Wolbachia are vertically transmitted, obligatory intracellular bacteria that infect a great number of species of arthropods and nematodes. In insects, they are mainly known for disrupting the reproductive biology of their hosts in order to increase their transmission through the female germline. In Drosophila melanogaster, however, a strong and consistent effect of Wolbachia infection has not been found. Here we report that a bacterial infection renders D. melanogaster more resistant to Drosophila C virus, reducing the load of viruses in infected flies. We identify these resistance-inducing bacteria as Wolbachia. Furthermore, we show that Wolbachia also increases resistance of Drosophila to two other RNA virus infections (Nora virus and Flock House virus) but not to a DNA virus infection (Insect Iridescent Virus 6). These results identify a new major factor regulating D. melanogaster resistance to infection by RNA viruses and contribute to the idea that the response of a host to a particular pathogen also depends on its interactions with other microorganisms. This is also, to our knowledge, the first report of a strong beneficial effect of Wolbachia infection in D. melanogaster. The induced resistance to natural viral pathogens may explain Wolbachia prevalence in natural populations and represents a novel Wolbachia–host interaction.

Wolbachia are intracellular bacterial symbionts that are able to protect various insect hosts from viral infections. This tripartite interaction was initially described in Drosophila melanogaster carrying wMel, its natural Wolbachia strain. wMel has been shown to be genetically polymorphic and there has been a recent change in variant frequencies in natural populations. We have compared the antiviral protection conferred by different wMel variants, their titres and influence on host longevity, in a genetically identical D. melanogaster host. The phenotypes cluster the variants into two groups — wMelCS-like and wMel-like. wMelCS-like variants give stronger protection against Drosophila C virus and Flock House virus, reach higher titres and often shorten the host lifespan. We have sequenced and assembled the genomes of these Wolbachia, and shown that the two phenotypic groups are two monophyletic groups. We have also analysed a virulent and over-replicating variant, wMelPop, which protects D. melanogaster even better than the closely related wMelCS. We have found that a ∼21 kb region of the genome, encoding eight genes, is amplified seven times in wMelPop and may be the cause of its phenotypes. Our results indicate that the more protective wMelCS-like variants, which sometimes have a cost, were replaced by the less protective but more benign wMel-like variants. This has resulted in a recent reduction in virus resistance in D. melanogaster in natural populations worldwide. Our work helps to understand the natural variation in wMel and its evolutionary dynamics, and inform the use of Wolbachia in arthropod-borne disease control.

The immune system has most likely evolved to limit the negative impact exerted by pathogens on host homeostasis. This defense strategy relies on the concerted action of innate and adaptive components of the immune system, which sense and target pathogens for containment, destruction or expulsion. Resistance to infection refers to these immune functions, which reduce the pathogen load of an infected host as the means to preserve homeostasis. Immune-driven resistance to infection is coupled to an additional, and arguably as important, defense strategy that limits the extent of dysfunction imposed to host parenchyma tissues during infection, without exerting a direct negative impact on pathogens.This defense strategy, called disease tolerance, relies on tissue damage control mechanisms that prevent the deleterious effects of pathogens, while uncoupling immunedriven resistance mechanisms from immunopathology and disease. Here we provide a unifying view of resistance and disease tolerance within the framework of immunity to infection.

In the last decade, bacterial symbionts have been shown to play an important role in protecting hosts against pathogens. Wolbachia, a widespread symbiont in arthropods, can protect Drosophila and mosquito species against viral infections. We have investigated antiviral protection in 19 Wolbachia strains originating from 16 Drosophila species after transfer into the same genotype of Drosophila simulans. We found that approximately half of the strains protected against two RNA viruses. Given that 40% of terrestrial arthropod species are estimated to harbour Wolbachia, as many as a fifth of all arthropods species may benefit from Wolbachia-mediated protection. The level of protection against two distantly related RNA viruses – DCV and FHV – was strongly genetically correlated, which suggests that there is a single mechanism of protection with broad specificity. Furthermore, Wolbachia is making flies resistant to viruses, as increases in survival can be largely explained by reductions in viral titer. Variation in the level of antiviral protection provided by different Wolbachia strains is strongly genetically correlated to the density of the bacteria strains in host tissues. We found no support for two previously proposed mechanisms of Wolbachia-mediated protection — activation of the immune system and upregulation of the methyltransferase Dnmt2. The large variation in Wolbachia's antiviral properties highlights the need to carefully select Wolbachia strains introduced into mosquito populations to prevent the transmission of arboviruses.

Pathogen entry route can have a strong impact on the result of microbial infections in different hosts, including insects. Drosophila melanogaster has been a successful model system to study the immune response to systemic viral infection. Here we investigate the role of the Toll pathway in resistance to oral viral infection in D. melanogaster. We show that several Toll pathway components, including Spätzle, Toll, Pelle and the NF-kB-like transcription factor Dorsal, are required to resist oral infection with Drosophila C virus. Furthermore, in the fat body Dorsal is translocated from the cytoplasm to the nucleus and a Toll pathway target gene reporter is upregulated in response to Drosophila C Virus infection. This pathway also mediates resistance to several other RNA viruses (Cricket paralysis virus, Flock House virus, and Nora virus). Compared with control, viral titres are highly increased in Toll pathway mutants. The role of the Toll pathway in resistance to viruses in D. melanogaster is restricted to oral infection since we do not observe a phenotype associated with systemic infection. We also show that Wolbachia and other Drosophila-associated microbiota do not interact with the Toll pathway-mediated resistance to oral infection. We therefore identify the Toll pathway as a new general inducible pathway that mediates strong resistance to viruses with a route-specific role. These results contribute to a better understanding of viral oral infection resistance in insects, which is particularly relevant in the context of transmission of arboviruses by insect vectors.