The parasite invasion marker SRPN6 reduces sporozoite numbers in salivary glands of Anopheles gambiae

For malaria transmission to occur, Plasmodium sporozoites must infect the salivary glands of their mosquito vectors. This study reports that Anopheles gambiae SRPN6 participates in a local salivary gland epithelial response against the rodent malaria parasite, Plasmodium berghei . We showed previously that SRPN6, an immune inducible midgut invasion marker, influences ookinete development. Here we report that SRPN6 is also specifically induced in salivary glands with the onset of sporozoite invasion. The protein is located in the basal region of epithelial cells in proximity to invading sporozoites. Knockdown of SRPN6 during the late phase of sporogony by RNAi has no effect on oocyst rupture but significantly increases the number of sporozoites present in salivary glands. Despite several differences between the passage of Plasmodium through the midgut and the salivary glands, this study identifies a striking overlap in the molecular responses of these two epithelia to parasite invasion.     

Anopheles gambiae SRPN2 facilitates midgut invasion by the malaria parasite Plasmodium berghei

We report on a phylogenetic and functional analysis of genes encoding three mosquito serpins (SRPN1, SRPN2 and SRPN3), which resemble known inhibitors of prophenoloxidase-activating enzymes in other insects. Following RNA interference induction by double-stranded RNA injection, knockdown of SRPN2 in adult Anopheles gambiae produced a notable phenotype: the appearance of melanotic pseudotumours, which increased in size and number with time, indicating spontaneous melanization and association with an observed lifespan reduction. Furthermore, knockdown of SRPN2 strongly interfered with the invasion of A. gambiae midguts by the rodent malaria parasite Plasmodium berghei. It did not affect ookinete formation, but markedly reduced oocyst numbers, by 97%, as a result of increased ookinete lysis and melanization.     

Morphological evidence for proliferative regeneration of the Anopheles stephensi midgut epithelium following Plasmodium falciparum ookinete invasion

Ookinetes are motile invasive stages of the malaria parasite that enter the midgut epithelium of the mosquito vector via an intracellular route. Ookinetes often migrate through multiple adjacent midgut epithelial cells, which subsequently undergo apoptosis/necrosis and are extruded from the midgut epithelium into the midgut lumen. Hundreds of ookinetes may simultaneously invade the midgut epithelium, causing destruction of an appreciable proportion of the total number of midgut epithelial cells. However, there is little evidence that ookinete invasion of the midgut epithelium per se is detrimental to the survival of the mosquito vector implying that efficient mechanisms exist to restore the damaged midgut epithelium following malaria parasite infection. Proliferation and differentiation of precursor stem cells could replace the midgut epithelial cells destroyed and lost as a consequence of ookinete invasion. Although the existence of so-called "regenerative" cells within the mosquito midgut epithelium has long been recognized, there has been no previously published evidence for proliferation/differentiation of these putative precursor midgut epithelial cells in mature adult female mosquitoes. In the current study, examination of Giemsa-stained histological sections from Anopheles stephensi mosquito midguts infected with the human malaria parasite Plasmodium falciparum provided morphological evidence that regenerative cells undergo division and subsequent differentiation into normal columnar midgut epithelial cells. Furthermore, the number of these putatively proliferating/differentiating regenerative cells was significantly higher in P. falciparum-infected compared to uninfected mosquitoes, and was positively correlated with both the level of malaria parasite infection and midgut epithelial cell destruction. The loss of invaded midgut epithelial cells associated with intracellular migration by ookinetes, therefore, appears to trigger, and to be compensated by, proliferative regeneration of the mosquito midgut epithelium.     

Morphological evidence for proliferative regeneration of the Anopheles stephensi midgut epithelium following Plasmodium falciparum ookinete invasion

Ookinetes are motile invasive stages of the malaria parasite that enter the midgut epithelium of the mosquito vector via an intracellular route. Ookinetes often migrate through multiple adjacent midgut epithelial cells, which subsequently undergo apoptosis/necrosis and are extruded from the midgut epithelium into the midgut lumen. Hundreds of ookinetes may simultaneously invade the midgut epithelium, causing destruction of an appreciable proportion of the total number of midgut epithelial cells. However, there is little evidence that ookinete invasion of the midgut epithelium per se is detrimental to the survival of the mosquito vector implying that efficient mechanisms exist to restore the damaged midgut epithelium following malaria parasite infection. Proliferation and differentiation of precursor stem cells could replace the midgut epithelial cells destroyed and lost as a consequence of ookinete invasion. Although the existence of so-called “regenerative” cells within the mosquito midgut epithelium has long been recognized, there has been no previously published evidence for proliferation/differentiation of these putative precursor midgut epithelial cells in mature adult female mosquitoes. In the current study, examination of Giemsa-stained histological sections from Anopheles stephensi mosquito midguts infected with the human malaria parasite Plasmodium falciparum provided morphological evidence that regenerative cells undergo division and subsequent differentiation into normal columnar midgut epithelial cells. Furthermore, the number of these putatively proliferating/differentiating regenerative cells was significantly higher in P. falciparum-infected compared to uninfected mosquitoes, and was positively correlated with both the level of malaria parasite infection and midgut epithelial cell destruction. The loss of invaded midgut epithelial cells associated with intracellular migration by ookinetes, therefore, appears to trigger, and to be compensated by, proliferative regeneration of the mosquito midgut epithelium.     

Plasmodium berghei: plasmodium perforin-like protein 5 is required for mosquito midgut invasion in Anopheles stephensi

During its life cycle the malarial parasite Plasmodium forms three invasive stages which have to invade different and specific cells for replication to ensue. Invasion is vital to parasite survival and consequently proteins responsible for invasion are considered to be candidate vaccine/drug targets. Plasmodium perforin-like proteins (PPLPs) have been implicated in invasion because they contain a predicted pore-forming domain. Ookinetes express three PPLPs, and one of them (PPLP3) has previously been shown to be essential for mosquito midgut invasion. In this study we show through phenotypic analysis of loss-of-function mutants that PPLP5 is equally essential for mosquito infection. Deltapplp5 ookinetes cannot invade midgut epithelial cells, but subsequent parasite development is rescued if the midgut is bypassed by injection of ookinetes into the hemocoel. The indistinguishable phenotypes of Deltapplp5 and Deltapplp3 ookinetes strongly suggest that these two proteins contribute to a common process.     

Infection-Induced Interaction between the Mosquito Circulatory and Immune Systems

Insects counter infection with innate immune responses that rely on cells called hemocytes. Hemocytes exist in association with the insect''s open circulatory system and this mode of existence has likely influenced the organization and control of anti-pathogen immune responses. Previous studies reported that pathogens in the mosquito body cavity (hemocoel) accumulate on the surface of the heart. Using novel cell staining, microdissection and intravital imaging techniques, we investigated the mechanism of pathogen accumulation in the pericardium of the malaria mosquito, Anopheles gambiae, and discovered a novel insect immune tissue, herein named periostial hemocytes, that sequesters pathogens as they flow with the hemolymph. Specifically, we show that there are two types of endocytic cells that flank the heart: periostial hemocytes and pericardial cells. Resident periostial hemocytes engage in the rapid phagocytosis of pathogens, and during the course of a bacterial or Plasmodium infection, circulating hemocytes migrate to the periostial regions where they bind the cardiac musculature and each other, and continue the phagocytosis of invaders. Periostial hemocyte aggregation occurs in a time- and infection dose-dependent manner, and once this immune process is triggered, the number of periostial hemocytes remains elevated for the lifetime of the mosquito. Finally, the soluble immune elicitors peptidoglycan and β-1,3-glucan also induce periostial hemocyte aggregation, indicating that this is a generalized and basal immune response that is induced by diverse immune stimuli. These data describe a novel insect cellular immune response that fundamentally relies on the physiological interaction between the insect circulatory and immune systems.     

The invasion of the midgut of the mosquito Culex (Culex) quinquefasciatus Say, 1823 by the helminth Litomosoides chagasfilhoi Moraes Neto, Lanfredi and De Souza, 1997

The Litomosoides chagasfilhoi helminth was studied as a model for microfilaria invasion of the midgut of Culex quinquefasciatus mosquito, vector of Wuchereria bancrofti helminth, causative agent of the human filariasis. Histology and transmission and scanning electron microscopy were utilized to show the topography of mosquito midgut invasion by the helminth. An analysis of midguts dissected at different time points after a blood meal demonstrated that the microfilariae interacted and crossed the peritrophic matrix and the midgut epithelium of C. quinquefasciatus. The microfilariae invaded preferentially the mosquito abdominal midgut and the invasion process occurred between 2 and 3h after the blood feeding. In some cases, microfilariae caused an opening in the midgut that separated the epithelial cells, while in others cases, the worms caused the detachment of cells from the epithelium. Ultimately, L. chagasfilhoi crossing activity appeared to damage the midgut. It was also observed that the microfilariae lost their sheaths during their passage through the fibrous material of the peritrophic matrix, before they reached the midgut epithelium. Since the exsheathment process is necessary for the continuity of larvae development, it seems that the passage through the peritrophic matrix is an important step for the parasite's life cycle. This experimental model revealed details of the interaction process of helminthes within the vector midgut, contributing to the knowledge of factors involved in the vector competence of C. quinquefasciatus as a vector of filariasis.     

The transmembrane isoform of Plasmodium falciparum MAEBL is essential for the invasion of Anopheles salivary glands

Malaria transmission depends on infective stages in the mosquito salivary glands. Plasmodium sporozoites that mature in midgut oocysts must traverse the hemocoel and invade the mosquito salivary glands in a process thought to be mediated by parasite ligands. MAEBL, a homologue of the transmembrane EBP ligands essential in merozoite invasion, is expressed abundantly in midgut sporozoites. Alternative splicing generates different MAEBL isoforms and so it is unclear what form is functionally essential. To identify the MAEBL isoform required for P. falciparum (NF54) sporozoite invasion of salivary glands, we created knockout and allelic replacements each carrying CDS of a single MAEBL isoform. Only the transmembrane form of MAEBL is essential and is the first P. falciparum ligand validated as essential for invasion of Anopheles salivary glands. MAEBL is the first P. falciparum ligand experimentally determined to be essential for this important step in the life cycle where the vector becomes infectious for transmitting sporozoites to people. With an increasing emphasis on advancing vector-based transgenic methods for suppression of malaria, it is important that this type of study, using modern molecular genetic tools, is done with the agent of the human disease. Understanding what P. falciparum sporozoite ligands are critical for mosquito transmission will help validate targets for vector-based transmission-blocking strategies.     

SOAP,a novel malaria ookinete protein involved in mosquito midgut invasion and oocyst development

An essential, but poorly understood part of malaria transmission by mosquitoes is the development of the ookinetes into the sporozoite-producing oocysts on the mosquito midgut wall. For successful oocyst formation newly formed ookinetes in the midgut lumen must enter, traverse, and exit the midgut epithelium to reach the midgut basal lamina, processes collectively known as midgut invasion. After invasion ookinete-to-oocyst transition must occur, a process believed to require ookinete interactions with basal lamina components. Here, we report on a novel extracellular malaria protein expressed in ookinetes and young oocysts, named secreted ookinete adhesive protein (SOAP). The SOAP gene is highly conserved amongst Plasmodium species and appears to be unique to this genus. It encodes a predicted secreted and soluble protein with a modular structure composed of two unique cysteine-rich domains. Using the rodent malaria parasite Plasmodium berghei we show that SOAP is targeted to the micronemes and forms high molecular mass complexes via disulphide bonds. Moreover, SOAP interacts strongly with mosquito laminin in yeast-two-hybrid assays. Targeted disruption of the SOAP gene gives rise to ookinetes that are markedly impaired in their ability to invade the mosquito midgut and form oocysts. These results identify SOAP as a key molecule for ookinete-to-oocyst differentiation in mosquitoes.     

Plasmodium activates the innate immune response of Anopheles gambiae mosquitoes.

Innate immune-related gene expression in the major disease vector mosquito Anopheles gambiae has been analyzed following infection by the malaria parasite, Plasmodium berghei. Substantially increased levels of mRNAs encoding the antibacterial peptide defensin and a putative Gram-negative bacteria-binding protein (GNBP) are observed 20-30 h after ingestion of an infected blood-meal, at a time which indicates that this induction is a response to parasite invasion of the midgut epithelium. The induction is dependent upon the ingestion of infective, sexual-stage parasites, and is not due to opportunistic co-penetration of resident gut micro-organisms into the hemocoel. The response is activated following infection both locally (in the midgut) and systemically (in remaining tissues, presumably fat body and/or hemocytes). The observation that Plasmodium can trigger a molecularly defined immune response in the vector constitutes an important advance in our understanding of parasite-vector interactions that are potentially involved in malaria transmission, and extends knowledge of the innate immune system of insects to encompass responses to protozoan parasites.     

Do Plasmodium ookinetes invade a specific cell type in the mosquito midgut?

Recent debate in Plasmodium ookinete invasion has been centered on whether the parasite chooses a specific cell type to cross the midgut epithelium in the mosquito. A few publications have described the mosquito midgut being composed of complex surface-structures, histochemically and biochemically diverse cell types, and have proposed that Plasmodium gallinaceum ookinetes prefers a specific cell type (Ross cell) in Aedes aegypti for crossing the midgut epithelium. Two recent publications reported, however, that with differential interference contrast microscopy, all midgut epithelial cells in uninfected mosquitoes appear structurally similar and argued that ookinetes do not invade a specific cell type. These observations are discussed here in the context of the 'Ross cell' hypothesis.     

Molecular interactions between Anopheles stephensi midgut cells and Plasmodium berghei: the time bomb theory of ookinete invasion of mosquitoes

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 inflicts 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 inflicted is repaired efficiently by an actin purse-string-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.     

Penetration of the Mosquito (Aedes aegypti) Midgut Wall by the Ookinetes of Plasmodium gallinaceum

ABSTRACT We observed Plasmodium gallinaceum ookinetes in both intracellular and intercellular positions in the midgut epithelium of the mosquito Aedes aegypti. After epithelial cell invasion intracellular ookinetes lacked a parasitophorous vacuolar membrane and were surrounded solely by their own pellicle. Thus, the ookinete in the midgut epithelium of the mosquito differs from erythrocytic and hepatic stages in that the parasite in the vertebrate host is surrounded by a vacuole. The midgut epithelial cytoplasm around the apical end of invading ookinetes was replaced by fine granular material deprived of normal organelles. Membranous structure was observed within the fine granular area. Most ookinetes were seen intracellularly on the luminal side and intercellularly on the haemocoel side of the midgut epithelial cells. These observations suggest that the ookinete first enters into the midgut epithelial cell, then exits to the space between the epithelial cells and moves to the basal lamina where the ookinete develops to the oocyst.     

Malaria infection of the mosquito Anopheles gambiae activates immune-responsive genes during critical transition stages of the parasite life cycle.

Six gene markers have been used to map the progress of the innate immune response of the mosquito vector, Anopheles gambiae, upon infection by the malaria parasite, Plasmodium berghei. In addition to four previously reported genes, the set of markers included NOS (a nitric oxide synthase gene fragment) and ICHIT (a gene encoding two putative chitin-binding domains separated by a polythreonine-rich mucin region). In the midgut, a robust response occurs at 24 h post-infection, at a time when malaria ookinetes traverse the midgut epithelium, but subsides at later phases of malaria development. In contrast, the salivary glands show no significant response at 24 h, but are activated in a prolonged late phase when sporozoites are released from the midgut into the haemolymph and invade the glands, between 10 and 25 days after blood feeding. Furthermore, the abdomen of the mosquito minus the midgut shows significant activation of immune markers, with complex kinetics that are distinct from those of both midgut and salivary glands. The parasite evidently elicits immune responses in multiple tissues of the mosquito, two of which are epithelia that the parasite must traverse to complete its development. The mechanisms of these responses and their significance for malaria transmission are discussed.     

Penetration of the mosquito (Aedes aegypti) midgut wall by the ookinetes of Plasmodium gallinaceum.

We observed Plasmodium gallinaceum ookinetes in both intracellular and intercellular positions in the midgut epithelium of the mosquito Aedes aegypti. After epithelial cell invasion intracellular ookinetes lacked a parasitophorous vacuolar membrane and were surrounded solely by their own pellicle. Thus, the ookinete in the midgut epithelium of the mosquito differs from erythrocytic and hepatic stages in that the parasite in the vertebrate host is surrounded by a vacuole. The midgut epithelial cytoplasm around the apical end of invading ookinetes was replaced by fine granular material deprived of normal organelles. Membranous structure was observed within the fine granular area. Most ookinetes were seen intracellularly on the luminal side and intercellularly on the haemocoel side of the midgut epithelial cells. These observations suggest that the ookinete first enters into the midgut epithelial cell, then exists to the space between the epithelial cells and moves to the basal lamina where the ookinete develops to the oocyst.