MECHANISMS OF FLUID SHEAR‐INDUCED INHIBITION OF POPULATION GROWTH IN A RED‐TIDE DINOFLAGELLATE1

Net population growth of some dinoflagellates is inhibited by fluid shear at shear stresses comparable with those generated during oceanic turbulence. Decreased net growth may occur through lowered cell division, increased mortality, or both. The dominant mechanism under various flow conditions was determined for the red‐tide dinoflagellate Lingulodinium polyedrum (Stein) Dodge. Cell division and mortality were determined by direct observation of isolated cells in 0.5‐mL cultures that were shaken to generate unquantified fluid shear. Larger volume cultures were exposed to quantified laminar shear in Couette‐flow chambers (0.004–0.019 N·m ^{? 2} shear stress) and to unquantified flow in shaken flasks. In these larger cultures, cell division frequency was calculated from flow cytometric measurements of DNA·cell^?1. The mechanism by which shear inhibits net growth of L. polyedrum depends on shear stress level and growth conditions. Observations on the isolated cells showed that shaking inhibited growth by lowering cell division without increased mortality. Similar results were found for early exponential‐phase cultures exposed to the lowest experimental shear stress in Couette‐flow chambers. However, mortality occurred when a late exponential‐phase culture was exposed to the same low shear stress and was inferred to occur in cultures exposed to higher shear stresses. Elevated mortality in those treatments was confirmed using behavioral, morphological, and physiological assays. The results predict that cell division in L. polyedrum populations will be inhibited by levels of oceanic turbulence common for near‐surface waters. Shear‐induced mortality is not expected unless shear‐stress levels are unusually high or when cellular condition resembles late exponential/stationary phase cultures.     

MEASUREMENT OF MICROALGAL CELL VOLUME BY FLOW CYTOMETRY

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.     

CELL DIVISION IN THE DINOFLAGELLATE GAMBIERDISCUS TOXICUS IS PHASED TO THE DIURNAL CYCLE AND ACCOMPANIED BY ACTIVATION OF THE CELL CYCLE REGULATORY PROTEIN,CDC2 KINASE1

The cell division cycle in several pelagic dinoflagellate species has been shown to be phased with the diurnal cycle, suggesting that their cell cycle may be regulated by a circadian clock. In this study, we examined the cell cycle of an epibenthic dinoflagellate, Gambierdiscus toxicus Adachi and Fukuyo (Dinophyceae), and found that cell division was similarly phased to the diurnal cycle. Cell division occurred during a 3-h window beginning 6 h after the onset of the dark phase. Cell cycle progression in higher eukaryotes is regulated by a cell cycle regulatory protein complex consisting of cyclin and the cyclin-dependent kinase CDC2. In this report, we identified a CDC2-like kinase in G. toxicus that displays activity in vitro against a known substrate of CDC2 kinase, histone H1. As in higher eukaryotes, CDC2 kinase was expressed constitutively in G. toxicus throughout the cell cycle, but it was activated only late in the dark phase, concurrent with the presence of mitotic cells. These results indicate that cell division in G. toxicus is regulated by molecular controls similar to those found in higher eukaryotes.     

GROWTH AND CELL CYCLE OF TWO CLOSELY RELATED RED TIDE-FORMING DINOFLAGELLATES: GYMNODINIUM NAGASAKIENSE AND G. CF. NAGASAKIENSE1

Small-sized vegetative cells were found to co-occur with normal-sized cells in populations of the European bloom-forming dinoflagellate Gymnodinium cf. nagasakiense Takayama et Adachi, currently known as Gyrodinium aureolum Hulburt, but not in populations of the closely related Japanese species Gymnodiniumn agasakiense. We examined how cell size differentiation may influence growth and cell cycle progression under a 12:12-h light: dark cycle in the European taxon, as compared to the Japanese one. Cell number and volume and chlorophyll red fluorescence in both species varied widely during the photocycle. These variations generally appeared to be related lo the division period, which occurred at night, as indicated by the variations of the fraction of binucleated cells (mitotic index) as well as the distribution of cellular DNA content. “Small” cells of G. cf. nagasakiense divided mainly during the first part of the dark period, although a second minor peak of dividing cells could occur shortly before light onset. In contrast, “large” cells displayed a sharp division peak that occurred 9 h after the beginning of the dark period. The lower degree of synchrony of “small” cells could be a consequence of their faster growth. Alternatively, these data may suggest that cell division is lightly controlled by an endogenous clock in “large” cells and much more loosely controlled in “small” cells. Cells of the Japanese species, which were morphologically similar to “large” cells of the European taxon, displayed an intermediate growth pattern between the two cell types of G. cf. nagasakiense, with a division period that extended to most of the dark period.     

Species‐specific physiological response of dinoflagellates to quantified small‐scale turbulence1

Turbulence has been shown to alter different aspects of the physiology of some dinoflagellates. The response appears to be species‐specific and dependent on the experimental design and setup used to generate small‐scale turbulence. We examined the variability of the response of three dinoflagellate species to the turbulence, following the same experimental design used by Berdalet (1992) on Akashiwo sanguinea (Hirasaka) Ge. Hansen et Moestrup (=Gymnodinium nelsonii G. W. Martin). In all experiments, turbulence was generated by an orbital shaker at 100 rpm, which corresponded on bulk average, to dissipation rates (ε, quantified using an acoustic Doppler velocimeter) of ≈2 cm² · s^?3. Turbulence did not appreciably affect Gymnodinium sp., a small dinoflagellate. However, Alexandrium minutum Halim and Prorocentrum triestinum J. Schiller exhibited a reduced net growth rate (33% and 28%, respectively) when shaken during the exponential growth phase. Compared to the still cultures, the shaken treatments of A. minutum and P. triestinum increased the mean cell volume (up to 1.4‐ and 2.5‐fold, respectively) and the mean DNA content (up to 1.8‐ and 5.3‐fold, respectively). Cultures affected by turbulence recovered their normal cell properties when returned to still conditions. The swimming speed of the cells exposed to agitation was half that of the unshaken ones. Overall, the response of A. minutum and P. triestinum was similar, but with lower intensity, to that observed previously on A. sanguinea. We found no clear trends related to taxonomy or morphology.     

CELL DIVISION PERIODICITY IN 13 SPECIES OF MARINE PHYTOPLANKTON ON A LIGHT: DARK CYCLE1

The division rates of 26 clonal cultures representing 13 species of planktonic marine algae (6 diatoms, 2 flagellated chrysophytes, 2 coccolithophores, 1 cryptomonad flagellate, I dinoflagellate, 1 green alga) were determined every 2 h for 48 h during exponential growth on a 14:10 LD cycle in nutrient-replete batch culture. Cyclic oscillations in the division rate were detectable in 22 of these clones. Of 14 diatom clones examined, four displayed nearly constant division rates throughout the LD cycle and ten showed strong periodicity favoring division during the light periods. In contrast, all other algae (12 clones) exhibited division rate maxima during periods of darkness, and clearly detectable decreases in cell number for time intervals of 4–8 h during periods of illumination. Intraspecific differences in division periodicity were found among eight clones of the diatom Thalassiosira pseudonana (Hustedt) Hasle & Heimdal and six clones of the coccolithophore Emiliania huxleyi (Lohm.) Hay & Mohler.     

CHARACTERIZATION OF A DINOFLAGELLATE CRYPTOCHROME BLUE‐LIGHT RECEPTOR WITH A POSSIBLE ROLE IN CIRCADIAN CONTROL OF THE CELL CYCLE1

Karenia brevis (C. C. Davis) G. Hansen et Moestrup is a dinoflagellate responsible for red tides in the Gulf of Mexico. The signaling pathways regulating its cell cycle are of interest because they are the key to the formation of toxic blooms that cause mass marine animal die‐offs and human illness. Karenia brevis displays phased cell division, in which cells enter S phase at precise times relative to the onset of light. Here, we demonstrate that a circadian rhythm underlies this behavior and that light quality affects the rate of cell‐cycle progression: in blue light, K. brevis entered the S phase early relative to its behavior in white light of similar intensity, whereas in red light, K. brevis was not affected. A data base of 25,000 K. brevis expressed sequence tags (ESTs) revealed several sequences with similarity to cryptochrome blue‐light receptors, but none related to known red‐light receptors. We characterized the K. brevis cryptochrome (Kb CRY) and modeled its three‐dimensional protein structure. Phylogenetic analysis of the photolyase/CRY gene family showed that Kb CRY is a member of the cryptochrome DASH (CRY DASH) clade. Western blotting with an antibody designed to bind a conserved peptide within Kb CRY identified a single band at ～55 kDa. Immunolocalization showed that Kb CRY, like CRY DASH in Arabidopsis, is localized to the chloroplast. This is the first blue‐light receptor to be characterized in a dinoflagellate. As the Kb CRY appears to be the only blue‐light receptor expressed, it is a likely candidate for circadian entrainment of the cell cycle.     

EFFECTS OF TURBULENCE ON THE MARINE DINOFLAGELLATE GYMNODINIUM NELSONII1

Laboratory experiments were conducted to study the effects of agitation on growth, cell division, and nucleic acid dynamics of the dinoflagellate Gymnodinium nelsonii Martin. When cultures were placed on an orbital shaker at 100 rpm, cell division was prevented, cellular volume increased up to 1.5 times that of the nonperturbed cells, the form and location of the cell nucleus were modified, and the RNA and DNA concentrations per cell increased up to 10 times those of the controls. When shaking was stopped after 10 days, cells divided immediately at about 2/3 of the division rate of the unshaken populations, and all the altered parameters were restored. If the agitation continued for more than 20 days, total cell death and disintegration occurred. Several cellular types differing in size and shape were observed in the control and shaken cultures. One possible hypothesis for these results is that failure of the cell to divide results from physical disturbance of the microtubule assemblage associated with chromosome separation during mitosis. My study suggests that small-scale oceanic turbulence of sufficient intensity may inhibit growth of individual dinoflagellate cells, but immediate development of the population may continue when calm weather follows the active mixing period.     

Inhibition of cell proliferation by mechanical agitation involves transient cell cycle arrest at G1 phase in dinoflagellates

Summary.  Cell proliferation of dinoflagellates is negatively affected by mechanical agitation and red tides caused by members of the group have been correlated with periods of calm sea conditions. The mechanism involved in the mechanically transduced inhibition of cell proliferation is thought to involve the disruption of the cell division apparatus. In this study, we used highly synchronized cells and flow cytometry to study the effects of mechanical agitation on cell cycle progression. We observed that mechanical agitation induced transient cell cycle arrest at G₁ phase, in both the heterotrophic dinoflagellate Crypthecodinium cohnii and the photosynthetic dinoflagellate Heteroscapsa triquetra. Received March 12, 2002; accepted July 20, 2002; published online November 29, 2002     

INTERACTIONS OF LIGHT/DARK CYCLES AND NITROGEN PULSES ON THE TIMING OF CELL DIVISION IN THE NITROGEN-LIMITED MARINE DIATOM CYLINDROTHECA FUSIFORMIS (BACILLARIOPHYCEAE)1

Cell division in most eukaryotic algae grown on alternating periods of light and dark (LD) is synchronized or phased so that cell division occurs only during a restricted portion of the LD cycle. However, the phase angle of the cell division gate, the time of division relative to the beginning of the light period, is known to be affected by growth conditions such as nutrient status and temperature. In this study, it is shown that the phase angle of cell division in a diatom, Cylindrotheca fusiformis Reimann and Lewin, is affected by the N-limited growth rate; cell division occurred later in the dark period (12:12 h LD cycle) when the growth rate was infradian (D = 0.42 d^?1) than when it was ultradian (D = 1.0 d^?1). Nitrogen-pulses did not affect the phase angle of the division gate, but could shift the time of peak cell division activity within the division gate. The effects, if any, of N-pulses were dependent upon the growth rate and the time of day that the pulses were administered. These responses indicate that the timing of cell division in this diatom is not determined solely by the zeitgeber from the LD cycle, but rather that a LD cycle control mechanism and a N-mediated control mechanism are both involved and are somewhat interdependent. In addition, an increase in protein was observed immediately after administering a N-pulse to C. fusiformis in the ultradian growth mode indicating that the accumulation of protein can be uncoupled from the cell division cycle.     

EFFECTS OF SMALL-SCALE TURBULENCE ON PHOTOSYNTHESIS,PIGMENTATION, CELL DIVISION,AND CELL SIZE IN THE MARINE DINOFLAGELLATE GOMAULAX POLYEDRA (DINOPHYCEAE)1

Several experiments were conducted to understand better the physiological mechanisms underlying growth inhibition of the dinoflagellate Gonyaulax polyedra Stein due to small-scale turbulence shear. To measure photosynthetic ¹⁴C uptake, a “phytoplankton wheel” device for rotating cultures in closed bottles was used. Turbulence was quantified biologically in the bottles by comparing growth inhibition with that in cultures with constant shear between a fixed cylinder and an outer concentric rotating cylinder (a stable Couette flow). At saturating irradiances, particulate photosynthesis (P_sat) or photosynthesis per unit chlorophyll (P^B_sat) were not inhibited completely at the highest turbulence level (26.6 rad.s^?1), and photosynthesis was less sensitive than growth. Photosynthesis per cell (P^C_sat) was increased by turbulence. In three experiments on the effects of turbulence on photosynthesis versus irradiance curves, the slope of the curve, α, for particulate photosynthesis at limiting irradiances did not change. Photosynthesis per unit chlorophyll per unit irradiance (α^B) decreased at high (but not intermediate) turbulence levels. Photosynthesis per cell per unit irradiance, α^C, increased with turbulence, suggesting an increase in photosynthetic efficiency in turbulent cultures. In two of the three experiments, respiration rates increased with turbulence, and in one experiment excretion of photosynthetically fixed ¹⁴C was not affected by motion. Ratios of accessory pigments to chlorophyll a did not change with turbulence, but pigments per cell and per dry weight increased with turbulence. These findings suggest little or no disruption of the photosynthetic apparatus. When turbulence was applied for 1 week, β-carotene increased while peridinin and diadinoxanthin decreased, suggesting inhibition of synthesis of these latter pigments by prolonged turbulence. Since cell numbers did not increase or decreased during turbulent 72–h incubations, cell division was inhibited and also the cells were very much enlarged. Increases in α^C per cell suggest that, in the sea, photo synthetic metabolism can persist efficiently without cell division during turbulent episodes. After turbulence ceases or reaches low levels again, cells can then divide and blooms may form. Thus, blooms can come or go fairly rapidly in the ocean depending on the degree of wave- and wind-induced turbulence.     

DIEL IN SITU PICOPHYTOPLANKTON CELL DEATH CYCLES COUPLED WITH CELL DIVISION1

The diel variability in picophytoplankton cell death was analyzed by quantifying the proportion of dead cyanobacteria Prochlorococcus and Synechococcus cells along several in situ diel cycles in the open Mediterranean Sea. During the diel cycle, total cell abundance varied on average 2.8 ± 0.6 and 2.6 ± 0.4 times for Synechococcus and Prochlorococcus populations, respectively. Increasing percentages of dead cells of Prochlorococcus and Synechococcus were observed during the course of the day reaching the highest values around dusk and decreasing as the night progressed, indicating a clear pattern of diel variation in the cell mortality of both cyanobacteria. Diel cycles of cell division were also monitored. The maximum percentage of dead cells (Max % DC) and the G2 + M phase of the cell division occurred within a period of 2 h for Synechoccoccus and 4.5 h for Prochlorococcus, and the lowest fraction of dead cells occurred at early morning, when the maximum number of cells in G1 phase were also observed. The G1 maximum corresponded with the maximal increase in newly divided cells (minimum % dead cells), and the subsequent exposure of healthy daughter cells to environmental stresses during the day resulted in the progressive increase in dying cells, with the loss of these cells from the population when cell division takes place. The discovery of diel patterns in cell death observed revealed the intense dynamics of picocyanobacterial populations in nature.     

ACTIVE GROWTH OF THE OCEANIC DINOFLAGELLATE CERATIUM TERES IN THE CARIBBEAN AND SARGASSO SEAS ESTIMATED BY CELL CYCLE ANALYSIS1

The growth rate of an oceanic dinoflagellate, Ceratium teres Kofoid, was investigated in the Sargasso and Caribbean Seas from September 1989 to July 1990 using the cell cycle analysis method. Estimated growth rates ranged from 0.29 to 0.58 day^?1 and were 1.5–7.2 times higher than generally accepted rates for oceanic dinoflagellates. The higher rates in this report were mainly due to an improvement in techniques that determine the duration of a terminal cell cycle phase in situ. The day-to-day variation in growth rates was surprisingly small, but, from long-term measurements, a weak correlation was found among temperature, daily irradiance, and seasonal growth rate. The calculated species-specific primary production ranged from 0.5 to 1.8 mg C·m^?2·day^?1, about 1% of the estimated total production. Ceratium teres may be an important carbon source at the base of the grazing food chain.     

GALEIDINIIUM RUGATUM GEN. ET SP. NOV. (DINOPHYCEAE), A NEW COCCOID DINOFLAGELLATE WITH A DIATOM ENDOSYMBIONT1

A new sand‐dwelling dinoflagellate from Palau, Galeidinium rugatum Tamura et Horiguchi gen. et sp. nov., is described. The life cycle of this new alga consists of a dominant nonmotile phase and a brief motile phase. The motile cell transforms itself directly into the nonmotile cell after swimming for a short period, and cell division takes place in the nonmotile phase. The nonmotile cell possesses a dome‐like cell covering, which is wrinkled and equipped with a transverse groove on the surface. The cell has 10–20 chloroplasts and a distinct eyespot. The motile cell is Gymnodinium‐like in shape. The dinoflagellate possesses an endosymbiotic alga to which the chloroplasts belong and which is separated from the host (dinoflagellate) cytoplasm by a unit membrane. The endosymbiont cytoplasm also possesses its own eukaryotic nucleus and mitochondria. The eyespot is surrounded by triple membranes and is located in the host cytoplasm. Photosynthetic pigment analysis, using HPLC, revealed that G. rugatum possesses fucoxanthin as the principal accessory pigment instead of peridinin. The rbcL tree showed that G. rugatum is monophyletic with Durinskia baltica (Levander) Carty et Cox and Kryptoperidinium foliaceum (Stein) Lindemann and that this clade is closely related to the pennate diatom, Cylindrotheca sp. The endosymbiont of G. rugatum is therefore shown to be a diatom. Phylogenetic analysis based on small subunit rDNA sequences demonstrated that G. rugatum, D. baltica, and K. foliaceum, all of which are known to harbor an endosymbiont of diatom origin, are closely related.     

The reversible effect of flow on the morphology of ceratocorys horrida (PERIDINIALES,DINOPHYTA)*

Most cells experience an active and variable fluid environment, in which hydrodynamic forces can affect aspects of cell physiology including gene regulation, growth, nutrient uptake, and viability. The present study describes a rapid yet reversible change in cell morphology of the marine dinoflagellate Ceratocorys horrida Stein, due to fluid motion. Cells cultured under still conditions possess six large spines, each almost one cell diameter in length. When gently agitated on an orbital shaker under conditions simulating fluid motion at the sea surface due to light wind or surface chop, as determined from digital particle imaging velocimetry, population growth was inhibited and a short‐spined cell type appeared that possessed a 49% mean decrease in spine length and a 53% mean decrease in cell volume. The reduction in cell size appeared to result primarily from a 39% mean decrease in vacuole size. Short‐spined cells were first observed after 1 h of agitation at 20°C; after 8 to 12 d of continuous agitation, long‐spined cells were no longer present. The morphological change was completely reversible; in previously agitated populations devoid of long‐spined cells, cells began to revert to the long‐spined morphology within 1 d after return to still conditions. During morphological reversal, spines on isolated cells grew up to 10 μm·d^?1. In 30 d the population morphology had returned to original proportions, even though the overall population growth was zero during this time. The reversal did not occur as a result of cell division, because single‐cell studies confirmed that the change occurred in the absence of cell division and much faster than the 16‐d doubling time. The threshold level of agitation causing morphology change in C. horrida was too low to inhibit population growth in the shear‐sensitive dinoflagellate Lingulodinium polyedrum. At the highest level of agitation tested, there was negative population growth in C. horrida cultures, indicating that fluid motion caused cell mortality. Small, spineless cells constituted a small percentage of the population under all conditions. Although their abundance did not change, single‐cell studies and morphological characteristics suggest that the spineless cells can rapidly transform to and from other cell types. The sinking rate of individual long‐spined cells in still conditions was significantly less than that of short‐spined cells, even though the former are larger and have a higher cell density. These measurements demonstrate that the long spines of C. horrida reduce cell sinking. Shorter spines and reduced swimming would allow cells to sink away from turbulent surface conditions more rapidly. The ecological importance of the morphological change may be to avoid conditions that inhibit population growth and potentially cause cell damage.     

Influence of stem-cell cycle time on accelerated re-population during radiotherapy in head and neck cancer

Objectives

Tumour re‐population during radiotherapy was identified as an important reason for treatment failure in head and neck cancers. The process of re‐population is suggested to be caused by various mechanisms, one of the most plausible one being accelerated division of stem‐cells (i.e. drastic shortening of cell cycle duration). However, the literature lacks quantitative data regarding the length of tumour stem‐cell cycle time during irradiation.

Materials and methods

The presented work suggests that if accelerated stem‐cell division is indeed a key mechanism behind tumour re‐population, the stem‐cell cycle time can drop below 10 h during radiotherapy. To illustrate the possible implications, the mechanism of accelerated division was implemented into a Monte Carlo model of tumour growth and response to radiotherapy. Tumour response to radiotherapy was simulated with different stem‐cell cycle times (between 2 and 10 h) after the initiation of radiotherapy.

Results

It was found that very short stem‐cell cycle times lead to tumour re‐population during treatment, which cannot be overcome by radiation‐induced cell kill. Increasing the number of radiation dose fractions per week might be effective, but only for longer cell cycle times.

Conclusion

It is of crucial importance to quantitatively assess the mechanisms responsible for tumour re‐population, given that conventional treatment regimens are not efficient in delivering lethal doses to advanced head and neck tumours.     

Cohesins and condensins orchestrate the 4D dynamics of yeast chromosomes during the cell cycle

Duplication and segregation of chromosomes involves dynamic reorganization of their internal structure by conserved architectural proteins, including the structural maintenance of chromosomes (SMC) complexes cohesin and condensin. Despite active investigation of the roles of these factors, a genome‐wide view of dynamic chromosome architecture at both small and large scale during cell division is still missing. Here, we report the first comprehensive 4D analysis of the higher‐order organization of the Saccharomyces cerevisiae genome throughout the cell cycle and investigate the roles of SMC complexes in controlling structural transitions. During replication, cohesion establishment promotes numerous long‐range intra‐chromosomal contacts and correlates with the individualization of chromosomes, which culminates at metaphase. In anaphase, mitotic chromosomes are abruptly reorganized depending on mechanical forces exerted by the mitotic spindle. Formation of a condensin‐dependent loop bridging the centromere cluster with the rDNA loci suggests that condensin‐mediated forces may also directly facilitate segregation. This work therefore comprehensively recapitulates cell cycle‐dependent chromosome dynamics in a unicellular eukaryote, but also unveils new features of chromosome structural reorganization during highly conserved stages of cell division.     

LIFE CYCLE OF THE HETEROTROPHIC DINOFLAGELLATE PFIESTERIA PISCICIDA (DINOPHYCEAE)1

The putatively toxic dinoflagellate Pfiesteria piscicida (Steidinger et Burkholder) has been reported to have an unusual life cycle for a free‐living marine dinoflagellate. As many as 24 life cycle stages were originally described for this species. During a recent phylogenetic study in which we used clonal cultures of P. piscicida, we were unable to confirm many reported life cycle stages. To resolve this discrepancy, we undertook a rigorous examination of the life cycle of P. piscicida using nuclear staining techniques combined with traditional light microscopy, high‐resolution video microscopy, EM, and in situ hybridization with a suite of fluorescently labeled peptide nucleic acid (PNA) probes. The results showed that P. piscicida had a typical haplontic dinoflagellate life cycle. Asexual division occurred within a division cyst and not by binary fission of motile cells. Sexual reproduction of this homothallic species occurred via the fusion of isogamous gametes. Examination of tanks where P. piscicida was actively feeding on fish showed that amoebae were present; however, they were contaminants introduced with the fish. Whole cell probing using in situ hybridization techniques confirmed that these amoebae were hybridization negative for a P. piscicida‐specific PNA probe. Direct observations of clonal P. piscicida cultures revealed no unusual life cycle stages. Furthermore, the results of this study provided no evidence for transformations to amoebae. We therefore conclude that P. piscicida has a life cycle typical of free‐living marine dinoflagellates and lacks any amoeboid or other specious stages.     

Utilization of amino acids by Chromatium sp. strain D

Summary The duration of various morphologically distinct phases in the division cycle of the marine heterotroph Cryptothecodinium cohnii was measured in cultures initiated with synchronously excysted swarmer cells. Parent cysts were selectively isolated on plastic surfaces and progeny of a narrow age distribution harvested in a specifically conditioned medium. The swarmer phase, an interval of predivisional encystment, daughter cell formation and excystment were 5.0, 3.0 and 2.0 h respectively. Two major kinds of cytokinesis (production of 2 and 4 daughter cells) were observed resulting in a mean daughter cell number of 2.7 under these conditions. Other growth parameters for this dinoflagellate are described.     

Cell Cycle Dynamics of Cultured Coral Endosymbiotic Microalgae (Symbiodinium) Across Different Types (Species) Under Alternate Light and Temperature Conditions

Dinoflagellates of the genus Symbiodinium live in symbiosis with many invertebrates, including reef‐building corals. Hosts maintain this symbiosis through continuous regulation of Symbiodinium cell density via expulsion and degradation (postmitotic) and/or constraining cell growth and division through manipulation of the symbiont cell cycle (premitotic). Importance of premitotic regulation is unknown since little data exists on cell cycles for the immense genetic diversity of Symbiodinium. We therefore examined cell cycle progression for several distinct SymbiodiniumITS2‐types (B1, C1, D1a). All types exhibited typical microalgal cell cycle progression, G₁ phase through to S phase during the light period, and S phase to G₂/M phase during the dark period. However, the proportion of cells in these phases differed between strains and reflected differences in growth rates. Undivided larger cells with 3n DNA content were observed especially in type D1a, which exhibited a distinct cell cycle pattern. We further compared cell cycle patterns under different growth light intensities and thermal regimes. Whilst light intensity did not affect cell cycle patterns, heat stress inhibited cell cycle progression and arrested all strains in G₁ phase. We discuss the importance of understanding Symbiodinium functional diversity and how our findings apply to clarify stability of host‐Symbiodinium symbioses.