 10–100 among strains indicating high levels of kdr. Most of the strains had moderate resistance to d-phenothrin. Significant but much lower levels of resistance were detected for λ–cyalothrin, permethrin and cypermethrin. For z-cypermethrin and bifenthrin, only one strain exhibited resistance with RRKC50 values of 10- and 21-fold, respectively. None of the strains showed RRKC50 >10 with deltamethrin, and moderate resistance was seen in three strains, while the rest were susceptible. Mosquitoes from all strains exhibited some recovery from all pyrethroids except d-phenothrin. Regression analysis was used to analyze the relationship between RRLC50 and RRKC50. Both were highly correlated (R2 = 0.84 – 0.97) so that the slope could be used to determine how much additional pyrethroid was needed to insure lethality. Slopes ranged from 0.875 for d-phenothrin (RRLC50 ≃ RRKC50) to 8.67 for λ–cyalothrin (~8.5 fold more insecticide needed to kill). Both RRLC50 and RRKC50 values were highly correlated for all pyrethroids except bifenthrin indicating strong cross resistance. Bifenthrin appears to be an alternative pyrethroid without strong cross resistance that could be used as an alternative to the current widespread use of permethrin in Mexico.

Keywords: Aedes aegypti, pyrethroids, insecticide resistance, cross resistance

Aedes aegypti (L.) is the primary vector in Mexico of the four dengue virus serotypes (DENV1-4), the causative agents of dengue fever (DF) and dengue hemorrhagic fever (DHF). Although Ae. albopictus (Skuse) is present in many regions of the country; it still has not been incriminated as an important vector. Mexico is severely affected by DF and DHF because DENV1-4 viruses co-occur with Ae. aegypti in most states of Mexico. In 2007, 28 of 32 states registered cases of DF and DHF, in 2008, 26 states and in 2009, 25 states registered confirmed cases.

The coastal regions of Mexico are most affected by DF and DHF and the state of Veracruz, with an area of 72,815 km2 and a coastline of 684 km, has had the greatest numbers of DF and DHF cases. In 2007 alone, there were 15,253 DF cases of which 2,645 progressed to DHF. In 2008 there were 4,117 DF cases of which 2,051 progressed to DHF and in 2009 there were 6,390 DF cases of which 2, 978 eventually progressed to DHF (Boletin de Epidemiologia, Mexico 2007, 2008, 2009).

The official Mexican policy for the surveillance and control of vectors, NOM-032-SSA2-2002, recommended the use of the organophosphate larvicide temephos (Abate® granules) at a concentration of 1 ppm for the control of larval Ae. aegypti and a formulation of permethrin as an adulticide has now been used for more than 10 consecutive years (1999–2010). On June 1, 2011, a new policy was published (NOM-032-SSA2-2010) that established the characteristics that insecticides should have for the control of disease vectors, including Ae. aegypti, in Mexico without specifying which agents should be used, stating instead that it would depend on proven effectiveness, resistance and safety characteristics related to exposure.

In Mexican populations of Ae. aegypti, resistance to permethrin is conditioned in part by esterases (Flores et al. 2005, 2006). The presence of a new mutation (Ile1,016) in the voltage-gated sodium channel gene is also associated with kdr to pyrethroids. This mutation was originally found in a permethrin resistant strain from Isla Mujeres, off the coast of Cancun (Saavedra-Rodriguez et al. 2007). High frequencies of this resistance allele were subsequently found in collections of Ae. aegypti from 78 sites in Mexico with some of the highest frequencies detected in collections from Veracruz state (Ponce et al. 2009, Siller et al. 2011).

The practice of utilizing a single insecticide until the appearance of resistance has become a standard practice that quickly reduces the number of insecticides available for vector control. Rotations, mosaics and mixtures have instead been proposed as strategies for insecticide resistance management (Curtis 1985, Curtis et al. 1993, Roush 1989). Mathematical models have been applied for estimating how these tools could be used in an optimal manner (Tabashnik 1989). However, these models have been rarely tested under field conditions, especially for insect vectors, due to the difficulties in determining changes in frequencies of resistance genes in large samples of insects from resistant populations (Hemingway et al. 1997).

In Mexico, there was a large-scale field trial with Anopheles albimanus that used rotations or mosaics of insecticides substituting the simple use of DDT or of a specific pyrethroid (Hemingway et al. 1997; Penilla et al. 1998). Changes in the frequency of resistance genes were monitored for four years (Penilla et al. 1998). The results were promising and predicted that rotations or mosaics of insecticides are viable long-term strategies for the sustainable use of insecticides in disease control programs. With that goal in mind, the aim of the present study was to determine the levels of resistance to eight pyrethroids in seven collections of Ae. aegypti from the state of Veracruz. This knowledge will facilitate the selection of viable alternative pyrethroids for use in a rotation program for sustained control of Ae. aegypti at the local, regional and possibly state-wide levels.

Materials and Methods

Study area

Seven field collections of Ae. aegypti were collected in 2009 from the state of Veracruz (Fig. 1). The localities sampled were: Panuco (22°03′00.00″N, 98°10′59.92”W), Tantoyuca (21°20′54.44″N, 98°13′45.90″W ), Poza Rica (20°32′00.00″N, 97°26′59.84″W ), Martinez de la Torre (20°03′42.55″N, 97°03′06.97″W), Veracruz (19°10′21.48″N, 96°07′59.93″W), Coatzacoalcos (18°08′16.00″N, 94°26′07.49″W) and Cosoleacaque (18°00′03.16″N, 94°37′46.90″W). The New Orleans strain was used as a susceptible reference strain.

Figure 1.

Figure 1

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Map of the seven field populations of Ae. aegypti collected from state of Veracruz, Mexico.

Mosquitoes

Laboratory colonies were established from larvae collected from natural breeding sites and maintained at 25 ± 4°C and 12:12 (L:D). Upon pupation, they were placed in 250-ml flasks in cages (30×30 cm) until the adults emerged (parental generation). Adults were allowed to intermate. The male mosquitoes were fed a 10% sugar solution and the females were fed on rats (Rattus norvegicus) for the production of eggs, for which flasks with water, lined inside with filter paper, were provided. These eggs corresponded to the F1 generation, which were use for all bioassays.

Bioassays

F1 females 1–3 days of age were held without blood feeding for use in bioassays. The Brogdon and McAllister (1988) bottle bioassay was used in which a 250 mL Wheaton® bottle received a one mL solution of acetone containing technical grade insecticide (ChemService, West Chester, PA). The bottle was capped and shaken to insure uniform coverage and dried for an hour at room temperature. The insecticides tested were: d-(cis-trans)-phenothrin (6.3% cis, 91.7% trans), trans-permethrin (92% trans-6% cis), deltamethrin (99%), λ–cyhalothrin (99.1%), bifenthrin (99%), cypermethrin (99%), α-cypermethrin (99.5%), and z-cypermethrin (98%). Doses (μg/bottle) were predetermined that gave a range of knockdown rates and a 24h mortality rate from 0 – 99%. The number of different doses tested varied from 5 – 10, with three repetitions per dose and 20 females per repetition.

The numbers of knocked down mosquitoes were recorded every 10 min up to 1h. After 1h of exposure, all mosquitoes were gently transferred to a recovery container without insecticide and were offered a cotton ball soaked in a sugar solution. Mortality was recorded at 24 h. Both the bottles and recovery containers were kept at 24 ± 2 ° C and 70% RH.

Data analysis

Rates of kdr and 24 h recovery were analyzed by log-probit software (Raymond et al. 1985), according to Finney (1971). The Abbott (1925) correction formula for control mortality was applied. We determined the KC50 (concentration causing 50% knockdown) after one hour of exposure. The LC50 (concentration causing 50% mortality) was estimated from mortality data 24 h after recovery. The resistance ratio at 1 h of exposure (RRKC50) was calculated by dividing the KC50 of a F1 test population by the KC50 of the New Orleans reference susceptible strain. The RRLC50 was similarly calculated by dividing the LC50 of the test population by the LC50 of the New Orleans strain. The criteria of Mazzarri and Georghiou (1995) were used to classify RRs as high (> 10-fold), moderate (5- 10 fold), or low (< 5-fold). Linear regression was carried out between the RRLC50 vs RRKC50 for each insecticide in the seven strains. A R2 was estimated to test the strength of the correlation. The slope is the ratio of the change in LC50 relative to the change in KC50 and indicates how much pesticide concentration should be increased to cause lethality vs. knockdown. A slope ≃ 1 suggests RRLC50 ≃ RRKC50 or that most knocked down mosquitoes are dead 24 hours later. A slope > 1 suggests RRLC50 > RRKC50 or that recovery occurs and more insecticide is required to kill knocked down mosquitoes.

Cluster and principal component analyses are multivariate statistical techniques for summarizing relationships among collections in which many quantitative variables (e.g. LC50 for eight pyrethroids) have been measured. All 56 LC50 values were entered into a 7 (strains) × 8 (insecticides) matrix. The Pearson’s correlation coefficient (ρ) among LC50 values for each pesticide was calculated between each pair of collections to generate a symmetrical 8 × 8 correlation matrix. For cluster analysis, a second matrix of (1- ρ) values was calculated and subject to a UPGMA (Unweighted Pair Group Method with Arithmetic Mean - Sneath and Sokal, (1963)) cluster analysis using NEIGHBOR in PHYLIP3.6.7 (Felsenstein, 2005) to generate dendrograms indicating similarity among the eight pesticides.

Principal components were calculated as a linear combination of eight LC50 values multiplied by the eigenvector. There were 7 different principal components. The sum of the products of the eigenvectors and the LC50 values for each principal component is the eigenvalue. The eigenvalue is proportional to the magnitude of the general correlation in LC50 values among the 7 collections. If LC50 values for the eight pesticides covary among collections then the eigenvalue will be large but if LC50 values vary independently among collections then the eigenvalue will be small. The principal component with the largest eigenvector is defined as the first principal component while the principal component with the next largest eigenvector is defined as the second principal component. The first and second principal components should explain most of the covariance among collections in order to be useful. Bivariate plots of the first two principal components are useful in summarizing relationships among collections especially when they account for >70% of the covariance among LC50 values. Biplot analysis is a facet of principal components (Gabriel, 1971) that requires little additional computation and identifies the magnitude of variances and covariances among individual pesticides. Principal component and biplot analyses were performed on R version 2.11.1 (http://www.r-project.org/) using the prcomp and biplot functions.

There are three useful properties of biplots for analysis of insecticide resistance. First, the length of a biplot vector is proportional to the variance in LC50 values among and indicates how much a LC50 value varies among collections relative to other insecticides. Second, the cosine of the angle (in radians) between two biplot vectors equals the correlation in allele frequencies among collections at the two loci. If both vectors point in the same direction, then the angle between them approaches 0. The cosine of 0 radians is 1 which is also the correlation in LC50 values between those two insecticides. If both vectors form a right angle, cosine 90° (π/2 radians) = 0 indicating that LC50 values for two insecticides vary independently among collections. If vectors point in opposite directions, cosine 180° (π radians) = −1 indicating that LC50 values for two insecticides vary in opposite directions in collections. A third useful property of biplots is that they can be graphed alongside population principal components to identify which pesticides are causing specific patterns of differentiation among collections. All multivariate techniques were repeated for KC50, RRLC50, and RRKC50 values.

Results

Knockdown resistance, RRKC50

KC50 and RRKC50 values appear in Table 1. All strains showed >30 fold kdr to α-cypermethrin. Five strains showed from 7–62 fold resistance to d-phenothrin. Coatzacoalcos, Poza Rica and Veracruz showed kdr with λ–cyalothrin. Moderate resistance was found in Cosoleacaque. Mosquitoes from Panuco, Tantoyuca and M. de la Torre were susceptible to λ–cyalothrin. Poza Rica and M. de la Torre exhibited 12–18 fold resistance to permethrin, while Coatzacoalcos, Veracruz and Panuco exhibited moderate resistance. Only Cosoloeacaque and Tantoyuca were susceptible to permethrin. Similar findings were obtained for cypermethrin, in which Poza Rica and M. de la Torre exhibited high resistance, and moderate resistance was noted in Coatzacoalcos and Cosoleacaque. Only Coatzacoalcos exhibited 10 fold resistance to z-cypermethrin, while moderate resistance was exhibited by Poza Rica and Tantoyuca. Panuco, M. de la Torre, Veracruz and Cosoleacaque were susceptible to z-cypermethrin. For bifenthrin, Veracruz mosquitoes exhibited 21 fold resistance, while moderate resistance was seen in Cosoleacaque. Panuco, Tantoyuca, Poza Rica and Coatzacoalcos, were susceptible with RRKC50 values ≤ 2. No strains exhibited resistance to deltamethrin, and only Coatzacoalcos, Veracruz and Tantoyuca displayed moderate resistance. Panuco, Tantoyuca, M. de la Torre and Cosoleacaque were susceptible to deltamethrin.

Table 1.

Knockdown concentration (KC50 ) and resistance ratio (RRKC50) of 8 pyrethroids against Aedes aegypti females from Veracruz, Mexico.

STRAIN d-PHENOTHRIN PERMETHRIN DELTAMETHRIN CYPERMETHRIN α-CYPERMETHRIN Z-CYPERMETHRIN λ–CYHALOTHRIN BIFENTHRIN
Panuco 4211 413 311 342 359 294 480 480
KC50 2 8.13 (4.67–14.68) 2.28(1.71–2.96) 0.063(0.046–0.089) 0.263(0.190–0.368) 0.141(0.113–0.177) 0.223(0.180–0.282) 0.014(0.010–0.019) 0.09(0.060–0.140)
RRKC50 3 18.48 5.07 3.00 2.58 88.12 2.14 2.76 0.43
b (± SE)4 0.533 (0.068) 1.179 (0.121) 1.139 (0.123) 1.063 (0.111) 1.534 (0.158) 1.792 (0.177) 0.905 (0.077) 0.629 (0.062)
x2 (df)5 10.64 (4) 12.32 (3) 0.68 (3) 8.08 (4) 25.35 (4) 6.05 (3) 7.21 (6) 8.78 (6)
Tantoyuca 720 660 483 343 404 345 270 450
KC50 3.26(2.53–4.16) 1.48(1.27–1.70) 0.017(0.015–0.020) 0.108(0.076–0.148) 0.047(0.032–0.070) 0.561(0.432–0.727) 0.016(0.009–0.029) 0.18(0.140–0.230)
RRKC50 7.41 3.29 0.81 1.06 29.37 5.39 3.24 0.86
b (± SE) 0.998 (0.066) 1.889 (0.143) 1.992 (0.164) 1.045 (0.120) 0.807 (0.087) 1.338 (0.140) 0.484 (0.068) 1.540 (0.191)
x2 (df) 8.32 (10) 15.17 (9) 7.07 (6) 1.36 (4) 18.03 (5) 14.15 (4) 5.72 (5) 8.35 (4)
Poza Rica 664 600 299 325 339 318 480 405
KC50 27.29(24.54–29.96) 8.29(7.15–9.57) 0.146(0.130–0.160) 1.580(1.410–1.762) 0.462(0.385–0.560) 0.923(0.790–1.062) 0.090(0.070–0.120) 1.04(0.900–1.200)
RRKC50 62.02 18.42 6.95 15.49 288.75 8.87 18.00 4.95
b (± SE) 2.678 (0.226) 1.801 (0.144) 3.487 (0.325) 3.247 (0.350) 2.009 (0.184) 2.640 (0.268) 1.332 (0.129) 2.132 (0.180)
x2 (df) 67.46 (9) 8.27 (8) 0.021 (3) 22.52 (4) 1.94 (4) 9.42 (4) 11.62 (7) 9.22 (8)
M.de la Torre 300 300 379 418 287 341 369 517
KC50 14.01(10.60–18.30) 5.59(4.61–6.58) 0.019 (0.014–0.026) 1.145(1.00–1.294) 0.361(0.307–0.428) 0.260(0.199–0.339) 0.016(0.014–0.019) 0.153(0.127–0.187)
RRKC50 31.84 12.42 0.90 11.22 225.62 2.5 3.20 0.73
b (± SE) 1.461 (0.142) 2.258 (0.270) 1.112 (0.112) 2.824 (0.245) 2.319 (0.270) 1.405 (0.128) 1.749 (0.144) 1.810 (0.175)
x2 (df) 22.62 (3) 12.68 (3) 19.03 (4) 17.88 (6) 16.27 (3) 6.09 (4) 6.29 (6) 15.81 (4)
Veracruz 353 430 278 285 337 286 390 315
KC50 25.47(21.70–29.80) 2.66(2.23–3.14) 0.154(0.132–0.177) 0.281(0.231–0.339) 0.245(0.204–0.297) 0.333(0.259–0.426) 0.086(0.060–0.120) 4.40 (3.870–4.970)
RRKC50 57.89 5.91 7.33 2.75 153.12 3.20 17.20 20.95
b (± SE) 2.293 (0.220) 1.913 (0.164) 2.767 (0.291) 2.108 (0.214) 1.904 (0.186) 1.557 (0.167) 1.159 (0.118) 2.814 (0.266)
x2 (df) 15.10 (4) 4.18 (5) 5.35 (3) 5.10 (3) 9.80 (4) 8.38 (3) 7.38 (5) 20.10 (6)
Coatzacoalcos 380 361 302 370 334 283 225 270
KC50 18.40(15.84–21.19) 2.94(2.48–3.45) 0.168(0.149–0.189) 0.550(0.447–0.662) 0.435(0.344–0.562) 1.078(0.937–1.232) 0.120(0.080–0.180) 0.43(0.310–0.600)
RRKC50 41.82 6.53 8.00 5.39 271.87 10.36 24.00 2.05
b (± SE) 2.395 (0.229) 2.192 (0.198) 3.236 (0.318) 2.105 (0.179) 1.524 (0.147) 3.108 (0.387) 1.005 (0.117) 1.306 (0.168)
x2 (df) 6.62 (4) 11.22 (4) 7.51 (3) 16.09 (5) 10.82 (4) 33.15 (3) 6.40 (4) 15.96 (3)
Cosoleacaque 737 481 293 447 364 246 360 360
KC50 3.78(3.05–4.46) 0.68(0.57–0.80) 0.030(0.023–0.038) 0.256(0.198–0.319) 0.122(0.089–0.167) 0.203.(0.157–0.254) 0.045(0.030–0.060) 1.32(1.060–1.660)
RRKC50 8.59 1.51 1.43 5.39 76.25 1.29 9.00 6.29
b (± SE) 1.224 (0.086) 1.693 (0.154) 1.582 (0.181) 1.537 (0.130) 1.102 (0.102) 1.806 (0.207) 0.896 (0.087) 1.498 (0.174)
x2 (df) 9.10 (10) 5.77 (6) 15.62 (3) 14.78 (6) 8.00 (4) 3.43 (2) 21.63 (4) 19.51 (4)
New Orleans 287 300 340 375 426 370 360 360
KC50 0.44(0.38–0.49) 0.45(0.37–0.51) 0.021(0.017–0.024) 0.102(0.084–0.122) 0.0016(0.0013–0.0018) 0.104(0.081–0.135) 0.005(0.0002–0.001) 0.21(0.170–0.280)
b (± SE) 3.018 (0.327) 2.787 (0.330) 2.798 (0.298) 1.860 (0.174) 1.994 (0.167) 1.342 (0.125) 0.998 (0.103) 1.446 (0.140)
x2 (df) 10.97 (3) 13.51 (3) 10.14 (4) 19.55 (4) 15.18 (5) 1.18 (4) 15.00 (4) 45.17 (4)
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1

Number of females tested,

2

KC50, 50% knockdown concentration in μg/bottle, 95% confidence interval in parentheses,

3

RRKC50, resistance ratio: KC50 resistant strain/KC50 New Orleans strain,

4

Slope of regression line Probit-log, standard error (± SE) are in parentheses,

5

Chi-square (degree of freedom).

Surprisingly, high levels of resistance to α-cypermethrin were detected, with most strains exhibiting > 100 fold resistance. Moderate resistance (14–62 fold) was seen for d-phenothrin; for which two-thirds of the strains displayed resistance. Significant but much lower levels (17–24 fold ) of resistance were detected for λ–cyalothrin in half of the strains. Resistance was even lower for permethrin in which a third of the strains showed kdr. Similar results were obtained for cypermethrin with two of the strains showing 11–15 fold greater values. For the pyrethroids z-cypermethrin and bifenthrin, only one strain demonstrated resistance (RRKC50 10 – 21 fold, respectively). For deltamethrin, none of the strains had RRKC50 values >10, moderate resistance was found in three strains, while the rest were susceptible. RRKC50 is a useful statistic to describe the amount of kdr in a population. It would be interesting to relate the magnitude of the kdr with the frequency of Ile1,016 mutations. This could be very helpful when the necessary molecular tools are lacking, as the case in many countries in Latin America.

Resistance to 1 h exposure and 24 h post-recovery, RRLC50

Mortality 24 h following an initial 1 hour insecticide exposure provides a means of implicating other, possibly metabolic, forms or resistance. Table 2 lists LC50 and resistance ratio (RRLC50) beginning with the insecticides with the highest resistance values. Six strains exhibited resistance to α-cypermethrin, with RRLC50 values greater than 100 fold for five of the strains. Cosoleacaque was relatively susceptible to α-cypermethrin (4.5 fold). For λ–cyalothrin, Coatzacoalcos, Poza Rica, Veracruz and Cosoleacaque had RRLC50 values >10. Martinez de la Torre showed moderate resistance and Panuco and Tantoyuca and were susceptible. Poza Rica, Veracruz, Coatzacoalcos and M. de la Torre exhibited resistance to d-phenothrin, while Tantoyuca and Cosoleacaque were moderately resistant and Panuco and Tantoyuca were susceptible. Veracruz, Cosoleacaque and Poza Rica exhibited resistance to bifenthrin. Coatzacoalcos displayed moderate resistance and Panuco, Tantoyuca and M. de la Torre were susceptible. Coatzacoalcos was the most resistant to z-cypermethrin, followed by Poza Rica and Tantoyuca; Veracruz and M. de la Torre were moderately resistant and Panuco and Cosoleacaque were susceptible. In the case of deltamethrin, even though no strain showed kdr, recovery was observed at 24 h post-exposure in Coatzacoalcos, Veracruz and Poza Rica. Panuco, Tantoyuca and Cosoleacaque were susceptible. Poza Rica exhibited the greatest resistance to permethrin, followed by M. de la Torre and Veracruz. Coatzacoalcos and Tantoyuca were moderately resistant and Panuco and Cosoleacaque were susceptible to permethrin. Poza Rica and M. de la Torre were resistant to cypermethrin, while only Coatzacoalcos displayed moderate resistance and Panuco, Tantoyuca, Veracruz and Cosoleacaque were susceptible with RRLC50 <5.

Table 2.

Toxicity (LC50 ) and resistance ratio (RRLC50) of 8 pyrethroids against Aedes aegypti females from Veracruz, Mexico

STRAIN d-PHENOTHRIN PERMETHRIN DELTAMETHRIN CYPERMETHRIN α-CYPERMETHRIN Z-CYPERMETHRIN λ–CYHALOTHRIN BIFENTHRIN
Panuco 4211 413 311 342 359 294 480 480
LC502 0.24(0.13–0.41) 0.62(0.43–0.83) 0.023(0.016–0.031) 0.196(0.129–0.299) 0.171(0.135–0.222) 0.200(0.158–0.258) 0.006(0.003–0.114) 0.033(0.019–0.056)
RRLC503 0.71 2.82 2.56 2.97 155.45 3.92 1.43 0.61
b (± SE) 4 0.797 (0.070) 1.289 (0.115) 1.100 (0.125) 0.801 (0.099) 1.407 (0.157) 1.568 (0.167) 0.469 (0.065) 0.526 (0.059)
x2 (df)5 6.70 (4) 4.62 (3) 7.45 (3) 9.98 (4) 11.92 (4) 8.29 (3) 26.43 (6) 5.68 (6)
Tantoyuca 720 660 483 343 404 345 270 450
LC50 0.98(0.68–1.34) 1.24(1.05–1.44) 0.017(0.014–0.018) 0.124(0.085–0.177) 0.051(0.032–0.0825) 0.990(0.784–1.284) 0.001(0.0004–0.002) 0.147(0.113–0.188)
RRLC50 2.88 5.64 1.89 1.88 46.36 19.41 1.43 2.72
b (± SE) 0.814 (0.061) 1.727 (0.131) 2.141 (0.171) 0.921 (0.116) 0.651 (0.083) 1.519 (0.167) 0.344 (0.064) 1.540 (0.192)
x2 (df) 11.96 (10) 28.79 (9) 7.13 (6) 6.99 (4) 12.55 (5) 24.65 (4) 3.60 (5) 6.91 (4)
Poza Rica 664 600 299 325 339 318 480 405
LC50 17.40(15.16–19.64) 7.31(4.78–10.71) 0.150(0.130–0.170) 1.881(1.502–2.514) 0.743(0.595–0.972) 1.096(0.882–1.366) 0.109(0.081–0.143) 0.835(0.737–0.947)
RRLC50 51.18 33.23 16.67 28.50 675.45 21.49 155.71 15.46
b (± SE) 2.602 (0.206) 2.105 (0.157) 2.594 (0.268) 1.436 (0.282) 1.674 (0.179) 1.598 (0.218) 1.129 (0.120) 2.521 (0.193)
x2 (df) 23.07 (9) 47.30 (8) 9.61 (3) 29.98 (4) 9.61 (4) 6.30 (4) 15.32 (7) 37.92 (8)
M.de la Torre 300 300 379 418 287 341 369 517
LC50 4.74(3.39–6.37) 4.84(3.94–5.74) 0.001(0.0004–0.002) 1.234(1.055–1.444) 0.467(0.373–0.624) 0.337(0.262–0.434) 0.006(0.0042–0.0074) 0.131(0.110–0.158)
RRLC50 13.94 22.00 0.11 18.70 424.54 6.61 8.57 2.43
b (± SE) 1.378 (0.130) 2.223 (0.257) 0.521 (0.085) 2.073 (0.193) 1.604 (0.240) 1.528 (0.137) 1.104 (0.116) 1.903 (0.187)
x2 (df) 16.54 (3) 10.58 (3) 23.09 (4) 36.98 (6) 10.80 (3) 35.25 (4) 18.82 (6) 18.01 (4)
Veracruz 353 430 278 285 337 286 390 315
LC50 14.72(12.22–17.39) 2.41(2.03–2.83) 0.159(0.126–0.197) 0.276(0.227–0.333) 0.320(0.259–0.408) 0.376(0.267–0.538) 0.047(0.029–0.073) 1.908(1.508–2027)
RRLC50 43.29 10.95 17.67 4.18 290.91 7.37 67.14 35.33
b (± SE) 2.270 (0.211) 2.003 (0.166) 1.703 (0.242) 2.112 (0.214) 1.612 (0.178) 1.056 (0.151) 0.776 (0.104) 2.521 (0.279)
x2 (df) 6.26 (4) 2.98 (5) 16.09 (3) 7.73 (3) 7.63 (4) 9.38 (3) 28.51 (5) 37.09 (6)
Coatzacoalcos 380 361 302 370 334 283 225 270
LC50 9.97(8.16–11.82) 1.96(1.64–2.30) 0.175(0.156–0.195) 0.405(0.315–0.509) 0.579(0.448–0.776) 1.364(1.216–1.545) 0.138(0.084–0.243) 0.413(0.281–0.608)
RRLC50 29.32 8.91 19.44 6.14 526.36 26.74 197.14 7.65
b (± SE) 2.110 (0.201) 2.137 (0.191) 3.528 (0.331) 1.538 (0.135) 1.419 (0.148) 3.734 (0.502) 0.758 (0.108) 1.100 (0.160)
x2 (df) 12.03 (4) 26.54 (4) 4.11 (3) 21.74 (5) 10.78 (4) 6.34 (3) 9.35 (4) 20.89 (3)
Cosoleacaque 737 481 293 447 364 246 360 360
LC50 2.18(1.68–2.77) 0.43(0.35–0.51) 0.025(0.019–0.033) 0.155(0.108–0.207) 0.005(0.0008–0.014) 0.102(0.066–0.140) 0.019(0.010–0.033) 1.358(1.082–1.722)
RRLC50 6.41 1.95 2.78 2.35 4.54 2.00 27.14 25.15
b (± SE) 1.063 (0.078) 1.749 (0.162) 1.380 (0.175) 1.243 (0.121) 0.447 (0.088) 1.422 (0.194) 0.588 (0.081) 1.424 (0.168)
x2 (df) 10.96 (10) 7.14 (6) 20.68 (3) 41.69 (6) 20.05 (4) 0.189 (2) 44.37 (4) 22.66 (4)
New Orleans 287 300 340 375 426 370 360 360
LC50 0.34(0.28–0.39) 0.22(0.15–0.27) 0.009(0.003–0.015) 0.066(0.054–0.081) 0.0011(0.0009–0.0013) 0.051(0.041–0.064) 0.0007(0.0002–0.001) 0.054(0.040–0.071)
b (± SE) 2.521 (0.277) 1.737 (0.226) 1.616 (0.173) 1.771 (0.169) 2.403 (0.194) 1.612 (0.150) 0.494 (0.091) 1.378 (0.134)
x2 (df)5 12.87 (3) 17.62 (3) 9.87 (4) 30.55 (4) 43.11 (5) 44.65 (4) 31.02 (4) 21.83 (4)
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1

Number of females tested,

2

LC50, 50% lethal dose in μg/bottle, 95% confidence interval in parentheses,

3

RRLC50, resistance ratio: LC50 resistant strain/LC50 New Orleans strain,

4

Slope of regression line Probit-log, standard error (± SE) are in parentheses,

5

Chi-square (degree of freedom).

The results of mortality at 24 h demonstrated that the majority of the pyrethroid insecticides showed recovery of mosquitoes with the exception of d-phenothrin. For α-cypermethrin and cypermethrin, the results for both resistance after 24 h and kdr were similar. For permethrin, 3 strains exhibited resistance at 24 h, and two exhibited kdr. Surprisingly, three strains showed resistance to deltamethrin at 24 h, the same strains that had moderate kdr of RRKC50≤ 5. For z-cypermethrin, three strains showed resistance at 24 h, where one had already shown kdr and where two were moderately resistant. There was a large recovery rate in mosquitoes exposed to λ–cyalothrin.

Cluster analysis

Cluster analysis was performed on the LC50, KC50, RRLC50, RRKC50 for each of the 8 compounds (Fig. 2). The resulting dendrograms for LC50 and RRLC50 were almost identical so that only a single dendrogram is presented. The same was true for KC50 and RRKC50. The resistance profiles were highly correlated for deltamethrin and λ-cyhalothrin (ρ = 0.924), followed by permethrin with cypermethrin (ρ = 0.911) and d-phenothrin with α-cypermethrin (ρ = 0.796). The resistance statistics for bifenthrin were not correlated with any of the other 7 compounds.

Figure 2.

Figure 2

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UPGMA dendrograms arising cluster analysis of one minus Pearson correlation coefficient for LC50, KD50, RRLC50, RRKD50,for each of the eight pyrethroids.

Principal component and biplot analyses were performed on the LC50, KC50, RRLC50, and RRKC50 values. All four plots were extremely similar and so only the results for RRKC50 are shown (Fig. 3). The first and second principal components accounted for 58.9 and 23.4% (cumulatively 82.3%) of the covariance among LC50 values thus representing a good summary of resistance profiles among collections. The distribution of population principal components suggests that the first principal component represents overall resistance. Tantoyuca, Cosoleacaque, Panuco and M. de la Torre were more susceptible in general to all pyrethroids (except bifenthrin ) compared with Veracruz, Coatzacoalcos and Poza Rica. The second principal component represents resistance to bifenthrin. Veracruz displayed 21 fold resistance. The remainder exhibited moderate resistance or were susceptible. The biplot vectors for all pyrethroids, except bifenthrin, point in a similar direction. As noted in the cluster analysis, note that biplots vector are very similar for deltamethrin and λ-cyhalothrin, permethrin and cypermethrin and for d-phenothrin and α-cypermethrin.

Figure 3.

Figure 3

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Biplot analyses of RRKD50 values. The first and second principal components are represented as solid dots for each of the seven strains. Biplots are represent as arrows for each of the eight pyrethroids.

Regression analysis

Recovery 24 h after exposure provides evidence for additive, possibly metabolic resistance mechanisms. Regression analysis between RRLC50 and RRKC50 are shown in Fig. 4. For d-phenothrin R2= 0.93 with a slope of 0.875. This indicates that the majority of mosquitoes knocked down with d-phenothrin did not recover. In contrast for permethrin R2= 0.96 with a slope of 1.91. This indicates that approximately twice the concentration of permethrin sufficient to knockdown mosquitoes is required to eventually kill those mosquitoes. A similar pattern was seen for the other pyrethoids except for λ–cyalothrin where the slope was 8.67, indicating that approximately 9 fold higher concentration of λ–cyalothrin is necessary to eventually kill knocked down mosquitoes. However, this interpretation should be validated in laboratory bioassays with increased concentrations of permethrin and λ–cyalothrin.

Figure 4.

Figure 4

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Regression analysis between RRLC50 and RRKD50. The proportion of the total variance accounted for by the linear model is R2. The estimated slope is also provided.

Discussion

In Mexico, as in the majority of Latin American countries, various insecticides have been used for vector control. For example DDT, was used extensively in 1960s for the control of malaria, and was eventually followed by the use of organophosphate insecticides such as malathion (adulticide) and temephos (larvicide). For Ae. aegypti control, malathion was used until 1989 in spatial applications, and temephos granules were applied to water containers as potential breeding sites for Ae. aegypti larvae (Fernandez 1999). The use of temephos has been uninterrupted up to the present. Numerous reports have indicated that DDT has conferred cross resistance to pyrethroids (Hemingway et al 1989). In areas such as the Caribbean and South America, there are reports of resistance to pyrethroids in populations of Ae. aegypti in Venezuela (Field et al. 1984, Mazarri and Georgiou 1995), Puerto Rico (Hemingway et al. 1989) and Dominican Republic (Mekuria et al. 1991) associated with cross-resistance to DDT. Reports have shown that resistance associated with pyrethroids is due not only to mutations in the voltage-gated sodium channel gene, but also to enzymatic mechanisms (Brengues et al. 2003, Rodriguez et al. 2005). Similarly, there are numerous reports indicating that the prolonged use of temephos for more than 30 years has produced resistance in Ae. aegypti populations (Paeporn et al. 2003, Bisett et al. 2004, Braga et al. 2004, Rodriguez et al. 2004, Saelim et al. 2005, Montella et al. 2007). In Mexico cross-resistance to permethrin is mediated in part by esterases (Flores et al. 2005, 2006).

An unusual aspect of the present study was the comparison of RRLC50 and RRKC50 values. This aspect was studied following the observation that many of the mosquitoes that were knocked down following permethrin exposure in the bottle bioassay recovered when removed to the insectary away from the pesticide. With the exception of d-phenothrin, from 2–9 fold greater amounts of pyrethroids were required to eventually kill exposed mosquitoes. However, we are uncertain about the relevancy of these results to the field. Assuming a knocked down mosquito falls into an insecticide free environment, it seems to us very likely that the mosquito will die due to desiccation, predation or a greater likelihood of being crushed or damaged. However our results suggest that at least a proportion of knocked down mosquitoes may recover from their initial exposure. This may be especially important with the Ile1,016 resistance allele in which Saavedra-Rodriguez et al. (2007) observed that 90.6% (221/244) of Ile1,016/Val1,016 heterozygotes were knocked down after a 1 hour exposure to permethrin but that 57.4% (127/221) of knocked down heterozygotes eventually recovered. Thus the rate of recovery following knockdown is inherited as an additive genetic trait with heterozygotes exhibiting a intermediate phenotype. The rate of response to selection is predicted to be much more rapid for a trait with an additive genetic inheritance than for a trait with a strictly recessive inheritance (e.g. kdr). This may explain why Ile1,016 has spread so rapidly in Mexico (Ponce et al. 2009; Barbosa et al 2011).

Mosquitoes are capable of developing resistance to the majority of insecticides used for their control, such that the established effective dose suddenly fails. Generally, the immediate response to control failure is to increase the frequency of treatment or the concentration of the insecticide, thus aggravating the problem further. Instead a rational strategy for managing resistance requires understanding of the actual physiological, biochemical and genetic mechanisms involved. Early detection as a preventive method should be complemented with a strategy of rotation of active insecticides which permits the utilization of an alternative compound preferably with a different mode of action so that resistance does not continue to increase. The development of new formulations that reduce resistance or potentiate the action of the insecticide (Curtis et al. 1993, Roush 1989) would also be applicable. These strategies require constant monitoring of populations exposed to these compounds to predict increases in the resistance. Pyrethroid resistance is clearly increasing despite the initial optimism over their rapid action and novelty (Malcom, 1988). Evidence of resistance to permethrin in Ae. aegypti populations in Mexico due to enzymatic mechanisms (Flores et al, 2005, 2006, 2009) and target-site insensitivity (kdr) (Saavedra et al. 2007, 2008; Ponce et al. 2009) suggest that the widespread use of permethrin has conferred cross resistance to many other pyrethroids including those not commonly used in mosquito control in Mexico.

The analyses presented here show that the field strains from the state of Veracruz are resistant to most of the pyrethroids analyzed. Even though RRKC50 were < 10 fold (no knockdown resistance present) for deltamethrin, treated mosquitoes in three strains recovered after 24h. The results suggest that populations in the state of Veracruz have been exposed to strong selection pressure, resulting from the continuous application permethrin for more than 9 years, together with the effect of insecticides applied for the control of agricultural pests and other urban pests. In light of the prevalence of cross resistance to pyrethroid in Veracruz state, it is pertinent to ask if pyrethroids should continue to be insecticides of first choice in Mexico for controlling Ae. aegypti populations.

Acknowledgments

This study was supported by FOMIX CONACyT, Veracruz through Grant No. 68298 within the project “Estrategia de control del mosquito vector del dengue Aedes aegypti (L.) en base a su resistencia a insecticidas en Veracruz”, CONACyt Ciencia Basica Grant No. 102120 within the project “Mutacion’kdr’ asociada a la resistencia a piretroides en Aedes aegypti (L.) en Mexico” and UANL PAICYT. This work was also funded in part by NIH-NIAID U01 AI088647

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