Here we presented a national wide surveillance for evaluating the susceptibility of Ae. aegypti to the IGR pyriproxyfen and the OP malathion in Brazil, insecticides currently employed by the General Coordination of Arboviruses Surveillance - formerly PNCD. This monitoring was promoted by the Brazilian MoH and was the broadest evaluation ever carried out in a country of continental dimensions, resulting in the evaluation of populations from 132 cities in the time spam of one year, in which a total of 137,280 larvae and 131,000 adults were tested. To the top of our knowledge, it is also globally the largest surveillance round recorded of insecticide resistance monitoring in Ae. aegypti.
We evidenced the feasibility of conducting an insecticide resistance monitoring action in a standardized and strongly coordinated manner, in a model that could be of assistance to the implementation of national monitoring plans in other countries. A systematic literature review covering insecticide resistance data in Ae. aegypti field populations from Latin America showed that less than half of the countries in this continent have published some bioassay data between 2008 and 2018. Also, the number of populations representing each national surveillance was generally rather small [26].
The monitoring of susceptibility to temephos and deltamethrin carried out between 1999 and 2011 by the previous “National Network for Monitoring the Resistance of Ae. aegypti to Insecticides” generally used to evaluate between 25 and 74 populations, in every two years [16]. This time we were able to increase the number of tested populations due to at least three main factors.
Firstly, an increased funding for vector surveillance and control actions with the advent of Zika virus outbreak in 2015 and 2016, reserved the amount of around US$ 501,700 specifically for IR monitoring purposes. This financial resource was enough to supply all collection material to the municipalities, to temporarily hire laboratory technicians, to cover laboratory expenses with mosquito rearing and bioassays, to organize one workshop with participation of representatives from the 26 Brazilian States and to produce didactic material with instructions for collections. This awareness and training of at least two health agents of each State was crucial for a homogeneous sampling, maintenance and registration of the paddles with eggs, and a correct shipment to the central laboratory, according to standardized procedures. This task was not trivial, especially in a country such as Brazil, whose geographic dimensions and organization complexity are enormous.
In addition to the presential meeting with State representatives, an instructional video was made available on the institutional webpages of the MoH and IOC/Fiocruz [27]. Overall, these aspects contributed for the success of egg collection (96% of the selected cities collected eggs appropriately), from which 94% of the samples were good enough to perform the bioassays.
The third aspect that made it possible to evaluate this high number of populations was that we employed diagnostic-dose tests for a screening of insecticide resistant populations. These assays require a smaller number of insects reared, space and time spent in colonies maintenance and tests execution, compared to dose-response tests. We are aware that in order to obtain a robust profile of a population, dose-response assays are more informative, since by performing a quantitative test it is possible to inform about the resistance ratios and the homogeneity of tested populations. However, as the last official data on IR monitoring occurred in 2013, and there was no record about pyriproxyfen and malathion carried out on a large scale throughout the country, it was preferred to obtain at least the qualitative status of susceptibility/resistance to the current used insecticides in a broader territorial distribution as possible. Population here classified as resistant to pyriproxyfen, were finely investigated with dose-response tests to assess larvicide resistance [24]. In the case of malathion, where two DDs were employed, at least ten populations were not considered as susceptible even when exposed to the higher dose. This suggests that these populations might present the higher levels of resistance among the 132 evaluated Ae. aegypti populations. In the Brazilian Aedes control program routine, the OP larvicide temephos was alternatively replaced by the benzo-phenyl urea (BPU) chitin synthesis inhibitors diflubenzuron (wettable powder 25%) and novaluron (emulsifiable concentrate 10%) between 2009 and 2014, after almost 30 years of use (starting in the 1980`s). In an attempt to reduce selective pressure on BPUs, the Brazilian MoH introduced another class IGR, with a mode of action distinct from chitin synthesis inhibitors: the juvenile hormone analogue pyriproxyfen, applied at potable water reservoir tanks at 0.01 mg of active ingredient/liter [28]. The amount of pyriproxyfen and malathion distributed by the Brazilian MoH specifically for Ae. aegypti control between 2014 and 2019 are listed in supplementary file “Additional file 1: Table S1”.
Out of all Ae. aegypti populations herein evaluated, 99.3% were classified as susceptible to the IGR pyriproxyfen. The six resistant populations were from the same geographic region (Northeast), in the States of Bahia (Itabuna, Brumado, Serrinha) and Ceará (Quixadá, Icó, Juazeiro do Norte), suggesting the emergence of resistance to pyriproxyfen, with a regionalized distribution. Interestingly some of these populations exhibited discrepant RR50 and RR95 values in some populations, suggesting a heterogeneous response within the population, as represented by their low slope values of their dose-response Probit analyses. These populations are likely experiencing an initial process of selection where only few individuals are resistant so far. The pyriproxyfen RRs were low, when compared to those previously reported for temephos: Itabuna reported RR95 of 18.6 and 55.8 in 2004 and 2013, respectively; Serrinha had 254.9 in 2012 and Juazeiro do Norte showed RR95 of 10.4 in 2003 and 17.5 in 2004 [29, 3]. This regionalization should be related with differences in operational applications and quantity of insecticides used, but also with peculiarities in the genetic background of populations. Likewise, Ae. aegypti populations from the Northeast presented the highest levels of resistance to temephos in Brazil [8]. On the other hand, this same region presented the lowest levels of resistance to pyrethroids [8], accompanied by the lowest frequencies of kdr alleles [30].
Here we evidenced that the lowest concentration of malathion that killed 100% of Rockefeller females in 30 min was 20 µg/bottle, dose 2.5 times lower than that recommended by WHO in bottle assays (50 µg) [24]. We did not observe any populations resistant to malathion (mortalities less than 90%) when the WHO DD 50 µg/bottle was employed, while 73 populations (55.8% of the total evaluated) was classified as resistant under 20 µg/bottle exposition. The WHO-suggested DD is based on tests performed in reference laboratories and estimated from a variety of susceptible strains for resistance detection, seeking ease of testing and reliability. This DD should be considered as a guide that can be refined for the local situation whenever it is possible [31]. The local DD was more sensitive in early discriminating resistant individuals. This has an interesting approach in the sense of identifying decrease in susceptibility before it has reached levels that may mean loss of effectiveness of the insecticide in the field. The resistance monitoring program in Brazil seeks to detect changes in susceptibility early so that the product used can be changed in a timely manner. The early detection also permits management approaches in time that resistance is not so high that would be of difficult reversal.
The meaning of the laboratory observed resistance in correlation with effectiveness of the product under field conditions should be studied. Studies conducted two decades ago had already reported resistance of Ae. aegypti to malathion in Northeastern Brazilian populations, when OP was used to control both the larval phase (temephos) and the adult vector (malathion) [16].
The election of insecticides against Ae. aegypti in Brazil followed criteria established by the WHO, also indicating that change of the product should occur in places with a high RR (> 10.0) and with confirmed lack of efficacy in simulated field tests [10]. However, the substitution of an insecticide takes an average of two years to happen [2], since it depends on series of bureaucratic processes. Therefore, the time spent between the first detection of resistance in laboratory bioassays and the effective change of the compound in the field was not effective for precluding insecticide resistance to spread. In order to avoid reduction in the effectiveness of the insecticides in the field, a most sensitive criterion for its replacement was adopted since 2006. Management has started to be indicated in localities where mosquito populations presented mortality rates below 70% in DD assays or with RR95 > 3.0 [10]. Results obtained in São Paulo State were the basis of this arrangement, where simulated field trials with temephos and PY`s, demonstrated failures in the control of Ae. aegypti in populations with RR > 5.0, and acceptable with RR < 3.0. PY`s were ineffective in simulated field trials against populations with mortality rates below 70% to the DD in laboratory bioassays [32].
Most of the Ae. aegypti populations from Latin America tested for DDT showed resistance to this compound (86.7%). High frequencies of resistant populations were also observed for temephos and deltamethrin (75.7 and 33%, respectively). These patterns could be explained by the long and frequent use of these insecticides in the continent [26]. Considering that the pyriproxyfen larvicide has been used on a large scale in Brazil for more than six years, independently of the resistance status the necessity of discontinuity of its use is already evident, according to the insecticide rotation strategy adopted in this country.
Other larvicides allowed for use in water for human consumption with WHO pre-qualification are, in addition to the OP temephos, the IGRs novaluron and diflubenzuron, the biological Bacillus thuringiensis, a formulation of B. thuringiensis associated with B. sphaericus, and spinosad [33]. It is also worth considering the possibility of the return of temephos use since it has not been applied for about 11 years, and there are indications of a reduction in resistance in some locations evaluated [8].
In relation to adulticides, the situation is alarming, since there is just one alternative to PY`s and to the OP malathion, the association of prallethrin with imidacloprid [33]. In the most recent national evaluation for PY`s (2011 and 2012) high RRs to deltamethrin were indicated throughout the country [7]. Also, in São Paulo, localities with higher numbers of dengue incidence were those with higher levels of resistance to PY`s, although these compounds were no longer being applied by governmental campaigns against Ae. aegypti. This was correlated with the excessive use of insecticides by household, especially during arbovirus epidemic seasons and with PY`sapplication against other urban vectors, as it was the case in a locality where there was an intense campaign against vectors of Leishmania [34]. Herein we showed resistance to malathion in most of the populations evaluated with the 20 µg/bottle DD. Therefore, the chemical control against Ae. aegypti is crucially threatened in most of the country, as long as no other alternative compound is available.
Emerging resistance to all the main classes of neurotoxic insecticide (CA`s, OC`s, OP`s and PY`s) has been detected in Ae. aegypti from Americas, Africa, and Asia [29]. The occurrence of resistance to the IGR, most recently adopted class of insecticides, reinforces the importance of using integrated tools that can contribute to reducing the need for chemical vector control, modifying the determinants of arbovirus transmission in a sustainable manner, such as environmental management and education [35]. Consequently, lower use of chemical insecticides reduces the risk of factors associated such as ecological imbalance, outbreak of secondary pests, harmful effects to human health and to other non-target animals [36].
In line with the Vector Integrated Management Strategy, biological control acts on target species through the use of their natural predators or parasites, in the most target-specific manner [37]. The Bacillus thuringiensis var israelensis (Bti) is for long being referred as a promising Ae. aegypti larvicide alternative to neurotoxic compounds. However, the large-scale production of Bti under formulations sufficiently persistent in environmental conditions, especially in containers exposed to sunlight, is an important limitation [38, 39]. This avoids Bti to be a strategic option to be adopted in the routine of Ae. aegypti control in Brazil at a national scale. Even if implemented, biological control must be used rationally just as it is required for chemical insecticides. Despite the believed unlike emergence of resistance to Bti due to its multiple modes of toxicity, some mechanisms of resistance to this compound were described in laboratory selected strains [29, 40].
Still seeking to reduce the need for chemical vector control, national and local campaigns of vector control have to reinforce measures to improve sanitary infrastructure and to strengthen community engagement in the destruction or correct treatment of possible larval breading sites. In parallel, new alternative methods of vector surveillance and control endorsed by WHO should be implemented and monitored, considering regional peculiarities, as is the case of Wolbachia, Sterile insect technique SIT, Release of insects with dominant lethality (RIDL), toxic sugar baits, autodissemination of IGRs, spacial repellents [1, 2, 41].
Finally, an alert should be made about the high frequency of populations with the presence of Ae. albopictus mosquitoes (59.8%). Our collections were performed in the grounds of houses at urban territory, evidencing the high expansion of that species in the country, since its first record in 1986 in rural areas [42]. Further studies are recommended to better understand the role of Ae. albopictus in the transmission of arboviruses in Brazil. In parallel, the monitoring of insecticide resistance of Ae. aegypti should also consider Ae. albopictus populations.