Guadalupe Reyes-Solis

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.

Rhythms and synchronization patterns in gene expression in the Aedes aegypti mosquito

BackgroundAedes aegypti is arguably the most studied of all mosquito species in the laboratory and is the primary vector of both Dengue and Yellow Fever flaviviruses in the field. A large number of transcriptional studies have been made in the species and these usually report transcript quantities observed at a certain age or stage of development. However, circadian oscillation is an important characteristic of gene expression in many animals and plants, modulating both their physiology and behavior. Circadian gene expression in mosquito species has been previously reported but for only a few genes directly involved in the function of the molecular clock.ResultsHerein we analyze the transcription profiles of 21,494 messenger RNAs using an Ae. aegypti Agilent® microarray. Transcripts were quantified in adult female heads at 24 hours and then again at 72 hours and eight subsequent time points spaced four hours apart. We document circadian rhythms in multiple molecular pathways essential for growth, development, immune response, detoxification/pesticide resistance. Circadian rhythms were also noted in ribosomal protein genes used for normalization in reverse transcribed PCR (RT-PCR) to determine transcript abundance. We report pervasive oscillations and intricate synchronization patterns relevant to all known biological pathways.ConclusionThese results argue strongly that transcriptional analyses either need to be made over time periods rather than confining analyses to a single time point or development stage or exceptional care needs to be made to synchronize all mosquitoes to be analyzed and compared among treatment groups.

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.

Local Evolution of Pyrethroid Resistance Offsets Gene Flow among Aedes aegypti Collections in Yucatan State, Mexico

The mosquito Aedes aegypti is the major vector of the four serotypes of dengue virus (DENV1-4). Previous studies have shown that Ae. aegypti in Mexico have a high effective migration rate and that gene flow occurs among populations that are up to 150 km apart. Since 2000, pyrethroids have been widely used for suppression of Ae. aegypti in cities in Mexico. In Yucatan State in particular, pyrethroids have been applied in and around dengue case households creating an opportunity for local selection and evolution of resistance. Herein, we test for evidence of local adaptation by comparing patterns of variation among 27 Ae. aegypti collections at 13 single nucleotide polymorphisms (SNPs): two in the voltage-gated sodium channel gene para known to confer knockdown resistance, three in detoxification genes previously associated with pyrethroid resistance, and eight in putatively neutral loci. The SNPs in para varied greatly in frequency among collections, whereas SNPs at the remaining 11 loci showed little variation supporting previous evidence for extensive local gene flow. Among Ae. aegypti in Yucatan State, Mexico, local adaptation to pyrethroids appears to offset the homogenizing effects of gene flow.

Chikungunya Virus in Febrile Humans and Aedes aegypti Mosquitoes, Yucatan, Mexico.

Chikungunya virus (CHIKV) was isolated from 12 febrile humans in Yucatan, Mexico, in 2015. One patient was co-infected with dengue virus type 1. Two additional CHIKV isolates were obtained from Aedes aegypti mosquitoes collected in the homes of patients. Phylogenetic analysis showed that the CHIKV isolates belong to the Asian lineage.

Vacant Lots: Productive Sites for Aedes (Stegomyia) aegypti (Diptera: Culicidae) in Mérida City, México

ABSTRACT We assessed the potential for vacant lots and other nonresidential settings to serve as source environments for Aedes (Stegomyia) aegypti (L.) (Diptera: Culicidae) in Mérida City, México. Mosquito immatures were collected, during November 2011–June 2013, from residential premises (n = 156 site visits) and nonresidential settings represented by vacant lots (50), parking lots (18), and streets or sidewalks (28). Collections totaled 46,025 mosquito immatures of 13 species. Ae. aegypti was the most commonly encountered species accounting for 81.0% of total immatures, followed by Culex quinquefasciatus Say (12.1%). Site visits to vacant lots (74.0%) were more likely to result in collection of Ae. aegypti immatures than residential premises (35.9%). Tires accounted for 75.5% of Ae. aegypti immatures collected from vacant lots. Our data suggest that vacant lots should be considered for inclusion in mosquito surveillance and control efforts in Mérida City, as they often are located near homes, commonly have abundant vegetation, and frequently harbor accumulations of small and large discarded water-holding containers that we now have demonstrated to serve as development sites for immature mosquitoes. In addition, we present data for associations of immature production with various container characteristics, such as storage capacity, water quality, and physical location in the environment.

Urban Mosquito Fauna in Mérida City, México: Immatures Collected from Containers and Storm-Water Drains/Catch Basins

Abstract. We examined the species composition and temporal occurrence of immature mosquitoes in containers and storm-water drains/catch basins from November 2011 to June 2013 in Mérida City, México. A wide range of urban settings were examined, including residential premises, vacant lots, parking lots, and streets or sidewalks with storm-water drains/catch basins. In total, 111,776 specimens of 15 species were recorded. The most commonly collected species were Aedes (Stegomyia) aegypti (L.) (n = 60,961) and Culex quinquefasciatus Say (45,702), which together accounted for 95.4% of the immatures collected. These species were commonly encountered during both rainy and dry seasons, whereas most other mosquito species were collected primarily during the rainy season. Other species collected were Aedes (Howardina) cozumelensis Diaz Najera, Aedes (Ochlerotatus) taeniorhynchus (Wiedemann), Aedes (Ochlerotatus) trivittatus (Coquillett), Culex coronator Dyar and Knab, Culex interrogator Dyar and Knab, Culex lactator Dyar and Knab, Culex nigripalpus Theobald, Culex salinarius Coquillett, Culex tarsalis Coquillett, Culex thriambus Dyar, Haemagogus equinus Theobald, Limatus durhamii Theobald, and Toxorhynchites rutilus (Coquillett). The greatest number of species was recorded from vacant lots (n = 11), followed by storm-water drains/catch basins (nine) and residential premises (six). Our study demonstrated that the heterogeneous urban environment in Mérida City supports a wide range of mosquito species, many of which are nuisance biters of humans and/or capable of serving as vectors of pathogens affecting humans or domestic animals. We also briefly reviewed the medical importance of the encountered mosquito species.

QTL mapping of genome regions controlling temephos resistance in larvae of the mosquito Aedes aegypti.

Introduction The mosquito Aedes aegypti is the principal vector of dengue and yellow fever flaviviruses. Temephos is an organophosphate insecticide used globally to suppress Ae. aegypti larval populations but resistance has evolved in many locations. Methodology/Principal Findings Quantitative Trait Loci (QTL) controlling temephos survival in Ae. aegypti larvae were mapped in a pair of F3 advanced intercross lines arising from temephos resistant parents from Solidaridad, México and temephos susceptible parents from Iquitos, Peru. Two sets of 200 F3 larvae were exposed to a discriminating dose of temephos and then dead larvae were collected and preserved for DNA isolation every two hours up to 16 hours. Larvae surviving longer than 16 hours were considered resistant. For QTL mapping, single nucleotide polymorphisms (SNPs) were identified at 23 single copy genes and 26 microsatellite loci of known physical positions in the Ae. aegypti genome. In both reciprocal crosses, Multiple Interval Mapping identified eleven QTL associated with time until death. In the Solidaridad×Iquitos (SLD×Iq) cross twelve were associated with survival but in the reciprocal IqxSLD cross, only six QTL were survival associated. Polymorphisms at acetylcholine esterase (AchE) loci 1 and 2 were not associated with either resistance phenotype suggesting that target site insensitivity is not an organophosphate resistance mechanism in this region of México. Conclusions/Significance Temephos resistance is under the control of many metabolic genes of small effect and dispersed throughout the Ae. aegypti genome.

Aedes (Stegomyia) aegypti and Aedes (Howardina) cozumelensis in Yucatán State, México, with a summary of published collection records for Ae. cozumelensis

ABSTRACT: We collected mosquito immatures from artificial containers during 2010–2011 from 26 communities, ranging in size from small rural communities to large urban centers, located in different parts of Yucatán State in southeastern México. The arbovirus vector Aedes (Stegomyia) aegypti was collected from all 26 examined communities, and nine of the communities also yielded another container-inhabiting Aedes mosquito: Aedes (Howardina) cozumelensis. The communities from which Ae. cozumelensis were collected were all small rural communities (<6,000 inhabitants) in the north-central part of Yucatán State. These new collection records for Ae. cozumelensis demonstrate that this mosquito has a far broader geographic range in the Yucatán Peninsula than previously known. Ae. cozumelensis immatures were collected from both residential premises and cemeteries, with specimens recovered from rock holes as well as various artificial containers including metal cans, flower vases, buckets, tires, and a water storage tank The co-occurrence with Ae. aegypti in small rural communities poses intriguing questions regarding linkages between these mosquitoes, including the potential for direct competition for larval development sites. Additional studies are needed to determine how commonly Ae. cozumelensis feeds on human blood and whether it is naturally infected with arboviruses or other pathogens of medical or veterinary importance. We also summarize the published records for Ae. cozumelensis, which are restricted to collections from Mexicos Yucatán Peninsula and Belize, and uniformly represent geographic locations where Ae. aegypti can be expected to occur.

Ecological Niche Model for Predicting Distribution of Disease-Vector Mosquitoes in Yucatán State, México

Abstract The majority of the Yucatán State, México, presents subtropical climate that is suitable for many species of mosquitoes that are known to be vectors of diseases, including those from the genera Aedes and Culex. The objective of this study is to identify the geographic distribution of five species from these two genera and estimate the human population at risk of coming in contact with them. We compiled distributional data for Aedes aegypti (L.), Aedes (Howardina) cozumelensis (Diaz Najera), Culex coronator Dyar and Knab, Culex quinquefasciatus Say, and Culex thriambus Dyar from several entomological studies in Yucatán between March 2010 and September 2014. Based on these data, we constructed ecological niche models to predict the spatial distribution of each species using the MaxEnt algorithm. Our models identified areas with suitable environments for Ae. aegypti in most of Yucatán. A similar percentage of urban (97.1%) and rural (96.5%) populations were contained in areas of highest suitability for Ae. aegypti, and no spatial pattern was found (Morans I = 0.33, P = 0.38); however, we found an association of abundance of immature forms of this species with annual mean temperature (r = 0.19, P≤0.001) and annual precipitation (r = 0.21, P≤0.001). Aedes cozumelensis is also distributed in most areas of the Yucatán State; Cx. quinquefasciatus, Cx. coronator, and Cx. thriambus are restricted to the northwest. The information generated in this study can inform decision-making to address control measures in priority areas with presence of these vectors.