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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

Journal Article · · Microscopy and Microanalysis
 [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
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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.

Research Organization:
Lawrence Livermore National Laboratory (LLNL), Livermore, CA (United States)
Sponsoring Organization:
USDOE National Nuclear Security Administration (NNSA); USDOE Laboratory Directed Research and Development (LDRD) Program
Grant/Contract Number:
AC52-07NA27344; 011-LW-039
OSTI ID:
1647138
Report Number(s):
LLNL-JRNL-557111; 584713
Journal Information:
Microscopy and Microanalysis, Vol. 19, Issue 6; ISSN 1431-9276
Publisher:
Microscopy Society of America (MSA)Copyright Statement
Country of Publication:
United States
Language:
English
Citation Metrics:
Cited by: 31 works
Citation information provided by
Web of Science

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Cited By (9)

Farming, slaving and enslavement: histories of endosymbioses during kinetoplastid evolution journal June 2018
Autotrophic and heterotrophic acquisition of carbon and nitrogen by a mixotrophic chrysophyte established through stable isotope analysis journal May 2017
Correlative microscopy combining transmission electron microscopy and secondary ion mass spectrometry: A general review on the state-of-the-art, recent developments, and prospects journal June 2019
Decoding molecular interactions in microbial communities journal July 2016
Origin of termite eusociality: trophallaxis integrates the social, nutritional, and microbial environments: Origin of termite eusociality journal March 2015
Genome Evolution and Nitrogen Fixation in Bacterial Ectosymbionts of a Protist Inhabiting Wood-Feeding Cockroaches journal May 2016
Intracellular Isotope Localization in Ammonia sp. (Foraminifera) of Oxygen-Depleted Environments: Results of Nitrate and Sulfate Labeling Experiments journal February 2016
What Kills the Hindgut Flagellates of Lower Termites during the Host Molting Cycle? journal December 2017
Can 13 C stable isotope analysis uncover essential amino acid provisioning by termite-associated gut microbes? journal January 2015

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59 BASIC BIOLOGICAL SCIENCES
bacteria
bioenergy
biological and medical sciences
ecology
focused ion beam
SIMS
protists
scanning electron microscopy
stable isotopes
termites
transmission electron microscopy