Using green fluorescent malaria parasites to screen for permissive vector mosquitoes
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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.
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.
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.
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.
KeywordsSalivary Gland Mosquito Species Infected Mosquito Plasmodium Berghei Anopheles Stephensi
Plasmodium berghei is one of the most commonly studied Plasmodium species, particularly for elucidating the interactions between the parasites and their hosts [1, 2, 3, 4, 5]. The natural mammalian host of P. berghei is the African tree rat Grammomys surdaster and its natural mosquito vector is Anopheles dureni [6, 7]. P. berghei is a species of choice for studies employing genetic manipulations due to the relative ease of parasite transfection and the function of many parasite genes has already been investigated in this species . Recently, a method has been developed in P. berghei that will now permit the inactivation of essential genes specifically at pre-erythrocytic stages of the parasite . Furthermore, the use of fluorescently labeled parasites has given unprecedented insights into the behaviour of these parasites within living insects and mice [10, 11, 12, 13]. Although there are differences between the rodent and human-infecting Plasmodium parasites at the genomic, antigenic and cellular level [14, 15], it is nonetheless clear that the rodent parasites are useful for elucidating the molecular basis of the core biology of Plasmodium, which often cannot be addressed with human parasites.
Plasmodium spp that infect humans are transmitted by a range of different mosquito species. While one of the main malaria vectors in Asia is Anopheles stephensi, the main vector in Africa is Anopheles gambiae. Both mosquito species are commonly used in laboratory experiments to study host-parasite interactions . Far less common are studies using the main South American malaria vector, Anopheles albimanus . This might be partly due to the facts that P. berghei has been reported to not infect A. albimanus  and that Plasmodium yoelii sporozoites generated in A. albimanus have been described as being non-infectious to the rodent host . Since A. gambiae, A. stephensi and A. albimanus are all amenable to genetic modification [19, 20, 21], an A. albimanus – P. berghei system would be an interesting addition to the existing laboratory models. In addition, establishment of such a model, or similar models for other parasite species, would be valuable for host-vector-parasite interaction studies in countries where importation of A. gambiae and A. stephensi is not allowed. Here, using a fluorescent parasite line, A. albimanus mosquitoes were screened to check whether it was permissive for P. berghei development. It was found that P. berghei was able to infect A. albimanus and to develop into infectious sporozoites within this mosquito species.
For all infections, P. berghei (strain NK65 or ANKA) lines expressing the green fluorescent protein were used that allowed the detection of oocysts and sporozoites in living mosquitoes and dissected organs [11, 22, 29]. A. stephensi (strain Sda500), A. gambiae (strain Yaoundé) and A. albimanus (strain STECLA) mosquitoes were reared at the Center for Production and Infection of Anopheles (CEPIA) of the Institut Pasteur using standard procedures. Mosquitoes were fed on P. berghei-infected mice (parasitaemia >1%) 3–5 days (A. stephensi and A. gambiae) or 3–8 days (A. albimanus) after emergence, kept at elevated humidity (70% relative humidity) for up to 6 weeks in dedicated incubators or rooms at 21°C and feed on 10% (w/v) sucrose solution with or without supplements (see text) and received one additional non-infected blood meal after 1 or 2 weeks. The mosquitoes were allowed to lay their eggs on wet filter paper deposited in a plastic Petri dish.
Observation of infected mosquitoes
Infection of mice
All experiments using mice were approved by the committee of Institut Pasteur and were performed according to the applicable guidelines and regulations. For infection by mosquito bite or by intra-dermal injection, C57Bl/6 mice (Janvier, France) were anaesthetised with ketamine-xylazine. Rodents were injected in the food pad with 3 μl PBS containing sporozoites with a modified Hamilton micro-syringe (Precision Instruments, USA).
Results and discussion
Evaluation of infected A. albimanus mosquitoes fed on sucrose solution with or without supplemented PABA and Pen/Strep. In total 14 infections were performed with mosquitoes fed on sugar (3 of these resulted in no infected midguts) and 20 infections with mosquitoes fed on PAPA+Pen/Strep. During each infection between 50 and 250 mosquitoes were investigated. Data shown are mean ± standard deviation.
Sugar with PABA+Pen/Strep
Range, number of infections
3.9 ± 4.3%
0 – 16.6%, n = 14
19.2 ± 9.7%
4.4 – 44%, n = 20
% of infected midguts with < 5 oocysts
80.3 ± 25.3
49.5 ± 18.1
% of infected midguts with 5–20 oocysts
9.1 ± 16.8
28.1 ± 14.4
% of infected midguts with 20 – 50 oocysts
10.6 ± 23.9
7.3 ± 7.5
% of infected midguts with > 50 oocysts
5.3 ± 9.1
Mosquitoes with sporozoites in the haemolymph but no more oocysts in the midgut
9.4 ± 12.3%
It had been previously shown that the efficiency of P. falciparum, P. yoelii and P. berghei development in A. stephensi can be increased by adding para-aminobenzoic acid (PABA) to the sugar water prior to the infectious blood meal [24, 25]. It is also known that, as the presence of bacteria in the midgut of mosquitoes inhibits the infectivity of P. falciparum to A. gambiae, A. stephensi and A. albimanus, Plasmodium infection rates can be increased by adding antibiotics [25, 26, 27]. Therefore, in attempts to increase the efficiency of P. berghei development in A. albimanus, the sucrose solution was supplemented with 0.5 g/l PABA and 0.1 g/l penicillin/streptomycin (Pen/Strep). In this case, a higher number of A. albimanus females were infected by P. berghei and a higher number of oocysts per mosquito gut was found (Table 1). While in the absence of PABA and Pen/Strep, rarely more than one mosquito in a cage of 200 females was seen that had more than five oocysts, infection rates exceeding 20% were regularly achieved in the presence of supplements (Table 1). Of these infected mosquitoes, over 49% had more than five oocysts, while 22% showed more than 20 (Table 1).
To analyse P. berghei sporozoites in the A. albimanus salivary glands, the latter were incubated in cell culture medium containing foetal calf serum and visualized by time-lapse microscopy. Sporozoites within the glands were able to move (Figure 3a). Their main movement pattern was the "back-and-forth" type of motility previously observed for P. berghei sporozoites in A. stephensi salivary glands, in the absence or presence of serum . Next, it was investigated if the sporozoites would be able to glide on a solid substrate, a prerequisite for infectivity to the mammalian host . When incubated in medium containing foetal calf serum, P. berghei sporozoites isolated from infected A. albimanus glands moved on glass slides in a manner indistinguishable from P. berghei sporozoites isolated from infected A. stephensi glands (Figure 3b).
Next, the infectivity to the mammalian host of sporozoites isolated from A. albimanus salivary glands was investigated. Injection of 2,000 such sporozoites into the skin of mice caused red blood cell infection as determined by blood smear analysis (Figure 3c). This showed that P. berghei sporozoites, isolated from infected A. albimanus, were capable of invading both mosquito and mammalian tissues and to differentiate into red blood cell invading forms. However, in two separate experiments, intra-dermal injection of 20,000 sporozoites obtained from the haemolymph of A. albimanus mosquitoes failed to induce infection. Mice remained uninfected, which confirms that Plasmodium sporozoites undergo a maturation process in the salivary glands of Anopheles . Qualitatively similar results were obtained by an infection of A. albimanus with P. berghei ANKA parasites expressing the green fluorescent protein .
Finally, whether P. berghei sporozoites can be transmitted to the mammalian host by the natural bite of A. albimanus mosquitoes during the third week post infection, was tested when sporozoites were present in the salivary glands of A. albimanus. Mouse infection was never induced even when over 10 infected mosquitoes were allowed to bite. Additionally, when artificial salivation was induced in immobilized A. albimanus, no ejection of sporozoites through the proboscis of the mosquitoes could be detected. Whether this reflects a true natural barrier to sporozoites inside A. albimanus or just the small number of sporozoites within the salivary glands remains to be determined. During previous studies using P. berghei infections of A. stephensi, sporozoites were already being ejected at day 11 post infection, when mosquitoes were artificially stimulated to salivate . However, their numbers were very low (less than five in less than 20% of mosquitoes) and sporozoites were never observed within the first minutes during salivation. As those sporozoites ejected early during salivation are likely to be deposited in the skin [11, 13], it is not surprising that even the combined bites of hundred A. stephensi mosquitoes at day 11 after the infectious blood meal were unable to infect mice. A similar situation might occur during infections with A. albimanus, where only very few sporozoites are taking up residence in the salivary glands and none was observed in the narrow parts of the salivary ducts.
While the difference between the findings described here and those of Vaughan et al.  might be due to the different strains of mosquitoes (A-2) and parasites (ANKA) used, it is more likely that the low number of oocysts developing in the absence of supplements escaped detection in the earlier study. Indeed, no difference was seen between fluorescent NK65 and ANKA strains in A. albimanus. This shows the advantage of using fluorescent parasites, as a single oocyst can be detected by careful observation in living mosquitoes and easily in dissected midguts.
The feasibility to screen mosquito species for susceptibility to a malaria parasite species, using fluorescent parasites was demonstrated. Using this methodology, it was found that the rodent malaria parasite P. berghei is able to infect the major South American malaria vector A. albimanus and to develop into infectious sporozoites albeit at low frequency. This suggests that it should be possible to isolate A. albimanus lines that are highly susceptible to P. berghei infection and that such lines may provide useful new tools for studying the interaction between a model malaria parasite and a major malaria-transmitting mosquito.
We thank the members of the CEPIA for help with mosquito rearing, the members of the Plateforme d'imagerie dynamique http://www.pfid.org for help with microscopy, Patricia Baldacci for comments on the manuscript and Rogerio Amino for discussions. RM is a Howard Hughes International Scholar and FF was a recipient of a Human Frontier Science Program long-term fellowship. Work in RM's laboratory is sponsored by grants from the Institut Pasteur (Grand Programme Horizontal Anopheles), the BioMalPar Program of the EU, the Fondation Schlumberger, the Fondation pour la Recherche Médicale and the Howard Hughes Medical Institute. Work in FF's laboratory is sponsored by grants from the Deutsche Forschungsgemeinschaft (SFB 544) and the Bundesministerium für Bildung und Forschung (BioFuture Program).
Additional File 3: Movie 3. Sporozoites in the salivary gland of an Anopheles albimanus mosquito. The gland was carefully isolated from an infected mosquito and placed in RPMI containing 5% foetal calf serum in a glass-bottom well dish. Note the displacement of sporozoites, mainly in a typical back-and-forth fashion. 0.1 frames per second, movie length: 100 seconds. For best viewing loop the movie. Size: 2 MB. (MOV 2 MB)
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