Establishment of a Wolbachia Superinfection in Aedes aegypti Mosquitoes as a Potential Approach for Future Resistance Management

Abstract

Wolbachia pipientis is an endosymbiotic bacterium estimated to chronically infect between 40-75% of all arthropod species. Aedes aegypti, the principle mosquito vector of dengue virus (DENV), is not a natural host of Wolbachia. The transinfection of Wolbachia strains such as wAlbB, wMel and wMelPop-CLA into Ae. aegypti has been shown to significantly reduce the vector competence of this mosquito for a range of human pathogens in the laboratory. This has led to wMel-transinfected Ae. aegypti currently being released in five countries to evaluate its effectiveness to control dengue disease in human populations. Here we describe the generation of a superinfected Ae. aegypti mosquito line simultaneously infected with two avirulent Wolbachia strains, wMel and wAlbB. The line carries a high overall Wolbachia density and tissue localisation of the individual strains is very similar to each respective single infected parental line. The superinfected line induces unidirectional cytoplasmic incompatibility (CI) when crossed to each single infected parental line, suggesting that the superinfection would have the capacity to replace either of the single constituent infections already present in a mosquito population. No significant differences in fitness parameters were observed between the superinfected line and the parental lines under the experimental conditions tested. Finally, the superinfected line blocks DENV replication more efficiently than the single wMel strain when challenged with blood meals from viremic dengue patients. These results suggest that the deployment of superinfections could be used to replace single infections and may represent an effective strategy to help manage potential resistance by DENV to field deployments of single infected strains.

Fig 1. Wolbachia density and distribution in the Ae. aegypti superinfected line.

(A) Comparison of total Wolbachia density between wMelwAlbB (G18), wMel, wAlbB and wMelPop-CLA infected mosquitoes. Density is expressed as the mean ratio between the wMel or wAlbB wsp gene and the Ae. aegypti host rps17 gene. Standard error of the mean is indicated (n = 10). (B) Wolbachia distribution in the ovaries of wMel, wAlbB and wMelwAlbB infected adult female mosquitoes. Wolbachia was visualised using FISH with probes specific to wMel (red) and wAlbB (green). DNA is stained in blue using DAPI.

Fig 2. Survival plots comparing the adult lifespan of wild type Ae. aegypti wMel, wAlbB and wMelwAlbB females (A) and males (B).

Wild type mosquitoes are indicated in black, wMel in blue, wAlbB in green and wMelwAlbB in red. All experiments were conducted using mosquitoes from G8 of the superinfected line.

Fig 3. Fecundity and fertility of Wolbachia-infected Ae. aegypti.

(A) Fecundity of Wolbachia-infected and wild type females as determined by mean egg production from individual female mosquitoes. Statistical significant differences between all data sets were determined using a Kruskal-Wallis test with Dunn’s multiple comparisons test. Significant differences are indicated by **** (p < 0.0001) or *** (p < 0.001) and standard error of the mean is indicated. Non-significant differences are not indicated. (B) Mean hatch rate of Wolbachia-infected and wild type females as determined by percentage of eggs hatching over time. Statistical differences between all data sets were determined using a two-way ANOVA with Tukey’s multiple comparisons test. For simplicity, only the comparison between wMel (current release strain) and wMelwAlbB is shown in the figure. A small but significant difference between the hatch rates for wMel and wMelwAlbB was observed after two weeks (*, p = 0.0159), however, no significant differences could be found at the 4 and 8 week time points. All experiments were conducted using mosquitoes from G8 of the superinfected line and standard error of the mean is indicated.

Fig 4. DENV serotype 2 (ET300) genome copies in Wolbachia-infected and wild type mosquitoes.

(A) DENV-2 was injected at 10⁴ genome copies per ml into the thorax of 3–5 day old female mosquitoes. Virus replication was quantified 7 days post injection in whole mosquito bodies using qPCR. For each mosquito line the mean number of genome copies is plotted and standard error of the mean is indicated. Infection rates are indicated as percentages. (B) DENV-2 was administered in a blood meal at 10⁷ copies per ml. Virus replication was quantified in whole mosquito bodies 14 days post feeding. For each mosquito line the mean number of genome copies is plotted and standard error of the mean is indicated. For both A and B significant differences between wild type and Wolbachia infected mosquitoes are indicated by *** (p < 0.0001 and infection rates are indicated as percentages). Zero values were not plotted or included in determining the mean or standard error of the mean.

Fig 5. Proportion of mosquitoes infected with DENV after blood-feeding on 36 dengue patient blood samples, as a function of plasma viremia, and stratified by serotype.

Lines represent fits from logistic regression to the data. All three strains were assessed for every feed; for DENV-1 there were 23 blood feeds, and 13 for DENV-4. DENV-2 and DENV-3 are not represented due to small sample sizes (n = 3 and n = 2 respectively). (A) Each point represents the proportion of mosquitoes with a DENV-infected abdomen, stratified by strain, and pooled across all time points. (B) The proportion of mosquitoes, pooled across all time points, that expectorated infectious DENV in their saliva.

Fig 6. Box plots representing the viral load of DENV in tissues of DENV-infected mosquitoes.

Virus was detected at days 10, 14 and 18 post exposure to 41 patient-derived infectious blood meals. (A) Abdomen tissue. (B) Salivary gland tissue. As detailed in S2 Table, wMel and wMelwAlbB are associated with significantly lower viral loads in abdomen tissue and salivary glands compared to wild type, and wMelwAlbB is associated with significantly lower viral loads in salivary glands compared to wMel.