<i>E. cloacae</i>-mediated inhibition of <i>P. falciparum</i> development is reversed by SRPN6 silencing.

<p>(A) Semi-quantitative RT-PCR analysis was used to determine the effects of gene silencing on <i>An. stephensi</i> midgut samples after feeding with a <i>P. falciparum</i> gametocyte/<i>E. cloacae</i> (10⁶/ml) mixture. SRPN6 mRNA abundance in midguts of dsSRPN6-injected mosquitoes was suppressed when compared with dsGFP controls. Ribosomal protein S7 (rpS7) served as a loading control. (B) <i>An. stephensi</i> mosquitoes were fed on a <i>P. falciparum</i> gametocyte culture (control) or were injected with dsGFP or dsSRPN6 and fed a <i>P. falciparum</i> gametocyte/<i>E. cloacae</i> (10⁶/ml) mixture. Midgut oocyst numbers were determined after 8 days by staining with mercurochrome. Data were pooled from three independent experiments and analyzed using Kruskal-Wallis analysis and a Dunn’s post-test to determine significance. Median oocyst numbers are depicted by the red line and the total numbers (n) of individual mosquitoes analyzed are denoted below each sample. The presence (+) or absence (−) of <i>Enterobacter</i> feeding or dsRNA treatment are shown below each sample. <i>P</i>-values are denoted by asterisks (** = <i>P</i><0.01; *** = <i>P</i><0.001). (C) Bar graphs showing the prevalence of infection among samples shows that SRPN6-silencing increase the percentage of mosquitoes containing at least one <i>P. falciparum</i> oocyst, although the results are not significant when analyzed by Chi-squared analysis.