Susceptibility of Aedes aegypti populations to pyriproxyfen in the Federal District of Brazil

Abstract INTRODUCTION: In Brasilia, pyriproxyfen (PPF; 0.01 mg/L) has been used for the larval control of Aedes aegypti mosquitoes since 2016. Information on the susceptibility of Ae. aegypti to PPF, and the development of resistance in populations from the Federal District of Brazil (FD) is limited. It is essential to monitor the susceptibility of Ae. aegypti to insecticides in order to improve vector control strategies. This study aimed to evaluate the susceptibility of Ae. aegypti populations from five areas of Brasilia to PPF. METHODS: We performed dose-response tests to estimate the emergence inhibition and resistance ratio of each field population, including the Rockefeller reference population. We also analyzed egg positivity, and the density and mortality of larvae and pupae. RESULTS: Populations from Vila Planalto (RR50=1.7), Regiment Guards Cavalry (RR50=2.5), and Sub-secretary of Justice Complex (RR50=3.7) presented high susceptibility to PPF, while the RR values of populations from Lago Norte (RR50=7.7) and Varjão (RR50=5.9) were moderately high, suggesting the emergence of insipient resistance to PPF in Brasilia. At 30 ng/mL, the highest larvae mortality rate was 2.7% for the population from Lago Norte, while that of pupae was 92.1% for Varjão and Vila Planalto. CONCLUSIONS: The five populations of Ae. aegypti from the FD are susceptible to PPF and there is a need to monitor the susceptibility of Ae. aegypti in new areas of the FD.


INTRODUCTION
Arboviruses (Arthropod-borne viruses) are of great importance for public health due to their high impact on health and the economy 1,2,3 . The main viruses transmitted by mosquitoes are urban yellow fever (YFV), dengue (DENV), chikungunya (CHINKV), and Zika (ZIKV). Some researchers 4 have reported the occurrence of at least one of these viruses in 146 countries, for which the main vectors are mosquitoes Aedes aegypti (Linnaeus, 1762) and Aedes albopictus (Skuse, 1894).
Ae. aegypti is the primary vector of arboviruses in Brazil and is spread in all Federative Units of the country 5 . Environmental, socioeconomic, biological, and non-biological factors favor the dispersion and proliferation of this species, in addition to its urban habits, which are associated with anthropophilia, endophilia, endophagia, domiciliation, and oviposition strategy in artificial breeding sites. This increases the transmission of arboviruses 6,7,8,9,10,11 .
The continuous and systematic use of the same product over a long period can select resistant individuals, compromising vectorcontrol. Currently, resistance to organophosphates and pyrethroids has been reported in several populations of Ae. aegypti, including populations in the Federal District of Brazil (FD) 12,13,14,15,16,17 .
In 1999, the National Network for Monitoring the Resistance of Ae. aegypti to Insecticides (MoReNAa) began to monitor the insecticide resistance of Ae. aegypti in Brazil, leading to changes in the products used in the National Program for Dengue Control (PNCD) 18 .

Field populations
We installed 60 ovitraps 22 , with the addition of 10% hay to increase egg capture yield 23 . All traps remained in each area for 2 weeks in the peridomicile environment. The traps were installed in the grounds of houses, protected from rain, with limited human and animal movement 24 . A volume of 20 mL of feno solution (10%) was added per trap to attract gravid females. The traps were replaced at the end of the first week and collected in the second week. The pallets containing eggs were collected, identified, and stored vertically inside a polystyrene box to prevent the eggs from being crushed or damaged. We transported the boxes to the Laboratório de Entomologia Médica, Secretaria de Vigilância em Saúde, Ministério da Saúde/SVS/MS. Then, we identified 25 and counted eggs, which were classified as hatchlings, withered, and viable 26 , and estimated egg positivity index (EPI) and egg density (EDI). Pallets with eggs were submerged in dechlorinated water and hatched larvae were transferred to basins containing 1 L of dechlorinated water. Larvae were fed with 3 mg of natural Guabi® shredded cat food, which was added every 3 days. After the emergence of adults, we offered a 10% sugar solution to males and females.
Three-days after their emergence, mosquitoes were fed with bird blood (Gallus gallus domesticus) every 48 h, according to Protocol No. 85/2018 of the Commission for Ethics in Animal Use (CEUA) of the University of Brasilia. Insectaries were maintained under controlled temperature (27 ± 2°C) and humidity (70 ± 15%) conditions. We used F1, F2, or F3 Ae. aegypti generations for the trials, according to the guidelines of the World Health Organization (WHO) 27 .

Susceptible population
We used the Rockefeller population of Ae. aegypti, from the Laboratorio de Entomologia/Diretoria de Vigilância Ambiental/ DIVAL/SES/DF.

Biological tests
Biological bioassays were performed according as previously described 27 , using nine concentrations ranging from 0.001 to 30 ng/mL. For each dose, a total of 270 third-stage larvae were exposed, including the control group. Larvae were selected homogeneously to standardize their physiological and chronological age. Then, the larvae were placed in 400 mL cups containing 250 mL of distilled water, covered with a fine mesh net attached to the edge with an elastic alloy. All larvae remained at rest for approximately 30 min for acclimatization. Subsequently, we removed 1 mL of water from each beaker. Then, 1 ml of PPF solution was added in nine increasing concentrations and the mixture was homogenized with a glass rod. We fed the larvae with Guabi® Natural Feed every 72 h. Mortality was recorded every 48 h by a single researcher using a specific form; we completed this work when all pupae had emerged into adults. The mortality criteria were as follows: i) larvae and pupae unable to ascend to the surface or show diving reactions when the water was disturbed; ii) immobile larvae and pupae when stimulated with a needle in their siphon or cervical region 27,28 and, iii) adults that did not complete development and were unable to completely emerge from the pupa during the emergence phase. Live adults were considered as those totally free of their exuviae and able to fly or walk when gently touched. We performed all trials in triplicate on four different days and prepared an equal number of controls with the same amount of water and 1 mL of alcohol. Mortality, as well as the emergence of adults, was recorded when all the specimens in the control condition had emerged as adults. We discarded assays in which adult emergence was less than 90% in the control group. When inhibition was between 91 and 99%, the Abbott formula was used for correction 27 . We controlled temperature (25-30°C) and relative air humidity (70-80%) with a heater and a conventional air humidifier.

Statistical analysis
We used the Polo PC program (Polo-PC, LeOra Software, Berkeley, CA) 29 (Raymond 1985) 30 to estimate the emergence inhibition doses of adults from the reference and field lines. The resistance ratios (RR) were determined through the EI 50 quotient of the field population by the EI 50 of the susceptible population, as well as the 95% confidence interval (CI 95%) of each population 31 . We also estimated the mortality of larvae and pupae 32,33 . The angular coefficient of the dose-response curve was calculated for each population using Graph-Pad Prism version 6.1 for Windows 34 . The criterion adopted for resistance classification was RR <5, indicating a susceptible field population; an RR between 5 and 10 indicated moderate resistance; and an RR >10 indicated high resistance 27 .

RESULTS
The ovitraps obtained 5,966 eggs from Ae. aegypti, of which 4,171 were viable, 1,212 withered, and 583 hatched. The Figure 1 shows the OPI and EDI of the traps installed to obtain Ae. aegypti eggs. The highest OPI (95%) was recorded for the traps deployed in Vila Planalto, while the lowest values were recorded for those deployed in Varjão, whose OPI was 36%. Although Vila Planalto presented the highest OPI, the EDI was low (34).
We exposed a total of 14,580 Ae. aegypti larvae to PPF. The Lago Norte and Varjão Ae. aegypti populations presented moderate resistance, with RR 50 values of 7.7 and 5.9, respectively. The populations from Vila Planalto (RR 50 =1.7), RCG (RR 50 =2.5), and SUAG (RR 50 =3.7) presented high susceptibility to PPF, as shown in Table 1. Table 2 presents data on the Rockefeller reference population, which obtained the highest EI compared to other field populations; thus, at a 30 ng/mL dose, the researchers recorded an average 99% EI of adults in the Rockefeller reference lineage. At this dose, the mean EI of adults in the field populations of Ae. aegypti was 92% for Vila Planalto and Varjão, 90% for Lago Norte, 89% for SUAG, and 87% for RCG. Figure 2 shows the mortality rates of larvae and pupae. The mortality of larvae exposed to PPF was low, while it was high for pupae, with values above 90% in most field populations. The gradient values of the Ae. aegypti populations from five areas of the FD are shown in Figure 3. We observed gradient patterns similar to the reference population in those from Vila Planalto and SUAG. Although the Ae. aegypti populations from Varjão and Lago Norte were similar, when compared to the reference population, the RCG population showed less homogeneity.
In Brazil, monitoring the insecticide resistance of Ae. aegypti populations has had an important impact on arboviruses epidemiology. Populations of Ae. aegypti with high levels of resistance contribute to the emergence of dengue outbreaks with high magnitude. In Campo Grande, the highest RR values (above 50 for deltamethrin) revealed that the period with the highest incidence of dengue coincides with the detection of Ae. aegypti populations with high resistance 38 .
Large urban centers with a greater flow of people, a history of mosquitoes and arbovirus circulation, are also determining factors for the increased spread of resistance. In 2019, the municipality of Palmas (RR 50 =28) had the highest probable number of dengue cases, unlike Caseara (RR 50 =1.6), which is less urbanized and is remote of other major centers urban areas 17 .
Thus, the monitoring of insecticide resistance in Ae. aegypti populations should be continuous and periodic for the rational management of adulticides and larvicides used to control mosquito populations, and for reducing local or large-scale resistance.
In this study, the mortality of larvae from field populations ranged from 0.6 to 2.0%, and that of pupae ranged from 99 to 88%, at a 30 ng/mL dose. This can be explained by the activity of PPF during the pupal phase, when comparative studies of field simulations between the Rockefeller and Itabuna/Bahia populations are conducted, with mortality rates of 97.9 and 95.1%, respectively 39 . Conversely, the larval mortality rate o was only 2.1 and 4.9%, respectively. Others studies 40 have reported similar results under laboratory conditions, with higher mortality in Ae. aegypti pupae.
The mortality rate of Ae. aegypti pupae treated with PPF was 100% with the 0.2 and 1 ppm doses 41 . Another study using commercial PPF showed that doses lower than 0.01 ppb resulted in 98.5% pupal mortality 42 . Therefore, PPF is highly effective at inhibiting the emergence of adults, hindering the formation of wings, and the development of reproductive organs and external genitalia 43 .
Currently, the Brazilian Ministry of Health 44 uses the Larval Index Rapid Assay for Aedes aegypti (LIRAa) to direct actions for the control of Ae. aegypti, based on the detection of mosquito larval foci. Thus, breeding sites treated with PPF, in which the larvae remain alive, may lead to incorrect estimates of mosquito infestation levels, as well as the possibility of overlapping treatment of Ae. aegypti larval foci.
The limitations of this study included delay in obtaining the F1 generation of all populations, due to the very cold weather in 2017, which affected estimates of the dose-response curve. No new assays could be performed with PPF sub-doses to improve standardization of the diagnostic dose curve assays.
New areas of the FD need to be monitored for changes in the susceptibility of Ae. aegypti. Field bioassays with Ae. aegypti populations from DF will also contribute to understanding the effectiveness of PPF in the field.