Wolbachia infections in natural Anopheles populations affect egg laying and negatively correlate with Plasmodium development

Abstract

The maternally inherited alpha-proteobacterium Wolbachia has been proposed as a tool to block transmission of devastating mosquito-borne infectious diseases like dengue and malaria. Here we study the reproductive manipulations induced by a recently identified Wolbachia strain that stably infects natural mosquito populations of a major malaria vector, Anopheles coluzzii, in Burkina Faso. We determine that these infections significantly accelerate egg laying but do not induce cytoplasmic incompatibility or sex-ratio distortion, two parasitic reproductive phenotypes that facilitate the spread of other Wolbachia strains within insect hosts. Analysis of 221 blood-fed A. coluzzii females collected from houses shows a negative correlation between the presence of Plasmodium parasites and Wolbachia infection. A mathematical model incorporating these results predicts that infection with these endosymbionts may reduce malaria prevalence in human populations. These data suggest that Wolbachia may be an important player in malaria transmission dynamics in Sub-Saharan Africa.

Figure 1. wAnga localizes to the female germline.

wAnga (in green, central panels) was visualized in the ovaries of 14- to 16-day-old A. coluzzii females by FISH. (a) wAnga is detected in ovarian follicles using a Cy3-labelled probe specific for 16S DNA (white arrows). (b,c) wAnga is absent from the follicles of tetracycline-treated control females (b) and in follicles of infected females in which the labelled probe was in competition with an identical unlabelled probe (1:20 labelled:unlabelled) (c). DNA is labelled with 4,6-diamidino-2-phenylindole (in blue, left panels). Scale bar, 20 μm.

Figure 2. Analysis of reproductive phenotypes induced by wAnga.

(a–c) A. coluzzii females and males collected as eggs or larvae from Vallée du Kou were bred to adulthood and forced mated, and after blood feeding the reproductive output of females was individually scored. wAnga-infected (green) and -uninfected (grey) females (right) and males (left) were identified by 16S nested PCR post hoc, and mating couples were divided into four groups (wAnga-negative couples, dark grey; wAnga-positive couples, purple; wAnga-positive females mated to wAnga-negative males, red; and wAnga-negative females mated to wAnga-positive males, blue). (a) Egg inviability was calculated by dividing the number of infertile and unhatched eggs in each brood by the total number of eggs 4 days after egg laying. CI, which would manifest as higher inviability in the cross between wAnga-negative females and wAnga-positive males, was not observed (Kruskal–Wallis test, χ²=1.25, d.f.=3, P>0.05). (b) The total number of eggs produced by each female was determined by adding the number of eggs laid to the number of eggs remaining in the ovaries at the time of dissection (Kruskal–Wallis, χ²=4.09, d.f.=3, P>0.05). (c) Hatched larvae from each brood in a were raised to adulthood and sex was scored. The sex ratio of each brood was calculated as the number of female progeny divided by the total progeny (Student's t-test on log₁₀-transformed ratios, t=0.257, d.f.=26, P>0.05). (d) A total of 221 blood-fed females were collected from the walls of houses in Vallée du Kou, and 2 days after collection placed in individual oviposition containers to record the timing of egg-laying. The cumulative proportion of females that laid (n=143) on each of 3 consecutive nights following access to an oviposition site is plotted. wAnga-infected females (green line) laid eggs more quickly than uninfected females (grey line) (log-rank test, χ²=32.36, d.f.=1, P<0.0001). In a and b, horizontal lines represent the medians. In c, vertical lines and error bars represent the geometric mean and the 95% confidence intervals, respectively. Numbers in parentheses indicate the sample size.

Figure 3. wAnga infections reduce malaria prevalence in mosquito populations and in models of human populations.

(a) Blood-fed females were collected from houses in Vallée du Kou, and allowed to develop eggs and oviposit. A minimum of 5 days post collection, thoraxes and abdomens were dissected and screened for the presence of wAnga and Plasmodium by 16S (or 16S nested) PCR and 18S quantitative PCR, respectively. Shown in red are the proportions of Plasmodium-infected females in both wAnga-infected (green, N=116) and uninfected (grey, N=105) females (Fisher's exact post hoc test on unnormalized data, two-tailed, P=0.0018; Supplementary Table 2). Numbers in parentheses indicate the sample size. (b) A modified Ross–Macdonald model incorporating Wolbachia-infected mosquito compartments was used to determine the impact of Wolbachia on malaria prevalence. In addition to variation in biting rate and susceptibility to malaria, Wolbachia-infected mosquitoes were either considered to have an identical daily mortality rate to uninfected mosquitoes (red line) or an enhanced daily mortality rate due to trade-offs with increased gonotrophic cycles (blue line). The shaded area represents the range in Wolbachia prevalence detected in our studies across multiple years.