Julien Martinez

Wolbachia Variants Induce Differential Protection to Viruses in Drosophila melanogaster: A Phenotypic and Phylogenomic Analysis

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.

Symbionts Commonly Provide Broad Spectrum Resistance to Viruses in Insects: A Comparative Analysis of Wolbachia Strains

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 Wolbachias antiviral properties highlights the need to carefully select Wolbachia strains introduced into mosquito populations to prevent the transmission of arboviruses.

Should Symbionts Be Nice or Selfish? Antiviral Effects of Wolbachia Are Costly but Reproductive Parasitism Is Not

Symbionts can have mutualistic effects that increase their host’s fitness and/or parasitic effects that reduce it. Which of these strategies evolves depends in part on the balance of their costs and benefits to the symbiont. We have examined these questions in Wolbachia, a vertically transmitted endosymbiont of insects that can provide protection against viral infection and/or parasitically manipulate its hosts’ reproduction. Across multiple symbiont strains we find that the parasitic phenotype of cytoplasmic incompatibility and antiviral protection are uncorrelated. Strong antiviral protection is associated with substantial reductions in other fitness-related traits, whereas no such trade-off was detected for cytoplasmic incompatibility. The reason for this difference is likely that antiviral protection requires high symbiont densities but cytoplasmic incompatibility does not. These results are important for the use of Wolbachia to block dengue virus transmission by mosquitoes, as natural selection to reduce these costs may lead to reduced symbiont density and the loss of antiviral protection.

Wolbachia Blocks Viral Genome Replication Early in Infection without a Transcriptional Response by the Endosymbiont or Host Small RNA Pathways

The intracellular endosymbiotic bacterium Wolbachia can protect insects against viral infection, and is being introduced into mosquito populations in the wild to block the transmission of arboviruses that infect humans and are a major public health concern. To investigate the mechanisms underlying this antiviral protection, we have developed a new model system combining Wolbachia-infected Drosophila melanogaster cell culture with the model mosquito-borne Semliki Forest virus (SFV; Togaviridae, Alphavirus). Wolbachia provides strong antiviral protection rapidly after infection, suggesting that an early stage post-infection is being blocked. Wolbachia does appear to have major effects on events distinct from entry, assembly or exit as it inhibits the replication of an SFV replicon transfected into the cells. Furthermore, it causes a far greater reduction in the expression of proteins from the 3´ open reading frame than the 5´ non-structural protein open reading frame, indicating that it is blocking the replication of viral RNA. Further to this separation of the replicase proteins and viral RNA in transreplication assays shows that uncoupling of viral RNA and replicase proteins does not overcome Wolbachia’s antiviral activity. This further suggests that replicative processes are disrupted, such as translation or replication, by Wolbachia infection. This may occur by Wolbachia mounting an active antiviral response, but the virus did not cause any transcriptional response by the bacterium, suggesting that this is not the case. Host microRNAs (miRNAs) have been implicated in protection, but again we found that host cell miRNA expression was unaffected by the bacterium and neither do our findings suggest any involvement of the antiviral siRNA pathway. We conclude that Wolbachia may directly interfere with early events in virus replication such as translation of incoming viral RNA or RNA transcription, and this likely involves an intrinsic (as opposed to an induced) mechanism.

Previous Exposure to an RNA Virus Does Not Protect against Subsequent Infection in Drosophila melanogaster

Background Immune priming has been shown to occur in a wide array of invertebrate taxa, with individuals exposed to a pathogen showing increased protection upon subsequent exposure. However, the mechanisms underlying immune priming are poorly understood. The antiviral RNAi response in Drosophila melanogaster is an ideal candidate for providing a specific and acquired response to subsequent infection. We exposed D. melanogaster to two challenges of a virus known to produce an antiviral RNAi response, to examine whether any protective effects of prior exposure on survival were observed. Results In this experiment we found no evidence that prior exposure to Drosophila C Virus (DCV) protects flies from a subsequent lethal challenge, with almost identical levels of mortality in flies previously exposed to DCV or a control. Conclusions Our results confirm the finding that ‘acquired’ immune responses are not ubiquitous across all invertebrate-pathogen interactions. We discuss why we may have observed no effect in this study, with focus on the mechanistic basis of the RNAi pathway.

Influence of the virus LbFV and of Wolbachia in a host-parasitoid interaction.

Symbionts are widespread and might have a substantial effect on the outcome of interactions between species, such as in host-parasitoid systems. Here, we studied the effects of symbionts on the outcome of host-parasitoid interactions in a four-partner system, consisting of the parasitoid wasp Leptopilina boulardi, its two hosts Drosophila melanogaster and D. simulans, the wasp virus LbFV, and the endosymbiotic bacterium Wolbachia. The virus is known to manipulate the superparasitism behavior of the parasitoid whereas some Wolbachia strains can reproductively manipulate and/or confer pathogen protection to Drosophila hosts. We used two nuclear backgrounds for both Drosophila species, infected with or cured of their respective Wolbachia strains, and offered them to L. boulardi of one nuclear background, either infected or uninfected by the virus. The main defence mechanism against parasitoids, i.e. encapsulation, and other important traits of the interaction were measured. The results showed that virus-infected parasitoids are less frequently encapsulated than uninfected ones. Further experiments showed that this viral effect involved both a direct protective effect against encapsulation and an indirect effect of superparasitism. Additionally, the Wolbachia strain wAu affected the encapsulation ability of its Drosophila host but the direction of this effect was strongly dependent on the presence/absence of LbFV. Our results confirmed the importance of heritable symbionts in the outcome of antagonistic interactions.

Prevalence of a virus inducing behavioural manipulation near species range border

The densities of conspecific individuals may vary through space, especially at the edge of species range. This variation in density is predicted to influence the diffusion of species‐specific horizontally transmitted symbionts. However, to date there is very little data on how parasite prevalence varies around the border of a host species. Using a molecular epidemiology approach, we studied the prevalence of a vertically and horizontally transmitted virus at the edge of the geographic range of its insect host, the Drosophila parasitoid wasp Leptopilina boulardi. L. boulardi is a Mediterranean parasitoid species showing a recent range expansion to the north (in France). The LbFV virus manipulates the behaviour of females, increasing their tendency to lay additional eggs in already parasitized Drosophila larvae (superparasitism). This is beneficial for the virus because it allows the virus to be horizontally transferred during superparasitism. We show that LbFV prevalence is very high in central populations, intermediate in marginal populations and almost absent from newly established peripheral populations of L. boulardi. We failed to detect any influence of temperature and diapause on viral transmission efficiency but we observed a clear relationship between prevalence and parasitoid density, and between parasitoid density and the occurrence of superparasitism, as predicted by our epidemiological model. Viral strains were all efficient at inducing the behavioural manipulation and viral gene sequencing revealed very low sequence variation. We conclude that the prevalence reached by the virus critically depends on density‐dependent factors, i.e. superparasitism, underlying the selective pressures acting on the virus to manipulate the behaviour of the parasitoid.

Addicted? Reduced host resistance in populations with defensive symbionts.

Heritable symbionts that protect their hosts from pathogens have been described in a wide range of insect species. By reducing the incidence or severity of infection, these symbionts have the potential to reduce the strength of selection on genes in the insect genome that increase resistance. Therefore, the presence of such symbionts may slow down the evolution of resistance. Here we investigated this idea by exposing Drosophila melanogaster populations to infection with the pathogenic Drosophila C virus (DCV) in the presence or absence of Wolbachia, a heritable symbiont of arthropods that confers protection against viruses. After nine generations of selection, we found that resistance to DCV had increased in all populations. However, in the presence of Wolbachia the resistant allele of pastrel—a gene that has a major effect on resistance to DCV—was at a lower frequency than in the symbiont-free populations. This finding suggests that defensive symbionts have the potential to hamper the evolution of insect resistance genes, potentially leading to a state of evolutionary addiction where the genetically susceptible insect host mostly relies on its symbiont to fight pathogens.

Wolbachia‐mediated protection against viruses in the invasive pest Drosophila suzukii

The maternally inherited bacterium Wolbachia is well known for spreading in natural populations by manipulating the reproduction of its arthropod hosts, but can also have mutualist effects that increase host fitness. In mosquitoes and Drosophila some Wolbachia strains can lead to an increase in survival of virus‐infected insects, and in most cases this is associated with reduced accumulation of the virus in host tissues. We investigated if the Wolbachia strain wSuz, which naturally infects Drosophila suzukii, is able to confer protection against Drosophila C virus and Flock House virus in different host genetic backgrounds. We found that this strain can increase host survival upon infection with these two viruses. In some cases this effect was associated with lower viral titres, suggesting that it confers resistance to the viruses rather than allowing the flies to tolerate infection. Our results indicate that, in D. suzukii, the antiviral protection provided by Wolbachia is not correlated to its density as found in other Drosophila species. This study demonstrates a phenotypic effect induced by wSuz on its native host which could explain its maintenance in natural populations of D. suzukii.

Wolbachia in European populations of the invasive pest Drosophila suzukii: regional variation in infection frequencies.

The invasive pest Drosophila suzukii is characterized by a specific fresh-fruit targeting behavior and has quickly become a menace for the fruit economy of newly infested North American and European regions. D. suzukii carries a strain of the endosymbiotic bacterium Wolbachia, named wSuz, which has a low infection frequency and no reproductive manipulation capabilities in American populations of D. suzukii. To further understand the nature of wSuz biology and assess its utility as a tool for controlling this pest’s populations, we investigated the prevalence of Wolbachia in 23 European D. suzukii populations, and compared our results with those available in American populations. Our data showed a highly variable infection frequency with a mean prevalence of 46%, which is significantly higher than the 17% found in American populations. Based on Multilocus Sequence Typing analysis, a single wSuz strain was diagnosed in all European populations of D. suzukii. In agreement with American data, we found no evidence of cytoplasmic incompatibility induced by wSuz. These findings raise two questions: a) why Wolbachia is maintained in field populations of D. suzukii and b) what are the selective forces responsible for the variation in prevalence within populations, particularly between European and American continents? Our results provide new insights into the D. suzukii-Wolbachia association and highlight regional variations that await further investigation and that should be taken into account for using Wolbachia-based pest management programs.