Caspar Controls Resistance to Plasmodium falciparum in Diverse Anopheline Species

Immune responses mounted by the malaria vector Anopheles gambiae are largely regulated by the Toll and Imd (immune deficiency) pathways via the NF-kappaB transcription factors Rel1 and Rel2, which are controlled by the negative regulators Cactus and Caspar, respectively. Rel1- and Rel2-dependent transcription in A. gambiae has been shown to be particularly critical to the mosquito's ability to manage infection with the rodent malaria parasite Plasmodium berghei. Using RNA interference to deplete the negative regulators of these pathways, we found that Rel2 controls resistance of A. gambiae to the human malaria parasite Plasmodium falciparum, whereas Rel 1 activation reduced infection levels. The universal relevance of this defense system across Anopheles species was established by showing that caspar silencing also prevents the development of P. falciparum in the major malaria vectors of Asia and South America, A. stephensi and A. albimanus, respectively. Parallel studies suggest that while Imd pathway activation is most effective against P. falciparum, the Toll pathway is most efficient against P. berghei, highlighting a significant discrepancy between the human pathogen and its rodent model. High throughput gene expression analyses identified a plethora of genes regulated by the activation of the two Rel factors and revealed that the Toll pathway played a more diverse role in mosquito biology than the Imd pathway, which was more immunity-specific. Further analyses of key anti-Plasmodium factors suggest they may be responsible for the Imd pathway–mediated resistance phenotype. Additionally, we found that the fitness cost caused by Rel2 activation through caspar gene silencing was undetectable in sugar-fed, blood-fed, and P. falciparum-infected female A. gambiae, while activation of the Toll pathway's Rel1 had a major impact. This study describes for the first time a single gene that influences an immune mechanism that is able to abort development of P. falciparum in Anopheline species. Further, this study addresses aspects of the molecular, evolutionary, and physiological consequences of the observed phenotype. These findings have implications for malaria control since broad-spectrum immune activation in diverse anopheline species offers a viable and strategic approach to develop novel malaria control methods worldwide.
Author Summary The relationship between malaria parasites and the mosquitoes that transmit them to humans comprises complex molecular interactions including mosquito immune responses. Anopheles can mount potent anti-Plasmodium immune responses; we show here that the gene caspar, which encodes a negative regulator of the immune signaling pathway Imd, controls mosquito resistance to the human malaria parasite. Silencing of this Imd pathway regulator results in complete resistance to human Plasmodium in three divergent Anopheline malaria vector species, yet does not cause complete resistance to a rodent Plasmodium species, indicating conservation of defense function among mosquito but not diverse parasite species. We also identify a panel of genes that are transcriptionally regulated by silencing of the caspar gene. Some of these genes contribute directly to parasite resistance. Finally, we show that the transient immune activation that renders mosquitoes resistant to the human malaria parasite has little to no effect on mosquito fitness as a measure of survival or fecundity under laboratory conditions. In sum, this study shows that the mosquito's immune pathway, Imd, can regulate resistance to Plasmodium through immune responses that entail several known anti-Plasmodium genes.