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Title: Correlated SEM, FIB-SEM, TEM, and NanoSIMS Imaging of Microbes from the Hindgut of a Lower Termite: Methods for In Situ Functional and Ecological Studies of Uncultivable Microbes

Abstract

The hindguts of lower termites harbor highly diverse, endemic communities of symbiotic protists, bacteria, and archaea essential to the termite's ability to digest wood. Despite over a century of experimental studies, ecological roles of many of these microbes are unknown, partly because almost none can be cultivated. Many of the protists associate with bacterial symbionts, but hypotheses for their respective roles in nutrient exchange are based on genomes of only two such bacteria. To show how the ecological roles of protists and nutrient transfer with symbiotic bacteria can be elucidated by direct imaging, we combined stable isotope labeling ( 13C-cellulose) of live termites with analysis of fixed hindgut microbes using correlated scanning electron microscopy, focused ion beam-scanning electron microscopy (FIB-SEM), transmission electron microscopy, and high resolution imaging mass spectrometry (NanoSIMS). We developed methods to prepare whole labeled cells on solid substrates, whole labeled cells milled with a FIB-SEM instrument to reveal cell interiors, and ultramicrotome sections of labeled cells for NanoSIMS imaging of 13C enrichment in protists and associated bacteria. Our results show these methods have the potential to provide direct evidence for nutrient flow and suggest the oxymonad protist Oxymonas dimorpha phagocytoses and enzymatically degrades ingested wood fragments, andmore » may transfer carbon derived from this to its surface bacterial symbionts.« less

Authors:
 [1];  [1];  [1];  [1];  [2];  [3]
  1. Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States). Physical and Life Sciences Directorate
  2. Univ. of California, Berkeley, CA (United States). Division of Organisms and the Environment, Environmental Science, Policy and Management
  3. Univ. of British Columbia, Vancouver, BC (Canada). Dept. of Botany, Canadian Inst. for Advanced Research
Publication Date:
Research Org.:
Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)
Sponsoring Org.:
USDOE National Nuclear Security Administration (NNSA); USDOE Laboratory Directed Research and Development (LDRD) Program
OSTI Identifier:
1647138
Report Number(s):
LLNL-JRNL-557111
Journal ID: ISSN 1431-9276; 584713
Grant/Contract Number:  
AC52-07NA27344; 011-LW-039
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Microscopy and Microanalysis
Additional Journal Information:
Journal Volume: 19; Journal Issue: 6; Journal ID: ISSN 1431-9276
Publisher:
Microscopy Society of America (MSA)
Country of Publication:
United States
Language:
English
Subject:
bacteria; bioenergy; biological and medical sciences; ecology; focused ion beam; SIMS; protists; scanning electron microscopy; stable isotopes; termites; transmission electron microscopy

Citation Formats

Carpenter, Kevin J., Weber, Peter K., Davisson, M. Lee, Pett-Ridge, Jennifer, Haverty, Michael I., and Keeling, Patrick J. Correlated SEM, FIB-SEM, TEM, and NanoSIMS Imaging of Microbes from the Hindgut of a Lower Termite: Methods for In Situ Functional and Ecological Studies of Uncultivable Microbes. United States: N. p., 2013. Web. doi:10.1017/s1431927613013482.
Carpenter, Kevin J., Weber, Peter K., Davisson, M. Lee, Pett-Ridge, Jennifer, Haverty, Michael I., & Keeling, Patrick J. Correlated SEM, FIB-SEM, TEM, and NanoSIMS Imaging of Microbes from the Hindgut of a Lower Termite: Methods for In Situ Functional and Ecological Studies of Uncultivable Microbes. United States. doi:10.1017/s1431927613013482.
Carpenter, Kevin J., Weber, Peter K., Davisson, M. Lee, Pett-Ridge, Jennifer, Haverty, Michael I., and Keeling, Patrick J. Fri . "Correlated SEM, FIB-SEM, TEM, and NanoSIMS Imaging of Microbes from the Hindgut of a Lower Termite: Methods for In Situ Functional and Ecological Studies of Uncultivable Microbes". United States. doi:10.1017/s1431927613013482. https://www.osti.gov/servlets/purl/1647138.
@article{osti_1647138,
title = {Correlated SEM, FIB-SEM, TEM, and NanoSIMS Imaging of Microbes from the Hindgut of a Lower Termite: Methods for In Situ Functional and Ecological Studies of Uncultivable Microbes},
author = {Carpenter, Kevin J. and Weber, Peter K. and Davisson, M. Lee and Pett-Ridge, Jennifer and Haverty, Michael I. and Keeling, Patrick J.},
abstractNote = {The hindguts of lower termites harbor highly diverse, endemic communities of symbiotic protists, bacteria, and archaea essential to the termite's ability to digest wood. Despite over a century of experimental studies, ecological roles of many of these microbes are unknown, partly because almost none can be cultivated. Many of the protists associate with bacterial symbionts, but hypotheses for their respective roles in nutrient exchange are based on genomes of only two such bacteria. To show how the ecological roles of protists and nutrient transfer with symbiotic bacteria can be elucidated by direct imaging, we combined stable isotope labeling (13C-cellulose) of live termites with analysis of fixed hindgut microbes using correlated scanning electron microscopy, focused ion beam-scanning electron microscopy (FIB-SEM), transmission electron microscopy, and high resolution imaging mass spectrometry (NanoSIMS). We developed methods to prepare whole labeled cells on solid substrates, whole labeled cells milled with a FIB-SEM instrument to reveal cell interiors, and ultramicrotome sections of labeled cells for NanoSIMS imaging of 13C enrichment in protists and associated bacteria. Our results show these methods have the potential to provide direct evidence for nutrient flow and suggest the oxymonad protist Oxymonas dimorpha phagocytoses and enzymatically degrades ingested wood fragments, and may transfer carbon derived from this to its surface bacterial symbionts.},
doi = {10.1017/s1431927613013482},
journal = {Microscopy and Microanalysis},
issn = {1431-9276},
number = 6,
volume = 19,
place = {United States},
year = {2013},
month = {10}
}

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    Works referencing / citing this record:

    Can 13 C stable isotope analysis uncover essential amino acid provisioning by termite-associated gut microbes?
    journal, January 2015

    • Ayayee, Paul A.; Jones, Susan C.; Sabree, Zakee L.
    • PeerJ, Vol. 3
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