SPECIES BOUNDARIES IN THE TOXIC DINOFLAGELLATE PROROCENTRUM LIMA (DINOPHYCEAE,PROROCENTRALES), BASED ON MORPHOLOGICAL AND PHYLOGENETIC CHARACTERS1

Wild and cultured specimens of Prorocentrum lima (Ehrenb.) F. Stein from 26 widely different areas in 13 countries were examined in order to determine consistent characters for delimiting species boundaries in this taxon. The morphological characters valve shape, valve size, valve ornamentation, number and shape of valve pores, number and shape of marginal pores, and periflagellar platelets were observed using LM and SEM, and two molecular genetic regions were sequenced. We identified stable morphological characters that were consistent among wild specimens and all cultures, which were valve shape, valve ornamentation, and number and arrangement of periflagellar platelets. All cultures of P. lima identified by these characters formed a monophyletic group in phylogenetic analyses based on the two genes, which, however, included the species Prorocentrum arenarium. P. arenarium was determined to be within the range of morphological variation of P. lima, and therefore we synonymize the two taxa. Within this monophyletic group, P. lima was divided into several subclades in the all phylogenetic analyses. There were no morphological characters specifically related to any one subclade. The subclades appeared to correlate broadly to sample collection regions, suggesting that geographically separated populations may have become genetically distinct within this epi‐benthic species. We have emended species boundaries in P. lima.     

NOTE PROROCENTRUM LIMA (DINOPHYCEAE) IN WATERS OF THE GREAT SOUTH CHANNEL NEAR GEORGES BANK

A 0–10 m plankton tow collected at one station in the Great South Channel near Georges Bank in June 1994 was rich in dinoflagellates, with the usually epibenthic Prorocentrum lima (Ehrenberg) Dodge present as the dominant species. Temperature and salinity profiles suggest that the phytoplankton population at this station may have been advected from waters on the southern flank of Georges Bank. This is the first unequivocal report of P. lima in the plankton community of the Great South Channel and its vicinity.     

MORPHOLOGICAL AND BIOCHEMICAL VARIABILITY OF THE TOXIC DINOFLAGELLATE PROROCENTRUM LIMA ISOLATED FROM THREE LOCATIONS AT HERON ISLAND, AUSTRALIA

Seventeen clones of the toxic, epiphytic-benthic dinoflagellate Prorocentrum lima ( Ehrenberg) Dodge isolated from three separate sites on the reef flats of Heron Island, Australia, were acclimated under the same set of environmental conditions. Morphological features examined for each clone included cell surface configuration, size, and dry weight. Physiological and biochemical features determined for each clone included reproduction rates, pigments (chlorophyll a, chlorophyll c₂, peridinin, and other xanthophylls), toxins (okadaic acid and methyl-okadaic acid), and macromolecular compounds (total protein, lipid, and carbohydrate). Variation in morphological features and reproduction rates of clones within and between sites was minimal and not significant. Also, variation in biochemical features within an individual site was low, but pronounced differences existed among sites, the most notable of which was toxin content (okadaic acid and methyl-okadaic acid). The greatest difference in biochemical features was between clones isolated from the southern site and clones isolated from the northern and southeastern sites. Results of a cluster analysis of clonal characters support the view that these two groups represent distinct genotypes. We suggest that these groups originated from separate seed sources and that the genetic integrity of each is maintained through asexual reproduction .     

A SINGLE-CELL IMMUNOASSAY FOR PHOSPHATE STRESS IN THE DINOFLAGELLATE PROROCENTRUM MINIMUM (DINOPHYCEAE)

Current techniques for studying phytoplankton physiology in the field, such as measurements of biochemical activities, nutrient addition bioassays, and determination of photosynthetic efficiency, are useful for assessing the physiology of the bulk community but suffer from a lack of specificity. This would be improved by the development of single-cell methods for monitoring in situ physiology. Here we develop and test an antibody-based assay for identifying phosphate stress in the model dinoflagellate Prorocentrum minimum (Pavillard) Schiller. Antiserum was raised against a cell-surface alkaline phosphatase purified from P. minimum. Western screening indicated that the antiserum reacted with phosphate-stressed cells but not nitrate-stressed or phosphate-replete cells in culture. Immunodepletion confirmed the identification of this protein as an alkaline phosphatase. Based on Western blots, the antiserum appeared to be specific for phosphate-regulated proteins in P. minimum because there is no discernible cross-reaction with closely related P. micans. A whole-cell immunofluorescence assay was used to identify phosphate stress in field populations of P. minimum from Narragansett Bay, Rhode Island. The percentage of labeled P. minimum cells in this environment during the summer of 1998 decreased through time as the inorganic phosphate concentration increased. The percentage of antibody-labeled cells significantly correlated with the percentage of ELF-97-labeled cells determined as another single-cell assay of phosphate stress. This is the first antibody-based method developed for monitoring cell-specific physiology in a dinoflagellate, and the method described here may serve as a model for developing similar tools in other species of phytoplankton.     

THE IDENTIFICATION AND PURIFICATION OF A CELL-SURFACE ALKALINE PHOSPHATASE FROM THE DINOFLAGELLATE PROROCENTRUM MINIMUM (DINOPHYCEAE)1

Two cell-surface proteins were identtjied in the dinoflagellate Prorocentrum minimum (Pavillard) Schilkr strain CCMP 1329 that are evident in phosphate-limited cultures, but not in nitrate-limited cultures or cultures growing exponentially in complete media. These proteins were detected by labeling cell-surface proteins with the biotinylating reagent succinimidyl 6-(biotinamido) hexanoate. One protein, of appoximately 200,000 daltons was purified using differential centrifugation, detergent extraction, and gel filtration chromatography. This purified protein was able to hydrolyze orthophosphate groups from p-nitrophenylphosphate at pH 8, indicating it is an alkaline phosphatase, although it is larger than other alkaline phosphatases isolated to date tom most microorganisms. This protein may be induced to help P. minimum cleave orthophosphate groups from organic forms of phosphate in marine environments. Ultimately, this protein could represent a unique antigen for developing an antibody probe for examining the relationships between phosphate stress and bloom formation in P. minimum, and perhaps other dinoflagellates, in the field.     

PRODUCTION OF 7-DEOXY-OKADAIC ACID BY A NEW CALEDONIAN STRAIN OF PROROCENTRUM LIMA (DINOPHYCEAE)

7-Deoxy-okadaic acid and okadaic acid were identified as the major diarrhetic shellfish poisoning (DSP) toxins produced by a New Caledonian strain of Prorocentrum lima Ehrenberg. Dinophysistoxin-1 was not produced by this strain. The cellular concentrations of 7-deoxy-okadaic acid were about one tenth that of okadaic acid and were maximal (∼1.4 pg·cell ^{− 1}) during the stationary growth phase of batch culture. Autolytic hydrolysis of cell extracts did not increase the concentrations of 7-deoxy-okadaic acid, whereas okadaic acid production increased more than 4-fold, indicating that 7-deoxy-okadaic acid, unlike okadaic acid, is not directly derived from large sulfated precursors. 7-Deoxy-okadaic acid could be detected by liquid chromatography-selected reaction monitoring mass spectrometry, HPLC-fluorescence detection after derivatization with 9-anthryldiazomethane (ADAM), and inhibition of protein phosphatases. The solvent washes currently used for solid-phase clean-up of ADAM-derivatized DSP samples elute derivatized 7-deoxy-okadaic acid, indicating that the current sample clean-up protocol for HPLC-fluorescence detection would miss any contamination by this toxin.     

COMPLEX GENE STRUCTURE OF THE FORM II RUBISCO IN THE DINOFLAGELLATE PROROCENTRUM MINIMUM (DINOPHYCEAE)1

We analyzed the unusually complex organization of the nuclear‐encoded (form II) RUBISCO gene in the dinoflagellate Prorocentrum minimum (Parvillard) Schiller by intensive genomic DNA and cDNA sequencing and Western blotting. Over 10 transcribed units (TUs) were detected, which varied dramatically in their 3′ untranslated region. Each TU appeared to contain four tandem copies of the RUBISCO coding region (1.46 kb each; coding unit, or CU) interspersed by a 63‐bp spacer; the four CUs in each TU were cotranscribed and apparently cotranslated to a tetrameric polyprotein that may undergo successive cleavage steps to yield mature RUBISCO. By means of real‐time PCR analysis, it was estimated that each of the P. minimum genome harbored 148±16 CUs. Although nucleotide sequences varied by 1%–9% among the detected CUs, their inferred amino acid sequences were essentially identical. Our results suggest that the complex structure of Pmrbc has been derived from extensive and repeated gene duplications, an evolutionary process that has also been observed for other dinoflagellate genes.     

PHYLOGENETIC ANALYSIS OF NINE SPECIES OF PROROCENTRUM (DINOPHYCEAE) INFERRED FROM 18S RIBOSOMAL DNA SEQUENCES, MORPHOLOGICAL COMPARISONS, AND DESCRIPTION OF PROROCENTRUM PANAMENSIS, SP. NOV.

Sequences of 18S rRNA genes were obtained from eight species of Prorocentrum Ehrenberg: P. minimum (Pavillard) Schiller, P. mexicanum Osorio Tafall, P. emarginatum Fukuyo, P. lima (Ehrenberg) Dodge, P. arenarium Faust, P. maculosum Faust, P. concavum Fukuyo, and P. panamensis, sp. nov. Prorocentrum panamensis is a new species of tropical dinoflagellate isolated from a benthic coral reef on the Pacific coast of Panama and described here using scanning electron microscopy. Cells are heart shaped, 46–52 μm long and 43–46 μm wide. The valve surfaces are areolate except in the central area. Pores of 0.15 μm in diameter are scattered in areolae, mainly around the periphery of the cell. The right valve has a specific ovoid depression with numerous appressed pores; we named this structure the sieve-like depression. The periflagellar area is nearly ovoid, located in a shallow depression, and almost equally set into both valves. It is unornamented (no apical expansion) but has numerous depressions in platelets. The flagellar and auxiliary pores are different in size and shape. The intercalary band is transversally striated. Phylogenetic relationships of gonyaulacoid, peridinioid, gymnodinioid, and prorocentroid dinoflagellates were inferred from complete 18S rDNA sequences. Two distinct phylogenetic analyses are presented for armored and unarmored Dinophyceae in an attempt to make the phylogenetic relationships between these different kinds of organisms clearer. The Prorocentrales appear to have a common origin, although two groups of Prorocentrum spp. are apparent. The first group includes benthic, symmetrical species (P. lima, P. arenarium, P. maculosum, and P. concavum). The second group contains planktonic and bentho-planktonic species (P. micans Ehrenberg, P. minimum, P. mexicanum, and P. panamensis sp. nov.). Genetic distances between species within these two groups were high; however, the divergence between the two groups seems to have occurred late in dinoflagellate evolution. In addition, the bentho-planktonic P. emarginatum appeared distantly related to both groups; however,its 18S rDNA sequence shares specific nucleotide substitutions with the two groups, suggesting an older origin of this species compared to the others. A morphological interpretation of this phylogenetic analysis is made on the basis of the specific structure of the periflagellar area. Finally, genetic data and morphological observations support the hypothesis that the genus Prorocentrum is rather heterogeneous; several species could be considered to constitute distinct genera.     

MORPHOLOGY AND TAXONOMY OF PROROCENTRUM MEXICANUM AND REINSTATEMENT OF PROROCENTRUM RHATHYMUM (DINOPHYCEAE)1

Dinoflagellates collected during red tide events in Bahia Mazatlan, Mexico during the early spring of 1999 and 2000 appeared under LM to belong to Prorocentrum mexicanum Osorio‐Tafall. Observations with SEM of those populations showed marked differences in shape and microornamentation from the related species, Prorocentrum rhathymum Loeblich III, Sherley and Schmidt. In P. mexicanum, the presence and dimensions of poroids, the uneven distribution of trichocyst pores not located in depressions, and the general architecture of the periflagellar region are more closely related to Prorocentrum caribbaeum Faust. Also, P. mexicanum has a three‐horned (sometimes two‐horned) spine and is deeper in the anterior than the posterior region, whereas P. rhathymum has a simple small spine and its sagittal view is oval. Furthermore, the number and distribution of trichocyst pores in the periflagellar area is different between the two species, being located on both valves in P. mexicanum and only on the right valve in P. rhathymum. To date, true P. mexicanum has been described only from plankton sampling, whereas P. rhathymum was frequently mentioned associated with floating detritus (macroalgae) but also forming red tides. Altogether, the evidence presented demonstrates that P. mexicanum (planktonic) and P. rhathymum (epibenthic) are distinct species and are not synonyms, as is often accepted.     

POTENTIAL TOLERANCE MECHANISMS OF PROROCENTRUM MICANS (DINOPHYCEAE) TO SUBLETHAL LEVELS OF COPPER1

We investigated how Prorocentrum micans Ehrenberg, a planktonic dinoflagellate common in Portuguese coastal waters, is able to tolerate and recover from sublethal concentrations of copper(II). The experimental design simulated events in inshore waters, where P. micans is subjected to high levels of pollutants, including copper. Decrease in growth rate, induction of a growth lag phase, temporary loss of motility, and potassium leakage were the effects induced in P. micans cultures by 90 nM labile copper. A 10–20-fold increase in cellular copper concentration was observed in toxicity experiments. Copper efflux (representing a 50% decrease in cellular metal content) was a short-term tolerance mechanism. A 25-kDa protein was detected after only 3 h of exposure to copper, but there was no evidence of phytochelatin synthesis. Ultracytochemical labeling of metals with the sulfide-silver procedure showed that copper was associated with the thecal plates, starch grains, and, to a lesser extent, lipid droplets. High values affixation capacities and average conditional stability constants for copper binding by starch, amylopectin, and cellulose support the location of copper in thecal plates and starch grains. We conclude that P. micans responds rapidly to copper toxicity and has two tolerance mechanisms for copper: copper efflux and sequestration in polymeric substances.     

ECTOCELLULAR GLUCOSIDASE AND PEPTIDASE ACTIVITY OF THE MIXOTROPHIC DINOFLAGELLATE PROROCENTRUM MINIMUM (DINOPHYCEAE)1

The activities of the enzymes α‐ and β‐glucosidase, and leucine aminopeptidase were measured in cultures of the dinoflagellate Prorocentrum minimum (Pavill.) J. Schiller and in field samples collected during dinoflagellate blooms occurring in tributaries of the Chesapeake Bay, Maryland, USA. Activities were measured using fluorogenic artificial substrates and partitioned among the >5 μm size fraction, small microbes fraction (0.1–5 μm), and dissolved phase (<0.1 μm). P. minimum and most other photosynthetic dinoflagellates are >5 μm in size and thus can be separated from the small microbes fraction, which contains most bacteria. Little to no glucosidase activity was detected associated with the >5 μm size fraction in cultures or in field samples, with most of the activity (67% to 93% in cultures, 54% to 100% in field samples) in the small microbes size fraction for both α and β glucosidase. In contrast, 67% to 90% of the total leucine aminopeptidase (LAP) activity in cultures was measured in the >5 μm fraction. Within a culture, LAP activity in the size fraction containing P. minimum decreased in response to ammonium and urea additions, but not in response to nitrate. In field samples, LAP activity was positively correlated with dinoflagellate abundance and chl a, and negatively correlated with ammonium concentration. During blooms, up to 34% of LAP activity was associated with the >5 μm fraction, indicating that when abundant, dinoflagellates may make a substantial contribution to ectocellular LAP activity in the water column.     

LIGHT AND ELECTRON MICROSCOPICAL OBSERVATIONS OF THE DINOFLAGELLATE ACTIMSCUS PESTASTERIAS (DINOPHYCEAE)1

I examined the heterotrophic non-armored dinoflaget-late Actiniscus pentasterias (Ehrenberg) Ehrenberg by light and electron microscopy. Actiniscus pentasterias contains an internal skeleton consisting of two star-like siliceous elements. Special emphasis is given to the flagellar apparatus, the nucleus, and a new type of extrusome, named a docidosome. A three dimensional model of the flagerllar apparatus includes a fibrous nuclear connnective, a posterior striated root, and a dorsal striated component of the longitudinal microtabular root. The nucleus is surrounded by a conspicuous fibrous lamina, also visible in the light microscope. The nuclear pores are situated in annulated invaginations of the nuclear envelope, increasing the nuclear surface area by 15–25%. The docidosomes are rod-shaped membrane-bound structures that terminate in a distinct proximal head. They show very complex substructure, consisting of an inner medulla with highly ordered paired ribbons and an outer cortex.     

PROROCENTRUM BELIZEANUM,PROROCENTRUM ELEGANS,AND PROROCENTRUM CARIBBAEUM,THREE NEW BENTHIC SPECIES (DINOPHYCEAE) FROM A MANGROVE ISLAND,TWIN CAYS,BELIZE1

Three new benthic dinoflagellate species, Prorocentrum belizeanum, Prorocentrum elegans, and Prorocentrum caribbaeum, from mangrove floating detritus are described from scanning electron micrographs. Species were identified based on shape, size, surface micromorphology, ornamentation of thecal plates, and architecture of the periflagellar area and intercalary band. Cells of P. belizeanum are round to slightly oval with a cell size of 55–60 μm long and 50–55 μm wide. Areolae are round and numerous (853–1024 per valve) and range from 0.66 to 0.83 μm in size. The periflagellar area of P. belizeanum is a broad V-shaped depression; it accommodates a flagellar and an auxiliary pore and a flared, curved apical collar. The intercalary band of P. belizeanum is horizontally striated. Prorocentrum elegans is a small species 15–20 μm long and 10–14 μm wide, with an ovate cell shape. The thecal surface is smooth. Two sizes of valve pores were recognized: large, round pores (20–22 per valve) arranged in a distinct pattern and smaller pores situated in an array along the intercalary band. The periflagellar area is V-shaped; it accommodates an uneven sized flagellar pore, an auxiliary pore, and an angled protuberant flagellar plate. The intercalary band is transversely striated. It is a bloom-forming species. Prorocentrum caribbaeum cells are heart-shaped with a rounded anterior end and a pointed posterior end. Cells range from 40 to 45 μm long and 30 to 35 μm wide. Thecal surface has two different-sized pores: large, round pores (145–203 per valve) arranged perpendicularly from the posterior margins, and small, round pores unevenly distributed on the thecal surface. The periflagellar area is ornate. It is V-shaped with a curved apical collar located next to the auxiliary pore; a smaller protuberant apical plate is adjacent to the flagellar pore. The intercalary band is transversely striated and sinuous. Cells are active swimmers.     

MORPHOLOGIC DETAILS OF SIX BENTHIC SPECIES OF PROROCENTRUM (PYRROPHYTA) FROM A MANGROVE ISLAND,TWIN CAYS,BELIZE, INCLUDING TWO NEW SPECIES1

Two new dinoflagellate species, Prorocentrum hoffmannianum and Prorocentrum ruetzlerianum, and four known species, Prorocentrum emarginatum Fukuyo 1981, Prorocentrum mesicanum Tafall 1942, Prorocentrum concavum Fukuyo 1981, and Prorocentrum lima (Ehr.) Dodge 1975, from floating detritus and sediments in a subtropical mangrove island, Twin Cays, Belize, Central America are described from scanning electron micrographs. Differences in the following characters of surface micromorphology separated the species: ornamentation of thecal plates (shape, size, and number of valve pores and areolae) and the architecture of the periflagellar area and intercalary band.     

MORPHOLOGY AND MOLECULAR PHYLOGENY OF PROROCENTRUM CONSUTUM SP. NOV. (DINOPHYCEAE), A NEW BENTHIC DINOFLAGELLATE FROM SOUTH BRITTANY (NORTHWESTERN FRANCE)1

A new marine benthic Prorocentrum species from sandy habitats of South Brittany (northwestern France), P. consutum sp. nov., is described using LM and SEM and molecular phylogenetic analyses. Cells have a subcircular to broadly ovoid shape and are plainly flattened. They are 57–61 μm long and 52–55 μm wide. A central pyrenoid is present, and the kidney‐shaped nucleus is positioned in the posterior region. In right valve view, the periflagellar area is deeply excavated, and the left valve forms a prominent apical ridge. The periflagellar area consists of nine platelets, and a small narrow collar is present around the flagellar pore. The ornamentation of this new species is very peculiar and is characterized by a ring of round areolae located at the periphery of the valves, each areola containing three or four pores. Apart from this ring of areolae, the cell surface is smooth and with scattered pores. Pores are not present in the center of the right or left valve. The intercalary band is generally narrow and faintly striated horizontally. The molecular phylogenetic position of P. consutum sp. nov. was inferred using SSU and LSU rDNA. In both analyses, this species branched with high support in the clade comprising species with a symmetric shape and appeared to be a sister group to that formed by P. lima and other tropical benthic species, such as P. arenarium, P. belizeanum, P. hoffmannianum, and P. maculosum.     

POTENTIAL UTILITY OF MITOCHONDRIAL CYTOCHROME B AND ITS MRNA EDITING IN RESOLVING CLOSELY RELATED DINOFLAGELLATES: A CASE STUDY OF PROROCENTRUM (DINOPHYCEAE)1

Difficulties often occur in separating closely related dinoflagellate species. In this study, the potential utility of mitochondrial cytochrome b (cob) gene sequence and mRNA editing characteristics was assessed using Prorocentrum Ehrenberg as a model. The cob sequences and the patterns of their mRNA editing were analyzed for several Prorocentrum taxa. Results revealed little difference in cob sequence and mRNA editing characteristics between geographic populations of P. minimum (Pavillard) Schiller, while a notable difference was detected between different species (P. minimum and P. micans Ehrenberg). Furthermore, these P. minimum populations consistently formed a tight cluster on phylogenetic trees inferred from cob sequences as well as mRNA editing characteristics, whereas different Prorocentrum species were well separated, with a genetic distance of 0.0042±0.0024 for the former and 0.0141±0.0012 for the latter (P<0.01; two‐tailed t‐test). When the analysis was applied to the case of P. donghaiense Lu et Goebel and CCMP1517 strain of P. dentatum Stein, no differences were detected between these two taxa with respect to cob mRNA editing pattern and only small differences equivalent to those between P. minimum populations were detected in terms of cob sequence. On the cob sequence‐ and editing‐based phylogenetic trees, P. donghaiense and P. dentatum CCMP1517 consistently clustered together at a position sister to P. minimum. The results suggest that cob, combined with its mRNA editing, can potentially be a useful delineator of Prorocentrum species, and that P. donghaiense and P. dentatum CCMP1517 are most likely the same species and both are closely related to P. minimum.     

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.