Malaria remains an important public health problem worldwide affecting mainly underdeveloped and developing countries in Africa, Asia and Latin America. The World Health Organization (WHO) estimated that 214 million cases of malaria occurred worldwide in 2015 [1]. Malaria elimination and eradication are present themes on WHO’s agenda for infectious diseases [2, 3]. Research institutes and policy makers have made great efforts worldwide in order to achieve significant reduction in malaria incidences, with the ambitious long-term aim of global eradication [4–7]. Approaches designed to progress towards malaria elimination must rely on the detection and treatment of infections, rather than fever, and comprise the concomitant use of different tools concerning health surveillance improvement through technologies, applying transmission blocking by development of vaccines, high sensibility new generation rapid tests, insecticides and drugs that can, among other features, circumvent the resistance issue [8].

Strategies focused on mass screening and treatment (MSAT) and variations of it, such as focused screening and treatment (FSAT) and reactive case detection (RCD), sometimes are described as success cases, but these strategies depend on several factors that can drive for failure, such as logistics, public health policies, population coverage, and even diagnostic tool sensitivity [9]. Likewise, mass drug administration (MDA) using artemisinin-based combination therapy (ACT) has been shown to be an effective strategy, as well as MSAT, for high-incidence scenarios. However, issues like community acceptance and drug resistance increasing are still relevant concerns [9]. These are potential control measures that can be improved by integration with effective vector control interventions. Extensive use of long lasting impregnated nets and indoor residual spray has led to a change in the vector comportment from indoor to outdoor feeding and resting behaviour [10, 11]. This shift brings a new challenge to target outdoor malaria transmission in a sustainable way in order to achieve elimination [12].

Ivermectin has proven to be effective against a range of Anopheles species [13–16]. Ivermectin can impact four of five vectorial capacity variables, including daily probability of adult mosquito survivorship, daily probability a mosquito feeds on a human, vector competence, and vector density in relation to the host [17–20]. Treating hosts with a systemic insecticide, such as ivermectin, could circumvent the issue of outdoor transmission, as it would target the vector regardless of feeding habit location and time [17]. In addition of having an excellent safety profile in humans, ivermectin has proven to be effective against a range of other neglected diseases, such as filariasis and helminthiasis [21]. Furthermore, the drug presents features in agreement with some of the malaria eradication research agenda (malERA) initiative recommendations, such as reducing adult mosquito survival rates, shifting age structure, reducing the proportion of older females, and targeting outdoor feeding and resting [6]. Moreover, if livestock are treated with ivermectin for malaria control, then this is coherent with the One Health concept since it acts against livestock parasites, improving both economic output and nutrient availability [22].

Ivermectin MDA, even when a single round is applied, reduces the survivorship of mosquitoes, shifts the mosquito population age structure, and decreases sporozoite rate [23]. Modelling suggests that adding ivermectin as an adjunct during ACT MDA could reduce malaria transmission and significantly reduce the number of MDAs and time to elimination [24]. Ivermectin has been used in MDA in Latin America for onchocerciasis control [25] and this infection has been eliminated in four of the six endemic countries. This illustrates that ivermectin MDA can be effectively implemented in Latin America for disease elimination. Indigenous populations are currently under ivermectin MDA intervention for onchocerciasis control in the Brazil-Venezuela border [26]. Variations in the mosquitocidal effect between anopheline species [27] and blood meals [28] make essential local studies regarding these features that directly affect the timing of ivermectin administration, a crucial parameter to form a useful addition to anti-malarial drugs [29].

Anopheles aquasalis seems to play an important role in malaria transmission in coastal regions of Latin America. Infection rates due to Plasmodium vivax were previously reported ranging from 0.5 to 1.7 %, both in outdoor and indoor resting mosquitoes in Venezuela [30], and in Brazil the infection rate was estimated 1.18 % [31]. Since mosquito colonies have been established, the species has been used as a model for assessing vector-parasite interactions [32]. Anopheles aquasalis has been described as presenting variable feeding behaviour, both anthropophilic and zoophilic [30, 31]. It was also designated as a widely distributed and abundant species [32, 33], being reported both at Atlantic and Pacific coasts, from Central America to southern Brazil [32]. It has been demonstrated that the species has both indoor and outdoor feeding and resting behaviour as well [27, 34, 35]. Furthermore, An. aquasalis has been described as zoophilic species in Amazon region [30, 36]. Such features allow to classify the species as of great importance for the Latin America.

Even though much evidence has been generated regarding ivermectin effects on malaria transmission, some questions remain unanswered regarding its effects on the vector’s biology [21, 37]. Here the ivermectin effects on the survivorship and reproductive fitness of the American malaria vector An. aquasalis were assessed. The differences of ivermectin effect on mosquito survivorship using membrane feeding assay (MFA) and direct feeding assay (DFA) from drug-treated volunteers were also evaluated.

Malaria elimination is an ambitious objective that has now been seriously considered and embraced both by the public health community and scientists worldwide. In this scenario, ivermectin has appeared as a potential complementary tool for elimination as it effectively targets outdoor transmission, has a novel mechanism of action that might bypass occurrence of resistance and could utilize implementation mechanisms that are already functional because of efforts to control other diseases, such as onchocerciasis and lymphatic filariasis [21]. Moreover, the drug has been reported to reduce vectorial capacity for Plasmodium transmission, both by reducing mosquito survival and possibly inhibiting Plasmodium falciparum sporogony [38, 39]. Even so, and despite recent discoveries, little is known about the effects of the drug on the biology of different vectors, especially from Latin America [27].

In this study, the effects of ivermectin on An. aquasalis survivorship and reproduction are showed for the first time. Ivermectin was shown to increase mortality and reduce reproductive capacity of An. aquasalis. The An. aquasalis ivermectin lethal concentrations (LC₅₀ = 47.03 ng/ml, LC₂₅ = 31.92 ng/ml, LC₅ = 18.28 ng/ml) are higher than calculated previously for Anopheles gambiae [17, 20] but still within human relevant range following oral treatment with 150–200 μg/kg [39, 40]. It must be noted that the methods used here and firstly used by Kobilinsky et al. [17] for LC estimates differ from others since an in vitro mixing of drug and blood was used instead of blood from treated subjects and this method could be influencing the higher LC values found here. Because single doses of 200 μg/kg can only keep blood concentrations compatible with this lethal concentrations for a short period, using higher or repeated doses or slow release formulations of ivermectin should be considered as a feasible strategy. These data allow to infer that ivermectin treatment of humans should impart a lethal effect on An. aquasalis.

In vivo data revealed that mosquitoes fed on volunteer blood containing ivermectin (200 µg/kg) at 4 h, 2, 4, 7, and 14 days post drug ingestion significantly reduced survivorship compared to those fed on untreated control individual blood. These findings are similar to Foley et al. [15] which showed survivorship reduction for Anopheles farauti until 14 days post ivermectin ingestion (250 μg/kg) by DFA. Ivermectin seems to have great affinity for adipose tissue. Strongly lipid binding may cause its slow release, thereby increasing its persistence in the body, as suggested previously [41, 42, 43]. This phenomenon may explain why mosquito lethal effects were observed as late as 14 days post drug ingestion. Increasing the dose of ivermectin would likely impart a greater effect against An. aquasalis for a longer period of time.

Ivermectin sub-lethal effects on the reproductive fitness of Anopheles mosquitoes were first reported by Gardner et al. [44] in Anopheles quadrimaculatus specimens fed canine blood containing ivermectin. Two studies indicate that ivermectin treatment of cattle reduces mosquito fecundity for Anopheles coluzzii [45] and An. gambiae s.s. [46]. A complete inhibition of An. gambiae fecundity when mosquitoes fed on human blood 24 h post treatment with a 150–200 µg/kg dosage was shown by Derua et al. [47]. The findings support and extend studies since was demonstrated that ivermectin effects on eggs/female proportion, eggs hatchability and even on pupae/larvae proportion under a low concentration dosage. Additionally it should be appreciated that human pharmacokinetic may differ from those in animals, as in the first three studies, domesticated animals were injected with doses varying from 6 to 600 µg/kg. These findings reinforce the hypothesis that even sub-lethal doses of ivermectin could play an important role on altering the vectorial capacity.

Studies conducted on ivermectin effects over mosquitoes are usually carried out through MFA [19, 42, 44]. As described previously, since ivermectin is lipophilic, it usually binds to fatty tissue where it may lead to higher concentrations in different compartments. This feature, in turn, led to believe that mosquitoes fed by DFA on sub-dermal capillaries may ingest higher ivermectin concentrations than mosquitoes fed by MFA with venous blood, imparting a greater mosquito lethal effect, as suggested by Chaccour et al. [27]. Here was also showed significant differences between MFA and DFA HRs (1.54 [1.406–1.684] p < 0.001) adjusting for volunteer gender. Although the limited number of volunteers (3 males and 3 females) may be a limitation for the study, these are exciting findings since previous results obtained from MFA studies may be underestimates of the real effects that occur during direct feeding after ivermectin MDA during a malaria elimination campaign. Regression model also revealed an increased risk for mosquitoes feeding on women volunteers independent of the blood feeding assay in accordance with a recent study reporting a greater availability of ivermectin in female human and in higher body mass indices volunteers [42]. Since only one single time point (4 h post-ingestion) was evaluated, additional studies must be carried out in order to assess these differences in later time-points where the effect of ivermectin decreases.

Ivermectin has proven to be effective against a range of malaria vectors worldwide. The drug affects many aspects of both vector biology and its vectorial capacity as well. Considering the diversity of environment in the Amazon region and consequently of entomological and epidemiological scenarios, malaria elimination campaigns in Amazon must resort to concomitant multiple strategies. Here a gap of knowledge regarding ivermectin effects on an important Amazon vector species, An. aquasalis was filled. It was demonstrated that ivermectin impacts mosquito survivorship for up to 14 days post-ingestion and has a deleterious effect on the vector reproductive fitness. Significant difference between MFA and DFA was found and no difference concerning blood meal volume comparing MFA and DFA was shown. Considering the findings, malaria elimination strategies in the Amazon could benefit from having ivermectin as an additional tool, which would readily complement the effect of the use of drugs for population treatment, or other vector control mechanism. Since outdoor transmission in Amazon has a relevant contribution to the overall malaria transmission and the ivermectin way of action influences this, the drug would likely have an impact on the incidence of disease in the region. Furthermore, since An. aquasalis is incriminated both as zoophilic and anthropophilic, has a widespread distribution and is implicated in malaria transmission as well, it seems to be feasible the deployment of strategies focused on cattle and/or human treatment. Future investigation concerning ivermectin effects on other important Amazonian species, such as Anopheles darlingi and Anopheles albitarsis, should be assessed prior to widespread adoption of ivermectin as a malaria elimination tool in the Amazon.