Virophage replication mode determines ecological and evolutionary changes in a host-virus-virophage system

Giant viruses can control their eukaryotic host populations, shaping the ecology and evolution of aquatic microbial communities. Understanding the impact of the viruses own parasites, the virophages, on their control of microbial communities remains a challenge. Most virophages have two modes of host infection and replication. They can exist as free particles that co-infect a host cell with the virus and replicate but inhibit viral replication. Virophages can also integrate into the host genome and remain dormant until the host is infected with a virus, leading to virophage reactivation and replication that does not immediately inhibit viral replication. Both replication modes are present within host-virus-virophage communities, and their relative contributions are expected to be context dependent and dynamic over time. The consequences of this dynamic regime for ecological and evolutionary dynamics remain unexplored. Here, we test whether and how the relative contribution of virophage replication modes influences the ecological dynamics of an experimental host-virus-virophage system and the evolutionary responses of the virophage. To do this, we indirectly manipulated the level of virophage (Mavirus) integration into the host (Cafeteria burkhardae) in the presence of the giant Cafeteria roenbergensis virus (CroV) (later identified as Cafeteria burkhardae, Schoenle et al. 2020). Our results show that higher virophage integration is positively correlated with host survival, but negatively correlated with virophage reactivation. In addition, communities with higher virophage integration were characterised by lower population densities and reduced fluctuations in both host and viral populations, whereas virophage fluctuations were increased. This study reveals the complex interplay between virophages, viruses and hosts, in which the virophage dual replication mode is a dynamic and reactive mechanism contributing to persistence of the microbial community.