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Applied and Environmental Microbiology, March 2008, p. 1740-1747, Vol. 74, No. 6
0099-2240/08/$08.00+0     doi:10.1128/AEM.01438-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Molecular Characterization of Ciliate Diversity in Stream Biofilms{triangledown} ,{dagger}

Andrew Dopheide,1 Gavin Lear,1 Rebecca Stott,2 and Gillian Lewis1*

School of Biological Sciences, University of Auckland, 3a Symonds Street, Auckland, New Zealand,1 National Institute for Water and Atmospheric Research, P.O. Box 11-115, Hamilton, New Zealand2

Received 28 June 2007/ Accepted 7 January 2008

Free-living protozoa are thought to be of fundamental importance in aquatic ecosystems, but there is limited understanding of their diversity and ecological role, particularly in surface-associated communities such as biofilms. Existing eukaryote-specific PCR primers were used to survey 18S rRNA gene sequence diversity in stream biofilms but poorly revealed protozoan diversity, demonstrating a need for protozoan-targeted primers. Group-specific PCR primers targeting 18S rRNA genes of the protozoan phylum Ciliophora were therefore designed and tested using DNA extracted from cultured protozoan isolates. The two most reliable primer combinations were applied to stream biofilm DNA, followed by cloning and sequencing analysis. Of 44 clones derived from primer set 384F/1147R, 86% were of probable ciliate origin, as were 25% of 44 clones detected by primer set 121F/1147R. A further 29% of 121F/1147R-detected clones matched sequences from the closely related phylum Apicomplexa. The highly ciliate-specific primer set 384F/1147R was subsequently used in PCRs on biofilm DNA from four streams exhibiting different levels of human impact, revealing differences in ciliate sequence diversity in samples from each site. Of a total of 240 clones, 73% were of probable ciliate origin; 54 different putative ciliate sequences were detected from throughout seven taxonomic ciliate classes. Sequences from Oligohymenophorea were most commonly detected in all samples, followed by either Spirotrichea or Phyllopharyngea. Restriction fragment length polymorphism profile-based analysis of clones suggested a potentially higher level of diversity than did sequencing. Nevertheless, newly designed PCR primers 384F/1147R were considered to provide an effective molecular basis for characterization of ciliate diversity in stream biofilms.

* Corresponding author. Mailing address: School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand. Phone: 64 (9) 373 7599. Fax: 64 (9) 373 7416. E-mail: gd.lewis{at}auckland.ac.nz

{triangledown} Published ahead of print on 25 January 2008.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

Applied and Environmental Microbiology, March 2008, p. 1740-1747, Vol. 74, No. 6
0099-2240/08/$08.00+0     doi:10.1128/AEM.01438-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Dopheide, A., Lear, G., Stott, R., Lewis, G. (2009). Relative Diversity and Community Structure of Ciliates in Stream Biofilms According to Molecular and Microscopy Methods. Appl. Environ. Microbiol. 75: 5261-5272 [Abstract] [Full Text]  
  • Vaerewijck, M. J. M., Sabbe, K., Bare, J., Houf, K. (2008). Microscopic and Molecular Studies of the Diversity of Free-Living Protozoa in Meat-Cutting Plants. Appl. Environ. Microbiol. 74: 5741-5749 [Abstract] [Full Text]  


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