Identification and quantification of the toxic dinoflagellate Gymnodinium sp. with competitive enzyme-linked immunosorbent assay (cELISA)

Polyclonal antibodies were raised against Gymnodinium sp. by immunizing rabbits with cells of the axenic strain. Based on the species-specific antiserum, an indirect competitive enzyme-linked immunosorbent assay (cELISA) was developed to identify and quantify Gymnodinium sp. A standard curve was established to correlate the cELISA signal to cell amount on a logit-log basis in the linear range between 24 and 6,250,000 cells, and the equation deducted was ln[A/(A₀ − A)]= 4.9193 − 1.1006 log[cell amount] (R² = 0.9948, n = 5). The detection limit was found to be 12 cells. The intra-assay and inter-assay coefficients of variation (CVs) were 5.8% and 9.7%, respectively. Field samples collected from Jiaozhou Bay, China were used to assess the robustness of the method. The results showed high agreement with that of cell-counting with a light microscope. The good reproducibility and precision of the cELISA implied that this new technique could be used for fast quantification of Gymnodinium sp.     

Gymnoxanthella radiolariae gen. et sp. nov. (Dinophyceae), a dinoflagellate symbiont from solitary polycystine radiolarians

The symbiotic dinoflagellate Gymnoxanthella radiolariae T. Yuasa et T. Horiguchi gen. et sp. nov. isolated from polycystine radiolarians is described herein based on light, scanning and transmission electron microscopy as well as molecular phylogenetic analyses of SSU and LSU rDNA sequences. Motile cells of G. radiolariae were obtained in culture, and appeared to be unarmored. The cells were 9.1–11.4 μm long and 5.7–9.4 μm wide, and oval to elongate oval in the ventral view. They possessed an counterclockwise horseshoe‐shaped apical groove, a nuclear envelope with vesicular chambers, cingulum displacement with one cingulum width, and the nuclear fibrous connective; all of these are characteristics of Gymnodinium sensu stricto (Gymnodinium s.s.). Molecular phylogenetic analyses also indicated that G. radiolariae belongs to the clade of Gymnodinium s.s. However, in our molecular phylogenetic trees, G. radiolariae was distantly related to Gymnodinium fuscum, the type species of Gymnodinium. Based on the consistent morphological, genetic, and ecological divergence of our species with the other genera and species of Gymnodinium s.s., we considered it justified to erect a new, separate genus and species G. radiolariae gen. et sp. nov. As for the peridinioid symbiont of radiolarians, Brandtodinium has been erected as a new genus instead of Zooxanthella, but the name Zooxanthella is still valid. Brandtodinium is a junior synonym of Zooxanthella. Our results suggest that at least two dinoflagellate symbiont species, peridinioid Zooxanthella nutricula and gymnodinioid G. radiolariae, exist in radiolarians, and that they may have been mixed and reported as “Z. nutricula” since the 19th century.     

Phylogenetic study of Gymnodinium dorsalisulcum comb. nov. from tropical Australian coastal waters (Dinophyceae)

A new species of the dinoflagellate genus Gymnodinium Stein, previously considered a member of Katodinium Fott, is characterized from two marine benthic habitats in tropical northern Australia. Gymnodinium dorsalisulcum comb. nov. was found to be very abundant at times, and in culture produced large quantities of mucus. We analyzed two regions of ribosomal DNA from this species (partial large subunit and complete small subunit sequences), using Bayesian analysis and phylogenetic models appropriate to alignments of ribosomal RNA genes. We compared it to eight species of the ‘true’Gymnodinium clade and to other dinoflagellates. The results show that it is a member of the Gymnodinium clade, and is closely related to Gymnodinium impudicum and G. chlorophorum. Katodinium was originally defined as having cells with an epitheca that is much larger than the hypotheca. However, this character is clearly inadequate, and the genus requires a re‐investigation to determine the apomorphies of the type species.     

Gymnodinium blooms in the Helgoland bight (North Sea) during August, 1968

Summary 1. As in other parts of the North Sea, dinoflagellate red tides occurred in Helgoland waters in August, 1968. Measurements of plankton, and physical and chemical water properties at the permanent station Helgoland Roads were analyzed to describe the blooms. In addition, planktological and hydrographical investigations at three areas south, southwest and northwest of Helgoland on 27 and 28 August, as well as at two drifting stations off the mouths of the Elbe and Eider rivers on 6 and 8 August, were used for this work.2.Gymnodinium sp. was abundant at all these localities, forming blooms near Helgoland from 14 to 30 August, with a maximum of 3 to 3.25 × 10⁶ cells/l (and 18 to 19µg chlorophylla/l) on 28 and 30 August at Helgoland Roads. The primary production was as high as 0.98 mg C/l in 6 hours in a suspension of 3.7 × 10⁶ Gymnodinium/l, where diatoms had been removed. This means that one millionGymnodinium produced 0.265 mg C in 6 hours. At Helgoland Roads nitrate and nitrite were depleted at times, but not phosphate.3. About 3 × 10⁶ Gymnodinium sp./l were found in the upper 16.5 m of water investigated around Helgoland on 27 and 28 August (maximal 7.8 × 10⁶ cells/l at 3 m). There was a marked vertical stratification ofGymnodinium with a concentration towards the surface during the day. This was particularly the case in the turbid water off the Elbe estuary, where numbers up to 0.3 × 10⁶/l were counted.Gymnodinium sp. formed 96 to 99 % of the phytoplankton biomass during maximal development. The chlorophylla content of one millionGymnodinium was only 3.5µg. An extinktion of E=0.083/1 m was measured in a suspension of 10⁶ Gymnodinium/l in samples with minimal other plankton and detritus.4. The water masses containingGymnodinium blooms, which reached Helgoland after a change of wind direction, were characterized by lower salinity and higher temperature. This indicates that blooms developed in the coastally influenced water masses east of Helgoland. The red tides occurred during a period of minimal discharge of Elbe river water and of relatively high salinity of the coastal water in the Inner German Bight. They developed after a long period of calm winds. There were no records of reported fish or shellfish poisoning.

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Gymnodinium-Wucherungen in der Helgoländer Bucht (Nordsee) im August 1968

Kurzfassung Wie in anderen Teilen der Nordsee traten auch in der Helgoländer Bucht im August 1968 starke Wucherungen von Dinoflagellaten auf. Untersuchungen des Planktons und der Wassereigenschaften auf der Helgoland-Reede sowie auf weiteren Stationen in der Helgoländer Bucht wurden für die Erklärung dieser Erscheinung ausgewertet. In allen untersuchten Gebieten (Fig. 1) fand sichGymnodinium sp. als dominante Planktonform. Gymnodinien-Wucherungen traten auf der Helgoland-Reede in der Zeit vom 14.–30. August auf und erreichten Zellzahlen von über 3 × 10⁶/l am 28. und 30. August, bei nur 18–19µg Chlorophylla/l. Eine Million Gymnodinien produzierten 0,265 mg C in 6 Stunden im Inkubator. Etwa 3 × 10⁶ Gymnodinium sp. wurden in den obersten 16,5 m der Wassersäule in der Nähe Helgolands während des Blüte-Maximums gemessen (maximal 7,8 × 10⁶/l in 3 m). Die Gymnodinien waren tagsüber in den obersten Metern konzentriert. Eine solche Vertikalschichtung war besonders im Küstenwasser vor der Elbmündung ausgeprägt, wo bis zu 0,3 × 10⁶ Zellen/l gefunden wurden.Gymnodinium sp. bildete 96–99 % der Biomasse während der Blüte bei Helgoland. Es wurden ein Chlorophyll-a-Gehalt von nur 3,5µg für 10⁶ Zellen ermittelt und eine Extinktion von E=0,083/1 m für eine Suspension von 10⁶ Gymnodinien/l in Proben mit minimalem Gehalt an übrigem Plankton und Detritus gemessen. Der Nitrat- und Nitritgehalt im Wasser war zeitweise erschöpft, nicht aber das Phosphat. Wenn Wassermassen mitGymnodinium-Blüten nach Helgoland gedriftet wurden, fiel der Salzgehalt, und die Temperatur stieg an, was auf eine Entstehung der Wucherungen im brackwasser-beeinflußten Gebiet östlich Helgolands hindeutet. DieGymnodinium-Blüten entwickelten sich nach einer außergewöhnlich langen windarmen Periode und während eines minimalen Süßwasserzuflusses aus der Elbe. Vergiftungserscheinungen an Seetieren wurden bei Helgoland nicht festgestellt.

     

Cyst‐motile stage relationship and molecular phylogeny of a new freshwater dinoflagellate Gymnodinium plasticum from Plastic Lake,Canada

The dinophyceaen genus Gymnodinium was established with the freshwater species G. fuscum as type. According to Thessen et al. (2012), there are 268 species, with the majority marine species. In recently published molecular phylogenies based on ribosomal DNA sequences, Gymnodinium is polyphyletic. Here, a new freshwater Gymnodinium species, G. plasticum, is described from Plastic Lake, Ontario, Canada. Two strains were established by incubating single cysts, and their morphology was examined with light microscopy and scanning electron microscopy. The cyst had a rounded epicyst and hypocyst with a wide cingulum and smooth surface. Vegetative cells were characterized by an elongated nucleus running vertically and a deep sulcal intrusion. The apical structure complex was horseshoe‐shaped and consisted of two pronounced ridges with a deep internal groove, encircling 80% of the apex. Small subunit ribosomal DNA (SSU rDNA), large subunit ribosomal DNA (LSU rDNA) and internal transcribed spacer (ITS) sequences were obtained from cultured strains. Molecular phylogeny based on concatenated SSU, LSU and ITS sequences supports the monophyly of the Gymnodiniales sensu stricto clade but our results suggest that many Gymnodinium species might need reclassification. Gymnodinium plasticum is closest to Dissodinium pseudolunula in our phylogeny but distant from the type species G. fuscum, as are the other gymnodiniacean taxa.     

Gymnodinium smaydae n. sp., a New Planktonic Phototrophic Dinoflagellate from the Coastal Waters of Western Korea: Morphology and Molecular Characterization

The marine phototrophic dinoflagellate Gymnodinium smaydae n. sp. is described from cells prepared for light, scanning, and transmission electron microscopy. Also, sequences of the small (SSU) and large subunits (LSU) and the internal transcribed spacer region (ITS1–5.8S–ITS2) of ribosomal DNA were analyzed. This newly isolated dinoflagellate possessed nuclear chambers, nuclear fibrous connective, an apical groove running in a counterclockwise direction around the apex, and a major accessory pigment peridinin, which are four key features for the genus Gymnodinium. The epicone was conical with a round apex, while the hypocone was ellipsoid. Cells growing photosynthetically were 6.3–10.9 μm long and 5.1–10.0 μm wide, and therefore smaller than any other Gymnodinium species so far reported except Gymnodinium nanum. Cells were covered with polygonal amphiesmal vesicles arranged in 11 horizontal rows, and the vesicles were smaller than those of the other Gymnodinium species. This dinoflagellate had a sharp and elongated ventral ridge reaching half way down the hypocone, unlike other Gymnodinium species. Moreover, displacement of the cingulum was 0.4–0.6 × cell length while in other known Gymnodinium species it is less than 0.3 × cell length. In addition, the new species possessed a peduncle, permanent chloroplasts, pyrenoids, trichocysts, pusule systems, and small knobs along the apical furrow, but it lacked an eyespot, nematocysts, and body scales. The sequence of the SSU, ITS1–5.8S–ITS2, and LSU rDNA region differed by 1.5–3.8%, 6.0–17.4%, and 9.1–17.5%, respectively, from those of the most closely related species. The phylogenetic trees demonstrated that the new species belonged to the Gymnodinium clade at the base of a clade consisting of Gymnodinium acidotum, Gymnodinium dorsalisulcum, Gymnodinium eucyaneum, etc. Based on morphological and molecular data, we suggest that the taxon represents a new species, Gymnodinium smaydae n. sp.     

Environment-dependent metabolic investments in the mixotrophic chrysophyte Ochromonas

Mixotrophic protists combine photosynthesis and phagotrophy to obtain energy and nutrients. Because mixotrophs can act as either primary producers or consumers, they have a complex role in marine food webs and biogeochemical cycles. Many mixotrophs are also phenotypically plastic and can adjust their metabolic investments in response to resource availability. Thus, a single species's ecological role may vary with environmental conditions. Here, we quantified how light and food availability impacted the growth rates, energy acquisition rates, and metabolic investment strategies of eight strains of the mixotrophic chrysophyte, Ochromonas. All eight Ochromonas strains photoacclimated by decreasing chlorophyll content as light intensity increased. Some strains were obligate phototrophs that required light for growth, while other strains showed stronger metabolic responses to prey availability. When prey availability was high, all eight strains exhibited accelerated growth rates and decreased their investments in both photosynthesis and phagotrophy. Photosynthesis and phagotrophy generally produced additive benefits: In low-prey environments, Ochromonas growth rates increased to maximum, light-saturated rates with increasing light but increased further with the addition of abundant bacterial prey. The additive benefits observed between photosynthesis and phagotrophy in Ochromonas suggest that the two metabolic modes provide nonsubstitutable resources, which may explain why a tradeoff between phagotrophic and phototrophic investments emerged in some but not all strains.     

Über den Feinbau von Theka,Pusule und Golgi-Apparat bei dem DinoflagellatenGymnodinium spec.

Zusammenfassung Das Pusulensystem vonGymnodinium spec. besteht aus röhrenförmigen Invaginationen des Plasmalemmas mit zarten Rippen auf der Lumen-Seite, die eng von einer besonderen, perforierten Zisterne umschlossen sind. Die Degeneration der Pusule, der Feinbau der Theka mit Trichocysten-ähnlichen Vesikeln und Kragengruben, die Bildung der Cystenwand, das Stigma und seine Verbindung mit der Geißelbasis und der Golgi-Apparat mit verschiedenen Vesikeltypen werden beschrieben.

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Ultrastructural studies on the theca, pusule, and golgi apparatus in the dinoflagellateGymnodinium spec.

Summary The pusule system ofGymnodinium spec. consists of tubes formed by the invaginated plasmalemma which is ornamented with delicate ribs on its luminal surface and tightly surrounded by a special, perforated cisterna. The degeneration of the pusule, the fine structure of the theca with some peculiar structures (trichocyst-like vacuoles, collared pits, the developing cyst wall), the stigma and its connection with the flagellar base, and the Golgi apparatus with different types of vesicles are described.

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Wir danken Herrn Dr. W. Koch, Göttingen, für die Überlassung derGymnodinium-Kultur und der Deutschen Forschungsgemeinschaft für Sachbeihilfen.     

Inducible Mixotrophy in the Dinoflagellate Prorocentrum minimum

Prorocentrum minimum is a neritic dinoflagellate that forms seasonal blooms and red tides in estuarine ecosystems. While known to be mixotrophic, previous attempts to document feeding on algal prey have yielded low grazing rates. In this study, growth and ingestion rates of P. minimum were measured as a function of nitrogen (‐N) and phosphorous (‐P) starvation. A P. minimum isolate from Chesapeake Bay was found to ingest cryptophyte prey when in stationary phase and when starved of N or P. Prorocentrum minimum ingested two strains of Teleaulax amphioxeia at higher rates than six other cryptophyte species. In all cases ‐P treatments resulted in the highest grazing. Ingestion rates of ‐P cells on T. amphioxeia saturated at ~5 prey per predator per day, while ingestion by ‐N cells saturated at 1 prey per predator per day. In the presence of prey, ‐P treated cells reached a maximum mixotrophic growth rate (μ_max) of 0.5 d^?1, while ‐N cells had a μ_max of 0.18 d^?1. Calculations of ingested C, N, and P due to feeding on T. amphioxeia revealed that phagotrophy can be an important source of all three elements. While P. minimum is a proficient phototroph, inducible phagotrophy is an important nutritional source for this dinoflagellate.     

Phagotrophic Ingestion of a Blue-Green Alga by Ochromonas*†

Anacystis nidulans disappeared rapidly from culture in the presence of an unidentified species of Ochromonas. Disappearance was light-independent and could be induced neither by bacteria associated with, nor by soluble products released from the flagellate. Electronmicrographs of mixed cultures revealed numerous A. nidulans cells in various stages of digestion within vacuoles of Ochromonas. Evidently the disappearance of the alga from culture resulted from phagotrophy by the chrysomonad. A 2-stage digestive process is suggested whereby A. nidulans cells are initially sequestered in the posterior “leucosin” vacuole and then undergo the terminal stages of digestion and elimination in smaller, peripheral vacuoles.     

DEGRADATION OF THE CELL-WALL POLYSACCHARIDE OF PORPHYRIDIUM SP. (RHODOPHYTA) BY MEANS OF ENZYMATIC ACTIVITY OF ITS PREDATOR,GYMNODINIUM SP. (PYRROPHYTA)1

The dinoflagellate Gymnodinium sp., which preys specifically on cells of the red microalga Porphyridium sp., possesses enzymes that degrade exocellular polysaccharides of the Porphyridium sp. A crude extract of Gymnodinium sp. was applied to this polysaccharide, and the degradation products were characterized by charge and size separations. Charge separation revealed the presence of a fraction that was not found in the native polysaccharide. This fraction, which was eluted from an anion-exchange resin with water alone, was composed mostly of glucose and xylose (in a 1:1 weight ratio). Size separation of the degradation products revealed three fractions; the molecular weight of the main one was 5 × 10⁶ daltons, whereas that of the native polysaccharide was 7 × 10⁶ daltons. The carbohydrate composition of these fractions was determined. Although the main product of degradation had a relatively high molecular weight, its viscosity was significantly reduced relative to the native polysaccharide. Additional enzymatic degradation is required for further exploration of the structure of the exocellular polymer of Porphyridium sp.     

Fourteen FITC-conjugated lectins as a tool for the recognition and differentiation of some harmful algae in Chinese coastal waters

In order to test the use of lectins as a tool for the differentiation of harmful algal species, 13 species and 23 strains of algae were tested with 14 fluorescein isothiocyanate (FITC)-conjugated lectins, and the results examined using flow cytometry (FCM), epifluorescence microscopy (EFM) and spectrofluorometry (SFM). The lectin probes SBA, WGA, GSL I, DBA and PHA-E could distinguish between morphologically similar Gymnodinium-like species, such as Karenia mikimotoi (GMDH01), Takayama pulchellum (TPXM01) and Gymnodinium sp. (GspXM01), by their different binding activities. With the precise quantitative measurements of binding obtained using SFM and FCM, lectins appeared to be useful in distinguishing different strains of the same species. The results also showed that PHA-E could differentiate Alexandrium tamarense (ATDH04) from other strains of this species, and SJA could distinguish A. tamarense (ATMJ02) from other strains of this species (including ATMJ01). Similarly, PNA could identify A. tamarense (ATDH01, 02, 03); UEA I could recognize A. tamarense (ATCI01-JN, ATCI01); and RCA120 could differentiate Alexandrium sp (AspGX01) from strain AspGX02, which was shown to produce different levels of paralytic shellfish poisoning toxin. Lectin probes could also bind these target cells in mixed algal samples. Positive cells identified by FCM were clearer than negative cells thus, in EFM, both GspXM01 and TPXM01 labeled with a WGA lectin probe could be distinguished from target cells of K. mikimotoi, Prorocentrum donghaiense and P. minimum (PMDH01, PMXM01) in mixed algal samples. FCM, EFM and SFM analysis could clearly distinguish lectin-probe-bound cells from negative cells in culture.     

On the Fine Structure of Trypanosoma cruzi in Tissue Cultures of Pigment Epithelium from the Chick Embryo. Uptake of Melanin Granules by the Parasite*

Trypanosoma cruzi has been cultured in pigment epithelial cells of the iris from the chick embryo. Melanin granules, identical with those of the host cells were found in the intracellular, amastigote (leishmania) forms. In many of the intracellular forms cytostome-like structures were seen, often in intimate contact with the pigment granules of the host cells, which suggests the uptake of melanin granules through the cytostomes by the process of intracellular phagotrophy.     

Cytochalasin D inhibits completion of cytokinesis and affects theca formation in dinoflagellates

Summary In the presence of cytochalasin D, dinoflagellates undergo mitosis and the cells begin to divide, but the completion of cell division is inhibited. InPausenella (dinospore formation),Gymnodinium andProrocentrum, Siamese twins arise which remain connected at the epicones whereas the hypocones, containing the nuclei, are separated. InScripsiella where the nucleus is centrally located, irregular binucleate cell bodies result. Cyst divisions which give rise to secondary or tertiary cysts inPaulsenella are not affected. In the athecatesPaulsenella andGymnodinium the morphogenesis of the separated cell portions is not or nearly not, respectively, disturbed by cytochalasin D. In the thecatesScripsiella andProrocentrum morphogenesis is heavily affected. InProrocentrum, wrinkled theca material is deposited instead of complete valvae. Doubling of the flagellar apparatus is not inhibited. It is concluded that the first phase of cytokinesis does not depend on actin. The daughter cells begin to separate by a mechanism which seems to be associated with the mitotic apparatus. Actin, however, is involved in the further constriction of the cleavage furrow in the second phase of cytokinesis and in the morphogenesis of the theca.     

GRAZING AND GROWTH OF THE MIXOTROPHIC CHRYSOMONAD POTERIOOCHROMONAS MALHAMENSIS (CHRYSOPHYCEAE) FEEDING ON ALGAE

The growth and grazing characteristics of Poterioochromonas malhamensis (Pringsheim) Peterfi (= Ochromonas malhamensis Pringsheim) (ca. 8 μm) feeding on phytoplankton, including the cyanobacteria Synechococcus sp. (ca. 2 μm) and Microcystis viridis (A. Brown) Lemmermann (ca. 6 μm) and the green alga Chlorella pyrenoidosa Chick (ca. 13 μm), were investigated in laboratory experiments involving the following treatments: (1) light without added algal prey (autotrophy), (2) light with added algal prey (mixotrophy), and (3) dark with added algal prey (phagotrophy). There were significantly higher cell numbers under mixotrophic and phagotrophic growth than under autotrophic growth. With phytoplankton as food, growth rates under both mixotrophy and phagotrophy were about two or three times higher than those under autotrophy, indicating that the algal diets were readily able to support the population growth of P. malhamensis. There were no significant differences in growth rate between mixotrophic and phagotrophic cultures during exponential growth. The ingestion rate of P. malhamensis with algal prey was also similar under both continuous light and dark. Poterioochromonas malhamensis ingested on average 0.27 M. viridis cells·flagellate^{− 1} ·h^{− 1} and 0.18 C. pyrenoidosa cells·flagellate^{− 1} ·h^{− 1} in continuous light and 0.25 M. viridis cells·flagellate^{− 1} ·h^{− 1} and 0.18 C. pyrenoidosa cells·flagellate^{− 1} ·h^{− 1} in continuous dark during exponential growth. The results showed that light had no effect on the growth and ingestion rates of P. malhamensis for phagotrophy during exponential growth. However, phagotrophic populations of P. malhamensis were incapable of growth in continuous darkness for longer than 5 days. Populations of P. malhamensis showed no increase when prey was added again after 4 days in continuous darkness, indicating that light is necessary for sustained phagotrophic growth of P. malhamensis. The study suggests that P. malhamensis, which has strong tolerance for light, is light dependent for phagotrophy.     

Morphology,ultrastructure, and molecular phylogeny of Wangodinium sinense gen. et sp. nov. (Gymnodiniales,Dinophyceae) and revisiting of Gymnodinium dorsalisulcum and Gymnodinium impudicum

The genus Gymnodinium includes many morphologically similar species, but molecular phylogenies show that it is polyphyletic. Eight strains of Gymnodinium impudicum, Gymnodinium dorsalisulcum and a novel Gymnodinium‐like species from Chinese and Malaysian waters and the Mediterranean Sea were established. All of these strains were examined with light microscopy, scanning electron microscopy and transmission electron microscopy. SSU, LSU and internal transcribed spacers rDNA sequences were obtained. A new genus, Wangodinium, was erected to incorporate strains with a loop‐shaped apical structure complex (ASC) comprising two rows of amphiesmal vesicles, here referred to as a new type of ASC. The chloroplasts of Wangodinium sinense are enveloped by two membranes. Pigment analysis shows that peridinin is the main accessory pigment in W. sinense. Wangodinium differs from other genera mainly in its unique ASC, and additionally differs from Gymnodinium in the absence of nuclear chambers, and from Lepidodinium in the absence of Chl b and nuclear chambers. New morphological information was provided for G. dorsalisulcum and G. impudicum, e.g., a short sulcal intrusion in G. dorsalisulcum; nuclear chambers in G. impudicum and G. dorsalisulcum; and a chloroplast enveloped by two membranes in G. impudicum. Molecular phylogeny was inferred using maximum likelihood and Bayesian inference with independent SSU and LSU rDNA sequences. Our results support the classification of Wangodinium within the Gymnodiniales sensu stricto clade and it is close to Lepidodinium. Our results also support the close relationship among G. dorsalisulcum, G. impudicum, and Barrufeta. Further research is needed to assign these Gymnodinium species to Barrufeta or to erect new genera.     

Phagotrophy of fluorescently labeled bacteria by an oceanic phytoplankter

Using fluorescently-labeled bacteria and detection by flow cytometry and epifluorescence microscopy, we demonstrate inducible mixotrophy in a marine photosynthetic flagellate, Ochromonas sp. (class Chrysophyceae). Phagotrophic uptake of bacteria increases under conditions of low or limiting light and nutrients, but deceases in periods of prolonged darkness; sustained phagotrophy may require light. In addition, this alga appears to discriminate between and preferentially ingest different types of bacteria. Although this clone is primarily photosynthetic, phagotrophy contributes to its nutrition, especially when light or nutrients limit photosynthesis.Correspondence to: M.D. Keller     

Morphology,phylogeny and toxin profiles of Gymnodinium inusitatum sp. nov., Gymnodinium catenatum and Gymnodinium microreticulatum (Dinophyceae) from the Yellow Sea,China

Four Gymnodinium species have previously been reported to produce microreticulate cysts. Worldwide, Gymnodinium catenatum strains are conservative in terms of larger subunit (LSU) rDNA and internal transcribed spacer region (ITS) sequences, but only limited information on the molecular sequences of other species is available. In the present study, we explored the diversity of Gymnodinium by incubating microreticulate cysts collected from the Yellow Sea off China. A total of 18 strains of Gymnodinium, from three species, were established. Two of these were identified as Gymnodinium catenatum and Gymnodinium microreticulatum, and the third was described as a new species, Gymnodinium inusitatum. Motile cells of G. inusitatum are similar to those of Gymnodinium trapeziforme, but they only share 82.52% similarity in LSU sequences. Cysts of G. inusitatum are polygonal in shape, with its microreticulate wall composed of approximately 14 concave sections. G. microreticulatum strains differ from each other at 69 positions (88.00% similarity) in terms of ITS sequences, whereas all G. catenatum strains share identical ITS sequences and belonged to the global populations. Phylogenetic analyses, based on LSU sequences, revealed that Gymnodinium species that produce microreticulate cysts are monophyletic. Nevertheless, the genus as a whole appears to be polyphyletic. Paralytic shellfish toxins (PSTs) were found in all G. catenatum strains tested (dominated by 11-hydroxysulfate benzoate analogs and N-sulfocarmaboyl analogs) but not in any of the G. microreticulatum and G. inusitatum strains. Our results support the premise that cyst morphology is taxonomically informative and is a potential feature for subdividing the genus Gymnodinium.     

Description of a New Planktonic Mixotrophic Dinoflagellate Paragymnodinium shiwhaense n. gen., n. sp. from the Coastal Waters off Western Korea: Morphology,Pigments, and Ribosomal DNA Gene Sequence

ABSTRACT. The mixotrophic dinoflagellate Paragymnodinium shiwhaense n. gen., n. sp. is described from living cells and from cells prepared by light, scanning electron, and transmission electron microscopy. In addition, sequences of the small subunit (SSU) and large subunit (LSU) rDNA and photosynthetic pigments are reported. The episome is conical, while the hyposome is hemispherical. Cells are covered with polygonal amphiesmal vesicles arranged in 16 rows and containing a very thin plate‐like component. There is neither an apical groove nor apical line of narrow plates. Instead, there is a sulcal extension‐like furrow. The cingulum is as wide as 0.2–0.3 × cell length and displaced by 0.2–0.3 × cell length. Cell length and width of live cells fed Amphidinium carterae were 8.4–19.3 and 6.1–16.0 μm, respectively. Paragymnodinium shiwhaense does not have a nuclear envelope chamber nor a nuclear fibrous connective (NFC). Cells contain chloroplasts, nematocysts, trichocysts, and peduncle, though eyespots, pyrenoids, and pusules are absent. The main accessory pigment is peridinin. The sequence of the SSU rDNA of this dinoflagellate (GenBank AM408889) is 4% different from that of Gymnodinium aureolum, Lepidodinium viride, and Gymnodinium catenatum, the three closest species, while the LSU rDNA was 17–18% different from that of G. catenatum, Lepidodinium chlorophorum, and Gymnodinium nolleri. The phylogenetic trees show that this dinoflagellate belongs within the Gymnodinium sensu stricto clade. However, in contrast to Gymnodinium spp., cells lack nuclear envelope chambers, NFC, and an apical groove. Unlike Polykrikos spp., which have a taeniocyst–nematocyst complex, P. shiwhaense has nematocysts without taeniocysts. In addition, P. shiwhaense does not have ocelloids in contrast to Warnowia spp. and Nematodinium spp. Therefore, based on morphological and molecular analyses, we suggest that this taxon is a new species, also within a new genus.