Final Activity Report Summary - MIXED INFECTIONS (Virulence Evolution in Mixed-Species Infections.) The dynamic nature of disease evolution emphasises the need to improve understanding of what determines the harm, i.e. virulence, which parasites cause to their hosts. Interest is growing regarding mixed-species parasite infections. Host condition, the environment and the genetic make-up of hosts and parasites can all influence parasite growth and virulence. The way these factors interact with each other to affect competition between different parasite species within a single host remains unclear. Although previous studies investigating the relationship between different strains of the same parasite revealed that patterns of growth of the participants were affected by the presence of a competitor, the nature of competition between different parasite species within the same host may be very different. Different parasite species may use different host resources and have diverse modes and timing of transmission. We addressed these issues experimentally by investigating competition between two different parasite species of the mosquito aedes aegypti. The objectives of the proposal were: 1. to identify virulence levels of mixed infections on host life-history traits and 2. to identify how within-host competition in a mixed infection affected parasite growth in different host environments. We explored how the competition between two parasite species affected levels of virulence in the host, probability of infection and parasite growth for the examined parasite species at different host food levels. The parasites were virulent under experimental conditions, corresponding to smaller than 70 % pre-adult mortality. Levels of virulence in a mixed-species infection were similar to those of the more virulent parasite. Competition and host food levels however differentially affected the two parasite species. The probability of infection and parasite growth for the least virulent parasite was negatively affected by the presence of a competitor. Intriguingly, the probability of infection for this parasite increased when alone in conditions in which host food was abundant, but decreased in competition. In contrast, the probability of infection for the more virulent parasite was unaffected by the presence of a competitor or by the host food level. It was apparent that this parasite beneficially altered its development according to its environment. This more virulent parasite produced two types of transmission spores, one that was transmitted from mother to offspring in the egg and another that was released in to the environment from dead, infected mosquitoes. The incidence of mosquitoes harbouring this latter type of spore was higher in the presence of a competitor and in case food for the mosquito was scarce. These conditions reduced the likelihood of a mosquito reaching adulthood and the parasite being able to transmit from mother to offspring. Therefore, the aims of both proposal objectives were successfully met. We also compared the expression of proteins in mosquitoes with a mixed-species parasite infection versus mosquitoes that were infected with a single infection of each parasite. A number of proteins were identified, whose expression was dependent upon being infected with a single parasite, and some whose expression was only observed when the mosquito harboured a mixed-species infection. 68 proteins with their expression being differentially affected by one or several parasite treatments were identified by mass spectroscopy. The function that these proteins expressed remained under investigation by the time of the project completion. In particular, it was investigated whether they were known to be involved in immune responses or to a more general stress response. This would shed light on the scope and specificity of the immune response elicited in invertebrates. Therefore, the partial aims of the first proposal objective were also met by this project component. Moreover, we investigated whether the sequence in which the mosquito became infected with each of the parasites affected the levels of virulence for the host and the probability of infection and growth for each of the parasites. Another experiment aimed to demonstrate how growth of each parasite species was affected by a competitor and host food availability. Finally, we explored how suppression and enhancement of nitric oxide, a component in the immune response, affected host virulence and infection and growth of each parasite. We were still collecting data for these experiments by the time of the fellowship completion.