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Molecular investigation of protozoan diversity in stream biofilms.

The objective of this study was to develop a molecular method for characterizing protozoan diversity in stream biofilms. Protozoa are defined as single celled eukaryotes possessing animal-like traits of motility and a heterotrophic mode of nutrition (an extremely diverse group). They are thought to be of fundamental importance in aquatic ecosystems, but understanding of their ecological role is very limited. This can be partly attributed to traditional visual-based methods for studying protozoa, which are difficult, tedious and often unreliable. Recent molecular biological studies have resolved long-outstanding questions of protozoan classification and evolution, and this data has provided an opportunity for development of molecular biology-based methods for protozoan study. In this project, PCR primers and associated molecular methods were evaluated for potential to detect protozoan sequence diversity in stream biofilms. Micromanipulation was used to isolate individual protozoan cells for development of pure protozoan cultures, to provide material for testing of molecular methods. Pure cultures of one amoeba, five ciliates, and four flagellate species were thus established and maintained on an ongoing basis. New PCR primers were designed to target 18S ribosomal RNA genes in the protozoan phylum Ciliophora, and tested in reactions on cultured protozoa. The most reliable primer combinations were applied to stream biofilm DNA extracts. Cloning and sequencing analysis showed about 80 % of sequences detected by primers 384F/1147R were of probable ciliate origin, while most of the remainder did not closely match any known sequences. About 30 % of sequences detected by primer set 121F/1147R were identified as belonging to Ciliophora, and a similar proportion as belonging to the closely related phylum Apicomplexa. T-RFLP analysis using primers 384F/1147R, combined with calculation of diversity indices, resulted in identification of differences in ciliate diversity between streams in different states of degradation. Diversity was highest in samples from a moderately impacted stream, and usually lowest in samples from a nearly pristine Waitakere Ranges stream. Diversity in a highly degraded urban stream was moderate, but highly skewed toward several extremely abundant sequences. Diversity in a slightly impacted stream was either low or moderate, depending upon the enzyme digestion treatment used. Some T-RFLP profile peaks were consistent between different samples, while others were not. Also tested were two sets of “universal” PCR primers targeting the eukaroyote 18S ribosomal RNA gene, which had detected protozoan sequences in environmental samples in prior studies. These performed poorly when applied to cultured protozoan template material, and were ineffective at detecting protozoan sequences in stream biofilms.

Use of PCR primers 384F and 1147R, along with cloning, sequencing, and T-RFLP analysis, was considered to provide an effective means for describing ciliate diversity in stream biofilms. To provide a molecular method for characterising total protozoan diversity requires development of PCR primers specific to other monophyletic protozoan taxa; this can be achieved using the methodology used in this study.

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