1. Nutritive Function of The Rainbow Trout (Oncorhynchus mykiss) Gastrointestinal tract Microbiomes
Omolola C. Betiku1,2*, Carl J. Yeoman2, T. Gibson Gaylord1, Suzanne L. Ishaq2, Glenn C. Duff3, Aurelien Mazurie4, and Wendy M. Sealey1
1United States Fish and Wildlife Service, Bozeman Fish Technology Center, Bozeman, MT 59715, USA.
2Department of Animal and Range Science, Montana State University, Bozeman, MT 59717, USA.
3Department of Animal and Range Sciences, New Mexico State University, Las Cruces, NM , USA.
4Information Technology Center, Montana State University, Bozeman, MT, 59717, USA.

2. Determination of GIT microbes
Culture-Dependent Methods: have been used by microbiologist for over 150 years to study bacteria diversity.
Disadvantages: revealed limited range of microbial diversity (<1% of the population).
Culture-Independent Methods (Metagenomics):
High-throughput sequencing technologies
Led to the discovery of vast new lineages of microbes.

3. Host-Microbial Ecosystem
Identify microbiota and gene functions associated with alternative diets and how they differ between mid- and hind-gut sections of the rainbow trout intestine

4. Ingredient Composition
(CON)
(CON)

5. 16 Week Feeding Trial Temperature:15 oC 2400-L Tank
Initial weight: 126 g
75 fish tank
Fed 2 daily to satiation

6. Growth at 16 weeks

7. No effects of diet on whole body proximate composition

8. Trout Intestinal Tract Sections

9. Shotgun Metagenomics Analysis
Midgut section: Luminal and Mucosal
DNA Isolation
Illumina sequencing: Illumina HiSeqTM 2005
Data analysis: Various Bioinformatics tools

10. Microbial Composition
Figure 2. Overall Taxonomic Compositions of Rainbow Trout GIT Microbiota.
Central pie chart shows the domain-level composition of the rainbow trout GIT, while outer pies show the major Phyla or Orders (for viruses) of each domain. Black segments of pie charts indicate all other less abundant taxa including reads that were unable to be annotated.
Overall, bacteria were predominant in the two regions of the trout GIT microbiome (avg. 74 ± 36% of all assignable contigs), but eukaryotic microbes (25 ± 37%), viruses (0.8 ± 1.6%),
and archaea (0.3 ± 0.4%) were also evident (Fig. 2).
Bacteria were largely from the Firmicutes, Actinobacteria, Proteobacteria, Bacteroidetes, Tenericutes and Fusobacterial phyla (Fig. 2).
Eukaryotes and archaea were almost exclusively from the Ascomycota and Euryarchaeota phyla, respectively (Fig. 2), while viruses were predominantly bacteriophage of the Caudovirales

11. Microbial Composition
Tenericutes
Euryarchaeota
Crenarchaeota
Figure 5. Phylum-level variation among rainbow trout GIT fed fishmeal-, animal- and
plant-based diets. Dual heatmaps show the relative abundances of phyla by diet and GIT
location (left) and the significance of any observed differences by gut location, or diet, as
stratified by gut location (right).
Midgut has greater diversity than the hindgut section of the gut.
Comparisons of species-level classifications and functional profiles revealed GIT location and location X diet relationships. Hind-, but not mid-GIT samples varied with diet.
This may be partially explained by the finding that dietary treatments caused a significant shift in hind-GIT microbiota (ANOSIM R=0.32, p<0.005), but no measurable effect in the trout mid-GIT.
The inclusion of plant protein in the diet led to 7.5 and 60-fold increases in archaea (0.6 ± 0.04%) within the hindgut relative to CON (0.08 ± 0.07%) and APD diets (0.01 ± 0.01%), respectively (Fig. 5).
Among bacterial constituents, there were trending enrichments in Bacteroidetes within the hind-GIT of PPD-consuming fish, while the CON diet caused trending increases in Firmicutes relative to other dietary treatments in the hind-GIT (Fig.
5).

12. Microbial Gene Functions
Functions related to protein (8 ± 3%), amino acid (17 ± 4%) and nitrogen (0.37 ± 0.28%) metabolisms collectively represented the highest fraction of annotated genes within the metagenomes (Fig. 3). Genes dedicated to carbohydrate (15 ± 4%)
and vitamin, cofactor, and prosthetic group metabolism (6 ± 1.6%) were also abundant (Fig. 3).

13. Microbial Gene Functions
Central aromatic intermediates
Organic sulfur metabolism
Fatty acids
Microbial genes related to sulfur, potassium, and aromatic metabolism were enriched in the midgut while carbohydrate, fatty acids and lipid metabolisms gene were enriched in the hindgut.
The hind-GIT microbes had a greater number of genes involved in carbohydrate, and fatty acid, lipid, or isoprenoid metabolisms. While the mid-GIT microbes were enriched for genes involved in sulfur, potassium, and aromatic compound metabolisms.
Polysaccharides

14. Conclusions Midgut has greater microbial diversity than the hindgut.
Comparisons of species-level classifications and functional profiles revealed GIT location and location X diet relationships. Hind-, but not mid-GIT samples varied with diet.
These results may support a nutritive role for trout GIT microbes and indicate a potential division of nutritive function between the mid- and hind- GIT microbiomes. These differences in functional division between the mid- and hind-GIT appear to be modulated by, and in some respects, adaptable to the fish diet.

15. Acknowledgements
BFTC Team
Yeoman’s Lab, MSU

16. THANK YOU!

17. In total, we observed 3,267 unique microbial species that collectively
encoded 6,054 unique annotatable gene functions.
By sample, 343±130 microbial species were observed in the mid- (377±142) and
hind-GIT (310±111). Good’s estimates of coverage indicated we had captured >96% of species and gene functions in all samples.
Comparisons of species-level classifications and functional profiles revealed GIT location and location X diet relationships. Hind-, but not mid-GIT samples varied with diet.
The hind-GIT microbes had a greater number of genes involved in carbohydrate, and fatty acid, lipid, or isoprenoid metabolisms. While the mid-GIT microbes were enriched for genes involved in sulfur, potassium, and aromatic compound metabolisms.
These results support a nutritive role for trout GIT microbes and indicate a potential division of nutritive function between the mid- and hind- GIT microbiomes. These differences in functional division between the mid- and hind-GIT appear to be modulated by, and in some respects, adaptable to the fish diet.

18. Fillet Color was altered by diet