^{*}

Conceived and designed the experiments: JCK. Performed the experiments: IB. Analyzed the data: IB. Wrote the paper: IB JCK.

The authors have declared that no competing interests exist.

Microsporidian parasites of mosquitoes offer a possible way of controlling malaria, as they impede the development of

We evaluated the possibility that larval infection by a microsporidian primes the immune system of adult mosquitoes in a way that enables a more effective anti-

The microsporidian-infected mosquitoes were less likely to harbour oocysts (58.5% vs. 81.8%), harboured fewer oocysts (8.9 oocysts vs. 20.7 oocysts) if the malaria parasite did develop and melanised the Sephadex bead to a greater degree (73% vs. 35%) than the controls. While the isolates differed in the number of oocysts and in the melanisation response, the stimulation of the immune response was not correlated with either measure of malaria development. Nevertheless, the consistent difference between microsporidian-infected and –uninfected mosquitoes — more effective melanisation and less successful infection by malaria — suggests that microsporidians impede the development of malaria by priming the mosquito's immune system.

Microsporidian parasites of mosquitoes offer a possibility of effective malaria control, as they target several factors that determine the epidemiology of malaria: they reduce mosquito populations by increasing larval and pupal mortality and by decreasing fecundity

The mechanism involved in achieving this interference is unknown. Possibilities include that the microsporidians use resources required for the development of malaria and that microsporidians block molecular targets used by malaria parasites to invade the mosquito's midgut. In this study we consider the possibility that a microsporidian infection primes the mosquito's immune system in a way that helps it to defend itself against a later infection by

We investigate the role of immune-priming in the interaction between microsporidians and malaria by exposing larvae of the mosquito

Our measure of the immune response was the degree to which a CM-25 Sephadex bead injected into the mosquito's thorax was melanised. As the melanisation response is genetically correlated (i.e. shares part of its genetic basis) with the antibacterial response (at least in terms of the phenotypic outcome: the extent to which bacteria are cleared)

We reared 1200 larvae individually in 12-well plates, in two consecutive blocks of 600 mosquitoes. 240 of these were uninfected controls, the others were infected with one of 4 microsporidian isolates. Out of the 701 mosquitoes that survived to adulthood, 327 were female and lived long enough to be used in the experiments. The numbers of females within treatments (59 in the control treatment; 60, 64, 70 and 74 for the four isolates) were similar (χ^{2} = 3.50, p = 0.478) and blocking had no effect on survival, so that our results were not biased because of larval mortality due to the microsporidian.

Of the 159 adult females exposed to the ^{2} = 0.03, p = 0.860), but the proportion of mosquitoes that fed did depend on the microsporidian isolate that infected them (χ^{2} = 7.55, p = 0.056), ranging from 64% to 87%. Of the 116 fed mosquitoes, 73 harboured oocysts 10 days later. Blocking had no effect on the feeding efficiency of the mosquitoes, except for treatment with isolate 3, where more mosquitoes from block one took a blood meal than block two (χ^{2} = 6.046, p = 0.0139). Microsporidian-infected mosquitoes were less likely (58.5%) to harbour oocysts than microsporidian-uninfected controls (81.8%) (χ^{2} = 4.40, p = 0.036) (^{2} = 0.65, p = 0.885). The mean number of oocysts in the 73 mosquitoes with at least 1 oocyst was 11.9, ranging from 4.8 to 20.7 among the five treatments (_{5,67} = 7.82, p<0.001). Block had no effect on the number of oocysts harboured by mosquitoes in each treatment group. Mosquitoes infected by a microsporidian harboured an average of 8.9 oocysts; controls harboured 20.7 oocysts (F_{1,69} = 30.93, p<0.001). The mean number of oocysts in mosquitoes infected by different microsporidian isolates ranged from 4.8 to 13.8 (F_{1,47} = 5.45, p = 0.003).

(a) The proportion of mosquitoes that harboured at least one oocyst 10 days after blood feeding. (b) The mean number of oocysts in mosquitoes with at least one oocyst. In both panels, the vertical lines show the standard errors of the estimates, the horizontal, dotted lines show the means of the controls, and the numbers in the bars indicate the number of mosquitoes analyzed. The isolates are numbered in order of increasing melanisation efficacy (see

Of the 168 adult females inoculated with CM-25 Sephadex beads, 138 (80%) survived. Overall, the five treatments did not differ in their survival (χ^{2} = 4.33, p = 0.363), though there was a slight difference in survival between blocks one and two for mosquitoes infected with isolate 4 (χ^{2} = 3.873, p = 0.0491) and 1 (χ^{2} = 3.949, p = 0.0469). In eleven mosquitoes we could not find the bead, so that we analyzed 127 beads. The degree to which a bead was melanised ranged from 35% in the uninfected control mosquitoes to 73% in the mosquitoes infected with one of the microsporidian isolates (_{1,123} = 11.47, p<0.001), and the four isolates differed in the degree to which they stimulated the melanisation response (F_{3,95} = 2.78 p = 0.004).

Each point represents the mean proportion of a Sephadex bead melanized by a mosquito. The vertical lines represent the standard errors of the means and the horizontal, dotted line shows the mean of the controls. Again, the numbers in the bars indicate the number of mosquitoes sampled.

Thus, larval infection by _{1,5} = 0.64, p = 0.459) or the intensity of infection (F_{1,5} = 1.63, p = 0.258) by malaria. Note, however, that with only four isolates in the experiment, the power to detect these correlations was weak (likelihood of infection: power = 0.1; intensity of infection: power = 0.18).

Each point shows the mean within a block of the control or the microsporidian-infected mosquitoes (where the four isolates were pooled), and the horizontal and vertical lines show the standard errors of the estimates. (a) Association between the melanization response and the proportion of mosquitoes harbouring at least one oocyst 10 days after bloodfeeding. (b) Association between the melanization response and the mean number of oocysts.

Larval infection by any of four isolates of the microsporidian

That

That

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

Another possible mechanism for the interference of microsporidian infection with

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

Overall, our results suggest that microsporidians prime the immune response of mosquitoes in a way that impedes the development of malaria parasites.

The experiment was conducted in 2 blocks (2 consecutive clutches, 1 week apart), each comprising 600 mosquitoes. We exposed 120 larvae per block to one of four isolates of

We used the G3 strain of

The microsporidian

Spores harboured by

The four

We exposed 2-day old

The

The degree to which Sephadex beads were melanised was used as a measure of immunity.

The beads range from 40–120 µm in diameter, of which we selected the smallest ones (estimated to range from 40 to 60 µm) visually. The beads were rehydrated in saline solution containing 1.3 mM NaCl, 0.5 mM KCl, 0.2 mM CaCl2 and 0.001% methyl green (pH 6.8)

Statistical analyses were performed with JMP version 6.0 (

To control for possible biases, we analysed the likelihood that a mosquito survived to become an adult, blood-fed and survived the infection by malaria or survived the bead injection with logistic analyses. The likelihood of a successful malaria infection (i.e. the likelihood that a mosquito harboured at least one oocyst) was examined with a logistic analysis. The number of oocysts was analysed only in the mosquitoes where the malaria-infection was successful (i.e. where at least one oocyst was found). The square-root of the number of oocysts was evaluated with an ANOVA and gave normally distributed residuals. The proportion of a bead that was melanised was arc-sine transformed and analysed with an ANOVA. Each analysis included block as a nominal factor. For each trait, we tested for a difference between control and microsporidian-infected mosquitoes and for a difference among the microsporidian isolates with two separate analyses. We estimated the association between melanisation and the two measures of malaria infection by calculating the correlation of the average values per treatment and block, using block as a fixed factor in the analysis of covariance.

We thank Lena Lorenz for preparing the isolates of