INTERACTIVE EFFECTS OF COPPER AND SILICIC ACID ON RESTING SPORE FORMATION AND VIABILITY IN A MARINE DIATOM1

The toxic effects of copper on resting spore formation and viability in the marine diatom Chaetoceros protuberans Lauder were determined both with and without silicic acid added to the medium. With silicic acid available, partial inhibition of resting spore formation occurred only at the highest cupric ion activity (pCu 8.6), while the percentage of cells forming spores at pCu's 10.2 and 11.3 was nearly the same as in the controls. Without silicic acid added to the medium, sporulation was completely inhibited at pCu 8.6 and greatly inhibited at pCu 10.2. At pCu 11.3 and in the controls, the rate of spore formation was less than 50%. The results indicate that the inhibition of resting spore formation by copper is related to the concentration of silicic acid available to cells of C. protuberans. This is consistent with previous studies which show that copper toxicity during vegetative growth involves interference with silicification in diatoms and is a function of the silicic acid concentration of the medium. Viable resting spores of C. protuberans were still present in cultures following exposure to elevated copper concentrations during a 100-day incubation period. This indicates that resting spores can serve to enhance diatom survival in areas polluted by heavy metals.     

EFFECT OF COPPER ON GROWTH,SILICIC ACID UPTAKE AND SOLUBLE POOLS OF SILICIC ACID IN THE MARINE DIATOM,THALASSIOSIRA WEISFLOGII (BACILLARIOPHYCEAE)1

The toxicity of Cu to Thalassiosira weissflogii (Grunow) was investigated, focusing on the internal soluble pool of silicic acid. Silicic acid uptake and growth rates were found to be functions of both the cupric ion activity and the concentration of silicic acid in the growth medium. The soluble pool of Si per cell depended on the balance between the uptake rate and the division rate. The soluble pool in non-dividing cultures reflected simply the uptake rate (and inhibition by copper of the uptake rate), but in dividing cultures the soluble pools had complex patterns with time depending on uptake rates and timing of division. Intracellular soluble pools of silicic acid are a good indicator for the relative inhibition of uptake and growth processes.     

KINETICS OF SILICIC ACID UPTAKE AND RATES OF SILICA DISSOLUTION IN THE MARINE DIATOM THALASSIOSIRA PSEUDONANA1,2

Tracer techniques using the stable isotope ³⁰Si were used to measure rates of silicic acid uptake and silica dissolution in silicon replete and silicon depleted populations of 2 clones of the marine diatom Thalassiosira pseudonana Hasle & Heimdal. Uptake kinetics were describable using the Michaelis-Menten equation for enzyme kinetics, and no threshold concentration for uptake was evident. The maximum specific uptake rate of the estuarine clone 3H (0.062–0.092 · h^?1) was higher than that of the Sargasso Sea clone 13-1 (0.028–0.031 · h^?1), but half-saturation constants for uptake by the 2 clones were not measurably different (0.8–2.3 μM for 3H; 1.4–1.5 μM for 13-1). There was little or no light dependence of uptake in populations grown under optimal light conditions prior to the experiment. Exponentially growing populations released silicic acid to the medium by dissolution of cellular silica at rates ranging from 6.5 to 15% of the maximum uptake rate.     

EFFECTS OF COPPER TOXICITY ON SILICIC ACID UPTAKE AND GROWTH IN THALASSIOSIRA PSEUDONANA11

The toxicity of Cu to Thalassiosira pseudonana (Hustedt) Hasle and Heimdal was investigated by examining both short and long term effects of Cu on cellular processes. Toxic levels of Cu (pCu* < 13) were found to inhibit short term Si(OH)₄ uptake rates with kinetics characteristic of irreversible inhibition at a hypothetical Cu-sensitive Si(OH)₄ transport site. Residual Si(OH)₄ concentrations (those below which no uptake could occur) were found to increase with increasing levels of Cu, and the toxic effects of Cu could be reversed by increasing the concentrations of Si(OH)₄ in the medium. The actual uptake of Cu by the cells was found to vary inversely with the ambient Si(OH)₄ concentration. Copper did not inhibit the uptake of NO₃^? or PO₄^3-. The long term inhibition of growth rate by Cu in this species was shown not to be a result of Si deficiency caused by the inhibition of Si(OH)₄ uptake. Cu inhibited cells were found to have higher Si cell quotas (including a sizable soluble pool) than the control cultures. They were, however, observed to have aberrant frustules, significantly larger than the control cells, suggesting interference with the silification process as a possible mechanism for inhibition of growth by Cu. A conceptual model is proposed for the Cu-Si(OH)₄-growth relationship. It includes a Cu sensitive Si(OH)₄ transport site that may also serve to transport Cu into the cell, and growth inhibition mediated by intracellular Cu concentrations which may block cell division or cause general cellular disfunction.     

DIATOM MINERALIZATION OF SILICIC ACID IV. KINETICS OF SOLUBLE Si POOL FORMATION IN EXPONENTIALLY GROWING AND SYNCHRONIZED NAVICULA PELLICULOSA1

Silicic acid taken up from the growth medium by Navicula pelliculosa (Bréb.) Hilse was shown to enter at least two compartments: i) soluble pools; ii) insoluble fraction comprised predominantly of the silica frustule. Soluble Si pools were extracted by a variety of agents from cells uniformly labeled for ten generations in medium containing ⁶⁸Ge-Si(OH)₄. 100 C water soluble and 0 C perchloric acid (PCA) soluble Si pools of 680 mM Si·l^?1 and 490 mM Si·l^?1 cell water represented 13 and 9%, respectively, of total, cell Si in exponential growth phase cells. Uniformly labeled cells synchronized by the combined synchronization technique accumulate at the cell cycle stage where silica frustule development is initiated. These cells contain water and PCA soluble pools of 10 nmol Si·10⁶ cells^?1 and-8.8 nmol Si·10⁶ cells^?1, respectively. On addition of Si(OH)₄, a rapid uptake ensues allowing the Si pool to expand 2.5-fold, apparently to provide precursors of the silica frustule.     

ULTRASTRUCTURE OF CHAETOCEROS MUELLERI (BACILLARIOPHYCEAE): AUXOSPORE,RESTING SPORE AND VEGETATIVE CELL MORPHOLOGY1

The structure of the frustule of auxospores, resting spores and vegetative cells of Chaetoceros muelleri Lemm. are described with LM and SEM. Vegetative frustules are relatively small and lightly silicified, are not united into filaments, and appear unornamented under LM and SEM. The setae are circular to subcircular in transverse section with spines and puncta arranged in a spiral pattern. The resting spore and auxospore frustules are more silicified than the vegetative frustules and appear unornamented under LM and SEM. The auxospores of C. muelleri were previously unknown.     

OBSERVATIONS ON THE SURVIVAL AND GERMINATION OF RESTING SPORES OF THREE CHAETOCEROS (BACILLARIOPHYCEAE) SPECIES1,2

Resting spores (hypnospores) of Chaetoceros diadema (Ehrenberg) Gran, Chaetoceros vanheurckii Gran, and Chaetoceros didymus Ehrenberg were collected from a large plastic enclosure moored in Saanich Inlet, B.C., Canada. The effects of combinations of temperature and irradiance on the germination of these resting spores were investigated. Nutrient uptake, carbon fixation, and changes in the photosynthetic capacity of the germinating spores were also examined. Resting spores germinated optimally at combinations of temperature and irradiance similar to those in the environment during sporulation. They did not germinate at irradiances 1.3 μEin m^?2 s^?1 or temperatures >25.3° C. Nitrate, phosphate and silicate were taken up after the resting spores had germinated and resumed vegetative growth. Chlorophyll a fluorescence in vivo, and the DCMU-induced increase in in vivo fluorescence also increased after the resting spores had germinated. Resting spores began to fix carbon as soon as they were placed in light. Spores remained viable for at least 645 d. The length of time between first exposure to light and germination did not change during this period; however, the percentage of viable resting spores decreased markedly. None of the Chaetoceros spores germinated after 737 d of storage at 2–4° C in darkness.     

THE ULTRASTRUCTURE AND CYTOCHEMISTRY OF RESTING CELL FORMATION IN AMPHORA COFFAEFORMIS (BACILLARIOPHYCEAE)1

The ultrastructure of logarithmic-growing cells and of resting cells in laboratory cultures of Amphora coffaeformis (Ag.) Kütz. isolated from deep ocean water was examined using electron and light microscopy. The acid Phosphatase activity, chlorophyll a and lipid content were assessed at weekly intervals of resting cell formation during cold-dark treatment, simulating deep ocean water. Approximately 4 wk are required to complete resting cell formation. During the first week, the cytoplasm undergoes extensive transformation and lysosomal activity is observed. Large vacules decrease in size and many small ones develop, the mitochondria become fewer and one or more massive mitochondria appear possibly by fusion of smaller ones; the cytoplasm becomes densely granular. During the second and third week, the cytoplasm continues to contract, lipid bodies begin to develop and the plastid becomes densely stained. At the fourth week, the mature resting cell is formed containing one or more massive mitochondria, a well-formed plastid, and granular cytoplasm containing occasional lipid droplets. There is no change in frustule morphology and the cytoplasm does not produce a protective layer. The variation in chemical constituents correlates with microscopic structure of the cells. The fine structure of cells during growth resumption when exposed to light at 25 c is presented. Previous reports of viable, chlorophyll-containing cells at great depths in the ocean may be explained by the results reported in this paper.     

DIATOM MINERALIZATION OF SILICIC ACID. VI. THE EFFECTS OF MICROTUBULE INHIBITORS ON SILICIC ACID METABOLISM IN NAVICULA SAPROPHILA1

The role of microtubules in silicon metabolism leading to valve formation was investigated in the pennate diatom Navicula saprophila Lange-Bertalot & Bonik. By using synchronized cells blocked after mitosis and cytokinesis but prior to cell wall formation, effects due to inhibition of mitosis were eliminated. Cells were treated with three anti-microtubule drugs to assess the role of microtubules. Chemical analogs to two of the drugs provided controls for inhibition not related to microtubule disruption. Although all three anti-microtubule drugs reduced cell separation at high concentrations (1 × 10^?3 M), podophyllotoxin was the only drug which reduced cell separation at concentrations lower than 1 × 10^?5 M. None of the drugs at any concentration tested affected cell viability. There was no differential inhibitory effect between the active and inactive drugs on silicic acid transport, total uptake, incorporation, or pool formation. There was no qualitative difference between silica incorporated in treated and untreated cells. A colchicine binding component was isolated from N. saprophila. The characteristics of colchicine binding suggest this component may be tubulin. Microtubules do not appear to be involved in any of the steps of silicon metabolism leading to valve formation and yet they have profound influence on the symmetry and pattern of the mineralized product, the siliceous valve.     

SPORE FORMATION IN THE LIFE CYCLES OF THE DIATOMS CHAETOCEROS DIADEMA AND LEPTOCYLINDRUS DANICUS1

A nitrogen limitation technique elicited the entire life cycle of the marine centric diatoms Chaetoceros diadema (Ehr.) Gran and Leptocylindrus danicus Cleve. In C. diadema the sexual cycle followed the same pattern as in the previously investigated C. didymus. Sexuality took place in narrow diameter cells, only at 2 and 5° C, and was seldom seen. Resting spore formation took place in cells of all sizes and at all temperatures at which the species grew vegetatively (2–15° C). The L. danicus life cycle is probably unique among diatoms. Nitrogen depletion induced sexuality in the entire culture at 10 and 15° C if the cell diameter was narrow (3–8 μm). Auxospore formation was followed by resting spore formation directly within the auxospore. In C. diadema, as in most centric diatoms, resting spores are not an obligate part of the life cycle, but they are in L. danicus. Resting spore formation is a versatile adaptive response in C. diadema, depending only on nitrogen depletion, although promoted by low temperatures. In L. danicus the linkage to the sexual process sharply limits conditions under which resting spores can form.     

THE EFFECTS OF IRON AND COPPER AVAILABILITY ON THE COPPER STOICHIOMETRY OF MARINE PHYTOPLANKTON1

We studied the interactive effects of iron (Fe) and copper (Cu) availability on the growth rates, Cu quotas, and steady‐state Cu‐uptake rates (ρ_ssCu) of 12 phytoplankton (from four classes and two marine environments). A mixed‐effect statistical model indicated that low Fe significantly decreased phytoplankton growth rates. In contrast, lowering Cu levels only decreased the growth rates of the oceanic phytoplankton. Under Fe/Cu sufficiency, the Cu quotas ranged from 0.36 to 3.8 μmol Cu · mol^?1 C. Copper levels in the growth medium had a significant positive effect on the Cu quotas, and this effect was dependent on the algal class. Under Fe/Cu sufficiency, the highest average Cu quotas were observed for the Bacillariophyceae, followed by the Cyanophyceae, Prymnesiophyceae, and lastly the Dinophyceae. Similar taxonomic trends were observed for the ρ_ssCu. Although the Cu:C ratios were not significantly higher in oceanic strains, there are five independent lines of evidence supporting a more important role of Cu in the physiology of the oceanic phytoplankton. The mixed‐effect model indicated a significant Cu effect on the growth rates and ρ_ssCu of the oceanic strains, but not the coastal strains. In addition, lowering the Cu concentration in the media decreased the Cu quotas and ρ_ssCu of the oceanic strains to a greater extent (5.5‐ and 5.4‐fold, respectively) than those of the coastals (3.8‐ and 4.7‐fold, respectively). Iron limitation only had a significant effect on the Cu quotas of the oceanic strains, and this effect was dependent on Cu level and taxonomic class. Our results highlight a complex physiological interaction between Fe and Cu in marine phytoplankton.     

EFFECTS OF VARIATION IN TEMPERATURE. II. ON THE FATTY ACID COMPOSITION OF EIGHT SPECIES OF MARINE PHYTOPLANKTON1

Eight species of marine phytoplankton showed significant variation in the relative amount of some fatty acids (FAs) in response to variation in temperature. Large changes in relative amounts of certain FAs occurred as a result of a 15° C change in growth temperature. For example, 14:0 increased from ?4% of total FAs at 10° C to > 20% at 25° C for Chaetoceros simplex and Isochrysis aff. galbana but decreased for Phaeodactylum tricornutum. The percentage of the polyunsaturated fatty acid (PUFA) 16:ω1 was consistently greater at 10° C than at 25° C, and the converse was usually true for 16: 4ω3. Calculated over all eight species, there was a modest but significant inverse relationship between the percentage of PUFAs and temperature. Only for Thalassiosira pseudonana was the percentage of either of the PUFAs and nutritionally essential fatty acids (EFAs) also an inverse function of temperature. For T. pseudonana, the percentage of the EFA 22:6ω3 decreased linearly with increasing temperature over the range from 10 to 25° C. For three species, the ratio of unsaturated/saturated FAs was correlated with growth rate when growth rate was controlled by variation in irradiance and temperature. Only for Thalassiosira pseudonana was the ratio of unsaturated/saturated FAs also an inverse function of temperature alone.     

EFFECTS OF ULTRAVIOLET RADIATION ON PHOTOSYNTHESIS IN THE SUBTROPICAL MARINE DIATOM,CHAETOCEROS GRACILIS (BACILLARIOPHYCEAE)1

Acclimation to ambient ultraviolet radiation (UVR) was examined in a subtropical marine diatom, Chaetoceros gracilis Schutt. Short-term exposure to UVR (<24 h) reduced the efficiency of photosynthetic energy conversion, carbon fixation, activity of 1,5-bisphosphate carboxylase-loxygenase (RUBISCO), and the rapid turnover of the putative Dl reaction center (32 kda) protein, whereas longer-term exposure to ambient UVR (24–48 h) revealed a steady-state acclimation, defined as recovery of carbon fixation and RUBISCO activity to rates equivalent to treatments without exposure to UVR. The turnover of D1 and chlorophyll a (Chl a) remained high during exposure to UVR. Efficiency of energy conversion by photosystem II, measured with double flash (pump and probe) fluorometry, increased by 24% in cells acclimated to UVR. Acclimation to UVR had no detectable effect on the functional absorption cross-section or cellular concentrations of Chl a, Chl c, or total carotenoids. However, the maximum rate of carbon fixation was reduced by UVR on a Chl a basis but remained unaffected on a per-cell basis. Response to UVR exposure in this subtropical diatom has two components: a short-term inhibitory response and a longer-term acclimation process that ameliorates the inhibition of carbon fixation.     

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.     

DIATOM MINERALIZATION OF SILICIC ACID. VII. INFLUENCE OF MICROTUBULE DRUGS ON SYMMETRY AND PATTERN FORMATION IN VALVES OF NAVICULA SAPROPHILA DURING MORPHOGENESIS1

Microtubule involvement in diatom valve symmetry and pattern formation was investigated using cells synchronized subsequent to mitosis and cytokinesis but prior to cell wall formation. Two analog drug pairs, each consisting of an active and an inactive microtubule drug, were used to distinguish inhibitory effects related or unrelated to microtubule disruption. The active anti-microtubule drug of each analog pair produced significantly higher percentages of aberrant valves than did the respective inactive analogs. High frequencies of aberrant valves also were caused by N-isophenlpropylcarbamate, which disorganizes rather than disrupts microtubules. Valves could be placed into different classes based upon characteristic aberrations. Formation of these classes was not random but was instead a function of both the drug and the drug concentration. The central nodule and the raphe were the principal valve components affected by anti-microtubule drugs. Stria alterations appeared as a secondary result of alterations in the central nodule/raphe. Valve aberrations occurred at very low drug concentrations in the range 1 × 10^?6 to 1 × 10^?9M.     

EFFECTS OF SILICON DEFICIENCY ON LIPID COMPOSITION AND METABOLISM IN THE DIATOM CYCLOTELLA CRYPTICA1

The effects of silicon deficiency on the metabolism and composition of lipids in Cyclotella cryptica T13L Reimann, Lewin, and Guillard were examined. Silicon-deficient cells had higher levels of neutral lipids (primarily triacylglycerols) and higher proportions of saturated and monounsaturated fatty acids than silicon-replete cells. After 4 h of silicon deficiency, the percentage of newly assimilated NaH¹⁴CO₃ partitioned into lipids increased from 27.6% to 54.1%, whereas the percentage partitioned into chrysolaminarin decreased from 21.6% to 10.6%. In addition, pulse-chase experiments with NaH¹⁴CO₃ indicated that the amount of ¹⁴C in the total cellular lipid fraction increased by 32% after 12 h of silicon deficiency despite the absence of additional photoassimilable ¹⁴C. Therefore, the accumulation of lipids in response to silicon deficiency appears to be due to two distinct processes: (a) an increase in the proportion of newly assimilated carbon partioned into lipids, and (2) a slow conversion of previously assimilated carbon from non-lipid compounds into lipids     

A MORPHOMETRIC STUDY OF LEAD AND COPPER EFFECTS ON DIATOMA TENUE VAR. ELONGATUM (BACILLARIOPHYTA)1

Polyphosphate bodies containing lead were induced in laboratory cultures of Diatoma tenue var. elongatum Lyngb. by the addition of phosphorus and 0.05 μg-atoms/l Pb to P deficient medium. Morphometric analysis of cells exposed to Pb showed a significant decrease in number of mitochondria with a concomitant increase in their volume and an increase in membranous organelles in the vacuole compared to phosphorus starved and phosphorus sufficient controls. Exposure of cultures to 0.08 μg-atoms/l copper resulted in reduction of the number of polyphosphate bodies formed during luxury uptake but no other significant morphological changes in cellular organelles. Ecological implications of the interactions between nutrients and low level trace metal contamination are discussed.     

EFFECT OF BLUE-GREEN LIGHT ON PHOTOSYNTHETIC PIGMENTS AND CHLOROPLAST STRUCTURE IN THE MARINE DIATOM STEPHANOPYXIS TURRIS1

Blue-green light increased the chlorophyll concentration and chloroplast number of cells of Stephanopyxis turris (Grev.) Ralfs, compared to white light controls. Light fields for growth were 400 μW·cm^?2 (12:12 LD cycles). Chlorophyll increased up to 100%/cell, but no change in the ratio of chlorophylls to major carotenoids occurred. The effect was, therefore, not that of complementary chromatic adaptation. At the same time, blue-green light enhanced the photosynthetic fixation of CO₂. At the ultrastructure level, an increase in, and rearrangement of, the thylakoid system occurred.     

ROLE OF LIGHT AND THE CELL CYCLE ON THE INDUCTION OF SPERMATOGENESIS IN A CENTRIC DIATOM1

The centric diatom, Thalassiosira weissflogii Grun., can be induced to undergo spermatogenesis by exposing cells maintained at saturating levels of continuous light to either dim light or darkness. Using flow cytometry to determine the relative DNA and chlorophyll content per cell, the number of cells within a population that responded to and induction signal was measured. From 0 to over 90% of a population differentiated into male gametes depending upon both the induction trigger and the population examined, regardless of the average cell size of the population. Through the use of synchromized cultures, we demonstrated that responsiveness to an induction trigger was a function of cell cycle stage; cells in early G₁ were not yet committed to complete mitosis and were induced to form male gametes, whereas cells further along in their cell cycle were unresponsive to these same cues. A simple model combining the influence of light on the mitotic cell cycle and on the induction of spermatogenesis is proposed to explain the observed diversity in population responses to changes in light conditions.