Study populations
The sampling points applied herein considered several areas throughout the Brazilian territory, covering a large number of close towns, in urban conglomerates with high population density, as suggested by Chediak et al (2016) [19], preferentially in sites previously evaluated during the 12-year period MoReNAa Network effort, as described by Valle et al. (2019) [9]. This proposal was also adjusted considering the operational capacity of the municipal sampling teams, resulting in the selection of 146 cities for Ae. aegypti samplings over the course of 17 months (Table 1, Fig. 1). Field Ae. aegypti populations were collected by the Endemic Control Agents of each city, using between 100 oviposition traps (ovitraps) in cities with up to 50,000 houses and 300 ovitraps in cities with over 500,000 houses, following the MoReNAa Network 2008 methodology [20].
To install the traps, houses evenly distributed in a grid pattern with full coverage of the urban territory were selected, in order to include regions presenting different infestation levels, and one trap was installed in a shaded area on the grounds of each selected house. A 0.04% yeast extract solution was used as an attractant for gravid females. In order to facilitate the preparation of this solution in the field, the agents were provided with a 50 mL conical tube containing 6 g of a commercial yeast extract (Arma Zen®). During the trap installation, the tubes were filled with tap water to the 50 mL mark and homogenized. With the aid of plastic Pasteur pipettes, 1 mL of this solution was added to the trap, which was then then filled with tap water to the 300 mL mark. The traps were maintained in the households for 15 days, with one paddle and an attractive solution change at the end of the first week. The paddles containing the eggs were air-dried for 2 to 3 days prior to being sent to the laboratories.
The samplings were carried out between August 2017 and December 2018, following a staggered schedule so as not to overload the laboratories. Three preferred months were chosen for the samplings in each region of the country, observing the most adequate climatic conditions in order to obtain higher egg densities. The field-collected samples were initially sent to a central entomology laboratory in each respective state, which then confirmed the correct sampling registration at the origin sites and adequate paddle storage. The paddles were then shipped to the Physiology and Arthropod Vector Control Laboratory (Laboratório de Fisiologia e Controle de Artrópodes Vetores - LAFICAVE), at the Oswaldo Cruz Institute (IOC/Fiocruz), Rio de Janeiro/RJ, where the arrivals were recorded, forms were stored and populations labeled with a code known only by the study director, in order to maintain origin confidentiality. Half of the populations remained at the LAFICAVE, while the other half was sent to the Applied Entomology Laboratory (Laboratório de Entomologia Aplicada - LEnA), at the Endemic Control Superintendence (Superintendência de Controle de Endemias - SUCEN), Marília, SP. Ae. aegypti specimen sorting, colony maintenance and bioassays were performed by the LAFICAVE and LEnA laboratories.
Mosquito rearing
Paddles containing eggs were submerged in dechlorinated water and hatched larvae were transferred to basins (33 x 24 x 8 cm) containing 1 L of dechlorinated water and 100 mg of fish food (TetraMin®, Tetra Marine Granules) added every three days. The resulting adult Ae. aegypti mosquitoes were identified to the species level and sorted gender, with 500 females and 500 males maintained in cylindrical carton cages (16 cm diameter X 18 cm high), where a 10% sucrose solution was offered, ad libtum. When the number of females were insufficient for producing an F1 generation (less than 100 females), new field collections were requested.
In order to produce eggs for the next generation, females were additionally fed blood from guinea pigs (Cavia Porcellus - Linnaeus, 1758) three days post-emergence (Fiocruz Ethics Committee on the Use of Animals authorizations LW-20/14 and L-004/2018). Alternatively, females were offered to feed on citrated rabbit blood through a Hemotek reservoir membrane feeder (Discovery Workshops, Accrington, UK), containing 6 mL of blood covered with a parafilm membrane, sealed with a rubber ring, at 37˚C for 1 hour. F1 generation mosquitoes were employed in the bioassays, although an F2 generation was required whenever the number of F1 generation individuals to perform all larvae and adult assays was insufficient.
Insectaries were maintained under controlled temperature (26 ± 2°C) and humidity (70 ± 10%) following the Fiocruz biosafety manual for vector insectaries and infectories [21]. About 50 specimens of the parental generation were cryopreserved for the creation of a DNA bank for future genetic analyses. Only male mosquitoes were cryopreserved, eliminating the need to extract the female's abdomen to prevent possible DNA amplification from spermatozoa present in their spermateca. The Rockefeller [22] reference strain concerning insecticide susceptibility and vigor under laboratory conditions was employed for the determination of diagnostic-doses (DD), and was exposed in parallel in each assay, as an assay quality control. Standardizations of the biological tests performed on both adults and larvae were carried out using this susceptible strain.
DD estimations
Before the susceptibility evaluations of field Ae. aegypti populations, the DD for pyriproxyfen and malathion were estimated, respectively, in larvae and adults, under our local conditions. It is important to note that a WHO reference for a pyriproxyfen DD is still not available so far. The locally established DDs were obtained by dose-response (DR) assays using the Rockefeller strain. The Rockefeller colony maintained at the LEnA was used for the tests in both laboratories.
DD estimation for pyriproxyfen
Larval bioassays were conducted with an IGR pyriproxyfen analytical standard (Sigma Pestanal®), pre-dissolved in acetone (Sigma Aldrich®) and further diluted in ethanol (Merck®). Following procedures described in the WHO Guidelines for larvicide bioassays, with some modifications [23], third stage larvae (L3 stage) were submitted to a gradient of 13 product concentrations (0.0667 to 0.2337µg/L), where adult emergence inhibition (EI) percentages were evaluated at the end of 7 to 10 days, when all control larvae had emerged into adults. Four replicates comprising 10 L3 larvae each were prepared for each concentration, and an equal number of controls were prepared using only ethanol. The larvae were fed 10 mg of fish food (TetraMin®, Tetra Marine Granules) on the first day and 5 mg on the third day after initial exposure. The assays were followed daily until complete adult emergence in the control group.
Assays were discarded if the EI of the control group was > 10%. If not, they were corrected by Abbott's formula when EI ranged between 5% and 10% [23]. Four tests were performed at different times. When pupae began to develop, cups were covered with a mesh to avoid eventual adult escapes. Mortality and adult emergence were recorded when all the specimens under the control condition had emerged. Live adults were considered as those totally free of their exuviae and able to fly when gently touched, and the other individuals were considered dead. The EI were calculated using Probit (Polo-PC, LeOra Software, Berkeley, CA) and logistic regression analyses [24]. Finally, the pyriproxyfen DD was determined as twice the dose that inhibited the emergence of adults in 99% (EI99) of Rockefeller larvae exposed to the compound.
DD estimation for malathion
To perform the bioassays, aliquots of OP stock solutions at a concentration of 3000 mg/L were prepared from a malathion analytical standard (Sigma Pestanal®) dissolved in acetone (Sigma Aldrich®) and stored at -80°C. Glass bottles (250 mL) (Wheaton) were coated on the inside with 1 mL of malathion dissolved in acetone at four concentrations (12, 15, 18 and 20 µg/bottle) prepared from the stock solution 24 h before the test. Two bottles per concentration and one control (coated on the inside with 1 mL of acetone only) were employed for each test, with each bottle containing 25 females aged 3 to 5 days old. Six tests with each dose were performed, on distinct days. Mosquitoes were exposed to the insecticide for up to 30 min, and mortality rates were recorded every 10 min. The dose that caused 100% mortality in 30 min was considered as the DD, as recommended by the WHO [23]. The DD tests with field populations consisted of 25 females aged 3 to 5 days old gently blown with a Castor aspirator inside the bottles: four bottles coated with the malathion DD and two controls coated with acetone only. Addition tests were conducted applying the WHO recommended DD (50 µg/bottle) [25]. Three independent assays were performed for each population and using both lab-determined and WHO recommended DDs.
Evaluation of pyriproxyfen susceptibility in field populations
First screening with DD
Once DD of the pyriproxyfen was obtained, larvae from each field population (16 replicates of 10 larvae, totaling 160 larvae) were exposed to the IGR DD, while 80 larvae from the same population (8 replicates of 10 larvae) were used as the negative control (ethanol only). In parallel, 80 Rockefeller larvae (8 replicas of 10 larvae) were also exposed to the DD, as the internal control of assay conditions. Only healthy larvae exhibiting normal movement and from the same breeding site were selected for each test. The IGR solutions were prepared from a pyriproxyfen analytical standard (Sigma Pestanal®) pre-dissolved in acetone (Sigma Aldrich®) and further diluted in ethanol (Merck®). Aliquots containing 15 µL of the IGR at a concentration of 100,000 mg/L were prepared and stored at -80°C. These aliquots were then used to prepare 5 mL stock solutions at a concentration of 300 mg/L and were stored in a refrigerator for up to 30 days. A new dilution was prepared on the same day of the tests from these stock solutions, at a final concentration from which 1 mL would result in the desired DD in the 250 mL test cups. Each population was tested four independent times. The EI of each population was established as the means of these four assays. A total of 240 larvae from the evaluated field population (including their replicates) were necessary for each dose-diagnostic test, totaling 960 larvae in the four repetitions performed in different rounds. WHO criteria were applied to classify the populations as susceptible, exhibiting suggested resistance or resistant, when EI were ≥ 98%, between 90 and 97.9% and < 90%, respectively [23].
Resistance Ratio Estimation
Field populations not susceptible to pyriproxyfen (EI < 98%) in DD assays were submitted to a DR assay in order to quantify their resistance levels. Larvae were exposed to a range of 10 concentrations (0.008–0.45 µg/L) in four replicates comprising 10 L3 larvae each and four control replicates using ethanol only. The Rockefeller strain was run in parallel, consisting of four replicates, with larvae exposed to the DD only. Mortality and metamorphosis rates were recorded until the emergence of all adults in the control condition. A total of 440 larvae were evaluated in each DR test, including their replicates, requiring 1,760 larvae from each field population to perform the repetitions of the four different rounds.
The inhibition of 50% and 95% adult emergence (EI50 and EI95) of each population were obtained by a Probit analysis [26]. Resistance ratios were obtained by dividing the EI (50 and 95) of each population by the equivalent EI of the Rockefeller reference strain. Populations were classified as suggested by Mazzarri and Georghiou [27] into low, moderate, or high resistance respectively for RR95 < 5, between 5.0 and 10.0, and > 10.0.
Evaluation of malathion susceptibility in field populations
The Ae. aegypti populations were tested using adult females, three to five days post emergence and not blood-fed, from the F1 or F2 generations. Each test consisted of the exposure of 20 to 25 females per bottle, with four bottles coated on the inside with each DD (the DD evaluated herein and 50 µg/bottle) in addition to two bottles coated on the inside with acetone only as the negative control. The reference Rockefeller strain was run in parallel with two bottles coated with each DD. Mortality rates were recorded every 15 min, and mosquitoes that could not stand were considered dead. Mortality rates for the replicas of each DD were calculated at the diagnosis time (30 min) in each assay. A total of four bioassays were performed for each population, and the final result considered the mean mortality of these bioassays. A total of 1,000 females from each field population were used to carry out four different rounds of these tests, comprising 250 females in each, including replicates.
The DD and DR assays for both the IGR and adulticide compounds were performed under test-insectary conditions, with controlled temperature (26 ± 2ºC) and humidity (70 ± 10%).
Data analysis
The percentages of adult emergence inhibition, lethal doses (LD), their respective confidence intervals (IC 95%) and the population slope were calculated by the Polo-PC software, employing a Probit analysis [26]. Resistance ratios (RR) were obtained by the quotient between the LD of a population by the Rockefeller reference strain values. Maps were constructed using the QGIZ version 2.18.6 and GIMP version 2.10.14 software packages [20, 24].