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Malaria parasites in mosquitoes: laboratory models, evolutionary temptation and the real world Christophe Boe¨te1,2,3 1

Institut de Recherche pour le De´veloppement, Laboratoire Ge´ne´tique et Evolution des Maladies Infectieuses, 911 Avenue Agropolis, BP 64501 34394, Montpellier Cedex 05, France 2 Laboratory of Entomology, Wageningen University, PO Box 8031, Binnenhaven 7, 6700 EH Wageningen, The Netherlands 3 Joint Malaria Programme, Kilimanjaro Christian Medical Centre, PO Box 2228, Moshi, Kilimanjaro, Tanzania

A recent study describing the effect of Plasmodium berghei infection on some Anopheles gambiae immune genes demonstrates that P. berghei is responsible for the upregulation of several genes involved in the immune response that affect parasitic development differently during the ookinete-to-oocyst developmental transition. It is important to question the relevance of such results, which are based on a laboratory model system, when discussing host–parasite interactions and, especially, the development of novel control strategies for malaria.

Infection and immunity Osta et al. [1] recently described the effect of Plasmodium berghei infection on some Anopheles gambiae immune genes. Using gene silencing, the authors demonstrated that P. berghei is responsible for activating several immune genes that affect parasitic development differently during the ookinete-to-oocyst developmental transition. Thus, the activation of a leucine-rich repeat immune (LRIM) gene leads to the death of some parasites, whereas the activation of two C-type lectin (CTL) genes favours parasite development by suppressing the melanization response. The results of Osta et al. have been hailed the ‘clearest example yet of coevolution between the parasite and its insect vector’ by Hemingway and Craig [2]. At first, it is tempting to give the results of Osta et al. evolutionary explanations and link them with previous investigations of malaria–mosquito interactions. The temptation of an evolutionary view Activation of an LRIM gene by P. berghei reduces the number of ookinetes crossing the midgut epithelium by w75%. This seems to be counterintuitive, although such a drastic (but not total) reduction could be explained by the possible selection of a mechanism by both the mosquito and the parasite that limits the parasite load. Reducing competition between parasites (particularly if they are highly genetically related) seems to benefit them. Indeed, reducing the number of parasites might lead to a lower cost of infection for the insect host, enabling Corresponding author: Boe¨te, C. ([email protected]).

it to survive until the parasite becomes transmissible to a vertebrate host. As already shown, a high A. gambiae survival rate has been associated with a low number of oocysts in natural conditions [3]; this could explain the selection of a mechanism that limits the number of parasites. However, it might be argued that there is no benefit to the mosquito in sustaining even reduced parasite development. Considering this point from a different angle raises the question of whether a strong immune response might have detrimental effects on the mosquito. Indeed, the insect circulatory system is open, and immune cytotoxic molecules might harm the parasite and the host [4]. This hypothesis has already found support; inducing an immune response in undernourished bumblebees leads to a reduction in survival rates [5]. Moreover, immunosuppression by the malaria parasite Plasmodium gallinaceum reduces the mortality rate of its host Aedes aegypti [6]. Put simply, avoiding the burden of infection benefits the parasite and the host, but an excessive immune response might be detrimental for both. Protective immunoreduction Activation of CTLs protects the remaining parasites against melanization after they form ookinetes. It should be noted that the absence of CTLs does not lead to melanization if LRIM1 is inactivated; suppressing LRIM1 after ookinete formation might protect malaria parasites from melanization. Thus, malaria infection leads to a decrease in the ability of mosquitoes to mount a strong, efficient melanization response. This mechanism of immunosuppression could explain previous field results in which A. gambiae was able to encapsulate CM-25 Sephadex beads but rarely malaria parasites [7]. It also corroborates other results obtained with a laboratory model of A. aegypti and P. gallinaceum, in which immunosuppression was shown to be systemic and stage specific [6,8]. Indeed, suppression of the mosquito melanization response when the parasite has developed into an ookinete or early oocyst has been demonstrated [6,8]. Such characteristics of the malaria parasite resemble the immunosuppression strategy of parasitoid wasps that lay their eggs within host insects – the immunosuppression is stage specific and systemic [6]. Even if the immunosuppression by parasitoids is due to the action of polydnavirus, this suggests that parasitoids and malaria

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parasites might have evolved similar mechanisms. Because virus-like particles are used by the parasitoid to destroy the immune cells (hemocytes) of its host [9], it is tempting to consider that such a mechanism could affect mosquito hemocytes. It has been shown that phenoloxidase (a key enzyme involved in the melanization process) is present within the hemocytes of A. aegypti [10] and that these cells are attached to the midgut in infected and uninfected A. gambiae (E.A. Levashina and F.C. Kafatos, unpublished). Being close to the infection site might render them highly susceptible to the actions of the parasite. Comparing the immunosuppression between malaria parasites and parasitoids might seem far fetched but previous microscopy work [11,12] has revealed the existence of crystalloid bodies in malaria ookinetes that resemble virus-like particles. Conclusions from these results remain highly speculative but they might raise important issues about host–parasite interactions. Moreover, it has been revealed that the protective immunosuppression is due to both direct and indirect effects of the parasite because even the bloodmeal of an infected host containing dead parasites drastically reduces melanization [6]. Thus, although Osta et al. provide insightful information about parasite development in mosquito vectors, their article does not enable specific definition of the effect of infected bloodmeal or the direct action of the parasite [1]. The dependency of the mosquito immune response on bloodmeal status [6,13] highlights the need for further studies that investigate possible immunosuppression by Plasmodium falciparum in its vectors and the causes of this phenomenon. The laboratory model and the real world If immunosuppression by malaria parasites can explain the low frequency of melanized parasites in the field [7], can evolutionary insights be drawn from the results of Osta et al.? Regarding the article by Osta et al., Hemingway and Craig mention the coevolution between P. berghei and A. gambiae [2]. Of course, both organisms exist in nature but do they ever meet? Probably not – P. berghei is a parasite of tree rats and is found (apart from in many Western laboratories) only in gallery forest in the Congo at high altitude (1000–1700 m), where it is transmitted by Anopheles dureni, not A. gambiae. Thus, it is extremely dubious to evoke an evolutionary history between A. gambiae and P. berghei. Another worrying point is the environmental conditions under which the experiments of Osta et al. were performed [1]. Whereas P. falciparum completes its extrinsic incubation period in A. gambiae at w27–308C, P. berghei can be maintained in mosquito cycles only at w218C. The expression of immune genes might be affected by temperature, and measurements of the reaction norms of gene expression in mosquitoes, with and without parasites, should be performed [14]. If one of the aims of investigating interactions of malaria mosquitoes is to find, using Plasmodium-resistant mosquitoes, tools that affect malaria transmission, it must be kept in mind that laboratory models might be limited in terms of advancing the understanding of relationships between P. falciparum and its vectors, and www.sciencedirect.com

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Box 1. Outstanding questions † How do environmental conditions affect the vectorial capacity of mosquitoes? † Is there adequation between laboratory models and natural host– parasite combinations? † How does mosquito immunity affect malaria transmission in natural conditions? † How does malaria epidemiology affect the selection of mosquito resistance? † Does Plasmodium falciparum suppress the mosquito melanization response in natural conditions?

the impact of these relationships on malaria epidemiology. Tahar et al. [15] have shown that results from P. berghei and A. gambiae do not correspond with the interactions between P. falciparum and A. gambiae. Moreover, as indicated by Hemingway and Craig [2], the SM1 peptide shows high efficiency against P. berghei [16] but not P. falciparum. Thus, at present, it seems that we are a long way from the successful deployment of modified mosquitoes that can impair malaria transmission, and ecological and evolutionary insights are needed to understand this transmission better (Box 1). Finally, even if a vision of transgenic mosquitoes brings excitement to the scientific community, one should bear in mind that available resources already exist to combat malaria, and they should be used efficiently and without delay. Acknowledgements I thank Caroline Ellson, Daniel Keates, Jacob C. Koella, Richard E.L. Paul and the four anonymous referees for helpful comments about previous versions of the manuscript. This work was supported by a Marie Curie Intra-European Fellowship at Wageningen University and by the Institut de Recherche pour le De´veloppement.

References 1 Osta, M.A. et al. (2004) Effects of mosquito genes on Plasmodium development. Science 303, 2030–2032 2 Hemingway, J. and Craig, A. (2004) New ways to control malaria. Science 303, 1984–1985 3 Lyimo, E.O. and Koella, J.C. (1992) Relationship between body size of adult Anopheles gambiae s.l. and infection with the malaria parasite Plasmodium falciparum. Parasitology 104, 233–237 4 Schmid-Hempel, P. (2003) Variation in immune defence as a question of evolutionary ecology. Proc. Biol. Sci. 270, 357–366 5 Moret, Y. and Schmid-Hempel, P. (2000) Survival for immunity: the price of immune system activation for bumblebee workers. Science 290, 1166–1168 6 Boe¨te, C. et al. (2004) Direct and indirect immunosuppression by a malaria parasite in its mosquito vector. Proc Biol. Sci. 271, 1611–1615 7 Schwartz, A. and Koella, J.C. (2002) Melanization of Plasmodium falciparum and C-25 Sephadex beads by field caught Anopheles gambiae (Diptera: Culicidae) from Southern Tanzania. J. Med. Entomol. 39, 84–88 8 Boe¨te, C. et al. (2002) Reduced efficacy of the immune melanisation response in mosquitoes infected by malaria parasites. Parasitology 125, 93–98 9 Rizki, R.M. and Rizki, T.M. (1990) Parasitoid virus-like particles destroy Drosophila cellular immunity. Proc. Natl. Acad. Sci. U. S. A. 87, 8388–8392 10 Hillyer, J.F. and Christensen, B.M. (2002) Characterization of hemocytes from the yellow fever mosquito, Aedes aegypti. Histochem. Cell Biol. 117, 431–440 11 Trefiak, W.D. and Dessert, S.S. (1973) Crystalloid inclusions in species of Leucocytozoon, Parahaemoproteus and Plasmodium. J. Protozool. 20, 73–80 12 Terzakis, J.A. (1969) A protozoan virus. Mil. Med. 134, 916–921

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13 Chun, J. et al. (1995) Effect of mosquito age and reproductive status on melanization of Sephadex beads in Plasmodium-refractory and -susceptible strains of Anopheles gambiae. J. Invertebr. Pathol. 66, 11–17 14 Tabachnick, W.J. (2003) Reflections on the Anopheles gambiae genome sequence, transgenic mosquitoes and the prospect for controlling malaria and other vector borne diseases. J. Med. Entomol. 40, 597–606

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15 Tahar, R. et al. (2002) Immune response of Anopheles gambiae to the early sporogonic stages of the human malaria parasite Plasmodium falciparum. EMBO J. 21, 6673–6680 16 Ito, J. et al. (2002) Transgenic anopheline mosquitoes impaired in transmission of a malaria parasite. Nature 417, 452–455