INFLUENCE OF CELL VOLUME ON THE GROWTH AND SIZE REDUCTION OF MARINE AND ESTUARINE DIATOMS1

The maximal growth rate (μ_max) of 19 marine and estuarine diatoms decreased with increasing cell volume (V). The relationship between log μ_max (Y) and log V (X) was calculated. Statistical analyses showed that the slope of the equation was not significantly different from those obtained by other researchers and that the 95% confidence intervals of mean μ_max at cell volumes of 10³–10⁵μm³ were not significantly different from those cited in most studies. A new regression line for diatoms was calculated as follows: log μ_max= 0.47–0.14 log V; r =–0.69. The rate of size reduction per generation of the 19 diatom species ranged from 0.03 to 0.87 μm per generation. The rate increased with increasing cell length and cell volume and with decreasing maximum division rate. Statistical analyses showed that the rate was closely related to the cell volume and to the reciprocal of the growth rate. The relationships between maximal growth rate and cell volume and between rate of size reduction and cell volume showed that a diatom with a large volume had a smaller maximal growth rate and a larger rate of size reduction than a diatom with a small volume. The estimates using the equation for the regression line between the rate of size reduction and the reciprocal of maximum division rate indicated that a diatom with a high maximum division rate would need more generation equivalents for a certain size reduction than a diatom with a low maximum division rate, but the periods required for reduction would be approximately equal irrespective of maximum division rate.     

用流式细胞光度术研究PGE2对小鼠骨髓CFU—GM细胞周期的影响

在一定PGE_2浓度(4.8×10~(-9)mol/L)作用下,小鼠骨髓细胞CFU-GM经4.5d和7d培养后,其增殖状态下的细胞G_n/G_r期细胞数均比对照组增加,S期细胞数减少,G_2 M期细胞数也有下降,但不明显。在不同PGE_2浓度(2.8×10~(-9)～2.8×10~(-6)mol/L)作用下,经4d培养,随着PGE_2浓度增加,G_n/G_1期细胞数递增,而S期细胞数却随PGE_2浓度增加而减少,G_2 M细胞数也减少,但与PGE_2剂量关系不明显。以上实验结果提示,PGE_2主要抑制G_1期细胞向S期细胞的转化,阻断S期细胞生长。 此外,通过流式细胞光度术(FCM)对小鼠骨髓细胞CFU-GM集落细胞的前向角和90°散射光测定,与对照组比较,前者无变化,后者变化较明显。此结果表明,PGE_2对细胞内部颗粒的折光度有影响。     

CD11b/DNA双参数流式细胞术检测HL-60细胞分化的细胞周期

目的采用双参数流式细胞术研究全反式维甲酸(alltransretinoidacid,ATRA)诱导人类急性早幼粒白血病细胞HL-60细胞分化的细胞周期。方法HL-60细胞经分化诱导剂ATRA(终浓度为1μmol/L)诱导不同时间点后,利用CD11b/DNA双参数流式细胞术同时检测分化细胞表面抗原CD11b的表达及分化细胞DNA含量。结果HL-60细胞经ATRA诱导后,细胞表面分化抗原CD11b表达明显升高,细胞阻滞于G0/G1期,且CD11b阳性细胞主要位于G0/G1期。结论CD11b/DNA双参数流式细胞术能简便,快速,直观地检测细胞分化的细胞周期。     

COMPARATIVE PHYSIOLOGICAL STUDY OF MARINE DIATOMS AND DINOFLAGELLATES IN RELATION TO IRRADIANCE AND CELL SIZE. I. GROWTH UNDER CONTINUOUS LIGHT1,2

Cell division rates and chlorophyll a and protein contents for ten diatom and dinoflagellate species were measured. Species were chosen to include a wide range of cell size in terms of both cell volume and cell protein: from 0.004 ng protein/cell for a small Chaetoceros sp. to 2.2 ng protein/cell for Prorocentrum micans Ehrenberg. Experiments were conducted in batch or semi-continuous cultures at 21 C under continuous illumination from 8–256 μEin ^.m^-2'^.s^-1. Light saturation of cell division occurred at 32–80 μEin m^-1 s^-1 for all species, with no observable difference between the two phylogenetic groups. When the light-saturated cell division rates were plotted against cell size as protein/cell, the diatoms and dinoflagellates fell on two separate lines with the diatoms having higher rates. Chl a /protein ratios (μg/μg) decreased with increasing irradiance. The diatoms had higher chl a per unit protein. The relationship between cell division rate and the chl a/protein ratio is discussed.     

FLOW CYTOMETRY AND MICROSCOPY OF GAMETOGENESIS IN NITZSCHIA PUNGENS,A TOXIC,BLOOM-FORMING,MARINE DIATOM1

The domoic acid-producing diatom Nitzschia pungens Grunow f. multiseries Hasle, which is responsible for amnesic shellfish poisonings in Prince Edward Island, Canada, underwent gametogenesis when senescent cells (i.e. in stationary growth phase for more than 290 days) were subcultured into fresh FE medium and light intensity was increased from 33 to 530 μE · m^?2· s^?1. The number of gametes produced varied with the salinity of the medium, with a maximum at 23.5‰. Cells in the exponential growth phase (0.8 div · d^?1) did not produce gametes, nor did senescent cells when transferred without change in light intensity. Anisogamous gametes, probably haploid, were isolated by combining conventional microscopy with flow cytometry. Zygotes resulting from syngamy yielded cigar-shaped naviculoid cells, morphologically different from parent cells (heteromorphism). These cells, with a division rate of 1.9 div · d^?1, could serve as a seed population and explain the origin and rapid progression of the toxic blooms of red-water proportions that have been a public health problem in Eastern Canada. Production of domoic acid by postexponential and moribund cells but not by gametes, zygotes, or immediately resulting cells, provides an insight into the dependence of toxicity on the developmental history of this diatom.     

RELATIONSHIPS BETWEEN NITRATE REDUCTASE ACTIVITY AND RATES OF GROWTH AND NITRATE INCORPORATION UNDER STEADY-STATE LIGHT OR NITRATE LIMITATION IN THE MARINE DIATOM THALASSIOSIRA PSEUDONANA (BACILLARIOPHYCEAE)1

Although activity of the enzyme nitrate reductase (NR) can potentially be used to predict the rate of nitrate incorporation in field assemblages of marine phytoplankton, application of this index has met with little success because the relationship between the two rates is not well established under steady-state conditions. To provide a basis for using NR activity measurements, the relationships among NR activity, growth rate, cell composition, and nitrate incorporation rate were examined in cultures of Thalassiosira pseudonana (Hustedt)Hasle and Heimdal, growing a) under steady-state light limitation, b) during transitions between low and high irradiance (15 or 90 μmol quanta.m^?2.s^?1), and c) under steady-state nitrate limitation. Using a modified assay for NR involving additions of bovine serum albumin to stabilize enzyme activity, NR activity in light-limited cultures was positively and quantitatively related to calculated rates of nitrate incorporation, even in cultures that were apparently starved of selenium. During transitions in irradiance, growth rates acclimated to new conditions within 1 day; through the transition, the relationship between NR activity and nitrate incorporation rate remained quantitative. In nitrate-limited chemostat cultures, NR activity was positively correlated with growth rate and with nitrate incorporation rates, but the relationship was not quantitative. NR activity exceeded nitrate incorporation rates at lower growth rates (<25% of nutrient-replete growth rates), but chemostats operating at such low dilution rates may not represent ecologically relevant conditions for marine diatoms. The strong relationship between NR activity and nitrate incorporation provides support for the idea that NR is rate-limiting for nitrate incorporation or is closely coupled to the rate-limiting step. In an effort to determine a suitable variable for scaling NR activity, relationships between different cell components and growth rate were examined. These relationships differed depending on the limiting factor. For example, under light limitation, cell volume and cell carbon content increased significantly with increased growth rate, while under nitrate limitation cell volume and carbon content decreased as growth rates increased. Despite the differences found between cell composition and growth rate under light and nitrate limitation, the relationships between NR activity scaled to different compositional variables and growth rate did not differ between the limitations. In field situations where cell numbers are not easily determined, scaling NR activity to particulate nitrogen content may be the best alternative. These results establish a strong basis for pursuing NR activity measurements as indices of nitrate incorporation in the field.     

COMBINATION OF FLOW CYTOMETRY AND SINGLE CELL ABSORPTION SPECTROSCOPY TO STUDY THE PHYTOPLANKTON STRUCTURE AND TO CALCULATE THE CHL A SPECIFIC ABSORPTION COEFFICIENTS AT THE TAXON LEVEL1

The phytoplankton community structure of a hypertrophic lake was quantitatively determined with the aid of flow cytometry. The flow cytometry signals were calibrated to obtain cell‐specific information, such as the chl a content and the biovolume per cell. The reliability of this method was tested with laboratory cultures. The results of the phytoplankton structure in a hypertrophic lake with respect to chl distribution in the different algal groups obtained by flow cytometry were compared with the results from HPLC pigment fingerprinting. Both methods yield the percentage contribution of the different algal groups to total chl a. The chl a specific absorption coefficient of the phytoplankton (a^*_Phy) was determined via visible (VIS) spectroscopy of samples taken from a hypertrophic lake (Auensee) in 2003. The results indicated that a^*_Phy of the total cell suspension is dependent on the phytoplankton structure as well as on environmental factors. The linear relationship between a^*_Phy at 675 nm and the product of the chl a content per cell and the biovolume offered the possibility to normalize phytoplankton absorption spectra to acquire the taxon‐specific a^*_Phy. The estimated a^*_Phy (675 nm) values were used to normalize single cell absorption spectra at this wavelength to obtain the a^*_Phy between 400 and 750 nm for representatives of the major algal groups. Our measurements show that the absorption coefficient for the whole phytoplankton community varies within the season. Finally, we used the a^*_Phy and the chl a distribution to calculate the light absorption of each algal group in the hypertrophic lake.     

STUDIES ON THE AUTECOLOGY OF THE MARINE DIATOM THALASSIOSIRA NORDENSKIOELDII. II. THE INFLUENCE OF CELL SIZE ON GROWTH RATE,AND CARBON,NITROGEN, CHLOROPHYLL a AND SILICA CONTENT1

The effect of cell size on growth rates and some cellular contents of Thalassiosira nordenskioeldii Cleve has been measured at 0 and 10 C. At 0 C the growth rate did not vary with cell size. The 2 smallest clones at this temperature had reduced growth rates because of the induction of sexuality in that size range. The clones grown at 10 C showed a significant negative relationship between growth rate and valve diameter with the cell surface area/volume ratio positively related to growth rate. At both temperatures the smaller cells had proportionately more carbon and nitrogen/unit cell volume. The amount of chlorophyll a and silica/unit cell surface area increased with increasing cell surface area at both 0 and 10 C. Both the C/N and C/chl a ratios showed no significant change with cell size at either temperature but there was a significant increase in the C/chl a ratio at 0 C. The C/Si ratio decreased with increasing cell size at both 0 and 10 C.     

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.     

INHIBITORY EFFECTS OF CONCANAVALIN A AND TUNICAMYCIN ON SEXUAL ATTACHMENT OF ALEXANDRIUM CATENELLA (DINOPHYCEAE)1

The effect of concanavalin A (a lectin) and tunicamycin (an inhibitor of protein glycosylation) on sexual attachment of gamete pairs of the dinoflagellate Alexandrium catenella Whedon & Kofoid was studied. Concanavalin A inhibited sexual attachment at a concentration of 0.005%, and this inhibition was released by the addition of glucose or mannose at 10 mM. Mating type plus cells of A. catenella treated with tunicamycin for 12 h were not capable of sexual attachment. These results are the first to suggest the existence of a cell–cell recognition system in sexual attachment in A. catenella.     

CELL AND GROWTH CHARACTERISTICS OF TYPES A AND B OF EMILIANIA HUXLEYI (PRYMNESIOPHYCEAE) AS DETERMINED BY FLOW CYTOMETRY AND CHEMICAL ANALYSES1

Two morphotypes of Emiliania huxleyi (Lohmann 1902) Hay et al. 1967, types A and B, known to be unequally distributed in the oceans, were grown in dilution cultures at a range of photon flux densities (PFDs) (1.5–155 μmol photons·m^?2·s^?1) and two temperatures (10° and 15° C). Calcite carbon and organic carbon content of the cells as well as instantaneous growth rate, cell size, chlorophyll fluorescence, and light-scatter properties clearly depended on growth conditions and differed considerably for the two morphotypes. The ratio between calcite carbon and organic carbon production showed an optimum of 0.65 in E. huxleyi type A cells at PFD = 17.5. The ratio increased slightly with a temperature increase from 10° to 15°C but remained < 1.0 at both temperatures in light-limited cells. In contrast, calcite carbon production exceeded organic carbon production (ratio: 1.4–2.2) in phosphate-deprived cultures. Emiliania huxleyi type B generally showed a higher calcite carbon/organic carbon ratio than E. huxleyi type A, but the relation with PFD was similar. The content of calcite carbon and organic carbon as well as the instantaneous growth rate, cell size, chlorophyll fluorescence, and light-scatter properties showed large diel variations that were closely related to the division cycle. Our results show the importance of mapping the structure of any sampled cell population with respect to the phase in the cell division cycle, as this largely determines the outcome of not only “per cell” measurements but also short time (less than 24 h) flux measurements. For instance, dark production of calcite by E. huxleyi was negatively affected by cell division. Slowly growing (phosphate-stressed) cultures produced calcite in the light and in the dark. In contrast, rapidly growing cultures at 10°C produced calcite only in the light, whereas in the dark there was a significant loss of calcite due to dissolution.     

THE EFFECT OF LABELING INTENSITY,ESTIMATED BY REAL-TIME CONFOCAL LASER SCANNING MICROSCOPY,ON FLOW CYTOMETRIC APPEARANCE AND IDENTIFICATION OF IMMUNOCHEMICALLY LABELED MARINE DINOFLAGELLATES1

Two different fluorescein isothiocyanate (FITC) conjugates were used to analyze the effect of labeling intensity on the flow cytometric appearance of marine dinoflagellates labeled with antibodies that specifically recognized the outer cell wall. Location of the labeling was revealed by epifluorescence and real-time confocal laser scanning microscopy using an anti-rabbit IgG/FITC-conjugated secondary antiserum. Flow cytometric measurements showed that cells of Prorocentrum species labeled this way could not always be distinguished from unlabeled cells. The labeling intensity increased several times when a biotinylated anti-rabbit IgG secondary antiserum was used in combination with a streptavidin/FITC conjugate. Flow cytometry indicated that the labeling intensity had increased 50%, which resulted in an improved separation of clusters of labeled and unlabeled cells.