**5. Insecticide resistance in** *Ae. aegypti* **— A threat to its control**

The extensive use of DDT to control *Ae. aegypti* in Mexico and other parts of the Americas during the 1950s and 1960s resulted in the development of resistance [36]. This action was unfortunate because both DDT and pyrethroids target voltage-gated sodium channels in the insect nerve sheath where structure-related interactions occur in specific regions of the sodium channels that prolong their opening and produce paralysis. Indeed, the similar mode of action probably produced cross-resistance to pyrethroids in DDT-resistant *Ae. aegypti* [37–41].

Pyrethroid resistance is clearly increasing despite the initial optimism over their rapid action and novelty [42]. Evidence of resistance to permethrin insecticide used in Mexico for more than 10 years in *Ae. aegypti* populations in Mexico due to enzymatic mechanisms such as α- aand β-esterases was reported in Baja California North and South [43], in Quintana Roo, south f Mexico [31], and some states of northeast Mexico [44]. More recently, Aponte et al. [45] found increased levels of esterases and glutathione S-transferase related with resistance to DDT, permethrin, and deltamethrin in *Ae. aegypti* populations from the state of Guerrero located on the west coast of Mexico.

The presence of a kdr mutation V1016I in the voltage-gated sodium channel gene is also associated with resistance to pyrethroids. This mutation was originally found in a permethrin resistant strain from Isla Mujeres, off the coast of Cancun [46,47]. 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 [48,49].

Flores et al. [50] reported an extensive monitoring of the frequency of kdr Ile1,016 in *Ae. aegypti* populations from Merida, Yucatan, south of Mexico, as part of the "Casa Segura" project. *Ae. aegypti* collections were characterized by both molecular kdr and biochemical resistance to pyrethroid insecticides such as permethrin and deltamethrin. Ile1,016 allele frequencies varied among collection sites ranging from 0.14 to 0.98. Within Merida City, fifteen collection sites had medium to high homozygote frequencies. The lowest Ile/Ile homozygote frequencies corresponded to small towns nearby Merida City.

A second mutation F1534C on the IIIS6 domain of the same gene was also detected in *Ae. aegypti* populations from Guerrero state located on the west coast of Mexico [45] and the Yucatan Peninsula [51] conferring resistance to pyrethroids.

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 [52–54]. Mathematical models have been applied for estimating how these tools could be used in an optimal manner [55]. 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 popula‐ tions [56].

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 specific pyrethroids [56,57]. Changes in the frequency of resistance genes were monitored for 4 years [57]. The results were promising and predicted that rotations or mosaics of insecticides are viable long-term strat‐ egies for the sustainable use of insecticides in disease control programs.

With that goal in mind [58], the resistance to eight pyrethroids in collections of *Ae. aegypti* from the state of Veracruz located on the east coast of Mexico was examined, considering that this knowledge would facilitate the selection of viable alternative pyrethroids besides permethrin for use in a rotation program for sustained control of *Ae. aegypti* at the local, regional, and possibly statewide levels. The results obtained showed that the strains analyzed were resistant to δ-phenothrin, deltamethrin, cypermethrin, α-cypermethrin, z-cypermethrin, λ-cyhalothrin, bifenthrin, as well as permethrin and suggested that populations in the state of Veracruz have been exposed to strong selection pressure, resulting from the continuous application of permethrin for more than a decade. They also evaluated resistance to chlorpyrifos [59] in the same strains, and overall, the populations in this study were less resistant to chlorpyrifos than to pyrethroids, so the rotation of insecticides in the control activities is suggested to delay or minimize the occurrence of high levels of resistance to chlorpyrifos among local populations of *Ae. aegypti*.

insect nerve sheath where structure-related interactions occur in specific regions of the sodium channels that prolong their opening and produce paralysis. Indeed, the similar mode of action probably produced cross-resistance to pyrethroids in DDT-resistant *Ae. aegypti* [37–41].

Pyrethroid resistance is clearly increasing despite the initial optimism over their rapid action and novelty [42]. Evidence of resistance to permethrin insecticide used in Mexico for more than 10 years in *Ae. aegypti* populations in Mexico due to enzymatic mechanisms such as α- aand β-esterases was reported in Baja California North and South [43], in Quintana Roo, south f Mexico [31], and some states of northeast Mexico [44]. More recently, Aponte et al. [45] found increased levels of esterases and glutathione S-transferase related with resistance to DDT, permethrin, and deltamethrin in *Ae. aegypti* populations from the state of Guerrero located on

The presence of a kdr mutation V1016I in the voltage-gated sodium channel gene is also associated with resistance to pyrethroids. This mutation was originally found in a permethrin resistant strain from Isla Mujeres, off the coast of Cancun [46,47]. 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 [48,49].

Flores et al. [50] reported an extensive monitoring of the frequency of kdr Ile1,016 in *Ae. aegypti* populations from Merida, Yucatan, south of Mexico, as part of the "Casa Segura" project. *Ae. aegypti* collections were characterized by both molecular kdr and biochemical resistance to pyrethroid insecticides such as permethrin and deltamethrin. Ile1,016 allele frequencies varied among collection sites ranging from 0.14 to 0.98. Within Merida City, fifteen collection sites had medium to high homozygote frequencies. The lowest Ile/Ile homozygote

A second mutation F1534C on the IIIS6 domain of the same gene was also detected in *Ae. aegypti* populations from Guerrero state located on the west coast of Mexico [45] and the

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 [52–54]. Mathematical models have been applied for estimating how these tools could be used in an optimal manner [55]. 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 popula‐

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 specific pyrethroids [56,57]. Changes in the frequency of resistance genes were monitored for 4 years [57]. The results were promising and predicted that rotations or mosaics of insecticides are viable long-term strat‐

With that goal in mind [58], the resistance to eight pyrethroids in collections of *Ae. aegypti* from the state of Veracruz located on the east coast of Mexico was examined, considering that this

egies for the sustainable use of insecticides in disease control programs.

frequencies corresponded to small towns nearby Merida City.

Yucatan Peninsula [51] conferring resistance to pyrethroids.

the west coast of Mexico.

102 Insecticides Resistance

tions [56].

Saavedra-Rodriguez et al. [60] examined changes in gene expression before, during and after five generations of permethrin laboratory selection in five strains of *Ae. aegypti* collections from the Yucatan Peninsula of Mexico. Changes in expression of 290 metabolic detoxification genes were measured using the *Aedes Detox* microarray. Selection simultaneously increased the LC50, KC50, and Ile1,016 frequency. Ten to eight genes were differentially transcribed after selection, and it was an inverse relationship between the Ile1,016 frequency and the numbers of differ‐ entially transcribed genes. Some genes were differential transcribed among field strains, but interestingly a few cytochrome P450 genes complex were overexpressed. The authors estab‐ lished that adaptation to permethrin in *Ae. aegypti* laboratory strain is conditioned presumably by geographic origin and extant target site insensitivity in the *para* gene. The lack of uniformity in the genes that responded to artificial selection as well as differences in the direction of their responses challenges the assumption that one or a few genes control permethrin metabolic resistance.

The selection pressure by the prolonged use of pyrethroids in Mexico had resulted in resistance to all of this kind of chemicals recommended for vector control in Mexico. All studies have shown the prevalence of cross-resistance caused by metabolic mechanisms and/or point mutations. Saavedra et al. [51] demonstrated that even in the absence of barriers to gene flow, local insecticide pressure, rather than the migration of mosquitoes with kdr-conferring mutations, is the primary determinant of the local kdr profile for *Ae. aegypti*. Thus, the early detection of insecticide resistance is highly relevant to establish a rotation program for insecticide resistance management in *Ae. aegypti* in Mexico. In an attempt to establish the importance of evaluating the strength of available techniques to assess the insecticide sus‐ ceptibility in *Ae. aegypti*, Lopez et al. (in press) conducted a study establishing the intensity of insecticide resistance through the Resistance Intensity Rapid Diagnostic Test (I-RDT) [61]. The RDT-I consists of exposing vector populations 1, 2, 5 and ten times the diagnostic dose previously established at a diagnosis time. For this study, they used four populations of *Ae. aegypti* from the state of Yucatan, south of Mexico, and three population from the state of Nuevo Leon, northeastern Mexico. They were exposed to the diagnostic dose (DD) of permethrin, bifenthrin, and d-(cis-trans)-phenothrin and enhanced DD at 2, 5, and 10 times. All populations resulted resistant to the pyrethroids evaluated according to WHO recommendations for assessing the significance of detected resistance (<90%) even when the DD was enhanced 5 times. To correlate these results with pyrethroid molecular resistance mechanisms, DNA from mosquitoes of each population were used to detect V1016I and F1534C mutations. The allelic frequency of Ile1,016 varied from 0.43 to 0.90 in the populations studied. For the 1534 locus, there was a predominance of homozygous mutant genotype in all populations with high frequencies of the mutant allele (0.75–1), showing that the F1534C mutation was more common than V1016I mutation. They also analyzed the co-occurrence of both V1016I and F1534C mutations, and results showed that more than 50% of mosquitoes genotyped expressed both mutations (double homozygous mutants).
