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Interactions between unicellular eukaryotes and bacteria are difficult to characterize in the environment owing to their large number and inherently microscopic scale. Although particular co-occurrences can be recovered through targeted approaches, e.g. single-cell sequencing or fluorescence in situ hybridization, the vast majority of the interactions remain unseen. Here, we discuss Emulsion, Paired Isolation and Concatenation polymerase chain reaction (epicPCR) as a tool to uncover these interactions in very high throughput. Originally developed for taxonomy-to-function linkage in bacterial communities, epicPCR has the potential to recover the complete interaction network in a given environment at single-cell resolution. This approach relies on the encapsulation of protistan single cells in emulsion droplets that can subsequently be gelified into beads. In this way, encapsulated cells can be exposed to lysis reagents and further phylogenetic paired marker amplification. A bacterium that physically co-occurs with the eukaryote will be jointly trapped, and the amplification will generate a concatenated PCR product containing physically coupled taxonomic markers from both partners, creating a link. Further amplification and sequencing enable the construction of an association pattern with statistically verified physical co-occurrences. Here, we discuss the potential, challenges and limitations of epicPCR. We argue that the microscopic scale at which epicPCR operates, the high throughput it delivers and its exploratory nature make it an unparalleled approach to unravel associations between microbes directly from environmental samples. This article is part of a discussion meeting issue ‘Single cell ecology’.

Microeukaryote predation on bacteria is a fundamental phenomenon to understand energy and nutrient dynamics at the base of the aquatic food web. To date, the most prevalent way to estimate grazing rates is by using epifluorescence microscopy to enumerate ingestion events of fluorescently labelled tracers (FLTs) after short-term incubation experiments. However, this approach can be sensitive to the type of FLT, requires skillful preparation of the samples and is limited to small sample sizes. We tested the susceptibility of rate estimates to the choice of prey and made a side-by-side comparison between microscopy and flow cytometry when recording ingestion by a bacterivorous flagellate. Short-term uptake experiments were established using 5 types of FLTs differing in quality (living, dead or inert) and size (large or small), with Ochromonas triangulata as a model flagellate. The experiments showed that (1) each of the different prey types yielded different clearing rates, ranging from 0.5 to 3.6 nl cell-1 h-1, with the largest differences (3-fold or higher) between small prey (lower rates) and large prey (higher rates); (2) the cytometry estimate differed significantly from the microscopy estimate in 3 out of 4 experimental configurations; and (3) the precision of the cytometric analysis was greater, with >3-fold higher uncertainty associated with microscopy counting. Our results validate that flow cytometry provides a more precise bacterivory estimate, and that the choice of FLT influences the grazing rate estimate to a high extent regardless of the analytical method used.