Recent outbreaks of various infectious diseases have highlighted the ever-present need to understand the drivers of the outbreak and spread of disease. Although much of the research investigating diseases focuses on single infections, natural systems are dominated by multiple infections. These infections may occur simultaneously, but are often acquired sequentially, which may alter the outcome of infection. Using waterfleas (Daphnia magna) as a model organism, we examined the outcome of sequential and simultaneous multiple infections with 2 microsporidian parasites (Ordospora colligata and Hamiltosporidium tvaerminnensis) in a fully factorial design with 9 treatments and 30 replicates. We found no differences between simultaneous and sequential infections. However, H. tvaerminnensis fitness was impeded by multiple infection due to increased host mortality, which gave H. tvaerminnensis less time to grow. Host fecundity was also reduced across all treatments, but animals infected with O. colligata at a younger age produced the fewest offspring. As H. tvaerminnensis is both horizontally and vertically transmitted, this reduction in offspring may have further reduced H. tvaerminnensis fitness in co-infected treatments. Our findings suggest that in natural populations where both species co-occur, H. tvaerminnensis may evolve to higher levels of virulence following frequent co-infection by O. colligata.

The microsporidian parasite Hamiltosporidium tvaerminnensis can infect Daphnia magna both horizontally (through environmental spores) and vertically (through parthenogenetic and sexually produced eggs). The spores of H. tvaerminnensis come in three distinguishable morphologies, which are thought to have different roles in the transmission of the parasite. In this study, we examined the role of the two most common spore morphologies (i.e. oval-shaped spores and pear-shaped spores) in horizontal transmission of H. tvaerminnensis. To this end, we infected hosts with solutions consisting of either mostly oval- or mostly pear-shaped spores, and quantified infection rates, parasite-induced host mortality and mean number of parasite spores produced per host. We found that spore morphology by itself did not influence infection rates and parasite-induced host mortality. Instead, host clone and parasite isolate interacted with spore morphology in shaping infection outcome and mortality. Thus, there appear to be strong genotype-by-genotype (G × G) interactions in this system. While there is no dispute that H. tvaerminnensis can transmit both vertically and horizontally, our findings do not support theoretical predictions that different spore morphologies hold different roles in horizontal transmission of H. tvaerminnensis.

Many parasites survive harsh periods together with their hosts. Without the possibility of horizontal transmission during host diapause, parasite persistence depends entirely on host survival. We therefore hypothesize that a parasite should be avirulent during its host's diapausing stage. In contrast, the parasite may express higher virulence, i.e. parasite-induced fitness reduction of the host, during host life stages with good opportunities for horizontal transmission. Here we study the effects of a vertically and horizontally transmitted microsporidium parasite, Hamiltosporidium tvaerminnensis, on the quantity and survival of resting eggs of its host Daphnia magna. We find that the parasite did not affect egg volume, hatching success and time to hatching of the Daphnia's resting eggs, although it did strongly reduce the number of resting eggs produced by infected females, revealing high virulence during the non-diapause phase of the host's life cycle. These results also explain another aspect of this system – namely the strong decline in natural population prevalence across diapause. This decline is not caused by mortality in infected resting stages, as was previously hypothesized, but because infected female hosts produce lower rates of resting eggs. Together, these results help explain the epidemiological dynamics of a microsporidian disease and highlight the adaptive nature of life stage-dependent parasite virulence.