Fz2 and Cdc42 Mediate Melanization and Actin Polymerization but Are Dispensable for Plasmodium Killing in the Mosquito Midgut

Abstract

The midgut epithelium of the mosquito malaria vector Anopheles is a hostile environment for Plasmodium, with most parasites succumbing to host defenses. This study addresses morphological and ultrastructural features associated with Plasmodium berghei ookinete invasion in Anopheles gambiae midguts to define the sites and possible mechanisms of parasite killing. We show by transmission electron microscopy and immunofluorescence that the majority of ookinetes are killed in the extracellular space. Dead or dying ookinetes are surrounded by a polymerized actin zone formed within the basal cytoplasm of adjacent host epithelial cells. In refractory strain mosquitoes, we found that formation of this zone is strongly linked to prophenoloxidase activation leading to melanization. Furthermore, we identify two factors controlling both phenomena: the transmembrane receptor frizzled-2 and the guanosine triphosphate–binding protein cell division cycle 42. However, the disruption of actin polymerization and melanization by double-stranded RNA inhibition did not affect ookinete survival. Our results separate the mechanisms of parasite killing from subsequent reactions manifested by actin polymerization and prophenoloxidase activation in the A. gambiae–P. berghei model. These latter processes are reminiscent of wound healing in other organisms, and we propose that they represent a form of wound-healing response directed towards a moribund ookinete, which is perceived as damaged tissue. A dangerous journey awaits malaria Plasmodium parasites ingested by a mosquito. Most parasites are destroyed by host responses in the midgut, and in parasite-resistant refractory strains of mosquito the mortality can reach 100%. This midgut “bottleneck” represents an appealing target for reducing malaria transmission by the genetic control of wild mosquitoes. However, the killing mechanisms are still unclear. In this study, electron microscopical analyses followed the entire midgut invasion process in mosquitoes to identify the major site(s) and ultrastructural features of Plasmodium killing. The authors found that invasion can be divided into two steps: a swift passage through a midgut cell, followed by establishment of the parasite in the basal extracellular space, where it becomes an important target for destruction by soluble immunity factors. In refractory mosquitoes, dead parasites are associated with the formation of organelle-free zones of actin in adjacent midgut cells, and melanin deposition on the parasite surface. The authors identify two genes, called frizzled-2 and cell division cycle 42, that control these phenomena. Actin zone formation and melanization are generally thought to be killing mechanisms; however, the authors show by gene silencing that neither is lethal to Plasmodium. Instead, these mechanisms may represent a form of mosquito wound-healing response that is triggered by the presence of a moribund parasite.