Effect of E1-A226V substitution on CHIKV dissemination to salivary glands of <i>Ae. aegypti</i> (AAPT) and <i>Ae. albopictus</i> (ALPROV). <i>Ae. aegypti</i> (light-grey) and <i>Ae. albopictus</i> (dark-grey) were orally infected with a blood-meal containing both E1-226A and E1-226V provided at the same titer, 10^6.5 pfu/mL.

<p>Every day, 5 mosquitoes were sacrificed to isolate salivary glands. The proportion of E1-226V compared to E1-226A (A) and the number of infectious particles (B) were determined. Error bars show the confidence intervals (95%) for % CHIKV E1-226V and the standard deviations for Log10 pfu/midgut.</p

Effects of intrathoracic injection compared to blood-meal ingestion on CHIKV dissemination and transmission in <i>Ae. aegypti</i> (AAPT) and <i>Ae. albopictus</i> (ALPROV).

<p><i>Ae. aegypti</i> and <i>Ae. albopictus</i> were orally infected or inoculated with E1-226A and E1-226V provided at the same titer, 10^6.5 pfu/mL. At day 7 post-infection, dissemination (A) and transmission (B) were examined by estimating the proportion of E1-226V in wings and saliva. Error bars show the confidence intervals (95%).</p

Unbalanced proportions of E1-226V and E1-226A provided in blood-meals to <i>Ae. aegypti</i> (AAPT) and <i>Ae. albopictus</i> (ALPROV). <i>Ae. aegypti</i> and <i>Ae. albopictus</i> were given an infectious blood-meal containing unbalanced proportions of two clones E1-A and E1-V isolated from the strains E1-226A and E1-226V, respectively.

<p>Two mixes were tested: one containing 1∶9 (E1-A:E1-V) and one containing a mix of 9∶1 (E1-A:E1-V). At day 7 post-infection, dissemination (A) and transmission (B) were examined by estimating the proportion of E1-V in wings and saliva, respectively, of five individual females. Error bars show the confidence intervals (95%).</p

Effect of the E1-A226V substitution on CHIKV transmission in <i>Ae. aegypti</i> (AAPT) and <i>Ae. albopictus</i> (ALPROV).

<p><i>Ae. aegypti</i> (light-grey) and <i>Ae. albopictus</i> (dark-grey) were orally infected with E1-226A and E1-226V provided at the same titer, 10^6.5 pfu/mL. At days 3, 5 and 7 post-infection, mosquitoes were prepared for salivation. Collected saliva was used to inoculate monolayers of Vero cells. After 3 days at 37°C, clones were identified by sequencing. The proportion of E1-226V compared to E1-226A (A) and the number of infectious particles (B) were determined. Error bars show the confidence intervals (95%) for % CHIKV E1-226V and the standard deviations for Log10 pfu/midgut.</p

Effect of E1-A226V substitution on CHIKV dissemination to wings of <i>Ae. aegypti</i> (AAPT) and <i>Ae. albopictus</i> (ALPROV).

<p><i>Ae. aegypti</i> (light-grey) and <i>Ae. albopictus</i> (dark-grey) were orally infected with a blood-meal containing both E1-226A and E1-226V provided at the same titer, 10^6.5 pfu/mL. Every day, 5 mosquitoes were sacrificed to isolate the wings. The proportion of E1-226V compared to E1-226A (A) and the number of infectious particles (B) were determined. Error bars show the confidence intervals (95%) for % CHIKV E1-226V and the standard deviations for Log10 pfu/midgut.</p

Effect of E1-A226V substitution on CHIKV infection in <i>Ae. aegypti</i> (AAPT) and <i>Ae. albopictus</i> (ALPROV).

<p><i>Ae. aegypti</i> (light-grey) and <i>Ae. albopictus</i> (dark-grey) were orally infected with a blood-meal containing both E1-226A and E1-226V provided at the same titer, 10^6.5 pfu/mL. Every day, 5 mosquitoes were sacrificed to isolate the midgut. The proportion of E1-226V compared to E1-226A (A) and the number of infectious particles (B) were determined. Error bars show the confidence intervals (95%) for % CHIKV E1-226V and the standard deviations for Log10 pfu/midgut.</p

<i>Wolbachia</i> densities in <i>Ae. albopictus</i> after a DENV-2 blood-meal provided at different titers.

<p>At days 2, 8 and 14 pi, 4–9 mosquitoes were individually used for DNA extraction. q-PCR was conducted using primers targeting the <i>w</i>AlbA (A) and <i>w</i>AlbB (B) genes. The <i>Wolbachia</i> copy number was normalized with the <i>Ae. albopictus</i> actin gene. A plasmid (pQuantAlb) containing the three loci <i>w</i>AlbA, <i>w</i>AlbB, and the <i>Ae. albopictus</i> actin gene was serially diluted to build standard curves. Error bars represent standard errors.</p

Viral dynamics in <i>Wolbachia</i>-infected and <i>Wolbachia</i>-uninfected <i>Ae. albopictus</i> after exposure to DENV-2.

<p>Batches of mosquitoes were exposed to an infectious blood-meal at two viral titers: 10⁷ FFU/mL (A, C) and 10⁵ FFU/mL (B, D). Every day, 4–9 mosquitoes were killed for RNA extraction and the number of DENV-2 genome copies was determined by qRT-PCR using primers targeting the C gene. Lines indicate the median. Significance was determined using the Mann-Whitney test (p<0.05).</p

Localization of <i>Wolbachia</i> and DENV-2 in <i>Ae. albopictus.</i>

<p>At 14 days post-DENV-2 infection, salivary glands were dissected, fixed, and then incubated simultaneously with two <i>Wolbachia</i> probes and two DENV specific probes. In panels A, B, and C, DENV-2 (green) is labeled with FITC. In panels D, E, and F, <i>Wolbachia</i> (red) is stained with Rhodamine. In panels G, H, and I, the red and green channels are merged. A co-localization of <i>Wolbachia</i> and DENV-2 was detected in some cells (panel H). DENV-uninfected and <i>Wolbachia</i>-uninfected controls are presented in panel C and F, respectively. Scale bars: 50 µm.</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