Large-subunit ribosomal RNA-targeted probes forPseudo-nitzschia australis Frenguelli, P. multiseries (Hasle) Hasle, P. pseudodelicatissima (Hasle) Hasle, and P. pungens (Grunow) Hasle were applied to cultured and natural samples using whole-cell and sandwich hybridization. Testing of the latter method is emphasized here, and technique refinements that took place during 1996-1997 are documented. Application of the sandwich hybridization test showed that the signal intensity obtained for a given number of target cells remained constant as batch cultures of these organisms progressed from active through stationary growth phases. This suggests that cellular rRNA content for each target species remained relatively stable despite changes in growth state. Application of whole-cell and sandwich hybridization assays to natural samples showed that both methods could be used to detect wild P. australis, P. pseudodelicatissima, and to a lesser degree P. multiseries, but detection of P. pungens was prone to error. A receptor-binding assay for domoic acid (DA) enabled detection of this toxin activity associated with a particulate fraction of the plankton and provided a context in which to view results of the rRNA probe tests. In one case, the probe for P. australis crossreacted with P. cf. delicatissima. The sample that contained the latter species also contained a low amount of DA activity. Under certain field conditions, results of whole-cell and sandwich hybridization tests disagreed. Detailed analysis of selected field samples illustrates how such situations arose. Collectively, the rRNA probe and toxin analyses suggest that manifestation of DA in the environment is possible in the absence of readily recognizable intact cells.

Pseudo-nitzschia austrulis Frenguelli is a marine pennatc diatom associated with the production of domoic acida neurocxcitatory amino acid linked to illness and mortality of humans and wildlife, Distinguishing P. austrulis from its co-occurring congeners is labor intensive and time consuming because of a requirement for scanning electron microscopy. Hcrc, we apply large-subunit ribosomal RNA (LSU rRNA)-targeted oligonucleotides in wholecell and sandwich hybridization formats to identify and enumerate this species collected from pure cultures and natural populations. Whole-cell hybridization employed fluorescently labeled probes, filter-based sample processing, and epifluorescence microscopy to enumerate labeled cells. In contrast, sandwich hybridization was accomplished by homogenizing cells in a chaotropic solution and performing two hybridization reactions: capture of LSU rRNA using an oligonuclcotide coupled to a macroscopic solid support and binding of signal probe to a region of LSU rRNA near that of the capture site. Sandwich hybrids were detected calorimetrically; color intensity was proportional to the abundance of target species in the original sample. The sandwich hybridization assay was semiautomated with a robotic processor. Both whole-cell and sandwich hybridization are useful techniques for identifying P. australis as it occurs in nature. Sandwich hybridization potentially offers the most rapid and simple means to accomplish this task when screening large numbers of environmental samples.Fundamental challenges common to all studies of harmful algal blooms (HABs) are identifying, enumerating, and mapping the distributions of toxic species as they occur in nature. Recent workshops on HABs have recognized these problems and the need to develop novel techniques to speed and ease these tasks (Anderson et al. 1993;ECOHAB 1995). At present such operations are difficult because of the time and labor required to count potentially toxic species collected in discrete samples and the need to repeat such procedures hundreds of times in order to chart organisms' growth and movement. In this contribution we apply large-subunit ribosomal RNA (LSU rRNA)-targeted oligonucleotide probes to detect and quantify Pseudo-nitzschia austrulis Frenguelli, a marine pennate diatom, collected from pure cultures and natural populations. This species is linked to the production of domoic acid (DA), a neuroexcitatory amino acid responsible for a human disorder known as amnesic shellfish poisoning (ASP: Per1 et al. 1990; Todd 1993).

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