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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

Published online by Cambridge University Press:  11 October 2013

Kevin J. Carpenter ,
Peter K. Weber ,
M. Lee Davisson ,
Jennifer Pett-Ridge ,
Michael I. Haverty  and
Patrick J. Keeling
Kevin J. Carpenter*
Affiliation:
Physical and Life Sciences Directorate, Lawrence Livermore National Laboratory, P.O. Box 808, L-231, Livermore, CA 94551, USA
Peter K. Weber*
Affiliation:
Physical and Life Sciences Directorate, Lawrence Livermore National Laboratory, P.O. Box 808, L-231, Livermore, CA 94551, USA
M. Lee Davisson
Affiliation:
Physical and Life Sciences Directorate, Lawrence Livermore National Laboratory, P.O. Box 808, L-231, Livermore, CA 94551, USA
Jennifer Pett-Ridge
Affiliation:
Physical and Life Sciences Directorate, Lawrence Livermore National Laboratory, P.O. Box 808, L-231, Livermore, CA 94551, USA
Michael I. Haverty
Affiliation:
Division of Organisms and the Environment, Environmental Science, Policy and Management, University of California at Berkeley, 1301 South 46th Street, Building 478, Richmond, CA 94804-4698, USA
Patrick J. Keeling
Affiliation:
Canadian Institute for Advanced Research, Department of Botany, University of British Columbia, 3529-6270 University Boulevard, Vancouver, BC V6T 1Z4, Canada
*
**Corresponding author.kjcarpenter@lbl.gov; kevin@kevinjcarpenter.com
*Corresponding author.weber21@llnl.gov
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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, and may transfer carbon derived from this to its surface bacterial symbionts.

Keywords

bacteriabioenergyecologyfocused ion beamSIMSprotistsscanning electron microscopystable isotopestermitestransmission electron microscopy
Type
Biomedical and Biological Applications
Information
Microscopy and Microanalysis , Volume 19 , Issue 6 , December 2013 , pp. 1490 - 1501
Copyright
Copyright © Microscopy Society of America 2013 

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Footnotes

†

Current address: Life Sciences Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Mail Stop Donner, Berkeley, CA 94720, USA

References

Behrens, S., Losekann, T., Pett-Ridge, J., Weber, P.K., Ng, W.O., Stevenson, B.S., Hutcheon, I.D., Relman, D.A. & Spormann, A.M. (2008). Linking microbial phylogeny to metabolic activity at the single-cell level by using enhanced element labeling-catalyzed reporter deposition fluorescence in situ hybridization (EL-FISH) and NanoSIMS. Appl Environ Microbiol 74(10), 3143–3150.CrossRefGoogle ScholarPubMed
Berchtold, M., Chatzinotas, A., Schönhuber, W., Brune, A., Amann, R., Hahn, D. & König, H. (1999). Differential enumeration and in situ localization of microorganisms in the hindgut of the lower termite Mastotermes darwinensis by hybridization with rRNA-targeted probes. Arch Microbiol 172, 407–416.CrossRefGoogle Scholar
Bloodgood, R.A. & Fitzharris, T.P. (1976). Specific associations of prokaryotes with symbiotic flagellate protozoa from the hindgut of the termite Reticulitermes and the wood-eating roach Cryptocercus. Cytobios 17(66), 103–122.Google Scholar
Boschker, H.T.S., Nold, S.C., Wellsbury, P., Bos, D., de Graaf, W., Pel, R., Parkes, R.J. & Cappenberg, T.E. (1998). Direct linking of microbial populations to specific biogeochemical processes by C-13-labelling of biomarkers. Nature 392(6678), 801–805.CrossRefGoogle Scholar
Brugerolle, G. & Bordereau, C. (2004). The flagellates of the termite Hodotermopsis sjoestedti with special reference to Hoplonympha, Holomastigotes and Trichomonoides trypanoides n. comb. Eur J Protistol 40, 163–174.CrossRefGoogle Scholar
Brugerolle, G. & Lee, J.H. (2000a). Order oxymonadida. In The Illustrated Guide to the Protozoa, 2nd ed., Lee, J.J., Leedale, G.F. & Bradbury, P. (Eds.), pp. 1186–1195. Lawrence, KS: Allen Press, Inc. Google Scholar
Brugerolle, G. & Lee, J.J. (2000b). Phylum Parabasalia. In An Illustrated Guide to the Protozoa, 2nd ed., Lee, J.J., Leedale, G.F. & Bradbury, P. (Eds.), pp. 1196–1250. Lawrence, KS: Allen Press Inc. Google Scholar
Brune, A. & Ohkuma, M. (2011). Role of the termite gut microbiota in symbiotic digestion. In Biology of Termites: A Modern Synthesis, Bignell, D.E., Roisin, Y. & Lo, N. (Eds.), pp. 439–475. London: Springer.Google Scholar
Brune, A. & Stingl, U. (2006). Prokaryotic symbionts of termite gut flagellates: Phylogenetic and metabolic implications of a tripartite symbiosis. Prog Mol Subcell Biol 41, 39–60.CrossRefGoogle ScholarPubMed
Carpenter, K.J., Chow, L. & Keeling, P.J. (2009). Morphology, phylogeny, and diversity of Trichonympha (Parabasalia: Hypermastigida) of the wood-feeding cockroach Cryptocercus punctulatus . J Eukaryot Microbiol 56(4), 305–313.CrossRefGoogle ScholarPubMed
Carpenter, K.J., Horak, A., Chow, L. & Keeling, P.J. (2011). Symbiosis, morphology, and phylogeny of Hoplonymphidae (Parabasalia) of the wood-feeding roach Cryptocercus punctulatus . J Eukaryot Microbiol 58(5), 426–436.CrossRefGoogle ScholarPubMed
Carpenter, K.J., Horak, A. & Keeling, P.J. (2010). Phylogenetic position and morphology of spirotrichosomidae (parabasalia): New evidence from Leptospironympha of Cryptocercus punctulatus . Protist 161(1), 122–132.CrossRefGoogle ScholarPubMed
Carpenter, K.J. & Keeling, P.J. (2007). Morphology and phylogenetic position of Eucomonympha imla (Parabasalia: Hypermastigida). J Eukaryot Microbiol 54(4), 325–332.CrossRefGoogle ScholarPubMed
Carpenter, K.J., Waller, R.F. & Keeling, P.J. (2008). Surface morphology of Saccinobaculus (Oxymonadida): Implications for character evolution and function in oxymonads. Protist 159(2), 209–221.CrossRefGoogle ScholarPubMed
Cleveland, L.R. (1925). The effects of oxygenation and starvation on the symbiosis between the termite Termopsis and its intestinal flagellates. Biol Bull 48, 309–325.CrossRefGoogle Scholar
Cleveland, L.R. & Grimstone, A.V. (1964). The fine structure of the flagellate Mixotricha paradoxa and its associated microorganisms. Proc R Soc 159, 668–686.Google Scholar
Cleveland, L.R., Hall, S.R., Sanders, E.P. & Collier, J. (1934). The wood-feeding roach Cryptocercus, its protozoa, and the symbiosis between protozoa and roach. Mem Am Acad Arts Sci 17, 1–342.Google Scholar
Giberson, R.T., Demaree, R.S. & Nordhausen, R.W. (1997). Four hour processing of clinical/diagnostic specimens for electron microscopy using microwave technique. J Vet Diag Invest 9, 61–67.CrossRefGoogle ScholarPubMed
Grassi, B. (1917). Flagellati viventi nei Termiti. Mem R Accad Lincei 12(5), 331–394.Google Scholar
Hollande, A. & Carruette-Valentin, J. (1971). Les atractophores, l'induction du fuseau et la division cellulaire chez les Hypermastigines Étude infrastructurale et révision systématique desTrichonymphines et des Spirotrichonymphines. Protistologica 7, 5–100.Google Scholar
Hollande, A. & Valentin, J. (1968). Infrastructure du complexe rostral et origine du fuseau chez Staurojoenina caulleryi. Comptes Rendus Hebdomadaires des Seances de l'academie de sciences Series D 266, 1283–1286.Google Scholar
Hongoh, Y., Ohkuma, M. & Kudo, T. (2003). Molecular analysis of bacterial microbiota in the gut of the termite Reticulitermes speratus (Isoptera: Rhinotermitidae). FEMS Microbiol Ecol 44(2), 231–242.CrossRefGoogle ScholarPubMed
Hongoh, Y., Sharma, V.K., Prakash, T., Noda, S., Taylor, T.D., Kudo, T., Sakaki, Y., Toyoda, A., Hattori, M. & Ohkuma, M. (2008a). Complete genome of the uncultured termite group 1 bacteria in a single host protist cell. Proc Natl Acad Sci USA 105(14), 5555–5560.CrossRefGoogle Scholar
Hongoh, Y., Sharma, V.K., Prakash, T., Noda, S., Toh, H., Taylor, T.D., Kudo, T., Sakaki, Y., Toyoda, A., Hattori, M. & Ohkuma, M. (2008b). Genome of an endosymbiont coupling N2 fixation to cellulolysis within protist cells in termite gut. Science 322(5904), 1108–1109.CrossRefGoogle ScholarPubMed
Inward, D., Beccaloni, G. & Eggleton, P. (2007). Death of an order: A comprehensive molecular phylogenetic study confirms that termites are eusocial cockroaches. Biol Lett 3(3), 331–335.CrossRefGoogle Scholar
Kirby, H. (1926). On Staurojoenina assimilis sp. nov. an intestinal flagellate from the termite Kalotermes minor Hagen. Univ Calif Publ Zool 29, 25–102.Google Scholar
Kirby, H. (1932). Flagellates of the genus Trichonympha in termites. Univ Calif Publ Zool 37, 349–476.Google Scholar
Kiuchi, I., Moriya, S. & Kudo, T. (2004). Two different size-distributions of engulfment-related vesicles among symbiotic protists of the lower termites, Reticulitermes speratus . Microb Environ 19, 211–214.CrossRefGoogle Scholar
Koidzumi, M. (1921). Studies on the intestinal protozoa found in the termites of Japan. Parasitol 13, 235–309.CrossRefGoogle Scholar
Leadbetter, J.R., Schmidt, T.M., Graber, J.R. & Breznak, J.A. (1999). Acetogenesis from H2 plus CO2 by spirochetes from termite guts. Science 283(5402), 686–689.CrossRefGoogle ScholarPubMed
Leander, B.S. & Keeling, P.J. (2004). Symbiotic innovation in the oxymonad Streblomastix strix . J Eukaryot Microbiol 51(3), 291–300.CrossRefGoogle ScholarPubMed
Lechene, C., Hillion, F., McMahon, G., Benson, D., Kleinfeld, A.M., Kampf, J.P., Distel, D., Luyten, Y., Bonventre, J., Hentschel, D., Park, K.M., Ito, S., Schwartz, M., Benichou, G. & Slodzian, G. (2006). High-resolution quantitative imaging of mammalian and bacterial cells using stable isotope mass spectrometry. J Biol 5(6), 20.CrossRefGoogle ScholarPubMed
Leidy, J. (1877). On intestinal parasites of Termes flavipes . Proc Acad Nat Sci Philadelphia 29, 146–149.Google Scholar
Lilburn, T.G., Kim, K.S., Ostrom, N.E., Byzek, K.R., Leadbetter, J.R. & Breznak, J.A. (2001). Nitrogen fixation by symbiotic and free-living spirochetes. Science 292(5526), 2495–2498.CrossRefGoogle ScholarPubMed
Lo, N., Tokuda, G., Watanabe, H., Rose, H., Slaytor, M., Maekawa, K., Bandi, C. & Noda, H. (2000). Evidence from multiple gene sequences indicates that termites evolved from wood-feeding cockroaches. Curr Biol 10(13), 801–804.CrossRefGoogle ScholarPubMed
Maaß, A. & Radek, R. (2006). The gut flagellate community of the termite Neotermes cubanus with special reference to Staurojoenina and Trichovina hrdyi nov. gen. nov. sp. Eur J Protistol 42, 125–141.CrossRefGoogle Scholar
Manefield, M., Whiteley, A.S., Griffiths, R.I. & Bailey, M.J. (2002). RNA stable isotope probing, a novel means of linking microbial community function to phylogeny. Appl Environ Microbiol 68(11), 5367–5373.CrossRefGoogle ScholarPubMed
Mayali, X., Weber, P.K., Brodie, E.L., Mabery, S., Hoeprich, P.D. & Pett-Ridge, J. (2012). High-throughput isotopic analysis of RNA microarrays to quantify microbial resource use. ISME J 6, 1210–1221.CrossRefGoogle ScholarPubMed
Miller, M. (1969). Caste differentiation in lower termites. In Biology of Termites, Krishna, K. and Weesner, F. (Eds.), pp. 283–307. New York: Academic Press.CrossRefGoogle Scholar
Murrell, J.C. & Whiteley, A.S. (Eds.) (2011). Stable Isotope Probing and Related Technologies. Washington, DC: ASM Press.Google Scholar
Musat, N., Halm, H., Winterholler, B., Hoppe, P., Peduzzi, S., Hillion, F., Horreard, F., Amann, R., Jorgensen, B.B. & Kuypers, M.M.M. (2008). A single-cell view on the ecophysiology of anaerobic phototrophic bacteria. Proc Natl Acad Sci USA 105(46), 17861–17866.CrossRefGoogle ScholarPubMed
Nakashima, K.I., Watanabe, H. & Azuma, J.I. (2002). Cellulase genes from the parabasalian symbiont Pseudotrichonympha grassii in the hindgut of the wood-feeding termite Coptotermes formosanus . Cell Mol Life Sci 59(9), 1554–1560.CrossRefGoogle ScholarPubMed
Noda, S., Iida, T., Kitade, O., Nakajima, H., Kudo, T. & Ohkuma, M. (2005). Endosymbiotic Bacteroidales bacteria of the flagellated protist Pseudotrichonympha grassii in the gut of the termite Coptotermes formosanus . Appl Environ Microbiol 71(12), 8811–8817.CrossRefGoogle ScholarPubMed
Noda, S., Inoue, T., Hongoh, Y., Kawai, M., Nalepa, C.A., Vongkaluang, C., Kudo, T. & Ohkuma, M. (2006). Identification and characterization of ectosymbionts of distinct lineages in Bacteroidales attached to flagellated protists in the gut of termites and a wood-feeding cockroach. Environ Microbiol 8(1), 11–20.CrossRefGoogle Scholar
Ohkuma, M. (2003). Termite symbiotic systems: Efficient bio-recycling of lignocellulose. Appl Microbiol Biotechnol 61(1), 1–9.CrossRefGoogle ScholarPubMed
Ohkuma, M. & Brune, A. (2011). Diversity, structure, and evolution of the termite gut microbial community. In Biology of Termites: A Modern Synthesis, Bignell, D.E., Roisin, Y. & Lo, N. (Eds.), pp. 413–438. London: Springer.Google Scholar
Ohkuma, M. & Kudo, T. (1996). Phylogenetic diversity of the intestinal bacterial community in the termite Reticulitermes speratus . Appl Environ Microbiol 62(2), 461–468.CrossRefGoogle ScholarPubMed
Ohkuma, M., Sato, T., Noda, S., Ui, S., Kudo, T. & Hongoh, Y. (2007). The candidate phylum “Termite Group 1” of bacteria: Phylogenetic diversity, distribution, and endosymbiont members of various gut flagellated protists. FEMS Microbiol Ecol 60(3), 467–476.CrossRefGoogle ScholarPubMed
Pett-Ridge, J. & Weber, P. (2012). NanoSIP: NanoSIMS applications for microbial biology. In Microbial Systems Biology: Methods and Protocols, Navid, A. (Ed.), pp. 375–408. New York: Humana Press.CrossRefGoogle Scholar
Popa, R., Weber, P.K., Pett-Ridge, J., Finzi, J.A., Fallon, S.J., Hutcheon, I.D., Nealson, K.H. & Capone, D.G. (2007). Carbon and nitrogen fixation and metabolite exchange in and between individual cells of Anabaena oscillarioides . ISME J 1(4), 354–360.CrossRefGoogle ScholarPubMed
Radajewski, S., Ineson, P., Parekh, N.R. & Murrell, J.C. (2000). Stable-isotope probing as a tool in microbial ecology. Nature 403(6770), 646–649.CrossRefGoogle ScholarPubMed
Radek, R., Hausmann, K. & Breunig, A. (1992). Ectobiotic and endocytobiotic bacteria associated with the termite flagellate Joenia-Annectens. Acta Protozoologica 31(2), 93–107.Google Scholar
Rother, A., Radek, R. & Hausmann, K. (1999). Characterizaion of surface structures covering termite flagellates of the family oxymonadidae and ultrastructure of two oxymonad species, Microrhopalodina multinucleata and Oxymonas sp. Eur J Protistol 35, 1–16.CrossRefGoogle Scholar
Stingl, U., Maaß, A., Radek, R. & Brune, A. (2004). Symbionts of the gut flagellate Staurojoenina sp. from Neotermes cubanus represent a novel, termite-associated lineage of Bacteroidales: Description of “Candidatus Vestibaculum illigatum”. Microbiol 150(Pt 7), 2229–2235.CrossRefGoogle ScholarPubMed
Trager, W. (1934). The cultivation of a cellulose-digesting flagellate, Trichomonas termopsidis, and of certain other termite protozoa. Biol Bull 66, 182–190.CrossRefGoogle Scholar
Weber, P.K., Graham, G.A., Teslich, N.E., Chan, W.M., Ghosal, S., Leighton, T.J. & Wheeler, K.E. (2010). NanoSIMS imaging of Bacillus spores sectioned by focused ion beam. J Microsc 238(3), 189–199.CrossRefGoogle ScholarPubMed
Woebken, D., Burow, L.C., Prufert-Bebout, L., Bebout, B.M., Hoehler, T.M., Pett-Ridge, J., Spormann, A.M., Weber, P.K. & Singer, S.W. (2012). Identification of a novel cyanobacterial group as active diazotrophs in a coastal microbial mat using NanoSIMS analysis. ISME J 6, 1427–1439.CrossRefGoogle Scholar
Yamaoka, I. (1979). Selective ingestion of food by the termite protozoa, Trichonympha agilis . Zoolog Mag (Tokyo) 88, 174–179.Google Scholar
Yamin, M.A. (1979). Flagellates of the orders Trichomonadida Kirby, Oxymonadida Grasse, and Hypermastigida Grassi and Foa reported from lower termites (Isoptera families Mastotermitidae, Kalotermitidae, Hodotermitidae, Termopsidae, Rhinotermitidae, and Serritermitidae) and from the wood-feeding roach cryptocercus (Dictyoptera, Cryptocercidae). Sociobiology 4(1), 3–119.Google Scholar
Yamin, M.A. (1981). Cellulose metabolism by the flagellate trichonympha from a termite is independent of endosymbiotic bacteria. Science 211(4477), 58–59.CrossRefGoogle ScholarPubMed
Yoshimura, T. (1995). Contribution of the protozoan fauna to nutritional physiology of the lower termite Coptotermes formosanus Shiraki (Isoptera: Rhinotermitidae). Wood Res 82, 68–129.Google Scholar
Yoshimura, T., Fujino, T., Itoh, T., Tsunodo, K. & Takahashi, M. (1996). Ingestion and decomposition of wood and cellulose by the protozoa in the hindgut of Coptotermes formosanus Shiraki (Isoptera: Rhinotermitidae) as evidenced by polarizing and transmission electron microscopy. Holzforschung 50, 99–104.CrossRefGoogle Scholar