Phenotypic and genotypic profi le of pyrethroid resistance in populations of the mosquito Aedes aegypti from Goiânia , Central West Brazil

Introduction: The mosquito Aedes aegypti has evolved resistance to pyrethroid insecticides. The present study evaluated Ae. aegypti from Goiânia for the resistant phenotype and for mutations associated with resistance. Methods: Insecticide dose-response bioassays were conducted on mosquitoes descended from fi eld-collected eggs, and polymerase chain reaction (PCR) was used to genotype 90 individuals at sites implicated in pyrethroid resistance. Results: All mosquito populations displayed high levels of resistance to deltamethrin, as well as high frequencies of the 1016Ilekdr and 1534Cyskdr mutations. Conclusions: Aedes aegypti populations in the Goiânia area are highly resistant to deltamethrin, presumably due to high frequencies of kdr (knockdown-resistance) mutations.

In tropical areas, Aedes aegypti is the main vector of severe diseases, including some, such as dengue and chikungunya, for which effective vaccines or treatments do not exist.Vector control is therefore critical for public health (1) .Worldwide, pyrethroids (PYs) are the most common class of insecticides used to control adult insects (2) .However, prolonged use of these insecticides has resulted in the evolution of PY-resistant mosquito populations in many countries, including Brazil (3) (4) .
Pyrethroids act by binding to subunits of voltage-gated sodium channels in insects' central nervous systems, keeping these channels in the open state.In Aedes aegypti, substitution mutations in the gene coding for these channels (AaNa(V)) can alter the conformation of the site where PYs typically bind, leading to resistant mosquitoes (5) .In Aedes aegypti, several studies have documented genetic polymorphisms in AaNa(V) (4) (5) (6) (7) (8) (9) , but only two substitutions (Val1016Ile and Phe1534Cys) are clearly related to resistance to PYs (9) .
The State of Goiás, located in Central-West Brazil, is home to approximately 6.5 million people.It is one of the states in Brazil most affected by dengue, with 163,808 cases reported in 2013 alone Secretária de Saúde do Estado de Goiás (SES-GO), 2014, unpublished data).More recently, sporadic cases of chikungunya have been reported and it is expected that the disease will soon become epidemic in Goiás.
For over 20 years, the PY deltamethrin has been the main Ae.aegypti adulticide in the State of Goiás.Little is known about tolerance to deltamethrin, or about genetic variation in the AaNa(V) gene, in populations of Ae. aegypti.We evaluated deltamethrin tolerance of Ae. aegypti individuals from three populations in the City of Goiânia, Goiás, and determined the frequency of genetic polymorphisms in residues 1016 and 1534 of the AaNa(V) gene in these populations.The study was approved by the Committee on the Ethics of Animal Use (ECAU) of the Federal University of Goiás [Universidade Federal de Goiás (UFG)], fi led under number 031/13.Aedes aegypti eggs were collected using ovitraps from three neighborhoods (Vila Finsocial, Sudoeste, and Jardim América) between March and April 2013, according to the protocol of the Brazilian Program for Dengue Control.Egg hatching and species identifi cation followed the procedures of Lima (10) .The larvae were used to establish three laboratory colonies, one from the eggs collected from each of the three neighborhoods.When these larvae reached adulthood, thirty adult males (F 0 generation) were genotyped at positions 1016 and 1534 of the AaNa(V) locus.For position 1016, conventional polymerase chain reaction (PCR) amplifi cation was used, and amplicon  sizes were verifi ed in polyacrylamide gels, following previously published protocols (4) .For position 1534, allele-specific real time polymerase chain reaction (RT-PCR) was used, as described previously (6) .The remaining F 0 adults were allowed to breed, and their offspring (F 1 generation) were assayed for resistance to deltamethrin.Nine concentrations of insecticide were used: 2.5mg/m 2 , 5mg/m 2 , 15mg/m 2 , 30mg/m 2 , 40mg/m 2 , 50mg/m 2 , 75mg/m 2 , 100mg/m 2 , and 125mg/m 2 .From each of the three colonies, 20 F 1 females 3-5 days post-eclosion were selected for each of these treatments, and placed in cylindrical plastic tubes containing paper impregnated in permethrin at the indicated concentration.In this sense, around 60 females were exposed to each concentration and to the untreated control, totalizing around 600 females/population.Tolerance to deltamethrin was estimated by comparing mortality of the Goiânia mosquitoes to those of the insecticide-susceptible Rockefeller strain, as recommended by the World Health Organization (WHO) (11) , insecticidal the same concentrations and the same number of female population were used to Rockfeller Probit analysis was performed with Polo-PC (12) , with α = 0.05, to obtain lethal concentracion LC 50 and LC 95 values for the three Goiânia colonies and the Rockefeller strain.The resistance ratio (RR) for each of the Goiânia populations was calculated as the ratio between the LC 50 or LC 95 of the Goiânia colonies and that of the Rockefeller strain.
A total of 7,400 eggs were collected.Of the 180 traps, 121 (67%) had fertile eggs.From the total number of eggs in each trap, an Ovitrap Positivity Index (OPI) was calculated.The OPIs of the Vila Finsocial, Sudoeste and Jardim América were 78.3%, 70%, and 53%, respectively.Successful hatching rates ranged from 64% to 75.5%.All populations showed the expected sex ratio of 1:1.
All of the Goiânia populations of Ae. aegypti showed high levels of resistance to deltamethrin (Figure 1).This is similar to trends seen in other regions of Brazil (3) (4) (9) .As suggested by Chandrer (13) , this is likely to be a result of the selection pressure imposed by the application of large amounts of the same group of insecticides over a long period of time.kdr (knockdown resistant) substitutions at both site 1016 and 1534 were seen at high frequencies in the three districts surveyed.Frequencies for the 1016Ile kdr substitution ranged from 0.47 to 0.72, with homozygotes for this substitution ranging from 0.23 to 0.60.The frequency of the 1534Cys kdr substitution was nearly 1.0 in all of the Goiânia populations; only one individual was heterozygous for this substitution (Table 1).A signifi cant fraction of mosquitoes carried two copies of both substitutions (Table 2), which may confer higher levels of resistance to PYs (9) .
In conclusion, having characterized three Ae.aegypti populations from Goiânia in Central-West Brazil for their tolerance to deltamethrin and for mutations in the AaNa(V) gene that are known to be implicated in insecticide resistance, we found that all populations exhibit high resistance to deltamethrin, consistent with the high frequency of mutations that we observed in the AaNa(V) gene.Our data support the position that pyrethroids are no longer effective for suppressing Ae. aegypti in Goiás.Due to the competence and effi ciency of this vector in the transmission of diseases such as yellow fever, dengue, and chikungunya, we conclude that there is an urgent demand for alternative and/or complementary methods of vector control.

FIGURE 1 -
FIGURE 1 -Deltamethrin resistance ratios for three Aedes aegypti populations from the City of Goiânia, Central-West Brazil.Resistance ratios for each population were calculated from the ratio between the LC 50 or LC 95 of the fi eld sample and that of the susceptible Rockefeller strain.Bars represent the average RRs resulting from three independent experiments.RR: resistance ratios; LC: lethal concentration.

TABLE 1 -Genotypic and allelic frequencies for each of the 1016 and 1534 AaNa(V) sites of Aedes aegypti populations from Goiânia, Central-West, Brazil.
N: total number of samples.