Primers used in qPCR determination of bacterial density.

<p>Primers used in qPCR determination of bacterial density.</p

Immune transcript analyses in <i>D. melanogaster</i> infected with <i>w</i>MelPop and <i>w</i>Mel.

<p>Transcripts are ranked by biological process and/or molecular function. Gene identifiers (Gene ID), Description and Symbol were compiled from Flybase. AFC, Absolute Fold Change, ND, No Detection. Asterisks indicate a statistically significant difference (n = 10, Mann-Whitney <i>U</i> test with q-value adjustment, *: q<0.05, **: q<0.01).</p

Dengue blocking in <i>D. melanogaster</i> and <i>A. aegypti</i> infected by <i>Wolbachia</i> strain <i>w</i>Mel.

<p>69 µl of 10⁷ pfu/ml of DENV2 strain 92T (grey circles) and DENV2 strain ET300 (black circles) were injected into flies (<i>w¹¹¹⁸w</i>Mel) and mosquitoes (MGYP2) infected by <i>w</i>Mel and their tetracycline-treated uninfected counterparts (<i>w¹¹¹⁸</i>tet and MGYP2tet). Dengue levels in individual insects were determined 8 days post-infection by RT-PCR using a TaqMan assay specific to dengue in 1 µg of total RNA. The fraction of flies that had detectable dengue infections is shown above each set of data points. (n = 15, Mann-Whitney <i>U</i> test, **: p<0.01, ***:p<0.001, ****:p<0.0001).</p

<i>Wolbachia</i>-Associated Bacterial Protection in the Mosquito <i>Aedes aegypti</i>

<div><p>Background</p><p><i>Wolbachia</i> infections confer protection for their insect hosts against a range of pathogens including bacteria, viruses, nematodes and the malaria parasite. A single mechanism that might explain this broad-based pathogen protection is immune priming, in which the presence of the symbiont upregulates the basal immune response, preparing the insect to defend against subsequent pathogen infection. A study that compared natural <i>Wolbachia</i> infections in <i>Drosophila melanogaster</i> with the mosquito vector <i>Aedes aegypti</i> artificially transinfected with the same strains has suggested that innate immune priming may only occur in recent host-<i>Wolbachia</i> associations. This same study also revealed that while immune priming may play a role in viral protection it cannot explain the entirety of the effect.</p><p>Methodology/Findings</p><p>Here we assess whether the level of innate immune priming induced by different <i>Wolbachia</i> strains in <i>A. aegypti</i> is correlated with the degree of protection conferred against bacterial pathogens. We show that <i>Wolbachia</i> strains <i>w</i>Mel and <i>w</i>MelPop, currently being tested for field release for dengue biocontrol, differ in their protective abilities. The <i>w</i>MelPop strain provides stronger, more broad-based protection than <i>w</i>Mel, and this is likely explained by both the higher induction of immune gene expression and the strain-specific activation of particular genes. We also show that <i>Wolbachia</i> densities themselves decline during pathogen infection, likely as a result of the immune induction.</p><p>Conclusions/Significance</p><p>This work shows a correlation between innate immune priming and bacterial protection phenotypes. The ability of the Toll pathway, melanisation and antimicrobial peptides to enhance viral protection or to provide the basis of malaria protection should be further explored in the context of this two-strain comparison. This work raises the questions of whether <i>Wolbachia</i> may improve the ability of wild mosquitoes to survive pathogen infection or alter the natural composition of gut flora, and thus have broader consequences for host fitness.</p></div

Survival curves of <i>Wolbachia</i>-infected (circle) and <i>Wolbachia</i>-uninfected (square) Drosophila infected with pathogenic bacteria (solid) or mock infected with PBS (open).

<p>(A&amp;E) <i>E. carotovora</i>, (B&amp;F) <i>B. cepacia</i>, (C&amp;G) <i>S. typhimurium</i> and (D&amp;H) <i>M. marinum</i>. Error bars are SEM calculated from the three replicates. * P-value&lt;0.05, ** P-value&lt;0.01, *** P-value&lt;0.001 denote differences in survival between <i>Wolbachia</i> infected and uninfected lines by Log-rank statistics (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002362#pntd.0002362.s001" target="_blank">Table S1A</a>).</p

Median (with interquartile range) relative <i>Wolbachia</i> density after infection in mosquitoes.

<p>Five pairs of individuals were used for <i>E. carotovora</i> (A), <i>B. cepacia</i> (B), <i>S. typhimurium</i> (C) and <i>M. marinum</i> (D). (Mann-Whitney U-test; * P-value&lt;0.05, ** P-value&lt;0.01).</p

Gene Ontology (GO) terms over-represented among gene transcripts significantly up-regulated in <i>w</i>MelPop-CLA-infected <i>A. aegypti</i>.

<p>Adjusted <i>P</i>-values are the <i>P</i>-values generated by the Ontologizer program <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002548#ppat.1002548-Grossmann2" target="_blank">[38]</a>, using the Benjamini-Hochberg method.</p

Gene Ontology (GO) terms over-represented among gene transcripts significantly up-regulated in <i>w</i>Mel-infected <i>A. aegypti</i>.

<p>Adjusted <i>P</i>-values are the <i>P</i>-values generated by the Ontologizer program <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002548#ppat.1002548-Grossmann2" target="_blank">[38]</a>, using the Benjamini-Hochberg method.</p

Venn diagram showing significant expression change in response to infection in <i>A. aegypti</i> infected with <i>w</i>MelPop-CLA or <i>w</i>Mel.

<p>The overlap region corresponds to <i>A. aegypti</i> gene transcripts significantly up- and down-regulated in response to both strains. Numbers indicate gene transcripts up-regulated/gene transcripts down-regulated.</p

Survival curves of <i>Wolbachia</i>-infected (circle) and <i>Wolbachia</i>-uninfected (square) mosquitoes infected with pathogenic bacteria (solid) or mock infected with PBS (open).

<p>(A&amp;E) <i>E. carotovora</i>, (B&amp;F) <i>B. cepacia</i>, (C&amp;G) <i>S. typhimurium</i> and (D&amp;H) <i>M. marinum</i>. Error bars are SEM calculated from the three replicates. * P-value&lt;0.05, ** P-value&lt;0.01, *** P-value&lt;0.001 denote differences in survival between <i>Wolbachia</i> infected and uninfected lines by Log-rank statistics (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002362#pntd.0002362.s001" target="_blank">Table S1B</a>). The relative risk ratio [EXP(B)] of <i>Wolbachia</i> uninfected to infected lines with 95% confidence intervals shown in parentheses is reported on graphs where significant.</p