Activation of Akt Signaling Reduces the Prevalence and Intensity of Malaria Parasite Infection and Lifespan in Anopheles stephensi Mosquitoes

Malaria (Plasmodium spp.) kills nearly one million people annually and this number will likely increase as drug and insecticide resistance reduces the effectiveness of current control strategies. The most important human malaria parasite, Plasmodium falciparum, undergoes a complex developmental cycle in the mosquito that takes approximately two weeks and begins with the invasion of the mosquito midgut. Here, we demonstrate that increased Akt signaling in the mosquito midgut disrupts parasite development and concurrently reduces the duration that mosquitoes are infective to humans. Specifically, we found that increased Akt signaling in the midgut of heterozygous Anopheles stephensi reduced the number of infected mosquitoes by 60–99%. Of those mosquitoes that were infected, we observed a 75–99% reduction in parasite load. In homozygous mosquitoes with increased Akt signaling parasite infection was completely blocked. The increase in midgut-specific Akt signaling also led to an 18–20% reduction in the average mosquito lifespan. Thus, activation of Akt signaling reduced the number of infected mosquitoes, the number of malaria parasites per infected mosquito, and the duration of mosquito infectivity.     

Inhibition of Malaria Infection in Transgenic Anopheline Mosquitoes Lacking Salivary Gland Cells

Malaria is an important global public health challenge, and is transmitted by anopheline mosquitoes during blood feeding. Mosquito vector control is one of the most effective methods to control malaria, and population replacement with genetically engineered mosquitoes to block its transmission is expected to become a new vector control strategy. The salivary glands are an effective target tissue for the expression of molecules that kill or inactivate malaria parasites. Moreover, salivary gland cells express a large number of molecules that facilitate blood feeding and parasite transmission to hosts. In the present study, we adapted a functional deficiency system in specific tissues by inducing cell death using the mouse Bcl-2-associated X protein (Bax) to the Asian malaria vector mosquito, Anopheles stephensi. We applied this technique to salivary gland cells, and produced a transgenic strain containing extremely low amounts of saliva. Although probing times for feeding on mice were longer in transgenic mosquitoes than in wild-type mosquitoes, transgenic mosquitoes still successfully ingested blood. Transgenic mosquitoes also exhibited a significant reduction in oocyst formation in the midgut in a rodent malaria model. These results indicate that mosquito saliva plays an important role in malaria infection in the midgut of anopheline mosquitoes. The dysfunction in the salivary glands enabled the inhibition of malaria transmission from hosts to mosquito midguts. Therefore, salivary components have potential in the development of new drugs or genetically engineered mosquitoes for malaria control.     

Rhoptry neck protein 11 has crucial roles during malaria parasite sporozoite invasion of salivary glands and hepatocytes

The malaria parasite sporozoite sequentially invades mosquito salivary glands and mammalian hepatocytes; and is the Plasmodium lifecycle infective form mediating parasite transmission by the mosquito vector. The identification of several sporozoite-specific secretory proteins involved in invasion has revealed that sporozoite motility and specific recognition of target cells are crucial for transmission. It has also been demonstrated that some components of the invasion machinery are conserved between erythrocytic asexual and transmission stage parasites. The application of a sporozoite stage-specific gene knockdown system in the rodent malaria parasite, Plasmodium berghei, enables us to investigate the roles of such proteins previously intractable to study due to their essentiality for asexual intraerythrocytic stage development, the stage at which transgenic parasites are derived. Here, we focused on the rhoptry neck protein 11 (RON11) that contains multiple transmembrane domains and putative calcium-binding EF-hand domains. PbRON11 is localised to rhoptry organelles in both merozoites and sporozoites. To repress PbRON11 expression exclusively in sporozoites, we produced transgenic parasites using a promoter-swapping strategy. PbRON11-repressed sporozoites showed significant reduction in attachment and motility in vitro, and consequently failed to efficiently invade salivary glands. PbRON11 was also determined to be essential for sporozoite infection of the liver, the first step during transmission to the vertebrate host. RON11 is demonstrated to be crucial for sporozoite invasion of both target host cells – mosquito salivary glands and mammalian hepatocytes – via involvement in sporozoite motility.     

Plasmodium falciparum Mating Patterns and Mosquito Infectivity of Natural Isolates of Gametocytes

Plasmodium falciparum infections in malaria endemic areas often harbor multiple clones of parasites. However, the transmission success of the different genotypes within the mosquito vector has remained elusive so far. The genetic diversity of malaria parasites was measured by using microsatellite markers in gametocyte isolates from 125 asymptomatic carriers. For a subset of 49 carriers, the dynamics of co-infecting genotypes was followed until their development within salivary glands. Also, individual oocysts from midguts infected with blood from 9 donors were genotyped to assess mating patterns. Multiplicity of infection (MOI) was high both in gametocyte isolates and sporozoite populations, reaching up to 10 genotypes. Gametocyte isolates with multiple genotypes gave rise to lower infection prevalence and intensity. Fluctuations of genotype number occurred during the development within the mosquito and sub-patent genotypes, not detected in gametocyte isolates, were identified in the vector salivary glands. The inbreeding coefficient Fis was positively correlated to the oocyst loads, suggesting that P. falciparum parasites use different reproductive strategies according to the genotypes present in the gametocyte isolate. The number of parasite clones within an infection affects the transmission success and the mosquito has an important role in maintaining P. falciparum genetic diversity. Our results emphasize the crucial importance of discriminating between the different genotypes within an infection when studying the A. gambiae natural resistance to P. falciparum, and the need to monitor parasite diversity in areas where malaria control interventions are implemented.     

A putative kinase-related protein (PKRP) from Plasmodium berghei mediates infection in the midgut and salivary glands of the mosquito

The completion of the Plasmodium (malaria) life cycle in the mosquito requires the parasite to traverse first the midgut and later the salivary gland epithelium. We have identified a putative kinase-related protein (PKRP) that is predicted to be an atypical protein kinase, which is conserved across many species of Plasmodium. The pkrp gene encodes a RNA of about 5300 nucleotides that is expressed as a 90 kDa protein in sporozoites. Targeted disruption of the pkrp gene in Plasmodium berghei, a rodent model of malaria, compromises the ability of parasites to infect different tissues within the mosquito host. Early infection of mosquito midgut is reduced by 58-71%, midgut oocyst production is reduced by 50-90% and those sporozoites that are produced are defective in their ability to invade mosquito salivary glands. Midgut sporozoites are not morphologically different from wild-type parasites by electron microscopy. Some sporozoites that emerged from oocysts were attached to the salivary glands but most were found circulating in the mosquito hemocoel. Our findings indicate that a signalling pathway involving PbPKRP regulates the level of Plasmodium infection in the mosquito midgut and salivary glands.     

Mosquito-Bacteria Symbiosis: The Case of Anopheles gambiae and Asaia

The symbiotic relationship between Asaia, an α-proteobacterium belonging to the family Acetobacteriaceae, and mosquitoes has been studied mainly in the Asian malaria vector Anopheles stephensi. Thus, we have investigated the nature of the association between Asaia and the major Afro-tropical malaria vector Anopheles gambiae. We have isolated Asaia from different wild and laboratory reared colonies of A. gambiae, and it was detected by PCR in all the developmental stages of the mosquito and in all the specimens analyzed. Additionally, we have shown that it localizes in the midgut, salivary glands and reproductive organs. Using recombinant strains of Asaia expressing fluorescent proteins, we have demonstrated the ability of the bacterium to colonize A. gambiae mosquitoes with a pattern similar to that described for A. stephensi. Finally, fluorescent in situ hybridization on the reproductive tract of females of A. gambiae showed a concentration of Asaia at the very periphery of the eggs, suggesting that transmission of Asaia from mother to offspring is likely mediated by a mechanism of egg-smearing. We suggest that Asaia has potential for use in the paratransgenic control of malaria transmitted by A. gambiae.     

Blood Feeding and Plasmodium Infection Alters the miRNome of Anopheles stephensi

Blood feeding is an integral process required for physiological functions and propagation of the malaria vector Anopheles. During blood feeding, presence of the malaria parasite, Plasmodium in the blood induces several host effector molecules including microRNAs which play important roles in the development and maturation of the parasite within the mosquito. The present study was undertaken to elucidate the dynamic expression of miRNAs during gonotrophic cycle and parasite development in Anopheles stephensi. Using next generation sequencing technology, we identified 126 miRNAs of which 17 were novel miRNAs. The miRNAs were further validated by northern hybridization and cloning. Blood feeding and parasitized blood feeding in the mosquitoes revealed regulation of 13 and 16 miRNAs respectively. Expression profiling of these miRNAs revealed that significant miRNAs were down-regulated upon parasitized blood feeding with a repertoire of miRNAs showing stage specific up-regulation. Expression profiles of significantly modulated miRNAs were further validated by real time PCR. Target prediction of regulated miRNAs revealed overlapping targeting by different miRNAs. These targets included several metabolic pathways including metabolic, redox homeostasis and protein processing machinery components. Our analysis revealed tight regulation of specific miRNAs post blood feeding and parasite infection in An. stephensi. Such regulated expression suggests possible role of these miRNAs during gonotrophic cycle in mosquito. Another set of miRNAs were also significantly regulated at 42 h and 5 days post infection indicating parasite stage-specific role of host miRNAs. This study will result in better understanding of the role of miRNAs during gonotrophic cycle and parasite development in mosquito and can probably facilitate in devising novel malaria control strategies at vector level.     

Plasmodium cynomolgi: the comparative infectiousness of individual rhesus monkeys

Five rhesus monkeys were infected with the malaria parasite Plasmodium cynomolgi Mayer. Anopheles stephensi Liston mosquitoes were fed on each monkey over the period of prepremunitive gametocytemia. Individual monkeys did not differ significantly in either mean daily gametocyte count (median = 1300 gametocytes per mosquito blood meal volume per day) or mean daily oocyst production (median = 34 oocysts per mosquito per day). Significant differences among monkeys in daily oocyst/gametocyte conversion ratio were attributable to essentially random correlation effects. The observed range in duration of the period of prepremunitive gametocytemia was 14–43 days. Total oocyst production over this period, as calculated for a unit mosquito biting rate of one per day, ranged from 130 to 2800 oocysts. The overall efficiency of conversion of gametocytes to oocysts in A. stephensi was estimated at 0.02 oocysts per gametocyte.     

Multiple blood feeding in mosquitoes shortens the Plasmodium falciparum incubation period and increases malaria transmission potential

Many mosquito species, including the major malaria vector Anopheles gambiae, naturally undergo multiple reproductive cycles of blood feeding, egg development and egg laying in their lifespan. Such complex mosquito behavior is regularly overlooked when mosquitoes are experimentally infected with malaria parasites, limiting our ability to accurately describe potential effects on transmission. Here, we examine how Plasmodium falciparum development and transmission potential is impacted when infected mosquitoes feed an additional time. We measured P. falciparum oocyst size and performed sporozoite time course analyses to determine the parasite’s extrinsic incubation period (EIP), i.e. the time required by parasites to reach infectious sporozoite stages, in An. gambiae females blood fed either once or twice. An additional blood feed at 3 days post infection drastically accelerates oocyst growth rates, causing earlier sporozoite accumulation in the salivary glands, thereby shortening the EIP (reduction of 2.3 ± 0.4 days). Moreover, parasite growth is further accelerated in transgenic mosquitoes with reduced reproductive capacity, which mimic genetic modifications currently proposed in population suppression gene drives. We incorporate our shortened EIP values into a measure of transmission potential, the basic reproduction number R₀, and find the average R₀ is higher (range: 10.1%–12.1% increase) across sub-Saharan Africa than when using traditional EIP measurements. These data suggest that malaria elimination may be substantially more challenging and that younger mosquitoes or those with reduced reproductive ability may provide a larger contribution to infection than currently believed. Our findings have profound implications for current and future mosquito control interventions.     

Acetylcholinesterases from the Disease Vectors Aedes aegypti and Anopheles gambiae: Functional Characterization and Comparisons with Vertebrate Orthologues

Mosquitoes of the Anopheles (An.) and Aedes (Ae.) genus are principal vectors of human diseases including malaria, dengue and yellow fever. Insecticide-based vector control is an established and important way of preventing transmission of such infections. Currently used insecticides can efficiently control mosquito populations, but there are growing concerns about emerging resistance, off-target toxicity and their ability to alter ecosystems. A potential target for the development of insecticides with reduced off-target toxicity is the cholinergic enzyme acetylcholinesterase (AChE). Herein, we report cloning, baculoviral expression and functional characterization of the wild-type AChE genes (ace-1) from An. gambiae and Ae. aegypti, including a naturally occurring insecticide-resistant (G119S) mutant of An. gambiae. Using enzymatic digestion and liquid chromatography-tandem mass spectrometry we found that the secreted proteins were post-translationally modified. The Michaelis-Menten constants and turnover numbers of the mosquito enzymes were lower than those of the orthologous AChEs from Mus musculus and Homo sapiens. We also found that the G119S substitution reduced the turnover rate of substrates and the potency of selected covalent inhibitors. Furthermore, non-covalent inhibitors were less sensitive to the G119S substitution and differentiate the mosquito enzymes from corresponding vertebrate enzymes. Our findings indicate that it may be possible to develop selective non-covalent inhibitors that effectively target both the wild-type and insecticide resistant mutants of mosquito AChE.     

Overexpression of phosphatase and tensin homolog improves fitness and decreases Plasmodium falciparum development in Anopheles stephensi

The insulin/insulin-like growth factor signaling (IIS) cascade is highly conserved and regulates diverse physiological processes such as metabolism, lifespan, reproduction and immunity. Transgenic overexpression of Akt, a critical regulator of IIS, was previously shown to shorten mosquito lifespan and increase resistance to the human malaria parasite Plasmodium falciparum. To further understand how IIS controls mosquito physiology and resistance to malaria parasite infection, we overexpressed an inhibitor of IIS, phosphatase and tensin homolog (PTEN), in the Anopheles stephensi midgut. PTEN overexpression inhibited phosphorylation of the IIS protein FOXO, an expected target for PTEN, in the midgut of A. stephensi. Further, PTEN overexpression extended mosquito lifespan and increased resistance to P. falciparum development. The reduction in parasite development did not appear to be due to alterations in an innate immune response, but rather was associated with increased expression of genes regulating autophagy and stem cell maintenance in the midgut and with enhanced midgut barrier integrity. In light of previous success in genetically targeting the IIS pathway to alter mosquito lifespan and malaria parasite transmission, these data confirm that multiple strategies to genetically manipulate IIS can be leveraged to generate fit, resistant mosquitoes for malaria control.     

Evaluation of Insecticides Susceptibility and Malaria Vector Potential of Anopheles annularis s.l. and Anopheles vagus in Assam,India

During the recent past, development of DDT resistance and reduction to pyrethroid susceptibility among the malaria vectors has posed a serious challenge in many Southeast Asian countries including India. Current study presents the insecticide susceptibility and knock-down data of field collected Anopheles annularis sensu lato and An. vagus mosquito species from endemic areas of Assam in northeast India. Anopheles annularis s.l. and An. vagus adult females were collected from four randomly selected sentinel sites in Orang primary health centre (OPHC) and Balipara primary health centre (BPHC) areas, and used for testing susceptibility to DDT, malathion, deltamethrin and lambda-cyhalothrin. After insecticide susceptibility tests, mosquitoes were subjected to VectorTest^™ assay kits to detect the presence of malaria sporozoite in the mosquitoes. An. annularis s.l. was completely susceptible to deltamethrin, lambda-cyhalothrin and malathion in both the study areas. An. vagus was highly susceptible to deltamethrin in both the areas, but exhibited reduced susceptibility to lambda-cyhalothrin in BPHC. Both the species were resistant to DDT and showed very high KDT₅₀ and KDT₉₉ values for DDT. Probit model used to calculate the KDT₅₀ and KDT₉₉ values did not display normal distribution of percent knock-down with time for malathion in both the mosquito species in OPHC (p<0.05) and An. vagus in BPHC (χ² = 25.3; p = 0.0), and also for deltamethrin to An. vagus in BPHC area (χ² = 15.4; p = 0.004). Minimum infection rate (MIR) of Plasmodium sporozoite for An. vagus was 0.56 in OPHC and 0.13 in BPHC, while for An. annularis MIR was found to be 0.22 in OPHC. Resistance management strategies should be identified to delay the expansion of resistance. Testing of field caught Anopheles vectors from different endemic areas for the presence of malaria sporozoite may be useful to ensure their role in malaria transmission.     

Anopheles Gambiae PRS1 Modulates Plasmodium Development at Both Midgut and Salivary Gland Steps

Background

Invasion of the mosquito salivary glands by Plasmodium is a critical step for malaria transmission. From a SAGE analysis, we previously identified several genes whose expression in salivary glands was regulated coincident with sporozoite invasion of salivary glands. To get insights into the consequences of these salivary gland responses, here we have studied one of the genes, PRS1 (Plasmodium responsive salivary 1), whose expression was upregulated in infected glands, using immunolocalization and functional inactivation approaches.

Methodology/Principal Findings

PRS1 belongs to a novel insect superfamily of genes encoding proteins with DM9 repeat motifs of uncharacterized function. We show that PRS1 is induced in response to Plasmodium, not only in the salivary glands but also in the midgut, the other epithelial barrier that Plasmodium has to cross to develop in the mosquito. Furthermore, this induction is observed using either the rodent parasite Plasmodium berghei or the human pathogen Plasmodium falciparum. In the midgut, PRS1 overexpression is associated with a relocalization of the protein at the periphery of invaded cells. We also find that sporozoite invasion of salivary gland cells occurs sequentially and induces intra-cellular modifications that include an increase in PRS1 expression and a relocalization of the corresponding protein into vesicle-like structures. Importantly, PRS1 knockdown during the onset of midgut and salivary gland invasion demonstrates that PRS1 acts as an agonist for the development of both parasite species in the two epithelia, highlighting shared vector/parasite interactions in both tissues.

Conclusions/Significance

While providing insights into potential functions of DM9 proteins, our results reveal that PRS1 likely contributes to fundamental interactions between Plasmodium and mosquito epithelia, which do not depend on the specific Anopheles/P. falciparum coevolutionary history.     

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.     

The Puf-family RNA-binding protein Puf2 controls sporozoite conversion to liver stages in the malaria parasite

Malaria is a vector-borne infectious disease caused by unicellular, obligate intracellular parasites of the genus Plasmodium. During host switch the malaria parasite employs specialized latent stages that colonize the new host environment. Previous work has established that gametocytes, sexually differentiated stages that are taken up by the mosquito vector, control expression of genes required for mosquito colonization by translational repression. Sexual parasite development is controlled by a DEAD-box RNA helicase of the DDX6 family, termed DOZI. Latency of sporozoites, the transmission stage injected during an infectious blood meal, is controlled by the eIF2alpha kinase IK2, a general inhibitor of protein synthesis. Whether RNA-binding proteins participate in translational regulation in sporozoites remains to be studied. Here, we investigated the roles of two RNA-binding proteins of the Puf-family, Plasmodium Puf1 and Puf2, during sporozoite stage conversion. Our data reveal that, in the rodent malaria parasite P. berghei, Puf2 participates in the regulation of IK2 and inhibits premature sporozoite transformation. Inside mosquito salivary glands puf2(-) sporozoites transform over time to round forms resembling early intra-hepatic stages. As a result, mutant parasites display strong defects in initiating a malaria infection. In contrast, Puf1 is dispensable in vivo throughout the entire Plasmodium life cycle. Our findings support the notion of a central role for Puf2 in parasite latency during switch between the insect and mammalian hosts.     

Protein Kinase C-Dependent Signaling Controls the Midgut Epithelial Barrier to Malaria Parasite Infection in Anopheline Mosquitoes

Anopheline mosquitoes are the primary vectors of parasites in the genus Plasmodium, the causative agents of malaria. Malaria parasites undergo a series of complex transformations upon ingestion by the mosquito host. During this process, the physical barrier of the midgut epithelium, along with innate immune defenses, functionally restrict parasite development. Although these defenses have been studied for some time, the regulatory factors that control them are poorly understood. The protein kinase C (PKC) gene family consists of serine/threonine kinases that serve as central signaling molecules and regulators of a broad spectrum of cellular processes including epithelial barrier function and immunity. Indeed, PKCs are highly conserved, ranging from 7 isoforms in Drosophila to 16 isoforms in mammals, yet none have been identified in mosquitoes. Despite conservation of the PKC gene family and their potential as targets for transmission-blocking strategies for malaria, no direct connections between PKCs, the mosquito immune response or epithelial barrier integrity are known. Here, we identify and characterize six PKC gene family members – PKCδ, PKCε, PKCζ, PKD, PKN, and an indeterminate conventional PKC − in Anopheles gambiae and Anopheles stephensi. Sequence and phylogenetic analyses of the anopheline PKCs support most subfamily assignments. All six PKCs are expressed in the midgut epithelia of A. gambiae and A. stephensi post-blood feeding, indicating availability for signaling in a tissue that is critical for malaria parasite development. Although inhibition of PKC enzymatic activity decreased NF-κB-regulated anti-microbial peptide expression in mosquito cells in vitro, PKC inhibition had no effect on expression of a panel of immune genes in the midgut epithelium in vivo. PKC inhibition did, however, significantly increase midgut barrier integrity and decrease development of P. falciparum oocysts in A. stephensi, suggesting that PKC-dependent signaling is a negative regulator of epithelial barrier function and a potential new target for transmission-blocking strategies.