Adriana E. Flores

A mutation in the voltage-gated sodium channel gene associated with pyrethroid resistance in Latin American Aedes aegypti

Pyrethroids are commonly used as mosquito adulticides and evolution of resistance to these compounds is a major threat to public health. ‘Knockdown resistance’ to pyrethroids (kdr) is frequently caused by nonsynonymous mutations in the voltage‐gated sodium channel transmembrane protein (para) that reduce pyrethroid binding. Early detection of kdr is critical to the development of resistance management strategies in mosquitoes including Aedes aegypti, the most prevalent vector of dengue and yellow fever viruses. Brengues et al. described seven novel mutations in hydrophobic segment 6 of domain II of para in Ae. aegypti. Assays on larvae from strains bearing these mutations indicated reduced nerve sensitivity to permethrin inhibition. Two of these occurred in codons Iso1011 and Val1016 in exons 20 and 21 respectively. A transition in the third position of Iso1011 encoded a Met1011 replacement and a transversion in the second position of Val1016 encoded a Gly1016 replacement. We have screened this same region in 1318 mosquitoes in 32 additional strains; 30 from throughout Latin America. While the Gly1016 allele was never detected in Latin America, we found two new mutations in these same codons. A transition in the first position of codon 1011 encodes a Val replacement while a transition in the first position of codon 1016 encodes an Iso replacement. We developed PCR assays for these four mutations that can be read either on an agarose gel or as a melting curve. Selection experiments, one with deltamethrin on a field strain from Santiago de Cuba and another with permethrin on a strain from Isla Mujeres, Mexico rapidly increased the frequency of the Iso1016 allele. Bioassays of F3 offspring arising from permethrin susceptible Val1016 homozygous parents and permethrin resistant Iso1016 homozygous parents show that Iso1016 segregates as a recessive allele in conferring kdr. Analysis of segregation between alleles at the 1011 and 1016 codons in the F3 showed a high rate of recombination even though the two codons are only separated by a ~250 bp intron. The tools and information presented provide a means for early detection and characterization of kdr that is critical to the development of strategies for resistance management.

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.

Wide spread cross resistance to pyrethroids in Aedes aegypti (Diptera: Culicidae) from Veracruz state Mexico.

ABSTRACT Seven F1 strains of Aedes aegypti (L.) were evaluated by bottle bioassay for resistance to the pyrethroids d-phenothrin, permethrin, deltamethrin, &lgr;-cyalothrin, bifenthrin, cypermethrin, á-cypermethrin, and z-cypermethrin. The New Orleans strain was used as a susceptible control. Mortality rates after a 1 h exposure and after a 24 h 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. The RRKC50 with &agr;-cypermethrin varied from 10 to 100 among strains indicating high levels of knockdown resistance. Most of the strains had moderate resistance to (d-phenothrin. Significant but much lower levels of resistance were detected for &lgr;-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 RRKC50 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 ensure lethality. Slopes ranged from 0.875 for d-phenothrin (RRLC50 RRKC50) to 8.67 for ë-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.

Effect of different densities of the twospotted spider mite Tetranychus urticae on CO2 assimilation, transpiration, and stomatal behaviour in rose leaves.

The effect of population density of Tetranychusurticae Koch on CO2 assimilation, transpiration and stomatal behaviour in rose leaves and on the diameter and length of stems and flower buds was investigated under greenhouse conditions. The investigation was performed in order to gain more insight into integrated control systems in rose crops grown under greenhouse conditions. Physiological processes, such as photosynthesis and transpiration, as well as stomatal behaviour and chlorophyll content, were studied as they form part of the plants nutrition mechanism and therefore affect the quantity and quality of the flowers. Information related to the effect of spider mite population density on bloom quality, diameter and length of stems and flower buds was also collected. The data indicate that increased mite density coincides with a decrease in the net photosynthetic rate, transpiration and chlorophyll content. Higher mite densities on leaves cause stomata to remain open for longer periods, which allows a greater loss of water. Spider mite densities of 10 and 50 mites per leaf cause a reduction in flower stem length of 17 and 26%, respectively, as compared to plants with no mites present.

PRICKLY PEAR CACTI PESTS AND THEIR CONTROL IN MEXICO

Opuntia spp., known by Mexicans as nopal, represents historically one of the most important biotic elements of Mexico. This natural resource has been and is being used for multiple purposes. Some of the current uses include: food for humans as both vegetable and fruit, forage for animals, source for alcoholic beverages, sweetener, live fences, industrial products such as cosmetics and dye, and as a medical source against diabetes and other diseases. Its cultural and natural values have been reflected in paintings, ancestral Indian codes, and old writings; thus its historic relevance is quite apparent. Furthermore, it is depicted both in the Mexican national seal and flag where it represents the very characteristic feature of Mexican culture and society. Opuntia spp. are distributed throughout the American continent and Mexico is considered a center of diversity as these species are well adapted to the arid and semiarid conditions of Mexico. Here we summarize and discuss briefly the most important insect pest species and one snail species which currently are considered as serious pests of cultivated Opuntia spp. in Mexico, and, thus require control measures. The control of these pest species is mainly through chemical pesticides and currently at least a dozen types of insecticides are being applied.

Differential transcription profiles in Aedes aegypti detoxification genes after temephos selection.

The mosquito Aedes aegypti is the main vector of Dengue and Yellow Fever flaviviruses. The organophosphate insecticide temephos is a larvicide that is used globally to control Ae. aegypti populations; many of which have in turn evolved resistance. Target site alteration in the acetylcholine esterase of this species has not being identified. Instead, we tracked changes in transcription of metabolic detoxification genes using the Ae. aegypti ‘Detox Chip’ microarray during five generations of temephos selection. We selected for temephos resistance in three replicates in each of six collections, five from Mexico, and one from Peru. The response to selection was tracked in terms of lethal concentrations. Uniform upregulation was seen in the epsilon class glutathione‐S‐transferase (eGST) genes in strains from Mexico prior to laboratory selection, while eGSTs in the Iquitos Peru strain became upregulated after five generations of temephos selection. While expression of many carboxyl/cholinesterase esterase (CCE) genes increased with selection, no single esterase was consistently upregulated and this same pattern was noted in the cytochrome P450 monooxygenase (CYP) genes and in other genes involved in reduction or oxidation of xenobiotics. Bioassays using glutathione‐S‐transferase (GST), CCE and CYP inhibitors suggest that various CCEs instead of GSTs are the main metabolic mechanism conferring resistance to temephos. We show that temephos‐selected strains show no cross resistance to permethrin and that genes associated with temephos selection are largely independent of those selected with permethrin in a previous study.

Frequency of V1016I and F1534C mutations in the voltage-gated sodium channel gene in Aedes aegypti in Venezuela.

BACKGROUND The V1016I and F1534C mutations in the voltage-gated sodium channel gene have been associated with resistance to pyrethroids and DDT in Aedes aegypti mosquitoes. A study was carried out to determine the frequency of I1016 and C1534 by real-time PCR in five natural populations of Ae. aegypti in Venezuela during 2008, 2010 and 2012, as well as in a strain selected with 0.14 µg of deltamethrin for 15 generations. RESULTS In natural populations, frequencies of I1016 varied between 0.01 and 0.37, and frequencies of C1534 between 0.35 and 1.0. In the Pampanito strain, the frequency of I1016 increased from 0.02 in F1 up to 0.5 in F15 and from 0.35 up to fixation for C1534 after selection with deltamethrin. CONCLUSION The results showed that C1534 frequencies are higher than I1016 frequencies in natural populations of Ae. aegypti in Venezuela, and that deltamethrin selected the C1534 more rapidly than I1016.

Resistance to malathion and deltamethrin in Aedes aegypti (Diptera: Culicidae) from western Venezuela.

ABSTRACT Resistance to the insecticides deltamethrin and malathion and the enzymes associated with metabolic resistance mechanisms were determined in four field 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 field 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 significantly 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 &agr;-esterases for 2008 and 2010 and &bgr;-esterases for 2008.

Update On the Frequency of Ile1016 Mutation In Voltage-Gated Sodium Channel Gene of Aedes aegypti In Mexico

Abstract We analyzed 790 Aedes aegypti from 14 localities of Mexico in 2009 to update information on the frequency of the Ile1016 allele in the voltage-gated sodium channel gene that confers resistance to pyrethroids and DDT. The Ile1016 mutation was present in all 17 collections, and was close to fixation <]?>in Acapulco (frequency  =  0.97), Iguala (0.93), and San Nicolas (0.90). Genotypes at the 1016 locus were not in Hardy–Weinberg proportions in collections from Panuco, Veracruz, Cosoleacaque, Coatzacoalcos, Tantoyuca, and Monterrey due in every case to an excess of homozygotes. The high frequencies of this mutation in Ae. aegypti are probably due to selection pressure from pyrethroid insecticides, particularly permethrin, which has been used in mosquito control programs for >10 years in Mexico.