Graphical AbstractHighlights d Human malaria parasites interact non-competitively with their mosquito vectors d Mosquito hormone signaling co-regulates egg and parasite development d Parasites use host lipids for their growth via a mosquito lipid transporter d Parasites respond to mosquito metabolism with consequences for vector controlThe development of the malaria-causing parasite Plasmodium falciparum depends on its ability to exploit the sexual cycle of its mosquito host in a noncompetitive manner. SUMMARYTransmission of malaria parasites occurs when a female Anopheles mosquito feeds on an infected host to acquire nutrients for egg development. How parasites are affected by oogenetic processes, principally orchestrated by the steroid hormone 20hydroxyecdysone (20E), remains largely unknown.Here we show that Plasmodium falciparum development is intimately but not competitively linked to processes shaping Anopheles gambiae reproduction. We unveil a 20E-mediated positive correlation between egg and oocyst numbers; impairing oogenesis by multiple 20E manipulations decreases parasite intensities. These manipulations, however, accelerate Plasmodium growth rates, allowing sporozoites to become infectious sooner. Parasites exploit mosquito lipids for faster growth, but they do so without further affecting egg development. These results suggest that P. falciparum has adopted a non-competitive evolutionary strategy of resource exploitation to optimize transmission while minimizing fitness costs to its mosquito vector. Our findings have profound implications for currently proposed control strategies aimed at suppressing mosquito populations.

Conversion from asexual proliferation to sexual differentiation initiates the production of the gametocyte, which is the malaria parasite stage required for human-to-mosquito transmission. This protocol describes an assay designed to probe the effect of drugs or other perturbations on asexual replication, sexual conversion and early gametocyte development in the major human malaria parasite Plasmodium falciparum. Synchronized asexually replicating parasites are induced for gametocyte production by the addition of conditioned medium, and they are then exposed to the treatment of interest during sexual commitment or at any subsequent stage of early gametocyte development. Flow cytometry is used to measure asexual proliferation and gametocyte production via DNA dye staining and the gametocyte-specific expression of a fluorescent protein, respectively. This screening approach may be used to identify and evaluate potential transmission-blocking compounds and to further investigate the mechanism of sexual conversion in malaria parasites. The full protocol can be completed in 11 d.

Many mosquito species, including the major malaria vector Anopheles gambiae, naturally undergo multiple reproductive cycles of blood feeding, egg development and egg laying in their lifespan. Such complex mosquito behavior is regularly overlooked when mosquitoes are experimentally infected with malaria parasites, limiting our ability to accurately describe potential effects on transmission. Here, we examine how Plasmodium falciparum development and transmission potential is impacted when infected mosquitoes feed an additional time. We measured P. falciparum oocyst size and performed sporozoite time course analyses to determine the parasite’s extrinsic incubation period (EIP), i.e. the time required by parasites to reach infectious sporozoite stages, in An. gambiae females blood fed either once or twice. An additional blood feed at 3 days post infection drastically accelerates oocyst growth rates, causing earlier sporozoite accumulation in the salivary glands, thereby shortening the EIP (reduction of 2.3 ± 0.4 days). Moreover, parasite growth is further accelerated in transgenic mosquitoes with reduced reproductive capacity, which mimic genetic modifications currently proposed in population suppression gene drives. We incorporate our shortened EIP values into a measure of transmission potential, the basic reproduction number R0, and find the average R0 is higher (range: 10.1%–12.1% increase) across sub-Saharan Africa than when using traditional EIP measurements. These data suggest that malaria elimination may be substantially more challenging and that younger mosquitoes or those with reduced reproductive ability may provide a larger contribution to infection than currently believed. Our findings have profound implications for current and future mosquito control interventions.

Anopheles coluzzii females, important malaria vectors in Africa, mate only once in their lifetime. Mating occurs in aerial swarms with a high male-to-female ratio, where traits underlying male mating success are largely unknown. Here, we investigated whether cuticular hydrocarbons (CHCs) influence mating success in natural mating swarms in Burkina Faso. As insecticides are widely used in this area for malaria control, we also determined whether CHCs affect insecticide resistance levels. We find that mated males have higher CHC abundance than unmated controls, suggesting CHCs could be determinants of mating success. Additionally, mated males have higher insecticide resistance under pyrethroid challenge, and we show a link between resistance intensity and CHC abundance. Taken together, our results suggest that CHC abundance may be subject to sexual selection in addition to selection by insecticide pressure. This has implications for insecticide resistance management, as these traits may be sustained in the population due to their benefits in mating even in the absence of insecticides.