We present a detailed analysis of the interactions between Anopheles stephensi midgut epithelial cells and Plasmodium berghei ookinetes during invasion of the mosquito by the parasite. In this mosquito, P.berghei ookinetes invade polarized columnar epithelial cells with microvilli, which do not express high levels of vesicular ATPase. The invaded cells are damaged, protrude towards the midgut lumen and suffer other characteristic changes, including induction of nitric oxide synthase (NOS) expression, a substantial loss of microvilli and genomic DNA fragmentation. Our results indicate that the parasite in¯icts extensive damage leading to subsequent death of the invaded cell. Ookinetes were found to be remarkably plastic, to secrete a subtilisin-like serine protease and the GPI-anchored surface protein Pbs21 into the cytoplasm of invaded cells, and to be capable of extensive lateral movement between cells. The epithelial damage in¯icted is repaired ef®ciently by an actin pursestring-mediated restitution mechanism, which allows the epithelium to`bud off' the damaged cells without losing its integrity. A new model, the time bomb theory of ookinete invasion, is proposed and its implications are discussed.

Malaria transmission depends on the competence of some Anopheles mosquitoes to sustain Plasmodium development (susceptibility). A genetically selected refractory strain of Anopheles gambiae blocks Plasmodium development, melanizing, and encapsulating the parasite in a reaction that begins with tyrosine oxidation, and involves three quantitative trait loci. Morphological and microarray mRNA expression analysis suggest that the refractory and susceptible strains have broad physiological differences, which are related to the production and detoxification of reactive oxygen species. Physiological studies corroborate that the refractory strain is in a chronic state of oxidative stress, which is exacerbated by blood feeding, resulting in increased steady-state levels of reactive oxygen species, which favor melanization of parasites as well as Sephadex beads.T he natural transmission cycle of the malaria parasite, Plasmodium, requires completion of a complex developmental cycle in the midgut and salivary glands of the Anopheles mosquito vector (1). However, after its entry with the blood meal, the parasite encounters the innate immune responses of the mosquito, which are often robust and coincide with major parasite losses (2-4). At the extreme, the vector is refractory and completely blocks transmission of the parasite. Genetically selected susceptible and refractory strains (4A r͞r and L3-5; henceforth S and R, respectively) have been described in the African mosquito, Anopheles gambiae. The R strain blocks parasite development in the midgut, oxidatively converting tyrosine to melanin, which crosslinks proteins into a capsule assembled around the parasite (5, 6). Melanotic encapsulation largely depends on three quantitative trait mosquito loci; the Plasmodium encapsulation genes Pen1, Pen2, and Pen3 that have been mapped (7), although not yet identified with particular sequences. The major locus, Pen1, has also been associated with the ability of the R strain to melanize CM-Sephadex beads (8).When the malaria ookinete passes the refractory mosquito midgut, the melanotic capsule first appears, and is significantly thicker, on the ookinete's apical end facing the hemolymph (6). This observation indicates that key components of the melanization reaction derive from the hemolymph. In a histological and ultrastructural survey of the R and S strains, we noted pronounced differences in their pericardial cells. These are scavenging nephrocytes, which are present alongside the dorsal vessel and that harbor numerous peroxisomes, catalase-rich organelles that are active in detoxification and neutralization of reactive oxygen species (ROS). The pericardial cells of S mosquitoes contain numerous peroxisomes, including some very large ones (Fig. 1A), whereas the cells of the R strain possess significantly fewer and smaller peroxisomes (Fig. 1B). These morphological differences suggested that the refractory phenotype may result from a systemic deficiency in ROS detoxification.We have previously used cDNA microarrays to explore the ...

SummaryDespite the fact that glycine-rich RNA-binding proteins (GRPs) have been implicated in the responses of plants to environmental stresses, their physiological functions and mechanisms of action in stress responses remain largely unknown. Here, we assessed the functional roles of GRP7, one of the eight GRP family members in Arabidopsis thaliana, on seed germination, seedling growth, and stress tolerance under high salinity, drought, or cold stress conditions. The transgenic Arabidopsis plants overexpressing GRP7 under the control of the cauliflower mosaic virus 35S promoter displayed retarded germination and poorer seedling growth compared with the wild-type plants and T-DNA insertional mutant lines under high salinity or dehydration stress conditions. By contrast, GRP7 overexpression conferred freezing tolerance in Arabidopsis plants. GRP7 is expressed abundantly in the guard cells, and has been shown to influence the opening and closing of the stomata, in accordance with the prevailing stress conditions. GRP7 is localized to both the nucleus and the cytoplasm, and is involved in the export of mRNAs from the nucleus to the cytoplasm under cold stress conditions. Collectively, these results provide compelling evidence that GRP7 affects the growth and stress tolerance of Arabidopsis plants under high salt and dehydration stress conditions, and also confers freezing tolerance, particularly via the regulation of stomatal opening and closing in the guard cells.