High-throughput DNA sequencing of the moose rumen from different geographical location reveals a core ruminal methanogenic archaeal diversity and a differential ciliate protozoal diversity.

This project expanded upon my work with moose bacteria from three geographic locations, to explore whether there were differences in methanogenic archaea or ciliated protozoa based on location.

Archaea are microorganisms in their own Domain, as they are neither Bacteria nor Eukaryota, although they often have similarities to organisms found in the other two domains.  Archaea are found in many extreme environments, but those found in the digestive tract of animals and humans come from the phylum Euryarchaeota.   Methanogens require hydrogen to make energy for themselves, and in that process (methanogenesis) methane is created as a byproduct.  In the digestive tract, especially in ruminants where the fermentation of plants creates a lot of hydrogen, the presence of methanogens acts a hydrogen sink and can prevent the build up of hydrogen which would otherwise lower the gut pH and be detrimental to both host and microbes.  To date, it is unclear if methanogens have any other health effect.

Protozoa are single-celled eukaryotes, and depending on which species they are, can be beneficial or pathogenic.  Typically, protozoa in the digestive tract of humans or other monogastrics are pathogens obtained from drinking contaminated water.  However, the digestive tracts of monogastrics (ex. humans) and ruminants (ex. moose) are very different, and the later can support a much different microbial community.  Specifically, protozoa found in ruminants that have cilia to move around (i.e. ciliated protozoa or ciliates) can have a number of roles, including fermentation of fiber or starch, or predation of bacteria and fungi.  As they are so difficult to maintain in culture and study in the lab, the role of protozoa in contributing to host health or methanogenesis is understudied.

Moose methanogen communities were significantly different between moose in Vermont, Norway, and Alaska, but maintained a core of shared taxa across all populations.  This implies that the moose rumen environment (pH, salt content, turnover, host-microbe interactions, etc.) is suitable for only a small number of methanogen species, and that this regulates the community as much as diet might.  Methanogen communities were also different based on sex of the moose, and age/weight.

mgen000034-f2
Figure 2: Diversity of moose rumen methanogens. Members of the RO clade are coloured in blues; members of the SGMT clade are coloured in reds. Mbr., Methanobrevibacter.

On the other hand, protozoal communities were dramatically different between moose in Vermont, Norway, and Alaska, and shared far fewer taxa.  This was surprising, as previous studies on deer had shown a core protozoal community across multiple geographically-separated populations.  These moose populations had not been geographically isolated long, but we hypothesized that diet was a much stronger driver of rumen protozoal diversity than previously thought.

mgen000034-f3
Figure 3: Diversity of the moose rumen protozoa.

Ishaq, S.L., Sundset, M.A., Crouse, J., Wright, A-D.G. 2015. High-throughput DNA sequencing of the moose rumen from different geographical location reveals a core ruminal methanogenic archaeal diversity and a differential ciliate protozoal diversity. Microbial Genetics, 2015(1).  Article

 

Featured Image; Figure 1: PCoA for moose methanogens (A, C, E) and protozoa (B, D, F). PCoA is coloured by (A, B) gender: female, red; male, blue; (C, D) location: Alaska, red; Norway, green; Vermont, blue; and (E, F) weight class: 1–100 kg, red triangle; 101–200 kg, yellow triangle; 201–300 kg, green down-facing triangle; 301–400 kg, green right-facing triangle, >400 kg (live weight), light blue circle; not available, blue square.

Dissertation: A Comparative Analysis Of The Moose Rumen Microbiota And The Pursuit Of Improving Fibrolytic Systems

As a Ph.D. student, I worked in the laboratory of Dr. André-Denis Wright in the Department of Animal Science at the University of Vermont. My thesis work investigated the microorganisms (bacteria, archaea, and protozoa) in the digestive tract of the moose from several geographical locations. In addition to identifying bacteria and ciliate protozoa using high-throughput sequencing, […]

High-throughput DNA sequencing of the ruminal bacteria from moose (Alces alces) in Vermont, Alaska, and Norway.

For the second project of my Ph.D., I expanded upon my findings in Vermont moose.  Following the collection of samples from moose in Vermont, I was able to obtain samples from moose in Alaska and Norway, as well.  The Alaskan moose were part of the Moose Research Station herd in Soldotna, Alaska, where they were allowed to roam and graze freely in a large 1 mi sq enclosure.  During sample collection for another project, Dr. John Crouse and Dr. Kimberlee Beckmen, both of the Alaska Department of Fish & Game, were able to intubate the sedated moose and collect rumen digesta samples.

A colleague in Norway, Dr. Monica Sunset, of the University of Tromsø, was able to facilitate sample collection and storage of moose rumen samples from two hunters; Drs. Even Jørgensen and Helge K. Johnsen, of the University of Tromsø.  As mailing rumen samples across country lines is restricted to prevent the potential spread of livestock diseases, it was actually easier to send me to Norway to extract DNA to ship home.  While in Norway, Dr. Sundset taught me how to culture microorganisms anaerobically – without the presence of oxygen.

For this project, we used high-throughput sequencing using the Roche 454 pyrosequencing platform, required me to learn the fine art of bioinformatics.  We were surprised to find that rumen bacterial communities in moose were different for each geographical location.  While we did not identify the diet that moose were eating, we speculated that these differences were driven by slightly different diets at the time points that each location was sampled in.  Plants often become more fibrous and less nutritious as the growing season develops and passes, and this nutritional change in substrate can select for different bacterial communities.  It has since been confirmed by a number of studies that geographic differences exist in the microbiome, driven by changes in site-specific diet, as well as food- and waterbourne microbial influences.


Ishaq, S.L., Wright, A-D.G. 2014. High-throughput DNA sequencing of the ruminal bacteria from moose (Alces alces) in Vermont, Alaska, and Norway. Microbial Ecology, 68(2):185-195. Article

Abstract

In the present study, the rumen bacteria of moose (Alces alces) from three distinct geographic locations were investigated. Moose are large, browsing ruminants in the deer family, which subsist on fibrous, woody browse, and aquatic plants. Subspecies exist which are distinguished by differing body and antler size, and these are somewhat geographically isolated. Seventeen rumen samples were collected from moose in Vermont, Alaska, and Norway, and bacterial 16S ribosomal RNA genes were sequenced using Roche 454 pyrosequencing with titanium chemistry. Overall, 109,643 sequences were generated from the 17 individual samples, revealing 33,622 unique sequences. Members of the phylum Bacteroidetes were dominant in samples from Alaska and Norway, but representatives of the phylum Firmicutes were dominant in samples from Vermont. Within the phylum Bacteroidetes, Prevotellaceae was the dominant family in all three sample locations, most of which belonged to the genus Prevotella. Within the phylum Firmicutes, the family Lachnospiraceae was the most prevalent in all three sample locations. The data set supporting the results of this article is available in the Sequence Read Archive (SRA), available through NCBI [study accession number SRP022590]. Samples clustered by geographic location and by weight and were heterogenous based on gender, location, and weight class (p < 0.05). Location was a stronger factor in determining the core microbiome than either age or weight, but gender did not appear to be a strong factor. There were no shared operational taxonomic units across all 17 samples, which indicates that these moose may have been isolated long enough to preclude a core microbiome among moose. Other potential factors discussed include differences in climate, food quality and availability, gender, and life cycle.

Keywords

Proteobacteria Unique Sequence Firmicutes Bacteroidetes Weight Class 

Ishaq poster FEMS 2013

Featured Image credit: U.S. National Park Service.