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.

The Rhabdoviridae is one of the most ecologically diverse families of RNA viruses with members infecting a wide range of organisms including placental mammals, marsupials, birds, reptiles, fish, insects and plants. The availability of complete nucleotide sequences for an increasing number of rhabdoviruses has revealed that their ecological diversity is reflected in the diversity and complexity of their genomes. The five canonical rhabdovirus structural protein genes (N, P, M, G and L) that are shared by all rhabdoviruses are overprinted, overlapped and interspersed with a multitude of novel and diverse accessory genes. Although not essential for replication in cell culture, several of these genes have been shown to have roles associated with pathogenesis and apoptosis in animals, and cell-to-cell movement in plants. Others appear to be secreted or have the characteristics of membrane-anchored glycoproteins or viroporins. However, most encode proteins of unknown function that are unrelated to any other known proteins. Understanding the roles of these accessory genes and the strategies by which rhabdoviruses use them to engage, divert and re-direct cellular processes will not only present opportunities to develop new anti-viral therapies but may also reveal aspects of cellar function that have broader significance in biology, agriculture and medicine.

Insect-symbiont interactions are known to play key roles in host functions and fitness. The common insect endosymbiont Wolbachia can reduce the ability of several human pathogens, including arboviruses and the malaria parasite, to replicate in insect hosts. Wolbachia does not naturally infect Aedes aegypti, the primary vector of dengue virus, but transinfected Ae. aegypti have antidengue virus properties and are currently being trialled as a dengue biocontrol strategy. Here, we assess the impact of Wolbachia infection of Ae. aegypti on the microbiome of wild mosquito populations (adults and larvae) collected from release sites in Cairns, Australia, by profiling the 16S rRNA gene using next-generation sequencing. Our data indicate that Wolbachia reduces the relative abundance of a large proportion of bacterial taxa in Ae. aegypti adults, that is in accordance with the known pathogen-blocking effects of Wolbachia on a variety of bacteria and viruses. In adults, several of the most abundant bacterial genera were found to undergo significant shifts in relative abundance. However, the genera showing the greatest changes in relative abundance in Wolbachia-infected adults represented a low proportion of the total microbiome. In addition, there was little effect of Wolbachia infection on the relative abundance of bacterial taxa in larvae, or on species diversity (accounting for species richness and evenness together) detected in adults or larvae. These results offer insight into the effects of Wolbachia on the Ae. aegypti microbiome in a native setting, an important consideration for field releases of Wolbachia into the population.insect, microbiome, mosquito, symbiosis, Wolbachia | INTRODUCTIONInteractions between insect hosts and their associated microbiome can have substantial impact on host physiology. Broad changes to the microbiome can affect functions including development, fecundity, metabolism and immunity/susceptibility to pathogens (Coon, Vogel, Brown, & Strand, 2014;de O Gaio et al., 2011;Gall et al., 2016;Ridley, Wong, Westmiller, & Douglas, 2012). It is also well known that specific symbiotic bacteria of insects can play key roles in host functions (Douglas, 2011). Two well-studied systems include the aphids where the obligate symbiont Buchnera aphidicola synthesizes amino acids that are essential for aphid nutrition (Akman G€ und€ uz & Douglas, 2009) More broadly, the overall structure of the microbiome can also be important in the fitness of insects such as bees and mosquitoes, with changes to the composition of the microbiome shown to increase *These authors contributed equally to this study. the mortality of bees following exposure to bacterial pathogens (Raymann, Shaffer, & Moran, 2017) and to alter mosquito fecundity (de O Gaio et al., 2011;Gendrin et al., 2015;). It is evident in cases such as these where significant microbiome-host interactions occur, disturbing the insect bacterial population can adversely affect host fitness (Douglas, 2011). These include a recent study that has exami...

Wolbachia pipientis is an insect endosymbiont known to limit the replication of viruses including dengue and Zika in their primary mosquito vector, Aedes aegypti. Wolbachia is being released into mosquito populations globally in a bid to control the diseases caused by these viruses. It is theorized that Wolbachia’s priming of the insect immune system may confer protection against subsequent viral infection. Other hypotheses posit a role for competition between Wolbachia and viruses for host cellular resources. Using an A. aegypti cell line infected with Wolbachia, we tested the effects of targeting siRNAs against the major innate immune pathways on dengue virus loads. We show that while Wolbachia infection induces genes in the Toll, JAK/STAT and RNAi pathways, only reduced expression of RNAi leads to a rebound of dengue virus loads in Wolbachia-infected cells. The magnitude of the effect explained less than 10% of the total DENV load, demonstrating that blocking must be dependent on other factors in addition to the expression of RNAi. The findings bode well for the long-term stability of blocking given that immunity gene expression would likely be highly plastic and susceptible to rapid evolution.