Cytoplasmic incompatibility.

<p><b>(A)</b> When a <i>Wolbachia</i>-infected male (red) mates with an uninfected female (black), a sperm–egg incompatibility means that some or all of the embryos die. Therefore, infected females produce more offspring than uninfected females (red versus black mosquitoes). <b>(B)</b> This reproductive advantage depends on the prevalence of <i>Wolbachia</i> in the population, because when <i>Wolbachia</i> is rare, females are unlikely to mate with infected males. The <i>Wolbachia</i> strain in <i>Aedes aegypti</i> carries a physiological cost, reducing the fecundity of infected females. If this cost exceeds the advantage of cytoplasmic incompatibility, then the infection is lost from the population. This creates a threshold prevalence below which <i>Wolbachia</i> is lost and above which it invades the population. This cartoon assumes infected females transmit <i>Wolbachia</i> to all their offspring. <i>Image credit</i>: <a href="https://doi.org/10.1093/gbe/evw018" target="_blank">https://doi.org/10.1093/gbe/evw018</a>.</p

Titres of DAV and DCV in infected <i>Ge-1</i> transgenic flies.

<p>(A) Relative viral titres in DAV infected flies. (B) Relative viral titres in DCV infected flies. Error bars are standard errors.</p

Identification of <em>Wolbachia</em> Strains in Mosquito Disease Vectors

<div><p><em>Wolbachia</em> bacteria are common endosymbionts of insects, and some strains are known to protect their hosts against RNA viruses and other parasites. This has led to the suggestion that releasing <em>Wolbachia-</em>infected mosquitoes could prevent the transmission of arboviruses and other human parasites. We have identified <em>Wolbachia</em> in Kenyan populations of the yellow fever vector <em>Aedes bromeliae</em> and its relative <em>Aedes metallicus,</em> and in <em>Mansonia uniformis</em> and <em>Mansonia africana,</em> which are vectors of lymphatic filariasis. These <em>Wolbachia</em> strains cluster together on the bacterial phylogeny, and belong to bacterial clades that have recombined with other unrelated strains. These new <em>Wolbachia</em> strains may be affecting disease transmission rates of infected mosquito species, and could be transferred into other mosquito vectors as part of control programs.</p> </div

Survival following challenge.

<p>Survival of flies inoculated with a low initial dose of DCV or control solution (uninfected cell culture medium) followed by a secondary infection with a high dose of DCV or control solution.</p

Resistance assay of resistant and susceptible recombinants.

<p>(A) Recombinants (left) were selected in the region mapped in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005387#ppat.1005387.g001" target="_blank">Fig 1</a> using two eye-colour markers, injected with sigma virus and tested for paralysis after exposure to CO₂. This identified an ~8 kb region containing a whole gene called <i>Ge-1</i> and the flanking 3’ untranslated regions of the genes <i>CG4705</i> and <i>Reps</i>. (B) relative viral titre in 20 resistant and 16 susceptible recombinants at 6 days post infection and 12 days post infection. (C) Domain organization of <i>Ge-1</i> (adapted from [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005387#ppat.1005387.ref038" target="_blank">38</a>]). The C-terminal domain is highlighted in blue with the highly conserved C-terminal region in dark blue. Red indicates a 26 amino acid deletion that differs between the resistant and susceptible alleles. Stars represent non-synonymous differences between <i>D</i>. <i>melanogaster</i> and <i>D</i>. <i>simulans</i>. Red stars represent changes that occurred on the lineage leading to <i>D</i>. <i>melanogaster</i> and green stars represent changes on the lineage leading to <i>D</i>. <i>simulans</i>. Serine-rich linker sequences from <i>D</i>. <i>simulans</i> and <i>D</i>. <i>melanogaster</i> susceptible line 22a and resistant line EME are shown. (D) <i>Ge-1</i> expression in 20 resistant and 16 susceptible recombinants (described in Fig 2B) at 6 days post infection and 12 days post infection. Error bars are standard errors. Viral RNA loads and gene expression were measured by quantitative PCR and standardised to the reference gene <i>Actin5c</i>.</p

The effect of <i>Ago2</i> and <i>DCP1</i> on susceptibility to DMelSV.

<p>(A) and (B) are the effect of <i>Ge-1</i> on susceptibility in <i>Ago2</i>^<i>51B</i> mutant <i>flies</i>. Blue represents flies with the resistant <i>Ge-1</i> allele and <i>Ago2</i> mutant, black the resistant <i>Ge-1</i> allele and <i>Ago2</i> wild-type, red the <i>Ge-1</i> susceptible allele and <i>Ago2</i> mutant, and orange the susceptible <i>Ge-1</i> allele and <i>Ago2</i> wild-type. (A) CO₂ sensitivity assay. (B) Relative viral titre estimated by quantitative PCR relative to <i>RpL32</i> was used as reference gene. (C) The effect of knocking down <i>DCP1</i> on viral titre in <i>Ge-1</i> susceptible backgrounds (red bar) and <i>Ge-1</i> heterozygous backgrounds (blue bar). Grey bars are controls for the RNAi. Error bars are standard errors.</p

Linkage map of the gene controlling susceptibility.

<p>(A) The susceptibility of the resistant (EME) and the susceptible (22a) parental lines was measured by injecting the flies with the DMelSV and testing whether they become paralysed after exposure to CO2 (a symptom of infection). Panel B, C and D are the three successive experiments where recombinants in the region known to contain the gene were selected using molecular markers and made homozygous. On the left are the genotypes of the recombinants, with chromosomal regions from the resistant parent in blue and the susceptible parent in yellow, and the location on chromosome 2 in the <i>D</i>. <i>melanogaster</i> genome R5. The infection rate is shown on the right, with lines classed as resistant in blue and susceptible in yellow. There is a near-perfect association between genotype and phenotype in the regions between the vertical lines. Experiment (B) narrowed down the region to 735 kb using 31 recombinant lines. (C) Further narrowed down the region to 298 kb using 28 recombinant lines. (D) Narrowed down the region to 89 kb using 15 recombinant lines.</p

Transgenic flies with the 26 amino acid deletion in <i>Ge-1</i> are resistant to DMelSV.

<p>Flies were transformed with BAC clones carrying <i>Ge-1</i> that differs only in the presence (red) or absence (blue) of the deletion. The four genotypes are independent transformants. (A) The proportion of flies that was paralysed or dead after exposure to CO₂. Each point is a vial of flies. Horizontal bars represent the mean of each line. (B) Mean viral titre in transgenic lines. Viral RNA loads were measured by quantitative PCR and standardised to the reference gene <i>RpL32</i>. Error bars are standard errors.</p

The effect of knocking down <i>Ge-1</i> and <i>Reps</i> by RNAi on susceptibility to DMelSV.

<p>(A) Proportion of flies that were paralysed after exposure to CO₂ in control, <i>Ge-1-RNAi</i> and <i>Reps-RNAi</i> lines. Flies were kept at 25°C. (B) Viral titres in control, <i>Ge-1-RNAi</i> and <i>Reps-RNAi</i> lines standardised to reference gene <i>Ef1alpha100E</i>. This experiment was repeated at 25°C and 29°C. Error bars are standard errors.</p

Prevalence of <i>Wolbachia</i> in mosquitoes from Kenya.

<p>The prevalence is shown with the 95% confidence interval in parentheses.</p