Development of a cob-18S rRNA gene real-time PCR assay for quantifying Pfiesteria shumwayae in the natural environment

Abstract

Despite the fact that the heterotrophic dinoflagellate Pfiesteria shumwayae is an organism of high interest due to alleged toxicity, its abundance in natural environments is poorly understood. To address this inadequacy, a real-time quantitative PCR assay based on mitochondrial cytochrome b (cob) and 18S rRNA gene was developed and P. shumwayae abundance was investigated in several geographic locations. First, cob and its 5'-end region were isolated from a P. shumwayae culture, revealing three different copies, each consisting of an identical cob coding region and an unidentified region (X) of variable length and sequence. The unique sequences in cob and the X region were then used to develop a P. shumwayae-specific primer set. This primer set was used with reported P. shumwayae-specific 18S primers in parallel real-time PCRs to investigate P. shumwayae abundance from Maine to North Carolina along the U.S. east coast and along coasts in Chile, Hawaii, and China. Both genes generally gave similar results, indicating that this species was present, but at low abundance (mostly <10 cells x ml(-1)), in all the American coast locations investigated (with the exception of Long Island Sound, where which both genes gave negative results). Genetic variation was detected by use of both genes in most of the locations, and while cob consistently detected P. shumwayae or close genetic variants, some of the 18S PCR products were unrelated to P. shumwayae. We conclude that (i) the real-time PCR assay developed is useful for specific quantification of P. shumwayae, and (ii) P. shumwayae is distributed widely at the American coasts, but normally only as a minor component of plankton even in high-risk estuaries (Neuse River and the Chesapeake Bay).

FIG. 1.

PCR amplification of mitochondrial cytochrome b and its 5′ flanking region of P. shumwayae and organization of the gene fragments. (A) PCR amplification using primers PPCOX3 and PSCOBR1 (Table 2). Arrows on the left indicate the molecular sizes of two of the bands in the l-kb DNA ladder. (B) Organization of the three PCR-amplified gene fragments. cox3, cytochrome c oxidase subunit 3; cob, cytochrome b; X, sequence that does not show similarity to known genes in GenBank DNA databases; bidirectional arrows, regions that are identical in all three copies. Coding regions are shown as solid boxes.

FIG. 2.

Alignment of the mitochondrial gene fragments that contain (5′ to 3′) cox3 (uppercase), the X region (lowercase), and cob (uppercase) in the two Pfiesteria species. Dots indicate nucleotides in P. piscicida that are identical to corresponding position in P. shumwayae. xxxxxx, X region (nucleotide sequence not shown); arrows, regions used to design primers for real-time PCR analysis. The potential start codon ATG of cob is boxed.

FIG. 3.

Real-time quantitative PCR standard curves of P. shumwayae, using primer sets PSMTF2-PSCOBR1 (A) and PS18SF1-PS18SR1 (B). P. shumwayae genomic DNAs equivalent to 1,000, 100, 10, 1, and 0.1 cells were used as templates. The standard curve was constructed as log of P. shumwayae cell number versus number of threshold cycles. Shown are results of duplicates (two data points overlap in cases where only one circle is shown).

FIG. 4.

Temporal variation in P. shumwayae abundance at the five stations in Neuse River, N.C. P. shumwayae cell concentration was measured with PSCOB primers for natural seawater before (Natural) and after (Fed) fed incubation using Rhodomonas sp. as prey. UPP, FB, FHM, MBM, and MM, Union Point Park, Flanners Beach, Fairfield Harbour Marina, Minnesott Beach Marina, and Matthews Marina stations, respectively.

FIG. 5.

Relationship between P. shumwayae abundance (quantified with PSCOB primers) and temperature (A), salinity (B), and chlorophyll concentration (C) in the Neuse River alone and other locations (pooled). A high cell concentration is out of the scale and indicated by the number.

FIG. 6.

Polymorphism of the clones obtained for field samples. The complete sequence shown is the 326-bp PSCOB fragment (Pscob) in the documented P. shumwayae mtDNA sequence (accession numbers AY746979 to AY746981). Numbers on the left indicate positions of the first nucleotide of each line. All possible nucleotide substitutions (19) found in all the field samples (Field) are shown above the corresponding sites in the P. shumwayae sequence. Blank spaces in Field indicate nucleotide identical to those in Pscob. Typically, 0 to 4 of these substitutions occur in one sample.

FIG. 7.

Unrooted neighbor-joining tree of P. shumwayae mtDNA clones (PSCOB, 326 bp) from natural environments. For each sampling station, four to eight of the resulting clones were sequenced; numbers in parentheses indicate the number of clones with identical sequence. Clone codes: Pfiesteria shumwayae, sequence in the P. shumwayae cob coding region 5′ upstream region, which is identical in all three clones (Pscox3-cob1, Pscox3-cob2, and Pscox3-cob3); UPP, FB, FHM, and MBM, Union Point Park, Flanners Beach, Fairfield Harbour Marina, and Minnesott Beach Marina stations in Neuse River, N.C., respectively (Table 1); RI, Narragansett Bay in Rhode Island; BH, Boston Harbor in Massachusetts; ME, embayment in Maine; CHILE, Puerto Mont, Chile (see Table 1). In some cases, the name of the sampling station is followed by a number indicating the substation and/or a string of numbers indicating the month, day, and year of the sampling event (e.g., BH1 070203, Boston Harbor sampling station 1 sampled on 2 July 2003).