Gene drive mosquitoes can aid malaria elimination by retarding Plasmodium sporogonic development

Abstract

Gene drives hold promise for the genetic control of malaria vectors. The development of vector population modification strategies hinges on the availability of effector mechanisms impeding parasite development in transgenic mosquitoes. We augmented a midgut gene of the malaria mosquito Anopheles gambiae to secrete two exogenous antimicrobial peptides, magainin 2 and melittin. This small genetic modification, capable of efficient nonautonomous gene drive, hampers oocyst development in both Plasmodium falciparum and Plasmodium berghei. It delays the release of infectious sporozoites, while it simultaneously reduces the life span of homozygous female transgenic mosquitoes. Modeling the spread of this modification using a large-scale agent-based model of malaria epidemiology reveals that it can break the cycle of disease transmission across a range of transmission intensities.

Fig. 1.. Generation of gene drive effector strains expressing AMPs.

(A) Schematic showing the design and integration strategy of the effector cassette coding for magainin 2 and melittin at the endogenous loci Gam1 and CP. AMP integration is targeted to the C terminus of Gam1 and the N terminus of CP, respectively. The gRNA target sequences (red) or gRNA module (red circle) is indicated, including the protospacer adjacent motif (bold) and the stop and start codons (underlines). Coding sequences (CDS) and signal peptides (CDS-SP) are indicated by light shading. Endogenous secretion signals of the A. gambiae Cecropin 1 and 2 genes (Cec1 SP and Cec2 SP) and ribosomal skipping signals (P2A and T2A) are indicated. Half arrows indicate primer binding sites for genomic PCR and RT-PCR. (B) PCR on genomic DNA (gDNA) of 15 pooled homozygous Gam1-MM, MM-CP, or wild-type (WT) individuals. (C) RT-PCR of midguts from WT, Gam1-MM or MM-CP mosquitoes that were either non–blood-fed (NBF) or dissected 3 hours post blood feeding (3 hours PBF), respectively. (D) Analysis of cDNA amplicons over the splice site subjected to next-generation sequencing showing the predicted splicing outcomes for strains Gam1-MM and MM-CP.

Fig. 2.. Plasmodium infection experiments.

(A) Schematic overview of Plasmodium infection experiments. (B) P. falciparum oocyst intensity 7 days pi (dpi) in midguts from WT, MM-CP and Gam1-MM mosquitoes dissected. Data from three biological replicates was pooled, and statistical analysis was performed using the Mann-Whitney test. (C) Bright-field images of midguts showing typical oocysts in WT and MM-CP mosquitoes at 7 dpi. (D) Quantification of P. falciparum oocyst diameter in WT and MM-CP mosquitoes 7, 9, and 15 dpi from three pooled biological replicates. Note that many oocysts in WT mosquitoes had ruptured on day 15 pi. Quantification of oocyst diameter (E) and fluorescent imaging (F) of oocysts in WT and MM-CP mosquitoes infected with P. berghei at 14 dpi. Sporozoite prevalence 10 to 16 dpi (G) and infection intensity across all days (H) was measured by qPCR of the P. falciparum Cyt-B gene in dissected heads and thoraces of individual MM-CP and WT mosquitoes (10 to 16 dpi). Only mosquitoes positive for oocyst DNA in the midgut were included in the analysis performed in two biological replicates. Statistical analysis in (D), (E), and (H) was performed by a t test assuming unequal variance. Statistical analysis in (G) was performed using a generalized linear model with a quasibinomial error structure where strain (P = 8.35 × 10⁻⁶) and dpi (P = 7.15 × 10⁻⁴) but not blood meal status (P = 0.0894) were found to be significant coefficients. In all panels, the provision of a supplemental blood meal 4 dpi is indicated by an additional blood drop. **P ≤ 0.01 and ***P ≤ 0.001; ns, not significant.

Fig. 3.. Life history traits and midgut transcriptome of MM-CP mosquitoes.

(A) Fecundity of individual homozygous MM-CP females compared to the WT and corresponding (B) larval hatching rates obtained during the first gonotrophic cycle. Data from three pooled biological replicates are shown. Statistical significance was determined by a t test assuming unequal variance. (C) Pupal sex ratios of MM-CP and WT strains analyzed using the chi-square test for equality. (D) Survival analysis of MM-CP and WT male and female mosquitoes maintained on sugar and (E) of F2 genotyped MM-CP female mosquitoes following backcrossing to the Ifakara strain, intercrossing of F1 mosquitoes, and provision of blood meals. Statistical significance was determined with a Mantel-Cox log rank test. Data from three biological replicates are pooled, and the mean and 95% confidence intervals are plotted. *P ≤ 0.05 and ***P ≤ 0.001. (F) Volcano plots of an RNA sequencing (RNA-seq) experiment performed on midguts dissected before or 6 and 20 hours after blood meal. Differentially expressed genes between MM-CP and WT mosquitoes (P ≤ 0.01) are indicated, and genes belonging to enriched GO groups are highlighted in red.

Fig. 4.. Gene drive and predicted epidemiological impact of strain MM-CP deployment.

(A) Assessment of nonautonomous gene drive in the progeny of male and female hemizygous MM-CP mosquitoes in the presence or absence of a vasa-Cas9 driver crossed to the WT. Larval offspring were subjected to multiplex PCR genotyping, and the mean and standard error (SEM) from three biological replicates is plotted, and the total number n is indicated. Statistical significance was determined using a one-way analysis of variance (ANOVA) with Tukey’s correction. ***P ≤ 0.001. (B) Heatmaps depicting elimination probabilities (top) and number of clinical cases reduced (bottom) at the end of 6 years following a single release of 1000 homozygous MM-CP mosquitoes that also carry a Cas9 IGD. Three transmission scenarios with varying EIRs for P. falciparum as a measure of exposure to infectious mosquitoes were explored. Homozygous transgenic mosquitoes are released 6 months after the start of the simulation in highly seasonal transmission settings of varying intensities. The parameter range that we explored for the reduction of the number of infectious sporozoites is represented on the major y axis, while the range for the average increase in time until sporozoites are released is represented on the major x axis. Parameter range estimates based on the experimental data for strain MM-CP are indicated next to the axes (gray bars).