Identification and enumeration of cultured and wild Pseudo-nitzschia (Bacillariophyceae) using species-specific LSU rRNA-targeted fluorescent probes and filter-based whole cell hybridization

Efforts to understand the ecologic and environmental parameters that govern harmful algal blooms (HABs) require rapid and specific identification of causative species. Traditional methods of species identification using light and electron microscopy are useful in this regard but are also time consuming, making routine analysis of a large number of samples difficult. Techniques that speed and ease the detection of HAB species as they occur in natural populations are therefore desirable. In this paper, we continue efforts to develop species-specific large subunit ribosomal RNA (LSU rRNA)-targeted fluorescent DNA probes for a variety of Pseudo-nitzschia H. Peragallo species, a group of marine pennate diatoms that includes representatives linked to production of domoic acid and amnesic shellfish poisoning (ASP). A custom filter tube and filtration manifold that has utility for both whole cell (in situ) hybridization as well as preparing samples for scanning electron microscopy (SEM) is described. Filter-based whole cell hybridization was used to identify a variety of newly isolated Pseudo-nitzschia clones, and probe results were confirmed using SEM. Some isolates of P. pungens (Grunow) Hasle exhibited variable (intraclonal) reactivity toward the P. pungens-specific probe. Three isolates of P. subpacifica (Hasle) Hasle were found to cross-react with probes designed for P. fraudulenta (Cleve) Hasle and P. heimii Manguin. Four isolates did not react with any species-specific probes; this group comprised three distinct morphotypes whose fine-scale morphologic features did not agree with published descriptions of Pseudo-nitzschia species. Evaluation of the filter method using cultured cells added to natural (whole water) samples indicated quantitative recovery of target species. Confirming results of probe assays using SEM was difficult when the target species was less than 10(4) cells.L-1 in the presence of greater than 10(6) cells.L-1 of other nontarget diatom species. A variety of Pseudo-nitzschia, including P. australis Frenguelli, P. fraudulenta, P. heimii, P. pseudodelicatissima (Hasle) Hasle, P. pungens, P. multiseries (Hasle) Hasle, and Nitzschia americana Hasle, were identified using whole cell hybridization in a variety of field samples containing mixed assemblages of plankton, and these results were confirmed using SEM. The filter tube method of applying probes was used onboard ship for near real-time identification and enumeration of a variety of Pseudo-nitzschia species.