Limits...
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.

Show MeSH

Related in: MedlinePlus

Parasite development in the mosquito midgut epithelium.(A) Number of live vs. lysing parasites in the midgut epithelium of An. gambiae (top) and C. quinquefasciatus (bottom) 30 hours after a Plasmodium falciparum-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 An. gambiae and C. quinquefasciatus were compared combining data from the five experiments and using the van Elteren test and were significantly lower in C. quinquefasciatus compared to the An. gambiae 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 An. gambiae and C. quinquefasciatus. The median oocyst number in An. gambiae was 6 oocysts per midgut, whereas in C. quinquefasciatus no oocysts could be found (P<0.0001, van Elteren test). (C) Representative images of mercurochrome stained mosquito midguts 8 days after P. falciparum NF54 infection. The Anopheles gambiae midgut contains oocysts (orange circles), whereas in Culex quinquefasciatus no oocysts can be found.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3643899&req=5

pone-0063387-g005: Parasite development in the mosquito midgut epithelium.(A) Number of live vs. lysing parasites in the midgut epithelium of An. gambiae (top) and C. quinquefasciatus (bottom) 30 hours after a Plasmodium falciparum-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 An. gambiae and C. quinquefasciatus were compared combining data from the five experiments and using the van Elteren test and were significantly lower in C. quinquefasciatus compared to the An. gambiae 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 An. gambiae and C. quinquefasciatus. The median oocyst number in An. gambiae was 6 oocysts per midgut, whereas in C. quinquefasciatus no oocysts could be found (P<0.0001, van Elteren test). (C) Representative images of mercurochrome stained mosquito midguts 8 days after P. falciparum NF54 infection. The Anopheles gambiae midgut contains oocysts (orange circles), whereas in Culex quinquefasciatus no oocysts can be found.

Mentions: To determine how many live and lysing parasites are present in the midguts of C. quinquefasciatus compared to An. gambiae, we counted the parasites in five independent experiments thirty hours after an infectious blood feeding (Figure 5A). In An. gambiae, we mainly observe live parasites, only a few midguts contained some lysing parasites. Medians of live parasites range from one to seven parasites per midgut. In contrast, midguts of C. quinquefasciatus mainly contained lysing parasites and only a few live parasites could be seen. Taking into account the results from all five infection experiments, we find significantly fewer live parasites in the midgut epithelium of C. quinquefasciatus than in An. gambiae (P<0.0001, van Elteren test). The main difference we observed here is the presence of lysed parasites in C. quinquefasciatus, which we rarely find in An. gambiae mosquitoes (Figure 5A).


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)

Parasite development in the mosquito midgut epithelium.(A) Number of live vs. lysing parasites in the midgut epithelium of An. gambiae (top) and C. quinquefasciatus (bottom) 30 hours after a Plasmodium falciparum-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 An. gambiae and C. quinquefasciatus were compared combining data from the five experiments and using the van Elteren test and were significantly lower in C. quinquefasciatus compared to the An. gambiae 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 An. gambiae and C. quinquefasciatus. The median oocyst number in An. gambiae was 6 oocysts per midgut, whereas in C. quinquefasciatus no oocysts could be found (P<0.0001, van Elteren test). (C) Representative images of mercurochrome stained mosquito midguts 8 days after P. falciparum NF54 infection. The Anopheles gambiae midgut contains oocysts (orange circles), whereas in Culex quinquefasciatus no oocysts can be found.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3643899&req=5

pone-0063387-g005: Parasite development in the mosquito midgut epithelium.(A) Number of live vs. lysing parasites in the midgut epithelium of An. gambiae (top) and C. quinquefasciatus (bottom) 30 hours after a Plasmodium falciparum-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 An. gambiae and C. quinquefasciatus were compared combining data from the five experiments and using the van Elteren test and were significantly lower in C. quinquefasciatus compared to the An. gambiae 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 An. gambiae and C. quinquefasciatus. The median oocyst number in An. gambiae was 6 oocysts per midgut, whereas in C. quinquefasciatus no oocysts could be found (P<0.0001, van Elteren test). (C) Representative images of mercurochrome stained mosquito midguts 8 days after P. falciparum NF54 infection. The Anopheles gambiae midgut contains oocysts (orange circles), whereas in Culex quinquefasciatus no oocysts can be found.
Mentions: To determine how many live and lysing parasites are present in the midguts of C. quinquefasciatus compared to An. gambiae, we counted the parasites in five independent experiments thirty hours after an infectious blood feeding (Figure 5A). In An. gambiae, we mainly observe live parasites, only a few midguts contained some lysing parasites. Medians of live parasites range from one to seven parasites per midgut. In contrast, midguts of C. quinquefasciatus mainly contained lysing parasites and only a few live parasites could be seen. Taking into account the results from all five infection experiments, we find significantly fewer live parasites in the midgut epithelium of C. quinquefasciatus than in An. gambiae (P<0.0001, van Elteren test). The main difference we observed here is the presence of lysed parasites in C. quinquefasciatus, which we rarely find in An. gambiae mosquitoes (Figure 5A).

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