Phototaxis by the Dinoflagellate Gymnodinium splendens Lebour*

A microscope-television system was used to monitor quantitatively the behavior of Gymnodinium splendens Lebour in response to light. The predominant behavioral sequence upon stimulation is (a) an initial 2–5 sec cessation of movement (stop-response) followed by (b) positive phototaxis. The action spectra for each response are identical, having maxima at 450 and 280 nm. Upon measuring the percent response to a range of stimulus intensities, it is apparent that a stop-response is not a behavioral prerequisite for phototaxis. An identical circadian rhythm in photoresponsiveness is observed for phototaxis and for the stop-response with greatest light sensitivity occurring during the first 4 hr of the entrained light period. The implication of phototactic sensitivity and the phototactic circadian rhythm in diurnal vertical migration is discussed.     

Isolation and Chemical Composition of Dinoflagellate Nuclei*

Nuclei were isolated from Peridinium cinctum. Peridinium trochoideum, Gyrodinium cohnii (Cryptothecodinium cohnii) and Gymnodinium nelsoni. The nuclei of G. cohnii, P. trochoideum and G. nelsoni were found to contain ～ 6.9, 34 and 143 picograms of DNA respectively. The ratios to DNA of RNA, acid-soluble and acid-insoluble protein for G. cohnii were 0.32, 0.13 and 0.99, respectively. The corresponding values for P. trochoideum were 0.22, 0.08 and 1.22, while those for G. nelsoni were 0.21, 0.10 and 1.09. The chemical composition of dinoflagellate nuclei is compared and contrasted with that of typical eukaryotic nuclei. The culture of these difficult organisms also is discussed.     

The Nutrition of the Freshwater Dinoflagellate Ceratium hirundinella*

A defined medium was devised for a freshwater isolate of the dinoflagellate Ceratium hirundinella. Highest cell yields were produced at 7,700–10,000 lux. The optimum pH range was between 7.0 and 7.5: the optimum temperature 21°C. Ceralium hirundinella tolerated a wide range (per liter) of Ca (0.1–100 mg) and Mg (0.1–50 mg) ion concentrations. The optimum range for growth was 20–30 rns Ca and 10–30 mg Mg. Cells cultured in media lacking Ca often became teratological yet motilp and viable. Variations in the Ca:Mg ratio had little effect on cell yield if the sum of the concentrations of the 2 ions remained the same. Organic as well as inorganic sources of N and P were utilized. NH₄ sources became toxic at elevated levels (7 mg N liter^-1). Methionine was not used as N source. Cells could not be completely depleted of P, but concentrations ≤ 0.01 mg P liter^-1 resulted in poor growth. Vitamin B₁₂, but not thiamine or biotin, was required. Highest cell yields were at a PII-metals concentration of 30 ml liter^-1; a t 100-ml liter^-1 cell yield was very low. Additions (per liter) of Fe (0.5 mg) and Mo (0.1 mg) to the basal medium produced higher cell yields, but Cu (0.1 mg) and V (0.1 mg) inhibited growth.     

Cell Division in Gonyaulax catenella,A Marine Catenate Dinoflagellate*

Mitosis and cytokinesis are described and illustrated for the first time in the mesokaryotic, catenate dinoflagellate Gonyaulax catenella. A structure similar to the central body of G. tamarensis and G. monilata is shown by light and electron microscopy to be situated intranuclearly near the arms of the U-shaped interphase nucleus, and is suggested to function in the segregation of daughter chromosomes. This structure has the fine structure of a nucleolus, and it is suggested that the term central body be replaced by persistent nucleolus (= endosome). The time required for the completion of mitosis is 4–6 hr, while cytokinesis requires at least 2 hr. Cytokinesis begins during the mitotic cycle, and the plane of fission is perpendicular to the mitotic plane of division. Parental fission moieties are retained and shared by the daughter cells while either a new antero-sinistral moiety or a posterodextral moiety is synthesized by the dividing cell.     

Development and Evaluation of a PCR Based Assay for Detection of the Toxic Dinoflagellate, <Emphasis Type="Italic">Gymnodinium catenatum</Emphasis>(Graham) in Ballast Water and Environmental Samples

Gymnodinium catenatum is a bloom forming dinoflagellate that has been known to cause paralytic shellfish poisoning (PSP) in humans. It is being reported with increased frequency around the world, with ballast water transport implicated as a primary vector that may have contributed to its global spread. Major limitations to monitoring and management of its spread are the inability for early, rapid, and accurate detection of G. catenatum in plankton samples. This study explored the feasibility of developing a PCR-based method for specific detection of G. catenatumin cultures and heterogeneous ballast water and environmental samples. Sequence comparison of the large sub unit (LSU) ribosomal DNA locus of several strains and species of dinoflagellates allowed the design of G. catenatum specific PCR primers that are flanked by conserved regions. Assay specificity was validated through screening a range of dinoflagellate cultures, including the morphologically similar and taxonomically closely related species G. nolleri. Amplification of the diagnostic PCR product from all the strains of G. catenatum but not from other species of dinoflagellates tested imply the species specificity of the assay. Sensitivity of the assay to detect cysts in ballast water samples was established by simulated spiked experiments. The assay could detect G. catenatum in all ‘blank’ plankton samples that were spiked with five or more cysts. The assay was used to test environmental samples collected from the Derwent river estuary, Tasmania. Based on the results we conclude that the assay may be utilized in large scale screening of environmental and ballast water samples.     

Morphological variation and phylogenetic analysis of the dinoflagellate Gymnodinium aureolum from a tributary of Chesapeake Bay

Cultures of four strains of the dinoflagellate Gymnodinium aureolum (Hulburt) G. Hansen were established from the Elizabeth River, a tidal tributary of the Chesapeake Bay, USA. Light microscopy, scanning electron microscopy, nuclear-encoded large sub-unit rDNA sequencing, and culturing observations were conducted to further characterize this species. Observations of morphology included: a multiple structured apical groove; a peduncle located between the emerging points of the two flagella; pentagonal and hexagonal vesicles on the amphiesma; production and germination of resting cysts; variation in the location of the nucleus within the center of the cell; a longitudinal ventral concavity; and considerable variation in cell width/length and overall cell size. A fish bioassay using juvenile sheepshead minnows detected no ichthyotoxicity from any of the strains over a 48-h period. Molecular analysis confirmed the dinoflagellate was conspecific with G. aureolum strains from around the world, and formed a cluster along with several other Gymnodinium species. Morphological evidence suggests that further research is necessary to examine the relationship between G. aureolum and a possibly closely related species Gymnodinium maguelonnense.     

Purification of Plastids from the Dinoflagellate Lingulodinium

Peridinin-containing dinoflagellates are a group of generally marine and photosynthetic protists whose plastids display a number of unusual features. In particular, the plastid genome may be reduced to as few as a dozen genes, and it is not clear if all these genes are expressed. To begin to characterize the plastid proteins, we attempted to purify chloroplasts from the dinoflagellate Lingulodinium polyedrum. We tested several different protocols and found that the organelles were inherently fragile and difficult to isolate intact. In particular, standard purification protocols as described for higher plants produced only broken plastids, as judged by complete loss of the stromal protein RuBisCO. We found that small amounts of RuBisCO could be retained in the plastids if the cells were treated with cellulase prior to lysis. Finally, we report that almost all RuBisCO was retained in plastids prepared from cells subjected to a heat shock treatment, although cellular proteins were denatured by the treatment.     

Illumination or confusion? Dinoflagellate molecular phylogenetic data viewed from a primarily morphological standpoint

Recent molecular sequencing results involving multiple genes require evaluation in the light of preexisting morphological data, particularly as different methodologies and genes produce trees that are incongruent in some respects or have major areas with poorly supported branch resolution. The present paper summarizes the current situation, primarily from a morphologist's perspective. Most of the tabulation‐based groups are coherent in small subunit (SSU) and large subunit (LSD) trees; but some, notably the prorocentroids and peridinioids, are not. In prorocen‐troids this is primarily because of intrinsic inadequacies of the molecules to resolve their phylogeny. In peridinioids it seems to be because of paraphyly of the group. Other artefacts are noted, such as the drastically different positions of Oxyrrhis in phylogenetic trees based on SSU and protein genes, and of Noctiluca in SSU trees that include analyses with different numbers of nucleotides. Polyphyly in non‐tabulate or poorly known groups has been confirmed, as has been the presence of cryptic thecae in members of those groups (group misattribution). Whether or not some extant groups of athecate, wholly dinokaryotic forms originated prior to polytabulate groups, like the suessioids, peridinioids and gonyaula‐coids, remains unclear. Gymnodinioids with a spiral acrobase seem to have given rise to the more complex athecate forms, whereas morphological features of the genus Gymnodinium are consistent with it being a sister group to polytabulate taxa such as Woloszynskia and the suessioids. Peridinioids and gonyaulacoids appear to have originated after that split. Dinophysoid and prorocentroid dinoflagellates appear to be derived from peridinioid forms. Trees based on protein genes, such as actin or α‐ and β‐tubulin, may help resolve some of the positions of key groups, but they do not include enough taxa to be widely useful as yet.     

Virus-like Particles Suppress Growth of the Red-Tide-Forming Marine Dinoflagellate <Emphasis Type="Underline">Gymnodinium mikimotoi</Emphasis>

We isolated 2 virus-like agents that suppressed growth of Gymnodinium mikimotoi from coastal waters of the Uwa Sea, Japan. The agents found in the flagellate cells, named GM6 and GM7, were filterable in a 0.22-lm-pore filter with approximately 100-nm shapes. Electron microscopic observation showed the presence of virus-like particles in severely damaged G. mikimotoi cells infected by GM6. The growth-suppression activity of the agents (GM6 or GM7) was lost by heating at 50°C, with treatments of DNase and protease, and filtration through a 0.05-lm filter. Our results suggest that the agents are DNA viruses infectious to and virulent for G. mikimotoi. This is the first report of a virus-like agent specific to G. mikimotoi.     

Dinoflagellate cyst distribution along a shelf to slope transect of an oligotrophic tropical sea (Sunda Shelf, South China Sea)

From 51 surface samples collected along a shelf to slope transect of the Sunda Shelf, South China Sea, 36 taxa of organic‐walled dinoflagellate cysts are identified. Oligotrophic tropical shelf assemblages on the Sunda Shelf are dominated by gonyaulacoids such as Spiniferites species, Operculodinium centmcarpum and Operculodinium israelianum. Concentrations of dinoflagellate cysts in the shelf sediments are generally low and correlate well with the content of fine‐grained (clay and silt fraction) sediments. Detailed comparisons of sediment grain‐size distributions to concentrations of dominant dinoflagellate taxa (Spiniferites species, 0. centmcarpum and 0. israelianum) in the shelf sediments indicate that these taxa behave in water like sediment particles with size range φ 5.75–6.25 (13–18 μm). In contrast, slope assemblages in fine‐grained sediments are dominated by protoperidinioids. This may reflect higher nutrient availability as a result of weak winter upwelling. The concentrations of dinoflagellate cysts in the shelf sediments are mainly controlled by transport and winnowing processes and are probably not representative of surface water conditions.     

Dinoflagellate expressed sequence tag data indicate massive transfer of chloroplast genes to the nuclear genome

The peridinin-pigmented plastids of dinoflagellates are very poorly understood, in part because of the paucity of molecular data available from these endosymbiotic organelles. To identify additional gene sequences that would carry information about the biology of the peridinin-type dinoflagellate plastid and its evolutionary history, an analysis was undertaken of arbitrarily selected sequences from cDNA libraries constructed from Lingulodinium polyedrum (1012 non-redundant sequences) and Amphidinium carterae (2143). Among the two libraries 118 unique plastid-associated sequences were identified, including 30 (most from A. carterae) that are encoded in the plastid genome of the red alga Porphyra. These sequences probably represent bona fide nuclear genes, and suggest that there has been massive transfer of genes from the plastid to the nuclear genome in dinoflagellates. These data support the hypothesis that the peridinin-type plastid has a minimal genome, and provide data that contradict the hypothesis that there is an unidentified canonical genome in the peridinin-type plastid. Sequences were also identified that were probably transferred directly from the nuclear genome of the red algal endosymbiont, as well as others that are distinctive to the Alveolata. A preliminary report of these data was presented at the Botany 2002 meeting in Madison, WI.     

Shimiella gen. nov. and Shimiella gracilenta sp. nov. (Dinophyceae,Kareniaceae), a Kleptoplastidic Dinoflagellate from Korean Waters and its Survival under Starvation

A small dinoflagellate, ~13 μm in cell length, was isolated from Jinhae Bay, Korea. Light microscopy showed that it was similar to the kleptoplastidic dinoflagellate Gymnodinium gracilentum nom. inval. rDNA sequences were obtained and its anatomy and morphology described using light and scanning and transmission electron microscopy. Phylogenetic analyses indicated that it belonged to the family Kareniaceae. However, its large subunit (LSU) rDNA sequences were 5.2–9.5% different from those of the other five genera in the family, and its clade was clearly divergent from that of each genus. Its overall morphology was different from those of the other five genera in the family and from Gymnodinium. Unlike Gymnodinium, this dinoflagellate did not have a horseshoe‐shaped apical groove, nuclear envelope chambers, or a nuclear fibrous connective (NFC). It had an apical line of narrow amphiesmal vesicles and an elongated apical furrow crossing the apex. Cells were covered with polygonal amphiesmal vesicles arranged in 16 rows. Starved cells did not contain their own plastids, eyespots, pyrenoids, peridinin, or fucoxanthin. However, they could survive without added prey for approximately one month using chloroplasts from the cryptophyte prey Teleaulax amphioxeia, indicating kleptoplastidy. Because this taxon is genetically distinct at the generic rank from the other genera in Kareniaceae, it is placed in Shimiella gen. nov., and because G. gracilentum was invalid, the new bionomial S. gracilenta sp. nov. is proposed.     

Origin of the plastid in the anomalously pigmented dinoflagellate Gymnodinium mikimotoi (Gymnodiniales, Dinophyta) as inferred from phylogenetic analysis based on the gene encoding the large subunit of form I-type RuBisCO

The dinoflagellate Gymnodinium mikimotoi Miyake et Kominami ex Oda possesses an anomalously pigmented plastid which contains 19′‐hexanoyloxyfucoxanthin, 19′‐butanoyloxyfucoxanthin and fucoxanthin instead of peridinin as the major carotenoids. Previously, we have shown that the plastid of G. mikimotoi belongs to the rhodoplast lineage as inferred from phylogenetic analyses based on the amino acid sequences deduced from psbA and psaA and the nucleotide sequence of the plastid small subunit ribosomal RNA. Furthermore, in the present study, we cloned and sequenced an additional representative plastid gene, rbcL, encoding the large subunit of ribulose 1–5 bisphosphate carboxylase/oxygenase (RuBisCO LSU) from G. mikimotoi. The amino acid sequence deduced from the rbcL gene of G. mikimotoi apparently revealed the conventional form I RuBisCO LSU, which is present in most photosynthetic organisms, and not the divergent form II existing in typically pigmented dinofl age Nates with plastids containing peridinin as the main carotenoid. This finding supports the hypothesis that the origins of the plastids in G. mikimotoi and peridinin‐type dinoflagellates are not related to each other. Molecular phylogenetic analysis based on the amino acid sequence deduced from the rbcL gene further showed that the plastid of G. mikimotoi belongs to the rhodoplast lineage. In particular, G. mikimotoi clustered with haptophytes in the phylogenetic tree. From this result, two hypotheses with respect to the origin of the plastid in G. mikimotoi can be proposed: G. mikimotoi may have engulfed a haptophyte‐like cell (tertiary symbiosis) or englulfed a rhodophyte‐like cell that was closely related to the origin of the plastid in the haptophyte (secondary symbiosis).     

Carotenes from Mutants of the Dinoflagellate, Crypthecodinium cohnii

SYNOPSIS.
The carotenoid compositions of 15 nitrosoguanidine-induced mutants of Crypthecodinium cohnii , a heterotrophic dinoflagellate, were determined by chromatographic and mass spectral analyses. Wild-type C. cohnii grown with irradiation of 250 W/cm² visible light at 27 C synthesizes β-carotene (33%) and γ-carotene (67%) amounting to 0.083 mg/g dry wt. There are 4 types of carotenoid-deficient mutants: (I) albinos which synthesize no C₄₀-carotonoids: (II) albinos blocked at the level of phytoene desaturation; (III) cream-colored cells which accumulate mainly §–carotene, with phytoene and/or β-zeacarotene also present; and (IV) light-orange strains which synthesize reduced amounts of β-carotene and γ-carotene.
Dark-grown wild-type cells produced 35% as much carotenoids as light-grown cells. Inhibition studies revealed that diphenylamine (3 γ) caused phytoene accumulation; nicotine at 0.9 mM blocked the final cyclization, to cause γ-carotene to accumulate in wild-type cells. Inhibition by adenine and guanine (1.5 mM) of carotenogenesis was demonstrated for the first time in any system. The effect of these purines was similar to that of diphenylamine addition: phytoene desaturation was largely inhibited.
The carotenogenic system in this dinoflagellate is similar to that of green algae and higher plants, and is under nuclear genetic control.     

High Speed Cinemicrography of the Direct Photophobic Response of Euglena and the Mechanism of Negative Phototaxis*

SYNOPSIS. The shock reaction of Euglena gracilis strain Z to a sudden increase in light intensity (the “direct photophobic response”) was examined by high speed cinemicrography. The response is expressed as a turning reaction toward the dorsal side of the cell, after a transduction time of 0.1–0.5 sec after the onset of stimulation. Transduction times, turning rates, and flagellar beat frequencies were measured by analyzing the filmed sequences. The experimental data are consistent with a mechanism of directional homeostasis in negative phototaxis that is based upon shading of the photoreceptor by the cell's posterior end.     

Molecular Phylogeny of the Parasitic Dinoflagellate Chytriodinium within the Gymnodinium Clade (Gymnodiniales,Dinophyceae)

The dinoflagellate genus Chytriodinium, an ectoparasite of copepod eggs, is reported for the first time in the North and South Atlantic Oceans. We provide the first large subunit rDNA (LSU rDNA) and Internal Transcribed Spacer 1 (ITS1) sequences, which were identical in both hemispheres for the Atlantic Chytriodinium sp. The first complete small subunit ribosomal DNA (SSU rDNA) of the Atlantic Chytriodinium sp. suggests that the specimens belong to an undescribed species. This is the first evidence of the split of the Gymnodinium clade: one for the parasitic forms of Chytriodiniaceae (Chytriodinium, Dissodinium), and other clade for the free‐living species.     

中国渤海海域甲藻孢囊的种类多样性和生态地理分布

在渤海湾24个站位采集0–10 cm表层沉积物144份样品, 进行甲藻孢囊的种类多样性和生态地理分布研究, 并分析渤海海域水体的富营养化趋势。在渤海海域共采集鉴定出6类32种甲藻孢囊, 包括原多甲藻类孢囊11种、裸甲藻类孢囊6种、膝沟藻类孢囊7种、钙质类孢囊5种、翼藻类孢囊2种及Tuberculodinioid类孢囊1种。其中, 自养型甲藻孢囊17种, 优势种为锥状斯氏藻(Scrippsiella trochoidea)孢囊, 丰富的孢囊库为锥状斯氏藻赤潮提供种源, 可作为该海域发生锥状斯氏藻赤潮的参考依据; 异养型甲藻孢囊15种, 优势种为无纹多沟藻(Polykrikos schwartzii)和锥形原多甲藻(Protoperidinium conicum)孢囊; 发现1种产麻痹性贝类毒素的孢囊--塔玛亚历山大藻(Alexandrium tamarense)孢囊, 但是仅在S6、S8和S14三个站位发现, 且孢囊数量较少。渤海甲藻孢囊组成与世界其它海域相似, 异养型甲藻孢囊种类丰富, 提示渤海海域富营养化程度较高。每个站位沉积物样品分为3层(每层约3 cm), 春夏两季表层、中层和底层之间孢囊组成差异不大, 平均种类数为5.58–7.29, 孢囊的平均密度范围在58.82–103.57 cysts·g^–1 DW之间; 孢囊种类丰富的站点, 孢囊密度也较高, 如S9、S14站位。对渤海海区表层沉积物中甲藻孢囊的生物多样性进行了统计分析, 发现表、中、底3层甲藻孢囊的平均种类多样性指数在春季(5月)分别为1.69、1.61和1.52; 略低于夏季(8月)的1.83、1.89和1.60。     

Dinoflagellate cysts as indicators of water quality and productivity in British Columbia estuarine environments

Palynological analyses of 60 surface sediment samples from estuarine environments near Vancouver Island, including the Georgia Strait (GS), the Effingham (EFF) and the Seymour–Belize (SB) Inlets were performed in order to document the distribution of dinoflagellate cyst assemblages and their relationship to hydrographic conditions, productivity and nutrient concentrations. We tested transfer functions using the analogue method, and suggest that dinoflagellate cyst assemblages can be used to reconstruct primary productivity, temperature and salinity. The EFF and SB Inlets are characterized by a dominance of autotrophic taxa, particularly Operculodinium centrocarpum, whereas the Protoperidinioid and gymnodinial heterotrophic taxa such as Quinquecuspis concreta and Brigantedinium spp. dominate the assemblages of the GS. Multivariate analysis shows that this distribution is closely linked to primary productivity, sea-surface temperature (SST) and spring silica concentration. The abundance of autotrophic taxa in the EFF and SB Inlets is associated with high primary productivity and low summer SST, indicating summer upwelling of coastal British Columbia, whereas the heterotrophic taxa that characterizes the GS assemblages are related to low productivity, high summer SST and high silica concentration during spring. Multivariate analysis shows that the most important environmental parameters related to dinocyst distribution in the restricted embayment of the GS, are distance to the shore, distance to Vancouver Harbor, spring sea surface salinity (SSS), spring phosphate concentration and spring productivity. The autotrophic taxa are generally more common in coastal and shallow waters, but Spiniferites ramosus and Pentapharsodinium dalei show an opposite correlation to spring productivity and salinity. P. dalei is particularly abundant around Vancouver Harbor, near highly urbanized shores and within the Fraser River plume, where salinity is low and spring productivity and continental runoff are high. S. ramosus shows its highest abundance on the western coast of GS. Protoperidinioid and gymnodinial cysts characterize distal zones within the central and southern GS that are associated with a mixture of brackish waters coming from the Fraser River and deep upwelling waters entering the GS via Juan de Fuca Strait. The relationship between dinoflagellate cyst assemblages and primary productivity in these estuarine systems differs from that in oceanic and outer neritic zones, where the abundance of heterotrophic taxa is commonly associated with upwelling and high productivity.