Do Ruminal Ciliates Select Their Preys and Prokaryotic Symbionts?

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

Interactions between Entodinium caudatum and an amino acid-fermenting bacterial consortium: fermentation characteristics and protozoal population in vitro

Ruminal protozoa, especially entodiniomorphs, engulf other members of the rumen microbiome in large numbers; and they release oligopeptides and amino acids, which can be fermented to ammonia and volatile fatty acids (VFAs) by amino acid-fermenting bacteria (AAFB). Studies using defaunated (protozoa-free) sheep have demonstrated that ruminal protozoa considerably increase intraruminal nitrogen recycling but decrease nitrogen utilization efficiency in ruminants. However, direct interactions between ruminal protozoa and AAFB have not been demonstrated because of their inability to establish axenic cultures of any ruminal protozoan. Thus, this study was performed to evaluate the interaction between Entodinium caudatum, which is the most predominant rumen ciliate species, and an AAFB consortium in terms of feed degradation and ammonia production along with the microbial population shift of select bacterial species (Prevotella ruminicola, Clostridium aminophilum, and Peptostreptococcus anaerobius). From an Ent. caudatum culture that had been maintained by daily feeding and transfers every 3 or 4 days, the bacteria and methanogens loosely associated with Ent. caudatum cells were removed by filtration and washing. An AAFB consortium was established by repeated transfers and enrichment with casamino acids as the sole substrate. The cultures of Ent. caudatum alone (Ec) and AAFB alone (AAFB) and the co-culture of Ent. caudatum and AAFB (Ec + AAFB) were set up in three replicates and incubated at 39°C for 72 h. The digestibility of dry matter (DM) and fiber (NDF), VFA profiles, ammonia concentrations, pH, and microscopic counts of Ent. caudatum were compared among the three cultures. The co-culture of AAFB and Ent. caudatum enhanced DM degradation, VFA production, and Ent. caudatum cell counts; conversely, it decreased acetate: propionate ratio although the total bacterial abundance was similar between Ec and the Ec + AAFB co-culture after 24 h incubation. The ammonia production and relative abundance of C. aminophilum and P. anaerobius did not differ between AAFB alone and the Ec + AAFB co-culture. Our results indicate that Ent. caudatum and AAFB could have a mutualistic interaction that benefited each other, but their interactions were complex and might not increase ammoniagenesis. Further research should examine how such interactions affect the population dynamics of AAFB

Dietary bioactive lipid compounds rich in menthol alter Interactions among members of ruminal microbiota in sheep

This study aimed to investigate the effects of two practically relevant doses of menthol-rich plant bioactive lipid compounds (PBLC) on fermentation, microbial community composition, and their interactions in sheep rumen. Twenty-four growing Suffolk sheep were divided into three treatments and were fed hay ad libitum plus 600 g/d of concentrate containing no PBLC (Control) or PBLC at low dose (80 mg/d; PBLC-L) or high dose (160 mg/d; PBLC-H). After 4 weeks on the diets, samples of ruminal digesta were collected and analyzed for short-chain fatty acid (SCFA), ammonia, and microbiota; microbiota being analyzed in the solid and the liquid digesta fractions separately. Ruminal SCFA and ammonia concentrations were not affected by the PBLC treatments. The microbiota in the solid fraction was more diverse than that in the liquid fraction, and the relative abundance of most taxa differed between these two fractions. In the solid fraction, phylogenetic diversity increased linearly with increased PBLC doses, whereas evenness (lowest in PBLC-L) and Simpson diversity index (greatest in PBLC-H) changed quadratically. In the liquid fraction, however, the PBLC supplementation did not affect any of the microbial diversity measurements. Among phyla, Chloroflexi (highest in PBLC-L) and unclassified_bacteria (lowest in PBLC-L) were altered quadratically by PBLC. Lachnospiraceae, Bacteroidaceae (increased linearly), BS11 (increased in PBLC-L), Christensenellaceae (decreased in PBLC treatments), and Porphyromonadaceae (increased in PBLC treatments) were affected at the family level. Among genera, Butyrivibrio increased linearly in the solid fraction, YRC22 increased linearly in the liquid fraction, whereas Paludibacter increased and BF311 increased linearly with increasing doses of PBLC in both fractions. The PBLC treatments also lowered methanogens within the classes Thermoplasmata and Euryarchaeota. Correlation network analysis revealed positive and negative correlations among many microbial taxa. Differential network analysis showed that PBLC supplementation changed the correlation between some microbial taxa and SCFA. The majority of the predicted functional features were different between the solid and the liquid digesta fractions, whereas the PBLC treatments altered few of the predicted functional gene categories. Overall, dietary PBLC treatments had little influence on the ruminal fermentation and microbiota but affected the associations among some microbial taxa and SCFA

Single ruminal ciliate cells (Sequence file and its metadata)

<div>This project contains MiSeq 16S gene sequences retrieved from 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 to verify the prokaryotic population in and outside of ciliate cells.</div><div><br></div><div>Metadata related to those sequences was uploaded as well.</div><div><br></div

Data_Sheet_1_Do Ruminal Ciliates Select Their Preys and Prokaryotic Symbionts?.pdf

<p>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.</p

Metagenomic investigation of gastrointestinal microbiome in cattle

The gastrointestinal (GI) tract, including the rumen and the other intestinal segments of cattle, harbors a diverse, complex, and dynamic microbiome that drives feed digestion and fermentation in cattle, determining feed efficiency and output of pollutants. This microbiome also plays an important role in affecting host health. Research has been conducted for more than a century to understand the microbiome and its relationship to feed efficiency and host health. The traditional cultivation-based research elucidated some of the major metabolism, but studies using molecular biology techniques conducted from late 1980’s to the late early 2000’s greatly expanded our view of the diversity of the rumen and intestinal microbiome of cattle. Recently, metagenomics has been the primary technology to characterize the GI microbiome and its relationship with host nutrition and health. This review addresses the main methods/techniques in current use, the knowledge gained, and some of the challenges that remain. Most of the primers used in quantitative real-time polymerase chain reaction quantification and diversity analysis using metagenomics of ruminal bacteria, archaea, fungi, and protozoa were also compiled

Data_Sheet_2_Do Ruminal Ciliates Select Their Preys and Prokaryotic Symbionts?.PDF

<p>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.</p

Table_1_Do Ruminal Ciliates Select Their Preys and Prokaryotic Symbionts?.DOCX

<p>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.</p