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

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 laband 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. *Corresponding author: [email protected] Acknowledgments This work was supported by the National Science Foundation (OCE0221137). Additional ship time was provided by the Long Island Sound Integrated Coastal Observing System (LISICOS; NOAA Grant NOAA NA04NOS4730256). We thank Huan Zhang and Senjie Lin for helpful advice, and Amy N.S. Siuda, Peter Boardman, Donald Schoener, Katharine Haberlandt, and the crew of the R/V Connecticut for sampling assistance. Andrew Payson helped with extraction of samples and running of numerous PCR reactions. Limnol. Oceanogr.: Methods 5, 2007, 163–173 © 2007, by the American Society of Limnology and Oceanography, Inc. LIMNOLOGY and OCEANOGRAPHY: METHODS Petroni et al. 2003; Stine et al. 2003; Agatha et al. 2004; Hosoi-Tanabe and Sako 2005). Recent progress in cultivation of oligotrich and choreotrich ciliates, the most abundant ciliate groups in the plankton, has led to a growing database of DNA sequences from the ribosomal and other genes. To date, these have been used principally to address questions of local and global diversity (Snoeyenbos-West et al. 2002). This study reports on the use of DNA sequences to design species-specific primers for amplification of ciliate DNA from environmental samples. Preliminary data show this method is reliable, sensitive, and time-efficient, compared with microscope techniques. It thus has the ability to provide information on distribution and abundance of individual species on spatial and temporal scales commensurate with ciliate movement and growth (meters and hours, respectively). Materials and procedures Development of primers—Snoeyenbos-West et al. (2002) identified the internal transcribed spacer region (ITS) of the ribosomal gene (ITS1-5.8S rDNA-ITS2) as an area of the ciliate genome that appears to be highly conserved across populations within a species. This was the area we focused on for primer design. Our initial target species, Laboea strobila and Favella ehrenbergii, were chosen for three reasons: (1) we had ITS sequence data from multiple populations (isolated from different locations and times), (2) both species are readily identifiable in Lugol’s preserved samples, allowing us to use microscopy to compare with the DNA-based method, and (3) both have been cultured in our lab for extended periods, allowing us to test the method extensively before working with field samples. A third target represents one species of the genus Strombidinopsis, frequently isolated from Long Island Sound (LIS), which we have not identified to species level using traditional methodology. All three target species were isolated from LIS and grown in 6-well plates, using various phytoplankton species as food. The initial step in primer development was performing a multiple alignment of all available sequences for each target species (collected from different locations and times), using Clustal X (http://www.ebi.ac.uk/clustalw/). The sequences were reviewed to identify areas of the ITS that are conserved across all available populations. A second sequence alignment, using all of our oligotrich and choreotrich sequences, was then used to identify those regions within the target species’ conserved areas that differed from all other species within our database. These sites were identified as potential target primer sites. Potential primer sites were evaluated using Beacon Designer 3.0 software (PREMIER Biosoft International) to find a primer pair where at least one was speciesspecific, produced a product at least 250 base pairs in length, minimized cross-dimer, and had a low self-dimer potential. The candidate primers were then compared with all oligotrich and choreotrich sequences within our database, including some sequences not yet published on GenBank (> 150 sequences in total). This step was intended to discover any potential conflicts with other species. They were then compared with all published sequences using the National Center for Biotechnology Information (NCBI) database Basic Local Alignment Search Tool (BLAST). If BLAST indicated a 100% match to a sequence from any other known ciliate, the potential primer was eliminated. We used a criterion of > 75% match to identify non-target sequences that might be amplified with a potential primer. The only marine planktonic organism that exceeded that level with any of our potential primers was the tintinnid Metacylis angulata, which was a potential match for F. ehrenbergii. As a result, M. angulata DNA from our library was added to our testing protocol. Testing primers—Primers were tested to verify that they amplified the target species, did not amplify DNA from nontarget ciliates, and did not amplify other planktonic DNA. This included testing them against DNA from multiple population isolates of each target species, including both cultures and ciliates picked from natural populations. Next, the primers were tested on DNA from other ciliate species, including testing the F. ehrenbergii primers on DNA from M. angulata. Then we tested with DNA from common phytoplankton species, including isolates of Tetraselmis sp., Rhodomonas sp., Gymnodinium sp., Cyclotella cryptica, Ditylum sp., Scrippsiella sp., Prorocentrum minimum, and Akashiwo sp. Finally, we tested the primers on DNA from mesozooplankton commonly found in LIS, including a mixed sample from a 200 μm net tow in LIS (September 2004), and from cultures of Acartia tonsa and A. hudsonica. Only those primer sets that passed these specificity tests were moved to the next phase of testing. DNA amplification can be inhibited in samples from chemically complex natural waters (Toranzos 1997). Thus, we also tested the primers on DNA extracted from a natural seawater sample amended by the addition of various cultured planktonic organisms, including the target species, to make an artificial planktonic community. This test was also used to evaluate optimal sample volumes to filter for extraction, optimal DNA concentrations for PCR, and detection limits for each species. For each target species, four 40 L samples were collected from LIS. These were passed first through a 20 μm mesh to eliminate the target ciliate species (verified microscopically) and combined into a 60 L container (Nalgene). Then, approximately 5000 ciliates from various cultures were added to the water. These included Strombidium stylifer, a Strobilidium sp., and a Tintinnopsis sp. In addition, F. ehrenbergii was added in tests of the primers for L. strobila and Strombidinopsis sp., L. strobila added in tests of the primers for F. ehrenbergii and Strombidinopsis sp., and Strombidinopsis sp. added in tests of the primers for F. ehrenbergii and L. strobila. Copepods (Acartia hudsonica and A. tonsa) and phytoplankton (Rhodomonas sp., Prorocentrum minimum, Thalassiosira weisflogii, Isochrysis sp., and Tetraselmis sp.) were also added to the water at typical in situ concentrations, to replace DNA removed during the 20 μm screening. The target ciliate species from Costas et al. Species-specific ciliate PCR primers