Effect of AgESP silencing on <i>Plasmodium berghei</i> infection.

<p>AgESP mRNA (A) and protein (B) silencing in the mosquito midgut by systemic dsRNA injection. (C) Effect of AgESP silencing on the number of <i>P. berghei</i> oocysts present in the midgut 7 days post infection. (D) Effect of <i>P. berghei</i> infection on SRPN6 expression. SRPN6 mRNA levels in midguts of mosquitoes fed on a healthy mouse (C, control; grey bar) or a <i>P. berghei-</i>infected mouse (I, infected; red bar). (E) Effect of AgESP silencing on expression of AgESP (blue bars) or SRPN6 (red bars) 24 h after ingestion of a <i>Plasmodium</i>-infected blood meal. (F) A significant reduction in number of parasites that invaded mosquito midgut was already observed in AgESP-silenced mosquitoes 28 h post feeding (PF). (G) Immunostaining of the ookinete surface with anti-Pb28 antibodies (red) and nuclear staining with DAPI (blue). There are fewer ookinetes in AgESP-silenced midguts, but no evidence of parasite fragmentation was observed. (H) Effect of AgESP silencing in the refractory <i>Anopheles gambiae</i> L35 strain on the number of melanized parasites (black dots). Each dot represents the number of parasites on an individual midgut. Live parasites are shown in green and melanized parasites in black. The red line indicates the median. Distributions were compared using the Mann-Whitney test. (I) Effect of silencing AgESP (blue bars) on the expression of gelsolin, Ciboulot, Wiskott-Aldrich syndrome protein (WASP), and F-actin capping protein (CP) 24 h PF on a <i>P. berghei</i>-infected mouse. The unpaired two-tailed <i>t</i>-test was used to compare the different experimental groups (*, <i>P</i> < 0.05; **, <i>P</i> < 0.01; ***, <i>P</i> < 0.001). All expression analysis was confirmed in 2–3 independent biological replicates. Infection phenotypes were confirmed in 2–3 independent experiments.