Single-cell genomics reveals complex microbial and viral associations in ciliates and testate amoebae

Protists play important roles in nutrient cycling across ecosystems, yet the composition and function of their associated microbiomes remain poorly studied. Here, we use cultivation-independent single-cell isolation and genome-resolved metagenomics to investigate the microbiomes and viromes of more than 100 uncultivated ciliates and amoebae from diverse environments. Our findings reveal unique microbiome structures and complex associations with bacterial symbionts and viruses, with stark differences between ciliates and amoebae. We recover 117 microbial genomes affiliated with known eukaryotic endosymbionts, including Holosporales, Rickettsiales, Legionellales, Chlamydiae, and Babelota, and 258 genomes linked to host-associated Patescibacteriota. Many show genome reduction and genes related to toxin-antitoxin systems and nucleotide parasitism, indicating adaptation to intracellular lifestyles. We also identify more than 80 giant viruses from diverse lineages, some actively expressing genes in single-cell transcriptomes, along with other viruses predicted to infect eukaryotes or symbiotic bacteria. The frequent co-occurrence of giant viruses and microbial symbionts, especially in amoebae, suggests multipartite interactions. Together, our study highlights protists as hubs of microbial and viral associations and provides a broad view of the diversity, activity, and ecological importance of their hidden partners.