Denaturing gradient gel electrophoresis (DGGE) as a tool for identification of marine nematodes

Abstract

Many phyla of marine invertebrates are difficult to identify using conventional morphological taxonomy. Larvae of a wider set of phyla are also difficult to identify as a result of conservation of morphology between species or because morphological characters are destroyed during sampling and preservation. DNA sequence analysis has the potential for identification of marine organisms to the species level. However, sequence analysis of specimens is time-consuming and impractical when species diversity is very high and densities of individuals huge, as they are in many marine habitats. The effectiveness of the 18S rRNA gene sequences for identification of one speciesrich marine group, the Nematoda, is analysed. Following identification of variable regions of the 18S rRNA gene, primers were designed to amplify a small segment of sequences suitable for denaturing gradient gel electrophoresis (DGGE). The effectiveness of DGGE for identifying individual species is analysed. DGGE analysis of natural communities of nematodes detected less than 2 ⁄ 3 of the species present. This fraction of the community probably represents the abundant species in the original samples. It is concluded that DGGE is not a useful tool for analysis of species richness in marine communities as it fails to detect rare species of which there are usually many in the marine benthic environment. However, DGGE may be a useful method for detecting changes in communities that influence the abundance of the most common taxa. KEY WORDS: Marine nematodes · 18S ribosomal DNA · Denaturing gradient gel electrophoresisResale or republication not permitted without written consent of the publisher

Rapid decline of PCR amplification from genomic extracts of DESS-preserved, slide-mounted nematodes

“…Previous studies have indicated that nematode degradation in glycerin is unpredictable and DNA can be affected at times ranging from days to months (Cook et al, 2005). We have also found similar variation in success: DNA has degraded after only a few weeks on slides for some specimens, whilst for other nematodes PCR amplifications and sequencing have been successful even though specimens were kept stored in slide mounts for several months.…”

“…Previous work suggested that standard taxonomic slide preparations of nematodes mounted in glycerin could be adversely affecting DNA integrity, thus reducing subsequent PCR success (Meldal, 2004;Cook et al, 2005). We have also encountered frequent problems in obtaining reliable and consistent PCR results from DESS-preserved specimens stored in slide mounts and determined that a formal study was needed to assess the impact of standard methodology.…”

Community structure of marine sedimentary protists in relation to flow and grain size

“…Piceno et al 1999, Webster et al 2003, but has rarely been used to assess protistan diversity in such samples (Coyne et al 2001). Because of our particular interest in the ciliate communities, and the fact that molecular assessments of community structure can disagree with morphological assessments due to extraction and PCR biases (Savin et al 2004, Cook et al 2005, we also examined ciliates microscopically using quantitative Protargol staining (QPS; Montagnes & Lynn 1987). …”

“…We obtained many bands representing diatoms, but no bands representing ciliates. DGGE can fail to reveal relatively less abundant taxa, because the PCR reaction generally amplifies the most abundant rDNA sequences in a sample of extracted DNA (Cook et al 2005). DGGE has been found to miss rare species of bacteria (Koizumi et al 2003), phytoplankton (Savin et al 2004), and metazoans (Cook et al 2005).…”

“…DGGE can fail to reveal relatively less abundant taxa, because the PCR reaction generally amplifies the most abundant rDNA sequences in a sample of extracted DNA (Cook et al 2005). DGGE has been found to miss rare species of bacteria (Koizumi et al 2003), phytoplankton (Savin et al 2004), and metazoans (Cook et al 2005). Besides overall cell abundance, the amount of template available for amplification from each organism also depends on the number of repeats of the target 18S rDNA sequences in the genome.…”