Exposing Anopheles mosquitoes to antimalarials blocks Plasmodium parasite transmission

Abstract

Bites of Anopheles mosquitoes transmit Plasmodium falciparum parasites that cause malaria, which kills hundreds of thousands of people every year. Since the turn of this century, efforts to prevent the transmission of these parasites via the mass distribution of insecticide-treated bed nets have been extremely successful, and have led to an unprecedented reduction in deaths from malaria¹. However, resistance to insecticides has become widespread in Anopheles populations^2-4, which has led to the threat of a global resurgence of malaria and makes the generation of effective tools for controlling this disease an urgent public health priority. Here we show that the development of P. falciparum can be rapidly and completely blocked when female Anopheles gambiae mosquitoes take up low concentrations of specific antimalarials from treated surfaces-conditions that simulate contact with a bed net. Mosquito exposure to atovaquone before, or shortly after, P. falciparum infection causes full parasite arrest in the midgut, and prevents transmission of infection. Similar transmission-blocking effects are achieved using other cytochrome b inhibitors, which demonstrates that parasite mitochondrial function is a suitable target for killing parasites. Incorporating these effects into a model of malaria transmission dynamics predicts that impregnating mosquito nets with Plasmodium inhibitors would substantially mitigate the global health effects of insecticide resistance. This study identifies a powerful strategy for blocking Plasmodium transmission by female Anopheles mosquitoes, which has promising implications for efforts to eradicate malaria.

Extended Data Figure 1:. Effects of ATQ exposure on survival and post blood-feeding egg production in An. gambiae females.

a) ATQ exposure has no effect on the acute or long-term survival of An. gambiae females (2-sided Log-Rank Mantel-Cox, n = 189, df = 1, χ² = 0.00, p = 0.9951). The sigmoidal fit used for subsequent modeling is shown. b) The production of eggs after an infections blood meal is unaffected by ATQ exposure (2-sided, unpaired Student’s t, n = 75, df = 1, t = 0.826, p = 0.4115). Means and 95% CI of the mean are indicated. Where relevant, statistical significance is indicated as so: ns = not significant, * = p < 0.05, ** = p < 0.01, *** = < 0.001, **** = p < 0.0001; n indicates the number of biologically independent mosquito samples.

Extended Data Figure 2:. Model Structure and Population Parameters.

(a) Schematic representation of the mosquito life cycle model with the time step of one day. Mosquitoes spend three days as eggs (E_i), ten days as larvae (L_i), which includes the pupal stage. Adult female mosquito compartments fall within the dashed box and begin with a rest day (R₀) followed by mating (M) or feeding (F). After feeding, females undergo two days of rest (R_i) followed by a day for egg laying (EL). Then the cycle repeats. Shaded boxes denote when exposure to insecticide or ATQ could occur. These are the same compartments were mosquitoes can become infected or transmit infections, assuming they have been infected for a period longer than the incubation time. (b) Survival of the mosquito population as a function of age. The curve is a Gompertz distribution with scale parameter b = 0.1868 and shape parameter η = 0.0293. (c) Functions relating human and mosquito infection levels with risk of infection. (i) The risk of a human becoming infected, β_H, as a function of the number of infectious feeders, f. (ii) The risk of a mosquito becoming infected, β_M, as a function of the fraction of the human population that is infected, I_H.

Extended Data Figure 3:. Sensitivity of model results to variation in prevalence, coverage and insecticide resistance.

The graphs show the enhanced effectiveness of insecticide combined with ATQ (relative to insecticide alone) in reducing human prevalence under varying levels of coverage (across panels), prevalence (along x-axis), coverage and insecticide resistance (bar color). The enhanced effectiveness of the interventions is defined as (the quantity of human prevalence with only insecticide - human prevalence with insecticide and ATQ over human prevalence with only insecticide) and is represented by positive values when the addition of ATQ is beneficial. Prevalence is quantified after ten years of simulation. The coverage is varied from 20%−80% (upper left panel 20%; upper right panel 20%; lower left panel 60%; and lower right panel 80%). In each panel, the position of the bars determines the malaria prevalence under no intervention, from 20–80%. The bar color represents insecticide resistance levels (dark green 0%; green 20%; light green 40%; yellow 60%; orange 80%; and red 100%). In the complete absence of insecticide resistance all mosquitoes that contact insecticide are killed, and thus, all dark green bars equal zero.

Extended Data Figure 4:. Malaria transmission model predicting the effects of adding ATQ to insecticide-treated nets in additional malaria prevalence settings.

The heat maps show changes in malaria transmission for bed net-like interventions using insecticide alone or insecticide plus an ATQ-like compound, relative to no intervention at varying coverage and varying insecticide resistance levels. The model considers both (a) 20% and (b) 70% prevalence of malaria. The effectiveness of the interventions is defined as (1 - proportion reduction in malaria transmission relative to no intervention) and is represented as colors ranging from yellow (no change in malaria transmission) to dark blue (elimination of malaria transmission) at varying levels of coverage (x-axis) and insecticide resistance (y-axis). Insecticide resistance is the percentage of mosquitoes that are impervious to insecticide. Coverage is the probability of a mosquito encountering an intervention during a single feeding episode. The model output demonstrates that addition of ATQ significantly increases the ability of an LLIN-like intervention to reduce and even eliminate malaria transmission.

Extended Data Figure 5:. Testing additional compounds for fitness costs and transmission blocking activity through tarsal contact.

(a) Mosquito survival relative to an untreated control after 48 h following exposure to ATQ, DEC, PYR, HYD, ACE and permethrin (PER). The proportion of female An. gambiae surviving exposure to each compound (1 mmol/m², 60 minutes) relative to the proportion of individuals surviving exposure to an untreated control is shown. PER exposure causes almost complete mortality (proportionate survival relative to controls = 0.055, Pairwise, 2-sided Chi² w/ Bonferroni correction, n = 80, df = 1, χ² = 76.10, p < 0.0001), while all other compounds behave comparably to controls. (b) Neither PYR nor DEC (1 mmol/m², 6 min) are capable of reducing the prevalence P. falciparum through tarsal contact, relative to controls (Pairwise Chi² w/ Bonferroni correction, DEC: n= 93, df = 1, χ² = 2.42, p = 0.12. PYR: n = 92, df = 1, χ² = 0.55, p = 0.46). Similarly, DEC and PYR had no impact on the intensity of infection, compared to a mock-treated control (Wilcoxon with Dunn’s post hoc, n = 183, df = 3, p = 0.31 (DEC) and p = 0.99 (PYR)). Letters indicate groups that are statistically different from one another. Statistical significance is indicated as **** = p < 0.0001. Medians are indicated; n denotes the number of biologically independent mosquito samples.

Extended Data Figure 6:. ATQ exposure via a netting substrate completely inhibits P. falciparum development.

An. gambiae females were allowed to rest for 60 min on 100 denier polyester netting that had been treated with either a 0.5 mg/ml (0.05% w/v) solution of ATQ in acetone or acetone alone. Females exposed to ATQ in this way failed to become infected after an infectious P. falciparum blood meal, demonstrating that a netting substrate is also capable of delivering sufficiently high doses of ATQ to inhibit infection (2-sided Chi², n = 98, df = 1, χ² = 75.55, p < 0.0001). Medians are indicated; n denotes the number of biologically independent mosquito samples.

Figure 1:. An. gambiae exposure to atovaquone (ATQ) aborts P. falciparum development.

(a) P. falciparum parasites are completely eliminated (0% oocyst intensity, and 0% prevalence of infection, shown in the pie charts) in females exposed to 1 mmol/m² ATQ for 60 minutes immediately prior to infection (Prevalence: Two-sided Chi², n = 166, df = 1, χ² = 155.14, p < 0.0001). The exposure method is shown in the graphic: green represents ATQ coated onto a glass surface. (b) Dose-dependent inhibition (range: 100 μmol/m² - 100 nmol/m²) of P. falciparum infection by exposure to ATQ. Significant reductions in prevalence and intensity were observed at doses as low as 1 μmol/m² (Prevalence: Two-sided Chi². 100 μmol/m²: n = 118, df = 1, χ² = 95.42, p < 0.0001. 10 μmol/m²: n = 239, df = 1, χ² = 117.6, p < 0.0001. 1 μmol/m²: n = 139, df = 1, χ² = 9.85, p = 0.0017. Intensity: Two-sided Mann-Whitney: 10 μmol/m²: n = 239, df = 1, U = 287.5, p = 0.0004. 1 μmol/m²: n = 139, df = 1, U = 686, p = 0.0104). (c) Dose-response curve fit for ATQ exposure (Non-linear regression, n = 13, df = 12, Sum of Squares = 1003, R² = 0.9441). The IC₅₀ for ATQ pre-infection exposure, calculated by interpolation, is indicated. Mean inhibition relative to control prevalence is indicated. Error bars are 95% CI. Dashed portions of the sigmoidal fit are estimated. In all panels where relevant, statistical significance is indicated as so: ns = not significant, * = p < 0.05, ** = p < 0.01, *** = < 0.001, **** = p < 0.0001. Medians are indicated. For (a) and (b), n indicates the number of biologically independent mosquito samples. For (c), n indicates the relative inhibition observed in ATQ-treated mosquitoes in independent experiments.

Figure 2:. The transmission blocking activity of ATQ is maintained at shorter exposure times and at time points of exposure before and after infection.

(a) P. falciparum parasites are completely eliminated (0% oocyst intensity, and 0% prevalence of infection, shown in the pie charts) in females exposed to either 1 mmol/m² or 100 μmol/m² ATQ for 6 min (Prevalence: Two-sided Chi². 1 mmol/m²: n = 113, df = 1, χ² = 91.00, p < 0.0001. 100 μmol/m²: n = 102, df = 1, χ² = 80.59, p < 0.0001). At 10 μmol/m², prevalence of infection (10 μmol/m²: n = 149, df = 1, χ² = 55.58, p < 0.0001) and median oocyst intensity (2-sided Mann-Whitney, n = 149, df = 1, U = 258, p = 0.0349) are significantly reduced in the ATQ-treated group. Medians are indicated. (b) IFAs of mosquito midgut lumens 21 h post P. falciparum infection using parasite-specific antibodies (anti-PfS25, green) and DNA (DAPI, blue) staining. Example images from 14 independent mosquito midgut samples (7 control, 7 ATQ-treated); P. falciparum forms are shown. Left panel: mature ookinete in controls. Right panel: zygote (asterisk) and retort forms (white arrows) in ATQ-treated females. ATQ-treated females have few ookinetes (1.2% total parasites) and a large proportion of zygotes (88.5% total parasites), indicating parasite arrest, while controls contain a significantly larger proportion of normal ookinetes (40.1%, Nominal Logistic Regression, n = 5091, df = 14, χ² = 1620.88, p < 0.0001). Scale bar: 10 μm. (c, d) P. falciparum parasites are completely eliminated also when females are exposed to ATQ (1 mmol/m², 6 min) either (c) 24 h prior (2-sided Chi² w/Bonferroni correction, n= 152, df = 1, χ² = 116.74, p < 0.0001) or (d) 12 h after (2-sided Chi² w/ Bonferroni correction, n = 141, df = 1, χ² = 75.11, p < 0.0001) an infectious blood meal. Medians are indicated. Where relevant, statistical significance is indicated as so: * = p < 0.05, ** = p < 0.01, *** = < 0.001, **** = p < 0.0001. For (a), (c) and (d), n indicates the number of biologically independent mosquito samples. For (b), n indicates the number of independent parasite forms.

Figure 3:. Malaria transmission model predicts that adding ATQ to insecticide-treated nets would increase bed net effectiveness.

(a) Heat maps of changes in malaria transmission for bed net-like interventions using insecticide alone or insecticide plus an ATQ-like compound, relative to no intervention at varying coverage and varying insecticide resistance levels. The model considers an intermediate 45% prevalence of human infection (effects at lower and higher malaria prevalence are described in Extended Fig. 4). The “effectiveness” of the interventions is defined as (1 - proportion reduction in malaria transmission relative to no intervention) and is represented as colors ranging from yellow (no change in malaria transmission) to dark blue (elimination of malaria transmission) at varying levels of coverage (x-axis) and insecticide resistance (y-axis). Insecticide resistance is the percentage of mosquitoes that are impervious to insecticide; coverage is the probability of a mosquito encountering an intervention during a single feeding episode. (b) Predicted effects of adding ATQ to existing insecticide-treated nets in 567 African locations with available insecticide resistance data (indicated by black dots on the map of Africa). For each location, the model considers the estimated bed net coverage and P. falciparum prevalence in 2–10 year old children reported in 2015, and insecticide resistance levels reported between 2013 and 2018. The graphs show mean malaria prevalence for insecticide/ATQ combination bed nets (INS+ATQ), relative to insecticide only bed nets (dotted line at y = 1), for sampled sites in West and East Africa (red boxes, n = 186 and n = 381, respectively). Error bars represent one standard deviation from the mean prevalence. In both (a) and (b), the model outputs demonstrate that addition of ATQ substantially increases the ability of treated nets to reduce malaria transmission across a broad range of transmission settings.

Figure 4:. Other cytochrome B inhibitors have P. falciparum transmission-blocking activity.

An. gambiae females exposed to 1 mmol/m² of the arthropod cytochrome B inhibitors acequinocyl (ACE) and hydramethylnon (HYD), as well as ATQ, for 6 minutes show strongly reduced prevalence (pie charts) of P. falciparum relative to controls (Pairwise, 2-sided Chi² w/ Bonferroni correction: ATQ: n = 141, df = 1, χ² = 75.11, p < 0.0001. HYD: n = 132, df = 1, χ² = 23.85, p < 0.0001. ACE: n = 141, df = 1, χ² = 26.00, p < 0.0001). HYD and ACE had no impact on the intensity of infection (Wilcoxon with Dunn’s post hoc, n = 282, df = 3, HYD: p = 0.99, ACE: p = 0.19). Letters indicate groups that are statistically different from one another **** = p < 0.0001; n indicates the number of biologically independent mosquito samples.