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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  w AlbB,  w Mel and  w MelPop-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  w Mel-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,  w Mel and  w AlbB. 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  w Mel 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.

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