The microbiome composition of <i>Aedes aegypti</i> is not critical for <i>Wolbachia</i>-mediated inhibition of dengue virus

<div><p>Background</p><p>Dengue virus (DENV) is primarily vectored by the mosquito <i>Aedes aegypti</i>, and is estimated to cause 390 million human infections annually. A novel method for DENV control involves stable transinfection of <i>Ae</i>. <i>aegypti</i> with the common insect endosymbiont <i>Wolbachia</i>, which mediates an antiviral effect. However, the mechanism by which <i>Wolbachia</i> reduces the susceptibility of <i>Ae</i>. <i>aegypti</i> to DENV is not fully understood. In this study we assessed the potential of resident microbiota, which can play important roles in insect physiology and immune responses, to affect <i>Wolbachia</i>-mediated DENV blocking.</p><p>Methodology/Findings</p><p>The microbiome of <i>Ae</i>. <i>aegypti</i> stably infected with <i>Wolbachia</i> strain <i>w</i>Mel was compared to that of <i>Ae</i>. <i>aegypti</i> without <i>Wolbachia</i>, using 16s rDNA profiling. Our results indicate that although <i>Wolbachia</i> affected the relative abundance of several genera, the microbiome of both the <i>Wolbachia-</i>infected and uninfected mosquitoes was dominated by <i>Elizabethkingia</i> and unclassified <i>Enterobacteriaceae</i>. To assess the potential of the resident microbiota to affect the <i>Wolbachia-</i>mediated antiviral effect, we used antibiotic treatment before infection with DENV by blood-meal. In spite of a significant shift in the microbiome composition in response to the antibiotics, we detected no effect of antibiotic treatment on DENV infection rates, or on the DENV load of infected mosquitoes.</p><p>Conclusions/Significance</p><p>Our findings indicate that stable infection with <i>Wolbachia</i> strain <i>w</i>Mel produces few effects on the microbiome of laboratory-reared <i>Ae</i>. <i>aegypti</i>. Moreover, our findings suggest that the microbiome can be significantly altered without affecting the fundamental DENV blocking phenotype in these mosquitoes. Since <i>Ae</i>. <i>aegypti</i> are likely to encounter diverse microbiota in the field, this is a particularly important result in the context of using <i>Wolbachia</i> as a method for DENV control.</p></div

Relative abundance of bacterial genera corrected for <i>Wolbachia</i>.

<p>Relative abundance of genera from 19 (- antibiotics) or 20 (+ antibiotics) <i>w</i>Mel mosquitoes after OTU assigned to <i>Wolbachia</i> were removed; ‘unclassified’ indicates OTU that were not classified at the genus level.</p

The effect of <i>Wolbachia</i> infection and antibiotic treatment on the relative abundance (<i>Wolbachia</i>-corrected) of specific genera.

<p>The effect of <i>Wolbachia</i> infection and antibiotic treatment on the relative abundance (<i>Wolbachia</i>-corrected) of specific genera.</p

Infection rate and DENV genome copy number are unaffected by manipulation of the microbiome using antibiotic treatment.

<p>(A) Percent of infected mosquitoes as determined by qRT-PCR; total numbers of mosquitoes tested per group are listed below each chart. (B) Total number of DENV genome copies per body for all mosquitoes identified as DENV-positive in (A). Statistical analysis was performed using a one-way ANOVA, only the effect of <i>Wolbachia</i> was significant. Data shows median and interquartile range.</p

Microbiome composition of wt and <i>w</i>Mel <i>Ae</i>. <i>aegypti</i> mosquitoes.

<p>Relative abundance of microbiota present in wt (A) or <i>w</i>Mel (B) <i>Ae</i>. <i>aegypti</i> lines as determined by sequencing of 16s rDNA following a DENV-infectious blood-meal. Where indicated (+ antibiotics) mosquitoes were treated with a combination of penicillin-streptomycin-kanamycin for 3 generations before profiling. Each bar represents a single mosquito. OTU are grouped by genus; ‘unclassified’ indicates OTU that were not classified at the genus level.</p

Intergenic regions (IGR) selected for 5’RACE experiments^a and name and position of the two putative<i>Wolbachia</i> small non-coding RNAs we identified.

<p>^aNote that we did not demonstrate sRNA-like intergenic-specific transcription for most of these regions</p><p>^bIGR ID as designed in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118595#pone.0118595.s001" target="_blank">S1 Data</a></p><p>^cDetermined by 5’RACE</p><p>^dDetermined by RT-PCR with downstream CDS</p><p>^eAlso predicted by the bioinformatic approach.</p><p>Intergenic regions (IGR) selected for 5’RACE experiments^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118595#t004fn001" target="_blank">a</a>} and name and position of the two putative<i>Wolbachia</i> small non-coding RNAs we identified.</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

The WIG profile of a highly conserved feature.

<p>This profile shows the probability that each position in the region belongs to the most conserved class. The conserved non-coding region is positioned in wMel coordinates 1,039,579–1,039,870. The top 3 bars containing single letter amino acid codes show 3 possible protein translation phases. At the bottom, coding region WD1082 is shown (in light blue with arrows). There are no previously annotated non-coding regions corresponding to this region.</p

Intergenic regions predicted by <i>changept</i> to be highly conserved.

<p>Intergenic regions predicted by <i>changept</i> to be highly conserved.</p

Summary of the RNA-Seq data obtained.

<p>Summary of the RNA-Seq data obtained.</p