Functional changes in the gut microbiota across the hibernation cycle examined by stable isotope‐assisted labeling
Authors: Gugel Sadie R. , Chiang Edna , Regan Matthew D. , Assadi‐Porter Fariba M. , Carey Hannah V. 
Published: June 2021

Hibernation in ground squirrels is a complex annual cycle of physiological adaptations characterized by high metabolic rate and body temperature (Tb) in summer, and bouts of depressed metabolism (torpor) with periodic returns to high metabolic rate and Tb (interbout arousal) in winter. Our previous results showed that the dynamic nature of the hibernation cycle in 13‐lined ground squirrels drives changes in the taxonomic composition of the gut microbial community (microbiota). These taxonomic shifts are likely due to changes in host diet, particularly the lack of ingested food during the hibernation season. Relative abundances of taxa that degrade plant‐derived glycans decrease during hibernation, while taxa that thrive on host‐derived substrates such as mucin increase. However, the functional significance of these shifts in microbiota composition is unknown due to a paucity of tools to measure such changes directly in hosts. To address this, we utilized cavity ring‐down spectroscopy (CRDS) to trace changes in 13CO2/ 12CO2 ratio (δ13C) in exhaled breath from ground squirrels as a real time functional output for microbial activities. Ground squirrels were orally gavaged with 13C‐labeled substrates that can only be metabolized by bacterial and not mammalian enzymes. These include inulin, a complex plant glycan, and mannitol, a simple sugar alcohol. Changes in breath δ13C reflect changes in bacterial metabolism of a labeled substrate. Baseline δ13C (δ13Cbase) before substrate gavage in all squirrels was −20 ± 2 ‰ (N=17). Administration of 13C‐labeled mannitol (150 mg/kg) in summer squirrels produced a δ13C peak of 897 ± 601 ‰ (mean±SD, N=5), a 178‐fold increase over δ13Cbase, reached ~2 h after gavage. Summer squirrels gavaged with 13C‐labeled inulin (500 mg/kg) had a δ13C peak of 2201 ± 913 ‰ (N=5), a 463‐fold increase over δ13Cbase, reached between 3–4 h after gavage. Further validation was provided when squirrel microbiotas were depleted using an orally administered antibiotic cocktail (ABX). Relative to untreated squirrels, peak δ13C values were lower after gavage of mannitol (−17 ± 0.9 ‰, 0.9‐fold increase over δ13Cbase, N=4, P<0.05) and inulin (258 ± 105 ‰, 86‐fold increase over δ13Cbase, N=3, P<0.01) administration. These results demonstrate that stable isotope‐assisted labeling with CRDS is a powerful technique to assess the functional capacities of ground squirrel gut microbiotas to metabolize different substrates in vivo, in real time. The reduced δ13C peaks of ABX‐treated squirrels (whose cecal contents had reduced bacterial DNA yield) confirm that the breath responses are attributable to gut microbes. To test whether seasonal changes in squirrel microbiotas produce parallel changes in microbiota functional activity, the results from summer squirrels will be compared to interbout arousal hibernating squirrels and to spring squirrels two weeks after refeeding.Support or Funding InformationThis study was supported by funds from UW‐Madison Hilldale Undergraduate/Faculty Research Fellowship and NSF award IOS1558044.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.