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An impossible journey? The development of Plasmodium falciparum NF54 in Culex quinquefasciatus.

Knöckel J, Molina-Cruz A, Fischer E, Muratova O, Haile A, Barillas-Mury C, Miller LH - PLoS ONE (2013)

Bottom Line: Our results reveal that ookinetes develop in the midgut lumen of C. quinquefasciatus in slightly lower numbers than in Anopheles gambiae G3.Eight days after the mosquito's blood meal, no oocysts can be found in C. quinquefasciatus.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland, United States of America. julia.knoeckel@gmail.com

ABSTRACT
Although Anopheles mosquitoes are the vectors for human Plasmodium spp., there are also other mosquito species-among them culicines (Culex spp., Aedes spp.)-present in malaria-endemic areas. Culicine mosquitoes transmit arboviruses and filarial worms to humans and are vectors for avian Plasmodium spp., but have never been observed to transmit human Plasmodium 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 Plasmodium is not able to overcome it. To investigate how human malaria parasites develop in culicine mosquitoes, we infected Culex quinquefasciatus with Plasmodium falciparum 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 C. quinquefasciatus in slightly lower numbers than in Anopheles gambiae 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 C. quinquefasciatus are alive, most of them are lysed. Eight days after the mosquito's blood meal, no oocysts can be found in C. quinquefasciatus. 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.

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Development of Plasmodium falciparum NF54 in the mosquito blood meal.(A) Epifluorescence images of parasite stages observed in the blood meal of Anopheles gambiae G3 (left panel) and Culex quinquefasciatus (right panel) 20 hours after the mosquitoes were fed on a P. falciparum 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).
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pone-0063387-g001: Development of Plasmodium falciparum NF54 in the mosquito blood meal.(A) Epifluorescence images of parasite stages observed in the blood meal of Anopheles gambiae G3 (left panel) and Culex quinquefasciatus (right panel) 20 hours after the mosquitoes were fed on a P. falciparum 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).

Mentions: For statistical analyses of the ookinete conversion rate (Figure 1B), data from three independent experiments were combined. Because some samples could not be used for parasite counting (blood meals tend to flake off the glass slide during the staining procedure and parasites could not be counted), not all groups in each experiment had the same number of samples. For combination of the data, the group with the lowest sample number from each experiment was taken in full. The data for each of the remaining groups was randomized using Microsoft Excel 2010 software and equal numbers of samples from those groups were taken after randomization and combined with the data of the two other feeds (6 samples each were taken from Exp. #1, 9 from #2 and 10 from #3). To compare the number of ookinetes in the blood meals of An. gambiae and C. quinquefasciatus (Figure 1C), data from two independent feeds were combined. The medians were compared using the Mann-Whitney U test and Graph Pad Prism 5 (GraphPad Software, Inc., La Jolla, California, USA).


An impossible journey? The development of Plasmodium falciparum NF54 in Culex quinquefasciatus.

Knöckel J, Molina-Cruz A, Fischer E, Muratova O, Haile A, Barillas-Mury C, Miller LH - PLoS ONE (2013)

Development of Plasmodium falciparum NF54 in the mosquito blood meal.(A) Epifluorescence images of parasite stages observed in the blood meal of Anopheles gambiae G3 (left panel) and Culex quinquefasciatus (right panel) 20 hours after the mosquitoes were fed on a P. falciparum 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).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3643899&req=5

pone-0063387-g001: Development of Plasmodium falciparum NF54 in the mosquito blood meal.(A) Epifluorescence images of parasite stages observed in the blood meal of Anopheles gambiae G3 (left panel) and Culex quinquefasciatus (right panel) 20 hours after the mosquitoes were fed on a P. falciparum 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).
Mentions: For statistical analyses of the ookinete conversion rate (Figure 1B), data from three independent experiments were combined. Because some samples could not be used for parasite counting (blood meals tend to flake off the glass slide during the staining procedure and parasites could not be counted), not all groups in each experiment had the same number of samples. For combination of the data, the group with the lowest sample number from each experiment was taken in full. The data for each of the remaining groups was randomized using Microsoft Excel 2010 software and equal numbers of samples from those groups were taken after randomization and combined with the data of the two other feeds (6 samples each were taken from Exp. #1, 9 from #2 and 10 from #3). To compare the number of ookinetes in the blood meals of An. gambiae and C. quinquefasciatus (Figure 1C), data from two independent feeds were combined. The medians were compared using the Mann-Whitney U test and Graph Pad Prism 5 (GraphPad Software, Inc., La Jolla, California, USA).

Bottom Line: Our results reveal that ookinetes develop in the midgut lumen of C. quinquefasciatus in slightly lower numbers than in Anopheles gambiae G3.Eight days after the mosquito's blood meal, no oocysts can be found in C. quinquefasciatus.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland, United States of America. julia.knoeckel@gmail.com

ABSTRACT
Although Anopheles mosquitoes are the vectors for human Plasmodium spp., there are also other mosquito species-among them culicines (Culex spp., Aedes spp.)-present in malaria-endemic areas. Culicine mosquitoes transmit arboviruses and filarial worms to humans and are vectors for avian Plasmodium spp., but have never been observed to transmit human Plasmodium 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 Plasmodium is not able to overcome it. To investigate how human malaria parasites develop in culicine mosquitoes, we infected Culex quinquefasciatus with Plasmodium falciparum 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 C. quinquefasciatus in slightly lower numbers than in Anopheles gambiae 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 C. quinquefasciatus are alive, most of them are lysed. Eight days after the mosquito's blood meal, no oocysts can be found in C. quinquefasciatus. 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.

Show MeSH
Related in: MedlinePlus