Dengue has enormous health impacts globally. A novel approach to decrease dengue incidence involves the introduction of Wolbachia endosymbionts that block dengue virus transmission into populations of the primary vector mosquito, Aedes aegypti. The wMel Wolbachia strain has previously been trialed in open releases of Ae. aegypti; however, the wAlbB strain has been shown to maintain higher density than wMel at high larval rearing temperatures. Releases of Ae. aegypti mosquitoes carrying wAlbB were carried out in 6 diverse sites in greater Kuala Lumpur, Malaysia, with high endemic dengue transmission. The strain was successfully established and maintained at very high population frequency at some sites or persisted with additional releases following fluctuations at other sites. Based on passive case monitoring, reduced human dengue incidence was observed in the release sites when compared to control sites. The wAlbB strain of Wolbachia provides a promising option as a tool for dengue control, particularly in very hot climates.

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. Mosquito-borne viral diseases represent an increasing threat to human and animal health globally. The mosquito species Aedes aegypti, a primary vector of the most significant human arboviral infections including the dengue, Zika and Chikungunya viruses, is highly invasive and is almost ubiquitous in tropical urban areas. Mosquito control remains the main approach for preventing and controlling outbreaks. A novel control strategy that is currently being trialed in several countries utilizes Ae. aegypti mosquitoes artificially infected with a bacterial symbiont known as Wolbachia pipientis. Although many insect species harbor native Wolbachia infections, Ae. aegypti is naturally uninfected. Wolbachia lives within host cells and is passed-on from mother to offspring, and can block virus transmission; once released it can invade and persist in host populations. Here we present the infection and assessment of two novel Wolbachia strains in Ae. aegypti. We show that one of the strains, wAu, provides particularly strong blocking of dengue and Zika virus transmission and offers greater stability at higher temperatures when compared to wMel—currently the most widely used strain for field releases. These results suggest that wAu is promising option for arbovirus control, especially in hot climates.

Wolbachiaare intracellular maternally inherited bacteria that can spread through insect populations and block virus transmission by mosquitoes, providing an important approach to dengue control. To better understand the mechanisms of virus inhibition, we here perform proteomic quantification of the effects ofWolbachiainAedes aegyptimosquito cells and midgut. Perturbations are observed in vesicular trafficking, lipid metabolism and in the endoplasmic reticulum that could impact viral entry and replication.Wolbachia-infected cells display a differential cholesterol profile, including elevated levels of esterified cholesterol, that is consistent with perturbed intracellular cholesterol trafficking. Cyclodextrins have been shown to reverse lipid accumulation defects in cells with disrupted cholesterol homeostasis. Treatment ofWolbachia-infectedAe.aegypticells with 2-hydroxypropyl-β-cyclodextrin restores dengue replication inWolbachia-carrying cells, suggesting dengue is inhibited inWolbachia-infected cells by localised cholesterol accumulation. These results demonstrate parallels between the cellularWolbachiaviral inhibition phenotype and lipid storage genetic disorders.

A possible malaria control approach involves the dissemination in mosquitoes of inherited symbiotic microbes to block Plasmodium transmission. However, in the Anopheles gambiae complex, the primary African vectors of malaria, there are limited reports of inherited symbionts that impair transmission. We show that a vertically transmitted microsporidian symbiont (Microsporidia MB) in the An. gambiae complex can impair Plasmodium transmission. Microsporidia MB is present at moderate prevalence in geographically dispersed populations of An. arabiensis in Kenya, localized to the mosquito midgut and ovaries, and is not associated with significant reductions in adult host fecundity or survival. Field-collected Microsporidia MB infected An. arabiensis tested negative for P. falciparum gametocytes and, on experimental infection with P. falciparum, sporozoites aren’t detected in Microsporidia MB infected mosquitoes. As a microbe that impairs Plasmodium transmission that is non-virulent and vertically transmitted, Microsporidia MB could be investigated as a strategy to limit malaria transmission.

Certain strains of the intracellular endosymbiontWolbachiacan strongly inhibit or block the transmission of viruses such as dengue virus (DENV) byAedesmosquitoes, and the mechanisms responsible are still not well understood. Direct infusion and liquid chromatography-Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry-based lipidomics analyses were conducted usingAedes albopictusAa23 cells that were infected with thewMel andwMelPop strains ofWolbachiain comparison to uninfected Aa23-T cells. Substantial shifts in the cellular lipid profile were apparent in the presence ofWolbachia. Most significantly, almost all sphingolipid classes were depleted, and some reductions in diacylglycerols and phosphatidylcholines were also observed. These lipid classes have previously been shown to be selectively enriched in DENV-infected mosquito cells, suggesting thatWolbachiamay produce a cellular lipid environment that is antagonistic to viral replication. The data improve our understanding of the intracellular interactions betweenWolbachiaand mosquitoes.IMPORTANCEMosquitoes transmit a variety of important viruses to humans, such as dengue virus and Zika virus. Certain strains of the intracellular bacterial genus calledWolbachiafound in or introduced into mosquitoes can block the transmission of viruses, including dengue virus, but the mechanisms responsible are not well understood. We found substantial shifts in the cellular lipid profiles in the presence of these bacteria. Some lipid classes previously shown to be enriched in dengue virus-infected mosquito cells were depleted in the presence ofWolbachia, suggesting thatWolbachiamay produce a cellular lipid environment that inhibits mosquito-borne viruses.

Aedes aegypti mosquitoes carrying the w AlbB Wolbachia strain show a reduced capacity to transmit dengue virus. w AlbB has been introduced into wild Ae. aegypti populations in several field sites in Kuala Lumpur, Malaysia, where it has persisted at high frequency for more than 2 years and significantly reduced dengue incidence. Although these encouraging results indicate that w AlbB releases can be an effective dengue control strategy, the long-term success depends on w AlbB maintaining high population frequencies and virus transmission inhibition, and both could be compromised by Wolbachia– host coevolution in the field. Here, w AlbB-carrying Ae. aegypti collected from the field 20 months after the cessation of releases showed no reduction in Wolbachia density or tissue distribution changes compared to a w AlbB laboratory colony. The w AlbB strain continued to induce complete unidirectional cytoplasmic incompatibility, showed perfect maternal transmission under laboratory conditions, and retained its capacity to inhibit dengue. Additionally, a field-collected w AlbB line was challenged with Malaysian dengue patient blood, and showed significant blocking of virus dissemination to the salivary glands. These results indicate that w AlbB continues to inhibit currently circulating strains of dengue in field populations of Ae. aegypti , and provides additional support for the continued scale-up of Wolbachia wAlbB releases for dengue control. This article is part of the theme issue ‘Novel control strategies for mosquito-borne diseases’.