Comparison of blood intake of <i>Anopheles gambiae</i> and <i>Culex quinquefasciatus</i> during a blood meal.

<p>Total hemoglobin content of female mosquitoes fed on a 40% hematocrit blood solution was determined by hemoglobinometry at different time points after a blood meal. Ten mosquitoes were analyzed for each time point and the average amount of ingested blood calculated using a standard curve. Values are shown as mean ± standard deviation. The volume of blood corresponding to the determined hemoglobin amount was compared between <i>An. gambiae</i> (--•--) and <i>C. quinquefasciatus</i> ( —▪— ).</p

An Impossible Journey? The Development of <i>Plasmodium falciparum</i> NF54 in <i>Culex quinquefasciatus</i>

<div><p>Although <i>Anopheles</i> mosquitoes are the vectors for human <i>Plasmodium</i> spp., there are also other mosquito species–among them culicines (<i>Culex</i> spp., <i>Aedes</i> spp.)–present in malaria-endemic areas. Culicine mosquitoes transmit arboviruses and filarial worms to humans and are vectors for avian <i>Plasmodium</i> spp., but have never been observed to transmit human <i>Plasmodium</i> spp. When ingested by a culicine mosquito, parasites could either face an environment that does not allow development due to biologic incompatibility or be actively killed by the mosquito’s immune system. In the latter case, the molecular mechanism of killing must be sufficiently powerful that <i>Plasmodium</i> is not able to overcome it. To investigate how human malaria parasites develop in culicine mosquitoes, we infected <i>Culex quinquefasciatus</i> with <i>Plasmodium falciparum</i> NF54 and monitored development of parasites in the blood bolus and midgut epithelium at different time points. Our results reveal that ookinetes develop in the midgut lumen of <i>C. quinquefasciatus</i> in slightly lower numbers than in <i>Anopheles gambiae</i> G3. After 30 hours, parasites have invaded the midgut and can be observed on the basal side of the midgut epithelium by confocal and transmission electron microscopy. Very few of the parasites in <i>C. quinquefasciatus</i> are alive, most of them are lysed. Eight days after the mosquito’s blood meal, no oocysts can be found in <i>C. quinquefasciatus</i>. Our results suggest that the mosquito immune system could be involved in parasite killing early in development after ookinetes have crossed the midgut epithelium and come in contact with the mosquito hemolymph.</p></div

Parasite development in the mosquito midgut epithelium.

<p>(A) Number of live vs. lysing parasites in the midgut epithelium of <i>An. gambiae</i> (top) and <i>C. quinquefasciatus</i> (bottom) 30 hours after a <i>Plasmodium falciparum</i>-infected blood meal. Parasites were stained with a monoclonal anti-Pfs25 antibody and counted using a DEMIRE 2 Epifluorescence microscope. The results of five independent infections are shown here (Exp. #1 - #5). Each dot represents one midgut and the number of live vs. lysing parasites in the given midgut. The medians are given as red lines on the axes of the graphs and the sample size (n) is indicated for each group. Infection intensities in <i>An. gambiae</i> and <i>C. quinquefasciatus</i> were compared combining data from the five experiments and using the van Elteren test and were significantly lower in <i>C. quinquefasciatus</i> compared to the <i>An. gambiae</i> control (P<0.0001). (B) Number of oocysts found on the midgut epithelium eight days after the infection in five independent infections (same as for 30 hours). Midguts were stained with mercurochrome and oocysts counted in a light microscope at 40× magnification. Infection intensities were compared between <i>An. gambiae</i> and <i>C. quinquefasciatus</i>. The median oocyst number in <i>An. gambiae</i> was 6 oocysts per midgut, whereas in <i>C. quinquefasciatus</i> no oocysts could be found (P<0.0001, van Elteren test). (C) Representative images of mercurochrome stained mosquito midguts 8 days after <i>P. falciparum</i> NF54 infection. The <i>Anopheles gambiae</i> midgut contains oocysts (orange circles), whereas in <i>Culex quinquefasciatus</i> no oocysts can be found.</p

Development of <i>Plasmodium falciparum</i> NF54 in the mosquito blood meal.

<p>(A) Epifluorescence images of parasite stages observed in the blood meal of <i>Anopheles gambiae</i> G3 (left panel) and <i>Culex quinquefasciatus</i> (right panel) 20 hours after the mosquitoes were fed on a <i>P. falciparum</i> NF54 gametocyte culture. Immunostaining was done using a monoclonal anti-Pfs25 antibody to stain the parasites (red) and DAPI to visualize the nuclei (blue). (B) Ookinete conversion rate at 20 and 30 hours after the mosquito blood meal. One hundred parasites were counted for each sample and the percentage of ookinetes calculated. Each dot represents the percentage of ookinetes in one blood meal and the lines are the medians for all samples. Three independent experiments were performed and the combined data from the three infections is shown here. The groups were compared using a Mann-Whitney U test. P-values for each comparison are indicated in the graph. (C) Total number of ookinetes in the mosquito blood meal 30 hours after infection. Each dot represents the number of ookinetes found in a given blood meal, the medians are indicated as lines. Two independent experiments were performed and the combined data is shown here. Similarity was tested using a Mann-Whitney U test, which revealed that the two groups are not significantly different (P = 0.972).</p

Transmission electron microscopy of infected midguts 24 hours post feed on <i>Plasmodium falciparum</i> NF54-infected blood.

<p>Shown are overviews including the entire midgut epithelial layer (left) and magnifications of the parasite (right). (A) Parasite in the midgut epithelium of <i>Anopheles gambiae</i> G3, which is located on the basal side of the midgut epithelium underneath the basal lamina. (B) <i>P. falciparum</i> NF54 in the midgut of <i>Culex quinquefasciatus</i>. Parasites are located on the basal side of the midgut epithelium (overview left panel) outside the midgut cells. Parasite 1 (top) is located underneath the basal lamina outside the midgut cell, as it is surrounded by two membranes, one belonging to a midgut cell (arrow, M) and one of parasite origin (arrow, P) (see insets in right panel). The organelles inside the parasite are less pronounced than in <i>An. gambiae</i> (A), indicating lysis of the parasite (right panel). Parasite 2 (bottom) is located between two adjacent midgut cells toward the basal side of the epithelium. Two membranes can be seen (right panel inset, arrows M, P), showing an extracellular location of the parasite. Note here that one midgut epithelial cell (asterisk) is not connected to the basal lamina and lacks microvilli and most organelles, indicating apoptosis. MV: microvilli; BL: Basal lamina.</p

Confocal imaging of parasites in the mosquito midgut epithelium.

<p>Midgut epithelial tissue was collected 30 hours after the mosquito blood meal. (A) <i>Plasmodium falciparum</i> NF54 parasite in <i>Anopheles gambiae</i> G3. (B–D) <i>Culex quinquefasciatus</i> midgut epithelium containing (B) a live <i>P. falciparum</i> parasite and (C, D) parasites in different stages of lysis. Parasites in (C) and (D) have lost their even rim staining, which now appears dotted. Some parasites still contain nuclei (C, yellow and white arrow), but most parasites do not contain nuclei anymore (D, white arrows). A midgut cell is “budding off” into the midgut lumen (C, orange arrow). Shown is a section of the z-stack in the location of the parasite (left) and a side view of the midgut epithelium to localize the parasites (right). (E) Epifluorescence imaging of a parasite in <i>C. quinquefasciatus</i>. Note the black pigment associated with the parasite, which is visible in all fluorescent channels (arrows). Parasites were stained with a monoclonal anti-Pfs25 antibody (red), actin was stained using Phalloidin (green), and nuclei were visualized with DAPI (blue). MF: Muscle fibers on the basal side of the midgut; MV: microvilli. The scale bar indicates 5 µm.</p