Publications: Research reports and publications
Molecular detection of bacterial and fungal communities in non-wadeable rivers - a pilot study for ecological health assessment
Wood SA, Biessy L, Atalah J, Pochon X, Clapcott J 2015. Molecular detection of bacterial and fungal communities in non-wadeable rivers - a pilot study for ecological health assessment. Cawthron Report 2740.
Non-wadeable rivers are waterways where water depth and/or velocity restrict or prevent the sampling of biota from representative habitats using standard protocols. Sampling of non-wadeable waterways for bioassessment presents a range of challenges, compared with wadeable waterways, including the need for increased equipment, personnel and sampling time (Clapcott et al. 2012). Alternative assessment measures such as the rate of organic matter decomposition and river metabolism offer potential for monitoring the ecological condition of these rivers (Clapcott & Young 2009, Collier et al. 2013a).
Cotton strip assays measure cellulose decomposition potential and have shown promise for assessing ecological health in non-wadeable rivers (Collier et al. 2013b). Cotton strips are made up of cellulose, a major building block in leaves and woody debris, and the assay evaluates the contribution of microbial decomposition to organic matter processing (Parkyn et al. 2010, Tiegs et al. 2013). The end point of the assay is a measure of tensile strength loss. To our knowledge, the microbial communities responsible for decomposition have not been evaluated. The species and community structure of these microbial communities is likely to vary among rivers of differing ecological health. Knowledge on community composition may be useful for understanding cotton strip assay results (i.e. identifying and quantifying the organisms that cause decomposition), or for making bioassessments of these rivers.
Previous research has shown the utility of using molecular techniques to characterise microbial communities in stream biofilms (Lewis et al. 2011). Such studies have demonstrated that molecular assessment of microbial communities can provide a cost effective way to analyse large sample sets. They can provide information on stream health and insights into base-level carbon and nutrient cycling in these ecosystems. However, analysis of a large dataset from across New Zealand found only limited correlation with bacterial community structure and land use or water quality (Lewis et al. 2011). Variations in substrate types sampled among streams may have accounted for some of this discrepancy. Using a standardised substrate, such as cotton strips, may improve the applicability of these techniques as biomonitoring tools.
In this study we used a high-throughput DNA fingerprinting method known as automated ribosomal intergenic spacer analysis (ARISA; Fisher & Triplett 1999). This polymerase chain reaction (PCR)-based method exploits the length difference of a region between two nuclear genes (16S and 23S ribosomal RNA genes). Total community DNA is extracted from the environmental sample, and PCR amplified using a fluorescently labelled primer. An automated system in which a laser detects the fluorescent DNA fragments is then used to determine the lengths of PCR amplicons within a sample. Different fragment lengths are assumed to be indicative of a species or strain. In this study we used two different ARISA assays – bacterial and fungal specific. While the technique does not provide taxonomic data, it is cost-effective (ca. $20 per sample for DNA extraction, PCR and analysis, labour excluded), and results can be obtained rapidly. This technique has proven to be very robust and has been used in studies of microbial populations in a variety of habitats including freshwater (Lear & Lewis 2009), marine (Hewson et al. 2006), and soil (Schloss et al. 2003).
The aims of this study were:
• To evaluate four DNA extraction methods to determine which provided the highest quality DNA from cotton strips for use in ARISA PCRs. The selected method needed to be easily upscalable and cost effective.
• To determine if bacterial and fungal communities within cotton strips can be used to provide information on ecosystem health in non-wadeable rivers in the Waikato region.