Characterization of Plasmodium developmental transcriptomes in Anopheles gambiae midgut reveals novel regulators of malaria transmission.

The passage through the mosquito is a major bottleneck for malaria parasite populations and a target of interventions aiming to block disease transmission. Here, we used DNA microarrays to profile the developmental transcriptomes of the rodent malaria parasite Plasmodium berghei in vivo, in the midgut of Anopheles gambiae mosquitoes, from parasite stages in the midgut blood bolus to sporulating oocysts on the basal gut wall. Data analysis identified several distinct transcriptional programmes encompassing genes putatively involved in developmental processes or in interactions with the mosquito. At least two of these programmes are associated with the ookinete development that is linked to mosquito midgut invasion and establishment of infection. Targeted disruption by homologous recombination of two of these genes resulted in mutant parasites exhibiting notable infection phenotypes. GAMER encodes a short polypeptide with granular localization in the gametocyte cytoplasm and shows a highly penetrant loss-of-function phenotype manifested as greatly reduced ookinete numbers, linked to impaired male gamete release. HADO encodes a putative magnesium phosphatase with distinctive cortical localization along the concave ookinete periphery. Disruption of HADO compromises ookinete development leading to significant reduction of oocyst numbers. Our data provide important insights into the molecular framework underpinning Plasmodium development in the mosquito and identifies two genes with important functions at initial stages of parasite development in the mosquito midgut.

Paternal effect of the nuclear formin-like protein MISFIT on Plasmodium development in the mosquito vector.

Malaria parasites must undergo sexual and sporogonic development in mosquitoes before they can infect their vertebrate hosts. We report the discovery and characterization of MISFIT, the first protein with paternal effect on the development of the rodent malaria parasite Plasmodium berghei in Anopheles mosquitoes. MISFIT is expressed in male gametocytes and localizes to the nuclei of male gametocytes, zygotes and ookinetes. Gene disruption results in mutant ookinetes with reduced genome content, microneme defects and altered transcriptional profiles of putative cell cycle regulators, which yet successfully invade the mosquito midgut. However, developmental arrest ensues during the ookinete transformation to oocysts leading to malaria transmission blockade. Genetic crosses between misfit mutant parasites and parasites that are either male or female gamete deficient reveal a strict requirement for a male misfit allele. MISFIT belongs to the family of formin-like proteins, which are known regulators of the dynamic remodeling of actin and microtubule networks. Our data identify the ookinete-to-oocyst transition as a critical cell cycle checkpoint in Plasmodium development and lead us to hypothesize that MISFIT may be a regulator of cell cycle progression. This study offers a new perspective for understanding the male contribution to malaria parasite development in the mosquito vector.

Conserved mosquito/parasite interactions affect development of Plasmodium falciparum in Africa.

In much of sub-Saharan Africa, the mosquito Anopheles gambiae is the main vector of the major human malaria parasite, Plasmodium falciparum. Convenient laboratory studies have identified mosquito genes that affect positively or negatively the developmental cycle of the model rodent parasite, P. berghei. Here, we use transcription profiling and reverse genetics to explore whether five disparate mosquito gene regulators of P. berghei development are also pertinent to A. gambiae/P. falciparum interactions in semi-natural conditions, using field isolates of this parasite and geographically related mosquitoes. We detected broadly similar albeit not identical transcriptional responses of these genes to the two parasite species. Gene silencing established that two genes affect similarly both parasites: infections are hindered by the intracellular local activator of actin cytoskeleton dynamics, WASP, but promoted by the hemolymph lipid transporter, ApoII/I. Since P. berghei is not a natural parasite of A. gambiae, these data suggest that the effects of these genes have not been drastically altered by constant interaction and co-evolution of A. gambiae and P. falciparum; this conclusion allowed us to investigate further the mode of action of these two genes in the laboratory model system using a suite of genetic tools and infection assays. We showed that both genes act at the level of midgut invasion during the parasite's developmental transition from ookinete to oocyst. ApoII/I also affects the early stages of oocyst development. These are the first mosquito genes whose significant effects on P. falciparum field isolates have been established by direct experimentation. Importantly, they validate for semi-field human malaria transmission the concept of parasite antagonists and agonists.

The developmental migration of Plasmodium in mosquitoes.

Migration of the protozoan parasite Plasmodium through the mosquito is a complex and delicate process, the outcome of which determines the success of malaria transmission. The mosquito is not simply the vector of Plasmodium but, in terms of the life cycle, its definitive host: there, the parasite undergoes its sexual development, which results in colonization of the mosquito salivary glands. Two of the parasite's developmental stages in the mosquito, the ookinete and the sporozoite, are invasive and depend on gliding motility to access, penetrate and traverse their host cells. Recent advances in the field have included the identification of numerous Plasmodium molecules that are essential for parasite migration in the mosquito vector.

Functional genomic analysis of midgut epithelial responses in Anopheles during Plasmodium invasion.

BACKGROUND: The malaria parasite Plasmodium must complete a complex developmental life cycle within Anopheles mosquitoes before it can be transmitted into the human host. One day after mosquito infection, motile ookinetes traverse the midgut epithelium and, after exiting to its basal site facing the hemolymph, develop into oocysts. Previously, we have identified hemolymph factors that can antagonize or promote parasite development. RESULTS: We profiled on a genomic scale the transcriptional responses of the A. gambiae midgut to P. berghei and showed that more than 7% of the assessed mosquito transcriptome is differentially regulated during invasion. The profiles suggested that actin- and microtubule-cytoskeleton remodeling is a major response of the epithelium to ookinete penetration. Other responses encompass components of innate immunity, extracellular-matrix remodeling, and apoptosis. RNAi-dependent gene silencing identified both parasite antagonists and agonists among regulators of actin dynamics and revealed that actin polymerization is inhibitory to the invading parasite. Combined transcriptional and reverse-genetic analysis further identified an unexpected dual role of the lipid-trafficking machinery of the hemolymph for both parasite and mosquito-egg development. CONCLUSIONS: We conclude that the determinants of malaria-parasite development in Anopheles include components not only of systemic humoral immunity but also of intracellular, local epithelial reactions. These results provide novel mechanistic insights for understanding malaria transmission in the mosquito vector.

Cell biological analysis of mosquito midgut invasion: the defensive role of the actin-based ookinete hood.

Successful completion of the Plasmodium lifecycle in the mosquito vector is critical for malaria transmission. It has been documented that the fate of Plasmodium in the mosquito ultimately depends on a fine interplay of molecular mosquito factors that act as parasite agonists and antagonists. Here we investigate whether the cellular responses of the invaded midgut epithelium can also determine the parasite fate and development. We show that the parasite hood, an actin-rich structure formed around the ookinete as it exits the epithelium, is a local epithelial defence reaction observed around 60% of invading parasites. The hood co-localizes with WASP, a promoter of actin filament nucleation, suggesting that it is an active reaction of the invaded cell against invading parasites. Importantly, depletion of WASP by RNAi leads to a significant reduction in hood formation, which is consistent with the previously documented role of this gene as a potent parasite antagonist. Indeed, in mosquitoes that are either genetically selected or manipulated by RNAi to be refractory to Plasmodium, most dead parasites exhibit an actin hood. In these mosquitoes, invading ookinetes are killed by lysis or melanization while exiting the midgut epithelium. Silencing WASP in these mosquitoes inhibits the formation of the hood and allows many parasites to develop to oocysts. These data in conjunction with fine microscopic observations suggest that the presence of the hood is linked to ookinete killing through lysis.

Microglia in the developing brain: from immunity to behaviour.

For decades, microglia, the resident macrophages of the brain, have been recognized mostly for their role in several, if not all, pathologies affecting the brain. However, several studies under physiological conditions demonstrate that microglial function is indispensable also in the healthy brain. Indeed, microglia implement key functions already during development, such as the clearance of the huge amount of neurons that are produced in large excess in the embryo and later die of apoptosis. Beside these classical functions, however, novel roles are emerging that strikingly link microglia with higher order brain functions and show that these cells can ultimately influence behaviour. Therefore a detailed understanding of microglia under physiological conditions may open unprecedented perspectives in the prevention and treatment of neuropsychiatric diseases.