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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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Confocal imaging of parasites in the mosquito midgut epithelium.Midgut epithelial tissue was collected 30 hours after the mosquito blood meal. (A) Plasmodium falciparum NF54 parasite in Anopheles gambiae G3. (B–D) Culex quinquefasciatus midgut epithelium containing (B) a live P. falciparum 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 C. quinquefasciatus. 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.
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pone-0063387-g003: Confocal imaging of parasites in the mosquito midgut epithelium.Midgut epithelial tissue was collected 30 hours after the mosquito blood meal. (A) Plasmodium falciparum NF54 parasite in Anopheles gambiae G3. (B–D) Culex quinquefasciatus midgut epithelium containing (B) a live P. falciparum 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 C. quinquefasciatus. 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.

Mentions: In the positive control–An. gambiae infected with P. falciparum NF54–ookinetes and young oocysts can be observed on the basal side of the midgut epithelium (Figure 3A, Figure S1 A-E). In C. quinquefasciatus, we also find ookinetes that appear normal (Figure 3B, Figure S1 H). Some parasites are round, indicating they started to transition into early oocyst stage (Figure S1 I). However, the observation of healthy appearing parasites is very rare in C. quinquefasciatus. In contrast to An. gambiae, many of the midguts contain parasites that are lysing. Figure 3C shows a parasite in the midgut epithelium of C. quinquefasciatus that resembles a young oocyst (yellow arrow). Parasites have also been seen in the more elongated banana-shape characteristic for ookinetes (Figure S1 K). In contrast to the even rim staining of Pfs25 in An. gambiae, in C. quinquefasciatus the parasite staining appears uneven and dotted. In some parasites, the nuclear staining is prominent inside the parasite (Figure 3C; xy, yellow arrow). However, the majority of parasites lack a nucleus. Parasite staining most of the times appears as single circles or accumulations of small circles, some with DNA staining, others without (Figure 3C, D, white arrows, Figure S1 L). Circular shapes with even or dotted rims stained with Pfs25 are smaller than parasites observed in An. gambiae and have diameters of 1.64±0.2 (n = 23) to 2.58±0.34 µm (n = 25). Only a few of the larger sized circles were observed to contain nuclei.


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)

Confocal imaging of parasites in the mosquito midgut epithelium.Midgut epithelial tissue was collected 30 hours after the mosquito blood meal. (A) Plasmodium falciparum NF54 parasite in Anopheles gambiae G3. (B–D) Culex quinquefasciatus midgut epithelium containing (B) a live P. falciparum 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 C. quinquefasciatus. 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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3643899&req=5

pone-0063387-g003: Confocal imaging of parasites in the mosquito midgut epithelium.Midgut epithelial tissue was collected 30 hours after the mosquito blood meal. (A) Plasmodium falciparum NF54 parasite in Anopheles gambiae G3. (B–D) Culex quinquefasciatus midgut epithelium containing (B) a live P. falciparum 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 C. quinquefasciatus. 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.
Mentions: In the positive control–An. gambiae infected with P. falciparum NF54–ookinetes and young oocysts can be observed on the basal side of the midgut epithelium (Figure 3A, Figure S1 A-E). In C. quinquefasciatus, we also find ookinetes that appear normal (Figure 3B, Figure S1 H). Some parasites are round, indicating they started to transition into early oocyst stage (Figure S1 I). However, the observation of healthy appearing parasites is very rare in C. quinquefasciatus. In contrast to An. gambiae, many of the midguts contain parasites that are lysing. Figure 3C shows a parasite in the midgut epithelium of C. quinquefasciatus that resembles a young oocyst (yellow arrow). Parasites have also been seen in the more elongated banana-shape characteristic for ookinetes (Figure S1 K). In contrast to the even rim staining of Pfs25 in An. gambiae, in C. quinquefasciatus the parasite staining appears uneven and dotted. In some parasites, the nuclear staining is prominent inside the parasite (Figure 3C; xy, yellow arrow). However, the majority of parasites lack a nucleus. Parasite staining most of the times appears as single circles or accumulations of small circles, some with DNA staining, others without (Figure 3C, D, white arrows, Figure S1 L). Circular shapes with even or dotted rims stained with Pfs25 are smaller than parasites observed in An. gambiae and have diameters of 1.64±0.2 (n = 23) to 2.58±0.34 µm (n = 25). Only a few of the larger sized circles were observed to contain nuclei.

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