Monitoring Euplotes species dynamics in mixed experiments using genetic markers for quantitative polymerase chain reaction

Abstract

Ciliates are widespread and play a major role in ecosystems as they form an important link between primary producers and higher trophic levels. They have been used as a classic model to study predator–prey interactions of co-evolutionary processes. In our experimental system, predators and prey interact dynamically, with prey exhibiting predator-induced defenses and predators potentially adapting through offensive strategies, both of which influence population dynamics. When analyzing population dynamics in similar ciliate species, individuals must be accurately identified and counted. Morphologically, the genus Euplotes is generally identified by the arrangement of cilia and cirri and the position of the macronucleus and micronucleus. This requires expensive and laborious cell counters and time-consuming staining methods. Furthermore, staining methods are not ideal for determining cell numbers, as individual cells may be lost during staining processes. As ciliates are unicellular organisms, we used DNA quantity to determine the number of individuals. We identified unique sequences of three Euplotes species: Euplotes octocarinatus, Euplotes daidaleos, and Euplotes aediculatus using random amplified polymorphic DNA (RAPD) fingerprinting. Using these sequences, we designed species-specific primers for quantitative polymerase chain reaction and generated corresponding standard curves based on microscopic cell counts. Using this method, we are now able to determine cell counts in unknown samples of different Euplotes species within a single experimental system and monitor population growth rates of one or even several species simultaneously. Additionally, using RAPD fingerprinting enables the identification of unique genetic sequences, allowing differentiation between clones of the same species and facilitating measurement of their population growth rates in mixed experiments.