Hostname: page-component-78c5997874-xbtfd Total loading time: 0 Render date: 2024-11-19T12:37:59.019Z Has data issue: false hasContentIssue false
  • English
  • Français

Application of denaturing gradient gel electrophoresis for analysing the gut microflora of Lumbricus rubellus Hoffmeister under different feeding conditions

Published online by Cambridge University Press:  28 April 2008

B.A. Knapp ,
J. Seeber ,
S.M. Podmirseg ,
E. Meyer  and
H. Insam
B.A. Knapp*
Affiliation:
University of Innsbruck, Institute of Microbiology, Technikerstrasse 25, 6020 Innsbruck, Austria
J. Seeber
Affiliation:
University of Innsbruck, Institute of Ecology, Technikerstrasse 25, 6020 Innsbruck, Austria
S.M. Podmirseg
Affiliation:
University of Innsbruck, Institute of Microbiology, Technikerstrasse 25, 6020 Innsbruck, Austria
E. Meyer
Affiliation:
University of Innsbruck, Institute of Ecology, Technikerstrasse 25, 6020 Innsbruck, Austria
H. Insam
Affiliation:
University of Innsbruck, Institute of Microbiology, Technikerstrasse 25, 6020 Innsbruck, Austria
*
*Author for correspondence Fax: 00435125072928 E-mail: B.Knapp@uibk.ac.at
Get access Rights & Permissions [Opens in a new window]

Abstract

The earthworm, Lumbricus rubellus, plays an essential role in soil ecosystems as it affects organic matter decomposition and nutrient cycling. By ingesting a mixture of organic and mineral material, a variety of bacteria and fungi are carried to the intestinal tract of the earthworm. To get a better understanding of the interactions between L. rubellus and the microorganisms ingested, this study tried to reveal if the diet affects the composition of the gut microflora of L. rubellus or if its intestinal tract hosts an indigenous, species-specific microbiota. A feeding experiment with L. rubellus was set up; individuals were collected in the field, transferred to a climate chamber and fed with food sources of different quality (dwarf shrub litter, grass litter or horse dung) for six weeks. DNA was extracted from the guts of the earthworms, as well as from the food sources and the surrounding soil, and further analysed by a molecular fingerprinting method, PCR-DGGE (Polymerase Chain Reaction – Denaturing Gradient Gel Electrophoresis). We were able to demonstrate that the gut microbiota was strongly influenced by the food source ingested and was considerably different to that of the surrounding soil. Sequencing of dominant bands of the bacterial DGGE fingerprints revealed a strong occurrence of y-Proteobacteria in all gut samples, independent of the food source. A specific microflora in the intestinal tract of L. rubellus, robust against diet changes, could not be found.

Keywords

Lumbricus rubellus Hoffmeisterpolymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysisgut microbiotamicrobial community compositionband identification
Type
Research Paper
Information
Bulletin of Entomological Research , Volume 98 , Issue 3 , June 2008 , pp. 271 - 279
Copyright
Copyright © 2008 Cambridge University Press

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

Bardgett, R.D., Wardle, D.A. & Yea, G.W. (1998) Linking above-ground and below-ground interactions: How plant responses to foliar herbivory influences soil organisms. Soil Biology Biochemistry 30, 1867–1878.CrossRefGoogle Scholar
Brown, G.G. & Doube, B.M. (2004) Functional interactions between earthworms, microorganisms, organic matter, and plants. pp. 213–239in Edwards, C.A. (Ed.) Earthworm Ecology. Boca Raton, FL, USA, CRC Press LLC.Google Scholar
Brown, G.G., Barois, I. & Lavelle, P. (2000) Regulation of soil organic matter dynamics and microbial activity in the drilosphere and the role of interactions with other edaphic functional domains. European Journal of Soil Biology 36, 177–198.CrossRefGoogle Scholar
Cai, H., Zarda, B., Mattison, G.R., Schönholzer, F. & Hahn, D. (2002) Fate of protozoa transiting the digestive tract of the earthworm Lumbricus terrestris L. Pedobiologia 46, 161–175.CrossRefGoogle Scholar
Chapman, S.K., Langley, J.A., Hart, S.C. & Koch, G.W. (2006) Plants actively control nitrogen cycling: uncorking the microbial bottleneck. New Phytologist 169, 27–34.CrossRefGoogle ScholarPubMed
Curry, J.P. & Schmidt, O. (2007) The feeding ecology of earthworms – A review. Pedobiologia 50, 463–477.CrossRefGoogle Scholar
Drake, H.L., Schramm, A. & Horn, M.A. (2006) Earthworm gut microbial biomes: Their importance to soil microorganisms, denitrification, and the terrestrial production of the greenhouse gas N2O. pp. 65–87in König, H. & Varma, A. (Eds) Intestinal Microorganisms of Termites and Other Invertebrates. Berlin, Springer-Verlag.CrossRefGoogle Scholar
Egert, M., Marhan, S., Wagner, B., Scheu, S. & Friedrich, M.W. (2004) Molecular profiling of 16S rRNA genes reveals diet-related differences of microbial communities in soil, gut, and casts of Lumbricus terrestris L. (Oligochaeta: Lumbricidae). FEMS Microbiology Ecology 48, 187–197.CrossRefGoogle ScholarPubMed
Furlong, M.A., Singleton, D.R., Coleman, D.C. & Whitman, W.B. (2002) Molecular and culture-based analyses of prokaryotic communities from an agricultural soil and the burrows and casts of the earthworm Lumbricus rubellus. Applied and Environmental Microbiology 68, 1265–1279.CrossRefGoogle ScholarPubMed
Gonzalez, J.M., Ortiz-Martinez, A., Gonzalez-Delvalle, M.A., Laiz, L. & Saiz-Jimenez, C. (2003) An efficient strategy for screening large cloned libraries of amplified 16S rDNA sequences from complex environmental communities. Journal of Microbiological Methods 55, 459–463.CrossRefGoogle ScholarPubMed
Görres, J.H., Savin, M.C. & Amador, J.A. (2001) Soil micropore structure and carbon mineralization in burrows and casts of an anecic earthworm (Lumbricus terrestris). Soil Biology and Biochemistry 33, 1881–1887.CrossRefGoogle Scholar
Grayston, S.J., Campbell, C.D., Bardgett, R.D., Mwadsley, J.L., Clegg, C.D., Ritz, K., Griffiths, B.S., Rodwell, J.S., Edwards, S.J., Davies, W.J., Elston, D.J. & Millard, P. (2004) Assessing shifts in microbial community structure across a range of grasslands of differing management intensity using CLPP; PFLA and community DNA techniques. Applied Soil Ecology 25, 63–84.CrossRefGoogle Scholar
Heuer, H., Krsek, M., Baker, P., Smalla, K. & Wellington, E.M.H. (1997) Analysis of actinomycete communities by specific amplification of genes encoding 16S rRNA and gel-electrophoretic separation in denaturing gradients. Applied and Environmental Microbiology 63, 3233–3241.CrossRefGoogle ScholarPubMed
Jolly, J.M., Lappinscott, H.M., Anderson, J.M. & Clegg, C.D. (1993) Scanning electron-microscopy of the gut microflora of two earthworms – Lumbricus terrestris and Octolasion cyaneum. Microbial Ecology 26, 235–245.CrossRefGoogle ScholarPubMed
Juen, A. & Traugott, M. (2005) Detecting predation and scavenging by DNA gut-content analysis: a case study using a soil insect predator-prey system. Oecologia 142, 344–352.CrossRefGoogle ScholarPubMed
Karsten, G.R. & Drake, H.L. (1995) Comparative assessment of the aerobic and anaerobic microfloras of earthworm guts and forest soils. Applied and Environmental Microbiology 61, 1039–1044.CrossRefGoogle ScholarPubMed
Karsten, G.R. & Drake, H.L. (1997) Denitrifying bacteria in the earthworm gastrointestinal tract and in vivo emission of nitrous oxide (N2O) by earthworms. Applied and Environmental Microbiology 63, 1878–1882.CrossRefGoogle Scholar
Kisand, V., Cuadros, R. & Wikner, J. (2002) Phylogeny of culturable estuarine bacteria catabolizing riverine organic matter in the northern Baltic sea. Applied and Environmental Microbiology 68, 379–388.CrossRefGoogle ScholarPubMed
Kowalchuk, G.A. & Stephen, J.R. (2001) Ammonia-oxidizing bacteria: A model for molecular Microbial Ecology. Annual Review of Microbiology 55, 485–529.CrossRefGoogle Scholar
Kowalchuk, G.A., Stephen, J.R., de Boer, W., Prosser, J.I., Embley, T.M. & Woldendorp, J.W. (1997) Analysis of ammonia-oxidizing bacteria of the ß subdivision of the class Proteobacteria in coastal sand dunes by denaturing gradient gel electrophoresis and sequencing of PCR-amplified 16S ribosomal DNA fragments. Applied and Environmental Microbiology 63, 1489–1497.CrossRefGoogle Scholar
Krištůfek, V., Ravasz, K. & Pižl, V. (1993) Actinomycete communities in earthworm guts and surrounding soil. Pedobiologia 37, 379–384.CrossRefGoogle Scholar
Liang, Z., Drijber, R.A., Lee, D.J., Dwiekat, I.M., Harris, S.D. & Wedin, D.A. (2008) A DGGE-cloning method to characterize arbuscular mycorrhizal community structure in soil. Soil Biology and Biochemistry 40, 956–966.CrossRefGoogle Scholar
Liu, W.-T., Marsh, T.L., Cheng, H. & Forney, L.J. (1997) Characterisation of microbial diversity by determining terminal restriction length polymorphisms of genes encoding 16S rDNA. Applied and Environmental Microbiology 63, 4516–4522.CrossRefGoogle Scholar
Lowe, C.N. & Butt, K.R. (2005) Culture techniques for soil dwelling earthworms: A review. Pedobiologia 49, 401–413.CrossRefGoogle Scholar
Marchesi, J.R., Sato, T., Weightman, A.J., Martin, T.A., Fry, J.C., Hiom, S.J. & Wade, W.G. (1998) Design and evaluation of useful bacterium-specific PCR primers that amplify genes coding for bacterial 16S rRNA. Applied and Environmental Microbiology 64, 795–799.CrossRefGoogle ScholarPubMed
Marhan, S. & Scheu, S. (2005) The influence of mineral and organic fertilisers on the growth of the endogeic earthworm Octolasion tyrtaeum (Savigny). Pedobiologia 49, 239–249.CrossRefGoogle Scholar
Mendez, R., Borges, S. & Betancourt, C. (2003) A microscopical view of the intestine of Onychochaeta borincana (Oligochaeta: Glossoscolecidae). Pedobiologia 47, 900–903.Google Scholar
Monciardini, P., Sossio, M., Cavaletti, L., Chiocchini, C. & Donadio, S. (2002) New PCR primers for the selective amplification of 16S rDNA from different groups of actinomycetes. FEMS Microbiology Ecology 42, 419–429.Google ScholarPubMed
Muyzer, G. & Smalla, K. (1998) Application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology. Antonie van Leeuwenhoek 73, 127–141.CrossRefGoogle Scholar
Muyzer, G., de Waal, E.C. & Uitterlinden, A.G. (1993) Profiling of complex microbial populations by denaturing gradient gel electrophoresis analyses of polymerase chain reaction-amplified genes for 16S rRNA. Applied and Environmental Microbiology 59, 695–700.CrossRefGoogle Scholar
Pedersen, J.C. & Hendriksen, N.B. (1993) Effect of passage through the intestinal-tract of detritivore earthworms (Lumbricus spp.) on the number of selected Gram-negative and total bacteria. Biology and Fertility of Soils 16, 227–232.CrossRefGoogle Scholar
Phillips, C.J., Harris, D., Dollhopf, S.L., Gross, K.L., Prosser, K.L., Prosser, J.I. & Paul, E.A. (2000) Effects of agronomic treatments on structure and function of ammonia-oxidizing communities. Applied and Environmental Microbiology 66, 5410–5418.CrossRefGoogle ScholarPubMed
Reeson, A.F., Jankovic, T., Kasper, M.L., Rogers, S. & Austin, A.D. (2003) Application of 16S rDNA-DGGE to examine the microbial ecology associated with a social wasp Vespula germanica. Insect Molecular Biology 12, 85–91.CrossRefGoogle ScholarPubMed
Sampedro, L. & Whalen, J.K. (2007) Changes in the fatty acid profiles through the digestive tract of the earthworm Lumbricus terrestris L. Applied Soil Ecology 35, 226–236.CrossRefGoogle Scholar
Sanguinetti, C.J., Net, E.D. & Simpson, A.J.G. (1994) Rapid silver staining and recovery for PCR products separated on polyacrylamide gels. BioTechniques 17, 915–919.Google ScholarPubMed
Schabereiter-Gurtner, C., Pinar, G., Lubitz, W. & Rolleke, S. (2001) An advanced molecular strategy to identify bacterial communities on art objects. Journal of Microbiological Methods 45, 77–87.CrossRefGoogle ScholarPubMed
Schönholzer, F., Hahn, D., Zarda, B. & Zeyer, J. (2002) Automated image analysis and in situ hybridization as tools to study bacterial populations in food resources, gut and cast of Lumbricus terrestris L. Journal of Microbiological Methods 48, 53–68.CrossRefGoogle ScholarPubMed
Schwieger, F. & Tebbe, C.C. (1998) A new approach to utilize PCR-single-strand conformation polymorphism for 16S rRNA gene-based microbial community analysis. Applied and Environmental Microbiology 64, 4870–4876.CrossRefGoogle ScholarPubMed
Simpson, J.M., McCracken, V.J., White, B.A., Gaskins, H.R. & Mackie, R.I. (1999) Application of denaturant gradient gel electrophoresis for the analysis of the porcine gastrointestinal microbiota. Journal of Microbiological Methods 36, 167–179.CrossRefGoogle ScholarPubMed
Singleton, D.R., Hendrix, P.F., Coleman, D.C. & Whitman, W.B. (2003) Identification of uncultured bacteria tightly associated with the intestine of the earthworm Lumbricus rubellus (Lumbricidae, Oligochaeta). Soil Biology and Biochemistry 35, 1547–1555.CrossRefGoogle Scholar
Suzuki, M. & Giovannoni, S. (1996) Bias caused by template annealing in the amplification of mixtures of 16S rRNA genes by PCR. Applied and Environmental Microbiology 62, 625–630.CrossRefGoogle ScholarPubMed
Tanaka, H., Aoyagi, H., Shina, S., Dodo, Y., Yoshimura, T., Nakamura, R. & Uchiyama, H. (2006) Influence of the diet components on the symbiotic microorganisms community in hindgut of Coptotermes formosanus Shiraki. Applied Microbiology and Biotechnology 71, 907–917.CrossRefGoogle ScholarPubMed
V. Wintzingerode, F., Göbel, U.B. & Stackebrandt, E. (1997) Determination of microbial diversity in environmental samples: pitfalls of PCR-based rRNA analysis. FEMS Microbiology Reviews 21, 213–229.CrossRefGoogle Scholar
Vainio, E.J. & Hantula, J. (2000) Direct analysis of wood-inhabiting fungi using denaturing gradient gel electrophoresis of amplified ribosomal DNA. Mycological Research 104, 927–936.CrossRefGoogle Scholar
Vanhoutte, T., Huys, G., De Brandt, E. & Swings, J. (2004) Temporal stability analysis of the microbiota in human faeces by denaturing gradient gel electrophoresis using universal and group-specific 16S rRNA gene primers. FEMS Microbiology Ecology 48, 437–446.CrossRefGoogle ScholarPubMed
Ward, J.H. (1963) Hierarchical grouping to optimize an objective function. Journal of the American Statistical Association 58, 236–244.CrossRefGoogle Scholar
Williamson, N., Brian, P. & Wellington, E.M.H. (2000) Molecular detection of bacterial and streptomycete chitinases in the environment. Antonie van Leeuwenhoek 78, 315–321.CrossRefGoogle ScholarPubMed