Evolution of virus and virophage facilitates persistence in a tripartite microbial system

Tripartite biotic interactions are inherently complex, and the strong interdependence of species and high levels of exploitation can make these systems short-lived and vulnerable to extinction. The persistence of species depends then on the balance between exploitation and avoidance of exploitation of the resource beyond the point where sustainable exploitation is no longer possible. We used this general prediction to test the potential for long-term persistence in a recently discovered tripartite microbial system in which a eukaryotic host is preyed upon by a giant virus that is in turn parasitized by a virophage. Host and virophage may benefit from this interaction because the virophage reduces the harmful effects of the giant virus on the host population over time and the virophage can survive integrated into the host genome when giant viruses are scarce. Here, we grew hosts in the presence and absence of the giant virus and virophage over ~280 host generations. We found that the three players persisted, but that the beneficial effect of the virophage for the host population diminished over time. We further tested whether the level of exploitation and replication evolved in the giant virus and/or virophage population over the course of the experiment and whether the changes were such that they avoid overexploitation. We found that the giant virus evolved towards lower replication levels and the virophage towards increased replication but decreased giant virus exploitation. These changes are predicted to facilitate persistence by lowering giant virus and host exploitation and consequently reducing the protective effect of the virophage. ### Competing Interest Statement The authors have declared no competing interest.