Evaluation and optimization of membrane feeding compared to direct feeding as an assay for infectivity

<p>Abstract</p> <p>Background</p> <p>Malaria parasite infectivity to mosquitoes has been measured in a variety of ways and setting, includind direct feeds of and/or membrane feeding blood collected from randomly selected or gametocytemic volunteers. <it>Anopheles gambiae s.l </it>is the main vector responsible of <it>Plasmodium falciparum </it>transmission in Bancoumana and represents about 90% of the laboratory findings, whereas <it>Plasmodium malariae </it>and <it>Plasmodium ovale </it>together represent only 10%.</p> <p>Materials and methods</p> <p>Between August 1996 and December 1998, direct and membrane feeding methods were compared for the infectivity of children and adolescent gametocyte carriers to anopheline mosquitoes in the village of Bancoumana in Mali. Gametocyte carriers were recruited twice a month through a screening of members of 30 families using Giemsa-stained thick blood smears. F1 generation mosquitoes issued from individual female wild mosquitoes from Bancoumana were reared in a controlled insectary conditions and fed 5% sugar solution in the laboratory in Bamako, until the feeding day when they are starved 12 hours before the feeding experiment. These F1 generation mosquitoes were divided in two groups, one group fed directly on gametocyte carriers and the other fed using membrane feeding method.</p> <p>Results</p> <p>Results from 372 <it>Plasmodium falciparum </it>gametocyte carriers showed that children aged 4–9 years were more infectious than adolescents (p = 0.039), especially during the rainy season. Data from 35 carriers showed that mosquitoes which were used for direct feeding were about 1.5 times more likely to feed (p < 0.001) and two times more likely to become infected, if they fed (p < 0.001), than were those which were used for membrane feeding. Overall, infectivity was about three-times higher for direct feeding than for membrane feeding (p < 0.001).</p> <p>Conclusion</p> <p>Although intensity of infectivity was lower for membrane feeding, it could be a surrogate to direct feeding for evaluating transmission-blocking activity of candidate malaria vaccines. An optimization of the method for future trials would involve using about three-times more mosquitoes than would be used for direct feeding.</p

Exceptional Diversity, Maintenance of Polymorphism, and Recent Directional Selection on the APL1 Malaria Resistance Genes of Anopheles gambiae

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

Wild Anopheles funestus mosquito genotypes are permissive for infection with the rodent malaria parasite, Plasmodium berghei.

Malaria parasites undergo complex developmental transitions within the mosquito vector. A commonly used laboratory model for studies of mosquito-malaria interaction is the rodent parasite, P. berghei. Anopheles funestus is a major malaria vector in sub-Saharan Africa but has received less attention than the sympatric species, Anopheles gambiae. The imminent completion of the A. funestus genome sequence will provide currently lacking molecular tools to describe malaria parasite interactions in this mosquito, but previous reports suggested that A. funestus is not permissive for P. berghei development.An A. funestus population was generated in the laboratory by capturing female wild mosquitoes in Mali, allowing them to oviposit, and rearing the eggs to adults. These F1 progeny of wild mosquitoes were allowed to feed on mice infected with a fluorescent P. berghei strain. Fluorescence microscopy was used to track parasite development inside the mosquito, salivary gland sporozoites were tested for infectivity to mice, and parasite development in A. funestus was compared to A. gambiae.P. berghei oocysts were detectable on A. funestus midguts by 7 days post-infection. By 18-20 days post-infection, sporozoites had invaded the median and distal lateral lobes of the salivary glands, and hemocoel sporozoites were observed in the hemolymph. Mosquitoes were capable of infecting mice via bite, demonstrating that A. funestus supports the complete life cycle of P. berghei. In a random sample of wild mosquito genotypes, A. funestus prevalence of infection and the characteristics of parasite development were similar to that observed in A. gambiae-P. berghei infections.The data presented in this study establish an experimental laboratory model for Plasmodium infection of A. funestus, an important vector of human malaria. Studying A. funestus-Plasmodium interactions is now feasible in a laboratory setting. This information lays the groundwork for exploitation of the awaited genome sequence of A. funestus

<i>Anopheles funestus</i> and <i>Anopheles gambiae</i> mosquitoes display equivalent susceptibility to <i>Plasmodium berghei</i> infection.

<p><i>A. gambiae</i> and <i>A. funestus</i> mosquitoes were fed on the same infected <i>P. berghei</i>-infected mouse in two replicate experiments, and midgut oocyst infections were quantified at 7 days post-infection. Each circle represents the number of midgut oocysts in an individual mosquito.</p

Light and GFP epi-fluorescence microscopy show fluorescent <i>Plasmodium berghei</i> parasites developing in <i>Anopheles funestus</i> mosquitoes and murine erythrocytes.

<p><b>A... </b><i>A. funestus</i> midgut with greater than 300 <i>P. berghei</i> oocysts (e.g., arrows) at 7 days post-infection. <b>B... </b><i>A. funestus</i> midgut showing normal oocysts (e.g., arrow) and oocysts that have recently undergone rupture (e.g., arrowhead) at 20 days post-infection. <b>C.</b> Imaging of fluorescent parasites through the cuticle of a live <i>A. funestus</i> showing parasite development in the midgut (MG) and salivary glands (SG). Note that tissues presented in panels B, D, and E originated from this mosquito. <b>D–E... </b><i>A. funestus</i> salivary glands showing that sporozoites preferentially invade the median (M) and distal lateral (DL) lobes. <b>F–G.</b> Blood smear from a mouse exposed to <i>P. berghei</i> via mosquito bite showing infected erythrocytes, indicating that <i>A. funestus</i> salivary gland sporozoites are infective to the vertebrate host and that the parasite can complete its life cycle inside the insect vector. Bars: A–C = 500 µm; D–E = 200 µm; F = 100 µm; G = 20 µm.</p