Ookinete destruction within the mosquito midgut lumen explains Anopheles albimanus refractoriness to Plasmodium falciparum (3D7A) oocyst infection

Previous studies have shown that the central American mosquito vector, Anopheles albimanus, is generally refractory to oocyst infection with allopatric isolates of the human malaria parasite Plasmodium falciparum. However, the reasons for the refractoriness of A. albimanus to infection with such isolates of P. falciparum are unknown. In the current study, we investigated the infectivity of the P. falciparum clone 3D7A to laboratory-reared A. albimanus and another natural vector of human malaria, Anopheles stephensi. Plasmodium falciparum gametocytes grown in vitro were simultaneously fed to both mosquito species and the progress of malaria infection compared. In 22 independent paired experimental feeds, no mature oocysts were observed on the midguts of A. albimanus 10days after bloodfeeding. In contrast, high levels of oocyst infection were found on the midguts of simultaneously fed A. stephensi. Direct immunofluorescence microscopy and light microscopical examination of Giemsa-stained histological sections were used to identify when the P. falciparum clone 3D7A failed to establish mature oocyst infections in A. albimanus. Similar densities of macrogametes/zygotes, and immature retort-form and mature ookinetes were found within the bloodmeals of both mosquito species. However, in A. albimanus, ookinetes were seldom associated with the peritrophic matrix, and were neither observed in the ectoperitrophic space nor the midgut epithelium. In contrast, ookinetes were frequently observed in these midgut compartments in A. stephensi. Additionally, young oocysts were observed on the midguts of A. stephensi but not A. albimanus 2days after bloodfeeding. Vital staining of the immature retort-form and mature ookinetes found within the luminal bloodmeal, demonstrated that a significantly greater proportion of these malaria parasite stages were non-viable in A. albimanus compared with A. stephensi. Overall, our observations indicate that ookinetes of the P. falciparum clone 3D7A are destroyed within the bloodmeal of A. albimanus and that the midgut lumen, rather than the midgut epithelium, is the site of mosquito refractoriness in this particular malaria parasite-mosquito vector combination.     

Selection of Anopheles stephensi for refractoriness and susceptibility to Plasmodium falciparum

Variation in susceptibility of the vector Anopheles stephensi Liston to the human malaria parasite Plasmodium falciparum (Welch) was demonstrated using twelve strains of mosquitoes and one strain of parasites cultured in vitro. The Beech strain of An. stephensi exhibited greatest natural refractoriness, but with high intrapopulation variability. By selection for the required characteristic, two refractory lines of the Punjab strain and one highly susceptible line of the Sind strain were obtained. The median number of oocysts in the two refractory lines was less than 4% of that in the unselected line, whilst the highly susceptible line yielded about twice as many oocysts as the unselected line. Selection progressed more by keeping the descendants of individual females separate and selecting between them (individual selection) rather than pooling the progeny of all selected mosquitoes (mass selection). Using the former procedure many lines were lost due to inbreeding depression, but the outcome was more successful.     

Engineered resistance to Plasmodium falciparum development in transgenic Anopheles stephensi

Transposon-mediated transformation was used to produce Anopheles stephensi that express single-chain antibodies (scFvs) designed to target the human malaria parasite, Plasmodium falciparum. The scFvs, m1C3, m4B7, and m2A10, are derived from mouse monoclonal antibodies that inhibit either ookinete invasion of the midgut or sporozoite invasion of salivary glands. The scFvs that target the parasite surface, m4B7 and m2A10, were fused to an Anopheles gambiae antimicrobial peptide, Cecropin A. Previously-characterized Anopheles cis-acting DNA regulatory elements were included in the transgenes to coordinate scFv production with parasite development. Gene amplification and immunoblot analyses showed promoter-specific increases in transgene expression in blood-fed females. Transgenic mosquito lines expressing each of the scFv genes had significantly lower infection levels than controls when challenged with P. falciparum.     

Current scenario of malaria in India

The Indian National Malaria Eradication Programme (NMEP) is reporting 2.5 to 3 million malaria cases, and about 1,000 malaria deaths annually. Malaria in the northeastern states is stable and in the peninsular India unstable. There are six major and three minor malaria vectors, of which Anopheles culicifacies transmits malaria in rural areas and An. stephensi in the towns. Other vectors are of local importance. Plasmodium vivax is the dominant infection and accounts for 60-65% cases whereas P. falciparum contributes 30-35% cases. Field operations to control malaria are impeded by resistance and/or exophilic vector behavior, parasite resistance to antimalarial drugs, operational problems in spraying, failure to search breeding of mosquitoes at weekly intervals, staff shortages and financial constraints. Resurgent malaria invaded new ecotypes created by green revolution, industrial growth and urban development resulting in paradigm shift towards man-made malaria. NMEP has launched a world bank-assisted enhanced malaria control project with primary emphasis to protect 62.2 million high risk population in 7 states.     

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.     

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

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

A Plasmodium whole-genome synteny map: indels and synteny breakpoints as foci for species-specific genes

Whole-genome comparisons are highly informative regarding genome evolution and can reveal the conservation of genome organization and gene content, gene regulatory elements, and presence of species-specific genes. Initial comparative genome analyses of the human malaria parasite Plasmodium falciparum and rodent malaria parasites (RMPs) revealed a core set of 4,500 Plasmodium orthologs located in the highly syntenic central regions of the chromosomes that sharply defined the boundaries of the variable subtelomeric regions. We used composite RMP contigs, based on partial DNA sequences of three RMPs, to generate a whole-genome synteny map of P. falciparum and the RMPs. The core regions of the 14 chromosomes of P. falciparum and the RMPs are organized in 36 synteny blocks, representing groups of genes that have been stably inherited since these malaria species diverged, but whose relative organization has altered as a result of a predicted minimum of 15 recombination events. P. falciparum-specific genes and gene families are found in the variable subtelomeric regions (575 genes), at synteny breakpoints (42 genes), and as intrasyntenic indels (126 genes). Of the 168 non-subtelomeric P. falciparum genes, including two newly discovered gene families, 68% are predicted to be exported to the surface of the blood stage parasite or infected erythrocyte. Chromosomal rearrangements are implicated in the generation and dispersal of P. falciparum-specific gene families, including one encoding receptor-associated protein kinases. The data show that both synteny breakpoints and intrasyntenic indels can be foci for species-specific genes with a predicted role in host-parasite interactions and suggest that, besides rearrangements in the subtelomeric regions, chromosomal rearrangements may also be involved in the generation of species-specific gene families. A majority of these genes are expressed in blood stages, suggesting that the vertebrate host exerts a greater selective pressure than the mosquito vector, resulting in the acquisition of diversity.     

Biological problems with the replacement of a vector population by Plasmodium-refractory mosquitoes

Attempts are being made to backcross into Anopheles gambiae s.s. the gene(s) which cause zoophily in Anopheles quadriannulatus. Such a backcrossed strain might be preferable to a Plasmodium-refractory strain as a basis for genetic control because a refractory strain could select for evasion of refractoriness in the wild Plasmodium population. The species composition of the malaria vector population in several Tanzanian villages was overwhelmingly An. gambiae s.s. in a normal rainy season, but consisted of four species, all proved by ELISA and/or PCR to carry P. falciparum sporozoites, at the time of the heavy rains associated with El Ni?o. Thus any scheme, for malaria transmission control by replacement of vectors by genetically-manipulated non-vectors, would have to be able to replace more than one species.     

Seasonal distribution, parity, resting, host-seeking behavior and association of malarial parasites of Anopheles stephensi Liston in Kolkata, West Bengal

Indoor resting and human-landing mosquito collections were conducted at selected localities in Kolkata, India to determine resting and host-seeking behavior, night biting activity, seasonal distributions and malaria infection rates. During a two-year study (2006–2007), 5123 and 1716 female mosquitoes were captured in indoor-resting and human-landing collections, respectively, from two types of residences (brick built rooms, temporary huts). Regression analysis demonstrated that the abundance of indoor resting An. stephensi was positively correlated with ambient temperature and relative humidity. The average duration of the gonotrophic cycle for laboratory-reared An. stephensi was about 4 days. Average proportion of parous An. stephensi , daily survival and daily mortality rates were 46%, 82% and 18%, respectively. Plasmodium vivax sporozoite infections were detected in the salivary glands of two wild-caught An. stephensi (sporozoite rate 2.2%) and one An. annularis (sporozoite rate 1.5%). No P. falciparum infections were detected. Oocyst infections were observed in one An. annularis mosquito (oocyst rate 1.5%).     

Allelic polymorphisms in apical membrane antigen-1 are responsible for evasion of antibody-mediated inhibition in Plasmodium falciparum

Apical membrane antigen-1 (AMA-1) is a target of antibodies that inhibit invasion of Plasmodium falciparum into human erythrocytes and is a candidate for inclusion in a malaria vaccine. We have identified a line of P. falciparum (W2mef) less susceptible to anti-AMA1 antibodies raised to the protein from a heterologous parasite line (3D7). We have constructed transgenic P. falciparum expressing heterologous AMA-1 alleles. In vitro invasion assays show that these transgenic parasites differ from parental lines in susceptibility to inhibitory antibodies, providing direct evidence that sequence polymorphisms within AMA-1 are responsible for evasion of immune responses that inhibit parasite invasion. We also generated a parasite line that would express a chimeric AMA-1 protein, in which highly polymorphic residues within domain 1 were exchanged. Inhibition assays suggest that these residues are not sufficient for inhibition by invasion-blocking antibodies. This study is the first to use P. falciparum allelic exchange to examine the relationship between genetic diversity and susceptibility to protective antibodies. The findings have important implications for the development of an AMA-1-based malaria vaccine.     

Cloning and characterization of ferredoxin and ferredoxin-NADP+ reductase from human malaria parasite

The human malaria parasite (Plasmodium falciparum) possesses a plastid-derived organelle called the apicoplast, which is believed to employ metabolisms crucial for the parasite's survival. We cloned and studied the biochemical properties of plant-type ferredoxin (Fd) and Fd-NADP+ reductase (FNR), a redox system that potentially supplies reducing power to Fd-dependent metabolic pathways in malaria parasite apicoplasts. The recombinant P. falciparum Fd and FNR proteins were produced by synthetic genes with altered codon usages preferred in Escherichia coli. The redox potential of the Fd was shown to be considerably more positive than those of leaf-type and root-type Fds from plants, which is favourable for a presumed direction of electron flow from catabolically generated NADPH to Fd in the apicoplast. The backbone structure of P. falciparum Fd, as solved by X-ray crystallography, closely resembles those of Fds from plants, and the surface-charge distribution shows several acidic regions in common with plant Fds and some basic regions unique to this Fd. P. falciparum FNR was able to transfer electrons selectively to P. falciparum Fd in a reconstituted system of NADPH-dependent cytochrome c reduction. These results indicate that an NADPH-FNR-Fd cascade is operative in the apicoplast of human malaria parasites.