SYNCHRONIZATION OF THE MARINE DINOFLAGELLATE AMPHIDINIUM CARTERI IN DENSE CULTURES1

Under improved conditions, Amphidinium carteri Hulburt can be obtained as dense cultures (5–9 × 10⁵ cells · ml^?1) 5 days after inoculation. The possible association of a marine bacterium with the alga is discussed. A method to synchronize this photosynthetic organism is described which is based on the endogenous rhythm of division and the effect of light-dark alternating periods.     

PHAGOTROPHY IN THE FRESHWATER,PHOTOSYNTHETIC DINOFLAGELLATE AMPHIDINIUM CRYOPHILUM1

The cold-water, photosynthetic dinoflagellate Amphidinium cryophilum Wedemayer, Wilcox & Graham feeds phagotrophically on other dinoflagellate species. Food is ingested through a feeding tube, termed here the “phagopod,” which extends from the antapex. The peduncle of this organism plays no observable role in the feeding process. The phagopod is essentially a hollow cylinder composed electron-opaque material that is possibly deposited on a membrane. No Amphidinium cytoplasmic components, including microtubules or other cytoskeletal elements, were observed in the phagopod. Prefeeding cells aggregate, in small clumps near prey organisms with their phagopods extended. Eventually some cells commence feeding, first inserting the phagopod through the prey cell-covering and then slowly, over a period of 10 min or more, drawing cytoplasm through the phagopod and into a nascent food vacuole. Both light and electron microscopy suggest that one or more prey cell amphiesmal membranes remain intact during the feeding process. Upon completion of feeding, the Amphidinium cell swims off with a prominent food vacuole in the hypocone, leaving at least part of the phagopod attached to the prey cell. Phagotrophy in A. cryophilum seems to vary with light intensity. At low light intensities, cells feed phagotrophically and are nearly colorless, whereas at high light levels they feed much less frequently, if at all, and are brightly pigmented.     

OCCURRENCE OF CHOLINE ESTERS IN THE MARINE DINOFLAGELLATE AMPHIDINIUM CARTERI12

Acrylylcholine, choline O-sulfate, and a yet unidentified choline ester have been isolated from cells of Amphidinium carteri. Natural and synthetic acrylyl choline had approximately 1/4,000 the activity of acetylcholine and choline O-sulfate about 1/20,000 the activity on isolated Mercenaria mercenaria heart.     

RECYCLING OF METABOLIZED IRON BY THE MARINE DINOFLAGELLATE AMPHIDINIUM CARTERAE1

Senescent, iron-limited cultures of Amphidinium carterae Hulburt were used to detect the presence of iron made available for phytoplankton growth by recycling of metabolized iron from disrupted cells. Both physical disruption of cells and physical disruption plus exposure to a low pH were tested. The test cultures remained viable over long periods of time, and were stimulated to full growth by the addition of Fe, but not by the addition of any other nutrients. Simple physical disruption of cells caused only a very slow release of available Fe, while disruption of cells plus exposure to a low pH resulted in a rapid release of available Fe. It is suggested that the digestive processes of herbivores are instrumental in the rapid regeneration of Fe as a nutrient available for phytoplankton growth.     

IDENTIFICATION OF A CYCLIC AMP-DEPENDENT PROTEIN KINASE IN THE DINOFLAGELLATE AMPHIDINIUM OPERCULATUM

Single cell analysis by flow cytometry is a powerful tool that has been employed to identify many different characteristics of phytoplankton populations. Cell volume is an important physiological component of many cellular processes. We have used a Coulter EPICS XL flow cytometer to measure cell volume in the spheroid dinoflagellate Amphidinium operculatum as a function of forward scatter. Cell volume measurements of this alga were quantified as equivalent spherical diameters from a standard curve obtained with latex beads of known diameter. This parameter was used to monitor cell diameter throughout the cell division cycle. In log phase cultures, A. operculatum showed increasing cell volumes throughout the light phase and a maximum cell volume concurrent with the onset of cell division late in the light phase. The maximum equivalent spherical diameter measured 14 μm, while the minimum equivalent spherical diameter was 10 μm that occurred late in the dark phase. Stationary phase cultures of A. operculatum did not exhibit oscillating cell volumes throughout the diel cycle. Chemical inhibition of the cell cycle using 100 μM olomoucine diminished cell volume changes during the light phase. These results suggest a coupling of size control to the cell division cycle.     

CAROTENOID PIGMENTS OF THE DINOFLAGELLATE GLENODINIUM FOLIACEUM STEIN12

The marine dinoflagellate, Glenoclinium foliaceum Stein, has been shown to contain fucoxanthin, instead of peridinin, as the major xanthophyll. In addition, 2 carotenes–β-carotene and a compound with spectral properties reminiscent of isomerized y-carotene–and 2 xanthophylls–diadinoxanthin and an unidentified compound–were also isolated. These results support an earlier work that indicated the possible presence of fucoxanthin in some members of the Pyrrophyta.     

COMPARATIVE ANALYSES OF THE DINOFLAGELLATE FLAGELLAR APPARATUS. III. FREEZE SUBSTITUTION OF AMPHIDINIUM RHYNCHOCEPHALUM1

The flagellar apparatus of the marine dinoflagellate Amphidinium rhynchocephalum Anissimowa was examined using the techniques of rapid freezing/freeze substitution and serial thin section three dimensional reconstruction. The flagellar apparatus is composed of two basal bodies that are offset from one another and lie at an angle of approximately 150° The transverse basal body is associated with two individual microtubules that extend from the proximal end of the basal body toward the flagellar opening. One of these microtubules is closely appressed to a striated fibrous root that also extends from the proximal base of the transverse basal body. The longitudinal basal body is associated with a nine member microtubular root that extends from the proximal end of the basal body toward the posterior of the cell. The longitudinal microtubular root and the transverse striated fiber are connected by a striated connective fiber. In addition to the microtubules associated with the transverse and longitudinal basal bodies, a group of microtubules originates adjacent to one of the transverse flagellar roots and extends into the cytoplasm. Vesicular channels extend from the flagellar openings to the region of the basal bodies where they expand to encompass the various connective structures of the flagellar apparatus. The possible function and evolutionary importance of these structures is discussed.     

SEXUALITY AND CYST FORMATION IN THE DINOFLAGELLATE GONYAULAX TAMARENSIS: CYST YIELD IN BATCH CULTURES1

Encystment of the toxic dinoflagellate Gonyaulax tamarensis Lebour (var. excavata) was monitored in batch cultures exposed to a variety of nutritional and environmental treatments. Limitation by nitrogen (as ammonium or nitrate) or phosphorus (as phosphate) resulted in cyst formation. When the initial concentration of limiting nutrient was varied, total cyst yield (mL^?1) was directly proportional to the cell yield at all but the highest nutrient concentrations (where encystment was minimal). Encystment efficiency was relatively constant (0.1–0.2 cysts · cell^?1) over a 5-fold range of cell densities, indicating that 20 to 40% of the vegetative populations successfully encysted. Cyst formation was negligible in nutrient-replete medium, even with a significant reduction in growth rate due to non-optimal light, temperature, or to high batch culture cell densities. Low light levels did decrease cyst yield once encystment was initiated by nutrient limitation, but this was probably linked to smaller motile cell yield and not to a specific inhibition of encystment. In contrast, encystment was more sensitive to temperature than was growth rate: optimal cyst production occurred over a relatively narrow temperature range and no cysts were formed at [Page missing]     

CIRCADIAN REGULATION OF BIOLUMINESCENCE IN THE DINOFLAGELLATE PYROCYSTIS LUNULA1

In the unicellular algae Pyrocystis lunula Schütt and Gonyaulax polyedra Stein, bioluminescence and its circadian regulation are similar in several respects, but there are also several important differences. As in G. polyedra, P. lunula emits light both as bright flashes and as a low intensity glow. At 20° C, the individual flashes are considerably brighter than in G. polyedra, and their durations are typically less than 500 ms. Both species show a circadian rhythm in the frequency of spontaneous flashes, which peaks in the night-phase under light–dark cycles and continues in both continuous light and dark. However, compared to G. polyedra, the circadian system in P. lunula is more sensitive to light: 10 min exposures (500 μmol · m^–2· s^–1 white light) can shift the phase of the rhythm by more than 8 h, and rhythmicity is completely suppressed at an irradiance above 20 μmol · m^–2· s^–1, where the G. polyedra rhythym persists for weeks. Like G. polyedra, period length increases with increasing irradiance of continuous red light but decreases with increasing intensity of continuous blue light. The glow in P. lunula differs markedly from that in G. polyedra in that it occurs at about the same intensity at all times during the circadian cycle; thus, it is not under circadian control but may fluctuate 5–10-fold in intensity within a time frame of seconds. This suggests that the glow may differ in its physiological basis in the two organisms. The results also indicate that the circadian regulation of luciferase activity differs in the two species. In G. polyedra, the organelle responsible for bioluminescence and luciferase is lost and then reformed on a daily basis; in P. lunula, the luciferase is conserved and localized elsewhere during the nonbioluminescent phase of the cycle.     

INTRACELLULAR LOCALIZATION OF SAXITOXINS IN THE DINOFLAGELLATE GONYAULAX TAMARENSIS1

The potent neurotoxin saxitoxin, and possibly several of its derivatives, are localized in two types of sites within the marine dinoflagellate Gonyaulax tamarensis Lebour. Immunocytochemical techniques using a polyclonal antibody and epifluorescence microscopy demonstrate toxin localization within the nucleus as well as on the periphery of small granules thought to be starch grains. In the nuclear region, the labelling occurred on or close to the permanently-condensed chromosomes as well as in an area within the two arms of the nucleus in the vicinity of the nucleous. No binding was observed in a closely-related, non-toxic dinoflagellate. Different binding affinities were observed between the nucleus and the grains at high and low antibody dilutions. This may relate to the polyclonal nature of the antiserum and to the presence of multiple toxins within the G. tamarensis isolate studied. Mechanistic interpretations of these labelling patterns remain speculative, especially the localization of the antigen at the outer edge of starch grains, but the distinct labelling in the nuclear region suggests that saxitoxin, with its two positively charged guanidinium groups, may bind to nucleic acids or nuclear proteins in a manner analogous to the polyamines and other cations. The labelling patterns reported here suggest that the saxitoxins may not simply be secondary metabolites but instead could be important compounds involved in the structure and function of the G. tamarensis genome.     

AMPHIDINIUM REVISITED. II. RESOLVING SPECIES BOUNDARIES IN THE AMPHIDINIUM OPERCULATUM SPECIES COMPLEX (DINOPHYCEAE), INCLUDING THE DESCRIPTIONS OF AMPHIDINIUM TRULLA SP. NOV. AND AMPHIDINIUM GIBBOSUM. COMB. NOV.1

Amphidinium operculatum Claparède et Lachmann, the type species of the dinoflagellate genus Amphidinium, has long had an uncertain identity. It has been considered to be either difficult to distinguish from other similar species or a morphologically variable species itself. This has led to the hypothesis that A. operculatum represents a “species complex.” Recently, the problem of distinguishing A. operculatum from similar species has become particularly acute, because several morphologically similar species have been found to produce bioactive compounds of potential interest to the pharmaceutical industry. In this study, we cultured and examined existing cultures of several species of Amphidinium, most of which have been previously identified as A. operculatum or as species considered by some to be synonyms or varieties of A. operculatum. Thirty strains were examined using comparative LM, SEM, and partial large subunit (LSU) rDNA sequence data. Through morphological and molecular phylogenetic analyses, six distinct species were identified, including Amphidinium trulla sp. nov. and Amphidinium gibbosum comb. nov. Amphidinium operculatum was redescribed based on four cultures. Genetic variability within the examined Amphidinium species varied greatly. There was little difference among strains in partial LSU rDNA for most species, but strains of A. carterae and A. massartii Biencheler differed by as much as 4%. In both A. carterae and A. massartii, three distinct genotypes based on partial LSU rDNA were found, but no morphological differences among strains could be observed using LM or SEM. In the case of A. carterae, no biogeographically related molecular differences were found.     

INTRASPECIFIC VARIATION IN THE DINOFLAGELLATE PERIDINIUM VOLZIP1

Clonal isolates of Peridinium volzii Lemmerman were analyzed morphologically and biochemically. Morphological observations at the light microscope level show the clones to be different varieties and forms of the same species. Biochemical analysis by enzyme electrophoresis and flow cytometric determination of nuclear DNA quantities indicates that these isolates are genetically heterogeneous without any clear correlation existing between morphological variation and biochemical variation. Isozyme analysis, however, indicates that strains from the same location are generally more related to each other than they are to isolates with other geographical origins. In general, our results suggest the presence of genetic redundancy and a multiclonal origin for individuals of the same species present in the same locale.     

TOPOGRAPHY OF CELL DIVISION IN THE STRUCTURALLY COMPLEX DINOFLAGELLATE GENUS ORNITHOCERCUS1

The dinophysoid dinoflagellates (to which Ornithocercus belongs) achieve growth both by increase in size of individual wall elements and, during rapid lateral expansion associated with cell division, by the formation of a semimeridional band of material termed the megacytic zone (MZ). The MZ maintains mother cell wall integrity during complete cytokinesis of the cell body and enclosure with new wall elements. The lists, extensive wing-like extensions of the wall, can only be reformed after dissolution of the MZ. Beginning near the ventral region (which is the last region of the wall to be duplicated), the MZ dislocates and its material is apparently resorbed. The last region of attachment is invariably dorsal and in several, but not all species, the daughter cells may remain attached during early list formation by a special remnant of the MZ, termed here the dorsal megacytic bridge (DMB). After full separation of daughter cells remnants of the DMB persist for an unknown but presumably short period. The topography of this process, involving radical ontogenetic alterations in the appearance of the daughter cells and some wall surfaces, is illustrated here by the scanning electron microscope. In addition 2 aberrant types of division are shown, one of which results in a double individual, termed a geminoid.     

EFFECT OF TEMPERATURE,LIGHT AND pH ON GROWTH,PHOTOSYNTHESIS AND RESPIRATION OF THE DINOFLAGELLATE PERIDINIUM CINCTUM FA. WESTII IN LABORATORY CULTURES1

Optimum light, temperature, and pH conditions for growth, photosynthetic, and respiratory activities of Peridinium cinctum fa. westii (Lemm.) Lef were investigated by using axenic clones in batch cultures. The results are discussed and compared with data from Lake Kinneret (Israel) where it produces heavy blooms in spring. Highest biomass development and growth rates occurred at ca. 23° C and ≥50 μE· m^?2·s¹ of fluorescent light with energy peaks at 440–575 and 665 nm. Photosynthetic oxygen release was more efficient in filtered light of blue (BG 12) and red (RG 2) than in green (VG 9) qualities. Photosynthetic oxygen production occurred at temperatures ranging from 5° to 32° C in white fluorescent light from 10 to 105 μE·m^?2·s^?1 with a gross maximum value of 1500 × 10^?12 g·cell^?1·h^?1 at the highest irradiance. The average respiration amounted to ca. 12% of the gross production and reached a maximum value of ca. 270·10^?12 g·cell^?1·h^?1 at 31° C. A comparison of photosynthetic and respiratory Q₁₀-values showed that in the upper temperature range the increase in gross production was only a third of the corresponding increase in respiration, although the gross production was at maximum. Short intermittent periods of dark (>7 min) before high light exposures from a halogen lamp greatly increased oxygen production. Depending on the physiological status of the alga, light saturation values were reached at 500–1000 μE·m^?2·s^?1 of halogen light with compensation points at 20–40 μE·m^?2·s^?1 and I_k-values at 100–200 μE·m^?2·s^?1. The corresponding values in fluorescent light in which it was cultured and adapted, were 25 to 75% lower indicating the ability of the alga to efficiently utilize varying light conditions, if the adaptation time is sufficient. Carbon fixation was most efficient at ca. pH 7, but the growth rates and biomass development were highest at pH 8.3.     

DEVELOPMENT OF THE ENDOPARASITIC DINOFLAGELLATE AMOEBOPHRYA CERATII WITHIN HOST DINOFLAGELLATE SPECIES1

The endoparasitic dinoflagellate Amoebophrya ceratii Koeppen occurs in coastal waters of Nova Scotia within cells of two dinoflagellate hosts, a Scrippsiella species (probably S. trochoidea (Stein) Loeb. III) and Dino-physis norvegica Claparede & Lachman. We describe the development of the endoparasitic stage (the trophont) of A. ceratii within host cells using light and electron microscopy. After entry into the host, the trophont grows and expands until most of the host cell is occupied by the parasite. Growth is marked by a proliferation of trophont nuclei and flagella and by the formation of numerous lobes, each of which possesses a characteristic dinoflagellate amphiesma. The mature endoparasitic trophont is recognized at the light microscopic level as a beehive-shaped structure that consists of numerous lobes of the developing motile sporont cells and a mastigocoel cavity containing the sporont flagella.     

THE MICROTUBULAR CYTOSKELETON OF AMPHIDINIUM RHYNCHOCEPHALUM (DINOPHYCEAE)1

The sub-thecal microtubular cytoskeleton of Amphidinium rhynchocephalum Anissimowa was investigated using indirect immunofluorescence microscopy and transmission electron microscopy. The majority of sub-thecal microtubules are longitudinally oriented and radiate from one of two sub-thecal transverse microtubular bands that lie adjacent to the anterior and posterior edge of the cingulum.Both transverse bands consist of 3–5 microtubules and are loop shaped with one end adjacent to the cell's right edge of the sulcus and the other end adjacent to the fibrous ventral ridge. The posterior transverse microtubular band (PTB) defines the posterior edge of the cingulum and gives rise to numerous posteriorly directed longitudinal microtubular bundles that consist of 1–3 microtubules per bundle. These bundles end at the posterior end of the cell. The PTB also gives rise to the cingular longitudinal microtubules that underlie the cingular groove and terminate at the anterior transverse microtubular band (ATB). The ATB defines the anterior edge of the cingulum and loops around the base of the epicone. This band gives rise to anteriorly directed longitudinal microtubular bundles that terminate in the small epicone of the cell. The longitudinal microtubular root of the flagellar apparatus is directed posteriorly and lies immediately beneath the theca but is distinct from the subthecal microtubule system. A narrow fibrous ridge is ventrally located to the cell's left between the exit apertures of the transverse and longitudinal flagella. In this position, the ventral ridge lies between and also connects with the anterior and posterior transverse microtubular bands. The ventral ridge is also associated with three microtubules that are distinct from other cytoskeletal microtubules. Our results demonstrate that the majority of sub-thecal microtubules originate from one of two microtubular bands associated with the cingulum. The possible role of the fibrous ventral ridge and its associated microtubules is also discussed.