The Wolbachia strain wAu provides highly efficient virus transmission blocking in Aedes aegypti

Abstract

Introduced transinfections of the inherited bacteria Wolbachia can inhibit transmission of viruses by Aedes mosquitoes, and in Ae. aegypti are now being deployed for dengue control in a number of countries. Only three Wolbachia strains from the large number that exist in nature have to date been introduced and characterized in this species. Here novel Ae. aegypti transinfections were generated using the wAlbA and wAu strains. In its native Ae. albopictus, wAlbA is maintained at lower density than the co-infecting wAlbB, but following transfer to Ae. aegypti the relative strain density was reversed, illustrating the strain-specific nature of Wolbachia-host co-adaptation in determining density. The wAu strain also reached high densities in Ae. aegypti, and provided highly efficient transmission blocking of dengue and Zika viruses. Both wAu and wAlbA were less susceptible than wMel to density reduction/incomplete maternal transmission resulting from elevated larval rearing temperatures. Although wAu does not induce cytoplasmic incompatibility (CI), it was stably combined with a CI-inducing strain as a superinfection, and this would facilitate its spread into wild populations. Wolbachia wAu provides a very promising new option for arbovirus control, particularly for deployment in hot tropical climates.

Fig 1. Wolbachia densities and tropism in Aedes mosquitoes.

(A) Total Wolbachia densities were measured by qPCR in wAlbA, wAlbB, wAu, and wMel carrying Aedes aegypti 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 Wolbachia density, edges show upper and lower quartiles, and whiskers indicate upper and lower extremes. (B) Total Wolbachia 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.

Fig 2. Virus inhibition in Wolbachia-infected Aedes aegypti lines.

(A) Semliki Forest virus (SFV) genome copies per host cell following thoracic injection into Wolbachia-infected lines and wild-type Ae. aegypti. 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 wAlbA, wMel, wAu, wAlbB and wt, respectively. Statistical analysis was performed using a one-way ANOVA with a Dunnett’s post-hoc test. Dengue-2 (DENV) (B and C) and Zika (ZIKV) (D and E) 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 RpS17 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.

Fig 3. High temperature results in reduced Wolbachia densities and maternal leakage.

(A) Larvae from the wAlbA, wAlbB, wAu, and wMel strains were reared at constant 27°C (C) or with temperature fluctuating between 27–37°C (12hours:12hours) (H) and assessed for Wolbachia density by qPCR upon adult emergence. Each point represents a pool of 3 adult mosquitoes. The centre of a box plot shows median Wolbachia 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) 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.

Fig 4. Fitness assessment of Wolbachia-infected and wild-type Ae. aegypti.

(A) Survival of adult females of Wolbachia-infected lines. Curves show percentage survival with shaded areas indicating 95% confidence intervals from 4 replicate cages for each line each containing a starting number of 25 adult females. (B) Fecundity of females from Wolbachia-infected lines and wild-type over the first gonotrophic cycle. 20 females were individualized for oviposition. Bars show average egg number per female. Error bars show SD. (C) Percentage hatch rates of eggs from Wolbachia-infected lines and wild-type mosquitoes after 5, 10, 20, 35 and 50 days of desiccated quiescence. For each time-point the number of eggs assessed varied from 200–500. Shaded areas around lines indicate 95% confidence intervals.

Fig 5. Generation of a wAu – wAlbB superinfection.

(A) Crosses between wAuwAlbB 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) Total Wolbachia densities measured by qPCR in wAlbB, wAu, and wAuwAlbB carrying Aedes aegypti females at ten days post adult eclosion. Each bar represents 10 biological replicates, with pools of 5 females per replicate. Error bars show SD. (C) wAu and wAlbB strain-specific densities in the ovaries of wAlbB, wAuwAlbB, and wAu carrying Ae. aegypti. 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. (D) Fluorescent in situ hybridization showing distributions of wAu (green) and wAlbB (red) in ovaries of the wAu, wAlbB, wAuwAlbB 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 S4 Fig. Blue stain is DAPI.