Viruses are an abundant component of aquatic systems, but their detection and quantification remain a challenge. Virophages co-replicate with giant viruses in the shared host cell, and can inhibit the production of new giant virus particles, thereby increasing the survival of the infected host population. Here, we present a protocol for Droplet Digital PCR (ddPCR) to quantify simultaneously giant virus and virophage in a mixed sample, enabling the rapid, culture-free and high throughput detection of virus and virophage. As virophage can be present as free virus particles or integrated into the virus host’s genome as well as associated with organic particles, we developed a simple method that enables discrimination between free and particle-associated virophages. The latter include aggregated virophage particles as well as virophage integrated into the host genome. We used, for our experiments, a host-virus-virophage system consisting of Cafeteria burkhardae, CroV and mavirus. Our results show that ddPCR can be an efficient method to quantify virus and virophage, and we discuss potential applications of the method for studying ecological and evolutionary processes of virus and virophages.
Chemostats and other flow through culture systems are a powerful tool for the study of microbial and plankton communities in experimental ecology and evolutionary studies. Commercially available chemostat systems allow the control of a large number of parameters (e.g., ph, pressure, CO 2 concentration) but are often expensive and offer a high level of control that is often not needed for many experimental studies. Non-commercial chemostats are more cost efficient, easily set up and flexible in volumes used. Different from semi-continuous culture systems, the flow through conditions of chemostats allow a constant inflow of resources from a reservoir (medium bottle) and outflow of unused nutrients, waste products and organisms, which are all collected in a waste bottle. Nutrient levels in the reservoir and the flow rate of the chemostat system (often referred to as dilution rate and presented as the fraction of the volume of the chemostat that is replaced per day) determines population growth rates and dynamics (for the theory behind chemostats see Smith & Waltman, 1995 and Weitz, 2015). Chemostats have been used in a number of studies and with different organisms and combinations of organisms. For example, Boer et al. (2010) studied how growth-limiting intracellular metabolites control yeast growth under diverse nutrient availability (Saccharomyces cerevisiae growing under five different nutrient supply of nitrogen: carbon: phosphorus). Becks et al. (2005) used chemostat systems to show how changes in the flow rate of a chemostat system influences the population dynamics of a three species microbial system. Frickel et al. (2016) used chemostats to investigate eco-evolutionary dynamics in a coevolving host-virus system (algae Chlorella variabilis and Chlorovirus strain PBCV-1) for 90 days. We present here an instruction for a cost efficient and flexible chemostat systems. These chemostats are composed of four main parts: a syringe unit, a glass bottle (i.e. the chemostat), a medium bottle and a waste bottle, all connected by tubing. A peristaltic pump and a low overpressure in the system allow the flow of medium from the reservoir to the chemostat bottle and of unused nutrients, waste products and organisms to the waste bottle. Chemostats are put on stirring plates to create a homogenosus environment within.
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.
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