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Brevetoxins produced by the marine dinoflagellate Karenia brevis (C. C. Davis) G. Hansen et Moestrup cause neurotoxic shellfish poisoning (NSP) in human consumers and also endanger a variety of coastal wildlife. In the eastern Gulf of Mexico the presence and abundance of this species have traditionally been monitored using light microscopy (LM) observations of whole water samples. Various molecular probe methods now enable detection of multiple species from a single sample, allowing rapid sample analysis. We describe the development of sandwich hybridization assays (SHAs) for Karenia brevis, K. selliformis Haywood, Steid. et L. MacK., K. mikimotoi (Miyake et Kominami ex M. Oda) G. Hansen et Moestrup, K. papilionacea Haywood et Steid., the Karlotoxin-producer Karlodinium veneficum (D. Ballant.) J. Larsen (=K. micrum), and Gymnodinium aureolum (Hulburt) G. Hansen, comb. nov. The assays require no nucleic acid purification and use LSU rRNA-targeted probes and a semiautomated, 96-well plate format. Probes tested in matrix format were specific relative to rRNAs of all nontarget species used. The response of the SHA for a constant number of K. brevis cells per unit volume of homogenate depended on the growth status of a culture, decreasing for senescent cells relative to actively growing cells. The results of preliminary field tests of the K. brevis SHA indicated that cells collected from natural populations tended to return a lower signal than those harvested from laboratory cultures, but these results are nonetheless very encouraging. These preliminary field studies show that robust standards are required for cell identification and enumeration, with which new methods can be compared.

Many species within the diatom genus Pseudo-nitzschia are difficult to distinguish without applying molecular analytical or microscopy-based methods. DNA, antibody and lectin probes have previously been used to provide rapid and specific detection of species and strains in complex field assemblages. Recently, however, well-documented cryptic genetic diversity within the group has confounded results of DNA probe tests in particular. Moreover, the number of species descriptions within the genus continues to increase, as do insights into toxin production by both new and previously described species. Therefore, a combination of classical morphological techniques and modern molecular methodologies is needed to resolve ecophysiological traits of Pseudo-nitzschia species. Here, we present an approach to recover and identify frustules from sample collection filters used for toxin analysis onboard the Environmental Sample Processor (ESP), an in situ sample collection and analytical platform. This approach provides a new and powerful tool for correlating species presence with toxin detected remotely and in situ by the ESP, and has the potential to be applied broadly to other sampling configurations. This new technique will contribute to a better understanding of naturally occurring Pseudo-nitzschia community structure with respect to observed domoic acid outbreaks.

Large-subunit ribosomal RNA-targeted probes for Pseudo-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 cross-reacted 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.