Herein we showed that Ae. aegypti populations from Roraima State, Brazil collected in 2016 and 2018 were resistant to the pyrethroid deltamethrin and under the process of becoming resistant to the organophosphate malathion. Remarkably, only Ae. aegypti was present in these collections, regardless of a recent register of Ae. albopictus in a rural area of Rorainopolis.
Pyrethroid resistance in Ae. aegypti from Roraima State had already been high in previous evaluations. The first registers were in 2007 and 2010 when the capital Boa Vista was detected as pyrethroid resistant [4, 17]. In 2011, Pacaraima presented the second highest resistance ratio (RR95 = 60.3) in the whole country [7]. Herein we demonstrate that even two years after pyrethroid governmental application was substituted by malathion, pyrethroid resistance has been maintained in the four localities evaluated. It is noteworthy that a new diagnostic dose was established for deltamethrin (0.03%) in WHO paper tests [13]. In this study we adopted a higher dosage (0.05%) signifying that the rate of mortality would probably be even lower if tested with 0.03% deltamethrin papers.
The Brazilian MoH started replacing pyrethroids with the organophosphate malathion in ultra-low volume-based applications against Ae. aegypti in 2010 [7] although pyrethroids are still used in campaigns against other vectors such as anophelines, phlebotomines and tritomines. In Roraima State the first stock of malathion was received in December 2015. Hereing we declare that all four Ae. aegypti populations collected in 2016, although not 100% killed by malathion 0.7%, had mortality above 90%, then not classified as resistant. The rate of mortality decreased to under 90% in Bonfim and Pacaraima two years afterwards, the mosquitoes therefore being classified as resistant to malathion. The diagnostic dose for malathion indicated by WHO is a bit higher, 0.8% [13]. Had we used 0.8% instead of 0.7%, the decrease in the mortality levels from 2016 to 2018 might have also been noticeable.
Different from that which occurs with pyrethroids, the active ingredient of household sprays and other governmental campaigns, the only probable source of organophosphate pressure was the governmental campaigns with malathion at that moment. The larvicide temephos has in theory not been applied in Roraima since 2013, when it was definitively substituted by IGRs [7]. The most recent data about temephos resistance in Roraima indicated resistance ratios (RR50) of 2.0 in Boa Vista (2010) and 4.3 in Pacaraima (2011), which were not considered high levels of resistance [4, 17]. Anyway, we cannot reject the possibility that mechanisms prior selected by temephos and pyrethroids are inducing cross resistance to malathion, as reported in some classical studies. For instance, a laboratory strain of Culex quinquefasciatus selected for temephos resistance in larvae developed cross resistance to several organophosphate adulticides, including malathion [18]. On the other hand, in Guadeloupe and Saint Martin Caribbean islands, Ae. aegypti populations developed high levels of resistance to temephos (8.9 to 33.1-fold) but low levels to malathion (1.7 to 4.4-fold) [19]
Concerning the possible mechanisms selected for insecticide resistance, alterations in the activity of GST and Esterase enzymes were detected in Boa Vista (2007) and Pacaraima (2011). Reduced activity of the Acetylcholinesterase enzyme was also observed in Pacaraima (2011) [7]. Lineages of Ae. aegypti Brazilian populations that acquired resistance to malathion through selection pressure in the laboratory exhibited increased activity of GST, multi-function Oxidases (MFO P450) and Estereses, as determined by bichoemical analyses [20]. Over expression of genes related to metabolic resistance was detected in Ae. aegypti populations from the Caribbean, such as French Guyana and French West Indies islands [19, 21]. On the other hand, those high levels of resistance to pyrethroids in Roraima might be partially justified by the absence of the wild-type NaVS haplotype, already reported in 2010 and 2011 in Boa Vista and Pacaraima [8]. The wild-type NaVS haplotype is still missing with predominance of the double kdr NaVR2 (1016Ile + 1534Cys), except in Rorainópolis, where the NaVR1 (1016Val + 1534Cys) predominates. We corroborated that NaVR2 leads to higher levels of resistance to pyrethroids [14] once homozygote R2R2 insects were only present among the survivors in the bioassays with Boa Vista and Bonfim. The remaining high levels of resistance to pyrethroids even after the substitution by malathion in Roraima may be associated with the high prevalence of domestic use of insecticides, all composed of pyrethroids and easily acquired in local markets, as reported in other Brazilian states [16, 22]. In addition, we cannot neglect the migration of Ae. aegypti resistant populations from the neighboring countries and measures adopted against other vector borne diseases. On the border between Pacaraima (Brazil) and Santa Helena (Venezuela) there is an intense control of malaria where pyrethroids are employed against Anopheles even in the urban area, thus also submitting Ae. aegypti to this selection pressure. In Bonfim, it is common to find pyrethroid sprays acquired in Lethen City, on the Guyanese side of the border.
We speculate that the kdr NaVR2 haplotype in Ae. aegypti populations in this region must have migrated mostly from Venezuela as it has been detected in Roraima State at least since 2010, when it either absent or at low frequencies in the neighboring States, Amazonas and Pará [8]. Interestingly, Rorainópolis is the only municipality in Roraima with Ae. albopictus colonization, however encountered in areas on the border with Amazonas State where the species has been recorded since 2015 (CGVS-SESAU-RR). Therefore, it seems that the transition between the biomes, Amazon Forest in the south and the savannah-like “Lavrado" in the north, has been limiting the dispersion of Ae. aegypti from Roraima downwards to Amazonas State. On the other hand, Ae. albopictus from Amazonas has avoided invasion of the capital Boa Vista in the Lavrado zone.
Besides new promising tools of Ae. aegypti control currently being tested in Brazil, such as Wolbachia, RIDL (Release of Insects carrying a Dominant Lethal gene) and pyriproxyfen autodissemination stations [23] [24, 25, 26], insecticides continue to play an important role on a long term basis in order to rapidly decrease the densities of a target population and consequently, mitigate the cycle of the arboviruses. Therefore, it is of prime importance that a constant surveillance of insecticide resistance be inherent to the chemical control strategy so as to guide authorities about product efficacy.