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.     

Midgut Microbiota of the Malaria Mosquito Vector Anopheles gambiae and Interactions with Plasmodium falciparum Infection

The susceptibility of Anopheles mosquitoes to Plasmodium infections relies on complex interactions between the insect vector and the malaria parasite. A number of studies have shown that the mosquito innate immune responses play an important role in controlling the malaria infection and that the strength of parasite clearance is under genetic control, but little is known about the influence of environmental factors on the transmission success. We present here evidence that the composition of the vector gut microbiota is one of the major components that determine the outcome of mosquito infections. A. gambiae mosquitoes collected in natural breeding sites from Cameroon were experimentally challenged with a wild P. falciparum isolate, and their gut bacterial content was submitted for pyrosequencing analysis. The meta-taxogenomic approach revealed a broader richness of the midgut bacterial flora than previously described. Unexpectedly, the majority of bacterial species were found in only a small proportion of mosquitoes, and only 20 genera were shared by 80% of individuals. We show that observed differences in gut bacterial flora of adult mosquitoes is a result of breeding in distinct sites, suggesting that the native aquatic source where larvae were grown determines the composition of the midgut microbiota. Importantly, the abundance of Enterobacteriaceae in the mosquito midgut correlates significantly with the Plasmodium infection status. This striking relationship highlights the role of natural gut environment in parasite transmission. Deciphering microbe-pathogen interactions offers new perspectives to control disease transmission.     

Morphological evidence for proliferative regeneration of the Anopheles stephensi midgut epithelium following Plasmodium falciparum ookinete invasion

Ookinetes are motile invasive stages of the malaria parasite that enter the midgut epithelium of the mosquito vector via an intracellular route. Ookinetes often migrate through multiple adjacent midgut epithelial cells, which subsequently undergo apoptosis/necrosis and are extruded from the midgut epithelium into the midgut lumen. Hundreds of ookinetes may simultaneously invade the midgut epithelium, causing destruction of an appreciable proportion of the total number of midgut epithelial cells. However, there is little evidence that ookinete invasion of the midgut epithelium per se is detrimental to the survival of the mosquito vector implying that efficient mechanisms exist to restore the damaged midgut epithelium following malaria parasite infection. Proliferation and differentiation of precursor stem cells could replace the midgut epithelial cells destroyed and lost as a consequence of ookinete invasion. Although the existence of so-called “regenerative” cells within the mosquito midgut epithelium has long been recognized, there has been no previously published evidence for proliferation/differentiation of these putative precursor midgut epithelial cells in mature adult female mosquitoes. In the current study, examination of Giemsa-stained histological sections from Anopheles stephensi mosquito midguts infected with the human malaria parasite Plasmodium falciparum provided morphological evidence that regenerative cells undergo division and subsequent differentiation into normal columnar midgut epithelial cells. Furthermore, the number of these putatively proliferating/differentiating regenerative cells was significantly higher in P. falciparum-infected compared to uninfected mosquitoes, and was positively correlated with both the level of malaria parasite infection and midgut epithelial cell destruction. The loss of invaded midgut epithelial cells associated with intracellular migration by ookinetes, therefore, appears to trigger, and to be compensated by, proliferative regeneration of the mosquito midgut epithelium.     

Plasmodium yoelii-Infected A. stephensi Inefficiently Transmit Malaria Compared to Intravenous Route

It was recently reported that when mosquitoes infected with P. berghei sporozoites feed on mice, they deposit approximately 100–300 sporozoites in the dermis. When we inoculate P. yoelii (Py) sporozoites intravenously (IV) into BALB/c mice, the 50% infectious dose (ID₅₀) is often less than 3 sporozoites, indicating that essentially all Py sporozoites in salivary glands are infectious. Thus, it should only take the bite of one infected mosquito to infect 100% of mice. In human subjects, it takes the bite of at least 5 P. falciparum-infected mosquitoes to achieve 100% blood stage infection. Exposure to 1–2 infected mosquitoes only leads to blood stage infection in approximately 50% of subjects. If mosquitoes carrying Py sporozoites inoculate 100–300 sporozoites per bite, and 1 to 2 mosquito bites achieve 50% blood stage infection rates, then this would suggest that the majority of sporozoites inoculated by mosquitoes into the dermis are not responsible for a productive infection, or that a significant number of sporozoite-infected mosquitoes do not inoculate any sporozoites. The objective of this study was to determine if this is the case. We therefore studied the infectivity to mice of the bites of 1, 2, 4, or 5–8 Py-infected mosquitoes. The bite of one Py sporozoite-infected mosquito caused blood stage infection in 41.4% (12/29) of mice, two bites infected 66.7% (22/33), four bites infected 75% (18/24), and five to eight bites infected 100% (21/21). These findings demonstrate that inoculation of sporozoites by mosquito bite is much less efficient than IV inoculation of Py sporozoites by needle and syringe. Such data may have implications for determining the best route and dose of administration to humans of our attenuated P. falciparum sporozoite vaccine, the scientific basis of which is immunity by bites from irradiated infected mosquitoes, and suggest that the challenge is to develop a method of administration that approximates IV inoculation, not one that mimics mosquito bite.     

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.     

Morphological evidence for proliferative regeneration of the Anopheles stephensi midgut epithelium following Plasmodium falciparum ookinete invasion

Ookinetes are motile invasive stages of the malaria parasite that enter the midgut epithelium of the mosquito vector via an intracellular route. Ookinetes often migrate through multiple adjacent midgut epithelial cells, which subsequently undergo apoptosis/necrosis and are extruded from the midgut epithelium into the midgut lumen. Hundreds of ookinetes may simultaneously invade the midgut epithelium, causing destruction of an appreciable proportion of the total number of midgut epithelial cells. However, there is little evidence that ookinete invasion of the midgut epithelium per se is detrimental to the survival of the mosquito vector implying that efficient mechanisms exist to restore the damaged midgut epithelium following malaria parasite infection. Proliferation and differentiation of precursor stem cells could replace the midgut epithelial cells destroyed and lost as a consequence of ookinete invasion. Although the existence of so-called "regenerative" cells within the mosquito midgut epithelium has long been recognized, there has been no previously published evidence for proliferation/differentiation of these putative precursor midgut epithelial cells in mature adult female mosquitoes. In the current study, examination of Giemsa-stained histological sections from Anopheles stephensi mosquito midguts infected with the human malaria parasite Plasmodium falciparum provided morphological evidence that regenerative cells undergo division and subsequent differentiation into normal columnar midgut epithelial cells. Furthermore, the number of these putatively proliferating/differentiating regenerative cells was significantly higher in P. falciparum-infected compared to uninfected mosquitoes, and was positively correlated with both the level of malaria parasite infection and midgut epithelial cell destruction. The loss of invaded midgut epithelial cells associated with intracellular migration by ookinetes, therefore, appears to trigger, and to be compensated by, proliferative regeneration of the mosquito midgut epithelium.     

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.     

Reverse genetics in the mosquito Anopheles gambiae: targeted disruption of the Defensin gene

Anopheles gambiae, the major vector of human malaria parasite, is an important insect model to study vector–parasite interactions. Here, we developed a simple in vivo double-stranded RNA (dsRNA) knockout approach to determine the function of the mosquito antimicrobial peptide gene Defensin. We injected dsRNA into adults and observed efficient and reproducible silencing of Defensin. Analysis of the knockdown phenotype revealed that this peptide is required for the mosquito antimicrobial defense against Gram-positive bacteria. In contrast, in mosquitoes infected by Plasmodium berghei, no loss of mosquito viability and no significant effect on the development and morphology of the parasite midgut stages were observed in the absence of Defensin. We conclude that this peptide is not a major antiparasitic factor in A. gambiae in vivo. Our results open new perspectives for the study of mosquito gene function in vivo and provide a basis for genome-scale systematic functional screens by targeted gene silencing.     

Wolbachia increases susceptibility to Plasmodium infection in a natural system

Current views about the impact of Wolbachia on Plasmodium infections are almost entirely based on data regarding artificially transfected mosquitoes. This work has shown that Wolbachia reduces the intensity of Plasmodium infections in mosquitoes, raising the exciting possibility of using Wolbachia to control or limit the spread of malaria. Whether natural Wolbachia infections have the same parasite-inhibiting properties is not yet clear. Wolbachia–mosquito combinations with a long evolutionary history are, however, key for understanding what may happen with Wolbachia-transfected mosquitoes after several generations of coevolution. We investigate this issue using an entirely natural mosquito–Wolbachia–Plasmodium combination. In contrast to most previous studies, which have been centred on the quantification of the midgut stages of Plasmodium, we obtain a measurement of parasitaemia that relates directly to transmission by following infections to the salivary gland stages. We show that Wolbachia increases the susceptibility of Culex pipiens mosquitoes to Plasmodium relictum, significantly increasing the prevalence of salivary gland stage infections. This effect is independent of the density of Wolbachia in the mosquito. These results suggest that naturally Wolbachia-infected mosquitoes may, in fact, be better vectors of malaria than Wolbachia-free ones.     

Human IGF1 Regulates Midgut Oxidative Stress and Epithelial Homeostasis to Balance Lifespan and Plasmodium falciparum resistance in Anopheles stephensi

Insulin and insulin-like growth factor signaling (IIS) regulates cell death, repair, autophagy, and renewal in response to stress, damage, and pathogen challenge. Therefore, IIS is fundamental to lifespan and disease resistance. Previously, we showed that insulin-like growth factor 1 (IGF1) within a physiologically relevant range (0.013–0.13 µM) in human blood reduced development of the human parasite Plasmodium falciparum in the Indian malaria mosquito Anopheles stephensi. Low IGF1 (0.013 µM) induced FOXO and p70S6K activation in the midgut and extended mosquito lifespan, whereas high IGF1 (0.13 µM) did not. In this study the physiological effects of low and high IGF1 were examined in detail to infer mechanisms for their dichotomous effects on mosquito resistance and lifespan. Following ingestion, low IGF1 induced phosphorylation of midgut c-Jun-N-terminal kinase (JNK), a critical regulator of epithelial homeostasis, but high IGF1 did not. Low and high IGF1 induced midgut mitochondrial reactive oxygen species (ROS) synthesis and nitric oxide (NO) synthase gene expression, responses which were necessary and sufficient to mediate IGF1 inhibition of P. falciparum development. However, increased ROS and apoptosis-associated caspase-3 activity returned to baseline levels following low IGF1 treatment, but were sustained with high IGF1 treatment and accompanied by aberrant expression of biomarkers for mitophagy, stem cell division and proliferation. Low IGF1-induced ROS are likely moderated by JNK-induced epithelial cytoprotection as well as p70S6K-mediated growth and inhibition of apoptosis over the lifetime of A. stephensi to facilitate midgut homeostasis and enhanced survivorship. Hence, mitochondrial integrity and homeostasis in the midgut, a key signaling center for IIS, can be targeted to coordinately optimize mosquito fitness and anti-pathogen resistance for improved control strategies for malaria and other vector-borne diseases.     

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.     

Differential expression of proteins in the midgut of Anopheles albimanus infected with Plasmodium berghei

The main vector for transmission of malaria in Mexico is the Anopheles albimanus mosquito. The midgut of disease-transmitting mosquitoes carries out a variety of functions that are related to blood feeding. We analyzed the midgut of A. albimanus infected with Plasmodium berghei (resistant mosquito) using a proteomic approach to identify putative short peptides that are enriched in the midgut after blood feeding. Mosquito midguts were analyzed by two-dimensional electrophoresis to determine the changes in protein profiles. We identified 21 spot proteins that are differentially expressed in the blood of mosquitoes during the immune challenge. Molecular weight of the spots varied from 13 to 36 kDa, with a broad isoelectric point range of 3.92–8.90. We identified the differentially expressed proteins using mass spectrometry and constructed a proteomic data base of the A. albimanus midgut with diverse functions, some of them proteins with digestive and immunologic functions. Identification of these proteins may have important implications for understanding the blood meal digestion process, as well as developing novel vector control strategies and understanding parasite vector interactions.     

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.     

The transmission-blocking effects of antimalarial drugs revisited: fitness costs and sporontocidal effects of artesunate and sulfadoxine-pyrimethamine

Assays used to evaluate the transmission-blocking activity of antimalarial drugs are largely focused on their potential to inhibit or reduce the infectivity of gametocytes, the blood stages of the parasite that are responsible for the onward transmission to the mosquito vector. For this purpose, the drug is administered concomitantly with gametocyte-infected blood, and the results are evaluated as the percentage of reduction in the number of oocysts in the mosquito midgut. We report the results of a series of experiments that explore the transmission-blocking potential of two key antimalarial drugs, artesunate and sulfadoxine-pyrimethamine, when administered to mosquitoes already infected from a previous blood meal. For this purpose, uninfected mosquitoes and mosquitoes carrying a 6 day old Plasmodium relictum infection (early oocyst stages) were allowed to feed either on a drug-treated or an untreated host in a fully factorial experiment. This protocol allowed us to bypass the gametocyte stages and establish whether the drugs have a sporontocidal effect, i.e. whether they are able to arrest the ongoing development of oocysts and sporozoites, as would be the case when a mosquito takes a post-infection treated blood meal. In a separate experiment, we also explored whether a drug-treated blood meal impacted key life history traits of the mosquito relevant for transmission, and if this depended on their infection status. Our results showed that feeding on an artesunate- or sulfadoxine-pyrimethamine-treated hosts has no epidemiologically relevant effects on the fitness of infected or uninfected mosquitoes. In contrast, when infected mosquitoes fed on an sulfadoxine-pyrimethamine-treated host, we observed both a significant increase in the number of oocysts in the midgut, and a drastic decrease in both sporozoite prevalence (?30%) and burden (?80%) compared with the untreated controls. We discuss the potential mechanisms underlying these seemingly contradictory results and contend that, provided the results are translatable to human malaria, the potential epidemiological and evolutionary consequences of the current preventive use of sulfadoxine-pyrimethamine in malaria-endemic countries could be substantial.     

Naturally Occurring Triggers that Induce Apoptosis-Like Programmed Cell Death in Plasmodium berghei Ookinetes

Several protozoan parasites have been shown to undergo a form of programmed cell death that exhibits morphological features associated with metazoan apoptosis. These include the rodent malaria parasite, Plasmodium berghei. Malaria zygotes develop in the mosquito midgut lumen, forming motile ookinetes. Up to 50% of these exhibit phenotypic markers of apoptosis; as do those grown in culture. We hypothesised that naturally occurring signals induce many ookinetes to undergo apoptosis before midgut traversal. To determine whether nitric oxide and reactive oxygen species act as such triggers, ookinetes were cultured with donors of these molecules. Exposure to the nitric oxide donor SNP induced a significant increase in ookinetes with condensed nuclear chromatin, activated caspase-like molecules and translocation of phosphatidylserine that was dose and time related. Results from an assay that detects the potential-dependent accumulation of aggregates of JC-1 in mitochondria suggested that nitric oxide does not operate via loss of mitochondrial membrane potential. L-DOPA (reactive oxygen species donor) also caused apoptosis in a dose and time dependent manner. Removal of white blood cells significantly decreased ookinetes exhibiting a marker of apoptosis in vitro. Inhibition of the activity of nitric oxide synthase in the mosquito midgut epithelium using L-NAME significantly decreased the proportion of apoptotic ookinetes and increased the number of oocysts that developed. Introduction of a nitric oxide donor into the blood meal had no effect on mosquito longevity but did reduce prevalence and intensity of infection. Thus, nitric oxide and reactive oxygen species are triggers of apoptosis in Plasmodium ookinetes. They occur naturally in the mosquito midgut lumen, sourced from infected blood and mosquito tissue. Up regulation of mosquito nitric oxide synthase activity has potential as a transmission blocking strategy.     

The Neovolcanic Axis Is a Barrier to Gene Flow among Aedes aegypti Populations in Mexico That Differ in Vector Competence for Dengue 2 Virus

Background

Aedes aegypti is the main mosquito vector of the four serotypes of dengue virus (DENV). Previous population genetic and vector competence studies have demonstrated substantial genetic structure and major differences in the ability to transmit dengue viruses in Ae. aegypti populations in Mexico.

Methodology/Principal Findings

Population genetic studies revealed that the intersection of the Neovolcanic axis (NVA) with the Gulf of Mexico coast in the state of Veracruz acts as a discrete barrier to gene flow among Ae. aegypti populations north and south of the NVA. The mosquito populations north and south of the NVA also differed in their vector competence (VC) for dengue serotype 2 virus (DENV2). The average VC rate for Ae. aegypti mosquitoes from populations from north of the NVA was 0.55; in contrast the average VC rate for mosquitoes from populations from south of the NVA was 0.20. Most of this variation was attributable to a midgut infection and escape barriers. In Ae. aegypti north of the NVA 21.5% failed to develop midgut infections and 30.3% of those with an infected midgut failed to develop a disseminated infection. In contrast, south of the NVA 45.2% failed to develop midgut infections and 62.8% of those with an infected midgut failed to develop a disseminated infection.

Conclusions

Barriers to gene flow in vector populations may also impact the frequency of genes that condition continuous and epidemiologically relevant traits such as vector competence. Further studies are warranted to determine why the NVA is a barrier to gene flow and to determine whether the differences in vector competence seen north and south of the NVA are stable and epidemiologically significant.     

Blocking of malaria parasite development in mosquito and fecundity reduction by midgut antibodies in Anopheles stephensi (Diptera: Culicidae)

Rabbits were immunized three times with extracts of Anopheles stephensi midgut. Immunized rabbits showed a high titer of antibodies when characterized by ELISA. We investigated the effect of anti-mosquito midgut antibodies on mosquito fecundity, longevity, mortality, engorgement, and the development of the malaria parasite in mosquitoes. Fecundity was reduced significantly (38%) and similarly hatchability by about 43.5%. There was no statistically significant effect on mortality, longevity, and engorgement. When the mosquito blood meal contained anti-midgut antibodies, fewer oocysts of Plasmodium vivax developed in the mosquito midgut and the proportion of mosquitoes becoming infected was significantly reduced. We also found that the midgut antibodies inhibit the development and/or translocation of the sporozoites. Antisera raised against midgut of A. stephensi recognized eight polypeptides (110, 92, 70, 45, 38, 29, 15, 13 kDa) by Western blotting. Cross-reactive antigens/epitopes present in other tissues of A. stephensi were also examined both by Western blotting and in vivo ELISA. Together, these observations open an avenue for research toward the development of a vector-based malaria parasite transmission blocking vaccine and/or anti-mosquito vaccine.     

Potential Benefits,Limitations and Target Product-Profiles of Odor-Baited Mosquito Traps for Malaria Control in Africa

Background

Traps baited with synthetic human odors have been proposed as suitable technologies for controlling malaria and other mosquito-borne diseases. We investigated the potential benefits of such traps for preventing malaria transmission in Africa and the essential characteristics that they should possess so as to be effective.

Methods and Principal Findings

An existing mathematical model was reformulated to distinguish availability of hosts for attack by mosquitoes from availability of blood per se. This adaptation allowed the effects of pseudo-hosts such as odor-baited mosquito traps, which do not yield blood but which can nonetheless be attacked by the mosquitoes, to be simulated considering communities consisting of users and non-users of insecticide-treated nets (ITNs), currently the primary malaria prevention method. We determined that malaria transmission declines as trap coverage (proportion of total availability of all hosts and pseudo hosts that traps constitute) increases. If the traps are more attractive than humans and are located in areas where mosquitoes are most abundant, 20–130 traps per 1000 people would be sufficient to match the impact of 50% community-wide ITN coverage. If such traps are used to complement ITNs, malaria transmission can be reduced by 99% or more in most scenarios representative of Africa. However, to match cost-effectiveness of ITNs, the traps delivery, operation and maintenance would have to cost a maximum of US$4.25 to 27.61 per unit per year.

Conclusions and Significance

Odor-baited mosquito traps might potentially be effective and affordable tools for malaria control in Africa, particularly if they are used to complement, rather than replace, existing methods. We recommend that developers should focus on super-attractive baits and cheaper traps to enhance cost-effectiveness, and that the most appropriate way to deploy such technologies is through vertical delivery mechanisms.     

Midgut specific immune response of vector mosquito Anopheles stephensi to malaria parasite Plasmodium

Innate immune-related polypeptides expression in midgut in the ageing vector mosquito A. stephensi following infection by malaria parasite, Plasmodium yoelii yoelii has been studied. Twenty polypeptides were induced by an infected blood meal during various stages of adult life. A 24 kDa polypeptide was induced generally in most of the stages. Maximum parasite induced polypeptides i.e. 22, 33, 111, 122, 127, 140, 143 and 146 kDa were found in 5 days of post blood feeding (PBF) which coincides with the presence of oocysts on the midgut. However, in addition, three polypeptides in 11 days PBF and 8 polypeptides in 20 days PBF were also induced due to parasite infection in aged mosquitoes. Quantitatively, the amount of soluble proteins in the midgut in oocyst-sporozoite-positive mosquitoes was always less as compared to their normal counterparts. The parasite evidently elicits defined immune responses by inducing specific polypeptides in the midgut of the mosquito.     

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.