Mosquito-borne diseases such as malaria and dengue fever represent a significant burden on global health, with millions of cases occurring annually. Current mosquito control strategies have not been able to prevent the recent resurgence of these diseases, necessitating the development of alternative strategies. One such strategy involves infecting key mosquito species, including the primary dengue vector Aedes aegypti, with the bacterial endosymbiont Wolbachia pipientis. In mosquitoes, Wolbachia cause pathogen blocking and restrict the reproduction of dengue and many other pathogens, leading to a decrease in vector competence. A further Wolbachia-induced phenotype, cytoplasmic incompatibility, allows Wolbachia to spread through uninfected insect populations in a self-sustaining manner. These phenotypes make Wolbachia a promising option for mosquito biocontrol. Wolbachia can affect many other aspects of the insect host’s physiology, including altering lifespan, feeding habits, locomotor behaviour and fecundity, but the basis of these effects is unclear. Interestingly, the Wolbachia genome is suggestive of nutritional parasitism. Many basic metabolic pathways are missing or incomplete and this indicates that Wolbachia must rely on a number of host resources. In this thesis I assess the hypothesis that competition over these key resources contributes to some of the physiological changes seen in Wolbachia-infected insects.
Cholesterol is a critical resource involved in many biological processes, which both Wolbachia and infecting viruses require for their replication and survival. Host cholesterol is likely to be scarce, as insects cannot synthesis it themselves. Competition for cholesterol could potentially underlie many Wolbachia-induced phenotypes including pathogen blocking. In Chapter 2, the role of cholesterol in pathogen blocking was examined using the model organism Drosophila melanogaster. Flies were reared on cholesterol-enriched food for multiple generations and then challenged with the pathogenic Drosophila C virus. Overall, there was a weaker pathogen blocking effect for high cholesterol flies and this translated to a decrease in average survival time of 2-5 days and an increase in the viral accumulation rate compared to Wolbachia-infected flies on standard food. In Chapter 4, the role of bloodmeal cholesterol in pathogen blocking was considered in A. aegypti. Mosquitoes were provided with a range of cholesterol-enriched bloodmeals, dosed with dengue virus. After virus quantification it was clear that there was no difference in viral titre of Wolbachia-infected mosquitoes regardless of bloodmeal cholesterol level. The one exception was for a rat blood control diet, which had the lowest cholesterol level. Here no Wolbachia-infected mosquitoes became infected with dengue, suggesting that low bloodmeal cholesterol levels may contribute in some way to pathogen blocking strength.
In Chapter three, cholesterol and amino acids, both key resources in egg development, were considered in the context of Wolbachia-induced changes to A. aegypti fecundity seen after feeding a non-human bloodmeal. Wolbachia-infected A. aegypti had 15-25% lower levels of cholesterol suggesting that Wolbachia is utilising and depleting the supply of host cholesterol. Through dietary manipulation, we increased the bloodmeal cholesterol provided to Wolbachia-infected mosquitoes, but observed no change to either fecundity or egg viability. Amino acids are another vital resource involved in all manner of physiological processes including protein synthesis and regulation of egg development. After feeding on a non-human bloodmeal Wolbachia-infected mosquitoes fed an amino acid supplemented diet laid approximately 15-20 more eggs than those fed a standard diet. Egg viability for these mosquitoes was also increased by 30-40%, and the majority of eggs appeared to be properly developed. These results suggest that Wolbachia sequesters host amino acids at the expense of developing eggs.
In Chapter 5, the metabolic changes caused by two different Wolbachia infections of A. aegypti through 1H NMR profiling were considered. The results revealed extensive manipulation of the amino acid pathways with increased levels of methionine, lysine and tryptophan and decreased levels of histidine. There were also significant effects on the host’s energy network and electron transport chain as evidenced by higher levels of ATP, NAD+/NADH and lower levels of pyruvate seen with infection. Wolbachia infection also causes significant changes to the host neurotransmitter profile, with infected mosquitoes exhibiting decreased levels of key neurotransmitters serotonin and acetylcholine, changes to key compounds in the dopamine biosynthesis pathway and increased levels of GABA. Together these changes represent a heavy metabolic burden due to infection, and this could contribute to some of the major physiological effects of infection.
It is clear from these results that competition between host and symbiont for key resources is occurring and that this competition can explain manipulations of host physiology by Wolbachia. Due to the importance of host fitness for Wolbachia-based biocontrol these findings represent a critical advance in the understanding of Wolbachia-host interactions.