Do Ruminal Ciliates Select Their Preys and Prokaryotic Symbionts?

Abstract

Ruminal ciliates both preys on and form symbiotic relationships with other members of the ruminal microbiota for their survival. However, it remains elusive if they have selectivity over their preys or symbionts. In the present study, we investigated the above selectivity by identifying and comparing the free-living prokaryotes (FLP) and the ciliate-associated prokaryotes (CAP) using Illumina MiSeq sequencing of 16S rRNA gene amplicons. We used single ciliates cells of both monocultures of Entodinium caudatum and Epidinium caudatum and eight different ciliate genera isolated from fresh rumen fluid of dairy cows. Irrespective of the source (laboratory monocultures vs. fresh isolates) of the single ciliate cells, the CAP significantly differed from the FLP in microbiota community profiles. Many bacterial taxa were either enriched or almost exclusively found in the CAP across most of the ciliate genera. A number of bacteria were also found for the first time as ruminal bacteria in the CAP. However, no clear difference was found in methanogens between the CAP and the FLP, which was confirmed using methanogen-specific qPCR. These results suggest that ruminal ciliates probably select their preys and symbionts, the latter of which has rarely been found among the free-living ruminal prokaryotes. The bacteria enriched or exclusively found in the CAP can be target bacteria to detect and localize using specific probes designed from their 16S rRNA sequences, to characterize using single-cell genomics, or to isolate using new media designed based on genomic information.

Keywords: ciliate-associated prokaryotes; free-living prokaryotes; preys; ruminal ciliates; symbionts.

Figure 1

Principal coordinates analysis based on unweighted UniFrac distances in (A) both the FLP and CAP of two ciliate monocultures [Ent. caudatum (red squares) and Epi. caudatum (blue spheres)] and (B) the CAP of fresh isolates of ruminal ciliate singles cells and the FLP of the rumen fluid collected from Jersey dairy cows. Based on ANOSIM, FLP differed (P < 0.001) from CAP of both the monocultures and the rumen fluid.

Figure 2

Temporal changes in species richness, phylogenetic diversity, and relative abundance of select taxa after feeding. (A) α-diversity measurements in the CAP of Ent. caudatum. (B) Sediminibacterium in the CAP of Ent. caudatum, and (C) Succinivibrio found in- and outside of Epi. caudatum cells. No significant temporal changes of α-diversity measurements were found in Epi. caudatum.

Figure 3

Relative abundance of bacterial phyla, classes and known genera in the CAP and the FLP of the monocultures of Ent. caudatum (A) and Epi. caudatum (B) Relative abundance did not add up to 100% because unclassified bacterial taxa were not included.

Figure 4

A maximum-likelihood tree showing CAP-specific bacterial 16S rRNA gene sequences shared by ciliate single cells [18 sequences from the freshly isolated ciliate single cells and 24 sequences from Ent. caudatum (Ent.) and Epi. caudatum (Epi.) in their monocultures]. Relative abundance of each taxon among CAP-specific bacteria was shown in parentheses. Minor taxa (< 0.5% of total sequences) were excluded.

Figure 5

Relative abundance of major bacterial phyla (A) and genera (B) (each representing >1% of total sequences) of the CAP of freshly isolated ruminal ciliate single cells (average of 8 genera) and the FLP of the rumen fluid collected from Jersey dairy cows. Relative abundance of the genera did not add up to 100% because unclassified bacterial taxa were not included.