Use of species-specific primers and PCR to measure the distributions of planktonic ciliates in coastal waters

Barbara A. Costas, George McManus, Mary Doherty, Laura A. Katz

Limnol. Oceanogr. Methods 5:163-173 (2007) | DOI: 10.4319/lom.2007.5.163

ABSTRACT: We developed a method to extract environmental DNA and amplify target portions of the internal transcribed spacer region (ITS) of the ribosomal gene (ITS1-5.8S-ITS2) from individual species of oligotrich and choreotrich ciliate microzooplankton. To date, we have lab- and field-tested primers specific to the tintinnid Favella ehrenbergii, the oligotrich Laboea strobila, and the choreotrich Strombidinopsis sp. For all three species, the primers were both species-specific (not producing PCR product from non-target DNA) and comprehensive (able to amplify from different populations of the target species). The method is both time-efficient and sensitive, compared with microscopy. In seawater samples amended with both target and non-target DNA, we were able to detect the targets at < 1 cell L–1. Some difficulties we encountered resulted from PCR-inhibitory compounds that co-extracted with the environmental DNA, and the rarity of the target DNA within natural plankton assemblages. Comparisons with microscopic counts were qualitatively similar to PCR (presence/absence of the species in different amounts of extract). We are evaluating ways to make the method fully quantitative by investigating the degree to which copy number for this gene may vary among individuals.