Figure 1.
Components of the Imd pathway explored in this study or others are represented by different colored shapes. Black arrows or lines indicate known interactions or translocations. Gray arrows indicate potential interactions based on D. melanogaster studies. The gray bracketed area indicates the molecules possibly involved in other responses, but not the responses against P. falciparum. Numbers and arrows within colored blocks indicate the -fold change in P. falciparum infection that results when the corresponding pathway member is silenced. The list of genes inside the nucleus portion of the diagram shows those known to be active against Plasmodium and whose expression has been shown by our studies to be REL2-regulated.
Figure 2.
Some members of the Imd pathway have an effect on P. falciparum infection.
(A–G) Dots represent individual oocyst counts following the indicated RNAi treatment; horizontal red bars represent the median number of oocysts per gut. P-values were derived from Mann-Whitney statistical tests and appear above each treatment and refer to that treatment as compared to the GFP dsRNA-treated control. Additional statistical analyses appear in Table S1) Filled portion of bars represent the % of all mosquitoes harboring at least one oocyst; open portion represents those in the group that were uninfected. All assays represent two to three independent biological replicate. Cpr, Caspar. (H) Prevalence of P. falciparum infection following the indicated RNAi treatment.
Figure 3.
Caspar silencing also influences early and late oocysts.
(A) Time course of caspar-silencing efficiency, quantified using real-time quantitative PCR. Gray bars represent the % caspar expression at each given time point as an average of two replicates, and error bars reflect the standard error between those replicates. Cpr, Caspar. (B) Infection intensity of mosquitoes silenced for caspar at 3 days post-infection (dpi). Since ookinetes invade the midgut at 24 hours post-infection this time point targets early oocysts. (C) Infection intensity of mosquitoes silenced for caspar at 6 days post-infection. Since sporozoites begin to emerge from the oocyst at 7–8 days post infection, this time point targets late oocysts. For both, dots represent individual oocyst counts following the indicated RNAi treatment; horizontal red bars represent the median number of oocysts per gut. Assays represent two to three independent biological replicates and were subject to Mann-Whitney statistical tests. P-values appear above each treatment and refer to that treatment as compared to the GFP dsRNA-treated control. Additional statistical analyses appear in Table S2. Filled portion of bars represent the % of all mosquitoes harboring at least one oocyst; open portion represents those in the group that were uninfected. Cpr, Caspar.
Figure 4.
Caspar-mediated killing of P. falciparum is not dependent on midgut bacteria.
(A) Blue bars represent bacteria colony-forming unit (CFUs) from midguts of mosquitoes undergoing the indicated treatments. Pluses and minuses indicate the presence or absence of antibiotic in GFP or Cpr dsRNA treated group. Each bar represents the average of at least 15 mosquitoes tested, with each mosquito's CFU count determined by averaging counts from three serial dilutions. Bars represent the standard deviation for all mosquitoes in a given treatment group. Cpr, Caspar. (B) Dots represent individual oocyst counts following the indicated RNAi treatment; horizontal red bars represent the median number of oocysts per gut. Pluses and minuses indicate the presence or absence of antibiotic in the GFP or Cpr dsRNA-treated group. Assays represent three independent biological replicates and were subject to Mann-Whitney statistical tests. P-values appear below each treatment and refer to that treatment as compared to the GFP dsRNA-treated control. Additional statistical analyses appear in Table S3. Filled portion of bars represent the % of all mosquitoes harboring at least one oocyst; open portion represents those in the group that were uninfected. Cpr, Caspar.
Figure 5.
Imd pathway components and effectors differ in their ability to affect P. falciparum infections of high, medium, and low infection intensities.
Intensity of P. falciparum oocysts in A. gambiae silenced for given genes and subjected to low (A), medium (C) or high (E) infection exposures. Bars represent median numbers of oocysts per midgut, and dots represent individual midgut oocyst counts. Assays represent at least three independent biological replicates and were subject to Mann-Whitney statistical tests. P-values appear above each treatment and refer to that treatment as compared to the GFP dsRNA-treated control. Non-significant p-values were not included in the figure. Additional statistical analyses appear in Table S4. D-F: Prevalence of infection in A. gambiae subjected to low (B), medium (D) and high (F) loads of P. falciparum. Filled portion of bars represent the % of all mosquitoes harboring at least one oocyst; open portion represents those in the group that were uninfected. wAPL1 (whole APL1) dsRNA – dsRNA for a conserved region of APL1 genes, which results in the silencing of all three APL1 proteins (APL1A, APL1B and APL1C).