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Knockdown resistance (kdr) genotypes and collection information for Aedes aegytpi from Iquitos, Peru (2000 - 2017)

Citation

Baltzegar, Jennifer (2021), Knockdown resistance (kdr) genotypes and collection information for Aedes aegytpi from Iquitos, Peru (2000 - 2017), Dryad, Dataset, https://doi.org/10.5061/dryad.2fqz612pc

Abstract

This study describes the evolution of knockdown resistance (kdr) haplotypes in Aedes aegypti in response to pyrethroid insecticide use over the course of 18 years in Iquitos, Peru. Based on the duration and intensiveness of sampling (~10,000 samples), this is the most thorough study of kdr population genetics in Ae. aegypti to date within a city. We provide evidence for the direct connection between programmatic citywide pyrethroid spraying and the increase in frequency of specific kdr haplotypes by identifying two evolutionary events in the population. The relatively high selection coefficients, even under infrequent insecticide pressure, emphasize how quickly Ae. aegypti populations can evolve. In our examination of the literature on mosquitoes and other insect pests, we could find no cases where a pest evolved so quickly to so few exposures to low or non-residual insecticide applications. The observed rapid increase in frequency of resistance alleles might have been aided by the incomplete dominance of resistance-conferring alleles over corresponding susceptibility alleles. In addition to dramatic temporal shifts, spatial suppression experiments reveal that genetic heterogeneity existed not only at the citywide scale, but also on a very fine scale within the city.

Methods

Entomological Surveys

Control History. During the past two decades, researchers have preserved mosquito specimens collected throughout the city of Iquitos in a repository (e.g. Cavany et al., 2020; Cromwell et al., 2017; Getis et al., 2003; Gunning et al., 2018; LaCon et al., 2014; Lenhart et al., 2020; Morrison et al., 2004a; Morrison et al., 2004b; Morrison et al., 2006; Morrison et al., 2008; Reiner et al., 2019; Schneider et al., 2004; Tun-Lin et al., 2009). This repository holds samples that were collected prior to pyrethroid application (2000 – 2002), during citywide pyrethroid use (2002 – 2014), and after pyrethroids were discontinued by the Ministry of Health (2014 – 2017). Prior to 2002, no citywide insecticide spraying targeted to control Ae. aegypti occurred. Once targeted control began, multiple sub-classes of pyrethroid insecticides were sprayed by the Iquitos Ministry of Health inside homes from 2002 – 2014. These sub-classes included deltamethrin, cypermethrin, alpha-cypermethrin, lambda-cyhalothrin, and alpha-cypermethrin + pyriproxyfen (Suppl. Tbl. 1). It was uncommon for residents to spray their own homes (A. Morrison, personal communication). In 2014, pyrethroids were discontinued in favor of malathion due to the development of phenotypic pyrethroid resistance (Gunning et al., 2018). During the years of pyrethroid applications, spraying occurred at an average of 3.25 treatments per year, with spraying occurring within a one-month period in most years. Because the applications were Ultra Low Volume space sprays with no residual, most generations of the mosquitoes in any year were not exposed to insecticide and mosquitoes were collected independent of whether sprays had been conducted (Ritchie et al. 2021).

Temporal Collections. Aedes aegypti were collected and stored at -80˚C by NAMRU-6 and University of California at Davis personnel since the late 1990s. Specimens dating back to the year 2000 were available for study. Mosquitoes were collected by backpack aspirator (Clark et al., 1994) prior to June 2009 and by Prokopack Aspirator following June 2009 (Vasquez-Prokopec et al., 2009; Reiner et al., 2019). Each mosquito in the repository was identified to species, sex, collection date, and collection site. Each collection site, typically an individual household, was associated with GPS coordinates (Fig. 1).

Spatial Collections. Intense suppression experiments based on pyrethroid spraying were conducted in 2013 and 2014 (Gunning et al., 2018) to test the predictions of a detailed Ae. aegypti population dynamics model (Magori et al., 2009). In brief, two areas of the city were identified as having relatively high densities of Ae. aegypti and were configured spatially in a way that allowed for a central spray sector with an outer buffer sector to act as an experimental control region (Fig. 2). To limit the impact of migration on resistance allele frequency, site dimensions were selected to be 3 – 5 times larger than the expected Ae. aegypti lifetime flight distance of approximately 150 m (Harrington et al., 2005).

The 2013 study site covered approximately 750 m x 450 m and contained 1,163 houses. Baseline samples were collected in January 2013. Systematic sampling began on 22 April 2013 and continued for 16 weeks until 8 August 2013. From 29 April 2013 to 3 June 2013, six weekly non-residual, indoor ultra-low volume (ULV) cypermethrin treatments were applied in the treatment sector.

The 2014 study site was larger and covered an approximate 600 m x 600 m area and contained 2,166 houses. Systematic sampling was conducted over a longer period of 44 weeks. ULV spraying of cypermethrin was performed from 28 April 2014 through 2 June 2014 in a similar manner as in the 2013 study. In addition to the study spray in 2014, a citywide spray was conducted in response to a dengue outbreak in February 2014, during which homes in both the experimental and buffer sectors were sprayed with pyrethroids. Throughout both suppression experiments, mosquitoes were collected and stored as described above.

DNA Extractions and Quantification

Male mosquitoes were chosen for genetic analysis throughout this study because female mosquitoes were typically the focus of virology and epidemiological studies and, therefore more males were available in the repository. Using females would also have brought the risk of genomic contamination from male mosquitoes (via insemination) and from humans (via human blood feeding). Males are expected to share similar allele frequencies with females because the VGSC is not sex-linked.

Whole mosquitoes were transferred from Iquitos, Peru to Raleigh, North Carolina, USA with permits from Peruvian and US authorities. Samples were stored at -80˚C prior to genomic DNA (gDNA) isolation and at -20˚C after gDNA isolation. Genomic DNA was extracted from whole male Ae. aegypti by one of two methods: Qiagen DNeasy blood and tissue kit (cat: 69582) or Canadian Center for DNA Barcoding protocol. In brief, for the Qiagen DNeasy kit protocol, whole male mosquitoes were homogenized and incubated in lysis buffer and proteinase K overnight at 55˚C. Following incubation and removal of chitinous material, RNase A treatment was performed to remove RNA contamination for both isolation methods. Then, the standard Qiagen protocol of washes was followed. Final samples were eluted two times in 150 µl warm dH2O (Invitrogen Cat #: 10977-015). A modified Canadian Center for DNA Barcoding (2020) protocol was also used for some mosquito DNA isolations to reduce costs while maintaining quality genomic DNA extractions. Samples were homogenized, incubated, and RNase A treated as described above before the lysate was passed through the filter of an AcroPrep™ PALL2 plate (Cat #: PALL 5053) to bind the gDNA. The filter was washed with Protein Wash Buffer to remove remaining proteins and then washed with cold Wash Buffer to remove additional contaminates. The filter was allowed to dry to ensure that no ethanol remained to interfere with DNA yield. Finally, two washes of 75 µl warm dH2O (Invitrogen Cat #: 10977-015) were performed to elute a final volume of 175 µl gDNA.

Quantification of gDNA was performed using a Quant-iT PicoGreen dsDNA assay (ThermoFisher Scientific - P11496) and samples were read on a Synergy H1 Hybrid Plate Reader (BioTek Instruments, Inc.) in the Genomic Sciences Laboratory at North Carolina State University (GSL).

Genotyping

Allele-specific quantitative PCR and melting curve analysis (AS-PCR) was used to genotype all mosquitoes in duplicate for each of the mutations most commonly found in Central and South America (V1016I and F1534C). If the two reactions were not scored identically, the sample was discarded from further analysis. Mismatches were rare and typically due to non-amplification of a sample or because certain criteria for scoring were not met; i.e., melting peak did not cross threshold. A smaller number (n=92) of individuals were additionally genotyped at the V410L locus to verify the strong linkage disequilibrium that has been previously reported between it and locus V1016I (Saavedra-Rodriguez et al., 2018). Each mosquito genotyped at the V410L locus was also genotyped twice to ensure accuracy.

Genotyping of V1016I. AS-PCR for the V1016I locus was based on the method reported by Saavedra-Rodriguez et al. (2007) and modifications to the I1016R primer made by the Entomology Branch at the Centers for Disease Control and Prevention (CDC), Atlanta, USA (A. Lenhart, personal communication). The PCR volume was reduced to 10 µl per reaction and contained 2.5 µl of dH2O, 0.5 µl of each primer at 10 µM (V1016F, I1016F, I1016R), 5 µl of PerfeCTa SYBR Green Supermix (Quanta – 95054-02K), and 1 µl of template. The primer sequences for V1016F and I1016F used are reported in Saavedra-Rodriquez et al. (2007), but the primer sequence for I1016R was modified to: 5’ - TGA TGA ACC SGA ATT GGA CAA AAG C – 3’ (CDC, personal communication). Samples were genotyped on a BioRad CFX384 Real-Time PCR machine in the GSL, with the following thermal conditions: step 1 - 95˚C for 3 minutes, step 2 – 95˚C for 10 seconds, step 3 – 60˚C for 10 seconds, step 4 – 72˚C for 10 seconds, step 5 - Go to step 2, 39 times, step 6 – 95˚C for 10 seconds, step 7 - Melting Curve 65˚C - 95˚C, increment 0.2˚C per 10 sec plus a plate read.

Genotyping of F1534C. AS-PCR for the F1534C locus was performed following the method reported by Yanola et al. (2011) with the following modifications. The PCR volume was reduced to a total of 10 µl per reaction and contained 5 µl dH2O, 0.2 µl C1534F primer, 0.4 µl F1534F primer, 0.4 µl F1534R primer, 3.0 µl of PerfeCTa SYBR Green Supermix (Quanta – 95054-02K), and 1 µl of template. Samples were genotyped on a BioRad CFX384 Real-Time PCR machine in the GSL, with the following thermal conditions: step 1 - 95˚C for 2 minutes, step 2 – 95˚C for 30 seconds, step 3 – 60˚C for 30 seconds, step 4 – 72˚C for 30 seconds, step 5 - Go to step 2, 34 times, step 6 – 72˚C for 2 minutes, step 7 - Melting Curve 65˚C - 95˚C, increment 0.2˚C per 10 sec plus a plate read.

Genotyping of V410L. The AS-PCR for the V410L locus was based on a protocol developed by K. Saavedra and shared via the Entomology Branch, CDC (A. Lenhart, personal communication). The total volume for each reaction was reduced to 10 µl: 3.8 µl dH2O, 0.05 µl Val410 primer (50 µM) 5’ – GCG GGC AGG GCG GCG GGG GCG GGG CCA TCT TCT TGG GTT CGT TCT ACC GTG – 3’, 0.05 µl Leu410 primer (50 µM) 5’ – GCG GGC ATC TTC TTG GGT TCG TTC TAC CAT T – 3’, 0.1 µl Rev410 primer (50 µM) 5’ – TTC TTC CTC GGC GGC CTC TT – 3’, 5.0 µl PerfeCTa SYBR Green SuperMix (Quanta – 95054-02K), and 1 µl template. Thermal conditions were performed on the BioRad CFX384 Real-Time PCR machine in the GSL, with the following thermal conditions: 95˚C for 3:00, 40 cycles of (95˚C for 0:10, 60˚C for 0:10, 72˚C for 0:30), 95˚C for 0:10, melting curve 65˚C – 95˚C increasing in increments of 0.2˚C per 10 sec plus a plate read.

Analysis for AS-PCR. Melt Curve peak calls were determined using the CFX Maestro Software (Bio-Rad - 12004110), verified by eye, and exported to a customized C++ script to quickly convert melt curve peak calls to genotypes for each sample. Melt curve genotypes were then read into a customized R script (R Core Team 2019) for allele frequency determination and other statistical analysis. 

Usage Notes

The datafiles are formatted for input used by the scripts in our github repository (https://github.com/jenbaltzegar/IQTmosq). But, please note that it is important to make sure that IsolationLog_IQT-Mosq.csv file has the "date" column in the MM-DD-YYYY format. We have found that if opened in Microsoft Excel, this format can be changed automatically and will cause problems for the downstream analysis.

Funding

NIH, Award: 1R01AI139085-01

NIH, Award: 1R01AI139085-01