1. Temperature plays a fundamental role in the dynamics of host-pathogen interactions.2. Wolbachia is an endosymbiotic bacteria that infects about 40% of arthropod species, which can affect host behaviour and reproduction. Yet, the effect of Wolbachia on host thermoregulatory behaviour is largely unknown, despite its use in disease vector control programs in thermally variable environments.3. Here, we used a thermal gradient to test whether Drosophila melanogaster infected with Wolbachia strain wMelCS exhibit different temperature preferences (T p ) to uninfected flies. 4. We found that Wolbachia-infected flies preferred a cooler mean temperature (T p = 25.06±0.25˚C) than uninfected flies (T p = 25.78±0.24˚C).5. This finding suggests that Wolbachia-infected hosts might seek out cooler microclimates to reduce exposure to and lessen the consequences of high temperatures. This finding has generated hypotheses that will be fruitful in areas of research for exploring the mechanisms by which the change in T p occurs in this complex and significant host-pathogen-environment interaction.

The route of pathogen entry can have a major effect on the ability of a virus to induce a prolific infection, but it can also affect the ability of the host organism to induce an immune response to fight the infection. Transmission of arboviruses that cause serious diseases in humans often begin by an insect ingesting a virus, which then disseminates through the internal organs and tissues and ultimately culminates in virus transmission to a human host. Understanding the effect of a natural route of infection on the host-pathogen interaction may facilitate development of approaches to prevent viral dissemination. Drosophila has been a useful model organism for understanding host-virus interactions; however, most studies have achieved infection by artificially injecting the virus into the host. Here, we developed a single-stranded quantitative PCR able to detect only actively replicating Drosophila C virus (DCV) to study the effect of viral feeding at the early stages of larval development. Exposure of newly hatched larvae to DCV led to 20 % of larvae becoming infected within 12 h post-contamination, and caused a 14 % egg-to-adult mortality. This is the first time, to the best of our knowledge, that it has been shown experimentally that DCV is able to establish a prolific infection following larval feeding. Using these newly developed tools, the results suggest that larvae that become infected die before adult eclosion.

Understanding viral dynamics in arthropods is of great importance when designing models to describe how viral spread can influence arthropod populations. The endosymbiotic bacterium Wolbachia spp., which is present in up to 40% of all insect species, has the ability to alter viral dynamics in both Drosophila spp. and mosquitoes, a feature that in mosquitoes may be utilized to limit spread of important arboviruses. To understand the potential effect of Wolbachia on viral dynamics in nature, it is important to consider the impact of natural routes of virus infection on Wolbachia antiviral effects. Using adult Drosophila strains, we show here that Drosophila-Wolbachia associations that have previously been shown to confer antiviral protection following systemic viral infection also confer protection against virus-induced mortality following oral exposure to Drosophila C virus in adults. Interestingly, a different pattern was observed when the same fly lines were challenged with the virus when still larvae. Analysis of the four Drosophila-Wolbachia associations that were protective in adults indicated that only the w1118-wMelPop association conferred protection in larvae following oral delivery of the virus. Analysis of Wolbachia density using quantitative PCR (qPCR) showed that a high Wolbachia density was congruent with antiviral protection in both adults and larvae. This study indicates that Wolbachia-mediated protection may vary between larval and adult stages of a given Wolbachia-host combination and that the variations in susceptibility by life stage correspond with Wolbachia density. The differences in the outcome of virus infection are likely to influence viral dynamics in Wolbachia-infected insect populations in nature and could also have important implications for the transmission of arboviruses in mosquito populations.A rthropods harbor a wide range of viruses that can be transmitted between individuals or populations of the same species or can bridge the interspecies gap to infect plants or other animals. The outcome of viral infections can be modulated by tripartite interactions between arthropods, viruses, and bacteria (1). One such interaction is the tripartite interaction between insects, viruses, and the endosymbyotic bacterium Wolbachia pipientis.Wolbachia spp. have gained much attention due to the antiviral effects they confer to their host. The impact of Wolbachia spp. on virus infection was first described in the Drosophila melanogaster host, where it was shown to protect against mortality induced by diverse viruses, including Drosophila C virus (DCV), cricket paralysis virus, and Flock House virus (2, 3). Since that discovery, Wolbachia-mediated antiviral effects have been demonstrated in a number of insect hosts and are being investigated as a way of limiting spread of arboviruses (reviewed in references 1, 4, 5, and 6). Notably, Wolbachia-mediated antiviral effects have been demonstrated in adult mosquitoes artificially infected with Wolbachia; in mosquitoes, Wolbachia can interfere with accu...

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Understanding antiviral processes in infected organisms is of great importance when designing tools targeted at alleviating the burden viruses have on our health and society. Our understanding of innate immunity has greatly expanded in the last 10 years, and some of the biggest advances came from studying pathogen protection in the model organism Drosophila melanogaster. Several antiviral pathways have been found to be involved in antiviral protection in Drosophila however the molecular mechanisms behind antiviral protection have been largely unexplored and poorly characterized. Host-virus interaction studies in Interestingly, Wolbachia-mediated protection was life-stage dependent as oral feeding of L1stage larvae resulted in a loss of Wolbachia-mediated protection in 3 out of 4 DrosophilaWolbachia associations shown to be protective at the adult stages. Loss of protection was associated with lower Wolbachia densities at larval compared to adult stages in the same three Drosophila-Wolbachia associations. These results will aid in understanding the effects of Wolbachia on viral dynamics in natural populations and will contribute to our understanding of life-stage susceptibility in Drosophila.In the 4 th chapter the role of apoptosis in mediating antiviral protection was studied following both viral injections (systemic infection) and oral infections using FHV as a model.Using altered gene expression of key genes involved in apoptosis I investigated the importance of apoptosis on antiviral protection. Knocking-out a pro-apoptotic transcription factor dP53 in adult flies lead to an increase in viral titers following a systemic FHV infection and resulted in earlier mortality of infected individuals compared to wild type flies. Contrary to systemic infection, oral FHV infection of the same fly line showed no effect on viral accumulation but led to earlier mortality of infected individuals. Over-expressing a proapoptotic gene reaper lead to a reduction in FHV viral titers following both systemic and oral infections, however while a reduction in viral titers lead to a delay in virus-induced mortality in systemically infected flies, no differences in mortality was observed between wild type and mutant flies following oral infection. Oral infection with FHV caused 30 % mortality in both wild type and P53 and reaper over-expressing flies. The similarities in the number of flies succumbing to oral infection indicates that apoptosis does not impact the outcome of initial challenge, but that it likely functions in protection following primary infection. Depending on whether apoptosis was suppressed or enhanced lead to differences in the effects of apoptosis on resistance (ability of the host to control virus accumulation) and tolerance (ability of the host to endure infection), which lead to the idea that viral tissue tropism could be responsible for the differences in resistance versus tolerance.Taken together the results enhance our understanding of antiviral mechanisms in Drosophila and show that both the route of...