Insecticide resistance and genetic variability in natural populations of Aedes ( Stegomyia ) aegypti ( Diptera : Culicidae ) from Colombia

Mosquito control prevails as the most efficient method to protect humans from the dengue virus, despite recent efforts to find a vaccine for this disease. We evaluated insecticide resistance and genetic variability in natural populations of Aedes aegypti (Linnaeus, 1762) from Colombia. This is the first Colombian study examining kdr mutations and population structure. Bioassays with larvae of three mosquito populations (Armenia, Calarcá and Montenegro) were performed according to the World Health Organization (WHO) guidelines, using Temephos. For the analysis of the Val1016Ile mutation and genetic diversity, we sampled recently-emerged adults from four mosquito populations (Armenia, Calarcá, Montenegro and Barcelona). Following the WHO protocol, bioassays implemented with larvae showed resistance to Temephos in mosquito populations from Armenia (77% ± 2) and Calarcá (62% ± 14), and an incipient altered susceptibility at Montenegro (88% ± 8). The RR95 of mosquito populations ranged from 3.7 (Montenegro) to 6.0 (Calarca). The Val1016Ile mutation analysis of 107 genotyped samples indicates that 94% of the specimens were homozygous for the wild allele (1016Val) and 6% were heterozygous (Val1016Ile). The 1016Ile allele was not found in Barcelona. Genetic variability analysis found three mitochondrial lineages with low genetic diversity and gene flow. In comparison with haplotypes from the American continent, those from this study suggest connections with Mexican and North American populations. These results confirm that a continuous monitoring and managing program of A. aegypti resistance in the state of Quindío is required.

ABSTRACT.Mosquito control prevails as the most efficient method to protect humans from the dengue virus, despite recent efforts to find a vaccine for this disease.We evaluated insecticide resistance and genetic variability in natural populations of Aedes aegypti (Linnaeus, 1762) from Colombia.This is the first Colombian study examining kdr mutations and population structure.Bioassays with larvae of three mosquito populations (Armenia, Calarcá and Montenegro) were performed according to the World Health Organization (WHO) guidelines, using Temephos.For the analysis of the Val1016Ile mutation and genetic diversity, we sampled recently-emerged adults from four mosquito populations (Armenia, Calarcá, Montenegro and Barcelona).Following the WHO protocol, bioassays implemented with larvae showed resistance to Temephos in mosquito populations from Armenia (77% ± 2) and Calarcá (62% ± 14), and an incipient altered susceptibility at Montenegro (88% ± 8).The RR 95 of mosquito populations ranged from 3.7 (Montenegro) to 6.0 (Calarca).The Val1016Ile mutation analysis of 107 genotyped samples indicates that 94% of the specimens were homozygous for the wild allele (1016Val) and 6% were heterozygous (Val1016Ile).The 1016Ile allele was not found in Barcelona.Genetic variability analysis found three mitochondrial lineages with low genetic diversity and gene flow.In comparison with haplotypes from the American continent, those from this study suggest connections with Mexican and North American populations.These results confirm that a continuous monitoring and managing program of A. aegypti resistance in the state of Quindío is required.KEY WORDS.Bioassays; gene flow; knockdown resistance; mitochondrial DNA; ND4 gene.Insecticide resistance and genetic variability in natural populations of Aedes (Stegomyia) aegypti ZOOLOGIA 32 (1): 14-22, February 2015 mologous domains (I-IV), each with six hydrophobic segments (S1-S6) (CATTERALL 2000).'Knockdown resistance' (kdr) is a generic term applied to insects that fail to lose coordinated activity immediately following PY exposure.
Kdr mutations in the Na V (associated or not with PY resistance) have been observed in a range of insects, including A. aegypti (SAAVEDRA-RODRIGUEZ et al. 2007).In populations of A. aegypti from Latin America and Southeast Asia, the mutations Val1016Ile, Val1016Gly, Phe1534Cys and Asp1794Tyr, all in the IIS6 and IIIS6 segment, are correlated with insecticide resistance (BRENGUES et al. 2003, SAAVEDRA-RODRIGUEZ et al. 2007, CHANG et al. 2009, HARRIS et al. 2010, LINSS et al. 2014).However, only one of these, a valine to isoleucine substitution at codon 1016, has been clearly linked to insecticide resistance; selection pressure under laboratory conditions, bioassays with adults, biochemical assays and molecular screening have confirmed this finding (RODPRADIT et al. 2005, SAAVEDRA-RODRIGUEZ et al. 2007, STRODE et al. 2008, GARCÍA et al. 2009, MARTINS et al. 2009, LUMJUAN et al. 2011, MARCOMBE et al. 2012).
Understanding the patterns of genetic structure and gene flow among A. aegypti populations is pivotal for the development of rational dengue control programs (URDANETA-MARQUEZ & ANNA-BELLA 2011).The current trend is to use microsatellites (MONTEIRO et al. 2014) and/or SNPs (single nucleotide polymorphism) (RASIC et al. 2014).Nevertheless, mitochondrial DNA (mtDNA) has been widely used in population genetics studies of A. aegypti from different geographic points and dengue endemic regions (GONÇALVES et al. 2012).The ND4 mitochondrial gene, which codifies the subunit 4 of the NADH dehydrogenase enzyme, is an effective tool to analyze genetic population structure and colonization events in A. aegypti.Such analyses were carried out in mosquito populations from Brazil (TWERDOCHLIB et al. 2012), Bolivia (PAUPY et al. 2012), Peru (YÁÑEZ et al. 2013), Venezuela (URDANETA- MARQUEZ et al. 2008) and Mexico (GORROCHOTEGUI-ESCALANTE et al. 2002).So far, only RAPDs (Random Amplified Polymorphic DNA) (OCAMPO & WESSON 2004, MEJÍA et al. 2011) and more recently the mtDNA (CALDERA et al. 2013) have been used to analyze the genetic structure of A. aegypti populations in Colombia.
Here, we evaluated the insecticide resistance and genetic variability in natural populations of A. aegypti from Colombia.This is the first Colombian study to look at insecticide resistance (OP and PY (kdr mutation)) and population structure.Our findings have shown that insecticide resistance is spreading in the country owing to the 1016Ile kdr allele (in low frequency), and that OP resistance is found in most populations of A. aegypti studied by us.Genetic variability analysis shows that the vector population has low genetic diversity and limited gene flow.

MATERIAL AND METHODS
We collected A. aegypti larvae in 2011, from three municipalities in the state of Quindío: Armenia (4°32'0"N, 75°40'0"W, 1,483 m), Montenegro (4°34'23"N, 75°45'20"W, 1,292 m), Calarcá (4°31'55"N, 75°39'1"W, 1,573 m) and Barcelona (4°25'53", 75°43'26", 1,573 m), a district of Calarcá.We followed the standard methods of the Pan-American Health Organization (PAHO) for determining the infestation rate of A. aegypti (OPS 1995).At each municipality, we randomly collected immatures from at least 25 different containers located in selected residences in the urban area.This included domestic breeding sites such as water storage vessels, plastic pails, tires, and cans.Each container was located at least 100 m away from the others.Larvae from the same municipality were pooled in the laboratory and stored until adults emerged under controlled conditions (25 ± 1°C, humidity 80 ± 10% and photoperiod 12:12 hours) in the medical entomology laboratory of the Center for the Study of Tropical Diseases (Centro de Investigaciones en Enfermedades Tropicales -CINTROP), Industrial University of Santander (UIS), Santander, Colombia.We collected recentlyemerged adults from each population for the analysis of the Val1016Ile mutation and genetic diversity.The mosquitoes were individually placed in absolute ethanol (99.5%) and stored in a freezer at -20°C.The remaining F 0 adults were used to produce the F 1 generation.Aedes aegypti F 1 larvae were used as the source in bioassays to determine Temephos susceptibility.Adults were fed a 10% honey solution and blood meals that were provided by rats, Rattus norvegicus (Berkenhout, 1769), twice a week to induce oviposition.
Bioassays were carried out with A. aegypti natural populations from Armenia, Montenegro and Calarcá.We were unable to run the bioassay with the population from Barcelona, since it was not possible to establish the colony base because there were few immatures on the field.Bioassays included F1 generation larvae and the insecticide Temephos pestanal 250 mg 97.5% (Sigma-Aldrich) following the World Health Organization (WHO) guidelines (WHO 1998).Bioassays were calibrated with Rockefeller, a susceptible strain of A. aegypti (Centers for Disease Control, CDC), using a diagnostic concentration of 0.0162 ppm Temephos.This is twice the LC 99 (lethal concentration that kills 99% of the larvae) of the susceptible strain.The results from larvae were expressed as mortality rates 24h after exposure to Temephos.The following criteria proposed by the WHO (1998) guidelines were adopted to classify population susceptibility status: susceptible (percentage of mortality > 98%), susceptibility incipiently altered (80-98%), or resistant (< 80%).Dose-response bioassays followed the WHO guidelines to determine larval susceptibility to Temephos (WHO 1981).In these experiments, third instar or initial fourth instar larvae were exposed to 10 concentrations of the insecticide to determine larval mortality between 5 and 95%.At each concentration and in the control, four replicates containing 20 larvae each were tested.Larval mortality was checked 24 h after exposure.All tests were repeated three times on different days.
Mortality data (expressed as a number of dead specimens per dose) was applied to calculate lethal concentrations to 50 and 95% (LC 50 and LC 95 ) of exposed individuals, and were analyzed by the log-probit method of FINNEY (1971) using the Probit software by RAYMOND (1993).Resistance ratios (RR 50 and RR 95 ) were obtained by dividing the lethal concentration of the population by the equivalent lethal concentration of the Rockefeller population.
DNA extraction followed BONA et al. (2012).Individual mosquitoes from each locality were genotyped at position1016 of the genomic DNA using allele-specific PCR (AS-PCR).We used three primers to determine the presence of the Val1016Ile mutation, one for the 1016Val allele: 5'-GCG GGC AGG GCG GCG GGG GCG GGG CCA CAA ATTGTT TCC CAC CCG CAC CGG -3', one for the 1016Ile allele: 5'-GCG GGC ACA AAT TGT TTC CCA CCC GCA CTG A -3', and a third common to both alleles: 5'-GGA TGA ACC GAA ATT GGA CAA AAG C -3'.PCR reactions followed the protocol described by SAAVEDRA-RODRIGUEZ et al. (2007) andMARTINS et al. (2009).Amplified PCR products were checked in 10% polyacrylamide gel.Using the gel results, we calculated genotypic and allelic frequencies, and Hardy-Weinberg equilibrium (HW) (SALMAN 2007, HARTL 2008).The Rockefeller strain of A. aegypti, a standard for insecticide susceptibility reared in the laboratory, and life-history trait parameters, were used as reference for the wild-type alleles (1016Val) of the Nav gene.
Consensus sequences were obtained using the Staden software version 1.5, and were aligned with the program BioEdit version 7.0 (HALL 2004), using the ClustalW tool (THOMPSON et al. 1994).The sequences were compared with others available on GenBank using Tblastx to verify the amplified fragment.The Mantel test was used to estimate the correlation between genetic (F ST ) and geographic (km) distances.The GenAlEx6 software was used to test isolation by distance (PEAKALL & SMOUSE 2012).Geographical distances for this analysis were obtained using Google Earth 6.0.The software Mega ver.5.05 (TAMURA et al. 2007) was used to create a tree with the Neighbor-Joining method, following the Jukes-Cantor genetic distance model.Bootstrap support was estimated with 1,000 replicates.Aedes (Stegomyia) albopictus (Skuse, 1894) (GenBank#EF153761) was used as the external group.
The haplotypes of this study were deposited in the GenBank under accession KF241755 -KF241757.In order to estimate gene flow among the populations analyzed, they were compared with the haplotypes available from America, published by GONÇALVES et al. (2012); these haplotypes are free of nuclear mitochondrial pseudogenes (NUMTs).Samples containing mixtures of mtDNA and NUMT sequences are expected to significantly affect the outcome of genealogy-and frequencybased analyses.This is because mtDNA and NUMTs have separate genealogies and thus different evolutionary histories (GONÇALVES et al. 2012, RIBEIRO 2012).To reduce the error caused by NUMTs in the samples two analyses were carried out: 1.We searched for heterozygous sites in the chromatogram and additional termination codons (RIBEIRO 2012), and 2. We compared the haplotypes with a list of NUMTs verified by BLACK &BERNHARDT (2009) andHLAING et al. (2009).If any NUMT was found, it was removed from the analysis.

RESULTS
We

Diagnostic concentration (% mortality)
The results of bioassays with larvae following the WHO protocol showed that A. aegypti populations (Mortality rate ± SD) from Armenia (77% ± 2) and Calarcá (62% ± 14) are resistant to OP Temephos and that the Montenegro population has incipient, altered susceptibility to the insecticide (88% ± 8).

Multiple concentrations (RR)
Dose-response bioassays following the WHO protocol resulted in resistance ratios (RR 95 ) greater than three, and were the highest in the A. aegypti populations from Calarcá.In general, the slope values of the A. aegypti populations studied were lower than those obtained from the Rockefeller strain, confirming their heterogeneity in comparison to the reference strain and the differences in their response to OP Temephos.The LC 50 and LC 95 of all population studied are presented for comparison in Table I.

Genotyping the 1016 fragment of Na V
A total of 107 A. aegypti individuals were genotyped for the Val1016Ile mutation.Of these individuals, 94% were homozygous dominant (Val/Val); 6% were heterozygous (Val/Ile), and homozygous recessive genotypes (Ile/Ile) were not found.In all populations, the genotypic frequencies of Val/Val were greater than the frequencies of Val/Ile and Ile/Ile (Table II).The frequencies of the allele 1016Val were greater than the frequencies of the 1016Ile allele in all populations (Fig. 1).The populations from Armenia, Calarcá and Montenegro are in Hardy-Weinberg equilibrium.Nevertheless, bioassays with adults should be carried out to confirm the susceptibility status in these populations.

Genetic diversity -fragment of mitochondrial gene ND4
The amplified product of the ND4 gene was 311bp.There were four polymorphic sites and 307 monomorphic sites.The analysis of the amplified fragment of 42 individuals resulted in three haplotypes without NUMTs: H1-Col (GenBank# KF241755), H2-Col (GenBank# KF241756) and H3-Col Haplotype diversity was 0.67 ± 0.036 (mean ± SD, n = 42), nucleotide diversity was 0.0058 ± 0.0002 and there were 1.81 nucleotide differences on average.The neutral selectivity test results (p > 0.05) were in agreement with the assumptions of the neutral mutation model (p > 0.05, Tajima's D Test 2.24, Fu Fs Test 2.58).
1986) has been the cause of intensive insecticide use to reduce dengue cases, selecting resistant vector populations.

DISCUSSION
We found that insecticide resistance is present in most studied populations of A. aegypti and is determined by the1016Ilekdr allele (showing low frequency), which confers OP resistance.Even though resistance to Temephos had been previously documented in Colombia (GRISALES et al. 2013),our findings on Temephos resistance are important because it has been the main insecticide used to control the immature stages of natural A. aegypti populations in the country (OCAMPO et al. 2011).Additionally, Temephos pressure on larvae may generate cross-resistance to PY or other OP used in the control of the adult stages (RODRÍGUEZ et al. 2002, TIKAR et al. 2009).
The application of the OP Temephos on breeding sites is essential to control A. aegypti immatures worldwide (WHO 2013).However, long-term use of this insecticide has caused the emergence of resistance in several Latin American countries (RODRÍGUEZ et al. 2007, LIMA et al. 2011, BISSET et al. 2013).Colombia's participation in the continental vector control campaign led by OPS (SANTACOLOMA et al. 2010), and several dengue outbreaks in 1970 and in 1980 in most Colombian regions (BOSHELL et al.

3
lection pressure using PY insecticides under laboratory conditions have documented fixation of the 1016Ile kdr allele after only five generations (IRAC 2011, SAAVEDRA-RODRIGUEZ et al. 2012).In the last decade, the 1016Ile kdr allele has rapidly spread in A. aegypti populations from Mexico and Brazil, simultaneously with the intensification of PY usage due to the emergence of dengue outbreaks (GARCÍA et al. 2009, LINSS et al. 2014).Therefore, enhanced surveillance for resistance should be a priority in localities where the 1016Ile kdr allele is found, before new adaptive alleles can be selected for decreasing the deleterious effects of kdr (BRITO et al. 2013).Consequently, bioassays with adults should be performed to confirm the susceptibility status of these populations.
Our results suggest that alternative control strategies need to be found before Temephos resistance compromises operational control.An alternative to reduce selection pressure for resistance is to devise an insecticide swapping program implementing the criteria proposed by the Brazilian Ministry of Health.According to these guidelines, Temephos needs to be replaced with another insecticide with a different action mechanism in populations with RR 95 у 3 (MINISTÉRIO DA SAÚDE 2006).In studies conducted on populations of A. aegypti from Brazil, it was observed that when the application of Temephos is interrupted in locations where RR 95 is greater than 10, resistance declines only gradually, and several years are needed for Temephos to be effective again (MONTELLA et al. 2007).On the other hand, despite the fact that Colombia has its own vector control program, the susceptibility status of the populations we evaluated should be considered as "populations with susceptibility loss to OP Temephos"; hence continuous monitoring of susceptibility status is recommended in order to determine the accurate moment to change the active substance (MINISTERIO DE LA PROTECCIÓN SOCIAL 2011).Nevertheless, the RR 95 values in all populations were greater than three.
Chemical measures used in vector control programs could affect the genetic diversity of A. aegypti populations, and as a result, induce genetic changes through bottleneck and genetic drift effects (URDANETA-MARQUEZ & ANNA-BELLA 2011).Low genetic diversity is most likely a result of a decline in population size caused by insecticide use, as it was observed in vector populations from Trinidad andTobago, andVenezuela (YAN et al. 1998, HERRERA et al. 2006).However, some studies have revealed the presence of greater genetic diversity in areas that are frequently treated with insecticides, as shown for A. aegypti populations from French Polynesia andBrazil (PAUPY et al. 2000, AYRES et al. 2004).In our results, genetic diversity (Hd), number of haplotypes (N) and nucleotide diversity () were lower (N = 3, Hd = 0.67 and = 0.006) than in other studies on the ND4 gene of A. aegypti.For example, 36 locations in the Americas, Asia and Africa (N = 20, Hd = 0.82 and = 0.020) (BRACCO et al. 2007), five states in Brazil (N = 24, Hd = 0.80 and = 0.017) (PADUAN & RIBOLLA 2008) and two populations from Colombia (N = 10, Hd = 0.068 and = 0.009) (CALDERA et al. 2013).
The studied Colombian A. aegypti populations were genetically structured, a trend also found in other populations from South America and Central America (GORROCHOTEGUI-ESCALANTE et al. 2002, COSTA-DA-SILVA et al. 2005, URDANETA-MARQUEZ et al. 2008, PAUPY et al. 2012, TWERDOCHLIB et al. 2012, CALDERA et al. 2013).The genetic structure of the studied Colombian populations occurred between regions separated by less than 17 km, similar to the patterns observed in Venezuela (distance less than 15 km between them), which suggest that gene flow is restricted (HERRERA et al. 2006).Geographic barriers associated with Andean mountains in the state of Quindío probably obstruct gene flow.However, in Quindio state, where tourism is intense, there is more road traffic between the studied populations (GOBERNACIÓN DEL QUINDÍO 2013, INVIAS 2013), which ties the gene flow in populations of A. aegypti to human transport (HUBER et al. 2004, COSTA-DA-SILVA et al. 2005).
Overall, two mitochondrial lineages were present in the studied Colombian populations, and the most frequent haplotypes came from group I.This pattern is in agreement with previous studies (GORROCHOTEGUI-ESCALANTE 2002, BOSIO et al. 2005, HERRERA et al. 2006, BRACCO et al. 2007, PADUAN & RIBOLA 2008).The haplotypes found in this study indicate a relationship between the Mexican and North American populations.These connections result from passive dispersal of A. aegypti among different countries, and passive vector dispersal is likely to be the most common pattern world-wide (GORROCHOTEGUI-ESCALANTE et al. 2002, HUBER et al. 2004, BOSIO et al. 2005, GONÇALVES et al. 2012).Further studies are necessary to ascertain how the vector entered Colombia, since the connection among the populations in this study is clear.We conclude that continuous monitoring and managing programs are needed to control A. aegypti populations in Colombia.Given that insecticide resistance could potentially compromise vector control programs, a threshold of RR 95 у 3.0 should be established for swapping among insecticides with different modes of action.

LITERATURE CITED
AYRES C, MELO-SANTOS M, PROTA J, SOLÉ-CAVA A, FURTADO A (2004) Genetic structure of natural populations of Aedes aegypti at the micro-and macro geographic levels in Brazil.
Genetic diversity and neutrality tests were calculated using the program DnaSP, version 5.0 (LIBRADO & ROZAS 2009).Molecular variation analysis (AMOVA) was performed with the program Arlequin version 3.5 (EXCOFFIER & LISCHER 2010).Population structure was determined using the Wright fixation index (F ST , Wright 1921) and gene flow (Nm) was obtained by the program Arlequin 3.5 (EXCOFFIER & LISCHER 2010) followed by Bonferroni correction.

Figure 1 .
Figure 1.Allelic frequencies of 1016Val and 1016Ile in the Na v of four A. aegypti populations from Colombia.
Table IV.Variable sites in three haplotypes of ND4 mitochondrial gene in Aedes aegypti from Colombia. of individuals that share each haplotype.

Table I .
Temephos susceptibility profile from Colombian populations of Aedes aegypti, showing means (standard deviations) for slopes, LC and RR.

Table II .
Genotypic frequency of the mutation Val1016Ile in the NaV of four Aedes aegypti populations from Quindío, Colombia.

Table III .
Number of individuals observed for each haplotype in samples of four populations of Aedes aegypti from Colombia.

Table V .
Genetic distances (Fst values, above the diagonal) and effective number of migrants (Nm, below the diagonal) of four natural Aedes aegypti populations from Quindío, Colombia.