Interspecific competition during transmission of two sympatric malaria parasite species to the mosquito vector

The role of species interactions in structuring parasite communities remains controversial. Here, we show that interspecific competition between two avian malaria parasite species, Plasmodium gallinaceum and P. juxtanucleare, occurs as a result of interference during parasite fertilization within the bloodmeal of the mosquito. The significant reduction in the transmission success of P. gallinaceum to mosquitoes, due to the co-infecting P. juxtanucleare, is predicted to have compromised its colonization of regions occupied by P. juxtanucleare and, thus, may have contributed to the restricted global distribution of P. gallinaceum. Such interspecies interactions may occur between human malaria parasites and, thus, impact upon parasite species epidemiology, especially in regions of seasonal transmission.     

Culex saltanensis Dyar, 1928--natural vector of Plasmodium juxtanucleare in Rio de Janeiro, Brazil.

Searching for the natural vector of Plasmodium juxtanucleare in an enzootic locality: Granjas Calábria (33% of the chickens infected), Jacarepaguá, in Rio de Janeiro, Brazil, 13 comparative captures of mosquitoes were carried out, simultaneously on man (out-doors) and on chicken (in a poultry-yard), between 6 and 9 p.m., from September 1988 to March 1989. Culex saltanensis was the most frequent species in captures on chicken, accounting for 41.7% of the mosquitoes collected on this bait, showing to be highly ornithophilic (90% captured on chicken versus 10% on man). Seven specimens of Cx. saltanensis were found naturally infected in Granjas Calábria: five with mature pedunculate oocysts and two with sporozoites (one in the haemocoele and one in the salivary glands). These sporozoites produced an infection by P. juxtanucleare in a chick, which had parasitemia on day 41 after inoculation. One Cx. coronator was found with mature pedunculate oocysts. Culex saltanensis was regarded as primary vector of P. juxtanucleare in Rio de Janeiro for being highly ornithophilic and in enough density to maintain the transmission, having been found with infective sporozoites in its salivary glands, and being susceptible to the parasite and able to transmit experimentally it by the bite.     

An overview of malaria transmission from the perspective of Amazon Anopheles vectors

In the Americas, areas with a high risk of malaria transmission are mainly located in the Amazon Forest, which extends across nine countries. One keystone step to understanding the Plasmodium life cycle in Anopheles species from the Amazon Region is to obtain experimentally infected mosquito vectors. Several attempts to colonise Ano- pheles species have been conducted, but with only short-lived success or no success at all. In this review, we review the literature on malaria transmission from the perspective of its Amazon vectors. Currently, it is possible to develop experimental Plasmodium vivax infection of the colonised and field-captured vectors in laboratories located close to Amazonian endemic areas. We are also reviewing studies related to the immune response to P. vivax infection of Anopheles aquasalis, a coastal mosquito species. Finally, we discuss the importance of the modulation of Plasmodium infection by the vector microbiota and also consider the anopheline genomes. The establishment of experimental mosquito infections with Plasmodium falciparum, Plasmodium yoelii and Plasmodium berghei parasites that could provide interesting models for studying malaria in the Amazonian scenario is important. Understanding the molecular mechanisms involved in the development of the parasites in New World vectors is crucial in order to better determine the interaction process and vectorial competence.     

Getting down to malarial nuts and bolts: the interaction between Plasmodium vivax merozoites and their host erythrocytes

Of the four Plasmodium species that routinely cause malaria in humans, Plasmodium falciparum is responsible for the majority of malaria mortality and consequently gets most of the headlines. Outside Africa, however, more malaria cases are caused by its distant cousin Plasmodium vivax, resulting in a daunting morbidity and economic burden for countries across Asia and the Americas. Plasmodium life cycles are complex, but the symptoms and pathology of malaria occur during the blood phase, when merozoites recognize and invade erythrocytes, initiating a developmental programme that culminates in lysis of the erythrocyte and release of multiple daughter merozoites. P. vivax merozoites are dependent on a single host cell receptor for erythrocyte invasion, the Duffy antigen receptor for chemokines, and humans that do not express this receptor on the surface of their erythrocytes are immune to P. vivax infection. This essential receptor-ligand interaction is addressed from both the host and parasite side in two papers in this issue of Molecular Microbiology, with important implications for plans to develop a P. vivax vaccine.     

Molecular phylogenetic analysis of the avian malarial parasite Plasmodium (Novyella) juxtanucleare

Plasmodium (Novyella) juxtanucleare is a widely distributed parasite that primarily infects chickens (Gallus gallus domesticus). All species of Novyella are characterized by very small schizonts, which in the case of P. juxtanucleare are always found juxtaposed to the erythrocyte nucleus, hence its name. Nearly complete small-subunit ribosomal RNA sequences have been obtained from 2 isolates of this species, and comparisons with other Plasmodium species have been made. Phylogenetic analysis reveals that this parasite is closely related to other avian-infecting Plasmodium species and that molecular relationships among the avian-infecting plasmodia do not correspond to their morphology-based subgeneric classifications.     

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.     

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.     

Progression of Plasmodium berghei through Anopheles stephensi is density-dependent

It is well documented that the density of Plasmodium in its vertebrate host modulates the physiological response induced; this in turn regulates parasite survival and transmission. It is less clear that parasite density in the mosquito regulates survival and transmission of this important pathogen. Numerous studies have described conversion rates of Plasmodium from one life stage to the next within the mosquito, yet few have considered that these rates might vary with parasite density. Here we establish infections with defined numbers of the rodent malaria parasite Plasmodium berghei to examine how parasite density at each stage of development (gametocytes; ookinetes; oocysts and sporozoites) influences development to the ensuing stage in Anopheles stephensi, and thus the delivery of infectious sporozoites to the vertebrate host. We show that every developmental transition exhibits strong density dependence, with numbers of the ensuing stages saturating at high density. We further show that when fed ookinetes at very low densities, oocyst development is facilitated by increasing ookinete number (i.e., the efficiency of ookinete-oocyst transformation follows a sigmoid relationship). We discuss how observations on this model system generate important hypotheses for the understanding of malaria biology, and how these might guide the rational analysis of interventions against the transmission of the malaria parasites of humans by their diverse vector species.     

Heritability of P. falciparum and P. vivax Malaria in a Karen Population in Thailand

The majority of studies concerning malaria host genetics have focused on individual genes that confer protection against rather than susceptibility to malaria. Establishing the relative impact of genetic versus non-genetic factors on malaria infection and disease is essential to focus effort on key determinant factors. This relative contribution has rarely been evaluated for Plasmodium falciparum and almost never for Plasmodium vivax. We conducted a longitudinal cohort study in a Karen population of 3,484 individuals in a region of mesoendemic malaria, Thailand from 1998 to 2005. The number of P. falciparum and P. vivax clinical cases and the parasite density per person were determined. Statistical analyses were performed to account for the influence of environmental factors and the genetic heritability of the phenotypes was calculated using the pedigree-based variance components model. The genetic contribution to the number of clinical episodes resulting from P. falciparum and P. vivax were 10% and 19% respectively. There was also moderate genetic contribution to the maximum and overall parasite trophozoite density phenotypes for both P. falciparum (16%&16%) and P. vivax (15%&13%). These values, for P. falciparum, were similar to those previously observed in a region of much higher transmission intensity in Senegal, West Africa. Although environmental factors play an important role in acquiring an infection, genetics plays a determinant role in the outcome of an infection with either malaria parasite species prior to the development of immunity.     

Susceptibility of species A, B, and C of Anopheles culicifacies complex to Plasmodium yoelii yoelii and Plasmodium vinckei petteri infections

The comparative susceptibilities of colonized species A, B, and C of Anopheles culicifacies complex and Anopheles stephensi were determined for 2 rodent malaria parasites Plasmodium vinckei petteri and Plasmodium yoelii yoelii. All the 3 members of the complex were found to support complete sporogony with varying success. Controls, A. stephensi, become readily infected, with >70% developing oocysts. Of the test groups, species A had the highest percentage of mosquitoes with oocysts (>25%) and sporozoites (>15%). Anopheles culicifacies species B were least susceptible; less than 10% had oocysts and sporozoites in the salivary glands. The results demonstrate that A. culicifacies species A is most susceptible and species B is least susceptible to infections with both the parasites.     

The effect of Plasmodium yoelii nigeriensis infection on the feeding persistence of Anopheles stephensi Liston throughout the sporogonic cycle.

Vector-borne parasites such as malaria have been shown to modify the feeding behaviour of their invertebrate hosts so as to increase the probability of transmission. However, evolutionary consideration of developmental changes in malaria within Anopheles mosquitoes suggests that the nature of altered feeding by mosquitoes should differ depending on the developmental stage of the parasite. We present laboratory evidence that the feeding persistence of female Anopheles stephensi towards a human host is decreased in the presence of Plasmodium yoelii nigeriensis oocysts (which cannot be transmitted), but increased when the malaria has developed into transmissible sporozoites in the salivary glands. In ten-minute trials, 33% of uninfected mosquitoes gave up their feeding attempt before the test period had ended, 53% of those harbouring oocysts had given up, but only 20% of those infected with sporozoites gave up by this time. We conclude that changes in feeding behaviour of mosquitoes mediated by parasite infection are sensitive to the developmental stage of the parasite and that these changes have important implications for malaria epidemiology.     

Plasmodium subtilisin-like protease 1 (SUB1): insights into the active-site structure, specificity and function of a pan-malaria drug target

Release of the malaria merozoite from its host erythrocyte (egress) and invasion of a fresh cell are crucial steps in the life cycle of the malaria pathogen. Subtilisin-like protease 1 (SUB1) is a parasite serine protease implicated in both processes. In the most dangerous human malarial species, Plasmodium falciparum, SUB1 has previously been shown to have several parasite-derived substrates, proteolytic cleavage of which is important both for egress and maturation of the merozoite surface to enable invasion. Here we have used molecular modelling, existing knowledge of SUB1 substrates, and recombinant expression and characterisation of additional Plasmodium SUB1 orthologues, to examine the active site architecture and substrate specificity of P. falciparum SUB1 and its orthologues from the two other major human malaria pathogens Plasmodium vivax and Plasmodium knowlesi, as well as from the rodent malaria species, Plasmodium berghei. Our results reveal a number of unusual features of the SUB1 substrate binding cleft, including a requirement to interact with both prime and non-prime side residues of the substrate recognition motif. Cleavage of conserved parasite substrates is mediated by SUB1 in all parasite species examined, and the importance of this is supported by evidence for species-specific co-evolution of protease and substrates. Two peptidyl alpha-ketoamides based on an authentic PfSUB1 substrate inhibit all SUB1 orthologues examined, with inhibitory potency enhanced by the presence of a carboxyl moiety designed to introduce prime side interactions with the protease. Our findings demonstrate that it should be possible to develop 'pan-reactive' drug-like compounds that inhibit SUB1 in all three major human malaria pathogens, enabling production of broad-spectrum antimalarial drugs targeting SUB1.     

Rodent Plasmodium: population dynamics of early sporogony within Anopheles stephensi mosquitoes

Early sporogony of Plasmodium parasites involves 2 major developmental transitions within the insect vector, i.e., gametocyte-to-ookinete and ookinete-to-oocyst. This study compared the population dynamics of early sporogony among murine rodent Plasmodium (Plasmodium berghei, Plasmodium chabaudi, Plasmodium vinckei, and Plasmodium yoelii) developing within Anopheles stephensi mosquitoes. Estimates of absolute densities were determined for gametocytes, ookinetes, and oocysts for 108 experimental infections. Total losses throughout early sporogony were greatest in P. vinckei (ca. 250,000-fold loss), followed by P. yoelii (ca. 70,000-fold loss), P. berghei (ca. 45,000-fold loss), and P. chabaudi (ca. 15,000-fold loss). The gametocyte-to-ookinete transition represented the most severe population bottleneck. Numerical losses during this transition (ca. 3,000- to 30,000-fold, depending on species) were orders of magnitude greater than losses incurred during the ookinete-to-oocyst transition (3- to 14-fold). There were no significant correlations between gametocyte and ookinete densities. Significant correlations between ookinete and oocyst densities existed for P. berghei, P. chabaudi, and P. yoelii (but not for P. vinckei), and were best described by nonlinear functions (P. berghei = sigmoid, P. chabaudi = hyperbolic, P. yoelii = sigmoid), indicating that conversion of ookinetes to oocysts in these species is density dependent. The upper theoretical limit for oocyst density on the mosquito midgut for P. chabaudi and P. yoelii (ca. 300 oocysts per midgut) was higher than for P. berghei (ca. 30 oocysts per midgut). This study provides basic information about population processes that occur during the early sporogonic development of some common laboratory model systems of malaria.     

The remarkable journey of adaptation of the Plasmodium falciparum malaria parasite to New World anopheline mosquitoes

Plasmodium falciparum originated in Africa, dispersed around the world as a result of human migration and had to adapt to several different indigenous anopheline mosquitoes. Anophelines from the New World are evolutionary distant form African ones and this probably resulted in a more stringent selection of Plasmodium as it adapted to these vectors. It is thought that Plasmodium has been genetically selected by some anopheline species through unknown mechanisms. The mosquito immune system can greatly limit infection and P. falciparum evolved a strategy to evade these responses, at least in part mediated by Pfs47, a highly polymorphic gene. We propose that adaptation of P. falciparum to new vectors may require evasion of their immune system. Parasites with a Pfs47 haplotype compatible with the indigenous mosquito vector would be able to survive and be transmitted. The mosquito antiplasmodial response could be an important determinant of P. falciparum population structure and could affect malaria transmission in the Americas.     

Comparative susceptibility of introduced forest-dwelling mosquitoes in Hawai'i to avian malaria, Plasmodium relictum

To identify potential vectors of avian malaria in Hawaiian native forests, the innate susceptibility of Aedes albopictus, Wyeomyia mitchellii, and Culex quinquefasciatus from 3 geographical sites along an altitudinal gradient was evaluated using local isolates of Plasmodium relictum. Mosquitoes were dissected 5-8 and 9-13 days postinfective blood meal and microscopically examined for oocysts and salivary-gland sporozoites. Sporogony was completed in all 3 species, but prevalence between species varied significantly. Oocysts were detected in 1-2% and sporozoites in 1-7% of Aedes albopictus that fed on infected ducklings. Wyeomyia mitchellii was slightly more susceptible, with 7-19% and 7% infected with oocysts and sporozoites, respectively. In both species, the median oocyst number was 5 or below. This is only the second Wyeomyia species reported to support development of a malarial parasite. Conversely, Culex quinquefasciatus from all 3 sites proved very susceptible. Prevalence of oocysts and sporozoites consistently exceeded 70%, regardless of gametocytemia or origin of the P. relictum isolate. In trials for which a maximum 200 oocysts were recorded, the median number of oocysts ranged from 144 to 200. It was concluded that Culex quinquefasciatus is the primary vector of avian malaria in Hawai'i.     

‘Manipulation’ without the parasite: altered feeding behaviour of mosquitoes is not dependent on infection with malaria parasites

Previous studies have suggested that Plasmodium parasites can manipulate mosquito feeding behaviours such as probing, persistence and engorgement rate in order to enhance transmission success. Here, we broaden analysis of this ‘manipulation phenotype’ to consider proximate foraging behaviours, including responsiveness to host odours and host location. Using Anopheles stephensi and Plasmodium yoelii as a model system, we demonstrate that mosquitoes with early stage infections (i.e. non-infectious oocysts) exhibit reduced attraction to a human host, whereas those with late-stage infections (i.e. infectious sporozoites) exhibit increased attraction. These stage-specific changes in behaviour were paralleled by changes in the responsiveness of mosquito odourant receptors, providing a possible neurophysiological mechanism for the responses. However, we also found that both the behavioural and neurophysiological changes could be generated by immune challenge with heat-killed Escherichia coli and were thus not tied explicitly to the presence of malaria parasites. Our results support the hypothesis that the feeding behaviour of female mosquitoes is altered by Plasmodium, but question the extent to which this is owing to active manipulation by malaria parasites of host behaviour.     

Circumsporozoite protein gene from Plasmodium brasilianum. Animal reservoirs for human malaria parasites?

We describe here the sequence of the circumsporozoite protein gene of the monkey malaria parasite Plasmodium brasilianum and show that the immunodominant repeat domain is the same as that of the human malaria parasite, Plasmodium malariae. The immunodominant epitope on the surface of sporozoites of a third species of human malaria parasite has, therefore, been identified. This genetic based data and the biological similarities between P. brasilianum and P. malariae support their putative zoonotic/anthroponotic relationship. We also show that an ape malaria parasite, Plasmodium reichenowi, and the human malaria parasite, Plasmodium falciparum, have a similar relationship. The implications of these observations are discussed with respect to vaccine development.     

Plasmodium yoelii: contribution of oocysts melanization to natural refractoriness in Anopheles dirus

It is well known that Anopheles dirus is naturally refractory to rodent malaria parasite, Plasmodium yoelii, but the mechanism is still largely unknown. Here, we found that some P. yoelii taken into An. dirus could develop into oocysts, but oocysts were partially melanized at 7 days and completely melanized at 15 days post-infectious blood meal. Transmission electronic microscopy could find the melanized P. yoelii oocysts in An. dirus as early as 5 days post-infection, with a few haemocytes attaching to the melanized oocysts, indicating a typical humoral melanization reaction. Although the change of protein pattern at 24h post-infection suggested that other unknown mechanisms and/or factors might be involved in killing ookinetes, our data implied that oocysts melanization was one of the mechanisms of An. dirus to block P. yoelii development. In addition, activity of phenoloxidase, such as monophenol oxidase and o-diphenoloxidase, in haemolymph of An. dirus fed on infectious blood meal was much higher than that of mosquitoes fed on 5% glucose or normal mouse blood (p<0.05), implying the possible role of PO in oocysts melanization by An. dirus.     

Plasmodium hermani: Experimental transmission by Culex salinarius and comparison with other susceptible Florida mosquitoes

Culex salinarius is susceptible to Plasmodium hermani, a malarial parasite of wild turkeys in Florida. The sporogonous cycle was completed and mosquitoes with infected salivary glands transmitted the parasite by bites. Transmission was also achieved by intraperitoneal and intravenous injections of whole body slurry. This is the third species found to be susceptible to turkey malaria in Florida. A comparison of C. salinarius with two other susceptible Florida mosquitoes, Culex nigripalpus and Wyeomyia vanduzeei, revealed that C. salinarius was more susceptible to P. hermani based on oocyst counts. C. nigripalpus has previously been demonstrated as an experimental and a natural vector of P. hermani, whereas W. vanduzeei has been designated as an experimental host only. In W. vanduzeei at least 30% of the oocysts were melanized (“black bodies”) and this mosquito did not transmit the parasite via bites. Additional detailed comparisons of comparative susceptibility and transmission potentials of these three species to turkey malaria, P. hermani, have been made.