LIFE HISTORY AND IN SITU GROWTH RATES OF ALEXANDRIUM TAYLORI (DINOPHYCEAE, PYRROPHYTA)

Alexandrium taylori Balech is a phototrophic marine dinoflagellate. It produced recurrent blooms during the summer months (July and August) of 1994 to 1997 in La Fosca beach (NW Mediterranean). In addition to a motile vegetative form, A. taylori had two benthic forms: temporary cysts and resting cysts. Temporary cysts were a temporally quiescent stage produced from the ecdysis of the vegetative cell in both natural populations and laboratory cultures. Temporary cysts may divide to form motile cells. Resting cysts had a thicker wall than the temporary cysts and had a red accumulation body. Gametes and planozygotes were also observed in laboratory cultures. Alexandrium taylori showed in situ diurnal vertical migration with an increase of vegetative cells in the water column in the morning through midday, with concentrations peaking in the afternoon followed by lower levels at night. Most vegetative cells lost their thecae and flagella, and with them their motility, turning into temporary cysts that settled in the early evening. The number of temporary cysts in the water column rose in the evening and at night. The temporary cysts gave rise to motile cells the following morning. Synthesis of DNA occurred in vegetative cells at night, and a preferential period of cell division occurred at sunrise. The estimated division rate in the field was 0.4–0.5 vegetative cells·day⁻¹. Temporary cysts had twice the DNA of a G₁ vegetative cell. The minimum in situ division rate of the temporary cysts was 0.14 day⁻¹. The role of the resting and temporary cyst population in the annual recurrence and maintenance of the A. taylori bloom is discussed.     

IDENTIFICATION OF THE TOXIC DINOFLAGELLATES ALEXANDRIUM CATENELLA AND A. TAMARENSE (DINOPHYCEAE) USING DNA PROBES AND WHOLE-CELL HYBRIDIZATION1

Fluorescent DNA probes (cCAT-F1 and cTAM-Fl) complementary to the 3′ end of ribosomal RNA (rRNA) internal transcribed spacer 1 sequences (ITS 1: positions 154–176) of toxic species of Alexandrium catenella (Whedon and Kofoid) Taylor and A. tamarense (Lebour) Taylor were applied to various cultures of the genus Alexandrium and several other phytoplankters using whole-cell fluorescent in situ hybridization. cCAT-F1 and cTAM-F1 reacted with targeted strains of A. catenella (catenella type) and A. tamarense (tamarense type), respectively, and did not react with isolates of A. affine (Inoue et Fukuyo) Balech, A. fraterculus (Balech) Balech, A. insuetum Balech, A. lusitanicum Balech, A. pseudogonyaulux (Biecheler)Horiguchi ex Yuki et Fukuyo comb. nov., nor isolates of Prorocentrum micans Ehrenberg, Amphidinium carterae Hulburt, Heterocapsa triquetra (Ehrenberg) Stein, Gymnodinium mikimotoi Miyake et Kominami ex Oda, Skeletonema costatum (Greville) Cleve, Heterosigma akashiwo (Hada) Hada, and Chattonella antiqua (Hada) Ono. DNase I and RNase A treatment showed that probes hybridized to ribosomal DNA, not rRNA. Probes were localized at the bottom of the U-shaped nucleus, a region that corresponds to the nucleolus. The probes are highly specific for particular strains of A. catenella and A. tamarense and are applicable for identifying these species collected from cultured and possibly natural populations.     

VEGETATIVE AND SEXUAL ASPECTS OF DINOPHYSIS PAVILLARDI (DINOPHYCEAE)

Phases in the life history of the dinoflagellate Dinophysis pavillardi Schroeder from cultured phytoplankton assemblages are described. Under stressful conditions, induced in the laboratory through substantial thermic and nutritive changes, vegetative cells divided repeatedly. Scanning electron and light microscopy of dividing specimens showed that thecal fission began with the separation of the sulcal and ventral epithecal plates and the simultaneous dislocation of the pore plates from the right cell half. The posterior progression of the division led to pairs of cells attached antapically, which produced a new wall of reduced size. This phase of the life cycle coincided with the appearance and development of small forms of D. pavillardi, which displayed cytological features and behavior typical of male gametes, suggesting a process of gametogenesis through depauperating mitotic divisions. Anisogamy occurred at the time of the maximum production of small cells and involved the shedding of thecal components by the smaller gamete and subsequent cytoplasmic fusion and formation of planozygotes. Although the dormancy aspects of this species remain unknown, these observations provide the first evidence of sexuality .     

IDENTIFICATION OF GROUP- AND STRAIN-SPECIFIC GENETIC MARKERS FOR GLOBALLY DISTRIBUTED ALEXANDRIUM (DINOPHYCEAE). II. SEQUENCE ANALYSIS OF A FRAGMENT OF THE LSU rRNA GENE1

A fragment of the large-subunit (LSU) ribosomal RNA gene (rDNA) from the marine dinoflagellates Alexandrium tamarense (Lebour) Balech, A. catenella (Whedon et Kofoid) Balech, A. fundyense Balech, A. affine (Fukuyo et Inoue) Balech, A. minutum Halim, A. lusitanicum Balech, and A. andersoni Balech was cloned and sequenced to assess inter- and intraspecific relationships. Cultures examined were from North America, western Europe, Thailand, Japan, Australia, and the ballast water of several cargo vessels and included both toxic and nontoxic isolates. Parsimony analyses revealed eight major classes of sequences, or “ribotypes,” indicative of both species- and strain-specific genetic markers. Five ribotypes subdivided members of the A. tamarense/catenella/ fundyense species cluster (the “tamarensis complex”) but did not correlate with morphospecies designations. The three remaining ribotypes were associated with cultures that clearly differ morphologically from the tamarensis complex. These distinct sequences were typified by 1) A. affine, 2) A. minutum and A. lusitanicum, and 3) A. andersoni. LSU rDNA from A. minutum and A. lusitanicum was indistinguishable. An isolate's ability to produce toxin, or lack thereof, was consistent within phylogenetic terminal taxa. Results of this study are in complete agreement with conclusions from previous work using restriction fragment-length polymorphism analysis of small subunit rRNA genes, but the LSU rDNA sequences provided finer-scale species and population resolution. The five divergent lineages of the tamarensis complex appeared indicative of regional populations; representatives collected from the same geographic region were the most similar, regardless ofmorphotype, whereas those from geographically separated populations were more divergent even when the same morphospecies were compared. Contrary to this general pattern, A. tamarense and A. catenella from Japan were exceptionally heterogeneous, displaying sequences associated with Australian, North American, and western European isolates. This diversity may stem from introductions of A., tamarense to Japan from genetically divergent sources in North America and western Europe. Alexandrium catenella from Japan and Australia appeared identical, suggesting that these two regional populations share a recent, common ancestry. One explanation for this genetic continuity was suggested by A. catenella cysts transported from Japan to Australia via ships' ballast water: the cysts contained LSU rDNA sequences that were indistinguishable from those of known populations of A. catenella in both Japan and Australia. Ships ballasted in South Korea and Japan have also fostered a dispersal of viable A. tamarense cysts to Australia, but their LSU rDNA sequences indicated they are genetically distinct from A. tamarense/catenella previously found in Australia and genetically distinct from each other, as well. Human-assisted dispersal is a plausible mechanism for inoculating a region with diverse representatives of the tamarensis complex from geographically and genetically distinct source populations. The D1-D2 region of Alexandrium LSU rDNA is a valuable taxo-nomic and biogeographic marker and a useful genetic reference for addressing dispersal hypotheses.     

GROWTH AND PHOSPHORUS UPTAKE BY THE TOXIC DINOFLAGELLATE ALEXANDRIUM CATENELLA (DINOPHYCEAE) IN RESPONSE TO PHOSPHATE LIMITATION1

Alexandrium catenella (Whedon et Kof.) Balech has exhibited seasonal recurrent blooms in the Thau lagoon (South of France) since first reported in 1995. Its appearance followed a strong decrease (90%) in phosphate (PO₄^3?) concentrations in this environment over the 1970–1995 period. To determine if this dinoflagellate species has a competitive advantage in PO₄^3?‐limited conditions in terms of nutrient acquisition, semicontinuous cultures were carried out to characterize phosphorus (P) uptake by A. catenella cells along a P‐limitation gradient using different dilution rates (DRs). Use of both inorganic and organic P was investigated from measurements of ³³PO₄^3? uptake and alkaline phosphatase activity (APA), respectively. P status was estimated from cellular P and carbon contents (Q_P and Q_C). Shifts in trends of Q_P/Q_C and Q_P per cell (Q_P·cell?1) along the DR gradient allowed the definition of successive P‐stress thresholds for A. catenella cells. The maximal uptake rate of ³³PO₄^3? increased strongly with the decrease in DR and the decrease in Q_P/Q_C, displaying physiological acclimations to PO₄^3? limitation. Concerning maximal APA per cell, the observation of an all‐or‐nothing pattern along the dilution gradient suggests that synthesis of AP was induced and maximized at the cellular scale as soon as PO₄^3? limitation set in. APA variations revealed that the synthesis of AP was repressed over a PO₄^3? threshold between 0.4 and 1 μM. As lower PO₄^3? concentrations are regularly observed during A. catenella blooms in Thau lagoon, a significant portion of P uptake by A. catenella cells in the field may come from organic compounds.     

INHIBITORY EFFECTS OF CONCANAVALIN A AND TUNICAMYCIN ON SEXUAL ATTACHMENT OF ALEXANDRIUM CATENELLA (DINOPHYCEAE)1

The effect of concanavalin A (a lectin) and tunicamycin (an inhibitor of protein glycosylation) on sexual attachment of gamete pairs of the dinoflagellate Alexandrium catenella Whedon & Kofoid was studied. Concanavalin A inhibited sexual attachment at a concentration of 0.005%, and this inhibition was released by the addition of glucose or mannose at 10 mM. Mating type plus cells of A. catenella treated with tunicamycin for 12 h were not capable of sexual attachment. These results are the first to suggest the existence of a cell–cell recognition system in sexual attachment in A. catenella.     

DOES DINOPHYSIS (DINOPHYCEAE) HAVE A SEXUAL LIFE CYCLE?1

A variety of morphotypes (of two size classes) within two wild populations of Dinophysis acuta Ehrenberg and D. cf. acuminata are described. The observation that these two cell types engaged in the formation of couplets (possibly leading to the engulfment of the smaller by the larger cell) suggests that these species may undergo a process of sexual reproduction involving the fusion of anisogamous gametes. This behavior and asexual cell division involved a small portion of the population at any one time and took place rapidly at specific subsurface depths and time of day, the former during the evening, the latter during the morning. Even though definite proof of sexuality by Dinophysis was not obtained, the possiblity that sexual dimorphism exists in several species of the genus is discussed.     

IDENTIFICATION OF GROUP- AND STRAIN-SPECIFIC GENETIC MARKERS FOR GLOBALLY DISTRIBUTED ALEXANDRIUM (DINOPHYCEAE). I. RFLP ANALYSIS OF SSU rRNA GENES1

Two distinct small-subunit ribosomal RNA genes (SSU rDNAs), termed the “A gene” and “B gene,” were recently found in the toxic dinoflagellate Alexandrium fundyense Balech. A restriction fragment length polymorphism (RFLP) assay was developed to rapidly detect the A and B genetic markers. SSU rDNA from 58 cultures with species designations of A. tamarense (Lebour) Balech, A. catenella (Whedon et Kofoid) Balech, A. fundyense, A. affine (Fukuyo et Inoue)Balech, A. minutum Halim, A. lusitanicum Balech, and A. andersoni Balech were screened. These cultures represent toxic and non-toxic isolates from North America, western Europe, Thailand, Japan, Australia, and the ballast water of several cargo ships. The RFLP assay revealed five distinct groups. Three subdivided the A. tamarense/catenella/fundyense“species complex” into clusters defined by geographic origin, not by morphospecies designations. The fourth group consisted of A. affine, whereas the fifth group was represented by A. minutum, A. lusitanicum, and A. andersoni. The B gene was only found in A. tamarense, A. catenella, and A. fundyense, but not in all isolates. However, all North American isolates of this closely related group harbored this gene, and it also was found in some A. tamarense from scattered locations in Japan and in the ballast water of one ship that operated exclusively between Japan and Australia. Isolates without the B gene appeared to have only a single class of SSU rDNA. The B sequence was not essential for toxin production, but thus far those organisms harboring it were toxic. The A. tamarense/catenella/fundyense complex is composed of genetically distinct populations, within which may exist two or all three of the mophotypically defined species. The B gene is a promising taxonomic and biogeographic marker and may be useful for tracking the regional and/or global dispersal of particular populations.     

CONTROL OF GERMINATION OF ALEXANDRIUM TAMARENSE (DINOPHYCEAE) CYSTS FROM THE LOWER ST. LAWRENCE ESTUARY (CANADA)

Cysts of the toxic dinoflagellate Alexandrium tamarense (Lebour) Balech 1992 from the lower St. Lawrence estuary were used in a test of the following hypotheses: (1) cyst germination is triggered by a change in temperature, and (2) germination rate varies throughout the year and is controlled by a circannual internal biological clock. Results show that cyst germination was not affected significantly by temperature of incubation over the range 1°–16° C, and light showed no significant stimulation of germination. This is supported by the lack of effect of cyst incubation conditions during evaluation of the seasonal changes in germination rate (two temperatures: 4° and 15° C, and two light conditions: darkness and 150 μmol photons·m^?2·s^?1). Thus, direct environmental control through short-term increases in temperature and exposure to light has no effect on the germination of the cysts tested. The rate of germination, observed monthly over a 16-month period, showed low germination (<20%) over most of the period tested, except for a maximum reaching more than 50% germination in August to October of the second year of the experiment. This pattern was observed for cysts both from monthly field collections and from laboratory-stored cysts kept under constant environmental conditions (4° C, in the dark). The peak in germination observed under constant environmental conditions (in the laboratory), the almost coincidental increase in cyst germination observed for the field-collected cysts, and the absence of effects of temperature and light during incubation could be explained either by a temperature-controlled cyst maturation period (the time-temperature hypothesis of Huber and Nipkow 1923) or by the presence of an internal biological clock. However, the large decline in the rate of germination 2 months after the maximum provides strong support for the biological clock hypothesis. The ca. 12-month maturation (dormancy) period observed for the laboratory-stored cysts is the longest reported for this species to our knowledge; this might be related to the low storage temperature (4° C), which is close to bottom temperatures generally encountered in this environment (0° to 6° C). Similar field and laboratory storage temperatures could explain the coincidental increase in germination rate in the fall of the second year if cyst maturation is controlled by temperature. A fraction of the laboratory-stored cysts did not follow a rhythmic pattern: A rather constant germination rate of about 20% was observed throughout the year. This continuous germination of likely mature cysts may supplement the local blooms of this toxic dinoflagellate, as these often occur earlier than peak germination observed in late summer. It seems that two cyst germination strategies are present in the St. Lawrence: continuous germination after cyst maturation, with temperature controlling the length of the maturation period, and germination controlled by a circannual internal rhythm.     

GROWTH STIMULATION OF ALEXANDRIUM TAMARENSE (DINOPHYCEAE) BY HUMIC SUBSTANCES FROM THE MANICOUAGAN RIVER (EASTERN CANADA)1

In the St. Lawrence Estuary, annual recurrent blooms of the toxic dinoflagellate Alexandrium tamarense L. Balech are associated with brackish waters. Riverine inputs are suspected to favor bloom development by increasing water column stability and/or by providing growth stimulants such as humic substances (HS). A 17‐day culture experiment was conducted to evaluate the importance of HS as growth factors for A. tamarense. Nonaxenic cultures were exposed to four HS extracts from three different sources: humic and fulvic acids isolated from the Manicouagan River, Quebec, Canada; humic acids from the Suwannee River, Georgia, United States; and a desalted alkaline soil extract. For each extract, four concentrations were tested as supplements to the artificial Keller medium, a nitrate‐rich algal culture medium. Additions of HS from all sources significantly enhanced the overall growth rates relative to the controls. Concentrations of HS, estimated by UV spectrophotometry, remained constant throughout the exponential growth phase, suggesting that the HS were acting mainly as growth promoters during our experiment. Dose–response curves indicated that HS could increase the growth rate of A. tamarense even at low concentrations, such as those encountered in the St. Lawrence Estuary. Our results support the hypothesis that HS from the Manicouagan River plume can stimulate the development of toxic dinoflagellate blooms.     

CYST FORMATION IN THE RED TIDE DINOFLAGELLATE ALEXANDRIUM TAMARENSE (DINOPHYCEAE): EFFECTS OF IRON STRESS1

The toxic red tide dinoflagellate Alexandrium tamarense (Lebour) Balech (synonymous with Protogonyaulax tamarensis (Lebour) Taylor) was subjected to iron stress in batch culture over a 24-day time course. Monitoring of life history stages indicated that iron stress induced formation of both temporary (= pellicular) and resting (= hypnozygotic) cysts. Our experimental induction of sexuality appeared to be associated with iron limitation rather than the total depletion of biologically available iron. Degenerative changes in organelle (i.e. chloroplast, mitochondrion and chromosome) ultrastructure were largely restricted to pellicular cysts, suggesting that these temporary cysts were more susceptible to short-term iron stress effects than were hypnozygotes. These results are consistent with the hypothesized ecological roles of cysts in maintaining viability over brief (pellicular cysts) and extended (hypnozygotes) exposure to adverse environmental conditions.     

LSU rDNA-BASED RFLP ASSAYS FOR DISCRIMINATING SPECIES AND STRAINS OF ALEXANDRIUM (DINOPHYCEAE)1

In a previous study large-subunit ribosomal RNA gene (LSU rDNA) sequences from the marine dinoflagellates Alexandrium tamarense (Lebour) Balech, A. catenella (Whedon et Kofoid) Balech, A. fundyense Balech, A. affine (Fukuyo et Inoue) Balech, A. minutum Halim, A. lusitanicum Balech, and A. andersoni Balech were compared to assess inter- and intraspecific relationships. Many cultures compared in that study contained more than one class of LSU rDNA. Sequencing pooled clones of rDNA from single cultures revealed length heterogeneities and sequence ambiguities. This complicated sequence comparisons because multiple rDNA clones from a single culture had to be sequenced individually to document the different classes of molecules present in that culture. A further complication remained as to whether or not the observed intraculture sequence variations were reliable genetic markers or were instead artifacts of the polymerase chain reaction (PCR) amplification, cloning, and/or sequencing methods employed. The goals of the present study were to test the accuracy of Alexandrium LSU rDNA sequences using restriction fragment-length polymorphism (RFLP) analysis and to devise RFLP-based assays for discriminating among representatives of that group. Computer-assisted examination of the sequences allowed us to identify a set of restriction enzymes that were predicted to reveal species, strain, and intraculture LSU rDNA heterogeneities. All groups identified by sequencing were revealed independently and repeatedly by RFLP analysis of PCR-amplified material. Five ambiguities and one length heterogeneity, each of which ascribes a unique group of Alexandrium species or strains, were confirmed by restriction digests. Observed intraculture LSU rDNA heterogeneities were not artifacts of cloning and sequencing but were instead a good representation of the spectrum of molecules amplified during PCR reactions. Intraculture LSU rDNA heterogeneities thus serve as unique genetic markers for particular strains of Alexandrium, particularly those of A. tamarense, A. catenella, and A. fundyense. However, some of these “signature heterogeneities” represented a smaller portion of PCR product than was expected given acquired sequences. Other deviations from predicted RFLP patterns included incomplete digestions and appearance of spurious products. These observations indicate that the diversity of sequences in PCR product pools were greater than that observed by cloning and sequencing. The RFLP tests described here are useful tools for characterizing Alexandrium LSU rDNA to define the evolutionary lineage of cultures and are applicable at a fraction of the time, cost, and labor required for sequencing.     

OBSERVATIONS ON THE MORPHOLOGY AND SEXUAL REPRODUCTION OF COOLIA MONOTIS (DINOPHYCEAE)1

The surface morphology of the dinoflagellate Coolia monotis Meunier was compared with the surface morphology of Ostreopsis, The apical pore of C. monotis is similar in architecture to that of Ostreopsis but considerably longer (12 μm) than in O. heptagona (8–9 μm) and O. ovata (6–7 μm). A ventral pore in C. monotis is located on the right ventral margin between apical plate l′ and precingular plate 6″ and is similar in appearance and location to the ventral pore of O. ovata. The longitudinal flagellum (20 μm) in C. monotis is longer than in O. ovata (12 μ). Although Coolia and Ostreopsis appear to be distinctly different and should remain as two separate genera, they appear to be related. Cells of C. monotis divided by binary fission. Doubling time was 3–4 days in the logarithmic phase of growth at 23°C, 12:12 h L:D, 30–90 μE-m^?2·s^?1, and a salinity of 36%. Cultures reached cell densities of 2.5 × 10³ cells·L^?1 after 15 days of growth. The sexual process in C. monotis occurred in Erdschreiber's medium when Danish soil extract was substituted with mangrove sediment extract under the culture conditions described above. Gamete fusion produced large biflagellated planozygotes (70–75 μm diam). Planozygote maturation involved cytoplasmic reorganization, loss of motility, development of a spherical shape (80–90 μm diam), and two to three orange accumulation bodies. The cells at this stage appeared to be thin-walled cysts. Further development included reorganization of cyst contents, emergence of non-motile gametes, and development of chloroplasts, sulcus, and girdle. The nucleus of the newly formed cells occupied 50% or more of the total cell volume. Meiosis occurred in the cyst, but nuclear cyclosis was not observed. Four daughter cells were produced within 36–48 h, and motile gametes developed. The gametes exhibited sexuality for 2 months and completed the sexual life cycle by going through a thin-walled cyst stage.     

ALEXANDRIUM SATOANUM SP. NOV. (DINOPHYCEAE) FROM MATOYA BAY,CENTRAL JAPAN1

The gonyaulacoid dinofiagellate Alexandrium satoanum Yuki et Fukuyo sp. nov. is described from Matoya Bay, Pacific coast of central Japan. The species is distinctive in its conical epitheca with almost straight sides and dorsal concavity of the hypotheca. The plate formula is Po, pc, 4′, 6″, 6c, 10s, 5″″, and 2″″, including two accessory plates inside the sulcus. The apical pore plate is triangular and possesses an anterior attachment pore at the right margin. The first apical plate does not make contact with the apical pore plate and lacks a ventral pore. A posterior attachment pore lies at the center of the posterior sulcal plate. In Matoya Bay, vegetative cells occur as solitary cells or sometimes in pairs during late spring and early summer in low concentrations. In connection with this study, the following new combination is proposed: Alexandrium pseudogonyaulax (Biecheler) Horiguchi ex Yuki et Fukuyo comb. nov.     

ULTRASTRUCTURE OF GLOEODINIUM MONTANUM (DINOPHYCEAE)1

The freshwater dinoflagellate Gloeodinium montanum Klebs (1912) was examined with transmission and scanning electron microscopy. Micrographs of ultrathin sections revealed a series of membrane layers rather than the usual dinoflagellate theca in vegetative cysts and in legates. Swarmers had distinct pellicles but appeared to be devoid of thecal plates and vesicles. The organization of cysts and swarmers appeared remarkably similar. All cell types had typical dinoflagellate nuclei with condensed chromosomes. Chloraplasts had girdle lamellae. One pyrenoid per cell was also present in chloroplasts of vegetative cysts. Starch grains and oil globules were distributed throughout the cytoplasm. Large accumulation bodies and polyvesicular vacuoles were found in aging cysts. Trichocysts and flagellar hairs were absent. Two types of intra-cellular prokaryotic organisms were discovered.     

DETECTION OF THE TOXIC DINOFLAGELLATE ALEXANDRIUM FUNDYENSE (DINOPHYCEAE) WITH OLIGONUCLEOTIDE AND ANTIBODY PROBES: VARIABILITY IN LABELING INTENSITY WITH PHYSIOLOGICAL CONDITION

The toxic dinoflagellate Alexandrium fundyense Balech was grown under temperature- and nutrient-limited conditions, and changes in labeling intensity on intact cells were determined for two probe types: an oligonucleotide probe targeting rRNA and a monoclonal antibody (MAb) targeting a cell surface protein. In nutrient-replete batch culture, labeling with the rRNA probe was up to 400% brighter during exponential phase than during stationary phase, whereas MAb labeling did not change significantly with growth stage at the optimal growth temperature. In cultures grown at suboptimal, low temperatures, there was a significant difference between labeling intensity in stationary versus exponential phase for both probe types, with exponential cells labeling brighter with the rRNA probe and slightly weaker with the MAb. The decrease in rRNA probe labeling with increasing culture age was likely due to lower abundance of the target nucleic acid, as extracted RNA varied in a similar manner. With the MAb and the rRNA probes, slower growing cultures at low, nonoptimal temperature labeled 35% and 50% brighter than cells growing faster at warmer temperatures. Some differences in labeling intensity per cell disappeared when the data were normalized to surface area or volume, which indicated that the number of target antigens or rRNA molecules was relatively constant per unit area or volume, respectively. Slow growth accompanying phosphorus and nitrogen limitation resulted in up to a 400% decrease in labeling intensity with the rRNA probe compared to nutrient-replete levels, whereas the MAb labeling intensity increased by a maximum of 60%. With both probes, labeling was more intense under phosphorus limitation than under nitrogen limitation, and for all conditions tested, labeling intensity was from 600% to 3600% brighter with the MAb than with the rRNA probe. Thus, it is clear that significant levels of variability in labeling intensity can be expected with both probe types because of the influence of environmental conditions and growth stage on cellular biochemistry, cell size,rRNA levels, and the number or accessibility of cell surface proteins. Of the two probes tested, the rRNA probe was the most variable, suggesting that in automated, whole-cell assays, it can be used only in a semiquantitative manner. For manual counts, the human eye will likely accommodate the labeling differences. The MAb probe was less variable, and thus should be amenable to both manual and automated counts.     

PHASED OSCILLATIONS IN CELL NUMBERS AND NITRATE IN BATCH CULTURES OF ALEXANDRIUM TAMARENSE (DINOPHYCEAE)1

Alexandrium tamarense (M. Lebour) Balech strains isolated in spring 2007 from a single bloom in Thau lagoon have been grown in nonaxenic artificial media. For three strains showing large oscillations in biomass (crashes followed by recoveries) on a scale of several days, a significant relationship was observed between changes in cell densities (as in vivo fluorescence) and changes in nitrate concentrations. Increases in cell densities were accompanied by decreases in nitrate, while decreases in cell densities corresponded to increases in nitrate, presumably due to nitrification. Net increases in nitrate could reach up to 15 μmol N · L^?1 · d^?1 indicating a very active nitrifying archaeal/bacterial population. However, following population crashes, algal cells can recover and attain biomass levels similar to those reached during the first growth phase. This finding indicates that those archaea/bacteria do not compete for nutrients or do not hamper algal growth under those conditions. In contrast to diatoms, dinoflagellates such as A. tamarense do not excrete/exude dissolved organic matter, thus preventing excessive bacterial growth. This mechanism could help explain the recovery of this species in the presence of bacteria.     

RESTRICTION FRAGMENT LENGTH POLYMORPHISM OF RIBOSOMAL DNA INTERNAL TRANSCRIBED SPACER AND 5.8S REGIONS IN JAPANESE ALEXANDRIUM SPECIES (DINOPHYCEAE)1

The 5.8S ribosomal RNA (rDNA) gene and flanking internal transcribed spacers (ITS1 and ITS2)from 9 isolates of Alexandrium catenella (Whedon and Kofoid) Taylor, 11 isolates of A. tamarense (Lebour) Taylor, and single isolates of A. affine (Inoue et Fukuyo) Balech, A. insuetum Balech, and A. pseudogonyaulax (Biecheler) Horiguchi ex Yuki et Fukuyo comb. nov. from various locations in Japan were amplified using the polymerase chain reaction (PCR) and subjected to restriction fragment-length polymorphism (RFLP) analysis. PCR products from all strains were approximately 610 bp, inclusive of a limited region of the 18S and 28S rRNA coding regions. RFLP analysis using four restriction enzymes revealed six distinct classes of rDNA (“ITS types”). Restriction patterns of A. catenella were uniform at the intra-specific level and clearly distinguishable from those of A. tamarense. The patterns associated with A. tamarense (“tamarense group”) were also uniform except for one strain, WKS-1. Some restriction fragments from WKS-1 were in common with those of A. catenella or A. tamarense, whereas some were distinct from all Alexandrium species tested. Alexandrium affine, A. insuetum, and A. pseudogonyaulax carry unique ITS types. The ITSs of the “tamarense group” exhibit sequence heterogeneity. In contrast, the ITSs of all other isolates (including WKS-1) appear homogeneous. RFLP analysis of the 5.8S rDNA and flanking ITSs regions from Alexandrium species reveals useful taxonomic and genetic markers at the species and/or population levels.     

NITROGENOUS NUTRITION OF ALEXANDRIUM CATENELLA (DINOPHYCEAE) IN CULTURES AND IN THAU LAGOON,SOUTHERN FRANCE1

Alexandrium catenella (Whedon et Kofoid) Balech was isolated from Thau lagoon (northern Mediterranean) and its growth and uptake characteristics measured for nitrate, ammonium, and urea. Although affinity constants did not indicate a preference for ammonium over nitrate, there was a strong inhibition of nitrate uptake by ammonium when both nitrogen (N) sources were present. Nitrogen budgets during growth in cultures revealed major imbalances between decreases in dissolved N and increases in particulate N, indicating excretion of dissolved organic N during the early part of the growth phase and uptake during the later part. A quasi‐unialgal bloom in November 2001 (4×10⁶ cells·L^?1) allowed measurements of uptake of nitrate, nitrite, ammonium, and urea; net and gross growth rate of A. catenella; and grazing rates on this organism. The affinity constants indicate that it is not a strong competitor for the N nutrients tested when these are in low concentrations (<10 μgat N·L^?1), compared with other members of the phytoplankton community. Indirect evidence from cultures indicate that dissolved organic N compounds could be important in triggering those blooms. Finally, the strongly unbalanced growth observed in the field indicates that A. catenella exhibits a storage rather than a growth response to a nutrient pulse and is adapted to low frequency events such as the passage of frontal disturbances. The disappearance of A. catenella was due to grazing that balanced growth at the peak of the bloom.