Synthesize palladium nanoparticles from the macroalgae Sargassum fusiforme: An eco-friendly tool in the fight against Plasmodium falciparum?

Although numerous drugs are practiced to control malaria and its vectors, more recently, eco-friendly control tools have been proposed to battle its etiologic agents. Thus, using green bionanotechnology approaches, we aimed to synthesize palladium nanoparticles (Pd NPs) from the macroalgae Sargassum fusiforme (Sf), its potential antiparasitic activity against P. falciparum, as well as its possible cytotoxicity, in HeLa cells. After the biosynthesis of the PdSf NPs, their characterization was carried out by UV-Vis, FESEM, and EDX analyses, and their hydrodynamic size, zeta potential, and surface area were determined. Furthermore, the functional groups of the PdSf NPs were analyzed by FT-IR and GC-MS. While PdSf NPs had an IC50 of 7.68 µg/mL (Chloroquine (CQ)-s) and 16.42 µg/mL, S. fusiforme extract had an IC50 of 14.38 µg/mL (CQ-s) and 35.27 µg/mL (CQ-r). With an IC50 value of 94.49 µg/mL, PdSf NPs exhibited the least toxic effect on the HeLa cells. The Lipinski rule of five and ADMET prediction were used to assess the in silico models of caffeine acid hexoside and quercetin 7-O-hexoside for the presence of drug-like properties. Pathogenic proteins, primarily responsible for motility, binding, and disease-causing, were the target of the structurally based docking studies between plant-derived compounds and pathogenic proteins. Thus, our study pioneered promising results that support the potential antiplasmodial activity of eco-friendly synthesized PdSf NPs using S. fusiforme extract against P. falciparum, opening perspectives for further exploration into the use of these NPs in malaria therapy.

Standard Selection Treatments with Sulfadiazine Limit Plasmodium yoelii Host-to-Vector Transmission.

Some antimalarial drugs that have lost clinical usefulness have been repurposed for experimental applications. One example is sulfadiazine, an analog of p-aminobenzoic acid (pABA), which inhibits the parasite's folate synthesis pathway to block DNA synthesis. Sulfadiazine treatment of mice infected with Plasmodium yoelii and P. berghei is routinely used to enrich for gametocytes by killing asexual blood-stage parasites, but it is not well known if there are downstream effects on transmission. To determine if there was a significant effect of sulfadiazine exposure upon transmission, we transmitted Plasmodium yoelii (17XNL strain) parasites to Anopheles stephensi mosquitoes and evaluated the prevalence and intensity of infection under different sulfadiazine treatment conditions. We observed that there was a reduction in both the number of mosquitoes that became infected and in the intensity of infection if parasites were exposed to sulfadiazine in the mouse host or mosquito vector. Sulfadiazine treatment could be marginally overcome if mosquitoes were provided fresh pABA. In contrast, we determined that gametocytes exposed to sulfadiazine could develop into morphologically mature ookinetes in vitro, thus sulfadiazine exposure in the host may be reversible if the drug is washed out and the parasites are supplemented with pABA in the culture media. Overall, this indicates that sulfadiazine dampens host-to-vector transmission and that this inhibition can only be partially overcome by exposure to fresh pABA in vivo and in vitro. Because gametocytes are of great interest for developing transmission-blocking interventions, we recommend the use of less disruptive approaches for gametocyte enrichment. IMPORTANCE In this work, we have uncovered a substantial problem with how many studies of the sexual stages of rodent malaria parasites are conducted. Briefly, the isolation of sexual blood-stage Plasmodium parasites, or gametocytes, is essential to study pretransmission and transmission-stage biology of malaria. A routine method for the isolation of this specific stage in rodent-infectious malaria models is drug treatment with sulfadiazine, an antifolate that selectively kills actively replicating asexual blood-stage parasites but not gametocytes. Thus, researchers use this as a convenient way to produce highly enriched gametocyte samples. However, in this work, we describe how this standard drug selection with sulfadiazine not only kills asexual blood-stage parasites but also substantially impacts host-to-vector transmission.

Evaluating putative repellent 'push' and attractive 'pull' components for manipulating the odour orientation of host-seeking malaria vectors in the peri-domestic space.

BACKGROUND: Novel malaria vector control approaches aim to combine tools for maximum protection. This study aimed to evaluate novel and re-evaluate existing putative repellent 'push' and attractive 'pull' components for manipulating the odour orientation of malaria vectors in the peri-domestic space. METHODS: Anopheles arabiensis outdoor human landing catches and trap comparisons were implemented in large semi-field systems to (i) test the efficacy of Citriodiol® or transfluthrin-treated fabric strips positioned in house eave gaps as push components for preventing bites; (ii) understand the efficacy of MB5-baited Suna-traps in attracting vectors in the presence of a human being; (iii) assess 2-butanone as a CO2 replacement for trapping; (iv) determine the protection provided by a full push-pull set up. The air concentrations of the chemical constituents of the push-pull set-up were quantified. RESULTS: Microencapsulated Citriodiol® eave strips did not provide outdoor protection against host-seeking An. arabiensis. Transfluthrin-treated strips reduced the odds of a mosquito landing on the human volunteer (OR 0.17; 95% CI 0.12-0.23). This impact was lower (OR 0.59; 95% CI 0.52-0.66) during the push-pull experiment, which was associated with low nighttime temperatures likely affecting the transfluthrin vaporisation. The MB5-baited Suna trap supplemented with CO2 attracted only a third of the released mosquitoes in the absence of a human being; however, with a human volunteer in the same system, the trap caught < 1% of all released mosquitoes. The volunteer consistently attracted over two-thirds of all mosquitoes released. This was the case in the absence ('pull' only) and in the presence of a spatial repellent ('push-pull'), indicating that in its current configuration the tested 'pull' does not provide a valuable addition to a spatial repellent. The chemical 2-butanone was ineffective in replacing CO2. Transfluthrin was detectable in the air space but with a strong linear reduction in concentrations over 5 m from release. The MB5 constituent chemicals were only irregularly detected, potentially suggesting insufficient release and concentration in the air for attraction. CONCLUSION: This step-by-step evaluation of the selected 'push' and 'pull' components led to a better understanding of their ability to affect host-seeking behaviours of the malaria vector An. arabiensis in the peri-domestic space and helps to gauge the impact such tools would have when used in the field for monitoring or control.

Plasmodium's journey through the Anopheles mosquito: A comprehensive review.

The malaria parasite has an extraordinary ability to evade the immune system due to which the development of a malaria vaccine is a challenging task. Extensive research on malarial infection in the human host particularly during the liver stage has resulted in the discovery of potential candidate vaccines including RTS,S/AS01 and R21. However, complete elimination of malaria would require a holistic multi-component approach. In line with this, under the World Health Organization's PATH Malaria Vaccine Initiative (MVI), the research focus has shifted towards the sexual stages of malaria in the mosquito host. Last two decades of scientific research obtained seminal information regarding the sexual/mosquito stages of the malaria. This updated and comprehensive review would provide the basis for consolidated understanding of cellular, biochemical, molecular and immunological aspects of parasite transmission right from the sexual stage commitment in the human host to the sporozoite delivery back into subsequent vertebrate host by the female Anopheles mosquito.

Lactic Acid Supplementation Increases Quantity and Quality of Gametocytes in Plasmodium falciparum Culture.

Malaria infection by Plasmodium falciparum continues to afflict millions of people worldwide, with transmission being dependent upon mosquito ingestion of the parasite gametocyte stage. These sexually committed stages develop from the asexual stages, yet the factors behind this transition are not completely understood. Here, we found that lactic acid increases gametocyte quantity and quality in P. falciparum culture. Low-passage-number NF54 parasites exposed to 8.2 mM lactic acid for various times were monitored using blood film gametocyte counts and RNA analysis throughout 2 weeks of gametocyte development in vitro for a total of 5 biological cohorts. We found that daily continuous medium exchange and 8.2 mM lactic acid supplementation increased gametocytemia approximately 2- to 6-fold relative to controls after 5 days. In membrane feeding mosquito infection experiments, we found that gametocytes continuously exposed to 8.2 mM lactic acid supplementations were more infectious to Anopheles stephensi mosquitoes, essentially doubling prevalence of infected midguts and oocyst density. Supplementation on days 9 to 16 did not increase the quantity of gametocytes but did increase quality, as measured by oocyst density, by 2.4-fold. Lactic acid did not impact asexual growth, as measured by blood film counts and luciferase quantification, as well as radioactive hypoxanthine incorporation assays. These data indicate a novel role for lactic acid in sexual development of the parasite.

Comparative demography elucidates the longevity of parasitic and symbiotic relationships.

Parasitic and symbiotic relationships govern vast nutrient and energy flows, yet controversy surrounds their longevity. Enduring relationships may engender parallel phylogenies among hosts and parasites, but so may ephemeral relationships when parasites colonize related hosts. An understanding of whether symbiont and host populations have grown and contracted in concert would be useful when considering the temporal durability of these relationships. Here, we devised methods to compare demographic histories derived from genomic data. We compared the historical growth of the agent of severe human malaria, Plasmodium falciparum, and its mosquito vector, Anopheles gambiae, to human and primate histories, thereby discerning long-term parallels and anthropogenic population explosions. The growth history of Trichinella spiralis, a zoonotic parasite disseminated by swine, proved regionally specific, paralleling distinctive growth histories for wild boar in Asia and Europe. Parallel histories were inferred for an anemone and its algal symbiont (Exaiptasia pallida and Symbiodinium minutum). Concerted growth in potatoes and the agent of potato blight (Solanum tuberosum and Phytophthora infestans) did not commence until the age of potato domestication. Through these examples, we illustrate the utility of comparative historical demography as a new exploratory tool by which to interrogate the origins and durability of myriad ecological relationships. To facilitate future use of this approach, we introduce a tool called C-PSMC to align and evaluate the similarity of demographic history curves.

Screening for potential prophylactics targeting sporozoite motility through the skin.

BACKGROUND: Anti-malarial compounds have not yet been identified that target the first obligatory step of infection in humans: the migration of Plasmodium sporozoites in the host dermis. This movement is essential to find and invade a blood vessel in order to be passively transported to the liver. Here, an imaging screening pipeline was established to screen for compounds capable of inhibiting extracellular sporozoites. METHODS: Sporozoites expressing the green fluorescent protein were isolated from infected Anopheles mosquitoes, incubated with compounds from two libraries (MMV Malaria Box and a FDA-approved library) and imaged. Effects on in vitro motility or morphology were scored. In vivo efficacy of a candidate drug was investigated by treating mice ears with a gel prior to infectious mosquito bites. Motility was analysed by in vivo imaging and the progress of infection was monitored by daily blood smears. RESULTS: Several compounds had a pronounced effect on in vitro sporozoite gliding or morphology. Notably, monensin sodium potently affected sporozoite movement while gramicidin S resulted in rounding up of sporozoites. However, pre-treatment of mice with a topical gel containing gramicidin did not reduce sporozoite motility and infection. CONCLUSIONS: This approach shows that it is possible to screen libraries for inhibitors of sporozoite motility and highlighted the paucity of compounds in currently available libraries that inhibit this initial step of a malaria infection. Screening of diverse libraries is suggested to identify more compounds that could serve as leads in developing 'skin-based' malaria prophylactics. Further, strategies need to be developed that will allow compounds to effectively penetrate the dermis and thereby prevent exit of sporozoites from the skin.

Sustainable manufacture of insect repellents derived from Nepeta cataria.

Malaria devastates sub-Saharan Africa; the World Health Organization (WHO) estimates that 212 million people contract malaria annually and that the plasmodium virus will kill 419 000 in 2017. The disease affects rural populations who have the least economic means to fight it. Impregnated mosquito nets have reduced the mortality rate but the Anopheles mosquitoes are changing their feeding patterns and have become more active at dusk and early morning rather than after 22h00 as an adaptation to the nets. Everyone is susceptible to the Anopheles at these times but infants and pregnant women are the most vulnerable to the disease. Plant-based mosquito repellents are as effective as synthetic repellents that protect people from bites. They are sustainable preventative measures against malaria not only because of their efficacy but because the local population can produce and distribute them, which represents a source of economic growth for rural areas. Here, we extract and test the essential oil nepetalactone from Nepeta cataria via steam distillation. Families in endemic areas of Burundi found them effective against bites but commented that the odor was pungent. An epidemiological study is required to establish its clinical efficacy.

A novel model fitted to multiple life stages of malaria for assessing efficacy of transmission-blocking interventions.

BACKGROUND: Transmission-blocking interventions (TBIs) aim to eliminate malaria by reducing transmission of the parasite between the host and the invertebrate vector. TBIs include transmission-blocking drugs and vaccines that, when given to humans, are taken up by mosquitoes and inhibit parasitic development within the vector. Accurate methodologies are key to assess TBI efficacy to ensure that only the most potent candidates progress to expensive and time-consuming clinical trials. Measuring intervention efficacy can be problematic because there is substantial variation in the number of parasites in both the host and vector populations, which can impact transmission even in laboratory settings. METHODS: A statistically robust empirical method is introduced for estimating intervention efficacy from standardised population assay experiments. This method will be more reliable than simple summary statistics as it captures changes in parasite density in different life-stages. It also allows efficacy estimates at a finer resolution than previous methods enabling the impact of the intervention over successive generations to be tracked. A major advantage of the new methodology is that it makes no assumptions on the population dynamics of infection. This enables both host-to-vector and vector-to-host transmission to be density-dependent (or other) processes and generates easy-to-understand estimates of intervention efficacy. RESULTS: This method increases the precision of intervention efficacy estimates and demonstrates that relying on changes in infection prevalence (the proportion of infected hosts) alone may be insufficient to capture the impact of TBIs, which also suppress parasite density in secondarily infected hosts. CONCLUSIONS: The method indicates that potentially useful, partially effective TBIs may require multiple infection cycles before substantial reductions in prevalence are observed, despite more rapidly suppressing parasite density. Accurate models to quantify efficacy will have important implications for understanding how TBI candidates might perform in field situations and how they should be evaluated in clinical trials.

Toxicity of ar-curcumene and epi-ß-bisabolol from Hedychium larsenii (Zingiberaceae) essential oil on malaria, chikungunya and St. Louis encephalitis mosquito vectors.

Mosquitoes act as vectors of key pathogens and parasites. Plant essential oils have been recognized as important sources of biopesticides, which do not induce resistance and have limited toxic effects on human health and non-target organisms. In this research, we evaluated the larvicidal and oviposition deterrence activity of Hedychium larsenii essential oil (EO) and its major compounds ar-curcumene and epi-ß-bisabolol. Both molecules showed high toxicity against early third instars of Anopheles stephensi (LC50=10.45 and 14.68µg/ml), Aedes aegypti (LC50=11.24 and 15.83µg/ml) and Culex quinquefasciatus (LC50=12.24 and 17.27µg/ml). In addition, low doses of ar-curcumene and epi-ß-bisabolol were effective as oviposition deterrents against the three tested mosquito species. Notably, the acute toxicity of H. larsenii oil and its major compounds against the mosquito biocontrol agent Poecilia reticulata was low, with LC50 higher than 1500ppm. Overall, the results from this study revealed that ar-curcumene and epi-ß-bisabolol from the H. larsenii oil can be considered for the development of novel and effective mosquito larvicides.

Paleopathology and Paleomicrobiology of Malaria.

Malaria is a disease caused by parasites of the genus Plasmodium, transmitted through the bites of female anopheles flies. Plasmodium falciparum causes severe malaria with undulating high fever (malaria tropica). Literary evidence of malarial infection dates back to the early Greek period, when Hippocrates described the typical undulating fever highly suggestive of plasmodial infection. Recent immunological and molecular analyses describe the unambiguous identification of malarial infections in several ancient Egyptian mummies and a few isolated cases in Roman and Renaissance Europe. Although the numbers of cases are low, there is evidence that the overall infection rates may have been relatively high and that this infectious disease may have had a significant impact on historical populations.

Supplementation with Abscisic Acid Reduces Malaria Disease Severity and Parasite Transmission.

Nearly half of the world's population is at risk for malaria. Increasing drug resistance has intensified the need for novel therapeutics, including treatments with intrinsic transmission-blocking properties. In this study, we demonstrate that the isoprenoid abscisic acid (ABA) modulates signaling in the mammalian host to reduce parasitemia and the formation of transmissible gametocytes and in the mosquito host to reduce parasite infection. Oral ABA supplementation in a mouse model of malaria was well tolerated and led to reduced pathology and enhanced gene expression in the liver and spleen consistent with infection recovery. Oral ABA supplementation also increased mouse plasma ABA to levels that can signal in the mosquito midgut upon blood ingestion. Accordingly, we showed that supplementation of a Plasmodium falciparum-infected blood meal with ABA increased expression of mosquito nitric oxide synthase and reduced infection prevalence in a nitric oxide-dependent manner. Identification of the mechanisms whereby ABA reduces parasite growth in mammals and mosquitoes could shed light on the balance of immunity and metabolism across eukaryotes and provide a strong foundation for clinical translation.

The Role of Oxidative Stress in the Longevity and Insecticide Resistance Phenotype of the Major Malaria Vectors Anopheles arabiensis and Anopheles funestus.

Oxidative stress plays numerous biological roles, both functional and pathological. The role of oxidative stress in various epidemiologically relevant biological traits in Anopheles mosquitoes is not well established. In this study, the effects of oxidative stress on the longevity and insecticide resistance phenotype in the major malaria vector species An. arabiensis and An. funestus were examined. Responses to dietary copper sulphate and hydrogen peroxide were used as proxies for the oxidative stress phenotype by determining the effect of copper on longevity and hydrogen peroxide lethal dose. Glutathione peroxidase and catalase activities were determined colorimetrically. Oxidative burden was quantified as protein carbonyl content. Changes in insecticide resistance phenotype were monitored by WHO bioassay. Insecticide resistant individuals showed an increased capacity for coping with oxidative stress, mediated by increased glutathione peroxidase and catalase activity. This effect was observed in both species, as well as in laboratory strains and F1 individuals derived from wild-caught An. funestus mothers. Phenotypic capacity for coping with oxidative stress was greatest in strains with elevated Cytochrome P450 activity. Synergism of oxidative stress defence enzymes by dietary supplementation with haematin, 3-Amino-1, 2, 4-triazole and Sodium diethyldithiocarbamate significantly increased pyrethroid-induced mortality in An. arabiensis and An. funestus. It is therefore concluded that defence against oxidative stress underlies the augmentation of the insecticide resistance phenotype associated with multiple blood-feeding. This is because multiple blood-feeding ultimately leads to a reduction of oxidative stress in insecticide resistant females, and also reduces the oxidative burden induced by DDT and pyrethroids, by inducing increased glutathione peroxidase activity. This study highlights the importance of oxidative stress in the longevity and insecticide resistance phenotype in malaria vectors.

Multipurpose effectiveness of Couroupita guianensis-synthesized gold nanoparticles: high antiplasmodial potential, field efficacy against malaria vectors and synergy with Aplocheilus lineatus predators.

Mosquito-borne diseases represent a deadly threat for millions of people worldwide. According to recent estimates, about 3.2 billion people, almost half of the world's population, are at risk of malaria. Malaria control is particularly challenging due to a growing number of chloroquine-resistant Plasmodium and pesticide-resistant Anopheles vectors. Newer and safer control tools are required. In this research, gold nanoparticles (AuNPs) were biosynthesized using a cheap flower extract of Couroupita guianensis as reducing and stabilizing agent. The biofabrication of AuNP was confirmed by UV-vis spectrophotometry, Fourier transform infrared (FTIR) spectroscopy, transmission electron microscopy (TEM), energy-dispersive X-ray (EDX) spectroscopy, X-ray diffraction (XRD), zeta potential, and particle size analysis. AuNP showed different shapes including spheres, ovals, and triangles. AuNPs were crystalline in nature with face-centered cubic geometry; mean size was 29.2-43.8 nm. In laboratory conditions, AuNPs were toxic against Anopheles stephensi larvae, pupae, and adults. LC50 was 17.36 ppm (larva I), 19.79 ppm (larva II), 21.69 ppm (larva III), 24.57 ppm (larva IV), 28.78 ppm (pupa), and 11.23 ppm (adult). In the field, a single treatment with C. guianensis flower extract and AuNP (10 × LC50) led to complete larval mortality after 72 h. In standard laboratory conditions, the predation efficiency of golden wonder killifish, Aplocheilus lineatus, against A. stephensi IV instar larvae was 56.38 %, while in an aquatic environment treated with sub-lethal doses of the flower extract or AuNP, predation efficiency was boosted to 83.98 and 98.04 %, respectively. Lastly, the antiplasmodial activity of C. guianensis flower extract and AuNP was evaluated against CQ-resistant (CQ-r) and CQ-sensitive (CQ-s) strains of Plasmodium falciparum. IC50 of C. guianensis flower extract was 43.21 µg/ml (CQ-s) and 51.16 µg/ml (CQ-r). AuNP IC50 was 69.47 µg/ml (CQ-s) and 76.33 µg/ml (CQ-r). Overall, our results showed the multipurpose effectiveness of C. guianensis-synthesized AuNPs, since they may be proposed as newer and safer tools in the fight against CQ-r strains of P. falciparum and for field control of malaria vectors, in synergy with wonder killifish predators.

Discovery of Dual-Stage Malaria Inhibitors with New Targets.

Malaria remains a major global health problem, with more than half of the world population at risk of contracting the disease and nearly a million deaths each year. Here, we report the discovery of inhibitors that target multiple stages of malaria parasite growth. To identify these inhibitors, we took advantage of the Tres Cantos Antimalarial Compound Set (TCAMS) small-molecule library, which is comprised of diverse and potent chemical scaffolds with activities against the blood stage of the malaria parasite, and investigated their effects against the elusive liver stage of the malaria parasite using a forward chemical screen. From a screen of nearly 14,000 compounds, we identified and confirmed 103 compounds as dual-stage malaria inhibitors. Interestingly, these compounds show preferential inhibition of parasite growth in liver- versus blood-stage malaria parasite assays, highlighting the drug susceptibility of this parasite form. Mode-of-action studies were completed using genetically modified and drug-resistant Plasmodium parasite strains. While we identified some compound targets as classical antimalarial pathways, such as the mitochondrial electron transport chain through cytochrome bc1 complex inhibition or the folate biosynthesis pathway, most compounds induced parasite death through as yet unknown mechanisms of action. Importantly, the identification of new chemotypes with different modes of action in killing Plasmodium parasites represents a promising opportunity for probing essential and novel molecular processes that remain to be discovered. The chemical scaffolds identified with activity against drug-resistant Plasmodium parasites represent starting points for dual-stage antimalarial development to surmount the threat of malaria parasite drug resistance.

Plasmodium transmission blocking activities of Vernonia amygdalina extracts and isolated compounds.

BACKGROUND: Medicinal plants are a validated source for discovery of new leads and standardized herbal medicines. The aim of this study was to assess the activity of Vernonia amygdalina leaf extracts and isolated compounds against gametocytes and sporogonic stages of Plasmodium berghei and to validate the findings on field isolates of Plasmodium falciparum. METHODS: Aqueous (Ver-H2O) and ethanolic (Ver-EtOH) leaf extracts were tested in vivo for activity against sexual and asexual blood stage P. berghei parasites. In vivo transmission blocking effects of Ver-EtOH and Ver-H2O were estimated by assessing P. berghei oocyst prevalence and density in Anopheles stephensi mosquitoes. Activity targeting early sporogonic stages (ESS), namely gametes, zygotes and ookinetes was assessed in vitro using P. berghei CTRPp.GFP strain. Bioassay guided fractionation was performed to characterize V. amygdalina fractions and molecules for anti-ESS activity. Fractions active against ESS of the murine parasite were tested for ex vivo transmission blocking activity on P. falciparum field isolates. Cytotoxic effects of extracts and isolated compounds vernolide and vernodalol were evaluated on the human cell lines HCT116 and EA.hy926. RESULTS: Ver-H2O reduced the P. berghei macrogametocyte density in mice by about 50% and Ver-EtOH reduced P. berghei oocyst prevalence and density by 27 and 90%, respectively, in An. stephensi mosquitoes. Ver-EtOH inhibited almost completely (>90%) ESS development in vitro at 50 µg/mL. At this concentration, four fractions obtained from the ethylacetate phase of the methanol extract displayed inhibitory activity >90% against ESS. Three tested fractions were also found active against field isolates of the human parasite P. falciparum, reducing oocyst prevalence in Anopheles coluzzii mosquitoes to one-half and oocyst density to one-fourth of controls. The molecules and fractions displayed considerable cytotoxicity on the two tested cell-lines. CONCLUSIONS: Vernonia amygdalina leaves contain molecules affecting multiple stages of Plasmodium, evidencing its potential for drug discovery. Chemical modification of the identified hit molecules, in particular vernodalol, could generate a library of druggable sesquiterpene lactones. The development of a multistage phytomedicine designed as preventive treatment to complement existing malaria control tools appears a challenging but feasible goal.

L-Arginine supplementation in mice enhances NO production in spleen cells and inhibits Plasmodium yoelii transmission in mosquitoes.

BACKGROUND: The life cycle of Plasmodium is complex, requiring invasion of two different hosts, humans and mosquitoes. In humans, initiation of an effective Th1 response during early infection is critical for the control of parasite multiplication. In mosquitoes, inhibition of the development of sexual-stage parasites interrupts the parasite transmission. In this study, we aim to investigate whether dietary supplementation of L-arginine (L-Arg) in mice affects Plasmodium yoelii 17XL (Py17XL) transmission in mosquitoes. METHODS: BALB/c mice were orally administered with 1.5 mg/g L-Arg daily for 7 days and infected with Py17XL. The mRNA levels of inducible nitric oxide synthase (iNOS) and arginase 1 in spleen cells were determined by real-time RT-PCR. The amount of nitric oxide (NO) released by spleen cells in vitro was determined by the Griess method. The effect of L-Arg supplementation on subsequent development of P. yoelii gametocytes was evaluated by an in vitro ookinete culture assay and mosquito feeding assay. RESULTS: Pretreatment of mice with L-Arg significantly increased the transcript level of iNOS in spleen cells and the amount of NO synthesized. Dietary L-Arg supplementation also significantly reduced the number of zygotes and ookinetes formed during in vitro culture and the number of oocysts formed on mosquito midguts after blood feeding. CONCLUSIONS: L-Arg enhances host immunity against blood-stage parasites as well as suppressing subsequent parasite development in mosquitoes. L-Arg as an inexpensive and safe supplement may be used as a novel adjunct treatment against malarial infection.

A combination of new screening assays for prioritization of transmission-blocking antimalarials reveals distinct dynamics of marketed and experimental drugs.

OBJECTIVES: The development of drugs to reduce malaria transmission is an important part of malaria eradication plans. We set out to develop and validate a combination of new screening assays for prioritization of transmission-blocking molecules. METHODS: We developed high-throughput assays for screening compounds against gametocytes, the parasite stages responsible for onward transmission to mosquitoes. An existing gametocyte parasitic lactate dehydrogenase (pLDH) assay was adapted for use in 384-well plates, and a novel homogeneous immunoassay to monitor the functional transition of female gametocytes into gametes was developed. A collection of 48 marketed and experimental antimalarials was screened and subsequently tested for impact on sporogony in Anopheles mosquitoes, to directly quantify the transmission-blocking properties of antimalarials in relation to their effects on gametocyte pLDH activity or gametogenesis. RESULTS AND CONCLUSIONS: The novel screening assays revealed distinct stage-specific kinetics and dynamics of drug effects. Peroxides showed the most potent transmission-blocking effects, with an intermediate speed of action and IC50 values that were 20-40-fold higher than the IC50s against the asexual stages causing clinical malaria. Finally, the novel synthetic peroxide OZ439 appeared to be a promising drug candidate as it exerted gametocytocidal and transmission-blocking effects at clinically relevant concentrations.

Ambient temperature and dietary supplementation interact to shape mosquito vector competence for malaria.

The extent to which environmental factors influence the ability of Anopheles mosquitoes to transmit malaria parasites remains poorly explored. Environmental variation, such as change in ambient temperature, will not necessarily influence the rates of host and parasite processes equivalently, potentially resulting in complex effects on infection outcomes. As proof of principle, we used Anopheles stephensi and the rodent malaria parasite, Plasmodium yoelii, to examine the effects of a range of constant temperatures on one aspect of host defense (detected as alterations in expression of nitric oxide synthase gene - NOS) to parasite infection. We experimentally boosted mosquito midgut immunity to infection through dietary supplementation with the essential amino acid l-Arginine (l-Arg), which increases midgut nitric oxide (NO) levels by infection-induced NOS catalysis in A. stephensi. At intermediate temperatures, supplementation reduced oocyst prevalence, oocyst intensity, and sporozoite prevalence suggesting that the outcome of parasite infection was potentially dependent upon the rate of NOS-mediated midgut immunity. At low and high temperature extremes, however, infection was severely constrained irrespective of supplementation. The effects of l-Arg appeared to be mediated by NO-dependent negative feedback on NOS expression, as evidenced by depressed NOS expression in l-Arg treated groups at temperatures where supplementation decreased parasite infection. These results suggest the need to consider the direct (e.g. effects of mosquito body temperature on parasite physiology) and indirect effects (e.g. mediated through changes in mosquito physiology/immunity) of environmental factors on mosquito-malaria interactions in order to understand natural variation in vector competence.