Larval infection by any of four isolates of the microsporidian V. culicis enhanced the melanisation response of adult A. gambiae and reduced the likelihood and intensity of infection by P. berghei. These results suggest that microsporidians impede the development of malaria in their mosquito vector by priming its immune system.
That V. culicis
enhanced the immune (and in particular the melanisation) response of adult A. gambiae
is far from trivial. Indeed, as V. culicis
depletes the resources of infected larvae, leaving them with fewer lipids, sugars and glycogen reserves than uninfected individuals 
, the body condition of emerging adults is worse and one should therefore expect their immune response to be weak. This, indeed, seems to be the case in Tenebrio molitor
, where infection by an (unnamed) microsporidian does not enhance the immune response, and in particular does not stimulate or enhance immune system parameters associated with melanisation 
. In contrast, and similar to our study, in a proteomic study of V. culicis
in another mosquito, Aedes aegypti
, the antibacterial response was stimulated up to 15 days after infection 
. Thus, both the melanisation pathway and the anti-Plasmodium
response are primed, suggesting that either both these pathways are used in attempts to clear V.culicis
, or that V.culicis
infection leads to a general priming of the entire immune system.
That V. culicis
impeded the development of malaria in its mosquito vector corroborates several studies, in which microsporidian infection reduced the proportion of mosquitoes developing oocysts and the number of oocysts 
and decreased the density 
and quality 
of sporozoites. This suppression appears to be a general characteristic of microsporidian infection, as similar results were found in all studied systems, including non-human malaria (e.g. P. yoelii 
) as well as P. falciparum
developing in A. stephensi 
or the main African vector, A. gambiae 
. What this study adds is that isolates impede development to different degrees, most likely caused by differences in their genetic make-up. This divergence is reflected in the microsporidian's and mosquito's phenotypic traits, e.g. the intensity of microsporidian infection, the mortality of larvae and pupae and the longevity of adults (Lorenz and Koella, unpublished data). Further experiments will investigate whether these traits are correlated with the mosquito's immune response and its reaction to malaria.
Both responses to microsporidian infection — more effective melanisation of a Sephadex bead and less successful infection by malaria — differed among the four microsporidian isolates. While it would be interesting to evaluate whether this variation is due to genetic differences among the isolates or other differences; e.g. maternal effects, this is not within the scope of this study. More importantly, for each isolate, microsporidian infection enhanced the melanisation response (and perhaps other immune responses) and impeded development of malaria, which suggests immune-priming as a mechanism. Stronger support would have been a negative correlation between enhanced melanisation by microsporidian isolates and interference with malaria by the same isolate, which we did not observe. However; one should not over-interpret this lack of association. First, the power to detect any such correlation was low, as we used only four isolates and the variation of the likelihood of malaria infection among the mosquitoes infected by the four isolates was low. Second, it is a prioiri not clear that an association should exist. A certain level of immune capability, for example, may eliminate the Plasmodium parasite or reduce its numbers. Beyond this threshold any increase in immune capability may have little effect on malarial infection, yet still be evident in the increasing melanisation response.
Another possible mechanism for the interference of microsporidian infection with Plasmodium might be competition for healthy gut cells. As microsporidians are intracellular parasites a possible immune response would be to shut down the cell through apoptosis, thus killing the microsporidian within. Plasmodium parasites, on the other hand, must pass through the midgut to form oocysts. It is therefore conceivable, that at this stage in the development process Plasmodium parasites that enter microsporidian-infected cells may be killed through apoptosis. Alternatively, Plasmodium parasites may not be able to develop in the presence of microsporidians, because of competition for a resource required by both parasites. It is possible that a more definitive answer to this question could have been reached by including a second set of controls in the experiment. Immune priming was only achieved with concurrent microsporidian infection, therefore a non-infective treatment such as lipopolysaccharide injections could have been used to induce non-infectious immune priming, allowing the comparison between microsporidian presence and absence to be made. However, these two possibilities do not explain the increased melanisation response associated with less effective infection by malaria. Therefore, immune priming remains the most likely explanation.
Further support for our suggestion that microsporidians impede the development of malaria by priming the immune response comes from a previous study, showing that microsporidian infection leads to an enhanced antibacterial response 
. This not only corroborates the idea mentioned in the introduction — that the melanisation response reflects more general immune priming — but may be a direct immunological mechanism, as antimicrobial responses appear to be largely responsible for the clearing of malaria parasites 
. Thus, immune-priming appears to stimulate the mosquito's immune system and prepare it for subsequent Plasmodium
challenge, thus making the infected mosquito partially refractory to malarial infection. A similar immune system boost occurs in mosquitoes following bacterial challenge. If mosquitoes are infected with bacteria before being fed on malarious blood, they are less likely to be infected my malaria than if they are not infected by the bacteria 
, whereas mosquitoes treated with antibiotics display lower expression of immune genes and are more susceptible to Plasmodium
Overall, our results suggest that microsporidians prime the immune response of mosquitoes in a way that impedes the development of malaria parasites.