Generation of a <i>w</i>Au – <i>w</i>AlbB superinfection.

<p>(<b>A</b>) Crosses between <i>w</i>Au<i>w</i>AlbB and wild-type lines. Eggs are from crosses of 20 males and 20 females. Numbers show percentage hatch rates with total numbers of eggs counted in parentheses. (<b>B</b>) Total <i>Wolbachia</i> densities measured by qPCR in <i>w</i>AlbB, <i>w</i>Au, and <i>w</i>Au<i>w</i>AlbB carrying <i>Aedes aegypti</i> females at ten days post adult eclosion. Each bar represents 10 biological replicates, with pools of 5 females per replicate. Error bars show SD. (<b>C</b>) <i>w</i>Au and <i>w</i>AlbB strain-specific densities in the ovaries of <i>w</i>AlbB, <i>w</i>Au<i>w</i>AlbB, and <i>w</i>Au carrying <i>Ae</i>. <i>aegypti</i>. Each bar represents the average densities from 5 biological replicates each containing ovaries of 10 adult females. Error bars show SD. Statistical analysis was performed using a one-way ANOVA. (<b>D</b>) Fluorescent <i>in situ</i> hybridization showing distributions of <i>w</i>Au (green) and <i>w</i>AlbB (red) in ovaries of the <i>w</i>Au, <i>w</i>AlbB, <i>w</i>Au<i>w</i>AlbB and wild-type (wt) lines. For all images ovaries were treated with both red and green probes. A no-probe control showing some green auto-fluorescence in wild-type ovaries is shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006815#ppat.1006815.s004" target="_blank">S4 Fig</a>. Blue stain is DAPI.</p

<i>Wolbachia</i> densities and tropism in <i>Aedes</i> mosquitoes.

<p>(<b>A</b>) Total <i>Wolbachia</i> densities were measured by qPCR in <i>w</i>AlbA, <i>w</i>AlbB, <i>w</i>Au, and <i>w</i>Mel carrying <i>Aedes aegypti</i> females at varying time points post adult eclosion. Each box represents 10 biological replicates, with pools of 5 females per replicate. The centre of a box plot shows median <i>Wolbachia</i> density, edges show upper and lower quartiles, and whiskers indicate upper and lower extremes. (<b>B</b>) Total <i>Wolbachia</i> densities in dissected tissues measured by qPCR. Each bar represents the average density of 5 biological replicates. For each of the tissue-specific replicates 5 biological replicates of 5 sets of salivary glands, 5 midguts, or 5 ovary pairs were assessed. Error bars show SD. Statistical analyses were performed using a two-tailed Student’s t-test.</p

Crosses between <i>Wolbachia</i>-infected lines.

<p>Crosses between <i>Wolbachia</i>-infected lines.</p

Virus inhibition in <i>Wolbachia</i>-infected <i>Aedes aegypti</i> lines.

<p>(<b>A</b>) Semliki Forest virus (SFV) genome copies per host cell following thoracic injection into <i>Wolbachia</i>-infected lines and wild-type <i>Ae</i>. <i>aegypti</i>. Females were left for 10 days prior to total RNA extraction and virus quantification by qPCR. Levels of target RNA sequences were normalized against the RPS17 house-keeping gene. 17, 16, 18, 17 and 17 females were PCR’d for the <i>w</i>AlbA, <i>w</i>Mel, <i>w</i>Au, <i>w</i>AlbB and wt, respectively. Statistical analysis was performed using a one-way ANOVA with a Dunnett’s post-hoc test. Dengue-2 (DENV) (<b>B</b> and <b>C</b>) and Zika (ZIKV) (<b>D</b> and <b>E</b>) viruses were orally administered to 5-day old females. After an incubation period of 12 days, females were salivated (Zika only) and salivary glands and abdomens dissected. Viral RNA in salivary glands (SG) and abdomens were quantified by reverse-transcriptase qPCR, with viral RNA levels normalized to host RNA using the <i>RpS17</i> house-keeping gene. A value of zero for normalized virus levels, indicates no amplification for virus cDNA in that sample. Zika viral titers in saliva were quantified by fluorescent focus assay with results show focus forming units (FFU). Proportions underneath each graph indicate the infection rate for a given strain. Statistical analyses for panels B, C, D and E were performed using a one-tailed Fisher’s exact test comparing rates of virus-positive to virus-negative samples. Black lines indicate median of non-zero values.</p

High temperature results in reduced <i>Wolbachia</i> densities and maternal leakage.

<p>(<b>A</b>) Larvae from the <i>w</i>AlbA, <i>w</i>AlbB, <i>w</i>Au, and <i>w</i>Mel strains were reared at constant 27°C (C) or with temperature fluctuating between 27–37°C (12hours:12hours) (H) and assessed for <i>Wolbachia</i> density by qPCR upon adult emergence. Each point represents a pool of 3 adult mosquitoes. The centre of a box plot shows median <i>Wolbachia</i> density, edges show upper and lower quartiles, and whiskers indicate upper and lower extremes. Statistical analyses were performed using a two-tailed Student’s t-test. (<b>B</b>) Females reared under larval temperature cycling conditions were allowed to recover upon emergence at a constant 27°C and were crossed to wild-type males with infection rates in resulting progeny assessed (1 Gen). Females reared under heat treatment were mated with wild-type males, and resulting progeny were also reared under high temperature conditions—resulting in two consecutive generations of high temperature treatment. Infection rates were then assessed in the pupae resulting from the second round of larval heating (2 Gen). Error bars show binomial 95% confidence intervals.</p

Hatch rate and fecundity of Uju.wMel.

<p>Egg hatch (A) and fecundity or mean number of eggs produced per female per gonotrophic cycle (B) of Uju.wMel was assessed at generation sixteen. Females were blood fed at six days post eclosion, individualized for laying, and eggs hatched after five days. Second instar larvae were counted to calculate percent hatch (A) and eggs per batch per female counted to give fecundity (B). A: Uju.wMel n = 452, UjuT n = 858, Uju.wt n = 508. B: Uju.wMel n = 16, UjuT n = 14, Uju.wt n = 20. Error bars represent the SEM.</p

Mating competitiveness of Uju.wMel males.

<p>Competitiveness of Uju.wMel males was assessed using three independent replicates of 50 male Uju.wMel : 50 male Uju.wt (<i>w</i>AlbA/B) : 50 females of either Uju.wMel or Uju.wt (total of 300 females in six cages). Hatching embryos indicated a compatible cross where both male and female parents were infected with the same <i>Wolbachia</i>. Error bars show the SEM. No significant differences in male mating competitiveness were found between the two lines with Chi-squared analysis using a likelihood framework.</p

CHIKV challenge.

<p>Mosquitoes were allowed to feed on artificial blood meals containing virus suspension and 7 days post infection 35–50 females were used for forced salivation. Samples were titrated by focus fluorescent assay on <i>Ae. albopictus</i> C6/36 cells. The transmission rate was estimated as the percentage of mosquitoes with infectious saliva among tested mosquitoes (A). Saliva samples were titrated by focus fluorescent assay on C6/36 <i>Ae. albopictus</i> cell culture. The total number of plaques was counted and the titer was calculated as FFU/saliva (B). No significant difference was found between Uju.wt and UjuT viral titers using a Wilcoxon rank sum test.</p

Knockdown analysis of <i>CPIJ005623</i> in <i>C. quinquefasciatus</i> Pel females.

<p><b>A</b>. Diagram representing experimental design of knockdown (KD) experiments, d-day post pupal eclosion, BF-Blood feed, T-time point. <b>B</b>. KD assessment of i<i>CPIJ005623</i> in Pel ovaries. Reduction of <i>CPIJ005623</i> mRNA levels similar to uninfected ovaries was observed in infected ovaries when CI developmental progression was seen. <b>C</b>. Increased early embryo (stageI) developmental arrest is detected after i<i>CPIJ005623</i> in compatible Pel male cross.</p

Transcription analysis of <i>CPIJ005623</i> in the <i>Culex pipiens</i> complex.

<p><b>A–B</b>. <i>CPIJ005623</i> transcription in adult female (A) and male (B) <i>Culex quinquefasciatus</i>, Pel (<i>Wolbachia</i>-infected) line relative to the Wolbachia-uninfected PelU, over time (days post pupal eclosion). Similar decreasing expression dynamics was seen in both sexes. <b>C–D</b>. Tissue analysis of <i>CPIJ005623</i> transcription at 4 d in ovaries (C) and 1 d in testes (D) in <i>C. quinquefasciatus Wolbachia</i>-infected lines Pel and Cxq (<i>30</i>), their <i>Wolbachia</i>-free counterparts PelU and CxqT, and <i>Wolbachia</i>-infected Italy line <i>C. molestus</i>. Upregulation of <i>CPIJ005623</i> expression is seen in all <i>Wolbachia</i>-infected lines. Average of the mean values of four biological repeats (+/− standard error-SE) are presented. Two-way ANOVA statistical analysis was used to determine effect of <i>Wolbachia</i> infection status (wis) and time on <i>CPIJ005623</i> expression. Wilcoxon rank-sum test was used to determine differences between <i>Wolbachia</i> infection status in <i>CPIJ005623</i> expression (C–D): *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001.</p