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Comparative Study
, 5, 23

Using Green Fluorescent Malaria Parasites to Screen for Permissive Vector Mosquitoes

Comparative Study

Using Green Fluorescent Malaria Parasites to Screen for Permissive Vector Mosquitoes

Friedrich Frischknecht et al. Malar J.


Background: The Plasmodium species that infect rodents, particularly Plasmodium berghei and Plasmodium yoelii, are useful to investigate host-parasite interactions. The mosquito species that act as vectors of human plasmodia in South East Asia, Africa and South America show different susceptibilities to infection by rodent Plasmodium species. P. berghei and P. yoelii infect both Anopheles gambiae and Anopheles stephensi, which are found mainly in Africa and Asia, respectively. However, it was reported that P. yoelii can infect the South American mosquito, Anopheles albimanus, while P. berghei cannot.

Methods: P. berghei lines that express the green fluorescent protein were used to screen for mosquitoes that are susceptible to infection by P. berghei. Live mosquitoes were examined and screened for the presence of a fluorescent signal in the abdomen. Infected mosquitoes were then examined by time-lapse microscopy to reveal the dynamic behaviour of sporozoites in haemolymph and extracted salivary glands.

Results: A single fluorescent oocyst can be detected in live mosquitoes and P. berghei can infect A. albimanus. As in other mosquitoes, P. berghei sporozoites can float through the haemolymph and invade A. albimanus salivary glands and they are infectious in mice after subcutaneous injection.

Conclusion: Fluorescent Plasmodium parasites can be used to rapidly screen susceptible mosquitoes. These results open the way to develop a laboratory model in countries where importation of A. gambiae and A. stephensi is not allowed.


Figure 1
Figure 1
Detecting development of oocysts in Anopheles midguts. (A) Abdomen and dissected midgut of an infected A. stephensi mosquito with green fluorescent P. berghei. Note that single oocysts can be detected in the intact mosquito, while multiple oocysts give a blurred signal. (B) Oocyst derived fluorescence detected in a well infected living A. albimanus mosquito (left) and an isolated midgut (right) 26 days post infection. In the mosquito the fluorescence appears blurred due to the opaque nature of the abdomen's chitin. (C) Four representative photographs from midguts of infected A. albimanus, A. gambiae and A. stephensi mosquitoes. The days after the infectious blood meals are indicated.
Figure 2
Figure 2
In vivo imaging of sporozoites in the haemolymph. (A) Left panel: An A. stephensi mosquito immobilized on a glass-slide for microscopy observation. Note the fluorescent signal at the base of the wings indicating haemolymph sporozoites (arrowhead). Right panel: An enlarged view of an A. stephensi mosquito viewed from the abdominal side indicating the fluorescent signal from sporozoites in the salivary gland (arrow) and from sporozoites in the veins of the wing (arrowhead). (B) Detection of individual sporozoites in the haemolymph. A vein of the wing imaged with a red filter (568 nm excitation) shows the auto-fluorescent mosquito tissue. The same region imaged with 488 nm excitation light shows the specific green fluorescence of the sporozoites (arrows) as well as the auto-fluorescent tissue. See also movie 1. (C) Three time-lapse images taken 3 seconds apart show the passive movement of sporozoites within the haemolymph of the mosquito tibia. The color image represents three images taken 3 seconds apart, pseudo-colored and overlayed to illustrate the movement of the sporozoites (see also movie 2). (D) Unusually many sporozoites (green) in the haemolymph of an A. albimanus thorax at 26 days post infection.
Figure 3
Figure 3
Infectious sporozoites in A. albimanus. (A) Isolated salivary gland of an infected A. albimanus mosquito at 26 days post infection. Sporozoites are shown in the left panel, while the movement occurring over 200 seconds is shown in the middle panel. This panel represents the projected standard deviation between time frames of a movie that spans 200 seconds with one image taken every 2 seconds. The insets show a typical back-and-forth moving sporozoite (arrowheads indicate ends of sporozoite). The time between frames is indicated in seconds. The right panel shows a merge of the static sporozoites (green) and the movement (red). See also movie 3. (B) An isolated sporozoite glides on a glass surface in the presence of 5% foetal calf serum. The asterix indicates the apical end of the sporozoites at 0 seconds, while the arrowhead indicates the apical end at the respective time frame. Time between frames is indicated in seconds. Scale bar: 10 μm. (C) Blood smear at 13 days after the injection of 2.000 salivary gland sporozoites shows an early trophozoite.

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