Can Wolbachia be used to control malaria?

Malaria is a mosquito-borne infectious disease caused by Plasmodium parasites transmitted by the infectious bite of Anopheles mosquitoes. Vector control of malaria has predominantly focused on targeting the adult mosquito through insecticides and bed nets. However, current vector control methods are often not sustainable for long periods so alternative methods are needed. A novel biocontrol approach for mosquito-borne diseases has recently been proposed, it uses maternally inherited endosymbiotic Wolbachia bacteria transinfected into mosquitoes in order to interfere with pathogen transmission. Transinfected Wolbachia strains in Aedes aegypti mosquitoes, the primary vector of dengue fever, directly inhibit pathogen replication, including Plasmodium gallinaceum, and also affect mosquito reproduction to allow Wolbachia to spread through mosquito populations. In addition, transient Wolbachia infections in Anopheles gambiae significantly reduce Plasmodium levels. Here we review the prospects of using a Wolbachia-based approach to reduce human malaria transmission through transinfection of Anopheles mosquitoes.     

Plasmodium sex determination and transmission to mosquitoes

In order to be transmitted by their mosquito vector, malaria parasites undergo sexual reproduction, which occurs between specialized male and female parasites (gametes) within the blood meal in the mosquito. Nothing was known about how Plasmodium determines the sex of its gametocytes (gamete precursors), which are produced in the vertebrate host. Recently, erythropoietin, the vertebrate hormone controlling erythropoiesis in response to anaemia, was implicated in Plasmodium sex determination in animal models of malaria. This review examines the available information and addresses the relevance of such a sex determining mechanism for Plasmodium falciparum transmission to mosquitoes, with special reference to low gametocytaemias.     

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.     

Seasonal changes in the feeding pattern of Culex pipiens pallens govern the transmission dynamics of multiple lineages of avian malaria parasites in Japanese wild bird community

Heterogeneity in the transmission of mosquito-borne pathogens is determined largely by distribution patterns of mosquito bites among wild animal populations. Although mosquitoes are crucial for transmission of avian malaria parasites, little is known about the ecology of natural vectors. We examined bloodmeal and parasite incidence in Culex pipiens pallens by a polymerase chain reaction (PCR)-based procedure to determine how the feeding pattern of mosquitoes govern transmission dynamics of avian malaria parasites in Japanese wild birds. We collected 881 unfed and 486 blood-fed Cx. pipiens pallens resting on vegetation in a park in Tokyo. The mosquitoes were separated into abdomen and thorax prior to PCR screening. Abdomens of unfed mosquitoes were combined into 95 pools. From these, we amplified Plasmodium DNA in 32 (33.7%) pools. Among blood-fed mosquitoes, 371 individuals were screened for blood-sources and Plasmodium parasites. Plasmodium DNA was amplified from mosquitoes fed on 6 of 13 avian species identified as blood-sources. Ten Plasmodium lineages were identified on the basis of 478 bp of the cytochrome b gene, with 0.2-10% sequence divergence. The three commonest Plasmodium lineages (CXPIP09, SGS1 and PADOM02) were detected in both the abdomens and thoraxes of mosquitoes, strongly suggesting transmission of these lineages. Jungle crow (Corvus macrorhynchos) served as a natural host for the three commonest Plasmodium lineages and made up 63.8% of blood-sources. As a significant increase in feeding of vector mosquitoes on jungle crows coincided with their breeding season, jungle crows were considered to be the primary reservoir of Plasmodium transmission in this study.     

The effects of age, exposure history and malaria infection on the susceptibility of Anopheles mosquitoes to low concentrations of pyrethroid

Chemical insecticides are critical components of malaria control programs. Their ability to eliminate huge numbers of mosquitoes allows them to swiftly interrupt disease transmission, but that lethality also imposes immense selection for insecticide resistance. Targeting control at the small portion of the mosquito population actually responsible for transmitting malaria parasites to humans would reduce selection for resistance, yet maintain effective malaria control. Here, we ask whether simply lowering the concentration of the active ingredient in insecticide formulations could preferentially kill mosquitoes infected with malaria and/or those that are potentially infectious, namely, old mosquitoes. Using modified WHO resistance-monitoring assays, we exposed uninfected Anopheles stephensi females to low concentrations of the pyrethroid permethrin at days 4, 8, 12, and 16 days post-emergence and monitored survival for at least 30 days to evaluate the immediate and long-term effects of repeated exposure as mosquitoes aged. We also exposed Plasmodium chabaudi- and P. yoelii-infected An. stephensi females. Permethrin exposure did not consistently increase mosquito susceptibility to subsequent insecticide exposure, though older mosquitoes were more susceptible. A blood meal slightly improved survival after insecticide exposure; malaria infection did not detectably increase insecticide susceptibility. Exposure to low concentrations over successive feeding cycles substantially altered cohort age-structure. Our data suggest the possibility that, where high insecticide coverage can be achieved, low concentration formulations have the capacity to reduce disease transmission without the massive selection for resistance imposed by current practice.     

Local adaptation and vector-mediated population structure in Plasmodium vivax malaria

Plasmodium vivax in southern Mexico exhibits different infectivities to 2 local mosquito vectors, Anopheles pseudopunctipennis and Anopheles albimanus. Previous work has tied these differences in mosquito infectivity to variation in the central repeat motif of the malaria parasite's circumsporozoite (csp) gene, but subsequent studies have questioned this view. Here we present evidence that P. vivax in southern Mexico comprised 3 genetic populations whose distributions largely mirror those of the 2 mosquito vectors. Additionally, laboratory colony feeding experiments indicate that parasite populations are most compatible with sympatric mosquito species. Our results suggest that reciprocal selection between malaria parasites and mosquito vectors has led to local adaptation of the parasite. Adaptation to local vectors may play an important role in generating population structure in Plasmodium. A better understanding of coevolutionary dynamics between sympatric mosquitoes and parasites will facilitate the identification of molecular mechanisms relevant to disease transmission in nature and provide crucial information for malaria control.     

Synthetic propeptide inhibits mosquito midgut chitinase and blocks sporogonic development of malaria parasite

Incessant transmission of the parasite by mosquitoes makes most attempts to control malaria fail. Blocking of parasite transmission by mosquitoes therefore is a rational strategy to combat the disease. Upon ingestion of blood meal mosquitoes secrete chitinase into the midgut. This mosquito chitinase is a zymogen which is activated by the removal of a propeptide from the N-terminal. Since the midgut peritrophic matrix acts as a physical barrier, the activated chitinase is likely to contribute to the further development of the malaria parasite in the mosquito. Earlier it has been shown that inhibiting chitinase activity in the mosquito midgut blocked sporogonic development of the malaria parasite. Since synthetic propeptides of several zymogens have been found to be potent inhibitors of their respective enzymes, we tested propeptide of mosquito midgut chitinase as an inhibitor and found that the propeptide almost completely inhibited the recombinant or purified native Anopheles gambiae chitinase. We also examined the effect of the inhibitory peptide on malaria parasite development. The result showed that the synthetic propeptide blocked the development of human malaria parasite Plasmodium falciparum in the African malaria vector An. gambiae and avian malaria parasite Plasmodium gallinaceum in Aedes aegypti mosquitoes. This study implies that the expression of inhibitory mosquito midgut chitinase propeptide in response to blood meal may alter the mosquito's vectorial capacity. This may lead to developing novel strategies for controlling the spread of malaria.     

Controlling malaria transmission with genetically-engineered, Plasmodium-resistant mosquitoes: milestones in a model system

We are developing transgenic mosquitoes resistant to malaria parasites to test the hypothesis that genetically-engineered mosquitoes can be used to block the transmission of the parasites. We are developing and testing many of the necessary methodologies with the avian malaria parasite, Plasmodium gallinaceum, and its laboratory vector, Aedes aegypti, in anticipation of engaging the technical challenges presented by the malaria parasite, P. falciparum, and its major African vector, Anopheles gambiae. Transformation technology will be used to insert into the mosquito a synthetic gene for resistance to P. gallinaceum. The resistance gene will consist of a promoter of a mosquito gene controlling the expression of an effector protein that interferes with parasite development and/or infectivity. Mosquito genes whose promoter sequences are capable of sex- and tissue-specific expression of exogenous coding sequences have been identified, and stable transformation of the mosquito has been developed. We now are developing the expressed effector portion of the synthetic gene that will interfere with the transmission of the parasites. Mouse monoclonal antibodies that recognize the circumsporozoite protein of P. gallinaceum block sporozoite invasion of mosquito salivary glands, as well as abrogate the infectivity of sporozoites to a vertebrate host, the chicken, Gallus gallus, and block sporozoite invasion and development in susceptible cell lines in vitro. Using the genes encoding these antibodies, we propose to clone and express single-chain antibody constructs (scFv) that will serve as the effector portion of the gene that interferes with transmission of P. gallinaceum sporozoites.     

A mitogen-activated protein kinase regulates male gametogenesis and transmission of the malaria parasite Plasmodium berghei

Differentiation of malaria parasites into sexual forms (gametocytes) in the vertebrate host and their subsequent development into gametes in the mosquito vector are crucial steps in the completion of the parasite's life cycle and transmission of the disease. The molecular mechanisms that regulate the sexual cycle are poorly understood. Although several signal transduction pathways have been implicated, a clear understanding of the pathways involved has yet to emerge. Here, we show that a Plasmodium berghei homologue of Plasmodium falciparum mitogen-activated kinase-2 (Pfmap-2), a gametocyte-specific mitogen-activated protein kinase (MAPK), is required for male gamete formation. Parasites lacking Pbmap-2 are competent for gametocytogenesis, but exflagellation of male gametocytes, the process that leads to male gamete formation, is almost entirely abolished in mutant parasites. Consistent with this result, transmission of mutant parasites to mosquitoes is grossly impaired. This finding identifies a crucial role for a MAPK pathway in malaria transmission.     

The complex interplay between mosquito positive and negative regulators of Plasmodium development

The malaria parasite, Plasmodium, requires sexual development in the mosquito before it can be transmitted to the vertebrate host. Mosquito genes are able to substantially modulate this process, which can result in major decreases in parasite numbers. Even in susceptible mosquitoes, haemolymph proteins implicated in systemic immune reactions, together with local epithelial responses, cause lysis of more than 80% of the ookinetes that cross the mosquito midgut. In a refractory mosquito strain, immune responses lead to melanisation of virtually all parasites. Conversely, certain mosquito genes have an opposite effect: they are used by the parasite to evade defence reactions. Detailed understanding of the interplay between positive and negative regulators of parasite development could lead to the generation of novel approaches for malaria control through the vector.     

Temperature-Dependent Pre-Bloodmeal Period and Temperature-Driven Asynchrony between Parasite Development and Mosquito Biting Rate Reduce Malaria Transmission Intensity

A mosquito needs to bite at least twice for malaria transmission to occur: once to acquire parasites and, after these parasites complete their development in their mosquito host, once to transmit the parasites to the next vertebrate host. Here we investigate the relationship between temperature, parasite development, and biting frequency in a mosquito-rodent malaria model system. We show that the pre-bloodmeal period (the time lag between mosquito emergence and first bloodmeal) increases at lower temperatures. In addition, parasite development time and feeding exhibit different thermal sensitivities such that mosquitoes might not be ready to feed at the point at which the parasite is ready to be transmitted. Exploring these effects using a simple theoretical model of human malaria shows that delays in infection and transmission can reduce the vectorial capacity of malaria mosquitoes by 20 to over 60%, depending on temperature. These delays have important implications for disease epidemiology and control, and should be considered in future transmission models.     

Calcium and a calcium-dependent protein kinase regulate gamete formation and mosquito transmission in a malaria parasite

Transmission of malaria parasites to mosquitoes is initiated by the obligatory sexual reproduction of the parasite within the mosquito bloodmeal. Differentiation of specialized transmission stages, the gametocytes, into male and female gametes is induced by a small mosquito molecule, xanthurenic acid (XA). Using a Plasmodium berghei strain expressing a bioluminescent calcium sensor, we show that XA triggers a rapid rise in cytosolic calcium specifically in gametocytes that is essential for their differentiation into gametes. A member of a family of plant-like calcium dependent protein kinases, CDPK4, is identified as the molecular switch that translates the XA-induced calcium signal into a cellular response by regulating cell cycle progression in the male gametocyte. CDPK4 is shown to be essential for the sexual reproduction and mosquito transmission of P. berghei. This study reveals an unexpected function for a plant-like signaling pathway in cell cycle regulation and life cycle progression of a malaria parasite.     

Mosquito immunity against Plasmodium

Understanding the molecular mechanisms of the innate immune responses of Anopheles gambiae against Plasmodium parasites is of great importance for current efforts to develop novel strategies for malaria disease control. The parasite undergoes substantial stage-specific losses during its development in the mosquito, which in some cases lead to complete refractoriness of the mosquito against the parasite. The underlying genetics of refractoriness are complex and multifactorial. Completion of the genome sequence of An. gambiae 2 years ago, together with the development of DNA microarrays in this species and the extension of the RNAi technique to adult mosquitoes, has allowed comparative and functional genomic approaches of the mosquito innate immune system. A variety of factors were shown to negatively affect the development of Plasmodium parasites in the mosquito, in some cases leading to complete transmission blockage. In addition, mosquito factors have been identified that play positive roles and are required for successful transmission of the parasite. These findings indicate a highly complex interplay between parasite and vector. Research is continuing to identify new factors involved in this interaction and to decipher the interplay of these molecules and their regulation.     

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.     

The malaria parasite,Plasmodium falciparum,increases the frequency of multiple feeding of its mosquito vector,Anopheles gambiae.

It has often been suggested that vector-borne parasites alter their vector''s feeding behaviour to increase their transmission, but these claims are often based on laboratory studies and lack rigorous testing in a natural situation. We show in this field study that the malaria parasite, Plasmodium falciparum, alters the blood-feeding behaviour of its mosquito vector, Anopheles gambiae s.l., in two ways. First, mosquitoes infected with sporozoited, the parasite stage that is transmitted from the mosquito to a human, took up larger blood meals than uninfected mosquitoes. Whereas 72% of the uninfected mosquitoes had obtained a full blood meal, 82% of the infected ones had engorged fully. Second, mosquitoes harbouring sporozoites were more likely to bite several people per night. Twenty-two per cent of the infected mosquitoes, but only 10% of the uninfected mosquitoes, contained blood from at least two people. We conclude that the observed changes in blood-feeding behaviour allow the parasite to spread more rapidly among human hosts, and thus confirm that the parasite manipulates the mosquito to increase its own transmission.     

Investigations on anopheline mosquitoes close to the nest sites of chimpanzees subject to malaria infection in Ugandan Highlands

ABSTRACT: BACKGROUND: Malaria parasites (Plasmodium sp.), including new species, have recently been discovered as low grade mixed infections in three wild chimpanzees (Pan troglodytes schweinfurthii) sampled randomly in Kibale National Park, Uganda. This suggested a high prevalence of malaria infection in this community. The clinical course of malaria in chimpanzees and the species of the vectors that transmit their parasites are not known. The fact that these apes display a specific behaviour in which they consume plant parts of low nutritional value but that contain compounds with anti-malarial properties suggests that the apes' health might be affected by the parasite. The avoidance of the night-biting anopheline mosquitoes is another potential behavioural adaptation that would lead to a decrease in the number of infectious bites and consequently malaria. METHODS: Mosquitoes were collected over two years using suction-light traps and yeast-generated CO2 traps at the nesting and the feeding sites of two chimpanzee communities in Kibale National Park. The species of the female Anopheles caught were then determined and the presence of Plasmodium was sought in these insects by PCR amplification. RESULTS: The mosquito catches yielded a total of 309 female Anopheles specimens, the only known vectors of malaria parasites of mammalians. These specimens belonged to 10 species, of which Anopheles implexus, Anopheles vinckei and Anopheles demeilloni dominated. Sensitive DNA amplification techniques failed to detect any Plasmodium-positive Anopheles specimens. Humidity and trap height influenced the Anopheles capture success, and there was a negative correlation between nest numbers and mosquito abundance. The anopheline mosquitoes were also less diverse and numerous in sites where chimpanzees were nesting as compared to those where they were feeding. CONCLUSIONS: These observations suggest that the sites where chimpanzees build their nests every night might be selected, at least in part, in order to minimize contact with anopheline mosquitoes, which might lead to a reduced risk in acquiring malaria infections.     

Exceptional diversity, maintenance of polymorphism, and recent directional selection on the APL1 malaria resistance genes of Anopheles gambiae

The three-gene APL1 locus encodes essential components of the mosquito immune defense against malaria parasites. APL1 was originally identified because it lies within a mapped QTL conferring the vector mosquito Anopheles gambiae natural resistance to the human malaria parasite, Plasmodium falciparum, and APL1 genes have subsequently been shown to be involved in defense against several species of Plasmodium. Here, we examine molecular population genetic variation at the APL1 gene cluster in spatially and temporally diverse West African collections of A. gambiae. The locus is extremely polymorphic, showing evidence of adaptive evolutionary maintenance of genetic variation. We hypothesize that this variability aids in defense against genetically diverse pathogens, including Plasmodium. Variation at APL1 is highly structured across geographic and temporal subpopulations. In particular, diversity is exceptionally high during the rainy season, when malaria transmission rates are at their peak. Much less allelic diversity is observed during the dry season when mosquito population sizes and malaria transmission rates are low. APL1 diversity is weakly stratified by the polymorphic 2La chromosomal inversion but is very strongly subdivided between the M and S "molecular forms." We find evidence that a recent selective sweep has occurred at the APL1 locus in M form mosquitoes only. The independently reported observation of a similar M-form restricted sweep at the Tep1 locus, whose product physically interacts with APL1C, suggests that epistatic selection may act on these two loci causing them to sweep coordinately.     

High prevalence and lineage diversity of avian malaria in wild populations of great tits (Parus major) and mosquitoes (Culex pipiens)

Avian malaria studies have taken a prominent place in different aspects of evolutionary ecology. Despite a recent interest in the role of vectors within the complex interaction system of the malaria parasite, they have largely been ignored in most epidemiological studies. Epidemiology of the disease is however strongly related to the vector's ecology and behaviour, and there is a need for basic investigations to obtain a better picture of the natural associations between Plasmodium lineages, vector species and bird hosts. The aim of the present study was to identify the mosquito species involved in the transmission of the haemosporidian parasites Plasmodium spp. in two wild populations of breeding great tits (Parus major) in western Switzerland. Additionally, we compared Plasmodium lineages, based on mitochondrial DNA cytochrome b sequences, between the vertebrate and dipteran hosts, and evaluated the prevalence of the parasite in the mosquito populations. Plasmodium spp. were detected in Culex pipiens only, with an overall 6.6% prevalence. Among the six cytochrome b lineages of Plasmodium identified in the mosquitoes, three were also present in great tits. The results provide evidence for the first time that C. pipiens can act as a natural vector of avian malaria in Europe and yield baseline data for future research on the epidemiology of avian malaria in European countries.     

Quantitative trait loci controlling refractoriness to Plasmodium falciparum in natural Anopheles gambiae mosquitoes from a malaria-endemic region in western Kenya

Natural anopheline populations exhibit much variation in ability to support malaria parasite development, but the genetic mechanisms underlying this variation are not clear. Previous studies in Mali, West Africa, identified two quantitative trait loci (QTL) in Anopheles gambiae mosquitoes that confer refractoriness (failure of oocyst development in mosquito midguts) to natural Plasmodium falciparum parasites. We hypothesize that new QTL may be involved in mosquito refractoriness to malaria parasites and that the frequency of natural refractoriness genotypes may be higher in the basin region of Lake Victoria, East Africa, where malaria transmission intensity and parasite genetic diversity are among the highest in the world. Using field-derived F2 isofemale families and microsatellite marker genotyping, two loci significantly affecting oocyst density were identified: one on chromosome 2 between markers AG2H135 and AG2H603 and the second on chromosome 3 near marker AG3H93. The first locus was detected in three of the five isofemale families studied and colocalized to the same region as Pen3 and pfin1 described in other studies. The second locus was detected in two of the five isofemale families, and it appears to be a new QTL. QTL on chromosome 2 showed significant additive effects while those on chromosome 3 exhibited significant dominant effects. Identification of P. falciparum-refractoriness QTL in natural An. gambiae mosquitoes is critical to the identification of the genes involved in malaria parasite transmission in nature and for understanding the coevolution between malaria parasites and mosquito vectors.     

Malaria renders mice susceptible to mosquito feeding when gametocytes are most infective

Mice infected with Plasmodium berghei, P. chabaudi, or P. yoelii became lethargic and ceased to display normal antimosquito behavior. Periods of reduced defensiveness corresponded with maximum mosquito engorgement and with periods of maximum gametocyte infectivity to mosquitoes. Increased feeding success of mosquitoes during periods of peak gametocyte infectivity may be important to the natural maintenance of these malaria parasites.