CTRP is essential for mosquito infection by malaria ookinetes

The malaria parasite suffers severe population losses as it passes through its mosquito vector. Contributing factors are the essential but highly constrained developmental transitions that the parasite undergoes in the mosquito midgut, combined with the invasion of the midgut epithelium by the malaria ookinete (recently described as a principal elicitor of the innate immune response in the Plasmodium-infected insect). Little is known about the molecular organization of these midgut-stage parasites and their critical interactions with the blood meal and the mosquito vector. Elucidation of these molecules and interactions will open up new avenues for chemotherapeutic and immunological attack of parasite development. Here, using the rodent malaria parasite Plasmodium berghei, we identify and characterize the first microneme protein of the ookinete: circumsporozoite- and TRAP-related protein (CTRP). We show that transgenic parasites in which the CTRP gene is disrupted form ookinetes that have reduced motility, fail to invade the midgut epithelium, do not trigger the mosquito immune response, and do not develop further into oocysts. Thus, CTRP is the first molecule shown to be essential for ookinete infectivity and, consequently, mosquito transmission of malaria.     

PbCap380, a novel oocyst capsule protein, is essential for malaria parasite survival in the mosquito

An essential requisite for transmission of Plasmodium, the causative agent of malaria, is the successful completion of a complex developmental cycle in its mosquito vector. Of hundreds of ookinetes that form in the mosquito midgut, only few transform into oocysts, a loss attributed to the action of the mosquito immune system. However, once oocysts form, they appear to be resistant to mosquito defences. During oocyst development, a thick capsule forms around the parasite and appears to function as a protective cover. Little information is available about the composition of this capsule. Here we report on the identification and partial characterization of the first Plasmodium oocyst capsule protein (PbCap380). Genetic analysis indicates that the gene is essential and that PbCap380(-) mutant parasites form oocysts in normal numbers but are gradually eliminated. As a result, mosquitoes infected with PbCap380(-) parasites do not transmit malaria. Targeting of the oocyst capsule may provide a new strategy for malaria control.     

Plasmodium gallinaceum: a novel morphology of malaria ookinetes in the midgut of the mosquito vector

Malaria ookinetes invade midgut epithelial cells of the mosquito vector from the bloodmeal in the lumen of the mosquito midgut, but the cellular interactions of ookinetes with the mosquito vector remain poorly described. We describe here a novel morphology of Plasmodium gallinaceum ookinetes in which the central portion of the ookinete is an elongated narrow tube or stalk joining the anterior and posterior portions of the parasite. We propose that the previously undescribed stalkform ookinete may be an adaptation to facilitate parasite locomotion through the cytoplasm of mosquito midgut epithelial cells.     

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.     

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.     

Plasmodium yoelii: semiquantitative analyses of circumsporozoite protein gene expression during the sporogonic development of P. y. yoelii and P. y. nigeriensis in the mosquito vector Anopheles stephensi

Malaria infection in the mosquito vector can be modulated by the vertebrate host, mosquito factors, and interactions between different parasite populations. Modulation of parasite development can be assessed through the study of gene expression. The present report describes a specific, sensitive, and nonradioactive method that permits assessment of parasite load and quantification of circumsporozoite protein gene expression during the sporogonic stages of Plasmodium yoelii yoelii and P. y. nigeriensis. A decrease in parasite load was observed when comparing DNA of oocysts on day 7 postinfection with that of oocysts and sporozoites on day 19. On day 7, parasites (oocysts) showed a marked increase of circumsporozoite protein expression when compared with that (sporozoites and oocysts) on day 19. The method developed in this work can be a valuable tool to understand parasite interaction mechanisms that are involved in mosquito malaria infections.     

Selection of Anopheles dirus for refractoriness and susceptibility to Plasmodium yoelii nigeriensis

Two lines of the Oriental malaria vector mosquito Anopheles dirus species A (Diptera: Culicidae), one fully refractory and one fully susceptible to Plasmodium yoelii nigeriensis (an African rodent malaria parasite), were established after 17 generations of mass selection, followed by single female selection for one or two generations. Prior to selection, the stock colony of An. dirus was 17% refractory. Both lines of An. dirus produced abundant ookinetes that started to invade the midgut within 24h post-infection, as seen in histological sections. In most of the refractory mosquitoes, oocysts stopped development <12 h post-invasion, indicating a rapid defence mechanism. Dead P. y. nigeriensis parasites were apparently localized as small melanized spots (2-5 microm) seen in wet preparations of mosquito midguts dissected 5-7 days post infective bloodmeal. In some refractory An. dirus females, apart from the spots, a small number of totally encapsulated oocysts (c. 10 microm) were also present. These larger melanized parasites predominated in a few females: they appeared 2-3 days post-infection as a secondary delayed defence mechanism. The progeny of reciprocal matings between susceptible and refractory lines had approximately 50% susceptibility. Backcrosses of F1 hybrids with susceptible or refractory lines increased or decreased the susceptibility of backcross progeny accordingly. Overall, these results suggest polygenic control of susceptibility to P. y. nigeriensis infection. The refractory line of An. dirus showed normal susceptibility to natural infections of the human malarias P. falciparum and P. vivax from local patients.     

A comparison of Anopheles gambiae and Plasmodium falciparum genetic structure over space and time

Population genetic structure and subdivision are key factors affecting the evolution of organisms. In this study, we analysed and compared the population genetic structure of the malaria parasite Plasmodium falciparum and its mosquito vector Anopheles gambiae over space and time in the Nianza Province, near Victoria Lake in Kenya. The parasites were collected from mosquitoes caught in six villages separated by up to 68 km in 2002 and 2003. A total of 545 oocysts were dissected from 122 infected mosquitoes and genotyped at seven microsatellite markers. Five hundred and forty-seven mosquitoes, both infected and uninfected, were genotyped at eight microsatellites. For the parasite and the vector, the analysis revealed no (or very little) genetic differentiation among villages. This may be explained by high local population sizes for the parasite and the mosquito. The small level of genetic differentiation observed between populations may explain the speed at which antimalarial drug resistance and insecticide resistance spread into the African continent.     

Plasmodium vivax: ookinete destruction and oocyst development arrest are responsible for Anopheles albimanus resistance to circumsporozoite phenotype VK247 parasites

Anopheles albimanus and An. pseudopunctipennis differ in their susceptibilities to Plasmodium vivax circumsporozoite phenotypes. An. pseudopunctipennis is susceptible to phenotype VK247 but almost refractory to VK210. In contrast, An. albimanus is almost refractory to VK247 but susceptible to VK210. To investigate the site in the mosquito and the parasite stage at which resistance mechanisms affect VK247 development in An. albimanus, parasite development was followed in a series of experiments in which both mosquitoes species were simultaneously infected with blood from patients. Parasite phenotype was determined in mature oocysts and salivary gland sporozoites by use of immunofluorescence and Western blot assays and/or gene identification. Ookinete maturation and their densities within the bloodmeal bolus were similar in both mosquito species. Ookinete densities on the internal midgut surface of An. albimanus were 4.7 times higher than those in An. pseudopunctipennis; however, the densities of developing oocysts on the external midgut surface were 6.12 times higher in the latter species. Electron microscopy observation of ookinetes in An. albimanus midgut epithelium indicated severe parasite damage. These results indicate that P. vivax VK247 parasites are destroyed at different parasite stages during migration in An. albimanus midguts. A portion, accumulated on the internal midgut surface, is probably destroyed by the mosquito's digestive enzymes and another portion is most likely destroyed by mosquito defense molecules within the midgut epithelium. A third group, reaching the external midgut surface, initiates oocyst development, but over 90% of them interrupt their development and die. The identification of mechanisms that participate in parasite destruction could provide new elements to construct transgenic mosquitoes resistant to malaria parasites.     

A NIMA-related protein kinase is essential for completion of the sexual cycle of malaria parasites

The molecular mechanisms regulating the sexual development of malaria parasites from gametocytes to oocysts in their mosquito vector are still largely unexplored. In other eukaryotes, NIMA-related kinases (Neks) regulate cell cycle progression and have been implicated in the regulation of meiosis. Here, we demonstrate that Nek-4, a new Plasmodium member of the Nek family, is essential for completion of the sexual cycle of the parasite. Recombinant Plasmodium falciparum Nek-4 possesses protein kinase activity and displays substrate preferences similar to those of other Neks. Nek-4 is highly expressed in gametocytes, yet disruption of the nek-4 gene in the rodent malaria parasite P. berghei has no effect on gamete formation and subsequent fertilization. However, further differentiation of zygotes into ookinetes is abolished. Measurements of nuclear DNA content indicate that zygotes lacking Nek-4 fail to undergo the genome replication to the tetraploid level that precedes meiosis. Cell cycle progression in the zygote is identified as a likely precondition for its morphological transition to the ookinete and for the successful establishment of a malaria infection in the mosquito.     

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.     

The Plasmodium bottleneck: malaria parasite losses in the mosquito vector

Nearly one million people are killed every year by the malaria parasite Plasmodium. Although the disease-causing forms of the parasite exist only in the human blood, mosquitoes of the genus Anopheles are the obligate vector for transmission. Here, we review the parasite life cycle in the vector and highlight the human and mosquito contributions that limit malaria parasite development in the mosquito host. We address parasite killing in its mosquito host and bottlenecks in parasite numbers that might guide intervention strategies to prevent transmission.     

Interactions between malaria parasites and their mosquito hosts in the midgut

This review examines what is presently known of the molecular interactions between Plasmodium and Anopheles that take place in the latter's midgut upon ingestion of the parasites with an infectious blood meal. In order to become 'established' in the gut and to transform into a sporozoite-producing oocyst, the malaria parasite needs to undergo different developmental steps that are often characterized by the use of selected resources provided by the mosquito vector. Moreover, some of these resources may be used by the parasite in order to overcome the insect host's defence mechanisms. The molecular partners of this interplay are now in the process of being defined and analyzed for both Plasmodium and mosquito and, thus, understood; these will be presented here in some detail.     

Levels of circumsporozoite protein in the Plasmodium oocyst determine sporozoite morphology

The sporozoite stage of the Plasmodium parasite is formed by budding from a multinucleate oocyst in the mosquito midgut. During their life, sporozoites must infect the salivary glands of the mosquito vector and the liver of the mammalian host; both events depend on the major sporozoite surface protein, the circumsporozoite protein (CS). We previously reported that Plasmodium berghei oocysts in which the CS gene is inactivated do not form sporozoites. Here, we analyzed the ultrastructure of P.berghei oocyst differentiation in the wild type, recombinants that do not produce or produce reduced amounts of CS, and corresponding complemented clones. The results indicate that CS is essential for establishing polarity in the oocyst. The amounts of CS protein correlate with the extent of development of the inner membranes and associated microtubules underneath the oocyst outer membrane, which normally demarcate focal budding sites. This is a first example of a protein controlling both morphogenesis and infectivity of a parasite stage.     

Estimation of survival rate and oviposition interval of Anopheles balabacensis mosquitoes from mark-recapture experiments in Sabah, Malaysia

An exophilic population of the vector mosquito Anopheles balabacensis Baisas was investigated in two mark-recapture studies (16.ix-13.x.1986 and 6-26.i.1987) at an inland, foothill village in Sabah, Malaysia. Wild female mosquitoes were intercepted as they came to feed on man or buffalo, given a bloodmeal, marked with fluorescent dust and released. The recapture rate was about 12%. A new method of analysis is proposed which uses cross-correlation and a time series model. The estimated survival per oviposition cycle was 0.48-0.54 and the oviposition cycle interval 2-3 days.     

Population dynamics of sporogony for Plasmodium vivax parasites from western Thailand developing within three species of colonized Anopheles mosquitoes

Background

The population dynamics of Plasmodium sporogony within mosquitoes consists of an early phase where parasite abundance decreases during the transition from gametocyte to oocyst, an intermediate phase where parasite abundance remains static as oocysts, and a later phase where parasite abundance increases during the release of progeny sporozoites from oocysts. Sporogonic development is complete when sporozoites invade the mosquito salivary glands. The dynamics and efficiency of this developmental sequence were determined in laboratory strains of Anopheles dirus, Anopheles minimus and Anopheles sawadwongporni mosquitoes for Plasmodium vivax parasites circulating naturally in western Thailand.

Methods

Mosquitoes were fed blood from 20 symptomatic Thai adults via membrane feeders. Absolute densities were estimated for macrogametocytes, round stages (= female gametes/zygotes), ookinetes, oocysts, haemolymph sporozoites and salivary gland sporozoites. From these census data, five aspects of population dynamics were analysed; 1) changes in life-stage prevalence during early sporogony, 2) kinetics of life-stage formation, 3) efficiency of life-stage transitions, 4) density relationships between successive life-stages, and 5) parasite aggregation patterns.

Results

There was no difference among the three mosquito species tested in total losses incurred by P. vivax populations during early sporogony. Averaged across all infections, parasite populations incurred a 68-fold loss in abundance, with losses of ca. 19-fold, 2-fold and 2-fold at the first (= gametogenesis/fertilization), second (= round stage transformation), and third (= ookinete migration) life-stage transitions, respectively. However, total losses varied widely among infections, ranging from 6-fold to over 2,000-fold loss. Losses during gametogenesis/fertilization accounted for most of this variability, indicating that gametocytes originating from some volunteers were more fertile than those from other volunteers. Although reasons for such variability were not determined, gametocyte fertility was not correlated with blood haematocrit, asexual parasitaemia, gametocyte density or gametocyte sex ratio. Round stages and ookinetes were present in mosquito midguts for up to 48 hours and development was asynchronous. Parasite losses during fertilization and round stage differentiation were more influenced by factors intrinsic to the parasite and/or factors in the blood, whereas ookinete losses were more strongly influenced by mosquito factors. Oocysts released sporozoites on days 12 to 14, but even by day 22 many oocysts were still present on the midgut. The per capita production was estimated to be approximately 500 sporozoites per oocyst and approximately 75% of the sporozoites released into the haemocoel successfully invaded the salivary glands.

Conclusion

The major developmental bottleneck in early sporogony occurred during the transition from macrogametocyte to round stage. Sporozoite invasion into the salivary glands was very efficient. Information on the natural population dynamics of sporogony within malaria-endemic areas may benefit intervention strategies that target early sporogony (e.g., transmission blocking vaccines, transgenic mosquitoes).     

Plasmodium falciparum produce lower infection intensities in local versus foreign Anopheles gambiae populations

Both Plasmodium falciparum and Anopheles gambiae show great diversity in Africa, in their own genetic makeup and population dynamics. The genetics of the individual mosquito and parasite are known to play a role in determining the outcome of infection in the vector, but whether differences in infection phenotype vary between populations remains to be investigated. Here we established two A. gambiae s.s. M molecular form colonies from Cameroon and Burkina Faso, representing a local and a foreign population for each of the geographical sites. Experimental infections of both colonies were conducted in Cameroon and Burkina Faso using local wild P. falciparum, giving a sympatric and allopatric vector-parasite combination in each site. Infection phenotype was determined in terms of oocyst prevalence and intensity for at least nine infections for each vector-parasite combination. Sympatric infections were found to produce 25% fewer oocysts per midgut than allopatric infections, while prevalence was not affected by local/foreign interactions. The reduction in oocyst numbers in sympatric couples may be the result of evolutionary processes where the mosquito populations have locally adapted to their parasite populations. Future research on vector-parasite interactions must take into account the geographic scale of adaptation revealed here by conducting experiments in natural sympatric populations to give epidemiologically meaningful results.     

Transmission Blocking Immunity in the Malaria Non-Vector Mosquito Anopheles quadriannulatus Species A

Despite being phylogenetically very close to Anopheles gambiae, the major mosquito vector of human malaria in Africa, Anopheles quadriannulatus is thought to be a non-vector. Understanding the difference between vector and non-vector mosquitoes can facilitate development of novel malaria control strategies. We demonstrate that An. quadriannulatus is largely resistant to infections by the human parasite Plasmodium falciparum, as well as by the rodent parasite Plasmodium berghei. By using genetics and reverse genetics, we show that resistance is controlled by quantitative heritable traits and manifested by lysis or melanization of ookinetes in the mosquito midgut, as well as by killing of parasites at subsequent stages of their development in the mosquito. Genes encoding two leucine-rich repeat proteins, LRIM1 and LRIM2, and the thioester-containing protein, TEP1, are identified as essential in these immune reactions. Their silencing completely abolishes P. berghei melanization and dramatically increases the number of oocysts, thus transforming An. quadriannulatus into a highly permissive parasite host. We hypothesize that the mosquito immune system is an important cause of natural refractoriness to malaria and that utilization of this innate capacity of mosquitoes could lead to new methods to control transmission of the disease.