The dynamics of interactions between Plasmodium and the mosquito: a study of the infectivity of Plasmodium berghei and Plasmodium gallinaceum,and their transmission by Anopheles stephensi,Anopheles gambiae and Aedes aegypti

Knowledge of parasite-mosquito interactions is essential to develop strategies that will reduce malaria transmission through the mosquito vector. In this study we investigated the development of two model malaria parasites, Plasmodium berghei and Plasmodium gallinaceum, in three mosquito species Anopheles stephensi, Anopheles gambiae and Aedes aegypti. New methods to study gamete production in vivo in combination with GFP-expressing ookinetes were employed to measure the large losses incurred by the parasites during infection of mosquitoes. All three mosquito species transmitted P. gallinaceum; P. berghei was only transmitted by Anopheles spp. Plasmodium gallinaceum initiates gamete production with high efficiency equally in the three mosquito species. By contrast P. berghei is less efficiently activated to produce gametes, and in Ae. aegypti microgamete formation is almost totally suppressed. In all parasite/vector combinations ookinete development is inefficient, 500-100,000-fold losses were encountered. Losses during ookinete-to-oocyst transformation range from fivefold in compatible vector parasite combinations (P. berghei/An. stephensi), through >100-fold in poor vector/parasite combinations (P. gallinaceum/An. stephensi), to complete blockade (>1,500 fold) in others (P. berghei/Ae. aegypti). Plasmodium berghei ookinetes survive poorly in the bloodmeal of Ae. aegypti and are unable to invade the midgut epithelium. Cultured mature ookinetes of P. berghei injected directly into the mosquito haemocoele produced salivary gland sporozoites in An. stephensi, but not in Ae. aegypti, suggesting that further species-specific incompatibilities occur downstream of the midgut epithelium in Ae. aegypti. These results show that in these parasite-mosquito combinations the susceptibility to malarial infection is regulated at multiple steps during the development of the parasites. Understanding these at the molecular level may contribute to the development of rational strategies to reduce the vector competence of malarial vectors.     

Anopheles and Plasmodium: from laboratory models to natural systems in the field

Parasites that cause malaria must complete a complex life cycle in Anopheles vector mosquitoes in order to be transmitted from human to human. Previous gene-silencing studies have shown the influence of mosquito immunity in controlling the development of Plasmodium. Thus, parasite survival to the oocyst stage increased when the parasite antagonist gene LRIM1 (leucine-rich repeat immune protein 1) of the mosquito was silenced, but decreased when the C-type lectin agonist gene CTL4 or CTLMA2 (CTL mannose binding 2) was silenced. However, such effects were shown for infections of the human mosquito vector Anopheles gambiae with the rodent parasite Plasmodium berghei. Here, we report the first results of A. gambiae gene silencing on infection by sympatric field isolates of the principal human pathogen P. falciparum. In contrast with the results obtained with the rodent parasite, silencing of the same three genes had no effect on human parasite development. These results highlight the importance of following up discoveries in laboratory model systems with studies on natural parasite-mosquito interactions.     

Immune response of Anopheles gambiae to the early sporogonic stages of the human malaria parasite Plasmodium falciparum

Deciphering molecular interactions between the malaria parasite and its mosquito vector is an emerging area of research that will be greatly facilitated by the recent sequencing of the genomes of Anopheles gambiae mosquito and of various Plasmodium species. So far, most such studies have focused on Plasmodium berghei, a parasite species that infects rodents and is more amenable to studies. Here, we analysed the expression pattern of nine An.gambiae genes involved in immune surveillance during development of the human malaria parasite P.falciparum in mosquitoes fed on parasite-containing blood from patients in Cameroon. We found that P.falciparum ingestion triggers a midgut-associated, as well as a systemic, response in the mosquito, with three genes, NOS, defensin and GNBP, being regulated by ingestion of gametocytes, the infectious stage of the parasite. Surprisingly, we found a different pattern of expression of these genes in the An.gambiae-P.berghei model. Therefore, differences in mosquito reaction against various Plasmodium species may exist, which stresses the need to validate the main conclusions suggested by the P.berghei-An.gambiae model in the P.falciparum-An.gambiae system.     

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.     

Proteomic profiling of Plasmodium sporozoite maturation identifies new proteins essential for parasite development and infectivity

Plasmodium falciparum sporozoites that develop and mature inside an Anopheles mosquito initiate a malaria infection in humans. Here we report the first proteomic comparison of different parasite stages from the mosquito -- early and late oocysts containing midgut sporozoites, and the mature, infectious salivary gland sporozoites. Despite the morphological similarity between midgut and salivary gland sporozoites, their proteomes are markedly different, in agreement with their increase in hepatocyte infectivity. The different sporozoite proteomes contain a large number of stage specific proteins whose annotation suggest an involvement in sporozoite maturation, motility, infection of the human host and associated metabolic adjustments. Analyses of proteins identified in the P. falciparum sporozoite proteomes by orthologous gene disruption in the rodent malaria parasite, P. berghei, revealed three previously uncharacterized Plasmodium proteins that appear to be essential for sporozoite development at distinct points of maturation in the mosquito. This study sheds light on the development and maturation of the malaria parasite in an Anopheles mosquito and also identifies proteins that may be essential for sporozoite infectivity to humans.     

Plasmodium berghei ookinetes bind to Anopheles gambiae and Drosophila melanogaster annexins

Using a proteomic approach we identified polypeptides from Anopheles gambiae and Drosophila melanogaster protein extracts that selectively bind purified Plasmodium berghei ookinetes in vitro; these were two and three distinct polypeptides, respectively, with an apparent molecular weight of about 36 kDa. Combining two-dimensional electrophoresis and MALDI-TOF (matrix-associated laser desorption ionization time of flight) mass spectrometry we determined that the polypeptides correspond to isomorphs of the annexin B11 protein of the fruit fly. When protein extracts derived from A. gambiae and D. melanogaster tissue culture cells were further fractionated, the binding activity matching the annexin protein could be localized in the fraction derived from cell membranes in both diptera. Antibody staining showed that annexin also binds to ookinetes during the invasion of the mosquito midgut. Finally, inclusion of antiannexin antisera in a mosquito blood meal impaired parasite development, suggesting a facilitating role for annexins in the infection of the mosquito by Plasmodium.     

Ookinete destruction within the mosquito midgut lumen explains Anopheles albimanus refractoriness to Plasmodium falciparum (3D7A) oocyst infection

Previous studies have shown that the central American mosquito vector, Anopheles albimanus, is generally refractory to oocyst infection with allopatric isolates of the human malaria parasite Plasmodium falciparum. However, the reasons for the refractoriness of A. albimanus to infection with such isolates of P. falciparum are unknown. In the current study, we investigated the infectivity of the P. falciparum clone 3D7A to laboratory-reared A. albimanus and another natural vector of human malaria, Anopheles stephensi. Plasmodium falciparum gametocytes grown in vitro were simultaneously fed to both mosquito species and the progress of malaria infection compared. In 22 independent paired experimental feeds, no mature oocysts were observed on the midguts of A. albimanus 10days after bloodfeeding. In contrast, high levels of oocyst infection were found on the midguts of simultaneously fed A. stephensi. Direct immunofluorescence microscopy and light microscopical examination of Giemsa-stained histological sections were used to identify when the P. falciparum clone 3D7A failed to establish mature oocyst infections in A. albimanus. Similar densities of macrogametes/zygotes, and immature retort-form and mature ookinetes were found within the bloodmeals of both mosquito species. However, in A. albimanus, ookinetes were seldom associated with the peritrophic matrix, and were neither observed in the ectoperitrophic space nor the midgut epithelium. In contrast, ookinetes were frequently observed in these midgut compartments in A. stephensi. Additionally, young oocysts were observed on the midguts of A. stephensi but not A. albimanus 2days after bloodfeeding. Vital staining of the immature retort-form and mature ookinetes found within the luminal bloodmeal, demonstrated that a significantly greater proportion of these malaria parasite stages were non-viable in A. albimanus compared with A. stephensi. Overall, our observations indicate that ookinetes of the P. falciparum clone 3D7A are destroyed within the bloodmeal of A. albimanus and that the midgut lumen, rather than the midgut epithelium, is the site of mosquito refractoriness in this particular malaria parasite-mosquito vector combination.     

Reactive oxygen species modulate Anopheles gambiae immunity against bacteria and Plasmodium

The involvement of reactive oxygen species (ROS) in mosquito immunity against bacteria and Plasmodium was investigated in the malaria vector Anopheles gambiae. Strains of An. gambiae with higher systemic levels of ROS survive a bacterial challenge better, whereas reduction of ROS by dietary administration of antioxidants significantly decreases survival, indicating that ROS are required to mount effective antibacterial responses. Expression of several ROS detoxification enzymes increases in the midgut and fat body after a blood meal. Furthermore, expression of several of these enzymes increases to even higher levels when mosquitoes are fed a Plasmodium berghei-infected meal, indicating that the oxidative stress after a blood meal is exacerbated by Plasmodium infection. Paradoxically, a complete lack of induction of catalase mRNA and lower catalase activity were observed in P. berghei-infected midguts. This suppression of midgut catalase expression is a specific response to ookinete midgut invasion and is expected to lead to higher local levels of hydrogen peroxide. Further reduction of catalase expression by double-stranded RNA-mediated gene silencing promoted parasite clearance by a lytic mechanism and reduced infection significantly. High mosquito mortality is often observed after P. berghei infection. Death appears to result in part from excess production of ROS, as mortality can be decreased by oral administration of uric acid, a strong antioxidant. We conclude that ROS modulate An. gambiae immunity and that the mosquito response to P. berghei involves a local reduction of detoxification of hydrogen peroxide in the midgut that contributes to limit Plasmodium infection through a lytic mechanism.     

Engineered resistance to Plasmodium falciparum development in transgenic Anopheles stephensi

Transposon-mediated transformation was used to produce Anopheles stephensi that express single-chain antibodies (scFvs) designed to target the human malaria parasite, Plasmodium falciparum. The scFvs, m1C3, m4B7, and m2A10, are derived from mouse monoclonal antibodies that inhibit either ookinete invasion of the midgut or sporozoite invasion of salivary glands. The scFvs that target the parasite surface, m4B7 and m2A10, were fused to an Anopheles gambiae antimicrobial peptide, Cecropin A. Previously-characterized Anopheles cis-acting DNA regulatory elements were included in the transgenes to coordinate scFv production with parasite development. Gene amplification and immunoblot analyses showed promoter-specific increases in transgene expression in blood-fed females. Transgenic mosquito lines expressing each of the scFv genes had significantly lower infection levels than controls when challenged with P. falciparum.     

Motility and infectivity of Plasmodium berghei sporozoites expressing avian Plasmodium gallinaceum circumsporozoite protein

Avian and rodent malaria sporozoites selectively invade different vertebrate cell types, namely macrophages and hepatocytes, and develop in distantly related vector species. To investigate the role of the circumsporozoite (CS) protein in determining parasite survival in different vector species and vertebrate host cell types, we replaced the endogenous CS protein gene of the rodent malaria parasite Plasmodium berghei with that of the avian parasite P. gallinaceum and control rodent parasite P. yoelii. In anopheline mosquitoes, P. berghei parasites carrying P. gallinaceum and rodent parasite P. yoelii CS protein gene developed into oocysts and sporozoites. Plasmodium gallinaceum CS expressing transgenic sporozoites, although motile, failed to invade mosquito salivary glands and to infect mice, which suggests that motility alone is not sufficient for invasion. Notably, a percentage of infected Anopheles stephensi mosquitoes showed melanotic encapsulation of late stage oocysts. This was not observed in control infections or in A. gambiae infections. These findings shed new light on the role of the CS protein in the interaction of the parasite with both the mosquito vector and the rodent host.     

Use of a Drosophila model to identify genes regulating Plasmodium growth in the mosquito

We performed a forward genetic screen, using Drosophila as a surrogate mosquito, to identify host factors required for the growth of the avian malaria parasite, Plasmodium gallinaceum. We identified 18 presumed loss-of-function mutants that reduced the growth of the parasite in flies. Presumptive mutation sites were identified in 14 of the mutants on the basis of the insertion site of a transposable element. None of the identified genes have been previously implicated in innate immune responses or interactions with Plasmodium. The functions of five Anopheles gambiae homologs were tested by using RNAi to knock down gene function followed by measuring the growth of the rodent parasite, Plasmodium berghei. Loss of function of four of these genes in the mosquito affected Plasmodium growth, suggesting that Drosophila can be used effectively as a surrogate mosquito to identify relevant host factors in the mosquito.     

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.     

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.     

Interactions of human malaria parasites, Plasmodium wVaxand P.falciparum, with the midgut of Anopheles mosquitoes

Abstract Present understanding of the development of sexual stages of the human malaria parasites Plasmodium vivax and P.falciparum in the Anopheles vector is reviewed, with particular reference to the role of the mosquito midgut in establishing an infection. The sexual stages of the parasite, the gametocytes, are formed in human erythrocytes. The changes in temperature and pH encountered by the gametocyte induce gametogenesis in the lumen of the midgut. Macromolecules derived from mosquito tissue and second messenger pathways regulate events leading to fertilization. In An.tessellatus the movement of the ookinete from the lumen to the midgut epithelium is linked to the release of trypsin in the midgut and the peritrophic matrix is not a firm barrier to this movement. The passage of the P. vivax ookinete through the peritrophic matrix may take place before the latter is fully formed. The late ookinete development in P.falciparum requires chitinase to facilitate penetration of the peritrophic matrix. Recognition sites for the ookinetes are present on the midgut epithelial cells. N-acetyl glucosamine residues in the oligosaccharide side chains of An.tessellatus midgut glycoproteins and peritrophic matrix proteoglycan may function as recognition sites for P.vivax and P.falciparum ookinetes. It is possible that ookinetes penetrating epithelial cells produce stress in the vector. Mosquito molecules may be involved in oocyst development in the basal lamina, and encapsulation of the parasite occurs in vectors that are refractory to the parasite. Detailed knowledge of vector-parasite interactions, particularly in the midgut and the identification of critical mosquito molecules offers prospects for manipulating the vector for the control of malaria.