NITRATE REDUCTASE ACTIVITY OF AMPHIDINIUM CARTERI AND CACHONINA NIEI (DINOPHYCEAE) IN BATCH CULTURE: DIEL PERIODICITY AND EFFECTS OF LIGHT INTENSITY AND AMMONIA1,2

The activity of extracted NADH-NO₃^? reductase was measured in the marine dinoflagellates Amphidinium carteri Hulburt and Cachonina niei Loeblich. Its activity showed a diel periodicity and was ca. twice as great at midday as at midnight. The enzyme activity was unstable, with an in vitro half-life of 2–3 h. Values of enzyme activity were low or undetectable during lag phase but paralleled the instantaneous growth rate value during log phase. Nitrate reductase activity was not found in the stationary phase of growth, but additions of NO₃^? resulted in enzyme activity after 24h. When A. carteri was exposed to a series of light intensities for several weeks, the division rate and enzyme activity increased with increasing light intensity up to saturating intensities. In 6 h exposures, enzyme activity decreased with decreasing light intensities below light intensities saturating division rate. Additions of NH₄⁺ (0.5–50 μm) to A. carteri cultures decreased the amount of extractable enzyme. The in vitro activity was not inhibited by similar NH⁺₄ concentrations.     

PHOSPHATE ACCUMULATION AND METABOLISM BY HETEROSIGMA AKASHIWO (RAPHIDOPHYCEAE) DURING DIEL VERTICAL MIGRATION IN A STRATIFIED MICROCOSM1

Diel vertical migration by Heterosigma akashiwo (Hada) Hada (Raphidophyceae) was monitored in a 1.5 in tall microcosm. Vertical stratification, with low salinity and low orthophosphate (P_i) concentration in the upper layer and high salinity and high P_i concentration in the lower layer, was simulated in the tank, analogous to summer stratification in the Seto Inland Sea. The phosphate metabolism of H. akashiwo during this vertical migration was studied using ³¹P-NMR spectroscopy. At night this species migrated to the lower phosphate-rich layer and took up inorganic phosphate (P_i) which then was accumulated as polyphosphate (PP_i) by an increase in the chain length of PP_i During the daytime this species migrated to the phosphate-depleted surface water and utilized the accumulated PP_i for photophosphorylation by decreasing the chain length of PP_i During the first night after the phosphorus was introduced to the previously impoverished waters, the cells took up inorganic phosphate, accumulating the new phosphorus nutrient internally as P_i But the cells did not convert P_i to PP_i presumably due to their lack of ATP. After the second day of the experiment, conversion of P_i to PP_i at night was much more rapid than on the first day, presumably due to increased ATP availability. Then the cycle continued, with uptake of P_i and conversion to PP_i at night at the bottom and its utilization during the day at the surface. These data suggest that the role of PP_i in the metabolism of this species appears to be as a phosphate pool which regulates the level of P_i and ATP in the cell. Diel vertical migration allows this red tide species to shuttle between the phosphate-rich lower layer and the photic upper layer in stratified waters. ³¹P-NMR is shown to be a valuable tool in studying the phosphorus metabolism in migrating organisms.     

HEMOLYTIC ACTIVITY OF HETEROCAPSA CIRCULARISQUAMA (DINOPHYCEAE) AND ITS POSSIBLE INVOLVEMENT IN SHELLFISH TOXICITY

Heterocapsa circularisquama Horiguchi is lethal to shellfish, particularly bivalves such as pearl oysters ( Pinctada fucata Gould). No detrimental effects of this flagellate on fish have been observed thus far. In this study, we found that H. circularisquama causes mammalian erythrocytes to lyse. Among the erythrocytes tested, rabbit erythrocytes showed the highest susceptibility, whereas erythrocytes from cattle, sheep, and human were relatively insensitive. Heterocapsa triquetra Stein, which is morphologically similar to H. circularisquama but not toxic to bivalves, showed no hemolytic activity toward rabbit erythrocytes. Culture supernatant or ultrasonic-ruptured cells of H. circularisquama showed only weak hemolytic activity. Hemolytic activity was found in the ethanol extract of H. circularisquama cells, suggesting that the hemolytic agents may be more stable in ethanol than in aqueous solution. Both an intact flagellate cell suspension and the ethanol extract caused morphological changes and eventual collapse of unfertilized eggs of Pacific oyster. Furthermore, the ethanol extract was lethal to the microzooplankton rotifer Brachionus plicatilis Müller, which is highly sensitive to H. circularisquama. Our results suggest that a hemolytic toxin produced by H. circularisquama may be one of the causative agents responsible for the shellfish toxicity.     

DIEL CHANGES IN PHASED-DIVIDING CULTURES OF CERATIUM FURCA (DINOPHYCEAE): NUCLEOTIDE TRIPHOSPHATES,ADENYLATE ENERGY CHARGE,CELL CARBON,AND PATTERNS OF VERTICAL MIGRATION1

The diel pattern of cell division, cell carbon, adenine nucleotides and vertical migration was determined for laboratory cultures of the photosynthetic marine dinoflagellate, Ceratium furca (Ehr.) Clap. & Lachm., entrained on an alternating 12:12 LD schedule at 20 C. Cell division was initiated during the latter portion of the dark period with ca. 30% of the population undergoing division. Cell C increased during the light period and exhibited a linear decrease with a loss of 33% during the dark period. ATP · cell^?1 increased during the light period and decreased by ca. 40–50% during the dark period. The diel patterns of cell C and ATP tended to “buffer” the magnitude of the change in C:ATP ratios around an overall mean value of 89. There was no obvious trend in the concentration of [GTP + UTP] · cell^?1 over the cell cycle. The cellular adenylate energy charge was maintained at values between 0.8 to 0.9 throughout the 24 h LD cycle, despite a ca. 40% decrease in total adenylates (A_T= ATP + ADP + AMP) during the dark period on 12:12 LD, and over a 68% decrease in ATP during 42 h of continuous darkness. These data lend experimental support to the theory of cellular metabolic control by the adenine nucleotides. With lateral illumination on 12:12 LD cycles, the cells began to concentrate at the surface of the experimental tubes shortly before the lights were turned on, and at the bottom of the tubes shortly before the lights were extinguished. This pattern continued for 6 days in continuous darkness, suggesting that the vertical migration pattern is independent of a phototactic response and may be under the control of an endogenous rhythm.     

DIEL PATTERNS IN LIGHT ABSORPTION AND ABSORPTION EFFICIENCY FACTORS OF ISOCHRYSIS GALBANA (PRYMNESIOPHYCEAE)1

The chl a specific absorption coefficients [a* (λ), m²·mg chl a ^{? 1}] were examined in chemostat culture of the Prymnesiophyceae Isochrysis galbana (Parke) under a 12:12‐h light:dark cycle at low light (75 μmol photons·m ^{? 2}·s ^{? 1}) and high light (500 μmol photons· m ^{? 2}·s ^{? 1}) conditions. Other associated measurements such as pigment composition, cell density, and diameter as the measure of cell size were also made at the two light regimes every 2 h for 2 days to confirm the periodicity. A distinct diel variability was observed for the a* (λ) with maxima near dawn and minima near dusk. The magnitude of diel variation in a* (440) was 15% at low light and 22% at high light. Pronounced diel patterns were observed for cell size with minima near dawn and maxima near dusk. The magnitude of diel variation in cell size was 9.3% at low light and 21% at high light. The absorption efficiency factors [Q _a (440)] were determined by reconstruction using intracellular concentrations of pigments and cell size. The Q _a (440) also showed a distinct diel variability, with minima near dawn and maxima near dusk. The diel variation in a* (λ) and Q _a (λ) was primarily caused by changes in cell size due to growth, although there was some influence from diel variations in the intracellular pigment concentrations. The results presented here indicated that diel variation in a* (λ) was an important component of the optical characterization of phytoplankton.     

HETEROCAPSA ARCTICA SUBSP. FRIGIDA SUBSP. NOV. (PERIDINIALES,DINOPHYCEAE)—DESCRIPTION OF A NEW DINOFLAGELLATE AND ITS OCCURRENCE IN THE BALTIC SEA1

Characteristics important in identification of Heterocapsa species (i.e., thecal plate pattern, body scale structure, and shape and position of the nucleus and pyrenoid) are practically identical in the dinoflagellate investigated here and in Heterocapsa arctica T. Horig. described from the Canadian Arctic. Analysis of internal transcribed spacer (ITS) sequences confirms that the two dinoflagellates are very closely related; however, there is a clear difference in their size and shape. Our experiments show that the low‐salinity Baltic Sea brackish water does not reduce the size of the marine H. arctica to match that of the Baltic Sea morphotype. On the basis of these dissimilarities in general morphology and its geographic isolation in the Baltic Sea, we consider our material sufficiently differentiated from the typical H. arctica to warrant the status of a new subspecies, H. arctica subsp. frigida subsp. nov. Being of a distinct cell shape, the occurrence of subsp. frigida has been recorded in Algaline phytoplankton monitoring data collected since 1993. Although it has never been responsible for high biomass blooms, it commonly occurs in spring in the Northern Baltic Proper and in the western Gulf of Finland, when the water temperatures are <5°C.     

THE RESPONSE OF GROWTH AND BIOCHEMICAL COMPOSITION TO VARIATIONS IN DAYLENGTH, TEMPERATURE, AND IRRADIANCE IN THE MARINE DIATOM THALASSIOSIRA PSEUDONANA (BACILLARIOPHYCEAE)

The relationships between the growth rate of the marine diatom Thalassiosira pseudonana (Hustedt) and irradiance, daylength, and temperature were determined in nutrient-sufficient semicontinuous cultures. The initial slopes of the growth versus total daily irradiance curves were not affected by temperature or daylength. Growth versus irradiance was best modeled as a hyperbolic function at short daylengths and better modeled as an exponential function at longer daylengths. The maximum or light-saturated growth rates at each daylength were modeled as a hyperbolic function of daylength. This model was extended in a novel manner to include temperature dependence providing a framework that can be used to interpret other experimental data on growth rate versus daylength. The resulting model should be useful in global models of phytoplankton growth. Carbon, nitrogen, and chl a quotas were influenced by daylength, irradiance, and temperature. Both C and N quotas were positive exponential functions of irradiance, whereas N and chl a quotas were significantly greater for cells grown at the lower temperature. The ratio chl a :C quota (chl a :Q_c) was a strong negative exponential function of total daily irradiance. Cells grown at 10° C had significantly greater chl a :Q_c ratios than those grown at 18° C, and daylength also had a significant positive influence on chl a :Q_c. The apparent effect of daylength on chl a :Q_c was removed by standardizing chl a :Q_c to growth rate (μ), resulting in a temperature-dependent relationship between chl a :Q_c·μ⁻¹ and irradiance that accounted for 95% of the variation in the data.     

PHYSIOLOGICAL AND OPTICAL PROPERTIES OF RHIZOSOLENIA FORMOSA (BACILLARIOPHYCEAE) IN THE CONTEXT OF OPEN-OCEAN VERTICAL MIGRATION1

Cultures of Rhizosolenia formosa H. Peragallo were studied to assess whether or not physiological and optical characteristics of this large diatom were consistent with the ability to migrate vertically in the open ocean. Time-course experiments examined changes in chemical composition and buoyancy of R. formosa during nitrate (N)–replete growth, N starvation, and recovery. Cells could maintain unbalanced growth for at least 53 h after depletion of ambient nitrate. Increases in C:N and carbohydrate: protein ratios observed during N starvation reversed within 24 h of reintroduction of nitrate to culture medium. Buoyancy was related to nutrition: Upon N depletion, the percentage of positively buoyant cells decreased to 4% from 11% but reverted to 9% within 12 h of nitrate readdition. Cells took up nitrate in the dark. Nitrogen-specific uptake rates averaged 0.48 d^?1; these rates were higher than N-specific growth rates (0. 15 d^?1), indicating the potential for luxury consumption of nitrate, which can be stored for later use. Measurements of photosynthesis vs. irradiance, chlorophyll-specific absorption (a_ph*(λ)), and pigment composition showed that cells may be adapted for growth under a wide range of irradiances. Values of a_ph*(λ) were lower for N-depleted cells than for N-replete cells, and N-depleted cells had higher ratios of total carotenoids to chlorophyll a. Aggregation of chloroplasts was more pronounced in N-depleted cells. These are possibly photoprotective mechanisms that would be an advantage to N-depleted cells in surface waters. Compounds that absorb in the ultraviolet region were detected in N-replete cells but were absent in N-depleted cultures. Overall, these results have important implications for migrations of Rhizosolenia in nature. Cells may survive fairly long periods in N-depleted surface waters and will continue to take up carbon; then they can resume nitrate uptake and revert to positive buoyancy upon returning to deep, N-rich water. Uncoupled uptake of carbon and nitrogen during migrations of Rhizosolenia is a form of new production that may result in the net removal of carbon from oceanic surface waters.     

CHEMICAL COMPOSITION AND PHOTOSYNTHETIC CHARACTERISTICS OF ETHMODISCUS REX (BACILLARIOPHYCEAE): EVIDENCE FOR VERTICAL MIGRATION1

Ethmodiscus rex (Rattray) Wiseman and Hendey cells from near surface net tows in the Southwest Atlantic Ocean and Caribbean Sea were examined for chemical composition, internal nutrient pool concentrations, and oxygen evolution characteristics. Elemental ratios indicated nitrogen limitation with C:N:P ratios of 125:9:1 (atoms), and carbon: chlorophyll (chl) ratios of 129:1 (weight). However, internal nitrate pools (1.4–27.1 mM) suggested that cells were not N-limited. Intracellular NO₃^? accounted for up to 54% (range = 3–54%) of the total N quota in some samples. Photosynthetic parameters were consistent with a high-light-adapted population and suggested an instantaneous maximum chl-specific photosynthetic rate (P^B_max) of 4.8–12.4 nmol O₂·μg chl^?1· h^?1. Respiration rates varied ten-fold and were inversely related to P^B_max Ethmodiscus chemical composition and buoyancy characteristics are similar to vertically migrating Rhizosolenia mats and the non-motile dinoflagellate Pyrocystis noctiluca Murray (Schuett). The presence of internal NO₃^? pools in Ethmodiscus suggests that this genus is also vertically migrating to exploit sub-surface nitrogen pools. Such behavior may be widespread in large, non-motile oceanic phytoplankton. Based on ascent rate data, chemical composition, and photosynthetic rates, we estimate that the entire division–migration cycle for Ethmodiscus requires at least 7–12 days.     

PHYSICAL AND CHEMICAL CONDITIONS SURROUNDING THE DIURNAL VERTICAL MIGRATION OF CRYPTOMONAS SPP. (CRYPTOPHYCEAE) IN A SEASONALLY STRATIFIED MIDWESTERN RESERVIOR (USA)

Cross Reservoir, a small mesotrophic reservoir located at the University of Kansas Ecological Reserves (Kansas, USA), contained a dense metalimnetic community of algae and photosynthetic bacteria between early July and late October 1997–1999. Within this community, various Cryptomonas species, primarily C. erosa (Ehrenberg), C. erosa var. reflexa (Marsson), and C. rostratisformis (Skuja), diurnally migrated as indicated by in situ fluorescence monitoring and direct phytoplankton enumeration. The Cryptomonas spp. typically resided near the oxic–anoxic boundary of the water column; however, they actively migrated upward during the day and descended to lower anoxic locations at night, apparently responding to diurnal changes in their local habitat. Their nocturnal environment had moderate levels of sulfide, elevated secondary nutrients, and a community of anoxygenic phototrophic bacteria, whereas their daytime environment had higher light, lower nutrients, and no local photosynthetic bacteria. Monitoring indicated that the Cryptomonas spp. migration was generally linked to daily variations in absolute light intensity (e.g. sunny vs. cloudy days) and the level of other potentially growth-limiting resources, particularly nitrogen and phosphorus. However, further analyses showed that the primary factor that determined whether the Cryptomonas spp. migrated or not on a given day was the slope of the light gradient immediately above the Cryptomonas spp. peak.     

CA2+ BINDING PROTEIN CALRETICULIN IN CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA): BIOCHEMICAL CHARACTERIZATION, DIFFERENTIAL EXPRESSION DURING SEXUAL REPRODUCTION, AND PHYLOGENETIC ANALYSIS

The occurrence of calreticulin, the main Ca²⁺ binding protein in the endoplasmic reticulum of eukaryotic cells, was investigated in the unicellular green alga Chlamydomonas reinhardtii Dangeard. The biochemical characterization of a diethylaminoethyl purified extract highlighted the presence, on SDS-PAGE, of a 55-kDa protein that stained blue with the Stains All dye, a diagnostic feature of acidic Ca²⁺ binding proteins. Immunoblot analyses revealed a strong cross-reaction of the Chlamydomonas reinhardtii protein with antibodies to plant calreticulins and the endoplasmic reticulum retention signal HDEL. Furthermore, the 55-kDa protein bound [⁴⁵Ca²⁺] and had an acidic isoelectric point (pI = 4.9) but was neither glycosylated nor phosphorylated. N-terminal sequencing revealed strong amino acid sequence similarity to calreticulin from other sources. The presence of calreticulin in Chlamydomonas reinhardtii suggested that an endoplasmic reticulum Ca²⁺ buffering mechanism was present in this unicellular chlorophyte. The data suggest an early origin and high conservation of endoplasmic-reticulum-mediated Ca²⁺ functions in eukaryotes, whereby specific posttranslational modifications of the proteinhave been specifically acquired in different lineages of photosynthetic eukaryotes. Moreover, northern and western blot analysis experiments showed a regulation of calreticulin expression during Chlamydomonas sexual reproduction with a high abundance of calreticulin mRNA and protein in reproductive cells.