Disrupting Mosquito Reproduction and Parasite Development for Malaria Control

Abstract

The control of mosquito populations with insecticide treated bed nets and indoor residual sprays remains the cornerstone of malaria reduction and elimination programs. In light of widespread insecticide resistance in mosquitoes, however, alternative strategies for reducing transmission by the mosquito vector are urgently needed, including the identification of safe compounds that affect vectorial capacity via mechanisms that differ from fast-acting insecticides. Here, we show that compounds targeting steroid hormone signaling disrupt multiple biological processes that are key to the ability of mosquitoes to transmit malaria. When an agonist of the steroid hormone 20-hydroxyecdysone (20E) is applied to Anopheles gambiae females, which are the dominant malaria mosquito vector in Sub Saharan Africa, it substantially shortens lifespan, prevents insemination and egg production, and significantly blocks Plasmodium falciparum development, three components that are crucial to malaria transmission. Modeling the impact of these effects on Anopheles population dynamics and Plasmodium transmission predicts that disrupting steroid hormone signaling using 20E agonists would affect malaria transmission to a similar extent as insecticides. Manipulating 20E pathways therefore provides a powerful new approach to tackle malaria transmission by the mosquito vector, particularly in areas affected by the spread of insecticide resistance.

Fig 1. Disrupting steroid hormone signaling by topical application of the 20E agonist methoxyfenozide (DBH) affects oviposition, mating, longevity and P. falciparum development.

(A) DBH-treated mated females failed to oviposit or laid a significantly lower number of eggs after a blood meal in a dose-response manner; whereas oviposition occurred in 100% of controls (Kruskal-Wallis test, p < 0.0001). Letters indicate post hoc significance (Dunn’s post hoc test). (B) The ability of virgin DBH-treated females to become inseminated was significantly reduced compared to control females (Fisher’s exact test for 2 μg, 0.5 μg, and 0.125 μg DBH: p < 0.001, p < 0.001, p = 0.024) (C) The median survival time of DBH-treated females was significantly lower than control-treated females (Log-rank test for 2 μg, 0.5 μg, and 0.125 μg DBH: p < 0.0001, p = 0.0044 and p = 0.0141, Median survival: 2 μg, 0.5 μg, and 0.125 μg DBH = 11,14, and 16d; Control = 19d). Data are presented as the percentage of survival of 4 replicates with Standard Error. Arrow indicates day of DBH application. (D) Females treated with 2 μg or 0.5 μg DBH showed an 87% and 56% reduction in P. falciparum infection prevalence (measured as number of females with oocysts 7 days post-infectious blood meal compared to controls), respectively (Fisher’s exact test for 2 μg and 0.5 μg DBH: p < 0.0001 and p < 0.0001). The number (n) of females analyzed is indicated in each panel.

Fig 2. Schematic of mosquito life cycle model.

Mosquitoes progress through the egg, larval, and pupal stages. Upon emergence, all mosquitoes rest outdoors for one day before mating first (50%) or feeding as virgins (50%). Some virgin feeders may subsequently mate, or, if exposed to and affected by DBH, may never mate. Afterwards, regardless of mating, all mosquitoes participate in up to 6 gonotrophic cycles, each cycle consisting of feeding (one day), resting indoors (two days), and ovipositing (one day). In our model, we assume that mosquitoes are exposed to DBH or insecticide only during their first feed (yellow circle) if applied via bed nets or only during their first indoor rest (blue circles) if applied via indoor spraying, and that mosquitoes exposed to DBH or insecticide via bed nets are nonetheless able to feed. Note that for indoor spraying, the bottom-most compartments are not used since in our model exposure always occurs after all mosquitoes have mated. In all gonotrophic cycles, DBH's effect on egg development reduces the number of eggs laid (no eggs are laid in the case of virgin mosquitoes). DBH also changes the age-dependent mortality starting on the day of first exposure and lasting throughout the mosquito's life. Insecticide increases mortality as well, but only on the day of exposure.

Fig 3. Effect of bed net-based interventions on the adult female mosquito population and individual age classes.

(A) The adult female mosquito population varies non-linearly under increasing coverage with different DBH doses (pink lines) or insecticide efficacy (blue lines). The vertical yellow bar indicates 85% coverage, which results in the largest female adult population and for which the age composition of the population is considered in (B). The population size shown is relative to the total female population in the absence of any interventions (black line). (B) The age composition of female mosquitoes in the presence of 2.0 μg DBH (pink) or the absence of any intervention (gray) at 85% coverage, indicated by a yellow bar in (A) and (C). The highlighted days in the x-axis indicate the age range of mosquitoes that are old enough to transmit malaria if infected. (C) The potentially infectious adult mosquito population under increasing levels of coverage with different DBH doses (pink lines) or insecticide efficacy (blue lines). This includes females at least 12 days after a blood meal, and in the case of DBH exposure, a proportion of these females are excluded due to reduced Plasmodium susceptibility. The yellow bar indicates 85% coverage, for which the age composition of the population is considered in (B). Without intervention, the proportion of mosquitoes at least 12 days after their first feed (black line) is 0.22. Insecticide of 60%, 80%, or 100% efficacy and DBH of experimentally determined efficacy (Fig 1) are used.

Fig 4. Effect of interventions on malaria under varying coverage.

Effectiveness against malaria considering changes in both coverage (x-axis) and efficacy (line style). In low (A), moderate (B), and high (C) transmission settings, the effectiveness (reduction in malaria relative to pre-intervention prevalence) of both DBH and insecticides increases as efficacy and coverage increase. DBH applied at 2 μg (solid pink line) had effectiveness greater than or comparable to 100% insecticide efficacy (solid blue line), while 0.5 μg (dashed pink line) and 0.125 μg DBH (dotted pink line) had effectiveness similar to that of 80% (dashed blue line) and 60% (dotted blue line) insecticide efficacy, respectively. The maximal DBH efficacy considered is our experimentally determined effects on egg development, mating, mortality, and Plasmodium susceptibility (Fig 1).