Gustavo Ponce García

Recent Rapid Rise of a Permethrin Knock Down Resistance Allele in Aedes aegypti in México

Background Aedes aegypti, the ‘yellow fever mosquito’, is the primary vector to humans of dengue and yellow fever flaviviruses (DENV, YFV), and is a known vector of the chikungunya alphavirus (CV). Because vaccines are not yet available for DENV or CV or are inadequately distributed in developing countries (YFV), management of Ae. aegypti remains the primary option to prevent and control outbreaks of the diseases caused by these arboviruses. Permethrin is one of the most widely used active ingredients in insecticides for suppression of adult Ae. aegypti. In 2007, we documented a replacement mutation in codon 1,016 of the voltage-gated sodium channel gene (para) of Ae. aegypti that encodes an isoleucine rather than a valine and confers resistance to permethrin. Ile1,016 segregates as a recessive allele conferring knockdown resistance to homozygous mosquitoes at 5–10 µg of permethrin in bottle bioassays. Methods and Findings A total of 81 field collections containing 3,951 Ae. aegypti were made throughout México from 1996 to 2009. These mosquitoes were analyzed for the frequency of the Ile1,016 mutation using a melting-curve PCR assay. Dramatic increases in frequencies of Ile1,016 were recorded from the late 1990s to 2006–2009 in several states including Nuevo León in the north, Veracruz on the central Atlantic coast, and Yucatán, Quintana Roo and Chiapas in the south. From 1996 to 2000, the overall frequency of Ile1,016 was 0.04% (95% confidence interval (CI95) = 0.12%; n = 1,359 mosquitoes examined). The earliest detection of Ile1,016 was in Nuevo Laredo on the U.S. border in 1997. By 2003–2004 the overall frequency of Ile1,016 had increased ∼100-fold to 2.7% (±0.80% CI95; n = 808). When checked again in 2006, the frequency had increased slightly to 3.9% (±1.15% CI95; n = 473). This was followed in 2007–2009 by a sudden jump in Ile1,016 frequency to 33.2% (±1.99% CI95; n = 1,074 mosquitoes). There was spatial heterogeneity in Ile1,016 frequencies among 2007–2008 collections, which ranged from 45.7% (±2.00% CI95) in the state of Veracruz to 51.2% (±4.36% CI95) in the Yucatán peninsula and 14.5% (±2.23% CI95) in and around Tapachula in the state of Chiapas. Spatial heterogeneity was also evident at smaller geographic scales. For example within the city of Chetumal, Quintana Roo, Ile1,016 frequencies varied from 38.3%–88.3%. A linear regression analysis based on seven collections from 2007 revealed that the frequency of Ile1,016 homozygotes accurately predicted knockdown rate for mosquitoes exposed to permethrin in a bioassay (R2 = 0.98). Conclusions We have recorded a dramatic increase in the frequency of the Ile1,016 mutation in the voltage-gated sodium channel gene of Ae. aegypti in México from 1996 to 2009. This may be related to heavy use of permethrin-based insecticides in mosquito control programs. Spatial heterogeneity in Ile1,016 frequencies in 2007 and 2008 collections may reflect differences in selection pressure or in the initial frequency of Ile1,016. The rapid recent increase in Ile1,016 is predicted by a simple model of positive directional selection on a recessive allele. Unfortunately this model also predicts rapid fixation of Ile1,016 unless there is negative fitness associated with Ile1,016 in the absence of permethrin. If so, then spatial refugia of susceptible Ae. aegypti or rotational schedules of different classes of adulticides could be established to slow or prevent fixation of Ile1,016.

MECHANISMS OF INSECTICIDE RESISTANCE IN FIELD POPULATIONS OF AEDES AEGYPTI (L.) FROM QUINTANA ROO, SOUTHERN MEXICO

ABSTRACT Potential insecticide-resistance mechanisms were studied with the use of biochemical assays in Aedes aegypti (L.) collected from 5 municipalities representing the north part of Quintana Roo: Benito Juarez, Cozumel, Isla Mujeres, Lazaro Cardenas, and Solidaridad. The activities of &agr; and &bgr; esterases, mixed-function oxidases (MFO), glutathione-S-transferase (GST), acethylcholinesterase (AChE), and insensitive acethylcholinesterase (iAChE) were assayed in microplates. Three replicates were performed for each enzyme and 60 males and 60 females were analyzed in each population. The New Orleans (NO) susceptible strain of Ae. aegypti was used as a susceptible reference and the threshold criteria for each enzyme were the highest NO absorbance values. In none of the 6 tests were absorbance values correlated in males and females. &agr; esterases were elevated in Benito Juarez, Cozumel females and in Lazaro Cardenas males and females. &bgr; esterases were elevated in Benito Juarez, Cozumel females and in Cozumel and Lazaro Cardenas males. Elevated esterases suggest potential insecticide-resistance mechanisms against organophosphate, carbamate, and some pyrethroid insecticides. Slightly elevated levels of MFOs appeared in Lazaro Cardenas females and in Cozumel, Isla Mujeres, and Solidaridad males. Mechanisms involving iAChE or GST were not apparent.

Resistance to Permethrin in Aedes aegypti (L.) in Northern Mexico

Abstract. Diagnostic dose (DD) of permethrin was determined in Aedes aegypti (L.), from three states (Coahuila, Sonora, and Tamaulipas) in northern Mexico. After exposing 10 groups of 100 females to the DD obtained and producing 50% mortality, individuals were divided into two categories: survivors and dead. Mosquitoes in each of these categories were dissected to separate the head, thorax, and abdomen. Biochemical tests were done on the head and thorax to determine activity by resistance-related enzymes including: &agr; and &bgr;-esterases, mixed-function oxidases (MFOs), glutathione-S-transferase (GST) and insensitive acetyl cholinesterase (iAChE). Results were compared with those for a susceptible strain of Ae. aegypti from New Orleans. A population from Coahuila showed iAChE as the only enzyme activity that surpassed the threshold established with the susceptible strain, however, this mechanism is not associated with resistance to pyrethroids, but rather with resistance to organophosphate and carbamate insecticides. For the populations from Tamaulipas, none of the mechanisms studied were important in conferring resistance to permethrin. MFOs were present at elevated levels of activity, appearing as the main detoxifying mechanism, in the population from Sonoita, Sonora state.