PHAGOTROPHY IN GYMNODINIUM FUNGIFORME (PYRRHOPHYTA): THE PEDUNCLE AS AN ORGANELLE OF INGESTION1

The non-photosynthetic phagotrophic dinoflagellate, Gymnodinium fungiforme Anissimova, ingests prey cytoplasm through a highly extensible structure called the peduncle. Although the peduncle is not observable when G. fungiforme is swimming, it protrudes 8–12 μm from the sulcal-angular vicinity of the cell during feeding, and is approximately 3.3 μm wide when the cytoplasm of its prey is flowing through it. A circular-oval ring of overlapping microtubules, the ‘microtubular basket’ may be seen in transmission electron microscope sections of G. fungiforme and it is inferred that this structure is a cross section of a retracted peduncle. The microtubular basket-peduncle complex is discussed in relation to similar structures in other dinoflagellates and to the tentacle of the suctorian ciliates which have a homologous ingestion system.     

A dinoflagellate CDK5-like cyclin-dependent kinase

Background information. Mitosis during the dinoflagellate cell cycle is unusual in that the nuclear envelope remains intact and segregation of the permanently condensed chromosomes uses a cytoplasmic mitotic spindle. To examine regulation of the dinoflagellate cell cycle in the context of these unusual nuclear features, it is necessary to isolate and characterize cell cycle regulators such as CDK (cyclin‐dependent kinase). Results. We report the characterization of a CDK from the dinoflagellate Lingulodinium polyedrum. This CDK reacts with an anti‐PSTAIRE antibody and was identified by protein microsequencing after partial purification. The protein microsequence shows homology toward the Pho85/CDK5 clade of CDKs. Neither the amount nor the phosphorylation state changed over the course of the cell cycle, in agreement with results reported for CDK5 family members in other systems. Conclusions. We conclude we have probably isolated a dinoflagellate CDK5‐like protein. The data reported here support the identification of this protein as a CDK5 homologue, and suggest that dinoflagellates may contain several CDK families.     

A dinoflagellate with both a mesocaryotic and a eucaryotic nucleus I. Fine structure of the nuclei

Summary Glenodinium foliaceum Stein (Pyrrophyta) has a single mesocaryotic nucleus which contains numerous typically dinoflagellate chromosomes and one or more nucleoli with a structure similar to that of nucleoli in higher organisms. In addition this organism possesses another nucleus-like organelle which is here termed the eucaryotic nucleus. This is a polymorphic body which varies in shape from ovoid to a branched filamentous form. As with the mesocaryotic nucleus it is surrounded by a perforated envelope. The organelle contains granular material and usually several nucleoli which again appear to have the typical form of nucleoli. No other dinoflagellate is known in which two nuclei of differing types are found. The function and significance of the presence of the two nuclei is discussed.     

THE ULTRASTRUCTURE OF GLENODINIOPSIS STEINII (DINOPHYCEAE)

The freshwater dinoflagellate Glenodiniopsis steinii Wolsoszyńska was examined using computer-assisted three-dimensional reconstruction of serially sectioned cells observed with the transmission electron microscope and images from the scanning electron microscope. Vegetative cells contain ultrastructure typical of freshwater dinoflagellates including trichocysts, mitochondria, Golgi bodies, starch grains, and lipid bodies. The chloroplast is a single, multilobed structure, not multiple discoid chloroplasts as previously described. The “C” shape of the nucleus is apparently due in part to the size and location of the pusule.     

An electron microscope study of nuclear and cell division in a dinoflagellate

Summary Light microscopical observations on the cell division of the small dinoflagellate Woloszynskia micra are correlated for the first time with an electron microscopical study. In prophase, whilst the nucleus enlarges and becomes pearshaped, the chromosomes divide to give pairs of chromatids. This process starts at one end and works to the other giving Y- and V-shaped chromosomes as it occurs. Cytoplasmic invaginations pass through the nucleus and by the end of prophase these are seen to contain a number of microtubules of about 180 Å diameter. There is no connection between the microtubules in the nuclear in vagination and either the flagellar bases or the chromosomes. At anaphase the nucleus expands laterally and the sister chromatids move towards opposite ends. The cell hypocone is now partially divided and the two longitudinal flagella well separate. The nucleus completes its division into two daughter nuclei and for a time portions of the cytoplasmic invaginations remain visible. Cell cleavage is completed by the division of the epicone. The nuclear membrane remains intact throughout division and the nucleolus does not break down.The mitotic division in this organism, which is unusual in comparison with the mitosis of higher organisms, is discussed in the light of other types of mitosis which have been reported and of earlier light microscopical observations on dinoflagellates.     

Telomere maintenance in liquid crystalline chromosomes of dinoflagellates

The organisation of dinoflagellate chromosomes is exceptional among eukaryotes. Their genomes are the largest in the Eukarya domain, chromosomes lack histones and may exist in liquid crystalline state. Therefore, the study of the structural and functional properties of dinoflagellate chromosomes is of high interest. In this work, we have analysed the telomeres and telomerase in two Dinoflagellata species, Karenia papilionacea and Crypthecodinium cohnii. Active telomerase, synthesising exclusively Arabidopsis-type telomere sequences, was detected in cell extracts. The terminal position of TTTAGGG repeats was determined by in situ hybridisation and BAL31 digestion methods and provides evidence for the linear characteristic of dinoflagellate chromosomes. The length of telomeric tracts, 25–80 kb, is the largest among unicellular eukaryotic organisms to date. Both the presence of long arrays of perfect telomeric repeats at the ends of dinoflagellate chromosomes and the existence of active telomerase as the primary tool for their high-fidelity maintenance demonstrate the general importance of these structures throughout eukaryotes. We conclude that whilst chromosomes of dinoflagellates are unique in many aspects of their structure and composition, their telomere maintenance follows the most common scenario.     

The Monophyletic Origin of the Peridinin‐, and Fucoxanthin‐Containing Dinoflagellate Plastid Through Tertiary Replacement

The dinoflagellates contain diverse plastids of uncertain origin. To determine the origin of the peridinin‐ and fucoxanthin‐containing dinoflagellate plastid, we sequenced the plastid‐encoded psaA, psbA, and rbcL genes from various red and dinoflagellate algae. The psbA gene phylogeny, which was made from a dataset of 15 dinoflagellates, 22 rhodophytes, five cryptophytes, seven haptophytes, seven stramenopiles, two chlorophytes, and a glaucophyte as the outgroup, supports monophyly of the peridinin‐, and fucoxanthin‐containing dinoflagellates, as a sister group to the haptophytes. The monophyletic relationship with the haptophytes is recovered in the psbA + psaA phylogeny, with stronger support. The rubisco tree utilized the ‘Form I’ red algal type of rbcL and included fucoxanthin‐containing dinoflagellates. The dinoflagellate + haptophyte sister relationship is also recovered in this analysis. Peridinium foliaceum is shown to group with the diatoms in all the phylogenies. Based on our analyses of plastid sequences, we postulate that: (1) the plastid of peridinin‐, and fucoxanthin‐containing dinoflagellates originated from a common ancestor; (2) the ancestral dinoflagellate acquired its plastid from a haptophyte though a tertiary plastid replacement; (3) ‘Form II’ rubisco replaced the ancestral rbcL after the divergence of the peridinin‐, and fucoxanthin‐containing dinoflagellates; and (4) we confirm that the plastid of P. foliaceum originated from a Stramenopiles endosymbiont.     

Fates of Evolutionarily Distinct,Plastid‐type Glyceraldehyde 3‐phosphate Dehydrogenase Genes in Kareniacean Dinoflagellates

The ancestral kareniacean dinoflagellate has undergone tertiary endosymbiosis, in which the original plastid is replaced by a haptophyte endosymbiont. During this plastid replacement, the endosymbiont genes were most likely flowed into the host dinoflagellate genome (endosymbiotic gene transfer or EGT). Such EGT may have generated the redundancy of functionally homologous genes in the host genome—one has resided in the host genome prior to the haptophyte endosymbiosis, while the other transferred from the endosymbiont genome. However, it remains to be well understood how evolutionarily distinct but functionally homologous genes were dealt in the dinoflagellate genomes bearing haptophyte‐derived plastids. To model the gene evolution after EGT in plastid replacement, we here compared the characteristics of the two evolutionally distinct genes encoding plastid‐type glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) in Karenia brevis and K. mikimotoi bearing haptophyte‐derived tertiary plastids: “gapC1h” acquired from the haptophyte endosymbiont and “gapC1p” inherited from the ancestral dinoflagellate. Our experiments consistently and clearly demonstrated that, in the two species examined, the principal plastid‐type GAPDH is encoded by gapC1h rather than gapC1p. We here propose an evolutionary scheme resolving the EGT‐derived redundancy of genes involved in plastid function and maintenance in the nuclear genomes of dinoflagellates that have undergone plastid replacements. Although K. brevis and K. mikimotoi are closely related to each other, the statuses of the two evolutionarily distinct gapC1 genes in the two Karenia species correspond to different steps in the proposed scheme.     

Phototactic responses of four marine dinoflagellates with different types of eyespot and chloroplast

Great structural variety is seen in the eyespot of dinoflagellates, a structure involved in phototaxis. Although there are several works on the phototactic responses in some species of dinoflagellates, none of the dinoflagellates used in these studies possessed an eyespot and, therefore, we have no knowledge of the relationship between eyespot type and phototactic response. In this study, we determined wavelength dependency curves for phototaxis in four marine dinoflagellates that possess a different type of either eyespot or chloroplast. These include: (i) a dinoflagellate possessing a peridinin-containing ohioroplast with an eyespot (Scrippsiella hexapraecingula Horiguchi et Chihara); (ii) a dinoflagellate containing a diatom endosymbiont and with the type B eyespot sensu Dodge (1984; (Peridinium foli-aceum (Stein) Biecheler); (iii) a dinoflagellate with peri-dinin-containing chloroplasts, but lacking an eyespot (Atexandrium hiranoi Kita et Fukuyo); and (iv) a dinoflagellate with fucoxanthin, 19′-hexanoyloxyfucoxanthin and 19′-butanoyloxyfucoxanthin, but lacking an eyespot (Gymnodinium mikimotoi Miyabe et Kominami ex Oda), Regardless of the eyespot or the chloroplast type, all four dinoflagellates showed similar wavelength dependency curves for phototaxis, with sensitivity between 380 and 520 nm, the highest peak at approximately 440 or 460 nm and smaller peaks or shoulders at 400–420 nm and 480–500 nm. Substantial peaks have also been noted in the ultraviolet range (260–280 nm). The ultrastructural study of the eye-spot of Scrippsiella hexapraecingula revealed that the eyespot consists of two layers of lipid globules and probably acts as a quarter-wave stack antenna.     

The encystment of a freshwater dinoflagellate: A light and electron-microscopical study

The process of encystment, or resting spore formation, in a freshwater dinoflagellate (Woloszynskia tylota nov. comb.) has been studied with both light and electron microscopy. The main features of the process are as follows: (i) the replacement of the theca by a thin, amorphous outer wall, which gradually thickens by the deposition of material on its inner face; (ii) the appearance of a layer of closely-packed lipid droplets at the cytoplasmic margin of the mature cyst, resembling a granular ‘inner wall’ in the light microscope; (iii) the reduction in size or disappearance of cytoplasmic structures such as chloroplasts, Golgi bodies and pusule; and (iv) the enlargement of a central ‘accumulation body’ and cytoplasmic vacuoles containing crystals. Comparisons are made with light-microscope studies of encystment of other dinoflagellates, with ultrastructural studies of non-motile division stages, with zooxanthellae and with fossil dinoflagellate cysts or hystrichospheres.     

An unusual mitotic mechanism in the parasitic protozoan Syndinium sp

Syndinium and related organisms which parasitize a number of invertebrates have been classified with dinoflagellates on the basis of the morphology of their zoospores. We demonstrate here that with respect to chromosome structure and chemistry as well as nuclear division, they differ fundamentally from free-living dinoflagellates. Alkaline fast green staining indicates the presence of basic proteins in Syndinium chromosomes. Chromatin fibers are about 30 Å thick and do not show the arrangement characteristic of dinoflagellate chromosomes. The four V-shaped chromosomes are permanently attached at their apexes to a specific area of the nuclear membrane through a kinetochore-like trilaminar disk inserted into an opening of the membrane. Microtubules connect the outer dense layer of each kinetochore to the bases of the two centrioles located in a pocket-shaped invagination of the nuclear envelope. During division kinetochores duplicate, and each sister kinetochore becomes attached to a different centriole. As the centrioles move apart, apparently pushed by a bundle of elongating microtubules (central spindle), the daughter chromosomes are passively pulled apart. During the process of elongation of the central spindle, the cytoplasmic groove on the nuclear surface which contains the central spindle sinks into the nuclear space and is transformed into a cylindrical cytoplasmic channel. A constriction in the persisting nuclear envelope leads to the formation of two daughter nuclei.     

Effects of the bacterial algicide IRI-160AA on cellular morphology of harmful dinoflagellates

The algicide, IRI-160AA, induces mortality in dinoflagellates but not other species of algae, suggesting that a shared characteristic or feature renders this class of phytoplankton vulnerable to the algicide. In contrast to other eukaryotic species, the genome of dinoflagellates is stabilized by high concentrations of divalent cations and transition metals and contains large amounts of DNA with unusual base modifications. These distinctions set dinoflagellates apart from other phytoplankton and suggest that the nucleus may be a dinoflagellate-specific target for IRI-160AA. In this study, morphological and ultrastructural changes in three dinoflagellate species, Prorocentrum minimum, Karlodinium veneficum and Gyrodinium instriatum, were evaluated after short-term exposure to IRI-160AA using super resolution structured illumination microscopy (SR-SIM) and transmission electron microscopy (TEM). Exposure to the algicide resulted in cytoplasmic membrane blebbing, differing chloroplast morphologies, nuclear expansion, and chromosome expulsion and/or destabilization. TEM analysis showed that chromosomes of algicide-treated K. veneficum appeared electron dense with fibrous protrusions. In algicide-treated P. minimum and G. instriatum, chromosome decompaction occurred, while for P. minimum, nuclear expulsion was also observed for several cells. Results of this investigation demonstrate that exposure to the algicide destabilizes dinoflagellate chromosomes, although it was not clear if the nucleus was the primary target of the algicide or if the observed effects on chromosomal structure were due to downstream impacts. In all cases, changes in cellular morphology and ultrastructure were observed within two hours, suggesting that the algicide may be an effective and rapid approach to mitigate dinoflagellate blooms.     

Bispinodinium angelaceum gen. et sp. nov. (Dinophyceae), a new sand‐dwelling dinoflagellate from the seafloor off Mageshima Island,Japan1

A new athecate dinoflagellate, Bispinodinium angelaceum N. Yamada et Horiguchi gen. et sp. nov., is described from a sand sample collected on the seafloor at a depth of 36 m off Mageshima Island, subtropical Japan. The dinoflagellate is dorsiventrally compressed and axi‐symmetric along the sulcus. The morphology resembles that of the genus Amphidinium sensu lato by having a small epicone that is less than one third of the total cell length. However, it has a new type of apical groove, the path of which traces the outline of a magnifying glass. The circular component of this path forms a complete circle in the center of the epicone and the straight “handle” runs from the sulcus to the circular component. Inside the cell, a pair of elongated fibrous structure termed here the “spinoid apparatus” extends from just beneath the circular apical groove to a point near the nucleus. Each of two paired structures consists of at least 10 hyaline fibers and this is a novel structure found in dinoflagellates. Phylogenetic analyses based on the SSU and LSU RNA genes did not show any high bootstrap affinities with currently known athecate dinoflagellates. On the basis of its novel morphological features and molecular signal, we conclude that this dinoflagellate should be described as a new species belonging to a new genus.     

PLANKTONIC SARCODINES: MICROHABITAT FOR OCEANIC DINOFLAGELLATES1

Two morphologically distinct species of free-swimming dinoflagellates belonging to the genus Gyrodinium utilize the spine and rhizopodial environments of planktonic foraminifera and colonial radiolaria as microhabitats. Up to 84% of the sarcodines examined in a given population were associated with these dinoflagellates at densities up to 20,000 cells per sarcodine in some radiolarian colonies. Both dinoflagellate species possess chloroplasts, indicating they are capable of autotrophy. ¹⁴C-labelling experiments with the radiolarian-associated dinoflagellate demonstrate that it can take up inorganic carbon under both light and dark conditions. Ultrastructural evidence suggests the foraminiferal dinoflagellate may be capable of phagotrophy. Hence, these algae should be considered mixotrophs. An unusual cytoplasmic extension used for attachment and possibly feeding occurs in the foraminiferal-associated Gyrodinium and is documented with electron microscopy. Ultrastructural examination suggests this organelle may be hydrostatically controlled and may be an extension of the sac pusule.     

Feeding by Phototrophic Red-Tide Dinoflagellates on the Ubiquitous Marine Diatom Skeletonema costatum

ABSTRACT We investigated feeding by phototrophic red‐tide dinoflagellates on the ubiquitous diatom Skeletonema costatum to explore whether dinoflagellates are able to feed on S. costatum, inside the protoplasm of target dinoflagellate cells observed under compound microscope, confocal microscope, epifluorescence microscope, and transmission electron microscope (TEM) after adding living and fluorescently labeled S. costatum (FLSc). To explore effects of dinoflagellate predator size on ingestion rates of S. costatum, we measured ingestion rates of seven dinoflagellates at a single prey concentration. In addition, we measured ingestion rates of the common phototrophic dinoflagellates Prorocentrum micans and Gonyaulax polygramma on S. costatum as a function of prey concentration. We calculated grazing coefficients by combining field data on abundances of P. micans and G. polygramma on co‐occurring S. costatum with laboratory data on ingestion rates obtained in the present study. All phototrophic dinoflagellate predators tested (i.e. Akashiwo sanguinea, Amphidinium carterae, Alexandrium catenella, Alexandrium tamarense, Cochlodinium polykrikoides, G. polygramma, Gymnodinium catenatum, Gymnodinium impudicum, Heterocapsa rotundata, Heterocapsa triquetra, Lingulodinium polyedrum, Prorocentrum donghaiense, P. micans, Prorocentrum minimum, Prorocentrum triestinum, and Scrippsiella trochoidea) were able to ingest S. costatum. When mean prey concentrations were 170–260 ng C/ml (i.e. 6,500–10,000 cells/ml), the ingestion rates of G. polygramma, H. rotundata, H. triquetra, L. polyedrum, P. donghaiense, P. micans, and P. triestinum on S. costatum (0.007–0.081 ng C/dinoflagellate/d [0.2–3.0 cells/dinoflagellate/d]) were positively correlated with predator size. With increasing mean prey concentration of ca 1–3,440 ng C/ml (40–132,200 cells/ml), the ingestion rates of P. micans and G. polygramma on S. costatum continuously increased. At the given prey concentrations, the maximum ingestion rates of P. micans and G. polygramma on S. costatum (0.344–0.345 ng C/grazer/d; 13 cells/grazer/d) were almost the same. The maximum clearance rates of P. micans and G. polygramma on S. costatum were 0.165 and 0.020 μl/grazer/h, respectively. The calculated grazing coefficients of P. micans and G. polygramma on co‐occurring S. costatum were up to 0.100 and 0.222 h, respectively (i.e. up to 10% and 20% of S. costatum populations were removed by P. micans and G. polygramma populations in 1 h, respectively). Our results suggest that P. micans and G. polygramma sometimes have a considerable grazing impact on populations of S. costatum.     

Molecular data and the evolutionary history of dinoflagellates

We have sequenced small-subunit (SSU) ribosomal RNA (rRNA) genes from 16 dinoflagellates, produced phylogenetic trees of the group containing 105 taxa, and combined small- and partial large-subunit (LSU) rRNA data to produce new phylogenetic trees. We compare phylogenetic trees based on dinoflagellate rRNA and protein genes with established hypotheses of dinoflagellate evolution based on morphological data. Protein-gene trees have too few species for meaningful in-group phylogenetic analyses, but provide important insights on the phylogenetic position of dinoflagellates as a whole, on the identity of their close relatives, and on specific questions of evolutionary history. Phylogenetic trees obtained from dinoflagellate SSU rRNA genes are generally poorly resolved, but include by far the most species and some well-supported clades. Combined analyses of SSU and LSU somewhat improve support for several nodes, but are still weakly resolved. All analyses agree on the placement of dinoflagellates with ciliates and apicomplexans (=Sporozoa) in a well-supported clade, the alveolates. The closest relatives to dinokaryotic dinoflagellates appear to be apicomplexans, Perkinsus, Parvilucifera, syndinians and Oxyrrhis. The position of Noctiluca scintillans is unstable, while Blastodiniales as currently circumscribed seems polyphyletic. The same is true for Gymnodiniales: all phylogenetic trees examined (SSU and LSU-based) suggest that thecal plates have been lost repeatedly during dinoflagellate evolution. It is unclear whether any gymnodinialean clades originated before the theca. Peridiniales appear to be a paraphyletic group from which other dinoflagellate orders like Prorocentrales, Dinophysiales, most Gymnodiniales, and possibly also Gonyaulacales originated. Dinophysiales and Suessiales are strongly supported holophyletic groups, as is Gonyaulacales, although with more modest support. Prorocentrales is a monophyletic group only in some LSU-based trees. Within Gonyaulacales, molecular data broadly agree with classificatory schemes based on morphology. Implications of this taxonomic scheme for the evolution of selected dinoflagellate features (the nucleus, mitosis, flagella and photosynthesis) are discussed.     

123 Permanent Fluorescent Nuclear Staining of Binucleate Dinoflagellates

Typically, fluorescent microscopy of dinoflagellate nuclei is of poor resolution, due mainly to visual obstruction of the nuclei by plastids, pigment granules, and thecal plates. Moreover, the usual slide mounts using buffered glycerol are temporary, and fade after a week or so. We have developed a procedure to clear pigments from dinoflagellates, followed by fluorescent staining of the nuclei. The cells are then prepared as permanent mounts using an ultraviolet light-catalyzed resin to produce stained samples which may be kept for at least three years with little loss of fluorescence. This procedure can also be used to prepare plastic embedded dinoflagellate cells which can then be sectioned at 1–2 nm, fluorescent stained, and permanently mounted. Suitable nuclear stains are DAPI, Hoechst 33258, ethidium bromide and acridine orange. The dinoflagellate (dinokaryotic), and endosymbiont (eukaryotic) nuclei are clearly visualized, revealing individual chromosomes in the dinoflagellate nucleus, and a highly lobed morphology of the endosymbiont nucleus.     

THE SMALLEST DINOFLAGELLATE GENOME IS YET TO BE FOUND: A COMMENT ON LAJEUNESSE ET AL. “SYMBIODINIUM (PYRRHOPHYTA) GENOME SIZES (DNA CONTENT) ARE SMALLEST AMONG DINOFLAGELLATES”1

LaJeunessse and colleagues (LaJeunesse et al. 2005) have recently documented small genome sizes of Symbiodinium and concluded that Symbiodinium is a dinoflagellate lineage with the smallest genome. The conclusion is inconsistent with recent discoveries of picoplanktonic dinoflagellates. The search for the smallest genome and the effort to understand the evolutionary history of dinoflagellate genome should be an area of research in the years to come, which can be greatly aided by an understanding on the current hypotheses regarding mechanisms of genome size evolution. Even the smallest dinoflagellate genome documented to date is too large to be sequenced with current technology, but sequencing of chromosomes or expressed genes of key representative species is feasible and can be very insightful for understanding genome composition and function in this important lineage of eukaryotes.     

A TECHNIQUE FOR COUNTING CHROMOSOMES OF ARMORED DINOFLAGELLATES,AND CHROMOSOME NUMBERS OF SIX FRESHWATER DINOFLAGELLATE SPECIES

A technique is presented to stain, squash, and enumerate thecate dinoflagellate chromosomes using a cellulose incubation and propionocarmine stain. Chromosome numbers for six freshwater armored dinoflagellates (Peridinium cinctum (O.F.M.) Ehrenberg, P. inconspicuum Lemm., P. limbatum (Stokes) Lemm., P. volzii Lemm., P. willei Huit.-Kaas, and Peridiniopsis polonicum (Wolosz.) Bourrelly) range from 41 (P. inconspicuum) to 210 (P. cinctum). Evidence is presented for dinoflagellate aneuploidy in culture.