Discovery of an Undocumented Symbiosis Between Arcellinida and a Flagellate Using Single-Cell Transcriptomics.

Symbiotic interactions involving single-celled eukaryotes have been studied for over a century. This is in part because these microbes occupy nearly all environments and have adapted to survive through interactions with other species. Among them, Arcellinida-amoebae enclosed in a test (i.e., shell)-have been studied extensively since the 1800s. The preservability of their tests, and sensitivity to environmental conditions, has made them important in studies of modern and paleoenvironments. However, despite centuries of observation, we have seen no record of protists living within Arcellinida tests. Here, we investigate a symbiosis involving an unknown flagellate found swimming inside the test of multiple Arcellinida species from a site in Acadia National Park (Maine, USA). We analyzed 125 single-cell Arcellinida transcriptomes from five genera, of which 10 individuals had a flagellate living inside the test at the time of sequencing. By combining microscopy, molecular, and bioinformatic methods, we identify this novel flagellate as belonging to the Prokinetoplastid clade. We also make inferences on the nature of the relationship through analysis of the host gene expression. Overall, our results provide a framework for studying microbial symbiosis in uncultivable groups and adds to our knowledge of interspecific interactions at the microbial level.