DOI: 10.5061/dryad.2fqz612pc
title: Knockdown resistance (kdr) genotypes and collection information for Aedes aegytpi from Iquitos, Peru (2000 - 2017)

description: 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.
description: <strong>Entomological Surveys</strong>
 <i>Control History.</i> During the past two decades,
 researchers have preserved mosquito specimens collected throughout the
 city of Iquitos in a repository (e.g. Cavany <i>et al.</i>,
 2020; Cromwell <i>et al.</i>, 2017; Getis <i>et
 al.,</i> 2003; Gunning <i>et al.,</i> 2018; LaCon
 <i>et al.,</i> 2014; Lenhart <i>et al.</i>, 2020;
 Morrison <i>et al.,</i> 2004a; Morrison <i>et
 al.,</i> 2004b; Morrison <i>et al.,</i> 2006; Morrison
 <i>et al.,</i> 2008; Reiner <i>et al.</i>, 2019;
 Schneider <i>et al.</i>, 2004; Tun-Lin <i>et
 al.</i>, 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 <i>Ae. aegypti </i>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 <i>et al., </i>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 <i>et al. </i>2021).
 <i>Temporal Collections. Aedes aegypti </i>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 <i>et al., </i>1994) prior to June 2009 and
 by Prokopack Aspirator following June 2009 (Vasquez-Prokopec <i>et
 al.</i>, 2009; Reiner <i>et al.</i>, 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).
 <i>Spatial Collections.</i> Intense suppression
 experiments based on pyrethroid spraying were conducted in 2013 and 2014
 (Gunning <i>et al.,</i> 2018) to test the predictions of a
 detailed <i>Ae. aegypti</i> population dynamics model (Magori
 <i>et al.,</i> 2009). In brief, two areas of the city were
 identified as having relatively high densities of <i>Ae.
 aegypti</i> 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<i>, </i>site dimensions were
 selected to be 3 – 5 times larger than the expected <i>Ae. aegypti
 </i>lifetime flight distance of approximately 150 m (Harrington
 <i>et al.</i>, 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. <strong>DNA Extractions and
 Quantification</strong> 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 <i>VGSC</i> 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
 <i>Ae. aegypti</i> 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 dH<sub>2</sub>O
 (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 dH<sub>2</sub>O (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).
 <strong>Genotyping</strong>
 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 <i>et al.,</i>
 2018). Each mosquito genotyped at the V410L locus was also genotyped twice
 to ensure accuracy. <i>Genotyping of
 V1016I.</i> AS-PCR for the V1016I locus was based on the method
 reported by Saavedra-Rodriguez <i>et al.</i> (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 dH<sub>2</sub>O, 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 <i>et
 al.</i> (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. <i>Genotyping of
 F1534C.</i> AS-PCR for the F1534C locus was performed following the
 method reported by Yanola <i>et al.</i> (2011) with the
 following modifications. The PCR volume was reduced to a total of 10 µl
 per reaction and contained 5 µl dH<sub>2</sub>O, 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. <i>Genotyping of V410L.</i> 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 dH<sub>2</sub>O, 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. <i>Analysis for
 AS-PCR.</i> 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. 
description: 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.