Skip to main content
SpringerLink
Log in

Application of ARDRA and PLFA analysis in characterizing the bacterial communities of the food, gut and excrement of saprophagous larvae ofPenthetria holosericea (Diptera: Bibionidae): a pilot study

  • Published:
Folia Microbiologica Aims and scope Submit manuscript

Abstract

Amplified ribosomal DNA restriction analysis (ARDRA) was used to compare the bacterial communities of the food, the gut sections (ceca, anterior and posterior midgut, hindgut) and the excrement of the litter feeding bibionid larvae ofPenthetria holosericea. For universal eubacterial primers ARDRA patterns were complex with only minor differences among samples. Taxon specific primers were also applied to characterize the samples. Fragment composition was transformed to presence/absence binary data and further analyzed. Cluster analysis revealed that bacterial communities of gut highly resembled each other with the exception of the ceca. ARDRA patterns of consumed leaves clustered together with the intact leaves but differed from those of the excrement. ARDRA results were compared with microbial community structure based on phospholipid fatty acid (PLFA) fingerprints. The cluster analysis of PLFA (presence/absence binary) data resulted in a pattern similar to the ARDRA data. The PCA analysis of PLFA relative content separated microbial communities into five groups: (1) anterior and posterior midgut, (2) hindgut, (3) ceca, (4) consumed and intact litter, (5) excrement. Both methods indicated that conditions in the larval gut result in formation of a specific microbial community which differs from both the food and excrement ones. Particularly ceca — (blind appendages, harbor very specific microbial community) are divided from the rest of the gut by perithropic membrane.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

ARDRA:

amplified ribosomal DNA restriction analysis

CFB:

Cytophaga-Flexibacter-Bacteroides

CLSU:

community level substrate utilization

DAPI:

4′,6-diamidino-2-phenylindole

DGGE:

denaturing gradient gel electrophoresis

DHA:

docosahexanoic acid

MUFA:

monounsaturated fatty acids

PCA:

principal component analysis

PLFA:

phospholipid fatty acid

PUFA:

oligounsaturated (‘polyunsaturated’) fatty acids

SDS:

sodium dodecyl sulfate

SRB:

sulfate-reducing bacteria

References

  • Amann R., Stromley J., Devereux R., Key R., Stahl D.A.: Molecular and microscopic identification of sulfate-reducing bacteria in multi-species biofilm.Appl.Environ.Microbiol. 58, 614–623 (1992).

    PubMed  CAS  Google Scholar 

  • Anderson J.M.: Spatiotemporal effects of invertebrates on soil processes.Biol.Fertil.Soils 6, 216–227 (1988).

    Article  CAS  Google Scholar 

  • Andrews J.E., Ohirogge J.: Fatty acid and lipid biosynthesis and degradation, pp. 339–352 in D.T. Dennis, D.H. Turpin (Eds):Plant Physiology. Biochemistry and Molecular Biology. John Wiley & Sons, New York 1990.

    Google Scholar 

  • Berchtold M., Chatzionas A., Schónhuber W., Brune A., Amann R., Hahn D., König H.: Differential enumeration andin situ localization of microorganisms in the hindgut of the lower termiteMastotermes darwiniensis by hybridization with rRNA-targed probes.Arch.Microbiol. 172, 407–416 (1999).

    Article  PubMed  CAS  Google Scholar 

  • Bligh E.G., Dryer W.J.: A rapid method of total lipid extraction and purification.Can.J.Biochem.Physiol. 37, 911–917 (1959).

    PubMed  CAS  Google Scholar 

  • Brennan P.J.:Mycobacterium and other actinomycetes, pp. 204–298 in C. Ratledge, S.G. Wilkinson (Eds):Microbial Lipids. Academic Press, London 1988.

    Google Scholar 

  • Dadd R.H.: Essential fatty acids for mosquitoes, other insects and vertebrates, pp. 189–214 in G. Bhaskaran, S. Friedman, J.G. Rodriguez, (Eds):Current Topics in Insect Endocrinology and Nutrition. Plenum Press, New York 1981.

    Google Scholar 

  • Dojka M.A., Harris J.K., Pace N.R.: Expanding the known diversity and environmental distribution of an uncultured phylogenetic division of bacteria.Appl.Environ.Microbiol. 66, 1617–1621 (2000).

    Article  PubMed  CAS  Google Scholar 

  • Dowling N.J.E., Widdel F., White D.C.: Phospholipid ester-linked fatty acid biomarkers of acetate-oxidizing sulphate-reducers and other sulphide-forming bacteria.J.Gen.Microbiol. 132, 1815–1825 (1986).

    CAS  Google Scholar 

  • Dunger W.:Tiere im Boden. A. Ziemsen-Verlag, Wittemberg (Germany) 1983.

    Google Scholar 

  • Ensign J.C.: Introduction to the actinomycetes, pp. 811–815 in A. Balows, H.G. Trüper, M. Dworkin, W. Harder, K.-H. Schleifer (Eds):The Prokaryotes. Vol. 41. Springer-Verlag, New York 1992.

    Google Scholar 

  • Erwin J.A.: Fatty acids in eukaryotic microorganisms, pp. 41–143 in J.A. Erwin (Ed.):Lipids and Biomembranes of Eukaryotic Microorganisms. Academic Press, New York 1973.

    Google Scholar 

  • Federle T.W.: Microbial distribution in soil — new techniques, pp. 493–498 in F. Megusar, M. Gantar (Eds):Perspectives in Microbial Ecology. Slovenian Society for Microbiology, Ljubljana 1986.

    Google Scholar 

  • Findlay R.H.: The use of phospholipid fatty acids to determine microbial community structure, pp. 1–17 in A.D.L. Akkermanns, J.D. van Elsas, F. de Bruijn (Eds):Molecular Microbial Ecology Manual. Kluwer Academic Press, Dordrecht (The Netherlands) 1996.

    Google Scholar 

  • Frostegård Å., Tunlid A., Bååth E.: Phospholipid fatty acid composition, biomass, and activity of microbial communities from two soil types experimentally exposed to different heavy metals.Appl.Environ.Microbiol. 59, 3605–3617 (1993).

    PubMed  Google Scholar 

  • Frouz J., Šantrůčková H., Elhottová D.: The effect of bibionid larvae feeding on the microbial community of litter and on reconsumed excrement.Pedobiologia 43, 221–230 (1999).

    Google Scholar 

  • Frouz J., Elhottova D., Šustr V., Krištůfek V., Hubert J.: Preliminary data about compartmentation of the gut of saprophagous bibionid larvaePenthetria holosericea.Eur.J.Soil Biol. 38, 53–57 (2002).

    Article  Google Scholar 

  • Frouz J., Krištůfek V., Li X., Šantrůčková H., Šustr V., Brune A.: Changes in amount of bacteria during gut passage of leaf litter and during coprophagy in three species ofBibionidae (Diptera) larvae.Folia Microbiol. 48, 535–542 (2003).

    Article  CAS  Google Scholar 

  • Holmes B.: The generaFlavobacterium, Sphingobacterium andWeeksella, pp. 3620–3630 in A. Balows, H.G. Trüper, M. Dworkin, W. Harder, K.-H. Schleifer (Eds):The Prokaryotes, Vol. 4. Springer-Verlag, New York 1992.

    Google Scholar 

  • Holt J.G., Krieg N.R., Sneath P.H., Staley J.T., Williams S.T. (Eds):Bergey’s Manual of Determinative Bacteriology, 9th ed. Williams & Wilkins, Baltimore 1994.

    Google Scholar 

  • Kaneda T.: Fatty acids in the genusBacillus. — 1.iso- andanteiso-fatty acids as characteristic constituents of lipids in 10 species.J.Bacteriol. 93, 894–903 (1967).

    PubMed  CAS  Google Scholar 

  • Kaneda T.:Iso- andanteiso-fatty acids in bacteria: biosynthesis, function, and taxonomic significance.Microbiol.Rev. 55, 288–302 (1991).

    PubMed  CAS  Google Scholar 

  • Karpachevsky L.O., Perel T.S., Bartsevich V.V.: The role ofBibionidae larvae in decomposition of forest litter.Pedobiologia 8, 146–149 (1968).

    Google Scholar 

  • Kroppenstedt R.M.: Fatty acid and menaquinone analysis of actinomycetes and related organisms, pp. 173–199 in M. Goodfellow, D.E. Minnikin (Eds):Chemical Methods in Bacterial Systematics. Academic Press, London 1985.

    Google Scholar 

  • Laczko E., Rudaz A., Aragno M.: Diversity of anthropogenically influenced or disturbed soil microbial communities, pp. 57–67 in H. Insam, A. Rangger (Eds):Microbial Communities Functional versusStructural Approaches. Springer-Verlag, Berlin 1997.

    Google Scholar 

  • Lane D.J.: 16S/23S rRNA sequencing, pp. 115–175 in E. Stackebrandt, M. Goodfellow (Eds):Nucleic Acid Techniques in Bacterial Systematics. John Wiley & Sons, New York 1991.

    Google Scholar 

  • Lösel D.M., Sancholle M.: Fungal lipids, pp. 28–62 in R. Prasad, M.A. Ghannoum M. (Eds):Lipids of Pathogenic Fungi. CRC Press, boca Raton (USA) 1996.

    Google Scholar 

  • Marchesi J.R., Sato T., Weightman A.J., Martin T.A., Fry J.C., Hiom S.J., Dymock D., Wade W.G.: Design and evaluation of useful bacterium-specific PCR primers that amplify genes coding for bacterial 16S rRNA.Appl.Environ.Microbiol. 64, 795–799 (1998).

    PubMed  CAS  Google Scholar 

  • Martin-Laurent F., Philippot L., Hallet S., Chaussod R., Germon J.C., Soulas G., Catroux G.: DNA extraction from soils: old bias for new microbial diversity analysis methods.Appl.Environ.Microbiol. 67, 2354–2359 (2001).

    Article  PubMed  CAS  Google Scholar 

  • Massol-Deya A.A., Odelson D.A., Hickey R.F., Tiedje J.M.: Bacterial community fingerprinting of amplified 16S and 16–23S ribosomal DNA gene sequences and restriction endonuclease analysis (ARDRA), pp. 1–8 in A.D.L. Akkermanns, J.D. van Elsas, F. de Bruijn (Eds):Molecular Microbial Ecology Manual. Kluwer Academic Press, Dordrecht (The Netherlands) 1995.

    Google Scholar 

  • Minnikin D.E., Goodfellow M.: Lipid composition in the classification and identification of nocardiae and related taxa, pp. 160–219 in M. Goodfellow, G.H. Brownell, J.A. Serrano (Eds):The Biology of the Nocardiae. Academic Press, London 1976.

    Google Scholar 

  • Moss C.W., Samuels S.B., Liddle J., McKinney R.M.: Occurrence of branched-chain hydroxy fatty acids inPseudomonas maltophilia.J.Bacteriol. 114, 1018–1024 (1973).

    PubMed  CAS  Google Scholar 

  • O’Leary W.M., Wilkinson S.G.: Gram-positive bacteria, pp. 117–201 in C. Ratlege, S.G. Wilkinson (Eds):Microbial Lipids. Academic Press, London 1988.

    Google Scholar 

  • Oravecz O., Krištůfek V.: Amplified ribosomal DNA restriction analysis: experimental approach and procedures, pp. 5–7 in O. Ďugová (Ed.):Life in Soil. Czechoslovak Society for Microbiology-Institute of Landscape Ecology-Slovak Academy of Sciences, Bratislava (Slovakia) 2002.

    Google Scholar 

  • Oyaizu H., Komagata K.: Chemotaxonomic and phenotypic characterisation of the strains of species in theFlavobacterium-Cytophaga complex.J.Gen.Appl.Microbiol. 27, 57–107 (1981).

    Article  CAS  Google Scholar 

  • Panikov N.S., Gorbenko A.Y., Zvyagintsev D.G.: Quantitative estimation of the effect of mesofauna on the rate of litterfall decomposition.Moscow Univ.Soil Sci.Bull. 3, 37–45 (1985).

    Google Scholar 

  • Pobozsny M.: On the feeding biology of larval St.Mark’s flyBibio marci L. (Diptera: Bibionidae).Acta Zool.Acad.Sci.Hung. 28, 355–360 (1982).

    Google Scholar 

  • Podani J.: Multivariate data analysis in ecology and systematics. A methodological guide to the SYN-TAX 5.0 package. SPB Publishing, The Hague (The Netherlands) 1994.

    Google Scholar 

  • Ramsing N.B., Fossing H., Ferdelman T.G., Andersen F., Thamdrup B.: Distribution of bacterial populations in a stratified fjord (Mariager Fjord, Denmark) quantified byin situ hybridization and related to chemical gradients in the water column.Appl. Environ.Microbiol. 62, 1391–1404 (2001).

    Google Scholar 

  • Reichenbach H.: The orderCytophagales, pp. 3631–3675 in A. Balows, H.G. Trüper, M. Dworkin, W. Harder, K.-H. Schleifer (Eds):The Prokaryotes, Vol. 4. Springer-Verlag, New York 1992.

    Google Scholar 

  • Russell N.J., Nichols D.S.: Polyunsaturated fatty acids in marine bacteria — a dogma rewritten.Microbiology 145, 767–779 (1999).

    Article  PubMed  CAS  Google Scholar 

  • Smit E., Leeflang P., Gommans S., Van Den Broek J., Van Mil S., Wernars K.: Diversity and seasonal fluctuations of the dominant members of the bacterial soil community in a wheat field as determined by cultivation and molecular methods.Appl. Environ.Microbiol. 67, 2284–2291 (2001).

    Article  PubMed  CAS  Google Scholar 

  • Staley J.T., Konopka A.: Measurement ofin situ activities of nonphotosyntetic microorganism in aquatic and terrestrial habitats.Ann.Rev.Microbiol. 39, 321–346 (1985).

    Article  CAS  Google Scholar 

  • Suzuki K., Goodfellow M., O’Donnell A.G.: Cell envelopes and classification, pp. 195–250 in M. Goodfellow, A.G. O’Donnell (Eds):Handbook of New Bacterial Systematics. Academic Press, San Diego 1993.

    Google Scholar 

  • Szabó I.M., Marton M., Buti I.: Intestinal microflora of the larvae of St.Mark’s fly. — IV. Studies on the intestinal bacterial flora of a larva-population.Acta Microbiol.Acad.Sci.Hung. 16, 381–397 (1969).

    Google Scholar 

  • Ter Braak C.J.F., Šmilauer P.:Canoco. Centre for Biometry, Wageningen (The Netherlands) 1998.

    Google Scholar 

  • Terra W.R.: Evolution of digestive systems of insects.Ann.Rev.Entomol. 35, 181–200 (1990).

    Article  Google Scholar 

  • Thimm T., Hoffmann A., Borkott H., Munch J.C., Tebbe C.: The gut of the soil microarthropodFolsomia candida (Collembola) is a frequently changeable but selective habitat and a vector for microorganisms.Appl.Environ.Microbiol. 67, 2660–2669 (1998).

    Google Scholar 

  • Vestal J.R., White D.C.: Lipid analysis in microbial ecology.Bioscience 39, 535–541 (1989).

    Article  PubMed  CAS  Google Scholar 

  • Voet D., Voet J.G.:Biochemistry. Lipids and Membranes, pp. 1360. John Wiley & Sons, New York 1995.

    Google Scholar 

  • Widmer F., Fliessbach A., Laczkoó E., Schulze-Aurich J., Zeyer J.: Assessing soil biological characteristics: a comparison of bulk soil community DNA-, PLFA-, and Biolog™-analyses.Soil Biol.Biochem. 33, 1029–1036 (2001).

    Article  CAS  Google Scholar 

  • Wirsen C.O., Jannasch H.W., Wakeham S.G., Canuel E.A.: Membrane lipids of a psychrophilic and barophilic deep-sea bacterium.Curr.Microbiol. 14, 319–322 (1987).

    Article  CAS  Google Scholar 

  • Yabuuchi E., Moss C.W.: Cellular fatty acid composition of strains of three species ofSphingobacterium gen.nov. andCytophaga johnsonae.FEMS Microbiol.Lett. 13, 87–91 (1982).

    Article  CAS  Google Scholar 

  • Yamanaka S., Fudo R., Kawaguchi A., Komagata K.: Taxonomic significance of hydroxy fatty acids in myxobacteria with special reference to 2-hydroxy fatty acids in phospholipids.J.Gen.Appl.Microbiol. 34, 57–66 (1988).

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Institute of Soil Biology, Academy of Sciences of the Czech Republic, 370 05, Česke Budějovice, Czechia

    O. Oravecz, D. Elhottová, V. Krištůfek, V. Šustr & J. Frouz

  2. Department of Microbiology, Eötvös Lorand University, 1117, Budapest, Hungary

    O. Oravecz & K. Márialigeti

  3. Institute of Landscape Ecology, Academy of Sciences of the Czech Republic, 370 05, České Budějovice, Czechia

    J. Tříska

Authors
  1. O. Oravecz
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. D. Elhottová
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. V. Krištůfek
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. V. Šustr
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. J. Frouz
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. J. Tříska
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. K. Márialigeti
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to V. Krištůfek.

Additional information

This work was supported by theGrant Agency of the Academy of Sciences of the Czech Republic, grant no. B6066903, theGrant Agency of the Czech Republic, no. 526/99/P033, theResearch Plan of the Institute of Soil Biology AS CR, project no. Z6066911 and by thePeregrinatio II. Foundation at the Eötvös Loránd University (Budapest, Hungary).

Rights and permissions

Reprints and permissions

About this article

Cite this article

Oravecz, O., Elhottová, D., Krištůfek, V. et al. Application of ARDRA and PLFA analysis in characterizing the bacterial communities of the food, gut and excrement of saprophagous larvae ofPenthetria holosericea (Diptera: Bibionidae): a pilot study. Folia Microbiol 49, 83–93 (2004). https://doi.org/10.1007/BF02931652

Download citation

  • Received:

  • Revised:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF02931652

Keywords

Search

Navigation