Single-Cell Amplicon Sequencing Reveals Community Structures and Transmission Trends of Protist-Associated Bacteria in a Termite Host

Abstract The hindgut protists of wood-feeding termites are usually colonized by prokaryotic symbionts. Many of the hurdles that have prevented a better understanding of these symbionts arise from variation among protist and termite host species and the inability to maintain prominent community members in culture. These issues have made it difficult to study the fidelity, acquisition, and differences in colonization of protists by bacterial symbionts. In this study, we use high throughput amplicon sequencing of the V4 region of 16S rRNA genes to determine the composition of bacterial communities associated with single protist cells of six protist species, from the genera Pyrsonympha , Dinenympha , and Trichonympha that are present in the hindgut of the termite Reticulitermes flavipes. By analyzing amplicon sequence variants (ASVs), the diversity and distribution of protist-associated bacteria was compared within and across these six different protist species. ASV analysis showed that, in general, each protist genus associated with a distinct community of bacterial symbionts which were conserved across different termite colonies. However, some ASVs corresponding to ectosymbionts (Spirochaetes) were shared between different Dinenympha species and to a lesser extent with Pyrsonympha and Trichonympha hosts. This suggested that certain bacterial symbionts may be cosmopolitan to some degree and perhaps acquired by horizontal transmission. Using a fluorescence-based cell assay, we could observe the horizontal acquisition of surface-bound bacteria. This acquisition was shown to be time-dependent, involve active processes, and was non-random with respect to binding locations on some protists.