The JNK Pathway and <i>Plasmodium berghei</i> infection in <i>Anopheles gambiae</i>.

<p>(A) Diagram representing the organization of the JNK signaling cascade based on functional studies from vertebrates and <i>Drosophila</i>. Five <i>An. gambiae</i> orthologs were functionally characterized, including two kinases, hemipterous <i>(hep)</i> and c-Jun N-terminal kinase <i>(jnk)</i>; a phosphatase, <i>puc</i>; and two transcription factors, Jun <i>(jun)</i> and Fos <i>(fos)</i> (B) Basal mRNA expression of putative genes from the JNK pathway in adult females. <i>Hemipterous (Hep)</i>, Jun N-terminal kinase <i>(JNK)</i>, <i>Jun</i> and <i>Fos</i> transcription factors and <i>puckered (puc)</i> mRNA levels in different organs of sugar-fed females. Mg, midgut; H, head; Th, thorax; Ab, abdomen; Hc, hemocyte; Ov, ovaries. Expression in different tissues relative to midgut levels, for which the mean was given a value of “1”. Error bars indicate SEM of two biological replicates. (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003622#ppat.1003622.s017" target="_blank">Table S11</a> for gene ID numbers and primer sequences) (C) Midgut expression of members of the JNK pathway in response to <i>Plasmodium</i> infection in three independent experiments. Ratio of expression in infected/control blood-fed mosquitoes of <i>Hep</i>, <i>JNK</i>, <i>Puc</i>, <i>Jun</i> and <i>Fos</i> mRNA levels in midguts of mosquitoes from 3 independent experiments (green, red and blue bars). Error bars indicate SEM of two technical replicates. The expression analysis in each biological replicate is shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003622#ppat.1003622.s009" target="_blank">Table S3</a>. P-values determined by Student's-T test after log 2 transformation; **, <i>p</i><0.01, ***, <i>p</i><0.001. *, p<0.05; **, p<0.01, ***, p<0.001. (D) Effect of silencing JNK pathway members on <i>P. berghei</i> infection. (E) Effect of silencing the transcription factor <i>jun</i> alone or and co-silencing <i>jun</i> and the negative regulator <i>puc</i> on <i>Plasmodium</i> infection. For (D) and (E), the green dots represent oocyst counts from individual midguts and the horizontal red bar indicates the median infection level. Groups were compared using the KS, Mann-Whitney and Kruskal-Wallis tests with Dunn's post test (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003622#ppat.1003622.s009" target="_blank">Table S3</a>). The P-values for the Mann-Whitney tests are shown. Graphs represent samples pooled from three biological replicates with comparable (not statistically different) medians in their <i>dsLacZ</i>-injected groups (n = number of midguts).