DNA PROBES AND A RECEPTOR-BINDING ASSAY FOR DETECTION OF PSEUDO-NITZSCHIA (BACILLARIOPHYCEAE) SPECIES AND DOMOIC ACID ACTIVITY IN CULTURED AND NATURAL SAMPLES

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.     

CHANGES IN DOMOIC ACID PRODUCTION AND CELLULAR CHEMICAL COMPOSITION OF THE TOXIGENIC DIATOM PSEUDO-NITZSCHIA MULTISERIES UNDER PHOSPHATE LIMITATION1

Production of domoic acid (DA), a neurotoxin, by the diatom Pseudo-nitzschia multiseries (previously Nitzschia pungens f. multiseries) Hasle and its cellular chemical composition were studied in phosphate-limited chemostat continuous cultures and in subsequent batch cultures. Under steady-state chemostat conditions, DA production increased from 0.01 to 0.26 pg DA · cell^?1· d^?1 as the growth rate decreased. When the nutrient supply was discontinued (to produce a batch culture), DA production was enhanced by a factor of ca. 3. DA production was temporarily suspended upon addition of phosphate to the batch cultures but resumed 1 d later at a higher rate coincident with the decline of phosphate uptake. In both steady-state continuous culture and batch culture, more DA was produced when alkaline phosphatase activity (APA) was high. The association of high DA production with high levels of APA and high cellular N:P ratios strongly suggests that phosphate limitation enhances DA production. Also, DA production was high when other primary metabolism (e.g. uptake of carbon, nitrogen, phosphorus and silicon, and cell division) was low, but chlorophyll a and adenosine triphosphate were generally high. This suggests that the synthesis of DA requires a substantial amount of biogenic energy.     

IDENTIFICATION OF CULTURED PSEUDO-NITZSCHIA (BACILLARIOPHYCEAE) USING SPECIES-SPECIFIC LSU rRNA-TARGETED FLUORESCENT PROBES1

Some, but not all, marine pennate diatoms of the genus Pseudo-nitzschia H. Peragallo are associated with the production of domoic acid, a naturally occurring amino acid responsible for amnesic shellfish poisoning. Distinguishing between potentially toxic and nontoxic representatives of this genus is time-consuming and difficult because it demands scanning electron microscopy of cleaned frustules. The objective of this work is to speed and ease identification of these organisms by using whole-cell (in situ) hybridization and species-specific large-subunit ribosomal RNA (LSU rRNA)-targeted oligonucleotide probes. Toward that end, cultures of P. australis Frenguelli, P. pungens (Grunow) Hasle, P. multiseries (Hasle) Hasle, P. fraudulenta (P. T. Cleve) Heiden, P. heimii Manguin, P. delicatissima (P. T. Cleve) Heiden, P. pseudo-delicatissima (Hasle) Hasle, and P. americana (Hasle) Fryxell were screened with a suite of 15 putative species-specific probes. Of those, a subset of eight probes was found that distinguished each species tested. In addition, Pseudo-nitzschia chloroplasts were labeled with a probe directed against a eubacterial-conserved sequence. Identification of new cultures based on their reactivity toward a set of probes agreed with species designations as defined by morphological criteria. Whole-cell hybridization is a rapid, simple, and cost-effective technique for discriminating among cultured Pseudo-nitzschia species.     

DOMOIC ACID PRODUCTION IN NITZSCHIA SP. (BACILLARIOPHYCEAE) ISOLATED FROM A SHRIMP-CULTURE POND IN DO SON, VIETNAM

Domoic acid (DA), a neuroexcitatory amino acid, was detected in batch culture of the newly recognized species Nitzschia navis-varingica Lundholm et Moestrup . The production of DA by this diatom was confirmed by electrospray ionization mass spectrometry. The diatom was collected from a shrimp-culture pond in Do Son, Vietnam. The production of DA (1.7 pg·cell ^{− 1}) is within the levels reported for Pseudo-nitzschia multiseries (Hasle) Hasle. The DA production started during the late exponential growth phase and reached a maximum during the early stationary growth phase. Maximum DA levels in the axenic culture decreased to about half that of the nonaxenic culture (0.9 pg·cell ^{− 1} vs. 1.7 pg·cell ^{− 1}), suggesting that DA production by the new species is influenced by bacteria.     

PSEUDO-NITZSCHIA PUNGENS AND P. MULTISERIES (BACILLARIOPHYCEAE): NOMENCLATURAL HISTORY,MORPHOLOGY, AND DISTRIBUTION1

Pseudo-nitzschia pungens f: multiseries is raised in rank from form to species based on morphological, physiological, and genetic features. Distinctive details of the valve face striae, the valve mantle, and the girdle of P. pungens and P. multiseries are outlined. The nomenclatural history and the distribution of the two species and their relationship to other species of the genus are discussed.     

MORPHOLOGY AND MOLECULAR ANALYSES OF THREE TOXIC SPECIES OF GAMBIERDISCUS (DINOPHYCEAE): G. PACIFICUS, SP. NOV., G. AUSTRALES, SP. NOV., AND G. POLYNESIENSIS, SP. NOV.

Three new dinoflagellate species, Gambierdiscus polynesiensis, sp. nov., Gambierdiscus australes, sp. nov., and Gambierdiscus pacificus, sp. nov., are described from scanning electron micrographs. The morphology of the three new Gambierdiscus species is compared with the type species Gambierdiscus toxicus Adachi et Fukuyo 1979, and two other species: Gambierdiscus belizeanus Faust 1995 and Gambierdiscus yasumotoi Holmes 1998. The plate formula is: Po, 3′, 7", 6C, 8S, 5‴, 1p, 2". Culture extracts of these three new species displayed both ciguatoxin- and maitotoxin-like toxicities. The following morphological characteristics differentiated each species. 1) Cells of G. polynesiensis are 68–85 μm long and 64–75 μm wide, and the cell’s surface is smooth. They are identified by a large triangular apical pore plate (Po), a narrow fish-hook opening surrounded by 38 round pores, and a large, broad posterior intercalary plate (1p) wedged between narrow postcingular plates 2‴ and 4‴. Plate 1p occupies 60% of the width of the hypotheca. 2) Cells of G. australes also have a smooth surface and are 76–93 μm long and 65–85 μm wide in dorsoventral depth. They are identified by the broad ellipsoid apical pore plate (Po) surrounded by 31 round pores and a long and narrow 1p plate wedged between postcingular plates 2‴ and 4‴. Plate 1p occupies 30% of the width of the hypotheca. 3) Cells of G. pacificus are 67–77 μm long and 60–76 μm wide in dorsoventral depth, and its surface is smooth. They are identified by the four-sided apical pore plate (Po) surrounded by 30 round pores. A short narrow 1p plate is wedged between the wide postcingular plates 2‴ and 4‴. Plate 1p occupies 20% of the width of the hypotheca. These three newly described species were also characterized by isozyme electrophoresis and DNA sequencing of the D8–D10 region of their large subunit (LSU) rRNA genes. The consistency between species designations based on SEM microscopy and classification inferred from biochemical and genetic heterogeneities was examined among seven isolates of Gambierdiscus. Their classification into four morphospecies was not consistent with groupings inferred from isozyme patterns. Three molecular types could be distinguished based on the comparison of their LSU rDNA sequences. Although G. toxicus TUR was found to be more closely related to G. pacificus, sp. nov. than to other G. toxicus strains, the molecular classification was able to discriminate G. polynesiensis, sp. nov. and G. australes, sp. nov. from G. toxicus. These results suggest the usefulness of the D8–D10 portion of the Gambierdiscus LSU rDNA as a valuable taxonomic marker.