Research ArticleLevels of Resistance to Pyrethroid among Distinct kdrAlleles in Aedes aegypti Laboratory Lines and Frequency of kdrAlleles in 27 Natural Populations from Rio de Janeiro Brazil

Luiz Paulo Brito12 Luana Carrara12 Rafael Maciel de Freitas3

Joseacute Bento Pereira Lima12 and Ademir J Martins 12

1Laboratorio de Fisiologia e Controle de Artropodes Vetores Instituto Oswaldo Cruz FIOCRUZ Rio de Janeiro Brazil2Laboratorio de Entomologia Instituto de Biologia do Exercito Rio de Janeiro Brazil3Laboratorio de Transmissores de Hematozoarios Instituto Oswaldo Cruz FIOCRUZ Rio de Janeiro Brazil

Correspondence should be addressed to Ademir J Martins ademirjriocfiocruzbr

Received 28 March 2018 Accepted 12 June 2018 Published 11 July 2018

Academic Editor Gelin Xu

Copyright copy 2018 Luiz Paulo Brito et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Background Severalmutations in voltage gated sodium channel (NaV) have been identified inAedes aegypti populations worldwideHowever only few are related to knockdown resistance to pyrethroids most of which with variations in the 1016 and 1534 NaV sitesIn Brazil at least two NaV alleles are known NaVR1 with a substitution in the 1534 (1016 Val+ + 1534 Ilekdr) and NaVR2 withsubstitutions in both 1016 and sites (1016Ilekdr + 1534Cyskdr) There is also the duplication in the NaV gene with one copy carryingthe substitution Ile1011Met although its effects on pyrethroid resistance remain to be clarified Our goals in this study were (1) todetermine the role of each kdr NaV allele and the duplication on pyrethroid resistance and (2) to screen the frequency of the kdralleles in 27 several natural Ae aegypti populations from the metropolitan region of Rio de JaneiroMethods Pyrethroid resistancewas evaluated by a knockdown time (KdT) assay an adaptation of the WHO test tubes with paper impregnated with deltamethrinWe used laboratory-selected Ae aegypti lineages R1R1 and R2R2 (homozygous for the kdr NaVR1 and NaVR2 alleles respectively)Dup (with duplication in theNaV gene) Rockefeller (the susceptibility reference control) and F1 hybrids among them Genotypingof both 1016 and 1534 NaV sites was performed in 811Ae aegypti sampled from 27 localities from Rio de Janeiro (17) Niteroi (6) andNova Iguacu (4) cities Rio de Janeiro State Brazil with a TaqMan real time PCR approach Results The laboratory lineages R1R1R2R2 and R1R2were the only ones that neededmore than 60minutes to knock down all the insects exposed to the pyrethroid beingthe KdT R2R2 gt R1R2 gt R1R1 corroborating the recessive nature of the kdr mutations Frequency of kdr alleles NaVR1 and NaVR2in field-caught mosquitoes varied from 0 to 52 and 43 to 86 respectively evidencing high levels of ldquoresistant genotypesrdquo (R1R1R1R2 and R2R2) which together summed 60 to 100 in Ae aegypti populations from Rio de Janeiro Conclusions The NaVR1 andNaVR2 kdr alleles confer resistance to the pyrethroid deltamethrin in homozygotes and R1R2 heterozygotes being the R2R2 mostresistant genotype The allele containing duplication in the NaV gene with a mutation in the 1011 site did not confer resistanceunder the tested conditions The frequencies of the ldquoresistant genotypesrdquo are elevated in Ae aegypti natural populations from Riode Janeiro

1 Introduction

Aedes aegypti is the primary vector of dengue chikungunyaandZika virus in tropical and subtropical regions of the globeThe incidence of dengue cases has dramatically increased inthe last decade with an estimation of 390 million dengueinfections per year [1] summedwith the recent re-emergence

of chikungunya and Zika New-born malformations andneurological complications associated with Zika led theWorld Health Organization (WHO) to declare the ldquoPub-lic Health Emergency of International Concernrdquo in 2016 [2]This situation worsened with a strong concern regarding apotential reurbanization of yellow fever virus in Brazil whichhave killedmore than 200 people in ruralmunicipalities from

HindawiBioMed Research InternationalVolume 2018 Article ID 2410819 10 pageshttpsdoiorg10115520182410819

2 BioMed Research International

July 2017 to March 2018 ie 8 months [3] This scenarioreinforces the need to strengthen vector control measures tomitigate disease transmission

New vector control strategies to reduce Ae aegypti popu-lation density below a critical threshold have been proposedwhich are expanding to vast open field application testswith support of local communities governments and stim-ulation by WHO [4] The dissemination of transmissionblocking mosquitoes carrying Wolbachia and the release oftransgenic-based sterile mosquitoes (RIDL) are among themost promising approaches developed so far [5 6] On theother hand the employment of insecticides will persist fora long time as a prime strategy for rapidly reduction onmosquito density especially during an outbreak In thissense it is important that these compounds are efficient overtarget populations Among the classes of insecticides recom-mended by theWorld Health Organization Pesticide Scheme(WHOPES) pyrethroids are themost employed againstAedesmosquitoes since they provoke the fast-acting knockdowneffect are cheaper and cause less nuisance to householdersindoor However the excessive and uncontrolled employ-ment of insecticides has been selecting Ae aegypti resistantpopulations worldwide [7 8] The principal physiologicalmechanisms selected for pyrethroid resistance are relatedto increase in the expression profile of metabolic enzymesespecially cyp P450 genes of the multifunction oxidases classand point mutation in its target site the voltage gated sodiumchannel (NaV) [8 9]

There are several mutations in insect NaV genes confer-ring resistance to pyrethroid with the L1014F kdr substitutionbeing the most common conserved among distinct insectorders This happens since there are few modifications per-mitted in the highly conserved NaV gene which respondsfor a central role in the neuron physiology of animals [10]Additionally the same kdr mutations may have multipleorigins in a species as evidenced for insects such as Mdomestica [11] and An gambiae [12] In Ae aegypti howeverthe L1014F kdrmutation is not found due to codon constraintto which the simultaneous selection of two mutations in thesame codon would be necessary which is unlikely to happen[13] Besides the fact that some kdr mutations are specificfor some species in Ae aegypti this number is uniquelyhigh [14] Other mutations are found in several positions ofthe Ae aegypti NaV where the relationship with resistanceto pyrethroid is better described to 1016 (Val to Ile inAmericas and Africa or Gly in Asia and Middle Eastern)and 1534 (Phe to Cys) NaV sites [15ndash18] In Ae aegypti LatinAmerican populations the allele containing a mutation inthe 1534 site is widely distributed meanwhile the alleleswith mutations in both 1016 + 1534 sites are increasing infrequency and dispersing [17 19] Considering these twosites in Brazil there is evidence of three alleles here calledNaVS (1016 Val

+ + 1534Phe+) NaVR1 (1016Val+ + 1534Cyskdr)

and NaVR2 (1016Ilekdr + 1534Cyskdr) [19 20] The I1011M isanother substitution found in Ae aegypti populations fromLatin American and is involved in a gene duplication event[21] Although it was proved to alter the sodium channelsensitivity to pyrethroids [22] its actual role in resistance

is controversial in natural populations where other kdrmutations occur [23 24] The S989P substitution (togetherwith V1016G and F1534C) also plays an important partfor pyrethroid resistance but its distribution seems to berestricted to Middle EastAsia [15] Genotyping of knownkdr single nucleotide polymorphisms (SNPs) in susceptibleand resistant individuals from laboratory-selected lineages aswell as natural populations and electrophysiological studiesevidencing altered NaV sensibility to pyrethroids have beencorroborating the hypothesis of such SNPs with knockdownresistance [14] Electrophysiological assays are important todetermine the role of only and combined mutations in thesensibility to pyrethroids Not less important is to evaluatethe whole organism making use of laboratory lines withhomogeneous genetic backgrounds

Here we evaluated the role of kdr mutations occurringin Brazilian Ae aegypti populations in response to thepyrethroid deltamethrin based on bioassays with laboratory-selected lines without interference of other known mech-anisms In addition the frequency of kdr mutations wasestablished for Ae aegypti natural populations from 27 local-ities in Rio de Janeiro State the most touristic city fromSouth America and likely the port of entry of dengue virusserotypes 1 2 and 3 in Brazil [25 26]

2 Methods

21 Laboratory Lineages Rockefeller is an Ae aegypti lin-eage reference for physiology experiments and constantlyemployed as a baseline of insecticide susceptibility [27] Inour laboratory it has been continuously maintained since1999 [28] Rockefeller is homozygous for theNaVS allele (1016Val+ + 1534 Phe+) The lineage here called R2R2 is the sameRock-kdr previously described in [29] homozygous for theallele NaVR2 (1016 Ile

kdr + 1534 Cyskdr) and maintained in thelaboratory since 2012 The lineage Dup does not harbour thekdrmutations in the 1016 and 1534 NaV sites but a duplicationin the NaV gene and a substitution at the 1011 site keepingboth variations 1011 Ile and 1011 Met [21]

For obtaining the R1R1 lineage we used insects main-tained in the laboratory originally collected at SantaremPA Brazil a city with high frequency of the NaVR1 alleleand no register of NaVR2 [19 30] We set up groups of onemale with three virgin females maintained together for threedays in 50 mL conical plastic tubes under the insectaryconditions Afterwards males were removed and genotypedfor the NaV 1534 site (see below) meanwhile females wereoffered to blood meal on anesthetized mice and three daysafter were individually induced to egg-laying in 6 cm Petridishes covered with a wet filter paper as described elsewhere[29] After egg-laying females were also genotyped Eggsfrom both parents revealed as R1R1 were induced to hatchresulting in a total of 86 larvae that gave origin to this firstR1R1 lineage

In order to homogenize the genetic background of thekdr lineages we further backcrossed that R1R1 new lineagewith the previous established R2R2 [29] To accomplish thatwe first mixed R1R1 males with R2R2 females obtaining anR1R2 offspring (F1) Males from this F1 (R1R2) were then

BioMed Research International 3

backcrossed with R2R2 females resulting in a F2 with thegenotypes R1R2 and R2R2 in an expected 11 proportionGroups of one F2 male with two R2R2 females were set upin conic tubes and carried out similarly as above Males weregenotyped and females were induced to lay eggs (F3) The F3eggs resulting from R1R2 males were used for producing thenext generation This procedure was repeated for two moregenerations until F5 Then similar groups were formed nowwith both males and females from this F5 The F6 resultingeggs used for moving forward belonged to the offspring ofmale and female both genotyped as R1R2 among which25 was expected to be R1R1 Finally new groups were setup among the F6 adults From the F7 resulting eggs thoseoriginated from R1R1 parental were used to finally establishthe new R1R1 lineage (supplementary figure S1)

Therefore a part of the NaV locus the original homozy-gote colonies (Rockefeller R1R1 and R2R2) had more similargenetic background with exception of the Dup lineage withduplication in the NaV which was not backcrossed with anyof these lineages

22 Bioassays An adaptation of WHO test tubes bioassays[31] was performed with Ae aegypti females exposed topapers impregnated in the laboratory with the pyrethroiddeltamethrin at 17 gcm2 (0034 solution) Deltamethrin(Sigma-Aldrich) was dissolved to a stock concentration at10 in acetone and then diluted to an intermediate solutionat 1 in silicone (Dow Corning) following a new dilution toa work solution at 0034 (340 mgL) also in the siliconeA total of 840 120583L of this work solution was then pipettedover a 12x14 cm2 filter paper sheet (Whatman Grade 1) withthe help of an electronic multichannel pipette (Eppendorf)and a frame which oriented the dispersion of 5 120583L at each96 equidistant spots At all batches of impregnation somepapers were filled only with silicone as negative control Thepaper sheetswere air-dried for two days before their use in thebioassays The bioassays proceeded as indicated [31] Around20 3-5 days old Ae aegypti females were transferred to theresting tube and then gently blown into the respective testtube where the knockdown was followed for up to 2 h inintervals of 2 or 5 minutes Each lineage was assayed withfour tubes in at least two independent times (females resultedfrom distinct batches of eggs and papers from differentimpregnation lots) Probit analysis [32] was performed inorder to infer the time necessary to knockdown 95 ofthe individuals of each lineage (KdT

95) The resistance ratio

(RR95) was taken by the quotient between the KdT

95of a

given lineage with the Rockefellerrsquos

23 Aedes aegypti from Rio de Janeiro State (RJ) We tookadvantage of a wide sampling previously performed in 2012 in27 localities inRJmetropolitan area comprising threemunic-ipalities Rio de Janeiro city (Tubiacanga Valqueire UrcaOlaria Gamboa Caju Pavuna Meier Grajau Paqueta VazLobo Jardim Guanabara Sao Cristovao Humaita Rio Com-prido Rio das Pedras and Taquara neighbourhoods) NiteroiCity (Jurujuba Itacoatiara Sao Franciso Fonseca PontaDrsquoAreia and Piratininga neighbourhoods) Nova Iguacu City(Cabucu Ceramica and Moqueta neighbourhoods) and

Belford Roxo City (Heliopolis neighbourhood) [33] Brieflymosquito eggs were collected during three consecutive weeksusing 60 ovitraps in a grid of 500 x 500 m2 per neighbour-hood Thus we believe our sample was representative of thegenetic variation presented in each neighbourhoodOvitrapsrsquopaddles were brought to the lab eggs were hatched andlarvae were reared until the adult stages when the DNA wasextracted

24 kdr Genotyping DNA was extracted from individualinsects tittered in 200 120583L squishing buffer (10 mM Tris-HCl pH 82 2 mM EDTA and 02 Triton X-100) and02 mgL Proteinase K (Promega) as described elsewhere[34] A customizedTaqMangenotyping assay (ThermoFisherScientific) was employed for 1016 (Val+ and Ilekdr) and 1534(Phe+ and Cyskdr) independently for each NaV site [20]Reactions were performed in 10 120583L containing 1120583L DNA1X TaqMan Genotyping Master Mix (Thermo Fisher Scien-tific) the mix of primers and probes Custom TaqMan SNPGenotyping Assay (1X for Val1016Ile AHS1DL6 and 05X forPhe1534CysAHUADFAThermoFisher Scientific) andH

2O

qs 10 120583L with the thermocycling program in accordancewith the manufacturerrsquos instructions in QuantStudio 6 qPCRequipment (Thermo Fisher Scientific)

For genotyping variations in the 1011 site (Ile or Met)we employed an allelic-PCR approach in which the specificproducts were detected through a dissociation curve analysisafter the amplification reaction as described elsewhere[13 21] Reactions contained 1X Sybr Green Master mix(ThermoFischer) 20 ng DNA 024 120583M primer 1011 forward51015840-GTCCTGTATTCCGTTCTTTTT-31015840common to both se-quences and 012 120583M of each two specific primers 1011Ile reverse 51015840-[long tail]-TACTTACTACTAGATTTGCC-31015840and 1011 Met reverse 51015840-[short tail]-TACTTACTACTAGAT-TTACT-31015840 and H

2O qs 125 120583L The specificity laid on the

31015840-end of the specific primers and the discrimination of theamplicons was possible due to a GC tail at the 51015840-end ofboth specific primers however with distinct sizesshort [GCGGGC] and long [GCGGGCAGGGCGGCGGG-GGCGGGGCC] providing TM of 77∘C and 82∘C respec-tively in a dissociation curve analysis For more detailsabout this method please check Saavedra-Rodriguez et al2007 [13] The thermocycling program consisted of 35 cycles(denaturation 94∘C 3010158401015840 annealing 57∘C 11015840and polymeraseextension 72∘C 4510158401015840) followed by the standard melt curveanalysis in a QuantStudio 6 qPCR equipment (ThermoFisher Scientific)

The 95 Confidence Intervals (CI95) of the allelicfrequencies were calculated using the exact binomial approxi-mation (httpwwwbiostathandbookcomconfidencehtml)Comparisons among genotypic frequencies pairs wereperformed with exact G test and Fisherrsquos method withdefault Markov chain parameters by Genepop version 42online version (httpgenepopcurtineduau)

3 Results

31 Ae aegypti Lineages and Bioassays The results of 1016and 1534 SNPs were merged to constitute the genotypes as

BioMed Research International 3

backcrossed with R2R2 females resulting in a F2 with thegenotypes R1R2 and R2R2 in an expected 11 proportionGroups of one F2 male with two R2R2 females were set upin conic tubes and carried out similarly as above Males weregenotyped and females were induced to lay eggs (F3) The F3eggs resulting from R1R2 males were used for producing thenext generation This procedure was repeated for two moregenerations until F5 Then similar groups were formed nowwith both males and females from this F5 The F6 resultingeggs used for moving forward belonged to the offspring ofmale and female both genotyped as R1R2 among which25 was expected to be R1R1 Finally new groups were setup among the F6 adults From the F7 resulting eggs thoseoriginated from R1R1 parental were used to finally establishthe new R1R1 lineage (supplementary figure S1)

Therefore a part of the NaV locus the original homozy-gote colonies (Rockefeller R1R1 and R2R2) had more similargenetic background with exception of the Dup lineage withduplication in the NaV which was not backcrossed with anyof these lineages

22 Bioassays An adaptation of WHO test tubes bioassays[31] was performed with Ae aegypti females exposed topapers impregnated in the laboratory with the pyrethroiddeltamethrin at 17 gcm2 (0034 solution) Deltamethrin(Sigma-Aldrich) was dissolved to a stock concentration at10 in acetone and then diluted to an intermediate solutionat 1 in silicone (Dow Corning) following a new dilution toa work solution at 0034 (340 mgL) also in the siliconeA total of 840 120583L of this work solution was then pipettedover a 12x14 cm2 filter paper sheet (Whatman Grade 1) withthe help of an electronic multichannel pipette (Eppendorf)and a frame which oriented the dispersion of 5 120583L at each96 equidistant spots At all batches of impregnation somepapers were filled only with silicone as negative control Thepaper sheetswere air-dried for two days before their use in thebioassays The bioassays proceeded as indicated [31] Around20 3-5 days old Ae aegypti females were transferred to theresting tube and then gently blown into the respective testtube where the knockdown was followed for up to 2 h inintervals of 2 or 5 minutes Each lineage was assayed withfour tubes in at least two independent times (females resultedfrom distinct batches of eggs and papers from differentimpregnation lots) Probit analysis [32] was performed inorder to infer the time necessary to knockdown 95 ofthe individuals of each lineage (KdT

95) The resistance ratio

(RR95) was taken by the quotient between the KdT

95of a

given lineage with the Rockefellerrsquos

23 Aedes aegypti from Rio de Janeiro State (RJ) We tookadvantage of a wide sampling previously performed in 2012 in27 localities inRJmetropolitan area comprising threemunic-ipalities Rio de Janeiro city (Tubiacanga Valqueire UrcaOlaria Gamboa Caju Pavuna Meier Grajau Paqueta VazLobo Jardim Guanabara Sao Cristovao Humaita Rio Com-prido Rio das Pedras and Taquara neighbourhoods) NiteroiCity (Jurujuba Itacoatiara Sao Franciso Fonseca PontaDrsquoAreia and Piratininga neighbourhoods) Nova Iguacu City(Cabucu Ceramica and Moqueta neighbourhoods) and

Belford Roxo City (Heliopolis neighbourhood) [33] Brieflymosquito eggs were collected during three consecutive weeksusing 60 ovitraps in a grid of 500 x 500 m2 per neighbour-hood Thus we believe our sample was representative of thegenetic variation presented in each neighbourhoodOvitrapsrsquopaddles were brought to the lab eggs were hatched andlarvae were reared until the adult stages when the DNA wasextracted

24 kdr Genotyping DNA was extracted from individualinsects tittered in 200 120583L squishing buffer (10 mM Tris-HCl pH 82 2 mM EDTA and 02 Triton X-100) and02 mgL Proteinase K (Promega) as described elsewhere[34] A customizedTaqMangenotyping assay (ThermoFisherScientific) was employed for 1016 (Val+ and Ilekdr) and 1534(Phe+ and Cyskdr) independently for each NaV site [20]Reactions were performed in 10 120583L containing 1120583L DNA1X TaqMan Genotyping Master Mix (Thermo Fisher Scien-tific) the mix of primers and probes Custom TaqMan SNPGenotyping Assay (1X for Val1016Ile AHS1DL6 and 05X forPhe1534CysAHUADFAThermoFisher Scientific) andH

2O

qs 10 120583L with the thermocycling program in accordancewith the manufacturerrsquos instructions in QuantStudio 6 qPCRequipment (Thermo Fisher Scientific)

For genotyping variations in the 1011 site (Ile or Met)we employed an allelic-PCR approach in which the specificproducts were detected through a dissociation curve analysisafter the amplification reaction as described elsewhere[13 21] Reactions contained 1X Sybr Green Master mix(ThermoFischer) 20 ng DNA 024 120583M primer 1011 forward51015840-GTCCTGTATTCCGTTCTTTTT-31015840common to both se-quences and 012 120583M of each two specific primers 1011Ile reverse 51015840-[long tail]-TACTTACTACTAGATTTGCC-31015840and 1011 Met reverse 51015840-[short tail]-TACTTACTACTAGAT-TTACT-31015840 and H

2O qs 125 120583L The specificity laid on the

31015840-end of the specific primers and the discrimination of theamplicons was possible due to a GC tail at the 51015840-end ofboth specific primers however with distinct sizesshort [GCGGGC] and long [GCGGGCAGGGCGGCGGG-GGCGGGGCC] providing TM of 77∘C and 82∘C respec-tively in a dissociation curve analysis For more detailsabout this method please check Saavedra-Rodriguez et al2007 [13] The thermocycling program consisted of 35 cycles(denaturation 94∘C 3010158401015840 annealing 57∘C 11015840and polymeraseextension 72∘C 4510158401015840) followed by the standard melt curveanalysis in a QuantStudio 6 qPCR equipment (ThermoFisher Scientific)

The 95 Confidence Intervals (CI95) of the allelicfrequencies were calculated using the exact binomial approxi-mation (httpwwwbiostathandbookcomconfidencehtml)Comparisons among genotypic frequencies pairs wereperformed with exact G test and Fisherrsquos method withdefault Markov chain parameters by Genepop version 42online version (httpgenepopcurtineduau)

3 Results

31 Ae aegypti Lineages and Bioassays The results of 1016and 1534 SNPs were merged to constitute the genotypes as

4 BioMed Research International

Table 1 List of genotypes based on SNP reactions for 1011 1016 and 1534 NaV sites of the Aedes aegypti laboratory lineages here evaluated

1011 1016 1534 GenotypelowastIleIle ValVal PhePhe SS

PheCys SR1CysCys R1R1

ValIle PhePhe SR3PheCys SR2 + R1R3CysCys R1R2

IleIle PhePhe R3R3PheCys R2R3CysCys R2R2

IleMet ValVal PhePhe DDlowastThe genotypes likely to occur based on the SNP genotype reactions are evidenced in bold

1011 1016 1534

NaVS Ile Val Phe

NaVR1 Ile Val Cys

NaVR2 Ile Ile Cys

NaVDIle Val PheMet Val Phe

Figure 1Voltage gated sodium channel (NaV) kdr alleles ofAedesagypti populations from Brazil Schematic representation of theNaV with its four domains each with six hydrophobic segmentsThe kdr sites 1011 and 1016 are in the IIS6 segment while the 1534site lies in the IIIS6 Each haplotype is represented by means of thevariation at each kdr site where wild-type and kdr aminoacids areindicated in black and red respectivelyThe colours of the alleles arethe same in the following figures

presented in Table 1 and were composed by the three allelesNaVS (1016 Val

+ + 1534Phe+) NaVR1 (1016Val+ + 1534Cyskdr)

and NaVR2 (1016Ilekdr + 1534Cyskdr) Therefore all insects1016 (ValIle) + 1534 (PheCys) were considered SR2 (seeTable 1) There was the additional NaVD which represents aduplication in the NaV gene similar to NaVS in relation to1016 and 1534 sites however with a copy 1011Ile+ and another1011 Metkdr (Figure 1)

The RR95

based on Rockefeller (SS) of each lineage andtheir hybrids are graphically represented in Figure 2 andthe KdT

95values expressed in minutes are detailed in the

supplementary Table S1 The only insects still flying after60 minutes of exposition to deltamethrin belonged to the

Figure 2 The knockdown time (KdT) profile of Aedes aegyptilaboratory lineages with distinct NaV genotypes The abscissa andordinate in the graph indicate respectively the time in minutes forknockdown 95 of the insects (KdT

95) from the respective lineages

and their resistant ratio (RR95) considering Rockefeller (SS) as

reference

genotypes R1R1 R1R2 and R2R2 ie homozygous for thekdr NaVR1 and NaVR2 alleles and the heterozygous hybridExcluding R1R2 the RR

95of hybrids genotypes (ie with

NaVS or NaVD alleles) varied between 17 and 38 confirmingthe recessive trait of kdrmutations regarding resistance to thepyrethroid deltamethrin

The R2R2 lineage homozygous for the kdr NaVR2 allelewas the most resistant (RR

95= 67) R2R2 individuals were

15-fold more resistance than R1R1 considering their KdT95

An exposition of 30 minutes to the insecticide was sufficientto knockdown 95 of Dup females

32 Kdr Genotyping of Ae aegypti Natural Populations fromRio de Janeiro State (RJ) The three NaV alleles NaVS NaVR1and NaVR2 were observed among the total of 811 genotypedinsects (Figure 3) Number of samples evaluated ranged from12 (Jurujuba Niteroi) to 73 (Ceramica Nova Iguacu) In totalaverage the NaVR2 kdr allele was the most frequent (654)followed by NaVR1 (275) and NaVS (72) Out of the 27localities only Urca presented theNaVR1 allele with the high-est frequency (525) (Supp Table S1) The wild-type NaVSwas far the less frequent ranging from 0 (Humaita Rio dasPedras and Sao Cristovao Rio de Janeiro) to 225 (Paquetaisland Rio de Janeiro) except Fonseca (Niteroi) where theNaVS frequency (206) was higher than the NaVR1 allele

BioMed Research International 5

Figure 3 Frequencies of kdr alleles considering 1016 and 1534 NaV sites of Aedes aegypti from Rio de Janeiro State Localities areindicated in the map The shadowed area in green pink and grey represent the regions Rio Niteroi and Baixada respectively

(176) Interestingly when pooling neighbourhoodrsquos datato their respective regions NaVS frequency was higher inNiteroi (142) than in Rio (40) a significant differenceby considering that there is no overlapping among theirsIC95 (supp material S2) Paqueta was removed from thisanalysis since it is an island distant fromboth Rio andNiteroioffshores These cities are separated by the Guanabara Bayconnected by a 13 Km bridge and ferry boats

The R2R2 genotype which would potentially accountfor higher levels of resistance to pyrethroids was the mostfrequent genotype (median 500) and the ldquoresistant geno-typesrdquo (R1R1 R2R2 and R1R2) together reached a median of884 among Ae aegypti from the sites evaluated (Table 2)The localities with the lowest frequency of the ldquoresistantgenotypesrdquo were Paqueta island (60) followed by five outof the six neighbourhoods evaluated from Niteroi (647 inFonseca-757 in Piratininga)

The genotypic frequencies among regions (Baixada RioNiteroi and Paqueta island) did not differ significantly

between Rio and Baixada as well as between Niteroi andPaqueta (both exact G test Pgt005) In their turn RioBaixadahighly differed from NiteroiPaqueta (see Table 3)

4 Discussion

Here we evaluated the difference in insecticide resistancelevels to the pyrethroid deltamethrin inAe aegypti laboratorylines with distinct kdr mutations introgressed from the fieldand free of other known resistance mechanism Severalrecords of increased levels of resistance to pyrethroid inparallel with dissemination of kdr alleles in natural vectorpopulations have been released in the last decade as anindirect correlation between kdr mutation and pyrethroidresistance inAe aegypti [8 35] Laboratory selection pressureexperiments with insecticide have also corroborated thiscorrelation when kdr frequencies have increased towardfixation [13] In addition electrophysiological tests based onnatural populations as well as samples that undergone site

6 BioMed Research International

Table 2 Kdr genotypic frequencies in field populations of Aedes aegypti from Rio de Janeiro State considering the 1016 and 1534 NaV sites

locality n observed genotype frequencies HWE testlowast ResistantgenotypesSS SR1 SR2 R1R1 R1R2 R2R2 Σ1205942 plowast

Cabucu 52 0 0019 0115 0135 0462 0269 88 0032 0865Ceramica 73 0014 0041 0041 0110 0411 0384 00 1000 0904Moqueta 65 0015 0015 0077 0077 0215 0600 315 0000 0892Heliopolis 36 0 0028 0111 0056 0111 0694 584 0000 0861Jurujuba 12 0 0 0250 0083 0583 0083 85 0036 0750Itacoatiara 24 0 0 0167 0208 0292 0333 163 0001 0833Sao Francisco 16 0 0 0250 0 0125 0625 34 0338 0750Fonseca 17 0059 0 0294 0118 0118 0412 855 0000 0647Ponta DAreia 14 0 0 0286 0 0 0714 34 0064 0714Piratininga 37 0081 0054 0108 0270 0189 0297 278 0000 0757Tubiacanga 43 0 0070 0047 0186 0163 0535 213 0000 0884Valqueire 31 0 0032 0097 0032 0290 0548 100 0019 0871Urca 20 0 0050 0050 0300 0400 0200 14 0708 0900Olaria 35 0 0000 0029 0086 0314 0571 28 0425 0971Gamboa 17 0 0 0059 0059 0529 0353 13 0718 0941Caju 23 0043 0 0043 0087 0087 0739 44 0224 0913Pavuna 34 0 0 0118 0118 0265 0500 207 0000 0882Meier 37 0 0054 0 0135 0297 0514 66 0086 0946Grajau 38 0 0 0053 0053 0316 0579 47 0194 0947Paqueta 20 0050 0 0350 0250 0150 0200 546 0000 0600Vaz Lobo 35 0 0143 0057 0143 0229 0429 130 0005 0800Jardim Guanab 32 0 0031 0 0094 0375 0500 08 0841 0969Sao Cirstovao 23 0 0 0 0043 0522 0435 12 0266 1000Humaita 18 0 0056 0111 0167 0500 0167 26 0454 0833Rio Comprido 16 0 0 0 0125 0313 0563 08 0364 1000Rio das Pedras 17 0 0 0 0118 0529 0353 02 0618 1000Taquara 26 0 0 0038 0077 0192 0692 79 0048 0962median 0 0 0059 0110 0292 0500 0884lowastHardy-Weinberg Equilibrium testlowastlowast Probability considering the chi-squared distribution for one or three degrees of freedom respectively for three or six genotypes

Table 3 Comparisons among kdr genotypic frequencies of Aedes aegypti from distinct regions of Rio de Janeiro State

Region pair 1205942 df P-valueBaixada x Niteroi 1008 2 0006Baixada x Rio 179 2 0409Niteroi x Rio infinity 2 lt0001Baixada x Paqueta 1330 2 0001Niteroi x Paqueta 209 2 0351Rio x Paqueta infinity 2 lt0001Baixada (Cabucu Ceramica and Moqueta ans Heliopolis) Niteroi (Jurujuba Itacoatiara Sao Francisco Fonseca Ponta DrsquoAreia and Piratininga) Rio(Tubiacanga Valqueire Urca Olaraia Gamboa Caju Pavuna Meier Grajau Vaz Lobo Jardim Guanabara Sao Cristovao Humaita Rio das Pedras andTaquara) and Paqueta (Paqueta island)

directed mutagenesis confirmed that kdr mutations found inAe aegypti alter sensibility to pyrethroids [36 37] Howeverto our knowledge this is the first study in vivo that evincesthe importance of such mutations in homogeneous kdrlaboratory lines of Ae aegypti except for the kdr locus

ie with minimum interference of other possibly selectedmechanisms as well as of pleiotropic effects that mightrespond differently to each distinct genetic background

WHO Pesticide Evaluation Scheme (WHOPES) recom-mends discriminating concentrations of insecticides for the

BioMed Research International 7

impregnated paper tests [38] The most recent WHOPESplan for detecting and monitoring IR in Aedes aegyptirecommended 003 as a discriminating concentration ofdeltamethrin [39] to where mortality is recorded 24 h after1 h of exposition to the insecticide Mortality under 90indicates resistance of the evaluated population as long as atleast 100 mosquitoes were tested This is a qualitative dose-diagnostic test good for determining the susceptibility statusof a given population although not suitable for comparinglevels of resistance among populations For this matter aquantitative dose-response test is indicated in which themortality rates over a range of insecticide concentrationsgenerate the lethal concentrations (LC) of each populationIn turn LC produces the resistance rations (RR) based ona reference lineage [40] However this sort of test requiresa large number of insects compared to the dose-responseapproach Here we applied a WHO-like dose-diagnostic testwith papers impregnated on our ownNevertheless instead ofevaluating mortality 24 h after 1h of exposition we followedthe knockdown rate for up to 2 h With this record over thetime a semiquantitative analysis was performed by extractingthe RR of the populations in this case based on their timeof knockdown It is worth noting that this knockdown timeRR displays a different scale generally shorter than thoseproduced by truly qualitative dose-response tests

We employed a very efficient TaqMan assay for the rapidgenotyping of the kdr alleles present in Ae aegypti LatinAmerican populations Our previous allele specific PCRalthough useful generally needed constant adjustments inthe number of cycles andor the concentration of specificprimers according to different thermal-cycle machinery orPCR kit employed [19 21] Sometimes unspecific shallowamplification also occurred pointing to the need of addi-tional confirmatory reactions The present TaqMan assayrenders the genotyping process more clear and straightfor-ward However one must be aware that any genotypingassay will only reveal the specific alleles available in thatassay For instance instead of a ValIle mutation found inAe aegypti from Latin American the kdr mutation in the1016 NaV site of Asian populations is a ValGly [14] towhich the assay employed here is unable to detect This isalso true for other potential mutations initially under lowfrequencies Therefore these allele specific methods shouldonly be used to evaluate genotypic frequencies of populationswith a previously well-explored genetic background of thetarget genes

The physiological importance of the kdr mutations andaltered sensitivity to pyrethroids have been evaluated withmutant insect NaV gene presenting punctual or combinedmutations in Xenopus oocites heterologous expression sys-tem followed by electrophysiological assays [22] Such assaysemploying direct mutagenesis of AaNaV cDNA demon-strated that the V1016I mutation alone (which we wouldcall NaVR3 allele) did not alter the channel sensitivityto pyrethroids while F1534C kdr mutation (NaVR1 allele)reduced the channel affinity of pyrethroid type I (perme-thrin) but not type II (deltamethrin) [37] In agreementthis same mutation reduced the affinity to type I but notto type II pyrethroids in the cockroach Blatella germanica

channel [41] However herein we found that although NaVR1conferred lower level of resistance than NaVR2 R1R1 insectswere resistant to deltamethrin a type II pyrethroid TheNaVR2 allele used to be absent in some deltamethrin resistantpopulations from the Northeast of Brazil where the NaVR1was found in high frequencies [19] More recent samplingsevidenced that the NaVR2 is disseminating and increasingin frequency also through those areas [20 30 42] Vera-Malof et al [17] proposed that the NaVR1 had emerged firstconferring low levels of resistance to pyrethroids and thenthe V1016I arose from that allele originating the NaVR2NaVR2 would have been rapidly selected and dispersed byconferring higher levels of resistance to pyrethroids Thepossible NaVR3 (1016Ilekdr + 1534Phe+) has not ever beenevidenced in Brazilian Ae aegypti populations and thereforewas not considered in our analysis Indeed we did not findany SR3 R2R3 and R3R3 individual

In the house flyMusca domestica the relationship of threeNaV alleles with resistance to pyrethroid was investigatedby evaluating the susceptibility of congenic strains and theirhybrids to a range of several pyrethroid compounds Thedouble mutant super-kdr allele (M918T + L1014F) conferredmore resistance than the classical kdr (L1014F) which in itsturn conferred more resistance than the kdr-his (L1014H)The heterozygotes kdrsuper-kdr and super-kdrkdr-his pre-sented intermediate resistance between the homozygouscharacterizing an incomplete recessive inherence partnerfor these M domestica NaV alleles [43] Ae aegypti fieldpopulations fromMalaysia showed increased resistancewhenpresenting kdrmutation in both 1016 and 1534 NaV sites [44]In that case however substitution in the 1016 site was Val toGly as common in Middle Eastern and Asian populations[15 45 46] Here a similar partner was observed in Aeaegypti The double mutant kdr allele NaVR2 conferred moreresistance to deltamethrin than NaVR1 evidenced by theKdT50

of the homozygote R2R2 (978 min) higher than thehomozygote R1R1 (674 min) corroborating the hypothesisthat the 1016 Ilekdr mutation synergises with 1534 Cyskdrproviding higher levels of resistance

The heterozygote R1R2 was intermediate (790 min)suggesting a synergistic effect of these two alleles The het-erozygotes with the wild-typeNaVS or the ldquoduplicatedrdquoNaVDalleles were all knocked down before 60 minutes howeverwith higher KdT

50than the homozygotes SS and DD charac-

terizing an incomplete recessive inherence of the kdr allelesThe I1011M mutation is frequent in Ae aegypti Brazilian

natural populations resistant to pyrethroids especially onthose where the NaVR2 allele is absent [30] A selectionpressure with pyrethroid in the laboratory increased thefrequency of 1011M where 100 of the insects had themutation but only heterozygotes were found [24] likewise infield populations [21] This finding raised the hypothesis thatI1011M mutation was part of a duplication event which wasevidenced by DNA sequencing and copy number variationqPCR assays [21] It is of note that this Dup lineage hadbeen originally selected from a field population and was notbackcrossed with Rockefeller differently from the processthat originated R1R1 and R2R2 colonies Although we cannot

8 BioMed Research International

assume the inexistence of any other resistance mechanismin the Dup lineage its KdT

50to deltamethrin (30 min) was

only twice the Rockefellerrsquos (145 min) not representing anexpressive tolerance It is possible to conclude that the 1011IleMet mutation in this ldquoheterozygousrdquo conformation is notimportant for knockdown resistance compared to those in1016 and 1534 NaV sites In agreement the same aforemen-tioned electrophysiological assays demonstrated that I1011Mreduced the sensitivity of the channel to permethrin but notto deltamethrin [37]

We took advantage of a large sampling of Ae aegypti inneighbourhoods from Rio de Janeiro city and surroundings[33] for exploring the frequency of the kdr alleles of 27 local-ities The predominance of the ldquoresistant genotypesrdquo (R1R1R1R2 and R2R2) ranged from 65 to 100 among the sampledlocalities Additionally the high frequency of ldquoresistant geno-typesrdquo matches the higher levels of resistance to pyrethroidsobserved from South-eastern Brazilian localities [8 30 47]When pooling the samples in Rio Baixada and Niteroi wedo not find significant difference in the genotypic frequenciesbetween Rio and Baixada but between Niteroi and bothRio and Baixada sites The distinct intensity of insecticideapplication and an unlikely active migration of Ae aegyptiamong these localities may reflect the observed differencesin the kdr genetic background A population genetic analysisbased on nuclear single nucleotide polymorphisms (SNP)and microssatelites revealed low overall spatial structuringamong 15 out of the same 27Ae aegypti populations fromRioherein evaluated for kdr genotyping The exception was thepopulation from Paqueta island the only which significantlydiffered from the other localities [33] likely due to thelimited gene flow island-continent Accordingly samplesfrom Paqueta presented the most divergent kdr frequenciesand presented the highest level of the wild-type NaVS alleleIn this island there is a preoccupation about natural andcultural heritage conservation in a way that the employmentof insecticides is supposedly better planned and controlledthan in the continent Even though still very high the lowerfrequency of kdr alleles was therefore expected in Paquetaisland In a recent study with Ae aegypti from five neighbourtowns aroundMerida Yucatan andMexico kdr frequenciesin both 1016 and 1534 NaV sites significantly varied amongtowns These differences were also significant in a finer scaleat the block levels in two of the evaluated towns [48]

5 Conclusion

Several mutations in the NaV gene are likely to conferpyrethroid resistance however due to significant fitnesscost they remain at very low frequencies Other mutationsconferring similar or higher levels of resistance neverthelesswith lower fitness cost are expected to evolve and disseminatein the population under pyrethroid selection pressure [10]As the selection exerted by governmental campaigns and byhousehold insecticide applications has been continuous overAe aegypti new other mutations are likely to be emergingfrom the current known wild-type and kdr alleles and pos-sibly conferring even higher resistance levels as recently evi-denced in the house flyM domestica [49]This highlights the

importance of monitoring not only the currently known kdrsites by direct genotyping technics but also the strategies ofwhole NaV gene sequencing associated with bioassays Herewe evidenced that the NaVR2 kdr allele confers higher levelof resistance to pyrethroid than this counterpart NaVR1 inAe aegypti laboratory lines with similar genetic backgroundsalso corroborating with the hypotheses of recessive inherentpattern of these kdr mutations Therefore the homozygouskdr genotypes as well as the heterozygous R1R2 are likely tobe the ones selected for pyrethroid resistance Additionallythe mutation in the 1011 NaV site was not important forresistance in the lineage with a duplication in the NaV geneconferring a heterozygous-like aspect to this mutation A kdrgenotyping survey of Ae aegypti from 27 distinct localitiesfrom Rio de Janeiro city and surroundings detected highfrequencies of ldquoresistant genotypesrdquo probably reflecting thehigh selection pressure exerted principally by householdinsecticide applicationsThis kind ofmolecularmonitoring isof relevance yet studies for unrevealing new markers relatedto resistance to other classes of insecticides are necessary

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

The authors thank Heloisa Diniz (IOCFiocruz) for takingcare of the figures This study was supported by the NationalInstitute of Health (NIH Grant no UO1 AI115595) InstitutoNacional de Ciencia e Tencologia em Entomologia Molecular(INCT-EM Grant no 4656782014-9) and Fundacao deAmparo a Pesquisa do Estado do Rio de Janeiro (FAPERJGrants nos E-261101682014 E-262018362017)

Supplementary Materials

Supplementary Figure S1 Scheme of backcrosses towardselection of Aedes aegypti kdr lineages highlighting theprocess of background homogenization between R2R2 andR1R1 Please follow detailed explanation in the Material andMethods Table Spp S1 Time of knockdown profile underexposition to the pyrethroid deltamethrin (003) in Aedesaegypti laboratory lineages with distinct genotypes for kdralleles Table Spp S2 Kdr allelic frequencies considering 1016and 1534 NaV sites in Aedes aegypti populations from Riode Janeiro State Supp Material S2 Kdr allelic frequenciesin Aedes aegypti populations from Rio de Janeito StateEach dot represents the frequency of the respective allelewith the confidence interval 95 amplitude indicated by thevertical bars A = Cabucu B = Ceramica C = Moqueta D =Heliopolis E = Jurujuba F = Itacoatiara G = Sao Francisco H= Fonseca I = Ponta Drsquoareia J = Piratininga K = TubiacanhaL = Valqueire M = Urca N = Olaria O = Gamboa

BioMed Research International 9

P = Caju Q = Pavuna R = Meier S = Grajau T = PaquetaU = Vaz Lobo V = Jardim Guanabara W = Sao Cristovao X= Rio Comprido Y = Humaita Z = Rio das Pesdras and 2 =Taquara (Supplementary Materials)

References

[1] S Bhatt PWGethingO J Brady et al ldquoThe global distributionand burden of denguerdquo Nature vol 496 no 7446 pp 504ndash5072013

[2] WHOWHO statement on the firstmeeting of the InternationalHealth Regulations (2005) (IHR 2005) Emergency Committeeon Zika virus and observed increase in neurological disor-ders and neonatal malformations 2016 Available from httpwwwwhointmediacentrenewsstatements20161st-emergen-cy-committee-zikaen

[3] D Couto-Lima Y Madec M I Bersot et al ldquoPotential riskof re-emergence of urban transmission of Yellow Fever virusin Brazil facilitated by competent Aedes populationsrdquo ScientificReports vol 7 no 1 2017

[4] WHO Mosquito (vector) control emergency response andpreparedness for Zika virus 2016 Available from httpwwwwhointneglected diseasesnewsmosquito vector control re-sponseen

[5] A Malavasi ldquoProject Aedes transgenic population control inJuazeiro and Jacobina Bahia Brazilrdquo BMC Proceedings vol 8no Suppl 4 p O11 2014

[6] L B Carrington B C N Tran N T H Le et al ldquoField-and clinically derived estimates of Wolbachia-mediated block-ing of dengue virus transmission potential in Aedes aegyptimosquitoesrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 115 no 2 pp 361ndash366 2018

[7] H Ranson J Burhani N Lumjuan and I V W C BlackldquoInsecticide resistance in dengue vectorsrdquo TropIKAnet no 12010

[8] C L Moyes J Vontas A J Martins et al ldquoContemporarystatus of insecticide resistance in the major Aedes vectorsof arboviruses infecting humansrdquo PLOS Neglected TropicalDiseases vol 11 no 7 Article ID e0005625 2017

[9] S Kasai O Komagata K Itokawa et al ldquoMechanisms ofPyrethroid Resistance in the Dengue Mosquito Vector Aedesaegypti Target Site Insensitivity Penetration andMetabolismrdquoPLOSNeglected Tropical Diseases vol 8 no 6 Article ID e29482014

[10] R H Ffrench-Constant B Pittendrigh A Vaughan and NAnthony ldquoWhy are there so few resistance-associatedmutationsin insecticide target genesrdquo Philosophical Transactions of theRoyal Society B Biological Sciences vol 353 no 1376 pp 1685ndash1693 1998

[11] F D Rinkevich S M Hedtke C A Leichter et al ldquoMultipleOrigins of kdr-type Resistance in the House Fly Musca domes-ticardquo PLoS ONE vol 7 no 12 Article ID e52761 2012

[12] J Pinto A Lynd J L Vicente et al ldquoMultiple origins ofknockdown resistance mutations in the afrotropical mosquitovector Anopheles gambiaerdquo PLoS ONE vol 2 no 11 Article IDe1243 2007

[13] K Saavedra-Rodriguez L Urdaneta-Marquez S Rajatileka etal ldquoA mutation in the voltage-gated sodium channel geneassociated with pyrethroid resistance in Latin American Aedesaegyptirdquo Insect Molecular Biology vol 16 no 6 pp 785ndash7982007

[14] Y Du Y Nomura B S Zhorov and K Dong ldquoSodium channelmutations and pyrethroid resistance in Aedes aegyptirdquo Insectsvol 7 no 4 2016

[15] A M Al Nazawi J Aqili M Alzahrani P J McCall and DWeetman ldquoCombined target site (kdr)mutations play a primaryrole in highly pyrethroid resistant phenotypes of Aedes aegyptifrom Saudi Arabiardquo Parasites amp Vectors vol 10 no 1 pp 1ndash102017

[16] H Kawada Y Higa K Futami et al ldquoDiscovery of PointMutations in the Voltage-Gated Sodium Channel from AfricanAedes aegypti Populations Potential Phylogenetic Reasons forGene Introgressionrdquo PLOS Neglected Tropical Diseases vol 10no 6 Article ID e0004780 2016

[17] F Z Vera-Maloof K Saavedra-Rodriguez A E Elizondo-Quiroga S Lozano-Fuentes and W C Black IV ldquoCoevolutionof the Ile1016 and Cys1534 Mutations in the Voltage GatedSodium Channel Gene of Aedes aegypti in Mexicordquo PLOSNeglected Tropical Diseases vol 9 no 12 Article ID e00042632015

[18] G Seixas L Grigoraki D Weetman et al ldquoInsecticide resis-tance is mediated by multiple mechanisms in recently intro-duced Aedes aegypti from Madeira Island (Portugal)rdquo PLOSNeglected Tropical Diseases vol 11 no 7 Article ID e00057992017

[19] J G B Linss L P Brito G A Garcia et al ldquoDistribution anddissemination of the Val1016Ile and Phe1534Cys Kdr mutationsin Aedes aegypti Brazilian natural populationsrdquo Parasites ampVectors vol 7 no 1 article 25 2014

[20] D F Bellinato P F Viana-Medeiros S C Araujo et alldquoResistance Status to the Insecticides Temephos Deltamethrinand Diflubenzuron in Brazilian Aedes aegypti PopulationsrdquoBioMed Research International vol 2016 Article ID 8603263pp 1ndash12 2016

[21] A J Martins L P Brito J G Linss et al ldquoEvidence for geneduplication in the voltage-gated sodium channel gene of Aedesaegyptirdquo Evolution Medicine and Public Health vol 2013 no 1pp 148ndash160 2013

[22] K Dong Y Du F Rinkevich et al ldquoMolecular biology of insectsodium channels and pyrethroid resistancerdquo Insect Biochemistryand Molecular Biology vol 50 no 1 pp 1ndash17 2014

[23] A JMartins RM Lins J G Linss et al ldquoVoltage-gated sodiumchannel polymorphism andmetabolic resistance in pyrethroid-resistant Aedes aegypti from Brazilrdquo The American Journal ofTropical Medicine and Hygiene vol 81 no 1 pp 108ndash115 2009

[24] A J Martins C D M Ribeiro D F Bellinato A A PeixotoD Valle and J B P Lima ldquoEffect of insecticide resistance ondevelopment longevity and reproduction of field or laboratoryselected Aedes aegypti populationsrdquo PLoS ONE vol 7 no 3Article ID e31889 2012

[25] R M R Nogueira M P Miagostovich A M B De FilippisM A S Pereira and H G Schatzmayr ldquoDengue Virus Type 3in Rio de Janeiro BrazilrdquoMemorias do Instituto Oswaldo Cruzvol 96 no 7 pp 925-926 2001

[26] R M R Nogueira M P Miagostovich E Lampe R W SouzaS M O Zagne andH G Schatzmayr ldquoDengue epidemic in thestate of Rio de Janeiro Brazil 1990ndash1 Co-circulation of dengue1 and dengue 2 serotypesrdquo Epidemiology and Infection vol 111no 1 pp 163ndash170 1993

[27] G Kuno ldquoEarly history of laboratory breeding of Aedes aegypti(Diptera Culicidae) focusing on the origins and use of selectedstrainsrdquo Journal of Medical Entomology vol 47 no 6 pp 957ndash971 2010

BioMed Research International 5

Figure 3 Frequencies of kdr alleles considering 1016 and 1534 NaV sites of Aedes aegypti from Rio de Janeiro State Localities areindicated in the map The shadowed area in green pink and grey represent the regions Rio Niteroi and Baixada respectively

(176) Interestingly when pooling neighbourhoodrsquos datato their respective regions NaVS frequency was higher inNiteroi (142) than in Rio (40) a significant differenceby considering that there is no overlapping among theirsIC95 (supp material S2) Paqueta was removed from thisanalysis since it is an island distant fromboth Rio andNiteroioffshores These cities are separated by the Guanabara Bayconnected by a 13 Km bridge and ferry boats

The R2R2 genotype which would potentially accountfor higher levels of resistance to pyrethroids was the mostfrequent genotype (median 500) and the ldquoresistant geno-typesrdquo (R1R1 R2R2 and R1R2) together reached a median of884 among Ae aegypti from the sites evaluated (Table 2)The localities with the lowest frequency of the ldquoresistantgenotypesrdquo were Paqueta island (60) followed by five outof the six neighbourhoods evaluated from Niteroi (647 inFonseca-757 in Piratininga)

The genotypic frequencies among regions (Baixada RioNiteroi and Paqueta island) did not differ significantly

between Rio and Baixada as well as between Niteroi andPaqueta (both exact G test Pgt005) In their turn RioBaixadahighly differed from NiteroiPaqueta (see Table 3)

4 Discussion

Here we evaluated the difference in insecticide resistancelevels to the pyrethroid deltamethrin inAe aegypti laboratorylines with distinct kdr mutations introgressed from the fieldand free of other known resistance mechanism Severalrecords of increased levels of resistance to pyrethroid inparallel with dissemination of kdr alleles in natural vectorpopulations have been released in the last decade as anindirect correlation between kdr mutation and pyrethroidresistance inAe aegypti [8 35] Laboratory selection pressureexperiments with insecticide have also corroborated thiscorrelation when kdr frequencies have increased towardfixation [13] In addition electrophysiological tests based onnatural populations as well as samples that undergone site

6 BioMed Research International

Table 2 Kdr genotypic frequencies in field populations of Aedes aegypti from Rio de Janeiro State considering the 1016 and 1534 NaV sites

locality n observed genotype frequencies HWE testlowast ResistantgenotypesSS SR1 SR2 R1R1 R1R2 R2R2 Σ1205942 plowast

Cabucu 52 0 0019 0115 0135 0462 0269 88 0032 0865Ceramica 73 0014 0041 0041 0110 0411 0384 00 1000 0904Moqueta 65 0015 0015 0077 0077 0215 0600 315 0000 0892Heliopolis 36 0 0028 0111 0056 0111 0694 584 0000 0861Jurujuba 12 0 0 0250 0083 0583 0083 85 0036 0750Itacoatiara 24 0 0 0167 0208 0292 0333 163 0001 0833Sao Francisco 16 0 0 0250 0 0125 0625 34 0338 0750Fonseca 17 0059 0 0294 0118 0118 0412 855 0000 0647Ponta DAreia 14 0 0 0286 0 0 0714 34 0064 0714Piratininga 37 0081 0054 0108 0270 0189 0297 278 0000 0757Tubiacanga 43 0 0070 0047 0186 0163 0535 213 0000 0884Valqueire 31 0 0032 0097 0032 0290 0548 100 0019 0871Urca 20 0 0050 0050 0300 0400 0200 14 0708 0900Olaria 35 0 0000 0029 0086 0314 0571 28 0425 0971Gamboa 17 0 0 0059 0059 0529 0353 13 0718 0941Caju 23 0043 0 0043 0087 0087 0739 44 0224 0913Pavuna 34 0 0 0118 0118 0265 0500 207 0000 0882Meier 37 0 0054 0 0135 0297 0514 66 0086 0946Grajau 38 0 0 0053 0053 0316 0579 47 0194 0947Paqueta 20 0050 0 0350 0250 0150 0200 546 0000 0600Vaz Lobo 35 0 0143 0057 0143 0229 0429 130 0005 0800Jardim Guanab 32 0 0031 0 0094 0375 0500 08 0841 0969Sao Cirstovao 23 0 0 0 0043 0522 0435 12 0266 1000Humaita 18 0 0056 0111 0167 0500 0167 26 0454 0833Rio Comprido 16 0 0 0 0125 0313 0563 08 0364 1000Rio das Pedras 17 0 0 0 0118 0529 0353 02 0618 1000Taquara 26 0 0 0038 0077 0192 0692 79 0048 0962median 0 0 0059 0110 0292 0500 0884lowastHardy-Weinberg Equilibrium testlowastlowast Probability considering the chi-squared distribution for one or three degrees of freedom respectively for three or six genotypes

Table 3 Comparisons among kdr genotypic frequencies of Aedes aegypti from distinct regions of Rio de Janeiro State

Region pair 1205942 df P-valueBaixada x Niteroi 1008 2 0006Baixada x Rio 179 2 0409Niteroi x Rio infinity 2 lt0001Baixada x Paqueta 1330 2 0001Niteroi x Paqueta 209 2 0351Rio x Paqueta infinity 2 lt0001Baixada (Cabucu Ceramica and Moqueta ans Heliopolis) Niteroi (Jurujuba Itacoatiara Sao Francisco Fonseca Ponta DrsquoAreia and Piratininga) Rio(Tubiacanga Valqueire Urca Olaraia Gamboa Caju Pavuna Meier Grajau Vaz Lobo Jardim Guanabara Sao Cristovao Humaita Rio das Pedras andTaquara) and Paqueta (Paqueta island)

directed mutagenesis confirmed that kdr mutations found inAe aegypti alter sensibility to pyrethroids [36 37] Howeverto our knowledge this is the first study in vivo that evincesthe importance of such mutations in homogeneous kdrlaboratory lines of Ae aegypti except for the kdr locus

ie with minimum interference of other possibly selectedmechanisms as well as of pleiotropic effects that mightrespond differently to each distinct genetic background

WHO Pesticide Evaluation Scheme (WHOPES) recom-mends discriminating concentrations of insecticides for the

BioMed Research International 7

impregnated paper tests [38] The most recent WHOPESplan for detecting and monitoring IR in Aedes aegyptirecommended 003 as a discriminating concentration ofdeltamethrin [39] to where mortality is recorded 24 h after1 h of exposition to the insecticide Mortality under 90indicates resistance of the evaluated population as long as atleast 100 mosquitoes were tested This is a qualitative dose-diagnostic test good for determining the susceptibility statusof a given population although not suitable for comparinglevels of resistance among populations For this matter aquantitative dose-response test is indicated in which themortality rates over a range of insecticide concentrationsgenerate the lethal concentrations (LC) of each populationIn turn LC produces the resistance rations (RR) based ona reference lineage [40] However this sort of test requiresa large number of insects compared to the dose-responseapproach Here we applied a WHO-like dose-diagnostic testwith papers impregnated on our ownNevertheless instead ofevaluating mortality 24 h after 1h of exposition we followedthe knockdown rate for up to 2 h With this record over thetime a semiquantitative analysis was performed by extractingthe RR of the populations in this case based on their timeof knockdown It is worth noting that this knockdown timeRR displays a different scale generally shorter than thoseproduced by truly qualitative dose-response tests

We employed a very efficient TaqMan assay for the rapidgenotyping of the kdr alleles present in Ae aegypti LatinAmerican populations Our previous allele specific PCRalthough useful generally needed constant adjustments inthe number of cycles andor the concentration of specificprimers according to different thermal-cycle machinery orPCR kit employed [19 21] Sometimes unspecific shallowamplification also occurred pointing to the need of addi-tional confirmatory reactions The present TaqMan assayrenders the genotyping process more clear and straightfor-ward However one must be aware that any genotypingassay will only reveal the specific alleles available in thatassay For instance instead of a ValIle mutation found inAe aegypti from Latin American the kdr mutation in the1016 NaV site of Asian populations is a ValGly [14] towhich the assay employed here is unable to detect This isalso true for other potential mutations initially under lowfrequencies Therefore these allele specific methods shouldonly be used to evaluate genotypic frequencies of populationswith a previously well-explored genetic background of thetarget genes

The physiological importance of the kdr mutations andaltered sensitivity to pyrethroids have been evaluated withmutant insect NaV gene presenting punctual or combinedmutations in Xenopus oocites heterologous expression sys-tem followed by electrophysiological assays [22] Such assaysemploying direct mutagenesis of AaNaV cDNA demon-strated that the V1016I mutation alone (which we wouldcall NaVR3 allele) did not alter the channel sensitivityto pyrethroids while F1534C kdr mutation (NaVR1 allele)reduced the channel affinity of pyrethroid type I (perme-thrin) but not type II (deltamethrin) [37] In agreementthis same mutation reduced the affinity to type I but notto type II pyrethroids in the cockroach Blatella germanica

channel [41] However herein we found that although NaVR1conferred lower level of resistance than NaVR2 R1R1 insectswere resistant to deltamethrin a type II pyrethroid TheNaVR2 allele used to be absent in some deltamethrin resistantpopulations from the Northeast of Brazil where the NaVR1was found in high frequencies [19] More recent samplingsevidenced that the NaVR2 is disseminating and increasingin frequency also through those areas [20 30 42] Vera-Malof et al [17] proposed that the NaVR1 had emerged firstconferring low levels of resistance to pyrethroids and thenthe V1016I arose from that allele originating the NaVR2NaVR2 would have been rapidly selected and dispersed byconferring higher levels of resistance to pyrethroids Thepossible NaVR3 (1016Ilekdr + 1534Phe+) has not ever beenevidenced in Brazilian Ae aegypti populations and thereforewas not considered in our analysis Indeed we did not findany SR3 R2R3 and R3R3 individual

In the house flyMusca domestica the relationship of threeNaV alleles with resistance to pyrethroid was investigatedby evaluating the susceptibility of congenic strains and theirhybrids to a range of several pyrethroid compounds Thedouble mutant super-kdr allele (M918T + L1014F) conferredmore resistance than the classical kdr (L1014F) which in itsturn conferred more resistance than the kdr-his (L1014H)The heterozygotes kdrsuper-kdr and super-kdrkdr-his pre-sented intermediate resistance between the homozygouscharacterizing an incomplete recessive inherence partnerfor these M domestica NaV alleles [43] Ae aegypti fieldpopulations fromMalaysia showed increased resistancewhenpresenting kdrmutation in both 1016 and 1534 NaV sites [44]In that case however substitution in the 1016 site was Val toGly as common in Middle Eastern and Asian populations[15 45 46] Here a similar partner was observed in Aeaegypti The double mutant kdr allele NaVR2 conferred moreresistance to deltamethrin than NaVR1 evidenced by theKdT50

of the homozygote R2R2 (978 min) higher than thehomozygote R1R1 (674 min) corroborating the hypothesisthat the 1016 Ilekdr mutation synergises with 1534 Cyskdrproviding higher levels of resistance

The heterozygote R1R2 was intermediate (790 min)suggesting a synergistic effect of these two alleles The het-erozygotes with the wild-typeNaVS or the ldquoduplicatedrdquoNaVDalleles were all knocked down before 60 minutes howeverwith higher KdT

50than the homozygotes SS and DD charac-

terizing an incomplete recessive inherence of the kdr allelesThe I1011M mutation is frequent in Ae aegypti Brazilian

natural populations resistant to pyrethroids especially onthose where the NaVR2 allele is absent [30] A selectionpressure with pyrethroid in the laboratory increased thefrequency of 1011M where 100 of the insects had themutation but only heterozygotes were found [24] likewise infield populations [21] This finding raised the hypothesis thatI1011M mutation was part of a duplication event which wasevidenced by DNA sequencing and copy number variationqPCR assays [21] It is of note that this Dup lineage hadbeen originally selected from a field population and was notbackcrossed with Rockefeller differently from the processthat originated R1R1 and R2R2 colonies Although we cannot

8 BioMed Research International

assume the inexistence of any other resistance mechanismin the Dup lineage its KdT

50to deltamethrin (30 min) was

only twice the Rockefellerrsquos (145 min) not representing anexpressive tolerance It is possible to conclude that the 1011IleMet mutation in this ldquoheterozygousrdquo conformation is notimportant for knockdown resistance compared to those in1016 and 1534 NaV sites In agreement the same aforemen-tioned electrophysiological assays demonstrated that I1011Mreduced the sensitivity of the channel to permethrin but notto deltamethrin [37]

We took advantage of a large sampling of Ae aegypti inneighbourhoods from Rio de Janeiro city and surroundings[33] for exploring the frequency of the kdr alleles of 27 local-ities The predominance of the ldquoresistant genotypesrdquo (R1R1R1R2 and R2R2) ranged from 65 to 100 among the sampledlocalities Additionally the high frequency of ldquoresistant geno-typesrdquo matches the higher levels of resistance to pyrethroidsobserved from South-eastern Brazilian localities [8 30 47]When pooling the samples in Rio Baixada and Niteroi wedo not find significant difference in the genotypic frequenciesbetween Rio and Baixada but between Niteroi and bothRio and Baixada sites The distinct intensity of insecticideapplication and an unlikely active migration of Ae aegyptiamong these localities may reflect the observed differencesin the kdr genetic background A population genetic analysisbased on nuclear single nucleotide polymorphisms (SNP)and microssatelites revealed low overall spatial structuringamong 15 out of the same 27Ae aegypti populations fromRioherein evaluated for kdr genotyping The exception was thepopulation from Paqueta island the only which significantlydiffered from the other localities [33] likely due to thelimited gene flow island-continent Accordingly samplesfrom Paqueta presented the most divergent kdr frequenciesand presented the highest level of the wild-type NaVS alleleIn this island there is a preoccupation about natural andcultural heritage conservation in a way that the employmentof insecticides is supposedly better planned and controlledthan in the continent Even though still very high the lowerfrequency of kdr alleles was therefore expected in Paquetaisland In a recent study with Ae aegypti from five neighbourtowns aroundMerida Yucatan andMexico kdr frequenciesin both 1016 and 1534 NaV sites significantly varied amongtowns These differences were also significant in a finer scaleat the block levels in two of the evaluated towns [48]

5 Conclusion

Several mutations in the NaV gene are likely to conferpyrethroid resistance however due to significant fitnesscost they remain at very low frequencies Other mutationsconferring similar or higher levels of resistance neverthelesswith lower fitness cost are expected to evolve and disseminatein the population under pyrethroid selection pressure [10]As the selection exerted by governmental campaigns and byhousehold insecticide applications has been continuous overAe aegypti new other mutations are likely to be emergingfrom the current known wild-type and kdr alleles and pos-sibly conferring even higher resistance levels as recently evi-denced in the house flyM domestica [49]This highlights the

importance of monitoring not only the currently known kdrsites by direct genotyping technics but also the strategies ofwhole NaV gene sequencing associated with bioassays Herewe evidenced that the NaVR2 kdr allele confers higher levelof resistance to pyrethroid than this counterpart NaVR1 inAe aegypti laboratory lines with similar genetic backgroundsalso corroborating with the hypotheses of recessive inherentpattern of these kdr mutations Therefore the homozygouskdr genotypes as well as the heterozygous R1R2 are likely tobe the ones selected for pyrethroid resistance Additionallythe mutation in the 1011 NaV site was not important forresistance in the lineage with a duplication in the NaV geneconferring a heterozygous-like aspect to this mutation A kdrgenotyping survey of Ae aegypti from 27 distinct localitiesfrom Rio de Janeiro city and surroundings detected highfrequencies of ldquoresistant genotypesrdquo probably reflecting thehigh selection pressure exerted principally by householdinsecticide applicationsThis kind ofmolecularmonitoring isof relevance yet studies for unrevealing new markers relatedto resistance to other classes of insecticides are necessary

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

The authors thank Heloisa Diniz (IOCFiocruz) for takingcare of the figures This study was supported by the NationalInstitute of Health (NIH Grant no UO1 AI115595) InstitutoNacional de Ciencia e Tencologia em Entomologia Molecular(INCT-EM Grant no 4656782014-9) and Fundacao deAmparo a Pesquisa do Estado do Rio de Janeiro (FAPERJGrants nos E-261101682014 E-262018362017)

Supplementary Materials

Supplementary Figure S1 Scheme of backcrosses towardselection of Aedes aegypti kdr lineages highlighting theprocess of background homogenization between R2R2 andR1R1 Please follow detailed explanation in the Material andMethods Table Spp S1 Time of knockdown profile underexposition to the pyrethroid deltamethrin (003) in Aedesaegypti laboratory lineages with distinct genotypes for kdralleles Table Spp S2 Kdr allelic frequencies considering 1016and 1534 NaV sites in Aedes aegypti populations from Riode Janeiro State Supp Material S2 Kdr allelic frequenciesin Aedes aegypti populations from Rio de Janeito StateEach dot represents the frequency of the respective allelewith the confidence interval 95 amplitude indicated by thevertical bars A = Cabucu B = Ceramica C = Moqueta D =Heliopolis E = Jurujuba F = Itacoatiara G = Sao Francisco H= Fonseca I = Ponta Drsquoareia J = Piratininga K = TubiacanhaL = Valqueire M = Urca N = Olaria O = Gamboa

BioMed Research International 9

P = Caju Q = Pavuna R = Meier S = Grajau T = PaquetaU = Vaz Lobo V = Jardim Guanabara W = Sao Cristovao X= Rio Comprido Y = Humaita Z = Rio das Pesdras and 2 =Taquara (Supplementary Materials)

References

[1] S Bhatt PWGethingO J Brady et al ldquoThe global distributionand burden of denguerdquo Nature vol 496 no 7446 pp 504ndash5072013

[2] WHOWHO statement on the firstmeeting of the InternationalHealth Regulations (2005) (IHR 2005) Emergency Committeeon Zika virus and observed increase in neurological disor-ders and neonatal malformations 2016 Available from httpwwwwhointmediacentrenewsstatements20161st-emergen-cy-committee-zikaen

[3] D Couto-Lima Y Madec M I Bersot et al ldquoPotential riskof re-emergence of urban transmission of Yellow Fever virusin Brazil facilitated by competent Aedes populationsrdquo ScientificReports vol 7 no 1 2017

[4] WHO Mosquito (vector) control emergency response andpreparedness for Zika virus 2016 Available from httpwwwwhointneglected diseasesnewsmosquito vector control re-sponseen

[5] A Malavasi ldquoProject Aedes transgenic population control inJuazeiro and Jacobina Bahia Brazilrdquo BMC Proceedings vol 8no Suppl 4 p O11 2014

[6] L B Carrington B C N Tran N T H Le et al ldquoField-and clinically derived estimates of Wolbachia-mediated block-ing of dengue virus transmission potential in Aedes aegyptimosquitoesrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 115 no 2 pp 361ndash366 2018

[7] H Ranson J Burhani N Lumjuan and I V W C BlackldquoInsecticide resistance in dengue vectorsrdquo TropIKAnet no 12010

[8] C L Moyes J Vontas A J Martins et al ldquoContemporarystatus of insecticide resistance in the major Aedes vectorsof arboviruses infecting humansrdquo PLOS Neglected TropicalDiseases vol 11 no 7 Article ID e0005625 2017

[9] S Kasai O Komagata K Itokawa et al ldquoMechanisms ofPyrethroid Resistance in the Dengue Mosquito Vector Aedesaegypti Target Site Insensitivity Penetration andMetabolismrdquoPLOSNeglected Tropical Diseases vol 8 no 6 Article ID e29482014

[10] R H Ffrench-Constant B Pittendrigh A Vaughan and NAnthony ldquoWhy are there so few resistance-associatedmutationsin insecticide target genesrdquo Philosophical Transactions of theRoyal Society B Biological Sciences vol 353 no 1376 pp 1685ndash1693 1998

[11] F D Rinkevich S M Hedtke C A Leichter et al ldquoMultipleOrigins of kdr-type Resistance in the House Fly Musca domes-ticardquo PLoS ONE vol 7 no 12 Article ID e52761 2012

[12] J Pinto A Lynd J L Vicente et al ldquoMultiple origins ofknockdown resistance mutations in the afrotropical mosquitovector Anopheles gambiaerdquo PLoS ONE vol 2 no 11 Article IDe1243 2007

[13] K Saavedra-Rodriguez L Urdaneta-Marquez S Rajatileka etal ldquoA mutation in the voltage-gated sodium channel geneassociated with pyrethroid resistance in Latin American Aedesaegyptirdquo Insect Molecular Biology vol 16 no 6 pp 785ndash7982007

[14] Y Du Y Nomura B S Zhorov and K Dong ldquoSodium channelmutations and pyrethroid resistance in Aedes aegyptirdquo Insectsvol 7 no 4 2016

[15] A M Al Nazawi J Aqili M Alzahrani P J McCall and DWeetman ldquoCombined target site (kdr)mutations play a primaryrole in highly pyrethroid resistant phenotypes of Aedes aegyptifrom Saudi Arabiardquo Parasites amp Vectors vol 10 no 1 pp 1ndash102017

[16] H Kawada Y Higa K Futami et al ldquoDiscovery of PointMutations in the Voltage-Gated Sodium Channel from AfricanAedes aegypti Populations Potential Phylogenetic Reasons forGene Introgressionrdquo PLOS Neglected Tropical Diseases vol 10no 6 Article ID e0004780 2016

[17] F Z Vera-Maloof K Saavedra-Rodriguez A E Elizondo-Quiroga S Lozano-Fuentes and W C Black IV ldquoCoevolutionof the Ile1016 and Cys1534 Mutations in the Voltage GatedSodium Channel Gene of Aedes aegypti in Mexicordquo PLOSNeglected Tropical Diseases vol 9 no 12 Article ID e00042632015

[18] G Seixas L Grigoraki D Weetman et al ldquoInsecticide resis-tance is mediated by multiple mechanisms in recently intro-duced Aedes aegypti from Madeira Island (Portugal)rdquo PLOSNeglected Tropical Diseases vol 11 no 7 Article ID e00057992017

[19] J G B Linss L P Brito G A Garcia et al ldquoDistribution anddissemination of the Val1016Ile and Phe1534Cys Kdr mutationsin Aedes aegypti Brazilian natural populationsrdquo Parasites ampVectors vol 7 no 1 article 25 2014

[20] D F Bellinato P F Viana-Medeiros S C Araujo et alldquoResistance Status to the Insecticides Temephos Deltamethrinand Diflubenzuron in Brazilian Aedes aegypti PopulationsrdquoBioMed Research International vol 2016 Article ID 8603263pp 1ndash12 2016

[21] A J Martins L P Brito J G Linss et al ldquoEvidence for geneduplication in the voltage-gated sodium channel gene of Aedesaegyptirdquo Evolution Medicine and Public Health vol 2013 no 1pp 148ndash160 2013

[22] K Dong Y Du F Rinkevich et al ldquoMolecular biology of insectsodium channels and pyrethroid resistancerdquo Insect Biochemistryand Molecular Biology vol 50 no 1 pp 1ndash17 2014

[23] A JMartins RM Lins J G Linss et al ldquoVoltage-gated sodiumchannel polymorphism andmetabolic resistance in pyrethroid-resistant Aedes aegypti from Brazilrdquo The American Journal ofTropical Medicine and Hygiene vol 81 no 1 pp 108ndash115 2009

[24] A J Martins C D M Ribeiro D F Bellinato A A PeixotoD Valle and J B P Lima ldquoEffect of insecticide resistance ondevelopment longevity and reproduction of field or laboratoryselected Aedes aegypti populationsrdquo PLoS ONE vol 7 no 3Article ID e31889 2012

[25] R M R Nogueira M P Miagostovich A M B De FilippisM A S Pereira and H G Schatzmayr ldquoDengue Virus Type 3in Rio de Janeiro BrazilrdquoMemorias do Instituto Oswaldo Cruzvol 96 no 7 pp 925-926 2001

[26] R M R Nogueira M P Miagostovich E Lampe R W SouzaS M O Zagne andH G Schatzmayr ldquoDengue epidemic in thestate of Rio de Janeiro Brazil 1990ndash1 Co-circulation of dengue1 and dengue 2 serotypesrdquo Epidemiology and Infection vol 111no 1 pp 163ndash170 1993

[27] G Kuno ldquoEarly history of laboratory breeding of Aedes aegypti(Diptera Culicidae) focusing on the origins and use of selectedstrainsrdquo Journal of Medical Entomology vol 47 no 6 pp 957ndash971 2010

6 BioMed Research International

Table 2 Kdr genotypic frequencies in field populations of Aedes aegypti from Rio de Janeiro State considering the 1016 and 1534 NaV sites

locality n observed genotype frequencies HWE testlowast ResistantgenotypesSS SR1 SR2 R1R1 R1R2 R2R2 Σ1205942 plowast

Cabucu 52 0 0019 0115 0135 0462 0269 88 0032 0865Ceramica 73 0014 0041 0041 0110 0411 0384 00 1000 0904Moqueta 65 0015 0015 0077 0077 0215 0600 315 0000 0892Heliopolis 36 0 0028 0111 0056 0111 0694 584 0000 0861Jurujuba 12 0 0 0250 0083 0583 0083 85 0036 0750Itacoatiara 24 0 0 0167 0208 0292 0333 163 0001 0833Sao Francisco 16 0 0 0250 0 0125 0625 34 0338 0750Fonseca 17 0059 0 0294 0118 0118 0412 855 0000 0647Ponta DAreia 14 0 0 0286 0 0 0714 34 0064 0714Piratininga 37 0081 0054 0108 0270 0189 0297 278 0000 0757Tubiacanga 43 0 0070 0047 0186 0163 0535 213 0000 0884Valqueire 31 0 0032 0097 0032 0290 0548 100 0019 0871Urca 20 0 0050 0050 0300 0400 0200 14 0708 0900Olaria 35 0 0000 0029 0086 0314 0571 28 0425 0971Gamboa 17 0 0 0059 0059 0529 0353 13 0718 0941Caju 23 0043 0 0043 0087 0087 0739 44 0224 0913Pavuna 34 0 0 0118 0118 0265 0500 207 0000 0882Meier 37 0 0054 0 0135 0297 0514 66 0086 0946Grajau 38 0 0 0053 0053 0316 0579 47 0194 0947Paqueta 20 0050 0 0350 0250 0150 0200 546 0000 0600Vaz Lobo 35 0 0143 0057 0143 0229 0429 130 0005 0800Jardim Guanab 32 0 0031 0 0094 0375 0500 08 0841 0969Sao Cirstovao 23 0 0 0 0043 0522 0435 12 0266 1000Humaita 18 0 0056 0111 0167 0500 0167 26 0454 0833Rio Comprido 16 0 0 0 0125 0313 0563 08 0364 1000Rio das Pedras 17 0 0 0 0118 0529 0353 02 0618 1000Taquara 26 0 0 0038 0077 0192 0692 79 0048 0962median 0 0 0059 0110 0292 0500 0884lowastHardy-Weinberg Equilibrium testlowastlowast Probability considering the chi-squared distribution for one or three degrees of freedom respectively for three or six genotypes

Table 3 Comparisons among kdr genotypic frequencies of Aedes aegypti from distinct regions of Rio de Janeiro State

Region pair 1205942 df P-valueBaixada x Niteroi 1008 2 0006Baixada x Rio 179 2 0409Niteroi x Rio infinity 2 lt0001Baixada x Paqueta 1330 2 0001Niteroi x Paqueta 209 2 0351Rio x Paqueta infinity 2 lt0001Baixada (Cabucu Ceramica and Moqueta ans Heliopolis) Niteroi (Jurujuba Itacoatiara Sao Francisco Fonseca Ponta DrsquoAreia and Piratininga) Rio(Tubiacanga Valqueire Urca Olaraia Gamboa Caju Pavuna Meier Grajau Vaz Lobo Jardim Guanabara Sao Cristovao Humaita Rio das Pedras andTaquara) and Paqueta (Paqueta island)

directed mutagenesis confirmed that kdr mutations found inAe aegypti alter sensibility to pyrethroids [36 37] Howeverto our knowledge this is the first study in vivo that evincesthe importance of such mutations in homogeneous kdrlaboratory lines of Ae aegypti except for the kdr locus

ie with minimum interference of other possibly selectedmechanisms as well as of pleiotropic effects that mightrespond differently to each distinct genetic background

WHO Pesticide Evaluation Scheme (WHOPES) recom-mends discriminating concentrations of insecticides for the

BioMed Research International 7

impregnated paper tests [38] The most recent WHOPESplan for detecting and monitoring IR in Aedes aegyptirecommended 003 as a discriminating concentration ofdeltamethrin [39] to where mortality is recorded 24 h after1 h of exposition to the insecticide Mortality under 90indicates resistance of the evaluated population as long as atleast 100 mosquitoes were tested This is a qualitative dose-diagnostic test good for determining the susceptibility statusof a given population although not suitable for comparinglevels of resistance among populations For this matter aquantitative dose-response test is indicated in which themortality rates over a range of insecticide concentrationsgenerate the lethal concentrations (LC) of each populationIn turn LC produces the resistance rations (RR) based ona reference lineage [40] However this sort of test requiresa large number of insects compared to the dose-responseapproach Here we applied a WHO-like dose-diagnostic testwith papers impregnated on our ownNevertheless instead ofevaluating mortality 24 h after 1h of exposition we followedthe knockdown rate for up to 2 h With this record over thetime a semiquantitative analysis was performed by extractingthe RR of the populations in this case based on their timeof knockdown It is worth noting that this knockdown timeRR displays a different scale generally shorter than thoseproduced by truly qualitative dose-response tests

We employed a very efficient TaqMan assay for the rapidgenotyping of the kdr alleles present in Ae aegypti LatinAmerican populations Our previous allele specific PCRalthough useful generally needed constant adjustments inthe number of cycles andor the concentration of specificprimers according to different thermal-cycle machinery orPCR kit employed [19 21] Sometimes unspecific shallowamplification also occurred pointing to the need of addi-tional confirmatory reactions The present TaqMan assayrenders the genotyping process more clear and straightfor-ward However one must be aware that any genotypingassay will only reveal the specific alleles available in thatassay For instance instead of a ValIle mutation found inAe aegypti from Latin American the kdr mutation in the1016 NaV site of Asian populations is a ValGly [14] towhich the assay employed here is unable to detect This isalso true for other potential mutations initially under lowfrequencies Therefore these allele specific methods shouldonly be used to evaluate genotypic frequencies of populationswith a previously well-explored genetic background of thetarget genes

The physiological importance of the kdr mutations andaltered sensitivity to pyrethroids have been evaluated withmutant insect NaV gene presenting punctual or combinedmutations in Xenopus oocites heterologous expression sys-tem followed by electrophysiological assays [22] Such assaysemploying direct mutagenesis of AaNaV cDNA demon-strated that the V1016I mutation alone (which we wouldcall NaVR3 allele) did not alter the channel sensitivityto pyrethroids while F1534C kdr mutation (NaVR1 allele)reduced the channel affinity of pyrethroid type I (perme-thrin) but not type II (deltamethrin) [37] In agreementthis same mutation reduced the affinity to type I but notto type II pyrethroids in the cockroach Blatella germanica

channel [41] However herein we found that although NaVR1conferred lower level of resistance than NaVR2 R1R1 insectswere resistant to deltamethrin a type II pyrethroid TheNaVR2 allele used to be absent in some deltamethrin resistantpopulations from the Northeast of Brazil where the NaVR1was found in high frequencies [19] More recent samplingsevidenced that the NaVR2 is disseminating and increasingin frequency also through those areas [20 30 42] Vera-Malof et al [17] proposed that the NaVR1 had emerged firstconferring low levels of resistance to pyrethroids and thenthe V1016I arose from that allele originating the NaVR2NaVR2 would have been rapidly selected and dispersed byconferring higher levels of resistance to pyrethroids Thepossible NaVR3 (1016Ilekdr + 1534Phe+) has not ever beenevidenced in Brazilian Ae aegypti populations and thereforewas not considered in our analysis Indeed we did not findany SR3 R2R3 and R3R3 individual

In the house flyMusca domestica the relationship of threeNaV alleles with resistance to pyrethroid was investigatedby evaluating the susceptibility of congenic strains and theirhybrids to a range of several pyrethroid compounds Thedouble mutant super-kdr allele (M918T + L1014F) conferredmore resistance than the classical kdr (L1014F) which in itsturn conferred more resistance than the kdr-his (L1014H)The heterozygotes kdrsuper-kdr and super-kdrkdr-his pre-sented intermediate resistance between the homozygouscharacterizing an incomplete recessive inherence partnerfor these M domestica NaV alleles [43] Ae aegypti fieldpopulations fromMalaysia showed increased resistancewhenpresenting kdrmutation in both 1016 and 1534 NaV sites [44]In that case however substitution in the 1016 site was Val toGly as common in Middle Eastern and Asian populations[15 45 46] Here a similar partner was observed in Aeaegypti The double mutant kdr allele NaVR2 conferred moreresistance to deltamethrin than NaVR1 evidenced by theKdT50

of the homozygote R2R2 (978 min) higher than thehomozygote R1R1 (674 min) corroborating the hypothesisthat the 1016 Ilekdr mutation synergises with 1534 Cyskdrproviding higher levels of resistance

The heterozygote R1R2 was intermediate (790 min)suggesting a synergistic effect of these two alleles The het-erozygotes with the wild-typeNaVS or the ldquoduplicatedrdquoNaVDalleles were all knocked down before 60 minutes howeverwith higher KdT

50than the homozygotes SS and DD charac-

terizing an incomplete recessive inherence of the kdr allelesThe I1011M mutation is frequent in Ae aegypti Brazilian

natural populations resistant to pyrethroids especially onthose where the NaVR2 allele is absent [30] A selectionpressure with pyrethroid in the laboratory increased thefrequency of 1011M where 100 of the insects had themutation but only heterozygotes were found [24] likewise infield populations [21] This finding raised the hypothesis thatI1011M mutation was part of a duplication event which wasevidenced by DNA sequencing and copy number variationqPCR assays [21] It is of note that this Dup lineage hadbeen originally selected from a field population and was notbackcrossed with Rockefeller differently from the processthat originated R1R1 and R2R2 colonies Although we cannot

8 BioMed Research International

assume the inexistence of any other resistance mechanismin the Dup lineage its KdT

50to deltamethrin (30 min) was

only twice the Rockefellerrsquos (145 min) not representing anexpressive tolerance It is possible to conclude that the 1011IleMet mutation in this ldquoheterozygousrdquo conformation is notimportant for knockdown resistance compared to those in1016 and 1534 NaV sites In agreement the same aforemen-tioned electrophysiological assays demonstrated that I1011Mreduced the sensitivity of the channel to permethrin but notto deltamethrin [37]

We took advantage of a large sampling of Ae aegypti inneighbourhoods from Rio de Janeiro city and surroundings[33] for exploring the frequency of the kdr alleles of 27 local-ities The predominance of the ldquoresistant genotypesrdquo (R1R1R1R2 and R2R2) ranged from 65 to 100 among the sampledlocalities Additionally the high frequency of ldquoresistant geno-typesrdquo matches the higher levels of resistance to pyrethroidsobserved from South-eastern Brazilian localities [8 30 47]When pooling the samples in Rio Baixada and Niteroi wedo not find significant difference in the genotypic frequenciesbetween Rio and Baixada but between Niteroi and bothRio and Baixada sites The distinct intensity of insecticideapplication and an unlikely active migration of Ae aegyptiamong these localities may reflect the observed differencesin the kdr genetic background A population genetic analysisbased on nuclear single nucleotide polymorphisms (SNP)and microssatelites revealed low overall spatial structuringamong 15 out of the same 27Ae aegypti populations fromRioherein evaluated for kdr genotyping The exception was thepopulation from Paqueta island the only which significantlydiffered from the other localities [33] likely due to thelimited gene flow island-continent Accordingly samplesfrom Paqueta presented the most divergent kdr frequenciesand presented the highest level of the wild-type NaVS alleleIn this island there is a preoccupation about natural andcultural heritage conservation in a way that the employmentof insecticides is supposedly better planned and controlledthan in the continent Even though still very high the lowerfrequency of kdr alleles was therefore expected in Paquetaisland In a recent study with Ae aegypti from five neighbourtowns aroundMerida Yucatan andMexico kdr frequenciesin both 1016 and 1534 NaV sites significantly varied amongtowns These differences were also significant in a finer scaleat the block levels in two of the evaluated towns [48]

5 Conclusion

Several mutations in the NaV gene are likely to conferpyrethroid resistance however due to significant fitnesscost they remain at very low frequencies Other mutationsconferring similar or higher levels of resistance neverthelesswith lower fitness cost are expected to evolve and disseminatein the population under pyrethroid selection pressure [10]As the selection exerted by governmental campaigns and byhousehold insecticide applications has been continuous overAe aegypti new other mutations are likely to be emergingfrom the current known wild-type and kdr alleles and pos-sibly conferring even higher resistance levels as recently evi-denced in the house flyM domestica [49]This highlights the

importance of monitoring not only the currently known kdrsites by direct genotyping technics but also the strategies ofwhole NaV gene sequencing associated with bioassays Herewe evidenced that the NaVR2 kdr allele confers higher levelof resistance to pyrethroid than this counterpart NaVR1 inAe aegypti laboratory lines with similar genetic backgroundsalso corroborating with the hypotheses of recessive inherentpattern of these kdr mutations Therefore the homozygouskdr genotypes as well as the heterozygous R1R2 are likely tobe the ones selected for pyrethroid resistance Additionallythe mutation in the 1011 NaV site was not important forresistance in the lineage with a duplication in the NaV geneconferring a heterozygous-like aspect to this mutation A kdrgenotyping survey of Ae aegypti from 27 distinct localitiesfrom Rio de Janeiro city and surroundings detected highfrequencies of ldquoresistant genotypesrdquo probably reflecting thehigh selection pressure exerted principally by householdinsecticide applicationsThis kind ofmolecularmonitoring isof relevance yet studies for unrevealing new markers relatedto resistance to other classes of insecticides are necessary

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

The authors thank Heloisa Diniz (IOCFiocruz) for takingcare of the figures This study was supported by the NationalInstitute of Health (NIH Grant no UO1 AI115595) InstitutoNacional de Ciencia e Tencologia em Entomologia Molecular(INCT-EM Grant no 4656782014-9) and Fundacao deAmparo a Pesquisa do Estado do Rio de Janeiro (FAPERJGrants nos E-261101682014 E-262018362017)

Supplementary Materials

Supplementary Figure S1 Scheme of backcrosses towardselection of Aedes aegypti kdr lineages highlighting theprocess of background homogenization between R2R2 andR1R1 Please follow detailed explanation in the Material andMethods Table Spp S1 Time of knockdown profile underexposition to the pyrethroid deltamethrin (003) in Aedesaegypti laboratory lineages with distinct genotypes for kdralleles Table Spp S2 Kdr allelic frequencies considering 1016and 1534 NaV sites in Aedes aegypti populations from Riode Janeiro State Supp Material S2 Kdr allelic frequenciesin Aedes aegypti populations from Rio de Janeito StateEach dot represents the frequency of the respective allelewith the confidence interval 95 amplitude indicated by thevertical bars A = Cabucu B = Ceramica C = Moqueta D =Heliopolis E = Jurujuba F = Itacoatiara G = Sao Francisco H= Fonseca I = Ponta Drsquoareia J = Piratininga K = TubiacanhaL = Valqueire M = Urca N = Olaria O = Gamboa

BioMed Research International 9

P = Caju Q = Pavuna R = Meier S = Grajau T = PaquetaU = Vaz Lobo V = Jardim Guanabara W = Sao Cristovao X= Rio Comprido Y = Humaita Z = Rio das Pesdras and 2 =Taquara (Supplementary Materials)

References

[1] S Bhatt PWGethingO J Brady et al ldquoThe global distributionand burden of denguerdquo Nature vol 496 no 7446 pp 504ndash5072013

[2] WHOWHO statement on the firstmeeting of the InternationalHealth Regulations (2005) (IHR 2005) Emergency Committeeon Zika virus and observed increase in neurological disor-ders and neonatal malformations 2016 Available from httpwwwwhointmediacentrenewsstatements20161st-emergen-cy-committee-zikaen

[3] D Couto-Lima Y Madec M I Bersot et al ldquoPotential riskof re-emergence of urban transmission of Yellow Fever virusin Brazil facilitated by competent Aedes populationsrdquo ScientificReports vol 7 no 1 2017

[4] WHO Mosquito (vector) control emergency response andpreparedness for Zika virus 2016 Available from httpwwwwhointneglected diseasesnewsmosquito vector control re-sponseen

[5] A Malavasi ldquoProject Aedes transgenic population control inJuazeiro and Jacobina Bahia Brazilrdquo BMC Proceedings vol 8no Suppl 4 p O11 2014

[6] L B Carrington B C N Tran N T H Le et al ldquoField-and clinically derived estimates of Wolbachia-mediated block-ing of dengue virus transmission potential in Aedes aegyptimosquitoesrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 115 no 2 pp 361ndash366 2018

[7] H Ranson J Burhani N Lumjuan and I V W C BlackldquoInsecticide resistance in dengue vectorsrdquo TropIKAnet no 12010

[8] C L Moyes J Vontas A J Martins et al ldquoContemporarystatus of insecticide resistance in the major Aedes vectorsof arboviruses infecting humansrdquo PLOS Neglected TropicalDiseases vol 11 no 7 Article ID e0005625 2017

[9] S Kasai O Komagata K Itokawa et al ldquoMechanisms ofPyrethroid Resistance in the Dengue Mosquito Vector Aedesaegypti Target Site Insensitivity Penetration andMetabolismrdquoPLOSNeglected Tropical Diseases vol 8 no 6 Article ID e29482014

[10] R H Ffrench-Constant B Pittendrigh A Vaughan and NAnthony ldquoWhy are there so few resistance-associatedmutationsin insecticide target genesrdquo Philosophical Transactions of theRoyal Society B Biological Sciences vol 353 no 1376 pp 1685ndash1693 1998

[11] F D Rinkevich S M Hedtke C A Leichter et al ldquoMultipleOrigins of kdr-type Resistance in the House Fly Musca domes-ticardquo PLoS ONE vol 7 no 12 Article ID e52761 2012

[12] J Pinto A Lynd J L Vicente et al ldquoMultiple origins ofknockdown resistance mutations in the afrotropical mosquitovector Anopheles gambiaerdquo PLoS ONE vol 2 no 11 Article IDe1243 2007

[13] K Saavedra-Rodriguez L Urdaneta-Marquez S Rajatileka etal ldquoA mutation in the voltage-gated sodium channel geneassociated with pyrethroid resistance in Latin American Aedesaegyptirdquo Insect Molecular Biology vol 16 no 6 pp 785ndash7982007

[14] Y Du Y Nomura B S Zhorov and K Dong ldquoSodium channelmutations and pyrethroid resistance in Aedes aegyptirdquo Insectsvol 7 no 4 2016

[15] A M Al Nazawi J Aqili M Alzahrani P J McCall and DWeetman ldquoCombined target site (kdr)mutations play a primaryrole in highly pyrethroid resistant phenotypes of Aedes aegyptifrom Saudi Arabiardquo Parasites amp Vectors vol 10 no 1 pp 1ndash102017

[16] H Kawada Y Higa K Futami et al ldquoDiscovery of PointMutations in the Voltage-Gated Sodium Channel from AfricanAedes aegypti Populations Potential Phylogenetic Reasons forGene Introgressionrdquo PLOS Neglected Tropical Diseases vol 10no 6 Article ID e0004780 2016

[17] F Z Vera-Maloof K Saavedra-Rodriguez A E Elizondo-Quiroga S Lozano-Fuentes and W C Black IV ldquoCoevolutionof the Ile1016 and Cys1534 Mutations in the Voltage GatedSodium Channel Gene of Aedes aegypti in Mexicordquo PLOSNeglected Tropical Diseases vol 9 no 12 Article ID e00042632015

[18] G Seixas L Grigoraki D Weetman et al ldquoInsecticide resis-tance is mediated by multiple mechanisms in recently intro-duced Aedes aegypti from Madeira Island (Portugal)rdquo PLOSNeglected Tropical Diseases vol 11 no 7 Article ID e00057992017

[19] J G B Linss L P Brito G A Garcia et al ldquoDistribution anddissemination of the Val1016Ile and Phe1534Cys Kdr mutationsin Aedes aegypti Brazilian natural populationsrdquo Parasites ampVectors vol 7 no 1 article 25 2014

[20] D F Bellinato P F Viana-Medeiros S C Araujo et alldquoResistance Status to the Insecticides Temephos Deltamethrinand Diflubenzuron in Brazilian Aedes aegypti PopulationsrdquoBioMed Research International vol 2016 Article ID 8603263pp 1ndash12 2016

[21] A J Martins L P Brito J G Linss et al ldquoEvidence for geneduplication in the voltage-gated sodium channel gene of Aedesaegyptirdquo Evolution Medicine and Public Health vol 2013 no 1pp 148ndash160 2013

[22] K Dong Y Du F Rinkevich et al ldquoMolecular biology of insectsodium channels and pyrethroid resistancerdquo Insect Biochemistryand Molecular Biology vol 50 no 1 pp 1ndash17 2014

[23] A JMartins RM Lins J G Linss et al ldquoVoltage-gated sodiumchannel polymorphism andmetabolic resistance in pyrethroid-resistant Aedes aegypti from Brazilrdquo The American Journal ofTropical Medicine and Hygiene vol 81 no 1 pp 108ndash115 2009

[24] A J Martins C D M Ribeiro D F Bellinato A A PeixotoD Valle and J B P Lima ldquoEffect of insecticide resistance ondevelopment longevity and reproduction of field or laboratoryselected Aedes aegypti populationsrdquo PLoS ONE vol 7 no 3Article ID e31889 2012

[25] R M R Nogueira M P Miagostovich A M B De FilippisM A S Pereira and H G Schatzmayr ldquoDengue Virus Type 3in Rio de Janeiro BrazilrdquoMemorias do Instituto Oswaldo Cruzvol 96 no 7 pp 925-926 2001

[26] R M R Nogueira M P Miagostovich E Lampe R W SouzaS M O Zagne andH G Schatzmayr ldquoDengue epidemic in thestate of Rio de Janeiro Brazil 1990ndash1 Co-circulation of dengue1 and dengue 2 serotypesrdquo Epidemiology and Infection vol 111no 1 pp 163ndash170 1993

[27] G Kuno ldquoEarly history of laboratory breeding of Aedes aegypti(Diptera Culicidae) focusing on the origins and use of selectedstrainsrdquo Journal of Medical Entomology vol 47 no 6 pp 957ndash971 2010

BioMed Research International 7

impregnated paper tests [38] The most recent WHOPESplan for detecting and monitoring IR in Aedes aegyptirecommended 003 as a discriminating concentration ofdeltamethrin [39] to where mortality is recorded 24 h after1 h of exposition to the insecticide Mortality under 90indicates resistance of the evaluated population as long as atleast 100 mosquitoes were tested This is a qualitative dose-diagnostic test good for determining the susceptibility statusof a given population although not suitable for comparinglevels of resistance among populations For this matter aquantitative dose-response test is indicated in which themortality rates over a range of insecticide concentrationsgenerate the lethal concentrations (LC) of each populationIn turn LC produces the resistance rations (RR) based ona reference lineage [40] However this sort of test requiresa large number of insects compared to the dose-responseapproach Here we applied a WHO-like dose-diagnostic testwith papers impregnated on our ownNevertheless instead ofevaluating mortality 24 h after 1h of exposition we followedthe knockdown rate for up to 2 h With this record over thetime a semiquantitative analysis was performed by extractingthe RR of the populations in this case based on their timeof knockdown It is worth noting that this knockdown timeRR displays a different scale generally shorter than thoseproduced by truly qualitative dose-response tests

We employed a very efficient TaqMan assay for the rapidgenotyping of the kdr alleles present in Ae aegypti LatinAmerican populations Our previous allele specific PCRalthough useful generally needed constant adjustments inthe number of cycles andor the concentration of specificprimers according to different thermal-cycle machinery orPCR kit employed [19 21] Sometimes unspecific shallowamplification also occurred pointing to the need of addi-tional confirmatory reactions The present TaqMan assayrenders the genotyping process more clear and straightfor-ward However one must be aware that any genotypingassay will only reveal the specific alleles available in thatassay For instance instead of a ValIle mutation found inAe aegypti from Latin American the kdr mutation in the1016 NaV site of Asian populations is a ValGly [14] towhich the assay employed here is unable to detect This isalso true for other potential mutations initially under lowfrequencies Therefore these allele specific methods shouldonly be used to evaluate genotypic frequencies of populationswith a previously well-explored genetic background of thetarget genes

The physiological importance of the kdr mutations andaltered sensitivity to pyrethroids have been evaluated withmutant insect NaV gene presenting punctual or combinedmutations in Xenopus oocites heterologous expression sys-tem followed by electrophysiological assays [22] Such assaysemploying direct mutagenesis of AaNaV cDNA demon-strated that the V1016I mutation alone (which we wouldcall NaVR3 allele) did not alter the channel sensitivityto pyrethroids while F1534C kdr mutation (NaVR1 allele)reduced the channel affinity of pyrethroid type I (perme-thrin) but not type II (deltamethrin) [37] In agreementthis same mutation reduced the affinity to type I but notto type II pyrethroids in the cockroach Blatella germanica

channel [41] However herein we found that although NaVR1conferred lower level of resistance than NaVR2 R1R1 insectswere resistant to deltamethrin a type II pyrethroid TheNaVR2 allele used to be absent in some deltamethrin resistantpopulations from the Northeast of Brazil where the NaVR1was found in high frequencies [19] More recent samplingsevidenced that the NaVR2 is disseminating and increasingin frequency also through those areas [20 30 42] Vera-Malof et al [17] proposed that the NaVR1 had emerged firstconferring low levels of resistance to pyrethroids and thenthe V1016I arose from that allele originating the NaVR2NaVR2 would have been rapidly selected and dispersed byconferring higher levels of resistance to pyrethroids Thepossible NaVR3 (1016Ilekdr + 1534Phe+) has not ever beenevidenced in Brazilian Ae aegypti populations and thereforewas not considered in our analysis Indeed we did not findany SR3 R2R3 and R3R3 individual

In the house flyMusca domestica the relationship of threeNaV alleles with resistance to pyrethroid was investigatedby evaluating the susceptibility of congenic strains and theirhybrids to a range of several pyrethroid compounds Thedouble mutant super-kdr allele (M918T + L1014F) conferredmore resistance than the classical kdr (L1014F) which in itsturn conferred more resistance than the kdr-his (L1014H)The heterozygotes kdrsuper-kdr and super-kdrkdr-his pre-sented intermediate resistance between the homozygouscharacterizing an incomplete recessive inherence partnerfor these M domestica NaV alleles [43] Ae aegypti fieldpopulations fromMalaysia showed increased resistancewhenpresenting kdrmutation in both 1016 and 1534 NaV sites [44]In that case however substitution in the 1016 site was Val toGly as common in Middle Eastern and Asian populations[15 45 46] Here a similar partner was observed in Aeaegypti The double mutant kdr allele NaVR2 conferred moreresistance to deltamethrin than NaVR1 evidenced by theKdT50

of the homozygote R2R2 (978 min) higher than thehomozygote R1R1 (674 min) corroborating the hypothesisthat the 1016 Ilekdr mutation synergises with 1534 Cyskdrproviding higher levels of resistance

The heterozygote R1R2 was intermediate (790 min)suggesting a synergistic effect of these two alleles The het-erozygotes with the wild-typeNaVS or the ldquoduplicatedrdquoNaVDalleles were all knocked down before 60 minutes howeverwith higher KdT

50than the homozygotes SS and DD charac-

terizing an incomplete recessive inherence of the kdr allelesThe I1011M mutation is frequent in Ae aegypti Brazilian

natural populations resistant to pyrethroids especially onthose where the NaVR2 allele is absent [30] A selectionpressure with pyrethroid in the laboratory increased thefrequency of 1011M where 100 of the insects had themutation but only heterozygotes were found [24] likewise infield populations [21] This finding raised the hypothesis thatI1011M mutation was part of a duplication event which wasevidenced by DNA sequencing and copy number variationqPCR assays [21] It is of note that this Dup lineage hadbeen originally selected from a field population and was notbackcrossed with Rockefeller differently from the processthat originated R1R1 and R2R2 colonies Although we cannot

8 BioMed Research International

assume the inexistence of any other resistance mechanismin the Dup lineage its KdT

50to deltamethrin (30 min) was

only twice the Rockefellerrsquos (145 min) not representing anexpressive tolerance It is possible to conclude that the 1011IleMet mutation in this ldquoheterozygousrdquo conformation is notimportant for knockdown resistance compared to those in1016 and 1534 NaV sites In agreement the same aforemen-tioned electrophysiological assays demonstrated that I1011Mreduced the sensitivity of the channel to permethrin but notto deltamethrin [37]

We took advantage of a large sampling of Ae aegypti inneighbourhoods from Rio de Janeiro city and surroundings[33] for exploring the frequency of the kdr alleles of 27 local-ities The predominance of the ldquoresistant genotypesrdquo (R1R1R1R2 and R2R2) ranged from 65 to 100 among the sampledlocalities Additionally the high frequency of ldquoresistant geno-typesrdquo matches the higher levels of resistance to pyrethroidsobserved from South-eastern Brazilian localities [8 30 47]When pooling the samples in Rio Baixada and Niteroi wedo not find significant difference in the genotypic frequenciesbetween Rio and Baixada but between Niteroi and bothRio and Baixada sites The distinct intensity of insecticideapplication and an unlikely active migration of Ae aegyptiamong these localities may reflect the observed differencesin the kdr genetic background A population genetic analysisbased on nuclear single nucleotide polymorphisms (SNP)and microssatelites revealed low overall spatial structuringamong 15 out of the same 27Ae aegypti populations fromRioherein evaluated for kdr genotyping The exception was thepopulation from Paqueta island the only which significantlydiffered from the other localities [33] likely due to thelimited gene flow island-continent Accordingly samplesfrom Paqueta presented the most divergent kdr frequenciesand presented the highest level of the wild-type NaVS alleleIn this island there is a preoccupation about natural andcultural heritage conservation in a way that the employmentof insecticides is supposedly better planned and controlledthan in the continent Even though still very high the lowerfrequency of kdr alleles was therefore expected in Paquetaisland In a recent study with Ae aegypti from five neighbourtowns aroundMerida Yucatan andMexico kdr frequenciesin both 1016 and 1534 NaV sites significantly varied amongtowns These differences were also significant in a finer scaleat the block levels in two of the evaluated towns [48]

5 Conclusion

Several mutations in the NaV gene are likely to conferpyrethroid resistance however due to significant fitnesscost they remain at very low frequencies Other mutationsconferring similar or higher levels of resistance neverthelesswith lower fitness cost are expected to evolve and disseminatein the population under pyrethroid selection pressure [10]As the selection exerted by governmental campaigns and byhousehold insecticide applications has been continuous overAe aegypti new other mutations are likely to be emergingfrom the current known wild-type and kdr alleles and pos-sibly conferring even higher resistance levels as recently evi-denced in the house flyM domestica [49]This highlights the

importance of monitoring not only the currently known kdrsites by direct genotyping technics but also the strategies ofwhole NaV gene sequencing associated with bioassays Herewe evidenced that the NaVR2 kdr allele confers higher levelof resistance to pyrethroid than this counterpart NaVR1 inAe aegypti laboratory lines with similar genetic backgroundsalso corroborating with the hypotheses of recessive inherentpattern of these kdr mutations Therefore the homozygouskdr genotypes as well as the heterozygous R1R2 are likely tobe the ones selected for pyrethroid resistance Additionallythe mutation in the 1011 NaV site was not important forresistance in the lineage with a duplication in the NaV geneconferring a heterozygous-like aspect to this mutation A kdrgenotyping survey of Ae aegypti from 27 distinct localitiesfrom Rio de Janeiro city and surroundings detected highfrequencies of ldquoresistant genotypesrdquo probably reflecting thehigh selection pressure exerted principally by householdinsecticide applicationsThis kind ofmolecularmonitoring isof relevance yet studies for unrevealing new markers relatedto resistance to other classes of insecticides are necessary

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

The authors thank Heloisa Diniz (IOCFiocruz) for takingcare of the figures This study was supported by the NationalInstitute of Health (NIH Grant no UO1 AI115595) InstitutoNacional de Ciencia e Tencologia em Entomologia Molecular(INCT-EM Grant no 4656782014-9) and Fundacao deAmparo a Pesquisa do Estado do Rio de Janeiro (FAPERJGrants nos E-261101682014 E-262018362017)

Supplementary Materials

Supplementary Figure S1 Scheme of backcrosses towardselection of Aedes aegypti kdr lineages highlighting theprocess of background homogenization between R2R2 andR1R1 Please follow detailed explanation in the Material andMethods Table Spp S1 Time of knockdown profile underexposition to the pyrethroid deltamethrin (003) in Aedesaegypti laboratory lineages with distinct genotypes for kdralleles Table Spp S2 Kdr allelic frequencies considering 1016and 1534 NaV sites in Aedes aegypti populations from Riode Janeiro State Supp Material S2 Kdr allelic frequenciesin Aedes aegypti populations from Rio de Janeito StateEach dot represents the frequency of the respective allelewith the confidence interval 95 amplitude indicated by thevertical bars A = Cabucu B = Ceramica C = Moqueta D =Heliopolis E = Jurujuba F = Itacoatiara G = Sao Francisco H= Fonseca I = Ponta Drsquoareia J = Piratininga K = TubiacanhaL = Valqueire M = Urca N = Olaria O = Gamboa

BioMed Research International 9

P = Caju Q = Pavuna R = Meier S = Grajau T = PaquetaU = Vaz Lobo V = Jardim Guanabara W = Sao Cristovao X= Rio Comprido Y = Humaita Z = Rio das Pesdras and 2 =Taquara (Supplementary Materials)

References

[1] S Bhatt PWGethingO J Brady et al ldquoThe global distributionand burden of denguerdquo Nature vol 496 no 7446 pp 504ndash5072013

[2] WHOWHO statement on the firstmeeting of the InternationalHealth Regulations (2005) (IHR 2005) Emergency Committeeon Zika virus and observed increase in neurological disor-ders and neonatal malformations 2016 Available from httpwwwwhointmediacentrenewsstatements20161st-emergen-cy-committee-zikaen

[3] D Couto-Lima Y Madec M I Bersot et al ldquoPotential riskof re-emergence of urban transmission of Yellow Fever virusin Brazil facilitated by competent Aedes populationsrdquo ScientificReports vol 7 no 1 2017

[4] WHO Mosquito (vector) control emergency response andpreparedness for Zika virus 2016 Available from httpwwwwhointneglected diseasesnewsmosquito vector control re-sponseen

[5] A Malavasi ldquoProject Aedes transgenic population control inJuazeiro and Jacobina Bahia Brazilrdquo BMC Proceedings vol 8no Suppl 4 p O11 2014

[6] L B Carrington B C N Tran N T H Le et al ldquoField-and clinically derived estimates of Wolbachia-mediated block-ing of dengue virus transmission potential in Aedes aegyptimosquitoesrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 115 no 2 pp 361ndash366 2018

[7] H Ranson J Burhani N Lumjuan and I V W C BlackldquoInsecticide resistance in dengue vectorsrdquo TropIKAnet no 12010

[8] C L Moyes J Vontas A J Martins et al ldquoContemporarystatus of insecticide resistance in the major Aedes vectorsof arboviruses infecting humansrdquo PLOS Neglected TropicalDiseases vol 11 no 7 Article ID e0005625 2017

[9] S Kasai O Komagata K Itokawa et al ldquoMechanisms ofPyrethroid Resistance in the Dengue Mosquito Vector Aedesaegypti Target Site Insensitivity Penetration andMetabolismrdquoPLOSNeglected Tropical Diseases vol 8 no 6 Article ID e29482014

[10] R H Ffrench-Constant B Pittendrigh A Vaughan and NAnthony ldquoWhy are there so few resistance-associatedmutationsin insecticide target genesrdquo Philosophical Transactions of theRoyal Society B Biological Sciences vol 353 no 1376 pp 1685ndash1693 1998

[11] F D Rinkevich S M Hedtke C A Leichter et al ldquoMultipleOrigins of kdr-type Resistance in the House Fly Musca domes-ticardquo PLoS ONE vol 7 no 12 Article ID e52761 2012

[12] J Pinto A Lynd J L Vicente et al ldquoMultiple origins ofknockdown resistance mutations in the afrotropical mosquitovector Anopheles gambiaerdquo PLoS ONE vol 2 no 11 Article IDe1243 2007

[13] K Saavedra-Rodriguez L Urdaneta-Marquez S Rajatileka etal ldquoA mutation in the voltage-gated sodium channel geneassociated with pyrethroid resistance in Latin American Aedesaegyptirdquo Insect Molecular Biology vol 16 no 6 pp 785ndash7982007

[14] Y Du Y Nomura B S Zhorov and K Dong ldquoSodium channelmutations and pyrethroid resistance in Aedes aegyptirdquo Insectsvol 7 no 4 2016

[15] A M Al Nazawi J Aqili M Alzahrani P J McCall and DWeetman ldquoCombined target site (kdr)mutations play a primaryrole in highly pyrethroid resistant phenotypes of Aedes aegyptifrom Saudi Arabiardquo Parasites amp Vectors vol 10 no 1 pp 1ndash102017

[16] H Kawada Y Higa K Futami et al ldquoDiscovery of PointMutations in the Voltage-Gated Sodium Channel from AfricanAedes aegypti Populations Potential Phylogenetic Reasons forGene Introgressionrdquo PLOS Neglected Tropical Diseases vol 10no 6 Article ID e0004780 2016

[17] F Z Vera-Maloof K Saavedra-Rodriguez A E Elizondo-Quiroga S Lozano-Fuentes and W C Black IV ldquoCoevolutionof the Ile1016 and Cys1534 Mutations in the Voltage GatedSodium Channel Gene of Aedes aegypti in Mexicordquo PLOSNeglected Tropical Diseases vol 9 no 12 Article ID e00042632015

[18] G Seixas L Grigoraki D Weetman et al ldquoInsecticide resis-tance is mediated by multiple mechanisms in recently intro-duced Aedes aegypti from Madeira Island (Portugal)rdquo PLOSNeglected Tropical Diseases vol 11 no 7 Article ID e00057992017

[19] J G B Linss L P Brito G A Garcia et al ldquoDistribution anddissemination of the Val1016Ile and Phe1534Cys Kdr mutationsin Aedes aegypti Brazilian natural populationsrdquo Parasites ampVectors vol 7 no 1 article 25 2014

[20] D F Bellinato P F Viana-Medeiros S C Araujo et alldquoResistance Status to the Insecticides Temephos Deltamethrinand Diflubenzuron in Brazilian Aedes aegypti PopulationsrdquoBioMed Research International vol 2016 Article ID 8603263pp 1ndash12 2016

[21] A J Martins L P Brito J G Linss et al ldquoEvidence for geneduplication in the voltage-gated sodium channel gene of Aedesaegyptirdquo Evolution Medicine and Public Health vol 2013 no 1pp 148ndash160 2013

[22] K Dong Y Du F Rinkevich et al ldquoMolecular biology of insectsodium channels and pyrethroid resistancerdquo Insect Biochemistryand Molecular Biology vol 50 no 1 pp 1ndash17 2014

[23] A JMartins RM Lins J G Linss et al ldquoVoltage-gated sodiumchannel polymorphism andmetabolic resistance in pyrethroid-resistant Aedes aegypti from Brazilrdquo The American Journal ofTropical Medicine and Hygiene vol 81 no 1 pp 108ndash115 2009

[24] A J Martins C D M Ribeiro D F Bellinato A A PeixotoD Valle and J B P Lima ldquoEffect of insecticide resistance ondevelopment longevity and reproduction of field or laboratoryselected Aedes aegypti populationsrdquo PLoS ONE vol 7 no 3Article ID e31889 2012

[25] R M R Nogueira M P Miagostovich A M B De FilippisM A S Pereira and H G Schatzmayr ldquoDengue Virus Type 3in Rio de Janeiro BrazilrdquoMemorias do Instituto Oswaldo Cruzvol 96 no 7 pp 925-926 2001

[26] R M R Nogueira M P Miagostovich E Lampe R W SouzaS M O Zagne andH G Schatzmayr ldquoDengue epidemic in thestate of Rio de Janeiro Brazil 1990ndash1 Co-circulation of dengue1 and dengue 2 serotypesrdquo Epidemiology and Infection vol 111no 1 pp 163ndash170 1993

[27] G Kuno ldquoEarly history of laboratory breeding of Aedes aegypti(Diptera Culicidae) focusing on the origins and use of selectedstrainsrdquo Journal of Medical Entomology vol 47 no 6 pp 957ndash971 2010

8 BioMed Research International

assume the inexistence of any other resistance mechanismin the Dup lineage its KdT

50to deltamethrin (30 min) was

only twice the Rockefellerrsquos (145 min) not representing anexpressive tolerance It is possible to conclude that the 1011IleMet mutation in this ldquoheterozygousrdquo conformation is notimportant for knockdown resistance compared to those in1016 and 1534 NaV sites In agreement the same aforemen-tioned electrophysiological assays demonstrated that I1011Mreduced the sensitivity of the channel to permethrin but notto deltamethrin [37]

We took advantage of a large sampling of Ae aegypti inneighbourhoods from Rio de Janeiro city and surroundings[33] for exploring the frequency of the kdr alleles of 27 local-ities The predominance of the ldquoresistant genotypesrdquo (R1R1R1R2 and R2R2) ranged from 65 to 100 among the sampledlocalities Additionally the high frequency of ldquoresistant geno-typesrdquo matches the higher levels of resistance to pyrethroidsobserved from South-eastern Brazilian localities [8 30 47]When pooling the samples in Rio Baixada and Niteroi wedo not find significant difference in the genotypic frequenciesbetween Rio and Baixada but between Niteroi and bothRio and Baixada sites The distinct intensity of insecticideapplication and an unlikely active migration of Ae aegyptiamong these localities may reflect the observed differencesin the kdr genetic background A population genetic analysisbased on nuclear single nucleotide polymorphisms (SNP)and microssatelites revealed low overall spatial structuringamong 15 out of the same 27Ae aegypti populations fromRioherein evaluated for kdr genotyping The exception was thepopulation from Paqueta island the only which significantlydiffered from the other localities [33] likely due to thelimited gene flow island-continent Accordingly samplesfrom Paqueta presented the most divergent kdr frequenciesand presented the highest level of the wild-type NaVS alleleIn this island there is a preoccupation about natural andcultural heritage conservation in a way that the employmentof insecticides is supposedly better planned and controlledthan in the continent Even though still very high the lowerfrequency of kdr alleles was therefore expected in Paquetaisland In a recent study with Ae aegypti from five neighbourtowns aroundMerida Yucatan andMexico kdr frequenciesin both 1016 and 1534 NaV sites significantly varied amongtowns These differences were also significant in a finer scaleat the block levels in two of the evaluated towns [48]

5 Conclusion

Several mutations in the NaV gene are likely to conferpyrethroid resistance however due to significant fitnesscost they remain at very low frequencies Other mutationsconferring similar or higher levels of resistance neverthelesswith lower fitness cost are expected to evolve and disseminatein the population under pyrethroid selection pressure [10]As the selection exerted by governmental campaigns and byhousehold insecticide applications has been continuous overAe aegypti new other mutations are likely to be emergingfrom the current known wild-type and kdr alleles and pos-sibly conferring even higher resistance levels as recently evi-denced in the house flyM domestica [49]This highlights the

importance of monitoring not only the currently known kdrsites by direct genotyping technics but also the strategies ofwhole NaV gene sequencing associated with bioassays Herewe evidenced that the NaVR2 kdr allele confers higher levelof resistance to pyrethroid than this counterpart NaVR1 inAe aegypti laboratory lines with similar genetic backgroundsalso corroborating with the hypotheses of recessive inherentpattern of these kdr mutations Therefore the homozygouskdr genotypes as well as the heterozygous R1R2 are likely tobe the ones selected for pyrethroid resistance Additionallythe mutation in the 1011 NaV site was not important forresistance in the lineage with a duplication in the NaV geneconferring a heterozygous-like aspect to this mutation A kdrgenotyping survey of Ae aegypti from 27 distinct localitiesfrom Rio de Janeiro city and surroundings detected highfrequencies of ldquoresistant genotypesrdquo probably reflecting thehigh selection pressure exerted principally by householdinsecticide applicationsThis kind ofmolecularmonitoring isof relevance yet studies for unrevealing new markers relatedto resistance to other classes of insecticides are necessary

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

The authors thank Heloisa Diniz (IOCFiocruz) for takingcare of the figures This study was supported by the NationalInstitute of Health (NIH Grant no UO1 AI115595) InstitutoNacional de Ciencia e Tencologia em Entomologia Molecular(INCT-EM Grant no 4656782014-9) and Fundacao deAmparo a Pesquisa do Estado do Rio de Janeiro (FAPERJGrants nos E-261101682014 E-262018362017)

Supplementary Materials

Supplementary Figure S1 Scheme of backcrosses towardselection of Aedes aegypti kdr lineages highlighting theprocess of background homogenization between R2R2 andR1R1 Please follow detailed explanation in the Material andMethods Table Spp S1 Time of knockdown profile underexposition to the pyrethroid deltamethrin (003) in Aedesaegypti laboratory lineages with distinct genotypes for kdralleles Table Spp S2 Kdr allelic frequencies considering 1016and 1534 NaV sites in Aedes aegypti populations from Riode Janeiro State Supp Material S2 Kdr allelic frequenciesin Aedes aegypti populations from Rio de Janeito StateEach dot represents the frequency of the respective allelewith the confidence interval 95 amplitude indicated by thevertical bars A = Cabucu B = Ceramica C = Moqueta D =Heliopolis E = Jurujuba F = Itacoatiara G = Sao Francisco H= Fonseca I = Ponta Drsquoareia J = Piratininga K = TubiacanhaL = Valqueire M = Urca N = Olaria O = Gamboa

BioMed Research International 9

P = Caju Q = Pavuna R = Meier S = Grajau T = PaquetaU = Vaz Lobo V = Jardim Guanabara W = Sao Cristovao X= Rio Comprido Y = Humaita Z = Rio das Pesdras and 2 =Taquara (Supplementary Materials)

References

[1] S Bhatt PWGethingO J Brady et al ldquoThe global distributionand burden of denguerdquo Nature vol 496 no 7446 pp 504ndash5072013

[2] WHOWHO statement on the firstmeeting of the InternationalHealth Regulations (2005) (IHR 2005) Emergency Committeeon Zika virus and observed increase in neurological disor-ders and neonatal malformations 2016 Available from httpwwwwhointmediacentrenewsstatements20161st-emergen-cy-committee-zikaen

[3] D Couto-Lima Y Madec M I Bersot et al ldquoPotential riskof re-emergence of urban transmission of Yellow Fever virusin Brazil facilitated by competent Aedes populationsrdquo ScientificReports vol 7 no 1 2017

[4] WHO Mosquito (vector) control emergency response andpreparedness for Zika virus 2016 Available from httpwwwwhointneglected diseasesnewsmosquito vector control re-sponseen

[5] A Malavasi ldquoProject Aedes transgenic population control inJuazeiro and Jacobina Bahia Brazilrdquo BMC Proceedings vol 8no Suppl 4 p O11 2014

[6] L B Carrington B C N Tran N T H Le et al ldquoField-and clinically derived estimates of Wolbachia-mediated block-ing of dengue virus transmission potential in Aedes aegyptimosquitoesrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 115 no 2 pp 361ndash366 2018

[7] H Ranson J Burhani N Lumjuan and I V W C BlackldquoInsecticide resistance in dengue vectorsrdquo TropIKAnet no 12010

[8] C L Moyes J Vontas A J Martins et al ldquoContemporarystatus of insecticide resistance in the major Aedes vectorsof arboviruses infecting humansrdquo PLOS Neglected TropicalDiseases vol 11 no 7 Article ID e0005625 2017

[9] S Kasai O Komagata K Itokawa et al ldquoMechanisms ofPyrethroid Resistance in the Dengue Mosquito Vector Aedesaegypti Target Site Insensitivity Penetration andMetabolismrdquoPLOSNeglected Tropical Diseases vol 8 no 6 Article ID e29482014

[10] R H Ffrench-Constant B Pittendrigh A Vaughan and NAnthony ldquoWhy are there so few resistance-associatedmutationsin insecticide target genesrdquo Philosophical Transactions of theRoyal Society B Biological Sciences vol 353 no 1376 pp 1685ndash1693 1998

[11] F D Rinkevich S M Hedtke C A Leichter et al ldquoMultipleOrigins of kdr-type Resistance in the House Fly Musca domes-ticardquo PLoS ONE vol 7 no 12 Article ID e52761 2012

[12] J Pinto A Lynd J L Vicente et al ldquoMultiple origins ofknockdown resistance mutations in the afrotropical mosquitovector Anopheles gambiaerdquo PLoS ONE vol 2 no 11 Article IDe1243 2007

[13] K Saavedra-Rodriguez L Urdaneta-Marquez S Rajatileka etal ldquoA mutation in the voltage-gated sodium channel geneassociated with pyrethroid resistance in Latin American Aedesaegyptirdquo Insect Molecular Biology vol 16 no 6 pp 785ndash7982007

[14] Y Du Y Nomura B S Zhorov and K Dong ldquoSodium channelmutations and pyrethroid resistance in Aedes aegyptirdquo Insectsvol 7 no 4 2016

[15] A M Al Nazawi J Aqili M Alzahrani P J McCall and DWeetman ldquoCombined target site (kdr)mutations play a primaryrole in highly pyrethroid resistant phenotypes of Aedes aegyptifrom Saudi Arabiardquo Parasites amp Vectors vol 10 no 1 pp 1ndash102017

[16] H Kawada Y Higa K Futami et al ldquoDiscovery of PointMutations in the Voltage-Gated Sodium Channel from AfricanAedes aegypti Populations Potential Phylogenetic Reasons forGene Introgressionrdquo PLOS Neglected Tropical Diseases vol 10no 6 Article ID e0004780 2016

[17] F Z Vera-Maloof K Saavedra-Rodriguez A E Elizondo-Quiroga S Lozano-Fuentes and W C Black IV ldquoCoevolutionof the Ile1016 and Cys1534 Mutations in the Voltage GatedSodium Channel Gene of Aedes aegypti in Mexicordquo PLOSNeglected Tropical Diseases vol 9 no 12 Article ID e00042632015

[18] G Seixas L Grigoraki D Weetman et al ldquoInsecticide resis-tance is mediated by multiple mechanisms in recently intro-duced Aedes aegypti from Madeira Island (Portugal)rdquo PLOSNeglected Tropical Diseases vol 11 no 7 Article ID e00057992017

[19] J G B Linss L P Brito G A Garcia et al ldquoDistribution anddissemination of the Val1016Ile and Phe1534Cys Kdr mutationsin Aedes aegypti Brazilian natural populationsrdquo Parasites ampVectors vol 7 no 1 article 25 2014

[20] D F Bellinato P F Viana-Medeiros S C Araujo et alldquoResistance Status to the Insecticides Temephos Deltamethrinand Diflubenzuron in Brazilian Aedes aegypti PopulationsrdquoBioMed Research International vol 2016 Article ID 8603263pp 1ndash12 2016

[21] A J Martins L P Brito J G Linss et al ldquoEvidence for geneduplication in the voltage-gated sodium channel gene of Aedesaegyptirdquo Evolution Medicine and Public Health vol 2013 no 1pp 148ndash160 2013

[22] K Dong Y Du F Rinkevich et al ldquoMolecular biology of insectsodium channels and pyrethroid resistancerdquo Insect Biochemistryand Molecular Biology vol 50 no 1 pp 1ndash17 2014

[23] A JMartins RM Lins J G Linss et al ldquoVoltage-gated sodiumchannel polymorphism andmetabolic resistance in pyrethroid-resistant Aedes aegypti from Brazilrdquo The American Journal ofTropical Medicine and Hygiene vol 81 no 1 pp 108ndash115 2009

[24] A J Martins C D M Ribeiro D F Bellinato A A PeixotoD Valle and J B P Lima ldquoEffect of insecticide resistance ondevelopment longevity and reproduction of field or laboratoryselected Aedes aegypti populationsrdquo PLoS ONE vol 7 no 3Article ID e31889 2012

[25] R M R Nogueira M P Miagostovich A M B De FilippisM A S Pereira and H G Schatzmayr ldquoDengue Virus Type 3in Rio de Janeiro BrazilrdquoMemorias do Instituto Oswaldo Cruzvol 96 no 7 pp 925-926 2001

[26] R M R Nogueira M P Miagostovich E Lampe R W SouzaS M O Zagne andH G Schatzmayr ldquoDengue epidemic in thestate of Rio de Janeiro Brazil 1990ndash1 Co-circulation of dengue1 and dengue 2 serotypesrdquo Epidemiology and Infection vol 111no 1 pp 163ndash170 1993

[27] G Kuno ldquoEarly history of laboratory breeding of Aedes aegypti(Diptera Culicidae) focusing on the origins and use of selectedstrainsrdquo Journal of Medical Entomology vol 47 no 6 pp 957ndash971 2010

BioMed Research International 9

P = Caju Q = Pavuna R = Meier S = Grajau T = PaquetaU = Vaz Lobo V = Jardim Guanabara W = Sao Cristovao X= Rio Comprido Y = Humaita Z = Rio das Pesdras and 2 =Taquara (Supplementary Materials)

References

[1] S Bhatt PWGethingO J Brady et al ldquoThe global distributionand burden of denguerdquo Nature vol 496 no 7446 pp 504ndash5072013

[2] WHOWHO statement on the firstmeeting of the InternationalHealth Regulations (2005) (IHR 2005) Emergency Committeeon Zika virus and observed increase in neurological disor-ders and neonatal malformations 2016 Available from httpwwwwhointmediacentrenewsstatements20161st-emergen-cy-committee-zikaen

[3] D Couto-Lima Y Madec M I Bersot et al ldquoPotential riskof re-emergence of urban transmission of Yellow Fever virusin Brazil facilitated by competent Aedes populationsrdquo ScientificReports vol 7 no 1 2017

[4] WHO Mosquito (vector) control emergency response andpreparedness for Zika virus 2016 Available from httpwwwwhointneglected diseasesnewsmosquito vector control re-sponseen

[5] A Malavasi ldquoProject Aedes transgenic population control inJuazeiro and Jacobina Bahia Brazilrdquo BMC Proceedings vol 8no Suppl 4 p O11 2014

[6] L B Carrington B C N Tran N T H Le et al ldquoField-and clinically derived estimates of Wolbachia-mediated block-ing of dengue virus transmission potential in Aedes aegyptimosquitoesrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 115 no 2 pp 361ndash366 2018

[7] H Ranson J Burhani N Lumjuan and I V W C BlackldquoInsecticide resistance in dengue vectorsrdquo TropIKAnet no 12010

[8] C L Moyes J Vontas A J Martins et al ldquoContemporarystatus of insecticide resistance in the major Aedes vectorsof arboviruses infecting humansrdquo PLOS Neglected TropicalDiseases vol 11 no 7 Article ID e0005625 2017

[9] S Kasai O Komagata K Itokawa et al ldquoMechanisms ofPyrethroid Resistance in the Dengue Mosquito Vector Aedesaegypti Target Site Insensitivity Penetration andMetabolismrdquoPLOSNeglected Tropical Diseases vol 8 no 6 Article ID e29482014

[10] R H Ffrench-Constant B Pittendrigh A Vaughan and NAnthony ldquoWhy are there so few resistance-associatedmutationsin insecticide target genesrdquo Philosophical Transactions of theRoyal Society B Biological Sciences vol 353 no 1376 pp 1685ndash1693 1998

[11] F D Rinkevich S M Hedtke C A Leichter et al ldquoMultipleOrigins of kdr-type Resistance in the House Fly Musca domes-ticardquo PLoS ONE vol 7 no 12 Article ID e52761 2012

[12] J Pinto A Lynd J L Vicente et al ldquoMultiple origins ofknockdown resistance mutations in the afrotropical mosquitovector Anopheles gambiaerdquo PLoS ONE vol 2 no 11 Article IDe1243 2007

[13] K Saavedra-Rodriguez L Urdaneta-Marquez S Rajatileka etal ldquoA mutation in the voltage-gated sodium channel geneassociated with pyrethroid resistance in Latin American Aedesaegyptirdquo Insect Molecular Biology vol 16 no 6 pp 785ndash7982007

[14] Y Du Y Nomura B S Zhorov and K Dong ldquoSodium channelmutations and pyrethroid resistance in Aedes aegyptirdquo Insectsvol 7 no 4 2016

[15] A M Al Nazawi J Aqili M Alzahrani P J McCall and DWeetman ldquoCombined target site (kdr)mutations play a primaryrole in highly pyrethroid resistant phenotypes of Aedes aegyptifrom Saudi Arabiardquo Parasites amp Vectors vol 10 no 1 pp 1ndash102017

[16] H Kawada Y Higa K Futami et al ldquoDiscovery of PointMutations in the Voltage-Gated Sodium Channel from AfricanAedes aegypti Populations Potential Phylogenetic Reasons forGene Introgressionrdquo PLOS Neglected Tropical Diseases vol 10no 6 Article ID e0004780 2016

[17] F Z Vera-Maloof K Saavedra-Rodriguez A E Elizondo-Quiroga S Lozano-Fuentes and W C Black IV ldquoCoevolutionof the Ile1016 and Cys1534 Mutations in the Voltage GatedSodium Channel Gene of Aedes aegypti in Mexicordquo PLOSNeglected Tropical Diseases vol 9 no 12 Article ID e00042632015

[18] G Seixas L Grigoraki D Weetman et al ldquoInsecticide resis-tance is mediated by multiple mechanisms in recently intro-duced Aedes aegypti from Madeira Island (Portugal)rdquo PLOSNeglected Tropical Diseases vol 11 no 7 Article ID e00057992017

[19] J G B Linss L P Brito G A Garcia et al ldquoDistribution anddissemination of the Val1016Ile and Phe1534Cys Kdr mutationsin Aedes aegypti Brazilian natural populationsrdquo Parasites ampVectors vol 7 no 1 article 25 2014

[20] D F Bellinato P F Viana-Medeiros S C Araujo et alldquoResistance Status to the Insecticides Temephos Deltamethrinand Diflubenzuron in Brazilian Aedes aegypti PopulationsrdquoBioMed Research International vol 2016 Article ID 8603263pp 1ndash12 2016

[21] A J Martins L P Brito J G Linss et al ldquoEvidence for geneduplication in the voltage-gated sodium channel gene of Aedesaegyptirdquo Evolution Medicine and Public Health vol 2013 no 1pp 148ndash160 2013

[22] K Dong Y Du F Rinkevich et al ldquoMolecular biology of insectsodium channels and pyrethroid resistancerdquo Insect Biochemistryand Molecular Biology vol 50 no 1 pp 1ndash17 2014

[23] A JMartins RM Lins J G Linss et al ldquoVoltage-gated sodiumchannel polymorphism andmetabolic resistance in pyrethroid-resistant Aedes aegypti from Brazilrdquo The American Journal ofTropical Medicine and Hygiene vol 81 no 1 pp 108ndash115 2009

[24] A J Martins C D M Ribeiro D F Bellinato A A PeixotoD Valle and J B P Lima ldquoEffect of insecticide resistance ondevelopment longevity and reproduction of field or laboratoryselected Aedes aegypti populationsrdquo PLoS ONE vol 7 no 3Article ID e31889 2012

[25] R M R Nogueira M P Miagostovich A M B De FilippisM A S Pereira and H G Schatzmayr ldquoDengue Virus Type 3in Rio de Janeiro BrazilrdquoMemorias do Instituto Oswaldo Cruzvol 96 no 7 pp 925-926 2001

[26] R M R Nogueira M P Miagostovich E Lampe R W SouzaS M O Zagne andH G Schatzmayr ldquoDengue epidemic in thestate of Rio de Janeiro Brazil 1990ndash1 Co-circulation of dengue1 and dengue 2 serotypesrdquo Epidemiology and Infection vol 111no 1 pp 163ndash170 1993

[27] G Kuno ldquoEarly history of laboratory breeding of Aedes aegypti(Diptera Culicidae) focusing on the origins and use of selectedstrainsrdquo Journal of Medical Entomology vol 47 no 6 pp 957ndash971 2010

10 BioMed Research International

[28] J B Lima M P Da-Cunha R C Da Silva et al ldquoResistanceof Aedes aegypti to organophosphates in several municipalitiesin the State of Rio de Janeiro and Espirito SantordquoThe AmericanJournal of TropicalMedicine andHygiene vol 68 no 3 pp 329ndash333 2003

[29] L P Brito J G B Linss T N Lima-Camara et al ldquoAssessing theeffects of Aedes aegypti kdr mutations on pyrethroid resistanceand its fitness costrdquo PLoS ONE vol 8 no 4 Article ID e608782013

[30] G d Garcia M R David A d Martins et al ldquoThe impactof insecticide applications on the dynamics of resistance Thecase of four Aedes aegypti populations from different Brazilianregionsrdquo PLOS Neglected Tropical Diseases vol 12 no 2 pe0006227 2018

[31] WHO Monitoring and managing insecticide resistance inAedes mosquito populations - Interim guidance for entomol-ogists Geneva World Health Organization 2016

[32] M Raymond ldquoPresentation drsquoune programme drsquoanalyse log-probit pour microordinateur cahiers Orstromrdquo Ser Ent MedParasitol vol 22 pp 117ndash121 1985

[33] G Rasic R Schama R Powell et al ldquoContrasting geneticstructure between mitochondrial and nuclear markers in thedengue fever mosquito from Rio de Janeiro Implications forvector controlrdquo Evolutionary Applications vol 8 no 9 pp 901ndash915 2015

[34] A J Martins J B P Lima A A Peixoto and D Valle ldquoFre-quency of Val1016Ile mutation in the voltage-gated sodiumchannel gene of Aedes aegypti Brazilian populationsrdquo TropicalMedicine amp International Health vol 14 no 11 pp 1351ndash13552009

[35] G P Garcıa A E Flores I Fernandez-Salas et al ldquoRecent rapidrise of a permethrin knock down resistance allele in Aedesaegypti in Mexicordquo PLOS Neglected Tropical Diseases vol 3 no10 article e531 2009

[36] C Brengues N J Hawkes F Chandre et al ldquoPyrethroid andDDT cross-resistance in Aedes aegypti is correlated with novelmutations in the voltage-gated sodium channel generdquo Medicaland Veterinary Entomology vol 17 no 1 pp 87ndash94 2003

[37] Y Du Y Nomura G Satar et al ldquoMolecular evidence for dualpyrethroid-receptor sites on a mosquito sodium channelrdquo Pro-ceedings of the National Acadamy of Sciences of the United Statesof America vol 110 no 29 pp 11785ndash11790 2013

[38] WHO Discriminating concentrations of insecticides for adultmosquitoes In Organization WH editor WHO PesticideEvaluation Scheme 2016

[39] WHO Monitoring and managing insecticide resistance inAedes mosquito populations Interi guidance for entomolo-gists World Health Organization 2016 page 11

[40] W R Halliday and K P Burnham ldquoChoosing the optimal diag-nostic dose for monitoring insecticide resistancerdquo Journal ofEconomic Entomology vol 83 no 4 pp 1151ndash1159 1990

[41] Z Hu Y Du Y Nomura and K Dong ldquoA sodium channelmutation identified in Aedes aegypti selectively reduces cock-roach sodium channel sensitivity to type I but not type IIpyrethroidsrdquo Insect Biochemistry and Molecular Biology vol 41no 1 pp 9ndash13 2011

[42] P F Viana-Medeiros D F Bellinato A J Martins and DValle ldquoInsecticide resistance associated mechanisms and fit-ness aspects in two Brazilian Stegomyia aegypti (= Aedesaegypti) populationsrdquoMedical and Veterinary Entomology vol31 no 4 pp 340ndash350 2017

[43] H Sun K P Tong S Kasai and J G Scott ldquoOvercoming super-knock down resistance (super-kdr) mediated resistance multi-halogenated benzyl pyrethroids are more toxic to super-kdrthan kdr house fliesrdquo Insect Molecular Biology vol 25 no 2 pp126ndash137 2016

[44] I H Ishak Z Jaal H Ranson and C S Wondji ldquoContrastingpatterns of insecticide resistance and knockdown resistance(kdr) in the dengue vectors Aedes aegypti and Aedes albopictusfrom Malaysiardquo Parasites amp Vectors vol 8 no 1 article no 1812015

[45] S A Stenhouse S Plernsub J Yanola et al ldquoDetection of theV1016G mutation in the voltage-gated sodium channel gene ofAedes aegypti (Diptera Culicidae) by allele-specific PCR assayand its distribution and effect on deltamethrin resistance inThailandrdquo Parasites amp Vectors vol 6 no 1 article no 253 2013

[46] C Chang W-K Shen T-T Wang Y-H Lin E-L Hsu andS-M Dai ldquoA novel amino acid substitution in a voltage-gatedsodiumchannel is associatedwith knockdown resistance to per-methrin in Aedes aegyptirdquo Insect Biochemistry and MolecularBiology vol 39 no 4 pp 272ndash278 2009

[47] M L G Macoris M T M Andrighetti D M V Wanderleyand P E M Ribolla ldquoImpact of insecticide resistance on thefield control of Aedes aegypti in the State of Sao Paulordquo Journalof the Brazilian Society of Tropical Medicine vol 47 no 5 pp573ndash578 2014

[48] R Deming P Manrique-Saide A Medina Barreiro et al ldquoSpa-tial variation of insecticide resistance in the dengue vectorAedes aegypti presents unique vector control challengesrdquo Para-sites amp Vectors vol 9 no 1 article no 67 2016

[49] S Kasai H Sun and J G Scott ldquoDiversity of knockdownresistance alleles in a single house fly population facilitatesadaptation to pyrethroid insecticidesrdquo Insect Molecular Biologyvol 26 no 1 pp 13ndash24 2017

Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Disease Markers

Hindawiwwwhindawicom Volume 2018

BioMed Research International

OncologyJournal of

Hindawiwwwhindawicom Volume 2013

Hindawiwwwhindawicom Volume 2018

Oxidative Medicine and Cellular Longevity

Hindawiwwwhindawicom Volume 2018

PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Computational and Mathematical Methods in Medicine

Hindawiwwwhindawicom Volume 2018

Behavioural Neurology

OphthalmologyJournal of

Hindawiwwwhindawicom Volume 2018

Diabetes ResearchJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Research and TreatmentAIDS

Hindawiwwwhindawicom Volume 2018

Gastroenterology Research and Practice

Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom

Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Disease Markers

Hindawiwwwhindawicom Volume 2018

BioMed Research International

OncologyJournal of

Hindawiwwwhindawicom Volume 2013

Hindawiwwwhindawicom Volume 2018

Oxidative Medicine and Cellular Longevity

Hindawiwwwhindawicom Volume 2018

PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Computational and Mathematical Methods in Medicine

Hindawiwwwhindawicom Volume 2018

Behavioural Neurology

OphthalmologyJournal of

Hindawiwwwhindawicom Volume 2018

Diabetes ResearchJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Research and TreatmentAIDS

Hindawiwwwhindawicom Volume 2018

Gastroenterology Research and Practice

Hindawiwwwhindawicom Volume 2018

Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom