REGULATION OF GAS VESICLE CONTENT AND BUOYANCY IN LIGHT- OR PHOSPHATE-LIMITED CULTURES OF APHANIZOMENON FLOS-AQUAE (CYANOPHYTA)1

Measurements of the gas vesicle space in steady-state light or phosphate-limited cultures of Aphanizomenon flos-aquae Ralfs, strain 7905 showed that gas vesicle content decreased as energy-limited growth rate increased hut was the same at several phosphate-limited growth rates. Upon a decrease in growth irradiance, gas vesicle content did increase in phosphate-limited cultures, hut the cultures remained nonbuoyant as long as P was limiting. Buoyant, energy-limited cultures lost their buoyancy in less than 2 h when exposed to higher irradiances. The primary mechanism for buoyancy loss was the accumulation of polysaccharide as ballast. Collapse of gas vesicles by turgor pressure played a minor role in the loss of buoyancy. When cultures were exposed to higher irradiances, cells continued to synthesize gas vesicles at the same rate as before the shift for at least 1 generation time. The amount of ballast required to make individual filaments in the population sink varied 4-fold. This variation appears to be due to differences in gas vesicle content among individual filaments.     

OXYGEN-INDUCED CHANGES IN MORPHOLOGY OF AGGREGATES OF APHANIZOMENON FLOS-AQUAE (CYANOPHYCEAE): IMPLICATIONS FOR NITROGEN FIXATION POTENTIALS1

The filamentous nitrogen-fixing cyanobacterium Aphanizomenon flos-aquae (L.) Ralfs forms bundle or fake shaped aggregates which can provide buoyancy control, protection against intense illumination, enhancement of phycosphere nutrient regeneration, and which may result from size-selective herbivory by zooplankton. The dimensions of aggregates can change quickly. In this study, after a period of darkness, illumination caused aggregates to elongate approximately five-fold over a 10-15 min period. The metamorphosis was reversible upon cessation of illumination and through successive light-dark cycles. Manipulations of environmental oxygen concentration and photosystem Ü activity (via DCMU amendment), together with measurements made inside flakes with O₂-sensitive microelectrocles, showed that the metamorphosis was a response to oxygen concentration and operated to enhance diffusive efflux of photosynthetically produced oxygen during illumination. During darkness oxygen concentration within contracted aggregates became severely depleted relative to the environment. We propose that metamorphic minimization of local oxygen concentration is an adaptation that enhances the ability of Aphanizomenon flos-aquae to fix atmospheric nitrogen via the oxygen-labile nitrogenase enzyme system.     

STORAGE OF PHOSPHORUS IN NITROGEN-FIXING ANABAENA FLOS-AQUAE (CYANOPHYCEAE)1

P accumulation and metabolic pathway in N₂-fixing Anabaena flos-aquae (Lyngb.) Bréb were investigated in P-sufficient (20 μMP) and P-limited (2 μMP) turbidostats in combined N-free medium. The cyanobacterium grew at its maximum rate (μ_max, 1.13 d^?1) at the high P concentration and at 65% of μ_max under P limitation, with total cell P concentrations (Q_P) at steady states of 12.0 and 5.2 fmol·cell^?1, respectively. At steady state, polyphosphates (PP_i) accounted for only 3% of Q_P (0.4 fmol·cell^?1) in P-rich cells. Its concentration in P-limited cells was 5.8% (0.3 fmol·cell^?1). On the other hand, sugar P was very high at 22% of Q_P in P-rich cells and was undetectable in P-limited cells. Pulse chase experiments with ³²P showed that P-rich cells initially incorporated the labeled P into the acid-soluble PP_i fraction within the first few minutes and to a lesser extent into nucleotide P. Radioactivity in the PP_i then declined rapidly with concomitant increases in sugar P and nucleotide P fractions. In contrast, in P-limited cells, no radiolabel was detected in acid-soluble PP_i, and ³²P was initially incorporated into nucleotide P, sugar P, and ortho P fractions. The latter two fractions then subsequently declined. Therefore, under N₂-fixing conditions the cyanobacteria appeared to store P as sugar P and also utilize P through different pathways under P-rich and -limited conditions. When nitrate was supplied as the N source under P-sufficient conditions, PP_i accounted for about 15% of steady-state Q_P, but no sugar P was detected. Therefore, the same organism stored P in different cell P fractions depending on its N sources.     

EFFECT OF SODIUM AND NITRATE ON GROWTH OF ANABAENA FLOS-AQUAE (CYANOPHYTA)1

The growth response of a nitrogen fixing cyanobacterium, Anabaena flos-aquae (Lyng) Bréb., to sodium and nitrate was examined in batch culture under controlled laboratory conditions. Sodium (range 0-12 mg Na⁺· L^-1) enhanced growth of the cyanobacterium under nitrate-sufficient (5.7 mg NO³-N · L^-1) but not nitrate-limited (0.49 mg NO³- N · L^-1) conditions. The magnitude of the growth response was related to the nutritional history of the culture. No significant effect of sodium on nitrate utilization was observed. The increase in ambient sodium levels in many lakes may provide a competitive advantage to cyanobacteria.     

DYNAMICS OF CELLULAR AND EXTRACELLULAR cAMP IN ANABAENA FLOS-AQUAE (CYANOPHYTA): INTRINSIC CULTURE VARIABILITY AND CORRELATION WITH METABOLIC VARIABLES1

The production and extracellular release of cyclic adenosine 3′: 5′-monophosphate (cAMP) by the blue-green alga Anabaena flos-aquae (Lyngb.) Breb. varied greatly within and between active growth phase and stationary phase and under differing nutrient regimes. Enhanced cellular cAMP production was found in actively growing Anabaena inoculated into media deficient in nitrate or phosphate, or into fresh media containing non-limiting nutrient concentrations. In stationary phase Anabaena, but not actively growing cells, the concentrations of intra-cellular cAMP present in cells grown under a variety of nutrient regimes could be significantly correlated to [¹⁴C]-bicarbonate uptake by an exponential relationship.     

CYANOBACTERIAL BUOYANCY REGULATION: THE PARADOXICAL ROLES OF CARBON1

In stratified lakes, dominance of the phytoplankton by cyanobacteria is largely the result of their buoyancy and depth regulation. Bloom-forming cyanobacteria regulate the gas vesicle and storage polymer contents of their cells in response to interactive environmental factors, especially light and nutrients. While research on the roles of nitrogen and phosphorus in cyanobacterial buoyancy regulation has reached a consensus, evaluations of the roles of carbon have remained open to dispute. We investigated the various effects of changes in carbon availability on cyanobacterial buoyancy with continuous cultures of Microcystis aeruginosa Kuetz. emend. Elenkin (1924), a notorious bloom-former. Although CO₂ limitation of photosynthesis can promote buoyancy in the short term by preventing the collapse of turgor-sensitive gas vesicles and/or by limiting polysaccharide accumulation, we found that sustained carbon limitation restricts buoyancy regulation by limiting gas vesicle as well as polysaccharide synthesis. These results provide an explanation for the positive effects of bicarbonate enrichment on cyanobacterial nitrogen uptake and bloom formation in lake experiments and may help to explain the pattern of cyanobacterial dominance in phosphorus-enriched, low-carbon lakes.     

NOTE ESTABLISHMENT OF AXENIC CULTURES OF ANABAENA FLOS-AQUAE AND APHANOTHECE NIDULANS (CYANOBACTERIA) BY LYSOZYME TREATMENT

A new method using lysozyme for the production of axenic cultures of Anabaena flos-aquae De Brebisson and Aphanothece nidulans P. Richter was developed. Cyanobacterial growth was not inhibited at concentrations up to 1.2 g·L⁻¹ of lysozyme, whereas the growth of heterotrophic bacteria was suppressed. At concentrations up to 0.8 g·L⁻¹ of lysozyme, ampicillin caused a reduction of heterotrophic bacteria. The axenic cultures of these strains were acquired through a simple treatment using 1.0 g·L⁻¹ of lysozyme without ampicillin. These cyanobacteria resisted digestion by lysozyme at our experimental concentrations, whereas bacteria were digested selectively. This method of purification seems to be especially useful with cyanobacterial species that are sensitive to antibiotics or other germicidal agents.     

KINETICS OF GROWTH AND DEATH IN ANABAENA FLOS-AQUAE (CYANOBACTERIA) UNDER LIGHT LIMITATION AND SUPERSATURATION

The kinetics of population growth and death were investigated in Anabaena flos-aquae (Lyngb.) Bréb grown at light intensities ranging from limitation to photoinhibition (5 W·m⁻² to 160 W·m⁻²) in a nutrient-replete turbidostat. Steady-state growth rate (μ, or dilution rate, D) increased with light intensity from 0.44·day⁻¹ at a light intensity of 5 W·m⁻² to 0.99·day⁻¹ at 20 W·m⁻² and started to decrease above about 22 W·m⁻², reaching 0.56·day⁻¹ at 160 W·m⁻². The Haldane function of enzyme inhibition fit the growth data poorly, largely because of the unusually narrow range of saturation intensity. However, it produced a good fit (P < 0.001) for growth under photoinhibition. Anabaena flos-aquae died at different specific death rates (γ) below and above the saturation intensity. When calculated as the slope of a v_x⁻¹ and D⁻¹ plot, where v_x and D are cell viability (or live cell fraction) and dilution rate, respectively; γ was 0.047·day⁻¹ in the range of light limitation and 0.103·day⁻¹ under photoinhibition. Live vegetative cells and heterocysts, either in numbers or as a percentage of the total cells, showed a peak at the saturation intensity and decreased at lower and higher intensities. The ratio of live heterocysts to live vegetative cells increased with intensity when light was limiting but decreased when light was supersaturating. In cells growing at the same growth rate, the ratio was significantly lower under light inhibition than under subsaturation and the cell N:C ratio was also lower under inhibition. The steady-state rate of dissolved organic carbon (DOC) production increased with light intensity. However, its production as a percentage of the total C fixation was lowest at the optimum intensity and increased as the irradiance decreased or increased. The rate and percentage was significantly higher under photoinhibition than limitation in cells growing at the same growth rate. About 22% of the total fixed carbon was released as DOC at the highest light intensity. No correlation was found between the number of dead cells and DOC.     

CARBOXYSOME CONTENT OF SYNECHOCOCCUS LEOPOLIENSIS (CYANOPHYTA) IN RESPONSE TO INORGANIC CARBON1

The carboxysome content of chemostat grown Synechococcus leopoliensis (Racib.) Komarek increases under inorganic carbon limitation. At growth rates of ca. 85%μ_max the carboxysome content (±SE) was 0.57 ± 0.09 carboxysomes·cell section^?1. Under severe carbon limitation (ca. 13%μ_max) this increased to 3.4 · 0.3 carboxysomes·cell section^?1. Corresponding to this change is a three order of magnitude decrease in the half-saturation constant of photosynthesis for dissolved inorganic carbon. Nitrogen and phosphorus limitation had no effect on carboxysome content or the kinetics of photosynthesis with respect to inorganic carbon. These results are discussed in light of the apparent lack of photorespiration in these organisms.     

KINETICS OF CELL DEATH IN THE CYANOBACTERIUM ANABAENA FLOS-AQUAE AND THE PRODUCTION OF DISSOLVED ORGANIC CARBON1

Kinetics of cell death and the production of dissolved organic carbon (DOC) were investigated in Anabaena flos-aquae (Lyngb.) Bréb grown on three different N sources (N₂nitrate, and ammonium) in a phosphorus (P)-limited chemostat. The fraction of live cells in the total population increased as growth rate increased with decreasing P limitation. Cell death was less in nitrate and ammonium media than in N₂. The specific death rate (γ), when calculated as the slope ofv^?1_x vs. D^?1, where v_xand D are live cell fraction (or cell viability) and dilution rate, respectively, was 0. 0082 day^?1 in N₂and 0.0042 day^?1 in nitrate. The slope of the plot in ammonium culture was not significant; however, the value of the live cell fraction was within the range for the NO^?₃culture. The fraction of live vegetative cells in N₂ culture was constant at all growth rates and the increase in the overall live cell fraction with growth rate was due entirely to an increase in live heterocysts. Live heterocysts comprised 3.5% of the total cells at a growth rate of 0.25 day^?1 and increased to 6.3% at 0.75 day^?1 with the ratio of live heterocysts to live vegetative cells linearly increasing with growth rate. The fraction of live vegetative cells was invariant in nitrate cultures us in N₂cultures. The live heterocysts fraction also increased with growth rate in nitrate cultures, along with the live heterocysts : live vegetative cells ratio, but the level was lower than in N₂cultures. DOC released from dead cells increased inversely with growth rate in N₂from 36.4% of the total DOC at a growth rate of 0.75 day^?1 to 54.15% at 0.25 day^?1. The contribution of cell death to the total DOC production in nitrate and ammonium media was significantly less than that under N₂DOC from dead cells consisted mainly of high-molecular-weight compounds, whereas DOC excreted from live cells was largely of low molecular weight.     

BUOYANCY REGULATION IN THE COLONIAL DIAZOTROPHIC CYANOBACTERIUM TRICHODESMIUM TENUE: ULTRASTRUCTURE AND STORAGE OF CARBOHYDRATE,POLYPHOSPHATE, AND NITROGEN1

Trichodesmium tenue Wille (1904) was examined using transmission electron microscopy to determine the role of carbohydrate, phosphorus, and nitrogen storage in buoyancy regulation. Carbohydrate storage area (mean = 2.06 ± 0.61 [SE] μm²; 6.62% of total cell area) in negatively buoyant colonies (NBCs) was significantly higher (P < 0.001) than in positively buoyant colonies (PBCs) (mean = 0.38 ± 0.06 μm²; 0.73%). Distinct diel periodicity of carbohydrate content was found in NBCs demonstrated by an increase from darkness to afternoon. Polyphosphate content was significantly higher (P < 0.001) in NBCs, with a mean of 0.44± 0.10 μm² (1.54%), as compared to PBCs, with a mean of 0.14 ± 0.05 μm² (0.24%). Polyphosphate content increased in NBCs from morning to evening, and PBCs had a 10% decrease from morning to afternoon. Calculations indicated that averaged effects of polyphosphate on increased cell density is approximately 20% of that from carbohydrate accumulation. Density contribution due to ballast weight of carbohydrate and polyphosphate indicated that NBCs were 12 times more dense than PBCs. Mean area of cyanophycin granules (N storage) was not significantly different between PBCs and NBCs. In conclusion, Trichodesmium tenue can regulate buoyancy by carbohydrate ballasting similar to that noted in limnetic cyanobacteria. Polyphosphate storage and possibly nitrogen storage products play a significant role in buoyancy regulation.     

FACTORS INFLUENCING THE TOXICITY AND ANIMAL SUSCEPTIBILITY OF ANABAENA FLOS-AQUAE (CYANOPHYTA) BLOOMS1

Biological factors have been found which can cause variable toxicity of colony and clonal isolates of Anabaena flos-aquae (Lyngb.) de Bréb. when cultured in the laboratory. These factors help to explain some of the variable toxicity of and animal susceptibility to A. flos-aquae blooms in nature. Two bacteria in the Enterobacteriaceae isolated from a toxic waterbloom, depressed toxin production in selected bacteria-free toxic clones of A. flos-aquae. These toxin-depressing bacteria decreased culture toxicity 3-fold from 80 to 240 mg/kg (intra-peritoneal in male mice). Many colony isolates from a toxic bloom had minimum lethal dosages (LD_min) greater than 240 mg/kg. This was because they were composed of mixtures of toxic and nontoxic filaments. The oral LD_min of the toxin from A. flos-aquae clonal isolate NRC-44-1 varied significantly for 6 different animal species. Using these oral LD_min it is estimated that a surface-concentrated bloom of toxic A. flos-aquae, having a biomass density of 20 mg/ml dry weight, would cause death of ducks or calves when 20 ml/kg was consumed whereas a monogastric animal such as a rat would require 80 ml/kg.     

SEASONAL ANALYSIS OF VOLATILE ORGANIC BIOGENIC SUBSTANCES (VOBS) IN FRESHWATER PHYTOPLANKTON POPULATIONS DOMINATED BY DINOBRYON,MICROCYSTIS AND APHANIZOMENON1

Over the course of one year the volatile organic biogenic substances (VOBS) found in the water of a eutrophied shallow lake were determined by gas chromatographic and mass spectrometric methods. The substances detected belonged to nor-carotenoids, terpenoids, unsaturated hydrocarbons, ketones, and aldehydes. The occurrence of particular VOBS correlated with the frequency with which certain phytoplankton species were recorded. The latter showed a well developed succession in that year. The following correspondences were found: β-cyclocitral and Microcystis; heptadec-cis 5-ene and Oscillatoria redekei; geosmin and argosmin and Aphanizomenon gracile; hepta-trans, cis 2,4-dienal, deca-trans, cis 2,4-dienal and Dinobryon; octa-trans, cis 1,3,5-triene and Asterionella formosa. Dictyopterenes and ectocarpene were only detected in high amounts in one sample obtained in August. The VOBS exhibited marked dynamics in the lake and usually were rapidly eliminated from the water body.     

REGULATION OF GAMETOGENESIS IN SCENEDESMUS OBLIQUUS (CHLOROPHYCEAE)1

The effects of nutrients, temperature and light on gametogenesis in Scenedesmus obliquus (Turp.) Kütz, were studied in culture. Concentrations of nitrogen in the medium employed showed a marked influence on gamete production. Gametogenesis is inhibited by N excess but is not a response to N starvation or depletion. A drop in N level from that of the growth medium is not required, nor does it per se trigger gametogenesis. The N concentration satisfying growth requirements insufficiently low to permit sexual differentiation. Nitrogen level in the growth medium has no effect on subsequent N to maintain a typical culture. Number of gametes present at maximum production time is inversely related to N concentration, but neither time of onset of gametogenesis, nor time of maximum gamete production is affected by N concentration. Cultures incubated at 15 C in medium lacking N take a minimum of 20—24 h to develop cells irreversibly committed to gamete formation. At the concentrations tested, no medium component other than the N-containing salt affected gametogenesis. Temperature influences both time of maximum production and numbers present at maximum production time. Time of maximum production is inversely related to incubation temperature; a 15 C incubation temperature yielded highest gamete production. Light enhances gametogenesis but gamete formation can occur in absence of light. Achievement of a light-saturated response is dependent upon illumination given at two critical periods: one occurs shortly after N withdrawal; the other occurs later, when cells are becoming irreversibly committed to gamete formation. Ability to produce gametes diminished with prolonged laboratory culture.     

CHANGES IN THE MORPHOLOGY AND POLYSACCHARIDE CONTENT OF MICROCYSTIS AERUGINOSA (CYANOBACTERIA) DURING FLAGELLATE GRAZING1

To investigate the changes in the morphology and polysaccharide content of Microcystis aeruginosa (Kütz.) Kütz. during flagellate grazing, cultures of M. aeruginosa were exposed to grazing Ochromonas sp. for a period of 9 d under controlled laboratory conditions. M. aeruginosa responded actively to flagellate grazing and formed colonies, most of which were made up of several or dozens of cells, suggesting that flagellate grazing may be one of the biotic factors responsible for colony formation in M. aeruginosa. When colonies were formed, the cell surface ultrastructure changed, and the polysaccharide layer on the surface of the cell wall became thicker. This change indicated that synthesis and secretion of extracellular polysaccharide (EPS) of M. aeruginosa cells increased under flagellate grazing pressure. The contents of soluble extracellular polysaccharide (sEPS), bound extracellular polysaccharide (bEPS), and total polysaccharide (TPS) in colonial cells of M. aeruginosa increased significantly compared with those in single cells. This finding suggested that the increased amount of EPS on the cell surface may play a role in keeping M. aeruginosa cells together to form colonies.     

斑马鱼脑细胞凋亡基因对束丝藻毒素致毒的响应

水华束丝藻是淡水湖泊中常见的水华蓝藻,是我国滇池冬春季节常见的优势种群,因其产生麻痹性贝类毒素,损伤人和动物的神经系统而倍受关注。但有关该毒素对动物神经系统损伤的研究较少,特别是对水生脊椎动物中枢神经系统损伤的研究尚无报道,为此本研究通过腹腔注射5.3μg STXeq/kg bw束丝藻毒素,研究了24h内该藻毒素对斑马鱼脑组织超微结构损伤及脑细胞凋亡基因表达的影响,以揭示该毒素对脑组织的损伤及其脑细胞在基因水平对该毒素的响应机理。研究表明,束丝藻毒素引起斑马鱼脑组织超微结构损伤,出现细胞膜发泡和形成凋亡小体等典型的细胞凋亡结构;从分子水平进一步分析显示,该毒素引起脑细胞p53、bax、caspase-3和c-jun等凋亡相关基因的表达上调,其上调量分别是对照组表达上调量的1.92、1.55、1.63和1.55倍,且具有时间-效应关系。这说明该毒素能通过引起脑细胞凋亡基因的表达异常,使脑细胞出现凋亡性形态损伤而导致脑细胞死亡;斑马鱼脑细胞可通过启动p53→bax→caspase-3线粒体径路实现其对该毒素的响应机制;束丝藻毒素具有损伤鱼类脑的神经毒性;这是束丝藻毒素引起脑细胞凋亡基因表达异常及超微结构损伤的直接证据,也是脑细胞在基因水平对束丝藻毒素积极响应分子机理的首次报道。     

REGULATION OF ALTERNATIVE PATHWAY RESPIRATION IN CHLAMYDOMONAS REINHARDTH (CHLOROPHYCEAE)1

The inhibitor propyl gallate was used to estimate partitioning of respiratory electron flow between the cytochrome amd alternative pathways in Chlamydomonas reinhardtii Dangeard. Nutrient limitation (nitrogen or phosphorus resulted in a large increase in alternative pathway capacity relative to cytochrome pathway activity, without regulating in engagement of the alternative pathway. High rates of respiration, which could be induced in phosphate-starved cells by a combination of phosphate addition and uncoupler, resulted in alternative pathway activity. Osmotic stress resulted in decreased electron flow through the cytochrome pathway and increased flow through the alternative pathway, while high temperature also resulted in alternative pathway engagement. Incubation with exogenous carbon sources could increase the rate of respiratory O₂ consumption; the increase was mediated entirely by the alternative pathway. We suggest that the alternative pathway functions in these cells both to maintain respiration during environmentally induced stress and as on energy overflow.     

RELATIONSHIP BETWEEN PHOTOSYNTHETIC OXYGEN CYCLING AND CARBON ASSIMILATION IN SYNECHOCOCCUS WH7803 (CYANOPHYTA)1

Mass spectrometric analysis of oxygen uptake and evolution in the light by marine Synechococcus WH7803 indicated that the respiration rate was near zero at low irradiance levels but increased significantly at high irradiances. The light intensity (I_r) at which oxygen uptake began to increase with increasing light intensity depended on the growth irradiance of the culture. In each case, I_r coincided with the minimum light intensity for saturation of carbon assimilation (I_k). At irradiances >I_r, net oxygen evolution rates paralleled carbon assimilation rates. Oxygen uptake at high light intensities was inhibited by DCMU, indicating that oxygen uptake was due to Mehler reaction activity. The onset of Mehler activity at I_k supports the idea that oxygen becomes an alternative sink for electrons from photosystem I when NADPH turnover is limited by the capacity of the dark reactions to utilize reductant.     

BIOCHEMICAL AND CYTOLOGICAL EVIDENCE FOR THE STIMULATION OFCLATHRIN-COATED PIT (VESICLE) FORMATION BY EXOGENOUS FOLIC ACID IN DUNALIELLA SALINA (CHLOROPHYTA)1

The effects of two kinds of molecules, Pb²⁺ and folic acid, on the formation of clathrin-coated pits and vesicles were studied in the unicellular green alga, Dunaliella salina Teod. Measurable amounts of algal clathrin were obtained in the postmicrosomal fraction from cells treated with folic acid. In contrast, algal clathrin heavy chains were below the detection limit in the postmicrosomal fraction from control and lead nitrate-treated cells. Consistent with the biochemical evidence, electron microscopy showed more clathrin-coated pits and vesicles in folic acid-treated cells compared to control cells or cells treated with lead nitrate alone. Observations of folic acid/lead nitrate-treated cells confirmed the endocytosis of Pb²⁺ through clathrin-coated pits and vesicles. As additional evidence for clathrin in the folic acid-stimulated cells of Dunaliella salina, clathrin was isolated and, for the first time in algae, the calcium-dependent reconstitution of clathrin cages was successfully obtained in vitro.