CONFIRMATION OF DOMOIC ACID PRODUCTION BY PSEUDONITZSCHIA AUSTRALIS (BACILLARIOPHYCEAE) CULTURES1

Single done isolates of Pseudonitzschia australis Frenguelli (= Nitzschia pseudoseriata Hasle) isolated from a toxic bloom in Monterey Bay, California produced domoic acid in culture. Although long-term historical records do not indicate previous blooms of this species on the Pacific coast, this is probably because it has been often misidentified as Nitzschia seriata Hasle; previous evidence for toxicity is lacking. Hydrographic data suggest that areas such as Monterey Bay might be “hot spots” for domoic acid-producing blooms.     

MORPHOLOGY OF THE MARINE DIATOM NITZSCHIA NAVIS-VARINGICA, SP. NOV. (BACILLARIOPHYCEAE), ANOTHER PRODUCER OF THE NEUROTOXIN DOMOIC ACID

A new marine diatom, Nitzschia navis-varingica , sp. nov., isolated from Vietnamese waters, is described by light, transmission, and scanning electron microscopy, including thin sectioning. The new species has been found to produce the neurotoxin domoic acid (DA), better known from several species of Pseudo-nitzschia Peragallo and one species of Amphora Ehrenberg. Production of DA is therefore more widespread among diatoms than previously thought. Taxonomically, the genus Nitzschia Hassall is exceptionally difficult, with about 900 described taxa. Grunow (in Cleve and Grunow 1880 divided the genus into 24 sections, and this system is still used with modifications. Nitzschia navis-varingica , sp. nov. fits best into a group of sections that includes Dubiae, Bilobatae , most of the Lanceolatae , and Lineares , all sensu Grunow, as the cell is slightly indented in the middle in girdle view and has a moderately eccentric raphe and a weak longitudinal fold on the valve. Many species within these sections have features similar to N. navis-varingica , but no species seems to be identical. Because both Pseudo-nitzschia and Nitzschia belong to the family Bacillariaceae, it seems reasonable to look for further producers of DA in this family, including freshwater species, which mainly comprise species within the sections Dubiae, Bilobatae, Lanceolatae , and Lineares.     

FLOW CYTOMETRY AND MICROSCOPY OF GAMETOGENESIS IN NITZSCHIA PUNGENS,A TOXIC,BLOOM-FORMING,MARINE DIATOM1

The domoic acid-producing diatom Nitzschia pungens Grunow f. multiseries Hasle, which is responsible for amnesic shellfish poisonings in Prince Edward Island, Canada, underwent gametogenesis when senescent cells (i.e. in stationary growth phase for more than 290 days) were subcultured into fresh FE medium and light intensity was increased from 33 to 530 μE · m^?2· s^?1. The number of gametes produced varied with the salinity of the medium, with a maximum at 23.5‰. Cells in the exponential growth phase (0.8 div · d^?1) did not produce gametes, nor did senescent cells when transferred without change in light intensity. Anisogamous gametes, probably haploid, were isolated by combining conventional microscopy with flow cytometry. Zygotes resulting from syngamy yielded cigar-shaped naviculoid cells, morphologically different from parent cells (heteromorphism). These cells, with a division rate of 1.9 div · d^?1, could serve as a seed population and explain the origin and rapid progression of the toxic blooms of red-water proportions that have been a public health problem in Eastern Canada. Production of domoic acid by postexponential and moribund cells but not by gametes, zygotes, or immediately resulting cells, provides an insight into the dependence of toxicity on the developmental history of this diatom.     

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.     

GROWTH AND DOMOIC ACID PRODUCTION BY PSEUDO‐NITZSCHIA SERIATA (BACILLARIOPHYCEAE) UNDER PHOSPHATE AND SILICATE LIMITATION1

Pseudo‐nitzschia seriata (Cleve) H. Peragallo isolated from Scottish west coast waters was studied in batch culture with phosphate (P) or silicate (Si) as the yield‐limiting nutrient at 15°C. This species produced the neurotoxin domoic acid (DA) when either nutrient was limiting but produced more when stressed by Si limitation during the stationary phase. Under P‐limiting conditions, exponential growth stopped after P was reduced to a low threshold concentration. Under Si‐limiting conditions, fast exponential growth was followed by a period of slower exponential growth, until Si became exhausted. A stationary phase was observed in the P‐limited but not the Si‐limited cultures, the latter showing a rapid decrease in cell density after the second exponential growth phase. Si‐limited cultures exhibited a further period of active metabolism (as indicated by increases in chl and carbon per cell) late in the experiment, presumably fueled by regenerated Si. DA production was low in exponential phase under both conditions. In P‐limited cultures, most DA was produced during the immediate postexponential phase, with little or no new DA produced during later cell senescence. In contrast, although a substantial amount of DA was produced during the slower exponential phase of the Si‐limited cultures, DA production was even greater near the end of the experiment, coincident with the period of chl synthesis and increase in carbon biomass. Comparison of the magnitude of toxin production in the two nutrient regimes indicated a greater threat of P. seriata‐generated amnesic shellfish poisoning events under Si rather than P nutrient limitation.     

CONFIRMATION OF DOMOIC ACID PRODUCTION BY PSEUDO‐NITZSCHIA AUSTRALIS (BACILLARIOPHYCEAE) ISOLATED FROM IRISH WATERS1

A nonaxenic isolate of the potentially toxic diatom Pseudo‐nitzschia australis (Frenguelli) from Irish waters was tested in two separate batch culture experiments. When grown under a low irradiance (～12 μmol photons·m ^{? 2}·s ^{? 1} 1 Received 20 March 2001. Accepted 21 August 2002.
; 16:8‐h light:dark cycle) for up to 40 days, the culture produced only trace amounts of the neurotoxin domoic acid (DA) during late stationary phase. Growth at a higher irradiance (～115 μmol photons·m ^{? 2}·s ^{? 1} 1 Received 20 March 2001. Accepted 21 August 2002.
; 12:12‐h light:dark cycle) resulted in DA production starting during late exponential phase and reaching a maximum concentration of 26 pg DA·cell ^{? 1} 1 Received 20 March 2001. Accepted 21 August 2002.
during late stationary phase. Liquid chromatography coupled to mass spectrometry was used to confirm the identity of DA in the culture. Irradiance and photoperiod could be important factors that contribute directly or indirectly to the control of DA production in P. australis. This is the first record of a DA‐producing diatom in Irish waters, and results indicate P. australis may have been the source of DA that has recently contaminated shellfisheries in this area.     

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.     

MORPHOGENESIS OF THE LABIATE PROCESS IN THE ARAPHID PENNATE DIATOM DIATOM A VULGARE1

Each valve of the araphid pennate diatom Diatoma has a labiate process (LP) at one end; in a frustule, the LPs are at diagonally opposite ends. After mitosis is over, an elongated dense body detaches from the spindle pole and migrates to one end of the daughter cell, always diagonally opposite the LP of the parental valve. This dense body trails a cone-shaped array of microtubules (MTs). Meanwhile, the new valve has begun to form within the Silica Deposition Vesicle (SDV). Having reached the end of the cell, this dense body moves back slightly and then settles onto the SDV, developing a layered substructure as it does so. Immediately beneath it, the LP of the new daughter valve differentiates. This dense object is clearly the homologue of the fibrous Labiate Process Apparatus (LPA) involved in the differentiation of the LP in several centric diatoms. In a few cases, these LPAs also hair been shown to originate from some component of the spindle pole. Thus, the homologue of the LPA of centric diatoms has now been found in an araphid pennate diatom; in each case, the LPA apparently comes from the pole of the spindle and presumably uses a cytoskeleton of MTs to locate the LP in its correct position. These observations support the possibility that the raphe evolved from the LP.     

IDENTIFICATION AND ASSESSMENT OF DOMOIC ACID PRODUCTION IN OCEANIC PSEUDO‐NITZSCHIA (BACILLARIOPHYCEAE) FROM IRON‐LIMITED WATERS IN THE NORTHEAST SUBARCTIC PACIFIC1

We identified and investigated the potential toxicity of oceanic Pseudo‐nitzschia species from Ocean Station Papa (OSP), located in a high‐nitrate, low‐chlorophyll (HNLC) region of the northeast (NE) subarctic Pacific Ocean. Despite their relatively low abundances in the indigenous phytoplankton assemblage, Pseudo‐nitzschia species richness is high. The morphometric characteristics of five oceanic Pseudo‐nitzschia isolates from at least four species are described using SEM and TEM. The species identified are Pseudo‐nitzschia dolorosa Lundholm et Moestrup, P. granii Hasle, P. heimii Manguin, and P. cf. turgidula (Hust.) Hasle. Additional support for the taxonomic classifications based on frustule morphology is provided through the sequencing of the internal transcribed spacer 1 (ITS1) rDNA. Pseudo‐nitzschia species identification was also assessed by the construction of ITS1 clone libraries and using automated ribosomal intergenic spacer analysis (ARISA) for environmental samples collected during the Subarctic Ecosystem Response to Iron Enrichment Study (SERIES), conducted in close proximity to OSP in July of 2002. Based on ITS1 sequences, the presence of P. granii, P. heimii, P. cf. turgidula, and at least five other putative, unidentified Pseudo‐nitzschia ITS1 variants was confirmed within iron‐enriched phytoplankton assemblages at OSP. None of the oceanic isolates produced detectable levels of particulate domoic acid (DA) when in prolonged stationary phase due to silicic acid starvation. The lack of detectable concentrations of DA suggests that either these strains produce very little or no toxin, or that the physiological conditions required to promote particulate DA production were not met and thus differ from their coastal, toxigenic congeners.     

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.     

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.     

Comparison of two domoic acid-producing diatoms: a review

In the past five years, awareness of domoic acid has increased from localized problems in Canada to outbreaks along both North American coasts. The phycotoxin domoic acid causes Amnesic Shellfish Poisoning (ASP) in humans and can be fatal. The known species of phytoplankton responsible for production of domoic acid include some pennate diatom species of the genus Nitzschia, sensu latu, which form stepped chains typical of the Pseudonitzschia. These diatoms are widely distributed, but their life histories and population dynamics are poorly understood. This review addresses histories of occurrences, morphology, geographical distributions, seasonal patterns, growth requirements, domoic acid production, and trophic interactions, with emphasis on a comparison of Pseudonitzschia pungens f. multiseries (Hasle) Hasle and Pseudonitzschia australis Frenguelli. Through continued research it will become possible to provide guidelines for regulatory agencies that protect both the consumer and the seafood industry.     

EFFECT OF COPPER ON GROWTH,SILICIC ACID UPTAKE AND SOLUBLE POOLS OF SILICIC ACID IN THE MARINE DIATOM,THALASSIOSIRA WEISFLOGII (BACILLARIOPHYCEAE)1

The toxicity of Cu to Thalassiosira weissflogii (Grunow) was investigated, focusing on the internal soluble pool of silicic acid. Silicic acid uptake and growth rates were found to be functions of both the cupric ion activity and the concentration of silicic acid in the growth medium. The soluble pool of Si per cell depended on the balance between the uptake rate and the division rate. The soluble pool in non-dividing cultures reflected simply the uptake rate (and inhibition by copper of the uptake rate), but in dividing cultures the soluble pools had complex patterns with time depending on uptake rates and timing of division. Intracellular soluble pools of silicic acid are a good indicator for the relative inhibition of uptake and growth processes.     

THE DIATOM GENERA ELLERBECKIA And ACTINOCYCLUS: AN APPRAISAL OF KOCIOLEK AND SPAULDING1

The contention that the diatom genera Ellerbeckia and Actinocyclus are congeneric on the basis of images of valves of the latter lying inside those of the former is disputed. It is suggested that conclusive evidence is lacking and that too few questions were asked to allow for the possibility that the two genera, one incorrectly identified to species and the other not identified at all, were lying together simply because they happened to fit.     

GROWTH RESPONSES OF THE DIATOM,CYCLOTELLA CRYPTICA (BACILLARIOPHYCEAE), TO GIBBERELLIC ACID1

The plant hormone, gibberellic acid (GA), stimulated growth of a marine diatom, Cyclotella cryptica Reimann, Lewin and Guillard. Four concentrations of GA (5 × 20 × 25 × and 35 × 10^?6 g/mL) were added to axenic cultures of C. cryptica. Changes in cell densities, measured by cell counts and turbidimetric readings, confirmed that GA at 20 × 10^?6 g/mL produced maximum stimulation. There was an increase in the total number of cells produced and a shorter lag phase of growth at this concentration. Coulter counter measurements of cell size, as well as ocular micrometer measurements, indicated there was no significant variation in cell volumes of GA grown cells over that of the controls.     

DIATOM MINERALIZATION OF SILICIC ACID. VIII. METABOLIC REQUIREMENTS AND THE TIMING OF PROTEIN SYNTHESIS1

We have examined the influence of temperature, protein synthesis, and energy metabolism on the process of silicon biomineralization in synchronized cultures of the diatom Navicula saprophilia Lange-Bertalot & Bonik (1976). Temperature effects on silicon polymerization were compared in vitro and in vivo. In vivo incorporation was very temperature dependent with a Q₁₀ of 7.53. In contrast, the Q₁₀ for in vitro polymerization was 1.42, indicating much lower temperature dependence. This difference in Q10 values suggests that in vivo polymerization involves more than autopolycondensation. Cycloheximide addition to synchronized cultures up to, but not later than one hour after the addition of silicic acid depressed total uptake, incorporation, but not pool size. Developing valves demonstrated morphological abnormalities with cycloheximide additions from 0 to 2 h following silicic acid addition. These data suggest that de nova proteins are required in biomineralization and that they are synthesized during or just after cytokinesis. Biomineralization is not coupled to energy derived directly from photosystem II or photosynthesis, since neither darkness nor DCMU had an effect on any aspect of silicification.     

DIATOM MINERALIZATION OF SILICIC ACID. II. REGULATION OF Si (OH)4 TRANSPORT RATES DURING THE CELL CYCLE OF NAVICULA PELLICULOSA1

Synchronized populations of Navicula pelliculosa (Bréb.) Hilse show a 10-fold increase in Si(OH)₄ transport rate during traverse through the cell division cycle. The transport activity pattern is similar to a “peak enzyme.” Kinetic analysis showed there was a significant change in K_s values, indicating increased “affinity” for Si(OH)₄ as cells neared maximal uptake rates. However, the dramatic changes in transport rate at various cell cycle stages were also reflected by alterations in the V_max, values of the transport process, suggesting a change in the number of functional transport “sites” in the plasma membrane. Cells in the wall forming stage, arrested from further development by Si(OH)₄ deprivation, maintained high transport rates for as long as 7 h. The rates decreased rapidly if protein synthesis were blocked or if Si(OH)₄ was added, the latter allowing the cells to traverse the rest of the cycle. The half-life of the transport activity ranged from 1.0 to 2.2 h when protein synthesis was inhibited at various cell cycle stages and during the natural decline of activity late in the cycle. The transport system appears to be metabolically unstable as is typical for a “peak protein.” The rise in transport rate through the cell cycle did not depend on the presence of Si(OH)₄ in the medium; therefore, the transport system does not appear to be induced by its substrate. The rise in transport is also observed in L:D synchronized cells developing in the presence of Si(OH)₄; neither does the transport system appear to be derepressed. The transport rate was strongly cell cycle-stage dependent; the data appeared to fit the “dependent pathway” model proposed by Hart-well to explain oscillations in enzyme synthesis during the cell cycle.     

MASS SEXUAL REPRODUCTION IN THE TOXIGENIC DIATOMS PSEUDO‐NITZSCHIA AUSTRALIS AND P. PUNGENS (BACILLARIOPHYCEAE) ON THE WASHINGTON COAST,USA1

Sexual reproduction is documented for the first time in field populations of the pennate diatoms Pseudo‐nitzschia australis Freng. and P. pungens (Grunow ex Cleve) Hasle (var. cingulata Villac and hybrids between var. cingulata and var. pungens). A bloom dominated by these species began on June 26, 2006, along Kalaloch Beach, Washington, USA, coincident with a drop in the Si(OH)₄:NO₃ ratio to below two. Multimodal size distributions were detected for both species, and synchronous auxosporulation occurred within the smallest size class during a 3‐week window. Auxospores and initial cells created a new class of large cells, and cells in the intermediate size classes increased in abundance during auxosporulation. Mating cells of both species were attached to colonies of surf‐zone diatoms. Paired gametangia, gametes, zygotes, auxospores, and large initial cells were found. Auxosporulation began first for P. pungens (June 30), apparently once a critical, high cell concentration was reached, followed by P. australis (July 5), when the total Pseudo‐nitzschia cell concentration reached 929,000 cells · L^?1. Low frequencies of auxosporulation occurred throughout the bloom but increased 4‐fold for P. australis and 3‐fold for P. pungens when macronutrients were reduced to low levels on July 11. A 2‐year life cycle was estimated for P. australis and 3 years for P. pungens, both with annual auxosporulation. Domoic acid (DA) in razor clams reached a maximum of 38 μg DA · g^?1 on July 18. A significant relationship existed between the percent of cells within the new size range and DA concentrations in razor clams on the same beach.